Introduction

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Endogenous and exogenous estrogens undergo oxidative metabolism by hepatic microsomal cytochrome P-450 (P-450; Martucci and Fishman, 1993). Aromatic hydroxylation at either the C2 or C4 position is a major route of estrogen metabolism in humans and other mammals, although there is less 4-hydroxylation than 2-hydroxylation. Estrogen 2- or 4-hydroxylation is catalyzed primarily by the CYP3A family with some contribution by the CYP1A family. Recently, a specific estrogen 4-hydroxylase has been identified in MCF-7 breast cancer cells. This activity has been attributed to a newly identified member of P-450, CYP1B1 (Hayes et al., 1996; Liehr and Ricci, 1996).

The carcinogenicity of estrogens, such as 17-estadiol (estradiol), is known to be related to their metabolism to reactive catechols as well as to their action as agonists of estrogen receptors (Yager and Liehr, 1996). The 2- and 4-hydroxylated metabolites of estrogens can directly or indirectly damage DNA, proteins, and lipids. The catechol metabolites generate active oxygen by reductive-oxidative cycling (Han and Liehr, 1995). However, in spite of much work over many years, it is still not clear whether this metabolic activation to catechols is really responsible for the carcinogenicity of estrogens.

We have already reported that the substituent elimination of various para-substituted phenols to afford hydroquinone, which is a novel metabolic pathway catalyzed by P-450, occurs in a rat liver microsomal system (Ohe et al., 1997) as well as in a P-450 chemical model system (Ohe et al., 1995). However, in the case of p-cresol, p-toluquinol was formed instead of hydroquinone because the methyl group is difficult to eliminate and the reaction stopped before elimination. This finding suggests that the metabolic quinol formation might occur in various para-alkylphenols other than p-cresol. We hypothesized that estrone and estradiol, both of which contain para-alkylphenol moiety, could be oxidized into the corresponding quinols by P-450 (Fig. 1). Many metabolic pathways of these estrogens by P-450 have been reported so far (Cheng and Schenkman, 1984; Martucci and Fishman, 1993), but hydroxylation at the C10 position, namely quinol formation, is not known. In addition, the quinol formation from estrogens might be linked to the mechanism for estrogen-induced carcinogenicity because quinol contains ,-unsaturated ketone that is an electrophilic moiety and a Michael reaction acceptor, which can bind covalently to DNA, RNA, and other cellular macromolecules and may lead to genotoxicity and cytotoxicity (Witz, 1989; Feron et al., 1991; Eder et al., 1993). In this study, we examined the quinol formation from estrone and estradiol by using various human recombinant P-450 enzymes.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   The quinol formation from estrone and estradiol catalyzed by P-450.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results and Discussion References

Materials. 10-Hydroxy-1,4-estradiene-3,17-dione and 10,17-dihydroxy-1,4-estradiene-3-one were prepared by the photooxygenation of estrone and 17-estradiol in the presence of Rose Bengal according to the method of Pylar Lupon et al. (1983) These two compounds were identified on the basis of ¹H-NMR and mass spectra (Numazawa et al., 1989).

Microsomal Incubations. The incubation mixture containing NADP⁺ (final 0.4 mM), substrate (0.1 mM), KCl (60 mM), MgCl₂ (4 mM), G-6-P (4 mM), and G-6-P dehydrogenase (5 U) in 1 ml of 0.1 M sodium phosphate buffer (pH 7.4) was preincubated for 2 min at 37°C. The reaction was initiated by adding microsomes (final 1 mg protein). NADPH reductase was not added in this assay. After incubation for 60 min at 37°C, the mixture was treated with 0.5 ml of 1 N aqueous HCl solution to stop the reaction and the products were extracted with 2 ml of ethyl acetate. The organic phase was separated and concentrated by argon flushing. The residue was used for qualitative analysis and quantitative analysis in the following manner.

Qualitative Analysis. Separation of 10-hydroxy-1,4-estradiene-3,17-dione or 10, 17-dihydroxy-1,4-estradiene-3-one was achieved using HPLC. The residue was dissolved in a small amount of methanol/H₂O/acetic acid (4/5/1) and injected into HPLC (JASCO TWINCLE, with a 6.0 × 250 mm Perkin-Elmer C₁₈ reversed-phase column, eluted with methanol/H₂O/acetic acid (52:47:1) at a flow rate of 1 ml/min. The eluent was monitored for absorbance at 280 nm). The desired fraction was collected and dissolved in a small amount of ethyl acetate. Each product was identified by gas chromatography-single ion monitor (GC-SIM) on the basis of the m/z peak ratio 286.3/268.3/145.0/124.0/123.0 or 288.3/270.3/147.0/124.0/123.6, respectively (Shimadzu QP5000; capillary column DB-5 30 m; J & W Scientific, Folsom, CA). Injection temperature was 250°C. The initial column temperature was 250°C for 3 min. It was raised in 5°C/min increments to 300°C and then held isothermally at this temperature.

Quantitative Analysis. The residue was dissolved in a small amount of acetone, and epiandrosterone was added as an internal standard. Each product was determined by GC-SIM on the basis of m/z 286.3 or 288.3, respectively (Shimadzu QP5000; capillary column DB-5 30 m; J & W Scientific). The injection temperature was 250°C. The initial column temperature was 260°C for 3 min. It was then raised in 5°C/min increments to 300°C, and held at that temperature.

	Results and Discussion

Top Abstract Introduction Experimental Procedures Results and Discussion References

To determine whether P-450 catalyzes quinol formation from estrone and estradiol, estrone or estradiol were incubated with microsomes prepared from the human CYP1A1-expressed B-lymphoblastoid cell line. Incubations were carried out for 60 min at 37°C and stopped by the addition of aqueous HCl solution, and the products were extracted with ethyl acetate. The extracts were separated by HPLC, and the desired fraction was analyzed by GS-MS. As a result, the corresponding quinol, namely 10-hydroxy-1,4-estradiene-3,17-dione or 10, 17-dihydroxy-1,4-estradiene-3-one, was detected. It was identified on the basis of its retention time and m/z peak ratio of 286.3/268.3/145.0/124.0/123.0 or 288.3/270.3/147.0/124.0/123.6 in GC-SIM mode, compared with those of the synthesized authentic compound (Table 1). When microsomes or NADP⁺ were omitted from the complete system, the quinols were not obtained. These results demonstrate that estrone and estradiol were converted into the corresponding quinols by CYP1A1.

According to the report of Pylar Lupon et al. (1983), the 10-isomer is formed only as a minor product during chemical synthesis, which implies that a  attack of oxygen at the 10-position predominates over an  attack stereochemically or thermodynamically. The same predominance might apply to the metabolic quinol formation by P-450, although the 10-isomer formation was not examined in the present study.

To further characterize the P-450 enzyme dependence of quinol formation, we determined the catalytic activities of various human P-450 enzymes, namely CYP1A1, 1A2, 2B6, 2C9, 2E1, and 3A4. Table 2 shows the quinol formation during a 60-min reaction. Incubations containing cDNA-expressed CYP1A1, 2B6, and 2E1 were found to convert estrone into the quinol. Likewise, these three isozymes also catalyzed quinol formation from estradiol. The products were not detected in the incubation with control (minus cDNA insert) microsomes or microsomes containing cDNA-expressed CYP1A2, 2C9, and 3A4. It is noteworthy and interesting that CYP2E1 is involved in this reaction, because CYP2E1 is known to catalyze relatively small compounds such as acetone, benzene, and ethanol. On the other hand, CYP3A4, which contributes to the aromatic hydroxylation of estrogens, did not catalyze the quinol formation.

In the present study, we discovered quinol formation from estrogens accompanied by 10-hydroxylation as a novel metabolic pathway. However, the amounts formed may be underestimated because the quinols can bind covalently to microsomal proteins, resulting in poor recovery. This might be one of the reasons why the quinols have not been found to be metabolites of estrogens so far, although their metabolism has been extensively investigated.

As described above, quinols can bind covalently to cellular macromolecules. Therefore, quinol formation from estrogens could be a kind of metabolic activation. However, it is unclear whether the quinol metabolites actually act as carcinogens by damaging cellular macromolecules, because their biological effects are not known. The biological actions of the quinol metabolites formed are therefore of interest.

In conclusion, we have shown that the quinol formation from estrone and estradiol, which is a novel metabolic pathway, is catalyzed by certain kinds of P-450 isozymes. We consider the present study to be one of the examples where quinol formation can occur in a variety of compounds that contain a phenolic hydroxy group. Additional studies on the application of this novel metabolic reaction are in progress.