Eugene G. DeMaster

Stable Compounds of Cigarette Smoke Induce Endothelial Superoxide Anion Production via NADPH Oxidase Activation

Objective—Endothelial dysfunction is an early manifestation of cigarette smoke (CS) toxicity. We have previously demonstrated that CS impairs nitric oxide (NO)-mediated endothelial function via increased generation of superoxide anion (SYMBOL). In these studies, we investigated whether stable compounds present in CS activate specific pathways responsible for the increased endothelial SYMBOL production. Methods and Results—Short exposure of bovine pulmonary artery endothelial cells (BPAECs), human pulmonary artery endothelial cells, and rat pulmonary arteries to CS extracts (CSEs) resulted in a large increase in SYMBOL production (20-fold, 3-fold, and 2-fold increase, respectively; P< 0.05 versus control), which was inhibited by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors diphenyleneiodinium, apocynin, and gp91 docking sequencetat peptide but not by oxypurinol, the NO synthase inhibitor NG-nitro-L-arginine methyl ester, or the mitochondrial respiration inhibitor rotenone. Exposure of BPAECs to acrolein, a stable thiol-reactive agent found in CS, increased SYMBOL production 5-fold, which was prevented by prior inhibition of NADPH oxidase. Conclusions—These studies demonstrate that thiol-reactive stable compounds in CS can activate NADPH oxidase and increase endothelial SYMBOL production, thereby reducing NO bioactivity and resulting in endothelial dysfunction. Clinically, these studies may contribute to the development of agents able to mitigate CS-mediated vascular toxicity.

Detoxification of hydrogen sulfide and methanethiol in the cecal mucosa

Colonic bacteria liberate large quantities of the highly toxic gases hydrogen sulfide (H(2)S) and methanethiol (CH(3)SH). The colonic mucosa presumably has an efficient means of detoxifying these compounds, which is thought to occur through methylation of H(2)S to CH(3)SH and CH(3)SH to dimethylsulfide (CH(3)SCH(3)). We investigated this detoxification pathway by incubating rat cecal mucosal homogenates with gas containing H(2)S, CH(3)SH, or CH(3)SCH(3). Neither CH(3)SH nor CH(3)SCH(3) was produced during H(2)S catabolism, whereas catabolism of CH(3)SH liberated H(2)S but not CH(3)SCH(3). Thus, H(2)S and CH(3)SH are not detoxified by methylation to CH(3)SCH(3). Rather, CH(3)SH is demethylated to H(2)S, and H(2)S is converted to nonvolatile metabolites. HPLC analysis of the homogenate showed the metabolite to be primarily thiosulfate. Analysis of cecal venous blood obtained after intracecal instillation of H(2)(35)S revealed that virtually all absorbed H(2)S had been oxidized to thiosulfate. The oxidation rate of H(2)S by colonic mucosa was 10,000 times greater than the reported methylation rate. Conversion to thiosulfate appears to be the mechanism whereby the cecal mucosa protects itself from the injurious effects of H(2)S and CH(3)SH, and defects in this detoxification possibly could play a role in colonic diseases such as ulcerative colitis.

Cigarette smoke-induced endothelium dysfunction: role of superoxide anion.

Objectives Cigarette smoking is strongly associated with coronary artery disease and atherosclerosis. While smoking has been shown to impair endothelium-dependent vasorelaxation, the mechanisms involved are not completely understood. We investigated the role of superoxide anion and vasoconstricting prostanoids in cigarette smoke induced endothelial dysfunction. Methods Endothelial function was assessed in rat aortic rings exposed to cigarette smoke-treated Krebs buffer, by measuring agonist stimulated endothelium-dependent vasorelaxation. Treatment with superoxide dismutase (SOD) as well as ifetroban, thromboxane A2/prostaglandin endoperoxide H2 (TxA2/PGH2) receptor blocker and indomethacin (cyclooxygenase inhibitor) was used to investigate the role of superoxide anion and vasoconstricting eicosanoids on cigarette smoke-induced endothelial dysfunction. The effect of cigarette smoke on endothelial nitric oxide synthase (eNOS) catalytic activity was measured by conversion of l-arginine to l-citrulline in rat aortas and rat endothelial cell homogenates supplemented with eNOS cofactors. Results Relaxations to receptor-dependent agonists, acetylcholine and adenosine diphosphate (ADP), as well as to a receptor-independent agonist, A23187 (Ca2+ ionophore) were significantly impaired by cigarette smoke. Cigarette smoke did not impair relaxations to sodium nitroprusside, indicating preserved guanylate cyclase activity. Further, cigarette smoke did not affect eNOS catalytic activity in homogenates from either endothelial cells or aortas previously exposed to cigarette-smoke-treated Krebs buffer. Treatment with SOD or ifetroban and in a lesser degree by indomethacin prevented cigarette-smoke-induced endothelial dysfunction. Conclusions Taken together, our results suggest that cigarette smoking causes an increase in vascular superoxide production which results in decreased nitric oxide (NO) bioactivity and concomitantly increases production of cyclooxygenase dependent and independent vasoconstricting eicosanoids.

Oxidation of hydrogen sulfide and methanethiol to thiosulfate by rat tissues: a specialized function of the colonic mucosa

Colonic bacteria release large quantities of the highly toxic thiols hydrogen sulfide (H(2)S) and methanethiol (CH(3)SH). These gases rapidly permeate the colonic mucosa, and tissue damage would be expected if the mucosa could not detoxify these compounds rapidly. We previously showed that rat cecal mucosa metabolizes these thiols via conversion to thiosulfate. The purpose of the present study in rats was to determine if this conversion of thiols to thiosulfate is (a) a generalized function of many tissues, or (b) a specialized function of the colonic mucosa. The tissues studied were mucosa from the cecum, right colon, mid-colon, ileum, and stomach; liver; muscle; erythrocytes; and plasma. The metabolic rate was determined by incubating homogenates of the various tissues with H(2)(35)S and CH(3)(35)SH and measuring the rate of incorporation of (35)S into thiosulfate and sulfate. The detoxification activity of H(2)S (expressed as nmol/mg per min) that resulted in thiosulfate production was at least eight times greater for cecal and right colonic mucosa than for the non-colonic tissues. Thiosulfate production from CH(3)SH was at least five times more rapid for cecal and right colonic mucosa than for the non-colonic tissues. We conclude that colonic mucosa possesses a specialized detoxification system that allows this tissue to rapidly metabolize H(2)S and CH(3)SH to thiosulfate. Presumably, this highly developed system protects the colon from what otherwise might be injurious concentrations of H(2)S and CH(3)SH. Defects in this detoxification pathway possibly could play a role in the pathogenesis of various forms of colitis.

Mechanisms of Inhibition of Aldehyde Dehydrogenase by Nitroxyl, the Active Metabolite of the Alcohol Deterrent Agent Cyanamide

Nitroxyl, produced in the bioactivation of the alcohol deterrent agent cyanamide, is a potent inhibitor of aldehyde dehydrogenase (AIDH); however, the mechanism of inhibition of AlDH by nitroxyl has not been described previously. Nitroxyl is also generated from Angelis salt (Na2N2O3) at physiological pH, and, indeed, Angelis salt inhibited yeast AlDH in a time- and concentration-dependent manner, with IC50 values under anaerobic conditions with and without NAD+ of 1.3 and 1.8 microM, respectively. Benzaldehyde, a substrate for AlDH, competitively blocked the inhibition of this enzyme by nitroxyl in the presence of NAD+, but not in its absence, in accord with the ordered mechanism of this reaction. The sulfhydryl reagents dithiothreitol (5 mM) and reduced glutathione (10 mM) completely blocked the inhibition of AlDH by Angelis salt. These thiols were also able to partially restore activity to the nitroxyl-inhibited enzyme, the extent of reactivation being dependent on the pH at which the inactivation occurred. This pH dependency indicates the formation of two inhibited forms of the enzyme, with an irreversible form predominant at pH 7.5 and below, and a reversible form predominant at pH 8.5 and above. The reversible form of the inhibited enzyme is postulated to be an intra-subunit disulfide, while the irreversible form is postulated to be a sulfinamide. Both forms of the inhibited enzyme are derived via a common N-hydroxysulfenamide intermediate produced by the addition of nitroxyl to active site cysteine thiol(s).

Hydroxylamine is a vasorelaxant and a possible intermediate in the oxidative conversion of L-arginine to nitric oxide

Our objective was to determine whether hydroxylamine is a possible intermediate in the oxidative conversion of L-arginine to nitric oxide. Vasorelaxation by hydroxylamine is known to be mediated by nitric oxide. The vasorelaxant properties of hydroxylamine were examined using rat aortic rings and an isolated rat lung perfusion model. Hydroxylamine and acetylcholine were equally effective in relaxing norepinephrine-contracted intact aortic rings, whereas only hydroxylamine relaxed aortic rings with endothelium removed. This endothelium-independent vasorelaxation by hydroxylamine indicated that the hydroxylamine-converting enzyme is not localized solely within endothelial cells. Catalase, an enzyme known to oxidize hydroxylamine to nitric oxide, was present in homogenates of intact and endothelium-denuded rings. Cyanamide, another catalase substrate and a known precursor of nitroxyl (HNO), was not a vasorelaxant of aortic rings or of isolated, hypoxia-constricted lungs. These results suggest that free nitroxyl is not an intermediate in the oxidation of hydroxylamine to nitric oxide. An overall pathway for the oxidative conversion of L-arginine through an hydroxylamine intermediate to nitric oxide is proposed.

Induction of Endothelial Cell Injury by Cigarette Smoke

Cigarette smoke contains different populations of free radicals which may be responsible for endothelial cell (EC) injury of smokers. The purpose of this study was to examine the effects of gas-phase cigarette smoke on EC endothelium-derived relaxing factor (EDRF)/NO-guanylate cyclase (GC)-cGMP pathway and on EC detachment-type injury after incubation with smoke. Furthermore, we examined whether different kind of antioxidants can prevent smoke-caused EC injury. We measured cGMP pathway using direct (sodium nitroprusside, SNP) and indirect (A23187, the calcium ionophore and bradykinin, BK) activators of GC. Directly and indirectly stimulated EC cGMP production dose-dependently decreased and EC detachment increased after incubation with smoke. Externally added thiols (glutathione, GSH; D-Penicillamine, DP; N-acetylcysteine, NAC) protected EC from damage of cGMP production and cell detachment. Other antioxidants (catalase, deferoxamine and superoxide dismutase) were ineffective. These results suggest that the thiol containing GC in EC is destroyed or inactivated or thiol like species responsible for activation of GC is incomplete in EC after incubation with smoke. It is also possible that externally added thiols bind an unknown component of smoke and this way, EC is protected. EC injury may contribute to vascular diseases associated with cigarette smoking.

First-pass gastric mucosal metabolism of ethanol is negligible in the rat.

Ethanol metabolism by gastric alcohol dehydrogenase (ADH) is thought to be an important determinant of peripheral ethanol time-concentration curves (AUCs) in rats and humans. We quantitated this metabolism in rats by measuring the gastric absorption of oral ethanol (0.25 g/kg) and the gastric venous-arterial (V-A) difference of ethanol versus ethanol metabolites (acetate, acetaldehyde, and bicarbonate). Over 1 h, approximately 20% of the ethanol was absorbed from the stomach and 70% was emptied into the duodenum. The gastric V-A difference of ethanol metabolites was less than 4% of that of ethanol. Thus, gastric metabolism accounted for less than 1% (less than 4% of 20% absorbed) of the dose. This negligible metabolism was predictable from the low affinity of gastric ADH for ethanol. In contrast, gastric ADH has a high affinity for octanol, and 66% of this compound was metabolized during gastric absorption. Evidence supporting gastric metabolism of ethanol largely derives from the lower AUCs observed after oral than after intravenous administration; however, we observed increasingly higher AUCs with increasingly rapid portal vein infusions of identical ethanol doses. We conclude that gastric metabolism of ethanol is negligible in the rat, and differences in AUCs ascribed to gastric metabolism may reflect differences in ethanol absorption.

Analysis of hepatic reduced glutathione, cysteine and homocysteine by cation-exchange high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method employing a mercury-based electrochemical detector and a cation-exchange column is described for the simultaneous measurement of reduced glutathione, cysteine, and homocysteine in liver homogenates. Sample preparation involves precipitation of protein with perchloric acid, removal of perchlorate by precipitation as its potassium salt and dilution with mobile phase. Mercaptoethylglycine is used as the internal standard. Using this procedure, the sum of the individual hepatic thiols agreed well with the total thiols determined with Ellmans reagent. Comparisons were made with (a) control rats, (b) rats depleted of hepatic thiols by pargyline pretreatment, and (c) rats administered L-cysteine.

Use of measurements of ethanol absorption from stomach and intestine to assess human ethanol metabolism

Controversy exists concerning the site (stomach vs. liver) and magnitude of first-pass metabolism of ethanol. We quantitated gastric and total ethanol absorption rates in five male subjects and utilized these measurements to evaluate first-pass metabolism. Gastric emptying of ethanol (0.15 g/kg) was determined via a gamma camera and gastric absorption from the ratio of gastric ethanol to [14C]polyethylene glycol. Gastric absorption accounted for 30% and 10% of ethanol administered with food and water, respectively. With food, estimated gastric mucosal ethanol concentrations fell from 19 to 5 mM over 2 h. Calculations using these concentrations and kinetic data for gastric alcohol dehydrogenase showed <2% of the dose underwent gastric metabolism. Application of observed ethanol absorption rates to a model of human hepatic ethanol metabolism indicated that only 30% and 4% of the dose underwent first-pass metabolism when administered with food and water, respectively. We conclude that virtually all first-pass ethanol metabolism occurs in the liver and first-pass metabolism accounts for only a small fraction of total clearance.Controversy exists concerning the site (stomach vs. liver) and magnitude of first-pass metabolism of ethanol. We quantitated gastric and total ethanol absorption rates in five male subjects and utilized these measurements to evaluate first-pass metabolism. Gastric emptying of ethanol (0.15 g/kg) was determined via a gamma camera and gastric absorption from the ratio of gastric ethanol to [14C]polyethylene glycol. Gastric absorption accounted for 30% and 10% of ethanol administered with food and water, respectively. With food, estimated gastric mucosal ethanol concentrations fell from 19 to 5 mM over 2 h. Calculations using these concentrations and kinetic data for gastric alcohol dehydrogenase showed <2% of the dose underwent gastric metabolism. Application of observed ethanol absorption rates to a model of human hepatic ethanol metabolism indicated that only 30% and 4% of the dose underwent first-pass metabolism when administered with food and water, respectively. We conclude that virtually all first-pass ethanol metabolism occurs in the liver and first-pass metabolism accounts for only a small fraction of total clearance.