Introduction

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trans-4-Phenyl-3-buten-2-one (PBO¹; trans-methyl styryl ketone, trans-benzylideneacetone, trans-benzalacetone) has been used as an industrial material for synthesis of chemicals and drugs, and as a flavoring additive for cosmetics, soaps, detergents, and cigarettes. It is also used as a food additive in gelatin, candy, puddings, beverages, and baked goods. However, Prival et al. (1982) reported that PBO produced a positive mutagenic response in the Ames Salmonella typhimurium assay with strains TA 100 and TA 1537, with S9 activation. In contrast, an antimutagenic effect of PBO on the UV-induced mutagenesis of Escherichia coli was observed (Motohashi et al., 1997). It has also been reported that PBO induces glutathione S-transferase and quinone reductase in animals and human cell lines (Prestera et al., 1993). The acute toxicity of PBO has been discussed (Opdyke, 1973).

Sauer et al. (1997a,b) detected the carbonyl-reduced metabolite of PBO, trans-4-phenyl-3-buten-2-ol (PBOL) in the blood of rats and mice given PBO i.v., and isolated N-phenylacetylglycine, N-benzylglycine, and mercapturate conjugates from the urine. They suggested that the lack of toxicity of PBO could be ascribed to its extensive metabolism and rapid excretion. However, they did not detect the double bond-reduced metabolite of PBO, 4-phenyl-2-butanone (PBA) in these animals.

In our recent study, an NADPH-linked double bond reductase responsible for the double bond reduction of a variety of ,-ketoalkenes to the corresponding ketoalkanes was purified to homogeneity from rat liver cytosol (Kitamura and Tatsumi, 1990). The double bond of PBO was also reduced by this enzyme. In this paper, we report on the in vivo metabolism of PBO in rats and dogs, focusing on the reductive metabolism of the compound.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results and Discussion References

Materials. PBO, PBA, and 4-phenyl-2-butanol were purchased from Nacalai Tesque Inc. (Kyoto, Japan). Aroclor 1254-induced rat liver S-9 and the cofactors were obtained from Oriental Yeast Co. (Tokyo, Japan). PBOL was prepared by the method of Chaikin and Brown (1949). S. typhimurium TA 100 was obtained from the Institute for Fermentation (Osaka, Japan).

Animals and Drug Administration. Male Wistar (Slc:Wistar/ST) rats, 7 to 8 weeks old (weighing 210-240 g), were purchased from Japan SLC, Inc. (Shizuoka, Japan). Two female beagle dogs, 10 and 11 months old and weighing 10.7 and 12.5 kg (Shimidzu Jikkenzairyo Inc., Kyoto, Japan), were used.

Identification of Reductive Metabolites of PBO in Rats. The reductive metabolites (M-1 and M-2) of PBO were extracted with 20 ml of diethyl ether from 10 ml of the pooled hemolyzed blood of rats that had received PBO. The supernatant was evaporated to about 50 µl at 0°C and mixed with 100 µl of ethanol. The solution was subjected to HPLC and gas chromatography (GC)-Mass analysis.

Determination of PBA, PBOL, and 4-Phenyl-2-butanol in Blood of Rats and Dogs. Reductive metabolites were determined in blood of rats and dogs given PBO or PBA. To determine the amounts of PBO, PBA, PBOL, and 4-phenyl-2-butanol in the blood, 0.6 ml of hemolyzed blood containing 10 µg of methyl p-aminobenzoate (an internal standard) was extracted with 5 ml of diethyl ether. The extract was evaporated to about 50 µl at 0°C and mixed with 100 µl of ethanol, because PBA and 4-phenyl-2-butanol were volatile. An aliquot of the extract was subjected to HPLC. If the extracts were dried by vacuum centrifugation at room temperature, as in the earlier work (Sauer et al., 1997a,b), the amounts of these metabolites were markedly decreased.

HPLC. The system for separation and determination of PBO and its metabolites in rat and dog blood consisted of a Hitachi 655A HPLC system (Tokyo, Japan) equipped with an ODS column (Inertsil ODS-2, 150- × 4.6-mm i.d., GL Science, Tokyo, Japan). The mobile phase was 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 260 nm. The elution times of PBOL, 4-phenyl-2-butanol, PBO, and PBA were 14.5, 15.6, 19.2, and 23.0 min, respectively.

GC-Mass. GC-mass 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, then increased at the rate of 20°C/min to 200°C. The retention times of PBA, PBOL, and PBO were 5.9, 6.5, and 6.8 min, respectively.

Mutation Assay. The assay was carried out as described by Ames et al. (1975) with a slight modification. A test compound was preincubated for 30 min at 37°C with the tester strain in the presence of S-9 mix in 0.1 M phosphate buffer. Soft agar was added, and the mixture was poured into an agar plate. After incubation for 2 days at 37°C, the numbers of revertants were counted. The numbers on control plates and plates with 0.1 µg of AF-2 (positive control) were 164 and 748/plate, respectively.

	Results and Discussion

Top Abstract Introduction Experimental Procedures Results and Discussion References

Reductive Metabolites of PBO in Rats and Dogs. When PBO was administered i.v. to rats and dogs, two metabolites (M-1 and M-2) were detected in the HPLC chromatograms of the extract of the blood of rats and dogs. These peaks were not observed in the chromatograms of control blood. M-1 and M-2 gave retention times corresponding to those of PBA and PBOL, respectively (Fig. 1). M-1 and M-2 were isolated from the blood of rats as described in Experimental Procedures. The mass spectrum of M-1 showed the molecular ion at m/z 148 and fragment ions at m/z 133, 105, 91, and 77 (Fig. 2). The UV spectrum of the metabolite revealed an absorption maximum at 260 nm with a shoulder at 280 nm. The mass spectrum of M-2 gave the molecular ion at m/z 148 and fragment ions at m/z 115, 105, 91, and 77. The UV spectrum of the metabolite revealed an absorption maximum at 250 nm with shoulders at 283 and 296 nm. The mass and UV spectra and the HPLC behaviors of these metabolites were identical with those of authentic samples of PBA and PBOL (data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   HPLC of the metabolites of PBO in blood after i.v. administration of 25 mg/kg of PBO to rats (A) and dogs (B).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Mass spectra of a metabolite (M-1) of PBO in rat blood and authentic PBA.

Blood Concentration of PBA and PBOL in Rats and Dogs. PBA was detected in blood after i.v. administration of PBO to male rats. The area under the curve (AUC_0-120) of PBA was 392 µg · min/ml. PBOL was detected in a small amount, in addition to the unchanged PBO. The AUC_0-120 values of unchanged PBO and PBOL after dosing of PBO were 72.1 and 4.1 µg · min/ml, which correspond to 18.4 and 1.0% of that of PBA, respectively. The C_max values for PBA and PBOL at 5 min were 9.46 and 0.12 µg/ml, respectively. V_d for PBO was 2449 ml/kg. In this case, 4-phenyl-2-butanol, the reduction product of both the carbonyl group and the double bond of PBO, was not detected in the blood (Fig. 3A). However, when PBA was administered i.v. to rats, 4-phenyl-2-butanol was detected in a small amount, in addition to unchanged PBA. The AUC_0-60, C_max, and T_1/2 for the metabolite were 80.9 µg · min/ml, 2.8 µg/ml, and 43.3 min, respectively. The AUC_0-60 and V_d for PBA were 304 µg · min/ml and 1215 ml/kg, respectively (Fig. 3B).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Blood concentrations of the reduced metabolites of PBO or PBA in rats and dogs. A, levels of PBA () and PBOL () in the blood after i.v. administration of PBO () at a dose of 25 mg/kg to rats. B, levels of 4-phenyl-2-butanol () in the blood after i.v. administration of PBA () at a dose of 25 mg/kg to rats. C, PBA () and PBOL () in the blood after i.v. administration of PBO () at a dose of 25 mg/kg to dogs. Each value in rats represents the mean ± S.D. of three experiments. Each value in dogs represents the mean of two experiments.

Mutagenicity of PBO, PBA, and PBOL. The mutagenic activities of PBO, PBA, and PBOL were examined with the Ames test using. S. typhimurium TA 100. PBO was mutagenic when S-9 mix was added. At the amount of 100 µg/plate, 190% revertants were observed compared with the control. PBA and PBOL showed no mutagenicity even in the presence of S-9 mix (data not shown). Thus, the double bond and carbonyl reductions of PBO appear to be detoxification pathways in vivo.

Metabolic Pathway of PBO In Vivo. This study has demonstrated that the double bond of the ,-ketoalkene compound PBO is reduced more efficiently than the carbonyl group in rats and dogs. Previously, we purified ,-ketoalkene double bond reductase from rat liver. 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 toward the double bond was detected with trans-stilbene or styrene, which have no carbonyl group adjacent to the double bond. The double bond of PBO may be reduced by this ,-ketoalkene double bond reductase in the animal body.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Postulated metabolic pathways of PBO in rats and dogs.