Metabolic activation, DNA adducts, and H-ras mutations in human neoplastic and non-neoplastic laryngeal tissue

Metabolic activation, DNA adducts, and H-ras mutations were examined in human laryngeal tissue (n = 16) from both smoker and non/ex-smoker patients with laryngeal cancer. DNA adducts detected by 32P-postlabelling were evident only in smokers (n = 13); in fact, smoking cessation for as little as 10 months resulted in no DNA adducts detected (n = 3). Total DNA adduct levels in these samples were significantly correlated with levels of cytochromes P-4502C and 1A1 in laryngeal microsomes. Moreover, the P-4501A1 levels represent the highest yet found in human tissues. In contrast, laryngeal microsomes did not have detectable P-4501A2 activity, while laryngeal cytosols showed appreciable N-acetyltransferase activity for p-aminobenzoic acid (NAT1) but not sulfamethazine (NAT2). DNA was extracted from laryngeal specimens and amplified by PCR. Nylon filter dot or slot blots were hybridized with 32P-labelled probes for codons 12, 13, and 61 of the H-ras gene. Sixty percent of specimens demonstrated mutations in either codon 12, 13, or 61; a single common and specific mutation was a Gln-->Glu transversion in codon 61. This mutation appeared in 5 laryngeal specimens, all from smokers. These results implicate cigarette smoke components, bioactivated by CYP1A1 and/or CYP2C, in DNA adduct formation. These results also demonstrate a probable smoking-related H-ras Gln-->Glu transversion in codon 61.