International Dairy Journal 22 (2012) 3e14

Contents lists available at SciVerse ScienceDirect

International Dairy Journal

journal homepage: www.elsevier.com/locate/idairyj

Review

Impact of cows milk estrogen on cancer risk

Peter W. Parodi*
Human Nutrition and Health Research, Dairy Australia, Melbourne, Australia

a r t i c l e i n f o a b s t r a c t

Article history: Estrogens have been implicated in cancer at hormone-responsive sites, such as the breast, ovaries,
Received 20 May 2011 endometrium and prostate. Because cows milk contains estrogens, some authors have suggested that its
Received in revised form consumption may contribute to the risk of cancer at these sites. However, these reports do not recognize
12 August 2011
the complex mechanisms these cells possess to regulate estradiol levels. Hormone-responsive and many
Accepted 14 August 2011
peripheral cells contain all the necessary steroidogenic enzymes necessary for in situ synthesis of bioactive
estradiol from abundant androgenic precursors and for inactivation of unwanted estrogens. Estradiol from
dairy products is extensively inactivated in the gastrointestinal tract and only about 5% survives the rst
pass to the liver. Thus daily dairy product intake would supply only about 0.25% of the FAO/WHO upper
acceptable daily intake of estradiol. Available epidemiological evidence does not suggest an association
between dairy product consumption and risk of cancer of the breast, ovaries and endometrium.
2011 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
2. Estrogens and cancer risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
2.1. Observational studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.2. Experimental studies and mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3. Serum estrogen levels and the risk of cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
3.1. Breast cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.2. Endometrial cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.3. Ovarian cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.4. Prostate cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4. Relationship between circulating estrogen levels and levels in breast tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
5. Relationship between urinary estrogens and estrogen metabolites in breast tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
6. Formation and transformation of tissue estrogen levels in the breast and other estrogen-responsive tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
7. Breast tissue estrogen content: uptake from the circulation versus synthesis in situ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
8. Estrogen content of cows milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
8.1. Factors influencing the hormone content of milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
9. Absorption and metabolism of estrogens from milk and its products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
10. Physiological influence of various exogenous sources of estrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
11. Epidemiological evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
11.1. Breast cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
11.2. Ovarian cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
11.3. Endometrial cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
11.4. Prostate cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
11.5. Colon cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
12. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

* Tel.: 617 3359 6110.

E-mail address: peterparodi@uqconnect.net.

0958-6946/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.idairyj.2011.08.006
4 P.W. Parodi / International Dairy Journal 22 (2012) 3e14
1. Introduction
It is generally accepted that estrogens are involved in the
development of cancer at hormone-responsive sites, such as the
breast, ovary and endometrium and, in conjunction with testos-
terone, the prostate (Bernstein & Ross, 1993; Kaaks, Lukanova, &
Kurzer, 2002; Lukanova & Kaaks, 2005; Risbridger, Bianco, Ellem,
& McPherson, 2003). Even so, the mechanisms whereby estrogens
inuence carcinogenesis remain largely unresolved. A number of
publications have hypothesized that estrogen present in cows milk
may be responsible for the development of cancer in estrogen-
responsive organs (Farlow, Xu, & Veenstra, 2009; Ganmaa & Sato,
2005; Outwater, Nicholson, & Barnard, 1997; Qin, Wang, Kaneko,
Hoshi, & Sato, 2004). However, these hypotheses fail to take into
account the complex mechanisms involved in the synthesis and
metabolism of estrogens within the cell and the metabolic
processes associated with the absorption of exogenous estrogens.
These aspects, together with a brief outline of relevant aspects of
estrogen carcinogenesis, are discussed in this review.
2. Estrogens and cancer risk
2.1. Observational studies
The study of breast cancer has received more attention than
cancer at the other hormone-responsive sites. Nevertheless, the
causes of breast cancer are still largely undetermined. Evidence
from a number of observational studies suggests that several
reproductive factors, which indicate an increased exposure to
estrogens, increase breast cancer risk. Early menarche, late meno-
pause, nulliparity and rst pregnancy late in life are associated with
increased risk. Bilateral oophorectomy, especially at an early age,
reduces risk. On the other hand, oral contraceptive use that
prevents ovulation and decreases serum estrogen levels results in
a slight increase in risk. In contrast, breast-feeding (particularly for
long durations, which also prevents ovulation) is associated with
lower risk, but this protection may result from cell differentiation
(Bernstein & Ross, 1993; Chodosh et al., 1999; Harris, Lippman,
Veronesi, & Willett, 1992; Kuller, 1995).
Evidence for a role of estrogens in endometrial cancer is robust
and it is considered that exposure to estrogens, unopposed by
progesterone, is a major etiological factor. Early menarche and late
menopause are risk factors, whereas parity is protective. A low level
of physical activity and obesity increase risk, possibly due to
a reduction in progesterone levels. There is an increased risk of
endometrial cancer in users of estrogen-only oral contraceptives
and hormone replacement therapy, but no increased risk when
progesterone is included in the preparation (Kaaks et al., 2002;
Pike, Spicer, Dahmoush, & Press, 1993).
The cause of ovarian cancer is largely unknown. Observational
evidence on the role of estrogens is conicting and often open to
alternative interpretation. There is some support for the concept
that continual ovulation may predispose women to development of
ovarian cancer. Pregnancy, breast-feeding and oral contraceptive
use are protective, but hormone replacement therapy is associated
with increased risk (Lukanova & Kaaks, 2005; Risch, 1998).
Although androgens are considered to have an important role in
prostate cancer development, animal and human cell culture
studies now suggest a role for estrogens (Bosland, 2004; Harkonen
& Makela, 2004). Recent studies with genetically modied mouse
models show that malignant changes to the prostate gland are
dependent on both androgen and estrogen responses, and that
neither hormone alone can produce an aberrant growth pattern
that leads to malignancy (Bosland, 2004; Risbridger et al., 2003).
2.2. Experimental studies and mechanisms
There is ample evidence that administration of estradiol (17b-
estradiol) to laboratory animals induces tumors and that castration
and use of antiestrogen drugs inhibits tumor development
(Bernstein & Ross, 1993; Liehr, 2000). The mechanisms whereby
estrogens cause cancer have not been established conclusively.
A commonly held hypothesis is that circulating estrogens diffuse
passively through cellular membranes of estrogen-responsive cells
and bind to nuclear estrogen receptors (ERs) of which there are two
forms, ERa and ERb. Binding to ERs stimulates transcription of
genes involved in cell proliferation. Rapidly proliferating cells are
susceptible to genetic errors during DNA replication, which if
uncorrected may ultimately lead to cancer. Estradiol may also
increase proliferation in cells already having mutations due to other
sources of DNA damage (Santen, 2002; Yager & Davidson, 2006;
Yue et al., 2003).
It has also been proposed that non-receptor-mediated mutagen-
esis induced by oxidized estrogen metabolites may contribute to
carcinogenesis (Cavalieri, Devanesan, Bosland, Badawi, & Rogan,
2002a; Cavalieri et al., 2002b; Rogan et al., 2003; Yager & Davidson,
2006). Estrogen metabolites are formed by a series of oxidative
biotransformations, both in the liver and in extrahepatic hormone-
responsive organs by the activities of a series of cytochrome P450
enzymes. There are two major mutually exclusive pathways for
the transformation of estradiol and estrone. In the rst pathway
the A ring is hydroxylated at position 2 or 4 to produce the
catechol estrogens 2-hydroxyestradiol and 2-hydroxyestrone or 4-
hydroxyestradiol and 4-hydroxyestrone, respectively. These four
metabolites may be inactivated in the liver and extrahepatic
estrogen-responsive tissue by glucuronidation and sulfation and
particularly in extrahepatic tissues by the enzyme catechol-O-
methyltransferase (COMT), which forms 2- and 4-methoxyestradiols,
and methoxyestrones. In certain conditions catechol estrogens may
be oxidized further to reactive catechol estrogen semiquinones then
to catechol estrogen-2,3-quinones and catechol estrogen-3,4-
quinones. Protection against the catechol estrogen-quinones occurs
through conjugation with glutathione catalyzed by glutathione-S-
transferases. Another protection mechanism results when catechol
estrogen-quinones are re-cycled to catechol estrogens by the action of
quinone reductases (Cavalieri, Frenkel, Liehr, Rogan, & Roy, 2000;
Lakhani, Sarkar, Venitz, & Figg, 2003; Yager & Davidson, 2006). The
second major oxidation pathway occurs at the D ring with the
formation of 16a-hydroxyestradiol and 16a-hydroxyestrone. These
16a-hydroxyderivatives may be further oxidized to estriol (Clemons &
Goss, 2001; Mueck, Seeger, & Lippert, 2002).
Estrogenic and genotoxic properties of the various estrogen
metabolites vary from one and other and from their parent estro-
gens. For instance, the 2-hydroxyestrogens bind to ERs with afnity
comparable with that for estradiol, but have limited mitogenic
effect and are considered antiestrogenic and anticarcinogenic
(Bradlow, Telang, Sepkovic, & Osborne, 1996; Mueck et al., 2002). 2-
Methoxyestradiol has very low afnity for ERs, but exhibits potent
apoptotic activity against rapidly growing cancer cells, possesses
antiangiogenic properties and has been trailed on patients
with breast and prostate cancer (Lakhani et al., 2003). On the other
hand, 16a-hydroxyestrogens that bind to ERs with lower afnity
than estradiol are thought to have a carcinogenic inuence (Mueck
et al., 2002).
Attention has focused on the estrogen-2,3- and estrogen-3,4-
quinones because of their ability to act both as electrophiles and
oxidants. As electrophiles, catechol quinones can form covalent
adducts with the purine bases adenine and guanine of DNA, these
depurinating adducts generate apurinic sites that may lead to
mutations and in turn cancer. As oxidants, catechol quinines redox
 P.W. Parodi / International Dairy Journal 22 (2012) 3e14
cycle with their semiquinones, producing the superoxide anion and
reactive oxygen species that may damage lipids and DNA (Cavalieri
et al., 2000; Yager & Davidson, 2006). The proposed ER- and non-
ER-mediated pathways may act in concert in an additive or
synergistic manner to cause cancer (Yue et al., 2003). However,
under normal physiological conditions deactivation and conjuga-
tion processes should ensure that insufcient reactive quinines are
produced to cause signicant DNA damage, whereas normal
cellular antioxidants should prevent oxidative damage. High levels
of hydroxy- and methoxyestrogens are found in blood (Eliasssen,
Missmer, Tworoger, & Hankinson, 2008) and urine (Eliassen et al.,
2009). Indeed, hydroxy- and methoxyestrogens can represent up
to 95% of the total estrogens in normal breast tissue (Castagnetta
et al., 2002; Rogan et al., 2003). Overall, no studies have demon-
strated that estrogen metabolites contribute to cancer in humans
(Yager & Davidson, 2006).
3. Serum estrogen levels and the risk of cancer
Many studies have examined the associations between circu-
lating estrogen levels and the risk of breast, endometrial, ovarian
and prostate cancer. Because these cancers produce estrogens
during their development only studies where blood was drawn
prospectively will be considered here. Estradiol circulates bound to
carrier proteins with around 70% bound to sex hormone-binding
globulin (SHBG), which inhibits it bioactivity, and 30% is more
loosely bound to albumin. Only about 1% circulates in the free form
(Grow, 2002). Eliassen and Hankinson (2008) recently reviewed
seroepidemiology studies of breast, endometrial and ovarian
cancer, and the Endogenous Hormones and Prostate Cancer
Collaborative Group (2008) conducted a pooled analysis of 18
prospective studies, which examined the associations between sex
hormone concentrations and the risk of prostate cancer.
3.1. Breast cancer
The Endogenous Hormones and Breast Cancer Collaborative
Group (2002) analyzed data from nine prospective studies of 663
postmenopausal women who developed breast cancer. The relative
risk (RR) with 95% condence intervals (CI) for the highest
compared with the lowest quintile of serum estradiol, free estra-
diol, estrone and estrone sulfate was 2.00 (1.47e2.71), 2.58
(1.76e3.78), 2.19 (1.48e3.22) and 2.00 (1.26e3.16), respectively. For
SHBG, the RR was 0.66 (0.43e1.00). Since this analysis, results for
combined data from the Swedish Malmo Diet and Cancer Study
(MDCS) and the Northern Sweden Health and Disease Study
(NSHDS), the New York University Womens Health Study
(NYUWHS), the Nurses Health Study (NHS), and the European
Prospective Investigation into Cancer and Nutrition (EPIC), were
reported and are reviewed by Eliassen and Hankinson (2008)
Results from the Studies of Hormones and Diet in the Etiology of
Breast Tumors (ORDET) were recently reported (Sieri et al., 2009).
In all these studies, there was roughly a two-fold increase in risk of
breast cancer with elevated levels of estrogens.
Only a few studies have reported associations between circu-
lating estrogens and the risk of premenopausal breast cancer. This
is undoubtedly due to the difculty in obtaining a representative
sample for menstruating women. Five small studies found no
association between estrogen levels and the risk of breast cancer.
Results from two recent large studies indicate that in the EPIC
cohort there was no association between estradiol or estrone levels
and the risk of breast cancer. On the other hand, in NHS II there was
a signicant positive association between breast cancer risk and
estradiol levels in blood collected during the follicular phase, but
not for luteal estradiol. There was no association between follicular
5
or luteal estrone levels and the risk of breast cancer (Eliassen &
Hankinson, 2008).
Two prospective studies found positive associations between
urinary estrogen levels and risk of postmenopausal breast cancer
(Key et al., 1996; Onland-Moret et al., 2003). However, the rele-
vance of urinary estrogens to functions in target cells is uncertain.
Seven prospective studies examined associations between either
serum or urinary estrogen metabolites and risk of breast cancer in
both pre- and postmenopausal women. These studies measured
only 2-hydroxyestrone and 16a-hydroxyestrone and overall there
were no statistically signicant associations between these two
metabolites or the ratio of 2-hydroxyestrone: 16a-hydroxyestrone
and the risk of breast cancer (Arslan et al., 2009; Eliassen, Missmer,
Tworoger, & Hankinson, 2008).
3.2. Endometrial cancer
There has been only one large prospective study on circulating
sex hormone levels and the risk of endometrial cancer (Lukanova
et al., 2004). This study combined data for postmenopausal
women from the NYUWHS, ORDET and NSHDS cohorts. Estradiol
and estrone levels were strongly associated with endometrial
cancer risk with RRs of 4.13 (1.76e9.72) and 3.67 (1.71e7.88),
respectively. SHBG was negatively associated with risk of endo-
metrial cancer, RR 0.46 (0.02e1.05).
3.3. Ovarian cancer
Eliassen and Hankinson (2008) reviewed the few prospective
studies that investigated associations between serum sex hormone
concentrations and the risk of ovarian cancer. Overall, there is no
convincing evidence for an association between circulating levels of
estrogens or androgens and the risk of ovarian cancer in pre- or
postmenopausal women.
3.4. Prostate cancer
The Endogenous Hormones and Prostate Cancer Collaborative
Group (2008) conducted a collaborative analysis of worldwide
data from 18 cohort studies that investigated the association
between endogenous hormone concentrations and the risk of
prostate cancer in 3886 men with incident prostate cancer and
6438 control subjects. There were no statistical signicant associ-
ations between serum concentrations of any androgens or estradiol
and the risk of prostate cancer. For estradiol the RR for the highest
versus lowest level of estradiol was 0.93 (0.77e1.11) and 0.95
(0.79e1.15) for free estradiol. An investigation of the possibility that
a combination of estrogens and androgens may be more strongly
associated with the risk of prostate cancer than either estrogen or
androgen alone, found no evidence of any interaction.
4. Relationship between circulating estrogen levels
and levels in breast tissue
To date, the strongest consistent evidence for a role of circu-
lating estrogens in cancer is for postmenopausal breast cancer.
However, circulating hormone levels may not reect hormone
concentrations in target cells. Even so, epidemiological associations
cannot be used to ascribe causality and improved knowledge of
estrogen metabolism at the cellular level is required.
Several studies have compared estrogen levels in normal and
malignant breast tissue with the levels in blood from pre- and
postmenopausal women (Chetrite, Cortes-Prieto, Philippe, Wright,
& Pasqualini, 2000; Geisler, 2003; van Landeghem, Poortman,
Nabuurs, & Thijssen, 1985; Pasqualini et al., 1996; Thijssen, van
6 P.W. Parodi / International Dairy Journal 22 (2012) 3e14

Landeghem, & Poortman, 1986). Even though serum estrogen levels
are up to 50-fold lower in postmenopausal than in premenopausal
women, their unconjugated estrogen levels in normal and malig-
nant breast tissue are similar. Estradiol levels are some 2.5-fold
higher in breast cancer tissue than in normal tissue for both pre-
and postmenopausal women. In breast cancer tissue estradiol
levels are about 10- to 20-fold higher and estrone levels 2- to 10-
fold higher than their corresponding serum levels. Available
studies suggest there is no simple correlation between the levels of
estrogens in serum and their level in tissue. These data suggest that
postmenopausal breast tissue has the ability to selectively accu-
mulate estrogens from the circulation or to synthesize them within
the tissue.
5. Relationship between urinary estrogens and estrogen
metabolites in breast tissue
Only one study was found that compared the levels of estrogens
and estrogen metabolites in breast tissue with those in urine.
In a small study, Taioli et al. (2010) found that total estrogen
metabolite levels were similar between breast tissue and urine.
However, the levels of individual components were quite different
between the two sources. The sum of estrone and estradiol was
signicantly higher in breast tissue than urine, while the
metabolites of the 2- and the 16-hydroxylation pathways were
lower, the latter signicantly lower. The ratio of 2:16 hydroxylation
products was nonsignicantly higher in breast tissue than in urine.
There were large inter-individual variations in tissue to urine ratios
of estrogens and their metabolites. It is possible that not all urinary
estrogens and their derivatives originated from breast tissue.
6. Formation and transformation of tissue estrogen levels
in the breast and other estrogen-responsive tissues
There is now substantial evidence that normal and malignant
breast tissue contains all the necessary steroidogenic enzymes
necessary for the synthesis of estradiol from circulating inactive
androgenic precursors (Labrie, 2003; Labrie et al., 2003; Pasqualini,
2004; Perel, Wilkins, & Killinger, 1980). Dehydroepiandrosterone
(DHEA) sulfate (S) is by far the predominant adrenal-derived
circulating precursor hormone in men and women. Its concentra-
tion is from 1000 to 10,000 times higher than that of estradiol and
100e500 times higher than that of testosterone. There is also
considerable estrogen sulfate and lesser amounts of androstene-
dione and androstenediol in serum. Initially, DHEA-S is converted
to DHEA by the enzyme steroid sulfatase (Fig. 1). DHEA can
then take part in two pathways. Reduction by members of the
17b-hydroxysteroid dehydrogenase (17b-HSD) family produces

Fig. 1. Outline of the in situ formation of estrogens from circulating precursors, DHEA-S and estrone sulfate (adapted from Labrie et al., 2003). DHEA, dehydroepiandrosterone;
DHEA-S, DHEA sulfate; 17b-HSD, 17b-hydroxysteroid dehydrogenase; 3b-HSD, 3b-hydroxysteroid dehydrogenase.
 P.W. Parodi / International Dairy Journal 22 (2012) 3e14
androstenediol (17b-HSD types 1,3,5 and 7 are involved in reduc-
tion reactions, whereas 17b-HSD types 2,4 and 6 are involved in
oxidation reactions). The enzyme 3b-hydroxysteroid dehydroge-
nase (3b-HSD) converts both DHEA and androstenediol to andros-
tenedione and testosterone, respectively, which, in turn, are
converted to estrone and estradiol by the aromatase enzyme. The
DHEA / androstenediol, androstenedione / testosterone and
estrone / estradiol conversions are interconvertable, depending
on the oxidative/reductive forms of 17b-HSD available, but the
action of aromatase is irreversible. Estrone and estradiol may also
be formed by the action of steroid sulfatase on estrone sulfate and
estradiol sulfate, respectively.
Although not studied to the same extent as breast tissue,
endometrial and ovarian tissue also contain a range of steroido-
genic enzymes capable of synthesizing estrogens from circulating
precursors (Ito, Utsunomiya, Yaegashi, & Sasano, 2007; Sasano &
Harada, 1998). The prostate likewise possesses all the necessary
enzymes to convert circulating DHEA into estradiol and testos-
terone, as well as 5a-reductase to convert testosterone to the more
bioactive dihydrotestosterone (Takase et al., 2006).
Normal cells possess mechanisms to ensure estradiol homeo-
stasis. ER expression in normal breast epithelial cells is inversely
related to circulating estradiol levels (Khan et al., 1999; Soderqvist,
von Schoultz, Tani, & Skoog, 1993). Estrone and estradiol can be
converted to inactive sulfates by the action of steroid sulfo-
transferase and either excreted from the cell or retained for future
supply of estrone and estradiol. Conjugation of estrone and estra-
diol by uridine diphosphate-glucuronosyltransferases inactivates
and produces the more soluble estrogen glucuronides that are
excreted to bile and urine. O-methylation of hydroxyestrogens by
COMT and conjugation of estrogen quinones with glutathione
facilitates removal of estrogen metabolites from cells.
The expression and level of the various steroidogenic and
metabolizing enzymes in each cell of hormone-responsive tissues
may inuence their estrogen content and potential to transform.
For instance, a higher activity of the reducing forms of 17b-HSD
may lead to higher levels of estradiol. Aromatase is important for
the conversion of androstenedione to estrone and testosterone to
estradiol. Aromatase inhibitors were shown to suppress blood and
breast cancer tissue estrogen levels in postmenopausal women by
80e95% (Geisler, 2003). However, in both pre- and postmenopausal
breast cancer patients estrone sulfatase activity was found to be
50e200 times higher than aromatase activity, with activity of both
enzymes greater in post- than in premenopausal women
(Pasqualini et al., 1996). Enzyme levels were higher in tumors than
in adjacent tissue, which, in turn, were higher than in normal tissue
(Chetrite et al., 2000). Tissue levels of estrone, estradiol and their
sulfates reected the enzyme activities. It is considered that
formation of estradiol from estrone sulfate is far more important
than aromatization in breast tissue (Chetrite et al., 2000; Pasqualini
et al., 1996; Santner, Feil, & Santen, 1984).
Several lines of evidence suggest that abnormal regulation of
the opposing action of estrogen sulfatases and estrogen sulfo-
transferases in breast tissue may be responsible for carcinogenesis.
Falany and Falany (1996) found that estrogen sulfotransferase was
present in human normal mammary epithelial cells, but was
absent in ER and ER breast cancer cell lines. Qian, Deng, and
Song (1998) showed that the estrogen-responsive human MCF-7
breast cancer cell line lacks estrogen sulfotransferase activity.
When this cell line was transfected with estrogen sulfotransferase
cDNA, estrogen-stimulated DNA synthesis and cell proliferation
was inhibited (Qian et al., 1998). Estrogen sulfotransferase
immunoreactivity in human breast carcinoma was signicantly
associated with a decreased risk of recurrence or improved
prognosis. On the other hand, steroid sulfatase immunoreactivity
7
was signicantly associated with increased risk of recurrence and
worsened prognosis (Suzuki et al., 2003).
Steroid sulfatase inhibitors blocked the ability of estrogen
sulfatase to stimulate the growth of carcinogen-induced mammary
tumors in ovariectomized rats (Purohit, Woo, Potter, & Reed, 2000).
MCF-7 breast cancer cells over-expressing steroid sulfatase injected
into the anks of ovariectomized nude mice were used to
demonstrate the effect of steroid sulfatase inhibitors in reducing
steroid sulfatase activity and tumorogenicity (Foster et al., 2006).
A recent initial trial in postmenopausal women with ER metastatic
breast cancer demonstrated that a steroid sulfatase inhibitor almost
completely blocked steroid sulfatase activity in peripheral blood
lymphocytes and tumor tissue (Stanway et al., 2006). A seroepi-
demiology study by Dorgan et al. (1996) did not detect an associ-
ation between serum estrone or estrone sulfate levels and the risk
of breast cancer. However, the ratio of estrone sulfate to estrone
was signicantly inversely associated with risk, suggesting that
women who develop breast cancer may be less able to metabolize
estrone to its inactive form. How estrogen activation and deacti-
vation mechanisms in cells become unbalanced is unknown at
present.
7. Breast tissue estrogen content: uptake from the circulation
versus synthesis in situ
Levels of estradiol in normal and in malignant breast tissue are
similar for pre- and postmenopausal women, despite the large
differences in serum levels (Thijssen et al., 1986; van Landeghem
et al., 1985). High estradiol levels in postmenopausal breast tissue
may result from enhanced uptake from the circulation or from in
situ aromatization of androgens and from estrogen sulfate via the
sulfatase/17b-HSD pathway. It is estimated that about 75% of
estrogens in premenopausal women are synthesized in peripheral
tissues and close to 100% after menopause (Labrie, 1991, 2003).
However, it is extremely difcult to calculate the relative contri-
butions of uptake from the circulation versus in situ synthesis for an
individual organ or tissue, such as the breast.
A number of lines of indirect evidence suggest a role for in situ
estrogen synthesis in tumor development that include:
 The presence of the necessary steroidogenic enzymes in breast
tissue (Labrie, 2003; Pasqualini, 2004).
 Levels of DHEA, the androgen precursor of estrogens, are many-
fold higher in normal and malignant breast tissue for both pre-
and postmenopausal women than in their serum (Labrie, 1991;
Thijssen et al., 1986).
 ER breast tumor tissue contains estradiol (Edery et al., 1981).
 There is little correlation between serum estradiol levels and
breast epithelia proliferation (Khan et al., 1999).
Yue, Wang, Hamilton, Demers, and Santen (1998) developed an
animal model where aromatase (A)- and sham (A)-transfected
human MCF-7 breast cancer cells were inoculated into ovariecto-
mized nude mice, which lack peripheral aromatase activity. With
the injection of androstenedione and the use of Silastic implants to
produce various estradiol levels, it was determined that under
physiological conditions, reecting those in postmenopausal
women, in situ aromatization made a major contribution to estra-
diol levels in breast cancer tissue. Previously, this group showed
that rats bearing chemically induced hormone-dependent tumors
synthesized about 25% of their estrogen content in situ by the
estrogen sulfatase pathway (Masamura, Santner, & Santen, 1996).
A few studies have determined the contribution that local in situ
estrone synthesis made to the estrogen content of normal and
malignant breast tissue in postmenopausal women using a double
8 P.W. Parodi / International Dairy Journal 22 (2012) 3e14
isotopic technique. Miller et al. (1998) infused 11 patients with
14
C-labeled estrone and 3H-labeled androstenedione, 18 h before
breast tissue and peripheral blood were taken for analysis. There
was considerable variation between breasts with the contribution
of in situ synthesis ranging from 0 to 75%. In a subsequent study
this group measured the percentage contribution of local
estrone synthesis in both normal and tumor tissue from 24 post-
menopausal women. Again, there were marked variations between
specimens from different patients and the relative proportion of
synthesis to uptake was higher in tumors (0e92%, median value
29%) than in normal tissue (0e58%, median value 15%). There was
a positive correlation between uptake in normal and malignant
tissue (Larionov, Berstein, & Miller, 2002). A small study by Reed
et al. (1989) using a similar isotopic infusion technique found that
in 3 of 7 subjects there was no or little in situ formation of estrone.
For the other 4 subjects in situ estrone synthesis appeared to
contribute around 85% of total estrone content in tumor tissue
while the value for normal tissue was 71%.
Breast tissue can also obtain signicant quantities of estradiol
from inltrating macrophages. Macrophages are present in normal
resting, pregnant, and lactating breast tissue with levels
increasing after involution, but probably do not exceed 20% of the
total cell population (Ferguson, 1985). A study by Kelly, Davison,
Bliss, and McGee (1988) found malignant tissue contained the
highest concentrations of macrophages with levels around 50% of
the tumor mass. Levels in benign tissue were around one-half this
value. Macrophages contain the necessary steroidogenic enzymes,
including aromatase, to convert the ubiquitous adrenal hormone
DHEA into estradiol (Mor et al., 1998; Schmidt, Kreutz, Lofer,
Scholmerich, & Straub, 2000). Estrogen production by macro-
phages is sufcient to stimulate the proliferation of adjacent
epithelial cells (Mor et al., 1998).
8. Estrogen content of cows milk
Estrogen levels in cows milk and factors that inuence them
have been inadequately studied. The objective of early studies was
to use estrogen levels as a tool to determine estrus and pregnancy.
Early methods that utilized bioassays, colorimetry and spectro-
uorimetry were crude and had a high limit of detection. Intro-
duction of radioimmunoassay (RIA) improved sensitivity, but
factors such as loss of hormones during removal of triglycerides,
cross reactivity of antibodies with other hormones, incomplete
hydrolysis of hormone conjugates and lack of pure standards still
affected results. Recently, improvement in extraction techniques
coupled with improved RIA assays and measuring techniques like
liquid chromatography (LC) or gas chromatography tandem mass
spectrometry (GCeMS/MS) have further advanced sensitivity and
specicity. However, these techniques are complex, time
consuming and expensive, which has limited sample numbers.
Only studies that utilized these improved techniques are reviewed
here. Unfortunately sample sizes for the different types of milk
were generally small.
Hartmann, Lacorn, and Steinhart (1998) measured the hormone
content of German whole milk (sample numbers unknown) using
GCeMS. The level of total estradiol was less than 20 pg mL1, which
was the limit of detection. Total estrone was present at around
130 pg mL1. Four samples of milk from non-pregnant cows of the
Utrecht University herd were analyzed by LC-MS/MS. Values for
free estrone were 23, 22, 27 and 24 pg mL1, whereas values for
total estrone (free conjugated) were 162, 208, 174, and
243 pg mL1. Free estradiol values were 5 pg mL1, not detected
(limit of detection 5 pg mL1), 5 pg mL1, and not detected, while
total estradiol values were 10, 10, 9 and 11 pg mL1 (Malekinejad,
Scherpenisse, & Bergwerff, 2006). Pape-Zambito, Magliaro, and
Kensinger (2007) measured individual estradiol levels in milk
from 206 cows in the Pennsylvania State University dairy herd
using RIA. Mean estradiol levels ranged from undetectable (limit of
quantication 0.7 pg mL1) to 22.9 pg mL1, with a mean value
1.4  0.2 pg mL1. Twelve samples of milk (4 whole, 5 half-
skimmed, 3 skimmed) were collected from a French supermarket
and analyzed using GCeMS/MS (Courant et al., 2007). The sum of
free plus conjugated estradiol ranged from 9.8 to 44.3 pg mL1, with
a mean value of 23.0 pg mL1. For estrone the corresponding values
were 75.8e277.5 pg mL1 and 152.8 pg mL1, respectively. Eighty
four percent of estradiol and 96% of estrone were present in the
conjugated form. Vicini et al. (2008) conducted the most compre-
hensive survey, with the analysis of 334 samples of retail whole
milk collected from 48 states in the USA during a three-week
period. RIA, with a sensitivity of 0.8 pg mL1, was used for the
assay. The mean value for estradiol was 4.97  0.239 pg mL1.
Recently, Farlow et al. (2009) measured estrogen levels in single
samples of whole, 2% fat and skim milk in the US using LC-MS/MS
(limit of quantitation 0.2 pg mL1). Levels of free estradiol and
estrone were 1.0, 1.8, and 2.4 pg mL1 and 10.3, 6.8 and 3.8 pg mL1,
respectively. Corresponding values for total estradiol and estrone
were 22.1, 22.2, 20.4 pg mL1 and 85.5, 84.6, 65.0 pg mL1,
respectively.
8.1. Factors inuencing the hormone content of milk
A number of factors can inuence the hormone content of milk.
Both estradiol and estrone levels increase during the estrous cycle,
reaching peak values at estrus, thereafter decreasing to the end of
the cycle (Gyawu & Pope, 1990; Monk, Erb, & Mollett, 1975).
Hormone levels can also vary with stage of lactation (Gyawu &
Pope, 1990). There is considerable inter-individual variation
within a heard and values for individual cows uctuate markedly
from week to week (Gyawu & Pope, 1990; Henderson, Karanikolas,
Kenealy, & Macmillan, 1994a).
The estrogen level in milk increases during pregnancy.
Henderson et al. (1994a) measured the levels of estrone sulfate in
milk from four herds. Levels rose progressively during pregnancy
from a mean value of 80e100 pg mL1 at 60e80 d of pregnancy to
a plateau value of around 1000 pg mL1 at 181e200 d. For non-
pregnant cows in the herds, estrone sulfate concentration ranged
from non-detected to 110 pg mL1 with a mean value of 59 pg mL1
Malekinejad et al. (2006) reported that the level of free estrone in
raw milk from non-pregnant cows and cows in the rst, second and
third trimester of gestation was 6.2, 9.2, 57 and 118 pg mL1,
respectively. Corresponding values for free estradiol were 5.6, 10.2,
20.4 and 21.3 pg mL1, respectively. For total estrogens
(free conjugated and for different cows from the above), estrone
levels in the rst, second and third trimester milk were 7.9, 452 and
1266 pg mL1, and for estradiol 18.6, 51.4 and 51.2 pg mL1,
respectively.
Pape-Zambito et al. (2007) found that mean free estradiol levels
of milk from non-pregnant cows, early pregnant cows (1e140 d)
and mid-pregnant cows (141e210 d) were 1.3, 0.9 and 3.0 pg mL1,
respectively. In a further study (Pape-Zambito, Magliaro, &
Kensinger, 2008) a group of cows was followed throughout preg-
nancy. Milk free estrone concentrations averaged 0.6, 7.9 and
27.1 pg mL1 during the rst, second and third trimester, respec-
tively. Corresponding values for estradiol were 0.3, 0.9 and
5.0 pg mL1, respectively.
Free estrogens are lipophilic and Pape-Zambito et al. (2008) and
Pape-Zambito, Roberts, and Kensinger (2010) found both estrone
and estradiol levels were signicantly correlated with the fat
content of whole milk. Malekinejad et al. (2006) found that for 0%,
1.5% and 3.5% fat milk the free estrone content was 8.2, 17.1 and
 P.W. Parodi / International Dairy Journal 22 (2012) 3e14
20.0 pg mL1, respectively. Corresponding values for estradiol were
10.3, 13.9 and 20.6 pg mL1, respectively. On the other hand,
another study found no relationship between total estrogen
content and the fat content of milk (Courant et al., 2007). This is
undoubtedly due to the fact that conjugated estrogens, by far the
major component of milk estrogens, are more soluble in aqueous
solution. Pasteurization and homogenization of raw milk do not
appear to affect the estrogen content (Pape-Zambito et al., 2010).
Ganmaa and Sato (2005), Ganmaa, Wang, Qin, Hoshi, and Sato
(2001), Maruyama, Oshima, and Ohyama (2010), Qin et al. (2004)
and others have suggested that compared with milk produced 100
years ago, modern herd bulk milk contains a larger proportion of
milk from cows in the latter half of pregnancy, and thus has elevated
levels of estrogens, which may contribute to reproductive disorders
and cancer at hormone-responsive sites. However, as noted above,
the increased estrogen levels during late pregnancy is mostly due to
estrone sulfate, which is largely biologically inactive. In addition,
during the past 60e70 years milk production per cow has increased
about 5-fold due to improved genetics for milk production coupled
with changes in nutritional management (Capper, Cady, & Bauman,
2009). Estradiol concentrations are negatively correlated with milk
yield (Pape-Zambito et al., 2008). This effect is probably due to the
fact that high feed intake increases liver blood ow and metabolic
clearance rate of estradiol by the liver (Sangsritavong, Combs,
Sartori, Armentano, & Wiltbank, 2002).
Pape-Zambito et al. (2008) estimate that the majority of cows
producing milk for consumption would have less than 2 pg mL1 of
estradiol. This estimate is based on their studies with herd milk and
milk from pregnant cows and because early lactation cows have
high milk yields and farmers generally dry off cows at 60 days
before the next expected calving. Thus, there is no evidence that the
levels of estrogen in modern commercial milk are higher than 100
years ago.
9. Absorption and metabolism of estrogens from
milk and its products
Estradiol in consumed dairy products is absorbed from the
intestine. The intestinal mucosa contains enzymes capable of the
transformation of estradiol to estrone and the formation of estrogen
sulfate and glucuronide conjugates. These metabolites and any free
estradiol pass to the portal circulation and rapidly pass to the liver
where near-complete metabolism to estrone, estrone sulfate (the
major conjugate) and some estrogen glucuronides and hydroxyl-
ated conjugates occurs (Lobo & Cassidenti, 1992; Longcope et al.,
1985). Part of the conjugated estrogens pass to the kidneys and
are excreted in urine. Most of the remainder is excreted with bile to
the intestinal lumen. Here, some of the conjugated estrogens may be
deconjugated by bacterial b-glucuronidases, but most of these are
re-conjugated by intestinal steroid sulfotransferases and glucur-
onosyltransferases and along with the majority of the remaining
bile-derived estrogen conjugates are re-absorbed and pass to the
liver (enterohepatic circulation). A portion escapes this enter-
ohepatic circulation and is excreted in the faeces. Any free estradiol
will be conjugated at the next pass of the liver (Adlercreutz &
Martin, 1980; Adlercreutz, Martin, Jarvenpaa, & Fotsis, 1979). Over-
all, it is estimated that as a result of metabolism by the intestinal
mucosa and rst pass to the liver, the bioavailability of oral estradiol
is only in the range of 2e5% (Dusterberg, Schmidt-Gollwitzer, &
Humpel, 1985; Kuhnz, Gansau, & Mahler, 1993).
As part of a German market basket survey, Hartmann et al.
(1998) determined the natural occurrence of steroid hormones in
food. Based on previously published national nutritional data they
estimated the daily intake of estrogens (estrone plus estradiol)
from milk products was 0.06 mg for men and 0.05 mg for women.
9
These values were due largely to the relatively bio-inactive estrone
and both estrone and estradiol were predominantly present as
inactive conjugates. Based on recent studies with sensitive assays
(Farlow et al., 2009; Malekiunejad et al., 2006; Pape-Zambito et al.,
2007, 2008, 2010; Vicini et al., 2008), it appears unlikely that free
estradiol levels in commercial milk would exceed 5.0 pg mL1.
Thus, consumption of 1.5 L of milk or equivalent dairy products per
day would result in a daily estradiol intake of not more than 7.5 ng,
of which no more than about 0.38 ng would survive rst pass
metabolism to the liver. This value must be considered in the
context of daily excretion rates for estradiol in humans. In
premenopausal women the daily production of estradiol varies
between 0.08 and 1.00 mg, depending upon the phase of the
menstrual cycle (Hsueh & Billig, 1995). With the cessation of
ovarian function after menopause, daily estradiol production
decreases to around 12 mg, which is mainly derived from estrone,
now the predominant estrogen and produced in peripheral tissues
tissue from androstenedione by the action of the aromatase
enzyme (Golden & Monroe, 1986). Production rates for estrone
range from 40 mg d1 for slender women to 200 mg d1 for obese
women (ODell, 1995). In men estradiol production is about
45 mg d1 (Kaufman & Vermeulen, 2005). Estradiol production in
prepubertal children has not been determined directly, but it is
considered values will be low (Andersson & Skakkebaek, 1999).
Thus, in the case of premenopausal women, intake of around
7.5 ng d1 of estradiol from 1.5 L of milk or equivalent would
represent merely 0.00076e0.0094% of their daily production and
only 5% of this intake would survive the rst pass of the liver in
a bioactive form. The percentages for postmenopausal women,
prepubertal children and men would be higher.
The Joint FAO/WHO Expert Committee on Food Additives (WHO,
2000) established an acceptable daily intake for estradiol of
0e50 ng kg1 of body weight. This gure was based on changes in
several hormone-dependent parameters in postmenopausal
women. A safety factor of 10 was used to account for normal
variation among individuals, and an additional factor of 10 was
added to protect sensitive individuals. Accordingly, estradiol
consumption from dairy products should account for only around
0.25% of the FAO/WHO upper acceptable daily intake value.
Milk contains estrone of which around 90% is estrone sulfate
(Henderson, Camberis, Simmons, Starrs, & Hardie, 1994b). Estrone
has low biological activity and estrone sulfate is inactive. It is
possible that some of the small quantity of estrone sulfate in milk
that survives metabolism during rst pass to the liver may be
converted to estrone by sulfatases then to estradiol by 17b-HSD.
However, milk-derived estrones are but a small fraction of a large
circulating pool. For instance, for postmenopausal women in the
Nurses Health study (Hankinson et al., 1995) mean concentrations
of estrone and estrone sulfate were 18- and 98-fold higher than
bioavailable estradiol. In addition, there are vastly higher levels of
circulating DHEA-S (Labrie, 2003), and even higher levels in breast
tissue (Thijssen et al., 1986), which can be bioconverted to estrone
and estradiol if required by the cell. Overall, estrogen-sensitive cells
possess multiple mechanisms to obtain estradiol for their growth
and maintenance and to inactivate surplus to requirements.
Coupled with the relatively minute amount of dairy product-
derived estradiol surviving intestinal absorption and rst pass
liver metabolism, this makes it unlikely that estradiol from this
source would inuence cancer development.
10. Physiological inuence of various exogenous
sources of estrogen
The estrogen content of milk, its contribution to the bodys
estrogen pool and its physiological signicance may be viewed in
10 P.W. Parodi / International Dairy Journal 22 (2012) 3e14
relation to the inuence of a number of external factors on serum
estrogen levels. Many factors have been studied but BMI appears to
be the most important determinant. Data from the Nurses Health
Study (Hankinson et al., 1995) showed that in postmenopausal
women serum estrogen levels were positively associated with BMI.
Mean serum estradiol levels ranged from 4.7 pg mL1 in the lowest
quintile of BMI (<21 kg m2) to 10.0 pg mL1 in the highest quintile
(29 kg m2). For bioavailable estradiol, the corresponding values
were 0.8 and 3.3 pg mL1. A pooled re-analysis of individual data
from 8 prospective studies showed similar variations (Endogenous
Hormones and Breast Cancer Collaborative Group, 2003).
Dietary components may inuence intestinal function and the
bacterial population. Changes in the number of b-glucuronidase-
producing bacteria can inuence deconjugation of estrogens, affect
the enterohepatic circulation and the level of estrogens excreted in
faeces and urine and their level in blood (Goldin & Gorbach, 1994).
Many studies investigated the role of dietary components such as
fat, protein and ber, on serum estrogen levels, but most suffered
from methodological faults, small sample size and lacked controls.
The Nurses Health Study found associations between different
dietary patterns and serum estrogen levels in postmenopausal
women, but the associations were lost after adjusting for BMI
(Fung, Hu, Barbieri, Willett, & Hankinson, 2007). The effect of
animal products and their nutrient components on circulating
estrogens levels in postmenopausal women was investigated in the
large Melbourne Collaborative Study (Brinkman et al., 2010). None
of the food items or nutrients explained more than 1% of the total
variation in concentration of any steroid hormone.
For clinical intervention studies, Carruba et al. (2006) random-
ized postmenopausal women to receive their normal diet or
a traditional Mediterranean diet (intervention). After 6 months, the
urinary total estrogen concentration in the intervention group
decreased by 40%. This decrease was largely driven by the estrogen
metabolites 2-hydroxy- and 16-ketoestradiol and 17-epiestriol.
Estradiol represented only 1.16% of total estrogens and this level
was signicantly higher than the baseline value. However, during
the study, subjects in the intervention group consumed signi-
cantly less calories than at baseline. The intervention group of
postmenopausal women in the large Womens Health Initiative
randomized controlled dietary modication trial consumed a low-
fat diet with increased intake of fruit, vegetables and grains
compared to their normal diet. Serum estrogen levels were
measured in a sub-group of women at baseline and after 1 year of
intervention. Estradiol concentrations decreased from 7.6 to
6.7 pg mL1. However, during this period there was a decrease of
2.4 kg in the weight of women in the intervention group (Prentice
et al., 2006). A change in diet to one low in animal fat and rened
carbohydrates and rich in low-glycemic-index foods, mono-
unsaturated and n-3 polyunsaturated fatty acids and phytoes-
trogens in a small group of postmenopausal women resulted in
a fall in serum estradiol levels from 8.62 to 7.07 pg mL1 after 4.5
months intervention. During this time, their BMI decreased from
26.88 to 25.26 kg m2 (Berrino et al., 2001).
Based on the limited data available, different dietary patterns
may be responsible for a 10e20% change in serum estradiol in
postmenopausal women, but about one-half of this effect may be
due to weight change. Similar changes in serum estradiol levels as
a result of dietary modication in premenopausal women may also
apply (Goldin et al., 1981).
The effect of oral contraceptive use and hormone replacement
therapy (HRT) on serum and tissue estradiol concentrations is also
informative and illustrates one of the paradoxes associated with the
role of estradiol in cancer development. A sub-group of post-
menopausal women in the Womens Health Initiative randomized
controlled clinical trial of estrogen therapy that received
0.625 mg d1 of conjugated equine estrogens (CEE) had serum
estrogen levels measured at baseline and after 1 year of therapy
(Edlefsen et al., 2010). Estradiol levels increased from 13.3 to
35.5 pg mL1, bioavailable estradiol from 8.9 to 16.1 pg mL1 and
free estradiol from 0.4 to 0.6 pg mL1. Estrone levels increased from
41.4 to 149.9 pg mL1. After a mean of 10.7 years of follow-up in the
intervention phase of the Womens Health Initiative randomized
controlled trial (LaCroix et al., 2011), the hazard ratio for breast
cancer in postmenopausal women who used 0.625 mg d1 of CEE
for a median of 5.9 years compared to non-users was 0.77
(0.62e0.95) Three other small randomized controlled trials of
estrogen-only therapy reviewed by Collins, Blake, and Crosignani
(2005), found no excess risk compared with placebo. Epidemio-
logical evidence from the large Nurses Health Study suggested that
women who had undergone a hysterectomy and used estrogen-
only therapy for less than 20 years did not have an increased risk
of developing breast cancer. However, longer duration of usage was
associated with higher risk, primarily for ER and progesterone
receptor positive breast cancers (Chen et al., 2006).
Use of oral contraceptives by premenopausal women causes
a suppression of ovarian function. As a result the elevated levels of
estradiol during mid-cycle and again during the mid-luteal phase
are suppressed several fold to levels at or below levels found in the
rst half of the follicular phase in non-users. A similar suppression
occurs for estrone (Carr & Bradshaw, 1998; Gaspard, Dubois, Gillian,
Franchimont, & Duviver, 1984). In addition, in breast tissue, oral
contraceptive use suppressed estradiol levels 7.8-fold and estrone
levels 2.9-fold compared to non-users (OBrien, Anandjiwala, &
Price, 1997). However, epidemiological evidence for women using
oral contraceptives and were subjected to these large reductions in
serum and tissue estrogens does not show a reduced risk for
subsequent breast cancer (Hunter et al., 2010; Kahlenborn,
Modugno, Potter, & Severs, 2006).
Around 30 years ago, several observation studies suggested that
hormonal changes in women might be associated with the risk of
colon cancer. However, the results of these studies were not
consistent. During the period 1961e1990 the sex-specic rates for
colon cancer incidence in men increased by 16%, whereas in women
the incidence declined by 21% (Potter, 1995). This inequality was
considered to be due to hormone differences. HRT use was
considered a possible determinant of the benecial effect in
women, and a number of epidemiological studies investigated this
relationship. Hebert-Croteau (1998) conducted a meta-analysis of
11 case-control studies, 7 cohort studies and a randomized
controlled trial. A summary RR of 0.85 (0.73e0.99) was found
between ever versus never users of HRT and the risk of colon
cancer. The estimated RR was lower among current and recent
users, RR 0.69 (0.52e0.91) as compared to short-term users, RR
0.88 (0.64e1.21). This meta-analysis did not include the 14-year
follow-up data from the large Nurses Health Study (Grodstein
et al., 1998), which showed current use of postmenopausal HRT
was associated with a RR of 0.65 (0.50e0.83). This association was
attenuated to a RR of 0.84 (0.67e1.05) in past users of HRT. A
subsequent meta-analysis that included the up-dated data from the
Nurses Health Study (Grodstein, Newcomb, & Stampfer, 1999)
found that women who had ever taken postmenopausal HRT,
compared to never users had a RR of 0.80 (0.74e0.86) for colon
cancer and a RR of 0.81 (0.72e0.92) for rectal cancer. Much of the
reduction in colorectal cancer was limited to current HRT users (RR,
0.66, 0.59e0.74).
In the Womens Health Initiative Randomized Controlled Trial
(Rossouw et al., 2002), the HR for participants who received
0.625 mg d1 CEE plus 2.5 mg d1 medroxyprogesterone acetate
compared with placebo was 0.63 (0.43e0.92). On the other hand, in
the CEE-only arm (LaCroix et al., 2011), the HR for CEE compared
 P.W. Parodi / International Dairy Journal 22 (2012) 3e14
with placebo was 1.15 (0.081e1.64). However, the number of colon
cancer cases during the trial was small. A large case-control study
by Hoffmeister, Raum, Krtschil, Chang-Claude, and Brenner (2009)
found the risk of colorectal cancer decreased in both estrogen-only
therapy, RR 0.42 (0.23e0.78) and in combination therapy, RR 0.60
(0.41e0.87).
The benet of estradiol for colorectal carcinogenesis was
conrmed in animal studies. For instance, Smirnoff, Liel, Gnainsky,
Shany, and Schwartz (1999) demonstrated that tumor numbers in
ovariectomized mice induced by dimethylhydrazine, were reduced
by 72% when treated with estradiol. Reduced cell growth and
increased apoptotic activity, in a does-dependent manner, was
noted in young adult mouse colonocytes when treated with phys-
iological levels of estradiol. In addition, estradiol treatment in
ovariectomized mice exhibited a signicant protection against
preneoplastic lesions (Weige, Allred, & Allred, 2009).
11. Epidemiological evidence
Because it is not possible to conduct appropriate clinical trials to
prove unequivocally that the small quantity of estrogens in dairy
products do not inuence cancer development, by default, epide-
miological studies, despite their limitations, may provide evidence
on the safety of dairy product consumption.
11.1. Breast cancer
Missmer, Smith-Warner, and Spiegelman (2002) conducted
a pooled analysis of 8 cohort studies that provided 351,041 subjects,
7379 of whom were diagnosed with breast cancer. No signicant
association was found between dairy product consumption and
breast cancer risk. A review of 10 cohort and 36 case-control studies
by Moorman and Terry (2004) concluded that the epidemiological
evidence did not support a strong association between consump-
tion of milk or other dairy products and the risk of breast cancer. In
later major prospective studies McCullough et al. (2005), from the
Cancer Prevention Study II, reported that dairy product consump-
tion was inversely associated with postmenopausal breast cancer
risk. The European Prospective Investigation into Cancer and
Nutrition (Pala et al., 2009), which recruited cohorts from 10
European countries, did not nd dairy product consumption a risk
factor for breast cancer.
Exposure to initiating events during childhood, adolescence and
early adulthood, when the mammary gland is attaining adult stage
morphology, may inuence the risk of breast cancer later in life.
A review of 4 case-control and 3 cohort studies (Parodi, 2005) did
not nd an association between adolescent dairy product
consumption and subsequent breast cancer development. A later
large study of participants from the Nurses Health Study and the
Nurses Health Study II found that consumption of whole milk as
part of a preschool diet was associated with a slightly decreased
risk of adult breast cancer (Michels, Rosner, Chumlea, Colditz, &
Willett, 2006). Examination of adolescence diet in the Nurses
Health Study II found no overall association for total milk or total
dairy intake and risk of breast cancer; however, a nonsignicant
inverse trend between breast cancer and low-fat milk and low-fat
dairy was noted (Linos, Willett, Cho, & Frazier, 2010). Sixty-ve
year follow-up of the Boyd Orr cohort showed that a family diet
rich in dairy products during childhood was not associated with
adult breast cancer risk (van der Pols et al., 2007). Epidemiology
suggests that dairy product consumption during various stages of
the life cycle is safe and not associated with a risk of breast cancer.
However, because dairy products contain calcium together with
a range of lipid anticancer agents (Parodi, 2005), such analyses
11
cannot unequivocally exonerate estrogens in milk and its products
as possible carcinogens.
11.2. Ovarian cancer
Genkinger et al. (2006) conducted a pooled analysis of 12 cohort
studies representing 553,217 women among whom 2132 epithelial
ovarian cancer cases were identied. No associations were
observed between intake of specic dairy foods and ovarian cancer
risk. Subsequently, it was also found that there were no associations
between intakes of various dairy foods and the risk of ovarian
cancer among participants in the Netherlands Cohort Study on Diet
and Cancer (Mommers, Schouten, Goldbohm, & van den Brandt,
2006). Higher intakes of total dairy foods were associated with
a statistically signicant decreased risk of ovarian cancer in the
Breast Cancer Detection Demonstration Project cohort (Koralek
et al., 2006).
11.3. Endometrial cancer
The role of dairy products in endometrial cancer has not been
studied widely. A systematic literature review and meta-analysis of
the available evidence by Bandera, Kushi, Moore, Gifkins, and
McCullough (2007) found no support for an association between
dairy product consumption and risk of endometrial cancer.
11.4. Prostate cancer
The World Cancer Research Fund/American Institute for Cancer
Research (WCRF/AICR) 2007 report on Food, Nutrition, Physical
Activity and the Prevention of Cancer (The World Cancer Research
Fund/American Institute for Cancer Research, 2007) considered
that there was limited evidence suggesting that high consumption
of milk and dairy products is a cause of prostate cancer. The WCRF/
AICR Panel suggested that high calcium intake from dairy products
down-regulates the formation of 1,25-dihydroxyvitamin D3 from
Vitamin D, thereby increasing cell proliferation in the prostate.
Also, consumption of milk products increases blood levels of
insulin-like growth factor-1, which has been associated with
increased prostate cancer risk in some studies. The Panel did not
implicate estrogens in prostate cancer risk.
Since the WCRF/AICR report, numerous prospective studies
have presented results that included sub-group analyses of asso-
ciations between types of milk and dairy products and stage of
prostate cancer. There were often contradictory results, but overall
the more recent studies do not provide strong support for an
increased risk of prostate cancer with dairy product consumption
(Parodi, 2009).
11.5. Colon cancer
Numerous epidemiological studies have demonstrated a nega-
tive association between dairy product consumption, particularly
milk, and the risk of colorectal cancer. Huncharek, Muscat, and
Kupelnick (2009) conducted a meta-analysis of 26,335 cases from
60 observational studies. The summary RR for high milk and dairy
product intake, respectively, on colon cancer risk was 0.78
(0.67e0.92) and 0.84 (0.75e0.95). Milk intake was not associated
with rectal cancer risk and thus in studies where colorectal cancer
was the end point; summary RR was attenuated to 0.90 (0.81e1.00).
Calcium in milk is believed to be the principal agent responsible for
its protective action (Holt, 2008; Pufulete, 2008), although lipid
components like conjugated linoleic acid, butyric acid and sphin-
gomyelin (Larsson, Bergkvist, & Wolk, 2005; Parodi, 1997) and milk
proteins, especially whey proteins (Parodi, 2007) may also
12 P.W. Parodi / International Dairy Journal 22 (2012) 3e14
contribute. Whether the small amount of bioactive estradiol in milk
has any impact on colon carcinogenesis is unknown, but epidemi-
ology suggests it causes no harm.
12. Conclusions
Although several lines of evidence suggest a role for estrogens in
cancer at hormone-responsive sites, such as the breast, ovaries,
endometrium and the prostate, the mechanisms remain elusive.
This may result from estrogens acting in concert with other
hormones, growth factors and cytokines and presents a major
research problem. Many studies have determined associations
between serum estrogen levels and the risk of cancer at estrogen-
responsive sites. The association is robust for postmenopausal
breast cancer, but less so for premenopausal women, strong for
endometrial cancer and null for ovarian and prostate cancer.
However, there is no correlation between circulating levels of
estrogens and their precursors with their levels in tissues. It is now
recognized that the hormone-responsive tissues contain all the
necessary steroidogenic enzymes for the synthesis of estrogens
from androgen precursors. In addition, the tissues contain enzymes
responsible for conjugation of bioactive estrogens to inactive
conjugates in order to maintain estradiol homeostasis.
A few studies have measured the contributions of estrogens
from in situ synthesis and uptake from the circulation in normal
and malignant breast tissue. Results show a large inter-individual
variation for in situ synthesis with values ranging from 0 to over
90%, overall values were higher in cancer tissue. Reasons for, and
the signicance of, the large variation in source of tissue estrogens,
in conjunction with a study of the levels and activity of metabo-
lizing enzymes require further investigation.
Cows milk contains estrogens. Recent high sensitivity assays
suggest that commercial milk would not contain more than 5 pg mL
of free estradiol. On ingestion only 2e5% of this bioactive form
survives metabolism in the intestinal mucosa and rst pass to the
liver. This amount is only a minute fraction of daily production
levels in women and men and is far less than possible uctuations
in serum levels due to diet, weight excess and use of hormone
therapy. Expected daily estradiol intake from dairy products
represents only about 0.25% of the FAO/WHO upper acceptable
daily intake of exogenous estradiol. Given the multiple mechanisms
cells possess to obtain estradiol for their function, it is most unlikely
that the small amount of exogenous estradiol provided by dairy
products would inuence carcinogenesis at estrogen-responsive
sites. Epidemiological studies do not show an association
between dairy product consumption and risk of cancer of the
breast, ovaries and endometrium. There is a slight positive associ-
ation with prostate cancer, but estrogens have not been implicated
as an etiological factor.
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