The mouse uterotrophic assay: a reevaluation of its validity in assessing the estrogenicity of bisphenol A.

The prevalence of synthetic chemicals in our environment that are capable of mimicking the female hormone estrogen is a growing concern. One such chemical, bisphenol A (BPA), has been shown to leach from a variety of resin-based and plastic products, including dental sealants and food and beverage containers, in concentrations that are sufficient to induce cell proliferation in vitro. The response to BPA in vivo has been varied; thus the aims of this study were to investigate a) whether BPA has an estrogenic effect in CD-1 mice, a strain that is useful for developmental studies; and b) whether the uterotrophic assay is a valid means of determining the estrogenicity of BPA by comparing it with other end points measured in the uterus. Immature female CD-1 mice were exposed to BPA in concentrations ranging from 0.1 to 100 mg/kg body weight for 3 days. Results showed that BPA induced a significant increase in the height of luminal epithelial cells within the uterus at concentrations of 5, 75, and 100 mg/kg and that BPA induced lactoferrin at concentrations of 75 and 100 mg/kg. A uterotrophic response (increase in uterine wet weight) was induced by 100 mg/kg BPA only. Further, the proportion of mice showing vaginal opening was greater after exposure to 0.1 and 100 mg/kg BPA, relative to the control animals and those receiving intermediate doses of BPA. These results demonstrate that BPA induces changes in the mouse uterus that differ depending on the exposure dose and the end point measured, and reveal that certain tissue effects show a nonmonotonic relationship with dose. These data also demonstrate that BPA induces estrogenic changes in the uterus of the CD-1 mouse, and highlight the need to reevaluate the validity of the mouse uterotrophic assay as an end point for determining the estrogenicity of suspected environmental estrogens.

Assays to measure estrogen and androgen agonists and antagonists.

Substantial evidence has surfaced on the hormone-like effects of many xenobiotics in fish, wildlife and humans (Colborn et al., 1993). The endocrine and reproductive effects of xenobiotics are believed to be due to their 1) mimicking the effects of endogenous hormones such as estrogens and androgens, 2) antagonizing the effects of normal, endogenous hormones, 3) altering the pattern of synthesis and metabolism of natural hormones, and 4) modifying the hormone receptor levels. Estrogen mimics (xenoestrogens) are among the environmental chemicals found to cause reproductive impairment in wildlife and humans. All of these compounds were found to be estrogenic long after they had been released into the environment. A single causal agent can be identified in cases in which humans have had occupational exposures, whereas in cases where wildlife have shown signs of reproductive damage the exposure is usually a combination of endocrine disruptors that may have acted cumulatively. It has been hypothesized that environmental estrogens may play a role in the decrease of human semen quantity and quality of human semen during the last 50 years. They may also be partly responsible for the increased incidence of testicular cancer and cryptorchidism in males and breast cancer incidence in both females and males in the industrialized word. Testing this hypothesis will require: 1) the identification of xenoestrogens among the chemicals present in the environment, 2) the development of a methodology to assess the interactions among mixtures of xenoestrogens to which humans are exposed, and 3) the discovery of markers of estrogen exposure. The development of fast and sensitive bioassays is central to the achievement of these three goals.

Human serum albumin shares the properties of estrocolyone-I, the inhibitor of the proliferation of estrogen-target cells.

The control of cell proliferation by estrogens was examined under the premises of the indirect-negative hypothesis, which states that estradiol cancels the proliferative inhibition exerted by a serum-borne protein on estrogen-target cells. Fractionation protocols resulted in the co-elution of the inhibitory activity with serum albumin. Removal of human albumin (HA) from charcoal-dextran stripped serum by hexyl-S agarose chromatography resulted in a preparation lacking inhibitory effect. HA inhibited the proliferation of human estrogen-target MCF-7 cells. Human non-estrogen-target MDA-MB231 cells were impervious to the effect of HA. MCF-7 cells were exposed to recombinant human albumin (rHA) and to its rDomain I and rDomains I + II. Inhibition of proliferation was maximal with rHA and with rDomains I + II; rDomain I was less inhibitory. The inhibitory effect of albumin was cell type and protein specific. Only estrogens cancelled the albumin inhibition; recombinant growth factors and other hormones were ineffective. These data suggest that: (a) albumin or a portion of it (most likely within Domains I and II) is the specific inhibitory signal for the proliferation of human estrogen-target, serum-sensitive cells; (b) estrogens specifically cancel this inhibition; (c) inhibitory signals prevail over putative growth factors; and (d) the default state in these cells is proliferation.

Domains of differential cell proliferation and formation of amnion folds in chick embryo ectoderm.

Patterns of cell proliferation in ectoderm epithelium that will form avian amnion correlate with morphogenesis, but not in an obvious pattern with respect to large-scale folding. At sites where the pre-axial amnion folds will first appear in 4- to 8-somite embryos, patterns of proliferation do not separate into domains that presage location of the single pre-axial fold that is commonly described in embryology texts. Instead, increased cell proliferation occurs in a significant, bilateral pattern. In stages with 13 to 27 somites, when lateral amnionic folds are prominent, five paraxial domains of cell proliferation correlate with morphology and show decreasing levels of cell proliferation with distance from the neural axis. Slowly growing areas surround rapidly growing areas and could assist buckling of epithelium by providing constraints on expansion of faster growing areas. Proliferation domains in ectoderm correlate with morphology and morphological events when localized changes in cell shape are lacking and suggest a role for differential cell proliferation in formation of large-scale epithelial folds in early chick embryos.