Mechanisms of N-acetyl-p-benzoquinone imine cytotoxicity

N-Acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen, rapidly reacts at physiological pH with glutathione (GSH) forming an acetaminophen-glutathione conjugate and stoichiometric amounts of acetaminophen and glutathione disulfide (GSSG). The same reaction products are formed in isolated hepatocytes incubated with NAPQI. In hepatocytes which have been treated with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) in order to inhibit glutathione reductase, the initial rise in GSSG concentration in the presence of NAPQI is maintained, whereas GSSG is rapidly reduced back to GSH in untreated hepatocytes. Oxidation by NAPQI of GSH to GSSG and the reduction of GSSG back to GSH by the NADPH-dependent glutathione reductase appear to be responsible for the rapid oxidation of NADPH that occurs in hepatocytes incubated with NAPQI in that the effect is blocked by pretreatment of cells with BCNU. When added to hepatocytes, NAPQI not only reacts with GSH but also causes a loss in protein thiol groups. The loss in protein thiols occurs more rapidly in cells pretreated with BCNU or diethylmaleate. Whereas both of these treatments enhance cytotoxicity caused by NAPQI, BCNU pretreatment has no effect on the covalent binding of [14C-ring]NAPQI to cellular proteins. Furthermore, dithiothreitol added to isolated hepatocytes after maximal covalent binding of [14C-ring]NAPQI but preceding cell death protects cells from cytotoxicity and regenerates protein thiols. Thus, the toxicity of NAPQI to isolated hepatocytes may result primarily from its oxidative effects on cellular proteins.     

The influence of selenium intake on chronic adriamycin toxicity and lipid peroxidation in rats

This paper reports on the influence of selenium intake on antioxidant protective systems during chronic adriamycin (AM) treatment in rats. Rats were kept for 14 weeks on a selenium deficient (Se-) diet or a diet containing selenium (Se+). No significant differences were found in any group with regard to the cardiac content of total and reduced glutathione (GSH) and heart superoxide dismutase specific activity. AM treatment did not modify lipid peroxidation as measured by cardiac malondialdehyde (MDH) formation in rats receiving either the Se- or the Se+ diet. In the Se+ rats AM had no effect on the exhalation of ethane or pentane but decreased the exhalation of ethane and increased that of pentane in the SE- rats. In Se- AM-treated rats mortality was higher. Since this did not seem to be correlated with modifications of any of the biochemical parameters taken into consideration, it is suggested that the better resistance of Se+ animals to AM treatment is related to some factors not yet identified.     

Effects of di(2‐ethylhexyl)phthalate on testicular oxidant/antioxidant status in selenium‐deficient and selenium‐supplemented rats

Di(ethylhexyl)phthalate (DEHP), the most widely used plasticizer, was investigated to determine whether an oxidative stress process was one of the underlying mechanisms for its testicular toxicity potential. To evaluate the effects of selenium (Se), status on the toxicity of DEHP was further objective of this study, as Se is known to play a critical role in testis and in the modulation of intracellular redox equilibrium. Se deficiency was produced in 3‐weeks‐old Sprague–Dawley rats feeding them ≤0.05 mg Se /kg diet for 5 weeks, and Se‐supplementation group was on 1 mg Se/kg diet. DEHP‐treated groups received 1000 mg/kg dose by gavage during the last 10 days of the feeding period. Activities of antioxidant selenoenzymes [glutathione peroxidase 1 (GPx1), glutathione peroxidase 4 (GPx4), thioredoxin reductase (TrxR)], catalase (CAT), superoxide dismutase (SOD), and glutathione S‐transferase (GST); concentrations of reduced glutathione (GSH), oxidized glutathione (GSSG), and thus the GSH/GSSG redox ratio; and thiobarbituric acid reactive substance (TBARS) levels were measured. DEHP was found to induce oxidative stress in rat testis, as evidenced by significant decrease in GSH/GSSG redox ratio (>10‐fold) and marked increase in TBARS levels, and its effects were more pronounced in Se‐deficient rats with ～18.5‐fold decrease in GSH/GSSG redox ratio and a significant decrease in GPx4 activity, whereas Se supplementation was protective by providing substantial elevation of redox ratio and reducing the lipid peroxidation. These findings emphasized the critical role of Se as an effective redox regulator and the importance of Se status in protecting testicular tissue from the oxidant stressor activity of DEHP. © 2011 Wiley Periodicals, Inc. Environ Toxicol 29: 98–107, 2014.     

Formation and reduction of glutathione-protein mixed disulfides during oxidative stress. A study with isolated hepatocytes and menadione (2-methyl-1,4-naphthoquinone)

Incubation of isolated rat hepatocytes with menadione (2-methyl-1,4-naphthoquinone) resulted in a dose-dependent depletion of intracellular reduced glutathione (GSH), most of which was oxidized to glutathione disulfide (GSSG). Menadione metabolism was also associated with a dose- and time-dependent inhibition of glutathione reductase, impairing the regeneration of GSH from GSSG produced during menadione-induced oxidative stress. Inhibition of glutathione reductase by pretreatment of hepatocytes with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) greatly potentiated both GSH depletion and GSSG formation during the metabolism of low concentrations of menadione. Concomitant with GSH oxidation, mixed disulfides between glutathione and protein thiols were formed. The amount of mixed disulfides produced and the kinetics of their formation were dependent on both the intracellular GSH/GSSG ratio and the activity of glutathione reductase. The mixed disulfides were mainly recovered in the cytosolic fraction and, to a lesser extent, in the microsomal and mitochondrial fractions. The removal of glutathione from protein mixed disulfides formed in hepatocytes exposed to oxidative stress was dependent on GSH and/or cysteine and appeared to occur predominantly via a thiol-disulfide exchange mechanism. However, incubation of the microsomal fraction from menadione-treated hepatocytes with purified glutathione reductase in the presence of NADPH also resulted in the reduction of a significant portion of the glutathione-protein mixed disulfides present in this fraction. Our results suggest that the formation of glutathione-protein mixed disulfides occurs as a result of increased GSSG formation and inhibition of glutathione reductase activity during menadione metabolism in hepatocytes.     

Nitrofurantoin-mediated oxidative stress cytotoxicity in isolated rat hepatocytes

Freshly isolated rat hepatocytes were used to study the mechanism(s) of toxicity of the antimicrobial drug nitrofurantoin. This 5-nitrofuran derivative stimulated hepatocyte oxygen uptake in the presence of the mitochondrial respiration inhibitors KCN or antimycin A. This could indicate the formation of O2- and H2O2, following intracellular nitrofurantoin reduction. Addition of nitrofurantoin to suspensions of isolated rat hepatocytes produced a dose- and time-dependent decrease of cell viability. H2O2 probably plays a significant role in the cytotoxic effects of nitrofurantoin as the catalase inhibitors azide or aminotriazole markedly enhanced cytotoxicity. The loss of cell viability was preceded by glutathione (GSH) depletion and a concomitant and nearly stoichiometric formation of oxidised glutathione (GSSG) that did not occur in hepatocytes lacking glutathione peroxidase activity isolated from rats fed a low-selenium diet. This indicates that H2O2 and the seleno-enzyme glutathione peroxidase are responsible for GSH oxidation. Furthermore, addition of nitrofurantoin to isolated rat hepatocytes produced a reversible inactivation of hepatocyte glutathione reductase activity and explains the maintenance of high GSSG levels. The compromised hepatocytes were also highly susceptible to H2O2. The hepatocyte toxicity of nitrofurantoin may, therefore, be attributed to oxidative stress caused by redox-cycling mediated oxygen activation.     

Role of glutathione reductase during menadione-induced NADPH oxidation in isolated rat hepatocytes

Metabolism of menadione (2-methyl-1,4-naphthoquinone) results in the rapid oxidation of NADPH within isolated rat hepatocytes. The glutathione redox cycle is thought to play a major role in the consumption of NADPH during menadione metabolism, chiefly through glutathione reductase (GSSG-reductase). This enzyme reduces oxidized glutathione (GSSG), formed via the glutathione-peroxidase reaction, with the concomitant oxidation of NADPH. To explore the relationship between GSSG-reductase and the consumption of NADPH during menadione metabolism, isolated rat hepatocyte suspensions were exposed to non-lethal and lethal menadione concentrations (100 and 300 microM respectively) following the inhibition of GSSG-reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Menadione produced a concentration-related depletion of GSH (measured as non-protein sulfhydryl content) which was potentiated markedly by BCNU. Menadione toxicity was potentiated at either concentration by BCNU based on lactate dehydrogenase leakage at 2 hr. In addition, the NADPH content of isolated hepatocytes rapidly declined following exposure to either concentration of menadione. However, at the lower menadione concentration (100 microM), the NADPH content returned to control values or above by 60 min, whereas the NADPH content of cells exposed to 300 microM menadione with or without BCNU remained depressed for the duration of the incubation. These data suggest that, although NADPH is required by GSSG-reductase for the reduction of GSSG to GSH during quinone-induced oxidative stress, this pathway does not appear to be the major route by which NADPH is consumed during the metabolism of menadione in isolated hepatocytes.     

Metabolism of acetaminophen by cultured rat hepatocytes. Depletion of protein thiol groups without any loss of viability

Over the course of 4 hr, the metabolism of acetaminophen (APAP) by cultured rat hepatocytes resulted in a depletion of protein thiols and an accumulation of oxidized glutathione (GSSG) in the medium. With 20 mM APAP, arylation and the formation of glutathione mixed disulfides accounted for a loss of 22% of the total protein thiols in the absence of any loss of viability. With 20 mM APAP and an inhibition of glutathione reductase by 1.3-(2-chloroethyl)-1-nitrosourea (BCNU), protein thiols were depleted by 40% by arylation and the formation of glutathione mixed disulfides, again without a loss of viability. With 20 mM APAP and BCNU in the presence of 20 mM deferoxamine, there was still little or no cell killing after 8 hr despite a loss now of almost 60% of the total protein thiols. These data do not support the hypothesis that a depletion of protein thiols is related to the toxicity of APAP. One millimolar APAP and BCNU killed 60% of the hepatocytes within 4 hr. In this circumstance, the loss of protein thiols was not attributable to either arylation by APAP metabolites or the formation of glutathione mixed disulfides. The antioxidant N,N'-diphenyl-phenylenediamine prevented the cell killing and the loss of protein thiols, a result implicating a role for lipid peroxidation in the depletion of protein-bound thiols. However, protein thiol depletion under these circumstances is not necessarily related to the lethal cell injury and most likely represents an epiphenomenon of the peroxidation of cellular lipids.     

1,2-Dichlorobenzene-mediated hepatocellular oxidative stress in Fischer-344 and Sprague-Dawley rats

1,2-Dichlorobenzene (1,2-DCB) is a potent hepatotoxicant in male Fischer 344 (F-344) rats but not in Sprague-Dawley (SD) rats. While Kupffer cell-dependent oxidative stress plays a role in the progression of 1,2-DCB-mediated liver injury, we hypothesize that initiation of liver injury is due to oxidative events within the hepatocyte. This study compared hepatocellular oxidative stress marked by glutathione disulfide (GSSG) and glutathione (GSH) production in either bile, liver, or isolated hepatocytes of F-344 and SD rats following 1,2-DCB administration. Hepatic GSH concentrations were depleted at a greater rate in F-344 than in SD rats within 12 h of 1,2-DCB administration (3.6 mmol/kg ip). In bile, GSSG concentrations were threefold greater in F-344 rats compared to SD rats by 9 h of 1,2-DCB treatment. Moreover, 1-aminobenzotriazole but not gadolinium chloride pretreatment blocked the rise in biliary GSSG concentrations following 1,2-DCB administration. In in vitro studies, isolated hepatocytes of F-344 rats had a 15% increase in cellular GSSG concentrations following 1 h of 1,2-DCB (3.55 nmol) exposure, while GSH decreased 22% by 6.5 h compared to controls. In contrast, isolated SD hepatocytes exposed to 1,2-DCB had no increase in GSSG and only an 8% reduction in GSH. Furthermore, parameters of lipid peroxidation were increased in F-344 rats and not in SD rats. Collectively, these data suggest that hepatocellular oxidative stress is dependent upon bioactivation and the enhanced oxidative stress in the F-344 rat may explain its susceptibility to 1,2-DCB compared to the SD rat.     

Inhibition of the activity of glutathione peroxidase by tertiary-butylhydroperoxide in cultured Chinese hamster cells and the role of cellular glutathione in the recovery of the activity.

Treatment of Chinese hamster cells with 1 mM tertiary-butylhydroperoxide (t-BuOOH) for 1 h markedly inhibited the activity of glutathione peroxidase (GSH-Px). However, the activity returned to pre-inhibition levels within 1-2 h with post-treatment incubation. The inhibition of the activity of GSH-Px by t-BuOOH was enhanced by depletion of levels of cellular GSH with the addition of L-buthionine sulfoximine (BSO) or diethylmaleate (DEM) and no subsequent recovery of the activity was observed for at least 4 h. The ratio of levels of GSSG to total GSH increased as a result of treatment with 1 mM t-BuOOH or diamide for 1 h but not as a result of treatment with hydrogen peroxide. After treatment with t-BuOOH, the level of GSH rapidly increased to more than twice the control level during 15-40 min of post-treatment incubation. Depletion of GSH by DEM after treatment with t-BuOOH reduced the rate of the recovery of the activity of GSH-Px, suggesting a role of cellular GSH in the recovery. However, the decrease in the rate of the recovery caused by DEM was small and in no way equivalent to the extent of depletion of GSH, suggesting that the rapid increase in the level of GSH after treatment with t-BuOOH is not closely related to the rapid recovery of the activity of GSH-Px.     

Hepatic glutathione metabolism and lipid peroxidation in response to excess dietary selenomethionine and selenite in mallard ducklings

Studies were conducted with mallard (Anas platyrhynchos) ducklings to determine the effects of excess dietary selenium (Se) on hepatic glutathione concentration and associated enzymes, and lipid peroxidation. Day-old ducklings were fed 0.1, 10, 20, or 40 ppm Se as seleno-DL-methionine or sodium selenite for 6 wk. Selenium from selenomethionine accumulated in a dose-dependent manner in the liver, resulting in a decrease in the concentration of hepatic-reduced glutathione (GSH) and total hepatic thiols (SH). These effects were accompanied by a dose-dependent increase in the ratio of oxidized glutathione (GSSG) to GSH, and an increase in malondialdehyde concentration as evidence of lipid peroxidation. Hepatic and plasma GSH peroxidase activity was initially elevated at 10 ppm Se as selenomethionine, whereas GSSG reductase activity was elevated at higher dietary concentrations of Se. Selenium from sodium selenite accumulated in the liver to an apparent maximum at 10 ppm in the diet, resulting in an increase in hepatic GSH and GSSG accompanied by a small decrease in total hepatic SH. Sodium selenite resulted in an increase in hepatic GSSG reductase activity at 10 ppm and in plasma GSSG reductase activity at 40 ppm. A small increase in lipid peroxidation occurred at 40 ppm. These findings indicate that excess dietary Se as selenomethionine has a more pronounced effect on hepatic glutathione metabolism and lipid peroxidation in ducklings than does selenite, which may be related to the pattern of accumulation. Effects of Se as selenite appear to be less pronounced in ducklings than reported in laboratory rodents. The effects of selenomethionine, which occurs in vegetation, are of particular interest with respect to the health of wild aquatic birds in seleniferous locations.     

1,3-(2-Chloroethyl)-1-nitrosourea potentiates the toxicity of acetaminophen both in the phenobarbital-induced rat and in hepatocytes cultured from such animals

The toxicity of acetaminophen was studied in hepatocytes cultured from phenobarbital-induced male rats. Such cells were less sensitive to acetaminophen than similar ones cultured from animals induced with 3-methylcholanthrene. In both cases, the toxicity of acetaminophen depended on its metabolism. Inhibition of glutathione reductase with 1,3-(2-chloroethyl)-1-nitrosourea (BCNU) potentiated the toxicity of acetaminophen in the presence or absence of 100 mM acetone, an agent that activates the mixed function oxidation of the toxin. BCNU enhanced the rate and extent of the depletion of GSH in the presence or absence of acetone. Pretreatment of the hepatocytes with the ferric iron chelator deferoxamine or addition to the culture medium of the antioxidant N,N'-diphenyl-p-phenylenediamine prevented the toxicity of acetaminophen in the presence of BCNU whether or not there was acetone in the cultures. BCNU similarly potentiated the hepatotoxicity of acetaminophen in the intact, phenobarbital-induced rat. These data indicate that the mechanism of the killing of hepatocytes induced with phenobarbital is similar to that reported previously with hepatocytes prepared from animals induced with 3-methylcholanthrene. In both cases it would seem that the liver cells are killed by acetaminophen as a result of an oxidative stress that accompanies the metabolism of this hepatotoxin.     

Influence of selenium and/or magnesium on alleviation alcohol induced oxidative stress in rats, normalization function of liver and changes in serum lipid parameters

The aim of this study was to evaluate the attenuating effect of given selenium and/or magnesium on ethanol-induced oxidative stress, disturbances of liver function and cholesterol metabolism. Forty male rats were divided into five groups: C - control, Et - intoxicated with alcohol (15% solution in drinking water), Et + Mg, Et + Se, Et + Mg + Se - intoxicated with alcohol and supplemented with selenium (0.4 mg Se/l water), magnesium (100 mg Mg/l water) and combination of Se and Mg, respectively. The experiment was carried out over the 3 months. The results show that the chronic ingestion of alcohol induces lipid peroxidation and histopathological changes in liver. Supplementation with magnesium only partially alleviates oxidative stress and damages in this tissue. The both selenium alone and combination of magnesium and selenium significantly elevated total antioxidant status (TAS) in serum, activity of glutathione peroxidase and ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) in liver and retarded oxidative stress and histopathological changes in this tissue. Chronic administration of ethanol (alone and with magnesium) resulted in significant decrease in the serum total cholesterol and retardation in the body weight gain in comparison with the control group. In the groups supplemented with selenium and selenium and magnesium simultaneously, concentration of total cholesterol in serum and body gains was similar to the control group. Supplementation of Se or selenium and magnesium simultaneously significantly enhances antioxidant defence and is more effective against alcohol-induced oxidative stress, disturbance of liver function and cholesterol metabolism than the separate use of magnesium.     

Selenium and drug metabolism--II. Independence of glutathione peroxidase and reversibility of hepatic enzyme modulations in deficient mice

Male mice were fed a diet containing less than 0.01 ppm selenium (Se-) for 6 months. A control group received the same diet containing 0.5 ppm selenium (Se+). In the livers of the Se- animals a drastic decrease in glutathione peroxidase (GSH-Px) activity was observed. It reached undetectable levels after 17 days of the Se- diet. At that time, GSH-transferase activity began to increase significantly, followed by changes in many other enzyme activities. After the 60th day, these enzyme modulations had reached a plateau with the following percentage changes compared to controls: GSH-transferases: 320% (1,2-dichloro-4-nitrobenzene), 218% (1-chloro-2,4-dinitrobenzene); glutathione reductase: 160%; ethoxycoumarin deethylase: 330%; cytochrome P-450-hydroperoxidase: 230%; heme oxygenase: 240%; UDP-glucuronyltransferase: 200%; GSH-thioltransferase: 64%; sulphotransferase: 62%; NADPH-cytochrome-P-450-reductase: 65%; flavin-containing mono-oxygenase: 57%. No significant changes were observed for GSH-transferase activity assayed with ethacrynic acid or for microsomal H2O2 formation and aniline hydroxylase activity. In single-pulse repletion experiments by injection of 250 micrograms selenium/kg body wt, different individual time constants for the recovery process of the enzymatic perturbations were observed. The half-times for the recovery ranged from 5.7 hr for the microsomal NADPH-cytochrome-P-450 reductase to over 29 hr for GSH-Px up to 44 hr for part of the GSH-transferase activity. 250 micrograms selenium/kg body wt were needed to restore 50% of GSH-Px activity in the long-term Se- mice compared to Se+ controls. All other enzymatic changes in the Se- mice needed a dose of 7 micrograms selenium/kg body wt for 50% restorage . The results demonstrate that processes other than those related to GSH-Px take place in a later phase of selenium deficiency in mouse liver with a chronologically common beginning. The different repletion and depletion kinetics as well as the different need of these processes for the trace element are discussed with respect to the existence of two separate selenium pools.     

Changes in redox ratio and protein glycation in precataractous lens from fructose-fed rats: effects of exogenous L-carnitine

1. Rats fed high dietary fructose are documented to form an acquired model of insulin resistance. The present study measured the effects of administration of L-carnitine (CA) on lens protein glycation, oxidative stress and redox homeostasis in rats fed a high-fructose diet. 2. Animals were divided into four groups: (i) an untreated control group (fed starch diet); (ii) an untreated fructose-group (fed a high-fructose diet); (iii) a CA-treated (300 mg/kg per day), fructose-fed group; and (iv) a CA-treated, starch-fed group. After 60 days treatment, lenses were dissected and multiple oxidative stress markers, glycation of proteins and the ratio of oxidized to reduced glutathione (GSSG/GSH) were determined. 3. A significant decline in enzyme and non-enzyme anti-oxidants and an increase in lipid peroxidation products, protein oxidation, protein glycation, GSSG/GSH ratio and aldehyde formation were observed in lens samples obtained from fructose-fed rats. Administration of CA to fructose-fed rats significantly attenuated oxidative damage and protein glycation and returned levels of anti-oxidants to near those seen in the control group. 4. The results of the present study indicate that dietary fructose disturbs lens integrity and exogenous CA may safeguard the lens by preventing glycation and oxidative stress.     

2-Amino-5-chlorophenol toxicity in renal cortical slices from Fischer 344 rats: effect of antioxidants and sulfhydryl agents

A rapid inhibition of protein synthesis is observed when isolated rat hepatocytes are incubated in the presence of 0.25-0.5 mM of tert-butyl hydroperoxide (tBOOH). Such an inhibition occurs in the absence of a cytolytic effect by tBOOH. Iron chelators (o-phenanthroline and desferrioxiamine), protected against oxidative cell death, but they did not modify the inhibition of protein synthesis caused by tBOOH (0.5 mM), suggesting that free radicals are less implicated in such an impairment. Electron micrographs of hepatocytes under oxidative stress show disaggregation of polyribosomes but not oxidative alterations, such as blebs or mitochondrial swelling. Protein synthesis inhibition is accompanied by a decrease in reduced glutathione (GSH) and an increase in glutathione disulfide (GSSG) and the level of protein S-thiolation (protein mixed disulfides formation). Such an increase of GSSG appears as a critical event since diethylmaleate (DEM) at 0.2 mM reduced GSH content by more than 50% but did not affect either GSSG content or protein synthesis. The addition of exogenous GSH and N-acetylcysteine (NAC) to tBOOH-treated hepatocytes significantly reduced the formation of protein mixed disulfides and restored the depressed protein synthesis either completely or partially. We suggest that S-thiolation of some key proteins may be involved in protein synthesis inhibition by tBOOH.     

Oxygen dependence of oxidative stress. Rate of NADPH supply for maintaining the GSH pool during hypoxia

NADPH supply for oxidized glutathione (GSSG) reduction was studied in hepatocytes under different steady-state O2 concentrations with controlled infusions of diamide, a thiol oxidant. When bis-chloro-nitrosourea (BCNU) was used to inhibit GSSG reductase, the rate of GSH depletion approximated the rate of diamide infusion, showing that diamide reacted preferentially with GSH under these experimental conditions. Under aerobic conditions without BCNU treatment, the GSH and NADPH pools were largely unaffected and little diamide accumulation or protein thiol oxidation occurred with diamide infusion rates up to 5.3 nmol/10(6) cells per min. However, at greater infusion rates, GSH and NADPH decreased, diamide and GSSG concentrations increased, and protein thiols were oxidized. This critical infusion rate was easily discernible and provided a convenient means to assess the capacity of cells to reduce GSSG as a function of O2 concentration. As the O2 concentration was decreased below 15 microM, the critical infusion rate decreased from the aerobic value of 5.3 to less than 2 nmol/10(6) cells per min in anoxic cells; half-maximal change occurred at 5 microM O2. Although cells could not maintain normal thiol and NADPH pools at infusion rates above the critical value, analysis of the rates of thiol depletion showed that the maximal NADPH supply rate for GSSG reduction under aerobic conditions was 7-8 nmol/10(6) cells per min and was affected by hypoxia to the same degree as the critical value. Thus, hypoxia and anoxia impair the capability of cells to supply NADPH for the reduction of thiol oxidants. This could be an important factor in the sensitivity of hypoxic and ischemic tissues to oxidative injury.     

Subchronic hepatotoxicity of selenomethionine ingestion in mallard ducks

Two-year-old male mallards (Anas platyrhynchos) received a control diet (0.2 ppm Se) or diets containing 1, 2, 4, 8, 16, or 32 ppm Se as selenomethionine for 14 wk. Se accumulated readily in the liver in a dose-dependent manner, reaching a mean concentration of 29 ppm (wet weight) in the 32 ppm group. Dietary Se of 2 ppm or greater increased plasma glutathione peroxidase activity. Mortality (10%) and histopathological effects, including bile duct hyperplasia and hemosiderin pigmentation of the liver and spleen, occurred in the 32 ppm group. These histopathological effects were accompanied by lower hemoglobin concentrations (16 and 32 ppm groups) and hematocrit (32 ppm group), and elevated plasma alkaline phosphatase activity (32 ppm group) indicative of cholestatic liver injury. Other manifestations of hepatotoxicity included significant linear dose responses for hepatic oxidized glutathione (GSSG) concentrations and ratio of GSSG to reduced glutathione (GSH). Means for both of these responses differed from controls in groups receiving 8-32 ppm Se. Mean hepatic GSH and malondialdehyde (a measure of lipid peroxidation) concentrations were significantly elevated in the 16 and 32 ppm groups. Subchronic effects of selenomethionine, which occurs in vegetation, are of particular interest with respect to the health of wild aquatic birds in seleniferous locations.     

Differential effect of dietary selenium on the long-term neurotoxicity induced by MDMA in mice and rats

We examined the effect of dietary selenium (Se) on the long-term effect of 3,4-methylenedioxymethamphetamine (MDMA) on dopamine (DA) and 5-hydroxytryptamine (5-HT) containing neurons in the brain of mice and rats. Animals were fed either a Se-deficient (<0.02 ppm) or Se-replete (0.2 ppm) diet for 8 weeks. On the seventh week mice received three injections of MDMA (15 mg/kg, i.p. 3 h apart) or saline and rats a single dose of MDMA (12.5 mg/kg i.p.) or saline. All animals were sacrificed 7 days later. MDMA administration to mice depleted striatal DA concentration in both dietary groups, although depletion was considerably larger in the Se-deficient mice (64%) than Se-replete mice (30%). In addition, a decrease in 5-HT (17-32%) occurred in brain regions of Se-deficient but not Se-replete mice. In rats, MDMA decreased cortical [(3)H]-paroxetine binding (62%) and 5-HT content, the depletion being similar in the Se-deficient and Se-replete groups. No DA loss occurred in either group. There was no difference in the hyperthermic response induced by MDMA in Se-deficient or Se-replete animals. The Se-deficient diet decreased glutathione peroxidase (GPx) activity by 30% in mouse striatum and cortex and increased the degree of lipid peroxidation in cortical synaptosomes. Se-deficient rats also showed a decrease in brain GPx activity compared with the Se-replete group, but the degree of lipid peroxidation in synaptosomes was similar in both dietary groups. These results suggest that the antioxidant capacity of rats and mice differ leading to a differential susceptibility to the oxidative stress caused by MDMA in situations of low dietary Se.     

Selenium Ameliorates Ibuprofen Induced Testicular Toxicity by Redox Regulation: Running Head: Se protects against NSAID induced testicular toxicity

Despite the Cox inhibitory anti-inflammatory and antipyretic effects of most widely used non-steroidal anti-inflammatory drugs (NSAIDs), such as Ibuprofen, their chronic use is associated with a plethora of patho-physiological insults. One such toxic effect on testicular tissues is not well studied and the underlying molecular mechanisms remain unexplored. Thus, the current study is designed to evaluate the antioxidant properties of essential trace element selenium (Se) to ameliorative Ibuprofen associated testicular toxic effects. Adult male Wistar rats were divided into 3 groups and fed on diets containing different concentrations of sodium selenite, viz. 0.01 mg/kg (Se- deficient), 0.2 mg/kg (Se-adequate), or 0.5 mg/kg (Se- supplemented) for 8 weeks. After diet feeding schedule, each group was divided into two subgroups i.e., with or without the treatment of Ibuprofen (120 mg/kg Bw). The protective effect of Se was evaluated by measuring testicular Se and selenoproteins status, spermatogenic markers, histopathology and testicular redox status. Ibuprofen diminished seminal volume, sperm count, sperm motility, which correlated well increased testicular reactive oxygen species. Se deficiency exacerbated these detrimental effects of ibuprofen by increasing oxidative stress. Alternatively, Se supplementation through antioxidant enzymes mediated protective effects. Se as essential antioxidant selenoproteins ameliorates Ibuprofen induced male reproductive toxicity.     

Relationships between ascorbic acid and alpha-tocopherol during diquat-induced redox cycling in isolated rat hepatocytes

The effects of diquat-induced redox cycling on the levels of cellular ascorbic acid and alpha-tocopherol were investigated in isolated rat hepatocytes. In untreated hepatocytes, the metabolism of 1 or 2 mM diquat resulted in the depletion of cellular ascorbic acid and glutathione, but not of alpha-tocopherol, in association with the induction of cell death during the experimental period. In 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) pretreated cells, 1 mM diquat induced cell death accompanied by glutathione was rapid (to 9% of controls by 15 min) and cell ascorbate was completely consumed by 2 hr of incubation. In contrast, cellular alpha-tocopherol levels were stable for the first 30 min, but were depleted in association with the onset of lipid peroxidation. Supplementation of 0.1 or 1.0 mM ascorbic acid in the incubation medium delayed the onset of diquat-induced alpha-tocopherol loss, lipid peroxidation and cytotoxicity. When the concentration of exogenous cellular ascorbic acid was consumed to below that of endogenous ascorbic acid, alpha-tocopherol loss and lipid peroxidation were initiated. The results indicate that untreated hepatocytes have an effective multicomponent antioxidant system against diquat-induced oxidative stress. However, when glutathione is depleted from hepatocytes by treatment with BCNU and diquat, ascorbic acid plays a vital role in maintaining cellular alpha-tocopherol levels and survival of the cell.