A mathematical model of the in vitro micronucleus assay predicts false negative results if micronuclei are not specifically scored in binucleated cells or in cells that have completed one nuclear division

A mathematical model is described that predicts the effect of altered nuclear/cell division kinetics and cytotoxicity on micronucleus expression in vitro when the micronucleus assay is performed without discriminating between cells that have divided once and cells that have not divided after genotoxic insult. The model is based on the probabilities of: (i) a viable cell completing nuclear division; (ii) micronucleus expression in a cell that completes nuclear division after genotoxic insult; (iii) a cell not dividing and surviving as a mononuclear cell; (iv) a cell dying by necrosis or apoptosis. The model predicts: (i) false negative results for relatively weak chromosome damaging agents that also inhibit nuclear division, if micronuclei are scored in mononucleated cells without discriminating between divided and non-divided cells; (ii) this tendency for a false negative result when scoring micronuclei without discriminating between non-divided and once-divided mononuclear cells increases with cell lines and culture conditions that do not result in optimal rates of nuclear division (i.e. >90% of dividing cells); (iii) the absolute increment in micronucleus frequency in binucleated cells is at least 2-fold greater than that observed in mononucleated cells when nuclear division is not inhibited and this difference increases with increasing nuclear inhibition. The number of dead cells does not influence the micronucleus frequency if only viable cells are considered when determining the micronucleus frequency ratio. The results from this model suggest that the micronucleus assay when performed by scoring mononucleated cells, without restricting the score to those cells that have divided once after genotoxic insult, is prone to produce false negative results and, therefore, cannot be considered reliable or conclusive. Scoring of micronuclei in cytokinesis-blocked binucleated cells is predicted by the model to provide consistent results under all culture conditions and based on these theoretical results should be considered the preferred choice.