Jason Hatch and Dr. R. Ward Rhees, Zoology

In recent years, phytoestrogens (nonsteroidal, estrogen-like substances found in plant sources such as beans, rice, soy and wheat) have received extensive investigative research. However, current information about the amount of phytoestrogens in human diets is limited (4). Scientific research has alluded to possible benefits of diets high in phytoestrogens based on epidemiological studies done on Asian and Eastern European cultures (2). These studies indicate that consumption of a phytoestrogen-rich diet, as seen in traditional Asiatic societies, is possibly associated with a lower risk of the so-called “Western” diseases such as breast cancer, prostate cancer, cardiovascular disease and osteoporosis (3). Epidemiological, animal and in vitro data especially encourage further assessment of the role of phytoestrogens in cancer prevention due to the lower incidence of hormone dependent tumors in Asia and Eastern Europe compared with western countries (1, 5). It is difficult to extract a single component of a total lifestyle from an epidemiological study, where several other significant lifestyle factors are operative, and expect to see a distinct correlation with disease without isolating a specific component, such as diet, and analyzing the experimental data. It may be that phytoestrogen ingestion needs to be life-long and combined with other dietary constituents and behaviors for the protective effects to be significantly manifest. Hence, the need to isolate a high phytoestrogen diet relating to cancer prevention warrants examination (5).

The aim of this investigation was to study the influence of phytoestrogen diets on cancerous growth in reproductive tissue of Noble (Nb) rats. The Nb rat has a genetic predisposition for spontaneous tumor growth in reproductive tissue (6). Thus, preventative benefits of a phytoestrogen diet in suppressing spontaneous tumor development in Nb rats was analyzed. To evaluate the impact of a specially maintained diet, 30 Nb rats (10 males, 20 females) were obtained and allowed to acclimate. Half of the males and half of the females (Group 1) were fed laboratory rat chow free of any phytoestrogens (PhytoFree). Whereas the remaining animals (Group 2) were given a diet of laboratory rat chow with 600 μg phytoestrogen per gram rat chow (Phyto600). The animals were then mated. Litters consisting of more than 10 pups were culled to five males and five females when possible. Pups were raised with their mothers for 30 days at which point they were separated into isolated cages. Pups from Group 1 were kept on PhytoFree diets and Group 2 on Phyto600 diets. Pup weight, food and water intake, and anal-genital distances (AGD) were measured daily. The data did not present any large discrepancies between the groups, except that the PhytoFree diet rats were on average heavier and consumed less water than their phytoestrogen diet counterparts.

As the experiment proceeded, unexpected problems arose while trying to raise the animals. For one, the females being raised on Phyto600 diets were difficult to get pregnant. Duration of female to male interaction was twice as long for these animals as compared to non-phytoestrogen diet females. Although no testing was conducted to analyze why this was the case, it is speculated that diets high in phytoestrogens possibly led to difficulties with the females’ ability to cycle properly. A second unexpected quandary resulted with none of the females in both groups bearing sufficient offspring in order to collect a large number of animals for sacrificing. On average the litters were 7 to 9 pups, whereas in other species of rat average litter size ranges from 10 to 13 pups. Again, this question was not looked into quantitatively; however, qualitative speculation leads one to suppose that Nb rats are not as hearty as other species, minimizing their ability to carry large litters. This may be due to frequent inbreeding, necessary for obtaining animals that possess certain genetic tendencies.

Pups were raised exclusively on diets indicated above, depending on the mother’s diet before pup birth. Pups at 100 days old were randomly selected from each group until 32 total animals were obtained. These were then sacrificed, perfused, and analyzed for tissue plasticity and other irregularities. Autopsies looked at all major organs (liver, pancreas, kidney, testicle, prostate, ovary, uterus, etc.) that are consistently under hormonal influence. Autopsies revealed that possible cancerous growth is evident, which was unexpected at 100 days (6). Two animals from Group 2 were found to have irregular tissue clusters, and no animals sacrificed from Group 1 had any sign of abnormal tissue. Anomalous tissue in the two animals from Group 2 was found in the epididymis of a male, and the bladder of a female. It is of interest to know that the male’s epididymis not only contained a tumorous growth, but also had irregular testicle sizes; one testicle measuring almost two times as large as the other. At this point, it can be deduced that the tissues contained definite tumor-like growths. Pending pathohistological analysis will substantiate these claims. The fact that cyst-like, fibrous tissue appeared in only animals on nonphytoestrogen diets warrants further examination. Therefore, this study will continue with periodic sacrificing on 150 and 200-day-old animals in order to determine whether aging has an impact on the extent, frequency, and amount of cancerous tumors present in the Nb rat. Premature conclusions speculate that indeed the effects of diets high in phytoestrogen compounds can markedly repress the instances of hormone-dependent cancers. Thus, it anticipated that data generated from this experiment would be concluded by mid-December.

Study of phytoestrogens and their relationship with cancer is providing valuable information that will help qualify suggestions in current scientific literature. At this point, the prospects are promising. By the end of this study, I anticipate having a model for analyzing how phytoestrogens influence spontaneous tumor development based on currently obtained data. I am confident that more cancerous growths will be detected in older Nb rats. It would also further indicate that high phytoestrogen diets are a factor in cancer prevention.

Additional Information: In order to maximize resources, this study was conducted with a three-fold purpose. The first was to study the effects of the phytoestrogen diets on cancer. Secondly, however, it was determined that no research had been done on Nb species with regard to its sexually dimorphic nucleus (SDN), a region of the rat brain which heavily influences sexual behavior in rats. When rat autopsies are taking place, brains are also removed for slicing and staining to look at the Nb rat’s SDN. Third, this study will use collected data and rat tissue for possible phytoestrogen effects on calbindin binding. It is presently anticipated that these studies will be published before the summer of 2002.

References

1. Murkies A, Wilcox G, et al. 1997 Clinical Review: Phytoestrogens. J Clin Endocrinol Metab. 83:297-303.
2. Adlercreutz H, 1990 Western diet and western diseases: hormonal and biochemical mechanisms & associations. Scand J Clin Lab Invest. 210:3-23.
3. Lephart E, et al. 1999 Brain regulatory behaviors and testosterone levels in adult rats on phytoestrogen diets. Soc Exper Bio and Med.221:131 135.
4. Lephart E, Rhees RW, et al. 2000 Neuroendocrine regulation of sexu ally dimorphic brain structure. Soc Exper Bio and Med. 1-36.
5. Adlercreutz H, Goldin BR, Gorbach SL. 1995 Soybean phytoestrogen intake and cancer risk. J Nutr. 125:757S- 770S.
6. Noble, Robert. 1982 Prostate Carcinoma of the Noble Rat in Relation to Hormones. Inter Review of Exper Pathology. 3:113-158.