Reductive Metabolism of an , -ketoalkyne, 4-phenyl-3-butyn-2-one, by Rat Liver Preparations

The reduction of the triple bond and carbonyl group of an , ketoalkyne, 4-phenyl-3-butyn-2-one (PBYO), by rat liver microsomes and cytosol was investigated. The triple-bond-reduced product trans-4-phenyl-3-buten-2-one (PBO) and the carbonyl-reduced product 4-phenyl-3-butyn-2-ol (PBYOL) were formed when PBYO was incubated with rat liver microsomes in the presence of NADPH. The triple bond of 1-phenyl-1-butyne, deprenyl, ethynylestradiol, ethinamate, and PBYOL, in which the triple bond is not adjacent to a carbonyl group, were not reduced by liver microsomes even in the presence of NADPH. PBO was further reduced to 4-phenyl-2-butanone (PBA) by liver cytosol with NADPH. PBYOL was also formed from PBYO by liver cytosol in the presence of NADPH or NADH. The microsomal triple-bond reductase activity was inhibited by disulfiram, 7-dehydrocholesterol, and 18 -glycyrrhetinic acid but not -diethylaminoethyldiphenylpropylacetate or carbon monoxide. The triple-bond reductase activity in liver microsomes was not enhanced by several inducers of the rat cytochrome P450 system. These results suggested that the triple-bond reduction is caused by a new type of reductase, not cytochrome P450. The microsomal and cytosolic carbonyl reductase activities were not inhibited by quercitrin, indomethacin, or phenobarbital. Only S-PBYOL was formed from PBYO by liver cytosol. In contrast, liver microsomes produced R-PBYOL together with the S-enantiomer to some extent. Ethoxyresorufin-O-dealkylase activity in rat liver microsomes was markedly inhibited by PBYO and PBO, partly by PBYOL, but not by PBA. , -Ketoalkynes, in which the triple bond is adjacent to the carbonyl group, have various pharmacological and toxicological effects and are classified as , -unsaturated carbonyl compounds. The , unsaturated carbonyl moiety is found in many naturally occurring compounds, such as plant allelochemicals, insect hormones, and pheromones (Wadleigh and Yu, 1987). , -Unsaturated carbonyl compounds have been used as industrial materials for the synthesis of various chemicals, including plastics, resins, pesticides, dyes, and pharmaceuticals. They are also used as flavoring additives for cosmetics, soaps, and cigarettes and as food additives in gelatins, candies, and beverages (Opdyke, 1973). These compounds are also present in automobile exhausts and tobacco smoke. They have toxic effects, such as genotoxicity and mutagenicity, and pharmacological effects, such as gastric anti-ulcer activity and an anti-carcinogenic effect (Eder et al., 1993; Czerny et al., 1998; Maria et al., 2000; Pan et al., 2000). In the Ames Salmonella typhimurium assay, some , -unsaturated carbonyl compounds produced a positive mutagenic response in strain TA 100 and TA 1537 with S9 activation (Prival et al., 1982). It has also been reported that these compounds induce drug-metabolizing enzymes, such as glucose 6-phosphatase, glutathione S-transferase, and quinone reductase in humans and animals (Jørgensen et al., 1992; Prestera et al., 1993). However, inhibitory effects of , -unsaturated carbonyl compounds on glutathione S-transferase was also reported (Chien et al., 1994). Compounds containing a triple bond have often been used as drugs, such as ethynylestradiol, ethinamate, pargyline, deprenyl, and desogestrel. During the development of new drugs, a triple bond may be introduced to increase the hydrophobicity. In contrast, some nondrug-like small molecules containing a triple bond, such as 1-ethynylpyrene and 1-ethynylnaphthalene, show an inhibitory effect on the cytochrome P450 system (Beebe et al., 1996; Foroozesh et al., 1997; Roberts et al., 1998). In contrast, an inducing effect of a triple-bond-containing compound, an antiglucocorticoid mifepristone, on the cytochrome P450 3A subfamily was also reported (Williams et al., 1997). , -Ketoalkynes also have an antimutagenic effect. For example, 4-phenyl-3-butyn-2-one (PBYO) showed an antimutagenic effect on UV-induced mutagenesis in Escherichia coli, and this compound was the most effective among various tripleand double-bond compounds examined (Motohashi et al., 1997). We previously reported the purification from rat liver cytosol of an NADPH-linked double-bond reductase responsible for the doublebond reduction of a variety of , -ketoalkenes to the corresponding ketoalkanes (Kitamura and Tatsumi, 1990). The enzyme exhibited reductase activity toward the double bond adjacent to the carbonyl group of , -ketoalkenes, including 15-ketoprostaglandins (Kitamura This work was supported by a grant-in-aid for Scientific Research (C13672343) from the Japan Society for the Promotion of Science. Address correspondence to: Dr. Shigeyuki Kitamura, Institute of Pharmaceutical Sciences, Hiroshima University, School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan. E-mail: [email protected] 1 Abbreviations used are: PBYO, 4-phenyl-3-butyn-2-one; PBO, trans-4-phenyl-3-buten-2-one; PBA, 4-phenyl-2-butanone; PBOL, trans-4-phenyl-3-buten-2ol; EROD, ethoxyresorufin-O-dealkylase; PBAOL, 4-phenyl-2-butanol; PBYOL, 4-phenyl-3-butyn-2-ol; HPLC, high-performance liquid chromatography; GC, gas chromatography; SKF 525-A, -diethylaminoethyldiphenylpropylacetate. 0090-9556/02/3004-414–420$7.00 DRUG METABOLISM AND DISPOSITION Vol. 30, No. 4 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 590/969725 DMD 30:414–420, 2002 Printed in U.S.A. 414 at A PE T Jornals on N ovem er 6, 2017 dm d.aspurnals.org D ow nladed from et al., 1993; 1996). trans-4-Phenyl-3-buten-2-one (PBO) was reduced to 4-phenyl-2-butanone (PBA) by the enzyme. In contrast, we found that the double-bond-reduced compound was a major metabolite in the blood of rats and dogs dosed with PBO (Kitamura et al., 1999). The level of PBA in blood was much higher than that of the carbonylreduced metabolite trans-4-phenyl-3-buten-2-ol (PBOL). Furthermore, we demonstrated that the double bond of , -ketoalkenes was reduced to afford the corresponding ketoalkanes by reductases in fish and bacteria (Tatsumi et al., 1992; Ishida et al., 1996). Fraser et al. (1967) also reported that the double bond of PBO was reduced by dog blood. The double-bond reduction of , -ketoalkenes seems to be common. However, enzymatic reduction of the triple bond of , ketoalkynes has not been reported yet. Carbonyl reduction of , -ketoalkene has been reported. Recently, we demonstrated that the carbonyl group of PBO was reduced by rat liver microsomes in the presence of NADPH, but not by liver cytosol. A novel type of microsomal carbonyl reductase was responsible for the reduction of PBO (Okamoto et al., 1999). Sauer et al. (1997a,b) reported that PBO was rapidly reduced to carbonyl-reduced metabolites in vivo in rats and mice. They suggested that the lack of toxicity of PBO in vivo might be ascribed to its extensive metabolism and rapid excretion. However, the reduction of the carbonyl group of , -ketoalkynes has not been studied. As noted above, PBO has often been used as a model , -ketoalkene to study metabolism. In the present study, triple-bond reduction and carbonyl reduction by rat liver microsomes and cytosol are examined using PBYO as a model , -ketoalkyne. Furthermore, the inhibitory effect of PBYO and its metabolites on the ethoxyresorufinO-dealkylase (EROD) activity in rat liver microsomes was examined. Experimental Procedures Materials. PBYO, 1-phenyl-1-butyne, 1-phenyl-2-propyn-1-ol, 18 -glycyrrhetinic acid, and 1-phenyl-1-propyne were obtained from Aldrich Chemical Co. (Milwaukee, WI). Propargylglycine (2-amino-4-pentynoic acid) was obtained from Sigma Chemical Co. (St. Louis, MO). Ethynylestradiol, deprenyl, ethinamate, PBO, PBA, and 4-phenyl-2-butanol (PBAOL) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). 4-Phenyl-3-butyn-2-ol (PBYOL) and PBOL were prepared by the method of Chaikin and Brown (1949) and were resolved to Rand S-enantiomers by lipase phosphatidylserine, as previously described (Takeshita et al., 1993). Animals. Male Wistar (Slc:Wistar/ST) rats (weighing 210–240 g) were purchased from Japan SLC, Inc. (Shizuoka, Japan). In some experiments, rats were administered phenobarbital, 3-methylcholanthrene, dexamethasone, or clofibrate intraperitoneally once daily for 3 days at 80, 25, 100, and 200 mg/kg, respectively, or acetone orally once at 3 g/kg at 24 h before sacrifice. Tissue Preparations. Tissues of interest were excised and homogenized in four volumes of 1.15% KCl. The homogenate of liver was centrifuged for 20 min at 9,000g and for 60 min at 105,000g successively to prepare the microsomal and cytosolic fractions. The microsomal fraction was washed by resuspension in the KCl solution and resedimentation. The microsomal and cytosolic fractions of other tissues were similarly obtained from the homogenates. Protein contents were determined by the method of Lowry et al. (1951) with bovine serum albumin as a standard protein. Identification of Reductive Metabolites of PBYO by Rat Liver Preparations. Two reductive metabolites of PBYO were isolated from an incubation mixture, which consisted of 0.2 mol of PBYO, 1 mol of NADPH, and 0.1 ml of microsomes in a total volume of 1 ml of 0.1 M Tris-HCl buffer, pH 7.4. After incubation for 20 min, the mixture was extracted with 5 ml of diethyl ether. The supernatant was evaporated to about 50 l at 0°C, and 0.1 ml of methanol was added. The solution was injected into an HPLC instrument equipped with a photodiode array UV detector (Beckman Instruments, Inc., Fullerton, CA) and a GC-mass spectrometer. Assay of Triple-Bond and Carbonyl Reductase Activities in Rat Liver Preparations. The incubation mixture consisted of 0.2 mol of PBYO, 1 mol of NADPH or NADH, and a liver preparation (microsomes, 1.5–1.6 mg of protein; cytosol, 0.5 mg of protein) in a final volume of 1 ml of 0.1 M Tris-HCl buffer, pH 7.4. The incubation was performed for 20 min at 37°C in air. Another sample was incubated under nitrogen or carbon monoxide in a Thunberg tube (Shibata Scientific Technology Ltd., Tokyo, Japan). The side arm contained the PBYO, and the body contained all other components. The tube was gassed for 3 min with nitrogen or carbon monoxide, evacuated with an aspirator, and again gassed with nitrogen or carbon monoxide. The reaction was started by mixing the components of the side arm and the body. In the experiment of the substrate specificity of the triple-bond reduction, PBYO was incubated with liver microsomes and NADPH in nitrogen using a Thunberg-type cuvette, and the absorbance at 340 nm was monitored. In some experiments for the determination of the triple-bond reduction product of PBYO, 0.1 M Tris HCl buffer prepared with D2O was used. The reaction mixture, following the addition of 10 g of methyl p-aminobenzoate as an internal standard, was extracted once with 5 ml of ether, the ether extract was then evaporated to about 50 l at 0°C, and finally 0.1 ml of methanol was added. An aliquot of the extract was injected into the HPLC system. Assay for Microsomal Drug-Metabolizing Activity in Rat Liver. EROD activity in rat liver microsomes, a highly specific reaction of the cytochrome P450 1A subfamily, was assayed by a fluorophotometric method (Burke et al., 1985). HPLC for Determination of Reductase Activity. HPLC was performed in a Hitachi 655A HPLC system (Tokyo, Japan) equipped with an ODS column (Inertsil ODS-3, 150 4.6-mm i.d.; GL Science, Tokyo, Japan). The mobile phase consisted of acetonitrile/water (40:60, v/v), and the flow rate was 0.5 ml/min. The chromatogram was monitored with a UV detector set at 254 nm. The elution times of PBYOL, PBO, PBA, and PBYO were 15.2, 19.3, 23.2, and 31.3 min, respectively. The amounts of reduction products were determined from the peak areas. HPLC for Determination of Rand S-PBYOL. To determine the enantiomers of R,S-PBYOL formed from PBYO by liver microsomes or cytosol, an aliquot of R,S-PBYOL extracted from the incubation mixture was subjected to HPLC on a Hitachi L-6000 chromatograph fitted with a chiral separation column (Chiralcel OD, 250 4.6-mm i.d.; Daicel Chemical Industries Ltd., Tokyo, Japan). The mobile phase consisted of n-hexane/2-propanol (96:4, v/v) and was delivered at a flow rate of 0.5 ml/min. The chromatograph was operated at a wavelength of 254 nm. The elution times of Rand S-PBYOL were 19.7 and 23.8 min, respectively. The amounts of reduction products were determined from the peak areas. GC-Mass Spectrometry. The GC-mass spectrometry was performed using a Shimadzu GC-17A/QP-5000 (Kyoto, Japan) equipped with a DB-5 fusedsilica capillary column (30-m 0.25-mm i.d.; J&W Scientific, Inc., Folsom, CA). The column temperature was held at 50°C for 1 min and then increased at the rate of 20°C/min to 200°C. The retention times of PBYOL and PBO were 6.2 and 6.5 min, respectively.