In vitro metabolism of 4-vinylcyclohexene in rat and mouse liver, lung, and ovary.

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.

Toxicity of methylating agents in isolated hepatocytes.

To investigate the pathogenesis of hepatotoxicity by methylating agents, we exposed isolated hepatocytes to N-nitrosodimethylamine (NDMA), N-methyl-N'-nitro-N nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (MNU), or methyl methanesulfonate (MMS). Although NDMA is a potent in vivo hepatotoxicant in rats, no evidence of hepatocyte injury, measured by the leakage of lactate dehydrogenase (LDH) activity into the medium, was observed following exposure to a 1-100 mM concentration of either NDMA or MNU. In contrast, exposure of hepatocytes to MMS or MNNG resulted in greater than or equal to 90% LDH release. These differences in toxicity were not related to the extent of covalent binding to hepatocytes. Following MMS or MNNG, but not MNU or NDMA exposure, a significant rise in the generation of thiobarbiturate (TBA)-reactive species was observed. When hepatocytes were exposed to the antioxidant promethazine prior to the addition of MMS or MNNG, the formation of TBA-reactive species was inhibited completely. Although promethazine blocked MNNG-mediated cell injury, the antioxidant had no effect on MMS intoxication. These data suggest that methylating agents can cause hepatotoxicity by more than a single mechanism. For MNNG, lipid peroxidation may be involved in the pathogenesis of acute hepatotoxicity.

Calcium transport, thiol status, and hepatotoxicity following N-nitrosodimethylamine exposure in mice.

The hepatotoxicant N-nitrosodimethylamine (NDMA) is presumed to exert toxicity through reactive metabolites. NDMA is similar in this respect to numerous other hepatotoxicants, for which hepatotoxicity is also associated with a rapid depletion of soluble and/or protein thiols, and an inhibition of calcium transport systems. We examined the hypothesis that hepatotoxicity for NDMA is preceded by thiol depletion and/or inhibition of calcium transport in isolated liver subcellular fractions. Centrizonal liver necrosis in mice was evident at 24 but not at 12 h subsequent to intraperitoneal administration of 40 mg NDMA/kg. Hepatotoxicity was not preceded by depletion of liver protein-free sulfhydryls, nor by protein sulfhydryl depletion in liver whole homogenate, microsomal, or plasma membrane fractions. NDMA-mediated toxicity was also not preceded by inhibition of calcium uptake capability by microsomal, mitochondrial, or plasma membrane fractions. In contrast, carbon tetrachloride produced the expected rapid decrease in microsomal calcium uptake capability, followed by a centrizonal necrosis that was maximal at about 24 h. These studies suggest that the mechanism of NDMA hepatotoxicity may differ from that of a number of other hepatotoxicants (e.g., carbon tetrachloride, acetaminophen, bromobenzene) for which toxicity is also mediated through reactive metabolites.

Influence of diet on the expression of hepatotoxicity from carbon tetrachloride in ICR mice.

Carbon tetrachloride-mediated hepatotoxicity in mice was influenced by two standard, commercially available diets and by a corn oil treatment vehicle. Animals maintained on Purina 5001 diet were less sensitive than animals maintained on Teklad LM-485 diet to hepatic intoxication by carbon tetrachloride (CCl4). Lower sensitivity of the Purina group was evidenced by significantly lower plasma alanine aminotransferase (ALT) levels and higher hepatic cytochrome P-450 levels at all dosages of CCl4. In addition to the diets, the nature of the corn oil vehicle affected toxicological responses of mice to CCl4. When the vehicle from which tocopherols had been extracted was used, CCl4 elicited about twice the levels of plasma ALT than when nonextracted corn oil was used. In conclusion, the nature of the animal diet and treatment vehicle not only can influence toxicological response, but also can be important considerations in the interpretation of toxicological data.

Protection against carbon tetrachloride hepatotoxicity by pretreatment with indole-3-carbinol.

The effects of administering indole-3-carbinol (I-3-C) on carbon tetrachloride (CCl4)-induced hepatotoxicity were examined. Mice received by gavage 0-150 mg I-3-C/kg body wt in methanol-extracted corn oil, followed 1 h later by 15 microliters CCl4/kg body wt in corn oil. Animals were sacrificed 24 h after receiving CCl4. Pretreatment with I-3-C reduced the degree of centrolobular necrosis, as observed histologically. Additionally, CCl4-mediated elevated serum enzymes were reduced by I-3-C. Although I-3-C induced elevated levels of cytochrome P-450 and associated mixed-function oxidase activity, the CCl4 depression of these parameters was not clearly reversed by I-3-C. However, CCl4 produced decreases in hepatic levels of glutathione (GSH), total reducing equivalents, and protein sulfhydryls, all of which were restored to control levels by I-3-C. Using mouse liver microsomes in an NADPH-fortified reaction mixture, I-3-C inhibited, in a concentration-dependent manner, CCl4-initiated lipid peroxidation, with 50% inhibition at 35-40 microM I-3-C. When mice were treated by gavage with 50 mg [14C]I-3-C/kg body wt, concentrations of radiolabel in the liver were greater than 100 microM after 1 hr. This was five times the level of radioactivity measured in blood and three times the concentration of I-3-C necessary for 50% inhibition of CCl4-mediated lipid peroxidation in vitro. The data are consistent with the hypothesis that I-3-C intervenes in CCl4-mediated hepatic necrosis by combining with reactive free radical metabolites of CCl4, thereby protecting critical cellular target sites.