QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.105.003806v1 33/8/1144    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hoffler, U. Articles by Ghanayem, B. I. Search for Related Content PubMed PubMed Citation Articles by Hoffler, U. Articles by Ghanayem, B. I.

INCREASED BIOACCUMULATION OF URETHANE IN CYP2E1-/- VERSUS CYP2E1+/+ MICE

Department of Pharmacology, Meharry Medical College, Nashville, Tennessee; and Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina

(Received January 24, 2005; accepted May 3, 2005)

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Urethane is a fermentation by-product and a potent animal carcinogen. Human exposure to urethane occurs through consumption of alcoholic beverages and fermented foods. Recently, CYP2E1 was identified as the primary enzyme responsible for the metabolism of [¹⁴C]carbonyl-labeled urethane. Subsequently, attenuation of urethane-induced cell proliferation and genotoxicity in CYP2E1-/- mice was reported. The present work compares the metabolism of single versus multiple exposures of CYP2E1-/- and CYP2E1+/+ mice to ¹⁴C-ethyl-labeled urethane. Urethane was administered as a single 10 or 100 mg/kg gavage dose or at 100 mg/kg/day for 5 consecutive days. CYP2E1+/+ mice administered single or multiple doses exhaled 78 to 88% of dose as ¹⁴CO₂/day. CYP2E1-/- mice eliminated 30 to 38% of a single dose as ¹⁴CO₂ in 24 h and plateaued after day 3 at 52% of dose/day. The concentrations of urethane-derived radioactivity in plasma and tissues were dose-dependent, increased as a function of the number of doses administered, and were significantly higher in CYP2E1-/- versus CYP2E1+/+ mice. Whereas urethane was the main chemical found in the plasma and tissues of CYP2E1-/- mice, it was not detectable in CYP2E1+/+ mice. In conclusion, multiple dosing led to considerable bioaccumulation of urethane in mice of both genotypes; however, greater retention occurred in CYP2E1-/- versus CYP2E1+/+ mice. Furthermore, greater bioaccumulation of ¹⁴C-ethyl-labeled than [¹⁴C]carbonyl-labeled urethane was observed in mice. Comparison of the metabolism of ethyl-versus carbonyl-labeled urethane was necessary for tracing the source of CO₂ and led us to propose for the first time that C-hydroxylation is a likely pathway of urethane metabolism.

Urethane is a potent animal carcinogen that was classified as "reasonably anticipated to be a human carcinogen" ([NTP], 2000). Previous studies have shown that independent of animal species or strain and regardless of the route of exposure, urethane undergoes rapid systemic distribution, and greater than 85% of an administered dose was eliminated in the expired air as CO₂, within 8 h in genetically intact mice (Skipper et al., 1951; IARC, 1974; Yamamoto et al., 1988, 1990; Nomeir et al., 1989; Hoffler and Ghanayem, 2003; Hoffler et al., 2003). Until recently, the accepted hypothesis regarding urethane metabolism presumed that enzymatic hydrolysis via esterase was the primary metabolic route, whereas N-hydroxylation and pathways mediated by cytochromes P450 (P450s) played minor roles (Fig. 1). Recent studies conducted in this laboratory using CYP2E1-/- mice, however, demonstrated for the first time that cytochrome P4502E1 (CYP2E1), and not esterase, was the principal enzyme responsible for greater than 96% of [¹⁴C]carbonyl-labeled urethane metabolism to ¹⁴CO₂ (Hoffler et al., 2003). It was also shown that P450s other than CYP2E1 and esterase contributed approximately 3 and less than 1%, respectively (Hoffler et al., 2003). In the absence of CYP2E1-mediated metabolism, the half-life of urethane was considerably increased (0.8 h in CYP2E1+/+ versus 22 h in CYP2E1-/- mice), and significantly higher blood and tissue concentrations of urethane-derived radioactivity were detected in CYP2E1-/- versus CYP2E1+/+ mice (Hoffler et al., 2003).

View larger version (26K): [in this window] [in a new window]   FIG. 1. A proposed scheme showing the metabolism of ethyl (*)- and carbonyl (+)-labeled urethane.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Chemicals. [Ethyl-1-¹⁴C]urethane, specific activity 56 mCi/mmol, was obtained from American Radiolabeled Chemicals (St. Louis, MO). Unlabeled urethane with purity greater than 98% was purchased from TCI America (Portland, OR). Using high performance liquid chromatography (HPLC), the radiochemical purity of ethyl-labeled urethane was determined to be greater than 99%.

Animals and Treatment. Male CYP2E1+/+ (wild-type) and CYP2E1-/- (CYP2E1-null) mice were 8 to 9 months old and ranged in weight from 28 to 42 g. Mice were obtained from a colony developed at the National Cancer Institute (Bethesda, MD) and were re-derived and bred at Charles River Laboratories, Inc. (Wilmington, MA) (Lee et al., 1996; Hoffler et al., 2003). Animals were individually housed in an animal facility with a 12-h light/dark cycle and fed NIH #31 diet and water ad libitum throughout the study. All animal care and experimentation were conducted according to National Institutes of Health guidelines (U.S. Department of Health and Human Services, 1986). Dosing solutions were made in tap water using a mixture of radiolabeled and unlabeled urethane. All urethane-dosing solutions were made fresh and administered by gavage at either 10 or 100 mg/kg, delivering 100 µCi/kg in a dose volume of 10 ml/kg.

Experimental Design. Groups (4-8 animals each) of CYP2E1-/- and CYP2E1+/+ mice were administered urethane by gavage as follows. 1) CYP2E1-/- and CYP2E1+/+ mice received a single 10- or 100- mg dose of ¹⁴C-ethyl-labeled urethane/kg and were euthanized 24 h after urethane administration. 2) CYP2E1-/- mice received 100 mg of ¹⁴C-ethyl-labeled urethane/kg/day for 5 consecutive days and were euthanized 24 h after the last dose.

Immediately after urethane administration, mice were housed in individual glass metabolism cages (Wyse Glass Specialties, Inc., Freeland, MI) for 24 or 120 h, which allowed for the separate collection of urine, feces, and exhaled radioactivity. A vacuum system was attached to the glass cages that permitted the passage of air through the cage at a flow rate of 0.6 to 0.8 l/min. Air passing through the cage was initially passed through solid calcium sulfate and soda lime to reduce moisture and CO₂ content, thus extending the efficiency of the trapping solutions over time. Air exiting the cage was passed through a series of traps. The first trap was an activated charcoal trap (SKC Inc., Eighty Four, PA) used to adsorb organic volatiles exhaled by urethane-treated mice. A second trap containing 400 ml of ethanol was used to capture exhaled organic volatiles that escape adsorption by the charcoal. Air was then passed through a trap containing approximately 400 ml of a 7:3 (v/v) mixture of ethylene glycol monomethyl ether and ethanolamine for collection of expired ¹⁴CO₂. CO₂ traps were changed at 1, 2, 4, 6, 8, and 24 h after urethane administration in the single-dose study. For the 5-day study, ¹⁴CO₂ traps were changed at 2, 4, 8, 16, and 24 h after each daily dose of urethane. Ethanol traps were changed every 24 h. Urine, feces, and charcoal traps were collected every 24 h after dosing. Urine and charcoal traps were stored at -80°C to be analyzed at a later time.

At the end of the holding periods, mice were euthanized by CO₂ asphyxiation, blood was collected from animals by cardiac puncture, and major tissues were dissected and stored at -60 to -80°C for analysis at a later time. Tissue and whole blood samples weighing between 25 and 50 mg were sampled in triplicates and ¹⁴C content was quantitated via oxidation to ¹⁴CO₂ using a PerkinElmer Tri-Carb sample oxidizer (PerkinElmer Life and Analytical Sciences, Boston, MA). Collected blood from 24 and 120 h was centrifuged to separate red blood cells and plasma, and urethane-derived radioactivity was also quantitated using the tissue oxidizer. Feces were air-dried, ground to a fine powder, weighed, and similarly analyzed in triplicate. Charcoal traps were opened; then, charcoal contents were weighed and analyzed in triplicates using the sample oxidizer. Recovery of radioactivity from the sample oxidizer was greater than 95%. Oxidized samples as well as triplicate aliquots of the ¹⁴CO₂ and organic volatile trapping solutions (1 ml) and urine (50 µl) were mixed with Ecolume (ICN Biomedicals, Costa Mesa, CA) and counted using a Beckman Coulter Model LS 9800 scintillation counter (Beckman Coulter, Fullerton, CA).

HPLC Analysis of Plasma, Liver Homogenates, and Urine. The metabolite profiles of plasma, liver homogenates, and urine samples from CYP2E1+/+ and CYP2E1-/- mice treated with 100 mg of ¹⁴C-ethyl-labeled urethane/kg for 24 h or daily for 5 consecutive days were analyzed by HPLC. Individual plasma, liver homogenates, and urine samples were centrifuged at 14,000g for 20 min at 4°C, and 100 µl of supernatant were directly injected into the HPLC apparatus. Small aliquots (10 µl) of plasma and urine supernatant were also analyzed for total radioactivity using a Beckman Coulter scintillation counter as previously described. The HPLC system consisted of a Waters 2690 Separations Module (Waters, Milford, MA) connected on-line with a UV detector, followed by a 515T Radiomatic Flow Scintillation Analyzer (PerkinElmer Life and Analytical Sciences). Samples were analyzed using a 4.6 x 250 mm C18 Microsorb-MV column (Rainin Instruments, Woburn, MA) with a Security guard column (Phenomenex, Torrance, CA) using a gradient consisting of 0.1% trifluoroacetic acid to 70% acetonitrile over 25 min at a flow rate of 1 ml/min. UV-absorbing peaks were monitored at 210 nm. Radioactive peaks were detected using a 500-µl flow cell with Ultima Flo-M scintillation fluid (PerkinElmer Life and Analytical Sciences) at 3 ml/min. Parent urethane was identified in the urine, plasma, and liver homogenates by comparing the retention times of these biological samples to the retention time of ¹⁴C-ethyl-labeled urethane standard. Furthermore, biological samples were spiked with ¹⁴C-ethyl-labeled urethane and analyzed using HPLC to confirm the presence of parent compound.

Statistical Analysis. Group mean comparisons were performed using Student's t test, two-tailed, assuming equal variances. Values were considered statistically significant at P 0.05.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Metabolism and Disposition of a Single Dose of ¹⁴C-Ethyl-Labeled Urethane. Within 24 h after a single administration of either 10 or 100 mg/kg ¹⁴C-ethyl-labeled urethane, approximately 80% of the total administered dose was eliminated in the expired air of CYP2E1+/+ mice as ¹⁴CO₂ (Table 1; Fig. 2). In contrast, CYP2E1-/- mice receiving the same treatment eliminated less than 40% of the total dose as ¹⁴CO₂ in 24 h (Table 1; Fig. 2). Whereas ¹⁴C-ethyl-labeled urethane-derived radioactivity, eliminated as organic volatiles in CYP2E1+/+ mice, comprised less than 1% of either the high or low doses, elimination in CYP2E1-/- mice via this route accounted for 3 and 4.2%, respectively (Table 1). In both CYP2E1+/+ and CYP2E1-/- mice, fecal excretion of ¹⁴C-ethyl-labeled urethane-derived radioactivity was negligible and accounted for less than 1% of the total administered urethane doses (Table 1). No significant differences were evident in the percentage of dose excreted as ¹⁴C-ethyl-labeled urethane-derived radioactivity in the urine regardless of the genotype or dose administered, 3.1 to 4.7% of dose (Table 1). However, HPLC analysis of urine identified greater than 62% of the ¹⁴C-ethyl-labeled radioactivity as unmetabolized urethane in CYP2E1-null mice, whereas in CYP2E1+/+ mice, the majority of the urinary radioactivity (>70%) was metabolites, with parent urethane accounting for the remainder.

View larger version (17K): [in this window] [in a new window]   FIG. 2. Effects of dose and time on cumulative 14CO2 exhalation in CYP2E1-/- and CYP2E1+/+ mice after gavage administration of 14C-ethyl-labeled urethane. Values are presented as cumulative percentage of dose and are the mean ± S.E. of four to eight mice. a and b denote significant differences in 14CO2 elimination by the two genotypes of mice at 100 and 10 mg/kg; c denotes a significant difference of 14CO2 elimination at the 2nd hour between the 10 and 100 mg/kg treated CYP2E1+/+ mice.

Notable differences and similarities were evident when comparing the current data on the metabolism and disposition of a single dose of ¹⁴C-ethyl-versus our earlier work using ¹⁴C-carbonyl-labeled urethane in CYP2E1-/- and CYP2E1+/+ mice (Hoffler et al., 2003). Regardless of the location of the radiolabel and independent of dose, exhalation of urethane-derived CO₂ in CYP2E1+/+ mice increased in a dose-dependent manner and plateaued within 8 h of exposure (Fig. 2; Hoffler et al., 2003). An approximately 50% decrease in urethane-derived ¹⁴CO₂ exhalation was determined at both the 10 and 100 mg/kg doses of either ¹⁴C-ethyl- or -carbonyl-treated CYP2E1-/- mice (Fig. 2; Hoffler et al., 2003). Although the average rate of ¹⁴CO₂ exhalation (percentage of dose per hour) remained relatively constant in CYP2E1-/- mice during the entire 24 h after exposure to either the high or low ¹⁴C-ethyl- or -carbonyl-labeled urethane doses, CYP2E1+/+ mice administered ¹⁴C-ethyl-labeled urethane experienced a minor delay in the production of exhaled ¹⁴CO₂ in the first 4 h after treatment (data not shown) in comparison to [¹⁴C]carbonyl-labeled urethane (Hoffler et al., 2003). At the end of 24 h, it was evident that the cumulative percentage of doses eliminated as exhaled ¹⁴CO₂ slightly decreased in mice treated with ¹⁴C-ethyl-labeled urethane in contrast to mice similarly treated with the ¹⁴C-carbonyl-labeled chemical (Table 1; Hoffler et al., 2003). Regardless of the location of the radiolabel, elimination of urethane-derived radioactivity as exhaled organic volatiles, or as urinary or fecal metabolites, was considered to be via minor pathways. However, significant differences in the disposition of ¹⁴C-ethyl-labeled urethane-derived radioactivity were observed in both CYP2E1+/+ and CYP2E1-/- mice administered 10 mg/kg in comparison to mice exposed to [¹⁴C]carbonyl-labeled urethane at the same dose. CYP2E1+/+ mice treated with 10 mg/kg ¹⁴C-ethyl-labeled urethane exhibited a 3-fold increase in the cumulative percentage of dose eliminated as organic volatiles, in comparison to CYP2E1+/+ mice administered 10 mg/kg ¹⁴C-carbonyl-labeled urethane (Table 1; Hoffler et al., 2003). Furthermore, 10 mg/kg ¹⁴C-ethyl-treated CYP2E1+/+ mice eliminated a 2-fold higher percentage of dose as urinary metabolites than did its [¹⁴C]carbonyl-treated counterparts. On the other hand, CYP2E1-/- mice administered the same ¹⁴C-ethyl-labeled urethane treatment exhibited a 2-fold decrease in the percentage of dose eliminated via renal excretion in comparison to 10 mg/kg [¹⁴C]carbonyl-treated CYP2E1-null mice (Table 1; Hoffler et al., 2003). With regard to the cumulative percentage of dose excreted as fecal metabolites, discernible differences were not apparent in mice treated with either ¹⁴C-ethyl- or [¹⁴C]carbonyl-labeled urethane (Table 1; Hoffler et al., 2003).

In general, the concentration of ¹⁴C-ethyl-labeled urethane-derived radioactivity detected in the blood and tissues increased in a dose-dependent manner in both mouse genotypes, but were considerably higher in CYP2E1-/- mice, independent of the location of the radiolabel (Table 2). Significant distinctions, however, were observed in the blood and tissues of CYP2E1+/+ mice. Twenty-four hours after exposure to ¹⁴C-ethyl-labeled urethane, a 3- to 16-fold increase in the concentration of ¹⁴C-ethyl-labeled urethane-derived radioactivity was detected in the blood and tissues of these mice versus CYP2E1+/+ mice treated with [¹⁴C]carbonyl-labeled urethane (Table 2; Hoffler et al., 2003).

Metabolism and Disposition of ¹⁴C-Ethyl-Labeled Urethane Administered Daily for 5 Consecutive Days. Inhibition of urethane metabolism to ¹⁴CO₂ continued after multiple doses of urethane (Fig. 3). Exhalation of ¹⁴CO₂ in CYP2E1+/+ mice after each daily dose of 100 mg of ¹⁴C-ethyl-labeled urethane/kg exhibited a pattern similar to that seen after a single dose (Fig. 3). Maximum ¹⁴CO₂ exhalation was achieved in CYP2E1+/+ mice after the second day of dosing, and steady-state levels were maintained with approximately 85% of the daily dose eliminated as respired ¹⁴CO₂. In CYP2E1-/- mice, maximum exhalation was delayed and observed after 3 days (approximately 52% of the daily doses) (Fig. 3). An average of 28 and 13% of the five cumulative ¹⁴C-ethyl-labeled urethane doses was eliminated as urethane-derived radioactivity in the urine and organic volatiles of CYP2E1-/- mice, respectively. CYP2E1+/+ mice, however, eliminated merely 10 and 3.5% of the five doses, respectively, via these routes.

View larger version (18K): [in this window] [in a new window]   FIG. 3. Effects of dose and time on the daily pattern of cumulative 14CO2 exhalation in CYP2E1-/- and CYP2E1+/+ mice after gavage administration of 100 mg/kg 14C-ethyl-labeled urethane/day for 5 consecutive days. Values are presented as percentage of the cumulative doses administered and are the mean ± S.E. of four to eight mice. All values from CYP2E1-/- mice are significantly different in comparison to CYP2E1+/+ values.

View larger version (23K): [in this window] [in a new window]   FIG. 4. Representative HPLC radiochromatograms of 14C-ethyl-labeled urethane-derived radioactivity in the urine of CYP2E1-/- and CYP2E1+/+ mice after daily gavage administration of 100 mg of urethane/kg for 5 days.

Plasma and tissue concentrations of urethane-derived radioactivity in both CYP2E1+/+ and CYP2E1-/- mice were markedly higher in mice administered five ¹⁴C-ethyl-labeled urethane exposures than in those receiving a single treatment (Table 2). HPLC analysis showed that most of the urethane-derived radioactivity detected in the plasma and liver homogenates of CYP2E1-/- mice that received a single ¹⁴C-ethyl-labeled urethane dose was parent compound (Fig. 5). In CYP2E1-/- mice administered five daily doses, approximately 83 to 88% of the urethane-derived radioactivity identified in the plasma and liver homogenates was unmetabolized urethane (Fig. 5). Parent urethane was not detected in plasma and tissues of CYP2E1+/+ mice administered either a single or multiple urethane dose. In comparing the current data on the bioaccumulation of ¹⁴C-ethyl-labeled versus [¹⁴C]carbonyl-labeled urethane (Hoffler et al., 2003), it was clear that ¹⁴C-ethyl-labeled urethane has a greater tendency to be retained in mice of both genotypes.

View larger version (13K): [in this window] [in a new window]   FIG. 5. Representative HPLC radiochromatograms of 14C-ethyl-labeled urethane-derived radioactivity in the plasma and liver homogenates of CYP2E1-/- mice 24 h after either a single or daily gavage administration of 100 mg of urethane/kg for 5 days.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Numerous studies investigating urethane's metabolism and disposition have suggested the involvement of two pathways. The first entails hydrolysis via esterase and was thought to be responsible for greater than 90% of urethane's metabolism to ¹⁴CO₂ (Fig. 1; Skipper et al., 1951; Kaye, 1960; Nomeir et al., 1989; Salmon and Zeise, 1991). The second pathway, which was considered minor, proposed dehydrogenation of urethane to form vinyl carbamate (VC) followed by P450-mediated oxidation of this intermediate to form vinyl carbamate epoxide (VCE) (Fig. 1; Dahl et al., 1978, 1980; Guengerich and Kim, 1991). Contrary to earlier assertions, a recent report from this laboratory using [¹⁴C]carbonyl-labeled urethane showed for the first time that CYP2E1, and not esterase, was the principal enzyme responsible for greater than 96% of urethane's metabolism (Hoffler et al., 2003). The contributions of other P450s and esterase were estimated at approximately 3 and 0.5% of the dose, respectively (Hoffler et al., 2003). Lawson and Pound (1973) suggested that covalent binding to mouse liver DNA in vivo requires the oxidation of the ethyl moiety of urethane. This finding, in addition to our recent report demonstrating an essential role for CYP2E1 in urethane metabolism and an increase in the half-life of urethane in CYP2E1-/- versus CYP2E1+/+ (Hoffler et al., 2003) mice, prompted this investigation. The objectives of the current studies included 1) investigating the metabolism and disposition of ¹⁴C-ethyl-labeled urethane in CYP2E1-/- and CYP2E1+/+ mice and comparing these data to our earlier results using [¹⁴C]carbonyl-labeled urethane (Hoffler et al., 2003); 2) assessing the consequences of lack of CYP2E1 on the metabolism and disposition of single versus multiple doses of ¹⁴C-ethyl-labeled urethane in mice of both genotypes; and 3) characterizing the bioaccumulation of ¹⁴C-ethyl-labeled urethane in mice of both genotypes after multiple doses.

After gavage administration, urethane was rapidly absorbed and distributed to all major tissues. Current work showed that metabolism and elimination of ¹⁴C-ethyl-labeled urethane via CO₂ was the predominant pathway in CYP2E1+/+ mice, although to a lesser measure than in CYP2E1+/+ mice treated with a similar dose of [¹⁴C]carbonyl-labeled urethane (Hoffler et al., 2003). In CYP2E1+/+ mice administered ¹⁴C-ethyl-labeled urethane, 73 to 77% of the dose was exhaled as ¹⁴CO₂ within 8 h (Fig. 2), whereas CYP2E1+/+ mice treated with [¹⁴C]carbonyl-labeled urethane eliminated 90 to 94% of the dose via the same route in 8 h (Hoffler et al., 2003). In CYP2E1-/- mice and regardless of the location of the label, animals exhaled 15 to 20% of the administered dose of urethane as ¹⁴CO₂ within 8 h after dosing. On average, 38% of a single ¹⁴C-ethyl- or [¹⁴C]carbonyl-labeled urethane dose was exhaled as ¹⁴CO₂ in 24 h after exposure of CYP2E1-/- mice. Variations in the percentage of dose eliminated as organic volatiles, or urinary and fecal metabolites were minor when comparing the disposition of ¹⁴C-ethyl-versus [¹⁴C]carbonyl-labeled urethane. These data confirmed our earlier findings that CYP2E1 plays an essential role in the metabolism of urethane. Furthermore, these data showed that a greater percentage of [¹⁴C]carbonyl-labeled urethane doses was metabolized to CO₂ versus ¹⁴C-ethyl-labeled urethane. The decrease in CO₂ elimination was associated with greater retention of ¹⁴C-ethyl-labeled urethane in blood and tissues of CYP2E1+/+ mice. In the current work, 24 h after a single ¹⁴C-ethyl-labeled urethane exposure of CYP2E1+/+ mice, the majority of tissues known to be targets of urethane had exceedingly higher concentrations of radioactivity than did CYP2E1+/+ mice treated with ¹⁴C-carbonyl-labeled urethane. These tissues included the brain (16-fold), spleen (14-fold), liver and thymus (13-fold), and lung (12-fold). These data are in agreement with those from the earlier work of Lawson and Pound (1973), which concluded that metabolites of the ethyl- and not the carbonyl-carbons of urethane bind to mouse DNA in vivo.

Bioaccumulation of ¹⁴C-ethyl-labeled urethane-derived radioactivity increased as a function of the number of doses administered to CYP2E1+/+ and CYP2E1-/- mice. However, in the absence of CYP2E1-mediated metabolism, a significantly greater retention of ¹⁴C-ethyl-labeled urethane-derived radioactivity was detected in CYP2E1-/- versus CYP2E1+/+ mice. HPLC analysis showed that whereas urethane was the primary chemical in the plasma and liver homogenates of CYP2E1-/- mice, it was not identifiable in CYP2E1+/+ mice. In a subsequent study using doses as high as 1000 mg/kg, significant prolongation of urethane-induced anesthesia was observed only in CYP2E1-/- mice administered urethane (unpublished data), confirming that the anesthesia was caused by unmetabolized urethane. Recent studies in this laboratory also demonstrated significant inhibition of urethane-induced genotoxicity and cell proliferation in CYP2E1-/- versus CYP2E1+/+ mice (Hoffler et al., 2005). This finding supports the hypothesis that CYP2E1-mediated metabolism, presumably via epoxidation across the ethyl carbons, is a prerequisite for the induction of genotoxicity and carcinogenicity by urethane.

Metabolism of both ¹⁴C-ethyl- and [¹⁴C]carbonyl-labeled urethane in CYP2E1+/+ mice produced considerable amounts of CO₂. However, until recently, it was thought that enzymatic hydrolysis of urethane via esterase to ethanol and subsequent metabolism via alcohol and aldehyde dehydrogenases was the primary route for the formation of CO₂ (Fig. 1). Because our recent findings indicated that the contribution of esterase to urethane metabolism was less than 1% of the dose (Hoffler et al., 2003), it was concluded that esteric cleavage was insufficient to justify the high percentage of ¹⁴C-ethyl-labeled urethane that converted to ¹⁴CO₂, as well as to explain the potent carcinogenicity of this chemical. As a result, we proposed for the first time that C-hydroxylation, catalyzed primarily by CYP2E1, is an important contributor to urethane metabolism (Fig. 1). This pathway explains ¹⁴CO₂ production in mice treated with ¹⁴C-ethyl-labeled urethane and presents a second and/or alternate metabolic route for the generation of ¹⁴CO₂ from [¹⁴C]carbonyl-labeled urethane (Fig. 1). It was reported that P450s could oxidatively cleave esters, leading to the formation of aldehydes or ketones from the alcohol moiety of the C-hydroxylated metabolite (Guengerich, 1987, 2001; Guengerich et al., 1988; Peng et al., 1995). Cleavage of many alkyl esters such as ethyl acetate, ethyl formate, and ethyl propionate via CYP2E1 has also been reported (Peng et al., 1995). Additionally, C-hydroxylation may provide another route for the formation of vinyl carbamate via dehydration of -hydroxyurethane (Fig. 1).

Bioactivation of chemicals via dehydrogenation and subsequent P450-mediated oxidation is not unique to urethane. Aflatoxin B₂ and some alkenylbenzene carcinogens such as safrole are metabolized via this pathway (Miller and Miller, 1983). The dehydrogenation product of urethane, VC, was shown to be a more potent carcinogen and mutagen than either ethyl N-hydroxycarbamate (N-hydroxyurethane) or urethane (Dahl et al., 1978, 1980). Initial studies by Dahl et al. (1978, 1980) demonstrated difficulties in identifying VC as a metabolite of urethane either in vivo or in vitro. However, subsequent studies using gas chromatography-mass spectrometry were successful in characterizing VC as a microsomal oxidation product of urethane (Guengerich and Kim, 1991). At present, isolation of VC in vivo has not been achieved. Oxidation of the ethyl moiety enables binding of the ethyl carbons to tissue macromolecules that may in turn foster adduct formation, and subsequent mutagenesis and tumorigenesis, presumably through VCE. Because of the highly reactive nature of epoxides, VCE has been termed as the ultimate carcinogenic metabolite of urethane. Numerous studies have identified etheno adducts of DNA and RNA in target tissues of urethane-induced carcinogenicity (Ribovich et al., 1982; Park et al., 1990; Fernando et al., 1996). In comparison to urethane, VC- and VCE-derived adducts were increased in target versus nontarget tissues (Ribovich et al., 1982; Miller and Miller, 1983; Park et al., 1990; Fernando et al., 1996). Collectively, these studies established that the formation of adducts resulted from the bioactivation of urethane at the ethyl carbons and not its hydrolysis product, ethanol.

In conclusion, current work supports our earlier findings demonstrating the predominance of CYP2E1 in urethane metabolism. In comparison to our earlier work using ¹⁴C-carbonyl-labeled urethane (Hoffler et al., 2003), current data demonstrated that there is greater retention of ¹⁴C-ethyl-labeled urethane-derived radioactivity in the blood and tissues of CYP2E1+/+ mice in association with a decrease in the exhalation of ¹⁴CO₂. Furthermore, bioaccumulation of ¹⁴C-ethyl-labeled urethane increased as a function of the number of administered urethane doses. Regardless of the position of the radio-label or the number of urethane doses, there was significantly greater bioaccumulation in CYP2E1-/- versus CYP2E1+/+ mice. Whereas the majority of ¹⁴C-ethyl-labeled urethane-derived radioactivity found in the blood and tissues of CYP2E1-/- mice was unmetabolized urethane, parent compound was not identified in CYP2E1+/+ mice. Collectively, our current and earlier studies suggested that both carbonyl- and ethyl-carbons were extensively incorporated into urethane-derived CO₂. We therefore proposed for the first time that urethane undergoes CYP2E1-mediated C-hydroxylation. This pathway explains CO₂ production from the ethyl or carbonyl carbons of urethane and leads to the formation of VCE. Recent studies in this laboratory demonstrated significant attenuation of urethane-induced genotoxicity and cell proliferation in CYP2E1-/- versus CYP2E1+/+ mice (Hoffler et al., 2005). Additionally, 1,N⁶-ethenoadenosine adduct formation in the lungs of CYP2E1-/- mice exposed to urethane was reduced in comparison to urethane-treated CYP2E1+/+ mice (our unpublished data). These findings support the hypothesis that CYP2E1-mediated metabolism, presumably via the epoxidation across the ethyl carbons, is a prerequisite for the induction of genotoxicity and carcinogenicity by urethane.

	Acknowledgments

 
We thank Dr. Tom Burka for invaluable insight. In addition, we sincerely appreciate the suggestions offered by Dr. Raj Chhabra, the dialogue concerning HPLC with Dr. Jay Breaux, and the technical assistance of Brian Chanas.

	Footnotes

 
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.105.003806.

ABBREVIATIONS: P450, cytochrome P450; HPLC, high performance liquid chromatography; VC, vinyl carbamate; VCE, vinyl carbamate epoxide.

Address correspondence to: Dr. Burhan I. Ghanayem, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709. E-mail: ghanayem{at}niehs.nih.gov

	References

Top Abstract Materials and Methods Results Discussion References

 


Benson RW and Beland FA (1997) Modulation of urethane (ethyl carbamate) carcinogenicity by ethyl alcohol: a review. Int J Toxicol 16: 521-544.[CrossRef]

Dahl G, Miller EC, and Miller JA (1980) Comparative carcinogenicities and mutagenicities of vinyl carbamate, ethyl carbamate, and ethyl-N-hydroxycarbamate. Cancer Research 40: 1194-1203.[Abstract/Free Full Text]

Dahl G, Miller JA, and Miller EC (1978) Vinyl carbamate as a promutagen and a more carcinogenic analog of ethyl carbamate. Cancer Res 38: 3793-3804.[Medline]

Fernando RC, Nair J, Barbin A, Miller JA, and Bartsch H (1996) Detection of 1,N⁶-ethenodeoxyadenosine and 3,N⁴-ethenodeoxycytodine by immunoaffinity/³²P-postlabelling in liver and lung DNA of mice treated with ethylcarbamate (urethane) or its metabolites. Carcinogenesis 17: 1711-1718.[Abstract/Free Full Text]

Guengerich FP (1987) Oxidative cleavage of carboxylic esters by cytochrome P450. J Biol Chem 262: 8459-8462.[Abstract/Free Full Text]

Guengerich FP (2001) Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity. Chem Res Toxicol 14: 612-640.

Guengerich FP and Kim DH (1991) Enzymatic oxidation of ethyl carbamate to vinyl carbamate and its role as an intermediate in the formation of 1, N⁶-ethenoadenosine. Chem Res Toxicol 4: 413-421.[CrossRef][Medline]

Guengerich FP, Peterson LA, and Böcker RH (1988) Cytochrome P450-catalyzed hydroxylation and carboxylic acid ester cleavage of Hantzsch pyridine esters. J Biol Chem 263: 8176-8183.[Abstract/Free Full Text]

Hoffler U, Dixon D, Peddada S, and Ghanayem BI (2005) Inhibition of urethane-induced genotoxicity and cell proliferation in CYP2E1-/- mice. Mutat Res 572: 58-72.[Medline]

Hoffler U, El-Masri HA, and Ghanayem BI (2003) Cytochrome P4502E1 (CYP2E1) is the principal enzyme responsible for urethane metabolism: comparative studies using CYP2E1-/- and CYP2E1+/+CYP2E1+/+ mice. J Pharmocol Exp Ther 305: 557-564.

Hoffler U and Ghanayem BI (2003) Inhibition of urethane metabolism and bioaccumulation in cytochrome P4502E1-null mice. Toxicol Sci 72 (Suppl): 1546.

IARC (1974) Urethane, in Monographs on the Carcinogenic Risk of Chemicals to Man: Some Antithyroid and Related Substances, Nitrofurans and Industrial Chemicals, vol 7, pp 111-140, International Agency for Research on Cancer, Lyon, France.

Kaye AM (1960) A study of the relationship between the rate of ethyl carbamate (urethan) catabolism and urethan carcinogenesis. Cancer Res 20: 237-241.

Lawson TA and Pound AW (1973) The interaction of carbon-14-labelled alkyl carbamates, labelled in the alkyl and carbonyl positions, with DNA in vivo. Chem-Biol Interact 6: 99-105.[CrossRef][Medline]

Lee SST, Buters JTM, Pineau T, Fernandez-Salguero P, and Gonzalez FJ (1996) Role of CYP2E1 in the hepatotoxicity of acetaminophen. J Biol Chem 271: 12063-12067.[Abstract/Free Full Text]

Nomeir AA, Ioannou YM, Sanders JM, and Matthews HB (1989) Comparative metabolism and disposition of ethyl carbamate (urethane) in male Fischer 344 rats and male B6C3F1 mice. Toxicol Appl Pharmacol 97: 203-215.[CrossRef][Medline]

Miller JA and Miller EC (1983) The metabolic activation and nucleic acid adducts of naturally occurring carcinogens: recent results with ethyl carbamate and the spice flavors safrole and estragole. Br J Cancer 48(1): 1-15.[Medline]

[NTP] National Toxicology Program (2000) Report on Carcinogens, 9th ed. National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC.

Park KK, Surh YJ, Stewart BC, and Miller JA (1990) Synthesis and properties of vinyl carbamate epoxide, a possible ultimate electrophilic and carcinogenic metabolite of vinyl carbamate and ethyl carbamate. Biochem Biophys Res Commun 169: 1094-1098.[CrossRef][Medline]

Peng HM, Raner GM, Vaz AD, and Coon MJ (1995) Oxidative cleavage of esters and amides to carbonyl products by cytochrome P450. Arch Biochem Biophys 318: 333-339.[CrossRef][Medline]

Ribovich ML, Miller JA, Miller EC, and Timmins LG (1982) Labeled 1,N⁶-ethenoadenosine and 3,N⁴-ethenocytidine in hepatic RNA of mice given [ethyl-1,2-³H or ethyl-1-¹⁴C] ethyl carbamate (urethan). Carcinogenesis 3: 593-546.[Abstract/Free Full Text]

Salmon A and Zeise L, eds (1991) Risks of Carcinogenesis from Urethane Exposure. CRC Press, Inc., Boca Raton, FL.

Skipper HE, Bennett LL Jr, Bryan CE, White L Jr, Newton MA, and Simpson L (1951) Carbamates in the chemotherapy of leukemia. VIII. Overall tracer studies on carbonyl-labeled urethan, methylene-labeled urethan, and methylene-labeled ethyl alcohol. Cancer Res 11: 46-51.

U.S. Department of Health and Human Services, National Institutes of Health (1986) Guide for the Care and Use of Laboratory Animals. NIH Publication no. 86-23, National Institutes of Health, Bethesda, MD.

Yamamoto T, Pierce WM Jr, Hurst HE, Chen D, and Waddell WJ (1988) Inhibition of the metabolism of urethane by ethanol. Drug Metab Dispos 16: 355-358.[Abstract]

Yamamoto T, Pierce WM Jr, Hurst HE, Chen D, and Waddell WJ (1990) Ethyl carbamate metabolism—in vivo inhibitors and in vitro enzymatic systems. Drug Metab Dispos 18: 276-280.[Abstract]

Zimmerli B and Schlatter J (1991) Ethyl carbamate: analytical methodology, occurrence, formation, biological activity and risk assessment. Mutat Res 259: 325-350.[CrossRef][Medline]

This article has been cited by other articles:

				B. I. Ghanayem Inhibition of Urethane-Induced Carcinogenicity in Cyp2e1-/- in Comparison to Cyp2e1+/+ Mice Toxicol. Sci., February 1, 2007; 95(2): 331 - 339. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.105.003806v1 33/8/1144    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hoffler, U. Articles by Ghanayem, B. I. Search for Related Content PubMed PubMed Citation Articles by Hoffler, U. Articles by Ghanayem, B. I.