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Sulfur mustard intoxication, oxidative stress, and antioxidants.

Sulfur Mustard (SM) is a potent alkylating agent with electrophilic property which has been used as a chemical warfare agent in at least 12 conflicts. It has reemerged as a major threat in recent years. Medical attention is primarily concerned with its action on the skin, eyes, and respiratory tract which may be complicated by damage to ophthalmic, pulmonary, and gasterointestinal systems, followed by bone marrow depression. The cytotoxicity of SM and production of reactive oxygen substances (ROS) has been proposed to result from electrophilic or oxidative stress with depletion of cellular detoxifying thiol levels including glutathione. Also, ROS are transformed by iron-requiring reactions into highly toxic oxidants that cause a chain reaction with membrane phospholipids to form lipid peroxides, leading to loss of membrane function, membrane fluidity, and finally membrane integrity. Provision and availability of scavengers of ROS and electrophilic compounds such as glutathione, sulfhydryls compounds, antioxidants, and substances that will increase production of endogenous scavengers may be considered protective and useful. Thereby, the role of substances such as selenium, copper, zinc, and antioxidants including vitamin E, vitamin C, and compounds like beta-carotene against SM cytotoxicity and lipid peroxidation might be interesting to be investigated in experimental animal models.

Antioxidants↗

Dithisone diabetes in rabbits and its prevention by sulfhydryl and imidazole containing compounds.

Diabetogenic action of dithisone was investigated in a total of 368 adult rabbits and 53 young animals between 10 h and 31 days of age. The diabetes was found in 95% of animals injected with dithisone and various forms of this disease were observed: 1. long-term diabetes without any signs of normalization of glycemia; 2. diabetes with periodical remissions; 3. several cases with a definite remission. The diabetes did not appear in young animals during certain periods of life in which the concentration of zinc in pancreatic islets was very low. It was possible to prevent the development of this disease by the administration of some compounds containing sulfhydryl or imidazole groups (cysteine, SH-glutathione, dimercaptopropanol (BAL), unithiol (sodium 2,3-dimercaptopropansulfonate), (histidine) and the diabetes also did not appear in such animals in which a majority of zinc was removed by glybenclamide. From these observations it was concluded that the development of diabetes after dithisone depends on the formation of dithisone complex with zinc in beta-cells.

Animals↗

The respiratory metabolism of Mycobacterium lepraemurium.

The respiratory metabolism of Mycobacterium lepraemurium isolated from Sprague-Dawley rats lepromata using several substrates was investigated. None of the intermediates of the glycolysis cycle as well as of the tricarboxylic acid cycle except succinate was oxidized by purified whole suspensions of M. lepraemurium. Likewise, many sulfur compounds such as cystine, thiourea, thioacetate, thiodiglycol, mercaptoact and some sulfhydryl compounds, e.g., cysteine, dithioerythritol, dithiorthritol, and penicillamine were readily oxidized by murine bacillary suspensions, whereas thioglycolate, thioglucose, and reduced glutathione were oxidized at a slow rate. Succinate was not or was very poorly oxidized by normal cells probably because of impermeability of the cell wall but the addition of succinate to the cell suspensions frozen for 1 min at -40 degrees C considerably enhanced oxygen uptake over the endogenous value. The oxidation of succinate was unaffected by inhibitors rotenone, atabrine, and amytal but was markedly inhibited by thenoyltrifluoroacetone, antimycin A, 2-N-heptyl-4-hydroxyquinoline-N-oxide, and cyanide. The thiol-binding agents, p-hydroxymercuribenzoate and N-ethylmaleimide were also effective inhibitors of succinate oxidation but the process was not affected by uncouplers dinitrophenol, dibromophenol, pentachlorophenol, and carbonyl-cyanide-m-chlorophenylhydrazone. The results indicated that succinate oxidation by M. lepraemurium was mediated by oxidative enzymes involving an electron transport chain with oxygen as the terminal electron acceptor.

Antimycin A↗

Structure-activity relationship of the preservation and restoration of glucocorticoid receptors in rat hepatocytes and rat liver homogenates by sulfhydryl, phosphorothioate and disulfide compounds.

The purpose of this work was to examine whether the ability of dithiothreitol to preserve the steroid-binding capacity of glucocorticoid receptors in subcellular preparations is specific or a general property of sulfhydryl compounds and selected phosphorothioate and disulfide derivatives. A further goal was to see if this effect could be demonstrated in intact cells. The ability to preserve the steroid-binding capacity of the glucocorticoid receptor is not a universal property of all sulfhydryl compounds since many of the compounds tested were inactive. The steroid-binding capacity of the glucocorticoid receptor of the 100,000 g supernatant of rat liver homogenate is preserved/restored by sulfhydryl compounds containing a mercaptoethylamine or mercaptopropylamine subunit. However, small changes in the structure of the sulfhydryl compound such as the rearrangement of a methylene group significantly alter its effectiveness. All of the phosphorothioates examined are derivatives of active sulfhydryl compounds and are effective in preserving steroid-binding. The extent of metabolism of the phosphorothioates and their failure to restore steroid-binding capacity after short-time exposure to receptor preparations are consistent with the sulfhydryl form being the active form of the phosphorothioates. S-2-(3-Amino-propylamino)ethylphosphorothioic acid (WR 2721) preserved steroid-binding capacity in isolated intact rat hepatocytes down to 25 microM demonstrating that concentrations obtainable in whole animals are effective with intact cells. Disulfide derivatives of active sulfhydryl compounds are either immediately toxic or ineffective except for 1,18-diamino-6,13-diaza-9,10-dithiaoctadecane (WR 149,024) which is more effective than its corresponding sulfhydryl. The demonstration that some sulfhydryl-forming compounds preserve the steroid-binding capacity of glucocorticoid receptors in intact cells at potentially physiologically obtainable concentrations suggests a potential role for these or similar compounds to bolster the efficacy of conventional glucocorticoid therapy.

Adrenalectomy↗

In vitro effects of various heterocyclic thiol compounds and beta-lactam antibiotics on vitamin K-dependent gamma-glutamylcarboxylation activity in liver microsomes.

The in vitro effect of N-methyltetrazolethiol (NMTT), one of the common substituents at the 3'-position of the cephem in various beta-lactam antibiotics, on liver microsomal gamma-glutamylcarboxylation (gamma-carboxylation) activity was examined using solubilized rat liver enzyme. The enzyme activity was inhibited by coexisting with NMTT and NADH, and this inhibitory activity could be suppressed by the addition of a sulfhydryl compound such as dithiothreitol (DTT), glutathione or cysteine. Various five-membered heterocyclic thiol compounds exhibited concentration-dependent inhibition of microsomal gamma-carboxylation activity. These inhibitory actions diminished markedly in the presence of 1 mM DTT. In vitro gamma-carboxylation activity also decreases upon addition of various beta-lactam antibiotics at 1 or 10 mM, depending upon the concentration of the drug. Among the heterocyclic thiol compounds, there is a correlation between their inhibitory activities and hydrophobicities. Thus, the in vitro inhibitory activity of heterocyclic thiol compounds and beta-lactam antibiotics on microsomal gamma-carboxylation activity is not correlated with their molecular structures, but rather depends on their hydrophobicities and with the concentrations in the reaction mixture.

Animals↗

Hemin-induced membrane sulfhydryl oxidation: possible involvement of thiyl radicals.

Sublytic levels (microM) of hemin destabilized RBC membrane as indicated by ghost fragmentation pattern using a laser viscodiffractometer. Furthermore, electron microscopic study shows that 5 microM of hemin induced echinocytic transformation whereas higher hemin concentration (40 microM) induced spherocytic transformation. In addition, hemin oxidized sulfhydryl groups in a dose dependent fashion and Electron Spin Resonance study suggests that such oxidation may involve a thiyl radical. Moreover, sulfhydryl compounds enhanced hemin-induced lipid peroxidation. Desferroxamine could prevent hemin-induced sulfhydryl oxidation as well as hemin-induced decrease in membrane stability. In contrast, vitamin E could effectively prevent hemin-induced lipid peroxidation but could not prevent hemin-mediated membrane destabilization.

Deferoxamine↗

Alcohol and gastrointestinal bleeding.

Ethanol has experimentally been shown to be "ulcerogenic," independent of gastric intraluminal pH. Ethanol remains ulcerogenic despite antisecretory doses of H2-receptor antagonists. Low-dose alcohol stimulates acid secretion in man, an effect possibly mediated by histamine or gastrin. High-dose alcohol reduces intraluminal acid by damaging mucosa, thereby enhancing back diffusion of hydrogen ion, and also by direct damage to oxyntic (parietal) cells. Ethanol is capable of increasing gastric mucosal permeability as evidenced by the increase in back diffusion (increases intraluminal pH) and by the characteristic fall in transmucosal potential difference which reflects surface cell layer exfoliation. This exfoliation may offer an explanation for the potentiating effect of alcohol on gastric mucosal injury when it is ingested simultaneously with other gastric irritants. Ethanol of greater than 20% concentration can rapidly destroy the gastric mucus-bicarbonate layer, which may be a defense layer for both the inhibition of back diffusion and bicarbonate neutralization of existing acid. Ethanol depletes sulfhydryl compounds in gastric mucosa. These sulfhydryls may be necessary for stabilization of cell membranes as well as for binding free radicals. Ethanol is damaging to the mucosal microcirculation. The rapidity of ethanol-induced damage makes it unlikely that the process is purely ischemic. The cytoprotective phenomenon, as this brief literature review suggests, is a multifactorial, dynamic process. The complex interplay of mucosal defense factors, endogenous and exogenous stimuli, induction of humoral responses, and ultimately the success or failure of cellular repair, is unlikely to be solely mediated by endogenous prostaglandins. Although prostaglandins are unquestionably significant to cytoprotection, the supporting and, perhaps, major roles of leukotrienes, sulfhydryls, histamines, and like substances cannot be ignored. Several innovative therapeutic agents directly derived from these research efforts have already entered the clinician's formulary. The significance of the concept of cytoprotection is only now being realized in clinical applications. Alcoholic hemorrhagic gastritis, although a significant clinical entity, remains a rather elusive diagnosis for the emergency physician. As a diagnosis of suspicion, therapeutic trials of antacids, sucralfate, or perhaps synthetic prostaglandin analogues are acceptable, pending endoscopic verification or short-term resolution of symptoms. All patients presenting with true hemorrhage or with persistent symptoms should undergo gastroenterologic referral and endoscopic evaluation. The Mallory-Weiss syndrome has long been associated with acute and chronic alcohol abuse.(ABSTRACT TRUNCATED AT 400 WORDS)

Alcoholism↗

Effects of thiol compounds on methotrexate uptake by murine lymphocytes from thymus and thymic lymphosarcoma.

The characteristics of methotrexate (MTX) uptake and the effects of exogenous sulfhydryl compounds (i.e. reduced glutathione and cysteine) on MTX accumulation in thymocytes and thymic lymphosarcoma cells has been studied. Significant differences in the rate and the extent of MTX uptake between normal and neoplastic cells were demonstrated. MTX accumulation was found to be more efficient in thymic lymphosarcoma cells as compared with parental cells. In the cells examined, MTX uptake was not affected by membrane impermeable GSH. In contrast, short-term exposure to cysteine revealed heterogeneity in MTX uptake systems between normal and neoplastic thymocytes. Thus, cysteine was demonstrated to enhance the rate and extent of MTX accumulation exclusively in thymic lymphosarcoma cells. The results obtained indicate that in neoplastic thymocytes biochemical changes had developed in the membrane redox state around the MTX uptake system. These alterations are chemically distinguishable by their characteristic response to cysteine. The findings suggest that the plasma membrane changes could be exploited for preferential enhancement of MTX uptake by neoplastic thymocytes.

Animals↗

Angiotensin-converting enzyme inhibitors. New orally active antihypertensive (mercaptoalkanoyl)- and [(acylthio)alkanoyl]glycine derivatives.

A variety of N-substituted (mercaptoalkanoyl)- and [(acylthio)alkanoyl]glycine derivatives was synthesized and their ability in inhibiting the activity of angiotensin-converting enzyme (ACE) was examined in vitro and in vivo. The acylthio derivatives prepared are assumed to act as prodrugs since they are much less active than the corresponding free SH compounds in vitro and can be expected to act in vivo only after conversion to the free sulfhydryl compounds. A number of these compounds are potent ACE inhibitors that lowered blood pressure in Na-deficient, conscious spontaneously hypertensive rats (SHR), a high renin model. One of the most active members of the series was (S)-N-cyclopentyl-N-[3-[(2,2-dimethyl-1-oxopropyl)thio]-2-methyl-1 -oxopropyl]glycine (REV 3659-(S), pivopril). Structure-activity relationships are discussed.

Administration, Oral↗

Inhibition of hemin-induced hemolysis by desferrioxamine: binding of hemin to red cell membranes and the effects of alteration of membrane sulfhydryl groups.

Hemin binds to red cell membranes during hemin-induced hemolysis but the precise mechanism of hemolysis has not been characterized. Desferrioxamine (DFO), an iron chelator, inhibited hemin-induced hemolysis. DFO partially prevented hemin binding to red cell membranes and partially removed previously bound hemin. Glutathione, an intracellular sulfhydryl compound, also inhibited hemin-induced hemolysis but was only about one tenth as potent as DFO. Decrease of membrane sulfhydryl groups by treatment of cells with either N-ethylmaleimide (NEM) or diamide (azodicarboxylic acid bis [dimethylamide]) enhanced hemin-induced hemolysis. Enhancement of hemin-induced hemolysis by NEM and diamide and inhibition of hemolysis by DFO were independent with no evidence of synergism or interference between the two processes. Red cell membranes were saturated with hemin at approximately 75 nmol per mg protein. DFO decreased the hemin saturation level to 25 nmol per mg protein. In the presence of DFO, hemin was bound as the DFO-hemin complex since membranes preferentially removed DFO-hemin complexes from mixtures of complexed and free hemin while free DFO was not bound by the membranes. Access to the inner surface of the membrane was required for binding of the DFO-hemin complex since DFO completely prevented hemin binding in intact cells but not in cells undergoing hemolysis or red cell ghosts. Approximately 50 x 10(6) molecules of hemin were bound to the membrane of one red cell following hemin-induced hemolysis.

Deferoxamine↗

Protective role of sulfhydryl reagents in oxidant lung injury.

Recently there has been a great deal of interest in exploring possible ways to protect the lung from oxidant damage. Since sulfhydryl compounds are among the most important endogenous antioxidants, their therapeutic use has been proposed. Glutathione (GSH), the main intracellular nonprotein sulfhydryl, plays an important role in the maintenance of cellular proteins and lipids in their functional state. With oxidant stress, GSH acts to protect cell constituents as evidenced by increased turnover to GSSG, formation of mixed disulfides with proteins, utilization of NADPH, and utilization of glucose in the pentose pathway. When GSH is experimentally lowered (e.g., by protein deficiency or with diethylmaleate) the toxic effects of oxidant stress are exacerbated as evidenced by increased membrane and cell damage, pulmonary edema, and mortality. Several recent investigations have shown that sulfhydryl reagents (particularly N-acetyl cysteine, a cell-permeable GSH precursor) can provide significant protection against certain pulmonary toxins. N-acetyl cysteine reduced the lethal effects of 100% O2 in rats by 65%. Therefore, the therapeutic potential of sulfhydryl reagents in the treatment and prevention of oxidant injury and the mechanisms involved are an important direction for lung research.

Animals↗

A novel diazonium-sulfhydryl reaction in the inactivation of yeast alcohol dehydrogenase by diazotized 3-aminopyridine adenine dinucleotide.

Diazotized 3-aminopyridine adenine dinucleotide has been found to modify four sulfhydryl groups per molecule of enzyme during the complete inactivation of yeast alcohol dehydrogenase. The reaction of sulfhydryl groups was indicated by titration studies with 5,5-dithiobis(2-nitrobenzoic acid) as well as isolation and quantitation of the cysteinyl derivative released by acid hydrolysis of the modified enzyme. The cysteinyl derivative was identified as S-(3-pyridyl)cysteine. Authentic S-(3-pyridyl)cystein was synthesized and structurally characterized for these studies. Diazonium-sulfhydryl reactions were demonstrated for a number of diazonium derivatives with cysteine, homocysteine, glutathione, and mercaptoethanol at 0-4 degrees and neutral pH. Second order rate constants were determined in reactions of these sulfhydryl compounds with diazotized 1-methyl-3-aminopyridinium chloride, diazotized 3-aminopyridine adenine dinucleotide, and diazotized 3-aminopyridine adenine dinucleotide phosphate.

Alcohol Oxidoreductases↗

Cigarette smoke: protection of alveolar macrophages by glutathione and cysteine.

Phagocytosis of bacteria by rabbit alveolar macrophages is inhibited quantitatively by cigarette smoke in vitro. This phagocytoxic effect was abolished by addition of 0.2 to 0.4 micromole of glutathione or cysteine per milliliter of cigarette smoke. Serum protein was required to obtain both the toxic effect of the smoke and the protective action of the sulfhydryl compounds. The protective role of the sulfhydryl agents suggests an oxidant action of the cigarette smoke on these pulmonary cells.

Animals↗

Studies on rat liver nuclear DNA damaged by chemical carcinogen (3'-Me DAB) and AP DNA endonuclease. II. Kinetic properties of AP DNA endonucleases in rat liver chromatin.

An experiment was designed to investigate the reaction mechanism of AP (apurinic or apyrimidinic) DNA endonucleases (APcI, APcII, APcIII) purified from rat liver chromatin. Sulfhydryl compounds (2-mercaptoethanol, dithiothreitol) brought about optimal activities of AP DNA endonucleases and N-ethylmaleimide or HgCl2 inhibited the enzyme activities, indicating the presence of sulfhydryl group at or near the active sites of the enzymes. Mg2+ was essential and 4mM of Mg2+ was sufficient for the optimal activities of AP DNA endonucleases. Km values of APcI, APcII and APcIII for the substrate (E. coli chromosomal AP DNA) were 0.53, 0.27 and 0.36 microM AP sites, respectively. AMP was the most potent inhibitor among adenine nucleotides tested and the inhibition was uncompetitive with respective to the substrate. The Ki values of APcI, APcII and APcIII were 0.35, 0.54 and 0.41mM, respectively. The degree of nick translation of AP DNAs nicked by APcI, APcII and APcIII with Klenow fragment in the presence and absence of T4 polynucleotide kinase or alkaline phosphatase were the same, suggesting that all 3 AP DNA endonucleases excise the phosphodiester bond of AP DNA strand to release 3-hydroxyl nucleotides and 5-phosphomonoester nucleotides.

Animals↗

Involvement of membrane sulfhydryl groups in apomorphine-induced inhibition of neutrophil migration.

Apomorphine inhibits fMet-Leu-Phe stimulated migration of rabbit neutrophils, in the absence of albumin, in the concentration range of 1-20 microM. In the presence of albumin inhibition by apomorphine occurs at higher concentrations. Casein-stimulated migration, and non-stimulated migration are equally inhibited by apomorphine. The presence or absence of extracellular Ca2+ has no effect on the degree of inhibition by apomorphine. The ability of albumin to interfere with apomorphine inhibition of migration is diminished after treatment of albumin with the sulfhydryl reagent N-ethylmaleimide. Sulfhydryl compounds, such as glutathione and dithiothreitol, strongly interfere with apomorphine inhibition. The results suggest that the inhibiting effect of apomorphine is due to a reaction with sulfhydryl groups, the intactness of which is required for neutrophil migration, and which are probably located on the plasma membrane.

Albumins↗

The enhanced sensitivity of mammalian cells to killing by X rays after prolonged exposure to several nitroimidazoles.

Electron affinic compounds, such as misonidazole, preferentially sensitize hypoxic cells to killing by X rays, and are also preferentially cytotoxic to cells deficient in oxygen. Prolonged exposure of cells to misonidazole prior to irradiation results in an increased radiosensitization. This is expressed as the Extra Enhancement Ratio (EER), defined as the ratio of the doses delivered immediately after the addition of the sensitizer or after prolonged incubation, that produce a given biological effect. Chinese hamster V79 cells have been used to investigate this prolonged incubation effect for a variety of 2-nitroimidazoles including misonidazole, desmethylmisonidazole and SR-2508 and as well as two ortho-substituted-4-nitroimidazoles with a bromine or sulfonamide group substituted in the 5-position. A considerable variation was observed in the magnitude of the Extra Enhancement Ratio (EER) produced by pre-incubation with different compounds at concentrations that produce the same sensitizing effect. There is a good correlation between the EER and the measured rate at which the various sensitizers deplete cells of non-protein sulfhydryl compounds. There is also a good correlation between the EER and the fraction of cells killed by the pre-incubation period in the drug.

Animals↗

In vitro oxidation of vitamin E, vitamin C, thiols and cholesterol in rat brain mitochondria incubated with free radicals.

The kinetics of oxidation of endogenous antioxidants such as vitamins C and E and thiols as well as membrane cholesterol in isolated rat brain mitochondria were studied. Oxidation was induced by incubating the mitochondria at 37 degrees C with the free radical generators 2,2' azobis (2'-amidinopropane) dihydrochloride (ABAPH) and 2,2' azobis (2,4-dimethyl) valeronitrile (ABDVN) which undergo thermal decomposition to yield free radicals. An approximate order for the in vitro ease of oxidation was: ascorbate >> alpha-tocopherol > sulfhydryls >> cholesterol. However, small amounts of ascorbate were present in the mitochondria when alpha-tocopherol and sulfhydryl compounds were getting oxidized. This observation is different from those with more homogeneous biological substrates like blood plasma or serum. The order of oxidation of the various compounds is a function of not only the redox potentials but also the (a) concentrations of the oxidized and reduced species, (b) compartmentation of the compounds and (c) enzymatic and nonenzymatic systems for the repair or regeneration of the individual antioxidants. Even though ascorbate levels are quite low within mitochondria this nutrient may play a major role as a first line of defense against oxidative stress. The lipid-soluble ABDVN was much more potent in oxidizing membrane alpha-tocopherol and thiols than the water-soluble ABAPH. With both free radical generators the rate of oxidation of the antioxidants consisted of two phases. The initial phase, that is more rapid, may represent a pool of antioxidant that is involved in immediate antioxidant protection of the organelle with the slower compartment being responsible for replenishing the faster pool whenever needed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amidines↗