MUTATIONAL SPECTRA OF A 100-BASE PAIR MITOCHONDRIAL-DNA TARGET SEQUENCE IN BRONCHIAL EPITHELIAL-CELLS - A COMPARISON OF SMOKING AND NONSMOKING TWINS

Seventeen separate mitochondrial hot spot mutations in a 100-bp target sequence (mitochondrial bp 10,030-10,130) were detected and measured in bronchial epithelial cell samples isolated from smokers and nonsmok ers, Among the individuals sampled were three pairs of monozygotic twi ns in which one twin had never smoked and had a nonsmoking spouse, and the other had a smoking history of >10 pack-years, Individual point m utations present at frequencies as low as 10(-6) were detected. Partia lly denaturing electrophoresis was used to separate mutant from nonmut ant sequences on the basis of their melting temperatures, and the targ et sequence was subsequently amplified via high-fidelity PCR with Pfu DNA polymerase. Tests were performed to determine whether mismatch int ermediates or DNA adducts present in the cellular DNA were converted t o mutants during PCR, Hot spot mutations were clearly observed in bron chial epithelial cells, and the same hot spots were observed consisten tly in different samples, Significant numerical variability in the mut ant fractions for individual mutants was observed among samples and ar e ascribed to unequal mitochondrial segregation in stem and transition cells, The mutational spectra in smokers' samples did not differ sign ificantly fi om the mutational spectra in nonsmokers' samples for this 100 bp of mitochondrial DNA, No smoking-specific hot spots were detec ted, The overall mutant fractions in smokers' samples were not elevate d compared to those of nonsmokers, As much variability was observed be tween two samples from the same individual's lung as between a sample from a smoker and a sample from a nonsmoker, These findings demonstrat e that inhaled tobacco smoke does not induce prominent point mutations in this 100-bp target mitochondrial sequence in smokers' bronchial ep ithelial cells, Endogenous factors (e.g., DNA replication errors or DN A damage by endogenous reactive chemicals) are suggested to be more li kely to represent the most important contributors to mitochondrial mut agenesis.