The mutagenicity of the atmospheric transformation products 2-nitrofluoranthene (2-NF) and 2-nitrodibenzopyranone (2-NDBP), as well as a related isomer 3-nitrodibenzopyranone (3-NDBP), was measured in quantitative forward mutation assays with bacteria (Salmonella typhimurium TM677) and in two metabolically competent human cell lines (MCL-5 and h1A1v2) that differ in their complement of cytochrome P450s and microsomal epoxide hydrolase. 2-NF was a potent mutagen in Salmonella TM677 both in the absence and presence of rat liver postmitochondrial supernatant (PMS). 2-NDBP was non-mutagenic in the absence of PMS, but was mutagenic in its presence. The converse result was obtained for 3-NDBP. The mutagenic potency series in Salmonella in the absence of PMS, expressed as the minimum detectable mutagen concentration (MDMC) in nmol/ml, was: 2-NF, 2.5; 3-NDBP, 16.9; and 2-NDBP, > 415. With PMS, the potency series was: 2-NF, 1.2; 2-NDBP, 15.1; 3-NDBP, 208. Neither 2-NDBP nor 3-NDBP were mutagenic at the tk locus in MCL-5 or h1A1v2 cells at up to 200 nmol/ml. 2-NF was also inactive in MCL-5 cells, but was a potent mutagen in h1A1v2 cells with an MDMC of 0.02 nmol/ml. Cytochrome P450 CYP1A1, present constitutively only in h1A1v2 cells, was implicated in 2-NF activation because mutagenicity was reduced by 55-80% when alpha-naphthoflavone (ANF) was present during incubation. The lack of mutagenicity in MCL-5 cells was attributed to the inability of 2-NF to induce CYP1A1 activity in this cell line. These data indicate a primary role for ring oxidation in 2-NF activation. Previous emphasis placed upon 2-NDBP as a major mutagen in ambient air may need to be modified in view of the negative results for this compound in the human cell assays and in the absence of PMS in Salmonella TM677. However, these findings support the concern that 2-NF may be a risk to human health.

Air Pollutants↗

PubMed1997

Use of cDNA-expressed human cytochrome P450 enzymes to study potential drug-drug interactions.

Complementary DNA (cDNA)-expressed human cytochrome P450 enzymes provide a reproducible, consistent source of single enzymes for many types of studies. The use of single enzymes systems, relative to multienzyme systems, has distinct advantages and disadvantages depending on the specific application. cDNA-expressed materials have advantages in the analysis of cytochrome P450 form-selective metabolism of a drug or drug candidate. This analysis can be accomplished by direct incubation of the drug with microsomes prepared from cells expressing a single cytochrome P450 form coupled with analysis of either metabolite formation or loss of parent compound. This approach allows the unambiguous assignment of specific biotransformations to specific enzymes. However, extending these data to the balance of enzymes present in human liver microsomes can be problematic. New approaches for relating rates of metabolism for cDNA-expressed enzymes to human liver microsomes metabolism are being developed (Crespi, 1995). In addition, cDNA-expressed enzymes can be used to study the cytochrome P450 form-selective inhibition by drugs or drug candidates. This analysis is accomplished through the study of the inhibition of the metabolism of a model substrate by the drug or drug candidate. Through these analyses, apparent Ki values can be obtained and compared to Ki values for known, clinically significant inhibitors of the same enzyme. For this application, cDNA-expressed, single enzyme systems have distinct advantages because of greater flexibility in the choice of model substrates and the lack of competing pathways of metabolism. Specific data for the use of cDNA-expressed CYP2C9, CYP2D6, and CYP3A4 are presented.

Aryl Hydrocarbon Hydroxylases↗

PubMed1997

Development of a human lymphoblastoid cell line constitutively expressing human CYP1B1 cDNA: substrate specificity with model substrates and promutagens.

An AHH-1 TK+/- cell derivative was developed that stably expresses human cytochrome P4501B1 (CYP1B1) cDNA in an extrachromosomal vector which confers resistance to 1-histidinol and co-expresses NADPH cytochrome P450 oxidoreductase (OR). The CYP1B1-expressing cell line was designated h1B1/OR. Microsomes prepared from CYP1B1 cDNA expressing cells exhibit elevated levels of 7-ethoxy-resorufin deethylase (EROD), 7-ethoxy-4-trifluoromethyl-coumarin deethylase (EFCD), benzo(alpha)pyrene hydroxylase (BPH), bufuralol 1'-hydroxylase, testosterone hydroxylase activities and spectrally quantifiable cytochrome P450. CYP1B1-containing microsomes did not contain detectable coumarin 7-hydroxylase, p-nitrophenol hydroxylase, lauric acid hydroxylase, (S)-mephenytoin 4'-hydroxylase or diclofenac 4'-hydroxylase activities. Kinetic parameters for selected substrates were compared among CYP1B1 and the two additional members of the CYP1 family, CYP1A1 and CYP1A2. For BPH and EFCD, the rank order of rates of substrate metabolism were CYP1A1 > CYP1B1 > CYP1A2. For EROD, the rank order of substrate metabolism was CYP1A1 > CYP1A2 > CYP1B1. For both EROD and EFCD the apparent K(m) values for CYP1B1 were more similar to CYP1A1 than to CYP1A2. In order to begin to characterize the promutagen activating ability of CYP1B1, the mutagenicity of selected chemicals was examined in h1B1/OR cells; there was increased sensitivity (CYP1B1-expressing relative to control cells) to the mutagenicity of benzo(a)pyrene, cyclopenta(c,d)pyrene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and aflatoxin B1 (AFB). CYP1B1, expressed in this system, appears to be particularly efficient at activating AFB.

Aflatoxin B1↗

PubMed1997

The R144C change in the CYP2C9*2 allele alters interaction of the cytochrome P450 with NADPH:cytochrome P450 oxidoreductase.

We have examined the kinetics of substrate metabolism by cDNA-expressed human CYP2C9 and the R144C variant. Both enzymes exhibited similar apparent K(m) values for (S)-warfarin 7-hydroxylation, diclofenac 4'-hydroxylation and lauric acid 11-hydroxylation. In contrast, the R144C variant (relative to CYP2C9) had slower rates of metabolism for all three substrates. The difference was most pronounced for (S)-warfarin. Surprisingly, the magnitude of the difference was found to be dependent on the cytochrome P450 to NADPH-cytochrome P450 reductase (OR) ratio in the system (the difference being more pronounced at higher OR to P450 ratios) implying that the R144C change affects interaction of the P450 with OR. The rates of (S)-warfarin 7-hydroxylation by CYP2C9 and the R144C variant also exhibited differential dependence on salt concentration which further supported a difference in interaction with OR. When OR was bypassed and the hydroxylation was supported by cumene hydroperoxide, no difference in the rates of diclofenac 4'-hydroxylation was observed for CYP2C9 and the R144C variant regardless of OR to P450 ratio. However, for (S)-warfarin 7-hydroxylation, some OR-dependence was maintained even when the reaction was supported by cumene hydroperoxide. Finally, we compared CYP2C9 activity and CYP2C9 protein levels for human lymphoblast expressed (high OR to P450 ratio) to human liver microsomes using immunoblotting and enzyme selective substrates. Human liver microsomal CYP2C9 and human lymphoblast-expressed CYP2C9 showed comparable amounts of activity per unit enzyme. This final observation indicates that the high OR to P450 ratio is the preferred model and predicts that the R144C change in human liver microsomal CYP2C9 should markedly reduce the rates of substrate metabolism. The implications of these observations for the interpretation of results with cDNA-expressed enzymes is discussed.

Alleles↗

PubMed1997

Quantitative RT-PCR measurement of cytochromes p450 1A1, 1B1, and 2B7, microsomal epoxide hydrolase, and NADPH oxidoreductase expression in lung cells of smokers and nonsmokers.

Bronchial epithelial cells (BEC) are the progenitors of bronchogenic carcinomas and are exposed to polycyclic aromatic hydrocarbon (PAH) procarcinogens through inhalation of combustion products. PAH are converted to carcinogenic molecules through a combination of monoxygenation by cytochrome p450 (CYP) enzymes in the presence of NADPH oxidoreductase (OR) and hydrolysis by microsomal epoxide hydrolase (mEH). In artificial systems, the relative expression of these genes determines whether carcinogenic or noncarcinogenic species are generated during metabolism. This relationship was explored in humans by using quantitative competitive reverse transcriptase polymerase chain reaction amplification to determine the range of expression of CYP1A1, CYP1B1, mEH, and NADPH OR in BEC recovered from 10 nonsmokers and 9 smokers. CYP2B7 expression was evaluated because, although little is known of its substrate specificity, it is expressed at high levels in human lung tissue. CYP1A1 and CYP1B1 were expressed in BEC at significantly different levels (P < 0.05) in the 9 smokers at 1.4 +/- 2.3 x 10(4) and 2.4 +/- 3.2 x 10(3) molecules/10(6) beta-actin molecules (mean +/- STD), respectively, but each was measurable in only one of the 10 nonsmokers. There was significant inter-individual variation (P < 0.05) in both CYP1A1 and CYP1B1 expression among the subjects for whom sufficient data were obtained. The inducibility of human BEC CYP1A1 gene by PAH exposure was confirmed in vitro by incubating cultured immortalized human BEC with beta-naphthoflavone and observing a > 6-fold induction of CYP1A1 after 24 h. In contrast to BEC, alveolar macrophages expressed CYP1A1 at low (30-70 molecules/10(6) beta-actin molecules) to unmeasurable levels in both smokers and nonsmokers. There was no significant difference in expression of mEH, CYP2B7, or NADPH OR in smokers compared with nonsmokers. The inter-individual variation in absolute and relative expression of PAH metabolism enzymes in BEC reported here supports the hypothesis that inter-individual variation in ability to activate/inactivate inhaled PAH carcinogens accounts for at least some of the inter-individual variation in risk for bronchogenic carcinoma.

Adult↗

PubMed1997

Coexpression of cytochrome P4502A6 and human NADPH-P450 oxidoreductase in the baculovirus system.

Heterologous expression using baculovirus vectors has become a popular method for the production of catalytically active cytochrome P450s (CYPs). We have systematically optimized the multiplicity of infection (MOI) for a coinfection approach for the coexpression of CYP2A6 (viral vector designated v2A6) and NADPH-P450 oxidoreductase (OR; viral vector designated vOR) using Sf9 insect cells. A 3000-fold range of MOI was examined in stationary culture and stirred suspension culture. Surprisingly, our results indicate that the best CYP2A6 catalytic activity (850-1300 pmol/ min/mg total lysate protein as measured by coumarin 7-hydroxylase activity) was obtained only when using a low MOI of v2A6 (1.5-3 x 10(-2)) and a vOR of 10- to 20-fold less. This activity was approximately 7- to 11-fold higher than the best activity obtained when infecting cells with v2A6 alone. At this level of coinfection, the P450 content ranged from 180 to 250 pmol/mg total lysate protein, and the NADPH cytochrome c reductase activity ranged from 350 to 520 nmol/min/mg total lysate protein. Increasing the MOI of both viruses to 50-fold higher resulted in lower overall activity with the optimum (250 pmol/min/mg total lysate protein) being seen earlier postinfection (60 vs. 72 hr). Increasing the MOI of vOR to levels comparable with those of v2A6, decreased coumarin 7-hydroxylase activity 14-fold. These results suggest that the best CYP2A6 catalytic activity depends on properly posttranslationally modified proteins accumulating in a right ratio as a result of primary, secondary, and possibly tertiary infection of both viruses. These results also suggest that high OR expression results in degradation of P450.

Animals↗

PubMed1997

Human cytochrome P4502B6: interindividual hepatic expression, substrate specificity, and role in procarcinogen activation.

The level of expression and interindividual variation in human hepatic microsomal cytochrome P450 (CYP) 2B6 was characterized using a polyclonal antibody (WB-2B6) raised against rat CYP2B1. Immunoblot analysis using cDNA-expressed human CYPs revealed strong cross-reactivity of this antibody with CYP2B6 (limit of detection < 0.05 pmol) and only minor cross-reactivities with human CYP2A6, CYP2D6, and CYP2E1, all of which could be resolved from CYP2B6 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of human liver microsomes using this antibody revealed immunodetectable CYP2B6 protein in a majority of individual liver samples, with levels up to 74 pmol/mg protein in the CYP2B6-positive samples. Kinetic analysis of cDNA-expressed CYPs identified many of these enzymes as catalysts of 7-ethoxy-4-trifluoromethylcoumarin (7EFC) O-deethylation, but with significantly different apparent K(M) values (CYP1A2 < CYP2B6 approximately CYP1A1 < CYP2C19 < CYP2C9 < CYP2E1 < CYP2A6). By assaying liver microsomal 7EFC O-deethylase activity at a low 7EFC concentration (5 microM) and preincubating human liver microsomes with anti-CYP1A, anti-CYP2C, and anti-CYP2E1 antibodies, we were able to monitor CYP2B6-dependent 7EFC O-deethylase activity in a panel of 17 human liver microsomes and observe a significant correlation (r2 = 0.80) between this activity and CYP2B6 protein content. The ability of CYP2B6 to activate prodrugs and procarcinogens was examined using gene locus mutation assays in CYP2B6-expressing human lymphoblast cells. CYP2B6-expressing cells were found to be more sensitive than control cells to the cytotoxicity and mutagenicity of cyclophosphamide, aflatoxin B1, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. CYP2B6 is thus a widely expressed human liver microsomal CYP that can contribute to a broad range of drug metabolism and procarcinogen activation reactions.

Adolescent↗

PubMed1996

Human cell mutagenicity of oxygenated, nitrated and unsubstituted polycyclic aromatic hydrocarbons associated with urban aerosols.

Polycyclic aromatic compounds (PAC) are ubiquitous pollutants in urban air that may pose risks to human health. In order to better assess the health risks associated with this class of compounds, a total of 67 PAC that either have been identified (55) or are suspected to be present (12) in urban aerosol samples were tested for mutagenicity in a forward mutation assay based on human B-lymphoblastoid cells. The cell line used (designated h1A1v2) constitutively expresses the cytochrome P4501A1, which is known to be necessary for the metabolism of many promutagens. The PAC tested included 39 polycyclic aromatic hydrocarbons (PAH). 19 oxygen-containing PAH (oxy-PAH) and nine NO2-substituted PAH (nitro-PAH). A total of 26 PAH were mutagenic. In comparing the minimum mutagenic concentrations of the mutagenic PAH with that of benzo[a]pyrene (B[a]P) it was found that dibenzo[a,l]pyrene (DB[al]P), cyclopenta[c,d]pyrene (CPP), naphtho[2,1-a]pyrene, dibenzo[a,e]pyrene (B[a]P) and 1-methylbenzo[a]pyrene were 24 +/- 21, 6.9 +/- 4.2, 3.2 + 3.0, 2.9 +/- 2.9 and 1.6+/- 1.4 times, respectively, more mutagenic than B[a]P, and that dibenzo[a,k]fluoranthene and B[a]P were approximately equally mutagenic. The 19 other mutagenic PAH were between approximately 2 and approximately 1800 times less mutagenic than B[a]P. Of the oxy-PAH tested only phenalenone, 7H-benz[d,e]anthracen-7-one, 3-nitro-6H-dibenzo[b,d]pyran-6-one, cyclopenta[c,d]pyren-3(4H)-one, 6H-benzo[c,d]pyren-6-one (BPK) and anthanthrenequinone were mutagenic; however, with the exception of BPK, these were over 50 times less active than B[a]P, BPK was approximately 3 times less active than B[a]P. Seven of the nitro-PAH were mutagenic including 9-nitroanthracene, 1-nitropyrene, 2-nitrofluoranthene, 3-nitrofluoranthene, 1,3-dinitropyrene, 1,6-dinitropyrene (1,6-DNP) and 1,8-dinitropyrene. 1,6-DNP was approximately 4 times less active than B[a]P; the six other mutagenic nitro-PAH were between 20 and 380 times less active than B[a]P. These results are discussed in terms of their relevance for determining the most important mutagens in ambient air. Based on reported concentrations of PAC in ambient aerosols, it is possible that CPP, DB[ae]P, DB[al]P and BPK could account for a greater proportion of the mutagenicity than B[a]P in some aerosols.

Aerosols↗

PubMed1996

Development of Caco-2 cells expressing high levels of cDNA-derived cytochrome P4503A4.

PURPOSE: To develop Caco-2 cell derivatives expressing high levels of human cytochrome P450 drug metabolizing enzymes. METHODS: The cDNAs for two cytochrome P450 forms, CYP2A6 and CYP3A4, were introduced into an extrachromosomal vector under control of the cytomegalovirus early intermediate promoter. Vector-bearing cells were selected via resistance to hygromycin B. RESULTS: Transfected cells exhibited high levels of cDNA-derived protein as measured by Western blot, spectrophotometric P450 determination and/or cytochrome P450 form-selective enzyme assay. CYP3A4 and CYP2A6 catalytic activities were about 100 fold higher than in control cells. cDNA-expressing cells were found to form tight monolayers and were suitable for study of xenobiotic transport and metabolism. The permeabilities of cephalexin, phenylalanine, mannitol and propranolol across transfected monolayers were found to be similar to those across untransfected monolayers. The appropriate transfected monolayers metabolized the CYP2A6 substrate coumarin and the CYP3A4 substrates testosterone and nifedipine. CONCLUSIONS: A Caco-2 cell system to simultaneously study drug transport and metabolism has been developed.

Aryl Hydrocarbon Hydroxylases↗

PubMed1995

Mutagenicity of benzo[a]pyrene and dibenzopyrenes in the Salmonella typhimurium TM677 and the MCL-5 human cell forward mutation assays.

The mutagenicity of benzo[a]pyrene (B[a]P), dibenzo[ae]pyrene (DB[ae]P), dibenzo[ah]pyrene (DB[ah]P), dibenzo[ai]pyrene (DB[ai]P), and dibenzo[al]pyrene (DB[al]P) was measured in quantitative forward mutation assays with bacteria (Salmonella typhimurium TM677) and a metabolically competent cell line derived from human B-lymphoblastoid cells (MCL-5) that contained activity for five cytochrome P450s and microsomal epoxide hydrolase found in human liver. DB[al]P and B[a]P, both potent animal carcinogens, were the most mutagenic substances in both assays. DB[al]P was nearly 50-fold more potent than B[a]P in human cells, but only 60% more mutagenic in Salmonella. The carcinogenic isomer DB[ah]P, though nonmutagenic in bacteria, was active in human cells. The following mutagenic potency series, expressed as the minimum detectable mutagen concentration (MDMC) in nmol/ml, was obtained with Salmonella in the presence of rat liver postmitochondrial supernatant (PMS): DB[al]P (3.7), B[a]P (5.8), DB[ae]P (6.9), DB[ai]P (14.9), DB[ah]P (> 100). None of the compounds were mutagenic in the absence of PMS. In human MCL-5 cells the potency series was: DB[al]P (3.1 x 10(-4)), B[a]P (1.5 x 10(-2)), DB[ae]P (2.5 x 10(-2)), DB[ah]P (0.5), DB[ai]P (3.2). The human cell assay thus exhibited over a 10,000-fold range between the most mutagenic and least mutagenic compound, whereas in the bacterial assay there was only a corresponding four-fold difference if the nonmutagenic DB[ah]P was excluded. The results were discussed in terms of their concordance with animal carcinogenicity studies.

Benzo(a)pyrene↗

PubMed1995

Comparison of substrate metabolism by wild type CYP2D6 protein and a variant containing methionine, not valine, at position 374.

We have analysed kinetic parameters of cDNA-derived CYP2D6 proteins derived from the original CYP2D6 cDNA isolate (Gonzalez FJ et al. Nature 1988: 331, 442-446) which contains methionine at position 374 (CYP2D6-Met) and a modified cDNA which contains valine at position 374 (CYP2D6-Val). This latter protein is predicted from the CYP2D6 genomic sequence. Several quantitative differences, but no qualitative differences in metabolism were observed. CYP2D6-Met was found to have a two-fold lower Km and a three-fold lower turnover rate for (R)(+)-bufuralol 1'-hydroxylation as compared to CYP2D6-Val. In contrast, CYP2D6-Met and CYP2D6-Val had a similar Km for debrisoquine 4-hydroxylation while CYP2D6-Val had an 18-fold higher turnover rate. CYP2D6-Val and CYP2D6-Met had similar Kms for metoprolol but CYP2D6-Val showed a three-fold higher capacity for the O-demethylation reaction compared to alpha-hydroxylation which is more similar to that seen in human liver. In the case of sparteine, CYP2D6-Val and CYP2D6-Met showed similar capacities for formation of the 2-dehydrosparteine metabolite but the Km value for CYP2D6-Met was six-fold higher than that for CYP2D6-Val. Kinetic differences between CYP2D6-Met and CYP2D6-Val were further probed by examination of apparent Ki for inhibition of (R,S)(+/-)-bufuralol 1'-hydroxylation. Similar Ki values (within a factor of three) were observed for perhexiline and (R,S)-propranolol while quinidine and dextromethorphan were 8.5-fold and 21-fold more effective inhibitors of CYP2D6-Val relative to CYP2D6-Met. An allele specific polymerase chain reaction assay was developed for the CYP2D6-Met allele. The CYP2D6-Met allele was not found among 83 individuals. In the aggregate, these data indicated that the CYP2D6-Val allele is the more common allele in human populations. The quantitative kinetic differences between these two enzymes appears most pronounced for substrates/inhibitors with rigid structures. CYP2D6-Val more often has a substantially lower Km and/or a substantially higher capacity to metabolize those substrates.

B-Lymphocytes↗