Biotransformation of Benzo[a]pyrene in Ahr Knockout Mice Is

Feb 16, 2009 - Section for Toxicology, The National Institute of Occupational Health, P.O. ... N-0033 Oslo, Norway, and Institute of Cancer Research, ...
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Chem. Res. Toxicol. 2009, 22, 584–591

Biotransformation of Benzo[a]pyrene in Ahr Knockout Mice Is Dependent on Time and Route of Exposure Carlos Sagredo,*,† Steen Mollerup,† Kathleen J. Cole,‡ David H. Phillips,‡ Heidi Uppstad,† and Steinar Øvrebø† Section for Toxicology, The National Institute of Occupational Health, P.O. Box 8149 Dep., N-0033 Oslo, Norway, and Institute of Cancer Research, Brookes Lawley Building, Sutton SM2 5NG, United Kingdom ReceiVed October 3, 2008

Benzo[a]pyrene (BP) is an ubiquitous environmental pollutant with potent mutagenic and carcinogenic properties. The Ah receptor (Ahr) is important in the metabolic activation of BP and is therefore central to BP-induced carcinogenesis. Although Ahr(-/-) mice are refractory to BP-induced carcinogenesis, higher levels of BP-DNA and -protein adducts were formed in them than in wild-type mice. These results indicated the presence of an Ahr-independent and/or a slower biotransformation of BP in Ahr knockout mice. To address this issue further, we have now performed a time-course experiment, with mice receiving a single oral dose of BP (100 mg/kg). Wild-type mice have an effective clearance of BP metabolites, mainly through 3-hydroxybenzo[a]pyrene and 9-hydroxybenzo[a]pyrene in the feces with reduced levels of DNA and protein adducts in the examined tissues. On the other hand, the Ahr(-/-) mice appear to have a lower metabolic clearance of BP resulting in increased levels of DNA and protein adducts and of unmetabolized BP. In addition, we have performed an administration route experiment and found that skin-exposed Ahr(-/-) mice showed lower levels of protein adducts along with markedly reduced P450 1B1 expression, but only in the exposed area, as compared with the wild-type mice. In addition, the systemic uptake of BP is increased in the Ahr(-/-) mice as compared with the wild-type mice. Hence, the lack of a functional Ah receptor results in an Ahr-independent biotransformation of BP with a slower clearance of BP and higher levels of DNA and protein adducts, but the distribution and levels of BP and BP-protein adducts are clearly dependent on the route of exposure. Introduction Polycyclic aromatic hydrocarbons (PAHs) are formed during incomplete combustion of organic matter and fossil fuels in industrial processes, automobile exhaust, cigarette smoke, and charbroiled food (1, 2). They are ubiquitous environmental contaminants found in air, water, and soil. Exposure to PAHs is high in some occupational environments where workers are exposed mainly through inhalation and skin absorption. For the general population, exposure is from smoking, diet, and air pollution (3-5). Benzo[a]pyrene (BP) is an important member of the PAH family and has served as a model for the study of the metabolic pathways and carcinogenic effects of PAHs (6-10). BP acts as a ligand for the aryl hydrocarbon receptor (Ahr), which upregulates the expression of phase I bioactivation genes and phase II conjugation genes (11, 12). This leads to an induction of important biotransformation enzymes, like P450 1A1 and P450 1B1, that metabolically activate BP to reactive intermediates, among them the carcinogenic BP diol epoxides (BPDEs). The BPDEs are known to form DNA and protein adducts. The formation of DNA adducts is considered to be the first step in the initiation of PAH-induced carcinogenesis (13, 14). Furthermore, it has been suggested that adduct levels may be predictive of cancer risk (15, 16). High levels of BPDE-DNA adducts have been found in human lung and stomach cancer tissue (13). * To whom correspondence should be addressed. Tel: +47 23 19 51 00. Fax +47 23 19 52 00. E-mail: [email protected]. † National Institute of Occupational Health. ‡ Institute of Cancer Research.

The discovery of the involvement of the cytochrome P450 1A1 and the Ahr signaling pathways in the metabolism of PAHs has led to several studies with Ah-responsive/-nonresponsive mice and P450 and Ahr knockout mouse models (17-28). Ah-nonresponsive mice have been shown to be at a lower risk of mutagenesis and carcinogenesis than Ah-responsive mice, when exposed to PAH by topical application or intraperitoneal (i.p.) injection. On the other hand, when the PAH was administered orally, Ah-nonresponsive mice experienced higher toxicity and tumor formation at organs distal to the site of administration and reduced survival time as compared with Ah-responsive mice (24). The latter showed higher risk of toxicity and tumor induction in organs at the site of administration, and also longer survival time, than Ah nonresponsive mice. Similar observations were made with knockout mouse models of P450 1A1, P450 1A2, and P450 1B1. These studies show that the Ahr-inducible P450 1A1 and P450 1B1 play important and possibly different roles in the activation and the detoxification of BP (17). In general, the knockout mice experienced higher levels of toxic lesions and higher mortality rates, together with a slower clearance of BP and increased levels of BP-DNA adducts than the wild-type mice (17, 26-28). In Ahr knockout mouse models, Ahr(-/-) mice have been found to be resistant to TCDD-induced toxicity and BP-induced carcinogenicity in skin (18, 25, 29, 30). When a single i.p. dose of BP was administered to Ahr(-/-) and wild-type mice, the formation and distribution of DNA adducts, as measured by 32P-postlabeling, were different, but the total sum of hepatic BP-DNA adducts was the same (21). End responses, such as toxicity, lethality, and DNA

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adduct formation, are clearly dependent on both the route of administration and the dose (24, 31). In our previous report, we studied the relationship between Ahr genotype and bioactivation of BP in internal mouse organs, after a single oral dose of BP. We reported higher levels of protein adducts and BP metabolites in Ahr(-/-) mice tissue as compared with wild-type mice, indicative of an Ahr-independent and/or a slower biotransformation of BP in mice lacking Ahr (32). In the present study, we have performed a time-course experiment for 6 months in mice that received a single oral dose of BP. The levels of protein and DNA adducts in internal organs and the levels of P450 1A1 and P450 1B1 gene expression, in addition to the clearance of BP and metabolites in urine and feces, are reported. In addition, we have compared the effect of the route of administration on the formation of protein adducts in skin and internal organs after a single topical or i.p. dose of BP.

Materials and Methods Chemicals. Standards for (()-benzo[a]pyrene-r-7,t-8,t-9,c-10tetrahydrotetrol (BP-tetrol I-1), (()-benzo[a]pyrene-r-7,t-8,t-9,10tetrahydrotetrol (BP-tetrol I-2), (()-benzo[a]pyrene-r-7,t-8,c-9,t10-tetrahydrotetrol (BP-tetrol II-1), (()-benzo[a]pyrene-r-7,t-8,c9,c-10-tetrahydrotetrol (BP-tetrol II-2), benzo[a]pyrene-4,5-dihydrodiol, benzo[a]pyrene-7,8-dihydrodiol, benzo[a]pyrene-9,10-dihydrodiol, benzo[a]pyrene-3-phenol, and benzo[a]pyrene-9-phenol were purchased from the National Cancer Institute (NCI), Chemical Carcinogen Repository (Midwest Research Institute, Kansas City, MO). Standards for the two remaining tetrols, benzo[a]pyrene-r7,c-8,t-9,c-10-tetrahydrotetrol (BP-tetrol III-1) and benzo[a]pyrener-7,c-8,t-9,t-10-tetrahydrotetrol (BP-tetrol III-2), were prepared in our laboratory as described earlier (33). BP was purchased from Sigma (St. Louis, MO). HPLC grade methanol was obtained from Fluka (Buchs, Switzerland). Water was obtained from a Milli-Q ultrapure water purification system (Millipore, Bedford, MA). Corn oil was purchased from a local food store. Animals and Treatment. The Ahr heterozygote model (C57BL6) was a kind gift from Dr. T. Ishikawa, Department of Pathology, Graduate School of Medicine, University of Tokyo, Japan. The animals were maintained in a germ-free facility using an air-filtered controlled environment. The Ahr(+/-) were interbred to generate Ahr(+/+) (wild-type), Ahr(+/-), and Ahr(-/-) mice. Genotyping was carried out as described in ref 25. To verify genotypes, realtime RT-PCR measurement of Ahr expression was carried out on tissue samples at the end of the experiments. There were no observed differences in growth rate and appearance between the different genotypes. We also did not observe any signs of illness or toxicity among the mice during the exposure studies. The animals were fed a standard diet (B&K Universal A/S, Norway) and given water ad libitum. All animal experiments were conducted in accordance with the standards for care and use of experimental animals approved by the Norwegian Reference Centre for Laboratory Animal Science & Alternatives. The weight of the animals ranged between 20 and 40 g at the start of the experiment and did not vary significantly throughout the study. Each group of animals consisted of one female mouse and two male mice of same genotype, and they were matched according to their weight. The time-course experiment included three Ahr(-/-) and three wild-type mice for each time point, that is, day 1, day 3, day 5, day 7, day 21, 2 and 6 months, and in total 36 animals. The animals were exposed to a single oral dose of BP (100 mg/kg) solubilized in corn oil (10 mg/mL). The animals were placed in metabolism cages for 24 h, for collection of urine and feces, prior to each time point of sacrifice. The animals were then sacrificed, and lung, liver, spleen, kidney, and heart were isolated and collected at each time point. The route-of-administration experiments included three mice of each genotype, for both i.p. and skin treatment, in total 12

Figure 1. Levels of the protein adducts (PAs) in internal mouse organs at different times after one oral dose of BP (100 mg/kg). The levels of PA are higher in all of the tissues of Ahr(-/-) mice (A) as compared with wild-type mice (B). Values are given as means ( SDs (n ) 3).

Figure 2. Comparison of DNA adduct levels in mouse lung between Ahr(-/-) and wild-type mice at different times after oral exposure to a single dose of BP (100 mg/kg). Values are given as means ( SDs (n ) 3).

animals. One group of mice was exposed to a single i.p. dose of BP (100 mg/kg) solubilized in corn oil (10 mg/mL). The other group was exposed to a single topical application with BP (100 mg/kg) solubilized in tetrahydrofuran (10 mg/mL). The animals were sacrificed after 24 h, and lung, liver, spleen, kidney, heart, and skin were harvested. For the topically exposed groups, in addition, skin samples were collected from the application site (defined as exposed skin) and from a site distant to it (defined as distal skin). Protein and DNA Isolation. Harvested organs were homogenized in a phosphate buffer solution using a Polytron homogenizer followed by brief centrifugation. The pellet was used for DNA

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Figure 3. Comparison of excretion in feces between Ahr(-/-) and wild-type mice treated with one oral dose of BP (100 mg/kg): BPphenol levels (A), BP-diol levels (B), and unmetabolized BP levels (C). Values are given as means ( SDs (n ) 3).

isolation, and the supernatant was used for protein quantification and isolation. Protein concentrations were determined by the Lowry method (34). Protein Adduct Purification. The ultimate carcinogenic diol epoxide, BPDE-I, gives rise to two tetrols, BP-tetrol-I-1 and BPtetrol-I-2. The less carcinogenic diol epoxide, BPDE-II, gives rise to two different tetrols, BP-tetrol-II-1 and BP-tetrol-II-2. BP-protein adducts were measured as released BP-tetrols after acid hydrolysis. The use of tetrols as surrogate markers for diol epoxides is a highly specific and valuable biomarker method (35, 36). To 900 µL of protein solution was added 100 µL of 1 M HCl, and this solution was incubated at 70 °C for 3 h. Water and methanol were added to a final volume of 5 mL with 10% methanol. The Sep-Pak C18 cartridges were conditioned with 10 mL of methanol and 10 mL of water prior to use. The protein solution was then applied to a preconditioned Sep-Pak C18 cartridge (Millipore, Milford, MA), followed by washing with 10 mL of water, and then, the tetrols were eluted with 5 mL of methanol. The eluate was evaporated at 45 °C under a nitrogen stream, and the residue redissolved in 500 µL of 10% methanol. Samples were stored at -20 °C. Urine and Feces. The amounts of collected urine and feces were weighed and processed immediately. The samples were treated as described by Jongeneelen et al. (37). In brief, urine samples were

Figure 4. Comparison of excretion in urine between Ahr(-/-) and wild-type mice treated with one oral dose of BP (100 mg/kg): BPphenol levels (A), BP-diol levels (B), and unmetabolized BP levels (C). Values are given as means ( SDs (n ) 3).

diluted with acetate buffer (0.1 M, pH 5), β-glucoronidase/ arylsulphatase was added, and the mixture was incubated overnight at 37 °C for deconjugation. Fecal samples were homogenized in acetate buffer (0.1 M, pH 5) and centrifuged (1200g for 10 min). β-Glucoronidase/arylsulphatase was then added to the supernatant for deconjugation and incubated overnight at 37 °C. The samples were then applied to preconditioned Sep-Pak C18 cartridges, using the same procedure as for tetrol purification. HPLC-Fluorescence Quantitation. The analysis was performed on an Agilent 1100 LC system using a Hypersil ODS, 5 µm, 3.9 mm × 150 mm column (Agilent) attached to an Agilent 1100 fluorescence detector. The injection volume was typically 20 µL, and the samples were separated by a linear gradient of methanol: water (30-100% methanol over 40 min; flow rate, 1 mL/min). The column temperature was 40 °C. The excitation and emission wavelengths were 341 and 381 nm, respectively. The tetrols and metabolites were identified and quantified as described previously (32, 33).

Biotransformation of Benzo[a]pyrene in Ahr Knockout Mice

Figure 5. Effect of i.p. exposure to BP on the expression of P450 1A1 and P450 1B1 levels in lung and liver. The levels of mRNA were measured by real-time RT-PCR and normalized to the expression of β-actin. 32

P-Postlabeling Analysis of DNA Adducts. DNA was extracted from lung tissue, and BP-DNA adducts were measured by 32Ppostlabeling analysis with the nuclease P1 modification as described previously (38). Labeled BP-DNA adducts were resolved by thinlayer chromatography, and results are expressed as adducts/108 nucleotides. Gene Expression. Gene expression measurements were carried out by quantitative real-time RT-PCR on an ABI PRISM 7900 (Applied Biosystems) as described previously (39). In brief, total RNA was reverse transcribed by the aid of random primers. PCR primers for β-actin (Actb), P450 1A1, and P450 1B1 were as in ref 39, and those for Ahr were as in ref 32. The amount of target cDNA in each sample was established by determining a fractional PCR threshold cycle number (Ct value). Specific gene expression levels were normalized to the expression of β-actin and calculated by the formula: 2-(Ct gene Ct β-actin). Statistical Analysis. For the analysis of gene expression, protein adducts, and metabolites, means were compared by the independent samples t-test with Welch’s correction.

Results We performed a time-course experiment, which lasted up to 6 months, with groups of Ahr(-/-) and wild-type mice, exposed to a single oral dose of BP. In general, we found higher protein adduct levels in all of the tissues of Ahr(-/-) as compared with wild-type mice (Figure 1). The levels were highest after day 1 in all organs except spleen, which had a peak level at day 3, for both mouse strains. In addition, spleen had the highest levels of BP-protein adducts, while kidney had the lowest for both genotypes (Figure 1). The BP adduct levels in the Ahr(-/-) remained high and tended to fall below 400 fmol/mg protein after day 7, except in spleen, which had levels of about 900 fmol/mg protein at day 7 and about 400 fmol/mg protein at day 21. For the wild-type mice, the BP adduct levels in spleen fell below 200 fmol/mg protein at day 5, while in all other tissues they were below 200 fmol/mg protein even at day 1. After 2 months (60 days), there were still traces of BP adduct levels in both Ahr(-/-) and wild-type mice (Figure 1). We did not detect any BP-protein adducts in any tissues 6 months after treatment (data not shown). The formation of BP-DNA adducts was measured in lung by the 32P-postlabeling method. Significant levels of DNA adducts were detected throughout the period of 1-60 days (Figure 2). Peak levels were detected after 5-7 days in the

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Ahr(-/-) mice, whereas the highest levels were observed after 3-5 days in the wild-type. Throughout the time period from 1 to 21 days, BP-DNA adduct levels were higher in Ahr(-/-) as compared to wild-type. A faster rate of decay was possibly observed in the knockouts, whose adduct levels were approaching the levels of the wild-type mice after 60 days. We did not observe a difference in the DNA adduct pattern between the two mouse strains. In both cases, nearly all of the radioactivity eluted as a single spot, corresponding to the BPDE-dG adduct. The excretion of BP, BP diols, and BP phenols in feces and urine between day 1 and day 21 is shown in Figures 3 and 4. The wild-type mice had higher BP-phenol levels in feces at day 1 and day 2, with a peak excretion at day 2. Between day 3 and day 7, the BP-phenol levels were lower in the wild-type mice, while at day 21, the levels were the same. The excretion of BP diols remained higher in the Ahr(-/-) relative to the wild-type mice, between day 1 and day 7. At day 21, the excretion levels were the same for both strains (Figure 3). Although levels of excreted BP and BP metabolites were higher (but not significantly different) in the Ahr(-/-) than in the wild-type mice, only a small proportion of the total excretion was observed in urine (Figure 4). Other groups of Ahr(-/-) and wild-type mice were exposed to a single topical or a single i.p. dose of BP for 24 h. The levels of P450 1A1 and P450 1B1 gene expression were measured in lung and liver after i.p. exposure. In the Ahr(-/-) mice, lower expression of P450 1A1 was found in both lung and liver as compared with wild-type mice, while P450 1B1 was lower only in the lung (Figure 5). The levels of P450 1A1 and P450 1B1 gene expression in the liver, lung, and skin after dermal application of BP are shown in Figure 6. Expression of P450 1A1 was lower in lung, liver, and in both exposed and unexposed skin area from Ahr(-/-) mice, as compared to the wild-type. Expression levels for P450 1B1 were lower only in the lung and the treated area of skin of the knockout mice, when compared with corresponding wild-type mice. Protein adduct levels after i.p. treatment with BP were significantly higher in livers of Ahr(-/-) than in those of wild-type mice (Figure 7A). For the rest of the measured internal organs, adduct levels were higher, but not statistically significantly, in the knockout mice. The total levels of protein adducts were highest in spleen, with the rest of the tissues showing approximately the same level of adducts (Figure 7A). After dermal application of BP, the differences in BP-protein adducts levels in the internal organs between Ahr(-/-) and wild-type mice were more prominent (Figure 7B). Liver and spleen showed significantly higher protein adduct levels in the Ahr(-/-) than in the wild-type mice. In contrast, the BP-protein adduct levels in the exposed skin area were considerably lower in the Ahr(-/-) mice than in the wildtype mice, while the BP-protein adduct levels in the unexposed skin were opposite, higher in the Ahr(-/-) as compared with the wild-type (Figure 7B). The levels of unmetabolized BP in the internal organs after i.p. exposure were not statistically different between the two strains, except in lung, where they were a factor of 6 lower in the Ahr(-/-) mice. In both strains, the lung and spleen showed the highest levels of unmetabolized BP (Table 1). In the skin-exposed group, the levels of unmetabolized BP were in general higher in the internal organs of the Ahr(-/-) as compared to the wild-type but not in the skin. The BP levels in lung, liver, and kidney were significantly higher in the Ahr(-/-) than in the wild-type mice. In unexposed skin, the BP levels were approximately equal in

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Figure 6. Effect of dermal exposure to BP on the expression of P450 1A1 and P450 1B1 levels in lung, liver, and skin. The levels of mRNA were measured by real-time RT-PCR and normalized to the expression of β-actin.

both strains. The BP levels in lung and liver were 10 and 30 times higher in the Ahr(-/-) than in the lung and liver, respectively, of the wild-type mice. Furthermore, the lung and unexposed skin showed the highest levels of unmetabolized BP in both strains (Table 1).

Discussion In a previous study, we found higher levels of protein adducts in the internal organs of the Ahr(-/-) mice as compared with the wild-type mice, when the mice received a single oral dose

of BP. These results could be explained by an Ahr-independent and/or a slower biotransformation of BP in mice lacking the Ahr (32). Time Study. In this study, we have shown that Ahr(-/-) mice exhibit higher levels of BP protein adducts over time as compared to wild-type mice. The levels of protein adducts in the Ahr(-/-) tend to approach the levels of protein adducts in the wild-type mice after 21 days, showing evidence of a slower biotransformation of BP in the Ahr(-/-). The excretion profiles in urine and feces for both genotypes are in agreement with other studies (40), but there are differences regarding the levels

Biotransformation of Benzo[a]pyrene in Ahr Knockout Mice

Figure 7. Total sum (fmol/mg proteins) of the protein adducts in kidney, heart, lung, liver, spleen, and skin after i.p. (A) and skin (B) exposure to a single dose of BP (100 mg/kg). Values are given as means ( SDs (n ) 3). The levels of significance used were p e 0.05.

Table 1. Levels of Unmetabolized BP (fmol/mg Protein) in Internal Organs after i.p. and Skin Exposure to BP (100 mg/kg) IP-exposed lung spleen kidney liver skina heart

skin-exposed

(+/+)

(-/-)

(+/+)

(-/-)

1199 ( 219 600 ( 284 84 ( 16 44 ( 19 44 ( 64 11 ( 5

166 ( 51 387 ( 54 69 ( 11 23 ( 20 163 ( 106 38 ( 30

66 ( 51 0.7 ( 0.2 0.4 ( 0.1 2.6 ( 0.5 249 ( 390 1.3 ( 1.1

1064 ( 396 10 ( 4 11 ( 4 75 ( 28 280 ( 123 8(3

a Each value represents the mean ( SD. For the i.p.-exposed animals, only lung (p ) 0.015) shows statistically differences between genotypes. For skin-exposed animals, lung (p ) 0.049), kidney (p ) 0.044), and liver (p ) 0.048) show statistically significant differences between genotypes. The level of significance used was p e 0.05. Measurements were done only in skin distal from the application site.

of BP and BP metabolite excretion between the Ahr(-/-) and the wild-type mice. Although not statistically significant, the wild-type mice showed higher levels of excretion of BP and BP phenols, especially 3OH-BP, a known metabolite produced from P450 1A1, in feces than did Ahr(-/-) mice. In addition, organs distal to the stomach (the site of administration) like lung and spleen in the Ahr (-/-) mice showed the highest levels of unmetabolized BP, as compared with the other organs in Ahr (-/-) mice, and to the lung and spleen in the wild-type mice. The high levels of BP in several organs in the Ahr (-/-) mice can be explained by reduced metabolism in the liver and therefore less presystemic elimination through the gut (20, 24).

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This has also been observed with Ahr-nonresponsive mice that were orally exposed to BP. The systemic exposure to BP increased in organs distal to the site of administration, as spleen and bone marrow, resulting in higher toxicity of BP and increased cell turnover (20, 24). Route of Exposure. We have compared two other routes of administration, that is, topical and i.p. exposure to BP. When the dose was applied topically, a change in BP-protein adducts pattern between the Ahr(-/-) and the wild-type mice was observed. In this case, the wild-type mice showed higher levels of protein adducts, but only in the exposed skin area, as compared with the Ahr(-/-) mice. The protein adducts levels were a factor of 6 higher in the exposed skin of wild-type as compared with the exposed skin of Ahr(-/-) mice. Similarly, the expression of P450 1A1 and P4501B1 was equally and markedly increased but only when exposed skin was compared with skin distal from the application site in wild-type mice. In addition, there were differences in levels of unmetabolized BP between the Ahr(-/-) and the wild-type mice. The levels of unmetabolized BP where higher in all of the tissues of the Ahr(-/-), with exception of the skin, as compared with the wild-type mice. Shimizu et al. (25) reported that BP carcinogenicity in skin was lost in mice lacking the Ahr. These observations were done on mice receiving a daily topical dose of BP and on mice receiving subcutaneous doses of BP. Our findings show that the skin of the wild-type mice is able to metabolize BP to a much higher extent than the skin of the knockouts, with a resulting increase in the BP-protein adduct levels locally and a reduced systemic uptake of BP. Because BP-protein adducts may be used as cancer risk markers, our result regarding topical exposure to BP supports the findings of Shimizu (25). When a single i.p. dose was applied, the Ahr(-/-) and wildtype mice showed similar levels of BP-protein adducts in the internal organs and the skin. In addition, the BP levels in the internal organs were almost the same between Ahr(-/-) and wild-type mice, with the exception of the lung. The lung of the wild-type mice after i.p. treatment showed higher levels of unmetabolized BP than the lung of the Ahr(-/-) mice. Although a puzzling result, it is reasonable to assume that BP is being metabolized by P450 1A1 and P450 1B1, which are expressed at high levels in the lung of the wild-type mice. Ultimately, we found lower, but not statistically significantly different, protein adduct levels in lung tissue in the wild-type mice than the knockout mice. Our protein adduct measurements are in agreement with the findings of Kondraganti et al. (21), who observed that the total hepatic BP-DNA adduct levels, as measured by 32P-postlabeling, were equal in Ahr (-/-) and wild-type mice after a single i.p. dose of BP. The metabolism of BP observed in our Ahr knockout mice study resembles the metabolism of BP observed with the Cyp1a1 knockout mice and Ah-nonresponsive mice (19, 20, 26). Uno et al. (26) reported an increase of BP-DNA adducts by 32Ppostlabeling when Cyp1a1 was knocked out, together with a slow clearance of BP. The Cyp1a1 (-/-) that received an oral dose of 125 mg/kg/day of BP died within 30 days. In contrast, the wild-type mice survived a whole year without displaying any sign of toxicity. This apparently paradoxical observation was explained by the protective effect of a functional Cyp1a1 gene (11). The follow-up studies by Uno et al. (27, 28) and later Dragin et al. (17) with different P450 knockout mouse lines that received daily doses of BP (i.p. and oral) illustrated the different roles of Cyp1a1 and Cyp1b1 genes in the metabolism of BP. In mice, the Ahr-inducible P450 1A1 appears

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to be more important in the detoxification than metabolic activation of BP (41); in another mouse model in which total cytochrome P450 activity was absent in the liver only, the role of P450 metabolism in general was also shown to be more important for detoxification of BP than for its activation (42). Meanwhile, P450 1B1 may be more important in metabolic activation, immune damage, and adduct formation in spleen and bone marrow (11). The role of P450 1B1 in enhancing the toxicity of BP in bone marrow has also been shown by Galvan et al. (19, 20) in studies with Ah-nonresponsive mice. Hence, in our skin-exposed wild-type mice, the increased expression of P450 1A1 and P450 1B1 resulted in both an activation, that is, increase, of BP-protein adducts levels and in a detoxification, that is, clearance, of systemic BP. Ahr(-/-) mice appear to have significant constitutive levels of P450 1B1, as found in our previous study (32). This could partly explain the observed BP metabolism and the higher formation of protein adducts found in the Ahr(-/-) mice, although the expression of other phase I and II enzymes may also affect the metabolic outcome (41, 43, 44). Our present results show that the Ahr(-/-) in general have higher levels of BP-DNA and -protein adducts, and unmetabolized BP, in all examined tissues as compared with the wild-type mice. These Ahr-mediated observations also appear to follow a gene dose effect as found in our previous study (32). Constitutive P450 1A1 expression level showed an Ahr gene-dose relationship in both the liver and the lung. However, protein adduct levels after exposure to BP showed an inverse relationship as compared to the Ahr gene dose, with Ahr (-/-) > Ahr (+/-) > Ahr (+/+) (32). The lack of a functional Ah receptor results in an Ahrindependent biotransformation of BP with a slower clearance of BP and higher levels of DNA and protein adducts. In the wild-type mice, the liver and skin represent a first-pass effect that reduces the systemic uptake of BP and subsequent formation of DNA adducts (24). Our results also show that the distribution and levels of BP and BP-protein adducts are clearly dependent on the route of exposure (45). Orally exposed Ahr(-/-) mice have higher levels of BP and protein adducts than i.p.- and skin-treated Ahr(-/-) mice (32). Ultimately, this would also affect the risk of both toxic depletion and cancer development. Acknowledgment. We thank Dr. T. Ishikawa for providing the Ahr knockout mouse model. We also thank Einar Eilertsen and Frøydis Kristoffersen for advice and help in animal treatment and Ingrid V. Botnen and Rita Bæra for excellent technical assistance. This study was supported by the Norwegian Research Council, the Norwegian Cancer Society, and by Cancer Research UK.

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