ARTICLE

4-Aminobiphenyl–Induced Liver and Urinary Bladder DNA Adduct Formation in Cyp1a2(–/–) and Cyp1a2(+/+) Mice

Yutaka Tsuneoka, Timothy P. Dalton, Marian L. Miller, Corey D. Clay, Howard G. Shertzer, Glenn Talaska, Mario Medvedovic, Daniel W. Nebert

Affiliation of authors: Department of Environmental Health and Center for Environmental Genetics (CEG), University of Cincinnati Medical Center, Cincinnati, OH.

Correspondence to: Daniel W. Nebert, MD, Department of Environmental Health, University of Cincinnati Medical Center, P.O. Box 670056, Cincinnati, OH 45267–0056 (e-mail: dan.nebert{at}uc.edu).


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Metabolites of the potent human carcinogen 4-aminobiphenyl (ABP) induce oxidative stress and form DNA adducts that are associated with hepatic and urinary bladder toxicity and bladder tumorigenesis. Results of in vitro and cell culture studies have suggested that cytochrome P450 1A2 (CYP1A2) is the major metabolic activator of ABP. We used Cyp1a2(–/–) knockout mice to examine the role of CYP1A2 in ABP–DNA adduct formation in the liver and the bladder. Methods: Cyp1a2(+/+) wild-type and Cyp1a2(–/–) mice (total of four mice per group) were treated topically with 10 mg/kg ABP for various times, with or without pretreatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an inducer of CYP1A2 activity. We evaluated ABP-induced toxicity by carrying out quantitative histology (of the liver, skin, and bladder), oxidative stress by measuring hepatic thiol levels, and liver and bladder DNA adduct formation by using 32P-postlabeling. Data were analyzed by general linear models and analysis of variance. All statistical tests were two-sided. Results: At the experimental times selected, we observed no histologic evidence of toxicity in the liver, skin, or bladder. Overall, Cyp1a2(+/+) mice had fewer DNA adducts 24 hours after ABP treatment than similarly treated Cyp1a2(–/–) mice. Compared with male mice, female mice had more DNA adducts in the liver but fewer adducts in the bladder, regardless of Cyp1a2 genotype. TCDD pretreatment was associated with a decrease in ABP–DNA adduct levels overall. After 2 hours of ABP treatment, hepatic thiol levels underwent statistically significant declines of severalfold in Cyp1a2(+/+) and Cyp1a2(–/–) males and in Cyp1a2(–/–) females. Conclusions: Contrary to our expectations, CYP1A2 expression was not associated with ABP-induced hepatic oxidative stress or with ABP–DNA adduct formation. Either CYP1A2 is not the major metabolic activator of ABP or other enzymes metabolically activate ABP in mice in the absence of CYP1A2.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The arylamine 4-aminobiphenyl (ABP) was formerly used as a dye intermediate, rubber antioxidant, and reagent for detecting sulfates but is no longer used commercially in most countries. An International Agency for Research on Cancer Working Group noted three decades ago that there was sufficient evidence to implicate ABP as a carcinogen in laboratory animals and humans [reviewed in (1)]. Oral administration of ABP to mice, rats, rabbits, or dogs produced various types of neoplasms, including carcinoma of the urinary bladder (2). On the basis of results from a number of clinical and epidemiologic studies, ABP is now an established human carcinogen. For example, a survey of cancer mortality among chemical plant workers found that 19 of 171 men exposed to ABP developed bladder tumors (3).

The primary routes for human exposure to ABP are skin contact, inhalation, and ingestion. Epidermal exposure to ABP still occurs in a number of countries during the manufacture of arylamine dyes (4). ABP might also be present in foods containing certain additives (1). However, today the major source of human exposure to ABP is via inhaled cigarette smoke. The amounts of ABP in mainstream and sidestream smoke are 4.6 ng and 140 ng per cigarette, respectively; a dose of 33 µg of ABP per year has been associated with at least a doubling in the bladder cancer rate in human populations (5).

ABP is metabolized to the N-hydroxy-ABP intermediate principally in the liver, and this intermediate is a known precursor to the formation of ABP–DNA adducts in both liver and bladder, as well as ABP–hemoglobin adduct formation in the blood (1,2,4). ABP–DNA adducts represent DNA damage that can ultimately result in mutation and tumor initiation.

Cytochrome P450 (CYP) enzymes are expressed in all tissues of the body (6,7), catalyze oxidative metabolism, and are most likely responsible for N-hydroxy-ABP formation. For the past decade, CYP1A2 has been regarded as the P450 enzyme primarily responsible for the metabolic activation of arylamines, including ABP (8). A major pathway for detoxifying N-hydroxy-ABP is N-acetyltransferase-2 (NAT2) (9). Polymorphisms in the CYP1A2 (10) and NAT2 (11) genes are associated with variation in the activities of the enzymes they encode in human populations. Moreover, expression of the CYP1A2 gene is induced in cigarette smokers, leading to even higher CYP1A2 enzyme activity (8). An ABP-exposed individual exhibiting high CYP1A2 activity and slow NAT2 acetylator activity would thus be expected to have increased levels of N-hydroxy-ABP, and therefore higher levels of ABP–hemoglobin and liver and bladder ABP–DNA adducts, than an individual with low CYP1A2 activity and rapid NAT2 acetylator activity.

Numerous studies support this metabolic activation/detoxification model. For example, there are strong associations between bladder ABP–DNA adduct levels and levels of ABP–hemoglobin adducts, excretion of urinary mutagenic metabolites, and number of pack-years of cigarettes smoked (12,13). ABP–DNA adduct levels in exfoliated urothelial cells and in human bladder biopsies are about five times higher in smokers than in nonsmokers (14,15), further supporting the likelihood of CYP1A2 involvement. In addition, the human exfoliated urothelial cells of slow NAT2 acetylators show more ABP–DNA adducts than those of rapid NAT2 acetylators (1623).

Because enzymes in the CYP1 family (6,7) are those primarily induced by cigarette smoke [reviewed in (7,8)] and because a balance appears to exist in the liver between metabolic activation principally by CYP1A2 and detoxification by NAT2, a model (Fig. 1Go) has been proposed to explain how ABP causes mammalian bladder cancer. The basic tenet of this model is that oxidative metabolism in the liver ultimately controls the level of ABP–DNA adducts in the bladder. Several lines of evidence support this theory. First, no detectable levels of the CYP1A2 enzyme are found in the urinary bladder of rodents, dogs, or humans (26,27). Second, when dogs were given ABP orally, more than 20% of the total dose was bound to hemoglobin, suggesting that there are high circulating levels of the N-hydroxy-ABP intermediate; moreover, instillation of ABP directly into the bladder was not associated with measurable levels of ABP– DNA adducts in the urothelium, whereas instillation of N-hydroxy-ABP was (28). Third, evidence that individuals who are slow NAT2 acetylators are at higher risk for ABP–DNA adduct formation than individuals who are rapid NAT2 acetylators has begun to accumulate (1618). The model in Fig. 1Go thus shows that hepatic CYP1A2 and NAT2 activities are competing reactions for the activation and detoxification, respectively, of arylamines such as ABP.



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Fig. 1. Commonly held view of ABP metabolism in the liver and urinary bladder, leading to ABP–DNA adducts. According to this scheme, CYP1A2 is the major player in the formation of arylamine reactive intermediates that cause liver toxicity and urinary bladder toxicity and tumorigenesis (24). N-acetylation (NAT2) and UDP glucuronosyltransferase (UGT) are regarded as the two major detoxication enzymes, whereas CYP1A2-mediated N-hydroxylation leads to formation of the proximal carcinogen, N-hydroxy-ABP. A lowered urinary pH also appears to play a major role in the ultimate rate of tumor initiation and progression (25). R-NH2 = any arylamine; R-NH-Ac = acetylated product; R-NH-Gluc = glucuronidated product; R-NH-OH = N-hydroxylated product; R-N(OH)-Ac = acetylated N-hydroxy product.

 
Polycyclic hydrocarbons are known to induce CYP1A2 activity. Expression of the CYP1A2 gene is induced via the aryl hydrocarbon receptor (AHR), by dioxin, and by polycyclic and halogenated aromatic hydrocarbons, including those found in cigarette smoke (6,8). Polycyclic hydrocarbon treatment of isolated rat hepatocytes was associated with a fivefold increase in N-hydroxylation (presumably reflecting CYP1A2 activity), a 22-fold increase in glucuronidation (presumably reflecting an inducible uridine diphosphate [UDP] glucuronosyltransferase activity), a 15-fold decrease in N-acetylation (presumably reflecting decreased NAT2 participation), and a threefold increase in ABP–DNA adducts (29). These data appear to further underscore the importance of AHR-mediated CYP1A2 induction in altering the balance between liver CYP1A2 and NAT2 activities (Fig. 1Go) and are consistent with epidemiologic data showing that arylamine-exposed individuals who smoke cigarettes have a greater risk of bladder cancer than arylamine-exposed individuals who do not smoke (5,6,8,9,1215,23,24).

In this study, we examined ABP–DNA adduct formation in the intact mouse. On the basis of the model shown in Fig. 1Go, we hypothesized that mice expressing high levels of CYP1A2 would form more ABP–DNA adducts than mice expressing low levels of CYP1A2 and that mice expressing no CYP1A2 might not form any ABP–DNA adducts.


    MATERIALS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chemicals

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was purchased from AccuStandard (New Haven, CT). All other chemicals and reagents were obtained from Aldrich Chemical Company (Milwaukee, WI) or Sigma Chemical Company (St. Louis, MO) as the highest available grades. ABP was a generous gift from Fred Kadlubar (National Center for Toxicology Research, Jefferson, AR).

Mice

Generation of the Cyp1a2(–/–) mouse line, starting from the C57BL/6J and 129/J inbred strains, has been described (30), and the Cyp1a2(–/–) mouse colony that we maintain has been backcrossed into C57BL/6J mice for eight generations, ensuring that the CYP1A2 knockout genotype resides in a genetic background that is greater than 99.8% C57BL/6J (31). Age-matched C57BL/6J Cyp1a2(+/+) (i.e., wild-type) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The CYP1A2 genotypes of all mice used in these studies were verified using established polymerase chain reaction–based genotyping procedures (30). All animal experiments were approved by the University of Cincinnati Medical Center Institutional Animal Care and Use Committee and conducted in accordance with the National Institutes of Health standards for the care and use of experimental animals.

We painted ABP on the skin of each mouse to mimic the usual occupational route of exposure in humans. In preliminary experiments, we tested a range of ABP doses (1–25 mg/kg of body weight; three wild-type mice per group) and 2–48 hours of ABP exposure times to determine the dose and duration of exposure that was associated with maximal ABP–DNA adduct formation with negligible toxic effects (data not shown). On the basis of the results of these preliminary experiments, we applied ABP (at 10 mg/kg of body weight, in 25 µL of acetone) topically to the dorsal skin (shaved 48 hours beforehand) of 8-week-old male and female mice (four mice per group) and killed the mice by CO2 gas inhalation 2, 24, or 48 hours later. Some mice were injected intraperitoneally with one dose of TCDD (15 µg/kg of body weight, in corn oil) 48 hours prior to ABP administration to induce CYP1A2 activity and protein expression to maximal levels (6,8). Control mice received the vehicles alone. The animals had access to rodent food (Harlan Teklad, Madison, WI) and water ad libitum. All mouse tissues were harvested between 9:00 AM and 10:00 AM to exclude any circadian rhythm effects.

Western Blot Analysis of CYP1A2 Protein Expression

Twenty-four hours after topical ABP treatment and, in some cases, TCDD pretreatment 48 hours prior to ABP treatment, hepatic microsomes were prepared as previously described (32). Protein concentrations were determined using BioRad protein reagent (Bio-Rad Laboratories, Hercules, CA). Microsomal proteins (0.5 µg/lane) were separated on sodium dodecylsulfate (0.1%)–polyacrylamide (10%) mini-gels, transferred to nitrocellulose membranes (Promega, Madison, WI), and visualized by staining with 2% Ponceau S to verify equivalent loading across lanes. Western blot analysis was performed using a goat polyclonal anti-CYP1A1/1A2 antibody (BD Gentest, Woburn, MA); this antibody recognizes both the CYP1A1 and CYP1A2 proteins. We used a horseradish peroxidase–conjugated secondary antibody (Dako, Carpenteria, CA) and the enhanced chemiluminescence system (ECL; Amersham, Piscataway, NJ) to detect primary antibody binding, with exposure times ranging from 1 to 30 seconds.

Mouse Tissue Histology

Mouse tissues (liver, skin, and bladder) were fixed in an iso-osmolar paraformaldehyde/glutaldehyde–phosphate-buffered solution, post-fixed in 1% phosphate-buffered osmium tetroxide, dehydrated in graded ethanol solutions ranging from 30% to 100%, treated with propylene oxide, and embedded in Spurr’s resin, as previously described (33). One-micron-thick sections were stained with toluidine blue to determine the extent of toxicity by examining tissue and cellular morphometrics. The volume density (Vd) of lipid, glycogen, and interstitial components in individual liver sections was examined by using a 75-point grid overlaid on x1250 microscopic fields. Grid intersections overlaying each structure (lipid, glycogen, and interstitial components) were counted, and the Vd for that structure was determined by dividing the number of counts over that particular structure by the total number of points over all structures. Vd is therefore similar to a percentage. The thickness of the skin epidermis and the epithelium of the bladder were determined by measuring a line drawn perpendicular to and beginning at the basement membrane and terminating at the keratin (for skin) or bladder lumen (for bladder). The length of each of these lines was digitized with the use of a Summagraphics tablet and SigmaScan Pro software (SigmaScan, Chicago, IL) and calibrated at the same magnification as the image (in microns) using a micrometer slide as a reference.

Measurement of Hepatic Thiols

We used previously published procedures to measure the levels of reduced glutathione (GSH) (34) and cysteine (35) in mouse liver at zero time, 2 hours, and 24 hours after topical ABP treatment. Experiments were also performed with intraperitoneal TCDD pretreatment 48 hours before ABP, but the results were not statistically significantly different (P>.05) and are therefore not shown. Duplicate determinations from four mice per group were carried out, and the experiment was repeated once (with four mice per group).

Measurement of ABP–DNA Adduct Levels

We measured ABP–DNA adduct levels in mouse liver samples and in urinary bladders with the use of a 32P-postlabeling technique, as previously described (36). We used the entire bladder for ABP–DNA adduct analysis because the urothelium, which constitutes only approximately 1% of the bladder, has six times more of the enzymes involved in the metabolic activation and conjugation of ABP and other arylamines than the smooth muscle and lamina propria combined, which constitutes approximately 90% and approximately 9% of the bladder, respectively (16,24). DNA was extracted from mouse tissue with the use of a Wizard genome DNA isolation kit (Promega, Madison, WI) and hydrolyzed (0.5–1.3 µg of DNA per sample) to 3'-phosphodeoxynucleotides by digestion with micrococcal endonuclease and spleen phosphodiesterase (both from Sigma). Following N-butanol extraction to remove most of the non-adducted 3'-phosphodeoxynucleotides, the 3'-phosphodeoxynucleosides were labeled at the 5' positions with 32P-ATP (NEN Life Science Products, Boston, MA) and T4 polynucleotide kinase (USB, Cleveland, OH). We used two-dimensional thin-layer chromatography on polyethyleneimine cellulose sheets to resolve the 32P-labeled DNA adducts (EM Sciences, Gibbstown, NJ). Visualization and analysis were done by scintillation counting (Packard 1900 CA; Packard, Downers Grove, IL) and autoradiography, respectively. Adduct levels in each DNA sample were calculated from the background-corrected adduct counts and the specific activity of the radiolabeled ATP and were expressed as relative adduct labeling (RAL). The RAL is defined as the minimal estimate of the number of nucleotides containing adducts per 109 nucleotides in the sample; this value is based on the amount of DNA in each sample and the specific radioactivity of the 32P-ATP used in the assay. Duplicates or triplicates of each DNA sample were analyzed independently.

Statistical Methods

Mean values and 95% confidence intervals (CIs) for the tissue and cellular morphometrics were generated with the use of the general linear model of SAS statistical software (SAS Institute, Cary, NC). Results of analysis of variance (ANOVA) for RAL were compared between groups of mice with the use of statistical linear models and Student’s t test. All assays were performed in duplicate or triplicate, and the average values were considered as one independent determination. Statistical differences between mean values for specific groups were compared with the use of factorial ANOVA. All statistical hypothesis testing was performed at the {alpha} = .05 significance level, and all statistical tests were two-sided. Statistical analyses were performed with the use of SAS statistical software. Pearson correlation coefficients were determined between liver and bladder ABP–DNA adducts from individual males and individual females.

Biohazard Precaution

TCDD and ABP are both highly toxic; ABP is a human carcinogen, whereas TCDD is a likely human carcinogen. All personnel were instructed on safe handling procedures. Lab coats, gloves, and masks were worn at all times, and contaminated materials were collected separately for disposal by the Hazardous Waste Unit of the University of Cincinnati Medical Center or by independent contractors. TCDD-treated mice were housed separately, and their carcasses were treated as contaminated biologic materials.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In preliminary experiments, we found that ABP–DNA adduct levels in mouse liver and urinary bladder were highest 24 hours after the mice were treated topically with 10 mg/kg ABP, that TCDD pretreatment 48 hours before ABP treatment caused the highest levels of inducible CYP1A2 activity, and that higher doses of either ABP or TCDD or longer treatment times did not produce any dramatically greater effects (data not shown). The ABP dose we used in our experiments was two to five times lower than that used to produce cancer in laboratory animal studies. We also knew from previous experiments that statistically significant (P<.05) histologic changes in liver and bladder do not develop until well beyond 48 hours after exposure to topical ABP (37). We therefore evaluated liver, skin, and bladder tissue histologically at 48 hours after ABP treatment, measured hepatic thiol levels at 2 and 24 hours after ABP treatment, and examined ABP–DNA adduct formation at 24 hours after topical ABP treatment. TCDD pretreatment, when included, was always given 48 hours before ABP treatment.

CYP1A2 Protein Levels in Cyp1a2(+/+) and Cyp1a2(–/–) Mice

We determined hepatic CYP1A2 and CYP1A1 protein expression in ABP-treated Cyp1a2(+/+) and Cyp1a2(–/–) mice, in some cases with TCDD pretreatment. The results from female Cyp1a2(+/+) and Cyp1a2(–/–) mice (Fig. 2Go) were similar to those from male mice of the same genotype (data not shown). We wanted to be certain that topical ABP or vehicle treatment did not cause any changes in liver CYP1A1 or CYP1A2 protein levels, and this was what we found (data not shown). By contrast, TCDD pretreatment induced hepatic CYP1A2 and CYP1A1 protein expression in Cyp1a2(+/+) mice and induced CYP1A1 protein expression in Cyp1a2(–/–) mice.



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Fig. 2. Western blot analysis of CYP1A1 and CYP1A2 expression in liver microsomes from eight individual 4-aminobiphenyl (ABP)–treated Cyp1a2(+/+) wild-type mice (top) and eight individual ABP-treated Cyp1a2(–/–) knockout mice (bottom) used in this study. The last four lanes show samples obtained from mice pretreated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). All lanes were loaded with 0.5 µg of protein.

 
Hepatic constitutive CYP1A1 protein levels were negligible in untreated mice (data not shown) and remained unchanged after topical ABP treatment (Fig. 2Go). By contrast, there were substantial levels of constitutively expressed CYP1A2 protein in ABP-treated Cyp1a2(+/+) mice; as expected, there was no constitutive CYP1A2 expression in Cyp1a2(–/–) knockout mice. In TCDD-pretreated Cyp1a2(+/+) mice, induced CYP1A1 (56.0 kd) and increases in CYP1A2 (54.5 kd) appeared as electrophoretically distinct bands. In TCDD-pretreated Cyp1a2(–/–) knockout mice, as expected, induced CYP1A1, but not CYP1A2, was found. We detected a modest amount of a faster-migrating protein that was recognized by the anti-CYP1A1/1A2 antibody in some samples, which we presumed was a proteolytic product of CYP1A1. Whether such proteolysis occurred in vivo or as a result of sample preparation was not determined. The data in Fig. 2Go thus confirm the absence of CYP1A2 in Cyp1a2(–/–) mice, the presence of substantial amounts of basal CYP1A2 protein levels in ABP-treated Cyp1a2(+/+) mice, the induction of CYP1A1 expression in both genotypes after pretreatment with TCDD, and the induction of CYP1A2 expression only in Cyp1a2(+/+) mice pretreated with TCDD.

Histologic Assessment of Liver, Skin, and Bladder Toxicity in ABP-Treated and TCDD-Pretreated Mice

We performed quantitative morphometrics in the liver, skin, and bladder to rule out toxic effects of ABP treatment and/or TCDD pretreatment. The amounts and the distribution of glycogen and interstitial components were similar among all groups. Interestingly, whereas Cyp1a2(+/+) mice in all groups generally appeared to have more intrahepatocyte fat than Cyp1a2(–/–) mice in all groups (Fig. 3Go), the interstitium of Cyp1a2(–/–) mice in all groups contained more fat droplets than that of Cyp1a2(+/+) mice in all groups. ABP treatment, TCDD pretreatment alone, or ABP treatment plus TCDD pretreatment appeared not to substantially alter the liver histology of mice of either genotype.



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Fig. 3. Light microscopy of representative liver sections from untreated (top row), 4-aminobiphenyl (ABP)–treated (second row), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-pretreated (third row), and TCDD pretreated and ABP-treated (bottom row) Cyp1a2(+/+) and Cyp1a2(–/–) mice. Histologic analysis was performed 48 hours after topical ABP treatment. Because TCDD pretreatment was always carried out 48 hours before ABP, histologic analysis of the "TCDD only" group occurred 96 hours after intraperitoneal TCDD treatment. The white arrows indicate fat in interstitial cells. The black arrows indicate fat in hepatocytes. Bar = 20 µm; original magnification = x100.

 
Quantitation and statistical analysis of these results are shown in Table 1Go. Statistical analysis was performed on square-root–transformed data, which stabilized variances across different experimental groups. Using the backward elimination of nonsignificant terms in a three-way ANOVA, we found that TCDD pretreatment of Cyp1a2(+/+) mice was associated with a statistically significant increase in the Vd of intrahepatocyte fat (mean increase = 0.76, 95% CI = 0.02 to 1.34) and that TCDD pretreatment of Cyp1a2(–/–) mice was associated with a statistically significant increase in the Vd of intrahepatocyte fat (mean increase = 1.79, 95% CI = 0.13 to 3.40). Neither ABP treatment nor any interaction effects were statistically significant.


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Table 1. Effect of 4-aminobiphenyl (ABP), with or without 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) pretreatment, on volume density (Vd) of intrahepatocyte fat versus interstitial fat in Cyp1a2(+/+) and Cyp1a2(-/-) mice*
 
Using the backward elimination of nonsignificant terms in a three-way ANOVA, we found that all groups of Cyp1a2(–/–) mice had statistically significantly more interstitial fat than the corresponding groups of Cyp1a2(+/+) mice (mean increase = 1.00, 95% CI = 0.26 to 1.74). The effects of TCDD pretreatment and ABP treatment and all interaction effects were not statistically significant.

Despite these subtle differences in lipid distribution within the liver of mice of different genotypes, these data suggest that the dose of ABP used in our experiments, either alone or in combination with TCDD pretreatment, was not acutely hepatotoxic. These findings suggest that the ABP–DNA adducts measured at the 24-hour time point represent a transitory burst of ABP exposure and metabolism with basically no liver or hepatocyte damage.

Skin was not regarded as a target organ in this study. However, because ABP was applied topically, we wanted to be certain that any toxic effects in skin 48 hours after ABP treatment would not complicate any effects seen in the liver or bladder. Furthermore, the greatest histologic response—in terms of mitotic index and inflammation—is known to occur approximately 96 hours after topical ABP treatment (37). We found that ABP-treated mice of both genotypes displayed more epidermal thickening and a higher mitotic index (33) than untreated mice of both genotypes (data not shown), but these differences were not statistically significant (P = .15). Intraperitoneal TCDD pretreatment had no substantial effect on skin histology, consistent with the lack of CYP1A2 expression in mouse skin (8).

We found no inflammation in the lamina propria of the bladders of treated or control mice. However, following combined ABP treatment and TCDD pretreatment, both Cyp1a2(–/–) and Cyp1a2(+/+) mice displayed some increased thickening of the bladder epithelium with no substantial differences between genotypes (data not shown).

Effect of ABP Treatment on Hepatic Thiol Levels

A decline in the levels of certain thiol-containing compounds, such as GSH and cysteine, is a strong indicator of oxidative stress (38). We observed statistically significant decreases (P<.001 in all cases) in hepatic GSH and cysteine levels in male Cyp1a2(+/+) and Cyp1a2(–/–) mice and female Cyp1a2(–/–) mice 2 hours after ABP treatment compared with untreated mice of the same sex and genotype (Fig. 4Go). We found that genotype, ABP, ABP-by-genotype, and ABP-by-sex interactions all had statistically significant effects on GSH levels at the 2-hour time points compared with the zero time points. Treatment with ABP was associated with an overall decrease in GSH level by 0.22-fold (95% CI = 0.19- to 0.28-fold); the overall difference in GSH levels between Cyp1a2(+/+) and Cyp1a2(–/–) genotypes was 1.6-fold (95% CI = 0.4- to 1.4-fold); the difference in decreased GSH levels associated with ABP treatment between male and female mice was 0.36-fold (95% CI = 0.21- to 1.00-fold); and the difference in decreased GSH levels associated with ABP treatment between Cyp1a2(+/+) and Cyp1a2(–/–) genotypes was 0.37-fold (95% CI = 0.22- to 1.25-fold). In the context of this analysis, we had at least 80% power to detect absolute interaction effects of 0.38- and 0.19-fold for the genotype-by-sex and genotype-by-sex-by-ABP interactions, respectively, at a statistical significance level of .05.



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Fig. 4. Levels of reduced glutathione (GSH) (top) and cysteine (bottom) in the livers of 4-aminobiphenyl (ABP)–treated Cyp1a2(+/+) and Cyp1a2(–/–) male (M) and female (F) mice. At zero time (open bar), 2 hours (closed bar), or 24 hours (gray bar) following treatment with topical ABP, the livers were examined for these thiols. Duplicate determinations from four mice per group were carried out (total of 48 mice), and the entire experiment was repeated once (total of 96 mice). Bars represent mean value, and error bars represent 95% confidence intervals. *, statistically significantly difference (P<.001) in all cases for untreated control mice of the same sex and genotype, using the backward elimination of statistically nonsignificant terms in a three-way analysis of variance to analyze the zero (control) and 2-hour-after-exposure data for GSH and cysteine levels.

 
We found that genotype, ABP, and ABP-by-sex interactions were all statistically significantly associated with cysteine concentrations measured at the 2-hour time points compared with those at the zero time points. Treatment with ABP was associated with an overall decrease in cysteine levels by 0.40-fold (95% CI = 0.28- to 0.51-fold); the overall difference in cysteine levels between Cyp1a2(+/+) and Cyp1a2(–/–) genotypes was 0.13-fold (95% CI = 0.03- to 0.24-fold); and the difference in decreased cysteine levels associated with ABP treatment between male and female mice was 0.25-fold (95% CI = 0.03- to 0.47-fold). In the context of this analysis, we had at least 80% power to detect absolute interaction effects of 0.3-, 0.3-, and 0.6-fold for the genotype-by-ABP, genotype-by-sex, and genotype-by-sex-by-ABP interactions, respectively, at a statistical significance level of .05.

If ABP-induced oxidative stress was dependent on hepatic expression of CYP1A2, we would have expected a greater decrease in the levels of these thiols in Cyp1a2(+/+) mice than in Cyp1a2(–/–) mice, but this was not observed. Curiously, female Cyp1a2(+/+) mice did not have statistically significant decreases in hepatic GSH or cysteine levels 2 hours after ABP treatment. However, the fact that we found that Cyp1a2(+/+) mice did not have a substantially greater decrease in the levels of either of these thiols than Cyp1a2(–/–) mice suggests that ABP-induced oxidative stress is not dependent on hepatic CYP1A2 expression. In each of the other three groups of mice (i.e., Cyp1a2(+/+) males, Cyp1a2(–/–) males, and Cyp1a2(–/–) females), GSH and cysteine concentrations returned to normal at the 24-hour time point after ABP treatment, indicating that the animals were able to respond successfully to this temporary depletion of hepatic thiols.

Relationship Between ABP–DNA Adduct Formation and CYP1A2 Genotype

Overall, Cyp1a2(+/+) mice had fewer DNA adducts 24 hours after ABP treatment than similarly treated Cyp1a2(–/–) mice. Fig. 5Go, A, shows ABP–DNA adducts in the liver. Interestingly, we found a striking sex difference in the levels of ABP–DNA formation, with females generally having much higher levels of liver ABP–DNA adducts than males. Cyp1a2(–/–) female mice had statistically significantly more hepatic ABP–DNA adducts than Cyp1a2(+/+) female mice, with (P = .028) or without (P = .014) TCDD pretreatment. Using the backward elimination of nonsignificant terms in a three-way ANOVA, we found that TCDD, sex, and genotype-by-sex interactions all had a statistically significant effect on liver adduct levels. Female Cyp1a2(–/–) mice had statistically significantly higher adduct levels than male Cyp1a2(–/–) mice (5.8-fold increase, 95% CI = 3.0- to 11.2-fold; P<.001), whereas female and male Cyp1a2(+/+) mice had virtually identical adduct levels (ratio of adduct levels in females to males = 1.7, 95% CI = 0.5 to 1.8; P = .88). Mice that were not pretreated with TCDD had statistically significantly higher adduct levels than mice that were pretreated (2.1-fold increase, 95% CI = 1.3- to 3.3-fold; P = .003). In the context of this analysis, we had at least 80% power of detecting 4.1-, 4.1-, and 14.9-fold effects of the genotype-by-TCDD, sex-by-genotype, and sex-by-genotype-by-TCDD interactions, respectively, at the {alpha} = .05 significance level.



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Fig. 5. 4-Aminobiphenyl (ABP)–DNA adduct formation in the liver (A) and the urinary bladder (B) of Cyp1a2(+/+) and Cyp1a2(–/–) mice, with (+) or without (–) 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) pretreatment, 24 hours after topical ABP treatment. We used four mice of each sex (M = male; F = female) and genotype for each treatment group (total of 32 mice); the experiment was repeated once (total of 64 mice). Bars indicate the mean relative adduct level (RAL); error bars represent 95% confidence intervals. A) *, P =.014 compared with Cyp1a2(+/+) females not pretreated with TCDD; {dagger}, P =.028 compared with Cyp1a2(+/+) females pretreated with TCDD. B) *, P =.013 compared with Cyp1a2(+/+) females pretreated with TCDD.

 
Fig. 5Go, B, shows ABP–DNA adducts in the urinary bladder. In the TCDD pretreatment group, the bladder ABP–DNA adduct levels in Cyp1a2(–/–) females were statistically significantly greater than those in Cyp1a2(+/+) females (P = .04). Using the backward elimination of nonsignificant terms in a three-way ANOVA, we found that TCDD pretreatment and sex had statistically significant effects on bladder adduct levels. For example, mice not pretreated with TCDD had higher adduct levels than mice that were pretreated (2.2-fold increase, 95% CI =1.5- to 3.3-fold; P<.001), and male mice had higher adduct levels than female mice (3.1-fold increase, 95% CI = 2.1- to 4.5-fold; P<.001). In the context of this analysis we had at least 80% power of detecting a 1.8-fold change in adduct levels due to the genotype. We also had at least 80% power of detecting 3.3-, 3.0-, 3.0-, and 9.0-fold effects of the sex-by-TCDD, genotype-by-TCDD, sex-by-genotype, and sex-by-genotype-by-TCDD interactions, respectively, at the {alpha} = .05 significance level.

Finally, we examined whether mice with high liver ABP–DNA adduct levels also had high bladder ABP–DNA adduct levels. Fig. 6Go shows the correlation of bladder adducts with liver adducts for individual mice in each of the four treatment groups. The correlation coefficients were 0.73 (P = .002) for male mice and 0.57 (P = .03) for female mice.



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Fig. 6. Correlation between the relative 4-aminobiphenyl (ABP)–DNA adduct levels using relative adduct labeling (RAL) in the urinary bladder and the liver of individual male (A) and female (B) mice. ABP–DNA adduct levels were measured in Cyp1a2(+/+) and Cyp1a2(–/–) mice 24 hours after topical treatment with ABP, with or without 2,3,7,8-tetrachlorodibenzo-p-dioxin pretreatment. Data shown are from 16 males and 16 females (the same 32 mice used to generate the data presented in Fig. 5Go), from one of the two (N = 32) mouse experiments. Correlation coefficients (r) and P values were determined using the Pearson correlation coefficient test.

 

    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The importance and relevance of an association between ABP–DNA adducts and urinary bladder toxicity as a prelude to cancer has been previously discussed (1223). If hepatic CYP1A2 were the primary enzyme involved in ABP metabolic activation to reactive intermediates (Fig. 1Go), we would predict that Cyp1a2(–/–) mice would have no liver ABP–DNA adducts or fewer adducts than Cyp1a2(+/+) mice. Furthermore, we would expect that TCDD-pretreated Cyp1a2(+/+) mice would have even more ABP–DNA adducts than Cyp1a2(+/+) mice not pretreated with TCDD, because TCDD causes an approximately fivefold induction of hepatic CYP1A2 protein expression (Fig. 2Go). However, we observed the opposite result in both instances (Fig. 5Go, A). First, the Cyp1a2(–/–) knockout mice had similar or higher levels of ABP–DNA adducts than their Cyp1a2(+/+) wild-type counterparts. Second, in mice pretreated with TCDD, the level of liver adducts was either lowered or did not change compared with that in the corresponding group of mice that did not receive TCDD.

The model shown in Fig. 1Go suggests that hepatic CYP1A2–mediated metabolic activation of ABP leads to the accumulation of reactive N-hydroxy metabolite in the bladder. On the basis of this model, we predicted that, after ABP treatment, Cyp1a2(+/+) mice would have more bladder ABP–DNA adducts than would Cyp1a2(–/–) mice. We also predicted that TCDD pretreatment, which magnified the differences in CYP1A2 protein expression between these two genotypes (Fig. 2Go), would be associated with even more bladder ABP–DNA adducts in Cyp1a2(+/+) mice than in Cyp1a2(–/–) mice. For both predictions, we observed the opposite (Fig. 5Go, B). First, in all four of the treatment groups, Cyp1a2(+/+) mice did not display statistically significantly higher levels of bladder ABP–DNA adducts than the Cyp1a2(–/–) mice. Notably, however, among female mice in the TCDD pretreatment group, Cyp1a2(–/–) mice had statistically significantly (P = .013) more bladder ABP–DNA adducts than Cyp1a2(+/+) mice. Second, we found that, similar to what we observed in liver, TCDD-pretreated mice had lower levels of bladder ABP–DNA adducts than the corresponding mice that did not receive TCDD. However, this difference was statistically significant only in females. Intriguingly, we found a sex difference in the bladder that was the opposite of what we observed in liver: males had higher levels of bladder ABP–DNA adducts than females. This finding might be related to urinary pH, which is known to be lower in male mice than in female mice (39).

There are more than 60-fold differences in CYP1A2 activity between individuals in any human population studied; these differences reflect altered gene transcription rates, mRNA and protein levels, and/or enzyme activity (6,8). By contrast, several studies have demonstrated an association between high CYP1A2 activity or protein levels and ABP-mediated N-hydroxy metabolite and adduct formation (28,4043). In the present study, however, we found no association between CYP1A2 levels and either ABP-induced oxidative stress or ABP–DNA adduct formation.

The findings described in the present study thus contradict the generalizations that have been made on the basis of results from both cell culture and in vitro studies, in which either microsomes or purified or cDNA-expressed P450 enzymes were used. For example, ABP is metabolically activated in vitro to the N-hydroxy form by CYP1A2 to a greater extent than any other P450 enzyme (8,28,4043). N-hydroxy-ABP is metabolized further by one or more UDP glucuronosyltransferases, N-acetyltransferases, and/or sulfotransferases to yield nitrenium ions that bind to DNA and proteins (4347). The involvement of CYP1A2 (or, for that matter, any enzyme) in causing toxicity or protecting against toxicity depends on a number of variables: the route of administration, the target organ, the cellular and subcellular location of the enzyme, the tissue specificity of enzyme expression, the dose and duration of exposure to the test chemical, and the extracellular and intracellular accumulation and distribution of the parent chemical and its metabolites. Our results suggest that studies in the intact animal—rather than in vitro or in cell culture—are absolutely necessary in evaluating the ultimate role of CYP1A2 (or any other enzyme) in environmental chemical–induced toxicity versus protection.

How relevant might these findings in mice be to clinical populations? Species differences in ABP metabolism are well known [e.g., (48)]. To address possible human–mouse differences in ABP metabolism, we used the modified vaccinia virus–T7 RNA polymerase system (49) to overexpress the human CYP1A2 and mouse CYP1A2 proteins. ABP N-hydroxylation, as well as ABP–DNA adduct formation, by human CYP1A2 was found to be approximately three times higher than that by mouse CYP1A2 (Dalton TP, Talaska G, Nebert DW: unpublished data). These data therefore suggest that differences in liver and bladder ABP–DNA levels between high and low expressors of human CYP1A2 might be greater than those between high and low expressors of mouse CYP1A2.

In the present study, we have shown that CYP1A2-mediated ABP metabolic activation to toxic and reactive intermediates in vitro or in cell culture thus cannot be extrapolated to ABP–DNA adduct formation in intact mice exposed to topical ABP. One previous clinical study (50) also found no association between high CYP1A2 levels and ABP–hemoglobin adduct formation in cigarette smokers. Two other recent reports (51, 52) using Cyp1a2(–/–) knockout mouse lines have come to similar conclusions. Kimura and coworkers (51) showed that Cyp1a2(–/–) and Cyp1a2(+/+) mice do not exhibit substantial differences in ABP-induced hepatocellular adenoma, carcinoma, or preneoplastic foci. Shertzer et al. (52) found that ABP-induced methemoglobinemia was higher in Cyp1a2(–/–) knockout mice than in Cyp1a2(+/+) mice—the opposite of what had been expected.

Additional arylamines that are proven CYP1A2 substrates in vitro include two cooked meat–derived heterocyclic amines, 2-amino-3-methylimidazo[4,5f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP) (8). Recent studies (53,54) comparing Cyp1a2(–/–) mice with Cyp1a2(+/+) wild-type mice also imply that CYP1A2 in the intact animal differs from CYP1A2 in vitro with regard to IQ and PhIP metabolism. These studies include the effect of intraperitoneal administration of IQ or PhIP on DNA adducts in liver, kidney, mammary gland, and colon (53) and the effect of PhIP on the incidence of several types of malignancies (54).

In the present study, we found that female mice had more hepatic ABP–DNA adducts than male mice, whereas male mice had more urinary bladder ABP–DNA adducts than female mice (Fig. 5Go). These findings are consistent with those of Flammang et al. (55). These observations are interesting because a sex difference for human bladder cancer has been reported: The risk appears to be higher in women than in men who have smoked a comparable number of cigarettes (56).

Urinary pH may also play a pivotal role in promulgating the N-hydroxy intermediate, as well as the glucuronide, of arylamines to react with urothelial DNA and protein. The importance of acidic urinary pH (Fig. 1Go) in causing urothelial cell arylamine-DNA adduct formation has been demonstrated not only in laboratory animals (25) but also in exposed worker populations (20).

Recent studies have suggested that NAT2 might not be a major factor in ABP detoxication. For example, in mouse lines congenic for rapid versus slow acetylator (NAT2) status, liver ABP–DNA adduct levels are independent of acetylator status, whereas urinary bladder ABP–DNA adduct levels are higher in rapid acetylators than in slow acetylators (55). Other studies in hamsters (57) and in humans (58) also found no association between the slow-NAT2-acetylator status and increased ABP metabolic activation or adduct formation. Studies in vitro with N-acetyltransferase activity thus also contradict studies with this detoxication pathway in the intact animal. Instead of the previously widely accepted scheme illustrated in Fig. 1Go, therefore, we suggest a more accurate representation of the possible mechanism for how liver and bladder ABP–DNA adduct formation might occur (Fig. 7Go).



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Fig. 7. Proposed scheme to explain 4-aminobiphenyl (ABP)–induced liver and urinary bladder ABP–DNA adducts, oxidative stress and, possibly, bladder cancer. Hepatic cytochrome P450 (CYP) enzymes other than CYP1A2, and perhaps non-P450 enzymes (e.g., prostaglandin H synthase-1 and/or synthase-2), presumably play an important role in the metabolic activation of ABP to N-hydroxy-ABP and, ultimately, to the electrophilic nitrenium ion (lower left), which is capable of forming DNA and protein adducts. If N-acetyltransferase-1 (NAT1) plays an important role in ABP activation in the urinary bladder (64,65), a nitrenium ion at acid pH would be formed from the unstable O-acetate metabolite, as shown, rather than the conventional N-acetyl-ABP, which is a stable hydroxylamide at acid pH. Several studies (5557) suggest that N-acetyltransferase-2 (NAT2) is not the principal enzyme involved in ABP detoxication. CYP(?) = one or more unknown cytochromes P450; non-CYP(?) = one or more unknown enzymes other than a cytochrome P450; CYP = cytochrome P450 that provides a ring hydroxylation; UGT = uridine diphosphate (UDP) glucuronosyltransferase; NAT2 = N-acetyltransferase-2; GST = glutathione S-transferase; Gluc = glucuronide conjugate; -COCH3 = acetyl group; -SG = glutathione conjugate; SULT = sulfotransferase.

 
If hepatic CYP1A2 is not the primary enzyme responsible for the metabolic activation of ABP in the intact mouse, what enzyme(s) might be? It is possible that other P450 cytochromes, such as CYP4B, prostaglandin H synthases-1 and -2 (59), and/or other enzymes might be involved (Fig. 7Go). For example, CYP4B activity is another P450 expressed in the urothelium, and its capacity to cause mutagenic activation of arylamines has been described (60). Although a peroxidase-mediated activation of N-hydroxy-ABP (61) and a microsomal NADH-dependent reductase that converts the N-hydroxy-ABP back to the parent amine (62) have been reported, these reactions appear to be relatively minor pathways. Even though CYP1A2 usually plays a major role in arylamine metabolic activation, at least in vitro and in cell culture, it should be noted that the cytochromes P450 CYP1A1 and CYP1B1––particularly in extrahepatic tissues—are also able to metabolize ABP at some level (63). CYP1A1 and CYP1B1 are induced by TCDD, as is CYP1A2, and our finding that TCDD pretreatment always lowered ABP–DNA adduct levels (Fig. 5Go) is consistent with TCDD-induced CYP1A1 and CYP1B1 participation in ABP detoxication. Other P450 enzymes in the CYP2 and CYP3 gene families have been shown not to metabolically activate ABP (46).

If NAT2 does not play a major role in ABP detoxication, what about the NAT1 enzyme? Following the initial report that NAT1 activity in the urinary bladder might be a causative factor in ABP–DNA adduct formation in the bladder (64), Taylor et al. (65) suggested that the slow-NAT2-acetylator status, together with a high NAT1 activity in the urinary bladder, might be the most important combination for ABP–DNA adduct formation and tumorigenesis in the bladder (Fig. 7Go).

If ABP–DNA adducts form in both liver and bladder, why do tumors sometimes occur in bladder and sometimes in liver? Urinary pH appears to be one important factor (20,25). The observation that ABP increases the turnover of bladder urothelial cells (66) might also be relevant.

Finally, the sudden decrease, followed by a return to normal, in hepatic thiol status (Fig. 4Go) confirms that the dose of ABP chosen for our studies was ideal for answering the questions that we posed. It is, however, possible that a smaller or larger dose of ABP, chronic instead of acute exposure, or a different route of administration might produce different results. Under the paradigm we chose to study, however, CYP1A2 does not play a critical role in ABP metabolic activation to form DNA adducts in either mouse liver or urinary bladder.

Regardless of the hepatic enzyme(s) involved in the N-hydroxy-ABP formation, it is possible that one or more sulfotransferases or UDP glucuronosyltransferases might be the pivotal step in catalyzing the N-hydroxy intermediate to the O-sulfate or O-glucuronide, either of which would promote the formation of nitrenium ions (Fig. 7Go, lower left). Urinary bladder NAT1-mediated O-acetylation (64,65) would also produce an unstable intermediate that has the capacity to form highly reactive nitrenium ions. The nitrenium ion is a potent electrophile that is capable of binding to hydroxyl and sulfhydryl groups, including those of GSH and cysteine. Thus, the nitrenium ion is a likely reason for the ABP-induced depletion of liver GSH and cysteine (Fig. 4Go). Because it is a highly reactive electrophile, the nitrenium ion is the most likely candidate leading to ABP–DNA adduct formation (47). One or more P450 enzymes are likely to perform the ring hydroxylation in the ABP detoxication pathway (Fig. 7Go). N-glucuronide, acetate, and glutathione (15) conjugation are also detoxication steps likely to be important. Because TCDD is known to induce several UDP glucuronosyltransferases and glutathione S-transferases (67), the effect of TCDD-mediated lowering of ABP–DNA adducts in both liver and bladder (Fig. 5Go) might reflect induction of these phase II enzymes. Which of these enzymes plays the critical role in ABP detoxication, however, remains to be determined.


    NOTES
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Present address: Y. Tsuneoka, Yokohama Hospital, Yokohama, Japan.

Supported in part by Public Health Service grants R01 ES06321 and P30 ES06096 (D. W. Nebert) from the National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services.

We thank Lucia Jorge-Nebert for valuable discussions, Stacey Andringa for help with the microscopy, and Kathleen LaDow and Brenda Schumann for technical assistance. These data were presented at the 21st Annual Meeting of the Society of Toxicology, San Francisco, CA (March 2001).


    REFERENCES
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Manuscript received December 23, 2002; revised June 4, 2003; accepted June 19, 2003.


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