Xuefeng Ren

Involvement of N-6 Adenine-Specific DNA Methyltransferase 1 (N6AMT1) in Arsenic Biomethylation and Its Role in Arsenic-Induced Toxicity

Background In humans, inorganic arsenic (iAs) is metabolized to methylated arsenical species in a multistep process mainly mediated by arsenic (+3 oxidation state) methyltransferase (AS3MT). Among these metabolites is monomethylarsonous acid (MMAIII), the most toxic arsenic species. A recent study in As3mt-knockout mice suggests that unidentified methyltransferases could be involved in alternative iAs methylation pathways. We found that yeast deletion mutants lacking MTQ2 were highly resistant to iAs exposure. The human ortholog of the yeast MTQ2 is N-6 adenine-specific DNA methyltransferase 1 (N6AMT1), encoding a putative methyltransferase. Objective We investigated the potential role of N6AMT1 in arsenic-induced toxicity. Methods We measured and compared the cytotoxicity induced by arsenicals and their metabolic profiles using inductively coupled plasma–mass spectrometry in UROtsa human urothelial cells with enhanced N6AMT1 expression and UROtsa vector control cells treated with different concentrations of either iAsIII or MMAIII. Results N6AMT1 was able to convert MMAIII to the less toxic dimethylarsonic acid (DMA) when overexpressed in UROtsa cells. The enhanced expression of N6AMT1 in UROtsa cells decreased cytotoxicity of both iAsIII and MMAIII. Moreover, N6AMT1 is expressed in many human tissues at variable levels, although at levels lower than those of AS3MT, supporting a potential participation in arsenic metabolism in vivo. Conclusions Considering that MMAIII is the most toxic arsenical, our data suggest that N6AMT1 has a significant role in determining susceptibility to arsenic toxicity and carcinogenicity because of its specific activity in methylating MMAIII to DMA and other unknown mechanisms.

Arsenic responsive microRNAs in vivo and their potential involvement in arsenic-induced oxidative stress.

Arsenic exposure is postulated to modify microRNA (miRNA) expression, leading to changes of gene expression and toxicities, but studies relating the responses of miRNAs to arsenic exposure are lacking, especially with respect to in vivo studies. We utilized high-throughput sequencing technology and generated miRNA expression profiles of liver tissues from Sprague Dawley (SD) rats exposed to various concentrations of sodium arsenite (0, 0.1, 1, 10 and 100mg/L) for 60days. Unsupervised hierarchical clustering analysis of the miRNA expression profiles clustered the SD rats into different groups based on the arsenic exposure status, indicating a highly significant association between arsenic exposure and cluster membership (p-value of 0.0012). Multiple miRNA expressions were altered by arsenic in an exposure concentration-dependent manner. Among the identified arsenic-responsive miRNAs, several are predicted to target Nfe2l2-regulated antioxidant genes, including glutamate-cysteine ligase (GCL) catalytic subunit (GCLC) and modifier subunit (GCLM) which are involved in glutathione (GSH) synthesis. Exposure to low concentrations of arsenic increased mRNA expression for Gclc and Gclm, while high concentrations significantly reduced their expression, which were correlated to changes in hepatic GCL activity and GSH level. Moreover, our data suggested that other mechanisms, e.g., miRNAs, rather than Nfe2l2-signaling pathway, could be involved in the regulation of mRNA expression of Gclc and Gclm post-arsenic exposure in vivo. Together, our findings show that arsenic exposure disrupts the genome-wide expression of miRNAs in vivo, which could lead to the biological consequence, such as an altered balance of antioxidant defense and oxidative stress.

Inhibition of monomethylarsonous acid (MMAIII)-induced cell malignant transformation through restoring dysregulated histone acetylation

Inorganic arsenic (iAs) and its high toxic metabolite, monomethylarsonous acid (MMA(III)), are able to induce malignant transformation of human cells. Chronic exposure to these chemicals is associated with an increased risk of developing multiple cancers in human. However, the mechanisms contributing to iAs/MMA(III)-induced cell malignant transformation and carcinogenesis are not fully elucidated. We recently showed that iAs/MMA(III) exposure to human cells led to a decreased level of histone acetylation globally, which was associated with an increased sensitivity to arsenic cytotoxicity. In the current study, it demonstrated that prolonged exposure to low-level MMA(III) in human urothelial cells significantly increased the expression and activity of histone deacetylases (HDACs) with an associated reduction of histone acetylation levels both globally and lysine specifically. Administration of the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), at 4 weeks after the initial MMA(III) treatment inhibited the MMA(III)-mediated up-regulation of the expression and activities of HDACs, leading to increase histone acetylation and prevention of MMA(III)-induced malignant transformation. These new findings suggest that histone acetylation dysregulation may be a key mechanism in MMA(III)-induced malignant transformation and carcinogenesis, and that HDAC inhibitors could be targeted to prevent or treat iAs-related cancers.

Toxicity of Trimethyltin and Dimethyltin in Rats and Mice

Extensive uses of methyltin compounds in polyvinyl chloride (PVC) production have led to a dramatic increase of occupational-related methyltin poisoning accidents and the widespread contamination of methyltins in various environmental media. Here, we conducted studies to compare the acute toxicity induced by trimethyltin (TMT) and dimethyltin (DMT), and investigated the cumulative toxic effects of TMT in rats and mice. Neurobehavioral changes were observed in rats and mice treated with either DMT or TMT, but we also observed that both TMT and DMT exposure in rats significantly lowered the blood potassium level. Moreover, the cumulative toxic coefficient factor of TMT was 1.7 in rats versus 3.8 in mice, suggesting a high cumulative risk for rats and a moderate risk for mice. In summary, we demonstrated that acute and chronic exposure to methyltin compounds induced neurotoxicity and hypokalemia. Moreover, our study suggests that TMT can accumulate in the body and pose a risk for workers chronically exposed to a low dose of TMT.

Chronic low level trimethyltin exposure and the risk of developing nephrolithiasis

Objectives Nephrolithiasis (kidney stones) is a common disease with the prevalence that is increasing globally. We previously found that trimethyltin (TMT), a by-product of plastic stabilisers, can inhibit the H+/K+ ATPase activity in renal intercalated cells and alter urinary pH, which is a known risk factor for nephrolithiasis. In this study, we conducted a cross-sectional analysis to evaluate the impact of chronic low level occupational TMT exposure on nephrolithiasis. Methods This study included 216 healthy workers with TMT exposure and 119 workers as controls with no TMT exposure. All study participants were administered a questionnaire and underwent a routine clinical examination including an ultrasonographic screening for kidney stones. Exposures were assessed by measuring TMT concentrations in personal air samples, blood and urine. Logistic regression analysis was used to estimate the ORs and 95% CIs for the risk of kidney stones. Results TMT exposed workers had a higher prevalence of kidney stones (18.06%) in comparison with control workers (5.88%). High TMT concentrations in personal air samples, blood and urines were positively associated with increased prevalence of kidney stones in workers exposed to TMT compared with controls workers (p-trend values=0.005, 0.008 and 0.002, respectively). The length of employment in plants with elevated TMT levels (duration of the exposure) was significantly associated with the increased prevalence of kidney stones (p trend=0.001). The ORs were 2.66 for <3 years, 3.73 for 3–<10 years and 7.89 for 10+ years of employment compared with control workers. Conclusions To our knowledge, this is the first report to demonstrate that occupational exposure to TMT is a potential risk factor for nephrolithiasis.

Interactive Influence of N6AMT1 and As3MT Genetic Variations on Arsenic Metabolism in the Population of Inner Mongolia, China.

Chronic arsenic exposure via drinking water has become a worldwide public health concern. In humans, inorganic arsenic (iAs) is metabolized to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) mainly mediated by arsenic (+3 oxidation state) methyltransferase (As3MT). We reported recently that N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) was involved in arsenic metabolism, and examined its interactive effect with As3MT on arsenic metabolism in vitro. To further evaluate the interactive effect of N6AMT1 and As3MT on arsenic biomethylation in humans, we conducted a human population-based study including 289 subjects living in rural villages in Inner Mongolia, China, and assessed their urinary arsenic metabolites profiles in relation to genetic polymorphisms and haplotypes of N6AMT1 and As3MT. Five N6AMT1 single nucleotide polymorphisms (SNPs; rs1003671, rs7282257, rs2065266, rs2738966, rs2248501) and the N6AMT1 haplotype 2_GGCCAT were significantly associated with the percentage of iAs (% iAs) in urine (e.g., for rs7282257, mean was 9.62% for TT, 6.73% for AA). Rs1003671 was also in a significant relationship with urinary MMA and DMA (the mean of %MMA was 24.95% for GA, 31.69% for GG; the mean of % DMA was 69.21% for GA, 59.82% for GG). The combined effect of N6AMT1 haplotype 2_GGCCAT and As3MT haplotype 2_GCAC showed consistence with the additive significance of each haplotype on % iAs: the mean was 5.47% and 9.36% for carriers with both and null haplotypes, respectively. Overall, we showed that N6AMT1 genetic polymorphisms were associated with arsenic biomethylation in the Chinese population, and its interaction with As3MT was observed in specific haplotype combinations.

Interactive Effects of N6AMT1 and As3MT in Arsenic Biomethylation.

In humans, arsenic is primarily metabolized by arsenic (+3 oxidation state) methyltransferase (As3MT) to yield both trivalent and pentavalent methylated metabolites. We recently reported that the putative N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) can biotransform monomethylarsonous acid (MMA(III)) to dimethylarsinic acid, conferring resistance of human cells to arsenic exposure. To further decipher the role of N6AMT1 and its interaction with As3MT in arsenic biomethylation, we examined the relative contribution of N6AMT1 and As3MT in metabolizing arsenic using several newly modified UROtsa human urothelial cells, ie, UROtsa cells with either a constant level of N6AMT1 or As3MT in combination with an inducible level of As3MT or N6AMT1, respectively. Our analysis confirmed the involvement of N6AMT1 in MMA(III) biomethylation but not for inorganic arsenic. In a comparable level of N6AMT1 and As3MT, the effect of N6AMT1 mediated MMA(III) biomethylation was obscured by the action of As3MT. Furthermore, we showed that the levels of N6AMT1 and As3MT proteins varied among and within human normal and cancerous tissues. Overall, the data showed that N6AMT1 has a role in MMA(III) biomethylation, but its effect is relatively minor and limited compared with As3MT. In addition, the varied levels and distributions of N6AMT1 and As3MT among human tissues may potentially contribute to the tissue specificity and susceptibility to arsenic toxicity and carcinogenicity.

Do environmental pollutants increase obesity risk in humans

Obesity has become a global epidemic and threat to public health. A good understanding of the causes can help attenuate the risk and spread. Environmental pollutants may have contributed to the rising global obesity rates. Some research reported associations between chemical pollutants and obesity, but findings are mixed. This study systematically examined associations between chemical pollutants and obesity in human subjects.

Application of human haploid cell genetic screening model in identifying the genes required for resistance to environmental toxicants: Chlorpyrifos as a case study.

INTRODUCTION High-throughput loss-of-function genetic screening tools in yeast or other model systems except in mammalian cells have been implemented to study human susceptibility to chemical toxicity. Here, we employed a newly developed human haploid cell (KBM7)-based mutagenic screening model (KBM7-mu cells) and examined its applicability in identifying genes whose absence allows cells to survive and proliferate in the presence of chemicals. METHODS KBM7-mu cells were exposed to 200 μM Chlorpyrifos (CPF), a widely used organophosphate pesticide, a dose causing approximately 50% death of cells after 48h of treatment. After a 2-3 week period of continuous CPF exposure, survived single cell colonies were recovered and used for further analysis. DNA isolated from these cells was amplified using Splinkerette PCR with specific designed primers, and sequenced to determine the genomic locations with virus insertion and identify genes affected by the insertion. Quantitative realtime reverse transcription PCR (qRT-PCR) was used to confirm the knockdown of transcription of identified target genes. RESULTS We identified total 9 human genes in which the cells carrying these genes conferred the resistance to CPF, including AGPAT6, AIG1, ATP8B2, BIK, DCAF12, FNBP4, LAT2, MZF1-AS1 and PPTC7. MZF1-AS1 is an antisense RNA and not included in the further analysis. qRT-PCR results showed that the expression of 6 genes was either significantly reduced or completely lost. There were no changes in the expression of DCAF12 and AGPAT6 genes between the KBM7-mu and the control KBM7 cells. DISCUSSION The KBM7-mu genetic screening system can be modified and applied to identify novel susceptibility genes in response to environmental toxicants, which could provide valuable insights into potential mechanisms of toxicity.

Mobilization and removing of cadmium from kidney by GMDTC utilizing renal glucose reabsorption pathway.

Chronic exposure to cadmium compounds (Cd(2+)) is one of the major public health problems facing humans in the 21st century. Cd(2+) in the human body accumulates primarily in the kidneys which leads to renal dysfunction and other adverse health effects. Efforts to find a safe and effective drug for removing Cd(2+) from the kidneys have largely failed. We developed and synthesized a new chemical, sodium (S)-2-(dithiocarboxylato((2S,3R,4R,5R)-2,3,4,5,6 pentahydroxyhexyl)amino)-4-(methylthio) butanoate (GMDTC). Here we report that GMDTC has a very low toxicity with an acute lethal dose (LD50) of more than 10,000mg/kg or 5000mg/kg body weight, respectively, via oral or intraperitoneal injection in mice and rats. In in vivo settings, up to 94% of Cd(2+) deposited in the kidneys of Cd(2+)-laden rabbits was removed and excreted via urine following a safe dose of GMDTC treatment for four weeks, and renal Cd(2+) level was reduced from 12.9μg/g to 1.3μg/g kidney weight. We observed similar results in the mouse and rat studies. Further, we demonstrated both in in vitro and in animal studies that the mechanism of transporting GMDTC and GMDTC-Cd complex into and out of renal tubular cells is likely assisted by two glucose transporters, sodium glucose cotransporter 2 (SGLT2) and glucose transporter 2 (GLUT2). Collectively, our study reports that GMDTC is safe and highly efficient in removing deposited Cd(2+) from kidneys assisted by renal glucose reabsorption system, suggesting that GMDTC may be the long-pursued agent used for preventive and therapeutic purposes for both acute and chronic Cd(2+) exposure.