National Toxicology Program

SUMMARY

Many edible plants are known to produce nonsteroidal compounds that can either mimic or antagonize the effects of endogenous steroid hormones. These compounds that are produced by plants themselves are called plant estrogens or phytoestrogens. Isoflavone, a phytoestrogen, such as genistein is found in high levels in soybean products1. It is unequivocally established that these compounds can bind to the estrogen receptor and can produce estrogen like effects in animals and cultured cells.

Methoxychlor is one of five endocrine disruptor compounds nominated by the Genistein is one of five endocrine disrupter compounds nominated by the National Toxicology Program (NTP) for reproductive assessment studies, neurotoxicological and immunotoxicological studies, and cancer bioassay studies. The title of this project is "Effects of Endocrine Disrupting Chemicals on Fertility and Reproductive Tract Cancers". These studies were carried out as an interagency agreement between the National Environmental Health Science (NIEHS) and the National Center for Toxicology Research (NCTR) with the immunotoxicology component being the only portion conducted outside of the NCTR facility in Jefferson, Arkansas. Thus, the purpose of these reported studies was to determine the potential effects of genistein on the immune system. The genistein/immunotoxicology code identification from NCTR is E2122.14

The exposure times and the doses, 0, 25, 250 and 1250 PPM, were determined by NCTR. Genistein was procured and administered in feed by personnel at NCTR in Jefferson, Arkansas. The control rats received casein NIH-31C as the feed. The studies were conducted in male and female Sprague Dawley rats. The F0 generation female rats received the test article for 65 days beginning 7 days into gestation. The F1 generation male and female rats received the test article beginning in utero for 14 days and continuing postpartum until day 77. On the day of sacrifice, whole spleens or bone marrow cells were placed in tubes with the appropriate medium, packed in wet ice and shipped to the Medical College of Virginia Campus/Virginia Commonwealth University (MCV/VCU), Richmond, Virginia, for assay evaluation on the following day. The assay days of the study were conducted between 13 January 1998 and 7 April 1998. Data included in the text refer to data generated in the assays conducted at MCV, unless otherwise specified. Included in Data Appendix B are data from the pathology studies conducted at NCTR with 6 doses ranging from 5 to 1250 PPM.

Exposure to genistein resulted in a statistically significant decrease in terminal body weight both in the F1 generation male and female rats as seen in the NCTR pathology study (n=15). A decrease of 9% (male) and 12% (female) as compared to the vehicle controls was observed at the highest dose of 1250 PPM. Although the decrease trend was present, no significant alteration was observed in the F0 generation females nor the F1 generation males in data from the body weight of rats assigned to immunotoxicology study (n=10). A statistically significant decrease was observed in terminal body weight in the F1 generation females of 11% at 1250 PPM.

Exposure to genistein resulted in a statistically significant increase in the relative spleen weight (% body weight) in the NCTR pathology study and MCV studies with the F1 generation males. The increase at the high dose, 1250 PPM, was 14% (NCTR) and 17% (MCV) as compared to the vehicle control. A statistically significant increase at all doses was observed in the F1 generation females in the MCV study of up to 24%. In the NCTR study a significant decrease in absolute thymus weight was observed in the F1 generation male rats at the 100, 625 and 1250 PPM doses.

The NCTR pathology study provided hematology and clinical chemistry data included in Data Appendix C. Hematology data that are germane to the immunology study include slight decreases in the white blood cell count, platelet count, hemoglobin and hematocrit for both the F1 generation male and female rats.

The F1 generation male and female rats were evaluated for genistein exposure effects on bone marrow cell number, colony-forming units and DNA synthesis. A statistically significant dose-dependent decrease of up to 33% in CFU/GM x 10⁵ cells was seen in the F1 generation male rats at 250 and 1250 PPM. A statistically significant decrease of 41% in cells/femur was observed in the F1 generation female rats at the high dose.

The baseline toxicology studies are summarized in Table ES-1.

For the F1 generation male rats exposed to genistein, a statistically significant increase of 31 % was observed in splenic B cells at the high dose of 1250 PPM. For both the F1 generation male and female rats, a statistically significant increase of up to 48% was observed in the splenic T cell and T cell subsets in the 250 and 1250 PPM doses. In the F0 generation female rats, no alterations were seen in the splenic surface markers.

In the spleen antibody-forming cell (AFC) response to T-dependent antigen, sheep erythrocytes, the only significant alteration was an increase observed in the F1 generation female rat response in IgM AFC/10⁶ spleen cells (122%) and IgM AFC/spleen x 103 (110%) at the high dose.

In spleen cells from F1 generation male and female rats, stimulation with the anti-CD3 receptor-mediated proliferation produced a statistically significant dose-dependent increase in proliferation up to 68% as compared to the vehicle control. In spleen cells from the F0 generation female rats, a basal proliferative stimulation was seen of 58% and 77% at 250 and 1250 PPM doses, respectively.

In the F0 generation female rats, a statistically significant increase in natural killer cell activity was observed with all effector-to-target ratios at the 250 and 1250 PPM doses. The observed increases range from 29% to 113%. No biologically relevant alterations were seen in the F1 generation male and female rats.

In conclusion, the results from this immunotoxicological evaluation demonstrate that, under the experimental conditions used, exposure to genistein did not impact on the immunocompetence of the F0 generation female rats with exception of an increased natural killer cell activity. However, for the F1 generation male and female rats, exposure to genistein for 77 days resulted in increases of both T cell number and T cell function.

Table ES-2 summarizes the immunology studies.