Effects of cow*s milk consumption on estrogen levels in human male volunteers

It is known since long that cow*s milk contains hormones, such as estrogens,
progesterone and growth factors. However, hormone levels in commercial milk
production have increased because lactation in new breeds of cows continues
throughout almost the entire pregnancy.
Because of the increased levels of estrogens in cow*s milk and the high
consumption of milk products in the Dutch population, it is of concern whether
intake of hormones via cow*s milk consumption could result in endocrine effects
in humans. Beside the possibility of endocrine disruption, relations between
milk consumption and prevalence of hormone-dependent cancers (e.g. breast- and
prostate cancers) have been suggested. Although clear epidemiological evidence
is currently lacking, it is worth investigating which hormones in milk are most
critical for these adverse effects. Exposure to estrogens appears to be
important in this possible relationship between milk consumption and
hormone-dependent cancers, as estrogens are critically involved in
hormone-dependent cancers. On one hand estrogens interact with the estrogen
receptor to induce cell proliferation. On the other hand, metabolites of
estrogens interact with the estrogen receptor and the DNA, thereby inducing
genotoxicity. Both mechanisms explain the carcinogenic properties of estrogens.
Preliminary data from a very recent Japanese study indicates increases in
systemic estrogen concentration after a single consumption of 600 ml commercial
milk.
In the Dutch situation can be expected that an additional intake of estrogens
takes place by means of the consumption of dairy products. It remains however
the question whether this additional exposure to estrogens from dietary intake
results in changes in systemic (internal) concentrations, and whether this can
be responsible for the observed changes in growth and development of juveniles
and the higher incidence of hormone-dependent cancers. The association between
the high dairy consumption in our country and before mentioned changes in
growth and development as well as higher tumor incidence is at present only
based on speculation. Other lifestyle factors, dietary factors and improved
medical care during the last fifty years could also be involved.
To reveal a possible causal connection between dietary estrogens and changes in
growth and development or higher tumor incidence, it must first be investigated
whether Dutch dairy consumption can lead to increased systemic estrogen
concentrations. If internal concentrations are not significantly influenced by
cow*s milk consumption, the risk of these hormones can be considered then as
nil in this specific situation. It is known from pharmacology that natural
estrogens are taken up poorly after oral intake. It is not unlikely that a
potential increase of systemic concentrations is reduced by first pass
metabolism in the liver. Phase II sulfate and glucuronidic metabolites are
formed, that are rapidly excreted from the body. It can however not be excluded
that re-uptake of estrogens via the entero-hepatic pathway also occurs,
possibly resulting in increases in systemic concentrations in blood and
tissues. Estrogens taken up from the cow*s milk may also be metabolized to
hydroxylated metabolites in the liver. Some of these metabolites also exhibit
estrogenic activity and are possibly carcinogenic.

The aim of this study is to investigate whether weeklong dietary intake of
estrogens via high cow*s milk consumption in the Dutch population increases
systemic estrogen concentrations. To this aim, estrogen levels are measured in
urine samples of volunteers.
In the urine samples, estrogen concentrations (17β-estradiol en
estrone) are measured at the division Veterinary and Public Health (VPH) at the
Institute for Risk Assessment Sciences (IRAS) at Utrecht University (Faculty
Veterinary Medicine). Measurements of estrogen metabolites are also included.
Because of the close association between estrogens and androgens, testosterone
measurements are also included. To investigate total amount of estrogenic
compounds excreted via urine, sum estrogenic activity will be investigated by
using in vitro screening of urine extracts (see below in study design).

Volunteers will be asked to drink 1.5 l cow*s low-fat (*halfvolle*) milk per
day for 7 days. The resulting daily intake of 1.5 l is considered a high-intake
scenario, while average daily intake of cow*s milk in the Netherlands is
estimated to be 270 ml (in 19-30 year olds). Before the study, a screening
questionnaire and an informed consent form have to be filled out. 3 days prior
to the intervention and during the intervention, the volunteers will remove
dairy products from their diet, as well as other foodstuffs containing large
amounts of phytoestrogens (specified in volunteer*s information). It is
expected that dietary estrogens are cleared from the body within this period.
Examples for meals appropriate during the study are suggested. Supplied food
questionnaires (qualitative) have to be filled out daily. At 8 days during the
study, volunteers are asked to collect morning urine, and they are invited to
the IRAS in the morning. At these meetings, urine sample vials and milk are
distributed, and urine samples handed in. The volunteers receive a simple
breakfast to-go. The volunteers will be asked for their body weight and length.

Urinary excretion of hormones is used in this study to examine changes in
internal concentrations. In the urine, 17β-estradiol and estron are measured as
a measure for systemic exposure. To investigate possible effects on the
estrogen/androgen balance, testosterone is also measured in the urine samples.
The hormones in the urine will be measured by LC-MS/MS at the division
Veterinary and Public Health of the IRAS. Hormone extracts of the urine samples
are also tested in two in vitro bio-assays for detection of estrogenic and
androgenic activity. These are ER-Calux and AR-Calux, in vitro assays using a
genetically modified mammal cell line. Analysis of estrogen metabolites will
also be included. The results of this research will be published in the open
scientific literature. The data will also be made available to the RIVM for
possible pharmacokinetic modeling. To this aim, the volunteers will be asked
for their body weight and length.

Volunteers will be asked to drink 1.5 l cow*s low-fat (*halfvolle*) milk per
day for 7 days. 3 days prior to the intervention and during the intervention,
the participants will remove dairy products from their diet, as well as other
foodstuffs containing large amounts of phytoestrogens. Supplied food
questionnaires (qualitative) have to be filled out daily (in total 31 days). At
8 days during the study, volunteers are asked to collect morning urine, and
they are invited to the IRAS. At these meetings, urine sample vials and milk
are distributed, and urine samples handed in. The volunteers receive a simple
breakfast to-go. A compensation of 150 ¤ will be given to the volunteers after
the study. Milk will be supplied by the research team to the volunteers. The
volunteers will be asked to inform the researchers about their body weight and
length.

The inclusion and exclusion criteria are designed to avoid health risk related
to milk consumption (mainly allergic reactions). Prior to and during the
intervention, volunteers will have to avoid certain foodstuffs. These are
listed in the information for volunteers and example meal recipes are
suggested. During the study, volunteers will have to fill in daily food
questionnaires (in total 31 days). Furthermore, only non-invasive (urine)
sampling will occur during this study.