Assessment of nicotine-induced DNA damage in a genotoxicological test battery

The role of the tobacco-alkaloid nicotine in tumour biology is widely discussed in the literature. Due to a strong capacity to induce angiogenesis, a pro-mutagenic potential in non-tumour and cancer cells, and a pro- and anti-apoptotic influence, nicotine seems to promote the growth of established tumours. However, results indicating DNA damage and genetic instability associated with nicotine have been contradictory thus far. A variety of markers and endpoints of genotoxicity are required to characterize the genotoxic potential of nicotine. Induction of DNA single- and double-strand breaks, the formation of micronuclei, and the induction of sister chromatid exchange and chromosome aberrations represent possible genotoxicological endpoints at different cellular levels.Human lymphocytes were exposed to nicotine concentrations between 1 μM and 1 mM for 24 h in vitro. The comet assay, the cytokinesis-block micronucleus test, the chromosome aberration (CA) test, and the sister chromatid exchange (SCE) test were then applied. Viability and apoptosis were measured by flow cytometry in combination with the annexin V-propidium iodide staining test.In this test setting, no enhanced DNA migration was measured by the comet assay. An increase in the micronucleus frequency was detected at a concentration of 100 μM nicotine without affecting the frequency of apoptotic cells. A distinct genotoxic effect was determined by the CA test and the SCE test, with a significant increase in CA and SCE at a concentration of 1 μM. In the annexin V test, nicotine did not influence the proportion of apoptotic or necrotic cells.The current data indicating the induction of CA by nicotine underscore the necessity of ongoing investigations on the potential of nicotine to initiate mutagenesis and tumour promotion. Taking into account the physiological nicotine plasma levels in smokers or in nicotine-replacement therapy, particularly the long-term use of nicotine should be critically discussed.

► Genotoxic potential of nicotine was investigated in human lymphocytes.
► In vitro exposure to nicotine at a concentration between 1 μM and 1 mM for 24 h.
► No detection of DNA migration in the comet assay.
► Detection of micronuclei, chromosome aberrations and sister chromatid exchanges.
► Nicotine was reported to induce permanent DNA damage.