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Background: The inhibitory effects of N-(4-hydroxyphenyl)-retinamide (4HPR) on tumorigenesis and tumor growth may result from its ability to induce apoptosis (programmed cell death). Since antioxidants inhibit 4HPR-induced apoptosis, experiments were planned to determine whether the levels of reactive oxygen species increase in cells undergoing apoptosis after exposure to 4HPR. Methods: Cells of the human cervical carcinoma cell line C33A and normal human cervical epithelial cells were treated with 4HPR and analyzed for survival, induction of apoptosis, generation of reactive oxygen species, and expression of the apoptosis-related proteins Bcl-2 and Bax. Results: Treatment with 4HPR decreased C33A cell number by inducing apoptosis in a time- and dose-dependent fashion. DNA fragmentation typical of apoptosis was observed in cells exposed to 4HPR at concentrations of 3 [mu]M or higher for 6-24 hours. The generation of reactive oxygen species was enhanced by 1.85-fold to 4.5-fold after a 1.5-hour treatment with 0.4-10 [mu]M 4HPR. Pyrrolidine dithiocarbamate, an oxygen radical scavenger, suppressed the rate of generation of reactive oxygen species and inhibited 4HPR-induced apoptosis. 4HPR failed to modulate cellular levels of the Bcl-2 and Bax proteins. N-(4-Methoxyphenyl)-retinamide, the major 4HPR metabolite, and several other retinoids that bind to nuclear retinoic acid receptors or retinoid X receptors failed to enhance the generation of reactive oxygen species and to induce apoptosis. 4HPR was much less effective in generating reactive oxygen species and in inducing apoptosis in normal human cervical epithelial cells than in C33A cervical carcinoma cells. Conclusions: Enhancement of the generation of reactive oxygen species may be involved in apoptotic pathway induction by 4HPR. [J Natl Cancer Inst 1997;89:1191-8]