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Bromate-induced Changes in p21 DNA Methylation and Histone Acetylation in Renal Cells
Ramya T Kolli1,2,3, Travis C Glenn2,4, Bradley T Brown5
1Department of Pharmaceutical and Biomedical Sciences.
Abstract:
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.
Insights
Bromate (BrO3-) exposure alters kidney cell histone acetylation, while epigenetic drugs like 5-Aza and TSA impact DNA methylation and p21 gene expression, revealing species-specific epigenetic differences in nephrotoxicity.
Area of Science:
- Environmental Toxicology
- Epigenetics
- Renal Cell Biology
Background:
- Bromate (BrO3-) is a nephrotoxic water disinfection byproduct.
- Epigenetic modifications, including DNA methylation and histone acetylation, play crucial roles in cellular regulation.
- Previous studies suggest epigenetic inhibitors like 5-aza-2'-deoxycytidine (5-Aza) and trichostatin A (TSA) can exacerbate BrO3- induced nephrotoxicity by altering p21 expression.
Purpose of the Study:
- To investigate the epigenetic regulation of the p21 gene in human (HEK293) and rat (NRK) kidney cells following exposure to BrO3- and epigenetic inhibitors.
- To compare the epigenetic landscape and toxicant-induced changes between human and rat p21 genes.
Main Methods:
- Sub-chronic exposure of HEK293 and NRK cells to BrO3-, 5-Aza, or TSA for 18 days, followed by a 9-day withdrawal period.
- Targeted gene bisulfite sequencing to analyze DNA methylation patterns in the p21 promoter region.
- Assessment of histone acetylation levels at the p21 transcription start site (TSS).
Main Results:
- Basal DNA methylation levels in the human p21 promoter were lower than in the rat p21 promoter.
- 5-Aza decreased DNA methylation in both human and rat cells, with persistent effects after withdrawal.
- BrO3- exposure altered histone acetylation in rat cells but not human cells, with transient effects after withdrawal.
Conclusions:
- Novel epigenetic regulatory sites within the p21 gene were identified.
- Significant interspecies differences exist in the epigenetic regulation of the p21 gene, particularly concerning toxicant-induced histone acetylation.
- These findings highlight the complex interplay between environmental toxicants, epigenetic modifications, and species-specific responses in nephrotoxicity.
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