Abstract
The mechanism of non-genotoxic hepatocarcinogenicity of the antihistaminic drug methapyrilene was studied using rat hepatocytes. Optimal survival of rat hepatocytes in culture was obtained when cells were plated in serum containing (10% FCS) medium, left to attach for 2 hours and then maintained in serum-free medium containing EGF, insulin and dexamethasone. At 48 hours of culture there was maximal stimulation of DNA synthesis by EGF and 0.5 μM methapyrilene and 1 mM phenobarbital stimulated DNA synthesis further. Methapyrilene was demonstrated to be an uncoupler of respiration as it produced a concentration-dependent increase in State 4 respiration rate of isolated rat hepatocytes. Low concentrations of MP produced stimulation of mitochondrial membrane potential and elevation of intracellular Ca2+ levels when isolated rat hepatocytes were incubated with methapyrilene for 1 to 5 minutes. Prolonged exposure to methapyrilene or high concentrations (3-10 mM) produced a toxic effect, inhibiting mitochondrial membrane potential and inducing cell death. The effect of methapyrilene on programmed cell death (apoptosis) was studied with a fluorescent dye (Hoechst 33342), which stained DNA. The number of apoptotic cells with condensed chromatin was counted in hepatocytes cultured in medium with and without TGF-β1 and several concentrations of test chemical. Methapyrilene did not induce apoptosis in serum-containing medium, whereas in the presence of EGF and hormones apoptosis was induced. The effect of methapyrilene on apoptosis was similar to the effect of phenobarbitone and it was dependent on culture conditions (presence of serum or EGF and hormones). Methapyrilene produced an increase in release of intracellular Ca2+ into the culture medium after 24 hours in culture and a dose-dependent decrease in triglyceride content after 48 hours in culture. The mechanism of hepatocarcinogenicity of methapyrilene can be explained by the modulation of cell proliferation and apoptosis through signal transduction pathways. Intracellular Ca2+ would be one of the second messengers involved as an increase in intracellular Ca2+ precedes both cell proliferation and apoptosis. An optimal method of cryopreserving hepatocytes was investigated. Cooling rates between -10 and -50 C/min appear optimal. The temperature of release of heat of fusion was the critical point in the cryopreservation protocols. Rapid cooling rates which reduced the release of heat of fusion were optimal. Hepatocytes after cryopreservation maintained similar levels of DNA synthesis to freshly prepared cultures and it was also stimulated by EGF after 48 hours in culture. However, Neutral Red uptake was decreased and cryopreserved hepatocytes were more sensitive to the toxic effect of methapyrilene. Cryopreservation had an adverse effect on mitochondrial function because there were more cells with lower mitochondrial membrane potential compared to freshly isolated cells and an impairment of the respiration function was demonstrated.