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(CANCER RESEARCH 49, 5999-6007. November I. 1989]
Influence of the Type of Dietary Fat on Developmental Growth of the MammaryGland in Immature and Mature Female BALB/c Mice1
Clifford W. Welsch2 and Deborah H. O'Connor
Department of Pharmacology and Toxicology, Michigan State L'nirersity. East Lansing Michigan 48824
ABSTRACT
The purpose of this study was to determine whether or not the type ofdietary fat can affect mammary gland growth processes in the immatureand mature female BALB/c mouse. Croups of immature and mature micewere fed one of the following purified semisynthetic diets containingdifferent types of fat, i.e., five vegetable oil diets (5% corn oil, 20% cornoil, 20% olive oil, 20% linseed oil, 19% coconut oil-1% corn oil); twoanimal fat diets (20% lard, 19% beef tallow-1% corn oil); and one fishoil diet (19% Menhaden oil-1% corn oil). In addition, fish-corn oil diets(20%) containing three different levels of corn oil (15%, 10%, 4.5%) andfish oil (5%, 10%, 15.5%) were also examined in these studies. Immaturemice were fed these diets from 21 to 45 days of age, ovariectomized at35 days of age, given injections daily of 17/3-estradiol (1 Mg)and progesterone (1 mg) on Days 42 to 44, and sacrificed on Day 45. Mammaryductal expansion through the mammary fat-pad (mm, nipple to farthestend bud) was determined on the inguinal (No. 4) mammary glands.Mature mice were fed these diets from 28 to 128 days of age. Half ofthese mice were sacrificed between 118 and 128 days of age during thestage of estrus of the estrous cycle. The remaining half were giveninjections daily of 17|8-estradiol (1 fig) and progesterone ( 1 mg) from 118to 127 days of age and sacrificed on Day 128. Mammary developmentalgrowth was assessed on inguinal mammary glands by ascription ofdevelopment scores, determination of epithelial area (mm2), and deter
mination of total DNA levels. In both immature mice and mammotrophichormone-treated mature mice fed the fish oil diet (19% Menhaden oil-1% corn oil, 15.5% Menhaden oil-4.5% corn oil), significantly (P < 0.05)reduced developmental growth of the mammary gland was observed whencompared to mice fed the 19 to 20% vegetable oil or animal fat diets. Nosignificant difference in mammary gland developmental growth was observed among the groups of mice fed the 19 to 20% vegetable oil oranimal fat diets. In immature mice and mammotrophic hormone-treatedmature mice, significantly (/' < 0.05) reduced mammary gland develop
mental growth was observed in mice fed the 5% corn oil diet comparedwith mice fed the 20% corn oil diet. In mature mice not treated withexogenous mammotrophic hormones, no significant effect of diet onmammae development was observed. Mean body weight gains among thedietary groups of mice were not significantly influenced by diet. Thus,among the fat diets examined in this study, only the animals fed the lowfat diet (5% corn oil) or the fish oil diet (19% Menhaden oil-1% corn oil,15.5% Menhaden oil-4.5% corn oil) had altered (suppressed) mammaedevelopment. Furthermore, dietary induced suppression of mammarygland developmental growth was observed only in mice whose mammaewere in a state of intense proliferation (immature mice and mature micetreated with mammotrophic hormones); the type or amount of dietary fatdid not affect mammae development in mice possessing a relativelyquiescent mammary gland (during estrous cycle).
INTRODUCTION
It has been reported by numerous laboratories that theamount and type of dietary fat can significantly influence thedevelopment and/or growth of mammary tumors in rodents(1). In general, diets rich in certain vegetable oils (e.g., corn oil.
Received 1/26/89; revised 5/3/89. 7/14/89; accepted 7/25/89.The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' This research supported by NIH Research Grant CA-42876.2To whom requests for reprints should be addressed, at Department of
Pharmacology and Toxicology, Michigan State University, East Lansing. MI48824.
safflower seed oil, sunflower seed oil) markedly enhance mammary tumorigenesis, while diets rich in certain animal fats (e.g.,beef tallow, lard) are often less efficacious in stimulating thisneoplastic process (2-6). In contrast, diets rich in certain fishoils (e.g.. Menhaden oil) often do not appear to enhance mammary tumorigenesis in experimental animals (7-11).
In contrast, considerable less effort has been directed towarddetermining whether or not the amount or type of dietary fatcan affect normal mammary gland developmental processes inexperimental animals. Recently, we reported (12) that theamount of dietary fat can affect normal mammary gland growthprocesses in mature female BALB/c mice; i.e., as the fat content(corn oil) of the diet was increased from 0% to 5% to 20%, asignificant increase in hormone-induced mammary glandgrowth was observed. In this paper, we have extended thesestudies to determine whether or not the amount and type ofdietary fat can affect normal mammary gland growth processesin female BALB/c mice. Furthermore, we have examined boththe prepubertal (immature) and early postpubertal (mature)female BALB/c mouse as early alterations in developmentalgrowth of the mammary gland may have profound effects uponthe susceptibility of this tissue to initiating events in mammarygland neoplasia (13, 14). It is generally recognized that factorswhich increase normal mammae proliferative processes oftenenhance the vulnerability of this tissue to dysplasia and/orneoplasia (14, 15). For these studies we have chosen to examinefour vegetable oils (corn oil, olive oil, coconut oil, and linseedoil), two animal fats (beef tallow and lard), and one fish oil(Menhaden oil). Each of these fats has marked differences infatty acid composition and often differs sharply in its ability toinfluence neoplastic mammary gland growth processes.
MATERIALS AND METHODS
A total of 599 nulliparous female BALB/c mice were used in thesestudies. The mice were obtained from Charles River Breeding Laboratories, Inc., Wilmington, MA. They were housed in a temperature-controlled (25.5 ±0.5°C)and light-controlled (14 h/day) room.
Diet Composition
The diets used in these studies are purified semisynthetic dietsprepared in our laboratory every 2 wk (fish diets weekly), stored inplastic bags containing nitrogen gas, and kept frozen prior to feeding.The composition of the diets and percentage by weight are as follows.The low fat diet consists of corn oil (5%), casein (17.10%), DL-methi-onine (0.30%), dextrin (45.50%), sucrose (22.70%), AIN salt mixture(3.50%), AIN vitamins (1.00%), and cellulose (5.00%). The high fatdiets (20%) consist of 4 vegetable oils, i.e., corn oil (20%). olive oil(20%), linseed oil (20%), coconut oil (19%)-corn oil (1%); 2 animalfats, i.e., lard (20%), beef tallow (19%)-corn oil (1%); and a series offish oil (Menhaden oil)-corn oil diets, i.e., fish (19%)-corn oil (1%), fish(15.5%)-corn oil (4.5%), fish (10%)-corn oil (10%), and fish (5%)-cornoil (15%). The components of the high fat diets (20%) included casein(20.17%), DL-methionine (0.35%), dextrin (32.18%), sucrose (16.09%),AIN salt mixture (4.13%), AIN vitamins (1.18%), and cellulose(5.90%). One very high fish oil-corn oil diet (3090) was used, i.e., fishoil ( 19.75%) and corn oil (10.25%); the components of this diet includedcasein (22.23%), DL-methionine (0.39%), dextrin (23.47%), sucrose
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DIETARY FAT AND MAMMARY GLAND DEVELOPMENT
(11.56%), A1N salt mixture (4.55%), AIN vitamins (1.30%). and cellulose (6.50%). All the diets are isocaloric; i.e., the proportions ofprotein, minerals, vitamins, and fiber were adjusted to keep their ratioconstant with respect to calories. All mice were fed ad libitum daily;nonconsumed diet was discarded daily. All of the dietary constituentswere obtained from U.S. Biochemical Corp. (Cleveland, OH) exceptbeef tallow and sucrose which were obtained from ICN Biomedicals,Inc. (Costa Mesa. CA) and fish oil (Menhaden) which was obtainedfrom Zapata Haynie Corp. (Reedville, VA). The concentrations (percentage) of the predominant fatty acids (1% or greater, manufacturer's
specifications) of the dietary oils and fats are shown in Table 1.
Dietary Fat and Mammary Gland Developmental Growth in ImmatureMice
Table 3 Interaction between dietary corn oil and Menhaden oil on mammarygiunti development in immature female BALB/c mice
Treatment"Corn
oil (5)*
Corn oil (20)Corn oil (15)/Menhaden oil (5)Corn oil (10)/Menhaden oil (10)Corn oil (4.5)/Menhaden oil
(15.5)Corn oil (19.75)/Menhaden oil
(10.25)No.
ofmice10
10109
910Body
wt(termination)17.9
+ 0.4'
17.8 ±0.518.3 ±0.418.2 ±0.618.1±0.618.3
±0.5Mammary
glanddevelopmental growth
(nipple to endbud distance,mm)10.91
±0.78(3)''
12.79 ±0.47 (b)12.12 ±0.6210.73 + 0.61 (a)10.93 ±0.65(a)11.
30 ±0.58
fl Female BALB/c mice were fed the diets from 21 to 45 days of age. At 35
days of age, all mice were ovariectomized. From 42 to 44 days of age, all micewere given injections s.c. daily of 17/i-estradiol (1 ng) and progesterone (1 mg).
Experiment 1. The experimental design of this study is provided inTable 2. Diets were fed to mice 21 to 45 days of age. At 35 days of age.each mouse was ovariectomized. Commencing at 42 days of age, eachmouse received 3 consecutive days of hormone treatment (17/j-estradiolplus progesterone, injected s.c) for the purpose of providing a uniformovarian hormonal milieu. The steroids (Sigma Chemical Co., St. Louis.MO) were mixed with gum arabic (Sigma Chemical Co.); the hor-mone:gum arabic mixture was dissolved in 0.9% NaCI solution. At 45days of age, all mice were sacrificed, and their inguinal (No. 4) mammary glands were excised, fixed in glacial acetic acid: 100% ethanol(1:3, v/v), and stained with alum carmine (Sigma Chemical Co.) forwhole-mount evaluation. The extent of mammary duct expansionthrough the mammary fat-pad of each gland was determined.
Experiment 2. The experimental design of this study is provided inTable 3. The design of this study is identical to that described inExperiment 1 except that only 2 dietary fats (oils) are examined (cornoil and Menhaden oil); such fats were blended together in 4 differentratios. Certain diets were formulated to ensure equal linoleic acidlevels.Table
1 Predominant fatty acids in oils and fats (percentage)Fatty acid concentrations of less than 1''i are notincluded.Coco-
Lin- Men-Corn Olive nut seed Beef haden
Fatty acids oil oil oil oil Lard tallowoilCaprylic
(8:0) 5.8Capric(10:0) 6.6Laurie (12:0) 53.5Myristic(14.0) 18.4 1.5 3.4 8.0Palmitic (16:0) 10.1 11.3 7.9 5.4 25.3 25.5 28.9Palmitoleic(16:l) 2.5 2.8 7.9Stearici 18:0) 1.6 2.2 1.6 3.5 15.0 24.9 4.0Oleicd 8:1) 31.4 78.5 5.0 19.0 44.5 35.7 13.4Linoleic (18:2) 56.3 7.2 1.0 24.0 9.3 1.6 1.1Linolenic(18:3) 47.0 1.0Eicosapentaenoic (20.5) 10.2Docosahexaenoic (22:6)12.8Table
2 Influence of dietary fat on mammary gland development in immaturefemale BALB/c miceMammary
gland developmentalNo. of Body wt growth (nipple to end
Treatment" mice (termination) bud distance,mm)Corn
oil (5)* 20 19.1 0.4C(a)" 8.94 ±0.45 (a)
Corn oil (20) 20 20.1 0.4 (a) 10.59 ±0.43 (a)Olive oil (20) 20 19.8 0.8 (a) 9.51 ±0.79 (a)Linseed oil (20) 20 19.8 0.7 (a) 9.31 ±0.58 (a)Coconut oil ( 19) + 20 20.2 0.5 (a) 9. 18 ±0.59 (a)
corn oil ( 1)Lard (20) 20 19.9 0.6 (a) 8.99 ±0.61 (a)Beef tallow (19)+ 18 20.0 0.3 (a) 10.32 ±0.63 (a)
corn oil (1)Menhaden oil ( 19) + 20 18.0 ±0.4 (b) 6. 18 ±0.60 (b)
corn oil(1)°Female BALB/c mice were fed the diets from 21 to 45 davs of age. At35days,
all mice were ovariectomized. From 42 to 44 days of age. all mice weregiven injections s.c. daily of 17|i-eslradiol (1 /jg) and progesterone (1 mg). Themice »eresacrificed at 45 days of age. and both inguinal mammary glands (No.4) were excised and examined for developmental growth.
* Numbers in parentheses, percentage.' Mean ±SE.rfa/b. P < 0.05.The
mice were sacrificed at 45 days of age. and both inguinal mammary glands(No. 4) were excised and examined for developmental growth.
* Numbers in parentheses, percentage.c Mean ±SE."a/b, P <0.05.Table
4 Influence of dietary fat on mammary gland development in maturefemale BALB/c miceMammary
Body wt glandNo. of (termin- epithelial Mammary gland
Treatment" mice ation) area (mm2) developmentscoresCorn
oil (5)* 10 24.4 ±0.8C 33.7 ±3.5 2.45 [2.0-3.5]'' (a)'Corn oil (20) 9 23.9 + 0.7 36.9 ±2.1 2.50 [1.5-3.5] (a)Olive oil (20) 10 23.7+1.0 36.4 ±2.7 2.50 [1.5-3.5] (a)Linseed oil (20) 8 23.7 ±0.6 31.7 ±1.4 2.13 [1.5-3.0)Coconut oil (19)+ 9 24.8 + 0.5 34.4 ±3.3 2.10(1.5-3.0)
corn oil (1)Lard (20) 10 23.4 ±0.7 34.5+1.6 2.70 [1.5-4.0] (a)Beef tallow (19)+ 9 23.5 ±0.7 37.8 ±3.1 2.30(1.5-3.5]
corn oil (1)Menhaden oil (19)+ 9 22.5 ±0.8 37.9 ±2.1 1.83 [1.5-2.0] (b/
corn oil(1)°Female BALB/c mice were fed the diets from 28 days of age to 118 to 128
days of age. All mice were sacrificed in estrus between 118 and 128 days of age,and both inguinal mammary glands (No. 4) were excised and examined fordevelopmental growth.
* Numbers in parentheses, percentage.c Mean ±SE.**Numbers in brackets, range.' a/b, P < 0.05.rSee"Results."i.e.,
the 5% corn oil/4.5% corn oil-15.5% Menhaden oil diets and the20% corn oil/19.75% corn oil-10.25% Menhaden oildiets.Dietary
Fat and Mammary Gland Developmental Growth in MatureMice: No HormoneTreatmentExperiment
1. The experimental design of this study is provided inTable 4. Diets were fed from 28 to 118-128 days of age. Between 118and 128 days of age, all mice were sacrificed during the stage of estrusof the estrous cycle, and their inguinal (No. 4) mammary glands wereexcised and prepared for whole-mount evaluation. Developmentalgrowth of the mammary glands was assessed on each gland by ascriptionof mammary gland development scores and determination of mammarygland epithelialarea.Dietary
Fat and Mammary Gland Developmental Growth in MatureMice: Mammotrophic HormoneTreatmentExperiment
1. The experimental design of this study is provided inTable 5. Diets were fed from 28 to 128 days of age. Commencing at118 days of age, 17/i-estradiol plus progesterone was injected s.c. dailyfor 10 days; all mice were sacrificed at 128 days of age. At sacrifice,theinguinal
(No. 4) mammary glands were excised and prepared for whole-mount evaluation. Developmental growth of the mammary glands wasassessed on each gland by ascription of mammary gland developmentscores, determination of mammary gland epithelial area, and determination of mammary gland DNA levels.
Experiment 2. The experimental design of this study is provided in
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DIETARY FAT AND MAMMARY GLAND DEVELOPMENT
Table 5 Influence of dietary fat on mammary gland development in mature female BALB/c mice treated with mammotrophic hormones
Treatment"Corn
oil (5)*
Corn oil (20)Olive oil (20)Linseed oil (20)Coconut oil ( 19) + corn oil ( 1)Lard (20)Beef tallow (19) + corn oil ( 1)Menhaden oil (19) + corn oil
(1)No.
ofmice20
20192020202020Body
wt (termination)27.6
0.5C
26.8 0.926.8 0.927.0 1.428.0 0.928.1 1.127.8 0.826.5 0.8Mammary
glandDNA(^g/gland)126.9
16.6123.1 15.5117.9 16.9114.7 11.4114.6 10.5123.3 17.9135.4 20.394.8 14.6Mammary
gland epithelialarea(mm2)51.0
±3.658.7 ±2.8 (a)60.2 4.152.3 2.663.4 2.5 (a)58.8 3.460.7 4.048.9 ±2.4 (b)Mammary
glanddevelopmentscores2.95
12.5-4.5]" (a)'3.63 [2.5-5.0] (b)3.32 [2.0-5.0] (b)3. 18 [2.0-5.0] (b)3.43 [2.5-5.0] (b)3. 10 [2.0-5.0] (b)3.55 [2.0-5.0] (b)2.38 12.0-3.5) (c)
" Female BALB/c mice were fed the diets from 28 to 128 days of age. 17/i-Estradiol (1 ng) and progesterone (1 mg) »ereinjected s.c. daily from 118 to 127 days
of age. The mice were sacrificed at 128 days of age. and both inguinal mammary glands (No. 4) were excised and examined for developmental growth.* Numbers in parentheses, percentage.' Mean ±SE.d Numbers in brackets, range.' a/b. b/c. P < 0.05.
Table 6. The design is identical to that described in Experiment 1(above), except that only 2 dietary fats (oils) are examined (corn oil andMenhaden oil); such fats were blended together in 4 different ratios.Mammary gland growth was assessed on each gland by ascription ofmammary gland development scores.
Immature Mice—Measurement of Mammary Duct Expansion throughthe Mammary Fat-Pad
Developmental growth of the mammary ducts was assessed in codedwhole-mount preparations by measuring the linear distance (mm) ofduct expansion through the mammary fat-pad, from the nipple to themost distant mammary end bud (see Fig. I). Distance of duct expansion(penetration) through the mammary fat-pad was determined by a computer-assisted image analysis system (R & M Biometrics Corp.. Nashville, TN).
Mature Mice—Ascription of Mammary Gland Development Scores
Development of the mammary ductal-alveolar epithelium was assessed in whole-mount preparations by a standard procedure (16).Mammary glands were coded and rated for development according tothe following criteria: 1, few ducts, few or no end ducts; 2, moderateduct growth, moderate number of end ducts; 3, numerous ducts ano"
branches, many end ducts; 4. numerous ducts and branches, minimallobuloalveolar growth; 5. numerous ducts and branches, moderatelobuloalveolar growth; and 6, numerous ducts and branches, denselobuloalveolar growth as in late pregnancy.
Mature Mice—Determination of Mammary Gland Epithelial Area
Mammary ductal-alveolar epithelial area (mm2) was determined incoded whole mounts of mouse mammary glands by a computer-assistedimage analysis system (R & M Biometrics Corp.) (12). A television
Table 6 Interaction between dietary corn oil and Menhaden oil on mammarygland development in mature female BALB/c mice treated »-¡Ihmammolrophic
hormones
Treatment"Corn
oil(5)*Corn
oil(20)Cornoil (15)/Menhadenoil(5)Corn
oil (I0)/Menhadenoil(10)Corn
oil (4.5)/Menhadenoil(15.5)Corn
oil(l9.75)/Menhadenoil(10.25)No.
ofmice101010101010Body
wt (termination)28.3
±0.7C29.5
±0.927.8±0.629.1
±0.828.2
±0.826.3
±0.9Mammary
glanddevelopmentsocres3.58
[2.0-5.0]''(a)'3.92[2.0-5.5](b)3.72
[2.0-4.5]3.36
[2.0-5.0]2.18
[1.5-3.5](c)3.00
(2.0-5.0] (d)
°Female BALB/c mice were fed the diets from 28 to 128 days of age. \lfi-
Estradiol (I Mg)and progesterone (1 mg) were injected s.c. daily from 118 to 127days of age. The mice were sacrificed at 128 days of age. and both inguinalmammary glands (No. 4) were excised and examined for developmental growth.
* Numbers in parentheses, percentage.' Mean ±SE.* Numbers in brackets, range.' a/c. b/c. b/d. P < 0.05.
camera attached to a low-power microscope generates an image of thetissue section consisting of light (fat-pad) and dark (epithelium) points.
This image is relayed to a computer (Zenith Data Systems), whichcounts the number of dark points. These data are then converted toarea (mm2) measurements. The total epithelial area for each gland was
determined. Mouse mammary glands are ideal for such analysis as theepithelium (ducts and alveoli) of the mammae stain dark red (alumcarmine), contrasting exceptionally well with the nonstaining surrounding adipose tissue (fat-pad). The positive aspect of this methodology inthe assessment of mammae developmental growth is its objectivity. Thedisadvantages of this technique are 3-fold; i.e., it is relatively timeconsuming (=45 min/gland), occasionally the computer will recorddark points that do not represent epithelium (e.g., blood vessels), andthe ductal epithelium in the lower depths of the fat-pad may yieldslightly fewer numbers of dark spots than the ductal epithelium thatlies in the upper fat-pad regions. A skilled technician can correct, to
large extent, for these difficulties.
Mature Mice—Determination of Mammary Gland DNA Levels
Mammary gland DNA levels were determined on coded wholemounts of mouse mammary glands by a standard procedure (17). Thelymph nodes were excised from each gland, and water and lipids wereextracted from trichloroacetic acid-precipitated materials by sequentialwashings (4°C)with l M sodium acetate in methanol, metha-
nohchloroform (2:1, v/v), absolute ethanol, and ethyl ether. The resulting dry fat-free tissue was washed with cold 10% perchloric acid and
then treated with hot 5% perchloric acid (30 min); the DNA content inthe supernatant was determined by a diphenylamine reaction. TotalDNA content of the mammary gland in the mature rodent is an indexof mammae growth when the ductal structures are in an active state ofproliferation and expansion, i.e., after the administration of exogenousmammotrophic hormones or during the physiological state of pregnancy (18). In the virgin mouse, only =20% of the DNA in the wholemammary gland (inguinal minus lymph nodes) is derived from ductalepithelium; after hormonal stimulation (e.g., late pregnancy), >90% ofthe DNA of the gland is derived from the ductal-alveolar component.The amount of mammary epithelial DNA, from the virgin state to thestate of late pregnancy, increases approximately 27 times (19). Thus,this method of quantitatively assessing mammary ductal-alveolar developmental growth is useful when examining the mammae of hormon-ally stimulated mature female mice.
Statistics
Multigroup mean comparisons were analyzed by one-way analysis ofvariance in concert with the Newman-Keuls multiple comparison test.Student's t test was used when comparing paired group means. Mam
mary gland development score means were analyzed by the Wilcoxinsigned rank test. Unless otherwise noted, significance was set at P <0.05.
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DIETARY FAT AND MAMMARY GLAND DEVELOPMENT
distance fromnipple to farthestend bud (mm) lymph
node
nipple
Fig. I. Diagramatic sketch of an inguinal (No. 4) mammary gland from animmature female mouse. The length (mm) of the curved line, from the nipple,through the lymph node and extending to the farthest end bud was determinedfor each inguinal (No. 4) mammary gland (immature mice). Fig. 2. Representative whole mount of an inguinal (No. 4) mammary gland
from a fish oil (19% Menhaden oil-1% corn oil)-fed immature mouse. Distancefrom nipple to farthest end bud is 7.4 mm.
Fig. 3. Representative whole mount of an inguinal (No. 4) mammary glandfrom a vegetable oil (20% corn oil)-fed immature mouse. Distance from nipple tofarthest end bud is 10.9 mm.
Fig. 4. Whole mount of an inguinal (No. 4) mammary gland from a fish oil(19% Menhaden oil-1% corn oil)-fed immature mouse. Distance from nipple tofarthest end bud is 3.4 mm. Approximately 15% of the fish oil-fed immature micehad profound inhibition of mammary duct expansive growth as shown in thisphotograph. Such inhibition of duct growth was observed only in fish oil-fedmice.
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DIETARY FAT AND MAMMARY GLAND DEVELOPMENT
RESULTS
Effect of Dietary Fat on Developmental Growth of the Mammary Gland in Immature Mice. Developmental growth of themammary gland in immature mice was assessed by measuringthe expansive growth of the ductal system through the fat-pad.This was accomplished by determining the distance (mm) fromthe nipple to the farthest extended end bud (see Fig. 1). Therewas no significant difference among the groups of mice fed thevegetable oil and/or animal fat diets in mean nipple to end buddistance (range, 8.99 to 10.59 mm) (Table 2). Ductal expansivegrowth in mice fed the fish oil diet, however, was significantly(P < 0.05) reduced (mean, 6.18 mm) when compared withductal growth in animals fed the vegetable oil or animal fatdiets (Table 2) (Fig. 2 to 4). In mice fed the low fat diet (cornoil, 5%), ductal growth was less (mean, 8.94 mm) than that ofmice fed the high fat diet (corn oil, 20%) (mean, 10.59 mm),but this difference just missed the 5% level of statistical probability (P < 0.07) (Table 2).
The corn oil/fish oil diet group components of this studywere repeated (10 mice/group). Nipple to end bud distances(mm) were 7.80 ±0.49, 11.31 ±0.47, and 9.50 ±0.52 in the5% corn oil, 20% corn oil, and 19% Menhaden oil-1% corn oildietary groups, respectively. Mean body weights (g) at thetermination of the study were 16.7 ±0.4, 17.0 ±0.6, and 17.8±0.6, respectively, for the mice in these dietary groups. Nippleto end bud distance was significantly (P< 0.05) less in mice fedthe 5% corn oil diet and fish oil diet compared with mice fedthe 20% corn oil diet. Combining both of these experiments,nipple to end bud distances in the 5% corn oil diet group andfish oil diet groups were significantly (P < 0.05) less than thatobserved in the 20% corn oil group; no significant difference innipple to end bud distance was observed between the 5% cornoil and fish oil diet groups.
The effects of different ratios of dietary corn oil and fish oilon expansive growth of the ductal system in immature mice areshown in Table 3. Corn oil/fish oil dietary ratios of 10%/10%or4.5%/15.5%, respectively, significantly (P< 0.05) decreasednipple to end bud distance when compared to mice fed a 20%corn oil diet. Mice fed the 5% corn oil diet had significantly (P< 0.05) reduced nipple to end bud distances when compared tomice fed the 20% corn oil diet.
Effect of Dietary Fat on Developmental Growth of the Mammary Gland in Mature Mice: No Hormone Treatment. Developmental growth of the mammary gland in mature mice havingnormal estrous cycles was assessed by ascription of mammarygland development scores and determination of mammarygland epithelial area. There was no significant difference amongthe groups of mice fed the vegetable oil and/or animal fat dietsin development scores or epithelial areas (Table 4). Mammarygland development scores were significantly (P< 0.05) reducedin mice fed the fish oil diet compared with mice fed the 20%corn oil, olive, oil, and lard diets although no significant different in mammary gland epithelial areas was observed amongany of the groups of mice.
The corn oil/fish oil diet group components of this studywere repeated (10 mice/group). Mean mammary gland development scores were 2.05 (1.5 to 2.5), 2.10 (1.5 to 2.5), and 1.9(1.5 to 2.5) for the 5% corn oil, 20% corn oil, and 19%Menhaden oil-1% corn oil dietary groups, respectively. Meanmammary gland epithelial areas (mm2) were 28.7 ±1.3, 27.0
±2.0, and 30.3 ±1.8, respectively, for these dietary groups.Mean body weights (g) at the termination of the study were25.5 ±0.7, 23.2 ± 1.0, and 24.7 ±0.8, respectively. No
significant effect of these diets on mammary gland developmentscores or epithelial areas was observed. Combining both ofthese studies, no significant effect on developmental growth ofthe mammary gland was observed among any of these groupsof mice, irrespective of the amount or type of dietary fat.
Effect of Dietary Fat on Developmental Growth of the Mammary Gland in Mature Mice: Treatment with MammotrophicHormones. Developmental growth of the mammary gland inmature mice treated with mammotrophic hormones (17/i-estra-diol and progesterone) was assessed by ascription of development scores (Figs. 5 to 8), determination of epithelial area,and/or determination of total DNA levels. Mean mammarygland development scores or mean mammae epithelial areaswere not significantly different among the groups of mice fedthe vegetable oil or animal fat diets (Table 5). Mice fed the fishoil diet had significantly (P < 0.05) reduced mammary glanddevelopment scores when compared to mice fed the vegetableoil or animal fat diets; mean mammary gland epithelial areawas significantly (P < 0.05) less in mice fed the fish oil dietscompared with mice fed the 20% corn oil or coconut oil diets.Mean total mammary gland DNA level was numerically thelowest in mice fed the fish oil diet; however, no significantdifferences among the dietary groups were observed when utilizing this growth parameter. Mammary gland developmentscores were significantly (P < 0.05) reduced in mice fed the 5%corn oil diet compared with mice fed the 20% corn oil diet;mean mammae epithelial area was less in the 5% corn oil dietgroup compared with the 20% corn oil group, but this differencedid not reach the 5% level of statistical probability.
The corn oil/fish oil diet group components of this studywere repeated (10 mice/group). Mean mammary gland development scores for the 5% corn oil, 20% corn oil, and 19%Menhaden-1% corn oil dietary groups were 2.60 (2.0 to 3.0),3.40 (2.5 to 4.5), and 2.45 (2.0 to 3.5), respectively. The meanmammary gland epithelial areas (mm2) for these dietary groups
were 59.2 ±2.9, 69.7 ±5.8, and 59.8 ±3.6, respectively. Meanmammary gland DNA (//g/gland) levels for these dietary groupswere 235.4+17.1, 234.9 ±31.4,and 184.3 ±20.1, respectively.Mean body weights (g) at the termination of the study were28.2 ±0.8, 28.5 ±0.6, and 27.5 ±0.5 Mean mammary glanddevelopment scores were significantly (P< 0.05) reduced in the5% corn oil and fish oil dietary groups, compared with the 20%corn oil group; mean mammary gland epithelial areas were alsoreduced in these groups, compared with the 20% corn oil group,but this difference did not quite reach the 5% level of statisticalprobability (P < 0.10). The mean mammae DNA level wasnumerically less in the fish oil diet group compared with the20% corn oil group, but this difference was not statisticallysignificant. Combining both of these experiments, mean mammary gland development scores and mean mammae epithelialareas were significantly (P < 0.05) less in the 5% corn oil andfish oil diet groups when compared with the 20% corn oil dietgroup.
The effects of the different ratios of dietary corn oil and fishoil on developmental growth of the mammae in mammotrophichormone-treated mature mice are shown in Table 6. Meanmammary gland development scores were significantly (P <0.05) reduced in the mice fed a corn oil/fish oil dietary ratio of4.5%/15.5% or 19.75%/10.25%, respectively, compared withmice fed a 20% corn oil diet. Mice fed a corn oil/fish oil dietaryratio of 4.5%/15.5%, respectively, had significantly (P < 0.05)less mammae development than mice fed a 5% corn oil diet.Mammae epithelial areas or total DNA levels were not assessedin this study.
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6
Fig. 5. Whole mount of an inguinal (No. 4) mammary gland from a mam-motrophic hormone-treated mature female mouse. Mammary gland developmentscore is 2.0. This gland is characterized as having moderate duct growth and amoderate number of end ducts. This gland was obtained from a fish oil (19%Menhaden oil-1% corn oil)-fed mouse.
Fig. 6. Whole mount of an inguinal (No. 4) mammary gland from a mammotrophic hormone-treated mature female mouse. Mammary gland developmentscore is 3.0. This gland is characterized as having numerous ducts and branchesand many end ducts. This gland was obtained from a com oil (5%)-fed mouse.
8
Fig. 7. Whole mount of an inguinal (No. 4) mammary gland from a mam-motrophic hormone-treated mature female mouse. The mammary gland development score is 4.0. This gland is characterized as having numerous ducts andbranches with minimal lobuloalveolar development. This gland was obtained froma corn oil (20%)-fed mouse.
Fig. 8. Whole mount of an inguinal (No. 4) mammary gland from a 111:1111motrophic hormone-treated mature female mouse. The mammary gland development score is 5.0. This gland is characterized as having numerous ducts andbranches with moderate lobuloalveolar development. This gland was obtainedfrom a corn oil (20%)-fed mouse.
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DIETARY FAT AND MAMMARY GLAND DEVELOPMENT
Body Weight Gains. There was no significant difference inbody weight gains among any of the dietary groups of mice(immature mice, mature mice, or mammotrophic hormone-
treated mature mice) with but one exception. The group ofimmature mice fed the fish oil diet in Experiment 1 (Table 2)had significantly (P < 0.05) less body weight gains than did thegroups of mice fed the 20% vegetable or animal fat diets. Bodyweight gains among the groups of mice fed the fish oil diets ineach of the other experiments were not significantly altered.
DISCUSSION
In this study, high levels of corn oil, olive oil, coconut oil,linseed oil, lard, beef tallow, and fish oil were fed to groups ofimmature and mature female BALB/c mice. Only the mice fedthe fish oil diets showed altered (suppressed) mammary glanddevelopment. Furthermore, fish oil-induced, suppressed mammae development was observed only in mice whose mammaryepithelium was in a state of intense proliferation (immaturemice and mammotrophic hormone-treated mature mice). Importantly, no significant difference in mammae developmentwas observed in groups of immature or mature mice fed theother fat (oil) diets; mammary gland development and growthamong these groups of mice were indistinguishable.
The first laboratory to study and report the effect of type ofdietary fat on normal mammary gland developmental processeswas that of Abraham et al. (20). They reported that femaleBALB/c mice fed a high level of corn oil had considerably moremammary gland ductal epithelial growth than did mice fed acomparable level of hydrogenated cottonseed oil. The hydro-genated cottonseed oil is deficient in essential fatty acids; essential fatty acid deficiency in mice results in suppressed mammarygland development (21, 22), a phenomenon that can be reversedby the sole administration of linoleic acid (21). To circumventthis problem, we added a small amount of corn oil, which isrich in linoleic acid, to each of our high fat diets (coconut oil,beef tallow, and Menhaden oil) that contain marginal levels ofthis essential nutrient. It is our experience that mice fed beeftallow or fish oil diets (20%), as their sole source of dietary fat,have significantly reduced body weight gains. In the presentstudy, body weight gains were comparable among all dietarygroups, in immature mice as well as mature mice. Thus, thesignificant inhibitory effect of dietary fish oil on mammarygland development in immature and mature mice was observedin animals with a normal rate of body weight gain. In ourstudies we also observed a suppression of mammary glanddevelopment in mice fed a low fat diet (5% corn oil) comparedwith mice fed a high fat diet (20% corn oil). This growthdifferential was observed only in mice bearing a proliferatingmammary gland (immature mice and mammotrophic hormone-treated mature mice) and is consistent with a previous reportfrom our laboratory (12) and by others (23).
The mechanism by which dietary fish oil suppresses mammary gland developmental processes is uncertain. The resultsof our study clearly demonstrate that the rate of ductal proliferation (expansive growth through the mammary fat-pad) issignificantly reduced in mice fed a diet rich in fish oil whencompared with mice fed, e.g., a diet rich in corn oil. Thus, ourdata clearly support the concept that changes in dietary fatcomposition act by influencing mammae ductal cell proliferation. It is germane to point out that Abraham et al. could notdemonstrate any significant difference in DNA synthesis or cellcycle kinetics (3, 11, 24) of mouse mammary tumors as afunction of the type of dietary fat (e.g., corn oil versus hydro
genated cottonseed oil or fish oil); diet was shown only to affectthe rate of tumor cell loss (11, 24). Thus, according to theconcept proposed by Abraham et al., increased mammary tumorsize, as a function of type of dietary fat, is a reflection primarilyof cell loss, not proliferative processes. This is a very importantconcept, one that clearly needs to be confirmed and is indirectlysupported by a number of laboratories who report that the fatcontent of the diet can significantly affect immune systemmechanisms (25-28). In our study, utilizing the normal mammary gland, dietary fish oil clearly suppressed mammae proliferative processes. It is unlikely that immune system dynamicswould modulate normal mammae development; an interactionwith neoplastic mammae, in contrast, is far more likely.
Altered mammae proliferative processes, as a function of thefat content of the diet, can be explained by a number ofmechanisms. Potential mechanisms include the generation oflipid peroxy radicals and/or oxygen radicals (29), alteration inmembrane fluidity (30), changes in intercellular communication(31), alterations in mammotrophic hormone secretion (32, 33),and enhancement of hormone and/or growth factor responsiveness (12). In recent years, Abraham and coworkers (3, 11, 20,22, 24, 34) and others (35-38) have offered the hypothesis thatdietary fat, at least in part, may affect normal (and neoplastic)mammary gland growth processes by influencing, directly orindirectly, prostaglandin biosynthesis. Certain dietary fish oils,e.g., Menhaden oil, are especially rich in long chain n-3 fattyacids such as EPA1 (20:5) and DHA (22:6). EPA and DMA
modify linoleic acid (18:2) and arachidonic acid (20:4) metabolism, thus sharply interfering with prostaglandin biosynthesis(39-41). It is conceivable, therefore, that the inhibitory effectof dietary Menhaden oil on the developmental growth of themouse mammary gland, as observed in our study, is manifestedvia an inhibition of linoleic acid utilization. Although ourMenhaden oil diet was supplemented with corn oil (1%), ensuring adequate recommended daily allowance levels of linoleicacid, the rather large amounts of EPA and DHA in this dietcould interfere with linoleic acid utilization. Indeed, in ourstudy, suppressed normal mammae developmental growth wasobserved in mice fed dietary corn oil/Menhaden ratios of 1:3(4.5% corn oil:15.5% Menhaden oil) and even 2:1 (19.75% cornoil: 10:25% Menhaden oil). Thus, if the EPA-DH A/linoleic acidutilization concept is correct, then EPA and/or DHA appearsto be effective even when diets contain an abundance of linoleicacid. It is important to point out, in addition, that our observedinhibitory effect of dietary Menhaden oil on mouse mammaedevelopmental growth cannot solely be attributed to the broadclass of n-3 fatty acids, as high dietary levels of linseed oil didnot, whatsoever, affect this developmental process. Linseed oilis rich (47%) in linolenic acid (18:3, n-3)). Although linolenicacid can inhibit the metabolism of arachidonic acid, via cycloox-ygenase, and the conversion of linoleic acid to arachidonic acid,this n-3 fatty acid does not appear to be as effective as EPA/DHA in the suppression of eicosanoid formation from arachidonic acid (42).
It is important to point out that we could find no correlationbetween the linoleic acid content of the diet and developmentalgrowth of the mouse mammary gland. Widely varying linoleicacid levels (percentage) of s 11.2, 1.4, 0.8, 4.8, 1.9, and 0.9 arefound in our corn oil, olive oil, coconut oil, linseed oil, lard,and beef tallow, respectively, high fat diets; such diets did notdifferentially affect the developmental growth of the mousemammary gland. These results are in contrast with those of a
3The abbreviations used are: EPA. eicosapentaenoic acid; DHA, docosahex-
aenoic acid.
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number of laboratories that report a direct relationship betweenthe level of dietary linoleic acid and rodent mammary tumordevelopmental growth stimulation (36, 43-47). It is conceivable, therefore, that the neoplastic mammary gland is moresensitive to dietary linoleic acid than the proliferating mousemammary gland. It is also interesting to note that mice fed thelow fat diet (5% corn oil) had less mammary gland developmental growth than did mice fed any of the high (20%) fat diets(except Menhaden oil). The linoleic acid content of the low fatdiet is 2.8%, a level of linoleic acid that is higher than most ofthe high fat diets used in our study. It is difficult to explain thisdifference on the basis of calories, as these diets were formulatedto be isocaloric. The mechanism by which diets low in fat butcontaining adequate levels of essential fatty acids suppressmammae developmental processes remains to be determined.
Factors which influence developmental growth processes ofthe normal mammary gland can markedly influence the susceptibility of this tissue to neoplastic transformation. This is particularly apparent in young or immature rodents, where theadministration of mammotrophic hormones, or an early pregnancy, can significantly alter (enhance or suppress) chemicalcarcinogenesis of the mammary gland (14, 48, 49). In general,those factors which enhance mammae epithelial proliferationand/or inhibit mammae differentiation, increase the susceptibility of this epithelium to a carcinogenic stimulus (14, 15). Itbecomes important, therefore, to identify those factors whichhave the ability to influence developmental growth processes ofthe normal mammary gland. The results of our study provideevidence that developmental growth of the mouse mammarygland can be significantly affected (suppressed) by diet, but byonly rather extreme dietary intervention, i.e., by reducing fatconsumption by 75% (20% to 5%) or by feeding very high levelsof certain fish (Menhaden)-derived oils that are extremely richin n-3 polyunsaturated fatty acids such as EPA and/or DHA.The feeding of high dietary levels of four different vegetableoils (corn oil, olive oil, coconut oil, linseed oil) and two differentanimal fats (lard and beef tallow), all strikingly different in fattyacid composition, did not differentially affect developmentalgrowth of the mouse mammary gland.
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1989;49:5999-6007. Cancer Res Clifford W. Welsch and Deborah H. O'Connor Micethe Mammary Gland in Immature and Mature Female BALB/c Influence of the Type of Dietary Fat on Developmental Growth of
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