The antioxidant and hepatoprotective effects of zinc are related to hepatic cytochrome P450 depression and metallothionein induction in rats with experimental cirrhosis.

The aim of the present study was to investigate the time-course of changes in hepatic lipid peroxidation, cytochrome P450 and metallothionein concentrations, and superoxide dismutase and catalase activities in relation to the onset and development of cirrhosis in CCl4-treated rats. Further, the effects of oral zinc administration on these parameters were assessed. Cirrhosis was induced in 120 rats by intraperitoneal injections of CCl4 twice weekly over 9 weeks. Controls were 120 additional animals. Both groups were further subdivided to receive either a standard diet or one supplemented with zinc. Subsets of 10 animals each were euthanized at weeks 1, 2, 3, 5, 7 and 9 from the start of the study. Results indicated that zinc administration delayed the cirrhotic process and the increase in lipid peroxidation. These changes, consistently maintained during the first 5 weeks of the study, were associated with a significant decrease in the hepatic concentration of cytochrome P450 and an increase in the hepatic concentration of metallothioneins. Zinc supplementation did not produce any significant change in superoxide dismutase and catalase activities. These results suggest that cytochrome P450 and metallothioneins may play an important role in the hepato-protective effects of zinc against lipid peroxidation in experimental cirrhosis.

Time-course of changes in hepatic lipid peroxidation and glutathione metabolism in rats with carbon tetrachloride-induced cirrhosis.

1. The aims of the present study were to assess: (i) the temporal relationships between hepatic lipid peroxidation, changes in the glutathione detoxification system and the onset/development of cirrhosis in CCl4-treated rats; and (ii) the effects of oral zinc administration on these parameters. 2. Cirrhosis was induced in 120 rats by intraperitoneal injections of CCl4 twice a week over 9 weeks. One hundred and twenty additional animals were used as controls. Both groups were further subdivided to receive either a standard diet or one supplemented with zinc. Subsets of 10 animals each were killed at weeks 1, 2, 3, 5, 7 and 9 from the start of the study. 3. Induction of cirrhosis produced a decrease in the components of the hepatic glutathione anti-oxidant system: glutathione transferase activity decreased from week 1, the concentration of reduced glutathione (GSH) decreased from week 5 and glutathione peroxidase (GPx) activity decreased from week 7. This impairment was chronologically related to an increase in free radical generation. Hepatic lipid peroxidation was significantly correlated with GPx activity (r = -0.47; P < 0.001) in CCl4-treated rats. Zinc administration did not produce any significant improvement of the hepatic glutathione system. 4. In conclusion, cirrhosis induction in rats by CCl4 administration produced a decrease in the hepatic glutathione antioxidant system that was related to an increase in free radical production. Furthermore, zinc supplementation produced a reduction in the degree of hepatic injury and a normalization of lipid peroxidation, but not an improvement of the hepatic GSH anti-oxidant system.

Hepatic production of apolar aldehydes in rats with carbon tetrachloride-induced cirrhosis.

The aim of this study was to identify apolar aldehydes in liver homogenates from rats with CCl4-induced cirrhosis and, as a corollary, the antioxidant effect of zinc administration. The study was performed in five control rats and in ten cirrhotic rats which were further sub-divided into two groups to receive either a standard diet or one supplemented with zinc. The percentage of hepatic fibrosis, plasma malondialdehyde concentration and alanine aminotransferase activity were measured as well as the following aldehydes: hexanal, octanal, decanal, 2-hexenal, 2-octenal, 2-nonenal, 2,4-heptadienal and 2,4-decadienal. Of the 10 cirrhotic rats, 4 had elevated concentrations of the highly toxic 2,4-dialkenals which coincided with a higher percentage of fibrosis and plasma alanine aminotransferase activity. These aldehydes were not observed in the control group. Zinc administration was associated with a reduction of the hepatic malondialdehyde concentration and an amelioration on the degree of hepatic injury. In conclusion, this study demonstrates the presence of the highly toxic 2,4-dialkenals in hepatic tissue of rats whith CCl4-induced cirrhosis. Results obtained would suggest that these particular aldehydes may be related to the severity of the hepatic injury.

Inhibition of hepatic cell nuclear DNA fragmentation by zinc in carbon tetrachloride-treated rats.

BACKGROUND/AIMS: The aims of this study were to ascertain: 1) whether hepatic cell DNA fragmentation is increased in rats with early stages of liver disease induced by carbon tetrachloride; 2) whether the inhibition of DNA cleavage is involved in the hepatoprotective effects of zinc; and 3) if relationships exist between DNA fragmentation and the onset of fibrosis in this experimental model. METHODS: Twenty-one treated rats and 23 controls were divided into two groups to receive either a standard diet or one supplemented with zinc. All the animals were sacrificed 1 week later for histological and biochemical assessments, which included a DNA fragmentation index, hepatic zinc and metallothionein concentrations, fibrosis measured by hepatic hydroxyproline concentration and plasma alanine aminotransferase activity. RESULTS: Hepatic cell DNA fragmentation was increased in rats with early hepatic fibrosis and the increase was independent of hepatocytolysis, as measured by alanine aminotransferase activity. Oral zinc administration inhibited hepatic cell DNA fragmentation in the treated rats and was proportional to the hepatic concentration of the metal. The mechanism of the zinc-mediated decrease in DNA cleavage was related to an increase in the hepatic metallothionein concentration. Hepatic cell DNA fragmentation was related to hydroxyproline concentration. CONCLUSIONS: Our results suggest that apoptosis may be involved in the early transformations occurring in the liver and which can lead to the initiation of cirrhosis. As such, the potential therapeutic use of zinc supplementation would warrant further investigation.

Influence of zinc intake on hepatic lipid peroxidation and metallothioneins in alcoholic rats: relationship to collagen synthesis.

Hepatic lipid peroxidation, metallothioneins, collagen and proline hydroxylase activity were investigated in 16 ethanol-fed rats and in 16 control animals. The rats were further divided into three groups to receive either a standard diet, a zinc-deficient diet or a zinc-supplemented diet. The animals were sacrificed at week 12 of the experiment for histological and biochemical assessments. Hepatic tissue examination indicated that oral zinc supplementation was associated with a decrease in lipid peroxidation, collagen deposition and proline hydroxylase activity together with an increase in metallothionein concentration in alcoholic rats. There were no significant differences in lipid peroxidation in the control group in relation to the diet. Zinc supplementation was associated with increased concentrations of hepatic metallothioneins together with decreased concentrations of proline-hydroxylase and collagen but to a lesser degree than in alcoholic animals. These results indicate that zinc is an efficient hepato-protective agent against lipid peroxidation in alcoholic rats and its effect may be, in part, mediated by the activation of metallothionein synthesis. Also, lipid peroxidation may be related to changes in hepatic collagen synthesis.

Treatment of acute lead intoxication. A quantitative comparison of a number of chelating agents.

The efficacy of several chelating agents in alleviating acute lead intoxication has been investigated in male Swiss mice. The relative effectiveness of diethylenetriaminepentaacetic acid (DTPA), ethyleneglycolbis-(beta-amino-ethylether)-N,N'-tetraacetic acid (EGTA), cyclohexanediaminetetraacetic acid (CDTA), L-cysteine, N-acetyl-L-cysteine (NAC), ascorbic acid, sodium diethyldithiocarbamate (DDC), 2,3-dimercaptosuccinic acid (DMSA) and sodium 2,3-dimercapto-1-propanesulfonate (DMPS) in reducing lethality of lead was examined. Significant increases in survival were noted with CDTA, ascorbic acid, DMSA, and DMPS. Therapeutic effectiveness (TEF) was determined for these compounds; TEF for ethylenediaminetetraacetic acid (EDTA) and for 2,3-dimercaptopropanol (BAL) was also determined; CDTA (2.33) and EDTA (1.73) showed the highest values. In subsequent experiments, the effect of the chelating agents on the distribution and excretion of lead was investigated. Lead acetate trihydrate was administered subcutaneously at doses of 37.8 mmol/kg (LD50), and fifteen minutes later, chelators were given intraperitoneally at doses approximately equal to one-fourth of their respective LD50 values. EDTA, DTPA and CDTA were the most effective agents in increasing the urinary excretion of lead, whereas DTPA, CDTA, and DDC increased significantly the fecal excretion of lead. EDTA, DDC, and CDTA were the most effective chelators in reducing the concentration of lead found in various tissues. On the basis of these results, CDTA may be considered as an alternative in the treatment of acute lead poisoning.

Evaluation of the perinatal and postnatal effects of uranium in mice upon oral administration.

Perinatal and postnatal studies were performed in Swiss mice given uranium--as uranyl acetate dihydrate--at daily dosages of 0, 0.05, 0.5, 5, and 50 mg/kg from day 13 of pregnancy until weaning of the litters on day 21 post-birth. Postnatal development was monitored after 0, 4, and 21 d of lactation. At doses of 0, 0.05, 0.5, and 5 mg/kg.d, treatment with uranium had no significant effect on sex ratios, mean litter size, pup body weight, or pup body length throughout lactation. Significant decreases in the mean litter size on postnatal day 21, and in the viability and lactation indices were observed at the 50 mg/kg.d dose level. When comparing the "no observable effect level" (NOEL) for reproductive effects of uranium, with the concentrations of the metal usually ingested by men, a safety factor below 1,000 can be estimated.

The effects of uranium on reproduction, gestation, and postnatal survival in mice.

Uranyl acetate dihydrate was tested for its effects on reproduction, gestation, and postnatal survival in Swiss mice. Four groups of animals, each of which consisted of 25 males and 25 females, were administered 0, 5, 10, and 25 mg/kg/day of uranyl acetate dihydrate. Mature male mice were treated orally for 60 days prior to mating with mature virgin female mice treated orally for 14 days prior to mating. Treatment of the females continued throughout mating, gestation, parturition, and nursing of the litters. One-half of the dams in each group were sacrificed on Day 13 of gestation and the remaining dams were allowed to deliver and wean their offspring. Postnatal development was monitored after 0, 4, and 21 days of lactation. No adverse effects on fertility were evident at the doses employed in this study. Nevertheless, embryolethality could be observed in the 25 mg/kg/day group. Significant increases in the number of dead young per litter were seen at birth and at Day 4 of lactation in the 25 mg/kg/day group. The growth of the offspring was always significantly lower for the uranium-treated animals. However, the present results suggest that uranium does not cause any adverse effects on fertility, general reproductive parameters, or offspring survival at the concentrations usually ingested by man.

The effects of repeated parenteral administration of chelating agents on the distribution and excretion of uranium.

The effects of repeated ip administration of gallic acid, 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron), diethylenetriaminepentaacetic acid (DTPA), and 5-aminosalicylic acid (5-AS) on the distribution and excretion of uranium were assessed in male Swiss mice. Only Tiron significantly increased the amount of uranium excreted into urine and feces. A significant decrease in the concentration of uranium in liver, spleen and bone was observed after administration of Tiron, whereas injection of gallic acid or DTPA resulted in a significant decrease in the concentration of the metal in the liver. The results show that Tiron was consistently the most effective chelator of those tested in the treatment of uranium poisoning after repeated daily administration of the metal.

The developmental toxicity of uranium in mice.

To evaluate the developmental toxicity of uranium, 5 groups of 20 pregnant Swiss mice were given by gavage daily doses of 0, 5, 10, 25 and 50 mg/kg of uranyl acetate dihydrate on gestational days 6-15. Cesarean sections were performed on all females on gestation day 18. Fetuses were examined for external, visceral, and skeletal abnormalities. The results indicated that such exposure resulted in maternal toxicity as evidenced by reduced weight gain and food consumption during treatment, and increased relative liver weight. There were no treatment-related effects on the number of implantation sites per dam, or on the incidence of postimplantation loss (resorptions plus dead fetuses). The number of live fetuses per litter and the fetal sex ratio were not affected by the treatment. However, dose-related fetal toxicity, consisting primarily of reduced fetal body weight and body length, and an increased incidence of abnormalities was observed. Malformations (cleft palate, bipartite sternebrae) and developmental variations (reduced ossification and unossified skeletal variations) were noted at the 25 and 50 mg/kg per day test levels. Therefore, administration of uranyl acetate dihydrate during organogenesis in mice produced maternal toxicity at 5, 10, 25 and 50 mg/kg per day. The "no observable effect level" (NOEL) for fetotoxicity including teratogenicity was below 5 mg/kg per day, as some anomalies were observed at this dose. There was no evidence of embryolethality at any dosage level used in this study.

Effectiveness of chelation therapy with time after acute vanadium intoxication.

The effect of increasing the time interval between vanadium exposure and chelation therapy was studied in male Swiss mice. The following chelating or reducing agents were administered i.p. at 0, 0.5, 2 and 8 h after i.p. administration of 0.16 mmol kg-1 sodium metavanadate: ascorbic acid, deferoxamine mesylate (DFOA) and 4,5-dihydroxy-1,3-benzene-disulphonic acid (Tiron). These agents were given at doses equal to one-quarter of their respective LD50 values. Daily elimination of vanadium into urine and faeces was determined for four days. The excretion of vanadium was especially rapid in the first 24 h. Treatment with Tiron increased significantly the urinary elimination of vanadium in all four groups during Day 1, whereas DFOA significantly increased the faecal excretion during the same period. Treatment with DFOA or Tiron resulted in a significant decrease in the concentration of vanadium in the kidney four days after sodium metavanadate administration. The magnitude of the increased elimination of vanadium, as well as the decreased tissue concentration of the metal, was remarkably attenuated by increasing the time interval between vanadium injection and administration of the chelators.

Acute zinc intoxication: comparison of the antidotal efficacy of several chelating agents.

Four zinc compounds (acetate, nitrate, chloride and sulfate) were administered po or ip to rats and mice. The LD50 values were determined. Animals were observed for 14 days. The majority of deaths occurred during the first 48 hr. The clinical and physical signs appearing after intoxication included miosis, conjunctivitis, decreased food and water consumption and hemorrhages and hematomas in the tail. These changes decreased with time which would suggest a quick elimination of zinc. To determine the effect of 6 chelating agents on the toxicity of zinc, various doses of zinc acetate (66-330 mg/kg) were given ip to male mice followed by the injection of one of the chelators. DTPA, D-PA, CDTA and EDTA were the most effective. CDTA and DTPA were also the most effective in increasing the urinary excretion of zinc. DTPA appears to be the most effective agent of those tested in the prevention of acute zinc intoxication. However, CDTA may be considered as a possible alternative.