Introduction

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Aliphatic nitriles are a high volume class of chemicals used in the preparation of homopolymers and copolymers that are used for the manufacture of elastomers, coatings, and plastics (Budavari, 1989). They are also used in the preparation of acids, amines, amides, esters, and other nitriles (Budavari, 1989). Acrylonitrile (AN) is a major member of this class and is a known mutagen, an animal carcinogen, and a suspected human carcinogen (World Health Organization, 1983; US Environmental Protection Agency, 1983; US Department of Health and Human Services, 1990). AN is thought to be metabolized by cytochrome P-450 (CYP) 2E1 to 2-cyanoethylene oxide (Guengerich et al., 1981, 1991; Kedderis et al., 1993). Furthermore, AN is a known potent depletor of tissue glutathione (Ghanayem et al., 1985). Many of the toxic effects of AN are attributed to the epoxide intermediate, cyanide release, and/or glutathione depletion. In addition, 2-cyanoethylene oxide has been shown to react with DNA (Guengerich et al., 1981; Hogy and Guengerich, 1986) and is highly mutagenic (Recio and Skopec, 1988). It was therefore suggested that 2-cyanoethylene oxide may play a role in the carcinogenic potential of AN.

Methacrylonitrile (MAN) is another major member of the aliphatic nitrile class of chemicals. Products containing MAN are reported to produce toxic effects in laboratory animals, and some cases of human exposure and toxicity have been reported. Exposure to aliphatic nitriles have been shown to adversely affect the hepatic, cardiovascular, renal, gastrointestinal, and central nervous systems (Farooqui and Mumtaz, 1991). MAN has been shown to be toxic in rats, dogs, and rabbits by dermal, inhalation, intraperitoneal, ocular, and gavage routes (McOmie, 1949; Smyth et al., 1962; Pozzani et al., 1968; Tanii and Hashimoto, 1984). Symptoms of MAN toxicity include trembling, salivation, vasodilatation, diarrhea, irregular breathing, and convulsions (Pozzani, 1968; Farooqui and Mumtaz, 1991). Gavage administration of MAN to pregnant Sprague-Dawley rats during the first 2 weeks of gestation resulted in abortion (Farooqui and Villarreal, 1992). Additionally, MAN caused a reduction in maternal body weight gain and the development of edema in the fallopian tubes (Farooqui and Villarreal, 1992). In contrast, more recent gavage studies in rats and rabbits indicated that MAN administration during organogenesis was devoid of maternal or developmental toxicities (George et al., 1996).

Metabolite identification studies suggested that MAN is metabolized via the cytochrome P-450 enzymes to the epoxide intermediate 1-cyano-1-methoxyloxirane (Ghanayem and Burka, 1996). Although the epoxide intermediate was not identified in vivo, strong evidence originating from the identity of MAN metabolites in bile, urine, and expired air supports its formation in rats and mice. This epoxide intermediate may interact with reduced glutathione, presumably via glutathione transferases, resulting in the formation of 1-(S-glutathionyl)-2-propanone (SGTP), which was identified in the bile of rats receiving MAN (Fig. 1). Catabolism of SGTP results in the formation of N-acetyl-S-(2-hydroxypropyl)-L-cysteine (NAHPC), which was identified in the urine of MAN-treated rats (Ghanayem et al., 1992, 1994). SGTP may also be the subject of a nucleophilic attack of glutathione on the sulfur atom resulting in formation of glutathione disulfide and acetone (Ghanayem et al., 1992, 1994). Alternatively, 1-cyano-1-methyloxirane may undergo reductive metabolism leading to the formation of acetone (Ghanayem et al., 1992, 1994). Furthermore, the epoxide intermediate may be metabolized via the epoxide hydrolases (Fig. 1). Epoxidation and subsequent metabolism of the epoxide intermediate are considered to be the main contributors for cyanide and CO₂ formation from AN (Abreu and Ahmed, 1980; Burka et al., 1994) and are very likely responsible for their production from MAN.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   A metabolism scheme for MAN. Underlined metabolites have been identified. Italicized abbreviations are enzymes or enzyme-catalyzed transformations: EH, epoxide hydrolase; GST, glutathione-S-transferase; MAP, mercapturic acid pathway; P450, cytochrome P-450; Red'n, reduction.

A second metabolic pathway constitutes direct conjugation of MAN with reduced glutathione resulting in the formation of 1-(S-glutathionyl)-2-cyanopropane (Fig. 1). Catabolism of this metabolite accounts for the formation of N-acetyl-S-(2-cyanopropyl)cysteine (NACPC), which we previously identified in the urine of MAN-treated rats (Ghanayem et al., 1992, 1994).

The toxicity/carcinogenicity of AN has been, at least partially, attributed to its oxidation via an epoxide intermediate. The current work was undertaken as part of an effort to assess the role of oxidative metabolism in MAN-induced toxicity.

	Materials and Methods

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Animals and Dose Preparation. Male and female wild-type (CYP2E1^+/+) and knockout (CYP2E1^/; CYP2E1-null) mice, ranging in weight from 21 to 29 g, were obtained from a colony developed at the National Cancer Institute (Bethesda, MD) (Lee et al., 1996). Dosing solutions were prepared by diluting 2-methyl-[2-¹⁴C]AN (1.8 mCi/mmol; Amersham Corp., Arlington Heights, IL) with unlabeled MAN (Aldrich Chemical Co., Milwaukee, WI) in water to deliver 40 to 80 µCi/kg at a concentration of 12 mg/kg b.wt. in a dose volume of 10 ml/kg. The radiochemical purity of MAN was determined by HPLC (system described below) to be approximately 84%. The remaining radioactivity was retained on the HPLC precolumn and was thought to be mostly polymers. The chemical purity of the unlabeled compound was 99%. 1-Aminobenzotriazole (ABT) (99% pure; Sigma Chemical Co.) was administered approximately 2 h before ¹⁴C-MAN at 50 mg/kg i.p. in a dose volume of 2.5 ml of saline/kg.

Disposition Experiments. The rates and routes of elimination of MAN-derived radioactivity were determined through collection and analysis of excreta (exhaled organic volatiles, ¹⁴CO₂, urine, and feces) after the administration of single doses of MAN by gavage to mice (n = 3-5/treatment group). NIH rodent chow no. 31 and water were provided ad libitum at all times throughout the experiments. All animal care and procedures were performed according to the National Institutes of Health guidelines (U.S. Department of Health and Human Services, 1985). After dosing, each animal was housed in a glass metabolism cage (Wyse Glass Specialties, Inc., Freeland, MI) that was attached to a vacuum system that pulled air through the cage at a flow rate of 0.4 to 0.6 liter/min. Air exiting the cage was passed through an activated charcoal trap (SKC Inc., Eighty Four, PA) for collection of organic volatile ¹⁴C and then through a trap containing 200 to 400 ml of a 7:3 (v/v) mixture of ethylene glycol monomethyl ether and ethanolamine for collection of expired ¹⁴CO₂. The efficiency of the CO₂ trapping solution was increased by passing the incoming cage air through calcium sulfate and soda lime to reduce moisture content. All traps were changed and sampled at 4, 8, and 24 h after dosing. Urine and feces were collected at 24 h after dosing, at which time all mice were euthanized by CO₂ asphyxiation. The percentage of the total dose excreted in urine over time was determined by counting ¹⁴C contained in triplicate 20-µl aliquots of each sample directly in Ecolume (ICN, Research Products Division, Costa Mesa, CA) in a model LS 5801 or 9800 scintillation counter (Beckman Instruments, Inc., Fullerton, CA). Total exhaled ¹⁴CO₂ was determined by counting triplicate 1-ml aliquots of the trapping solution in Ecolume in the scintillation counter. The percentage of the total dose excreted in feces and as organic volatiles was determined after combusting triplicate 50-mg samples of feces (weighed, air dried, and ground to a fine powder) and triplicate 25-mg samples of the activated charcoal traps in a model 306 biological sample oxidizer (Packard Instruments Co., Meriden, CT). Additionally, mice underwent necropsies, and selected tissues were weighed, sampled, and oxidized as described previously (Ghanayem et al., 1994). All oxidized samples were analyzed for radioactivity content directly in the scintillation counter.

HPLC Analysis of MAN-Derived Radioactivity Contained in Urine and Charcoal Traps. The relative amounts of MAN and radiolabeled metabolites in urine and in solvent extracts of the activated charcoal traps (traps were washed with 0.75-1.0 ml ethanol) were determined using a Waters HPLC system (Waters Corp., Milford, MA) equipped with a Waters model 481 UV detector (230 nm) and a Radiomatic model A-280 radiochemical detector (Packard Instrument Co., Meriden, CT). The system used a 5-µm Rainin Microsorb C₁₈ column (Varian Associates, Palo Alto, CA) with a mobile phase of 100% 0.01 M sodium phosphate buffer (pH = 6) for 5 min., then a linear gradient from 0% to 10% acetonitrile over 25 min (total run time = 30 min). Aliquots (50-100 µl) of either urine or charcoal trap extracts were injected directly into the HPLC system after centrifugation for 5 min in an Eppendorf model 5412 microfuge (Brinkmann Instruments, Westbury, NY).

Urinary Metabolite Identification. Urine from either MAN-treated male wild-type or CYP2E1-null mice was pooled and injected into the HPLC system for the collection and identification of MAN-derived metabolites. Fractions (collected by time) of the effluent containing selected radiolabeled peaks with the same R_T were then combined, concentrated to dryness, resuspended in water, and further purified using a Waters Bondapack C₁₈ column with a linear gradient of 100% trifluoroacetic acid (0.1%) to 10% acetonitrile in 20 min. The radiolabeled peaks underwent a third isolation using the same column with a linear gradient of 100% trifluoroacetic acid (0.05%) to 7.5% methanol in 15 min. The identity of two major MAN-derived metabolites, NAHPC and NACPC, were confirmed by comparison of NMR spectra with those of previously isolated metabolites or synthesized standards.

Statistical Analysis. Group mean comparisons were performed using one-way ANOVA (Tukey's or Tukey-Kramer honest significant difference) with JMP software (SAS Institute Inc., Cary, NC). Values were considered statistically significant at P  .05.

	Results

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Consistent with our earlier studies (Ghanayem et al., 1994), the present work showed that ¹⁴C-MAN is well absorbed from the gastrointestinal tract and distributed to most major tissues after gavage administration to CYP2E1-null and wild-type mice of either sex. However, significant differences were found in the elimination of ¹⁴C-MAN derived radioactivity between CYP2E1-null and wild-type mice. Exhalation of ¹⁴C-MAN-derived organic volatiles in the 24-h period after dosing was 34% versus 1 to 3% of the administered dose in CYP2E1-null versus wild-type mice, respectively (Fig. 2). HPLC analysis revealed that parent MAN is the only constituent of the exhaled organic volatiles detectable by all animals (data not shown). Minimal differences in the exhalation of parent MAN were observed in male and female mice of respective groups (Fig. 2). Exhalation of CO₂ derived from metabolism of MAN was 3- to-5 fold greater in wild-type compared with CYP2E1-null animals (Fig. 3). No significant difference in the elimination of ¹⁴CO₂ by male and female mice of either genotype was observed (Fig. 3). A significant portion of the administered MAN dose was eliminated in the feces of mice of each genotype (Fig. 4). However, no significant quantitative differences were found in the fecal excretion of MAN-derived radioactivity between CYP2E1-null and wild-type mice of either sex (Fig. 4).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Cumulative percentage of the total dose of MAN-derived radioactivity exhaled as organic volatiles by male CYP2E1-null () and wild-type (), female CYP2E1-null () and wild-type () mice, and male CPY2E1-null mice pretreated with ABT (). Each value represents the mean ± S.D. of three to five mice. bValues that are statistically different from wild-type mice values. cValues of ABT-pretreated CYP2E1-null mice that are statistically different from CYP2E1-null mice values.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Cumulative percentage of the total dose of MAN-derived radioactivity exhaled as 14CO2 by male CYP2E1-null () and wild-type (), female CYP2E1-null () and wild-type () mice, and male CYP2E1-null mice pretreated with ABT (). Each value represents the mean ± S.D. of three to five mice. bValues Which are statistically different from wild-type mice values. cValues of ABT-pretreated CYP2E1-null mice that are statistically different from CYP2E1-null mice values.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   A summary of 14C-MAN disposition in (A) male and (B) female CYP2E1-null () and wild-type () mice and ABT-pretreated CYP2E1-null mice () within 24 h after MAN administration by gavage. Each value represents the mean ± S.D. of three to five mice. bValues that are statistically different from wild-type mice values. cValues of ABT-pretreated CYP2E1-null mice that are statistically different from CYP2E1-null mice values.

Urinary excretion is the main pathway for the elimination of MAN-derived radioactivity (Fig. 4). Furthermore, the urinary excretion of MAN-derived radioactivity was significantly smaller in CYP2E1-null versus wild-type male mice. HPLC analysis of these urine samples revealed significant qualitative differences (Fig. 5, Table 1). An approximate 50% decrease in the urinary excretion of NAHPC was found in CYP2E1-null compared with wild-type mice (Table 1). NAHPC is formed via cytochrome P-450-mediated metabolism (Ghanayem et al., 1994) (Fig. 1). In contrast, a significant increase in the excretion of NACPC was seen in CYP2E1-null mice versus wild-type animals (Table 1). NACPC is formed via metabolic pathways that do not involve cytochrome P-450 enzymes (Ghanayem et al., 1994) (Fig. 1). Similar patterns of the urinary excretion of MAN metabolites were observed in male and female mice (Table 1).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Representative HPLC radiochromatograms of MAN urinary metabolites excreted in 24-h cumulative urine of male (A) CYP2E1-null, (B) wild-type mice, and (C) ABT-pretreated CYP2E1-null mice. Peak 1 is presently unidentified, peak 2 is NAHPC, and peak 3 is NACPC. MAN elutes at an RT of 22.5 min. in this HPLC system.

Because current data revealed that CYP2E1-null mice were capable of forming metabolites (NACPC) originating from the oxidative metabolism of MAN, we hypothesized that other cytochrome P-450 enzymes may be involved. We examined this possibility by treating CYP2E1-null mice with the nonspecific P-450 inhibitor ABT before MAN administration. Gavage administration of 12 mg MAN/kg to male CYP2E1-null animals pretreated with ABT showed that these mice exhaled a significantly greater portion of the dose as parent MAN (Fig. 2) and a significantly smaller portion of the dose as ¹⁴CO₂ (Fig. 3). Furthermore, although ABT resulted in a significant decrease in the excretion of MAN urinary metabolites, it had no significant effect on MAN fecal excretion (Fig. 4). HPLC analyses of urine from ABT + MAN-treated animals showed that less than 1% of the administered MAN dose was eliminated as NAHPC (Fig. 5, Table 1); this was associated with a significant increase in urinary NACPC excretion.

Determination of the tissue distribution of MAN-derived radioactivity in CYP2E1-null, CYP2E1-null mice pretreated with ABT, and wild-type mice is shown in Table 2. The concentration of MAN-derived radioactivity is consistently greater in the tissues of wild-type versus CYP2E1-null mice. Furthermore, with the exception of the zymbals gland, pretreatment of CYP2E1-null mice with ABT resulted in an additional decline in the concentration of tissue MAN-derived radioactivity.

	Discussion

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Recent work in our laboratory and others suggested that MAN may be metabolized via two major pathways (Fig. 1). First, MAN is metabolized to an epoxide intermediate in a reaction catalyzed by the cytochrome P-450 enzymes; this intermediate is subsequently conjugated with glutathione. Second, MAN is directly conjugated with glutathione (Fig. 1). Glutathione conjugation may occur via the glutathione transferases or by a direct nonenzymatic reaction.

Significant differences in the metabolism of MAN were reported between rats and mice (Ghanayem et al., 1994). Compared with rats, mice are more efficient metabolizers of MAN via the epoxide intermediate and its subsequent conjugation with glutathione (Ghanayem et al., 1994). Other studies also demonstrated that mice are more efficient metabolizers of AN via the epoxide intermediate than rats (Roberts et al., 1991). Elimination of MAN-derived radioactivity in rats occurred primarily in expired air as organic volatiles and as CO₂. In contrast, mice were less efficient in eliminating MAN via these pathways (Ghanayem et al., 1994). Unchanged MAN and acetone were the primary components of the exhaled organic volatiles.

Evidence was reported suggesting that the CYP2E1 is the main P-450 enzyme responsible for the oxidation of AN, a chemical structurally similar to MAN (Guengrich et al., 1991; Kedderis et al., 1993). MAN oxidation was also shown to proceed via the P-450 enzymes (Ghanayem and Burka, 1996). Because oxidative metabolism is thought to play a significant role in the toxicity and carcinogenicity of AN, present work was undertaken to determine whether CYP2E1 is involved in the oxidative metabolism of MAN using CYP2E1-null and wild-type mice.

Exhalation of MAN-derived organic volatiles (mostly parent MAN) was significantly greater in female and male CYP2E1-null versus wild-type mice, respectively. This clearly suggested that MAN was metabolized at a slower rate in CYP2E1-null versus wild-type animals, resulting in higher MAN blood levels and subsequent exhalation. Exhalation of CO₂ derived from the metabolism of MAN was 3- to 5-fold greater in wild-type than CYP2E1-null animals. Collectively, increased parent MAN and decreased CO₂ exhalation suggest that CYP2E1 is a major enzyme responsible for the oxidative metabolism of MAN (Fig. 1). This conclusion is further supported by the nature of the urinary metabolites identified in CYP2E1-null and wild-type mice. A significant reduction in the formation and excretion of NAHPC (originating through metabolism to the epoxide intermediate) in conjunction with a significant increase in NACPC (originating from the direct MAN-glutathione conjugate) was seen in CYP2E1-null mice compared to wild-type animals.

These data clearly showed that CYP2E1 was not the only enzyme responsible for MAN oxidative metabolism, as evident from the fact that a significant portion of the administered MAN dose was eliminated by CYP2E1-null mice as urinary NAHPC and exhaled as CO₂. These metabolites are most likely formed through the epoxide intermediate (Fig. 1). We therefore hypothesized that additional cytochrome P-450 enzymes may be involved in the oxidative metabolism of MAN. To examine this hypothesis, we administered MAN to male CYP2E1-null mice pretreated with the CYP inhibitor ABT. Results of this experiment showed that although MAN exhalation was increased, CO₂ exhalation was reduced to negligible levels compared with that seen in CYP2E1-null mice. Furthermore, total urinary elimination of MAN-derived metabolites significantly decreased. Analyses of urine from CYP2E1-null mice pretreated with ABT also revealed a drastic reduction in urinary NAHPC (less than 1% of the dose). Although the contribution of various cytochrome P-450 enzymes may vary with the dose used, it is clear that under the current experimental conditions, CYP2E1 is the primary enzyme responsible for MAN oxidative metabolism. However, other cytochrome P-450 enzymes also contribute to the oxidation of this chemical. Earlier in vitro studies suggested that although human CYP2E1 is the main catalyst of AN epoxidation, other P-450 isozymes are also involved in the epoxidation of this chemical (Kedderis et al., 1993).

A comparison of the tissue concentrations of MAN-derived radioactivity in mice tissues revealed that MAN-derived radioactivity is generally higher in wild-type > CYP2E1-null mice > CYP2E1-null mice pretreated with ABT. Because the overall metabolic picture of MAN in the three groups of mice indicates that oxidative metabolism of MAN follows the same pattern, the present data suggest that a direct relationship may exist between tissue concentrations of MAN-derived radioactivity and oxidation of MAN. These data may also suggest that the oxidative metabolites derived from MAN have a greater tissue half-life than the parent compound. Whether this observation means that oxidative MAN metabolism results in greater binding and subsequent greater toxicity remains unclear at this time. However, because oxidative metabolism of AN is thought to play a role in its toxicity, future studies will focus on the characterization of the toxicity of MAN in CYP2E1-null versus wild-type mice.

In summary, although CYP2E1 plays a major role in the oxidative metabolism of MAN, other cytochrome P-450 enzymes are also involved in this pathway. Furthermore, the present data demonstrate a direct relationship between MAN oxidative metabolism and the tissue concentrations of MAN-derived radioactivity. This data may point to a greater tissue binding of the oxidative metabolites of MAN such as the epoxide intermediate compared with the parent chemical. Finally, this work points to no obvious differences in the metabolism of MAN in male and female mice.