Research

Reproductive 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).

Mr. 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, estradiol-17b 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).  

The 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.

In 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.   Our initial results have shown that genistein is present in relatively high concentrations in oviductal fluid within 24 hours of dosing. 

Analysis of Herbal Supplements

Collaboration 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) .

Cardiovascular Effects of Ephedrine and Nicotine

Collaboration with Drs. Bill Gurley & Stephanie Gardner, UAMS College of Pharmacy, & Drs. Richard Grippo and Stan Trauth, ASU

            Funding through the ASU-Arkansas Biotechnology Institute is allowing us to explore the biological effects of tobacco, specifically nicotine, in combination with ephedrine, the active agent of the plant ephedra, which was contained in popular dietary supplements.  Nicotine has potent cardiovascular effects such as increased heart rate and blood pressure.  Like nicotine, ephedrine stimulates the cardiovascular system through the sympathetic nervous system.  These two agents are likely to be taken together by smokers, users of smokeless tobacco or nicotine patch users who are also trying to lose weight, and the drugs may be additive in their effects on the cardiovascular system.  To study any interactive effects of nicotine and ephedrine, an animal model has been established at ASU that  allows manipulation of dosages and routes of administration of both drugs while monitoring heart rate, blood pressure and EKG patterns.  To substantiate drug concentrations in the animals, analytical procedures will be implemented at ASU to measure blood nicotine, ephedrine and metabolites.  At UAMS, simultaneous studies in human subjects focus on the cardiovascular effects of nicotine and ephedra taken alone and together, as well as with other additives to common dietary supplements.  In these studies, the same cardiovascular parameters will be monitored, along with blood chemistry and drug/metabolite analyses.  It is expected that this information will be of interest to government health-related industries, to better understand some of the harmful effects of ephedra-containing dietary supplements which have been reported.  These supplements have recently been publicized as having dangerous side effects, in particular with respect to toxic effects to the heart.  Work completed by Mr. Chris Brown has shown that ephedrine, caffeine or nicotine don't alone significantly effect the heart; however, administration of a combination of these agents seemed to increase the number of non-cardiac cells within the heart, attributed to cardiac inflammation.

References:  See CV

This website is maintained by A. Grippo (agrippo@astate.edu)

06/23/09