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.

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.

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.