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)

Peroxyl radical- and cytochrome P-450-dependent metabolic activation of (+)-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene in mouse skin in vitro and in vivo.

The role of peroxyl radicals and cytochrome P-450 in the metabolic activation of the (+)-enantiomer of 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene [(+)-BP-7,8-diol] was investigated in the epidermis of CD-1 mice. In skin homogenates from untreated or acetone-pretreated animals [7-14C]-(+)-BP-7,8-diol (20 microM) was metabolized primarily to 7 alpha,8 beta-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydroBP [(-)-anti-BPDE] as detected by high performance liquid chromatography of the stable tetraol hydrolysis products. The amounts of anti-BPDE-tetraols increased with the length of time of incubation (0-90 min). Only small amounts of 7 beta, 8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydroBP [(+)-syn-BPDE]-tetraols were detected. Epoxidation was not dependent upon NADPH. The addition of butylated hydroxyanisole (BHA, a free radical scavenger) decreased the formation of both anti- and syn-BPDE-tetraols (I50 less than 1 microM). In epidermal homogenates from animals pretreated with beta-naphthoflavone (beta-NF, an inducer of cytochrome P-450c), (+)-BP-7,8-diol was metabolized almost exclusively to (+)-syn-BPDE. The amounts of syn-BPDE-tetraols also increased with time of incubation with only small amounts of anti-BPDE-tetraols being detected. Epoxidation was NADPH-dependent and was not inhibited by the addition of BHA. The addition of alpha-naphthoflavone (an inhibitor of cytochrome P-450) inhibited syn-BPDE-tetraol formation (I50 approximately 2.5 microM). The DNA adducts formed in mouse epidermis after topical application of [1,3-3H]-(+)-BP-7,8-diol (200 nmol/mouse, 50 microCi/mouse) were analyzed by high performance liquid chromatography. The hydrocarbon-modified deoxyribonucleosides were identified by comparison with standards of (+)-syn-BPDE-dGuo and (-)-anti-BPDE-dGuo. In animals that received no pretreatment, similar amounts of (-)-anti-BPDE-dGuo and (+)-syn-BPDE-dGuo were formed after 3 h of exposure to (+)-BP-7,8-diol, whereas in beta-NF-pretreated animals larger proportions of (+)-syn-BPDE-dGuo were formed. Analysis of the tetraol hydrolysis products and DNA adducts formed from (+)-BP-7,8-diol in mouse skin demonstrates that two independent pathways of metabolic activation occur in vivo: in control animals peroxyl radical-mediated pathways are important contributors to metabolic activation, whereas in beta-NF-pretreated animals cytochrome P-450 is the major oxidizing agent. These results provide the first evidence that peroxyl radicals play a role in xenobiotic metabolism in vivo.

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.

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.

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.

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.

Roles of individual human cytochrome P-450 enzymes in the bioactivation of benzo(a)pyrene, 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene, and other dihydrodiol derivatives of polycyclic aromatic hydrocarbons.

Human liver microsomes oxidized 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene [B(a)P-7,8-diol] to products that yield DNA adduct formation and umu gene expression in the tester system Salmonella typhimurium TA1535/pSK1002. The umu response is correlated to levels of microsomal cytochrome P-450NF (P-450NF) and nifedipine oxidation in different human liver samples used for activation, and both the (+)- and (-)-enantiomers of B(a)P-7,8-diol gave similar results in these and other assays. The microsomal umu response was inhibited by antibodies raised against P-450NF. 7,8-Benzoflavone stimulated the B(a)P-7,8-diol-dependent umu response observed with purified P-450NF and human liver and lung microsomes. Thus, P-450NF appears to be the major enzyme involved in the activation of B(a)P-7,8-diol in human liver and possibly lung. Similar results were obtained for the activation of trans-9,10-dihydroxy-9,10-dihydrobenzo(b)fluoranthene and trans-3,4-dihydroxy-3,4-dihydro-7,12-dimethylbenz(a)anthracene, compounds that are known to form highly tumorigenic diol-epoxides. The major product of the oxidation of (+)-B(a)P-7,8-diol was the cis-syn isomer of benzo(a)pyrene-7,8,9,10-tetraol[7 beta, 8 alpha, 9 beta, 10 beta-tetrahydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]. Studies on the nature of the human liver enzymes involved in the formation of B(a)P-7,8-diol [from benzo(a)pyrene] indicate that neither P-450NF, P-450PA, P-450j, P-450DB, nor P-450MP is involved. The correlation of 7,8-diol formation with phenacetin O-deethylation in a set of liver samples and the partial inhibition of the reaction by 7,8-benzoflavone and anti-rat P-450 beta NF-B suggest that the enzyme involved may be P1-450, the human ortholog of rat P-450 beta NF-B, which catalyzes both the formation of B(a)P-7,8-diol and its subsequent oxidation in tissues of polycyclic hydrocarbon-treated rats. The differential effects of inhibitors indicate that benzo(a)pyrene 3-hydroxylation, 4,5-epoxidation, and 9,10-epoxidation are catalyzed by an enzyme(s) distinct from that which forms the 7,8-epoxide. The roles of the human P-450 enzymes differ from the rodent orthologs in the paradigm for bioactivation of polycyclic hydrocarbons; further, flavones appear to have opposing effects on diol formation and further epoxidation in both human liver and lung.

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)

Reactivity and tumorigenicity of bay-region diol epoxides derived from polycyclic aromatic hydrocarbons.

During the past decade substantial progress has been made in elucidating factors that determine the tumorigenic activity of bay-region diol epoxides, major ultimate carcinogenic metabolites derived from polycyclic aromatic hydrocarbons. Neither high nor low chemical reactivity of the diol epoxides (as measured by rates of uncatalyzed solvolysis) is required for high tumorigenic response. In contrast, aspects of molecular structure such as conformation and absolute configuration strongly influence tumorigenic activity. The role of conformation is illustrated by the observation that those diol epoxides whose hydroxyl groups are pseudoaxial are weak or inactive as tumorigens. Absolute configuration is an important determinant of biological activity of bay-region diol epoxides: in all cases studied to date, the predominantly formed (R,S)-diol-(S,R)-epoxides are generally the most tumorigenic of the four metabolically possible configurational isomers. In the course of investigating the effects of structural factors on tumorigenic activity, we identified the (4R,3S)-diol-(2S,1R)-epoxide of benzo(c)phenanthrene as the most potent tumorigen (in initiation-promotion experiments on mouse skin) of the diol epoxides studied to date. Studies of all four configurationally isomeric diol epoxides derived from benzo(c)phenanthrene led to the striking observation that these diol epoxides exhibit an exceptionally high efficiency of covalent binding, relative to hydrolysis, when allowed to react with calf thymus DNA in aqueous solution. Thus, these diol epoxides should provide an excellent tool for the detailed study of such binding. When the four isomeric benzo(c)phenanthrene diol epoxides are compared, there appears to be no simple correlation between tumorigenic response and either the extent of binding to DNA or the major types of deoxyribonucleoside adducts formed. Deoxyribonucleoside adducts of benzo(c)phenanthrene diol epoxide have also been identified from the DNA of cultured rodent embryo cells after treatment of the cells with tritium-labeled benzo(c)phenanthrene. The distribution of adducts is consistent with predominant metabolic formation of the (4R,3S)-diol-(2S,1R)-epoxide; deoxyadenosine is the major site in the cellular DNA attacked by this epoxide, just as it is in DNA in solution. Further experiments are in progress which we hope will identify more subtle aspects of the DNA binding of benzo(c)phenanthrene diol epoxides that may be uniquely correlated with their tumorigenic activity.

Instability of (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BaPDE)-DNA adducts formed in benzo[a]pyrene-treated Wistar rat embryo cell cultures.

One of the peaks present in HPLC profiles of [3H]benzo[a]-pyrene (BaP)-deoxyribonucleosides prepared by enzymatic degradation of [3H]BaP-DNA isolated from Wistar rat embryo cell cultures exposed to [G-3H]BaP was found to be r-7,c-9,c-10,t-8-tetrahydroxy-7,8,9,10-tetrahydroBaP, a BaP-DNA adduct decomposition product (Pruess-Schwartz, D. and Baird, W.M., Cancer Res., 46, 545-552, 1986). To investigate the stability of the hydrocarbon-deoxyribonucleoside linkages in intact BaP-modified DNA, DNA was isolated from Wistar rat embryo cells that had been exposed to [G-3H]BaP and incubated in darkness at 37 degrees C at a range of pH values from 5 to 11 for 72 h or for 1-150 h at pH 7. The rate of breakdown of [3H]BaP-DNA adducts (0.25%/h) was linear over 150 h. The amounts of the two major BaP-DNA adduct decomposition products, I and II (present in a ratio of 1:3), increased with length of time of incubation. Formation of I was not affected by pH, whereas, formation of II was highest at acidic and neutral pH. Analysis of the decomposition products by immobilized boronate chromatography and reverse-phase HPLC demonstrated that both I and II contained cis-vicinal hydroxyl groups and decomposition product II cochromatographed with r-7,c-9, c-10,t-8-tetrahydroxy-7,8,9,10-tetrahydroBaP, a (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydroBaP (syn-BaPDE)-derived tetraol. At neutral pH [3H](+/-)-syn-BaPDE-modified calf thymus DNA formed a decomposition product identical to II. Analysis of the BaP-DNA adducts that remained covalently bound to the DNA after the above incubations demonstrated that the amounts of both major syn-BaPDE-deoxyguanosine adducts decreased with length of time of incubation. Thus, syn-BaPDE-deoxyribonucleoside adducts formed in the DNA of [3H]BaP-treated Wistar rat embryo cells are unstable and breakdown spontaneously in the absence of light to yield syn-BaPDE-tetraol decomposition products.

Separation of (+)-syn- and (-)-anti-benzo[a]pyrene dihydrodiol epoxide-DNA adducts in 32P-postlabeling analysis.

The (+)-enantiomer of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) is a diagnostic probe for cytochrome P-450 and non-cytochrome P-450 pathways of dihydrodiol epoxidation. The principle products of epoxidation are the (+)-syn-dihydrodiol epoxide [(+)-syn-BPDE] and the (-)-anti-dihydrodiol epoxide [(-)-anti-BPDE]. Chromatographic conditions are described that separate the major deoxynucleoside 3',5'-bisphosphate adducts derived from these dihydrodiol epoxides on commercial poly(ethylenimine) thin-layer plates. Inclusion of boric acid and magnesium chloride in the D4 solvent is a key feature of the separation. Reasonable separation of these bisphosphate adducts from the major deoxynucleoside 3',5'-bisphosphate adduct derived from (+)-anti-BPDE is also observed. 32P-Postlabeling analysis of DNA adducts produced following topical administration of benzo[a]pyrene to mouse skin suggests that cytochrome P-450 plays a major role in its metabolism to DNA binding derivatives.

32P-postlabeling analysis of DNA adduction in mouse skin following topical administration of (+)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene.

32P-Postlabeling was employed for analysis of DNA adducts produced in mouse skin following topical administration of enantiomers of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol). Deoxynucleoside 3'-monophosphates were isolated by digestion of epidermal DNA with micrococcal endonuclease and spleen phosphodiesterase and phosphorylated with [gamma-32P]ATP. 32P-Labeled deoxynucleoside 3',5'-bisphosphate adducts to diastereomeric benzo[a]pyrene dihydrodiol epoxides (BPDE) were separated by four-directional thin-layer chromatography on poly(ethylenimine)-cellulose plates using a recently described solvent system [Reddy, A. P., Pruess-Schwartz, D., and Marnett, L. J. (1992) Chem. Res. Toxicol. (preceding paper in this issue)]. When (+)-BP-7,8-diol was topically administered, a major adduct spot was detected that cochromatographed with a standard produced by reaction of 7(S),8(R)-dihydroxy-9-(S),10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-syn-BPDE] with DNA. The level of this adduct increased in a dose- and time-dependent fashion and was elevated in animals pretreated with beta-naphthoflavone. Relatively small amounts of radioactivity cochromatographed with standards of deoxynucleoside 3',5'-bisphosphate adducts derived from 7(S),8(R)-dihydroxy-9(R),10(S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-anti-BPDE]. Following topical administration of (-)-BP-7,8-diol, a single adduct spot was detected that cochromatographed with a standard of the major deoxyguanosine adduct derived from 7(R),8(S)-dihydroxy-9-(S),10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE]. The stereochemistry of epoxidation of the enantiomers of BP-7,8-diol indicates that cytochrome P-450 catalyzes the terminal activation step of benzo[a]pyrene activation to an ultimate carcinogen in mouse skin, a target organ for its carcinogenic activity.

Oxidation of polycyclic hydrocarbons by oxygen radicals.

Dihydrodiolepoxides (BPDE) are reactive derivatives that appear to mediate the genotoxic effects of benzo[a]pyrene (BP) and several other polycyclic hydrocarbons. Their formation requires sequential epoxidation, hydration, and epoxidation steps. The first epoxidation step is catalyzed by cytochrome P-450 isoenzymes whereas the second is catalyzed by cytochromes P-450 and assorted species of oxygen radicals (in particular peroxyl radicals). The stereochemistry of epoxidation of the procarcinogen 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) is a useful tool with which to dissect the contributions of cytochrome P-450-dependent and independent pathways. [32P]-Postlabeling techniques were developed to analyze the diastereomeric composition of BPDE-deoxynucleoside-bis-phosphate adducts recovered from DNA of target tissues for BP tumorigenesis. Application of these techniques revealed that cytochrome P-450 is the major contributor to BPDE production in mouse skin in vivo. Following administration of BP or (+)-BP-7,8-diol to female CD-1 mice, only minor amounts of adducts derived from peroxyl radical epoxidation were detected. In contrast, pretreatment of the animals with the tumor promoter tetradecanoyl phorbol acetate (TPA) led to the formation of sizeable amounts of non-cytochrome P-450-dependent adducts following coincident administration of (+)-BP-7,8-diol with a second dose of TPA. The results demonstrate that although oxygen radicals appear to play a minor role in tumor initiation by BP, they may play a major role in tumor promotion by phorbol esters.