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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The VNTR of the AS3MT gene is associated with brain activations during a memory span task and their training-induced plasticity.

BACKGROUND: The Arsenic (+3 oxidation state) methyltransferase (AS3MT) gene has been identified as a top risk gene for schizophrenia in several large-scale genome-wide association studies. A variable number tandem repeat (VNTR) of this gene is the most significant expression quantitative trait locus, but its role in brain activity in vivo is still unknown. METHODS: We first performed a functional magnetic resonance imaging (fMRI) scan of 101 healthy subjects during a memory span task, trained all subjects on an adaptive memory span task for 1 month, and finally performed another fMRI scan after the training. After excluding subjects with excessive head movements for one or more scanning sessions, data from 93 subjects were included in the final analyses. RESULTS: The VNTR was significantly associated with both baseline brain activation and training-induced changes in multiple regions including the prefrontal cortex and the anterior and posterior cingulate cortex. Additionally, it was associated with baseline brain activation in the striatum and the parietal cortex. All these results were corrected based on the family-wise error rate method across the whole brain at the peak level. CONCLUSIONS: This study sheds light on the role of AS3MT gene variants in neural plasticity related to memory span training.

Association between the polymorphism of three genes involved in the methylation and efflux of arsenic (As3MT, MRP1, and P-gp) with lung cancer in a Mexican cohort.

Lung cancer is the most common neoplasm and the primary cause-related mortality in developed and in most of nondeveloped countries. Epidemiological studies have demonstrated that even at low arsenic doses, the lungs are one of the main target organs and that chronic arsenic exposure has been associated with an increase in lung cancer development. Among the risk factors for cancer, arsenic methylation efficiency (As3MT) and the clearance of arsenic from cells by two members of the ATP-binding cassette (ABC) transporter family (multidrug resistance protein 1 [MRP1] and P-glycoprotein [P-gp]) play an important role in processing of arsenic and decreasing its intracellular levels. This study aimed to evaluate the association between chronic exposure to arsenic with polymorphism of three proteins involved in arsenic metabolism and efflux of the metalloid in subjects with lung cancer. Polymorphism in As3MT, MRP1, and P-gp modified the arsenic metabolism increasing significantly the AsV urinary levels. A significant association between MRP1 polymorphisms with an increase in the risk for cancer was found. The high inorganic arsenic urinary levels registered in the studied subjects suggest a reduction in the efficiency of As3MT, MRP1, and P-gp firstly because of gene polymorphisms and secondarily because of high internal inorganic arsenic levels. MRP1 polymorphism was associated with a twofold increase in the risk of lung cancer.

Synonymous single nucleotide polymorphism in arsenic (+3) methyltransferase of the Western mosquitofish (Gambusia affinis) and its gene expression among field populations.

Naturally occurring arsenic is toxic at extremely low concentrations, yet some species persist even in high arsenic environments. We wanted to test if these species show evidence of evolution associated with arsenic exposure. To do this, we compared allelic variation across 872 coding nucleotides of arsenic (+3) methyltransferase (as3mt) and whole fish as3mt gene expression from three field populations of Gambusia affinis, from water sources containing low (1.9 ppb), medium-low (3.3 ppb), and high (15.7 ppb) levels of arsenic. The high arsenic site exceeds the US EPA's Maximum Contamination Level for drinking water. Medium-low and high populations exhibited homozygosity, and no sequence variation across all animals sampled. Eleven of 24 fish examined (45.8%) in the low arsenic population harbored synonymous single nucleotide polymorphisms (SNPs) in exons 4 and/or 10. SNP presence in the low arsenic population was not associated with differences in as3mt transcript levels compared to fish from the medium-low site, where SNPs were noted; however, as3mt expression in fish from the high arsenic concentration site was significantly lower than the other two sites. Low sequence variation in fish populations from sites with medium-low and high arsenic concentrations suggests greater selective pressure on this allele, while higher variation in the low population suggests a relaxed selection. Our results suggest gene regulation associated with arsenic detoxification may play a more crucial role in influencing responses to arsenic than polymorphic gene sequence. Understanding microevolutionary processes to various contaminants require the evaluation of multiple populations across a wide range of pollution exposures.

Inorganic arsenic-mediated upregulation of AS3MT promotes proliferation of nonsmall cell lung cancer cells by regulating cell cycle genes.

Long-term arsenic exposure can promote cancer through epigenetic mechanisms, and arsenite methyltransferase (AS3MT) plays an important role in this process. However, the expression patterns and mechanisms of AS3MT in arsenic carcinogenesis remain unclear. In this study, we found that the AS3MT was overexpressed in arsenic exposed population, non-small cell lung cancer (NSCLC) tissues, and A549 cells with sodium arsenite (NaAsO2 ) treatment for 48 hours. Besides, the level of AS3MT expression was positively correlated with the concentrations of urinary total arsenic (tAs), inorganic arsenic (iAs), methanearsonic acid (MMA), and dimethylarsinic acid (DMA) in all subjects. Functional experiments demonstrated that siRNA-mediated knockdown of AS3MT significantly inhibited proliferation of A549 cells. Mechanism investigation revealed that silencing of AS3MT inhibited proliferation by increasing mRNA expression levels of p21 and E2F1, and inhibiting CDK1, CDK2, CDK4, CDK6, Cyclin A2, Cyclin E1, Cyclin E2, and PCNA mRNA expression. Therefore, arsenic increased AS3MT expression in vivo and in vitro, which could directly act on the cell and affect the progression of NSCLC by regulating cell cycle genes.

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.

Role of As3mt and Mth1 in the genotoxic and carcinogenic effects induced by long-term exposures to arsenic in MEF cells.

DNA damage plays a crucial role in the transforming potential of the human carcinogen arsenic. The arsenic biotransformation enzyme AS3MT is known to participate in the generation of ROS after arsenic exposure, whereas MTH1 sanitizes oxidized dNTP pools to prevent the incorporation of damaged bases into DNA. In this work, we sought to assess the role of these two enzymes in the genotoxic and carcinogenic effects of arsenic exposure. Thus, mouse embryonic fibroblasts (MEF), transformed by chronic arsenite exposure, were monitored for DNA damage by the comet and the micronucleus assays at different time-of-exposure intervals lasting for 50 weeks. Results indicate that the oxidative and DNA damage of chronically exposed MEF cells increased time-dependently up to the point of transformation. As3mt expression followed a pattern like that of DNA damage, and its forced inhibition by shRNA technology before transformation resulted in a DNA damage decrease. On the other hand, Mth1 mRNA levels increased after the transformation point, and its forced knock-down increased significantly the levels of DNA damage and decreased the aggressiveness of the oncogenic phenotype. Thus, As3mt and Mth1 have important differential roles in the accumulation of DNA damage linked to the transformation process: while As3mt contributes to the genotoxic effects before the transformation, Mth1 prevents the DNA damage fixation after the acquisition of the oncogenic phenotype. This study demonstrates the influence of As3mt and Mth1 in arsenic DNA damage induction and it is the first to present Mth1 as a candidate modulator biomarker of the tumoral phenotype.

Polymorphisms in arsenic (+ 3 oxidation state) methyltransferase (AS3MT) predict the occurrence of hyperleukocytosis and arsenic metabolism in APL patients treated with As2O3.

Polymorphisms in arsenic (+ 3 oxidation state) methyltransferase (AS3MT) have been shown to be related to interindividual variations in arsenic metabolism and to influence adverse health effects in acute promyelocytic leukemia (APL) patients treated with arsenic trioxide (As2O3). The occurrence of hyperleukocytosis with As2O3 treatment seriously affects the early survival rate of APL patients, but no definite explanation for such a complication has been clearly established. To clarify the causes of this situation, AS3MT polymorphisms 14215 (rs3740390), 14458 (rs11191439), 27215 (rs11191446), and 35991 (rs10748835) and profiles of plasma arsenic metabolites were evaluated in a group of 54 newly diagnosed APL patients treated with single-agent As2O3. High-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) was used to determine the concentrations of plasma arsenic metabolites. Plasma arsenic methylation metabolism capacity was evaluated by the percentage of inorganic arsenic (iAs), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), primary methylation index (PMI, MMA/iAs), and secondary methylation index (SMI, DMA/MMA). The results showed that APL patients who developed hyperleukocytosis had a higher plasma iAs%, but a lower MMA% and PMI than those who did not develop hyperleukocytosis during As2O3 treatment. In addition, patients with the AS3MT 14215 (rs3740390) CC genotype had significantly higher plasma iAs% and incidence of hyperleukocytosis, but lower PMI than patients with the CT + TT genotype. Conversely, we did not observe statistically significant associations between the occurrence of hyperleukocytosis and AS3MT 14458 (rs11191439), 27215 (rs11191446), and 35991 (rs10748835) polymorphisms in our study subjects. These results indicated that AS3MT 14215 (rs3740390) might be used as an indicator for predicting the occurrence of hyperleukocytosis in APL patients treated with As2O3.

Association of arsenic-related AS3MT gene and antioxidant SOD2 gene expression in industrial workers occupationally exposed to arsenic.

Increasing anthropogenic activities related to industrialization and exposure to different toxicants increases the health hazards of industrial workers. Arsenic (As) exposure induces DNA damage and generates reactive oxygen species, which may result in many disease phenotypes. Present study explores the expression variation of As 3 methyltransferase (AS3MT) and superoxide dismutase (SOD2) genes in blood samples of industrial workers of different industries (brick kiln, paint, welding, pesticide, and furniture) using quantitative real-time polymerase chain reaction. A total of 250 blood samples of industrial workers were collected along with age- and gender-matched controls. Relative expression of AS3MT (p < 0.05) and SOD2 (p < 0.01) genes was found significantly downregulated in exposed workers compared to controls. Significant low levels of AS3MT and SOD2 gene expression were observed in workers in the paint and pesticide industry compared to other industries. Similarly, reduced expression of AS3MT (p < 0.05) and SOD2 (p < 0.01) was observed in smokers of industrial workers compared to smokers of the control group. Workers with >10 years of exposure had less AS3MT expression compared to workers with <10 years of exposure. Additionally, a positive Spearman correlation was observed between AS3MT versus SOD2 (r = 0.742; p < 0.0001) in industrial workers. This study suggests that decreased AS3MT and SOD2 expression levels may lead to bioaccumulation of As in the body accompanied by increased oxidative stress ultimately inducing DNA damage.

Influence of AS3MT polymorphisms on arsenic metabolism and liver injury in APL patients treated with arsenic trioxide.

Arsenic-induced side effects limit its application in the treatment of acute promyelocytic leukemia (APL). We recently demonstrated that AS3MT 14215 (rs3740390) genotypes were associated with urinary arsenic metabolites and hematological and biochemical values. To further decipher the role of AS3MT genotypes on arsenic metabolism and toxicity, AS3MT 27215 (rs11191446), 35587 (rs11191453), 35991 (rs10748835), and their interactive effects were examined in fifty APL patients treated with arsenic trioxide (As2O3) for the first time. Urinary arsenic metabolites and methylation capacity indexes were evaluated by the percentage of inorganic arsenic (iAs), monomethylarsonate (MMA), dimethylarsinate (DMA), primary methylation index (PMI, MMA/iAs), secondary methylation index (SMI, DMA/MMA), and total methylation index (TMI, [MMA+DMA]/iAs). Results showed 27215 (rs11191446) genotypes had no statistical significance in arsenic metabolism, as only 5 (10%) patients were the non-wild-type genotypes. 35587 (rs11191453) genotypes were significantly associated with MMA%, DMA%, and SMI. 35991 (rs10748835) genotypes were significantly associated with iAs%, DMA%, PMI, TMI, and the level of ALT and AST. Patients with both 35587 (rs11191453) TT and 35991 (rs10748835) AG+GG genotypes were significantly associated with DMA% and SMI. In addition, patients with both 35991 (rs10748835) AA and 35587 (rs11191453) TC+CC genotypes had the highest DMA%, SMI, and TMI, but the lowest iAs%, ALT and AST level, indicating that additive effects exist on arsenic metabolism and liver function. Our data promotes the realization that AS3MT 35587 (rs11191453), 35991 (rs10748835), especially their joint genotypes 35991 (rs10748835) AA / 35587 (rs11191453) TC+CC, is a novel predictive biomarker for the therapeutic efficacy of As2O3 in the treatment of APL.

Arsenic metabolites; selenium; and AS3MT, MTHFR, AQP4, AQP9, SELENOP, INMT, and MT2A polymorphisms in Croatian-Slovenian population from PHIME-CROME study.

The relationships between inorganic arsenic (iAs) metabolism, selenium (Se) status, and genetic polymorphisms of various genes, commonly studied in populations exposed to high levels of iAs from drinking water, were studied in a Croatian-Slovenian population from the wider PHIME-CROME project. Population consisted of 136 pregnant women in the 3rd trimester and 176 non-pregnant women with their children (n = 176, 8-9 years old). Their exposure to iAs, defined by As (speciation) analyses of biological samples, was low. The sums of biologically active metabolites (arsenite + arsenate + methylated As forms) for pregnant women, non-pregnant women, and children, respectively were: 3.23 (2.84-3.68), 1.83 (1.54-2.16) and 2.18 (1.86-2.54) ng/mLSG; GM (95 CI). Corresponding plasma Se levels were: 54.8 (52.8-56.9), 82.3 (80.4-84.0) and 65.8 (64.3-67.3) ng/mL; GM (95 CI). As methylation efficiency indexes confirmed the relationship between pregnancy/childhood and better methylation efficiency. Archived blood and/or saliva samples were used for single nucleotide polymorphism (SNP) genotyping of arsenic(3+) methyltransferase - AS3MT (rs7085104, rs3740400, rs3740393, rs3740390, rs11191439, rs10748835, rs1046778 and the corresponding AS3MT haplotype); methylene tetrahydrofolate reductase - MTHFR (rs1801131, rs1801133); aquaporin - AQP 4 and 9 (rs9951307 and rs2414539); selenoprotein P1 - SELENOP (rs7579, rs3877899); indolethylamine N-methyltransferase - INMT (rs6970396); and metallothionein 2A - MT2A (rs28366003). Associations of SNPs with As parameters and urine Se were determined through multiple regression analyses adjusted using appropriate confounders (blood As, plasma Se, ever smoking, etc.). SNPs' influence on As methylation, defined particularly by the secondary methylation index (SMI), confirmed the 'protective' role of minor alleles of six AS3MT SNPs and their haplotype only among non-pregnant women. Among the other investigated genes, the carriers of AQP9 (rs2414539) were associated with more efficient As methylation and higher urine concentration of As and Se among non-pregnant women; poorer methylation was observed for carriers of AQP4 (rs9951307) among pregnant women and SELENOP (rs7579) among non-pregnant women; MT2A (rs28366003) was associated with higher urine concentration of AsIII regardless of the pregnancy status; and INMT (rs6970396) was associated with higher As and Se concentration in non-pregnant women. Among confounders, the strongest influence was observed for plasma Se; it reduced urine AsIII concentration during pregnancy and increased secondary methylation index among non-pregnant women. In the present study of populations with low As exposure, we observed a few new As-gene associations (particularly with AQPs). More reliable interpretations will be possible after their confirmation in larger populations with higher As exposure levels.