Cytotoxicity, genotoxicity, and mutagenicity of 1-chloro-2-hydroxy-3-butene and 1-chloro-3-buten-2-one, two alternative metabolites of 1,3-butadiene

Abstract

The cytotoxicity, genotoxicity, and mutagenicity of 1-chloro-2-hydroxy-3-butene (CHB), a known in vitro metabolite of the human carcinogen 1,3-butadiene, have not previously been investigated. Because CHB can be bioactivated by alcohol dehydrogenases to yield 1-chloro-3-buten-2-one (CBO), a bifunctional alkylating agent that caused globin-chain cross-links in erythrocytes, in the present study we investigated the cytotoxic and genotoxic potential of CHB and CBO in human normal hepatocyte L02 cells using the MTT assay, the relative cloning efficiency assay and the comet assay. We also investigated the mutagenic potential of these compounds with the Ames test using Salmonella strains TA1535 and TA1537. The results provide clear evidence for CHB and CBO being both cytotoxic and genotoxic with CBO being approximately 100-fold more potent than CHB. Interestingly, CHB generated both single-strand breaks and alkali-labile sites on DNA, whereas CBO produced only alkali-labile sites. CHB did not directly result in DNA breaks, whereas CBO was capable of directly generating breaks on DNA. Interestingly, both compounds did not induce DNA cross-links as examined by the comet assay. The Ames test results showed that CHB induced point mutation but not frameshift mutation, whereas the toxic effects of CBO made it difficult to reliably assess the mutagenic potential of CBO in the two strains. Collectively, the results suggest that CHB and CBO may play a role in the mutagenicity and carcinogenicity of 1,3-butadiene.

Keywords: %Tail DNA; 1,2,3,4-diepoxybutane; 1,3-Butadiene; 1,3-butadiene; 1-Chloro-2-hydroxy-3-butene; 1-Chloro-3-buten-2-one; 1-chloro-2-hydroxy-3-butene; 1-chloro-3-buten-2-one; 3,4-epoxy-1,2-butanediol; 3,4-epoxy-1-butene; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; ALS; BD; CBO; CHB; Comet assay; DEB; EB; EBD; FBS; GSH; Genotoxicity; MMS; MTT; Mutagenicity; RCE; SD; SSB; alkali-labile sites; fetal bovine serum; glutathione; methyl methanesulfonate; percentage of DNA in the tail; relative cloning efficiency; single-strand breaks; standard deviation.

Fig. 1

Cytotoxicity of CBO on human hepatocyte L02 cells as measured by the MTT assay. Cells were exposed to CBO in FBS-free media at 37 °C for 1 h. Results are shown as the mean ± SD of the values obtained from six independent samples (*p ≤ 0.05, ***p ≤ 0.001).

Fig. 2

The effects of CHB (A) and CBO (B) on the long-term survival (relative cloning efficiency) of human hepatocyte L02 cells. Cells were exposed to CHB and CBO in FBS-free media at 37 °C for 1 h. Results are shown as the mean ± SD of the values obtained from three independent samples (*p ≤ 0.05, ***p ≤ 0.001).

Fig. 3

DNA migration caused by different concentrations of CHB (A) and CBO (B) on human hepatocyte L02 cells as determined by the standard comet assay. Cells were exposed to CHB and CBO in FBS-free media at 37 °C for 1 h. Results are shown as the mean ± SD of the values obtained from three independent samples (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Fig. 4

DNA migration caused by different concentrations of CHB in human hepatocyte L02 cells as determined by the pH 11.9 and pH 9 comet assays to distinguish SSB from ALS. Cells were exposed to CHB in FBS-free media at 37 °C for 1 h. Results are shown as the mean ± SD of the values obtained from three independent samples (*p ≤ 0.05, ***p ≤ 0.001).

Fig. 5

DNA migration caused by different concentrations of CBO on lysed cells as determined by the acellular comet assay. Cells were first lysed and then exposed to CBO in 400 mM pH 7 phosphate buffer at 37 °C for 1 h. Results are shown as the mean ± SD of the values obtained from three independent samples (*p ≤ 0.05).

Fig. 6

The numbers of revertant colonies per plate observed for strain Salmonella TA1535 after being treated with different doses of CHB as described in the Materials and methods section. EB (5 mg per plate) was used as a positive control. Results are shown as the mean ± SD of the values obtained from three independent samples (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).