Inhibition Of Hepatic Aldehyde Dehydrogenase Research Articles

Novel prodrugs of cyanamide that inhibit aldehyde dehydrogenase in vivo.

S-Methylisothiourea (4), when administered to rats followed by a subsequent dose of ethanol, gave rise to a 119-fold increase in ethanol-derived blood acetaldehyde (AcH) levels-a consequence of the inhibition of hepatic aldehyde dehydrogenase (A1DH)-when compared to control animals not receiving 4. The corresponding O-methylisourea was totally inactive under the same conditions, suggesting that differential metabolism may be a factor in this dramatic difference between the pharmacological effects of O-methylisourea and 4 in vivo. The S-n-butyl- and S-isobutylisothioureas (8 and 9, respectively) at doses equimolar to that of 4 were nearly twice as effective in raising ethanol-derived blood AcH, while S-allylisothiourea (10) was slightly less active. However, blood ethanol levels of all experimental groups were indistinguishable from controls. Pretreatment of the animals with 1-benzylimidazole, a known inhibitor of the hepatic mixed function oxidases, followed sequentially by either 8, 9, or 10 plus ethanol, reduced blood AcH levels by 66-88%, suggesting that the latter compounds were being oxidatively metabolized to a common product that led to the inhibition of AcH metabolism. In support of this, when 8 was incubated in vitro with rat liver microsomes coupled to catalase and yeast A1DH, the requirement for microsomal activation for the inhibition of A1DH activity was clearly indicated. We suggest that S-oxidation is involved and that the S-oxides produced in vivo inhibited A1DH directly, or spontaneously released cyanamide, an inhibitor of A1DH. Indeed, incubation of 8 with rat liver microsomes and NADPH gave rise to cyanamide as metabolite, identified as its dansylated derivative. Cyanamide formation was minimal in the absence of coenzyme. Extending the side chain was detrimental, since S-benzylisothiourea (11) and S-n-hexadecylisothiourea (12) were toxic, the latter producing extensive necrosis of the liver and kidneys when administered to rats.

S‐Methyl‐N,N‐Diethylthiocarbamate Sulfoxide and S‐Methyl‐N,N‐Diethylthiocarbamate Sulfone, Two Candidates for the Active Metabolite of Disulfiram

The mechanism of action of disulfiram involves inhibition of hepatic aldehyde dehydrogenase (ALDH). Although disulfiram inhibits ALDH in vitro, it is believed that the drug is too short-lived in vivo to inhibit the enzyme directly. The ultimate inhibitor is thought to be a metabolite of disulfiram. In this study, we examined the effects of S-methyl-N,N-diethylthiocarbamate (MeDTC) sulfoxide and S-methyl-N,N-diethylthiocarbamate sulfone (confirmed and proposed metabolites of disulfiram, respectively) on rat liver mitochondrial low K(m) ALDH. MeDTC sulfoxide and MeDTC sulfone, in 10-min incubations with detergent-solubilized mitochondria, inhibited ALDH activity with an IC50 (mean +/- SD) of 0.93 +/- 0.04 and 0.53 +/- 0.11 microM, respectively, compared with 7.4 +/- 1.0 microM for the parent drug disulfiram. Inhibition by MeDTC sulfone and MeDTC sulfoxide, both at 0.6 microM, was time-dependent, following apparent pseudo-first-order kinetics with a t1/2 of inactivation of 3.5 and 8.8 min, respectively. Dilution of ALDH inhibited by either sulfoxide or sulfone did not restore activity, an indication of irreversible inhibition. Addition of glutathione (50 to 1000 microM) to ALDH before the inhibitors did not alter the inhibition by MeDTC sulfoxide. In contrast, the inhibition by MeDTC sulfone was decreased > 10-fold (IC50 = 6.3 microM) by 50 microM of glutathione and almost completely abolished by 500 microM of glutathione. The cofactor NAD, in a concentration-dependent manner, protected ALDH from inhibition by MeDTC sulfoxide and MeDTC sulfone. In incubations with intact mitochondria, the potency of the two compounds was reversed (IC50 of 9.2 +/- 3.6 and 0.95 +/- 0.30 microM for the MeDTC sulfone and sulfoxide, respectively). Our results suggest that MeDTC sulfone is highly reactive with normal cellular constituents (e.g., glutathione), which may protect ALDH from inhibition, unless this inhibitor is formed very near the target enzyme. In contrast, MeDTC sulfoxide is a better candidate for the ultimate active metabolite of disulfiram, because it is more likely to be sufficiently stable to diffuse from a distant site of formation, such as the endoplasmic reticulum, penetrate the mitochondria, and react with ALDH located in the mitochondrial matrix.

Metabolic activation of n-butyraldoxime by rat liver microsomal cytochrome P450 : A requirement for the inhibition of aldehyde dehydrogenase

n-Butyraldoxime ( n-BO) is known to cause a disulfiram/ethanol-like reaction in humans, a manifestation of the inhibition of hepatic aldehyde dehydrogenase (AlDH). As with a number of other in vivo inhibitors of AlDH, n-BO does not inhibit purified AlDH in vitro, suggesting that a metabolite of n-BO is the actual inhibitor of this enzyme. In re-examination of the effect of n-BO on blood acetaldehyde levels following ethanol in the Sprague-Dawley rat, we found that pretreatment with substrates and/or inhibitors of cytochrome P450 blocked the n-BO-induced rise in blood acetaldehyde in the following order of decreasing potency: 1-benzylimidazole (0.1 mmol/kg) > 3-amino-1,2,4-triazole (1.0 g/kg) > ethanol (3.0 g/kg) > phenobarbital (0.1% in the drinking water, 7 days) > SKF-525A (40 mg/kg). Rat liver microsomes were shown to catalyze the conversion of n-BO to an active metabolite that inhibited yeast AlDH. This reaction was dependent on NADPH and molecular oxygen and was inhibited by CO and 1-benzylimidazole. Hydroxylamine, postulated by others to be a metabolite of n-BO, inhibited AlDH via a catalase-mediated reaction and not through an NADPH-supported microsome-catalyzed reaction. Using GLC-mass spectrometry, 1-nitrobutane (an N-oxidation product) and butyronitrile (a dehydration product) were identified as metabolites from microsomal incubations of n-BO. However, neither of these metabolic products inhibited AlDH directly or in the presence of liver microsomes and NADPH. We conclude that another NADPH-dependent, cytochrome P450-catalyzed metabolic product of n-BO is responsible for the inhibition of AlDH by n-BO.

Inhibition of hepatic aldehyde dehydrogenase by carbon tetrachloride: An in vitro study

1. 1. In vitro inhibition of rat liver mitochondrial and microsomal aldehyde dehydrogenase (ALDH) under conditions of active CCl 4 metabolism was investigated. 2. 2. Incubation of microsomes or mitochondria in the presence of NADPH alone caused significant, time-dependent inhibition of mitochondrial and microsomal ALDH. EDTA partially protected ALDH from inhibition. 3. 3. Incubation of microsomes or microsomes plus mitochondria in the presence of NADPH and CCl 4 resulted in marked inhibition of microsomal and mitochondrial ALDH activity. The inhibition was both dose- and time-dependent and was relatively less in the presence of EDTA. 4. 4. It is proposed that the inhibition of membrane-bound ALDH may be one of the early events responsible for the genesis of CCl 4-hepatotoxicity.

Structure vs. activity in the sulfonylurea-mediated disulfiram-ethanol reaction

The oral hypoglycemic agents, chlorpropamide (CP) and tolbutamide (TB) are known to elicit a clinical disulfiram-ethanol reaction (DER) when consumed with alcohol. In rats, this DER is manifested in vivo by the elevation of blood acetaldehyde (AcH) levels, a consequence of the inhibition of hepatic aldehyde dehydrogenase (AIDH). Administration of CP or TB to rats (1.0 mmol/kg, IP), followed by ethanol one hour before sacrifice, raised blood AcH levels 12- and 2-times that of control animals, respectively for CP and TB when measured at 3 hours, and 20-fold and 8-fold at 16 hours post drug administration. CP and TB had no effect on AIDH activity when incubated with either intact or osmotically disrupted rat liver mitochondria, indicating that a metabolite of CP or TB is responsible for the inhibition of AIDH in vivo. Hydrolysis products of CP, the 2′-hydroxylated products of CP, tolpropamide and tolethamide, or the 3′-hydroxylated analogs of CP and tolpropamide, were uniformly inactive in elevating ethanol-derived blood AcH. Pretreatment of rats with 3-amino-1,2,4-triazole or SKF-525A had no effect on the elevation of blood AcH mediated by CP or TB, while phenobarbital pretreatment decreased blood AcH by 69%. Although our results clearly indicated that side chain hydroxylation and subsequent oxidation do not play a role in AIDH inhibition by CP or TB, the nature of the side chain attached to the sulfonylurea moiety appears to influence this inhibitory activity in vivo. Thus, the order of activity in the homologous series was, chlorpropamide > chlorbutamide > chlorethamide ⪢ chlormethamide, chloriso propamide = 0.

Inhibition of hepatic aldehyde dehydrogenases in the rat by calcium carbimide (calcium cyanamide).

Oral administration of 7.0 mg/kg calcium carbimide (calcium cyanamide, CC) to the rat produced differential inhibition of hepatic aldehyde dehydrogenase (ALDH) isozymes, as indicated by the time-course profiles of enzyme activity. The low-Km mitochondrial ALDH was most susceptible to inhibition following CC administration, with complete inhibition occurring at 0.5 h and return to control activity at 96 h. The low-Km cytosolic and high-Km mitochondrial, cytosolic, and microsomal ALDH isozymes were inhibited to a lesser degree and (or) for a shorter duration compared with the mitochondrial low-Km enzyme. The time course of carbimide, the hydrolytic product of CC, was determined in plasma following oral administration of 7.0 mg/kg CC to the rat. The maximum plasma carbimide concentration (102 ng/mL) occurred at 1 h and the apparent elimination half-life in plasma was 1.5 h. Carbimide was not measurable in the liver during the 6.5 h time interval when carbimide was present in the plasma. There were negative, linear correlations between plasma carbimide concentration and hepatic low-Km mitochondrial, low-Km cytosolic, and high-Km microsomal ALDH activities. In vitro studies demonstrated that carbimide, at concentrations obtained in plasma following oral CC administration, produced only 19% inhibition of low-Km mitochondrial ALDH and no inhibition of low-Km cytosolic and high-Km microsomal ALDH isozymes. These data demonstrate that carbimide, itself, is not primarily responsible for hepatic ALDH inhibition in vivo following oral CC administration. It would appear that carbimide must undergo metabolic conversion in vivo to inhibit hepatic ALDH enzymes, which is supported by the observation of no measurable carbimide in the liver when ALDH was maximally inhibited following oral CC administration.