Bioactivation of pentaerythrityl tetranitrate by mitochondrial aldehyde dehydrogenase

Mitochondrial aldehyde dehydrogenase (ALDH2) contributes to vascular bioactivation of the antianginal drugs nitroglycerin (GTN) and pentaerythrityl tetranitrate (PETN), resulting in cGMP-mediated vasodilation. Although continuous treatment with GTN results in the loss of efficacy that is presumably caused by inactivation of ALDH2, PETN does not induce vascular tolerance. To clarify the mechanisms underlying the distinct pharmacological profiles of GTN and PETN, bioactivation of the nitrates was studied with aortas isolated from ALDH2-deficient and nitrate-tolerant mice, isolated mitochondria, and purified ALDH2. Pharmacological inhibition or gene deletion of ALDH2 attenuated vasodilation to both GTN and PETN to virtually the same degree as long-term treatment with GTN, whereas treatment with PETN did not cause tolerance. Purified ALDH2 catalyzed bioactivation of PETN, assayed as activation of soluble guanylate cyclase (sGC) and formation of nitric oxide (NO). The EC(50) value of PETN for sGC activation was 2.2 ± 0.5 μM. Denitration of PETN to pentaerythrityl trinitrate was catalyzed by ALDH2 with a specific activity of 9.6 ± 0.8 nmol · min(-1) · mg(-1) and a very low apparent affinity of 94.7 ± 7.4 μM. In contrast to GTN, PETN did not cause significant inactivation of ALDH2. Our data suggest that ALDH2 catalyzes bioconversion of PETN in two distinct reactions. Besides the major denitration pathway, which occurs only at high PETN concentrations, a minor high-affinity pathway may reflect vascular bioactivation of the nitrate yielding NO. The very low rate of ALDH2 inactivation, presumably as a result of low affinity of the denitration pathway, may at least partially explain why PETN does not induce vascular tolerance.     

Comparative vasodilator effects of nitroglycerin,pentaerythritol trinitrate and biometabolites,and other organic nitrates

Previous studies in man have shown pentaerythritol (PE) trinitrate, given either sublingually or orally, produces a prolonged hypotensive effect. The coronary vasodilator and systemic vasoddepressor activities of PE trinitrate and its metabolites, PE dinitrate, PE mononitrate and PE, were evaluated in dogs to determine whether the metabolites were active and contributory. Coronary vasodilator activity was estimated with a flow transducer placed on the left anterior descending artery, and reduction of arterial pressure was determined directly via the femoral artery. Quantitative comparisons were made from dose—response curves established for nitroglycerin (NG), PE nitrates, and other common organic nitrates after intrajugular administration. Increase of coronary blood flow and reduction of arterial pressure were proportionally related, and the proportionality was the same for all drugs. Relative to NG, the potency of PE trinitrate was about 20%, erythrityl tetranitrate 12%, and isosorbide dinitrate 3.5%. The ratios of vasodilator activity of PE trinitrate and its metabolites were: PE trinitrate 100; PE dinitrate 1.5; PE mononitrate 0.5; and PE 0. Tachyphylaxis was observed after close-order injections of NG or PE trinitrate. In addition, there was cross tolerance between NG and PE trinitrate and also between PE trinitrate and its less active metabolites.     

Nitrate tolerance as a model of vascular dysfunction: Roles for mitochondrial aldehyde dehydrogenase and mitochondrial oxidative stress

Organic nitrates are a group of very effective anti-ischemic drugs. They are used for the treatment of patients with stable angina, acute myocardial infarction and chronic congestive heart failure. A major therapeutic limitation inherent to organic nitrates is the development of tolerance, which occurs during chronic treatment with these agents. The mechanisms underlying nitrate tolerance remain incompletely defined and are likely multifactorial. One mechanism seems to be a diminished bioconversion of nitroglycerin, another seems to be the induction of vascular oxidative stress, and a third may include neurohumoral adaptations. Recent studies have revealed that mitochondrial reactive oxygen species (ROS) formation and a subsequent oxidative inactivation of nitrate reductase, the mitochondrial aldehyde dehydrogenase (ALDH-2), play an important role in the development of nitrate and crosstolerance. The present review focus first on the role of oxidative stress and second on the role of ALDH-2 in organic nitrate bioactivation leading to the development of tolerance and cross-tolerance (endothelial dysfunction) in response to nitroglycerin treatment. Recently, the role of mitochondrial oxidative stress in the development of nitrate tolerance was demonstrated in a mouse model with a heterozygous deletion of manganese superoxide dismutase (MnSOD^+/?), which is the mitochondrial isoform of this enzyme. Studies from our own laboratory have provided evidence for cross-talk between mitochondrial and cytosolic (Nox-dependent) sources of ROS. We close this review by focusing on the protective properties of the organic nitrate pentaerithrityl tetranitrate, which upregulates enzymes that have strong antioxidative activity, such as heme oxygenase-1 and ferritin, thereby preventing the development of tolerance and endothelial dysfunction.     

Potency and in vitro tolerance of organic nitrates: partially denitrated metabolites contribute to the tolerance-devoid activity of pentaerythrityl tetranitrate

Neither therapeutic dosage of nitrovasodilators nor the development of tolerance correlates with nitrate groups in these molecules. Clinically, low dosages of glyceryl trinitrate (GTN) develop tolerance, but 100-fold higher dosages of pentaerythrityl tetranitrate (PETN) do not. Vasorelaxation was studied on prostaglandian F2alpha (PGF2alpha)-precontracted porcine pulmonary arteries in organ bath procedure. In vitro tolerance was induced by incubating the arteries with different nitrate concentrations and thereafter concentration-response curves were repeated. Furthermore, 14 mg/kg PETN were daily administered to rats by gavage; PETN and metabolites were measured in feces and blood. In vitro, the vasodilator potencies increased from mononitrates to tetranitrates (pD2: 4.14 to 8.18); PETN was the most potent vasodilator. In vitro tolerance was found with PETN and trinitrates but not with dinitrates and mononitrates. Thus, in vitro tolerance correlated with the in vitro potency of nitrates but not with the vasodilator potency of NO donors in general, because S-nitroso-N-aectyl-D-penicillamine and N-phenylpiperazin-NONOate were more potent than GTN but did not induce tolerance. After feeding of rats with PETN, pentaerythrityl dinitrate (PEdiN) and mononitrate (PEmonoN) but neither PETN nor PEtriN (both detected in feces) were found in the blood. The missing systemic bioavailability of PETN and PEtriN may explain the discrepancy between in vitro and in vivo findings. We conclude that the partially denitrated metabolites PEdiN and PEmonoN contribute to the moderate and tolerance-devoid clinical activity of PETN.     

Time course of the carbon tetrachloride-induced decrease in mitochondrial aldehyde dehydrogenase activity

Hepatic microsomal enzymes like cytochrome P-450 and glucose 6-phosphatase are inhibited after exposure to CCl4 in vivo. Since comparatively less is known about the effects of CCl4 on nonmicrosomal enzymes, we investigated the rapidity by which CCl4 inhibits the low Km mitochondrial aldehyde dehydrogenase (ALDH) isozyme, an enzyme known to be inhibited 24 hr after CCl4 treatment. The activity of this ALDH isozyme was significantly lowered 6 and 12 hr after a single 1 ml/kg intragastric dose of CCl4. The mitochondrial low Km ALDH specific activities exhibited a similar pattern of destruction/inhibition to the documented target enzyme microsomal cytochrome P-450 in that lowest values were observed 6 hr after CCl4. These values were 44 and 37% of control for cytochrome P-450 content and the low Km ALDH activity, respectively. Alcohol dehydrogenase activity, expressed as activity per gram liver, was depressed 12 hr after CCl4 dosing. Finally, the activity of the low Km cytosolic ALDH, the isozyme that metabolizes malondialdehyde at low concentrations, was not affected by CCl4 treatment. The CCl4-induced decline in the activity of the matrix ALDH isozyme occurs earlier than previously reported mitochondrial damage. The study of sensitive enzymes like the low Km ALDH may provide valuable information by which it may be possible to determine the relationship of the truly rapid biochemical effects of CCl4 such as microsomal lipid peroxidation with later effects on nonmicrosomal components.     

Identification of human liver mitochondrial aldehyde dehydrogenase as a potential target for microcystin-LR

Microcystins (MCs) are hepatotoxins produced by a variety of freshwater cyanobacteria. The toxicity of these hepatotoxins is a severe health issue for both humans and livestock; MCs have been implicated in the development of liver cancer, necrosis, and even deadly intrahepatic bleeding. Microcystin-LR (MC-LR) is the MC variant most commonly encountered in a contaminated aquatic system. Thus far, MC-LR has only been shown to target the serine/threonine protein phosphatases 1 and 2A (PP1 and PP2A) and it is still unknown whether MC-LR can bind and inhibit any other protein targets inside the cell. To find potential MC-LR targets, we screened a phage display library for peptide ligands that specifically recognize MC-LR. Using these peptide sequences as guides, we performed a series of bioinformatics analyses revealing that MC-LR binds human liver aldehyde dehydrogenase 2 (ALDH2) at residues 447-451. We confirmed MC-LR binding of ALDH2 via automated docking computation, which yielded results matching our experimental and bioinformatics analyses. ALDH2 dysfunction may lead to aldehyde-induced reactive oxygen species (ROS) generation and, in turn, apoptosis. Therefore, ALDH2 could potentially be a target of MC-LR associated with the process of ROS-induced apoptosis. Our current study presents a new approach to the study of interactions of biological molecules by combining phage display technology with computational methods.     

Developmental expression of aldehyde dehydrogenase in rat: a comparison of liver and lung development.

Metabolism is one of the major determinants for age-related changes in susceptibility to chemicals. Aldehydes are highly reactive molecules present in the environment that also can be produced during biotransformation of xenobiotics and endogenous metabolism. Although the lung is a major target for aldehyde toxicity, early development of aldehyde dehydrogenases (ALDHs) in lung has been poorly studied. The expression of ALDH in liver and lung across ages (postnatal day 1, 8, 22, and 60) was investigated in Wistar-Han rats. In adult, the majority of hepatic ALDH activity was found in mitochondria, while cytosolic ALDH activity was the highest contributor in lung. Total aldehyde oxidation capability in liver increases with age, but stays constant in lung. These overall developmental profiles of ALDH expression in a tissue appear to be determined by the different composition of ALDH isoforms within the tissue and their independent temporal and tissue-specific development. ALDH2 showed the most notable tissue-specific development. Hepatic ALDH2 was increased with age, while the pulmonary form did not. ALDH1 was at its maximum value at postnatal day 1 (PND1) and decreased thereafter both in liver and lung. ALDH3 increased with age in liver and lung, although ALDH3A1 was only detectible in lung. Collectively, the present study indicates that, in the case of aldehyde exposure, the in vivo responses would be tissue and age dependent.     

Oxidative stress and mitochondrial impairment after treatment with anti-HIV drugs: clinical implications

Thirty years after the discovery of HIV infection, there are numerous antiretroviral drugs that control the disease when administered in a potent combination referred to as Highly Active Antiretroviral Therapy (HAART). This therapy reduces the viral load and improves immune system reconstitution, leading to a significant reduction of HIV-related morbidity and mortality. However, HAART does not completely eliminate HIV, so treatment must continue throughout the patient's life. Prolonged use of HAART has been related to long-term adverse events that can compromise patient health. These deleterious effects have been reported for the majority of antiretroviral drugs and are the most common causes for therapy discontinuation. In most of these adverse events, such as diabetes, cardiovascular diseases, neurological disorders and metabolic alterations, oxidative stress and mitochondrial impairment play important roles. This review covers the implication of antiretroviral drugs in the overproduction of reactive oxygen species and the reduction of antioxidant defences, and in the consequent mitochondrial dysfunction, focusing on the molecular mechanisms involved and the clinical implications for HIV-infected patients.     

Structure of daidzin, a naturally occurring anti-alcohol-addiction agent, in complex with human mitochondrial aldehyde dehydrogenase

The ALDH2*2 gene encoding the inactive variant form of mitochondrial aldehyde dehydrogenase (ALDH2) protects nearly all carriers of this gene from alcoholism. Inhibition of ALDH2 has hence become a possible strategy to treat alcoholism. The natural product 7-O-glucosyl-4'-hydroxyisoflavone (daidzin), isolated from the kudzu vine ( Peruraria lobata), is a specific inhibitor of ALDH2 and suppresses ethanol consumption. Daidzin is the active principle in a herbal remedy for "alcohol addiction" and provides a lead for the design of improved ALDH2. The structure of daidzin/ALDH2 in complex at 2.4 A resolution shows the isoflavone moiety of daidzin binding close to the aldehyde substrate-binding site in a hydrophobic cleft and the glucosyl function binding to a hydrophobic patch immediately outside the isoflavone-binding pocket. These observations provide an explanation for both the specificity and affinity of daidzin (IC50 =80 nM) and the affinity of analogues with different substituents at the glucosyl position.     

Blood levels of the metabolites of glyceryl trinitrate and pentaerythritol tetranitrate after administration of a two-step preparation.

Blood levels and urinary excretion rates of glyceryl trinitrate- pentaerythritol tetranitrate, and their less nitrate containing metabolites have been determined in ten human volunteers after a single dose of a two- step preparation containing glyceryl trinitrate and pentaerythritol tetranitrate. Blood levels accounted for peak levels of about 40% of the glyceryl trinitrate and 0.4% of the pentaerythritol tetranitrate metabolites, respectively. Within the first 24 h post administration 22% of the glyceryl trinitrate and 19% of the pentaerythritol tetranitrate were excreted as nitrate metabolites, chiefly in form of conjugates. The determinations were obtained by gas chromatography on extremely inactive columns and electron capture detection by means of derivatives.     

Brain aldehyde dehydrogenase activity in rat strains with high and low ethanol preferences

The activity of aldehyde dehydrogenase in subcellular fractions of whole brain homogenates from the AA and ANA rat strains developed respectively for high and low ethanol preferences has been studied. No significant strain or sex differences between naive AA and ANA rats were found. In ethanol-experienced rats some strain and sex differences were found, the most consistent being higher enzyme activity in AA females than in males both with aliphatic and aromatic aldehyde substrates. However, contrary to previous findings no relation between brain aldehyde dehydrogenase activity and drinking behavior was found in the AA and ANA rat strains.     

Inhibition of rat hepatic mitochondrial aldehyde dehydrogenase isozymes by repeated cyanamide administration: Pharmacokinetic-pharmacodynamic relationships

The inhibition of rat hepatic mitochondrial aldehyde dehydrogenase (ALDH) isozymes was studied in apparent steady-state conditions after repeated intra-peritoneal cyanamide administration. The low-K_m mitochondrial ALDH isozyme was more susceptible to cyanamide-induced inhibition (DI₅₀ = 0.104 mg kg^?1) than the high-K_m isozyme (DI₅₀ = 8.52 mg kg^?1), with almost complete inhibition occurring at 0.35 mg kg^?1 total cyanamide administered for the low-K_m isozyme. The relationships between plasma and liver cyanamide concentrations and the inhibition of high-K_m ALDH were established by means of the sigmoid I_max model. The effect of dosing rate on the plasma concentration of cyanamide at apparent steady-state showed non-linearity, indicating that clearance or first-pass metabolism of cyanamide during its absorption after intraperitoneal administration did not remain constant throughout the range of doses studied.     

Increase of hepatic and serum aldehyde dehydrogenase activity after TCDD treatment.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a contaminant of some herbicides, is an extremely potent enzyme inducer. Male albino rats of two genetically different substrains developed for the inducibility (RR) and noninducibility (rr) of aldehyde dehydrogenase by phenobarbital were given TCDD 80 microgram/kg as a single dose 6 days before analysis. rr-Animals having no induction of the hepatic soluble high-Km aldehyde dehydrogenase by phenobarbital showed an approximate 25-fold increase in activity after TCDD treatment. The increase in aldehyde dehydrogenase activity could be detected only when measured with a millimolar substrate concentration. The aldehyde dehydrogenase activity in serum was increased 3-fold after TCDD administration in both substrains.     

Disulfiram metabolism as a requirement for the inhibition of rat liver mitochondrial low Km aldehyde dehydrogenase.

In humans and animals, disulfiram produces a disulfiram-ethanol reaction after an ethanol challenge, the basis of which is the inhibition of liver aldehyde dehydrogenase (ALDH). Disulfiram and the metabolites diethyldithiocarbamate (DDTC), diethyldithiocarbamate-methyl ester (DDTC-Me), and S-methyl-N,N-diethylthiolcarbamate (DETC-Me) were studied in order to determine the role of bioactivation in disulfiram's action as an inhibitor of rat liver mitochondrial low Km ALDH (RLM low Km ALDH). In in vitro studies, disulfiram and DDTC (0.01 to 2.0 mM) both inhibited RLM low Km ALDH in a concentration-dependent manner. The addition of rat liver microsomes to the mitochondrial incubation did not further increase disulfiram-induced RLM low Km ALDH inhibition. However, DDTC-induced RLM low Km ALDH inhibition was increased further, but only at DDTC concentrations less than 0.05 mM. DDTC-Me and DETC-Me (2.0 mM) similarly exhibited an increased RLM low Km ALDH inhibition after the addition of liver microsomes. In in vivo studies, disulfiram (75 mg/kg), DDTC (114 mg/kg), DDTC-Me (41.2 mg/kg) or DETC-Me (18.6 mg/kg) administered i.p. to female rats inhibited RLM low Km ALDH. Inhibition of drug metabolism by pretreatment of rats with the cytochrome P450 inhibitor N-octylimidazole (NOI) (20 mg/kg, i.p.) prior to either disulfiram, DDTC, DDTC-Me or DETC-Me administration blocked the inhibition of RLM low Km ALDH. The in vitro and in vivo data support the conclusion that bioactivation of disulfiram to a reactive chemical species is required for RLM low Km ALDH inhibition and a disulfiram-ethanol reaction.     

Alcohol and aldehyde dehydrogenase activity in the stomach and small intestine of rats poisoned with methanol

The activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) were measured with fluorogenic naphthaldehydes in the stomach and small intestine homogenates of rats dosed with 6 g methanol/kg bw after 6, 12, 24 h and 2, 5, 7 days. After intoxication with a sublethal dose, the ADH activity measured with these naphthaldehydes and ALDH activities in the stomach and small intestine were significantly decreased. This inhibition is stronger in the stomach and probably depends on cell damage and protein denaturation. We conclude that the activity measured with 6-methoxy-2-naphthaldehyde (MONAL-62) may be due to the activity of rat ADH-1 isoenzyme, and the activity detected with 4-methoxy-1-naphthaldehyde (MONAL-41) to the activity of rat ADH-2 isoenzyme.     

Comparing the role of glutathione-S-transferase and mitochondrial aldehyde dehydrogenase in nitroglycerin biotransformation and the correlation with calcitonin gene-related peptide

Both glutathione-S-transferase (GST) and mitochondrial aldehyde dehydrogenase (ALDH-2) have been reported to participate in the biotransformation of nitroglycerin. In this study, we explored which is the major player in nitroglycerin biotransformation. In vivo, rats were treated with nitroglycerin, the blood pressure and plasma calcitonin gene-related peptide (CGRP) were measured. The inhibitor of GST (ethacrynic acid) or ALDH-2 (cyanamide) was given before nitroglycerin treatment; In vitro, the isolated aorta rings were incubated with nitroglycerin to obtain the concentration–response curve. Ethacrynic acid or cyanamide was pre-incubated with the rings before nitroglycerin treatment. The release of CGRP from the aorta rings was determined. Both ethacrynic acid and cyanamide were able to reverse the depressant action of nitroglycerin while the inhibitory effect of cyanamide was more profound. However, combined administration of both inhibitors did not produce an additive effect. The change of plasma CGRP level positively correlated with the change of nitroglycerin-induced hypotensive effects. In the isolated aorta rings, vasodilator responses to nitroglycerin were reduced in the presence of ethacrynic acid or cyanamide while the inhibitory effect of cyanamide was more profound. However, combined administration of both inhibitors did not produce an additive effect. The change of CGRP release from the rings positively correlated with the nitroglycerin-induced vasodilator responses. The present results suggest that both GST and ALDH-2 are involved in nitroglycerin action while ALDH-2 plays a major role, and the change of CGRP contents closely correlates with the biotransformation of nitroglycerin.     

Role of propiolaldehyde and other metabolites in the pargyline inhibition of rat liver aldehyde dehydrogenase

The metabolism of pargyline proceeds by way of three separate cytochrome P-450 catalyzed N-dealkylation reactions: N-depropargylation, N-demethylation and N-debenzylation. Propiolaldehyde, a product of N-depropargylation, is a potent inhibitor of aldehyde dehydrogenase (AlDH). The formation of pargyline-derived propiolaldehyde by isolated rat liver microsomes in vitro was confirmed using gas chromatographic/mass spectrometric techniques. The measured rates of propiolaldehyde formation for uninduced and phenobarbital-induced microsomes in vitro were 0.2 +/- 0.03 and 0.9 +/- 0.2 mumole/30 min/g wet weight liver respectively. However, these rates may have been artificially low due to competition between semicarbazide, the trapping agent, and microsomal proteins for the generated propiolaldehyde. CO significantly inhibited the microsome-catalyzed N-depropargylation reaction in vitro, whereas CoCl2 pretreatment of rats partially blocked the pargyline-induced rise in blood acetaldehyde after ethanol. Inhibition of the low Km liver mitochondrial AlDH by propiolaldehyde in vitro exhibited first-order kinetics, which is consistent with irreversible inhibition. Acetaldehyde did not attenuate the inhibition of AlDH by propiolaldehyde in vitro or by pargyline in vivo. Propargyl alcohol, a substance which is metabolized to propiolaldehyde by alcohol dehydrogenase, also inhibited AlDH in vivo and caused a quantitatively similar rise in blood acetaldehyde after ethanol as pargyline. Other putative metabolites of pargyline, namely benzylamine and propargylamine, inhibited AlDH in vivo, albeit to a lesser degree than pargyline, but neither of these amines inhibited AlDH directly. Monoamine oxidase was implicated in the conversion of benzylamine to an active inhibitory species, possibly an imine. From these studies, we conclude that propiolaldehyde was the primary metabolite responsible for the pargyline inhibition of AlDH in vivo; however, certain amine metabolites may have contributed to a lesser degree by conversion to yet unknown inhibitory forms.