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The associations between serum perfluorinated chemicals and thyroid function in adolescents and young adults
Journal of Hazardous Materials 244–245 (2013) 637–644
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The associations between serum perfluorinated chemicals and thyroid function in adolescents and young adults Chien-Yu Lin a,b , Li-Li Wen c , Lian-Yu Lin d , Ting-Wen Wen e , Guang-Wen Lien e , Sandy H.J. Hsu f , Kuo-Liong Chien g , Chien-Chang Liao h , Fung-Chang Sung i , Pau-Chung Chen e,j,k,∗∗ , Ta-Chen Su d,∗ a
Department of Internal Medicine, En Chu Kong Hospital, New Taipei City 237, Taiwan School of Medicine, Fu Jen Catholic University, Taipei County 242, Taiwan c Department of Clinical Laboratory, En Chu Kong Hospital, New Taipei City 237, Taiwan d Department of Internal Medicine, National Taiwan University Hospital, Taipei 100, Taiwan e Institute of Occupational Medicine and Industrial Hygiene, College of Public Health, National Taiwan University, Taipei 100, Taiwan f Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan g Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei 100, Taiwan h Department of Anesthesiology, Taipei Medical University Hospital, Taipei, Taiwan i Institute of Environmental Health, College of Public Health, China Medical University, Taichung 404, Taiwan j Department of Public Health, College of Public Health, National Taiwan University, Taipei 100, Taiwan k Department of Environmental and Occupational Medicine, National Taiwan University College of Medicine and National Taiwan University Hospital, Taipei, Taiwan b
h i g h l i g h t s We determine the association between PFCs and thyroid function in a cohort. The concentrations of PFNA associated with free T4 significantly. The association was greater in male aged 20–30, active smokers and higher BMI.
a r t i c l e
i n f o
Article history: Received 14 August 2012 Received in revised form 25 October 2012 Accepted 26 October 2012 Available online 2 November 2012 Keywords: PFC PFNA Thyroid Thyroxine Humans
a b s t r a c t Perfluorinated chemicals (PFCs) have been widely used in a variety of products worldwide for years. However, the effect of PFCs on thyroid function has not yet been clearly defined. We recruited 567 subjects (aged 12–30 years) in a population-based cohort of adolescents and young adults with abnormal urinalysis in the childhood to determine the relationship between serum level of PFCs and the levels of serum free thyroxine (T4) and thyroid stimulating hormone (TSH). The geometric means and geometric standard deviation concentrations of perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorononanoic acid (PFNA) and perfluoroundecanoic acid (PFUA) were 2.67 (2.96) ng/ml, 7.78 (2.42) ng/ml, 1.01 (3.48) ng/ml and 5.81 (2.92) ng/ml, respectively. Differences in the levels of free T4 and TSH across different categories of PFOA, PFOS and PFUA were insignificant. After controlling for confounding factors, multiple linear regression analyses revealed mean serum level of free T4 increased significantly across categories (<60th, 60–89 and >90th percentiles) of PFNA (P for trend =0.012 in the full model). The association between PFNA and free T4 was more significant in male subjects in age group 20–30, active smokers and in those with higher body mass index in stratified analysis. Serum concentrations of PFNA were associated with serum free T4 levels in adolescents and young adults. © 2012 Elsevier B.V. All rights reserved.
Abbreviations: BMI, body mass index; BP, blood pressure; EBP, elevated blood pressure; ETS, environmental tobacco smoke; NHANES, National Health and Nutrition Examination Survey; NTD, New Taiwan dollar; PFCs, perfluorinated chemicals; PFHxS, perfluorohexane sulfonic acid; PFNA, perfluorononanoic acid; PFOA, perfluorooctanoic acid; PFOS, perfluorooctane sulfonate; PFUA, perfluoroundecanoic acid; TSH, thyroid stimulating hormone; T4, free thyroxine; T3, triiodothyronine. ∗ Corresponding author at: Department of Internal Medicine, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei 10055, Taiwan. Tel.: +886 2 23123456x66719; fax: +886 2 23712361. ∗∗ Co-corresponding author at: Institute of Occupational Medicine and Industrial Hygiene, College of Public Health, National Taiwan University, Taipei 100, Taiwan. E-mail addresses: [email protected] (P.-C. Chen), [email protected](T.-C. Su). 0304-3894/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jhazmat.2012.10.049
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1. Introduction Perfluorinated chemicals (PFCs) are a class of highly stable man-made compounds composed of a variable length fluorinated carbon backbone and a carboxylate or sulfonate functional group. PFCs are uniquely hydrophobic, oleophobic and lipophobic, and because of this unusual character profile, they are widely used in industrial applications as surfactants and emulsifiers and in consumer products such as food packaging, non-stick pan coatings, fire-fighting foams, paper and textile coatings, and personal care products. Despite the production and use of PFCs for the past sixty years, concerns regarding environmental hazards related to these compounds arose only recent, and a growing body of literature is now building regarding human and wildlife exposure [1]. The half-life of serum elimination of PFCs in humans appears to be years. The longer the carbon chain length, the longer PFCs persists in the body. For example, perfluorobutane sulfonate (a 4carbon PFC) is eliminated, on average, in a little over one month in humans [2], while perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) (8-carbon compounds, referred to as C8 compounds) are eliminated in 3.5 (95% CI, 3.0–4.1) and 4.8 (95% CI, 4.0–5.8) years, respectively. However, perfluorohexane sulfonic acid (PFHxS), a 6-carbon compound, is an exception to the rule as it is eliminated in 7.3 years (95% CI, 5.8–9.2) [3]. Half-life of nine-carbon perfluorononanoic acid (PFNA) in humans has not been estimated. As the manufacturing emission of PFOA and PFOS dropped, PFNA demonstrated an obvious increase in concentrations in the environment and in the tissues of humans and wildlife [4,5]. Toxicological studies in rats and mice have shown that repeated dosing with PFOS at high concentrations of ppm level suppressed serum thyroxine (T4) and triiodothyronine (T3) [4]. Reduced T4 and T3 were also found in monkeys treated with PFOA [5]. However, the concomitant increase in thyroid stimulating hormone (TSH) that would be expected through feedback stimulation was not found. These findings are consistent with the hypothesis advanced by Gutshall et al. [6], suggesting that PFCs may act by displacing thyroid hormones from their binding proteins in circulation. In contrast to PFOS and PFOA, although there was no report about PFNA exposure and thyroid hormones in mammals, a recent study demonstrated significantly elevated plasma T3 levels in zebrafish after long-term PFNA exposure [7]. In humans, the causal biochemical mechanisms of thyroid toxicity after exposure to PFCs are not clearly defined. In the occupational population, several studies have reported an association between exposure to PFOA and thyroid function. One study detected no association [8] while another found a weak negative association with free thyroxine (FT4) and positive association for T3 [9]. Regarding studies on the population surrounding a chemical plant that was exposed to PFOA at levels greater than in the general population (the C8 Health Project), the results showed PFOA and PFOS were associated with elevations in serum total T4 and a significant reduction in T3 uptake in adults and positive associations of PFOS and PFNA with total T4 and of PFOA and hypothyroidism in children [10,11]. In the general population, one study indicated only serum perfluorotridecanoic acid was negatively correlated with total T4 and positively with TSH levels after measuring for 13 PFCs [12]. In Inuit adults, PFOS concentrations were negatively associated with TSH, T3, and thyroxine-binding globulin and positively with FT4 concentrations [13]. Another study in pregnant women showed no association of PFOA, PFOS and PFHxS with thyroid functions [14]. A recent cross sectional analysis of self-reported thyroid disease in the U.S. National Health and Nutrition Examination Survey adults population reported a significant association for PFOA in females, and a similar but much less significant association in males, but no association for PFOS [15].
Since the results of published epidemiological studies are not consistent, have mostly focused on PFOS and PFOA, mostly used total T4 instead of FT4 as an outcome, mostly enrolled adults as the study subjects. The aim of the current study was to screen for potential associations between serum concentrations of 12 measured PFCs with thyroid function in a subsample of community-based adolescents and young adults in Taiwan previously sampled in a mass urine screening. We used FT4 and TSH as outcomes since FT4 represent immediately available hormone, of which is thought to yield more comprehensive information concerning the thyroid regulatory system than total T4 [16]. 2. Materials and methods 2.1. Participants and study design From 1992 to 2000, the Chinese Foundation of Health in Taipei, Taiwan, conducted an annual urine screening of approximately 2,615,000 to 2,932,000 school age children in grades 1–12 in Taiwan. A urine strip was used for the screening. School-age children with positive results on two tests for proteinuria, glucosuria or hematuria underwent a third urine screening test and a general health check-up with the same protocol. The check-up included anthropometric measures, fasting blood tests and blood pressure (BP). A total of 103,756 school children received the health checkups and the third urine screen and also had complete data profiles after detailed data checking. Among these children, 9227 had elevated blood pressure (EBP) and 94,529 had normal BP based on the American Heart Association criteria [17]. This campaign has been detailed in a previous report [18]. From 2006 to 2008 we established a cohort, the Young Taiwanese Cardiovascular Cohort Study, based on these students with and without childhood EBP selected from the 1992–2000 mass urine screening population. In the follow-up, we mailed invitation letters to eligible students in the Taipei area. After 3–5 days, 12 trained assistants and nurses conducted telephone interviews inviting these subjects with childhood EBP to come in for a followup health examination. No telephone interview contact was made with normotensive students. Among the 707 subjects with EBP in childhood, 303 completed the follow-up health examinations, for a response rate of 42.9%. Among the 6390 subjects with normal BP in childhood, 487 completed the follow-up health examinations, for a response rate of 7.6%. The detailed information is available in recent reports [19,20]. This study was approved by the ethics committee of the National Taiwan University Hospital (Research Ethics Committee, NTUH). Informed consent was obtained by writing from each participant and their parents (for children and adolescents participants) when they joined the cohort follow-up study. Because of limited serum samples, we did not measure serum PFCs or thyroid function in all 790 subjects. Of the potential subjects, 144 were eliminated due to lack of sufficient serum sample volume for serum tests for PFCs while another 95 subjects were eliminated due to the same reason that prevented the measurement of thyroid functions. A detailed flow chart of the selection process is shown in Fig. 1. Finally, 551 participants were enrolled for final analysis. Among the 551 subjects for the final analysis, 214 subjects were male and 337 subjects were female while 510 subjects were normotensive and 41 subjects were hypertensive. 2.2. Anthropometric and biochemical data Sociodemographic information such as age, gender, history of medication and household income was recorded during the interview. The degree of alcohol intake was determined by questionnaire and categorized into current alcohol consumption and no alcohol drinking. Smoking status was subdivided into active
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Fig. 1. Flowchart of recruitment of participants in the Young Taiwanese Cardiovascular Cohort Study. 790 subjects with previous mean age of 12.5 year-old adolescents were recruited to NTUH after a mean of 9 years later for during 2006–2008.
smoker, passive smoker or has never smoked. Household income was categorized as either above 50,000 New Taiwan Dollars (NTD) (equivalent to 1600 USD) per month or below. Weight and height were measured by standard methods. The body mass index (BMI) was calculated as weight (in kg) divided by the square of height (in m). Two seated blood pressure and heart rate measurements were made at least 1 min apart after 3 min of rest by using a mercury manometer and the appropriate cuff size. Hypertension was defined as self-reported current use of anti-hypertensive medication or an average BP greater than 140/90 mmHg. Childhood EBP was defined as systolic blood pressure, diastolic blood pressure, or both greater than or equal to the modified sex- and agespecific criteria for blood pressure values [17]. Diabetes mellitus was defined as a fasting serum glucose >126 mg/dl or self-reported current use of oral hypoglycemic agents or insulin. We use sensitive TSH assays and FT4 as markers of thyroid function tests because these tests has simplified the interpretation of thyroid function tests [16]. Serum TSH and FT4 were measured by immunoluminometric assay in an architect random access assay system (Abbott Diagnostics, Abbott Park, IL). Inter-assay coefficient of variation for FT4 and TSH were 1.37% and 2.09%, respectively. Inter-assay coefficient of variation for FT4 and TSH were 4.51% and 3.34%, respectively. The normal range of FT4 is 0.7–1.48 ng/dl while TSH is 0.35–4.94 m IU/l in this study. Estimated glomerular filtration rate (eGFR) was estimated by using the simplified modification of diet in renal disease study equation for subjects aged ≥20 [21] and using Bedside Schwartz equation for subjects aged < 20 [22], chronic renal failure was defined as eGFR below 60 ml/min/1.73 m2 . 2.3. Measurement of PFCs concentration The plasma samples were stored at −80 ◦ C before analysis. Twelve PFCs were analyzed in our study. However, in eight, over 70% of the PFCs were below the limits of quantitation. Therefore, we used plasma samples of PFOA, PFOS, PFNA and
perfluoroundecanoic acid (PFUA) for analysis in this study. We analyzed plasma samples using a Waters ACQUITY UPLC system (Waters Corporation, Milford, MA) coupled with a Waters Quattro Premier XE triple quadrupole mass spectrometer (Waters Corporation, Milford, MA). Details of the analytical method have been described elsewhere [23]. Briefly, frozen samples were thawed at room temperature and then were vortexed for 30 s to ensure homogeneity. An aliquot of 100 l plasma sample was further vortexed for 30 s with 100 l of 1% formic acid (pH = 2.8). The solution was mixed with 80 l of methanol and 20 l of 0.375 ng/ml internal standard (13C8-PFOA) in methanol, and was sonicated for 20 min before being centrifuged at 14,000 rpm for 20 min. The collected supernatant (about 150 l) was then filtered through a 0.22 m PVDF syringe filter before the instrumental analysis. The calibration standard solutions were prepared in 100 l of bovine plasma and went through sample preparation under the same procedure; the concentrations of all analytes were equivalent to 0.05 in 300 ng/ml bovine plasma containing a fixed amount of internal standard (75 ng/ml). In our previous study, the background levels of the analytes in methanol, reagent and bovine plasma blank were examined. The existing backgrounds of PFOA, PFNA and PFUA in these blank samples were found and their levels were less than 1.5, 0.75 and 3.0 ng/ml, respectively. Because we observed similar signal intensities of these three analytes in the three types of blanks, the possible sources of contamination could be polytetrafluoroethylene and perfluoroalkoxy components originated from the LC system instead of labware [23]. Consequently, the reported concentrations of PFOA, PFNA and PFUA had subtracted from them the background levels found in the blank of the batch. The backgrounds of these chemicals did not significantly influence the quantification of these compounds in plasma samples. The limits of quantitation for PFOA, PFOS, PFNA and PFUA were 1.5, 0.22, 0.75 and 1.5 ng/ml, respectively. For concentrations below the detection limits (38.8% for PFOA, 1.6% for PFOS, 58.3% for PFNA
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and 24.9% for PFUA), a value half the detection limit was used. All laboratory analyses were conducted by investigators blinded to the characteristics of study subjects. 2.4. Statistical analysis SPSS for Windows (version 16.0, SPSS Inc., Chicago, IL) was used for all statistical analyses. PFC concentrations were expressed as geometric mean and geometrics standard deviation. The relation of PFC variables to categorical variables was tested using the Mann–Whitney U test or Kruskal–Wallis test (for three or more groups). Since there were high percentages of concentrations below the detection limits for PFOA, PFNA and PFUA (38.8% for PFOA, 1.6% for PFOS, 58.3% for PFNA and 24.9% for PFUA), we divided each PFC into different categories for linear regression models, cut-off values of PFOA were set at the 50th (the reference category), 75th, and 90th percentiles; cut-off values of PFOS and PFUA were set at the 25th (the reference category), 50th and 75th, percentiles; cut-off values of PFNA were set at the 60th (the reference category), and 90th percentiles. Analyses were conducted using linear regression with TSH and FT4 as the outcome. TSH was log-normally distributed and thus be natural log-transformed in regression analyses. The subjects were calculated separately for male and female in two age groups: 12–19 and 20–30. For linear regression models, we used age, gender, lifestyle factors (smoking status and drinking status) as covariates to adjust for potential confounders. Both hypo- and hyperthyroidism can affect blood pressure [24,25]. Thyroid functions may also have influence on BMI, glucose homeostasis [26], rather than vice versa. We supposed that neither BMI, glucose nor blood pressure is a confounder and adjusting for these covariates may result in overadjustment. Each PFC was modeled separately. In
logistic regression, we used the same model as confounders to test exposures to PFCs related to the odds ratio of being hypothyroid (TSH above normal range). Since the subjects’ metabolic function, obesity status, and renal function might affect the metabolism and accumulation of the studied lipophilic compounds and our previous reports showed the effects of PFOA may vary in obese subjects [27], we calculated the regressions stratified by BMI, smoking status and hypertension in Table 4. Diabetes Mellitus and chronic renal failure were not calculated due to limited cases (n = 12 and 18, respectively). We also conducted formal tests of interaction (done by including cross-product terms in models). We also measured thyroid functions across categories of four PFCs by stratifying two groups (normal vs. elevated blood pressure in the childhood) in linear regression analyses, since these two subpopulations were quite different with such varying participation rates. 3. Results The demographic characteristics of the sample population are outlined in Table 1. The study sample consisted of 214 males and 337 females. Among the 551 study subjects in the manuscript, 221 (40.1%) subjects had EBP in the childhood, but only 41 (7.4%) subjects currently have hypertension and only 12 (2.2%) subjects have diabetes mellitus and 18 (3.3%) have chronic renal failure. The geometric means and geometric standard deviation of concentrations of PFOA, PFOS, PFNA and PFUA were 2.67 (2.96) ng/ml, 7.78 (2.42) ng/ml, 1.01 (3.48) ng/ml and 5.81 (2.92) ng/ml, respectively. Males had a higher average concentration of PFOS than females (P < 0.001). Subjects older than 20 years had a higher median serum concentration of PFOS than younger ones (P = 0.008). Subjects who were active smokers had higher median serum concentrations
Table 1 Basic demographics of the sample subjects including geometric mean (geometric SD) of PFC concentrations.
Total Sex Male Female Age (years) 12–19 20–30 Household income <50000NTD per month ≥50000NTD per month Smoking status Never smoked Passive smoker Active smoker Current alcohol consumption No Yes Body mass index (kg/m2 ) <24 ≥24 Chronic renal failure No Yes Current hypertension Yes No Diabetes mellitus Yes No
No.
PFOA (ng/ml)
PFOS (ng/ml)
PFNA (ng/ml)
PFUA (ng/ml)
551
2.67 (2.96)
7.78 (2.42)
1.01 (3.48)
5.81 (2.92)
*
214 337
2.71 (2.94) 2.64 (2.98)
8.82 (2.60) 7.18 (2.29)*
1.14 (3.84) 0.93 (3.24)
6.06 (2.99) 5.65 (2.88)
212 339
2.80 (2.90) 2.59 (3.00)
7.04 (2.38)** 8.28 (2.44)**
0.91 (3.34) 1.08 (3.56)
5.80 (2.98) 5.81 (2.90)
228 322
2.60 (3.02) 2.71 (2.93)
7.53 (2.41) 7.95 (2.43)
0.95 (3.45) 1.05 (3.50)
5.81 (2.98) 5.78 (2.89)
464 16 65
2.69 (2.99) 2.43 (2.81) 2.71 (2.79)
7.66 (2.43) 9.94 (1.73) 8.31 (2.44)
0.95 (3.38)∞ 1.39 (4.25)∞ 1.42 (3.86)∞
5.70 (2.90) 5.60 (3.53) 6.65 (2.97)
496 54
2.64 (2.99) 2.82 (2.73)
7.70 (2.40) 8.51 (2.66)
0.98 (3.43) 1.29 (3.90)
5.72 (2.93) 6.50 (2.91)
414 137
2.79 (1.08) 2.34 (3.01)
7.42 (2.43)# 8.95 (2.37)#
1.01 (3.49) 1.01 (3.47)
5.76 (2.92) 5.96 (2.94)
553 18
2.66 (2.96) 3.04 (3.05)
7.91 (2.34) 4.70 (4.55)
1.01 (3.48) 0.99 (3.81)
5.68 (2.92)## 11.07 (2.64)##
41 510
2.29 (3.28) 2.70 (2.94)
7.25 (3.36) 7.82 (2.35)
1.06 (3.41) 1.01 (3.49)
5.82 (3.15) 5.81 (2.91)
12 539
2.63 (2.61) 2.67 (2.97)
5.93 (4.27) 7.82 (2.38)
0.63 (2.33) 1.02 (3.50)
6.17 (2.42) 5.80 (2.94)
PFC, perfluorinated chemicals; PFOA, perfluorooctanoic acid; PFOS, perfluorooctane sulfonate; PFNA, perfluorononanoic acid; PFUA, perfluoroundecanoic acid; NTD, new Taiwan dollars. Chronic renal failure was defined as eGFR < 60 ml/min/m2 . * Males had a higher average concentration of PFOS than females (P < 0.001). ** Subjects older than 20 years had a higher mean serum concentration of PFOS than younger ones (P = 0.008). ∞ Active smokers had higher mean serum concentrations of PFNA than passive smokers or non-smokers (P = 0.031). # PFOS concentrations were higher in those with higher BMI (P = 0.004). ## PFUA concentrations were higher in those with chronic renal failure (P = 0.014).
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Table 2 Mean and standard error of FT4 across categories of serum PFC levels in linear regression models (n = 545). Free T4 (ng/dl) Total (n = 545)
Male 12–19 (n = 65)
Female 12–19 (n = 144)
Male 20–30 (n = 144)
Female 20–30 (n = 192)
PFOA (ng/ml) <3.64 (<50th) ≤6.66 (50th–75th) ≤9.71 (75th–90th) >9.71 (>90th)
1.07 (0.01) 1.08 (0.02) 1.10 (0.02) 1.06 (0.02)
1.03 (0.06) 1.01 (0.07) 1.08 (0.07) 1.00 (0.08)
1.04 (0.04) 1.05 (0.04) 1.02 (0.04) 1.03 (0.05)
1.11 (0.02) 1.13 (0.03) 1.15 (0.03) 1.05 (0.04)
1.08 (0.03) 1.09 (0.03) 1.15 (0.04) 1.11 (0.04)
PFOS (ng/ml) <5.47 (<25th) ≤8.62 (25th–50th) ≤13.14 (50th–75th) >13.14 (>75th)
1.07 (0.02) 1.09 (0.02) 1.07 (0.02) 1.09 (0.02)
1.04 (0.07) 1.07 (0.07) 1.04 (0.06) 1.02 (0.06)
1.05 (0.04) 1.06 (0.04) 1.03 (0.04) 1.09 (0.04)
1.10 (0.03) 1.08 (0.03) 1.13 (0.03) 1.14 (0.02)
1.07 (0.03) 1.11 (0.03) 1.07 (0.03) 1.09 (0.03)
PFNA (ng/ml) <1.2 (<60th) ≤6.46 (60th–90th) >6.46 (>90th)
1.07 (0.01)* 1.06 (0.02)* 1.12 (0.02)*
1.04 (0.06) 1.01 (0.06) 1.09 (0.07)
1.05 (0.04) 1.04 (0.04) 1.11 (0.05)
1.11 (0.02)* 1.07 (0.03)* 1.17 (0.03)*
1.08 (0.03) 1.10 (0.03) 1.12 (0.04)
PFUA (ng/ml) <1.50 (<25th) ≤6.46 (25th–50th) ≤13.91 (50th–75th) >13.91 (>75th)
1.08 (0.02) 1.07 (0.02) 1.07 (0.02) 1.09 (0.02)
1.08 (0.06) 1.03 (0.07) 0.99 (0.06) 1.03 (0.06)
1.05 (0.04) 1.04 (0.04) 1.04 (0.04) 1.04 (0.04)
1.10 (0.03) 1.12 (0.03) 1.12 (0.03) 1.13 (0.03)
1.09 (0.33) 1.07 (0.03) 1.07 (0.03) 1.10 (0.03)
Model: adjusted for age, gender, and lifestyle factors (smoking status, drinking status). PFC, perfluorinated chemicals; T4, free thyroxine; PFOA, perfluorooctanoic acid; PFOS, perfluorooctane sulfonate; PFNA, perfluorononanoic acid; PFUA, perfluoroundecanoic acid. * P for trend < 0.05.
of PFNA than passive smokers or non-smokers (P = 0.031). In addition, PFOS concentrations were higher in those with higher BMI (P = 0.004) and PFUA concentrations were higher in those with chronic renal failure (P = 0.014). In this 551 study subjects, 18 subjects are hypothyroid (TSH above normal range) while 5 subjects are hyperthyroid (TSH below normal range). Moreover, only 2 subjects are hyperthyroxinemic (Free T4 above normal range) and no subject is hypothyroxemic (Free T4 below normal range). No differences were found between the exposures to PFCs related to the odds ratio of being hypothyroid in logistic regression while the number of participants with other thyroid dysfunction is insufficient to conduct such an analysis (see Supplementary Table S1). Not all four PFCs were correlated with one another (see Supplementary Table S2). Of these, PFNA and PFUA were most strongly correlated, with a Spearman correlation coefficient of 0.51 (P < 0.001).
The potential association of FT4 across different categories of serum levels of PFOA, PFOS, PFNA and PFUA after adjustment for other potential covariates is listed in Table 2. Mean levels (95% CI) of FT4 increased significantly across categories of PFNA (1.07 (1.04–1.10) ng/ml; 1.06 (1.03–1.09) ng/ml, 1.12 (1.08–1.16) ng/ml; P for trend = 0.012). There was a significant positive association between serum PFNA and FT4 in male in age group 20–30, with mean levels (95% CI) of FT4 increased significantly across categories of PFNA (1.11 (1.07–1.15) ng/ml; 1.07 (1.02–1.12) ng/ml, 1.17 (1.11–1.22) ng/ml; P for trend = 0.017). Concentrations of PFOA, PFOS and PFUA were not associated with levels of FT4. Concentrations of all four PFCs were not associated with levels of TSH (see Table 3). PFNA was also associated with FT4 when continuous exposure metric was used while concentrations of other three PFCs were not associated with levels of FT4 or TSH when expressed
Table 3 Mean and standard error of natural log transformed TSH across categories of serum PFC levels in linear regression models (n = 545). log TSH (m IU/l) Total (n = 545)
Male 12–19 (n = 65)
Female 12–19 (n = 144)
Male 20–30 (n = 144)
Female 20–30 (n = 192)
PFOA (ng/ml) <3.64 (<50th) ≤6.66 (50th–75th) ≤9.71 (75th–90th) >9.71 (>90th)
0.48 (0.08) 0.45 (0.09) 0.36 (0.11) 0.41 (0.12)
0.35 (0.26) 0.19 (0.31) 0.24 (0.31) −0.15 (0.33)
0.46 (0.18) 0.54 (0.18) 0.38 (0.20) 0.11 (0.24)
0.45 (0.08) 0.63 (0.11) 0.57 (0.12) 0.54 (0.15)
0.30 (0.24) 0.05 (0.25) −0.03 (0.31) 0.38 (0.31)
PFOS (ng/ml) <5.47 (<25th) ≤8.62 (25th–50th) ≤13.14 (50th–75th) >13.14 (>75th)
0.45 (0.09) 0.41 (0.09) 0.57 (0.09) 0.37 (0.09)
0.27 (0.30) 0.18 (0.32) 0.42 (0.28) 0.27c(0.29)
0.52 (0.19) 0.36 (0.19) 0.54 (0.18) 0.40 (0.22)
0.44 (0.12) 0.50 (0.11) 0.64 (0.10) 0.49 (0.09)
0.24 (0.26) 0.20 (0.25) 0.35 (0.27) 0.01 (0.26)
PFNA (ng/ml) <1.2 (<60th) ≤6.46 (60th–90th) >6.46 (>90th)
0.43 (0.08) 0.40 (0.09) 0.58 (0.12)
0.32 (0.27) 0.33 (0.28) 0.43 (0.32)
0.53 (0.18) 0.39 (0.19) 0.61 (0.27)
0.54 (0.08) 0.41 (0.10) 0.57 (0.11)
0.15 (0.24) 0.20 (0.25) 0.48 (0.35)
PFUA (ng/ml) <1.50 (<25th) ≤6.46 (25th–50th) ≤13.91 (50th–75th) >13.91 (>75th)
0.51 (0.09) 0.45 (0.10) 0.45 (0.09) 0.39 (0.09)
0.39 (0.30) 0.24 (0.31) 0.31 (0.30) 0.31 (0.28)
0.54 (0.19) 0.59 (0.20) 0.47 (0.19) 0.37 (0.19)
0.51 (0.10) 0.44 (0.11) 0.50 (0.10) 0.59 (0.10)
0.29 (0.26) 0.20 (0.27) 0.26 (0.27) 0.10 (0.25)
Model: adjusted for age, gender, and lifestyle factors (smoking status, drinking status). PFC, perfluorinated chemicals; TSH, thyroid stimulating hormone; PFOA, perfluorooctanoic acid; PFOS, perfluorooctane sulfonate; PFNA, perfluorononanoic acid; PFUA, perfluoroundecanoic acid.
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Table 4 Mean and standard error of free T4 (ng/dl) across different serum levels of PFNA in subpopulations of the sample subjects. No. Body mass index (kg/m2 ) <24 ≥24* Smoking status Never smoked Has smoked* Current hypertension Yes* No*
<60th
60th–89th
≥90th
410 135
1.06 (0.02) 1.11 (0.03)*
1.07 (0.02) 1.06 (0.03)*
1.11 (0.02) 1.16 (0.04)*
464 81
1.09 (0.02) 1.08 (0.03)
1.10 (0.02) 1.04 (0.03)
1.13 (0.02) 1.16 (0.04)
40 505
1.23 (0.08) 1.06 (0.01)
1.09 (0.07) 1.06 (0.02)
1.19 (0.08) 1.12 (0.02)
Model: adjusted for age, gender, and lifestyle factors (smoking status, drinking status). T4, free thyroxine; PFNA, perfluorononanoic acid. eGFR: estimated glomerular filtration rate. * P for trend < 0.05.
continuously (see Supplementary Table S3). Considering that the concentrations of four PFCs were correlated with each other, all PFCs were forced into the model. In this analysis, entering all four PFCs into a single disease model did not alter the results. The result was shown in Supplementary Table S4. Mean and standard error of serum FT4 with categories of PFNA in the different subpopulations of the sample subjects are shown in Table 4. The association between FT4 and PFNA was significant for active smokers and those with higher BMI but the tests of interaction are insignificant. The association between FT4 and PFOA, PFOS and PFUA for all subgroups was insignificant, so did the association between TSH and PFNA (see Supplementary Table S5–S8). Mean and standard error of serum FT4 and TSH across categories of PFCs in the two subpopulations (normal vs. elevated blood pressure in the childhood) are shown in Supplementary Table S9, mean levels of FT4 increased significantly across categories of PFNA in both groups. 4. Discussion To our knowledge, our report is the first to link serum PFNA levels to thyroid functions in a cohort survey. In this study, we found that increased serum PFNA concentrations are associated with elevated serum FT4. However, the association is small and subclinical. Our analysis showed that the serum PFOA, PFNA and PFUA concentrations were not different between adolescents and young adults. Unlike other lipophilic persistent pollutants that display increasing serum concentrations as individuals age, the lack of this general trend in PFOA, PFNA and PFUA could be explained by intrauterine transfer, exposure early in life with ongoing exposures being much higher than earlier historical exposures, or a combination of these factors [28]. We report a median concentration of PFUA of 6.95 ng/ml in men and 6.31 ng/ml in women in this study. Our finding is compatible with another study which analyzed the levels of perfluorinated chemicals in umbilical cord blood in Taiwan [23]. These concentrations are 10 times higher than a previous report in the biomonitoring literature for PFUA, although levels of PFOA, PFOS and PFNA were comparable to other reports [5]. High PFUA concentration may be unique to Taiwan. One study investigated the influence of the semiconductor and electronics industries on PFC contamination in receiving rivers in Taiwan. PFUA was detected but not determined to be the major PFC in wastewater [29]. The source of exposure is not clear and needs further investigation. PFNA is used as a surfactant in the production of fluoropolymer polyvinylidene fluoride. Since the manufacturing emission of PFOA and PFOS dropped, concerns about the possible health impact of PFNA exposure have been raised. One animal study demonstrated
that, like the 8-carbon PFOA, the 9-carbon PFNA is a developmental toxicant and an immune system toxicant [30]. However, longer chain perfluorinated carboxylic acids are considered more bioaccumulative and toxic [31]. One recent animal study reported the first evidence of the thyroid-disrupting effect of long-term PFNA exposure in zebrafish. In that study, Zebrafish were exposed to different concentrations of PFNA (0, 0.05, 0.1, 0.5, and 1 mg/l) from their early life stages, and the exposure period lasted for 180 days. Significantly elevated plasma T3 levels were observed in both F0 and F1 adults, as well as PFNA-induced histological changes in the thyroid follicles of F0 male zebrafish. The authors found that PFNA exposure triggered a hyperstimulation of thyroid hormone synthesis/secretion. Moreover, the inhibited expression of sulfate conjugation and the glucuronide conjugation pathway lead to reduced biliary elimination of thyroid hormone, which may have accounted for the increased plasma thyroid hormone levels [7]. However, it is difficult to detect the same metabolic effect in the low exposure group of the general population and in the animal studies presented with a high concentration level. Recent studies have shown that environmental persistent organic pollutants can modify human epigenetic status such as global DNA methylation, probably via glutathione depletion. DNA methylation mediates transcription repression and, plays a critical role in development, differentiation and disease. Recently, prenatal exposure to PFOS has been associated with glutathione-Stransferase gene promoter hypermethylation in rats [32]. In utero PFOA exposure was inversely correlated with cord serum global DNA methylation in humans [33]. It is possible that the environment exposed amounts of PFNA induce thyroid toxicity by altering methylation profile as an epimutagen. However, there is no published study to investigate the association between PFNA and DNA methylation till now. In humans, a few studies have investigated the impact of PFNA exposure on thyroid function. One study investigated the serum concentrations of major PFCs among the general population in Korea, among the studied PFCs (including PFNA), they only found the concentrations of perfluorotridecanoic acid were negatively correlated with total T4, and positively with TSH levels [12]. Another study researched thyroid function and PFCs in children living near a chemical plant [11], interquartile range shifts in serum PFOS (15–28 ng/ml) and serum PFNA (1.2–2.0 ng/ml) were both associated with a 1.1% increase in total T4 in children 1–17 years old. Our study provides the first evidence of an association between serum PFNA levels and FT4 in adolescent and young adults. Serum PFNA from lowest to highest category (1.07–1.12 ng/ml) were associated with a 4.7% increase in FT4 in this study. In our study, we found elevated serum FT4 without the concomitant decrease in TSH that would be expected through feedback stimulation. One possible explanation is thyroid resistance, a syndrome of reduced end-organ responsiveness to thyroid hormone. The common causes of the syndrome including mutations of the  form of the thyroid hormone receptor, thyroid hormone cell transporter defect and thyroid hormone metabolism defect [34]. Another explanation is an autonomous TSH hypersecretion from pituitary gland. However, it is not clear PFNA may interfere in which step of hypothalmic-pituitary-thyroid axis. Moreover, we observed a trend that FT4 decreased going from the “<60” category to the “60th–89th” category. However, the pairwise comparisons between these two groups were insignificant. The non-monotonic relation between PFNA categories and outcomes has been found in our previous study [20], which is a common finding for other endocrine-active chemicals [35], for which high doses inhibit the low-dose response system while initiating a wide array of other adverse effects via different response mechanisms [36]. The possible explanation is that the dose–response effects of PFNA on FT4 may not be a linear relationship in humans. PFNA had no consistent or potentially relevant changes at a low level and but may start to exert its effects at a higher dose.
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In subgroup analysis, the association between FT4 and PFNA was significant in male subjects in age group 20–30. Other gender/age groups showed the same trend but were insignificant. Our previous studies have reported that the effects of PFNA on humans may vary in gender, age and obese subjects [20]. Other study has also found gender/PFCs interaction for thyroid functions [10]. One possible explanation is that the effect of PFNA on FT4 is much weaker than the effect of gender, age. When considering the thyroid toxic effect of PFNA in the above populations, the trend is too small to be statistically significant. Moreover, we also found the association between FT4 and PFNA was significant in those with higher BMI, and active smokers. Active smokers had higher mean serum concentrations of PFNA than passive smokers or non-smokers in our study. However, when we consider epidemiological matters, it is possible that selection bias, multiple comparisons, and chance may also contribute to the results. Our study has several limitations. First, the cross-sectional design does not permit causal inference due to time ordering. Second, our study population is composed of adolescents and young adults with abnormal urinalysis in the childhood and living in Taipei area. We cannot infer the same association in the general population. Third, we did not take into account all medications that may have impact on thyroid functions which will be a confounding variable. However, more than 95% of participants self-reported no significant clinical diseases and no medication history, particularly in thyroid disease. Fourth, unmeasured confounding factors (such as other environmental pollutants) may influence both serum PFCs and FT4 rather than exposure affecting outcome. Finally, the status of the thyroid tissue was not available to determine thyroid pathology. So we cannot further investigate the possible pathophysiology between PFNA and thyroid function. In conclusion, in a Taiwan adolescent and young adult population, we found that a higher serum concentration of PFNA was associated with elevated serum FT4. These findings hint at an effect for low-dose PFNA in humans, although the potential biological significance between PFNA and FT4 is small and subclinical in the Taiwan population. Further studies are needed to confirm these findings and to clarify whether these associations are causal. Conflict of interest The authors declare that they have no competing interests. Acknowledgments This study was supported by grants from National Health Research Institute of Taiwan (EX97-9721PC, EX97-9821PC, X979921PC, EX95-9531PI, EX95-9631PI and EX95-9731PI) and from National Science Council of Taiwan (99-2314-B-385-001-MY3). No funding organization or sponsor played any part in the design or conduct of the study; in the analysis or interpretation of the data; or in the preparation, review, or approval of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat. 2012.10.049. This material is available free of charge via the Internet at http://pubs.acs.org/. References [1] M. Houde, J.W. Martin, R.J. Letcher, K.R. Solomon, D.C. Muir, Biological monitoring of polyfluoroalkyl substances: a review, Environ. Sci. Technol. 40 (2006) 3463–3473.
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The associations between serum perfluorinated chemicals and thyroid function in adolescents and young adults Chien-Yu Lin a,b , Li-Li Wen c , Lian-Yu Lin d , Ting-Wen Wen e , Guang-Wen Lien e , Sandy H.J. Hsu f , Kuo-Liong Chien g , Chien-Chang Liao h , Fung-Chang Sung i , Pau-Chung Chen e,j,k,∗∗ , Ta-Chen Su d,∗ a
Department of Internal Medicine, En Chu Kong Hospital, New Taipei City 237, Taiwan School of Medicine, Fu Jen Catholic University, Taipei County 242, Taiwan c Department of Clinical Laboratory, En Chu Kong Hospital, New Taipei City 237, Taiwan d Department of Internal Medicine, National Taiwan University Hospital, Taipei 100, Taiwan e Institute of Occupational Medicine and Industrial Hygiene, College of Public Health, National Taiwan University, Taipei 100, Taiwan f Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan g Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei 100, Taiwan h Department of Anesthesiology, Taipei Medical University Hospital, Taipei, Taiwan i Institute of Environmental Health, College of Public Health, China Medical University, Taichung 404, Taiwan j Department of Public Health, College of Public Health, National Taiwan University, Taipei 100, Taiwan k Department of Environmental and Occupational Medicine, National Taiwan University College of Medicine and National Taiwan University Hospital, Taipei, Taiwan b
h i g h l i g h t s We determine the association between PFCs and thyroid function in a cohort. The concentrations of PFNA associated with free T4 significantly. The association was greater in male aged 20–30, active smokers and higher BMI.
a r t i c l e
i n f o
Article history: Received 14 August 2012 Received in revised form 25 October 2012 Accepted 26 October 2012 Available online 2 November 2012 Keywords: PFC PFNA Thyroid Thyroxine Humans
a b s t r a c t Perfluorinated chemicals (PFCs) have been widely used in a variety of products worldwide for years. However, the effect of PFCs on thyroid function has not yet been clearly defined. We recruited 567 subjects (aged 12–30 years) in a population-based cohort of adolescents and young adults with abnormal urinalysis in the childhood to determine the relationship between serum level of PFCs and the levels of serum free thyroxine (T4) and thyroid stimulating hormone (TSH). The geometric means and geometric standard deviation concentrations of perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorononanoic acid (PFNA) and perfluoroundecanoic acid (PFUA) were 2.67 (2.96) ng/ml, 7.78 (2.42) ng/ml, 1.01 (3.48) ng/ml and 5.81 (2.92) ng/ml, respectively. Differences in the levels of free T4 and TSH across different categories of PFOA, PFOS and PFUA were insignificant. After controlling for confounding factors, multiple linear regression analyses revealed mean serum level of free T4 increased significantly across categories (<60th, 60–89 and >90th percentiles) of PFNA (P for trend =0.012 in the full model). The association between PFNA and free T4 was more significant in male subjects in age group 20–30, active smokers and in those with higher body mass index in stratified analysis. Serum concentrations of PFNA were associated with serum free T4 levels in adolescents and young adults. © 2012 Elsevier B.V. All rights reserved.
Abbreviations: BMI, body mass index; BP, blood pressure; EBP, elevated blood pressure; ETS, environmental tobacco smoke; NHANES, National Health and Nutrition Examination Survey; NTD, New Taiwan dollar; PFCs, perfluorinated chemicals; PFHxS, perfluorohexane sulfonic acid; PFNA, perfluorononanoic acid; PFOA, perfluorooctanoic acid; PFOS, perfluorooctane sulfonate; PFUA, perfluoroundecanoic acid; TSH, thyroid stimulating hormone; T4, free thyroxine; T3, triiodothyronine. ∗ Corresponding author at: Department of Internal Medicine, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei 10055, Taiwan. Tel.: +886 2 23123456x66719; fax: +886 2 23712361. ∗∗ Co-corresponding author at: Institute of Occupational Medicine and Industrial Hygiene, College of Public Health, National Taiwan University, Taipei 100, Taiwan. E-mail addresses: [email protected] (P.-C. Chen), [email protected](T.-C. Su). 0304-3894/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jhazmat.2012.10.049
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1. Introduction Perfluorinated chemicals (PFCs) are a class of highly stable man-made compounds composed of a variable length fluorinated carbon backbone and a carboxylate or sulfonate functional group. PFCs are uniquely hydrophobic, oleophobic and lipophobic, and because of this unusual character profile, they are widely used in industrial applications as surfactants and emulsifiers and in consumer products such as food packaging, non-stick pan coatings, fire-fighting foams, paper and textile coatings, and personal care products. Despite the production and use of PFCs for the past sixty years, concerns regarding environmental hazards related to these compounds arose only recent, and a growing body of literature is now building regarding human and wildlife exposure [1]. The half-life of serum elimination of PFCs in humans appears to be years. The longer the carbon chain length, the longer PFCs persists in the body. For example, perfluorobutane sulfonate (a 4carbon PFC) is eliminated, on average, in a little over one month in humans [2], while perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) (8-carbon compounds, referred to as C8 compounds) are eliminated in 3.5 (95% CI, 3.0–4.1) and 4.8 (95% CI, 4.0–5.8) years, respectively. However, perfluorohexane sulfonic acid (PFHxS), a 6-carbon compound, is an exception to the rule as it is eliminated in 7.3 years (95% CI, 5.8–9.2) [3]. Half-life of nine-carbon perfluorononanoic acid (PFNA) in humans has not been estimated. As the manufacturing emission of PFOA and PFOS dropped, PFNA demonstrated an obvious increase in concentrations in the environment and in the tissues of humans and wildlife [4,5]. Toxicological studies in rats and mice have shown that repeated dosing with PFOS at high concentrations of ppm level suppressed serum thyroxine (T4) and triiodothyronine (T3) [4]. Reduced T4 and T3 were also found in monkeys treated with PFOA [5]. However, the concomitant increase in thyroid stimulating hormone (TSH) that would be expected through feedback stimulation was not found. These findings are consistent with the hypothesis advanced by Gutshall et al. [6], suggesting that PFCs may act by displacing thyroid hormones from their binding proteins in circulation. In contrast to PFOS and PFOA, although there was no report about PFNA exposure and thyroid hormones in mammals, a recent study demonstrated significantly elevated plasma T3 levels in zebrafish after long-term PFNA exposure [7]. In humans, the causal biochemical mechanisms of thyroid toxicity after exposure to PFCs are not clearly defined. In the occupational population, several studies have reported an association between exposure to PFOA and thyroid function. One study detected no association [8] while another found a weak negative association with free thyroxine (FT4) and positive association for T3 [9]. Regarding studies on the population surrounding a chemical plant that was exposed to PFOA at levels greater than in the general population (the C8 Health Project), the results showed PFOA and PFOS were associated with elevations in serum total T4 and a significant reduction in T3 uptake in adults and positive associations of PFOS and PFNA with total T4 and of PFOA and hypothyroidism in children [10,11]. In the general population, one study indicated only serum perfluorotridecanoic acid was negatively correlated with total T4 and positively with TSH levels after measuring for 13 PFCs [12]. In Inuit adults, PFOS concentrations were negatively associated with TSH, T3, and thyroxine-binding globulin and positively with FT4 concentrations [13]. Another study in pregnant women showed no association of PFOA, PFOS and PFHxS with thyroid functions [14]. A recent cross sectional analysis of self-reported thyroid disease in the U.S. National Health and Nutrition Examination Survey adults population reported a significant association for PFOA in females, and a similar but much less significant association in males, but no association for PFOS [15].
Since the results of published epidemiological studies are not consistent, have mostly focused on PFOS and PFOA, mostly used total T4 instead of FT4 as an outcome, mostly enrolled adults as the study subjects. The aim of the current study was to screen for potential associations between serum concentrations of 12 measured PFCs with thyroid function in a subsample of community-based adolescents and young adults in Taiwan previously sampled in a mass urine screening. We used FT4 and TSH as outcomes since FT4 represent immediately available hormone, of which is thought to yield more comprehensive information concerning the thyroid regulatory system than total T4 [16]. 2. Materials and methods 2.1. Participants and study design From 1992 to 2000, the Chinese Foundation of Health in Taipei, Taiwan, conducted an annual urine screening of approximately 2,615,000 to 2,932,000 school age children in grades 1–12 in Taiwan. A urine strip was used for the screening. School-age children with positive results on two tests for proteinuria, glucosuria or hematuria underwent a third urine screening test and a general health check-up with the same protocol. The check-up included anthropometric measures, fasting blood tests and blood pressure (BP). A total of 103,756 school children received the health checkups and the third urine screen and also had complete data profiles after detailed data checking. Among these children, 9227 had elevated blood pressure (EBP) and 94,529 had normal BP based on the American Heart Association criteria [17]. This campaign has been detailed in a previous report [18]. From 2006 to 2008 we established a cohort, the Young Taiwanese Cardiovascular Cohort Study, based on these students with and without childhood EBP selected from the 1992–2000 mass urine screening population. In the follow-up, we mailed invitation letters to eligible students in the Taipei area. After 3–5 days, 12 trained assistants and nurses conducted telephone interviews inviting these subjects with childhood EBP to come in for a followup health examination. No telephone interview contact was made with normotensive students. Among the 707 subjects with EBP in childhood, 303 completed the follow-up health examinations, for a response rate of 42.9%. Among the 6390 subjects with normal BP in childhood, 487 completed the follow-up health examinations, for a response rate of 7.6%. The detailed information is available in recent reports [19,20]. This study was approved by the ethics committee of the National Taiwan University Hospital (Research Ethics Committee, NTUH). Informed consent was obtained by writing from each participant and their parents (for children and adolescents participants) when they joined the cohort follow-up study. Because of limited serum samples, we did not measure serum PFCs or thyroid function in all 790 subjects. Of the potential subjects, 144 were eliminated due to lack of sufficient serum sample volume for serum tests for PFCs while another 95 subjects were eliminated due to the same reason that prevented the measurement of thyroid functions. A detailed flow chart of the selection process is shown in Fig. 1. Finally, 551 participants were enrolled for final analysis. Among the 551 subjects for the final analysis, 214 subjects were male and 337 subjects were female while 510 subjects were normotensive and 41 subjects were hypertensive. 2.2. Anthropometric and biochemical data Sociodemographic information such as age, gender, history of medication and household income was recorded during the interview. The degree of alcohol intake was determined by questionnaire and categorized into current alcohol consumption and no alcohol drinking. Smoking status was subdivided into active
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Fig. 1. Flowchart of recruitment of participants in the Young Taiwanese Cardiovascular Cohort Study. 790 subjects with previous mean age of 12.5 year-old adolescents were recruited to NTUH after a mean of 9 years later for during 2006–2008.
smoker, passive smoker or has never smoked. Household income was categorized as either above 50,000 New Taiwan Dollars (NTD) (equivalent to 1600 USD) per month or below. Weight and height were measured by standard methods. The body mass index (BMI) was calculated as weight (in kg) divided by the square of height (in m). Two seated blood pressure and heart rate measurements were made at least 1 min apart after 3 min of rest by using a mercury manometer and the appropriate cuff size. Hypertension was defined as self-reported current use of anti-hypertensive medication or an average BP greater than 140/90 mmHg. Childhood EBP was defined as systolic blood pressure, diastolic blood pressure, or both greater than or equal to the modified sex- and agespecific criteria for blood pressure values [17]. Diabetes mellitus was defined as a fasting serum glucose >126 mg/dl or self-reported current use of oral hypoglycemic agents or insulin. We use sensitive TSH assays and FT4 as markers of thyroid function tests because these tests has simplified the interpretation of thyroid function tests [16]. Serum TSH and FT4 were measured by immunoluminometric assay in an architect random access assay system (Abbott Diagnostics, Abbott Park, IL). Inter-assay coefficient of variation for FT4 and TSH were 1.37% and 2.09%, respectively. Inter-assay coefficient of variation for FT4 and TSH were 4.51% and 3.34%, respectively. The normal range of FT4 is 0.7–1.48 ng/dl while TSH is 0.35–4.94 m IU/l in this study. Estimated glomerular filtration rate (eGFR) was estimated by using the simplified modification of diet in renal disease study equation for subjects aged ≥20 [21] and using Bedside Schwartz equation for subjects aged < 20 [22], chronic renal failure was defined as eGFR below 60 ml/min/1.73 m2 . 2.3. Measurement of PFCs concentration The plasma samples were stored at −80 ◦ C before analysis. Twelve PFCs were analyzed in our study. However, in eight, over 70% of the PFCs were below the limits of quantitation. Therefore, we used plasma samples of PFOA, PFOS, PFNA and
perfluoroundecanoic acid (PFUA) for analysis in this study. We analyzed plasma samples using a Waters ACQUITY UPLC system (Waters Corporation, Milford, MA) coupled with a Waters Quattro Premier XE triple quadrupole mass spectrometer (Waters Corporation, Milford, MA). Details of the analytical method have been described elsewhere [23]. Briefly, frozen samples were thawed at room temperature and then were vortexed for 30 s to ensure homogeneity. An aliquot of 100 l plasma sample was further vortexed for 30 s with 100 l of 1% formic acid (pH = 2.8). The solution was mixed with 80 l of methanol and 20 l of 0.375 ng/ml internal standard (13C8-PFOA) in methanol, and was sonicated for 20 min before being centrifuged at 14,000 rpm for 20 min. The collected supernatant (about 150 l) was then filtered through a 0.22 m PVDF syringe filter before the instrumental analysis. The calibration standard solutions were prepared in 100 l of bovine plasma and went through sample preparation under the same procedure; the concentrations of all analytes were equivalent to 0.05 in 300 ng/ml bovine plasma containing a fixed amount of internal standard (75 ng/ml). In our previous study, the background levels of the analytes in methanol, reagent and bovine plasma blank were examined. The existing backgrounds of PFOA, PFNA and PFUA in these blank samples were found and their levels were less than 1.5, 0.75 and 3.0 ng/ml, respectively. Because we observed similar signal intensities of these three analytes in the three types of blanks, the possible sources of contamination could be polytetrafluoroethylene and perfluoroalkoxy components originated from the LC system instead of labware [23]. Consequently, the reported concentrations of PFOA, PFNA and PFUA had subtracted from them the background levels found in the blank of the batch. The backgrounds of these chemicals did not significantly influence the quantification of these compounds in plasma samples. The limits of quantitation for PFOA, PFOS, PFNA and PFUA were 1.5, 0.22, 0.75 and 1.5 ng/ml, respectively. For concentrations below the detection limits (38.8% for PFOA, 1.6% for PFOS, 58.3% for PFNA
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and 24.9% for PFUA), a value half the detection limit was used. All laboratory analyses were conducted by investigators blinded to the characteristics of study subjects. 2.4. Statistical analysis SPSS for Windows (version 16.0, SPSS Inc., Chicago, IL) was used for all statistical analyses. PFC concentrations were expressed as geometric mean and geometrics standard deviation. The relation of PFC variables to categorical variables was tested using the Mann–Whitney U test or Kruskal–Wallis test (for three or more groups). Since there were high percentages of concentrations below the detection limits for PFOA, PFNA and PFUA (38.8% for PFOA, 1.6% for PFOS, 58.3% for PFNA and 24.9% for PFUA), we divided each PFC into different categories for linear regression models, cut-off values of PFOA were set at the 50th (the reference category), 75th, and 90th percentiles; cut-off values of PFOS and PFUA were set at the 25th (the reference category), 50th and 75th, percentiles; cut-off values of PFNA were set at the 60th (the reference category), and 90th percentiles. Analyses were conducted using linear regression with TSH and FT4 as the outcome. TSH was log-normally distributed and thus be natural log-transformed in regression analyses. The subjects were calculated separately for male and female in two age groups: 12–19 and 20–30. For linear regression models, we used age, gender, lifestyle factors (smoking status and drinking status) as covariates to adjust for potential confounders. Both hypo- and hyperthyroidism can affect blood pressure [24,25]. Thyroid functions may also have influence on BMI, glucose homeostasis [26], rather than vice versa. We supposed that neither BMI, glucose nor blood pressure is a confounder and adjusting for these covariates may result in overadjustment. Each PFC was modeled separately. In
logistic regression, we used the same model as confounders to test exposures to PFCs related to the odds ratio of being hypothyroid (TSH above normal range). Since the subjects’ metabolic function, obesity status, and renal function might affect the metabolism and accumulation of the studied lipophilic compounds and our previous reports showed the effects of PFOA may vary in obese subjects [27], we calculated the regressions stratified by BMI, smoking status and hypertension in Table 4. Diabetes Mellitus and chronic renal failure were not calculated due to limited cases (n = 12 and 18, respectively). We also conducted formal tests of interaction (done by including cross-product terms in models). We also measured thyroid functions across categories of four PFCs by stratifying two groups (normal vs. elevated blood pressure in the childhood) in linear regression analyses, since these two subpopulations were quite different with such varying participation rates. 3. Results The demographic characteristics of the sample population are outlined in Table 1. The study sample consisted of 214 males and 337 females. Among the 551 study subjects in the manuscript, 221 (40.1%) subjects had EBP in the childhood, but only 41 (7.4%) subjects currently have hypertension and only 12 (2.2%) subjects have diabetes mellitus and 18 (3.3%) have chronic renal failure. The geometric means and geometric standard deviation of concentrations of PFOA, PFOS, PFNA and PFUA were 2.67 (2.96) ng/ml, 7.78 (2.42) ng/ml, 1.01 (3.48) ng/ml and 5.81 (2.92) ng/ml, respectively. Males had a higher average concentration of PFOS than females (P < 0.001). Subjects older than 20 years had a higher median serum concentration of PFOS than younger ones (P = 0.008). Subjects who were active smokers had higher median serum concentrations
Table 1 Basic demographics of the sample subjects including geometric mean (geometric SD) of PFC concentrations.
Total Sex Male Female Age (years) 12–19 20–30 Household income <50000NTD per month ≥50000NTD per month Smoking status Never smoked Passive smoker Active smoker Current alcohol consumption No Yes Body mass index (kg/m2 ) <24 ≥24 Chronic renal failure No Yes Current hypertension Yes No Diabetes mellitus Yes No
No.
PFOA (ng/ml)
PFOS (ng/ml)
PFNA (ng/ml)
PFUA (ng/ml)
551
2.67 (2.96)
7.78 (2.42)
1.01 (3.48)
5.81 (2.92)
*
214 337
2.71 (2.94) 2.64 (2.98)
8.82 (2.60) 7.18 (2.29)*
1.14 (3.84) 0.93 (3.24)
6.06 (2.99) 5.65 (2.88)
212 339
2.80 (2.90) 2.59 (3.00)
7.04 (2.38)** 8.28 (2.44)**
0.91 (3.34) 1.08 (3.56)
5.80 (2.98) 5.81 (2.90)
228 322
2.60 (3.02) 2.71 (2.93)
7.53 (2.41) 7.95 (2.43)
0.95 (3.45) 1.05 (3.50)
5.81 (2.98) 5.78 (2.89)
464 16 65
2.69 (2.99) 2.43 (2.81) 2.71 (2.79)
7.66 (2.43) 9.94 (1.73) 8.31 (2.44)
0.95 (3.38)∞ 1.39 (4.25)∞ 1.42 (3.86)∞
5.70 (2.90) 5.60 (3.53) 6.65 (2.97)
496 54
2.64 (2.99) 2.82 (2.73)
7.70 (2.40) 8.51 (2.66)
0.98 (3.43) 1.29 (3.90)
5.72 (2.93) 6.50 (2.91)
414 137
2.79 (1.08) 2.34 (3.01)
7.42 (2.43)# 8.95 (2.37)#
1.01 (3.49) 1.01 (3.47)
5.76 (2.92) 5.96 (2.94)
553 18
2.66 (2.96) 3.04 (3.05)
7.91 (2.34) 4.70 (4.55)
1.01 (3.48) 0.99 (3.81)
5.68 (2.92)## 11.07 (2.64)##
41 510
2.29 (3.28) 2.70 (2.94)
7.25 (3.36) 7.82 (2.35)
1.06 (3.41) 1.01 (3.49)
5.82 (3.15) 5.81 (2.91)
12 539
2.63 (2.61) 2.67 (2.97)
5.93 (4.27) 7.82 (2.38)
0.63 (2.33) 1.02 (3.50)
6.17 (2.42) 5.80 (2.94)
PFC, perfluorinated chemicals; PFOA, perfluorooctanoic acid; PFOS, perfluorooctane sulfonate; PFNA, perfluorononanoic acid; PFUA, perfluoroundecanoic acid; NTD, new Taiwan dollars. Chronic renal failure was defined as eGFR < 60 ml/min/m2 . * Males had a higher average concentration of PFOS than females (P < 0.001). ** Subjects older than 20 years had a higher mean serum concentration of PFOS than younger ones (P = 0.008). ∞ Active smokers had higher mean serum concentrations of PFNA than passive smokers or non-smokers (P = 0.031). # PFOS concentrations were higher in those with higher BMI (P = 0.004). ## PFUA concentrations were higher in those with chronic renal failure (P = 0.014).
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Table 2 Mean and standard error of FT4 across categories of serum PFC levels in linear regression models (n = 545). Free T4 (ng/dl) Total (n = 545)
Male 12–19 (n = 65)
Female 12–19 (n = 144)
Male 20–30 (n = 144)
Female 20–30 (n = 192)
PFOA (ng/ml) <3.64 (<50th) ≤6.66 (50th–75th) ≤9.71 (75th–90th) >9.71 (>90th)
1.07 (0.01) 1.08 (0.02) 1.10 (0.02) 1.06 (0.02)
1.03 (0.06) 1.01 (0.07) 1.08 (0.07) 1.00 (0.08)
1.04 (0.04) 1.05 (0.04) 1.02 (0.04) 1.03 (0.05)
1.11 (0.02) 1.13 (0.03) 1.15 (0.03) 1.05 (0.04)
1.08 (0.03) 1.09 (0.03) 1.15 (0.04) 1.11 (0.04)
PFOS (ng/ml) <5.47 (<25th) ≤8.62 (25th–50th) ≤13.14 (50th–75th) >13.14 (>75th)
1.07 (0.02) 1.09 (0.02) 1.07 (0.02) 1.09 (0.02)
1.04 (0.07) 1.07 (0.07) 1.04 (0.06) 1.02 (0.06)
1.05 (0.04) 1.06 (0.04) 1.03 (0.04) 1.09 (0.04)
1.10 (0.03) 1.08 (0.03) 1.13 (0.03) 1.14 (0.02)
1.07 (0.03) 1.11 (0.03) 1.07 (0.03) 1.09 (0.03)
PFNA (ng/ml) <1.2 (<60th) ≤6.46 (60th–90th) >6.46 (>90th)
1.07 (0.01)* 1.06 (0.02)* 1.12 (0.02)*
1.04 (0.06) 1.01 (0.06) 1.09 (0.07)
1.05 (0.04) 1.04 (0.04) 1.11 (0.05)
1.11 (0.02)* 1.07 (0.03)* 1.17 (0.03)*
1.08 (0.03) 1.10 (0.03) 1.12 (0.04)
PFUA (ng/ml) <1.50 (<25th) ≤6.46 (25th–50th) ≤13.91 (50th–75th) >13.91 (>75th)
1.08 (0.02) 1.07 (0.02) 1.07 (0.02) 1.09 (0.02)
1.08 (0.06) 1.03 (0.07) 0.99 (0.06) 1.03 (0.06)
1.05 (0.04) 1.04 (0.04) 1.04 (0.04) 1.04 (0.04)
1.10 (0.03) 1.12 (0.03) 1.12 (0.03) 1.13 (0.03)
1.09 (0.33) 1.07 (0.03) 1.07 (0.03) 1.10 (0.03)
Model: adjusted for age, gender, and lifestyle factors (smoking status, drinking status). PFC, perfluorinated chemicals; T4, free thyroxine; PFOA, perfluorooctanoic acid; PFOS, perfluorooctane sulfonate; PFNA, perfluorononanoic acid; PFUA, perfluoroundecanoic acid. * P for trend < 0.05.
of PFNA than passive smokers or non-smokers (P = 0.031). In addition, PFOS concentrations were higher in those with higher BMI (P = 0.004) and PFUA concentrations were higher in those with chronic renal failure (P = 0.014). In this 551 study subjects, 18 subjects are hypothyroid (TSH above normal range) while 5 subjects are hyperthyroid (TSH below normal range). Moreover, only 2 subjects are hyperthyroxinemic (Free T4 above normal range) and no subject is hypothyroxemic (Free T4 below normal range). No differences were found between the exposures to PFCs related to the odds ratio of being hypothyroid in logistic regression while the number of participants with other thyroid dysfunction is insufficient to conduct such an analysis (see Supplementary Table S1). Not all four PFCs were correlated with one another (see Supplementary Table S2). Of these, PFNA and PFUA were most strongly correlated, with a Spearman correlation coefficient of 0.51 (P < 0.001).
The potential association of FT4 across different categories of serum levels of PFOA, PFOS, PFNA and PFUA after adjustment for other potential covariates is listed in Table 2. Mean levels (95% CI) of FT4 increased significantly across categories of PFNA (1.07 (1.04–1.10) ng/ml; 1.06 (1.03–1.09) ng/ml, 1.12 (1.08–1.16) ng/ml; P for trend = 0.012). There was a significant positive association between serum PFNA and FT4 in male in age group 20–30, with mean levels (95% CI) of FT4 increased significantly across categories of PFNA (1.11 (1.07–1.15) ng/ml; 1.07 (1.02–1.12) ng/ml, 1.17 (1.11–1.22) ng/ml; P for trend = 0.017). Concentrations of PFOA, PFOS and PFUA were not associated with levels of FT4. Concentrations of all four PFCs were not associated with levels of TSH (see Table 3). PFNA was also associated with FT4 when continuous exposure metric was used while concentrations of other three PFCs were not associated with levels of FT4 or TSH when expressed
Table 3 Mean and standard error of natural log transformed TSH across categories of serum PFC levels in linear regression models (n = 545). log TSH (m IU/l) Total (n = 545)
Male 12–19 (n = 65)
Female 12–19 (n = 144)
Male 20–30 (n = 144)
Female 20–30 (n = 192)
PFOA (ng/ml) <3.64 (<50th) ≤6.66 (50th–75th) ≤9.71 (75th–90th) >9.71 (>90th)
0.48 (0.08) 0.45 (0.09) 0.36 (0.11) 0.41 (0.12)
0.35 (0.26) 0.19 (0.31) 0.24 (0.31) −0.15 (0.33)
0.46 (0.18) 0.54 (0.18) 0.38 (0.20) 0.11 (0.24)
0.45 (0.08) 0.63 (0.11) 0.57 (0.12) 0.54 (0.15)
0.30 (0.24) 0.05 (0.25) −0.03 (0.31) 0.38 (0.31)
PFOS (ng/ml) <5.47 (<25th) ≤8.62 (25th–50th) ≤13.14 (50th–75th) >13.14 (>75th)
0.45 (0.09) 0.41 (0.09) 0.57 (0.09) 0.37 (0.09)
0.27 (0.30) 0.18 (0.32) 0.42 (0.28) 0.27c(0.29)
0.52 (0.19) 0.36 (0.19) 0.54 (0.18) 0.40 (0.22)
0.44 (0.12) 0.50 (0.11) 0.64 (0.10) 0.49 (0.09)
0.24 (0.26) 0.20 (0.25) 0.35 (0.27) 0.01 (0.26)
PFNA (ng/ml) <1.2 (<60th) ≤6.46 (60th–90th) >6.46 (>90th)
0.43 (0.08) 0.40 (0.09) 0.58 (0.12)
0.32 (0.27) 0.33 (0.28) 0.43 (0.32)
0.53 (0.18) 0.39 (0.19) 0.61 (0.27)
0.54 (0.08) 0.41 (0.10) 0.57 (0.11)
0.15 (0.24) 0.20 (0.25) 0.48 (0.35)
PFUA (ng/ml) <1.50 (<25th) ≤6.46 (25th–50th) ≤13.91 (50th–75th) >13.91 (>75th)
0.51 (0.09) 0.45 (0.10) 0.45 (0.09) 0.39 (0.09)
0.39 (0.30) 0.24 (0.31) 0.31 (0.30) 0.31 (0.28)
0.54 (0.19) 0.59 (0.20) 0.47 (0.19) 0.37 (0.19)
0.51 (0.10) 0.44 (0.11) 0.50 (0.10) 0.59 (0.10)
0.29 (0.26) 0.20 (0.27) 0.26 (0.27) 0.10 (0.25)
Model: adjusted for age, gender, and lifestyle factors (smoking status, drinking status). PFC, perfluorinated chemicals; TSH, thyroid stimulating hormone; PFOA, perfluorooctanoic acid; PFOS, perfluorooctane sulfonate; PFNA, perfluorononanoic acid; PFUA, perfluoroundecanoic acid.
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Table 4 Mean and standard error of free T4 (ng/dl) across different serum levels of PFNA in subpopulations of the sample subjects. No. Body mass index (kg/m2 ) <24 ≥24* Smoking status Never smoked Has smoked* Current hypertension Yes* No*
<60th
60th–89th
≥90th
410 135
1.06 (0.02) 1.11 (0.03)*
1.07 (0.02) 1.06 (0.03)*
1.11 (0.02) 1.16 (0.04)*
464 81
1.09 (0.02) 1.08 (0.03)
1.10 (0.02) 1.04 (0.03)
1.13 (0.02) 1.16 (0.04)
40 505
1.23 (0.08) 1.06 (0.01)
1.09 (0.07) 1.06 (0.02)
1.19 (0.08) 1.12 (0.02)
Model: adjusted for age, gender, and lifestyle factors (smoking status, drinking status). T4, free thyroxine; PFNA, perfluorononanoic acid. eGFR: estimated glomerular filtration rate. * P for trend < 0.05.
continuously (see Supplementary Table S3). Considering that the concentrations of four PFCs were correlated with each other, all PFCs were forced into the model. In this analysis, entering all four PFCs into a single disease model did not alter the results. The result was shown in Supplementary Table S4. Mean and standard error of serum FT4 with categories of PFNA in the different subpopulations of the sample subjects are shown in Table 4. The association between FT4 and PFNA was significant for active smokers and those with higher BMI but the tests of interaction are insignificant. The association between FT4 and PFOA, PFOS and PFUA for all subgroups was insignificant, so did the association between TSH and PFNA (see Supplementary Table S5–S8). Mean and standard error of serum FT4 and TSH across categories of PFCs in the two subpopulations (normal vs. elevated blood pressure in the childhood) are shown in Supplementary Table S9, mean levels of FT4 increased significantly across categories of PFNA in both groups. 4. Discussion To our knowledge, our report is the first to link serum PFNA levels to thyroid functions in a cohort survey. In this study, we found that increased serum PFNA concentrations are associated with elevated serum FT4. However, the association is small and subclinical. Our analysis showed that the serum PFOA, PFNA and PFUA concentrations were not different between adolescents and young adults. Unlike other lipophilic persistent pollutants that display increasing serum concentrations as individuals age, the lack of this general trend in PFOA, PFNA and PFUA could be explained by intrauterine transfer, exposure early in life with ongoing exposures being much higher than earlier historical exposures, or a combination of these factors [28]. We report a median concentration of PFUA of 6.95 ng/ml in men and 6.31 ng/ml in women in this study. Our finding is compatible with another study which analyzed the levels of perfluorinated chemicals in umbilical cord blood in Taiwan [23]. These concentrations are 10 times higher than a previous report in the biomonitoring literature for PFUA, although levels of PFOA, PFOS and PFNA were comparable to other reports [5]. High PFUA concentration may be unique to Taiwan. One study investigated the influence of the semiconductor and electronics industries on PFC contamination in receiving rivers in Taiwan. PFUA was detected but not determined to be the major PFC in wastewater [29]. The source of exposure is not clear and needs further investigation. PFNA is used as a surfactant in the production of fluoropolymer polyvinylidene fluoride. Since the manufacturing emission of PFOA and PFOS dropped, concerns about the possible health impact of PFNA exposure have been raised. One animal study demonstrated
that, like the 8-carbon PFOA, the 9-carbon PFNA is a developmental toxicant and an immune system toxicant [30]. However, longer chain perfluorinated carboxylic acids are considered more bioaccumulative and toxic [31]. One recent animal study reported the first evidence of the thyroid-disrupting effect of long-term PFNA exposure in zebrafish. In that study, Zebrafish were exposed to different concentrations of PFNA (0, 0.05, 0.1, 0.5, and 1 mg/l) from their early life stages, and the exposure period lasted for 180 days. Significantly elevated plasma T3 levels were observed in both F0 and F1 adults, as well as PFNA-induced histological changes in the thyroid follicles of F0 male zebrafish. The authors found that PFNA exposure triggered a hyperstimulation of thyroid hormone synthesis/secretion. Moreover, the inhibited expression of sulfate conjugation and the glucuronide conjugation pathway lead to reduced biliary elimination of thyroid hormone, which may have accounted for the increased plasma thyroid hormone levels [7]. However, it is difficult to detect the same metabolic effect in the low exposure group of the general population and in the animal studies presented with a high concentration level. Recent studies have shown that environmental persistent organic pollutants can modify human epigenetic status such as global DNA methylation, probably via glutathione depletion. DNA methylation mediates transcription repression and, plays a critical role in development, differentiation and disease. Recently, prenatal exposure to PFOS has been associated with glutathione-Stransferase gene promoter hypermethylation in rats [32]. In utero PFOA exposure was inversely correlated with cord serum global DNA methylation in humans [33]. It is possible that the environment exposed amounts of PFNA induce thyroid toxicity by altering methylation profile as an epimutagen. However, there is no published study to investigate the association between PFNA and DNA methylation till now. In humans, a few studies have investigated the impact of PFNA exposure on thyroid function. One study investigated the serum concentrations of major PFCs among the general population in Korea, among the studied PFCs (including PFNA), they only found the concentrations of perfluorotridecanoic acid were negatively correlated with total T4, and positively with TSH levels [12]. Another study researched thyroid function and PFCs in children living near a chemical plant [11], interquartile range shifts in serum PFOS (15–28 ng/ml) and serum PFNA (1.2–2.0 ng/ml) were both associated with a 1.1% increase in total T4 in children 1–17 years old. Our study provides the first evidence of an association between serum PFNA levels and FT4 in adolescent and young adults. Serum PFNA from lowest to highest category (1.07–1.12 ng/ml) were associated with a 4.7% increase in FT4 in this study. In our study, we found elevated serum FT4 without the concomitant decrease in TSH that would be expected through feedback stimulation. One possible explanation is thyroid resistance, a syndrome of reduced end-organ responsiveness to thyroid hormone. The common causes of the syndrome including mutations of the  form of the thyroid hormone receptor, thyroid hormone cell transporter defect and thyroid hormone metabolism defect [34]. Another explanation is an autonomous TSH hypersecretion from pituitary gland. However, it is not clear PFNA may interfere in which step of hypothalmic-pituitary-thyroid axis. Moreover, we observed a trend that FT4 decreased going from the “<60” category to the “60th–89th” category. However, the pairwise comparisons between these two groups were insignificant. The non-monotonic relation between PFNA categories and outcomes has been found in our previous study [20], which is a common finding for other endocrine-active chemicals [35], for which high doses inhibit the low-dose response system while initiating a wide array of other adverse effects via different response mechanisms [36]. The possible explanation is that the dose–response effects of PFNA on FT4 may not be a linear relationship in humans. PFNA had no consistent or potentially relevant changes at a low level and but may start to exert its effects at a higher dose.
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In subgroup analysis, the association between FT4 and PFNA was significant in male subjects in age group 20–30. Other gender/age groups showed the same trend but were insignificant. Our previous studies have reported that the effects of PFNA on humans may vary in gender, age and obese subjects [20]. Other study has also found gender/PFCs interaction for thyroid functions [10]. One possible explanation is that the effect of PFNA on FT4 is much weaker than the effect of gender, age. When considering the thyroid toxic effect of PFNA in the above populations, the trend is too small to be statistically significant. Moreover, we also found the association between FT4 and PFNA was significant in those with higher BMI, and active smokers. Active smokers had higher mean serum concentrations of PFNA than passive smokers or non-smokers in our study. However, when we consider epidemiological matters, it is possible that selection bias, multiple comparisons, and chance may also contribute to the results. Our study has several limitations. First, the cross-sectional design does not permit causal inference due to time ordering. Second, our study population is composed of adolescents and young adults with abnormal urinalysis in the childhood and living in Taipei area. We cannot infer the same association in the general population. Third, we did not take into account all medications that may have impact on thyroid functions which will be a confounding variable. However, more than 95% of participants self-reported no significant clinical diseases and no medication history, particularly in thyroid disease. Fourth, unmeasured confounding factors (such as other environmental pollutants) may influence both serum PFCs and FT4 rather than exposure affecting outcome. Finally, the status of the thyroid tissue was not available to determine thyroid pathology. So we cannot further investigate the possible pathophysiology between PFNA and thyroid function. In conclusion, in a Taiwan adolescent and young adult population, we found that a higher serum concentration of PFNA was associated with elevated serum FT4. These findings hint at an effect for low-dose PFNA in humans, although the potential biological significance between PFNA and FT4 is small and subclinical in the Taiwan population. Further studies are needed to confirm these findings and to clarify whether these associations are causal. Conflict of interest The authors declare that they have no competing interests. Acknowledgments This study was supported by grants from National Health Research Institute of Taiwan (EX97-9721PC, EX97-9821PC, X979921PC, EX95-9531PI, EX95-9631PI and EX95-9731PI) and from National Science Council of Taiwan (99-2314-B-385-001-MY3). No funding organization or sponsor played any part in the design or conduct of the study; in the analysis or interpretation of the data; or in the preparation, review, or approval of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat. 2012.10.049. This material is available free of charge via the Internet at http://pubs.acs.org/. References [1] M. Houde, J.W. Martin, R.J. Letcher, K.R. Solomon, D.C. Muir, Biological monitoring of polyfluoroalkyl substances: a review, Environ. Sci. Technol. 40 (2006) 3463–3473.
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