Epigenetic events determine tissue-specific toxicity of inhalational exposure to the genotoxic chemical 1,3-butadiene in male C57BL/6J mice

Abstract

1,3-Butadiene (BD), a widely used industrial chemical and a ubiquitous environmental pollutant, is a known human carcinogen. Although genotoxicity is an established mechanism of the tumorigenicity of BD, epigenetic effects have also been observed in livers of mice exposed to the chemical. To better characterize the diverse molecular mechanisms of BD tumorigenicity, we evaluated genotoxic and epigenotoxic effects of BD exposure in mouse tissues that are target (lung and liver) and non-target (kidney) for BD-induced tumors. We hypothesized that epigenetic alterations may explain, at least in part, the tissue-specific differences in BD tumorigenicity in mice. We evaluated the level of N-7-(2,3,4-trihydroxybut-1-yl)guanine adducts and 1,4-bis-(guan-7-yl)-2,3-butanediol crosslinks, DNA methylation, and histone modifications in male C57BL/6 mice exposed to filtered air or 425 ppm of BD by inhalation (6 h/day, 5 days/week) for 2 weeks. Although DNA damage was observed in all three tissues of BD-exposed mice, variation in epigenetic effects clearly existed between the kidneys, liver, and lungs. Epigenetic alterations indicative of genomic instability, including demethylation of repetitive DNA sequences and alterations in histone-lysine acetylation, were evident in the liver and lung tissues of BD-exposed mice. Changes in DNA methylation were insignificant in the kidneys of treated mice, whereas marks of condensed heterochromatin and transcriptional silencing (histone-lysine trimethylation) were increased. These modifications may represent a potential mechanistic explanation for the lack of tumorigenesis in the kidney. Our results indicate that differential tissue susceptibility to chemical-induced tumorigenesis may be attributed to tissue-specific epigenetic alterations.

Keywords: 1,3-butadiene; epigenetics; genotoxicity.

FIG. 1.

Amounts of THB-Gua-BD adducts (A) and Bis-N7G-BD crosslinks (B) in tissues from mice exposed to 425 ppm of BD. Data are presented as mean ± SD (n = 3). Asterisk and pound (* and #) denote significant (P < 0.05) differences in the amount of the same adduct between the lung and liver, and lung and kidney, respectively.

FIG. 2.

Effects of BD exposure on the extent of DNA methylation in mouse tissues. Loss of methylation at SINE B1, SINE B2, major and minor satellite repetitive elements in the tissues of BD-exposed mice as measured by McrBC-methylation sensitive quantitative PCR (qPCR). The results are presented as the average fold change in the degree of DNA hypomethylation relative to the control values of the corresponding tissues. Data are presented as mean ± SD (n = 3). Asterisks (*) denote significant (P < 0.05) differences from the controls.

FIG. 3.

Effects of BD exposure on the extent of 5-hydroxymethylcytosine in mouse tissues. Loss of methylation at SINE B1, SINE B2, major and minor satellite repetitive elements in the tissues of BD-exposed mice as determined by hydroxymethylated DNA immunoprecipitation (hMeDIP). The results are presented as the average fold change in the degree of DNA hypomethylation relative to the control values of the corresponding tissues. Data are presented as mean ± SD (n = 3). Asterisks (*) denote significant (P < 0.05) differences from the controls.

FIG. 4.

Effects of BD exposure on histone modifications. H3K27me3, H4K20me3, H3K9me3, H3K27ac, H3K56ac, and H4K16ac levels were assessed by immunostaining using specific antibodies against trimethylated or acetylated histones. Equal sample loading was confirmed by immunostaining against total histone H3 or H4. Densitometry analysis of the immunostaining results is shown as change in methylation or acetylation level relative to the tissue-matched controls after correction for the total amount of each histone in the individual samples. Results are presented as the average fold change relative to the control values of the corresponding tissues. Data are presented as mean ± SD (n = 3). Asterisks (*) denote significant (P < 0.05) differences from the controls.

FIG. 5.

Effects of BD exposure on the expression of DNA methylation and DNA demethylation genes. mRNA levels of DNA methyltransferase genes (A) and methylcytosine dioxygenase genes (B) were evaluated by qPCR. Results are presented as the average fold change relative to the control values of the corresponding tissue. All experimental genes were run at least in triplicate. Data are presented as mean ± SD (n = 3). Asterisks (*) denote significant (P < 0.05) differences from the controls.

FIG. 6.

Effects of BD exposure on the expression of histone modifying genes. mRNA levels of histone methyltransferase genes (A) and genes related to histone acetylation (B) were evaluated by qPCR. Results are presented as the average fold change relative to the control values of the corresponding tissue. All experimental genes were run at least in triplicate. Data are presented as mean ± SD (n = 3). Asterisks (*) denote significant (P < 0.05) differences from the controls.

FIG. 7.

Summary of genotoxic and epigenetic changes in the tissues of BD-exposed mice. This chart highlights the significant alterations observed in each of the tissues as presented in Figs. 1–6. Arrows indicate a direction of the effect (increase or decrease) observed for each of the marks listed in the same box, when compared with the same tissues in control mice.