Expression and inducibility of drug-metabolizing enzymes in novel murine liver epithelial cell lines and their ability to activate procarcinogens

Four novel nontransformed epithelial cell lines, isolated from fetal or adult mouse liver, were tested: (a) to determine the profile of xenobiotic metabolizing enzymes; (b) to evaluate the inducibility of the polysubstrate (cytochrome P-450-dependent) monooxygenase system by various classes of inducers; and (c) to assess the capacity of the cells to metabolize structurally different procarcinogens. With regard to the phase I pathway, the cells expressed various P-450 (class IA, IA2, IIB, IIE1, IIIA) and flavin adenine dinucleotide-containing monooxygenase-dependent bio-transformation enzyme activities at levels (in lines C2.8 and C6) comparable with those present in murine adult liver preparations. The expression of various P-450s was demonstrated also by immunoprecipitation assays using rabbit polyclonal antibodies. For the phase II pathway, cells expressed substantial levels of glutathione S-transferase, glutathione S-epoxide transferase, and UDP-glucuronosyltransferase. Low expression of epoxide hydrolase was observed. Induction of P-450 function by sodium phenobarbital, beta-naphthoflavone, isosafrole, ethanol, and pregnenolone 16 alpha-carbonitrile, monitored using specific P-450-linked activities, was considerably elevated (over 5-fold in class IIB with the C2.8 and C6 cell lines). The most competent C2.8 and C6 cell lines were able to activate benzo(a)pyrene, cyclophosphamide, dimethylnitrosamine, diethylstilbestrol, and 2-naphthylamine as shown by the significantly increased frequencies of mitotic gene conversion, mitotic crossing-over, and point [reverse] mutation in the diploid D7 strain of Saccharomyces cerevisiae after 4 [cyclophosphamide], 24 [benzo(a)pyrene,2-naphthylamine, dimethylnitrosamine] or 48 [diethylstilbestrol], h of exposure in the presence of 3 x 10(6) cells/flask. The degree of conservation and the inducibility of representative oxidative and postoxidative reactions in the novel epithelial cell lines C2.8 and C6, together with their ability to activate a wide spectrum of procarcinogens, offers a means to study the potential of chemicals for inducing DNA damage in short-term genotoxicity testing. In addition the cells may be suitable for analyzing the metabolic disposition of compounds and the multistage process of carcinogenesis.     

Development of a human lymphoblastoid cell line constitutively expressing human CYP1A1 cDNA: substrate specificity with model substrates and promutagens

AHH-1 TK+/–cell derivatives were developed that stablyexpress human CYP1A1 cDNA, and an AHH-1 TK+/–derivativeexpressing higher levels of CYP1A2 cDNA in extrachromosomalvectors which confer resistance to I-histidinol. The CYP1A1-expressingcell lines, designated h1A1 and h1A1v2, differ by containingone and two CYP1A1 cDNA expression units per vector. The CYP1A2-expressingcell line, designated h1A2v2, also has two CYP1A2 cDNA expressionunits per vector. Microsomes prepared from CYP1A1 cDNA expressingcells exhibit high, constitutive levels of 7-ethoxyresorufindeethylase (EROD), 7-ethoxycoumarin deethylase (ECD), 7-ethoxy-4-trifluoromethylcoumarindeethylase (EFCD), benzo[a]-pyrene hydroxylase (BPH) activitiesand spectrally quantifiable cytochrome P450. Kinetic comparisonsbetween cDNA-expressed CYP1A1 and CYP1A2 indicate that CYP1A1is more active than CYP1A2 for EROD, ECD, EFCD and BPH. CYP1A2was more active than CYP1A1 for acetanilide hydroxylation andactivation of aflatoxin B₁ (AFB₁). The mutagenicity of selectedpromutagens were examined in h1A1 cells and control cells. Relativeto control cells, the h1A1 cell line exhibits increased sensitivityto the mutagenicity of benzo[a]pyrene, cyclopenta[c,d]pyrene,4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and AFB₁.     

Wide spectrum detection of precarcinogens in short-term bioassays by simultaneous superinduction of multiple forms of cytochrome P450 isoenzymes

The use of Aroclor 1254 to induce S9 liver fractions is a standard method for conducting short-term genotoxicity assays. An alternative induction procedure, using beta-naphthoflavone (beta-NF), as a safe (non-carcinogenic) substitute for polychlorinated biphenyls, combined with sodium phenobarbital (PB), was found to be equally effective. The aim of this work is to realize a novel schedule of induction for the preparation of metabolizing systems containing a wider spectrum of induced cytochrome P450s. Five inducers of different 'classes' such as PB (class IIB P450s), beta-NF (IA), isosafrol (IA2), ethanol (IIE1) and pregnenolone 16 alpha-carbonitrile (IIIA) were injected daily both separately (to achieve maximal monooxygenase induction) in male and female mice. Induction was monitored using specific P450-linked activities. In the optimal schedule for complete induction, the various monooxygenases were greater (2- to 4-fold) than those achieved by the classical schedule. More than a 14-fold increase of total P450 and 3.3-fold increase of NADPH-cytochrome (P450) c-reductase activity, over those uninduced, account for the above increase. For example, there was a marked increase in the deethylation of ethoxyresorufin (37-fold) compared to the uninduced mice that was considerably higher than classical induction (8-fold over uninduced). On the contrary, phase II reactions i.e. epoxide hydrolase, glutathione S-transferase, glutathione S-epoxide transferase and UDP-glucuronosyl transferase, examined to compare the phase I/phase II ratios in the traditional and proposed procedures, were increased to a lesser extent (2-fold over uninduced). No significant sex differences were seen. Five precarcinogens specifically metabolized by each of the induced P450s elicited a higher mutagenicity response in the presence of superinduced fractions with respect to the classical one, when tested on Salmonella typhimurium (cyclophosphamide, benzo[alpha]pyrene, 2-naphthylamine and dimethylnitrosamine) or Saccharomyces cerevisiae D7 strain (diethylstilbestrol). These novel metabolizing biosystems, with an enhanced spectrum of induced P450s and oxidative/post-oxidative reaction rates, are recommended for detecting unknown xenobiotics in genotoxicity studies.     

Expression of mouse cytochrome P450IA1 cDNA in repair-deficient and repair-proficient CHO cells.

Recombinant DNA techniques have been used to develop Chinese hamster ovary cell lines for studying chemically induced genotoxicity. These cell lines express a specific cytochrome P450 isozyme responsible for the metabolism of polycyclic aromatic hydrocarbons and exhibit defined differences in DNA repair capacity. A bacterial gene (neo) conferring resistance to gentamicin was inserted into the pcD expression vector containing the mouse cytochrome P1450 (P450IA1) cDNA to facilitate the selection of transformed cells. This plasmid was introduced into the nucleotide-excision-repair-deficient UV5 cell line by electroporation. Transformed clonal isolates expressing the P1450 cDNA were identified by differential cytotoxicity assays using benzo[a]pyrene (B[a]P). One such clone, termed UV5P1, was mutagenized with ethyl methanesulfonate and selected for resistance to killing by UV radiation to derive a repair-competent clone that expresses similar P1450 activity to that of the parental cell line. Two repair-competent clones were selected and called 5P1R1 and 5P1R3. The resulting cell lines (UV5P1, 5P1R1, and 5P1R3) expressed arylhydrocarbon hydroxylase activity. UV5P1 and 5P1R3 were compared in terms of cytotoxicity and mutagenicity after exposure to B[a]P. Induced mutations were measured at the adenine phosphoribosyltransferase (aprt) and hypoxanthine guanine phosphoribosyltransferase (hprt) loci. Repair-deficient UV5P1 cells were killed by B[a]P at concentrations below 0.1 microM, while the repair-proficient 5P1R3 cells showed no toxicity up to 60 microM. Mutation induction at both loci was also much more efficient in UV5P1 cells. These new cell lines provide a more sensitive system that can be used in a battery of assays to evaluate the genotoxicity of chemicals requiring P450IA1 metabolic activation and to assess the role of DNA repair in modulating the biological effects of DNA damage.     

Transfection of a human cytochrome P-450 gene into the human lymphoblastoid cell line, AHH-1, and use of the recombinant cell line in gene mutation assays

We have demonstrated that the human cytochrome P₁-450 gene canbe transfected into the AHH-1 human lymphoblastoid cell lineusing the pHEBo vector and hygromycin selection. The transfectedgene was expressed when regulatory sequences derived from theherpes simplex virus thymidine kinase gene were incorporatedin appropriate orientations. Gene expression was monitored atthe enzyme level using assays for 7-ethoxyresorufin deethylase,7-ethoxycoumarin deethylase and benzo(a)pyrene hydroxylase activities.Bulk transformed cell populations had 2- to 3-fold more of theseenzyme activities compared with control populations. Subclonesof the bulk population expressing still higher levels of 7-ethoxyresorufindeethylase activity were also obtained. Expression of the transfectedcytochrome P₁-450 gene was stable for 20–30 days in thepresence of hygromycin B. The transformed cell populations werefound to be suitable for use in gene locus mutation assays andthe mutagenicity of aflatoxin-B₁ and 2-acetylaminofluorene (AAF)were examined. Aflatoxin-B₁ was found to be 2–3 timesmore mutagenic to cells bearing the transfected cytochrome P₁-450activity as compared with control cells. In contrast, no differencein AAF mutagenicity was observed. Analysis of the AAF metaboliteprofile indicated that cells expressing the transfected cytochromeP₁-450 gene produced 8-fold more N- and 7-hydroxy-AAF than controlcells. The similarity in mutagenic responses between controlcells and cells bearing the transfected cytochrome P₁-450 genemay be due to the low deacetylase activity of AHH-1 cells. Theseobservations indicate that this vector and expression systemare suitable for introducing novel metabolic activities intothe AHH-1 cell line.     

Smokeless tobacco extracts mutate human cells

Two commercial brands of smokeless tobacco were extracted with water and these extracts were tested in human cell mutation assays. Using the human cell line TK-6 which expresses no cytochrome P450, the two extracts tested were found to be detectably mutagenic in the range 1-3 mg/ml extractable solids. In AHH-1 cells which constitutively express cytochrome P450IAI, a similar result was found for both brands tested. The two extracts were treated with neutral nitrite solutions to mimic physiologic oral conditions or acidic conditions or acidic conditions with nitrite to mimic physiologic gastric conditions. The mutagenicity of both extracts for both TK-6 and AHH-1 cells was markedly decreased by treatment at neutral pH with sodium nitrite (0.25 mM) and by acidic treatment (2 h, pH 3.0). Treatment of extracts with sodium nitrite at pH 3.0 did not have any effect on the mutagenicity of the untreated extracts for TK-6 cells. The mutagenicity of both the extracts destroyed by acidic treatment however, seemed to be restored to a level equivalent to the mutagenicity of the untreated extracts for the TK-6 cells. The same series of experiments with P450-proficient AHH-1 cells showed uniform reduction of mutagenic activity. Since the two cell lines are equally sensitive to mutation by aqueous tobacco extracts it is concluded that mutagenicity is not cytochrome P450 mediated. It would further appear that the extract mutagen(s) is acid and neutral nitrite labile.     

Stable expression of human cytochrome P450IA2 cDNA in a human lymphoblastoid cell line: role of the enzyme in the metabolic activation of aflatoxin B1

A human lymphoblastoid cell line stably expressing a human cytochrome P450IA2 cDNA was developed. This recombinant cell line displayed P450IA2 protein and estradiol 2-hydroxylase activity, neither of which was detected in the parental cell line. The recombinant cell line was also approximately 1000-fold more sensitive to the cytotoxicity and mutagenicity of the carcinogenic mycotoxin aflatoxin B1 than was the parent cell line. The increase in mutagenicity was supported by a corresponding increase in the level of aflatoxin B1 binding to DNA in cells expressing P450IA2 relative to control cells.     

The role of 12 cDNA-expressed human,rodent, and rabbit cytochromes P450 in the metabolism of benzo[a]pyrene and benzo[a]pyrene trans-7, 8-dihydrodiol

The potent carcinogen benzo[a]pyrene (B[a]P) and its metabolite B[a]P trans-7, 8-dihydrodiol (7, 8-diol) require metabolic activation by the microsomal cytochrome P450s (P450s) to exert several adverse biological effects, including binding to DNA, toxicity, mutagenicity, and carcinogenicity. In the study reported here, we defined the role of each of 12 individual cDNA-expressed cytochrome P450s in the metabolism of B[a]P and 7, 8-diol. Human P450s 1A1 and 1A2 were expressed in the absence or presence of epoxide hydrolase (EH) in a human lymphoblastoid cell line, and six human and five rodent and rabbit P450s were expressed from cDNA with vaccinia virus vectors in the hepatoma cell line Hep G2. B[a]P metabolism resulted in nine metabolites (three diols, three quinones, and three phenols), which were separated, identified, and quantitated by high-pressure liquid chromatography. In the human lymphoblastoid cells, human 1A1 metabolized B[a]P at a rate 4.5 times greater than that for 1A2. EH was shown to be directly involved in B[a]P activation, since increasing the amount of EH resulted in less 7-hydroxybenzo[a]pyrene and more 7, 8-diol formation. Of the human P450s expressed with the vaccinia virus vectors in Hep G2 cells, 1A2 and 2C9 showed the highest activity and 2B6 showed moderate activity for B[a]P metabolism. Mouse 1A1 had activity 40 times higher than any human, rabbit, or rodent P450s, indicating the potential pitfalls of extrapolating P450 activity across species. Metabolism of the 7, 8-diol resulted in six metabolites (four tetrols and two triols). In the lymphoblastoid cells, human 1A1 was shown to be 4.2 times more active than 1A2 for 7, 8-diol metabolism. Among human P450s expressed from vaccinia virus, 1A2, 2E1, and 2C9 gave the highest activity, and 2C8 and 3A4 showed moderate activity for 7, 8-diol metabolism to the diol epoxides. Again, mouse 1A1 was much more active than any other P450. These studies, in which we determined the capacity of individual P450 in the metabolism and activation of B[a]P and 7, 8-diol, may thus lead to a better understanding of how P450s control the detoxification and activation of polycyclic aromatic hydrocarbons. © 1994 Wiley-Liss, Inc.     

Metabolism of 2-acetylaminofluorene and benzo(a)pyrene and activation of food-derived heterocyclic amine mutagens by human cytochromes P-450

The human P-450 CYP1A1 gene and a P450IA2 complementary DNA have been expressed in Cos-1 cells and the expressed proteins were assayed for their capacity to metabolize the carcinogens 2-acetylaminofluorene (AAF), benzo(a)pyrene, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was determined. The expressed human P450IA1 and P450IA2 proteins, when run on a 7.5% sodium dodecyl sulfate-polyacrylamide gel, migrated with different mobilities, with the former displaying the lower molecular weight. In human liver microsomes from 18 subjects, only a protein band corresponding to P450IA2 was detectable. Cos-1 cell-expressed P450IA1 and P450IA2 were capable of N-hydroxylating AAF and these activities were inhibited by alpha-naphthoflavone. In human liver microsomes, a correlation of r = 0.76 (P less than 0.05; n = 18) was obtained between AAF N-hydroxylase activity and P450IA2 content. AAF N-hydroxylase activity of human liver microsomes was also strongly inhibited by alpha-naphthoflavone. Except in the case of PhIP, where both proteins exhibited similar activities, P450IA2 was at least an order of magnitude more efficient than P450IA1 in activating IQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline to mutagens as measured in the Ames test. Statistically significant correlations were obtained between IQ activation and P450IA2 content (r = 0.75, r2 = 0.56) and PhIP activation and P450IA2 content (r = 0.71, r2 = 0.5) in human liver microsomes. The activation of both IQ and PhIP by expressed proteins and human liver microsomes was strongly inhibited by alpha-naphthoflavone. The above data suggest a major role for P450IA2 in activation (N-hydroxylation) of aromatic amides and amines in human liver. When benzo(a)pyrene hydroxylase activity was determined, only Cos-1 cell-expressed P450IA1 exhibited appreciable activity. While alpha-naphthoflavone inhibited Cos-1 cell-expressed P450IA1 benzo(a)pyrene hydroxylase activity, it caused a marked stimulation of this activity in human liver microsomes, which lack P450IA1 protein. The lack of a role for P450IA proteins in benzo(a)pyrene metabolism is further supported by the poor correlation (r = 0.43, P greater than 0.05) between this activity and P450IA2 content of human liver microsomes. However, when P450IIIA3 content of the above human liver microsomes was determined by using the Western blot technique and correlated with benzo(a)pyrene metabolism, an r value of 0.70 (P less than 0.5) was obtained. These data suggest that human P450IIIA proteins are involved in benzo(a)pyrene metabolism.     

Genotoxicity of tamoxifen epoxide and toremifene in human lymphoblastoid cells containing human cytochrome P450s

The clastogenicity of tamoxifen and toremifene was tested insix human lymphoblastoid cell lines each expressing increasedmonooxygenase activity associated with a specific transfectedhuman cytochrome P450 cDNA (CYP1A1, CYP1A2, CYP2D6, CYP2E1 orCYP3A4). The chemicals were also tested in a cell line (MCL-5)expressing elevated native CYP1A1 and containing transfectedCYP1A2, CYP2A6, CYP2E1 and CYP3A4 and epoxide hydrolase, andin a cell line containing only the viral vector (Ho1). Dose-relatedincreases in micronuclel were observed when cells expressing2E1, 3A4, 2D6 or MCL-5 cells were exposed to tamoxifen. Thepositive responses in the cell lines were in the order MCL-5> 2E1 > 3A4 > 2D6. Toremifene also gave positive resultswith 2E1, 3A4 and MCL-5 cells, although the responses were lessmarked and the positive effects required higher doses than withtamoxifen. A synthesized epoxide of tamoxifen was also testedin these cell lines and produced similar increases in the incidencesof micronucleated cells. The increases in the responses observedwith the epoxide were greater than with tamoxifen or toremifene.The P450 isoenzyme activities in these cells were in a rangesimilar to those of human tumour-derived cell lines. Microsomes(1A1, 2A2, 2A6, 2B6, 2E1, 3A4 and 2D6) from these cells allmetabolized tamoxifen. The major metabolite detected by HPLCwas N-desmethyltamoxifen, and 4-hydroxytamoxifen was also detectedin cells with cytochrome P450 2E1 and 2D6. These results areconsistent with the following conclusions. (1) Tamoxifen requiresmetabolic activation to DNA-reactive species by specific CYPmonooxygenases in order to exert its genotoxic effects. (2)The positive clastogenic effects elicited in lymphoblastoidcells by tamoxifen epoxide suggest that the genotoxic (and possiblythe carcinogenic) effects of tamoxifen may be due to one ormore epoxide metabolites that are generated intracellularly,probably in close proximity to the nudeus. (3) Tamoxifen ismore genotoxic than toremifene.     

Characterization of a novel cytochrome P450 from the transformable cell line, C3H/10T1/2

The cytochrome P450 in the transformable C3H/10T1/2 (10T1/2) cell line has been characterized and compared to the major polycyclic aromatic hydrocarbon (PAH)-inducible hepatic form, cytochrome P450IA1 (P450IA1). The mouse hepatoma cell line, Hepa-1, was used as an in vitro model for P450IA1 expression and regulation by PAH. Microsomes from uninduced and benz[a]anthracene (BA)-induced 10T1/2 cells provided PAH mono-oxygenated product profiles that were totally different from metabolite profiles produced by microsomes from uninduced and BA-induced Hepa-1 cells even though total activities were similar. The proximate carcinogen, 7,12-dimethylbenz[a]anthracene-3,4-diol (DMBA-3,4-diol) was a major product for the 10T1/2 microsomes, while Hepa-1 formed less than 2% of this metabolite. Hepa-1 converted benzo[a]pyrene (BP) to BP-4,5-diol and DMBA to 7-hydroxymethyl-12-methyl-BA, while 10T1/2 did not produce either product. Polyclonal antibody to rat hepatic P450IA1 did not inhibit metabolism of either PAH substrate by 10T1/2 microsomes, but totally inhibited such metabolism by Hepa-1 microsomes. Western immunoblot analysis of BA-induced 10T1/2 microsomes showed that less than 1% of total P450 was P450IA1. The PAH-metabolizing activity of 10T1/2 microsomes was highly inducible (14-fold) by pre-treatment of non-confluent intact cells with BA, but was only half as inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin. In contrast, the P450IA1 activity of Hepa-1 cells was highly inducible by both compounds. The distinct metabolite profiles, antibody inhibition data and lack of immunoreactivity all indicate that PAH metabolism in 10T1/2 cells is catalyzed by a form of P450 distinct from P450IA1. The anomalous induction patterns suggest that this novel isozyme is predominantly regulated by a mechanism other than the Ah receptor.     

Human cytochrome P450IIA3: cDNA sequence, role of the enzyme in the metabolic activation of promutagens, comparison to nitrosamine activation by human cytochrome P450IIE1

We report that, in a human cell line, human cytochrome P450IIA3 is capable of metabolizing aflatoxin B1, benzo[a]-pyrene, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) to cytotoxic and mutagenic species. Cytochrome P450IIA3-mediated activation of NDMA and NDEA was compared with human cytochrome P450IIE1-mediated activation in the same cell system. P450IIE1 was more effective at activating NDMA than P450IIA3, while P450IIA3 was more effective at activating NDEA than P450IIE1. Whole cells and microsomal fractions obtained from control cells and from cells expressing the P450IIA3 cDNA were characterized for expression of P450IIA3. Microsomal coumarin 7-hydroxylase activity was some 40 times greater in the transfected cells than in the control cells and was catalyzed by a protein that was immunochemically related to the rat liver cytochrome P450IIA gene family. Immunoblot analysis demonstrated that this protein was readily detectable in transfected cells but barely detectable in control cells. We also report the DNA and deduced amino acid sequence of the P450IIA3 cDNA isolate used in this study. Our isolate encodes a protein 489 amino acids that is five amino acids shorter at the N terminus but otherwise identical to a previously reported human P450IIA3 cDNA sequence.     

A tobacco smoke-derived nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, is activated by multiple human cytochrome P450s including the polymorphic human cytochrome P4502D6

We have developed a human B-lymphoblastoid cell line, designated 2D6/Hol, which stably expresses human cytochrome P450 CYP2D6 cDNA. This cell line exhibits bufuralol 1'-hydroxylase activity and immunologically detectable CYP2D6 protein. The specific activity of (+)-bufuralol 1'-hydroxylase in microsomes from 2D6/Hol cells was comparable to that observed in human liver microsomes. This cell line was used to examine the mutagenicity activation of three tobacco smoke-derived nitrosamines, N-nitrosonornicotine (NNN), 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal) (NNA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), by CYP2D6. Exposure of 2D6/Hol cells to NNK concentrations of 30-90 micrograms/ml induced a concentration-dependent decrease in relative survival and increase in mutant fraction at the hypoxanthine guanine phosphoribosyl transferase (hprt) locus. In contrast, NNK was non-mutagenic and non-cytotoxic to control cells at exposure concentrations up to 150 micrograms/ml. NNK mutagenicity in 2D6/Hol cells was compared to the responses observed in isogenic cell lines expressing human CYP1A2 (1A2/Hol), human CYP2A3 (2A3/Hol) and human CYP2E1 (2E1/Hol). These three additional human cytochrome P450-expressing cell lines were also found to be sensitive to NNK-induced mutagenicity and cytotoxicity. We found no evidence for CYP2D6-mediated activation of NNN or NNA. NNN was non-cytotoxic and non-mutagenic to both control and 2D6/Hol cells. NNA was equally cytotoxic and mutagenic to control cells and 2D6/Hol cells. The activation of NNA to a mutagen may have been carried out by P450 native to the AHH-1 TK +/- cell line. The 2D6/Hol cell line, in conjunction with the control cell line and other isogenic cell lines expressing other human cytochrome P450 cDNAs provides a useful system for the examination of the role of the polymorphic CYP2D6 in human procarcinogen activation and drug metabolism.     

Metabolism of benzo[a]pyrene by brain microsomes of fetal and adult rats and mice. Induction by 5,6 benzoflavone, comparison with liver and lung microsomal activities

Using brain, lung and liver microsomes as the enzyme sourcein in vitro assays, benzo[a]pyrene (B[a]P) metabolism was studiedin fetuses and dams of mice (C57B1/6) and rats (WAG). Separationand quantitation of B[a]P metabolites were performed by h.p.l.c.Microsomal preparations were tested for cytochrome P-450 dependentO-dealkylatlon of 7-ethoxycoumarin and epoxide hydrolase activities.Another parameter measured included the conjugation of 1-chloro-2,4dinitrobenzene to glutathione by cytosolic glutathione-S-transferaseactivity. The induction of B[a]P metabolism was studied aftertreatment of animals with 5,6-benzoflavone (BF). Mixed functionoxygenase, epoxide hydrolase and glutathione-S-transferase activiteswere transpiacentally inducible after dams were treated withBF. Metabolic activation of B[a]P by fetal brain microsomeswas lower in both species than that by fetal lung and livermicrosomes, but it was higher in fetuses than in adults. Allmetabolites of B[a]P increased after BF treatment; the productionof 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (7,8-dihydrodiolB[a]P) was higher in brain microsomes from BF-treated rats thanthat in mice. In stimulated rats, the formation of 7,8-dihydrodiolB[a]P by fetal brain microsomes was higher than that by fetallung microsomes, whereas in mice, the opposite was observed.These data suggest that initiation could occur in utero, andpartially explain the species-specific differences in susceptibilityto transplacental tumorigenesis by polycyclic aromatic hydrocarbonsby differences in biotransformation in the target organ.     

High Susceptibility to Lung Cancer Analyzed in Terms of Combined Genotypes of P450IA1 and Mu-class Glutathione S-Transferase Genes

Lung cancer is closely associated with cigarette smoking. Aromatic hydrocarbons in smoke, including benzo[n]pyrene, first require metabolic activation by Phase I enzymes, cytochrome P450, to their ultimate forms, and these activated forms are then subjected to detoxification by Phase II enzymes, especially glutathione S-transferases. Thus, genetically determined susceptibility to lung cancer may depend on the metabolic balance between Phase I and Phase II enzymes. In this study, we identified individuals genetically at high risk of lung cancer in terms of polymorphisms of the P450IA1 gene and GST1 gene. The relative risk of individuals with a combination of the genotypes of both a homozygous rare allele of the P450IA1 gene and the nulled GST1 gene was remarkably high at 5.8 for lung cancer and 9.1 for squamous cell carcinoma compared with other combinations of genotypes.     

High susceptibility to lung cancer analyzed in terms of combined genotypes of P450IA1 and Mu-class glutathione S-transferase genes.

Lung cancer is closely associated with cigarette smoking. Aromatic hydrocarbons in smoke, including benzo[a]pyrene, first require metabolic activation by Phase I enzymes, cytochrome P450, to their ultimate forms, and these activated forms are then subjected to detoxification by Phase II enzymes, especially glutathione S-transferases. Thus, genetically determined susceptibility to lung cancer may depend on the metabolic balance between Phase I and Phase II enzymes. In this study, we identified individuals genetically at high risk of lung cancer in terms of polymorphisms of the P450IA1 gene and GST1 gene. The relative risk of individuals with a combination of the genotypes of both a homozygous rare allele of the P450IA1 gene and the nulled GST1 gene was remarkably high at 5.8 for lung cancer and 9.1 for squamous cell carcinoma compared with other combinations of genotypes.     

In vitro activation of the dioxin receptor to a DNA-binding form by food-borne heterocyclic amines.

The carcinogenic heterocyclic amines, which are formed upon cooking of protein-rich food, are activated in the body mainly by cytochrome P450 IA1 and IA2. Several of these co-called food mutagens have, by enzymatic and immunoblotting techniques, been shown to be weak inducers of cytochrome P450 IA in the rat. To elucidate whether this induction occurs via the dioxin receptor, the capacity of the heterocyclic amines to activate in vitro the dioxin receptor to a DNA-binding form was determined. The activation of the receptor in Hepa cell cytosol was analyzed with a gel-retardation assay using a [32P]3'-end-labeled synthetic oligonucleotide, corresponding to nucleotides -1026 to -999 of the rat cytochrome P450 IA1 gene (XRE1), as probe. Five out of 14 heterocyclic amines had the ability to induce specific DNA-binding activity in the Hepa cell cytosol in a dose-dependent manner. The concentrations eliciting 50% of the maximal effect (EC50) were found to be between 30 and 135 microM. Thus, in comparison to high-affinity ligands such as benzo[a]pyrene (B[a]P; EC50 11.2 nM), and 2,3,7,8-tetrachloro-1,6-dibenzo-p-dioxin (TCDD; EC50 1.9 nM), the activating capacity of the heterocyclic amines is low. Binding to the dioxin receptor has been shown to be dependent on the three-dimensional size of a molecule in relation to a 6.8 x 13.7 A rectangle. The relatively low EC50 values found for the heterocyclic amines might be explained by the smaller size of these molecules when compared to that of B[a]P and TCDD.     

Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by human cytochrome P450 1B1

Benzo[a]pyrene (B[a]P), a ubiquitous environmental, tobacco and dietary carcinogen, has been implicated in human cancer etiology. The role of human cytochrome P450 1B1 in the metabolism of B[a]P is poorly understood. Using microsomal preparations of human P450 1A1, 1A2 and 1B1 expressed in baculovirus-infected insect cells, as well as human and rat P450 1B1 expressed in yeast, we have determined the metabolism of B[a]P, with and without the addition of exogenous epoxide hydrolase, and B[a]P-7,8-dihydrodiol (7,8-diol), each substrate at a concentration of 10 microM. HPLC analysis detected eight major metabolites of B[a]P and four metabolites of the 7,8-diol. The results of these studies indicate that cytochrome P450 1B1 carries out metabolism of B[a]P along the pathway to the postulated ultimate carcinogen, the diol epoxide 2, at rates much higher than P450 1A2 but less than P450 1A1. The rates of formation of the 7,8-diol metabolite in incubations with epoxide hydrolase are 0.17 and 0.38 nmol/min/nmol P450 for human P450 1B1 and 1A1, respectively, and undetectable for 1A2. The rates of total tetrol metabolite formation from the 7,8-diol, which are indicative of diol epoxide formation, are 0.60, 0.43 and 2.58 nmol/min/nmol P450 for 1B1, 1A2 and 1A1 respectively. In agreement with other reports of rat P450 1B1 activity, our data show this rat enzyme to be very active for B[a]P and 7,8-diol, with rates higher than human P450 1B1. In addition to the established role of P450 1A1 in B[a]P metabolism, P450 1B1 may significantly contribute to B[a]P and 7,8-diol metabolism and carcinogenesis in rodent tumor models and in humans.      

Relative contribution of various forms of cytochrome P450 to the metabolism of benzo[a]pyrene by human liver microsomes

A variety of cytochromes P450 have been implicated in the hepaticmetabolism of benzo[a]pyrene (BP), including forms that areconstitutively expressed and those that are highly inducible.In the present study the metabolism of BP to organic solvent-solublederivatives by eight forms of cytochrome P450 isolated fromrat liver and by a series of 11 human liver microsomal sampleswas investigated. The relative contribution of specific P450forms to the human hepatic metabolism was evaluated. A 4-foldvariation in formation of total organic solvent-soluble BP metaboliteswas observed, as well as differences in the regio- and stereo-selectivityof this metabolism between the three individuals studied. Thelevels of expression of cytochromes P450 from five gene sub-families,as determined by Western blot analysis, did not show any correlationwith the rate of BP metabolism to organic solvent-soluble derivativesin these livers. No reduction in metabolism was observed inthree livers in which either the debrisoqulne P450 (P450IID1)was not expressed or bufuralol 1-hydroxylase activity was low.Of six different antibodies to forms of rat liver P450 tested,only those to P450s MC_la (P450IA2), MC_lb (P450IA1) and UT (P450IIA1)consistently inhibited BP metabolism. This inhibition was generallylimited and rarely exceeded 30%. An antibody to cytochrome P450PB (P450 did, however, inhibit the formation of metabolitesat the 4,5- and 9,10-positions of BP by microsomal fractionsof livers from one individual who had been receiving the drugphenytoin. These data indicate that several forms of P450 inhuman liver are involved in the metabolism of BP and that bothconstitutively expressed as well as inducible forms are importantin its disposition in man.