Molecular effects of arsenic and smokeless tobacco aqueous extract in cultured cells

Chronic arsenic exposure can lead to various health issues including cancer. There has been a growing concern about co-exposure to various prevalent lifestyle habits and their role in the enhancement of arsenic toxicity. Smokeless tobacco (SLT) products are extensively consumed in many South Asian countries, where their use frequently co-occurs with exposure to arsenic from contaminated groundwater. To decipher the oral epithelial cell responses to arsenic and SLT alone and in co-exposure, we performed multi-omics analyses of DNA methylome, transcriptomic reprogramming and genotoxic effects in controlled experimental settings. Chronic exposure studies revealed hypomethylation of genes involved in inflammation response and apoptosis, further corroborated by the upregulation of genes involved in these processes due to arsenic and the combined treatment in acute exposure setting. Next, to validate the omics results at the phenotypic level, we observed a dose dependent decrease in cell viability, induction of DNA damage, cell cycle changes, and an increase in apoptotic cells, with the most pronounced effects observed under arsenic and SLT co-exposure conditions. The observed DNA damage was likely the result of apoptosis induction, as chronic exposure experiments based on whole-exome sequencing did not reveal increased mutagenicity following the arsenic and/or SLT exposure. Our integrative omics study provides insights into both chronic and acute responses to arsenic and SLT co-exposure, with both types of responses converging on some of the same mechanisms. We identified large-scale epigenomic and transcriptomic reprograming associated with arsenic and SLT co-exposure, alongside genotoxic effects presumably manifesting as consequences of apoptosis induction. The findings point to a role of arsenic and SLT in altering key molecular responses, especially in the context of the co-exposure, and call for further studies in humans in the areas of exposure, to validate the observed mechanisms. htert-immortalized normal oral keratinocytes (NOK) cells underwent the respective, controlled treatment protocols. Briefly, each week the cells were exposed for 24 hr, and following treatment, the exposure medium was replaced with normal media and cells were left for recovery. Exposures were repeated in for a period of 5 weeks. Following the repeated exposure protocol, genomic DNA was extracted from the cells using standard protocol. For each sample, 500ng DNA were bisulfite converted to enable quantitative assessment of methylation level over 850K CpG sites. Following this treatment, methylation profiles were obtained with Infinium® Methylation EPIC bead arrays (Illumina), using recommended protocols for amplification, labelling, hybridisation, and scanning. For each treatment condition triplicate wells were analysed. Samples: Samples: Parental (4 biological replicates), NT (Non treated time matched control, 3 biological replicates), SA (Sodium arsenite treated: 10µM, 3 biological replicates), SLT (smokeless tobacco aqueous extract treated: 3000 µg/mL, 3 biological replicates), SA+SLT [co-exposed: (SA: 7µm)+(SLT: 1000 µg/mL), 3 biological replicates].