In vitro genotoxicity assay of sidestream smoke using a human bronchial epithelial cell line.

Genotoxic effects of air contaminants, such as gaseous or particulate compounds, have been difficult to investigate due to inefficient methods for exposing cell cultures directly to these substances. New cultivation and exposure techniques enable treatment of epithelial cells with sample atmospheres with subsequent in vitro assays, as demonstrated by a new system called CULTEX (CULTEX: patent No. DE 19801763; PCT/EP99/00295), which uses a transwell membrane technique for direct exposure of complex mixtures, for example sidestream cigarette smoke, at the air/liquid interface. The sensitivity and susceptibility of human bronchial epithelial cells to this complex mixture have already been shown for cytotoxic endpoints. In this study, genotoxic effects of sidestream cigarette smoke at different concentrations were assessed using the alkaline comet assay. HFBE 21 cells were exposed for 1 h to clean air, nitrogen dioxide or sidestream smoke. Exposure of the cells to sidestream cigarette smoke induced DNA strand breaks in a dose-dependent manner. The combination of gas phase exposure and the comet assay provides a realistic and efficient model for sensitive detection of DNA strand breaks induced by airborne and inhalable compounds.

Comparison of cytochrome P450 2A6 polymorphism frequencies in Caucasians and African-Americans using a new one-step PCR-RFLP genotyping method.

CYP2A6 (cytochrome P450 2A6), which was first identified as the human coumarin 7-hydroxylase, is the most important enzyme in nicotine C-oxidation. The enzyme also metabolically activates the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in vitro. Polymorphisms in the CYP2A6 gene may thus impact on both smoking behavior and lung cancer susceptibility. Several different genotyping methods have been reported with conflicting results in the frequencies of CYP2A6 polymorphic variants. Thus we decided to perform a sequence analysis of the entire CYP2A6 gene. Sequencing confirmed the published CYP2A6 cDNA sequence. However, intron sequences differed considerably from the reported sequence of the CYP2A6*3 (v2) variant. Our analyses revealed that parts of introns shared homologies with the published sequence of CYP2A13. Based on our sequence data we developed a one step protocol for specific amplification of exon 3 of CYP2A6. The resulting PCR product can be used directly for restriction endonuclease digestion with XcmI and DdeI to determine the frequencies of the reported variant alleles CYP2A6*2 and CYP2A6*3. In a population of 305 African-Americans and 145 Caucasians, we found allele frequencies of 0.003 (2/610) for CYP2A6*2 and 0 (0/610) for CYP2A6*3 in African-Americans and allele frequencies of 0.014 (4/290) and 0 (0/290) in Caucasians. We conclude that both alleles are considerably less frequent in populations than previously reported.