Bromate-induced Changes in p21 DNA Methylation and Histone Acetylation in Renal Cells

Bromate (BrO3-) is a water disinfection byproduct (DBP) previously shown to induce nephrotoxicity in vitro and in vivo. We recently showed that inhibitors of DNA methyltransferase 5-aza-2-deoxycytidine (5-Aza) and histone deacetylase trichostatin A (TSA) increased BrO3- nephrotoxicity whereas altering the expression of the cyclin-dependent kinase inhibitor p21. Human embryonic kidney cells (HEK293) and normal rat kidney (NRK) cells were sub-chronically exposed to BrO3- or epigenetic inhibitors for 18 days, followed by 9 days of withdrawal. DNA methylation was studied using a modification of bisulfite amplicon sequencing called targeted gene bisulfite sequencing. Basal promoter methylation in the human p21 promoter region was substantially lower than that of the rat DNA. Furthermore, 5-Aza decreased DNA methylation in HEK293 cells at the sis-inducible element at 3 distinct CpG sites located at 691, 855, and 895 bp upstream of transcription start site (TSS). 5-Aza also decreased methylation at the rat p21 promoter about 250 bp upstream of the p21 TSS. In contrast, sub-chronic BrO3- exposure failed to alter methylation in human or rat renal cells. BrO3- exposure altered histone acetylation in NRK cells at the p21 TSS, but not in HEK293 cells. Interestingly, changes in DNA methylation induced by 5-Aza persisted after its removal; however, TSA- and BrO3--induced histone hyperacetylation returned to basal levels after 3 days of withdrawal. These data demonstrate novel sites within the p21 gene that are epigenetically regulated and further show that significant differences exist in the epigenetic landscape between rat and human p21, especially with regards to toxicant-induced changes in histone acetylation.