Differential effects of non-genotoxic carcinogens and proliferating agentson cell growth, survival and apoptosis in hepatic cells in vitro

Many nongenotoxic carcinogen's (ngc) produce hyperplastic lesions from whic h neoplastic foci may arise. Modulation of the rate of apoptosis by some ng c's within these lesions may be critical to their mechanism of tumour promo tion but some may be cytotoxic. To establish if these compounds are apoptot ic or necrotic in vitro, three ngc's (12-0-tetradecanoyl phorbol-13-acetate (TPA); nickel, and di(2-ethylhexyl-phthalate (DEHP), two noncarcinogenic h epatoproliferating agents (1,4-dichlorobenzene (DCB; HGF) and an in vitro g enotoxic reference compound (7-hydroxy-2-acetylaminofluorene (70H2AAF) were used to induce mitogenic or growth responses in two liver cell-lines HepG2 and JTC-15. MTT and H-3-thymidine incorporation assays were used to measur e cell growth and DNA replicative activity respectively. Rates of apoptosis were assayed using FITC-annexin V with propidium iodide staining and flow cytometry. Responses in HepG2 cells were HGF (proliferation at greater than or equal to 3 ng/ml), TPA (cell growth at greater than or equal to 8 ng/ml ), DEHP (proliferation at greater than or equal to 0.05 mug/ml). NiCl2 and 70H-2AAF were cytotoxic above 0.001 mug/ml and 100 ng/ml respectively. An e quivocal result was obtained for DCB. Responses in JTC-15 cells were HGF (p roliferation, 3 ng/ml), TPA (DNA replication, 10 ng/ml), and DEHP (cell mas s, 2.5 mul/ml). NiCl2 and 70H-2AAF were cytotoxic above 0.01 mug/ml and I 1 0 mg/ml respectively. Equivocal results were obtained for DCB. In flow cyto metry assays apoptotic and necrotic populations were not clearly separable. Approximate rates of apoptosis in HepG2 were: control 8.7%; DEHP, 10.19%. NiCl2, 12.67%; 70H2AAF, 16.56%; TPA, 19.72%; HGF, 23.73%; DCB, 24.59%; posi tive apoptotic control (taxol) 26.94%. These data show apoptosis was increa sed in chemically activated populations of HepG2. The ngc, DEHP, unexpected tly produced proliferation in HepG2 and almost totally suppressed apoptosis in vitro in HepG2 relative to the non-carcinogenic hepatoproliferators. Th e rate of apoptosis induced by the ngc TPA was not considered to be suffici ently different to the rates of apoptosis induced by the noncarcinogenic he patoproliferators. The results emphasize the importance of considering necr otic reactions from effects on apoptosis in detecting non-genotoxic carcino gens. (C) 2001 Elsevier Science Inc. All rights reserved.