Effect of anisotonic cell-volume modulation on glutathione-S-conjugate release, t-butylhydroperoxide metabolism and the pentose-phosphate shunt in perfused rat liver.

1. Addition of 1-chloro-2,4-dinitrobenzene to isolated perfused rat liver results in the rapid formation of its glutathione-S-conjugate [S-(2,4-dinitrophenyl)glutathione], which is released into both, bile and effluent perfusate. Anisotonic perfusion did not affect total S-conjugate formation, but release of the S-conjugate into the perfusate was increased (decreased) following hypertonic (hypotonic) exposure at the expense of excretion into bile. Stimulation of S-conjugate release into the perfusate following hypertonic exposure paralleled the time course of volume-regulatory net K+ uptake. 2. Basal steady-state release of oxidized glutathione (GSSG) into bile was 1.30 +/- 0.12 nmol.g-1.min-1 (n = 18) during normotonic (305 mOsmol/l) perfusion and was 3.8 +/- 0.3 nmol.g-1.min-1 in the presence of t-butylhydroperoxide (50 mumol/l). Hypotonic exposure (225 mOsmol/1) lowered both, basal and t-butylhydroperoxide (50 mumol/l)-stimulated GSSG release into bile by 35% and 20%, respectively, whereas hypertonic exposure (385 mOsmol/l) increased. Anisotonic exposure was without effect on t-butylhydroperoxide removal by the liver. GSSG release into bile also decreased by 33% upon liver-cell swelling due to addition of glutamine plus glycine (2 mmol/l, each). 3. Hypotonic exposure led to a persistent stimulation 14CO2 production from [1-14C]glucose by about 80%, whereas 14CO2 production from [6-14C]glucose increased by only 10%. Conversely, hypertonic exposure inhibited 14CO2 production from [1-14C]glucose by about 40%, whereas 14CO2 production from [6-14C]glucose was unaffected. The effect of anisotonicity on 14CO2 production from [1-14C]glucose was also observed in presence of t-butylhydroperoxide (50 mumol/l), which increased 14CO2 production from [1-14C]glucose by about 40%. 4. t-Butylhydroperoxide (50 mumol/l) was without significant effect on volume-regulatory K+ fluxes following exposure to hypotonic (225 mOsmol/l) or hypertonic (385 mOsmol/l) perfusate. Lactate dehydrogenase release from perfused rat liver under the influence of t-butylhydroperoxide was increased by hypertonic exposure compared to hypotonic perfusions. 5. The data suggest that hypotonic cell swelling stimulates flux through the pentose-phosphate pathway and diminishes loss of GSSG under conditions of mild oxidative stress. Hypotonically swollen cells are less prone to hydroperoxide-induced lactate dehydrogenase release than hypertonically shrunken cells. Hypertonic cell shrinkage stimulates the excretion of glutathione-S-conjugates into the sinusoidal circulation at the expense of biliary secretion.     

Age-related changes of antioxidant enzyme activities, glutathione status and lipid peroxidation in rat erythrocytes after heat stress

The aim of this study was to determine whether exposure to heat stress would lead to oxidative stress and whether this effect varied with different exposure periods. We kept 1-, 6- and 12-month-old male Wistar rats at an ambient temperature of either 22 degrees C or 40 degrees C for 3 and 7 days and measured glucose-6-phosphate dehydrogenase (G-6-PD), Cu,Zn-superoxide dismutase (Cu,Zn-SOD), catalase (CAT), selenium-dependent glutathione peroxidase (Se-GSH-Px) and glutathione-S-transferase (GST) activities and levels of thiobarbituric acid-reactive substances (TBARS), reduced glutathione (GSH) and oxidized glutathione (GSSG) in erythrocytes and determined GSH/GSSG ratio, total glutathione and the redox index. G-6-PD and CAT activities were found to be significantly increased in 1- and 6-month-old rats after 3 and 7 days of heat stress, but G-6-PD activities decreased in 12-month-old rats. Cu, Zn-SOD activity decreased in 1-month-old rats after heat stress, whereas it increased in 6- and 12-month-old rats. GST activity increased in all groups. GSH and total GSH levels and GSH/GSSG ratios decreased in 1- and 6-month-old rats but they increased in 12-month-old rats after heat stress. GSSG levels increased in 1- and 6-month-old rats but decreased in 12-month-old rats after heat stress. TBARS levels increased in all groups. Seven days of stress is more effective in altering enzyme activities and levels of GSH, GSSG and TBARS. When the effects of both heat stress and aging were examined together, it was interesting to note that they mostly influenced G-6-PD activity.     

Lipid peroxidation and activity of antioxidant enzymes in diabetic rats

We hypothesized that oxygen free radicals (OFRs) may be involved in pathogenesis of diabetic complications. We therefore investigated the levels of lipid peroxidation by measuring thiobarbituric acid reactive substances (TBARS) and activity of antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT)] in tissues and blood of streptozotocin (STZ)-induced diabetic rats. The animals were divided into two groups: control and diabetic. After 10 weeks (wks) of diabetes the animals were sacrificed and liver, heart, pancreas, kidney and blood were collected for measurement of various biochemical parameters. Diabetes was associated with a significant increase in TBARS in pancreas, heart and blood. The activity of CAT increased in liver, heart and blood but decreased in kidney. GSH-Px activity increased in pancreas and kidney while SOD activity increased in liver, heart and pancreas. Our findings suggest that oxidative stress occurs in diabetic state and that oxidative damage to tissues may be a contributory factor in complications associated with diabetes.     

Inhibition of biliary taurocholate excretion during menadione metabolism in perfused rat liver

In perfused rat liver menadione elicits substantial oxidation in both the NADPH and GSH redox systems. Biliary excretion of GSSG is increased several-fold. Menadione derivatives appear in the bile predominantly as the menadione-S-glutathione conjugate, thiodione (60%), or as conjugates derived therefrom (17%). About 10% appear as menadione glucuronides. The excretion of taurocholate into bile is strongly inhibited upon menadione infusion. The inhibition of taurocholate excretion is small in livers with a low content of Se-GSH-peroxidase and in glutathione-depleted livers. In these livers intracellular GSSG and biliary GSSG release remain at low values, although menadione still imposes oxidative stress as indicated by an oxidation of intracellular NADPH. Under anoxic conditions menadione has little influence on both the NADPH and GSH redox systems and also on biliary taurocholate excretion. The amount of thiodione released into bile is similar to that found under normoxia, whereas the amount of glucuronidated products almost doubled. We conclude (a) that intracellular formation of GSSG by menadione occurs via the generation of hydrogen peroxide; (b) that the inhibition of biliary taurocholate excretion by menadione is related to the increased formation of glutathione disulfide; and (c) that menadione derivatives show little, if any, contribution to the inhibition of taurocholate excretion.     

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.     

Formation of activated oxygen in the hypoxic rat liver

The biliary GSSG efflux rate of normoxic perfused rat liver was 1.5 +/- 0.2 nmol/min/g liver wet weight. The GSSG efflux rate as indicator for the flux through the glutathione peroxidase reaction and, therefore, for an oxidative loading increased with the extent of hypoxia. 2.6 +/- 0.5 nmol/min/g were released from the severely hypoxic liver. The hydroxyl radical scavenger formate as well as the xanthine oxidase inhibitor allopurinol reduced the efflux rate of GSSG. GSH was released from the perfused liver at a rate of 15.5 nmol/min/g which was nearly unchanged in severe hypoxia. The high rate of glucose liberation from the hypoxic liver declined to almost that of the normoxic organ in the presence of formate. There is an 'oxidative stress' during hypoxic liver perfusion which probably originates from increased generation of activated oxygen species in the degradation of purine nucleotides.     

Liver and biliary levels of glutathione and thiobarbituric acid reactants after acute lindane intoxication

The i.p. administration of 60 mg kg-1 body weight of lindane, the gamma-isomer of hexachlorocyclohexane, to fed rats led to an enhancement of hepatic lipid peroxidation after 24 h of treatment. This was evidenced by significant increases in the hepatic production and biliary release of thiobarbituric acid reactive substances, and in the biliary release of glutathione disulphide. Under these conditions, the content of cytochrome P450 was enhanced concomitantly with increases in the total microsomal oxygen uptake, superoxide radical generation and (+)-catechin (cyanid-3-ol) sensitive respiration. The glutathione status of hepatocytes was altered by lindane as the content and biliary release of glutathione disulphide was drastically augmented, leading to a decrease in the cellular and biliary GSH/GSSG ratios. It is suggested that lindane treatment leads to an induced oxidative capacity, which, in turn, alters the glutathione status of the liver tissue.     

Ameliorative potential of S-allyl cysteine on oxidative stress in STZ induced diabetic rats

Increased oxidative stress and impaired antioxidant defense mechanism are important factors in the pathogenesis and progression of diabetes mellitus and other oxidant-related diseases. The present study was undertaken to evaluate the possible protective effects of S-allyl cysteine (SAC) against oxidative stress in streptozotocin (STZ) induced diabetic rats. SAC was administered orally for 45 days to control and STZ induced diabetic rats. The effects of SAC on glucose, plasma insulin, thiobarbituric acid reactive substances (TBARS), hydroperoxide, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), oxidized glutathione (GSSG) and GSH/GSSG ratio were studied. The levels of glucose, TBARS, hydroperoxide, and GSSG were increased significantly whereas the levels of plasma insulin, reduced glutathione, GSH/GSSG ratio, superoxide dismutase, catalase and GPx were decreased in STZ induced diabetic rats. Administration of SAC to diabetic rats showed a decrease in plasma glucose, TBARS, hydroperoxide and GSSG. In addition, the levels of plasma insulin, superoxide dismutase, catalase, GPx and reduced glutathione (GSH) were increased in SAC treated diabetic rats. The above findings were supported by histological observations of the liver and kidney. The antioxidant effect of SAC was compared with glyclazide, a well-known antioxidant and antihyperglycemic drug. The present study indicates that the SAC possesses a significant favorable effect on antioxidant defense system in addition to its antidiabetic effect.     

Oxidative Stress in the Heart of Rats Infected with Trypanosoma evansi

This study was conducted to investigate the occurrence of oxidative stress in the heart tissue of rats infected with Trypanosoma evansi. Rats were divided into 2 groups (A and B) with 12 animals each, and further subdivided into 4 subgroups (A1 and A2, 6 animals/each; and B1 and B2, 6 animals/each). Animals in the groups B1 and B2 were subcutaneously inoculated with T. evansi. Thiobarbituric acid reactive substances (TBARS), superoxide dismutase activity (SOD), glutathione S-transferase activity (GST), reduced glutathione activity (GSH), and non-protein thiols (NPSH) in the heart tissue were evaluated. At day 5 and 15 post-infection (PI), an increase in the TBARS levels and a decrease in the SOD activity (P<0.05) were observed. GSH and GST activities were decreased in infected animals at day 15 PI (P<0.05). Considering the proper functioning of the heart, it is possible that the changes in the activity of these enzymes involved in the oxidative stress may be related, at least in part, in the pathophysiology of rats infected with T. evansi.     

Factors influencing the inhibition of biliary glutathione efflux induced by biliary obstruction

The aim of this study was to investigate mechanisms responsible for the inhibition of biliary glutathione efflux in rats with secondary biliary cirrhosis. Rats were studied after bile duct obstruction for 28 days. The biliary secretion of reduced glutathione (GSH), oxidised glutathione (GSSG) and cysteine were completely inhibited in biliary obstructed rats. Hepatic gamma glutamyltranspeptidase (gamma-GT) activity increased significantly, but following its inhibition by acivicin administration GSH, GSSG and cysteine were still absent in bile. Biliary obstruction resulted in a significant increase of the permeability of the paracellular pathway, as shown by the higher bile/plasma ratio and hepatic clearance of [14C]sucrose. GSH and GSSG were, however, significantly lower in the carotid artery and hepatic vein of obstructed animals and the arteriovenous difference across the liver was reduced. The concentration of GSH was significantly reduced and that of GSSG increased in the liver of obstructed rats. Biliary obstruction induced an increase in the hepatic concentration of cysteine and an inhibition of both gamma glutamylcysteine synthetase and methionine adenosyl transferase activities. Dichlorofluorescein (DCF) and the GSSG/GSH ratio and thiobarbituric acid reactive substances (TBARS) concentration, markers of reactive oxygen species production and lipid peroxidation, respectively, were significantly increased. Our data indicate that increased degradation or blood reflux of glutathione do not participate in the disruption of its secretion into bile and support the view that impairment of glutathione synthesis and oxidative stress could contribute to the decline in biliary glutathione output.     

Rat lung glutathione release: response to oxidative stress and selenium deficiency

We performed experiments to characterize the glutathione-dependent metabolism occurring during tert-butyl hydroperoxide infusion in isolated perfused rat lungs and to examine the effect of selenium deficiency on this metabolism. Selenium deficiency resulted in decreased lung glutathione peroxidase activity but normal glutathione reductase activity and glutathione content. Infusion of the hydroperoxide into control lungs caused a proportional increase in tissue glutathione disulfide (GSSG) concentration and release of GSSG into the perfusate up to an infusion rate of 250 nmol of tert-butyl hydroperoxide X min-1 X 100 g body wt-1. Infusion rates greater than this resulted in continued rise of tissue GSSG concentrations but GSSG release into the perfusate plateaued. Infusion of tert-butyl hydroperoxide into selenium-deficient rat lungs resulted in much lower concentrations of tissue GSSG and GSSG release into the perfusate; however, release in the selenium-deficient rat lung was also found to be saturable at infusion rates of 450 nmol of tert-butyl hydroperoxide X min-1 X 100 g of body wt-1. Selenium deficiency in the rat decreases the rate of reduction of infused tert-butyl hydroperoxide by glutathione and may predispose the lung to free radical damage.     

Effects of aging on the susceptibility to the toxic effects of cyclosporin A in rats. Changes in liver glutathione and antioxidant enzymes

Free radicals are involved in aging and cyclosporin A-induced toxicity. The age-related changes in the liver oxidative status of glutathione, lipid peroxidation, and the activity of the enzymatic antioxidant defense system, as well as the influence of aging on the susceptibility to the hepatotoxic effects of cyclosporin (CyA) were investigated in rats of different ages (1, 2, 4, and 24 months). The hepatic content of reduced glutathione (GSH) increased with aging, peaked at 4 months, and decreased in senescent rats. By contrast, glutathione disulfide (GSSG) and thiobarbituric acid-reactive substances (TBARS) concentrations and superoxide dismutase, catalase, and glutathione peroxidase activities were higher in the oldest than in the youngest rats. CyA treatment, besides inducing the well-known cholestatic syndrome, increased liver GSSG and TBARS contents and the GSSG/GSH molar ratio, and altered the nonenzymatic and enzymatic antioxidant defense systems. The CyA-induced cholestasis and hepatic depletion of GSH, and the increases in the GSSG/GSH ratio, and in GSSG and TBARS concentrations were higher in the older than the mature rats. Moreover, superoxide dismutase and catalase activities were found to be significantly decreased only in treated senescent rats. The higher CyA-induced oxidative stress, lipoperoxidation, and decreases in the antioxidant defense systems in the aged animals render them more susceptible to the hepatotoxic effects of cyclosporin.     

Pathophysiological consequences of enhanced intracellular superoxide formation in isolated perfused rat liver.

The potential toxicity of enhanced intracellular reactive oxygen formation was investigated in isolated perfused livers of male Fischer rats. The presence of the redox-cycling agent diquat in the perfusate (200 microM) increased the basal efflux of glutathione disulfide (GSSG) into bile (2.65 +/- 0.26 nmol GSH-equivalents/min per g liver wt.) and perfusate (0.55 +/- 0.15 nmol/min per g) approximately 10-fold. Since no evidence was found for degradation of GSSG in the biliary tract of these animals, it could be estimated that diquat induced a constant O2- generation of approximately 1000 nmol/min per g liver wt for 1 h. Thus, reactive oxygen formation under these conditions was 1-2 orders of magnitude higher than under various pathophysiological conditions. Only minor liver injury (release of lactate dehydrogenase activity) was observed. To increase the susceptibility of the liver to the oxidant stress, animals were pretreated in vivo with 200 mg/kg body wt. phorone, which caused a 90% depletion of the hepatic glutathione content, 100 mg/kg ferrous sulfate, a combination of phorone and ferrous sulfate, or 40 mg/kg BCNU, which caused a 60% inhibition of hepatic GSSG reductase. Only the combined treatment of phorone + ferrous sulfate or BCNU caused a significant increase of the diquat-induced liver injury. Our results demonstrated an extremely high resistance of the liver against intracellular reactive oxygen formation (even with impaired detoxification systems) and can serve as reference for the evaluation of potential contributions of reactive oxygen to liver injury in various disease states.     

Hepatic response to the oxidative stress induced by E. coli endotoxin: Glutathione as an index of the acute phase during the endotoxic shock

Reactive oxygen species are important mediators of cellular damage during endotoxic shock. In order to investigate the hepatic response to the oxidative stress induced by endotoxin, hepatic and plasma glutathione (total, GSH and GSSG), GSSG/GSH ratio as well as Mn-superoxide dismutase and catalase activities were determined during the acute and recovery phases of reversible endotoxic shock in the rat. A significant increase in liver and plasma total glutathione content was observed 5 h after endotoxin treatment (acute phase), followed by a diminution of these parameters below control values at 48 h (recovery phase). The significant increases of GSSG levels and GSSG/GSH ratio are indicative of oxidative stress occurring during the acute phase. Liver Mn-SOD activity showed a similar time dependency as the GSSG/GSH ratio; however, a marked decrease in the liver catalase activity was observed during the process. These results indicate the participation of liver glutathione in the response to endotoxin and the possible use of plasma glutathione levels and GSSG/GSH ratio as indicators of the acute phase during the endotoxic process. (Mol Cell Biochem 159: 115-121, 1996)     

Effect of low flow ischemia-reperfusion injury on liver function

The release of liver enzymes is typically used to assess tissue damage following ischemia-reperfusion. The present study was designed to determine the impact of ischemia-reperfusion on liver function and compare these findings with enzyme release. Isolated, perfused rat livers were subjected to low flow ischemia followed by reperfusion. Alterations in liver function were determined by comparing rates of oxygen consumption, gluconeogenesis, ureagenesis, and ketogenesis before and after ischemia. Lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP) activities in effluent perfusate were used as markers of parenchymal and endothelial cell injury, respectively. Trypan blue staining was used to localize necrosis. Total glutathione (GSH + GSSG) and oxidized glutathione (GSSG) were measured in the perfusate as indicators of intracellular oxidative stress. LDH activity was increased 2-fold during reperfusion compared to livers kept normoxic for the same time period whereas PNP activity was elevated 5-fold under comparable conditions. Rates of oxygen consumption, gluconeogenesis, and ureagenesis were unchanged after ischemia, but ketogenesis was decreased 40% following 90 min ischemia. During reperfusion, the efflux rates of total glutathione and GSSG were unchanged from pre-ischemic values. Significant midzonal staining of hepatocyte nuclei was observed following ischemia-reperfusion, whereas normoxic livers had only scattered staining of individual cells. Reperfusion of ischemic liver caused release of hepatic enzymes and midzonal cell death, however, several major liver functions were unaffected under these experimental conditions. These data indicate that there were negligible changes in liver function in this model of ischemia and reperfusion despite substantial enzyme release from the liver and midzonal cell death.     

Blood glutathione homeostasis as a determinant of resting and exercise-induced oxidative stress in young men

Abstract

Although the importance of glutathione in protection against oxidative stress is well recognised, the role of physiological levels of glutathione and other endogenous antioxidants in protecting against exercise-induced oxidative stress is less clear. We evaluated the role of glutathione and selected antioxidant enzymes as determinants of lipid peroxidation at rest and in response to exercise in men (n = 13–14) aged 20–30 years, who cycled for 40 min at 60% of their maximal oxygen consumption (VO_2max). Levels of plasma thiobarbituric acid reactive substances (plasma TBARS) and blood oxidised glutathione (GSSG) increased by about 50% in response to exercise. Mean blood reduced glutathione (GSH)decreased by 13% with exercise. Of the measured red blood cell (RBC)antioxidant enzyme activities, only selenium-dependent glutathione peroxidase (Se-GPX) activity rose following exercise. In univariate regression analysis, plasma TBARS levels at rest predicted postexercise plasma TBARS and the exercise-induced change in total glutathione (TGSH). Blood GSSG levels at rest were strongly determinant of postexercise levels. Multiple regression analysis showed blood GSH to be a determinant of plasma TBARS at rest. The relative changes in TGSH were determinant of postexercise plasma TBARS. In summary, higher blood GSH and lower plasma TBARS at rest were associated with lower resting, and exercise-induced, lipid peroxidation. Subjects with a favourable blood glutathione redox status at rest maintained a more favourable redox status in response to exercise-induced oxidative stress. Changes in blood GSH and TGSH in response to exercise were closely associated with both resting and exercise-induced plasma lipid peroxidation. These results underscore the critical role of glutathione homeostasis in modulating exercise-induced oxidative stress and, conversely, the effect of oxidative stress at rest on exercise-induced changes in glutathione redox status.     

Blood glutathione homeostasis as a determinant of resting and exercise-induced oxidative stress in young men

Although the importance of glutathione in protection against oxidative stress is well recognized, the role of physiological levels of glutathione and other endogenous antioxidants in protecting against exercise-induced oxidative stress is less clear. We evaluated the role of glutathione and selected antioxidant enzymes as determinants of lipid peroxidation at rest and in response to exercise in men (n = 13-14) aged 20-30 years, who cycled for 40 min at 60% of their maximal oxygen consumption (VO2max). Levels of plasma thiobarbituric acid reactive substances (plasma TBARS) and blood oxidised glutathione (GSSG) increased by about 50% in response to exercise. Mean blood reduced glutathione (GSH) decreased by 13% with exercise. Of the measured red blood cell (RBC) antioxidant enzyme activities, only selenium-dependent glutathione peroxidase (Se-GPX) activity rose following exercise. In univariate regression analysis, plasma TBARS levels at rest predicted postexercise plasma TBARS and the exercise-induced change in total glutathione (TGSH). Blood GSSG levels at rest were strongly determinant of postexercise levels. Multiple regression analysis showed blood GSH to be a determinant of plasma TBARS at rest. The relative changes in TGSH were determinant of postexercise plasma TBARS. In summary, higher blood GSH and lower plasma TBARS at rest were associated with lower resting, and exercise-induced, lipid peroxidation. Subjects with a favourable blood glutathione redox status at rest maintained a more favourable redox status in response to exercise-induced oxidative stress. Changes in blood GSH and TGSH in response to exercise were closely associated with both resting and exercise-induced plasma lipid peroxidation. These results underscore the critical role of glutathione homeostasis in modulating exercise-induced oxidative stress and, conversely, the effect of oxidative stress at rest on exercise-induced changes in glutathione redox status.     

Dapsone induces oxidative stress and impairs antioxidant defenses in rat liver

Dapsone (DDS) is currently used in the treatment of leprosy, malaria and in infections with Pneumocystis jirovecii and Toxoplasma gondii in AIDS patients. Adverse effects of DDS involve methemoglobinemia and hemolysis and, to a lower extent, liver damage, though the mechanism is poorly characterized. We evaluated the effect of DDS administration to male and female rats (30 mg/kg body wt, twice a day, for 4 days) on liver oxidative stress through assessment of biliary output and liver content of reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation, and expression/activities of the main antioxidant enzymes glutathione peroxidase, superoxide dismutase, catalase and glutathione S-transferase. The influence of DDS treatment on expression/activity of the main DDS phase-II-metabolizing system, UDP-glucuronosyltransferase (UGT), was additionally evaluated. The involvement of dapsone hydroxylamine (DDS-NHOH) generation in these processes was estimated by comparing the data in male and female rats since N-hydroxylation of DDS mainly occurs in males. Our studies revealed an increase in the GSSG/GSH biliary output ratio, a sensitive indicator of oxidative stress, and in lipid peroxidation, in male but not in female rats treated with DDS. The activity of all antioxidant enzymes was significantly impaired by DDS treatment also in male rats, whereas UGT activity was not affected in any sex. Taken together, the evidence indicates that DDS induces oxidative stress in rat liver and that N-hydroxylation of DDS was the likely mediator. Impairment in the activity of enzymatic antioxidant systems, also associated with DDS-NHOH formation, constituted a key aggravating factor.     

Age-dependent changes in rat liver lipid peroxidation and glutathione content induced by acute ethanol ingestion

The study of the influence of the age of animals (13 to 53 weeks) on total liver thiobarbituric acid reactive substances (TBAR) content showed an increase which is maximal in rats of 39 weeks of age compared to young animals (13 weeks), followed by a dimunition in the 53 weeks old group. In this situation, the content of hepatic GSH and total GSH equivalents as well as the GSH/GSSG ratio were decreased with ageing, while GSSG levels were enhanced in the oldest group studied. Acute ethanol intoxication resulted in a marked increase in liver TBAR content in young animals, together with a decline in GSH, total GSH equivalents and GSH/GSSG ratio, and an enhancement in GSSG. These changes elicited by ethanol intake were reduced with ageing. It is concluded that ethanol-induced oxidative stress in the liver is diminished during ageing, despite the progressive decrease in the glutathione content of the tissue observed in control animals.     

Acetaminophen-induced liver oxidative stress and hepatotoxicity: influence of Kupffer cell activity assessed in the isolated perfused rat liver

Summary

The influence of acetaminophen (APAP) treatment (400 mg/kg) on Kupffer cell function was studied in the isolated perfused liver by colloidal carbon infusion, concomitantly with parameters related to oxidative stress (thiobarbituric acid reactants (TBARS) formation and glutathione (GSH) content) and tissue injury (sinusoidal efflux of lactate dehydrogenase (LDH)). APAP led to increased rates of hepatic TBARS formation, GSH depletion, and higher sinusoidal LDH efflux compared to control values, without changes in the basal rate of O₂ consumption. In addition, APAP significantly enhanced the rate of carbon uptake by perfused livers and the associated carbon-induced O₂ consumption, with carbon-induced LDH effluxes being increased by 411% over control values or by 124% compared to basal LDH release in APAP-treated rats. APAP-induced changes in liver TBARS formation and GSH levels were attenuated by gadolinium chloride (GdCl₃) pretreatment, whereas those in carbon uptake, carbon-induced respiration, and LDH efflux were abolished. GdCl₃ pretreatment decreased liver O₂ consumption irrespectively of APAP treatment, an effect that seems to be due to depression of mitochondrial respiration. It is concluded that APAP intoxication enhances Kupffer cell function as assessed in the intact liver, which may represent an important source of reactive O₂ species and chemical mediators conditioning the increased oxidative stress status and the tissue injury which developed.