In mice depleted of GSH by treatment with buthionine sulfoximine (BSO), methimazole (2-mercapto-1-methylimidazole, MMI) causes liver injury characterized by centrilobular necrosis of hepatocytes and an increase in serum alanine transaminase (SALT) activity. MMI requires metabolic activation by both P450 monooxygenase and flavin-containing monooxygenase (FMO) before it produces the hepatotoxicity. MMI and its analogues were examined for the ability to increase SALT activity in GSH-depleted mice. Saturation of the C-4,5 double bond in MMI resulted in a complete loss of hepatotoxicity. Similarly, ring fusion of a benzene nucleus to the C-4,5 double bond, forming 2-mercapto-1-methylbenzimidazole, abolished the toxic potency. As for MMI, 2-mercapto-1,4,5-trimethylimidazole, and 2-mercapto-1-methyl-4, 5-di-n-propylimidazole, the toxic potency decreased with the increasing bulk of the 4- and 5-alkyl substituents. Furthermore, methylation of the thiol group of MMI totally reduced its toxicity. These structural requirements and the known toxicity of thiono-sulfur compounds led us to the hypothesis that MMI would undergo epoxidation of the C-4,5 double bond by P450 enzymes and, after being hydrolyzed, the resulting epoxide would be then decomposed to form N-methylthiourea, a proximate toxicant. Before N-methylthiourea would produce toxicity, it would be further biotransformed to its S-oxidized metabolites mainly by FMO. Evidence for this hypothesis was provided by the facts that N-methylthiourea and glyoxal as the accompanying fragment were identified as urinary metabolites in mice treated with MMI and that N-methylthiourea caused a marked increase in SALT activity when administered to mice in combination with BSO.
To study the antioxidant activity of quercetin 3-O-beta-D-glucuronide (Q3GA), which is one of the quercetin metabolites in the blood after intake of quercetin-rich food, the inhibitory effect of Q3GA on lipid peroxidation was estimated using phosphatidylcholine large unilamellar vesicles (PC LUV) as a biomembrane model. Iron ion, an aqueous peroxyl radical generator, a peroxynitrite generator, or lipoxygenase was used as the inducer of lipid peroxidation. In all cases, Q3GA inhibited lipid peroxidation significantly, although its inhibitory effect was lower than that of quercetin aglycon. The ultrafiltration of PC LUV containing Q3GA revealed that Q3GA has low but significant affinity with the membranes of phospholipid bilayers. It is therefore likely that Q3GA acts as an efficient antioxidant in membranous lipid peroxidation through its localization in the phospholipid bilayer. This conjugated quercetin metabolite seems to retain the ability to protect cellular and subcellular membranes from peroxidative attack by reactive oxygen species and peroxidative enzymes.
To assess the efficacy of conjugated quercetin metabolites as attenuators for oxidative stress in the central nervous system, we measured the 13-hydroperoxyoctadecadienoic acid (13-HPODE)-dependent formation of reactive oxygen species (ROS) in pheochromocytoma PC-12 cells in the presence of quercetin 3-O-beta-glucuronide (Q3GA) and related compounds. A 2',7'-dichlorofluorescin (DCFH) assay showed that Q3GA significantly suppressed the formation of ROS, when it was coincubated with 13-HPODE (coincubation system). However, it was less effective than quercetin aglycon in the concentration range from 0.5 to 10 microM. In an experiment in which the cells were incubated with the test compounds for 24 h before being exposed to 13-HPODE, Q3GA was also effective in suppressing the formation of ROS in spite that little Q3GA was taken up into the cells. These results suggest that antioxidative metabolites of quercetin are capable of protecting nerve cells from attack of lipid hydroperoxides.
Effect of quercetin and its conjugated metabolite quercetin 3-O-beta-D-glucuronide (Q3GA), on peroxynitrite-induced consumption of lipophilic antioxidants in human plasma low-density lipoprotein (LDL) was measured to estimate the role of dietary flavonoids in the defense system against oxidative modification of LDL based on the reaction of nitric oxide and superoxide anion. Synthesized peroxynitrite-induced consumption of endogenous lycopene beta-carotene and alpha-tocopherol was effectively suppressed by adding quercetin aglycone into LDL solution. Q3GA also inhibited the consumption of these antioxidants effectively. These results indicate that dietary quercetin is capable of inhibiting peroxynitrite-induced oxidative modification of LDL in association with lipophilic antioxidants present within this lipoprotein particle.
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