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. 2014 Jun;139(2):371-80.
doi: 10.1093/toxsci/kfu044. Epub 2014 Mar 10.

Dose- and time-dependent epigenetic changes in the livers of Fisher 344 rats exposed to furan

Aline de Conti  1 , Tetyana Kobets, Claudia Escudero-Lourdes, Beverly Montgomery, Volodymyr Tryndyak, Frederick Alan Beland, Daniel R Doerge, Igor Petrovych Pogribny
Affiliations

Dose- and time-dependent epigenetic changes in the livers of Fisher 344 rats exposed to furan

Aline de Conti et al. Toxicol Sci. 2014 Jun.

Abstract

The presence of furan in common cooked foods along with evidence from experimental studies that lifetime exposure to furan causes liver tumors in rats and mice has caused concern to regulatory public health agencies worldwide; however, the mechanisms of the furan-induced hepatocarcinogenicity remain unclear. The goal of the present study was to investigate whether or not long-term exposure to furan causes epigenetic alterations in rat liver. Treating of male Fisher 344 rats by gavage 5 days per week with 0, 0.92, 2.0, or 4.4 mg furan/kg body weight (bw)/day resulted in dose- and time-dependent epigenetic changes consisting of alterations in DNA methylation and histone lysine methylation and acetylation, altered expression of chromatin modifying genes, and gene-specific methylation. Specifically, exposure to furan at doses 0.92, 2.0, or 4.4 mg furan/kg bw/day caused global DNA demethylation after 360 days of treatment. There was also a sustained decrease in the levels of histone H3 lysine 9 and H4 lysine 20 trimethylation after 180 and 360 days of furan exposure, and a marked reduction of histone H3 lysine 9 and H3 lysine 56 acetylation after 360 days at 4.4 mg/kg bw/day. These histone modification changes were accompanied by a reduced expression of Suv39h1, Prdm2, and Suv4-20h2 histone methyltransferases and Ep300 and Kat2a histone acetyltransferases. Additionally, furan at 2.0 and 4.4 mg/kg bw/day induced hypermethylation-dependent down-regulation of the Rassf1a gene in the livers after 180 and 360 days. These findings indicate possible involvement of dose- and time-dependent epigenetic modifications in the furan hepatotoxicity and carcinogenicity.

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Figures

FIG. 1.
FIG. 1.
DNA methylation and the expression of DNA methyltransferases in the livers of control rats and rats treated with furan. (A) The extent of DNA methylation. The results are presented as an average percentage change in the degree of DNA hypomethylation in the livers of rats from the furan-treated group relative to that in the control group, which was assigned a value of 100%. The expression of Dnmt1 (B), Dnmt3a (C), and Dnmt3b (D) genes was determined by qRT-PCR as detailed in the Materials and Methods section. The results are presented as an average fold change in the expression of each gene in the livers of rats from each experimental group relative to that in the control group, which were assigned a value 1. Values are mean ± SD (n = 5). * denotes a significant (p < 0.05) difference from the control rats; † denotes significant (p < 0.05) trend.
FIG. 2.
FIG. 2.
Westernblot analysis of histone H3K9, and H4K20 trimethylation and H3K9 and H3K56 acetylation in the livers of control rats and rats treated with furan. (A) Representative Western blot images. (B) Densitometric analysis of the immunostaining results, shown as percent change in histone modification level in the each experimental group relative to the corresponding values in the control group. Values are mean ± SD (n = 5). * denotes a significant (p < 0.05) difference from the control rats; † denotes significant (p < 0.05) trend.
FIG. 3.
FIG. 3.
The expression of chromatin-modifying genes in the livers of control rats and rats treated with furan. The expression of Prdm2, Suv39h1, Suv420h2, Ehmt2, Ep300, and Kat2a genes was determined by qRT-PCR as detailed in the Materials and Methods section. The results are presented as an average fold change in the expression of each gene in the livers of rats from each experimental group relative to that in the control group, which were assigned a value 1. Values shown are mean ± SD (n = 5). * denotes a significant (p < 0.05) difference from the control rats; † denotes significant (p < 0.05) trend.
FIG. 4.
FIG. 4.
Promoter methylation and expression of Rassf1a, and Cdh1 tumor suppressor genes in the livers of control rats and rats treated with furan. (A) Methylation-specific PCR analysis of Rassf1a promoter methylation. Bisulfite-modified DNA was amplified with two sets of primers specific to unmethylated (U) and methylated (M) CpG sites in the promoter region. Presence of methylated PCR product band after amplification with methylation-specific primers indicates appearance of de novo methylation. (B) The expression of Rassf1a as determined by qRT-PCR. (C) Methylation-specific PCR analysis of p16INK4a and Cdh1 promoter methylation. (D) The expression of p16INK4a as determined by qRT-PCR. (D) The expression of Cdh1 as determined by qRT-PCR. Values are mean ± SD (n = 5). * denotes a significant (p < 0.05) difference from the control rats; † denotes significant (p < 0.05) trend.
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