CORRESPONDENCE/REBUTTAL pubs.acs.org/est
Comment on “Effects of Triclocarban, Triclosan, and Methyl Triclosan on Thyroid Hormone Action and Stress in Frog and Mammalian Culture Systems” n Hinther et al.,1 two in vitro assays (a cultured frog tadpole tail fin biopsy (C-fin) assay and a GH3 cell assay) for thyroid hormone action were used to evaluate the effects of triclosan (TCS), methyltriclosan (mTCS), and triclocarban (TCC) on thyroid hormone signaling and cellular stress. Neither model has been validated nor accepted by an authoritative body. Hinther et al.2,3 developed the C-fin assay in 2010 and thus far appear to be the only investigators using this model.4 The GH3 cell assay for thyroid hormone action is a relatively new high throughput screen with the potential to provide information on chemicals that activate thyroid receptors (TR). However, confirmation studies with a validated assay should have been conducted. OPPTS 890.1100 Amphibian Metamorphosis Assay5 is a whole animal model validated by the U.S. Environmental Protection Agency and the Organisation for Economic Co-operation and Development for use in screening for thyroid axis disrupting chemicals. It was disappointing that positive or negative controls were not used to evaluate assay performance and to establish a baseline for determining the magnitude of the effects reported. The stress end points measured in the C-fin assay do not represent a validated method for measuring cellular stress and may not definitively correlate with cellular stress. Furthermore, a change at the transcriptional level does not necessarily correspond to a change in protein translation requiring proteomic investigation. Hinther’s Master’s Thesis, which can be found on the University of Victoria, Canada Web site, goes into greater depth describing the results. For example, on page 127, she states: “It is interesting to note, although TCS did not alter the THresponsive gene transcript levels, in either models tested, it did elicit a cellular stress response in the biopsies as indicated by the altered CAT, HSP30, and HSP70 transcript levels. This suggests a possible mechanism for TCS accelerating metamorphosis in intact Rana catesbeiana tadpoles through a stress response.” She further explains that these effects may actually be unrelated to TCS exposure, but result from the stress conditions of the assay. Amphibians do not express deiodinase (DI) type I which is a mammalian DI6 but Xenopus do express DI-II and DI-III, however. Fort et al.7 found that TCS exposure during metamorphosis did not alter DI-II or DI-III expression. Biotransformation of xenobiotics is generally poor in amphibians. Although anurans are capable of CYP oxidation reactions such as O- and N-demethylation it is unlikely that they can readily methylate xenobiotics such as TCS. We have not detected mTCS in amphibian tissue during our studies.8 While it is slightly more plausible that bacteria could methylate TCS in the culture three factors make it unlikely. First, the formation of mTCS even in bacteria is improbable from an energetic viewpoint since mTCS has a higher enthalpy than TCS. Second, the exposure we provide is
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flow-through with complete volume exchange every 2.7 h, so it is unlikely that any accumulation would occur. Third, no mTCS was detected in the exposure solutions collected from the various tanks even at the highest concentration tested (50 μg TCS/L nominal). “Non-target” (or nonspecific) effects may or may not be tied directly to either homeostatic control of stress or control of the thyroid axis. Most xenobiotics have a “non-specific” effect at some concentration. To be considered an endocrine modulator or disruptor, the chemical in question must have a site and mode of action by EDSP/OECD definition (Kavlock, 1999).9 It is quite possible that TCC, mTCS, or TCS do not have any direct acting effects on either system and by definition are not endocrine active. Overall, it is possible to look for a myriad of “non-target” effects of TCC, TCS, and mTCS and eventually find one that produces a response. In contrast, weight-of-evidence (WOE) ensures that a proper evaluation of multiple end points is used. Any evaluation of TCC, TCS, and mTCS should be based on a WOE. Since, TCC induced down-regulation of RLK1, but did not alter TRβ it should not necessarily be considered a disruptor of thyroid axis without further evidence. We are not convinced that the experimental program described was based on sufficiently established scientific principles necessary to produce the information needed for the conclusions purported in Hinther et al.1 Lastly, these findings, given the number of caveats provided to couch these results, do not establish any reason to counter the already validated dose response assessments for TCS with respect to potential thyroid activity as well as other biological effects. Paul DeLeo, Ph.D.* American Cleaning Institute
Sascha Pawlowski, Ph.D. BASF SE
Charles Barton, Ph.D., DABT Georgia-Pacific LLC
Douglas J. Fort, Ph.D. Fort Environmental Laboratories
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
’ REFERENCES (1) Hinther, A.; Bromba, C. M.; Wulff, J. E.; Helbing, C. C. Effects of triclocarban, triclosan, and methyl triclosan on thyroid hormone action
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CORRESPONDENCE/REBUTTAL
and stress in frog and mammalian culture systems. Environ. Sci. Technol. 2011, 45, 5395–5402. (2) Hinther, A.; Vawda, S.; Skirrow, R. C.; Veldhoen, N.; Collins, P.; Cullen, J. T.; Helbing, C. C. Nanometals induce stress and alter thyroid hormone action in amphibian at or below North American water quality guidelines. Environ. Sci. Technol. 2010, 44, 8314–8321. (3) Hinther, A.; Domanski, D.; Vawda, S.; Helbing, C. C. C-FIN: a cultured frog tadpole tail fin biopsy approach for the detection of thyroid hormone-disrupting chemicals . Environ. Toxicol. Chem. 2010, 29, 380– 388. (4) US National Library of Medicine and National Institutes of Health, PubMed, http://www.ncbi.nlm.nih.gov/pubmed?term=c-fin (assessed August 1, 2011). (5) OPPTS. 890.1100 Amphibian Metamorphosis (Frog), EPA 740C-09-002; United State Environmental Protection Agency: Washington DC, October 2009. (6) Fort, D. J.; Degitz, S.; Tietge, J.; Touart, L. W. The Hypothalamic-pituitary-thyroid (HPT) axis in frogs and its role in frog development and reproduction. Crit. Rev. Toxicol. 2007, 37, 117–161. (7) Fort, D. J.; Mathis, M. B.; Hanson, W.; Fort, C. E.; Navarro, L. T.; Peter, R.; B€uche, C.; Unger, S.; Pawlowski, S.; Plautz, J. R. Triclosan and thyroid-mediated metamorphosis in Anurans: differentiating growth effects from thyroid-driven metamorphosis in Xenopus laevis. Toxicol. Sci. 2011, 121, 292–302. (8) Fort, D. J., Mathis M., Todhunter, B., Fort, C. E., Fort, H. M., Pawlowski, S. Accumulation and depuration of triclosan in larval Xenopus laevis during metamorphosis. in preparation. (9) Kavlock, R. J. Overview of endocrine disruptor research activity in the United States. Chemoshere 1999, 39, 1227–1236.
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