Combination chemoprevention of complex mixtures of fruits and vegetables

People who eat sufficient amounts of fruits and vegetables as part of their
daily diet have a reduced risk of a number of chronic diseases, such as cancer,
cardiovascular disease and type II diabetes mellitus. There is ample scientific
evidence suggesting that these health benefits are the consequence of the
combined action of different phytochemicals present in fruits and vegetables.
However, the number of studies in humans is limited. More research is needed to
unravel the most optimal combination of phytochemicals in fruits and vegetables
that can protect against disease risk. Furthermore, the underlying mechanisms
by these phytochemicals exert their effect remain unclear. In addition, people
might respond differently to dietary changes due to their genetic make-up. It
was shown that subjects with specific genetic characteristics may benefit more
from certain combinations of phytochemicals than others. More combinations of
fruits and vegetables should be tested in humans with different genetic
backgrounds at the level of appropriate phenotypic markers of effect, including
whole genome gene expression changes in order to provide insight into the
underlying molecular mechanisms. Therefore, the aim of the present human
dietary intervention study is to evaluate the effect of various combinations of
vegetables and fruit containing different complex mixtures of phytochemicals in
healthy volunteers on the level of phenotypic markers of disease risk in
combination with whole genome gene expression analyses, and taking genetic
variability between subjects into account.

The main objective of the human dietary intervention study is to investigate
the beneficial health effects of food products containing various combinations
of an equivalent of 400 grams vegetables and fruits in healthy volunteers. This
will be evaluated in different subgroups with specific genetic characteristics
(max. 20 SNPs) on the level of different phenotypical markers, combined with
gene expression profiling.

This human dietary intervention study has a randomized controlled cross-over *
repeated measures design, including only healthy volunteers. After a two week
run-in period, subjects will be randomized into one of the 9 study groups. Each
group will follow two intervention periods of two weeks in which at each period
one of nine different food product containing different combinations of
vegetables and fruits will be consumed, separated by a one week washout period.
At baseline and after each intervention period, blood and urine is collected,
and a photograph is taken from the fundus of the right eye for analyses of
markers of oxidative stress, DNA damage, biomarkers of cardiovascular disease
risk and type II diabetes, gene expression analyses and determination of
maximum 20 genetic polymorphisms.

Nine different food products containing different combination of fruits and
vegetables will be evaluated in this human dietary intervention study. Seven of
these 9 different food products consist of a smoothie, containing 400 grams of
vegetables and fruits which will be consumed during the day: Smoothies 1-4
contain a specific selection of vegetables and fruit resulting in an
overrepresentation of a specific class of phytochemicals. Smoothies 5-7 will
consist of a combination of the four different classes overrepresented in
smoothies 1-4, with increasing biodiversity. Food product 8 and 9 will consist
of a pearl which is a crouton-like product, consisting of a core of oats and
riceflour, and will be either coated or non-coated with the most diverse
mixture of vegetables and fruits as used in smoothie number 7. Each subject
will be randomized into one of the nine study groups, which will start of a two
week run-in period followed by two intervention periods of two weeks separated
by a washout-period. At each intervention period, one of the nine different
food products will be consumed. At baseline, and after each intervention
period, blood and urine will be collected, as well as a photographic image of
the fundus of the right eye will be taken.

Healthy volunteers will have to sign an informed consent and must follow a
strict diet in consultation with a dietician. The food products contain
different combination of vegetables and fruits, at the level of
recommendations, which are freely available and are considered to be healthy.
Participants will have to come to the university 8 times. The first visit only
comprises signing of the informed consent and oral explanation of the study
design by the principle investigator. At baseline, and after the first and
second intervention period, subjects will come to the university after an
overnight fast and donate urine and blood. Also a photograph of the fundus of
the right eye is taken. In total, subjects will have to collect three times 24
hour urine, at 3 times 34 mL of blood divided over 4 vacutainers will be drawn,
and 3 images of the fundus of the right eye will be taken. The remaining visits
are for collection of the food products.The risk of collection of these samples
is considered to be minimal. However, the studyperiod of 7 weeks is relatively
and will have an impact on the daily life of the volunteers. This discomfort is
expected to be moderate. During the whole study period, subjects have to keep
track of their diet using a food diary.