Mechanistic investigations of low dose exposures to the genotoxic compounds bisphenol-A and rotenone

Abstract A complete hazard and risk assessment of any known genotoxin requires the evaluation of the mutagenic, clastogenicand aneugenicpotential of the compound. In the case of aneugenicchemicals, mechanism of action (MOA) and quantitative responses may be investigated by studying their effects upon the fidelity of functioning of components of the cell cycle. These present studies have demonstrated that the plastics component bisphenol-A (BPA) and the natural pesticide rotenoneinduce micronuclei and modify the functioning of the microtubule organising centres(MTOCs) of the mitotic spindles of cultured mammalian cells in a dose-dependent manner. BPA and rotenonewere used as model compounds in an investigation of dose response relationships for the hazard/risk assessment of aneugens. Thresholds of action for the induction of aneuploidy have been predicted for spindle poisonson the basis of the multiple targets, which may need disabling before a quantitative response can be detected. The cytokinesis blocked micronucleusassay (CBMA) methodology was utilised in the human lymphoblastoid cell lines AHH-1, MCL-5 and Chinese hamster V79 cell lines. A no observable effect level (NOEL) at 10.8 μg/ml BPA was observed for MN induction. Rotenoneshowed a small increase in MN induction with the first significant effect at 0.25 ng/ml in V79 cells but there was no significant effect in the metabolically competent cell line, MCL-5. For a mechanistic evaluation of the aneugeniceffects of BPA and rotenone, fluorescently labelled antibodies were used to visualise microtubules (α-tubulin) and MTOCs (γ-tubulin). The NOELs for tripolar mitotic spindle induction in V79 cells were 7 μg/ml for BPA and 80 pg/ml for rotenone(concentrations which produced similar changes to mitotic index(M.I.)). Interestingly there was close proximity to the NOEL of 10.8 μg/ml BPA for micronucleus(MN) induction in the human lymphoblastoid AHH-1 cell. Multiple MTOCs can therefore be predicted as a possible mechanism for MN induction. The similarity in concentration inducing tripolar mitosis, M.I. and MN changes suggests immunofluorescence analysis to be a useful dose setting assay with emphasis on the mechanism.