Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6
Received July 28, 2000; accepted September 27, 2000
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ABSTRACT |
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Key Words: carboxylesterase; CYP2E1; DNA adducts; epoxide; lung tumors; p-nitrophenol hydroxylase; vinyl carbamate.
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INTRODUCTION |
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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 [14C-ethyl]-EC and [14C-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.
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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.
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MATERIALS AND METHODS |
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Treatment of animals.
Female A/J mice of 2025 g body weight were purchased from Jackson Laboratories (Bar Harbor, MA). Female C57BL/6 and CD-1 mice, also of 2025 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 [14C-carbonyl]-VC to lung proteins, CD-1 mice were treated with VC (20 µCi/kg [14C-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 µCi/kg [14C-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 K2HPO4 buffer, and aliquots (100 µl) 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
).
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 µM). The membrane was then incubated overnight at room temperature with one of the following antibodies: anti-CYP2E1, anti-hydrolase A, or anti-hydrolase B2. 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 [14C-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 µl) of the dialyzed samples were solubilized overnight with 1 M NaOH (1 ml). After addition of glacial acetic acid (300 µl) 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, N6-ethenodeoxyadenosine 3'-monophosphate (dAMP) and 3, N4-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 µl) of the synthesized adducts were then purified using a polymeric reverse-phase column (25 x 0.46 cm, 5 µm 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 KitTM. 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 µg 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 µg) 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 µl. 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 (25 x 0.46 cm, 5 µm 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.
32P-postlabeling/thin layer chromatography.
32P-postlabeling was carried out as described previously (Nair et al., 1995), with modifications. Fractions of the adducts were pooled, lyophilized, and radiolabeled with 25 µCi [
32P]-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 [
32P]-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 [
32P]-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 [
32P]-ATP for the specified adduct. Final values were expressed as number of adducts per 108 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.
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RESULTS |
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Effects of VC on Carboxylesterase Enzymes
The effects of VC on carboxylesterase enzymes are summarized in Figure 3. Microsomal carboxylesterase activity, as 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.
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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.
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DISCUSSION |
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In this study, we have used the 32P-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 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.
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ACKNOWLEDGMENTS |
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NOTES |
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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.
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REFERENCES |
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Barbin, A., and Bartsch, H. (1986). Mutagenic and promutagenic properties of DNA adducts formed by vinyl chloride metabolites. In The Role of Cyclic Nucleic Acid Adducts in Carcinogenesis and Mutagenesis (B. Singer and H. Bartsch, Eds.), pp. 345358, IARC Scientific Publications No. 70, International Agency for Research on Cancer, Lyon.
Basu, A. K., Wood, M. L., Neidernhofer, L. J., Ramos, L. A., and Essigmann, J. M. (1993). Mutagenic and genotoxic effects of three vinyl chloride-induced DNA lesions: 1,N6-ethenoadenine, 3,N4-ethenocytosine and 4-amino-5-(imidazol-2-yl)imidazole. Biochemistry 32, 1279312801.[ISI][Medline]
Bochner, B. R., and Ames, B. N. (1982). Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography. J. Biol. Chem. 237, 97599769.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72, 248254.[ISI][Medline]
Dahl, G. A., Miller, J. A., and Miller, E. C. (1978). Vinyl carbamate as a promutagen and a more carcinogenic analog of ethyl carbamate. Cancer Res. 38, 37933804.[Abstract]
Dahl, G. A., Miller, E. C., and Miller, J. A. (1980). Comparative carcinogenicities and mutagenicities of vinyl carbamate, ethyl carbamate, and ethyl N-hydroxycarbamate. Cancer Res. 40, 11941203.[ISI][Medline]
Fernando, R. C., Nair, J., Barbin, A., Miller, J. A., and Bartsch, H. (1996). Detection of 1,N6- ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine by immunoaffinity32P-post-labelling in liver and lung DNA of mice treated with ethyl carbamate (urethane) or its metabolites. Carcinogenesis 17, 17111718.[Abstract]
Forkert, P. G. (1995). CYP2E1 is preferentially expressed in Clara cells of murine lung: Localization by in situ hybridization and immunohistochemical methods. Am. J. Resp. Cell. Mol. Biol. 12, 589596.[Abstract]
Forkert, P. G., and Lee, R. P. (1997). Metabolism of ethyl carbamate by pulmonary cytochrome P-450 and carboxylesterase isozymes: Involvement of CYP2E1 and hydrolase A. Toxicol. Appl. Pharmacol. 146, 245254.[ISI][Medline]
Forkert, P. G., Stringer, V., and Troughton, K. M. (1986). Pulmonary toxicity of , 1-dichloroethylene: Correlation of early changes with covalent binding. Can. J. Physiol. Pharmacol. 164, 112121.
Guengerich, F. P., Kim, D.-H., and Iwasaki, M. (1991). Role of human cytochrome P-450 IIE1 in the oxidation of many low molecular weight cancer suspects. Chem. Res. Toxicol. 4, 168179.[ISI][Medline]
Guengerich, F. P., and Kim, D. H. (1991). Enzymatic oxidation of ethyl carbamate and its role as an intermediate in the formation of 1,N6-ethenoadenosine. Chem. Res. Toxicol. 4, 413421.[ISI][Medline]
Guichard, Y., Nair, J., Barbin, A., and Bartsch, H. (1993). Immunoaffinity clean-up combined with 32P-postlabelling analysis of 1, N6-ethenoadenine and 3, N4-ethenocytosine in DNA. In Postlabelling Methods for Detection of DNA Adducts (D. H. Phillips, M. Castegnaro, and H. Bartsch, Eds.), pp. 263269. IARC Scientific Publications No. 124, International Agency for Research on Cancer, Lyon.
Gupta, R. C. (1985). Enhanced sensitivity of 32P-postlabelling analysis of aromatic carcinogen: DNA adducts. Cancer Res. 45, 56565662.[Abstract]
Hughes, N. C., and Phillips, D. H. (1990). Covalent binding of dibenzpyrenes and benzo[a]pyrene to DNA: Evidence for synergistic and inhibitory interactions when applied in combination to mouse skin. Carcinogenesis 11, 16111619.[Abstract]
Kato, S., Shields, P. G., Caporaso, N. E., Hoover, R. N., Trump, B. F., Sugimura H., Weston, A., and Harris, C. C. (1992). Cytochrome P450IIE1 genetic polymorphisms, racial variation, and lung cancer risk. Cancer Res. 52, 67126715.[Abstract]
Koop, D. R. (1986). Hydroxylation of p-nitrophenol by rabbit ethanol-inducible cytochrome P-450 isozyme 3a. Mol. Pharmacol. 29, 399404.[Abstract]
Lee, R. P., and Forkert, P. G. (1999). Inactivation of cytochrome P-450 (CYP2E1) and carboxylesterase (hydrolase A) enzymes by vinyl carbamate in murine pulmonary microsomes. Drug. Metab. Dispos. 27, 233239.
Leithauser, M. T., Liem, A., Stewart, B. C., Miller, E. C., and Miller, J. A. (1990). 1, N6,-ethenoadenosine formation, mutagenicity and murine tumor induction as indicators of the generation of an electrophilic epoxide metabolite of the closely related carcinogens ethyl carbamate (urethane) and vinyl carbamate. Carcinogenesis 11, 463473.[Abstract]
Malkinson, A. M. (1991). Genetic studies on lung tumor susceptibility and histogenesis in mice. Environ. Health Perspect. 93, 149159.[ISI][Medline]
Miller, J. A., and Miller, E. C. (1983). The metabolic activation and nucleic acid adducts of naturally occurring carcinogens: Recent results with ethyl carbamate and the spice flavors safrole and estragole. Brit. J. Cancer 48, 115.[ISI][Medline]
Mirvish, S. S. (1968). The carcinogenic action and metabolism of urethan and N-hydroxyurethan. Adv. Cancer Res. 11, 142.[Medline]
Morgan, E. W., Yan, B., Greenway, D., Petersen, D. R., and Parkinson, A. (1994). Purification and characterization of two rat liver microsomal carboxylesterases (hydrolase A and B). Arch. Biochem. Biophys. 315, 495512.[ISI][Medline]
Nair, J., Barbin, A., Guichard, Y., and Bartsch, H. (1995). 1, N6-ethenodeoxyadenosine and 3, N4 -ethenodeoxycytidine in liver DNA from humans and untreated rodents detected by immunoaffinity 32P-postlabeling. Carcinogenesis 16, 613617.[Abstract]
Park, K. K., Liem, A., Stewart, B. C., and Miller, J. A. (1993). Vinyl carbamate epoxide, a major strong electrophilic, mutagenic and carcinogenic metabolite of vinyl carbamate and ethyl carbamate (urethane). Carcinogenesis 14, 441450.[Abstract]
Park, K.-K., Surh, Y.-J., Stewart, B. C., and Miller J. A. (1990). Synthesis and properties of vinyl carbamate epoxide, a possible ultimate electrophilic and carcinogenic metabolites of vinyl carbamate and ethyl carbamate. Biochem. Biophys. Res. Commun. 169, 10941098.[ISI][Medline]
Rannug, A., Alexandrie, A. K., Persson, I., and Ingelman-Sundberg, M. (1995). Genetic polymorphism of cytochrome P450 1A1, 2D6 and 2E1: Regulation and toxicological significance. J. Occup. Environ. Med. 37, 2536.[ISI][Medline]
Ribovich, M. L., Miller, J. A., Miller, E. C., and Timmins, L. G. (1982). Labeled 1,N6-ethenoadenosine and 3,N4-ethenocytidine in hepatic RNA of mice given [ethyl-1,23H] or [ethyl-114C]ethyl carbamate (urethan). Carcinogenesis 3, 539546.[ISI][Medline]
Scherer, E., Winterwerp, H., and Emmelot, P. (1986). Modification of DNA and metabolism of ethyl carbamate in vivo: Formation of 7-(2-oxoethyl)guanine and its sensitive determination by reductive tritiation using 3H sodium borohydride. In The Role of Cyclic Nucleic Acid Adducts in Carcinogenesis and Mutagenesis (B. Singer and H. Bartsch, Eds.), pp. 109125. IARC Scientific Publications No. 70, International Agency for Research on Cancer, Lyon.
Shimkin, M. B., and Stoner, G. D. (1975). Lung tumors in mice: Application to carcinogenesis bioassay. Adv. Cancer Res. 21, 158.[Medline]
Shultz, M. A., Choudary, P. V., and Buckpitt, A. R. (1999). Role of murine cytochrome P-450 2F2 in metabolic activation of naphthalene and metabolism of other xenobiotics. J. Pharmacol. Exp. Ther. 290, 281288.
Uematsu, F., Kikuchi, H., Motomiya, M., Abe, T., Sagami, I., Ohmachi, T., Wakui, A., Kanamaru, R., and Wantanabe, M. (1991). Association between restriction fragment length polymorphism of the human cytochrome P450 IIE1 gene and susceptibility to lung cancer. Jpn. J. Cancer Res.87, 254256.
Watson, W. P., and Crane, A. E. (1989). HPLC-32P-postlabelling analysis of 1, N6-ethenodeoxyadenosine and 3, N4-ethenodeoxycytidine. Carcinogenesis 4, 7577.
You, M., Candrian, U., Maronpot, R., Stoner, G., and Anderson, M. (1989). Activation of the K-ras protooncogene in spontaneously occurring and chemically induced lung tumors of the strain A mouse. Proc. Natl. Acad. Sci. U. S. A. 86, 30703074.[Abstract]