Scholarly article on topic 'The relationship between mercury and autism: A comprehensive review and discussion'

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Abstract of research paper on Clinical medicine, author of scientific article — Janet K. Kern, David A. Geier, Lisa K. Sykes, Boyd E. Haley, Mark R. Geier

Abstract The brain pathology in autism spectrum disorders (ASD) indicates marked and ongoing inflammatory reactivity with concomitant neuronal damage. These findings are suggestive of neuronal insult as a result of external factors, rather than some type of developmental mishap. Various xenobiotics have been suggested as possible causes of this pathology. In a recent review, the top ten environmental compounds suspected of causing autism and learning disabilities were listed and they included: lead, methyl-mercury, polychorinated biphenyls, organophosphate pesticides, organochlorine pesticides, endocrine disruptors, automotive exhaust, polycyclic aromatic hydrocarbons, polybrominated diphenyl ethers, and perfluorinated compounds. This current review, however, will focus specifically on mercury exposure and ASD by conducting a comprehensive literature search of original studies in humans that examine the potential relationship between mercury and ASD, categorizing, summarizing, and discussing the published research that addresses this topic. This review found 91 studies that examine the potential relationship between mercury and ASD from 1999 to February 2016. Of these studies, the vast majority (74%) suggest that mercury is a risk factor for ASD, revealing both direct and indirect effects. The preponderance of the evidence indicates that mercury exposure is causal and/or contributory in ASD.

Academic research paper on topic "The relationship between mercury and autism: A comprehensive review and discussion"

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Journal of Trace Elements in Medicine and Biology

ELSEVIER journal homepage: www.elsevier.com/locate/jtemb

Review

The relationship between mercury and autism: A comprehensive review and discussion

Janet K. Kerna b c'*, David A. Geiera c, Lisa K. Sykesc, Boyd E. Haley1d, Mark R. Geier

a Institute of Chronic Illnesses, Inc., 14 Redgate Court, Silver Spring, MD, 20905 USA b Council for Nutritional and Environmental Medicine, Mo i Rana, Norway c CoMeD, Inc., 14 Redgate Court, Silver Spring, MD, 20905 USA d University of Kentucky, 410 Administration Drive, Lexington, KY, 40506 USA

ARTICLE INFO

Article history: Received 21 March 2016 Received in revised form 17 May 2016 Accepted 1 June 2016

Keywords:

Autism spectrum disorders (ASD)

Autism

Mercury

Human studies

Relationship

ABSTRACT

The brain pathology in autism spectrum disorders (ASD) indicates marked and ongoing inflammatory reactivity with concomitant neuronal damage. These findings are suggestive of neuronal insult as a result of external factors, rather than some type of developmental mishap. Various xenobiotics have been suggested as possible causes of this pathology. In a recent review, the top ten environmental compounds suspected of causing autism and learning disabilities were listed and they included: lead, methyl-mercury, polychorinated biphenyls, organophosphate pesticides, organochlorine pesticides, endocrine disruptors, automotive exhaust, polycyclic aromatic hydrocarbons, polybrominated diphenyl ethers, and perfluorinated compounds. This current review, however, will focus specifically on mercury exposure and ASD by conducting a comprehensive literature search of original studies in humans that examine the potential relationship between mercury and ASD, categorizing, summarizing, and discussing the published research that addresses this topic. This review found 91 studies that examine the potential relationship between mercury and ASD from 1999 to February 2016. Of these studies, the vast majority (74%) suggest that mercury is a risk factor for ASD, revealing both direct and indirect effects. The preponderance of the evidence indicates that mercury exposure is causal and/or contributory in ASD.

© 2016 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY

license (http://creativecommons.org/licenses/by/4.0/).

Contents

1. Introduction .............................................................................................................................................. 9

2. Brain biomarkers and mercury levels in children with ASD.............................................................................................9

3. Human tissue mercury levels and ASD symptom severity.............................................................................................10

4. Body tissues studies that examine mercury levels in ASD vs. controls ................................................................................ 11

5. Porphyrin biomarkers of mercury body burden and ASD severity.....................................................................................11

6. Human tissue studies that show an increased susceptibility to mercury (or "pro-oxidant environmental toxins") in ASD.........................13

7. Epidemiological studies that examine Thimerosal in vaccines as a risk factor for ASD ............................................................... 14

8. Epidemiological studies that examine mercury in RhoGam as a risk factor for ASD..................................................................16

9. Epidemiological studies that examine mercury in the air as a risk factor for ASD .................................................................... 17

10. Epidemiological studies that examine mercury from other sources as a risk factor for ASD........................................................19

11. Discussion.............................................................................................................................................19

11.1. Mercurial compounds and toxicity............................................................................................................19

11.2. Other neurotoxicants..........................................................................................................................19

11.3. Brain pathology and susceptibility............................................................................................................19

11.4. Neurodevelopmental disorders in general....................................................................................................20

* Corresponding author at: Institute of Chronic Illnesses, Inc., 14 Redgate Court, Silver Spring MD, 20905 USA.

E-mail address: jkern@dfwair.net (J.K. Kern).

http://dx.doi.org/10.1016/jjtemb.2016.06.002

0946-672X/© 2016 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

11.5. Governmental policies and neurodevelopmental disorders in general.......................................................................20

12. Conclusion.............................................................................................................................................21

Author contributions...................................................................................................................................21

Conflicts of interest.....................................................................................................................................21

Acknowledgments......................................................................................................................................21

Appendix A. Supplementary data...................................................................................................................21

References..............................................................................................................................................21

1. Introduction

Autism spectrum disorders (ASD) is defined by persistent deficits in social communication and social interaction, and restricted, repetitive patterns of behavior, interests, or activities [1]. Although an ASD diagnosis is defined behaviorally by the American Psychiatric Association, other features, more physical or health related, are associated with an ASD diagnosis.

ASD is considered to be heritable with complex inheritance and genetic heterogeneity [2]; however, a consensus is emerging that the total fraction of ASD attributable to genetic inheritance may only be 30-40% [3]. Chromosomal microarray testing reveals that approximately 80% of children with ASD have a normal genome [4]. Of the remaining 20%, approximately half of those have various polymorphisms of unknown significance and the other half of those have de novo mutations with little or no commonality. These findings suggest that non-genetic factors have a significant role in the etiology of ASD.

In addition, many brain pathology studies indicate marked and ongoing neuroinflammation in ASD [5-14]. This type of reactive pathology is suggestive of insult and with concomitant neuronal damage [15] rather than some type of developmental mishap as has been suggested [16,17]. A developmental mishap does not explain the evidence of neuroinflammatory reactivity and neuronal damage within the brain in ASD which includes: (1) activated microglia (immune macrophages within the brain); (2) activated astrocytes (a broad class of cells that support neurons within the brain); (3) elevated levels of glial fibrillary acidic protein (GFAP; an intermediate filament protein that is expressed by astrocytes possibly to maintain structural integrity, known to be upregulated in response to injury); (4) increased oxidative stress (e.g., elevated neurotrophin-3, elevated 3-nitrotyrosine, and oxidized glutathione levels, etc.); (5) elevated levels of 8-oxo-guanosine (a product of oxidative damage to DNA); (6) elevated proinflammatory cytokines (e.g., tumor necrosis factor alpha, interleukin 6, and granulocyte-macrophage colony-stimulating factor); (7) aberrant expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB, a protein complex that regulates transcription and reflects the cellular response to stress); and (8) neuronal cell loss [8,15,18-21]. Nor does it explain the classic regression found in autism that occurs around 15-22 months of age where these children lose previously acquired neurological function, such as language and other interactive skills and abilities [22].

Various xenobiotics have been suggested as causal agents in the pathology of ASD. In a highly-cited review, Grandjean and Lan-drigan [23] identified five industrial chemicals as developmental neurotoxicants based on epidemiological evidence: lead, methyl-mercury, polychlorinated biphenyls, arsenic, and toluene. In an Environmental Health Perspectives editorial, Landrigan et al. [3] note that neurodevelopmental disabilities affect over 10% of children born in the US each year and listed the top ten environmental compounds suspected of causing autism and learning disabilities: lead, methylmercury, polychorinated biphenyls, organophosphate pesticides, organochlorine pesticides, endocrine disruptors such as

phthalates, automotive exhaust, polycyclic aromatic hydrocarbons, polybrominated diphenyl ethers (brominated flame retardants), and perfluorinated compounds. Both teams specify "methylmercury" rather than the broader class "mercury", possibly because more studies exist on the methylmercury form (found in fish), and possibly because ethylmercury (found in Thimerosal-containing vaccines) and mercury vapor (released from dental amalgams) are unpopular targets.

Of the numerous studies that have been conducted over the last three decades that examine the relationship between mercury exposure and ASD, the majority of the studies found that mercury is a risk factor for ASD. However, there are also several studies that suggest mercury is not a risk factor for ASD, therefore evaluating the totality of the evidence is not easy.

This review will focus on mercury exposure and ASD by conducting a comprehensive literature search of original studies in humans that examine the potential relationship between mercury and ASD from 1999 to February 2016, including studies of human tissue levels of mercury, studies of biomarkers for mercury exposure, and epidemiological studies. The literature search includes published original research studies on mercury and ASD, from PubMed and Google Scholar; however, references cited in identified publications were also searched to locate additional studies. Search words included: autism, autism spectrum disorders, ASD, pervasive developmental disorders, PDD, mercury, Hg, Thimerosal, metals, methyl-mercury, ethyl-mercury, inorganic-Hg, mercury chloride.

This review will categorize, summarize, and discuss the published research that addresses this topic. Each section of this paper will present an area of scientific inquiry on the issue and the studies which have been published on it. An associated table(s) in each section will briefly describe the pertinent studies and their findings. This review will begin with studies that examine brain biomarkers and mercury levels in children with ASD.

2. Brain biomarkers and mercury levels in children with ASD

Many studies show production of numerous auto-antibodies which react with specific brain proteins and brain tissues in children with ASD. These auto-antibodies can also act to alter the function of the respective brain tissue [24]. In addition, studies show that anti-brain antibodies are associated with more severe cognitive and behavioral profiles in children with ASD [25]. Moreover, recent studies (see Table 1 ) have found that certain brain auto-antibodies correlate with mercury levels in children with ASD [26,27].

This finding is biologically plausible since studies show that mercury exposure, especially to the mercury-based compound Thimerosal, can cause autoimmune dysfunction. For example, Voldani et al. [28] conducted a study that demonstrated certain dietary peptides, bacterial toxins, and xenobiotics, such as Thimerosal, can bind to lymphocyte receptors and/or tissue enzymes, resulting in autoimmune reaction in children with autism. Havarinasab et al. [29] also found that Thimerosal can induce (in genetically susceptible mice) a systemic autoimmune syndrome.

Table 1

Studies that show brain biomarkers correlate with mercury levels in children with ASD.

Biomarker Authors and Journal N Age Purpose ofStudy Findings

Brain Tissue Mostafa and Al-Ayadhi [26] ASD 40 Serum antineuronal Higher seropositivity for

Autoantibodies Egypt. J. Pediatr. Allergy Controls 40 antibodies and blood antineuronal antibodies and

(antineuronal antibodies) Immunol. 3-8 y mercury levels were estimated between autism and controls higher blood mercury in autism vs. controls. Seropositivity of antineuronal antibodies had positive association with elevated blood mercury (found in 70% of autistic children). Both markers positively associated with behavioral abnormalities, autistic regression, EEG abnormalities

Brain Tissue Mostafa and Refai [27] ASD 50 Blood mercury levels and Serum levels of blood mercury

Autoantibodies Clin. Cell. Immunol. Controls 30 seropositivity ofanti-MBP were significantly higher in

(anti-MBP auto-antibodies) 5-12 y autoantibodies in autistic children autistic children than healthy controls; increased levels of blood mercury were found in 48% of autistic patients, and 72% of autistic children had anti-MBP auto-antibodies. There was a significant positive association between the elevated levels of blood mercury and anti-MBP auto-antibodies in autistic children.

Brain Neuropeptides in Mostafa et al. [30] ASD 84 Examined significant and positive linear

Serum Metab. Brain Dis. Controls 84 pro-inflammatory relationship between levels of

3-10 y neuropeptides and mercury in serum serum neurokinin A and blood mercury in moderate and severe ASD, but not controls

Brain Oxidative Stress - Sajdel-Sulkowska et al. [31] ASD 9 Oxidative stress marker Significant increases in the

3-NT Am. J. Biochem. Biotechnol. Controls 10 3-NT, mercury, and the mean cerebellar levels of 3-NT

5-37 y antioxidant selenium in autism and controls and in the ratio of mercury/selenium in the brains of subjects diagnosed with autism when compared to controls; there was a significant dose-dependent positive correlation between oxidative stress markers and total mercury levels

ASD = autism spectrum disorders; anti-MBP = anti myelin basic protein; 3-NT=3-nitrotyrosine; EEG = electroencephalogram; y = years of age.

In a recent study, Mostafa et al. [30] found a significant and positive linear relationship between levels of serum neurokinin A (a pro-inflammatory neuropeptide) and blood mercury levels in children with moderate and severe ASD, but not in healthy control children. They found that 78.3 % of the children with ASD with increased serum levels of neurokinin A had elevated blood mercury levels.

In addition, Sajdel-Sulkowska et al. [31] reported that mercury levels in the cerebellar areas of the brain correlate with the oxidative marker neurotrophin-3 (NT-3) in the brains of those with a diagnosis of ASD. In contrast, Khan et al. [32] measured brain levels of mercury and the oxidative stress marker, 3-nitrotyrosine (3-NT) in both male and female control and ASD cases, age 4-16 years. The researchers found that although 3-NT was increased in overall ASD, mercury levels measured only in the extracortical regions (brain stem and cerebellum) were not different between cases and controls (see Table 2 ). This finding may suggest that the same levels of mercury may promote oxidative stress only in susceptible individuals.

Pamphlett and Kum Jew [33] examined the human locus ceruleus, a region of the brain that has been implicated in ASD. They found that controls were significantly more likely to have mercury in their locus ceruleus than the individuals with ASD. It is difficult to interpret the meaning of that finding, so that study was catego-

rized as not showing a relationship between mercury and ASD and placed in Table 2.

3. Human tissue mercury levels and ASD symptom severity

This section looks specifically at human tissue studies which examine the relationship between mercury and symptom severity in ASD. In the studies that examine blood (whole blood and red blood cells) and nails, results show that the higher the mercury levels, the worse the autism symptoms. However, hair levels are not simple to interpret. The first study showing a relationship between autism severity and hair mercury levels was published by Holmes et al. [34]. They originally hypothesized that the higher the mercury levels in the hair, the greater the autism severity (due to greater exposure). However, they found that the more severely affected the child was, the lower the hair mercury levels. When the researchers found that hair mercury levels in the children with autism were lower than controls (to be discussed more in the following section), they proposed the "poor excretor theory" suggesting that children with autism have more difficulty excreting mercury than typically developing children and are more prone to accumulate the mercury. Other studies, however, have found that the higher the hair mercury concentrations, the worse the autism symptoms [35]. In addition, evidence suggests that the time of hair growth analysis is likely important because younger children with ASD show lower

Table 2

Studies that did not show brain mercury levels were different in children with ASD vs. controls.

Biomarker Authors and Journal N Age Purpose of Study Findings

Brain Khan et al. [32] ASD 10 Brain mercury levels Brain mercury levels

J. Physiol. Pharmacol. Controls 11 measured in extracortical measured in extracortical

4-16 y regions autism vs. controls regions in children with

autism vs. controls were

not different

Brain- Pamphlett and Kum Jew ASD 6 Levels on mercury in the Higher levels of mercury

Locus ceruleus [33] Controls 11 locus ceruleus were found in the controls

16-48 y

ASD = autism spectrum disorders; y=years of age.

Table 3

Studies that show that human tissue mercury levels are associated with ASD symptom severity.

Body Tissue Authors and Journal N Age Purpose of Study Findings

Hair Holmes et al. [34] ASD 94 Relationship between autism Mercury levels inversely

Int J Toxicol Controls 45 1-2 y and hair mercury levels correlated with symptom severity

Hair and nails Lakshmi Priya and Geetha [35] ASD 45 Lead and mercury in hair and The elevation was much

Biol. Trace Elem. Res. Controls 50 4-12 y nails autism vs. controls pronounced in LFA group subjects when compared among autistic groups MFA and HFA

Hair Elsheshtawy et al. [37] ASD 32 Investigated the relationship Positive correlation of CARS

Middle East Curr. Psychiatry Controls 32 between autism and mercury, scores with mercury from hair

Children lead, copper, zinc samples

Hair Geieretal. [38] ASD 18 Hair toxic metal concentrations Increasing hair mercury

Int. J. Environ. Res. Public 1-6 y and ASD severity concentrations significantly

Health correlated with increased ASD severity

Whole blood and RBC Adams et al. [39] ASD 51 Investigated toxic metals in Found a strong association in

Biol. Trace Elem. Res. Controls 40 autism and autism severity in the degree of severity of

3-15 y whole blood, RBCs, and urine autism for all the severity scales with mercury (whole blood and RBC)

Red Blood Cells (RBC) Alabdali et al. [40] ASD 30 Concentration of lead and Levels of mercury GST, and

Behav. Brain Funct. Controls 30 mercury were measured in red vitamin E were correlated with

3-12y blood cells, plus GST and vitamin E severity of social and cognitive impairment measures

GST=glutathione-s-transferase; CARS = Childhood Autism Rating Scale; ASD = autism spectrum disorders; ATEC = Autism Treatment Evaluation Checklist; LFA = low functioning autism; MFA = mid functioning autism; HFA= high functioning autism; RBC = red blood cells; y=years of age.

levels of hair mercury, while older children with ASD show higher levels than their respective controls [36]. These findings suggest two competing variables: (1) susceptibility (poor excretors) and (2) exposure (higher exposure). Furthermore, these two variables may change over time, as detoxification pathways become blocked or cleared, and as exposures change, such that an absence of clear associations between tissues levels and symptoms would be unsurprising. Nonetheless, based on the studies reviewed, some trends can be observed. Table 3 lists human tissue (hair, nails, blood) studies that show a correlation between tissue mercury levels and ASD symptom severity [34,35,37-40].

No studies were found during our literature search that examined tissue mercury levels and autism severity that did not find a correlation. Thus, none are presented.

4. Body tissues studies that examine mercury levels in ASD vs. controls

This section differs from the previous section because it does not address symptom severity, but rather whether children with ASD have different levels of mercury in tissues than do control children. The majority of studies find differences in mercury levels between children with ASD and healthy controls [34-37,40-52]. These studies are presented in Table 4. However, many do not find differences [56-63]. These studies are presented in Table 5 .

Worth noting is that the studies in Table 6 which compare the mercury levels of children with ASD versus children with other neu-rodevelopmental disorders show no differences in mercury levels [64-66]. In many instances neurodevelopmental disorders are difficult to clearly separate, because of frequent overlap in core and associated features. This finding suggests that it is difficult to separate these children based on biomarkers and/or xenobiotic levels as well.

5. Porphyrin biomarkers of mercury body burden and ASD severity

Studies have shown that urinary porphyrins (heme precursors formed in the heme synthesis pathway) can afford a measure of xenobiotic exposure and of tissue toxic metal body-burden, particularly with respect to mercury [67-69]. Mercury toxicity has been demonstrated to be associated with elevations in urinary coproporhyrin (cP), pentacoproporphyrin (5cxP), and an atypical porphyrin, called precoprpophyrin (prcP) which is not found in the urine of unexposed controls. As such, prcP is considered to be a specific porphyrin marker for mercury exposure [67-69], and elevated urinary prcP is suggestive of mercury body burden [70]. The higher the urinary prcp levels, the higher the mercury body burden.

Six of the seven studies in this section found a relationship between the porphyrin biomarkers of mercury body burden and ASD severity (Table 7 ) [63,61-76]. However, one did not find a dif-

Table 4

Human Tissue Studies that Show Significantly Different Mercury Levels in ASD vs. Healthy Controls.

Body Tissue Authors and Journal N Age Purpose of Study Findings

Hair Holmes et al. [34] ASD 94 Relationship between The level of mercury was significantly

Int. J. Toxicol. Controls 45 autism and first baby hair lower in cases 0.47 ppm versus

1-2 y mercury levels 3.63 ppm in controls

Hair and nails Lakshmi Priya and Geetha [35] ASD 45 Lead and mercury in hair Significant elevation in the levels of

Biol. Trace Elem. Res. Controls 50 and nails autism vs. mercury in both hair and nail samples

4-12 y controls in autism vs. controls

Hair Majewska et al. [36] ASD 91 Levels of hair mercury in Autistic children significantly differed

Acta Neurobiol Exp Controls 75 autism vs. controls from healthy peers in the

3-9 y concentrations of mercury in hair

Hair Elsheshtawy et al. [37] ASD 32 Investigated the The level of mercury was significantly

Middle East Curr. Psychiatry Controls 32 relationship between lower in cases (0.55 ± 0.06 |xg/mg)

3-4 y autism and mercury, lead, than in controls (3.2 ± 0.2 |xg/mg)

copper, zinc

Hair Mohamed et al. [41] ASD 100 Assess the levels and Hair mercury levels were higher in

Behav. Neurol. Controls 100 possible environmental children with ASD than controls

2-15 y risk factors from metals

Hair Tabatadze et al. [42] ASD 30 Evaluation levels of High contamination to mercury in ASD

Georgia Med. News Controls 30 essential trace elements children compared to controls

4-5 y and heavy metals in ASD

vs. controls

Hair Adams et al. [43] ASD 78 First baby haircuts Children with lower levels of mercury

Toxicol. Environ. Chem. Controls 31 evaluated for mercury and in hair were 2.5 times more likely to

Born between 1988 and 99 ASD have ASD

Hair DeSoto and Hitlan [44] ASD 82 Examine mercury levels in Significant relation does exist between

J. Child Neurol. Controls 55 hair and blood the blood levels of mercury and ASD

ASD mean 7.2 y with ASD having higher blood mercury

Controls mean 7.8 y levels

Hair Blaurock-Busch et al., [45] ASD 44 Assessed the levels of ten Elevated hair concentrations were

Maedica (Buchar) 3-9 y toxic metals and essential noted for mercury in autism vs.

elements in hair samples of controls

children with autism

Hair Hodgson et al. [46] ASD 27 Investigated hair mercury Mercury levels were markedly

Exp. Biol. Med. (Maywood) Controls 27 levels in autism and elevated in the hair of autistic subjects

ASD mean 5.3 y Controls mean 5.5 controls vs. control subjects

Hair Obrenovich et al. [47] y ASD 22 Hair toxic metals in autism Significant alteration in deposition of

Biol. Trace Elem. Res. Controls 39 vs. controls several heavy metal species, including

<6y mercury in hair samples between the

groups

Hair Al-Ayadhi 2005 [48] ASD 77 Hair metals in autism vs. Higher levels of mercury in ASD vs.

Neurosciences (Riyadh) Controls 77 controls controls

Hair Fido and Al-Saad [49] <14 y ASD 40 Toxic metals in the hair of Children with autism had significantly

Autism Controls 40 children with autism vs. (p < 0.001) higher in-hair concentration

4-8 y controls levels of lead, mercury and uranium.

Blood and hair Yassa [50] ASD 45 Blood and hair samples High level of mercury and lead among

Environ. Toxicol. Pharmacol. Controls 45 from 45 children from those kids with autism, with

2-10 y Upper Egypt with autism significant decline in the blood level of

vs. controls lead and mercury with the use of

DMSA as a chelating agent

Red Blood Cells (RBC) Alabdali et al. [40] ASD 20 Concentration of lead and ASD had significantly higher lead and

Behav. Brain. Funct. Controls 20 mercury were measured in mercury levels and lower GST activity

3-15 y red blood cells, plus GST and vitamin E concentrations

and vitamin E compared to controls

RBC Geieretal. [51] ASD 83 Mercury levels in children Mean mercury levels were 1.9-fold

Acta Neurobiol. Exp. (Wars) Controls 89 with ASD vs. controls significantly increased in ASD (21.4

ASD mean 7.3 y microg/L) vs. controls (11.4 microg/L).

Controls mean 11.4 y

Blood (plasma) El-Ansary [52] ASD 20 Mercury levels Blood mercury levels were higher in

Data in Brief Controls 20 ASD

3-15 y

Urine Bradstreet et al. [53] ASD 221 Children with ASD and Children with ASD excreted six-fold

J. Am. Phys. Surg. Controls 18 controls treated with greater mercury than controls in their

3-16 multiple doses of DMSA urine

Urine Blaurock-Busch et al. [54] ASD 44 Exposure to mercury and Statistically significant differences in

Maedica (Buchar) Controls 146 other heavy metals in the mean urine levels of mercury

3-9 y children with autism

spectrum disorders versus

controls

Baby Teeth Adams et al. ASD 15 Level of mercury, lead, and Children with autism had significantly

J. Toxicol. Environ. Health A [55] Controls 11 zinc in baby teeth in (2.1-fold) higher levels of mercury

4-9 y autism vs. controls

RBC+ red blood cells; GST=glutathione-s-transferase; DMSA = 2,3-dimercaptosuccinic acid; y=years of age.

Table 5

Body tissue studies that did not find significantly different mercury levels in ASD vs. healthy controls.

Type of Study Authors and Journal N Purpose ofStudy Findings

Hair De Palma et al. [56] ASD 44 Hair toxic metals in autism vs. Found no association between

J. Aut. Dev. Disord. Controls 61 controls autism and hair mercury

Children

Hair and blood Ipetal. [57] ASD 82 Hair and blood mercury levels No difference in the mean

J. Child. Neurol. Controls 55 and autism mercury levels

ASD mean 7.2 y

Controls mean 7.8 y

Blood Hertz-Picciotto et al. [58] ASD 452 Blood mercury levels in autism After accounting for dietary

Environ. Health Perspect. 2-5 y vs. controls and other differences in

mercury exposures, total

mercury in blood not

statistically different

Blood Yau et al. [59] ASD 84 Prenatal and early-life Total mercury in serum

Environ. Res. Controls 159 exposures to mercury collected from mothers during

<1y mid-pregnancy and newborn

bloodspots were not

significantly associated with

Blood Rahbar et al. [60] ASD 109 Investigate the association Did not find a significant

Neurotox. Res. Controls 109 between blood mercury difference (P = 0.61) between

2-8 y concentrations in control blood mercury concentrations

children and ASDs and ASDs

Blood/toxicokinetic model McKean etal. [61] ASD 164 Using methyl-mercury Cumulative methyl-mercury

Environ. Health DD 35 concentration estimates from exposure does not appear to

Controls 58 toxicokinetic model, detectably elevate the risk of

Mother infant pairs 274 methyl-mercury exposure autism or developmental delay

<1 y estimated in autism, DD, and

controls

Urine Soden et al. [62] ASD 15 24-h provoked urine excretion Excess chelatable body burden

Clin. Toxicol. (Phila) Controls 4 test for heavy metals in ofAs, Cd, Pb, or mercury is zero

3-7 y children with autism

Urine Woods et al. [63] ASD 64 Mean mercury levels were No differences were found

Environ. Health Perspect. PDD 19 evaluated between autism, between NT and autism in

Controls 114 PDD, and NT urinary mercury levels or in

2-12 y past mercury exposure

DD = developmental delay; NT = neurotypical; PDD = pervasive developmental disorder; As = arsenic; Cd = cadmium; Pb = lead; y=years of age.

Table 6

Body Tissue Studies that did not Find Significantly Different Mercury Levels in ASD vs. Children with Neurological Disorders, Neuropsychiatric Disorders, or Learning

Disabilities.

Type of Study Authors and Journal N Purpose of Study Findings

Blood Macedoni-Luksic et al. [64] ASD 52 Levels of metals in blood No significant difference in

Biol. Trace Elem. Res. Other neurologic disorders 22 aluminum, lead, mercury in blood levels of metals between

1-16y ASD compared to children with the groups was found

neurological disorders

Blood, urine and hair Albizzati et al. [65] ASD 17 Metals in blood, urine and hair No difference was found

Przegl. Epidemiol. Neuropsychiatric disorders 17 samples from children with between children with autism

6-16 y autism and children with and children with

neuropsychiatric disorders, neuropsychiatric disorders,

unspecified unspecified

Urine Wright et al. [66] ASD 56 Urinary mercury levels No statistically significant

PloS One Siblings 42 between children with ASD differences were found

Controls 121 and controls- normal and with between children with ASD

Delayed 34 learning disabilities and controls

5-16 y

ASD = autism spectrum disorders; y=years of age.

ference between the ASD cases and controls or a relationship with ASD severity (see Table 8 ) [77].

6. Human tissue studies that show an increased susceptibility to mercury (or "pro-oxidant environmental toxins") in ASD

Earlier in Section 2, the issue of susceptibility to mercury in ASD was mentioned. Many studies suggest that children with ASD represent a population vulnerable to the adverse effects of mercury [78]. This section covers studies which examine susceptibility

to mercury in ASD, listed in Table 9 . These studies use a variety of tissues, including brain tissue, lymphoblastoid cell lines (LCLs), and blood samples [52,79-92]. A major focus is the transmethyla-tion/transsulfuration concentrations, which are consistently found to be abnormal in ASD [52,78,81-88,90]. Several of these studies suggest that children with ASD have limited thiol availability and decreased glutathione (GSH) reserve capacity, resulting in a compromised detoxification capacity and increased oxidative stress [52,78,81-88,90]. Four of these studies are on brain tissue, showing increased susceptibility to toxic substances in the brains of subjects with ASD. Most of these studies show susceptibility to

Table 7

Studies that Show that Mercury Body Burden Biomarkers Correlate with ASD Severity.

Biomarker Authors and Journal N Age Purpose of Study Findings

Heme Synthesis Pathway Natafet al. [71] ASD 106 Examined urinary porphyrin The atypical molecule

Metabolite Toxicol. Appl. Pharmacol. Controls 163 levels in children with precoproporphyrin, a specific

(precoproporphyrin) 2-15 y neurodevelopmental disorders indicator of heavy metal

toxicity, was also elevated in

autistic disorder but not

significantly in Asperger's

Heme Synthesis Pathway Geier and Geier [72] ASD 37 Examined urinary porphyrin An apparent dose-response

Metabolite Neurotox. Res. 7-22 y pattern indicative of mercury effect was observed between

(coproporphyrins) toxicity autism severity and increased

urinary coproporphyrins

Heme Synthesis Pathway Geier etal. [73] ASD 26 Urinary porphyrins and Mercury

Metabolites J. Neurol. Sci. 2-13 y transsulfuration metabolites in intoxication-associated urinary

(pentacarboxyporphyrin, ASD were examined porphyrins were significantly

precoproporphyrin, and correlated with increasing

coproporphyrin) CARS scores and GSSG levels.

Heme Synthesis Pathway Geier etal. [74] ASD 71 Evaluated relationship Participants with severe ASD

Metabolites J. Toxicol. Environ. Health A Controls 14 between ASD severity and had significantly increased cP I,

(coproporphyrins) ASD 3-22 y urinary porphyrins cP III, and total cP levels in

Controls 3-59 y comparison to participants

with mild ASD. A significant

correlation was observed

between increasing cP levels

and CARS scores.

Heme Synthesis Pathway Kernet al. [75] ASD 24 Urinary porphyrins and The results of the study

Metabolites Biometals 2-13 y specific domains of the ATEC indicated that the overalll

(pentacarboxyporphyrin, ATEC scores and each of the

precoproporphyrin, and ATEC subscales

coproporphyrin) (Speech/Language, Sociability,

Sensory/Cognitive Awareness,

Health/Physical/Behavior)

were linearly related to urinary

porphyrins associated with

mercury toxicity.

Heme Synthesis Pathway Heyer etal. [76] ASD 30 Evaluated penta and ASD and PDD childrenhad

Metabolite (pentacarboxyl Autism Res. PDD14 coproporphyrins as biological higher mean urinary penta and

(penta) and coproporphyrins) Controls 32 indicators of ASD, PDD-NOS, copro-porphyrin

2-12 y neurotypical (NT) controls concentrations compared with

same-aged NT children.

Combined Z-score measure

had 33% and 21% sensitivity for

autism and PDD-NOS,

respectively, with 100%

specificity.

Heme Synthesis Pathway Woods et al. [63] ASD 100 Mean porphyrin and mercury Elevated copro-, hexacarboxyl-

Metabolite pentacarboxyl-, Environ. Health Perspect. PDD 27 levels were evaluated between and pentacarboxyl- porphyrin

precopro- and Controls 117 autism, PDD and NT concentrations were associated

copro-porphyrins 2-12 y with autism but not PDD-NOS.

cP = coproporphyria cP l = coproporphyrin I; cP lll = coproporphyrin III; penta= pentacarboxyl ASD = autism spectrum disorders; ATEC=Autism Treatment Evaluation Checklist; GSSG = oxidized glutathione; NT = neurotypical; PDD-NOS = pervasive developmental disorder -not otherwise specified; y=years of age.

Table 8

Studies that did not Show that Mercury Body Burden Biomarkers Correlate with ASD Severity.

Biomarker Authors and Journal N Age Purpose of Study Findings

Heme Synthesis Pathway Shandley et al. [77] ASD 70 Investigated whether Analyses did not to find

Metabolite Autism Res. Siblings 36 porphyrin profiles can reliably support for the hypotheses

Controls 54 be used to (a) differentiate ASD that porphyrin levels could

2-6 y cases from healthy controls; be used as a valid tool to

and (b) predict ASD severity detect ASD cases or predict

severity

ASD = autism spectrum disorders; y=years of age.

mercury because they show a suboptimal detoxification capacity, however, some of the studies show direct evidence of increased mercury damage in individuals with autism as compared to controls [79-81,89].

7. Epidemiological studies that examine Thimerosal in vaccines as a risk factor for ASD

One of the most controversial areas of study is the epidemiological investigation of mercury in Thimerosal - containing vaccines (TCVs) as a risk factor for ASD. The controversial nature of these studies is reflected by the fact that most of the studies that are conducted without public health and/or industry support arrive

Tissue Studies that Examine Susceptibility to Mercury (or "pro-oxidant environmental toxins") in ASD.

Body Tissue/Substance

Authors and Journal

Purpose of Study

Findings

Cerebellum and temporal cortex

Cerebellum

Plasma

Plasma

Plasma

Plasma

B-lymphocytes

Rose et al. [79] J. Toxicol.

Rose et al. [80] Transl. Psychiatry

James et al. [81] FASEBJ.

Rose et al. [82] Transl. Psychiatry

Chauhanetal. [83] Neurochem. Res.

Guetal. [84] Free Radic. Biol. Med.

Fryeet al. [85] Transl. Psychiatry

Geieretal. [86] J. Neurol. Sci.

James etal. [87] Am. J. Clin. Nutr.

James et al. [88] Am. J. Med. Genet. B Neuropsychiatr. Genet.

Sharpe et al. [89] J. Toxicol.

Peripheral blood mononuclear cells Rose et al. [90]

Autism Res. Treat.

Human serum albumin Blood RNA, Gene Expression Blood (plasma)

Vojdanietal. [91]

Int. J. Immunopathol. Pharmacol.

Stamova et al. [92] Neurotox. Res.

El-Ansary [52] Data in Brief

ASD 16 Controls 16 ASD 5-13 y Controls 5-37 y ASD 22 Controls 14 ASD mean 7.8 y Controls mean 27.7 y ASD 10 Controls 10 ASD mean 7.8 y Controls mean 27.7 y ASD 15 Controls 15 ASD 4-39 y Controls 4-36 y ASD 10 Controls 10

4-39 y ASD 10 Controls 10 ASD mean 11.1 y Controls mean 11.3 y ASD 18

Controls 18 ASD mean 8.5 y Controls mean 8.8 y ASD 28 2-16 y

ASD 40 Controls 42

ASD 80 Controls 73

3-14 y

ASD 4 Controls 4

5-13 y ASD 43 Controls 41 3-10 y ASD 50 Controls 50 3-14 y ASD 33 Controls 51

2-5 y ASD 20 Controls 20

3-15 y

Human LCL in autism vs. controls exposed to TM

Human LCL in autism and mitochondrial reserve capacity

LCLs derived from autistic children and controls, effects of TM on and GSH levels

Examined cerebellum and temporal cortex (Brodmann area 22(BA22))

Compared GSH redox status in postmortem brain samples from cerebellum and frontal, temporal, parietal and occipital cortex of subjects with ASD vs. controls Activities of GSH-related enzymes in the cerebellum tissues from autism and controls

Plasma markers of oxidative stress and measures of cognitive and language development and ASD behavior

Examined plasma transsulfuration metabolites

Plasma concentrations of

transmethylation/transsulfuration metabolites and glutathione redox status in autistic children

Plasma concentrations of

transmethylation/transsulfuration metabolites and glutathione redox status in autistic children

TM exposure in B-lymphocytes taken from individuals with autism, their nonautistic twins, and their nontwin siblings

Quantified the intracellular glutathione redox couple (GSH/GSSG) in resting peripheral blood mononuclear cells

Measured IgG, IgM and IgA antibodies against CD26, CD69, streptokinase, gliadin and casein peptides and against ethyl mercury bound to human serum albumin in autism Correlations between gene expression and mercury levels in blood of boys with and without autism

Levels of GSH/GSSG

Autism LCLs exhibited greater reduction in ATP-linked respiration, maximal respiratory capacity, and reserve capacity, compared to control LCLs exposed to TM

Depletion of reserve capacity making them more vulnerable to pro-oxidant environmental toxins

TM resulted in greater decrease in GSH/GSSG ratio and increase in free radical generation in autism vs. control cells

GSH was significantly decreased in both the cerebellum and BA22 in autism vs. controls; decreased GSH/GSSG redox/antioxidant capacity and increased oxidative stress in the autism brain

Levels of reduced GSH were significantly decreased in autism compared to controls: redox ratio of GSH to GSSG was also significantly decreased

GPx, GST, GR, and GCL activity were significantly decreased in autism compared to that of the control group.

ASD groups demonstrated lower fGSH and fGSH/GSSG

Decreased plasma levels of reduced glutathione (GSH), cysteine, and sulfate were observed among study participants relative to controls Transmethylation/transsulfuration pathway concentrations in autistic children were significantly different from values in the control; and decreased glutathione redox status in autism vs. controls Plasma levels of cysteine, glutathione, and the ratio of reduced to oxidized glutathione, an indication of antioxidant capacity and redox homeostasis, were significantly decreased

Exposure to TM resulted in four of the families showed thimerosal hypersensitivity, whereas none of the control individuals displayed this response Both glutathione and cysteine redox ratios were decreased in autistic compared to control children

TM binds to lymphocyte receptors and/or tissue enzymes, resulting in autoimmune reaction in children with autism

Findings suggest different genetic transcriptional programs associated with mercury levels in autism compared to controls Blood GSH levels were lower in ASD

LCL = lymphoblastoid cell lines; TM =Thimerosal; GSH = reduced glutathione; fGSH = reduced free glutathione; GSSG = oxidized glutathione; GPx=glutathione peroxidase; GST=glutathione-S-transferase; GR=glutathione reductase; GCL=glutamate cysteine ligase; y = years of age.

Table 10

Epidemiological studies that show thimerosal in vaccines is a risk factor for ASD.

Type ofStudy/Source of Exposure Authors and Journal Database Age or Time Frame Purpose ofStudy Findings

Epidemiology Gallagher and Goodman [93] NH1S database Examine NH1S 1997-2002, boys U.S. male neonates vaccinated

TCVs J. Toxicol. Environ. Health A 3-17 y 3-17 years old, born before 1999 with the hepatitis B vaccine

and exposure to TM priorto 1999 (from vaccination

record) had a threefold higher

risk for parental report of

autism diagnosis compared to

boys not vaccinated as

neonates during that same

time period

Epidemiology Gallagher and Goodman [94] NHANES database Examine National Health and Boys given Thimerosal (Hep B)

TCVs Toxicol. Environ. Chem. 1-9y Nutrition Examination Survey more susceptible to

1999-2000, children aged 1-9 developmental disability than

years unvaccinated boys

Epidemiology Young et al. [95] VSD database Ecological study of TM containing Increased risk of an ASD

TCVs J. Neurosci. Birth to 13 months vaccines and risk of NDs diagnosis with TCVs

Epidemiology Geier and Geier [96] VAERS database Dose (50 vs. 25 micrograms) of 1ncreased odds ratios for

TCVs J. Toxicol. Environ. Health A 1994-1998 mercury from TM in VAERS autism with higher doses of TM

Epidemiology Geier and Geier [97] VAERS database Association between TCVs DTaP Exposure to mercury from

TCVs Med. Sci. Monit. 1997-2001 comparison to TM-free DTaP and TCVs administered in the US

VSD database autism in VAERS and VSD was a consistent significant

1992-1997 risk factor for autism

Epidemiology Geier and Geier [98] VAERS database Dose of TCVs and autism in VAERS Dose-response curves showed

TCVs Pediatr. Rehabil. USDE 2001 and USDE data increases in odds ratios ofNDs

(autism) from both VAERS and

USDE closely linearly

correlated with increasing

doses of TM-containing

childhood vaccines

Epidemiology Geier and Geier [99] VAERS database TM-DTaP and NDs in VAERS An association was found

TCVs Exp. Biol. Med. 1992-2000 between TM-DTaP and autism

Epidemiology Geier and Geier [100] BSS-CDC database Mercury doses from TCVs on Evidence showing a direct

TCVs Med. Sci. Monitor. USDE population prevalence of autism relationship between

CDC yearly live birth estimates increasing doses of mercury

from TCVs and autism

Epidemiology Geier et al. [101] VSD database Relationship between Evidence supporting TCVs as a

TCVs Biol. Trace Elem. Res. PDD 534 Thimerosal-containing Hib and the risk factor for PDD

PDD mean 4.1 y risk for PDD

Epidemiology Geier et al. [102] VSD database NDs/PDD and Thimerosal dose Evidence supporting TCVs as a

TCVs IJERPH 1991-2000 risk factor for NDs/PDD that is

dose dependent

Epidemiology Geier et al. [104] VAERS database Risk of ASD following TCVs Evidence supporting TCVs as a

TCVs Transl. Neurodegener. 1998-2000 risk factor for ASD

VSD database

1991-1999

Epidemiology Geier et al. [104] VAERS database Risk of NDs following Evidence supporting TCVs as a

TCVs J. Biochem. Pharmacol. Res. 1997-1999 Thimerosal-preserved DTaP risk factor for NDs/PDD

2004-2006

HepB = Hepatitis B vaccine; TCVs = Thimerosal containing vaccines; ASD = autism spectrum disorders; PDD = pervasive developmental disorders; ND = neurodevelopmental disorder; TM = Thimerosal; DTaP = Diphtheria, Tetanus, acellular Pertussis; HepB = Hepatitis B vaccine; Hib = Haemophilus influenzae Type b; RhoGAM = Rho (D) Immune Globulin; VAERS = Vaccine Adverse Events Reporting System; VSD = Vaccine Safety Datalink; USDE = US Department of Education; TCVs = Thimerosal-containing vaccines; EPA = Environmental Protection Agency; RBC = red blood cells; DD = developmental disability; NHANES = National Health and Nutrition Examination; NH1S = National Health Interview Survey; BSS-CDC = Biological Surveillance Summaries of the Centers for Disease Control; y=years of age.

at conclusions that stand in sharp contrast to most of the studies conducted or supported by public health entities and/or industry. Table 10 presents studies which find Thimerosal in vaccines to be a significant risk factor for ASD [93-104]. Table 11 presents studies which find Thimerosal in vaccines is not a significant risk factor for ASD [105-112].

1t should be mentioned, that Thimerosal was included in this review even though the mercury in Thimerosal is part of a compound: sodium ethyl-mercury thiosalicylate. This is because Thimerosal is 49.55% mercury by weight and rapidly decomposes in aqueous saline solutions into ethyl-mercury hydroxide and ethyl-mercury chloride. Thimerosal is estimated to contribute to about half of the mercury exposure of infants [113]. Thimerosal is still used in many vaccines to date, particularly in developing countries [78].

1t should also be mentioned that a review of the potential relationship between Thimerosal and ASD by Schultz [114] did not

include studies that used the VAERS database. Schultz stated that the VAERS system is a passive reporting system to which anyone can report and thus is a bias dataset. However, VAERS-based studies were included in this review because the Centers for Disease Control (CDC) states that the reports of possible vaccine-associated events to the VAERS are submitted by informed and conscientious healthcare professionals and that despite the limitations of spontaneous reports, the VAERS database provides vital information of clinical importance. The VAERS Working Group of the CDC and the Food and Drug Administration (FDA) have published epidemiologic studies based upon the VAERS [115].

8. Epidemiological studies that examine mercury in RhoGam as a risk factor for ASD

Rho(D) immune globulin (Trade names include RhoGAM) is given to a woman to prevent the formation of antibodies to Rh

Table 11

Epidemiology Studies that did not Find a Relationship between Thimerosal in Vaccines is a Risk Factor for ASD.

Type of Study Source of Exposure

Authors and Journal

Database

Age or Time Frame

Purpose of Study

Findings

Epidemiology TCVs

Epidemiology TCVs

Epidemiology TCVs

Epidemiology TCVs

Epidemiology TCVs

Epidemiology TCVs

Epidemiology TCVs

Epidemiology TCVs

Verstraeten et al. [105] Pediatrics Madsenetal [106] Pediatrics

Stehr-Green etal. [107] Am. J. Pre. Med.

Hviid et al. [108] JAMA

Andrews etal. [109] Pediatrics

Price et al. [110] Pediatrics

Schechter and Grether [111] Arch. Gen. Psychiatry

Mrozek-Budzyn et al. [112] Przegl. Epidemiol.

VSD database 1991-1998

Danish Psychiatric Central Research Register 2-10 y

US - Special Education Services Sweden - National database Denmark- National registry Mid 1980s - mid 1990s Danish

National registry 1990-1996

United Kingdom National registry 1988-1997

1994-1999 ASD 256 Controls 752 6-13 y

California Department of Developmental Services

1995-2007

Medical Records ASD 96 Controls 193 Children

Assessed the possible toxicity of TCVs among infants TCVs in Denmark and incidence of autism

TCVs and autism

To determine whether vaccination with a TM-containing vaccine is associated with autism Relationship between the amount of TM an infant receives via DTP or DT vaccine and NDs (autism) TCVs and autism

No analyses found significant increased risks for autism Data do not support a correlation between TCVs and autism

No correlation between TCVs and autism

Results do not support a causal relationship between TCVs and ASD

No evidence of an association with TM exposure

No findings of increased risk for any of the three ASD outcomes

Autism prevalence in California Data do not support the

after removal of TM from most childhood vaccines

To determine an association of TCVs exposure with the risk of

hypothesis that exposure to TCVs during childhood is a primary cause of autism No evidence of an association between TCVs and autism

TCVs = Thimerosal containing vaccines; ASD = autism spectrum disorders; ND = sis; VSD=Vaccine Safety Datalink; y=years of age.

neurodevelopmental disorder; TM =Thimerosal; DTaP = Diphtheria, Tetanus, acellular Pertus-

autism

Table 12

Epidemiological Studies that Show Thimerosal in RhoGam is a Risk Factor for ASD.

Type of Study Source of Authors and Journal N Age or Time Frame Purpose of Study Findings

Exposure

Epidemiology Geier etal. [116] NDs 298 Maternal Increases in maternal

RhoGAM Neuro Endocrin. Lett. Controls 124 Rh-negativity/TM-containing Rh-negativity among children

Controls 2021 RhoGAM with NDs, autism spectrum

1980-2001 disorders, and attention-

Controls 2002+ deficit-disorder/attention-

deficit-hyperactivity-disorder

Epidemiology Geier and Geier [117] ASD 53 Maternal Significant dose-response

RhoGAM J. Matern. Fetal. Neonatal. Med. Controls 926 Rh-negativity/TM-containing relationship between the

ASD 1987-2001 RhoGAM severity ofthe regressive ASDs

Controls 1980-1989 and total mercury dose

children received from

RhoGAM

RhoGAM = Rho(D) Immune Globulin; NDs = neurodevelopmental disorders.

Table 13

Epidemiological Studies that did not Show Thimerosal in RhoGam is a Risk Factor for ASD.

Type of Study Source of Authors and Journal N Age or Time Frame Purpose of Study Findings

Exposure

Epidemiology Miles and Takahashi [118] ASD 214 Association between Rh status, No association was found

RhoGam Am. J. Med. Genet. A Children diagnosed RhoGam use in pregnancy and between maternal RhoGam use

between1995-2005 autism and autism

RhoGAM = Rho(D) Immune Globulin.

positive blood. It is an injection given at around 28 weeks of pregnancy to Rh negative mothers. For many years RhoGam was preserved with Thimerosal. Three epidemiological studies have been conducted to examine the safety of Rhogam preserved with Thimerosal. Two studies (Table 12 ) conducted by independent investigators, found Thimerosal in RhoGam to be a risk factor for ASD [116,117]. The third study (Table 13 ), sponsored by the

RhoGam manufacturer, Johnson and Johnson, Inc., did not find Thimerosal in RhoGam to be a risk factor for ASD [113]. In 2001, Thimerosal was removed from Rh immune globulin [118].

Table 14

Epidemiological Studies that Show Mercury in Air Pollution is a Risk Factor for ASD.

Type of Study Source of Authors and Journal N Age or Time Frame Purpose of Study Findings

Exposure.

Epidemiology Zhang andWong[119] Total mercury emission based Examined mercury exposure Evidence suggests an increase

Prenatal Mercury Exposure Environ. Int. on the data of1999 in China increases in China in autism related to increasing mercury exposure

Epidemiology Palmeret al. [120] Texas Education Department Mercury release, special Association between

Coal-burning power plants Health Place 2000-2001 education rates, and autism environmentally released

EPA- Toxic Release Inventory disorder mercury and special education

2004 4 million children rates were fully mediated by

enrolled in grades K through 12 increased autism rates.

Epidemiology Palmeret al. [121] EPA-Toxic Release Inventory Proximity to mercury release Association between

Coal-burning power plants Health Place 1998 and autism prevalence proximaty of released mercury and autism

Epidemiology Blanchard etal. [122] US EPA National Scale Air Occurrence of autism related to Risk of autism is greater in the

Coal burning power plants Rev. Environ. Health Toxins distribution of mercury in geographic areas of higher

Assessment 2002 ambient air levels of ambient mercury

Texas Education Association

Epidemiology Dickerson et al. [123] US EPA- Toxics Release ASD prevalence and proximity Association between urban

Industrial facilities releasing Sci. Total Environ. Inventory1991-1999 to industrial facilities releasing residential proximity to

arsenic, lead or mercury into ADDM 2000-2008 arsenic, lead or mercury industrial facilities emitting air

air pollutants and higher ASD prevalence

Epidemiology Windham et al. [124] HAP concentrations 1996 ASD and environmental Increased risk of ASD

Air Pollution Environ. Health Perspect. ASD 284 exposures, ambient air, San associations included mercury,

Controls 657 Francisco Bay cadmium, nickel,

Born in 1994 trichloroethylene, and vinyl chloride

Epidemiology Roberts etal. [125] US EPA Associations between U.S. EPA Overall measure of metals

Air Pollution Environ. Health Perspect. Nurse's Health Study II - levels of hazardous air were significantly associated

ASD 325 pollutants at time and place of with ASD, with odds ratios

Controls 22,101 birth and ASD ranging from 1.5 (for overall metals measure) to 2.0 (for diesel and mercury)

EPA = Environmental Protection Agency; ASD = autism spectrum disorders; ADDM = Autism and Developmental Disabilities Monitoring; HAP = hazardous air pollutant.

Table 15

Epidemiological studies that did not show mercury in air pollution is a risk factor for ASD.

Type of Study Source of Authors and Journal N Purpose of Study Findings

Exposure Age or Time Frame

Epidemiology Lewandowski et al. [126] Texas Toxic Release Inventory Mercury exposure from Analysis suggests mercury

Coal J. Toxicol. Environ. Health A School District Autism coal-fired power plants and emissions not consistently

Prevalence autism in Texas associated with autism

2001-2007 prevalence in Texas school

districts

Table 16

Epidemiological studies that show mercury from other sources is a risk factor for ASD.

Type of Study Source of Authors and Journal N Purpose ofStudy Findings

Exposure Age or Time Frame

Epidemiology Geier etal. [128] ASD 100 Maternal dental amalgams Subjects with > or=6

mercury dental amalgams Acta Neurobiol. Exp. (Wars) 7-13 y during pregnancy and risk of autism amalgams were 3.2-fold significantly more likely to be diagnosed with autism (severe) in comparison to ASD (mild) than subjects with<or=5 amalgams.

Epidemiology Shandley and Austin [129] Australian Pink Disease Tested the hypothesis that Prevalence rate of ASD among

General/Pink Disease J. Toxicol. Environ. Health Support Group individuals with a known the grandchildren of pink

2009 hypersensitivity to mercury (pink disease survivors) may be more likely to have descendants with an ASD disease survivors (1 in 22) to be significantly higher than the comparable general population prevalence rate (1 in 160).

ASD »autism spectrum disorders; y »years of age.

9. Epidemiological studies that examine mercury in the air as a risk factor for ASD

Several studies suggest that mercury in air pollution is a risk factor for ASD, as shown in Table 14 [119-125]. The Palmer et al. study [120] which found an association between mercury release

and ASD rates was partially replicated by Lewandowski et al. [126]; however, Lewandowski and colleagues concluded that mercury emissions were not consistently associated with autism prevalence in Texas school districts, thus their study was placed in the category of not finding a relationship between mercury and ASD (Table 15). Overall, a 2014 meta-analysis of the evidence of the impact of pre-

natal and early infancy mercury exposures on autism risk found a significant association between increasing environmental mercury exposures and an increasing ASD risk (odds ratio = 1.66, 95% confidence interval = 1.14-2.17) [127]. It was observed that this effect remained similar after excluding studies not adjusted for con-founders . Table 16 presents studies that show mercury from other sources is a risk factor for ASD.

10. Epidemiological studies that examine mercury from other sources as a risk factor for ASD

Geier et al. [128] found that maternal mercury fillings during pregnancy were a risk factor for ASD. Austin and Shandley [129] hypothesized and found that descendents of pink disease (mercury poisoning) survivors would have a higher rate of ASD. 1n contrast, however, van Wijngaarden et al. [130] found no relationship between prenatal methyl-mercury exposure and ASD phenotypic behaviors (Table 17 ).

11. Discussion

As mentioned in the 1ntroduction, numerous studies have been conducted over the last three decades that examine the relationship between mercury and ASD. This comprehensive search for human studies that examined the potential relationship between mercury and ASD found 91 studies between 1999 to February 2016. The findings from the vast majority (74%) of those studies suggest that mercury is a risk factor for ASD. These studies reveal both direct and indirect effects of mercury exposure. How these effects may interact in ASD is summarized in Fig. 1. Fig. 1 illustrates mercury effects on the brain in ASD as suggested by the research, showing both causal and correlative findings. The figure shows, starting from the top, that mercury causes (purple arrows) autoimmune activation, oxidative stress, neuroinflammation, neuronal damage, and loss of neuronal connectivity. These are direct effects from mercury exposure. In the next line from the top, the figure shows that autoimmune activation, oxidative stress, neuroinflammation can then also cause (purple arrows) neuronal damage and loss of neuronal connectivity. These are indirect effects from mercury exposure. The lower part of the figure shows the relevant correlations (green arrows), such as autism symptom severity correlates with neuronal damage and neuronal loss of connectivity. 1n addition, autism symptom severity also correlates with mercury levels, which, in turn, correlate with autoimmune activation, oxidative stress levels, and neuroinflammatory biomarkers.

11.1. Mercurial compounds and toxicity

Mercury exists in three forms: elemental mercury (e.g., vapor from dental amalgams), inorganic mercury compounds (e.g., mercuric chloride), and organic mercury compounds (e.g., methyl and ethyl mercury). According to the US Environmental Protection Agency, all forms of mercury are quite toxic [131]. Organic compounds generally exert stronger cytotoxic effects as compared to inorganic mercury [132]. Methyl mercury can be formed by the reaction of metallic mercury with organic molecules. Bacteria can facilitate the formation of methyl mercury. An example of methyl mercury poisoning is the Minamata tragedy where fish were contaminated with methyl mercury from the dumping of mercury-tainted waste into water in Minamata, Japan. Exposed newborns showed delayed neurodevelopmental toxicity [133]. Ethyl mercury, which is used in Thimerosal (ethyl mercury thiosalicylate) and as a fungicide, is man-made. An example of ethyl mercury poisoning is the 1960 Iraq ethyl mercury tragedy where many families

suffered illness and death from eating grains treated with ethyl mercury [134].

Exposure to ethyl mercury is said to be safer than exposure to methyl mercury because the blood half-life of intramuscular ethyl mercury from Thimerosal in vaccines in infants has been found to be substantially shorter than that of oral methyl mercury in adults [135]. However, it is important to note that mercury from Thimerosal is found in the brain and kidney and that even when mercury levels are decreased in the blood, the mercury levels have been found to be unchanged in the brain [136]. It is also important to note that once the mercury from Thimerosal enters the brain, some of it remains in the form of ethyl mercury, and some is found as methyl mercury and inorganic mercury. As stated by Rodrigues et al. [137], of the total mercury found in the brain after Thimerosal exposure, 63% is in the form of inorganic mercury, 13.5% is ethyl mercury, and 23.7% is methyl mercury. They further stated that mercury in the tissues and blood following Thimerosal treatment is predominantly found as inorganic mercury, but a considerable amount of ethyl mercury is also found in the liver and brain.

Thimerosal is sometimes referred to as an adjuvant, a substance (as one added to a vaccine) that enhances the immune response to an antigen [138]. More commonly, however, it is considered a preservative, while aluminum salts are considered the most common adjuvant [139]. Both Thimerosal and aluminum are considered xenobiotics and toxic, with Thimerosal being the more toxic of the two [140]. In the US Thimerosal is still in over 50% of the flu vaccines which are recommended for infants, children, and pregnant women. It is also in tetanus and in one version of the multidose meningococcal vaccine in the US. In the developing countries around the world Thimerosal is still present in many of the childhood vaccines [141].

H.2. Otherneurotoxicants

Evidence suggests other possible causal or contributory exposures, such as lead [35], organophosphate insecticides [142], phthalates [143], glyphosates [144], and pyrethroids [145]. Exposure to these other suggested xenobiotic exposures may also be causal and/or contributory in ASD; however, some research suggests that their level of exposure and contribution may conceivably be less than mercury [146]. Future studies could include further analysis of the attributable risk from neurotoxins.

H.3. Brain pathology and susceptibility

As mentioned in the Introduction, the brain pathology in ASD indicates marked and ongoing inflammatory reactivity with concomitant neuronal damage. When brain inflammation is sustained, as is seen in ASD, there is loss of neuronal connectivity, which is also seen in ASD. It is conceivable that all of the suggested neurotoxi-cants are able to bring about loss of connectivity through direct insult as well as by activating a neuroinflammatory process from which collateral damage is likely.

When the brain pathology in ASD is examined, it matches the brain pathology found in mercury intoxication [18]. Kern et al. [18] describe 20 different brain pathologies found in both ASD and mercury intoxication. Lead and organophosphates have also been shown to be capable of producing some of the same pathology [147], although not as similar as that produced by mercury [18]. It is also important to mention that toxicants can work synergisti-cally by depleting the sulfur dependent detoxification system, and glutathione reserves in particular.

Many neurotoxicants, and particularly mercury, are sequestered or detoxified by cellular thiols (organo-sulfur compound that contains a carbon-bonded sulfhydryl (—C—SH or R—SH) group, such as glutathione) and thiol availability is known to be limited in ASD

Table 17

Epidemiological studies that did not show mercury from other sources is a risk factor for ASD.

Type of Study Source of Exposure Authors and Journal N Age or Time Frame Purpose ofStudy Findings

Epidemiology van Wijngaarden et al. [130] Seychelles Child Development Evaluated the association Prenatal exposure to

Maternal Epidemiology Study between prenatal methylmercury was not

1986-1990 methylmercury exposure and associated with ASD

ASD phenotype phenotypic behaviors

ASD = autism spectrum disorders.

Fig. 1. This figure illustrates mercury effects on the brain in ASD suggested by the research, both causal and correlative findings.

making this population of individuals more susceptible to toxicants. For example, Chauhan et al. [83] reported that in cerebellum and temporal cortex samples from subjects with ASD, glutathione levels were significantly decreased. Several other studies show that children diagnosed with an ASD have abnormal sulfation chemistry, limited thiol availability, and decreased glutathione (GSH) reserve capacity, with a resulting and subsequent compromised oxidation/reduction (redox) and detoxification capacity [81,87,88] and a concomitant vulnerability to brain insult [78].

11.4. Neurodevelopmental disorders in general

The findings from this collection and review of the literature on the relationship between ASD and mercury may have broader implications. ASD has increased over the past three decades, but so have other neurodevelopmental disorders [148-156]. Some examples are as follows: In the 1980s, 1 child in 1000 developed autism, and by 2013, 1 child in 45 developed autism [149-152]. In 1976, 1 child in 30 was learning disabled, while by 2013, 1 child in 6 was learning disabled [148,149]. In 1996, 1 in 18 children developed attention deficit/hyperactivity disorder (ADHD), and by 2012, 1 in 8 children developed ADHD, an increase of about 75 percent [153]. The rate among children three to four years old with an ADHD diagnosis has almost doubled since 1997 [153]. Tic disorder, once considered rare, is now considered to be the most common movement disorder, with 0.2-46.3% of school children experiencing tics during his/her lifetime [154]. Similarly, obsessive compulsive dis-

order, also once considered rare, now affects at least 1 in 50-100 children depending on the estimates [155]. The needs and numbers of emotionally disturbed youth are also growing such that by 2004, about 1 in 11 children are were diagnosed with emotional disturbances [156]. Overall, as of 2011,1 in 6 children in the United States had a neurodevelopmental disorder, which also represents a dramatic increase in the last few decades [147,149].

11.5. Governmental policies and neurodevelopmental disorders in general

It appears that lack of governmental intervention and regulation may be a contributing factor to the epidemic of neurodevelopmen-tal disorders. Limited government action has taken place in regard to reducing prenatal and postnatal exposure to mercury and other neurotoxicants resulting in an ever increasingly toxic environment, possibly due to conflict-of-interest issues and conflict between areas of government. For example, the 2008 Obama/Biden Plan for a Healthy America included the reduction of toxicants such as mercury, stating that, "More than five million women of childbearing age have high levels of toxic mercury in their blood, and approximately 630,000 newborns are born at risk every year." However, in January 2012 when the US Food and Drug Administration (FDA) drafted a rule prohibiting the use of mercury-based dental fillings in pregnant women, nursing mothers, children aged less than 6 years of age, and other sensitive groups, the United States Department of

Health & Human Services (HHS), under President Obama, failed to release it [157,158]. As a consequence nothing was accomplished.

12. Conclusion

The research that examines the relationship between mercury and ASD is extensive. One of the purposes of this review of the scientific literature was to bring together and organize the plethora of studies to make it easier for researchers to examine and interpret the evidence. It is a compilation of every original study with any investigation of ASD and any potential exposure to mercury from any source, at any time point, in the human population. In order to limit the introduction of any bias, the authors of this review did not analyze each study, evaluate bias or study quality, or discuss similarities and difference between the studies.

From this inventory of the available research, it is clear that the vast majority of the research, conducted by multiple research groups, from many different countries, using many different methodologies, supports a link between mercury exposure and a diagnosis of ASD. In this evaluation, it was found that 74% of studies support a link between mercury exposure and ASD, which corroborates a previous evaluation of the same issue conducted in 2010. In that study, Desoto and Hitlan also found that 74% of studies support a link between mercury exposure and ASD [159]. This agreement in science six years later is compelling and supports the validity of the finding.

The compilation of the evidence indicates that children with ASD are more susceptible to mercury than typically developing children, and that is reflected in significantly different levels of mercury, or biomarkers indicative of mercury, in the brain, blood, urine, baby teeth, hair, and nails. In addition, many of these studies have found that the mercury, or biomarkers indicative of mercury, correlate with symptom severity such that the higher the mercury levels the worse the autism symptom severity. The majority of the epi-demiological research also support the hypothesis that mercury is a risk factor for ASD. Based on the preponderance of the evidence, mercury exposure is causal and/or contributory in ASD. With the increase in neurodevelopmental disorders in general, and especially ASD, the evidence suggests that governmental/public policy changes are urgently needed.

Author contributions

Dr. Kern and Mr. Geier conceptualized the design of the study. Dr. Kern wrote the majority of the initial draft of the paper. Mr. Geier, Reverend Sykes, Dr. Haley, and Dr. Geier critically reviewed and revised the manuscript.

All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.

Conflicts of interest

The authors have been involved in vaccine/biologic litigation.

Acknowledgments

This study was supported by the non-profit Institute of Chronic Illnesses (ICI), Inc. and the non-profit CoMeD, Inc. The funding sources were not involved in study design, data collection, analysis, interpretation of data, writing of the manuscript, or in the decision to submit the manuscript for publication.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.Org/10.1016/j.jtemb.2016.06. 002.

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