Arsenic Impairs Wound Healing Processes in Dermal Fibroblasts and Mice.

Inorganic arsenic (NaAsO2) is a naturally occurring metalloid found in water resources globally and in the United States at concentrations exceeding the U.S. Environmental Protection Agency Maximum Contamination Level of 10 ppb. While exposure to arsenic has been linked to cancer, cardiovascular disease, and skin lesions, the impact of arsenic exposure on wound healing is not fully understood. Cultured dermal fibroblasts exposed to NaAsO2 displayed reduced migration (scratch closure), proliferation, and viability with a lowest observable effect level (LOEL) of 10 µM NaAsO2 following 24 h exposure. An enrichment of Matrix Metalloproteinase 1 (MMP1) transcripts was observed at a LOEL of 1 µM NaAsO2 and 24 h exposure. In vivo, C57BL/6 mice were exposed to 10 µM NaAsO2 in their drinking water for eight weeks, then subjected to two full thickness dorsal wounds. Wounds were evaluated for closure after 6 days. Female mice displayed a significant reduction in wound closure and higher erythema levels, while males showed no effects. Gene expression analysis from skin excised from the wound site revealed significant enrichment in Arsenic 3-Methyltransferase (As3mt) and Estrogen Receptor 2 (Esr2) mRNA in the skin of female mice. These results indicate that arsenic at environmentally relevant concentrations may negatively impact wound healing processes in a sex-specific manner.

Effects of Intestinal Microbiota on the Biological Transformation of Arsenic in Zebrafish: Contribution and Mechanism.

Both the gut microbiome and their host participate in arsenic (As) biotransformation, while their exact roles and mechanisms in vivo remain unclear and unquantified. In this study, as3mt-/- zebrafish were treated with tetracycline (TET, 100 mg/L) and arsenite (iAsIII) exposure for 30 days and treated with probiotic Lactobacillus rhamnosus GG (LGG, 1 × 108 cfu/g) and iAsIII exposure for 15 days, respectively. Structural equation modeling analysis revealed that the contribution rates of the intestinal microbiome to the total arsenic (tAs) and inorganic As (iAs) metabolism approached 44.0 and 18.4%, respectively. Compared with wild-type, in as3mt-/- zebrafish, microbial richness and structure were more significantly correlated with tAs and iAs, and more differential microbes and microbial metabolic pathways significantly correlated with arsenic metabolites (P < 0.05). LGG supplement influenced the microbial communities, significantly up-regulated the expressions of genes related to As biotransformation (gss and gst) in the liver, down-regulated the expressions of oxidative stress genes (sod1, sod2, and cat) in the intestine, and increased arsenobetaine concentration (P < 0.05). Therefore, gut microbiome promotes As transformation and relieves As accumulation, playing more active roles under iAs stress when the host lacks key arsenic detoxification enzymes. LGG can promote As biotransformation and relieve oxidative stress under As exposure.

Association Between Arsenic Toxicity, AS3MT Gene Polymorphism and Onset of Type 2 Diabetes.

Arsenic (As) exposure in drinking water has become a serious public health issue. AS3MT gene is involved in the metabolism of arsenic, so a single nucleotide polymorphism in this gene may lead to the development of type 2 diabetes in arsenic-exposed areas. This study aimed to evaluate the association of the AS3MT gene with the development of type 2 diabetes in highly arsenic-exposed areas of Punjab, Pakistan. Total 200 samples equal in number from high arsenic exposed-areas of Lahore (Nishtar) and Kasur (Mustafa Abad) were collected. rs11191439 was utilized as an influential variable to evaluate the association between arsenic metabolism and diabetes status to find a single nucleotide polymorphism in the AS3MT gene. We observed the arsenic level in drinking water of the arsenic-exposed selected areas 115.54 ± 1.23 µg/L and 96.88 ± 0.48 µg/L, respectively. The As level in the urine of diabetics (98.54 ± 2.63 µg/L and 56.38 ± 12.66 µg/L) was higher as compared to non-diabetics (77.58 ± 1.8 µg/L and 46.9 ± 8.95 µg/L) of both affected areas, respectively. Correspondingly, the As level in the blood of diabetics (6.48 ± 0.08 µg/L and 5.49 ± 1.43 µg/L) and non-diabetics (6.22 ± 0.12 µg/L and 5.26 ± 0.24 µg/L) in the affected areas. Genotyping showed significant differences in the frequencies of alleles among cases and controls. Nevertheless, notable disparities in genotype distribution were observed in SNPs rs11191439 (T/C) (P < 0.05) and when comparing T2D patients and non-diabetic control subjects. The AS3MT gene and clinical parameters show a significant association with the affected people with diabetes living in arsenic-exposed areas.

Maternal Exposure to Arsenic and Its Impact on Maternal and Fetal Health: A Review.

Arsenic exposure is a significant public health issue, with harmful effects caused by its use in commercial products such as car batteries, pesticides, and herbicides. Arsenic has three main compounds: inorganic, organic, and arsine gas. Inorganic arsenic compounds in water are highly toxic. The daily intake of arsenic from food and beverages is between 20 and 300 mcg/day. Arsenic is known for its carcinogenic properties and is classified as a human carcinogen by different institutions. Exposure can lead to oxidative stress, DNA damage, and epigenetic deregulation, which can cause endocrine disorders, altered signal transduction pathways, and cell proliferation. In addition, arsenic can easily cross the placenta, making it a critical concern for maternal and fetal health. Exposure can lead to complications such as gestational diabetes, anemia, low birth weight, miscarriage, and congenital anomalies. Female babies are particularly vulnerable to the negative impact of arsenic exposure, with a higher risk of low weight for gestational age and congenital cardiac anomalies. Therefore, it is crucial to monitor and regulate the levels of arsenic in drinking water and food sources to prevent these adverse health outcomes. Further research is necessary to fully understand the impact of arsenic exposure on human health, especially during pregnancy and infancy, by implementing preventative measures and monitoring the levels of arsenic in the environment.

Association between arsenic (+3 oxidation state) methyltransferase gene polymorphisms and arsenic methylation capacity in rural residents of northern China: a cross-sectional study.

Arsenic is a toxic metal-like element. The toxic reaction of the body to arsenic is related to the ability of arsenic methylation metabolism. As the rate-limiting enzyme of arsenic methylation metabolism, the genetic single nucleotide polymorphisms (SNPs) of arsenic (+ 3 oxidation state) methyltransferase (AS3MT) gene are related to capacity of arsenic methylation. In this paper, we investigated the association of five SNPs (rs7085104, rs3740390, 3740393, rs10748835, and rs1046778) in AS3MT with arsenic methylation metabolizing using the data and samples from a cross-sectional case-control study of arsenic and Type 2 diabetes mellitus conducted in Shanxi, China. A total of 340 individuals were included in the study. Urinary total arsenic (tAs, µg/L) was detected by liquid chromatography-atomic fluorescence spectrometry (LC-AFS). According to "safety guidance value of urinary arsenic for population" as specified in WS/T665-2019 (China), participants were divided into the control group (tAs ≤ 32 µg/L, n = 172) and arsenic-exposed group (tAs > 32 µg/L, n = 168). iAs%, MMA%, and DMA% are as the indicator of arsenic methylation capacity. The genotypes of AS3MT SNPs were examined by Multiple PCR combined sequencing. Linear regression analysis showed that AG + GG genotype in rs7085104 was associated with decreased iAs% and increased DMA%. Moreover, AG + AA genotype in rs10748835 and TC + CC genotype in rs1046778 were associated with decreased iAs% and MMA% and increased DMA%. The interaction between rs7085104 and arsenic is associated with iAs% and DMA%. The interaction of rs3740390 and rs10748835 with arsenic is associated with iAs%. Haplotype CTAC (rs3740393-rs3740390-rs10748835-rs1046778) was associated with lower iAs% and higher DMA%, but this association disappeared after adjusting for age, gender, drink, smoking, BMI and tAs. Haplotype GCAC was associated with decreased MMA%. Our study provides additional support for revealing the factors influencing the metabolic capacity of arsenic methylation and might be helpful to identify the population susceptible to arsenic exposure through individualized screening in the future.

In silico comparative structural and functional analysis of arsenite methyltransferase from bacteria, fungi, fishes, birds, and mammals.

BACKGROUND: Arsenic, a ubiquitous toxic metalloid, is a threat to the survival of all living organisms. Bioaccumulation of arsenic interferes with the normal physiological pathway. To overcome arsenic toxicity, organisms have developed arsenite methyltransferase enzyme, which methylates inorganic arsenite to organic arsenic MMA (III) in the presence of S-adenosylmethionine (SAM). Bacteria-derived arsM might be horizontally transported to different domains of life as arsM or as3mt (animal ortholog). A systematic study on the functional diversity of arsenite methyltransferase from various sources will be used in arsenic bioremediation. RESULTS: Several arsenite methyltransferase protein sequences of bacteria, fungi, fishes, birds, and mammals were retrieved from the UniProt database. In silico physicochemical studies confirmed the acidic, hydrophilic, and thermostable nature of these enzymes. Interkingdom relationships were revealed by performing phylogenetic analysis. Homology modeling was performed by SWISS-MODEL, and that was validated through SAVES-v.6.0. QMEAN values ranged from - 0.93 to - 1.30, ERRAT score (83-96), PROCHECK (88-92%), and other parameters suggested models are statistically significant. MOTIF and PrankWeb discovered several functional motifs and active pockets within the proteins respectively. The STRING database showed protein-protein interaction networks. CONCLUSION: All of our in silico studies confirmed the fact that arsenite methyltransferase is a cytosolic stable enzyme with conserved sequences over a wide range of organisms. Thus, because of its stable and ubiquitous nature, arsenite methyltransferase could be employed in arsenic bioremediation.

Arsenite Methyltransferase Is an Important Mediator of Hematotoxicity Induced by Arsenic in Drinking Water.

Chronic arsenic exposures via the consumption of contaminated drinking water are clearly associated with many deleterious health outcomes, including anemia. Following exposure, trivalent inorganic arsenic (AsIII) is methylated through a series of arsenic (+III oxidation state) methyltransferase (As3MT)-dependent reactions, resulting in the production of several intermediates with greater toxicity than the parent inorganic arsenicals. The extent to which inorganic vs. methylated arsenicals contribute to AsIII-induced hematotoxicity remains unknown. In this study, the contribution of As3MT-dependent biotransformation to the development of anemia was evaluated in male As3mt-knockout (KO) and wild-type, C57BL/6J, mice following 60-day drinking water exposures to 1 mg/L (ppm) AsIII. The evaluation of hematological indicators of anemia revealed significant reductions in red blood cell counts, hemoglobin levels, and hematocrit in AsIII-exposed wild-type mice as compared to unexposed controls. No such changes in the blood of As3mt-KO mice were detected. Compared with unexposed controls, the percentages of mature RBCs in the bone marrow and spleen (measured by flow cytometry) were significantly reduced in the bone marrow of AsIII-exposed wild-type, but not As3mt-KO mice. This was accompanied by increased levels of mature RBCS in the spleen and elevated levels of circulating erythropoietin in the serum of AsIII-exposed wild-type, but not As3mt-KO mice. Taken together, the findings from the present study suggest that As3MT-dependent biotransformation has an essential role in mediating the hematotoxicity of AsIII following drinking water exposures.

As3MT is related to relative RNAs and base modifications of p53 in workers exposed to arsenic.

As3MT is the key enzyme involved in the methylation metabolism of arsenic. It is associated with DNA methylation closely also. This study is to explore the relationships between As3MT and epigenetic changes, and how p53 and relative ncRNAs and mRNAs play roles in the process. In this study, workers from four arsenic plants and individuals who resided in villages far away from the four plants were recruited. Arsenic compounds, relative indices, 28 relative RNAs, and base modifications of exons 5-8 of p53 were detected separately. Several methods were used to analyze the associations between them. Results shown that As3MT RNA was closely associated with all selected lncRNAs, miRNAs, and mRNAs related to miRNA production and maturation, tumorigenesis, and base modifications of p53. There probably exists causal relationship. Base modifications of exons 7 and 8 of p53 had significant synergistic effects on the expression of As3MT RNA and a series of genetic indices. But miR-190, miR-548, and base modifications of exon 5 of p53 had substantial inhibitory effects. Arsenic compounds and relative indices of metabolic transformation may have limited roles. The main novel finding in the present study is that As3MT play special and significant roles in the genotoxicity and carcinogenesis which could be coordinated operation with p53, and influenced by epigenetic factors largely, such as lncRNAs and miRNAs. P53 and relative ncRNAs and mRNAs may regulate the process by interacting with As3MT. The changes may initiate by arsenic, but probability through indirect relationship.

Arsenic metabolism, N6AMT1 and AS3MT single nucleotide polymorphisms, and their interaction on gestational diabetes mellitus in Chinese pregnant women.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in N6AMT1 and AS3MT are associated with arsenic (As) metabolism, and efficient As methylation capacity has been associated with diabetes. However, little is known about the gene-As interaction on gestational diabetes mellitus (GDM). OBJECTIVE: This study aimed to explore the individual and combined effects of N6AMT1 and AS3MT SNPs with As metabolism on GDM. METHODS: A cross-sectional study was performed among 385 Chinese pregnant women (86 GDM and 299 Non-GDM). Four SNPs in N6AMT1 (rs1997605 and rs1003671) and AS3MT (rs1046778 and rs11191453) were genotyped. Urinary inorganic arsenic (iAs), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) were determined, and the percentages of As species (iAs%, MMA%, and DMA%) were calculated to assess the efficiency of As metabolism. RESULTS: Pregnant women with N6AMT1 rs1997605 AA genotype had lower iAs% (B: 2.11; 95% CI: 4.08, -0.13) and MMA% (B: 0.21; 95% CI: 0.39, -0.04) than pregnant women with GG genotype. The AS3MT rs1046778 and rs11191453 C alleles were negatively associated with iAs% and MMA% but positively associated with DMA%. Higher urinary MMA% was significantly associated with a lower risk of GDM (OR: 0.54; 95% CI: 0.30, 0.97). The A allele in N6AMT1 rs1997605 (OR: 0.46; 95% CI: 0.26, 0.79) was associated with a decreased risk of GDM. The additive interactions between N6AMT1 rs1997605 GG genotypes and lower iAs% (AP: 0.50; 95% CI: 0.01, 0.99) or higher DMA% (AP: 0.52; 95% CI: 0.04, 0.99) were statistically significant. Similar additive interactions were also found between N6AMT1 rs1003671 GG genotypes and lower iAs% or higher DMA%. CONCLUSIONS: Genetic variants in N6AMT1 and efficient As metabolism (indicated by lower iAs% and higher DMA%) can interact to influence GDM occurrence synergistically in Chinese pregnant women.

AS3MT facilitates NLRP3 inflammasome activation by m6A modification during arsenic-induced hepatic insulin resistance.

N6-methyladenosine (m6A) messenger RNA methylation is the most widespread gene regulatory mechanism affecting liver functions and disorders. However, the relationship between m6A methylation and arsenic-induced hepatic insulin resistance (IR), which is a critical initiating event in arsenic-induced metabolic syndromes such as type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD), remains unclear. Here, we showed that arsenic treatment facilitated methyltransferase-like 14 (METTL14)-mediated m6A methylation, and that METTL14 interference reversed arsenic-impaired hepatic insulin sensitivity. We previously showed that arsenic-induced NOD-like receptor protein 3 (NLRP3) inflammasome activation contributed to hepatic IR. However, the regulatory mechanisms underlying the role of arsenic toward the post-transcriptional modification of NLRP3 remain unclear. Here, we showed that NLRP3 mRNA stability was enhanced by METTL14-mediated m6A methylation during arsenic-induced hepatic IR. Furthermore, we demonstrated that arsenite methyltransferase (AS3MT), an essential enzyme in arsenic metabolic processes, interacted with NLRP3 to activate the inflammasome, thereby contributing to arsenic-induced hepatic IR. Also, AS3MT strengthened the m6A methylase association with NLRP3 to stabilize m6A-modified NLRP3. In summary, we showed that AS3MT-induced m6A modification critically regulated NLRP3 inflammasome activation during arsenic-induced hepatic IR, and we identified a novel post-transcriptional function of AS3MT in promoting arsenicosis.

Involvement of a AS3MT/c-Fos/p53 signaling axis in arsenic-induced tumor in human lung cells.

Arsenite methyltransferase (AS3MT) is an enzyme that catalyzes the dimethylation of arsenite (+3 oxidation state). At present, the studies on arsenic carcinogenicity mainly focus on studying the polymorphisms of AS3MT and measuring their catalytic activities. We recently showed that AS3MT was overexpressed in lung cancer patients who had not been exposed to arsenic. However, little is known about the molecular mechanisms of AS3MT in arsenite-induced tumorigenesis. In this study, we showed that AS3MT protein expression was higher in the arsenic-exposed population compared to the unexposed population. AS3MT was also overexpressed in human lung adenocarcinoma (A549) and human bronchial epithelial (16HBE) cells exposed to arsenic (A549: 20-60 µmol/L; 16HBE: 2-6 µmol/L) for 48 h. Furthermore, we investigated the effects of AS3MT on cell proliferation and apoptosis using siRNA. The downregulation of AS3MT inhibited the proliferation and promoted the apoptosis of cells. Mechanistically, AS3MT was found to specifically bind to c-Fos, thereby inhibiting the binding of c-Fos to c-Jun. Additionally, the siRNA-mediated knockdown of AS3MT enhanced the phosphorylation of Ser392 in p53 by upregulating p38 MAPK expression. This led to the activation of p53 signaling and the upregulated expression of downstream targets, such as p21, Fas, PUMA, and Bax. Together, these studies revealed that the inorganic arsenic-mediated upregulation of AS3MT expression directly affected the proliferation and apoptosis of cells, leading to arsenic-induced toxicity or carcinogenicity.

A Role of DNA Methylation within the CYP17A1 Gene in the Association of Genetic and Environmental Risk Factors with Stress-Related Manifestations of Schizophrenia.

As genetic and environmental influences on schizophrenia might converge on DNA methylation (DNAm) within loci which are both associated with the disease and implicated in response to environmental stress, we examined whether DNAm within CYP17A1, a hypothalamus-pituitary-adrenal axis gene which is situated within the schizophrenia risk locus 10q24.32, would mediate genetic and environmental effects on stress-related schizophrenia symptoms. DNAm within an exonic-intronic fragment of CYP17A1 was assessed in the blood of 66 schizophrenia patients and 63 controls using single-molecule real-time bisulfite sequencing. Additionally, the VNTR polymorphism of the AS3MT gene, a plausible causal variant within the 10q24.32 locus, was genotyped in extended patient and control samples (n = 700). The effects of local haplotype, VNTR and a polyenviromic risk score (PERS) on DNAm, episodic verbal memory, executive functions, depression, and suicidality of patients were assessed. Haplotype and PERS differentially influenced DNAm at four variably methylated sites identified within the fragment, with stochastic, additive, and allele-specific effects being found. An allele-specific DNAm at CpG-SNP rs3781286 mediated the relationship between the local haplotype and verbal fluency. Our findings do not confirm that the interrogated DNA fragment is a place where genetic and environmental risk factors converge to influence schizophrenia symptoms through DNAm.

As3MT-mediated SAM consumption, which inhibits the methylation of histones and LINE1, is involved in arsenic-induced male reproductive damage.

Studies have demonstrated that arsenic (As) induces male reproductive injury, however, the mechanism remains unknown. The high levels of arsenic (3) methyltransferase (As3MT) promote As-induced male reproductive toxicity. For As-exposed mice, the germ cells in seminiferous tubules and sperm quality were reduced. Exposure to As caused lower S-adenosylmethionine (SAM) and 5-methylcytosine (5 mC) levels, histone and DNA hypomethylation, upregulation of long interspersed element class 1 (LINE1, or L1), defective repair of double-strand breaks (DSBs), and the arrest of meiosis, resulting in apoptosis of germ cells and lower litter size. For GC-2spd (GC-2) cells, As induced apoptosis, which was prevented by adding SAM or by reducing the expression of As3MT. The levels of LINE1, affected by SAM content, were involved in As-induced apoptosis. Furthermore, folic acid (FA) and vitamin B12 (VB12) supplements restored SAM, 5 mC, and LINE1 levels and blocked impairment of spermatogenesis and testes and lower litter size. Exposed to As, mice with As3MT knockdown showed less impairment of spermatogenesis and testes and greater litter size compared to As-exposed wild-type (WT) mice. Thus, the high As3MT levels induced by As consume SAM and block histone and LINE1 DNA methylation, elevating LINE1 expression and evoking impairment of spermatogenesis, which causes male reproductive damage. Overall, we have found a mechanism for As-induced male reproductive damage, which provides biological insights into the alleviation of reproductive injury induced by environmental factors.

Functional characterization of the schizophrenia associated gene AS3MT identifies a role in neuronal development.

Genome-wide association studies (GWAS) have identified multiple genomic regions associated with schizophrenia, although many variants reside in noncoding regions characterized by high linkage disequilibrium (LD) making the elucidation of molecular mechanisms challenging. A genomic region on chromosome 10q24 has been consistently associated with schizophrenia with risk attributed to the AS3MT gene. Although AS3MT is hypothesized to play a role in neuronal development and differentiation, work to fully understand the function of this gene has been limited. In this study we explored the function of AS3MT using a neuronal cell line (SH-SY5Y). We confirm previous findings of isoform specific expression of AS3MT during SH-SY5Y differentiation toward neuronal fates. Using CRISPR-Cas9 gene editing we generated AS3MT knockout SH-SY5Y cell lines and used RNA-seq to identify significant changes in gene expression in pathways associated with neuronal development, inflammation, extracellular matrix formation, and RNA processing, including dysregulation of other genes strongly implicated in schizophrenia. We did not observe any morphological changes in cell size and neurite length following neuronal differentiation and MAP2 immunocytochemistry. These results provide novel insights into the potential role of AS3MT in brain development and identify pathways through which genetic variation in this region may confer risk for schizophrenia.

Human adaptation to arsenic in Bolivians living in the Andes.

Humans living in the Andes Mountains have been historically exposed to arsenic from natural sources, including drinking water. Enzymatic methylation of arsenic allows it to be excreted more efficiently by the human body. Adaptation to high-arsenic environments via enhanced methylation and excretion of arsenic was first reported in indigenous women in the Argentinean Andes, but whether adaptation to arsenic is a general phenomenon across native populations from the Andes Mountains remains unclear. Therefore, we evaluated whether adaptation to arsenic has occurred in the Bolivian Andes by studying indigenous groups who belong to the Aymara-Quechua and Uru ethnicities and have lived in the Bolivian Andes for generations. Our population genetics methods, including genome-wide selection scans based on linkage disequilibrium patterns and allele frequency differences, in combination with targeted and whole-genome sequencing and genotype-phenotype association analyses, detected signatures of positive selection near the gene encoding arsenite methyltransferase (AS3MT), the main arsenic methylating enzyme. This was among the strongest selection signals (top 0.5% signals via locus-specific branch length and extended haplotype homozygosity tests) at a genome-wide level in the Bolivian study groups. We found a large haplotype block of 676 kb in the AS3MT region and identified candidate functional variants for further analysis. Moreover, our analyses revealed associations between AS3MT variants and the fraction of mono-methylated arsenic in urine and showed that the Bolivian study groups had the highest frequency of alleles associated with more efficient arsenic metabolism reported so far. Our data support the idea that arsenic exposure has been a driver for human adaptation to tolerate arsenic through more efficient arsenic detoxification in different Andean populations.

Gender difference in arsenic biotransformation is an important metabolic basis for arsenic toxicity.

BACKGROUND: Arsenic metabolism enzymes can affect the toxic effects of arsenic. However, the effects of different genders on the metabolites and metabolic enzymes in liver arsenic metabolism is still unclear. This study analyzed the gender differences of various arsenic metabolites and metabolic enzymes and further explored the effects of gender differences on arsenic metabolism in liver tissues of rats. METHODS: Rats were treated with high/medium/low doses of iAs3+ or iAs5+. Liver pathological changes were observed with electron microscopy. The monomethyl aracid (MMA) and dimethyl aracid (DMA) was determined by high performance liquid chromatography-hydride generation atomic fluorescence spectroscopy. S-adenosylmethionine (SAM), arsenate respiratory reductase (ARR), nicotinamide adenine dinucleotide (NAD), purine nucleoside phosphorylase (PNP), pyruvate kinase (PK), and myeloperoxidase (MPO) SAM, ARR, NAD, PNP, PK, and MPO were determined by enzyme-linked immunoassay. RT-qPCR was used to determine Arsenic (+ 3 oxidation state) methyltransferase (AS3MT). RESULTS: The iAs3+ and iAs5+ at high doses induced pathological changes in the liver, such as increased heterochromatin and lipid droplets. Compared within the same group, MMA and DMA were statistically significant in iAs3 + high, iAs3 + medium and iAs5+ low dose groups (P < 0.05). MMA of male rats in iAs3+ high and medium groups was higher than that of female rats, and the DMA of male rats was lower than that of female rats. As3MT mRNA in the male iAs3+ high group was higher than that of females. Besides, compared between male and female, only in iAS3+ low dose, iAS3+ medium dose, iAS5+ low dose, and iAS5+ medium dose groups, there was significant difference in SAM level (P < 0.05). Compared within the same group, male rats had significantly higher PNP and ARR activities while lower PK activity than female rats (P < 0.05). Between the male and female groups, only the iAS3+ high dose and medium dose group had a statistically significant difference (P < 0.05). The NAD activity of females in iAS3+ high dose group was higher than that of males. CONCLUSION: The gender differences in the arsenic metabolism enzymes may affect the biotransformation of arsenic, which may be one of the important mechanisms of arsenic toxicity of different sexes and different target organs.

Arsenic 3 methyltransferase (AS3MT) automethylates on cysteine residues in vitro.

Arsenic toxicity is a global concern to human health causing increased incidences of cancer, bronchopulmonary, and cardiovascular diseases. In human and mouse, inorganic arsenic (iAs) is metabolized in a series of methylation steps catalyzed by arsenic (3) methyltransferase (AS3MT), forming methylated arsenite (MAsIII), dimethylarsenite (DMAIII) and the volatile trimethylarsine (TMA). The methylation of arsenic is coordinated by four conserved cysteines proposed to participate in catalysis, namely C33, C62, C157, and C207 in mouse AS3MT. The current model consists of AS3MT methylating iAs in the presence of the cofactor S-adenosyl-L-methionine (SAM), and the formation of intramolecular disulfide bonds following the reduction of MAsV to MAsIII. In the presence of endogenous reductants, these disulfide bonds are reduced, the enzyme re-generates, and the second round of methylation ensues. Using in vitro methylation assays, we find that AS3MT undergoes an initial automethylation step in the absence of iAs. This automethylation is enhanced by glutathione (GSH) and dithiothreitol (DTT), suggesting that reduced cysteines accept methyl groups from SAM to form S-methylcysteines. Following the addition of iAs, automethylation of AS3MT is decreased. Furthermore, using a Flag-AS3MT immunoprecipitation coupled to MS/MS, we identify both C33 and C62 as acceptors of the methyl group in vivo. Site-directed mutagenesis (C to A) revealed that three of the previously described cysteines were required for AS3MT automethylation. In vitro experiments show that automethylated AS3MT can methylate iAs in the presence of SAM. Thus, we propose that automethylated may represent an active conformation of AS3MT.

Regulation of arsenic methylation: identification of the transcriptional region of the human AS3MT gene.

The human enzyme As(III) S-adenosylmethionine methyltransferase (AS3MT) catalyzes arsenic biotransformations and is considered to contribute to arsenic-related diseases. AS3MT is expressed in various tissues and cell types including liver, brain, adrenal gland, and peripheral blood mononuclear cells but not in human keratinocytes, urothelial, or brain microvascular endothelial cells. This indicates that AS3MT expression is regulated in a tissue/cell type-specific manner, but the mechanism of transcriptional regulation of expression of the AS3MT gene is not known. In this study, we define the DNA sequence of the core promoter region of the human AS3MT gene. We identify a GC box in the promoter to which the stress-related transcription factor Sp1 binds, indicating involvement of regulatory elements in AS3MT gene expression.

AS3MT Polymorphism: A Risk Factor for Epilepsy Susceptibility and Adverse Drug Reactions to Valproic Acid and Oxcarbazepine Treatment in Children From South China.

Epilepsy is a common neurologic disorder characterized by intractable seizures, involving genetic factors. There is a need to develop reliable genetic markers to predict the risk of epilepsy and design effective therapies. Arsenite methyltransferase (AS3MT) catalyzes the biomethylation of arsenic and hence regulates arsenic metabolism. AS3MT variation has been linked to the progression of various diseases including schizophrenia and attention deficit or hyperactivity disorder. Whether genetic polymorphism of AS3MT contributes to epilepsy remains unclear. In this study, we investigated the association of AS3MT gene polymorphism with susceptibility to epilepsy in children from south China. We also explored the effect of AS3MT variation on the safety of antiepileptic drugs. Genotypic analysis for AS3MT rs7085104 was performed using samples from a Chinese cohort of 200 epileptic children and 244 healthy individuals. The results revealed a genetic association of AS3MT rs7085104 with susceptibility to pediatric epilepsy. Mutant homozygous GG genotype exhibited a lower susceptibility to childhood epilepsy than AA genotype. Carriers of AS3MT rs7085104 AA genotype exhibited a higher risk of digestive adverse drug reactions (dADRs) in children when treated with valproic acid (VPA) or oxcarbazepine (OXC). Additionally, bioinformatics analysis identified eight AS3MT target genes related to epilepsy and three AS3MT-associated genes in VPA-related dADRs. The effects of AS3MT on epilepsy might involve multiple targets including CNNM2, CACNB2, TRIM26, MTHFR, GSTM1, CYP17A1, NT5C2, and YBX3. This study reveals that AS3MT may be a new gene contributing to epileptogenesis. Hence, analysis of AS3MT polymorphisms will help to evaluate susceptibility to pediatric epilepsy and drug safety.

Polymorphisms of the AS3MT gene are associated with arsenic methylation capacity and damage to the P21 gene in arsenic trioxide plant workers.

Epidemiological evidence suggests that the metabolic profiles of each individual exposed to arsenic (As) are related to the risk of cancer, coronary heart disease, and diabetes. The arsenite methyltransferase (AS3MT) gene plays a key role in As metabolism. Several single nucleotide polymorphisms in the AS3MT gene may affect both enzyme activity and gene transcription. AS3MT polymorphisms are associated with the proportions of monomethylarsenic acid (MMA) and dimethylarsenic acid (DMA) in urine as well as the incidence of cancer. P21 protein is a cyclin-dependent kinase inhibitor. Mutations of the P21 gene have been found in cancer patients. In our study, we investigate whether polymorphisms of the AS3MT gene alter As methylation capacity and adversely affect the P21 gene in arsenic trioxide plant workers. The DNA damage was examined by the quantitative polymerase chain reaction. Restriction fragment length polymorphism was used to analyze the genotype of the AS3MT gene. The results showed that DNA damage in P21 gene fragments was greater in those individuals exposed to high levels of As. There was a strong positive correlation between the DNA damage to P21 gene fragments and the percentage of MMA in urine. However, DNA damage in P21 gene fragments was negatively associated with the percentage of DMA in urine (%uDMA), primary methylation index (PMI), and secondary methylation index. We found that subjects with the rs7085104 GG or GA allele were associated with higher %uDMA and PMI and less DNA damage. The subjects with the rs11191454 GG+GA or GA allele were also associated with higher %uDMA and PMI and less DNA damage. Our results suggest that rs1191454 and rs7085104 in the AS3MT gene affect the As-induced DNA damage by altering individual metabolic efficiency.