Article pubs.acs.org/est
Contribution of Persistent Organic Pollutant Exposure to the Adipose Tissue Oxidative Microenvironment in an Adult Cohort: A Multipollutant Approach Francisco Artacho-Cordón,†,‡ Josefa León,‡,§ José M. Sáenz,‡ Mariana F. Fernández,†,‡,∥ Piedad Martin-Olmedo,⊥ Nicolás Olea,†,‡,∥ and Juan P. Arrebola*,‡,⊥,# †
Radiology and Physical Medicine Department, University of Granada, Granada, 18012, Spain Instituto de Investigación Biosanitaria (ibs.GRANADA), Hospitales Universitarios de Granada, Granada, 18012, Spain § CIBER en Enfermedades Hepáticas y Digestivas (CIBEREHD), 28029 Madrid, Spain ∥ CIBER en Epidemiología y Salud Pública (CIBERESP), 28029 Madrid, Spain ⊥ Escuela Andaluza de Salud Pública, Granada, 18011, Spain # Oncology Unit, Virgen de las Nieves University Hospital, Granada, 18012 Spain
Environ. Sci. Technol. 2016.50:13529-13538. Downloaded from pubs.acs.org by TRINITY COLG DUBLIN on 11/01/18. For personal use only.
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S Supporting Information *
ABSTRACT: Despite growing in vitro and in vivo evidence of the putative role of persistent organic pollutants (POPs) in the induction of oxidative damage in cell structures, this issue has been poorly addressed from an epidemiologic perspective. The aim of this study was to explore associations between adipose tissue POP concentrations and the in situ oxidative microenvironment. A cross-sectional study was conducted in a subsample (n = 271) of a previously established cohort, quantifying levels of eight POPs and four groups of oxidative stress biomarkers in adipose tissue. Associations were explored using multivariate linear regression analyses adjusted for potential confounders. We assessed the combined effect of POPs on oxidative stress/glutathione system biomarkers using weighted quantile sum regression (WQS). Increased concentrations of p,p′-DDE, HCB, β-HCH, dicofol, and PCBs (congeners −138, −153, and −180) were predominantly associated with higher lipid peroxidation (TBARS) [exp(β) = 1.09−1.78, p < 0.01−0.04)] and SOD activity [exp(β) = 1.13−1.48, p < 0.01−0.05)] levels. However, only a few associations were observed with glutathione system biomarkers, e.g., PCB-180 with total glutathione [exp(β) = 1.98, p = 0.03]. The WQS index was found to be positively associated with SOD activity, and PCB-138, PCB-180, and β-HCH were the main contributors to the index. Likewise, the WQS index was positively associated with TBARS levels, with the three PCBs acting as the main contributors. This is the first epidemiological evidence of the putative disruption by POPs of the adipose tissue oxidative microenvironment. Our results indicate that POP exposure may enhance alternative pathways to the glutathione detoxification route, which might result in tissue damage. Further research is warranted to fully elucidate the potential health implications.
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INTRODUCTION There is increasing evidence of the role of free radicals in the pathogenesis of human diseases, including carcinogenesis, atherosclerosis, diabetes mellitus, and cardiovascular, hepatic, Alzheimer, and Parkinson diseases.1,2 The most abundant of these free radicals are reactive oxygen species (ROS), which include the superoxide free radical (O2−), hydrogen peroxide (H2O2), and hydroxyl free radical (OH−). These compounds are able to induce cell damage, and humans have developed complex mechanisms to keep them at bay. The best known pathway to remove intracellular ROS is the redox cycle. Briefly, O2− is transformed into H2O2 by superoxide dismutase (SOD), which is then neutralized and converted into water and oxygen by catalase (CAT) and glutathione peroxidase (GPX). For this © 2016 American Chemical Society
purpose, GPX needs to transform reduced glutathione (GSH) into oxidized glutathione (GSSG), which is finally restored by glutathione reductase (GRd).2 However, if the concentrations of these antioxidant activities become insufficient in the course of metabolism to decompose all of the H2O2 formed, this may undergo metal ion-catalyzed cleavage by the Fenton reaction to generate the even more toxic OH− radical, which is dependent upon the availability of iron and copper.3,4 Although it has been suggested that the glutathione cycle protects cell structures Received: Revised: Accepted: Published: 13529
July 28, 2016 November 2, 2016 November 16, 2016 November 16, 2016 DOI: 10.1021/acs.est.6b03783 Environ. Sci. Technol. 2016, 50, 13529−13538
Article
Environmental Science & Technology
semirural area). Participants were over 16 years old, with no hormone-related disease or cancer, and had lived in one of the two study areas for ≥10 years. Out of 409 individuals who were contacted, 387 (94.6%) agreed to participate in the study, although 116 (30.0%) additional individuals were excluded from the present study because an inadequate biological sample was available for the measurement of oxidative stress markers. No statistically significant differences in baseline characteristics were found between the initial (N = 387) and final study population (N = 271) (Supporting Information Table S1). All subjects signed their informed consent to participate in the study, which was approved by the Ethics Committee of each hospital. Measurement of Oxidative Stress Biomarkers. All biomarkers were analyzed using commercial available kits (Enzo Life Sciences, Inc., Farmingdale, NY, USA) in an automatic microplate reader (TRIAD MRX II series, Dynex Technologies Inc., Chantilly, Virginia, USA). Samples of adipose tissue were thawed slowly on ice and repeatedly washed with cold PBS to remove blood clots and other debris. Tissues were then homogenized in the appropriate buffer at the proportion specified by the kits, using a pestle. Total SOD activity [isozymes SOD1 (cytosolic Cu/Zn SOD), SOD2 (mitochondrial Mn SOD), and SOD3 (extracellular Cu/Zn SOD)] was measured with an assay in which superoxide ions are generated from the conversion of xanthine and oxygen to uric acid and hydrogen peroxide by xanthine oxidase. The superoxide anion then converts WST-1 (2-(4iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) to WST-1 formazan, a colored product that absorbs light at 450 nm. SOD reduces the superoxide ion concentration and thereby lowers the rate of WST-1 formazan formation. SOD was expressed as units per milligram protein. Hemeoxygenase-1 (HO-1) was assayed by a quantitative sandwich immunoassay. A mouse monoclonal antibody specific for HO-1 is precoated on the wells of the provided HO-1 immunoassay plate. HO-1 is captured by the immobilized antibody and detected with an HO-1 specific rabbit polyclonal antibody. The polyclonal antibody is subsequently bound by a horseradish peroxidase conjugated antirabbit IgG secondary antibody. The assay is developed with tetramethylbenzidine substrate, and a blue color develops in proportion to the amount of captured HO-1. The color development is stopped with an acid stop solution that converts the end point color to yellow, and the color intensity is then measured at 450 nm. The assay is specific for human HO-1 and does not cross-react with human HO-2 or HO-3. GPx and GRd activities were obtained by monitoring the oxidation of NADPH at 340 nm. The rate of decrease in the absorbance at 340 nm is directly proportional to the GPx and GRd activities in the sample, which were expressed as micromoles per minute milligram protein. GSH and GSSG were quantified by adding DTNB (5,5′dithiobis-2-nitrobenzoic acid, Ellman’s reagent) to produce a yellow-colored 5-thio-2-nitrobenzoic acid (TNB) that absorbs at 405 nm. The TNB production rate is directly proportional to the concentration of glutathione in the sample. GSH and GSSG levels were expressed in nanomoles per minute milligram protein. Measurement of lipid peroxidation (TBARS) was based on the reaction of 2-thiobarbituric acid with MDA (compound resulting from decomposition of polyunsaturated fatty acid lipid peroxides) at 25 °C to yield a chromophore with absorbance
from oxidation by H2O2, it does not inhibit the production of radicals by the Fenton reaction.5 A number of endogenous and exogenous sources of free radicals have been identified.6 The mitochondrion constitutes the main endogenous source, due to the generation of substantial levels of free radicals throughout the electron transport chain, while a wide variety of exogenous sources have been identified over recent decades.2 Exogenous sources include specific dietary and lifestyle patterns, physical exercise, some drugs (anesthetics or chemotherapeutics), as well as inadvertent exposure to certain environmental pollutants, such as ionizing radiation or chemical agents.6−9 In this regard, increasing evidence has been published to support the putative role in oxidative stress of widely used pesticides and industrial byproducts.10−12 Persistent organic pollutants (POPs) constitute a heterogeneous group of synthetic organic compounds used worldwide until the 1980s for insect control and sanitary purposes as well as in agriculture and industry. Although the majority of the countries have banned or severely restricted the use of POPs, considerable research efforts have raised awareness about the ongoing and continuous exposure of the general population, attributed to the high persistence and lipophilia of these compounds and their consequent bioaccumulation and biomagnification in the food chain.13 Long-term exposure to certain POPs has been acknowledged to induce adverse health outcomes at doses traditionally considered safe, including reproductive disorders, carcinogenicity, metabolic disruption, and epigenetic modulation.14−18 Suspected mechanisms of action include the POP-mediated induction of ROS. In fact, in vitro and in vivo studies have reported disturbances in oxidative stress biomarkers after acute exposure, mainly to organochlorine pesticides (OCPs) such as DDT19 or lindane20−23 and to polychlorinated biphenyls (PCBs).24 However, only a limited number of epidemiological studies have addressed the relationship between POPs and oxidative stress in humans.25 In this regard, it was recently reported that serum POP levels were inversely associated with the glutathione-related markers GSSG and GSSG/GSH ratio26 and positively correlated with biomarkers of cellular damage, such as malondialdehyde (MDA)27 and oxidized low-density lipoprotein.26 Considering that increased oxidative stress in adipose tissue has been acknowledged as an early pathogenic mechanism of obesity-related diseases, e.g. metabolic syndrome,28 it is therefore of interest to elucidate environmental factors affecting the local production of ROS, including chemicals that are preferentially stored in adipose tissue such as POPs. Thus, the objective of this study was to explore potential associations between long-term POP accumulation in adipose tissue and the oxidative microenvironment of this compartment in a human cohort from Southern Spain, where we have previously reported associations between accumulated levels of POPs in the adipose tissue and the risk of hypertension, obesity, or type 2 diabetes.
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MATERIAL AND METHODS Study Population: GraMo Cohort. The study population is a subsample of a previously established adult cohort that has been extensively characterized elsewhere.16,29−31 Briefly, the population was recruited between July 2003 and June 2004 among patients undergoing noncancer-related surgery at two public hospitals from Southern Spain: San Cecilio University hospital (Granada, urban area) and Santa Ana Hospital (Motril, 13530
DOI: 10.1021/acs.est.6b03783 Environ. Sci. Technol. 2016, 50, 13529−13538
Article
Environmental Science & Technology
Table 1. Characteristics of the Study Population and Adipose Tissue Levels of Oxidative Stress Biomarkers and POPs (n = 271) percentiles n (%) age (y) sex male female BMI (kg/m2) normal weight (BMI < 25.0) overweight (25.0 < BMI < 30.0) obese (BMI > 30.0) residence urban semirural educational level less than primary schooling primary schooling secondary schooling/university perceived weight loss current smoker ocupational class nonmanual worker manual worker retired origin of adipose tissue pelvic waist front abdominal wall limbs a
51.5 ± 11.4 123 (45.4) 148 (54.6) 27.7 ± 5.1b 88 (32.5) 114 (42.1) 69 (25.5) 136 (50.2) 135 (49.8) 80 (29.5) 126 (46.5) 64 (23.6) 113 (41.7) 84 (31.0) 46 (17.0) 206 (76.0) 19 (7.0)
n (%) > LOD
mean
55 (20.3) 252 (93.0) 271 (100) 62 (22.9) 234 (86.3) 244 (90.0) 255 (94.1) 254 (93.7)
