Phthalate-induced oxidative stress and association with asthma-related airway inflammation in adolescents

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Phthalate-induced oxidative stress and association with asthma-related airway inflammation in adolescents Carmen Franken a,b,∗ , Nathalie Lambrechts a , Eva Govarts a , Gudrun Koppen a , Elly Den Hond c , Daniëlla Ooms a , Stefan Voorspoels a , Liesbeth Bruckers d , Ilse Loots e , Vera Nelen c , Isabelle Sioen f,g , Tim S. Nawrot h,i , Willy Baeyens j , Nicolas Van Larebeke j,k , Greet Schoeters a,b,l a

Flemish Institute for Technological Research (VITO), Mol, Belgium Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium c Provincial Institute for Hygiene, Antwerp, Belgium d Interuniversity Institute for Biostatistics and Statistical Bioinformatics, Hasselt University, Hasselt, Belgium e Political and Social Sciences, University of Antwerp, Antwerp, Belgium f Department of Public Health, Ghent University, Ghent, Belgium g Department of Food Safety and Food Quality, Ghent University, Ghent, Belgium h Centre for Environmental Sciences, Hasselt University, Diepenbeek, Belgium i Department of Public Health & Primary Care, Leuven University, Leuven, Belgium j Department of Analytical and Environmental Chemistry, Vrije Universiteit Brussel, Brussels, Belgium k Department of Radiotherapy and Nuclear Medicine, Ghent University, Ghent, Belgium l Department of Environmental Medicine, Institute of Public Health, University of Southern Denmark, Odense, Denmark b

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Article history: Received 8 August 2016 Received in revised form 24 January 2017 Accepted 24 January 2017 Keywords: Human biomonitoring Oxidative stress Phthalate Doctor-diagnosed asthma 8-hydroxydeoxyguanosine

a b s t r a c t Background: In Belgium, around 8.5% of the children have asthmatic symptoms. Increased asthma risk in children has been reported in relation to exposure to phthalate plasticizers but the underlying mechanisms are largely unknown. Aim: The aim of this study was to identify if oxidative stress, assessed by excision of 8hydroxydeoxyguanosine (8-OHdG) from damaged DNA, is an intermediate marker for the association between phthalate exposure and doctor-diagnosed asthma. Material and methods: In 418 14–15-year-old youngsters, recruited as a representative sample of residents of Flanders (Belgium), personal exposure to phthalates was assessed by measuring phthalate metabolites in urine: mono(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono-n-butyl phthalate (MnBP), mono-benzyl phthalate (MBzP), mono-isobutyl phthalate (MiBP) and mono-ethyl phthalate (MEP). Analysis of 8-OHdG in urine was used as a sensitive biomarker of oxidative stress at the level of DNA. The presence of doctordiagnosed asthma was elicited by a self-administered questionnaire. Associations were assessed using multiple linear and logistic regression models. Mediation was tested using Baron and Kenny’s regression approach. Results: A significant increased risk of a youngster being diagnosed with asthma was found for both urinary MnBP (metabolite of dibutyl phthalate (DBP)) and the sum of the three di(2-ethylhexyl) phthalate metabolites (DEHP = MEHP + MEHHP + MEOHP), with respective odds ratio of 1.84 [95% CI: 1.02, 3.32] for MnBP and 1.94 [95% CI: 1.07, 3.51] for DEHP. In addition, we observed significant associations between all urinary phthalate metabolites and increased urinary levels of 8-OHdG. The associations were stronger

Abbreviations: 8-OHdG, 8-hydroxydeoxyguanosine; BBzP, butylbenzyl phthalate; BMI, Body Mass Index; CI, confidence interval; DBP, dibutyl phthalate; DEHP, di(2ethylhexyl) phthalate; DEP, diethyl phthalate; DIBP, diisobutyl phthalate; DIDP, diisodecyl phthalate; DINP, diisononyl phthalate; DNOP, di-n-octyl phthalate; FLEHS, Flemish Environment and Health Study; HMW, high molecular weight; IL-1␤, interleukin ␤; ISAAC, International Study of Asthma and Allergy in Childhood; LMW, low molecular weight; LOQ, limit of quantification; MBzP, mono-benzyl phthalate; MCNP, mono(carboxynonyl) phthalate; MCOP, mono(carboxyoctyl) phthalate; MEHP, mono(2-ethylhexyl) phthalate; MEHHP, mono(2-ethyl-5-hydroxyhexyl) phthalate; MEOHP, mono(2-ethyl-5-oxohexyl) phthalate; MEP, mono-ethyl phthalate; MiBP, mono-isobutyl phthalate; MnBP, mono-n-butyl phthalate; OR, odds ratio; PPAR, peroxisome proliferator-activated receptor; ROS, reactive oxygen species; SG, specific gravity; Th2, T helper 2-type; TNF-␣, tumor necrosis factor ␣. ∗ Corresponding author at: Flemish Institute for Technological Research (VITO), Boeretang 200, B-2400 Mol, Belgium. E-mail address: [email protected] (C. Franken). http://dx.doi.org/10.1016/j.ijheh.2017.01.006 1438-4639/© 2017 Elsevier GmbH. All rights reserved.

Please cite this article in press as: Franken, C., et al., Phthalate-induced oxidative stress and association with asthma-related airway inflammation in adolescents. Int. J. Hyg. Environ. Health (2017), http://dx.doi.org/10.1016/j.ijheh.2017.01.006

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in girls than in boys. We did not found evidence that 8-OHdG was associated with doctor-diagnosed asthma. Conclusion: The results of our study are in line with other findings from epidemiological surveys and raise further concern about DEHP and DBP as risk factors for asthma, however, the underlying mechanisms are not yet well understood. © 2017 Elsevier GmbH. All rights reserved.

1. Introduction Asthma has become one of the most common chronic diseases among children and is one of the major causes of hospitalization among those younger than 15 years of age (WHO, 2013). As there is no unique marker for asthma, epidemiological studies of risk factors for asthma often use the answers to the International Study of Asthma and Allergy in Childhood (ISAAC) questionnaire to categorize children as asthma patients with doctor’s diagnosed asthma as a robust outcome parameter (Ku et al., 2015; Toren et al., 2005). In Belgium, around 8.5% of the children aged 13–14 years have self-reported asthmatic symptoms (WHO, 2007). Exposure to chemicals has been associated with allergic sensitization of the respiratory tract and with rhinitis and asthma, mainly in the occupational setting (Kimber et al., 2014). Recently, epidemiological studies of the general population have linked exposure to commonly used phthalate plasticizers, such as di(2ethylhexyl) phthalate (DEHP), to increased risks for developing allergies and asthma; however, the conclusions are not always consistent (Bornehag and Nanberg, 2010). Phthalates are chemical plasticizers widely used in consumer products such as building materials, toys, food packaging, cosmetics, and medical devices (Schettler, 2006). Because phthalates are not chemically bound to products, they can easily diffuse within materials, leach out, and then disperse into the air or adhere to airborne particles and settled dust (Bamai et al., 2014; Fujii et al., 2003). Contaminated food and polyvinyl chloride (PVC) containing building materials are considered to be the most important sources of exposure to high molecular weight (HMW) phthalates (e.g. DEHP and butylbenzyl phthalate (BBzP); metabolites >250 Da) for the general population (Koch et al., 2013; Schettler, 2006; Wittassek et al., 2011). The use of cosmetics and personal care products may increase exposure to low molecular weight (LMW) phthalates (e.g. dibutyl phthalate (DBP), diisobutyl phthalate (DIBP) and diethyl phthalate (DEP)) (Bertelsen et al., 2013; Just et al., 2010; Koch et al., 2013; Silva et al., 2004; Wittassek et al., 2011). Phthalates are rapidly metabolized and excreted in urine and faeces; most of the metabolites are excreted in urine within 24 h (Anderson et al., 2001; Koch et al., 2005). Concentrations of phthalate metabolites in urine provide an integrated measure of phthalate exposure from all possible sources (Hogberg et al., 2008). Although some phthalates are best known for their action as endocrine disruptors, there is also evidence, mainly from in vitro and animal studies, that phthalates may impact immune and allergic responses. One of the hypotheses is that phthalates act as adjuvants; this means that they have no intrinsic sensitizing properties but may enhance immune responsiveness (Bornehag and Nanberg, 2010). However, the mode of action at exposure levels that reflect current human exposures is not yet well understood. Oxidative damage by release of reactive oxygen species (ROS) and/or impairing antioxidant defenses has been attributed to some phthalates, such as DEHP and its main metabolite mono(2ethylhexyl)-phthalate (MEHP), by in vitro and animal studies (Erkekoglu et al., 2010; Tetz et al., 2013). Only two studies examined this association in humans. Both Guo et al. (2014b) and Ferguson

et al. (2015) observed positive associations between both HMW and LMW phthalate metabolites and systemic markers of oxidative stress including 8-hydroxydeoxyguanosine (8-OHdG) in adults. Oxidative stress is a well-accepted mechanism in the pathogenesis of asthma since it can initiate and augment inflammation (Finkel and Holbrook, 2000; Peterson et al., 1998; Sugiura and Ichinose, 2008). However, to our knowledge, no human study so far investigated if oxidative stress, by excision of 8-OHdG from damaged DNA, may act as a mediator in the association between phthalate exposure and doctor-diagnosed asthma. Therefore, we investigated in 14–15-year-old Flemish adolescents whether phthalate exposure is associated with oxidative stress and an increased risk of being diagnosed with asthma. We focused on youngsters as they are uniquely vulnerable at the pubertal stage and are increasingly exposed to personal care products at that age. In a next step, we evaluated if oxidative stress is the mechanism underlying the association between phthalate exposure and doctor-diagnosed asthma. 2. Material and methods 2.1. Study population Within the 2nd and 3rd Flemish Environment and Health Study (FLEHS II: N = 210 [May 2008-July 2009]; FLEHS III: N = 208 [March 2013-December 2013]), two cohorts of 14–15-year-old adolescents were recruited from the general population of Flanders. The recruitments resulted in a total number of 418 youngsters. Both study populations have been described before (De Craemer et al., 2016; Geens et al., 2014). The study protocols were approved by the Ethical Committee of the University of Antwerp, and informed consent was obtained from all participants. 2.2. Anthropometric data and collection of urine Body weight and height of the children was measured with standardized equipment. Body Mass Index (BMI) was calculated as the weight/height2 (kg/m2 ) and classes were based on Belgian growth curves, specific for age and sex (http://www.vub.ac.be/ groeicurven/groei-curven.html). Each participant donated a urine sample of about 200 mL for subsequent analysis. In the FLEHS II study, morning urine was collected at home on the day of the examination. In FLEHS III, a spot urine sample was collected at field examination during school hours. All samples were transported to the central laboratory within one day after sampling. 2.3. Questionnaires All participants completed an extensive questionnaire at home, providing information about educational level, housing, life style, food intake, tobacco-smoke, health status and medication. Questions on respiratory health (asthma, wheezing, hay fever, rhinitis and eczema) were based on the ISAAC questionnaire (ISAAC, 1993). We decided to use the question “Has a doctor ever diagnosed your child as asthmatic?” as the gold standard for asthma. Additionally, a

Please cite this article in press as: Franken, C., et al., Phthalate-induced oxidative stress and association with asthma-related airway inflammation in adolescents. Int. J. Hyg. Environ. Health (2017), http://dx.doi.org/10.1016/j.ijheh.2017.01.006

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short questionnaire on recent exposure during the past three days was completed. 2.4. Urinary phthalate metabolites Urine samples were frozen and stored at −20 ◦ C in the dark until analysis. The recipients were tested prior to urine collection and were found to be phthalate free. Seven urinary phthalate metabolites (four HMW metabolites (MEHP: mono(2ethylhexyl) phthalate, MEHHP: mono(2-ethyl-5-hydroxyhexyl) phthalate, MEOHP: mono(2-ethyl-5-oxohexyl) phthalate, and MBzP: mono-benzyl phthalate); and three LMW metabolites (MnBP: mono-n-butyl phthalate, MEP: mono-ethyl phthalate, and MiBP: mono-isobutyl phthalate)) were measured by ultraperformance liquid chromatography-tandem mass spectrometry after enzymatic cleavage of the conjugated compounds as described by Servaes et al. (2013). The two LMW metabolites, MEP and MiBP respectively, were only measured in the FLEHS III study population. Analytical performance was described in Geens et al. (2014). Biomarker values were reported if they were above the limit of quantification (LOQ) as determined during validation in the laboratory. The LOQ for all phthalate metabolites ranged from 0.1 to 10 ␮g/L in the FLEHS II study and from 0.1 to 0.5 ␮g/L in the FLEHS III study. Values below the LOQ were replaced by LOQ/2. 2.5. Oxidative stress biomarker 8-hydroxydeoxyguanosine (8-OHdG) in urine Urine samples were frozen and stored at −80 ◦ C until 8-OHdG analysis. After thawing, they were centrifuged at 2000 × g for 15 min and 50 ␮L of the supernatant was used for the determination of 8-OHdG with a commercial competitive enzyme-linked immunosorbent assay kit (New 8-OHdG check ELISA kit; Japan Institute for the Control of Aging, Shizuoka, Japan) according to the manufacturer’s instructions. The anti-8-OHdG mouse monoclonal antibody (clone N45.1), with an established specificity, was used as a primary antibody (Toyokuni et al., 1997). The values from each urine sample were calculated based on calibration sigmoid plots of absorbance (450 nm) of an 8-OHdG standard at various concentrations. The determination range was 0.5–200 ng/mL. The LOQ of urinary 8-OHdG was for both studies 0.5 ␮g/L. The analytical procedure was qualified as demonstrated by participation to an international ring test (Rossner et al., 2016). 2.6. Statistical analysis Statistical analyses were done with IBM SPSS Statistics Version 23 (IBM Corp, Armonk, NY, US) and R Version 3.1.2 (R foundation for Statistical Computing, Vienna, Austria). Due to a non-normal distribution, the urinary biomarker concentrations were ln-transformed and described as geometric means with 95% confidence intervals (95% CI). For each of the biomarkers, the total number may be lower than 210 adolescents (FLEHS II) or 208 adolescents (FLEHS III) due to technical analytical problems or inadequate urinary volumes. To control for differences in urine dilution of spot urine samples, the urinary phthalate metabolite and 8-OHdG concentrations were adjusted for specific gravity (SG) using the following formula: Pc = P[(1.024-1)/(SG-1)], where Pc is the corrected biomarker concentration, P is the raw concentration, and SG is the specific gravity for the sample (Duty et al., 2005; Meeker et al., 2009). All phthalate metabolite concentrations were converted from micrograms per liter to micromoles per liter using molecular weights (MEHP: 278.34; MEHHP: 293.33; MEOHP: 292.33; MnBP: 222.24; MBzP: 256.25; MEP: 194.18; MiBP: 222.24 g/mol). In addition, phthalate metabolites derived from the same parent compound were combined creating the sum parameter DEHP (sum of the three

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di(2-ethylhexyl) phthalates MEHP, MEHHP and MEOHP). In line with previous studies (e.g. (Wolff et al., 2008)), we summed the HMW phthalate metabolites (HMW: sum of DEHP and MBzP), LMW phthalate metabolites (LMW sum of MnBP, MEP and MiBP) and all phthalate metabolites (HMW + LMW: sum of DEHP, MBzP, MnBP, MEP and MiBP). The Chi-square test was used to compare differences between the two studies for a discrete variable. Independent samples t-test was performed to compare means of biomarker concentrations between boys and girls.

2.6.1. Regression and mediation analyses To estimate the change in effect as a function of the exposure independent of other factors, single pollutant multiple linear and logistic regression models were fitted, adjusted for predefined literature-based influencing factors, independently of their statistical significance level i.e. sex, age, smoking status, cohort and familial asthma for doctor-diagnosed asthma. Effect estimates and their 95% CI were calculated. Effect modification by sex and familial asthma was analyzed in models including main effects and cross-product terms. A p-value less than 0.20 for the effect of the cross-product was taken as an indication of interaction. Mediation analysis was performed based on the procedure described by Baron and Kenny (1986) to investigate the mediating effect of 8-OHdG levels in urine, in the association between phthalate exposure and doctor-diagnosed asthma. A stepwise approach was followed in which four conditions must be met: 1) an established association between the urinary phthalate metabolite and doctor-diagnosed asthma; 2) an established relationship between the urinary phthalate metabolite and urinary 8-OHdG; 3) an association between urinary 8-OHdG and doctor-diagnosed asthma; and 4) after adjustment for urinary 8-OHdG, the association between the urinary phthalate metabolite and doctor-diagnosed asthma is no longer significant.

3. Results The adolescent cohorts from FLEHS II and III comprise 418 youngsters. There were 216 (51.7%) boys and 202 (48.3%) girls, with a mean age of 14.8 ± 0.5 years for boys and girls respectively. The main characteristics for both groups are presented in Table 1. Of all participants 11.2% smoked. In comparison with the FLEHS II study, twice as many individuals in the FLEHS III study were underweighted (BMI range: 13.9-16.8 kg/m2 for boys, 14.7–17.7 kg/m2 for girls, p-value = 0.004). Because of the selection of participants through schools, individuals were not recruited during the summer months. A prevalence of 15.6% familial asthma was reported by questionnaire survey; 14.8% adolescents reported asthma in the past year and 8.6% had asthma diagnosed by a doctor. Concentrations of urinary phthalate metabolites and oxidative stress biomarkers, corrected for SG and stratified by sex, of the two study populations are presented in Table 2. Except for DEHP and MnBP, all samples had concentrations above the LOQ. Notably, for the biomarkers measured in both studies, concentrations in FLEHS III were significantly lower compared to FLEHS II (p-value < 0.001). When phthalate metabolites and oxidative stress biomarker concentrations were compared by sex, we observed significantly higher urinary concentrations of MnBP, MBzP and 8-OHdG for boys compared with girls (geometric mean concentrations in boys vs. girls: MnBP: 0.17 vs. 0.14 ␮mol/L, MBzP: 0.07 vs. 0.05 ␮mol/L, 8-OHdG: 15.6 vs. 14.0 ␮g/L; p-value = 0.003–0.01). However, the reverse was seen for urinary MEP (0.15 vs. 0.36 ␮mol/L; pvalue < 0.001).

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Table 1 The main characteristics of the two adolescent populations by sex.

Variable Age ≤14.5 year 14.5–15.5 year >15.5 year Current smoking Missing BMI classa Underweight Normal weight Overweight Alcohol consumption Never Less than monthly Less than weekly Weekly Missing Educational level Artistic Vocational Technical General Missing Season of sampling Autumn Winter Spring Summer Familial occurrence of asthma Missing Doctor-diagnosed asthma Missing Current asthma Missing Hay fever ever Missing

Adolescents FLEHS II (N = 210)

Adolescents FLEHS III (N = 208)

All adolescents

Boys (N = 121)

Girls (N = 89)

Boys (N = 95)

Girls (N = 113)

(N = 418)

39 (32.2) 70 (57.9) 12 (9.9) 9 (7.4) 1 (0.8)

28 (31.5) 53 (59.6) 8 (9.0) 9 (10.1) 2 (2.2)

16 (16.8) 74 (77.9) 5 (5.3) 12 (12.6) 0 (0.0)

39 (34.5) 60 (53.1) 14 (12.4) 17 (15.0) 3 (2.7)

122 (29.2) 257 (61.5) 39 (9.3) 47 (11.2) 6 (1.4)

8 (6.6) 100 (82.6) 13 (10.7)

12 (13.5) 68 (76.4) 9 (10.1)

20 (21.1) 69 (72.6) 6 (6.3)

24 (21.2) 74 (65.5) 15 (13.3)

64 (15.3) 311 (74.4) 43 (10.3)

16 (13.2) 69 (57.0) 28 (23.1) 5 (4.1) 3 (2.5)

14 (15.7) 57 (64.0) 14 (15.7) 3 (3.4) 1 (1.1)

48 (50.5) 24 (25.3) 17 (17.9) 6 (6.3) 0 (0.0)

59 (52.2) 31 (27.4) 15 (13.3) 6 (5.3) 2 (1.8)

137 (32.8) 181 (43.3) 74 (17.7) 20 (4.8) 6 (1.4)

0 (0.0) 15 (12.4) 61 (50.4) 43 (35.5) 2 (1.7)

0 (0.0) 5 (5.6) 25 (28.1) 58 (65.2) 1 (1.1)

2 (2.1) 13 (13.7) 24 (25.3) 56 (58.9) 0 (0.0)

8 (7.1) 22 (19.5) 44 (38.9) 39 (34.5) 0 (0.0)

10 (2.4) 55 (13.2) 154 (36.8) 196 (46.9) 3 (0.7)

40 (33.1) 27 (22.3) 54 (44.6) 0 (0.0) 19 (15.7) 6 (5.0) 14 (11.6) 4 (3.3) 20 (16.5) 5 (4.1) 32 (26.4) 2 (2.2)

7 (7.9) 35 (39.3) 47 (52.8) 0 (0.0) 12 (13.5) 13 (14.6) 4 (4.5) 14 (15.7) 11 (12.4) 13 (14.6) 18 (20.2) 12 (13.5)

34 (35.8) 13 (13.7) 48 (50.5) 0 (0.0) 13 (13.7) 7 (7.4) 8 (8.4) 7 (7.4) 15 (15.8) 7 (7.4) 19 (20.0) 8 (8.4)

59 (52.2) 23 (20.4) 31 (27.4) 0 (0.0) 21 (18.6) 0 (0.0) 10 (8.8) 0 (0.0) 16 (14.2) 2 (1.8) 21 (18.6) 3 (2.7)

140 (33.5) 98 (23.4) 180 (43.1) 0 (0.0) 65 (15.6) 26 (6.2) 36 (8.6) 25 (6.0) 62 (14.8) 27 (6.5) 90 (21.5) 28 (6.7)

Data are numbers and percentages [N (%)]. Abbreviations: N, number of adolescents. a Cut-off values for BMI classes were based on Belgian growth curves, specific for age and sex (http://www.vub.ac.be/groeicurven/groeicurven.html).

Table 2 Descriptive statistics of the different phthalate metabolites and oxidative stress biomarker stratified by sex, measured in individual urine samples from both study populations. Adolescents FLEHS II Biomarker DEHP (␮mol/L) Boys Girls MnBP (␮mol/L) Boys Girls MBzP (␮mol/L) Boys Girls MEP (␮mol/L) Boys Girls MiBP (␮mol/L) Boys Girls 8-OHdG (␮g/L) Boys Girls

Adolescents FLEHS III

All adolescents

% < LOQ

N

Geometric mean (95% CI)

% < LOQ

N

Geometric mean (95% CI)

Geometric mean (95% CI)

7.4% 12.4%

118 89

0.22 (0.20–0.24) 0.21 (0.19–0.24)

2.1% 5.3%

94 113

0.07 (0.06–0.08) 0.07 (0.07–0.08)

0.13 (0.12–0.15) 0.12 (0.11–0.13)

0% 5.6%

119 89

0.21 (0.19–0.24) 0.19 (0.16–0.21)

0% 0%

94 113

0.13 (0.11–0.15) 0.12 (0.10–0.13)

0.17 (0.16–0.19) 0.14 (0.13–0.16)

0% 0%

119 89

0.16 (0.13–0.19) 0.12 (0.10–0.15)

0% 0%

94 113

0.03 (0.02–0.03) 0.02 (0.02–0.03)

0.07 (0.06–0.09) 0.05 (0.04–0.06)

0% 0%

0% 0%

94 113

0.15 (0.12–0.18) 0.36 (0.27–0.48)

0.15 (0.12–0.18) 0.36 (0.27–0.48)

0% 0%

0% 0%

94 113

0.13 (0.11–0.15) 0.13 (0.12–0.15)

0.13 (0.11–0.15) 0.13 (0.12–0.15)

0% 0%

94 113

14.1 (13.1–15.2) 12.5 (11.5–13.6)

15.6 (14.8–16.4) 14.0 (13.1–15.0)

0% 0%

117 85

16.9 (15.8–18.1) 16.4 (14.9–18.1)

Urinary biomarkers were corrected for SG to take into account variations in urine dilution. MEHP, MEHHP and MEOHP were summed to create DEHP. Abbreviations: LOQ: limit of quantification; N: number of adolescents; CI: confidence interval; DEHP: di(2-ethylhexyl) phthalate; MnBP: mono-n-butyl phthalate; MBzP: mono-benzyl phthalate; MEP: mono-ethyl phthalate; MiBP: mono-isobutyl phthalate; 8-OHdG: 8-hydroxydeoxyguanosine.

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3.1. Exposure-response associations 3.1.1. Between urinary phthalate metabolites and doctor-diagnosed asthma The change in odds for being diagnosed with asthma in association with an increase from P25 to P75 in phthalate metabolite concentration is illustrated in Fig. 1. Based on the pooled data of FLEHS II and FLEHS III adolescents, a statistically significant association was observed between DEHP (OR = 1.94 [95% CI: 1.07, 3.51]) and MnBP (OR = 1.84 [95% CI: 1.02, 3.32]) and doctor-diagnosed asthma. Doctor-diagnosed asthma was not significantly associated with urinary concentrations of MBzP, after analyzing the two study populations separately or taking them together. Also, the two metabolites MEP and MiBP, which were only measured in FLEHS III, showed no significant associations with asthma diagnosed by a doctor. Combining the data from the two studies tends to give more precise estimates of effects, hence with narrower confidence intervals. The sum of HMW, LMW or all phthalate metabolites showed no significant associations with doctor-diagnosed asthma (Supplemental Table S1). There was no effect modification by sex in the association between individual phthalate metabolites or their sums, and doctor-diagnosed asthma (p-interaction > 0.20). Effect modification by familial asthma was only observed for DEHP (pinteraction = 0.19), where a significant association was obtained with this pollutant only in the adolescents without familial asthma (OR = 2.46 [95% CI: 1.24, 4.88]). 3.1.2. Between urinary phthalate metabolites and 8-OHdG Fig. 2 shows the effect estimate with urinary 8-OHdG concentrations for an increase from P25 to P75 in phthalate metabolite concentration. All phthalate metabolites were significantly associated with higher 8-OHdG concentrations in the two separate populations, but also when both populations were combined. The largest percent change with an increase from P25 to P75 in exposure was observed after pooling the data of the phthalate metabolites measured in both cohorts i.e. 25% (95% CI: 16, 34%) for DEHP, 23% (95% CI: 17, 30%) for MnBP and 25% (95% CI: 16, 35%) for MBzP respectively. After taking the sum of the HMW, LMW or all metabolites, a statistically significant association was observed for each sum component with 8-OHdG (HMW: 16% [95% CI: 11, 20%]; LMW: 9% [95% CI: 7, 12%]; HMW + LMW: 7% [95% CI: 5, 9%]) (Supplemental Table S1). Sex modified the association between the individual phthalate metabolites and urinary levels of 8-OHdG (p-interaction ≤ 0.0010.13). The effect estimate of 8-OHdG for each metabolite was higher in girls than in boys. The associations were statistically significant in both sexes (p-value ≤ 0.001-0.02) except for MEP where significance was reached only in girls (p-value(girls) < 0.001). 3.2. Oxidative stress as a mediator between phthalates and asthma To verify whether the associations between urinary MnBP and DEHP, and the risk of being diagnosed with asthma is mediated through urinary 8-OHdG, we tested if the requirements to demonstrate mediation were met according to Baron and Kenny (Fig. 3). Doctor-diagnosed asthma showed a positive association with both DEHP and MnBP (Fig. 3, model 1: OR(DEHP) = 1.94 [95% CI: 1.07, 3.51]; OR(MnBP) = 1.84 [95% CI: 1.02, 3.32]) (Fig. 1). The associations between these two metabolites and urinary 8-OHdG (Fig. 2) were highly significant (Fig. 3, model 2: ␤(DEHP) = 1.25 [95% CI: 1.16, 1.34]; ␤(MnBP) = 1.23 [95% CI: 1.17, 1.30]). However, there was no significant association between 8-OHdG and doctor-diagnosed asthma (Fig. 3, model 3: p-value = 0.46). After adjustment for urinary 8-OHdG, the associations between the two urinary phthalate metabolites DEHP and MnBP and

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doctor-diagnosed asthma remained significant (Fig. 3, model 4: OR(DEHP) = 2.06 [95% CI: 1.09, 3.90]; OR(MnBP) = 1.97 [95% CI: 1.04, 3.72]). Additionally, the OR for doctor-diagnosed asthma in model 4 was slightly higher compared to model 1. Based on the results of model 3 and 4, 8-OHdG could not be considered as a (partial) mediator in the associations between DEHP and MnBP, and the risk of being diagnosed with asthma. 4. Discussion Epidemiologic findings suggest that phthalates may be associated with an increased risk of allergies and being diagnosed with asthma in children. In our study we explored the links between phthalate exposure, oxidative stress and doctor-diagnosed asthma. 4.1. Phthalate exposure and asthma We observed significant associations between the three urinary DEHP metabolites and urinary MnBP, and doctor-diagnosed asthma, which were not influenced by sex. There was effect modification by familial asthma as a significant association between DEHP and doctor-diagnosed asthma was obtained only in adolescents without familial asthma. This indicates that phthalate exposure may exert a significant effect on the risk of asthma when there is no genetic burden. Several studies in literature examined the association of phthalates with asthma. Among Swedish children 3–8 years of age, house dust concentrations of DEHP were associated with physician-confirmed asthma (Bornehag et al., 2004). Similar results were found in Bulgaria and Denmark, where positive associations between house dust DEHP concentrations and child asthma (Kolarik et al., 2008) and current wheeze (Callesen et al., 2014) were reported. Also increasing levels of DBP in dust were found to be associated with diagnosed eczema. Additionally, its urinary metabolite MnBP was related to diagnosed asthma (Hsu et al., 2012). The studies, that measured individual phthalate metabolites in urine and examined their relations with allergic diseases, were not always consistent. For example, positive associations were found between two urinary phthalate metabolites of DINP and DIDP, mono(carboxyoctyl) phthalate (MCOP) and mono(carboxynonyl) phthalate (MCNP), and current asthma in a cross-sectional study of 623 Norwegian 10-year-old children (Bertelsen et al., 2013). Beko et al. (2015) observed a lack of association for MnBP and DEHP in relation to allergic sensitization in a case-control study (N = 500) of 3–5 year old Danish children. Another recent cross-sectional study among participants of NHANES (National Health and Nutrition Examination Study) found increasing urinary concentrations of MBzP, but not DEHP or MnBP, to be significantly associated with current asthma, wheeze, hay fever, and rhinitis in adults; this was not observed in children 6–17 years of age (Hoppin et al., 2013). 4.2. Phthalate exposure and oxidative stress Since asthma is a complex disease that may include several endotypes characterized by different biological processes, we have tested whether oxidative stress may be a component that mediates the association between exposure to phthalates and increased occurrence of asthma in exposed children. We observed significant associations between all individual and summed up urinary phthalate metabolites, and urinary levels of 8-OHdG. Urinary 8-OHdG has been commonly used as a systemic biomarker of oxidative stress for establishing associations with long-term health outcomes including asthma, neurological and reproductive effects, and cancer (Il’yasova et al., 2012; Loft et al., 1992). The formation of 8-OHdG is a result of direct interactions

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Fig. 1. Change in odds for being diagnosed with asthma in association with an increase from P25 to P75 in SG-corrected phthalate metabolite concentration (␮mol/L) measured in adolescents of the FLEHS II and III study. Interpretation OR: OR for increase of the urinary phthalate metabolite concentration from P25 to P75 with doctor-diagnosed asthma. The model was adjusted for gender, age, smoking status and familial asthma; in the pooled analysis cohort was included. * Statistically significant association (p-value < 0.05). Statistically significant interaction with sex if p-interaction < 0.20. MEHP, MEHHP and MEOHP were summed to create DEHP. Abbreviations: OR, odds ratio; CI, confidence interval; N, number of cases included in the model; DEHP, di(2-ethylhexyl) phthalate; MnBP, mono-n-butyl phthalate; MBzP, mono-benzyl phthalate; MEP, mono-ethyl phthalate; MiBP, mono-isobutyl phthalate

Fig. 2. Change in SG-corrected urinary 8-OHdG concentrations (␮g/L) in association with an increase from P25 to P75 in SG-corrected phthalate metabolite level (␮mol/L) measured in adolescents of the FLEHS II and III study. Interpretation estimate: multiplicative factor for increase of the urinary phthalate metabolite concentration from P25 to P75 with urinary 8-OHdG concentration. * Statistically significant association (p-value < 0.05). Statistically significant interaction with sex if p-interaction < 0.20. The model was adjusted for gender, age and smoking status; in the pooled analysis cohort was included. MEHP, MEHHP and MEOHP were summed to create DEHP. Abbreviations: CI, confidence interval; N, number of cases included in the model; DEHP, di(2-ethylhexyl) phthalate; MnBP, mono-n-butyl phthalate; MBzP, mono-benzyl phthalate; MEP, mono-ethyl phthalate; MiBP, mono-isobutyl phthalate.

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Fig. 3. Mediation analysis to investigate the mediating effect of urinary 8-OHdG in the associations between urinary (A) DEHP and (B) MnBP, and doctor-diagnosed asthma. Interpretation ␤: multiplicative factor for increase of the urinary phthalate metabolite concentration from P25 to P75 with urinary 8-OHdG concentration. Interpretation OR: OR for increase of the urinary phthalate metabolite concentration from P25 to P75 with doctor-diagnosed asthma. Model 2 was adjusted for cohort, gender, age and smoking status. Model 1, 3 and 4 were adjusted for cohort, gender, age, smoking status and familial asthma; in model 4 8-OHdG was included. Urinary biomarkers were corrected for SG to take into account variations in urine dilution. MEHP, MEHHP and MEOHP were summed to create DEHP. Abbreviations: DEHP, di(2-ethylhexyl) phthalate; MnBP, mono-n-butyl phthalate; 8-OHdG, 8-hydroxydeoxyguanosine; OR, odds ratio; ␤, estimate.

between ROS and DNA (precursors). Upon DNA repair or oxidation of the DNA pool present in the cell, 8-OHdG is excreted in urine (Cooke et al., 2008; Poulsen et al., 2014). In line with our study in adolescents, recent epidemiologic studies in adults have reported links between urinary phthalate metabolites with increased levels of urinary markers of oxidative stress, e.g. 8-OHdG (Ferguson et al., 2015; Guo et al., 2014b), malondialdehyde (Kim et al., 2013; Kim et al., 2014) and 8-isoprostane (Ferguson et al., 2014). We observed an influence of sex on the association between urinary phthalate metabolites and 8-OHdG concentrations. For MEP, there was a strong association in girls, but this was not observed for boys. It may be related to the smaller range in MEP concentration in boys. Indeed girls reported to use more personal care products, and DEP, the parent compound of MEP, is an ingredient in many personal care products like colognes, deodorants, and fragranced products (Den Hond et al., 2013; Duty et al., 2005; Koo and Lee, 2004). For the other phthalates, urinary phthalate metabolite concentrations, both in unadjusted and density adjusted samples, were higher in boys compared to girls, while concentrations were equally distributed between boys and girls. Nevertheless, from the

higher estimates and smaller p-values, we could deduce that the associations between the individual urinary phthalate metabolites and 8-OHdG concentrations were stronger in girls than in boys. Also Guo et al. (2014b) reported sex-specific effects on phthalate-associated oxidative stress; more significant associations between urinary phthalate metabolites and 8-OHdG were found in females. Recent studies among heavy smokers and coke oven plant workers reported significantly higher levels of 8-OHdG and 8-iso-PGF2a in women and also lower levels of erythrocyte antioxidant enzymes than observed in men (Guo et al., 2014a; Hakim et al., 2012). Emerging evidence indicates that if the exposures are the same, women may be at greater risk for adverse health effects like lung cancer than men. This is probably because the elevated activity of CYP-enzymes in women may convert chemicals to potent oxidants and oxidizers, and in turn produce higher levels of DNA adducts. In addition, women were found to have a DNA repair capacity that is 10–15% lower than that in men, which could also contribute to the gender differences in oxidative stress response (Mollerup et al., 2006; Thomas et al., 2005; Uppstad et al., 2011; Wei et al., 2000).

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4.3. Oxidative stress-mediated link between phthalate exposure and asthma Despite an inconclusive debate about whether the enhanced oxidative stress observed in asthma subjects is caused by inflammation or is a causative factor in the pathogenesis of the disease, many reports have supported the critical role of oxidative stress in the development of various chronic immunologic diseases. ROS such as hydrogen peroxide (H2O2) transfer stimulating signals as a critical intracellular second messenger, resulting in the modulation of immune responses (Cho and Moon, 2010). In our study we could not demonstrate the mediating effect of oxidative stress in the association between phthalate exposure and asthma, since no significant association was found between urinary 8-OHdG concentration and doctor-diagnosed asthma. There are a number of possible explanations for the lack of this association. Our study included 10% asthmatics, which were not asked to refrain from using asthma medication. The (potential) use of medication may have resulted in suppressing (the systemically measured) oxidative stress. Furthermore, urinary 8-OHdG might not be the optimal marker for assessing oxidative stress reported to occur in asthmatic patients. Recently, Ben Anes et al. (2016) reported changes in asthmatic patients of some systemic antioxidant molecules (total thiols, protein sulfhydryls, glutathione in plasma) and some systemically measured (in plasma or blood) oxidative stress markers, such as lipid peroxidation products (malondialdehyde), but not in protein oxidation and DNA damage markers (comet assay). 4.4. Possible mechanisms of phthalate-induced asthma Identifying the mechanisms whereby phthalate exposure is associated with doctor-diagnosed asthma remains an important area of research. More knowledge would increase the evidence base for the association. Despite associations between all phthalate metabolites and the oxidative stress biomarker, only the sum of the three DEHP metabolites and MnBP were associated with doctor’s diagnosed asthma. It is clear from in vitro and in vivo animal studies, that phthalates have different modes of action by which they may influence immune and inflammatory parameters that may play a role in complex respiratory diseases such as asthma. Depending on the concentration, exposure route and the length of the carbon side chain, phthalates stimulated in mice Th2 differentiation, produced Th2 cytokines and enhanced levels of Th2 promoted immunoglobulins (mainly IgG1 but also IgE) (Larsen et al., 2002; Larsen et al., 2003; Larsen and Nielsen, 2008). A limited amount of data also suggest phthalate-induced enhancement of mast cell degranulation and eosinophilic infiltration which are important parts in the early phase of inflammatory response (Bornehag and Nanberg, 2010; Kimber and Dearman, 2010). Phthalates have been found to affect the immune system by increasing the production of inflammatory cytokines, including tumor necrosis factor ␣ (TNF-␣), interleukin 1␤ (IL-1␤), IL-18, and IL-6 (Nishioka et al., 2012; Wang et al., 2012). Pro-IL-1␤ needs to be cleaved to become biologically active. This is achieved by the engagement of the inflammasome. Inflammasomes are multi-protein signaling complexes that trigger the activation of inflammatory caspases and additionally the maturation of IL-1␤. Therefore, they play an important role in regulating innate immunity and inflammatory responses. Certain physiological events including ion fluxes, endosomal rupture, production of ROS and mitochondrial dysfunction have been repeatedly shown to trigger the activation of the inflammasome (Abderrazak et al., 2015; Ni et al., 2016). As phthalate metabolites are capable of producing ROS and consequently oxidative stress in various cell types, they may contribute to this pathway as well (Erkekoglu et al., 2010; Tetz et al., 2013).

The prevalence of asthma is greater in boys than in girls during prepubescent ages; however, after puberty, this trend reverses, suggesting that the adolescent period may be a sensitive window for developing asthma symptoms (Almqvist et al., 2008; Choi, 2011; Osman et al., 2007). A possible explanation for the switch around puberty may include hormonal changes. Female hormones (i.e. estrogens) appear to play a significant role, e.g. by acting on intermediary factors, in allergic disease. Estrogen’s influences on immune cells favor the allergic response promoting Th2 polarization, encouraging class switching of B cells to IgE production and prompting mast cell and basophil degranulation. In addition, there is evidence that not only endogenous estrogens exert effects in allergic disease, but also that exogenous compounds with estrogenic activity (xenoestrogens) may play a role in asthma and other allergic disorders (Bonds and Midoro-Horiuti, 2013; Narita et al., 2007). Due to the fact that testosterone is an immunosuppressant and is likely to be protective against immunological and inflammatory processes that trigger asthma, selective androgen receptor modulators, such as some phthalates, could have an impact on the risk of asthmatic attacks (Canguven and Albayrak, 2011). Finally, toxicological studies have shown that phthalates can activate a subset of peroxisome proliferator-activated receptors (PPARs), that have been identified as important immunomodulators and potentially novel anti-inflammatory targets for airway diseases. Possible modulatory effects of phthalates on PPARs may include partial antagonist effects, which may interrupt anti-inflammatory signaling and therefore possibly contribute to inflammatory effects; dysregulation of gene transcription, causing a different transcriptional activation as compared to the endogenous PPAR ligands; and/or cross talk with other signaling pathways (Kocbach et al., 2013). 4.5. Strengths and limitations of the study The strength of this study was that by pooling the data, a larger sample size (N = 418) was obtained to explore the association between specific urinary phthalate metabolites and doctor-diagnosed asthma, and urinary 8-OHdG. Although the exposure levels were considerably different between the two study populations, similar results were obtained in both campaigns, indicating independent validation. Pooling data of the two cohort studies was done by including cohort as a fixed factor to the regression model. However, this was probably not enough to describe fully the heterogeneity in the statistical models. Due to the cross-sectional nature of this study, reverse causality cannot be ruled out; however, and as mentioned below, the use of population-based prospective cohort studies may help to solve or minimize this problem. Phthalates have also been shown to be correlated with personal life style characteristics, food consumption habits and product uses, and exposure often occurs together with other environmental pollutants (Smit et al., 2015). Therefore, we have to bear in mind that the observed associations with urinary 8-OHdG and doctor-diagnosed asthma might also result from combined exposures. Hence, future studies should consider the entire exposome. Another limitation of the study was the collection of spot urine samples, that are relatively easy to collect, but significant variability may exist in the data with respect to exposure prediction. This may result from fluctuations in time in liquid consumption and kidney function, short biological half-lives of phthalate metabolites and their low intraclass-correlation coefficients (Aylward et al., 2016; Johns et al., 2015). More urine samples would be needed to obtain more robust exposure information. In addition, urinary biomarkers were measured in both spot urine collected during the day (FLEHS III study) and morning (FLEHS II study) urine samples. The latter might be (for some phthalates) of minor importance as Wittassek

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et al. (2007) have demonstrated that spot urine samples and 24h urine samples produce comparable estimates of daily phthalate intake, i.e. for DEHP due to a ‘chronic exposure pattern’. On the other hand, identification of the causal path might be still complicated by linking current exposure biomarkers, that reflect exposure over the last day (e.g. urinary phthalate metabolites), with effects that have developed earlier in life (e.g. asthma), or by using a biomarker (8-OHdG) that is measured in urine at the same time as the exposure. Indeed, several studies suggest that perinatal and early childhood are susceptible periods for asthma onset later in life (Gascon et al., 2015; Ku et al., 2015; Smit et al., 2015; Whyatt et al., 2014). In addition, biomarker-related differences in physiological processes and metabolism of asthma patients are commonly observed. Therefore, prospective studies are required to investigate further causality of the association between phthalate exposures and doctor-diagnosed asthma. 5. Conclusion Overall our findings demonstrate that in Western-European adolescents, exposed to detectable levels of phthalates, asthma diagnosed by a doctor was significantly associated with urinary MnBP and the sum of the three DEHP metabolites. This finding adds to the increasing literature on DEHP exposure and asthma. However, no significant association was found between doctordiagnosed asthma and the individual (or summed up) phthalate metabolites MBzP, MEP and MiBP. A highly significant association of phthalate exposure with oxidative stress was observed in our study for both HMW and LMW phthalates. This finding adds to the increasing evidence that phthalates activate oxidative stress and immune responses. The fact girls seem more responsive than boys may indicate that hormonal systems may be implied, however, the exact biological pathways are not yet well understood. The use of single spot urine samples and the cross-sectional nature of this study as well as life style associated co-exposures warrant cautious interpretation of the results. Acknowledgments This study was commissioned, financed and steered by the Ministry of the Flemish Community (Department of Economics, Science and Innovation; Flemish Agency for Care and Health; and Department of Environment, Nature and Energy). Carmen Franken was supported by a PhD fellowship from the University of Antwerp. The authors would like to thank all co-workers and participants in the study. 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.ijheh.2017.01. 006. References Abderrazak, A., Syrovets, T., Couchie, D., El, H.K., Friguet, B., 2015. Simmet T, et al. NLRP3 inflammasome: from a danger signal sensor to a regulatory node of oxidative stress and inflammatory diseases. Redox Biol. 4, 296–307, http://dx. doi.org/10.1016/j.redox.2015.01.008, S2213-2317(15)00009-9 {pii]. Almqvist, C., Worm, M., Leynaert, B., 2008. Impact of gender on asthma in childhood and adolescence: a GA2LEN review. Allergy 63, 47–57, http://dx.doi. org/10.1111/j.1398-9995.2007.01524.x, ALL1524 [pii]. Anderson, W.A., Castle, L., Scotter, M.J., Massey, R.C., Springall, C., 2001. A biomarker approach to measuring human dietary exposure to certain phthalate diesters. Food Addit. Contam. 18, 1068–1074, http://dx.doi.org/10. 1080/02652030110050113. Aylward, L.L., Hays, S.M., Zidek, A., 2016. Variation in urinary spot sample, 24 h samples, and longer-term average urinary concentrations of short-lived environmental chemicals: implications for exposure assessment and reverse

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Please cite this article in press as: Franken, C., et al., Phthalate-induced oxidative stress and association with asthma-related airway inflammation in adolescents. Int. J. Hyg. Environ. Health (2017), http://dx.doi.org/10.1016/j.ijheh.2017.01.006