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

Mutat Res Genet Toxicol Environ Mutagen. 2019 Jan:837:65-72. doi: 10.1016/j.mrgentox.2018.10.007. Epub 2018 Oct 29.


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 extrapolating the in vitro responses of these test agents to animal models and humans. The Pig-a 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-a as a reporter of gene mutation both in vitro, in L5178Y/Tk+/- cells, and in vivo, in Sprague-Dawley rats. NNK significantly increased Pig-a mutant frequency in L5178Y/Tk+/- cells, but only at concentrations of 100 μg/ml and greater, and only in the presence of S9 activation. Pig-a 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-a assays were negative. Thus, while NNK induces mutations in the in vitro Pig-a assay, the in vivo Pig-a assay has limited ability to detect NNK mutagenicity under conditions relevant to NNK exposure in smokers.

Keywords: In vitro to in vivo extrapolation; Inhalation; Mutation spectra; Sprague-Dawley rat; Tobacco constituent.

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Female
  • Male
  • Membrane Proteins / genetics*
  • Mice
  • Mutagenicity Tests
  • Mutation / drug effects*
  • Mutation / genetics
  • Mutation Rate
  • Nicotiana / chemistry
  • Nitrosamines / toxicity*
  • Rats
  • Rats, Sprague-Dawley


  • Membrane Proteins
  • Nitrosamines
  • phosphatidylinositol glycan-class A protein
  • 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone