Induction of apoptosis and inhibition on proliferation by mixtures of pesticides in human keratinocytes

489: We have recently shown that combined exposure of human keratinocytes (KCs) to pesticides at low concentrations resulted in more than additive cytotoxic effects (neutral red assay). To further investigate the mechanisms of action of pesticide mixtures in primary human foreskin KCs and a human KC cell line (HaCaT), apoptosis and proliferation were studied simultaneously using a novel triple-staining technique (with TUNEL and Hoechst 33342 staining as markers for apoptosis and with Ki67- immunoreactivity as a marker of proliferation). 12-O-tetradecanoylphorbol-13-acetate (TPA), a known skin toxicant, was used as a positive control.Generally, TUNEL- and Ki67- positive staining was inversely expressed: low numbers of apoptotic cells and high scores of proliferating cells have been found in solvent controls. In contrast, high scores of apoptotic cells and low numbers of proliferating cells were observed in TPA- treated cultures.Following short-term exposure (5 hours) to the pesticides benomyl, bendiocarb or paraquat as single substances or in binary mixtures, TUNEL-positive cells were markedly increased and Ki67-immunoreactive KCs were decreased. Interestingly, long-term treatment (24 hours) with single substances resulted in moderate effects, whereas binary mixtures of pesticides induced a pronounced increase in the number of TUNEL-positive KCs and a strong decrease in the number of Ki67-positive KCs compared to controls.In addition, HaCaT cells which were treated with the same pesticide mixtures for 24 hours, generally showed less apoptosis than primary KCs.The results suggest a time-dependent induction of apoptosis which may be critically involved in the higher cytotoxicity of pesticide mixtures compared to exposure to single substances in primary human KCs.