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Concentration- and time-dependent genotoxicity profiles of isoprene monoepoxides and diepoxide, and the cross-linking potential of isoprene diepoxide in cells - PubMed

  • ️Wed Jan 01 2014

Concentration- and time-dependent genotoxicity profiles of isoprene monoepoxides and diepoxide, and the cross-linking potential of isoprene diepoxide in cells

Yan Li et al. Toxicol Rep. 2014.

Abstract

Isoprene, a possible carcinogen, is a petrochemical and a natural product being primarily produced by plants. It is biotransformed to 2-ethenyl-2-methyloxirane (IP-1,2-O) and 2-(1-methylethenyl)oxirane (IP-3,4-O), both of which can be further metabolized to 2-methyl-2,2'-bioxirane (MBO). MBO is mutagenic, but IP-1,2-O and IP-3,4-O are not. While IP-1,2-O has been reported being genotoxic, the genotoxicity of IP-3,4-O and MBO, and the cross-linking potential of MBO have not been examined. In the present study, we used the comet assay to investigate the concentration- and time-dependent genotoxicity profiles of the three metabolites and the cross-linking potential of MBO in human hepatocyte L02 cells. For the incubation time of 1 h, all metabolites showed positive concentration-dependent profiles with a potency rank order of IP-3,4-O > MBO > IP-1,2-O. In human hepatocellular carcinoma (HepG2) and human leukemia (HL60) cells, IP-3,4-O was still more potent in inducing DNA breaks than MBO at high concentrations (>200 μM), although at low concentrations (≤200 μM) IP-3,4-O exhibited slightly lower or similar potency to MBO. Interestingly, their time-dependent genotoxicity profiles (0.5-4 h) in L02 cells were different from each other: IP-1,2-O and MBO (200 μM) exhibited negative and positive profiles, respectively, with IP-3,4-O lying in between, namely, IP-3,4-O-caused DNA breaks did not change over the exposure time. Further experiments demonstrated that hydrolysis of IP-1,2-O contributed to the negative profile and MBO induced cross-links at high concentrations and long incubation times. Collectively, the results suggested that IP-3,4-O might play a significant role in the toxicity of isoprene.

Keywords: %Tail DNA, percentage of DNA in the tail; 2-(1-Methylethenyl)oxirane; 2-Ethenyl-2-methyloxirane; 2-Methyl-2,2′-bioxirane; Comet assay; DEB, 1,2,3,4-diepoxybutane; DMEM, Dulbecco's Modified Eagle's Medium; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; Genotoxicity; HL60, human leukemia cells; HepG2, human hepatocellular carcinoma cells; IMDM, Iscove's Modified Dulbecco's Medium; IP-1,2-O, 2-ethenyl-2-methyloxirane; IP-3,4-O, 2-(1-methylethenyl)oxirane; Isoprene; Isoprene metabolites; MBO, 2-methyl-2,2′-bioxirane; MMS, methyl methanesulfonate; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; PBMCs, peripheral blood mononuclear cells; SD, standard deviation; mCPBA, m-chloroperoxybenzoic acid.

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Figures

Fig. 1
Fig. 1

The metabolism pathways of isoprene.

Fig. 2
Fig. 2

Cytotoxicity of IP-3,4-O and MBO at different concentrations with different exposure times as measured by the MTT assay (IP-1,2-O did not exhibit cytotoxicity up to 1000 μM and 12 h, and thus its data were not shown) (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Fig. 3
Fig. 3

The rates of DNA migration caused by three metabolites of isoprene (IP-1,2-O, IP-3,4-O, and MBO) at different concentrations with the incubation time being 1 h in human hepatocyte L02 cells as measured by the standard comet assay (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Fig. 4
Fig. 4

The rates of DNA migration caused by IP-1,2-O, IP-3,4-O, and MBO at different concentrations with the incubation time being 1 h in HepG2 and HL60 cells as measured by the standard comet assay (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Fig. 5
Fig. 5

The rates of DNA migration induced by 1000 μM IP-1,2-O, 200 μM IP-3,4-O, and 200 and 1000 μM MBO in human hepatocyte L02 cells with the incubation time being 0.5, 1, 2, and 4 h (an additional time point of 6 h was tested for 1000 μM MBO) as measured by the standard comet assay (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Fig. 6
Fig. 6

The decrease in the rate of DNA migration caused by IP-1,2-O with the increase in the incubation time could be attributed to rapid hydrolysis of IP-1,2-O. Human hepatocyte L02 cells were incubated with 1000 μM IP-1,2-O for 1, 2, and 4 h. For comparison, cells were also incubated with 1000 μM IP-1,2-O for 2 and 4 h, but the IP-1,2-O-containing media were replaced with freshly prepared media every hour (which were indicated by 2R and 4R) (*p ≤ 0.05, **p ≤ 0.01).

Fig. 7
Fig. 7

Exposure to high concentrations of MBO for long time led to decreases in the rates of DNA migration caused by MMS in human hepatocyte L02 cells. Cells were exposed to different concentrations of MBO for 6 h and then 500 μM MMS for 1 h, and subsequently were subjected to the standard comet assay. Inset: Cells were incubated with 1000 μM MBO for 4 h and then 500 μM MMS for 1 h, and were subjected to the standard comet assay. Lower concentrations of MBO did not show any statistically significant effects, as a result, the data were not shown. As a positive control, cells were incubated with DEB (500 μM, 1 h) and then MMS (*p ≤ 0.05, **p ≤ 0.01).

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