MECHANISTIC LINKAGE BETWEEN DNA-ADDUCTS, MUTATIONS IN ONCOGENES AND TUMORIGENESIS OF CARCINOGENIC ENVIRONMENTAL POLYCYCLIC AROMATIC-HYDROCARBONS IN STRAIN A J MICE/

Five polycyclic aromatic hydrocarbons (PAHs), benzo[a]pyrene (B[a]P), benzo[b]fluoranthene (B[b]F), dibenz[a,h]anthracene (DBA), 5-methylchr ysene (5MC), and cyclopenta[cd]pyrene (CPP) were examined for their lu ng tumorigenic activities in strain A/J mice, their ability to form PA H-DNA adducts in lung tissues, and their ability to mutate the Ki-ras oncogene in PAH-induced tumors. PAHs dissolved in tricaprylin were adm inistered 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 surfa ce adenomas enumerated. DBA produced maximal tumor multiplicity at the highest dose, 10 mg/kg, giving 32.2 lung adenomas per mouse. At 100 m g/kg, B[a]P, B[b]F, 5MC, and CPP gave 12.8, 5.3, 93.1, and 32.2 lung a denomas per mouse, respectively. The dose response data for each PAH w as fit to y = 0.6 + bx(1.6), where y is the observed mean lung adenoma s per mouse at dose x (in mg/kg), 0.6 is the observed background of lu ng adenomas per mouse, and b is the fitted constant representing the p otency 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 relati ve potencies of B[b]F, DBA, 5MC, and CPP compared to B[a]P were PAH (r elative activity): DBA (118); 5MC (8.8); CPP (2.9); B[a]P (1.0); B[b]F (0.43). DNA adducts were measured by P-32-postlabeling techniques on DNA from lungs of mice treated with these PAHs. Adducts identified by cochromatography with standards were: from B[a]P, -(N-2-2'-deoxyguanos yl)-7,8,9,10-tetrahydro-B[a]P, and two adducts resulting from the meta bolic activation of 9-hydroxy-B[a]P and trans-7,8-dihydroxy-7,8-dihydr o-B[a]P; from B[b]F, oxy-B[b]F-9,10-diol-11,12-oxide-2'-deoxyguanosine ; from DBA, three adducts from the metabolic activation of s-3,4,10,11 ,-tetrahydroxy-3,4,10,11-tetrahydro-DBA and two nti-DBA-3,4-diol-1,2-o xide-N-2-[2'-deoxyguanosine] adducts; from 5MC, y-4-(N-2-2'-deoxyguano syl)-1,2,3,4-tetrahydro-5MC; from CPP, four CPP-3,4-oxide-2'-deoxyguan osine adducts. Ki-ras codon 12 mutation analysis of PAH-induced tumors was performed using PCR and dideoxy sequencing methods. Mutations fro m lung tumors from tricaprylin-treated mice were GGT --> GAT, GGT --> CGT, and GGT --> GTT. DBA produced no mutations in Ki-ras codon 12 abo ve spontaneous levels. High proportions (greater than or equal to 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 statisti cally 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 w ith the GGT --> TGT mutations, and cyclopenta-ring oxide-2'-deoxyguano sine adducts associated with the GGT --> CGT mutations.