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Published on: April 7, 2014
In vitro metabolism of 4-vinylcyclohexene in rat and mouse liver, lung, and ovary
D A Keller1, S C Carpenter, S Z Cagen
1E.I. du Pont de Nemours and Company, Haskell Laboratory for Toxicology and Industrial Medicine, Newark, Delaware 19714, USA.
Abstract:
4-Vinylcyclohexene (4-VCH), the dimer of 1,3-butadiene, is an ovarian toxicant in mice due to the formation of a diepoxide metabolite, but the tissue-specific site of formation of the metabolites is unknown. Microsomal preparations from liver, lung, and ovaries obtained from female Crl:CD BR rats and female B6C3F1 mice were tested for their ability to metabolize the following reactions: 4-VCH to 4-VCH-1,2-epoxide and 4-VCH-7,8-epoxide; 4-VCH-1,2-epoxide to 4-VCH diepoxide and 4-VCH-1,2-diol; 4-VCH-7,8-epoxide to 4-VCH diepoxide and 4-VCH-7,8-diol; and hydrolysis of 4-VCH diepoxide. Microsomes were incubated with the test chemical and the reaction products were analyzed by gas chromatography. Rat liver and lung microsomes and mouse liver and lung microsomes metabolized 4-VCH to 4-VCH-1,2-epoxide at detectable rates. Mouse liver had a Vmax for the reaction that was 56-fold higher than that for rat liver (11.1 and 0.20 nmol/min/mg protein, respectively). The Vmax for mouse lung was 2-fold higher than that for rat lung. 4-VCH-1,2-epoxide formation was not detected in ovarian microsomes from rats or mice. Metabolism of 4-VCH to 4-VCH-7,8-epoxide was detected in microsomes from rat liver and mouse liver and lung, at rates very low compared to those for metabolism to the 1,2-epoxide. Rat and mouse liver had very similar K(m) and Vmax values for metabolism of 4-VCH-1,2-epoxide to 4-VCH diepoxide. The Vmax for rat liver was 3.69 and for mouse liver was 5.35 nmol/min/mg protein. Rat and mouse ovaries did not have detectable capacity to metabolize 4-VCH-1,2-epoxide to the diepoxide. Rat and mouse liver and lung have very similar K(m) and Vmax values for metabolism of 4-VCH-7,8-epoxide to the diepoxide, while ovaries did not have detectable rates for this reaction. Hydrolysis of 4-VCH-1,2-epoxide to 4-VCH-1,2-diol was at similar rates in rat and mouse liver microsomes. Hydrolysis of 4-VCH-7,8-epoxide to 4-VCH-7,8-diol was detected only in rat liver microsomes. Hydrolysis of 4-VCH diepoxide was detected in rat and mouse liver and lung, and in rat ovary microsomes. The Vmax for rat liver was 9-fold greater than that for mouse liver (5.51 and 0.63 nmol/min/mg protein, respectively), and lung and ovary tissues were not as active as rat liver. The balance of activation versus detoxication reactions in rats and mice suggests that the mouse may be more susceptible to 4-VCH toxicity because of generation of high levels of epoxide metabolites. In general, the mouse is more efficient at metabolism of 4-VCH to epoxides than is the rat. In contrast, the rat may be more efficient at hydrolysis of epoxides. Thus, the rat would tend to produce a lower concentration of epoxide metabolites than the mouse, at equal doses of 4-VCH.
Insights
Mice are more susceptible to 4-vinylcyclohexene (4-VCH) ovarian toxicity because their livers generate higher levels of toxic epoxide metabolites. Rats, however, are more efficient at detoxifying these epoxides through hydrolysis, leading to lower metabolite concentrations.
Area of Science:
- Toxicology
- Metabolism
- Biochemistry
Background:
- 4-Vinylcyclohexene (4-VCH) is an ovarian toxicant in mice, linked to a diepoxide metabolite.
- The specific tissues responsible for 4-VCH metabolite formation remain unidentified.
Purpose of the Study:
- To investigate the tissue-specific metabolism of 4-vinylcyclohexene (4-VCH) in rats and mice.
- To determine the sites of formation and hydrolysis of 4-VCH epoxide metabolites.
Main Methods:
- Microsomal preparations from rat and mouse liver, lung, and ovaries were used.
- Enzyme kinetics (Vmax, Km) were analyzed using gas chromatography to quantify metabolite formation and hydrolysis.
- Specific reactions studied included 4-VCH epoxidation, epoxide hydrolysis, and diepoxide formation.
Main Results:
- Mouse liver microsomes exhibited significantly higher Vmax for 4-VCH to 4-VCH-1,2-epoxide conversion (56-fold) compared to rat liver.
- Neither rat nor mouse ovarian microsomes formed 4-VCH-1,2-epoxide or the diepoxide metabolite.
- Mice showed higher efficiency in epoxide formation, while rats demonstrated greater efficiency in epoxide hydrolysis, particularly in the liver.
Conclusions:
- Liver and lung tissues are the primary sites for 4-VCH epoxidation, not ovaries.
- Differences in metabolic activation (epoxide formation) and detoxification (hydrolysis) pathways contribute to species-specific susceptibility to 4-VCH toxicity.
- Mice are potentially more susceptible to 4-VCH toxicity due to higher epoxide metabolite generation, whereas rats may be protected by more efficient epoxide hydrolysis.
