Laboratory of Pharmacology and Chemistry, National Toxicology Program, National Institute of Environmental Health Sciences, MD C3-02, P.O. Box 12233, Research Triangle Park, North Carolina 277092233
Received December 28, 1999; accepted January 18, 2001
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ABSTRACT |
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Key Words: transgenic mouse; Tg.AC mouse; p53+/mouse; metabolism; cytochrome P450; GST-; ethoxyquin; benzene; methacrylonitrile.
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INTRODUCTION |
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Stewart et al. (1984) first reported induction of tumors in transgenic animals, leading to the development of the Tg.AC transgenic mouse line derived from the FVB/N wild type (Leder et al., 1990). Tg.AC mice contain a v-Ha-ras oncogene fused to the promoter region of the zeta-globin gene. Activation of the Ha-ras gene has been correlated with the initiation step of two-stage epidermal tumorigenesis (Brown et al., 1986
); therefore, Tg.AC mice appear to be sensitive to carcinogenic activity of nongenotoxic compounds as well as certain genotoxic carcinogens (Tennant et al., 1995
). The p53+/ mouse line, arising from the C57BL/6 wild type, has one disrupted allele for the p53 tumor suppressor gene (Donehower et al., 1992
). Genotoxic chemicals may subsequently alter the remaining functional allele leading to tumor formation.
Chemical carcinogenesis is frequently dependent on metabolism of xenobiotics through reactive intermediates. Therefore, for purposes of carcinogenicity testing, it is critical to establish that the xenobiotic metabolizing enzyme systems of potential transgenic animal models are comparable to those of their parent strains. It is generally assumed that the genetic manipulation used to create Tg.AC and p53+/ mice will not interfere with xenobiotic metabolizing enzyme systems, but to our knowledge this assumption has not been tested and reported in the literature. In the present study, the well-characterized metabolism of 3 xenobiotics, benzene (Mathews et al., 1998), ethoxyquin (Burka et al., 1996
), and methacrylonitrile (Ghanayem et al., 1994
) are compared in the Tg.AC and p53+/ transgenic mouse lines with their respective wild types, the FVB/N and C57BL/6 mouse strains. The metabolic pathways that are addressed include arene oxide formation, aromatic ring opening, hydroxylation, epoxidation, and O-deethylation, plus major phase II biotransformation reactions.
Cytochrome P450 (CYP) enzymes from the CYP1, CYP2, and CYP3 families play important roles in activation of procarcinogens administered to mammalian species (Ioannides, 1996). Therefore, it is extremely critical that these enzymes have normal expression and function in the proposed Tg.AC and p53+/ mouse carcinogen testing models. Consequently, representative isoforms (CYP1A2, CYP2E1, and CYP3A) from these 3 CYP families have been chosen for comparison of protein expression levels in each of the 4 test mouse genotypes. CYP2E1 is the apparent major oxidase in the metabolism of two of the present test substrates: benzene (Valentine et al., 1996
), a known human carcinogen, and methacrylonitrile (Ghanayem et al., 1999
), structurally related to the rodent carcinogen acrylonitrile. The specific enzyme involved in the monooxygenase-mediated O-deethylation of the third test substrate, ethoxyquin, is presently unknown. However, both CYP1A2 and CYP2E1 have been shown to function as O-deethylases in the metabolism of compounds that are structurally similar to ethoxyquin. CYP1A2 includes among its substrates the aromatic amines, e.g., the metabolically activated carcinogen 2-acetylaminofluorene. Hence, CYP1A2 was chosen to represent the CYP1 family because of its importance in procarcinogen activation and its potential for metabolizing ethoxyquin. Finally, expression of CYP3A protein was determined in the 4 mouse genotypes. Although the involvement of CYP3A in the metabolism of the 3 test substrates is equivocal, this subfamily plays an important role among CYP3 enzymes in the metabolism of endogenous steroid hormones and activation of xenobiotics such as aflatoxin to carcinogens.
A survey of the expression of representative metabolic enzymes in Tg.AC and p53+/ mice would not be complete without considering levels of phase II type biotransformation enzymes in these genetically altered animals. One such enzyme, glutathione S-transferase (GST), plays an important role in the detoxification and elimination of many compounds, including methacrylonitrile (Ghanayem et al., 1994). Furthermore, GST is quite active in ethoxyquin metabolism (Burka et al., 1996
) and, to a lesser extent, benzene metabolism (Mathews et al., 1998
). It is uncertain which classes (or class) of GSTs are prominent in catalyzing glutathione conjugation to benzene, ethoxyquin, or methacrylonitrile in the mouse; therefore, GST-
was arbitrarily chosen for investigation of GST protein expression.
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MATERIALS AND METHODS |
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Animal experimental protocol.
Benzene-, ethoxyquin-, or methacrylonitrile-treated mice were placed individually in glass metabolism cages (Wyse Glass Specialties, Inc., Freeland MI) to determine the rates and routes of radioactivity excreted in urine, feces, and in the case of benzene and methacrylonitrile, expired air. All mice received NIH-31 rodent diet and water for ad libitum consumption. The amount of radiolabel eliminated in expired air was determined by pulling air through the cage by means of a vacuum system using a flow rate of 0.40.6 l/min. Air exiting the cage was passed through a trap containing 200400 ml of ethanol for collection of volatile 14C, then through a trap containing a mixture of ethylene glycol monomethyl ether and ethanolamine (7:3, v/v) for collection of expired 14CO2. Traps were changed at time points of 12, 24, and 30 h following benzene administration and at time points of 4, 8, 12, and 24 h following methacrylonitrile administration. Collection of expired air was halted when sampled traps contained little or no radiolabeled material. The trapping efficiency of the solutions was maintained over the longer time points by treating the intake air with calcium sulfate and soda lime to reduce moisture and CO2 content. Urine and feces were collected from the cages at 24, 48, and 72 h postdosing. All mice were euthanized by CO2 asphyxiation 72 h following dose administration. Urine and feces were analyzed for radioactivity content as follows: Feces were air-dried, weighed, and ground to a fine powder using a mortar and pestle. Triplicate feces samples (2550 mg each) were combusted in a Packard Instruments Co. (Meriden, CT) model 306 biological tissue oxidizer and counted in either a Beckman Instruments Inc. (Fullerton, CA) model 5801 or 9800 liquid scintillation counter. Triplicate 20- to 50-µl aliquots of urine, as well as triplicate 1-ml aliquots of the organic volatiles and CO2 traps, were added to Ecolume (ICN, Research Products Division, Costa Mesa, CA) and counted directly in the scintillation counter.
HPLC analysis of radioactivity in urine.
Urine samples (up to 50 µl) from all treatment groups were injected onto a Waters Corp. (Milford, MA) HPLC system equipped with a Waters model 481 UV detector and a Radiomatic model A-280 radiochemical detector (Packard Instruments Co., Meriden, CT). All urine samples were prepared for injection by centrifugation for 5 min in an Eppendorf model 5412 microfuge (Brinkman Instruments, Westbury, NY). A 10-µm Waters µBondapak C18 column was used to analyze urine from benzene- and ethoxyquin-treated mice. A linear gradient of 90% 35 mM tetrabutylammonium hydrogen sulfate to 90% methanol in 35 min was used for resolving benzene-derived urinary metabolites. Urine from ethoxyquin-treated mice was analyzed using a linear gradient of 100% 0.01 M sodium phosphate buffer (pH 6) to 80% acetonitrile in 30 min. Methacrylonitrile-derived urinary metabolites were resolved on a Rainin Dynamax C18 column (Varian Chromatography Systems, Walnut Creek, CA) using a linear gradient of 100% 0.01 M sodium phosphate buffer (pH 6) to 25% acetonitrile in 15 min. All analyses were performed at a flow rate of 1.5 ml/min. Metabolites were identified on either the basis of comparative retention times with authentic standards or the comparison of retention times and peak profiles from co-chromatography of radiolabeled urine and purified radiolabeled metabolites obtained from previous studies of benzene, ethoxyquin, and methacrylonitrile metabolism.
Expression of representative xenobiotic metabolizing enzymes in liver.
Microsome preparation (at 4°C; pH of all buffer solutions = 7.4): livers were removed from euthanized naive mice from the 4 genotype populations (n = 56/group), homogenized individually in 50 mM Tris-HCl buffer containing 1.15% KCl, 1 mM EDTA, and 0.5 mM phenylmethyl sulfonyl fluoride, then fractionated by centrifugation at 10,000 x g for 30 min and 105,000 x g for 70 min. Aliquots of the cytosolic fraction were stored at 80°C, then microsomal pellets were suspended in a wash buffer of 100 mM Na pyrophosphate containing 0.1 mM EDTA and recentrifuged at 105,000 x g for 70 min. Microsomal protein was stored at 80°C following resuspension in 10 mM KPO4 buffer containing 0.25 M sucrose. The total protein concentration in microsomal and cytosolic fractions was determined using the Bio-Rad (Hercules, CA) protein assay based on the Bradford dye-binding procedure (Bradford, 1976).
For the Western blot analysis, mouse liver microsomal and cytosolic proteins were electrophoretically separated on NuPage 412% Bis-Tris gels (Invitrogen, Carlsbad, CA), and transferred to 0.45 µm pore size nitrocellulose membranes according to the manufacturer's instructions. Microsomal proteins were loaded (per well) at either 15 or 25 µg, cytosolic protein was loaded at 50 µg, and CYP-induced positive control rat microsomal proteins and purified GST- (isozyme 1-1) were loaded at 15 µg. Relative expression levels of CYP proteins were determined using ECL Western Blotting Kits purchased from Amersham Pharmacia Biotech (Piscataway, NJ). These kits contained both the antirat CYP1A2, CYP2E1, and CYP3A antibodies and the immunodetection system. Purified GST-
and antirat GST-
antibody used for determining GST expression in liver cytosol were purchased from Oxford Biomedical Research, Inc. (Oxford, MI). Each antirat antibody was cross-reactive with the respective mouse protein. Antibody incubations and immunodetection procedures were carried out according to manufacturer's instructions. Optical densities of protein bands were determined and compared using NIH Image V.1.62 software (National Institutes of Health, Bethesda, MD).
Statistical analysis.
Statistical analysis was performed using either one-way ANOVA or the Tukey-Kramer HSD test for pairwise comparisons (JMP Statistical Software, SAS Institute Inc., Cary, NC). Values were considered significantly different at p < 0.05.
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RESULTS |
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DISCUSSION |
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Neither qualitative nor quantitative differences were observed between the wild types and their genetically altered lines for any major metabolic markers in urine for any of the 3 test compounds. However, significant quantitative differences in marker metabolites were observed between wild-type strains for all 3 xenobiotics. This fact increases the confidence that our analytical system would have detected major differences between the wild-type strains and their corresponding transgenic lines. A few minor nonmarker urinary metabolites were qualitatively different between transgenic lines and wild-type strains. However, in all cases, the concentrations of these minor metabolites were near background levels and found in only a few individual mice. These metabolites may, in fact, exist in all animals of the relevant treatment groups, but are below the HPLC analytical system's limits of detection for some animals.
It is apparent from the results of the present study that metabolism of 3 orally administered test compounds, benzene, ethoxyquin, and methacrylonitrile, was not affected by the altered genomes of either Tg.AC or p53+/ mice. Benzene undergoes the most diverse metabolism of the three, with CYP2E1 apparently responsible for the formation of the initial epoxide metabolite (benzene oxide) and the subsequent formation of a hydroquinone from phenol (Valentine et al., 1996). The oxidase responsible for the O-deethylation of ethoxyquin is presently unknown. Either CYP2E1 or CYP1A2 could be involved; both O-deethylate compounds structurally similar to ethoxyquin (Ioannides, 1996
). CYP2E1 is purported to be the major oxidase in methacrylonitrile metabolism (Ghanayem et al., 1999
). However, other monoxygenases can metabolize methacrylonitrile, as well as benzene, as demonstrated in CYP2E1-/- mice (Valentine et al., 1996
; Ghanayem et al., 1999
). Further, CYP2B1 has been shown to metabolize benzene in rat liver microsomes (Snyder et al., 1993
).
Only CYP2E1 of the CYPs surveyed for protein expression in this work has been definitively linked with major monooxygenase activity toward any of the 3 test compounds. However, this fact neither diminishes the importance of CYPs other than CYP2E1 in xenobiotic metabolism nor diminishes the importance of normal function of those enzymes in animals that may be used to evaluate a broad range of potential procarcinogens. Results of the present investigation should be reassuring, in that representative enzymes of all 3 prominent CYP xenobiotic metabolizing families appear to have comparable expression in Tg.AC and p53+/ genetically altered mice to that of the corresponding parental mouse strain.
Other oxidative enzymes, such as epoxide hydrolase and aldehyde dehydrogenase, appear to retain normal function and activity in Tg.AC and p53+/ mice, as inferred by the quantitative and qualitative similarities of specific metabolite markers in urine of benzene-treated mice. Additionally, the presence and similarities of glucuronide and sulfate conjugates in urine of ethoxyquin- and benzene-treated mice indicate normal expression and function of glucuronosyl- and sulfotransferases in Tg.AC and p53+/ mice.
GST was chosen as the representative phase II enzyme for determination of protein expression in the 4 mouse genotypes because of its importance in biotransformation of many xenobiotics, including all 3 test substrates investigated in the present work. Although not represented in the abbreviated metabolic scheme in Figure 3 (constructed for identification of the origins of the specific metabolite markers only), GST is quite active in ethoxyquin metabolism, as evidenced by the presence of large amounts of GSH conjugates in bile of ethoxyquin-treated rats (Burka et al., 1996
). No GSH-conjugation products have been characterized in urine of ethoxyquin-treated rats or mice; therefore, this material is either excreted in feces and/or undergoes degradation, enterohepatic circulation, and urinary excretion as non-GSH-derived conjugates. GST is also active in benzene metabolism. Metabolites arising from GSH conjugation are excreted in urine of benzene-treated mice; however, they are minor and are not included as metabolite markers in the present study. Normal GST function and activity was inferred by the qualitative and quantitative similarities in the mercapturic acid metabolite present in the urine of methacrylonitrile-treated mice. Further, GST protein expression was found to be comparable in naive genetically altered mice and corresponding wild type.
The functional allele of p53+/ mice may be mutated by genotoxic compounds, resulting in tumor formation (Donehower et al., 1992). Results of the present study provide direct evidence that protein expression and function of some key hepatic metabolizing enzymes important in activation of procarcinogens remain normal in these animals. The same apparently holds true for Tg.AC mice; the insertion of v-Ha-ras into the genome does not appear to affect expression of hepatic enzymes associated with xenobiotic metabolism. Chemically induced expression of the v-Ha-ras gene in skin may result in higher than normal incidence of skin papillomas and carcinomas following dermal exposure to various xenobiotics (Eastin et al., 1998
). However, v-Ha-ras initiated tumorigenesis in other injured tissues may be impeded by lack of v-Ha-ras expression in those tissues. For instance, Delker, et al. (1999) reports no expression of this transgene in liver of Tg.AC mice following oral administration of chloroform. However, cyclosporin A administered by gavage did increase the incidence of skin papillomas, lymphomas, and forestomach tumors in Tg.AC mice (Eastin et al., 1998
), indicating that this transgenic mouse model may be appropriate for compounds administered by routes other than dermal exposure. It is possible that first pass metabolic activation of carcinogens in the liver could result in tumorigenesis at sites other than liver, such as epidermal tissue. Therefore, the present work in which test compounds were administered orally should be considered relevant to the validation of the use of Tg.AC mouse as a model for carcinogenicity testing.
Skin, the major target organ in Tg.AC mice, contains enzymes from all 3 important xenobiotic-metabolizing CYP families, including CYP1A1, CYP2E1, and CYP3A (Jugert et al., 1994), all potentially involved in metabolic activation of compounds administered dermally. Recent evidence in one transgenic mouse line expressing v-Ha-ras in skin indicated that CYP1A1 induction by TCDD (as measured by 7-ethoxyresorufin O-deethylase activity in epidermal microsomes) was significantly lower than CYP1A1 induction in littermates not expressing v-Ha-ras in skin (Reiners et al., 1997
). It is uncertain if v-Ha-ras expression has an effect on Ah receptor mediated CYP1A1 induction in epidermal tissue of Tg.AC mice. The current work, investigating metabolism of test substrates following single-dose administration, was not designed to investigate differential enzyme induction in these animals.
In conclusion, xenobiotic metabolism of 3 orally administered, structurally different test substrates does not appear to be appreciably affected by the altered genomes in either the Tg.AC or p53+/ transgenic mouse lines. Further, the constitutive expression of several representative biotransformation enzymes in liver of these transgenic mouse lines does not differ from their parental mouse strains. This suggests that metabolism of most, if not all, procarcinogens administered to the p53+/ mouse would be comparable to that in the parental C57BL/6 mouse. The same apparently holds true for the Tg.AC mouse for orally administered compounds undergoing metabolism in the liver. However, further studies may be needed to characterize potential effects of v-Ha-ras expression on Ah receptormediated induction of metabolizing enzymes in skin of dermally treated Tg.AC mice.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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