Impact of Arsenite on Transient and Persistent Histone H3 Modifications and Transcriptional Response

Abstract

Arsenite-contaminated groundwater poses a major health concern affecting millions of people. Chronic exposure to elevated levels of inorganic arsenic is implicated in carcinogenesis, with impaired DNA repair and dysregulated DNA and histone modifications as key factors. Using human A549 lung carcinoma cells, we investigated the persistence of acute arsenite-induced cellular stress at the epigenetic and transcriptional levels after 24 h of exposure to 1-25 μM NaAsO₂, reflecting low to high acute exposure scenarios, followed by a 48 h arsenite-free postincubation period. The primary objective was to analyze alterations in acetylation and methylation marks on both bulk histone H3 and specific DNA repair gene loci. We conducted immunochemical and proteomic analyses to assess alterations in histone modification patterns. Transient effects were observed at both methylated and acetylated residues, with hypoacetylation specifically detected at promoters of certain DNA repair genes, including MLH1, MSH2, MPG, and XPA. Among all modifications analyzed, H3K18ac exhibited the most pronounced decline, suggesting its preferential sensitivity toward arsenite. H3 hypoacetylation was further observed in noncancerous human BEAS-2B lung cells, indicating that this effect is not cancer cell-specific. Mechanistically, in A549 cells, increased total HDAC or decreased HAT activity could be excluded. Instead, a persistent moderate decline in HDAC activity and a delayed, pronounced induction of HAT activity suggest targeted arsenite interactions with specific enzymes of the histone acetylation regulatory network.

1

Assessment of arsenite cytotoxicity, uptake, and impact on cell cycle progression in A549 cells. Cells were treated with NaAsO₂ for 24 h, followed by either no (0 h) or a 48 h postincubation period in arsenite-free cell culture medium. ATP content was analyzed immediately after arsenite treatment (A) or 48 h afterward (B). The relative cell count (RCC) was assessed using CASY cell count analysis (C), and the cellular uptake of arsenic was quantified by AAS (D). Moreover, the cell cycle distribution was analyzed by flow cytometry using DAPI staining (E,F). Shown are mean values ± SD from three independent experiments, each conducted in duplicate. Statistical analysis was performed using a one-way ANOVA followed by a Dunnett’s post hoc test to evaluate which of the observed changes in exposed cells reached statistical significance compared to the untreated control: *­(p < 0.05), **­(p < 0.01), ****­(p < 0.0001). To assess the effect of postincubation, statistical significance was determined using ANOVA with Šídák’s post hoc test: #### (p < 0.0001).

2

Effect of arsenite and postincubation on the gene expression profile of A549 cells. Cells were exposed to 25 μM NaAsO₂ for 24 h and subsequently subjected to either a 0 or 48 h arsenite-free recovery phase. The mRNA levels were assessed by high-throughput RT-qPCR in 95 genes. Displayed is the relative change in gene expression (RGE) compared to the untreated control against the p-value generated by two-way ANOVA, followed by Dunnett’s post hoc test. For simplification purposes, all p-values <0.0001 are displayed on the line of p = 0.0001.

3

Gene expression profiles of arsenite-treated and postincubated A549 cells. Cells were exposed to NaAsO₂ in the dose range of 1 μM to 25 μM for 24 h and subsequently subjected to either a 0 or 48 h arsenite-free recovery period. Gene expression was determined using high-throughput RT-qPCR. The results are presented as log₂-fold change of the relative gene expression (RGE). Gene induction is represented in red color; gene repression is depicted in blue. Shown are mean values from at least three independent experiments, each conducted in duplicate.

4

Basal versus arsenite-modified hPTM levels at selected DNA repair genes. A549 cells were incubated with different concentrations from 5 μM to 20 μM NaAsO₂ for 24 h. (A) and (B) show the ChIP-qPCR results for H3K4me3 and H3K27me3 at 20 μM. (C) Shows the results for H3K18ac at 20 μM and (D) at 5 μM and 10 μM. (E) Displays the ChIP-qPCR data of H3K9ac in the whole dose range. As positive and negative control locus RPL30, GAPDH, MB, and MYOD1 were determined. In addition, an IgG isotype control antibody was used to detect the nonspecific background. The corresponding ChIP controls for the enrichment of histone H3 at the positive control locus RPL30 can be found in the Supporting Information Figure S2. A representative example of the fragmentation pattern is given in Supporting Information Figure S3. Shown are the mean values ± SD of at least three independent experiments. Statistical analysis was performed using Welch’s test to evaluate which of the observed changes in exposed cells reached statistical significance compared to the untreated control: *­(p < 0.05), **­(p < 0.01), ***­(p < 0.001).

5

Effects of arsenite exposure and postincubation on the levels of various post-translational histone modifications in A549 cells. Cells were exposed to NaAsO₂ for 24 h and subsequently subjected to either 0 h (A,C,E,G) or 48 h (B,D,F,H) arsenite-free recovery period. Relative modification abundance was detected using Western blotting. For the semiquantitative analysis presented here, the results were normalized to the respective loading controls (Ponceau, histone H3, and β-Actin) and averaged. Shown are mean values ± SD from at least three independent experiments. Statistical analysis was performed using a one-way ANOVA followed by a Dunnett’s post hoc test to evaluate which of the observed changes in exposed cells reached statistical significance compared to the untreated control: *­(p < 0.05), **­(p < 0.01), ***­(p < 0.001), ****­(p < 0.0001).

6

Effects of acute arsenite exposure on the level of H3K9ac and H3K18ac in BEAS-2B cells. PTM levels were detected using Western blotting. The values were normalized to the respective loading controls (Ponceau, histone H3, and β-Actin) and averaged. Shown are mean values ± SD from at least three independent experiments performed. Statistical analysis was performed using a one-way ANOVA followed by a Dunnett’s post hoc test to evaluate which of the observed changes in exposed cells reached statistical significance compared to the untreated control: *­(p < 0.05), *** (p < 0.001), **** (p < 0.0001).

7

Relative abundances of the variably modified forms of selected peptides from histone H3 upon treatment of A549 cells with increasing arsenite concentrations. The cells were incubated with 5 μM to 20 μM NaAsO₂ for 24 h. Post-translational histone modifications were analyzed by LC–MS/MS. The data are shown for all modified forms of peptide spanning residues K9-R17 (A), K18-R26 (C), K27-R40 from canonical H3 (E) and variant H3.3 (G), and the respective zoomed in versions show PTM combinations at K9/K14 (B), K18/K23 (D), and K27/K36 (F,H) of these sequences significantly changing in abundance. The quantitative data obtained from biological triplicates were plotted as individual dots. The bar heights correspond to the mean ± confidence interval at 95% confidence level. The histograms of the other analyzed peptides from H3 are represented in Supporting Information Figure S7.

8

Effect of arsenite exposure and postincubation on the total HDAC and HAT activity in A549 cells. Cells were exposed to NaAsO₂ for 24 h and subsequently subjected to either 0 h (A,C,E) or 48 h (B,D,F) recultivation in the absence of arsenite. HDAC and HAT activity were determined by ELISA-based methods. The HDAC activity is shown in (A,B), while (C,D) display the results with an additional 2 μM Trichostatin A (TSA) HDAC inhibitor treatment of the nuclear extracts. The HAT activity is displayed in (E,F). Shown are mean values ± standard deviation from at least three independent experiments performed in duplicate. Statistical analysis was performed using a one-way ANOVA followed by a Dunnett’s post hoc test to evaluate which of the observed changes in exposed cells reached statistical significance compared to the untreated control: *­(p < 0.05), **­(p < 0.01), *** (p < 0.001).