Mechanistic linkage between DNA adducts, mutations in oncogenes and tumorigenesis of carcinogenic environmental polycyclic aromatic hydrocarbons in strain A/J mice

Abstract

Five polycyclic aromatic hydrocarbons (PAHs), benzo[ a]pyrene (B[ a]P), benzo[ b]fluoranthene (B[ b]F), dibenz[ a, h]anthracene (DBA), 5-methylchrysene (5MC), and cyclopenta[ cd]pyrene (CPP) were examined for their lung tumorigenic activities in strain A/J mice, their ability to form PAH-DNA adducts in lung tissues, and their ability to mutate the Ki- ras oncogene in PAH-induced tumors. PAHs dissolved in tricaprylin were administered by single intraperitoneal injection to male strain A/J mice (20 mice/dose) at doses up to 200 mg/kg depending on the PAH. Animals were sacrificed 8 months later and the lungs removed, fixed, and surface adenomas enumerated. DBA produced maximal tumor multiplicity at the highest dose, 10 mg/kg, giving 32.2 lung adenomas per mouse. At 100 mg/kg, B[ a]P, B[ b]F, 5MC, and CPP gave 12.8, 5.3, 93.1, and 32.2 lung adenomas per mouse, respectively. The dose response data for each PAH was fit to y = 0.6 + bx 1.6, where y is the observed mean lung adenomas per mouse at dose x (in mg/kg), 0.6 is the observed background of lung adenomas per mouse, and b is the fitted constant representing the potency of each PAH. Statistical analysis indicated that the fit of the data to the equation was extremely high with adjusted R 2 values > 0.985 and small fit standard errors. Based on this equation, the relative potencies of B[ b]F, DBA, 5MC, and CPP compared to B[ a]P were PAH (relative activity): DBA (118); 5MC (8.8); CPP (2.9); B[ a]P (1.0); B[ b]F (0.43). DNA adducts were measured by 32P-postlabeling techniques on DNA from lungs of mice treated with these PAHs. Adducts identified by cochromatography with standards were: from B[ a]P, 7 R,8 S,9 S-trihydroxy-10 R-( N 2-2′-deoxyguanosyl)-7,8,9,10-tetrahydro-B[ a]P, and two adducts resulting from the metabolic activation of 9-hydroxy-B[ a]P and trans-7,8-dihydroxy-7,8-dihydro-B[ a]P; from B[ B]F, 5-hydroxy-B[ b]F-9,10-diol-11,12-oxide-2′-deoxyguanosine; from DBA, three adducts from the metabolic activation of trans,trans-3,4,10,11-tetrahydroxy-3,4,10,11-tetrahydro-DBA and two anti-DBA-3,4-diol-1,2-oxide- N 2-[2′-deoxyguanosine] adducts; from 5MC, 1 R,2 S,3 S-trihydroxy-4-( N 2-2′-deoxyguanosyl)-1,2,3,4-tetrahydro-5MC; from CPP, four CPP-3,4-oxide-2′-deoxyguanosine adducts. Ki- ras codon 12 mutation analysis of PAH-induced tumors was performed using PCR and dideoxy sequencing methods. Mutations from lung tumors from tricaprylin-treated mice were GGT → GAT, GGT → CGT, and GGT → GTT. DBA produced no mutations in Ki- ras codon 12 above spontaneous levels. High proportions (≥ 50%) of GGT → TGT mutations from B[ a]P, B[ b]F and 5MC induced tumors and GGT → CGT mutations from CPP tumors were observed and were statistically significant compared to mutations in tricaprylin control tumors. We conclude from the DNA adduct and Ki-ras mutation studies that bay region diol-epoxide-2′-deoxyguanosine PAH-DNA adducts are associated with the GGT → TGT mutations, and cyclopenta-ring oxide-2′-deoxyguanosine adducts associated with the GGT → CGT mutations.