Glutathione Depletion In Liver Research Articles

Alcohol spiked with zolpidem and midazolam potentiates inflammation, oxidative stress and organ damage in a mouse model.

Crime-related spiking of alcoholic drinks with prescription drugs is quite common and has been happening for centuries. This study, therefore, evaluated the effects of oral administration of alcohol spiked with the zolpidem and midazolam potent sedatives on inflammation, oxidative stress and various organ damage in male Swiss albino mice. Mice were randomly assigned into six treatment groups; the first group constituted the normal control, the second group received 50mg/kg body weight of zolpidem only, the third group received 50mg/kg body weight zolpidem dissolved in 5g/kg alcohol, the fourth group received 50mg/kg midazolam only, the fifth group received midazolam (50mg/kg) dissolved in 5g/kg alcohol and the sixth group received 5g/kg alcohol. Alcohol-induced significant reduction in neurological function and altered blood hematological indicators. Such neurological impairment and negative effects on blood were exacerbated in mice administered with spiked alcohol. Additionally, midazolam and zolpidem enhanced alcohol-driven elevation of liver function markers; the serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) gamma glutamyltransferase (GGT), total bilirubin and alkaline phosphatase. Exposure to alcohol and/or spiked alcohol led to significant augmentation of nitric oxide and malonaldehyde, with concomitant depletion of liver glutathione (GSH) levels. Similarly, serum levels of pro-inflammatory cytokines tumor necrosis factor alpha and interferon-gamma were increased by co-exposure with midazolam or zolpidem. Alcohol-induced hepatotoxicity and nephrotoxicity were amplified by exposure to alcohol spiked with midazolam/zolpidem. Exposure to alcohol spiked with midazolam or zolpidem appears to exacerbate neurological deficits, inflammation, oxidative stress, and organ damage.

NF‐κB and AMPK/PI3K/Akt signaling pathways are involved in the protective effects of Platycodon grandiflorum saponins against acetaminophen‐induced acute hepatotoxicity in mice

Acute liver injury (ALI) induced by acetaminophen (APAP) overdose is the most common cause of drug-induced liver injury. Saponins from Platycodon grandiflorum (PGSs) ameliorate alcohol-induced hepatotoxicity and enhance human lung carcinoma cell death via AMPK signaling pathway. However, whether PGS could protect from APAP-induced ALI through AMPK activation and its downstream signals is still poorly elucidated. This work investigated the protective effect and the underlying mechanisms of PGS against APAP-induced liver toxicity in mouse. PGS was administered at 15 or 30mg/kg i.g./day for 1week before a single injection of APAP (250mg/kg, i.p.) 1hr after last treatment of PGS. Serum alanine/aspartate aminotransferases, liver tumor necrosis factor-α and interleukin-1β levels, liver malondialdehyde formation, liver glutathione depletion, cytochrome P450 E1, and 4-hydroxynonenal levels were measured to demonstrate the protective efficacy of PGS against APAP-induced ALI. Liver histological observation provided further evidence on PGS's protective effects. PGS treatment altered the phosphorylation of AMPK and PI3K/Akt, as well as the downstream signals including Bcl-2 family, caspase, and NF-κB in a dose-dependent manner. In conclusion, we demonstrate that PGS exhibits a significant liver protection against APAP-induced ALI, mainly through NF-κB and AMPK/PI3K/Akt signaling pathways.

Regulatory Effect of Iranian Hypericum Scabrum Essential Oils on Hepatic Metabolizing Enzymes in Rats Treated by Acetaminophen

Hypericum Scabrum is a traditional herbal drug with various therapeutic effects. The aim of the present study was to assess the modulating effect of Iranian Hypericum Scabrum essential oils on hepatic metabolizing enzymes in rats treated by acetaminophen. For this purpose, the activities of cytochrome P450 (CYP450), glutathione s-transferase (GST) and liver function markers (ALT, AST, ALP) together with level of GSH measured analytically in time intervals (2, 4, 8, 16 & 24 h) after treatments confirmed by histophatological consideration in rat livers. The results indicated that acetaminophen (500 mg/kg bw, i.p) was associated with extensive liver damages as judged by histological examinations. These results were corroborated with induction of CYP450 and reduction of GST activities concomitant with glutathione depletion in liver. Hepatic function markers (ALT, AST, ALP) was also significantly increased in treated rats. Treatment of Hypericum Scabrum essential oil (100 and 200 mg/kg b.w, i.p) concomitant with acetaminophen could modulate these parameters to normal value. These data confirmed by histopathological findings. Based on these data it appears that Hypericum Scabrum essential oil can modulate liver damages induce by acetaminophen through modulating its metabolism.

Hepatoprotective and antioxidant activity of Karisalai Karpam, a polyherbal Siddha formulation against acetaminophen-induced hepatic damage in rats.

Background:The usage of Siddha medicine in Tamil Nadu and several parts of Southern India has considerably increased over the past two decades and it is steadily crossing the various geographies owing to its inexpensiveness compared to conventional medicines and has fairly high acceptance rates because of its herbal origin and therefore its nontoxic nature.Aim:This study aims to investigate the anti-hepatotoxic and antioxidant potential of the Karisalai Karpam formulation.Materials and Methods:Karisalai Karpam tablet at 50, 100, and 200 mg/kg/day, p.o. doses were administered orally to rats for three consecutive days. Single dose of acetaminophen (3 g/kg, p.o.) was administered on the 3rd day. Animals were sacrificed 48 h after the administration of acetaminophen, and their serum bilirubin, different hepatic enzymes and in vivo antioxidant activity were estimated.Statistical Analysis:Data were evaluated using analysis of variance, followed by Tukey tests. A level of P < 0.05 was considered statistically significant.Results:Pretreatment with Karisalai Karpam tablet showed dose-dependent hepatoprotective activity. Karisalai Karpam tablet (200 mg/kg) reduces serum glutamic oxaloacetate transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase and total bilirubin, direct bilirubin by 67.8%, 72.3%, 47.6%, 61.3% and 62.9% respectively compared to disease control group. A significant increase (P < 0.001) in antioxidant enzyme level was observed in Karisalai Karpam treated animals. At higher doses, Karisalai Karpam prevented the depletion of glutathione in liver tissue.Conclusion:Results confirmed that Karisalai Karpam tablet could protect the liver against acetaminophen-induced oxidative damage possibly by increasing the antioxidant defence mechanism in rats.

Efficacy of crude extract of Emblica officinalis (amla) in arsenic-induced oxidative damage and apoptosis in splenocytes of mice.

Introduction:Arsenic, an environmental contaminant naturally occurred in groundwater and has been found to be associated with immune-related health problems in humans.Objective:In view of increasing risk of arsenic exposure due to occupational and non-occupational settings, the present study has been focused to investigate the protective efficacy of amla against arsenic-induced spleenomegaly in mice.Results:Arsenic exposures (3 mg/kg body weight p.o for 30 days) in mice caused an increase production of ROS (76%), lipid peroxidation (84%) and decrease in the levels of superoxide dismutase (53%) and catalase (54%) in spleen as compared to controls. Arsenic exposure to mice also caused a significant increase in caspases-3 activity (2.8 fold) and decreases cell viability (44%), mitochondrial membrane potential (47%) linked with apoptosis assessed by the cell cycle analysis (subG1-28.72%) and annexin V/PI binding in spleen as compared to controls. Simultaneous treatment of arsenic and amla (500 mg/kg body weight p.o for 30 days) in mice decreased the levels of lipid peroxidation (33%), ROS production (24%), activity of caspase-3 (1.4 fold), apoptosis (subG1 12.72%) and increased cell viability (63%), levels superoxide dismutase (80%), catalase (77%) and mitochondrial membrane potential (66%) as compared to mice treated with arsenic alone.Conclusions:Results of the present study indicate that the effect of arsenic is mainly due to the depletion of glutathione in liver associated with enhanced oxidative stress that has been found to be protected following simultaneous treatment of arsenic and amla.

NFR2 EXPRESSION IN APAP ACUTE INTOXICATION

Acetaminophen (APAP) overdose promotes the formation of NAPQI, the depletion of liver glutathione and oxidative stress. N‐acetilcysteine (NAC) is a cysteine donator that is a precursor for the synthesis of GSH. Nrf2 is a transcription factor, important in antioxidant response pathway, promoting the expression of phase II enzyme and the regulation of liver GSH. Male C57 mices, were fasted for 16h prior to the application of APAP (250mg/kg). The treatment with NAC (204mg/kg) occurred 1h after APAP challenge. The collection of liver tissue occurred 24h after treatment. Analysis: Histopathology by score of necrosis fields; hepatic levels of GSH by spectrophotometry and hepatic levels of Vitamin E (VE) by HPLC; the gene expression of NFR2 by RT‐qPCR (Taqman probes). Statistic Analysis: Student. t test (Graphpad Prism 5.0). The group (ANAC) that received treatment with NAC 1h after APAP challenge, showed a reduction of 28% of necrosis (p &lt;0.05). ANAC GSH levels were 1.5 higher than the APAP levels (p &lt;0.05), although VE presents no differences between the groups. The Nfr2 gene expression fold change had almost doubled compared with APAP group (p &lt;0.05). The results show that after the treatment with NAC, the increase of NFR2 gene expression promotes the increase of GSH levels, saving the reduction of VE and consequently, the oxidative stress, protecting the liver from necrosis. Support by FAPESP (2011/12749–0 &amp; 2011/17616–9)

Liver preconditioning induced by iron in a rat model of ischemia/reperfusion

AimsLiver preconditioning against ischemia–reperfusion (IR) injury is a major area of experimental research, in which regulation of gene expression with cytoprotective responses due to transient oxidative stress development has been reported. Considering that significant cytoprotection occurs after exposure to low levels of iron (Fe), we tested the hypothesis that sub-chronic administration of Fe to rats underlying transient oxidative stress preconditions the liver against IR injury. Main methodsAnimals received six doses (50mg Fe–dextran/kg ip) every second day during 10days, before partial IR (vascular clamping) or sham laparotomy (control). Transient oxidative stress was defined by liver glutathione and protein carbonyl contents (24, 48, and 72h after Fe treatment). Plasma and liver Fe status and ferritin content (western blot) were assessed in animals not subjected to IR. Liver injury and inflammatory response were evaluated by serum transaminases, liver morphology and serum TNF-α. Fe preconditioning against IR injury was correlated with liver glutathione content and the redox-sensitive NF-κB signaling pathway (EMSA) and western blot analysis of haptoglobin. Key findingsSignificant hepatoprotection against IR injury, underlying transient oxidative stress and enhancement in the total and labile Fe pools, was achieved by Fe administration. Abrogation of IR injury is related to reduced TNF-α response (91%), abolishment of the IR-induced liver glutathione depletion and recovery of the NF-κB signaling pathway (75%), lost during IR. SignificanceSub-chronic Fe administration protects the liver against IR injury through antioxidant and anti-inflammatory responses, with recovery of NF-κB activation and related acute-phase response signaling.

Does inflammation play a role in kava hepatotoxicity?

The pathophysiology of kava hepatotoxicity remains inconclusive. There is circumstantial evidence for the roles of toxic metabolites, inhibition of cyclooxygenase (COX) enzymes and depletion of liver glutathione. Pharmacogenomic effects are likely, particularly for Cytochrome P450 genes. Experimental and clinical cases of hepatotoxicity show evidence of hepatitis. The question remains whether this inflammation is caused by components of kava directly, or indirectly due to the downstream effects.

Liver glutathione depletion after preservation and reperfusion in human liver transplantation

The oxidative stress is an important mechanism responsible for dysfunction after orthotopic liver transplantation (OLT). Glutathione (GSH) low levels after cold storage render the grafts vulnerable to reperfusion injury. Aim of this study was to evaluate GSH and oxidized glutathione (GSSG) liver concentrations, the hepatocellular injury and function in optimal and suboptimal grafts after human OLT. Liver biopsies were taken in 33 patients before the implant and two hours after reperfusion, allowing determination of GSH, GSSG and oxidative stress ratio (GSH/GSSG). Serum transaminases, prothrombin activity (PT) and factor V were measured to evaluate injury and function respectively. Histopathological injury was analyzed by an index of five parameters. There was a decrease in GSH (p<0.01) after reperfusion (0.323 +/- 0.062 ìmol/g to 0.095 +/- 0.01 ìmol/g and 0.371 +/- 0.052 ìmol/g to 0.183 +/- 0.046 ìmol/g) in suboptimal and optimal groups, respectively. An increase of GSSG (p<0.05) occurred after reperfusion (0.172 +/- 0.038 ìmol/g to 0.278 +/- 0.077 ìmol/g and 0.229 +/- 0.048 ìmol/g to 0.356 +/- 0.105 ìmol/g) in suboptimal and optimal groups, respectively. A decrease (p<0.01) occurred in the GSH/GSSG ratio after reperfusion (2.23 +/- 0.31 to 0.482 +/- 0.042 and 2.47 +/- 0.32 to 0.593 +/- 0.068) in suboptimal and optimal groups, respectively. Histopathological injury scores were higher (p<0.05) in the suboptimal group than in optimal (6.46 +/- 0.4 vs. 5.39 +/- 1.1) and showed correlation with PT and factor V in the optimal group (p<0.05). Multivariate analysis pointed steatosis as an independent risk factor to histopathological injury (p<0.05). There was a significant GSH depletion and GSSG formation after cold storage and reperfusion due to a similar oxidative stress in optimal and suboptimal grafts, but these levels were not related to graft viability.

The acute-phase response of the liver in relation to thyroid hormone-induced redox signaling

Recently, we reported that 3,3′,5-triiodothyronine (T 3) induces the expression of redox-sensitive genes as a nongenomic mechanism of T 3 action. In this study, we show that T 3 administration to rats (daily doses of 0.1 mg/kg ip for 3 consecutive days) induced a calorigenic response and liver glutathione depletion as an indication of oxidative stress, with higher levels of interleukin (IL)-6 in serum (ELISA) and hepatic STAT3 DNA binding (EMSA), which were maximal at 48–72 h after treatment. Under these conditions, the protein expression of the acute-phase proteins haptoglobin and β-fibrinogen is significantly augmented, a change that is suppressed by pretreatment with α-tocopherol (100 mg/kg ip) or gadolinium chloride (10 mg/kg iv) before T 3. It is concluded that T 3 administration induces the acute-phase response in rat liver by a redox mechanism triggered at the Kupffer cell level, in association with IL-6 release and activation of the STAT3 cascade, a response that may contribute to reestablishing homeostasis in the liver and extrahepatic tissues exhibiting oxidative stress.

Homocysteine Prevents Total Parenteral Nutrition (TPN)–Induced Cholestasis Without Changes in Hepatic Oxidative Stress in the Rat

The role of oxidative stress in total parenteral nutrition (TPN)-associated cholestasis with liver glutathione depletion was recently shown. The aims of this study were to test the appearance of cholestasis and oxidative stress during TPN, and the hypothesis that reducing oxidative stress with a precursor of glutathione (GSH), homocysteine, would restore bile flow. Three groups of rats (weight, 179-278 g) were studied: 1) D/aa group received dextrose and amino acids (3.4 g/d); 2) D/aa/L group received the same amount of amino acids, and lipids were added on an equicaloric basis (50 kcal/d) with a lowered amount of dextrose; and 3) a control group, which received dextrose perfusion and had free access to chow. A subgroup of D/aa/L rats (n = 6) received a TPN solution containing homocysteine. After 5 days of TPN, bile was collected during 2 hours. In liver homogenates, GSH, thiobarbituric acid reactive substances (TBARS), and carbonyl content of proteins (Prot-CO) were measured to test the level of oxidative stress and hepatic lipid and protein oxidation. After TPN, bile flow was significantly lower in the D/aa group than in the control group. Addition of lipids further decreased bile flow. Addition of homocysteine to TPN with lipids significantly increased bile flow. Aspartate aminotransferase increased significantly in both TPN groups compared with the control group. gamma-Glutamyl transpeptidase was not different among TPN groups. An increased hepatic lipid oxidation was demonstrated by TBARS level in both TPN groups when compared with the control group. However, the liver GSH contents were not different. Protein oxidation was also significantly increased by TPN. The addition of homocysteine to TPN solution increased bile flow without liver injury or changes of lipid and protein oxidation. This study shows that TPN administered to rats induces a decrease of bile flow and an oxidative stress but that the two changes are not directly correlated. Addition of lipids further impairs bile flow but does not increase the occurrence of liver injury. Consequently, it seems more likely that TPN primarily induces a cholestatic effect that in turn induces an oxidative stress rather than inducing an oxidative stress that leads to cholestasis. However, an association of both mechanisms is not totally excluded.

Homocysteine Prevents Total Parenteral Nutrition (TPN)–Induced Cholestasis Without Changes in Hepatic Oxidative Stress in the Rat

ABSTRACTBackgroundThe role of oxidative stress in total parenteral nutrition (TPN)–associated cholestasis with liver glutathione depletion was recently shown. The aims of this study were to test the appearance of cholestasis and oxidative stress during TPN, and the hypothesis that reducing oxidative stress with a precursor of glutathione (GSH), homocysteine, would restore bile flow.MethodsThree groups of rats (weight, 179–278 g) were studied: 1) D/aa group received dextrose and amino acids (3.4 g/d); 2) D/aa/L group received the same amount of amino acids, and lipids were added on an equicaloric basis (50 kcal/d) with a lowered amount of dextrose; and 3) a control group, which received dextrose perfusion and had free access to chow. A subgroup of D/aa/L rats (n = 6) received a TPN solution containing homocysteine. After 5 days of TPN, bile was collected during 2 hours. In liver homogenates, GSH, thiobarbituric acid reactive substances (TBARS), and carbonyl content of proteins (Prot‐CO) were measured to test the level of oxidative stress and hepatic lipid and protein oxidation.ResultsAfter TPN, bile flow was significantly lower in the D/aa group than in the control group. Addition of lipids further decreased bile flow. Addition of homocysteine to TPN with lipids significantly increased bile flow. Aspartate aminotransferase increased significantly in both TPN groups compared with the control group. γ‐Glutamyl transpeptidase was not different among TPN groups. An increased hepatic lipid oxidation was demonstrated by TBARS level in both TPN groups when compared with the control group. However, the liver GSH contents were not different. Protein oxidation was also significantly increased by TPN. The addition of homocysteine to TPN solution increased bile flow without liver injury or changes of lipid and protein oxidation.DiscussionThis study shows that TPN administered to rats induces a decrease of bile flow and an oxidative stress but that the two changes are not directly correlated. Addition of lipids further impairs bile flow but does not increase the occurrence of liver injury. Consequently, it seems more likely that TPN primarily induces a cholestatic effect that in turn induces an oxidative stress rather than inducing an oxidative stress that leads to cholestasis. However, an association of both mechanisms is not totally excluded.

In Vivo Effects of Sambucus nigra L. on CCl4-Induced Hepatotoxicity in Rats

ABSTRACT The effect of different extracts of elder (Sambucus nigra L.) flowers on biochemical reactions in rats before and after CCl4-in-duced hepatotoxicity was examined. Administration of elder flowers impaired antioxidant defense mechanisms, particularly by depletion of liver glutathione (GSH) content and reduction of activities of catalase (CAT) and peroxidase (Px). Rats treated with CCl4 had an expected increase in lipid peroxidation (LP) and a decrease in glutathione reductase (GSH-R), CAT and Px activity. Pretreatment of the rats with elder flower extracts before treatment with CCl4 amplified the CCl4-induced liver injury, increasing LP and reducing GSH content. Treatment with both CCl4 and flower extracts significantly increased the serum trans-aminases: glutamic pyruvic transaminase (SGPT) and glutamic oxalacetic transaminase (SGOT).

Depletion of liver glutathione potentiates the oxidative stress and decreases nitric oxide synthesis in a rat endotoxin shock model.

To verify the effects of liver glutathione depletion on redox status and nitric oxide system in a rat endotoxic shock model. Prospective, randomized, controlled study on rats. A cardiocirculatory research laboratory. A total of 28 Sprague-Dawley male rats (200-250 g body weight) were divided into four experimental groups. Arterial blood, liver, and lung samples were taken from each animal under sodium pentobarbital (40 mg/kg i.p.) anesthesia 4 hrs after lipopolysaccharide (LPS group: 5 mg/kg i.p.; n = 7) or vehicle (control group: isotonic NaCl sterile solution i.p.; n = 7) treatments. Phorone (250 mg/kg i.p.) was injected to deplete glutathione in another two experimental groups of rats 30 mins before LPS (phorone+LPS group; n = 7) or vehicle (phorone group; n = 7) treatments, and 4 hrs later the same samples as in LPS and control groups were taken under anesthesia. Compared with the control group, the LPS group presented higher plasma concentration of end products of nitric oxide metabolism nitrites/nitrates, higher lung activity of inducible nitric oxide synthase, and oxidative stress defined by increased plasma concentration of the lipid peroxides malonaldehyde and 4-hydroxynonenal, and decreased plasma total antioxidant capacity. Treatment with phorone depleted liver glutathione (80% to 90%). In the liver glutathione-depleted animals, the oxidative stress induced by LPS was potentiated and blunted the increases in inducible nitric oxide synthase and plasma nitrites/nitrates. These results show that depletion of the liver glutathione increases the oxidative stress and decreases nitric oxide synthesis of LPS-induced shock in rats.

Sonochemistry of quinones in argon-saturated aqueous solutions: Enhanced cytochrome C reduction: [formula omitted], Raphael C. Lawson and Wilmarie F. Santana, Department of Chemistry, University of Puerto Rico, Humacao, P. R. 00791

Liver preconditioning against ischemia–reperfusion (IR) injury is a major area of experimental research, in which regulation of gene expression with cytoprotective responses due to transient oxidative stress development has been reported. Considering that significant cytoprotection occurs after exposure to low levels of iron (Fe), we tested the hypothesis that sub-chronic administration of Fe to rats underlying transient oxidative stress preconditions the liver against IR injury.Animals received six doses (50 mg Fe–dextran/kg ip) every second day during 10 days, before partial IR (vascular clamping) or sham laparotomy (control). Transient oxidative stress was defined by liver glutathione and protein carbonyl contents (24, 48, and 72 h after Fe treatment). Plasma and liver Fe status and ferritin content (western blot) were assessed in animals not subjected to IR. Liver injury and inflammatory response were evaluated by serum transaminases, liver morphology and serum TNF-α. Fe preconditioning against IR injury was correlated with liver glutathione content and the redox-sensitive NF-κB signaling pathway (EMSA) and western blot analysis of haptoglobin.Significant hepatoprotection against IR injury, underlying transient oxidative stress and enhancement in the total and labile Fe pools, was achieved by Fe administration. Abrogation of IR injury is related to reduced TNF-α response (91%), abolishment of the IR-induced liver glutathione depletion and recovery of the NF-κB signaling pathway (75%), lost during IR.Sub-chronic Fe administration protects the liver against IR injury through antioxidant and anti-inflammatory responses, with recovery of NF-κB activation and related acute-phase response signaling.

Dose dependence of covalent binding of acrylonitrile to tissue protein and globin in rats.

The dose dependence of acrylonitrile (AN) covalent binding to tissue protein, following a single acute exposure over a 100-fold range in dose, was measured. Covalent binding was a linear function of AN dose in the lower dose range (0.02-0.95 mmol AN/kg). The slopes of the dose-response curves indicated that tissues varied by nearly 10-fold in their reactivity with AN. The relative order of covalent binding was as follows: blood > > kidney = liver > forestomach = brain > glandular stomach > > muscle. Similar dose-response behavior was observed for globin total covalent binding and for globin N-(2-cyanoethyl) valine (CEValine) adduct formation. The latter adduct was found to represent only 0.2% of the total AN adduction to globin. Regression of tissue protein binding versus globin total covalent binding or globin CEValine adduct indicated that both globin biomarkers could be used as surrogates to estimate the amount of AN bound to tissue protein. At higher AN doses, above approximately 1 mmol/kg, a sharp break in the covalent binding dose-response curve was observed. This knot value is explained by the nearly complete depletion of liver glutathione and the resultant termination of AN detoxification. The toxicity of AN is known to increase sharply above this dose. The data suggest that a comparison of specific tissue proteins labeled by AN above and below this threshold dose may provide some insight into the mechanism of AN-induced toxicity.

Dose Dependence of Covalent Binding of Acrylonitrile to Tissue Protein and Globin in Rats

LI, J., AND CORBETT, D. (1997). Fundam. Appl. ToxicoL 36, 149-156. The dose dependence of acrylonitrile (AN) covalent binding to tissue protein, following a single acute exposure over a 100-fold range in dose, was measured. Covalent binding was a linear function of AN dose in the lower dose range (0.02-0.95 mmol AN/ kg). The slopes of the dose-response curves indicated that tissues varied by nearly 10-fold in their reactivity with AN. The relative order of covalent binding was as follows: blood S> kidney = liver > forestomach = brain > glandular stomach *> muscle. Similar dose-response behavior was observed for globin total covalent binding and for globin N-(2-cyanoethyl)valine (CEValine) adduct formation. The latter adduct was found to represent only 0.2% of the total AN adduction to globin. Regression of tissue protein binding versus globin total covalent binding or globin CEValine adduct indicated that both globin biomarkers could be used as surrogates to estimate the amount of AN bound to tissue protein. At higher AN doses, above approximately 1 mmol/kg, a sharp break in the covalent binding dose-response curve was observed. This knot value is explained by the nearly complete depletion of liver glutathione and the resultant termination of AN detoxification. The toxicity of AN is known to increase sharply above this dose. The data suggest that a comparison of specific tissue proteins labeled by AN above and below this threshold dose may provide some insight into the mechanism of AN-induced toxicity. O 1997 Soctety of Toxicology. Acrylonitrile (AN) is a vinyl monomer used in the production of synthetic fibers, rubber, and plastics. The estimated production of AN in the United States in 1995 was 3.21 billion pounds, ranking it 40th among manufactured chemicals (Kirschner, 1996).

Diurnal variability of cysteine and glutathione content in the pancreas and liver of the mouse

The sensitivity of organs such as the liver to injury by certain drugs is modulated by the endogenous capacity to synthesize glutathione during periods of increased demand. Recent experimental evidence suggests that glutathione availability could also play an important role in preventing pancreatic injury as well. To better understand the role of cysteine availability in regulating glutathione homeostasis in the pancreas and liver under normal conditions, the diurnal variation in cysteine in mouse pancreas and liver was measured and compared with corresponding measurements of organ glutathione content. Pancreatic cysteine varied significantly over a 24-hr period, dropping to 21 nmol/g at 2 p.m. and rising to 68 nmol/g at 10 P.M. Fasting prevented this diurnal variation in pancreatic cysteine. Pancreatic glutathione was at its lowest at 10 P.M. and rose sharply to a peak at 2 A.M. Fasting had no effect on this diurnal pattern. In contrast to the pancreas, fasting did not prevent the diurnal change in liver cysteine, whereas it caused substantial depletion of liver glutathione. Together, these findings suggest that under normal conditions, pancreatic and liver glutathione content are not determined solely by tissue cysteine availability. Moreover, basal glutathione content is under differing homeostatic mechanisms in the pancreas and the liver.

Glutathione- S-transferase is a target for covalent modification by a halothane reactive intermediate in the guinea pig liver

The anesthetic halothane is bioactivated by the liver cytochrome P450 system to the reactive intermediate, trifluoroacetyl chloride, which can acylate liver protein. Cytosolic glutathione- S-transferase (GST) was identified as a major target for protein adduct formation in guinea pig liver slices exposed to halothane. To determine if GST is also a target in vivo, male Hartley guinea pigs were exposed to 1% halothane in 40% O 2 for 4 h. At 10 h post exposure, livers were removed and microsomal and cytosolic fractions prepared. Past studies have shown these conditions resulted in maximal covalent binding of halothane intermediates to hepatic protein. Protein was isolated by ethanol precipitation and washed with trichloroacetic acid to remove unbound metabolites. Cytosolic GST was isolated by gel filtration and S-hexyl-glutathione affinity chromatography to electrophoretic purity. Protein adducts were quantified using a covalently bound fluorine assay. Covalent binding of a halothane intermediate to cytosolic and microsomal protein was determined as 2.0 ± 0.4 and 13.2 ± 2.3 nmol F/mg protein, respectively. Liver glutathione depletion by buthionine sulfoximine pretreatment produced an increase in covalent binding only to cytosolic proteins (3.3 ± 0.4 nmol F/mg protein). Adduct formation to cytosolic GST was determined to be 4.7 ± 1.6 nmol F/mg protein. Glutathione- S-transferase is a target for covalent modification in the liver following an inhalation exposure to halothane.

Hispidulin protection against hepatotoxicity induced by bromobenzene in mice

The effects of the natural flavonoid hispidulin (6-methoxy-5, 7, 4′-trihydroxyflavone) on bromobenzene-induced hepatotoxicity in mice were investigated. We found a correlation between liver injury and hepatic lipid peroxidation besides a strong liver glutathione depletion due to the toxicant. Hispidulin at doses between 50 and 150 mg/kg i.p. compared favourably with the reference compound N-acetyl-L-cysteine for inhibition of liver injury and lipid peroxidation. The flavonoid at the highest dose tested was also able to counteract reduced glutathione depletion induced by bromobenzene in starved mice. These hepatoprotective effects can be related to the antioxidant properties of hispidulin.