Flow cytometric recognition of clastogen induced chromatin damage in G0/G1 lymphocytes by non-stoichiometric Hoechst fluorochrome binding

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Abstract
Changes in chromatin structure were induced in human peripheral blood lymphocytes. Resting G0/G1 cells were exposed to either X‐rays, mitomycin C, or bleomycin and stimulated with PHA. Exposure to such agents provokes an increase in the non‐cycling cell fraction; and a distinctive, non‐cycling G‐/G1 subpopulation appears which is characterized by a 23% reduced Hoechst fluorescence intensity. This novel subpopulation was found as early as 24 h after PHA stimulation; it was still present in 72 h cultures. Bromodeoxyuridine (BrdUrd/Hoechst) 33258‐ethidium bromide (EB) flow cytometric analysis revealed increments of this subpopulation from 2% of the non‐cycling cell fraction in the control culture to 29% (X‐rays), 15% (mitomycin C), and 24% (bleomycin) after clastogen exposure. In the presence of the ligase inhibitor 3‐aminobenzamide, this aberrant cell population increased significantly after X‐ray treatment. With the aid of a viable BrdUrd/Hoechst staining assay, the newly identified non‐cycling subpopulation with decreased Hoechst 33258 binding was identified as a distinctive signal cluster. Other than the regular non‐cycling and cycling cell fractions, this subpopulation with non‐stoichiometric Hoechst dye binding showed progressive uptake of ethidium bromide; however, by such criteria 44% of the subpopulation was still viable. It is concluded that the clastogen induced subpopulation of noncycling cells represents damaged cells with altered dye binding properties.