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Cultured lymphocytes from autistic children and non-autistic siblings up-regulate heat shock protein RNA in response to thimerosal challenge

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Abstract

There are reports suggesting that some autistic children are unable to mount an adequate response following exposure to environmental toxins. This potential deficit, coupled with the similarity in clinical presentations of autism and some heavy metal toxicities, has led to the suggestion that heavy metal poisoning might play a role in the etiology of autism in uniquely susceptible individuals. Thimerosal, an anti-microbial preservative previously added routinely to childhood multi-dose vaccines, is composed of 49.6% ethyl mercury. Based on the levels of this toxin that children receive through routine immunization schedules in the first years of life, it has been postulated that thimerosal may be a potential triggering mechanism contributing to autism in susceptible individuals. One potential risk factor in these individuals may be an inability to adequately up-regulate metallothionein (MT) biosynthesis in response to presentation of a heavy metal challenge. To investigate this hypothesis, cultured lymphocytes (obtained from the Autism Genetic Resource Exchange, AGRE) from autistic children and non-autistic siblings were challenged with either 10 microM ethyl mercury, 150 microM zinc, or fresh media (control). Following the challenge, total RNA was extracted and used to query "whole genome" DNA microarrays. Cultured lymphocytes challenged with zinc responded with an impressive up-regulation of MT transcripts (at least nine different MTs were over-expressed) while cells challenged with thimerosal responded by up-regulating numerous heat shock protein transcripts, but not MTs. Although there were no apparent differences between autistic and non-autistic sibling responses in this very small sampling group, the differences in expression profiles between those cells treated with zinc versus thimerosal were dramatic. Determining cellular response, at the level of gene expression, has important implications for the understanding and treatment of conditions that result from exposure to neurotoxic compounds.

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... If retinoids modulate CD38 transcription in the brain, which in turn mediates oxytocin release, then the relationship we have shown [72] between cognitive function and CD38 mRNA levels in LBC cells may be reflecting common state characteristics of OT-CD38-RA pathways in different tissues. Indeed, various studies have employed LBC lines to model brain dysfunctions in autism [90][91][92] and other neuropsychiatric disorders [93][94][95][96][97]. ...
... Most of the studies to date have focused on human lymphocytes gene expression profiling, comparison between illness groups and normal controls, and cross-matching with human postmortem brain gene expression data. A number of studies have specifically examined gene expression patterns in ASD [90][91][92][114][115][116][117][118][119][120][121][122][123]. It should be noted that predating this gush of expression studies in ASD, hyperserotonemia was observed in one third of patients and platelet serotonin was suggested as a marker for this disorder [124][125][126][127]. ...
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Increasing evidence suggests that the nonapeptide, oxytocin (OT), helps shape social and affiliative behaviors not only in lower mammals but also in humans. Recently, an essential mediator of brain OT release has been discovered, ADP-ribosyl cyclase and/or CD38. We have subsequently shown that polymorphisms across the CD38 gene are associated with autism spectrum disorders (ASD). Notably, CD38 expression in lymphoblastoid cells (LBC) is reduced in cell lines derived from ASD subjects compared to parental cell lines. Intriguingly, a correlation was observed between CD38 expression and measures of social function in ASD. Finally, we have shown that all-trans retinoic acid (ATRA), a known inducer of CD38 transcription, can rescue low CD38 expressing LBC lines derived from ASD subjects and restore normal levels of transcription of this ectoenzyme providing 'proof of principle' in a peripheral model that retinoids are potential therapeutic agents in ASD.
... Venous blood lymphocytes were used because they serve as an excellent system for studying different types of genetic endpoints in many diseases including autism [14,15,[20][21][22][23][24]. Blood cells are distributed throughout the entire organism via the circulatory system, hence, genomic damage are not just the indicators of the state of the lymphocytes in the circulation, but can also be induced in any part of the organism. ...
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... No apparent reports on stress protein behavior, specifically in etHg/thimerosal damaged kidney tissues, were found. In other cell types, human lymphocytes exposed to thimerosal responded by upregulating numerous Hsp transcripts, but not metallothioneins (Walker, Segal, and Aschner 2006). These findings were corroborated by Singh and Hanson (2006) who also did not find marked differences between serum levels of metallothioneins in normal and autistic children exposed to thimerosal through vaccination. ...
Article
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... While the first large-scale gene expression analysis of autism was performed on postmortem brain tissues from autistic and nonautistic individuals [Purcell, Jeon, Zimmerman, Blue, & Pevsner, 2001], several subsequent gene expression studies, including our earlier study on monozygotic twins discordant for autism, have utilized LCL or primary blood lymphocytes from autistic probands and controls with the goal of identifying expressed biomarkers for diagnostic purposes [Baron, Liu, Hicks, & Gregg, 2006;Gregg et al., 2008;Hu et al., 2006;Nishimura et al, 2007a]. It is further postulated that these surrogate experimental models of autism may reveal clues to the pathobiology of this neurobiological disorder [Walker, Segal, & Aschner, 2006]. Indeed, gene expression profiles of different brain regions have been shown to exhibit the highest similarity to whole blood [Sullivan, Fan, & Perou, 2006], while a metaanalysis of studies performed in blood and postmortem brain demonstrated convergent gene expression changes [Middleton et al., 2005]. ...
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... The first DNA microarray study of autism identified ,30 genes as differentially expressed in the cerebellum from autopsy tissue of autistic and normal subjects, and focused on the abnormal expression of the glutamate receptor as a potential pathogenic mechanism [51]. Several recent applications of global gene expression analysis to autism have evaluated gene expression in lymphoblastoid cell lines (LCL) and in whole blood with the goal of identifying differentially expressed genes in a peripherally-derived tissue which may serve as diagnostic biomarkers for ASD, or surrogate markers for dysregulated metabolic and signaling pathways in peripheral and central tissues, which may provide clues to the pathophysiology of the disorder [43,44,[52][53][54]. Our previous study on LCL from monozygotic twins discordant in severity of ASD revealed differentially expressed genes that function in nervous system development as well as genes that mapped in silico to autism susceptibility regions on chromosomes that were previously identified by numerous genetic analyses [43]. ...
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Despite the identification of numerous autism susceptibility genes, the pathobiology of autism remains unknown. The present "case-control" study takes a global approach to understanding the molecular basis of autism spectrum disorders based upon large-scale gene expression profiling. DNA microarray analyses were conducted on lymphoblastoid cell lines from over 20 sib pairs in which one sibling had a diagnosis of autism and the other was not affected in order to identify biochemical and signaling pathways which are differentially regulated in cells from autistic and nonautistic siblings. Bioinformatics and gene ontological analyses of the data implicate genes which are involved in nervous system development, inflammation, and cytoskeletal organization, in addition to genes which may be relevant to gastrointestinal or other physiological symptoms often associated with autism. Moreover, the data further suggests that these processes may be modulated by cholesterol/steroid metabolism, especially at the level of androgenic hormones. Elevation of male hormones, in turn, has been suggested as a possible factor influencing susceptibility to autism, which affects approximately 4 times as many males as females. Preliminary metabolic profiling of steroid hormones in lymphoblastoid cell lines from several pairs of siblings reveals higher levels of testosterone in the autistic sibling, which is consistent with the increased expression of two genes involved in the steroidogenesis pathway. Global gene expression profiling of cultured cells from ASD probands thus serves as a window to underlying metabolic and signaling deficits that may be relevant to the pathobiology of autism.
... On the transcriptional level, heme oxygenase, tubulin alpha (TUBA) and carnitine palmitoyltransferase 1 (Cpt1) all showed an effect of MeHg exposure in the liver, suggesting that MeHg may affect oxidoreductase activity, structural protein activity and acyltransferase activity. Several studies have shown that mercury compounds may induce the transcription of heme oxygenase in mammals (Korashy and El-Kadi, 2004;Walker et al., 2006;Kawata et al., 2007;Koizumi et al., 2007), in line with the current finding. In liver there was also a significant effect of dietary lipids on heme oxygenase transcription. ...
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This experiment aimed to study the molecular toxicity of methylmercury (MeHg) in liver, brain and white muscle of Atlantic salmon fed a diet based on fish oil (FO, high dietary n-3/n-6 ratio) compared to an alternative diet mainly based on vegetable oil (VO, low dietary n-3/n-6 ratio). Juvenile salmon were fed decontaminated diets or the FO and VO diets enriched with 5 mg Hg/kg (added as MeHg) for three months. The dietary lipid composition affected the fatty acid composition in the tissues, especially in liver and white muscle. After 84 days of exposure, the liver accumulated three times as much MeHg as the brain and white muscle. Vitamin C content and heme oxygenase, tubulin alpha (TUBA) and Cpt1 transcriptional levels all showed significant effects of MeHg exposure in the liver. TBARS, α-tocopherol, γ-tocopherol, and the transcriptional levels of thioredoxin, heme oxygenase, TUBA, PPARB1, D5D and D6D showed an effect of dietary lipid composition in liver tissue. Effects of dietary lipids were observed in brain tissue for MT-A, HIF1, Bcl-X and TUBA. Interaction effects between MeHg exposure and dietary lipid composition were observed in all tissues. Our data suggest that dietary fats have modulating effects on MeHg toxicity in Atlantic salmon.
... transcription in the brain, which in turn mediates oxytocin release, then the relationship we have shown (Riebold et al., 2011) between cognitive function and CD38 mRNA levels in LBC cells may be reflecting common state characteristics of OT-CD38-RA pathways in different tissues. Indeed, various studies have employed LBC lines to model brain dysfunctions in autism (Hu et al., 2009;Nishimura et al., 2007;Walker et al., 2006) and other neuropsychiatric disorders (Chagnon et al., 2008;De Luca et al., 2008;Kuratomi et al., 2008;Pandey et al., 2008;Tseng et al., 2008). ...
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Arginine vasopressin (AVP) and oxytocin (OXT) are social hormones and mediate affiliative behaviors in mammals and as recently demonstrated, also in humans. There is intense interest in how these simple nonapeptides mediate normal and abnormal behavior, especially regarding disorders of the social brain such as autism that are characterized by deficits in social communication and social skills. The current review examines in detail the behavioral genetics of the first level of human AVP-OXT pathway genes including arginine vasopressin 1a receptor (AVPR1a), oxytocin receptor (OXTR), AVP (AVP-neurophysin II [NPII]) and OXT (OXT neurophysin I [NPI]), oxytocinase/vasopressinase (LNPEP), ADP-ribosyl cyclase (CD38) and arginine vasopressin 1b receptor (AVPR1b). Wherever possible we discuss evidence from a variety of research tracks including molecular genetics, imaging genomics, pharmacology and endocrinology that support the conclusions drawn from association studies of social phenotypes and detail how common polymorphisms in AVP-OXT pathway genes contribute to the behavioral hard wiring that enables individual Homo sapiens to interact successfully with conspecifics. This article is part of a Special Issue entitled Oxytocin, Vasopressin, and Social Behavior.
... Large population genome-wide association studies (GWAS), and copy number variation (CNV) technologies have led to identification of multiple genetic hotspots for ASDs ( Glessner et al., 2009;Ma et al., 2009;Merikangas et al., 2009;Pinto et al., 2010;Weiss et al., 2009). Expression microarray analysis revealed ASDs candidate genes that could result from the interaction of genetic background and environmental factors (Enstrom et al., 2009; V. W. Walker et al., 2006). However, the evidence for ASDs pathogenesis is still inconclusive, largely because of heterogeneity of patient populations and diverse model systems employed among different studies. ...
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Autism spectrum disorders (ASDs) are a group of diseases exhibiting impairment in social drive, communication/language skills and stereotyped behaviors. Though an increased number of candidate genes and molecular interactions have been identified by various approaches, the pathogenesis remains elusive. Based on clinical observations, data from accessible GWAS and expression datasets we identified ASDs gene candidates. Integrative gene network and a novel CNV-centric Node Network (CNN) analysis method highlighted ASDs-associated key elements and biological processes. Functional analysis identified neurological functions including synaptic cholinergic receptor (CHRNA) families, dopamine receptor (DRD2), and correlations between social behavior and oxytocin related pathways. CNN analysis of genome-wide genetic and expression data identified inheritance-related clusters related to PTEN/TSC1/FMR1 and mTor/PI3K regulation. Integrative analysis identified potential regulators of networks, specifically TNF and beta-estradiol, suggesting a potential central role in ASDs. Our data provide information on potential disease mechanisms, and key regulators that may generate novel postulations, and diagnostic molecular biomarkers.
... Cultured lymphocytes from autistic children when challenged with 10 μM ethyl mercury responded in a different pattern than those of non-autistic siblings. Following the challenge, autistic cultured lymphocytes responded by upregulating numerous heat shock protein transcripts, but not MTs [28]. ...
Article
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Objectives The neurobiological basis for autism remains poorly understood. However, research suggests that environmentalfactors and neuroinflammation, as well as genetic factors, are contributors. This study aims to test the role that might be played by heat shock protein (HSP)70, transforming growth factor (TGF)-β2, Caspase 7 and interferon-γ (IFN-γ)in the pathophysiology of autism. Materials and methods HSP70, TGF-β2, Caspase 7 and INF-γ as biochemical parameters related to inflammation were determined in plasma of 20 Saudi autistic male patients and compared to 19 age- and gender-matched control samples. Results The obtained data recorded that Saudi autistic patients have remarkably higher plasma HSP70, TGF-β2, Caspase 7 and INF-γ compared to age and gender-matched controls. INF-γ recorded the highest (67.8%) while TGF-β recorded the lowest increase (49.04%). Receiver Operating Characteristics (ROC) analysis together with predictiveness diagrams proved that the measured parameters recorded satisfactory levels of specificity and sensitivity and all could be used as predictive biomarkers. Conclusion Alteration of the selected parameters confirm the role of neuroinflammation and apoptosis mechanisms in the etiology of autism together with the possibility of the use of HSP70, TGF-β2, Caspase 7 and INF-γ as predictive biomarkers that could be used to predict safety, efficacy of a specific suggested therapy or natural supplements, thereby providing guidance in selecting it for patients or tailoring its dose.
... HE understanding of the contribution of environmental exposure to the development of autism and autism-like disorders is an active research area [1], [2]. However, neither the associated risk factor nor the pathophysiological Manuscript received March 13, 2009. ...
Article
Autism is one of the major disorders associated with brain development that presents early in life, as early as the fetal development phases to the early stages post birth. About 1 in 150 eight-year-old children in multiple areas of the United States had an autism spectrum disorder (ASD). Environmental agents and genetics are likely players but no specific target or cure has yet been identified. In this study, we present the role of the interaction between the water channel proteins aquaporins (AQP) and cations in autism. AQPs have six transmembrane domains interconnected via five loops (labeled A-E). Metal ions in water recognize specific motifs in the loop structure thereby controlling the 3D structure, pore constriction and hence modulating water permeability. AQP11, is expressed in Purkinje cells which are target cells of autism. In addition, AQP11 is the only mammalian aquaporin that has tri-cysteine motif, a high affinity mercury ion binding site. A dataset of frequency of amino acid binding to metal ions termed Protein-Metal Ion Site-frequency (ProMIS) was generated from Protein Data Bank (PDB). A score was then used to evaluate the likelihood of common mono/bivalent metal ions interacting with the 20 amino acids. ProMIS Scores are predictive of metal ion binding sites in AQPs. Scores of Loop E has higher sensitivity, specificity and predictive values. Loops C and E are predicted to bind better with bivalent cations and loop B to monovalent cation binding. The current study has identified/evaluated several cation binding sites in human AQPs and provides valuable insight for the discovery of novel therapeutics for Autism and Autism spectrum disorders.
... Interestingly, brain excitotoxicity as an important process in autism etiology can be attenuated through many steps; one of them is up-regulation of HSP70 (Walker et al. 2006). This hypothesis may be supported by the consideration of the recent study by El-Ansary et al. (2011). ...
Article
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To identify neuroinflammatory biomarkers in patients with various severity of autism spectrum disorder (ASD) increases the insight about the pathogenesis and pathophysiology of this neurodevelopmental disorder. The aim of the present study was to analyze the levels in plasma of TGFβ2, Heat shock protein 70 (HSP70), and hematopoietic prostaglandin D2 synthase (H-PGDS) in Saudi ASD children and healthy age-matched neurotypical controls. Also, it was in the present study examined the correlation among these neuroinflammatory biomarkers and the sensory deficit exhibited by the ASD children. Blood samples from 38 Saudi children with ASD and 32 age-matched neurotypical controls were withdrawn after an overnight fast. For the blood taking 3 mL EDTA containing blood collection tubes was used. The samples were centrifuged for 20 min (4 °C; 3000×g) directly after the blood sampling. The harvested plasma was used for in vitro quantification of TGF-β2, HSP70, and H-PGDS by using the sandwich enzyme immunoassay. Receiver operating characteristic (ROC) analysis and predictiveness curves showed that each of TGF-β2, HSP70 or H-PGDS alone could not be used as a predictive neuroinflammatory biomarker for ASD. However, when TGF-β2 and HSP70 were combined in one ROC curve, the AUC was increased to an appreciable value that makes them together robust predictors of variation between the ASD and neurotypical control groups. Overall, it was in the present study found significant differences for TGF-β2 and HSP70 when the ASD and neurotypical control groups were compared, independently of the sensory deficit level. In conclusion, the present study highlights the usefulness of TGF-β2, HSP70, and H-PGDS as diagnostic tools to differentiate between ASD and neurotypical control children, but not among subgroups of ASD children exhibiting different severity levels of sensory dysfunction. The presented data also suggest the effectiveness of ROC as a powerful statistical tool, which precisely can measure a combined effect of neuroinflammatory biomarkers intended for diagnostic purposes.
... Autism may result from a combination of genetic susceptibility (perhaps in the form of reduced ability to remove mercury or other neurotoxins from the system) and environmental exposure at key times in development. [5][6][7] This would mean a generalized increase in mercury levels would be expected to co-occur with a generalized increase in autism, but some people The question of what is leading to the apparent increase in autism is of great importance. Like the link between aspirin and heart attack, even a small effect can have major health implications. ...
Article
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The question of what is leading to the apparent increase in autism is of great importance. Like the link between aspirin and heart attack, even a small effect can have major health implications. If there is any link between autism and mercury, it is absolutely crucial that the first reports of the question are not falsely stating that no link occurs. We have reanalyzed the data set originally reported by Ip et al. in 2004 and have found that the original p value was in error and that a significant relation does exist between the blood levels of mercury and diagnosis of an autism spectrum disorder. Moreover, the hair sample analysis results offer some support for the idea that persons with autism may be less efficient and more variable at eliminating mercury from the blood.
... A significant homology over a stretch of 15 amino acids was also found between an N-terminal region of herpes simplex virus type 2 UL14 protein and the substrate-binding domain of HSP70 family proteins, and the protein shared some functions of these molecular chaperones, such as nuclear translocation upon heat shock, ATP deprivation and osmotic shock [229] (Table 15). Similar homology and activation of HSPs both bioevents associated with increased immune cross-reactivity may be responsible for: a) abnormal measles-mumps-rubella (MMR) antibodies and autoimmunity in autistic children receiving MMR vaccine [111,230], b) the fact that cultured lymphocytes from autistic children and non-autistic siblings upregulated HSP RNA in response to thimerosal challenge [231], and c) the finding that HSP and gliadin peptide promote development of peptidase antibodies in children with autism and patients with autoimmune disease [105,110,120]. [Nb. ...
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T. gondii is an obligate intracellular parasite estimated to infect approximately 30-50% of the world’s population. Markedly elevated levels of antitoxoplasmal IgG antibodies reported in the patients with first-onset schizophrenia, migraine, Parkinson’s disease, AD, obsessive-compulsive syndrome, attention-deficit hyperactive disease, facial nerve palsy, may suggest that the parasite is responsible for development of all these clinical entities. Because the main target antigens of IgG are the surface antigens of the parasite, one cannot exclude that T. gondii may also be responsible for generation of IgG antibodies and autoantibodies specific for nervous system proteins, and production of maternal anti-fetal brain antibodies, in patients with ASD and several other neurodevelopmental disorders. This suggestion may be supported by the finding that T. gondii invades and replicates in a wide variety of circulating nucleated cells, such as macrophages, leukocytes, erythrocytes, lymphocytes, natural killer, and dendritic cells using them as a “Trojan horse” to gain access to privileged sites in the brain. T. gondii tachyzoites infects also a miscellaneous human brain cells, including vascular endothelial cells, microglial cells, astrocytes, neurons, and plexus choroideus, while bradyzoite development was observed in Purkinje cells in the cerebellum, and within human fibroblasts. After entry to the host cells, tachyzoites, bradyzoites, and sporozoites form different parasitophorus vacuoles (PV) within these anatomical structures, including human astrocytes and plexus choroideus. Invasion of the brain by the parasite may have pathophysiologic consequences for the host because, for example, it was reported that some autistic children had markedly increased glial fibrillary acidic protein (GFAP) in brain samples and the cerebrospinal fluid, and glial filaments (detected by immunostaining of the GFAP) were found to accumulate around the perimeter of the T. gondii cysts, as they developed in mouse brain astrocytes. Also, the negatively charged outer leaflet of T. gondii plasma membranes may participate in the marked reduction in the net positive charge of myelin basic protein (MBP) (18-19 kDa in size) and in generation of the enhanced MBP autoantibodies found in autism. The great variety of the brain cells and structures/regions attacked by T. gondii is consistent with the large number of elevated serum antibodies to brain proteins found in patients with ASD and other neurodevelopmental disorders. This review provided literature findings on identification, characterization and role of serum/plasma IgG antibodies and/or autoantibodies found in patients with ASD, as well as the IgG antibodies in the blood of mothers, which recognize fetal brain proteins, and their similarities and differences with IgG antibodies and/or excreted/secreted antigens reported in humans and mice during acute and chronic T. gondii infection. Several differences in molecular weights and range and numbers of resolved T. gondii proteins reported in various animal and human studies may be explained, at least in part, by various laboratory procedures used for these investigations, for example sodium dodecyl sulfate-polyacrylamide gel electrophoresis conditions and concentrations of polyacrylamide gel used. Some other factors, such as various intra-, interindividual and intergroup degrees of oxidative stress characteristic for ASD patients (eg. reduced vs. nonreduced antigen bands) and/or hydrophobicity, antioxidative treatment, T. gondii strain and its virulence, biologic material-supernatant preservation conditions, temperature, methods of antigen analysis and detection in sera, etc. Finally, the presence of tight protein-protein interaction complexes between T. gondii parasitophorus vacuole membrane (PVM) and host cell mitochondrium and endoplasmic reticulum, and the fact that mitochondrial-associated membranes are the site for decarboxylation of phosphatidylserine to phosphatidylethanolamine may explain why children with autism had markedly decreased plasma levels of phosphatidylethanolamine and why these of phosphatidylserine were increased. In the light of all these findings one cannot exclude that T. gondii infection and glycosylphosphatidylinositol-anchored brain protein antigens are, at least partially, responsible for the alteration in amino-glycerophospholipids levels and generation of several antibodies and autoantibodies present in sera of patients with ASD and other neuropsychiatric diseases. T. gondii PVM exhibited a remarkable, tight association with host mitochondria and endoplasmic reticulum, mediated by the parasite protein ROP2. One cannot exclude that this T. gondii PVM-host cell organelle complex remains quite stable for the whole host cell life span, even after necrosis or apoptosis of the parasite/host cell, and functions as an important energy source, and an abundant source of antigens acting as a triggering factor for NF-B. It may act as a perpetuum mobile-like machinery responsible for persistency of neuroinflammation characteristic for individuals with ASD, DS, and AD, as well as in mice with chronic T. gondii infection. This reasoning may be supported by the finding that PVM pore permits free bidirectional access to the host cytoplasm for molecules of up to 1300-1900 Da, including nucleotides and amino acids, and mean molecular weight of an amino acid is about 120 Da. Moreover, the parasite dense granule protein14 (GRA14) has a special topology in the PVM with its C terminus facing the host cell cytoplasm and its N terminus facing the vacuolar lumen, thus possibly ensuring trafficking of GRA proteins to their ultimate destination and contact with antigen presenting cells. Voltage-dependent anion channels also provides similar biologic function. Moreover, antigen peptides are about 4 to 9 amino acids in length, and only as little as 1-2 ng of peptides bind heat shock protein70 (HSPs), GRP94 and other HSPs and elicit a cellular immune response. Endoplasmic reticulum protein retrotranslocation machinery during antigen cross-presentation by dendritic cells and HSPs-peptide association in antigen processing and presentation by MHC class I, II, and III molecules, may play an important role in these immune processes. These arguments may be further supported by the finding of association of MHC genes with autism, and the fact that the activation of NFB by T. gondii important for this process correlated with the increased expression of antiapoptotic genes and localization of phosphorylated IB to the PVM. In addition, HSPs, especially Hsp90, have a potent endosome-targeting capability in professional antigen presenting cells. It was found that Hsp90-chaperoned proteins were presented much more selectively through the MHC class I pathway (early endosomal pathway) than through the MCH class II pathway (late endosomal pathway). In contrast, free proteins are presented preferentially through the MCH class II pathway, but not through the MCH class I pathway, resulting in antibody rather than cytolytic T lymphocyte responses. Because T. gondii-derived Hsp70 functions as a B cell mitogen, one may suggest that the immune dysfunction in patients with ASD and their families associated with the presence of several antibodies and autoantibodies directed to brain proteins is due to congenital/acquired T. gondii infection of the central nervous system. In addition, the existence of qualitative and quantitative age-specific patterns in innate immune reactivity in response to Toll-like receptors characteristic for children even up to 2 years old, as well as the parasite inhibition of IFN--induced MHC class II expression in macrophages, may be responsible for the delayed ASD diagnosis until approximately 1.5 to 2 years of age despite evidence of prenatal changes in the brain. Development of innate immunity to T. gondii LPS antigens approximately at this age as compared with much earlier efficacious immune reaction to its peptide antigens may strongly support this suggestion. Oxidative stress characteristic for ASD and several other neurodegenerative diseases induces Hsp70 (and probably other HSPs) expression, and therefore increases the net quantity of chaperones that can associate immunogenic peptides. This may affect direct T. gondii antigens presentation by MHC classes I, II, and III molecules, and therefore can serve as an explanation for the increased generation of various antibodies and autoantibodies against brain proteins reported in those patients and their families. This view is also in line with the finding that N-glycosylated structures are involved in T. gondii-host cell interactions. Finally, female autistic children with a markedly severe autism course than males had a significantly higher serum level of anti-myelin-associated glycoprotein antibodies as compared with males and controls. This may be explained by a markedly higher glucocorticosteroid receptor binding potency in the males than females associated with alterations in the receptor interaction with Hsp70 and Hsp90, which contribute to gender-related differences in vulnerability to stress-related disorders.
... CREBBP and EP300 (Histone acetyltransferase p300) acetylate histone and non-histone proteins for transcriptional regulation of antiviral interferons [40][41][42][43][44][45]. JUN is proposed to help promote IL1 and IL12 expression in influenza A. Microarray studies of cultured lymphocytes show that the vaccine ingredient thimerosal increased expression of JUN [46]. Figure 2 Functional interaction network of Rheumatoid Arthritis associated genes. ...
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Background Near universal administration of vaccines mandates intense pharmacovigilance for vaccine safety and a stringently low tolerance for adverse events. Reports of autoimmune diseases (AID) following vaccination have been challenging to evaluate given the high rates of vaccination, background incidence of autoimmunity, and low incidence and variable times for onset of AID after vaccinations. In order to identify biologically plausible pathways to adverse autoimmune events of vaccine-related AID, we used a systems biology approach to create a matrix of innate and adaptive immune mechanisms active in specific diseases, responses to vaccine antigens, adjuvants, preservatives and stabilizers, for the most common vaccine-associated AID found in the Vaccine Adverse Event Reporting System.ResultsThis report focuses on Guillain-Barre Syndrome (GBS), Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), and Idiopathic (or immune) Thrombocytopenic Purpura (ITP). Multiple curated databases and automated text mining of PubMed literature identified 667 genes associated with RA, 448 with SLE, 49 with ITP and 73 with GBS. While all data sources provided valuable and unique gene associations, text mining using natural language processing (NLP) algorithms provided the most information but required curation to remove incorrect associations. Six genes were associated with all four AIDs. Thirty-three pathways were shared by the four AIDs. Classification of genes into twelve immune system related categories identified more ¿Th17 T-cell subtype¿ genes in RA than the other AIDs, and more ¿Chemokine plus Receptors¿ genes associated with RA than SLE. Gene networks were visualized and clustered into interconnected modules with specific gene clusters for each AID, including one in RA with ten C-X-C motif chemokines. The intersection of genes associated with GBS, GBS peptide auto-antigens, influenza A infection, and influenza vaccination created a subnetwork of genes that inferred a possible role for the MAPK signaling pathway in influenza vaccine related GBS.Conclusions Results showing unique and common gene sets, pathways, immune system categories and functional clusters of genes in four autoimmune diseases suggest it is possible to develop molecular classifications of autoimmune and inflammatory events. Combining this information with cellular and other disease responses should greatly aid in the assessment of potential immune-mediated adverse events following vaccination.
... In our study, we found no difference in MT3 mRNA levels between autistic children and healthy controls, and among the gender subgroups. Discrepancies of MT expression have been reported in cultured autistic cell lines (Walker et al. 2006). In addition, metallothionein gene expression is induced by a high variety of stimuli other than mercury and different forms of MT can be regulated differently by mercury in different cell types (Aschner et al. 1997). ...
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Genetic susceptibility to high mercury body burden has been suggested as an autism risk factor in children. Metallothionein III (MT3) is the brain-specific form of the metallothionein family, which plays a key role in metal metabolism. We therefore looked for genetic variations in the MT3 gene that might increase the predisposition to autism. DNA was extracted from 132 autistic children and 132 age and gender-matched unrelated controls. All the samples were analyzed for nine single nucleotide polymorphisms (SNPs) with minor allele frequency > 10% in the MT3 gene. The mRNA levels of MT3 in white blood cells were evaluated by real-time PCR. We did not detect any association between these MT3 polymorphisms and the mRNA levels of MT3. We did not detect any association between MT3 polymorphisms and autism risk. However, we detected four novel MT3 SNPs that are not in the human SNP database. The clinical importance of these SNPs needs further investigation. Our data suggest that MT3 gene polymorphisms are not associated with autism.
... These results are uncontroversial in the empirical research on mercury as a toxin. Although some defenders of mercury in medical applications have split hairs over the impact of ethyl versus methyl mercury, both are known to be highly neurotoxic (e.g., see Geier & Geier, 2006; also Walker et al., 2006). This conference focuses primary attention on research bearing on the causes of autism because until the causes are known, treatment protocols must operate somewhat in the dark. ...
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Why is the number of new severe diagnoses still skyrocketing? It can't be all genetic. Human genes doen't change that fast. Childhood autism is now more common that spina bifida, cancer, Down syndrome, or any other childhood disorder (Muhle, et alo., 2004, Pediatrics 113). Data through 2007 shows that the autism diagnosis is still climbing. CDC also reports that about 40% of the new cases are severe. Meantime, criteria for diagnosis have hardly changed in 50 years.
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Autism spectrum disorder (ASD) is a group of complex neurodevelopmental disorders with diverse clinical manifestations and symptoms. In the last 10 years, there have been significant advances in understanding the genetic basis for ASD, critically supported through the establishment of ASD bio-collections and application in research. Here, we summarise a selection of major ASD bio-collections and their associated findings. Collectively, these include mapping ASD candidate genes, assessing the nature and frequency of gene mutations and their association with ASD clinical subgroups, insights into related molecular pathways such as the synapses, chromatin remodelling, transcription and ASD-related brain regions. We also briefly review emerging studies on the use of induced pluripotent stem cells (iPSCs) to potentially model ASD in culture. These provide deeper insight into ASD progression during development and could generate human cell models for drug screening. Finally, we provide perspectives concerning the utilities of ASD bio-collections and limitations, and highlight considerations in setting up a new bio-collection for ASD research.
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Emerging evidence supports the theory that some autism spectrum disorders (ASDs) may result from a combination of genetic/biochemical susceptibility, specifically a reduced ability to excrete mercury (Hg), and exposure to Hg at critical developmental periods. Elemental/inorganic Hg is released into the air/water where it becomes methylated and accumulates in animal tissues. The US population is primarily exposed to methyl-Hg by fish consumption. In addition, many pharmaceuticals have been, and some continue to be, a ubiquitous source of danger because they contain mercurials. Mercurials may be found in drugs for the eye, ear, nose, throat, and skin; in bleaching creams; as preservatives in cosmetics, tooth pastes, lens solutions, vaccines, allergy test and immunotherapy solutions; in antiseptics, disinfectants, and contraceptives; in fungicides and herbicides; in dental fillings and thermometers; and many other products. Hg has been found to cause immune, sensory, neurological, motor, and behavioural dysfunctions similar to traits defining/associated with ASDs, and that these similarities extend to neuroanatomy, neurotransmitters, and biochemistry. Furthermore, a review of molecular mechanisms indicates that Hg exposure can induce death, disorganization and/or damage to selected neurons in the brain similar to that seen in recent ASD brain pathology studies, and this alteration may likely produce the symptoms by which ASDs are diagnosed. Finally, a review of treatments suggests that ASD patients who undergo protocols to reduce Hg and/or its effects show significant clinical improvements in some cases. In conclusion, the overwhelming preponderance of the evidence favours acceptance that Hg exposure is capable of causing some ASDs.
Article
The frequency of zinc deficiency, copper toxicity and low zinc/copper in children with autism spectrum disorders (ASDs) may indicate decrement in metallothionein system functioning. A retrospective review of plasma zinc, serum copper and zinc/copper was performed on data from 230 children with autistic disorder, pervasive developmental disorder-NOS and Asperger's syndrome. The entire cohort's mean zinc level was 77.2 microg dl(-1), mean copper level was 131.5 microg dl(-1), and mean Zn/Cu was 0.608, which was below the 0.7 cut-off of the lowest 2.5% of healthy children. The plasma zinc/serum copper ratio may be a biomarker of heavy metal, particularly mercury, toxicity in children with ASDs.
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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.
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Historically, entities with a vested interest in a product that critics have suggested is harmful have consistently used research to back their claims that the product is safe. Prominent examples are: tobacco, lead, bisphenol A, and atrazine. Research literature indicates that about 80–90% of studies with industry affiliation found no harm from the product, while only about 10–20% of studies without industry affiliation found no harm. In parallel to other historical debates, recent studies examining a possible relationship between mercury (Hg) exposure and autism spectrum disorder (ASD) show a similar dichotomy. Studies sponsored and supported by industry or entities with an apparent conflict of interest have most often shown no evidence of harm or no “consistent” evidence of harm, while studies without such affiliations report positive evidence of a Hg/autism association. The potentially causal relationship between Hg exposure and ASD differs from other toxic products since there is a broad coalition of entities for whom a conflict of interest arises. These include influential governmental public health entities, the pharmaceutical industry, and even the coal burning industry. This review includes a systematic literature search of original studies on the potential relationship between Hg and ASD from 1999 to August 2015, finding that of the studies with public health and/or industry affiliation, 86% reported no relationship between Hg and ASD. However, among studies without public health and/or industry affiliation, only 21% find no relationship between Hg and ASD. The discrepancy in these results suggests a bias indicative of a conflict of interest.
Conference Paper
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This document contains the abstracts of presentations and writings contributed by the named persons and others at the Sertoma International Conference on Autism Spectrum Disorders. The purpose of the conference was to address the following question: Why is the number of new severe diagnoses still skyrocketing? It can't be all genetic. Human genes don't change that fast. Childhood autism is now more common than spina bifida, cancer, Down syndrome, or any other childhood disorder (Muhle, et aI., 2004, Pediatrics 1/3). The graph on the front page shows data through 2003 but the CDC reported February 9, 2007 that the red line of autism is still climbing in 2007. About 40% of the new cases are severe and criteria for those cases have hardly changed in 50 years. [Follow up research and publications show that at the time of this writing, July 22, 2016, the autism epidemic has only gotten more intense. All efforts to make it go away by creative statistics, burying the number of real cases by performing pretend searches that find "no evidence" that the number of severe cases is increasing, and so forth, have failed. The fact is that diagnoses placing individuals on the autism spectrum continue to increase. More importantly, issues raised in the presentations at this conference pointed to causal factors that are not plausibly deniable.]
Chapter
Mercury is a ubiquitous environmental contaminant. Exposures to mercury occur globally and pose significant threats to human health. Mercury toxicity to the nervous system has been extensively studied, and risks to human health as a result of mercury exposure have been evaluated on this basis, especially for exposures at high doses. Data from experimental models, such as rodent systems, suggest that mercury may also have a significant effect on the function of the immune system. However, little is known about the risks posed to human health as a result of mercury immunotoxicity, mainly due to variations in dose, route of exposure, and differences between the rodent and human immune systems. The evidence for mercury as an immunotoxic agent is reviewed here, specifically in the context of human exposures to mercury and the relevance of models of mercury immunotoxicity to human health. In light of evidence that mercury may affect the immune system, the influence of the immune system in other organ systems targeted by mercury is also reviewed.
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Autism spectrum disorders are a set of neurodevelopmental disorders that are highly hereditable. Increased genomic instability has been observed in other heritable paediatric neurobiological disorders; therefore, the aim of our study was to test the hypothesis that DNA damage is increased in children with autism and that B vitamin status may explain variations in genome integrity between autistic and normal children. We compared 35 children with autism, 27 of their siblings without autism and 25 age- and gender-matched community controls for genomic stability using the cytokinesis-block micronucleus cytome (CBMN-cyt) assay, B vitamins and homocysteine, as well as autism-related behaviours. It was found that there were no differences in CBMN-cyt biomarkers between the three groups. Vitamin B2 was significantly raised in children with autism and their siblings compared with controls (P = 0.027 and P = 0.016 respectively) but there was no difference in other B vitamins or homocysteine. In conclusion, although replication using a larger cohort is needed, it appears unlikely that genomic instability is a feature of the aetiology of autism. We cannot rule out in utero effects or other types of DNA damage not measured by the CBMN-cyt assay. Autism Res 2014, ●●: ●●–●●. © 2014 International Society for Autism Research, Wiley Periodicals, Inc.
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Historically, entities with a vested interest in a product that critics have suggested is harmful have consistently used research to back their claims that the product is safe. Prominent examples are: tobacco, lead, bisphenol A, and atrazine. Research literature indicates that about 80-90 % of studies with industry affiliation found no harm from the product, while only about 10-20 % of studies without industry affiliation found no harm. In parallel to other historical debates, recent studies examining a possible relationship between mercury (Hg) exposure and autism spectrum disorder (ASD) show a similar dichotomy. Studies sponsored and supported by industry or entities with an apparent conflict of interest have most often shown no evidence of harm or no "consistent" evidence of harm, while studies without such affiliations report positive evidence of a Hg/autism association. The potentially causal relationship between Hg exposure and ASD differs from other toxic products since there is a broad coalition of entities for whom a conflict of interest arises. These include influential governmental public health entities, the pharmaceutical industry, and even the coal burning industry. This review includes a systematic literature search of original studies on the potential relationship between Hg and ASD from 1999 to date, finding that of the studies with public health and/or industry affiliation, 86 % reported no relationship between Hg and ASD. However, among studies without public health and/or industry affiliation, only 19 % find no relationship between Hg and ASD. The discrepancy in these results suggests a bias indicative of a conflict of interest.
Article
Multiple observations indicate that environmental and epigenetic factors play an important role in the emergence of autism spectrum disorders (ASD). Growing ASD incidence rates, the incomplete penetrance of many rare variants linked to autism, and increased exposure to environmental contaminants all strongly support the role of gene × environment interactions in a substantial fraction of autistic patients. Within this framework, genetically susceptible individuals exposed to detrimental environmental factors at critical times during neurodevelopment might undergo disrupted brain morphogenesis, neuronal connectivity, and synaptic functioning consequently yielding ASD. Several teratogenic drugs and prenatal viral infections are able to cause autism in humans, as supported by case reports, cohort studies, and animal models. Moreover, recent studies have shown that some newly identified potential neurotoxicants may negatively affect developmental trajectories, leading to altered cognitive, attentive, behavioral, and motor performances, as well as to systemic abnormalities frequently seen in autistic individuals. A variety of mechanisms are potentially involved, ranging from oxidative and inflammatory brain damage to altered gene expression and impaired signal transduction. More research is needed to thoroughly investigate the effects of these compounds on neurodevelopment, to validate their involvement specifically in ASD, to study gene × environment interactions in potentially susceptible individuals, and to plan targeted prevention strategies.
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The propaganda dispensed by Public health care and vaccine apologists is, at best, a weak attempt to rationalize the healthcare establishment's positions using all the tools of doublespeak or, as George Orwell's called it in his book 1984, "newspeak", to: (a) mislead, (b) distort reality, (c) pre-tend to communicate, (d) make the bad seem good, (e) avoid and/or shift responsibility, (f) make the negative appear positive, (g) create a false ver-bal map of the world, and (h) create dissonance between reality and what their narrative said or did not say. Such propaganda often relies on half-truths and/or superficially logical, but foundationally flawed, phrasing. However, this propaganda is funda-mentally flawed and based on pseudo-science or non-reviewable statistical studies of medical records, where, contrary to ethical science, the study design, data selection/rejection criteria, exact approach used to evaluate the data, and/or the original data set itself is kept confidential making inde-pendent evaluation/verification of the published findings impossible.doc/1G1-170731919.html) triggered this presentation of the factual realities that rebut the myths/misconceptions pre-sented in that article and/or in similar articles published and/or underwritten by the purveyors of vaccines and vaccination recommendations. Each myth/misconception is summarized in a short statement and then addressed by presenting the factual reality and when appropriate, providing peer-reviewed references that support this reality.
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Autism spectrum disorders are a heterogeneous group of neurodevelopmental conditions characterised by impairments in reciprocal social interaction, communication and stereotyped behaviours. As increased DNA damage events have been observed in a range of other neurological disorders, it was hypothesised that they would be elevated in lymphoblastoid cell lines (LCLs) obtained from children with autism compared with their non-autistic siblings. Six case-sibling pairs of LCLs from children with autistic disorder and their non-autistic siblings were obtained from the Autism Genetic Resource Exchange (AGRE) and cultured in standard RPMI-1640 tissue culture medium. Cells were exposed to medium containing either 0, 25, 50, 100 and 200 µM hydrogen peroxide (an oxidative stressor) or 0, 5, 10, 20 and 40 µM s-nitroprusside (a nitric oxide producer) for 1h. Following exposure, the cells were microscopically scored for DNA damage, cytostasis and cytotoxicity biomarkers as measured using the cytokinesis-block micronucleus cytome assay. Necrosis was significantly increased in cases relative to controls when exposed to oxidative and nitrosative stress (P = 0.001 and 0.01, respectively). Nuclear division index was significantly lower in LCLs from children with autistic disorder than their non-autistic siblings when exposed to hydrogen peroxide (P = 0.016), but there was no difference in apoptosis, micronucleus frequency, nucleoplasmic bridges or nuclear buds. Exposure to s-nitroprusside significantly increased the number of micronuclei in non-autistic siblings compared with cases (P = 0.003); however, other DNA damage biomarkers, apoptosis and nuclear division did not differ significantly between groups. The findings of this study show (i) that LCLs from children with autism are more sensitive to necrosis under conditions of oxidative and nitrosative stress than their non-autistic siblings and (ii) refutes the hypothesis that children with autistic disorder are abnormally susceptible to DNA damage.
Chapter
Mercury occurs as elemental mercury and as inorganic and organic compounds (mercury vapor, mercury liquid, mercury salts, short-chain alkylmercury compounds, alkoxyalkylmercury compounds, and phenylmercury compounds), all with different toxicological properties. Total mercury can be analyzed in water, air, and biological material by cold vapor atomic absorption methods and by neutron activation analysis, and can be detected down to concentrations of a tenth of a nanogram per gram in biological material. Methylmercury (MeHg) can be detected in biological material at levels of a few nanograms by extraction with benzene after strong acidification with hydrochloric acid, followed by gas chromatographic analysis of MeHg chloride. Other analytical methods for speciating inorganic mercury and several of the organomercurial forms have also been published. These methods include isotope dilution mass spectrometry, time-of-flight mass spectrometry, high performance liquid chromatography inductively coupled (ICP) plasma mass spectrometry, capillary electrophoresis-ICP, gas chromatography-ICP, and X-ray absorption fine structure spectroscopy.
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Although autism spectrum conditions (ASCs) are defined behaviorally, they also involve multileveled disturbances of underlying biology that find striking parallels in the physiological impacts of electromagnetic frequency and radiofrequency exposures (EMF/RFR). Part I of this paper will review the critical contributions pathophysiology may make to the etiology, pathogenesis and ongoing generation of core features of ASCs. We will review pathophysiological damage to core cellular processes that are associated both with ASCs and with biological effects of EMF/RFR exposures that contribute to chronically disrupted homeostasis. Many studies of people with ASCs have identified oxidative stress and evidence of free radical damage, cellular stress proteins, and deficiencies of antioxidants such as glutathione. Elevated intracellular calcium in ASCs may be due to genetics or may be downstream of inflammation or environmental exposures. Cell membrane lipids may be peroxidized, mitochondria may be dysfunctional, and various kinds of immune system disturbances are common. Brain oxidative stress and inflammation as well as measures consistent with blood-brain barrier and brain perfusion compromise have been documented. Part II of this paper will review how behaviors in ASCs may emerge from alterations of electrophysiological oscillatory synchronization, how EMF/RFR could contribute to these by de-tuning the organism, and policy implications of these vulnerabilities. Changes in brain and autonomic nervous system electrophysiological function and sensory processing predominate, seizures are common, and sleep disruption is close to universal. All of these phenomena also occur with EMF/RFR exposure that can add to system overload ('allostatic load') in ASCs by increasing risk, and worsening challenging biological problems and symptoms; conversely, reducing exposure might ameliorate symptoms of ASCs by reducing obstruction of physiological repair. Various vital but vulnerable mechanisms such as calcium channels may be disrupted by environmental agents, various genes associated with autism or the interaction of both. With dramatic increases in reported ASCs that are coincident in time with the deployment of wireless technologies, we need aggressive investigation of potential ASC - EMF/RFR links. The evidence is sufficient to warrant new public exposure standards benchmarked to low-intensity (non-thermal) exposure levels now known to be biologically disruptive, and strong, interim precautionary practices are advocated.
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Synopsis Two previous epidemiological studies of autistic twins suggested that autism was predominantly genetically determined, although the findings with regard to a broader phenotype of cognitive, and possibly social, abnormalities were contradictory. Obstetric and perinatal hazards were also invoked as environmentally determined aetiological factors. The first British twin sample has been re-examined and a second total population sample of autistic twins recruited. In the combined sample 60% of monozygotic (MZ) pairs were concordant for autism versus no dizygotic (DZ) pairs; 92% of MZ pairs were concordant for a broader spectrum of related cognitive or social abnormalities versus 10% of DZ pairs. The findings indicate that autism is under a high degree of genetic control and suggest the involvement of multiple genetic loci. Obstetric hazards usually appear to be consequences of genetically influenced abnormal development, rather than independent aetiological factors. Few new cases had possible medical aetiologies, refuting claims that recognized disorders are common aetiological influences.
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Expression of the metallothionein (MT) gene in frozen human lymphocytes has been developed as a new molecular biomarker of heavy metal exposure. Workers at a Polish battery factory with high exposure to cadmium were monitored for airborne exposure and blood cadmium levels. A novel quantitative reverse transcription-PCR (RT-PCR) technique, making use of a homologous internal standard, was used to assess the level of MT-specific mRNA in frozen stored aliquots of blood samples taken from exposed and control workers. Results from this assay showed a statistically significant 2.5-fold increase in MT mRNA in exposed compared to control workers. The RT-PCR results also showed significant correlation with airborne cadmium, as registered on personal monitors and with blood cadmium levels. The results suggest that gene induction measured by quantitative RT-PCR is a promising approach for application as a biomarker of biologically effective dose in small samples of frozen tissues or cells.
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Molecular Psychiatry publishes work aimed at elucidating biological mechanisms underlying psychiatric disorders and their treatment
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Thimerosal is a preservative that has been used in manufacturing vaccines since the 1930s. Reports have indicated that infants can receive ethylmercury (in the form of thimerosal) at or above the U.S. Environmental Protection Agency guidelines for methylmercury exposure, depending on the exact vaccinations, schedule, and size of the infant. In this study we compared the systemic disposition and brain distribution of total and inorganic mercury in infant monkeys after thimerosal exposure with those exposed to MeHg. Monkeys were exposed to MeHg (via oral gavage) or vaccines containing thimerosal (via intramuscular injection) at birth and 1, 2, and 3 weeks of age. Total blood Hg levels were determined 2, 4, and 7 days after each exposure. Total and inorganic brain Hg levels were assessed 2, 4, 7, or 28 days after the last exposure. The initial and terminal half-life of Hg in blood after thimerosal exposure was 2.1 and 8.6 days, respectively, which are significantly shorter than the elimination half-life of Hg after MeHg exposure at 21.5 days. Brain concentrations of total Hg were significantly lower by approximately 3-fold for the thimerosal-exposed monkeys when compared with the MeHg infants, whereas the average brain-to-blood concentration ratio was slightly higher for the thimerosal-exposed monkeys (3.5 +/- 0.5 vs. 2.5 +/- 0.3). A higher percentage of the total Hg in the brain was in the form of inorganic Hg for the thimerosal-exposed monkeys (34% vs. 7%). The results indicate that MeHg is not a suitable reference for risk assessment from exposure to thimerosal-derived Hg. Knowledge of the toxicokinetics and developmental toxicity of thimerosal is needed to afford a meaningful assessment of the developmental effects of thimerosal-containing vaccines.
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Neurotoxic effects of methylmercury were investigated in vitro in primary cultures of human neurons and astrocytes isolated from human fetal brain and in the human neuroblastoma cell line SH-SY5Y. The protection provided by agents with antioxidant properties was tested in these cultures to examine the oxidative stress mechanism of methylmercury poisoning. After 24 h of exposure to methylmercury, LC50 values were 6.5, 8.1 and 6.9 muM for human neurons, astrocytes and neuroblastoma cells, respectively, and the degree of cell damage increased at longer exposure times. Depletion of the cellular pool of reduced glutathione (GSH) by treatment with buthionine sulfoximine potentiated methylmercury cytotoxicity in all three cell types; neuroblastoma cells were the most sensitive. Addition of GSH extracellularly blocked methylmercury neurotoxicity in all cell types. The major beneficial effect of GSH could be attributed to its capacity to form conjugates with methylmercury, which reduces the availability of these organometallic molecules to the cells and facilitates their efflux. Cysteine protected astrocytes and neuroblastoma cells from methylmercury neurotoxicity, while selenite, Vitamin E and catalase produced some minor protective effects in three cell types, particularly in neurons. The present study showed that the human neural cells tested had differential responses to methylmercury: astrocytes were resistant to methylmercury neurotoxicity and neurons were more most responsive to protection afforded by antioxidants among the three cell types.
Article
Chronic exposure to cadmium (Cd) via food and drinking water is a major human health concern. We have previously shown that metallothionein (MT), a metal-binding protein, plays an important role in protecting against Cd toxicity produced by repeated sc injections. However, it is unclear whether MT protects against Cd-induced nephrotoxicity following chronic oral exposure, a route with obvious human relevance. To clarify this issue, MT-I/II knockout (MT-null) and background-matched wild-type (WT) mice were allowed free access to drinking water containing CdCl 2 (30, 100, and 300 ppm Cd), or feed containing CdCl 2 (100 ppm Cd) for 6 months, and the resultant nephrotoxicity was examined. Chronic oral Cd exposure produced a dose-dependent accumulation of Cd in liver and kidney of WT mice, reaching levels up to 50 μg Cd/g tissue. Immunohistological localization of renal MT indicated that chronic oral Cd exposure in WT mice greatly increased MT in the proximal tubules and the medulla, with cellular localization in both the cytoplasm and nuclei. As expected, no MT was detected in kidneys of MT-null mice. After 6 months of Cd exposure, tissue Cd concentrations in MT-null mice were only about one-fifth of that in WT mice. Even though the renal Cd concentrations were much lower in the MT-null mice, they were more sensitive than WT mice to Cd-induced renal injury, as evidenced by more severe nephropathic lesions, increased urinary excretion of y-glutamyl-transferase and glucose, and elevated blood urea nitrogen. Six months of Cd exposure to MT-null animals resulted in greater increases in renal caspase-3 activity, an indicator of apoptosis, than to WT mice. In conclusion, this study demonstrates that lack of MT renders MT-null mice vulnerable to Cd-induced nephrotoxicity after chronic oral exposure, the primary route of human Cd exposure.
Article
Cadmium (Cd) is an environmental pollutant. Chronic exposure of humans to Cd results in various maladies, including anemia and altered immune function. Metallothionein (MT) has been proposed to play an important role in Cd detoxication. Thus, we hypothesized that intracellular MT protects against Cd-induced hematotoxicity and immunotoxicity. Control and MT-I/II knockout (MT-null) mice were given s.c. injections of CdCl2 over a wide range of doses, 6 times/week for up to 10 weeks. Cd-induced anemia was evident after 5 weeks of exposure and progressed with time. MT-null mice were about 10 times more susceptible to Cd-induced anemia, as evidenced by decreased erythrocytes (25%), hemoglobin concentration (30%), and hematocrit (35%) after 10 weeks of Cd injections. Cd produced dose- and time-dependent increases in neutrophils (7×), along with a marked elevation of serum IL-1β (6×) and TNF-α (20×) levels. MT-null mice were more susceptible than controls to Cd-induced alterations in peripheral leukocytes and cytokine levels. Chronic exposure to Cd also produced dose- and time-dependent splenomegaly (5×), with loss of lymphoid structure, inflammation, hyperplasia, appearance of giant cells, and fibrosis. Thymus weights were decreased by Cd in a dose-dependent manner (60%). MT-null mice were also approximately 10 times more susceptible than controls to these lesions. In conclusion, the present study demonstrates that repeated injections of Cd produces hematotoxic and immunotoxic effects, and intracellular MT protects against these chronic Cd-induced effects.
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A symposium on the role of brain metallothioneins (MTs) in physiology and pathology was held at the 1996 Annual Society of Toxicology Meeting in Anaheim, California. The objectives of this symposium were to: (1) review the physiologic function of MTs, (2) examine the distribution of brain MTs with particular emphasis on cell-specific localization (neurons vs neuroglia), (3) discuss MT gene responsiveness upon toxic insult with metals, and (4) discuss the potential role of MTs in the etiology of neurodegenerative disorders. Dr. Cherian discussed the biochemical properties of the MTs, emphasizing structural similarities and differences between the MTs. Dr. Klaassen addressed the expression and distribution of the MTs in brains with special reference to the cell-specific localization of MTs. Dr. Aschner provided data illustrating a potential role for MTs in attenuating the cytotoxicity caused by methylmercury (MeHg) in cultured neonatal astrocytes. Dr. Palmiter discussed the properties of MT-III and the increased sensitivity of MT-III knockout mice to kainate-induced seizures. Cerebral zinc metabolism, its relationship to MT homeostasis, and its pathogenic potential in Alzheimer's disease was addressed by Dr. Bush.
Article
In connection with the dealkylation of methyl mercury (MeHg) and ethyl Hg (EtHg) with reactive oxygen-producing systems, we examined the ability of phagocytic cells to degrade MeHg or EtHg into inorganic mercury in vitro by collecting them from blood or peritoneal cavity of several species of animal. EtHg was readily degraded by human polymorphonuclear leukocytes (PMN), rat PMN, guinea-pig PMN, rabbit PMN, guinea-pig macrophages (M phi), human monocytes and guinea-pig eosinophils. In contrast, rat hepatocytes and the M phi hybridoma clone 39 cells were weaker in their degrading ability. Degradation of MeHg by these cells was always much weaker than EtHg, under identical conditions; however, by increasing the cell numbers, MeHg was appreciably degraded by human PMN, rat PMN and rabbit PMN. The reactive oxygen species mainly responsible for alkyl Hg degradation seemed to be hydroxyl radicals produced by M phi, and hypochlorous acid produced by PMN, monocytes and eosinophils. It was also suggested that the degradation of alkyl Hg by these cells might be an intraphagosomal event.
Article
Human peripheral blood lymphocytes have the capacity to produce metallothioneins (MTs) as a protective response to cadmium exposure. To define the range of metal species inducing lymphocyte MTs, cellular proteins synthesized after exposure to each of 11 heavy metals were analyzed by gel electrophoresis. Toxic metals such as cadmium, mercury and silver were found to induce thioneins (apoproteins of MTs) at relatively low concentrations (maximum at approximately 10 microM), whereas less toxic metals such as zinc, copper and nickel were inductive at relatively high concentrations (maximum at approximately 200 microM). Tin, lead, iron, cobalt, and manganese did not induce thioneins. The heavy metal specificity of MT induction in the lymphocyte resembles that in the liver, and the regulatory mechanism of MT production seems to be similar in both of these tissues. In the cells exposed to highly toxic metals such as cadmium and mercury, expression of cytotoxicity (represented by decline of cysteine uptake) was remarkable at the metal concentrations higher than those saturating thionein induction, supporting the protective role of MTs against heavy metals.
Article
To help to evaluate the role of metallothionein (MT) in peripheral-blood leucocytes, we examined MT protein and mRNA levels in these cells before and after exposure to CdCl2 in culture. Protein was assayed by 109Cd2+ binding, and RNA by dot-blot hybridization. MT was induced in both lymphocytes and adherent monocytes about 10-fold with a 12 h exposure to 10 microM-CdCl2, but absolute levels were 3-fold higher in monocytes: 57 x 10(5) (+Cd2+) versus 6 x 10(5) (-Cd2+) molecules/cell for monocytes; 18 x 10(5) (+Cd2+) versus 2 x 10(5) (-Cd2+) for lymphocytes. Polymorphonuclear cells expressed relatively little MT (0.6 x 10(5) molecules/cell), and this did not change with phorbol ester stimulation or exposure to Cd2+, arguing against a direct protective role for MT in activated neutrophils. MT mRNA levels corresponded qualitatively to expression of protein in these cells. Our data provide quantitative comparisons of leucocyte MT expression and regulation in the human population. Variation in both basal and induced MT mRNA levels reflects environmental or experimental (intra-individual) and possibly genetic (inter-individual) differences.
Article
Metallothioneins are induced by both Zn2+ and dexamethasone in HeLa cells grown in serum-free medium. Dexamethasone is able to induce metallothionein synthesis in HeLa cells in virtually zincfree medium ([Zn2+] = 0.1 μM). The presence of dexamethasone does not shift the dose/response curve for metallothionein induction by Zn2+, further indicating that the two inducers work through independent mechanisms. Dexamethasone stimulates Zn2+ uptake 1.7-fold over 24 h. However, there is no increase in Zn2+ uptake during the first 4 h. In contrast, metallothionein synthesis in response to either Zn2+ or dexamethasone is clearly observable within 4 h of exposure to either inducer. The increased intracellular 65Zn2+ content observed at 24 h is completely accounted for by the increased level of metal bound to metallothionein. In a continuous labeling experiment the rate of synthesis of metallothionein reached a steady state after about 4 h, in response to either inducer. The lag period was identical for both dexamethasone and Zn2+, with similarly shaped induction curves. Induction by dexamethasone, but not by Zn2+, was inhibited by progesterone. Zn2+ and dexamethasone appear to induce metallothionein synthesis in HeLa cells by mechanisms independent of one another.
Article
Synopsis Personality characteristics of 87 parents of autistic probands and 38 parents of Down's syndrome probands were examined using a standardized personality interview. Using best-estimate ratings derived from subject and informant interviews, parents of autistic individuals were rated significantly higher than controls on three characteristics: aloof, untactful and undemonstrative. When ratings were based on interviews with subjects only, parents of autistic probands were rated as significantly more aloof, untactful and unresponsive. There were no significant differences between parent groups on ratings based on informant interviews only. The implications of these findings for future family studies of autism are discussed.
Article
Mercuric chloride (MC, Hg2+) and methylmercury (MeHg, CH3Hg+) significantly inhibited the initial rates of uptake of 86Rb (a tracer for K+), as well as the Na(+)-dependent uptake of [3H]-L-glutamate. Both mercury species were also found to increase [3H]-D-aspartate and 86Rb release from cultured astrocytes. Astrocytes were more sensitive to the effects of MC with IC50's for glutamate and Rb uptake an order of magnitude lower than those noted for the organic species (MeHg). Increased potency, and irreversibility relative to MeHg, were also noted for MC induced astrocytic D-aspartate and Rb release. These observations support the hypothesis that the astrocyte plasma membrane is an important target for mercurials and specifically that low concentrations of MC and MeHg inhibit the ability of astrocytes to maintain transmembrane ion gradients. The propensity of MC to interfere with astrocytic functions, relative to MeHg, was also corroborated by measurements on the inducibility of the astrocytic metalloprotein, metallothionein (MT). Whilst a dose-dependent increase in MT protein synthesis occurred upon exposure to either MC or MeHg, MC was shown to be the more potent of the mercurials. The greater susceptibility of astrocytes to MC compared with MeHg lends support, at the cellular level, to the hypothesis that accumulation of inorganic mercury (MC) at an order of magnitude lower concentration than MeHg, may be equally neurotoxic.
Article
The diagnostic boundaries of the behavioural phenotype for autism were examined in 28 MZ pairs and 20 DZ same-sex twin pairs, where one or both twins had autism. In the non-autistic cotwin (i.e. in twin pairs discordant for autism) it was common to find language impairments in childhood and social deficits persisting into adulthood. Concordance for this broader phenotype was much greater in MZ pairs than DZ pairs, indicating a strong genetic component. Behavioural and cognitive manifestations of autism were compared both within and between MZ twin pairs. The variation was as great within MZ twin pairs as between pairs, suggesting that it does not index genetic heterogeneity (although aetiological heterogeneity probably exists). Current diagnostic practices need re-evaluation.
Article
Studies of families ascertained through a single autistic proband suggest that the genetic liability for autism may be expressed in nonautistic relatives in a phenotype that is milder but qualitatively similar to the defining features of autism. The objective of this study was to examine behaviors that may define this broader phenotype in relatives ascertained through two autistic siblings. The authors used a semistructured family history interview to compare the rates of social and communication deficits and stereotyped behaviors in relatives ascertained through two autistic siblings (families with multiple-incidence autism; 25 families) with the rates in relatives of Down syndrome probands (30 families). Higher rates of social and communication deficits and stereotyped behaviors were found in the relatives in the families with multiple-incidence autism. These data suggest that further studies should be undertaken to delineate the boundaries of the broader autism phenotype and that this broader phenotype should be included in some future genetic analyses of this disorder.
Article
First-degree relatives of 99 autism probands and of 36 Down's syndrome controls were assessed with standardised tests of intellectual functioning, reading, and spelling. Higher mean verbal IQ scores, and discrepancies in favour of verbal scores, were characteristic of autism relatives. No consistent differences were found on performance scales, reading, and spelling tests. Among autism relatives, siblings affected with the broad phenotype of autism had significantly lower IQ scores and poorer reading and spelling performances than unaffected siblings. However, the small size of the cognitive difference and the lack of a distinctive cognitive profile indicates that standardised cognitive measures used in this study are unlikely to improve the operationalised definition of the broad phenotype of autism. The slightly superior verbal performance of relatives in the autism group might represent some form of heterozygote advantage.
Article
Metallothionein (MT) proteins play an important role in the detoxification of heavy metals. Since methylmercury (MeHg) preferentially accumulates in astrocytes, we investigated the ability of the astrocyte-specific MT isoform, MT-I, to attenuate MeHg-induced cytotoxicity. Increased astrocytic MT expression was achieved by 24-h pretreatment of neonatal rat primary astrocyte cultures with 100 microM zinc (ZnSO4). Subsequently, the astrocytes were treated with MeHg (10 microM), and its toxic effects on cell volume, Na+ uptake, and K+ release were investigated and compared to cells treated with or without MeHg, but in the absence of Zn pretreatment. Pretreatment of astrocytes with Zn was associated with a 2.9-fold increase in MT protein levels (P<0.02), and a 5.6-fold increase in MT mRNA levels (p<0.002) compared to control astrocytes. Astrocytes expressing increased MT protein levels were resistant to MeHg-induced swelling. In isotonic buffer the effect of MeHg on swelling was abolished (p<0.01) by 24-h Zn pretreatment, in such a way that volume profiles in these cells did not differ from controls. Zn-induced increased expression of MTs was also associated with significant attenuation of astrocytic Na+ uptake (p<0.01) and Rb+ (a marker for K+) release (p<0.001) in response to treatment with MeHg. These results demonstrate (1) that astrocytes can be induced to express high levels of MT proteins by pretreatment with Zn, and (2) that Zn confers resistance against the acute effect of MeHg on astrocytic swelling and the associated changes in ion (Na+ and K+) transport. Taken together, the data suggest that astrocytic MT induction offers effective cellular adaptation to MeHg cytotoxicity.
Article
Metallothionein-I (MT-I) was expressed in neonatal rat primary astrocyte cultures and an astrocytoma cell line by pGFAP-MT-I plasmid transfection under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Following transient transfection of the pGFAP-MT-I plasmid, MT-I mRNA and MT-I protein levels were determined by northern blot and immunoprecipitation analyses, respectively. The ability of cells over-expressing MT-I to withstand acute methylmercury (MeHg) treatment was measured by the release of preloaded Na251CrO4, an indicator of membrane integrity. Transfection with the pGFAP-MT-I plasmid led to increased mRNA (2. 5-fold in astrocytes and 7.4-fold in astrocytomas) and MT-I protein (2.4-fold in astrocytes and 4.0-fold in astrocytomas) levels compared with their respective controls. Increased expression of MT-I was associated with attenuated release of Na251CrO4 upon MeHg (5 microM) treatment. These results demonstrate that MT-I can be highly expressed both in primary astrocyte cultures and astrocytomas by pGFAP-MT-I plasmid transfection, and lend credence to the hypothesis that increased expression of MT-I affords protection against the cytotoxic effects of MeHg. Taken together, the data suggest that MT offer effective cellular adaptation to MeHg cytotoxicity.
Article
The mechanisms associated with metallothionein (MT) gene regulation are complex and poorly understood. Only a modest increase in brain MT expression levels is attained by exposure to metals, MT gene transfection, and MT gene knock-in techniques. Accordingly, in the present study, MT null astrocytes isolated from transgenic mice deficient in MT-I and MT-II genes were introduced as a zero background model of MT expression. MT protein levels were determined by western blot analysis. MT proteins in MT-I and MT-II null astrocytes were undetectable. Transient MT-I gene transfection increased the levels of foreign MT expression in MT-I and MT-II null astrocytes by 2.3-fold above basal levels in wild-type astrocytes. Intracellular Na(2)51CrO(4) efflux and D-[2,3-3H]aspartate uptake were studied as indices of acute methylmercury (MeHg) (5 microM) cytotoxicity. In MT-I and MT-II knockout astrocytes MeHg led to significant (p<0.01) increase in Na(2)51CrO(4) efflux and a significant (p<0.05) decrease in the initial rate (1 min) of D-[2, 3-3H]aspartate uptake compared to MT-I and MT-II knockout controls. Transfection of the MT-I gene in MT-I and MT-II null mice significantly (p<0.01) decreased the effect of MeHg on Na(2)51CrO(4) efflux in MT null, as well as wild-type astrocytes. MT-I gene transfection in MT-I and MT-II null astrocytes reversed the inhibitory effect of MeHg on D-[2,3-3H]aspartate uptake, such that initial rates of uptake in MT-I transfected cells in the presence and absence of MeHg (5 microM) were indistinguishable. These results demonstrate that: (1) astrocytes lacking MTs are more sensitive to MeHg than those with basal MT protein levels, (2) the MT-I gene can be overexpressed in MT-I and MT-II null astrocytes by transient MT-I gene transfection, and (3) that foreign MT expression endows astrocytes with increased resistance to MeHg.
Article
Zinc plays an important role in the maintenance of the immune system. While the mechanisms of zinc ions interaction with immune cells are still poorly understood, a striking concurrent effect of zinc is the induction of the biosynthesis of metallothioneins (MT), a group of low molecular weight, cysteine-rich metal-binding proteins, believed to play a role in zinc homeostasis. In humans, they are encoded by a family of genes, located at 16q13 containing 10 functional and 7 non-functional MT isoforms. In this work we analyzed the spectrum of different isoforms in human peripheral blood lymphocytes. It was demonstrated by RT-PCR that the MT-2a, MT-1a, MT-1e, MT-1f, MT-1g, MT-1h and MT-1x genes are expressed in these cells and that these isoforms are further upregulated by zinc, as examined by quantitative RT-PCR. Surprisingly, RT-PCR also showed the presence, even in unstimulated cells, of MT-3 transcripts, which are considered as brain-specific isoforms. In an effort to determine whether MTmRNA abundance is translated into MT protein, MT isolated from zinc-treated lymphocytes by gel chromatography was resolved into 7 metal-binding fractions by using RP-HPLC. Automatic Edman-degradation of the different fractions revealed the presence of MT-2a, MT-1a, MT-1e, MT-1f, MT-1g, MT-1h, MT-1x and MT-1k, an isoform which until now was only identified at the level of protein in human liver and kidney tissue.
Article
Cadmium (Cd) and arsenic (As) are important inorganic toxicants in the environment. Humans certainly have the potential to be exposed to the mixtures of Cd and As, but the toxicological interactions of these inorganic mixtures are poorly defined. Metallothionein (MT) is a cysteine-rich, metal-binding protein that plays an important role in Cd detoxication, but its role in As toxicity is less certain. To examine the role of MT in Cd- and/or As-induced nephrotoxicity, MT-I/II-knockout (MT-null) mice and background-matched wild-type (WT) mice were fed CdCl(2) (100 ppm Cd) in the diet, NaAsO(2) (22.5 ppm As) in the drinking water, or Cd plus As for 4 months. Subsequently, nephrotoxicity was examined by morphological and biochemical techniques. Chronic exposure to Cd produced more renal toxicity than As, and the combination of Cd and As produced even more renal injury than caused by either of the chemicals given alone. In mice receiving Cd plus As, proximal tubule degeneration and atrophy, glomerular swelling and interstitial fibrosis were more severe than those produced by either inorganic. Furthermore, lack of MT rendered MT-null mice more sensitive than WT mice to the nephrotoxicity produced by chronic Cd- and/or As-exposure. MT-null mice were especially susceptible to the toxicity produced by the combination of Cd and As, as evidenced by decreased body weight, enzymuria, glucosuria, proteinuria and nephropathy. In conclusion, this study indicates that As may potentiate Cd nephrotoxicity during the long-term, combined exposure, and that intracellular MT plays a role in decreasing the nephropathy of combined exposure to Cd and As.
Article
Chronic exposure to cadmium (Cd) via food and drinking water is a major human health concern. We have previously shown that metallothionein (MT), a metal-binding protein, plays an important role in protecting against Cd toxicity produced by repeated sc injections. However, it is unclear whether MT protects against Cd-induced nephrotoxicity following chronic oral exposure, a route with obvious human relevance. To clarify this issue, MT-I/II knockout (MT-null) and background-matched wild-type (WT) mice were allowed free access to drinking water containing CdCl(2) (30, 100, and 300 ppm Cd), or feed containing CdCl(2) (100 ppm Cd) for 6 months, and the resultant nephrotoxicity was examined. Chronic oral Cd exposure produced a dose-dependent accumulation of Cd in liver and kidney of WT mice, reaching levels up to 50 microg Cd/g tissue. Immunohistological localization of renal MT indicated that chronic oral Cd exposure in WT mice greatly increased MT in the proximal tubules and the medulla, with cellular localization in both the cytoplasm and nuclei. As expected, no MT was detected in kidneys of MT-null mice. After 6 months of Cd exposure, tissue Cd concentrations in MT-null mice were only about one-fifth of that in WT mice. Even though the renal Cd concentrations were much lower in the MT-null mice, they were more sensitive than WT mice to Cd-induced renal injury, as evidenced by more severe nephropathic lesions, increased urinary excretion of gamma-glutamyl-transferase and glucose, and elevated blood urea nitrogen. Six months of Cd exposure to MT-null animals resulted in greater increases in renal caspase-3 activity, an indicator of apoptosis, than to WT mice. In conclusion, this study demonstrates that lack of MT renders MT-null mice vulnerable to Cd-induced nephrotoxicity after chronic oral exposure, the primary route of human Cd exposure.
Article
Genes differentially expressed in association with disruption of the metallothionein gene were screened using two hepatic stellate cell lines isolated and established from the livers of normal 129/Sv (IMS/N cells) and transgenic mice deficient in the genes for metallothionein-I and -II (IMS/MT (-) cells). We found one cDNA (tentatively named NM31) that was expressed only in IMS/IN cells. Transfecting IMS/MT (-) cells with the genes for both metallothionein-I and -II resulted in NM31 expression. These results suggest that metallothionein is essential for NM31 gene expression. The nucleotide sequence of NM31 (294 bp) was identical to the 3' region of 3.1 mRNA (PTZ 17), which is abundant in the embryonic mouse brain and is related to chemically induced seizures. The present study indicates that metallothionein mediates the expression of specific genes. This is a novel explanation for some of the functions of metallothionein.
Article
On July 7, 1999, the American Academy of Pediatrics and the US Public Health Service issued a joint statement calling for removal of thimerosal, a mercury-containing preservative, from vaccines. This action was prompted in part by a risk assessment from the Food and Drug Administration that is presented here. The risk assessment consisted of hazard identification, dose-response assessment, exposure assessment, and risk characterization. The literature was reviewed to identify known toxicity of thimerosal, ethylmercury (a metabolite of thimerosal) and methylmercury (a similar organic mercury compound) and to determine the doses at which toxicity occurs. Maximal potential exposure to mercury from vaccines was calculated for children at 6 months old and 2 years, under the US childhood immunization schedule, and compared with the limits for mercury exposure developed by the Environmental Protection Agency (EPA), the Agency for Toxic Substance and Disease Registry, the Food and Drug Administration, and the World Health Organization. Delayed-type hypersensitivity reactions from thimerosal exposure are well-recognized. Identified acute toxicity from inadvertent high-dose exposure to thimerosal includes neurotoxicity and nephrotoxicity. Limited data on toxicity from low-dose exposures to ethylmercury are available, but toxicity may be similar to that of methylmercury. Chronic, low-dose methylmercury exposure may cause subtle neurologic abnormalities. Depending on the immunization schedule, vaccine formulation, and infant weight, cumulative exposure of infants to mercury from thimerosal during the first 6 months of life may exceed EPA guidelines. Our review revealed no evidence of harm caused by doses of thimerosal in vaccines, except for local hypersensitivity reactions. However, some infants may be exposed to cumulative levels of mercury during the first 6 months of life that exceed EPA recommendations. Exposure of infants to mercury in vaccines can be reduced or eliminated by using products formulated without thimerosal as a preservative.
Article
Autism is a syndrome characterized by impairments in social relatedness and communication, repetitive behaviors, abnormal movements, and sensory dysfunction. Recent epidemiological studies suggest that autism may affect 1 in 150 US children. Exposure to mercury can cause immune, sensory, neurological, motor, and behavioral dysfunctions similar to traits defining or associated with autism, and the similarities extend to neuroanatomy, neurotransmitters, and biochemistry. Thimerosal, a preservative added to many vaccines, has become a major source of mercury in children who, within their first two years, may have received a quantity of mercury that exceeds safety guidelines. A review of medical literature and US government data suggests that: (i) many cases of idiopathic autism are induced by early mercury exposure from thimerosal; (ii) this type of autism represents an unrecognized mercurial syndrome; and (iii) genetic and non-genetic factors establish a predisposition whereby thimerosal's adverse effects occur only in some children.
Article
The hepatic and the renal subcellular distribution of zinc, cadmium or mercury and induction of tissue metallothionein (MT) at 24, 48 and 72 h following an oral equimolar dose (15 micro;mol metal/kg) of zinc (II) chloride, cadmium (II) chloride or mercury (II) chloride in male albino mice were investigated. There was a moderate increase in hepatic and renal zinc levels mainly in their nuclear mitochondrial fraction (NMF) 24 h post zinc chloride administration. Subsequently, the hepatic zinc increased and the renal zinc declined with time. The zinc-induced hepatic MT level was maximum at 48 h, which decreased slightly thereafter, while there was no marked increase in renal MT level at any time interval. The cadmium was equally distributed in liver and kidney more in their supernatant cytosol fraction (SCF) than in their NMF at 24 h after a dose of cadmium chloride. The cadmium levels showed a decreasing trend in hepatic fractions and an increasing trend in renal fractions with time. The cadmium-induced hepatic and renal MT were substantial at 24 h post cadmium administration, the former decreased thereafter while the latter enhanced at 48 h before declining. The accumulation of mercury in kidney was 1.5 times that in liver, which was localised more in their SCF than in their NMF at 24 h in response to a dose of mercuric chloride. The mercury levels of hepatic and renal subcellular fractions started declining after 24 h and at 72 h they were significantly lower. The induction of hepatic and renal MT was maximum at 24 h after mercuric chloride administration, which declined thereafter concomitant with the decrease in their mercury levels. However, the MT levels in both the organs remained considerably higher than in normal animals at 72 h post exposure. The results show that the accumulation of metal in liver and kidney follows the order: Hg > Cd > Zn and the induction of MT follows Hg > Cd > Zn in liver and Cd > Hg > Zn in kidney. The alterations in zinc and copper homeostasis were more marked in liver than in kidney and follows the order: Hg > Cd > Zn.
Article
We gratefully acknowledge the families and individuals who have contributed their biomaterials, time, and financial resources to AGRE, especially Marianne Toedtman, AGRE family recruiter; Ed Berry, phlebotomist; Andrew Smith, M.D., pediatric neurologist; Paul Law, M.D., M.P.H., for database development; and Nancy Jones, AGRE Web master. We specifically thank Sallie and Tom Bernard, for their generous financial support of AGRE, and the Schering-Plough Research Institute and Pfizer Inc., for their contributions to AGRE. We also thank Maricela Alarcon, Ph.D., for her error checking and advice; Jianjun Liu, Ph.D., for updating of the genotyping data; and scientists who have started to utilize AGRE, for their helpful comments and criticism. The members of the AGRE Steering Committee are: W. Ted Brown, New York State Institute for Basic Research in Developmental Disabilities, Staten Island; Maya Bucan, University of Pennsylvania, Philadelphia; Joseph Buxbaum, Mt. Sinai School of Medicine, New York; T. Conrad Gilliam, Columbia University Genome Center, New York; David A. Greenberg, Mt. Sinai School of Medicine, New York; David H. Ledbetter, University of Chicago, Chicago; Bruce L. Miller, University of California, San Francisco; Stanley F. Nelson, UCLA School of Medicine, Los Angeles; Jonathan Pevsner, Kennedy Krieger Institute, Baltimore; Jerome I. Rotter, Cedars-Sinai Medical Center, Los Angeles; Carol Samango-Sprouse, Children’s National Medical Center, Baltimore; Gerard D. Schellenberg, University of Washington and Veterans Affairs Medical Center, Seattle; Rudolph E. Tanzi, Massachusetts General Hospital, Boston; and Kirk C. Wilhelmsen, University of California, San Francisco.
Article
Neurotoxic effects of methylmercury, were investigated in vitro in primary cultures of human neurons and astrocytes isolatedfrom human fetal brain and in the human neuroblastoma cell line SH-SY5Y. The protection provided by agents with antioxidant properties was tested in these cultures to examine the oxidative stress mechanism of methylmercury poisoning. After 24 h of exposure to methylmercury, LC50 values were 6.5, 8.1 and 6.9 microM for human neurons, astrocytes and neuroblastoma cells, respectively, and the degree of cell damage increased at longer exposure times. Depletion of the cellular pool of reduced glutathione (GSH) by treatment with buthionine sulfoximine potentiated methylmercury cytotoxicity in all three cell types; neuroblastoma cells were the most sensitive. Addition of GSH extracellularly blocked methylmercury neurotoxicity in all cell types. The major beneficial effect of GSH could be attributed to its capacity to form conjugates with methylmercury, which reduces the availability of these organometallic molecules to the cells and facilitates their efflux. Cysteine protected astrocytes and neuroblastoma cells from methylmercury neurotoxicity, while selenite, Vitamin E and catalase produced some minor protective effects in three cell types, particularly in neurons. The present study showed that the human neural cells tested had differential responses to methylmercury: astrocytes were resistant to methylmercury neurotoxicity and neurons were more most responsive to protection afforded by antioxidants among the three cell types.
Article
Metallothionein (MT) is a small-molecular weight, cysteine-rich protein that binds metals. The protective role of MT in Cd toxicity is well established but its ability to protect against toxicity of other metals remains unclear. In this study, wild-type and MT-I and -II null mice (MT-null mice) were used to determine whether MT is protective against the lethality of not only Cd but also Zn, Cu, Fe, Pb, Hg and As. Following daily subcutaneous administration of an increasing dose of each metal, starting with a low, non-toxic dose, we compared the cumulative median lethal dose (LD(50)) of each metal between wild-type and MT-null mice. The LD(50) of Cd for wild-type mice was 6.9-fold higher than for MT-null mice. The LD(50) of Zn was 2.4-fold higher for wild-type mice than for MT-null mice, and 1.4-fold higher for Cu and As. The LD(50) of Hg was 1.3-fold higher for wild-type mice than for MT-null mice, but this was not statistically significant. No difference in LD(50) values was observed between wild-type and MT-null mice following Pb and Fe administration. These results suggest that MT is an important protein in the cellular defense against Cd toxicity and lethality, but it provides much less protection against the lethality of the other metals.
Article
The major source of thimerosal (ethyl mercury thiosalicylate) exposure is childhood vaccines. It is believed that the children are exposed to significant accumulative dosage of thimerosal during the first 2 years of life via immunization. Because of health-related concerns for exposure to mercury, we examined the effects of thimerosal on the biochemical and molecular steps of mitochondrial pathway of apoptosis in Jurkat T cells. Thimerosal and not thiosalcylic acid (non-mercury component of thimerosal), in a concentration-dependent manner, induced apoptosis in T cells as determined by TUNEL and propidium iodide assays, suggesting a role of mercury in T cell apoptosis. Apoptosis was associated with depolarization of mitochondrial membrane, release of cytochrome c and apoptosis inducing factor (AIF) from the mitochondria, and activation of caspase-9 and caspase-3, but not of caspase-8. In addition, thimerosal in a concentration-dependent manner inhibited the expression of XIAP, cIAP-1 but did not influence cIAP-2 expression. Furthermore, thimerosal enhanced intracellular reactive oxygen species and reduced intracellular glutathione (GSH). Finally, exogenous glutathione protected T cells from thimerosal-induced apoptosis by upregulation of XIAP and cIAP1 and by inhibiting activation of both caspase-9 and caspase-3. These data suggest that thimerosal induces apoptosis in T cells via mitochondrial pathway by inducing oxidative stress and depletion of GSH.
Article
Thimerosol is an antiseptic containing 49.5% ethyl mercury that has been used for years as a preservative in many infant vaccines and in flu vaccines. Environmental methyl mercury has been shown to be highly neurotoxic, especially to the developing brain. Because mercury has a high affinity for thiol (sulfhydryl (-SH)) groups, the thiol-containing antioxidant, glutathione (GSH), provides the major intracellular defense against mercury-induced neurotoxicity. Cultured neuroblastoma cells were found to have lower levels of GSH and increased sensitivity to thimerosol toxicity compared to glioblastoma cells that have higher basal levels of intracellular GSH. Thimerosal-induced cytotoxicity was associated with depletion of intracellular GSH in both cell lines. Pretreatment with 100 microM glutathione ethyl ester or N-acetylcysteine (NAC), but not methionine, resulted in a significant increase in intracellular GSH in both cell types. Further, pretreatment of the cells with glutathione ethyl ester or NAC prevented cytotoxicity with exposure to 15 microM Thimerosal. Although Thimerosal has been recently removed from most children's vaccines, it is still present in flu vaccines given to pregnant women, the elderly, and to children in developing countries. The potential protective effect of GSH or NAC against mercury toxicity warrants further research as possible adjunct therapy to individuals still receiving Thimerosal-containing vaccinations.
Article
Environmental exposure to mercurials continues to be a public health issue due to their deleterious effects on immune, renal and neurological function. Recently the safety of thimerosal, an ethyl mercury-containing preservative used in vaccines, has been questioned due to exposure of infants during immunization. Mercurials have been reported to cause apoptosis in cultured neurons; however, the signaling pathways resulting in cell death have not been well characterized. Therefore, the objective of this study was to identify the mode of cell death in an in vitro model of thimerosal-induced neurotoxicity, and more specifically, to elucidate signaling pathways which might serve as pharmacological targets. Within 2 h of thimerosal exposure (5 microM) to the human neuroblastoma cell line, SK-N-SH, morphological changes, including membrane alterations and cell shrinkage, were observed. Cell viability, assessed by measurement of lactate dehydrogenase (LDH) activity in the medium, as well as the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, showed a time- and concentration-dependent decrease in cell survival upon thimerosal exposure. In cells treated for 24 h with thimerosal, fluorescence microscopy indicated cells undergoing both apoptosis and oncosis/necrosis. To identify the apoptotic pathway associated with thimerosal-mediated cell death, we first evaluated the mitochondrial cascade, as both inorganic and organic mercurials have been reported to accumulate in the organelle. Cytochrome c was shown to leak from the mitochondria, followed by caspase 9 cleavage within 8 h of treatment. In addition, poly(ADP-ribose) polymerase (PARP) was cleaved to form a 85 kDa fragment following maximal caspase 3 activation at 24 h. Taken together these findings suggest deleterious effects on the cytoarchitecture by thimerosal and initiation of mitochondrial-mediated apoptosis.
A family study of autism: cognitive patterns and levels in parents and siblings
  • E Fombonne
  • P Bolton
  • J Prior
  • H Jordan
  • M Rutter
Fombonne E, Bolton P, Prior J, Jordan H, Rutter M. A family study of autism: cognitive patterns and levels in parents and siblings. J Child Psychol Psychiatry 1997;38:667-83.
Degradation of methyl and ethyl mercury into inorganic mercury by hydroxyl radical produced from rat liver microsomes
  • I Suda
  • S Totoki
  • T Uchida
  • H Takahashi
Suda I, Totoki S, Uchida T, Takahashi H. Degradation of methyl and ethyl mercury into inorganic mercury by hydroxyl radical produced from rat liver microsomes. Arch Toxicol 1992;66:40-4.
Degradation of methyl and ethyl mercury into inorganic mercury by hydroxyl radical produced from rat liver microsomes
  • Suda
A family study of autism: cognitive patterns and levels in parents and siblings
  • Fombonne