Oxidative DNA Damage in XPC-Knockout and Its Wild Mice Treated

May 1, 2008 - Xeroderma pigmentosa complementation group C-knockout. (Xpc-KO) and wild-type mice were treated with equilenin (EN), and the formation ...
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Oxidative DNA Damage in XPC-Knockout and Its Wild Mice Treated with Equine Estrogen Yoshinori Okamoto,† Pei-Hsin Chou,‡ Sung Yeon Kim,†,§ Naomi Suzuki,† Y. R. Santosh Laxmi,† Kanako Okamoto,† Xiaoping Liu,† Tomonari Matsuda,‡ 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, and Research Center for EnVironmental Quality Management, Kyoto UniVersity, Otsu, Shiga, 520-0811, Japan ReceiVed December 6, 2007

Long-term hormone replacement therapy with equine estrogens is associated with a higher risk of breast, ovarian, and endometrial cancers. Reactive oxygen species generated through redox cycling of equine estrogen metabolites may damage cellular DNA. Such oxidative stress may be linked to the development of cancers in reproductive organs. Xeroderma pigmentosa complementation group C-knockout (Xpc-KO) and wild-type mice were treated with equilenin (EN), and the formation of 7,8-dihydro-8oxodeoxyguanosine (8-oxodG) was determined as a marker of typical oxidative DNA damage, using liquid chromatography electrospray tandem mass spectrometry. The level of hepatic 8-oxodG in wildtype mice treated with EN (5 or 50 mg/kg/day) was significantly increased by approximately 220% after 1 week, as compared with mice treated with vehicle. In the uterus also, the level of 8-oxodG was significantly increased by more than 150% after 2 weeks. Similar results were observed with Xpc-KO mice, indicating that Xpc does not significantly contribute to the repair of oxidative damage. Oxidative DNA damage generated by equine estrogens may be involved in equine estrogen carcinogenesis. Introduction 1

Hormone replacement therapy (HRT) is widely used by postmenopausal women to alleviate menopausal symptoms and to protect against osteoporosis. In the United States, more than 40% of women in this demographic currently receive HRT, with Premarin (Wyeth-Ayerst) being one of the most commonly used products (1). However, HRT is associated with a significantly increased risk of breast, ovarian, and endometrial cancers (2–4). Premarin, composed of approximately 30% equilin (EQ), 10% equilenin (EN), and other estrogens, is frequently used for this purpose (1). Like human estrogens, EN and EQ are hydroxylated to 4-hydroxyequilenin (4-OHEN) and 4-hydroxyequilin (4OHEQ), respectively (1) (Figure 1). 4-OHEN is rapidly autoxidized to an o-quinone, which reacts readily with DNA in vitro, resulting in the formation of bulky DNA adducts (1, 5, 6). 4-OHEQ is also autoxidized to an o-quinone that isomerizes to 4-OHEN-o-quinone; therefore, 4-OHEQ produces DNA adducts identical to those generated by 4-OHEN (7). Mutagenic events induced by 4-OHEQ were observed in a supF shuttle vector plasmid propagated in human cells (8). 4-OHEN-induced DNA adducts were highly miscoded during translesion synthesis catalyzed by human DNA polymerases (9–11). Equine estrogen* To whom correspondence should be addressed. Tel: 631-444-7849. Fax: 631-444-3218. E-mail: [email protected]. † SUNY at Stony Brook. ‡ Kyoto University. § Present address: Department of Pharmacy, Wonkwang University, Iksan Chonbuk 570-709, South Korea. 1 Abbreviations: HRT, hormone replacement therapy; EQ, equilin; EN, equilenin; 4-OHEQ, 4-hydroxyequilin; 4-OHEN, 4-hydroxyequilenin; 8-oxodG, 8-oxo-7,8-dihydro-2′-deoxyguanosine; dNTP, 2′-deoxynucleoside triphosphate; ROS, reactive oxygen species; Xpc, xeroderma pigmentosa complementation group C; PAGE, polyacrylamide gel electrophoresis; HPLC, high-performance liquid chromatography; LC/MS/MS, liquid chromatography electrospray tandem mass spectrometry.

derived DNA adducts have been detected in the mammary fat pads of rats treated with 4-OHEN (12), in breast tumor and adjacent normal tissues of several patients receiving HRT, and in paraffin-embedded breast tumor tissues (13). Redox cycling between the o-quinone of 4-OHEN and its semiquinone radical generates reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and ultimately reactive hydroxyl radicals (14) (Figure 1). When 4-OHEN was incubated with DNA or exposed to breast cancer cells in culture, increased formation of 7,8-dihydro-8-oxodeoxyguanosine (8-oxodG) lesions, a typical marker of oxidative DNA damage, was detected (14–18). Similar phenomena were observed when 4-OHEN was injected directly into the mammary fat pads of rats (12). 8-OxodG is a miscoding and mutagenic lesion (19–21). Therefore, oxidative DNA damage induced by equine estrogens may also be involved in carcinogenesis. If equine estrogens produce mutagenic DNA lesions in breast, ovarian, and endometrial tissues of women receiving HRT, such mutagenic DNA adducts may contribute to the initiation of these cancers. The equine estrogen-derived DNA adducts therefore provide biomarkers useful in evaluating the risk of HRT on an individual basis. Xpc-knockout (Xpc-KO) mice are deficient in both alleles of mouse xeroderma pigmentosum complementation group C, one of several factors involved in the recognition of a variety of bulky DNA-distorting lesions in nucleotide excision repair (22). Recently, a reduction in the rate of repair of 8-oxodG was observed in human keratinocytes and fibroblasts lacking Xpc, indicating that Xpc plays an unexpected and multifaceted role in cell protection from oxidative DNA damage (23). To explore the oxidative DNA damage induced by equine estrogens in animals, female mice were treated orally with EN. The levels of 8-oxodG in the liver and uterus were determined

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Equilenin-OxidatiVe DNA Damage

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Figure 1. DNA damage and redox cycling mediated through 4-OHEN.

using liquid chromatography electrospray tandem mass spectrometry (LC/MS/MS). Liver was chosen as a major organ metabolizing EN, and uterus was chosen as one of target reproductive organs developing cancer. To evaluate the contribution of Xpc in the removal of oxidative DNA damage, EN was also administered to Xpc-KO mice. The level of 8-oxodG observed in Xpc-KO mice was compared with that observed in wild-type B6129F1 mice.

Materials and Methods Materials. EN was purchased from Steraloids Inc. (Wilton, NH). 4-OHEN was prepared as described previously (10). Desferrioxamine and alkaline phosphatase were obtained from Sigma-Aldrich (St. Louis, MO). 15N5-dG was purchased from Cambridge Isotope Laboratory (Andover, MA). Nuclease P1 was obtained from Roche Applied Science (Indianapolis, IN). Animal Studies. The use of animals was in compliance with the guidelines established by the NIH Office of Laboratory Animal Welfare. Xpc-KO mice and mice of the wild-type progenitor strain, B6129F1 (female, 6 weeks old), were purchased from Taconic (Germantown, NY). Animals were acclimated in temperature (22 ( 2 °C) and humidity (55 ( 5%) controlled rooms with a 12 h light-dark cycle for at least 1 week prior to use. Regular laboratory chow and tap water were allowed ad lib. Mice were treated orally (p.o.) with EN (5 or 50 mg/kg/day) for 1 or 2 weeks. Control mice were treated with an identical volume of corn oil. Mice were euthanized by CO2 asphyxiation at 24 h after the final treatment and then subjected to open thoracotomy. The liver and uterus were removed quickly, frozen, and stored at -80 °C until DNA extraction. Preparation of DNA Samples. DNA was extracted from the tissue by a modified method of Wang et al. (24). To prevent

artifactual 8-oxodG formation during DNA extraction, desferrioxamine was used as an antioxidant, as demonstrated in interlaboratory validation study organized by the European Standards Committee on Oxidative DNA Damage (25). The tissue (100 mg) was homogenized at 4 °C in 0.2 mL of lysis solution A (320 mM sucrose, 5 mM MgCl2, 10 mM Tris-HCl, 0.1 mM desferrioxamine, pH 7.5, and 1% Triton X-100) using a disposable pellet pestle (Kimble/Kontes, Vineland, NJ). After the tissue was ground, the homogenate was subjected to centrifugation at 10000g for 20 s at 4 °C. One milliliter of lysis solution A was added to the pellet and agitated by vortexing. These steps were repeated twice. After centrifugation, 200 µL of solution B (10 mM Tris-HCl, 5 mM EDTA-Na2, and 0.15 mM desferrioxamine, pH 8.0) and 20 µL of 10% SDS were added to the pellet. The mixture was vortexed for 10 s and incubated at 37 °C for 10 min for suspension of the pellet and to allow for complete lysis of the nuclear membrane. After the addition of 2.7 µg of RNase T1 and 10 µg of RNase A in a buffer (10 mM Tris-HCl, 1 mM EDTA, and 2.5 mM desferrioxamine, pH 7.4), the samples were incubated at 50 °C for 15 min. Following incubation, 10 µL of Qiagen proteinase K solvent was added, and the samples were incubated for 1 h at 37 °C. After the addition of 0.3 mL of NaI solution (7.6 M NaI, 40 mM Tris-HCl, 20 mM EDTA-Na2, and 0.3 mM desferrioxamine, pH 8.0), DNA was precipitated by the addition of 500 µL of 2-propanol. The DNA lump was fished out and washed twice with 40% 2-propanol. Finally, the recovered DNA was dried and dissolved in 500 µL of distilled water. The concentration of DNA was determined by UV spectroscopy as 50 µg/mL ) O.D.260nm 1.0. Determination of 8-OxodG by LC/MS/MS Analysis. The level of 8-oxodG was determined using LC/MS/MS [high-performance liquid chromatography (HPLC), a Shimadzu LC-10ADvp pump and SIL-10AD autoinjector; MS/MS, Waters-Micromass Quattro Ultima Pt Triple Quadrupole mass spectrometer]. To quantify

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8-oxodG accurately, 15N5-8-oxodG was prepared from 15N5dG, whose all five Ns in the guanine base were replaced by 15N (Cambridge Isotope Laboratory), following a method developed previously (26), and used as an internal standard. The DNA (25 µg) was mixed with 15N5-8-oxodG (500 pg) and digested at 37 °C for 3 h with nuclease P1 (4 unit) in 114 µL of buffer mixture [100 µL of 30 mM sodium acetate buffer containing 10 mM 2-mercaptoethanol, pH 5.3; 5 µL of 20 mM ZnSO4; 5 µL of 15 N5-8-oxodG solution (5 ng/mL); 4 µL of nuclease P1 solution (1 unit/µL); and 3 units of alkaline phosphatase]. After incubation, 20 µL of 0.5 M Tris-HCl, pH 8.5, was added and incubated for 3 h. The enzymes were methanol-precipitated, and the supernatant containing the nucleoside was evaporated and reconstituted with 100 µL of water. The LC column was eluted over a gradient that began at a ratio of 2% methanol to 98% water and was changed to 40% methanol over a period of 40 min, changed to 80% methanol from 40 to 45 min, and finally returned to the original starting conditions, 2:98, for the remaining 15 min. The total run time was 60 min. Sample injection volumes of 50 µL each were separated on a Shim-pack FC-ODS column (150 mm × 4.6 mm) and eluted at a flow rate of 0.4 mL/min. Mass spectral analyses were carried out in positive ion mode with nitrogen as the nebulizing gas. The ion source temperature was 130 °C, the desolvation gas temperature was 380 °C, and the cone voltage was operated at a constant 40 V. Nitrogen gas was also used as the desolvation gas (700 L/h) and cone gas (35 L/h), and argon was used as the collision gas at a collision cell pressure of 1.5 × 10-3 mBar. Positive ions were acquired in MRM mode. The MRM transitions were monitored as follows: 15N5-8oxodG (m/z 288.8 f 172.8) and 8-oxodG (m/z 283.8 f 167.8), respectively. The level of 8-oxodG in the DNA samples was determined by comparing with 15N5-8-oxodG. The detection limit was approximately 0.1 adducts in 106 bases using 25 µg of DNA. The amount of dG in the DNA digest was monitored by a Simadzu SPD-10A UV-visible detector prior to MS/MS analysis. The level of 8-oxodG lesions was estimated by the following equation: the level of 8-oxodG lesion ) (amount of 8-oxodG)/[(amount of dG) × 4]. Statistical Analysis. Results were expressed as means ( standard deviations (SDs). A two-sided student’s t test was used to evaluate the difference. Values of p e 0.05 were considered statistically significant from the control group.

Okamoto et al.

Figure 2. LC/MS/MS analysis of 8-oxodG. The DNA (25 µg) was mixed with 15N5-8-oxodG (500 pg) and digested with nuclease P1 and alkaline phosphatase in a buffer. The resulting nucleosides were analyzed using LC/MS/MS, as described in the Materials and Methods. Quantitative analysis of 15N5-8-oxodG and 8-oxodG was achieved by monitoring the MS/MS transitions corresponding to the loss of deoxyribose from 15N5-8-oxodG (m/z 288.8 f 172.8) and 8-oxodG (m/z 283.8 f 167.8), respectively. By comparing with internal standard 15 N5-8-oxodG (A), the level of 8-oxodG in hepatic DNA samples from Xpc-KO mouse treated with a vehicle (B) or EN (5 mg/kg/day, p.o. for 2 weeks) (C) was determined.

Table 1. Levels of Hepatic and Uterine 8-OxodG in Xpc-KO and Wild-Type Mice Treated Orally with EN 8-oxodG (adducts/106 dNs) dose (mg/kg)

Results The level of 8-oxodG lesions in B6129F1 mice treated p.o. with EN was determined using LC/MS/MS analysis and an internal standard, 15N5-8-oxodG (Figure 2). The detection limit for 25 µg of DNA sample was approximately 0.1 adduct/106 nucleotides. The level of 8-oxodG in the livers of mice treated with 5 mg/kg EN was significantly increased, 220% after 1 week (6.3 adducts/106 nucleotides) and 170% after 2 weeks (4.9 adducts/106 nucleotides), as compared with that of mice treated with vehicle (2.9 adducts/106 nucleotides) (Table 1). With 50 mg/kg EN treatment, a high level of hepatic 8-oxodG (222%) (p < 0.01) was also observed after 1 week; however, no significant increase was detected after 2 weeks. In the uterus, the background level of 8-oxodG was 3.9 times higher than that observed in the liver (Table 1). Although no significant difference was observed after 1 week of treatment with 5 and 50 mg/kg EN, the levels of uterine 8-oxodG were significantly increased after 2 weeks of treatment, to 150 and 162%, respectively, of the background level. When Xpc-KO mice were treated for 1 week with 5 and 50 mg/kg EN, the hepatic 8-oxodG levels increased 155 and 283%, respectively (Table 1); however, no significant increase of 8-oxodG was detected after 2 weeks, as observed with the wild-type mice. The increases of 8-oxodG level in the wild-type and Xpc-KO mice treated for 1 week with 50 mg/

control EN

5.0 50

control EN

5.0 50

duration (weeks)

wild type

Xpc-KO

1 2 1 2

liver 2.9 ( 0.5a (100)b 6.3 ( 1.6* (220) 4.9 ( 2.0* (170) 6.4 ( 1.4** (222) 3.4 ( 1.4 (117)

4.8 ( 2.4a (100)b 7.4 ( 3.7 (155) 5.3 ( 0.9 (110) 13.6 ( 0.4** (283) 7.7 ( 3.9 (160)

1 2 1 2

uterine 11.2 ( 2.2a (100)b 12.9 ( 1.3 (115) 16.8 ( 3.9** (150) 11.8 ( 1.1 (105) 18.1 ( 5.4* (162)

9.9 ( 4.1a (100)b 12.1 ( 1.3 (122) 15.7 ( 0.7* (159) 10.9 ( 1.6 (110) 19.1 ( 6.4* (193)

a Data are expressed as mean values ( SD from three mice in the EN-treated groups and six mice in the control groups. t test: *p < 0.05 and **p < 0.01. b The numbers in brackets are % values relative to the control level.

kg EN were 3.5 ( 1.0 and 8.8 ( 0.4 adducts/106 nucleotides, respectively, when their control levels were subtracted. The 8-oxodG increase in Xpc-KO mice was 2.5 times higher than that observed in the wild-type mice (p < 0.01). The levels of uterine 8-oxodG were significantly increased to 159 (15.7 adducts/106 nucleotides) and 193% (19.1 adducts/106 nucleotides) after 2 weeks of treatment with 5 and 50 mg/kg EN, as observed with the wild-type mice.

Equilenin-OxidatiVe DNA Damage

Discussion Many chemicals, including polycyclic aromatic hydrocarbons and estrogens, are metabolically activated to form quinones. ROS such as superoxide, hydrogen peroxide, and, ultimately, reactive hydroxyl radicals are generated through redox cycling between the o-quinone and its semiquinone radical and attack cellular macromolecules, including DNA, to generate oxidative DNA damage such as 8-oxodG (27). Similarly, 4-OHEN, a major metabolite of EN, autoxidizes to the o-quinone without enzymatic or metal ion catalysis (7). ROS generated during redox cycling between 4-OHEN-o-quinones and their semiquinones cause oxidative DNA damage; indeed, increased formation of 8-oxodG was observed when calf thymus DNA was reacted directly with 4-OHEN (14). In mice treated orally with EN, significant increases of 8-oxodG were observed in the liver and uterus using LC/MS/MS (Table 1). A similar result was observed when 4-OHEN was injected directly into the mammary fat pads of rats (12). These results indicated that oxidative DNA damage occurs in reproductive organs. ROS generated by equine estrogen and its metabolite may contribute to the process of equine estrogen carcinogenesis. Women receiving HRT take Premarin at a dose 0.625 (∼0.01 mg/kg/day) or 1.25 mg/day (∼0.021 mg/kg/day). Because equine estrogens present in Premarin are approximately 40%, women get equine estrogens 0.004 or 0.0084 mg/kg/day. Although the doses (5.0 and 50 mg/kg/day) used in this animal experiment were in excess than the daily dose for women, women receive HRT over 5 years. Therefore, to determine the capability of forming EN-induced oxidative DNA damage in a short period (1 or 2 weeks), high doses were used in this experiment, as reported that other research groups treated a high dose of 4-OHEN (3.1 mg/kg) to rats (12). Although the increases of hepatic and uterine 8-oxodG level were observed with both the wild-type and the Xpc-KO mice, the 8-oxodG level for 50 mg/kg EN treatment was similar or slightly higher than that observed with 5 mg/kg EN treatment. Such excessive EN doses may give overcapacity for the metabolizing enzymes to produce 4-OHEN, a precursor of forming ROS. The high level of hepatic 8-oxodG observed after 1 week of treatment (5.0 or 50 mg/kg) was subsequently decreased at 2 week. The decrease of hepatic 8-oxodG over time may occur by repair enzymes induced after EN treatment. Our results encourage treating animals for longer periods with low EN doses for predicting the capability of forming oxidative damage in women. Xpc is a factor involved in the recognition of a variety of bulky DNA-distorting lesions in nucleotide excision repair (22) and may also be associated with repair of 8-oxodG (23). If Xpc is involved in the repair of 8-oxodG, then the lesions should remain in the tissues of Xpc-KO mice for considerably longer than in the tissues of wild-type mice. When EN was administered to both Xpc-KO and wild-type mice, a significantly higher formation of 8-oxodG was detected only in liver of Xpc-KO mice treated with 50 mg/kg; no significant difference in 8-oxodG levels was observed between them with other doses and/or tissues (Table 1). Our results indicate that Xpc may not have a primary role in the repair of 8-oxodG. In Xpc-KO and wild-type mice treated only with the vehicle, the levels of 8-oxodG in the uterus were 3.9 and 2.1 times, respectively, higher than that observed in the liver. The uterus is a principal producer of steroid hormones, including estrogen. Estrogen metabolites are known to produce free radicals during redox cycling, generating oxidative DNA damage, including

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8-oxodG (28). ROS generated by steroid metabolites may be the cause of the increased background level of 8-oxodG in the uterus. In conclusion, our results indicate that oxidative DNA damage induced by equine estrogen in reproductive organs may contribute to the process of equine estrogen carcinogenesis and increase the risk of developing reproductive cancer in women receiving HRT. Acknowledgment. This study was supported by Grant ES012408 from the National Institute of Environmental Health Sciences, Grants-in-Aid for scientific research 18101003 from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and Grants-in-Aid for scientific research from the Ministry of Health, Labor and Welfare of Japan.

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