Morphological transforming activity and metabolism of cyclopenta-fused isomers of benz[a]anthracene in mammalian cells

4 isomeric cyclopenta-derivatives of benz[e]anthracene (benz[a]aceanthrylene, benz[j]aceanthrylene, benz[l]aceanthrylene, and benz[k]acephenanthrylene) were examined for their ability to morphologically transform C3H10T1/2CL8 mouse-embryo fibroblasts. All of these polycyclic aromatic hydrocarbons studied except benz[k]acephenanthrylene transformed C3H10T1/2CL8 cells to both type II and type III foci in a concentration-dependent fashion. Benz[j]aceanthrylene was the most active, equivalent in activity to benzo[a]pyrene on a molar basis, in producing dishes of cells with transformed foci (94% at 1.0 microgram/ml). Benz[e]aceanthrylene, and benz[l]aceanthrylene produced 58% and 85% of the dishes with foci respectively at 10 micrograms/ml. Metabolism studies with [3H]benz[j]aceanthrylene in C3H10T1/2CL8 cells in which unconjugated, glucuronic acid conjugated, and sulfate conjugated metabolites were measured indicated that the dihydrodiol precursor to the bay-region diol-epoxide, 9,10-dihydroxy-9,10-dihydrobenz[j]aceanthrylene, was the major dihydrodiol formed (55%). Smaller quantities of the cyclopenta-ring dihydrodiol, 1,2-dihydroxy-1,2-dihydrobenz[j]aceanthrylene (14%), and the k-region dihydrodiol, 11,12-dihydroxy-11,12-dihydrobenz[j]aceanthrylene (5%) were also formed. Similar studies with [14C]benz[l]aceanthrylene indicated that the k-region dihydrodiol, 7,8-dihydroxy-7,8-dihydrobenz[l]aceanthrylene was the major metabolite formed (45%). The cyclopenta-ring dihydrodiol, 1,2-dihydroxy-1,2-dihydrobenz[l]aceanthrylene and 4,5-dihydroxy-4,5-dihydrobenz[l]aceanthrylene were formed in minor amounts (less than 6%). Therefore, metabolism at the cyclopenta-ring of B(j)A and B(l)A is a minor pathway in C3H10T1/2CL8 cells in contrast to previously reported studies with cyclopenta[cd]pyrene in which the cyclopenta-ring dihydrodiol was the major metabolite. These results suggest that routes of metabolic activation other than oxidation at the cyclopenta-ring such as bay region or k-region activation may play an important role with these unique polycyclic aromatic hydrocarbons in C3H10T1/2CL8 cells.     

Benz[j]aceanthrylene: a novel polycyclic aromatic hydrocarbon with bacterial mutagenic activity

Initial studies on the mutagenicity and metabolism of a novel cyclopenta-PAH, benz[j]aceanthrylene, are reported in the Salmonella bacterial system. The spectrum of activity of benz[j]aceanthrylene over the 5 Ames tester strains is similar to that of benzo[a]pyrene, and the dose-response curves for strain TA98 are comparable. Like other biologically active PAH, benz[j]aceanthrylene is a frame-shift mutagen requiring metabolic activation. An interesting feature of the S9 dependence of activity is the low concentration (congruent to 10-fold smaller than for benzo[a]pyrene) at which optimal activity is observed. The 1,2-dihydro-1,2-diol (product of metabolism of the cyclopenta-ring) appears to be the predominant metabolite, and implicates the 1,2-oxide as the ultimate mutagenic species.     

Bacterial mutagenicity of two cyclopentafused isomers of benzpyrene

Two novel cyclopentafused polycyclic aromatic hydrocarbons, naphtho(1,2,3-mno)acephenanthrylene (cyclopenta benzo[e]pyrene) and naphtho(2,1,8-hij)acephenanthrylene (cyclopenta(ij)benzo[a]pyrene) were evaluated for mutagenic activity in the Ames Salmonella typhimurium plate incorporation assay. Both compounds required S9 metabolic activation, and showed optimal activity at low S9 concentrations (below 0.6 mg/plate). Both compounds induced frameshift and base-pair substitution mutations, being active in strains TA98, TA100, TA1537, TA1538 and TA104, but not in strain TA1535. Cyclopenta(ij)benzo[a]pyrene was more active than cyclopentabenzo[e]pyrene, and both were more potent than their parent ring systems, benzo[a]pyrene and benzo[e]pyrene, respectively. Cyclopenta(ij)benzo[a]pyrene was more active in strain TA104 than in TA100 or TA98 (250-470, 340 and 80-100 rev/nmole) as was benzo[a]pyrene (120, 70 and 40 rev/nmole respectively); cyclopentabenzo[e]pyrene was more active in TA100 than TA104 or TA98 (70 versus 50 and 40 rev/nmole), and benzo[e]pyrene showed a similar pattern (4, 3.5 and 0.6 rev/nmole). The relative potencies of the four compounds are in accord with predictions based on perturbational molecular orbital calculations. The peak of activity at low S9 concentrations is consistent with epoxidation at the cyclopentafused ring being the major route of metabolic activation for both these cyclopentafused compounds.     

Mutagenicity of non-K-region diols and diol-epoxides of benz(a)anthracene and benzo(a)pyrene in S. typhimurium TA 100

When incubated with a 9,000 x g rat-liver supernatant, benzo(a)pyrene 7,8-diol and benz(a)anthracene 8,9-diol were more active than the parent hydrocarbons in inducing his⁺ revertant colonies of S. typhimurium TA 100. Benzo(a) pyrene 9,10-diol was less active than benzo(a)pyrene; the K-region diols, benz(a)anthracene 5,6-diol and benzo(a)pyrene 4,5-diol, were inactive. None of the diols was active when the cofactors for the microsomal mono-oxygenase were omitted. The diol-epoxides benzo(a)pyrene 7,8-diol 9,10-oxide, benz(a)anthracene 8,9-diol 10,11-oxide and 7-methylbenz(a)anthracene 8,9-diol 10,11-oxide and the K-region epoxides, benzo(a)pyrene 4,5-oxide and benz(a)anthracene 5,6-oxide, were mutagenic without further metabolism.     

CP-arene oxides: the ultimate, active mutagenic forms of cyclopenta-fused polycyclic aromatic hydrocarbons (CP-PAHs)

The bacterial mutagenic response (Ames-assay, Salmonella typhimurium strain TA98+/-S9-mix) of a series of monocyclopenta-fused polycyclic aromatic hydrocarbons (CP-PAHs) identified in combustion exhausts, viz. cyclopenta[cd]pyrene (1), acephenanthrylene (2), aceanthrylene (3) and cyclopenta[hi]chrysene (4), is re-evaluated. The mutagenic effects are compared with those exerted by the corresponding partially hydrogenated derivatives, 3,4-dihydrocyclopenta[cd]pyrene (5), 4,5-dihydroacephenanthrylene (6), 1,2-dihydroaceanthrylene (7) and 4,5-dihydrocyclopenta[hi]chrysene (8). It is shown that the olefinic bond of the externally fused five-membered ring of 1, 3 and 4 is of importance for a positive mutagenic response. In contrast, whilst CP-PAH 2 is found inactive, its dihydro analogue (6) shows a weak metabolism-dependent response. The importance of epoxide formation at the external olefinic bond in the five-membered ring is substantiated by the bacterial mutagenic response of independently synthesized cyclopenta[cd]pyrene-3,4-epoxide (9), acephenanthrylene-4,5-epoxide (10), aceanthrylene-1,2-epoxide (11) and cyclopenta[hi]chrysene-4,5-epoxide (12). Their role as ultimate, active mutagenic forms, when CP-PAHs 1, 3 and 4 exhibit a positive mutagenic response, is confirmed. Semi-empirical Austin Model 1 (AM1) calculations on the formation of the CP-arene oxides (9-12) and their conversion into the monohydroxy-carbocations (9a-12a and 9b-12b) via epoxide-ring opening support our results. For 2 and 4, which also possess a bay-region besides an annelated cyclopenta moiety, the calculations rationalize that epoxidation at the olefinic bond of the cyclopenta moiety is favoured.     

Benz[j]aceanthrylene: A novel polycyclic aromatic hydrocarbon with bacterial mutagenic activity

Initial studies on the mutagenicity and metabolism of a novel cyclopenta-PAH, benz[j]aceanthrylene, are reported in the Salmonella bacterial system. The spectrum of activity of benz[j]aceanthrylene over the 5 Ames tester strains is similar to that of benzo[a]pyrene, and the dose-response curves for strain TA98 are comparable. Like other biologically active PAH, benz[j]aceanthrylene is a frame-shift mutagen requiring metabolic activation. An interesting feature of the S9 dependence of activity is the low concentration (≅10-fold smaller than for benzo[a]pyrene) at which optimal activity is observed. The 1,2-dihydro-1,2-diol (product of metabolism of the cyclopenta-ring) appears to be the predominant metabolite, and implicates the 1,2-oxide as the ultimate mutagenic species.     

Mutagenicity of benzo[a]pyrene 7,8-dihydrodiol and 7,12-dimethylbenz[a]anthracene 3,4-dihydrodiol in S. typhimurium mediated by microsomes from rat liver and mouse skin

The mutagenic activities of trans-7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene (BP 7,8-diol) and of trans-3,4-dihydroxy-7,12-dimethylbenz[a]-anthracene (DMBA 3,4-diol) towards S. typhimurium TA100 were measured in assays that were carried out on a micro-scale in liquid medium in the presence of microsomal fractions prepared from mouse skin or rat liver. In the presence of an NADPH-generating system, microsomal enzymes converted both diols into mutagens that were probably the respective 'bay-region' diol-epoxides. The rate of the enzyme-catalysed conversion of the BP 7,8-diol into mutagens by microsomal preparations from mouse epidermis was similar to that occurring with microsomes from rat liver. Pretreatment of mice by the topical application of benz[a]anthracene (BA) or 7,12-dimethylbenz[a]-anthracene (DMBA) increased the mutagenic activity of BP 7,8-diol mediated by mouse skin microsomal preparations by 2-fold and this was paralleled by a 4-fold increase in epidermal aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity. The results are discussed in relation to the high susceptibility of mouse skin to polycyclic aromatic hydrocarbon (PAH) carcinogenesis.     

Chemical rearrangement of phenol-epoxide metabolites of polycyclic aromatic hydrocarbons to quinone-methides

Evidence of the involvement of triol-epoxide and phenol-epoxide metabolites in the metabolic activation of polycyclic hydrocarbons is accumulating. It is proposed that the phenolic OH-groups present in such epoxides will activate the epoxide moieties and permit their rearrangement to quinone-methides. These quinone-methides are highly reactive, potentially-isolable chemical entities with strong alkylating activity. In one resonance form they are resonance-stabilized carbonium ions. Only epoxides that also possess phenolic OH-groups in certain positions will form quinone-methides: these appear to include 9-hydroxybenzo [a] pyrene 4,5-oxide and the triol-epoxides 9-hydroxy-trans-1,2-dihydro-1,2-dihydroxychrysene 3,4-oxide and 2-hydroxy-trans-9,10-dihydro-9,10-dihydroxybenzo[a]pyrene 7,8-oxide.     

Induction of anchorage-independent growth in human diploid fibroblasts by the cyclopenta-polycyclic aromatic hydrocarbon, benz[l]aceanthrylene

Cyclopenta-fused polycyclic aromatic hydrocarbons are a class of environmental PAH that have been recently identified. Many of these chemicals have been found to be more active than benzo[a]pyrene in tests for genetic toxicity using bacterial and rodent cells. Benz[l]aceanthrylene, a cyclopenta-polycyclic aromatic hydrocarbon related to benz[a]anthracene, and benzo[a]pyrene were compared for their activity to induce cytotoxicity and anchorage-independent growth with normal human diploid fibroblasts. Both benz[l]aceanthrylene and benzo[a]pyrene were relatively non-cytotoxic to normal human diploid fibroblasts. However, benz[l]aceanthrylene was twice as active compared to benzo[a]pyrene over the concentration range examined as an inducer of anchorage-independent growth. The ability of benz[l]aceanthrylene to induce anchorage-independent colony growth in normal human cells, in combination with its demonstrated ability as a mouse-skin tumorigen, suggests this PAH to be a potential multi-species carcinogen.     

High mutagenicity and toxicity of a diol epoxide derived from benzo[a]pyrene

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP 7,8-diol-9,10-epoxide) is a suspected metabolite of benzo[a]pyrene that is highly mutagenic and toxic in several strains of $\text{Salmonella}\text{typhimurium}$ and in cultured Chinese hamster V79 cells. BP 7,8-diol-9,10-epoxide was approximately 5, 10 and 40 times more mutagenic than benzo[a]pyrene 4,5-oxide (BP 4,5-oxide) in strains TA 98 and TA 100 of $\text{S.}\text{typhimurium}$ and in V79 cells, respectively. Both compounds were equally mutagenic to strain TA 1538 and non-mutagenic to strain TA 1535 of $\text{S.}\text{typhimurium}$. The diol epoxide was toxic to the four bacterial strains at 0.5–2.0 nmole/plate, whereas BP 4,5-oxide was nontoxic at these concentrations. In V79 cells, the diol epoxide was about 60-fold more cytotoxic than BP 4,5-oxide.     

The metabolic activation of benz[a]anthracene in hamster embryo cells: evidence that diol-epoxides react with guanosine, deoxyguanosine and adenosine in nucleic acids

The principal nucleoside-hydrocarbon adducts present in hydrolysates of RNA and DNA isolated from hamster embryo cells treated with benz[a]anthracene (BA) were examined by chromatography on Sephadex LH 20 and by high pressure liquid chromatography (HPLC) on Spherisorb 5 ODS. The results extend the previous finding that a non-'bay-region' diol-epoxide, anti-BA-8,9-diol 10,11-oxide (r-8,t-9-dihydroxy-t-10,11-oxy-8,9,10,11-tetrahydrobenz[a] anthracene) is involved in the binding of BA to cellular nucleic acids and show that this diol-epoxide most probably reacts with guanosine and adenosine in RNA and with deoxyguanosine in DNA. The results also show that a 'bay-region' diol-epoxide anti-BA-3,4-diol 1,2-oxide (t-3,-4-dihydroxy-t-1,2-oxy-1,2,3,4-tetrahydrobenz[a]anthracene, which is thought to be involved in the binding of benz[a]anthracene, which is thought to be involved in the binding of benz[a]anthracene to DNA in some situations, reacts mainly with deoxyguanosine.     

Metabolic activation of 3-methylcholanthrene: mutagenic and transforming activities of the 9,10-dihydrodiol.

3-Methylcholanthrene and five related dihydrodiols have been tested for microsome-mediated mutagenicity towards $\text{Salmonella}\text{typhimurium}$ TA100 and for the induction of mutation to 8-azaguanine resistance in V79 Chinese hamster cells and malignant transformation in M2 mouse fibroblasts. In both mutagenicity test systems, the 9,10-diol was considerably more active than either the parent hydrocarbon, the related $\text{cis}$-2α,3-diol, the $\text{trans}$-4,5-, the $\text{trans}$-7,8- or the $\text{trans}$-11,12-dihydrodiols. At a non-toxic concentration (1μg/ml medium), the 9,10-diol induced the formation of more transformed malignant foci in cultures of M2 cells than 3-methylcholanthrene and the other diols were either inactive or only weakly active in this test system. The results obtained indicate that the 9,10-dihydrodiol derived from 3-methylcholanthrene is involved, presumably following conversion into the corresponding vicinal diol-epoxide, 9,10-dihydro-9,10-dihydroxy-3-methylcholanthrene 7,8-oxide, in the metabolic activation of this carcinogenic polycyclic hydrocarbon.     

Enhancement of DNA binding,mutagenicity and carcinogenicity of polycyclic aromatic hydrocarbons after induction of cytochrome P-450 by ellipticines in rats and mice

Pretreatment of rats by ellipticines enhanced the microsomal concentration of cytochrome P-450, benzo[a]pyrene (BP) metabolism and activation and, to a smaller extent, ethoxycoumarin deethylation, but not acetanilide hydroxylation. This increased BP biotransformation was essentially due to the formation of bay-region metabolites, BP 9,10-diol, BP 7,8-diol and 9-hydroxy-BP, or to the formation of BP 7,8-diol-9,10-epoxide- and of 9-hydroxy-BP 4,5-oxide-DNA adducts. In the ellipticine series, 9-fluoroellipticine (9-FE) presents a slight inducing potency compared with the parent and 9-hydroxy molecules. Pretreatment of mice with 9-hydroxyellipticine (9-OHE) led also to an increased mutagenicity of BP and to an augmentation of skin carcinogenesis by 7,12-dimethylbenz[a]anthracene (DMBA). These results clearly show that 9-OHE induces the biosynthesis of cytochrome P-450 which markedly stimulates the mutagenic and carcinogenic potentialities of polycyclic aromatic hydrocarbons (PAH).     

Bacterial mutagenicity of aceanthrylene: a novel cyclopenta-fused polycyclic aromatic hydrocarbon of low molecular weight

Aceanthrylene, a non-alternant cyclopenta-fused hydrocarbon, was shown to be weakly mutagenic without S9 and strongly mutagenic with S9 in the Ames Salmonella plate incorporation assay. The compound was most active in strain TA100 (35 revertants/nmole in the presence of 0.3 mg of S9 protein), and less active in strains TA98, TA1537 and TA1538 (20, 10 and 3.1 rev/nmole respectively, + S9). Strain TA1535 was unresponsive, suggesting that this compound induces frameshift mutations rather than base-pair substitutions. The mutagenic potency of aceanthrylene is consistent with predictions of its activity based on the relatively large delocalization energy (delta E deloc/beta = 0.931) of the carbonium ion which would result from oxirane ring opening of the 1,2-epoxide, a potential active metabolite.     

Bioactivation of cysteine conjugates of 1-nitropyrene oxides by cysteine conjugate beta-lyase purified from Peptostreptococcus magnus.

To determine the role of cysteine conjugate beta-lyase (beta-lyase) in the metabolism of mutagenic nitropolycyclic aromatic hydrocarbons, we determined the effect of beta-lyase on the mutagenicities and DNA binding of cysteine conjugates of 4,5-epoxy-4,5-dihydro-1-nitropyrene (1-NP 4,5-oxide) and 9,10-epoxy-9,10-dihydro-1-nitropyrene (1-NP 9,10-oxide), which are detoxified metabolites of the mutagenic compound 1-nitropyrene. We purified beta-lyase from Peptostreptococcus magnus GAI0663, since P. magnus is one of the constituents of the intestinal microflora and exhibits high levels of degrading activity with cysteine conjugates of 1-nitropyrene oxides (1-NP oxide-Cys). The activity of purified beta-lyase was optimal at pH 7.5 to 8.0, was completely inhibited by aminooxyacetic acid and hydroxylamine, and was eliminated by heating the enzyme at 55 degrees C for 5 min. The molecular weight of beta-lyase was 150,000, as determined by fast protein liquid chromatography. S-Arylcysteine conjugates were good substrates for this enzyme. As determined by the Salmonella mutagenicity test, 5 ng of beta-lyase protein increased the mutagenicity of the cysteine conjugate of 1-NP 9,10-oxide (10 nmol per plate) 4.5-fold in Salmonella typhimurium TA98 and 4.1-fold in strain TA100. However, beta-lyase had little effect on the cysteine conjugate of 1-NP 4,5-oxide (10 nmol per plate). Both conjugates exhibited only low levels of mutagenicity with nitroreductase-deficient strain TA98NR. In vitro binding of 1-NP oxide-Cys to calf thymus DNA was increased by adding purified beta-lyase or xanthine oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Host-mediated mutagenicity experiments with benzo[a]pyrene and two of its metabolites

Benzo[a]pyrene (BP) and two of its major metabolites, the ultimate mutagen BP-4,5-oxide and the proximate mutagen trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (BP-7,8-diol) were investigated for mutagenicity in Salmonella typhimurium TA1538, TA98 and TA100 using an intrasanguineous host-mediated assay. BP and BP-4,5-oxide were not mutagenic under any experimental conditions. BP-7,8-diol was inactive with the strain TA1538 but was mutagenic with the strains TA98 and TA100. The effect was potentiated by pretreatment of the host mice with the cytochrome P-450 inducer 5,6-benzoflavone. We conclude: (i) one of the reasons for the observed insensitivity of the intrasanguineous host-mediated assay towards BP is that BP-4,5-oxide, which contributes to the microsome-mediated mutagenicity of BP, is inactive in the host-mediated assay; (ii) the finding that BP-7,8-diol is mutagenic in the host-mediated assay demonstrates that the lack of mutagenicity of BP is not intrinsic; (iii) the potentiated mutagenicity after treatment of the hosts with 5,6-benzoflavone suggests that cytochrome P-450 is more important in the activation of BP-7,8-diol in this system than other enzymes (e.g. prostaglandin synthase) that can also activate this compound in vitro.     

Metabolic activation of benzo[a]pyrene in human skin maintained in short-term organ culture

The metabolic activation of benzo[a]pyrene (BP) was examined in six samples of human skin after topical application of the hydrocarbon to the skin in short-term organ culture. The results show that all of the samples were capable of metabolizing BP to water-soluble products and to ether-soluble products that included the 4,5-, 7,8- and 9,10-dihydrodiols and a product which had chromatographic properties identical with those of authentic trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (BP-11,12-diol). The major BP-deoxyribonucleoside adduct detected in each skin sample appeared to be formed from the reaction of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BP-7,8-diol 9,10-oxide) with deoxyguanosine residues in DNA.     

The relationship between the levels of DNA-hydrocarbon adducts and the formation of sister-chromatid exchanges in Chinese hamster ovary cells treated with derivatives of polycyclic aromatic hydrocarbons

The frequencies of the induction of sister-chromatid exchanges and the levels of deoxyribonucleoside-hydrocarbon adducts formed in Chinese hamster ovary cells that had been treated with either dihydrodiols or a diol-epoxide derived from polycyclic aromatic hydrocarbons were determined. Up to 6-fold increases in the incidence of these exchanges were observed when the cells were treated either with the dihydrodiols, trans-3,4-dihydro-3,4-dihydroxy-7-methylbenz[a]anthracene,trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene or the diol-epoxide, (±)-r-7, t-8dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a] pyrene but when the cells were transferred to media free of these compounds, there were rapid reductions in the frequency of these exchanges. When the exchanges were induced by the diol-epoxide, the decreases in frequency were paralleled by decreases in the levels of deoxyribonucleoside-diol-epoxide adducts that were present in hydrolysates of DNA isolated from the cells. There thus appears to be a close relationship between the frequency of sister-chromatid exchanges and the levels of deoxyribonucleoside-diol-epoxide adduct formation.     

Effect of fluorine substitution on benzo[j]fluoranthene genotoxicity.

The metabolism and mutagenic activity of 4-fluorobenzo[j]fluoranthene (4F-B[j]F) and 10-fluorobenzo[j]fluoranthene (10F-B[j]F) were evaluated and compared with benzo[j]fluoranthene (B[j]F) using an identical rat liver homogenate preparation. Previous studies have shown that the major genotoxic metabolites of B[j]F are the 4,5- and 9,10-dihydrodiol. The 9,10-dihydrodiol was the principal metabolite formed in the case of 4F-B[j]F, while the 4,5-dihydrodiol was the principal metabolite formed in the metabolism of 10F-B[j]F. Studies on the relative genotoxicity of these fluorinated derivatives were performed to indirectly determine the possible contribution of the 4,5- and 9,10-dihydrodiol in the activation of B[j]F to a genotoxic agent. In the presence of microsomal activation, both of these fluorinated derivatives of B[j]F were more mutagenic in S. typhimurium TA97a, TA98 and TA100 than B[j]F. However, differences in mutagenic potency were observed between 4F- and 10F-B[j]F. 10F-B[j]F had similar mutagenic potency to 4F-B[j]F in TA97a and TA98 at doses associated with the linear portion of the dose response curve. However, a slightly higher mutagenic response was observed with 10F-B[j]F in TA98 at doses above 5 nmol. In contrast, 4F-B[j]F was more active than 10F-B[j]F as a mutagen in TA100. The tumor-initiating activity of these analogs on mouse skin was assessed at doses of 2.0, 1.0 and 0.3 mumol. Skin irritation was observed with the fluorinated B[j]F derivatives at doses above 0.3 mumol. At a dose of 0.3 mumol, 4F-B[j]F exhibited tumorigenic activity which was similar to B[j]F. In contrast, 10F-B[j]F was less active than B[j]F at all three doses assayed. Both fluorinated derivatives of B[j]F formed higher levels of DNA adducts in vivo in mouse skin than B[j]F. A modified 32P-postlabeling method was required to detect fast migrating B[j]F:DNA adducts that went undetected in previous studies. The level of DNA adducts formed from 4F-B[j]F was considerably greater than the levels observed with 10F-B[j]F. This is consistent with the greater mutagenic activity in S. typhimurium TA100 and tumor-initiating activity exhibited by 4F-B[j]F. These studies suggest that fluorine substitution may significantly alter the intrinsic genotoxicity of the 4,5- and 9,10-dihydrodiol of B[j]F. These data also imply that B[j]F may be primarily activated via the formation of the 9,10-dihydrodiol metabolite. This pathway of activation is inconsistent with our previous studies which indicate that the 4,5-dihydrodiol is the most important pathway of activation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Formation of DNA-binding products from isolated benzo[a]pyrene metabolites in rat liver nuclei

Liver nuclei from 3-methylcholanthrene-treated rats in the presence of NADPH metabolized 3- and 9-hydroxybenzo[a]pyrene and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene to products that bound to DNA. Maximal binding was obtained with the dihydrodiol which was approximately 3-fold that with 9-hydroxybenzo[a]pyrene, and 60-fold that with 3-hydroxybenzo[a]pyrene, as substrates. Both 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene and 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene were also extensively metabolized by the nuclear fraction but did not give rise to DNA-binding products.

The available evidence suggests that the DNA binding species derived from 9-hydroxy-benzo[a]pyrene is 9-hydroxy-benzo[a]pyrene-4,5-oxide and from 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, as previously observed in different systems, 7,8-dihydro-7,8-dihydroxy-benzo[a]pyrene-9,10-oxide.