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Chem. Res. Toxicol. 2003, 16, 1138-1144
r-Hydroxylation of Tamoxifen and Toremifene by Human and Rat Cytochrome P450 3A Subfamily Enzymes Sung Yeon Kim, Naomi Suzuki, Y. R. Santosh Laxmi, Robert Rieger, and Shinya Shibutani* Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651 Received March 12, 2003
An increased risk of developing endometrial cancer is observed in breast cancer patients treated with tamoxifen (TAM) and in healthy women undergoing TAM chemoprevention therapy. TAM-DNA adducts were detected in the endometrium of women taking TAM (Shibutani, S., et al. (2000) Carcinogenesis 21, 1461-1467) and are formed primarily through O-sulfonation of R-hydroxytamoxifen (R-OHTAM). To explore the genotoxicic mechanisms of TAM, TAM was incubated with one of multiple human cytochrome P450 enzymes, i.e., P450 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 3A7, 4A11, 4F2, 4F3A, or 4F3B, in a NADPH regenerating system, and the metabolites were identified using HPLC/ UV analysis with authentic standards. Among the 18 human P450 enzymes, P450 3A4 generated a significant amount of R-OHTAM. When some rat P450 enzymes were examined, P450 3A2 also catalyzed R-hydroxylation of TAM. Similarly, human P450 3A4 and rat P450 3A1 and 3A2 converted toremifene (TOR, a chlorinated TAM analogue) to R-hydroxytoremifene (R-OHTOR). The formation of R-OHTAM and R-OHTOR by these P450 enzymes was confirmed by tandem mass spectroscopy. Only the P450 3A subfamily enzymes are able to R-hydroxylate TAM and TOR. Although the formation of R-OHTOR by these enzymes was much higher than that of R-OHTAM, TOR is known to be much less genotoxic than TAM. The results support our proposed mechanism that the lower genotoxicity of TOR is due to limited O-sulfonation of R-OHTOR by hydroxysteroid sulfotransferases, resulting in the poor formation of DNA adducts (Shibutani, S., et al. (2001) Cancer Res. 61, 3925-3931).
Introduction 1
TAM (the structure in Figure 1) is widely used as the first endocrine therapy for breast cancer patients (1, 2) and as a chemopreventive agent for healthy women at high risk of developing this disease (3). However, administration of TAM to breast cancer patients (4-9) and to women under chemopreventive trial (3) is associated with an increased risk of endometrial cancer. Treatment of rats with TAM induced hepatocellular tumors (10-12) that are associated with the formation of covalent DNA adducts (13-17) induced by the activated metabolites of this drug (18-20). TAM was listed as a human carcinogen by the International Agency for Research on Cancer (21). TAM is converted by phase I enzymes to several metabolites including R-OHTAM, N-desTAM, 4-OHTAM, and TAM N-oxide (22-26) (Figure 2). The P450s in the * To whom correspondence should be addressed. Tel: 631-444-8018. Fax: 631-444-3218. E-mail:
[email protected]. 1 Abbreviations: TAM, tamoxifen; R-OHTAM, R-hydroxytamoxifen; 4-OHTAM, 4-hydroxytamoxifen; N-desTAM, N-desmethyltamoxifen; TAM N-oxide, tamoxifen N-oxide; 4-OH-N-desTAM, 4-hydroxy-Ndesmethyltamoxifen; R-OH-N-desTAM, R-hydroxy-N-desmethyltamoxifen; dG-N2-TAM, R-(N2-deoxyguanosinyl)tamoxifen; dG-N2-N-desTAM, R-(N2-deoxyguanosinyl)-N-desmethyltamoxifen; dG-N2-TAM N-oxide, R-(N2-deoxyguanosinyl)tamoxifen N-oxide; TOR, toremifene; R-OHTOR, R-hydroxytoremifene; 4-OHTOR, 4-hydroxytoremifene; N-desTOR, Ndesmethyltoremifene; HPLC, high-performance liquid chromatography; MS/MS, tandem mass spectroscopy; P450, cytochrome P450 enzyme.
Figure 1. Structures of TAM and TOR.
liver and other tissues of animals and humans (27) catalyze the majority of TAM except for the formation of TAM N-oxide by flavin-containing monooxygenase (28-30). N-Demethylation of TAM is catalyzed primarily by P450 1A1 and the 3A family (31-34). Two enzymes, P450 2D6 and 2C9, play a predominant role in the 4-hydroxylation of TAM (34, 35). R-OHTAM was detected as a minor metabolite in the plasma of women treated with TAM (26). R-OHTAM can be produced by incubating TAM with rat or human liver microsomes (32, 36); the rate of formation of R-OHTAM by human liver microsome is approximately half that observed in rats (32). Sridar et al. (37) and Boocock et al. (38) recently indicated that P450 3A4 catalyzes the conversion of TAM to R-OHTAM. R-OHTAM is sulfonated by rat and human hydroxysteroid sulfotransferases (39, 40) and reacts with the exocyclic amino group of guanine in DNA, forming two trans (fr-1 and fr-2) and two cis (fr-3 and fr-4) diastereoisomers of dG-N2-TAM (Figure 2) (15,
10.1021/tx0300131 CCC: $25.00 © 2003 American Chemical Society Published on Web 08/20/2003
Formation of R-OHTAM and R-OHTOR by P450 Enzymes
Chem. Res. Toxicol., Vol. 16, No. 9, 2003 1139
Figure 2. Mechanism for the formation of TAM metabolites and TAM-DNA adducts.
41, 42). Mass spectroscopic analysis and 32P-postlabeling/ HPLC analysis demonstrated that dG-N2-TAM and dGN2-N-desTAM are the major hepatic DNA adducts of rodents treated with TAM (43-45). dG-N2-TAM N-oxide was also detected as a minor adduct in the liver of mice treated with TAM (44). The three different TAM-DNA adducts accounted for over 95% of DNA adducts induced by TAM (44, 45). dG-N2-TAM adducts were detected in the endometrium of women treated with TAM (46, 47). It is also possible that TAM-DNA adducts might be produced via oxidation of 4-OHTAM, as R-(N2-deoxyguanosinyl)-4-hydroxytamoxifen (dG-N2-4-OHTAM) adducts have been identified in vitro at the dG residues in DNA, induced by 4-OHTAM quinone methide (48). However, DNA adducts corresponding to 4-OHTAM have not been detected to any significant degree in animal studies (16, 49). Therefore, R-hydroxylation of TAM and its metabolites, followed by O-sulfonation and/or O-acetylation, is
a major pathway for the formation of TAM-DNA adducts (16, 23, 25, 41). A high frequency of mutations was observed in the hepatic DNA of λ/lac I transgenic rats treated with TAM (50). dG-N2-TAM adducts display a high miscoding and mutagenic potential in mammalian cells (51, 52). If TAM-DNA adducts are not repaired (53), mutations may occur at the adduct sites and initiate the development of endometrial cancers. TOR (the structure in Figure 1), a chlorinated TAM derivative, has been used for breast cancer therapy in 27 countries since 1987. TOR is metabolized by human liver microsomes to N-desTOR, 4-OHTOR, deaminohydroxytoremifene, and R-OHTOR (54, 55). The formation of N-desTOR and deamino-hydroxytoremifene is mediated by P450 1A and 3A4 enzymes (54). The Ndemethylated and 4-hydroxylated metabolites were detected in human blood (56) and are mainly excreted in rat feces (57, 58). Small amounts of R-OHTOR and
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R-hydroxy-N-desmethyltoremifene (R-OH-N-desTOR) were detected in rat urine (58). Unlike TAM, TOR produced two orders of magnitude lower DNA adducts in rat liver (12, 14, 59) and did not promote hepatocarcinoma in rats (12, 14). In the present study, to explore what P450 enzymes are involved in the R-hydroxylation of TAM and TOR, one of 20 human and rat P450 enzymes was incubated with TAM or TOR in vitro and their metabolites were analyzed using an HPLC/UV detector and MS/MS with authentic standards. We found that human and rat P450 3A subfamily enzymes are involved in the R-hydroxylation of TAM and TOR; interestingly, the formation of R-OHTOR by the P450 enzymes was much higher than that of R-OHTAM.
Materials and Methods Chemicals. TAM and TOR were purchased from Sigma (St. Louis, MO) and LKT Laboratories (St. Paul, MN), respectively. R-OHTAM (60), R-OHTOR (61), 4-OHTAM and 4-OHTOR (62), N-desTAM and N-desTOR (63), R-OH-N-desTAM (64), and 4-OH-N-desTAM (65) were synthesized by the established methods. Acetonitrile, triethylamine, and distilled water, all HPLC grade, were purchased from Fisher Scientific (Fair Lawn, NJ). Reaction of TAM or TOR with Human and Rat P450 Enzymes. Human and rat P450 enzymes and the NADPH regenerating system were purchased from Gentest (BD Biosciences Company, Woburn, MA). All products were from baculovirus-insect cells expressed from P450s cDNA and with supplemental cDNA expressed reductase. TAM or TOR (100 µM) was incubated at 37 °C for 2 h in 100 mM potassium phosphate buffer, pH 7.4 (200 µL), containing 50 pmol of P450 enzyme, 1.3 mM NADPH, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl2, and glucose-6-phosphate dehydrogenase (0.4 U/mL). The reaction mixture was prewarmed at 37 °C, and the reaction was started by the addition of P450 enzyme. After 2 h of incubation, the reaction was stopped by the addition of 200 µL of cold methanol. The samples were mixed using a vortex and centrifuged (15 000g) for 10 min. The supernatants were evaporated to dryness and solved with an appropriate amount of ethanol (50 µL) and subjected to HPLC. Human P450 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 3A7, 4A11, 4F2, 4F3A, and 4F3B and rat P450 3A1 and 3A2 were used for this assay. HPLC Analysis of TAM and TOR Metabolites. HPLC was performed using 515 HPLC pump, 996 photodiode array detector, and pump control module. The sample was subjected to a Waters symmetry C18 column (4.5 mm × 150 mm, 5 µm) and eluted at a flow rate of 1.0 mL/min with a linear gradient of 30-100% of acetonitrile in a 50 mM triethylammonium acetate buffer (pH 7.4) over 30 min, after which 100% acetonitrile was eluted for another 30 min. TAM or TOR metabolites were monitored by UV absorbance at 240 nm and quantified by comparing to the standards. The detection limit of TAM or TOR metabolites was approximately 5 ng. Determination of TAM and TOR Metabolites by MS/MS. When the reaction mixture of TAM or TOR with P450 enzymes was subjected to HPLC, the eluate containing metabolites was collected and evaporated to dryness. Analysis by mass spectroscopy was performed on a Quattro LCZ (Micromass, U.K.) outfitted with a nanospray source. The dried sample was diluted with 25 µL of 50% acetonitrile/H2O, and approximately 3 µL was transferred to the capillary tubes (New Objective Inc., MA) and mounted in the apparatus. The source block was set to 80 °C, and the high voltage applied to the capillary was set to 0.88 kV in the positive ion mode. The cone voltage was optimized for each experiment. Acquisitions in the MS mode were scanned from m/z 200 to 900 in 4 s. MS/MS experiments used argon at 4 × 10-4 mbar, and Q2 was scanned from m/z 50 to 500 in 4 s.
Figure 3. HPLC analysis of TAM metabolites. (A) A mixture of authentic TAM and TAM metabolites (1, R-OHTAM; 2, 4-OHTAM; 3, N-desTAM; and 4, TAM) was subjected to a Waters symmetry C18 column (4.5 mm × 150 mm, 5 µm) and eluted at a flow rate of 1.0 mL/min with a linear gradient of 30-100% of acetonitrile in 50 mM triethyamine buffer (pH 7.4) over 30 min, after which 100% acetonitrile was eluted for another 30 min. TAM metabolites were monitored by the UV absorbance at 240 nm using a photodiode array detector. TAM (100 µM) was incubated at 37 °C for 2 h in 100 mM potassium phosphate buffer, pH 7.4 (200 µL), containing 50 pmol of P450 3A4 (B) or P450 3A2 (C), 1.3 mM NADPH, 3.3 mM glucose-6phosphate, 3.3 mM MgCl2, and glucose-6-phosphate dehydrogenase (0.4 U/mL). The reaction mixture was subjected to an HPLC system for analysis of TAM metabolites.
Results To establish what P450 enzyme(s) is involved in the R-hydroxylation of TAM, TAM was incubated with a human P450 enzyme (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 3A7, 4A11, 4F2, 4F3A, or 4F3B) in an NADPH regenerating system. The formation of TAM metabolites increased when 10-50 pmol of P450 was used; however, the product of TAM metabolites decreased when 100 pmol of the enzyme was used (data not shown), as observed earlier by another laboratory (34). Therefore, we used 50 pmol of P450 enzyme for those experiments with TAM. The resulting TAM metabolites were analyzed using HPLC with a photodiode array detector. R-OHTAM, 4-OHTAM, N-desTAM, and TAM, can be resolved by HPLC and eluted at 12.6, 16.0, 18.5, and 24.5 min, respectively (Figure 3A). As shown in Table 1, most of the P450 enzymes used, except for P450 1B1, 2A6, 2E1, and the four family enzymes, converted TAM to N-desTAM to variable degrees. Rat P450 3A1 and 3A2 were also able to N-demethylate TAM (Table 1). Incuba-
Formation of R-OHTAM and R-OHTOR by P450 Enzymes Table 1. Formation of TAM Metabolites in Reactions Catalyzed by P450 Enzymesa
human
rat
P450 enzyme
TAM metabolites (pmol) R-OHTAM 4-OHTAM N-desTAM
1A1 1A2 1B1 2A6 2B6 2C8 2C9 2C18 2C19 2D6 2E1 3A4 3A5 3A7 4A11 4F2 4F3A 4F3B 3A1 3A2
3260 ( 370 720 ( 22
100 ( 15 630 ( 180
92 ( 4 1250 ( 300 550 ( 84 700 ( 50 660 ( 30 3500 ( 84
65 ( 49 97 ( 50
6170 ( 780 1820 ( 105 160 ( 63
300 ( 67
53 ( 15
130 ( 64
4090 ( 670 2870 ( 130
a TAM (100 µM) was incubated at 37 °C for 2 h in 100 mM potassium phosphate buffer, pH 7.4 (200 µL), containing 50 pmol of P450 enzyme, 1.3 mM NADPH, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl2, and glucose-6-phosphate dehydrogenase (0.4 U/mL). TAM metabolites were determined using an HPLC/UV system and authentic standards. Data are expressed as mean values ( SD from three reaction samples. Blank entries, not detectable.
Figure 4. MS/MS analysis of TAM and TOR metabolites. The elute of TAM or TOR metabolites was collected by HPLC as described in Figures 3 and 5 and evaporated to dryness. TAM (A) and TOR (B) metabolites were analyzed by MS/MS spectroscopy as described in the Materials and Methods.
tion of TAM with human P450 2D6, 2C9, and 2C19 resulted in the formation of 4-OHTAM (Table 1). The formation of N-desTAM and 4-OHTAM by P450 enzymes was also confirmed by mass spectroscopy (data not shown). Among the 18 human P450 enzymes, P450 3A4 (Figure 3B) and P450 2D6 (data not shown) promoted small amounts (53 and 56 pmol, respectively) of a product with a similar retention time (12.6 min) as R-OHTAM. No such products were observed when NADPH was not added into the reaction mixture containing P450 3A4 or P450 2D6 (data not shown). Using positive ion mass spectroscopy, the parent ion of the product obtained from P450 3A4 exhibited at m/z 388, identifying the molecular mass of this product as 387 Da. The product was identified as R-OHTAM by comparing the MS/MS spectra from authentic standards of R-OHTAM and 4-OHTAM with the sample spectrum (Figure 4A). The appearance of an ion at m/z 179 representing a fragment of 1,2-biphenyleth-
Chem. Res. Toxicol., Vol. 16, No. 9, 2003 1141 Table 2. Formation of TOR Metabolites in Reactions Catalyzed by P450 Enzymesa P450 enzyme human rat
3A4 3A1 3A2
TOR metabolites (pmol) R-OHTOR 4-OHTOR N-desTOR 332 ( 11 440 ( 42 500 ( 24
3350 ( 20 3180 ( 230 1900 ( 140
a TOR (100 µM) was incubated at 37 °C for 2 h in 100 mM potassium phosphate buffer, pH 7.4 (200 µL), containing 50 pmol of P450 enzyme, 1.3 mM NADPH, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl2, and glucose-6-phosphate dehydrogenase (0.4 U/mL). TOR metabolites were determined using an HPLC/UV system and authentic standards. Data are expressed as mean values ( SD from three reaction samples. Blank entries, not detectable.
ylene cation in the sample spectrum correlated with the R-OHTAM while this ion was not present in the MS/MS spectrum of 4-OHTAM. On the other hand, the parent ion of a product from P450 2D6 exhibited at m/z 374, showing the molecular mass as 373 Da (data not shown). No significant assignment of R-OHTAM (m/z 388) was detected in the product of P450 2D6. Because the molecular mass of both R-OHN-desTAM and 4-OH-N-desTAM was 373 Da, the authentic standards were synthesized and subjected to the HPLC system. The retention times of R-OH-N-desTAM and 4-OH-N-desTAM were 7.8 and 12.5 min, respectively. The retention time of the product of P450 2D6 was similar to that of 4-OH-N-desTAM. In addition, using µBondapak C18 (0.39 cm × 30 cm) with a linear gradient of 5-30% of acetonitrile over 15 min and subsequently 30-100% acetonitrile over 15 min in a 50 mM triethylammonium acetate buffer (pH 7.4), authentic R-OHTAM (tR ) 28.4 min) can be resolved from 4-OH-N-desTAM (tR ) 29.6 min) (data not shown). Using this system, the formation of R-OHTAM was not observed in the product of P450 2D6. Therefore, the product of P450 2D6 was identified as 4-OH-N-desTAM. Rat P450 3A1 and 3A2 catalyzed TAM to form a large amount of N-desTAM (Table 1). When P450 3A2 was used, significant amounts of products eluted at 12.6 and 22.8 min (Figure 3C). The product at 12.6 min exhibited at m/z 388, identifying the molecular mass of R-OHTAM as 387 Da, as observed for P450 3A4 (Figure 4A). The formation of R-OHTAM by rat P450 3A2 was 2.5 times higher than that of human P450 3A4. The product observed at 22.8 min was not identified. TOR metabolites can also be resolved by the HPLC system applied for analysis of TAM metabolites; the retention times of R-OHTOR, 4-OHTOR, N-desTOR, and TOR were 13.3, 14.3, 15.7, and 21.4 min, respectively (Figure 5A). Incubation of TOR with human P450 3A4 (Figure 5B) or rat P450 3A1 (data not shown) and 3A2 (Figure 5C) showed a large amount of N-desTOR formation, followed by a small amount of R-OHTOR (Table 2). The product at 20.7 min was not identified. The formation of R-OHTOR by these P450 enzymes was approximately 4-6 times higher than those observed for TAM. The products at 13.3 min generated by these P450 enzymes were determined using MS/MS mass spectroscopy. The parent ion of a product from P450 3A4, 3A1, or 3A2 exhibited at m/z 422, identifying the molecular mass of R-OHTOR as 421 Da. Daughter ions present at m/z 245 and 310 identified this product as the R-OHTOR (Figure 4B) because these ions were present in the MS/MS spectrum of the standard R-OHTOR but not
1142 Chem. Res. Toxicol., Vol. 16, No. 9, 2003
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Figure 6. Formation of TAM carbocation intermediate.
Figure 5. HPLC analysis of TOR metabolites. (A) A mixture of authentic TOR and TOR metabolites (1, R-OHTOR; 2, 4-OHTOR; 3, N-desTOR; and 4, TOR) was subjected to the HPLC system described in the legend of Figure 3. TOR (100 µM) was incubated at 37 °C for 2 h in 100 mM potassium phosphate buffer, pH 7.4 (200 µL), containing 50 pmol of P450 3A4 (B) or P450 3A2 (C), 1.3 mM NADPH, 3.3 mM glucose-6phosphate, 3.3 mM MgCl2, and glucose-6-phosphate dehydrogenase (0.4 U/mL). The reaction mixture was subjected to the HPLC system for analysis of TOR metabolites.
present in the 4-OHTOR. Our results indicated that human P450 3A4 and rat 3A1 and 3A2 are involved in the R-hydroxylation of TOR.
Discussion To detect the small amount of R-OHTAM formation by P450 enzymes, a longer incubation time was used in this experiment. Therefore, the amounts of TAM and TOR metabolites produced were not determined in the linear reaction range. However, when TAM was incubated with human P450 enzymes, P450 1A1, 2D6, and 3A4 demethylated TAM primarily; our results with human P450 enzymes expressed in baculovirus-insect cells were consistent with data reported by other laboratories using the enzymes expressed in other cell types (35, 37, 66). Rat P450 3A1 and 3A2 were also involved in N-demethylation of TAM. N-desTAM can be produced from TAM with a number of P450 enzymes. The formation of 4-OHTAM was also observed in reactions catalyzed by P450 2D6 and 2C9, as reported earlier (34, 35, 66). Among 18 different human P450 enzymes, only P450 3A4 could convert TAM to R-OHTAM (Table 1). Our result is consistent with recent papers by Sridar et al. (37) and Boocock et al. (38). Because the formation of
R-OHTAM by this enzyme was too low, the accurate kinetic parameters could not be determined using a photodiode detector. A sensitive analysis using a fluorescence detector could determine the kinetic parameters. However, there is a discrepancy in the level of R-OHTAM detected. Although the same enzyme source reported by Boocock et al. (38) was used in our experiments, the formation of TAM metabolites including R-OHTAM by P450 3A4 or 2D6 was much lower than that reported by Boocock et al. (38) It may be because our data were taken with the longer incubation time in the nonlinear reaction range. Especially, the rate of R-OHTAM formation (0.0088 pmol/min enzyme) detected using a NADPH regenerating system in our laboratory was 18 times lower than that (0.157 pmol/min enzyme) observed with a non-NADPH regenerating system by Boocock et al. (38). Further careful analysis is required to determine the rate of R-OHTAM formation. The P450 3A4 mRNA has been detected in human endometrium (67), suggesting that R-OHTAM could be produced in this tissue. Because dG-N2-TAM adducts have been detected in the endometrium of women treated with TAM (43, 44), the resulting R-OHTAM may be O-sulfonated and react with endometrial DNA, forming dG-N2-TAM adducts. We also found that rat P450 3A2 is involved in the R-hydroxylation of TAM. Because P450 3A subfamily enzymes including P450 3A2 were induced in the liver of rats after the TAM treatment (68), such enzyme induction may be associated with the development of hepatocarcinoma in rats. TOR was converted to R-OHTOR by human P450 3A4 and rat P450 3A1 and 3A2 (Table 2). Although kinetic study is required to determine the accurate formation of the R-hydroxylated metabolites, the formation of ROHTOR was much higher than that of R-OHTAM even when variable amounts of the enzymes were used. This reaction may occur through the formation of a carbocation at the ethyl moiety of both TAM and TOR, as proposed in Figure 6. The presence of the chlorine atom at the ethyl moiety of TOR induces a negative inductive effect on the carbocation. This effect may contribute to the greater reactivity of the carbocation generated from TOR than that of TAM, resulting in the higher amount of R-OHTOR formation. Using accelerator mass spectrometry, Boocock et al. (38) have demonstrated that carbocations generated during R-hydroxylation of TAM by P450 3A4 may bind irreversibly to DNA. The formation of R-OHTOR by P450 3A4, 3A1, and 3A2 was much higher than that of R-OHTAM. If this mechanism is primarily involved in the formation of DNA adducts, treatment of TOR should produce much higher amounts of DNA adducts than TAM. However, TOR is less genotoxic than TAM (12, 14, 59). In fact, using 32P-postlabeing analysis, we did not detect the significant formation of TAM-DNA adducts when calf thymus DNA was incubated with TAM and
Formation of R-OHTAM and R-OHTOR by P450 Enzymes
P450 3A4 or P450 3A2 (Kim, S. Y., and Shibutani, S. Unpublished data). Therefore, the mechanism proposed by Boocock et al. (38) may be a minor pathway for the formation of TAM-DNA adducts. TAM-DNA adducts are primarily formed from R-hydroxylated TAM metabolites including R-OHTAM via O-sulfonation catalyzed by hydroxysteroid sulfotransferases (39, 40). Because R-OHTOR and R-hydroxy-NdesTOR were detected in rat urine (58), such R-hydroxylated TOR metabolites could be precursors to the formation of TOR-DNA adducts. As observed in this study, the amount of R-OHTOR produced by the P450 3A family enzymes was higher than that of R-OHTAM, predicting that formation of TOR-DNA adducts could be higher than that of TAM-DNA adducts. However, the formation of hepatic DNA adducts by TOR in rats was two orders of magnitude less than that of TAM (12, 14, 59). No hepatocarcinoma was promoted in rats treated with TOR (12, 14). Because R-OHTOR is a poor substrate for rat and human hydroxysteroid sulfotransferases (61), the lower genotoxicity of TOR may be due to the limited O-sulfonation of R-OHTOR by this enzyme. Because clinical efficacy of TOR for breast cancer patients is similar to that of TAM (69), the use of TOR, instead of TAM, could reduce the risk of developing endometrial cancer in women treated with TAM.
Acknowledgment. This research was supported by the National Institute of Environmental Health Sciences Grant ES09418.
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(13) (14)
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