A Novel Arsenic Methyltransferase Splice Variant Expressed in Human Keratinocytes

Authors

Caitlin H. Reynolds, University of LouisvilleFollow
Alexandra N. Nail, University of LouisvilleFollow
Miroslav Styblo, University of North Carolina at Chapel HillFollow
Peter Cable, University of North Carolina at Chapel HillFollow
J. Christopher States, University of LouisvilleFollow

Program/Event

Arts and Research Showcase 2024

Abstract

Chronic inorganic arsenic (iAs) exposure from contaminated drinking water is a global issue that impacts over 220 million people worldwide. iAs is a Group 1 human carcinogen. Skin lesions, including skin cancer, are hallmarks of arsenic toxicity. Arsenic methyltransferase (AS3MT) is required for the biotransformation of iAs to monomethyl-As (MMA^V and MMA^III) and MMA to dimethyl-As (DMA^V and DMA^III), and the iAs:MMA:DMA ratio determines arsenic clearance and toxicity. Both trivalent MMA (MMA^III) and DMA (DMA^III) are exceedingly more toxic compared to iAs and may promote toxic and carcinogenic effects associated with chronic iAs exposure. HaCaT cells exposed to 100 nM iAs for 28 weeks undergo malignant transformation and represent an important tool to study iAs-induced skin carcinogenesis. However, whether these cells can metabolize iAs remains unknown. Our hypothesis is that HaCaT cells methylate iAs to promote malignant transformation. Human keratinocytes (HaCaT, Ker-CT, and HEKn) cells and a liver cell line (HepG2) were exposed to sodium arsenite (0 or 100 nM) for 24 h. iAs speciation within cells and media from 100 nM exposed cultures was determined by hydride-generation inductively coupled plasma mass spectrometry coupled with a cryotrap (HG-CT-ICP-MS). Total RNA and genomic DNA was isolated from 0 or 100 nM exposed cells. RNA was treated with DNAseI (-/+) and RNAseA (-/+) and AS3MT mRNA expression was determined by RT-PCR. Overall, human keratinocytes are poor iAs methylators compared to HepG2 cells. Among human keratinocytes, HaCaT cells contained the least amount of iAs metabolites compared to Ker-CT and HEKn cells. HaCaT cells also expressed the least amount of canonical AS3MT mRNA. In addition, we discovered a novel alternatively spliced AS3MT mRNA isoform that is expressed predominantly in human keratinocytes. Our conclusion is that arsenic-induced malignant transformation in HaCaT cells chronically exposed to iAs is likely driven by iAs rather than iAs metabolites. The novel AS3MT splice variant retains intron 2 of the AS3MT gene and this may promote nonsense-mediated mRNA decay of this transcript. Future work will determine whether chronic iAs exposure in human keratinocytes promotes upregulation of the canonical AS3MT mRNA and/or the novel AS3MT splice variant, and whether the new AS3MT splice variant is degraded by nonsense-mediated decay preventing translation of a truncated, non-functional AS3MT protein.

Recommended Citation

Reynolds, Caitlin H.; Nail, Alexandra N.; Styblo, Miroslav; Cable, Peter; and States, J. Christopher (2025) "A Novel Arsenic Methyltransferase Splice Variant Expressed in Human Keratinocytes," The Cardinal Edge: Vol. 3: Iss. 2, Article 29.
Available at: https://ir.library.louisville.edu/tce/vol3/iss2/29