Modulation of mammary gland development in prepubertal male rats exposed to genistein and methoxychlor.

The estrogenic isoflavone genistein is a common dietary component that has been shown to affect reproductive development in experimental animals at high doses. The objective of the present study was to examine interactions of genistein and the hormonally active pesticide methoxychlor on mammary gland development in juvenile rats. Timed-pregnant Sprague-Dawley rats were fed a soy- and alfalfa-free diet containing different combinations of genistein (300 and 800 ppm) and methoxychlor (800 ppm). Rats were fed these diets starting on gestation day (GD)1 and continuing through pregnancy and lactation until postnatal day (PND) 22, when the pups were killed. Inguinal mammary glands from both female and male pups were processed as whole-mount preparations for morphometric analysis. The total glandular area and the numbers of branch points, lateral buds, and terminal end buds in the male rats were found to be significantly greater in the groups exposed to methoxychlor than those exposed to genistein only. These effects were not observed in the female rats. In the male rats, methoxychlor had the most prominent effect on elongating the glandular ducts, while genistein enhanced the ductile branching. The 2 compounds in combination promoted the development of alveolar-lobular structure, an effect not observed with either compound alone. Immunostaining for proliferating cell nuclear antigen revealed a high percentage of immunopositive cells in the mammary epithelia of the males exposed to methoxychlor and genistein (800 ppm) compared to the controls. While no significant changes in serum levels of mammotrophic hormones were detected, increased immunostaining for insulin-like growth factor-1 receptor, estrogen receptor alpha, and progesterone receptor in the genistein + methoxychlor group suggested that local factors involved in regulating mammary growth may have played a role in propagating the endocrine effects of these two compounds. These results indicated that the mammary glands of juvenile male rather than juvenile female rats may be more sensitive to certain endocrine-active compounds and that high levels of phytoestrogens have the potential to alter the toxicological behaviors of other hormone mimics.

Combined effects of dietary phytoestrogen and synthetic endocrine-active compound on reproductive development in Sprague-Dawley rats: genistein and methoxychlor.

Humans and wildlife are frequently exposed to mixtures of endocrine active-compounds (EAC). The objective of the present study was to investigate the potential of the phytoestrogen genistein to influence the reproductive developmental toxicity of the endocrine-active pesticide methoxychlor. Three levels of genistein (0, 300, or 800 ppm) and two levels of methoxychlor (0 or 800 ppm) were used in this study. Sprague-Dawley rats were exposed to the two compounds, either alone or in combinations, through dietary administration to dams during pregnancy and lactation and to the offspring directly after weaning. Both compounds, methoxychlor in particular, were associated with reduced body growth at 800 ppm, but pregnancy outcome was not affected by either treatment. An acceleration of vaginal opening (VO) in the exposed female offspring was the only observed effect of genistein at 300 ppm. Exposure to 800 ppm genistein or 800 ppm methoxychlor caused accelerated VO and also altered estrous cyclicity toward persistent estrus in the female offspring. The estrogenic responses to genistein and methoxychlor administered together were apparently accumulative of the effects associated with each compound alone. Methoxychlor, but not genistein, delayed preputial separation (PPS) in the male rats. When administered with methoxychlor, genistein at 800 ppm enhanced the effect of methoxychlor on delaying PPS. Genistein and methoxychlor treatment did not change gender-specific motor activity patterns in either sex. To explore possible mechanisms for interaction between the two compounds on development, we performed estrogen receptor (ER)- and androgen receptor (AR)-based in vitro transcriptional activation assays using genistein and the primary methoxychlor metabolite 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE). While the in vitro assays supported the estrogenic effects of genistein and methoxychlor and the antiandrogenic effects of methoxychlor, the reactivity of these compounds with ERs alpha and beta could not predict the greater in vivo estrogenic potency of methoxychlor over genistein; nor could the potentiation of the methoxychlor effect on PPS by genistein be predicted based on in vitro HPTE and genistein reactions with the AR. Data from this study indicate that phytoestrogens are capable of altering the toxicological behaviors of other EACs, and the interactions of these compounds may involve complexities that are difficult to predict based on their in vitro steroid receptor reactivities.

Gene expression profiles in mammary gland of male rats treated with genistein and methoxychlor.

Humans and wildlife are frequently exposed to mixtures of natural and synthetic endocrine-active compounds. To understand the impact of dietary phytoestrogen on the susceptibility to synthetic chemicals in the environment, we studied the effects of a binary mixture consisting of the isoflavone genistein and the pesticide methoxychlor on the development of the mammary gland. Sprague-Dawley rats were exposed to genistein at 800ppm, methoxychlor at 800ppm, or their combination through dietary administration to dams during pregnancy and lactation and to the offspring directly after weaning. At post-natal day (PND) 90, offspring rats were killed and their inguinal mammary glands collected for gene expression analysis utilizing the Clontech Atlas Rat 1.2 cDNA array, which contains probes for 1176 genes. Treatment with both genistein and methoxychlor altered gene expression profiles of the mammary glands in male rats, and the effects were more prominent in the combination treatment than the single-compound groups. Specific gene changes suggested that treatments affected the stromal and epithelial compartments of the mammary, involving genes controlling growth factor signaling, apoptosis, and tissue remodeling. This study demonstrates that dietary phytoestrogens in combination with a synthetic endocrine-active chemical can cause unique effects in endocrine-responsive tissues and highlights the importance of studying the effects of chemical combinations on the multiple biological processes underlying toxicological responses.