Content uploaded by Lilian U Thompson
Author content
All content in this area was uploaded by Lilian U Thompson on Mar 11, 2014
Content may be subject to copyright.
Biochemical and Molecular Roles of Nutrients
Flaxseed and Its Lignan Precursor, Secoisolariciresinol Diglycoside, Affect
Pregnancy Outcome and Reproductive Development in Rats
1,2,3
Janet C. L. Tou, Jianmin Chen and Lilian U. Thompson
4
Department of Nutritional Sciences, University of Toronto, Toronto, ON, Canada M5S 3E2
ABSTRACT Flaxseed is the richest source of the mammalian lignan precursor secoisolariciresinol diglycoside
(SDG). Because lignans have estrogen agonist or antagonist properties, the objective of this study was to
determine whether feeding flaxseed to rats during a hormone-sensitive period has reproductive effects. Rat dams
were fed a basal diet or the basal diet supplemented with 10% flaxseed, 5% flaxseed or SDG at the level in 5%
flaxseed during pregnancy and lactation. At weaning, the offspring were fed the basal diet. Flaxseed had no effect
on pregnancy outcome except that the 10% flaxseed diet lowered birth weight (P , 0.05), compared with other
treatments, and produced hormonal effects. The female offspring had shortened anogenital distance, greater
uterine and ovarian relative weights, earlier age and lighter body weight at puberty, lengthened estrous cycle and
persistent estrus (P , 0.05), whereas the males had reduced postnatal weight gain and, at postnatal d 132, greater
sex gland and prostate relative weights (P , 0.05), suggesting estrogenic effects. In contrast, compared with the
basal diet, 5% flaxseed reduced immature ovarian relative weight by 29% (P , 0.05), delayed puberty by ;5d(P
, 0.05) and tended to lengthen diestrus, indicating an antiestrogenic effect. The SDG produced results similar to
those of 5% flaxseed, suggesting that lignans were responsible for the observed effects. Lignans were transferred
to the offspring via rat dam’s milk as indicated by the recovery of radioactivity in the offspring of lactating dams
given
3
H-SDG. Because flaxseed affects the reproductive development of offspring, caution is suggested when
consuming flaxseed during pregnancy and lactation. J. Nutr. 128: 1861–1868, 1998.
KEY WORDS:
●
flaxseed
●
lignans
●
secoisolariciresinol diglycoside
●
rats
●
reproduction
Phytoestrogens such as lignans and isoflavones, which act as
either estrogen agonists or antagonists (Bandbury and White
1954, Farnsworth et al. 1975), have generated interest because
of their potential use in hormone replacement therapy and
cancer prevention. Mammalian lignans are produced by the
action of gut microflora on precursors such as the plant lignan
secoisolariciresinol diglycoside (SDG).
5
Flaxseed, the richest
known source of SDG (Thompson et al. 1991 and 1997), has
been observed to decrease the mammary tumor incidence,
number and size in carcinogen-treated rats (Serraino and
Thompson 1992, Thompson et al. 1996a and 1996b). This was
attributed in part to the antiestrogenic properties of mamma-
lian lignans produced from SDG in flaxseed (Orcheson et al.
1998). However, along with health benefits, flaxseed and its
lignans may also have adverse effects because phytoestrogens
have been reported to produce infertility and hyperestrogeni-
zation in a number of species (Price and Fenwick 1985, Rick-
ard and Thompson 1997).
Dietary substances capable of altering hormone levels are of
concern during pregnancy, a hormone-sensitive period for
both the mother and offspring. During pregnancy, high estro-
gen levels are needed by the mother to establish and maintain
pregnancy by stimulating uterine changes to allow implanta-
tion, enhancing uterine growth to accommodate a growing
fetus and by acting as a trigger for parturition (Pasqualini et al.
1985). On the other hand, administration of estrogens to
pregnant animals has been found to cause detrimental effects
on pregnancy. These include decreased pregnancy weight gain
and food intake, which result in increased resorption or abor-
tion, prolonged gestation length and labor, leading to in-
creased stillbirth incidence, decreased litter size and decreased
birth weight, with the end result of decreased pup survival
(Zimmerman et al. 1991).
Pregnancy is also a hormone-sensitive period for the repro-
ductive development of the fetus. Sexual differentiation of the
genitalia and central nervous system (CNS) into either male
or female develops under the influence of hormones. In rats,
critical sexual differentiation occurs from gestation d 18 and
continues to postnatal day (PND) 10 (Manson and Kang
1989). Normally, the high maternal estrogen levels during
pregnancy are prevented from exerting hormone toxicity on
the fetus by both sex hormone-binding globulin (SHBG) and
a
-fetoprotein (AFP) in humans and by AFP in rats. By bind-
1
Presented in part at Experimental Biology 97, April 6–9, 1997, New
Orleans, LA [Tou, J.C.L. & Thompson, L.U. (1997) The effect of flaxseed on
reproductive development and function. FASEB J. 11: A420 (abs.)].
2
Support was provided by the Flax Council of Canada and the Natural
Sciences and Engineering Research Council of Canada.
3
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’’
in accordance with 18 USC section 1734 solely to indicate this fact.
4
To whom correspondence and reprint requests should be addressed.
5
Abbreviations used: AA, arachidonic acid; AFP,
a
-fetoprotein; AGD, ano-
genital distance; ALA,
a
-linolenic acid; CNS, central nervous system; EPA, eico-
sapentanoic acid; LA, linoleic acid; PG, prostaglandin; PND, postnatal day, SDG,
secoisolariciresinol diglycoside; SHBG, sex hormone binding globulin.
0022-3166/98 $3.00 © 1998 American Society for Nutritional Sciences.
Manuscript received 17 December 1997. Initial review completed 23 January 1998. Revision accepted 2 July 1998.
1861
by guest on June 7, 2013jn.nutrition.orgDownloaded from
ing estrogen, these proteins prevent the free estrogen from
interacting with receptors to produce hormonal effects. Those
estrogens not extensively bound to AFP tend to be potent
estrogenic toxins, capable of disrupting normal reproduction
in the rat (Pasqualini et al. 1985). Whitten et al. (1993 and
1995) reported that the phytoestrogen coumestrol could be
transferred to offspring postnatally via rat dam’s milk and
affect offspring reproduction at adolescence and later life.
Reproductive abnormalities include altered puberty onset, dis-
rupted estrous cycles and decreased fertility in female offspring,
and decreased sexual behavior in male offspring. Environmen-
tal estrogens to which humans are most likely exposed include
phytoestrogens in foods; there are trends toward their in-
creased consumption as a result of their reported health ben-
efits (Haumann 1997). The objectives of this study were to
determine the following: 1) the effect of feeding pregnant rats
5 or 10% flaxseed or its lignan precursor SDG at a level
equivalent to that in 5% flaxseed on rat dam’s pregnancy and
pregnancy outcome; 2) whether SDG can be transferred from
mother to offspring via the milk to produce reproductive
effects on the offspring; and 3) the long-term reproductive
effects and health implications of exposing offspring to flax-
seed during a period when hormone-dependent development
is occurring. The 5 and 10% flaxseed levels were chosen for
this study because they have been shown previously to be
colon and/or mammary cancer protective in rats (Jenab and
Thompson 1996, Serraino and Thompson 1991 and 1992,
Thompson et al. 1996a and 1996b).
MATERIALS AND METHODS
Chemicals. The SDG in flaxseed (Linott variety; Omega Prod-
ucts, Melfort, SK) was isolated and purified using a modification of
the Klosterman method (Bakke and Klosterman 1956, Klosterman
and Smith 1954) as described by Rickard et al. (1996). The benzyl
methylenes of purified SDG were labeled with tritium by Amersham
International (Little Chalfont, Buckinghamshire, UK) using a gas
exchange method, resulting in a specific radioactivity of 999 GBq/
mmol and radiochemical purity of .98.5%.
Animals. Twenty-eight female and seven male Sprague-Dawley
rats (77 d old; Charles River Canada, Montreal, Canada) were
individually housed in polycarbonate cages in 22–24°C rooms with
50% humidity and a 12 h light:dark cycle. Animals were acclimated
for 7 d during which they were given free access to water and fed a
phytoestrogen-free basal diet. At the end of acclimation, males
(353.71 6 21.15 g) and females (249.82 6 13.34 g) were mated. The
female rat, determined to be in estrus from microscopic examination
of vaginal smears, was placed with the male rat in a stainless steel cage
with a wire-mesh floor. One male was mated with one female from
each diet group. Successful mating was determined by the presence of
copulation plugs in the cage’s collection tray and confirmed by
microscopic observation of sperm in vaginal smears. Pregnant female
rats were individually housed in polycarbonate cages. Animal care
and use conformed to the published guidelines (Canadian Council on
Animal Care, 1984), and the experimental protocol was approved by
the University of Toronto Animal Care Committee.
Diets. Pregnant rat dams were randomly assigned to the basal diet
or basal diet supplemented with either 10% flaxseed, 5% flaxseed or
a daily gavage of 1.5 mg SDG dissolved in 1 mL distilled water. The
SDG level was estimated to be approximately equivalent to that
given to rats in the 5% flaxseed diets on the basis of a SDG concen-
tration of 2.93
m
mol/g flaxseed according to HPLC analysis (Thomp-
son et al. 1996a) and a diet intake of about 15 g/d. Rats not gavaged
with SDG were gavaged daily with 1 mL saline. The diet composition
was based on the AIN-93 G diet (Reeves et al. 1993). The basal diet
supplemented with flaxseed was corrected for protein, fat and fiber
contributed by flaxseed so that the energy value of the diets was the
same (Table 1). All ingredients were from Dyets, (Bethlehem, PA).
Diets were stored at 4°C and fresh diets were provided to the rats
every 2 d.
Experimental design. Pregnant rat dams (7 per diet group) were
given free access to their assigned diets, and pregnancy progression
was monitored by measuring daily maternal weight gain and food
intake throughout gestation. Parturition was classified as difficult if
delivery exceeded 2 h, blood was present after delivery and rat dams
failed to clean pups (Barrow 1990). Pregnancy outcomes measured
were litter size, birth weight and stillbirth incidence, calculated as
live birth index (a ratio of number of viable pups born/total number
of pups born 3 100) postnatal pup survival (the ratio of number of
viable pups at PND 21/total number of pups born 3 100), and
percentage of females (the number of female pups/total number of
pups born 3 100). After birth, rat dams continued to consume their
respective diets. Maternal weight, food intake and offspring weight
were measured every other day during the lactation period. At the
end of lactation, i.e., PND 21, all rat dams and two offspring (one
male and one female) per litter were killed by CO
2
inhalation. The
uterus and ovaries of female offspring, the testes and sex glands of the
male offspring and all of the major organs of offspring were removed,
blotted dry and weighed. Organ weights were expressed as relative
weight, i.e., wet organ weight/body weight. All remaining offspring
were weaned and fed the basal diet so that offspring were exposed to
flaxseed or SDG only during the rat dam’s pregnancy and lactation.
At PND 50, and again at PND 132, a subgroup of male offspring (n
5 6–10) and female offspring (n 5 6 –10) were killed by CO
2
inhalation to determine the effect of early flaxseed and SDG exposure
on reproductive and major organs at puberty and adulthood. As on
PND 21, each subgroup consisted of 2–3 offspring (1–2 of each sex)
per litter from each of the dams.
Lignan transfer to offspring. To determine whether mammalian
lignans from the rat dams could be transferred to the offspring
through the milk, on PND 20, lactating rat dams (n 5 3) from the
SDG-treated group were gavaged with
3
H-SDG (3.7 kBq/g body
weight) in 1 mL distilled water. A control group of lactating rat dams
from the SDG-treated group (n 5 3) were gavaged with 1.5 mg
unlabeled SDG in 1 mL distilled water. Two offspring (one male and
one female) per litter remained with their respective dam. After 24 h,
the rat dams and offspring were killed by CO
2
inhalation. Trunk
blood was collected and all tissues and gastrointestinal contents were
removed, weighed and stored frozen at 220°C. Blood and tissue
radioactivity were measured as described in detail by Rickard and
Thompson (1998). Briefly, 0.5 mL of a 9:1 (v/v) solution of hyamine
hydroxide (ICN Biomedicals, Aurora, Canada) in distilled water was
added to the tissues and incubated in a 60°C shaking water bath. For
blood samples, 300
m
L hyamine hydroxide in ethanol (1:2, v/v) was
added and samples incubated in a 60°C shaking water bath for 1 h.
Corrections for chemical and color quenching of samples were done
automatically by the LKB Wallac 1217 Rackbeta liquid scintillation
counter (Fisher Scientific, Ottawa, Canada) by using the sample
channels ratio technique. The ratio of counts for the sample in each
TABLE 1
Composition of the diets
Ingredient Basal
1
5% flaxseed
2
10% flaxseed
2
g/kg diet
Cornstarch 397.48 394.40 391.20
Casein 200.00 188.80 177.60
Soybean oil (TBHQ) 70.02 51.70 33.50
Dextrose 132.00 132.00 132.00
Sucrose 100.00 100.00 100.00
Cellulose 50.00 32.60 15.20
Mineral mix 93G AIN 35.00 35.00 35.00
Vitamin mix 93G AIN 10.00 10.00 10.00
L-Cystine 3.00 3.00 3.00
Choline bitartrate 2.50 2.50 2.50
Flaxseed — 50.00 100.00
1
Semipurified AIN-93 G (Reeves et al. 1993).
2
Modified semipurified AIN-93G.
TOU ET AL.1862
by guest on June 7, 2013jn.nutrition.orgDownloaded from
channel, i.e., section of the energy spectrum for tritium, varies lin-
early with efficiency for quenching solutions (Evans 1974). Extensive
color quenching of samples was avoided by bleaching colored tissue
samples with 100
m
L of 9.7 mol/L hydrogen peroxide and blood
samples with 0.5
m
L of 9.7 mol/L hydrogen peroxide and then
incubating the samples for another 30 min. After cooling, 15 mL of
the cocktail scintillator Cytoscint ES (ICN Biomedicals) and 0.5 mL
of 0.5 mol/L acetic acid were added. Tissue samples were placed in the
dark for 2 h; blood samples were placed in the dark for4dto
equilibrate before counting in the liquid scintillation counter. To
determine radioactivity in gastrointestinal contents, samples were
homogenized in 30 mL distilled water using a Polytron homogenizer
(Brinkman Instruments Canada, Mississauga, Canada). Aliquots (100
m
L) were added to 1 mL Cytoscint ES and incubated in a 60°C
shaking water bath for 2 h. After cooling, 15 mL of Cytoscint ES and
0.5 mL 0.5 mol/L acetic acid were added. Gastrointestinal samples
were counted after being in the dark for4dtoeliminate chemilu-
minescence from fecal porphyrins. Radioactivity values were adjusted
for background radioactivity and counting efficiency. Total body
radioactivity was calculated as the sum of tissue radioactivity 3
sample tissue weight/aliquot weight.
Growth and anogenital distance (AGD). Offspring weight gain
was measured every other day. Immature male rats were distinguished
from females by their longer AGD, the distance from the papilla to
the anus. Hormone imbalance in male or female offspring due to in
utero diet treatment was determined from the difference in the AGD
at PND 3 when compared with rats receiving the basal diet (con-
trols). Dietary effect on pup genitalia during lactation was determined
from the difference in AGD at PND 21 and at PND 3. AGD
measurements were also adjusted for body size by calculating relative
AGD, i.e., (AGD at PND 21 - AGD at PND 3)/body weight gain
from PND 3 to 21.
Puberty onset and estrous cycle. In female rats, the age and
weight at puberty onset as indicated by visible opening of the vagi-
nal aperture was determined. Vaginal smears were taken on PND
40–50 and PND 100 –132 and examined microscopically to deter-
mine estrous cycling. The estrous phase of animals was classified as
follows: 1) proestrus (mainly epithelial cells present); 2) estrus (main-
ly cornified cells present); 3) metestrus (cornified and leukocytes
present in large numbers); or 4) diestrus (mainly leukocytes present).
Rat estrous cycle length was determined as the number of days
required to complete the four phases of proestrus, estrus, metestrus
and diestrus. Rats were considered acyclic if they remained in one
phase for .10 d.
Histological examination of the prostate. The prostates of male
rats killed at PND 132 were fixed in 10% buffered formalin, processed
routinely and embedded in paraffin. Paraffin sections of 5
m
m thick-
ness were serially cut at 200-
m
m intervals. After deparaffinization in
toluene and rehydration in a graded series of ethyl alcohol, the
sections were stained with hematoxylin and eosin and then examined
under a light microscope.
Statistical analysis. All statistical analyses were done using Sig-
maStat Version 2.0 by Jandel Scientific (San Rafael, CA). Differ-
ences among diet treatment groups within the same age and gender
group were analyzed by one-way ANOVA. Two-way ANOVA was
used to determine the differences in weight due to diet treatment or
gender, the differences in AGD or relative AGD due to diet treat-
ment or gender, the difference in female relative sex organ weights
(i.e., uterine and ovarian weights) due to diet treatment or age and
the differences in male relative sex organ weights (i.e., testes and
accessory sex glands) due to diet treatment or age. Data not normally
distributed were analyzed nonparametrically using Kruskal-Wallis
one-way ANOVA on ranks. Post-hoc multiple comparison tests
included Tukey’s test (parametric) or Dunn’s test (nonparametric).
Differences were considered significant at P , 0.05. Results are
expressed as means 6
SEM.
RESULTS
Pregnancy outcomes. Compared with the basal diet (con-
trol), the 5% flaxseed, SDG at the level found in 5% flaxseed
or 10% flaxseed diet fed to pregnant rat dams throughout
gestation produced no significant differences in maternal food
intake, weight gain, gestation length or parturition and preg-
nancy outcomes such as litter size, live birth index, postnatal
pup survival and number of female vs. male offspring. An
exception was the 10% flaxseed diet, which caused signifi-
cantly lowered (P , 0.05) mean birth weight (4.73 6 0.07 g)
compared with the basal diet (5.14 6 0.10 g), 5% flaxseed
(5.06 6 0.08 g) and 1.5 mg SDG (5.24 6 0.07 g). All rat dams
killed at the end of lactation (PND 21) showed no organ
weight abnormalities, except for the significantly (P , 0.05)
larger relative uterine and ovarian weights in those fed 10%
flaxseed compared with the basal diet (Table 2).
Lignan transfer via milk. The mean total body radioactiv-
ity (995.98 6 201.61 Bq) in nursing male and female offspring
TABLE 2
The effect of exposure during pregnancy and lactation to
flaxseed or secoisolariciresinol diglycoside (SDG) on maternal
reproductive organ relative weights in rats
1
Dietary treatment
2
Uterus
3
Ovaries
3
g/100 g body wt
Basal diet 127.86 6 8.93
a
42.83 6 3.18
a
5% Flaxseed 152.64 6 6.49
ab
43.59 6 4.71
ab
SDG 155.80 6 10.15
ab
51.35 6 5.27
ab
10% Flaxseed 176.98 6 15.40
b
61.69 6 5.13
b
1
Data are means 6 SEM (n 5 7). Different letters indicate significant
differences (P , 0.05) by one-way ANOVA followed by Tukey’s test.
2
Pregnant rat dams were randomly assigned to basal diet, basal
diet supplemented with 10% flaxseed, 5% flaxseed or a daily gavage of
1.5 mg SDG in distilled water. Assigned diets were fed throughout 22 d
of gestation and 21 d of lactation.
3
At the end of lactation (postnatal d 21), rat dams were killed and
uterine and ovarian weights measured.
TABLE 3
The effect of exposure during pregnancy to flaxseed or
secoisolariciresinol diglycoside (SDG) on body weight,
anogenital distance (AGD) and relative AGD in
rats at postnatal day 3
1
Treatment
2
n Body weight AGD Relative AGD
3
g mm mm/g
Females
Basal diet 54 6.61 6 0.17 3.0 6 0.1
b
0.46 6 0.01
b
5% Flaxseed 54 6.55 6 0.17 3.1 6 0.1
b
0.48 6 0.02
b
SDG 44 6.75 6 0.20 3.2 6 0.1
b
0.49 6 0.02
b
10% Flaxseed 58 6.23 6 0.14 2.3 6 0.1
a
0.39 6 0.02
a
Males
Basal diet 42 6.50 6 0.10
b
5.0 6 0.1
b
0.78 6 0.02
5% Flaxseed 43 6.80 6 0.15
b
5.0 6 0.1
b
0.75 6 0.02
SDG 45 6.95 6 0.15
b
5.1 6 0.1
b
0.74 6 0.01
10% Flaxseed 47 5.84 6 0.12
a
4.2 6 0.1
a
0.73 6 0.02
1
Data are means 6 SEM. Different letters indicate significant differ-
ences (P , 0.05) within the same gender by one-way ANOVA followed
by Dunn’s test (nonparametric). Two-way ANOVA showed diet treat-
ment (P , 0.001) and gender effects (P , 0.001) on AGD and relative
AGD but insignificant treatment and gender interaction.
2
Pregnant rat dams were randomly assigned to basal diet, basal
diet supplemented with 10% flaxseed, 5% flaxseed or a daily gavage of
1.5 mg SDG in distilled water.
3
Relative AGD was calculated as anogenital distance/body weight.
FLAXSEED AND REPRODUCTION 1863
by guest on June 7, 2013jn.nutrition.orgDownloaded from
of rat dams gavaged with
3
H-SDG in distilled water was
significantly higher (P , 0.05) than that in the nursing
offspring of control rat dams gavaged with unlabeled SDG
(34.56 6 14.65 Bq), which in turn was not significantly
different than the value obtained for the distilled water con-
trols (32.56 6 8.23 Bq).
Body weight and anogenital distance. The body weight,
AGD and relative AGD measured at PND 3 were affected by
in utero exposure to flaxseed (Table 3). Female offspring had
shorter AGD compared with male offspring, and in utero, 10%
flaxseed exposure caused shortening (P , 0.05) of the AGD in
both sexes. Only the male offspring treated with 10% flaxseed
in utero had significantly (P , 0.05) lower body weights than
those in the basal diet, 5% flaxseed and 1.5 mg SDG diet
groups. Body weight was correlated to AGD (r 5 0.85, P
, 0.01); thus, when calculated as relative AGD (AGD/body
weight), the effect of 10% flaxseed on AGD persisted only in
the female offspring. A two-way ANOVA showed both diet
and gender effects (P , 0.001) on AGD and relative AGD.
Weight gain, change in AGD and change in relative AGD
from PND 3 to 21 were also affected by lactation exposure to
flaxseed (Table 4). Lactation exposure to 10% flaxseed signif-
icantly (P , 0.05) lowered body weight gain in male offspring
and shortened AGD in both sexes compared with other diet
groups. When adjusted for body weight by calculation of
relative change in AGD, diet effects on AGD persisted only in
females. Compared with the other diet treatments, female
offspring exposed to 10% flaxseed had lower relative AGD. In
contrast, rats consuming 5% flaxseed or SDG at levels equiv-
alent to that in the 5% flaxseed diet had higher relative AGD
compared with the basal diet and the 10% flaxseed treatment
groups. A two-way ANOVA indicated both gender and diet
effects (P , 0.001) on AGD and relative AGD.
Puberty onset. Flaxseed and SDG altered the timing of
puberty (Table 5). Exposure of female offspring to 10% flax-
seed during pregnancy and lactation resulted in puberty onset
at a significantly (P , 0.05) earlier age and lighter body
weight, whereas 5% flaxseed resulted in puberty onset at an
older age but at the same body weight as the basal diet group.
Effects similar to those produced by 5% flaxseed occurred
when SDG was given at the level found in 5% flaxseed.
Estrous cycles. All female offspring were cycling at PND
50 (Table 6). However, compared with other diet treatments,
those exposed to 10% flaxseed during pregnancy and lactation
had significantly (P , 0.05) lengthened estrous cycles because
of prolonged time in the estrus phase. By PND 132, 20% of the
10% flaxseed-treated rats were acyclic due to persistent estrus,
whereas 14.3% of the SDG-treated rats and 16.7% of the 5%
flaxseed-treated rats were acyclic as a result of persistent
diestrus. Of the animals still cycling at PND 132, only the 10%
flaxseed-treated animals had significantly lengthened estrous
cycles due to prolonged time in estrus phase compared with
the basal diet group.
Reproductive organ and major organ weights. Except for
the reproductive organs, there were no significant effects on
the major organ weights in the offspring killed at PND 21, 50
TABLE 4
The effect of exposure during pregnancy and lactation to
flaxseed or secoisolariciresinol diglycoside (SDG) on weight
gain, change in anogenital distance (DAGD) and
relative DAGD in rats
1
Treatment n Weight gain
2
DAGD
3
Relative DAGD
4
g mm mm/g
Females
Basal diet 54 35.55 6 0.94 6.8 6 0.1
b
0.20 6 0.01
b
5% Flaxseed 54 33.96 6 0.70 7.5 6 0.2
b
0.23 6 0.01
c
SDG 44 34.16 6 0.69 7.5 6 0.2
b
0.23 6 0.01
c
10% Flaxseed 58 33.07 6 0.86 5.4 6 0.2
a
0.17 6 0.01
a
Males
Basal diet 42 37.85 6 0.96
b
10.6 6 0.3
b
0.28 6 0.01
5% Flaxseed 43 35.90 6 1.06
b
10.8 6 0.4
b
0.31 6 0.02
SDG 45 37.22 6 1.43
b
11.2 6 0.4
b
0.34 6 0.02
10% Flaxseed 47 31.31 6 1.02
a
9.0 6 0.2
a
0.30 6 0.01
1
Data are means 6 SEM. Different letters indicate significant differ-
ences (P , 0.05) within the same gender by one-way ANOVA followed
by Dunn’s test (nonparametric). Two-way ANOVA showed diet treat-
ment (P , 0.001) and gender effects (P , 0.001) of AGD and relative
AGD but insignificant treatment and gender interaction.
2
Lactation period weight gain was determined as weight at post-
natal d 21 2 postnatal d 3.
3
Anogenital distance was calculated as anogenital distance at
postnatal d 21 2 postnatal d 3.
4
Relative AGD was calculated as anogenital distance (postnatal
d212 postnatal d 3)/body weight gain (postnatal d 21 2 postnatal d 3).
TABLE 5
The effect of exposure during pregnancy and lactation to
flaxseed or secoisolariciresinol diglycoside (SDG) on puberty
onset in female rat offspring
1
Dietary treatment n Age Weight
dg
Basal diet 13 30.1 6 0.8
b
93.69 6 1.85
b
5% Flaxseed 13 34.9 6 0.6
c
98.23 6 2.17
b
SDG 15 34.7 6 0.7
c
99.60 6 1.96
b
10% Flaxseed 17 25.5 6 0.3
a
77.77 6 1.84
a
1
Data are means 6 SEM. Different letters indicate significant differ-
ences (P , 0.05) by one-way ANOVA followed by Tukey’s test (para-
metric).
TABLE 6
The effect of exposure during pregnancy and lactation to
flaxseed or secoisolariciresinol diglycoside (SDG) on estrous
cycling and length in female rat offspring
1
Treatment
Postnatal day 40–50 Postnatal day 100–132
Rats
cycling
2
Cycle
length
3
Rats
cycling
2
Cycle
length
3
ndnd
Basal diet 13/13 4.9 6 0.2
a
7/7 5.4 6 0.2
a
5% Flaxseed 13/13 6.3 6 0.3
a
5/6 6.8 6 0.9
ab
SDG 15/15 6.5 6 0.3
a
6/7 7.3 6 0.2
ab
10% Flaxseed 17/17 7.5 6 0.3
b
8/10 7.7 6 0.5
b
1
Data are means 6 SEM. Different letters indicate significant differ-
ences (P , 0.05) by one-way ANOVA followed by Dunn’s test (non-
parametric).
2
Estrous cycles were determined by vaginal smears. More than
10 d in one phase indicated acyclicity.
3
Estrous cycles were determined by vaginal smears. Time required
to complete the proestrus, estrus, metestrus and diestrus phases indi-
cated estrous cycle length. Vaginal smears were taken at postnatal d
40–50 and in older rats at postnatal d 100–132.
TOU ET AL.1864
by guest on June 7, 2013jn.nutrition.orgDownloaded from
or 132. In female offspring (PND 21), the immature uterine
relative weight was higher in 10% flaxseed, 5% flaxseed and
SDG diet groups compared with the basal diet group, but this
difference disappeared by PND 50 and 132 (Table 7). At PND
21, offspring exposed to 5% flaxseed and SDG had lower
relative ovarian weights than the controls, but the difference
disappeared at PND 50. On the other hand, the relative
ovarian weight was greater in the 10% flaxseed diet group at
PND 50 compared with the other diet treatments, and this
persisted into adulthood (PND 132) relative to the basal diet
group. A two-way ANOVA indicated significant (P , 0.001)
diet and age effects on female reproductive organ weights.
Pregnancy and lactation exposure to flaxseed or SDG had
no effect on male accessory sex gland or testes weight at PND
21 or 50 but at a later age (PND 132), male offspring that had
been treated with 10% flaxseed had greater accessory sex gland
and prostate relative weights than those of the basal, 5%
flaxseed and 1.5 mg SDG diet groups (Table 8). A two-way
ANOVA indicated significant (P , 0.001) age and diet effects
on sex gland weight.
Prostate histology. The rat prostate consists of ventral lobe,
dorsal-lateral lobes and coagulating glands, but morphologic
alterations were detected only in the ventral lobe. Microscopic
examination of the ventral prostate of male offspring exposed
to the basal diet from pregnancy and lactation until killed at
PND 132 showed that prostate acini was composed mainly of
columnar epithelial cells surrounded by small stroma that
formed infoldings that projected into the lumen (Fig. 1A). At
PND 132, the ventral lobe of male offspring exposed to 10%
flaxseed during the pregnancy and lactation period exhibited
extensive cell proliferation (Fig. 1B). There were increased
amounts of secretory epithelial cells forming more papillary
infoldings and secondary projections. The epithelial cells of
the acini were taller columnar-shaped cells with some nuclei
located in apical regions. This proliferation was not restricted
to the focal region but occurred in most areas of the ventral
lobe. In contrast to the histologic appearance of the 10%
flaxseed group, the male offspring exposed to 5% flaxseed
during the pregnancy and lactation periods exhibited mild
inhibition of prostate growth at PND 132 (Fig. 1C). The
epithelial cells of the acini were cuboidal or flattened cuboidal
with fewer infoldings. In some areas, the acini had more
stroma. At PND 132, the histologic appearance of the ventral
TABLE 7
The effect of exposure during pregnancy and lactation to flaxseed or secoisolariciresinol diglycoside (SDG) on female rat offspring
reproductive organ relative weights at the different developmental stages
1
Treatment
2
n Body weight Uterus
2
Ovaries
2
g mg/100 g body weight
21 d
Basal diet 7 48.05 6 3.35 80.16 6 6.15
a
64.16 6 5.66
b
5% Flaxseed 7 40.70 6 2.88 125.53 6 14.34
b
43.96 6 1.95
a
SDG 7 46.07 6 1.90 138.03 6 8.82
b
44.01 6 4.54
a
10% Flaxseed 7 39.98 6 2.06 147.95 6 8.14
b
59.10 6 5.05
ab
50 d
Basal diet 6 179.92 6 4.51 195.81 6 21.89 58.31 6 3.40
a
5% Flaxseed 7 191.03 6 8.09 178.51 6 10.33 60.08 6 4.18
a
SDG 8 187.73 6 7.41 171.19 6 14.78 58.89 6 2.46
a
10% Flaxseed 7 191.83 6 9.39 170.34 6 14.31 87.23 6 5.82
b
132 d
Basal diet 7 283.17 6 15.86 175.79 6 18.10 37.15 6 2.79
a
5% Flaxseed 6 335.02 6 17.36 156.87 6 12.00 40.77 6 2.13
ab
SDG 7 326.66 6 12.79 159.43 6 16.71 41.11 6 2.34
ab
10% Flaxseed 10 304.94 6 8.24 168.96 6 6.34 47.51 6 2.50
b
1
Data are means 6 SEM. Means with different letters are significantly different (P , 0.05) within the same age groups by one-way ANOVA followed
by Tukey’s test (parametric).
2
Relative organ weights were calculated as wet weight/body weight.
TABLE 8
The effect of exposure during pregnancy and lactation to flaxseed or secoisolariciresinol diglycoside (SDG) on male rat offspring
reproductive organ relative weights at postnatal day 132
1
Treatment n Body weight Sex glands
2
Testes Seminal vesicle Prostate
g mg/100 g body weight
Basal diet 6 570.12 6 14.56 701.89 6 13.84
a
565.02 6 32.31 243.21 6 13.96 279.31 6 15.99
a
5% Flaxseed 6 584.03 6 23.19 708.37 6 32.84
a
571.47 6 28.48 258.66 6 14.81 270.12 6 16.97
a
SDG 10 578.91 6 14.12 706.30 6 26.18
a
543.46 6 20.40 244.50 6 11.88 290.31 6 13.95
a
10% Flaxseed 6 522.85 6 12.22 827.62 6 29.49
b
627.06 6 39.10 259.79 6 14.18 359.79 6 23.43
b
1
Data are means 6 SEM. Means with different letters are significantly different (P , 0.05) by one-way ANOVA followed by Tukey’s test (parametric).
2
Relative organ weights were calculated as wet weight/body weight.
FLAXSEED AND REPRODUCTION 1865
by guest on June 7, 2013jn.nutrition.orgDownloaded from
lobe of male offspring exposed to SDG was similar to that of
the basal diet group (Fig. 1D).
DISCUSSION
The 10 or 5% flaxseed or SDG at levels found in 5%
flaxseed had no apparent effect on rat dam’s pregnancy but
exerted reproductive changes in the offspring. Inadequate or
excessive estrogen disrupts pregnancy by altering the proper
estrogen balance required for the establishment and mainte-
nance of pregnancy. Estrogenization occurred in pregnant rat
dams fed 10% flaxseed as indicated by greater maternal uterine
and ovarian relative weights, but it was not enough to impair
pregnancy. In contrast, feeding the phytoestrogen coumestrol
at 150–900
m
g/kg diet estrogenized the reproductive tract of
rat dams and impaired pregnancy by increasing embryo degen-
eracy (Fredericks et al. 1981). The absence of pregnancy
effects by flaxseed or SDG suggests a weaker estrogenic prop-
erty of the mammalian lignans compared with coumestrol.
Coumestrol has the strongest estrogenic potency of the several
phytoestrogens that have been investigated (Mayr et al. 1992).
Pregnancy and lactation are more hormone-sensitive peri-
ods for offspring than for the pregnant rat dams because sexual
differentiation of the offspring’s reproductive tract and CNS is
occurring during those periods under the influence of hor-
mones (Manson and Kang 1989). Pregnant rat dams fed 10%
flaxseed had offspring with lower birth weights than those fed
the other diets. This suggests estrogenization because high
dose estrogen has been reported to inhibit pituitary growth
hormones (Wiedemann 1981). Continued growth suppression
in nursing offspring indicated that estrogen exposure occurred
via lignan transfer through rat dams’ milk. Lower body weight
in the male but not the female offspring suggests that de-
creased growth was not the result of decreased milk produc-
tion.
Other early evidence of estrogenization of female offspring
in response to flaxseed or SDG includes shortening of AGD,
which usually occurs in the presence of estrogen, and the
higher immature uterine growth, which is a classic measure of
estrogenic potency. Significant changes in timing of puberty
onset, ovarian relative weight, premature cycle irregularity and
acyclicity occurred after flaxseed or SDG exposure ended,
indicating that changes exerted during sexual differentiation
resulted in permanent effects. Whether reproductive effects
exerted were estrogenic or antiestrogenic depended on the
dosage of flaxseed given.
Compared with the other diet groups, the female offspring
exposed to 10% flaxseed had earlier age and lighter weight at
puberty onset, greater ovarian relative weight at PND 50,
which persisted into adulthood, lengthened estrous cycles, and
by PND 132, persistent estrus in 20% of the offspring. These
reproductive effects were determined to be estrogenic on the
basis of reports of similar results in neonatal female rats treated
with synthetic and endogenous estrogens (Sheehan et al.
1980). This was also in agreement with results of feeding
0.01% coumestrol to lactating rats dams whose offspring
showed persistent estrus at PND 132 (Whitten et al. 1993). It
is possible that 10% flaxseed may exert effects on the ovarian
weight and ovarian cycles indirectly through estrogenization of
the developing CNS. Normally, the CNS produces cyclical
release of hormones regulating the ovarian cycle but early
estrogenization of the CNS produces acyclic hormone release,
resulting in ovarian follicle growth but failure to ovulate and
lack of estrous cyclicity due to persistent estrous (MacLusky
and Naftolin 1981).
The 5% flaxseed or 1.5 mg SDG diet group had significantly
higher relative AGD, greater immature uterine relative weight
but lower ovarian relative weight compared with the basal diet
group. At a later stage, puberty onset was delayed and estrous
cycles were lengthened due to prolonged diestrus. By PND
132, ;15% of adult animals were acyclic due to persistent
diestrus. Diestrus occurs when estrogen stimulation of the
vaginal epithelium is blocked or stopped (Nalbandov 1976).
These reproductive effects were determined to be antiestro-
genic based on the Gellert and Wilson (1979) study, which
reported similar effects of delayed puberty onset, decreased
regularity of ovarian cycles and cessation of cyclicity at an
earlier age in neonatal androgenized female rats. Compared
with the basal diet group, the lower immature ovarian relative
weight and premature acyclicity in female offspring exposed to
5% flaxseed suggests that the mechanism whereby 5% flaxseed
exerted its antiestrogenic action was by direct effects on the
reproductive tract. According to Manson and Kang (1989),
FIGURE 1 Typical histologic sections of the ventral prostate at
postnatal d 132 in male rats exposed to either basal diet, basal diet
supplemented with 10% flaxseed, 5% flaxseed or secoisolariciresinol
diglycoside (SDG) at the level found in 5% flaxseed during pregnancy
and lactation. (A) Basal diet group: acini are composed of columnar
epithelial cells surrounded by small stoma; (B) 10% flaxseed group:
acini are lined by tall columnar cells with more infoldings and projecting
into the lumen. Some of the nuclei are located in the apical region of the
cell; (C) the 5% flaxseed group: acini are lined mainly with cuboidal or
flattened cuboidal epithelial cells with fewer infoldings and surrounded
by more stoma; (D) the SDG group: features are similar to those of basal
diet group (magnification X125).
TOU ET AL.1866
by guest on June 7, 2013jn.nutrition.orgDownloaded from
delayed puberty results from ovary damage and the delay in
puberty onset is proportional to the time required for the
follicle to repopulate. At a later age, this partial destruction of
the follicle pool can result in premature cessation of ovarian
cycles. Flaxseed had effects on the sex organs but no gross
effect on other major organs based on the absence of weight
changes.
Male offspring exposed to 10% flaxseed during the preg-
nancy and lactation periods were estrogenized as indicated by
shortened AGD. AGD was not significantly longer when
expressed relative to body weight. Immature testes and acces-
sory sex gland relative weights were also unaffected. By PND
132, there were greater accessory sex gland and prostate rela-
tive weights and cell proliferation in the prostate of male
offspring exposed to 10% flaxseed during pregnancy and lac-
tation compared with the other diet treatment groups. Evi-
dently, reproductive effects are not always immediately ob-
served but may manifest when the offspring develop
reproductive capacity upon reaching puberty or adulthood
(Whitten et al. 1993 and 1995).
In the rodent model, early exposure to estrogen has been
reported to alter prostatic growth and response to androgens
during adulthood, and to promote preneoplastic lesions and
tumor formation in the aging animals (Rajfer and Coffey 1978,
Santti et al. 1994). Similar observations of higher prostate
relative weight and hyperplasia at PND 132 in the 10%
flaxseed diet group suggest that early exposure to high dose
flaxseed may potentially increase the risk of prostate cancer. It
is noted, however, that soybean, a rich source of isoflavone
phytoestrogens, increased prostate weight when fed to un-
treated mice but decreased prostate dysplasia when fed to
neonatal mice that had been estrogenized by 3 d treatment
with diethylstilbestrol (Makela et al. 1995). If the lignans act
in the same way as the isoflavones, then 10% flaxseed in the
presence of a more potent estrogen may provide cancer pro-
tective effects by acting as an estrogen antagonist.
In contrast to 10% flaxseed, 5% flaxseed resulted in a mild
inhibition of prostate growth. A similar inhibition of epithelial
cell proliferation was observed in the mammary gland of rats
fed 5% flaxseed (Serraino and Thompson 1991). In addition,
lower nuclear aberration and decreased tumor number and size
were observed in the SDG- or 5% flaxseed–treated rats (Ser-
raino and Thompson 1992, Thompson et al. 1996a and
1996b). Female offspring exposed to 5% flaxseed during preg-
nancy and lactation had delayed puberty onset followed by
increased incidence of premature acyclicity due to diestrus.
Epidemiologic evidence suggests that late menarche, early
menopause and hormone deprivation are beneficial in reduc-
ing breast cancer risk (Kelsey et al. 1993). According to
Whitten et al. (1995) phytoestrogen exposure during sexual
differentiation can alter sex-specific patterns of development,
and these changes may be cancer protective. Therefore, de-
pending on the dose, flaxseed exposure during pregnancy and
lactation results in reproductive changes that may either re-
duce or increase cancer incidence in the offspring.
The long-term hormonal effects exerted by flaxseed may
also alter fertility. The 10% flaxseed–treated female offspring
had an earlier age at puberty onset, suggesting that the ani-
mal’s reproductive lifespan may increase. However, cycle ir-
regularity, premature cessation of cyclicity and increased inci-
dence of persistent estrus with 10% flaxseed treatment suggests
that, ultimately, the reproductive lifespan may be shortened.
The 5% flaxseed delayed puberty and produced early acyclic-
ity, which may compromise reproduction because this can
decrease the potential mating that can occur over an animal’s
lifespan. The similarity of the SDG results to 5% flaxseed on
reproductive markers indicates that the lignans produced from
SDG were the components responsible for the partial anties-
trogenic properties of 5% flaxseed. Unfortunately, SDG at the
level found in 10% flaxseed was not tested to determine
whether estrogenic effects observed were due to lignans.
The high (n-3) fatty acid,
a
-linolenic acid [18:3(n-3),
ALA] content of flaxseed (Cunnane 1995) has been suspected
to be responsible for the reproductive effects observed, partic-
ularly at the 10% flaxseed level. The (n-3) competes with the
(n-6) fatty acids for the same desaturase/elongase enzyme, with
the (n-3) fatty acids having greater affinity for these enzymes
(Drevon 1992). Therefore, raising the (n-3)/(n-6) ratio can
inhibit the elongation/desaturation of the (n-6) fatty acid
linoleic acid [18:2(n-6), LA] to arachidonic acid [20:4(n-6),
AA], the precursor for series-2 prostaglandins (PG) (Horrobin
1983, Willis 1981), which are important for normal reproduc-
tive function in males and females (Bygedeman et al.1987).
However, we believe that the (n-3) fatty acid was not primar-
ily responsible for the effects seen. First, the fatty acid com-
position of the liver of offspring in this study (unpublished
data) showed higher ALA (4.65 6 0.50 g/100 g) in the 10%
flaxseed group compared with ALA (1.36 6 0.18 g/100 g) in
the basal diet group. However, the greater (n-3)/(n-6) fatty
acid ratio produced by 10% flaxseed was not high enough to
compromise the elongation/desaturation of LA to AA, as
indicated by the absence of significant differences in liver LA
(19.01 6 0.85 g/100 g) and AA (12.18 6 0.90 g/100 g) in the
10% flaxseed group compared with the LA (21.20 6 0.82
g/100 g) and AA (14.73 6 0.78 g/100 g) in the control group.
Second, some other studies have shown that raising dietary
(n-3) fatty acids to levels that suppressed production of series-2
PG from AA improved rather than compromised reproduc-
tion. Truijillo and Broughton (1995) reported that increased
dietary (n-3) fatty acids increased the number of ova released
during ovulation, whereas raising dietary (n-6) fatty acids
reduced ovulation in rats. Pregnant women given a diet sup-
plemented with fish oil, a rich source of long-chain (n-3) fatty
acids, had decreased production of the series-2 PG from AA
but improved reproduction as indicated by a lower incidence
of toxemia, fewer premature deliveries and prolonged gesta-
tion, resulting in higher birth weights (Olsen et al 1986, Olsen
and Secher 1990). In contrast, pregnant rats treated with 10%
flaxseed in this study had offspring with lower birth weights
and no changes in pregnancy outcomes (i.e., offspring survival,
gestation length and littersize). Third, Pandalai et al. (1996)
found that high concentrations of eicosapentanoic acid [20:
5(n-3), EPA] inhibited, whereas low concentrations promoted
proliferation of both prostate cancer and normal cells. In
contrast, 10% flaxseed, which would have produced higher
EPA from ALA than the 5% flaxseed or basal diet, resulted in
higher prostate cell proliferation, whereas 5% flaxseed inhib-
ited prostate cell proliferation compared with the basal diet
group. This suggests that the effects in the prostate were not
due to the flaxseed oil but to some other component(s) present
in flaxseed such as the lignans.
In conclusion, feeding rat dams 5 or 10% flaxseed or SDG
at levels present in 5% flaxseed produced no significant effect
on pregnancy except for the lower birth weights of offspring in
those fed 10% flaxseed. However, feeding flaxseed or SDG
during pregnancy and lactation had hormone-related dose-
dependent effects on the offspring with potential implications
for reproduction and cancer risk in the long term. The effects
of flaxseed at the levels used are more likely due to the
mammalian lignans produced from SDG than from its ALA-
rich oil. Lignans likely were transferrred from the rat dams
through the milk. Thus, caution is suggested when consuming
FLAXSEED AND REPRODUCTION 1867
by guest on June 7, 2013jn.nutrition.orgDownloaded from
flaxseed at high doses during the hormone-sensitive periods of
pregnancy and lactation. It is also suggested that the dose and
timing of flaxseed exposure be considered when adopting flax-
seed for chronic disease prevention and therapeutic applica-
tion. Similar cautions have been suggested to nursing mothers
consuming soy products because soy can increase the isofla-
vone phytoestrogen concentration in their milk up to 10-fold
(Setchell et al. 1997) and daily exposure of infants to phy-
toestrogen-rich milk or soy-based infant formula containing
phytoestrogen may be sufficient to exert biological effects
(Haumann 1997, Sheehan 1998).
LITERATURE CITED
Bakke, J. E. & Klosterman, H. J. (1956) A new diglycoside from flaxseed. Proc.
Natl. Acad. Sci. U.S.A. 10: 18–22.
Barrow, P. (1990) Technical procedures in reproductive toxicology. Lab. Anim.
Handb. 11: 9 –25.
Bradbury, R. B. & White, D. C. (1954) Oestrogens and related substances in
plants. Vitam. Horm. 12: 207–233.
Bygedeman, M., Gottlieb, C., Swanborg, K. & Swahn, M. L. (1987) Role of
prostaglandins in human reproduction recent advances. In: Advances in
Prostaglandin, Thomboxane and Leukotriene Research, Vol. 17 (Samuelsson,
B., Paoletti, R. & Ramwell, P. W., eds.), pp. 1112–1116. Raven Press, New
York, NY.
Canadian Council on Animal Care (1984) Guide to the Care and Use of
Experimental Animals. Ottawa, ON, Canada.
Cunnane, S. C. (1995) Metabolism and function of alpha-linolenic acid in
humans. In: Flaxseed in Human Nutrition (Cunnane, S. C. & Thompson, L. U.,
eds.), pp. 99 –127. AOCS Press, Champaign, IL.
Drevon, C .A. (1992) Marine oils and their effects. Nutr. Rev. 50: 38–45.
Evans, E .A. (1974) Tritium and Its Compounds (Evans, E. A., ed.), 2nd ed., pp.
489–490. Wiley, New York, NY.
Farnsworth, N. R., Bingel, A. S., Cordell, G. A., Crane, F. A. & Wong, H. H. (1975)
Potential value of plants as sources of new antifertility agents II. J. Pharm. Sci.
64: 717–754.
Fredricks, G. R., Kincaid, R. L., Bondioli, K. R. & Wright, R. W., Jr. (1981)
Ovulation rates and embryo degeneracy in female mice fed the phytoestrogen
coumestrol. Proc. Soc. Exp. Biol. Med. 167: 237–241.
Gellert, R. J. & Wilson, C. (1979) Reproductive function in rats exposed
prenatally to pesticides and polychlorinated biphenyls (PCB). Environ. Res.
18: 437–443.
Haumann, B. F. (1997) Soy protein foods gain storage space. INFORM 6:
588–596.
Horrobin, D. F. (1983) The regulation of prostaglandin biosynthesis by the
manipulation of essential fatty acid metabolism. Rev. Pure Appl. Sci. 4:
339–383.
Jenab, M. & Thompson, L. U. (1996) The influence of flaxseed and lignans on
colon carcinogenesis and beta-glucuronidase radioactivity. Carcinogenesis
17: 1343–1348.
Kelsey, J. L., Gammon, M. D. & John, E. M. (1993) Reproductive factors and
breast cancer. Epidemiol. Rev. 15: 7–16.
Klosterman, H. J. & Smith, F. (1954) The isolation of
b
-hydroxy-
b
-methylglu-
taric acid from the seed of flax. J. Am. Chem. Soc. 76: 1229–1230.
MacLusky, N. J. & Naftolin, F. (1981) Sexual differentiation of the central
nervous system. Science (Washington, DC) 211: 1298–1306.
Makela, S. I., Pylkkanen, L. H., Santii, R.S.S. & Adlercruetz, H. (1995) Dietary
soybean may be antiestrogenic in male mice. J. Nutr. 124: 437–445.
Manson, J. M. & Kang, Y. J. (1989) Test methods for assessing female
reproductive and developmental toxicology. In: Principles and Methods of
Toxicology (Hayes, A. W., ed.), 2nd ed., pp. 311–359. Raven Press Ltd., New
York, NY.
Mayr, U., Butsch, A. & Schneider, S. (1992) Validation of two in vitro test
systems for estrogenic activities with zearalenone, phytoestrogens and cereal
extracts. Toxicology 74: 135–49.
Nalbandov, A. V. (1976) The estrous cycle. In: Reproductive Physiology of
Mammals and Birds: The Comparative Physiology of Domestic and Labora-
tory Animals and Man (Nalbanov, A. V., ed.), pp. 98 –124. W. H. Freeman &
Co., San Francisco, CA.
Olsen, S .F., Hansen, H. S., Sorensen, T. I., Jensen, B., Secher, N. J., Sommer, S.
& Knudsen, L. B. (1986) Intake of marine fat, rich in (n-3) polyunsaturated
fatty acids may increase birthweight by prolonged gestation. Lancet 2: 367–
369.
Olsen, S. F. & Secher, N. J. (1990) A possible preventative effect of low dose
fish oil on early delivery and pre-eclampsia indications from a 50 year old
controlled trial. Br. J. Nutr. 64: 599–609.
Orcheson, L. J., Rickard, S. E., Seidl, M. M. & Thompson, L. U. (1998) Flaxseed
and its mammalian lignan precursor cause lengthening or cessation of estrous
cycling in rats. Cancer Lett. 125: 69–76.
Pandalai, P. K., Pilat, M. J., Yamazaki, K., Naik, H. & Pienta, K. J. (1996) The
effects of omega-3 and omega-6 fatty acids on in vitro prostate cancer
growth. Anticancer Res. 16: 815– 820.
Pasqualini, J. R., Kind, F. A. & Sumida, C. (1985) The binding of homones in
maternal and fetal biological fluids. In: Hormones and the Fetus (Pasqualini,
J. R., ed.), pp. 1–50. Pergamon Press, New York, NY.
Price, K .R. & Fenwick, G. R. (1985) Naturally occurring oestrogens in foods—a
review. Food Addit. Contam. 2: 73–106.
Rajfer, J. & Coffey, D. S. (1978) Sex steroid imprinting of the immature
prostate. Investig. Urol. 16: 186 –192.
Reeves, P. G., Nielson, F. H. & Fahey, G. C., Jr. (1993) AIN-93 purified diets for
laboratory rodents: final report of the American Institute of Nutrition ad hoc
writing committee on the reformulation of the AIN-96A rodent diet. J. Nutr.
123: 1939–1951.
Rickard, S. E., Orcheson, L. J., Seidl, M. M., Luyengi, L., Fong, H.H.S. & Thomp-
son, L. U. (1996) Dose-dependent production of mammalian lignans in rats
and in vitro from the purified precursor secoisolariciresinol diglycoside in
flaxseed. J. Nutr. 126: 2012–2019.
Rickard, S. E. & Thompson, L. U. (1997) Phytoestrogens and lignans: effects
on reproduction and chronic disease. In: Antinutrients and Phytochemicals in
Food (Shahidi, F., ed.), pp. 273–293. American Chemical Society, Washing-
ton, DC.
Rickard, S. E. & Thompson, L. U. (1998) Chronic exposure to secoisolaricir-
esinol diglycoside alters lignan desposition in rats. J. Nutr. 128: 615–623.
Santti, R., Newbold, R. R., Makela, S., Pylkkanen, L. & Lachlan, J. A. (1994)
Developmental estrogenization and prostatic neoplasia. Prostate 24: 67–78.
Serraino, M. R. & Thompson, L. U. (1991) The effect of flaxseed supplemen-
tation on early risk markers for mammary carcinogenesis. Cancer Lett. 60:
135–142.
Serraino, M. R. & Thompson, L. U. (1992) The effect of flaxseed supplemen-
tation on the initiation and promotional stages of mammary tumorigenesis.
Nutr. Cancer 17: 153–159.
Setchell, K. D., Zimmer-Nechemias, L., Cai, J. & Heubi, J .E. (1997) Exposure
of infants to phyto-oestrogens from soy-based infant formula. Lancet 350:
23–27.
Sheehan, D. M. (1998) Herbal medicines, phytoestrogens and toxicity: risk:
benefit considerations. Proc. Soc. Exp. Biol. Med. 217: 379–385.
Sheehan, D. M., Branham, W. S., Medlock, K. L., Olson, M. E. & Zehr, D. (1980)
Estrogen plasma binding and regulation of development in the neonatal rat.
Teratology 21(2): 68A (abs).
Thompson, L. U., Rickard, S. E., Cheung, F., Kenaschuk, E. O. & Obermeyer,
W. R. (1997) Variability in anticancer lignan levels in flaxseed. Nutr. Cancer
27: 26–30.
Thompson, L. U., Rickard, S. E., Orcheson, L. J. & Seidl, M. M. (1996a)
Flaxseed and its lignan and oil components reduce mammary tumor growth
at a late stage of carcinogenesis. Carcinogenesis 17: 1373–1776.
Thompson, L. U., Robb, P., Serraino, M. & Cheung, F. (1991) Mammalian
lignan production from various foods. Nutr. Cancer 16: 43–52.
Thompson, L. U., Seidl, M. M., Rickard S. E., Orcheson, L. J. & Fong, H.H.S.
(1996b) Antitumorigenic effect of mammalian lignan precursor from flax-
seed. Nutr. Cancer 26: 159 –165.
Trujillo, E. P. & Broughton, K. S. (1995) Ingestion of n-3 polyunsaturated fatty
acids and ovulation in rats. Journal of Reproduction and Fertility 105: 197–
203.
Whitten, P .L., Lewis, C. & Naftolin, F. (1993) A phytoestrogen diet induces the
premature anovulatory syndrome in lactationally exposed female rats. Biol.
Reprod. 49: 1117–1121.
Whitten, P. L., Lewis, C., Russel, E. & Naftolin, F. (1995) Potential adverse
effects of phytoestrogens. J. Nutr. 125: 771S–776S.
Wiedemann E. (1981) Adrenal and gonadal steriods. In: Endocrine Control of
Growth (Daughaday, W. H., ed.), pp. 67–119. Elsevier, New York, NY.
Willis, A. L. (1981) Nutritional and pharmacological factors in eicosanoid
biology. Nutr. Rev. 39: 289 –301.
Zimmermann, S. A., Clavenger, W. R., Brimhall, B. B. & Bradshaw, W.S. (1991)
Diethylstilbestrol induced perinatal lethality in the rat. II. Perturbation of par-
turition. Biol. Reprod. 44: 583–589.
TOU ET AL.1868
by guest on June 7, 2013jn.nutrition.orgDownloaded from
