Comparison of mutagenesis and malignant transformation by dihydrodiols from benz[a]anthracene and 7,12-dimethylbenz[a]anthracene.

Five dihydrodiols derived from benz[a]anthracene (BA) and 4 dihydrodiols derived from 7,12-dimethylbenz[a]anthracene (DMBA) have been tested, together with the parent hydrocarbons, for their abilities to induce mutations to 8-azaguanine resistance in V79 (Chinese hamster cells and malignant transformation in M2 mouse fibroblasts. The syn- and anti-isomers of benz[a]anthracene 8,9-diol 10,11-oxide were also tested for biological activity in these two systems. The non-K-region 1,2- and 3,4-dihydrodiols of BA induced mutations but the non-K-region 8,9-dihydrodiol and the K-region 5,6-dihydrodiol were inactive as mutagens; none of these BA diols transformed M2 mouse fibroblasts. The 3,4- and the 8,9-dihydrodiols derived from 7,12-dimethylbenz[a]anthracene induced mutations in V79 cells and malignant transformation in M2 mouse fibroblasts and both were more active than the hydrocarbon itself. The K-region 5,6-dihydrodiol and the non-K-region 10,11-dihydrodiol of DMBA were inactive in both test systems. The results are not inconsistent with other data suggesting that the metabolic activation of both BA and DMBA occurs through conversion of the respective 3,4-dihydrodiols into the related vicinal diol-epoxides, although other dihydrodiols may also be involved in vivo. Both the BA diol-epoxides tested were mutagenic, but although the anti-isomer transformed M2 fibroblasts, the syn-isomer was inactive.     

The involvement of a 'bay-region' and a non-'bay-region' diol-epoxide in the metabolic activation of benz[a]anthracene in mouse skin and in hamster embryo cells

The principal nucleoside-hydrocarbon adducts present in hydrolysatesof DNA that had been isolated either from mouse skin or fromhamster embryo cells treated with benz(a)anthracene have beenexamined by chromatography on Sephadex LH20 and by high pressureliquid chromatography on Spherisorb 5 ODS. The results showthat one of the major adducts prepared from the DNA of mouseskin and of hamster embryo cells has chromato-graphic propertiessimilar to those of an adduct formed when anti-BA-8, 9-diol10, 11-oxide (r-8,t-9-dihydroxy-t10, 11-oxy-8, 9, 10, 11-tetrahydro-benz(a)anthracene)reacts with DNA, whilst a second major adduct has chromatographicproperties similar to those of an adduct prepared by reactinganti-BA-3, 4-diol 1, 2-oxide (t-3, r-4-dihydroxy-t-1, 2-oxy-1,2, 3, 4-tetrahydrobenz(a)anthracene) with DNA. On the basisof this and other evidence, it appears that both of these diol-epoxidesmay contribute to the biological activity of benz(a)anthracene.     

Characterization of the Ah receptor and aryl hydrocarbon hydroxylase induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin and benz(a)anthracene in the human A431 squamous cell carcinoma line

Certain human cell lines previously have been shown to exhibit substantial induction of aryl hydrocarbon hydroxylase (AHH, cytochrome P450IA1) when treated in culture with aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benz(a)anthracene. Yet the Ah receptor, which is known to mediate the AHH induction process in rodent cells and tissues, has not previously appeared to be present at a significant level in any human cell line. In the human A431 squamous cell carcinoma line we found that cytosolic Ah receptor was present in high concentration (approximately 200 fmol/mg cytosol protein at maximal saturation); this corresponds to approximately 10,000 Ah receptor sites per cell in the human A431 line compared with about 35,000 receptor sites per cell in the mouse Hepa-1 hepatoma cell line in which Ah receptor previously has been extensively characterized. Detection of Ah receptor in A431 cytosol required modification of assay techniques, especially reduction in the amount of charcoal used to adsorb nonspecifically bound radioligand. The specific binding peak from A431 cytosol sedimented approximately 9S on sucrose gradients, the same as the cytosolic receptor from the well-characterized mouse Hepa-1 hepatoma cell line. In addition to [3H]TCDD, specific binding to Ah receptor in A431 cytosol also was detected with [3H]3-methylcholanthrene and with [3H]benzo(a)pyrene as radioligands. A specific [3H]TCDD-Ah receptor complex was extracted from nuclei of A431 cells incubated in culture at 37 degrees C with [3H]TCDD. The nuclear form of Ah receptor sedimented approximately 5S, the same as the nuclear receptor from mouse Hepa-1 cells. AHH activity was induced in A431 cells treated in culture with TCDD or benz(a)anthracene. The maximum level of induced AHH activity that could be achieved in A431 cells was about 20% of the maximally induced level in the mouse Hepa-1 cell line. However, the dose-response curves for AHH induction by TCDD or benz(alpha)anthracene in A431 cells were shifted about one log unit to the right of the curves for Hepa-1 cells. The lower sensitivity of A431 cells to AHH inducers was in proportion to the lower affinity with which cytosolic Ah receptor in A431 cells bound [3H]TCDD. The saturation curve for binding of [3H]TCDD to cytosolic Ah receptor in A431 cells also was shifted about one log unit to the right of the curve for saturation of the cytosolic receptor from mouse Hepa-1 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and characterization of cytochrome P-450 induced by benz(a)anthracene in mouse skin microsomes

Topical application of benz(a)anthracene to mouse skin elicited a 2-fold increase in cytochrome P-450 content, with accompanying increases in monooxygenase activities such as benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and acetanilide 4-hydroxylation, in the microsomes. A major form of cytochrome P-450 was purified from skin microsomes of mice treated with polycyclic aromatic hydrocarbon. A specific content of 1.95 nmol/mg of protein, which corresponded to 48-fold purification from the microsomes, was observed. The purified protein produced a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a molecular weight of 55,000. Using Western blotting, the band immunochemically cross-reacted with antibody which had been raised against rat liver cytochrome P-450MC-1. The purified preparation efficiently catalyzed benzo(a)pyrene hydroxylation and 7-ethoxycoumarin O-deethylation when reconstituted with NADPH-cytochrome P-450 reductase. These activities were inhibited by 7,8-benzoflavone as well as anti-cytochrome P-450MC-1 antibody, but not by P-450PB-1 antibody. The results indicate that, in mouse skin microsomes, a cytochrome P-450 induced by benz(a)anthracene is enzymatically and immunochemically similar to rat liver cytochrome P-450MC-1. It is suggested that this enzyme plays an important role in the activation of carcinogenic polycyclic aromatic hydrocarbons.     

Tumorigenicity of nitrated derivatives of pyrene, benz[a]anthracene, chrysene and benzo[a]pyrene in the newborn mouse assay

Eight nitropolycyclic aromatic hydrocarbons (PAHs), including 1- and 4-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene, 7-nitrobenz[a]anthracene, 6-nitrochrysene and 6-nitrobenzo-[a]pyrene and their parent PAHs were tested fro tumorigenicity in the newborn mouse model by i.p. administration at 1, 8, and 15 days after birth. Both pyrene and 1-nitropyrene induced similar incidences of hepatic tumors in males, yielding a 12-15% and a 21-28% tumor incidence at total doses of 700 and 2800 nmol per mouse, respectively. Liver tumors did not occur in females and the 3-10% lung tumor yield in both sexes was similar to that found in solvent-treated controls. The presumed proximate carcinogen, 1-nitrosopyrene, administered at 700 nmol per mouse, caused liver tumors in 45% of the males and in 9% of the females. 4-Nitropyrene was more tumorigenic than pyrene or 1-nitropyrene; at a dose of 2800 nmol, it induced liver tumors in 83% of the males and 7% of the females, with a lung tumor yield of 38 and 31%, respectively. Female mice treated with 200 nmol of 1,3-, 1,6- or 1,8-dinitropyrene did not develop liver tumors but the hepatic tumor incidence in males was 20, 32 and 16%, respectively, which was significantly greater than that found in mice treated with pyrene. In male mice administered 2800 nmol of benz[a]anthracene, the hepatic tumor incidence was 79%, while treatment with 7-nitrobenz[a]anthracene showed an incidence of only 28%. Similarly, 560 nmol of benzo[a]pyrene caused a 49% liver tumor yield in males while those given 6-nitrobenzo[a]pyrene had a 28% incidence. Treatment with benzo[a]pyrene also induced a 35 and 48% lung tumor incidence in males and females while the comparable values in 6-nitrobenzo[a]pyrene-treated mice were 14 and 2%. Chrysene administered at 2800 nmol per mouse induced hepatic and lung tumors in 41% and 21% of the males, respectively; at the 700-nmol dose, it induced only liver tumors in 29% of the males and in none of the females. In contrast, treatment with 6-nitrochrysene at 700 nmol per mouse resulted in a 76 and 23% hepatic tumor incidence in males and females, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Exceptional carcinogenic activity of benz[a]anthracene 3,4-dihydrodiol in the newborn mouse and the bay region theory

Benz[a]anthracene and the five metabolically possible trans-dihydrodiols of benz[a]anthracene were tested for carcinogenicity in newborn Swiss-Webster mice. Four hundred, 800, and 1600 nmoles hydrocarbon i.p. were sequentially injected on Days 1, 8, and 15 of life. The mice were killed at 22 weeks of age. Of the mice treated with trans-3,4-dihydroxy-3, 4-dihydrobenz[a]anthracene, 24% developed malignant lymphoma, whereas 4% of the animals treated with benz[a]anthracene had malignant lymphoma. None of the animals treated with the trans-1,2-dihydroxy-1,2-dihydrobenz[a]anthracene, trans-5,6-dihydroxy-5,6-dihydrobenz[a]anthracene, trans-8,9-dihydroxy-8,9-dihydrobenz[a]anthracene, or trans-10,11-dihydroxy-10,11-dihydrobenz[a]anthracene had malignant lymphoma. trans-3,4-Dihydroxy-3,4-dihydrobenz[a]anthracene caused about 35-fold more pulmonary adenomas than did benz[a]anthracene, whereas the trans-1,2-dihydroxy-1,2-dihydrobenz[a]anthracene, trans-5,6-dihydroxy-5,6-dihydrobenz[a]anthracene, trans-8,9-dihydroxy-8,9-dihydrobenz[a]anthracene, and trans-10, 11-dihydroxy-10,11-dihydrobenz[a]anthracene had little or no activity. The exceptionally high carcinogenicity of trans-3,4-dihydroxy-3,4-dihydrobenz[a]anthracene is consistent with the metabolism of this compound to either or both of the diastereomeric bay region 3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthracenes, and the data support the bay region theory of polycyclic hydrocarbon carcinogenesis.     

Tumorigenicity of the diastereomeric benz[a]anthracene 3,4-diol-1,2-epoxides and the (+)- and (-)-enantiomers of benz[a]anthracene 3,4-dihydrodiol in newborn mice.

The tumorigenic activity of benz[a]anthracene (BA), the (+)- and (-)-enantiomers of trans-3,4-dihydroxy-3,4-dihydrobenz[a]anthracene (BA 3,4-dihydrodiol), and the racemic diastereomers of the BA 3,4-diol-1,2-epoxides [i.e., either or both of the diastereomeric 1,2-epoxides derived from BA 3,4-dihydrodiol in which the epoxide oxygen is cis (diol epoxide-1) or trans (diol epoxide-2) to the benzylic 4-hydroxyl group) was examined in newborn Swiss-Webster mice. The mice were administered ip a total dose of 280 nmoles of compound in divided doses consisting of 40 nmoles within 24 hours of birth, 80 nmoles at 8 days of age, and 160 nmoles at 15 days of age. The experiment was terminated when the animals were 26 weeks of age. BA 3,4-diol-1,2-epoxide-2 was the most potent compound tested. All animals treated with BA 3,4-diol-1,2-epoxide-2 developed pulmonary tumors with an average of 13.3 tumors per mouse. BA 3,4-diol-1,2-epoxide-1 produced pulmonary tumors in 42% of the mice with an average of only 0.56 tumors per mouse. The (-)-enantiomer of BA 3,4-dihydrodiol with [3R,4R] absolute stereochemistry was the second most tumorigenic derivative of BA tested; it produced pulmonary tumors in 71% of the mice with an average of 1.88 tumors per mouse. BA and the (+)-enantiomer of BA 3,4-dihydrodiol had little or no tumorigenic activity at the dose tested. A comparison of the average number of pulmonary tumors per mouse revealed that BA 3,4-diol-1,2-epoxide-2 was about 30-fold more tumorigenic than was BA 3,4-diol-1,2-epoxide-1, 8-fold more tumorigenic than was (-)-BA 3,4-dihydrodiol, and greater than 85-fold more tumorigenic than was BA. These data indicate that in newborn mice BA 3,4-dihydrodiol and a BA 3,4-diol-1,2-epoxide are proximate and ultimate carcinogenic metabolites of BA, respectively.     

The involvement of a non-‘bay-region’ diol-epoxide in the metabolic activation of benz[a]anthracene in hamster embryo cells

The major hydrocarbon-nucleoside adduct present in hydrolysates of DNA from hamster embryo cells that had been treated with ³H-labelled benz[a]anthracene in culture has been examined by chromatography on Sephadex LH-20 columns and by high-pressure liquid chromatography. The results show that this adduct most probably arises from r-8,t-9-hydroxy-t-10,11-oxy-8,9,10,11-tetrahydrobenz[a]anthracene (anti-BA-8,9-diol 10,11-oxide). On the basis of this and other evidence, this non-bay-region diol-epoxide appears to be a reactive intermediate involved in the metabolic activation of benz[a]anthracene.     

Time course of oxidative benz[a]anthracene metabolism by liver microsomes of normal and PCB-treated rats

The time course for the oxidative metabolism of benz-[a)anthraceneby liver microsomes of normal, 3,3',4,4' -tetrachlorobiphenyl-(TCBP)and polychlorinated biphenyl-(PCB) treated rats has been investigated.These are shown not to be linear in all cases. In normal microsomesthe 10, 11-dihydrodiol is the main metabolite, followed by the5,6- and 8,9-dihydrodiols. Secondary metabolism, i.e. formationof dihydrodiol epoxides, is observed only after 5 min. In contrast,TCBP microsomes produce predominantly the 5,6-dihy-drodiol followedby the 8,9-dihydrodiol, whereas the formation of the 10, 11-dihydrodiolis suppressed. Metabolism deriving from oxidation of the 5,6-positionis increased 15–20 fold; again secondary metabolites occurbetween the 5th and 10th min of incubation. Gas chromatographyand mass spectra data suggest the formation of the ultimatecarcinogen, 3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthracene,as concluded from detection of its rearrangement product, the2,3,4-triol. In PCB-treated rats secondary metabolism is observedwithin 2.5 min. 5,6-Oxidation is increased 27 fold, 8,9-oxidation10 fold, but 10,11-oxidation is completely suppressed. The above-mentionedultimate carcinogen is also formed. Moreover, a series of tetrolsis detected. Optimum incubation times dependent on the problemunder study are discussed.     

Effects of butylated hydroxyanisole and butylated hydroxytoluene on 7,12-dimethylbenz[a]anthracene—DNA adduct formation in mouse embryo cell cultures

Neither butylated hydroxyanisole (BHA) nor butylated hydroxytoluene (BHT) significantly reduced overall 7,12-dimethylbenz[a]anthracene (DMBA)—DNA adduct formation in mouse embryo cell cultures. However, analysis of DMBA—DNA adducts by Servacel DHB chromatography and high-pressure liquid chromatography showed that treatment of cells with BHA, but not with BHT, resulted in a decreased contribution from the syn bay region dihydrodiol epoxide to overall binding.     

Comparative regioselective and stereoselective metabolism of 7-chlorobenz[a]anthracene and 7-bromobenz[a]anthracene by mouse and rat liver microsomes

Quantitative metabolism of 7-chlorobenz[a]anthracene (7-Cl-BA) and 7-bromobenz[a]anthracene (7-Br-BA) by liver microsomes of uninduced mice and rats was studied. Both enzymatic systems metabolize 7-Cl-BA preferentially at the C-8 and C-9 aromatic double bond region, approximately 42 and approximately 56% respectively, of the total metabolites. 7-Cl-BA and 7-Br-BA were metabolized considerably at C-3 and C-4, C-5 and C-6, C-8 and C-9, and C-10 and C-11. While 7-Cl-BA trans-3,4-dihydrodiol was formed in a 7-8% yield of the total metabolites in both enzymatic systems, 7-Br-BA trans-3,4-dihydrodiol was formed 16.0 and 9.9% respectively, from the mouse and rat liver microsomal metabolism. In mutagenicity assays with the Salmonella typhimurium tester strain TA100 in the presence of S9 activation enzymes, both of these trans-3,4-dihydrodiols exhibited higher mutagenicity than 7-Cl-BA and 7-Br-BA, while the other trans-dihydrodiol metabolites were either essentially inactive or weaker than the parent compounds. These results suggest that 7-Cl-BA trans-3,4-dihydrodiol and 7-Br-BA trans-3,4-dihydrodiol are the proximate metabolites of 7-Cl-BA and 7-Br-BA. Metabolism of 7-Cl-BA and 7-Br-BA by mouse liver microsomes was also in a stereoselective manner, preferentially giving trans-dihydrodiol metabolites an R, R stereochemistry.     

Activated Ki-ras genes in bladder epithelial cell lines transformed by treatment of primary mouse bladder explant cultures with 7,12-dimethylbenz[a]anthracene

DNA from five lines of transformed bladder epithelial cells derived from cultures of primary cells that had been treated with 7,12-dimethylbenz[a]anthracene (DMBA) can transform NIH 3T3 mouse fibroblasts in DNA transfection experiments. Southern analysis of DNA from NIH 3T3 primary and secondary transformants established that four of the DMBA-transformed cell lines contained activated cellular Ki-ras, while the remaining cell line contained a transforming gene that is unrelated to Ki-ras, N-ras, and Ha-ras. The point mutations responsible for Ki-ras activation were detected using oligonucleotide probes following selective amplification of Ki-ras specific sequences using the polymerase chain reaction. The results showed that activation of Ki-ras invariably involved a GC----AT transition mutation of the first position of codon 12. Surprisingly, a Ki-ras gene that was activated by a GC----AT transition mutation at the same position was also detected in a single transformed bladder urothelial cell line derived from control cultures of mouse bladder cells. Together, our results indicate that Ki-ras activation in the DMBA-transformed bladder cell lines may not be a direct consequence of interaction of activated DMBA metabolites with the Ki-ras gene.     

Effects of cycloheximide on the induction of CYP1A1 gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in three human breast cancer cell lines

Treatment of MCF-7, MDA-MB-231 and Hs578-T human breast cancercell lines with 10_–9 M 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) induces CYP1A1 gene expression in the MCF-7 but not inthe MDA-MB-231 or Hs578-T cells. Pretreatment of the cells with10_–5 M cycloheximide results in significantly increasedP4501A1 mRNA levels in all three cells lines. However, in cellsco-treated with 10_–5 M cycloheximide plus 10_–9 MTCDD, an induced response by TCDD was observed in the MCF-7and MDA-MB-231 but not in Hs578-T cells. Gel-retardation assaysof nuclear extracts from the three cell lines complexed witha ³²P-labeled dioxin-responsive element (DRE) gave a TCDD-inducibleretarded band only in the MCF-7 and MDA-MB-231 cells. A retardedband with a similar mobility was observed in nuclear extractsfrom Hs578-T cells treated with either 10_–9 M TCDD orDMSO (solvent control). These results suggest that aryl hydrocarbonnon-responsive MDA-MB-231 and Hs578-T human breast cancer celllines contain the CYP1A1 gene and treatment with cycloheximideincreases both constitutive and TCDD-induced CYP1A1 gene expression.     

Metabolism and DNA adduct formation of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in fish cell lines in culture

The metabolic activation of the carcinogens benzo[a]anthracene(BP) and 7,12-dimethylbenz[a]anthracene (DMBA) was examinedin cell lines derived from bluegill fry (BF-2), rainbow trout(RTG-2) and brown bullhead (BB). All three cell lines metabolizedBP (0.5 µg/ml medium) almost completely to water-solublemetabolites within 120 h, but the maximum amount of BP boundto DNA ranged from only 5 pmol/mg DNA in the BF-2 cells to 17in the BB cells and 44 in the RTG-2 cells. The major BP-DNAadduct in the BB and BF-2 cells was that formed by reactionof (+)-anti-BP-7,8-diol-9,10 epoxide [(+)anti-BPDE] with deoxyguanosine.This adduct was also present in the RTG-2 cell DNA, but therewere larger amounts of unidentified polar BP-DNA adducts. Exposureof the cells to [³H]BP-7,8-diol, a metabolic precursor of (+)anti-BPDE,resulted in binding of 1.5, 12 and 35 pmol BP per mg DNA inthe BF-2, BB and RTG-2 cells, respectively. More than 90% ofthe BP-7,8-diol added to the BF-2 cultures was recovered asa glucuronic acid conjugate, but the RTG-2 cells formed moreglutathione conjugates than glucuronide conjugates. The BB cellsformed both types of conjugates at a slower rate for more than75% of the 7,8-diol was recovered unchanged after 24 h. Thethree cell lines differed in the proportion of a 0.1 µg/mldose of DMBA metabolized in 48 h: the values ranged from 47%in the BF-2 cells to 78% in the BB cells and 97% in the RTG-2cells. The amount of DMBA bound to DNA ranged from 4.7 to 8.6pmol/mg DNA in the three cell lines: DMBA-3,4-diol-1,2-epoxide(DMBADE) adducts were present in the BB cell DNA, but no significantamounts of DMBADE-DNA adducts were detected in the RTG-2 orBF-2 cell DNA. These results demonstrate that fish cell culturescan activate BP to an ultimate carcinogenic metabolite, (+)anti-BPDE,but the level of binding of this metabolite to DNA is much lowerthan that which occurs in rodent embryo cell cultures. In BF-2cell cultures formation of BP-7,8-diol-glucuronide effectivelyprevents the activation of this diol to (+)anti-BPDE. A substantialproportion of the BP-7,8-diol is also metabolized to glucuromdeand glutathione conjugates in BB and RTG-2 cells. DMBA alsobinds to DNA at very low levels in these fish cell cultures.Thus effective conjugation of diols and their metabolites byfish cell lines appears to greatly reduce metabolic activationof hydrocarbons through the bay-region diol epoxide pathwaythat predominates in mammalian cell cultures.     

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.     

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.