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Metabolic Activation of Benzo[c]phenanthrene by Cytochrome P450 Enzymes in Human Liver and Lung Matthias Baum, Shantu Amin,† F. Peter Guengerich,‡ Stephen S. Hecht,§ Werner Ko¨hl, and Gerhard Eisenbrand* Department of Chemistry, University of Kaiserslautern, Division of Food Chemistry and Environmental Toxicology, Erwin Schro¨ dinger Strasse, D-67663 Kaiserslautern, Germany, Division of Chemical Carcinogenesis, American Health Foundation, Valhalla, New York 10595, Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, and University of Minnesota Cancer Center, Minneapolis Minnesota 55455 Received November 24, 2000
The environmentally occurring polycyclic aromatic hydrocarbon (PAH) benzo[c]phenanthrene (B[c]PH) is a weak carcinogen in rodents. In contrast, the dihydrodiol-epoxides of B[c]PH are among the most carcinogenic PAH metabolites tested so far. In rodents, B[c]PH is predominantly metabolized to B[c]PH-5,6-dihydrodiol (B[c]PH-5,6-DH) and only to a minor extent to B[c]PH-3,4-DH, the proximate precursor of the highly potent ultimate carcinogen, B[c]PH3,4-DH-1,2-epoxide. This might explain why in rodents B[c]PH is a weak carcinogen. However, little is known about human metabolism of B[c]PH. Using microsomal preparations from human liver and lung, we investigated the metabolic activation of B[c]PH. In contrast to the findings in experimental animals, human liver microsomes predominantly generated B[c]PH-3,4-DH and only to a minor extent B[c]PH-5,6-DH. Only one lung tissue sample was found to be metabolically active, producing B[c]PH-5,6-DH together with small amounts of B[c]PH-3,4DH. Catalytic activities known to be associated with specific cytochrome P450 (P450) enzyme activities were determined and correlated with the spectrum of B[c]PH metabolites. The results indicate that B[c]PH-DH formation in human liver is mainly mediated by P450 1A2. Studies with P450 enzyme selective inhibitors confirmed these findings. Further support was obtained using preparations of the respective human recombinant P450 enzymes expressed in Escherichia coli and yeast. In addition to P450 1A2, P450 1B1 effectively mediated B[c]PH-metabolism. The umu-assay for induction of SOS repair response in Salmonella typhimurium TA 1535 pSK 1002 containing a umuC-lacZ reporter gene was used to study metabolic generation of genotoxic metabolites from B[c]PH-DHs in human microsomal preparations. B[c]PH-3,4-DH was activated by human liver microsomes to a potent genotoxic agent. Taken together, the results clearly demonstrate that human liver microsomes can effectively catalyze the biotransformation of B[c]PH into highly genotoxic metabolites. The results provide evidence that B[c]PH should be considered a potentially potent carcinogen in humans, and that rodent models may underestimate the risk.
Introduction Benzo[c]phenanthrene (B[c]PH),1 (Scheme 1) is a polycyclic aromatic hydrocarbon (PAH) that occurs in the environment as a product of incomplete combustion, and also has been identified in cigarette smoke, wastewater, and food (1-4). Data from a total diet study carried out in The Netherlands in 1984-86 showed that about 8% of the diets investigated contained some detectable B[c]PH (3). The daily intake was estimated to be similar to that of benzo[a]pyrene (B[a]P) (3). * To whom correspondence should be addressed. Phone: (+)49 631 2974. Fax: (+)49 631 3085. E-mail:
[email protected]. † Division of Chemical Carcinogenesis. ‡ Department of Biochemistry and Center in Molecular Toxicology. § University of Minnesota Cancer Center. 1 Abbreviations: PAH, polycyclic aromatic hydrocarbons; B[c]PH, benzo[c]phenanthrene; B[c]PH-3,4-DH, benzo[c]phenanthrene-3,4-dihydrodiol; B[c]PH-5,6-DH, benzo[c]phenanthrene-5,6-dihydrodiol; OHB[c]PH, hydroxybenzo[c]phenanthrene; R-NF, R-naphthoflavone; B[a]P, benzo[a]pyrene; B[a]P-7,8-DH, benzo[a]pyrene-7,8-dihydrodiol; Trol, troleandomycin; P450, cytochrome P450; 5-MeCHR, 5-methylchrysene; 5-MeCHR-1,2-DH, 5-methylchrysene-1,2-dihydrodiol.
B[c]PH is a weak carcinogen in rodents (5-8). In contrast to the apparently low biological activity of B[c]PH, evidence from in vivo and in vitro studies clearly show that fjord-region B[c]PH-3,4-DH-1,2-epoxides are potent ultimate carcinogens. Two enantiomeric B[c]PH3,4-DH (S,S or R,R-configuration, Scheme 1) are formed in the oxidation of B[c]PH and are then converted into four B[c]PH-DH-epoxides (Scheme 1). Both anti- and syn-DH-epoxides are potent inducers of SOS-response in the SOS-chromotest and potent mutagens in Salmonella typhimurium and V79 cells. In V79 cells, (-)-anti-B[c]PH-3,4-DH-1,2-epoxide is the more potent isomer. (+)-anti B[c]PH-3,4-DH-epoxide was found to be more potent in S. typhimurium (9, 10). All four B[c]PH-3,4-DH-epoxides were able to form DNA adducts in mouse epidermis when applied onto the skin with the (-)-anti-3,4-DH-1,2-epoxide being the most active (11, 12). In rodent embryo cells incubated with B[c]PH, DNA adducts resulting from formation of (-)-anti-B[c]PH-3,4-
10.1021/tx000240s CCC: $20.00 © 2001 American Chemical Society Published on Web 06/18/2001
Metabolic Activation of Benzo[c]phenanthrene Scheme 1. Activation of B[c]PH to 3,4-DH-1,2-epoxides
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that human P450s 1A1, 1A2, 1B1, 2A6, and 2E1 all are able to activate B[c]PH, primarily to the corresponding dihydrodiols. However, P450 1A1 and 1A2 were the most active enzymes in formation of B[c]PH-3,4-DH generating predominantly the R,R-isomer (20). In the present study, P450 dependent metabolism of B[c]PH was investigated, using a set of microsomes from 18 individual human liver tissue samples and 11 human lung tissue samples. Our aim was to clarify how B[c]PH is metabolized in a wide spectrum of human samples.
Materials and Methods
DH-1,2-epoxide were detected (13). On mouse skin B[c]PH-3,4-DH-1,2-epoxides are potent tumor-initiators (5, 14), with (-)-anti-B[c]PH-3,4-DH-1,2-epoxide being the more potent isomer (6). After injection into the mammary fat pads of rats, racemic anti-B[c]PH-3,4-DH-1,2-epoxide was a considerably more potent carcinogen than the corresponding anti-7,8-dihydrodiol-9,10-epoxide of B[a]P (15, 16). The low carcinogenicity of B[c]PH in rodents, in contrast to the high carcinogenicity of its fjord-region DH-epoxides, can be reconciled with rodent-specific metabolism. In rat liver microsomes, only minor amounts of B[c]PH-3,4-DH (89-94% R,R-isomer) are formed (17). In mouse skin in vivo, only small amounts of DNA adducts are formed upon application of B[c]PH, whereas treatment with B[c]PH-3,4-DH results in a 6-fold higher level of adduct formation (18). The formation of B[c]PH-3,4-DH is therefore the critical step in the formation of the ultimate carcinogen, B[c]PH-3,4-DH-epoxide. Activation of B[c]PH-3,4-DH by Aroclor-induced rat microsomes revealed a severalfold higher mutagenic potential in S. typhimurium TA 98 and TA 100 and in mammalian V79 cells as compared to parent B[c]PH and to the corresponding 1,2- and 5,6dihydrodiols (9). Furthermore, B[c]PH-3,4-DH was found to be a potent tumor initiator in the mouse, inducing local tumors after skin application and distant tumors after intraperitoneal injection, while B[c]PH-5,6-DH was nearly inactive (5, 6). Only limited data are available on biological activity and metabolic fate of B[c]PH in humans. Human mammary carcinoma cells MCF-7 activate B[c]PH into DNAbinding B[c]PH-3,4-DH-1,2-epoxides (18). Both B[c]PH and B[c]PH-3,4-DH were effectively activated, mainly generating (-)-anti-B[c]PH-3,4-DH-epoxide and (+)-synB[c]PH-3,4-DH-1,2-epoxide that covalently bind to DNA, forming predominantly adducts with deoxyadenosine. P450s 1A1 and 1B1 have been allocated a major role in B[c]PH activation (19). A study on regio- and stereoselective metabolism of B[c]PH in genetically engineered V79 cells expressing human P450 enzymes has shown
Chemicals. [G-3H]-B[c]PH (0,1Ci/mmol, prepared by catalytic tritium-exchange (Amersham), was purified by HPLC on a Li Chrosorb Si 60 (5 mm) column (Merck) with hexane/ dichloromethane as eluent. B[c]PH, B[c]PH-3,4-DH were synthesized as described (21). B[c]PH-5,6-DH was prepared as described in the literature (22). 1-, 2-, 3-, 4-, and 5-OH-B[c]PH were obtained from the corresponding methoxybenzo[c]phenanthrene.2 B[a]P-7,8-DH was synthesized by following the literature procedure (23). NADP, glucose 6-phosphate, glucose 6-phosphate-dehydrogenase, R-naphthoflavone, and troleandomycin were obtained from Sigma. Preparation of Microsomes. Microsomes were prepared from 18 human liver and 11 lung samples obtained from organ donors through the Tennessee Donor Services (Nashville, TN), and the Veterans Administration Medical Center (Little Rock, AR), as described elsewhere (24). No information on smoking habits was available. Causes of death were subarachnoid hemorrhages [5], motor vehicle accidents [8], gunshot wounds [3], closed head injury [1], braintumor [1]. Protein concentrations were estimated using Coomassie Blue G-250 (Pierce Coomassie Plus Protein Assay; Pierce Chemical Co). P450 determinations in liver microsomes were performed as described previously using the dithionite difference method (25, 26). Catalytic Assays. The following substrates were used to determine P450-linked catalytic activities in human hepatic microsomes: ethoxyresorufin (P450 1A2), nifedipine (P450 3A4), coumarin (P450 2A6), chlorzoxazone (P450 2E1), (s)-mephenytoin (P450 2C9), and bufuralol (P450 2D6). Details of experimental protocols are presented elsewhere (27, 28 and references therein). Metabolism Studies. [G-3H]-B[c]PH (0.1 Ci/mmol) was incubated at 37 °C in 0.5 mL of 100 mM phosphate-buffer (pH 7.4) containing 0.5 mg of microsomal protein, 3 mM MgCl2, and an NADPH-generating system consisting of 1 mM NADP+, 5 mM glucose 6-phosphate, and 0.5 units of glucose 6-phosphatedehydrogenase (Type XII from Torula yeast) (Sigma). Boiled microsomes were used as blanks. Each incubation was performed in duplicate. The reaction was initiated by adding the substrate in DMSO (final concentration 1% DMSO). Initial time course studies showed that metabolism of B[c]PH was linear up to 20 min. An incubation time of 20 min at 37 °C was therefore selected. In range finding studies, [G-3H]-B[c]PH concentrations from 5 to 30 µM were incubated. At 5 µM, the relative turnover rates were severalfold higher than at 30 µM. Since metabolite yields already approached the determination limit, the concentration of 5 µM was selected for all incubations. Incubations with lung microsomes, were run for 60 min because of the expected lower P450 activity. Reactions were terminated by addition of ice-cold acetone. Mixtures were extracted three times with ethyl acetate. The organic layers were transferred into appropriate vials and evaporated to dryness under a stream of N2. Recovery of radioactivity was g94%. Inhibition Studies. Inhibition of B[c]PH metabolism was studied by incubating hepatic microsomes with known P450 selective inhibitors: R-naphthoflavone (P450 1A1/1A2) and 2
Lin, J., and Amin, S., personal communication.
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Table 1. Metabolism of [G-3H] B[c]PH in Human Hepatic Microsomesa hepatic microsomal sample no. 39 98 100c 107 108 109 110 112 115 116 118 123c 126 129 130c 132c 133 134c mean min/max
B[c]PH-3,4-DH 11 22 2.5 27 22 20 10 25 22 13 3.7 6.6 11 18 4.1 16 19 12 15 2.5-27
B[c]PH metabolites (pmol/mg of protein/min)b ratio 4-OH B[c]PH-5,6-DH 3,4-DH/5,6-DH B[c]PH A 5.8 4.5 1.4 6.2 3.9 5.1 5.8 5.4 11 5.3 1.5 1.7 1.0 5.1 1.8 5.1 5.4 6.1 4.6 1.4-11
2.0 4.8 1.8 4.3 5.5 4.0 1.8 4.7 2.0 2.5 2.4 3.9 11 3.5 2.3 3.2 3.4 2.1
nd nd nd 0.7 0.8 1.2 nd 1.7 1.5 0.7 nd 0.7 0.7 1.3 nd 0.5 0.9 0.6 1.0 0.5-1.7
7.6 8.4 2.7 21 14 16 11 15 19 14 1.7 7.5 6.4 10 3.2 11 18 6.4 11 2.7-21
B
C
D
1.6 1.6 nd 3.8 2.2 3.0 nd 4.6 2.5 2.4 nd 3.9 1.4 2.0 1.2 1.9 4.4 1.4 2.1 1.2-4.6
nd 2.3 nd 2.2 3.1 2.5 nd 2.6 nd 2.0 nd nd 1.7 3.2 nd 3.5 nd 2.2 1.4 1.7-3.5
nd nd nd nd nd nd nd 1.8 7.5 4.3 nd nd nd 4.5 nd 4.1 1.6 3.9 1.5 1.6-7.5
a [G-3H] B[c]PH (5 µM) was incubated for 20 min at 37 °C with hepatic microsomes and cofactors as described in the Materials and Methods. Metabolites were analyzed by HPLC (see Figure 1). b Values represent the average of two determinations. c Microsomes from female donors. nd: Not detectable, detection limit: 0.2 pmol/mg of protein/min.
troleandomycin (P450 3A4). Inhibitors dissolved in DMSO were added to the incubations at concentrations of 1, 10, and 25 µM, as required. Recombinant P450. Incubations were carried out with membranes from Escherichia coli expressing human P450 1A2 or 3A4 together with human NADPH-P450 reductase (29 and references therein) and from yeast expressing P450 1B1 (30). [G-3H]-B[c]PH concentrations, assay conditions, sample workups were identical to those of liver microsomes. A total amount of 250 pmol recombinant P450 was used for each incubation. Analysis of Metabolites. Metabolites extracted into ethyl acetate were analyzed by HPLC using a L-6200A Intelligent Pump (Merck-Hitachi) with a 500 µL injection loop (Rheodyne) and a 4.6 × 250 mm Vydac 10 µm octadecylsilane C18-column (Separations Group). For UV detection (280 nm), a GATLCD501-detector (GAT-Analysentechnik) was used. Radioactivity was detected with a Radiomatic Flo-ONE Beta A-500Detector (Packard) by online-addition of scintillation cocktail (Ultima Flo M (Packard); 3 mL of scintillation-cocktail/mL solvent). Recovery was calculated by measuring total baselinecorrected counts of each incubation relative to the activity of unmetabolized substrate. The eluting solvent was methanol/ H2O, flow rate 1 mL/min. The elution program was: linear gradient from 40 to 50% methanol (10 min); isocratic conditions (25 min); second linear gradient to 70% methanol (10 min); isocratic period (15 min), third linear gradient to 100% methanol (10 min); a final isocratic period (10 min). Statistical Analyses. Spearman rank correlations were used to evaluate significance of the data. Umu-test. Bacterial Tester Strains. Tester strain S. typhimurium TA 1535/pSK 1002 containing the umuC regulatory sequence attached to the lacZ reporter gene was used to detect activation of PAH-DHs to ultimate genotoxic metabolites (31). Genotoxicity Assays. Activation of procarcinogens to genotoxic metabolites by liver microsomal P450 enzymes in S. typhimurium TA 1535/pSK 1002 was performed according to procedures described previously (32-35). Human hepatic microsomes (5-100 µg protein/mL depending on study) were incubated with dihydrodiols (50 µM) in 50 mM potassium phosphate buffer (pH 7.4), total volume 1.5 mL, containing an NADPH-generating system (5 mM glucose 6-phosphate, 0.5 mM NADP+ and 1 IU yeast glucose 6-phosphate dehydrogenase/mL), and a 1.1 mL of suspension of the bacterial tester strain S. typhimurium TA 1535/pSK 1002.
Figure 1. A representative HPLC-radiochromatogram of the organic extractable metabolites of B[c]PH obtained by incubation [G-3H]-B[c]PH (5 µM) with human hepatic microsomes (0.5 mg of protein/mL) in the presence of an NADPH-generating system as described in materials and methods. Peak “E” (unidentified) did not change during incubation and was invariably present in control incubations. (a) Counts per minute. The induction of umu-gene expression as a consequence of DNA damage was quantified by measuring β-galactosidaseactivity photometrically (420 nm) according to the method of Miller (36). After lysis of the bacteria, an aliquot of the incubation-mixture was incubated with o-nitrophenyl-β-Dgalactopyranoside as substrate. The induction of umu gene expression is calculated as units of β-galactosidase activity per minute per milligram of protein or of β-galactosidase activity per milliliter incubation mixture. The umu- gene expression was linear over 2 h under these conditions, an incubation time of 2 h was therefore selected. Control experiments using boiled microsomes were performed for all dihydrodiols tested. Results are represented as means of two independent incubations, each incubation being assayed four times.
Results Metabolism of B[c]PH by Human Hepatic Microsomes. A representative HPLC radiochromatogram of organic extractable metabolites obtained from incubation of [G-3H]-B[c]PH with human liver microsomes is shown in Figure 1. Qualitatively similar metabolite profiles were obtained with each of the 18 human hepatic microsomes. Levels of metabolites varied about 2-10-fold but profiles were similar. Formation rates of metabolites (Table 1), show B[c]PH to be a good substrate for human liver microsomes. All liver samples generated a similar me-
Metabolic Activation of Benzo[c]phenanthrene
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Table 2. P450 Enzyme Activities in Human Hepatic Microsmes P450
testa
activity (pmol/mg of protein)
1A1/1A2 2C19 2D6 2E1 3A4 2A6
EROD MP BUF CZ NF COUM
22-129 8-279 55-816 900-6800 100-23000 161-2220
a EROD, 7-ethoxyresorufin O-deethylation; MP, (S)-mephenytoin 4′-hdroxylation; BUF, bufuralol-1′-hydroxylation; CZ, chlorzoxazone-6-hydroxylation; NF, nifedipine oxidation; COUM, coumarin 7-hydroxylation.
Table 3. Correlation Coefficients [r] among P450-Dependent Activities and Hepatic B[c]PH-Metabolites P450-dependent activities metabolite
EROD
MP
B[c]PH-3,4-DH B[c]PH-5,6-DH 4-OH B[c]PH A B C D
0.78a
0.26 0.37 0.36 0.47 0.39 -0.27 0.25
a
0.59b 0.68a 0.87c 0.65a 0.51b 0.24
BUF
CZ
NF
-0.06 0.21 0.35 0.26 0.34 0.75a -0.30 0.09 0.19 -0.06 0.27 0.50b -0.11 0.22 0.21 -0.02 -0.01 -0.31 0.18 0.30 0.16
COUM 0.28 0.17 0.13 0.26 0.03 0.10 -0.02
p < 0.01. b p < 0.05. c p < 0.001.
tabolite pattern. B[c]PH-3,4-DH was identified as the main metabolite (2.5-27 pmol/mg of protein/min). In all samples, B[c]PH-5,6-DH (1.4-11 pmol/mg of protein/min) was also detected as a minor metabolite. A comparison of incubations at 5 µM with those at 30 µM showed that turnover rates for the formation of dihydrodiols at 5 µM were severalfold higher than at 30 µM. When B[c]PH3,4-DH formation was examined in one specific liver microsomal preparation with high activity of P450 1A2, relative B[c]PH 3,4-DH formation was even higher at 5 µM than at 30 µM (data not shown). Since at 5 µM incubation the analytical determination limit was already approached, 5 µM was selected as standard B[c]PH incubation concentration. In 12 of 18 samples, 4-OH B[c]PH was also identified. No indication of other phenolic metabolites was obtained. Metabolite profiles of female and male donors were similar. Four peaks were observed with retention times shorter than those of the dihydrodiols. The most prominent polar peak “A” was present in all 18 samples, while peaks “B”, “C”, and “D” were seen in most of the samples. Formation of peaks “A”, “B”, and “C” correlated with dihydrodiol formation which might suggest metabolites of higher polarity such as triols or tetraols that might have arisen from dihydrodiols. Involvement of Human Hepatic P450s in the Formation of B[c]PH Metabolites. Correlation Studies. Catalytic activities associated with specific enzyme activities were determined in all human hepatic samples by typing with the corresponding substrates. The assays used and ranges for the 18 samples are given in Table 2. The total P450 content of the samples varied 2-3-fold (results not shown). The results of the Spearman-correlation analysis between amounts of the different metabolites formed and P450 activities are given in Table 3. Significant correlations were found between EROD-activity (P450 1A1/1A2) and the formation of B[c]PH-3,4-DH, 4-OH-B[c]PH, and
Figure 2. Influence of P450 enzyme selective inhibitors on B[c]PH-DH formation. Human liver-microsomes (0.5 mg/mL) were incubated with [3H]B[c]PH (5 µM) and an NADPHgenerating system in the presence of the amounts of P450 enzyme selective inibitors as indicated in Materials and Methods. (a) Metabolite formation relative to the control without inhibitor; HL 133: human liver sample low in P450 3A4; HL 115: human liver sample rich in P450 3A4. (b) R-Naphthoflavone (P450 1A1/1A2-inhibitor). (c) troleandomycin (P450 3A4-inhibitor).
to a lesser extent with B[c]PH-5,6-DH. In addition, a significant correlation was also seen between B[c]PH-5,6DH formation and P450 3A4 activity. No evidence was found for the involvement of P450 2C19, 2D6, 2E1, or 2A6. These results clearly suggest that in human liver tissue P450 1A2 is mainly responsible for the formation of B[c]PH-DHs. P450 3A4 appears exclusively to be involved in the formation of B[c]PH-5,6-DH. Inhibition of B[c]PH Metabolism by P450 Inhibitors. We investigated the influence of P450 enzyme-specific inhibitors on B[c]PH metabolism in human liver samples with high P450 1A2 activity, having largely variable P450 3A4 activity (Figure 2). The P450 1A1/1A2-inhibitor R-naphthoflavone (R-NF) inhibited the formation of B[c]PH-3,4-DH in all samples in a concentration dependent manner [10 µM R-NF: 80% (HL115) to total inhibition (HL133)]. Consistent with these findings, formation of 4-OH-B[c]PH was completely inhibited even at 1 µM R-NF (data not shown). The formation of B[c]PH-5,6-DH was slightly inhibited in a sample with low P450 3A4 activity (HL133; 10 µM R-NF: 40% inhibition), whereas in a sample with high P450 3A4 activity (HL 115), B[c]PH-5,6-DH was enhanced by factors of 2.5-3.5 (10-25 µM R-NF). Interestingly, under these conditions, further metabolites were formed that coeluted with 3- and 5-OHB[c]PH, indicative of a shift in the position of preferential aromatic hydroxylation (data not shown). The P450 3A4 inhibitor troleandomycin had no influence on B[c]PH-3,4-DH formation (Figure 2), but slightly diminished B[c]PH-5,6-DH formation (10 µM: 16% inhibition) in a sample with high P450 3A4 activity (HL 115). Metabolism of B[c]PH-by Recombinant Human P450s. B[c]PH metabolite formation by human recombinant P450s is summarized in Table 4. Incubations were carried out in the absence of added epoxide hydrolase. Under these conditions, P450 1A2 hydroxylates B[c]PH mainly at position 4 and, to some extent, also at positions 3 or 5. In addition, substantial amounts of B[c]PH-3,4DH, B[c]PH-5,6-DH, and metabolites of increased polarity were formed. P450 1B1 was also active, forming mainly 4-OH-B[c]PH, together with 3- or 5-OH B[c]PH. In addition, B[c]-
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Table 4. Metabolism of [G-3H] B[c]PH by Human Recombinant P450-Enzymesa B[c]PH metabolites (pmol/nmol of P450/minb) recombinant P450
B[c]PH-3,4-DH
B[c]PH-5,6-DH
4-OH B[c]PH
3-OH or 5-OH-B[c]PH
A
B
C
D
1A2 1B1
7.4 6.2
8.0 10
59 23
23 19
3.0 38
6.6 12
6.8 1.1
9.0 4.5
a [G-3H]-B[c]PH (5 µM) was incubated for 20 min at 37 °C with membrane-preparations of microorganisms expressing human P450enzymes. Membrane-amounts corresponding to 250 pmol of P450/mL incubation mixture were used for each incubation. b Values represent the average of two determinations.
Table 5. Metabolism of [G-3H] B[c]PH in Hepatic Microsomes from Animals B[c]PH metabolites (pmol/mg of protein/min)
b
species
B[c]PH3,4-DH
B[c]PH5,6-DH
3-, 4-, 5OH-B[c]PH
rat (uninduced) rat (phenobarbitala) pig cow
10 nqb 3.9-9.0 84
17 54 7.6-9.3 75
2.1 ndb nd nd
a p.o. in drinking water (1 g of phenobarbital/L) over 5 days. nq, not quantified. nd, not detected.
PH-5,6-DH, together with smaller amounts of B[c]PH3,4-DH, could be identified. Polar peak "A“ metabolites were predominant, correlating with an increase of water soluble polar metabolites (based on radioactivity found in the water phase; data not shown). No hydroxylation at the 1- or 2-positions was observed. Metabolism of B[c]PH by Human Pulmonary Microsomes. Only 1 of 11 human lung samples showed detectable activity. In contrast to the liver microsomes, mainly B[c]PH-5,6-DH (3.0 pmol/mg of protein/min) was formed. B[c]PH-3,4-DH (1.5 pmol/mg of protein/min) was observed only as a minor component. Except for traceamounts of peak “A”, no other metabolites were seen. To ascertain the involvement of P450 1A1, a prominent P450 in lung (37), we incubated the active sample in the presence of the P450 1A1/1A2 inhibitor R-NF. Metabolism was completely inhibited, even at low R-NFconcentrations (1 µM). Metabolism of B[c]PH by Liver Microsomes from Rats, Pigs and Cows. B[c]PH metabolism was further investigated with liver microsomes from rats (uninduced and phenobarbital-induced), pigs, and cattle. Microsomes from phenobarbital-treated rats were more active than those from untreated rats. The rat liver microsomes generated a metabolite pattern as had already been described earlier by Ittah et al. (17) with B[c]PH-5,6-DH as the main and B[c]PH-3,4-DH as a minor metabolite. A similar metabolite spectrum was found in mirosomes from pig liver while bovine liver microsomes formed both DHs in equal amounts (Table 5). Genotoxicity of B[c]PH Dihydrodiols: Induction of SOS-Response in S. typhimurium TA 1535 pSK 1002 after Activation by Human Hepatic Microsomes. The two B[c]PH-dihydrodiols identified as metabolites generated in our studies in human hepatic and pulmonary microsomes, were tested in comparison to B[a]P-7,8-DH for their further activation to potentially genotoxic metabolites using the same set of human livermicrosome samples as for the above metabolism studies. B[c]PH-3,4-DH induced clearly umu gene expression, depending on the microsomal protein concentration (Figure 3). In a comprehensive study using all human microsomal preparations a close correlation (r ) 0.8; p
Figure 3. Induction of umu-gene expression in S. typhimurium TA 1535 pSK 1002 by B[c]PH-3,4-DH. Human liver microsomes were incubated with B[c]PH-3,4-DH (50 µM) in the presence of an NADPH-generating system as described in Materials and Methods. (a) umu-units/mL incubation mixture. (b) Microsomal protein/mL incubation mixture; HL 108: human liver sample low in P450 3A4; HL 110: human liver sample rich in P450 3A4.
< 0.001) was found between induced umu response and P450 3A4 activity (data not shown). B[c]PH-3,4-DH was highly potent, giving rise to a response of 23-104 umu units/mg of protein/min. By comparison, B[c]PH-5,6-DH was poorly active inducing a response of only
