The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.

 

Johns Hopkins School of Public Health

Abstract for TestSmart -- Endocrine Disruptors

Xenoestrogens: Pharmacokinetics and Strain Differences in Animal Bioassays

Robert M. Bigsby
Indiana University School of Medicine, 1001 W. Walnut St., Indianapolis, IN, 46202, USA

Certain organochlorine pesticide residues and other environmental contaminants exhibit estrogen-like behavior and thereby may pose a threat to human health. Epidemiologic data on exposure levels and putative health problems are inconclusive. Correlation between breast cancer incidence and high blood levels of organochlorines has been observed but others have found no such association (1-7). The debate is likely to continue because there are no "unexposed" cohorts to compare, and exposure information is difficult to obtain. For this reason we must rely on experimental animals studies to determine rational safety levels for humans.

We used an ovariectomized mouse model to study the relationships between blood levels of two orgcanochlorine pesticide residues and estrogenic responses (8). DDT is infamous as an endocrine disruptor in birds. The o,p'-isomer of DDT, which comprises about 15 % of the technical mixture, is estrogenic. The β-isomer of hexachlorcyclohexane (HCH), another pesticide compound, also exhibits estrogenic activity. Although both of these compounds were banned for agricultural use in the US in the 1970's, they are still in use in third world nations, particularly in Mexico, India and China and they remain ubiquitous as environmental contaminants throughout the world. In general, human blood serum levels of these two compounds range from non-detectable to 5 ng/ml but may be much higher, 30 - 240 ng/ml, depending upon locale or whether occupational exposure has occurred. Because of their high fat solubility, organochlorine compounds concentrate in body fat and blood levels represent the amount that has leached out of that body compartment. In order to simulate this equilibrium state in an animal model, small doses of o,p'-DDT or β-HCH were delivered continuously from Silastic capsules implanted subcutaneously in ovariectomized mice. Doses were adjusted over ranges of 120-fold or 60-fold, for o,p'-DDT or β-HCH, respectively. Blood and fat levels of compound were analyzed by gas chromatography/electron capture techniques. Vaginal epithelial thickness and uterine epithelial cell height were used as estrogenic endpoints.

Blood and fat levels of compound were correlated linearly, with fat concentrations being approximately 100-fold higher than that of blood. This ratio is consistent with that seen in human blood and fat samples. Estrogenic effects were observed at blood levels as low as 18.4 ± 4.87 ng/ml (mean ± SEM) or 40.9 ± 5.29 ng/ml for o,p'-DDT or β-HCH, respectively (Fig. 1). These observations contradict the notion that very large doses of these compounds, producing blood levels well above those seen in the general population, are required to produce estrogenic effects.

In addition, local tissue concentrations of organochlorines may be influenced by their release from fat reservoirs. We examined whether o,p'-DDT and β-HCH would be released from fat during periods of dietary restriction and thereby produce estrogenic effects (9). Ovariectomized mice were loaded with compound by 3 daily injections. Eight weeks later, animals were starved for two days and their uteri were examined for 3H-thymidine incorporation (Fig. 2). In the β-HCH-loaded animals, fasting increased thymidine incorporation; fasting had no effect in the animals that had been loaded with o,p'-DDT. These observations indicate that β-HCH, but not o,p'-DDT, can be released from fat deposits in sufficient quantities to produce systemic estrogenic effects. It is suggested that since o,p'-DDT is more lipophilic it remains in the fat during the fasting period, rather than being released into the blood. Since the epithelium of the mammary gland is embedded in fat tissue, these observations are especially relevant to the relationship between organochlorine compounds and breast cancer.

In addition to the above considerations, it is important to take into account differences in sensitivity due to animal strain and specific endpoints when assessing biological activity of putative xenoestrogens. We have examined the effects of bisphenol A (BPA) in the Sprague-Dawley (S-D) and Fisher 344 (F344) rat strains (10-12). BPA is a compound used in the manufacture of plastics; it can leach from plastic food containers into the food supply. In our studies, animals received either a single intraperitoneal injection of BPA or were treated continuously for 3 days via a subcutaneous Silastic implant. Serum prolactin levels were increased 7-fold (P < 0.05) by the 3-day BPA treatment in F344 rats but not in S-D rats. Similarly, uterine wet weight and epithelial cell height were increased (P < 0.001) in F344 rats but not in S-D rats. A single dose of BPA induced an increased rate of DNA synthesis in vaginal epithelium of F344 rats with an ED50 of 38 mg/kg; BPA had no effect on the epithelial proliferation in the vaginas of S-D rats, even at 150 mg/kg (Fig. 3). On the other hand, BPA induced expression of estrogen-responsive genes (c-fos, c-jun, VEGF) in vagina and uterus with equal potencies (ED50 = 38 mg/kg) in both strains of rat. Thus, determination of estrogenic activity of xenobiotics such as BPA may depend upon the endpoint and animal strain used in the bioassay.

References

  1. Wolff, MS, Toniolo, PG, Lee, EW, Rivera, M, Dubin, N. Blood levels of organochlorine residues and risk of breast cancer. J Natl Cancer Inst 85:648-652 (1993).
  2. Krieger, N, Wolff, MS, Hiatt, RA, Rivera, M, Vogelman, J, Orentreich, N. Breast cancer and serum organochlorines: a prospective study among white, black, and Asian women. J Natl Cancer Inst 86:589-599 (1994).
  3. MacMahon, B. Editorial: Pesticide residues and breast cancer. J Natl Cancer Inst 86:572-573 (1994).
  4. Hunter, DJ, Hankinson, SE, Laden, F, Colditz, GA, Manson, JE, Willett, WC, Speizer, FE, Wolff, MS. Plasma organochlorine levels and the risk of breast cancer. N Engl J Med 337:1253-1258 (1997).
  5. Høyer, AP, Grandhean, P, Jørgensen, T, Brock, JW, Hartvig, HB. Organochlorine exposure and risk of breast cancer. Lancet 352:1816-1820 (1998).
  6. Zheng, T., Holford, T. R., Mayne, S. T., Owens, P. H., Ward, B., Carter, D., Dubrow, R., Zahm, S. H., Boyle, P., and Tessari, J. Beta-benzene hexachloride in breast adipose tissue and risk of breast carcinoma. Cancer 85:2212-2218 (1999).
  7. Dorgan, J. F., Brock, J. W., Rothman, N., Needham, L. L., Miller, R., Stephenson, H. E. J., Schussler, N., and Taylor, P. R. Serum organochlorine pesticides and PCBs and breast cancer risk: results from a prospective analysis (USA). Cancer Causes Control 10:1-11 (1999).
  8. Ulrich, E. M., Caperell-Grant, A., Hites, R. A., and Bigsby, R. M. Environmentally relevant xenoestrogen tissue concentrations correlated to biological response in mice. Environ Health Perspect submitted2000.
  9. Bigsby, R. M., Caperell-Grant, A., and Madhukar, B. V. Xenobiotics released from fat during fasting produce estrogenic effects in ovariectomized mice. Cancer Res, 57:865-869, 1997.
  10. Steinmetz, R., Brown, N. G., Allen, D. L., Bigsby, R. M., and Ben-Jonathan, N. The Environmental Estrogen Bisphenol A Stimulates Prolactin Release in Vitro and in Vivo. Endocrinology 138:1780-1786, 1997.
  11. Steinmetz, R., Mitchner, N. A., Grant, A., Allen, D. L., Bigsby, R. M., and Ben-Jonathan, N. The xenoestrogen bisphenol A induces growth, differentiation, and c-fos gene expression in the female reproductive tract. Endocrinology 139:2741-2747, 1998.
  12. Long, X., Steinmetz, R., Ben-Jonathan, N., Caperell-Grant, A., Young, P. C., Nephew, K. P., and Bigsby, R. M. Strain differences in vaginal responses to the xenoestrogen bisphenol A [In Process Citation]. Environ Health Perspect 108:243-247, 2000.