Introduction

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,-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 (PBYO¹) showed an antimutagenic effect on UV-induced mutagenesis in Escherichia coli, and this compound was the most effective among various triple- and 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 double-bond 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 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 carbonyl-reduced 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 ethoxyresorufin-O-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 R- and 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 µmol of PBYO, 1 µmol 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 µl 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 µmol of PBYO, 1 µmol 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 µg 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 µl 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 R- and 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 R- and 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 fused-silica 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.

	Results

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Triple-Bond and Carbonyl Reduction of PBYO by Rat Liver Preparations. When PBYO was incubated with rat liver microsomes in the presence of NADPH, two metabolites (M-1 and M-2) were detected in the HPLC chromatogram of the extract of the incubation mixture. These peaks were not observed in the chromatogram of the control, which was incubated without addition of PBYO. The retention times of M-1 and M-2 corresponded to those of PBO and PBYOL, respectively (Fig. 1A). However, PBA, the further reduced product of the double bond of PBO, was not detected in this case. In contrast, when PBYO was incubated with rat liver cytosol, only M-2 was detected in the extract (Fig. 1B). The mass spectra of M-1 gave the molecular ion at m/z 146 and the fragment ions at m/z 131, 103, and 77. The mass spectra of M-2 also gave the molecular ion at m/z 146 and the fragment ions at m/z 131, 101, and 77. The UV spectra of M-1 and M-2 revealed absorption maxima at 285 and 240 nm, respectively. The mass and UV spectra and the HPLC behavior of M-1 and M-2 were identical to those of authentic samples of PBO and PBYOL, respectively.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   HPLC of the metabolites of PBYO by rat liver microsomes (A) or cytosol (B) with NADPH. PBYO was incubated with 0.1 ml of liver microsomes or cytosol in the presence of NADPH for 20 min. The extract was subjected to HPLC. The absorbance (full scale, 0.008 absorbance units) was measured at 254 nm. Other details are described under Experimental Procedures.

Triple-Bond Reductase Activity of Liver Microsomes. The time courses of NADPH-linked reduction of PBYO to PBO by liver microsomes from rats were essentially linear for 30 min. These reductase activities increased linearly with increasing amounts of liver microsomes, up to 3 mg of protein. When PBYO was incubated with liver microsomes in the presence of NADPH for 20 min at varying pH, the pH optimum for the formation of PBO was observed at pH 7.4 (data not shown). In the other experiments of this study, incubations were carried out for 20 min using 1.5 mg of protein of liver microsomes at pH 7.4. The liver microsomes exhibited the triple-bond reductase activity toward PBYO with NADPH under aerobic conditions. However, only weak reductase activity was observed with NADH. In contrast, liver cytosol exhibited only marginal reductase activity toward PBYO even in the presence of NADPH or NADH (Fig. 2A). Liver microsomes did not exhibit appreciable double-bond reductase activity toward PBO. On the contrary, the cytosol exhibited double-bond reductase activity toward PBO in the presence of NADPH (Fig. 2B). When the microsomes were boiled, the triple-bond reductase activity was no longer observed in the presence of NADPH (data not shown). The microsomal triple-bond reductase activity was examined in the presence of NADPH using rat lung, kidney, small intestine, stomach, large intestine, brain, heart, spleen, and the adrenal gland in addition to liver. The significant reductase activity was also observed in adrenal, brain, kidney, lung, stomach, as well as liver (Fig. 3A).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Reduction of PBYO to PBO (A) and PBO to PBA (B) by rat liver microsomes or cytosol. Each value represents the mean ± S.D. of four experiments. PBYO or PBO was incubated with 0.1 ml of liver microsomes (about 1.5 mg of protein) or 0.05 ml of liver cytosol (about 0.5 mg of protein) in the presence of NAD(P)H for 20 min. PBO or PBA formed were determined by HPLC. Other details are described under Experimental Procedures.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Tissue distribution of triple-bond reductase (A) and carbonyl reductase (B) activities toward PBYO. Each value represents the mean ± S.D. of four experiments. Reactions were conducted with 0.1 ml of tissue microsomes in the presence of NADPH for 20 min. PBO or PBYOL formed was determined by HPLC. Other details are described under Experimental Procedures. S.I., small intestine; L.I., large intestine.

Carbonyl Reductase Activity of Liver Microsomes and Cytosol. The carbonyl reductase activities of liver microsomes and cytosol of rats toward PBYO were examined. The time courses of NAD(P)H-linked reduction of PBYO to PBYOL by liver microsomes and cytosol from rats were essentially linear for 30 min. These reductase activities increased linearly with increasing amount of liver microsomes, up to 2.0 mg of protein, and to 1.2 mg of protein in liver cytosol (data not shown). In the following experiments on the carbonyl reduction of PBYO, incubations were carried out for 20 min using 1.5 mg of protein of liver microsomes or 0.5 mg of protein of cytosol. The microsomes exhibited a carbonyl reductase activity in the presence of NADH. The microsomal reductase activity of rat liver was also observed in the presence of NADPH, but the activity was about half of the NADH-linked one. In contrast, liver cytosol exhibited the reductase activity with NADPH and NADH at similar rates (Fig. 4). When the liver microsomes and cytosol were boiled, these reductase activities were abolished (data not shown). When the microsomal carbonyl bond reductase activity of some tissues was examined, the NADPH-linked activity in the adrenal gland was 2-fold higher than that in liver, but other tissues did not exhibit appreciable activity (Fig. 3B).

View larger version (51K): [in this window] [in a new window]   Fig. 4.   Carbonyl reduction of PBYO to PBYOL by rat liver microsomes and cytosol. Each value represents the mean ± S.D. of four experiments. Reactions were conducted with 0.05 ml of liver cytosol or 0.1 ml of liver microsomes in the presence of NAD(P)H for 20 min. Other details are described under Experimental Procedures.

Effect of Some Inhibitors and Inducers on PBYO Triple-Bond and Carbonyl Reductions. The NADPH-linked microsomal triple-bond reductase activity of rat liver was inhibited by the addition of disulfiram, 7-dehydrocholesterol, progesterone, and 18-glycyrrhetinic acid but not -diethylaminoethyldiphenylpropylacetate (SKF 525-A), sodium arsenite, potassium cyanide, or carbon monoxide. Dicumarol, which is a potent inhibitor of ,-ketoalkene double-bond reductase, had no effect on the triple bond reduction (Fig. 5A). The activities were similar under anaerobic conditions. Thus, the triple-bond reduction is independent on air (data not shown). The NADPH-linked carbonyl reductase activity in liver microsomes was not inhibited by 18-glycyrrhetinic acid, metyrapone, cortisone, or progesterone (1 × 10⁴ M), which are inhibitors of 11-hydroxysteroid dehydrogenase or 20-hydroxysteroid dehydrogenase (Rekka et al., 1996; Takada et al., 1998) (Fig. 5B). The NADH-linked reductase activity in liver microsomes was also not affected by these inhibitors. The carbonyl reductase activity of liver cytosol and microsomes toward PBYO in the presence of NADH or NADPH was also not inhibited by quercitrin, phenobarbital, pyrazole, disulfiram, indomethacin, or dicumarol at the concentration of 1 × 10⁴ M. However, the NADH-linked carbonyl reductase activity of liver cytosol toward PBYO was inhibited by propargylglycine at the concentration of 1 × 10⁴ M (data not shown). When rats were pretreated with phenobarbital, 3-methylcholanthrene, dexamethasone, acetone, or clofibrate, the triple-bond- and carbonyl-reducing activities of the liver microsomes were not enhanced (data not shown). These observations suggest that the triple-bond and carbonyl reduction activities are exhibited by different enzymes from cytochrome P450, and carbonyl reduction of PBYO is catalyzed by a novel carbonyl reductase in liver microsomes and cytosol.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Effect of some inhibitors on the triple-bond reduction of PBYO to PBO (A) and the carbonyl reduction of PBYO to PBYOL (B) by rat liver microsomes with NADPH. Each value represents the mean ± S.D. of four experiments. PBYO was incubated with liver microsomes and NADPH in the presence of an inhibitor (1 × 104 M) for 20 min. The activity without inhibitors was 1.5 ± 0.2 nmol/min/mg of protein in (A) and 3.2 ± 0.4 nmol/min/mg of protein in (B). Other details are described under Experimental Procedures.

Substrate Specificity of Triple-Bond Reduction by Rat Liver Microsomes. The ability of rat liver microsomes to reduce triple bonds in various compounds was spectrophotometrically estimated in the presence of NADPH. When deprenyl, ethinamate, ethynylestradiol, 1-phenyl-1-butyne, 1-phenyl-2-propyn-1-ol, 1-phenyl-1-propyne, or PBYOL, of which the triple bond was not conjugated with the carbonyl group, was incubated with liver microsomes in the presence of NADPH, the amount of NADPH was not changed. These facts suggest that a triple bond adjacent to a carbonyl group can be reduced by the microsomal enzyme(s).

Retro-Oxidation of PBO and PBYOL to PBYO by Liver Microsomes. The retro-oxidation reactions of PBO and PBYOL to PBYO by liver microsomes and cytosol of untreated rats were examined. When PBYOL was incubated with liver microsomes or cytosol in the presence of NADP⁺ or NAD⁺ (1 × 10³ or 1 × 10⁴ M), PBYO was not formed from PBYOL. However, when PBYOL was incubated with liver microsomes in the presence of NADPH (1 × 10⁴ M), PBYO was formed from PBYOL at the rate of 8.0 ± 1.2 nmol/20 min/mg of protein. Thus, oxidative metabolism of the hydroxyl group of PBYOL by the cytochrome P450 system may occur. When PBO was incubated with liver microsomes, PBYO was not formed even in the presence of NADP⁺ or NAD⁺ at the concentrations of 1 × 10⁴ and 1 × 10³ M. Thus, the reverse reaction of PBO to PBYO does not occur, similar to the case of double-bond reduction of ,-ketoalkenes to ketoalkynes (Kitamura and Tatsumi, 1990).

Stereoselectivity of Carbonyl Reduction. In the conversion of PBYO to PBYOL by liver cytosol with NADH or NADPH, the major product was S-PBYOL. In contrast, PBYO was reduced by liver microsomes with NADH or NADPH to both S- and R-PBYOL. In the case of NADPH-dependent reduction, R-PBYOL mainly was formed (Fig. 6). These facts suggest that the carbonyl reduction by liver microsomes and cytosol was catalyzed by different types of enzymes and the microsomal NADH- and NADPH-linked reductions also involved different enzymes.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Stereospecific carbonyl reduction of PBYO to PBYOL by rat liver microsomes and cytosol. A, HPLC of the extract of the incubation mixture of liver cytosol in the presence of NADPH; B, reduction of PBYO to R- and S-PBYOL by liver cytosol; C, HPLC of the extract of incubation mixture of liver microsomes in the presence of NADPH; D, reduction of PBYO to R- and S-PBYOL by liver microsomes. Each value in B and D represents the mean of three experiments. Reactions were conducted with 0.05 ml of liver cytosol or 0.1 ml of liver microsomes in the presence of NAD(P)H for 20 min. Reduction products were determined by HPLC with a chiral column. Other details are described under Experimental Procedures.

Effect of PBYO and Its Related Compounds on EROD Activity of Liver Microsomes. The comparative inhibitory effects of PBYO, PBYOL, PBO, PBOL, PBAOL, and PBA on EROD activity catalyzed by cytochrome P450 were determined in liver microsomes of 3-methylcholanthrene-treated rats. PBYO, PBO, and PBOL showed strong inhibitory effects on EROD activity, followed by PBYOL. The inhibitory effects of PBA and PBAOL were weaker than those of the above compounds. The lag time was not observed in the inhibition of EROD activity by these compounds. The IC₅₀ values of PBO, PBOL, PBYO, and PBYOL were about 50, 70, 100, and 200 µM, respectively (Fig. 7).

View larger version (29K): [in this window] [in a new window]   Fig. 7.   Inhibitory effect of PBYO and its metabolites on EROD activity. Each value represents the mean ± S.D. of four experiments. Reactions were conducted with 0.01 ml of liver microsomes (about 0.15 mg of protein) in the presence of NADPH in cuvettes at 37°C. Activities were measured fluorophotometrically. PBYO and its metabolites (PBYOL, PBO, PBOL, PBA, and PBAOL) dissolved in dimethyl sulfoxide (<1% of total incubation mixture) were added after a 2-min preincubation. Other details are described under Experimental Procedures.

	Discussion

Top Abstract Introduction Results Discussion References

This study demonstrated that the triple bond and carbonyl group of PBYO, an ,-ketoalkyne compound, were reduced by rat liver microsomes and cytosol, as shown in Fig. 8. PBYO was reduced to PBO (triple-bond reduction product) by liver microsomal enzyme(s) and to S- and R-PBYOL (carbonyl-reduced products) by liver microsomal enzyme. In the cytosolic reduction, S-PBYOL mainly was formed, accompanied with the R-enantiomer. Previously, we also demonstrated that the carbonyl group of PBO was reduced stereospecifically to R-PBOL by NAD(P)H-linked microsomal carbonyl reductase (Okamoto et al., 1999). The double bond of PBO was not further reduced to afford PBA in liver microsomes. PBO was reported to be reduced by ,-ketoalkene double-bond reductase in liver cytosol (Kitamura and Tatsumi, 1990). Thus, PBYO was reduced to PBA by the combination of liver microsomes and cytosol.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Postulated metabolic pathways of PBYO in rats. PBYO is metabolized to PBO, PBOL, PBA, PBYOL, and PBAOL by rat liver preparations. M, microsomes; C, cytosol.

The microsomal triple-bond reductase is different from ,-ketoalkene double-bond reductase in respect to cellular distribution and susceptibility to inhibitors. The microsomal triple-bond reductase activity was inhibited by disulfiram but not by dicumarol, which is a potent inhibitor of ,-ketoalkene reductase. Thus, the characteristics of the two multibond reductases are different. The microsomal triple-bond reductase activity was not inhibited by SKF 525-A or carbon monoxide. The microsomal triple-bond reductase activity was not stimulated by cytochrome P450 inducers. These facts suggest that the enzyme responsible for the reduction of the triple bond in microsomes is a new type of reductase, not cytochrome P450. The inhibitory effect of disulfiram on the microsomal triple-bond reduction suggests a contribution of cytochrome P450 2E1 to the reduction. We therefore examined the reducing ability of cytochrome P450 2E1 using the rat recombinant cytochrome P450 2E1 isoform (GENTEST, Woburn, MA). However, the triple bond of PBYO was not reduced. Solubilization of the enzyme from the microsomal membrane was attempted using detergents, such as deoxycholate, cholate, trypsin, Tween 20, and Emulgen 913. However, we could not solubilize the enzyme from the microsomal membrane, despite the complete solubilization of PBYO carbonyl reductase(s). It may be that the triple bond reductase is a multiple microsomal enzyme system like 7-dehydrocholesterol ⁷-reductase (Nishino and Ishibashi, 2000). Further purification of the microsomal enzyme responsible for the triple-bond reduction in rat liver is in progress.

Previously, we purified an ,-ketoalkene double-bond reductase from rat liver cytosol. The enzyme exhibited a significant double-bond reductase activity toward ,-ketoalkenes, including 15-ketoprostaglandins (Kitamura and Tatsumi, 1990; Kitamura et al., 1993; 1996). In contrast, no reductase activity was detected toward the double bond in trans-stilbene and diethylstilbestrol, which have no carbonyl group adjacent to the double bond. 1-Phenyl-1-butyne, 1-phenyl-2-propyn-1-ol, 1-phenyl-1-propyne, ethynylestradiol, ethinamate, deprenyl, and PBYOL, in which the triple bond is not adjacent to the carbonyl group, were not reduced by rat liver microsomes even in the presence of NADPH. Thus, an adjacent carbonyl group is also necessary for the reduction of a triple bond. The double bond of ,-ketoalkene is thought to be reduced by the nucleophilic attack of hydride anion on -carbon of the double bond catalyzed by ,-ketoalkene double-bond reductase, and the enol-type intermediate formed is transformed to the saturated carbonyl compound (Westbrook and Jarabak, 1978). The same mechanism can be adapted to the triple-bond reduction of ,-ketoalkynes. This triple-bond reduction mechanism is consistent with the fact that the reverse reaction did not proceed even in the presence of NADP⁺ or NAD⁺. Furthermore, it is also supported by the finding that when PBYO was incubated with liver microsomes in buffer composed by D₂O, PBO, which contained one deuterium in the molecule, was isolated from the mixture (data not shown). The properties of these enzymes may resemble each other in many respects.

Carbonyl reductase acting on PBO in rat liver microsomes and dog blood was reported (Fraser et al., 1967; Okamoto et al., 1999). Cytosolic carbonyl reductase acting on PBYO was also found in this study. The reductase produced the S-enantiomer, as is usual for cytosolic carbonyl reductases (Prelusky et al., 1982; Eyles and Pond, 1992; Testa, 1995). However, the enzyme could use either NADH or NADPH, unlike usual cytosolic carbonyl reductase. Thus, the NAD(P)H-linked carbonyl reductase found in rat liver cytosol in this study is a novel type of carbonyl reductase. In this study, it was also demonstrated that PBYO was reduced to PBYOL by liver microsomes in the presence of NADPH or NADH. The R-enantiomer was formed by the liver enzyme, especially in the presence of NADPH. The microsomal carbonyl reductase found in this study is also different from reported microsomal carbonyl reductases, 11-hydroxysteroid dehydrogenase, and 20-hydroxysteroid dehydrogenase in terms of inhibitors, electron donor requirement, and the stereospecificity of the product (Maser and Bannenberg, 1994; Maser, 1995; Takada et al., 1998).

Some compounds containing a triple bond are known to be potent irreversible inhibitors of the cytochrome P450 system. Recently, the mechanism of inhibition was suggested to be persistent binding with the heme moiety (Beebe et al., 1996; Roberts et al., 1998). We also examined the inhibitory effect of PBYO and its reduced metabolites on EROD activity of rat liver microsomes. PBYO and PBO showed inhibitory effects on the microsomal activity. On the other hand, the inhibitory effect of PBYOL was less than that of the parent PBYO. PBA and PBAOL showed no inhibitory effect on EROD activity at 10⁶ to 10⁴ M. These facts suggest that the multiple bonds of PBYO and PBO are related to the inhibitory effect against the cytochrome P450 system, and the metabolic pathways of triple- and double-bond reductions of PBYO and PBO and carbonyl reduction of PBYO seem to be significant as detoxification steps.