Hepatic Non-protein Sulfhydryl Content Research Articles

Increased hepatotoxicity of acetaminophen in Hsp70i knockout mice

The effect of the inducible forms of 70 kDa heat shock protein (Hsp70i) on acetaminophen (APAP) hepatotoxicity was assessed in an Hsp70i knockout mouse model. Absence of the Hsp70i protein in liver was verified by monitoring Hsp levels in knockout and control mice after heat stress (41.5 degrees C water bath immersion for 30 min). Hsp70i knockout mice were more susceptible to APAP-induced hepatotoxicity than controls, as indicated by elevated serum alanine aminotransferase activities 24 and 48 h after the APAP dose. Increased APAP hepatotoxicity in knockout mice was verified by morphological evaluation of liver sections. The difference in toxic response to APAP between knockout and control strain mice could not be attributed to differences in APAP bioactivation, assessed by measurement of CYP2E1 and glutathione S-transferase activities, hepatic nonprotein sulfhydryl content, or covalent binding of reactive APAP metabolites to proteins. Pretreatment with transient hyperthermia to produce a general upregulation of Hsps resulted in decreased APAP hepatotoxicity in both the knockout and control strains. Among thermally-pretreated mice, hepatotoxicity of APAP was greater in the knockouts compared with the control strain. These observations suggest that increased Hsp70i expression in response to APAP acts to limit the extent of tissue injury. Results further suggest that other factors related to heat stress can also contribute to protection against APAP toxicity.

The contribution of oxidation and deacetylation to acetaminophen nephrotoxicity in female Sprague-Dawley rats

Young adult female rats are more susceptible to acetaminophen (APAP) induced nephrotoxicity than are male rats. The purpose of the present study was to assess the contribution of oxidation and deacetylation to the expression of APAP nephrotoxicity. Male and female rats received APAP (1100 mg kg −1 i.p.) alone or following pretreatment with 1-aminobenzotriazole (ABT), a suicide inhibitor of cytochromes P450, or tri- o-tolylphosphate (TOTP), an irreversible carboxyesterase inhibitor. Rats were sacrificed 6 or 24 h following administration of 1100 mg APAP kg −1 containing [ring- 14C]APAP. Blood urea nitrogen (BUN) concentration was used as an index of nephrotoxicity. Renal and hepatic non-protein sulfhydryl (NPSH) contents and covalent binding of radiolabel derived from APAP were determined 6 h following APAP administration. Pretreating female rats with ABT, TOTP, or both compounds prevented the APAP-induced elevation in BUN concentration at 24 h. Pretreatment with ABT or ABT plus TOTP prevented APAP-induced depletion of both hepatic and renal NPSH content at 6 h in female rats. In male rats, APAP treatment did not significantly affect hepatic NPSH content. However, renal NPSH content in males was significantly decreased following APAP treatment and the decrease was prevented when rats were pretreated with ABT or ABT plus TOTP. Covalent binding of radiolabel derived from APAP was significantly greater in female kidney as compared to male kidney. Further, covalent binding in female kidney was significantly decreased when rats were pretreated with ABT, TOTP or both. These data suggest that both oxidative metabolism and deacetylation may contribute to APAP-induced nephrotoxicity in rats.

Subchronic dispositional and toxicological effects of arsenate administered in drinking water to mice.

Exposure to the drinking water contaminant arsenate is a daily occurrence and there are concerns that this exposure may lead to cancer. Although the acute dispositional effects of arsenate have been studied in detail, there is minimal information on the disposition and toxicological effects of it after continuous exposure. The objective of this study was to examine in mice the effect of a 4-wk treatment with arsenate administered in drinking water. Female B6C3F1 mice (3/cage) were housed in metabolism cages and given water and food ad libitum. Two groups (A, B) of mice were treated (4 cages/treatment/group) with distilled water (control, C) or water containing 0.025 mg/L (L) or 2.5 mg/L (H) arsenate. Group A was sacrificed on d 28 and plasma and urine samples were taken for determination of clinical chemistry parameters. Liver and kidney tissue samples were taken for histopathological analysis. The reduced nonprotein sulfhydryl (NPSH) content in several tissues was determined. Group B was gavaged with [73As]arsenate on d 28 and continued the arsenate drinking water exposure for 48 h. Excreta and tissues were collected and analyzed for 73As. Urine was further analyzed for arsenate and its metabolites. There were no effects on the mean daily amount of water and food consumed, whereas the mean daily urine volume excreted was significantly elevated by 10% in the H-treated animals compared to C and L. A dose-related hepatic vacuolar degeneration in the liver was observed, but no histological changes were evident in the kidney. Only clinical chemistry parameters in plasma were altered by the arsenate treatment. Glucose was significantly lower at the H dose compared to C and L, triglycerides were significantly greater in C than L and H, and creatinine was significantly greater in H than C. Hepatic NPSH content in the H animals was significantly lower than C and L animals, whereas no effects in lung and kidney were detected. The weights of liver, lung, and kidney, as well as their tissue/body weight ratios, were significantly decreased in the H animals. 73As was primarily eliminated in urine, and its elimination was not affected by dose. No effects on the 48-h 73As cumulative excretion (urine+fecal) were detected. The 73As distribution was low in amount and widely dispersed throughout the animal (< 3% of the 73As dose). The kidney had the highest 73As concentration of the tissues (0.01% 73As dose/g tissue). Dimethylarsinic acid was the major metabolite detected in urine, with lower amounts of arsenate arsenite, and monomethylarsonate. There were no differences between the treatment groups in the amount of urinary metabolites after a single dose of [73As]arsenate. Several toxicological effects were observed in animals administered arsenate in drinking water, but no changes in the disposition of this arsenical were detected at the doses used in this study.

Protection against Acetaminophen Hepatotoxicity by a Single Dose of Clofibrate: Effects on Selective Protein Arylation and Glutathione Depletion

Previous reports demonstrated that repeated administration of peroxisome proliferators protects against acetaminophen (APAP) hepatotoxicity in mice. This protection was associated with a decrease in APAP's selective protein arylation and glutathione depletion. This study was conducted to determine if a single dose of clofibrate (CFB), rather than repeated doses, would similarly prevent APAP toxicity. CD-1 male mice received a single dose of 500 mg CFB/kg and controls were given corn oil 24 hr prior to APAP challenge. After an 18-hr fast, mice were challenged with 800 mg APAP/kg (in 50% propylene glycol) and killed at 4 or 12 hr. Other mice similarly pretreated were killed without APAP challenge. The results showed that pretreatment with a single CFB dose significantly decreased APAP-induced hepatotoxicity. At 12 hr after APAP plasma sorbitol dehydrogenase activity and the severity of hepatocellular necrosis were decreased in CFB pretreated mice. Surprisingly, no differences in hepatic nonprotein sulfhydryl (NPSH) depletion or selective arylation of target proteins in cytosol were observed at 4 hr after APAP challenge. Neither did a single dose of CFB significantly alter hepatic NPSH content prior to APAP challenge. These results indicate that protection against APAP hepatotoxicity by CFB does not require repeated administration, and the absence of significant alterations in APAP's selective protein arylation or glutathione depletion suggests that the protection against APAP hepatotoxicity after a single treatment with CFB may differ mechanistically from the protection observed after repeated CFB dosing.

Protection against Acetaminophen Hepatotoxicity by a Single Dose of Clofibrate: Effects on Selective Protein Arylation and Glutathione Depletion

Previous reports demonstrated that repeated administration of peroxisome proliferators protects against acetaminophen (APAP) hepatotoxicity in mice. This protection was associated with a decrease in APAP's selective protein arylation and glutathione depletion. This study was conducted to determine if a single dose of clofibrate (CFB), rather than repeated doses, would similarly prevent APAP toxicity. CD-1 male mice received a single dose of 500 mg CFB/kg and controls were given corn oil 24 hr prior to APAP challenge. After an 18-hr fast, mice were challenged with 800 mg APAP/kg (in 50% propylene glycol) and killed at 4 or 12 hr. Other mice similarly pretreated were killed without APAP challenge. The results showed that pretreatment with a single CFB dose significantly decreased APAP-induced hepatotoxicity. At 12 hr after APAP plasma sorbitol dehydrogenase activity and the severity of hepatocellular necrosis were decreased in CFB pretreated mice. Surprisingly, no differences in hepatic nonprotein sulfhydryl (NPSH) depletion or selective arylation of target proteins in cytosol were observed at 4 hr after APAP challenge. Neither did a single dose of CFB significantly alter hepatic NPSH content prior to APAP challenge. These results indicate that protection against APAP hepatotoxicity by CFB does not require repeated administration, and the absence of significant alterations in APAP's selective protein arylation or glutathione depletion suggests that the protection against APAP hepatotoxicity after a single treatment with CFB may differ mechanistically from the protection observed after repeted CFB dosing.

Relationship between α-naphthylisothiocyanate-induced liver injury and elevations in hepatic non-protein sulfhydryl content

Acute administration of α-naphthylisothiocyanate (ANIT) to rats has been used as a model of intrahepatic cholestasis. The mechanism of toxicity of ANIT is unknown, although recent evidence suggests a causal or permissive role for glutahione (GSH) (Dahm LJ and Roth RA, Biochem Pharmacol 42: 1181–1188, 1991). In these studies, ANIT treatment elevated hepatic non-protein sulfhydryl (NPSH) content, an indicator of GSH content, when liver injury was evident. The purpose of the present study was to characterize the effects of ANIT on hepatic NPSH content and to relate these changes to the development of liver injury. In rats fasted for 24 hr, administration of ANIT (100 mg/kg, per os [p.o.]) did not change hepatic NPSH content, bile flow, or serum measurments of total bilirubin concentration, alanine aminotransferase (ALT) activity, or γ-glutamyltransferase (GGT) activity by 12 hr post-treatment relative to corn oil vehicle controls. However, by 24 hr after ANIT treatment, rats exhibited cholestasis and elevations in serum markers of liver injury. These markers were associated temporally with an increase in hepatic NPSH content, which consisted entirely of GSH. To determine whether the cholestasis caused by ANIT treatment might have caused the elevation of hepatic NPSH content, an extrahepatic cholestasis in rats was produced by ligation of the common bile duct. Bile duct ligation elevated hepatic NPSH content between 6 and 12 hr after ligation. Administration to rats of a non-hepatotoxic analog of ANIT, β-naphthylisothiocyanate, also elevated hepatic NPSH content 24 hr after treatment. Taken together, these results indicate that the elevation in hepatic NPSH content after ANIT treatment is associated temporally with the onset of liver injury, but this elevation does not appear to participate causally in the mechanism of injury.

Protection against α-naphthylisothiocyanate-induced liver injury by decreased hepatic non-protein sulfhydryl content

α-Naphthylisothiocyanate (ANIT) injures bile duct epithelium and hepatic parenchymal cells in rats. It is commonly believed that ANIT must undergo bioactivation by hepatic, cytochrome P450-dependent mixed-function oxidases (MFO), since agents which are inducers or inhibitors of hepatic MFO activity enhance or attenuate, respectively, the liver injury associated with ANIT. Several of these agents also affect hepatic glutathione (GSH) content and/or GSH S-transferase activity in a manner to suggest a causal role for GSH in ANIT-induced hepatotoxicity. To determine whether GSH might be involved in the mechanism of injury, buthionine sulfoximine (BSO), diethyl maleate (DEM), or phorone was used to reduce hepatic non-protein sulfhydryl (NPSH) content, an indicator of GSH content. Twenty-four hours after ANIT treatment, rats exhibited cholestasis and elevations in serum of total bilirubin concentration, total bile acid concentration, aspartate aminotransferase (AST) activity, and γ-glutamyltransferase activity. Cotreatment of rats with BSO decreased NPSH content by 70% at 24 hr and prevented the cholestasis and elevations in serum markers of liver injury caused by ANIT. Likewise, cotreatment of rats with DEM afforded protection against markers of liver injury. Phorone treatment attenuated ANIT-induced elevations in serum total bilirubin concentration and AST activity. Although BSO treatment afforded protection against ANIT-induced liver injury at 24 hr, the injury was evident at 48 hr, and it appeared to coincide with a return of hepatic NPSH content. These results suggest that GSH plays a causal or permissive role in the liver injury caused by ANIT.

Hepatic lipid peroxidation, sulfhydryl status, and toxicity of the blue‐green algal toxin microcystin‐LR in mice

Microcystin-LR (MCLR), a cyclic heptapeptide produced by the blue-green algae Microcystis aeruginosa, produces death in female mice treated with 100 micrograms MCLR/kg. Kupffer-cell hyperplasia was observed histologically after treatment with 50 or 100 micrograms MCLR/kg. No other changes or lethality were observed with the 50 micrograms MCLR/kg, while 100% lethality occurred in less than 2 h in mice treated with 100 micrograms/kg. In these animals liver weights increased by 45% and hepatic hemoglobin content increased 106% at 60 min posttreatment. Liver histology showed loss of hepatic architecture and necrosis 30 min after treatment, and congestion with blood became evident at 45 min after treatment. Serum enzymes were significantly increased 45 min posttreatment. Hepatic nonprotein sulfhydryl content decreased 19% when calculated on the basis of cytosolic protein and 39% when based upon the total protein content, respectively. The sulfhydryl content of the liver cytoskeletal fraction decreased 26% by 30 min after treatment. Decreased enzyme-mediated and increased non-enzyme-mediated lipid peroxidation were observed in hepatic microsomes following both in vivo and in vitro exposure of hepatic microsomes to MCLR. The toxicity of MCLR may be related to alterations in the sulfhydryl content of the cytoskeletal protein. Furthermore, MCLR may either directly or indirectly affect microsomes, suggesting alterations in structure and function of smooth endoplasmic reticulum.

The effect of combined treatment with potassium dichromate and maleic acid on renal function in the rat

Subthreshold doses of potassium dichromate (K 2Cr 2O 7) have been shown to enhance or potentiate the nephrotoxic effects of mercuric chloride and citrinin. The mechanism of this effect is unknown. The purpose of this study was to examine the phenomenon further by investigating the interaction of K 2Cr 2O 7 with maleic acid, a nephrotoxicant with an action thought to be different from those above. Male rats were housed in stainless steel metabolism cages and received a single intraperitoneal injection of maleic acid (300 mg/kg), K 2Cr 2O 7 (10 mg/kg s.c.) or the combination of maleic acid plus K 2Cr 2O 7. While treatment with either agent alone produced only minimal alterations in renal function, the combination of agents produced marked changes in 24 h urine volume, glucose excretion and osmolality. Water consumption was unaffected by the combined treatment. A marked decline in the capacity of renal slices to accumulate both organic anions ( p-aminohippurate) and cations (tetraethylammonium) was observed after treatment with both substances. Modifications in renal and hepatic non-protein sulfhydryl content also were observed. These results suggest that K 2Cr 2O 7, as was observed with both mercury and citrinin, enhanced or potentiated the nephrotoxic effects of maleic acid, probably in the proximal tubule, the principal site of glucose reabsorption and organic ion secretion.

Depletion of hepatic non-protein sulfhydryl content during exposure of rats and mice to butadiene

B6C3F1 mice, Sprague-Dawley and Wistar rats were exposed to 1,3-butadiene in a closed exposure system. Exposure concentrations were kept above 2000 ppm to ensure saturation of butadiene metabolism in both species ( V max conditions). Hepatic non-protein sulfhydryl (NPSH) content was determined in butadiene-exposed animals (and air-exposed controls) after exposures for 0, 7 and 15 h. Depletion of hepatic NPSH content was different for the species and strains investigated. In mice, hepatic NPSH content declined to about 20% after 7 h and was further depleted to about 4% at 15 h when signs of acute toxicity were observed. After a 7 h exposure of rats to butadiene, hepatic NPSH content was depleted to about 65% (Wistar) or 80% (Sprague-Dawley) of the corresponding controls but remained practically stable after a 15 h exposure to butadiene. The time-courses of depletion by butadiene of hepatic NPSH support previous findings on differences in butadiene metabolism between rats and mice and offer an additional explanation for the considerable species differences observed in the toxicity and carcinogenicity of this compound.

Subacute toxicity of vinyltoluene vapour: effects on the hepatic and renal drug biotransformation and the urinary excretion of thioether.

Male rats were exposed by inhalation to vinyltoluene at concentrations of 50, 100, or 300 p.p.m. for 8, 12 or 15 weeks. Vinyltoluene was found to be metabolized to glutathione conjugates via the formation of electrophilic intermediates. This metabolic pathway was suggested by the decreased hepatic non-protein sulfhydryl content, with a concomitant increase in the urinary excretion of thioethers. The excretion of thioethers showed no saturation phenomena, suggesting that the formation of electrophilic intermediates capable of conjugating with glutathione is fairly linear, at least with exposure to vinyltoluene vapour up to 300 p.p.m. The slight increase in the activities of hepatic drug biotransformation enzymes (7-ethoxycoumarin 0-deethylase. UDPglucuronosyltransferase) observed after 8 weeks of exposure to vinyltoluene vapour disappeared by week 15 irrespective of the continued intermittent inhalation of vinyltoluene. Histological study revealed that cell size had decreased in the rats exposed to vinyltoluene.

Vinyl Chloride-Induced Depression of Hepatic Nonprotein Sulfhydryl Content and Effects on Bromosulphalein (BSP) Clearance in Rats

Vinyl Chloride-Induced Depression of Hepatic Nonprotein Sulfhydryl Content and Effects on Bromosulphalein (BSP) Clearance in Rats

Vinyl chloride-induced depression of hepatic non-protein sulfhydryl conten and effects on bromosulphalen (BSP) clearance in rats

Rats were exposed to atmospheres of 2000, 1000, 250, 150, 50 and 10 ppm vinyl chloride (VC) for 1–7 h to determine the effect of VC on the hepatic non-protein sulfhydryl content. Exposure to 2000, 1000, 250 and 150 ppm VC caused a progressive depression of the hepatic non-protein sulfhydryl content. Following exposure to 50 ppm VC for 7 h to depression was inconsistent, and no depression was observed after 10 ppm VC for 7 h. Also, exposure to 1000 ppm VC did not alter the serum clearance of bromosulphalein (BSP).