Diploid human fibroblasts (IMR-90 cells), grown to confluency and growtharrested by serum starvation, were irradiated with a variety of doses of UV light (0.02540 Jim') or incubated with broad dose ranges of four direct-acting mutagens [ethyl methanesulfonate (EMS), ICR-170, methyl methanesulfonate (MMS), and 4-nitroquinoline oxide (4-NQO)] and pulsed with a thymidine analog, 5-bromodeoxyuridine (BrdUrd) to detect evidence of DNA repair. These cells and appropriate controls were immunochemically stained and subjected to flow cytometric analysis to quantify BrdUrd incorporation into DNA and simultaneously measure cellular DNA content. Since the maximal quantity of BrdUrd incorporated with repairing cells is profoundly less than the amount incorporated during replicative synthesis and flow cytometric analysis collects information on a cell-to-cell basis, data collection using linear histograms succeeded both in revealing repairing cellular populations and eliminating replicative cells from the analysis. Technical modifications necessary to achieve stoichiometry with UV-irradiated IMR-90 fibroblasts included a 48h repair (and pulse) period, followed by denaturing cellular DNA for 15 min at 90°C. The limit of detection was equal to or below the lowest dose investigated (0.025 J/m'). DNA repair was also detected with cultures incubated with low doses of all chemicals and pulsed with BrdUrd for a 24 h period. The limits of detection were near or below 500 pM EMS, 5 pM MMS, 0.25 pM 4-NQO, and 0.1 pM ICR-170. o 1993 Wiley-Liss, Inc.Key terms: Ethyl methanesulfonate, flow cytometry; EMS; ICR-170; IMR-90 cells; methyl methanesulfonate, MMS; 4-nitroquinoline oxide, 4-NQO; unscheduled DNA synthesis, UDS Mammalian cells possess a variety of mechanisms for restoring the integrity of DNA damaged by environmental or endogenous agents. Some instances of damage are repaired with minimal disturbance to the DNA molecule (ex. photoreactivation of UV-induced cyclobutane pyrimidine dimers by a dimer-specific endonuclease), whereas other repair processes, exemplified by several types of excision repair, remove not only the lesion, but excavate a considerable number of adjacent nucleotides in the process (26). With this latter circumstance, DNA integrity is restored by resynthesizing the excised domain and ligating this patch to the parental DNA strand. Various analytical methods have been developed to detect and quantify DNA excision repair in cells, typified by assays that measure 'Abbreviations: BrdUrd, 5-Bromodeoxyuridine; BU, BrdUrd con-

Gossypol acetic acid, a male anti-fertility drug, was evaluated for its effects on cell multiplication, chromosomes, scheduled and unscheduled DNA synthesis, and the surface ultrastructure in cultured murine erythroleukemia cells (clone 6A11A). Gossypol treatments inhibited cell multiplication at 10 and 20 micrograms/ml concentrations and this inhibitory effect increased with elevated dosage and prolonged treatment. Gossypol significantly depressed the mitotic index but did not alter chromosome numbers or increase the frequency of chromosomal structural abnormalities. Cell fraction techniques revealed that gossypol induced a negative effect on cellular DNA synthesis at concentrations as low as 3.3 micrograms/ml after 24 hr of treatment. The number of cells undergoing DNA synthesis decreased with increasing dosages and durations of drug exposure. An unscheduled DNA synthesis assay (UDS) found gossypol to be an active UDS-inducing agent at certain dose levels and treatment times, as measured by increase in net nuclear gain and percentage of UDS cells (ANOVA, Bonferroni test, P less than 0.05). A scanning electron microscope study revealed that 10 micrograms/ml treatment with gossypol caused changes in mouse erythroleukemia cell surface ultrastructure characterized by general balding and the appearance of holes, often after 48 hr of treatment.