The rodent uterotrophic assay: response to Ashby and Newbold et al. (Correspondence).

The comments submitted by Ashby et al. and Newbold et al. reflect disagreement with our statement that the classical mouse uterotrophic assay is of limited use in assessing the estrogenicity of chemicals. We restate here that the uterotrophic assay is a relatively insensitive test for determining whether a chemical is estrogenic, for establishing no-observed-effect levels (NOELs), and ultimately for predicting the level of exposure at which adverse effects may occur. Ashby and colleagues cite a recent publication by Newbold et al. (1) as "an example of the biological validity of this [uterotrophic] assay." The study to which Ashby et al. refer shows that exposure of neonatal mice to daily injections from postnatal days 1-5 of either genistein (50 mg/kg/day) or DES (1 [micro]g/kg/day) induces a uterotrophic response by the end of day 5 and uterine adenocarcinoma by 18 months of age. Ashby et al. interpret "this confluence of findings" as being "encouraging for the utility of the rodent uterotrophic assay for predicting adverse effects in rodents."

First, the study of Newbold et al. (1) does not conform to the classic use of the uterotrophic assay. This assay is characterized by a 3-day exposure of an adult ovariectomized or juvenile (prepubertal) rodent to a chemical, or in the case of the developmental uterotrophic assay, a 12-day exposure of a rodent from postnatal day (PND) 10 to PND 22 (2). Newbold et al. (3) cited Bern's study (40, which shows that the perinatal mouse is far more sensitive to the effects of estrogen exposure than the prepubertal or adult mouse, a widely held tenet in the field of endocrine disruption.

Second, by using a single dose of genistein and DES, the study of Newbold et al. (1) was not aimed at determining the minimal exposure that would cause an effect for the end points of uterine wet weight and uterine adenocarcinoma at these ages. To emphasize this point, previous work from Newbold's group has demonstrated that exposure of neonatal mice to a 10-fold lower dose of genistein (5 mg/kg) resulted in the development of multi-oocyte follicles by 2 months of age (5), an effect that is likely to compromise fertility. Yet, a 3-day exposure of prepubertal mice (days 17-19) to < 50 mg/kg genistein was insufficient to induce a uterotrophic response (6). Thus, for the parameter of multi-oocyte follicles, the uterotrophic assay is not predictive.

Third, induction of adenocarcinoma is the most extreme of biological end points to consider, particularly in a field that is attempting to tease out the subtleties of biological perturbations due to chemical exposure in populations characterized by variation. Again, to emphasize this point, Newbold (7) demonstrated that exposure of CD-1 mice to 100 [micro]g/kg DES on gestational days 9-16 induced uterine adenocarcinoma; yet a dose of DES that was 10,000-fold lower (0.01 [micro]g/kg) resulted in reduced reproductive capacity. By using the uterotrophic assay to establish the estrogenicity of bisphenol A (BPA), it can be concluded that exposure to a dose < 100 mg/kg/day is not detrimental to CD-1 mice. However, exposure of the same strain of mice during gestational days 9-20 to doses of BPA 4,000 times lower (25 [micro]g/kg/day) induced profound proliferative changes in the lobuloalveolar network of the adult mammary gland (8). We also observed the persistence of epithelial structures that have been shown to be sites of neoplasia in rodents and humans, and an alteration in DNA synthesis between the epithelium and stroma, which is the substratum of neoplasia in this tissue (8).

Ashby's statement that there is "insufficient genistein in soy-based infant formula to trigger a uterotrophic response in rodents" which "indicates the probable absence of a carcinogenic hazard" leads him to conclude that the dose of genistein found in infant formula is not of biological concern. This rationalization clearly demonstrates the dangerous misuse of the uterotrophic assay that we pointed out in our paper in Environmental Health Perspectives (9).

Newbold et al. support our findings that "many cellular and biochemical end points in the uterus ... are often more sensitive end points than wet weight increase," yet they disagree with our purported conclusion that the uterotrophic assay has limited value. We concluded that "the uterotrophic assay is of limited value in assessing the estrogenicity of a suspected environmental estrogen" (9), and we maintain this position on the basis of our work and that of others. Research by Newbold and colleagues dearly demonstrates this phenomenon (3). This group's study showed that exposure of prepubertal mice (days 17-19) to a variety of chemicals induced a uterotrophic response in all cases except with endosulfan and kepone. In spite of an observed increase in the number of uterine glands and PCNA labeling, these two chemicals were not able to induce a uterotrophic effect at any dose, even at those doses that were toxic to the animal (endosulfan). Although this work provides a seminal contribution to the field of endocrine disruption, it cannot claim to have "compared the sensitivity of different end points, for 10 compounds, over a large dose range for each chemical." Newbold et al. (3) described a dose-response curve for 10 chemicals, using the uterotrophic assay as the end point. Then, they tested the change in various cellular and biochemical end points at the dose that induced the maximal uterotrophic response.

We agree with the statement of Newbold et al. that the mouse uterotrophic assay should be "optimized so that meaningful and informative end points are included that cover a range of effects induced by the chemical under study." However, we propose that this modified assay be renamed so as not to be confused with the classical mouse uterotrophic assay, which is simply a measure of the increase in uterine wet weight after exposure to a chemical. In redefining the assay, we should consider the statement by Roy Hertz (1(9) who suggested that

   the sine qua non of estrogenic activity remains the mitotic stimulation of
   the tissues of the female genital tract. A substance which can directly
   elicit this response is an estrogen; one that cannot do this is not an
   estrogen.

In regard to the issue of measuring uterine wet weight versus "blotted" weight that was raised by Newbold et al. in their letter, we believe that a universally accepted technique should be used by all researchers to accommodate a valid comparison of data. In our study, we chose to define uterine weight as "blotted" because we believe that it is closer to the ideal of Hertz (10), as outlined above, which asserts that mitotic activity is the best index of an estrogenic effect.

In conclusion, we maintain our original position that the uterotrophic assay is not sufficiently sensitive to be used as a tool to determine whether or not a chemical is estrogenic or to establish NOELs. In the absence of other more sensitive end points, relying on the uterotrophic assay alone to make statements that exposure to certain estrogenic chemicals is not of biological concern, as appears to be the modus operandi of some researchers in the field, is hazardous to the health of all species.

Caroline M. Markey Cheryl L. Michaelson Electra C. Veson Carlos Sonnenschein Ana M. Soto Tufts University School of Medicine Boston, Massachusetts E-mail: [e-mail]

REFERENCES AND NOTES

(1.) Newbold RR, Banks EP, Bullock B, Jefferson WN. Uterine adenocarcinoma in mice treated neonatally with genistein. Cancer Res 61:4325-4328 (2001).

(2.) Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC): Final Report. Vol. 2. Washington, DC:U.S. Environmental Protection Agency, 1998.

(3.) Newbold RR, Jefferson WN, Padilla-Banks E, Walker VR, Pena DS. Cell response endpoints enhance sensitivity of the immature mouse uterotrophic assay Reprod Toxicol 15:245-252 (2001).

(4.) Bern HA. The fragile fetus. In: Chemically-induced Alterations in Sexual and Functional Development: The Wildlife/Human Connection. (Colborn T, Clement C, eds). Princeton, NJ:Princeton Scientific Publishing, 1992;9-15.

(5.) Jefferson WN, Padilla-Burgos E, Miller C, Newbold RR. Endocrine modulating effects of various phytoestrogens. In: Human Diet and Endocrine Modulation: Estrogenic and Carcinogenic Effects (Dunaif GE, Olin SS, Scimeca JA, Thomas JA, eds). Washington, DC:ILSI Press, 1998;286-289.

(6.) Jefferson WN, Newbold RR. Potential endocrine-modulating effects of various phytoestrogens in the diet. Nutrition 16:658-662 (2000).

(7.) Newbold RR. Cellular and molecular effects of developmental exposure to diethylstilbestrol: implications for other environmental estrogens. Environ Health Perspect 103(suppl 7):83-87 (1995).

(8.) Markey CM, Luque EH, Munoz de Toro M, Sonnenschein C, Soto AM. In utero exposure to bisphenol A alters the development and tissue organization of the mouse mammary gland. Biol Reprod 65:1215-1223 (2001).

(9.) Markey CM, Michaelson CL, Veson EC, Sonnenschein C, Soto AM. The mouse uterotrophic assay: a reevaluation of its validity in assessing the estrogenicity of bisphenol A. Environ Health Perspect 109:55-60 (2001).

(10.) Hertz R. The estrogen problem--retrospect and prospect. In: Estrogens in the Environment II-Influences on Development. (McLachlan JA, ed). New York:Elsevier, 1985;1-11.