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Reply to Comment on “Effects of Triclocarban, Triclosan, And Methyl Triclosan on Thyroid Hormone Action and Stress in Frog and Mammalian Culture Systems”
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n the comment by Fort et al regarding our recent publication by Hinther et al.,1 the authors expressed concern that our synopsis of their previous work2 was misrepresenting the results described therein and that acknowledgment or discussion of the supplemental data provided in their response to our letter to the editor regarding that work was lacking.3,4 We maintain that our synopsis of their manuscript was accurate. In this manuscript,2 tadpole developmental stage was reported as not significantly different compared to controls at day 21 of triclosan exposure. However, the data presented in the manuscript, when reanalyzed by us3 and subsequently again by the authors4 demonstrate that there indeed was a significant acceleration of Xenopus laevis tadpole development due to triclosan exposure. Our Chi square analysis of the frequency data presented in the Fort et al. paper2 showed clear significance in the three lower of the four concentrations tested (0.6, 1.5, 7.2, and 32.3 μg/L triclosan) compared to control animals (for a detailed rationale and analysis refer to ref 3). Using Mann Whitney U tests,4 Fort et al. showed a significant acceleration in development at 0.6 and 7.2 μg/L triclosan. We therefore agree with Fort et al that a statistical acceleration of Xenopus laevis tadpole development occurred. A significant increase in TRβ mRNA transcript levels were observed at 1.5 and 7.2 μg/L triclosan which were observed in stage-matched metamorphic climax animals.2 This observation was regarded as not biologically significant by the authors.2 Dismissing this observation as not biologically relevant due to the absence of a linear concentration response relationship disregards well-precedented hormone response phenomena:3 TRβ mRNA perturbation can be transitory (but biologically important) and present itself as an apparent “inverted U” rather than a linear response at a given time point.5,6 Therefore, the observation of a perturbation in Fort et al.2 is consistent with the observed developmental acceleration. The supplemental data4 referred to in the comment was not peer reviewed and provided insufficient information in order to properly assess the assertions presented therein. However, the work has now been published in Toxicological Sciences7 allowing for a more thorough assessment of the data. The original publication stated in the abstract that “End points measured to evaluate effects on thyroid-mediated metamorphosis including developmental stage, thyroid histology, TRβ expression, DI-2 and DI-3 expression, and thyroid gland T4 (thyroxine) and plasma T4 and T3 (triiodothyronine) levels were not affected by TCS exposure.” However, it is evident in the manuscript that three of these thyroid end points did show evidence of thyroid axis disruption (thyroid gland hypertrophy, follicle cell height changes, and alterations of plasma T4 levels). A detailed commentary on these findings can be found in ref 8. Fort et al. now confirm in the accompanying comment that reduced plasma T4 levels occur upon triclosan exposure. r 2011 American Chemical Society
Stage-matching is essential for many end points that were measured in the Fort et al. study.7 It is noteworthy that Fort et al. comment on an inability to completely stage-match all specimens in that work. More details regarding this issue would permit a more complete evaluation of the original data. Caren C. Helbing, Ph.D.* Department of Biochemistry and Microbiology, University of Victoria
Jeremy E. Wulff, Ph.D. Department of Chemistry, University of Victoria
Caleb M. Bromba Department of Chemistry, University of Victoria
Ashley Hinther, M.Sc. Department of Biochemistry and Microbiology, University of Victoria
Nik Veldhoen, Ph.D. Department of Biochemistry and Microbiology, University of Victoria
’ AUTHOR INFORMATION Corresponding Author
*E-mail: chelbing@ uvic.ca.
’ REFERENCES (1) Hinther, A.; Bromba, C. M.; Wulff, J. E.; Helbing, C. C. Effects of triclocarban, triclosan, and methyl triclosan on thyroid hormone action and stress in frog and mammalian culture systems. Environ. Sci. Technol. 2011, 45, 5395–402. (2) Fort, D.; Rogers, R.; Gorsuch, J.; Navarro, L.; Peter, R.; Plautz, J. Triclosan and anuran metamorphosis: No effect on thyroid-mediated metamorphosis in Xenopus laevis. Toxicol. Sci. 2010, 113, 392–400. (3) Helbing, C. C.; Van Aggelen, G.; Veldhoen, N. Triclosan affects thyroid hormone-dependent metamorphosis in anurans. Toxicol. Sci. 2011, 119, 417–418. (4) Fort, D. J.; Rogers, R. L.; Pawlowski, S.; Champ, S. Triclosan does not affect thyroid-mediated metamorphosis in Xenopus laevis Additional data. Toxicol. Sci. 2011, 119, 419–422. (5) Zhang, F.; Degitz, S. J.; Holcombe, G. W.; Kosian, P. A.; Tietge, J.; Veldhoen, N.; Helbing, C. C. Evaluation of gene expression endpoints in the context of a Xenopus laevis metamorphosis-based bioassay to detect thyroid hormone disruptors. Aquat. Toxicol. 2006, 76, 24–36. (6) Veldhoen, N.; Skirrow, R.; Osachoff, H.; Wigmore, H.; Clapson, D.; Gunderson, M.; van Aggelen, G.; Helbing, C. The bactericidal agent Triclosan modulates thyroid hormone-associated gene expression and Published: July 21, 2011 7600
dx.doi.org/10.1021/es202358r | Environ. Sci. Technol. 2011, 45, 7600–7601
Environmental Science & Technology
CORRESPONDENCE/REBUTTAL
disrupts postembryonic anuran development. Aquat. Toxicol. 2006, 80, 217–227. (7) Fort, D. J.; Mathis, M. B.; Hanson, W.; Fort, C. E.; Navarro, L. T.; Peter, R.; Buche, 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) Helbing, C. C.; Propper, C.; Veldhoen, N. Triclosan affects the thyroid axis of amphibians. Toxicol. Sci. 2011 (doi:10.1093/toxsci/ kfr127).
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dx.doi.org/10.1021/es202358r |Environ. Sci. Technol. 2011, 45, 7600–7601
