Peter Moldéus

Oxidation pathways for the intracellular probe 2',7'-dichlorofluorescin

The oxidation of 2′,7′-dichlorofluorescin (DCFH) to a fluorescent product is currently used to evaluate oxidant stress in cells. However, there is considerable uncertainty as to the enzymatic and nonenzymatic pathways that may result in DCFH oxidation. Iron/hydrogen peroxide-induced DCFH oxidation was inhibited by catalase or by the hydroxyl radical scavenger dimethylsulfoxide; however, superoxide dismutase (SOD) had no effect on DCFH oxidation. The formation of hydroxyl radical (indicated by the oxidation of salicylic acid to 2,3-dihydroxybenzoic acid) was proportional to DCFH oxidation, suggesting that the hydroxyl radical is responsible for the iron/peroxide-mediated oxidation of DCFH. Utilizing a superoxide generating system consisting of hypoxanthine/xanthine oxidase, oxidation of DCFH was unaffected by SOD, catalase or desferoxamine, and stimulated by removing hypoxanthine from the reaction mixture. In contrast, SOD or elimination of hypoxanthine abolished superoxide formation. In addition, potassium superoxide did not support the oxidation of DCFH. Thus, superoxide is not involved in DCFH oxidation. Boiling xanthine oxidase eliminated its concentration-dependent oxidation of 1 μM DCFH, indicating that xanthine oxidase ian enzymatically utilize DCFH as a high affinity substrate. Kinetic studies of the oxidation of DCFH by xanthine oxidase indicated a Km(app) of 0.62μM. Hypoxanthine competed with DCFH with a Ki(app) of 1.03 mM. These studies suggest that DCFH oxidation may be a useful indicator of oxidative stress. However, other types of cellular damage may produce DCFH oxidation. For example, conditions or chemicals that damage intracellular membranes may release to the cytoplasm oxidases or peroxidases that might use DCFH as a substrate, similar to xanthine oxidase

Methodologies for the application of monobromobimane to the simultaneous analysis of soluble and protein thiol components of biological systems.

A series of simple methodologies for the determination of the redox status of low molecular weight and protein thiols in biological systems is described. Based centrally upon the use of monobromobimane, we describe a standard in situ derivatisation procedure simultaneously resulting in maximal recovery of both free, reduced low molecular weight and bromobimane accessible protein thiols as their corresponding bimane adducts from intact biological systems. Test systems include isolated and cultured cells, tissue homogenates and body fluids such as blood plasma. Quantitation of the bimane adducts of cysteine and glutathione is achieved by reversed phase high performance liquid chromatography, whereas quantitation of the corresponding adducts of protein thiols is achieved by fluorescence spectroscopy following protein precipitation. Full validation data for quantitative estimates are described. Additionally we have coupled these procedures to prederivatization denaturation treatments of biological protein samples in order to quantitate pools of protein thiols which are inaccessible to bromobimane in samples of native protein. We have also coupled these procedures with prederivatization reductions of biological systems under study with dithiothreitol, rendering simultaneously both oxidized low molecular weight thiols and oxidized protein thiols accessible to derivatisation with monobromobimane. Thus, we have obtained quantitative determinations of cysteine and glutathione present in mixed disulfides with protein and in soluble low molecular weight disulfides and estimates of intraprotein disulfides in a number of test biological systems.

Lung Protection by a Thiol-Containing Antioxidant: N-Acetylcysteine

N-acetylcysteine (NAC) is a thiol-containing compound which nonenzymatically interacts and detoxifies reactive electrophiles and free radicals. NAC was shown to effectively protect human bronchial fibroblasts against the toxic effects of tobacco smoke condensates and the isolated perfused lung against the glutathione (GSH)-depleting effect of tobacco smoke. NAC was also shown to reduce the reactive oxygen intermediate hydrogen peroxide (H2O2) and protect against the toxic effects of H2O2. In vivo studies, however, demonstrated that NAC when administered orally has very low bioavailability due to rapid metabolism to GSH among other metabolites. Thus, even though NAC is very effective in protecting cells of different origins from the toxicity of reactive components in tobacco smoke and reactive oxygen species, a direct scavenging effect by NAC in vivo, particularly when administered orally, does not seem likely. The bioavailability of NAC itself is very low when given this route. A more relevant mechanism in vivo for any protective effect NAC may exert against toxic species may be due to NAC acting as a precursor of GSH and facilitating its biosynthesis. GSH will then serve as the protective agent and detoxify reactive species both enzymatically and nonenzymatically.

Paracetamol metabolism and toxicity in isolated hepatocytes from rat and mouse

Abstract Hepatocytes isolated from mouse and rat catalyzed the formation of glucuronide, sulphate, glutathione and cysteine conjugates of paracetamol. These metabolites were separated by high pressure liquid chromatography. 1. Sulphation had higher affinity for paracetamol than glucuronidation in hepatocytes from both mouse and rat, whereas glucuronidation had higher capacity. The maximal rate of glucuronidation was similar in hepatocytes from both species, the rate of sulphation was, however, several-fold less in hepatocytes from mouse. 2. Formation of the glutathione conjugate was directly correlated with loss of intracellular glutathione (GSH). The rate of glutathione conjugate formation increased about three times in rat hepatocytes after phenobarbital treatment. This induced rate was, however, only half of that in hepatocytes from control mouse. In both species the reaction was saturated only at very high paracetamol concentrations. The rate of formation of the cysteine conjugate was very low compared to the other reactions. 3. Only hepatocytes isolated from mouse lost integrity, measured as increased permeability of the cell membranes, upon incubation in the presence of paracetamol.

Metabolic activation and hepatotoxicity. Effect of cysteine, N-acetylcysteine, and methionine on glutathione biosynthesis and bromobenzene toxicity in isolated rat hepatocytes.

Abstract Freshly isolated rat hepatocytes contained a high level (30–40 nmol/10 6 cells) of reduced glutathione (GSH) which decreased steadily upon incubation in an amino acid containing medium lacking cysteine and methionine. This decrease in GSH level was prevented, and turned into a slight increase, when either cysteine, N -acetylcysteine, or methionine was also present in the medium. The amino acid uptake into hepatocytes was more rapid with cysteine than with methionine. Cystine was not taken up, or taken up very slowly, by the cells and could not be used to prevent the decrease in GSH level which occurred in the absence of cysteine and methionine. The level of GSH in hepatocytes freshly isolated from rats pretreated with diethylmaleate was markedly decreased (to ~5 nmol/10 6 cells) but increased rapidly upon incubation of the cells in a medium containing amino acids including either cysteine, N -acetylcysteine, or methionine. Again, cysteine was taken up into the cells more rapidly than methionine. The rate of uptake of cysteine was moderately enhanced in hepatocytes with a lowered level of intracellular GSH as compared to cells with normal GSH concentration. Exclusion of glutamate and/or glycine from the medium did not markedly affect the rate of resynthesis of GSH by hepatocytes incubated in the presence of exogenously added cysteine or methionine. Incubation of hepatocytes with bromobenzene in an amino acid-containing medium lacking cysteine and methionine resulted in accelerated cell damage. Addition of either cysteine, N -acetylcysteine, or methionine to the medium caused a decrease in bromobenzene toxicity. The protective effect was dependent, however, on the time of addition of the amino acid to the incubate; e.g., the effect on bromobenzene toxicity was greatly reduced when either cysteine or methionine was added after 1 h of preincubation of the hepatocytes with bromobenzene as compared to addition at zero time. This decrease in protective effect in bromobenzene-exposed cells was related to a similar decrease in the rate of uptake of cysteine and methionine into hepatocytes preincubated with bromobenzene. The rate of uptake, and incorporation into cellular protein, of leucine was also markedly inhibited in hepatocytes preincubated with bromobenzene. In contrast, there was no measurable change in the rate of release of leucine from cellular protein as a result of incubation of hepatocytes with bromobenzene. It is concluded that the presence of cysteine, N -acetylcysteine, or methionine in the medium protects hepatocytes from bromobenzene toxicity by providing intracellular cysteine for GSH biosynthesis and suggested that an inhibitory effect on amino acid uptake may contribute to the cytotoxicity of bromobenzene in hepatocytes.

The isolation of rat intestinal microsomes with stable cytochrome P-450 and their metabolism of benzo(α)pyrene

Abstract A procedure has been developed for the isolation of microsomes from rat intestinal mucosa with stable cytochrome P -450. Preservation of the hemoprotein has been obtained by including trypsin inhibitor, glycerol, and heparin in the homogenization medium. The spectral properties of the hemoprotein from control and phenobarbital (Pb)- and 3-methylcholanthrene (MC)-treated rats were examined. In fed animals, MC given orally resulted in a 30-fold stimulation of benzo(α)pyrene (BP) monooxygenase within 24 h with a simultaneous increase in cytochrome P -450 ( P -448) but had no effect on NADPH-cytochrome c reductase activity. The effect of MC on BP monooxygenase and cytochrome P -450 ( P -448) could be seen as early as 1.5 h after oral administration. Pb treatment increased cytochrome P -450 levels but had no effect on NADPH-cytochrome c reductase activity and comparatively little effect on BP monooxygenase. In fasted animals, MC also produced large increases in BP monooxygenase activity when compared to control animals. At 7 μ m α-naphthoflavone, BP monooxygenase was inhibited 96% in microsomes from MC-treated rats and stimulated 4.5-fold in microsomes from control animals. The pattern of BP metabolites was similar for intestinal microsomes from control and MC-treated rats but differed sharply from that produced by hepatic microsomes. The 4,5-oxide of BP constituted one of the major intestinal metabolites with only small amounts of dihydrodiols being formed after a 5-min incubation. It is concluded that the cytochrome P -450-linked monooxygenase system present in the intestinal mucosa differs markedly from the hepatic system with regard to induction properties, substrate specificity, and pattern of BP metabolites.

The multiple roles of glutathione in drug metabolism

Abstract The important role of glutathione in drug biotransformation and prevention of drug toxicity is well established. However, new information concerning the formation and degradation of glutathione conjugates, the oxidation of glutathione by free radical intermediates, and the possible involvement of glutathione in regulation of cellular calcium homeostasis has further stimulated interest in glutathione research. This review also highlights some very interesting observations: glutathione conjugation can, in some cases, result in toxicity, and glutathione oxidation during hydroperoxide metabolism may precipitate the early loss of Ca 2+ homeostatis associated with hydroperoxide-induced cytotoxicity.

Studies on the anti-inflammatory activity of Ebselen: Ebselen interferes with granulocyte oxidative burst by dual inhibition of NADPH oxidase and protein kinase C?

Ebselen (PZ51, 2-phenyl-1,2-benzoisoselenazol-3-(2H)-one) was shown to be an inhibitor of human granulocyte oxidative burst stimulated by phorbol myristate acetate (IC50 25 microM). Estimation of the primary oxygen metabolites of the burst was complicated by the redox chemistry of Ebselen. Ebselen inhibited NADPH-stimulated superoxide generation by a partially purified NADPH oxidase preparation with an IC50 of 0.5-1.0 microM. Ebselen was also shown to inhibit the activity of partially purified Ca2+- and phospholipid-dependent protein kinase C (IC50 ca. 0.5 microM). Phorbol ester-stimulated phosphorylation of protein in intact cells was inhibited by Ebselen (IC50 ca. 50 microM). These pharmacodynamic properties of Ebselen are discussed in terms of its anti-inflammatory activity in vivo. The findings are also discussed in terms of Ebselens known ability to interact with sulfhydryl components of cells, particularly critical thiol components of the enzymes studied.

Interaction of menadione (2-methyl-1,4-naphthoquinone) with glutathione

The interaction of menadione with reduced glutathione (GSH) led to a removal of menadione and formation of menadione-GSH conjugate and glutathione disulfide (GSSG). The changes in thiol level were essentially biphasic with an initial rapid decrease in GSH and appearance of GSSG (less than 1 min) followed by secondary less pronounced changes. The interaction of menadione and GSH caused an oxygen uptake and both superoxide anion radical and hydrogen peroxide were produced during the reaction, the amount dependent on the GSH/menadione ratio. Catalase did not protect against the initial decrease in GSH level but markedly inhibited the secondary changes while superoxide dismutase had little effect. These results suggest that the initial changes in thiol level are the result in part of a redox reaction between menadione and GSH as well as conjugate formation, whilst the secondary changes reflect conjugate formation and the activity of other oxidants such as hydrogen peroxide. The potential biological significance of this reaction was investigated using hepatocytes depleted of reduced pyridine nucleotides and thus not able to perform enzyme-catalyzed reduction of menadione. In these cells menadione induced GSSG formation at a rate similar to that observed in control cells. This suggests that quinone-induced oxidative challenge caused by the chemical interactions of a quinone and glutathione may have biological relevance.

The metabolism of N-acetylcysteine by human endothelial cells

When human umbilical endothelial cells were depleted of their glutathione by incubation in a sulfur amino acid-free medium, subsequent incubation of the cells with this deficient medium supplemented with N-acetylcysteine resulted in a dose-dependent stimulation of the synthesis of cellular glutathione. Similarly, the inclusion of N-acetylcysteine in the medium during the period of depletion of glutathione caused a dose-dependent retardation of the depletion kinetics. In contrast, the incubation of control cells in normal medium supplemented with N-acetylcysteine did not increase cellular glutathione levels above controls. These observations indicate the presence of an N-deacetylase in/on the cells with specificity for N-acetylcysteine. Due to the large surface area of the endothelium in the vasculature it seems likely that endothelial cell N-deacetylation plays a role in the metabolic disposition of N-acetylcysteine, particularly when administered intravenously. N-Acetylcysteine is, however, a relatively poor precursor to glutathione biosynthesis in comparison to cystine. Thus, any cytoprotective, antioxidant effect exerted by N-acetylcysteine on the human endothelium is likely to be due to direct scavenging of reactive intermediates rather than by stimulated glutathione synthesis in the endothelial cells themselves.