Toward Resolution of the Divergent Effects of Estrogens on the Prostate Gland of CF-1 Mice

In October 2000 an ad hoc panel of experts was convened by the National Toxicology Program and the U.S. Environmental Protection Agency to review conflicting reports of endocrine effects induced by low doses of synthetic estrogens in rodents. Of primary concern were reports by Nagel et al. (1) and vom Saal et al. (2) of increases in prostatic weight in CF-1 mice exposed in utero to comparatively low doses of either bisphenol A (BPA) or diethylstilbestrol (DES), and subsequent reports by Cagen et al. (3) and Ashby et al. (4) describing the absence of such effects for both chemicals. After the meeting it was realized that the panel had failed to consider the work of Nonneman et al. (5) from the vom Saal group. Nonneman et al. (5) described the influence of male intrauterine position (IUP) on prostatic weight and suggested that differences in estrogen levels between embryos, according to their IUP, was responsible for the differing prostatic weights observed. That observation led to the evaluation of synthetic estrogens in the CF-1 mouse. Because the results of Nonneman et al. (5) indicate that control prostate weights in the CF-1 mouse are more variable than hitherto thought, this variability should be taken into account in evaluating the four sets of mouse prostate data under discussion (1-4).

The panel noted, not for the first time, that the CF-1 mouse control prostatic weights reported by Ashby et al. (4) (49.1 mg, ~6 months of age) were significantly heavier than those reported by Nagel et al. (1) (~42 mg, ~6 months of age), vom Saal et al. (2) (~.42 mg, ~8 months of age), or Cagen et al. (3) (~39 mg, 3.5 months of age). In particular, the prostatic weights reported by Nagel et al. (1) for animals exposed to BPA (~55 mg) or reported by yom Saal et al. (2) for animals exposed to DES (~55 mg) were similar to the control prostate weights reported by Ashby et al. (4). In all of these studies, the IUP of the male pups was not known: either one male pup was selected randomly from the litter (1,2) or all male pups were retained without knowledge of IUP (3,4). vom Saal suggested to the panel that the prostatic weights of the mice used by Ashby et al. (4) may have been increased by some unknown experimental condition leading to a loss of sensitivity to the two estrogens tested (1,2). In fact, our animal body weights were higher than those described by Nagel et al. (1), but vom Saal et al. (6) reported that body weight is not related to prostatic weight in CF-1 mice of this age range (6). In contrast, the data described by Nonneman et al. (5) may be more relevant to our prostate weights. Nonneman et al. (5) recorded prostate weights for CF-1 mice at ~3.5 months of age according to the IUP of the male pups. These weights were as follows: OM (adjacent to no males) pups, 59.5 [+ or -] 3.3 mg (mean [+ or -] SE of seven pups); 1M pups (adjacent to one male), not reported; and 2M pups (adjacent to two males), 49.4 [+ or -] 3.0 mg (5). From these data, the average control prostate weight for CF-1 mice in the vom Saal's laboratory in 1992 must have been [is greater than] 50 mg, which is greater thhn any of the control values discussed above (1-4). In addition, the variability of control prostate weights in the study by Nagel et al. (1) (SE = 1.0) is much lower than would be expected based on the data of Nonneman et al. (5) (SE ~3), a difference made more surprising by the fact that knowledge of IUP would have been expected to reduce, rather than increase, the variability in IUP-specific prostatic weights.

The above considerations indicate two things. First, that control prostate weights of ~49 mg reported by Ashby et al.(4), although higher than recent values from the laboratory of yom Saal (1,2,6), agree with those reported in the only published reference to control CF-1 mouse prostate weights presented according to IUP (5). Second, given the dependency of prostate weight on IUP (5), it would be of value to statistically analyze the optimum study design for the detection of a 30% increase in prostatic weight, as reported for BPA (1). In particular, it would be of value to establish how the use of seven test and seven control pups, each selected randomly from 14 dams without knowledge of IUP, could have achieved such sensitivity. In conclusion, the divergent findings for the prostate gland of CF-1 mice (1-4) might be clarified by publishing individual prostate weights from earlier studies (1,2,5,6) in combination with an analysis of how IUPs of the pups under study might affect those data.

John Ashby
Syngenta Central Toxicology Laboratory
Alderley Park, Cheshire, United Kingdom
E-mail: john.ashby@syngenta.com

REFERENCES AND NOTES

(1.) Nagel SC, vom Saal FS, Thayer KA, Dhar M, Boechler M, Welshons WV. Relative binding affinity-serum modified access (RBA-SMA) assay predicts the relative in vivo bioactivity of the xenoestrogens bisphenol A and octylphenol. Environ Health Perspect 105:70-76 (1997).

(2.) Vom Saal FS, Timms BG, Montane MM, Palanza P, Thayer KA, Nagel SC, Dhar MD, Ganjam VK, Parmigiani S, Welshons WV. Prostate enlargement in mice due to fetal exposure to low doses of estradiol or diethylstilbestrol and opposite effects at high doses. Proc Natl Acad Sci USA 94:2056-2061 (1997).

(3.) Cagen SZ, Waechter JM Jr, Dimond SS, Breslin W J, Butala JH, Jekat FW, Joiner RL, Shiotsuka RN, Veenstra GE, Harris LR. Normal reproductive organ development in CF-1 mice following prenatal exposure to Bisphenol A. Toxicol Sci 50:36-44 (1999).

(4.) Ashby J, Tinwell H, Haseman J. Lack of effects for low dose levels of BPA and DES on the prostate gland of CF1 mice exposed in utero. Regul Toxicol Pharmacol 30:156-166 (1999).

(5.) Nonneman DJ, Ganjam VK, Welshons WV, vom Saal FS. Intrauterine position effects on steroid metabolism and steroid receptors of reproductive organs of male mice. Biol Reprod 47:723-729 (1992).

(6.) Welshons WV, Nagel SC, vom Saal FS. Response [Letter]. Environ Health Perspect 106:A316-A317 (1998).

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