Comparative Xenobiotic Metabolism between Tg.AC and p53+/– Genetically Altered Mice and Their Respective Wild Types

J. M. Sanders,1, L. T. Burka, B. Chanas and H. B. Matthews

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 27709–2233

Received December 28, 1999; accepted January 18, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The use of transgenic animals, such as v-Ha-ras activated (Tg.AC) and p53+/– mice, offers great promise for a rapid and more sensitive assay for chemical carcinogenicity. Some carcinogens are metabolically activated; therefore, it is critical that the altered genome of either of these model systems does not compromise their capability and capacity for metabolism of xenobiotics. The present work tests the generally held assumption that xenobiotic metabolism in the Tg.AC and p53+/– mouse is not inherently different from that of the respective wild type, the FVB/N and C57BL/6 mouse, by comparing each genotype's ability to metabolize benzene, ethoxyquin, or methacrylonitrile. Use of these representative substrates offers the opportunity to examine arene oxide formation, aromatic ring opening, hydroxylation, epoxidation, O-deethylation, and a number of conjugation reactions. Mice were treated by gavage with 14C-labeled parent compound, excreta were collected, and elimination routes and rates, as well as 14C-derived metabolite profiles in urine, were compared between relevant treatment groups. Results of this study indicated that metabolism of the 3 parent compounds was not appreciably altered between either FVB/N and Tg.AC mice or C57BL/6 and p53+/– mice. Further, expression of CYP1A2, CYP2E1, CYP3A, and GST-{alpha} in liver of naive genetically altered mice was similar to that of corresponding wild-type mice. Thus, these results suggest that the inherent ability of Tg.AC and p53+/– mice to metabolize xenobiotics is not compromised by their altered genomes and would not be a factor in data interpretation of toxicity studies using either transgenic mouse line.

Key Words: transgenic mouse; Tg.AC mouse; p53+/mouse; metabolism; cytochrome P450; GST-{alpha}; ethoxyquin; benzene; methacrylonitrile.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rodent bioassays conducted by the National Toxicology Program (NTP) are considered to be the best available method for identification of putative human carcinogenic agents (Tennant et al., 1995Go). However, cost and duration greatly limit the number of bioassays that are completed on a yearly basis. Many untested chemicals are used extensively in commercial operations or exist naturally in the environment. Therefore, improving the efficiency and accuracy of the current test methods is a major priority of the NTP. One tool possessing great promise for assessing chemical carcinogenicity is the use of genetically altered animals such as the Tg.AC and p53+/– mouse lines. Potential advantages offered by the use of transgenic animals to prescreen for suspected carcinogens and/or augment current testing techniques may include reduced time to tumor formation, fewer animals per study, and the collection of elucidative data at very low exposure levels of test compounds.

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., 1990Go). 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., 1986Go); therefore, Tg.AC mice appear to be sensitive to carcinogenic activity of nongenotoxic compounds as well as certain genotoxic carcinogens (Tennant et al., 1995Go). The p53+/– mouse line, arising from the C57BL/6 wild type, has one disrupted allele for the p53 tumor suppressor gene (Donehower et al., 1992Go). 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., 1998Go), ethoxyquin (Burka et al., 1996Go), and methacrylonitrile (Ghanayem et al., 1994Go) 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, 1996Go). 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., 1996Go), a known human carcinogen, and methacrylonitrile (Ghanayem et al., 1999Go), 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., 1994Go). Furthermore, GST is quite active in ethoxyquin metabolism (Burka et al., 1996Go) and, to a lesser extent, benzene metabolism (Mathews et al., 1998Go). 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-{alpha} was arbitrarily chosen for investigation of GST protein expression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals, substrates, and dose.
Male hemizygous Tg.AC and p53+/–mice and their respective wild types, FVB/N and C57BL/6 mice, were purchased from Taconic (Germantown, NY). At time of dosing, mice ranged in age from 2 to 3.5 months and weighed from 23 to 34 g. [U-14C] benzene, with a radiochemical purity of >= 97%, was procured from Chemsyn Science Laboratories (Lenexa, KS). [3-14C] ethoxyquin, approximately 96% pure, and [2-14C] methacrylonitrile, approximately 98% pure, were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Dosing solutions were made by diluting radiolabeled compound with unlabeled compound in vehicle to administer 37–57 µCi/kg. Unlabeled benzene, obtained from J. T. Baker (Phillipsburg, PA), and methacrylonitrile, obtained from Aldrich Chemical Co. (Milwaukee, WI), had chemical purities of 99% or better. The purity of unlabeled ethoxyquin (approximately 75%), obtained from Sigma Chemical Co. (St. Louis, MO), was enhanced by vacuum distillation prior to use. All chemicals were administered at doses previously shown to be in the linear range for metabolism and excretion. Benzene was administered at 0.1 mg/kg in corn oil; ethoxyquin was administered at 25 mg/kg in a 1:1:8 ratio (v/v/v) of ethanol, Emulphor EL-620 (GAF Corp., New York, NY), and water; and methacrylonitrile was administered in water at a concentration of 1.5 mg/kg. All 3 compounds were administered at dose volumes of 5 ml/kg by gavage.

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 0.4–0.6 l/min. Air exiting the cage was passed through a trap containing 200–400 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 (25–50 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 = 5–6/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, 1976Go).

For the Western blot analysis, mouse liver microsomal and cytosolic proteins were electrophoretically separated on NuPage 4–12% 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-{alpha} (isozyme 1-1) were loaded at 1–5 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-{alpha} and antirat GST-{alpha} 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cumulative excretion of benzene-, ethoxyquin-, and methacrylonitrile-derived radioactivity in urine, feces, and as 14CO2 following dose administration was compared between two mouse strains, FVB/N and C57BL/6 mice, and their respective transgenic lines, Tg.AC and p53+/– mice (Figs. 1 and 2GoGo). The percent total dose recovered in excreta was similar (not shown) between mouse strains for each test compound, therefore, direct comparisons of the data were made. Results indicated (with the minor exception of exhaled 14CO2 following administration of methacrylonitrile) few or no significant differences between treatment groups for either the route or amount of radioactivity excreted following administration of any of the 3 chemicals (Figs. 1 and 2GoGo). Both wild types exhaled slightly more methacrylonitrile-derived CO2 than their corresponding transgenic mouse lines. Further, C57BL/6 mice exhaled slightly more 14CO2 than did FVB/N mice following methacrylonitrile administration.



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FIG. 1. Cumulative excretion of radiolabeled dose in urine, feces, and as CO2 following administration of (A) benzene, (B) ethoxyquin, and (C) methacrylonitrile to either FVB/N wild-type (open bars) or Tg.AC (filled bars) mice by gavage. Each value represents the mean ± SD of 5–6 mice. *Significant difference between wild-type strains. #Significantly different from wild type.

 


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FIG. 2. Cumulative excretion of radiolabeled dose in urine, feces, and as CO2 following administration of (A) benzene, (B) ethoxyquin, and (C) methacrylonitrile to either C57BL/6 wild-type (open bars) or p53+/– (filled bars) mice by gavage. Each value represents the mean ± SD of 4–6 mice. *Significant difference between wild-type strains. #Significantly different from wild type.

 
Comparisons were also made between the amounts of selected benzene-, ethoxyquin-, and methacrylonitrile-derived metabolites excreted in urine of each treatment group. Table 1Go lists well-characterized pathways for benzene, ethoxyquin, and methacrylonitrile metabolism in rodent models (Burka et al., 1996Go; Ghanayem et al., 1994Go; Mathews et al., 1998Go). These pathways are inferred by the existence of specific metabolites excreted in urine following administration of the corresponding parent compounds. Abbreviated metabolic schemes containing the metabolites used as markers for the pathways are displayed in Figure 3Go.


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TABLE 1 Metabolic Pathways Leading to Major Metabolites Excreted in Urine of Mice Treated with Either Benzene, Ethoxyquin, or Methacrylonitrile
 


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FIG. 3. Metabolic schemes for (A) benzene (adapted from Sabourin et al., 1988), (B) ethoxyquin (adapted from Burka et al., 1996), and (C) methacrylonitrile (adapted from Ghanayem et al., 1994), showing selected metabolites excreted in urine. See Table 1Go for identification of metabolite markers and pathways.

 
In the present study, benzene-treated FVB/N wild-type mice and Tg.AC mice excreted similar concentrations of phenyl and hydroquinone glucuronides (PG and HQG, respectively), phenyl sulfate (PS), and muconic acid (MA) in urine (Fig. 4Go). Similar results were obtained when these metabolic markers were compared in C57BL/6 and p53+/– mice (Fig. 5Go). Although, the major metabolites of benzene were present in all treatment groups, C57BL/6 wild types excreted more HQG than did FVB/N wild types. Concurrently, FVB/N mice excreted more PG and PS than did C57BL/6 mice. There were no significant differences between each wild-type strain and the respective transgenic line in concentrations of major ethoxyquin metabolic markers, in this case, the major sulfate (DHTS) and glucuronide (DHTG) conjugates previously shown to be excreted by B6C3F1 mice (Burka et al., 1996Go). However, as with benzene, slight quantitative differences in ethoxyquin metabolism were observed between wild types. C57BL/6 mice excreted more DHTG than did FVB/N mice. A mercapturic acid (NAHPC) was used as the marker for methacrylonitrile metabolism and as with the former two test compounds, the concentration of the metabolite was not significantly different between each wild-type strain and its corresponding transgenic line. However, the concentration of NAHPC was significantly different between the two mouse strains. HPLC chromatograms of urine from either benzene-, ethoxyquin-, or methacrylonitrile-treated mice contained other mostly unidentified minor metabolites, not used as pathway markers, that in some instances showed slight quantitative or qualitative differences between relevant treatment groups. However, differences for the nonmarker metabolites between mouse strains and their respective transgenic mouse lines were minimal in all cases (data not shown).



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FIG. 4. Relative quantities of selected metabolites of (A) benzene, (B) ethoxyquin, and (C) methacrylonitrile excreted in cumulative 24-h urine by FVB/N wild-type (open bars) and Tg.AC (filled bars) mice. Each value represents the mean ± SD of 3–6 determinations. *Significant difference between wild-type strains. See Table 1Go for identification of metabolites and Figure 3Go for chemical structures.

 


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FIG. 5. Relative quantities of selected metabolites of (A) benzene, (B) ethoxyquin, and (C) methacrylonitrile excreted in cumulative 24-h urine by C57BL/6 wild-type (open bars) and p53+/- (filled bars) mice. Each value represents the mean ± SD of 4–6 determinations. *Significant difference between wild-type strains. See Table 1Go for identification of metabolites and Figure 3Go for chemical structures.

 
The relative expression of representative CYPs (CYP1A1, CYP2E1, and CYP3A) involved in xenobiotic metabolism was determined in liver of naive Tg.AC and p53+/– genetically altered mice and FVB/N and C57BL/6 wild types (Figs. 6 and 7GoGo). Expression of each of these enzymes, as measured by optical density of the protein bands, varied between individual mice of each genotype. However, no significant differences (statistical data not shown) were detected in expression of CYP1A2, CYP2E1, or CYP3A between either FVB/N and Tg.AC mice or C57BL/6 and p53+/– mice. Single protein bands were observed for reactivity of the CYP1A2 and CYP2E1 antibodies for the corresponding protein in mouse microsomal preparations. Double protein bands were detected on membranes probed with CYP3A antibody (Figs. 6C and 7CGoGo). CYP3A antibody strongly reacted with protein in the CYP3A positive control microsomes of lane 12 of each gel and with corresponding protein in lanes representing individual mice of each genotype, indicating those proteins to be of CYP3A origin. Anti-CYP3A also reacted with protein in the CYP2E1-induced microsomes of lane 11 of the same gel as well as protein in the corresponding bands of mice from each genotype. The identity of the protein(s) in those bands is equivocal.



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FIG. 6. Representative Western blot analysis of (A) CYP1A2, (B) CYP2E1, and (C) CYP3A in liver of naive Tg.AC (lanes 1–5) and FVB/N wild-type (lanes 6–10) mice. For lanes 1–10, each lane represents liver microsomal protein of individual mice loaded at 15 µg. Lane 11 of (B) and (C) contains approximately 5 µg liver microsomal protein from isoniazid-treated rats (positive control for CYP2E1). Lane 12 of (A) contains approximately 1.5 µg of liver microsomal protein from isosafrole-treated rats (positive control for CYP1A2). Lane 12 of (B) and (C) contains approximately 5 µg of liver microsomal protein from dexamethasone-treated rats (positive control for CYP3A). Positive controls were located between 51 and 58.1 kDa molecular weight markers (not shown).

 


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FIG. 7. Representative Western blot analysis of (A) CYP1A2, (B) CYP2E1, and (C) CYP3A in liver of naive p53+/– (lanes 1–5) and C57BL/6 wild-type (lanes 6–10) mice. For lanes 1–10, each lane represents liver microsomal protein of individual mice loaded at 15 µg. Lane 11 of (B) and (C) contains approximately 5 µg liver microsomal protein from isoniazid-treated rats (positive control for CYP2E1). Lane 12 of (A) contains approximately 1.5 µg of liver microsomal protein from isosafrole-treated rats (positive control for CYP1A2). Lane 12 of (B) and (C) contains approximately 5 µg of liver microsomal protein from dexamethasone-treated rats (positive control for CYP3A). Positive controls are located between 51 and 58.1 kDa molecular weight markers (not shown).

 
Figure 8Go demonstrates the relative expression of GST-{alpha} in each of the 4 mouse genotypes. As with results obtained for the surveyed CYP enzymes, the expression of GST-{alpha} varied within each genotypic group. However, there were no statistically significant differences in the means of the band densities of each transgenic line and its respective wild type. Further, there was no difference in GST-{alpha} expression between the two wild-type mouse strains (statistical data not shown).



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FIG. 8. Representative Western blot analysis of GST-{alpha} in liver of naive mice. (A) Microsomal protein from an FVB/N wild-type mouse was loaded in lane 1. Lanes 2–4 contain Tg.AC mouse cytosolic protein. Lanes 5–7 contain FVB/N wild-type mouse cytosolic protein. (B) Microsomal protein from a C57BL/6 wild-type mouse was loaded in lane 1. Lanes 2–4 contain p53+/– mouse cytosolic protein. Lanes 5–7 contain C57BL/6 wild-type mouse cytosolic protein. Microsomal protein loaded at 25 µg was from an individual mouse. Cytosolic protein from individual mice was loaded at 50 µg. Lane 8 of both (A) and (B) contains purified enzyme GST-{alpha} (1–1) loaded at 1 µg.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The FVB/N-derived Tg.AC transgenic mouse and the C57BL/6-derived p53+/–mouse show great promise as useful models for characterizing the carcinogenic potential of a wide range of chemicals. However, if these models are to be used, it is critical that xenobiotics are metabolized and excreted in a manner identical to that of the parent strain. Thus, the present work has tested the generally held assumption that genetic alterations in these two transgenic animal lines do not alter major biotransformation pathways found in the parent strains. Indeed, metabolism of 3 test xenobiotics through some of the more important phase I and II biotransformation pathways was not significantly different between the 2 transgenic mouse lines and their respective wild types. Of the parameters measured, the only variation seen was in cumulative 14CO2 excretion following methacrylonitrile administration. In this case, differences were marginally significant between either wild-type strain and corresponding transgenic line and between the wild-type strains themselves. It is likely, however, that these differences are the result of normal experimental variation between individual animals.

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., 1996Go). 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, 1996Go). CYP2E1 is purported to be the major oxidase in methacrylonitrile metabolism (Ghanayem et al., 1999Go). However, other monoxygenases can metabolize methacrylonitrile, as well as benzene, as demonstrated in CYP2E1-/- mice (Valentine et al., 1996Go; Ghanayem et al., 1999Go). Further, CYP2B1 has been shown to metabolize benzene in rat liver microsomes (Snyder et al., 1993Go).

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 3Go (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., 1996Go). 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., 1992Go). 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., 1998Go). 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., 1998Go), 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., 1994Go), 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., 1997Go). 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 receptor–mediated induction of metabolizing enzymes in skin of dermally treated Tg.AC mice.


    ACKNOWLEDGMENTS
 
We thank Mr. J. E. Fossett and Mr. P. J. Schupp for their excellent technical assistance.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (919) 541-1885. E-mail: sandersm{at}niehs.nih.gov. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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