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Response to Comment on “Intake of Iodine and Perchlorate and Excretion in Human Milk Gibbs begins with a kind statement that we have expanded the extant knowledge base of perchlorate, thiocyanate, and iodide excretion into human milk. However, that compliment is short-lived; after reading these lengthy comments, the reader is left with the impression that our primary purpose in undertaking the study must have been to significantly misrepresent his work. It is true that in a manuscript of ∼5000 words, 3 figures and 2 large tables, exactly 6 sentences cited one or more of 3 references in which Dr. Gibbs was a (co)author. It is also true that one of these sentences bore the innocuous statement (throughout, italics and references in square brackets connote excerpts from the original manuscript, ref (1)): There is no universal agreement on the extent of threat posed by perchlorate in breast milk [20-23]. One should also note that of the three papers of Gibbs which we have allegedly maligned, one [22] is merely another set of similar comments by Gibbs et al. on one of our previous papers (2). While commenting may not be an ideal pastime, we note that Gibbs et al. have previously written other commentaries accusing others of mischaracterizations (3) and have even written commentaries on their own papers, in the latter case to provide further evidence that the characterization on their original papers was on the mark. It seems fair to point out that the “Perchlorate Study Group” (PSG), the sponsor of the Gibbs comments (and much of his previous work on perchlorate), despite its academic sounding name, is actually an organization of perchlorate manufacturers and users (5, 6). Other seemingly unbiased but concerned observers have questioned whether respectable journals should publish PSG sponsored studies (that always find perchlorate not to be of concern) because of conflict of interest (7). While we do not claim that all that the PSG and its subset “Council for Water Quality” has sponsored is necessarily erroneous, the reader should know that these organizations have a vested interest in attempting to show that perchlorate is harmless or that the effect of perchlorate is overshadowed by other iodine transport inhibitors. If one cannot be directly proven wrong, it would be a reasonable obfuscation strategy to claim that one has cited some PSG sponsored work that has been completely misrepresented and mischaracterized, whether because of one’s incompetence or deliberate intent.
interpretation of how that study was done is better known by Gibbs than us. In the paper, the text states that Figure 1 plots RAIU as a function of the molar concentration whereas Figure 1 has an abscissa label of “Micromolar concentration”. In Table 1 of Tonacchera et al., all of the different experiments are listed in detail; the lowest perchlorate concentration listed, contrary to the present assertion of Gibbs, is 0.1 µM. Gibbs claims that we have mischaracterized their work by stating that the Chinese Hamster Ovary (CHO) cell line that they used was nonhuman while in fact it was transfected with human NIS (hNIS). We are perfectly aware of the pioneering work done by Ajjan et al. (13) who performed the original experiments with CHO cell lines that express hNIS. The fact remains that even transfected with human NIS, CHO cells are still not human. In fact no human thyroid cell line has
Let us now examine the points Gibbs makes. First, he contends that we confuse inhibition of uptake as inhibition of transport. While it is true that Tonacchera et al. [5] studied the inhibition of radioactive iodine uptake (RAIU), the work of Dohan et al. [6] and Tran et al. (9) have since shown unequivocally that perchlorate not only inhibits iodide transport but it is also itself transported. In fact, long before the study of Tonacchera et al., the work of Wyngaarden et al. (10, 11) and Stanbury and Wyngaarden (12) established that among perchlorate, thiocyanate, and nitrate the first is not only the most powerful in inhibiting radioiodine uptake, it is capable of discharging radioiodine loaded into isolated thyroidal tissues (10, 11) or the thyroid glands of living human subjects (12). Occam’s razor will dictate that the only way such discharge can take place is if perchlorate is in fact transported into the thyroid. Second, Gibbs contends that we misinterpreted the lowest perchlorate concentration used by Tonacchera et al. in their study and it was 0.01 µM. It may be true that a proper 2656
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FIGURE 1. (a) Histogram of frequency distribution of iodide concentrations of breastmilk samples at intervals of 0.10 log I(µg/L) units. The solid line shows the best fit to a Gaussian distribution. (b) Histogram of frequency distribution of perchlorate concentrations of breastmilk samples at intervals of 0.10 log I(µg/L) units. The solid line shows the best fit to a Gaussian distribution. 10.1021/es900052a CCC: $40.75
2009 American Chemical Society
Published on Web 02/27/2009
functional NIS. The only relevant human cell lines that can be maintained in cell culture are thyroid cancer cells that do not express NIS and do not transport iodide (9). The salient point was that what we observe in real breastfeeding mothers is quantitatively different from what has been reported on the basis of isolated cell or animal studies. Even though other methods (14, 15) have reported a different selectivity order for the NIS (I- > ClO3- > SCN- > NO3- > Br-) we have never disputed that the results reported by Tonacchera et al. are concordant with results reported earlier. Gibbs should be happy that we chose his paper to represent what has been done before. We did not criticize the value of this study or the methodology and do not understand why he feels a need to defend his work here. Third, Gibbs contends that we should have included the greater residence time of thiocyanate relative to iodide or perchlorate in the body. Depending on the purpose of what the data are used for, this can be a valid argument. Unfortunately he forgets that if the greater residence time is taken into account, the same dose of a longer residence time analyte will in fact translate to a greater concentration of that analyte and the discrepancy between what we observe and the selectivity factors reported by Tonacchera et al. will increase even further. We have no reason to dispute that babies get more thiocyanate in breast milk than perchlorate, nor is there any sinister intent in the fact that infant I and SCN intake in Table 1 were reported as µg/d while perchlorate intake was reported as µg/kg/d. The latter was done at the suggestion of a reviewer so that the figure could be readily compared with the NAS reference dose of 0.7 µg/kg/d. But after this, Gibbs launches into a remarkable exercise of convoluted logics whereas one of the important finds in our study was the relative iodine uptake inhibition potencies of perchlorate and thiocyanate were different from those previously reported, he uses the same old numbers to carry out meaningless numerical manipulations to try to prove that once again, one should be more concerned about thiocyanate than perchlorate! Fourth, Gibbs contends that we do not recognize published effect of thiocyanate on breast milk and cites the Laurberg study [45] that we had referred to. We had never disputed that increased serum thiocyanate concentrations will result in decreased expression of iodide in breast milk. Gibbs should have quoted the allegedly offending sentence in its entirety: While thiocyanate and/or nitrate have been discussed as posing greater risk of low iodine uptake [7, 8] based on the selectivity factors for the NIS determined in vitro [5] and projected dietary intake amounts, the merit of this argument, with specific reference to breastfed infants may be doubtful. The keyword here is dietary intake. It has been the position of the PSG that dietary intake of thiocyanate is far greater than that of perchlorate and even after accounting for the iodine uptake inhibition potency (as per Tonacchera et al.), thiocyanate should be of far greater concern. This is the argument that we question. Unlike perchlorate which is neither bound nor transformed in vivo in humans, there is more than enough evidence that thiocyanate is both bound and transformed. Early work on rabbit plasma experiments had shown that 30-40% of the thiocyanate, over a large range of thiocyanate concentrations, is bound by plasma alone (16). We had also noted earlier that especially with reference to thiocyanate present in milk, its lactoperoxidase mediated oxidation products are powerful antibacterial agents [16-18] and can thus benefit infants. Finally, we note that urinary thiocyanate cannot be taken as a direct indication of the total dietary intake or serum concentration of thiocyanate as thiocyanate is also indigenously formed as a metabolic product. To resolve this question it behooves Gibbs and the PSG to show by controlled experiments that dietary thio-
cyanate is translated anywhere near-quantitatively to a corresponding increase in free serum thiocyanate concentration. Fifth, Gibbs asserts that we have been deluded by visual illusions and there is no statistical basis to believe that in real mothers perchlorate does inhibit the transport of iodine into milk. We indeed appreciate the opportunity to present a more detailed analysis. Also, it is true that quirks in the graphing software resulted in the first abscissa tick mark to remain unlabeled in Figure 3b whereas it should have been labeled 50. However, the same data were presented in linear form in Figure 3a and it would have not been extraordinarily difficult to decipher what the unlabeled tick should actually have been labeled. As to breaking axes, it is presently standard practice to minimize white space if the distribution of data so justifies: Neither is there any hidden agenda nor is an explanation required. We have never disputed that perchlorate and/or iodine concentrations may be log-normally distributed; log-normal distribution is common in nature. Indeed, when Gibbs raised this issue previously [22], we had responded (17): Even if iodide concentration was also log-normally distributed in terms of occurrence, how does this preclude iodide from having a linear or inverse linear relationship [with perchlorate]? Readers of this journal are well aware, for example, that the concentrations of many species, such as ammonium and sulfate, in atmospheric aerosols are lognormally distributed. It is also widely recognized that these two species are often highly linearly correlated. Indeed, Gibbs will hopefully be pleased to learn that if we exclude the data for Donor 13 (who had by far the highest milk iodine values, with three samples exceeding 1 mg/L and some of whose data could not be plotted in Figure 3 to minimize white space), the data for the remaining donors comprising 358 samples (for which both sets of analyses were available) do indeed reasonably fit a log-normal distribution pattern. Figure 1a and b represent the iodide and perchlorate distributions as histograms. The solid curve in each figure represents best fits to a normal distribution (the concentrations in Figure 1 are already in logarithmic form) obtained by MS Excel Solver (18), the uncertainties in the value of the mean location and the standard deviation were computed by the Solveraid macro (19). For iodine (Figure 1a), the distribution was centered at log (Iodine, µg/L) ) 1.6640 (with a standard deviation of ( 0.0207), corresponding to an iodine concentration of 46.14 µg/L. The standard deviation (σI) of this distribution was 0.4057 in log I (µg/L) units, with the 95% uncertainty spanning ( 0.0207. Similarly, for perchlorate (Figure 1b), the distribution was centered at log (PC, µg/L) ) 0.8425 (with a standard deviation of ( 0.0150), corresponding to a PC concentration of 6.96 µg/L. The standard deviation of this distribution was 0.3355 (σPC) in log PC (µg/L) units, with the 95% uncertainty spanning ( 0.0150. For PC values above the mean of the distribution (>6.96 µg/L PC), there were 174 samples, as would be expected (50% of the 358 samples above the mean and 50% below). If perchlorate and iodine were random, independent, variables with no connection, then according to probability distribution tables (20), of these 174 samples bearing >6.96 µg/L PC, 50.00, 30.85, and 15.87% (amounting respectively to 87, 54, and 28 samples), would have had iodine concentrations >46.14 (mean), > 73.60 (mean + 0.5 σI), and >117.4 (mean + σI) µg/L, respectively. If on the other hand, elevated levels of perchlorate depress iodine concentrations, the actual number of samples that meet these criteria will be smaller and the difference from the random predictions will increase with the increased iodine concentration strata (up to a point that the predicted numbers become too few). Compared to the expected 87, 54, and 28, the number of samples that met these criteria were 86, 49, and 17. We VOL. 43, NO. 7, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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therefore stand by our statement that...This continues to suggest that in real mothers perchlorate does inhibit the transport of iodine into milk.
Literature Cited (1) Kirk, A. B.; Martinelango, K.; Dutta, A.; Tian, K.; Smith, E. E.; Dasgupta, P. K. Perchlorate and iodide in dairy and breast milk. Environ. Sci. Technol. 2005, 39, 2011–2017. (2) Dasgupta, P. K.; Kirk, A. B.; Dyke, J. V.; Ohira, S.-I. Intake of Iodine and Perchlorate and Excretion in Human Milk. Environ. Sci. Technol. 2008, 42, 8115–8121. (3) Gibbs, J. P.; Engel, A.; Lamm, S. H. The NAS perchlorate review: Second-guessing the experts. Environ. Health Perspect. 2005, 113, A727–A728. (4) Gibbs, J. P.; Narayanan, L.; Mattie, D. R. Study among school children in Chile: Subsequent urine and serum perchlorate levels are consistent with perchlorate in water in Taltal. J. Occup. Environ. Med. 2004, 46, 516–517. (5) Sourcewatch. Perchlorate Study group; http://www.sourcewatch.org/index.php?title)Perchlorate_Study_Group; accessed December 22, 2008. (6) Madsen, T. Jahagirdar, S. The politics of rocket fuel pollution. The perchlorate study group and its industry backers; Environment California: Los Angeles, CA, 2006; https://www. policyarchive.org/handle/10207/5475; accessed December 22, 2008. (7) Shamon, M.; Bahumian, M. Perchlorate and the thyroid. Deconstructing a questionable industry-funded study; August, 2005; http://thyroid.about.com/od/toxictriggers/a/ perchlorate.htm. (8) Pleus R. EWG press release irresponsible and alarmist; http://archives.foodsafety.ksu.edu/fsnet/2003/9-2003/fsnet_ september_21.htm#DR. (9) Tran, N.; Valentin-Blasini, L.; Blount, B. C.; McCuistion, C. G.; Fenton, M. S.; Gin, E.; Salem, A.; Hershman, J. M. Thyroidstimulating hormone increases active transport of perchlorate into thyroid cells. Am. J. Physiol. Endocrinol. Metab. 2008, 294, E802–E806. (10) Wyngaarden, J. B.; Wright, B. M.; Ways, P. The effect of certain anions on the accumulation and retention of iodide by the thyroid gland. Endocrinology 1952, 50, 537–549. (11) Wyngaarden, J. B.; Stanbury, J. B.; Rapp, B. The effects of iodide, perchlorate, thiocyanate and nitrate administration upon the iodide concentrating mechanism of the rat thyroid. Endocrinology 1953, 52, 568–574.
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(12) Stanbury, J. B.; Wyngaarden, J. B. Effect of perchlorate on the human thyroid gland. Metabolism 1952, 1, 533–539. (13) Ajjan, R. A.; Findlay, C.; Metcalfe, R. A.; Watson, P. F.; Crisp, M.; Ludgate, M.; Weetman, A. P. J. Clin. Endocrinol. Metabol. 1998, 83, 1217–1221. (14) De La Vieja, A.; Dohan, O.; Levy, O.; Carrasco, N. Molecular analysis of the sodium/iodide symporter: impact on thyroid and extrathyroid pathophysiology. Physiol. Rev. 2000, 80, 1083–1105. (15) Eskandari, S.; Loo, D. D.; Dai, G.; Levy, O.; Wright, E. M.; Carrasco, N. Thyroid Na/I symporter. Mechanism, stoichiometry, specificity. J. Biol. Chem. 1997, 272, 27230–27238. (16) Pollay, M.; Stevens, A.; Davis, C., Jr. Determination of plasmathiocyanate binding and the Donnan ratio under simulated physiological conditions. Anal. Biochem. 1966, 17, 192–200. (17) Kirk, A. B.; Martinelango, P. K.; Kang, T.; Dutta, A.; Smith, E. E.; Dasgupta, P. K. Response to Comment on “Perchlorate and Iodide in Dairy and Breast Milk”. Environ. Sci. Technol. 2005, 39, 5202–5203. (18) Walsh, S.; Diamond, D. Nonlinear curve fitting using MS Excel Solver. Talanta 1995, 42, 561–572. (19) de Levie, R. Advanced Excel for Scientific Data Analysis; Oxford University Press: New York, 2004. (20) National Institutes of Standards and Technology. Engineering Statistics Handbook. Section 1.3.6.7.1. Cumulative distribution function of the standard normal distribution; http://www.itl. nist.gov/div898/handbook/eda/section3/eda3671.htm.
Purnendu K. Dasgupta, Andrea B. Kirk, and Shin-Ichi Ohira Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, Texas 76019-006
Jason V. Dyke Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061 ES900052A
