María Cascales

Hepatotoxicity and aging: endogenous antioxidant systems in hepatocytes from 2-, 6-, 12-, 18- and 30-month-old rats following a necrogenic dose of thioacetamide

The influence of aging on the mechanisms of liver injury and regeneration was studied in a model of hepatotoxicity induced in 2-, 6-, 12-, 18- and 30-month-old rats by a sublethal dose of thioacetamide (500 mg/kg body weight), a soft nucleophilic and hepatotoxic compound metabolized by the hepatic microsomal FAD monooxygenase system. Samples-blood and hepatocytes-were obtained at 0, 12, 24, 48, 72 and 96 h following thioacetamide intoxication. Parameters of liver injury in serum (NADPH-isocitrate dehydrogenase (ICDH) activity) indicate that the severity of injury was significantly higher in the adult groups (6 and 12 months old) when compared either with the youngest (2 months old) or oldest (18 and 30 months old) groups. Parameters related to biotransformation, such as microsomal FAD monooxygenase, followed mainly the same pattern of age-dependent changes as those observed for injury. The profile of glutathione-S-transferase activity showed an initial induction parallel to liver injury and opposite to the levels of reduced glutathione and protein -SH groups. Enzyme activities and gene expression of the systems involved in the cell endogenous antioxidant defense, such as Mn- and Cu,Zn-superoxide dismutases (SOD), catalase and glutathione peroxidase (GPX) showed significant age-dependent changes that can be summarized as follows: an increase in all enzyme activities and gene expression and a decreased ability to restore the initial activities following 96 h of thioacetamide. We conclude, first, that the gene expression and activity of the enzymes involved in the intracellular antioxidant defense system increased with aging, which can be considered a consequence of the enhanced oxidative state of the cell (decreased in GSH level); and second, that the lower and delayed response in the aged groups significantly influenced the restoration towards normal of GSH and the antioxidant enzyme activities.

Age-dependent modifications in rat hepatocyte antioxidant defense systems

BACKGROUND/AIMS Age-dependent changes in the hepatic antioxidant systems were studied in hepatocytes from newly weaned (21 days) to 30-month-old rats. RESULTS Biphasic changes were observed in superoxide dismutase (SOD), glucose-6-phosphate dehydrogenase (G6PDH) and malic enzyme (ME), in which noticeable decreases were detected in hepatocytes from newly weaned to 6-month-old rats: Cu-Zn SOD decreased to 46% (p < 0.001), Mn SOD to 41% (p < 0.001), G6PDH to 71% and ME to 19% (p < 0,001), and significant increases were observed from 6 to 30 months. In hepatocytes from 6- to 30- month-old rats the enzymes involved in antioxidant defense underwent increases in their activities as well in their mRNA: Cu-Zn SOD (142%, p < 0.001), catalase (182%, p < 0.001) and glutathione peroxidase (325%, p < 0.001). However, chronological decreases were observed in the levels of reduced glutathione (69%, p < 0.001), in the GSH/GSSG ratio (78%) and in protein thiol groups (55%, p < 0.001), with concomitant increases in peroxides (155%, p < 0.001) and malondialdehyde (142%, p < 0.001) levels. DNA ploidy was also assayed by flow cytometry; a sharp increase in tetraploid (2.5-40.1%, p < 0.001) and octoploid (0.1-16.1%; p < 0.001) populations, and a noticeable decrease in diploid hepatocytes (92.9-34.3%; p < 0.001), were observed. Populations involved in 2C-->4C DNA synthesis decreased from 3.6 to 0.9% (p < 0.001), while those involved in 4C-->8C increased from 0.9% to 5.2% (p < 0.001). A hypodiploid population (apoptotic cells) was detected from 12 months, increasing thereafter. CONCLUSIONS These results show that the antioxidant cell defense system increases with age but the rate of reactive oxygen species generation exceeds the induced antioxidant ability, generating a situation that favors oxidative stress and peroxidation. The progressive polyploidization is accompanied by changes in the proliferative potential that decreases from 2C to 4C and increased from 4C to 8C. The relationship between the modifications of the oxidant/antioxidant system and increased polyploidy is not clear and may be interpreted as two independent manifestations of the aging process.

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.

Novel mechanism of Vitamin E protection against cyclosporine A cytotoxicity in cultured rat hepatocytes

Cyclosporine A (CsA) is the immunosuppressor most frequently used in transplant surgery and in the treatment of autoimmune diseases. It has been shown that CsA is able to generate reactive oxygen species and lipid peroxidation which are directly involved in the CsA hepatotoxicity. As antioxidant, Vitamin E (VitE) has been used to diminish the toxicity of CsA in vitro. Besides its direct action as the classical antioxidant implicated in preventing lipid peroxidation, we decided to investigate the effect of VitE on the endogenous antioxidant defense system, such as Mn and CuZn superoxide dismutase (MnSOD, CuZnSOD) catalase and glutathione peroxidase (GPx) on CsA cytotoxicity in primary cultures of rat hepatocytes. In cells incubated in the presence of CsA, there was an increase in the expression and activity of MnSOD and CuZnSOD but not in that of catalase and GPx. However, when hepatocytes were coincubated with CsA and VitE, an increase in the expression and activity in all antioxidant enzymes (MnSOD, CuZnSOD, catalase and GPx) was observed. In conclusion, we suggest (a) that the imbalance between SOD and catalase/GPx by the effect of CsA is the main mechanism responsible for peroxide accumulation and cell death in hepatocytes, and (b) that the presence of VitE in culture media reduces the oxidative stress through the inhibition of lipid peroxidation, but also through the increase of the expression and activity of catalase and GPx which allows the restoration of SOD and catalase/GPx coordination, indispensable for the correct cell defense against ROS.

Malic enzyme and glucose 6-phosphate dehydrogenase gene expression increases in rat liver cirrhogenesis.

The cirrhogenic ability of thioacetamide has been used to induce a model of chronic generalized liver disease that resembles the preneoplastic state of human fibrosis. Malic enzyme (ME) and glucose-6-phosphate dehydrogenase (G6PDH) are two cytosolic NADPH-generating enzymes; their activities significantly increased in liver when macronodular cirrhosis was induced by long-term thioacetamide administration to rats. The progressive increase in G6PDH and ME activities during the cirrhogenic process is parallel to the induction in gene expression of both enzymes detected by the increase in their mRNAs. These data indicate that NADPH-consuming mechanisms such as the microsomal oxidizing system and the maintenance of the cell redox state could be involved. A relationship between the extent of G6PD and ME gene expression and oxidative stress generated by the oxidative metabolism of thioacetamide is proposed as the hepatic concentration of malondialdehyde, a metabolite derived from lipid peroxidation, underwent a progressive and significant enhancement during thioacetamide-induced cirrhogenesis. These results led us to suggest that the enhanced activities of G6PDH and ME might be related to microsomal mechanisms of detoxification as well as to the maintenance of the cellular redox state. Furthermore, the noticeable increase in the hepatocyte population involved in DNA replication parallel to G6PDH activity suggests that G6PDH, through ribose-5-phosphate, might also be involved in the processes of DNA synthesis and repair.

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.

Necrogenic and regenerative responses of liver of newly weaned rats against a sublethal dose of thioacetamide

The hepatocellular necrogenic and regenerative responses of newly weaned rats (21 days old) to a sublethal dose of thioacetamide (6.6 mmol kg-1) were studied in comparison to adult (6-month old rats), in terms of liver injury, antioxidant defense systems and cell proliferation. Hepatocellular necrosis, detected by serum aspartate aminotransferase, was less severe in newly weaned rats than in adult animals and was parallel to previous changes in the activity of microsomal FAD monooxygenase system responsible for thioacetamide biotransformation. Liver damage in hepatocytes from newly weaned rats was also detected by the decreased levels of glutathione and protein thiol groups (47%, p < 0.001 and 52%, p < 0.001 vs. untreated, respectively) and by the enhanced malondialdehyde production (334%, p < 0.001) and glutathione S-transferase activity (384%, p < 0.001). No significant differences were detected in these values when compared to adults. Changes in cytosolic and mitochondrial superoxide dismutase and catalase activities in hepatocytes from newly weaned rats at 24 h, following thioacetamide (49%, p < 0.001; 50% and 53%, p < 0.001 vs. untreated, respectively), were less severe against those in adult hepatocytes at 48 h of intoxication, and the increases in glutathione peroxidase and glutathione reductase activities were significantly lowered: 25% (p < 0.001) and 41% (p < 0.001), respectively. Post-necrotic DNA synthesis in hepatocytes from newly weaned rats peaked at 48 h of intoxication, while in adults a more intense peak appeared at 72 h preceded by a sharp decrease in tetraploid population. These differences indicate that the lower necrogenic response against the same dose of thioacetamide in newly weaned rats may be due to the lower rate of thioacetamide biotransformation and to the earlier onset of cell division. Accordingly, the growing liver from newly weaned rats presents advantages against the necrogenic aggression of thioacetamide, first, because the diminished activity of its specific microsomal detoxification system, and second because the earlier increase in the proliferative response prevents the progression of injury permitting an earlier restoration of liver function.

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.

Intracellular calcium concentration impairment in hepatocytes from thioacetamide-treated rats. Implications for the activity of Ca2+-dependent enzymes

METHODS/RESULTS Thioacetamide induced a severe perivenous necrosis followed by a hepatocellular regenerative response, when administered in a single dose of 6.6 mmol/kg to rats. As (Ca2+)i plays an important role in both toxic cell killing and cell proliferation, the disturbances in the basal cytosolic calcium as well as the levels of Ca2+ sequestered in the endoplasmic reticulum were determined in hepatocytes isolated at 0, 12, 24, 48 and 72 h after thioacetamide administration. The basal Ca2+ increased progressively, reaching a maximum at 24 h of the intoxication (205%, p < 0.001), while the microsomal sequestered Ca2+ decreased at 24 h to 16% (p < 0.001) when compared with untreated controls. Changes in the activity of glycogen phosphorylase alpha paralleled those of basal free calcium and showed the maximum value also at 24 h (291%; p < 0.001). Moreover, there was a close association in time between the basal concentration of Ca2+ and the inhibition of microsomal Ca(2+)-dependent ATPase activity. CONCLUSIONS The significant decrease in the levels of GSH and protein thiols indicates that oxidative stress is involved in thioacetamide-induced cell injury, but these decreases did not precede changes in cytosolic Ca2+ level. In the sequence of events leading to hepatic cell injury and regeneration, thioacetamide mobilized hepatic (Ca2+)i via inhibition of microsomal Ca(2+)-ATPase which may have activated Ca(2+)-dependent mechanisms involved both in cell death and in acute mitogen response.

Involvement of nitric oxide synthesis in hepatic perturbations induced in rats by a necrogenic dose of thioacetamide

The biological actions of nitric oxide (NO), a highly diffusible and short‐lived radical, range from signal transduction to cytotoxicity. The present study investigated whether NO is released in the course of liver necrosis and regeneration induced by a single necrogenic dose of thioacetamide (6.6 mmol kg−1 body wt) to rats. Samples of liver were obtained at 0, 3, 12, 24, 48, 72 and 96 h after thioacetamide administration. Inducible nitric oxide synthase (iNOS) activity was determined in purified liver homogenates and a sharp 6 fold increase (P<0.001) in iNOS activity was recorded at 48 h of intoxication, followed by a slight but progressive increase at 72 and 96 h. Changes in the expression of iNOS, as detected by its mRNA levels, were parallel to the NOS enzyme activity. Hepatocyte NO synthesis showed a progressive increase at 24, 48 and 72 h, to 8 (P<0.001), 13 (P<0.001) and 13 (P<0.001) times the initial values, respectively. In isolated Kupffer cells, where initial NO release was ten fold higher than in hepatocytes, a progressive increase was detected from 48 h which reached two fold of initial at 72 h of intoxication (192%, P<0.001). Hepatic cyclic GMP concentration did not change significantly. However, mitochondrial aconitase activity decreased markedly at 12 and 24 h of intoxication showing a sharp increase towards normal values at 48 h which was maintained at 72 and 96 h. As protein kinase C (PKC) is one of the likely candidates to mediate iNOS expression, translocation (activation) of PKC was assayed in hepatocytes, and a significant two fold increase (P<0.001) between 48 and 96 h after thioacetamide intoxication was observed. When peritoneal macrophages from control rats were incubated with serum from thioacetamide‐treated rats, a sharp increase in NO release was detected with serum obtained at 48 h, reaching at 96 h a value four fold (P<0.001) that of the control. These results suggest that iNOS activity and NO release play a role in the pathophysiological mechanisms that trigger post‐necrotic hepatocellular regeneration following thioacetamide administration.