The mouse uterotrophic assay: other end points. (Correspondence).

In their EHP article titled "The Mouse Uterotrophic Assay: A Reevaluation of Its Validity," Markey et al. (1) argue against using the uterotrophic assay as an end point for determining estrogenicity of synthetic chemicals. They conclude (1),

   The uterotrophic assay is of limited value in determining the estrogenicity
   of a suspected environmental estrogen because changes at the cellular level
   were observed at significantly lower doses than those at which a change in
   wet weight occurred.

In agreement with their findings, we have similar data which show that many cellular and biochemical end points in the uterus, such as epithelial cell height, cell number, gland number, and lactoferrin induction, are often more sensitive end points than wet weight increase. We have reported this information at international meetings and have shared our findings with the U.S. Environmental Protection Agency's Endocrine Disruptor Screening and Testing Advisory Committee. We have also compared the sensitivity of different end points for 10 compounds over a large dose range for each chemical (2). However, we disagree with the conclusion of Markey et al. (1) that the uterotrophic assay has limited value. Our increasing knowledge about the various actions of estrogens now makes it feasible to expand the uterotrophic assay to include information about different pathways and end points. Thus, we suggest that the uterotrophic assay be expanded to encompass additional measures to the standard wet weight data to increase its sensitivity. The objective of studying these end points when the uterotrophic assay is negative is to eliminate false negatives. Further, mechanistic information can be gained by developing a "blueprint" of responses for various estrogenic compounds. The mouse uterotrophic assay is valid: a reevaluation of the assay should assure that the assay is optimized so that meaningful and informative end points are included that cover a range of effects induced by the chemicals under study.

Two other points are worthy of mention. Numerous studies appearing in the literature report data from the uterotrophic bioassay as wet weight increase, when in fact they are reporting "blotted" uterine weight. The true wet weight of the uterus includes uterine tissue plus luminal fluid. Recording only "blotted" weight overlooks the significant role estrogens play in uterine water imbibition, an early marker of estrogen action. This is an important end point that again makes the assay more sensitive.

Another important point is that proliferating cell nuclear antigen (PCNA), or any other marker of uterine cell proliferation, may give erroneous results if measured after 3 days of continuous treatment with an estrogen, as described by Markey et al. (1). Estrogens are known to increase mitosis, but they also act to inhibit mitosis if the estrogen dose is too high or if it is sustained (Table 1). This may explain why the authors found no significant difference in the expression of PCNA in the luminal epithelium between treatment groups (control, estradiol, and bisphenol A), although other studies have shown increases in PCNA expression following treatment with these same compounds (3). Using time-course experiments in the immature mouse, we have determined that excellent PCNA labeling occurs 18 hr after an initial dose of estradiol or diethylstilbestrol (DES) (2). The time for maximum stimulation of mitosis varies with the pharmacokinetics of the compound being tested (2), but most chemicals, including known estrogens that we have tested thus far, showed slight or no increase in mitosis after 3 days of treatment. In fact, mitosis is inhibited by 3 days of treatment with DES (Table 1).

In summary, we agree with Markey et al. (1) that the current uterotrophic bioassay (consisting of only wet weight) is not sensitive, and we encourage a reevaluation of the assay in the framework of the information presented.

Table 1. PCNA-labeled uterine epithelial cells
after treatment with DES or estradiol.

                      Percent
Treatment       PCNA-labeled cells

Control         5.92 [+ or -] 0.79
DES 1x         31.81 [+ or -] 3.78 *
Estradiol 1x   46.67 [+ or -] 3.68 *
DES 3x          1.23 [+ or -] 1.23
Estradiol 3x    9.60 [+ or -] 0.84

Immature outbred CD-1 mice were administered 10 [micro]g/kg
DES or 500 [micro]g/kg estradiol dissolved in corn oil by sc
injection for 1 day or 3 days and sacrificed the morning
following the last injection (2). These doses of DES and
estradiol have been previously determined to cause maximum
uterine wet weight increase (4). Uterine tissues
were collected and PCNA was determined by previously
described methods (2,5).

* p < 0.05; statistically significant difference from control
by ANOVA.
Retha R. Newbold
Wendy N. Jefferson
Elizabeth Padilla-Banks
National Institute of Environmental
Health Sciences
Research Triangle Park, North Carolina
E-mail: [e-mail]

REFERENCES AND NOTES

(1.) 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)

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

(3.) Klotz DM, Hewitt SC, Korach KS, DiAugustine RP. Activation of a uterine insulin-like growth factor I signaling pathway by clinical and environmental estrogens: requirements of estrogen receptor [alpha]. Endocrinology 141:3430-3439 (2000).

(4.) Shelby MD, Newbold RR, Tully DB, Chae K, Davis VL. Assessing environmental chemicals for estrogenicity using a combination of in vitro and in vivo assays. Environ Health Perspect 104:1296-1300 (1996).

(5.) Foley J, Ton T, Maronpot R, Butterworth B, Goldsworthy TL. Comparison of proliferating cell nuclear antigen to tritiated thymidine as a marker of proliferating hepatocytes in rats. Environ Health Perspect 101(suppl 5):199-205 (1993).