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ENDOCRINE DISRUPTOR DDE ASSOCIATED TO A HIGH-FAT DIET ENHANCES THE IMPAIRMENT OF THE LIVER FATTY ACID COMPOSITION IN RATS Luis Miguel Miguel Rodríguez-Alcalá, Carla Sá, Lígia L. Pimentel, Diogo Pestana, Diana Teixeira, Ana Faria, Conceição Calhau, and Ana Gomes J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03274 • Publication Date (Web): 08 Oct 2015 Downloaded from http://pubs.acs.org on October 9, 2015
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Journal of Agricultural and Food Chemistry
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ENDOCRINE DISRUPTOR DDE ASSOCIATED TO A HIGH-FAT DIET EN-
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HANCES THE IMPAIRMENT OF THE LIVER FATTY ACID COMPOSITION IN
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RATS
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Luis M. Rodríguez-Alcalá1†, Carla Sá2†, Lígia L. Pimentel1, Diogo Pestana2,3, Diana Teixei-
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ra2, Ana Faria4,5, Conceição Calhau2,3*, Ana Gomes1
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1
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or de Biotecnologia, Universidade Católica Portuguesa/Porto, Rua Arquiteto Lobão Vital,
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CBQF - Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superi-
Apartado 2511, 4202-401 Porto, Portugal
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2
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gação Médica, P-4200-450 Porto, Portugal
Department of Biochemistry, Faculty of Medicine, University of Porto, Centro de Investi-
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3
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4200-450 Porto, Portugal
CINTESIS—Center for Research in Health Technologies and Information Systems, Porto-
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4
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ty of Sciences, University of Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal
Chemistry Investigation Centre (CIQ), Department of Chemistry and Biochemistry, Facul-
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465 Porto, Portugal
Faculty of Nutrition and Food Sciences, University of Porto, Rua Dr. Roberto Frias, 4200-
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†
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*To whom correspondence should be addressed:
[email protected].
These authors contributed equally.
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ABSTRACT
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The banned pesticide dichlorodiphenyltrichloroethane (DDT) and its main metabolite p,p’-
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dichlorodiphenyldichloroethylene (DDE) are commonly found in the food chain and in all tissues of
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living organisms. DDE is associated to metabolic diseases acting as an endocrine disruptor and
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more recently to the obesity pandemic. This study focuses on using fatty acid analysis to relate
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DDE exposure and metabolic dysfunction: liver and adipose tissue (visceral and subcutaneous)
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composition from male Wistar rats fed a standard (STD) or high-fat (HF) diet versus the addition of
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DDE in water.
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DDE exposure increased liver levels of palmitic, stearic, oleic, trans fatty acids, and linoleic acid
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having altered the n6 and n3 pathways leading to high concentration of arachidonic acid and DHA
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(C22:6 n3). The results of this study confirm the close relationship between this pesticide metabo-
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lite and hepatic lipid dysfunction, underscoring its role as emerging targets for prevention and ther-
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apy of non-alcoholic fatty liver disease (NAFLD).
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Keywords: Liver, Adipose tissue, Fatty acids, Obesity, DDE, NAFLD.
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Journal of Agricultural and Food Chemistry
INTRODUCTION
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Dichlorodiphenyltrichloroethane (DDT) is an organochlorine insecticide first synthesized in 1874
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and widely used since 1945 for the control of malaria, typhus and agricultural and forest pests 1.
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However DDT is also toxic for marine microorganisms, fishes, amphibians and birds while in hu-
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mans it has been associated to liver, pancreatic and breast cancer 2–4 besides its identification as an
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endocrine disruptor 5. Despite being banned in western countries since the 70´s, it is still used in
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developing countries for vector-control (malaria) with the support of the World health organization
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(WHO) 6. Furthermore, although the European pesticide Regulation 1107/2009 aimed not to ap-
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prove the utilization of compounds with unacceptable risks to human/animal health and the envi-
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ronment, also encourage to the utilization of less harmful substances as biopesticides, crop-
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protection agents derived from natural materials according to the United States Environmental Pro-
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tection Agency (US EPA); however they have to overcome specific issues when compared to con-
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ventional agrochemicals as manufacturing costs, improvement of formulations (effectiveness and
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shelf life) and registration and commercialization problems 7. Thus, widespread utilization of DDT
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in agriculture and industry together with its slow biodegradation and high lipophilic nature, have
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resulted in accumulation in the food chain, in all tissues of living organisms and long-range atmos-
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pheric transport 8.
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Furthermore, the main in vivo metabolite of DDT is dichlorodiphenyldichloroethylene (DDE), one
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of the most commonly detected environmental pollutants in human tissues (as result of degradation
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by cytochrome P450) and therefore a marker for past exposure to DDT 9; however DDT can be also
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transformed into DDE and dichlorodiphenyldichloroethane (DDD) through photolysis and micro-
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bial biodegradation 10. These two compounds (DDT and DDE) show a half-life in humans of 4.2-6
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yr. and 7-8.6 yr respectively
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France reported levels in human plasma of 268 and 118 ng/g lipid, respectively
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focused in the hepatic induction of CYP2B by DDT and DDE through the feeding of rats as it is
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associated to liver carcinogenesis, tumor promotion and increment of the liver/body weight ratio
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while some studies recently performed in the United States and
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. Some studies
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reporteing EC50 values of 0.39 µg DDT/dL serum, 0.06 µg DDE/dL serum, 9.3 ng DDT/g liver and
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4.4 ng DDE/g liver 14.
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As stated above, these persistent pollutants act as endocrine disruptors mimicking or antagonizing
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endogenous estrogens and androgens, altering the pattern of synthesis and metabolism of natural
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hormones as well as modifying receptor levels 15. Many of these hormones regulate body weight
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homeostasis: while, high estrogens plasma levels in women have been positively associated with
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weight control 16,17, studies carried out in children found that in-utero exposure to DDT and its me-
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tabolite DDE increased the risk to develop obesity
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health problems and concomitantly a central feature for a vast array of other chronic diseases (insu-
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lin resistance, type 2 diabetes, fatty liver disease, atherosclerosis, hypertension, stroke, cancer,
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asthma), with significant morbidity and mortality 20. Therefore, the possible implications of DDT
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and DDE in the development of obesity have gained much attention in the last years. However, at
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the current moment there are no previous studies describing the impact of DDE associated to diet in
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the fatty acid (FA) composition of adipose tissue and liver. This is of high relevance since expan-
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sion of adipocytes has been associated to higher levels of unsaturated FA in order to maintain mem-
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brane fluidity
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linked to high caloric intake, physical inactivity, genetics, and certain medications' side effects 22.
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Furthermore, previous results in obese humans from our group showed that persistant organinc pol-
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lutants (mainly endrin, dieldrin, hexachlorobenzene, and DDT) were pervasive in the visceral (42.4
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ng DDT/g fat; 72.4 ng DDE/g fat) and subcutaneous adipose tissue (39.3 ng DDT/g fat; 18.5 ng
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DDE/g fat) of this population and their concentration increased with age and duration of obesity 23.
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It was also found a positive correlation between persistent organic pollutants (POPs) levels and
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metabolic syndrome, aggravating some of its features, such as glucose intolerance, hypertension,
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dyslipidaemia and inflammation.
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Taking into account the confirmed presence of p,p’DDE in animal tissues and their recognized en-
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docrine disruption ability, the present work aimed to evaluate the effect of p,p’DDE on changes in
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the fatty acid composition of adipose tissue and liver in rats.
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. This disease is currently one of the main
while some alterations as non-alcoholic fatty liver disease (NAFLD) have been
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Journal of Agricultural and Food Chemistry
MATERIAL AND METHODS
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Chemicals
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Hexane, dimethylformamide and methanol (HPLC grade) and sulphuric acid (98%) were purchased
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from VWR Scientific (West Chester, PA). Supelco 37 FAME mix, sodium methoxide (95%), DDE,
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ketamine, medetomidine and isoflurance were obtained from Sigma (Sigma-Aldrich, St. Louis, MO,
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USA), GLC-Nestlé36 FAME mix and tritridecanoin (99%) were from Nu-Chek Prep, inc. (Elysian,
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Minnesota, USA) while butterfat CRM-164 (EU Commission; Brussels, Belgium) was from
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Fedelco Inc. (Madrid, Spain).
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Animal Tissues
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Liver and Adipose tissue were collected from twenty-four male Wistar rats (Charles River Labora-
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tories, Barcelona, Spain), weighing 267±11.8 g (8 weeks), randomly divided into four groups of six
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animals each and subjected to different treatments for a total of 12 weeks: STD, Standard group;
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STD/DDE, Standard with p, p’-DDE group; HF, Hight-fat group; HF/DDE, high-fat with p,p’-DDE
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group, as described. The p,p’-DDE exposure treatment was applied in the water with the average
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concentration of 100 µg/kg/day (2.5 times less than LOAEL). DDE was first dissolved in ethanol
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and the concentrations of stock and final solution were corrected (STD/DDE and HF/DDE), accord-
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ing to water intake and animal average weight, in order to maintain the exposure concentration and
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ethanol volume. In the control groups, STD and HF, the same final volume of ethanol (0.01%) was
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added to the water. Added water and chow were supplied ad libitum and every 3-4 days renewed
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and the intake assessed. The diets were respectively Standard (STD) (Teklad 2014, Harlan Labora-
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tories, Santiga, Spain; 48% carbohydrate (w/w), 14.3% protein (w/w) and 4% lipid (w/w)) and High
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fat (HF) with 45% of energy from lipids (D145 Research Diets, New Brunswick, USA; 41% carbo-
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hydrate (w/w), 24% protein (w/w) and 24% lipid (w/w)). Animal handling and housing protocols
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followed European Union guidelines (86/609/EEC) and Portuguese Act (129/92) for the use of exACS Paragon Plus Environment
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perimental animals. At the end of the 12 weeks, animals were anesthetized with a mixture of keta-
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mine (50mg/kg) and medetomidine (1mg/kg) and maintained with isoflurance. Organs were dis-
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sected, pat dried and weighed before snap freezing in liquid nitrogen and storing at -80ºC until fur-
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ther analysis.
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Analysis of fatty acids
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For the analysis of the total fatty acid (FA) composition, 200 mg of liver tissue and 30 mg of adi-
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pose tissue, 200 mg of STD diet and 50 mg of HF diet were accurately weighed and FA methyl
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esters (FAME) prepared using methanolic solutions of sodium methoxide (0.5M; 80ºC, 10 min) and
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sulphuric acid (1M; 60ºC, 30 min) according to Castro-Gómez et al. 24. For quantification purposes
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samples were added with 100 µL of tritridecanoin as internal standard (1.34 mg/mL in hexane) pri-
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or to derivatization. FAME were analysed in a gas chromatrograph HP6890A (Hewlett-Packard,
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Avondale, PA, USA), equipped with a flame-ionization detector (GLC-FID) and a BPX70 capillary
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column (50m x 0.32 mm x 0.25 µm; SGE Europe Ltd, Courtaboeuf, France). Analysis conditions
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were as follows: injector (split 10:1; injection volume 1 µL) and detector temperatures were 250ºC
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and 270ºC, respectively; carrier gas was Hydrogen (11 psi) and the oven temperature program start-
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ed at 60ºC (hold 2 min), raised 10ºC/min to 135ºC (hold 2 min), then 10ºC/min to 165ºC (hold 2
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min) and finally 10ºC/min to 230ºC (hold 7 min). Supelco 37 and CRM-164 were used for identifi-
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cation of fatty acids. GLC-Nestlé36 was assayed for calculation of response factors and detection
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and quantification limits (LOD: 0.395 ng FA/mg liver, 2.73 ng FA/mg adipose tissue ; LOQ: 1.32
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ng FA/ mg liver, 8.8 ng FA/mg adipose tissue). Analysis were performed in triplicate
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Statistical analysis
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All results are expressed as mean and standard deviations. In a first instance, data were examined
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for the presence of outliers 25 and an exploratory analysis of data was performed to test normal dis-
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tribution and homogeneity of variance (Levene´s test). Data showing normal distribution were ana-
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lysed according to the ANOVA procedure with Bonferroni´s or Tamhane´s as post-hoc according to ACS Paragon Plus Environment
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homogeneity of variance. Otherwise, data were analysed according to the Kruskal-Wallis´ test using
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Mann-Whitney´s test as post-hoc. All the analyses were performed using the SPSS Statistics soft-
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ware v22.0 for Mac (IBM, Armonk, NY, USA). Level of significance was set at p
