Introduction

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p,p'-DDT [1,1,1-trichloro-2,2-bis(2-chlorophenyl-4-chlorophenyl)ethane] is a broad-spectrum insecticide, which was used from the 1940s in large quantities, but was banned in many countries in the 1970s because of its persistence in the environment. It was replaced in part by less persistent alternatives. This pesticide, however, is still used mainly in developing countries. p,p'-DDT and related compounds, many of which are carcinogenic and mutagenic, are also known to be environmental estrogens. p,p'-DDT is a phenobarbital-type inducer, which induces the cytochrome P450 (CYP¹) 2B and 3A subfamily (Lewis and Lake, 1997; Nims et al., 1998). p,p'-DDT and its metabolites accumulate in animal tissues and induce various enzymes (Esaac and Matsumura, 1980). The higher levels of p,p'-DDE [1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene] than p,p'-DDT in tissues reflect its stability (Morgan and Roan, 1971). These compounds mimic hormones and bind to the estrogen receptor and androgen receptor (Robinson et al., 1985; Kelce et al., 1995; Chen et al., 1997). p,p'-DDT and related compounds, p,p'-DDD [1,1-dichloro-2,2-bis(4-chlorophenyl)ethane], o,p'-DDT [1,1,1-trichloro-2,2-bis(2-chlorophenyl-4-chlorophenyl)ethane], and dicofol [1,1-bis(4-chlorophenyl)-2,2,2-trichloroethanol], are known to be xenobiotic estrogens, but p,p'-DDE is an antiandrogen (You, 2000). To assess the possible risks associated with human exposure to the pesticide, it is essential to thoroughly elucidate its metabolism in mammalian species, birds, and marine and freshwater species.

p,p'-DDT is converted by reductive dechlorination to p,p'-DDD in insects, birds, and animals (Hassall, 1972; Esaac and Matsumura, 1980). Several reports have indicated that p,p'-DDT is mainly metabolized to p,p'-DDE by dehydrochlorination in mammalian species, insects, and microorganisms, and p,p'-DDD is an intermediate of the reaction, which may proceed via -hydroxyl-DDD (Benitez et al., 1995; Fox et al., 1998). p,p'-DDT and p,p'-DDE are further oxidized to 2,2-bis(4-chlorophenyl)acetic acid (p,p'-DDA), the major excreted metabolite in animals (Wallcave et al., 1974; Gold and Brunk, 1982). The dechlorination is of considerable significance because this reaction is the first and also is the rate-limiting step in the metabolism of p,p'-DDT in mammals and microorganisms. A role of CYP in the microsomal reduction of p,p'-DDT was suggested (Esaac and Matsumura, 1980; Baker and Van Dyke, 1984; Kelner et al., 1986). However, the mechanism of microsomal reductive dechlorination of p,p'-DDT is not known in detail. Furthermore, the metabolism of o,p'-DDT, which contaminates technical-grade DDT about 20%, has not been established. In the present study, the in vitro metabolism of p,p'-DDT and o,p'-DDT by rat liver microsomes was examined, focusing on reductive dechlorination to DDD isomers.

	Experimental Procedures

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Materials. p,p'-DDT, p,p'-DDE, and p,p'-DDD were purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). o,p'-DDT, o,p'-DDD, p,p'-DDMU [1-chloro-2,2-bis(4-chlorophenyl)ethylene], p,p'-DDA, dicofol, and o,p'-DDE were obtained from Labor Dr. Ehrenstorfer-Schäfers (Augsburg, Germany). FMN and hematin (Fe³⁺) were from Sigma Chemical Co. (St. Louis, MO). Reduced FMN was prepared photochemically from FMN by the previously described method (Kitamura et al., 1999a). p,p'-DDT and o,p'-DDT were used after recrystallization from n-hexane. Microsomal preparations from recombinant rat CYP 2B1 and 3A1 expressed in insect cells and from nine recombinant human CYP isoforms (i.e., CYP 1A1, 1A2, 2A6, 2B6, 2C9, 2D6, 2E1, 3A4, and 4A11) expressed in a human B lymphoblastoid cell line were purchased from GENTEST (Woburn, MA).

Animals. Male rats (Slc:SD; 180-210 g; Slc:Japan; Shizuoka, Japan) were used. In some experiments, phenobarbital was administered to rats intraperitoneally at a dose of 80 mg/kg for 3 days, 3-methylcholanthrene at 25 mg/kg for 3 days, dexamethasone at 100 mg/kg for 4 days, clofibrate at 250 mg/kg for 3 days, and acetone at 3 g/kg orally for 1 day.

Preparation of Liver Microsomes. Rat livers were homogenized in 4 volumes of 1.15% KCl. The homogenate was centrifuged at 9000g for 20 min, and the supernatant fraction was separated into microsomes and cytosol by centrifugation at 105,000g for 60 min. The microsomes were washed by resuspension in 2 volumes of the KCl solution and by resedimentation at 105,000g for 60 min. The pellets of microsomes were resuspended in the solution to make 1 ml equivalent to 1 g of liver. Protein contents in the liver microsomal preparation of untreated, phenobarbital-treated, dexamethasone-treated, clofibrate-treated, acetone-treated, and 3-methylcholanthrene-treated rats were 12.6 to 14.8, 18.5 to 20.9, 14.5 to 16.7, 13.4 to 15.8, 12.5 to 14.4, and 13.4 to 17.2 mg of protein/ml, respectively, as determined by the method of Lowry et al. (1951).

Identification of Reductive Metabolites of p,p'-DDT Formed by Rat Liver Microsomes. Two reductive metabolites of p,p'-DDT or p,p'-DDD were determined from a large-scale incubation mixture. The large-scale incubation mixture consisted of 1 µmol of the substrate, 5 µmol of NADPH, and 1 ml of liver microsomes of untreated rats in a total volume of 5 ml of 0.1 M Tris-HCl buffer, pH 7.4. After incubation for 20 min, the mixture was extracted with 20 ml of n-hexane. The supernatant was evaporated to dryness, and 0.1 ml of methanol was added. The solution was injected into a high-performance liquid chromatograph and a gas chromatograph-mass spectrometer. Gas chromatograph-mass spectrometer was performed using a Shimadzu GC-17A/QP-5000 (Kyoto, Japan) in the electron impact mode. A DB-5 fused-silica capillary column (30-m × 0.25-mm i.d.; J & W Scientific, Inc., Folsom, CA) was used. The column temperature was held at 100°C for 3 min, then increased at a rate of 15°C/min to 240°C. One microliter of sample was injected into the injection port, which was held at 250°C. Splitless injection was used. The retention times of p,p'-DDD, p,p'-DDE, and p,p'-DDMU were 13.4, 13.0, and 12.4 min, respectively. The mass spectra of p,p'-DDD, p,p'-DDE, and p,p'-DDMU showed m/z 320 (M⁺), 235, and 165, m/z 318 (M⁺), 246, and 176, and m/z 284 (M⁺), 212, and 176, respectively.

Assay of Reductase Activity. The incubation mixture consisted of 0.1 µmol of p,p'-DDT or o,p'-DDT (50 µl of methanol solution), 1 µmol of NADPH or NADH, and 0.2 ml of liver microsomes in a final volume of 1 ml of 0.1 M K,Na-phosphate buffer, pH 7.4. Incubation was performed using a Thunberg tube under anaerobic conditions. The side arm contained NADPH or NADH, and the body contained all the other components. The tube was gassed for 3 min with nitrogen, evacuated with an aspirator for 5 min, and again gassed with nitrogen. The reaction was started by mixing the components of the side arm and the body together and was continued for 30 min at 37°C. The mixture, after addition of 1 µg of phenothiazine as an internal standard, was extracted once with 5 ml of n-hexane, and the extract was evaporated to dryness in vacuo. The residue was dissolved in 0.1 ml of methanol and then subjected to high-performance liquid chromatography (HPLC).

HPLC. HPLC was performed in a Hitachi L-6000 high-performance liquid chromatograph (Tokyo, Japan) equipped with an ultraviolet absorption detector. The instrument was fitted with a 4 × 125-mm LiChrospher RP-Select B (5 µm) (Merck, Darmstadt, Germany). The mobile phase was acetonitrile/H₂O (6:4). The chromatograph was operated at a flow rate of 1.0 ml/min at ambient temperature and at a wavelength of 254 nm. Elution times of p,p'-DDA, dicofol, p,p'-DDD, o,p'-DDD, p,p'-DDT, p,p'-DDMU, o,p'-DDT, p,p'-DDE, and o,p'-DDE were 4.5, 15.4, 16.5, 17.8, 27.7, 29.0, 35.3, and 36.7 min, respectively. The amounts of metabolites formed were determined from the peak areas.

Thin-Layer Chromatography (TLC). Silica gel plates (Kieselgel 60 GF254, 0.2 mm thick; Merck, Darmstadt, Germany) were developed in n-hexane/acetone (9:1, v/v). Spots were visualized under UV light (254 nm). The R_f values of authentic p,p'-DDA, dicofol, p,p'-DDD, p,p'-DDT, p,p'-DDMU, and p,p'-DDE were 0.12, 0.33, 0.36, 0.48, 0.52, and 0.63, respectively.

	Results

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Metabolism of p,p'-DDT by Rat Liver Microsomes. p,p'-DDT was incubated with liver microsomes of untreated rats in the presence of NADPH for the detection of metabolites, as described under Experimental Procedures. Two peaks were detected in an HPLC chromatogram of the extract of the incubation mixture (Fig. 1A). These peaks were not detected in the control, which was incubated without the substrate. The metabolites detected at 16.5 and 35.3 min correspond to p,p'-DDD and p,p'-DDE, respectively. In the case of boiled microsomes, these metabolites were not detected. The metabolites corresponding to p,p'-DDT and p,p'-DDE were isolated as described under Experimental Procedures. They were identified unequivocally as p,p'-DDT and p,p'-DDE by mass spectrometry, TLC, and HPLC comparison with authentic samples. The further metabolized products p,p'-DDMU and p,p'-DDA were not detected by HPLC or TLC as metabolites of p,p'-DDT with liver microsomes (data not shown). When p,p'-DDT was incubated with liver cytosol in the presence of NADPH, p,p'-DDD and p,p'-DDE were not detected in the chromatogram of the extract (Fig. 1B). In contrast, dicofol, an oxidized product of p,p'-DDT, and phenolic metabolites were also not detected in the HPLC chromatogram, even when p,p'-DDT was incubated with liver microsomes in the presence of NADPH under aerobic conditions (data not shown).

View larger version (30K): [in this window] [in a new window]   Fig. 1.   HPLC of the metabolites of p,p'-DDT generated by rat liver. A, extract of the incubation mixture of p,p'-DDT with rat liver microsomes. B, extract of the incubation mixture of p,p'-DDT with liver cytosol. Incubation was performed at 37°C for 30 min with liver microsomes or cytosol of untreated rats in the presence of NADPH under anaerobic conditions. After incubation, the mixture was extracted with n-hexane, and the extract was subjected to HPLC, as described under Experimental Procedures. The absorbance (full scale, 0.016 atomic units) was measured at 254 nm.

Dechlorinating Activity of Rat Liver Microsomes Toward p,p'-DDT. The time course of the dechlorination of p,p'-DDT to p,p'-DDD by liver microsomes from rats in the presence of NADPH was essentially linear for 45 min. The formation of p,p'-DDE from p,p'-DDT was also observed, but the amounts were about one-hundredth of those of p,p'-DDD (Fig. 2A). When the reductase activity was assayed with various amounts of microsomes under anaerobic conditions, the reductase activity increased linearly with increasing amount of liver microsomes up to 4.0 mg of protein. In contrast, very low activities were observed under aerobic conditions (Fig. 2B). In the other experiments of this study, incubations were carried out for 30 min using 0.2 ml of liver microsomes (about 3 mg of protein, equivalent to 200 mg of wet liver weight) under anaerobic conditions.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Dechlorination and dehydrochlorination of p,p'-DDT to p,p'-DDD and p,p'-DDE with rat liver microsomes. A, time course of the activity. B, liver microsome dependence. In A, a mixture containing 0.1 µmol of p,p'-DDT and 0.2 ml of liver microsomes from rats in 0.1 M phosphate buffer, pH 7.4 was incubated at 37o for 10, 20, 30, 45, and 60 min in the presence of NADPH under anaerobic conditions. The p,p'-DDD and p,p'-DDE formed were determined using HPLC. Other details are described under Experimental Procedures. In B, the same procedure as in A was used except that the amount of liver microsomes from rats (0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 mg of protein) was varied and the incubation time was 30 min. p,p'-DDD formed under aerobic or anaerobic conditions was determined by HPLC.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Dechlorination of p,p'-DDT by rat liver microsomes. Each bar represents the mean ± S.D. of four rats. The incubation was performed at 37°C for 30 min with 200 µl of liver microsomes (about 3 mg of protein) of untreated rats in the presence or absence of an inhibitor (1 × 104 M) under anaerobic conditions. The p,p'-DDD formed was determined by HPLC. Other details are described under Experimental Procedures. , incubation was performed in an atmosphere of carbon monoxide; ND, not detected.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Dechlorination of p,p'-DDT by liver microsomes of rats pretreated with inducers of the cytochrome P450 system. Each bar represents the mean ± S.D. of four rats. Incubation was performed at 37°C for 30 min with 200 µl of liver microsomes of rats pretreated with inducers in the presence of NADPH under anaerobic conditions. The p,p'-DDD formed was determined by HPLC. Other details are described under Experimental Procedures.

Identification of CYP Isoforms Involved in the Dechlorination. Identification of CYP isoforms involved in the dechlorination of p,p'-DDT in liver microsomes was attempted by using recombinant rat and human CYPs. Recombinant rat CYP 2B1 and 3A1 catalyzed this dechlorination in the presence of NADPH at rates of 1.16 and 1.03 nmol/min/nmol CYP, respectively. Recombinant human CYP 3A4 and 2B6 exhibited substantial activities for dechlorination of p,p'-DDT in the presence of NADPH. Human CYPs 1A1, 1A2, 2A6, 2C9, 2D6, 2E1, and 4A11 also showed some dechlorinase activity (Fig. 5). Control microsomes of human B lymphoblastoid cells exhibited no appreciable dechlorinase activity. These facts suggest that the dechlorination of p,p'-DDT is catalyzed predominantly by phenobarbital- and dexamethasone-inducible CYPs, but other CYPs also contribute to the dechlorination.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Dechlorination of p,p'-DDT by microsomes from human B lymphoblastoid cells expressing individual recombinant human cytochrome P450. Each bar represents the mean of duplicate determination. Incubation was performed at 37°C for 30 min with microsomal preparations in the presence of NADPH under anaerobic conditions. p,p'-DDD formed was determined by HPLC. Other details are described under Experimental Procedures.

Heme-Based Dechlorination in Liver Microsomes. The NADH- and NADPH-dependent dechlorinating activities in liver microsomes of untreated rats were enhanced by the addition of FMN. Even when the microsomal fraction was boiled, the activities with both NAD(P)H and FMN were not abolished but were enhanced to some extent (Table 1). The activity was sensitive to inhibition by carbon monoxide. When FMN was replaced with flavin adenine dinucleotide or riboflavin, a similar result was obtained in the dechlorination of p,p'-DDT (data not shown). These facts suggest that CYP is also involved in the nonenzymatic dechlorination of p,p'-DDT in the liver microsomes of rats.

Reductive Metabolism of o,p'-DDT by Liver Microsomes. o,p'-DDT, the isomer of p,p'-DDT, is known to contaminate technical-grade DDT and to have a higher estrogenic activity than p,p'-DDT. In this study, the dechlorinating activity toward o,p'-DDT by rat liver microsomes was compared with that toward p,p'-DDT. The liver microsomes exhibited reductase activity toward o,p'-DDT in the presence of NADPH, NADH, or both NADPH and NADH. The reductase activity in the presence of these cofactors toward o,p'-DDT was similar to that in the case of p,p'-DDT. Similar reducing activity of boiled liver microsomes was observed in the presence of NADPH and FMN (Fig. 6).

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Comparative study of dechlorination of p,p'-DDT and o,p'-DDT by rat liver microsomes. Each bar represents the mean ± S.D. of four rats. p,p'-DDT or o,p'-DDT was incubated with 200 µl of liver microsomes of untreated rats at 37°C for 30 min under anaerobic conditions. The p,p'-DDD or o,p'-DDD formed was determined by HPLC. Other details are described under Experimental Procedures. a, microsomes boiled for 5 min were used.

Metabolism of p,p'-DDD by Liver Microsomes. Metabolism of p,p'-DDD by rat liver microsomes was also examined. When p,p'-DDD was incubated with liver microsomes in the presence of NADPH under anaerobic conditions, two peaks corresponding to authentic p,p'-DDE and p,p'-DDMU were detected in the HPLC chromatogram of the extract of the incubation mixture (Fig. 7). The metabolites isolated by HPLC were identified unequivocally as p,p'-DDE and p,p'-DDMU by comparison with authentic samples (data not shown). When p,p'-DDE was incubated similarly with liver microsomes in the presence of NADPH, p,p'-DDMU was not detected (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 7.   HPLC of the metabolites of p,p'-DDD by rat liver microsomes. The incubation was performed at 37°C for 30 min with liver microsomes of untreated rats in the presence of NADPH under anaerobic conditions. The incubation mixture was extracted with n-hexane and the extract was subjected to HPLC, as described under Experimental Procedures. The absorbance (full scale, 0.016 atomic units) was measured at 254 nm.

	Discussion

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It is known that halogenated hydrocarbons, including p,p'-DDT, are metabolized to dehalogenated metabolites by reductive dehalogenation. Previous studies showed that the dehalogenation of carbon tetrachloride, hexachloroethane, halothane, or (-bromoisovaleryl)urea occurred in the liver microsomal fraction of mammalian species and that cytochrome P450 was involved in the microsomal dehalogenation (Fujii et al., 1981; Kubic and Anders, 1981; Ahr et al., 1982; Nastainczyk et al., 1982; Oka et al., 1996). Recent experiments have demonstrated that rat liver microsomes can directly transform halothane to dehalogenated metabolites in a reductive reaction involving the formation of a halothane-free radical intermediate (Baker and Van Dyke, 1984). In contrast, it has been shown that p,p'-DDT is mainly metabolized to p,p'-DDD and p,p'-DDE by reductive dechlorination and dehydrochlorination, respectively. In the reduction of p,p'-DDT to p,p'-DDD, Kelner et al. (1986) demonstrated that thiols stimulated the reductive dechlorination in liver microsomes. It was also reported that the pesticide covalently bound to microsomal protein and lipids owing to the formation of a DDT free-radical intermediate that led to the formation of p,p'-DDD (Baker and Van Dyke, 1984). However, the CYP isoform(s) catalyzing the reduction have not been identified.

Some studies on CYP isoforms involved in the reductive dehalogenation of halogenated hydrocarbons have been conducted. Sprackline et al. (1996) demonstrated that the dehalogenation of halothane is mainly catalyzed by CYP 2A6 and 3A4. Reductive dehalogenation of hexachloroethane is catalyzed CYP 1A2 (Yanagita et al., 1998). Oka et al. (1996) also demonstrated that debromination of (-bromoisovaleryl)urea is catalyzed by CYP 1A1 and 2B1. In the present study, the dechlorination of p,p'-DDT was demonstrated to be mainly mediated by CYP 2B1, 3A1, 2B6, and 3A4. It is known that p,p'-DDT induces CYP 2B1 and 4A1 in rat livers (Lewis and Lake, 1997; Nims et al., 1998). This suggests that the dechlorinating activity toward p,p'-DDT would be enhanced by the pretreatment of rats with p,p'-DDT. Indeed, in a preliminary study in which rats received intraperitoneal p,p'-DDT at a dose of 100 mg/kg for 3 days before sacrifice, the liver microsomes exhibited 5-fold higher activity than those of untreated rats. In contrast, in the liver microsomal reduction of p,p'-DDT, NADH was about half as effective as NADPH. We previously observed that NADH was about half as effective as NADPH in N-oxide reduction, nitro reduction, and zonisamide reduction by liver microsomes of animals (Kitamura and Tatsumi, 1984; Sugihara et al., 1996; Tatsumi et al., 1986). NADH seems to be generally effective in drug-reductive metabolism with liver microsomes. In the NADH-dependent reaction, the electron may be transferred through cytochrome b₅.

Clark and Shamaan (1984) reported that p,p'-DDT was metabolized to p,p'-DDE by glutathione-dependent dehydrochlorinase in the housefly. They demonstrated that the enzyme was identical to glutathione S-transferase. However, the identity of dehydrochlorinase and glutathione S-transferase has not yet been established. Dehydrochlorination of lindane was also reported to be catalyzed by glutathione-dependent dehydrochlorinase in the housefly (Tanaka et al., 1981). In our experiment, CYP 1A1, 2B1, 3A1, and 2E1 did not catalyze the dehydrochlorination (data not shown). We could not confirm a role of CYP in the dehydrochlorination of p,p'-DDT to p,p'-DDE. In contrast, p,p'-DDD was enzymatically metabolized to p,p'-DDE by rat liver microsomes or cytosol, but only to a small extent. Thus, p,p'-DDE may be formed via p,p'-DDD by an unknown enzyme system in rat liver. p,p'-DDMU was also formed from p,p'-DDD by liver microsomes. This dehydrochlorination may be catalyzed by liver dehydrochlorinase, similar to the case of p,p'-DDT. In contrast, it was reported that p,p'-DDE was dechlorinated to p,p'-DDMU by marine sediment microcosms (Quensen et al., 1998). However, this dechlorinating metabolism of p,p'-DDE to p,p'-DDMU did not occur in rat liver (Fig. 8). It is reported that the major metabolite of p,p'-DDT remaining in tissues of mammalians is p,p'-DDE (You, 2000). It remains to be determined whether p,p'-DDE is formed via p,p'-DDD, as suggested above or by dehydrochlorinase, as has been demonstrated in insects. We are conducting a further study on this point.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Postulated metabolic pathway of p,p'-DDT by rat liver microsomes.

Recent experiments have demonstrated that rat blood can transform p,p'-DDT to p,p'-DDD in a reductive reaction involving the heme moiety of hemoglobin (Sugihara et al., 1998). The microsomal NAD(P)H-dependent reduction seems to proceed enzymatically, catalyzed by the CYP system. However, when a flavin was added to the mixture, the reducing activity was enhanced and was nonenzymatic in character. The nonenzymatic dechlorination of p,p'-DDT in liver microsomes presented in this study seems to proceed in two steps. The first step is the reduction of a flavin by NADPH-cytochrome P450 reductase with NADPH or NADH-cytochrome b₅ reductase with NADH, whereas in boiled microsomes, the flavin seems to be reduced nonenzymatically by NADPH or NADH, as reported by Singer and Kearney (1950). The second step is the nonenzymatic reduction of p,p'-DDT to p,p'-DDD by the reduced flavin catalyzed by the heme group of hemoproteins, such as CYP and cytochrome b₅ (Fig. 7). In boiled microsomes, the activity was increased, possibly because the substrate and cofactor have easier access to the heme moiety of denatured hemoproteins. We proposed a similar mechanism for the debromination of (-bromoisovaleryl)urea in the blood of rats (Kitamura et al., 1999a). We also demonstrated that the major route of dechlorination of p,p'-DDT in fish was a nonenzymatic reaction by blood catalyzed by heme (Kitamura et al., 1999b). Other investigators demonstrated that reduced iron porphyrins catalyze the reductive dechlorination of p,p'-DDT (Castro, 1964; Zoro et al., 1974). Here, we suggest that the dechlorination of p,p'-DDT is also mediated by nonenzymatic reduction in liver microsomes separately from the enzymatic reduction by the CYP system.

Bitman et al. (1968) reported that indicators of estrogenic action increased after o,p'-DDT administration to rats and birds, the responses resembling those to 17-estradiol, whereas p,p'-DDT exhibited only slight activity. Metcalfe et al. (2000) also reported that gonadal development and endocrine responses were altered in fish exposed to o,p'-DDT. Furthermore, the relative potencies for intersex induction are o,p'-DDT > nonylphenol. However, the metabolism of o,p'-DDT has not been investigated except for a study in pigeon liver preparations (Hassall and Manning, 1972). In this study, we demonstrated that o,p'-DDT is dechlorinated by rat liver microsomes via enzymatic and nonenzymatic reactions in a similar ratio to p,p'-DDT. This fact suggests that o,p'-DDT is metabolized and degraded in the environment similar to p,p'-DDT.

It is reported that dicofol, -hydroxyl-p,p'-DDT, has a relatively high estrogenic activity among DDT analogs. Kapoor et al. (1972) reported that dicofol was detected as a minor urinary metabolite of p,p'-DDT in mice. We also examined the oxidation of p,p'-DDT to dicofol using rat liver microsomes as a metabolic activation pathway. However, we could not detect any oxidized metabolite of p,p'-DDT. The bridge site of p,p'-DDT may be protected from attack because of steric hindrance. The possibility remains that aromatic rings of DDTs may be hydroxylated by liver microsomal enzymes. However, Sundström et al. (1977) reported that only small amounts of 3-hydroxy-p,p'-DDE were formed from p,p'-DDT in pigs. In our experiments, it was demonstrated that the metabolism of p,p'-DDT or o,p'-DDT in rats is mainly reductive.