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Influence of aminoguanidine on parameters of liver injury and regeneration induced in rats by a necrogenic dose of thioacetamide

1 When aminoguanidine, a nucleophilic hydrazine compound, was administered to rats (50 mg kg−1 body wt) 30 min before a necrogenic dose of thioacetamide (500 mg kg−1 body wt), significant changes related to liver injury and hepatocellular regeneration were observed. 2 The extent of necrosis was noticeably less pronounced, as detected by the peak of serum aspartate aminotransferase activity. Depletion of hepatic glutathione (GSH) and the increase in malondialdehyde concentration as markers of oxidative stress, produced by thioacetamide metabolism, were significantly diminished. However, the activity of microsomal FAD monooxygenase, the system responsible for thioacetamide oxidation, did not show significant alterations. Antioxidant enzyme systems involved in the glutathione redox cycle, such as glutathione reductase and glutathione peroxidase activities, slightly decreased following aminoguanidine pretreatment. 3 Primary cultures of peritoneal macrophages from control rats, when incubated in the presence of serum collected following thioacetamide intoxication, showed a significant decrease in nitric oxide (NO) release at 24 h, that was more pronounced in the group pretreated with aminoguanidine. However, the sharp and progressive increase in macrophage NO release, when incubated in the presence of serum obtained at 48, 72 and 96 h, were increased by aminoguanidine‐pretreatment. 4 The cell population involved in DNA synthesis sharply increased in both groups at 48 h of intoxication, although the values at 0, 24, 72 and 96 h were markedly higher in the group pre‐treated with aminoguanidine. Polyploidy at 72 and 96 h of intoxication was delayed by the effect of aminoguanidine and a progressive increase in the hypodiploid hepatocyte population, which reached 16% of the total at 96 h, was observed. 5 These results indicate that a single dose of aminoguanidine before thioacetamide administration, markedly diminished the severity of the liver injury by decreasing oxidative stress and lipoperoxidation, but hepatocellular regeneration was apparently unaffected probably due to an enhanced mitogenic activity.

Potentiation of thioacetamide hepatotoxicity by phenobarbital pretreatment in rats. Inducibility of FAD monooxygenase system and age effect

The ability of phenobarbital to induce the expression and activity of microsomal drug monooxygenases in the liver presents one of the most important issues in the field of chemical interactions and in the toxicity of xenobiotics. The model of rat liver injury induced by a single dose of thioacetamide (500 mg/kg intraperitoneally) was used to study the effect of phenobarbital (80 mg/kg/day intraperitoneally) for 5 days prior to thioacetamide. Serum parameters of liver injury such as aspartate aminotransferase activity, gamma-glutamyl transferase activity and the total bilirubin levels, as well as the activities of hepatic FAD and cytochrome P450 microsomal monooxygenases, were assayed in 2- and 12-month-old rats. Samples of blood and liver were obtained from controls (injected at 0 h with 0.5 ml of 0.9% NaCl) and at 12, 24, 48, 72 and 96 h of thioacetamide intoxication either to non-treated or phenobarbital pretreated rats. Potentiation of thioacetamide hepatotoxicity by phenobarbital pretreatment was demonstrated at morphological level, and by significant increases in the activities of serum aspartate aminotransferase and gamma-glutamyl transferase, and in the levels of total bilirubin. The extent of potentiation of thioacetamide-induced liver injury by phenobarbital pretreatment was similar in both age groups. Microsomal FAD monooxygenase activity, the enzyme responsible for thioacetamide biotransformation, was significantly enhanced (twofold) by phenobarbital pretreatment, and also underwent a further increase following thioacetamide, preceding the peak of necrosis. Cytochrome P450 monooxygenases were induced by phenobarbital pretreatment more than sixfold, and sharply decreased when phenobarbital was withdrawn and thioacetamide administered, showing at 48 h intoxication values close to basal. Phenobarbital pretreatment potentiated thioacetamide necrogenicity, and this potentiation was parallel to the induction of the microsomal FAD monooxygenase system, both by phenobarbital and by thioacetamide itself. The extent of thioacetamide-induced liver injury was significantly higher in 12-month-old rats, but the effect of phenobarbital pretreatment was similar in both age groups.

Mitochondrial involvement in cocaine-treated rat hepatocytes: effect of N-acetylcysteine and deferoxamine.

The cytotoxicity of cocaine (0–1000 μM), was studied on parameters related to the mitochondrial role and the cascade of events that lead to apoptosis in hepatocyte cultures from phenobarbitone (PB) pretreated rats. Cytotoxicity was dose‐dependent and LDH leakage was significantly enhanced above 100 μM cocaine. Apoptosis was visualized by DNA fragmentation on agarose gel, and appeared at 50 and 100 μM cocaine. Cocaine induced biphasic changes in mitochondrial transmembrane potential and significantly increased the mitochondrial release of cytochrome c, the caspase‐3 like DEVDase activity and the level of 20 kDa subunit, a product of pro‐caspase‐3 cleavage. The protective effect of N‐acetylcysteine (NAC) and deferoxamine (DFO) on all these parameters confirmed the involvement of oxygen radicals in cocaine‐induced necrosis/apoptosis. We conclude: first, that the biphasic changes recorded in mitochondrial inner membrane potential by the effect of cocaine, were parallel to apoptosis; second, that caspase‐3 activity and cleavage to it p20 subunit increased sharply in parallel to the translocation of cytochrome c from mitochondria to cytosol; and third, that the antioxidants, NAC or DFO exerted a noticeable protective role in counteracting the cytotoxicity of cocaine, these effects being more pronounced in the case of DFO than NAC. These findings demonstrate that cocaine cytotoxicity involves mitochondrial damage.

Induction of cell proliferation by cyclosporine A in primary cultures of rat hepatocytes

Cyclosporine A (CsA) has been reported to be able to promote cell proliferation, although the precise mechanism by which CsA stimulates cell growth remains uncertain. In the present study, we examined, in hepatocyte cultures, the effect of CsA on parameters related to the cell cycle as well as the levels of proteins involved in the control and progression of the cycle. Flow cytometry analysis detected an increase in the percentage of cells involved in the S phase of the cycle, which correlated with increases in the levels of cyclins D1 and E (two G1-progression regulators), as well as in those of PCNA (proliferating cell nuclear antigen), and without modification in p27, an inhibitory protein of CDKs. We also examined in nucleus the levels of nuclear factor kappaB (a nuclear factor involved in the transcription of the cyclin D1 gene) and found that this transcription factor increased in the presence of CsA. We conclude that the increases in cyclin D1, PCNA, and cyclin E, together with the invariable level of p27, clearly show that CsA induces hepatocytes to proliferate. These results reinforce the idea of the growth-promoting effect of CsA in cultured hepatocytes.