The relevance of endocrine disruptors is being increasingly recognized.20
This is the first report demonstrating that a novel endocrine disruptor, TCC, induces androgen-like effects in intact immature males. Key findings in this study include demonstrations that exposure to TCC (1) results in an increase of accessory sex organ weight, protein content, and DNA content, (2) has no significant effect on histological appearance of these organs, and (3) has no significant effect on circulating LH levels, circulating T levels, and on testicular size or histological appearance of testes.
Previously, our group demonstrated that TCC amplified the androgenic action of exogenous androgens with respect to the weight of accessory sex organs. The data presented here demonstrate that TCC induced a prominent increase of weight in all accessory sex organs ( and ) in age-matched intact male rats (48-52 days old). These tissues rely on androgenic stimulation to develop, grow, and maintain function. In addition, but to a lesser degree, TCC exposure resulted in modest but statistically significant increases of body weight and liver size, and these effects are consistent with the proposed androgen-augmenting actions of TCC and not to direct toxic effects of TCC. Anabolic effects of androgens are well recognized; for example, a previous study using the Hershberger assay has shown that testosterone propionate (TP) injections alone in castrated rat increases both total body weight and liver weight.21
An increase of the body weight in the TCC-treated animals may be related to changes of androgen-binding protein (ABP) and/or effects of TCC on insulin action; future studies should address this issue.
In this study (in the presence of endogenous androgens) and our previous report (in the presence of exogenous androgens),8
TCC treatment consistently induced increases of accessory sex organs that were disproportionately greater than its effects on body and liver weight. These effects persist, when weights of accessory sex organs are expressed as percentage of terminal body weight (, columns on the right) or percentage of liver weight (not shown). Since the weights of accessory sex organs may be affected by their water content, as well as the size and the number of cells, this study also collected data regarding dry organ weight, percentage of water content, protein content, and DNA content (). Only the LABC had a significant increase in water content following treatment with TCC. However, all accessory sex organs, including LABC, exhibited an increase in weight suggestive of either cell hypertrophy or hyperplasia. Ventral prostate, LABC and glans penis had significantly increased protein and DNA content (); hence, in view of the latter finding, exposure to TCC resulted in increased number of cells per organ, that is, hyperplasia.
Despite the clear differences in organ weights in this study, there was no defining histological difference between accessory sex glands from treated and control rats. This suggests that the increased growth associated with TCC in this short exposure period was proportional in the epithelium and surrounding parenchyma of each organ. Androgen receptor expression was higher in the epithelial cells of both the vesicular glands and the prostate than in the surrounding smooth muscle, as reported previously for rats and mice.22,23
This difference in AR concentration did not result in an exaggerated or unbalanced growth response within the organ under the experimental conditions used. Tissue growth was apparently normal and organized, unlike that observed after neonatal exposure to estradiol.22
In our previous report,8
effects of TCC were studied in vitro using the cell-based human AR-mediated bioassay developed by Chen and associates.9
Triclocarban alone had no effect on AR-mediated transcriptional activity, but it significantly amplified effects induced by T.8
Furthermore, in that study, using the castrated immature rat model, TCC alone had no effect on accessory sex organs, but in the presence of TP, TCC significantly increased weight of all accessory sex organs above and beyond the effect of TP alone.8,21
Previously, we have shown the lack of effect of TCC in cells with no AR expression.8
To further investigate the AR-dependent mode of action of TCC, 2 types of human prostate cancer cells with different levels of AR expression were transiently transfected with reporter gene driven by various androgen response elements. Human LNCaP is a well-established model for studies on androgen-dependent prostate growth.24,25
C4-2 is a second generation of LNCaP subline.26
It was derived from a chimeric tumor induced by co-inoculating castrated mouse with C4 cells and a bone stromal cell line. Although having chromosomal markers similar to those of the parental LNCaP cells, C4–2B expressed lower steady-state levels of AR protein and messenger RNA (mRNA) transcript and the cells is less responsive to androgens.26
In this report, TCC and androgen (either T or DHT) cotreatment significantly increased luciferase activity compared to androgen alone in both LNCaP and C2–4B cells, although the effect was less pronounced in C4–2B (); this may be due to its relative low level of AR expression. In both models, the enhancement of signal by TCC was suppressed by biclutamide, indicating the potentiation effect of TCC is AR-dependent. Hence, both in vitro and in vivo data point at TCC acting as a novel and unique endocrine disruptor with no intrinsic agonist or antagonist properties, but only potentiating androgenic effects at the level of the target cell. This report verifies these effects and demonstrates that exposure to TCC in the presence of physiological levels of endogenous androgens increases androgenic effects.
Another interesting observation was the absence of significant effect of TCC on LH and T levels, as well as no significant change in size or histology of testes. Since TCC exerted androgen-potentiating actions in vitro and in castrated rats, one could expect that TCC would potentiate androgen-induced negative feedback on hypothalamo-pituitary action, reduce LH, and ultimately decrease testicular androgen production. Several possibilities could attribute to the lack of the effect. Although the blood of both treated and control animals was collected at the same time of the day, LH is secreted in pulses varying in frequency and amplitude, which also stimulates T secretion in a pulsatile manner.27
Consequently, the interpretation of the data regarding LH and T levels from a single blood collection should be cautious and future studies with serial blood collection during the treatment are warranted.
Peripubertal animals (48-52 days) were used in both castrated8
and current intact male study for TCC exposure. The use of immature castrated male rats in our previous study was based on Hershberger’s protocol.28
Consequently, the present report was designed to assess the effect of TCC exposure on age-matched intact males. It is known that in the rat, production of T is high during late gestation, decreases dramatically in the neonate, reaching a nadir at about 2 weeks after birth, and increases again to attain adult levels by 60 days of age29,30
accompanied by a shift from predominately androstenedione, observed in prepubertal rats, to testosterone seen in mature rats.31
This changing androgen secretion pattern may be critical for T to reach adult levels with decreasing sensitivity of the LH negative feedback system being a component of the underlying mechanism.31
It is therefore possible that the fully mature long-loop feedback has not yet been established in our rat model. Alternatively, the mechanism(s) by which TCC augments androgen action on growth of accessory sex organs may involve different AR coregulators than those involved in mediating T actions on the hypothalamus and pituitary. Thus, for example, TCC effects may be limited to direct AR-dependent mechanisms and may not influence the long-loop negative feedback, which requires aromatization and estrogen receptor signal transduction at the central level.32–35
Furthermore, it is not known whether TCC crosses the blood-brain barrier. Nevertheless, the absence of compensatory effects on LH in immature rats indicates that TCC exposure may be of potential clinical concern and warrants more investigation on interaction of age, dose, and length of exposure.
Another interesting point is the observation that the serum of control animals contained low but detectable levels of TCC. Hence, one may speculate that water or food may have contained TCC. However, the level of TCC in water and rat chow was not measured in this study.
The present observations raise concerns regarding potential significant health risks related to exposure to TCC.1–6
Indeed, widespread use of TCC-containing soaps and other personal hygiene products over several decades has led to repeated exposures, which may have induced important long-term effects. Some of these potential adverse effects including in utero and peripubertal period may be far-reaching and may have irreversible consequences because of the crucial role of hormones in directing the development and programming cells for later life.10–14
Furthermore, since TCC is remarkably stable and resistant to biological and chemical treatments, there is also a potential for exposure by ingestion of contaminated water and/or agricultural products exposed to TCC-containing sludge.1–6
Notably, this study only evaluated the effects of oral exposure to TCC, whereas a dominant route of human exposure is likely dermal due to the use of TCC-containing soaps. However, human oral exposure is also possible in view of the presence of TCC in municipal sludge and water.1–6
Further studies will need to include assessment of TCC absorption following use of TCC-containing products and determination of TCC content in drinking water and food.