Reply to 2nd Comment on “Effects of Triclocarban, Triclosan, And

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Reply to 2nd Comment on “Effects of Triclocarban, Triclosan, And Methyl Triclosan on Thyroid Hormone Action and Stress in Frog and Mammalian Culture Systems”

T

he letter to the editor by DeLeo et al. regarding our recent publication1 includes a number of misrepresentations that give the appearance of controversy over the findings. Both the cultured frog tail fin (C-fin) biopsy and the rodent GH3 cell assays were developed upon a solid scientific foundation dating back half a century. The C-fin assay is based upon Schaffer’s2 discovery that cultured tadpole tails maintain their responsiveness to thyroid hormone (TH). Tail culture has been used by many laboratories and shows demonstrated utility as a screen for the identification of TH disruptors (for example, refs 3
5). The C-fin assay, 6,7 first published in 2010, is a modification on this theme. The direct proliferative effect of THs on GH3 cells,8 developed in 1968,9 forms the basis of the T-screen published in 2005.10 GH3 cellbased assays have been employed to evaluate suspected TH disruptors by multiple laboratories with various modifications (for example, 11 and 12). The Amphibian Metamorphosis Assay (using Nieuwkoop and Faber13 (NF) stage 51 Xenopus laevis tadpoles and a 21-day exposure14) was used by Fort et al. to evaluate the effect of triclosan (TCS).15 This study was followed by a nonstandard modification of this assay that comprised a 32-day exposure beginning with NF stage 47 tadpoles.16 As indicated in previous correspondences, we disagree with the authors’ data interpretation and conclusions.17
19 The authors dismissed several instances where significant effects on thyroid axis end points were evident. Subsequent mTCS and TCC studies are not yet published in the peer-reviewed literature and, as such, the relevant data needed to evaluate the statements presented in DeLeo et al. are currently unavailable. The assertion that our study1 lacks positive or negative controls is incorrect. Extensive controls for all gene transcripts are in the publication. It is impossible to present fold-change data without first establishing baseline control levels. Changes in heat shock protein 30 (Hsp30) and catalase mRNA levels, are established indicators of cellular stress20,21 and were chosen because they also have a link to TH responsiveness.1 We measured mRNA levels because changes in transcript abundance comprise a sensitive biological end point associated with cellular stress. The statement: “a change at the transcriptional level does not necessarily correspond to a change in protein translation requiring proteomic investigation”, is moot since only the transcript level changes are germane to the study. DeLeo et al. quoted an excerpt from Hinther’s M.Sc. thesis followed by a statement that the ensuing commentary in the thesis “explains that these effects may actually be unrelated to TCS exposure, but result from the stress conditions of the assay”. This statement is incorrect and is misrepresentative of the actual thesis contents. Hinther establishes that stress induction could be a possible mechanism of TCS action r 2011 American Chemical Society

leading to the observed metamorphic effects. The thesis discussion following the highlighted quote provides welldocumented evidence from the literature that stress induction could contribute to accelerated metamorphosis. This is clearly different from stating that the observed effects may be unrelated to TCS exposure. A link between deiodinases and biotransformation of xenobiotics is puzzling in the context presented. THs are iodinated, while TCS is chlorinated. Deiodinases are incapable of metabolizing TCS and presenting the status of their mRNA transcripts is uninformative. DeLeo et al. cite unpublished data to argue that it is unlikely that amphibia can methylate xenobiotics. There are no published data to support or refute this claim although bioaccumulation of mTCS in fish has been reported.22
24 mTCS is believed to be produced through microbial methylation under aerobic conditions23
25 but the source of methylation remains unknown. The authors state: “the formation of mTCS even in bacteria is improbable from an energetic viewpoint since mTCS has a higher enthalpy than TCS”. There are several ways in which this statement is incorrect: (1) One cannot validly compare enthalpies of formation for molecules containing different numbers of atoms. The enthalpy of formation (ΔHf) for a molecule corresponds to the amount of energy that would be required to make that structure from the constituent atoms in their standard state.26 (2) In fact, an appropriately constructed isodesmic equation (eq 1, employing S-adenosyl methionine as the likely methyl group donor) reveals methylation of TCS to be thermodynamically favored by ca. 11 kJ/mol. For the purposes of this calculation, we used published calorimetry data for known methylation reactions27 and substituted the calculated enthalpies of formation for TCS and mTCS [calculated by four different methods: All calculations were performed with PM3, A., RM1 and MNDO methods. The final calculated ΔHrxn is taken from the average of all four methods, and was close to the value (
13 kJ/mol) obtained directly from PM3 calculations. The values for ΔHf shown in eq 1 are the PM3 values, since these are believed to be the most accurate] for the enthalpies of formation for the known substrates and products (calculated by the same four methods and validated by comparison with experimentally derived values28,29). Indeed, biological methylations of heteroatom-donors to provide neutral products are generally expected to be

Published: October 07, 2011 10285

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Environmental Science & Technology energetically favorable, since they relieve the positive charge in the sulfonium cofactor.30

(3) Living systems do not operate at an energetic minimum. Nature is able to deploy highly energetic molecules (e.g., ATP) with exquisite selectivity to accomplish any number of seemingly contra-thermodynamic chemical transformations. Consider the formation of glucose from carbon dioxide and water (eq 2). This reaction is disfavored by 2805 kJ/mol and yet it is readily accomplished through photosynthesis.

Our endeavors are geared toward augmenting the scientific knowledgebase through innovative and rigorous studies that are firmly grounded in known biological response paradigms. These methods have provided valuable information on the biological activity of TCS and other chemicals and it is possible that regulatory bodies may adopt our novel approaches in the future. However, acceptance or validation by an authoritative body is no replacement for well designed, executed, and interpreted scientific experiments. Our study presents an accurate report of our findings with careful, critical consideration of the available literature. Caren C. Helbing,†,* Ashley Hinther,† Jeremy E. Wulff,‡ Caleb M. Bromba,‡ and Nik Veldhoen† †

Department of Biochemistry and Microbiology, University of Victoria, PO Box 3055 Stn CSC, Victoria, B.C., Canada, V8W 3P6

‡

Department of Chemistry, University of Victoria, PO Box 3065 Stn CSC, Victoria, B.C., Canada, V8W 3 V6

’ AUTHOR INFORMATION Corresponding Author

*Phone: (250) 721-6146; fax: (250) 721-8855; e-mail: chelbing@ uvic.ca.

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(4) Schriks, M.; Zvinavashe, E.; Furlow, J. D.; Murk, A. J. Disruption of thyroid hormone-mediated Xenopus laevis tadpole tail tip regression by hexabromocyclododecane (HBCD) and 2,20 ,3,30 ,4,40 ,5,50 ,6-nona brominated diphenyl ether (BDE206). Chemosphere 2006, 65 (10), 1904–8. (5) Iwamuro, S.; Yamada, M.; Kato, M.; Kikuyama, S. Effects of bisphenol A on thyroid hormone-dependent up-regulation of thyroid hormone receptor alpha and beta and down-regulation of retinoid X receptor gamma in Xenopus tail culture. Life Sci 2006, 79 (23), 2165–71. (6) Hinther, A.; Domanski, D.; Vawda, S.; Helbing, C. C. C-fin: a cultured frog tadpole tail fin biopsy approach for detection of thyroid hormone-disrupting chemicals. Environ. Toxicol. Chem. 2010, 29 (2), 380–8. (7) Hinther, A.; Vawda, S.; Skirrow, R. C.; Veldhoen, N.; Collins, P.; Cullen, J. T.; van Aggelen, G.; Helbing, C. C. Nanometals induce stress and alter thyroid hormone action in amphibia at or below North American water quality guidelines. Environ. Sci. Technol. 2010, 44 (21), 8314–21. (8) Kirkland, W. L.; Sorrentino, J. M.; Sirbasku, D. A. Control of cell growth. III. Direct mitogenic effect of thyroid hormones on an estrogendependent rat pituitary tumor cell line. J Natl Cancer Inst 1976, 56 (6), 1159–64. (9) Tashjian, A. H., Jr.; Yasumura, Y.; Levine, L.; Sato, G. H.; Parker, M. L. Establishment of clonal strains of rat pituitary tumor cells that secrete growth hormone. Endocrinology 1968, 82 (2), 342–52. (10) Gutleb, A. C.; Meerts, I. A.; Bergsma, J. H.; Schriks, M.; Murk, A. J. T-Screen as a tool to identify thyroid hormone receptor active compounds. Environ. Toxicol. Pharmacol. 2005, 19 (2), 231–8. (11) Freitas, J.; Cano, P.; Craig-Veit, C.; Goodson, M. L.; Furlow, J. D.; Murk, A. J. Detection of thyroid hormone receptor disruptors by a novel stable in vitro reporter gene assay. Toxicol. In Vitro 2011, 25 (1), 257–66. (12) Taxvig, C.; Olesen, P. T.; Nellemann, C. Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay. Toxicol. Appl. Pharmacol. 2011, 250 (3), 263–9. (13) Nieuwkoop, P. D., Faber, F. Normal table of Xenopus laevis.; Garland Publishing,: New York, NY, 1994. (14) Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1100: Amphibian Metamorphosis (Frog), EPA 740-C-09-002; U.S. Environmental Protection Agency: Washington, DC, 2009. (15) Fort, D. J.; Rogers, R. L.; Gorsuch, J. W.; Navarro, L. T.; Peter, R.; Plautz, J. R. Triclosan and anuran metamorphosis: no effect on thyroid-mediated metamorphosis in Xenopus laevis. Toxicol. Sci. 2010, 113 (2), 392–400. (16) 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. (17) Helbing, C. C.; van Aggelen, G.; Veldhoen, N. Triclosan affects thyroid hormone-dependent metamorphosis in anurans. Toxicol. Sci. 2011, 119 (2), 417–8author reply 419
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