Introduction

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Vinyl carbamate (VC)² is a primary metabolite derived from metabolism of ethyl carbamate (EC, urethane), a naturally occurring dietary constituent that is present in fermented products and alcoholic beverages (Ough, 1976; Battaglia et al., 1990). Serious concern has been raised regarding the potential carcinogenic risk posed by exposure to EC and VC, particularly in view of the large amounts of EC used as a cosolvent in analgesic drugs in Japan between 1950 and 1975 (Nomura, 1975; Miller, 1991). VC but not EC was found to be mutagenic to Salmonella typhimurium (Dahl et al., 1978, 1980; Leithauser et al., 1990). VC also exhibited greater carcinogenic potency than EC at similar doses and produced numbers of lung tumors that were 20- to 50-fold greater than those elicited by EC, depending on the route of exposure (Dahl et al., 1978, 1980). Moreover, VC-induced formation of etheno-DNA adducts in livers and lungs of mice were at levels that were 3-fold higher than those produced by EC (Fernando et al., 1996). These findings indicated that the toxic and carcinogenic potential of EC is linked not only with the parent compound but also with its metabolic derivative VC.

The toxic and carcinogenic effects of EC and VC are believed to be associated with their metabolism to a reactive intermediate. It was postulated that oxidation of EC leads to the formation of VC, which is oxidized further to produce VC epoxide, a metabolite that has been proposed to be the ultimate carcinogenic species (Fig. 1) (Dahl et al., 1978, 1980; Park et al., 1990, 1993). This proposed metabolic pathway involving two oxidative steps has been supported by data from studies of EC and VC in human liver microsomes that implicated the cytochrome P-450 isozyme CYP2E1 in their metabolism (Guengerich and Kim, 1991; Guengerich et al., 1991). Although the liver is a major target of EC- and VC-induced carcinogenicity, the lung appears to be a highly susceptible tissue. A latent period of about 1 year was required for manifestation of hepatic tumors, whereas lung tumors developed more rapidly and were found 2 to 6 months after EC treatment (Tannenbaum, 1964; Mirvish, 1968; Shimkin and Stoner, 1975). It thus is of importance to determine the underlying basis for this particular susceptibility of the lung to EC-induced tumor formation and to identify the mechanisms involved in EC and VC bioactivation. As an initial step to this end, our previous studies have investigated the lung metabolism of EC (Forkert and Lee, 1997). Data from these studies supported a central role for CYP2E1 in catalyzing the metabolism of EC in murine lung microsomes. An objective of the present study is to determine whether lung CYP2E1 is also involved in VC metabolism and to confirm whether both steps of the oxidative pathway of EC metabolism are mediated by CYP2E1.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Proposed pathway of EC and VC metabolism.

Although metabolic activation is of importance in manifestation of toxicity and carcinogenicity, the detoxication pathway is also of relevance. In our recent studies, we reported that the catalytic activity of carboxylesterases, a class of enzymes that metabolize ester substrates, was significantly decreased in lung microsomes reacted with EC, suggesting that the carboxylesterases are involved in EC metabolism (Forkert and Lee, 1997). The end products of EC metabolism by the carboxylesterases are ethanol, ammonia, and CO₂, indicating that this metabolic pathway is associated with detoxication (Boyland and Rhoden, 1949; Kaye, 1960; Mirvish and Kaye, 1964; Nomier et al., 1989). This assertion is supported by studies that showed that, when carboxylesterase enzymes were inhibited with either paraoxon or phenylmethylsulfonyl fluoride (PMSF), CYP2E1-dependent oxidation of EC was enhanced and produced significantly higher levels of covalent binding of [¹⁴C-ethyl]-EC to microsomal proteins (Forkert and Lee, 1997). The structural and chemical characteristics common to EC and VC implicated the carboxylesterases in VC metabolism and, hence, detoxication. However, the extent and involvement of this enzyme system in VC metabolism in the lung has not been identified and characterized.

In the present study, we have used a microsomal incubation system to investigate the metabolism of VC in the lungs of mice. We have measured enzyme catalytic activities for CYP2E1 and carboxylesterase enzymes and used enzyme inhibition and immunoinhibition experiments to investigate the involvement of these enzyme systems in VC metabolism. Protein immunoblotting was used to further confirm the potential isozyme-selective metabolism of VC by cytochrome P-450 and carboxylesterase enzymes. In addition, covalent binding of [¹⁴C-carbonyl]-VC was measured to assess magnitudes of formation of reactive intermediates from VC.

	Materials and Methods

Top Abstract Introduction Materials and methods Results Discussion References

Treatment of Animals. Female CD-1 mice of 20 to 25 g body weight were purchased from Charles River Canada (St. Constant, Quebec, Canada), housed over hardwood bedding, and maintained on a 12-h light/dark cycle. Mice were given free access to food (Purina Rodent Chow; Ralston Purina International, Strathroy, Ontario, Canada) and drinking water and were acclimated for 7 days before being entered into an experimental group. Mice were sacrificed by cervical dislocation, and lungs were removed for preparation of microsomes.

Materials. Chemicals and reagents used in this study were purchased from suppliers as detailed in the following. American Radiolabeled Chemicals (St. Louis, MO): [¹⁴C-carbonyl]-vinyl carbamate (>98% radiochemical purity, specific activity 1.5 mCi/mmol); Bio-Rad Laboratories (Hercules, CA): p-nitroblue tetrazolium chloride, 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt, molecular weight standards; ICN Chemical Co. (Costa Mesa, CA): Eco-Lite scintillation fluid; Canberra-Packard Canada Ltd. (Mississauga, Ontario, Canada): Soluene; New England Nuclear Co. (Boston, MA): [¹⁴C]formaldehyde (>95% radiochemical purity, specific activity 10 mCi/mmol); Parish Chemical Co. (Orem UT): diallyl sulfone, purity 97%; Sigma Chemical Co. (St. Louis, MO): PMSF, p-nitrophenol, 4-nitrocatechol, p-nitrophenyl acetate; Spectrum Medical Industries Inc. (Los Angeles, CA): Spectrapor dialysis tubing, molecular weight cutoff 3500. [¹⁴C]-N-nitrosodimethylamine, formaldehyde-free (specific activity, 40 mCi/mmol) was generously donated by Dr. C. S. Yang (Rutgers University, Piscataway, NJ). Vinyl carbamate was a contribution from Dr. J. A. Miller (McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI). Monoclonal antibody (mAb) 1-91-3, a monoclonal CYP2E1 inhibitory antibody, and mAb HyHel 9, an antibody specific to egg white lysozyme, were contributions from Dr. S. S. Park (Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick, MD). Rabbit polyclonal antibodies raised against rat liver microsomal hydrolase A and hydrolase B were generously donated by Dr. A. Parkinson (Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS). A goat polyclonal antibody raised against rabbit microsomal CYP2E1 was obtained from Oxford Biomedical Research Inc. (Oxford, MS). All other chemicals were of reagent grade and were purchased from standard commercial suppliers.

Preparation of Microsomes. Lungs from 10 mice were pooled for each sample and were prepared using differential centrifugation as described previously (Lee and Forkert, 1995). Microsomes were resuspended in 100 mM K₂HPO₄ buffer containing 1.5 mM EDTA, pH 7.0. Aliquots (250 µl) were placed in Eppendorf tubes, layered over with argon, frozen in liquid nitrogen, and stored at 70°C. Protein concentrations were determined using the method of Lowry et al. (1951), with bovine serum albumin as the standard.

Microsomal Incubations with VC. In preliminary experiments, microsomal incubations performed at 37°C yielded results that were highly variable. This may have been due to the instability of VC at this temperature. Incubations performed at 25°C produced data that were highly reproducible. Therefore, all subsequent incubations with VC were performed at 25°C. Reaction mixtures consisted of 3 mg of lung microsomal protein suspended in 100 mM K₂HPO₄ buffer containing 1.5 mM EDTA, pH 7.0, and an NADPH-generating system (7.5 mM glucose 6-phosphate, 2 U of glucose 6-phosphate dehydrogenase, 5.0 mM MgCl₂, and 0.4 mM NADP⁺). The final incubation volume was 1 ml. The mixtures were preincubated for 3 min at 25°C with agitation, and the reaction was initiated by addition of VC dissolved in water. Incubation vessels were promptly sealed with Teflon stoppers and incubations were continued for an additional 30 min. Reactions were terminated by cooling the samples on ice. Microsomes were washed in 100 mM K₂HPO₄ buffer, pH 7.0, and centrifuged at 105,000g for 30 min at 0°C. The resulting microsomal pellet was resuspended in 1 ml of 100 mM K₂HPO₄ buffer, pH 7.0, and the microsomal suspension was used for the enzyme assays. To assess for linearity of reactions, a series of incubations with lung microsomes and VC was performed, with substrate concentrations ranging from 100 µM to 10 mM and incubation times ranging from 10 to 180 min. Based on the data obtained, an incubation time of 30 min and a VC concentration of 0.5 mM were used for subsequent incubations.

Microsomal Incubations with the Carboxylesterase Inhibitor PMSF. Incubation mixtures consisted of 3 mg of lung microsomal protein suspended in 1 ml of 100 mM K₂HPO₄ buffer containing 1.5 mM EDTA, pH 7.0, in a final incubation volume of 1 ml. The mixtures were preincubated for 3 min at 25°C with gentle agitation. The reaction was initiated by addition of 25 µM PMSF in dimethyl sulfoxide, and the incubation continued for an additional 20 min at 25°C. The reactions were terminated by cooling the samples on ice. The microsomes were then washed and centrifuged at 105,000g for 30 min at 0°C. The microsomal pellet was resuspended in buffer and incubated with VC and/or carboxylesterase activity was determined.

Microsomal Incubations with Diallyl Sulfone (DASO₂). Reaction mixtures consisted of 3.0 mg of lung microsomal protein suspended in 100 mM K₂HPO₄ buffer containing 1.5 mM EDTA, pH 7.0, in a final volume of 1 ml. Components of the NADPH-generating system were added and the mixtures were preincubated for 3 min at 37°C with gentle agitation. The reaction was initiated by the addition of 1 mM DASO₂ in water, and the mixtures were incubated for an additional 30 min at 37°C with agitation. The reaction was terminated by placing the vessels on ice. The microsomes subsequently were washed, recovered, and resuspended in 100 mM K₂HPO₄ buffer. The microsomal suspension was then used for incubation with VC and/or measurement of NDMA demethylase activity.

Enzyme Assays. Catalytic activity of CYP2E1 was determined by measuring demethylation of [¹⁴C]-N-nitrosodimethylamine (NDMA), as described previously (Hong et al., 1989). Reaction mixtures consisted of 3.0 mg of microsomal protein suspended in 1 ml of 100 mM K₂HPO₄ buffer containing 1.5 mM EDTA, pH 7.0, and components of an NADPH-generating system, as detailed previously. Samples were preincubated for 3 min at 37°C, and the reaction was initiated by addition of 40 µM [¹⁴C]NDMA (specific activity 40 mCi/mmol) in water. Reactions were carried out for 10 min at 37°C with agitation and were terminated by cooling the samples on ice. Radiolabeled formaldehyde ([¹⁴C]HCHO) was extracted into hexane; 2 ml of this hexane solution was added to 18 ml of Eco-lite scintillation fluid, and levels of radioactivity were determined by liquid scintillation spectroscopy. The formation of [¹⁴C]HCHO was quantified by relating sample cpm to those determined for known amounts of [¹⁴C]HCHO.

Immunoinhibition Studies. mAb 1-91-3, an inhibitory mAb specific for CYP2E1 (Park et al., 1986), was used to inhibit lung CYP2E1 (Lee and Forkert, 1995). mAb HyHel 9, a mAb specific for egg white lysozyme (Smith-Gill et al., 1982) was used as a control antibody to assess for nonspecific reactions. Incubations with antibodies were performed at a mAb protein to microsomal protein ratio of 0.5, as described in our previous studies (Lee and Forkert, 1995). Briefly, reaction mixtures containing 3.0 mg of microsomal protein in a volume of 1 ml 100 mM K₂HPO₄ buffer, pH 7.0, were incubated with the appropriate mAb at room temperature for 30 min with gentle agitation. Thereafter, components of the NADPH-generating system were added and the mixtures were preincubated for 3 min at 25°C. The reaction was initiated by addition of 0.5 mM VC in water, and the mixtures were incubated for an additional 30 min at 25°C. The reactions were terminated by cooling the samples on ice. The microsomes were then washed and recovered, and NDMA demethylase activity or levels of covalent binding were determined.

Protein Immunoblotting. After incubation with PMSF and/or VC, microsomal samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) on an 8.5% gel as described previously (Forkert et al., 1994). The protein samples were then transferred to a 0.45-µm nitrocellulose membrane for 1 h at 12 V in 25 mM Tris-HCl, 192 mM glycine, pH 8.3, and 20% (v/v) methanol. The membrane subsequently was incubated for 2 h in a blocking solution consisting of 5% nonfat dried milk in 20 mM Tris/500 mM NaCl, pH 7.5. After thorough rinsing in buffer, the membrane was incubated overnight with a goat anti-rabbit liver CYP2E1 polyclonal antibody (1:200) or a rabbit anti-rat liver hydrolase A or B² (1:1500) (Morgan et al., 1994). The membrane then was rinsed in buffer, reacted for 2 h with an IgG conjugated to alkaline phosphatase, and immersed in a solution containing p-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt. The apparent molecular weight of immunodetectable protein bands was estimated by reference to the position of prestained molecular weight standards.

Covalent Binding of [¹⁴C-carbonyl]-VC to Lung Microsomes. Covalent binding was determined by using the equilibrium dialysis method as described in our previous studies (Forkert et al., 1986). Reaction mixtures consisted of 3.0 mg of microsomal protein suspended in 1 ml of 100 mM K₂HPO₄ buffer containing 1.5 mM EDTA, pH 7.0, and components of an NADPH-generating system; the mixtures were preincubated for 3 min at 25°C. The reaction was initiated by addition of 0.5 mM VC (0.30 µCi [¹⁴C-carbonyl]-VC, specific activity 1.5 mCi/mmol) in water. Incubation vessels were capped with Teflon stoppers and incubated for an additional 30 min at 25°C with gentle agitation. The samples were cooled on ice, after which 0.5 ml of 4% SDS was added. The samples then were transferred to polypropylene tubes and placed into a boiling water bath for 15 min. The boiled microsomal proteins were cooled slowly at room temperature, transferred to dialysis tubing, and dialyzed overnight in 500 ml of 0.1% SDS/100 mM K₂HPO₄ buffer, pH 7.0. Aliquots (250 µl) of the dialyzed samples were solubilized overnight in 2 ml of Soluene. After the addition of glacial acetic acid (300 µl) and Eco-Lite scintillation fluid (15 ml), levels of radioactivity of dialyzed samples were determined by liquid scintillation spectroscopy. The difference in the levels of radioactivity of the dialysate and the buffer represented the amounts of covalently bound VC in the sample.

Statistical Analysis. All data are expressed as mean ± S.D. Data were analyzed by one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test to identify significant differences between experimental groups. The level of significance was set at p < .05.

	Results

Top Abstract Introduction Materials and methods Results Discussion References

Effects of PMSF and/or VC on NDMA Demethylase and Carboxylesterase Activities. Levels of NDMA demethylase activity were decreased significantly in lung microsomes incubated with VC in the presence of NADPH compared with those obtained in reactions in which NADPH was omitted (Fig. 2). The alterations were concentration-dependent. Demethylase activities decreased progressively in microsomes incubated with VC in amounts ranging from 100 µM to 1 mM compared with those in control incubations performed with VC alone (0.34 ± 0.02 nmol [¹⁴C]HCHO/mg protein/min) or with only NADPH (0.33 ± 0.09 nmol [¹⁴C]HCHO/mg protein/min). Saturation was achieved at a concentration of 1.0 mM VC, and further decreases detected at concentrations ranging from 1.0 mM to 10 mM were slight (Fig. 2). Time-dependent alterations in NDMA demethylase activity also were determined and were detected at incubation periods of up to 30 min in microsomes incubated with 0.5 mM VC in the presence of NADPH. No further changes in demethylase activity were identified at incubation times of 30 to 180 min (Fig. 3). Based on these data, a VC concentration of 0.5 mM and an incubation time of 30 min were used for subsequent experiments, unless otherwise noted.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Effect of VC concentrations on CYP2E1-dependent [14C]NDMA demethylase activity in lung microsomes. Reaction mixtures contained 3.0 mg of microsomal protein and an NADPH-generating system in a final incubation volume of 1 ml of 100 mM K2HPO4 buffer, pH 7.0. The mixtures were preincubated for 3 min at 25°C, and the reaction was initiated by addition of VC in water. The incubations were carried out for 30 min, after which the microsomes were washed and reharvested, and [14C]NDMA demethylase activity was determined. Data are expressed as mean ± S.D. of quadruplicate determinations performed on three to four different microsomal samples. Data were analyzed using one-way ANOVA followed by the Student-Newman-Keuls test; p < .05.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effect of incubation time on CYP2E1-dependent [14C]NDMA demethylase activity in lung microsomes reacted with VC. Reaction mixtures containing 3.0 mg of microsomal protein and an NADPH-generating system were preincubated for 3 min at 25°C; the reaction was initiated by addition of 0.5 mM VC in water. Samples were incubated for various times ranging from 10 to 180 min. The reactions subsequently were terminated, the microsomes were washed and reharvested, and [14C]NDMA demethylase activity was determined. Data are expressed as mean ± S.D. of quadruplicate determinations from three to four different microsomal preparations. Data were analyzed by one-way ANOVA, followed by the Student-Newman-Keuls test; p < .05.

Inhibition of CYP2E1 by an Inhibitory mAb and DASO₂. Results of the antibody inhibition experiments are summarized in Table 2. Preincubation of lung microsomes with the CYP2E1 inhibitory mAb (Park et al., 1986) significantly inhibited NDMA demethylase activity by about 70%, compared with levels in microsomes that were not reacted with the mAb. Lung microsomes incubated with VC and NADPH caused a significant decrease (50%) in demethylation, compared with the levels detected in microsomal incubations conducted in the absence of NADPH or VC; these data are consistent with those of the preceding experiments (Table 1). When microsomes were incubated with the mAb and VC, the level of demethylase activity was similar to that detected in microsomes that were incubated with the mAb alone. Incubations with the nonspecific mAb HyHel 9 (Smith-Gill et al., 1982) and VC produced levels of demethylase activity that were similar to those in microsomes incubated with only VC.

Covalent Binding of [¹⁴C-carbonyl]-VC to Lung Microsomal Proteins. Results of the covalent binding studies are summarized in Table 3. Covalent binding of [¹⁴C-carbonyl]-VC to lung microsomal proteins was significantly greater in preparations incubated in the presence of VC and NADPH compared with the level detected in incubations conducted with VC in the absence of NADPH. The magnitude of binding in microsomes that were preincubated with the CYP2E1 mAb was not significantly different from that obtained in incubations carried out with VC in the absence of both the mAb and NADPH. The amount of binding is significantly lower than the amount detected in microsomes incubated with VC and NADPH. In control incubations performed with the nonspecific mAb HyHel 9, the binding level was similar to that obtained in microsomes reacted with VC and NADPH.

Protein Immunoblotting. The CYP2E1 polyclonal antibody recognized a single protein band of M_r 51,000 in murine lung microsomes (Fig. 4A), and this protein species is consistent with that detected in our previous studies (Lee and Forkert, 1995). The content of immunodetectable CYP2E1 was diminished significantly in microsomal samples previously incubated with VC and NADPH compared with protein samples incubated with only the vehicle or only PMSF. When microsomes were incubated with PMSF and subsequently with VC, immunoreactivity for CYP2E1 was virtually abolished.

View larger version (43K): [in this window] [in a new window]   Fig. 4.   Protein immunoblotting of lung microsomal proteins for CYP2E1 and hydrolase A.  Microsomal proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and reacted with antibodies for CYP2E1 (A) or hydrolase A (B). A, lanes 6 to 9 and 2 to 5 contained 3.0 and 5.0 µg of protein, respectively. B, lanes 2 to 5 and 6 to 9 contained 0.5 and 1.0 µg of protein, respectively. A and B, microsomal proteins were loaded as follows: lane 1, molecular weight standards; lanes 2 and 6, control; lanes 3 and 7, PMSF (25 µM); lanes 4 and 8, VC (0.5 mM); lanes 5 and 9, PMSF (25 µM) and VC (0.5 mM). All incubations with VC were conducted in the presence of NADPH.

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

It was postulated first by Dahl et al. (1978) that EC is oxidized to VC, which undergoes an additional oxidation step to produce VC epoxide, a metabolite that has been proposed to be the ultimate carcinogenic species (Fig. 1). Because of the high susceptibility of the lung to EC- and VC-induced tumor formation, it was of interest to determine whether a similar oxidative pathway is also involved in the lung metabolism of both of these carbamates. As an initial step to this end, our previous studies have investigated the metabolism of EC in lung microsomal incubations (Forkert and Lee, 1997). Our findings showed that EC metabolism was mediated by cytochrome P-450, inasmuch as NADPH was required for production of a reactive metabolite(s) that binds to lung microsomal proteins. This binding was increased significantly in microsomes preincubated with paraoxon, an inhibitor of the carboxylesterases, and indicated that inhibition of the detoxication pathway led to enhancement of EC oxidation and activation (Forkert and Lee, 1997). In the present studies, we have investigated the lung metabolism of VC to establish whether the metabolic events involving VC oxidation and detoxication are similar to those identified for EC.

Data from the present studies showed that lung metabolism of VC is cytochrome P-450-dependent: substantial binding of VC to proteins was detected in lung microsomes incubated in the presence of NADPH compared with the level obtained in incubations conducted in the absence of NADPH (Table 3). Parallel studies performed to investigate the role of CYP2E1 in VC oxidation showed that levels of NDMA demethylase activity were decreased significantly in lung microsomes incubated with VC and NADPH, compared with those detected in incubations conducted in the absence of NADPH (Tables 1 and 2). This loss of enzyme activity coincided with a marked decrease in the content of immunodetectable CYP2E1 (Fig. 3A). These findings suggested activation of VC to a reactive metabolite capable of alkylating the protein moiety of CYP2E1, resulting in decreased immunoreactivity. These data implicating CYP2E1 in VC metabolism are supported by data from our experiments with enzyme inhibitors. Preincubation of lung microsomes with a CYP2E1 mAb or DASO₂ significantly inhibited NDMA demethylation as was detected in incubations with VC, thus confirming the involvement of CYP2E1 in VC oxidation (Table 2). Importantly, covalent binding of VC to lung proteins was about 50% lower in microsomes that were preincubated with the CYP2E1 mAb or DASO₂ compared with levels in microsomal incubations in which the mAb or DASO₂ was omitted (Table 3). These findings are consistent with a role for CYP2E1 in lung metabolism of VC. Similar alterations in NDMA demethylase activity and covalent binding were observed in previous studies with EC (Forkert and Lee, 1997). Hence, the oxidation of both EC and VC appears to be mediated by CYP2E1. These results are in agreement with those from studies in human liver microsomes that implicated CYP2E1 as the major isozyme catalyzing the oxidation of both EC and VC (Guengerich and Kim, 1991; Guengerich et al., 1991).

Previous studies have shown that more than 90% of a dose of EC is converted to expired CO₂, with ethanol as the primary product of catabolism (Boyland and Rhoden, 1949; Bryan et al., 1949). Findings from studies with liver homogenates and metabolic inhibitors implicated the microsomal carboxylesterases in the hydrolysis of EC to CO₂ (Mirvish, 1968). Moreover, data from more recent studies showed that porcine liver esterase was highly efficient in the metabolism and conversion of EC to CO₂ (Yamamoto et al., 1990). Hence, available data supported a role for the carboxylesterases in the hepatic metabolism and detoxication of EC. Our recent studies have investigated the involvement of the microsomal carboxylesterases in EC metabolism in lung microsomes (Forkert and Lee, 1997). Lung microsomes incubated with EC produced a significant decrease in the levels of carboxylesterase activity. The carboxylesterase inhibitors, paraoxon and PMSF, were used in lung microsomal incubations to examine the isozyme-selective metabolism of EC. Paraoxon is a broad-spectrum carboxylesterase inhibitor that inhibits both hydrolase A and B, whereas PMSF selectively inhibits hydrolase A (Morgan et al., 1994). Both of these agents inhibited microsomal carboxylesterase activity to such an extent that EC could not be metabolized by this enzyme system (Forkert and Lee, 1997). These data together with the loss of immunodetectable hydrolase A but not hydrolase B (Fig. 3) supported EC metabolism by microsomal carboxylesterases and, in particular, by hydrolase A.

An objective of the present studies is to determine whether, as in the case of EC, microsomal carboxylesterases have a role in lung metabolism of VC. These studies also were undertaken to identify the potential isozyme-selective metabolism of VC. Our results showed a slight but significant decrease (22%) in carboxylesterase activity in microsomes incubated with VC compared with the constitutive level in lung microsomes (Table 1). When the microsomes were reacted with PMSF and then with VC, this decrease was exacerbated and comprised 50% of the level detected in microsomes incubated with VC alone. These data suggested that the amount of carboxylesterase activity inhibited by PMSF was substantial and prevented hydrolysis of VC by the carboxylesterases. These findings coupled with the loss of immunodetectable hydrolase A supported lung metabolism of VC by this carboxylesterase isozyme; hydrolase B did not appear to be involved. Loss in hydrolase A immunoreactivity may be the result of interaction of VC with the protein, inducing a conformational change such that recognition by the antibody is reduced. For microsomal incubations involving the carboxylesterase inhibitor PMSF, the loss in immunodetectable hydrolase A may be due to binding of the inhibitor to active-site serine residues of the protein (Kitz and Wilson, 1962; Heymann et al., 1972). Our findings with VC, together with the results from our previous studies with EC, strongly supported contributions of the carboxylesterases toward both EC and VC metabolism. The similarity of the metabolic pathway involved is underscored by our finding that both of the carbamate compounds are selectively metabolized by hydrolase A.

The byproducts of carboxylesterase-mediated EC metabolism have been identified as ethanol, ammonia, and CO₂ (Boyland and Rhoden, 1949; Kaye, 1960; Mirvish and Kaye, 1964; Nomier et al., 1989). Based on these findings, an assumption has been made that this metabolic pathway represents detoxication, and this is supported by the results of studies using paraoxon and carbaryl as metabolic inhibitors (Yamamoto et al., 1990). Carboxylesterase activity was inhibited by these compounds and coincided with decreased covalent binding of EC to liver proteins. Because the extent of binding generally is regarded as a measure of reactive metabolite formation, these data suggested that activation is depressed by reaction with the carboxylesterase inhibitors, leading to decreased EC binding. More recent studies have reported decreased production of CO₂ from EC in lung microsomes incubated with paraoxon (Page and Carlson, 1994). However, CO₂ is also a product generated from P-450-mediated metabolism of EC (Park et al., 1993; Kemper et al., 1995), and it is not clear through which pathway the CO₂ is generated and what the relative contributions are from the carboxylesterase and oxidative pathways. In our recent studies with EC, it was shown that levels of covalent binding to lung proteins were increased by about 95 and 85% in microsomes preincubated with paraoxon and PMSF, respectively (Forkert and Lee, 1997). These increases produced by paraoxon and PMSF correlated with decreases of about 80 and 60% in levels of carboxylesterase activity, respectively. These data are consistent with a role of detoxication for the carboxylesterases, and inhibition of these enzymes led to augmentation of the activation pathway and, hence, increased EC binding. In this investigation with VC, carboxylesterase activity is decreased by about 50% in lung microsomes preincubated with PMSF (Table 1). This decrease corresponded with a significant increase of 35% in covalent binding of VC to lung microsomal proteins. These findings supported the involvement of the carboxylesterases in detoxication, and inhibition of these enzymes leads to enhanced activation, resulting in increased generation and binding of a VC metabolite(s). However, the carboxylesterases may not represent the only pathway of detoxication. Recent studies have reported that in vitro generation of 1,N⁶-ethenoadenosine from the VC epoxide is inhibited by glutathione; adduct formation is decreased further by the addition of glutathione S-transferases (Kemper et al., 1995). The relative extents to which the carboxylesterases and glutathione are involved in EC and VC detoxication are not known and remain to be investigated.

In summary, our results are consistent with important roles for CYP2E1 and hydrolase A in bioactivation and detoxication of VC, respectively, and modification of either metabolic pathway leads to alterations in the extent to which reactive intermediates are formed, as assessed by covalent binding of VC to lung proteins.