The uterotrophic response assay (using prepubertal or adult ovariectomized animals) has been considered the most reliable indicator of in vivo estrogenic activity (Laws et al., 2000) (EDSTAC, 1998). In this assay, females within a dose group for each chemical are housed 5 per cage starting on PND 21, and injected daily subcutaneously (s.c.) with a test chemical on PND 21, 22, and 23. The uterine response is assessed 5 hr after the last injection on PND 23 (Laws et al., 2000). ) by measuring uterine weight, epithelial cell height and cell number, and uterine gland number, as sensitive indicators of exposure to estrogenic chemicals (Shelby et al., 1996). Liver, kidney, uterus and spleen are examined for histopathology to assess acute toxicity.
In vivo pubertal assay
This assay examines the ability of estrogenic chemicals to alter the onset of vaginal opening and estrus in mice.
Typically, the assay uses 20 litters of females for each of 6 doses for each chemical being tested. Starting on PND 21, body weights are recorded and chemicals are administered daily subcutaneously (s.c.) until vaginal estrus is reached, typically within 18 days in controls in individually housed females located near males (Howdeshell et al., 1999). The age at vaginal opening is recorded, typically within 10 days in controls. To determine the date of vaginal estrus, daily vaginal lavage is performed following vaginal opening to examine vaginal epithelial cell type or for a maximum of 5 weeks (antiestrogenic chemicals delay puberty). The relationship between the first vaginal estrus and the first ovulation, which is the final event in the transition from the pubertal period to adolescence, is verified by examining the ovaries for evidence of ovulation and the fallopian tubes for the presence of ooctyes (vom Saal and Bronson, 1980). Body weight is recorded as estrogenic chemicals can alter growth curves (Howdeshell et al., 1999). Liver, kidney, uterus and spleen are examined for histopathology to assess acute toxicity.
In vivo prostate assay
Developing male prostates are much more sensitive to some ECs at environmentally relevant concentrations than tissues derived from female mammals (including humans). For example, the potency of bisphenol A (a weak EC) relative to DES (a strong EC) appears to be about 1000 times higher in the mouse prostate (Steinmetz et al. 1997; Gupta, 2000a,b) compared to MCF-7 breast cancer cells (Krishnan et al. 1993; Nagel et al. 1997). That is, the ratio of bisphenol A/DES potency is 100-200 in the male prostate and is about 100,000 in MCF-7 cells. To confirm the higher sensitivity of our in vivo prostate assay to some weak ECs, we have retested the effect of DES and bisphenol A on the prostate by measuring prostate budding, prostate size, and prostate weight. Our preliminary data (Figure 3, Table 3) or recently published data (Table 4 from Gupta, 2000a) confirm previous studies on CF-1 mice (Nagel et al., 1997; vom Saal et al., 1998) that a 10 ??g/kg/day dose of bisphenol A in CD-1 mouse fetuses produces about the same increase in prostate size as does a 0.1 ??g/kg/day dose of DES. These data are also consistent with other published data (Steinmetz et al. 1997). Note that the doses administered are comparable to those found in some human exposure studies (Schonfelder et al., 2000). Furthermore, this type of experimental paradigm focuses on permanent effects of "low dose" exposure of fetuses to ECs during critical periods in reproductive organ differentiation, as recommended by NIH (NTP, 2000).
In this assay, virgin females are paired with stud males for 4 h at the end of the dark phase of an L:D cycle. Mating is verified by the presence of a copulatory plug (gestation day 0), and females are separated from the stud male. Each test concentration of a test chemical is dissolved in 30 ??l of tocopherol-stripped corn oil (Cat# 901415, ICN, Aurora,OH) and injected from GD 14-18. Each pregnant female (15 per dose) receives a subcutaneous injection of one of six doses [0 (negative control), 10, 100, and 1000 mg/kg/day) of the test chemical. This assay also examines the effects of 1 and 10 ??g/kg/day of DES, as a positive control. [A 1 ??g/kg/day dose of DES is used because of the greater sensitivity of the prostate to estrogenic chemicals compared to the uterus.]. A total of 90 pregnant females are treated in this assay. Females deliver their litters normally on gestation day 19, and pups are weaned on postnatal day 21. Randomly selected males from each litter are individually housed until 2 months old.
At two months of age (PND 60), the males are sacrificed and body weights are recorded. The prostate is removed and weighed. The testes, epididymides, preputial glands and seminal vesicles are also removed and weighed, since in prior studies, prenatal exposure to low doses of estrogenic chemicals has affected these tissues (vom Saal et al., 1998; Gupta, 2000a).
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