Introduction

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Urethane (ethyl carbamate) has been used in humans as a hypnotic agent and for the treatment of varicose veins, chronic leukemia, and multiple myeloma (IARC, 1974). Commercially, urethane has been utilized as a cosolvent for pesticides, fumigants, and cosmetics, and as an agent to impart wash-and-wear properties to fabrics (Benson and Beland, 1997). Urethane is also formed as a by-product of fermentation processes and is found in tobacco leaves and smoke. Currently, the greatest risk of human exposure to urethane is through consumption of alcoholic beverages and fermented foods (Benson and Beland, 1997). Under normal dietary conditions, free of alcoholic beverages, urethane intake in adults was approximately 20 ng/kg b.wt., with bread serving as the main source (Zimmerli et al., 1986). In table wines, urethane concentrations exceeded 10 ng/g and in fruit brandies, its concentration ranged between 0.2 and 20.2 mg/g (Schlatter and Lutz, 1990). Currently, a Reference Concentration (RfC) or Reference Dose (RfD) for acceptable human exposure levels to urethane has not been established.

Urethane is a well established animal carcinogen. Nettleship et al. first discovered urethane's carcinogenicity in 1943. Lung tumors, principally lung adenomas, could be detected in C3H female mice within 2 to 3 months after one or two intraperitoneal (i.p.) injections of a minimal anesthetizing dose (1 ml/100g b.wt). Additionally, it was reported that repeated dosing of urethane produces a variety of tumor types in mice, rats, hamsters, guinea pigs, and toads (Schmahl et al., 1977). These findings also showed induction of tumors by urethane occurred regardless of the route of exposure. Mutagenic and teratogenic responses caused by urethane exposure have also been investigated (Schmahl et al., 1977). Nomura (1975) presented findings from a multigenerational study demonstrating that a single subcutaneous injection of urethane (1.0 mg/g b.wt.) on gestation day 17 caused tumor formation at multiple sites in first generation (F₁) offspring. Furthermore, mating among F₁ mice produced a second generation exhibiting the same pattern of tumor development. Because these and other findings showed urethane is a multisite carcinogen, it was classified as "reasonably anticipated to be a human carcinogen" (National Toxicology Program, 2000).

It is currently thought that metabolic activation is a prerequisite for the development of urethane-induced tumors. As shown in Fig. 1, earlier studies suggested that metabolism of urethane occurs via two major pathways (Yamamoto et al., 1990; Page and Carlson, 1994; Forkert and Lee, 1997; Lee et al., 1998). The first pathway is thought to entail oxidative metabolism of urethane catalyzed by cytochromes P450, leading to the formation of vinyl carbamate (VC) (Fig. 1). Subsequently, VC may undergo oxidation to produce vinyl carbamate epoxide (VCE) (Fig. 1). VCE is considered the ultimate carcinogen, binding to macromolecules; DNA, RNA, and proteins to produce adducts (Guengerich and Kim, 1991). The second pathway of urethane metabolism was thought to be catalyzed by esterase and leads to the formation of CO₂, ethanol, and NH₃ (Fig. 1). Interestingly, the final metabolite common to both pathways is CO₂. Studies conducted in rats (F344) and mice (A/Jax and B6C3F1) by Yamamoto et al. (1988) and Nomeir et al. (1989) using [carbonyl-¹⁴C]urethane demonstrated that greater than 90% of the administered dose was exhaled as ¹⁴CO₂ within 24 h. It has been proposed that the most dominant pathway for urethane metabolism to CO₂ (~95%) is hydrolysis via esterase (Skipper et al., 1951; Kaye, 1960; Mirvish, 1968; Nomeir et al., 1989; Salmon and Zeise, 1991).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   A proposed scheme of urethane metabolism

Dahl et al. (1978) proposed that cytochromes P450 were responsible for urethane's bioactivation. Studies by Guengerich and Kim (1991) focused specifically on the involvement of CYP2E1. Human liver microsomes were incubated with either urethane or VC in the presence of adenosine and an NADPH-generating system. 1,N⁶-Ethenoadenosine adducts developed as a result of exposure to these chemicals. In subsequent incubations that included the CYP2E1 inhibitor, diethyldithiocarbamate, or CYP2E1 antibodies, inhibition of adduct formation was observed. Moreover, 1,N⁶-ethenoadenosine and 3,N⁴-ethenocytidine adducts were also seen in vivo in hepatic RNA after a single injection of 0.5-0.6 mg/g b.wt. [ethyl-1,2-³H] or [ethyl-1-¹⁴C]urethane to 12-day-old and adult male mice (Ribovich et al., 1982). Generally, the current hypothesis states that while esterase is the primary enzyme responsible for urethane metabolism, CYP2E1 is responsible for urethane activation (Yamamoto et al., 1990; Forkert and Lee, 1997; Lee et al., 1998). In an attempt to address this hypothesis and assess the metabolic basis of urethane toxicity and carcinogenicity, the present work is designed to more directly characterize the enzymes responsible for urethane metabolism using CYP2E1-null versus wild-type mice.

	Materials and Methods

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Chemicals. [Carbonyl-¹⁴C]ethyl carbamate (urethane), specific activity 50 mCi/mmol, was obtained from PerkinElmer Life Science Products (Boston, MA). By using high-performance liquid chromatography (HPLC) the radiochemical purity was determined to be greater than 99%. 1-Aminobenzotriazole (ABT) and paraoxon (PAX) were purchased from Sigma-Aldrich (St. Louis, MO). All chemicals were of the best commercially available purity.

Animals and Treatments. CYP2E1-null mice were obtained from a colony developed at the Laboratories of Dr. Frank Gonzalez, National Cancer Institute, Bethesda, MD (Lee et al., 1996). J1 embryonic stem cells generated from 129/Sv mice were used to generate the CYP2E1 mutant null mice (Lee et al., 1996). Chimeric males were crossed with C57BL/6N females once to generate heterozygous mutant mice as the F₁ hybrid. Homozygous mutant mice for CYP2E1 were generated from an intercross of F₁ mice, then maintained at Charles River Laboratories (Wilmington, MA) by intercrossing homozygous mice. Wild-type littermates obtained from the F₁ intercross were also maintained by intercrossing at Charles River Laboratories to serve as age-matched, strain-matched, wild-type mice. No further backcross to either 129/Sv or C57BL/6N was performed at Charles River Laboratories. Furthermore, the nulizygosity of the CYP2E1-null mice was confirmed using Western blot analysis as previously described (Wang et al., 2002). In the present work, 7- to 8-month-old male wild-type and CYP2E1-null mice ranging in weight from 28 to 42 g were used. Animals were housed in facilities with a 12-h light/dark cycle and fed National Institutes of Health no. 31 diet and water. Both food and water were available ad libitum throughout the experiments. All animal care and experimentation were conducted according to National Institutes of Health guidelines (U.S. Department of Health and Human Services, 1985).

Experimental Design. Groups (4-8 animals each) of CYP2E1-null and WT mice were administered urethane by gavage as follows:

1.	CYP2E1-null and wild-type mice received 10 mg urethane/kg and held for 24 h;
2.	CYP2E1-null and wild-type mice received 100 mg urethane/kg and held for 24 h;
3.	CYP2E1-null and wild-type mice received ABT at 50 mg/kg i.p. followed by 10 mg urethane/kg and held for 24 h;
4.	CYP2E1-null and wild-type mice received PAX at 1 mg/kg i.p. followed by 10 mg urethane/kg and held for 24 h;
5.	CYP2E1-null mice received 10 or 100 mg urethane/kg and held for 72 h.

HPLC Analysis of Plasma and Liver Homogenates. The metabolite profile of plasma and liver samples from WT and CYP2E1-null mice treated with 100 mg/kg for 24 h were analyzed by HPLC. Individual plasma samples were centrifuged at 14,000g for 20 min at 4°C and 100 µl of supernatant were directly injected into the HPLC. Liver specimens of approximately 200 mg were homogenized in sodium phosphate buffer (pH 7) at a 2:3 ratio, centrifuged at 14,000g for 20 min at 4°C, and 100 µl of the supernatant were injected into the HPLC. The HPLC system consisted of a Waters 2690 separations module (Waters Corporation, Milford, MA) connected online with a UV detector followed by a 515T radiomatic flow scintillation analyzer for detection of radioactivity (PerkinElmer Life Sciences). Samples were analyzed using a 4.6 × 250 mm C₁₈ Microsorb-MV column (Rainin Instrument Co., Woburn, MA) preceded by a security guard column (Phenomenex, Torrance, CA) using a linear gradient consisting of 100% 0.1% trifluoroacetic acid to 70% acetonitrile/30% trifluoroacetic acid over 25 min at a flow rate of 1 ml/min. UV absorbance was monitored at 254 nm. Radioactive peaks were detected using a 500 µl flow cell with Ultima Flo-M scintillation fluid (PerkinElmer Life Sciences) at 3 ml/min.

Pharmacokinetic Analysis of Urethane Metabolism. A pharmacokinetic model was developed to assess the contribution of each enzymatic system to the metabolism of urethane. Mathematically, individual compartments depicted the production of CO₂ via specific pathways (Fig. 2). Each enzymatic reaction was modeled by a first-order equation. The first-order constants were set as k₁, k₂, and k₃ for metabolism by CYP2E1, other P450 enzymes, and esterase, respectively.

View larger version (9K): [in this window] [in a new window]   Fig. 2.   A proposed compartmentalized model describing urethane metabolism to CO2 via specific enzymatic pathways (including associated first-order kinetic constants).

Statistical Analysis. Group mean comparisons were performed using Student's t test. Values were considered statistically significant at P  0.05.

	Results

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Exhalation of Urethane-Derived ¹⁴CO₂. WT mice treated with either 10 mg/kg or 100 mg/kg [carbonyl-¹⁴C]urethane exhaled ¹⁴CO₂ in a dose-dependent manner (Fig. 3). Regardless of the dose, exhalation of urethane-derived ¹⁴CO₂ in wild-type mice plateaued at 6 to 8 h and was approximately 91-93% of the administered dose (Fig. 3B and Table 1). In comparison to wild-type mice, significant decreases in urethane-derived ¹⁴CO₂ exhalation were observed in both the 10 and 100 mg/kg-treated CYP2E1-null mice (Fig. 3). Furthermore, exhalation of ¹⁴CO₂ in KO mice was time-dependent (Table 2). Calculation of the rate of ¹⁴CO₂ exhalation (% dose/h) in mice showed that while the rate of ¹⁴CO₂ exhalation was less than 0.05% of dose/h between 8 and 24 h in WT mice, it remained greater than 1.2% of dose/h in CYP2E-null mice (data not shown). We therefore decided to assess urethane metabolism in KO mice over a 72-h period. Approximately 70% of the administered low and high urethane doses were eliminated as ¹⁴CO₂ over a 72-h period (Fig. 3A; Table 2). Furthermore, the rate of ¹⁴CO₂ elimination remained relatively high and was approximately 0.25% of dose/h in KO mice at the end of the 72-h holding period, especially at the high dose (Fig. 3A).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   A, effects of dose and genotype on the exhalation of 14CO2 in CYP2E1/ and CYP2E1+/+ mice after gavage administration of 14C carbonyl-labeled urethane as a function of time. All values are presented as cumulative percentage of dose. CYP2E1/ values for time points during the first 24 h are the mean ± S.E. of eight mice. CYP2E1+/+ values are the mean ± S.E. of three to four mice. a, statistical significance of 14CO2 elimination by mice at 100 mg/kg; b, statistical significance at 10 mg/kg; c, statistical difference in the second-hour value between the 10 and 100 mg/kg-treated wild-type mice. B, effects of dose and genotype on the exhalation of 14CO2 in CYP2E1/ and CYP2E1+/+ mice treated with 14C-carbonyl-labeled urethane by gavage as a function of time. All values are presented as cumulative percentage of dose. CYP2E1/ values for time points during the first 24 h are the mean ± S.E. of eight mice. CYP2E1+/+ values are the mean ± S.E. of three to four mice. (c) Statistical difference in the second-hour value between the 10 and 100 mg/kg-treated wild-type mice.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Effects of 1-aminobenzotriazole (ABT) and paraoxon (PAX) on the exhalation of 14CO2 in CYP2E1/ and CYP2E1+/+ mice after gavage administration of 10 mg/kg 14C-labeled urethane as a function of time. All values are presented as cumulative percentage of dose. Values from CYP2E1/ mice administered urethane only are the mean ± S.E. of eight mice. The remaining values are the mean ± S.E. of three to four mice. (a) Statistical significance of 14CO2 elimination by the two genotypes of mice at 10 mg/kg; (b) statistical comparison of values from KO mice with and without PAX; (c) statistical comparison of values from mice of both genotypes with and without ABT.

Estimations of the First-Order Pharmacokinetic Constants (k) and Half-Life for Each Metabolic Pathway. The pharmacokinetic model was simulated against ¹⁴CO₂ exhalation data from wild-type, CYP2E1-null, and ABT-pretreated mice. Whenever applicable, simulations for both administered doses, 10 and 100 mg carbonyl-labeled urethane/kg b.wt., were conducted against available experimental data (Fig. 5). Initial model fitting was performed on the data set for the ABT-pretreated mice. This data set depicted the production of ¹⁴CO₂ from urethane by esterase only, allowing for the estimation of k₃. Using the determined k₃ value, the data set for the CYP2E1-null mice was then used to calculate k₂. In CYP2E1-null mice, esterase and cytochromes P450 other than CYP2E1 were responsible for the production of ¹⁴CO₂ from urethane. Lastly, determination of k₁ was performed using data collected from the wild-type mice (CYP2E1, other cytochromes P450 and esterase were functional) and the previously determined k₂ and k₃. Model simulations against each data set are shown in Fig. 5, A-C. The inhibitory effects of ABT have diminished by 24 h according to Fig. 5C. With the exception of this time point, the model simulation exhibited an excellent fit with the data. The failure of the model simulation (in ABT-pretreated mice) to fit the 24 h time point may be attributed to the short half-life of ABT (8 h) (Meschter et al., 1994), which may indicate that the inhibitory effect of ABT declined at the 24 h time point. Using the ACSL software, k₁ (CYP2E1), k₂ (other cytochromes P450), and k₃ (esterase) were estimated to be 0.85 h¹, 0.028 h¹, and 0.0035 h¹, respectively. Half-lives of urethane were based on ¹⁴CO₂ exhalation data and were estimated to be 0.8 h for wild-type and 22 h for CYP2E1-null mice.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Pharmacokinetic model simulations of the experimental CO2 exhalation data from 100 mg/kg wild-type () and CYP2E1-null mice () (A); 10 mg/kg wild-type () and CYP2E1-null mice () (B); and ABT-pretreated wild-type () and CYP2E1-null mice () (C). The lines represent the results of the model simulations and the symbols represent the actual experimental CO2 production data from mice treated with urethane (U). n = 8 for the CYP2E1-null mice and 3 to 4 for all other groups.

Urethane-Derived Radioactivity Exhaled as Organic Volatiles. While less than 1% of the dose was eliminated as organic volatiles in the expired air in wild-type mice, CYP2E1-null mice eliminated a significantly greater portion of the dose (3-4%) via the same route (Table 1). In comparison, at 72 h after urethane administration of 10 or 100 mg/kg, 8 and 5% of the low and high doses were exhaled as organic volatiles, respectively. These values were significantly higher than those determined at 24 h (Table 2). Additionally, pretreatment of mice with ABT or PAX resulted in a small increase in the percentage of dose exhaled as organic volatiles (Table 1). Preliminary HPLC analysis of charcoal trap extracts suggested that urethane was present in the expired air of CYP2E1-null mice. Additional work is currently in progress to identify other exhaled organic volatiles.

Urinary and Fecal Excretion of Urethane-Derived Radioactivity. While there were no significant differences in the excretion of urethane-derived radioactivity in the urine of mice of either genotype treated with 100 mg/kg urethane for 24 h, a significant increase was observed in KO mice administered 10 mg/kg versus wild-type mice (2 versus 6% of dose; Table 1). Urinary excretion of urethane-derived radioactivity at 72 h (9-10% of dose) after dosing was statistically similar regardless of dose (Table 2). However, when compared with that at 24 h, the percentage of urethane dose excreted in the urine at 72 h was doubled (Table 2). The effect of ABT and PAX on the excretion of urethane-derived radioactivity in the urine of mice of both genotypes was shown in Table 1. Although ABT increased the urinary excretion of urethane-derived radioactivity in wild-type mice, it resulted in a negligible change in urethane urinary elimination in KO mice (Table 1). Inhibition of esterase in CYP2E1-null or wild-type mice by PAX showed a significant decrease in the amount of urethane-derived radioactivity excreted in the urine (Table 1). Using HPLC analyses, approximately 70% of the total amount of urethane-derived radioactivity excreted in the urine of CYP2E1-null mice was identified as parent urethane. However, HPLC analyses of urine collected from wild-type mice demonstrated that parent compound accounted for approximately 15% of total urethane-derived radioactivity. Furthermore, the decrease in urethane in the urine of wild-type mice was associated with an increase in an early eluting metabolite with an opposite pattern in CYP2E1-null mice (~65% of dose in wild-type versus 13% of dose in CYP2E1-null mice). Overall, fecal excretion of urethane-derived radioactivity was negligible in all groups (Tables 1 and 2).

Analysis and Identification of Urethane-Derived Radioactivity in Blood and Tissues. In general, tissue and blood concentrations of urethane-derived radioactivity increased in a dose-dependent manner, achieving a maximum within 24 h, and declining at 72 h after dosing in each of the two mouse genotypes (Table 3). Moreover, tissue and blood concentrations of urethane-derived radioactivity were significantly higher in CYP2E1-null versus wild-type mice in all treatment groups (Table 4). However, the effect of ABT was more pronounced than that of PAX (Table 4). Fractionation of blood and subsequent analysis demonstrated that the concentration of urethane-derived radioactivity in whole blood, plasma, and red blood cells (48-52 µg urethane/g blood or ml plasma) were essentially similar in the 100 mg/kg CYP2E1-null mice. In contrast, wild-type mice administered the same dose contained an approximately 2-fold increase in urethane-derived radioactivity in red blood cells versus plasma (1.75 versus 0.83 µg urethane/g or ml). HPLC analysis of the plasma of CYP2E1-null mice at 24 h after the administration at 100 mg/kg surprisingly showed that urethane was the only radiolabeled chemical detectable in the plasma. Subsequently, analysis of liver homogenates from these mice also showed that urethane was the only detectable radiolabeled chemical (data not shown).

	Discussion

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Urethane is a documented multisite carcinogen capable of inducing a host of tumor types in various organs and animal species. Potential human exposure via the consumption of foods containing urethane and its ability to cause tumorigenesis in animals has prompted extensive investigations into urethane's mechanism(s) of action. Currently, the accepted hypothesis centers on the assumption that while urethane metabolism via esterase is the primary pathway, activation of this chemical via CYP2E1 to vinyl carbamate, and subsequently to vinyl carbamate epoxide, is a prerequisite for tumor development. Most studies addressing this hypothesis, however, relied on the use of enzyme modulators. Generally, inhibitors/inducers of metabolism produce inconclusive results, affecting not only their intended targets, but also altering other enzymes and normal biochemical/physiological processes (Ghanayem et al., 2000). With the advent of genetically engineered mice, the effect of a single enzyme on the bioactivation and/or detoxification of a xenobiotic can be directly determined (Gonzalez, 1998; Gonzalez and Kimura, 1999; Ghanayem et al., 2000). Therefore, the overall objective of ongoing research in this laboratory is to assess the relationships between urethane metabolism and toxicity, mutagenicity, and carcinogenicity. Present work focuses on the assessment of the role of CYP2E1 in urethane metabolism using CYP2E1-null and wild-type mice.

Current results showed that urethane was rapidly absorbed and distributed to all major tissues of mice. However, significant differences in urethane metabolism were observed in wild-type and CYP2E1-null mice. Regardless of dose, 91 to 93% of administered urethane was metabolized to ¹⁴CO₂ and eliminated in expired air of wild-type mice within 6 h. In contrast, a greater than 6-fold decrease in exhaled ¹⁴CO₂ was observed in CYP2E1-null mice treated with urethane, indicating that CYP2E1 was the principal enzyme responsible for urethane metabolism. These findings are in stark contrast to previous studies reporting esterase as the primary enzyme responsible for urethane metabolism to ¹⁴CO₂. Although limited, intercrossing of 129/Sv and C57BL/6N in the current studies may have produced wild-type and CYP2E1-null mice with different genetic backgrounds, in turn affecting the present results; strong evidence suggested otherwise. Clear differences in urethane metabolism between wild-type and CYP2E1-null mice regardless of dose, small animal to animal variations within treated groups, and the strong agreement of the current results with earlier studies that used various mouse strains argues against nonuniform genetic backgrounds influencing urethane metabolism in the present study. Skipper et al. (1951) showed that within 24 h after a single radiolabeled urethane dose had been administered intraperitoneally to CFW mice, greater than 95% of the dose was eliminated as expired ¹⁴CO₂. Nomeir et al. (1989) demonstrated that greater than 90% of administered oral or i.v. doses of [carbonyl-¹⁴C]urethane was exhaled by B6CF31 mice as ¹⁴CO₂ within 4 h. Furthermore, elimination of ¹⁴CO₂ in the expired air of male A/Jax mice constituted approximately 85% of an oral dose of urethane (Yamamoto et al., 1988, 1990). An IARC review of available studies concluded that regardless of the route of exposure, urethane undergoes rapid systemic distribution and approximately 90% of a given dose would be excreted as exhaled ¹⁴CO₂ within 24 h (IARC, 1974). More recently, subsequent studies performed in this laboratory comparing the metabolism and disposition of carbonyl and ethyl-labeled urethane in CYP2E1-null and wild-type mice yielded relatively identical results to present data (Hoffler and Ghanayem, 2002). Clearly, present and earlier studies are in agreement that biotransformation of urethane to ¹⁴CO₂ in mice was not significantly affected by mouse strain.

Current results unquestionably showed CYP2E1 was the principal enzyme responsible for the metabolism of [carbonyl-¹⁴C]urethane. Furthermore, these data demonstrated that CYP2E1's absence caused a dramatic increase in urethane's half-life (0.8 h in wild-type and 22 h in CYP2E1-null mice) and may lead to urethane bioaccumulation upon multiple dosing. The present work also suggested that additional enzymes, other than CYP2E1, were participating in urethane metabolism. Using the universal P450 inhibitor, ABT, the role of all cytochromes P450 was examined. Pretreatment of mice with ABT resulted in significant reduction in urethane metabolism to CO₂ and rendered both genotypes of mice metabolically similar. However, these results suggested that the involvement of other cytochromes P450 was minimal compared with the contribution of CYP2E1. Because urethane's metabolism to CO₂ was not entirely inhibited by ABT, it was hypothesized that non-cytochromes P450 such as esterase were involved. Urethane is an aliphatic ester and is susceptible to esteric cleavage by esterases. Previous work by Yamamoto et al. (1990) showed that pretreatment of A/Jax mice with PAX, a cholinesterase inhibitor, caused a significant increase of urethane-derived radioactivity in the blood 2 h after urethane administration. In the present study, the rate of urethane metabolism to CO₂ was initially inhibited in PAX-pretreated mice. Recovery, however, was observed within 24 h, suggesting that PAX-mediated inhibition of esterase was transient. Ironically, urinary excretion of urethane-derived radioactivity was significantly reduced in both PAX-pretreated CYP2E1-null and wild-type mice. Whether this decrease means that the majority of urinary metabolites derived from urethane originate from pathways mediated by esterase remains to be determined. Preliminary HPLC analysis of urine showed significant presence of parent urethane in the urine of CYP2E1-null versus wild-type mice.

To assess the contributions of CYP2E1, other cytochromes P450, and esterase in the metabolism of urethane, a pharmacokinetic model was simulated against actual CO₂ exhalation data from wild-type, CYP2E1-null, and ABT-pretreated mice. First-order kinetic constants (k) were calculated for each metabolic pathway. Generally, the model simulation exhibited an excellent fit with the experimental data. Initial modeling was performed on the data from ABT-pretreated mice. This data set depicted the production of CO₂ from urethane via esterase and revealed that esterase contribution was negligible, accounting for less than 0.5% of an administered dose (k = 0.0035 h¹). The failure of the model prediction to fit the 24-h time point may be attributed to ABT's short half-life (8 h) in mice (Meschter et al., 1994). Therefore, the inhibitory effect of ABT may decline, resulting in an underestimation by the model. In CYP2E1-null mice, esterase and cytochromes P450 (other than CYP2E1) were responsible for the production of CO₂. The rate constant for these cytochromes P450 was calculated and revealed that the contribution of these enzymes to urethane metabolism to CO₂ was approximately 3.2% of an administered dose (k = 0.028 h¹). In wild-type mice CYP2E1, other cytochromes P450, and esterase were responsible for urethane metabolism to CO₂. Using the rate constants for esterase and other cytochromes P450, the rate constant for CYP2E1 was calculated (k = 0.85 h¹). Greater than 96% of urethane metabolism to CO₂ was attributed to CYP2E1.

Blood and tissue concentrations of urethane-derived radioactivity were dose-dependent in mice. Interestingly, tissue and blood levels of urethane remained elevated even at 72 h after chemical administration to CYP2E1-null mice. Therefore, in the absence of CYP2E1-mediated metabolism, urethane clearance was drastically inhibited and its half-life dramatically increased. Furthermore, retention of radioactivity in tissues and blood was potentiated in both CYP2E1-null and wild-type mice after pretreatment with either ABT or PAX. Notably, these results contradict the assumption that radioactivity in tissues was the result of re-incorporation of ¹⁴CO₂ and ethanol (Skipper et al., 1951; Salmon and Zeise, 1991). Moreover, these data may contradict the suggestion that elevated levels of urethane-derived radioactivity in tissues resulted from covalent binding of metabolites originating via CYP2E1-mediated oxidation (Salmon and Zeise 1991), especially since inhibition of urethane oxidation in CYP2E1-null and in ABT-pretreated mice was associated with higher tissue levels of urethane-derived radioactivity. In fact, HPLC analysis confirmed that urethane was the only chemical detectable in the blood and liver of CYP2E1-null mice treated with this chemical.

In conclusion, urethane was rapidly absorbed and distributed to all major tissues after gavage administration. The use of CYP2E1-null mice directly demonstrated for the first time that CYP2E1 and not esterase was the principal enzyme responsible for the metabolism of [carbonyl-¹⁴C]urethane to ¹⁴CO₂. Pharmacokinetic modeling of ¹⁴CO₂ exhalation data revealed that CYP2E1, other cytochromes P450, and esterase contribute 96.4, 3.2, and 0.4% to urethane metabolism to CO₂, respectively, in genetically intact mice. Although model estimates suggested that contributions of cytochromes P450 (other than CYP2E1) and esterase were minimal, the roles of these enzymes were exaggerated in CYP2E1-null mice, possibly due to the increased urethane blood concentration in these animals. Absence of CYP2E1 led to a significant increase in the half-life of urethane in CYP2E1-null mice (22 h versus 0.8 h in wild-type mice) and suggested that urethane bioaccumulation may occur upon multiple exposures. Polymorphisms in CYP2E1 may enhance or reduce a person's predisposition to cancer. As reported by Marchand et al. (1998), CYP2E1 RsaI and DraI polymorphisms were associated with a 10-fold decrease in the risk of overall lung cancer (RsaI variant) and adenocarcinoma (DraI variant) compared with homozygous wild-type genotypes. Therefore, urethane half-life may increase in humans with polymorphisms that decrease basal CYP2E1 metabolic activity. However, it remains to be determined whether a decrease in urethane metabolism in exposed humans results in the induction of toxic/carcinogenic effects by unmetabolized urethane or by metabolites formed via other pathways. Additional work is currently in progress to determine whether inhibition of urethane metabolism in CYP2E1-null mice may influence the mutagenicity and carcinogenicity of this chemical.