Final Report: Effects of an Endocrine Disruptor on Prostate Development and GrowthEPA Grant Number: R827403
Title: Effects of an Endocrine Disruptor on Prostate Development and Growth
Investigators: Timms, Barry G.
Institution: University of South Dakota
EPA Project Officer: Hahn, Intaek
Project Period: July 1, 1999 through June 30, 2002
Project Amount: $432,452
RFA: Endocrine Disruptors (1999) RFA Text | Recipients Lists
Research Category: Economics and Decision Sciences , Endocrine Disruptors , Health , Safer Chemicals
The long-term objective of this research project was to determine the consequences of exposure to endocrine disrupters during fetal prostate development, particularly environmental estrogens. The specific objective of the experiments was to understand the mechanisms associated with our initial finding of dose-dependent effects on prostate growth as a result of fetal exposure to environmental chemicals. Specifically, we have examined regional growth effects in the urogenital sinus (UGS) following exposure of mouse fetuses to a low, physiologically relevant dose of the pesticide methoxychlor (MXC). By comparison, the effects of a high dose of MXC have been examined and compared to diethylstilbestrol (DES) treatment as a positive estrogen control.
Pregnant female CD-1 mice were fed oil (control) or oil plus DES (0.1 µg/kg); low-dose MXC (100 µg/kg); and high-dose MXC (100 mg/kg) on gestation days (GD) 14-18. Caesarian delivery was performed on day 19. To preclude intrauterine position effects, one 1M male per litter was used for the studies. A total of five animals was taken for each group. The urogenital complex was fixed in situ according to previously published protocols. Serial sections were prepared on coded samples. Morphometric analysis was performed to provide data on the number of buds developing in the urogenital sinus (UGS). Three-dimensional (3-D) reconstructions were prepared for the control, positive control (DES), and MXC-treated animals (Winsurf, University of Hawaii).
The number of prostatic ducts developing in specific UGS regions was calculated from the reconstruction data sets. A summary of these findings is shown in Figure 1. Although statistical analysis of these data is not yet complete, there was a slight increase in the number of ducts in the ventral and lateral regions in the low-dose MXC-treated group and a trend for the low-dose treatment to result in slightly higher duct counts than from the higher dose of MXC. The regional differences in duct number seen in previous studies were not seen with these treatments, but these animals were taken by caesarian delivery at a slightly later time, which is equivalent to the day of birth (GD 19).
Although the ductal development data have not typically shown highly significant differences, our experience with another positive control (ethinylestradiol), and an endocrine disrupter (bisphenol A), indicate that the mean cross-sectional area measurement (an indicator of volume) is correlated with a more significant growth effect (vom Saal, et al., 2000; Kaiser, 2000).
Figure 1. Total Number of Ducts in Each Region of the Urogenital Sinus in Control, Low-, and High-Dose Treated GD 19 Mice. (Mean ± SEM). DLP: combined dorsolateral region.
In agreement with previous low-dose studies with other endocrine disruptors (Timms, et al., 2004), the most significant effect was an increase in the size of the dorsal prostate region and coagulating gland. An interesting finding was a similar increase in the tissue volume of the ventral prostate. This region is not normally affected by estrogenic endocrine disruptors, but the complexity of MXC effects (estrogenic agonist and antagonist) may be reflected in this response (Waters, et al., 2001). The regional volumetric data are summarized in Figure 2. Except for the seminal vesicle and utricle, a consistent pattern of higher tissue volume was seen in the low-dose treated mice. Of significance was the increased size of the ventral mesenchymal pad (VMP). This region of mesenchymal tissue is intimately associated with the process of branching morphogenesis in the distal tips of the ventral prostate ducts (Timms, et al., 1995). Whether this increase is a consequence of an increased growth stimulation of the stromal cells by a low dose of MXC, or a consequence of an increased number of cells in the ductal tip epithelium, is yet to be determined. The VMP also is associated with a specific expression of fibroblast growth factor (FGF-10), which also may be up-regulated by this treatment (Thomson, et al., 2002). Even though there were no striking differences in the number of developing prostatic outgrowths, the effects of the low-dose MXC were more pronounced when the volume (size) of specific UGS regions were analyzed.
Figure 2. Effect of Low- and High- Dose MXC Treatment on Regional Growth of the Prostatic Ducts and the Ventral Mesenchymal Pad (VMP), a Region of Stroma Associated With Branching Morphogenesis of the Ventral Prostate. SV: seminal vesicle; CG: coagulating gland; DP: dorsal prostate; LP: lateral prostate; DLP: dorsolateral prostate; VP: ventral prostate.
Regional Anatomy. Representative examples of the reconstructed UGS and associated structures are illustrated in Figure 3. Different anatomical effects on the ducts and the UGS were more readily observed in the 3-D reconstructions and showed differences that were not recognized by examining the number of prostatic outgrowths by low and high doses. The shape of the UGS in the region of the prostatic sulci (or furrows), along which the dorsal ducts develop, was much more pronounced in the low-dose MXC- and DES-treated mice. This confirms previous findings reported for other low-dose endocrine disruptor effects (Timms, et al., 2004). In addition, the reduced volume of the UGS structures (dorsal, lateral, ventral, and coagulating glands) in the high-dose MXC treatment is likely a consequence of ductal elongation and narrowing, as seen in Figure 3D. The overall volume was not dissimilar to that of the controls, but the morphology was distinctly different. Temporal and spatial interactions of the prostatic epithelium and surrounding mesenchyme are thought to play an important role in the development of ductal patterning (Timms, et al., 1995), and these interactions may have been more affected by the high-dose treatment. The long-term consequences of such morphological changes on the adult prostate requires further study.
Figure 3. Three-dimensional Reconstructions of the Serially Sectioned UGS and Associated Structures of Control (A), Low-Dose DES-Treated (B), Low-Dose MXC Treated (C), and High-Dose MXC-Treated (D) mice. Some anatomical effects not appreciated from the volume or budding data are more clearly seen in these surface-rendered images, including a shape change in the prostatic sulci of the UGS (white * in B and C) and narrowing of the ventral, lateral, and dorsal ducts in the high-dose MXC-treated mice (D).
To examine whether the fetal exposure to MXC caused a permanent effect on the growth characteristics of the prostate, the number of ductal tips was determined in 1-month and 2-month-old mice. The entire prostatic complex of control and treated mice was microdissected using techniques described by Sugimura, et al. (1986). The number of tips at the distal ends of the ducts in each prostatic lobe was counted. A summary of these data is shown in Figure 4. These data have not been subjected to statistical analyses, but the raw data show evidence of certain trends. First, the total number of ductal tips present at 1 month of age remains at a similar level in the control and low-dose treated mice. At the high-dose treatment, both DES- and MXC-treated mice have a reduced number of prostatic ductal tips, which does not differ between the ages studied. A slight increase in the number of tips in the low-dose MXC mice at 2 months of age likely reflects the increase in the number of tips observed in the dorsolateral prostates and the coagulating glands of the 2-month-old mice. This finding supports the concept that the increased volume associated with these regions in the fetal mice (Figure 2: DP; DLP; CG) results in a permanent change in growth parameters that are specific to certain regions of the UGS and associated structures that have been shown to be sensitive to estrogenic endocrine disruption (Timms, et al., 2004). By comparison, the high dose of MXC had an opposite growth-promoting effect on the ventral ductal system (Figure 4B; see also Figure 2: VP). Additional analyses of these data are in progress.
Figure 4. Effect of Low Dose Treatment on the Mean Total Number of Ductal Tips Formed in the Prostate of 1-Month- and 2-Month-Old Mice (A). The regional ductal tips counts for the ventral (B), dorsolateral (C), and coagulating glands (D) also are shown.
Microdissection and representative images of the microdissected dorsolateral prostate are illustrated in Figure 5. Overall, the major observed effect was that morphological changes were more apparent in the older mice (2 months). No distinct differences between the low-dose DES and low-dose MXC treatment were apparent, but at later stages of growth (2 months), there were more dissimilar changes in the ductal growth patterns. For example, compared to the pattern of ductal branching in the control dorsolateral ducts (a palmate pattern), the low-dose DES treated mice exhibited more swollen ductal tips. This reflects the increased volumetric changes seen in the dorsolateral prostate of the fetal mice, especially in the dorsal region (Figure 2).
Figure 5. Whole Mount Microdissected Dorsolateral Prostate Tissue Taken From 1-Month and 2-Month-Old Mice That Had Been Exposed In Utero to Low and High Doses of DES and MXC and Compared to Control Untreated Mice at the Same Age Points.
Based on published and current data, it is postulated that physiologically relevant factors (i.e., endocrine disruption effects) during critical periods of reproductive development in the male fetus are likely to cause alterations of growth parameters in the prostate, which may persist into adulthood. The consequences of these changes in growth patterns will need to be investigated and compared at the cellular level in the regions of the prostate exhibiting the most significant responses to the endocrine disruptors.
Kaiser J. Endocrine disrupters: panel cautiously confirms low-dose effects. Science 2000;290(5492):695-697.
Sugimura Y, Cunha GR, Donjacour AA. Morphogenesis of ductal networks in the mouse prostate. Biology Reproduction 1986;34(5):961-971.
Timms BG, Lee CW, Aumüller G, Seitz J. Instructive induction of prostate growth and differentiation by a defined urogenital mesenchyme. Microscopy Research and Technology 1995;30:319-332.
Waters KM, Safe S, Gaido KW. Differential gene expression in response to methoxychlor and estradiol through ERalpha, ERbeta, and AR in reproductive tissues of female mice. Toxicology Science 2001;63:47-56.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
|Other project views:||All 11 publications||4 publications in selected types||All 2 journal articles|
||Thomson AA, Timms BG, Barton L, Cunha GR, Grace OC. The role of smooth muscle in regulating prostatic induction. Development 2002;129(8):1905-1912.||
||Timms BG, Howdeshell KL, Barton L, Bradley S, Richter CA, Vom Saal FS. Estrogenic chemicals in plastic and oral contraceptives disrupt the fetal mouse prostate and urethra. Biology of Reproduction 2005;102(19):7014-7019. .||