Summary of Supportive Science
Regarding Thimerosal Removal
Updated December 2012
www.safeminds.orgScience Summary on Mercury in Vaccines (Thimerosal Only)
SafeMinds Update – December 2012
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Contents
ENVIRONMENTAL IMPACT ................................................................................................................................. 4
A PILOT SCALE EVALUATION OF REMOVAL OF MERCURY FROM PHARMACEUTICAL WASTEWATER USING GRANULAR ACTIVATED CARBON
(CYR 2002) .................................................................................................................................................................4
BIODEGRADATION OF THIOMERSAL CONTAINING EFFLUENTS BY A MERCURY RESISTANT PSEUDOMONAS PUTIDA STRAIN (FORTUNATO
2005) .........................................................................................................................................................................4
USE OF ADSORPTION PROCESS TO REMOVE ORGANIC MERCURY THIMEROSAL FROM INDUSTRIAL PROCESS WASTEWATER (VELICU 2007)5
HUMAN & INFANT RESEARCH ............................................................................................................................ 5
IATROGENIC EXPOSURE TO MERCURY AFTER HEPATITIS B VACCINATION IN PRETERM INFANTS (STAJICH 2000)..................................5
MERCURY CONCENTRATIONS AND METABOLISM IN INFANTS RECEIVING VACCINES CONTAINING THIMEROSAL: A DESCRIPTIVE STUDY
(PICHICHERO 2002).......................................................................................................................................................6
HAIR MERCURY IN BREAST‐FED INFANTS EXPOSED TO THIMEROSAL‐PRESERVED VACCINES (MARQUES 2007) ...................................6
MERCURY LEVELS IN NEWBORNS AND INFANTS AFTER RECEIPT OF THIMEROSAL‐CONTAINING VACCINES (PICHICHERO 2008)...............6
HEPATITIS B TRIPLE SERIES VACCINE AND DEVELOPMENTAL DISABILITY IN US CHILDREN AGED 1‐9 YEARS (GALLAGHER 2008) .............7
NEONATE EXPOSURE TO THIMEROSAL MERCURY FROM HEPATITIS B VACCINES (DOREA 2009)......................................................8
URINARY PORPHYRIN EXCRETION IN NEUROTYPICAL AND AUTISTIC CHILDREN (WOODS 2010).....................................................8
EMBRYONIC EXPOSURE TO THIMEROSAL, AN ORGANOMERCURY COMPOUND, CAUSES ABNORMAL EARLY DEVELOPMENT OF
SEROTONERGIC NEURONS. (IDA‐ETO 2011)........................................................................................................................9
SPECIATION OF METHYL‐ AND ETHYL‐MERCURY IN HAIR OF BREASTFED INFANTS ACUTELY EXPOSED TO THIMEROSAL‐CONTAINING
VACCINES. (DOREA 2011) ...............................................................................................................................................9
INTEGRATING EXPERIMENTAL (IN VITRO AND IN VIVO) NEUROTOXICITY STUDIES OF LOW‐DOSE THIMEROSAL RELEVANT TO VACCINES.
(DOREA 2011)............................................................................................................................................................10
NEONATAL EXPOSURE TO THIMEROSAL FROM VACCINES AND CHILD DEVELOPMENT IN THE FIRST 3YEARS OF LIFE. (MROZEK‐BUDZYN
2012) .......................................................................................................................................................................11
BREAST‐FEEDING AND RESPONSES TO INFANT VACCINES: CONSTITUTIONAL AND ENVIRONMENTAL FACTORS. (DOREA 2012).............11
THIMEROSAL EXPOSURE IN EARLY LIFE AND NEUROPSYCHOLOGICAL OUTCOMES 7‐10 YEARS LATER. (BARILE 2012).........................12
NON‐HUMAN PRIMATE INFANT RESEARCH .......................................................................................................12
COMPARISON OF BLOOD AND BRAIN MERCURY LEVELS IN INFANT MONKEYS EXPOSED TO METHYLMERCURY OR VACCINES CONTAINING
THIMEROSAL (BURBACHER 2005)...................................................................................................................................12
PEDIATRIC VACCINES INFLUENCE PRIMATE BEHAVIOR, AND AMYGDALA GROWTH AND OPIOID LIGAND BINDING (HEWITSON 2008).13
MICROARRAY ANALYSIS OF GI TISSUE IN A MACAQUE MODEL OF THE EFFECTS OF INFANT VACCINATION (WALKER 2008) ..............14
DELAYED ACQUISITION OF NEONATAL REFLEXES IN NEWBORN PRIMATES RECEIVING A THIMEROSAL‐CONTAINING HEPATITIS B VACCINE:
INFLUENCE OF GESTATIONAL AGE AND BIRTH WEIGHT. (HEWITSON 2010) ...............................................................................14
INFLUENCE OF PEDIATRIC VACCINES ON AMYGDALA GROWTH AND OPIOID LIGAND BINDING IN RHESUS MACAQUE INFANTS: A PILOT
STUDY. (HEWITSON 2010) ............................................................................................................................................15
ANIMAL RESEARCH ...........................................................................................................................................16
NEUROTOXIC EFFECTS OF POSTNATAL THIMEROSAL ARE MOUSE STRAIN DEPENDENT (HORNIG 2004) ...........................................16
EFFECT OF THIMEROSAL, A PRESERVATIVE IN VACCINES, ON INTRACELLULAR CA2+ CONCENTRATION OF RAT CEREBELLAR NEURONS
(UEHA‐ISHIBASHI 2004) ...............................................................................................................................................16Science Summary on Mercury in Vaccines (Thimerosal Only)
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THIMEROSAL DISTRIBUTION AND METABOLISM IN NEONATAL MICE: COMPARISON WITH METHYL MERCURY (ZAREBA 2007)..............16
EFFECTS OF LIPOPOLYSACCHARIDE AND CHELATOR ON MERCURY CONTENT IN THE CEREBRUM OF THIMEROSAL‐ADMINISTERED MICE
(MINAMI 2007)..........................................................................................................................................................17
GENDER‐SELECTIVE TOXICITY OF THIMEROSAL (BRANCH 2008).............................................................................................18
EFFECTS OF INTERMITTENT, VACCINATION‐LIKE SCHEME, THIMEROSAL ADMINISTRATION ON RAT DEVELOPMENT AND BEHAVIOR (OLCZAK
2008) .......................................................................................................................................................................18
EFFECTS OF POSTNATAL ADMINISTRATION ON THIMEROSAL ON RAT DEVELOPMENT AND BEHAVIOR (DUSZCZYK 2008).....................19
INDUCTION OF METALLOTHIONEIN IN MOUSE CEREBELLUM AND CEREBRUM WITH LOW‐DOSE THIMEROSAL INJECTION (MINAMI 2009)
.................................................................................................................................................................................20
NEONATAL ADMINISTRATION OF A VACCINE PRESERVATIVE, THIMEROSAL, PRODUCES LASTING IMPAIRMENT OF NOCICEPTION AND
APPARENT ACTIVATION OF OPOID SYSTEM IN RATS (OLCZAK 2009)........................................................................................21
IDENTIFICATION AND DISTRIBUTION OF MERCURY SPECIES IN RAT TISSUES FOLLOWING ADMINISTRATION OF THIMEROSAL OR
METHYLMERCURY. (RODRIGUES 2010).............................................................................................................................21
NEONATAL ADMINISTRATION OF THIMEROSAL CAUSES PERSISTENT CHANGES IN MU OPIOID RECEPTORS IN THE RAT BRAIN (OLCZAK
2010) .......................................................................................................................................................................22
CHRONIC METALS INGESTION BY PRAIRIE VOLES PRODUCES SEX‐SPECIFIC DEFICITS IN SOCIAL BEHAVIOR: AN ANIMAL MODEL OF AUTISM.
(CURTIS 2010)..........................................................................................................................................................23
NEUROLIGIN‐DEFICIENT MUTANTS OF C. ELEGANS HAVE SENSORY PROCESSING DEFICITS AND ARE HYPERSENSITIVE TO OXIDATIVE STRESS
AND MERCURY TOXICITY. (HUNTER 2010) ........................................................................................................................23
LASTING NEUROPATHOLOGICAL CHANGES IN RAT BRAIN AFTER INTERMITTENT NEONATAL ADMINISTRATION OF THIMEROSAL. (OLCZAK
2010) .......................................................................................................................................................................24
PERSISTENT BEHAVIORAL IMPAIRMENTS AND ALTERATIONS OF BRAIN DOPAMINE SYSTEM AFTER EARLY POSTNATAL ADMINISTRATION OF
THIMEROSAL IN RATS. (OLCZAK 2011) .............................................................................................................................24
MERCURY DISPOSITION IN SUCKLING RATS: COMPARATIVE ASSESSMENT FOLLOWING PARENTERAL EXPOSURE TO THIOMERSAL AND
MERCURIC CHLORIDE. (BLANUSA 2012) ...........................................................................................................................25
SEX‐DEPENDENT CHANGES IN CEREBELLAR THYROID HORMONE‐DEPENDENT GENE EXPRESSION FOLLOWING PERINATAL EXPOSURE TO
THIMEROSAL IN RATS. (KHAN 2012)................................................................................................................................25
PRENATAL EXPOSURE TO ORGANOMERCURY, THIMEROSAL, PERSISTENTLY IMPAIRS THE SEROTONERGIC AND DOPAMINERGIC SYSTEMS IN
THE RAT BRAIN: IMPLICATIONS FOR ASSOCIATION WITH DEVELOPMENTAL DISORDERS. (IDA‐ETO 2012) ........................................26
ADMINISTRATION OF THIMEROSAL TO INFANT RATS INCREASES OVERFLOW OF GLUTAMATE AND ASPARTATE IN THE PREFRONTAL CORTEX:
PROTECTIVE ROLE OF DEHYDROEPIANDROSTERONE SULFATE. (DUSZCZYK‐BUDHATHOKI 2012)....................................................27
MATERNAL THIMEROSAL EXPOSURE RESULTS IN ABERRANT CEREBELLAR OXIDATIVE STRESS, THYROID HORMONE METABOLISM, AND
MOTOR BEHAVIOR IN RAT PUPS; SEX‐ AND STRAIN‐DEPENDENT EFFECTS. (SULKOWSKI ZL 2012)..................................................27
CELLULAR RESEARCH.........................................................................................................................................28
BIOCHEMICAL AND MOLECULAR BASIS OF THIMEROSAL‐INDUCED APOPTOSIS IN T CELLS: A MAJOR ROLE OF MITOCHONDRIAL PATHWAY
(MAKANI 2002)..........................................................................................................................................................28
THIMEROSAL INDUCES MICRONUCLEI IN THE CYTOCHALASIN B BLOCK MICRONUCLEUS TEST WITH HUMAN LYMPHOCYTES (WESTPHAL
2003) .......................................................................................................................................................................28
THIMEROSAL INDUCES DNA BREAKS, CASPASE‐3 ACTIVATION, MEMBRANE DAMAGE, AND CELL DEATH IN CULTURED HUMAN NEURONS
AND FIBROBLASTS (BASKIN 2003)...................................................................................................................................29
ACTIVATION OF METHIONINE SYNTHASE BY INSULIN‐LIKE GROWTH FACTOR‐1 AND DOPAMINE: A TARGET FOR NEURODEVELOPMENTAL
TOXINS AND THIMEROSAL. (WALY 2004) ........................................................................................................................29
UNCOUPLING OF ATP‐MEDIATED CALCIUM SIGNALING AND DYSREGULATION INTERLEUKIN‐6 SECRETION IN DENDRITIC CELLS BY
NANAMOLAR THIMEROSAL (GOTH 2006) .........................................................................................................................30Science Summary on Mercury in Vaccines (Thimerosal Only)
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THIMEROSAL INDUCES NEURONAL CELL DEATH BY CAUSING CYTOCHROME C AND APOPTOSIS‐INDUCING FACTOR RELEASE FROM
MITOCHONDRIA (YEL 2005)...........................................................................................................................................30
IN VITRO UPTAKE OF GLUTAMATE IN GLAST AND GLT‐1 TRANSFECTED MUTANT CHO‐K1 CELLS IS INHIBITED BY THE ETHYLMERCURY‐
CONTAINING PRESERVATIVE THIMEROSAL (MUTKUS 2005) ..................................................................................................30
THIMEROSAL NEUROTOXICITY IS ASSOCIATED WITH GLUTATHIONE DEPLETION: PROTECTION WITH GLUTATHIONE PRECURSORS (JAMES
2005) .......................................................................................................................................................................31
THIMEROSAL INDUCES APOPTOSIS IN A NEUROBLASTOMA MODEL VIA THE CJUN‐N‐TERMINAL KINASE PATHWAY (HERDMAN 2006) .31
THIMEROSAL INDUCES TH2 RESPONSES VIA INFLUENCING CYTOKINE SECRETION BY HUMAN DENDRITIC CELLS (AGRAWAL 2007)........31
EFFECTS OF THIMEROSAL ON NGF SIGNAL TRANSDUCTION AND CELL DEATH IN NEUROBLASTOMA CELLS (PARRAN 2005).................32
GENOTOXICITY OF THIMEROSAL IN CULTURED HUMAN LYMPHOCYTES WITH AND WITHOUT METABOLIC ACTIVATION SISTER CHROMATID
EXCHANGE ANALYSIS PROLIFERATION INDEX AND MITOTIC INDEX (EKE 2008)...........................................................................32
ZINC IONS CAUSE THE THIMEROSAL‐INDUCED SIGNAL OF FLUORESCENT CALCIUM PROBES IN LYMPHOCYTES (HAASE 2008)...............32
CHARACTERIZATION OF EARLY EVENTS INVOLVED IN HUMAN DENDRITIC CELL MATURATION INDUCED BY SENSITIZERS: CROSS TALK
BETWEEN MAPK SIGNALLING PATHWAYS (TROMPEZINSKI 2008)............................................................................................33
MITOCHONDRIAL MEDIATED THIMEROSAL‐INDUCED APOPTOSIS IN A HUMAN NEUROBLASTOMA CELLINE (SK‐N‐SH)(HUMPHREY 2009)
.................................................................................................................................................................................34
MITOCHONDRIAL DYSFUNCTION, IMPAIRED OXIDATIVE‐REDUCTION ACTIVITY, DEGENERATION, AND DEATH IN HUMAN NEURONAL AND
FETAL CELLS INDUCED BY LOW‐LEVEL EXPOSURE TO THIMEROSAL AND OTHER METAL COMPOUNDS (GEIER 2009) ...........................34
SENSITIZATION EFFECT OF THIMEROSAL IS MEDIATED IN VITRO VIA REACTIVE OXYGEN SPECIES AND CALCIUM SIGNALING. (MIGDAL 2010)
.................................................................................................................................................................................35
EVALUATION OF CYTOTOXICITY ATTRIBUTED TO THIMEROSAL ON MURINE AND HUMAN KIDNEY CELLS. (PARK 2007)........................35
THE RELATIVE TOXICITY OF COMPOUNDS USED AS PRESERVATIVES IN VACCINES AND BIOLOGICS. (GEIER 2010)...............................36
LOW MOLECULAR WEIGHT THIOLS REDUCE THIMEROSAL NEUROTOXICITY IN VITRO: MODULATION BY PROTEINS. (ZIEMINSKA 2010) ..36
RESPONSIVENESS OF HUMAN MONOCYTE‐DERIVED DENDRITIC CELLS TO THIMEROSAL AND MERCURY DERIVATIVES. (MIGDAL 2010)...37
MERCURY INDUCES AN UNOPPOSED INFLAMMATORY RESPONSE IN HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS IN VITRO.
(GARDNER 2009)........................................................................................................................................................38
LUTEOLIN AND THIOSALICYLATE INHIBIT HGCL(2) AND THIMEROSAL‐INDUCED VEGF RELEASE FROM HUMAN MAST CELLS. (ASADI
2010) .......................................................................................................................................................................38
INTERMINGLED MODULATORY AND NEUROTOXIC EFFECTS OF THIMEROSAL AND MERCURIC IONS ON ELECTROPHYSIOLOGICAL RESPONSES
TO GABA AND NMDA IN HIPPOCAMPAL NEURONS. (WYREMBEK 2010)...............................................................................39
THIMEROSAL‐DERIVED ETHYLMERCURY IS A MITOCHONDRIAL TOXIN IN HUMAN ASTROCYTES: POSSIBLE ROLE OF FENTON CHEMISTRY
IN THE OXIDATION AND BREAKAGE OF MTDNA. (SHARPE 2012) ..........................................................................................40
CYTOPROTECTIVE EFFECT OF HYALURONIC ACID AND HYDROXYPROPYL METHYLCELLULOSE AGAINST DNA DAMAGE INDUCED BY
THIMEROSAL IN CHANG CONJUNCTIVAL CELLS. (YE 2012) ....................................................................................................40
ITPKC SUSCEPTIBILITY IN KAWASAKI SYNDROME AS A SENSITIZING FACTOR FOR AUTOIMMUNITY AND CORONARY ARTERIAL WALL
RELAXATION INDUCED BY THIMEROSAL'S EFFECTS ON CALCIUM SIGNALING VIA IP3. (YETER 2012)...............................................41
THIMEROSAL‐INDUCED APOPTOSIS IN MOUSE C2C12 MYOBLAST CELLS OCCURS THROUGH SUPPRESSION OF THE PI3K/AKT/SURVIVIN
PATHWAY (LI 2012).....................................................................................................................................................42Science Summary on Mercury in Vaccines (Thimerosal Only)
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Environmental Impact
A PILOT SCALE EVALUATION OF REMOVAL OF MERCURY FROM PHARMACEUTICAL
WASTEWATER USING GRANULAR ACTIVATED CARBON (CYR 2002)
Cyr PJ, Suri RP, Helmig ED. Water Res. 2002 Nov;36(19):4725‐34.
Thimerosal (an organic mercury compound) is widely used in the pharmaceutical industry and
hospitals. This study examines the removal of mercury (thimerosal and Hg(II)) from a
pharmaceutical wastewater using F‐400 granular activated carbon (GAC) at bench and pilot
scales. Bench scale dynamic column tests are conducted with 30, 60, 90 and 120 min empty bed
contact times (EBCTs). The pilot scale study is conducted using two GAC columns‐in‐series each
of 30 min EBCT. The capital and operational cost analysis for the treatment system is performed.
Simultaneous removal of copper, turbidity, phenol, and color from the wastewater by the pilot
scale system is also reported.
BIODEGRADATION OF THIOMERSAL CONTAINING EFFLUENTS BY A MERCURY RESISTANT
PSEUDOMONAS PUTIDA STRAIN (FORTUNATO 2005)
Raquel Fortunato, João G. Crespo and M.A.M. Water Research Volume 39, Issue 15, September 2005,
Pages 3511‐3522
Thiomersal, a toxic organomercurial with a strong bactericidal effect, is the most widely used
preservative in vaccine production. As a result, vaccine production wastewaters are frequently
polluted with thiomersal concentrations above the European limit for mercury effluent
discharges for which there is, presently, no remediation technology available. This work
proposes a biotechnological process for the remediation of vaccine production wastewaters
based on the biological degradation of thiomersal to metallic mercury, under aerobic conditions,
by a mercury resistant bacterial strain. The kinetics of thiomersal degradation by a pure culture
of Pseudomonas putida spi3 was firstly investigated in batch reactors using a thiomersal
amended mineral medium. Subsequently, a continuous stirred tank reactor fed with the same
medium was operated at a dilution rate of 0.05 h−1, and the bioreactor performance and
robustness was evaluated when exposed to thiomersal shock loads. In a second stage, the
bioreactor was fed directly with a real vaccine wastewater contaminated with thiomersal and
the culture ability to grow in the wastewater and remediate it was evaluated for dilution rates
ranging from 0.022 to 0.1 h−1.Science Summary on Mercury in Vaccines (Thimerosal Only)
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USE OF ADSORPTION PROCESS TO REMOVE ORGANIC MERCURY THIMEROSAL FROM
INDUSTRIAL PROCESS WASTEWATER (VELICU 2007)
Velicu M, Fu H, Suri RP, Woods K. J Hazard Mater. 2007 Sep 30;148(3):599‐605. Epub 2007 Mar 12.
Carbon adsorption process is tested for removal of high concentration of organic mercury
(thimerosal) from industrial process wastewater, in batch and continuously flow through
column systems. The organic mercury concentration in the process wastewater is about 1123
mg/L due to the thimerosal compound. Four commercially available adsorbents are tested for
mercury removal and they are: Calgon F‐400 granular activated carbon (GAC), CB II GAC,
Mersorb GAC and an ion‐exchange resin Amberlite GT73. The adsorption capacity of each
adsorbent is described by the Freundlich isotherm model at pH 3.0, 9.5 and 11.0 in batch
isotherm experiments. Acidic pH was favorable for thimerosal adsorption onto the GACs.
Columns‐in‐series experiments are conducted with 30‐180 min empty bed contact times
(EBCTs). Mercury breakthrough of 30 mg/L occurred after about 47 h (96 Bed Volume Fed
(BVF)) of operation, and 97 h (197 BVF) with 120 min EBCT and 180 min EBCT, respectively.
Most of the mercury removal is attributed to the 1st adsorbent column. Increase in contact
time by additional adsorbent columns did not lower the effluent mercury concentration below
30 mg/L. However, at a lower influent wastewater pH 3, the mercury effluent concentration
decreased to less than 7 mg/L for up to 90 h of column operation (183 BVF).
Human & Infant Research
IATROGENIC EXPOSURE TO MERCURY AFTER HEPATITIS B VACCINATION IN PRETERM
INFANTS (STAJICH 2000)
Stajich GV, Lopez GP, Harry SW, Sexson, SW. J Pediatr. 2000 May; 136(5):679‐81.
Stajich measured blood mercury levels in low birth weight and term newborns dministered the
Hepatitis B vaccine containing 12.5 mcg ethyl mercury. The investigation documented elevated
post‐immunization concentrations relative to pre‐immunization levels in all neonates studied.
Levels of blood mercury after exposure in low birth weight infants were 7.36 (± 4.99) mcg/L.
Note: One infant was found to have developed a mercury level of 23.6 mcg/L, thus meeting the
CDC criteria as a case of chemical poisoning from mercury defined as a blood level of 10mcg/L
or greater. Science Summary on Mercury in Vaccines (Thimerosal Only)
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MERCURY CONCENTRATIONS AND METABOLISM IN INFANTS RECEIVING VACCINES
CONTAINING THIMEROSAL: A DESCRIPTIVE STUDY (PICHICHERO 2002)
Pichichero ME, Cernichiari E, Lopreiato J and Treanor J. Lancet. 2002; 360:1737‐41.
Pichichero reported a mercury blood level in a 2‐month‐old infant of 20.55 nmol/L five days
after the infant received a 37.5 mcg dose of ethylmercury (the amount contained in one DTaP
and one Hepatitis B vaccine). Many infants, however, beginning in the early 1990’s and for the
next decade, received a 62.5 mcg dose of ethylmercury (adding in the Haemophilus influenzae
type b (Hib) vaccine) at the 2‐month well baby visit. A vaccine expert from the Johns Hopkins
Institute for Vaccine Safety estimated that these infants may have experienced peak blood
mercury levels of 48.3 nmol/L; well above the presumed EPA safety threshold of 29.0 nmol/L.
As a reference point, the CDC recently defined a toxic exposure to mercury in an adult as a
blood mercury level of >10mcg /L (50 nmol/L) ‐‐ approximately the same blood level that some
infants experienced at two months of age.
HAIR MERCURY IN BREAST‐FED INFANTS EXPOSED TO THIMEROSAL‐PRESERVED VACCINES
(MARQUES 2007)
Marques RC, Dorea JG, Fonseca MF, Bastos WR, Malm O. Eur J Pediatr. 2007 Jan 20;
Marques investigated the impact of thimerosal on the total mercury content of hair in breast
fed infants receiving thimerosal containing vaccines and found exposure to vaccine‐EtHg
represents 80% of that expected from total breast milk‐Hg in the first month but only 40% of
the expected exposure integrated in the 6 months of breastfeeding. However, the Hg exposure
corrected for body weight at the day of immunization was much higher from thimerosal‐ EtHg
(5.7 to 11.3 mugHg/kg b.w.) than from breastfeeding (0.266 mugHg/kg b.w.). While mothers
showed a relative decrease (‐57%) in total hair‐mercury during the 6 months lactation there
was substantial increase in the infant's hair‐mercury (446%).
MERCURY LEVELS IN NEWBORNS AND INFANTS AFTER RECEIPT OF THIMEROSAL‐
CONTAINING VACCINES (PICHICHERO 2008)
Pichichero ME, Gentile A, Giglio N, Umido V, Clarkson T, Cernichiari E, Zareba G, Gotelli C, Gotelli M, Yan
L, Treanor J Pediatrics. 2008 Feb;121(2):e208‐14
OBJECTIVES: Thimerosal is a mercurial preservative that was widely used in multidose vaccine
vials in the United States and Europe until 2001 and continues to be used in many countries
throughout the world. We conducted a pharmacokinetic study to assess blood levels and
elimination of ethyl mercury after vaccination of infants with thimerosal‐containing vaccines.
METHODS: Blood, stool, and urine samples were obtained before vaccination and 12 hours to
30 days after vaccination from 216 healthy children: 72 newborns (group 1), 72 infants aged 2Science Summary on Mercury in Vaccines (Thimerosal Only)
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months (group 2), and 72 infants aged 6 months (group 3). Total mercury levels were measured
by atomic absorption. Blood mercury pharmacokinetics were calculated by pooling the data on
the group and were based on a 1‐compartment first‐order pharmacokinetics model. RESULTS:
For groups 1, 2, and 3, respectively, (1) mean +/‐ SD weights were 3.4 +/‐ 0.4, 5.1 +/‐ 0.6, and
7.7 +/‐ 1.1 kg; (2) maximal mean +/‐ SD blood mercury levels were 5.0 +/‐ 1.3, 3.6 +/‐ 1.5, and
2.8 +/‐ 0.9 ng/mL occurring at 0.5 to 1 day after vaccination; (3) maximal mean +/‐ SD stool
mercury levels were 19.1 +/‐ 11.8, 37.0 +/‐ 27.4, and 44.3 +/‐ 23.9 ng/g occurring on day 5 after
vaccination for all groups; and (4) urine mercury levels were mostly nondetectable. The blood
mercury half‐life was calculated to be 3.7 days and returned to prevaccination levels by day 30.
CONCLUSIONS: The blood half‐life of intramuscular ethyl mercury from thimerosal in vaccines
in infants is substantially shorter than that of oral methyl mercury in adults. Increased mercury
levels were detected in stools after vaccination, suggesting that the gastrointestinal tract is
involved in ethyl mercury elimination. Because of the differing pharmacokinetics of ethyl and
methyl mercury, exposure guidelines based on oral methyl mercury in adults may not be
accurate for risk assessments in children who receive thimerosal‐containing vaccines.
HEPATITIS B TRIPLE SERIES VACCINE AND DEVELOPMENTAL DISABILITY IN US CHILDREN AGED
1‐9 YEARS (GALLAGHER 2008)
Carolyn Gallagher and Melody Goodman Toxicological & Environmental Chemistry Vol. 90, No. 5,
September–October 2008, 997–1008
This study investigated the association between vaccination with the Hepatitis B triple series
vaccine prior to 2000 and developmental disability in children aged 1–9 years (n¼1824), proxied
by parental report that their child receives early intervention or special education services (EIS).
National Health and Nutrition Examination Survey 1999–2000 data were analyzed and adjusted
for survey design by Taylor Linearization using SAS version 9.1 software, with SAS callable
SUDAAN version 9.0.1. The odds of receiving EIS were approximately nine times as great for
vaccinated boys (n¼46) as for unvaccinated boys (n¼7), after adjustment for confounders. This
study found statistically significant evidence to suggest that boys in United States who were
vaccinated with the triple series Hepatitis B vaccine, during the time period in which vaccines
were manufactured with thimerosal, were more susceptible to developmental disability than
were unvaccinated boys. Science Summary on Mercury in Vaccines (Thimerosal Only)
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NEONATE EXPOSURE TO THIMEROSAL MERCURY FROM HEPATITIS B VACCINES (DOREA 2009)
Dórea JG, Marques RC, Brandão KG. Universidade de Brasília, Brasília, DF, Brazil. Am J Perinatol. 2009
Mar 12.
Infant exposure to ethylmercury (EtHg) has not only increased but is starting earlier as a result
of the current immunization schedule that uses thimerosal‐containing vaccines (TCVs).
Although vaccination schedule varies considerably between countries, infants in less‐developed
countries continue to be exposed to EtHg derived from more affordable TCVs. We studied the
exposure of newborns to EtHg from hepatitis B vaccines; hospital records (21,685) were
summarized for the years 2001 to 2005 regarding date of birth, vaccination date, and birth
weight. Most of the vaccinations occurred in the first 24 hours postdelivery; over the 5 years,
there was an increase in vaccinations within hours of birth (same day), from 7.4% (2001) to
87.8% (2005). Nearly 94.6% of infants are now being vaccinated within the first 24hours. Range
of mercury exposure spread from 4.2 to 21.1 mug mercury/kg body weight for those receiving
TCVs with the highest thimerosal concentration; these exposure levels are conservative for 2%
of children receiving vaccines within 2 to 3 postnatal days, when they are still going through
physiological postnatal weight loss. Because of the particular timing (transitioning from in utero
to ex utero metabolism) and specific aspects of exposure (i.e., parenteral mode, bypassing
gastroenteric barriers) and dose (related to vaccine manufacturer and with variation in birth
weight), this study reveals critical issues that can modulate toxicokinetics and toxicodynamics
of organomercurials in neonates.
URINARY PORPHYRIN EXCRETION IN NEUROTYPICAL AND AUTISTIC CHILDREN (WOODS 2010)
James S. Woods; Sarah E. Armel; Denise I. Fulton; Jason Allen; Kristine Wessels; P. Lynne Simmonds;
Doreen Granpeesheh; Elizabeth Mumper; J. Jeffrey Bradstreet; Diana Echeverria; Nicholas J. Heyer;
James P.K. Rooney From Environmental Health Perspectives
Background: Increased urinary concentrations of pentacarboxyl‐, precopro‐ and copro‐
porphyrins have been associated with prolonged mercury (Hg) exposure in adults, and
comparable increases have been attributed to Hg exposure in children with autism (AU).
Objectives: This study was designed to measure and compare urinary porphyrin concentrations
in neurotypical (NT) children and same‐age children with autism, and to examine the
association between porphyrin levels and past or current Hg exposure in children with autism.
Methods: This exploratory study enrolled 278 children 2–12 years of age. We evaluated three
groups: AU, pervasive developmental disorder‐not otherwise specified (PDD‐NOS), and NT.
Mothers/caregivers provided information at enrollment regarding medical, dental, and dietary
exposures. Urine samples from all children were acquired for analyses of porphyrin, creatinine,
and Hg. Differences between groups for mean porphyrin and Hg levels were evaluated. LogisticScience Summary on Mercury in Vaccines (Thimerosal Only)
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regression analysis was conducted to determine whether porphyrin levels were associated with
increased risk of autism. Results: Mean urinary porphyrin concentrations are naturally high in
young children and decline by as much as 2.5‐fold between 2 and 12 years of age. Elevated
copro‐ (p < 0.009), hexacarboxyl‐ (p < 0.01) and pentacarboxyl‐ (p < 0.001) porphyrin
concentrations were significantly associated with AU but not with PDD‐NOS. No differences
were found between NT and AU in urinary Hg levels or in past Hg exposure as determined by
fish consumption, number of dental amalgam fillings, or vaccines received. Conclusions: These
findings identify disordered porphyrin metabolism as a salient characteristic of autism. Hg
exposures were comparable between diagnostic groups, and a porphyrin pattern consistent
with that seen in Hg‐exposed adults was not apparent.
EMBRYONIC EXPOSURE TO THIMEROSAL, AN ORGANOMERCURY COMPOUND, CAUSES
ABNORMAL EARLY DEVELOPMENT OF SEROTONERGIC NEURONS. (IDA‐ETO 2011)
Ida‐Eto M, Oyabu A, Ohkawara T, Tashiro Y, Narita N, Narita M. Neurosci Lett. 2011 Nov
14;505(2):61‐4. doi: 10.1016/j.neulet.2011.05.053. Epub 2011 Jun 6.
Even though neuronal toxicity due to organomercury compounds is well known, thimerosal, an
organomercury compound, is widely used in pediatric vaccine preservation. In the present
study, we examined whether embryonic exposure to thimerosal affects early development of
serotonergic neurons. Thimerosal (1mg Hg/kg) was intramuscularly administered to pregnant
rats on gestational day 9 (susceptible time window for development of fetal serotonergic
system), and fetal serotonergic neurons were assessed at embryonic day 15 using anti‐
serotonin antibodies. A dramatic increase in the number of serotonergic neurons localized to
the lateral portion of the caudal raphe was observed in thimerosal group (1.9‐fold increase,
p<0.01 compared to control). These results indicate that embryonic exposure to thimerosal
affects early development of serotonergic neurons.
SPECIATION OF METHYL‐ AND ETHYL‐MERCURY IN HAIR OF BREASTFED INFANTS ACUTELY
EXPOSED TO THIMEROSAL‐CONTAINING VACCINES. (DOREA 2011)
Dórea JG, Bezerra VL, Fajon V, Horvat M. Clin Chim Acta. 2011 Aug 17;412(17‐18):1563‐6. doi:
10.1016/j.cca.2011.05.003. Epub 2011 May 7.
BACKGROUND: Different chemical forms of mercury occur naturally in human milk. The most
controversial aspect of early post‐natal exposure to organic mercury is ethylmercury (EtHg) in
thimerosal‐containing vaccines (TCV) still being used in many countries. Thus exclusively
breastfed infants can be exposed to both, fish derived methylmercury (MeHg) in maternal diets
and to EtHg from TCV. The aim of the study is to evaluate a new analytical method for ethyl and
methyl mercury in hair samples of breastfed infants who had received the recommended
schedule of TCV. METHODS: The hair of infants (<12 months) that had been exposed to TCVScience Summary on Mercury in Vaccines (Thimerosal Only)
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(Hepatitis B and DTaP) was analysed. A method coupling isothermal gas chromatography with
cold‐vapor atomic fluorescence spectrometry was used for MeHg which can also speciate EtHg
in biological matrices. RESULTS: In 20 samples of infants' hair, all but two samples showed
variable amounts of MeHg (10.3 to 668 ng/g), while precise and reliable concentrations of EtHg
(3.7 to 65.0 ng/g) were found in 15 of the 20 samples. A statistically significant inverse
association (r=‐05572; p=0.0384) was found between hair‐EtHg concentrations and the time
elapsed after the last TCV shot. CONCLUSIONS: The analytical method proved sensitive
enough to quantify EtHg in babies' hair after acute exposure to thimerosal in vaccine shots.
Provided that the mass of hair was above 10mg, organic‐mercury exposure during early life can
be speciated, and quantified in babies' first hair, thus opening opportunities for clinical and
forensic studies.
INTEGRATING EXPERIMENTAL (IN VITRO AND IN VIVO) NEUROTOXICITY STUDIES OF LOW‐
DOSE THIMEROSAL RELEVANT TO VACCINES. (DOREA 2011)
Dórea JG. Neurochem Res. 2011 Jun;36(6):927‐38. doi: 10.1007/s11064‐011‐0427‐0. Epub 2011
Feb 25.
There is a need to interpret neurotoxic studies to help deal with uncertainties surrounding
pregnant mothers, newborns and young children who must receive repeated doses of
Thimerosal‐containing vaccines (TCVs). This review integrates information derived from
emerging experimental studies (in vitro and in vivo) of low‐dose Thimerosal (sodium ethyl
mercury thiosalicylate). Major databases (PubMed and Web‐of‐science) were searched for in
vitro and in vivo experimental studies that addressed the effects of low‐dose Thimerosal (or
ethylmercury) on neural tissues and animal behaviour. Information extracted from studies
indicates that: (a) activity of low doses of Thimerosal against isolated human and animal brain
cells was found in all studies and is consistent with Hg neurotoxicity; (b) the neurotoxic effect of
ethylmercury has not been studied with co‐occurring adjuvant‐Al in TCVs; (c) animal studies
have shown that exposure to Thimerosal‐Hg can lead to accumulation of inorganic Hg in brain,
and that (d) doses relevant to TCV exposure possess the potential to affect human neuro‐
development. Thimerosal at concentrations relevant for infants' exposure (in vaccines) is toxic
to cultured human‐brain cells and to laboratory animals. The persisting use of TCV (in
developing countries) is counterintuitive to global efforts to lower Hg exposure and to ban Hg in
medical products; its continued use in TCV requires evaluation of a sufficiently nontoxic level of
ethylmercury compatible with repeated exposure (co‐occurring with adjuvant‐Al) during early
life.Science Summary on Mercury in Vaccines (Thimerosal Only)
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NEONATAL EXPOSURE TO THIMEROSAL FROM VACCINES AND CHILD DEVELOPMENT IN THE
FIRST 3YEARS OF LIFE. (MROZEK‐BUDZYN 2012)
Mrozek‐Budzyn D, Majewska R, Kieltyka A, Augustyniak M. Neurotoxicol Teratol. 2012 Nov‐
Dec;34(6):592‐7. doi: 10.1016/j.ntt.2012.10.001. Epub 2012 Oct 13.
BACKGROUND: Despite the common use of Thimerosal as a preservative in childhood vaccines
since the 1930s, there are not many studies on ethylmercury toxicokinetics and toxicodynamics
in infants. The knowledge of ethylmercury's potential adverse effects is derived mostly from
parallel methylmercury research or from animal and theoretical models. AIM OF THE STUDY:
This study was designed to examine the relationship between neonatal exposure to Thimerosal‐
containing vaccine (TCV) and child development. MATERIAL AND METHODS: The study sample
consisted of 196 infants born between January 2001 and March 2003 to mothers attending
ambulatory prenatal clinics in the first and second trimesters of pregnancy in Krakow.
Vaccination history (date and the type of the vaccine) was extracted from physicians' records.
Child development was assessed using the Bayley Scales of Infant Development (BSID‐II)
measured in one‐year intervals over 3years. General Linear Model (GLM) and Generalized
Estimating Equation (GEE) models adjusted for potential confounders were used to assess the
association. RESULTS: An adverse effect of neonatal TCV exposure was observed for the
psychomotor development index (PDI) only in the 12th and 24th months of life (β=‐6.44,
p<0.001 and β=‐5.89, p<0.001). No significant effect of neonatal TCV exposure was found in the
36th month. The overall deficit in the PDI attributable to neonatal TCV exposure measured over
the course of the three‐year follow‐up (GEE) was significantly higher in TCV group (β=‐4.42,
p=0.001). MDI scores did not show the adverse association with neonatal TCV exposure.
BREAST‐FEEDING AND RESPONSES TO INFANT VACCINES: CONSTITUTIONAL AND
ENVIRONMENTAL FACTORS. (DOREA 2012)
Dórea JG. Am J Perinatol. 2012 Nov;29(10):759‐75. doi: 10.1055/s‐0032‐1316442. Epub 2012
Jul 6.
Neonates and nursing infants are special with regard to immune development and vulnerability
to infectious diseases. Although breast‐feeding is essential to modulate and prime immune
defenses, vaccines (an interventional prophylaxis) are crucial to prevent and control infectious
diseases. During nursing, the type of feeding influences infants' natural defenses (including gut
colonization) and their response to vaccines, both through cell‐mediated immunity and specific
antibody production. Given the variety and combination of vaccine components (antigens and
excipients, preservative thimerosal, and aluminum adjuvants) and route of administration,
there is a need to examine the role of infant feeding practices in intended and nonintended
outcomes of vaccination. Maternal factors related to milk constituents (nutrients andScience Summary on Mercury in Vaccines (Thimerosal Only)
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pollutants) and feeding practices can affect response to vaccines. Collectively, studies that
compared type of feeding (or used breast‐feeding‐adjusted statistical models) showed
significant influence on some vaccines taken during infancy. Nurslings deprived of the full
benefit of breast‐feeding could have altered immune responses affecting vaccine outcome. In
the absence of studies elucidating neurodevelopment (including excitoxicity) and
immunotoxicity issues, vaccination practices should promote and support breast‐feeding.
THIMEROSAL EXPOSURE IN EARLY LIFE AND NEUROPSYCHOLOGICAL OUTCOMES 7‐10 YEARS
LATER. (BARILE 2012)
Barile JP, Kuperminc GP, Weintraub ES, Mink JW, Thompson WW. J Pediatr Psychol. 2012 Jan‐
Feb;37(1):106‐18. doi: 10.1093/jpepsy/jsr048. Epub 2011 Jul 23.
OBJECTIVE: The authors used a public use data set to investigate associations between the
receipt of thimerosal‐containing vaccines and immune globulins early in life and
neuropsychological outcomes assessed at 7‐10 years. METHODS: The data were originally
created by evaluating 1,047 children ages 7‐10 years and their biological mothers. This study
developed seven latent neuropsychological factors and regressed them on a comprehensive set
of covariates and thimerosal exposure variables. RESULTS: The authors found no statistically
significant associations between thimerosal exposure from vaccines early in life and six of the
seven latent constructs. There was a small, but statistically significant association between early
thimerosal exposure and the presence of tics in boys. CONCLUSIONS: This finding should be
interpreted with caution due to limitations in the measurement of tics and the limited
biological plausibility regarding a causal relationship.
Non‐Human Primate Infant Research
COMPARISON OF BLOOD AND BRAIN MERCURY LEVELS IN INFANT MONKEYS EXPOSED TO
METHYLMERCURY OR VACCINES CONTAINING THIMEROSAL (BURBACHER 2005)
Burbacher TM, Shen DD, Liberato N, Grant KS, Cernichiari E, Clarkson T. Environmental Health
Perspectives. 2005 Aug;113(8):1015‐21.
Burbacher compared brain mercury levels in infant Macaca fascicularis primates exposed to
injected ethylmercury (thimerosal) and equal amounts of ingested methylmercury. The
ethylmercury more rapidly converted to inorganic mercury in the brains of the primates which
resulted in increasing levels of inorganic mercury and the primates exposed to ethylmercury
retained at least twice as much inorganic mercury in their brains compared to the primates
exposed to methylmercury. The relative concentrations in monkeys with detectable levels of
inorganic mercury were 16 ng/g in thimerosal‐treated monkeys and 7 ng/g in the
methylmercury‐treated monkeys in which inorganic mercury levels were detectable. InorganicScience Summary on Mercury in Vaccines (Thimerosal Only)
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mercury was below detectable levels in 8 out of 17 of the methylmercury‐treated monkeys.
Exposures to mercury during these critical periods of development disrupt the growth and
migration of neurons, with the potential to cause irreversible damage to the central nervous
system. Prior primate studies found inorganic mercury in the brain was associated with
microgliosis and neuroinflammation, recent finding also documented in autistic brain.
PEDIATRIC VACCINES INFLUENCE PRIMATE BEHAVIOR, AND AMYGDALA GROWTH AND
OPIOID LIGAND BINDING (HEWITSON 2008)
L. Hewitson B. Lopresti , C. Stott, J. Tomko , L. Houser , E. Klein , G. Sackett , S. Gupta , D. Atwood , L. Blue ,
E. R. White , A. Wakefield IMFAR (May 2008)
Background: Macaques are commonly used in pre‐clinical vaccine safety testing, but the
combined childhood vaccine regimen, rather than individual vaccines, has not been studied.
Childhood vaccines are a possible causal factor in autism, and abnormal behaviors and
anomalous amygdala growth are potentially inter‐related features of this condition. Objectives:
The objective of this study was to compare early infant cognition and behavior with amygdala
size and opioid binding in rhesus macaques receiving the recommended childhood vaccines
(1994‐1999), the majority of which contained the bactericidal preservative
thylmercurithiosalicylic acid (thimerosal). Methods: Macaques were administered the
recommended infant vaccines, adjusted for age and thimerosal dose (exposed; N=13), or saline
(unexposed; N=3). Primate development, cognition and social behavior were assessed for both
vaccinated and unvaccinated infants using standardized tests developed at the Washington
National Primate Research Center. Amygdala growth and binding were measured serially by
MRI and by the binding of the non‐selective opioid antagonist [11C]diprenorphine, measured
by PET, respectively, before (T1) and after (T2) the administration of the measles‐mumps‐
rubella vaccine (MMR). Results: Compared with unexposed animals, significant
neurodevelopmental deficits were evident for exposed animals in survival reflexes, tests of
color discrimination and reversal, and learning sets. Differences in behaviors were observed
between exposed and unexposed animals and within the exposed group before and after MMR
vaccination. Compared with unexposed animals, exposed animals showed attenuation of
amygdala growth and differences in the amygdala binding of [11C]diprenorphine. Interaction
models identified significant associations between specific aberrant social and non‐social
behaviors, isotope binding, and vaccine exposure. Conclusions: This animal model, which
examines for the first time, behavioral, functional, and neuromorphometric consequences of
the childhood vaccine regimen, mimics certain neurological abnormalities of autism. The
findings raise important safety issues while providing a potential model for examining aspects
of causation and disease pathogenesis in acquired disorders of behavior and development.Science Summary on Mercury in Vaccines (Thimerosal Only)
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MICROARRAY ANALYSIS OF GI TISSUE IN A MACAQUE MODEL OF THE EFFECTS OF INFANT
VACCINATION (WALKER 2008)
S. J. Walker , E. K. Lobenhofer , A. Wakefield , L. Hewitson IMFAR (May 2008)
Background: There has been considerable debate regarding the question of an interaction
between childhood vaccinations and adverse sequelae in the gastrointestinal tract, immune
system, and central nervous system of some recipients. These systems, either singly or in
combination, appear to be adversely affected in many ASD children. Although pre‐clinical tests
of individual vaccines routinely find the risk/benefit ratio to be low, previously there has not
been a study to examine the effects of the comprehensive vaccination regime currently in use
for infants. Objectives: This study was designed to evaluate potential alterations in normal
growth and development resulting from the vaccine regimen that was in use from 1994‐1999.
Specifically, this portion of the study was to compare the gene expression profiles obtained
from gastrointestinal tissue from vaccinated and unvaccinated infants. Methods: Infant male
macaques were vaccinated (or given saline placebo) using the human vaccination schedule.
Dosages and times of administration were adjusted for differences between macaques and
humans. Biopsy tissue was collected from the animals at three time points: (1) 10 weeks [pre‐
MMR1], (2) 14 weeks [post‐MMR1] and, (3) 12‐15 months [at necropsy]. Whole genome
microarray analysis was performed on RNA extracted from the GI tissue from 7 vaccinated and
2 unvaccinated animals at each of these 3 time points (27 samples total). Results:
Histopathological examination revealed that vaccinated animals exhibited progressively severe
chronic active inflammation, whereas unexposed animals did not. Gene expression
comparisons between the groups (vaccinated versus unvaccinated) revealed only 120 genes
differentially expressed (fc >1.5; log ratio p<0.001) at 10 weeks, whereas there were 450 genes
differentially expressed at 14 weeks, and 324 differentially expressed genes between the 2
groups at necropsy. Conclusions: We have found many significant differences in the GI tissue
gene expression profiles between vaccinated and unvaccinated animals. These differences will
be presented and discussed.
DELAYED ACQUISITION OF NEONATAL REFLEXES IN NEWBORN PRIMATES RECEIVING A
THIMEROSAL‐CONTAINING HEPATITIS B VACCINE: INFLUENCE OF GESTATIONAL AGE AND
BIRTH WEIGHT. (HEWITSON 2010)
Hewitson L, Houser LA, Stott C, Sackett G, Tomko JL, Atwood D, Blue L, White ER. J Toxicol Environ Health
A. 2010 Jan;73(19):1298‐313.
This study examined whether acquisition of neonatal reflexes in newborn rhesus macaques was
influenced by receipt of a single neonatal dose of hepatitis B vaccine containing the
preservative thimerosal (Th). Hepatitis B vaccine containing a weight‐adjusted Th dose was
administered to male macaques within 24 h of birth (n = 13). Unexposed animals received
saline placebo (n = 4) or no injection (n = 3). Infants were tested daily for acquisition of nineScience Summary on Mercury in Vaccines (Thimerosal Only)
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survival, motor, and sensorimotor reflexes. In exposed animals there was a significant delay in
the acquisition of root, snout, and suck reflexes, compared with unexposed animals. No
neonatal responses were significantly delayed in unexposed animals. Gestational age (GA) and
birth weight (BW) were not significantly correlated. Cox regression models were used to
evaluate main effects and interactions of exposure with BW and GA as independent predictors
and time‐invariant covariates. Significant main effects remained for exposure on root and suck
when controlling for GA and BW, such that exposed animals were relatively delayed in time‐to‐
criterion. Interaction models indicated there were various interactions between exposure, GA,
and BW and that inclusion of the relevant interaction terms significantly improved model fit.
This, in turn, indicated that lower BW and/or lower GA exacerbated the adverse effects
following vaccine exposure. This primate model provides a possible means of assessing adverse
neurodevelopmental outcomes from neonatal Th‐containing hepatitis B vaccine exposure,
particularly in infants of lower GA or BW. The mechanisms underlying these effects and the
requirements for Th requires further study
INFLUENCE OF PEDIATRIC VACCINES ON AMYGDALA GROWTH AND OPIOID LIGAND BINDING
IN RHESUS MACAQUE INFANTS: A PILOT STUDY. (HEWITSON 2010)
Hewitson L, Lopresti BJ, Stott C, Mason NS, Tomko J. Acta Neurobiol Exp (Wars). 2010;70(2):147‐64.
This longitudinal, case‐control pilot study examined amygdala growth in rhesus macaque
infants receiving the complete US childhood vaccine schedule (1994‐1999). Longitudinal
structural and functional neuroimaging was undertaken to examine central effects of the
vaccine regimen on the developing brain. Vaccine‐exposed and saline‐injected control infants
underwent MRI and PET imaging at approximately 4 and 6 months of age, representing two
specific timeframes within the vaccination schedule. Volumetric analyses showed that exposed
animals did not undergo the maturational changes over time in amygdala volume that was
observed in unexposed animals. After controlling for left amygdala volume, the binding of the
opioid antagonist [(11)C]diprenorphine (DPN) in exposed animals remained relatively constant
over time, compared with unexposed animals, in which a significant decrease in [(11)C]DPN
binding occurred. These results suggest that maturational changes in amygdala volume and the
binding capacity of [(11)C]DPN in the amygdala was significantly altered in infant macaques
receiving the vaccine schedule. The macaque infant is a relevant animal model in which to
investigate specific environmental exposures and structural/functional neuroimaging during
neurodevelopment.Science Summary on Mercury in Vaccines (Thimerosal Only)
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Animal Research
NEUROTOXIC EFFECTS OF POSTNATAL THIMEROSAL ARE MOUSE STRAIN DEPENDENT
(HORNIG 2004)
Hornig M, Chian D, Lipkin WI. Molecular Psychiatry. 2004 Sep;9(9):833‐45.
Hornig exposed autoimmune‐prone infant mice with thimerosal‐containing vaccines at the dose
given to human infants adjusted for mouse weight. This investigation reported a number of
observable effects including growth delay; reduced locomotion; exaggerated response to
novelty; and densely packed, hyperchromic hippocampal neurons with altered glutamate
receptors and transporters. Strains resistant to autoimmunity were not susceptible. These
findings implicate genetic influences and provide a model for investigating thimerosal‐related
neurotoxicity.
EFFECT OF THIMEROSAL, A PRESERVATIVE IN VACCINES, ON INTRACELLULAR CA2+
CONCENTRATION OF RAT CEREBELLAR NEURONS (UEHA‐ISHIBASHI 2004)
Ueha‐Ishibashi T, Oyama Y, Nakao H, Umebayashi C, Nishizaki Y, Tatsuishi T, Iwase K, Murao K, Seo H.
Toxicology 2004 Jan 15;195(1):77‐84.
Ueha‐Ishibashi investigated the effect of thimerosal on cerebellar neurons dissociated from 2‐
week‐old rats was compared with those of methylmercury. Both agents at 1 microM or more
similarly decreased the cellular content of glutathione in a concentration‐dependent manner,
suggesting an increase in oxidative stress and increased intercellular concentrations of Ca2+.
Thimerosal was also found to exert cytotoxic actions on cerebellar granule neurons and its
potency was similar to that of methylmercury. The FDA and EPA use methymercury as their
toxicity standard, so demonstration of equivalence shows the potential of thimerosal to cause
the same harm as methylmercury, for which more research exists.
THIMEROSAL DISTRIBUTION AND METABOLISM IN NEONATAL MICE: COMPARISON WITH
METHYL MERCURY (ZAREBA 2007)
Grazyna Zareba, Elsa Cernichiari, Rieko Hojo, Scott Mc Nitt, Bernard Weiss, Moiz M Mumtaz, Dennis E
Jones, Thomas W Clarkson Neurotoxicology. 2007 Feb 23; : 17382399
Thimerosal, which releases the ethyl mercury radical as the active species, has been used as a
preservative in many currently marketed vaccines throughout the world. Because of concerns
that its toxicity could be similar to that of methyl mercury, it is no longer incorporated in many
vaccines in the United States. There are reasons to believe, however, that the disposition and
toxicity of ethyl mercury compounds, including thimerosal, may differ substantially from thoseScience Summary on Mercury in Vaccines (Thimerosal Only)
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of the methyl form. The current study sought to compare, in neonatal mice, the tissue
concentrations, disposition and metabolism of thimerosal with that of methyl mercury. ICR
mice were given single intramuscular injections of thimerosal or methyl mercury (1.4 mg Hg
kg(‐1)) on postnatal day 10 (PND 10). Tissue samples were collected daily on PND 11‐14. Most
analysed tissues demonstrated different patterns of tissue distribution and a different rate of
mercury decomposition. The mean organic mercury in the brain and kidneys was significantly
lower in mice treated with thimerosal than in the methyl mercury‐treated group. In the brain,
thimerosal‐exposed mice showed a steady decrease of organic mercury levels following the
initial peak, whereas in the methyl mercury‐exposed mice, concentrations peaked on day 2
after exposure. In the kidneys, thimerosal‐exposed mice retained significantly higher inorganic
mercury levels than methyl mercury‐treated mice. In the liver both organic and inorganic
mercury concentrations were significantly higher in thimerosal‐exposed mice than in the
methyl mercury group. Ethyl mercury was incorporated into growing hair in a similar manner to
methyl mercury. The data showing significant kinetic differences in tissue distribution and
metabolism of mercury species challenge the assumption that ethyl mercury is toxicologically
identical to methyl mercury.
EFFECTS OF LIPOPOLYSACCHARIDE AND CHELATOR ON MERCURY CONTENT IN THE
CEREBRUM OF THIMEROSAL‐ADMINISTERED MICE (MINAMI 2007)
Takeshi Minami, Keisuke Oda, Naoya Gima, Hideo Yamazaki Environmental Toxicology and
Pharmacology Volume 24, Issue 3, November 2007, Pages 316‐320
Thimerosal is one of the best‐known preservative agents for vaccines in the world but a
relationship between its use and autism has long been suspected so that its effects on the brain
need more detailed research. We here examined the influence of lipopolysaccharide injury to
the blood–brain barrier on the penetration of mercury from thimerosal into mouse cerebrums,
as well as the effect of chelator of heavy metals on cerebrum mercury content. Mercury can be
expected to be detected in the cerebrum of normal mice, because the metal is present in
standard mouse chow. When 60 µg/kg of thimerosal was subcutaneously injected into the
mouse, the mercury content in the cerebrum was significantly higher 48 h after the thimerosal
injection with a maximum peak after 72 h. In addition, mercury content in the cerebrum was
still higher on day 7 than in the control group. When lipopolysaccharide was pre‐injected into
mice to induce damage on blood–brain barrier, the mercury content in the cerebrum was
significantly higher at 24 and 72 h after the injection of 12 µg/kg of thimerosal compared to the
control group, this dose alone does not cause any increase. The mercury content in the
cerebrums of mice was decreased to the control group level on day 7 when a chelator,
dimercaprol, was administered once a day from days 3 to 6 aftera 60 µg/kg, s.c. injection. In
addition, d‐penicillamine as a chelator decreased the mercury contents in the cerebrum afterScience Summary on Mercury in Vaccines (Thimerosal Only)
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the high dose administration. In conclusion, a physiological dose of thimerosal did not increase
the content of mercury in the cerebrum, but levels were increased when damage to the blood–
brain barrier occurred in mice injected with thimerosal. In addition, a chelator of heavy metals
may be useful to remove mercury from the cerebrum.
GENDER‐SELECTIVE TOXICITY OF THIMEROSAL (BRANCH 2008)
Branch DR Exp Toxicol Pathol. 2008 Sep 2. [Epub ahead of print]
A recent report shows a correlation of the historical use of thimerosal in therapeutic
immunizations with the subsequent development of autism; however, this association remains
controversial. Autism occurs approximately four times more frequently in males compared to
females; thus, studies of thimerosal toxicity should take into consideration gender‐selective
effects. The present study was originally undertaken to determine the maximum tolerated dose
(MTD) of thimersosal in male and female CD1 mice. However, during the limited MTD studies, it
became apparent that thimerosal has a differential MTD that depends on whether the mouse is
male or female. At doses of 38.4‐76.8mg/kg using 10% DMSO as diluent, seven of seven male
mice compared to zero of seven female mice tested succumbed to thimerosal. Although the
thimerosal levels used were very high, as we were originally only trying to determine MTD, it
was completely unexpected to observe a difference of the MTD between male and female mice.
Thus, our studies, although not directly addressing the controversy surrounding thimerosal and
autism, and still preliminary due to small numbers of mice examined, provide, nevertheless, the
first report of gender‐selective toxicity of thimerosal and indicate that any future studies of
thimerosal toxicity should take into consideration gender‐specific differences.
EFFECTS OF INTERMITTENT, VACCINATION‐LIKE SCHEME, THIMEROSAL ADMINISTRATION ON
RAT DEVELOPMENT AND BEHAVIOR (OLCZAK 2008)
Olczak M., Duszczyk M., Mierzejewski P. & Majewska M. D. Dept. Pharmacol. Inst. Psychiatry &
Neurology, Warsaw, Poland Publication ref.: FENS Abstr., vol.4, 083.19, 2008
Mercury from thimerosal, which was added to many child vaccines, is one of the agents
suspected to be responsible for autism epidemics observed in the past two decades. Data
analysis from Vaccine Adverse Event Reporting System of the Center for Disease Control and
Prevention (USA) documented that children immunized with vaccines containing thimerosal
were several times more likely do develop autism and other neurodevelopmental
diseases/disorders than those, who did not receive thimerosal. In this study we examined the
potential neurotoxic effects of different cumulative doses of thimerosal, from 0.040 mg/kg to
25 mg/kg, administered to rats s.c. or i. m. in four doses on postnatal days 7‐14. Three strains of
rats were tested: Wistar, Lewis and Brown Norway. Development and behaviour or the
experimental animals was monitored. At different developmental stages (between weeks 4 andScience Summary on Mercury in Vaccines (Thimerosal Only)
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22 of age) several behavioral tests were conducted, which included open field locomotor
activity, motor coordination, pain reaction (hot plate), water maze learning and memory test,
prepulse inhibition, and social interaction test. Brains of thimerosal treated rats accumulated a
significant amount of mercury. They were examined for histopathological changes. Generally,
rats appeared to be quite resistant to overt neurotoxic effects of thimerosal at doses tested,
although higher doses of this drug caused subtle changes on some behavioral measures, which
appear to be species and sex dependent. Significant thimerosal effects on pain reaction, certain
learning parameters and prepulse inhibition were observed. Also some aspects of social
interactions were altered. Behavioural and histopathological data will be presented in the
context of putative rat model of mercury‐mediated neurodevelopmental pathologies. Funded
by EC grant MEXC‐CT‐2006‐42371 to M. D. Majewska.
EFFECTS OF POSTNATAL ADMINISTRATION ON THIMEROSAL ON RAT DEVELOPMENT AND
BEHAVIOR (DUSZCZYK 2008)
Michalina Duszczyk, Mieszko Olczak, Pawe Mierzejewski, Dorota M. Majewska. Department of
Pharmacology and Physiology of the Central Nervous System, Institute of Psychiatry and Neurology,
Warsaw, Poland. Pharmacological Reports. 2008 60; p261‐262
Numerous clinical findings support hypothesis that mercury, which was added to many infant
vaccines in the form of thimerosal between 2000–2004, may be one of the factors responsible
for autism epidemics currently observed all over the world. Data from Adverse Event Reporting
of the Center for Disease Control and Prevention (USA) provide strong epidemiological evidence
for a link between vaccine‐thimerosal exposure and autism or other neurodevelopmental
disorders/diseases. The onset of autistic symptoms in children often follows the administration
of vaccine thimerosal and symptom emergence is consistent with the expression of
developmental mercury toxicity. In this study, we examined potential neurodevelopmental
outcomes following postnatal exposure of rats to thimerosal (Sigma‐Aldrich), administered sc or
im from 0.040 mg/kg to 50 mg/kg in four equal doses on days 7–14 after birth. Three strains of
rats were used in this experiment: Wistar, Lewis and Brown Norway. Development and
behavior of experimental animals was observed. Various behavioral tests were carried out,
which evaluated: open field locomotor and exploratory activity, motor coordination, pain
reaction (hot plate), learning and memory (water maze), prepulse inhibition, sociability (social
interaction test). Growth of animals was monitored and after animal sacrifice, weight of brains
was measured. Thimerosal had variable, often biphasic, effects on different measured
behaviors, which were strain‐ and dose‐dependent, but no dramatic behavioral impairments
were observed at doses tested. Data will be discussed in the context of rodent model of autism
following postnatal exposure to mercury. [Note: autism is 4 times more prevalent in boys thanScience Summary on Mercury in Vaccines (Thimerosal Only)
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girls, and no one has been able to identify why. The differential gender effects of thimerosal
and mercury might explain why.]
INDUCTION OF METALLOTHIONEIN IN MOUSE CEREBELLUM AND CEREBRUM WITH LOW‐
DOSE THIMEROSAL INJECTION (MINAMI 2009)
Minami T, Miyata E, Sakamoto Y, Yamazaki H, Ichida S. Department of Life Sciences, School of Science &
Engineering, Kinki University, 3‐4‐1 Kowakae, Higashi‐osaka, Osaka, 577‐8502, Japan,
minamita@life.kindai.ac.jp. Cell Biol Toxicol. 2009 Apr 9.
Thimerosal, an ethyl mercury compound, is used worldwide as a vaccine preservative. We
previously observed that the mercury concentration in mouse brains did not increase with the
clinical dose of thimerosal injection, but the concentration increased in the brain after the
injection of thimerosal with lipopolysaccharide, even if a low dose of thimerosal was
administered. Thimerosal may penetrate the brain, but is undetectable when a clinical dose of
thimerosal is injected; therefore, the induction of metallothionein (MT) messenger RNA (mRNA)
and protein was observed in the cerebellum and cerebrum of mice after thimerosal injection, as
MT is an inducible protein. MT‐1 mRNA was expressed at 6 and 9 h in both the cerebrum and
cerebellum, but MT‐1 mRNA expression in the cerebellum was three times higher than that in
the cerebrum after the injection of 12 microg/kg thimerosal. MT‐2 mRNA was not expressed
until 24 h in both organs. MT‐3 mRNA was expressed in the cerebellum from 6 to 15 h after the
injection, but not in the cerebrum until 24 h. MT‐1 and MT‐3 mRNAs were expressed in the
cerebellum in a dose‐dependent manner. Furthermore, MT‐1 protein was detected from 6 to
72 h in the cerebellum after 12 microg/kg of thimerosal was injected and peaked at 10 h. MT‐2
was detected in the cerebellum only at 10 h. In the cerebrum, little MT‐1 protein was detected
at 10 and 24 h, and there were no peaks of MT‐2 protein in the cerebrum. In conclusion, MT‐1
and MT‐3 mRNAs but not MT‐2 mRNA are easily expressed in the cerebellum rather than in the
cerebrum by the injection of low‐dose thimerosal. It is thought that the cerebellum is a
sensitive organ against thimerosal. As a result of the present findings, in combination with the
brain pathology observed in patients diagnosed with autism, the present study helps to support
the possible biological plausibility for how low‐dose exposure to mercury from thimerosal‐
containing vaccines may be associated with autism. Science Summary on Mercury in Vaccines (Thimerosal Only)
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NEONATAL ADMINISTRATION OF A VACCINE PRESERVATIVE, THIMEROSAL, PRODUCES
LASTING IMPAIRMENT OF NOCICEPTION AND APPARENT ACTIVATION OF OPOID SYSTEM IN
RATS (OLCZAK 2009)
Olczak M, Duszczyk M, Mierzejewski P, Majewska MD. Brain Res. 2009 Dec 8;1301:143‐51. Epub 2009
Sep 9.
Thimerosal (THIM), an organomercury preservative added to many child vaccines is a suspected
factor in pathogenesis of neurodevelopmental disorders. We examined the pharmacokinetics of
Hg in the brain, liver and kidneys after i.m. THIM injection in suckling rats and we tested THIM
effect on nociception. THIM solutions were injected to Wistar and Lewis rats in a vaccination‐
like mode on PN days 7, 9, 11 and 15 in four equal doses. For Wistar rats these were: 12, 48,
240, 720, 1440, 2160, 3000 microg Hg/kg and for Lewis: 54, 216, 540 and 1080 microg Hg/kg.
Pharmacokinetic analysis revealed that Hg from THIM injections accumulates in the rat brain in
significant amounts and remains there longer than 30 days after the injection. At the 6th week
of age animals were examined for pain sensitivity using the hot plate test. THIM treated rats of
both strains and sexes manifested statistically significantly elevated pain threshold (latency for
paw licking, jumping) on a hot plate (56 degrees C). Wistar rats were more sensitive to this
effect than Lewis rats. Protracted THIM‐induced hypoalgesia was reversed by naloxone (5
mg/kg, i.p.) injected before the hot plate test, indicative of involvement of endogenous opioids.
This was confirmed by augmented catalepsy after morphine (2.5 mg/kg, s.c.) injection. Acute
THIM injection to 6‐week‐old rats also produced hypoalgesia, but this effect was transient and
was gone within 14 days. Present findings show that THIM administration to suckling or adult
rats impairs sensitivity to pain, apparently due to activation the endogenous opioid system.
IDENTIFICATION AND DISTRIBUTION OF MERCURY SPECIES IN RAT TISSUES FOLLOWING
ADMINISTRATION OF THIMEROSAL OR METHYLMERCURY. (RODRIGUES 2010)
Rodrigues JL, Serpeloni JM, Batista BL, Souza SS, Barbosa F Jr. Arch Toxicol. 2010 Nov;84(11):891‐6. Epub
2010 Apr 13.
Methylmercury (Met‐Hg) is one the most toxic forms of Hg, with a considerable range of
harmful effects on humans. Sodium ethyl mercury thiosalicylate, thimerosal (TM) is an
ethylmercury (Et‐Hg)‐containing preservative that has been used in manufacturing vaccines in
many countries. Whereas the behavior of Met‐Hg in humans is relatively well known, that of
ethylmercury (Et‐Hg) is poorly understood. The present study describes the distribution of
mercury as (‐methyl, ‐ethyl and inorganic mercury) in rat tissues (brain, heart, kidney and liver)
and blood following administration of TM or Met‐Hg. Animals received one dose/day of Met‐Hg
or TM by gavage (0.5 mg Hg/kg). Blood samples were collected after 6, 12, 24, 48, 96 and 120 h
of exposure. After 5 days, the animals were killed, and their tissues were collected. Total bloodScience Summary on Mercury in Vaccines (Thimerosal Only)
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mercury (THg) levels were determined by ICP‐MS, and methylmercury (Met‐Hg), ethylmercury
(Et‐Hg) and inorganic mercury (Ino‐Hg) levels were determined by speciation analysis with LC‐
ICP‐MS. Mercury remains longer in the blood of rats treated with Met‐Hg compared to that of
TM‐exposed rats. Moreover, after 48 h of the TM treatment, most of the Hg found in blood was
inorganic. Of the total mercury found in the brain after TM exposure, 63% was in the form of
Ino‐Hg, with 13.5% as Et‐Hg and 23.7% as Met‐Hg. In general, mercury in tissues and blood
following TM treatment was predominantly found as Ino‐Hg, but a considerable amount of Et‐
Hg was also found in the liver and brain. Taken together, our data demonstrated that the
toxicokinetics of TM is completely different from that of Met‐Hg. Thus, Met‐Hg is not an
appropriate reference for assessing the risk from exposure to TM‐derived Hg. It also adds new
data for further studies in the evaluation of TM toxicity.
NEONATAL ADMINISTRATION OF THIMEROSAL CAUSES PERSISTENT CHANGES IN MU OPIOID
RECEPTORS IN THE RAT BRAIN (OLCZAK 2010)
Olczak M, Duszczyk M, Mierzejewski P, Bobrowicz T, Majewska MD. Neurochem Res. 2010 Aug
28.
Thimerosal added to some pediatric vaccines is suspected in pathogenesis of several
neurodevelopmental disorders. Our previous study showed that thimerosal administered to
suckling rats causes persistent, endogenous opioid‐mediated hypoalgesia. Here we examined,
using immunohistochemical staining technique, the density of μ‐opioid receptors (MORs) in the
brains of rats, which in the second postnatal week received four i.m. injections of thimerosal at
doses 12, 240, 1,440 or 3,000 μg Hg/kg. The periaqueductal gray, caudate putamen and
hippocampus were examined. Thimerosal administration caused dose‐dependent statistically
significant increase in MOR densities in the periaqueductal gray and caudate putamen, but
decrease in the dentate gyrus, where it was accompanied by the presence of degenerating
neurons and loss of synaptic vesicle marker (synaptophysin). These data document that
exposure to thimerosal during early postnatal life produces lasting alterations in the densities of
brain opioid receptors along with other neuropathological changes, which may disturb brain
development.Science Summary on Mercury in Vaccines (Thimerosal Only)
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CHRONIC METALS INGESTION BY PRAIRIE VOLES PRODUCES SEX‐SPECIFIC DEFICITS IN SOCIAL
BEHAVIOR: AN ANIMAL MODEL OF AUTISM. (CURTIS 2010)
Curtis JT, Hood AN, Chen Y, Cobb GP, Wallace DR. Behav Brain Res. 2010 Nov 12;213(1):42‐9. Epub 2010
Apr 28.
We examined the effects of chronic metals ingestion on social behavior in the normally highly
social prairie vole to test the hypothesis that metals may interact with central dopamine
systems to produce the social withdrawal characteristic of autism. Relative to water‐treated
controls, 10 weeks of chronic ingestion of either Hg(++) or Cd(++) via drinking water
significantly reduced social contact by male voles when they were given a choice between
isolation or contact with an unfamiliar same‐sex conspecific. The effects of metals ingestion
were specific to males: no effects of metals exposure were seen in females. Metals ingestion
did not alter behavior of males allowed to choose between isolation or their familiar cage‐
mates, rather than strangers. We also examined the possibility that metals ingestion affects
central dopamine functioning by testing the voles' locomotor responses to peripheral
administration of amphetamine. As with the social behavior, we found a sex‐specific effect of
metals on amphetamine responses. Males that consumed Hg(++) did not increase their
locomotor activity in response to amphetamine, whereas similarly treated females and males
that ingested only water significantly increased their locomotor activities. Thus, an ecologically
relevant stimulus, metals ingestion, produced two of the hallmark characteristics of autism ‐
social avoidance and a male‐oriented bias. These results suggest that metals exposure may
contribute to the development of autism, possibly by interacting with central dopamine
function, and support the use of prairie voles as a model organism in which to study autism.
NEUROLIGIN‐DEFICIENT MUTANTS OF C. ELEGANS HAVE SENSORY PROCESSING DEFICITS AND
ARE HYPERSENSITIVE TO OXIDATIVE STRESS AND MERCURY TOXICITY. (HUNTER 2010)
Hunter JW, Mullen GP, McManus JR, Heatherly JM, Duke A, Rand JB. Dis Model Mech. 2010 May‐Jun;3(5‐
6):366‐76. Epub 2010 Jan 18.
Neuroligins are postsynaptic cell adhesion proteins that bind specifically to presynaptic
membrane proteins called neurexins. Mutations in human neuroligin genes are associated with
autism spectrum disorders in some families. The nematode Caenorhabditis elegans has a single
neuroligin gene (nlg‐1), and approximately a sixth of C. elegans neurons, including some
sensory neurons, interneurons and a subset of cholinergic motor neurons, express a neuroligin
transcriptional reporter. Neuroligin‐deficient mutants of C. elegans are viable, and they do not
appear deficient in any major motor functions. However, neuroligin mutants are defective in a
subset of sensory behaviors and sensory processing, and are hypersensitive to oxidative stress
and mercury compounds; the behavioral deficits are strikingly similar to traits frequently
associated with autism spectrum disorders. Our results suggest a possible link between geneticScience Summary on Mercury in Vaccines (Thimerosal Only)
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defects in synapse formation or function, and sensitivity to environmental factors in the
development of autism spectrum disorders.
LASTING NEUROPATHOLOGICAL CHANGES IN RAT BRAIN AFTER INTERMITTENT NEONATAL
ADMINISTRATION OF THIMEROSAL. (OLCZAK 2010)
Olczak M, Duszczyk M, Mierzejewski P, Wierzba‐Bobrowicz T, Majewska MD. Folia Neuropathol.
2010;48(4):258‐69.
Thimerosal, an organomercurial added as a preservative to some vaccines, is a suspected
iatrogenic factor, possibly contributing to paediatric neurodevelopmental disorders including
autism. We examined the effects of early postnatal administration of thimerosal (four i.m.
injections, 12 or 240 μg THIM‐Hg/kg, on postnatal days 7, 9, 11 and 15) on brain pathology in
Wistar rats. Numerous neuropathological changes were observed in young adult rats which
were treated postnatally with thimerosal. They included: ischaemic degeneration of neurons
and "dark" neurons in the prefrontal and temporal cortex, the hippocampus and the
cerebellum, pathological changes of the blood vessels in the temporal cortex, diminished
synaptophysin reaction in the hippocampus, atrophy of astroglia in the hippocampus and
cerebellum, and positive caspase‐3 reaction in Bergmann astroglia. These findings document
neurotoxic effects of thimerosal, at doses equivalent to those used in infant vaccines or higher,
in developing rat brain, suggesting likely involvement of this mercurial in neurodevelopmental
disorders
PERSISTENT BEHAVIORAL IMPAIRMENTS AND ALTERATIONS OF BRAIN DOPAMINE SYSTEM
AFTER EARLY POSTNATAL ADMINISTRATION OF THIMEROSAL IN RATS. (OLCZAK 2011)
Olczak M, Duszczyk M, Mierzejewski P, Meyza K, Majewska MD. Behav Brain Res. 2011 Sep
30;223(1):107‐18. doi: 10.1016/j.bbr.2011.04.026. Epub 2011 Apr 28.
The neurotoxic organomercurial thimerosal (THIM), used for decades as vaccine preservative, is
a suspected factor in the pathogenesis of some neurodevelopmental disorders. Previously we
showed that neonatal administration of THIM at doses equivalent to those used in infant
vaccines or higher, causes lasting alterations in the brain opioid system in rats. Here we
investigated neonatal treatment with THIM (at doses 12, 240, 1440 and 3000 μg Hg/kg) on
behaviors, which are characteristically altered in autism, such as locomotor activity, anxiety,
social interactions, spatial learning, and on the brain dopaminergic system in Wistar rats of both
sexes. Adult male and female rats, which were exposed to the entire range of THIM doses
during the early postnatal life, manifested impairments of locomotor activity and increased
anxiety/neophobia in the open field test. In animals of both sexes treated with the highest
THIM dose, the frequency of prosocial interactions was reduced, while the frequency of
asocial/antisocial interactions was increased in males, but decreased in females. Neonatal THIMScience Summary on Mercury in Vaccines (Thimerosal Only)
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treatment did not significantly affect spatial learning and memory. THIM‐exposed rats also
manifested reduced haloperidol‐induced catalepsy, accompanied by a marked decline in the
density of striatal D₂ receptors, measured by immunohistochemical staining, suggesting
alterations to the brain dopaminergic system. Males were more sensitive than females to some
neurodisruptive/neurotoxic actions of THIM. These data document that early postnatal THIM
administration causes lasting neurobehavioral impairments and neurochemical alterations in
the brain, dependent on dose and sex. If similar changes occur in THIM/mercurial‐exposed
children, they could contribute do neurodevelopmental disorders.
MERCURY DISPOSITION IN SUCKLING RATS: COMPARATIVE ASSESSMENT FOLLOWING
PARENTERAL EXPOSURE TO THIOMERSAL AND MERCURIC CHLORIDE. (BLANUSA 2012)
Blanuša M, Orct T, Vihnanek Lazarus M, Sekovanić A, Piasek M. J Biomed Biotechnol.
2012;2012:256965. doi: 10.1155/2012/256965. Epub 2012 Jul 26.
Due to the facts that thiomersal‐containing vaccine is still in use in many developing countries,
and all forms of mercury have recognised neurotoxic, nephrotoxic, and other toxic effects,
studies on disposition of ethylmercury and other mercury forms are still justified, especially at
young age. Our investigation aimed at comparing mercury distribution and rate of excretion in
the early period of life following exposure to either thiomersal (TM) or mercuric chloride (HgCl₂)
in suckling rats. Three experimental groups were studied: control, TM, and HgCl₂, with 12 to18
pups in each. Both forms of mercury were administered subcutaneously in equimolar quantities
(0.81 μmol/kg b.w.) three times during the suckling period (on the days of birth 7, 9, and 11) to
mimic the vaccination regimen in infants. After the last administration of TM or HgCl₂, total
mercury retention and excretion was assessed during following six days. In TM‐exposed group
mercury retention was higher in the brain, enteral excretion was similar, and urinary excretion
was much lower compared to HgCl₂‐exposed sucklings. More research is still needed to
elucidate all aspects of toxicokinetics and most harmful neurotoxic potential of various forms of
mercury, especially in the earliest period of life.
SEX‐DEPENDENT CHANGES IN CEREBELLAR THYROID HORMONE‐DEPENDENT GENE
EXPRESSION FOLLOWING PERINATAL EXPOSURE TO THIMEROSAL IN RATS. (KHAN 2012)
Khan A, Sulkowski ZL, Chen T, Zavacki AM, Sajdel‐Sulkowska EM. J Physiol Pharmacol. 2012
Jun;63(3):277‐83.
Mammalian brain development is regulated by the action of thyroid hormone (TH) on target
genes. We have previously shown that the perinatal exposure to thimerosal (TM, metabolized
to ethylmercury) exerts neurotoxic effects on the developing cerebellum and is associated with
a decrease in cerebellar D2 activity, which could result in local brain T3 deficiency. We have also
begun to examine TM effect on gene expression. The objective of this study was to expand onScience Summary on Mercury in Vaccines (Thimerosal Only)
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our initial observation of altered cerebellar gene expression following perinatal TM exposure
and to examine additional genes that include both TH‐dependent as well as other genes critical
for cerebellar development in male and female neonates exposed perinatally (G10‐G15 and P5
to P10) to TM. We report here for the first time that expression of suppressor‐of‐white‐apricot‐
1 (SWAP‐1), a gene negatively regulated by T3, was increased in TM‐exposed males (61.1%
increase), but not in females; (p<0.05). Positively regulated T3‐target genes, Purkinje cell
protein 2 (Pcp2; p=0.07) and Forkhead box protein P4 (FoxP4; p=0.08), showed a trend towards
decreased expression in TM‐exposed males. The expression of deiodinase 2 (DIO2) showed a
trend towards an increase in TM‐exposed females, while deiodinase 3 (DIO3), transthyretin
(TTR), brain derived neurotrophic factor (BDNF) and reelin (RELN) was not significantly altered
in either sex. Since regulation of gene splicing is vital to neuronal proliferation and
differentiation, altered expression of SWAP‐1 may exert wide ranging effects on multiple genes
involved in the regulation of cerebellar development. We have previously identified activation
of another TH‐dependent gene, outer dense fiber of sperm tails 4, in the TM exposed male
pups. Together, these results also show sex‐dependent differences between the toxic impacts
of TM in males and females. Interestingly, the genes that were activated by TM are negatively
regulated by TH, supporting our hypothesis of local brain hypothyroidism being induced by TM
and suggesting a novel mechanism of action TM in the developing brain.
PRENATAL EXPOSURE TO ORGANOMERCURY, THIMEROSAL, PERSISTENTLY IMPAIRS THE
SEROTONERGIC AND DOPAMINERGIC SYSTEMS IN THE RAT BRAIN: IMPLICATIONS FOR
ASSOCIATION WITH DEVELOPMENTAL DISORDERS. (IDA‐ETO 2012)
Ida‐Eto M, Oyabu A, Ohkawara T, Tashiro Y, Narita N, Narita M. Brain Dev. 2012 May 31. [Epub
ahead of print]
Thimerosal, an organomercury compound, has been widely used as a preservative. Therefore,
concerns have been raised about its neurotoxicity. We recently demonstrated perturbation of
early serotonergic development by prenatal exposure to thimerosal (Ida‐Eto et al. (2011) [11]).
Here, we investigated whether prenatal thimerosal exposure causes persistent impairment
after birth. Analysis on postnatal day 50 showed significant increase in hippocampal serotonin
following thimerosal administration on embryonic day 9. Furthermore, not only serotonin,
striatal dopamine was significantly increased. These results indicate that embryonic exposure to
thimerosal produces lasting impairment of brain monoaminergic system, and thus every effort
should be made to avoid the use of thimerosal.Science Summary on Mercury in Vaccines (Thimerosal Only)
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ADMINISTRATION OF THIMEROSAL TO INFANT RATS INCREASES OVERFLOW OF GLUTAMATE
AND ASPARTATE IN THE PREFRONTAL CORTEX: PROTECTIVE ROLE OF
DEHYDROEPIANDROSTERONE SULFATE. (DUSZCZYK‐BUDHATHOKI 2012)
Duszczyk‐Budhathoki M, Olczak M, Lehner M, Majewska MD. Neurochem Res. 2012
Feb;37(2):436‐47. doi: 10.1007/s11064‐011‐0630‐z. Epub 2011 Oct 21.
Thimerosal, a mercury‐containing vaccine preservative, is a suspected factor in the etiology of
neurodevelopmental disorders. We previously showed that its administration to infant rats
causes behavioral, neurochemical and neuropathological abnormalities similar to those present
in autism. Here we examined, using microdialysis, the effect of thimerosal on extracellular
levels of neuroactive amino acids in the rat prefrontal cortex (PFC). Thimerosal administration
(4 injections, i.m., 240 μg Hg/kg on postnatal days 7, 9, 11, 15) induced lasting changes in amino
acid overflow: an increase of glutamate and aspartate accompanied by a decrease of glycine
and alanine; measured 10‐14 weeks after the injections. Four injections of thimerosal at a dose
of 12.5 μg Hg/kg did not alter glutamate and aspartate concentrations at microdialysis time
(but based on thimerosal pharmacokinetics, could have been effective soon after its injection).
Application of thimerosal to the PFC in perfusion fluid evoked a rapid increase of glutamate
overflow. Coadministration of the neurosteroid, dehydroepiandrosterone sulfate (DHEAS; 80
mg/kg; i.p.) prevented the thimerosal effect on glutamate and aspartate; the steroid alone had
no influence on these amino acids. Coapplication of DHEAS with thimerosal in perfusion fluid
also blocked the acute action of thimerosal on glutamate. In contrast, DHEAS alone reduced
overflow of glycine and alanine, somewhat potentiating the thimerosal effect on these amino
acids. Since excessive accumulation of extracellular glutamate is linked with excitotoxicity, our
data imply that neonatal exposure to thimerosal‐containing vaccines might induce excitotoxic
brain injuries, leading to neurodevelopmental disorders. DHEAS may partially protect against
mercurials‐induced neurotoxicity.
MATERNAL THIMEROSAL EXPOSURE RESULTS IN ABERRANT CEREBELLAR OXIDATIVE STRESS,
THYROID HORMONE METABOLISM, AND MOTOR BEHAVIOR IN RAT PUPS; SEX‐ AND STRAIN‐
DEPENDENT EFFECTS. (SULKOWSKI ZL 2012)
Sulkowski ZL, Chen T, Midha S, Zavacki AM, Sajdel‐Sulkowska EM. Cerebellum. 2012
Jun;11(2):575‐86. doi: 10.1007/s12311‐011‐0319‐5.
Methylmercury (Met‐Hg) and ethylmercury (Et‐Hg) are powerful toxicants with a range of
harmful neurological effects in humans and animals. While Met‐Hg is a recognized trigger of
oxidative stress and an endocrine disruptor impacting neurodevelopment, the developmental
neurotoxicity of Et‐Hg, a metabolite of thimerosal (TM), has not been explored. We
hypothesized that TM exposure during the perinatal period impairs central nervous system
development, and specifically the cerebellum, by the mechanism involving oxidative stress. ToScience Summary on Mercury in Vaccines (Thimerosal Only)
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test this, spontaneously hypertensive rats (SHR) or Sprague‐Dawley (SD) rat dams were exposed
to TM (200 μg/kg body weight) during pregnancy (G10‐G15) and lactation (P5‐P10). Male and
female neonates were evaluated for auditory and motor function; cerebella were analyzed for
oxidative stress and thyroid metabolism. TM exposure resulted in a delayed startle response in
SD neonates and decreased motor learning in SHR male (22.6%), in SD male (29.8%), and in SD
female (55.0%) neonates. TM exposure also resulted in a significant increase in cerebellar levels
of the oxidative stress marker 3‐nitrotyrosine in SHR female (35.1%) and SD male (14.0%)
neonates. The activity of cerebellar type 2 deiodinase, responsible for local intra‐brain
conversion of thyroxine to the active hormone, 3',3,5‐triiodothyronine (T3), was significantly
decreased in TM‐exposed SHR male (60.9%) pups. This coincided with an increased (47.0%)
expression of a gene negatively regulated by T3, Odf4 suggesting local intracerebellar T3
deficiency. Our data thus demonstrate a negative neurodevelopmental impact of perinatal TM
exposure which appears to be both strain‐ and sex‐dependent.
Cellular Research
BIOCHEMICAL AND MOLECULAR BASIS OF THIMEROSAL‐INDUCED APOPTOSIS IN T CELLS: A
MAJOR ROLE OF MITOCHONDRIAL PATHWAY (MAKANI 2002)
Makani S, Gollapudi S, Yel L, Chiplunkar S, Gupta S. Genes & Immunity. 2002 Aug;3(5):270‐8.
Makani found thimerosal, in micromolar concentration, causes cell death (apoptosis) in
immune cells (T cells). The data also suggested that the thimerosal induced apoptosis in T cells
occurred via mitochondrial pathways by inducing oxidative stress and depletion of glutathione.
THIMEROSAL INDUCES MICRONUCLEI IN THE CYTOCHALASIN B BLOCK MICRONUCLEUS TEST
WITH HUMAN LYMPHOCYTES (WESTPHAL 2003)
Westphal GA, Asgari S, Schulz TG, Bünger J, Müller M, Hallier E. Archives of Toxicology. 2003 Jan;
77(1):50 – 55.
Significant induction of micronuclei was seen at concentrations of thimerosal between 0.05‐0.5
µg/ml in 14 out of 16 experiments. Thus, genotoxic effects were seen even at concentrations
which can occur at the injection site. Toxicity and toxicity‐related elevation of micronuclei was
seen at and above 0.6 µg/ml thimerosal. Marked individual and intraindividual variations in the
in vitro response to thimerosal among the different blood donors occurred. However, there was
no association observed with any of the glutathione S‐transferase polymorphism investigated.
In conclusion, thimerosal is genotoxic in the cytochalasin B block micronucleus test with human
lymphocytes (immune cells). These data raise some concern on the widespread use of
thimerosal. Science Summary on Mercury in Vaccines (Thimerosal Only)
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THIMEROSAL INDUCES DNA BREAKS, CASPASE‐3 ACTIVATION, MEMBRANE DAMAGE, AND
CELL DEATH IN CULTURED HUMAN NEURONS AND FIBROBLASTS (BASKIN 2003)
Baskin DS, Ngo H, Didenko VV. Toxicological Sciences. 2003 Aug;74(2):361‐8.
Baskin documented that thimerosal disrupts cell membranes, damages DNA and alters cell
shape at concentrations only 4 times those expected from vaccines. Greater effects were seen
as the length of time of exposure grew, suggesting that under real conditions the concentration
needed for the observed alterations would be much lower. It has been documented in
subsequent research that exposure of cells to nanomolar levels of thimerosal after 24 hours
results in cell alterations.
ACTIVATION OF METHIONINE SYNTHASE BY INSULIN‐LIKE GROWTH FACTOR‐1 AND
DOPAMINE: A TARGET FOR NEURODEVELOPMENTAL TOXINS AND THIMEROSAL. (WALY 2004)
Waly M, Olteanu H, Banerjee R, Choi SW, Mason JB, Parker BS, Sukumar S, Shim S, Sharma A, Benzecry
JM, Power‐Charnitsky VA, Deth RC. Mol Psychiatry. 2004 Apr;9(4):358‐70.
Methylation events play a critical role in the ability of growth factors to promote normal
development. Neurodevelopmental toxins, such as ethanol and heavy metals, interrupt growth
factor signaling, raising the possibility that they might exert adverse effects on methylation. We
found that insulin‐like growth factor‐1 (IGF‐1)‐ and dopamine‐stimulated methionine synthase
(MS) activity and folate‐dependent methylation of phospholipids in SH‐SY5Y human
neuroblastoma cells, via a PI3‐kinase‐ and MAP‐kinase‐dependent mechanism. The stimulation
of this pathway increased DNA methylation, while its inhibition increased methylation‐sensitive
gene expression. Ethanol potently interfered with IGF‐1 activation of MS and blocked its effect
on DNA methylation, whereas it did not inhibit the effects of dopamine. Metal ions potently
affected IGF‐1 and dopamine‐stimulated MS activity, as well as folate‐dependent phospholipid
methylation: Cu(2+) promoted enzyme activity and methylation, while Cu(+), Pb(2+), Hg(2+)
and Al(3+) were inhibitory. The ethylmercury‐containing preservative thimerosal inhibited both
IGF‐1‐ and dopamine‐stimulated methylation with an IC(50) of 1 nM and eliminated MS activity.
Our findings outline a novel growth factor signaling pathway that regulates MS activity and
thereby modulates methylation reactions, including DNA methylation. The potent inhibition of
this pathway by ethanol, lead, mercury, aluminum and thimerosal suggests that it may be an
important target of neurodevelopmental toxins.Science Summary on Mercury in Vaccines (Thimerosal Only)
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UNCOUPLING OF ATP‐MEDIATED CALCIUM SIGNALING AND DYSREGULATION INTERLEUKIN‐6
SECRETION IN DENDRITIC CELLS BY NANAMOLAR THIMEROSAL (GOTH 2006)
Goth SR, Chu RA, Gregg JP, Cherednichenko G, Pessah IN. Environ Health Perspect. 2006
Jul;114(7):1083‐91.
Goth investigated adenosine triphosphate (ATP) mediated Ca2+ responses in dendritic cells
(responsible for initiating primary immune responses) exposed briefly to nanamolar
concentrations (100nM, 5 min) of thimerosal and found that dendritic cells were exquisitely
sensitive to thimerosal resulting in uncoupling of the positive and negative regulation of Ca2 +
signals.
THIMEROSAL INDUCES NEURONAL CELL DEATH BY CAUSING CYTOCHROME C AND
APOPTOSIS‐INDUCING FACTOR RELEASE FROM MITOCHONDRIA (YEL 2005)
Yel L, Brown LE, Su K, Gollapudi S, Gupta S. Int J Mol Med. 2005 Dec;16(6):971‐7.
Yel demonstrated that thimerosal, at nanamolar concentrations, induced neuronal cell death
through the mitochondrial pathway. The thimerosal induced apoptosis was associated with
depolarization of mitochondrial membranes, generation of reactive oxygen species and release
of cytochrome c and apoptosis‐inducing factor, suggesting that thimerosal cause apoptosis in
neuroblastoma cells by altering the mitochondrial microenvironment.
IN VITRO UPTAKE OF GLUTAMATE IN GLAST AND GLT‐1 TRANSFECTED MUTANT CHO‐K1 CELLS
IS INHIBITED BY THE ETHYLMERCURY‐CONTAINING PRESERVATIVE THIMEROSAL (MUTKUS
2005)
Mutkus L, Aschner JL, Syversen T, Shanker G, Sonnewald U, Aschner M. Bio Trace Elem Res. 2005
Summer;105(1‐3):71‐86
Mutkus determined that thimerosal caused significant and selective changes in both glutamate
transporter mRNA and protein expression in the CHO‐K1 cell line. This study suggests that
thimerosal accumulation in the central nervous system might contribute to dysregulation of
glutamate homeostasis. Glutamate is a neurotransmitter and is necessary for proper brain
functioning. Note: Yip (2007) documented decreased levels of glutamate in autistic cerebral
brain tissue and Hornig ( 2004) noted altered glutamate receptors in thimerosal exposed mice. Science Summary on Mercury in Vaccines (Thimerosal Only)
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THIMEROSAL NEUROTOXICITY IS ASSOCIATED WITH GLUTATHIONE DEPLETION: PROTECTION
WITH GLUTATHIONE PRECURSORS (JAMES 2005)
JAMES SJ, SLIKKER W 3RD, MELNYK S, NEW E, POGRIBNA M, JERNIGAN S. NEUROTOXICOLOGY. 2005
JAN;26(1):1‐8.
James notes that the viability of neuronal cell lines was decreased after just 3 hour exposure to
2.5 micromolar concentrations of thimerosal. Also noted was that cultured neuroblastoma cells
were found to have lower levels of GSH and increased sensitivity to thimerosal in comparison to
glioblastoma cells that contain higher levels of GSH. Furthermore, pretreatment with
glutathione ethyl ester or NAC prevented cytotoxicity with exposure up to 15 micromolar
thimerosal.
THIMEROSAL INDUCES APOPTOSIS IN A NEUROBLASTOMA MODEL VIA THE CJUN‐N‐
TERMINAL KINASE PATHWAY (HERDMAN 2006)
Herdman ML, Marcelo A, Huang Y, Niles RM, Dhar S, Kiningham KK. Toxicol Sci. 2006 Jul;92(1):246‐53.
Herdman notes that cJun N‐terminase kinase (JNK)‐signaling pathway activation has been
implicated in neuronal apoptosis. Herdman investigated the role that the JNK pathway plays in
neurotoxicity caused by thimerosal. SK‐N‐SH cells treated with thimerosal (0‐10 microM)
showed an increase in the phosphorylated (active) form of JNK and cJun with 5 and 10 microM
thimerosal treatment at 2 and 4 h.. To assess which components are essential to apoptosis,
cells were treated with a cell‐permeable JNK inhibitor and the downstream effectors of
apoptosis were analyzed. Results indicate that thimerosal‐induced neurotoxicity occurs through
the JNK‐signaling pathway, independent of cJun activation, leading to apoptotic cell death.
THIMEROSAL INDUCES TH2 RESPONSES VIA INFLUENCING CYTOKINE SECRETION BY HUMAN
DENDRITIC CELLS (AGRAWAL 2007)
Agrawal A, Kaushal P, Agrawal S, Gollapudi S, Gupta S. J Leukoc Biol. 2007 Feb;81(2):474‐82.
Agrawal documented that thimerosal exercised TH2‐promoting effects through modulation of
functions of human dendritic cells (DC) by inhibition of LPS induced proinflammatory cytokines
TNF‐alpha, IL‐6, and IL‐12p70 resulting in an increase TH2 (IL‐5, IL‐13 and decreased TH1 (IFN‐
gamma). Thimerosal exposure of DC led to depletion of intracellular glutathione (GSH) and the
addition of exogenous GSH to DC abolished the TH2 promoting effect of thimerosal. (Note
James has documented that children with autism have low levels of plasma glutathione)Science Summary on Mercury in Vaccines (Thimerosal Only)
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EFFECTS OF THIMEROSAL ON NGF SIGNAL TRANSDUCTION AND CELL DEATH IN
NEUROBLASTOMA CELLS (PARRAN 2005)
Parran DK, Barker A, Ehrich M. Toxicological Sciences. 2005 Jul;86(1):132‐40.
Parran documented that thimerosal causes DNA fragmentation of neuronal cells and disrupts
neuronal growth factor signaling at micromolar and even nanomolar concentrations. With and
without NGF, thimerosal caused elevated levels of fragmented DNA appearing at 0.01 microM
(apoptosis) to decrease at concentrations >1 microM (necrosis). These data demonstrate that
thimerosal could alter NGF‐induced signaling in neurotrophin¬treated cells at concentrations
lower than those responsible for cell death.
GENOTOXICITY OF THIMEROSAL IN CULTURED HUMAN LYMPHOCYTES WITH AND WITHOUT
METABOLIC ACTIVATION SISTER CHROMATID EXCHANGE ANALYSIS PROLIFERATION INDEX
AND MITOTIC INDEX (EKE 2008)
Eke D, Celik A. Mersin University, Faculty of Science and Letters, Department of Biology, 33343 Mersin,
Turkey. Toxicol In Vitro. 2008 Jun;22(4):927‐34. Epub 2008 Feb 1.
Thimerosal is an antiseptic containing 49.5% of ethyl mercury that has been used for years as a
preservative in many infant vaccines and in flu vaccines. Thimerosal is an organic mercurial
compound used as a preservative in biomedical preparations. In this study, we evaluated the
genotoxic effect of thimerosal in cultured human peripheral blood lymphocytes using sister
chromatid exchange analysis in culture conditions with and without S9 metabolic activation.
This study is the first report investigating the genotoxic effects of thimerosal in cultured human
peripheral blood lymphocyte cells using sister chromatid exchange analysis. An analysis of
variance test (ANOVA) was performed to evaluate the results. Significant induction of sister
chromatid exchanges was seen at concentrations between 0.2 and 0.6 microg/ml of thimerosal
compared with negative control. A significant decrease (p<0.001) in mitotic index (MI) and
proliferation index (PRI) as well as an increase in SCE frequency (p<0.001) was observed
compared with control cultures. Our results indicate the genotoxic and cytotoxic effect of TH in
cultured human peripheral blood lymphocytes at tested doses in cultures with/without S9
fraction.
ZINC IONS CAUSE THE THIMEROSAL‐INDUCED SIGNAL OF FLUORESCENT CALCIUM PROBES IN
LYMPHOCYTES (HAASE 2008)
Haase H, Hebel S, Engelhardt G, Rink L., Institute of Immunology, RWTH Aachen University Hospital,
Aachen, Germany. Cell Calcium. 2008 Oct 31.
Most fluorescent probes for the investigation of calcium signaling also detect zinc ions.
Consequently, changes in the intracellular zinc concentration could be mistaken for calciumScience Summary on Mercury in Vaccines (Thimerosal Only)
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signals. Thimerosal (TMS) is used as a calcium‐mobilizing agent and we analyzed the
contribution of zinc ions to the signal observed with fluorescent calcium probes after TMS
stimulation. Our findings show that the fluorescent signal in lymphocytes is entirely due to zinc
release. Experiments in the T lymphocyte cell line Jurkat and primary human lymphocytes show
that TMS and its active metabolite, ethyl mercury, cause an increase in signal intensity with
probes designed for the detection of either calcium or zinc ions. The TMS/ethyl mercury‐
induced signal of the calcium probes Fluo‐4 and FURA‐2 was completely absent when the zinc
chelator TPEN [N,N,N',N'‐tetrakis‐(2‐pyridyl‐methyl)ethylenediamine] was added. In contrast,
the signal caused by thapsigargin‐induced release of calcium from the endoplasmic reticulum
was unaffected by TPEN. In light of these observations, zinc may also contribute to calcium
signals caused by mercury‐containing compounds other than TMS, and a potential involvement
of zinc release in the immunomodulatory effects of these substances should be considered.
CHARACTERIZATION OF EARLY EVENTS INVOLVED IN HUMAN DENDRITIC CELL MATURATION
INDUCED BY SENSITIZERS: CROSS TALK BETWEEN MAPK SIGNALLING PATHWAYS
(TROMPEZINSKI 2008)
Trompezinski S, Migdal C, Tailhardat M, Le Varlet B, Courtellemont P, Haftek M, Serres M. Toxicol Appl
Pharmacol. 2008 Aug 1;230(3):397‐406. Epub 2008 Apr 8.
Dendritic cells (DCs), efficient‐antigen presenting cells play an important role in initiating and
regulating immune responses. DC maturation following exposure to nickel or DNCB induced an
up‐regulation of phenotypic markers and inflammatory cytokine secretion. Early intracellular
mechanisms involved in DC maturation required to be precise. To address this purpose, DCs
derived from human monocytes were treated with sensitizers (nickel, DNCB or thimerosal) in
comparison with an irritant (SDS). Our data confirming the up‐regulation of CD86, CD54 and
cytokine secretion (IL‐8 and TNFalpha) induced by sensitizers but not by SDS, signalling
transduction involved in DC maturation was investigated using these chemicals. Kinase activity
measurement was assessed using two new sensitive procedures (Facetrade mark and CBA)
requiring few cells. SDS did not induce changes in signalling pathways whereas NiSO(4), DNCB
and thimerosal markedly activated p38 MAPK and JNK, in contrast Erk1/2 phosphorylation was
completely inhibited by DNCB or thimerosal and only activated by nickel. A pre‐treatment with
p38 MAPK inhibitor (SB203580) suppressed Erk1/2 inhibition induced by DNCB or thimerosal
demonstrating a direct interaction between p38 MAPK and Erk1/2. A pre‐treatment with an
antioxidant, N‐acetyl‐L‐cysteine (NAC) markedly reduced Erk1/2 inhibition and p38 MAPK
phosphorylation induced by DNCB and thimerosal, suggesting a direct activation of p38 MAPK
via an oxidative stress and a regulation of MAPK signalling pathways depending on chemicals.
Because of a high sensitivity of kinase activity measurements, these procedures will be suitable
for weak or moderate sensitizer screening.Science Summary on Mercury in Vaccines (Thimerosal Only)
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MITOCHONDRIAL MEDIATED THIMEROSAL‐INDUCED APOPTOSIS IN A HUMAN
NEUROBLASTOMA CELLINE (SK‐N‐SH)(HUMPHREY 2009)
Humphrey ML, Cole MP, Pendergrass JC, Kiningham KK. Neurotoxicology. 2005 Jun;26(3):407‐16.
Humphrey noted that after only short (2 hour) exposures to thimerosal at 5 micromolar
concentrations in a human neuroblastoma cell line caused morphological changes including
membrane alterations and cell shrinkage leading to cell death. Cytochrome C was shown to
leak from the mitochondria followed by caspase 9 cleavage. These findings support
deleterious effects on cellular cytoarchitecture and initiation of mitochondrial‐mediated
apoptosis induced by thimerosal.
MITOCHONDRIAL DYSFUNCTION, IMPAIRED OXIDATIVE‐REDUCTION ACTIVITY,
DEGENERATION, AND DEATH IN HUMAN NEURONAL AND FETAL CELLS INDUCED BY LOW‐
LEVEL EXPOSURE TO THIMEROSAL AND OTHER METAL COMPOUNDS (GEIER 2009)
D.A. Geier et al. Toxicological & Environmental Chemistry. 2009, 1–15, iFirst
Thimerosal (ethylmercurithiosalicylic acid), an ethylmercury (EtHg)‐releasing compound
(49.55% mercury (Hg)), was used in a range of medical products for more than 70 years. Of
particular recent concern, routine administering of Thimerosal‐containing biologics/childhood
vaccines have become significant sources of Hg exposure for some fetuses/infants. This study
was undertaken to investigate cellular damage among in vitro human neuronal (SH‐SY‐5Y
neuroblastoma and 1321N1 astrocytoma) and fetal (nontransformed) model systems using cell
vitality assays and microscope‐based digital image capture techniques to assess potential
damage induced by Thimerosal and other metal compounds (aluminum (Al) sulfate, lead (Pb)(II)
acetate, methylmercury (MeHg) hydroxide, and mercury (Hg)(II) chloride) where the cation was
reported to exert adverse effects on developing cells. Thimerosal‐associated cellular damage
was also evaluated for similarity to pathophysiological findings observed in patients diagnosed
with autistic disorders (ADs). Thimerosal‐induced cellular damage as evidenced by
concentration‐and time‐dependent mitochondrial damage, reduced oxidative–reduction
activity, cellular degeneration, and cell death in the in vitro human neuronal and fetal model
systems studied. Thimerosal at low nanomolar (nM) concentrations induced significant cellular
toxicity in human neuronal and fetal cells. Thimerosal‐induced cytoxicity is similar to that
observed in AD pathophysiologic studies. Thimerosal was found to be significantly more toxic
than the other metal compounds examined. Future studies need to be conducted to evaluate
additional mechanisms underlying Thimerosal‐induced cellular damage and assess potential co‐
exposures to other compounds that may increase or decrease Thimerosal‐mediated toxicity. Science Summary on Mercury in Vaccines (Thimerosal Only)
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SENSITIZATION EFFECT OF THIMEROSAL IS MEDIATED IN VITRO VIA REACTIVE OXYGEN
SPECIES AND CALCIUM SIGNALING. (MIGDAL 2010)
Migdal C, Foggia L, Tailhardat M, Courtellemont P, Haftek M, Serres M. Toxicology. 2010 Jul‐Aug;274(1‐
3):1‐9. Epub 2010 May 10.
Thimerosal, a mercury derivative composed of ethyl mercury chloride (EtHgCl) and thiosalicylic
acid (TSA), is widely used as a preservative in vaccines and cosmetic products and causes
cutaneous reactions. Since dendritic cells (DCs) play an essential role in the immune response,
the sensitization potency of chemicals was studied in vitro using U937, a human
promyelomonocytic cell line that is used as a surrogate of monocytic differentiation and
activation. Currently, this cell line is under ECVAM (European Center for the Validation of
Alternative Methods) validation as an alternative method for discriminating chemicals.
Thimerosal and mercury derivatives induced in U937 an overexpression of CD86 and interleukin
(IL)‐8 secretion similarly to 1‐chloro‐2,4‐dinitrobenzene (DNCB), a sensitizer used as a positive
control for DC activation. Non‐sensitizers, dichloronitrobenzene (DCNB), TSA and sodium
dodecyl sulfate (SDS), an irritant, had no effect. U937 activation was prevented by cell
pretreatment with N‐acetyl‐L‐cysteine (NAC) but not with thiol‐independent antioxidants
except vitamin E which affected CD86 expression by preventing lipid peroxidation of cell
membranes. Thimerosal, EtHgCl and DNCB induced glutathione (GSH) depletion and reactive
oxygen species (ROS) within 15 min; another peak was detected after 2h for mercury
compounds only. MitoSOX, a specific mitochondrial fluorescent probe, confirmed that ROS
were essentially produced by mitochondria in correlation with its membrane depolarization.
Changes in mitochondrial membrane permeability induced by mercury were reversed by NAC
but not by thiol‐independent antioxidants. Thimerosal and EtHgCl also induced a calcium (Ca2+)
influx with a peak at 3h, suggesting that Ca2+ influx is a secondary event following ROS
induction as Ca2+ influx was suppressed after pretreatment with NAC but not with thiol‐
independent antioxidants. Ca2+ influx was also suppressed when culture medium was deprived
of Ca2+ confirming the specificity of the measure. In conclusion, these data suggest that
thimerosal induced U937 activation via oxidative stress from mitochondrial stores and
mitochondrial membrane depolarization with a primordial effect of thiol groups. A cross‐talk
between ROS and Ca2+ influx was demonstrated.
EVALUATION OF CYTOTOXICITY ATTRIBUTED TO THIMEROSAL ON MURINE AND HUMAN
KIDNEY CELLS. (PARK 2007)
Park EK, Mak SK, Kültz D, Hammock BD. J Toxicol Environ Health A. 2007 Dec;70(24):2092‐5.
Renal inner medullary collecting duct cells (mIMCD3) and human embryonic kidney cells
(HEK293) were used for cytoscreening of thimerosal and mercury chloride (HgCl2). Thimerosal
and HgCl2 acted in a concentration‐dependent manner. In mIMCD3 cells the 24‐h LC50 valuesScience Summary on Mercury in Vaccines (Thimerosal Only)
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for thimerosal, thiosalicylic acid, 2,2‐dithiosalicylic acid, and 2‐sulfobenzoic acid were 2.9, 2200,
>1000, and >10,000 microM, respectively. The 24‐h LC50 value for HgCl2 in mIMCD3 cells was
40 microM. In HEK293 cells, the 24‐h LC50 value for thimerosal was 9.5 microM. These data
demonstrate that the higher cytotoxicity produced by thimerosal on renal cells with respect to
similar compounds without Hg may be related to this metal content. The present study also
establishes mIMCD3 cells as a valuable model for evaluation of cytotoxicity of nephrotoxic
compounds.
THE RELATIVE TOXICITY OF COMPOUNDS USED AS PRESERVATIVES IN VACCINES AND
BIOLOGICS. (GEIER 2010)
Geier DA, Jordan SK, Geier MR. Med Sci Monit. 2010 Apr 28;16(5):SR21‐7.
BACKGROUND: In vaccines/biologics, preservatives are used to prevent microbial growth.
MATERIAL/METHODS: The present study examined: (1) the comparative toxicities of commonly
used preservatives in US licensed vaccines to human neurons; and (2) the relative toxicity index
of these compounds to human neurons in comparison to bacterial cells. RESULTS: Using human
neuroblastoma cells, the relative cytotoxicity of the levels of the compounds commonly used as
preservative in US licensed vaccines was found to be phenol <2‐phenoxyethanol <
benzethonium chloride < Thimerosal. The observed relative toxicity indices (human
neuroblastoma cells/bacterial cells) were 2‐phenoxyethanol (4.6‐fold) < phenol (12.2‐fold) <
Thimerosal (>330‐fold). In addition, for the compounds tested, except for 2‐phenoxyethanol,
the concentrations necessary to induce significant killing of bacterial cells were significantly
higher than those routinely present in US licensed vaccine/biological preparations.
CONCLUSIONS: None of the compounds commonly used as preservatives in US licensed
vaccine/biological preparations can be considered an ideal preservative, and their ability to fully
comply with the requirements of the US Code of Federal Regulations (CFR) for preservatives is
in doubt. Future formulations of US licensed vaccines/biologics should be produced in aseptic
manufacturing plants as single dose preparations, eliminating the need for preservatives and an
unnecessary risk to patients.
LOW MOLECULAR WEIGHT THIOLS REDUCE THIMEROSAL NEUROTOXICITY IN VITRO:
MODULATION BY PROTEINS. (ZIEMINSKA 2010)
Zieminska E, Toczylowska B, Stafiej A, Lazarewicz JW. Toxicology. 2010 Aug 7. [Epub ahead of print]
Thimerosal (TH), an ethylmercury complex of thiosalicylic acid has been used as preservative in
vaccines. In vitro neurotoxicity of TH at high nM concentrations has been reported. Although a
number of toxicological experiments demonstrated high affinity of mercury to thiol groups of
the extracellular amino acids and proteins that may decrease concentration of free TH in the
organism, less is known about the role of interactions between proteins and amino acids in
protection against TH neurotoxicity. In the present study we examined whether the presence ofScience Summary on Mercury in Vaccines (Thimerosal Only)
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serum proteins and of l‐cysteine (Cys), d,l‐homocysteine (Hcy), N‐acetyl cysteine (NAC), l‐
methionine (Met) and glutathione (GSH) in the incubation medium affects the TH‐induced
changes in the viability, the intracellular levels of calcium and zinc and mitochondrial
membrane potential in primary cultures of rat cerebellar granule cells. The cells were exposed
to 500nM TH for 48h or to 15‐25muM TH for 10min. Our results demonstrated a decrease in
the cells viability evoked by TH, which could be prevented partially by serum proteins, albumin
or in a dose‐dependent manner by 60, 120 or 600muM Cys, Hcy, NAC and GSH, but not by Met.
This neuroprotection was less pronounced in the presence of proteins. Incubation of neurons
with TH also induced the rise in the intracellular calcium and zinc concentration and decrease in
mitochondrial membrane potential, and these effects were abolished by all the sulfur
containing compounds studied and administered at 600muM concentration, except Met. The
loss of the ethylmercury moiety from TH as a result of interaction with thiols studied was
monitored by (1)H NMR spectroscopy. This extracellular process may be responsible for the
neuroprotection seen in the cerebellar cell cultures, but also provides a molecular pathway for
redistribution of TH‐derived toxic ethylmercury in the organism. In conclusion, these results
confirmed that proteins and sulfur‐containing amino acids applied separately reduce TH
neurotoxicity, while their combination modulates in more complex way neuronal survival in the
presence of TH.
RESPONSIVENESS OF HUMAN MONOCYTE‐DERIVED DENDRITIC CELLS TO THIMEROSAL AND
MERCURY DERIVATIVES. (MIGDAL 2010)
Migdal C, Tailhardat M, Courtellemont P, Haftek M, Serres M. Toxicol Appl Pharmacol. 2010 Jul;246(1‐
2):66‐73. Epub 2010 Apr 22.
Several cases of skin sensitization have been reported following the application of thimerosal,
which is composed of ethyl mercury and thiosalicylic acid (TSA). However, few in vitro studies
have been carried out on human dendritic cells (DCs) which play an essential role in the
initiation of allergic contact dermatitis. The aim of the present study was to identify the effect
of thimerosal and other mercury compounds on human DCs. To address this purpose, DCs
derived from monocytes (mono‐DCs) were used. Data show that thimerosal and mercury
derivatives induced DC activation, as monitored by CD86 and HLA‐DR overexpression associated
with the secretion of tumor necrosis factor alpha and interleukin 8, similarly to
lipopolysaccharide and the sensitizers, 1‐chloro‐2,4‐dinitrobenzene (DNCB) and nickel sulfate,
which were used as positive controls. In contrast, TSA, the non‐mercury part of thimerosal, as
well as dichloronitrobenzene, a DNCB negative control, and the irritant, sodium dodecyl sulfate,
had no effect. Moreover, oxidative stress, monitored by ROS induction and depolarization of
the mitochondrial membrane potential, was induced by thimerosal and mercury compounds, as
well as DNCB, in comparison with hydrogen peroxide, used as a positive control. The role ofScience Summary on Mercury in Vaccines (Thimerosal Only)
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thiol oxidation in the initiation of mono‐DC activation was confirmed by a pre‐treatment with
N‐acetyl‐l‐cysteine which strongly decreased chemical‐induced CD86 overexpression. These
data are in agreement with several clinical observations of the high relevance of thimerosal in
patch‐test reactions and prove that human mono‐DCs are useful in vitro tools for determining
the allergenic potency of chemicals.
MERCURY INDUCES AN UNOPPOSED INFLAMMATORY RESPONSE IN HUMAN PERIPHERAL
BLOOD MONONUCLEAR CELLS IN VITRO. (GARDNER 2009)
Gardner RM, Nyland JF, Evans SL, Wang SB, Doyle KM, Crainiceanu CM, Silbergeld EK. Environ Health
Perspect. 2009 Dec;117(12):1932‐8. Epub 2009 Aug 19.
BACKGROUND: The human immune response to mercury is not well characterized despite the
body of evidence that suggests that Hg can modulate immune responses, including the
induction of autoimmune disease in some mouse models. Dysregulation of cytokine signaling
appears to play an important role in the etiology of Hg‐induced autoimmunity in animal models.
OBJECTIVES: In this study, we systematically investigated the human immune response to Hg in
vitro in terms of cytokine release. METHODS: Human peripheral blood mononuclear cells
(PBMCs) were isolated from 20 volunteers who donated blood six separate times. PBMCs were
cultured with lipopolysaccharide and concentrations of mercuric chloride (HgCl(2)) up to 200
nM. Seven cytokines representing important pathways in physiologic and pathologic immune
responses were measured in supernatants. We used multilevel models to account for the
intrinsic clustering in the cytokine data due to experimental design. RESULTS: We found a
consistent increase in the release of the proinflammatory cytokines interleukin‐1beta (IL‐1beta)
and tumor necrosis factor‐alpha, and concurrent decrease in release of the antiinflammatory
cytokines interleukin 1‐receptor antagonist (IL‐1Ra) and IL‐10 in human PBMCs treated with
subcytotoxic concentrations of HgCl(2). IL‐4, IL‐17, and interferon‐gamma increased in a
concentration‐response manner. These results were replicated in a second, independently
recruited population of 20 different volunteers. CONCLUSIONS: Low concentrations of HgCl(2)
affect immune function in human cells by dysregulation of cytokine signaling pathways, with
the potential to influence diverse health outcomes such as susceptibility to infectious disease or
risk of autoimmunity.
LUTEOLIN AND THIOSALICYLATE INHIBIT HGCL(2) AND THIMEROSAL‐INDUCED VEGF RELEASE
FROM HUMAN MAST CELLS. (ASADI 2010)
Asadi S, Zhang B, Weng Z, Angelidou A, Kempuraj D, Alysandratos KD, Theoharides TC. Int J
Immunopathol Pharmacol. 2010 Oct‐Dec;23(4):1015‐20.
HgCl2 is a known environemental neurotoxin, but is also used as preservative in vaccines as
thimerosal containing ethyl mercury covalently linked to thiosalicylate. We recently reportedScience Summary on Mercury in Vaccines (Thimerosal Only)
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that mercury choloride (HgCl(2)) can stimulate human mast cells to release vascular endothelial
growth factor (VEGF), which is also vasoactive and pro‐inflammatory. Here we show that
thimerosal induces significant VEGF release from human leukemic cultured LAD2 mast cells (at
1 microM 326 ± 12 pg/106 cells and 335.5 ± 12 pg/106 cells at 10 microM) compared to control
cells (242 ± 21 pg/106 cells, n=5, p less than 0.05); this effect is weaker than that induced by
HgCl2 at 10 microM (448 ± 14 pg/106 cells) (n=3, p less than 0.05). In view of this finding, we
hypothesize that the thiosalicylate component of thimerosal may have an inhibitory effect on
VEGF release. Thimerosal (10 microM) added together with the peptide Substance P (SP) at 2
microM, used as a positive control, reduced VEGF release by 90 percent. Methyl thiosalicylate
(1 or 10 microM) added with either SP or HgCl2 (10 microM) inhibited VEGF release by 100
percent, while sodium salicylate or ibuprofen had no effect. Pretreatment for 10 min with the
flavonoid luteolin (0.1 mM) before HgCl2 or thimerosal compeletly blocked their effect. Luteolin
and methyl thiosalicylate may be useful in preventing mercury‐induced toxicity.
INTERMINGLED MODULATORY AND NEUROTOXIC EFFECTS OF THIMEROSAL AND MERCURIC
IONS ON ELECTROPHYSIOLOGICAL RESPONSES TO GABA AND NMDA IN HIPPOCAMPAL
NEURONS. (WYREMBEK 2010)
Wyrembek P, Szczuraszek K, Majewska MD, Mozrzymas JW. J Physiol Pharmacol. 2010
Dec;61(6):753‐8.
The organomercurial, thimerosal, is at the center of medical controversy as a suspected factor
contributing to neurodevelopmental disorders in children. Many neurotoxic effects of
thimerosal have been described, but its interaction with principal excitatory and inhibitory
neurotransmiter systems is not known. We examined, using electrophysiological recordings,
thimerosal effects on GABA and NMDA‐evoked currents in cultured hippocampal neurons. After
brief (3 to 10 min) exposure to thimerosal at concentrations up to 100 μM, there was no
significant effect on GABA or NMDA‐evoked currents. However, following exposure for 60‐90
min to 1 or 10 μM thimerosal, there was a significant decrease in NMDA‐induced currents
(p<0.05) and GABAergic currents (p<0.05). Thimerosal was also neurotoxic, damaging a
significant proportion of neurons after 60‐90 min exposure; recordings were always conducted
in the healthiest looking neurons. Mercuric chloride, at concentrations 1 μM and above, was
even more toxic, killing a large proportion of cells after just a few minutes of exposure.
Recordings from a few sturdy cells revealed that micromolar mercuric chloride markedly
potentiated the GABAergic currents (p<0.05), but reduced NMDA‐evoked currents (p<0.05).
The results reveal complex interactions of thimerosal and mercuric ions with the GABA(A) and
NMDA receptors. Mercuric chloride act rapidly, decreasing electrophysiological responses to
NMDA but enhancing responses to GABA, while thimerosal works slowly, reducing both NMDA
and GABA responses. The neurotoxic effects of both mercurials are interwoven with theirScience Summary on Mercury in Vaccines (Thimerosal Only)
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modulatory actions on GABA(A) and NMDA receptors, which most likely involve binding to
these macromolecules.
THIMEROSAL‐DERIVED ETHYLMERCURY IS A MITOCHONDRIAL TOXIN IN HUMAN
ASTROCYTES: POSSIBLE ROLE OF FENTON CHEMISTRY IN THE OXIDATION AND BREAKAGE OF
MTDNA. (SHARPE 2012)
Sharpe MA, Livingston AD, Baskin DS. J Toxicol. 2012;2012:373678. doi: 10.1155/2012/373678.
Epub 2012 Jun 28.
Thimerosal generates ethylmercury in aqueous solution and is widely used as preservative. We
have investigated the toxicology of Thimerosal in normal human astrocytes, paying particular
attention to mitochondrial function and the generation of specific oxidants. We find that
ethylmercury not only inhibits mitochondrial respiration leading to a drop in the steady state
membrane potential, but also concurrent with these phenomena increases the formation of
superoxide, hydrogen peroxide, and Fenton/Haber‐Weiss generated hydroxyl radical. These
oxidants increase the levels of cellular aldehyde/ketones. Additionally, we find a five‐fold
increase in the levels of oxidant damaged mitochondrial DNA bases and increases in the levels
of mtDNA nicks and blunt‐ended breaks. Highly damaged mitochondria are characterized by
having very low membrane potentials, increased superoxide/hydrogen peroxide production,
and extensively damaged mtDNA and proteins. These mitochondria appear to have undergone
a permeability transition, an observation supported by the five‐fold increase in Caspase‐3
activity observed after Thimerosal treatment.
CYTOPROTECTIVE EFFECT OF HYALURONIC ACID AND HYDROXYPROPYL METHYLCELLULOSE
AGAINST DNA DAMAGE INDUCED BY THIMEROSAL IN CHANG CONJUNCTIVAL CELLS. (YE
2012)
Ye J, Zhang H, Wu H, Wang C, Shi X, Xie J, He J, Yang J. Graefes Arch Clin Exp Ophthalmol. 2012
Oct;250(10):1459‐66. doi: 10.1007/s00417‐012‐2087‐4. Epub 2012 Jun 24.
BACKGROUND: To investigate genotoxicity of the preservative thimerosal (Thi), and the
cytoprotective and antioxidant effects of hyaluronic Acid (HA) and hydroxypropyl
methylcellulose (HPMC) on Chang conjunctival cells.
METHOD: Cells were divided into three groups. One group was exposed to Thi at various
concentrations (0.00001 %∼0.001 %) for 30 min; the other two groups were pretreated with
0.3 % HA or 0.3 % HPMC for 30 min before the Thi exposure. After cell viability was evaluated,
alkaline comet assay and detection of the phosphorylated form of the histone variant H2AX
(γH2AX) foci were used to determine DNA damage. Reactive oxygen species (ROS) production
was assessed by the fluorescent probe, 2', 7'‐dichlorodihydrofluorescein diacetate (DCFH‐DA).
RESULTS: A significant change of cell viability was observed after exposure to 0.001 % Thi for 30Science Summary on Mercury in Vaccines (Thimerosal Only)
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41 | P a g e
min. DNA single‐ and double‐strand breaks were significantly increased in a dose‐dependent
manner with Thi exposure. In addition, intracellular ROS induced by Thi was dose‐dependent,
except at 0.001 % less ROS was induced than at 0.0005 %. However, cells pretreated with 0.3 %
HA or 0.3 % HPMC showed significantly increased cell survival, decreased DNA damage, and
decreased ROS production compared to cells exposed to Thi alone. Pretreatment with 0.3 % HA
was found to be even more protective than 0.3 % HPMC. CONCLUSION: Thi can induce DNA
damage in human conjunctival epithelial cells, probably due to oxidative stress. HA and HPMC
are protective agents that have antioxidant properties and can decrease DNA damage induced
by Thi. Pretreatment of 0.3 % HA may be more protective of the ocular surface than 0.3 %
HPMC.
ITPKC SUSCEPTIBILITY IN KAWASAKI SYNDROME AS A SENSITIZING FACTOR FOR
AUTOIMMUNITY AND CORONARY ARTERIAL WALL RELAXATION INDUCED BY THIMEROSAL'S
EFFECTS ON CALCIUM SIGNALING VIA IP3. (YETER 2012)
Yeter D, Deth R. Autoimmun Rev. 2012 Oct;11(12):903‐8. doi: 10.1016/j.autrev.2012.03.006.
Epub 2012 Apr 1.
Recently, a single nucleotide polymorphism (SNP) of the inositol 1,4,5‐triphosphate kinase C
(ITPKC), rs28493229, was found to passively confer susceptibility for Kawasaki syndrome (KS)
and subsequent coronary arterial lesions. This association is believed to be the result of
defective phosphorylation of inositol 1,4,5‐triphosphate (IP3), which releases calcium from
intracellular stores, resulting from reduced genetic expression of ITPKC in carriers of the SNP.
Reduced ITPKC activity would increase IP3 levels, and thus, increase calcium release. We
hypothesized that an environmental agent which influences IP3‐mediated calcium release is
potentiated by the ITPKC SNP. This led us to an attractive candidate, thimerosal, an
organomercurial medical preservative still used in several pediatric vaccines. Thimerosal is well‐
known to sensitize IP3 receptors via its induction of oxidative stress, resulting in enhanced
release of intracellular calcium with distinctive consequences for various cell types.
Dysregulated calcium signaling in T cells and other immune cells can result in autoimmunity,
while hyperpolarization of vascular smooth muscle cells secondary to the stimulation of
calcium‐activated potassium channels can result in increased vascular permeability and arterial
relaxation. We propose that ITPKC susceptibility in KS is related to its synergy with
environmental triggers, such as thimerosal, which alter calcium homeostasis and promote
oxidative stress. Therefore, carriers of the ITPKC SNP are more susceptible to thimerosal‐
induced autoimmunity and coronary arterial lesions observed in KS. This would explain why
only a susceptible subset of children develops KS although pediatric thimerosal exposure is
nearly universal due to vaccination. As was experienced with the infantile acrodynia epidemic,
only 1 in 500 children developed the disease although pediatric mercury exposure was nearlyScience Summary on Mercury in Vaccines (Thimerosal Only)
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42 | P a g e
ubiquitous due to the use calomel teething powders. This hypothesis also mirrors the current
leading theory for KS in which a widespread infection only induces the disease in susceptible
children. We conclude that KS may be the acute febrile form of acrodynia.
THIMEROSAL‐INDUCED APOPTOSIS IN MOUSE C2C12 MYOBLAST CELLS OCCURS THROUGH
SUPPRESSION OF THE PI3K/AKT/SURVIVIN PATHWAY (LI 2012)
Li WX, Chen SF, Chen LP, Yang GY, Li JT, Liu HZ, Zhu W. PLoS One. 2012;7(11):e49064. doi:
10.1371/journal.pone.0049064. Epub 2012 Nov 7.
BACKGROUND: Thimerosal, a mercury‐containing preservative, is one of the most widely used
preservatives and found in a variety of biological products. Concerns over its possible toxicity
have reemerged recently due to its use in vaccines. Thimerosal has also been reported to be
markedly cytotoxic to neural tissue. However, little is known regarding thimerosal‐induced
toxicity in muscle tissue. Therefore, we investigated the cytotoxic effect of thimerosal and its
possible mechanisms on mouse C2C12 myoblast cells. METHODOLOGY/PRINCIPAL FINDINGS:
The study showed that C2C12 myoblast cells underwent inhibition of proliferation and
apoptosis after exposure to thimerosal (125‐500 nM) for 24, 48 and 72 h. Thimerosal caused S
phase arrest and induced apoptosis as assessed by flow cytometric analysis, Hoechst staining
and immunoblotting. The data revealed that thimerosal could trigger the leakage of
cytochrome c from mitochondria, followed by cleavage of caspase‐9 and caspase‐3, and that an
inhibitor of caspase could suppress thimerosal‐induced apoptosis. Thimerosal inhibited the
phosphorylation of Akt(ser473) and survivin expression. Wortmannin, a PI3K inhibitor, inhibited
Akt activity and decreased survivin expression, resulting in increased thimerosal‐induced
apoptosis in C2C12 cells, while the activation of PI3K/Akt pathway by mIGF‐I (50 ng/ml)
increased the expression of survivin and attenuated apoptosis. Furthermore, the inhibition of
survivin expression by siRNA enhanced thimerosal‐induced cell apoptosis, while overexpression
of survivin prevented thimerosal‐induced apoptosis. Taken together, the data show that the
PI3K/Akt/survivin pathway plays an important role in the thimerosal‐induced apoptosis in
C2C12 cells. CONCLUSIONS/SIGNIFICANCE: Our results suggest that in C2C12 myoblast cells,
thimerosal induces S phase arrest and finally causes apoptosis via inhibition of
PI3K/Akt/survivin signaling followed by activation of the mitochondrial apoptotic pathway
Summary of Supportive ScienceRegarding Thimerosal RemovalUpdated December 2012www.safeminds.orgScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 20121 | P a g eContentsENVIRONMENTAL IMPACT ................................................................................................................................. 4A PILOT SCALE EVALUATION OF REMOVAL OF MERCURY FROM PHARMACEUTICAL WASTEWATER USING GRANULAR ACTIVATED CARBON(CYR 2002) .................................................................................................................................................................4BIODEGRADATION OF THIOMERSAL CONTAINING EFFLUENTS BY A MERCURY RESISTANT PSEUDOMONAS PUTIDA STRAIN (FORTUNATO2005) .........................................................................................................................................................................4USE OF ADSORPTION PROCESS TO REMOVE ORGANIC MERCURY THIMEROSAL FROM INDUSTRIAL PROCESS WASTEWATER (VELICU 2007)5HUMAN & INFANT RESEARCH ............................................................................................................................ 5IATROGENIC EXPOSURE TO MERCURY AFTER HEPATITIS B VACCINATION IN PRETERM INFANTS (STAJICH 2000)..................................5MERCURY CONCENTRATIONS AND METABOLISM IN INFANTS RECEIVING VACCINES CONTAINING THIMEROSAL: A DESCRIPTIVE STUDY(PICHICHERO 2002).......................................................................................................................................................6HAIR MERCURY IN BREAST‐FED INFANTS EXPOSED TO THIMEROSAL‐PRESERVED VACCINES (MARQUES 2007) ...................................6MERCURY LEVELS IN NEWBORNS AND INFANTS AFTER RECEIPT OF THIMEROSAL‐CONTAINING VACCINES (PICHICHERO 2008)...............6HEPATITIS B TRIPLE SERIES VACCINE AND DEVELOPMENTAL DISABILITY IN US CHILDREN AGED 1‐9 YEARS (GALLAGHER 2008) .............7NEONATE EXPOSURE TO THIMEROSAL MERCURY FROM HEPATITIS B VACCINES (DOREA 2009)......................................................8URINARY PORPHYRIN EXCRETION IN NEUROTYPICAL AND AUTISTIC CHILDREN (WOODS 2010).....................................................8EMBRYONIC EXPOSURE TO THIMEROSAL, AN ORGANOMERCURY COMPOUND, CAUSES ABNORMAL EARLY DEVELOPMENT OFSEROTONERGIC NEURONS. (IDA‐ETO 2011)........................................................................................................................9SPECIATION OF METHYL‐ AND ETHYL‐MERCURY IN HAIR OF BREASTFED INFANTS ACUTELY EXPOSED TO THIMEROSAL‐CONTAININGVACCINES. (DOREA 2011) ...............................................................................................................................................9INTEGRATING EXPERIMENTAL (IN VITRO AND IN VIVO) NEUROTOXICITY STUDIES OF LOW‐DOSE THIMEROSAL RELEVANT TO VACCINES.(DOREA 2011)............................................................................................................................................................10NEONATAL EXPOSURE TO THIMEROSAL FROM VACCINES AND CHILD DEVELOPMENT IN THE FIRST 3YEARS OF LIFE. (MROZEK‐BUDZYN2012) .......................................................................................................................................................................11BREAST‐FEEDING AND RESPONSES TO INFANT VACCINES: CONSTITUTIONAL AND ENVIRONMENTAL FACTORS. (DOREA 2012).............11THIMEROSAL EXPOSURE IN EARLY LIFE AND NEUROPSYCHOLOGICAL OUTCOMES 7‐10 YEARS LATER. (BARILE 2012).........................12NON‐HUMAN PRIMATE INFANT RESEARCH .......................................................................................................12COMPARISON OF BLOOD AND BRAIN MERCURY LEVELS IN INFANT MONKEYS EXPOSED TO METHYLMERCURY OR VACCINES CONTAININGTHIMEROSAL (BURBACHER 2005)...................................................................................................................................12PEDIATRIC VACCINES INFLUENCE PRIMATE BEHAVIOR, AND AMYGDALA GROWTH AND OPIOID LIGAND BINDING (HEWITSON 2008).13MICROARRAY ANALYSIS OF GI TISSUE IN A MACAQUE MODEL OF THE EFFECTS OF INFANT VACCINATION (WALKER 2008) ..............14DELAYED ACQUISITION OF NEONATAL REFLEXES IN NEWBORN PRIMATES RECEIVING A THIMEROSAL‐CONTAINING HEPATITIS B VACCINE:INFLUENCE OF GESTATIONAL AGE AND BIRTH WEIGHT. (HEWITSON 2010) ...............................................................................14INFLUENCE OF PEDIATRIC VACCINES ON AMYGDALA GROWTH AND OPIOID LIGAND BINDING IN RHESUS MACAQUE INFANTS: A PILOTSTUDY. (HEWITSON 2010) ............................................................................................................................................15ANIMAL RESEARCH ...........................................................................................................................................16NEUROTOXIC EFFECTS OF POSTNATAL THIMEROSAL ARE MOUSE STRAIN DEPENDENT (HORNIG 2004) ...........................................16EFFECT OF THIMEROSAL, A PRESERVATIVE IN VACCINES, ON INTRACELLULAR CA2+ CONCENTRATION OF RAT CEREBELLAR NEURONS(UEHA‐ISHIBASHI 2004) ...............................................................................................................................................16Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 20122 | P a g eTHIMEROSAL DISTRIBUTION AND METABOLISM IN NEONATAL MICE: COMPARISON WITH METHYL MERCURY (ZAREBA 2007)..............16EFFECTS OF LIPOPOLYSACCHARIDE AND CHELATOR ON MERCURY CONTENT IN THE CEREBRUM OF THIMEROSAL‐ADMINISTERED MICE(MINAMI 2007)..........................................................................................................................................................17GENDER‐SELECTIVE TOXICITY OF THIMEROSAL (BRANCH 2008).............................................................................................18EFFECTS OF INTERMITTENT, VACCINATION‐LIKE SCHEME, THIMEROSAL ADMINISTRATION ON RAT DEVELOPMENT AND BEHAVIOR (OLCZAK2008) .......................................................................................................................................................................18EFFECTS OF POSTNATAL ADMINISTRATION ON THIMEROSAL ON RAT DEVELOPMENT AND BEHAVIOR (DUSZCZYK 2008).....................19INDUCTION OF METALLOTHIONEIN IN MOUSE CEREBELLUM AND CEREBRUM WITH LOW‐DOSE THIMEROSAL INJECTION (MINAMI 2009).................................................................................................................................................................................20NEONATAL ADMINISTRATION OF A VACCINE PRESERVATIVE, THIMEROSAL, PRODUCES LASTING IMPAIRMENT OF NOCICEPTION ANDAPPARENT ACTIVATION OF OPOID SYSTEM IN RATS (OLCZAK 2009)........................................................................................21IDENTIFICATION AND DISTRIBUTION OF MERCURY SPECIES IN RAT TISSUES FOLLOWING ADMINISTRATION OF THIMEROSAL ORMETHYLMERCURY. (RODRIGUES 2010).............................................................................................................................21NEONATAL ADMINISTRATION OF THIMEROSAL CAUSES PERSISTENT CHANGES IN MU OPIOID RECEPTORS IN THE RAT BRAIN (OLCZAK2010) .......................................................................................................................................................................22CHRONIC METALS INGESTION BY PRAIRIE VOLES PRODUCES SEX‐SPECIFIC DEFICITS IN SOCIAL BEHAVIOR: AN ANIMAL MODEL OF AUTISM.(CURTIS 2010)..........................................................................................................................................................23NEUROLIGIN‐DEFICIENT MUTANTS OF C. ELEGANS HAVE SENSORY PROCESSING DEFICITS AND ARE HYPERSENSITIVE TO OXIDATIVE STRESSAND MERCURY TOXICITY. (HUNTER 2010) ........................................................................................................................23LASTING NEUROPATHOLOGICAL CHANGES IN RAT BRAIN AFTER INTERMITTENT NEONATAL ADMINISTRATION OF THIMEROSAL. (OLCZAK2010) .......................................................................................................................................................................24PERSISTENT BEHAVIORAL IMPAIRMENTS AND ALTERATIONS OF BRAIN DOPAMINE SYSTEM AFTER EARLY POSTNATAL ADMINISTRATION OFTHIMEROSAL IN RATS. (OLCZAK 2011) .............................................................................................................................24MERCURY DISPOSITION IN SUCKLING RATS: COMPARATIVE ASSESSMENT FOLLOWING PARENTERAL EXPOSURE TO THIOMERSAL ANDMERCURIC CHLORIDE. (BLANUSA 2012) ...........................................................................................................................25SEX‐DEPENDENT CHANGES IN CEREBELLAR THYROID HORMONE‐DEPENDENT GENE EXPRESSION FOLLOWING PERINATAL EXPOSURE TOTHIMEROSAL IN RATS. (KHAN 2012)................................................................................................................................25PRENATAL EXPOSURE TO ORGANOMERCURY, THIMEROSAL, PERSISTENTLY IMPAIRS THE SEROTONERGIC AND DOPAMINERGIC SYSTEMS INTHE RAT BRAIN: IMPLICATIONS FOR ASSOCIATION WITH DEVELOPMENTAL DISORDERS. (IDA‐ETO 2012) ........................................26ADMINISTRATION OF THIMEROSAL TO INFANT RATS INCREASES OVERFLOW OF GLUTAMATE AND ASPARTATE IN THE PREFRONTAL CORTEX:PROTECTIVE ROLE OF DEHYDROEPIANDROSTERONE SULFATE. (DUSZCZYK‐BUDHATHOKI 2012)....................................................27MATERNAL THIMEROSAL EXPOSURE RESULTS IN ABERRANT CEREBELLAR OXIDATIVE STRESS, THYROID HORMONE METABOLISM, ANDMOTOR BEHAVIOR IN RAT PUPS; SEX‐ AND STRAIN‐DEPENDENT EFFECTS. (SULKOWSKI ZL 2012)..................................................27CELLULAR RESEARCH.........................................................................................................................................28BIOCHEMICAL AND MOLECULAR BASIS OF THIMEROSAL‐INDUCED APOPTOSIS IN T CELLS: A MAJOR ROLE OF MITOCHONDRIAL PATHWAY(MAKANI 2002)..........................................................................................................................................................28THIMEROSAL INDUCES MICRONUCLEI IN THE CYTOCHALASIN B BLOCK MICRONUCLEUS TEST WITH HUMAN LYMPHOCYTES (WESTPHAL2003) .......................................................................................................................................................................28THIMEROSAL INDUCES DNA BREAKS, CASPASE‐3 ACTIVATION, MEMBRANE DAMAGE, AND CELL DEATH IN CULTURED HUMAN NEURONSAND FIBROBLASTS (BASKIN 2003)...................................................................................................................................29ACTIVATION OF METHIONINE SYNTHASE BY INSULIN‐LIKE GROWTH FACTOR‐1 AND DOPAMINE: A TARGET FOR NEURODEVELOPMENTALTOXINS AND THIMEROSAL. (WALY 2004) ........................................................................................................................29UNCOUPLING OF ATP‐MEDIATED CALCIUM SIGNALING AND DYSREGULATION INTERLEUKIN‐6 SECRETION IN DENDRITIC CELLS BYNANAMOLAR THIMEROSAL (GOTH 2006) .........................................................................................................................30Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 20123 | P a g eTHIMEROSAL INDUCES NEURONAL CELL DEATH BY CAUSING CYTOCHROME C AND APOPTOSIS‐INDUCING FACTOR RELEASE FROMMITOCHONDRIA (YEL 2005)...........................................................................................................................................30IN VITRO UPTAKE OF GLUTAMATE IN GLAST AND GLT‐1 TRANSFECTED MUTANT CHO‐K1 CELLS IS INHIBITED BY THE ETHYLMERCURY‐CONTAINING PRESERVATIVE THIMEROSAL (MUTKUS 2005) ..................................................................................................30THIMEROSAL NEUROTOXICITY IS ASSOCIATED WITH GLUTATHIONE DEPLETION: PROTECTION WITH GLUTATHIONE PRECURSORS (JAMES2005) .......................................................................................................................................................................31THIMEROSAL INDUCES APOPTOSIS IN A NEUROBLASTOMA MODEL VIA THE CJUN‐N‐TERMINAL KINASE PATHWAY (HERDMAN 2006) .31THIMEROSAL INDUCES TH2 RESPONSES VIA INFLUENCING CYTOKINE SECRETION BY HUMAN DENDRITIC CELLS (AGRAWAL 2007)........31EFFECTS OF THIMEROSAL ON NGF SIGNAL TRANSDUCTION AND CELL DEATH IN NEUROBLASTOMA CELLS (PARRAN 2005).................32GENOTOXICITY OF THIMEROSAL IN CULTURED HUMAN LYMPHOCYTES WITH AND WITHOUT METABOLIC ACTIVATION SISTER CHROMATIDEXCHANGE ANALYSIS PROLIFERATION INDEX AND MITOTIC INDEX (EKE 2008)...........................................................................32ZINC IONS CAUSE THE THIMEROSAL‐INDUCED SIGNAL OF FLUORESCENT CALCIUM PROBES IN LYMPHOCYTES (HAASE 2008)...............32CHARACTERIZATION OF EARLY EVENTS INVOLVED IN HUMAN DENDRITIC CELL MATURATION INDUCED BY SENSITIZERS: CROSS TALKBETWEEN MAPK SIGNALLING PATHWAYS (TROMPEZINSKI 2008)............................................................................................33MITOCHONDRIAL MEDIATED THIMEROSAL‐INDUCED APOPTOSIS IN A HUMAN NEUROBLASTOMA CELLINE (SK‐N‐SH)(HUMPHREY 2009).................................................................................................................................................................................34MITOCHONDRIAL DYSFUNCTION, IMPAIRED OXIDATIVE‐REDUCTION ACTIVITY, DEGENERATION, AND DEATH IN HUMAN NEURONAL ANDFETAL CELLS INDUCED BY LOW‐LEVEL EXPOSURE TO THIMEROSAL AND OTHER METAL COMPOUNDS (GEIER 2009) ...........................34SENSITIZATION EFFECT OF THIMEROSAL IS MEDIATED IN VITRO VIA REACTIVE OXYGEN SPECIES AND CALCIUM SIGNALING. (MIGDAL 2010).................................................................................................................................................................................35EVALUATION OF CYTOTOXICITY ATTRIBUTED TO THIMEROSAL ON MURINE AND HUMAN KIDNEY CELLS. (PARK 2007)........................35THE RELATIVE TOXICITY OF COMPOUNDS USED AS PRESERVATIVES IN VACCINES AND BIOLOGICS. (GEIER 2010)...............................36LOW MOLECULAR WEIGHT THIOLS REDUCE THIMEROSAL NEUROTOXICITY IN VITRO: MODULATION BY PROTEINS. (ZIEMINSKA 2010) ..36RESPONSIVENESS OF HUMAN MONOCYTE‐DERIVED DENDRITIC CELLS TO THIMEROSAL AND MERCURY DERIVATIVES. (MIGDAL 2010)...37MERCURY INDUCES AN UNOPPOSED INFLAMMATORY RESPONSE IN HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS IN VITRO.(GARDNER 2009)........................................................................................................................................................38LUTEOLIN AND THIOSALICYLATE INHIBIT HGCL(2) AND THIMEROSAL‐INDUCED VEGF RELEASE FROM HUMAN MAST CELLS. (ASADI2010) .......................................................................................................................................................................38INTERMINGLED MODULATORY AND NEUROTOXIC EFFECTS OF THIMEROSAL AND MERCURIC IONS ON ELECTROPHYSIOLOGICAL RESPONSESTO GABA AND NMDA IN HIPPOCAMPAL NEURONS. (WYREMBEK 2010)...............................................................................39THIMEROSAL‐DERIVED ETHYLMERCURY IS A MITOCHONDRIAL TOXIN IN HUMAN ASTROCYTES: POSSIBLE ROLE OF FENTON CHEMISTRYIN THE OXIDATION AND BREAKAGE OF MTDNA. (SHARPE 2012) ..........................................................................................40CYTOPROTECTIVE EFFECT OF HYALURONIC ACID AND HYDROXYPROPYL METHYLCELLULOSE AGAINST DNA DAMAGE INDUCED BYTHIMEROSAL IN CHANG CONJUNCTIVAL CELLS. (YE 2012) ....................................................................................................40ITPKC SUSCEPTIBILITY IN KAWASAKI SYNDROME AS A SENSITIZING FACTOR FOR AUTOIMMUNITY AND CORONARY ARTERIAL WALLRELAXATION INDUCED BY THIMEROSAL'S EFFECTS ON CALCIUM SIGNALING VIA IP3. (YETER 2012)...............................................41THIMEROSAL‐INDUCED APOPTOSIS IN MOUSE C2C12 MYOBLAST CELLS OCCURS THROUGH SUPPRESSION OF THE PI3K/AKT/SURVIVINPATHWAY (LI 2012).....................................................................................................................................................42Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 20124 | P a g eEnvironmental ImpactA PILOT SCALE EVALUATION OF REMOVAL OF MERCURY FROM PHARMACEUTICALWASTEWATER USING GRANULAR ACTIVATED CARBON (CYR 2002)Cyr PJ, Suri RP, Helmig ED. Water Res. 2002 Nov;36(19):4725‐34.Thimerosal (an organic mercury compound) is widely used in the pharmaceutical industry andhospitals. This study examines the removal of mercury (thimerosal and Hg(II)) from apharmaceutical wastewater using F‐400 granular activated carbon (GAC) at bench and pilotscales. Bench scale dynamic column tests are conducted with 30, 60, 90 and 120 min empty bedcontact times (EBCTs). The pilot scale study is conducted using two GAC columns‐in‐series eachof 30 min EBCT. The capital and operational cost analysis for the treatment system is performed.Simultaneous removal of copper, turbidity, phenol, and color from the wastewater by the pilotscale system is also reported.BIODEGRADATION OF THIOMERSAL CONTAINING EFFLUENTS BY A MERCURY RESISTANTPSEUDOMONAS PUTIDA STRAIN (FORTUNATO 2005)Raquel Fortunato, João G. Crespo and M.A.M. Water Research Volume 39, Issue 15, September 2005,Pages 3511‐3522Thiomersal, a toxic organomercurial with a strong bactericidal effect, is the most widely usedpreservative in vaccine production. As a result, vaccine production wastewaters are frequentlypolluted with thiomersal concentrations above the European limit for mercury effluentdischarges for which there is, presently, no remediation technology available. This workproposes a biotechnological process for the remediation of vaccine production wastewatersbased on the biological degradation of thiomersal to metallic mercury, under aerobic conditions,by a mercury resistant bacterial strain. The kinetics of thiomersal degradation by a pure cultureof Pseudomonas putida spi3 was firstly investigated in batch reactors using a thiomersalamended mineral medium. Subsequently, a continuous stirred tank reactor fed with the samemedium was operated at a dilution rate of 0.05 h−1, and the bioreactor performance androbustness was evaluated when exposed to thiomersal shock loads. In a second stage, thebioreactor was fed directly with a real vaccine wastewater contaminated with thiomersal andthe culture ability to grow in the wastewater and remediate it was evaluated for dilution ratesranging from 0.022 to 0.1 h−1.Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 20125 | P a g eUSE OF ADSORPTION PROCESS TO REMOVE ORGANIC MERCURY THIMEROSAL FROMINDUSTRIAL PROCESS WASTEWATER (VELICU 2007)Velicu M, Fu H, Suri RP, Woods K. J Hazard Mater. 2007 Sep 30;148(3):599‐605. Epub 2007 Mar 12.Carbon adsorption process is tested for removal of high concentration of organic mercury(thimerosal) from industrial process wastewater, in batch and continuously flow throughcolumn systems. The organic mercury concentration in the process wastewater is about 1123mg/L due to the thimerosal compound. Four commercially available adsorbents are tested formercury removal and they are: Calgon F‐400 granular activated carbon (GAC), CB II GAC,Mersorb GAC and an ion‐exchange resin Amberlite GT73. The adsorption capacity of eachadsorbent is described by the Freundlich isotherm model at pH 3.0, 9.5 and 11.0 in batchisotherm experiments. Acidic pH was favorable for thimerosal adsorption onto the GACs.Columns‐in‐series experiments are conducted with 30‐180 min empty bed contact times(EBCTs). Mercury breakthrough of 30 mg/L occurred after about 47 h (96 Bed Volume Fed(BVF)) of operation, and 97 h (197 BVF) with 120 min EBCT and 180 min EBCT, respectively.Most of the mercury removal is attributed to the 1st adsorbent column. Increase in contacttime by additional adsorbent columns did not lower the effluent mercury concentration below30 mg/L. However, at a lower influent wastewater pH 3, the mercury effluent concentrationdecreased to less than 7 mg/L for up to 90 h of column operation (183 BVF).Human & Infant ResearchIATROGENIC EXPOSURE TO MERCURY AFTER HEPATITIS B VACCINATION IN PRETERMINFANTS (STAJICH 2000)Stajich GV, Lopez GP, Harry SW, Sexson, SW. J Pediatr. 2000 May; 136(5):679‐81.Stajich measured blood mercury levels in low birth weight and term newborns dministered theHepatitis B vaccine containing 12.5 mcg ethyl mercury. The investigation documented elevatedpost‐immunization concentrations relative to pre‐immunization levels in all neonates studied. Levels of blood mercury after exposure in low birth weight infants were 7.36 (± 4.99) mcg/L. Note: One infant was found to have developed a mercury level of 23.6 mcg/L, thus meeting theCDC criteria as a case of chemical poisoning from mercury defined as a blood level of 10mcg/Lor greater. Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 20126 | P a g eMERCURY CONCENTRATIONS AND METABOLISM IN INFANTS RECEIVING VACCINESCONTAINING THIMEROSAL: A DESCRIPTIVE STUDY (PICHICHERO 2002)Pichichero ME, Cernichiari E, Lopreiato J and Treanor J. Lancet. 2002; 360:1737‐41.Pichichero reported a mercury blood level in a 2‐month‐old infant of 20.55 nmol/L five daysafter the infant received a 37.5 mcg dose of ethylmercury (the amount contained in one DTaPand one Hepatitis B vaccine). Many infants, however, beginning in the early 1990’s and for thenext decade, received a 62.5 mcg dose of ethylmercury (adding in the Haemophilus influenzaetype b (Hib) vaccine) at the 2‐month well baby visit. A vaccine expert from the Johns HopkinsInstitute for Vaccine Safety estimated that these infants may have experienced peak bloodmercury levels of 48.3 nmol/L; well above the presumed EPA safety threshold of 29.0 nmol/L.As a reference point, the CDC recently defined a toxic exposure to mercury in an adult as ablood mercury level of >10mcg /L (50 nmol/L) ‐‐ approximately the same blood level that someinfants experienced at two months of age. HAIR MERCURY IN BREAST‐FED INFANTS EXPOSED TO THIMEROSAL‐PRESERVED VACCINES(MARQUES 2007)Marques RC, Dorea JG, Fonseca MF, Bastos WR, Malm O. Eur J Pediatr. 2007 Jan 20;Marques investigated the impact of thimerosal on the total mercury content of hair in breastfed infants receiving thimerosal containing vaccines and found exposure to vaccine‐EtHgrepresents 80% of that expected from total breast milk‐Hg in the first month but only 40% ofthe expected exposure integrated in the 6 months of breastfeeding. However, the Hg exposurecorrected for body weight at the day of immunization was much higher from thimerosal‐ EtHg(5.7 to 11.3 mugHg/kg b.w.) than from breastfeeding (0.266 mugHg/kg b.w.). While mothersshowed a relative decrease (‐57%) in total hair‐mercury during the 6 months lactation therewas substantial increase in the infant's hair‐mercury (446%).MERCURY LEVELS IN NEWBORNS AND INFANTS AFTER RECEIPT OF THIMEROSAL‐CONTAINING VACCINES (PICHICHERO 2008)Pichichero ME, Gentile A, Giglio N, Umido V, Clarkson T, Cernichiari E, Zareba G, Gotelli C, Gotelli M, YanL, Treanor J Pediatrics. 2008 Feb;121(2):e208‐14OBJECTIVES: Thimerosal is a mercurial preservative that was widely used in multidose vaccinevials in the United States and Europe until 2001 and continues to be used in many countriesthroughout the world. We conducted a pharmacokinetic study to assess blood levels andelimination of ethyl mercury after vaccination of infants with thimerosal‐containing vaccines.METHODS: Blood, stool, and urine samples were obtained before vaccination and 12 hours to30 days after vaccination from 216 healthy children: 72 newborns (group 1), 72 infants aged 2Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 20127 | P a g emonths (group 2), and 72 infants aged 6 months (group 3). Total mercury levels were measuredby atomic absorption. Blood mercury pharmacokinetics were calculated by pooling the data onthe group and were based on a 1‐compartment first‐order pharmacokinetics model. RESULTS:For groups 1, 2, and 3, respectively, (1) mean +/‐ SD weights were 3.4 +/‐ 0.4, 5.1 +/‐ 0.6, and7.7 +/‐ 1.1 kg; (2) maximal mean +/‐ SD blood mercury levels were 5.0 +/‐ 1.3, 3.6 +/‐ 1.5, and2.8 +/‐ 0.9 ng/mL occurring at 0.5 to 1 day after vaccination; (3) maximal mean +/‐ SD stoolmercury levels were 19.1 +/‐ 11.8, 37.0 +/‐ 27.4, and 44.3 +/‐ 23.9 ng/g occurring on day 5 aftervaccination for all groups; and (4) urine mercury levels were mostly nondetectable. The bloodmercury half‐life was calculated to be 3.7 days and returned to prevaccination levels by day 30.CONCLUSIONS: The blood half‐life of intramuscular ethyl mercury from thimerosal in vaccinesin infants is substantially shorter than that of oral methyl mercury in adults. Increased mercurylevels were detected in stools after vaccination, suggesting that the gastrointestinal tract isinvolved in ethyl mercury elimination. Because of the differing pharmacokinetics of ethyl andmethyl mercury, exposure guidelines based on oral methyl mercury in adults may not beaccurate for risk assessments in children who receive thimerosal‐containing vaccines.HEPATITIS B TRIPLE SERIES VACCINE AND DEVELOPMENTAL DISABILITY IN US CHILDREN AGED1‐9 YEARS (GALLAGHER 2008)Carolyn Gallagher and Melody Goodman Toxicological & Environmental Chemistry Vol. 90, No. 5,September–October 2008, 997–1008 This study investigated the association between vaccination with the Hepatitis B triple seriesvaccine prior to 2000 and developmental disability in children aged 1–9 years (n¼1824), proxiedby parental report that their child receives early intervention or special education services (EIS).National Health and Nutrition Examination Survey 1999–2000 data were analyzed and adjustedfor survey design by Taylor Linearization using SAS version 9.1 software, with SAS callableSUDAAN version 9.0.1. The odds of receiving EIS were approximately nine times as great forvaccinated boys (n¼46) as for unvaccinated boys (n¼7), after adjustment for confounders. Thisstudy found statistically significant evidence to suggest that boys in United States who werevaccinated with the triple series Hepatitis B vaccine, during the time period in which vaccineswere manufactured with thimerosal, were more susceptible to developmental disability thanwere unvaccinated boys. Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 20128 | P a g eNEONATE EXPOSURE TO THIMEROSAL MERCURY FROM HEPATITIS B VACCINES (DOREA 2009) Dórea JG, Marques RC, Brandão KG. Universidade de Brasília, Brasília, DF, Brazil. Am J Perinatol. 2009Mar 12.Infant exposure to ethylmercury (EtHg) has not only increased but is starting earlier as a resultof the current immunization schedule that uses thimerosal‐containing vaccines (TCVs).Although vaccination schedule varies considerably between countries, infants in less‐developedcountries continue to be exposed to EtHg derived from more affordable TCVs. We studied theexposure of newborns to EtHg from hepatitis B vaccines; hospital records (21,685) weresummarized for the years 2001 to 2005 regarding date of birth, vaccination date, and birthweight. Most of the vaccinations occurred in the first 24 hours postdelivery; over the 5 years,there was an increase in vaccinations within hours of birth (same day), from 7.4% (2001) to87.8% (2005). Nearly 94.6% of infants are now being vaccinated within the first 24hours. Rangeof mercury exposure spread from 4.2 to 21.1 mug mercury/kg body weight for those receivingTCVs with the highest thimerosal concentration; these exposure levels are conservative for 2%of children receiving vaccines within 2 to 3 postnatal days, when they are still going throughphysiological postnatal weight loss. Because of the particular timing (transitioning from in uteroto ex utero metabolism) and specific aspects of exposure (i.e., parenteral mode, bypassinggastroenteric barriers) and dose (related to vaccine manufacturer and with variation in birthweight), this study reveals critical issues that can modulate toxicokinetics and toxicodynamicsof organomercurials in neonates.URINARY PORPHYRIN EXCRETION IN NEUROTYPICAL AND AUTISTIC CHILDREN (WOODS 2010)James S. Woods; Sarah E. Armel; Denise I. Fulton; Jason Allen; Kristine Wessels; P. Lynne Simmonds;Doreen Granpeesheh; Elizabeth Mumper; J. Jeffrey Bradstreet; Diana Echeverria; Nicholas J. Heyer;James P.K. Rooney From Environmental Health PerspectivesBackground: Increased urinary concentrations of pentacarboxyl‐, precopro‐ and copro‐porphyrins have been associated with prolonged mercury (Hg) exposure in adults, andcomparable increases have been attributed to Hg exposure in children with autism (AU). Objectives: This study was designed to measure and compare urinary porphyrin concentrationsin neurotypical (NT) children and same‐age children with autism, and to examine theassociation between porphyrin levels and past or current Hg exposure in children with autism. Methods: This exploratory study enrolled 278 children 2–12 years of age. We evaluated threegroups: AU, pervasive developmental disorder‐not otherwise specified (PDD‐NOS), and NT.Mothers/caregivers provided information at enrollment regarding medical, dental, and dietaryexposures. Urine samples from all children were acquired for analyses of porphyrin, creatinine,and Hg. Differences between groups for mean porphyrin and Hg levels were evaluated. LogisticScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 20129 | P a g eregression analysis was conducted to determine whether porphyrin levels were associated withincreased risk of autism. Results: Mean urinary porphyrin concentrations are naturally high inyoung children and decline by as much as 2.5‐fold between 2 and 12 years of age. Elevatedcopro‐ (p < 0.009), hexacarboxyl‐ (p < 0.01) and pentacarboxyl‐ (p < 0.001) porphyrinconcentrations were significantly associated with AU but not with PDD‐NOS. No differenceswere found between NT and AU in urinary Hg levels or in past Hg exposure as determined byfish consumption, number of dental amalgam fillings, or vaccines received. Conclusions: Thesefindings identify disordered porphyrin metabolism as a salient characteristic of autism. Hgexposures were comparable between diagnostic groups, and a porphyrin pattern consistentwith that seen in Hg‐exposed adults was not apparent.EMBRYONIC EXPOSURE TO THIMEROSAL, AN ORGANOMERCURY COMPOUND, CAUSESABNORMAL EARLY DEVELOPMENT OF SEROTONERGIC NEURONS. (IDA‐ETO 2011)Ida‐Eto M, Oyabu A, Ohkawara T, Tashiro Y, Narita N, Narita M. Neurosci Lett. 2011 Nov14;505(2):61‐4. doi: 10.1016/j.neulet.2011.05.053. Epub 2011 Jun 6.Even though neuronal toxicity due to organomercury compounds is well known, thimerosal, anorganomercury compound, is widely used in pediatric vaccine preservation. In the presentstudy, we examined whether embryonic exposure to thimerosal affects early development ofserotonergic neurons. Thimerosal (1mg Hg/kg) was intramuscularly administered to pregnantrats on gestational day 9 (susceptible time window for development of fetal serotonergicsystem), and fetal serotonergic neurons were assessed at embryonic day 15 using anti‐serotonin antibodies. A dramatic increase in the number of serotonergic neurons localized tothe lateral portion of the caudal raphe was observed in thimerosal group (1.9‐fold increase,p<0.01 compared to control). These results indicate that embryonic exposure to thimerosalaffects early development of serotonergic neurons.SPECIATION OF METHYL‐ AND ETHYL‐MERCURY IN HAIR OF BREASTFED INFANTS ACUTELYEXPOSED TO THIMEROSAL‐CONTAINING VACCINES. (DOREA 2011)Dórea JG, Bezerra VL, Fajon V, Horvat M. Clin Chim Acta. 2011 Aug 17;412(17‐18):1563‐6. doi:10.1016/j.cca.2011.05.003. Epub 2011 May 7.BACKGROUND: Different chemical forms of mercury occur naturally in human milk. The mostcontroversial aspect of early post‐natal exposure to organic mercury is ethylmercury (EtHg) inthimerosal‐containing vaccines (TCV) still being used in many countries. Thus exclusivelybreastfed infants can be exposed to both, fish derived methylmercury (MeHg) in maternal dietsand to EtHg from TCV. The aim of the study is to evaluate a new analytical method for ethyl andmethyl mercury in hair samples of breastfed infants who had received the recommendedschedule of TCV. METHODS: The hair of infants (<12 months) that had been exposed to TCVScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201210 | P a g e(Hepatitis B and DTaP) was analysed. A method coupling isothermal gas chromatography withcold‐vapor atomic fluorescence spectrometry was used for MeHg which can also speciate EtHgin biological matrices. RESULTS: In 20 samples of infants' hair, all but two samples showedvariable amounts of MeHg (10.3 to 668 ng/g), while precise and reliable concentrations of EtHg(3.7 to 65.0 ng/g) were found in 15 of the 20 samples. A statistically significant inverseassociation (r=‐05572; p=0.0384) was found between hair‐EtHg concentrations and the timeelapsed after the last TCV shot. CONCLUSIONS: The analytical method proved sensitiveenough to quantify EtHg in babies' hair after acute exposure to thimerosal in vaccine shots.Provided that the mass of hair was above 10mg, organic‐mercury exposure during early life canbe speciated, and quantified in babies' first hair, thus opening opportunities for clinical andforensic studies.INTEGRATING EXPERIMENTAL (IN VITRO AND IN VIVO) NEUROTOXICITY STUDIES OF LOW‐DOSE THIMEROSAL RELEVANT TO VACCINES. (DOREA 2011)Dórea JG. Neurochem Res. 2011 Jun;36(6):927‐38. doi: 10.1007/s11064‐011‐0427‐0. Epub 2011Feb 25.There is a need to interpret neurotoxic studies to help deal with uncertainties surroundingpregnant mothers, newborns and young children who must receive repeated doses ofThimerosal‐containing vaccines (TCVs). This review integrates information derived fromemerging experimental studies (in vitro and in vivo) of low‐dose Thimerosal (sodium ethylmercury thiosalicylate). Major databases (PubMed and Web‐of‐science) were searched for invitro and in vivo experimental studies that addressed the effects of low‐dose Thimerosal (orethylmercury) on neural tissues and animal behaviour. Information extracted from studiesindicates that: (a) activity of low doses of Thimerosal against isolated human and animal braincells was found in all studies and is consistent with Hg neurotoxicity; (b) the neurotoxic effect ofethylmercury has not been studied with co‐occurring adjuvant‐Al in TCVs; (c) animal studieshave shown that exposure to Thimerosal‐Hg can lead to accumulation of inorganic Hg in brain,and that (d) doses relevant to TCV exposure possess the potential to affect human neuro‐development. Thimerosal at concentrations relevant for infants' exposure (in vaccines) is toxicto cultured human‐brain cells and to laboratory animals. The persisting use of TCV (indeveloping countries) is counterintuitive to global efforts to lower Hg exposure and to ban Hg inmedical products; its continued use in TCV requires evaluation of a sufficiently nontoxic level ofethylmercury compatible with repeated exposure (co‐occurring with adjuvant‐Al) during earlylife.Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201211 | P a g eNEONATAL EXPOSURE TO THIMEROSAL FROM VACCINES AND CHILD DEVELOPMENT IN THEFIRST 3YEARS OF LIFE. (MROZEK‐BUDZYN 2012)Mrozek‐Budzyn D, Majewska R, Kieltyka A, Augustyniak M. Neurotoxicol Teratol. 2012 Nov‐Dec;34(6):592‐7. doi: 10.1016/j.ntt.2012.10.001. Epub 2012 Oct 13.BACKGROUND: Despite the common use of Thimerosal as a preservative in childhood vaccinessince the 1930s, there are not many studies on ethylmercury toxicokinetics and toxicodynamicsin infants. The knowledge of ethylmercury's potential adverse effects is derived mostly fromparallel methylmercury research or from animal and theoretical models. AIM OF THE STUDY:This study was designed to examine the relationship between neonatal exposure to Thimerosal‐containing vaccine (TCV) and child development. MATERIAL AND METHODS: The study sampleconsisted of 196 infants born between January 2001 and March 2003 to mothers attendingambulatory prenatal clinics in the first and second trimesters of pregnancy in Krakow.Vaccination history (date and the type of the vaccine) was extracted from physicians' records.Child development was assessed using the Bayley Scales of Infant Development (BSID‐II)measured in one‐year intervals over 3years. General Linear Model (GLM) and GeneralizedEstimating Equation (GEE) models adjusted for potential confounders were used to assess theassociation. RESULTS: An adverse effect of neonatal TCV exposure was observed for thepsychomotor development index (PDI) only in the 12th and 24th months of life (β=‐6.44,p<0.001 and β=‐5.89, p<0.001). No significant effect of neonatal TCV exposure was found in the36th month. The overall deficit in the PDI attributable to neonatal TCV exposure measured overthe course of the three‐year follow‐up (GEE) was significantly higher in TCV group (β=‐4.42,p=0.001). MDI scores did not show the adverse association with neonatal TCV exposure.BREAST‐FEEDING AND RESPONSES TO INFANT VACCINES: CONSTITUTIONAL ANDENVIRONMENTAL FACTORS. (DOREA 2012)Dórea JG. Am J Perinatol. 2012 Nov;29(10):759‐75. doi: 10.1055/s‐0032‐1316442. Epub 2012Jul 6.Neonates and nursing infants are special with regard to immune development and vulnerabilityto infectious diseases. Although breast‐feeding is essential to modulate and prime immunedefenses, vaccines (an interventional prophylaxis) are crucial to prevent and control infectiousdiseases. During nursing, the type of feeding influences infants' natural defenses (including gutcolonization) and their response to vaccines, both through cell‐mediated immunity and specificantibody production. Given the variety and combination of vaccine components (antigens andexcipients, preservative thimerosal, and aluminum adjuvants) and route of administration,there is a need to examine the role of infant feeding practices in intended and nonintendedoutcomes of vaccination. Maternal factors related to milk constituents (nutrients andScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201212 | P a g epollutants) and feeding practices can affect response to vaccines. Collectively, studies thatcompared type of feeding (or used breast‐feeding‐adjusted statistical models) showedsignificant influence on some vaccines taken during infancy. Nurslings deprived of the fullbenefit of breast‐feeding could have altered immune responses affecting vaccine outcome. Inthe absence of studies elucidating neurodevelopment (including excitoxicity) andimmunotoxicity issues, vaccination practices should promote and support breast‐feeding.THIMEROSAL EXPOSURE IN EARLY LIFE AND NEUROPSYCHOLOGICAL OUTCOMES 7‐10 YEARSLATER. (BARILE 2012)Barile JP, Kuperminc GP, Weintraub ES, Mink JW, Thompson WW. J Pediatr Psychol. 2012 Jan‐Feb;37(1):106‐18. doi: 10.1093/jpepsy/jsr048. Epub 2011 Jul 23.OBJECTIVE: The authors used a public use data set to investigate associations between thereceipt of thimerosal‐containing vaccines and immune globulins early in life andneuropsychological outcomes assessed at 7‐10 years. METHODS: The data were originallycreated by evaluating 1,047 children ages 7‐10 years and their biological mothers. This studydeveloped seven latent neuropsychological factors and regressed them on a comprehensive setof covariates and thimerosal exposure variables. RESULTS: The authors found no statisticallysignificant associations between thimerosal exposure from vaccines early in life and six of theseven latent constructs. There was a small, but statistically significant association between earlythimerosal exposure and the presence of tics in boys. CONCLUSIONS: This finding should beinterpreted with caution due to limitations in the measurement of tics and the limitedbiological plausibility regarding a causal relationship.Non‐Human Primate Infant ResearchCOMPARISON OF BLOOD AND BRAIN MERCURY LEVELS IN INFANT MONKEYS EXPOSED TOMETHYLMERCURY OR VACCINES CONTAINING THIMEROSAL (BURBACHER 2005)Burbacher TM, Shen DD, Liberato N, Grant KS, Cernichiari E, Clarkson T. Environmental HealthPerspectives. 2005 Aug;113(8):1015‐21.Burbacher compared brain mercury levels in infant Macaca fascicularis primates exposed toinjected ethylmercury (thimerosal) and equal amounts of ingested methylmercury. Theethylmercury more rapidly converted to inorganic mercury in the brains of the primates whichresulted in increasing levels of inorganic mercury and the primates exposed to ethylmercuryretained at least twice as much inorganic mercury in their brains compared to the primatesexposed to methylmercury. The relative concentrations in monkeys with detectable levels ofinorganic mercury were 16 ng/g in thimerosal‐treated monkeys and 7 ng/g in themethylmercury‐treated monkeys in which inorganic mercury levels were detectable. InorganicScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201213 | P a g emercury was below detectable levels in 8 out of 17 of the methylmercury‐treated monkeys.Exposures to mercury during these critical periods of development disrupt the growth andmigration of neurons, with the potential to cause irreversible damage to the central nervoussystem. Prior primate studies found inorganic mercury in the brain was associated withmicrogliosis and neuroinflammation, recent finding also documented in autistic brain. PEDIATRIC VACCINES INFLUENCE PRIMATE BEHAVIOR, AND AMYGDALA GROWTH ANDOPIOID LIGAND BINDING (HEWITSON 2008)L. Hewitson B. Lopresti , C. Stott, J. Tomko , L. Houser , E. Klein , G. Sackett , S. Gupta , D. Atwood , L. Blue ,E. R. White , A. Wakefield IMFAR (May 2008)Background: Macaques are commonly used in pre‐clinical vaccine safety testing, but thecombined childhood vaccine regimen, rather than individual vaccines, has not been studied.Childhood vaccines are a possible causal factor in autism, and abnormal behaviors andanomalous amygdala growth are potentially inter‐related features of this condition. Objectives:The objective of this study was to compare early infant cognition and behavior with amygdalasize and opioid binding in rhesus macaques receiving the recommended childhood vaccines(1994‐1999), the majority of which contained the bactericidal preservative thylmercurithiosalicylic acid (thimerosal). Methods: Macaques were administered therecommended infant vaccines, adjusted for age and thimerosal dose (exposed; N=13), or saline(unexposed; N=3). Primate development, cognition and social behavior were assessed for bothvaccinated and unvaccinated infants using standardized tests developed at the WashingtonNational Primate Research Center. Amygdala growth and binding were measured serially byMRI and by the binding of the non‐selective opioid antagonist [11C]diprenorphine, measuredby PET, respectively, before (T1) and after (T2) the administration of the measles‐mumps‐rubella vaccine (MMR). Results: Compared with unexposed animals, significantneurodevelopmental deficits were evident for exposed animals in survival reflexes, tests ofcolor discrimination and reversal, and learning sets. Differences in behaviors were observedbetween exposed and unexposed animals and within the exposed group before and after MMRvaccination. Compared with unexposed animals, exposed animals showed attenuation ofamygdala growth and differences in the amygdala binding of [11C]diprenorphine. Interactionmodels identified significant associations between specific aberrant social and non‐socialbehaviors, isotope binding, and vaccine exposure. Conclusions: This animal model, whichexamines for the first time, behavioral, functional, and neuromorphometric consequences ofthe childhood vaccine regimen, mimics certain neurological abnormalities of autism. Thefindings raise important safety issues while providing a potential model for examining aspectsof causation and disease pathogenesis in acquired disorders of behavior and development.Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201214 | P a g eMICROARRAY ANALYSIS OF GI TISSUE IN A MACAQUE MODEL OF THE EFFECTS OF INFANTVACCINATION (WALKER 2008)S. J. Walker , E. K. Lobenhofer , A. Wakefield , L. Hewitson IMFAR (May 2008)Background: There has been considerable debate regarding the question of an interactionbetween childhood vaccinations and adverse sequelae in the gastrointestinal tract, immunesystem, and central nervous system of some recipients. These systems, either singly or incombination, appear to be adversely affected in many ASD children. Although pre‐clinical testsof individual vaccines routinely find the risk/benefit ratio to be low, previously there has notbeen a study to examine the effects of the comprehensive vaccination regime currently in usefor infants. Objectives: This study was designed to evaluate potential alterations in normalgrowth and development resulting from the vaccine regimen that was in use from 1994‐1999.Specifically, this portion of the study was to compare the gene expression profiles obtainedfrom gastrointestinal tissue from vaccinated and unvaccinated infants. Methods: Infant malemacaques were vaccinated (or given saline placebo) using the human vaccination schedule.Dosages and times of administration were adjusted for differences between macaques andhumans. Biopsy tissue was collected from the animals at three time points: (1) 10 weeks [pre‐MMR1], (2) 14 weeks [post‐MMR1] and, (3) 12‐15 months [at necropsy]. Whole genomemicroarray analysis was performed on RNA extracted from the GI tissue from 7 vaccinated and2 unvaccinated animals at each of these 3 time points (27 samples total). Results:Histopathological examination revealed that vaccinated animals exhibited progressively severechronic active inflammation, whereas unexposed animals did not. Gene expressioncomparisons between the groups (vaccinated versus unvaccinated) revealed only 120 genesdifferentially expressed (fc >1.5; log ratio p<0.001) at 10 weeks, whereas there were 450 genesdifferentially expressed at 14 weeks, and 324 differentially expressed genes between the 2groups at necropsy. Conclusions: We have found many significant differences in the GI tissuegene expression profiles between vaccinated and unvaccinated animals. These differences willbe presented and discussed.DELAYED ACQUISITION OF NEONATAL REFLEXES IN NEWBORN PRIMATES RECEIVING ATHIMEROSAL‐CONTAINING HEPATITIS B VACCINE: INFLUENCE OF GESTATIONAL AGE ANDBIRTH WEIGHT. (HEWITSON 2010)Hewitson L, Houser LA, Stott C, Sackett G, Tomko JL, Atwood D, Blue L, White ER. J Toxicol Environ HealthA. 2010 Jan;73(19):1298‐313.This study examined whether acquisition of neonatal reflexes in newborn rhesus macaques wasinfluenced by receipt of a single neonatal dose of hepatitis B vaccine containing thepreservative thimerosal (Th). Hepatitis B vaccine containing a weight‐adjusted Th dose wasadministered to male macaques within 24 h of birth (n = 13). Unexposed animals receivedsaline placebo (n = 4) or no injection (n = 3). Infants were tested daily for acquisition of nineScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201215 | P a g esurvival, motor, and sensorimotor reflexes. In exposed animals there was a significant delay inthe acquisition of root, snout, and suck reflexes, compared with unexposed animals. Noneonatal responses were significantly delayed in unexposed animals. Gestational age (GA) andbirth weight (BW) were not significantly correlated. Cox regression models were used toevaluate main effects and interactions of exposure with BW and GA as independent predictorsand time‐invariant covariates. Significant main effects remained for exposure on root and suckwhen controlling for GA and BW, such that exposed animals were relatively delayed in time‐to‐criterion. Interaction models indicated there were various interactions between exposure, GA,and BW and that inclusion of the relevant interaction terms significantly improved model fit.This, in turn, indicated that lower BW and/or lower GA exacerbated the adverse effectsfollowing vaccine exposure. This primate model provides a possible means of assessing adverseneurodevelopmental outcomes from neonatal Th‐containing hepatitis B vaccine exposure,particularly in infants of lower GA or BW. The mechanisms underlying these effects and therequirements for Th requires further studyINFLUENCE OF PEDIATRIC VACCINES ON AMYGDALA GROWTH AND OPIOID LIGAND BINDINGIN RHESUS MACAQUE INFANTS: A PILOT STUDY. (HEWITSON 2010)Hewitson L, Lopresti BJ, Stott C, Mason NS, Tomko J. Acta Neurobiol Exp (Wars). 2010;70(2):147‐64.This longitudinal, case‐control pilot study examined amygdala growth in rhesus macaqueinfants receiving the complete US childhood vaccine schedule (1994‐1999). Longitudinalstructural and functional neuroimaging was undertaken to examine central effects of thevaccine regimen on the developing brain. Vaccine‐exposed and saline‐injected control infantsunderwent MRI and PET imaging at approximately 4 and 6 months of age, representing twospecific timeframes within the vaccination schedule. Volumetric analyses showed that exposedanimals did not undergo the maturational changes over time in amygdala volume that wasobserved in unexposed animals. After controlling for left amygdala volume, the binding of theopioid antagonist [(11)C]diprenorphine (DPN) in exposed animals remained relatively constantover time, compared with unexposed animals, in which a significant decrease in [(11)C]DPNbinding occurred. These results suggest that maturational changes in amygdala volume and thebinding capacity of [(11)C]DPN in the amygdala was significantly altered in infant macaquesreceiving the vaccine schedule. The macaque infant is a relevant animal model in which toinvestigate specific environmental exposures and structural/functional neuroimaging duringneurodevelopment.Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201216 | P a g eAnimal ResearchNEUROTOXIC EFFECTS OF POSTNATAL THIMEROSAL ARE MOUSE STRAIN DEPENDENT(HORNIG 2004)Hornig M, Chian D, Lipkin WI. Molecular Psychiatry. 2004 Sep;9(9):833‐45.Hornig exposed autoimmune‐prone infant mice with thimerosal‐containing vaccines at the dosegiven to human infants adjusted for mouse weight. This investigation reported a number ofobservable effects including growth delay; reduced locomotion; exaggerated response tonovelty; and densely packed, hyperchromic hippocampal neurons with altered glutamatereceptors and transporters. Strains resistant to autoimmunity were not susceptible. Thesefindings implicate genetic influences and provide a model for investigating thimerosal‐relatedneurotoxicity.EFFECT OF THIMEROSAL, A PRESERVATIVE IN VACCINES, ON INTRACELLULAR CA2+CONCENTRATION OF RAT CEREBELLAR NEURONS (UEHA‐ISHIBASHI 2004)Ueha‐Ishibashi T, Oyama Y, Nakao H, Umebayashi C, Nishizaki Y, Tatsuishi T, Iwase K, Murao K, Seo H.Toxicology 2004 Jan 15;195(1):77‐84.Ueha‐Ishibashi investigated the effect of thimerosal on cerebellar neurons dissociated from 2‐week‐old rats was compared with those of methylmercury. Both agents at 1 microM or moresimilarly decreased the cellular content of glutathione in a concentration‐dependent manner,suggesting an increase in oxidative stress and increased intercellular concentrations of Ca2+. Thimerosal was also found to exert cytotoxic actions on cerebellar granule neurons and itspotency was similar to that of methylmercury. The FDA and EPA use methymercury as theirtoxicity standard, so demonstration of equivalence shows the potential of thimerosal to causethe same harm as methylmercury, for which more research exists.THIMEROSAL DISTRIBUTION AND METABOLISM IN NEONATAL MICE: COMPARISON WITHMETHYL MERCURY (ZAREBA 2007)Grazyna Zareba, Elsa Cernichiari, Rieko Hojo, Scott Mc Nitt, Bernard Weiss, Moiz M Mumtaz, Dennis EJones, Thomas W Clarkson Neurotoxicology. 2007 Feb 23; : 17382399Thimerosal, which releases the ethyl mercury radical as the active species, has been used as apreservative in many currently marketed vaccines throughout the world. Because of concernsthat its toxicity could be similar to that of methyl mercury, it is no longer incorporated in manyvaccines in the United States. There are reasons to believe, however, that the disposition andtoxicity of ethyl mercury compounds, including thimerosal, may differ substantially from thoseScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201217 | P a g eof the methyl form. The current study sought to compare, in neonatal mice, the tissueconcentrations, disposition and metabolism of thimerosal with that of methyl mercury. ICRmice were given single intramuscular injections of thimerosal or methyl mercury (1.4 mg Hgkg(‐1)) on postnatal day 10 (PND 10). Tissue samples were collected daily on PND 11‐14. Mostanalysed tissues demonstrated different patterns of tissue distribution and a different rate ofmercury decomposition. The mean organic mercury in the brain and kidneys was significantlylower in mice treated with thimerosal than in the methyl mercury‐treated group. In the brain,thimerosal‐exposed mice showed a steady decrease of organic mercury levels following theinitial peak, whereas in the methyl mercury‐exposed mice, concentrations peaked on day 2after exposure. In the kidneys, thimerosal‐exposed mice retained significantly higher inorganicmercury levels than methyl mercury‐treated mice. In the liver both organic and inorganicmercury concentrations were significantly higher in thimerosal‐exposed mice than in themethyl mercury group. Ethyl mercury was incorporated into growing hair in a similar manner tomethyl mercury. The data showing significant kinetic differences in tissue distribution andmetabolism of mercury species challenge the assumption that ethyl mercury is toxicologicallyidentical to methyl mercury. EFFECTS OF LIPOPOLYSACCHARIDE AND CHELATOR ON MERCURY CONTENT IN THECEREBRUM OF THIMEROSAL‐ADMINISTERED MICE (MINAMI 2007) Takeshi Minami, Keisuke Oda, Naoya Gima, Hideo Yamazaki Environmental Toxicology andPharmacology Volume 24, Issue 3, November 2007, Pages 316‐320 Thimerosal is one of the best‐known preservative agents for vaccines in the world but arelationship between its use and autism has long been suspected so that its effects on the brainneed more detailed research. We here examined the influence of lipopolysaccharide injury tothe blood–brain barrier on the penetration of mercury from thimerosal into mouse cerebrums,as well as the effect of chelator of heavy metals on cerebrum mercury content. Mercury can beexpected to be detected in the cerebrum of normal mice, because the metal is present instandard mouse chow. When 60 µg/kg of thimerosal was subcutaneously injected into themouse, the mercury content in the cerebrum was significantly higher 48 h after the thimerosalinjection with a maximum peak after 72 h. In addition, mercury content in the cerebrum wasstill higher on day 7 than in the control group. When lipopolysaccharide was pre‐injected intomice to induce damage on blood–brain barrier, the mercury content in the cerebrum wassignificantly higher at 24 and 72 h after the injection of 12 µg/kg of thimerosal compared to thecontrol group, this dose alone does not cause any increase. The mercury content in thecerebrums of mice was decreased to the control group level on day 7 when a chelator,dimercaprol, was administered once a day from days 3 to 6 aftera 60 µg/kg, s.c. injection. Inaddition, d‐penicillamine as a chelator decreased the mercury contents in the cerebrum afterScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201218 | P a g ethe high dose administration. In conclusion, a physiological dose of thimerosal did not increasethe content of mercury in the cerebrum, but levels were increased when damage to the blood–brain barrier occurred in mice injected with thimerosal. In addition, a chelator of heavy metalsmay be useful to remove mercury from the cerebrum. GENDER‐SELECTIVE TOXICITY OF THIMEROSAL (BRANCH 2008)Branch DR Exp Toxicol Pathol. 2008 Sep 2. [Epub ahead of print]A recent report shows a correlation of the historical use of thimerosal in therapeuticimmunizations with the subsequent development of autism; however, this association remainscontroversial. Autism occurs approximately four times more frequently in males compared tofemales; thus, studies of thimerosal toxicity should take into consideration gender‐selectiveeffects. The present study was originally undertaken to determine the maximum tolerated dose(MTD) of thimersosal in male and female CD1 mice. However, during the limited MTD studies, itbecame apparent that thimerosal has a differential MTD that depends on whether the mouse ismale or female. At doses of 38.4‐76.8mg/kg using 10% DMSO as diluent, seven of seven malemice compared to zero of seven female mice tested succumbed to thimerosal. Although thethimerosal levels used were very high, as we were originally only trying to determine MTD, itwas completely unexpected to observe a difference of the MTD between male and female mice.Thus, our studies, although not directly addressing the controversy surrounding thimerosal andautism, and still preliminary due to small numbers of mice examined, provide, nevertheless, thefirst report of gender‐selective toxicity of thimerosal and indicate that any future studies ofthimerosal toxicity should take into consideration gender‐specific differences.EFFECTS OF INTERMITTENT, VACCINATION‐LIKE SCHEME, THIMEROSAL ADMINISTRATION ONRAT DEVELOPMENT AND BEHAVIOR (OLCZAK 2008)Olczak M., Duszczyk M., Mierzejewski P. & Majewska M. D. Dept. Pharmacol. Inst. Psychiatry &Neurology, Warsaw, Poland Publication ref.: FENS Abstr., vol.4, 083.19, 2008Mercury from thimerosal, which was added to many child vaccines, is one of the agentssuspected to be responsible for autism epidemics observed in the past two decades. Dataanalysis from Vaccine Adverse Event Reporting System of the Center for Disease Control andPrevention (USA) documented that children immunized with vaccines containing thimerosalwere several times more likely do develop autism and other neurodevelopmentaldiseases/disorders than those, who did not receive thimerosal. In this study we examined thepotential neurotoxic effects of different cumulative doses of thimerosal, from 0.040 mg/kg to25 mg/kg, administered to rats s.c. or i. m. in four doses on postnatal days 7‐14. Three strains ofrats were tested: Wistar, Lewis and Brown Norway. Development and behaviour or theexperimental animals was monitored. At different developmental stages (between weeks 4 andScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201219 | P a g e22 of age) several behavioral tests were conducted, which included open field locomotoractivity, motor coordination, pain reaction (hot plate), water maze learning and memory test,prepulse inhibition, and social interaction test. Brains of thimerosal treated rats accumulated asignificant amount of mercury. They were examined for histopathological changes. Generally,rats appeared to be quite resistant to overt neurotoxic effects of thimerosal at doses tested,although higher doses of this drug caused subtle changes on some behavioral measures, whichappear to be species and sex dependent. Significant thimerosal effects on pain reaction, certainlearning parameters and prepulse inhibition were observed. Also some aspects of socialinteractions were altered. Behavioural and histopathological data will be presented in thecontext of putative rat model of mercury‐mediated neurodevelopmental pathologies. Fundedby EC grant MEXC‐CT‐2006‐42371 to M. D. Majewska. EFFECTS OF POSTNATAL ADMINISTRATION ON THIMEROSAL ON RAT DEVELOPMENT ANDBEHAVIOR (DUSZCZYK 2008)Michalina Duszczyk, Mieszko Olczak, Pawe Mierzejewski, Dorota M. Majewska. Department ofPharmacology and Physiology of the Central Nervous System, Institute of Psychiatry and Neurology,Warsaw, Poland. Pharmacological Reports. 2008 60; p261‐262Numerous clinical findings support hypothesis that mercury, which was added to many infantvaccines in the form of thimerosal between 2000–2004, may be one of the factors responsiblefor autism epidemics currently observed all over the world. Data from Adverse Event Reportingof the Center for Disease Control and Prevention (USA) provide strong epidemiological evidencefor a link between vaccine‐thimerosal exposure and autism or other neurodevelopmentaldisorders/diseases. The onset of autistic symptoms in children often follows the administrationof vaccine thimerosal and symptom emergence is consistent with the expression ofdevelopmental mercury toxicity. In this study, we examined potential neurodevelopmentaloutcomes following postnatal exposure of rats to thimerosal (Sigma‐Aldrich), administered sc orim from 0.040 mg/kg to 50 mg/kg in four equal doses on days 7–14 after birth. Three strains ofrats were used in this experiment: Wistar, Lewis and Brown Norway. Development andbehavior of experimental animals was observed. Various behavioral tests were carried out,which evaluated: open field locomotor and exploratory activity, motor coordination, painreaction (hot plate), learning and memory (water maze), prepulse inhibition, sociability (socialinteraction test). Growth of animals was monitored and after animal sacrifice, weight of brainswas measured. Thimerosal had variable, often biphasic, effects on different measuredbehaviors, which were strain‐ and dose‐dependent, but no dramatic behavioral impairmentswere observed at doses tested. Data will be discussed in the context of rodent model of autismfollowing postnatal exposure to mercury. [Note: autism is 4 times more prevalent in boys thanScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201220 | P a g egirls, and no one has been able to identify why. The differential gender effects of thimerosaland mercury might explain why.] INDUCTION OF METALLOTHIONEIN IN MOUSE CEREBELLUM AND CEREBRUM WITH LOW‐DOSE THIMEROSAL INJECTION (MINAMI 2009)Minami T, Miyata E, Sakamoto Y, Yamazaki H, Ichida S. Department of Life Sciences, School of Science &Engineering, Kinki University, 3‐4‐1 Kowakae, Higashi‐osaka, Osaka, 577‐8502, Japan,minamita@life.kindai.ac.jp. Cell Biol Toxicol. 2009 Apr 9.Thimerosal, an ethyl mercury compound, is used worldwide as a vaccine preservative. Wepreviously observed that the mercury concentration in mouse brains did not increase with theclinical dose of thimerosal injection, but the concentration increased in the brain after theinjection of thimerosal with lipopolysaccharide, even if a low dose of thimerosal wasadministered. Thimerosal may penetrate the brain, but is undetectable when a clinical dose ofthimerosal is injected; therefore, the induction of metallothionein (MT) messenger RNA (mRNA)and protein was observed in the cerebellum and cerebrum of mice after thimerosal injection, asMT is an inducible protein. MT‐1 mRNA was expressed at 6 and 9 h in both the cerebrum andcerebellum, but MT‐1 mRNA expression in the cerebellum was three times higher than that inthe cerebrum after the injection of 12 microg/kg thimerosal. MT‐2 mRNA was not expresseduntil 24 h in both organs. MT‐3 mRNA was expressed in the cerebellum from 6 to 15 h after theinjection, but not in the cerebrum until 24 h. MT‐1 and MT‐3 mRNAs were expressed in thecerebellum in a dose‐dependent manner. Furthermore, MT‐1 protein was detected from 6 to72 h in the cerebellum after 12 microg/kg of thimerosal was injected and peaked at 10 h. MT‐2was detected in the cerebellum only at 10 h. In the cerebrum, little MT‐1 protein was detectedat 10 and 24 h, and there were no peaks of MT‐2 protein in the cerebrum. In conclusion, MT‐1and MT‐3 mRNAs but not MT‐2 mRNA are easily expressed in the cerebellum rather than in thecerebrum by the injection of low‐dose thimerosal. It is thought that the cerebellum is asensitive organ against thimerosal. As a result of the present findings, in combination with thebrain pathology observed in patients diagnosed with autism, the present study helps to supportthe possible biological plausibility for how low‐dose exposure to mercury from thimerosal‐containing vaccines may be associated with autism. Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201221 | P a g eNEONATAL ADMINISTRATION OF A VACCINE PRESERVATIVE, THIMEROSAL, PRODUCESLASTING IMPAIRMENT OF NOCICEPTION AND APPARENT ACTIVATION OF OPOID SYSTEM INRATS (OLCZAK 2009)Olczak M, Duszczyk M, Mierzejewski P, Majewska MD. Brain Res. 2009 Dec 8;1301:143‐51. Epub 2009Sep 9.Thimerosal (THIM), an organomercury preservative added to many child vaccines is a suspectedfactor in pathogenesis of neurodevelopmental disorders. We examined the pharmacokinetics ofHg in the brain, liver and kidneys after i.m. THIM injection in suckling rats and we tested THIMeffect on nociception. THIM solutions were injected to Wistar and Lewis rats in a vaccination‐like mode on PN days 7, 9, 11 and 15 in four equal doses. For Wistar rats these were: 12, 48,240, 720, 1440, 2160, 3000 microg Hg/kg and for Lewis: 54, 216, 540 and 1080 microg Hg/kg.Pharmacokinetic analysis revealed that Hg from THIM injections accumulates in the rat brain insignificant amounts and remains there longer than 30 days after the injection. At the 6th weekof age animals were examined for pain sensitivity using the hot plate test. THIM treated rats ofboth strains and sexes manifested statistically significantly elevated pain threshold (latency forpaw licking, jumping) on a hot plate (56 degrees C). Wistar rats were more sensitive to thiseffect than Lewis rats. Protracted THIM‐induced hypoalgesia was reversed by naloxone (5mg/kg, i.p.) injected before the hot plate test, indicative of involvement of endogenous opioids.This was confirmed by augmented catalepsy after morphine (2.5 mg/kg, s.c.) injection. AcuteTHIM injection to 6‐week‐old rats also produced hypoalgesia, but this effect was transient andwas gone within 14 days. Present findings show that THIM administration to suckling or adultrats impairs sensitivity to pain, apparently due to activation the endogenous opioid system.IDENTIFICATION AND DISTRIBUTION OF MERCURY SPECIES IN RAT TISSUES FOLLOWINGADMINISTRATION OF THIMEROSAL OR METHYLMERCURY. (RODRIGUES 2010)Rodrigues JL, Serpeloni JM, Batista BL, Souza SS, Barbosa F Jr. Arch Toxicol. 2010 Nov;84(11):891‐6. Epub2010 Apr 13.Methylmercury (Met‐Hg) is one the most toxic forms of Hg, with a considerable range ofharmful effects on humans. Sodium ethyl mercury thiosalicylate, thimerosal (TM) is anethylmercury (Et‐Hg)‐containing preservative that has been used in manufacturing vaccines inmany countries. Whereas the behavior of Met‐Hg in humans is relatively well known, that ofethylmercury (Et‐Hg) is poorly understood. The present study describes the distribution ofmercury as (‐methyl, ‐ethyl and inorganic mercury) in rat tissues (brain, heart, kidney and liver)and blood following administration of TM or Met‐Hg. Animals received one dose/day of Met‐Hgor TM by gavage (0.5 mg Hg/kg). Blood samples were collected after 6, 12, 24, 48, 96 and 120 hof exposure. After 5 days, the animals were killed, and their tissues were collected. Total bloodScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201222 | P a g emercury (THg) levels were determined by ICP‐MS, and methylmercury (Met‐Hg), ethylmercury(Et‐Hg) and inorganic mercury (Ino‐Hg) levels were determined by speciation analysis with LC‐ICP‐MS. Mercury remains longer in the blood of rats treated with Met‐Hg compared to that ofTM‐exposed rats. Moreover, after 48 h of the TM treatment, most of the Hg found in blood wasinorganic. Of the total mercury found in the brain after TM exposure, 63% was in the form ofIno‐Hg, with 13.5% as Et‐Hg and 23.7% as Met‐Hg. In general, mercury in tissues and bloodfollowing TM treatment was predominantly found as Ino‐Hg, but a considerable amount of Et‐Hg was also found in the liver and brain. Taken together, our data demonstrated that thetoxicokinetics of TM is completely different from that of Met‐Hg. Thus, Met‐Hg is not anappropriate reference for assessing the risk from exposure to TM‐derived Hg. It also adds newdata for further studies in the evaluation of TM toxicity.NEONATAL ADMINISTRATION OF THIMEROSAL CAUSES PERSISTENT CHANGES IN MU OPIOIDRECEPTORS IN THE RAT BRAIN (OLCZAK 2010)Olczak M, Duszczyk M, Mierzejewski P, Bobrowicz T, Majewska MD. Neurochem Res. 2010 Aug28. Thimerosal added to some pediatric vaccines is suspected in pathogenesis of severalneurodevelopmental disorders. Our previous study showed that thimerosal administered tosuckling rats causes persistent, endogenous opioid‐mediated hypoalgesia. Here we examined,using immunohistochemical staining technique, the density of μ‐opioid receptors (MORs) in thebrains of rats, which in the second postnatal week received four i.m. injections of thimerosal atdoses 12, 240, 1,440 or 3,000 μg Hg/kg. The periaqueductal gray, caudate putamen andhippocampus were examined. Thimerosal administration caused dose‐dependent statisticallysignificant increase in MOR densities in the periaqueductal gray and caudate putamen, butdecrease in the dentate gyrus, where it was accompanied by the presence of degeneratingneurons and loss of synaptic vesicle marker (synaptophysin). These data document thatexposure to thimerosal during early postnatal life produces lasting alterations in the densities ofbrain opioid receptors along with other neuropathological changes, which may disturb braindevelopment.Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201223 | P a g eCHRONIC METALS INGESTION BY PRAIRIE VOLES PRODUCES SEX‐SPECIFIC DEFICITS IN SOCIALBEHAVIOR: AN ANIMAL MODEL OF AUTISM. (CURTIS 2010)Curtis JT, Hood AN, Chen Y, Cobb GP, Wallace DR. Behav Brain Res. 2010 Nov 12;213(1):42‐9. Epub 2010Apr 28.We examined the effects of chronic metals ingestion on social behavior in the normally highlysocial prairie vole to test the hypothesis that metals may interact with central dopaminesystems to produce the social withdrawal characteristic of autism. Relative to water‐treatedcontrols, 10 weeks of chronic ingestion of either Hg(++) or Cd(++) via drinking watersignificantly reduced social contact by male voles when they were given a choice betweenisolation or contact with an unfamiliar same‐sex conspecific. The effects of metals ingestionwere specific to males: no effects of metals exposure were seen in females. Metals ingestiondid not alter behavior of males allowed to choose between isolation or their familiar cage‐mates, rather than strangers. We also examined the possibility that metals ingestion affectscentral dopamine functioning by testing the voles' locomotor responses to peripheraladministration of amphetamine. As with the social behavior, we found a sex‐specific effect ofmetals on amphetamine responses. Males that consumed Hg(++) did not increase theirlocomotor activity in response to amphetamine, whereas similarly treated females and malesthat ingested only water significantly increased their locomotor activities. Thus, an ecologicallyrelevant stimulus, metals ingestion, produced two of the hallmark characteristics of autism ‐ social avoidance and a male‐oriented bias. These results suggest that metals exposure maycontribute to the development of autism, possibly by interacting with central dopaminefunction, and support the use of prairie voles as a model organism in which to study autism.NEUROLIGIN‐DEFICIENT MUTANTS OF C. ELEGANS HAVE SENSORY PROCESSING DEFICITS ANDARE HYPERSENSITIVE TO OXIDATIVE STRESS AND MERCURY TOXICITY. (HUNTER 2010)Hunter JW, Mullen GP, McManus JR, Heatherly JM, Duke A, Rand JB. Dis Model Mech. 2010 May‐Jun;3(5‐6):366‐76. Epub 2010 Jan 18.Neuroligins are postsynaptic cell adhesion proteins that bind specifically to presynapticmembrane proteins called neurexins. Mutations in human neuroligin genes are associated withautism spectrum disorders in some families. The nematode Caenorhabditis elegans has a singleneuroligin gene (nlg‐1), and approximately a sixth of C. elegans neurons, including somesensory neurons, interneurons and a subset of cholinergic motor neurons, express a neuroligintranscriptional reporter. Neuroligin‐deficient mutants of C. elegans are viable, and they do notappear deficient in any major motor functions. However, neuroligin mutants are defective in asubset of sensory behaviors and sensory processing, and are hypersensitive to oxidative stressand mercury compounds; the behavioral deficits are strikingly similar to traits frequentlyassociated with autism spectrum disorders. Our results suggest a possible link between geneticScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201224 | P a g edefects in synapse formation or function, and sensitivity to environmental factors in thedevelopment of autism spectrum disorders.LASTING NEUROPATHOLOGICAL CHANGES IN RAT BRAIN AFTER INTERMITTENT NEONATALADMINISTRATION OF THIMEROSAL. (OLCZAK 2010)Olczak M, Duszczyk M, Mierzejewski P, Wierzba‐Bobrowicz T, Majewska MD. Folia Neuropathol.2010;48(4):258‐69.Thimerosal, an organomercurial added as a preservative to some vaccines, is a suspectediatrogenic factor, possibly contributing to paediatric neurodevelopmental disorders includingautism. We examined the effects of early postnatal administration of thimerosal (four i.m.injections, 12 or 240 μg THIM‐Hg/kg, on postnatal days 7, 9, 11 and 15) on brain pathology inWistar rats. Numerous neuropathological changes were observed in young adult rats whichwere treated postnatally with thimerosal. They included: ischaemic degeneration of neuronsand "dark" neurons in the prefrontal and temporal cortex, the hippocampus and thecerebellum, pathological changes of the blood vessels in the temporal cortex, diminishedsynaptophysin reaction in the hippocampus, atrophy of astroglia in the hippocampus andcerebellum, and positive caspase‐3 reaction in Bergmann astroglia. These findings documentneurotoxic effects of thimerosal, at doses equivalent to those used in infant vaccines or higher,in developing rat brain, suggesting likely involvement of this mercurial in neurodevelopmentaldisordersPERSISTENT BEHAVIORAL IMPAIRMENTS AND ALTERATIONS OF BRAIN DOPAMINE SYSTEMAFTER EARLY POSTNATAL ADMINISTRATION OF THIMEROSAL IN RATS. (OLCZAK 2011)Olczak M, Duszczyk M, Mierzejewski P, Meyza K, Majewska MD. Behav Brain Res. 2011 Sep30;223(1):107‐18. doi: 10.1016/j.bbr.2011.04.026. Epub 2011 Apr 28.The neurotoxic organomercurial thimerosal (THIM), used for decades as vaccine preservative, isa suspected factor in the pathogenesis of some neurodevelopmental disorders. Previously weshowed that neonatal administration of THIM at doses equivalent to those used in infantvaccines or higher, causes lasting alterations in the brain opioid system in rats. Here weinvestigated neonatal treatment with THIM (at doses 12, 240, 1440 and 3000 μg Hg/kg) onbehaviors, which are characteristically altered in autism, such as locomotor activity, anxiety,social interactions, spatial learning, and on the brain dopaminergic system in Wistar rats of bothsexes. Adult male and female rats, which were exposed to the entire range of THIM dosesduring the early postnatal life, manifested impairments of locomotor activity and increasedanxiety/neophobia in the open field test. In animals of both sexes treated with the highestTHIM dose, the frequency of prosocial interactions was reduced, while the frequency ofasocial/antisocial interactions was increased in males, but decreased in females. Neonatal THIMScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201225 | P a g etreatment did not significantly affect spatial learning and memory. THIM‐exposed rats alsomanifested reduced haloperidol‐induced catalepsy, accompanied by a marked decline in thedensity of striatal D₂ receptors, measured by immunohistochemical staining, suggestingalterations to the brain dopaminergic system. Males were more sensitive than females to someneurodisruptive/neurotoxic actions of THIM. These data document that early postnatal THIMadministration causes lasting neurobehavioral impairments and neurochemical alterations inthe brain, dependent on dose and sex. If similar changes occur in THIM/mercurial‐exposedchildren, they could contribute do neurodevelopmental disorders.MERCURY DISPOSITION IN SUCKLING RATS: COMPARATIVE ASSESSMENT FOLLOWINGPARENTERAL EXPOSURE TO THIOMERSAL AND MERCURIC CHLORIDE. (BLANUSA 2012)Blanuša M, Orct T, Vihnanek Lazarus M, Sekovanić A, Piasek M. J Biomed Biotechnol.2012;2012:256965. doi: 10.1155/2012/256965. Epub 2012 Jul 26.Due to the facts that thiomersal‐containing vaccine is still in use in many developing countries,and all forms of mercury have recognised neurotoxic, nephrotoxic, and other toxic effects,studies on disposition of ethylmercury and other mercury forms are still justified, especially atyoung age. Our investigation aimed at comparing mercury distribution and rate of excretion inthe early period of life following exposure to either thiomersal (TM) or mercuric chloride (HgCl₂)in suckling rats. Three experimental groups were studied: control, TM, and HgCl₂, with 12 to18pups in each. Both forms of mercury were administered subcutaneously in equimolar quantities(0.81 μmol/kg b.w.) three times during the suckling period (on the days of birth 7, 9, and 11) tomimic the vaccination regimen in infants. After the last administration of TM or HgCl₂, totalmercury retention and excretion was assessed during following six days. In TM‐exposed groupmercury retention was higher in the brain, enteral excretion was similar, and urinary excretionwas much lower compared to HgCl₂‐exposed sucklings. More research is still needed toelucidate all aspects of toxicokinetics and most harmful neurotoxic potential of various forms ofmercury, especially in the earliest period of life.SEX‐DEPENDENT CHANGES IN CEREBELLAR THYROID HORMONE‐DEPENDENT GENEEXPRESSION FOLLOWING PERINATAL EXPOSURE TO THIMEROSAL IN RATS. (KHAN 2012)Khan A, Sulkowski ZL, Chen T, Zavacki AM, Sajdel‐Sulkowska EM. J Physiol Pharmacol. 2012Jun;63(3):277‐83.Mammalian brain development is regulated by the action of thyroid hormone (TH) on targetgenes. We have previously shown that the perinatal exposure to thimerosal (TM, metabolizedto ethylmercury) exerts neurotoxic effects on the developing cerebellum and is associated witha decrease in cerebellar D2 activity, which could result in local brain T3 deficiency. We have alsobegun to examine TM effect on gene expression. The objective of this study was to expand onScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201226 | P a g eour initial observation of altered cerebellar gene expression following perinatal TM exposureand to examine additional genes that include both TH‐dependent as well as other genes criticalfor cerebellar development in male and female neonates exposed perinatally (G10‐G15 and P5to P10) to TM. We report here for the first time that expression of suppressor‐of‐white‐apricot‐1 (SWAP‐1), a gene negatively regulated by T3, was increased in TM‐exposed males (61.1%increase), but not in females; (p<0.05). Positively regulated T3‐target genes, Purkinje cellprotein 2 (Pcp2; p=0.07) and Forkhead box protein P4 (FoxP4; p=0.08), showed a trend towardsdecreased expression in TM‐exposed males. The expression of deiodinase 2 (DIO2) showed atrend towards an increase in TM‐exposed females, while deiodinase 3 (DIO3), transthyretin(TTR), brain derived neurotrophic factor (BDNF) and reelin (RELN) was not significantly alteredin either sex. Since regulation of gene splicing is vital to neuronal proliferation anddifferentiation, altered expression of SWAP‐1 may exert wide ranging effects on multiple genesinvolved in the regulation of cerebellar development. We have previously identified activationof another TH‐dependent gene, outer dense fiber of sperm tails 4, in the TM exposed malepups. Together, these results also show sex‐dependent differences between the toxic impactsof TM in males and females. Interestingly, the genes that were activated by TM are negativelyregulated by TH, supporting our hypothesis of local brain hypothyroidism being induced by TMand suggesting a novel mechanism of action TM in the developing brain.PRENATAL EXPOSURE TO ORGANOMERCURY, THIMEROSAL, PERSISTENTLY IMPAIRS THESEROTONERGIC AND DOPAMINERGIC SYSTEMS IN THE RAT BRAIN: IMPLICATIONS FORASSOCIATION WITH DEVELOPMENTAL DISORDERS. (IDA‐ETO 2012)Ida‐Eto M, Oyabu A, Ohkawara T, Tashiro Y, Narita N, Narita M. Brain Dev. 2012 May 31. [Epubahead of print]Thimerosal, an organomercury compound, has been widely used as a preservative. Therefore,concerns have been raised about its neurotoxicity. We recently demonstrated perturbation ofearly serotonergic development by prenatal exposure to thimerosal (Ida‐Eto et al. (2011) [11]).Here, we investigated whether prenatal thimerosal exposure causes persistent impairmentafter birth. Analysis on postnatal day 50 showed significant increase in hippocampal serotoninfollowing thimerosal administration on embryonic day 9. Furthermore, not only serotonin,striatal dopamine was significantly increased. These results indicate that embryonic exposure tothimerosal produces lasting impairment of brain monoaminergic system, and thus every effortshould be made to avoid the use of thimerosal.Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201227 | P a g eADMINISTRATION OF THIMEROSAL TO INFANT RATS INCREASES OVERFLOW OF GLUTAMATEAND ASPARTATE IN THE PREFRONTAL CORTEX: PROTECTIVE ROLE OFDEHYDROEPIANDROSTERONE SULFATE. (DUSZCZYK‐BUDHATHOKI 2012)Duszczyk‐Budhathoki M, Olczak M, Lehner M, Majewska MD. Neurochem Res. 2012Feb;37(2):436‐47. doi: 10.1007/s11064‐011‐0630‐z. Epub 2011 Oct 21.Thimerosal, a mercury‐containing vaccine preservative, is a suspected factor in the etiology ofneurodevelopmental disorders. We previously showed that its administration to infant ratscauses behavioral, neurochemical and neuropathological abnormalities similar to those presentin autism. Here we examined, using microdialysis, the effect of thimerosal on extracellularlevels of neuroactive amino acids in the rat prefrontal cortex (PFC). Thimerosal administration(4 injections, i.m., 240 μg Hg/kg on postnatal days 7, 9, 11, 15) induced lasting changes in aminoacid overflow: an increase of glutamate and aspartate accompanied by a decrease of glycineand alanine; measured 10‐14 weeks after the injections. Four injections of thimerosal at a doseof 12.5 μg Hg/kg did not alter glutamate and aspartate concentrations at microdialysis time(but based on thimerosal pharmacokinetics, could have been effective soon after its injection).Application of thimerosal to the PFC in perfusion fluid evoked a rapid increase of glutamateoverflow. Coadministration of the neurosteroid, dehydroepiandrosterone sulfate (DHEAS; 80mg/kg; i.p.) prevented the thimerosal effect on glutamate and aspartate; the steroid alone hadno influence on these amino acids. Coapplication of DHEAS with thimerosal in perfusion fluidalso blocked the acute action of thimerosal on glutamate. In contrast, DHEAS alone reducedoverflow of glycine and alanine, somewhat potentiating the thimerosal effect on these aminoacids. Since excessive accumulation of extracellular glutamate is linked with excitotoxicity, ourdata imply that neonatal exposure to thimerosal‐containing vaccines might induce excitotoxicbrain injuries, leading to neurodevelopmental disorders. DHEAS may partially protect againstmercurials‐induced neurotoxicity.MATERNAL THIMEROSAL EXPOSURE RESULTS IN ABERRANT CEREBELLAR OXIDATIVE STRESS,THYROID HORMONE METABOLISM, AND MOTOR BEHAVIOR IN RAT PUPS; SEX‐ AND STRAIN‐DEPENDENT EFFECTS. (SULKOWSKI ZL 2012)Sulkowski ZL, Chen T, Midha S, Zavacki AM, Sajdel‐Sulkowska EM. Cerebellum. 2012Jun;11(2):575‐86. doi: 10.1007/s12311‐011‐0319‐5.Methylmercury (Met‐Hg) and ethylmercury (Et‐Hg) are powerful toxicants with a range ofharmful neurological effects in humans and animals. While Met‐Hg is a recognized trigger ofoxidative stress and an endocrine disruptor impacting neurodevelopment, the developmentalneurotoxicity of Et‐Hg, a metabolite of thimerosal (TM), has not been explored. Wehypothesized that TM exposure during the perinatal period impairs central nervous systemdevelopment, and specifically the cerebellum, by the mechanism involving oxidative stress. ToScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201228 | P a g etest this, spontaneously hypertensive rats (SHR) or Sprague‐Dawley (SD) rat dams were exposedto TM (200 μg/kg body weight) during pregnancy (G10‐G15) and lactation (P5‐P10). Male andfemale neonates were evaluated for auditory and motor function; cerebella were analyzed foroxidative stress and thyroid metabolism. TM exposure resulted in a delayed startle response inSD neonates and decreased motor learning in SHR male (22.6%), in SD male (29.8%), and in SDfemale (55.0%) neonates. TM exposure also resulted in a significant increase in cerebellar levelsof the oxidative stress marker 3‐nitrotyrosine in SHR female (35.1%) and SD male (14.0%)neonates. The activity of cerebellar type 2 deiodinase, responsible for local intra‐brainconversion of thyroxine to the active hormone, 3',3,5‐triiodothyronine (T3), was significantlydecreased in TM‐exposed SHR male (60.9%) pups. This coincided with an increased (47.0%)expression of a gene negatively regulated by T3, Odf4 suggesting local intracerebellar T3deficiency. Our data thus demonstrate a negative neurodevelopmental impact of perinatal TMexposure which appears to be both strain‐ and sex‐dependent.Cellular Research BIOCHEMICAL AND MOLECULAR BASIS OF THIMEROSAL‐INDUCED APOPTOSIS IN T CELLS: AMAJOR ROLE OF MITOCHONDRIAL PATHWAY (MAKANI 2002)Makani S, Gollapudi S, Yel L, Chiplunkar S, Gupta S. Genes & Immunity. 2002 Aug;3(5):270‐8.Makani found thimerosal, in micromolar concentration, causes cell death (apoptosis) inimmune cells (T cells). The data also suggested that the thimerosal induced apoptosis in T cellsoccurred via mitochondrial pathways by inducing oxidative stress and depletion of glutathione. THIMEROSAL INDUCES MICRONUCLEI IN THE CYTOCHALASIN B BLOCK MICRONUCLEUS TESTWITH HUMAN LYMPHOCYTES (WESTPHAL 2003)Westphal GA, Asgari S, Schulz TG, Bünger J, Müller M, Hallier E. Archives of Toxicology. 2003 Jan;77(1):50 – 55. Significant induction of micronuclei was seen at concentrations of thimerosal between 0.05‐0.5µg/ml in 14 out of 16 experiments. Thus, genotoxic effects were seen even at concentrationswhich can occur at the injection site. Toxicity and toxicity‐related elevation of micronuclei wasseen at and above 0.6 µg/ml thimerosal. Marked individual and intraindividual variations in thein vitro response to thimerosal among the different blood donors occurred. However, there wasno association observed with any of the glutathione S‐transferase polymorphism investigated.In conclusion, thimerosal is genotoxic in the cytochalasin B block micronucleus test with humanlymphocytes (immune cells). These data raise some concern on the widespread use ofthimerosal. Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201229 | P a g eTHIMEROSAL INDUCES DNA BREAKS, CASPASE‐3 ACTIVATION, MEMBRANE DAMAGE, ANDCELL DEATH IN CULTURED HUMAN NEURONS AND FIBROBLASTS (BASKIN 2003)Baskin DS, Ngo H, Didenko VV. Toxicological Sciences. 2003 Aug;74(2):361‐8. Baskin documented that thimerosal disrupts cell membranes, damages DNA and alters cellshape at concentrations only 4 times those expected from vaccines. Greater effects were seenas the length of time of exposure grew, suggesting that under real conditions the concentrationneeded for the observed alterations would be much lower. It has been documented insubsequent research that exposure of cells to nanomolar levels of thimerosal after 24 hoursresults in cell alterations. ACTIVATION OF METHIONINE SYNTHASE BY INSULIN‐LIKE GROWTH FACTOR‐1 ANDDOPAMINE: A TARGET FOR NEURODEVELOPMENTAL TOXINS AND THIMEROSAL. (WALY 2004)Waly M, Olteanu H, Banerjee R, Choi SW, Mason JB, Parker BS, Sukumar S, Shim S, Sharma A, BenzecryJM, Power‐Charnitsky VA, Deth RC. Mol Psychiatry. 2004 Apr;9(4):358‐70.Methylation events play a critical role in the ability of growth factors to promote normaldevelopment. Neurodevelopmental toxins, such as ethanol and heavy metals, interrupt growthfactor signaling, raising the possibility that they might exert adverse effects on methylation. Wefound that insulin‐like growth factor‐1 (IGF‐1)‐ and dopamine‐stimulated methionine synthase(MS) activity and folate‐dependent methylation of phospholipids in SH‐SY5Y humanneuroblastoma cells, via a PI3‐kinase‐ and MAP‐kinase‐dependent mechanism. The stimulationof this pathway increased DNA methylation, while its inhibition increased methylation‐sensitivegene expression. Ethanol potently interfered with IGF‐1 activation of MS and blocked its effecton DNA methylation, whereas it did not inhibit the effects of dopamine. Metal ions potentlyaffected IGF‐1 and dopamine‐stimulated MS activity, as well as folate‐dependent phospholipidmethylation: Cu(2+) promoted enzyme activity and methylation, while Cu(+), Pb(2+), Hg(2+)and Al(3+) were inhibitory. The ethylmercury‐containing preservative thimerosal inhibited bothIGF‐1‐ and dopamine‐stimulated methylation with an IC(50) of 1 nM and eliminated MS activity.Our findings outline a novel growth factor signaling pathway that regulates MS activity andthereby modulates methylation reactions, including DNA methylation. The potent inhibition ofthis pathway by ethanol, lead, mercury, aluminum and thimerosal suggests that it may be animportant target of neurodevelopmental toxins.Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201230 | P a g eUNCOUPLING OF ATP‐MEDIATED CALCIUM SIGNALING AND DYSREGULATION INTERLEUKIN‐6SECRETION IN DENDRITIC CELLS BY NANAMOLAR THIMEROSAL (GOTH 2006)Goth SR, Chu RA, Gregg JP, Cherednichenko G, Pessah IN. Environ Health Perspect. 2006Jul;114(7):1083‐91.Goth investigated adenosine triphosphate (ATP) mediated Ca2+ responses in dendritic cells(responsible for initiating primary immune responses) exposed briefly to nanamolarconcentrations (100nM, 5 min) of thimerosal and found that dendritic cells were exquisitelysensitive to thimerosal resulting in uncoupling of the positive and negative regulation of Ca2 +signals. THIMEROSAL INDUCES NEURONAL CELL DEATH BY CAUSING CYTOCHROME C ANDAPOPTOSIS‐INDUCING FACTOR RELEASE FROM MITOCHONDRIA (YEL 2005)Yel L, Brown LE, Su K, Gollapudi S, Gupta S. Int J Mol Med. 2005 Dec;16(6):971‐7.Yel demonstrated that thimerosal, at nanamolar concentrations, induced neuronal cell deaththrough the mitochondrial pathway. The thimerosal induced apoptosis was associated withdepolarization of mitochondrial membranes, generation of reactive oxygen species and releaseof cytochrome c and apoptosis‐inducing factor, suggesting that thimerosal cause apoptosis inneuroblastoma cells by altering the mitochondrial microenvironment. IN VITRO UPTAKE OF GLUTAMATE IN GLAST AND GLT‐1 TRANSFECTED MUTANT CHO‐K1 CELLSIS INHIBITED BY THE ETHYLMERCURY‐CONTAINING PRESERVATIVE THIMEROSAL (MUTKUS2005)Mutkus L, Aschner JL, Syversen T, Shanker G, Sonnewald U, Aschner M. Bio Trace Elem Res. 2005Summer;105(1‐3):71‐86Mutkus determined that thimerosal caused significant and selective changes in both glutamatetransporter mRNA and protein expression in the CHO‐K1 cell line. This study suggests thatthimerosal accumulation in the central nervous system might contribute to dysregulation ofglutamate homeostasis. Glutamate is a neurotransmitter and is necessary for proper brainfunctioning. Note: Yip (2007) documented decreased levels of glutamate in autistic cerebralbrain tissue and Hornig ( 2004) noted altered glutamate receptors in thimerosal exposed mice. Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201231 | P a g eTHIMEROSAL NEUROTOXICITY IS ASSOCIATED WITH GLUTATHIONE DEPLETION: PROTECTIONWITH GLUTATHIONE PRECURSORS (JAMES 2005)JAMES SJ, SLIKKER W 3RD, MELNYK S, NEW E, POGRIBNA M, JERNIGAN S. NEUROTOXICOLOGY. 2005JAN;26(1):1‐8.James notes that the viability of neuronal cell lines was decreased after just 3 hour exposure to2.5 micromolar concentrations of thimerosal. Also noted was that cultured neuroblastoma cellswere found to have lower levels of GSH and increased sensitivity to thimerosal in comparison toglioblastoma cells that contain higher levels of GSH. Furthermore, pretreatment withglutathione ethyl ester or NAC prevented cytotoxicity with exposure up to 15 micromolarthimerosal. THIMEROSAL INDUCES APOPTOSIS IN A NEUROBLASTOMA MODEL VIA THE CJUN‐N‐TERMINAL KINASE PATHWAY (HERDMAN 2006)Herdman ML, Marcelo A, Huang Y, Niles RM, Dhar S, Kiningham KK. Toxicol Sci. 2006 Jul;92(1):246‐53.Herdman notes that cJun N‐terminase kinase (JNK)‐signaling pathway activation has beenimplicated in neuronal apoptosis. Herdman investigated the role that the JNK pathway plays inneurotoxicity caused by thimerosal. SK‐N‐SH cells treated with thimerosal (0‐10 microM)showed an increase in the phosphorylated (active) form of JNK and cJun with 5 and 10 microMthimerosal treatment at 2 and 4 h.. To assess which components are essential to apoptosis,cells were treated with a cell‐permeable JNK inhibitor and the downstream effectors ofapoptosis were analyzed. Results indicate that thimerosal‐induced neurotoxicity occurs throughthe JNK‐signaling pathway, independent of cJun activation, leading to apoptotic cell death. THIMEROSAL INDUCES TH2 RESPONSES VIA INFLUENCING CYTOKINE SECRETION BY HUMANDENDRITIC CELLS (AGRAWAL 2007)Agrawal A, Kaushal P, Agrawal S, Gollapudi S, Gupta S. J Leukoc Biol. 2007 Feb;81(2):474‐82. Agrawal documented that thimerosal exercised TH2‐promoting effects through modulation offunctions of human dendritic cells (DC) by inhibition of LPS induced proinflammatory cytokinesTNF‐alpha, IL‐6, and IL‐12p70 resulting in an increase TH2 (IL‐5, IL‐13 and decreased TH1 (IFN‐gamma). Thimerosal exposure of DC led to depletion of intracellular glutathione (GSH) and theaddition of exogenous GSH to DC abolished the TH2 promoting effect of thimerosal. (NoteJames has documented that children with autism have low levels of plasma glutathione)Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201232 | P a g eEFFECTS OF THIMEROSAL ON NGF SIGNAL TRANSDUCTION AND CELL DEATH INNEUROBLASTOMA CELLS (PARRAN 2005)Parran DK, Barker A, Ehrich M. Toxicological Sciences. 2005 Jul;86(1):132‐40. Parran documented that thimerosal causes DNA fragmentation of neuronal cells and disruptsneuronal growth factor signaling at micromolar and even nanomolar concentrations. With andwithout NGF, thimerosal caused elevated levels of fragmented DNA appearing at 0.01 microM(apoptosis) to decrease at concentrations >1 microM (necrosis). These data demonstrate thatthimerosal could alter NGF‐induced signaling in neurotrophin¬treated cells at concentrationslower than those responsible for cell death. GENOTOXICITY OF THIMEROSAL IN CULTURED HUMAN LYMPHOCYTES WITH AND WITHOUTMETABOLIC ACTIVATION SISTER CHROMATID EXCHANGE ANALYSIS PROLIFERATION INDEXAND MITOTIC INDEX (EKE 2008)Eke D, Celik A. Mersin University, Faculty of Science and Letters, Department of Biology, 33343 Mersin,Turkey. Toxicol In Vitro. 2008 Jun;22(4):927‐34. Epub 2008 Feb 1.Thimerosal is an antiseptic containing 49.5% of ethyl mercury that has been used for years as apreservative in many infant vaccines and in flu vaccines. Thimerosal is an organic mercurialcompound used as a preservative in biomedical preparations. In this study, we evaluated thegenotoxic effect of thimerosal in cultured human peripheral blood lymphocytes using sisterchromatid exchange analysis in culture conditions with and without S9 metabolic activation.This study is the first report investigating the genotoxic effects of thimerosal in cultured humanperipheral blood lymphocyte cells using sister chromatid exchange analysis. An analysis ofvariance test (ANOVA) was performed to evaluate the results. Significant induction of sisterchromatid exchanges was seen at concentrations between 0.2 and 0.6 microg/ml of thimerosalcompared with negative control. A significant decrease (p<0.001) in mitotic index (MI) andproliferation index (PRI) as well as an increase in SCE frequency (p<0.001) was observedcompared with control cultures. Our results indicate the genotoxic and cytotoxic effect of TH incultured human peripheral blood lymphocytes at tested doses in cultures with/without S9fraction. ZINC IONS CAUSE THE THIMEROSAL‐INDUCED SIGNAL OF FLUORESCENT CALCIUM PROBES INLYMPHOCYTES (HAASE 2008)Haase H, Hebel S, Engelhardt G, Rink L., Institute of Immunology, RWTH Aachen University Hospital,Aachen, Germany. Cell Calcium. 2008 Oct 31.Most fluorescent probes for the investigation of calcium signaling also detect zinc ions.Consequently, changes in the intracellular zinc concentration could be mistaken for calciumScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201233 | P a g esignals. Thimerosal (TMS) is used as a calcium‐mobilizing agent and we analyzed thecontribution of zinc ions to the signal observed with fluorescent calcium probes after TMSstimulation. Our findings show that the fluorescent signal in lymphocytes is entirely due to zincrelease. Experiments in the T lymphocyte cell line Jurkat and primary human lymphocytes showthat TMS and its active metabolite, ethyl mercury, cause an increase in signal intensity withprobes designed for the detection of either calcium or zinc ions. The TMS/ethyl mercury‐induced signal of the calcium probes Fluo‐4 and FURA‐2 was completely absent when the zincchelator TPEN [N,N,N',N'‐tetrakis‐(2‐pyridyl‐methyl)ethylenediamine] was added. In contrast,the signal caused by thapsigargin‐induced release of calcium from the endoplasmic reticulumwas unaffected by TPEN. In light of these observations, zinc may also contribute to calciumsignals caused by mercury‐containing compounds other than TMS, and a potential involvementof zinc release in the immunomodulatory effects of these substances should be considered. CHARACTERIZATION OF EARLY EVENTS INVOLVED IN HUMAN DENDRITIC CELL MATURATIONINDUCED BY SENSITIZERS: CROSS TALK BETWEEN MAPK SIGNALLING PATHWAYS(TROMPEZINSKI 2008)Trompezinski S, Migdal C, Tailhardat M, Le Varlet B, Courtellemont P, Haftek M, Serres M. Toxicol ApplPharmacol. 2008 Aug 1;230(3):397‐406. Epub 2008 Apr 8.Dendritic cells (DCs), efficient‐antigen presenting cells play an important role in initiating andregulating immune responses. DC maturation following exposure to nickel or DNCB induced anup‐regulation of phenotypic markers and inflammatory cytokine secretion. Early intracellularmechanisms involved in DC maturation required to be precise. To address this purpose, DCsderived from human monocytes were treated with sensitizers (nickel, DNCB or thimerosal) incomparison with an irritant (SDS). Our data confirming the up‐regulation of CD86, CD54 andcytokine secretion (IL‐8 and TNFalpha) induced by sensitizers but not by SDS, signallingtransduction involved in DC maturation was investigated using these chemicals. Kinase activitymeasurement was assessed using two new sensitive procedures (Facetrade mark and CBA)requiring few cells. SDS did not induce changes in signalling pathways whereas NiSO(4), DNCBand thimerosal markedly activated p38 MAPK and JNK, in contrast Erk1/2 phosphorylation wascompletely inhibited by DNCB or thimerosal and only activated by nickel. A pre‐treatment withp38 MAPK inhibitor (SB203580) suppressed Erk1/2 inhibition induced by DNCB or thimerosaldemonstrating a direct interaction between p38 MAPK and Erk1/2. A pre‐treatment with anantioxidant, N‐acetyl‐L‐cysteine (NAC) markedly reduced Erk1/2 inhibition and p38 MAPKphosphorylation induced by DNCB and thimerosal, suggesting a direct activation of p38 MAPKvia an oxidative stress and a regulation of MAPK signalling pathways depending on chemicals.Because of a high sensitivity of kinase activity measurements, these procedures will be suitablefor weak or moderate sensitizer screening.Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201234 | P a g eMITOCHONDRIAL MEDIATED THIMEROSAL‐INDUCED APOPTOSIS IN A HUMANNEUROBLASTOMA CELLINE (SK‐N‐SH)(HUMPHREY 2009)Humphrey ML, Cole MP, Pendergrass JC, Kiningham KK. Neurotoxicology. 2005 Jun;26(3):407‐16. Humphrey noted that after only short (2 hour) exposures to thimerosal at 5 micromolarconcentrations in a human neuroblastoma cell line caused morphological changes includingmembrane alterations and cell shrinkage leading to cell death. Cytochrome C was shown toleak from the mitochondria followed by caspase 9 cleavage. These findings support deleterious effects on cellular cytoarchitecture and initiation of mitochondrial‐mediatedapoptosis induced by thimerosal. MITOCHONDRIAL DYSFUNCTION, IMPAIRED OXIDATIVE‐REDUCTION ACTIVITY,DEGENERATION, AND DEATH IN HUMAN NEURONAL AND FETAL CELLS INDUCED BY LOW‐LEVEL EXPOSURE TO THIMEROSAL AND OTHER METAL COMPOUNDS (GEIER 2009) D.A. Geier et al. Toxicological & Environmental Chemistry. 2009, 1–15, iFirstThimerosal (ethylmercurithiosalicylic acid), an ethylmercury (EtHg)‐releasing compound(49.55% mercury (Hg)), was used in a range of medical products for more than 70 years. Ofparticular recent concern, routine administering of Thimerosal‐containing biologics/childhoodvaccines have become significant sources of Hg exposure for some fetuses/infants. This studywas undertaken to investigate cellular damage among in vitro human neuronal (SH‐SY‐5Yneuroblastoma and 1321N1 astrocytoma) and fetal (nontransformed) model systems using cellvitality assays and microscope‐based digital image capture techniques to assess potentialdamage induced by Thimerosal and other metal compounds (aluminum (Al) sulfate, lead (Pb)(II)acetate, methylmercury (MeHg) hydroxide, and mercury (Hg)(II) chloride) where the cation wasreported to exert adverse effects on developing cells. Thimerosal‐associated cellular damagewas also evaluated for similarity to pathophysiological findings observed in patients diagnosedwith autistic disorders (ADs). Thimerosal‐induced cellular damage as evidenced byconcentration‐and time‐dependent mitochondrial damage, reduced oxidative–reductionactivity, cellular degeneration, and cell death in the in vitro human neuronal and fetal modelsystems studied. Thimerosal at low nanomolar (nM) concentrations induced significant cellulartoxicity in human neuronal and fetal cells. Thimerosal‐induced cytoxicity is similar to thatobserved in AD pathophysiologic studies. Thimerosal was found to be significantly more toxicthan the other metal compounds examined. Future studies need to be conducted to evaluateadditional mechanisms underlying Thimerosal‐induced cellular damage and assess potential co‐exposures to other compounds that may increase or decrease Thimerosal‐mediated toxicity. Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201235 | P a g eSENSITIZATION EFFECT OF THIMEROSAL IS MEDIATED IN VITRO VIA REACTIVE OXYGENSPECIES AND CALCIUM SIGNALING. (MIGDAL 2010)Migdal C, Foggia L, Tailhardat M, Courtellemont P, Haftek M, Serres M. Toxicology. 2010 Jul‐Aug;274(1‐3):1‐9. Epub 2010 May 10.Thimerosal, a mercury derivative composed of ethyl mercury chloride (EtHgCl) and thiosalicylicacid (TSA), is widely used as a preservative in vaccines and cosmetic products and causescutaneous reactions. Since dendritic cells (DCs) play an essential role in the immune response,the sensitization potency of chemicals was studied in vitro using U937, a humanpromyelomonocytic cell line that is used as a surrogate of monocytic differentiation andactivation. Currently, this cell line is under ECVAM (European Center for the Validation ofAlternative Methods) validation as an alternative method for discriminating chemicals.Thimerosal and mercury derivatives induced in U937 an overexpression of CD86 and interleukin(IL)‐8 secretion similarly to 1‐chloro‐2,4‐dinitrobenzene (DNCB), a sensitizer used as a positivecontrol for DC activation. Non‐sensitizers, dichloronitrobenzene (DCNB), TSA and sodiumdodecyl sulfate (SDS), an irritant, had no effect. U937 activation was prevented by cellpretreatment with N‐acetyl‐L‐cysteine (NAC) but not with thiol‐independent antioxidantsexcept vitamin E which affected CD86 expression by preventing lipid peroxidation of cellmembranes. Thimerosal, EtHgCl and DNCB induced glutathione (GSH) depletion and reactiveoxygen species (ROS) within 15 min; another peak was detected after 2h for mercurycompounds only. MitoSOX, a specific mitochondrial fluorescent probe, confirmed that ROSwere essentially produced by mitochondria in correlation with its membrane depolarization.Changes in mitochondrial membrane permeability induced by mercury were reversed by NACbut not by thiol‐independent antioxidants. Thimerosal and EtHgCl also induced a calcium (Ca2+)influx with a peak at 3h, suggesting that Ca2+ influx is a secondary event following ROSinduction as Ca2+ influx was suppressed after pretreatment with NAC but not with thiol‐independent antioxidants. Ca2+ influx was also suppressed when culture medium was deprivedof Ca2+ confirming the specificity of the measure. In conclusion, these data suggest thatthimerosal induced U937 activation via oxidative stress from mitochondrial stores andmitochondrial membrane depolarization with a primordial effect of thiol groups. A cross‐talkbetween ROS and Ca2+ influx was demonstrated.EVALUATION OF CYTOTOXICITY ATTRIBUTED TO THIMEROSAL ON MURINE AND HUMANKIDNEY CELLS. (PARK 2007)Park EK, Mak SK, Kültz D, Hammock BD. J Toxicol Environ Health A. 2007 Dec;70(24):2092‐5.Renal inner medullary collecting duct cells (mIMCD3) and human embryonic kidney cells(HEK293) were used for cytoscreening of thimerosal and mercury chloride (HgCl2). Thimerosaland HgCl2 acted in a concentration‐dependent manner. In mIMCD3 cells the 24‐h LC50 valuesScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201236 | P a g efor thimerosal, thiosalicylic acid, 2,2‐dithiosalicylic acid, and 2‐sulfobenzoic acid were 2.9, 2200,>1000, and >10,000 microM, respectively. The 24‐h LC50 value for HgCl2 in mIMCD3 cells was40 microM. In HEK293 cells, the 24‐h LC50 value for thimerosal was 9.5 microM. These datademonstrate that the higher cytotoxicity produced by thimerosal on renal cells with respect tosimilar compounds without Hg may be related to this metal content. The present study alsoestablishes mIMCD3 cells as a valuable model for evaluation of cytotoxicity of nephrotoxiccompounds.THE RELATIVE TOXICITY OF COMPOUNDS USED AS PRESERVATIVES IN VACCINES ANDBIOLOGICS. (GEIER 2010)Geier DA, Jordan SK, Geier MR. Med Sci Monit. 2010 Apr 28;16(5):SR21‐7.BACKGROUND: In vaccines/biologics, preservatives are used to prevent microbial growth. MATERIAL/METHODS: The present study examined: (1) the comparative toxicities of commonlyused preservatives in US licensed vaccines to human neurons; and (2) the relative toxicity indexof these compounds to human neurons in comparison to bacterial cells. RESULTS: Using humanneuroblastoma cells, the relative cytotoxicity of the levels of the compounds commonly used aspreservative in US licensed vaccines was found to be phenol <2‐phenoxyethanol <benzethonium chloride < Thimerosal. The observed relative toxicity indices (humanneuroblastoma cells/bacterial cells) were 2‐phenoxyethanol (4.6‐fold) < phenol (12.2‐fold) <Thimerosal (>330‐fold). In addition, for the compounds tested, except for 2‐phenoxyethanol,the concentrations necessary to induce significant killing of bacterial cells were significantlyhigher than those routinely present in US licensed vaccine/biological preparations.CONCLUSIONS: None of the compounds commonly used as preservatives in US licensedvaccine/biological preparations can be considered an ideal preservative, and their ability to fullycomply with the requirements of the US Code of Federal Regulations (CFR) for preservatives isin doubt. Future formulations of US licensed vaccines/biologics should be produced in asepticmanufacturing plants as single dose preparations, eliminating the need for preservatives and anunnecessary risk to patients.LOW MOLECULAR WEIGHT THIOLS REDUCE THIMEROSAL NEUROTOXICITY IN VITRO:MODULATION BY PROTEINS. (ZIEMINSKA 2010)Zieminska E, Toczylowska B, Stafiej A, Lazarewicz JW. Toxicology. 2010 Aug 7. [Epub ahead of print]Thimerosal (TH), an ethylmercury complex of thiosalicylic acid has been used as preservative invaccines. In vitro neurotoxicity of TH at high nM concentrations has been reported. Although anumber of toxicological experiments demonstrated high affinity of mercury to thiol groups ofthe extracellular amino acids and proteins that may decrease concentration of free TH in theorganism, less is known about the role of interactions between proteins and amino acids inprotection against TH neurotoxicity. In the present study we examined whether the presence ofScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201237 | P a g eserum proteins and of l‐cysteine (Cys), d,l‐homocysteine (Hcy), N‐acetyl cysteine (NAC), l‐methionine (Met) and glutathione (GSH) in the incubation medium affects the TH‐inducedchanges in the viability, the intracellular levels of calcium and zinc and mitochondrialmembrane potential in primary cultures of rat cerebellar granule cells. The cells were exposedto 500nM TH for 48h or to 15‐25muM TH for 10min. Our results demonstrated a decrease inthe cells viability evoked by TH, which could be prevented partially by serum proteins, albuminor in a dose‐dependent manner by 60, 120 or 600muM Cys, Hcy, NAC and GSH, but not by Met.This neuroprotection was less pronounced in the presence of proteins. Incubation of neuronswith TH also induced the rise in the intracellular calcium and zinc concentration and decrease inmitochondrial membrane potential, and these effects were abolished by all the sulfurcontaining compounds studied and administered at 600muM concentration, except Met. Theloss of the ethylmercury moiety from TH as a result of interaction with thiols studied wasmonitored by (1)H NMR spectroscopy. This extracellular process may be responsible for theneuroprotection seen in the cerebellar cell cultures, but also provides a molecular pathway forredistribution of TH‐derived toxic ethylmercury in the organism. In conclusion, these resultsconfirmed that proteins and sulfur‐containing amino acids applied separately reduce THneurotoxicity, while their combination modulates in more complex way neuronal survival in thepresence of TH.RESPONSIVENESS OF HUMAN MONOCYTE‐DERIVED DENDRITIC CELLS TO THIMEROSAL ANDMERCURY DERIVATIVES. (MIGDAL 2010)Migdal C, Tailhardat M, Courtellemont P, Haftek M, Serres M. Toxicol Appl Pharmacol. 2010 Jul;246(1‐2):66‐73. Epub 2010 Apr 22.Several cases of skin sensitization have been reported following the application of thimerosal,which is composed of ethyl mercury and thiosalicylic acid (TSA). However, few in vitro studieshave been carried out on human dendritic cells (DCs) which play an essential role in theinitiation of allergic contact dermatitis. The aim of the present study was to identify the effectof thimerosal and other mercury compounds on human DCs. To address this purpose, DCsderived from monocytes (mono‐DCs) were used. Data show that thimerosal and mercuryderivatives induced DC activation, as monitored by CD86 and HLA‐DR overexpression associatedwith the secretion of tumor necrosis factor alpha and interleukin 8, similarly tolipopolysaccharide and the sensitizers, 1‐chloro‐2,4‐dinitrobenzene (DNCB) and nickel sulfate,which were used as positive controls. In contrast, TSA, the non‐mercury part of thimerosal, aswell as dichloronitrobenzene, a DNCB negative control, and the irritant, sodium dodecyl sulfate,had no effect. Moreover, oxidative stress, monitored by ROS induction and depolarization ofthe mitochondrial membrane potential, was induced by thimerosal and mercury compounds, aswell as DNCB, in comparison with hydrogen peroxide, used as a positive control. The role ofScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201238 | P a g ethiol oxidation in the initiation of mono‐DC activation was confirmed by a pre‐treatment withN‐acetyl‐l‐cysteine which strongly decreased chemical‐induced CD86 overexpression. Thesedata are in agreement with several clinical observations of the high relevance of thimerosal inpatch‐test reactions and prove that human mono‐DCs are useful in vitro tools for determiningthe allergenic potency of chemicals.MERCURY INDUCES AN UNOPPOSED INFLAMMATORY RESPONSE IN HUMAN PERIPHERALBLOOD MONONUCLEAR CELLS IN VITRO. (GARDNER 2009)Gardner RM, Nyland JF, Evans SL, Wang SB, Doyle KM, Crainiceanu CM, Silbergeld EK. Environ HealthPerspect. 2009 Dec;117(12):1932‐8. Epub 2009 Aug 19.BACKGROUND: The human immune response to mercury is not well characterized despite thebody of evidence that suggests that Hg can modulate immune responses, including theinduction of autoimmune disease in some mouse models. Dysregulation of cytokine signalingappears to play an important role in the etiology of Hg‐induced autoimmunity in animal models. OBJECTIVES: In this study, we systematically investigated the human immune response to Hg invitro in terms of cytokine release. METHODS: Human peripheral blood mononuclear cells(PBMCs) were isolated from 20 volunteers who donated blood six separate times. PBMCs werecultured with lipopolysaccharide and concentrations of mercuric chloride (HgCl(2)) up to 200nM. Seven cytokines representing important pathways in physiologic and pathologic immuneresponses were measured in supernatants. We used multilevel models to account for theintrinsic clustering in the cytokine data due to experimental design. RESULTS: We found aconsistent increase in the release of the proinflammatory cytokines interleukin‐1beta (IL‐1beta)and tumor necrosis factor‐alpha, and concurrent decrease in release of the antiinflammatorycytokines interleukin 1‐receptor antagonist (IL‐1Ra) and IL‐10 in human PBMCs treated withsubcytotoxic concentrations of HgCl(2). IL‐4, IL‐17, and interferon‐gamma increased in aconcentration‐response manner. These results were replicated in a second, independentlyrecruited population of 20 different volunteers. CONCLUSIONS: Low concentrations of HgCl(2)affect immune function in human cells by dysregulation of cytokine signaling pathways, withthe potential to influence diverse health outcomes such as susceptibility to infectious disease orrisk of autoimmunity.LUTEOLIN AND THIOSALICYLATE INHIBIT HGCL(2) AND THIMEROSAL‐INDUCED VEGF RELEASEFROM HUMAN MAST CELLS. (ASADI 2010)Asadi S, Zhang B, Weng Z, Angelidou A, Kempuraj D, Alysandratos KD, Theoharides TC. Int JImmunopathol Pharmacol. 2010 Oct‐Dec;23(4):1015‐20.HgCl2 is a known environemental neurotoxin, but is also used as preservative in vaccines asthimerosal containing ethyl mercury covalently linked to thiosalicylate. We recently reportedScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201239 | P a g ethat mercury choloride (HgCl(2)) can stimulate human mast cells to release vascular endothelialgrowth factor (VEGF), which is also vasoactive and pro‐inflammatory. Here we show thatthimerosal induces significant VEGF release from human leukemic cultured LAD2 mast cells (at1 microM 326 ± 12 pg/106 cells and 335.5 ± 12 pg/106 cells at 10 microM) compared to controlcells (242 ± 21 pg/106 cells, n=5, p less than 0.05); this effect is weaker than that induced byHgCl2 at 10 microM (448 ± 14 pg/106 cells) (n=3, p less than 0.05). In view of this finding, wehypothesize that the thiosalicylate component of thimerosal may have an inhibitory effect onVEGF release. Thimerosal (10 microM) added together with the peptide Substance P (SP) at 2microM, used as a positive control, reduced VEGF release by 90 percent. Methyl thiosalicylate(1 or 10 microM) added with either SP or HgCl2 (10 microM) inhibited VEGF release by 100percent, while sodium salicylate or ibuprofen had no effect. Pretreatment for 10 min with theflavonoid luteolin (0.1 mM) before HgCl2 or thimerosal compeletly blocked their effect. Luteolinand methyl thiosalicylate may be useful in preventing mercury‐induced toxicity.INTERMINGLED MODULATORY AND NEUROTOXIC EFFECTS OF THIMEROSAL AND MERCURICIONS ON ELECTROPHYSIOLOGICAL RESPONSES TO GABA AND NMDA IN HIPPOCAMPALNEURONS. (WYREMBEK 2010)Wyrembek P, Szczuraszek K, Majewska MD, Mozrzymas JW. J Physiol Pharmacol. 2010Dec;61(6):753‐8.The organomercurial, thimerosal, is at the center of medical controversy as a suspected factorcontributing to neurodevelopmental disorders in children. Many neurotoxic effects ofthimerosal have been described, but its interaction with principal excitatory and inhibitoryneurotransmiter systems is not known. We examined, using electrophysiological recordings,thimerosal effects on GABA and NMDA‐evoked currents in cultured hippocampal neurons. Afterbrief (3 to 10 min) exposure to thimerosal at concentrations up to 100 μM, there was nosignificant effect on GABA or NMDA‐evoked currents. However, following exposure for 60‐90min to 1 or 10 μM thimerosal, there was a significant decrease in NMDA‐induced currents(p<0.05) and GABAergic currents (p<0.05). Thimerosal was also neurotoxic, damaging asignificant proportion of neurons after 60‐90 min exposure; recordings were always conductedin the healthiest looking neurons. Mercuric chloride, at concentrations 1 μM and above, waseven more toxic, killing a large proportion of cells after just a few minutes of exposure.Recordings from a few sturdy cells revealed that micromolar mercuric chloride markedlypotentiated the GABAergic currents (p<0.05), but reduced NMDA‐evoked currents (p<0.05).The results reveal complex interactions of thimerosal and mercuric ions with the GABA(A) andNMDA receptors. Mercuric chloride act rapidly, decreasing electrophysiological responses toNMDA but enhancing responses to GABA, while thimerosal works slowly, reducing both NMDAand GABA responses. The neurotoxic effects of both mercurials are interwoven with theirScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201240 | P a g emodulatory actions on GABA(A) and NMDA receptors, which most likely involve binding tothese macromolecules.THIMEROSAL‐DERIVED ETHYLMERCURY IS A MITOCHONDRIAL TOXIN IN HUMANASTROCYTES: POSSIBLE ROLE OF FENTON CHEMISTRY IN THE OXIDATION AND BREAKAGE OFMTDNA. (SHARPE 2012)Sharpe MA, Livingston AD, Baskin DS. J Toxicol. 2012;2012:373678. doi: 10.1155/2012/373678.Epub 2012 Jun 28.Thimerosal generates ethylmercury in aqueous solution and is widely used as preservative. Wehave investigated the toxicology of Thimerosal in normal human astrocytes, paying particularattention to mitochondrial function and the generation of specific oxidants. We find thatethylmercury not only inhibits mitochondrial respiration leading to a drop in the steady statemembrane potential, but also concurrent with these phenomena increases the formation ofsuperoxide, hydrogen peroxide, and Fenton/Haber‐Weiss generated hydroxyl radical. Theseoxidants increase the levels of cellular aldehyde/ketones. Additionally, we find a five‐foldincrease in the levels of oxidant damaged mitochondrial DNA bases and increases in the levelsof mtDNA nicks and blunt‐ended breaks. Highly damaged mitochondria are characterized byhaving very low membrane potentials, increased superoxide/hydrogen peroxide production,and extensively damaged mtDNA and proteins. These mitochondria appear to have undergonea permeability transition, an observation supported by the five‐fold increase in Caspase‐3activity observed after Thimerosal treatment.CYTOPROTECTIVE EFFECT OF HYALURONIC ACID AND HYDROXYPROPYL METHYLCELLULOSEAGAINST DNA DAMAGE INDUCED BY THIMEROSAL IN CHANG CONJUNCTIVAL CELLS. (YE2012)Ye J, Zhang H, Wu H, Wang C, Shi X, Xie J, He J, Yang J. Graefes Arch Clin Exp Ophthalmol. 2012Oct;250(10):1459‐66. doi: 10.1007/s00417‐012‐2087‐4. Epub 2012 Jun 24.BACKGROUND: To investigate genotoxicity of the preservative thimerosal (Thi), and thecytoprotective and antioxidant effects of hyaluronic Acid (HA) and hydroxypropylmethylcellulose (HPMC) on Chang conjunctival cells. METHOD: Cells were divided into three groups. One group was exposed to Thi at variousconcentrations (0.00001 %∼0.001 %) for 30 min; the other two groups were pretreated with0.3 % HA or 0.3 % HPMC for 30 min before the Thi exposure. After cell viability was evaluated,alkaline comet assay and detection of the phosphorylated form of the histone variant H2AX(γH2AX) foci were used to determine DNA damage. Reactive oxygen species (ROS) productionwas assessed by the fluorescent probe, 2', 7'‐dichlorodihydrofluorescein diacetate (DCFH‐DA).RESULTS: A significant change of cell viability was observed after exposure to 0.001 % Thi for 30Science Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201241 | P a g emin. DNA single‐ and double‐strand breaks were significantly increased in a dose‐dependentmanner with Thi exposure. In addition, intracellular ROS induced by Thi was dose‐dependent,except at 0.001 % less ROS was induced than at 0.0005 %. However, cells pretreated with 0.3 %HA or 0.3 % HPMC showed significantly increased cell survival, decreased DNA damage, anddecreased ROS production compared to cells exposed to Thi alone. Pretreatment with 0.3 % HAwas found to be even more protective than 0.3 % HPMC. CONCLUSION: Thi can induce DNAdamage in human conjunctival epithelial cells, probably due to oxidative stress. HA and HPMCare protective agents that have antioxidant properties and can decrease DNA damage inducedby Thi. Pretreatment of 0.3 % HA may be more protective of the ocular surface than 0.3 %HPMC.ITPKC SUSCEPTIBILITY IN KAWASAKI SYNDROME AS A SENSITIZING FACTOR FORAUTOIMMUNITY AND CORONARY ARTERIAL WALL RELAXATION INDUCED BY THIMEROSAL'SEFFECTS ON CALCIUM SIGNALING VIA IP3. (YETER 2012)Yeter D, Deth R. Autoimmun Rev. 2012 Oct;11(12):903‐8. doi: 10.1016/j.autrev.2012.03.006.Epub 2012 Apr 1.Recently, a single nucleotide polymorphism (SNP) of the inositol 1,4,5‐triphosphate kinase C(ITPKC), rs28493229, was found to passively confer susceptibility for Kawasaki syndrome (KS)and subsequent coronary arterial lesions. This association is believed to be the result ofdefective phosphorylation of inositol 1,4,5‐triphosphate (IP3), which releases calcium fromintracellular stores, resulting from reduced genetic expression of ITPKC in carriers of the SNP.Reduced ITPKC activity would increase IP3 levels, and thus, increase calcium release. Wehypothesized that an environmental agent which influences IP3‐mediated calcium release ispotentiated by the ITPKC SNP. This led us to an attractive candidate, thimerosal, anorganomercurial medical preservative still used in several pediatric vaccines. Thimerosal is well‐known to sensitize IP3 receptors via its induction of oxidative stress, resulting in enhancedrelease of intracellular calcium with distinctive consequences for various cell types.Dysregulated calcium signaling in T cells and other immune cells can result in autoimmunity,while hyperpolarization of vascular smooth muscle cells secondary to the stimulation ofcalcium‐activated potassium channels can result in increased vascular permeability and arterialrelaxation. We propose that ITPKC susceptibility in KS is related to its synergy withenvironmental triggers, such as thimerosal, which alter calcium homeostasis and promoteoxidative stress. Therefore, carriers of the ITPKC SNP are more susceptible to thimerosal‐induced autoimmunity and coronary arterial lesions observed in KS. This would explain whyonly a susceptible subset of children develops KS although pediatric thimerosal exposure isnearly universal due to vaccination. As was experienced with the infantile acrodynia epidemic,only 1 in 500 children developed the disease although pediatric mercury exposure was nearlyScience Summary on Mercury in Vaccines (Thimerosal Only)SafeMinds Update – December 201242 | P a g eubiquitous due to the use calomel teething powders. This hypothesis also mirrors the currentleading theory for KS in which a widespread infection only induces the disease in susceptiblechildren. We conclude that KS may be the acute febrile form of acrodynia.THIMEROSAL‐INDUCED APOPTOSIS IN MOUSE C2C12 MYOBLAST CELLS OCCURS THROUGHSUPPRESSION OF THE PI3K/AKT/SURVIVIN PATHWAY (LI 2012)Li WX, Chen SF, Chen LP, Yang GY, Li JT, Liu HZ, Zhu W. PLoS One. 2012;7(11):e49064. doi:10.1371/journal.pone.0049064. Epub 2012 Nov 7.BACKGROUND: Thimerosal, a mercury‐containing preservative, is one of the most widely usedpreservatives and found in a variety of biological products. Concerns over its possible toxicityhave reemerged recently due to its use in vaccines. Thimerosal has also been reported to bemarkedly cytotoxic to neural tissue. However, little is known regarding thimerosal‐inducedtoxicity in muscle tissue. Therefore, we investigated the cytotoxic effect of thimerosal and itspossible mechanisms on mouse C2C12 myoblast cells. METHODOLOGY/PRINCIPAL FINDINGS: The study showed that C2C12 myoblast cells underwent inhibition of proliferation andapoptosis after exposure to thimerosal (125‐500 nM) for 24, 48 and 72 h. Thimerosal caused Sphase arrest and induced apoptosis as assessed by flow cytometric analysis, Hoechst stainingand immunoblotting. The data revealed that thimerosal could trigger the leakage ofcytochrome c from mitochondria, followed by cleavage of caspase‐9 and caspase‐3, and that aninhibitor of caspase could suppress thimerosal‐induced apoptosis. Thimerosal inhibited thephosphorylation of Akt(ser473) and survivin expression. Wortmannin, a PI3K inhibitor, inhibitedAkt activity and decreased survivin expression, resulting in increased thimerosal‐inducedapoptosis in C2C12 cells, while the activation of PI3K/Akt pathway by mIGF‐I (50 ng/ml)increased the expression of survivin and attenuated apoptosis. Furthermore, the inhibition ofsurvivin expression by siRNA enhanced thimerosal‐induced cell apoptosis, while overexpressionof survivin prevented thimerosal‐induced apoptosis. Taken together, the data show that thePI3K/Akt/survivin pathway plays an important role in the thimerosal‐induced apoptosis inC2C12 cells. CONCLUSIONS/SIGNIFICANCE: Our results suggest that in C2C12 myoblast cells,thimerosal induces S phase arrest and finally causes apoptosis via inhibition ofPI3K/Akt/survivin signaling followed by activation of the mitochondrial apoptotic pathway
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