Toxicology of Phytoestrogens
My postdoctoral research at Penn State University at the J.O. Almquist Research Center focused on the physiological response of bull sperm to its immediate environment in the female. In these studies, the fluid from the bovine oviduct, the site of fertilization, was used to simulate gamete exposure conditions in vivo.
Sperm motility, capacitation/acrosome reaction (biochemical changes in preparation for fertilization) and the ability to fertilize bovine ova were studied following incubation in oviductal fluid collected during different stages of the estrous cycle of the cow (Grippo et al, 1995; Kavanaugh et al, 1992). These sperm properties have since been used in my lab at ASU to study the effect of toxicants, in particular phytoestrogens, on mammalian sperm physiology and fertilization success. Phytoestrogens are plant products which have been shown to be endocrine disruptive in many mammalian species, including sheep.
Ms. Yan Xie developed an HPLC assay to determine concentrations of three phytoestrogens: biochanin A, daidzein and genistein (Grippo et al., 1999). This method has been expanded and refined to include the phytoestrogen formononetin and the flavonoid quercetin (see below).
Devon Bryant tested the effect of genistein and estradiol on the viability and
acrosome reaction of ram sperm (Bryant et al., 2000). Using both bright-field and fluorescence in a dual staining
technique, we found no statistical difference between percent acrosome reacted
live sperm treated with genistein at 50, 100 or 200 mM
when compared to control. However,
increased acrosome reaction, particularly at 200 mM.
Similar results were obtained in bovine sperm:
Mr. Kevin Scarborough found no statistical difference in acrosome
reaction of bull sperm exposed to genistein, daidzein, zearalenol or estradiol,
compared to control (Scarborough, 2002).
oviduct and its fluid are of vital importance to successful fertilization.
In studies done by Mr. Yubin Luo (Grippo et al., 2000) an HPLC method was
used to determine whether daily or staged oviductal fluid differed in
amounts of monosaccharides, theorizing that such carbohydrates may play a role
in biochemical signaling to sperm within the oviduct.
We found no measurable amounts of monosaccharides Fuc, Gal, GlcN, Man,
Xyl in samples of bovine oviductal fluid collected daily from the whole oviduct,
nor from isthmic, nonluteal ODF.
order to influence gametes and fertilization directly, a toxicant might be
expected to be present at the site of fertilization, the oviduct.
To determine whether phytoestrogens ingested orally reach the oviduct,
Mr. Sam Snipes, an ASU honors student, has cannulated the oviducts of ewes and collected their
oviductal fluid and blood. After injecting biochanin A into the rumen, oviductal fluid
and blood samples were again collected, and all samples were analyzed by
HPLC for the presence and concentration of two phytoestrogens, the parent
compound, biochanin A, and its metabolite, genistein.
of Herbal Supplements
with Dr. Bill Gurley, UAMS College of Pharmacy
Herbal supplements are used by more than 45 million people in the US, but
are not under the regulatory jurisdiction of the Food and Drug Administration. They also exist in many forms (from plant material or crude
digests to extracted agents) that may contain compounds unrelated to those
expected by the consumer. Indeed,
the plants from which herbal supplements are produced contain many compounds
that have biological activities different from the “active” ingredients
promoted by the supplement manufacturer. Bioflavonoids
are ubiquitous to most plants, including those used in herbal supplements, and
may be found in the supplements themselves.
These compounds have endocrine disruptive activities that may be
important to the health of those consuming herbal supplements, if discovered in
physiologically active concentrations.
In work funded through the AR BRIN (http://brin.uams.edu), and undertaken
by Ms. Kayla Capps for her honors thesis, we used HPLC to separate and analyze herbal
and dietary supplements
to determine the concentrations of five bioflavonoids (formononetin, daidzein,
biochanin A, genistein, and quercetin)
(Grippo, Capps, Rougeau, Gurley 2006 in review).
In collaboration with Dr. Robyn Hannigan and her
students, we have analyzed all of these supplements (both traditional herbal
supplements and ephedra-containing dietary supplements) for their metals
concentrations (Grippo, Hamilton, Hannigan, Gurley 2006) .
Effects of Ephedrine and Nicotine
Collaboration with Drs. Bill Gurley & Stephanie Gardner, UAMS College of Pharmacy
This website is maintained by A. Grippo (firstname.lastname@example.org)