Oxidative DNA Damage and Mammary Cell Proliferation by Alcohol

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Oxidative DNA Damage and Mammary Cell Proliferation by AlcoholDerived Salsolinol Mariko Murata,*,† Kaoru Midorikawa,† and Shosuke Kawanishi†,‡ †

Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Mie 513-8670, Japan

‡

ABSTRACT: Drinking alcohol is a risk factor for breast cancer. Salsolinol (SAL) is endogenously formed by a condensation reaction of dopamine with acetaldehyde, a major ethanol metabolite, and SAL is detected in blood and urine after alcohol intake. We investigated the possibility that SAL can participate in tumor initiation and promotion by causing DNA damage and cell proliferation, leading to alcoholassociated mammary carcinogenesis. SAL caused oxidative DNA damage including 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), in the presence of transition metal ions, such as Cu(II) and Fe(III)EDTA. Inhibitory effects of scavengers on SAL-induced DNA damage and the electron spin resonance study indicated the involvement of H2O2, which is generated via the SAL radical. Experiments on scavengers and site specificity of DNA damage suggested ·OH generation via a Fenton reaction and copper-peroxide complexes in the presence of Fe(III)EDTA and Cu(II), respectively. SAL significantly increased 8-oxodG formation in normal mammary epithelial MCF-10A cells. In addition, SAL induced cell proliferation in estrogen receptor (ER)-negative MCF-10A cells, and the proliferation was inhibited by an antioxidant N-acetylcysteine and an epidermal growth factor receptor (EGFR) inhibitor AG1478, suggesting that reactive oxygen species may participate in the proliferation of MCF-10A cells via EGFR activation. Furthermore, SAL induced proliferation in estrogen-sensitive breast cancer MCF-7 cells, and a surface plasmon resonance sensor revealed that SAL significantly increased the binding activity of ERα to the estrogen response element but not ERβ. In conclusion, SAL-induced DNA damage and cell proliferation may play a role in tumor initiation and promotion of multistage mammary carcinogenesis in relation to drinking alcohol.

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INTRODUCTION Epidemiological studies showed a positive association between alcohol intake and the risk of breast cancer.1,2 Nelson et al. have recently demonstrated that the majority of alcohol-attributable female cancer deaths were from breast cancer (56% to 66%) in the United States.3 Since many breast cancer deaths are attributable to alcohol consumption, they strongly suggest that greater emphasis on the role of alcohol as an important risk factor for breast cancer is needed.3 International Agency for Research on Cancer (IARC)4 has evaluated that alcoholic beverages are carcinogenic to humans (Group 1) and that the occurrence of malignant tumors including those in the female breast is related to the consumption of alcoholic beverages. Recently, “acetaldehyde associated with consumption of alcoholic beverages” was also classified as Group 1 (carcinogenic to human).5 Oxidative stress is an essential mechanism of alcohol-associated carcinogenesis,6,7 whereas acetaldehyde forms DNA adduct. 8 Carcinogenic mechanisms of alcoholassociated breast cancer are not fully elucidated. Salsolinol (SAL) is endogenously generated by the condensation reaction between dopamine and acetaldehyde. SAL is detected in blood and urine after alcohol intake9,10 and positively related with blood aldehyde level.11 It is suggested by Collins et al.12 that © 2013 American Chemical Society

the urinary SAL after drinking alcohol reflects the trapping of peripheral dopamine by acetaldehyde and that the dopamine is not from the central nervous system because dopamine originating from the central nervous system cannot cross the blood−brain barrier. A recent review13 has indicated that peripheral dopamine is released from neuronal cells in peripheral tissues and that dopamine released from sympathetic nerves predominantly contributes to plasma dopamine levels. Therefore, SAL may be formed also in mammary tissues after drinking, so we need to pay attention to the possibility that SAL can participate in alcohol-associated breast carcinogenesis. To examine whether SAL can affect tumor initiation and promotion in relation to mammary carcinogenesis, we investigated DNA damage and cell proliferation induced by SAL using the cultured human nontumorigenic mammary epithelial MCF-10A cells and estrogen-dependent breast cancer MCF-7 cells. Formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative damage, in MCF-10A cells treated with SAL was measured by using an electrochemical detector coupled to HPLC (HPLC-ECD). To Received: May 17, 2013 Published: September 10, 2013 1455

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sensor.17 The running buffer used for immobilization and the binding assay was 25 mM Tricine, 160 mM KCl, 5 mM MgCl2, and 0.05% Tween 20 (pH 7.8). The biotinylation of oligonucleotide with sequence of human pS2 ERE was for immobilization to the streptavidin-treated sensor chip. The complementary oligonucleotide was annealed to the immobilized ERE. Human ERα and ERβ (2 × 10−7 M) were liganded with 10−7 M E2 or 10−5 M SAL by the incubation at 37 °C for 5 min. Then, the liganded ER was injected over the surfaces coated with double-stranded ERE via a sample loop, as described previously.14 The binding activity of liganded ER to ERE was expressed as % activity, that is, binding response with 100 nM E2 as 100% and that without chemical (DMSO 0.1%) as 0%. All samples contained 0.1% DMSO. Measurement of 8-oxodG in DNA of Cultured Human Mammary Epithelial Cells Treated with SAL. MCF-10A cells were trypsinized, and 5 × 105 cells were plated into a dish of 10 cm in diameter with seeding medium. Cells were allowed to attach and grow until 70−90% confluency for 3−4 days. Then, cells were treated with SAL at 37 °C for 2 h and trypsinized and washed three times with cold PBS. DNA was extracted by the modified method of Wang et al.,18 digested to component nucleosides with nuclease P1 and bacterial alkaline phosphatase, and analyzed by HPLC-ECD as previously described.19 Comet Assay in MCF-10A Cells Treated with SAL. To assess the relative yields of 8-oxodG and strand breaks, alkali comet assays in single cells with or without human 8-oxoguanine DNA glycosylase 1 (hOGG1) were performed according to instructions provided by the manufacturer (hOGG1 FLARE Assay Kit in conjunction with Comet Assay single cell gel electrophoresis kits, Trevigen). MCF-10A cells were incubated with 100 μM SAL at 37 °C for 1 h. The treated cells (3 × 103 cells) were suspended in 75 μL of low melting point agarose and placed on comet slides. After solidification of agarose, the slides were immersed in lysis solution at 4 °C for 1 h. Following lysis, slides were incubated with hOGG1 at 37 °C for 45 min. The slides were placed in a horizontal gel electrophoresis unit and incubated in alkali solution (0.3 M NaOH and 1 mM EDTA, pH > 13) for 20 min to allow DNA unwinding. Electrophoresis (1 V/cm) was performed in alkali solution for 20 min at 4 °C. The slides were immersed in ethanol (5 min), airdried, and stained with SYBR Gold Nucleic Acid Gel Stain (Molecular Probes Inc., Eugene, OR). The cells were observed under fluorescence microscopy (BX53, Olympus). Detection of DNA Damage Caused by SAL in the Presence of Metal Ions. 5′-End 32P-labeled DNA fragments were prepared as described previously.19−21 The standard reaction mixture contained SAL at various concentrations, 5′-end 32P-labeled DNA fragments, calf thymus DNA (20 μM per base), and 20 μM metal ions in 200 μL of 10 mM sodium phosphate buffer (pH 7.8) containing 5 μM DTPA. After incubation at 37 °C for 1 h, the DNA fragments were heated at 90 °C in 1 M piperidine for 20 min where indicated and treated as described previously.22 The preferred cleavage sites were determined by direct comparison of the positions of the oligonucleotides with those produced by the chemical reactions of the Maxam−Gilbert procedure23 using a DNA-sequencing system (LKB 2010 Macrophor, Pharmacia Biotech, Uppsala, Sweden). The relative amounts of oligonucleotides from the treated DNA fragments were measured with a laser densitometer (LKB 2222 UltroScan XL, Pharmacia Biotech). Analysis of Formation of 8-oxodG in Calf Thymus DNA by SAL. Calf thymus DNA fragments (100 μM) and 20 μM metal ions were incubated with SAL in 400 μL of 4 mM sodium phosphate buffer (pH 7.8) containing 5 μM DTPA at 37 °C for 1 h. After ethanol precipitation, DNA fragments were digested to individual nucleosides with nuclease P1 and calf intestine alkaline phosphatase, and analyzed with an HPLC-ECD. ESR Spectroscopic Measurements. ESR spectra using a JES-FE3XG spectrometer (JEOL) with 100 kHz field modulation detect the free radicals derived from SAL. The spectra were recorded utilizing a microwave power of 4 mW and a modulation amplitude of 0.05 mT.

elucidate the molecular mechanism of DNA damage, we measured 8-oxodG formation in calf thymus DNA and examined DNA damage using 5′-end 32P-labeled DNA fragments obtained from genes relevant to human cancer. We also analyzed radical species from SAL by the electron spin resonance (ESR) technique. We examined the effects on cell proliferation by the modified method of E-screen assay. Furthermore, to study interaction between SAL-liganded estrogen receptor (ER) and estrogen response element (ERE), we measured binding affinity by using the surface plasmon resonance (SPR) sensor.

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MATERIALS AND METHODS

Materials. SAL was from Toronto Research Chemicals Inc. (North York, ON, Canada). N-Acetylcysteine (NAC), CuCl2, Tween 20, and Fe(III)EDTA sodium salt were from Nacalai Tesque (Kyoto, Japan). Calf thymus DNA, alkaline phosphatase, superoxide dismutase (SOD, 3000 units/mg from bovine erythrocytes), catalase (45000 units/mg from bovine liver), phenol red free DMEM, insulin, hydrocortisone, charcoal (activated), and 4-hydroxytamoxifen (4-OH-tamoxifen) were from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum (FBS), horse serum (HS), epidermal growth factor (EGF), Dulbecco’s modified Eagle’s medium (DMEM), and DMEM/Ham’s F12 medium were purchased from Gibco (Grand Island, NY). 17-β-Estradiol (E2) was obtained from Calbiochem-Novabiochem Corporation (La Jolla, CA). AG1478 was obtained from Calbiochem (San Diego, CA). Nuclease P1 (400 units/mg), dimethyl sulfoxide (DMSO), and kanamycin sulfate were from Wako Chemical Co. (Osaka, Japan). LGlutamine was from ICN Biomedicals Inc. (Aurora, OH). Dextran T70 was from Pharmacia Biotech (Uppsala, Sweden). The BIAcore sensor chips SA (modified with streptavidin) were obtained from Biacore Inc. (Uppsala, Sweden). Human recombinant estrogen receptor α (ERα) and estrogen receptor β (ERβ) were obtained from Panvera (Madison, WI). Cell Culture. Human estrogen-sensitive breast cancer MCF-7 cells (ATCC No. HTB 22) and nontumorigenic mammary epithelial MCF10A cells (ATCC No. CRL 10317) were obtained from American Type Culture Collection. For routine maintenance, cells were grown in seeding medium (MCF-7 cells, DMEM supplemented with 100 ng/ mL kanamycin, and 5% FBS; MCF-10A cells, DMEM/F12 supplemented with 20 ng/mL EGF, 0.01 mg/mL insulin, 500 ng/ mL hydrocortisone, 100 ng/mL kanamycin, and 5% HS) at 37 °C in a humidified atmosphere of 5% CO2. Sex steroids in serum were removed by charcoal-dextran treatment for experimental medium by the method reported previously.14 The experimental medium was phenol red free medium supplemented with 5% charcoal-dextranserum, 100 ng/mL kanamycin, and 4 mM L-glutamine. The singlestranded biotinylated oligonucleotide (35mer, HPLC grade) with the sequence of human pS2 estrogen response element (ERE)15 (5′XGTCCAAAGTCAGGTCACGGTGGCCTGATCAAAGTT-3′, X indicates biotin-labeled) and the complementary unbiotinylated oligonucleotide (35mer, HPLC grade) were obtained from Invitrogen Corporation (Carlsbad, CA). Bioassay for Measuring Cell Proliferating Activity. The modified method of the E-screen assay16 was performed to measure cell proliferating activity. Briefly, MCF-7 cells and MCF-10A cells were trypsinized and plated into 12-well plates at an initial concentration of 3 × 104 cells per well with seeding medium. After the cells were allowed to attach for 24 h, the seeding medium was replaced with experimental medium. SAL and other compounds were added. The final solvent concentration in culture medium did not exceed 0.1% DMSO, as this concentration did not affect cell yields. The control condition also contained 0.1% DMSO. Cells were incubated for six days, and then trypsinized and harvested. Harvested cells were counted using a Coulter counter (Beckman Coulter, Tokyo, Japan). Analysis of ER-ERE Binding. The BIAcore-biosensor system (Biacore X, Pharmacia Biosensor, Uppsala, Sweden) permits the monitoring of macromolecular interaction in real time using an SPR 1456

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Figure 1. Autoradiogram of 32P-labeled DNA fragments incubated with SAL in the presence of metal ions. (A) The reaction mixture contained the 5′-end 32P-labeled 309-bp fragment from the human p16 tumor suppressor gene, 20 μM per base of calf thymus DNA, 100 μM SAL, and 20 μM metals in 10 mM sodium phosphate buffer (pH 7.8) containing 5 μM DTPA. (B,C) The reaction mixture contained the 5′-end 32P-labeled 337-bp fragment from the human c-Ha-ras-1 proto-oncogene, 20 μM per base of calf thymus DNA, (A) 5 μM SAL, and 20 μM CuCl2 or (B) 100 μM SAL and 20 μM Fe(III)EDTA, and a scavenger in 10 mM sodium phosphate buffer (pH 7.8) containing 5 μM DTPA. The concentrations of scavengers were as follows: 5% (v/v) ethanol; 0.1 M mannitol; 0.1 M sodium formate; 5% (v/v) DMSO; 0.1 M methional; 30 units of catalase; 30 units of heatinactivated catalase; 30 units of SOD; 20 or 50 μM bathocuproine; and 1 mM deferoxamine. After the incubation at 37 °C for 1 h, DNA fragments were treated with 1 M piperidine for 20 min at 90 °C and then electrophoresed on an 8% polyacrylamide/8 M urea gel. The autoradiogram was visualized by exposing an X-ray film to the gel.

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RESULTS Damage to 32P-Labeled DNA Fragments Induced by SAL in the Presence of Metal Ions and Effects of Scavengers. Figure 1A shows the autoradiogram of DNA fragments treated with SAL in the presence of various transition metals. Oligonucleotides from 32P-labeled DNA fragments were detected on the autoradiogram as a result of DNA damage. SAL induced DNA damage in the presence of Cu(II) and Fe(III)EDTA (Figure 1A). However, SAL did not induce DNA damage in the presence of Fe(III), Fe(III)citrate, Co(II), Ni(II), Mn(II), or Mn(III). Cu(II)-mediated DNA damage by SAL was much stronger than Fe(III)EDTA-mediated DNA damage. To speculate reactive species, we examined the effects of scavengers and metal chelators on DNA damage by SAL in the presence of metal ions. In the presence of Cu(II) (Figure 1B), typical free hydroxyl radical (·OH) scavengers such as ethanol, mannitol, sodium formate, and DMSO, showed no inhibitory effect on DNA damage, whereas methional inhibited the DNA damage. Catalase and bathocuproine, a Cu(I)-specific chelator, inhibited DNA damage, suggesting the involvement of H2O2 and Cu(I). It is reported that methional can scavenge both ·OH and the species like the Cu(I)-hydroperoxo complex that are less reactive than ·OH.24 These results indicate that the primary reactive species in Cu(II)-mediated DNA damage may not be · OH but a copper−oxygen complex such as the Cu(I)hydroperoxo complex [Cu(I)OOH]. In the case of Fe(III)EDTA-mediated DNA damage (Figure 1C), the inhibitory effects of free ·OH scavengers, methional, catalase, and deferoxamine, an iron chelating agent, were observed, indicating the involvement of ·OH through a Fenton reaction.

On the contrary, SOD enhanced SAL-induced DNA damage in the presence of either metal ion (Figure 1B and C). Site Specificity of DNA Damage by SAL. The preferred cleavage sites were determined by the modified method of the Maxam−Gilbert procedure, to clarify the site specificity of DNA damage by the reactive species. SAL caused Cu(II)mediated DNA cleavage frequently at thymine and cytosine residues (Figure 2A). However, SAL plus Fe(III)EDTA caused DNA cleavage at every nucleotide position without a marked site specificity (Figure 2B), suggesting the involvement of free · OH. It is well known that ·OH is a highly reactive species and directly abstracts a hydrogen atom from the DNA deoxyribosephosphate backbone, resulting in DNA cleavage at every nucleotide without a marked site specificity.24,25 However, site specificity of Cu(II)-mediated DNA damage may be determined by binding sites of Cu(II) in DNA since it can be speculated that Cu(II) bound to DNA at specific sites is reduced to Cu(I) to react with H2O2, resulting in the formation of the DNA-Cu(I)OOH complex, which immediately attacks DNA constituents adjacent to copper-binding sites before being scavenged by ·OH scavengers.25,26 Detection of SAL Radical. To confirm radical formation from SAL, the ESR spin-stabilization method using Zn(II)27 was applied to detect semiquinone radical formation from SAL without any spin-trapping agents such as DMPO. SAL produced a quartet 1:3:3:1 ESR signal in the presence of Zn(II) (Figure 3). The hyperfine splitting constant of the radical was aH = 0.56 mT. This radical is assigned as a SAL semiquinone radical, which has three equivalent hydrogens at C1, C5, and C8 positions, taking into consideration the hyperfine constants reported by Martinez-Alvarado et al.28 1457

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Figure 2. Site specificity of DNA damage induced by SAL in the presence of Cu(II) and Fe(III)EDTA. (A,B) The 5′-end 32P-labeled 343-bp fragment from the human p53 tumor suppressor gene in 10 mM sodium phoshate buffer at pH 7.8 containing 5 μM DTPA and 20 μM/base of calf thymus DNA was incubated with (A) 50 μM SAL, 20 μM CuCl2, or (B) 100 μM SAL and 20 μM Fe(III)EDTA at 37 °C for 1 h. The DNA fragments were analyzed as described in the legend to Figure 1. The relative amounts of oligonucleotides were measured by scanning the autoradiogram with a laser densitometer.

cultured human nontumorigenic mammary MCF-10A cells treated with SAL. The amount of 8-oxodG in calf thymus DNA increased with increasing concentrations of SAL in the presence of Cu(II) or Fe(III)EDTA (Figure 4A). Interestingly, SOD and Mn(II), which has SOD-mimic activity, enhanced SAL-induced DNA damage in the presence of either metal ion (Figure 4B). SAL significantly increased 8-oxodG formation in MCF-10A

Addition of H2O2 enlarged the signal, and the formation of the SAL radical was further enhanced in the presence of copper and iron ions. These results indicate that SAL can induce oxidative DNA damage in the presence of metal ions. Oxidative Damage to Cellular and Isolated DNA Treated with SAL. By using HPLC-ECD, we measured 8oxodG formation in isolated DNA and cellular DNA from 1458

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Figure 3. Detection of radicals derived from SAL in the presence of Zn(II). The reaction mixture contained 100 mM SAL and 250 mM ZnSO4 in the presence and absence of 200 mM H2O2, 4 mM CuCl2, or Fe(III)EDTA in 500 mM acetate buffer (pH 5.2). The ESR spectra were measured immediately after mixing. Spectra were recorded with a microwave power of 4 mW, a modulation amplitude of 0.05 mT, and a receiver gain of 1000.

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DISCUSSION The present study showed the possibility that SAL may play a role in the initiation and promotion of alcohol-related breast cancer through oxidative DNA damage and cell proliferation in human mammary cell lines. The proposed mechanisms of SALinduced DNA damage and cell proliferation are shown in Figure 7. We showed that SAL induced oxidative DNA damage in a dose-dependent manner in the presence of Cu(II) or Fe(III)EDTA. Iron is the most abundant transition metal in chromosome systems.29 This raises a possibility of specific metal complex with endogenous compounds, which is a redoxactive transition metal inside the cell.30 Chronic alcohol intake is associated with increased accumulation of iron.31 In addition, our result showed that Mn(II) enhanced metal-mediated DNA damage. Several metals including copper, iron, and manganese are known to be potent mutagens and carcinogens.32,33 In the presence of metal ions, oxidation of SAL to the semiquinone radical occurs with simultaneous reduction of metal (Mn; Cu(II) and Fe(III)) to Mn−1 (Cu(I) and Fe(II)). The semiquinone radical is further oxidized to the o-benzoquinone derivative with the formation of O2·−, which is dismutated to H2O2. Fe(II) makes H2O2 convert to ·OH via the Fenton reaction. Cu(I) may form a metal-hydroperoxo complex, such as Cu(I)OOH. The antioxidant enzyme SOD enhanced DNA damage instead of defense against oxidative stress. Several papers showed that SOD enhanced oxidative DNA damage.34,35 Medinas et al.36 demonstrated that Cu,Zn-SOD had peroxidase activity and that it was enhanced in bicarbonate buffer. We compared enhancing effect of SOD on 8-oxodG formation between bicarbonate buffer and phosphate buffer, but there was no significant difference (2.3-fold increase in bicarbonate buffer and 3.1-fold increase in phosphate buffer), suggesting little peroxidase activity under the condition used. Therefore, the enhancing effect is probably due to SOD-catalyzed con-

cells (Figure 4C). Alkaline comet assay without hOGG1 showed that SAL slightly increased the cells with a comet tail compared to those in the control, and hOGG1 treatment revealed the increase of damaged cells with 8-oxodG formation (Figure 4D). Cell Proliferating Activity of SAL in EstrogenIndependent MCF-10A Cells. To examine the activity of estrogen-independent cell proliferation, we used the human mammary cell line MCF-10A with neither ERα nor ERβ. SAL induced cell proliferation in MCF-10A cells, whereas E2 did not. SAL showed significant cell proliferation starting at 1 μM (data not shown) with maximal proliferating activity at 100 μM (P < 0.01) about 2-fold compared to that of the control (Figures 5A,B). N-Acetylcysteine (NAC), an antioxidant, significantly attenuated cell proliferating activity (Figure 5A), and epidermal growth factor receptor (EGFR) inhibitor, AG1478, efficiently inhibited cell proliferation (Figure 5B). Proliferating Activity of SAL in MCF-7 Cells and Binding Ability of Liganded ER to ERE. To examine the activity of cell proliferation via ER, cell number was counted using estrogen-dependent human breast cancer cell line MCF-7 treated with SAL. SAL induced maximal proliferating activity at 10 μM (P < 0.01) with significant differences relative to the solvent (0.1% DMSO) control (data not shown), and the intensity of maximal cell proliferating activity of SAL was about 50% of 100 pM 17β-estradiol (E2) in MCF-7 cells (Figure 6A). 4-OH-Tamoxifen, an antagonist for estrogen, significantly decreased SAL-mediated cell proliferation, similar to E2, suggesting estrogenic activity of SAL (Figure 6A). The binding activity of SAL-liganded ER to ERE was measured by an SPR sensor (Figure 6B). SAL-liganded ERα had significantly higher binding activity than the control. The binding activity was about 60% of E2. On the contrary, when ERβ was used, there was no significant difference between SAL and control (0.1% DMSO). 1459

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Figure 4. Oxidative damage to cellular and isolated DNA by SAL. (A) Calf thymus DNA (100 μM per base) was incubated with indicated concentrations of SAL in the presence of 20 μM CuCl2 or Fe(III)EDTA at 37 °C for 1 h. After ethanol precipitation, DNA was enzymatically digested to the component nucleosides and analyzed with an HPLC-ECD. Results are expressed as the means and SD of values obtained from three independent experiments. (B) Calf thymus DNA (100 μM per base) was incubated with 100 μM SAL, 20 μM CuCl2, or Fe(III)EDTA in the presence and absence of 30 unit SOD or 20 μM MnCl2 at 37 °C for 2 h. After ethanol precipitation, DNA was treated and analyzed. The amount of 8-oxodG/105dG was expressed as the mean value of SAL plus metal (= 1). Results are expressed as the means and SD of values obtained from three independent experiments. #, P < 0.05, and ##, P < 0.01, significant difference compared with the condition of SAL plus metal by Student’s t-test. (C) MCF-10A cells were treated with 100 μM SAL at 37 °C for 2 h. DNA was extracted and treated as described in Materials and Methods, and the amount of 8-oxodG was analyzed with an HPLC-ECD. Results are expressed as the means and SD of values obtained from 6 independent experiments. *, P < 0.05, and **, P < 0.01, significant difference compared with the control by Student t-test. (D) MCF-10A cells were treated with 100 μM SAL at 37 °C for 1 h. The alkaline comet assay was used to determine DNA strand breaks and alkali-labile DNA damage (hOGG1(−)) and 8-oxodG formation (hOGG1(+)).

Figure 5. Cell proliferating activity of SAL in MCF-10A cells. MCF-10A cells were incubated with 10 μM SAL or 100 pM estradiol (E2). (A) NAC (5 mM) or (B) AG1478 (2 μM) was added where indicated. Control condition contained 0.1% DMSO. After incubation at 37 °C for 6 days, cells were trypsinized, harvested, and then counted. Results are expressed as the means and SD of values obtained from six independent experiments. **, P < 0.01, significant difference compared with the control, and ##, P < 0.01, significant difference compared between the conditions with and without the indicated inhibitors by Student’s t-test. 1460

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Figure 6. Cell proliferating activity of SAL in MCF-7 cells and binding ability of SAL-liganded ER to ERE. (A) MCF-7 cells were incubated with 10 μM SAL or 100 pM estradiol (E2) in the presence and absence of 1 μM 4-OH-tamoxifen. The control condition contained 0.1% DMSO. After incubation at 37 °C for 6 days, cells were trypsinized, harvested, and then counted. Results are expressed as the means and SD of values obtained from six independent experiments. *, P < 0.01, significant difference compared with the control, and #, P < 0.01, significant difference compared between the conditions with and without 4-OH-tamoxifen by Student’s t-test. (B) Human ERα and ERβ (20 nM) were liganded with 100 nM E2 or 10 μM SAL by the incubation at 37 °C for 5 min. Then, the liganded ER was injected by a 40 μL-injection over the sensor chip surface immobilized with double-stranded human pS2 ERE. The binding activity of liganded ER to ERE was expressed as % activity, that is, binding response with 100 nM E2 as 100% and with no-ligand (DMSO 0.1%) as 0%. Results are expressed as the mean and SD of % activity obtained from 4 independent experiments. **, P < 0.01, significant difference compared with the control by Student’s t-test.

Figure 7. Proposed mechanisms of DNA damage and cell proliferation induced by SAL.

sumption of O2·−, resulting in accumulation of H2O2. It is also reported that Mn(II) catalytically scavenges O2·−.37 In our study, Mn(II) enhanced the DNA damage as well as SOD. Thus, H2O2 and transition metals play important roles in generating reactive species responsible for SAL-induced DNA damage including 8-oxodG formation. We tried to assess the

relative yields of 8-oxodG and strand breaks in single cell (MCF-10A) with or without the treatment of hOGG1 in conjunction with the alkaline comet assay. As the results show, SAL induced a little DNA damage and strand break without hOGG1 and a significant increase of positive cells with hOGG1, as reflected from the tail length of the comet. The 1461

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dismutase; ESR, electron spin resonance; 8-oxodG, 8-oxo-7,8dihydro-2′-deoxyguanosine; HPLC-ECD, electrochemical detector coupled to HPLC; hOGG1, human 8-oxoguanine DNA glycosylase 1; NAC, N-acetylcysteine; E2, 17β-estradiol; ER, estrogen receptor; ERE, estrogen response element; SPR, surface plasmon resonance

formation of 8-oxodG may be predominant rather than strand breakage in SAL-treated cells. 8-OxodG is known to cause misreplication of DNA, leading to mutation.38,39 SAL may play a role in oxidative DNA damage, as an initiation stage of carcinogenesis. We showed that SAL exerted cell proliferating activity on estrogen-sensitive MCF-7 cells. An estrogen receptor antagonist, 4-OH-tamoxifen inhibited the cell proliferation in MCF-7 cells, suggesting that SAL exerted cell proliferating activity via binding to ER. The SPR sensor revealed that SAL significantly increased the binding activity of ERα to ERE but not ERβ. Fan et al. reported that alcohol induced up-regulation of ER α and stimulated estrogen receptor signaling in human breast cancer cell lines.40 Considering this report and our results, SAL may induce mitogenic activity in mammary cells through estrogen receptor signaling. However, SAL exerted proliferating activity on estrogen-independent MCF-10A cells. Interestingly, an antioxidant NAC attenuated SAL-induced cell proliferation in MCF-10A cells. ROS can affect several growth factor receptors including EGFR, resulting in abnormal cell growth regulation.41 We also observed the attenuation of SAL-induced proliferation in MCF-10A cells by AG1478, suggesting the involvement of EGFR. Relevantly, Burdick et al. also showed that benzo[a]pyrene quinones transactivated the EGFR in breast epithelial cells via ROS, leading to cell proliferation.42 Therefore, SALderived ROS may participate in the proliferation of MCF-10A cells via EGFR activation. Collectively, SAL has cell proliferating activity in mammary cells via two pathways, ERα-ERE and ROS-mediated EGFR activation. Therefore, SAL may contribute to the promotion of mammary carcinogenesis. An understanding of the mechanisms is important for the development of appropriate strategies for the prevention and treatment of alcohol-associated cancers. Recently, Brooks and Zakhari1 reviewed possible mechanisms of breast cancer development for tumor initiation as the cumulative carcinogens such as acetaldehyde formation, ROS generation through CYP2E1 activation, and for tumor promotion such as elevated dehydroepiandrosterone sulfate (DHEAS), resulting in increase of estrogen level, and growth of ER-positive cells by alcohol drinking. In addition to those factors, the present study suggests a novel possibility that SAL-induced oxidative DNA damage and cell proliferation may lead to tumor initiation and promotion in alcohol-related mammary carcinogenesis.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*2-174 Edobashi, Tsu, Mie, 514-8507, Japan. Phone/Fax: +8159-231-5011. E-mail: [email protected]. Funding

This research was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Masaki Sawaya (Mie University) for his assistance with the experiments. ABBREVIATIONS SAL, salsolinol; IARC, International Agency for Research on Cancer; ROS, reactive oxygen species; SOD, superoxide 1462

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