Jacob C. Ong and Dr. Edwin D. Lephart, Zoology

Phytoestrogens are estrogen-like compounds derived from plants such as the soy plant and can mimic the function of the human estrogen hormone by binding to estrogen receptors. Estrogen plays a major role in the prevention of osteoperosis and cardiovascular disease in woman. Evidence also suggests that estrogen influences various parts of the memory process and that its impact on an organism is dependent on the developmental stage at which exposure occurs [1]. Women under the luteal cycle of menstruation, which is the period when estrogen is released into the bloodstream, performed better on tasks such as reading words out loud or naming colors [1]. A human Eastern diet, such as an Asian diet, contains a higher percentage of soybean and phytoestrogens than a Western diet. One area that still needs to be explored is the impact that dietary changes have on memory. The rat has historically been used in many maze studies in order to test memory. However, experimental rats are usually fed a phytoestrogen diet, which should impact their performance. This study will use controls to determine how much memory is increased by the addition of phytoestrogens to a diet. This study will also test the hypothesis that males on a phytoestrogen diet will perform better at maze tasks until the amount of phytoestrogens in their bodies exceeds an optimal level. The use of males simplifies the study because females also produce their own estrogen.

I have spent three months assisting Trent Lund on a portion of his dissertation project, which is similar to my proposed project. Due to some unforeseen circumstances, I will order the rats starting fall 2000. The tentative plan is to order female rats and put half of them on a phytoestrogen diet and the other half on a phytoestrogen-free diet. Then, they will be mated to male rats and the male progeny will continue to be fed the same diet as their mothers. When the rats are old enough they will receive less food in order to reach 90% of their normal weight. This will ensure that the rats are motivated to obtain the rewards in the maze. The rats will then be introduced to maze, which consists of a circular center with 8 arms branching off the center in a radial fashion, roughly similar to an eight-arm starfish. The rats will be trained to find Froot- Loops at the end of each arm, which are each 36 cm long. Once they have learned this task, they will then be tested. The initial eight-arm maze task will test their reference and working memory. They will be placed in the center of the maze and will be allowed to roam around the maze for three minutes or until they have entered each arm of the maze. An error is counted if the rat returns to an arm that it has already entered.

After this test, the diets will be changed. Half of the phytoestrogen group will be switched to the phytoestrogen-free diet, and half of the phytoestrogen-free group will be switched to the phytoestrogen diet. This will generate four groups that correspond to human populations. The first group will have eaten phytoestrogens their whole lives and will correspond to humans who have stayed on an Eastern diet their whole lives. The second group will have switched midway through their lives from a phytoestrogen to phytoestrogen-free diet, which will correspond with Easterners who switched to a Western diet. The third group will have switched from a phytoestrogen-free diet to a phytoestrogen diet. This group is potentially the most interesting because it represents people on a Western diet who switched to an Eastern diet. The final group, a control, consists of rats who stayed on a phytoestrogen-free diet their whole lives, corresponding to humans that stay on a Western diet (see Table 1). Once the four different groups have been generated, then their long-term memory can be tested with the four-arm task. In the eight-arm task, Froot-loops were placed in each arm, but in the four-arm task only half of the eight arms will contain the reward. The rats will need to relearn which of the arms contain the reward. Colorful posters will be placed on the walls surrounding the maze so that the rat can orient itself as soon as it enters the center of the maze. The rat can earn a total of four points on a trial if within four tries they enter each of the arms containing the reward. However, a point would be deducted from the maximum score of four if the rat entered an arm that did not contain a reward or if the rat re-entered an arm. The rats will then be sacrificed and the frontal cortex, which is involved in memory, will be tested for calbindin by Western blotting. Calbindin, i.e. calcium binding proteins, are thought to be involved in neuronal development as well as the development of sexually dimorphic neuronal structures [2]. If a phytoestrogen diet increases the memory of the rats, then a corresponding change in calbindin expression in the frontal cortex should be detected.

I learned from helping Trent Lund that this type of research is time consuming. At certain periods I would need to spend 3 or maybe 4 hours a day testing the animals. It would take 80 days from the day that I order the rats to the first day that I could start running the animals on the tests. Then, it could take another 57 days to complete the eight-arm maze and the four-arm maze tasks. Thus, this research projects needs approximately 4 ½ straight months. Planning is essential. Trent and I also found some preliminary results that are interesting. The males

performed better with the phytoestrogens. After they had been on the phytoestrogen past a certain amount of days they began to perform more poorly, and in the end performed worse than the males that had never been exposed to phytoestrogens. The purpose of my study is to determine the optimal level of phytoestrogen intake in order to increase memory. This information could be used in the prevention of Alzheimer’s disease.

References

• Sherwin, B.B., Estrogenic effects on memory in women. In Hormonal restructuring of the adult brain- basic and clinical perspectives, Luines, V.N. Harding, C. F., Eds., 743: 213, 1994.
• Lephart, E.D., Dimorphic expression of calbindin-D28K in the medial basal hypothalamus from perinatal male and female rats, Developmental Brain Research, 96: 281-284, 1996.
• Research supported by ORCA and BYU Multi-cultural Office, also much thanks to Dr. Edwin Lephart and Trent Lund for their training and guidance.