Evaluation of 4-(methylnitrosamino)-1-(3-pyridy1)-1-butanone (NNK) mutagenicity using in vitro and in vivo Pig-α assays

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a genotoxic carcinogen found in tobacco and tobacco smoke. Several in vitro and in vivo assays have been used for evaluating the genotoxicity of tobacco smoke and tobacco smoke constituents like NNK, yet it is not clear which in vitro assays are most appropriate for extra-polating the in vitro responses of these test agents to animal models and humans. The Pig-alpha gene mutation assay can be performed in vitro, in laboratory animals, and in humans, a potential benefit in estimating in vivo responses from in vitro data. In the current study we used Pig-alpha as a reporter of gene mutation both in vitro, in L5178Y/T1c(+/-) cells, and in vivo, in Sprague-Dawley rats. NNK significantly increased Pig-alpha mutant frequency in L5178Y/T1c(+/-) cells, but only at concentrations of 100 pg/ml and greater, and only in the presence of S9 activation. Pig-alpha mutations in L5178Y/Tk(+/-) cells were detected in 80% of the NNK-induced mutants, with the predominate mutation being G -> A transition; vehicle control mutants contained deletions. In the in vivo study, rats were exposed to NNK daily for 90 days by inhalation, a common route of exposure to NNK for humans. Although elevated mutant frequencies were detected, these responses were not clearly associated with NNK exposure, so that overall, the in vivo Pig-alpha assays were negative. Thus, while NNK induces mutations in the in vitro Pig-alpha assay, the in vivo Pig-alpha assay has limited ability to detect NNK mutagenicity under conditions relevant to NNK exposure in smokers.