Evaluation of the potential in vivo genotoxicity of quercetin.

Quercetin, a naturally occurring flavonol commonly detected in apples, cranberries, blueberries, and onions, has been reported to possess antioxidant, anti-carcinogenic, anti-inflammatory, and cardioprotective properties. While positive results have been consistently reported in numerous in vitro mutagenicity and genotoxicity assays of quercetin, tested in vivo, quercetin has generally produced negative results in such studies. Furthermore, no evidence of carcinogenicity related to the oral administration of quercetin was observed in chronic rodent assays. In order to further define the in vivo genotoxic potential of quercetin, a bone marrow micronucleus assay and an unscheduled DNA synthesis (UDS) assay were conducted in Wistar rats. Administered orally to male rats at dose levels of up to 2000 mg/kg body weight, quercetin did not increase the number of micronucleated polychromatic erythrocytes (MN-PCE) 24 or 48 h following dosing in the micronucleus assay. Likewise, orally administered quercetin (up to 2000 mg/kg body weight) did not induce UDS in hepatocytes of male or female rats. While measurable levels of metabolized quercetin were observed in rat plasma samples for up to 48 h after dosing, peaking at 1h following treatment administration, the unmetabolized aglycone was not identified in either plasma or bone marrow. With the exception of only a few rats, the aglycone was also not detected in liver tissue. These results demonstrate that quercetin is not genotoxic under the conditions of these assays and further support the negative results of previously conducted in vivo assays.

Studies comparing in vivo:in vitro metabolism of three pharmaceutical compounds in rat, dog, monkey, and human using cryopreserved hepatocytes, microsomes, and collagen gel immobilized hepatocyte cultures.

The in vivo metabolism of three pharmaceutical compounds, EMD68843, EMD96785, and EMD128130, was compared in fresh and cryopreserved hepatocyte (CPH) suspensions and microsomes from rat, dog, monkey, and human livers and fresh human and rat hepatocyte collagen gel immobilized cultures (GICs). Half of the major in vivo metabolites was produced by phase 1 (hydroxylation, oxidation, hydrolysis, N-dealkylation) and half by phase 2 metabolism (mostly glucuronidation but also sulfation and glycine conjugation). The identity and percentage of phase 1 and 2 metabolites from each compound produced in hepatocytes compared well with that in each species in vivo. Glucuronidation was more extensive in GICs than in CPHs. In contrast, CPHs but not GICs, produced sulfate metabolites. Microsomes (supplemented with NADPH only) produced most of the phase 1 but no phase 2 metabolites. Metabolism in CPHs was the same as in fresh hepatocyte suspensions. Discrete species differences in metabolism were detected by CPHs and microsomes. Cytochrome P450 and glucuronosyl S-transferase contents of CPHs did not account for species differences in the percentage of phase 1 and 2 metabolites or the rate of disappearance of the parent compounds in these cells. These data show a good correlation between major metabolites formed in vivo and in vitro. CPHs and GICs, unlike microsomes, carried out sequential phase 1 and 2 metabolism. Each in vitro system has its own advantages, however, for short-term metabolism studies CPHs may be more useful since they are readily available, easier and quicker to prepare than GICs, and have more comprehensive enzyme systems than microsomes.

Role of blood-brain barrier P-glycoprotein in limiting brain accumulation and sedative side-effects of asimadoline, a peripherally acting analgaesic drug.

Studies with knockout mice lacking mdr1a P-glycoprotein (P-gp) have previously shown that blood-brain barrier P-gp is important in preventing the accumulation of several drugs in the brain. Asimadoline (EMD 61753) is a peripherally selective kappa-opioid receptor agonist which is under development as a therapeutic analgaesic. From the structural characteristics of this drug and its peripheral selectivity, we hypothesized that it is transported by P-gp. Using a pig-kidney polarized epithelial cell line transfected with mdr cDNAs, we demonstrate that asimadoline is transported by the mouse mdr1a P-gp and the human MDR1 P-gp. Furthermore, we show that in mdr1a/1b double knockout mice, the absence of P-gp leads to a 9 fold increased accumulation of asimadoline in the brain. In line with this accumulation difference, mdr1a/1b (-/-) mice are at least 8 fold more sensitive to the sedative effect of asimadoline than wild-type mice. Interestingly, the oral uptake of asimadoline was not substantially altered in mdr1a/1b (-/-) mice. Our results demonstrate that for some drugs, P-gp in the blood-brain barrier can have a therapeutically beneficial effect by limiting brain penetration, whereas at the same time intestinal P-gp is not a significant impediment to oral uptake of the drug.

Formation of 4,4-dialkoxycyclohexa-2,5-dienone N-(thiol-S-yl)imine during reaction of 4-alkoxynitrosobenzenes with thiols in alcoholic solvents.

During the interaction of nitrosoarenes with glutathione in aqueous media, intermediate generation of a highly resonance-stabilized sulfenamide cation has been repeatedly suggested. Most intermediates and end products could be explained by reactions of this sulfenamide cation with different nucleophiles such as excess thiol, solvent water, and metabolically produced arylamine. The present paper presents evidence for adduct formation of the sulfenamide cation with solvent alcohol at neutral pH. Sulfenamide cations generated from 4-nitrosophenetole and 4-nitrosoanisole, respectively, are strongly suggested to form the metastable ketals 4-ethoxy-4-methoxycyclohexa-2,5-dienone N-(glutathion-S-yl)imine and 4,4-dimethoxycyclohexa-2,5-dienone N-(glutathion-S-yl)imine, respectively, during reaction with solvent methanol. Reaction of the two sulfenamide cations in ethanol yielded 4,4-diethoxycyclohexa-2, 5-dienone N-(glutathion-S-yl)imine and 4-ethoxy-4-methoxycyclohexa-2, 5-dienone N-(glutathion-S-yl)imine, respectively. Although the metastability of the ketals did not allow isolation of pure solid material, chromatographic and chemical behavior as well as tandem MS fragmentation substantiate a ketal structure of these intermediates. To confirm the proposed structure, new compounds, 2, 6-dimethyl-4-nitrosophenetole, 2,6-dimethyl-4-nitrophenetole, 2, 6-dimethyl-4-phenetidine, and N-(glutathion-S-yl)-N-hydroxy-4-aminoacetophenone, were synthesized and included in supportive experiments. In summary, the detection of ketals corroborates once more the occurrence of a sulfenamide cation which obviously not only reacts with soft nucleophiles such as GSH but, to a limited extent, also reacts with hard nucleophiles. The toxicological significance of this result is discussed.

Phellodonic acid, a new biologically active hirsutane derivative from Phellodon melaleucus (Thelephoraceae, Basidiomycetes).

A new hirsutane derivative, phellodonic acid (1), has been isolated from fermentations of Phellodon melaleucus strain 87113. Its structure was elucidated by spectroscopic methods. The compound exhibits antibiotic activities towards bacteria and fungi. 1 is the first bioactive metabolite from cultures of a species belonging to the family Thelephoraceae.