Parameters related to oxygen free radicals in erythrocytes, plasma and epidermis of the hairless rat.

The following parameters related to oxygen free radicals (OFR) were determined in erythrocytes and the epidermis of hairless rats: catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), reduced (GSH) and oxidized (GSSG) glutathione, glutathione S-transferase (GST), superoxide dismutase (SOD) and thiobarbituric acid reactive substances (TBARS). GSH, GSSG and TBARS were also analyzed in plasma. In erythrocytes, the Pearson correlation coefficients (r) were significant (p < 0.001) between glutathione and other parameters as follows: GSH correlated negatively with GSSG (r = -0.665) and TBARS (r = -0.669); GSSG correlated positively with SOD (r = 0.709) and TBARS (r = 0.752). Plasma GSSG correlated negatively with erythrocytic thermostable GST activity (r = -0.608; p=0.001) and with erythrocytic total GST activity (r = -0.677; p < 0.001). In epidermis (p < 0.001 in all cases), GSH content correlated with GSSG (r = 0.682) and with GPx (r = 0.663); GSSG correlated with GPx (r = 0.731) and with GR (r = 0.794). By multiple linear regression analysis some predictor variables (R(2)) were found: in erythrocytes, thermostable GST was predicted by total GST activity and GSSG, GSSG content was predicted by GSH and by the GSH/GSSG ratio and GPx activity was predicted by GST, CAT and SOD activities; in epidermis, GSSG was predicted by GR and SOD activities and GR was predicted by GSSG, TBARS and GPx. It is concluded that the hairless rat is a good model for studying OFR-related parameters simultaneously in blood and skin, and that it may provide valuable information about other animals under oxidative stress.

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.

Urinary levels of metallothioneins and metals in subjects from a semiindustrialized area in Tarragona Province of Spain.

The monitoring of heavy metals is important if adverse effects on health are to be avoided. In humans, metallothionein (MT) has been used as a biomonitor for the assessment of cadmium (Cd). In the present study, subjects drawn from the population of Tarragona Province (NE Spain) were investigated. Urinary MT, zinc (Zn), and copper (Cu) concentrations, corrected for creatine concentrations, were determined in 625 samples from healthy subjects aged between 10 and 65 yr. Mean values of MT and Cu in females were higher than those in males, with levels of 29.5 (23.8) vs. 22.7 (24.9) micrograms MT/creatinine (p < 0.001) and 4.8 (6.1) vs 3.4 (4.9) micrograms Cu/g creatinine (p < 0.001). No differences between males and females were observed with respect to urinary Zn: 78.0 (66.4) vs 73.0 (85.5) micrograms/g creatinine, respectively (p = 0.332). Significantly higher MT, Zn, and Cu values were observed in the females aged 15-19 yr and, in the age group of 50-54 yr, only in the Zn and Cu values, when compared with those in males. Significant positive correlations of MT vs Zn and Cu as well as correlations of Zn vs Cu levels were observed in both genders. The present findings confirm the proposed role of MT as a biomonitor of mineral status.

Metallothionein expression in human lung and its varying levels after lung transplantation. Toulouse Lung Transplantation Group.

The aim of this study was to determine the lung levels of metallothionein (MT), a free radical scavenger, because oxygen-derivated free radicals (ODFRs) have been involved both in reperfusion injury of transplanted lungs and in cardiac or renal allograft destruction. First, MT localization was evaluated in 14 normal human lung biopsy specimens. Then, in lung transplant recipients, MT content in BAL fluid (BALF) and its transcription rate in alveolar macrophages (AMs) were determined. The BALFs of 69 patients were separated into six groups: lung transplant recipients in clinically stable condition (CSR), those with acute rejection (AR), asymptomatic cytomegalovirus infection (ACMV), cytomegalovirus pneumonitis (CMVP), bronchiolitis obliterans syndrome (BOS), and patients without transplants who served as control subjects (NTCs). In normal lungs, 83% of AMs were positively stained. MT staining was also observed in pleural endothelial cells and basal cells from bronchial epithelium. In lung transplant recipients, MT levels in BALF were significantly higher in patients with CSR, AR, ACMV, and CMVP compared with NTCs, while during BOS, MT had a significantly lower level compared with other lung transplant groups. However, no difference among groups was found concerning MT-II messenger RNA expression in AMs, showing that, as in normal lung, AMs are not the only cells that produce MT. These data report for the first time to our knowledge MT cell distribution in human lung with specific emphasis on its enhanced levels after lung transplantation, even in the absence of complication. Possible correlation among MT levels, ODFRs, cytokine levels, and corticosteroid treatment during complications of lung transplantation are discussed.

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.

Oral meso-2,3-dimercaptosuccinic acid in pregnant Sprague-Dawley rats: teratogenicity and alterations in mineral metabolism. I. Teratological evaluation.

meso-2,3-Dimercaptosuccinic acid (DMSA), an effective antagonist for the treatment of lead, arsenic, mercury, and cadmium poisoning, was evaluated for developmental toxicity in pregnant Sprague-Dawley rats. DMSA was administered by gavage on d 6-15 of gestation at doses of 0, 100, 300, or 1000 mg DMSA/kg/d. At termination on d 20 of gestation, fetuses were examined for external, visceral, and skeletal malformations and variations. Maternal toxicity was observed at all doses, as evidenced by a significant decrease in body weight gain. There were no effects with respect to hematology or clinical chemistry. Increased early resorptions, increased percentage postimplantation loss, and reduced fetal body weight per litter were observed at 100, 300, and 1000 mg/kg/d. Examination of fetuses for gross external abnormalities, visceral and skeletal malformations, or ossification variations revealed that DMSA did not produce teratogenicity at any dosage level. However, significant fetotoxicity was observed at 100, 300, and 1000 mg/kg/d. The no-observable-effect level (NOEL) for maternal and developmental toxicity was less than 100 mg DMSA/kg/d.

Oral meso-2,3-dimercaptosuccinic acid in pregnant Sprague-Dawley rats: teratogenicity and alterations in mineral metabolism. II. Effect on mineral metabolism.

The effect of meso-2,3-dimercaptosuccinic acid (DMSA) on mineral metabolism was investigated in pregnant Sprague-Dawley rats. Animals were given by gavage doses of 0, 100, 300, or 1000 mg DMSA/kg/d on gestational d 6-15. On d 20 of gestation dams were killed and fetuses were removed from the uterus. The levels of calcium, magnesium, zinc, copper, and iron were measured in maternal liver, kidney, and intestine, as well as in whole fetus and in fetal liver. Mineral analysis of maternal and fetal tissues revealed pronounced effects of DMSA on mineral metabolism. The results of this investigation indicate a strong possibility that the negative effects of the drug on pregnancy are due in part to the changes in mineral metabolism.

Developmental toxicity of vanadium in mice after oral administration.

Vanadium, as vanadyl sulphate pentahydrate, was evaluated for its embryotoxic, fetotoxic and teratogenic potential in Swiss mice. The compound was administered by gavage to pregnant mice at doses of 0, 37.5, 75 or 150 mg kg-1 day-1 on days 6-15 of pregnancy. On gestation day 18, all live fetuses were examined for external, visceral and skeletal malformations and variations. Maternal toxicity was observed in the vanadium-treated animals, as evidenced by reduced weight gain, reduced body weight on gestation day 18 (corrected for gravid uterine weight) and decreased absolute liver and kidney weights at 75 and 150 mg kg-1 day-1. The number of total implants, live and dead fetuses, late resorptions, the sex ratio and the post-implantation losses were not significantly different between the vanadium-treated mice and the controls. However, there was a significant increase in the number of early resorptions per litter at all dose levels. Fetotoxicity was evidenced by lower fetal weights and fetal lengths, and the presence of developmental variations. Malformation incidence also was increased by the administration of vanadium. Thus, the 'no observable effect level' (NOEL) for maternal toxicity, embryofetotoxicity and teratogenicity for vanadyl sulphate pentahydrate under these test conditions was below 37.5 mg kg-1 day-1 for Swiss mice.

Evaluation of the developmental toxicity of 2,3-dimercapto-1-propanesulfonate (DMPS) in mice. Effect on mineral metabolism.

The sodium salt of 2,3-dimercapto-1-propanesulfonic acid (DMPS), a potent chelating agent used in the treatment of inorganic and organic heavy metal intoxications was evaluated for developmental toxicity in pregnant Swiss mice. DMPS was administered by gavage at doses of 0, 75, 150 and 300 mg/kg per day on gestational days 6-15. Females were evaluated for body weight gain, food consumption, appearance and behavior, survival rates and reproduction data. Cesarean sections were performed on gestation day 18. There were no maternal toxic effects, and no treatment-related changes were recorded in the number of total implants, resorption, the number of live and dead fetuses, fetal body weight or fetal sex distribution data. Gross external, soft tissue and skeletal examination of the DMPS-treated fetuses did not show significant differences at any dose in comparison with the controls. Mineral analysis of maternal and fetal tissues revealed slight effects of DMPS on metabolism of calcium, magnesium, zinc, copper and iron. The results of this study in mice indicate that DMPS is not a developmental toxicant at levels up to 300 mg/kg per day.

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.