Solutions to the kinetic problem in the micronucleus assay.

The standard micronucleus assay as applied to lymphocyte cultures does not discriminate between non-dividing cells and cells that have divided. Micronuclei can only be expressed after nuclear division and because the proportion of dividing cells may vary from one culture to another the standard micronucleus assay can be very imprecise. To overcome this problem we have developed four new methods for scoring micronuclei only in lymphocytes that have divided once only. In the stathmokinetic method the proportion of dividing cells was measured by blocking them at metaphase with colchicine and the number of micronuclei was determined in a parallel culture. The ratio of micronucleus index and metaphase index gave the number of micronuclei per metaphase. With the flow cytometric method, the cells in S phase were allowed to incorporate bromodeoxyuridine and then allowed to divide. By virtue of their reduced fluorescence after staining with Hoechst 33342 it was possible to sort these cells out with a fluorescence activated cell sorter. The autoradiographic method employed incorporation of tritiated thymidine in cells in S phase so that after division these cells could be recognised by their labelled nuclei. In the cytokinesis-block method, dividing cells were inhibited from performing cytokinesis by exposing them to 3.0 micrograms/ml cytochalasin-B. Dividing cells were easily recognised by their binucleate appearance. Results from the four methods for micronuclei induced by X-rays were in close agreement, but the simplest and most precise of the methods was the cytokinesis-block method.