Exposure to exogenous estrogens such as synthetic 17α-ethinyl estradiol (EE) occurs via multiple sources (i.e. hormonal contraceptives, environmental contamination, hormone replacement therapy). The natural estrogen, 17-estradiol (E2), is a well-studied immunomodulatory hormone at both environmental and pharmacologic levels. Conversely, little data exist regarding the immune effects of EE at either environmentally-relevant exposure levels or at pharmacological levels. Further, EE is delivered to patients in a clinical setting via different routes of exposure (e.g. subcutaneous or oral). Many key questions in relation to potential immunological effects of EE are unanswered. Important variables in estrogen-modulation of the immune system include: (i) dose, (ii) age, (iii) gender, and (iv) route of exposure. Thus, pertinent questions emerge. Does exposure to EE at low concentrations for a subacute duration affect the immune or reproductive systems? Are the effects similar in both hormones and between sexes? Are these effects similar in juvenile and aged mice? How do the effects compare across two common routes of exposure (subcutaneous versus oral)? To address these questions, three separate studies were performed. In the first study, we investigated whether very low, but environmentally relevant, doses of EE, E2 (10 ng/kg body weight), or vehicle orally administered every other day for 21 days to young (6 week-old) and aged (>15 month-old) C57BL/6 mice had immunomodulatory effects. As expected, significant gender and age-related differences were noted with regard to thymus weight, thymocyte recovery, spleen weight, and splenocyte recovery. However, low dose treatment of either E2 or EE had no marked effects on the thymus or spleen organ to body weight ratios, cell numbers, or lymphocyte subsets. Low dose oral estrogens did not alter the ability of activated splenocytes to induce interferon-γ or nitric oxide. No effects on male reproductive organ to BW ratios of young or aged mice were found. Similarly, with the exception of E2-stimulating effects on the female reproductive tract of young mice, there were no pronounced effects in females.
In separate studies, intact juvenile female and male C57BL/6 mice were given daily subcutaneous (second study) or oral (third study) doses of either EE or E2 (0.04, 0.4, or 4.0 μg per 25 g BW) for 21 days. In the subcutaneous exposure study, both EE and E2 morphologically altered uterine and seminal vesicle weights. However, EE had a more pronounced effect compared to E2, especially in males, even at the lowest dose administered. Additionally, like E2, EE induced thymic atrophy in both sexes. In female mice, thymic atrophy and thymic cellularity were significantly decreased by subcutaneous EE and E2 at doses of 0.4 and 4.0 μg/25 g body weights. EE elicited significantly more pronounced thymic atrophy-inducing effects compared to E2 at the 4.0 μg/25g dose. In males, thymocyte cellularity was decreased by both subcutaneous EE and E2 only at the highest dose tested (4.0 μg/25 g body weight), whereas only 4.0 EE significantly decreased thymus to body weight ratios. Neither splenic weights, splenic cellularity, nor splenic cell phenotype were affected by either estrogenic compound regardless of route of exposure. Oral exposure of EE or E2 did not induce marked immunological effects. Collectively, these data demonstrate that select thymic and reproductive endpoints are significantly altered following a 21-day subcutaneous exposure to either EE or E2 and that the thymus is a more sensitive target than the spleen with regard to subacute exposure to EE. In addition, EE at a comparable dose was more potent than E2 at exerting thymic and reproductive organ morphological alterations. Furthermore, route of administration is critical, as subcutaneous exposure induced far more dramatic thymic and reproductive morphological alterations than did oral administration. Future studies need to address the precise mechanism through which EE induces thymic atrophy and diminished thymus cellularity. Are these effects mediated directly through the thymus, perhaps through estrogen-induced increased thymocyte apoptosis or alterations to thymic epithelial cells? Or could EE be mediating alterations via bone marrow stem cells targeted for distribution to the thymus? Our novel findings regarding EE-induced effects on the thymus are of health significance and set the stage for future work.