Dose-dependent mutation profile in the c-Ha-ras proto-oncogene of skin tumors in mice initiated with benzo[a]pyrene

• Published in: Carcinogenesis (New York) 1999-09, Vol.20 (9), p.1689-1696
• Main Authors: Wei, Shu-Jing Caroline, Chang, Richard L., Merkler, Kathleen A., Gwynne, Mark, Cui, Xiao Xing, Murthy, Bindu, Huang, Mou-Tuan, Xie, Jian-Guo, Lu, Yao-Ping, Lou, You-Rong, Jerina, Donald M., Conney, Allan H.
• Format: Article
• Language: English
• Subjects: 10-epoxy-7; 10-tetrahydrobenzo[a]pyrene; 10R)-7; 12-dimethylbenz[a]anthracene; 12-O-tetradecanoylphorbol 13-acetate; 1R; 2-epoxy-1; 4-dihydroxy-1; 4-tetrahydrobenzo[c]phenanthrene; 4R)-3; 7R; 8-dihydroxy-9; Administration, Cutaneous; Amino Acid Substitution; Animals; B[c]PhDE; Benzo(a)pyrene - administration & dosage; Benzo(a)pyrene - toxicity; benzo[a]pyrene; Biological and medical sciences; BPDE; C-Ha-ras gene; Carcinogenesis, carcinogens and anticarcinogens; Carcinogens - administration & dosage; Carcinogens - toxicity; Carcinoma in Situ - chemically induced; Carcinoma in Situ - genetics; Carcinoma in Situ - pathology; Chemical agents; Cocarcinogenesis; Codon - genetics; DB[a; dibenzo[a; DMBA; DNA - genetics; DNA Mutational Analysis; DNA, Neoplasm - genetics; Dose-Response Relationship, Drug; Epidermis - drug effects; Epidermis - pathology; Exons - genetics; Female; Genes, ras; H&E; hematoxylin and eosin; hprt; Hyperplasia; hypoxanthine (guanine) phosphoribosyltransferase gene; Keratoacanthoma - chemically induced; Keratoacanthoma - genetics; Keratoacanthoma - pathology; l]P; l]pyrene; Medical sciences; Mice; Papilloma - chemically induced; Papilloma - genetics; Papilloma - pathology; Point Mutation; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Proto-Oncogene Proteins p21(ras) - genetics; selective UV radiation fractionation; single-strand conformation polymorphism; Skin Neoplasms - chemically induced; Skin Neoplasms - genetics; Skin Neoplasms - pathology; SSCP; SURF; Tetradecanoylphorbol Acetate - toxicity; TPA; Tumors
• ISSN: 0143-3334; 1460-2180
• DOI: 10.1093/carcin/20.9.1689

Female CD-1 mice were treated topically with a low (25–50 nmol) or high (800 nmol) dose of benzo[a]pyrene (BP) or acetone vehicle, followed by 5 nmol 12-O-tetradecanoylphorbol 13-acetate (TPA) twice a week for 26 weeks. Selective UV radiation fractionation followed by PCR methods were used to analyze histologically defined subsets of cells (~100–200 cells) on formalin-fixed, paraffin-embedded and H&E stained microscope sections. DNA samples from normal-appearing, hyperplastic or tumor regions from the skin of animals from each treatment group were isolated and amplified by PCR with c-Ha-ras-specific primers. Single-strand conformation polymorphism (SSCP) analyses were performed on both exon 1 and 2 products from each sample. DNA extracted from each aberrant band of SSCP analyses was amplified by PCR for further sequence analysis. The data indicate that c-Ha-ras mutations can be detected in normal-looking and hyperplastic epidermal cells as well as in tumor cells obtained from mice initiated with BP and promoted with TPA. The frequencies of c-Ha-ras mutations for normal-looking, hyperplastic and tumor samples were 3/20 (15%), 8/17 (47%) and 58/68 (85%), respectively, for the low dose group and 8/18 (44%), 10/20 (50%) and 64/86 (74%), respectively, for the high dose group. These observations indicate that there were no dose dependencies in the mutation frequencies for normal-looking, hyperplastic and tumor samples. For combined high dose and low dose samples, differences in mutation frequencies of the c-Ha-ras gene between the normal-looking, hyperplastic and tumor samples were highly significant (P < 0.0001, Fisher's exact test). All mutations detected were located at codons 12, 13 and 61 of the c-Ha-ras gene. With the numbers in parentheses indicating the nucleotide position in the coding sequence of the c-Ha-ras proto-oncogene, the distributions of mutations for G→A (35), G→T (35), G→C (37), G→T (38), C→A (181), A→T (182) and A→G (182) in the low dose tumors were 5, 2, 11, 74, 0, 7 and 2%, respectively, and the distribution of mutations in tumors from animals treated with a high dose of BP were 3, 7, 13, 61, 15, 1 and 0%, respectively. Differences in the global mutation spectra (site and kind of all mutations) for the c-Ha-ras gene between the high and low dose group tumors were statistically significant (P < 0.004, Fisher's exact test) and the major difference between these two groups was C→A (181) base substitutions. In summary, our data indicate that: