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  1. S. D. Blaschko,
  2. E. Willingham*,
  3. L. S. Baskin*
  1. University of California Irvine School of Medicine
  2. *University of California, San Francisco, San Francisco, CA


Purpose Because caffeine and endocrine-disrupting compounds are present in the human diet, the goal of this study was to assess the embryonic effects of low doses of caffeine and 17-beta estradiol (E2) via oral exposure in the mother. As endpoints of effects, we examined whether or not embryonic exposure to caffeine and E2 produced hypospadias, an endocrine-linked abnormality of the male genitalia, and we also assessed fetal mass. Exposure to androgens during development is thought to result in larger male offspring. Caffeine exposure is known to result in smaller offspring, and it served as a real-time control for mass effects with E2.

Materials and Methods Timed-pregnant CD1 mice were treated with caffeine in a water vehicle or E2 in a sesame oil vehicle via oral gavage. Mice received caffeine in doses of 5 mg/kg, 10 mg/kg, or 15 mg/kg or vehicle only or E2 in doses of 10 μg/kg, 100 μg/kg, or 200 μg/kg, or vehicle only on embryonic days 12 through 17. On embryonic day 19 the embryos were collected and weighed. Genital tubercles of the treated embryos were microdissected and sectioned for histological analysis. Treated and control embryonic urethral casts were also examined for developmental differences. Liquid plastic in the ratio of 1 mL:1 drop:1 drop Base Solution:Catalyst:Promotor was injected into the embryonic bladder to form a urethral cast. The liquid plastic was allowed to set at room temperature for 1 hour before the tissue was fixed in formalin overnight. The tissue was digested in 20% KOH in order to visualize the cast.

Results Caffeine-treated mice demonstrated a dose-dependent reduction in mass in both male and female fetuses. In contrast, E2-treated mice demonstrated a sex-specific reduction in mass. E2 dose was inversely correlated with mass overall. Male fetuses from the two highest E2 dosage groups were significantly smaller than their control male counterparts, and males from the highest E2 dosage group were significantly smaller than control females. E2 control males were significantly larger than all females, and there was no difference in mass among control and treated females. Serial histological sections and urethral cast data demonstrated no difference in development of the genital tubercle between caffeine-treated mice and controls or E2-treated mice and controls.

Conclusions Our results indicate a dose-dependent reduction in mass with administration of caffeine. We found a sex-specific fetal effect from low-dose orally administered E2, which appears to exert androgen-inhibiting effects on mass in males.

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