Molecular effects of arsenic and smokeless tobacco aqueous extract in cultured cells [RNA-seq]

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 Normal oral keratinocytes cells were grown in T25 flasks (seeding density ~ 3 - 5 × 105 cells). Upon reaching a confluency of around 70%, the cells were treated with media containing the test substances and were grouped into various time points based on exposure period (2 hrs, 4 hrs and 8 hrs). Two experimental control groups were included, the baseline untreated control (0 hr), and time-matched untreated control (harvested at 8 hrs). Each timepoint condition consisted of four experimental replicates. After exposure, the media containing the test substances were removed from the flasks and the cellular RNA was extracted using standard methods (TRIzol™ Reagent, Cat. No. 15596026, Invitrogen). Estimation of yield was done by Qubit analysis (Qubit 3.0 Fluorometer, Life Technologies, Invitrogen™, Thermo Scientific Fisher) . RNA integrity was analysed on High Sensitivity RNA ScreenTape (Cat. No. 5067- 5579, Agilent), using the 4200 TapeStation automated electrophoresis instrument (Agilent). All samples exhibited RNA Integrity Number (RIN) above 8.4. 500 ng RNA per sample was used to prepare libraries (QuantSeq 3’ mRNA-Seq Library Prep Kit for Illumina (FWD)), Cat. No. 015, Lexogen).