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Carcinogenesis Advance Access originally published online on December 13, 2006
Carcinogenesis 2007 28(6):1371-1378; doi:10.1093/carcin/bgl244
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

7H-dibenzo[c,g]carbazole metabolism by the mouse and human CYP1 family of enzymes

Howard G. Shertzer*, Mary B. Genter, Glenn Talaska, Christine P. Curran, Daniel W. Nebert and Timothy P. Dalton

Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati Medical Center, 3223 Eden Avenue, PO Box 670056, Cincinnati, OH 45267-0056, USA

* To whom correspondence should be addressed. Tel: +513 558 0522; Fax: +513 558 0925; Email: shertzhg{at}ucmail.uc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Found in tobacco smoke, fossil fuel and other organic combustion products, 7H-dibenzo[c,g]carbazole (DBC) is a potent mouse lung carcinogen and potential human carcinogen. Although the first hydroxylation is critical for determining activation versus detoxication, the enzymes responsible for site-specific hydroxylation of DBC are not known. We found that DBC-DNA adduct levels are significantly higher in aromatic hydrocarbon receptor null Ahr(–/–) mice, suggesting that the induction of Aromatic hydrocarbon receptor (AHR)-regulated genes, such as those in the CYP1 family, decrease DBC genotoxicity. Using knockout mice for Cyp1a1, Cyp1a2 and Cyp1b1, we showed that the major CYP1 enzymes that metabolize DBC are CYP1A1 in ß-naphthoflavone (BNF)-induced liver, CYP1A2 in non-induced liver, CYP1B1 and CYP1A1 in induced lung and none in non-induced lung. DBC metabolism by the human CYP1 enzymes was examined in vitro using SupersomesTM. Each mouse CYP1, as well as each human CYP1, has a unique DBC metabolite profile. Comparison of the metabolite profile in BNF-induced mice suggested that CYP1A1 primarily generates 1-OH, 2-OH and (5 + 6)-OH-DBC, whereas CYP1A2 generates primarily (5 + 6)-OH-DBC and CYP1B1 primarily generates 4-OH-DBC. This was similar to that observed in the human CYP1 enzymes. Most importantly, lung CYP1B1 is associated with forming 4-OH-DBC, the most potent metabolite leading to DBC-DNA adducts. These studies suggest that for non-pulmonary routes of exposure (i.e. skin, gastric, i.p.), low hepatic expression of CYP1A2 and CYP1A1, together with high expression levels of lung CYP1B1 and CYP1A1, may define a phenotype for high susceptibility to carcinogens such as DBC.

Abbreviations: BaP, benzo[a]pyrene; BNF, ß-naphthoflavone; DBC, 7H-dibenzo[c,g]carbazole


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Lung cancer due to chemicals in tobacco smoke is the primary cause of cancer deaths in both men and women in the USA, and is the most common lethal malignancy in the world (1). The potent mouse lung carcinogen 7H-dibenzo[c,g]carbazole (DBC) (2) is found in cigarette smoke and other complex pyrolysis mixtures such as those derived from fossil fuels. DBC is considered by the International Agency for Research on Cancer as a group 2B possible human carcinogen (3). Extremely hydrophobic, DBC circulates to all tissues following exposure by any route. For example, topical application led to dose-dependent increases in DBC concentrations in lung, liver and other tissues (4).

The general pathway for DBC metabolism is shown in Figure 1. The first step in DBC metabolism is monooxygenase-mediated formation of monohydroxylated derivatives (5,6). There are four potential DBC activation mechanisms that are initiated by oxidation and may lead to hydroxylation products. The first is the formation of an epoxide, followed by binding of the negatively charged DNA base heteroatoms to the charge-induced carbonium cation. Second, phenols of DBC may undergo further oxidation to diones (7). DBC-dione metabolites with {alpha},ß-unsaturated carbon–carbon bonds may act as Michael reaction acceptors for the nucleic acid bases and nucleosides (7). Third, DBC binding to DNA may occur directly through the reaction of DNA with the cation radical of DBC (2,8). Fourth, the seventh position nitrogen may be hydroxylated, followed by sulfation to produce a strong leaving group and production of the DNA-reactive nitrenium cation.


Figure 1
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  		Fig. 1. General scheme for DBC oxidative activation and detoxication. Adapted from Warshawsky et al. (2).

 
Although the chemistry for DBC metabolism is generally understood, the enzymes responsible for its metabolism are not. CYP1 enzymes appear to be involved in the metabolic activation of DBC (9,10). In Chinese hamster V79 cells, stable expression of CYP1A1 activates DBC to form mutagenic metabolites, whereas co-expression of CYP1A2 and N-acetyltransferase nearly abolished mutagenic activation of DBC (10). In another study, 3-methylcholanthrene induced both CYP1A1 and the production of most of the mono- and dihydroxylated metabolites of DBC (5). The production of total 14C-metabolites from radiolabeled DBC indicated that the oveall disappearance of parent compound may be increased (11) or unchanged (5), depending on the experimental conditions.

The detoxication pathway for DBC is not well known (2). Radiolabeled DBC, administered by aerosol inhalation, circulates throughout the body and, within a few hours, 95% of the dose appears in feces. Unexpectedly, no sulfation or glucuronidation metabolites were found in feces, even though monohydroxylated DBCs (primarily 5- and 3-substituted) are the major products of metabolism (12). These results suggest that excretion products represent either unconjugated DBC metabolites or other conjugation products, possibly N-acetates or glutathione conjugates. Because site-specific monohydroxylation products determine the pathway of DBC leading to activation or detoxication, this study evaluates mouse and human CYP1 enzymes in determining the DBC metabolite profile and elimination pharmacokinetics.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Chemicals and human CYP1 microsomes
DBC (supplied by Dr David Warshawsky, Department of Environmental Health, University of Cincinnati) was synthesized and purified as described previously (13). DBC metabolite standards were synthesized as described (6). All other chemicals and reagents were obtained from Sigma–Aldrich Chemical Company (St Louis, MO) as the highest available grades. Human CYP1 enzymes were obtained from BD Biosciences (Billerica, MA; www.bd.com/biosciences), as SupersomesTM. These microsomes, derived from baculovirus-infected insect cells, contain reduced nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome P450 oxidoreductase and CYP1A1 (Cat. # 456211), CYP1A2 (Cat. # 456203) or CYP1B1 (Cat. # 456220).

Animals and treatment
Experiments involving mice were performed according to the National Institutes of Health standards for care and use of experimental animals and the University of Cincinnati Institutional Animal Care and Use Committee. Animals were group-housed, maintained on a 12 h light–dark cycle and had access to standard rodent chow and water ad libitum. C57BL/6J inbred female mice (8–10 weeks of age) were purchased from Jackson Laboratories (Bar Harbor, ME). The Cyp1a1(–/–) (14), Cyp1a2(–/–) (15), Cyp1b1(–/–) (16) and aromatic hydrocarbon receptor null (Ahr(–/–)) (17) knockout lines on the C57BL/6J background, having disruption of the indicated gene, were generated as described. For induction via the AHR, mice were treated by i.p. injection for 3 consecutive days with either corn oil vehicle or with 80 mg ß-naphthoflavone (BNF)/kg body wt. For DNA adduct studies (Figure 2), mice were treated with a single i.p. injection of 8 mg DBC/kg body wt in corn oil 24 h after the last BNF treatment. For metabolism studies (Figures 3–6), mice were killed by carbon dioxide asphyxiation 24 h after the last treatment, and liver and lung microsomes were isolated by differential centrifugation as described (18). For pharmacokinetic studies (Figure 7 and Table I), mice were treated with a single i.p. injection of 10 mg DBC/kg body wt in corn oil 24 h after the last BNF treatment, and blood was collected from the saphenous vein. This mode of exposure for DBC has been validated for DBC-induced lung cancer (13,19).


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  		Table I. Area under the curve (AUC) mean values ± SEMs

 


Figure 2
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  		Fig. 2. DBC-DNA adducts. After treatment by i.p. injection for 3 consecutive days with 80 mg BNF/kg body wt, mice received 8 mg DBC/kg body wt by i.p. injection. Tissues were excised, frozen on dry ice and DBC-DNA adducts were determined using [32P]-post-labeling. Data are shown as mean values ± SEMs (N = 8). Data were evaluated statistically using a one-way analysis of variance, followed by Student–Newman–Keuls test for pairwise comparison. *P < 0.05 versus wild-type (Ahr(+/+)) genotype at the same time after DBC treatment.

 


Figure 3
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  		Fig. 3. Metabolism of DBC by mouse liver microsomes and human CYPs. Upper figures: mice were treated by i.p. injection for 3 consecutive days with either corn oil vehicle (open bars) or with BNF (black bars), and liver microsomes were isolated by differential centrifugation, 24 h after the last treatment. DBC metabolites were quantified as described in the legend to Figure 4. Overall metabolism was calculated as the sum of the rate of formation of individual fractions, representing various positions of hydroxylation and unknown hydrophilic and hydrophobic fractions. Non-metabolized DBC (parent compound) eluted independently after the elution of each of the shown fractions. Data are shown as mean values ± SEMs (N = 4). Data were evaluated statistically using a one-way analysis of variance, followed by Student–Newman–Keuls test for pairwise comparison. *P < 0.01 versus non-induced microsomes of the same mouse genotype. **P < 0.01 versus each of the other human CYP1s. Lower figures: the specific activities for human CYP1A1, CYP1A2 and CYP1B1 are shown with DBC (left panel) or BaP (right panel) as substrate. BaP is often used as a standard reference substrate for CYP1A1. Results shown as mean values ± SEMs (N = 4).

 


Figure 4
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  		Fig. 4. Metabolism of DBC by mouse liver microsomes. Mice were treated by i.p. injection for 3 consecutive days with either corn oil vehicle (open bars) or with 80 mg BNF/kg body wt (black bars), and liver microsomes were used to assess DBC metabolism. Early eluting fractions, at or near the void volume, were not individually identified, and referred to as ‘unknown hydrophilic’ (unknown hydrophilic metabolites). The sum of peaks eluting at later time are collectively referred to ‘unknown hydrophobic’. The % DBC metabolized is expressed as a percentage of the 60 µM added to the reaction mixture. The DBC recovery as HPLC peaks was between 90 and 105%. The results are the average for two independent trials, and the values for DBC metabolism shown are percentages of the total DBC metabolites represented by peaks on the chromatogram. Data are shown as mean values ± SEMs (N = 4).

 


Figure 5
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  		Fig. 5. Metabolism of DBC by human CYP microsomes (supersomes). The reaction mixture contained an NADPH-regenerating system, 60 µM DBC and 0.5 nmol human CYP1A1, CYP1A2 or CYP1B1 in 100 mM potassium phosphate buffer (pH 7.4). Otherwise, the conditions and determination of DBC metabolism were identical to the procedures described previously. Data are shown as mean values ± SEMs (N = 4).

 


Figure 6
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  		Fig. 6. Metabolism of DBC by mouse lung microsomes. Treatment of mice, harvesting of lung microsomes and determination of DBC metabolism and HPLC procedures were described previously. Open bars = vehicle-treated mice. Black bars = BNF-treated mice. Data are shown as mean values ± SEMs (N = 4).

 


Figure 7
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  		Fig. 7. Pharmacokinetics of DBC in Cyp1 knockout mice. Mice were treated by i.p. injection for 3 consecutive days with either corn oil vehicle (open circles) or 80 mg BNF/kg body wt (black circles). Twenty-four hours after the last BNF treatment, mice were administered i.p. 10 mg DBC/kg body wt. At the indicated times, 15 µl blood was collected and extracted three times with ethyl acetate, dried under argon and suspended in 100 µl methanol. Seventy-five microliters were applied to the C18 reverse-phase HPLC column, eluted with a methanol gradient and DBC was quantified from the molar absorbance at 280 nm of DBC standards (6). The limit of detection for DBC was 0.25–0.3 nmol. Since the equivalent volume of whole blood applied to the column was 11.25 µl, the lower limit of detection for blood is ~0.275 nmol/11.25 µl, or ~24 µM. That is, the lowest numbers are very close to the detection limit. Data are shown as mean blood concentrations ± SEMs (N = 4).

 
Microsomal metabolism of DBC
These procedures were done under yellow lighting to minimize photo-oxidation of DBC and its metabolites. The reaction mixture was equilibrated at 37°C and contained 1.6 mg induced or uninduced mouse microsomal protein (or 0.5 nmol human CYP1), and 60 µM DBC, in 0.1 M potassium phosphate buffer, pH 7.4. The reaction was initiated by addition of 100 µl NADPH-regenerating system (NADPH-RS; final concentrations of 240 µM NADP+, 5 mM glucose 6-phosphate, 1 mM MgCl2 and 1 U/ml glucose 6-phosphate dehydrogenase). After 30 min, another 100 µl NADPH-RS was added. The reaction was stopped at 60 min by the addition of 1 ml argon-saturated acetone. The following procedures were done under yellow lighting and argon-flushed solvents. The sample was extracted three times with argon-saturated ethyl acetate, and the ethyl acetate fractions were pooled and evaporated to dryness under argon. Samples were dissolved in 200 µl ethanol and 50 µl applied to a Waters high performance liquid chromatography (HPLC) system; separation was achieved using a C18 reverse-phase column, eluting at 1 ml/min with a methanol gradient consisting of 76–79% methanol in water for 6 min, 79–85% methanol for 3 min, 85–100% in 18 min and 100% methanol for 11 min (5). We used both UV detection at 280 nm, and fluorescence detection at 280:380 nm (excitation:emission). We used fluorescence to ensure that the identity of unknown peaks eluted with known metabolite standards, which were synthesized as described (6). Absorbance was used to quantify metabolites by using the molar absorbance values for the individual metabolite standards. Parent compound (DBC) eluted after the metabolites indicated in the figures.

Pharmacokinetics of DBC
At the time of killing, blood was collected from each mouse, immediately diluted 1:1 with acetone followed by the addition of an internal standard (t-butylhydroquinone) at fixed amount per gram tissue. Samples were prepared under yellow lighting and argon, and analyzed by HPLC, as described for DBC metabolism and the legend to Figure 3.

[32P]-post-labeling
We used a DNA post-labeling procedure that was slightly modified from the original published methods (7,13,20). Isolation of genomic DNA from tissues was performed using the ProMega Wizard® DNA purification kit (Fischer Scientific, Cincinnati, OH). Briefly, frozen tissue (~30 mg) was thawed in lysis buffer, then RNase A (3 µl of 10 mg/ml) was added and the sample incubated at 37°C for 20 min. Proteins were precipitated with the supplied buffer and the DNA was isolated from the supernatant using ice-cold isopropanol. DNA was washed with ethanol and diluted in re-suspension buffer to a final concentration of ~0.5 mg/ml. DNA was then hydrolyzed to 3'-phosphodeoxynucleotides using calf thymus phosphodiesterase and micrococcal endonuclease. Nucleotide digests were labeled with [32P]-ATP, and thin-layer chromatography was performed using a polyethyleneimine–cellulose anion exchanger. The [32P]-nucleotide adducts were located on the chromatography plate by autoradiography, scraped from the plate and counted in a scintillation counter (21). Relative adduct levels = (c.p.m. adducts/c.p.m. unadducted nucleotides) x 107 were calculated.

Statistics
Statistical significance of the differences between group sample mean values was determined as indicated in the legends to the tables and figures. Statistics were performed using SigmaStat Statistical Analysis software (SPSS, Chicago, IL).

Biohazard precautions
DBC is a potentially hazardous and possible human carcinogen. DBC was handled in accordance with the National Institutes of Health guidelines for carcinogens. Personnel were instructed in safe handling procedures, including the use of lab coats and gloves when using these compounds. Special precautions were taken to ensure that any contaminated materials were collected separately for disposal by the University of Cincinnati Environmental Health and Safety Unit.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
We first determined that the AHR influenced formation of DBC-DNA adducts in liver and lung, utilizing the Ahr(–/–) knockout mouse model (Figure 2). The data suggest that the AHR is involved in the regulation of genes that overall convey protection, by decreasing the DBC-DNA adduct levels in liver and in lung.

Some of the chemical pathways for DBC metabolism have been previously described (5,6; Figure 1). Although studies employing agents that induce by way of the AHR suggested the involvement of CYP1 family members in DBC metabolism, the specific activity and metabolite profile of DBC by individual CYP1 family members has never been conducted before. We therefore evaluated DBC metabolism in mouse liver microsomes isolated from mice ablated in only one Cyp1 gene. The overall rates of DBC metabolism for liver and lung, in wild-type and the knockout mouse lines, are shown (Figure 3, upper), and compared with metabolism using human CYP1 enzymes (Figure 3, lower left). The changes in DBC metabolism in the knockout lines suggest that CYP1A2 in non-induced liver and CYP1A1 in BNF-induced liver are primarily responsible for DBC metabolism. In lung, the CYP1 enzymes are not involved in DBC metabolism in non-induced mice, whereas in induced mice, CYP1B1––and to a somewhat lesser extent, CYP1A1––participate in DBC metabolism. It should be noted that the data for Figure 3 are expressed as ‘metabolism per amount of microsomal protein’, whereas the adduct data in Figure 2 are expressed ‘adducts per amount of DNA’. There is much less endoplasmic reticulum per cell in lung than in liver, hence, fewer adducts per number of DNA bases, but not much difference in DBC metabolism per mg microsomal protein.

Interestingly, the rates of DBC metabolism by CYP1A1, CYP1A2 and CYP1B1 are completely different from the well-known CYP1 substrate benzo[a]pyrene (BaP, Figure 3, lower right). Furthermore, although BNF and DBC are both substrates for the CYP1 enzymes, DBC does not induce CYP1 activity using the treatment regimen of 40 mg DBC/kg body wt (data not shown). This information is important for the evaluation of the elimination pharmacokinetic data (see below).

Quantification of individual DBC metabolites is shown in Figure 4 (mouse liver microsomes), Figure 5 (human supersomes) and Figure 6 (lung microsomes). These results are presented as both the percentage of individual metabolite relative to total metabolism (top panels) and as the actual amount of each metabolite (lower panels). Note that for liver (Figure 4) and lung (Figure 6) metabolism, the total amount of metabolites is small for uninduced mice, relative to metabolism in BNF-induced mice. For the purposes of evaluating the contribution of CYP1 enzymes to detoxication and excretion, versus activation and toxicity, the amount of metabolites are more important than the percent distribution of metabolites. In liver, the distribution of monohydroxylated DBC metabolites varied greatly between the mouse genotypes, and with BNF induction (Figure 4). In wild-type mice, BNF induction increased the percentage of 1-OH-DBC and 2-OH, at the expense of 4-OH; in Cyp1a1(–/–) mice, BNF induction increased 4-OH at the expense of (5 + 6)-OH; in Cyp1a2(–/–) mice, induction increased 1-OH, 2-OH and (5 + 6)-OH at the primary expense of 3-OH and in Cyp1b1(–/–) mice, induction increased 1-OH, 2-OH and 3-OH at the expense of 4-OH. Comparison of the metabolite profile in BNF-induced mice suggested that CYP1A1 primarily generates 1-OH, 2-OH and (5 + 6)-OH-DBC, CYP1A2 generates primarily (5 + 6)-OH-DBC and CYP1B1 primarily generates 4-OH-DBC. This was similar to that observed in the human CYP1 enzymes (Figure 5). The similar metabolite profile for wild-type and Cyp1b1(–/–) mice was expected, due to the very low levels of CYP1B1 in liver.

In order to determine the metabolic similarity of mouse and human metabolism of DBC, we evaluated the DBC metabolite profile for metabolism by human CYP1 family members (Figure 5). Human CYP1 enzymes metabolize DBC to form ~65–75% monohydroxylated products and ~25–35% unidentified products, which we have grouped as hydrophilic (eluting near the void volume) and hydrophobic (eluting at later time points). The majority of unidentified metabolites are hydrophilic, possibly N-hydroxy-DBC or dihydroxylated products. The pattern of metabolites co-eluting with known standards is very different for the different CYP1 enzymes. Human CYP1A1 produces (5 + 6)-OH-DBC > 3-OH > 1-OH = 4-OH > 2-OH. Human CYP1A2 produces (5 + 6)-OH-DBC > > 3-OH, with no 1-OH, 2-OH or 4-OH. Finally, human CYP1B1 produces 4-OH-DBC > (5 + 6)-OH >3-OH >1-OH, with no 2-OH.

Although liver metabolism may be quite important for DBC pharmacokinetics and overall systemic clearance, it is typical that the organ-specific metabolism of a toxicant or carcinogen is a critical factor in defining target-organ specificity. Therefore, we evaluated DBC metabolism by lung microsomes (Figure 6). A noticeable feature of DBC metabolism in the lung was the high percentage of unknown hydrophobic metabolites. Because this high percentage was present using lung microsomes from each of the Cyp1 knockout lines, it appears that CYP1 enzymes are not responsible for forming these hydrophobic products. Hydrophobic metabolites probably include DBC-diones or N-hydroxy-DBC (conjugation products would not be formed in this microsomal metabolism system). Furthermore, unlike in liver, (5 + 6)-OH-DBC is a relatively minor reaction product in lung. In BNF-induced lung microsomes, CYP1B1, and to a lesser extent CYP1A1, appear to form 4-OH-DBC. All three CYP1s apparently produce 3-OH-DBC.

A consistent finding with human CYP1 enzymes, and with mouse liver and lung microsomes, is that CYP1B1 is associated with the production of 4-OH-DBC. This metabolite is believed to be the most DNA reactive and promutagenic of the identified metabolites of DBC (2).

We next asked whether CYP1 enzymes are important in DBC metabolite clearance in mice, by examining the pharmacokinetic profile of DBC in each Cyp1 knockout mouse line. It should be noted that we did not observe DBC metabolites in blood, although blood metabolites may have been present at concentrations lower than the detection limit of ~24 µM. The results (Figure 7) show the blood concentrations of DBC, following administration of DBC. The legend to Figure 7 shows the area under the curve values for the depicted curves. In non-induced mice, DBC is cleared from the blood in 4–8 h. Knocking out the Cyp1a1(–/–) gene had no effect on blood clearance of DBC. This was an expected result, considering the low levels of CYP1A1 in non-induced mice. Knocking out the Cyp1a2 gene produced a small increase in the rate of blood clearance by unknown mechanisms, but possibly related to biotransformation genes constitutively up-regulated in the Cyp1a2(–/–) mouse (22). Knocking out the Cyp1b1 gene doubled peak blood concentration and area under the curve for DBC, suggesting that CYP1B1 is the dominant CYP1 that metabolizes DBC in non-induced mice. Although CYP1B1 is not well expressed in non-induced liver and lung (23), it may be highly expressed in tissues rich in squamous epithelium (24), vascular smooth muscle (25) and possibly adrenal gland and breast tissue (23). CYP1B1 is also highly inducible in lung, but not in liver.

In BNF-induced mice, DBC is essentially cleared from the blood much more rapidly than in non-induced mice, reflective by a 9-fold decrease in the area under the curve. CYP1A2 did not appear to be important in the overall clearance of DBC in BNF-induced mice, since clearance rates were not altered in induced Cyp1a2(–/–) mice. In Cyp1a1(–/–) mice, and to a lesser extent in Cyp1b1(–/–) mice, DBC showed greater persistence in blood, suggesting that CYP1A1 is the dominant CYP1 that metabolizes DBC in BNF-induced mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
DNA adducts
Although the absolute levels of DBC-DNA adducts shown in Figure 2 is lower in lung relative to liver, lung displays a much greater persistence of adducts that may contribute to the potency of DBC as a lung carcinogen. Since Ahr(–/–) mice are non-responsive to agents that induce drug-metabolizing enzymes, it is most likely that the protection afforded by the AHR against DNA adduct formation by DBC is due to a change in the pattern of metabolism of DBC in Ahr(–/–) mice. Although the Ahr null mouse model is useful as an indicator of metabolism directed by the AHR, results must be interpreted with caution. There may be AHR-regulated activities that are not directly linked to xenobiotic metabolism. Since these mice exhibit a high percentage of embryonic lethality, severe immune system toxicity, bile duct fibrosis and reduction in liver size (17), such physiological changes as in hepatic blood flow may contribute to overall systemic metabolism.

DBC metabolism and the AHR
We have shown that the metabolic pathways for DBC depend heavily on the tissue-specific expression profiles of CYP1A1, CYP1A2 and CYP1B1. Cyp1 family inducibility is under the control of the AHR. Numerous studies, mostly concerning the induction of CYP1A1 and/or CYP1B1, have documented inter-individual differences in the accumulation of human CYP1 family members in cells and tissues following PAH exposure (26–29). It is established that the inducibility phenotype can be delineated in human tissues/cells exposed to prototypic AHR agonists or from tissues isolated from smokers (30), and that a highly inducible CYP1 phenotype is associated with susceptibility to cigarette smoking-induced lung cancer. For example, ~10% of Caucasians exhibit a high CYP1-inducing AHR phenotype. A correlation in humans between CYP1 inducibility and various forms of toxicity and cancer have been demonstrated (31,32). Several independent laboratories have reported a greater risk for bronchogenic carcinoma, laryngeal carcinoma and cancer of the oral cavity in cigarette smokers with the high-CYP1-inducing AHR phenotype, compared with smokers with the poor CYP1-inducing AHR phenotype (33). It is clear that cigarette smoking is able to induce BaP hydroxylase activity in extra-pulmonary tissues such as placenta (30). Induction of placental BaP hydroxylase by cigarette smoke occurred only in a minority of the cohort; in the majority of people, smoking did not activate placental AHR. The contribution of individual components of cigarette smoke to smoking-related lung cancer is difficult to assess for minor components, even for such potent lung carcinogens as DBC.

The toxicological outcome for exposure to any chemical or chemical mixture, such as cigarette smoke, often depends heavily on the route of exposure. Cigarette smoke presents a direct exposure of the component chemicals, including BaP and DBC, to the lung, a tissue with much less capacity for detoxication than liver. In these studies, DBC was administered i.p., a mode of exposure that is justified by the development of lung tumors (2). However, with i.p. injection, first-pass liver metabolism may decrease the availability of chemicals to the lung. Thus, whereas overall systemic induction of CYP1 enzymes might increase pulmonary DNA adducts from DBC in cigarette smoke, CYP1s may decrease levels of DNA adducts from DBC in other pyrolysis products, where dermal or gastric exposure might occur.

A number of allelic forms of the human CYP1A1 and CYP1A2 genes are known (34); however, the functional toxicological, pharmacological and pathological consequences of these CYP polymorphisms are largely unknown (35). Nonetheless, it is clear that differences in human CYP1 expression levels have functional consequences relative to chemical toxicity and cancer (33,36). Variability in the human CYP1 inducibility phenotype shows linkage with the AHR locus (37).

CYP1A1 is induced in human lung in smokers and ex-smokers (29), and human CYP1A1 is clearly involved in the metabolic activation of DBC to form DBC-DNA adducts (38). Although basal CYP1A1 expression is very low or undetectable in most tissues, substantial levels of CYP1A1 mRNA, protein and enzyme activity appear following induction of the CYP1A1 gene by inducing agents, many of which are in turn metabolized by the gene product. The inducible CYP1A1 enzyme activity is ubiquitous, located in virtually every tissue of the body, although the levels of inducibility vary with AHR responsiveness (39). Substantial constitutive CYP1A2 activity occurs in mammalian liver, and the human CYP1A2 gene is inducible in liver, GI tract, nasal epithelium, brain (40) and possibly lung (41). There are >60-fold differences in hepatic CYP1A2 mRNA, protein and activity between individuals in any human population studied (42). CYP1B1 has high catalytic activity for BaP, many N-heterocyclic amines, arylamines, azo dyes and several other carcinogens (43). Unlike CYP1A1, CYP1B1 is constitutively expressed in extrahepatic tissues, and is induced in human lung of smokers and ex-smokers (29). CYP1B1 expression is high in squamous epithelium and in various types of tumors (44). The Cyp1b1(–/–) mouse shows a decreased susceptibility to 7,12-dimethylbenzo[a]anthracene-induced lymphomas (16), marrow toxicity and preleukemia (45) and 7,12-dimethylbenzo[a]anthracene-induced ovarian cancers (46).This knockout also has decreased susceptibility to dibenzo[a,l]pyrene-induced tumors (47). Thus, the expression of CYP1B1 appears to be generally associated with chemical carcinogenesis.

It is clear from the results of the present study that CYP1A1, CYP1A2 and CYP1B1 are involved in DBC metabolism. The availability of Cyp1a1, Cyp1a2 and Cyp1b1 knockout mouse lines has allowed us to rigorously evaluate the contribution of each individual P450 enzyme toward the metabolism and pharmacokinetics of DBC in the intact animal. This is important because CYP1-mediated metabolism of DBC could a priori either increase metabolic activation or increase detoxication and clearance. The situation is confounded by the tissue distribution and inducibility of these P450 enzymes, with CYP1A1 inducible in both liver and lung, CYP1B1 present and inducible in lung with little in liver and CYP1A2 present and inducible in liver with probably a small presence in lung. Although CYP1 (CYP1A1 and CYP1B1) activation may increase the carcinogenicity of DBC in the context of direct DBC exposure to the lung, such as with cigarette smoking, our results suggest that CYP1 enzymes are protective when exposure to DBC is via i.p. injection. When liver is involved in the pharmocokinetics of DBC (i.e. with dermal, gastric or i.p. exposure), then liver forms of CYP1A1 and CYP1A2 might be expected to participate in DBC metabolic clearance, since liver contains such a high level of activity for these CYP1s. The lung forms of CYP1A1 and CYP1B1 might be expected to be more important in the metabolic activation of DBC in the course of direct exposure (inhalation), and exposure via blood when exposure level is high and hepatic first-pass elimination is not complete. The dosage of DBC and the balance between metabolic clearance by the liver, and metabolic activation by the lung, would then determine pulmonary genotoxicity of DBC.

In summary, this study has shown that constitutive and inducible levels of expression of CYP1A1, CYP1A2 and CYP1B1 in liver and lung direct the first step in the oxidative metabolism of DBC. Due to differential metabolism, the consequence of DBC exposure may lead to metabolic activation, mutagenesis and carcinogenesis; alternatively, the differential metabolism may facilitate detoxication and excretion. The results from these studies provide information about the pathways leading to DBC-induced lung cancer, which would be an important component of a model for risk assessment. Such a model would help to identify individuals at risk for developing environmental and smoking-related lung cancer and who might benefit from dietary or chemical intervention, such as with the use of selective AHR modulators, or even antioxidant vitamins. Identifying tumor-susceptible individuals, especially young people or populations at risk, may allow for early intervention via counseling to discuss preventive strategies, including avoiding risky behavior and occupations, dietary and nutritional approaches and pharmaceutical agents (e.g. AHR antagonists).


   Acknowledgments
 
This work was supported in part by National Institutes of Health grants R01 ES10133 (H.G.S.), R01 ES08147 (D.W.N.), R01 ES12463 (T.P.D.) and National Institute of Environmental Health Sciences center grant P30 ES06096.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 7, 2006; revised November 8, 2006; accepted November 30, 2006.


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