Hepatic GSH Levels Research Articles

Comparative study of natural antioxidants – curcumin, resveratrol and melatonin – in cadmium-induced oxidative damage in mice

The present study was designed to examine the antioxidative effect of curcumin, resveratrol and melatonin pre-treatment on cadmium-induced oxidative damage and cadmium distribution in an experimental model in mice. Male CD mice were treated once daily for 3 days with curcumin (50 mg/kg b.w., p.o.), resveratrol (20 mg/kg b.w., p.o.) or melatonin (12 mg/kg, p.o.), dispersed in 0.5% methylcellulose. One hour after the last dose of antioxidants cadmium chloride was administered (7 mg/kg b.w., s.c.) to pre-treated animals and control animals receiving methylcellulose. At 24th h after Cd administration the lipid peroxidation (LP – expressed as malondialdehyde production), reduced glutathione (GSH), catalase (CAT) and glutathione peroxidase (GPx) were estimated in liver homogenates. Cadmium concentration was measured in the liver, kidneys, testes and brain by AAS. Cadmium chloride administration to mice induced hepatic lipid peroxidation (to 133%, p < 0.001), decreased GSH content (to 65%, p < 0.001) and inhibited catalase (to 68%, p < 0.001) and GPx activity (to 60%, p < 0.001) in the liver. Curcumin, resveratrol and melatonin oral pre-treatment completely prevented the Cd-induced lipid peroxidation and Cd-induced inhibition of GPx hepatic activity. Resveratrol was effective against Cd-induced inhibition of catalase activity ( p < 0.001). The decrease in hepatic GSH level was not prevented by curcumin, resveratrol or melatonin pre-treatment. In mice treated with antioxidants alone the level of LP, GSH, GPx or CAT was not different from control levels. The pre-treatment with antioxidants did not affect cadmium distribution in the tissues of Cd-intoxicated mice. The results demonstrate that curcumin, resveratrol and melatonin pre-treatment effectively protect against cadmium-induced lipid peroxidation and ameliorate the adverse effect of cadmium on antioxidant status without any reduction in tissue Cd burden.

Protective effect of Lygodium flexuosum (L.) Sw. (Lygodiaceae) against d-galactosamine induced liver injury in rats

The protective effect of Lygodium flexuosum n-hexane extract against d-galactosamine was evaluated in Wistar rats. In preventive groups extract was administered at 48, 24 and 2 h before d-galactosamine intoxication whereas in post-treatment groups extract were administered 2, 24 and 48 h after d-galactosamine intoxication. Rats pre-treated with n-hexane extract at a dose of 200 and 100 mg/kg of Lygodium flexuosum showed a significant prevention of elevated AST, ALT, LDH levels and hepatic malondialdehyde in d-galactosamine treated rats. Hepatic glutathione levels significantly upregulated by the extract treatment in d-galactosamine treated rats. Quantification of histopathological sections supported the preventive action of n-hexane extract of Lygodium flexuosum. Rats treated with the extract at a dose of 200 and 100 mg/kg Lygodium flexuosum after the establishment of d-galactosamine induced liver injury showed complete protection of liver as evidenced from normal AST, ALT and LDH levels, hepatic GSH and MDA levels and also by normal histological index of liver in treated rats. Rats treated with n-hexane extract of Lygodium flexuosum were comparable to that of Silymarin, the standard hepatoprotective drug.

Whey Feeding Suppresses the Measurement of Oxidative Stress in Experimental Burn Injury

Burns cause thermal injury to local tissue and trigger systemic acute inflammatory processes, which may lead to multiple distant organ dysfunction. We investigated the protective effect of dietary whey supplementation on distant organs in a rat model. Forty-eight rats were divided into six groups of eight: groups 1 and 2 were the controls, fed a standard diet and a whey-supplemented diet, respectively; groups 3 and 4 were fed a standard diet and subjected to burn injury; and groups 5 and 6 were fed a whey-supplemented diet and subjected to burn injury. We measured the oxidative stress variables, as well as glutathione in the liver and kidney, and histologically examined skin samples obtained 4 h (groups 3 and 5) and 72 h (groups 4 and 6) after burn injury. Glutathione (GSH) levels remained the same in the liver but were slightly elevated in the kidneys after burn injury in the rats fed a standard diet. Whey supplementation caused a significant increase in hepatic GSH levels 4 h after burn injury. Moreover, there was a significant rebound effect in the liver and kidney GSH levels after 72 h and whey supplementation potentiated this effect. Hepatic and renal lipid peroxide levels were also increased 4 h after burn injury in the rats fed a standard diet. Whey supplementation significantly suppressed the burn-induced increase in hepatic and renal lipid peroxide levels. Histological examination revealed that although whey supplementation resulted in decreased subepidermal inflammation, the indicators of wound healing and collagen deposition were not improved. Whey pretreatment suppressed hepatic and renal oxidative stress measurements after experimental burn injury.

Mitochondrial oxidative stress and permeability transition in Isoniazid and Rifampicin induced liver injury in mice

To evaluate the role of mitochondrial oxidative stress and permeability transition (MPT) in isoniazid (INH) and rifampicin (RMP) induced hepatotoxicity in mice. Liver damage was induced by co-treatment of INH (50 mg/kg) and RMP (100 mg/kg). Pre-treatment with either methionine or phorone was done to modulate hepatic GSH level. Liver cell injury was assessed biochemically and histologically. Evidence of apoptosis was sought by TUNEL test, caspase assay and histology. INH and RMP co-treatment caused steatosis and increased apoptosis of the hepatocytes, hepatic oxidative stress, particularly in the mitochondrial fraction with increased mitochondrial permeability transition (MPT). Mitochondrial oxidative stress as well as liver cell injury was increased by prior treatment with phorone. This was attenuated by pretreatment with methionine suggesting a glutathione (GSH) dependent phenomenon. Oxidative stress in the mitochondria and inappropriate MPT are important in the pathogenesis of apoptotic liver cell injury in INH-RMP hepatotoxicity. The phenomenon is GSH dependent and methionine supplementation might have a protective role.

Triploid and Diploid Rainbow Trout Do Not Differ in Their Stress Response to Transportation

AbstractWe examined the neuroendocrine and cellular stress responses of diploid and triploid rainbow trout Oncorhynchus mykiss to transportation. Juvenile diploid and triploid rainbow trout (28 and 26 g/fish average weight, respectively) were stocked at 100 g/L in replicate 70‐L tanks and subjected to transportation for an 8‐h period. Subsequent levels of plasma cortisol and glucose and of cellular hepatic glutathione (GSH) and heat shock protein 70 (Hsp70) were similar between ploidy groups, indicating that triploid fish respond to transportation in much the same way as diploid fish. A stationary treatment was also included that involved confinement of experimental fish in similar tanks without transport to determine to what extent high‐density containment contributed to the stress response in the absence of the noise and vibration of transport. Unexpectedly, fish in the stationary treatment had significantly higher plasma cortisol and glucose levels than the transported fish; however, this might be attributable to a confounding effect of hyperoxia, as oxygen levels fluctuated between 150% and 460% saturation in the stationary tank, while those in the transported tank remained within 100–200% saturation. We suggest that when long stops are necessary while transporting fish, water agitators be used to preclude the additional stress of excessive gas saturation. This may be particularly important for triploid fish, which had lower hepatic GSH levels than diploid fish as well as a low level of mortality in the stationary treatment, unlike the diploid fish.

The influence of curcumin and manganese complex of curcumin on cadmium-induced oxidative damage and trace elements status in tissues of mice

Curcumin (diferuoyl methane) from turmeric is a well-known biologically active compound. It has been shown to ameliorate oxidative stress and it is considered to be a potent cancer chemopreventive agent. In our previous study the antioxidative effects of curcumin in cadmium exposed animals were demonstrated. Also manganese exerts protective effects in experimental cadmium intoxication. The present study examined the ability of the manganese complex of curcumin (Mn-curcumin) and curcumin to protect against oxidative damage and changes in trace element status in cadmium-intoxicated male mice. Curcumin or Mn-curcumin were administered at equimolar doses (0.14 mmol/kg b.w.) for 3 days, by gastric gavages, dispersed in methylcellulose. One hour after the last dose of antioxidants, cadmium chloride (33 micromol/kg) was administered subcutaneously. Both curcumin and Mn-curcumin prevented the increase of hepatic lipid peroxidation -- expressed as MDA level, induced by cadmium intoxication and attenuated the Cd-induced decrease of hepatic GSH level. No change in hepatic glutathione peroxidase or catalase activities was found in Cd-exposed mice. A decreased GSH-Px activity was measured in curcumin and Mn-curcumin alone treated mice. Neither curcumin nor Mn-curcumin treatment influenced cadmium distribution in the tissues and did not correct the changes in the balance of essential elements caused by Cd-treatment. The treatment with Mn-curcumin increased the Fe and Mn content in the kidneys of both control and Cd-treated mice and Fe and Cu content in the brain of control mice. In conclusion, regarding the antioxidative action, introducing manganese into the curcumin molecule does not potentiate the studied effects of curcumin.

Protective Effect of MESNA (2-Mercaptoethane Sulfonate) Against Hepatic Ischemia/Reperfusion Injury in Rats

Reoxygenation of ischemic tissue generates various reactive oxygen metabolites (ROMs), which have a deleterious effect on various cellular functions. We evaluated the possible protective effect of 2-mercaptoethane sulfonate (MESNA) on hepatic ischemia/reperfusion (I/R) injury. Wistar albino rats were subjected to 45-min hepatic ischemia, followed by 60-min reperfusion. 2-Mercaptoethane sulfonate, 150 mg/kg, or saline was given intraperitoneally (i.p.) twice, 15 min before ischemia and immediately before reperfusioin. We measured serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels to assess liver function. Liver tissue samples were taken to measure the levels of malondialdehyde (MDA), an end-product of lipid peroxidation; glutathione (GSH), a key antioxidant; and myeloperoxidase (MPO) activity, as an indirect index of neutrophil infiltration. We also measured hepatic collagen content, as a fibrosis marker. Plasma ALT and AST levels were higher in the I/R group than in the control group, but this increase was significantly decreased by MESNA treatment. Hepatic GSH levels, which were significantly depressed by I/R, increased back to the control levels in the MESNA-treated I/R group. Increases in tissue MDA levels and MPO activity caused by I/R injury decreased back to the control levels after MESNA treatment. Similarly, the increased hepatic collagen content in the I/R group decreased to the level of the control group after MESNA treatment. The fact that MESNA alleviated I/R-induced injury of the liver and improved hepatic structure and function suggests that its antioxidant and oxidant scavenging properties may be of therapeutic value in protecting the liver against oxidative injury caused by I/R.

Protective Effect of Potato Peel Powder in Ameliorating Oxidative Stress in Streptozotocin Diabetic Rats

The potential of dietary potato peel (PP) powder in ameliorating oxidative stress (OS) and hyperglycemia was investigated in streptozotocin (STZ)-induced diabetic rats. In a 4-week feeding trial, incorporation of potato peel powder (5 and 10%) in the diet of diabetic rats was found to significantly reduce the plasma glucose level and also reduce drastically the polyuria of STZ diabetic rats. The total food intake was significantly reduced in the diabetic rats fed 10% PP powder compared to the control diabetic rats. However, the body weight gain over 28 days was nearly four times greater in PP powder supplemented diabetic rats (both at 5 and 10%) compared to the control diabetic rats. PP powder in the diet also decreased the elevated activities of serum transaminases (ALT and AST) and nearly normalized the hepatic MDA and GSH levels as well as the activities of specific antioxidant enzymes in liver of diabetic rats. The result of these studies clearly establishes the modulatory propensity of PP against diabetes induced alterations. Considering that potato peels are discarded as waste and not effectively utilized, these results suggest the possibility that PP waste could be effectively used as an ingredient in health and functional food to ameliorate certain disease states such as diabetes.

Supplementation with N-acetylcysteine and taurine failed to restore glutathione content in liver of streptozotocin-induced diabetics rats but protected from oxidative stress

Rats were rendered diabetic with streptozotocin and supplemented or not with N-acetylcysteine (NAC) and taurine (TAU). The liver was examined for the quantity of glutathione (GSH), both total and oxidised (GSSG), by HPLC assay. Moreover, the liver expression of gamma-glutamyl-cysteine synthetase, cysteine dioxygenase and heme oxygenase 1 was evaluated. Streptozotocin-diabetic rats showed decreased levels of liver glutathione (GSH); dietary supplementation with the antioxidants NAC and TAU failed to restore liver GSH to the level of control rats. Gamma-glutamyl-cysteine synthetase expression was not reduced in the diabetic rats, so the low hepatic GSH level in the supplemented diabetic rats cannot be ascribed to decreased expression of the biosynthetic key enzyme. Moreover, the diabetic rats showed no evidence of increased expression of cysteine dioxygenase, which could have indicated that NAC-derived cysteine was consumed in metabolic pathways different from GSH synthesis. However, NAC+TAU treatment provided partial protection from glutathione oxidation in the liver of diabetic rats; moreover, the antioxidant treatment reduced the hepatic overexpression of heme oxygenase 1 (HO-1) mRNA which was detected in the diabetic rats. In conclusion, although NAC was not able to restore liver GSH levels, the antioxidant treatment restrained GSH oxidation and HO-1 overexpression, which are markers of cellular oxidative stress: diabetic rats probably exploit NAC as an antioxidant itself rather than as a GSH precursor.

Oxytocin Protects Against Sepsis-Induced Multiple Organ Damage: Role of Neutrophils

Sepsis, commonly associated with enhanced generation of reactive oxygen metabolites, leads to multiple organ dysfunctions. The neurohypophyseal hormone oxytocin (OT), released during social contact, was recently shown to modulate the immune and inflammatory processes. We investigated the protective role of OT against sepsis-induced pelvic inflammation. Under anesthesia, sepsis was induced in female Sprague-Dawley rats (200-250 g) by cecal ligation and perforation method. Sham-operated rats served as controls. Either saline or OT (1 mg/kg) was given subcutaneously immediately after and at the 16th hour, and rats were decapitated at the 24th hour of sepsis induction. Colon, uterus, and liver samples were obtained for the histopathological analysis of damage and for the measurement of myeloperoxidase (MPO) activity, indicating neutrophil infiltration, malondialdehyde (MDA), indicating lipid peroxidation, and glutathione (GSH), a key antioxidant, levels. Colonic, uterine and liver MDA levels in the sepsis group were significantly increased (P < 0.01-P < 0.001), while colonic and uterine GSH levels were decreased (P < 0.05-P < 0.01) when compared to the control group. OT treatment reversed the MDA and GSH levels back to the control levels, while hepatic GSH levels were not altered. MPO activity in the colon and liver was increased by sepsis (P < 0.05-P < 0.001) while OT treatment abolished the elevated MPO activity. Collagen levels in the uterus and liver were increased by sepsis (P < 0.01) and OT treatment reduced the collagen levels in both tissues (P < 0.01-P < 0.05). Serum TNF-alpha levels were significantly increased by sepsis (P < 0.001) and OT treatment abolished the sepsis-induced increase in TNF-alpha levels. OT protects against sepsis-induced oxidative damage by acting as an antioxidant agent and its protective effect in the colon and liver appears to be dependent on its inhibitory effect on neutrophil infiltration. Our results suggest that OT may have a therapeutic value in limiting sepsis-associated multiple organ damage.

Protection of rats against 3-butene-1,2-diol-induced hepatotoxicity and hypoglycemia by N-acetyl- l-cysteine

3-Butene-1,2-diol (BDD), an allylic alcohol and major metabolite of 1,3-butadiene, has previously been shown to cause hepatotoxicity and hypoglycemia in male Sprague–Dawley rats, but the mechanisms of toxicity were unclear. In this study, rats were administered BDD (250 mg/kg) or saline, ip, and serum insulin levels, hepatic lactate levels, and hepatic cellular and mitochondrial GSH, GSSG, ATP, and ADP levels were measured 1 or 4 h after treatment. The results show that serum insulin levels were not causing the hypoglycemia and that the hypoglycemia was not caused by an enhancement of the metabolism of pyruvate to lactate because hepatic lactate levels were either similar (1 h) or lower (4 h) than controls. However, both hepatic cellular and mitochondrial GSH and GSSG levels were severely depleted 1 and 4 h after treatment and the mitochondrial ATP/ADP ratio was also lowered 4 h after treatment relative to controls. Because these results suggested a role for hepatic cellular and mitochondrial GSH in BDD toxicity, additional rats were administered N-acetyl- l-cysteine (NAC; 200 mg/kg) 15 min after BDD administration. NAC treatment partially prevented depletion of hepatic cellular and mitochondrial GSH and preserved the mitochondrial ATP/ADP ratio. NAC also prevented the severe depletion of serum glucose concentration and the elevation of serum alanine aminotransferase activity after BDD treatment without affecting the plasma concentration of BDD. Thus, depletion of hepatic cellular and mitochondrial GSH followed by the decrease in the mitochondrial ATP/ADP ratio was likely contributing to the mechanisms of hepatotoxicity and hypoglycemia in the rat.

Long-term administration of aqueous garlic extract (AGE) alleviates liver fibrosis and oxidative damage induced by biliary obstruction in rats

The aim of this study was to assess the antioxidant and antifibrotic effects of chronic administration of aqueous garlic extract on liver fibrosis induced by biliary obstruction in rats. Liver fibrosis was induced in male Wistar albino rats by bile duct ligation and scission (BDL). Aqueous garlic extract (AGE, 1 ml/kg, i.p., corresponding to 250 mg/kg) or saline was administered for 28 days. At the end of the experiment, rats were killed by decapitation. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels were determined to assess liver functions and tissue damage, respectively. Tumor necrosis factor-alpha (TNF-α) was also assayed in serum samples. Liver tissues were taken for determination of the free radicals, renal malondialdehyde (MDA) levels, an end product of lipid peroxidation; glutathione (GSH) levels, a key antioxidant; and myeloperoxidase (MPO) activity, as an indirect index of neutrophil infiltration. Hepatic collagen content, as a fibrosis marker was also determined. Serum AST, ALT, LDH, and TNF- α levels were elevated in the BDL group as compared to control group, while this increase was significantly decreased by AGE treatment. Hepatic GSH levels, significantly depressed by BDL, were elevated back to control levels in AGE-treated BDL group. Increases in tissue free radical and MDA levels and MPO activity due to BDL were reduced back to control levels by AGE treatment. Similarly, increased hepatic collagen content in the BDL rats was reduced to the level of the control group with AGE treatment. Since AGE administration alleviated the BDL-induced oxidative injury of the liver and improved the hepatic structure and function, it seems likely that AGE with its antioxidant and antifibrotic properties, may be of potential therapeutic value in protecting the liver fibrosis and oxidative injury due to biliary obstruction.

Effects of Antioxidant Vitamins on Glutathione Depletion and Lipid Peroxidation Induced by Restraint Stress in the Rat Liver

Stress as a cofactor has been reported to affect the progression and severity of several diseases. The influence of stress on the liver is of interest from the clinical point of view because stress plays a potential role in aggravating liver diseases in general and hepatic inflammation in particular, probably through generation of reactive oxygen species. The present study was undertaken to investigate the potential of the antioxidant vitamins A (retinol), E (tocopherol) and C (ascorbic acid) individually and in combination (vitamin E + C) to modulate restraint stress-induced oxidative changes. These effects were determined by measuring changes in hepatic levels of free radical scavenging enzymes such as superoxide dismutase (SOD), glutathione-S-transferase (GST) and catalase, as well as levels of total glutathione (GSH), malondialdehyde (MDA), aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Immobilisation was achieved by placing the animals in wire mesh cages of their size. The rats were orally administered vitamins A, E and C individually and in combination (E + C) prior to and after 6 hours of immobilisation stress exposure. The hepatic levels of SOD, GST, catalase, GSH and MDA were determined by spectrophotometric methods. Liver SOD activity was assayed by monitoring the amount of enzyme required to inhibit autoxidation of pyrogallol by 50%. Hepatic GST was monitored by following the increase in absorbance at 340 nm of CDNB-GSH conjugate generated due to GST catalysis between GSH and CDNB. Catalase activity in liver tissues was determined using peroxidase as the substrate. Lipid peroxidation was measured by determining the level of thiobarbituric acid reactive substances. ALT and AST were determined by commercial kits. Six hours of immobilisation stress caused a decrease in liver levels of SOD (p = 0.001), catalase (p = 0.031), GST (p = 0.021) and GSH (0.013), while levels of MDA (p = 0.0015), AST (p = 0.05) and ALT (p = 0.046) were increased compared with non-stressed control rats. Both pre-vitamin stress and post-vitamin stress treatments either alone or in combination were associated with increased normalisation of these parameters towards control values, with post-vitamin treatment being the more effective of the two. Vitamins E and C individually were found to be more effective in restoring the endogenous antioxidant system than vitamin A. The combined vitamin (E + C) post-stress treatment was found to be effective but not additive in combating hepatic oxidative stress. The beneficial effects of these vitamin treatments were also reflected in reversions of altered AST and ALT levels towards their control values. Vitamins E or C alone or in combination can be given as prophylactic/therapeutic supplements for combating scavenging free radicals generated in liver tissue. This approach may reduce oxidative stress caused by diseases such as cirrhosis.

Melatonin Improved the Disturbances in Hepatic Prooxidant and Antioxidant Balance and Hepatotoxicity Induced by a High Cholesterol Diet in C57BL/6J Mice

We examined the effect of melatonin in prooxidant and antioxidant state in the liver of C57BL/6J mice fed on a high cholesterol (HC) diet. Mice were fed with normal mice chow containing 1.5% cholesterol and 0.5% cholic acid for 4 months without and with melatonin (10 mg/L in drinking water) treatment. HC diet was observed to increase malondialdehyde (MDA) and diene conjugate (DC) levels in the liver. This diet lowered glutathione (GSH), alpha-tocopherol, and total ascorbic acid levels as well as glutathione peroxidase (GSH-Px) and glutathione transferase (GST) activities in the liver, but hepatic superoxide dismutase (SOD) activity remained unchanged. Although melatonin treatment did not affect these parameters in mice fed a normal diet, it reduced hepatic MDA and DC levels in mice fed an HC diet. Hepatic alpha-tocopherol and ascorbic acid levels increased, but hepatic GSH levels remained unchanged in the melatonin-treated HC group as compared to the HC group. Melatonin treatment was found to increase liver GSH-Px and GST activities in mice fed an HC diet. However, SOD activity did not alter in the liver of hypercholesterolemic mice following melatonin treatment. In addition, the histopathological lesions observed in the cholesterol-plus-melatonin group were less severe than those seen in the cholesterol group. According to these observations, we can say that melatonin treatment has an ameliorating effect on the disturbances in prooxidant and antioxidant balance and histopathological lesions in the liver of mice following cholesterol feeding.

Arsenic antagonism studies with monoisoamyl DMSA and zinc in male mice

Administration of zinc either alone or in combination with monoisoamyl dimercaptosuccinic acid (DMSA) during and post-arsenic exposure was investigated in male mice. The animals were administered 2 mg kg −1 arsenic as sodium arsenite, intraperitoneally, once daily for 5 days either alone or in combination with 10 mg kg −1, zinc (as zinc acetate, orally), 50 mg kg −1 monoisoamyl dimercaptosuccinic acid (MiADMSA) given orally (p.o.), 2 h after arsenic administration. Another group of arsenic treated animals was given both zinc (10 mg kg −1) and MiADMSA (50 mg kg −1, p.o.). Animals were sacrificed 24 h after the last dose. In another set of experimentation, arsenic pre-exposed mice (2 mg kg −1, i.p. for 5 days) were treated with saline, zinc, MiADMSA or zinc plus MiADMSA for next 3 days and sacrificed thereafter. Exposure to arsenic led to a significant inhibition of blood δ-aminolevulinic acid dehydratase (ALAD), depletion of glutathione (GSH) level and marginal elevations of zinc protoporphyrin (ZPP). Arsenic exposure caused a significant decrease in hepatic and renal GSH level and an increase in liver oxidized glutathione (GSSG) and liver and kidney thiobarbituric acid reactive substance (TBARS) levels. Concomitant administration of zinc with arsenic provided significant protection to blood ALAD activity while, GSH and ZPP levels remained unaltered. Co-administration of MiADMSA with arsenic significantly prevented accumulation of arsenic in blood, liver and kidney while, zinc had no effect on tissue arsenic concentration. Combined administration of zinc and MiADMSA had no major additional beneficial effects over their individual effects. Interestingly, post-arsenic exposure treatment with MiADMSA provided significant recovery in blood ALAD activity while, zinc supplementation alone had no effect. The best results however, were obtained when MiADMSA was administered along-with zinc. Most of the biochemical variables indicative of hepatic oxidative stress responded favorably to MiADMSA treatment while, zinc administration had no effect. Administration of MiADMSA significantly depleted arsenic concentration from the soft tissues while, combined zinc and MiADMSA had no additional beneficial effect over the individual effect of MiADMSA. The results thus lead us to conclude that in order to achieve best effects of chelation therapy, co-administration of zinc with chelator might be preferred. However, detailed experimental studies with variable doses and after chronic arsenic exposure are required.

The healing-promoting effect of saliva on skin burn is mediated by epidermal growth factor (EGF): role of the neutrophils

Local skin trauma induces inflammatory responses resulting in local tissue and distant organ injury. EGF, a polypeptide hormone, mainly produced in saliva, is one of the major accelerators in wound healing. Wistar albino rats of both sexes received either bovine serum albumin or EGF (10 μg/kg) subcutaneously before a circular (18 mm diameter) partial thickness burn was induced. Afterwards, some rats were placed in separate cages to prevent licking, while the others were caged together to allow wound-licking. Treatments were continued for 5 more days and on the 5th day animals were decapitated. Histopathological analysis of skin damage and dermal myeloperoxidase (MPO) activity, as an index for neutrophil activity, were evaluated. Oxidant injury to the liver and intestines was determined by measuring glutathione (GSH) and malondialdehyde (MDA) levels, as well as MPO activity. The results demonstrate that healing of the burn wound on the skin is accelerated by both wound-licking and EGF administration, which also attenuated tissue neutrophil accumulation, suggesting the role of neutrophils as the source of mediators involved in delayed epithelial regeneration. Moreover, local dermal burn results in oxidant injury to the liver, concomitant with significant elevations in hepatic and intestinal GSH levels. Exogenous administration of EGF at physiological doses had no effect on inflammatory responses of the distant organs, while allowing the rats to lick the wound reduced the oxidant injury to the liver. Since saliva or EGF enhances skin wound healing, topical use of EGF-rich artificial saliva merits consideration for its use in burn patients.

Influence of feeding schedule on 24-h rhythm of hepatotoxicity induced by acetaminophen in mice.

The influence of feeding schedule on the chronopharmacological aspects of acetaminophen (APAP) was examined in mice housed under 12-h light/dark cycle (lights on from 7:00 AM to 7:00 PM) with food and water ad libitum feeding (ALF) or under repeated time-restricted feeding (feeding time between 9:00 AM and 5:00 PM) for 2 weeks before the experiment. For the ALF group, there was a significant 24-h rhythm of mortality after APAP (600 mg/kg i.p.) injection. Peak mortality was observed after APAP injection at 9:00 PM and 1:00 AM, and nadir mortality was observed after drug injection at 9:00 AM. Hepatotoxicity after APAP (300 mg/kg i.p.) injection at 9:00 PM was significantly more severe than that after drug injection at 9:00 AM. Immunohistochemical staining using anti-APAP antibody 2 h after APAP injection was detected in centrilobular hepatocytes after drug injection at 9:00 PM but not after drug injection at 9:00 AM. CYP2E1 activity and hepatic glutathione (GSH) levels in untreated mice showed significant 24-h rhythms associated with APAP toxicity rhythm. The reduction in hepatic GSH levels after APAP injection at 9:00 PM was greater than that after drug injection at 9:00 AM. On the other hand, manipulation of the feeding schedule modified APAP hepatotoxicity rhythmicity, CYP2E1 activity, and GSH levels in the liver. Manipulation of the feeding schedule and choosing the most appropriate time of the day for drug administration may help to achieve rational chronopharmacology of some drugs including APAP in specific experimental and clinical situations.

Effects of cyclophosphamide and buthionine sulfoximine on ovarian glutathione and apoptosis

Treatment with the anticancer drug cyclophosphamide (CPA) destroys ovarian follicles. The active metabolites of CPA are detoxified by conjugation with glutathione (GSH). We tested the hypotheses that CPA causes apoptosis in ovarian follicles and that suppression of ovarian GSH synthesis before CPA administration enhances CPA-induced apoptosis. Proestrous rats were given two injections, 2 h apart, with (1) saline, then saline; (2) saline, then 50 mg/kg CPA; (3) saline, then 300 mg/kg CPA; or (4) 5 mmol/kg buthionine sulfoximine (BSO) to inhibit glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis, and then 50 mg/kg CPA. Statistically significantly increased DNA fragmentation by agarose gel electrophoresis and granulosa cell apoptosis by TUNEL were observed in the CPA-treated ovaries 24 h after the second injection, but BSO did not enhance the effect of 50 mg/kg CPA. We next tested the hypothesis that CPA depresses ovarian GSH concentration and expression of the rate-limiting enzyme in GSH synthesis, GCL. Proestrous rats were injected with 300 or 50 mg/kg CPA or vehicle and were sacrificed 8 or 24 h later. After CPA treatment, ovarian and hepatic GSH levels decreased significantly, and ovarian GCL subunit mRNA levels increased significantly. There were no significant changes in GCL subunit protein levels. Finally, we tested the hypothesis that GSH depletion causes apoptosis in ovarian follicles. Proestrous or estrous rats were injected with 5 mmol/kg BSO or saline at 0700 and 1900 h. There was a significant increase in the percentage of histologically atretic follicles and a nonsignificant increase in the percentage of apoptotic, TUNEL-positive follicles 24 h after onset of BSO treatment. Our results demonstrate that CPA destroys ovarian follicles by inducing granulosa cell apoptosis and that CPA treatment causes a decline in ovarian GSH levels. More pronounced GSH suppression achieved after BSO treatment did not cause a statistically significant increase in follicular apoptosis. Thus, GSH depletion does not seem to be the mechanism by which CPA causes follicular apoptosis.

Effects of cyclophosphamide and buthionine sulfoximine on ovarian glutathione and apoptosis

Treatment with the anticancer drug cyclophosphamide (CPA) destroys ovarian follicles. The active metabolites of CPA are detoxified by conjugation with glutathione (GSH). We tested the hypotheses that CPA causes apoptosis in ovarian follicles and that suppression of ovarian GSH synthesis before CPA administration enhances CPA-induced apoptosis. Proestrous rats were given two injections, 2 h apart, with (1) saline, then saline; (2) saline, then 50 mg/kg CPA; (3) saline, then 300 mg/kg CPA; or (4) 5 mmol/kg buthionine sulfoximine (BSO) to inhibit glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis, and then 50 mg/kg CPA. Statistically significantly increased DNA fragmentation by agarose gel electrophoresis and granulosa cell apoptosis by TUNEL were observed in the CPA-treated ovaries 24 h after the second injection, but BSO did not enhance the effect of 50 mg/kg CPA. We next tested the hypothesis that CPA depresses ovarian GSH concentration and expression of the rate-limiting enzyme in GSH synthesis, GCL. Proestrous rats were injected with 300 or 50 mg/kg CPA or vehicle and were sacrificed 8 or 24 h later. After CPA treatment, ovarian and hepatic GSH levels decreased significantly, and ovarian GCL subunit mRNA levels increased significantly. There were no significant changes in GCL subunit protein levels. Finally, we tested the hypothesis that GSH depletion causes apoptosis in ovarian follicles. Proestrous or estrous rats were injected with 5 mmol/kg BSO or saline at 0700 and 1900 h. There was a significant increase in the percentage of histologically atretic follicles and a nonsignificant increase in the percentage of apoptotic, TUNEL-positive follicles 24 h after onset of BSO treatment. Our results demonstrate that CPA destroys ovarian follicles by inducing granulosa cell apoptosis and that CPA treatment causes a decline in ovarian GSH levels. More pronounced GSH suppression achieved after BSO treatment did not cause a statistically significant increase in follicular apoptosis. Thus, GSH depletion does not seem to be the mechanism by which CPA causes follicular apoptosis.

Retinoid X receptor alpha regulates glutathione homeostasis and xenobiotic detoxification processes in mouse liver.

Retinoid X receptor alpha (RXRalpha) plays a pivotal role in regulating liver metabolism. RXRalpha-mediated gene expression involved in amino acid metabolism was examined using the NIA Mouse 15K cDNA microarray containing 15,000 different expressed sequence tags. Seven amino acid metabolic genes, three of which encode enzymes involved in phase II detoxification process, were identified as RXRalpha target genes in mouse liver. Glutamate-cysteine ligase catalytic subunit (GCLC), glutathione S-transferasemu, and glutathione peroxidase 1 were down-regulated in the liver of hepatocyte RXRalpha-deficient mice. The down-regulation of GCLC in RXRalpha-deficient mice led to 40% and 45% reductions in the rate of glutathione (GSH) synthesis and level of hepatic GSH, respectively. Primary hepatocytes from RXRalpha-deficient mice were more sensitive to t-butylhydroperoxide-induced oxidative stress. However, GSH diminished RXRalpha-deficient mice were resistant to acetaminophen (APAP)-induced hepatotoxicity. Analysis of phase I detoxification genes revealed that CYP1A2 and CYP3A11 were up-regulated in wild-type mice but down-regulated in RXRalpha-deficient mice after APAP administration. Taken together, the data indicate that RXRalpha centrally regulates both phase I and phase II drug metabolism and detoxification. Regulation of hepatic GSH levels by RXRalpha is essential to protect hepatocytes from oxidative stress, whereas up-regulation of phase I drug metabolism genes by RXRalpha may render the liver more sensitive to APAP-induced toxicity.