The Interactive Toxicity of Benzo(a)Pyrene and Ultraviolet Radiation - an In Vitro Investigation
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The work presented here adopted an in vitro approach with cell types from different species (fish: Epithelioma Papillosum Cyprini (EPCA1), Rainbow Trout Gonad (RTG-2); mammals: Chinese Hamster Ovary (CHO-K01), primary human fibroblast cells (84BR)) to elucidate the potential genotoxic effects of the interaction of the polycyclic aromatic hydrocarbon (PAH), benzo(a)pyrene (B(a)P) (0.0, 0.1, 1.0 and 3.2 µg mlˉ¹) with ultraviolet radiation (UVA/UVB) (typically 25, 50, 100, 200, 500, 1000, 2000, 4000, 6000, 8000 J mˉ²). Initially the experimental techniques and conditions were optimised and validated in the CHO-K1, EPCA1 and RTG-2 cell lines. It was shown that mammalian (CHO-Kl) and fish cells (EPCA1 and RTG-2) exhibited similar sensitivities to chemicals with different modes of action i.e. clastogenic ethyl methansulphonate (EMS) (0.0, 0.8, 1.6 and 3.2 mM) and aneugenic colchicine (COL) (0.0, 0.1, 1.0 and 1.8 µg mlˉ¹) following cytotoxicity experiments with neutral red retention (NRR). Similarly, using the micronucleus assay (Mn) all the cell lines tested showed a similar response to EMS and COL and the use of the anti-kinetochore stain provided a useful approach with which to distinguish between clastogenic and aneugenic effects in the cell. Following comet assay experiments the importance of optimising and validating variables was demonstrated. The optimal variables chosen for the comet assay were 20 minutes unwinding for fish cells (EPCA1 and RTG-2) and 40 minutes unwinding time for mammalian cells (CHO-K1 and 84BR) with 20 minutes electrophoresis for all cell types. Following these validation studies, the cytotoxic and genotoxic effects produced in cells of aquatic (EPCA1, RTG-2) and mammalian (CHO-K1, 84BR) origin following treatment with B(a)P and UVR was investigated. The incubation of all cells (EPCA1, RTG-2, CHO-K1) with B(a)P alone caused limited cytotoxicity (NRR), increased DNA damage (comet assay) and altered cellular functions that were from aneugenic and clastogenic mechanisms (Mn assay). EPCA1, RTG-2 and CHO-K1 cells irradiated with UVB displayed a significant increase in cytotoxicity (NRR) and DNA damage (comet assay). Cells irradiated with UVA (RTG-2, CHO-K1, 84BR) showed no significant increases in cytotoxicity and only CHO-K1 showed increased DNA damage (comet assay). There were significant increases in cellular alterations (Mn assay) following UVA irradiation. All cells (RTG-2, CHO-K1, 84BR) incubated with B(a)P followed by irradiation with UVA showed a synergistically increased cytotoxicity (NRR) and DNA damage (comet assay) from a 1.2-fold increase up to a 4-fold increase in DNA damage. There were also altered cellular mechanisms that may be due to both aneugenic and clastogenic mechanisms (Mn assay). Oxidative stress as a product of the formation of the hydroxyl radical was shown to be a key element in these processes (Electron Spin Resonance (ESR)). It is therefore concluded that the genotoxic effects of the PAH B(a)P and UVA irradiation are synergistically increased when both insults are experienced in combination. This worrying result was observed within both fish and mammalian cell types and appeared to be mediated via an oxidative stress mechanism which included the formation of the hydroxyl radical.
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