Cancer Letters 203 (2004) 139144 www.elsevier.

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Ethanol inhibits benzo[a]pyrene-DNA adduct removal and increases 8-oxo-deoxyguanosine formation in human mammary epithelial cells
Keith W. Singletarya,*, Sean L. Barnesa, Richard B. van Breemenb
Department of Food Science and Human Nutrition, University of Illinois, 467, Bevier Hall, 905 South Goodwin Avenue, Urbana-Champaign, Urbana, IL, USA b Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago, IL, USA Received 10 March 2003; received in revised form 8 September 2003; accepted 8 September 2003
a

Abstract The effect of ethanol and acetaldehyde treatment on the removal of benzo[a]pyrene diol-epoxide (BPDE)-DNA adducts in the immortalized, human mammary epithelial cell line MCF-10F was examined. Treatment of cells with 15mM and 25mM ethanol resulted in signicantly more BPDE-DNA adducts/unit DNA remaining at multiple time points, compared to controls. The half-life of BPDE-DNA adducts in cells exposed to both 15 and 25 mM ethanol were 11.9 and 12.3 h, respectively, compared to a half-life of 9.8 h for the control cells. In contrast, for cells exposed to acetaldehyde at doses of 2.5 and 5.0 mM no signicant trend in BPDE-DNA adduct persistence occurred, compared to controls. The inhibition of adduct removal for cells treated with ethanol was not associated with any changes in cell viability due to ethanol exposure. However, BP-treated cells exposed to 25mM ethanol exhibited a signicant 2-fold increase in 8-oxo-deoxyguanosine (8-oxo-deG) adducts compared to BP-treated cells alone. No signicant increase in 8-oxo-deG was observed for cells treated with BP and exposed to 5.0 mM acetaldehyde. Thus, ethanol exposure of human mammary epithelial cells is associated with a decreased capacity to remove BPDE-DNA adducts. This inhibitory effect of ethanol on adduct removal in part may be related to ethanol-associated oxidative stress. q 2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: Carcinogenesis; DNA adducts; Ethanol; 8-oxo-deoxyguanosine

1. Introduction Numerous epidemiologic studies have reported a positive association between alcohol intake and breast cancer risk among women [1 3]. Although it is
* Corresponding author. Tel.: 1-217-333-5549; fax: 1-217244-2455. E-mail address: kws@uiuc.edu (K.W. Singletary).

unlikely that ethanol itself is a genotoxic initiator of carcinogenesis, it has been reported that ethanol may alter multiple processes contributing to the initiation and promotion stages of carcinogenesis [4]. For example, treatment of breast cancer cells with ethanol was associated with changes in hormone receptor and tumor suppressor protein expression, and in cell proliferation and invasiveness, that taken together could enhance the promotion and progression of

0304-3835/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2003.09.004

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cancer [5 8]. Also, exposure of human mammary epithelial cells to physiologically relevant concentrations of ethanol and its metabolite acetaldehyde resulted in increased formation of benzo[a]pyrene diol-epoxide (BPDE)-DNA adducts [9]. However, little is known about the capacity of ethanol or acetaldehyde to affect the repair or removal of mammary cell DNA damage induced by polycyclic aromatic hydrocarbons such as BP. In this regard, ethanol intake has been found to suppress the liver activity of the DNA repair enzyme, O6-methylguanine transferase, which protects the cell from carcinogeninduced mutations [10,11]. Furthermore, in these studies administration to animals of disulram, an acetaldehyde dehydrogenase inhibitor, enhanced this suppressive effect of ethanol on DNA repair. Thus, both ethanol and acetaldehyde may be capable of interfering with DNA damage removal [12,13]. Of interest to impairment of DNA repair are the recent observations that oxidative stress can promote protein damage and can inactivate human DNA mismatch repair systems [14,15]. Numerous publications indicate that ethanol metabolism increases the production of reactive oxygen species [16 23], which could interfere with multiple cellular processes including DNA repair if not sufciently removed by the repair enzymes [13]. Therefore, the present experiments were initiated to examine the effects of exposure to ethanol and to acetaldehyde on removal of carcinogen DNA adducts in cultures of MCF-10F cells, and to ascertain whether concurrent treatment of cells with BP and ethanol or acetaldehyde augmented oxidative stress as measured by oxidative DNA damage.

exposure to several mammary carcinogens, including the environmental carcinogen, benzo(a)pyrene (BP) [24,25]. Cells were routinely plated in either 75 or 150 cm3 cell culture asks containing DMEM/F-12 medium (Gibco-BRL, Grand Island, NY) consisting of 1.05 mM calcium and supplemented with 100 IU/ml penicillin, 100 mg/ml streptomycin, 2.5 mg/ml amphotericin B, 0.5 mg/ml hydrocortisone, 0.02 mg/ml epidermal growth factor (all from SigmaAldrich, St. Louis), 0.1 mg/ml cholera enterotoxin, 10 mg/ml insulin and 5% equine serum. Cells used in all experiments were from the same passage and were incubated at 37 8C with 0.5% CO2. 2.2. Adduct removal measurements To examine the effect of ethanol on BPDE-DNA adduct removal, T150 asks were seeded with 5 104 cells and designated to be treated with ethanol at nal concentrations of 0, 15 or 25 mM. Cells were allowed to attach for 48 h after which the medium was replaced with fresh medium containing ethanol at the specied concentrations. Control and treatment media were changed once a day for a total of 6 days. Two hours prior to addition of BP, media in all groups were removed and replaced with control medium. This was done to minimize the effects of acute ethanol exposure that could be superimposed on the chronic effects. After this 2-h period, control media was replaced with medium containing BP at a nal concentration of 0.08 mg/ml. This concentration of BP has previously been determined to be in the linear dose-response range for BPDE-DNA adduct formation in these cells [9], and was chosen so that adequate amounts of BPDE-DNA adducts would be present to measure after 7 days. After 12 h of incubation, the same ethanol treatment regimen used prior to BP administration was resumed. Media in all groups was changed daily during the post-BP treatment period. Cells from four asks/group were harvested at 0, 12, 24 h, 3 days (72 h) and 7 days (168 h) post-BP-treatment. To evaluate the effect of acetaldehyde on BP-DNA adduct removal, the experimental design employed was the same as described above, except that, in light of the volatility of acetaldehyde, the acetaldehyde concentration in each treatment ask was replenished every 12 h. Cell

2. Materials and methods 2.1. Cell culture The MCF-10F cell line used in these experiments was obtained from the Michigan Cancer Foundation and were cultured according to procedures recommended by Soule et al. [24]. This immortalized human MCF-10F cell line has been used as a model for studying human mammary carcinogenesis in vitro, behaves like normal breast epithelial cells, and can be transformed to the neoplastic phenotype by short

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viability was determined by the trypan blue exclusion assay. DNA from cell pellets was isolated using a DNA extraction kit (Genomix by Test Test, Friendswood, Texas), and typically exhibited a A260/A280 ratio of at least 1.8. DNA digestion, 32P-post-labeling and TLC separation of adducts was performed according to procedures described by Barnes and Singletary [9]. 2.3. 8-oxo-dG measurement To determine the effect of concomitant exposure of cells to either ethanol or acetaldehyde plus BP on 8-oxo-deoxyguanosine (8-oxo-dG) adducts, cells were treated as described above with either 25 mM ethanol or 5 mM acetaldehyde for 6 days and then with BP at 0.08 (g/ml for 12 h. Thereafter cell pellets were collected, DNA isolated, digested, and 8-oxo-dG quantitated by LC MS MS as described as by Hua et al. [26]. Statistical comparisons among means were conducted using ANOVA with Fishers LSD test for posthoc comparisons. Differences between means were considered statistically signicant at the p , 0:05 level.

squares method) was characterized by the power function generally expressed as f x bxA : Calculation of the half-life of BP-DNA adducts for each group revealed a signicant increase in the time required for cells to eliminate 50% of the initial damage. There was a signicant 21 and 26% increase in the half-life value for cells treated with 15 or 25 mM ethanol, respectively, compared to controls. For cells exposed to acetaldehyde a trend toward higher levels of BP-DNA adducts remaining compared to controls was observed but the differences were not statistically different except for the 2.5 mM group at the 12 h time point (Fig. 2). There were no signicant differences in cell viability among control and treatment groups during the time periods studied. At the initiation of treatments and by day 7, viability was , 98% for all groups.

3. Results Incubation of cells with ethanol was associated with a signicant increase in prevalence of BPDEDNA adducts compared to controls (Fig. 1). The adduct formed has been identied previously as that resulting from the binding of BPDE to deoxyguanosine [9]. For cells treated with either 15 or 25 mM ethanol, signicantly higher (, 35%) levels of BPDE-DNA adducts persisted compared to controls at 12 h post-BP treatment, with the difference between treatments and controls being less apparent at 1 and 3 days post-BP treatment. At 7 days post-BP treatment, a signicantly higher level of BPDE-DNA adducts was observed only in the 15 mM-treated group. Disappearance of adducts was characterized (using a semi-log plot, Fig. 1B) as a biphasic curve in which the rate of adduct disappearance was quite rapid between 0 and 24 h post-BP (i.e. a phase), followed by a slower rate of removal beyond 24 h (i.e. b phase). The best-t curve (using the linear least

Fig. 1. Effect of ethanol treatment on BP-DNA adduct removal in MCF-10F cells (A). Semilog plot of data (B). Mean adduct values at 0 h were 379 nmol BP/mol DNA. Values represent means of four separate determinations. *Statistically signicant difference vs controls.

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Fig. 2. Effect of acetaldehyde on BP-DNA adduct removal in MCF10F cells. Mean adduct values at 0 h were 377 nmol BP/mol DNA. Values represent means of four separate determinations. *Statistically signicant difference vs controls.

The formation of 8-oxo-dG adducts was inuence by the combination of BP and ethanol but not by the combination of BP dosing with acetaldehyde treatment (Fig. 3). Treatment of cells with both ethanol and BP resulted in a signicant 2-fold increase in 8-oxo-dG adducts compared to cells treated with BP alone. However, there was no signicant increase in 8-oxo-dG adducts for cells treated with acetaldehyde plus BP compared to BP alone. For cells not treated with BP, 8-oxo-dG adduct values were 2.8 ^ 0.3/105 G and 4.2 ^ 0.7/105 G for controls and ethanol-exposed cells, respectively p 0:1:

these compounds that have been reported for women who consumed ethanol. For example, moderate ethanol intake has been reported to result in blood alcohol concentrations from 5 to 24 mM [28,29]. Also, blood concentrations of acetaldehyde up to 6 mM have been reported for alcohol-consuming women [30]. Our data indicate that exposure to ethanol had a greater inhibitory effect on adduct removal compared to cells treated with acetaldehyde. Whereas ethanol treatment at the 15 and 25 mM concentrations resulted in higher levels of adducts compared to controls, cells chronically exposed to acetaldehyde exhibited greater levels of BPDE-DNA adduct only at one time point and dose (12 h, 2.5 mM). The removal of DNA damage by the MCF-10F cells was not linear, but rather appeared to be biphasic based on semi-log plots. This is similar to the biphasic pattern of adduct repair reported for polycyclic aromatic hydrocarboninduced DNA damage in mouse skin following treatment with either BP or 7,12-dimethylbenz[a] anthracene [31,32]. Biphasic repair kinetics have been observed for other forms of DNA damage [14,33] and in part may be due to specic mechanisms and rates of action of individual repair proteins [34]. The decrease in BPDE-DNA adduct disappearance observed in MCF-10F cells exposed to ethanol appeared to be due to ethanols inuence on physiological processes other than ethanol-mediated toxicity and loss of viability. The insignicant effect of acetaldehyde

4. Conclusions The present studies provide evidence that physiologically relevant doses of ethanol have a modest but signicant inhibitory effect on BPDE-DNA adduct removal in cultures of human mammary epithelial cells. Since genomic repair of DNA damage is heterogeneous [27], the magnitude of ethanols effect on gene-specic DNA adduct removal may be different than that reected in our determination of overall disappearance. This warrants further study. No signicant inhibitory effect of acetaldehyde exposure on adduct removal was observed for the doses of BP and acetaldehyde used in these studies. The concentrations of ethanol and acetaldehyde used in these experiments were chosen based on blood levels of

Fig. 3. Effect of treatment with BP and ethanol or acetaldehyde on 8-oxo-deG adducts in MCF-10F cells. Values represent means 1025. *Statistically signicant difference vs BP only.

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treatment on adduct levels also suggests that the inhibition of adduct removal with ethanol treatment in these cells is likely not due simply to metabolism to the DNA- and protein-reactive [13] metabolite acetaldehyde. In contrast to our ndings with human mammary cells, both ethanol and acetaldehyde were reported to inhibit the liver activity of the DNA damage repair enzyme O6 methyl guanine transferase [6]. The differences between our mammary data and those from liver cells may partly be due to tissue-, repair system-, or ethanol dosing-specic differences. Of interest is our observation that co-treatment of BP-dosed cells with ethanol but not acetaldehyde enhances 8-oxo-deG formation. Also, ethanol treatment in the absence of BP exposure increased 8-oxodG levels by , 50%, compared to controls, although the difference was not signicant. It should be noted that the levels of 8-oxo-dG reported here are similar to levels measured by our tandem mass spectrometry method [26] in DNA from other cells in culture. Using this same procedure we have routinely detected 8-oxo-dG levels of , 0.5/105 G in human cells, comparable to those levels reported by the European Standards Committee on Oxidative DNA Damage [35]. Our data indicate that ethanol-induced generation of oxidative damage may be a contributing factor to the greater inhibitory effect of ethanol on adduct removal. Castro et al. [17] detected bioactivation of ethanol to acetaldehyde and 1-hydroxyethyl free radicals by cytosolic xanthine oxidoreductase in rat breast tissue, which suggests that ethanol metabolism can be mediated by other enzymes in addition to alcohol dehydrogenase and cytochrome P450, and can yield multiple free radical species. The pathway of ethanol metabolism that is generating the oxidative stress in the MCF-10F cells is not known. Others have reported that oxidative stress can damage cellular proteins [36 40] including the human DNA mismatch repair system [41] and thus can impair mechanisms that limit DNA damage [42]. The specic effect of ethanol on DNA repair proteins in human mammary cells warrants further study. Taken together, these results suggest that ethanol- and oxidative stress-associated inhibition of carcinogenDNA adduct removal in nonneoplastic human mammary cells may be another biological mechanism to explain the increased risk for breast cancer among women consuming alcohol.

Acknowledgements Supported in part by NIAAA PHS Grant #5F31AA05479.

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