RAPID AND EXTENSIVE LETHAL ACTION OF CLOFIBRATE ON HEPATOMA-CELLS IN-VITRO

Clofibrate, for a long time in use as a hypolipidemic drug, is a well known peroxisomal proliferator (PP) and hepatocarcinogen in rodents. W e show here that in vitro 1 mM clofibrate induces a rapid and massive death of rat AH-130 hepatoma cells. Cell death was prominent already a fter 4 h of treatment, with a characteristic 'apoptotic' pattern by co nventional microscopy. This was further supported by the pronounced ch romatin condensation detectable on 4',6-diamine-2'-phenylindole dihydr ochloride (DAPI) staining, the clearcut internucleosomal DNA fragmenta tion on agarose-gel electrophoresis (ladder patten), and the accumulat ion of markedly hypochromic cells observed in flow cytometric DNA hist ograms. Consistently with the apoptotic features of the process, some parameters commonly used to detect cell death, such as plasma membrane permeabilization to trypan blue or propidium iodide, lack of mitochon drial retention of rhodamine 123, or extracellular release of lactate dehydrogenase, were all virtually negative. However, these same parame ters became markedly positive after 24 h of treatment, which was sugge stive for the occurrence of 'secondary' necrosis among AH-130 cells, B y a combination of flow cytometric parameters, after 4 h on 1 mM clofi brate only 41% of the AH-130 cells could still be categorized as viabl e (i.e., non-apoptotic and non-necrotic), while 46% of cells appeared apoptotic and 13% necrotic. At 24 h, 67% of cells were necrotic, 20% a poptotic and only 13% non-apoptotic and nonnecrotic, Apoptosis was als o extensive in AH-130 cells treated with another PP such as nafenopin at 1 mM concentration and in human hepatoma HepG2 cells treated with c lofibrate, By contrast, clofibrate did not cause apoptosis on primary rat hepatocyte cultures, These observations indicate that: (i) apart f rom their well-known cell growth-promoting action, PPs such as clofibr ate or nafenopin may exert a substantial cytotoxic action on targets s uch as the AH-130 or HepG2 hepatoma cells; (ii) this cell death evolve s from an initial 'apoptotic' to an eventual 'necrotic' pattern; (iii) detection of cell death requires the adoption of a full panel of test s, adequate to cover the whole evolving death pattern, while such test s may even be substantially misleading whenever applied individually; (iv) the cytotoxicity of clofibrate and similar agents on normal and, particularly, tumoural cells may deserve careful reevaluation.