Folate exacerbates the effects of ethanol on peripubertal mouse mammary gland development

Alcohol 46 (2012) 285e292

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Folate exacerbates the effects of ethanol on peripubertal mouse mammary gland development Patricia A. Masso-Welch a, *, Menachem E. Tobias a, Shyam C. Vasantha Kumar a, MaryLou Bodziak a, Terry Mashtare Jr. b, Judith Tamburlin a, Stephen T. Koury a a b

Department of Biotechnical and Clinical Laboratory Sciences, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14214, USA Department of Biostatistics, School of Public Health and Health Professions, State University of New York at Buffalo, Buffalo, NY 14214, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 August 2010 Received in revised form 31 August 2011 Accepted 14 December 2011

Alcohol consumption is linked with increased breast cancer risk in women, even at low levels of ingestion. The proposed mechanisms whereby ethanol exerts its effects include decreased folate levels resulting in diminished DNA synthesis and repair, and/or acetaldehyde-generated DNA damage. Based on these proposed mechanisms, we hypothesized that ethanol would have increased deleterious effects during periods of rapid mammary gland epithelial proliferation, such as peripuberty, and that folate deficiency alone might mimic and/or exacerbate the effects of ethanol. To test this hypothesis, weightmatched 28e35 day old CD2F1 female mice were pair-fed liquid diets
3.2% ethanol,
0.1% folate for 4 weeks. Folate status was confirmed by assay of liver and kidney tissues. In folate deficient mice, no significant ethanol-induced changes to the mammary gland were observed. Folate replete mice fed ethanol had an increased number of ducts per section, due to an increased number of terminal short branches. Serum estrogen levels were increased by ethanol, but only in folate replete mice. These results demonstrate that folate deficiency alone does not mimic the effects of ethanol, and that folate deficiency in the presence of ethanol blocks proliferative effects of ethanol on the mammary ductal tree. Ó 2012 Elsevier Inc. All rights reserved.

Keywords: Mammary gland Folate Ethanol Mouse Puberty Development

Introduction A recently released comprehensive report concluded that the only significant, consistently clinically-supported single dietary modifier of human breast cancer risk is alcohol consumption (World Cancer Research Fund & American Institute for Cancer Research, 2007). Ethanol was shown to act by itself to increase both pre- and post-menopausal breast cancer incidence in women. Although the risk of breast cancer in women in the U.S. is high (1 in 8, or 12.5%), this risk is significantly increased (to 1 in 7, or 14.3%) by as little as one drink per day. With each additional drink per day, the risk is increased by 10% (World Cancer Research Fund & American Institute for Cancer Research, 2007). Ethanol is therefore a clinically relevant dietary modifier of breast cancer risk. Although these epidemiologic findings suggest that women should abstain

Supported by the Mark Diamond Research Foundation and the Dept. of Biotechnical and Clinical Laboratory Sciences in the University at Buffalo. * Corresponding author. Department of Biotechnical and Clinical Laboratory Sciences, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, 3435 Main Street, Buffalo, NY 14214, USA. Tel.: þ1 716 829 5191; fax: þ1 716 829 3601. E-mail address: [email protected] (P.A. Masso-Welch). 0741-8329/$ e see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.alcohol.2011.12.003

completely from ethanol throughout their lives to lower their overall breast cancer risk, there may be time points of greater sensitivity to the deleterious effects of ethanol, and timing of ethanol exposure and dietary conditions at that time of exposure may influence long-term risk. Multiple mechanisms of the deleterious effects of ethanol on breast cancer susceptibility have been previously proposed, including secondary effects from acetaldehyde generation, with ensuing DNA damage, and effects on folate metabolism, resulting in folate insufficiency for DNA synthesis and repair (reviewed in Dumitrescu & Shields, 2005; Mason & Choi, 2005; Singletary, 1997; Barnes, Singletary, & Frey, 2000). Dietary antioxidants such as folate may therefore modify the effects of ethanol on breast cancer risk (Dumitrescu & Shields, 2005; Mason & Choi, 2005). For example, high folate intake was shown to protect against the breast cancer risk induced by ethanol consumption in a prospective cohort study (Baglietto, English, Gertig, Hopper, & Giles, 2005). Other epidemiologic studies have shown no interaction of ethanol and folate levels in overall breast cancer risk (Feigelson et al., 2003; Hartman et al., 2005; Tjonneland et al., 2007). However, folate status at specific developmental times of rapid cell reproduction and tissue morphogenesis, such as peripuberty for the mammary gland, may have a disproportionate effect to modify risk. Therefore, the

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Table 1 Isocaloric diets used in these studies, with ethanol supplemented after reconstitution. Dietary group

Group 1

Group 2

Group 3

Group 4

Dyets catalog # Maltose dextran Cellulose (microcrystalline) Corn oil (stab 0.15% BHT) Salt mix # 210020 Sodium acetate L-amino acid premix Vitamin mix # 317759 (folate-free) Folic acid premix (5 g/kg) Succinyl sulfathiazole Choline chloride Xanthum gum Ethanol

717768 58.20 g/L 12.67 g/L 25.33 g/L 12.67 g/L 2.05 g/L 44.55 g/L 2.53 g/L 0.20 g/L 2.53 g/L 0.51 g/L 3.00 g/L 32 g/L

717769 152.90 g/L 12.67 g/L 25.33 g/L 12.67 g/L 2.05 g/L 44.55 g/L 2.53 g/L 0.20 g/L 2.53 g/L 0.51 g/L 3.00 g/L 0 g/L

717770 58.40 g/L 12.67 g/L 25.33 g/L 12.67 g/L 2.05 g/L 44.55 g/L 2.53 g/L 0 g/L 2.53 g/L 0.51 g/L 3.00 g/L 32 g/L

717771 153.10 g/L 12.67 g/L 25.33 g/L 12.67 g/L 2.05 g/L 44.55 g/L 2.53 g/L 0 g/L 2.53 g/L 0.51 g/L 3.00 g/L 0 g/L

purpose of this study was to test the hypothesis that ethanol consumption during puberty will induce morphologic changes in the mouse mammary gland which can predispose to breast cancer. We also hypothesize that, if ethanol’s deleterious effects on the mammary gland to increase breast cancer risk are partially mediated by its ability to decrease bioavailable folate during this time of rapid cell division, then effects of ethanol on the mammary gland may be mimicked or potentiated by folate deprivation. A second goal of this study was to refine a mouse model for dietary ethanol exposure, for the purpose of future studies examining the effects of this clinically relevant mammary carcinogen in genetically engineered mouse models. Although the rat mammary gland is more similar to the human breast than the mouse mammary gland, in terms of structure and composition, rat models of chemical mammary carcinogenesis are of unknown relevance to human breast cancer, both in terms of the etiology of breast cancer and the phenotype of resulting tumors (e.g. hormone dependence) (reviewed in Medina & Thompson, 2000). In contrast, genetically engineered mouse models offer us the opportunity to examine the effects of dietary interventions on mammary tumor incidence with a clinically relevant etiology, e.g. the Her2/neu overexpressing mouse model (reviewed in Medina, 2000). Materials and methods Materials Chloroform, folinic acid, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), sodium L-ascorbate, 2-mercaptoethanol, acid washed activated charcoal, CHES (N-Cyclohexyl-2-aminoethanesulfonic acid), K3Fe(CN)6, KCN and NP-40 were purchased from SigmaeAldrich (St. Louis, MO). Lactobacillus casei (subspecies rhamnosis, ATCC #7469) was obtained from ATCC (Rockville, MD).

a final liquid concentration of 0.1% folate (w/v). After one week on baseline liquid diet, mice were weighed and randomized by weight into one of 4 dietary groups using a 2  2 study design (N ¼ 8 mice per group), with the variables being folate at 0 or 0.1% (w/v) and ethanol at 0 or 3.2% (v/v). Amino acid defined diets were purchased from Dyets Inc. (Bethlehem, PA). Individual diet content is summarized in Table 1. Amino acid composition is summarized in Table 2. Diets were received in powder form and stored at 4  C in the dark until used. Ethanol was purchased as 95% ethyl alcohol from Pharmco (Brookfield, CT). Capped 30 ml graduated sipper feeding tubes (Dyets Inc., Cat #900012) supported by feeding tube holders (Dyets Cat #901100) were used to deliver liquid diet. Mice were weighed daily and dietary consumption was recorded to perform pair feeding, with each control mouse (receiving diets containing no ethanol) receiving the volume consumed by its respective ethanolfed control the previous day. Ethanol-fed groups were offered 24 ml of their respective diet every day. Diets were prepared and changed daily into clean sipper tubes. Mice to be placed on 3.2% (v/v) ethanol diets were first acclimated for three days with a lower ethanol dose 1.6% (v/v) (a 1:1 mixture of Dyets #717769 and #717771), before receiving the final diets containing 3.2% ethanol (v/v)
0.1% (w/v) folate for the following 4 weeks. The physiologic relevance of the folate dosage to human consumption is calculated as follows. Mice received liquid diets prepared with a final folate dose of 0 or 1 mg/ml (0.1% (w/v)). Average consumption of 10 ml per day of 1 mg/ml folate gives a daily

Table 2 Composition of amino acid mix used for all diets. Amino acid

Concentration (mg/kg)

L-Alanine

0.89 2.84 1.71 0.89 0.89 8.85 5.9 0.84 2.08 2.81 4.56 2.08 2.94 0.89 0.89 2.08 0.44 0.89 2.08 44.55 g/kg

L-Arginine

Animals Female CD2F1 mice were purchased from Harlan Sprague Dawley Labs Inc. (Indianapolis, IN) at day 21e28 of age. Animal rooms were air-conditioned and humidity controlled, with a light cycle of 12 h on and 12 h off. Animals were housed in accordance with the standards set by the NIH and the University at Buffalo Institute Animal Care and Use Committee. Feeding protocol Mice were weighed and individually housed in cages upon arrival, where they were acclimated to the baseline liquid Modified Clifford Koury Folate Deficient Liquid Diet (Bills, Koury, Clifford, & Dessypris, 1992) containing 4 mg folate per kg diet, resulting in

L-Asparagine$H2O L-Aspartic

acid

L-Cystine L-Glutaminc acid Glycine L-Histidine L-Isoleucine L-Leucine L-Lysine$HCl L-Methionine L-Phenylalanine L-Proline L-Serine L-Threonine L-Tryptophan L-Tyrosine L-Valine Total L-AA

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dosage of 10 mg folate/0.018 kg mouse (or a daily dose of 556 mg folate/kg mouse), which is the basal minimum daily folate dose based on a 2 mg folate/kg diet, consuming 5 g diet consumed per day (for a total dose of 10 mg/day) (Reeves, Nielsen, & Fahey, 1993). To convert mouse to Human Equivalent Dosage (HED), we can use the FDA recommendations from their web site (http://www.fda. gov/cber/gdlns/dose.htm#v). Based on weight, the 556 mg folate/ kg mouse dosage is multiplied by a conversion factor of 0.08 to obtain a 44 mg folate/kg human dosage. For a 65 kg human, this results in a dosage of 2889 mg folate per woman per day, which is 7.2 times the minimum recommended dosage for women of 400 mg per day (McNulty, 1997). Sacrifice and tissue collection Mice were sacrificed after 4 weeks of feeding by halothane euthanasia followed by cervical dislocation. Mice were weighed and vaginal smears were performed to determine estrous cycle stage. Thoracic blood was collected into heparinized Caraway tubes for manual hematocrit determination. Contralateral abdominal mammary glands (gland #4) were dissected for whole mount preparation or placed into 4% phosphate buffered formalin for paraffin embedding. Inguinal mammary glands, uterine horn, ovaries, kidney, spleen and liver were dissected, weighed, and divided into weighed fractions for RNA/protein by snap-freezing in liquid nitrogen and storage at 70  C, or placed into formaldehyde for paraffin embedding. Morphologic analysis of whole mounts Left abdominal mammary glands (gland #4) were prepared for whole mounts as described (Ip et al., 2007; Thompson et al., 1997). Mammary gland whole mounts were scanned at 4000 dpi using a Microtek ArtixScan 4000tf microscope slide scanner (Microtek USA, Compton, CA). Each whole mount was analyzed for the following criteria using NIH Image J software, by double-blind analysis. Total volume of the epithelial tree was determined as percentage of area, with the entire ductal tree defined as the nonadipose dense portion of the composite image, with the lymph node area subtracted. Length of fat pad was distance (in mm) from the terminal epithelial structure most distal from the nipple, to the edge of the lymph node. Ductal branching and terminal structures were evaluated from the distal edge of the lymph node to the edge of the fat pad as follows: Symmetric bifurcations were scored as terminal branches (TBr), and asymmetric bifurcations were scored as side branches (SBr). Terminal structures, analyzed under the light microscope using a 10 objective, were scored as terminal end buds (TEBs, terminal bulbous structures whose width > ductal width); terminal ductules (Td, terminal structures whose width  duct width); and alveolar buds (AB, terminal structures composed of multiple terminal ductules clustered). Analysis of paraffin sections Contralateral abdominal mammary gland (right gland #4) from each mouse was fixed in phosphate buffered formalin for preparation of hematoxylin and eosin stained paraffin sections. Scanned images of sections were overlaid with a 4 mm square grid overlay, and ducts intersecting the grid were used to score ductal frequency per unit area. The same ducts were then photographed under a 40 objective on an Olympus BHS-RFCA microscope connected to a SPOT RT color digital camera (Diagnostic Instruments, Sterling Heights, MI). Ducts were scored as single-layered (with a single layer of luminal epithelium overlaying the myoepithelium) or multilayered (with multiple layers of luminal epithelial cells).

287

Ductal area, stromal area and luminal area of the randomly selected ducts were calculated using Image J imaging program (NIH, Bethesda, MD), and converted from pixels2 to mM2 using a stage micrometer as a reference (Media Cybernetics, Bethesda, MD). Folate bioassay The folate microbiological assay was used to quantify bioavailable folate in liver and kidney as described (Horne & Patterson, 1988). Briefly, tissues were boiled for 10 min in 5 ml Extraction Buffer (0.25 mM HEPES, 0.25 mM CHES, 0.1 M sodium L-ascorbate, 29 mM 2-mercaptoethanol). Tissues were homogenized and spun down for 8 min at 6500  g. The defatted supernatant fraction was stored at 70  C under nitrogen. Folate-free serum conjugase was used to homogenize folate polyglutamates to monoglutamates for 3 h at 37  C. Samples were then boiled for 5 min, centrifuged at 13,000  g for 20 min, aliquoted, and stored at 70  C under nitrogen. Extracts were incubated for 24 h at 37  C with folate-dependent L. casei in saline solution, and OD595 was used as an index of L. casei cell number. Purified folinic acid was used as a standard. Final concentration was calculated per g tissue as well as per total organ. Estrogen assay Sera were assayed for estradiol levels using an inhibition EIA (Cat #ES-180S-100, CalBiotech, Spring Valley, CA). Briefly, 25 ml of sera, standards or controls were added to each anti-estradiol antibody-coated well, with 100 ml of estradioleenzyme conjugate. After 120 min, the wells were rinsed 3 times with 300 ml rinse solution per well. 100 ml of TMB reagent was added to develop the reaction for 30 min at room temperature, followed by addition of 50 ml of Stop Reaction solution. Absorbance at 450 nm was read using a BioTek EL808 ELISA plate reader. Absorbance values were used to generate the standard curve and calculate sample estradiol concentrations. Statistical analysis Statistical analyses were performed using SAS 9.2. Analysis of effects of dietary group on organ weights at sacrifice, whole mount and histological analyses of branching, structures, and areas, folate levels, hematocrit, and estradiol levels, were performed using a mixed linear model with ethanol, folate, and ethanol by folate interaction as fixed effects, and a variable to take pair feeding into account as a random effect. Weights were analyzed with data binned by week, as well as on a daily basis. To analyze effects of four dietary groups on weights of individual pair-fed mice over time, global contrast was performed to determine significance at each time point. For time points determined to be significant (p < 0.0014 at each of 36 time points), repeated measure ANOVAs (weights and dietary consumptions) were performed using time as a third variable (p < 0.05 as significant). To test for interactions between ethanol and folate, a two-by-two ANOVA model was performed, with ethanol/folate as main effects and the interaction. p < 0.0084 was used to determine significance of interactions between ethanol and folate. Pair-wise comparisons were performed in the event of significant effects. Results Effects of ethanol and folate on mouse growth and folate status Despite receiving the same total daily food volume through pair feeding, mice from the ethanol-fed groups showed significantly greater body weights (at daily weighing (data not shown), or when

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Table 3 Effects of ethanol
folate on mouse body weight and food consumption over time. Data represent average
S.E.M. of mouse weight or dietary consumption during a one-week period. Significant differences between dietary groups are indicated by different superscript letters in a row. N ¼ 8 mice per group. Dietary group Folate/ethanol

Group 1 þ/þ

Food consumption (ml) 3 day 11.98 acclimation Week 1 10.27 Week 2 11.63 Week 3 10.85 Week 4 11.52 Mouse weight (g) Day zero 18.29 3 day 18.88 acclimation Week 1 18.35 Week 2 20.15 Week 3 21.13 Week 4 22.26

Group 2 þ/

Group 3 /þ

Group 4 /


0.32

12.90
0.35

10.85
0.58

12.15
0.52







9.94 11.55 10.88 11.40

0.19 0.24 0.18 0.28


0.37
0.37a





0.34a 0.41a 0.50a 0.37a







0.15 0.25 0.25 0.26

18.27
0.35 17.55
0.40b 16.11 16.18 16.82 18.25







0.23b 0.18b 0.26b 0.33b

9.86 10.78 10.55 10.59







0.56 0.20 0.26 0.47

18.20
0.34 18.51
0.29a 17.74 18.50 19.75 20.14







0.60a 0.60a 0.62a 0.43b

9.58 10.59 10.81 10.19







0.49 0.24 0.19 0.31

18.16
0.33 17.62
0.39b 15.87 15.74 16.17 17.75







0.38b 0.44b 0.45b 0.28b

binned by average weekly weight (Table 3)), compared to their pairfed control, regardless of folate status. Similarly, ethanol significantly increased absolute liver and spleen weights at sacrifice, compared to pair-fed control, irrespective of folate status. Kidneys were enlarged by ethanol feeding, compared to pair-fed control, only in mice lacking dietary folate (Table 4). Normalization of organ weights to the animal’s total body weight showed a similar increase for liver and spleen relative weights. Relative kidney weight was unchanged by dietary group. To determine if there was a pathologic cause for changes in organ size, histologic analysis was performed on the livers, kidneys and spleens from all mice. The livers of ethanol-fed mice showed increased epithelial cell size and intracellular lipid accumulation (Fig. 1), which was exacerbated by lack of folate in the diet (Fig. 1C, arrow). However, no steatohepatitis was observed in any dietary group, as defined by lobular inflammation, fibroblast recruitment and collagen deposition (Kleiner et al., 2005). No frank pathologic changes were seen in the kidney or spleen sections of the ethanol-fed mice. Folate and ethanol showed a significant interactive effect on liver weight (p ¼ 0.034). Effects of diet on estrogen levels Because we were interested in effects of ethanol to alter sex steroid levels in the mouse, we performed an estrogen EIA on mouse sera. Ethanol induced a significant increase in estrogen levels, but only in folate replete mice (Table 5). Ethanol interacted with folate to cause a significant increase in estradiol levels,

Table 4 Tissue weights at time of sacrifice. Weights are presented as g/mouse and normalized to % mouse body weight. Data represent average
S.E.M. of mouse weight or dietary consumption during a one-week period. Significant differences between dietary groups are indicated by different superscript letters in a row. No letters in a row indicate no significant difference. Dietary group Folate/ethanol

Group 1 þ/þ

Absolute organ weights Liver weight (g) 1.14 Kidney weight (g) 0.30 Spleen weight (g) 0.11 Relative organ weights Liver weight % 4.90 Kidney weight % 1.28 Spleen weight % 0.46

Group 2 þ/

Group 3 /þ

Group 4 /


0.03a
0.01
0.01a

0.74
0.02b 0.27
0.021 0.07
0.00b

1.05
0.04a 0.30
0.01a 0.10
0.00a

0.76
0.02b 0.25
0.01b 0.05
0.00b


0.12a
0.02
0.02a

3.85
0.08b 1.43
0.01 0.35
0.01b

5.00
0.19a 1.46
0.10 0.46
0.02a

4.02
0.09b 1.32
0.04 0.27
0.01b

compared to all other groups. Vaginal smears taken at sacrifice demonstrated that mice were distributed throughout multiple estrus stages, independent of diet group. Confirmation that these mice had entered puberty by the time of sacrifice was obtained by histologic analysis of the ovary, which revealed follicles in all stages of maturation in every mouse (data not shown). These results suggest that mice in all four dietary groups were actively cycling. Effects of diets on folate status Bioavailable tissue folate levels were analyzed to determine the effects of ethanol feeding on folate status. As summarized in Table 5, ethanol increased folate status in the whole livers of mice fed ethanol in the presence of folate. When normalized to liver size, the folate per gram liver was indistinguishable between ethanol and control mice. Therefore, the four weeks of ethanol feeding produced here did not induce a significant decrease in folate levels per g tissue, and in fact, ethanol increased the total folate present in the enlarged livers of mice fed folate. Folate and ethanol showed a significant interaction on folate levels per whole liver (p ¼ 0.0024), but not when normalized to liver weight. As expected, mice fed for four weeks with diets with no folate (Table 5) showed a greatly decreased steady state level of folate in liver (Groups 3 and 4, 0.3 and 1.9 mg folate/g liver, respectively), compared to folate replete mice (Groups 1 and 2, 14.8 and 16.2 mg folate/g liver, respectively). Ethanol further decreased folate levels in folate-deprived mice per g liver and decreased hematocrit significantly, by 14% (Table 5). The interaction between folate and ethanol on hematocrit however, was not significant (p ¼ 0.053). Effects of ethanol and folate status on peripubertal mammary gland development In order to determine the effects of ethanol exposure
folate on mouse mammary gland development, we performed a systematic analysis of mammary gland whole mounts and paraffin sections for branching frequency, type of terminal structures, and histopathology. We hypothesized that ethanol feeding at peripuberty would alter mouse mammary gland development, and that this effect may be rescued by folate. We also anticipated that folate deprivation alone would mimic some of the effects of ethanol exposure. The results of the mammary gland whole mount analyses are summarized in Table 6. In folate replete mice, ethanol induced an increase in the total number of terminal structures, through increasing terminal short branches, rather than a change in side (lateral) or bifurcating longitudinal branches. Interestingly, none of these changes were observed in mice from which folate was withheld (Table 6, Groups 3 and 4). No increase in epithelium was seen. The interaction between folate and ethanol was statistically significant (p ¼ 0.0059). Paraffin sections were then analyzed for histologic changes in the mouse mammary gland. The results of these studies are summarized in Table 7. The largest effects of ethanol were seen in folate replete mice; ductal abundance (number of ducts per section) was significantly increased by ethanol (Group 1 versus 2). The interaction between folate and ethanol was statistically significant (p ¼ 0.0017). At the level of individual ducts, there was an increased epithelial area per duct, independent of lumenal diameter (discussed below) or stromal desmoplasia. This increase in area of the luminal epithelium reflects a trend toward increased incidence of cellular multilayering. No evidence of epithelial invasion outside of the basement membrane was seen; all multilayered epithelial cells were contained in situ within ducts (data not shown). In contrast, in the folate-deprived mice, ethanol had no significant effect on ductal

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289

Fig. 1. Histology of representative liver paraffin sections. Panel A, Group 1 (þethanol, þfolate), Panel B, Group 2 (no ethanol, þfolate), Panel C, Group 3 (þethanol, no folate), Panel D, Group 4 (no ethanol, no folate). Arrows indicate liver epithelial cells. Liver epithelial cells in Panels A and C show intracellular lipid accumulation. All images were taken using a 20 objective.

abundance (ducts per section), although again there was a trend toward increased multilayering, which was not significant (Table 7, Groups 3 and 4). Ethanol induced one consistent effect on the mouse mammary gland, regardless of folate feeding. An absolute loss of visible ductal secretions was seen in all mice fed ethanol, and this is reflected in the decreased luminal area of the patent ducts (Table 7, % ducts with secretions). Discussion Refinement of a short term ethanol feeding model in mice One goal of this study was to refine a mouse model for developmentally timed dietary ethanol exposure. We describe a dietary regimen whereby peripubertal mice, after an initial weight loss in the first week, maintain and increase their weight throughout four weeks of feeding, regardless of folate feeding. Because mice fed ethanol self-restrict their daily consumption of the liquid diets, it was necessary to use the ethanol-fed mice as the index for feeding their partners. Despite this restricted feeding, mice showed a Table 5 Effect of dietary treatments on bioavailable tissue folate status, hematocrit, and estradiol level at time of sacrifice. Weights are presented as g/mouse and normalized to % mouse body weight. Data represent average
S.E.M. of mouse weight or dietary consumption during a one-week period. Significant differences between dietary groups are indicated by different superscript letters in a row. Dietary group Ethanol/folate

Group 1 þ/þ

Estradiol (pg/ml) mg folate/liver mg folate/g liver mg folate/kidney mg folate/g kidney Hematocrit (%)

8.9 17.0 14.8 1.3 4.4 41.3









0.6a 1.5a 1.2a 0.1a 0.2a 0.9

Group 2 /þ 6.2 11.9 16.2 1.3 4.6 41.8









0.6b 0.7b 1.1a 0.1a 0.2a 1.2a

Group 3 þ/ 6.2 0.3 0.3 0.1 0.4 35.3









0.3b 0.7c 0.7b 0.0b 0.1b 1.1b

Group 4 / 5.8 1.5 1.9 0.2 0.6 41.1









0.3b 0.7c 0.9b 0.0b 0.1b 2.0a

plateauing growth curve similar to that reported by Harlan Labs for mice over 18 g of weight (http://www.harlan.com/). In addition, ethanol-fed mice weighed more than controls, regardless of folate status. This apparent increased body weight of ethanol-fed mice is due in part to feeding patterns and also some organ enlargement compared to pair-fed control. Mice were weighed before daily feeding, and because ethanol-fed mice feed continuously throughout the day, they were more likely to have recently eaten than the respective controls. Although other ethanol studies have utilized gavage or i.p. or i.v. injection for ethanol administration (Golovenko, Zhuk, Zin’kovskii, Zhuk, & Kopanitsa, 2001; Linsenbardt et al., 2009; Ramachandra, Phuc, Franco, & Gonzales, 2007), which would eliminate this variable in time of ethanol exposure, we chose to use the dietary approach for decreased stress compared to daily gavage, and physiologic relevance to human ethanol consumption.

Table 6 Effect of dietary treatments on mammary gland morphology by quantitative analysis of mammary gland whole mounts for branching and terminal structures. Data are presented as # of structures identified per whole mount or % of total structures. Data represent means
S.E.M. for N ¼ 8 mice per group. Significant differences between dietary groups are indicated by different superscript letters in a row. Dietary group Ethanol/folate

Group 1 þ/þ

Branching morphogenesis Total # branches 111.6 # Long. branches 52.1 # Side branches 32.4 # Term. short 27.1 branches Terminal structures Total # terminal 103.4 structures % Terminal end buds 67.0 % Terminal ductules 15.5 % Alveolar buds 17.5







Group 2 /þ 11.3 6.5 3.6 4.1a

77.6 42.0 21.5 14.1







10.5 4.9 4.0 2.1

Group 3 þ/ 79.6 50.1 17.1 12.4







18.2 9.4 6.0 3.7

Group 4 / 86.0 63.9 12.0 10.1







5.9 5.7 1.3 1.4b


11.3a

62.6
6.2b

68.9
15.2

77.0
4.5


3.0
3.3
3.7

77.0
4.9 13.0
4.8 10.0
3.3

73.5
3.2 12.5
4.3 14.0
4.3

64.5
4.1 26.5
3.5 9.00
1.5

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Table 7 Effects of ethanol
folate on mammary gland histology. Data are presented as # of structures identified per whole mount or % of total structures. Data represent means
S.E.M. for N ¼ 8 mice per group. Significant differences between dietary groups are indicated by different superscript letters in a row. Dietary group Ethanol/folate

Group 1 þ/þ

Ducts/section Area/section (mm2) Ductal area (mm2) % Ductal area/section % Epithel. area/duct % Stromal area/duct % Luminal area/duct % Ducts w/secretions % Incidence of luminal epithelial multilayering

17.6 44.47 0.40 0.96 37.8 56.1 6.1 0.0 12.7












Group 2 /þ 2.9a 5.48 0.11 0.25 1.4a 1.6 0.6a 0.0a 2.1

The liver and spleen weights of the ethanol-fed mice were increased compared to their pair-fed controls, in absolute weight as well as when normalized to animal body weight. With the exception of increased liver cell size due to intracellular lipid accumulation, no frank pathology was seen in the tissues. Folate status: folate replete diets It is noteworthy that, in mice fed folate replete diets, four weeks of ethanol feeding did not induce a significant decrease in tissue folate levels. Therefore, it appears that the dietary conditions for ethanol exposure used here, when folate is present in the diet at 0.1% (w/v) (equivalent to 4 mg/kg in the diet), are sufficient to maintain the folate levels similar to controls fed no ethanol. In a longer-term feeding study in weanling CD2F1 mice, folate replete diets (equivalent to 5 mg folate/kg diet) versus folate deficient diets (0.05 mg/kg) had no effect on mouse weight or hematocrit (Bills, Hinrichs, Morgan, & Clifford, 1992). In that study, folate replete mice had 18.2 mg/g liver (calculated from 41.1 nM/g liver); their low folate controls had 5.2 mg folate/g liver (calculated from 11.5 nM folate/g liver) (Bills, Hinrichs et al., 1992). In comparison, the mice in our study had lower levels of folate per g liver under both folate replete conditions (11.89 mg folate/g liver) and folate deficient conditions (1.48 mg folate/g liver). The short term nature of our feeding study (4 weeks), complete absence of folate in the deficient diet, and its administration around puberty may contribute to the lower tissue levels of folate observed in our study. Residual folate levels in the mice may result from the synthesis of folate by gut bacteria and incorporation into tissues, as described in the rat (Rong, Selhub, Goldin, & Rosenberg, 1991). However, in our studies residual folate was unlikely to be originating from endogenous gut bacteria, because the antibiotic succinyl sulfathiazole was included in our diets. Hematocrit was significantly suppressed (by 14%) by ethanol, but only in folate-deprived mice. Interaction between ethanol and folate We hypothesized that ethanol’s effects on the mouse mammary gland may be mediated at least partially through effects on tissue folate status, based on previous epidemiologic studies in women which showed an ability of high folate intake to mitigate ethanolinduced breast cancer risk (Baglietto et al., 2005; Sellers et al., 2001; Zaridze, Lifanova, Maximovitch, Day, & Duffy, 1991). Evidence of a clinically relevant association between folate status and breast cancer risk is suggested by studies in which women with polymorphisms in genes involved with folate metabolism are associated with breast and other cancers (Chen et al., 2005; Choi et al., 2003; Han, Hankinson, Zhang, De, & Hunter, 2004; Shrubsole et al., 2004; Zhang et al., 1999). It has been suggested that altered methylation patterns resulting from changes in one carbon

9.4 57.43 0.27 0.51 24.8 51.9 23.3 39.4 5.5












Group 3 þ/ 1.5b 4.64 0.08 0.16 1.3b 1.8a 2.4b 9.0b 1.7

7.6 37.21 0.15 0.46 29.4 62.2 8.4 0.0 22.7












Group 4 / 2.6b 5.57 0.06 0.16 2.4b 2.5b 2.0a 0.0a 11.5

9.4 50.44 0.13 0.27 28.0 55.3 16.7 17.4 14.9












1.2b 4.03 0.02 0.05 1.0b 2.81 2.4c 3.5b 4.6

metabolism can result in loss of expression of key regulatory genes, e.g. E cadherin, that can potentially contribute to breast cancer progression (Graff, Gabrielson, Fujii, Baylin, & Herman, 2000; Toyooka et al., 2001). Ethanol has been described as a folate antagonist, and as such, could represent an environmental stressor that increases the minimum daily requirement for folate (Hillman & Steinberg, 1982; Weir, McGing, & Scott, 1985). The ability of ethanol to deplete folate has also been seen in rat models (Collins, Eisenga, Bhandari, & McMartin, 1992; Fernandez, Carreras, & Murillo, 1998; Fernandez, Murillo, & Sanchez, 2000). Folate deficiency is commonly seen in chronic alcoholism, presenting as subclinical micronutrient malnutrition (Gloria et al., 1997). Further, folic acid is absolutely required for DNA repair to occur (Choi & Mason, 2000). Therefore, a goal of our study was to test the hypothesis that the effects of ethanol on mouse mammary gland development are mimicked or potentiated by folate deprivation. We predicted that the mammary gland may be particularly susceptible to folate deficiency as a tissue, because of its multiple developmental windows in which it undergoes epigenetic reprogramming and a burst of biosynthetic activity, including puberty, estrous cycling and pregnancy, as well as the remodeling which occurs during lactogenesis and involution (reviewed in Forsyth, 1991). Kidney was utilized as a peripheral tissue with limited folate storage capability, to act as a surrogate for peripheral steady state folate levels. Based on the lack of effect of folate levels when normalized to g liver and kidney after four weeks feeding in Groups 1 and 2 (folate replete diets), the effects of ethanol consumption on mammary tissue development are not likely to be due to ethanol-induced decreases in systemic folate levels. This is consistent with the observation that even low levels of ethanol consumption (1 drink per day), which would not be expected to impact folate status, induce a 10% increased risk of breast cancer (World Cancer Research Fund & American Institute for Cancer Research, 2007). In the absence of folate (Group 3), ethanol did not significantly alter the mammary gland structure compared to mammary gland of the pair-fed control (Group 4, ethanol-free, folate-free). In fact, we found that folate replete status in Group 1 exacerbated the effects of ethanol on the structure of the mouse mammary gland, in terms of increased epithelial density and a trend toward cellular multilayering. Multilayering of the epithelium occurs normally during mammary gland development and ductal morphogenesis from terminal end bud structures (reviewed in Humphreys, 1999). In order for the initial solid cord of cells to evolve into a single-layered duct with a patent lumen, the central cells normally die by apoptosis (Humphreys et al., 1996). The presence of the abundant folate in the folate replete diets used here, although similar to that used in other mouse studies (Bills, Hinrichs et al., 1992; Heid, Bills, Hinrichs, & Clifford, 1992; Walzem & Clifford, 2010), is calculated to be 7.2 the Human Equivalent Dosage (as described in Materials and Methods). This high Human Equivalent Dose of folate may

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block the apoptosis of the multilayered cells in the center of the duct from apoptosis during puberty and ductal morphogenesis, similar to the effects of antioxidants to prevent anoikis of the central cells in spheroids in three-dimensional culture (Schafer et al., 2009). This phenomenon may be clinically relevant to the development of ductal carcinoma in situ (DCIS), as a recent epidemiologic study likewise found an unexpected association between folate intake and breast cancer risk in post-menopausal women (Stolzenberg-Solomon et al., 2006). This raises the question of whether a diet containing high levels of folate can contribute to the ability of ethanol to increase breast cancer risk. A recent review article summarizes the potential risks associated with high levels of dietary supplementation (Moiseeva & Manson, 2009). Using an alternate approach, blocking folate through dietary depletion or pharmacologic antagonists can decrease preneoplastic intestinal lesions (Song, Medline, Mason, Gallinger, & Kim, 2000) or delay nerve sheath tumor onset (Bills, Hinrichs et al., 1992). In a rat mammary tumor model, in which a range of folate was fed from d21 of weaning throughout 9 months of age, showed a decreased incidence of N-methyl-N-nitrosourea (NMU-induced mammary tumors (Kotsopoulos et al., 2003)). More relevant to these studies, when folate deficiency was induced in rats from d27 of age to day 57, this 30 day diet was sufficient to delay NMU-induced tumor onset (Baggott et al., 1992), suggesting that folate deficiency at a key developmental window can have longterm effects on mammary tumor development and subsequent resistance or susceptibility to carcinogenesis. Potential mechanisms of action: alterations in hormone status In terms of defining the mechanism of action of ethanol to increase breast cancer risk, ethanol may also have direct effects on hormonal status, e.g. increased serum estrone sulfate (Dorgan et al., 2001). In the current study, ethanol induced a significant increase in serum estradiol levels, but only in the folate replete group. It is noteworthy that folate deprivation alone (Group 4), or in the presence of ethanol (Group 4) did not alter estradiol levels or ductal branching or the frequency of epithelial structures, compared to the folate replete group (Group 2). This suggests that the alterations in mammary gland structure require both ethanol and folate replete status to be manifested. This increased estradiol level may be sufficient to explain the increased ductal morphogenesis observed here. Morphologic alterations can affect mammary tumor risk A rat model was previously described for studying the effects of ethanol (folate was not examined) on susceptibility to chemical carcinogenesis in the mammary gland (reviewed in Singletary, 1997). Feeding rats with ethanol specifically induced an increase in abundance of and proliferation within the carcinogen-sensitive terminal end bud structures, although this effect was not linearly dose dependent (Singletary & McNary, 1992, 1994). Based on these studies, we expected that ethanol would induce similar morphologically recognizable changes in the mouse mammary gland. Distinct from our results, Singletary et al. found that ethanol feeding resulted in an increase in terminal end buds (Singletary & McNary, 1992), the highly proliferative, carcinogen-sensitive structures present in the mammary gland of nonparous animals (Russo, Tay, & Russo, 1982). We specifically saw an increase in the number of terminal short branches, which arise by the progesterone-dependent process of eosinophil- and macrophagedependent TEB cleavage (Gouon-Evans, Rothenberg, & Pollard, 2000). Ethanol feeding did not induce a nonspecific inflammatory state of the mammary gland however, based on the lack of

291

infiltrating white blood cells in the mammary adipose tissue (data not shown). There was one effect of ethanol that was observed independent of folate status. A lack of secretions in the lumen of ducts in all ethanol-fed mice, regardless of folate status, suggests that ethanol may have independent effects to suppress the limited secretory differentiation that normally occurs during estrous cycling. Folate deficiency has been previously shown to inhibit exocrine secretion in other tissues (pancreas) (Balaghi & Wagner, 1995). However, in this study the effect of ethanol to decrease secretions was independent of folate replete versus deficient status, suggesting a direct effect of ethanol on secretory differentiation. Summary and conclusions In summary, the hypothesis of this project was that folate depletion might mimic or exacerbate the effects of ethanol on peripubertal mouse mammary gland development. In contrast to our hypothesis, the effects of ethanol were distinct in the presence and absence of folate, and in fact, folate replete status induced a potentially worse morphologic appearance in the mouse mammary gland, with increased ductal abundance and increased epithelial area in individual ducts, due to increased area of the luminal epithelium and apparent epithelial multilayering. Estradiol was only increased by ethanol feeding when folate was not limiting. An inhibitory effect of ethanol on secretory activity in the mammary gland is suggested by the absence of luminal secretions in the ducts of animals fed ethanol, regardless of folate status. In conclusion, the effects of ethanol on mouse mammary gland morphology are unlikely to be due to a primary effect of ethanol on folate status, as folate deprivation alone does not mimic these effects, and folate deprivation combined with ethanol does not exacerbate the mammary gland phenotype. Further studies are required to determine whether these ethanol-induced mammary gland changes translate to mammary tumor susceptibility in the mouse. Given the direct positive correlation between ethanol intake and human breast cancer risk, it is essential to identify if developmental windows of acute mammary gland remodeling represent critical time points during which lifestyle interventions can have a broad impact on chemoprevention of breast cancer. Acknowledgments This work was supported by grants from the Mark Diamond Research Foundation to support the graduate research projects of M.E.T. and S.C.V.K. We would like to gratefully acknowledge Mark Koury for critiquing this manuscript, and Mary Vaughan and the Research Histology Core Laboratory of Roswell Park Cancer Institute for invaluable technical assistance. We would like to acknowledge Dawn M. Bowers for performing the estradiol ELISAs. S. Koury and P. Masso-Welch developed the experimental design, and S.C. Vasantha Kumar and M.E. Tobias refined it. M.E. Tobias, S.C. Vasantha Kumar and M.L. Bodziak performed the experimental procedures under the guidance of S. Koury and P. Masso-Welch. P. Masso-Welch wrote the manuscript and had primary responsibility for final manuscript content. J. Tamburlin assisted in project development and data interpretation. T. Mashtare performed statistical analysis and assisted with data interpretation. All authors critically analyzed and interpreted the data, and read and approved the final version of the article. References Baggott, J. E., Vaughn, W. H., Juliana, M. M., Eto, I., Krundieck, C. L., & Grubbs, C. L. (1992). Effects of folate deficiency and supplementation on methylnitrosourea-

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