1. Project Description:
Introduction The chemical and nutritional composition
of dry bean varies among cultivars and due to
environmental conditions. Moraghan and Grafton
[1] reported large differences in the ability
of bean plants to accumulate zinc, phosphorus,
iron and calcium in seeds among segregating populations
from crosses between low and high accumulators.
Bean seeds also vary widely for seed coat color
and characteristics, and colored bean types are
hypothesized to contain phytochemicals that have
been linked to positive health benefits such as
the inhibition of cellular oxidation [2;3]. Preliminary
data collected during the preparation of this
application showed a surprising result, where
total phenolics [mg gallic acid equivalents/100
g dry wt.] were higher in red beans (666 mg) than
black (442); and predictably lowest in white,
149. Scientists have attributed the health benefits
of beans, e.g. anti-cancer effects, to their high
concentrations of folate and fiber, as well as
to lower the glycemic index [4]. However, only
limited work has been done to determine if bean
phytochemicals have antioxidant properties in
vivo, and how such characteristics, if they exist,
relate to the health benefits of beans. The goal
of this proposal is to determine if beans with
high in vivo antioxidant activity and low glycemic
effects can be identified and whether such cultivars
have unique potential for promoting human health.
Background/Preliminary Work Henry Thompson joined
the faculty of Colorado State University (CSU)
in January 2003 and established the CSU Cancer
Prevention Laboratory (CPL) in the Department
of Horticulture. His decision to do this was based
in part on the results of three dietary intervention
studies in women at high risk for breast cancer
that he had conducted between 1998 and 2002. Approximately
450 women participated in studies designed to
discover the effects of plant food rich diets
on biomarkers for cancer risk. In that work, women
in the experimental groups generally consumed
an average of 12.4 servings per day of vegetables
and fruit, and the effects of different diets
on markers for oxidative cellular damage were
determined. Diets were formulated that varied
in the botanical families from which these vegetables
and fruits were selected. While statistically
significant reductions in levels of oxidative
cellular markers were observed (typically 15-20%
reductions), Thompson’s research team was
surprised that greater effects were not detected
among individuals consuming 3.4 vs 12.4 servings/day.
To investigate this further, discussions commenced
with plant breeders and producers of food crops
at CSU and led to the formulation of the hypothesis
that the most health beneficial cultivars of commonly
consumed plant foods are currently not available
to the consumer, in part because plant breeders
and biomedical scientists have not had the opportunity
to work together and establish “human health-related
characteristics” for which plant breeders
can select. These discussions ultimately led to
Thompson’s lab moving to CSU. The collaborative
research project described in this application
between the laboratory of Dr. Mark Brick and Thompson’s
CPL research team is designed to extend efforts
to enhance the value of the food supply for health
promotion and disease prevention to dry beans,
and for potential use in future breeding of dry
beans that have value added health benefits.
Why dry beans? In one of Thompson’s clinical
studies, all women (N=267) were placed on the
same diet (referred to as a run-in diet) for 2
weeks; during that time urinary excretion of a
whole body index of lipid peroxidation [5-9],
8-isoprostane F-2a, decreased from baseline levels
by 33%; the run-in diet was actually low in vegetables
and fruit (3.0 serving per day); consequently,
the magnitude of the beneficial effect was surprising
and not readily explained. However, in the run-in
diet, dry bean-based meals were consumed for lunch
or dinner on average 4-5 times per week (ave.
daily intake =0.5 servings). This raises the possibility
of a “bean-related effect” and led
to discussions between Brick and Thompson.
Why preclinical studies and not clinical investigations?
We have the experience, the patient population
(women at high risk for breast cancer and breast
cancer survivors) and the infrastructure in which
to conduct both pre-clinical and clinical investigations.
The Request for Proposals (RFP) favors clinical
studies, yet we propose pre-clinical studies.
Why? Unfortunately, there is little information
available to give solid answers to two questions
that the CPL team posed to the Brick lab, namely;
1) What bean cultivars have the highest antioxidant
activity in vivo and, 2) Can bean cultivars with
high antioxidant activity be identified that also
have a relatively low glycemic effect. In the
absence of such data, it is our judgment that
it is not advisable to propose clinical studies
of the health benefits of beans. Such studies
are expensive and time consuming; therefore they
should only be done when “defining”
pre-clinical evidence is available. Consequently,
pre-clinical studies are proposed in this application
because they represent the critical gateway through
which the most meaningful clinical studies can
proceed. It is essential to first establish the
rationale for the selection of bean cultivars
with the greatest potential health benefits, and
then proceed to investigate whether health benefits
are observed in clinical studies. Failure to do
so elevates the risk that the most promising genotypes,
vis a vie human health, will be overlooked. If
scientists simply test any available bean cultivar
without thoughtful science-based selection of
the varieties with the greatest potential to impact
human health, it could poorly serve those who
wish to define and promote the health benefits
of dry beans.
Definition of research problem and hypotheses
to be tested
Ho1: Dry bean market classes differ
in antioxidant activity and even among those with
the highest antioxidant activity in vitro, there
will be differences in in vivo activity.
We will evaluate 10 of the 12 most diverse recognized
USDA market classes of beans in the US, as well
as three land races (pinto, great northern and
small red) and two important market classes, yellow
bean cultivated in Mexico, and Nuna (or pop bean)
from South America for in vivo antioxidant activity.
This assortment of cultivars will test the most
diverse market types that vary for seed coat color,
origin (Middle vs Andean) and compare undeveloped
land races with cultivars bred for yield and pest
resistance but not health benefits.
Ho2: Dry bean cultivars with high in
vivo antioxidant activity can be identified that
also have low glycation reaction potential in
either control or pre-diabetic animals; collectively,
this will result in a reduction in diabetes-associated
inflammation and oxidation. Bean cultivars
with the highest in vivo antioxidant activity
from each market class and place of origin will
be evaluated for glycemic activity via monitoring
of hemoglobin A1c (Hb- A1c). Levels of Hb- A1c
are used clinically as a time averaged method
to monitor how effectively diabetics are managing
their blood glucose. Inflammation will be assessed
by measuring circulating levels of C-reactive
protein (CRP).
Ho3: Dry bean cultivars with high in
vivo antioxidant activity and low glycemic activity
in vivo will both have cancer inhibitory activity
against experimentally induced breast cancer.
The two most promising bean cultivars based on
in vivo assays will be compared with the two least
promising cultivars for potential health benefit
based on feeding trials. The AIN-93G diet formulation
will also serve as the positive control
2. Research approach and methodology:
Seed Sources: Seed for all entries and accessions
will be produced at the Colorado Agriculture Experiment
Station Research Facilities during summer 2004.
Based on previous experience all lines or land
races can be produced at this site or purchased
commercially.
Dietary approach In phytochemical research,
it is common not to use whole foods, but rather
to prepare an extract from the botanical of interest
and study the activity of the extract. However,
this presumes that it is known what chemical fraction(s)
of the plant food is responsible for the hypothesized
effect and that the same fraction, when incorporated
into the diet will have the same health benefits
as when the whole food is consumed. While there
is value in this approach for certain applications,
we are interested in working with the whole dry
bean as a food for subsequent use in clinical
studies. Therefore, we will adapt the approach
reported in [10]. The dry bean cultivar of interest
is first cooked under standardized conditions,
then the beans will be immediately freeze dried
and then ground into a powder which is subsequently
substituted in a modification of AIN 93G based
on the starch, fiber, protein, and fat content
of the bean. (Further details are not presented
because of space limitations). Using this dietary
approach, rats grow at the same rate as control
animals fed AIN93-G and as reported in [10], pinto
beans were shown to protect against experimentally
induced colon cancer using this methodology.
Ho1 : Screening cultivars for potential antioxidant
activity As noted in a recent review article [11],
many laboratories around the world are using chemical
analyses to determine the antioxidant capacity
of botanicals, particularly vegetables, fruits,
and grains. Yet, there is an absence of knowledge
about whether these test tube assays accurately
reflect the potential for antioxidants in plants
to affect in vivo levels of oxidative damage to
cellular macromolecules, i.e. lipids, proteins,
DNA. The work proposed begins to address this
deficit of knowledge as it relates to dry beans.
The Brick laboratory has access to over 1000
bean germ lines that can be screened for potentially
health beneficial traits. In this circumstance,
the value of an ex-vivo screening tool is obvious.
We will screen bean varieties listed under Ho1
. We have available to us a battery of six assays
generally applied to the assessment of plant materials
for characterization of antioxidant activity:
total phenolics, ABTS, ORAC, TRAP, FOX, and carotenoids-tocopherols.
These assays will be applied to extracts of cooked,
dry bean powder (the same material that would
be incorporated into experimental diets) in order
to develop and validate an in vitro approach that
predicts in vivo antioxidant activity of dry beans.
The assay or combination of assays that have the
highest correlation with in vivo antioxidant activity
(described below) will be used to evaluate the
cultivars in this project and in future projects.
In vitro antioxidant activity assays: Total
phenolics Total phenolics in an extract are measured
spectrophotometrically at 765nm based upon a color
reaction of phenolic compounds with Folin-Ciocalteu,
a phosphomolybdio-phosphotungstic reagent. ABTS
Assay for Antioxidant Activity is based upon oxidation
of 2,2' azinobis(3-ethylbenzothiazoline-6-sulfonic
acid) diammonium salt) (ABTS) to the activated
ABTS+· radical using MnO2 based upon [12].
A water-soluble analog of vitamin E, Trolox, (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid) is used as a standard. Total Peroxyl Radical-trapping
Potential (TRAP) Assay is based upon the potential
of antioxidants in extracts to scavenge peroxyl
radicals generated by thermal decomposition of
2,2' diazobis (2-amidinopropane) dihydrochloride
(AAPH) [13]. Detection of the oxidation product
is based upon colorimetric absorption of the oxidation
product, dichlorofluorescein (DCFH to DCF) at
504 nm. Oxygen Radical Absorbance Capacity (ORAC)
Assay generates peroxyl radicals [considered the
most abundant free radical in nature by Prior
and Cao (2000)], from 2,2'-azobis(2-amidopropane)
dihydrochloride (AAPH). FOX3 (Lipid Peroxidation)
Assay. Lipid hydroperoxides are detected by their
oxidation of Fe (II), in the presence of xylenol
orange, to a Fe (III)-xylenol orange complex.
Free radical-mediated chain oxidation is initiated
in micelles of lipid (Intralipid ®) by irradiation
with ultraviolet light. Color change is measured
at 570 nm against H2O2 standards and the data
are expressed as the amount of extract that inhibits
lipid peroxidation by 50% (IC 50) [14]. Tocopherols
and selected carotenoids Alpha- and gamma-tocopherol,
along with selected carotenoids (a-carotene, ß-carotene,
lutein, lycopene, cryptoxanphane) are measured
by the HPLC method of Hess et al [15], with modification.
Briefly, 200 mL plasma is stabilized with BHT
and deproteinated with ethanol. The analytes are
extracted with hexane and the hexane removed under
reduced pressure. The extract is reconstituted
with mobile phase and separated by isocratic reverse
phase HPLC with photo diode array detection[16]
.
In vivo antioxidant activity The ability to
detect the effects of antioxidants in vivo is
enhanced if oxidation is elevated above basal
levels. The induction of diabetes in the rat by
a single injection of streptozotocin is accompanied
by oxidation of lipids, proteins, and DNA. We
propose to study whole body lipid peroxidation
measured as urinary excretion of 8-isoprostane
F2-alpha (8-EPG). We are currently using this
assay in our clinical studies and have found that
levels of 8-EPG are responsive to dietary phytochemical
antioxidants.
Design of Experiment Female Sprague Dawley rats
will be obtained from Taconic Farms, Germantown,
NY. at 21 days of age. At 24 days of age, rats
will given a single injection of streptozotocin
[STZ] (60 mg/ kg body weight, i.p.) following
an overnight fast. Elevated blood glucose and
increased cellular oxidation are detected within
48 hr. Rats will be housed in metabolic cages
equipped with tunnel feeders and urine collection
funnels. The rats will be provided free access
to diet and water throughout the study. Experimental
diets will initiated beginning 48 hours post STZ
injection. Urine will be collected daily for 14
days for analysis of 8-isoprostane F-2a. Food
consumption will also be quantified daily. Study
termination is 14 days post STZ injection. Statistical
power: When the sample size in a treatment group
is 12 rats, a 0.050 level two-sided t-test of
the specified contrast in a one-way analysis of
variance will have 80% power to detect a difference
of .33 SD (overall effect size = 0.73).
Assessment of 8-isoprostane F2-alpha The 8-EPG
enzyme immunoassay kit used in our lab is produced
and sold by Cayman Scientific. The polyclonal
antibody employed by the kit is very specific
for 8-EPG, and the kit shows minimal cross reactivity
with numerous COX dependant and independent prostanoids.
Prostaglandin F1a does cross react in this assay,
exhibiting ~12% of the activity of 8-EPG.
Ho2-3 Dry bean cultivars have been reported
to have variable glycemic effects [17]. Our goal
is to identify bean cultivars with high in vivo
antioxidant activity from the tests of Ho1 and
that have a low glycemic effect. This will be
accomplished in a 4 week feeding study in which
effects of different bean cultivars on the glycation
of hemoglobin (Hb A1c) will be determined sequentially
in untreated and then pre-diabetic rats. Effects
on inflammation and oxidation also will be determined.
The positive control diet for this experimental
design is of AIN-93G[18;19]. Twelve rats/treatment
group (statistical power= 80%) will be fed experimental
diets for two weeks. Blood will be obtained at
baseline and after 2 weeks on treatment via retro-orbital
sinus bleeding. Urine also will be collected.
At two weeks, rats with be injected i.p. with
30 mg/kg STZ, a dose that induces a pre-diabetic
state. Blood will again be collected 14 days post
STZ. Urine will be collected throughout. Blood
will be assessed for HbA1c (10 µl) and C-reactive
protein (20 µl) at all time points. Urine
will be analyzed for 8-EPG. We hypothesize that
it will be possible to identify bean cultivars
that inhibit in vivo lipid peroxidation while
decreasing glycemic activity in untreated and
pre-diabetic rats, and that high antioxidant activity
combined with low glycemic effects will also result
in better control of the inflammatory response
that accompanies the onset of diabetes (assessed
via C-reactive protein).
Evaluation of dry bean cultivars on the promotion
phase of experimentally induced mammary carcinogenesis
Emerging evidence indicates that elevated oxidation,
inflammation, and diabetes (type-2) are predisposing
factors that contribute to the risk for breast
cancer. Consequently, we hypothesize that bean
cultivars with antioxidant, anti-inflammatory,
and low glycemic activity will have cancer inhibitory
activity relative to the control diet, AIN 93-G
or bean cultivars that have limited health beneficial
profiles as defined here.
Thompson’s lab has over 25 years experience
in experimental models for breast cancer [20-23];
methods are describe in brief. Animals will be
injected with 50 mg methylnitrosourea/kg body
weight at 21 days of age as described in published
procedures [23]. We anticipate that the experiment
will be terminated within 8 weeks of carcinogen
treatment. The bean cultivars will be selected
based on the work described above. Feeding of
experimental diets will be initiated 7 days post
carcinogen. Animals will be weighed and palpated
for detection of mammary tumors 3 times per week.
At necropsy all tumors will be excised and processed
for histopathological evaluation. The effects
of bean cultivars on the incidence, multiplicity
and latency of mammary cancer will be determined.
The primary hypothesis is that dry bean cultivars
with a favorable health profile will provide at
least 30% protection against neoplasia in comparison
to the control diet (AIN 93G). Since we expect
nearly 100% incidence of cancer in the control
group, we chose the number of animals per group
(N=30) that will give more than 80% power for
a test of trend where the difference between the
highest and lowest incidence levels is 30%.
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