The oxidative metabolism and toxicity of the para isomers of methylphenol (cresol), ethylphenol, and isopropylphenol were studied using male Sprague-Dawley rat liver microsomes and precision-cut liver slices. Reactive intermediates from each compound were trapped using radiolabeled glutathione and were detected and quantified by HPLC. Conjugates were collected and their structures determined by fast atom bombardment mass spectrometry and proton nuclear magnetic resonance. During microsomal incubations each test compound formed monoglutathione conjugates with structures which are consistent with the formation of quinone methide intermediates. In each case the glutathione moiety was attached to the benzylic carbon on the alkyl side chain of the phenol. With ethylphenol, which has a prochiral benzylic carbon, two isomeric conjugates were detected. The rate of formation of the glutathione conjugates in liver slice incubations was 4-isopropylphenol > 4-ethylphenol > 4-methylphenol. This correlated with the toxicity of the three compounds in liver slices. At equimolar concentrations 4-isopropylphenol was the most toxic while 4-methylphenol was the least toxic. Depletion of intracellular glutathione was observed in the presence of each test compound which preceded cell death. Enhancement of cellular thiol levels with N-acetylcysteine protected cells from the toxic effects of all three compounds as did inhibition of cytochrome P450 activity with metyrapone. These results suggest the formation of quinone methide intermediates from three alkylphenols during oxidative metabolism and demonstrate a correlation between the amount of reactive intermediate formed and toxicity observed in liver slices.

The effects of p-alkyl substituents on the relative cytotoxicity of 4-alkyl-2-methoxyphenols were investigated in isolated rat liver slices. The derivatives of 4-alkyl-2-methoxyphenol studied were 4-methyl- (creosol), 4-ethyl-, 4-propyl-, 4-isopropyl-, 4-allyl-2-methoxyphenol (eugenol), as well as 4-allyl-2,6-dimethoxyphenol. The data were correlated with previous microsomal experiments which showed that all of the 4-alkyl-2-methoxyphenols were converted to quinone methides (QMs; 4-methylene-2,5-cyclohexadien-1-ones) via a cytochrome P450-catalyzed process [Bolton, J. L. Comeau, E., and Vukomanovic, V. (1995) Chem.-Biol. Interact., in press]. The present investigation showed little correlation between the rate of QM formation in microsomes and the relative toxicities of the alkylphenols, unless the QMs formed were of similar reactivity. In contrast, a plot of alkylphenol toxicity versus the relative hydrolysis rates of QMs derived from these phenols fit a parabolic equation with a minimum at the data for 4-isopropyl-2-methoxyphenol. These data suggest that in vivo oxidation of phenols to QMs which have lifetimes in the 10 s-10 min range results in cytotoxicity. QMs with reactivities outside this window are less toxic since the electrophile is either too stable for reaction with cellular nucleophiles or too reactive for nucleophilic cellular macromolecules to compete with solvent. These data suggest that a reactivity window exists for QMs which is a primary determinant of the extent of cytotoxic injury caused by these reactive electrophiles.

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Estrogen receptor-beta (ERβ) is a drug target for memory consolidation in postmenopausal women. Herein is reported a series of potent and selective ERβ agonists (SERBAs) with in vivo efficacy that are A-C estrogens, lacking the B and D estrogen rings. The most potent and selective A-C estrogen is selective for activating ER relative to seven other nuclear hormone receptors, with a surprising 750-fold selectivity for the β over α isoform and with ECs of 20-30 nM in cell-based and direct binding assays. Comparison of potency in different assays suggests that the ER isoform selectivity is related to the compound's ability to drive the productive conformational change needed to activate transcription. The compound also shows in vivo efficacy after microinfusion into the dorsal hippocampus and after intraperitoneal injection (0.5 mg/kg) or oral gavage (0.5 mg/kg). This simple yet novel A-C estrogen is selective, brain penetrant, and facilitates memory consolidation.

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