Strain-Related Differences in Bioactivation of Vinyl Carbamate and Formation of DNA Adducts in Lungs of A/J, CD-1, and C57BL/6 Mice

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1 TOXICOLOGICAL SCIENCES 59, (2001) Copyright 2001 by the Society of Toxicology Strain-Related Differences in Bioactivation of Vinyl Carbamate and Formation of DNA Adducts in Lungs of A/J, CD-1, and C57BL/6 Mice A. Paul Titis and Poh-Gek Forkert 1 Department of Anatomy and Cell Biology, Queen s University, Kingston, Ontario, Canada K7L 3N6 Received July 28, 2000; accepted September 27, 2000 Inbred strains of mice exhibit differing susceptibilities to formation of lung tumors induced by procarcinogens including ethyl carbamate (EC) and vinyl carbamate (VC). Strain A/J mice are susceptible, whereas C57BL/6 mice are resistant to lung tumor development. In this study, we tested the hypothesis that differential susceptibilities of A/J, CD-1, and C57BL/6 mice to lung tumor development are associated, in part, with their capacities for VC bioactivation and with the extents of DNA adduct formation. Previous studies have shown that the P450 isozyme CYP2E1 and microsomal carboxylesterases are involved in activation and detoxication of VC, respectively. Bioactivation capacity, as estimated by ratios of p-nitrophenol hydroxylase, a CYP2E1 catalytic marker, to carboxylesterase activities, was greater in control A/J ( ) and CD-1 ( ) mice than in control C57BL/6 ( ) mice. The ratios were reduced in all three strains of mice treated with VC. Covalent binding of [ 14 C-carbonyl]-VC to lung proteins was time- and dosedependent, and was significantly higher in A/J and CD-1 mice than in C57BL/6 mice. Experiments using 32 P-postlabeling/thinlayer chromatography showed formation of the DNA adducts 1,N 6 -ethenodeoxyadenosine and 3,N 4 -ethenodeoxycytidine in lungs of mice treated with VC. The DNA adducts were detected at 30 min after treatment, peaked at 60 min, and declined thereafter. Levels of 1,N 6 -ethenodeoxyadenosine and 3,N 4 -ethenodeoxycytidine were about 70% higher in A/J and CD-1 mice than in C57BL/6 mice. These results indicated that formation of VC metabolites in these murine strains is linked to their bioactivation capacities, and suggested that this attribute may confer differing susceptibilities to lung tumor development. Key Words: carboxylesterase; CYP2E1; DNA adducts; epoxide; lung tumors; p-nitrophenol hydroxylase; vinyl carbamate. Ethyl carbamate (EC, urethane) induces tumors in a variety of tissues including the skin, lung, liver, mammary gland, lymphoid tissue, and Harderian glands (Mirvish, 1968). A long latent period of about 1 year is generally required for tumor development, with the exception of the lung, where tumors are formed more rapidly and are manifested in about 2 to 6 months (Mirvish, 1968; Shimkin and Stoner, 1975). Both EC and VC, 1 To whom correspondence should be addressed. Fax: (613) forkertp@post.queensu.ca. a primary metabolite derived from EC, induce the same spectrum of tumors in the lung, although VC appears to be a more potent carcinogen than EC. Lung tumors induced by VC were 20- to 50-fold greater in number than those induced by EC, depending on exposure conditions (Dahl et al., 1978, 1980). Moreover, VC but not EC was mutagenic to Salmonella typhimurium (Dahl et al., 1978, 1980; Leithauser et al., 1990). These findings indicated that the lung is an important and possibly primary target of EC- and VC-induced toxicity and carcinogenicity. The carcinogenicity of EC has been ascribed to its metabolism to VC and subsequently to VC epoxide (VCO), a metabolite that has been suggested to be the ultimate carcinogenic species (Dahl et al., 1978, 1980; Ribovich et al., 1982). Findings from previous studies with human liver microsomes are consistent with a role for CYP2E1 in oxidation of EC and VC to VCO (Guengerich et al., 1991; Guengerich and Kim, 1991). Data from our recent studies in the lungs of mice also supported involvement of CYP2E1 in EC and VC metabolism (Fig. 1) (Forkert and Lee, 1997; Lee and Forkert, 1999). Our studies indicated additionally that both EC and VC are metabolized by carboxylesterase enzymes (Forkert and Lee, 1997; Lee and Forkert, 1999), a pathway that generates the end products ethanol, ammonia, and carbon dioxide (Fig. 1). Covalent binding of [ 14 C-ethyl]-EC and [ 14 C-carbonyl]-VC to lung microsomal proteins occurred when incubations were performed in the presence of NADPH and was augmented under conditions in which carboxylesterase enzymes were inhibited (Forkert and Lee, 1997; Lee and Forkert, 1999). In contrast, binding was reduced when CYP2E1 was inhibited. These findings supported the premise that CYP2E1 and carboxylesterase enzymes mediated the activation and detoxication of EC or VC, respectively. Previous studies have investigated the targets of reactive intermediates formed from EC and VC metabolism. Both EC and VC produced 1,N 6 -ethenoadenosine and 3,N 4 -ethenocytidine adducts in hepatic RNA and 7-(2-oxoethyl)guanine adducts in hepatic DNA of rats and mice (Ribovich, 1982; Miller and Miller, 1983; Scherer et al., 1986). In subsequent studies, VCO was synthesized and found to react with calf thymus DNA in vitro to form two major guanine adducts, N 2,3-ethe- 82

2 DNA ADDUCT FORMATION FROM VINYL CARBAMATE 83 MATERIALS AND METHODS FIG. 1. Proposed scheme of EC and VC metabolism. nodeoxyguanosine and 7,-(2 -oxoethyl)deoxyguanosine, and a minor adenine adduct, 1,N 6 -ethenodeoxyadenosine ( da) (Park et al., 1993). The formation of da has been regarded as being biologically important because of its ability to miscode in transcription of DNA (Barbin and Bartsch, 1986; Basu et al., 1993). More recent studies have identified da and 3,N 4 - ethenodeoxycytidine ( dc) in liver and lung DNA of mice treated with EC, VC, or VCO (Fernando et al., 1996). Both of these etheno-dna adducts were formed at significantly higher levels after treatment with VC than with EC. Taken together, these data are consistent with the assertion that the electrophilic VCO is the ultimate carcinogenic species formed from EC and VC, leading to DNA alkylation and formation of adducts. It is of interest that laboratory strains of mice exhibit differing susceptibilities to lung tumor formation (Malkinson, 1991; Shimkin and Stoner, 1975). A high incidence of spontaneous and chemically induced lung tumors is found in Strain A mice, whereas the incidence of both is low in C57BL/6 mice, and they are resistant to the carcinogenic effects of chemicals including EC. We reasoned that, as bioactivation of EC and VC to a reactive intermediate is prerequisite to interaction with DNA, the enzymatic systems involved in the metabolic pathway are likely to have an important role in promoting formation of DNA adducts. In this investigation, we have used the susceptible A/J and resistant C57BL/6 mice to test the hypothesis that differences in the capacity for bioactivation affect the amounts of reactive metabolites and DNA adducts formed from VC. Swiss-Webster CD-1 mice, a strain commonly used in laboratory studies, were also included. These three strains of mice were used to determine: (1) the capacity for bioactivation by calculating the ratios of CYP2E1-dependent p-nitrophenol (PNP) hydroxylase to carboxylesterase activities, (2) levels of covalent binding of VC to lung proteins, and (3) levels of da and dc adducts formed in lung DNA. Materials. Chemicals and reagents used in this study were obtained as follows: BioRad Protein Assay Dye Reagent Concentrate (BioRad Laboratories), QIAamp Tissue Kit (QIAGEN Inc., Santa Clarita, CA); p-nitroblue tetrazolium chloride, 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt, molecular weight standards (Bio-Rad Laboratories, Hercules, CA); 2 deoxyadenosine 3 monophosphate, 2 deoxycytidine 3 monophosphate, 2 deoxyguanosine 3 monophosphate, chloroacetaldehyde, p-nitrophenol, p-nitrophenyl acetate, and 4-nitrocatechol (Sigma Chemical Co., St. Louis, MO); Eco- Lite and Universol scintillation fluids (ICN Chemical Co., Costa Mesa, CA); RNase A, micrococcal nuclease (from Staphylococcus aureus), calf spleen phosphodiesterase, T4 polynucleotide kinase (Pharmacia Biotech, Baie d Urfe, Quebec, Canada); Amersham Hyperfilm MP (Amersham Life Science, Arlington Heights, IL); polyethyleneimine cellulose membranes (Aldrich, Milwaukee, WI); polymeric reverse-phase column ( cm, 5 m PLRP-S 100 A, Phenomenex) [ - 32 P]-ATP (specific activity 6 mci/mmol) was purchased from Mandel Scientific, Boston, MA. [ 14 C-carbonyl]-vinyl carbamate ( 98% radiochemical purity, specific activity 1.5 mci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO); dialysis tubing, molecular weight cutoff 3500 Da, was purchased from Fisher Scientific (Nepean, Ontario, Canada). Vinyl carbamate was synthesized according to procedures described previously (Park et al., 1990). Rabbit polyclonal antibodies for rat liver microsomal hydrolase A and hydrolase B were generously contributed by Dr. A. Parkinson (Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS). Goat polyclonal antibodies directed against rabbit microsomal CYP2E1 were obtained from Oxford Biomedical Research Inc., (Oxford, MI). All other chemicals were purchased from standard commercial suppliers. Treatment of animals. Female A/J mice of g body weight were purchased from Jackson Laboratories (Bar Harbor, MA). Female C57BL/6 and CD-1 mice, also of g body weight, were purchased from Charles River Canada (St. Constant, Quebec, Canada). The mice were maintained on a 12-h light/dark cycle, and given unrestricted access to food (Mouse Diet 5015, PMI Feeds Inc.) and drinking water. For experiments undertaken to observe the effects of VC on microsomal CYP2E1 and carboxylesterase enzymes, groups of A/J, CD-1, and C57BL/6 mice (n 20) were treated with VC in saline (60 mg/kg, i. p.) and sacrificed 1 h later. For time-course and dose-response studies to determine covalent binding of [ 14 C-carbonyl]-VC to lung proteins, CD-1 mice were treated with VC (20 Ci/kg [ 14 C-carbonyl]-VC/60 mg/kg, i. p., specific activity, 1.5 mci/mmol) in saline. In experiments to determine binding levels in A/J, CD-1, and C57BL/6 mice, animals were treated with VC (20 Ci/kg [ 14 C-carbonyl]-VC/210 mg/kg, i. p., specific activity, 1.5 mci/mmol). All mice were sacrificed by cervical dislocation 1 h after VC treatment. The lungs were frozen in liquid nitrogen and stored at 70 C. For determination of strain differences in formation of DNA adducts, A/J, CD-1, and C57BL/6 mice were treated with VC (60 mg/kg, i.p.). The mice were sacrificed by cervical dislocation 1 h after VC treatment. The lungs were frozen in liquid nitrogen and stored at 70 C. In all the experiments, control mice received appropriate volumes of the vehicle. Preparation of microsomes. Lungs from 20 mice were pooled, and microsomes were prepared by differential centrifugation as described previously (Forkert, 1995). Microsomal pellets were resuspended in 100 mm K 2 HPO 4 buffer, and aliquots (100 l) were frozen in liquid nitrogen and stored at 70 C. Protein concentrations were determined by the Bradford method, using bovine serum albumin as the standard (Bradford, 1976). Enzyme assays. PNP hydroxylase activity was used as a catalytic marker for CYP2E1-dependent enzyme activity. Levels of PNP hydroxylase activity were determined according to procedures described previously (Forkert and Lee, 1997). Microsomal carboxylesterase activity was determined by measuring the hydrolysis of p-nitrophenyl acetate (PNA) to PNP as described in previous studies (Morgan et al., 1994; Forkert and Lee, 1997).

3 84 TITIS AND FORKERT Protein immunoblotting. Protein immunoblotting was carried out using methods described previously (Forkert, 1995). Lung microsomal proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) and transferred to a nitrocellulose membrane (0.45 M). The membrane was then incubated overnight at room temperature with one of the following antibodies: anti-cyp2e1, anti-hydrolase A, or anti-hydrolase B 2. The membrane was subsequently incubated for 2 h with IgG conjugated to alkaline phosphatase (1:1000). The protein bands were visualized by reaction with a solution containing p-nitroblue tetrazolium chloride and 5-bromo-4- chloro-3-indolylphosphate p-toluidine salt. Covalent binding of [ 14 C-carbonyl]-VC to lung microsomes. Covalent binding was determined by using equilibrium dialysis as described previously (Forkert et al., 1986). Lungs were homogenized in 0.01M sodium phosphate buffer, ph 7.0, containing 2% SDS. The samples were boiled for 15 min, cooled, and dialyzed overnight against 500 ml of 100 mm potassium phosphate buffer, ph 7.0, containing 0.1% SDS. Aliquots (250 l) of the dialyzed samples were solubilized overnight with 1 M NaOH (1 ml). After addition of glacial acetic acid (300 l) and aqueous scintillation fluid (15 ml), levels of radioactivity were determined. The difference in the amounts of radioactivity of the dialysate and the buffer was regarded as the quantity of covalently bound VC in the sample. Synthesis and purification of ethenodeoxynucleotides ( dnmps). The 1, N 6 -ethenodeoxyadenosine 3 -monophosphate ( damp) and 3, N 4 -ethenodeoxycytidine 3 -monophosphate ( dcmp) DNA adducts were synthesized as described previously (Guichard et al., 1993). The parent nucleotides deoxyadenosine 3 -monophosphate (damp) or deoxycytidine 3 -monophosphate (dcmp) (0.125 M) were reacted with chloroacetaldehyde (3 M) in 0.2 M ammonium acetate, ph 6.0, for 24 h at 37 C, with gentle agitation. Aliquots (10 l) of the synthesized adducts were then purified using a polymeric reverse-phase column ( cm, 5 m PLRP-S 100 A, Phenomenex). The etheno adducts were monitored at an absorbance of 260 nm, and were eluted isocratically with 0.01 M triethylammonium acetate and 3% acetonitrile, ph 7.1, as the mobile phase, at a flow rate of 1 ml/min over a duration of 20 min. Peaks corresponding to damp and dcmp were collected, lyophilized, and analyzed for confirmation of their identities. DNA was isolated from lung tissue using the QIAamp Tissue Kit. Lung samples from each mouse were pulverized in liquid nitrogen using a cold mortar and pestle, and about 25 mg of the powder derived from each lung was then lysed overnight with Proteinase K in a shaking waterbath set at 55 C. The samples were then incubated with 400 g of RNase A for 30 min at 70 C. The resultant gelatinous material was then sheared using a 22-gauge needle followed by shearing with a 26-gauge needle. The remainder of the procedure is based on adsorption of the DNA onto the QIAamp spin column silica membrane during a brief centrifugation step. The DNA was eluted from the spin column and quantitated at 260 nm. Aliquots of the purified DNA were digested to deoxynucleotide 3 -monophosphates (dnmp) according to methods described previously (Gupta, 1985), with modifications. The DNA sample (10 g) was incubated with 1 U micrococcal nuclease and 0.2 U calf spleen phosphodiesterase in 10 mm sodium succinate/5 mm calcium chloride, ph 6.0, in a final volume of 25 l. The reaction mixtures were incubated at 37 C for 3 h. The reaction was then terminated, and the samples were stored at 20 C. The digested DNA samples were subjected to HPLC analysis as described previously (Watson and Crane, 1989), with modifications. The nucleotides were separated using a polymeric reverse-phase column ( cm, 5 m PLRP-S 100 A, Phenomenex) and eluted isocratically with 0.01 M triethylammonium acetate and 3% acetonitrile, ph 7.1, as the mobile phase and a flow rate of 1 ml per min. Fractions containing damp and dcmp were identified and collected on the basis of retention times relative to damp and the synthesized damp and dcmp. 2 The antibodies were raised against rat liver microsomal hydrolase A and hydrolase B. For the sake of convenience, the murine lung microsomal proteins recognized by these antibodies are designated by the same nomenclature. 32 P-postlabeling/thin layer chromatography. 32 P-postlabeling was carried out as described previously (Nair et al., 1995), with modifications. Fractions of the adducts were pooled, lyophilized, and radiolabeled with 25 Ci [ 32 P]- ATP in the presence of 10 U T4-polynucleotide kinase and 125 mm Tris- HCl/25 mm magnesium chloride/25 mm dithiothreitol buffer, ph 6.8, for 1.5 h in a 37 C dry bath. The samples were then placed on ice. Known amounts of adduct standards (2, 50, and 75 fmol) were run in parallel with adduct samples as external standards in order to determine the specific activity of the [ 32 P]- ATP. Samples were then resolved by two-dimensional thin layer chromatography (TLC), as described in previous studies (Bochner and Ames, 1982; Fernando et al., 1996). Aliquots were spotted to predeveloped polyethyleneimine cellulose membranes at a point 2 cm from the bottom and left margin, and dried at room temperature. TLC plates were immersed in methanol for 5 min, dried, then developed in the first dimension using 1 M acetic acid, ph 3.5. The membranes were then soaked in methanol (10 min), dried, and trimmed to remove excess radioactive inorganic phosphate. Subsequently, the membranes were developed in the second dimension, using saturated ammonium sulfate, ph 3.5. Finally, plates were dried thoroughly at room temperature, and exposed to X-ray film in developing cassettes equipped with intensifying screens for 3 h at 86 C. Individual spots corresponding to deoxynucleotide 5 monophosphates and ethenodeoxynucleotide 5 -monophosphates were characterized by comparison with autoradiograms from purified standards. Quantification of DNA adducts. Quantification of formed DNA adducts was carried out by excising areas on the TLC membrane corresponding to adduct spots and measuring the amounts of radioactivity. DNA adduct levels were calculated as described previously (Hughes and Phillips, 1990). Counts derived from external standards run in parallel with adduct samples were converted to disintegrations per minute (DPM) values, which were then used to create a standard curve to calculate the specific activity of [ 32 P]-ATP. Amounts of unmodified parent nucleotides were calculated by converting the peak area compiled from individual HPLC chromatograms to microgram amounts. The counts derived from adduct spots corresponding to the ethenodeoxynucleotide 5 -monophosphates were also converted to DPM values. DPM values for each adduct were then divided by the microgram amount of the corresponding parent nucleotide found in the sample and divided by the specific activity of the [ 32 P]-ATP for the specified adduct. Final values were expressed as number of adducts per 10 8 parent nucleotides. Instrumentation. HPLC experiments were conducted on a Beckman System Gold Programmable Solvent Module 126 HPLC with a Beckman System Gold Module 168 UV detector. Spectra for all other assays were determined with a Beckman model DU640 diode array UV spectrophotometer. Radioactivity measurements were conducted on a Beckman model LS 1801 scintillation counter. Statistical analysis. Data are expressed as mean SD and were analyzed by one-way or two-way analysis of variance followed by pairwise multiple comparisons with the Student-Newman-Keuls test. Effects of VC on CYP2E1 RESULTS The effects of VC treatment on the CYP2E1 enzyme in CD-1 mice are summarized in Figure 2. Levels of CYP2E1- dependent PNP hydroxylase activity in lung microsomes from control A/J and CD-1 mice were significantly different from those in C57BL/6 mice (Fig. 2B). Levels of hydroxylase activity in control A/J mice were 10% and 40% higher than those in CD-1 and C57BL/6 mice, respectively. In mice treated with VC (60 mg/kg), PNP hydroxylase activities in lung microsomes were significantly decreased so that the residual amounts were similar in all strains of mice.

4 DNA ADDUCT FORMATION FROM VINYL CARBAMATE 85 estimated by determining the hydrolysis of PNA to PNP, differed significantly in control A/J, CD-1, and C57BL/6 mice. Levels of carboxylesterase activity in control C57BL/6 mice were higher than those in either control A/J or control CD-1 mice. Treatment with VC (60 mg/kg) produced significant decreases in carboxylesterase activity in all three strains of mice. The results of protein immunoblotting using antibodies specific for hydrolase A are depicted in Figure 3A. The hydrolase A antibody recognized a protein band of approximately 57 kda in lung microsomes from A/J, CD-1, and C57BL/6 mice. This is similar to the apparent molecular weight detected for hydrolase A in lung microsomes from CD-1 mice reported in previous studies (Forkert and Lee, 1997). Differences in the contents of immunodetectable hydrolase A in lung microsomal FIG. 2. Protein immunoblotting for CYP2E1 (A) and PNP hydroxylase (B) activity in lung microsomes of A/J, CD-1, and C57BL/6 mice. The mice were untreated or treated with VC (60 mg/kg, i.p.) and were sacrificed 1 h later. In the immunoblots, microsomal proteins were separated by SDS PAGE, transferred to a nitrocellulose membrane, and reacted with polyclonal antibodies for CYP2E1. Lanes 1 6 contained 0.7 g of microsomal proteins. Proteins from the three strains of mice were loaded as follows: lane 1, control A/J; lane 2, VC-treated A/J; lane 3, control CD-1; lane 4, VC-treated CD-1; lane 5, control C57BL/6; lane 6, VC-treated C57BL/6. Hydroxylase activity was determined spectrally by measuring the formation of 4-nitrocatechol as described in Materials and Methods. Data are expressed as mean SD of triplicate determinations using three to four different microsomal preparations. a p 0.05 compared with levels in control CD-1 and C57BL/6 mice; b p 0.05 compared with levels in control C57BL/6 mice; c p 0.05 compared with levels in the respective control mice. Protein immunoblotting analyses showed that the CYP2E1 antibody reacted with lung microsomal proteins and detected a band of about 51 kda (Fig. 2A). This is consistent with the molecular weight of the band observed for the lung CYP2E1 protein in previous studies (Forkert, 1995). The content of immunodetectable CYP2E1 was highest in lung microsomes from control A/J mice, whereas the amounts of CYP2E1 protein were lower in microsomal samples from control CD-1 and C57BL/6 mice. The contents of CYP2E1 protein from all three strains of mice treated with VC (60 mg/kg) were decreased 1 h after exposure; the diminution was most evident in CD-1 and C57BL/6 mice. Effects of VC on Carboxylesterase Enzymes The effects of VC on carboxylesterase enzymes are summarized in Figure 3. Microsomal carboxylesterase activity, as FIG. 3. Protein immunoblotting for hydrolase A (A) and carboxylesterase activity (B) in lung microsomes of A/J, CD-1, and C57BL/6 mice. The mice were untreated or treated with VC (60 mg/kg, i.p.) and were sacrificed 1 h later. In the immunoblots, microsomal proteins were separated by SDS PAGE, transferred to a nitrocellulose membrane, and reacted with polyclonal antibodies for hydrolase A. Lanes contained 0.1 g of microsomal proteins. Proteins from the three strains of mice were loaded as follows: lane 1, control A/J; lane 2, VC-treated A/J; lane 3, control CD-1; lane 4, VC-treated CD-1; lane 5, control C57BL/6; lane 6, VC-treated C57BL/6. Carboxylesterase activity was determined spectrally by measuring the formation of p-nitrophenyl acetate to PNP and was performed as described in Materials and Methods. Data are expressed as mean SD of triplicate determinations using three to four different microsomal preparations. a p 0.05 compared with levels in control A/J and CD-1 mice; b p 0.05 compared with levels in control CD-1 mice; c p 0.05 compared with levels in the respective control mice.

5 86 TITIS AND FORKERT treatment with a single dose of VC (60 mg/kg) (Fig. 5). Levels of binding increased after 30 min and reached a peak at 60 min; this was followed by a decline with amounts that were similar at 120 and 180 min. Treatment of mice with doses of [ 14 C- carbonyl]-vc ranging from 30 to 210 mg/kg produced levels of binding that were proportional to the dose given. Evaluation of magnitudes of binding to lung proteins in A/J, CD-1, and C57BL/6 mice revealed that levels in A/J and CD-1 mice were significantly higher than those found in C57BL/6 mice (Table 1). DNA Adduct Formation Relative retention times for da, dc, da, and dc standards were determined by HPLC analysis using a polymeric reverse- FIG. 4. Ratios of PNP hydroxylase to carboxylesterase activities in lung microsomes of A/J, CD-1, and C57BL/6 mice. The mice were untreated or treated with VC (60 mg/kg, i. p.) and were sacrificed 1 h later for preparation of microsomes. Data represent the mean SD of triplicate determinations from three to four microsomal preparations and were analyzed by two-way ANOVA and the Student-Newman-Keuls test. a p 0.05 compared with levels in control C57BL/6 mice; b p 0.05 compared with levels in the respective control mice; c p 0.05 compared with levels in VC-treated A/J and C57BL/6 mice; d p 0.05 compared with levels in VC-treated C57BL/6 mice. samples from control A/J, C57BL/6, and CD-1 mice were not obviously apparent (Fig. 3A, lanes 1, 3, and 5). Slight diminution in immunoreactivity of the hydrolase A protein was detected in lung microsomal samples from VC-treated mice (Fig. 3A, lanes 2, 4, and 6), and this effect was more apparent in samples from C57BL/6 than in either A/J or CD-1 mice. Nevertheless, the alterations in the contents of hydrolase A protein in all the strains of mice tested were not pronounced. Immunoblotting analysis for hydrolase B showed that this carboxylesterase isozyme was detectable in lung microsomal samples from all the strains of mice examined (data not shown). However, no alterations in protein contents were apparent after VC treatment, a finding that confirms data obtained in previous in vitro studies (Lee and Forkert, 1999). The potential capacities for bioactivation were determined by calculating the ratios of PNP hydroxylase activity to carboxylesterase activity in both control and VC-treated mice (Fig. 4). The ratios in control A/J and control CD-1 mice were significantly higher than that in control C57BL/6 mice. Those in all VC-treated mice were reduced significantly. However, the ratios in the three strains of VC-treated mice were different from one another, with C57BL/6 having the lowest ratio. Covalent Binding of [ 14 C-Carbonyl]-Vinyl Carbamate to Lung Proteins Covalent binding was determined to estimate the amounts of reactive metabolites formed from VC. Results from the timecourse studies showed that covalent binding of [ 14 C-carbonyl]- VC to lung proteins in CD-1 mice was detectable 15 min after FIG. 5. Time- and dose-dependent responses in covalent binding of [ 14 C- carbonyl]-vc to lung proteins in CD-1 mice. In the time-course experiments, mice were treated with [ 14 C-carbonyl]-VC (60 mg/kg, 20 Ci/kg, i.p.) of VC, and covalent binding measurements were determined from 0.25 to 3 h after VC treatment. In the dose-response experiments, mice were treated with VC at doses ranging from 30 to 210 mg/kg (20 Ci/kg, i.p.), and covalent binding was determined at 1 h after VC treatment. Data represent the mean SD of triplicate determinations from three to four lung homogenate preparations.

6 DNA ADDUCT FORMATION FROM VINYL CARBAMATE 87 TABLE 1 Covalent Binding of [ 14 C-Carbonyl]-VC to Lung Proteins in A/J, CD-1, and C57BL/6 Mice Strain nmol[ 14 C-carbonyl]-VC/mg protein A/J * CD * C57BL/ Note. Mice were treated with VC (20 Ci, 210 mg/kg), and were sacrificed 1 h later. Covalent binding of [ 14 C-carbonyl]-VC to lung proteins was performed in triplicate using three to four lung preparations, and was determined by equilibrium dialysis as described in Materials and Methods. Values are expressed as mean SD. *Significantly different from levels in C57BL/6 mice (p 0.05). phase column. The unmodified nucleotides da and dc eluted at about 3.5 and 5.5 min, respectively, whereas the etheno adducts dc and da eluted at about 10 and 12 min, respectively. Formation of the DNA adducts from VC metabolism was determined using the highly sensitive 32 P-postlabeling/TLC technique. Representative autoradiograms obtained from timecourse experiments are shown in Figure 6 (A and B). Data from these experiments revealed that treatment of CD-1 mice with VC (60 mg/kg) produced detectable levels of da and dc at 30 min (Fig. 6C). The levels reached a peak at 60 min and decreased from 90 to 180 min. Levels of da were at all times higher than those found for dc. The autoradiograms for the dose-response experiments are shown in Figure 7 (A and B). The results revealed that da and dc were both detectable 1 h after treatment of mice with 30 mg/kg of VC (Fig. 7C). Formation of da increased after treatment with 60 mg/kg of VC, decreased at the higher doses of 90 and 120 mg/kg, and was lowest at 120 mg/kg. The generation of dc was also evident 1 h after treatment with 30 mg/kg of VC, but the levels were considerably lower than those detected for da. The amounts increased at the 60 and 90 mg/kg doses, decreased when mice were treated with 120 mg/kg of VC, and reached levels similar to those for da at the same high dose. Levels of da were considerably higher than those found for dc at VC doses ranging from 30 to 90 mg/kg, but were similar at 120 mg/kg. The formation of DNA etheno adducts was determined in A/J, CD-1, and C57BL/6 mice to establish whether differences in generation of da and dc existed in these strains of mice under control conditions and after exposure to VC. Representative autoradiograms of lung DNA samples obtained after 32 P-postlabelling and 2D-TLC are depicted in Figure 8. The da and dc adducts were both detected in lung DNA obtained from control A/J, CD-1, and C57BL/6 mice (Figs. 8 and 9). Treatment with a single dose of VC (60 mg/kg) induced formation of both da and dcat1hinthelungs of all three strains of mice (Figs. 8 and 9). Significantly greater amounts of da were formed in lung DNA isolated from A/J and CD-1 mice than those isolated from C57BL/6 mice (Fig. 9). Levels of da formed in A/J and CD-1 mice were about 70% and 60% higher, respectively, than levels formed in C57BL/6 mice. The amounts of dc formed in A/J and CD-1 mice were both about 70% higher than levels formed in C57BL/6 mice. The quantities of the da adduct formed in all three strains of mice were considerably higher than those of the dc adduct formed. In summary, the da and dc adducts formed in A/J and CD-1 mice were significantly higher than those formed in C57BL/6 mice. However, the extents to which both DNA adducts were formed in the lungs of A/J and CD-1 mice were similar. DISCUSSION The magnitude of a toxic and/or carcinogenic lesion within a target tissue is associated, in part, with the amounts of FIG. 6. Time-dependent effects of VC on formation of DNA adducts in CD-1 mice. Identification of the DNA adducts was performed by the 32 P- postlabeling/tlc procedure and was determined between 30 to 180 min after treatment with VC (60 mg/kg, i.p.). Representative autoradiograms of PEI- TLC maps depict effects of VC on formation of the DNA adducts damp (1) and dcmp (2) at 30 (A) and 60 min (B) following VC administration. Data represent the mean SD of triplicate determinations from three to four lung DNA preparations (C).

7 88 TITIS AND FORKERT hydroxylase activity, whereas C57BL/6 mice had the lowest; CD-1 mice had intermediate amounts (Fig. 2). On the other hand, carboxylesterase activity was highest in C57BL/6 mice, intermediate in A/J mice, and lowest in CD-1 mice (Fig. 3). FIG. 7. Dose-response effects of VC treatment on formation of DNA adducts. Identification of the DNA adducts was determined by the 32 P-postlabeling/TLC procedure and was performed 1 h after treatment of CD-1 mice with VC. Representative autoradiograms of PEI-TLC maps depict the effects of 30 (A) and 60 mg/kg (B) of VC on formation of the DNA adducts damp (1) and dcmp (2). Data represent the mean SD of triplicate determinations from three to four lung DNA preparations (C). reactive intermediates formed, which is in turn linked to the final balance between activation and detoxication. Our previous in vitro studies have produced data consistent with involvement of lung CYP2E1 and carboxylesterase enzymes in activation and detoxication, respectively, of EC and VC in CD-1 mice (Forkert and Lee, 1997; Lee and Forkert, 1999). In the present in vivo study, we have determined and compared CYP2E1-mediated PNP hydroxylase and carboxylesterase activities in lung microsomes from control A/J, CD-1, and C57BL/6 mice. In this context, it should be noted that while PNP hydroxylation is regarded as an index of CYP2E1 catalytic activity (Koop, 1986), PNP is also a substrate for other P450 isozymes, including CYP2F2 (Shultz et al., 1999). Our results showed that A/J mice had the highest levels of PNP FIG. 8. Representative autoradiograms of PEI-TLC maps of lung DNA from A/J and C57BL/6 mice. Formation of the DNA adducts was measured at 1 h following treatment of mice with VC (60 mg/kg, i.p.). Aliquots of 10 g of 32 P-postlabeled DNA were spotted onto PEI-cellulose membranes and resolved by 2-D TLC. Individual spots corresponding to ethenodeoxynucleotide 5 -monophosphates were identified by comparison with autoradiograms from the purified standards. Standards: (A) damp (1) and dcmp (2); (B) control A/J mice; (C) VC-treated A/J mice; (D) control C57BL/6 mice; (E) VC-treated C57BL/6 mice. Other spots represent [ - 32 P]ATP and normal nucleotides present in the samples.

8 DNA ADDUCT FORMATION FROM VINYL CARBAMATE 89 FIG. 9. Formation of da (A) and dc (B) in lung DNA from control and VC-treated A/J, CD-1, and C57BL/6 mice. Levels of da and dc were determined at 1 h after treatment of mice with VC (60 mg/kg, i.p.). Control mice received only the vehicle. Data are expressed as mean SD from three to four different experiments for each strain of mice. *p 0.05 compared with levels in the respective control mice; **p 0.05 compared with levels in VC-treated A/J and CD-1 mice. Treatment of mice with VC significantly reduced PNP hydroxylase and carboxylesterase activities and protein contents (Figs. 2 and 3). Residual levels of activities of both enzymes were similar in all strains of mice treated with VC, suggesting that metabolism of VC differed between the strains and was dependent on enzyme levels expressed constitutively. In order to determine the magnitude of VC activation, we calculated the ratios of PNP hydroxylase to carboxylesterase activities in control and VC-treated mice. The ratios were significantly higher in control A/J and CD-1 mice than in control C57BL/6 mice, indicating that bioactivation capacities were potentially greater in A/J and CD-1 mice than in C57BL/6 mice. The ratios were significantly decreased in all three strains of mice treated with VC, with the ratio in C57BL/6 mice being the lowest (Fig. 4). These data suggested that activation of VC is likely to be greater in A/J and CD-1 mice than in C57BL/6 mice. This assertion is confirmed by data from the covalent binding studies, which demonstrated that amounts of binding of [ 14 C- carbonyl]-vc to lung proteins were significantly higher in A/J and CD-1 mice than in C57BL/6 mice (Table 1). These results indicated that greater bioactivation capacities are coincident with enhanced formation of VC metabolites. In this study, we have used the 32 P-postlabeling/TLC technique to investigate formation of da and dc adducts in lungs of untreated and VC-treated mice. Low background levels of da and dc were found in untreated mice, a finding also reported in previous studies, and may be related to the type of diet given to the mice (Fernando et al., 1996). Time-course studies in CD-1 mice revealed that formation of da and dc were detectable at 30 min after VC treatment, reached a peak at 60 min, and declined thereafter (Fig. 6C). However, the decreases for da occurred more rapidly and were more precipitous than those observed for dc. Dose-response experiments showed that formation of the major DNA adduct da was maximal at a dose of 60 mg/kg, and decreased rapidly after treatment with 90 and 120 mg/kg of VC (Fig. 7). On the other hand, levels of dc reached a plateau between 60 and 90 mg/kg, then declined after treatment with a higher dose of VC. These findings from the time-course and dose-response studies are in agreement, and suggested that, once oxidation of VC had achieved saturation, decreases in adduct formation seen at longer periods after VC treatment or at higher VC doses indicated lack of further metabolism. The underlying basis for the decreased adduct formation after saturation of VC metabolism is not clear. However, we speculated that repair processes may have been in place and were effective in reducing the amounts of adducts present in lung DNA. Other factors such as regenerative processes may also play a role. We have investigated potential differences in formation of da and dc in the lungs of A/J, CD-1, and C57BL/6 mice. Our results showed strain-related differences in the extents to which DNA adducts were formed (Figs. 8 and 9). As expected, the formation of da and dc was significantly higher in VC-treated mice than in the controls. The amounts of da and dc generated in A/J and CD-1 mice were significantly higher than those in C57BL/6 mice, although the quantities of da formed were substantially higher than dc in all strains of mice examined. Of significance in this context is the observation that formation of specific DNA adducts such as da and dc is associated with mutations in certain protooncogenes (Anderson and Reynolds, 1989; Basu et al., 1993; You et al., 1989). A question arises regarding what effects on protooncogenes accrue as a result of the differential formation of da and dc in the lungs of A/J, CD-1, and C57BL/6 mice, and this is an issue

9 90 TITIS AND FORKERT that remains to be investigated. The finding that CD-1 mice have a phenotype not unlike that of A/J mice in terms of bioactivation capacity and DNA adduct formation suggested that these mice might tentatively be classified as a susceptible strain. Taken together, our results demonstrated that VC produced higher levels of da and dc adducts in the susceptible A/J than in the resistant C57BL/6 mice, and this finding coincided with their potential capacities for bioactivation of VC. However, differences in CYP2E1 levels between the strains were not as profound as differences in DNA adduct formation, suggesting that other factors such as VC activation by other P450 enzymes may be involved. This assertion is supported by findings from previous studies that showed that covalent binding of VC to lung microsomal proteins was partially inhibited (50%) in microsomes preincubated with an inhibitory CYP2E1 monoclonal antibody (Lee and Forkert, 1999). Enzymatic systems responsible for the activation of chemicals and drugs in humans have shown wide interindividual variations in their levels of expression. These variations have been attributed, in part, to polymorphisms in genes encoding particular enzymes, leading to absence or altered activity levels. Enhanced gene expression associated with CYP2E1 polymorphisms is reported to be higher among the Japanese than among the Caucasian population (Kato et al., 1992). Furthermore, there was a positive correlation between CYP2E1 polymorphisms and lung cancer in a Japanese cohort (Uematsu et al., 1991). Overexpression of CYP2E1 through a different allele may produce elevated enzyme levels, resulting in increased bioactivation capacities for CYP2E1-selective substrates. Consequently, individuals with unfavorable genotypes with enhanced activation and reduced detoxication of xenobiotics might be at higher risk than with those with a more favorable combination (Rannug et al., 1995). Our findings therefore suggested that the susceptible A/J and resistant C57BL/6 mice are relevant models for investigating differences in response to chemical carcinogens in human populations. Taken together, our results supported the view that differences in bioactivation capacities are related, in part, to tumorigenic risk associated with exposure to VC and other potential carcinogens. ACKNOWLEDGMENTS We thank Tina Inalsingh for providing excellent technical assistance in the DNA adduct studies. We also thank Toufan Parman from the laboratory of Dr. Peter G. Wells, Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada, for valuable advice during the initial stages of this work. This research was supported by Grant from the National Cancer Institute of Canada (P.G.F.). REFERENCES Anderson, M. W., and Reynolds, S. H. (1989). Activation of oncogenes. In The Pathobiology of Neoplasia (A. Sirica, Ed.), pp Plenum Press, New York,. Barbin, A., and Bartsch, H. (1986). 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