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Home , Perfluorooctanoic acid

Environmental Research 152 (2017) 157–164

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Environmental Research

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Circulating levels of perfluoroalkyl substances (PFASs) and carotid artery atherosclerosis crossmark ⁎ P. Monica Linda, , Samira Salihovicb,c, Bert van Bavelc, Lars Lindd a Department of Medical Sciences, Occupational and Environmental Medicine, Uppsala University Uppsala, Sweden b Department of Medical Sciences, Molecular and Science for Life Laboratory, Uppsala University, Uppsala, Sweden c MTM Research Centre, School of Science and Technology, Örebro University, Örebro, Sweden d Department of Medical Sciences, Cardiovascular Epidemiology, Uppsala University, Uppsala, Sweden

ARTICLE INFO ABSTRACT

Keywords: Background and objective: During recent years, some persistent organic pollutants (POPs) have been linked to Atherosclerosis atherosclerosis. One group of POPs, the poly- and perfluoroalkyl substances (PFASs) have not been investigated Atherosclerotic plaques with regard to atherosclerotic plaques. fl Poly- and per uoroalkyl substances (PFASs) Methods: Carotid artery atherosclerosis was assessed by ultrasound in 1016 subjects aged 70 years in the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study. Eight PFASs were detected in > 75% of participants' plasma by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Results: No significant linear associations were observed between the PFASs and intima-media thickness (IMT), or the echogenicity in the intima-media complex (IM-GSM, a marker of lipid infiltration in the artery) when men and women were analyzed together. Neither was occurrence of carotid plaques related to PFASs levels. However, highly significant interactions were observed between some PFASs and sex regarding both IM- GSM and plaque prevalence. Perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), and perfluor- oundecanoic acid (PFUnDA), were all related to IM-GSM in a positive fashion in women (p=0.002–0.003), while these relationships were negative in men. The levels of PFUnDA were significantly related to carotid plaque in women (OR 1.59, 95%CI 1.03–2.43, p=0.03), but not in men (OR 0.93, 95%CI 0.62–1.42, p=0.75). Conclusions: In this cross-sectional study, a pronounced gender difference was observed regarding associations between some PFASs, especially the long-chain PFUnDA, and markers of atherosclerosis, with more pronounced relationships found in women. These findings suggest a sex-specific role for PFASs in athero- sclerosis.

1. Introduction perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA). PFOS and PFOA share the feature of being fully fluorinated,

Per- and polyfluoroalkyl substances (PFASs) are a huge class of eight-carbon chain organic acids (C8). However, numerous perfluor- compounds used to manufacture a wide variety of industrial and oalkyls with varying carbon chain length, from four carbons to fourteen household applications due to their unique properties of being both carbons (C4-C14), can be detected in various compartments in the water and oil repellent. The production of PFASs was initiated in the environment (Lau et al., 2007). late 1940s and ubiquitous amounts of PFASs have since then been Global regulatory actions to control the emissions and production released to the environment (Lindstrom et al., 2011). The most widely of PFASs have only recently been implemented for perfluorooctane used among the large group of PFASs are the perfluoroalkyl sulfonic sulfonate (PFOS) and its precursors which were included in the Annex acids (PFSAs) and perfluoroalkyl carboxylic acids (PFCAs), including B of the 2009 Stockholm Convention (UNEP, 2015). Furthermore,

Abbreviations: AMS, Artery Measurement Software; CV, cardiovascular; HRGC/ HRMS, high-resolution mass spectrometry; IM-GSM, grey scale of the carotid artery intima- media complex; IMT, intima media thickness; L-PFOS, linear isomer of perfluorooctane sulfonic acid; PIVUS, Prospective Investigation of the Vasculature in Uppsala Seniors; PCBs, polychlorinated biphenyls; PFASs, per- and polyfluoroalkyl substances; PFCAs, perfluoroalkyl carboxylic acids; PFDA, perfluorodecanoic acid; PFDS, perfluorodecane sulfonic acid; PFHpA, perfluoroheptanoic acid; PFHxS, perfluorohexane sulfonic acid; PFNA, perfluorononanoic acid; PFOSA, perfluorooctane sulfonamide; PFUnDA, perfluoroundecanoic acid; POPs, persistent organic pollutants; UPLC-MS/MS, ultra-performance liquid chromatography coupled to tandem mass spectrometry ⁎ Corresponding author. E-mail addresses: [email protected] (P.M. Lind), [email protected] (S. Salihovic), [email protected] (B. van Bavel), [email protected] (L. Lind). http://dx.doi.org/10.1016/j.envres.2016.10.002 Received 28 June 2016; Received in revised form 5 October 2016; Accepted 7 October 2016 Available online 20 October 2016 0013-9351/ © 2016 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by/4.0/). P.M. Lind et al. Environmental Research 152 (2017) 157–164 voluntary discontinuation agreements from major manufacturers of Table 1 PFOS and PFOA have been implemented in the United States and Basic characteristics and major cardiovascular risk factors in the sample (n=1016). Western Europe (US EPA, 2006, 2014) . Although recent temporal Means with SD or proportions are given in parenthesis. studies suggest that these phase-out policies have resulted in reduced Total sample Women Men emissions and decreasing concentrations of PFOS and PFOA in humans and wildlife (Bjerregaard-Olesen et al., 2016; Johansson n 1016 509 507 et al., 2014; Nost et al., 2014; Okada et al., 2013; Rigét et al., 2013; Variable Mean (SD) Mean (SD) Mean (SD) Height (cm) 168.96 (9.12) 162.14 175.79 (6.45) Wang et al., 2011; Yeung et al., 2013), it is yet unclear whether the (5.63) effect is due to a change in the global production as there is a dynamic Weight (kg) 77.32 (14.42) 71.17 83.49 (12.97) shift in the industrial production of PFASs. (13.12) The presence of PFASs in men, women, and children has been Waist circumference (cm) 91.16 (11.58) 87.6 (11.56) 94.74 (10.45) BMI (kg/m2) 27.03 (4.33) 27.08 (4.88) 26.99 (3.71) extensively reported (Bjerregaard-Olesen et al., 2016; Frisbee et al., SBP (mmHg) 149.63 153.3 (22.6) 145.95 2009; Wang et al., 2011) and an integral part of the exposure in the (22.68) (22.18) general population arises through the dietary exposure including DBP (mmHg) 78.68 (10.18) 78.03 79.33 (10.25) (Fromme et al., 2009). The and toxic (10.08) potential of perfluoroalkyls has been suggested to increase with cholesterol (mmol/l) 5.43 (1.02) 5.72 (0.97) 5.14 (0.98) LDL-cholesterol (mmol/l) 3.38 (0.88) 3.52 (0.86) 3.24 (0.87) increased carbon-chain lengths (Liao et al., 2009; Martin et al., HDL-cholesterol (mmol/l) 1.51 (0.43) 1.66 (0.43) 1.36 (0.37) 2003) and the most frequently detected compounds in the general Serum triglycerides (mmol/l) 1.28 (0.6) 1.27 (0.58) 1.29 (0.62) population are PFOS and PFOA followed by perfluorononanoic acid Fasting glucose (mmol/l) 5.3 (1.6) 5.2 (1.5) 5.4 (1.6) (PFNA), and perfluorohexane sulfonic acid (PFHxS) (Glynn et al., Current smoking (%) 0.11 (0.31) 0.11 (0.32) 0.1 (0.3) 2012; Karrman et al., 2010; Kato et al., 2011; Sundström et al., 2011; Statin treatment (%) 15 13 16 Antihypertensive treatment (%) 31 31 31 Wang et al., 2011). mellitus (%) 12 10 14 A number of adverse health effects have been linked to elevated Myocardial infarction (%) 7 3 11 levels of PFASs (Steenland et al., 2010a). However, there is no uniform Stroke (%) 4 2 5 picture on the cardiovascular effects in response to PFASs exposure, Heart failure (%) 4 3 5 and most studies up to date have been based on occupational or accidental settings (Borg et al., 2013; Sakr et al., 2007; Simpson et al., 2. Material and methods 2013; Steenland et al., 2010b; Winquist and Steenland, 2014) with a gender bias in favor of men. However, many studies show uniformly 2.1. Subjects that certain PFASs are related to high serum cholesterol levels (Nelson et al., 2010; Skuladottir et al., 2015; Winquist and Steenland, 2014). Eligible were all subjects aged 70 living in the community of Since high serum cholesterol levels are linked to atherosclerosis, it is of Uppsala, Sweden. The subjects were chosen from the register of importance to evaluate if cholesterol mediates possible relationships community living and were invited in a randomized order. The subjects between PFASs and cardiovascular diseases. received an invitation by letter within 2 months of their 70th birthday. At present, there is limited information on the relationship between Of the 2025 subjects invited, 1016 subjects participated giving a PFASs exposure and atherosclerosis in a gender-balanced sample of the participation rate of 50.1%. The investigation was conducted between general population. One study found a relationship between PFOS 2001 and 2004. levels and the carotid artery intima-media thickness (IMT), especially The study was approved by the Ethics Committee of the University in women (Lin et al., 2013). We have recently shown that polychlori- of Uppsala and the participants gave informed consent prior to the nated biphenyls (PCBs) are related to atherosclerosis in humans (Lind study. et al., 2012b). Animal experiments support that this finding is causal Basic characteristics of the sample is given in Table 1. (Dalton et al., 2001; Wu et al., 2011). Correspondingly, we hypothe- sized that also PFASs might have cardiovascular effects in the general population. We measured several PFASs in plasma from a population- 2.2. Basic characteristics and cardiovascular risk factors based sample of almost 1000 elderly men and women (50% women) in the Prospective Investigation of the Vasculature in Uppsala Seniors The participants were asked to answer a questionnaire about their (PIVUS-study) (Lind et al., 2005), with the hypothesis that elevated medical history, smoking habits and regular medication. Regarding levels of PFASs are related to atherosclerosis measured by ultrasound medical history, the participants were asked if they have been given a in the carotid arteries in this cross-sectional study. Since previous diagnosis by a physician regarding a list of diagnoses. The smoking studies have pointed towards a gender difference in the elimination of question was coded as current or no current smoking. All subjects were PFASs (Kudo et al., 2002), and that PFASs might effect sex investigated in the morning after an over-night fast. No medication or (Joensen et al., 2013), we paid special attention to the possibility that smoking was allowed after midnight. After recordings of height, weight, effects of PFASs on atherosclerosis are sex-specific. abdominal and hip circumference, an arterial cannula was inserted in We used three different indices of atherosclerosis in the present the brachial artery for blood sampling and later regional infusions of study. First, a local thickening of the intima-media complex, which is vasodilators (not dealt with in the present study). Blood pressure was considered an established atherosclerotic plaque. Second, the intima- measured by a calibrated mercury sphygmomanometer in the non- media thickness (IMT) in the common carotid artery, which is a cannulated arm to nearest mmHg after at least 30 min of rest and the common measure of early atherosclerosis, but that could also be average of three recordings was used. Lipid variables and fasting blood increased by media hypertrophy. Third, the echogenicity of the glucose were measured by standard laboratory techniques. intima-media complex (denoted the intima-media grey scale median, As the participation rate in baseline investigation of this cohort was IM-GSM), a measure of the structural composition of the arterial wall. only 50%, we carried out an evaluation of cardiovascular disorders and The latter two indices are used as indicators of early changes in the medications in 100 consecutive non-participants. The prevalence of carotid artery. cardiovascular drug intake, history of myocardial infarction, coronary revascularization, antihypertensive medication, statin use and insulin treatment were similar to those in the investigated sample, while the prevalence of diabetes, congestive heart failure and stroke tended to be

158 P.M. Lind et al. Environmental Research 152 (2017) 157–164 higher among the non-participants (see reference Lind et al., 2005 for 1.7 µm) analytical column by a 6-port column switch valve. details). Quantitative analysis of the PFASs was performed using the internal standard method; all standards (i.e., 13C labeled internal standards, 13C 2.3. Carotid artery ultrasound evaluation labeled recovery standards and native calibration standards) were purchased from Wellington Laboratories (Guelph, Ontario, Canada). The carotid artery was assessed by external B-mode ultrasound The method detection limits ranged between 0.01 and 0.17 ng mL−1 imaging (Acuson XP128 with a 10 MHz linear transducer, Acuson depending on the analyte. and 25th and 75th of the Mountain View, California, USA). The common carotid artery (CCA), eight different PFASs with detectable levels (ng mL−1) in > 75% of the the bulb and the internal carotid artery (ICA) were visualized and the subjects are given in Suppl Table 1. occurrence of plaque was recorded on both sides. A plaque was considered to be present if the intima media thickness (IMT) was 2.5. Statistical analyses locally thickened more than 50% compared to the surrounding IMT (Hulthe et al., 1997). The numbers of carotid arteries with plaques (0, 1 All eight PFASs were skewed towards high levels, but were normally or 2) were recorded. distributed following ln-transformation. Relationships between PFASs The images were digitized and imported into the AMS (Artery and number of carotid arteries with plaque (0, 1 or 2) were evaluated Measurement Software) automated software for dedicated analysis of by ordinal logistic regression models, first using the PFASs as linear intima-media thickness and the grey scale median of the intima-media variables, and thereafter using the squared form of the PFASs to search complex (Liang et al., 2000). The IMT was evaluated in the far wall in for non-linear effects. For the continuous analysis, two steps of the CCA 1–2 cm proximal to the bulb. A maximal 10 mm segment with adjustments were used. First, adjustment for gender only, and secondly good image quality was chosen for intima-media thickness -analysis multiple adjustment for gender, LDL and HDL-cholesterol and serum from the carotid artery. The programme automatically identifies the triglycerides, BMI, blood pressure, smoking and exercise habits, energy borders of the intima-media thickness of the far wall and the inner and alcohol intake, diabetes and educational level. Of the 12 variables diameter of the vessel and calculates intima-media thickness and the included in the model in the multiple adjustment, smoking and diameter from around 100 discrete measurements through the 10 mm exercise habits, energy and alcohol intake, and educational level are long segment. This automated analysis could be manually corrected if life-style factors with the potential to influence PFASs levels, as well as not found appropriate at visual inspection. The given value for carotid indices of atherosclerosis. LDL and HDL-cholesterol and serum artery intima-media thickness is the mean value from both sides. Thus, triglycerides, BMI, blood pressure and diabetes are classical risk factors the IMT is a measure of the thickness of the artery wall, not including for atherosclerosis, but it is less obvious that those variables could the adventitia, but only the intima and media layers of the wall. affect PFASs levels. Some of those, like cholesterol, have been linked to A region of interest was placed manually around the intima-media PFASs and could eventually be a mediator in the PFASs vs. athero- segment that was evaluated for intima-media thickness and the sclerosis relationship. programme calculates the echogenicity in the intima-media complex A similar approach was used when relating PFASs to either IMT or from analysis of the individual pixels within the region of interest on a IM-GSM. In this analysis, linear regression analysis was performed. scale from 0 (black) to 256 (white). The blood was used as the reference In order to search for sex-specific relationships, interaction terms for black and the adventitia was the reference for white. The grey scale between sex and the 8 PFASs were created and included in a separate median (GSM)-value given is the mean value from both sides (intima- set of models. If the interaction term was significant, analyses were also media grey scale median, IM-GSM). Thus, a low value of IM-GSM is performed in men and women separately. thought of as a marker of lipid infiltration in the carotid artery wall, the Structural equation models (SEM) were used to disentangle the role first step in carotid plaque formation. of mediation of serum cholesterol on the relationships between PFASs The mean length of the evaluated intima-media segments was 9.0 and indices of atherosclerosis. (SD 2.1) mm when subjects with a segment recording less than 5 mm STATA14 (Stata corp, College Station, TX, USA) was used for were excluded. The measurements of intima-media thickness were calculations. repeated in 30 random subjects giving a coefficient of variation of carotid artery intima-media thickness of 7.2% and 7.5% for IM-GSM. 3. Results

2.4. Methods for the chemical analysis of the PFASs The mean value for IMT was 0.88 mm (0.16 SD), while the corresponding mean value for IM-GSM was 79 (23 SD). In the total The analytical method used to determine plasma concentrations of sample, 273 subjects showed bilateral plaque, 343 showed unilateral PFASs in all samples (N=1, 016) was successfully validated in terms of plaque and 327 were free of carotid plaque. recovery, accuracy, and precision (Salihovic et al., 2015, 2013). Briefly, As could be seen in Table 2, no consistent significant linear 150 µL serum extracts were analyzed using an automated column- associations were seen between the PFASs and IMT or IM-GSM when switching ultra-performance liquid chromatography-tandem mass men and women were analyzed together (and adjusting for sex). spectrometry (HPLC-MS/MS) method for determination of PFASs. Neither was occurrence of plaques in the carotid arteries related to The current study evaluated the 8 PFASs for which > 80% of the PFASs levels (Table 4). population showed measurable levels above the lower level of detec- Introduction of a quadratic term for the PFASs in the model did not tion; perfluoroheptanoic acid (PFHpA), perfluorononanoic acid disclose any consistent significant non-linear relationships. (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid However, a highly significant interaction between sex and some of (PFUnDA), perfluorohexane sulfonic acid (PFHxS), linear isomer of the PFCAs were seen regarding IM-GSM (p=0.002 for PFNA and perfluorooctane sulfonic acid (L-PFOS), perfluorooctanoic acid (PFOA) PFDA, p=0.0004 for PFUnDA). When men and women were analyzed and perfluorooctane sulfonamide (PFOSA). The analytical procedure separately, PFNA, PFDA, and PFUnDA were all related to IM-GSM in a involves rapid precipitation using 96-well plates followed by positive fashion in women (beta 6.023, 95%CI (2.06, 9.986), p=0.003 instrumental analyses on a Acquity UPLC coupled to a Quattro Premier for PFNA, 7.546, (2.622, 12.47), p=0.003 for PFDA and 7.045, (2.602, XE HPLC-MS/MS system (Waters Corporation, Milford, USA) with an 11.488), p=0.002 for PFUnDA), while these relationships were nega- atmospheric electrospray interface operating in negative ion mode tive in men (although only significantly so for PFUnDA, beta −4.574, system by injecting a 250 µL aliquot of the sample onto a C18 (−9.092, −0.057), p=0.047, Fig. 1). Following adjustment for multiple (2.1×20 mm, 2.5 µm) trap column connected to a C18 (2.1×100 mm, risk factors these gender differences persisted, although at this stage

159 P.M. Lind et al. Environmental Research 152 (2017) 157–164

Table 2 Table 3 Relationships between eight different PFASs (all ln-transformed) and the intima-media Relationships between eight different PFASs (all ln-transformed) and the intima-media thickness (IMT) (at top) and the echogenicity of the intima-media complex (IM-GSM) thickness (IMT) (at top) and the echogenicity of the intima-media complex (IM-GSM) (below). Relationships are given both for sex-adjusted and multiple adjusted analysis (below) in sex-stratified analyses. Relationships are given for non-adjusted and multiple (gender, HDL- and LDL-cholesterol and serum triglycerides, BMI, blood pressure, adjusted analyses (HDL- and LDL-cholesterol and serum triglycerides, BMI, blood smoking exercise habits, energy and alcohol intake, diabetes and educational level). The pressure, smoking exercise habits, energy and alcohol intake, diabetes and educational regression coefficients (Beta), 95%CI and p-values are given for the linear models. level). The regression coefficients (Beta), 95%CI and p-values are given for the linear PFASs; perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), perfluorono- models. For abbreviations of PFASs, see Table 2. nanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUn- DA), perfluorohexane sulfonic acid (PFHxS), linear isomer of perfluorooctane sulfonic Non-adjusted Multiple adjusted acid (L-PFOS), as well as perfluorooctane sulfonamide (PFOSA). Beta (95% CI) P-value Beta (95% CI) P-value Sex adjusted Multiple adjusted IMT Beta (95% CI) P-value Beta (95% CI) P-value Males IMT PFHpA 0.001 (−0.018, 0.021) 0.90 −0.004 (−0.024, 0.71 PFHpA 0.001 (−0.014, 0.014) 0.99 0.002 (−0.012, 0.016) 0.78 0.017) PFHxS −0.006 (−0.021, 0.41 0.001 (−0.014, 0.016) 0.90 PFHxS −0.004 (−0.026, 0.019) 0.75 0.004 (−0.019, 0.027) 0.73 0.008) L-PFOS 0.001 (−0.021, 0.024) 0.90 0.004 (−0.019, 0.028) 0.72 L-PFOS 0.003 (−0.015, 0.021) 0.76 0.003 (−0.015, 0.022) 0.72 PFOA 0.006 (−0.025, 0.037) 0.72 0.013 (−0.02, 0.045) 0.44 PFOA −0.002 (−0.025, 0.84 0.007 (−0.017, 0.03) 0.58 PFNA 0.012 (−0.017, 0.04) 0.420 0.005 (−0.025, 0.035) 0.75 0.021) PFDA −0.005 (−0.041, 0.031) 0.80 −0.009 (−0.049, 0.64 PFNA 0.01 (−0.011, 0.03) 0.37 0.003 (−0.018, 0.025) 0.76 0.03) PFDA 0.001 (−0.026, 0.026) 0.99 0.003 (−0.0025, 0.03) 0.85 PFOSA 0.005 (−0.02, 0.031) 0.70 0.013 (−0.014, 0.039) 0.35 PFOSA 0.012 (−0.006, 0.031) 0.18 0.024 (0.006, 0.042) 0.01 PFUnDA −0.009 (−0.043, 0.025) 0.60 −0.015 (−0.053, 0.43 PFUnDA −0.004 (−0.027, 0.02) 0.77 −0.001 (−0.027, 0.96 0.023) 0.026) Females IM-GSM PFHpA −0.001 (−0.021, 0.018) 0.88 0.009 (−0.011, 0.029) 0.39 PFHpA 0.369 (−1.507, 2.245) 0.70 0.471 (−1.481, 2.424) 0.64 PFHxS −0.008 (−0.027, 0.011) 0.40 −0.001 (−0.02, 0.93 PFHxS 0.04 (−1.863, 1.943) 0.97 −0.541 (−2.522, 0.59 0.018) 1.441) L-PFOS 0.005 (−0.025, 0.036) 0.73 0.006 (−0.025, 0.037) 0.70 L-PFOS 1.443 (−0.789, 3.675) 0.21 1.238 (−1.103, 3.578) 0.30 PFOA −0.013 (−0.047, 0.021) 0.46 0.001 (−0.034, 0.035) 0.97 PFOA 1.475 (−1.501, 4.452) 0.33 0.933 (−2.206, 4.072) 0.56 PFNA 0.007 (−0.024,0 0.038) 0.68 0.003 (−0.029, 0.035) 0.84 PFNA 1.315 (−1.427, 4.056) 0.35 1.915 (−0.996, 4.825) 0.20 PFDA 0.005 (−0.031, 0.042) 0.77 0.021 (−0.019, 0.06) 0.30 PFDA 1.591 (−1.861, 5.043) 0.37 0.258 (−3.528, 4.044) 0.89 PFOSA 0.021 (−0.005, 0.047) 0.12 0.039 (0.012, 0.065) 0.004 PFOSA 1.675 (−0.45, 3.8) 0.12 0.455 (−1.741, 2.651) 0.68 PFUnDA 0.002 (−0.031, 0.035) 0.89 0.019 (−0.018, 0.056) 0.307 PFUnDA 1.071 (−2.116, 4.257) 0.51 −0.393 (−3.99, 3.205) 0.83 IM-GSM only the association between PFNA and IM-GSM was still significant in Males PFHpA −1.842 (−4.483, 0.799) 0.17 −0.859 (−3.648, 0.55 women (beta 5.268, (1.048, 9.489), p=0.014). For details, see Table 3. 1.93) Unadjusted analyses of the eight PFASs vs the three markers of PFHxS −0.991 (−3.868, 1.886) 0.50 −1.298 (−4.339, 0.40 atherosclerosis is given in Suppl Table 2. 1.742) Since PFASs repeatedly have been shown to be related to choles- L-PFOS 0.524 (−2.417, 3.465) 0.73 1.366 (−1.814, 4.545) 0.40 − − − − terol , we further investigated if the above-described PFOA 1.09 ( 5.191, 3.011) 0.60 0.582 ( 4.974, 0.80 3.81) relationship between PFUnDA and IM-GSM in women was mediated PFNA −2.569 (−6.344, 1.207) 0.18 −0.902 (−4.98, 0.66 by cholesterol in a SEM analysis. This analysis disclosed that 3.4% of 3.176) the total effect of PFUnDA on IM-GSM in women was mediated by PFDA −3.596 (−8.405, 1.214) 0.14 −3.303 (−8.672, 0.23 cholesterol (p=0.012). For men, the corresponding proportion of the 2.067) − − total effect of PFUnDA on IM-GSM being mediated by serum choles- PFOSA 0.915 ( 2.092, 3.923) 0.55 0.329 ( 2.875, 3.532) 0.84 PFUnDA −4.574 (−9.092, 0.05 −4.405 (−9.52, 0.71) 0.09 terol was 1.7% (p=0.014). −0.057) A highly significant interaction between sex and some of the PFASs were seen also regarding carotid plaques (p=0.006 for PFOSA, p=0.005 Females − for PFUnDA). In this case, PFUnDA was significantly related to carotid PFHpA 2.834 (0.178, 5.49) 0.04 2.18 ( 0.608, 4.967) 0.13 PFHxS 0.915 (−1.612, 3.442) 0.48 −0.085 (−2.737, 0.95 plaque in women (OR 1.59, 95%CI (1.03, 2.43), p=0.034, Fig. 2), but 2.567) not in men (0.93, (0.62, 1.42), p=0.74). A similar tendency was seen for L-PFOS 2.833 (−0.626, 6.291) 0.11 1.068 (−2.51, 4.647) 0.56 PFOSA (OR 1.28, (0.98, 1.66), p=0.07 in women and 0.99, (0.75, 1.29), PFOA 4.644 (0.313, 8.976) 0.04 2.72 (−1.875, 7.315) 0.25 p=0.91 in men). For details, see Table 5. PFNA 6.023 (2.06, 9.986) 0.003 5.268 (1.048, 9.489) 0.01 − We further investigated if the above-described relationship between PFDA 7.546 (2.622, 12.47) 0.0028 4.09 ( 1.353, 9.533) 0.14 PFOSA 2.639 (−0.373, 5.652) 0.09 0.438 (−2.646, 3.522) 0.78 PFUnDA and carotid plaques in women was mediated by cholesterol in PFUnDA 7.045 (2.602, 11.488) 0.002 3.948 (−1.23, 9.126) 0.14 a SEM analysis. This analysis disclosed no significant mediation of serum cholesterol on the PFUnDA vs. carotid plaques relationship (p=0.18). with 15% showing a GFR < 60 ml/in/m2. Including GFR in the models In order to evaluate if common cardio-metabolic medications used for the above-mentioned significant relationships did however only in the elderly influenced the above-mentioned significant relationship, have marginal effects on these significant relationships. we included statins, beta-blockers, calcium antagonists, ACE-inhibi- tors, diuretics, insulin, oral antidiabetic agents in the models, but 4. Discussion inclusion of those medications had only marginal effects of the fi signi cant relationships described above. The present cross-sectional study showed that the long-chain fi The mean glomerular ltration rate (GFR), as calculated by the PFASs, especially PFUnDA, were related to echogenicity of the carotid 2 Cockcroft-Gault equation, was 81 (SD 20) ml/in/m in the sample, artery wall and to overt atherosclerotic plaque in women. We have

160 P.M. Lind et al. Environmental Research 152 (2017) 157–164

Table 4 Relationships between eight different PFASs (all ln-transformed) and number of carotid arteries with atherosclerotic plaques. Relationships are given both for sex-adjusted and multiple adjusted analysis (sex, HDL- and LDL-cholesterol and serum triglycerides, BMI, blood pressure, smoking exercise habits, energy and alcohol intake, diabetes and educational level). The odds ratios (OR), 95%CI and p-values are given for the relationships. For abbreviations of PFASs, see Table 2.

Sex adjusted Multiple adjusted

OR (95% CI) P-value OR (95% CI) P-value

PFHpA 0.92 (0.79, 1.08) 0.32 0.95 (0.81, 1.13) 0.59 PFHxS 1.08 (0.94, 1.25) 0.28 1.11 (0.95, 1.29) 0.18 L-PFOS 1.07 (0.93, 1.21) 0.34 1.07 (0.94, 1.22) 0.32 PFOA 0.92 (0.75, 1.13) 0.45 0.95 (0.77, 1.18) 0.67 PFNA 1.02 (0.84, 1.24) 0.85 1.09 (0.88, 1.34) 0.43 PFDA 0.99 (0.74, 1.32) 0.94 1.1 (0.81, 1.51) 0.54 PFOSA 1.09 (0.91, 1.3) 0.36 1.13 (0.94, 1.37) 0.19 PFUnDA 1.06 (0.81, 1.39) 0.65 1.2 (0.9, 1.62) 0.22

Fig. 2. Relationship between circulating levels of PFUnDA (on a ln-scale, ng mL−1) and carotid artery plaque in women (p < 0.05). The predictive margins (and 95%CI) for the probability of have carotid artery plaque (and 95%CI) is given at the y-axis.

Table 5 Relationships between eight different PFASs (all ln-transformed) and number of carotid arteries with atherosclerotic plaques in sex-stratified analyses. Relationships are given for multiple adjusted analyses (HDL- and LDL-cholesterol and serum triglycerides, BMI, blood pressure, smoking exercise habits, energy and alcohol intake, diabetes and educational level). The odds ratios (OR), 95%CI and p-values are given for the relationships. For abbreviations of PFASs, see Table 2.

Males Females

Variable OR (95% CI) P-value OR (95% CI) P-value

PFHpA 0.83 (0.66, 1.05) 0.12 1.11 (0.86, 1.41) 0.42 PFHxS 1.19 (0.94, 1.51) 0.16 1.06 (0.87, 1.29) 0.56 L-PFOS 1.09 (0.89, 1.35) 0.41 1.05 (0.88, 1.25) 0.59 PFOA 0.95 (0.68, 1.31) 0.74 0.96 (0.72, 1.27) 0.77 PFNA 1.11 (0.81, 1.51) 0.52 1.07 (0.8, 1.42) 0.64 PFDA 0.97 (0.63, 1.49) 0.89 1.29 (0.82, 2.04) 0.27 PFOSA 0.99 (0.75, 1.29) 0.92 1.28 (0.98, 1.66) 0.07 PFUnDA 0.93 (0.62, 1.42) 0.75 1.59 (1.03, 2.43) 0.03

linked to atherosclerosis in the carotid arteries.

4.1. Carotid artery plaque

Presence of atherosclerotic plaques in the carotid arteries is a known predictor of future CV events (O'Leary et al., 1999). Atherosclerosis in the carotid arteries is also known to be predictive for plaques in the coronary circulation (Hulthe et al., 1997). Thus, subjects with carotid artery plaques are at increased risk also for myocardial infarction. The presently found association between PFUnDA levels and carotid artery plaque in women is therefore of concern. Why this relationship only is seen in women is not known, but it has recently been found that a high level of PFOS is related to sex levels (Joensen et al., 2013). Since sex hormones seem capable to modulate the development of atherosclerosis (Naessen et al., 2012) it is not unlikely that PFASs could affect men and women − Fig. 1. Relationship between circulating levels of PFUnDA (on a ln-scale, ng mL 1) and in different ways. the echogenicity of the intima-media complex (IM-GSM) in women (FIGURE 1A, p=0.002) and in men (FIGURE 1B, p < 0.05). The predictive margins (and 95%CI) for 4.2. Echogenicity of the intima-media complex IM-GSM is given at the y-axis.

The echogenicity of the intima-media complex is closely related to previously shown that atherosclerosis is associated with other environ- the echogenicity on overt plaques (Lind et al., 2007), and has been mental contaminants, like the PCBs (Lind et al., 2012b), some shown to be a powerful predictor of future CV mortality (Wohlin et al., (Lind and Lind, 2011), as well as some metals (Lind et al., 2009). The echogenicity of overt plaques has been shown to be related 2012a). The present study adds to that prior knowledge that several to the structural composition of the plaque (El-Barghouty et al., 1996), environmental contaminants with very different chemical properties, and it is therefore likely that also the echogenicity of the intima-media different mechanisms of action and different degrees of exposure are complex is a marker of composition of the vascular wall. In this case it

161 P.M. Lind et al. Environmental Research 152 (2017) 157–164 is likely that an echolucent (dark) intima-media complex is indicative because of its longer carbon chain PFUnDA is more bioaccumulative of lipid infiltration, while an echogenic (white) intima-media complex (Zhang et al., 2013). The few available mechanistic studies of PFUnDA possibly represent collagen disposition. found, in line with previous research of other PFASs, that PFUnDA In the present study, some of the PFASs, more specifically the long- activated the PPAR alpha receptor and inhibited the function of the chain PFCAs showed positive relationships vs. IM-GSM in women, but thyroid hormone (Long et al., 2013; Takahashi et al., 2014). Moreover, the opposite tendency in men. Thus, most likely these PFCAs are Long et al. (2013) also investigated the effect of PFAAs on the aryl related to an increased lipid infiltration in the vascular wall in men, but receptor and found that PFDA and PFDoDA elicited an more fibrous (collagen) tissue in women. How this translates in terms activating effect but not PFUnDA Thus, no PFUnDA-specificeffects of risk is hard to tell, since both a low and a high IM-GSM were related have been reported in the available literature. Overall, further experi- to an increased risk (Wohlin et al., 2009). mental studies with regard to PFUnDA and the other long-chain PFCAs are required to gain a deeper insight to the mechanistic pathway by 4.3. Intima-media thickness which PFUnDA exerts effects on the arterial wall. A number of alterations in the , such as T-cell- IMT is known as a predictor of CV events, and is usually regarded as dependent antibody responses (DeWitt et al., 2016), interferon-gamma a marker of atherosclerosis (O'Leary et al., 1999). However, athero- expression (Ryu et al., 2014), toll-like receptor 2 (TLR2) expression sclerosis is a disorder of the intima, but IMT is also incorporating the and activation of interleukins by the NF-κB pathway (Zhang et al., media in the measurement. In the present study, no associations were 2014), and a reduction in IgM antibodies (DeWitt et al., 2016), have seen between the PFASs and IMT. This is in contrast to the study by been linked to PFASs exposure. As reviewed by (Libby and Hansson, Lin et al who found an association between PFOS levels and IMT in 2015) an immune reaction against lipid infiltration and trapping in the younger subjects (Lin et al., 2013). The differences in age between the arterial wall is an early and very important event in the development of studies might have contributed to the diverse findings. Of interest is the atherosclerosis. Future experimental studies have to guide which of finding of Lin et al that the relationship between PFASs and IMT was these immunological effects of PFASs that could be involved in mainly found in women, in accordance with findings in the present atherosclerosis development. study. It has repeatedly been shown that PFASs levels in humans are related to cholesterol metabolism (Nelson et al., 2010; Skuladottir 4.4. Other environmental contaminants et al., 2015; Winquist and Steenland, 2014). In order to test if an effect of PFASs on atherosclerosis is mediated by the effect of PFASs on Since we previously have reported other environmental contami- cholesterol, we performed SEM analyses on the major findings in this nants, like the PCBs (Lind et al., 2012b), some phthalates (Lind and study. That analyses disclosed that the mediating role of serum Lind, 2011), as well as some metals (Lind et al., 2012a) to be related to cholesterol significant, but very small (1–3%), regarding the PFUnDA indices of atherosclerosis, we added PCB209, Cr, Ni, Al, mono-methyl vs. IM-GSM relationship, and not significant regarding the PFUnDA vs. (MMP) and bisphenol A (BPA) to the models investigating plaque relationship. Thus, in this study, serum cholesterol was not a the PFUnDA vs. IM-GSM or carotid plaque relationships, but addition major mediator of the PFASs vs. atherosclerosis relationship. of these other environmental contaminants did not change the In a very recent by Lin et al (Lin et al., 2016) it was shown PFUnDA vs. IM-GSM or carotid plaque relationships to any significant that PFOS was related to circulating microparticles (both endothelium degree. This is well in line with previous observations that these major and platelet-derived), as well as to IMT. Furthermore, the link between environmental contaminants are not closely related and in order to PFOS and IMT was closer in those with elevated levels of micropar- obtain information on the effect of environmental contaminants on a ticles. Endothelium and platelet-derived microparticles are markers of phenotype like atherosclerosis, many different contaminants have to be apoptosis and are linked to endothelial dysfunction, an early feature of evaluated (Lampa et al., 2012). atherosclerosis. Thus, with that study a link between PFASs and early atherosclerosis is established. 4.5. Potential mechanisms of action 4.6. Limitation of the study There is no well known obvious explanation for an effect of PFASs on atherosclerosis. PFASs are known to be agonists for the PPAR alpha The present sample is limited to Caucasians aged 70. Therefore, and gamma receptors (Lau et al., 2007), although the different PFASs caution should be made to draw conclusions to other ethnic and age differ regarding binding to these two types of PPAR receptors. groups. The present study had a moderate participation rate. However, Activation of PPAR alpha and gamma-receptors could be different an analysis of non-participants showed the present sample to be fairly mechanisms be involved in different steps in glucose homeostasis, such representative of the total population regarding most cardiovascular as influence on insulin resistance (Lebovitz and Banerji, 2001) and disorders and drug intake. Furthermore, many statistical tests were insulin secretion (Lupi et al., 2004), as well as influence circulating performed and thus the present findings since we liked to include not levels of lipids (Thomas et al., 2007) and altering the amount of only the most commonly analyzed PFASs, PFOS and PFOA, and we abdominal fat (Moon et al., 2011; Thomas et al., 2007). In line with this included not only the most commonly used marker of carotid athero- knowledge is our previous findings that some, but not all, of the sclerosis, IMT, but also information on overt plaques and also on the measured PFASs were related to diabetes in the present sample (Lind echogenicity of the arterial wall, IM-GSM, to investigate early changes et al., 2014). Diabetes, dyslipidemia and obesity are all known risk in the atherosclerotic process. Given the prior limited knowledge in this factors for atherosclerosis development, but in the present study the area on the possible different actions of different PFASs on the association between the PFASs and atherosclerosis was seen indepen- vasculature, it was very hard to make any specific prior hypothesis dently of these risk factors. on which PFASs that would be related to a specific marker of It was mainly the long-chained PFASs that were related to indices of atherosclerosis. Using this approach to give a more detailed picture atherosclerosis in the present study, despite that lower concentrations of the relationship between multiple PFASs and three markers of generally were seen for those PFASs. This is hard to explain from atherosclerosis must hopefully be more valuable than just analyzing experimental data, since it has been shown that PPAR alpha activation two PFASs and IMT, but could produce false positive results due to occurred in an additive fashion when different PFASs were added to multiple statistical testing. Our findings must therefore be regarded as PFOA (Wolf et al., 2014), a PFASs with higher concentration than exploratory and must therefore be taken with caution until reproduced PFUnDA. Structurally, PFUnDA is the 11 carbon version of PFOA and by others. The finding that two different markers of atherosclerosis,

162 P.M. Lind et al. Environmental Research 152 (2017) 157–164 overt plaque and IM-GSM were associated with some PFASs in a fairly Borg, D., et al., 2013. Cumulative health risk assessment of 17 perfluoroalkylated and fl fi poly uoroalkylated substances (PFASs) in the Swedish population. Environ. Int. 59, consistent manner does however make the present ndings likely not to 112–123. be merely chance findings. Dalton, T.P., et al., 2001. Dioxin exposure is an environmental risk factor for ischemic Cross-sectional studies could always be subjected to reverse causa- heart disease. Cardiovasc Toxicol. 1, 285–298. DeWitt, J.C., et al., 2016. Suppression of antigen-specific antibody responses in mice tion, so ideally prospective studies on plaque progression and devel- exposed to perfluorooctanoic acid: role of PPARalpha and T- and B-cell targeting. J. opment of atherosclerosis-related disorders, such as myocardial in- Immunotoxicol. 13, 38–45. farction and stroke, should be carried out. El-Barghouty, N.M., et al., 1996. Histological verification of computerised carotid plaque characterisation. Eur. J. Vasc. Endovasc. Surg. 11, 414–416. Frisbee, S.J., et al., 2009. The C8 health project: design, methods, and participants. 4.7. Conclusions Environ. Health Perspect. 117, 1873–1882. Fromme, H., et al., 2009. Perfluorinated compounds – Exposure assessment for the general population in western countries. Int. J. Hyg. Environ. Health 212, 239–270. In conclusion, in this cross-sectional study, a pronounced gender fl ff Glynn, A., et al., 2012. Per uorinated alkyl acids in blood serum from primiparous di erence was seen regarding associations between some of the PFASs, women in Sweden: serial sampling during pregnancy and nursing, and temporal especially the long-chain PFCAs such as PFUnDA, and markers of trends 1996–2010. Environ. Sci. Technol. 46, 9071–9079. atherosclerosis, with more pronounced relationships found in women. Hulthe, J., et al., 1997. Atherosclerotic changes in the carotid artery bulb as measured by fi B-mode ultrasound are associated with the extent of coronary atherosclerosis. Stroke These ndings suggest a role for PFASs in atherosclerosis. 28, 1189–1194. Joensen, U.N., et al., 2013. PFOS (perfluorooctanesulfonate) in serum is negatively Competing interests associated with testosterone levels, but not with , in healthy men. Hum. Reprod. 28, 599–608. Johansson, J.H., et al., 2014. Temporal trends (1999–2010) of perfluoroalkyl acids in The authors declare that they have no competing interests. commonly consumed items. Environ. Pollut. 188, 102–108. Karrman, A., et al., 2010. perfluorinated compounds in Catalonia, Spain: ’ concentrations and trends in human and milk samples. Environ. Sci. Pollut. Authors contributions Res. Int. 17, 750–758. Kato, K., et al., 2011. Trends in exposure to polyfluoroalkyl chemicals in the US PML, SS and LL drafted the manuscript. population: 1999–2008. Environ. Sci. Technol. 45, 8037–8045. fl PML contributed to critical revision of the manuscript for impor- Kudo, N., et al., 2002. Sex hormone-regulated renal transport of per uorooctanoic acid. Chem.-Biol. Interact. 139, 301–316. tant intellectual content and in addition finalized the manuscript. Lampa, E., et al., 2012. An investigation of the co-variation in circulating levels of a large SS contributed to critical revision of the manuscript for important number of environmental contaminants. J. Expo. Sci. Environ. Epidemiol. 22, – intellectual content. 476 482. Lau, C., et al., 2007. Perfluoroalkyl acids: a review of monitoring and toxicological LL, together with PML, conceived of, designed, and coordinated the findings. Toxicol. Sci. 99, 366–394. study and contributed to critical revision of the manuscript for Lebovitz, H.E., Banerji, M.A., 2001. Insulin resistance and its treatment by important intellectual content. . Recent Prog. Horm. Res. 56, 265–294. Liang, Q., et al., 2000. A multiscale dynamic programming procedure for boundary Also, LL is principal investigator of PIVUS and had full access to all detection in ultrasonic artery images. IEEE Trans. Med. Imaging 19, 127–142. the data in the study and takes responsibility for the integrity of the Liao, C., et al., 2009. Changes in synaptic transmission, calcium current, and neurite data and the accuracy of the data analysis. growth by perfluorinated compounds are dependent on the chain length and functional group. Environ. Sci. Technol. 43, 2099–2104. SS and BvB were responsible for the chemical analyses of PFASs Libby, P., Hansson, G.K., 2015. Inflammation and immunity in diseases of the arterial measurements and performed, acquisition, analysis and interpretation tree: players and layers. Circ. Res. 116, 307–311. of this data and contributed to critical revision of the manuscript for Lin, C.Y., et al., 2013. 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