CHARACTERIZATION OF THE IN-VITRO HEPATOCYTE MODEL FOR TOXICOLOGICAL EVALUATION - REPEATED GROWTH-STIMULATION AND GLUTATHIONE RESPONSE

Hepatocytes in culture represent a useful model for investigating the effects of toxic agents on liver cells. However, further development o f this model is hampered by the difficulty in promoting cell prolifera tion over prolonged periods and the lack of knowledge about the bioche mical status of the cells relevant to the toxic response under prolife ration conditions. In an effort to overcome these limitations, this wo rk focused on the establishment of conditions to ameliorate the promot ion of hepatocyte proliferation in vitro. It also examined the effects of growth stimuli on the levels of glutathione (GSH), a highly signif icant parameter influencing the resistance against toxic agents. In ad dition, albumin secretion was monitored as an indicator of liver-speci fic functions. Two modified L-15 media were developed: medium A for su pporting cell differentiation, and medium B for promotion of prolifera tion. Collagen and Matrigel were used as substrata. In medium A, the t ime course of GSH levels was comparable for both substrata, with an in itial increase followed by a plateau and then by a progressive decreas e from the second to the fourth week. Hepatocytes cultured on collagen and sequentially exposed to medium B (containing epidermal growth fac tor +/- norepinephrine) and medium A, showed repeated responsiveness t o stimulation of DNA synthesis. Moreover, for cultures on collagen, a higher GSH content was observed in parallel with DNA synthesis stimula tion, while albumin secretion was diminished. Although cells on Matrig el were refractory to DNA synthesis stimulation, GSH levels were still increased upon exposure to the growth factors, while under these cond itions, albumin synthesis remained unaltered. These results show the p ossibility of expanding growth stimulus applications in hepatocyte cul tures when it is of interest in cell pathology studies, such as those involving the effects of long-term exposure to xenobiotics, especially carcinogens. The results also suggest that hepatocytes subjected to a growth stimulus may have better protection against toxic injury.