Fungal oxidation of (+/-)-9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene: formation of diastereomeric benzo[a]pyrene 9,10-diol 7,8-epoxides.

The filamentous fungus Cunninghamella elegans oxidized (+/-) trans-9,10-dihydroxy-9,10-dihydrobenzo[a]-pyrene to a complex mixture of metabolites which were detected by high-pressure liquid chromatography. Two of the metabolites were identified as (+/-)7 beta, 8 alpha, 9 alpha, 10 beta-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene and (+/-)-7 beta,8 alpha,9 beta,10 alpha-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene. A third product gave absorption and mass spectra consistent with a diol-epoxide structure. Hydrolysis of this compound gave (+/-)-7 beta, 8 alpha, 9 beta, 10 alpha-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene as the major identifiable product with a minor unidentified tetraol. Synthetic (+/-)-9 alpha, 10 beta-dihydroxy-7 beta, 8 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene gave the same hydrolysis products and had the same retention time on high-pressure liquid chromatography as did the fungal metabolite. The trans-9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene recovered at the end of the experiment showed no optical activity, indicating that both enantiomers were metabolized by the fungus. the results suggest that C. elegans oxidizes (+/-)-trans-9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene to diastereomeric benzo[a]pyrene 9,10-diol 7,8-epoxides.     

(+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo (a)pyrene: a potent skin carcinogen when applied topically to mice.

(+/-)-trans-7,8-Dihydroxy-7,8-dihydrobenzo[a]-pyrene, a known metabolite of benzo [a]pyrene, has been tested for carcinogenic activity on mouse skin by topical application of 0.15 or 0.30 mumol every 2 weeks for 60 weeks. At the low dose (0.15 mumol), the compound was equipotent to the parent hydrocarbon, benzo[a]pyrene, and considerably more potent than its metabolic precursor, benzo[a]pyrene 7,8-oxide, in eliciting tumors, as determined by both the onset of tumors and the total number of animals developing carcinomas. Application of 7,8-epoxy-,8,9,10-tetrahydrobenzo[a]pyrene (0.30 mumol every 2 weeks), a compound related to the carcinogenic benzo[a]pyrene 7,8-oxide but with the double bond removed from the 9,10-position of the molecule, did not elicit any tumors. The above results indicate that the (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene is a more proximate carcinogen than benzo[a]pyrene 7,8-oxide and that the carcinogenicity of benzo[a]pyrene 7,8-oxide and (+/-)trans-7,8-digydrobenzo[a]pyrene may be due to metabolic conversion of these compounds to the highly reactive and mutagenic stereoisomers of 7,8-digydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.     

Epoxidation of (+/-)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene during (bi)sulfite autoxidation: activation of a procarcinogen by a cocarcinogen.

The (bi)sulfite ion undergoes extensive autoxidation in neutral aqueous media with the formation of sulfur trioxide radical anion that is detected by ESR. The radical anion subsequently reacts with molecular oxygen to form a peroxyl radical. We find that when (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) is included in this autoxidation system, BP-7,8-diol is converted to diolepoxides, ultimate carcinogenic derivatives of benzo[a]pyrene. This epoxidation occurs with a stereoselectivity consistent with either a peroxyl radical or a peracid as the epoxidizing agent. The epoxidation is dependent on the concentration of both (bi)sulfite and oxygen. In the presence of 10 microM butylated hydroxyanisole, which abolishes (bi)sulfite autoxidation, no (bi)sulfite-dependent epoxidation occurs. These results are discussed in regard to the mechanism of (bi)sulfite autoxidation, and in relationship to the cocarcinogenicity of sulfur dioxide [anhydrous (bi)sulfite] for benzo[a]pyrene-induced pulmonary neoplasia.     

Tumorigenicity of the optical enantiomers of the diastereomeric benzo[a]pyrene 7,8-diol-9,10-epoxides in newborn mice: exceptional activity of (+)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

The tumorigenicities of benzo[a]pyrene and each optical enantiomer of the diastereomeric benzo[a]pyrene 7,8-diol-9,10-epoxides derived from trans-7,8-dihydroxy-7,8-dihydrobenzol[a]pyrene were tested by sequential intraperitoneal injection of mice with 1,2, and 4 nmol, or with 2, 4, and 8 nmol of each compound on the 1st, 8th, and 15th day of life, respectively. The experiment was terminated when the animals were 34--37 weeks old. (+)-7beta, 8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzol[a]pyrene [(+)-BP-7beta,8alpha-diol-9alpha,10alpha-epoxide 2] had exceptional tumorigenicity, whereas benzo[a]-pyrene and the other three optically pure isomers of the benzo[a]pyrene 7,8-diol,9,10-epoxides had little or no activity. These results demonstrate differences in the carcinogenic activities of optically active isomers of a polycyclic hydrocarbon diol epoxide. Eleven percent of control mice had pulmonary tumors, whereas 71% and 100% of the mice treated with a total dose of 7 or 14 nmol of (+)-BP-7beta,8alpha-diol-9alpha,10alpha-epoxide 2, respectively, had pulmonary tumors. Control mice had an average of 0.12 pulmonary tumors per mouse, whereas mice treated with a total dose of 7 or 14 nmol of (+)-BP-7beta,8alpha-diol-9alpha,10alpha-epoxide 2 had 1.72 and 7.67 pulmonary tumors per mouse, respectively. Mice treated with 14 nmol of (-)-BP-7alpha,8beta-diol-9beta,10beta-epoxide 2, (-)-BP-7beta,8alpha-diol-9beta,10beta-epoxide 1, or (+)-BP-7alpha,8beta-diol-9alpha,10alpha-epoxide 1 had 0.13, 0.25, and 0.34 pulmonary tumors per animal, respectively.     

Carcinogenicity of benzo[a]pyrene 4,5-, 7,8-, and 9,10-oxides on mouse skin.

Benzo[a]pyrene and three arene oxides of benzo[a]pyrene (benzo[a]pyrene 4,5-, 7,8-, and 9,10-oxides) have been tested for carcinogenicity in mice by topical application of each compound (0.1 or 0.4 mumol) once every 2 weeks for 60 weeks. At the high dose, benzo[a]pyrene and the 7,8-oxide were highly carcinogenic, whereas the 4,5-oxide (K-region oxide) was weakly active and the 9,10-oxide was inactive. At the low dose, only benzo[a]pyrene was highly carcinogenic. The carcinogenic activities of the three arene oxides of benzo[a]pyrene were not correlated with their stabilities or mutagenic activities.     

Regio- and stereoselectivity of various forms of purified cytochrome P-450 in the metabolism of benzo[a]pyrene and (-) trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene as shown by product formation and binding to DNA

Highly purified cytochromes P-450(LM2) and P-450(LM4) and partially purified P-450(LM1), P-450(LM3b), and P-450(LM7) from rabbit liver microsomes exhibit different catalytic activities in the metabolism of benzo[a]pyrene (BzP) and (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene [(-)trans-7,8-diol] in a reconstituted enzyme system. The two highly purified cytochromes also exhibit differences in the activation of BzP and (-)trans-7,8-diol to intermediates that bind to DNA, as well as in the stereoselective conversion of (-)trans-7,8-diol to the highly mutagenic and carcinogenic diol-epoxides r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10- tetrahydrobenzo[a]pyrene (diol-epoxide I) and r - 7,t - 8 - dihydroxy - c - 9,10 - oxy - 7,8,9,10 - tetrahydrobenzo[a]pyrene (diol-epoxide II). P-450(LM2) is more active than P-450(LM4) in the metabolism of BzP and in its conversion to products that bind to DNA. In contrast, P-450(LM4) is more active than P-450(LM2) in the metabolism of (-)trans-7,8-diol and in its conversion to products that bind to DNA. The ratio of activity (percent substrate metabolized) with BzP relative to that with (-)trans-7,8-diol is 21 for P-450(LM2) and 0.3 for P-450(LM4); P-450(LM1), P-450(LM3b), and P-450(LM7) gave intermediate ratios. Marked stereoselectivity in the oxygenation of the (-)trans-7,8-diol to the highly mutagenic and putatively carcinogenic diol-epoxides I and II was observed with P-450(LM4), whereas the other preparations showed less selectivity. The ratio of diolepoxide I to diol-epoxide II ranges from 0.3 for P-450(LM7) to 11 for P-450(LM4). The substrate specificity and regio- and stereo-selectivity of the different forms of cytochrome P-450 may regulate the balance between activation and detoxification pathways of BzP and therefore determine the susceptibility of individual tissues, strains, and species to the carcinogenic action of BzP.     

Enzymatic conversion of benzo(a)pyrene leading predominantly to the diol-epoxide r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene through a single enantiomer of r-7, t-8-dihydroxy-7,8-dihydrobenzo(a)pyrene.

Benzo(a)pyrene is metabolically and stereospecifically converted by mixed-function oxidases of rat liver microsomes and epoxide hydratase (glycol hydro-lyase (epoxide-forming), EC 4.2.1.63)to the single enantiomer (-)r-7,t-8-dihydroxy-7,8-dihydrobenzol (A) pyrene. This enantiomer is further metabolized stereoselectively by the mixed-function oxidases to predominantly the diol-epoxide, r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzol(a)pyrene in which the 7-hydroxyl and the 9,10-epoxide are trans. Other unidentified metabolites are also formed from the r-7,t-8-dihydroxy-7,8-dihydrobenzo(a)pyrene. Racemic r-7,t-8-dihydroxy-7,8-dihydrobenzo(a)-pyrene is converted metabolically to both r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene and r-7,t-8-dihydroxy-c-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene. The diol-epoxides are unstable in aqueous medium, and their identification and characterization as r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene and r-7,t-8-dihydroxy-c-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene were accomplished by the identity of their tetrahydroxytetrahydrobenzo(a)pyrenes hydrolysis products with those of the authentic synthetic compounds with respect to mobility on high-pressure liquid chromatography and mass and ultraviolet absorption spectral analysis. The diol-epoxides were also reduced in the presence of NADPH to distinct trihydroxypentahydrobenzo(a)pyrenes. Since the synthetic racemic r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene is very highly mutagenic in mammalian cells, we suggest that it is the metabolically formed diol-epoxide that may be an ultimate carcinogenic form of benzo(a)pyrene.     

Mechanism of the inhibition of mutagenicity of a benzo[a]pyrene 7,8-diol 9,10-epoxide by riboflavin 5''-phosphate.

Riboflavin 5'-phosphate (flavin mononucleotide; FMN) inhibits the mutagenicity of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P diol epoxide), the only known ultimate carcinogenic metabolite of benzo[a]pyrene. Coincubation of 10, 25, and 50 nmol of FMN with strain TA100 of histidine-dependent Salmonella typhimurium inhibits the mutagenicity of 0.05 nmol of the diol epoxide by 50, 70, and 90%, respectively. Ribose 5-phosphate and riboflavin show no significant effects at comparable doses. Reaction of B[a]P diol epoxide with FMN in aqueous solution at neutral pH produces only tetraols, with no evidence for covalent adducts. At pH 7 the rate of hydrolysis of B[a]P diol epoxide in dioxane/water, 1:9 (vol/vol), at 25 degrees C is increased more than 10-fold in the presence of 100 muM FMN. Spectrophotometric studies and quantitative rate data for the reaction of the diol epoxide with FMN indicate that a complex is formed between the diol epoxide and the flavin moiety of FMN (Ke = 1,400-3,400 M-1) prior to general acid-catalyzed hydrolysis of the epoxide to tetraols by the phosphate monoanion of FMN. Comparable concentrations of ribose 5-phosphate and riboflavin do not significantly increase the rate of hydrolysis, although evidence for complex formation between riboflavin and the diol epoxide is observed. General acid-catalyzed hydrolysis of bay-region polycyclic hydrocarbon diol epoxides by compounds that have a high affinity for these ultimate carcinogens represents a potentially useful way of inhibiting their carcinogenic activity.     

Cyclopenta[c,d]pyrene: a highly mutagenic polycyclic aromatic hydrocarbon.

A polycyclic aromatic hydrocarbon recently isolated from carbon black and identified as cyclopenta[c,d]pyrene (CPP) is highly mutagenic. By the criteria of the Salmonella/mammalian-microsome mutagenicity test, the mutagenic potency of CPP is equalled by only two other naturally occurring polycyclic aromatic hydrocarbons--benzo[a]pyrene and dibenz[a,c]anthracene. The potent mutagenicity of CPP is noteworthy for two reasons: (i) CPP is a mutagenic polycyclic aromatic hydrocarbon without a "bay-region" and (ii) there is evidence that it is distributed widely in the environment. On the basis of experimental observations and perturbational molecular orbital calculations we propose that a mutagenic metabolite of CPP will be the 3,4-oxide. The carbonium ion derived from opening of CPP 3,4-oxide is identical to that derived from opening of benzo[a]pyrene 7,8-diol-9,10-oxide, the metabolite now thought to be an ultimate mutagenic and carcinogenic species.     

Crystal and molecular structure of a benzo[a]pyrene 7,8-diol 9,10-epoxide N2-deoxyguanosine adduct: absolute configuration and conformation

Benzo[a]pyrene 7,8-diol 9,10-epoxide adducts in DNA are implicated in mutagenesis, and their formation from the diol epoxides and subsequent incorrect replication by human DNA polymerases provide an attractive mechanism for the induction of cancer by this highly carcinogenic hydrocarbon and its diol epoxide metabolites. Here, we describe the crystal structure of such an adduct at the exocyclic amino group of a purine nucleoside. The present adduct derives from trans opening at C10 of the (-)-(7S,8R)-diol (9R,10S)-epoxide enantiomer by the exocyclic N(2)-amino group of deoxyguanosine. In the crystal, the pyrene rings of adjacent molecules stack with each other, but the guanine bases do not stack either intermolecularly with each other or intramolecularly with the pyrene. The most notable features of the molecular structure are (i) independent and unambiguous proof of the absolute configuration of the adduct based on the spatial relationship between the known chiral carbon atoms of the deoxyribose and the four asymmetric centers in the hydrocarbon moiety; (ii) visualization of the relative orientations of the pyrene and guanine ring systems as well as the conformation of the partially saturated hydrocarbon ring (comprising carbon atoms 7, 8, 9, and 10), both of which conformational features in the crystal are in good agreement with deductions from NMR and CD measurements in solution; and (iii) the presence in the crystal of a syn glycosidic torsion angle, a conformation that is unusual in B-DNA but that may be involved in error-prone replication of these benzo[a]pyrene 7,8-diol 9,10-epoxide deoxyguanosine adducts by DNA polymerases.     

Modification of DNA by the benzo[a]pyrene metabolite diol-epoxide r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

The structural modification of double-stranded circular DNA of simian virus 40 and plasmid ColE1 by in vitro binding of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene was studied. Stepwise hydrolysis with endonuclease S1 and DNase followed by DNA base analysis by thin-layer chromatography provided evidence that binding to adenine caused the local denaturation of DNA, whereas the more than 10-fold greater binding to guanine did not create such local denaturation. Of the two synthetic double-stranded polymers, poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC), bound to the diol-epoxide, only the former showed a marked hydrolysis after endonuclease S1 treatment, whereas binding occurred 24-fold more on the latter.     

Mutagenic and cytotoxic activity of benzol[a]pyrene 4,5-, 7,8-, and 9,10-oxides and the six corresponding phenols.

The benzo[a]pyrene 4,5-, 7,8-, and 9,10-oxides and the six corresponding phenols (4-, 5-, 7-, 8-, 9-, and 10-hydroxybenzo[a]pyrene) have been tested for mutagenic and cytotoxic activity in bacteria and in a mammalian cell culture system. Benzo[a]pyrene 4,5-oxide (K-region) was highly mutagenic in two histidine-dependent strains (TA1537 and TA1538) of Salmonella typhimurium which detect frameshift mutagens. In contrast, benzo[a]pyrene 7,8- and 9,10-oxides were less than 1% as mutagenic as the 4,5-oxide. Benzo[a]pyrene 7,8- and 9,10-oxides were unstable in aqueous media, whereas the 4,5-oxide was stable for several hours. This difference in stability could not account for the different mutagenic activities of the three arene oxides. The benzo[a]pyrene oxides were inactive in a strain (TA1535) that is reverted by base pair mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine or in a strain (TA1536) that detects framshift mutagens similar to the acridine half-mustard ICR-191. Benzo-[a]-pyrene and the six phenols were all stable in aqueous media, but they had little or no mutagenic activity in any of the four Salmonella strains. Conversion of 8-azaguanine-sensitive Chinese hamster V79 cells to 8-azaguanine-resistant variants was increased by benzo[a]pyrene 4,5-oxide, whereas the 9,10-oxide was considerably less active. Benzo[a]pyrene and the other derivatives had little or no effect. Benzo[a]yrene 4,5-oxide was more cytotoxic to the Chinese hamster V79 cells than the 7,8- and 9,10-oxides, while 8-hydroxybenzo[a]pyrene was the most cytotoxic of the six phenols.     

Polkappa protects mammalian cells against the lethal and mutagenic effects of benzo[a]pyrene

Several low-fidelity DNA polymerases have recently been discovered that are able to bypass DNA lesions during DNA synthesis in vitro. The efficiency and accuracy of lesion bypass is, however, both polymerase and lesion specific. For example, in vitro studies revealed that human DNA polymerase kappa (Polkappa) is unable to insert a base opposite a cis-syn thymine-thymine dimer or cisplatin adduct, yet can bypass some DNA lesions such as abasic site and acetylaminofluorene-adducted guanine in an error-prone manner. More importantly, Polkappa is able to bypass benzo[a]pyrene (B[a]P)-adducted guanine accurately and efficiently. To investigate the biological function of Polkappa, we have generated mouse embryonic stem (ES) cells deficient in the Polk gene encoding the enzyme. Polk-deficient ES cells grow normally and their sensitivities to UV and x-ray radiation are only slightly affected. In contrast, the mutant cells are highly sensitive to both killing and mutagenesis induced by B[a]P. Furthermore, the spectrum of mutations recovered in the Polk-deficient cells is different from that in the wild-type cells. Thus, our results indicate that Polkappa plays an important role in suppressing mutations at DNA lesions generated by B[a]P.     

Metabolism of benzo[a]pyrene by human mammary epithelial cells: toxicity and DNA adduct formation.

Pure cultures of human breast epithelial cells and of fibroblastic cells in early passage provided the opportunity to ask whether either cell type had the capability for metabolizing chemical carcinogens and, if so, was the fate of the metabolic products compatible with chemical carcinogens being a factor in the initiation of breast cancer in women. For this purpose, cells were exposed to benzo[a]pyrene (BaP), and (i) the influence on growth potential and (ii) the extent, type, and persistence of adducts between the metabolites of BaP and DNA were measured. Compared with fibroblasts, inhibition of growth by epithelial cells was 50-100 times more sensitive to BaP. Because of this differential sensitivity, epithelial cells were exposed to 0.4 microM BaP and fibroblasts were exposed to 4.0 microM BaP in the studies of DNA adduct formation. Separation by high-pressure liquid chromatography of adducts between (+/-)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP diol epoxide) and nucleosides from purified DNA revealed that epithelial cells contained modified DNA within 6 hr after adding BaP. Adducts between the 7R anti stereoisomer of BaP diol epoxide and deoxyguanosine predominated at all times. syn BaP diol epoxide adducts with deoxyguanosine and what appeared to be BaP diol epoxide adducts with deoxycytidine were consistently present but at much lower frequency. All three types of BaP diol epoxide--DNA adducts persisted in epithelial cells for 72 hr in BaP-free medium. No adducts were detected in fibroblastic cultures until 96 hr after first exposure to BaP. At this time, the type and extent of BaP diol epoxide--DNA adduct formation was similar to that in epithelial cells exposed to one-tenth the dose of BaP. The type, extent, rate of formation, and persistence of the adducts in human breast epithelial cells was similar to that in cells transformable by exposure to BaP, an indication that they may be targets for chemically induced carcinogenesis.     

Metabolic conversion of dibenz[a,h]anthracene (+/-)trans-1,2-dihydrodiol and chrysene (+/-)trans-3,4-dihydrodiol to vicinal dihydrodiol epoxides.

The hydroxyl groups of dibenz[a,h]anthracene trans-1,2-dihydrodiol and chrysene trans-3,4-dihydrodiol are known to be predominantly in quasi-axial conformations. These dihydrodiols were metabolized by liver microsomes from 3-methylcholanthrene-pretreated rats to form 1,2,3,4-tetrahydrotetrols as the major products. The major metabolites of the dihydrodiols were isolated by reversed-phase high-performance liquid chromatography and identified by ultraviolet--visible absorption and mass spectral analyses. The results indicate that axial hydroxyl groups of dibenz[a,h]anthracene trans-1,2-dihydrodiol and of chrysene trans-3,4-dihydrodiol do not direct metabolism away from their respective vicinal double bond.     

Identification of mutagenic metabolites of benzo(a)pyrene in mammalian cells.

The mutagenicity of benzo[a]pyrene and 15 of its derivatives, which included phenols, the benzo[a]yrene-4,5-epoxide (the K-region epoxide), dihydrodiols, two isomeric 7,8-diol-9,10-epoxides, a 6-methyl derivative, and a 6-hydroxymethyl derivative, were tested with Chinese hamster V79 cells in order to identify the mutagenic metabolites of benzo[a]pyrene. Mutations were characterized by resistance to ouabain or 8-azaguanine. Since V79 cells do not metabolize polycyclic hydrocarbons, mutagenesis was tested both in the presence and absence of benzo[a]pyrene-metabolizing normal golden hamster cells. All the tested phenols, 4,5-diols, trans-9,10-diol, 6-methyl, and 6-hydroxymethyl derivatives of benzo[a]pyrene showed little or no mutagenicity for both genetic markers. The (+/-)7alpha,8beta-dihydroxy-9alpha,10alpha-epoxy-7,8;9,10-tetrahydrobenzo[a]pyrene and K-region 4,5-epoxide exhibited similar and moderate mutagenicity in the absence of benzo[a]pyrene-metabolizing cells, but the (+/-)7alpha,8beta-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene showed a 2000- and 270-fold higher mutation frequency for ouabain and 8-azaguanine resistance, respectively, than did the K-region 4,5-epoxide. The trans-7,8-diol which was not mutagenic in the absence of benzo[a]pyrene-metabolizing cells was more mutagenic than benzo[a]pyrene after metabolism and mutagenesis by trans-7,8-diol in these cells was inhibited by 7,8-benzoflavone, an inhibitor of mixed-function oxidases. Metabolically formed trans-7,8-diol was isolated and incubated with rat liver microsomes in the presence of co-factors. High-pressure liquid chromatography analysis indicated that the major metabolite of trans-7,8-diol is 7alpha,8beta-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. The results indicate that the latter compound is metabolically formed and the major mutagenic intermediate of benzo[a]yrene metabolism.     

Carcinogenic epoxides of benzo[a]pyrene and cyclopenta[cd]pyrene induce base substitutions via specific transversions.

We have determined the spectrum of base-pair substitution mutations induced in the lacI gene of a uvrB- strain of Escherichia coli by two polycyclic aromatic hydrocarbons--(+/-)7 alpha,8 beta-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10 tetrahydrobenzo[a]pyrene (BPDE), and 3,4-epoxycylopenta[cd]pyrene (CPPE). Approximately 10% of all lacI mutations induced by either BPDE or CPPE are nonsense mutations, suggesting that base-pair substitutions are a large fraction of the mutational events induced by these agents in the uvrB- bacteria. Both carcinogens specifically induced the G . C leads to T . A and, to a lesser extent, the A . T leads to T . A transversions. One possible mechanism for transversion induction at G . C sites by BPDE might involve carcinogen binding to the exocyclic amino group of guanine in the template strand followed by a rotation of the modified base around its glycosylic bond from the anti to the syn conformation. This could allow specific pairing of modified bases with an imino tautomer of adenine.     

Immunohistochemical detection of anti-(±)-trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene-bound adduct in nuclei of cultured HeLa cells and mouse lung tissue

Summary Rabbit IgG against anti-BP-DE-modified calf thymus DNA was characterized and utilized for detection of adducts formed in cultured HeLa cells treated with anti-BP-DE or in lung tissues from mice given BP. Four weekly treatments with 1 mg of the modified (1.2% of total nucleotides) DNA were used to raise the rabbit antiserum, which even at 10⁶-fold dilution clearly recognized adducts in an ELISA. The detection limit was 25 fmol of anti-BP-DE adduct with 10³-fold diluted IgG fraction. The IgG did not cross-react with BP, BP tetraol, guanine, or 7-methyl-guanine, and only slightly with the syn form of BP-DE, (±)-4,5-dihydrobenzo(a)-pyrene-4,5-epoxide, aflatoxin B₁, and N-methyl-N-nitrosourea-modified DNA. Although syn-BP-DE-modified DNA inhibited the reaction between IgG and anti-BP-DE adduct, the inhibition rate was low, not correlating with the numbers of modified bases. Essentially similar values for level of bound adducts in HeLa cells treated with anti-BP-DE were generated by both ELISA and associated radioactivity derived from anti-³H-BP-DE. Immunohistochemical detection of adduct formation was dependent on the amounts of anti-BP-DE added to the culture medium of HeLa cells. Similar positive binding was obtained in mouse lung alveolar cell nuclei after intragastric administration of BP. These observations indicate that the prepared IgG is highly specific for anti-BP-DE-modified DNA and that should prove useful for detection of adducts formed in tissue samples exposed to anti-BP-DE or BP.Abbreviations used BP benzo(a)pyrene - BP-DE (±)-trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene - PBS phosphate-buffered saline - ELISA enzyme-linked immunosorbent assay - MEM minimum essential medium