Search

Environmental and endogenous electrophiles cause tissue damage through their high reactivity with endogenous nucleophiles such as DNA, proteins, and lipids. Protection against damage is mediated by glutathione (GSH) conjugation, which can occur spontaneously or be facilitated by the glutathione S-transferase (GST) enzymes. To determine the role of GST enzymes in protection against electrophiles as well as the role of specific GST families in mediating this protection, we exposed mutant mouse lines lacking the GSTP, GSTM, and/or GSTT enzyme families to the model electrophile acrylamide, a ubiquitous dietary contaminant known to cause adverse effects in humans. An analysis of urinary metabolites after acute acrylamide exposure identified the GSTM family as the primary mediator of GSH conjugation to acrylamide. However, surprisingly, mice lacking only this enzyme family did not show increased toxicity after an acute acrylamide exposure. Therefore, GSH conjugation is not the sole mechanism by which GSTs protect against the toxicity of this substrate. Given the prevalence of null GST polymorphisms in the human population (approximately 50% for GSTM1 and 20-50% for GSTT1), a substantial portion of the population may also have impaired acrylamide metabolism. However, our study also defines a role for GSTP and/or GSTT in protection against acrylamide mediated toxicity. Thus, while the canonical detoxification function of GSTs may be impaired in GSTM null individuals, disease risk secondary to acrylamide exposure may be mitigated through non-canonical pathways involving members of the GSTP and/or GSTT families., Competing Interests: The authors have declared that no competing interests exist.

Oxidative stress is suggested to play an important role in several pathophysiological conditions. A recent study showed that decreasing 5-oxoproline (pyroglutamate) concentration, an important mediator of oxidative stress, by over-expressing 5-oxoprolinase, improves cardiac function post-myocardial infarction in mice. The aim of the current study is to gain a better understanding of the role of the glutathione cycle in a mouse model of myocardial infarction by establishing quantitative relationships between key components of this cycle. We developed and validated an LC-MS method to quantify 5-oxoproline, L-glutamate, reduced glutathione (GSH) and oxidized GSH (GSSG) in different biological samples (heart, kidney, liver, plasma, and urine) of mice with and without myocardial infarction. 5-oxoproline concentration was elevated in all biological samples from mice with myocardial infarction. The ratio of GSH/GSSG was significantly decreased in cardiac tissue, but not in the other tissues/body fluids. This emphasizes the role of 5-oxoproline as an inducer of oxidative stress related to myocardial infarction and as a possible biomarker. An increase in the level of 5-oxoproline is associated with a decrease in the GSH/GSSG ratio, a well-established marker for oxidative stress, in cardiac tissue post-myocardial infarction. This suggests that 5-oxoproline may serve as an easily measurable marker for oxidative stress resulting from cardiac injury. Our findings show further that liver and kidneys have more capacity to cope with oxidative stress conditions in comparison to the heart, since the GSH/GSSG ratio is not affected in these organs despite a significant increase in 5-oxoproline., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Diosbulbin B (DIOB), a furanoid, is a major constituent of herbal medicine Dioscorea bulbifera L. Exposure to DIOB caused liver injury in humans and experimental animals. The mechanisms of DIOB-induced hepatotoxicities remain unknown. The present study demonstrated that DIOB induced hepatotoxicities in a time- and dose-dependent manner in mice. H&E stained histopathologic image showed the occurrence of necrosis in the liver obtained from the mice treated with DIOB at dose of 200 mg/kg. Pretreatment with KTC protected the animals from hepatotoxicities and hepatic GSH depletion induced by DIOB, increased area under the concentration-time curve of blood DIOB, decreased urinary excretion of GSH conjugates derived from DIOB, and increased urinary excretion of parent drug. Pretreatment with BSO exacerbated DIOB-induced hepatotoxicities. In order to define the role of furan moiety in DIOB-induced liver toxicities, we replaced the furan of DIOB with a tetrahydrofuran group by chemical hydrogenation of the furan ring of DIOB. No liver injury was observed in the animals given the same doses of tetrahydro-DIOB. The furan moiety was essential for DIOB-induced hepatotoxicities. The results implicate the cis-enedial reactive metabolite of DIOB was responsible for the observed toxicities. The observed modest depletion of hepatic GSH in DIOB-treated animals suggests the actions of one or more reactive metabolites, and the hepatic injury observed could be due at least in part to reactions of these metabolites with crucial biomolecules. Cytochrome P450 3A enzymes are implicated in DIOB-induced hepatotoxicities by catalyzing the formation of the reactive metabolite of DIOB.

Background: The effect of liver resection on acetaminophen metabolism and whether it is affected by residual liver volume is poorly understood., Methods: We investigated the effects of liver resection on acetaminophen metabolism in a single centre, prospective observational, case-control study of inpatients. Patients undergoing liver resection were administered therapeutic post-operative acetaminophen. Glutathione and urinary acetaminophen metabolites were measured over the first three post-operative days and compared between patients with low (Group A) and high (Group B) residual liver volume., Results: 41 patients (41% female, median age 62 [IQR 53-72] years) were included. Mean urinary cysteine levels increased significantly from post-operative day 1 to 2 (578.0 mg/day 95% CI 478.9-677.1 vs. 775.4 mg/day, 95% CI 625.7-925.1; p=0.03). Group A (n=11) had significantly higher median levels of cysteine (day 1, 464.3 mg/day [IQR 355.6-582.0]; day 3, 717.6 mg/day [IQR 423.5-1104.0]) compared to Group B (n=11): day 1, 545.4 mg/day (IQR 346.9-843.5); day 3, 508.1mg/day (IQR 390.8-788.4; p=0.048). No significant difference was observed in glutathione or 5-oxoproline levels between the groups., Conclusion: Low residual liver volume results in altered acetaminophen metabolism, however, no evidence of glutathione deficiency was observed. Therapeutic acetaminophen is safe after major liver resection provided liver function is adequate., (Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.)

Background: Within equine drug surveillance, there is significant interest in analyzing intact phase II conjugates of drugs in urine, but progress has been limited by a lack of reference material., Method: In this study, in vitro techniques using equine liver fractions were employed to produce glucuronide and sulfate conjugates of stanozolol, 16β-hydroxystanozolol and nandrolone, the glucuronide conjugate of morphine and the glutathione metabolite of chlordinitrobenzene for the first time in equine sports drug surveillance., Results: The glucuronide conjugate of the synthetic progestagen altrenogest was also produced in vitro, removing the requirement for sample hydrolysis during routine urinalyses., Conclusion: These results highlight the potential of in vitro studies for the production of phase II reference material, allowing the development of assays based on intact conjugates.

The role of K+ channels in bile acid-independent bile flow (BAIF) was studied in the isolated and bile duct-cannulated perfused rat liver by changing the driving force on K+ and by using a variety of K+ channel blockers. Bile flow rate, effluent perfusate K+ content, and portal pressure were measured. Increase in perfusate K+ from 5.9 to 80 mM caused inhibition of bile flow that could be fitted to a Boltzmann distribution, indicating partial dependence of bile formation on the K+ equilibrium potential and hence K+ channel activity. To investigate this further, the effects of compounds established as K+ channel blockers in liver or other tissues were surveyed. Ba2+ (1-5 mM) inhibited mean bile flow by 20%. Tetraethylammonium (TEA) inhibition of basal bile flow was biphasic with saturable (IC50 approximately 0.7 mM) and linear components. In contrast, infusion of the K+ channel blockers 4-aminopyridine (5 mM), cesium (2.5 mM), quinidine (0.1 mM), iberiotoxin (90 nM), or paxilline (100 nM) did not affect bile flow. As expected for a K+ channel blocker, Ba2+ caused a net K+ uptake. Conversely, TEA did not affect basal K+ fluxes, although TEA-induced cholestasis was accompanied by a 26% decrease in biliary glutathione excretion. These results suggest that the partial cholestasis induced by the K+ channel blockers Ba2+ and TEA occurs by significantly different mechanisms. Whereas the Ba2+ response implicates K+ channel activity as a significant driving force in BAIF, TEA-sensitive K+ channels are not present or are not involved in bile formation.

Acivicin (AT-125; 6.25-200 mumol/kg i.v.) inhibited hepatic, biliary and renal gamma-glutamyltranspeptidase (GGT) activity up to 88, 99 and 97%, respectively, in 4-week-old rats. This inhibition of GGT by acivicin resulted in a 10- to 12-fold increase in the biliary excretion of reduced (GSH) and oxidized glutathione. Because the biliary excretion of cysteinylglycine (Cys-Gly), Cys-Gly disulfide, cysteine (Cys) and cystine concomitantly decreased (63-99%), the biliary excretion rate of total glutathione-derived thiols and disulfides did not change. In contrast, acivicin treatment dramatically elevated the urinary excretion rate of glutathione-derived thiols in a dose-dependent fashion, resulting in a 390-fold increase at the highest dosage. This mainly originated from enhancement of urinary excretion of GSH (up to 7200-fold), although the excretion of Cys and Cys-Gly into urine was also increased. Acivicin treatment did not affect hepatic and renal levels of GSH but, at high dosages, reduced the concentration of Cys in these organs. GSH and oxidized glutathione concentrations in serum were increased, whereas cystine was diminished in acivicin-treated rats. Inhibition of GGT by acivicin (100 mumol/kg i.v.) failed to influence the biliary excretion of methylmercury but increased urinary excretion 34-fold. Even though the urinary thiol excretion was much higher than the biliary thiol excretion in the acivicin-treated rats, methylmercury was preferentially excreted into bile rather than urine, indicating the importance of the liver as an excretory organ for methylmercury.(ABSTRACT TRUNCATED AT 250 WORDS)

In spite of the importance of glutathione (GSH) in the detoxification of toxic metabolites of drugs, virtually nothing is known about the regulation of hepatic GSH homeostasis in man. In order to estimate the turnover of hepatic GSH and to assess the effect of different doses of acetaminophen (paracetamol) on the synthesis of GSH in man, [3H]cystine and varying doses of acetaminophen were administered to healthy volunteers, and the time course of the specific activity of the cysteine moiety of N-acetylcysteinyl-acetaminophen excreted in urine was followed. The fractional rate of turnover of the tracer in N-acetylcysteinyl-acetaminophen increased significantly from 0.031 +/- 0.007 h-1 after doses of acetaminophen ranging from 50 to 300 mg to 0.045 +/- 0.011 and 0.121 +/- 0.027 h-1 following 600 and 1200 mg of acetaminophen, respectively. The data indicate that therapeutic doses of acetaminophen markedly stimulate the rate of turnover of the pool of cysteine available for the synthesis of GSH, most likely due to an increased rate of synthesis of GSH which is required to detoxify the toxic metabolite of acetaminophen. Patients who are not able to respond to a similar demand on their stores of GSH by increasing the synthesis of GSH may be at higher risk of developing hepatic injury from drugs that require GSH for their detoxification.

Administration of toluene and xylenes to rats caused a decrease in liver glutathione concentration. The effect was most pronounced after the administration of o-xylene. 26% of the initial glutathione level was found three hours after treatment with o-xylene (4.0 mmoles/kg). No in vitro conjugation of o-xylene with glutathione was observed, neither spontaneously nor in the presence of 105,000 g supernatant from rat liver homogenate, containing glutathione S-transferases. Thus, a metabolite of o-xylene, which is not formed during incubation with 105,000 g supernatant, reacts with glutathione. A thioether was isolated from urine of rats given o-xylene; the compound was identified as o-methylbenzyl mercapturic acid by GC-MS and NMR. Chromatographic evidence was found for the presence of benzyl mercapturic acid in the urine of toluene-treated rats. The amounts of mercapturic acids excreted in the urine after administration of toluene, p-xylene, m-xylene, and o-xylene were 0.4-0.7,0.6,1.3, and 10-21% of the dose, respectively. These results demonstrate the involvement of a thusfar unknown pathway in the biotransformation of toluene and xylenes.

The metabolism and excretion of styrene oxide-glutathione conjugates were studied in the intact rat and by isolated perfusion techniques in rat lung, liver and kidney. The intact animals converted the styrene oxide-glutathione conjugates almost entirely to styrene oxide-n-acetylcysteine derivatives mercapturic acids), with much smaller amounts of cysteine, cysteinylglycine and (unchanged) glutathione conjugates. Only small amounts of biliary excretion (approximately 5%) and less than 1% of fecal excretion of radioactivity were found. The isolated perfused kidney retains the capacity to transform virtually all of the glutathione conjugates at the dose level studied (25-30 mumol/kidney) and the principal component in urine from the isolated kidney was the cysteine conjugate. Isolated perfused lung experiments demonstrated that this organ has a minor role in styrene oxide-thioether conjugate. Isolated perfused lung experiments demonstrated that this organ has a minor role in styrene oxide-thioether conjugate metabolism. The isolated perfused liver experiments showed that phenobarbital pretreatment has little effect on biliary excretion but barbital pretreatment has little effect on biliary excretion but that it causes profound qualitative and quantitative effects on metabolism of styrene oxide-glutathione conjugates in the perfusion medium. The high level of styrene oxide-cysteine conjugates in the perfusion medium of an isolated perfused liver, especially after phenobarbital treatment, is consistent with the theory that gamma-glutamyltransferase activity is extracellular. The high-pressure liquid chromatography system of analysis described offers the ability to simultaneously separate and quantitate the major thioether conjugates of styrene oxide and other electrophilic epoxides.