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The use of transgenic animals, such as v-Ha-ras activated (TG:AC) and p53+/- mice, offers great promise for a rapid and more sensitive assay for chemical carcinogenicity. Some carcinogens are metabolically activated; therefore, it is critical that the altered genome of either of these model systems does not compromise their capability and capacity for metabolism of xenobiotics. The present work tests the generally held assumption that xenobiotic metabolism in the TG:AC and p53+/- mouse is not inherently different from that of the respective wild type, the FVB/N and C57BL/6 mouse, by comparing each genotype's ability to metabolize benzene, ethoxyquin, or methacrylonitrile. Use of these representative substrates offers the opportunity to examine arene oxide formation, aromatic ring opening, hydroxylation, epoxidation, O-deethylation, and a number of conjugation reactions. Mice were treated by gavage with (14)C-labeled parent compound, excreta were collected, and elimination routes and rates, as well as (14)C-derived metabolite profiles in urine, were compared between relevant treatment groups. Results of this study indicated that metabolism of the 3 parent compounds was not appreciably altered between either FVB/N and TG:AC mice or C57BL/6 and p53+/- mice. Further, expression of CYP1A2, CYP2E1, CYP3A, and GST-alpha in liver of naive genetically altered mice was similar to that of corresponding wild-type mice. Thus, these results suggest that the inherent ability of TG:AC and p53+/- mice to metabolize xenobiotics is not compromised by their altered genomes and would not be a factor in data interpretation of toxicity studies using either transgenic mouse line.

Methacrylonitrile (MAN) is a widely used aliphatic nitrile and is structurally similar to the known rat carcinogen and suspected human carcinogen acrylonitrile (AN). There is evidence that AN is metabolized via the cytochrome P-450 (CYP) 2E1. Recently, we identified two biliary conjugates originating from the interaction of MAN and its epoxide with glutathione. Mercapturic acids formed via the degradation of the two conjugates were also identified in rat and mouse urine. Additionally, a significant portion of MAN was eliminated in the expired air as CO2 (formed via the epoxide pathway) and unchanged MAN. The objective of the present work was to determine whether CYP2E1 is involved in the oxidative metabolism of MAN as was suggested for AN. 2-14C-MAN was administered to CYP2E1-null or wild-type mice by gavage at 12 mg/kg. Although total urinary and fecal excretion of MAN-derived radioactivity was slightly different in CYP2E1-null versus wild-type mice, the ratio of mercapturic acids originating from the epoxide-glutathione versus MAN-glutathione conjugates were lower in urine of CYP2E1-null mice than in that of wild-type animals. Exhalation of MAN-derived organic volatiles (primarily parent MAN) was 12- and 42-fold greater in female and male CYP2E1-null mice than in wild-type mice, respectively. Additionally, exhalation of CO2 derived from metabolism of MAN via the CYP2E1 pathway was 3- to 5-fold greater in wild-type than in CYP2E1-null animals. Although these data indicate that CYP2E1 is the principal enzyme responsible for the oxidative metabolism of MAN, other cytochrome P-450 enzymes may be involved. Assessment of MAN metabolism in CYP2E1-null mice pretreated with 1-aminobenzotriazole (CYP inhibitor) resulted in a further decrease in oxidative metabolites of MAN. Comparison of the tissue concentrations of MAN-derived radioactivity in mouse tissues revealed that MAN-derived radioactivity is generally higher in wild-type > CYP2E1-null mice > CYP2E1-null mice pretreated with 1-aminobenzotriazole, suggesting a direct relationship between MAN oxidative metabolism and the half-life of MAN and/or its metabolites in various tissues. It is therefore concluded that MAN oxidative metabolites such as the epoxide intermediate have greater reactivity than parent MAN.

Methacrylonitrile (MAN) is structurally similar to the known carcinogen acrylonitrile (AN), has similar industrial uses, and is occasionally used as its replacement. In contrast to AN, minimal information is available on the toxicity, carcinogenicity, or metabolism of MAN. Earlier work in this laboratory demonstrated that, in rats, MAN is primarily eliminated in the expired air as unchanged MAN, acetone, and CO2. Two mercapturic acids were identified in the urine of rats and mice treated with MAN. In the present work, the common bile duct of anesthetized rats was cannulated. The animals received a single gavage dose of 58 mg/kg 2-[14C]MAN. Bile was collected before and after MAN administration. Bile flow and MAN-derived radioactivity were determined at each time point. MAN had a minimal effect on bile flow and 4 to 6% of the administered MAN dose was excreted in the bile within 6 hr after dosing. HPLC analysis of bile showed two major metabolites, which were identified as 1-(S-glutathionyl)-2-propanone and 1-(S-glutathionyl)-2-cyanopropane by using NMR spectra and chemical synthesis. The ratio of the two metabolites in MAN-treated rats was approximately 2:1. Pretreatment of rats with sodium phenobarbital caused minimal quantitative or qualitative changes in the biliary excretion of MAN metabolites. In contrast, pretreatment of rats with beta-diethylaminoethyl diphenylpropylacetate before MAN administration resulted in a significant decrease in the ratio of 1-(S-glutathionyl)-2-propanone to 1-(S-glutathionyl)-2-cyanopropane (1:2). These data, especially the identification of 1-(S-glutathionyl)-2-propanone, indirectly demonstrated that MAN is metabolized via an epoxide intermediate (1-cyano-1-methyloxirane). Modulation of MAN metabolism by the P450 modulators suggested that this metabolic pathway is catalyzed via the cytochrome P450 enzymes. This work also confirms that MAN undergoes direct reaction with reduced glutathione via a Michael addition. Because of earlier work demonstrated that AN undergoes similar metabolism, MAN might have a qualitative toxicity profile similar to that of acryonitrile.

Methacrylonitrile (MAN) is an industrial chemical used to manufacture plastics and elastomers. The reported p.o. LD50 of MAN is 200 to 230 mg/kg in rats and 17 mg/kg in mice. Present investigations were undertaken to further characterize MAN metabolism and to study the metabolic basis for the species differences in its toxicity. Male F344 rats and B6C3F1 mice received a single gavage dose of 11.5 or 1.15 mg of [14C]MAN/kg and were placed in glass metabolism cages. Elimination of MAN in rats occurred primarily in expired air as unchanged MAN, acetone and CO2. Three major urinary metabolites of MAN were identified as N-acetyl-S-(2-cyanopropyl)-L-cysteine, N-acetyl-S-(2-hydroxypropyl)-L-cysteine and a deoxyuridine isomer. Quantitatively, rats and mice excreted ca. 7 and 49% of the high MAN dose in urine as N-acetyl-S-(2-hydroxypropyl)-L-cysteine, respectively. In addition, rats eliminated significantly more MAN-derived CO2 and deoxyuridine than mice. MAN elimination was almost complete within 24 hr after dosing and the tissue concentrations of MAN-derived radioactivity were, with the exception of the urinary bladder, consistently higher in rats than in mice. In conclusion, a quantitative difference between rats and mice in forming the epoxide intermediate, higher efficiency of mice to conjugate this intermediate with glutathione and greater capacity of rats to degrade it to acetone and CO2 are thought to contribute to the higher sensitivity of mice to MAN acute toxicity.

Aliphatic nitriles are a class of chemicals used in high volumes in the production of plastics and elastomers, in organic synthesis, and in production of a number of food packaging containers. Toxicity and metabolism of acrylonitrile (AN) are well characterized. On the other hand, minimal information is available on the toxicity or fate of structurally related, methacrylonitrile (MAN). In an attempt to predict the toxicity of MAN, the present studies were designed to compare the disposition of both nitriles in rats. After gavage administration of equimolar doses (0.87 mmol/kg) of 2-14C-MAN or 2-14C-AN to male F344 rats, both chemicals were well absorbed from the GI tract and distributed to all major tissues. However, major differences in the disposition of the two nitriles were observed. While approximately 39% of the administered MAN dose was eliminated as CO2 in 24 h after dosing, only 11% of an equimolar dose of AN was eliminated as such. In addition, 31% of the MAN dose was exhaled as organic volatiles in 24 h compared to less than 2% of an equivalent AN dose. MAN and acetone were identified by HPLC analysis of expired organic volatiles from MAN-treated rats. HPLC analysis showed that AN is the only organic volatile exhaled by AN-treated rats. Urinary excretion of MAN was 22% compared to 67% of an equivalent dose of AN. The major urinary metabolite from AN results from direct conjugation with GSH, whereas the major urinary metabolite from MAN results from conjugation of the epoxide with GSH.(ABSTRACT TRUNCATED AT 250 WORDS)

Methacrylonitrile (MAN), an aliphatic nitrile used in the production of plastics and elastomers, is structurally related to the known animal carcinogen, acrylonitrile. Although MAN has potential to cause significant toxicity, minimal information is available on its toxicity or fate. Current studies were designed to investigate the biological fate of [2-14C]MAN in male F344 rats. Following gavage administration of 115, 11.5, or 1.15 mg MAN/kg in water, male F344 rats were placed in glass metabolism cages and urine, expired air, and feces were collected. Rats were sacrificed at various times, and the concentration of MAN-derived radioactivity in tissues was determined. MAN was rapidly absorbed from the gastrointestinal tract and distributed to all major tissues. After gavage administration of 1.15-115 mg/kg, [2-14C]MAN is primarily eliminated in the expired air. Sixty to 70% of the low and medium doses were exhaled as 14CO2 in 72 hr compared with 25% of the highest dose. Whereas 40% of the high dose was expired as organic volatiles in 72 hr, only 9-12% of the low and medium doses were exhaled as such. It is therefore apparent that saturation of MAN metabolism occurs at the high dose. HPLC analysis of expired organic volatiles from MAN-treated rats showed that it contained two components that were identified as unchanged MAN and acetone. The MAN:acetone ratio was directly proportional to dose and decreased as a function of time. Urinary excretion accounted for 20-30% of all MAN doses within 72 hr after dosing. Investigating the effect of dosing vehicle on MAN disposition in rats revealed that administration of 115 mg MAN/kg in oil resulted in the death of rats within 24 hr after treatment. Furthermore, monitoring the fate of MAN in these rats before death showed that a significantly higher percentage of the dose was eliminated in urine and expired air. Analysis of this expired air also revealed that significantly more acetone and less unchanged MAN were exhaled by these animals. It is apparent that administration of MAN to F344 rats in oil resulted in slower absorption, decreased elimination of unchanged MAN, and increased metabolism to acetone and/or decreased degradation of acetone to CO2. The combination of these effects of an oil vehicle may have contributed to the death of rats by MAN. Comparison of the metabolism and disposition of MAN in F344 and Sprague-Dawley rats showed minor differences between the two strains.(ABSTRACT TRUNCATED AT 400 WORDS)