Nuria Sanz

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.

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.

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.

Variations of hepatic antioxidant systems and DNA ploidy in rats aged 2 to 8 months

Oxygen-reactive species are by-products of biological redox reactions and are involved in the development and aging processes. In order to test whether the time-dependent changes in the hepatic antioxidant defense are related to changes in DNA ploidy, we studied in rats, aged 2-8 months, the enzymes and metabolites related to the primary cell defense against oxidative stress, as well as the distribution of DNA into the cell cycle phases. Catalase and glutathione peroxidase, together with glutathione reductase and mitochondrial superoxide dismutase, underwent progressive and significant time course increases. Although no temporal changes were observed in the concentration of protein thiol groups and malondialdehyde in rats in the same age period, glutathione redox state, detected by the GSH/GSSG ratio decreased significantly to 41% (P<0.001) of the initial value. DNA content was assayed by flow cytometry in isolated hepatocytes, and changes in DNA ploidy and distribution in the cell cycle phases were determined. A sharp decrease in diploid population from rats aged 1-8 months (92.9% --> 11.1%) and a pronounced increase in hepatocyte polyploid populations in the same age period (2.6% --> 87.3%) were observed. However, liver cell population involved in S phase (DNA synthesis) was unchanged. These results indicate that the cell defense mechanisms against oxygen toxicity increased in liver of rats from 2-8 months in order to suppress the oxidative imbalance. During the 6-month period of a rats life (2-8 months), the significant alterations of GSH/GSSG ratio to a more oxidative state have no influence on the proliferating capacity of the cells.