Comparative in vitro metabolism of benzo[a]pyrene by recombinant zebrafish CYP1A and liver microsomes from beta-naphthoflavone-treated rainbow trout.

The zebrafish (Danio rerio) is a sensitive non-mammalian model used for studying polycyclic aromatic hydrocarbon (PAH)-induced chemical carcinogenesis. The susceptibility of zebrafish to PAH-induced carcinogenesis may be related to the ability of the zebrafish P450s to bioactivate these procarcinogens. As a part of our overall effort to identify the various P450 enzymes that are involved in the activation and detoxification of PAHs in zebrafish, therefore, we have examined the ability of recombinant zebrafish CYP1A (zCYP1A) expressed in yeast to metabolize BaP in vitro. Comparison studies also were conducted with liver microsomes from beta-naphthoflavone (BNF)-treated rainbow trout (Oncorhynchus mykiss). Results demonstrated that the trout liver microsomes were almost twice as active as zCYP1A in oxidizing BaP, with Vmax values of 1.7 and 0.94 nmol/min/nmol P450 for trout and zebrafish preparations, respectively. Like trout CYP1A1, cDNA-expressed zCYP1A was found to oxidize BaP to phenols, quinones and diols (BaP-7,8-diol and BaP-9,10-diol) in the presence of exogenous human microsomal epoxide hydrolase (hEH). BaP-7,8-diol is the precursor of the ultimate carcinogen, BaP-7,8-diol-9,10-epoxide (BaPDE). The ability of zCYP1A to bioactivate BaP was confirmed by the formation of DNA adducts when calf thymus DNA was added to the incubation mixture. BaP-DNA binding was enhanced by the addition of hEH to the incubation mixture. HPLC analysis of the [33P]-postlabeled DNA adducts showed the formation of at least four adducts mediated by both zCYP1A and trout liver microsomes, and one of these adducts co-migrated with BaPDE-dG in HPLC analysis. The addition of hEH to the incubation mixture decreased the formation of BaPDE-dG by zCYP1A and by trout liver microsomes while increasing the formation of an unidentified DNA adduct in the case of zCYP1A. zCYP1A also mediated the binding of BaP to protein, providing further evidence that this enzyme is capable of oxidizing BaP to reactive metabolites that bind to macromolecules. It thus appears that zCYP1A may play an important role in BaP-induced carcinogenesis in the zebrafish model by catalyzing the sequential formation of the ultimate diol epoxide carcinogenic metabolite of BaP.

Benzo(a)pyrene:DNA adduct formation in early-passage Wistar rat embryo cell cultures: evidence for multiple pathways of activation of benzo(a)pyrene.

The benzo(a)pyrene (BaP):DNA adducts formed in cells are present at very low levels and are usually identified by reverse-phase high-performance liquid chromatography of tritium labeled BaP:deoxyribonucleoside adducts with known standards. To improve the identification of the BaP:DNA adducts formed, acid hydrolysis techniques were used to convert the BaP:deoxyribonucleoside adducts formed in Wistar rat embryo cell cultures to BaP:purine adducts and BaP:tetraols. Early passage Wistar rat embryo cell cultures were exposed to [3H]BaP. The BaP:deoxyribonucleoside adducts were isolated by immobilized boronate chromatography and reverse-phase high-performance liquid chromatography. Three adducts (MS1, MS2, MS3) bound to the immobilized boronate column indicating that they contained cis-vicinal hydroxyl groups, a configuration which would result from reaction of 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydroBaP (anti-BaPDE) with DNA. MS2 resulted from reaction of (+)-anti-BaPDE with deoxyguanosine (dGuo), for it cochromatographed with a [14C]-(+)-anti-BaPDE:dGuo marker at the deoxyribonucleoside level and after hydrolysis of MS2 and the marker to 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroBaP (BaPDE):guanine and BaPDE:tetraol. MS1, an adduct that eluted in the same region as a (-)-anti-BaPDE:dGuo marker, was not formed by reaction of anti-BaPDE with DNA. Exposure of (-)-anti-BaPDE:dGuo to 0.1 N HCl for 24 h at 37 degrees C resulted in cleavage of the glycosidic bond to give an enantiomer that cochromatographed with the (+)-anti-BaPDE:dGuo hydrolysis product. Hydrolysis of MS1 under the same conditions yielded a product that eluted earlier than the hydrolysis product of anti-BaPDE:dGuo. Hydrolysis of MS1 at 80 degrees C under conditions which produce tetraols from BaPDE:deoxyribonucleoside adducts resulted in the formation of a product which did not elute with either 7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydro-BaP (syn-BaPDE) or anti-BaPDE tetraols. MS1 was also not present in cells exposed to BaP-7,8-diol or 3-hydroxyBaP. These results demonstrated that MS1 is formed by a different mechanism of activation than a simple bay-region diol-epoxide. MS3 and its hydrolysis products had chromatographic properties identical to those of r-7,c-9,c-10,t-8-tetrahydroxy-7,8,9,10-tetrahydroBaP, a tetraol formed from syn-BaPDE. MS3 appears to result from spontaneous breakdown of a syn-BaPDE:DNA adduct to give a tetraol that contains cis-vicinal hydroxyls in the 9 and 10 positions and therefore binds to an immobilized boronate column.(ABSTRACT TRUNCATED AT 400 WORDS)

Benzo(e)pyrene-induced alterations in the stereoselectivity of activation of 7,12-dimethylbenz(a)anthracene to DNA-binding metabolites in hamster embryo cell cultures.

Benzo(e)pyrene [B(e)P], a weakly carcinogenic polycyclic aromatic hydrocarbon, modifies tumor induction in mouse skin and the induction of mutation in mammalian cells by carcinogenic hydrocarbons. To determine how B(e)P alters the activation of the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) to DNA-binding metabolites, the hydrocarbon-DNA adducts formed in Syrian hamster embryo cell cultures were analyzed after 24, 48, or 72 h of exposure to 0.1 microgram DMBA/ml medium in the presence of various doses of B(e)P. The total binding of DMBA to DNA was inhibited 3- to 4-fold by high doses of B(e)P, while the binding of DMBA to DNA was increased by low doses of B(e)P at 48 and 72 h of exposure. The amounts of the three major adducts tentatively identified as anti-DMBA-3,4-diol-1,2-epoxide (DMBADE): deoxyguanosine, syn - DMBADE: deoxyadenosine (dAdo), and anti-DMBADE:dAdo decreased in the presence of 1.5 micrograms B(e)P/ml. In contrast, exposure to low doses of B(e)P, 0.1 and 0.3 microgram/ml medium, resulted in an increase in the amount of both anti-DMBADE:deoxyribonucleoside adducts and a decrease in the amount of syn-DMBADE:deoxyribonucleoside adduct present after 48 and 72 h of exposure. Thus, low doses of B(e)P specifically enhanced the formation of anti-DMBA-diol-epoxide:deoxyribonucleoside adducts, and this resulted in an increase in the total amount of DMBA bound to DNA. High doses of B(e)P resulted in a decrease in the formation of all DMBA:DNA adducts and consequently a decrease in the total binding of DMBA to DNA. The amount of DMBA bound to DNA in cultures exposed to a higher dose of DMBA, 0.2 microgram DMBA/ml medium, for 48 h decreased in the presence of both low and high concentrations of B(e)P. This decrease resulted from a reduction in the formation of all three major DMBA-DNA adducts as the dose of B(e)P increased, but the decrease was larger for the syn-DMBADE:dAdo adduct than for the anti-DMBADE:deoxyguanosine and :dAdo adducts. These results demonstrate that the effects of B(e)P on the metabolic activation of DMBA depend upon both the ratio of B(e)P:DMBA and the dose of DMBA. The ability of B(e)P to alter the stereochemical selectivity of activation of DMBA as well as the total amount of activated metabolites also suggests that the ratio of B(e)P:DMBA may be an important factor in B(e)P-induced modifications of the induction of biological effects by DMBA.

Distribution, covalent binding, and DNA adduct formation of 7,12-dimethylbenz(a)anthracene in SENCAR and BALB/c mice following topical and oral administration.

SENCAR mice are much more susceptible to tumor initiation by 7,12-dimethylbenz(a)anthracene (DMBA) administered topically than p.o. and are also more susceptible to initiation by topically applied DMBA than are BALB/c mice. To determine how the distribution and metabolic activation of DMBA differed in these strains and with route of administration, BALB/c and SENCAR mice were exposed to [3H]DMBA topically and p.o., and the distribution and DNA binding of DMBA were analyzed. Both the amount of DMBA in skin and the covalent binding of DMBA to epidermal DNA were greater following topical administration than after p.o. administration in both strains. Differences in DMBA distribution and macromolecular binding were found between SENCAR and BALB/c mice, with the binding of DMBA to DNA in epidermis tending to be greater in BALB/c mice than in SENCAR mice when differences were observed. The formation of individual DMBA:DNA adducts in epidermis was also examined in SENCAR and BALB/c mice following topical administration of DMBA. No substantial qualitative or quantitative differences in DMBA:DNA adducts were found between SENCAR and BALB/c mice. The anti/syn-DMBA-diol-epoxide-DNA adduct ratios calculated from the three major DMBA:deoxyribonucleoside adducts increased with time in both SENCAR and BALB/c mice. The data suggest that differences in the distribution and macromolecular binding of DMBA are responsible for the much greater skin tumor initiating activity of DMBA applied topically than p.o. but do not account for the greater sensitivity of the SENCAR mouse to DMBA-induced epidermal tumorigenesis.

Comparison of the cellular DNA-bound products of benzo(alpha)pyrene with the products formed by the reaction of benzo(alpha)pyrene-4,5-oxide with DNA.

DNA isolated from mouse embryo cell cultures that had been treated with [3H]benzo(alpha)pyrene was degraded with enzymes to deoxyribonucleosides, and the hydrocarbon-deoxyribonucleoside products were isolated by chromatography on a Sephadex LH20 column eluted with a water: methanol gradient. The hydrocarbon-deoxyribonucleoside products were not identical to those found in similar chromatograms of enzyme digests of DNA that had been reacted with benzo(alpha)pyrene-4,5-oxide in aqueous ethanol solution. This finding suggests that the metabolic activation of benzo(alpha)pyrene that results in this hydrocarbon becoming covalently bound to DNA in mouse embryo cells in culture may be more complex than simply formation of a K-region epoxide and reaction of that compound with the cellular DNA.

Benzo(e)pyrene-induced alterations in the metabolic activation of benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene by hamster embryo cells.

Benzo(e)pyrene (BeP) is a cocarcinogen with benzo(a)pyrene (BaP) and an anticarcinogen with 7,12-dimethylbenz(a)anthracene (DMBA) in mouse skin initiation-promotion assays (Slaga, T.J., Jecker, L., Bracken, W.M. and Weeks C.E. Cancer Lett. 7: 51-59, 1979). We have investigated the effects of BeP on the metabolic activation of BaP and DMBA in early-passage cultures of Syrian hamster embryo cells. BeP had no effect on BaP-induced mutation frequencies in hamster embryo cell-mediated assays with V79 target cells. However, it inhibited the DMBA-induced mutagenesis by as much as 10-fold at the highest dose tested. Low doses of BeP did not affect the total amount of BaP metabolized, but the proportion of water-soluble metabolites was reduced, and the proportions of trans-7,8-dihydro-7,8-dihydroxybenzo(a)pyrene and trans-9,10-dihydro-9,10-dihydroxybenzo(a)pyrene were increased. Higher doses did decrease BaP metabolism and caused similar alterations in the metabolite profile. In cultures treated with trans-7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, BeP greatly reduced the oxidative metabolism of this diol. BeP inhibited DMBA metabolism at all doses tested; the proportion of water-soluble metabolites formed was decreased, and the proportions of trans-8,9-dihydro-8,9-dihydroxy-7,12-dimethylbenz(a)anthracene and trans-3,4-dihydro-3,4-dihydroxy-7,12-dimethylbenz(a)anthracene were increased. The results demonstrate that BeP is an effective inhibitor of the secondary oxidation of carcinogenic hydrocarbon diols required to convert diols which are proximate carcinogens to ultimate carcinogens such as diol-epoxides. The balance between (a) limited inhibition with consequent increase in total exposure to the ultimate carcinogenic form and (b) sufficient inhibition to reduce exposure to the ultimate carcinogenic form may determine whether BeP acts as a co- or anticarcinogen with a particular carcinogenic hydrocarbon.

Species- and length of exposure-dependent differences in the benzo(a)pyrene:DNA adducts formed in embryo cell cultures from mice, rats, and hamsters.

The activation of benzo(a)pyrene (BaP) to DNA-binding metabolites in early-passage embryo cell cultures prepared from various species of rodents was investigated by exposing cells from mice (BALB/c and Sencar), rats (Wistar and Fischer 344), and Syrian hamsters to [3H]BaP for various lengths of time. The BaP:DNA adducts containing cis-vicinal hydroxyl groups such as those formed from 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BaPDE) were separated from the other types of BaP:DNA adducts by immobilized boronate chromatography, and the individual adducts were analyzed by high-performance liquid chromatography. A number of BaP:DNA adducts were present in the DNA from the cultures from all three species after 5 h of BaP treatment. After a 24-h exposure to BaP, the mouse and hamster embryo cell DNA contained a large amount of the adduct formed by reaction of (+)-anti-BaPDE with the 2-amino group of deoxyguanosine (dGuo) and a small amount of a 7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:dGuo adduct. A large number of BaP:DNA adducts derived from 7 beta, 8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene and other unidentified BaP metabolites were present in rat embryo cell cultures at all times. Neither the Fischer 344 nor the Wistar rat embryo cell cultures had a significant amount of (+)-anti-BaPDE:dGuo adduct after 5 h of BaP treatment, and in the Wistar rat cells larger amounts of other adducts were present even after a 96-h exposure to BaP. In cell cultures from all three species the proportion of (+)-anti-BaPDE:dGuo adduct increased as the length of time of exposure to BaP increased. There are major differences in the metabolic activation of BaP to DNA binding metabolites in embryo cells from various species of rodents. However, the variations between cell cultures from different strains of rats or mice are not as great as the variations between cell cultures from different species. The time-dependent alterations in the BaP:DNA adducts indicate that analysis after various lengths of time of exposure to BaP is essential to characterize accurately the pathways of metabolic activation of BaP in cells from various species and tissues.

Benzo(e)pyrene-induced alterations in the binding of benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene to DNA in Sencar mouse epidermis.

Benzo(e)pyrene [B(e)P] cotreatment slightly increases the tumor-initiating activity of benzo(a)pyrene [B(a)P] and greatly decreases the tumor-initiating activity of 7,12-dimethylbenz(a)anthracene (DMBA) in Sencar mice (DiGiovanni et al., Carcinogenesis 3: 371-375, 1982). The effects of B(e)P on the binding of B(a)P and DMBA to Sencar mouse epidermis were investigated using a protocol similar to the mouse skin tumorigenicity studies. After 12 h of exposure to 50 nmol [3H]B(a)P and low or high doses of B(e)P, the level of [3H]B(a)P bound to mouse epidermal DNA increased by 30%. However, after 24 h exposure to 50 nmol [3H]B(a)P and after 12 or 24 h of exposure to 200 nmol [3H]B(a)P, B(e)P had no effect on the amount of [3H]B(a)P bound to DNA. The ration of anti-(the isomer with the epoxide and benzylic hydroxyl on opposite faces of the molecule) B(a)P-7,8-diol-9,10-epoxide [B(a)PDE]-deoxyribonucleoside adducts to syn- (the isomer with the epoxide and benzylic hydroxyl on the same face of the molecule) B(a)PDE-deoxyribonucleoside adducts did not change at either initiating dose of B(a)P or at any time regardless of the dose of B(e)P. After 12 h of exposure to high doses of B(e)P and a 50-nmol initiating dose of B(a)P the level of [3H]B(a)P bound to DNA increased but there was no change in the proportion of particular B(a)PDE-deoxyribonucleoside adducts present. In contrast, B(e)P inhibited the binding of initiating doses of DMBA (5 and 20 nmol) to DNA after 12 and 48 h of exposure to all dose ratios of B(e)P:DMBA tested. The three major adducts, tentatively identified as anti-DMBA-3,4-diol-1,2-epoxide (DMBADE):deoxyguanosine, syn-DMBADE:deoxyadenosine and anti-DMBADE:deoxyadenosine, decreased to the same relative extent as the dose of B(e)P increased. Thus, the effects of B(e)P on the total binding of these hydrocarbons to DNA in epidermis correlate with the cocarcinogenic and anticarcinogenic effects of B(e)P on B(a)P and DMBA, respectively, in a mouse skin initiation-promotion assay. These results indicate that the mechanism of the co- or anticarcinogenic action of hydrocarbons such as B(e)P involves alteration of the binding of carcinogenic hydrocarbons to DNA. They also suggest that measurement of carcinogenic hydrocarbon-DNA adducts formed during cotreatment with other hydrocarbons will provide a rapid method for predicting the co- or anticarcinogenic effect of the other hydrocarbons.

Comparison of cytochrome P450- and peroxidase-dependent metabolic activation of the potent carcinogen dibenzo[a,l]pyrene in human cell lines: formation of stable DNA adducts and absence of a detectable increase in apurinic sites.

The potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) has been reported to form both stable and depurinating DNA adducts upon activation by cytochrome P450 enzymes and/or cellular peroxidases. Only stable DB[a,l]P-DNA adducts were detected in DNA after reaction of DB[a,I]P-11,12-diol-13,14-epoxides in solution or cells in culture. To determine whether DB[a,l]P can be activated to metabolites that form depurinating adducts in cells with either high peroxidase (human leukemia HL-60 cell line) or cytochrome P450 activity (human mammary carcinoma MCF-7 cell line), cultures were treated with DB[a,l]P for 4 h, and the levels of stable adducts and apurinic (AP) sites in the DNA were determined. DNA samples from DB[a,l]P-treated HL-60 cells contained no detectable levels of either stable adducts or AP sites. MCF-7 cells exposed to 2 microM DB[a,l]P for 4 h contained 4 stable adducts per 10(6) nucleotides, but no detectable increase in AP sites. The results indicate that metabolic activation of DB[a,l]P by cytochrome P450 enzymes to diol epoxides that form stable DNA adducts, rather than one-electron oxidation catalyzed either by cytochrome P450 enzymes or peroxidases to form AP sites, is responsible for the high carcinogenic activity of DB[a,l]P.

Formation of glucuronic acid conjugates of 7,12-dimethylbenz(a)anthracene phenols in 7,12-dimethylbenz(a)anthracene-treated hamster embryo cell cultures.

Secondary cultures of hamster embryo cells exposed to 0.5 nmol [G-3H]7,12-dimethylbenz(a)anthracene (DMBA) per ml medium metabolized more than 90% of the DMBA within 48 hr. Samples of medium were extracted with chloroform, methanol, and water. The chloroform phases contained about one-third of the DMBA metabolites; the major chloroform-extractable metabolite was 8,9-dihydro-8,9-dihydroxy-7,12-dimethylbenz(a)anthracene. Beta-glucuronidase treatment of the aqueous methanol-soluble metabolites converted almost one-half of them to chloroform-soluble metabolites, of which more than 80% were identified as phenolic derivatives of DMBA. Similar metabolite profiles were obtained by treating the medium with beta-glucuronidase before chloroform extraction. Separation of the methyl group-hydroxylated derivatives of DMBA from the phenolic derivatives was accomplished by high-pressure liquid chromatography. Small amounts of hydroxymethyl derivatives were detected only in the chloroform-extractable material, whereas DMBA phenols were the major component of the beta-glucuronidase-released mateirla. These results indicate that the major pathway of DMBA metabolism in hamster embryo cells is oxidation of the aromatic rings and not oxidation of the methyl groups.

Analysis of benzo(a)pyrene:DNA adducts formed in cells in culture by immobilized boronate chromatography.

A chromatographic procedure using boronic acid residues linked to a cellulose support [(N-(N'-[m-(dihydroxyboryl)-phenyl]succinamyl)amino]ethyl cellulose), used by Sawicki et al. (Cancer Res., 43: 3212-3218, 1983) for analysis of 7,12-dimethylbenz(a)anthracene:DNA adducts, was modified to allow the analysis of benzo(a)pyrene (BaP):DNA adducts formed in cells in culture. Adducts resulting from reaction of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BaPDE) contain cis-vicinal hydroxyl groups that complex with the boronic acid residues; adducts resulting from 7 beta, 8 alpha-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (syn-BaPDE) do not. A mixture of [3H]-syn-BaPDE:deoxyguanosine (dGuo) adduct and [14C]-anti-BaPDE:dGuo adduct was completely resolved on a column of boronate:cellulose. Early-passage cultures of Sencar mouse, Syrian hamster, and Wistar rat embryo cells and a culture of a human hepatoma cell line (Hep G2) were exposed to [3H]BaP, and the BaP:DNA adducts were resolved by boronate chromatography and high-performance liquid chromatography. The Hep G2 cells and mouse embryo cells contained two major adducts, a (+)-anti-BaPDE:dGuo adduct and a syn-BaPDE:dGuo adduct. Boronate chromatography permitted the resolution of an additional minor syn-BaPDE:deoxyribonucleoside adduct in the mouse embryo cells. The hamster and rat embryo cells contained a number of major BaP-DNA adducts that were resolved by boronate chromatography followed by high-performance liquid chromatography. The rat embryo cells contained three syn-BaPDE:deoxyribonucleoside adducts and approximately equal amounts of two adducts tentatively identified as dGuo adducts of the (+) and (-) enantiomers of anti-BaPDE. The boronate chromatography-high-performance liquid chromatography procedure improves the separation of the BaP:DNA adducts formed in biological systems and facilitates the identification of the BaP metabolite(s) responsible for the formation of these adducts.

Comparison of the products of the reaction of 7-methylbenz(a)anthracene 5,6-oxide and RNA, with those formed in 7-methylbenz(a)anthracene-treated cells.

RNA was isolated by a phenol extraction method from mouse embryo cells treated in culture with either [G-3H]-7-methylbenz(a)anthracene or [G-3H]-7-methylbenz(a)anthracene 5,6-oxide (the K-region epoxide). The RNA was degraded to ribonucleosides, mixed with ultraviolet-absorbing quantities of the epoxide ribonucleoside products isolated from RNA that had reacted with 7-methylbenz(a)anthracene 5,6-oxide in aqueous ethanol solution, and chromatographed on a column of Sephadex LH-20 eluted with a methanol:water gradient. The 7-methyl-benz(a)anthracene 5,6-oxide ribonucleoside products formed in cells were identical to those formed in aqueous solution, although the relative amounts of the products varied. The majority of these epoxide-ribonucleoside products were not identical to the products formed in cells treated with the parent hydrocarbon. These results suggest that the major reactive form of 7-methylbenz(a)anthracene that binds to RNA in mouse embryo cells is not the K-region epoxide of this hydrocarbon.

Stereochemical specificity in the metabolic activation of benzo(c)phenanthrene to metabolites that covalently bind to DNA in rodent embryo cell cultures.

Benzo(c)phenanthrene (BcPh) has only weak carcinogenic activity in rodent bioassays. However, bay-region diol-epoxides of BcPh have the highest tumor-initiating activities of all hydrocarbon diol-epoxides tested to date. To determine whether BcPh is metabolically activated to bay-region diol-epoxides that bind to DNA in cells, Sencar mouse, Syrian hamster, and Wistar rat embryo cell cultures were exposed to [5-3H]-BcPh, and the BcPh-deoxyribonucleoside adducts formed were analyzed by immobilized boronate chromatography and reverse-phase high-performance liquid chromatography. Greater than 74% of the BcPh-deoxyribonucleoside adducts formed in all 3 species resulted from reaction of (4R,3S)-dihydroxy-(2S,1R)-epoxy-1,2,3,4-tetrahydro-BcPh [(-)-BcPhDE-2] with DNA to yield deoxyadenosine and deoxyguanosine adducts in a ratio of 3:1. A much smaller proportion of BcPh-deoxyribonucleoside adducts were formed by reaction of (4S,3R)-dihydroxy-(2S,1R)-epoxy-1,2,3,4-tetrahydro-BcPh [(+)-BcPhDE-1] with deoxyadenosine. No BcPh-deoxyribonucleoside adducts arising from either (+)-BcPhDE-2 or (-)-BcPhDE-1 were detected. The absence of adducts from these isomers of BcPhDE was not due to failure of these isomers to react with DNA in cells, for reaction of (+/-)-BcPhDE-1 or (+/-)-BcPhDE-2 with DNA in solution or in hamster embryo cell cultures resulted in the formation of DNA adducts from both the (+)- and (-)-enantiomers of each BcPhDE. These results indicate that both the (+)- and (-)-3,4-dihydrodiols of BcPh are formed and that their metabolic activation to diol-epoxides occurs with high stereospecificity in cells from all 3 species of rodents. The finding that the major DNA-binding metabolite is (-)-BcPhDE-2, the diol-epoxide with the (R,S)-diol-(S,R)-epoxide absolute configuration that is associated with high carcinogenic activity of diol-epoxides of other hydrocarbons, demonstrates that these cells are able to activate BcPh to an ultimate carcinogenic metabolite. The fact that a high proportion of the BcPh-DNA adducts are deoxyadenosine adducts suggests that BcPh has DNA-binding properties similar to those of the potent carcinogen 7,12-dimethylbenz(a)anthracene. The stereospecificity observed in the metabolic activation of BcPh to DNA-binding metabolites and the reaction of these metabolites with both deoxyguanosine and deoxyadenosine suggest that studies of the interactions of BcPh with DNA in vivo may be a valuable approach for establishing the role of specific activation pathways and DNA adducts in tumor induction.

Fate of 7,12-dimethylbenz(a)anthracene in the mouse mammary gland during initiation and promotion stages of carcinogenesis in vitro.

Metabolic conversion and distribution of the products of 7,12-dimethylbenz(a)anthracene (DMBA) were analyzed in the mouse mammary cell transformation model in organ culture. In order to determine the levels of uptake of DMBA, the glands were exposed to the transforming dosage of the procarcinogen (7.8 microM, 20 microCi/ml) for 24 h, and the level of uptake was determined to be 8 x 10(4) cpm/mg of tissue. Subsequently, the glands were incubated in DMBA-free medium, and distribution of the radioactivity in DNA and in the acid-insoluble materials was measured. Data showed that, in addition to the 24-h DMBA treatment period, the initiation stage extends for another 3 h when the incubation is continued in DMBA-free medium. A saturation level of uptake of [3H]DMBA into the whole gland was observed at 12 h, while DMBA was continually metabolized with the products being bound to DNA and to the acid-insoluble fractions throughout the entire incubation period with or without DMBA. The three major adducts identified were anti-DMBA-3,4-diol-1,2-epoxide (DMBADE):deoxyguanosine, syn-DMBADE:deoxyadenosine, and anti-DMBADE:deoxyadenosine. Qualitatively the adduct profiles remained similar. However, with the additional 3-h incubation in DMBA-free medium, the three major DMBA-DNA adducts increased slightly by 6.5 to 7.5%. Thus the total 27-h time period can be considered as the duration of the initiation stage in the DMBA-induced carcinogenesis of mouse mammary cells in organ culture. Therefore the subsequent 6-h incubation in DMBA-free medium may be considered as within the promotional stage, and at the same time period the levels of the three DNA adducts decreased significantly by 67.5 to 84.1% (P less than 0.001).

Interspecies differences in the major DNA adducts formed from benzo(a)pyrene but not 7,12-dimethylbenz(a)anthracene in rat and human mammary cell cultures.

Mammary epithelial cells from rats and humans show both quantitative and qualitative species- and carcinogen-specific differences in their abilities to activate benzo(a)pyrene (B(a)P) and 7,12-dimethylbenz(a)anthracene (DMBA). Previous studies of the DNA binding of these compounds in mammary epithelial cells demonstrated that rat cells bound relatively more DMBA than B(a)P to DNA under identical treatment conditions, while the opposite pattern was exhibited by human mammary epithelial cells. The specific DNA adducts formed in these cells after 24-h incubations with [3H]DMBA and [3H]B(a)P were analyzed to determine if there were qualitative as well as quantitative differences in the amounts of individual adducts. Similar proportions of specific DMBA-DNA adducts were found in both rat and human cells, although the total amount of adducts formed was significantly higher in the rat cells. In contrast, an essentially qualitative species-specific difference was observed in the major B(a)P-DNA adduct present in the rat and human cells. The major B(a)P adduct formed in the human mammary epithelial cells was identified as the (+)-anti-B(a)P-7,8-dihydrodiol-9, 10-epoxide(BPDE)-deoxyguanosine adduct. However, this adduct was formed at very low levels in the rat mammary epithelial cells. The rat cells contained a large proportion of syn-BPDE adducts, and other unidentified B(a)P-DNA adducts. The high level of the (+)-anti-BPDE-deoxyguanosine adduct in the human but not the rat mammary cells is consistent with the potential role of (+)-anti-BPDE in the high mutagenic activity of B(a)P in the cell-mediated mutagenesis assays using the human mammary cells as activators, and the low mutagenic activity of B(a)P when rat cells were used as activators. The quantitative differences in the activation of DMBA by cells from these two species are also consistent with the cell-mediated mutagenic activities of DMBA using these cells as activators. These results suggest that the higher carcinogenic activity of DMBA compared to B(a)P in the rat mammary gland may not be indicative of the relative carcinogenic potencies of these compounds for human mammary cells.

Metabolic activation of benzo[g]chrysene in the human mammary carcinoma cell line MCF-7.

Benzo[g]chrysene (BgC) is an environmental pollutant, and recent studies have demonstrated that anti- BgC-11,12-dihydrodiol 13,14-epoxide (anti-BgCDE) is a potent mammary carcinogen in rats. To determine whether BgC can be metabolically activated to anti-BgCDE in human cells, the human mammary carcinoma cell line MCF-7 was treated with BgC and with the racemic trans-3,4- and 11,12-dihydrodiols. The DNA adducts formed in these experiments were examined using 32P-postlabeling, and specific adducts were identified through comparisons with adducts obtained by the reaction of the racemic syn- and anti-BgCDEs with calf thymus DNA and with purine deoxyribonucleoside-3'-phosphates in vitro. It was found that BgC is metabolically activated in MCF-7 cells to form major DNA adducts through both the syn- and anti-11,12-dihydrodiol 13,14-epoxide metabolites. BgC is therefore a potential environmental risk to humans. The major BgC-DNA adducts formed from both the dihydrodiol-epoxide diastereomers were deoxyadenosine adducts. Thus, BgC has DNA-binding properties that are very similar to those of the potent mammary carcinogens 7,12-dimethylbenz[a]anthracene and dibenzo[a,l]pyrene.

The K-region trans-8,9-diol does not significantly contribute as an intermediate in the metabolic activation of dibenzo[a,l]pyrene to DNA-binding metabolites by human cytochrome P450 1A1 or 1B1.

Metabolic activation of the K-region trans-8,9-diol of the highly carcinogenic hexacyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P) by human cytochrome P-450 (P450) 1A1 and 1B1 was investigated in Chinese hamster V79 cell lines expressing human P450 1A1 or 1B1. P450 1A1 and 1B1 are the major P450s involved in metabolic activation of polycyclic aromatic hydrocarbons in human cells. The major DNA adducts formed by metabolism of DB[a,l]P in cultures expressing P450 1A1 or 1B1 resulted mainly from the fjord region (-)-anti-DB[a,l]P-11,12-diol 13,14-epoxide [(-)-anti-DB[a,l]PDE] and, to a lesser extent, (+)-syn-DB[a,l]PDE. In V79 cells expressing human P450 1A1, high amounts of as yet unidentified highly polar DNA adducts are formed in addition to the DNA adducts derived from DB[a,l]PDEs. Human P450 1A1 has been found to metabolize DB[a,l]P on its K-region to the trans-8,9-diol, and it has been proposed that the DNA binding of the parent compound in P450 1A1-expressing tissues may be partially mediated by activation of the K-region trans-8,9-diol to form bis-diol epoxides. V79 cells expressing human P450 1A1 or 1B1 formed only low amounts of DNA adducts after treatment with high doses of the K-region trans-8,9-diol. None of the adducts formed were identical to the main adducts formed in the same cell lines by metabolic activation of DB[a,l]P or (-)-DB[a,l]P-trans-11,12-diol. These results demonstrate that the K-region trans-8,9-diol does not significantly contribute to the genotoxicity of the very potent carcinogen DB[a,l]P in human cells or tissues expressing P450 1A1 or 1B1.

Benzo(a)pyrene metabolism in bovine aortic endothelial and bovine lung fibroblast-like cell cultures.

The metabolism of [3H]benzo(a)pyrene ([3H]BP) in bovine aortic endothelial and bovine lung fibroblast-like cells in vitro was investigated. Both cell types metabolized BP to organic solvent-extractable and water-soluble metabolites. The major organic solvent-extractable metabolites were 9-hydroxy-benzo(a)pyrene and 3-hydroxybenzo(a)pyrene; 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, 9,10-dihydro-9,10-dihydroxy-benzo(a)pyrene, and BP quinones were also formed. No glucuronide or sulfate conjugates of BP metabolites were detected. When exposed to [3H]-3-hydroxybenzo(a)pyrene, both cell types metabolized this phenol to water-soluble derivatives, probably through oxidation rather than conjugation of the molecule. These results demonstrate that endothelial cells metabolze BP to a proximate carcinogenic derivative, the 7,8-dihydrodiaol. Thus, efforts to predict the biological effects of hydrocarbons of an organism must take into account possible metabolic activation by endothelial cells as well as by other target tissues. The formation of unconjugated, phenolic hydrocarbon derivatives by bovine cells suggests their use as a model system for studying the contribution of phenols to the induction of biological effects by hydrocarbons.

The potent carcinogen dibenzo[a,l]pyrene is metabolically activated to fjord-region 11,12-diol 13,14-epoxides in human mammary carcinoma MCF-7 cell cultures.

Dibenzo[a,l]pyrene (DB[a,l]P), an environmental hydrocarbon and very potent carcinogen in rodent bioassays, could be activated to DNA-binding intermediates in cells through formation of three different regioisomeric bay- or fjord-region diol-epoxides or other more highly oxidized metabolites. The mechanism of metabolic activation of DB[a,l]P in the human mammary carcinoma cell line MCF-7 was elucidated by analyzing the DB[a,l]P-DNA adducts formed by [35S]phosphorothioate postlabeling, immobilized boronate chromatography, and high-performance liquid chromatography. Six DB[a,l]P-DNA adducts were detected. Comparison with those formed in cells by DB[a,l]P-11,12-diol and by reaction of DNA with syn- and anti-(benzylic hydroxyl and epoxide oxygen cis and trans, respectively) DB[a,l]P-11,12-diol-13,14-epoxide (DB[a,l]PDE) demonstrated that all DB[a,l]P-DNA adducts in MCF-7 cells were formed by these diol-epoxide isomers. Cellular DNA contained large amounts of two syn- and one anti-DB[a,l]PDE-DNA adducts and small amounts of one syn- and two anti-DB[a,l]PDE-DNA adducts. The ability of human cells to activate DB-[a,l]P to its fjord-region 11,12-diol 13,14-epoxides suggests that environmental exposure to DB[a,l]P could pose a risk for humans.

Benzo(a)pyrene:DNA adduct formation in normal human mammary epithelial cell cultures and the human mammary carcinoma T47D cell line.

The benzo(a)pyrene (BaP):DNA adducts formed in normal human mammary epithelial cell cultures and the human mammary carcinoma T47D cell line were analyzed by chromatography and acid hydrolysis of the BaP:deoxyribonucleoside adducts to BaP:purine adducts and BaP:tetraols. Human mammary epithelial cell cultures and human mammary carcinoma T47D cells were exposed to [3H]BaP for 24 h, and the levels of binding were 81 and 182 pmol BaP/mg DNA in normal and T47D cultures, respectively. Analysis of BaP:deoxyribonucleoside adducts resolved by immobilized boronate chromatography and reversephase high-performance liquid chromatography demonstrated the presence of three BaP:deoxyribonucleoside adducts in both cells: M2, MS1, and MS2 in a ratio of 1.6:1:14. Two adducts (MS1 and MS2) bound to the immobilized boronate column indicating the presence of cis-vicinal hydroxyl groups, a configuration which would result from reaction of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydroBaP (anti-BaPDE) with DNA. MS2 was identified as (+)-anti-BaPDE:deoxyguanosine (dGuo) for it cochromatographed with a [14C]-(+)-anti-BaPDE:dGuo marker, the BaP:purine hydrolysis product of MS2 cochromatographed with [14C]-(+)-anti-BaPDE:guanine, and the tetraol hydrolysis products cochromatographed with (+/-)-anti-BaPDE:tetraols. MS1 was identified as (-)-anti-BaPDE:dGuo for MS1 eluted in the same relative position as a (-)-anti-BaPDE:dGuo marker, the BaP:purine hydrolysis product of MS1 cochromatographed with [14C]-(+)-anti-BaPDE:guanine, and the tetraol hydrolysis products cochromatographed with (+/-)-anti-BaPDE:tetraols. Thus, both adducts that bound to the immobilized boronate column were formed from (+/-)-anti-BaPDE. One major adduct that did not contain cis-vicinal hydroxy groups, M2, was detected in both cell types. M2 was formed from (+/-)-7 beta, 8 alpha-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydroBaP (syn-BaPDE) as M2 eluted in the same relative position as a syn-BaPDE:dGuo adduct marker and the tetraol hydrolysis products of M2 cochromatographed with tetraols formed from (+/-)-syn-BaPDE. The isolation of the individual BaP:DNA adducts followed by acid hydrolysis allowed the identification of the BaP:DNA adducts formed in human mammary cell cultures and demonstrated the presence of (-)-anti-BaPDE:dGuo. Thus, this work provides the first evidence, other than cochromatography, that (-)-anti-BaPDE is formed in cell systems and reacts with DNA in cells to form (-)-anti-BaPDE:dGuo.(ABSTRACT TRUNCATED AT 400 WORDS)