Subcellular distribution and properties of a clofibrate-induced aldehyde dehydrogenase from rat liver

The influence of hypolipidemic drug clofibrate on the activity of aldehyde dehydrogenase with different substrates was studied in subcellular fractions of rat liver homogenate. It was shown that under the action of clofibrate the content of the enzyme was increased 2-3-fold in purified peroxisomal fraction as well as in microsomes and mitochondria. No difference was found in the cytoplasmic fraction. Partial purification of clofibrate-induced aldehyde dehydrogenase from microsomes was undertaken. The enzyme is apparently membrane-bound. It has a molecular weight of 187,000 and a subunit size of 47,000, indicating that the molecule is a tetramer. An induced aldehyde dehydrogenase is active with several aliphatic and aromatic aldehydes but not with formaldehyde and glyceraldehyde. The enzyme has Km-values in the millimolar range for acetaldehyde, propionaldehyde, benzaldehyde and phenylacetaldehyde and in the micromolar range for nonanal. Both NAD and NADP serve as coenzymes for the purified aldehyde dehydrogenase. According to substrate specificity, kinetic and molecular properties clofibrate-induced aldehyde dehydrogenase appears to be identical to normal liver microsomal enzyme.     

Metabolism of malondialdehyde by rat liver aldehyde dehydrogenase

Mammalian liver contains a group of pyridine nucleotide linked aldehyde dehydrogenases [E.C. 1.2.1.3] which are present in high specific activity and possess wide substrate specificities. Malondialdehyde (MDA), a difunctional three-carbon aldehyde thought to be toxic, is generated during membrane lipid peroxidation in hepatocytes. The role of aldehyde dehydrogenase (ALDH) in the metabolism of MDA was tested in vitro with subcellular fractions and semipurified cytosolic preparations from rat livers. The cytosolic fraction accounted for virtually all of the MDA (50 microM) metabolizing activity observed in the postnuclear supernatant fraction. The rate of MDA disappearance was relatively low in the mitochondrial fraction and was not detectable in reaction mixtures which contained microsomes. Rat liver cytosol contained two ALDHs with MDA metabolizing activity. These enzymes were separated by DEAE-cellulose ion exchange chromatography and had apparent Km values of 16 microM and 128 microM for malondialdehyde. Mitochondria contained an ALDH enzyme with lower affinity (Km of 7.3 mM with NAD+) for malondialdehyde. These data show that rat liver contains at least three ALDH enzymes which oxidize malondialdehyde.     

Subcellular distribution and properties of rabbit liver aldehyde dehydrogenases

In rabbit liver, both NAD⁺- and NADP⁺-dependent aldehyde dehydrogenases were identified. The activities were distributed among at least three major groups of isozymes identifiable by gel electrophoresis. These isozymes also differed in their substrate and coenzyme preferences, subcellular distributions, and/or responses to effectors. The NAD⁺-dependent aldehyde dehydrogenase activity was distributed among the mitochondrial, microsomal, and cytosolic fractions. The NADP⁺-dependent aldehyde dehydrogenase activity was largely microsomal, with little true cytosolic NADP⁺-dependent activity demonstrable. Aliphatic aldehydes were oxidized equally well by aldehyde dehydrogenases in all three fractions. Aromatic aldehydes, however, were preferentially oxidized by microsomal aldehyde dehydrogenases. Disulfiram significantly inhibited mitochondrial (45 per cent) and cytosolic (93 per cent) NAD⁺-dependent aldehyde dehydrogenase, but it did not cause significant inhibition of microsomal NAD⁺-dependent activity. Disulfiram inhibited the NADP⁺-dependent aldehyde dehydrogenase activity (>71 per cent) in all subcellular fractions. Diethylstilbestrol activated both NAD⁺- and NADP⁺-dependent aldehyde dehydrogenases in mitochondria and cytosol. Microsomal aldehyde dehydrogenases were not affected by diethylstilbestrol.     

Effect of almond and anis oils on mouse liver alcohol dehydrogenase, aldehyde dehydrogenase and heart lactate dehydrogenase isoenzymes

The effects of short-term intraperitoneal injection of diluted almond or anis oil on heart lactate dehydrogenase isoenzymes, liver alcohol dehydrogenase and subcellular aldehyde dehydrogenase were studied in the female mouse. Hepatic alcohol dehydrogenase was induced from control by administration of almond oil 3.2 g/kg/d for 7 days, or anis oil 1.6 g/kg/d for 7 days. Treatment with almond but not anis oil inhibited both cytoplasmic and mitochondrial liver aldehyde dehydrogenase. The mitochondrial isoenzyme with an apparently low Km was also inhibited by the almond oil trial. No significant changes occurred in heart lactate dehydrogenase isoenzymes by the treatments used. The enzymatic inhibition kinetics were found to be non-competitive. The apparent Km for almond-treated mouse aldehyde dehydrogenase was greater than the controls. This indicates lower substrate affinity for almond oil than for acetaldehyde. The results suggest adverse hepatic metabolic interaction between almond oil and alcohol.     

Effect of gossypol on kinetics of mouse liver alcohol and aldehyde dehydrogenase

1. Gossypol, an antifertility ingredient of the cotton plant, altered specific activity of mouse liver alcohol dehydrogenase (L-ADH) and subcellular aldehyde dehydrogenase (L-ALDH) in mice of both sexes. 2. Intraperitoneal injection of a single gossypol dose, 50 mg/kg, inhibited both male and female L-ADH and cytoplasmic L-ALDH from saline controls 21 hr after drug treatment. 3. Gossypol inhibited female but not male mouse mitochondrial L-ALDH isoenzymes. 4. Gossypol-produced enzyme inhibition was determined as noncompetitive. 5. The results suggest gender-dependent sensitivity of mitochondrial L-ALDH to the gossypol inhibition. A toxic metabolic interaction between ethanol and gossypol has been indicated which suggests the contraindication of alcoholic beverages during gossypol use.     

Enzymatic requirement for cyanamide inactivation of rat liver aldehyde dehydrogenase

The in vitro inactivation of aldehyde dehydrogenase (ALDH) by cyanamide in rat liver slices, in intact mitochondria, and at various stages of purity was characterized. Low-Km ALDH was more susceptible to cyanamide inactivation than was the high-Km form. In addition, the presence of NAD or NADH was necessary for cyanamide inhibition of the ALDH activity. Cyanamide at low concentrations required enzymatic conversion to a reactive derivative that could inhibit ALDH. The data in this study are consistent with the suggestion of DeMaster et al. [Biochem. biophys. Res. Commun., 122, 358 (1984)] that catalase is the cyanamide-converting enzyme. An inhibitor of catalase activity, malonate, decreased the rate of cyanamide inactivation of ALDH in intact mitochondria. Furthermore, affinity chromatography-purified ALDH, free of catalase activity, was not susceptible to cyanamide inactivation. This affinity-purified ALDH was only inactivated by high concentrations of cyanamide. Thus, an alternative pathway for ALDH inactivation may exist in which enzymatic modification of cyanamide is not necessary. It is more likely, however, that a contaminating enzyme in the ALDH preparation is capable of activating cyanamide.     

Presence of cytosolic aldehyde dehydrogenase isozymes in adult and fetal rat liver

A colony of Wistar-strain rats bred at Purdue University was composed of animals with two different isozyme patterns of liver cytosolic aldehyde dehydrogenase (EC 1.2.1.3, ALDH) as determined by isoelectric focusing. One cytosolic isozyme pattern had a major activity band with a pI = 5.8 and a minor activity band at pI = 6.2. The other pattern contained three major isozymes with pI values of 5.3, 5.4 and 5.6 along with the pI 6.2 isozyme and a trace of the 5.8 one. The 5.8 and 6.2 isozymes were recognized by antibodies produced against horse and beef liver cytosolic ALDH, whereas the set of three (5.3-5.6) were not. The cytosolic isozymes were inhibited by low levels of disulfiram and had Km values for acetaldehyde in the 100 microM range, properties typical for cytosolic ALDHs. All animals contained the same isozymes of liver mitochondrial ALDH. These were a major activity with a pI = 5.2 and minor activities associated with isozymes of pI = 6.4 and 6.6. These isozymes were recognized by antibodies produced against pure horse and beef liver mitochondrial ALDHs. Both cytosolic and mitochondrial ALDHs were found in fetal liver as early as day 15 of gestation. The total activity for mitochondrial ALDH increased between day 15 and day 21 whereas that for cytosolic ALDHs remained relatively constant during development. It appeared that both cytosolic and mitochondrial ALDH were present by at least the third trimester and could afford the fetus some protection against the toxic action of endogenous or exogenous aldehydes.     

Metabolism of isovanillin by aldehyde oxidase, xanthine oxidase, aldehyde dehydrogenase and liver slices

Aromatic aldehydes are good substrates of aldehyde dehydrogenase activity but are relatively poor substrates of aldehyde oxidase and xanthine oxidase. However, the oxidation of xenobiotic-derived aromatic aldehydes by the latter enzymes has not been studied to any great extent. The present investigation compares the relative contribution of aldehyde dehydrogenase, aldehyde oxidase and xanthine oxidase activities in the oxidation of isovanillin in separate preparations and also in freshly prepared and cryopreserved liver slices. The oxidation of isovanillin was also examined in the presence of specific inhibitors of each oxidizing enzyme. Minimal transformation of isovanillin to isovanillic acid was observed in partially purified aldehyde oxidase, which is thought to be due to residual xanthine oxidase activity. Isovanillin was rapidly metabolized to isovanillic acid by high amounts of purified xanthine oxidase, but only low amounts are present in guinea pig liver fraction. Thus the contribution of xanthine oxidase to isovanillin oxidation in guinea pig is very low. In contrast, isovanillin was rapidly catalyzed to isovanillic acid by guinea pig liver aldehyde dehydrogenase activity. The inhibitor studies revealed that isovanillin was predominantly metabolized by aldehyde dehydrogenase activity. The oxidation of xenobiotic-derived aromatic aldehydes with freshly prepared or cryopreserved liver slices has not been previously reported. In freshly prepared liver slices, isovanillin was rapidly converted to isovanillic acid, whereas the conversion was very slow in cryopreserved liver slices due to low aldehyde dehydrogenase activity. The formation of isovanillic acid was not altered by allopurinol, but considerably inhibited by disulfiram. It is therefore concluded that isovanillin is predominantly metabolized by aldehyde dehydrogenase activity, with minimal contribution from either aldehyde oxidase or xanthine oxidase.     

Role of disulfiram in the in vitro inhibition of rat liver mitochondrial aldehyde dehydrogenase

The alcohol aversion therapy drug disulfiram has been shown to inhibit hepatic aldehyde dehydrogenase (ALDH), one of the key enzymes involved in ethanol metabolism. It is believed by some that disulfiram could be one of the active inhibitors in vivo. However, the actual interaction between disulfiram and ALDH remains ambiguous. We report here that when disulfiram inhibited recombinant rat liver mitochondrial ALDH (rlmALDH) in vitro, no significant molecular mass increase was detected during the first 30 min as determined by on-line HPLC-electrospray ionization mass spectrometry (LC-MS). This indicated that the inhibition in vitro was not caused directly by covalent adduct formation on the enzyme. We subsequently subjected both control and disulfiram-inhibited rlmALDH to Glu-C proteolytic digestion. LC-MS analysis of the Glu-C digestion of disulfiram-inhibited enzyme revealed that one peptide of M(r) = 4821, which contained the putative active site of the enzyme, exhibited a mass decrease of 2 amu as compared with the same peptide found in the Glu-C digestion of the control (M(r) = 4823). We believe that the loss of 2 amu indicated that inhibition of rlmALDH in vitro was due to formation of an intramolecular disulfide bond between two of the three adjacent cysteines in the active site, possibly via a very rapid and unstable mixed disulfide interchange reaction. Further confirmation of the intramolecular disulfide bond formation came from the fact that by adding dithiothreitol (DTT) we were able to recover partial enzyme activity. In addition, the peptide of M(r) = 4821 observed in the Glu-C digestion of the disulfiram-treated ALDH reverted to M(r) = 4823 after treatment with DTT, which indicated that the disulfide bond was reduced. We, thereby, conclude that disulfiram inhibited rlmALDH by forming an intramolecular disulfide, possibly via a fast intermolecular disulfiram interchange reaction.     

Differences in kinetic characteristics and in sensitivity to inhibitors between human and rat liver alcohol dehydrogenase and aldehyde dehydrogenase

1. On the basis of kinetic properties and sensitivity to pyrazole inhibition, it is shown that liver alcohol dehydrogenase present in human mainly corresponded to class I and in rat to class ADH-3 which differed in a number of parameters. 2. Two different aldehyde dehydrogenase (ALDH) isoenzymes were detected in both human and rat liver. The human isoenzymes corresponded to the ALDH-I and ALDH-II type. 3. In the rat, one isoenzyme had low Km and showed similar activity than in human liver but differed in their sensitivity to both disulfiran and nitrofazole inhibition whereas the other presented high Km and showed greater activity than the human one. 4. Caution must be therefore paid when extrapolating to human subjects the data on ethanol metabolism obtained with rats.     

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.     

Metabolism of alpha-naphthoflavone by rat, mouse, rabbit, and hamster liver microsomes

The metabolism of alpha-naphthoflavone (ANF) was studied in hepatic microsomes from rats, mice, rabbits, and hamsters, species in which ANF exerts its biological activities. The major metabolites produced by all species were ANF-5,6-oxide, ANF-6-phenol, and ANF-7,8-dihydrodiol. Minor metabolites produced by all species were ANF-5,6-dihydrodiol, ANF-7-phenol, and ANF-9-phenol. In general, the total rates of metabolism were similar within all species: 22-32 nmol ANF metabolized/15 min/mg protein. Mouse liver microsomes were approximately 1.7 to 2.9 times as active as the other species on a nanomole of cytochrome P-450 basis. The major sites of enzymatic oxidation were the 5,6 and 7,8 bonds of ANF where for all species, 49-71% and 15-46% of the total metabolism occurred, respectively.     

Effects of long-term ethanol treatment on aldehyde and alcohol dehydrogenase activities in rat liver.

Administration of intoxicating doses of ethanol by gavage for 3 weeks caused weight loss and reduced hepatic aldehyde dehydrogenase activity in the soluble, mitochondrial and microsomal fractions. Rats receiving equivalent amounts of ethanol as a constituent of a liquid diet for 5 weeks gained weight and showed no changes in aldehyde dehydrogenase activity. Alcohol dehydrogenase activity was decreased in the rats treated by gavage and unchanged in those given ethanol in the diet, but in spite of this the rate of ethanol elimination was accelerated in both groups. In the livers of two strains of rats genetically selected for their difference in voluntary alcohol consumption, the mitochondrial and microsomal aldehyde dehydrogenase activities had previously been shown to be significantly higher in the alcohol-consuming (AA) than in the alcohol-avoiding (ANA) rats. Similar differences were now found after long-term intragastric ethanol administration, although in both strains the absolute levels of aldehyde dehydrogenase were reduced. Profound reduction of mitochondrial low-K_m aldehyde dehydrogenase activity and high blood acetaldehyde were observed, especially in the ANA rats. This suggests a possible connection between the low activity of this enzyme and the increased acetaldehyde level.     

Inactivation mechanism of low-KM rat liver mitochondrial aldehyde dehydrogenase by cyanamide in vitro.

The inactivation of low-KM rat liver mitochondrial aldehyde dehydrogenase (ALDH) by the alcohol-sensitizing agent cyanamide (H2NCN) has been studied in vitro. The effect of the concentrations of NAD+ at different concentrations of catalase on the inactivation of ALDH by cyanamide (20 and 200 microM) in vitro point to an ALDH-NAD(+)-catalase complex prior to the binding to cyanamide to form the holoenzyme-inhibitor complex. Cyanamide itself could be responsible for the inactivation of ALDH. The possibility that both irreversibly inactivated ALDH and cyanamide remain free at the end of the inactivation process is discussed. The effects of pH and ionic strength on the inactivation process are also described. The pseudo-first order rate constants for inactivation of low-KM ALDH depends on both effects, suggesting that electrostatic forces are involved in the process and that a group with pK approximately 6.8, presumably a histidine residue, at the active site of ALDH could be involved. A representative equation for the inactivation process of low-KM ALDH by cyanamide in vitro has been fitted to experimental kinetic data, involving both catalase and inhibitor concentrations.     

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.     

Inhibition of aldehyde dehydrogenase in brain and liver by cyanamide

Cyanamide (H₂N—C  N) is effective as an agent to treat alcoholism presumably because it inhibits aldehyde dehydrogenase. In this study it was found that, in vivo, cyanamide is a very potent inhibitor of liver aldehyde dehydrogenases. but less effective against the brain enzymes. The ED₅₀ for liver was found to be 8 mg/kg when given intraperitoneally. The inhibition diminishes with time but is measurable for at least 24 hr, even though the bulk of the ¹⁴C-labeled cyanamide is excreted within 6 hr. Cyanamide is not effective when added to the assay mixture in vitro, suggesting that a metabolite is the inhibitor in vivo. A urinary metabolite has been isolated and partially characterized. It is an acid with a pKa' of 3.9 and an extinction coefficient of 1.72 × 10³ in base at 219 nm; the compound apparently retains the cyano group. However, it does not inhibit the enzyme in vitro.     

Genetic control of responsiveness of rat liver supernatant aldehyde dehydrogenase to phenobarbital and 3-methylcholanthrene

The responsiveness of the hepatic supernatant NAD⁺-dependent aldehyde dehydrogenase with a high Km value (high Km-AldDH) to phenobarbital (PB) and 3-methylcholanthrene (3-MC) treatment was studied in male rats of three strains; Wistar, Long-Evans, and Sprague-Dawley.A remarkable strain difference in the response of the enzyme to PB or 3-MC was observed. In rats of the Wistar strain the enzyme activity remained unchanged (non-responsive) in all rats after treatment with PB while it increased (responsive) 5- to 19-fold in all rats after treatment with 3-MC. The enzyme activity increased 8- to 20-fold and 2- to 8-fold respectively after treatment with PB and 3-MC in all rats of the Long-Evans strain. In rats of the Sprague-Dawley strain the enzyme activity remained unchanged in half of all the rats treated with PB or 3-MC and increased 2- to 7-fold over the basal level in half of the treated rats. The non-responsive rats to PB were all responsive to 3-MC treatment while the responsive rats to PB were responsive in 65% and non-responsive in 35% to 3-MC treatment.     

The effects of cephem antibiotics and related compounds on the aldehyde dehydrogenase in rat liver mitochondria

The effects of cephem antibiotics and their related compounds on aldehyde dehydrogenase obtained from rat liver mitochondria were studied. A pH of 8.8 and reaction temperature 24 degrees were the conditions for measurement of enzyme activity. The apparent Michaelis constant Km values for NAD, acetaldehyde and propionaldehyde were 3.8 X 10(-5) M, 4.0 X 10(-5) M and 2.5 X 10(-5) M, respectively. Cefamandole, cefoperazone and cefmetazole, having a 1-methyl-5-thiotetrazol group at position 3 of the cephem ring, caused a relatively potent inhibition of aldehyde dehydrogenase. Cefmetazole and cefoperazone also showed a significant inhibition on highly purified yeast aldehyde dehydrogenase; the extent of inhibition on yeast enzyme was almost the same as that on rat mitochondrial aldehyde dehydrogenase. The decrease in enzyme activity effected by 1-methyl-1H-tetrazol-5-thiol (MTT) was greater than those of 1H-tetrazol (TZ), 1H-tetrazol-5-thiol and 1-(2-dimethylaminoethyl)-1H-tetrazol-5-thiol, but was, of course, less than that of disulfiram. Cefamandole, cefmetazole and MTT showed competitive inhibition with NAD, while TZ was uncompetitive inhibitor with respect to both NAD and acetaldehyde. Enzyme inhibition caused by disulfiram, cefmetazole and MTT increased time-dependently and the addition of 2-mercaptoethanol into the medium effectively and completely restored enzyme inhibition. These results suggest that thiol group at position 5 of 1H-tetrazol ring is responsible for the type of inhibition with NAD, and methyl group at position 1 of 1H-tetrazol ring is important to exhibit a potent inhibition on aldehyde dehydrogenase.