Mitochondrial biotransformation of omega-(phenoxy)alkanoic acids, 3-(phenoxy)acrylic acids, and omega-(1-methyl-1H-imidazol-2-ylthio)alkanoic acids: a prodrug strategy for targeting cytoprotective antioxidants to mitochondria.

Mitochondrial reactive oxygen species (ROS) generation and the attendant mitochondrial dysfunction are implicated in a range of disease states. The objective of the present studies was to test the hypothesis that the mitochondrial beta-oxidation pathway could be exploited to deliver and biotransform the prodrugs omega-(phenoxy)alkanoic acids, 3-(phenoxy)acrylic acids, and omega-(1-methyl-1H-imidazol-2-ylthio)alkanoic acids to the corresponding phenolic antioxidants or methimazole. 3- and 5-(Phenoxy)alkanoic acids and methyl-substituted analogs were biotransformed to phenols; rates of biotransformation decreased markedly with methyl-group substitution on the phenoxy moiety. 2,6-Dimethylphenol formation from the analogs 3-([2,6-dimethylphenoxy]methylthio)propanoic acid and 3-(2,6-dimethylphenoxy)acrylic acid was greater than that observed with omega-(2,6-dimethylphenoxy)alkanoic acids. 3- and 5-(1-Methyl-1H-imidazol-2-ylthio)alkanoic acids were rapidly biotransformed to the antioxidant methimazole and conferred significant cytoprotection against hypoxia-reoxygenation injury in isolated cardiomyocytes. Both 3-(2,6-dimethylphenoxy)propanoic acid and 3-(2,6-dimethylphenoxy)acrylic acid also afforded cytoprotection against hypoxia-reoxygenation injury in isolated cardiomyocytes. These results demonstrate that mitochondrial beta-oxidation is a potentially useful delivery system for targeting antioxidants to mitochondria.

The role of cell cholesterol and the cytoskeleton in the interaction between IK1 and maxi-K channels.

Recently, we demonstrated a novel interaction between large-conductance (maxi-K or K(Ca)1.1) and intermediate-conductance (IK1 or K(Ca)3.1) Ca(2+)-activated K channels: activation of IK1 channels causes the inhibition of maxi-K activity (Thompson J and Begenisich T. J Gen Physiol 127: 159-169, 2006). Here we show that the interaction between these two channels can be regulated by the membrane cholesterol level in parotid acinar cells. Depletion of cholesterol using methyl-beta-cyclodextrin weakened, while cholesterol enrichment increased, the ability of IK1 activation to inhibit maxi-K channels. Cholesterol's stereoisomer, epicholesterol, was unable to substitute for cholesterol in the interaction between the two K channels, suggesting a specific cholesterol-protein interaction. This suggestion was strengthened by the results of experiments in which cholesterol was replaced by coprostanol and epicoprostanol. These two sterols have nearly identical effects on membrane physical properties and cholesterol-rich microdomain stability, but had very different effects on the IK1/maxi-K interaction. In addition, the IK1/maxi-K interaction was unaltered in cells lacking caveolin, the protein essential for formation and stability of caveolae. Finally, disruption of the actin cytoskeleton restored the IK1-induced maxi-K inhibition that was lost with cell cholesterol depletion, demonstrating the importance of an intact cytoskeleton for the cholesterol-dependent regulation of the IK1/maxi-K interaction.

Conversion of tris(8-quinolinolato-N1, O8) aluminum to 8-hydroxyquinoline and activity in bacterial reverse mutation assays.

Tris(8-quinolinolato-N1, O8) aluminum (AlQ), an aluminum chelate of 8-hydroxyquinoline (8OHQ) is an important charge transfer molecule in semiconducting imaging devices. This study was conducted to evaluate AlQ and 8OHQ for the ability to induce reverse mutations, either in the presence or absence of mammalian microsomal enzymes, and to determine if AlQ decomposes or is metabolized to 8OHQ under assay conditions. The tester strains used in the mutation assay were Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2uvrA (pKM101). The assays were conducted in the presence and absence of S9. AlQ doses were 1-1000 microg per plate while 8OHQ doses were 0.947-947 microg per plate to maintain molar equivalency. Stability studies were carried out for 4h at 37 degrees C under conditions designed to mimic mutation assays. Samples were analyzed by HPLC and LC/MS to tentatively identify potential metabolites of AlQ and 8OHQ. The results of the bacterial mutagenicity assay indicate that in the presence of S9, both AlQ and 8OHQ, caused increases in the mean number of revertants per plate with tester strains TA100 and WP2uvrA (pKM101). No increases were observed with any of the remaining tester strain/activation condition combinations. The stability study showed that AlQ degrades readily to 8OHQ under standard mutagenicity test conditions, and the positive test result with AlQ is due to the bioactivation of 8OHQ. In the presence of S9, 8OHQ is metabolized to one detectable product with molecular weight indicative of a one-oxygen insertion. 8OHQ N-oxide and 2,8-quinolinediol were ruled out as possible metabolites; 8OHQ epoxides and other quinolinediols were neither confirmed nor ruled out. Bacterial mutagenicity tests have not been shown to predict in vivo effects of 8OHQ; these assays are similarly expected to be poorly predictive of in vivo genotoxic and carcinogenic potential of AlQ.