Biological effects of alpha-pinene in cultured mammalian cells

Abstract

In this work we report the effects of exposure of mammalian cells to α-pinene, a bicyclic monoterpene founded in essential oils and used in insecticides, solvents, perfumes, etc.. Morphological analysis, performed in V79 cells exposed to increasing doses(25μM up to 50μM) of α-pinene, indicated a increase of dose-related nuclear abnormalities; apoptotic cells were seen at higher doses. Immunofluorescence with anti β- tubulin antibody showed that monoterpene induced genomic instability by interfering with mitotic process; in fact, 50% (vs 19% in the control cells) of irregular mitosis with multipolar or not correctly localized spindles were detected, suggesting that α-pinene affects cell stability by disturbing chromosome segregation. Cytogenetic analysis demonstrated that frequency of hypodiploid metaphases increased in a dose-dependent manner and, moreover, α-pinene induced endoreduplicated cells and double strand breaks. Alkaline comet confirmed that monoterpene exposure induced DNA lesions; in fact, OTM increased significantly in a dose-dependent manner. In order to assess whether the severe DNA damage evidenced by comet assay was originated through the ROS production, cells were incubated with CM-H2DCFDA and then analysed by flow cytometry. Results demonstrated an increase in fluorescence intensity after α-pinene treatment indicating an increased oxidative stress. On the whole, these findings strongly suggest that α-pinene is able to compromise genome stability preferentially through mitotic alterations and to damage DNA through reactive oxigen species production.