Jerry R. Mitchell

Mitochondria and xanthine oxidase both generate reactive oxygen species in isolated perfused rat liver after hypoxic injury.

Hypoxia caused severe damage in isolated perfused livers from fasted male Fischer rats without evidence of the formation of reactive oxygen species during hypoxia. Reoxygenation caused a significant increase in intracellular oxygen species in the injured liver, as indicated by increases in sinusoidal GSSG efflux and tissue GSSG levels. Both parameters were elevated further by addition of KCN (100 microM) or antimycin A (8 microM). Sinusoidal GSSG efflux was suppressed in part by addition of allopurinol (500 microM) and enhanced by hypoxanthine (250 microM). Xanthine oxidase appears to be a partial source, and damaged mitochondria a continuous and quantitatively greater source, of reactive oxygen as a result of liver injury following hypoxia.

[83] Use of isolated perfused organs in hypoxia and ischemia/reperfusion oxidant stress

Publisher Summary This chapter discusses the use of isolated perfused organs in hypoxia and ischemia/reperfusion oxidant stress. Several methods are available to detect reactive oxygen species in isolated organs. A sensitive method without the necessity of additional chemicals interfering with the biological system or the use of expensive equipment is to monitor the formation and cellular release of glutathione disulfide (GSSG) as an index for the activity of the endogenous defense system against reactive oxygen species. The method is based on the very rapid enzymatic dismutation of intracellularly generated superoxide to molecular oxygen and hydrogen peroxide, which is then reduced to water through glutathione peroxidase. Glutathione (GSH) provides the reducing equivalents for this reaction and is oxidized to its disulfide (GSSG). Most of the GSSG formed is immediately reduced back to GSH through glutathione reductase with the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). In the liver, GSSG is secreted mainly into bile against a steep concentration gradient. The biliary portion of the total GSSG export is a relatively constant value of about 80 to 85% as long as no major interference occurs with bile formation. GSSG is released mainly into the perfusate in other organs such as heart and lung in contrast to the 15 to 20% released into the perfusate in the liver. Although, only a small percentage of the GSSG generated is released from the cells, any change in the overall GSSG formation is reflected by similar changes in the cellular efflux. Thus, a significant increase in the efflux of GSSG into perfusate or bile (liver) indicates an enhanced activity of the defense system against reactive oxygen species (oxidant stress).

Hypoxic damage generates reactive oxygen species in isolated perfused rat liver

The aim of the present study was to investigate the possible role of reactive oxygen species in the pathogenesis of hypoxic damage in isolated perfused rat liver. One hour of hypoxia caused severe cell damage (lactate dehydrogenase release of greater than 12,000 mU/min/g liver wt) and total irreversible cholestasis which was accompanied by a loss of cellular ATP and a marked decrease in lactate efflux. Tissue glutathione disulfide (GSSG) content and GSSG efflux as a measure of hepatic reactive oxygen formation was less than 1% of total glutathione before and during hypoxia. Upon reoxygenation, however, hepatic GSSG content increased sharply to about twice the control values and GSSG efflux increased several-fold to around 3-4 nmol GSH-equivalents/min/g. The release of lactate dehydrogenase decreased upon reoxygenation and tissue ATP content recovered partially. When livers were reoxygenated at an earlier time interval than 1 hr of hypoxia, i.e., before the onset of damage, no enhanced GSSG formation was observed. The results demonstrate that hypoxic damage is a prerequisite to reactive oxygen formation during the subsequent reoxygenation period. Thus, reactive oxygen species appear unlikely to play a crucial role in the pathogenesis of hypoxic liver damage in the hemoglobin-free, isolated perfused liver model.

High-performance liquid chromatography and gas chromatography-mass spectrometry determination of specific lipid peroxidation products in vivo

Peroxidation of membrane lipids has been implicated in the toxicity of reactive oxygen intermediates and of several hepatotoxins, but the specific products of this peroxidation in vivo have not been chemically identified. A method for the isolation, identification, and quantitation of specific lipid hydroperoxy and hydroxy acids formed in vivo has been developed. Hydroxylated derivatives of linoleic, arachidonic, and docosahexaenoic acids formed in mouse liver phosphatidylcholines following carbon tetrachloride administration were isolated by high-pressure liquid chromatography and identified as the trimethylsilyl ether methyl ester derivatives by gas chromatography-mass spectrometry. This methodology should be important for the investigation of the role of lipid peroxidation in a variety of normal physiologic and pathologic processes.

Quantitation of lipid peroxidation products by gas chromatography-mass spectrometry☆

A method for the quantitation of lipid peroxidation products in total hepatic lipid has been developed. Lipid extracts are reduced and subsequently transmethylated with sodium methoxide. The hydroxy fatty acid methyl esters are isolated by silicic acid chromatography and derivatized to their trimethylsilyl ethers prior to analysis by gas chromatography-mass spectrometry. Three isomers, 11-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE), are quantitated using selected ion monitoring techniques relative to the internal standard, methyl 15-hydroxyarachidate. In mice treated with carbon tetrachloride (2 ml/kg), the HETE levels in total hepatic lipid were 20-fold greater than those found in control animals. HETE levels were also elevated (5- to 10-fold) in hepatic lipid from rats treated with the same dose of carbon tetrachloride. Studies on subcellular fractions with this methodology show that these lipid peroxidation products are 5- to 6-fold higher in hepatic plasma membrane vesicles isolated from rats treated with carbon tetrachloride when compared with those isolated from control animals.

Alkylation of the liver plasma membrane and inhibition of the Ca2+ ATPase by acetaminophen

Acetaminophen is activated metabolically to yield reactive species that bind covalently to liver cell macromolecules. The extent of covalent binding correlates with the occurrence and severity of hepatic necrosis. We reported previously [J. O. Tsokos-Kuhn, E. L. Todd, J. B. McMillin-Wood and J. R. Mitchell, Molec. Pharmac. 28, 56 (1985)] that active Ca2+ accumulation of isolated liver plasma membranes is decreased 60-75% after a hepatotoxic dose of acetaminophen in vivo. We now report that the protein of isolated liver plasma membranes was substantially labeled with drug metabolites after administration of [3H]acetaminophen. There was no increase in passive membrane permeability that might cause diminished Ca2+ accumulation. Intravesicular volume and relative purity of the vesicle preparations after acetaminophen were not different from controls. However, (Ca2+,Mg2+)-ATPase, a possible biochemical expression of the Ca2+ pump, was decreased 31% (P less than 0.025) after acetaminophen treatment. ATPase activity in both control and treated groups was enhanced by isolating membranes in the presence of 5 mM reduced glutathione (GSH), but the effects of drug treatment were not reversed. A similar effect of GSH on Ca2+ accumulation was observed previously [J. O. Tsokos-Kuhn, E. L. Todd, J. B. McMillin-Wood and J. R. Mitchell, Molec. Pharmac. 28, 56 (1985)]. These data are consistent with a hypothesis wherein alkylation of membrane proteins by reactive acetaminophen metabolites is a factor in the onset of hepatic necrosis after acetaminophen. They are not consistent with an oxidative stress hypothesis where thiol S-thiolation of membrane components is postulated to produce altered membrane permeability or thiol-reversible alterations in membrane protein structure and enzymatic function.

Acetaminophen hepatotoxicity invivo is not accompanied by oxidant stress

Hepatotoxic doses of acetaminophen in Fischer 344 rats did not increase biliary efflux of oxidized glutathione. Pretreatment of the animals with bis(2-chloroethyl)-N-nitrosourea inhibited hepatic glutathione reductase by 73 percent but did not potentiate the hepatotoxicity of acetaminophen and did not produce an increase in biliary efflux of oxidized glutathione in response to acetaminophen. Hepatic protein thiol content was not depleted by acetaminophen. A proposed role for oxidant stress mechanisms mediated either by reactive oxygen species or by the direct oxidant action of a reactive metabolite in acetaminophen-induced hepatotoxicity is unsubstantiated and unlikely.

[75] Measurement of oxidant stress in Vivo

Publisher Summary In this chapter, oxidized glutathione, glutathione disulfide (GSSG), is used to monitor oxidant stress in vivo . GSSG can be measured in plasma as an index of drug-induced oxidant stress by a modification of the enzymatic cycling method of Tietze. The measurement of GSSG in plasma is convenient but requires caution to avoid assay artifacts or data misinterpretation. When released into the blood, glutathione (GSH) and GSSG have a half-life of less than 2 minutes. Both compounds are degraded mainly in the kidney. Thus, actual plasma GSSG concentrations are the result of cellular release and degradation and depend in part on the location of the blood vessel from which the blood is sampled. As various tissues such as the heart, lung, and liver and erythrocytes can detoxify reactive oxygen and release GSSG into the plasma, the source of GSSG is not always clear. The determination of GSSG in the arterial versus venous blood supply for an organ and the organ GSSG concentration can be helpful in identifying individual tissue contributions to plasma GSSG.

Therapeutic doses of acetaminophen stimulate the turnover of cysteine and glutathione in man

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.

Determinants of hepatic glutathione turnover: toxicological significance

The tripeptide glutathione participates in a number of critically imporlant cellular processes (Fig. I). The function best known to the pharmacologist is associated with the nucleophilic structure of the compound which enables it to form conjugates with xenobiotics. The formation of these glutathione adducts proceeds spontaneously with some electmphilie compounds but in general conjugation is greatly facilitated by the glutathione transferases’. This group of enzymes binds a bmad spectrum of compounds and in this respect is very similar to serum albumin. In addition. however, the enzymes have a specific binding site for glutathione thereby bringing the nucleophilic glutathione in close proximity to an electmphilic atom of the first ~b strate. The resulting conjugates are 1” ‘li;cursors of the generic group of exck9 . products known as mercapturic acids. C.Ilowing removal of the gamm&glutamyl moiety by gamma-glutamyltransferase mainly located in the kidneys and intestinal mucosa. the glycine moiety is split off by peptidases and the resulting cysteine adduct of the original compound is then N-acetylated to the N-acetyl-cysteine thioether designated as mercapturic acid. The glutathione adducts are more polar than the original compc>unds, which facilitates their excretion. ln addition, when dealing with chemically reactive metatmlites of senobiotics such as acetaminophen. conjo gate formation prevents the covalent binding to tissue macromolecules and the hcpatotoxicity of the metabolitesY. The availability of glutathione for conjugate formation, therefore, is a critical determinant of the toxicity of electrophilic drug metabolites capable of alkylating nucleophilic sites on vital hepatic molecules.