PubMed · 11458488
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N M Wright. 2001. Identifying alcohol dependency.. https://pubmed.ncbi.nlm.nih.gov/11458488/
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Recently we have shown that daidzin, the major active principle of an ancient herbal treatment for "alcohol addiction", suppresses ethanol intake in alcohol-preferring laboratory animals. Further, we have identified the monoamine oxidase (MAO)-aldehyde dehydrogenase (ALDH-2) pathway of the mitochondria as the potential site of action of daidzin. Daidzin analogues that potently inhibit ALDH-2 but have no or little effect on MAO are most antidipsotropic, whereas those that also inhibit MAO exhibit little, if any, antidipsotropic activity. Therefore, in the design and synthesis of more potent antidipsotropic analogues, structural features important for the inhibition of both ALDH-2 and MAO must be taken into consideration. To gain further information on the structure-activity relationships at the inhibitor binding sites of ALDH-2 and MAO, we prepared 44 analogues of daidzin and determined their potencies for ALDH-2 and MAO inhibition. Results indicate that a sufficient set of criteria for a potent antidipsotropic analogue is an isoflavone with a free 4'-OH function and a straight-chain alkyl substituent at the 7 position that has a terminal polar function such as -OH, -COOH, or -NH(2). The preferable chain lengths for the 7-O-omega-hydroxy, 7-O-omega-carboxy, and 7-O-omega-amino subsitutents are 2 < or = n < or = 6, 5 < or = n < or = 10, and n > or = 4, respectively. Analogues that meet these criteria have increased potency for ALDH-2 inhibition and/or decreased potency for MAO inhibition and therefore are likely to be potent antidipsotropic agents.
Extensive use for disulfiram (DSF) has been found in the aversion therapy treatment of recovering alcoholics. Although it is known to irreversibly inhibit hepatic aldehyde dehydrogenase (ALDH), the specific mechanism of in vivo inhibition of the enzyme by the drug has not been determined yet. We have demonstrated in this report a novel, but simple and rapid method for structurally characterizing in vivo derived protein-drug adducts by linking on-line sample processing to HPLC-electrospray ionization mass spectrometry (HPLC-MS) and HPLC-tandem mass spectrometry (HPLC-MS/MS). Employing this approach, rats were administered DSF, and their liver mitochondria were isolated and solubilized. Both native and in vivo DSF-treated mitochondrial ALDH (mALDH) were purified in one step with an affinity cartridge. The in vivo DSF-treated mALDH showed 77% inhibition in enzyme activity as compared with that of the control. Subsequently, the control and DSF-inhibited mALDH were both subjected to HPLC-MS analyses. We were able to detect two adducts on DSF-inhibited mALDH, as indicated by the mass increases of approximately 71 and approximately 100 Da. To unequivocally determine the site and structure of these adducts, on-line pepsin digestion-HPLC-MS and HPLC-MS/MS were performed. We observed two new peptides at MH(+) = 973.7 and MH(+) = 1001.8 in the pepsin digestion of DSF-inhibited enzyme. These two peptides were subsequently subjected to HPLC-MS/MS for sequence determination. Both peptides possessed the sequence FNQGQC(301)C(302)C(303), derived from the enzyme active site region, and were modified at Cys(302) by N-ethylcarbamoyl (+71 Da) and N-diethylcarbamoyl (+99 Da) adducts. These findings indicated that N-dealkylation may be an important step in DSF metabolism, and that the inhibition of ALDH occurred by carbamoylation caused by one of the DSF metabolites, most likely S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO). Finally, there was no evidence of the presence of an intramolecule disulfide bridge modification on the peptide FNQGQCCC.
Interaction between endogenous opioids and nitric oxide (NO) has been shown in different biological models and pharmacological evidence suggest that opioids can induce NO release in endothelium as well as in neural cells. Cholestasis is associated with NO overproduction. The reason for increased NO synthesis is not clearly known but it can potentiate development of gastric mucosal damage in cholestatic subjects. Based on increased plasma levels of endogenous opioids and existence of NO overproduction in cholestasis, the present experiments were performed to investigate the role of interaction between endogenous opioids and NO in generation of ethanol-induced gastric damage in cholestatic rats. Cholestasis was induced by surgical ligation of bile duct and sham-operated rats served as controls. The animals received either 20 mg/kg of naltrexone or saline for 6 days and then were fasted and received L-arginine (200 mg/kg), NG-nitro-L-arginine methylester (L-NAME; 2, 5 and 10 mg/kg) or saline. The ethanol-induced gastric mucosal damage was significantly more severe in cholestatic rats than in sham-operated animals (115 +/- 12 mm2 vs. 72 +/- 11 mm2, P < 0.05). L-NAME significantly enhanced the development of gastric mucosal lesions in sham-operated rats. But in cholestatic animals, L-NAME decreased and L-arginine enhanced the severity of gastric damage. Pretreatment of animals with naltrexone decreased severity of gastric mucosal damage in cholestatic rats. Concurrent administration of naltrexone with L-arginine was protective against ethanol-induced gastric damage in both normal and cholestatic groups. Administration of naltrexone with L-NAME had the same effect in cholestatic and control rats and increased severity of gastric damage. Plasma levels of NO2- + NO3- were significantly higher in cholestatic rats than control animals (72 +/- 6 microM vs. 39 +/- 3 microM, P < 0.05). Pretreatment of animals with naltrexone significantly reduced plasma levels of NO2- + NO3- in cholestatic animals, but not in control rats (33 +/- 6 microM vs. 32 +/- 4 microM). The protective effect of L-NAME against gastric damage in cholestatic rats can be explained by inhibition of NO overproduction and it seems that interaction between opioids and NO may have an important role in generation of NO overproduction and gastric complications in cholestatic rats.