Elsevier

Alcohol

Volume 46, Issue 3, May 2012, Pages 285-292
Alcohol

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

https://doi.org/10.1016/j.alcohol.2011.12.003Get rights and content

Abstract

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, weight-matched 28–35 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.

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 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 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).

Section snippets

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 Sigma–Aldrich (St. Louis, MO). Lactobacillus casei (subspecies rhamnosis, ATCC #7469) was obtained from ATCC (Rockville, MD).

Animals

Female CD2F1 mice were purchased from Harlan Sprague Dawley Labs Inc. (Indianapolis, IN) at day 21–28 of age. Animal rooms

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 binned by average weekly weight (Table 3)), compared to their pair-fed 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,

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

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

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,

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    Supported by the Mark Diamond Research Foundation and the Dept. of Biotechnical and Clinical Laboratory Sciences in the University at Buffalo.

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