Evaluation of the effects of cotinine and nicotine-N'-oxides on the development of tumors in rats initiated with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide.

The effects of cotinine and nicotine-N'-oxides on tumor development in F344 rats initiated with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide [(FANFT) CAS: 24554-26-5] were evaluated. When rats were 6 weeks old, FANFT in an agar diet was administered for a 6-week period. Subsequently, cotinine, trans-nicotine-N'-oxide, and a mixture of cis-nicotine-N'-oxide and trans-nicotine-N'-oxide in drinking water were given as promoters in concentrations of 0.1, 0.02, and 0.02%, respectively. These nicotine metabolites were offered ad libitum for 78 weeks. Control groups consisted of rats that received tap water with or without prior administration of FANFT. Cotinine, trans-nicotine-N'-oxide, and the mixture of cis- and trans-nicotine-N'-oxides were neither carcinogens nor promoters of urinary bladder tumors in rats initiated with FANFT. A reduced incidence of urinary bladder tumors was observed in FANFT-pretreated animals that also received a mixture of cis- and trans-nicotine-N'-oxides. FANFT administration increased the incidences of mesothelioma of the peritoneum and thyroid tumors. Tumor formation in the tongue and palate observed in FANFT-treated rats was not affected by administration of these nicotine metabolites. There was, however, a significant increase in the incidence of forestomach tumors in rats that were initiated with FANFT and subsequently received either trans-nicotine-N'-oxide or a mixture of cis- and trans-nicotine-N'-oxides.

Influence of bay-region methyl group on formation of 5-methylchrysene dihydrodiol epoxide:DNA adducts in mouse skin.

The binding of tritium-labeled 5-methylchrysene to DNA of CD-1 mouse skin 24 hr after treatment has been studied. DNA was isolated from the treated skin areas of mice and hydrolyzed enzymatically to deoxyribonucleosides, and the hydrolysate was chromatographed on a Sephadex LH-20 column using a methanol:water gradient. The major adducts eluted between 70 and 100 ml (Peak 1), 470 and 590 ml (Peaks 2A to C), and 750 and 850 ml (peak 3). For identification of these products, markers were prepared from 5-methylchrysene bay-region dihydrodiol epoxides. [5-14C]1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene and [5-14C]7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-5-methylchrysene were synthesized by reacting the corresponding metabolically formed [5-14C]1,2-dihydro-1,2-dihydroxy-5-methylchrysene and [5-14C]7,8-dihydro-7,8-dihydroxy-5-methylchrysene with m-chloroperoxybenzoic acid. The structures of the dihydrodiol epoxides were established by their mass spectra and by hydrolysis to tetrols. Peak 2B was chromatographically indistinguishable, both on Sephadex LH-20 and reverse-phase high-pressure liquid chromatography, from the adduct formed when [5-14C]1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene was reacted with salmon sperm DNA in solution. Similarly, Peak 2A was chromatographically inseparable from the [5-14C]7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-5-methylchrysene:DNA adduct. Adduct 2B was formed to a greater extent than adduct 2A by the ratio of 2.7 to 1. These data indicate that 5-methylchrysene preferentially forms DNA adducts from the bay-region dihydrodiol epoxide adjacent to the methyl group.

Identification of mutagenic dihydrodiols as metabolites of benzo(j)fluoranthene and benzo(k)fluoranthene.

The metabolism of the environmental agents benzo(j)-fluoranthene and benzo(k)fluoranthene was investigated using supernatants from the livers of Aroclor 1254-pretreated rats, which are effective in activating benzo(j)fluoranthene and benzo(k)fluoranthene to metabolites mutagenic toward Salmonella typhimurium TA 100. Six bands of metabolites of benzo(j)fluoranthene were separated by high-pressure liquid chromatography, and each band was tested for mutagenicity toward S. typhimurium TA 100 with activation. The major mutagenic band contained two dihydrodiols, one of which was identified as 9,10-dihydro-9, 10-dihydroxybenzo(j)fluoranthene by comparison to a synthetic reference standard. 9,10-Dihydro-9,10-dihydroxybenzo(j)fluoranthene was mutagenic toward S. typhimurium TA 100 with activation, presumably as a result of conversion to the corresponding dihydrodiol-epoxide. The major dihydrodiol metabolite of benzo(k)fluoranthene was identified, by comparison to a synthetic standard, as 8,9-dihydro-8,9-dihydroxybenzo(k)fluoranthene. This dihydrodiol, which could also be converted to a dihydrodiol-epoxide, was mutagenic toward S. typhimurium TA 100 with activation. The results of this study indicate that metabolism to dihydrodiols is one pathway in the activation of benzo(j)fluoranthene and benzo(k)fluoranthene to ultimate mutagens for S. typhimurium TA 100.

Mutagenicity, tumor-initiating activity, and metabolism of methylphenanthrenes.

The mutagenicity, in vitro metabolism, and tumor-initiating activity of methylphenanthrenes were evaluated. The only monomethyl isomers which were mutagenic toward Salmonella typhimurium were 1- and 9-methylphenanthrenes. Among the disubstituted phenanthrenes assayed for mutagenicity, only 1,4-dimethylphenanthrene was active in the presence of metabolic activation. Studies on the in vitro metabolism of methylphenanthrenes were performed by incubation of the various isomers with the 9000 X g supernatant from Aroclor-treated rat livers. Comparison of mutagenicity with metabolites formed in vitro indicated that inhibition of 9,10-dihydrodiol formation was positively associated with mutagenic activity. Among the metabolites of 1- and 9-methylphenanthrenes, significant mutagenic activity was associated only with the 3,4- and/or 5,6-dihydrodiol. Metabolism to the 1,2- or 7,8-dihydrodiol, the requisite dihydrodiols for formation of "bay-region" dihydrodiol-epoxides, was most significant in the case of 4-methylphenanthrene. None of the isomeric methylphenanthrenes were active when assayed as tumor initiators on mouse skin. In contrast, 1,4-dimethylphenanthrene was found to have potent tumorigenic activity. These results suggest that inhibition of 9,10-dihydrodiol formation, the influence of a 4-methyl substituent in directing dihydrodiol formation at the 1,2- or 7,8-positions, and the presence of a bay-region methyl group may be responsible for eliciting a tumorigenic response for 1,4-dimethylphenanthrene.

Tumor-initiating activity and metabolism of polymethylated phenanthrenes.

The tumor-initiating activity of several polymethylated phenanthrenes was determined in mouse skin. Among the compounds assayed were 1,4-, 1,9-, 2,7-, 3,6-, 4,5-, 4,9-, and 4,10-dimethylphenanthrene. Only the 1,4- and 4,10-dimethylphenanthrenes were active as tumor initiators. Initiating doses of 300 micrograms and 1.0 mg of 1,4-dimethylphenanthrene after promotion with tetradecanoylphorbol acetate induced 80 and 100% incidences of skin tumors in mice, respectively, 4,10-Dimethylphenanthrene assayed under identical conditions induced skin tumors in 35 and 55% of the mice. The in vitro metabolism of 1,4-, 3,6-, 4,9-, and 4,10-dimethylphenanthrene was studied by incubation of the compounds with the 9000 x g supernatant from the livers of Aroclor-pretreated rats. The major dihydrodiol metabolite of both 1,4- and 4,10-dimethylphenanthrene was the 7,8-dihydrodiol, the requisite precursor for the formation of bay-region dihydrodiol-epoxides. Dihydrodiols were not observed among the metabolites of 4,9-dimethylphenanthrene. In the case of 3,6-dimethyphenanthrene, the major diol metabolite formed in vitro was the 9,10-dihydrodiol. These results support previously proposed structural requirements which favor the carcinogenic activity of methylated polynuclear aromatic hydrocarbons. These studies indicate that tumorigenic activity of methylated phenanthrenes requires inhibition of dihydrodiol formation at the K-region (9,10-positions) in addition to a bay-region methyl group and a free peri position, both adjacent to an unsubstituted angular ring.

Tumorigenic activity of the 4,5- and 9,10-dihydrodiols of benzo[j]fluoranthene and their syn- and anti-diol epoxides in newborn mice.

Benzo[j]fluoranthene (B[j]F), trans-4,5-dihydro-4,5-dihydroxy-B[j]F, and trans-9,10-dihydro-9,10-dihydroxy-B[j]F were evaluated for tumorigenic activity in newborn CD1 mice. These dihydrodiols were assayed at doses of 1.10 and 0.275 mumol/mouse. B[j]F and the syn- and anti-diol epoxides derived from these dihydrodiols were evaluated at doses of 1.10, 0.275, and 0.110 mumol/mouse (80 mice/group). trans-4,5-Dihydro-4,5-dihydroxy-B[j]F was more potent than trans-9,10-dihydro-9,10-dihydroxy-B-[j]F in inducing pulmonary tumors in both female and male mice. Administration of 1.10 mumol of trans-4,5-dihydro-4,5-dihydroxy-B[j]F resulted in a 90-92% incidence of pulmonary tumors with an average of 3.6 and 4.2 tumors/mouse among female and male mice, respectively. A similar tumorigenic activity was observed for B[j]F in lung. trans-9,10-Dihydro-9,10-dihydroxy-B[j]F was significantly less tumorigenic (P < 0.05), producing a 44 and 64% incidence of pulmonary tumors at a dose of 1.10 mumol with an average of 0.8 and 1.0 tumor/mouse in female and male mice, respectively. A statistically significant (P < 0.001) incidence of hepatic tumors was also produced among male mice administered either B[j]F, trans-4,5-dihydro-4,5-dihydroxy-B[j]F, or trans-9,10-dihydro-9,10-dihydroxy-B[j]F at a dose of 1.10 mumol/mouse. In comparing the tumorigenicity of the diasteromeric diol epoxides derived from both trans-4,5-dihydro-4,5-dihydroxy-B[j]F and trans-9,10-dihydro-9,10-dihydroxy-B[j]F, the anti-diasteromers exhibited greater tumorigenic activity. The most tumorigenic diol epoxide was anti-4,5-dihydroxy-6,6a-epoxy-4,5,6,6a-tetrahydro-B[j]F. At a dose of 0.275 mumol, this diol epoxide induced a 96 and 100% incidence of pulmonary tumors in female and male mice, with an average of 8.6 and 5.0 tumors/mouse, respectively. anti-9,10-Dihydroxy-11,12-epoxy-9,10,11,12-tetrahydro-B[j]F at this dose produced a 56 and 95% incidence of pulmonary tumors in female and male mice with an average of 1.0 and 2.8 tumors/mouse, respectively. syn-4,5-Dihydroxy-6,6a-epoxy-4,5,6,6a-tetrahydro-B[j]F and syn-9,10-dihydroxy-11,12-epoxy-9,10,11,12-tetrahydro-B[j]F at a dose of 0.275 mumol did not induce a significant incidence (P > 0.05) of pulmonary tumors in female or male mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of tumorigenic metabolites of benzo[j]fluoranthene formed in vivo in mouse skin.

The metabolism of benzo[j]fluoranthene (BjF) in vivo in mouse skin was investigated. trans-4,5-Dihydro-4,5-dihydroxybenzo[j]fluoranthene (BjF-4,5-diol) and trans-9,10-dihydro-9,10-dihydroxybenzo[j]fluoranthene (BjF-9,10-diol) have been identified as major metabolites. In addition, 4- and 10-hydroxybenzo[j]fluoranthene and benzo[j]fluoranthen-4,5-dione have been tentatively identified among the metabolites formed in vivo in mouse skin. The enantiomeric purity of the metabolic dihydrodiols of BjF as formed in vivo in mouse skin was determined. The major enantiomer of BjF-4,5-diol was present in 57-62% enantiomeric excess while that of BjF-9,10-diol was present in 66-71% enantiomeric excess. In each case the later-eluting enantiomer on chiral stationary-phase high performance liquid chromatography predominated. The tumor-initiating activity of trans-2,3-dihydro-2,3-dihydroxybenzo[j]fluoranthene (BjF-2,3-diol), BjF-4,5-diol, BjF-9,10-diol, and BjF was evaluated on the skin of female CD-1 mice. As a total initiation dose of 3 mumol/mouse BjF-4,5-diol resulted in a 100% incidence of tumor-bearing mice with 5.0 tumors/mouse. In comparison, BjF-9,10-diol elicited a 60% incidence of tumor-bearing mice with 1.7 tumors/mouse, while BjF-2,3-diol was inactive. At the same dose, BjF gave rise to a 90% incidence of tumor-bearing mice with 7.8 tumors/mouse. At a 1-mumol dose, BjF-4,5-diol induced a 78% incidence of tumor-bearing mice with 4.3 tumors/mouse while BjF gave rise to a 70% tumor incidence with 3.4 tumors/mouse while BjF gave rise to a 70% tumor incidence with 3.4 tumors/mouse. These studies indicate that while BjF-9,10-diol could contribute to the overall tumorigenic activity of BjF in mouse skin, BjF-4,5-diol is a more potent tumor initiator in the target tissue.

Reduction of tumorigenicity and of dihydrodiol formation by fluorine substitution in the angular rings of dibenzo(a,i)pyrene.

The tumor-initiating activities on mouse skin and in vitro metabolism of dibenzo(a,i)pyrene, 2-fluorodibenzo(a,i)pyrene, 3-fluorodibenzo(a,i)pyrene, and 2, 10-difluorodibenzo(a,i)pyrene were compared. After an initiating dose of 500 micrograms, followed by promotion with tetradecanoylphorbol acetate, dibenzo(a,i)pyrene induced skin tumors in 85% of the mice and caused 5.8 skin tumors/mouse. The corresponding tumorigenic activities for the fluorinated compounds were: 2-fluorodibenzo(a,i)pyrene (85%; 1.7 tumors/mouse); 3-fluorodibenzo(a,i)pyrene (80%; 3.1 tumors/mouse); and 2,10-difluorodibenzo(a,i)pyrene (10%; 0.1 tumors/mouse). After an initiating dose of 100 micrograms, only dibenzo(a,i)pyrene showed significant tumor-initiating activity. 3,4-Dihydro-3,4-dihydroxydibenzo(a,i)pyrene was identified as a metabolite of dibenzo(a,i)pyrene formed by the 9000 X g supernatant from the livers of Aroclor 1254-pretreated rats. Another dihydrodiol was tentatively identified as 1,2-dihydro-1,2-dihydroxydibenzo(a,i)pyrene. The formation of these angular ring dihdrodiols was inhibited in the metabolism of 2-fluorodibenzo(a,i)pyrene and 3-fluorodibenzo(a,i)pyrene. Angular ring dihydrodiols were not detected in the metabolism of 2,10-difluorodibenzo(a,i)pyrene. These results suggest that an angular ring dihydrodiol, 3,4-dihydro-3,4-dihydroxydibenzo(a,i)pyrene, which can form a bay-region dihydrodiol epoxide, may be a proximate carcinogen of dibenzo(a,i)pyrene.

Identification of topoisomerase I as the cytotoxic target of the protoberberine alkaloid coralyne.

Protoberberine alkaloids (coralyne and its derivatives), which exhibit antileukemic activity in animal models, have been shown to be potent inducers of topoisomerase (topo) I-DNA cleavable complexes using purified recombinant human DNA topo I. Different from the structurally similar benzophenanthridine alkaloid nitidine (a dual poison of both topos I and II), coralyne and its derivatives have marginal poisoning activity against DNA topo II. Yeast cells expressing human DNA topo I are shown to be specifically sensitive to killing by coralyne derivatives and nitidine, suggesting that cellular DNA topo I is their cytotoxic target. Two human camptothecin-resistant cell lines, CPT-K5 and A2780/CPT-2000, which are known to express highly camptothecin-resistant topo I, are only marginally resistant to coralyne derivatives and nitidine. Purification of human topo I from Escherichia coli cells overexpressing CPT-K5 recombinant topo I has demonstrated similar marginal cross-resistance to nitidine. It seems possible to develop coralyne and nitidine derivatives as new topo I-targeted therapeutics to overcome aspects of camptothecin-related resistance.

Identification of mutagenic metabolites of indeno[1,2,3-cd]pyrene formed in vitro with rat liver enzymes.

Indeno[1,2,3-cd]pyrene (IP) is a major environmental pollutant which is carcinogenic on mouse skin and in rat lung. Unlike benzo(a)pyrene, IP is a nonalternant polycyclic aromatic hydrocarbon which is devoid of a bay region. IP was mutagenic in Salmonella typhimurium TA100 in the presence of a 9000 X g supernatant from the livers of Aroclor-pretreated rats. Using a similar activation system, the major metabolites of IP were isolated and identified by comparison with synthetic reference standards. trans-1,2-Dihydro-1,2-dihydroxy-IP, 8-, 9-, and 10-hydroxy-IP, 8- and 9-hydroxy-trans-1,2-dihydro-1,2-dihydroxy-IP, and IP-1,2-quinone are among the metabolites formed in vitro. The 1,2-epoxide of indeno[1,2,3-cd]pyrene is a potent direct-acting mutagen. 8- and 9-hydroxy-IP were mutagenic with metabolic activation. 1-,2-, and 6-hydroxy-IP and the trans-1,2-dihydrodiol had no significant mutagenic activity in S. typhimurium TA100 with metabolic activation. These data suggest that the K-region oxides of IP and of 8- and 9-hydroxy-IP are ultimately responsible for its mutagenic activity.

Mutagenicity and tumorigenicity of dihydrodiols, diol epoxides, and other derivatives of benzo(f)quinoline and benzo(h)quinoline.

The mutagenic activities of benzo[f]quinoline, benzo[h]quinoline, and a number of their derivatives, including dihydrodiols, K-region oxides, diol epoxides, and tetrahydroepoxides, were assessed in strain TA 100 of Salmonella typhimurium. The dihydrodiol derivatives of benzo[f]quinoline and benzo[h]quinoline were also tested for tumorigenic activity in newborn mice. Benzo[f]quinoline was metabolically activated in the presence of rat liver S-9 preparation to products mutagenic to the bacterial system to a greater extent than was benzo[h]quinoline. However, trans-7,8-dihydro-7,8-dihydroxybenzo[f]quinoline was less mutagenic compared to trans-7,8-dihydroxy-7,8-dihydrobenzo[h]quinoline in the presence of rat liver homogenate. The data on the mutagenic activity of the dihydrodiol derivatives of benzoquinolines were consistent with the intrinsic mutagenicity of the corresponding epoxide derivatives, in that the bay-region diol epoxides and tetrahydroepoxide of benzo[h]quinoline exhibited considerably higher mutagenic activities compared to those of the corresponding derivatives of benzo[f]quinoline at equivalent doses. The K-region oxides of benzo[f]quinoline and benzo[h]quinoline were significantly less mutagenic than their corresponding bay-region diol epoxide and tetrahydroepoxide derivatives. The demonstration that benzo[f]quinoline is significantly more mutagenic than trans-7,8-dihydro-7,8-dihydroxybenzo[f]quinoline, a precursor to the weakly mutagenic bay-region diol epoxide, suggests that the bay-region diol epoxide formation is not the principal pathway for the metabolic activation of benzo[f]quinoline to a mutagen. On the other hand, the isomeric benzo[h]quinoline appears to exert its mutagenic effect via the formation of its bay-region diol epoxide. These results indicate that the position of a nitrogen heteroatom in phenanthrene (the analogous carbocyclic aromatic hydrocarbon) not only has a marked effect on the mutagenic activities of the diol epoxide derivatives, but also can alter the metabolic activation pathways of the parent hydrocarbon. Benzo[f]quinoline, benzo[h]quinoline, and their dihydrodiol derivatives were not tumorigenic in newborn mice.

Synthesis and evaluation of terbenzimidazoles as topoisomerase I inhibitors.

The synthesis and pharmacological activity of a series of terbenzimidazoles are described. The ability of these derivatives to induce DNA cleavage in the presence of topoisomerase I was evaluated in vitro. These analogs were also assayed for their cytotoxicity in RPMI 8402 cells and the camptothecin-resistant CPT-K5 cells. In addition the potential for these compounds to serve as substrates for MDR1 was also determined. Several terbenzimidazoles exhibited similar cytotoxicity against variants of human tumor cells that either overexpress MDR1 or are camptothecin-resistant.

Terbenzimidazoles: influence of 2"-, 4-, and 5-substituents on cytotoxicity and relative potency as topoisomerase I poisons.

Terbenzimidazoles poison the nuclear enzyme topoisomerase I and possess significant cytotoxic activity against several human tumor cell lines. The relative pharmacological activity of 4,5- and 5,6-benzoterbenzimidazoles was compared to that of 5-phenylterbenzimidazole (3). 5,6-Benzoterbenzimidazole is inactive as a topoisomerase I poison and did not exhibit significant cytotoxic activity. In contrast, 4,5-benzoterbenzimidazole retained activity as a topoisomerase I poison but exhibited weak cytotoxic activity relative to 3. While 5-(1-naphthyl)terbenzimidazole is less potent than 3 as a topoisomerase I poison and cytotoxic agent, 5-(2-naphthyl)-terbenzimidazole has comparable activity to 3. The presence of a p-methoxy or p-chloro substituent on the phenyl moiety did not dramatically alter the pharmacological activity of 3. Several analogs of 3 were synthesized wherein the 2"-substituent varied from methyl, ethyl, propyl, isopropyl, phenyl to p-methoxyphenyl. Evaluation of the intrinsic activity of these analogs as topoisomerase I poisons indicates that topoisomerase I poisoning was not diminished by the presence of a methyl, ethyl, propyl, and isopropyl substituent at the 2"-position. Among the various 2"-substituted analogs evaluated, only in the case of 2"-(p-methoxyphenyl)-5-phenylterbenzimidazole was a significant decrease in cytotoxicity observed.

Substituted 2,5'-Bi-1H-benzimidazoles: topoisomerase I inhibition and cytotoxicity.

Several 2'-aryl-5-substituted-2,5'bi-1H-benzimidazole derivatives were synthesized and evaluated as topoisomerase I poisons and for their cytotoxicity toward the human lymphoblast cell line RPMI 8402. This study focused on 18 2,5'-bi-1H-benzimidazole derivatives which contained either a 5-cyano, a 5-(aminocarbonyl), or a 5-(4-methylpiperazinyl) group. Among these bibenzimidazoles, the pharmacological activity of 2'-phenyl derivatives and the influence of the different positional isomers of either a 2'-tolyl group or a 2'-naphthyl moiety on cytotoxicity and topoisomerase I inhibitory activity were determined.

Synthesis and pharmacological evaluation of isoindolo[1,2-b]quinazolinone and isoindolo[2,1-a]benzimidazole derivatives related to the antitumor agent batracylin.

The synthesis and pharmacological activity of isoindolo[1,2-b]quinazolin-12(10H)-ones and isoindolo[2,1-a]benzimidazoles related to batracylin are described. The acute toxicity of batracyclin has been associated with the formation of its N-acetyl metabolite which is a potent inducer of unscheduled DNA synthesis in rat hepatocytes. The desamino derivative and the 8-aza analog of batracylin retained the ability to inhibit topoisomerase II but did not induce unscheduled DNA synthesis. While less active than batracylin, these analogs were cytotoxic to CCRF CEM leukemia cells. The isoindolo[2,1-a]benzimidazole derivatives were inactive as topoisomerase II inhibitors and, in general, failed to exhibit comparable antitumor activity or to induce unscheduled DNA synthesis.

Nicotine: a precursor for carcinogens.

Eric Boyland and collaborator demonstrated about 20 years ago that N'-nitrosonornicotine (NNN), a suspected smoke constituent, was a lung carcinogen in mice and that thiocyanate, a major detoxification product of the smoke component hydrogen cyanide, catalyzes the endogenous formation of nitrosamines. Also, Boyland presumed that the enzymatic conversion of nicotine may contribute to the carcinogenic potential of cigarette smoke via reactive intermediates. Chemical, biochemical and bioassay data gathered since these first observations, support the concept that the nicotine-derived NNN and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) contribute significantly to the carcinogenic activity of snuff and cigarette smoke. Reactive metabolites of nicotine may also be carcinogenic factors. This hypothesis requires exploration.

Tumorigenic activity of non-alternant polynuclear aromatic hydrocarbons in newborn mice.

The tumorigenic activity of benzo[b]fluoranthene, benzo[j]fluoranthene, benzo[k]fluoranthene, and indeno-[1,2,3-cd]pyrene was evaluated in newborn CD-1 mice. The total doses of these non-alternant polycyclic aromatic hydrocarbons employed in this study ranged from 0.5 to 2.1 mumol per mouse. The results of this assay indicate that both benzo[b]fluoranthene and benzo[j]fluoranthene exhibit significant tumorigenic activity. In contrast to these results, neither benzo[k]fluoranthene nor indeno[1,2,3-cd]pyrene were tumorigenic under these assay conditions.

32P-postlabeling analysis of DNA adducts from non-alternant PAH using thin-layer and high performance liquid chromatography.

DNA adduct formation in vivo in mouse skin following a single topical application of benzo[a]fluoranthene (BbF), benzo[j]fluoranthene (BjF), benzo[k]fluoranthene (BkF), or indeno[1,2,3-cd]pyrene (IP) was investigated in female CD-1 mice using 32P-postlabeling analysis. Distinct adduct profiles were detected for each of the non-alternant hydrocarbons examined. Two adducts, one major and one minor, were detected using polyethyleneiminecellulose (PEI-cellulose) thin-layer chromatography (TLC) for BbF and BjF while a single major adduct was detected for BkF and IP. The relative extent of binding to mouse skin DNA was in the order BbF greater than BjF greater than BkF greater than IP. 32P-Postlabeled DNA adducts separated by PEI-cellulose TLC were further analyzed by high performance liquid chromatography (HPLC). A single radioactive peak was detected for 32P-labeled DNA adducts of BjF and BkF. Three general areas of radioactivity were detected when 32P-labeled DNA adducts of BbF were separated on HPLC.

Fluoranthene and pyrene enhance benzo[a]pyrene--DNA adduct formation in vivo in mouse skin.

Fluoranthene and pyrene are potent cocarcinogens when applied together with benzo[a]pyrene (BaP) on mouse skin. In this study the effect of fluoranthene, pyrene and phenanthrene on the formation of BaP--DNA adducts in mouse skin was investigated. Co-application of either fluoranthene or pyrene with [3H]BaP resulted in an average increase in the level of [3H]BaP--DNA adducts of 56% to 66%, respectively, as compared to [3H]BaP alone. Only minor differences were observed in the ratio of (+/-)anti- to (+/-)synbenzo[a]pyrene diol epoxide--DNA adducts between experimental groups. An average 17% decrease in the formation of [3H]BaP--DNA adducts was observed upon co-application of [3H]BaP on mouse skin with phenanthrene. These data suggest a correlation between the observed increase in tumorigenicity of BaP in the presence of either fluoranthene or pyrene and an increase in the formation of (+/-)anti-benzo[a]pyrene diol epoxide--DNA adducts.

Exceptional tumor-initiating activity of 4-fluorobenzo[j]-fluoranthene on mouse skin: comparison with benzo[j]-fluoranthene, 10-fluoro-benzo[j]fluoranthene, benzo[a]pyrene, dibenzo[a,l]pyrene and 7,12-dimethylbenz[a]anthracene.

Exceptional tumorigenic potency was observed with 4-fluorobenzo[j]fuoranthene (4-fluoroB[j]F) relative to benzo[j]fluoranthene (B[j]F) and 10-fluorobenzo[j]fluoranthene (10-fluoroB[j]F) in a mouse skin initiation promotion bioassay. Comparison of the tumorigenic response obtained at total initiating doses of 50, 100, and 1000 nmol firmly established the greater tumorigenic potency of 4-fluoroB[j]F. B[j]F produced a significant tumorigenic response only at total initiating doses of 100 and 1000 nmol per mouse. 10-FluoroB[j]F produced a significant tumorigenic response only at the highest initiating dose, 1000 nmol per mouse. In contrast, 4-fluoroB[j]F produced a significant tumorigenic response at all three doses. At a total initiating dose of 50 nmol, a 90% incidence of tumor-bearing mice with an average of 3.05 tumors per mouse was observed with 4-fluoroB[j]F. A second initiation promotion bioassay was performed to establish the tumorigenic potency of 4-fluoroB[j]F relative to benzo[a]-pyrene (B[a]P), 7,12-dimethylbenz[a]anthracene (DMBA), and dibenzo[a,l]pyrene (DB[a,l]P). 4-FluoroB[j]F did exhibit significant tumor-initiating activity at doses of 10 and 25 nmol per mouse, inducing a 45 and 60% incidence of tumor-bearing mice with an average of 0.75 and 1.65 tumors per mouse, respectively. While B[a]P was not tumorigenic at these doses, DMBA and DB[a,l]P exhibited significant tumorigenic activity at doses of 1, 4, 10, and 25 nmol per mouse. DB[a,l]P induced a 95% incidence of tumor-bearing mice with an average of 5.0 tumors per mouse at a total initiator dose of 1 nmol. DMBA at this dose produced an 85% incidence of tumor-bearing mice with an average of 1.30 tumors per mouse. The results of these initiation promotion bioassays clearly demonstrate that 4-fluoroB[j]F is significantly more active than B[j]F, 10-fluoroB[j]F and B[a]P and less active than either DMBA or DB[a,l]P as a tumor initiator on mouse skin.