Science of the Total Environment 458–460 (2013) 209–216

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Science of the Total Environment

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Human exposure to p,p′-dichlorodiphenyldichloroethylene (p,p′-DDE) in urban andsemi-rural areas in southeast Spain: A gender perspective

Juan P. Arrebola a,b,⁎, Mariana F. Fernández a,b, Nicolás Olea a,b, Rosa Ramos a,b, Piedad Martin-Olmedo b,c

a Laboratory of Medical Investigations, San Cecilio University Hospital, University of Granada, 18071 Granada, Spainb CIBER en Epidemiología y Salud Pública (CIBERESP), Spainc Escuela Andaluza de Salud Pública, Cuesta del Observatorio s/n. Campus Universitario de Cartuja s/n, 18080 Granada, Spain

H I G H L I G H T S

• 387 adipose tissue and serum samples were collected from an adult cohort.• p,p′-DDE was analyzed as a surrogate for historic exposure to DDT.• Independent variables were gathered by questionnaire.• Predictors of concentrations were assessed by using multivariable linear regression.• Gender and local environment played a key role in the exposure of the population.

Abbreviations: p,p′-DDE, p,p′-Dichlorodiphendichlorodiphenyltrichloroethane; POP, persistent oof detection; BMI, body mass index.⁎ Corresponding author at: Laboratory of Medical Inves

Hospital, University of Granada, 18071 Granada, Spain. Te958 249953.

E-mail address: jparrebola@ugr.es (J.P. Arrebola).

0048-9697/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.scitotenv.2013.04.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 2 October 2012Received in revised form 1 April 2013Accepted 1 April 2013Available online 4 May 2013

Keywords:p,p′-DichlorodiphenyltrichloroethaneAdipose tissueSerumGenderResidenceExposure

p,p′-Dichlorodiphenyldichloroethylene (p,p′-DDE) is the main metabolite of pesticide dichlorodiphenyl-trichloroethane and a marker of past exposure to this organochlorine pesticide. p,p′-DDE is considered a per-sistent organic pollutant with potential adverse effects on human health. The aims of the present study were toassess p,p′-DDE levels in serum and adipose tissue from a cohort of adults in southern Spain and to explore thepredictors of exposure from a gender perspective. Chemical analyseswere performedusing gas chromatography–coupled mass spectrometry in tandem mode. The study population (n = 387) was intraoperatively recruitedin two areas of Granada Province (southern Spain), and data on potential predictors of these concentrationswere gathered by questionnaire. The statistical analysiswas performed bymeans ofmultivariable linear regressionmodels.All participants showed detectable concentrations of p,p′-DDE in both serum and adipose tissue, with medianconcentrations of 175.7 and 93.0 ng/g lipid, respectively (p b 0.001), and the two measurements showed posi-tive correlations. Women showed higher concentrations than men (115.8 and 66.2 ng/g lipid, respectively,p b 0.001). Concentrations in both matrices were positively associated with fatty food consumption, as well aswith age and BMI, the latter only in adipose tissue. Themultivariatemodel showed that, amongwomen, adiposetissue concentrations were approximately 40% higher in those residing in the semi-rural area and 2-fold higherin those whose mothers had participated in agricultural activities during the pregnancy.In this study population, gender and place of residence play a key role in human exposure to p,p′-DDE and canbe used to identify subjects at special risk of long-term exposure.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

The pesticide dichlorodiphenyltrichloroethane (DDT, CAS # 107917-42-0) is one of the most representative organochlorine pesticides. It was

yldichloroethylene; DDT,rganic pollutant; LD, limit

tigations, San Cecilio Universityl.: +34 958 240758; fax: +34

rights reserved.

widely used in agriculture and public health from the 1940s until the1970s, when it was prohibited in most countries due to the potentialthreat to human health. However, an estimated 4–5000 metric tons ofDDT per year are still used worldwide, mainly for disease vector control(van den Berg, 2009). Other reported sources of exposure are leakagesfrom storage tanks (Hay and Focht, 2000) and the use of the pesticidedicofol (2,2,2-trichloro-1,1-bis(4-chlorophenyl)ethanol), which includesDDT in its composition (Turgut et al., 2012).

DDT is a persistent organic pollutant (POP), i.e., a lipophilic chemicalthat is highly resistant to biodegradation and can bioaccumulate inorganisms and biomagnify through the food chain, especially in

210 J.P. Arrebola et al. / Science of the Total Environment 458–460 (2013) 209–216

lipid-rich compartments, including fat-containing foods (Olea et al.,2001; UNEP, 2012). Diet is believed to be the main source of expo-sure to DDT and other POPs in the general population (Bosch deBasea et. al., 2011).

Once in the environment and in living organisms, DDT ismainlyme-tabolized to p,p′-dichlorodiphenyldichloroethylene (p,p′-DDE, CAS #82413-20-5). This metabolite tends to persist for much longer than itsparent compound and is considered a marker of past exposure to DDT(World Health Organization, 1979). Previous research has evidencedthat p,p′-DDE is a weak estrogen receptor agonist (Soto et al., 1995)and an androgen receptor antagonist (Li et. al., 2008), and human expo-sure to this chemical has been linked to adverse health outcomes such asperipheral arterial disease in obese subjects (Min et al., 2011), obesity,dyslipidemia, insulin resistance (Lee et al., 2011), type 2 diabetesmellitus (Airaksinen et al., 2011; Arrebola et al., 2013), and vitamin Ddeficiency (Yang et al., 2012).

There are considerable knowledge gaps concerning the influence ofgender on POP exposure (Porta et al., 2008). Previous reports have indi-cated thatmen andwomen can experience substantially different inter-nal exposures to environmental pollutants even when exposed tosimilar external levels, which have been attributed to a combinationof gender-specific, genetic, physiological, and sociological characteris-tics (Garcia, 2003; Garcia et al., 2004; Kennedy and Koehoorn, 2003;London et al., 2002; Vahter et al., 2007). In terms of occupational expo-sure, Kennedy and Koehoorn (2003) stated that gender differences inexposure might be “apparent” (due to dissimilar tasks and workschedules) or “true” (related to intrinsic gender characteristics). Inaddition, women usually possess a higher percentage of body fat andcan therefore potentially accumulate a higher burden of certain lipo-philic chemicals (Salihovic et al., 2012).

Adipose tissue concentrations of POPs can be considered an indicatorof long-term exposure to these compounds (Kohlmeier and Kohlmeier,1995; Pearce et al., 1995), because they remain stable over time in com-parison to levels in other matrices, such as serum (Archibeque-Engle etal., 1997; Schisterman et al., 2005; Waliszewski et al., 2004). Previousstudies have suggested that concentrations of POPs in human adiposetissue might be influenced by lifestyles and socio-demographic charac-teristics (Arrebola et al., 2012b; Brauner et al., 2012; Vaclavik et al.,2006). The investigation of long-term human exposure to the pesticideDDT is a relevant issue in southern Spain for two reasons: the extensiveagricultural activity in the regionmight have been responsible formuchof the historical exposure of the population to this persistent chemical;andDDT remains in use for vector control in neighboringMorocco, fromwhere it can be readily borne by air to our region (UNEP, 2012).

The aims of the present study were to assess p,p′-DDE levels inserum and adipose tissue from a cohort of adults in southern Spainand to explore the predictors of exposure from a gender perspective.

2. Material and methods

2.1. Study area and population

The study population and epidemiological design were extensivelydescribed elsewhere (Arrebola et al., 2010; Arrebola et al., 2009). Insummary, human samples and data were collected between July 2003and June 2004 in two areas of Granada province (Southern Spain) sep-arated by 70 km: a densely populated urban area, corresponding to thecity of Granada and metropolitan suburbs (economy based on the ser-vice sector and light industry, with high levels of traffic-related air pol-lution); and a semi-rural area, corresponding to the town of Motril andsurroundings (small towns and villages on the Mediterranean coast,with intensive agricultural activity, including greenhouse cultivation).The use of DDT has been illegal in Spain since the 1970s; therefore, ag-ricultural activity would only account for the historical exposure in thisregion. Participants were recruited from among subjects undergoingnon-cancer-related surgery (47% inguinal hernia or abdominal surgery;

17% gall bladder surgery, 12% varicose vein surgery, and 24% othersurgery) at San Cecilio University Hospital in Granada and SantaAna Hospital in Motril. Surgical treatment made it ethically and prac-tically feasible to obtain adipose tissue samples. Inclusion criteriawere: age over 16 years, absence of hormone-related disease or can-cer, no hormone therapy, and residence in one of the study areas for≥10 years. Out of the 409 eligible subjects meeting the inclusioncriteria and invited to participate, 387 (94.6%) accepted and were in-cluded in the study. All subjects signed their informed consent toparticipate in the study, which was approved by the Ethics Committeeof each hospital.

2.2. Independent variables

Independent variableswere gathered using an ad hoc questionnaire,which was completed by each participant before surgery and adminis-tered face-to-face by a trained interviewer during the hospital stay.Socio-demographic characteristics included information on age, educa-tion, drinking and smoking habits, occupation,medical history, medica-tion, and perceived exposure to chemicals.

A short dietary section was composed of a semi-quantitativequestionnaire on food consumption habits; it included the follow-ing food groups: meat, cold meats, fats, fish, eggs, dairy products(without milk and cheese), milk, cheese, vegetables, pulses, fruit,bread, and pasta. The frequency of food consumption was gatheredin 4 categories: [1] no consumption (b1 portion/week), [2] 1portion/week, [3] 2–6 portions/week, or [4] >7 portions/week.Each variable was tested in the models with all of the categoriesand, when it did not change the associations, it was dichotomized(e.g., “[1] non-consumer (b1 portion/week) or [2] consumer of≥1 portion/week” and “[1] consumer of b2 portions/week or [2]consumer of ≥2 portions/week”). Water consumption includesboth bottled and non-bottled water. Participants' height and weightwere measured, and their body mass index (BMI) was calculated asweight/height squared (kg/m2). Residence in the city of Granada atthe time of the surgery was considered “urban” and residence in thearea of Motril was considered “semi-rural”. A subject was considereda smoker (past or present) at any level of daily tobacco consumption(≥1 cig/day). The number of children and accumulated breastfeedingtime (months) were recorded for the women.

Subjects were classified into six occupational categories, followingGoldthorpe's proposal (Regidor, 2001), and three grouped categorieswere formed: social classes I + II + III (non-manual workers) andIV + V (manual workers).

Questionnaires and research procedures were standardized andvalidated in a pilot study of 50 subjects, in which adipose tissue concen-trations of p,p′-DDEwere quantified and questionnaireswere completed.Based on this experience, sample collection protocols, analyticalmethodologies, and data collection criteria were tested, refined, andstandardized.

2.3. Sampling and chemical analysis

A5–10 g sample of adipose tissue and 10 mLof bloodwere collectedduring surgery and immediately coded and stored at −80 °C untilchemical analysis. Blood samples were then centrifuged for 5 min at2500 rpm to separate the serum. Main sources of tissue were pelvicwaist (42%), front abdominal wall (39%), and limbs (13%). Sampleswere all collected under 12-hour fasting conditions.

Adipose tissue samples were extracted and processed as describedelsewhere (Martinez Vidal et al., 2002; Moreno-Frias et al., 2004).Briefly, 200 mg of adipose tissue was extracted using n-hexane, andthe solution was then purified through 200 mg alumina in a glasscolumn and kept in test tubes at −80 °C. From each serum sample,4 mL was extracted with acidified diethyl ether and n-hexane, and

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the cleaned-up extract was eluted through a solid phase silica extrac-tion column (Sep-Pak, Waters).

Chemical analyseswere performed at Laboratorio Analítico Bioclínico(LAB) in Almería (Spain). p,p′-DDE was quantified by high-resolutiongas chromatography with a mass spectrometry detector in tandemmode, using a Saturn 2000 ion trap system (Varian Inc., Walnut Creek,CA) and 2 m × 0.25 mm silica capillary column (Bellefonte, PA)coupled to a Factor Four VF-5MS 30 m × 0.25-mm i.d. analyticalcolumn (Varian Inc., Walnut Creek, CA). C13-labeled p,p′-DDE wasused as the internal standard. Laboratory blanks with only solventswere tested and always yielded a negative result. Inter- and intra-dayvariability was b20%. For the quality control, laboratory fortifiedmatrixsamples at different concentrations were used. The limit of detection(LD) was determined as the smallest amount of the analyte that gavea signal-to-noise ratio ≥3 and was set at 0.01 μg/L. Concentrationsbelow the LD were assigned a random value between zero and the LD,as recommended by Antweiler and Taylor (2008). Concentrations of p,p′-DDE were calculated using matrix-matched calibration andexpressed in both wet (nanograms per gram of adipose tissue) andlipid (nanograms per gram of lipid) basis.

The recovery of p,p′-DDE from adipose tissue was studied in orderto assess the extraction efficiency of the methods used, spiking 10blank samples with target analytes at an intermediate point on thecalibration curve and processing them as described above. Recoveriesranged from 90 to 98%. A double-blinded procedure was followed sothat neither the chemical analysts nor statistical staff knew the identityor characteristics of any study subject.

2.4. Statistical analysis

p,p′-DDE concentrations were log-transformed and median concen-trations and 25th and 75th percentiles were calculated. Bivariate analyseswere carried out using the Fisher's exact test,Mann–Whitney'sU test, andsimple linear regression.

The analysis of potential predictors of lipid-basis p,p′-DDE con-centrations was performed as follows. First, bivariate linear regres-sion models were constructed by gender with the log-transformedp,p′-DDE concentrations as dependent variable and the potentialpredictors as independent variables, both for adipose tissue andserum concentrations. Second, multivariable linear regression analyseswere performed by gender including only the variables that showedp-values of b0.20 in the bivariate analyses or changed the estimatesby >10%. Finally, step-wise multivariable linear regression wasperformed for the whole population, using a stepwise backward elimi-nation technique, based on p-values and change in R2, for adipose tissueand serum p,p′-DDE concentrations. Interaction termswere tested aftereliminating all variables with non-significant associations.

Diagnosis of the models was performed in order to ensure thegoodness of fit and the fulfillment of implementation conditions.Generalized standard-error inflation factors were used to verify theabsence of colinearity between independent variables, while homo-scedasticity was assured by plotting residual versus fitted values.The linearity of quantitative independent variables was checked withpartial regression plots, and the normality of errors was verified bynormal QQ plot with 95% confidence intervals (Fox, 2008). Becausethe dependent variables were log-transformed, β coefficients in themodels are also shown as exp(β), i.e., the quotient between the valueof the dependent variable for a subject with independent variable xand the value of the dependent variable for another subject with inde-pendent variable x − 1, keeping the remaining independent variablesconstant. R2 was computed as the percentage of the variability in con-centrations explained by the statistical model.

The significance level was set at p ≤ 0.05; however, we consid-ered p ≤ 0.10 as borderline, following the recommendations ofGreenland (1989) andMickey and Green-land (1989), who suggestedthat increasing the traditional level of significance of 0.05 would help

to detect true predictors. Age and BMI were centered by their meanvalues (50 years and 27 kg/m2, respectively) to assist the interpreta-tion of the β coefficients.

Datawere stored and processed using SPSS Statistics v20.0 (Chicago,IL, USA), and multivariable analyses were carried out using the R statis-tical computing environment v2.14.1 (http://www.r-project.org/).

3. Results

3.1. Description of the study population and bivariate analyses

The main characteristics of the study population are summarizedin Table 1. Median age and BMI were 52.0 years and 26.6 kg/m2, re-spectively, with no statistically significant differences betweenmales and females. Median age was higher in urban than in semiruralresidents, but there was no significant difference between them in BMI.Median time in the current area of residencewas 35.0 years andmedianwater consumption was 4.3 glasses/day.

All adipose tissue and serum samples showed detectable levels ofp,p′-DDE, with a median concentration of 93.0 ng/g lipid (range 2.0–2331.4) in adipose tissue and 175.7 ng/g lipid (range 2.1–2330.2) inserum (p b 0.001). p,p′-DDE concentrations in adipose tissue werehigher in females than inmales (115.8 and 66.2 ng/g lipid, respectively,p b 0.001), but there were no statistically significant differences inserum concentrations. Adipose tissue p,p′-DDE concentrations did notsignificantly differ between semi-rural and urban dwellers, whereasserum concentrations were higher in the urban population (210.2 vs.138.6 ng/g lipid, respectively, p b 0.001).

3.2. Linear regression models

Table 2 shows the results of the bivariate and multivariable linearregression analyses of the predictors of adipose tissue and serum con-centrations of p,p′-DDE, stratified by gender. In the adjusted models,age was positively associated with serum and adipose tissue concen-trations in both men and women. An increase in the BMI was linkedto an increase in the adipose tissue concentrations but not serum con-centrations in both genders. Adipose tissue concentrations in femaleswere positively associated with residence in the semi-rural area,cheese consumption, and mother's occupation in agriculture duringpregnancy. Furthermore, consumption of ≥2 meat portions/weekwas associated with higher adipose tissue p,p′-DDE concentrationsin men and the consumption of ≥2 fish portions/week with higherserum concentrations in both genders.

Table 3 shows the results of the global stepwise multivariable analy-ses for adipose tissue and serum p,p′-DDE concentrations. Thefinalmodelfor adipose tissue explained 43% of the variability of the p,p′-DDE concen-trations, while the model for serum explained 14%. In the adipose tissuemodel, age, BMI, water consumption, and semi-rural residence were as-sociated with higher p,p′-DDE concentrations, while vegetable consump-tionwas associatedwith lower concentrations. In addition, adipose tissueconcentrations were borderline and significantly lower in males than infemales (p = 0.079). Higher adipose tissue p,p′-DDE concentrationswere found in cheese consumers versus non-consumers, as well as insubjects whose mothers had worked in agriculture during pregnancy.In the stepwise model for p,p′-DDE serum concentrations, positiveassociations were found with age, BMI, and fish consumption.When the adipose tissue model was further stratified by gender,we observed a positive influence of the semi-rural residence on adiposetissue p,p′-DDE concentrations in females but not in males (Fig. 1); infact, a significant interaction was found between place of residenceand gender in the stepwise model (Table 3). This interaction was notobserved in the stepwise model for serum concentrations.

When log-transformed p,p′-DDE serum concentrations wereintroduced into the model for adipose tissue, and vice-versa, thepercentage of the variability in concentrations explained by the

Table 1Characteristics of the study population.

Total Females (n = 190) Males (n = 197) p-Value⁎ Urban (n = 186) Semi-rural (n = 201) p-Value⁎

n % n % n % n % n %

ResidenceUrban (Granada) 186 48.1 81 42.6 105 53.3 0.036 – – – – –

Semi-rural (Motril) 201 51.9 109 57.4 92 46.7 – – – – – –

Occupational class 0.039 b0..001Non-manual worker 71 18.3 36 18.9 35 17.8 35 18.8 36 17.9Manual worker 294 76.0 149 78.4 145 73.6 130 69.9 164 81.6Retired 22 5.7 5 2.6 17 8.6 21 11.3 1 0.5

Worked in agriculturein the last 10 years

145 37.5 62 32.6 83 42.1 0.009 42 22.6 109 54.2 b0.001

Perceived exposureto pesticides

111 28.8 31 16.4 80 40.8 b0.001 29 15.6 82 40.8 b0.001

Mother's occupation duringpregnancy = agriculture

51 13.2 27 14.2 24 12.2 0.652 10 5.4 41 20.4 b0.001

Smoker 126 32.6 41 21.6 85 43.1 b0.001 63 33.9 63 31.3 0.644Vegetable consumption(≥2 portions/week)

278 71.8 149 78.4 129 65.5 0.004 138 74.2 140 69.7 0.395

Cheese consumer 359 92.8 175 92.1 184 93.4 0.616 181 97.3 178 88.6 0.002Cold meat consumer 351 90.7 170 89.5 181 91.9 0.398 177 95.2 174 86.6 0.016Meat consumption(≥2 portions/week)

243 62.8 117 61.6 126 64.0 0.537 122 65.6 121 60.2 0.327

Fish consumption(≥2 portions/week)

268 69.3 141 74.2 127 64.5 0.043 140 75.3 128 63.7 0.024

Blue fish consumer 269 69.5 121 63.7 148 75.1 0.011 128 68.8 141 70.1 0.609

Median Percentiles Median Percentiles Median Percentiles p-Value⁎⁎ Median Percentiles Median Percentiles p-Value⁎⁎

25th 75th 25th 75th 25th 75th 25th 75th 25th 75th

Age (years) 52.0 37.0 63.0 51.0 37.8 63.0 53.0 36.5 63.5 0.864 54.5 40.8 66.3 48.0 34.0 61.0 0.004BMI (kg/m2) 26.6 23.8 29.7 26.2 23.3 30.1 27.0 24.4 29.4 0.231 26.5 23.8 29.5 26.7 23.9 29.8 0.851Beer consumption(glasses/week)

0.0 0.0 3.3 0.0 0.0 0.0 2.0 0.0 7.0 0.345 0.0 0.0 3.0 0.0 0.0 3.5 0.780

Number of pregnanciesa 3.0 2.0 4.0 3.0 2.0 4.0 – – – – 3.0 2.0 3.5 3.0 2.0 4.0 0.853Breastfeeding time(months)a

4.8 2.0 8.0 4.8 2.0 8.0 – – – – 4.0 2.0 8.0 4.9 1.5 10.0 0.287

Water consumption(glasses/day)

4.3 3.0 7.5 4.0 3.0 7.3 5.0 3.0 8.0 0.345 4.0 3.0 6.0 5.0 3.0 8.0 0.000

Milk consumption(glasses/day)

1.0 1.0 2.0 1.0 1.0 2.0 1.0 1.0 2.0 0.222 1.0 1.0 2.0 1.0 0.5 2.0 0.956

p,p′-DDE serum(ng/g lipid)

175.7 101.7 331.1 191.0 106.2 342.0 168.0 99.4 312.2 0.459 210.2 136.2 400.3 138.6 78.5 271.1 b0.001

p,p′-DDE adipose tissue(ng/g lipid)

93.0 32.9 210.2 115.8 39.4 291.2 66.2 29.5 173.6 0.001 90.3 36.0 214.6 93.3 31.7 209.4 0.967

⁎ Fisher's exact test.⁎⁎ Mann–Whitney's U test.a Only women.

212 J.P. Arrebola et al. / Science of the Total Environment 458–460 (2013) 209–216

predictors increased from 43% to 47% in the adipose tissue model andfrom 14% to 19% in the serum model (Data not shown in tables).

4. Discussion

All participants in the present study showed detectable concentra-tions of p,p′-DDE in serum and adipose tissue, and levels were withinthe medium-low range of reports from other regions of the world inwhich DDT had long been prohibited (CDC, 2009; Cerrillo et al.,2006; Glynn et al., 2003; Hardell et al., 2006; Jakszyn et al., 2009;Kang et al., 2008; Munoz-de-Toro et al., 2006; Porta et al., 2010). Incomparison to a similar study by our group in 2010 in a populationfrom Eastern Bolivia (Arrebola et al., 2012a, 2012b), the adipose tissueand serum concentrations were 4- and 1.5-fold lower, respectively, inthe present subjects, despite their markedly older age and thereforelonger period for potential bioaccumulation of the pesticide. The differ-encemay be attributable to the later banning of DDT in Bolivia (in 1996)than in Spain (in 1977).

Gender was found to play a major role in the adipose tissuebioaccumulation of p,p′-DDE in this study population from southernSpain. Median adipose tissue concentrations were 1.7-fold higher inwomen than in men, and this difference remained significant in the

multivariable models. This may be explained by a lower cytochromeP450 metabolism of POPs in females than in males (McTernan et al.,2002; Moser and McLachlan, 2001; Silbergeld and Flaws, 2002). Inaddition, the concentrations of lipophilic chemicals may be affectedby the percentage of body fat in women (López-Flores et al., 2009).Furthermore, women are widely involved in agricultural activities butusually receive lower salaries, lower-status jobs, and less education onsafety measures in comparison to men (Garcia, 2003; Kunstadteret al., 2001; London et al., 2002), and it has been proposed thatworkers on lower salaries are more likely to be at risk of pesticideexposure (Robinson et al., 2011). This occupational exposure mightalso be relevant to the offspring, and we found in our study thatsubjects whose mothers had worked in agriculture during pregnancyshowed higher concentrations of p,p′-DDE in their adipose tissues.Although recent occupational exposure does not appear to be relevantfor this cohort, it might have been for their parents, given that DDTwas banned in Spain in 1977 (Porta et al., 2002), and 92% of the studysubjects were born before its prohibition.

Previous research evidenced the presence of p,p′-DDE and other POPsin human placentas (Bergonzi et al., 2009; Lopez-Espinosa et al., 2007;Shen et al., 2008), underlining the importance of the mother-to-fetustransfer of these chemicals (Sapbamrer et al., 2008). Thus, breastfeeding

Table 2Bivariate and multivariable linear regression models for the predictors of log-transformed serum and adipose tissue p,p′-DDE concentrations.

Bivariate analysis Multivariable analysis

Adipose tissue Serum Adipose tissue Serum

Females Males Females Males Females Males Females Males

β (SE) Exp(β) β (SE) Exp(β) β (SE) Exp(β) β (SE) Exp(β) β (SE) Exp(β) β (SE) Exp(β) β (SE) Exp(β) β (SE) Exp(β)

Age (years) 0.04 (0.01) 1.04⁎⁎ 0.04 (0.00) 1.04⁎⁎ 0.03 (0.01) 1.03⁎⁎ 0.02 (0.00) 1.02⁎⁎ 0.04 (0.01) 1.05⁎⁎ 0.05 (0.01) 1.05⁎⁎ 0.02 (0.01) 1.02⁎⁎ 0.02 (0.01) 1.02⁎⁎

BMI (kg/m2) 0.08 (0.02) 1.09⁎⁎ 0.07 (0.02) 1.08⁎⁎ 0.04 (0.02) 1.04⁎⁎ 0.03 (0.02) 1.03⁎ 0.05 (0.02) 1.06⁎⁎ 0.05 (0.02) 1.05⁎⁎ 0.01 (0.02) 1.01 0.03 (0.02) 1.03Place of residence = semi-rural 0.28 (0.20) 1.32 −0.27 (0.19) 0.77 −0.67 (0.21) 0.51⁎⁎ −0.57 (0.18) 0.57⁎⁎ 0.37 (0.19) 1.45⁎⁎ −0.09 (0.18) 0.91 −0.73 (0.22) 0.48 −0.44 (0.19) 0.65Occupational class = manual

worker0.49 (0.24) 1.64⁎⁎ −0.35 (0.21) 0.70 0.10 (0.25) 1.10 −0.24 (0.21) 0.78 0.20 (0.23) 1.22 0.03 (0.19) 1.03 – – – – – –

Worked in agriculture during thelast 10 years

0.39 (0.20) 1.48⁎ 0.13 (0.19) 1.14 0.32 (0.22) 1.37 –0.09 (0.18) 0.92 –0.14 (0.22) 0.87 – – – 0.67 (0.25) 1.96 – – –

Mother's occupation duringpregnancy = agriculture

0.64 (0.28) 1.89⁎⁎ 0.25 (0.29) 1.28 0.37 (0.31) 1.44 −0.08 (0.28) 0.92 0.84 (0.27) 2.32⁎⁎ – – – – – – – – –

Number of pregnancies 0.25 (0.05) 1.28⁎⁎ – – – 0.14 (0.06) 1.15⁎⁎ – – – −0.10 (0.06) 0.90 – – – –0.09 (0.07) 0.91 – – –

Breastfeeding time (months) 0.04 (0.02) 1.04⁎⁎ – – – 0.01 (0.02) 1.01 – – – −0.02 (0.02) 0.98 – – – – – – – – –

Smoker = yes −0.77 (0.23) 0.46⁎⁎ −0.40 (0.19) 0.67⁎⁎ −0.22 (0.25) 0.80 −0.29 (0.18) 0.75 0.26 (0.24) 1.29 0.01 (0.17) 1.01 − − − −0.08 (0.19) 0.92Beer consumption (glasses/week) −0.09 (0.05) 0.92⁎ −0.02 (0.01) 0.98⁎⁎ −0.03 (0.05) 0.97 −0.01 (0.01) 0.99 0.03 (0.05) 1.03 −0.01 (0.01) 0.99 – – – – – –

Water consumption (glasses/day) −0.03 (0.03) 0.97 0.01 (0.02) 1.01 −0.06 (0.03) 0.94⁎ −0.03 (0.02) 0.97 – – – – – – 0.03 (0.04) 1.03 0.00 (0.02) 1.00Milk consumption (glasses/day) −0.04 (0.08) 0.96 −0.12 (0.08) 0.89 −0.03 (0.08) 0.97 −0.08 (0.07) 0.92 – – – −0.07 (0.06) 0.94 0.06 (0.29) 1.07 − – –

Cheese consumer = Yes 0.65 (0.37) 1.92⁎ 0.03 (0.40) 1.03 −0.04 (0.38) 0.96 0.04 (0.41) 1.04 0.80 (0.34) 2.23⁎⁎ – – – – – – – – –

Cold meat consumer = yes −0.20 (0.34) 0.82 −0.65 (0.37) 0.52⁎ −0.16 (0.38) 0.85 0.18 (0.35) 1.20 – – – −0.16 (0.32) 0.85 – – – – –

Meat consumption ≥2 portions/week

−0.29 (0.20) 0.74 −0.24 (0.20) 0.79 −0.11 (0.21) 0.89 −0.32 (0.19) 0.73 −0.10 (0.18) 0.90 0.44 (0.18) 1.55⁎⁎ – – – 0.06 (0.20) 1.07

Vegetable consumption ≥2portions/week

−0.15 (0.24) 0.86 0.05 (0.20) 1.05 −0.23 (0.26) 0.79 0.29 (0.19) 1.34 – – – – – – – – – 0.25 (0.19) 1.28

Fish consumer ≥2 portions/week 0.86 (0.22) 2.37⁎⁎ 0.23 (0.20) 1.26 0.85 (0.23) 2.35⁎⁎ 0.50 (0.19) 1.66⁎⁎ 0.29 (0.22) 1.34 – – 0.60 (0.24) 1.82⁎⁎ 0.33 (0.19) 1.39⁎

Blue fish consumer = yes −0.03 (0.21) 0.97 −0.06 (0.22) 0.94 −0.03 (0.22) 0.97 0.04 (0.22) 1.04 – – – – – – – – – – – –

Perceived exposure to pesticides= yes

0.29 (0.27) 1.34 0.33 (0.19) 1.40⁎ −0.49 (0.28) 0.61⁎ 0.02 (0.19) 1.02 – – – – −0.12 (0.18) 0.89 −0.35 (0.31) 0.71 – –

SE: standard error. The multivariate models were performed including the variables that showed p-values b 0.20 in the bivariate analyses or changed the estimates by >10%.⁎ p ≤ 0.10.

⁎⁎ p ≤ 0.05.

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Table 3Multivariable stepwise linear regression model for the predictors of log-transformed p,p′-DDE concentrations in adipose tissue and serum (ng/g lipid).

Adipose tissue (R2 = 0.43) Serum (R2 = 0.14)

β SE exp(β) p-Value β SE exp(β) p-Value

Intercept 3.93 0.29 50.94 b0.001 5.02 0.16 151.11 b0.001Age (years, centered by 50) 0.04 0.01 1.05 b0.001 0.02 0.01 1.02 b0.001Gender = male −0.28 0.16 0.76 0.079 0.05 0.13 1.05 0.713BMI (kg/m2, centered by 27) 0.05 0.01 1.05 b0.001 0.02 0.01 1.02 0.092Residence = semi-rural 0.36 0.16 1.44 0.026 – – – –

Mother's occupation during pregnancy = agriculture 0.35 0.18 1.42 0.050 – – – –

Water consumption (glasses/day) 0.04 0.02 1.04 0.017 – – – –

Vegetable consumption = ≥2 portions/week −0.31 0.13 0.73 0.016 – – – –

Cheese consumer = yes 0.49 0.23 1.63 0.039 – – – –

Fish consumption = ≥2 portions/week – – – – 0.47 0.15 1.61 0.001(Gender = male) × (residence = semi-rural) −0.49 0.22 0.61 0.031 – – – –

SE: standard error.

214 J.P. Arrebola et al. / Science of the Total Environment 458–460 (2013) 209–216

and deliveries have been reported to reduce the body burden of POPs(Elserougy et al., 2012; Ibarluzea et al., 2011). In the multivariablemodels, adipose tissue concentrations of p,p′-DDEwere negatively corre-lated with accumulated breastfeeding time and number of children, butthe associations were not statistically significant. However, this “clear-ance effect” could be expected to be less influentialwith a longer time in-terval since the last child, andmost of thewomen in the studypopulationwere unlikely to have given birth recently (median age = 51 years).

The place of residence also emerged as an important predictor oflong-term exposure to p,p′-DDE (adipose tissue concentrations) inwomen, with a significantly higher bioaccumulation in semi-ruralversus urban residents in the global stepwise model. This associationis supported by the highly stable residence pattern of our subjects,who had lived for a median of 35.0 years in the same study area,and is consistent with previous findings of higher p,p′-DDE concen-trations in residents of areas with evidence of past DDT use(Galvan-Portillo et al., 2002; Mercado et al., 2013). Interestingly,when we stratified the statistical analyses by gender, the observed as-sociation was only significant in women and not in men. In fact, wefound a statistically significant interaction between gender andplace of residence in the global stepwise model. Thus, the higherlevels found in semi-rural versus urban subjects were only significantamong the females, indicating that rural women are at special risk ofexposure. It is crucial for epidemiologic studies to consider potentialgender-related variations in POPs exposure, especially in rural areas,where these disparities might be exacerbated by the generallylower education levels, weaker workplace safety measures, andhigher historic and present exposures to pesticides. It should also betaken into account that, although the recent occupational exposure

* Model was adjusted for age, body mass index, mother´s occupation in agriculture, water consumption, vegetable consumption,and cheese consumer

Fig. 1. Predicted means⁎ of p,p′-DDE concentrations (ng/g lipid) in adipose tissue with95% confidence intervals.

reported by our population was not significantly associated withp,p′-DDE concentrations in these subjects, women may under-estimatetheir occupational exposure in comparison to men, which might mask apotential association (Joffe, 1992).

The remaining associations with exposure in the multivariablemodel are in agreement with previous reports. A positive associationof p,p′-DDE with age has frequently been found in populations withlong-term exposure to POPs; this has usually been attributed to thebioaccumulation of these compounds over time (Burgaz et al., 1994;Daglioglu et al., 2010) or to a “cohort effect”, by which subjects bornbefore the prohibition or control of POPs would start with a higherbody burden in comparison to younger people (Ahlborg et al.,1995). The positive association with BMI might result from dietaryexposure, given that individuals with higher BMI usually consumemore fatty foods, which have been described as the main vehicle forPOP exposure in non-occupationally exposed populations (Brauneret al., 2011; Ibarluzea et al., 2011). This explanation appears to besupported by our finding that p,p′-DDE concentrations were positivelyassociated with fatty food consumption (e.g., cheese and meat withadipose tissue and fish with serum concentrations). The association be-tween p,p′-DDE concentrations and BMI may also be attributable to thepotential obesogenic effect of POPs (Karmaus et al., 2009; Smink et al.,2008; Verhulst et al., 2009). However, reports on the association ofPOP concentrations with BMI have been controversial (Arrebola et al.,2012b; Brauner et al., 2012; Daglioglu et al., 2010; Vaclavik et al.,2006). Wolff et al. (2007) described a pharmacokinetic model inwhich a positive relationship might be expected if biological sampleswere collected around 10 years after the peak exposure occurred(DDT was banned in Spain in 1977). This is because, although there isa dilution effect of body fat, POPs tend to have longer half-lives inobese than in lean persons.

In the present study, we estimated human exposure to p,p′-DDE intwo matrices with different biological meanings. Once standardizedfor lipid content, serum concentrations were approximately 2-foldhigher than those in adipose tissue, and a positive correlation wasfound between the log-transformed values of the two measurements.This is consistent with previous findings that, under equilibrium condi-tions, the lipid-standardized serum concentration of POPs may serve asa proxy for the total body burden (Gaskins and Schisterman, 2009).However, a better prediction of the variability (R2) in p,p′-DDE concen-trationswas obtainedwhen adipose tissue concentrations served as thedependent variable in the stepwise models (vs. serum). Lipophilicchemicals have been shown to bioaccumulate in fatty tissues; therefore,adipose tissue concentrations have been considered as good estimatorsof the cumulative exposure of POPs and serum concentrations as moreindicative of ongoing exposure and/or the release of these chemicalsduring lipolysis (Arrebola et al., 2012b; Waliszewski et al., 2004).

The fact that many of the predictors found were consistent in thetwo multivariable approaches improves the validity of the results.

215J.P. Arrebola et al. / Science of the Total Environment 458–460 (2013) 209–216

The present study population was not entirely representative of thegeneral population, but the sample size was large enough to yield robustassociations. Furthermore, despite the cross-sectional study design, ourmeasurement of concentrations in adipose tissue allowed us to estimatethe subjects' accumulated exposure to p,p′-DDE. Althoughmost countrieshave banned or severely restricted the use and production ofmany POPs,human exposure is still of public health concern (Porta et al., 2008),and further research is warranted to clarify potential routes of exposureand to identify groups at special risk.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

The authors gratefully acknowledge scientific and technical assis-tance provided by Richard Davies. Dr JP Arrebola is under contractwith the PTA-MICINN program (Spanish Ministry of Science and In-novation). This study was supported in part by research grantsfrom the Spanish Ministry of Health (FIS 02/974, EUS2008-03574);CIBER de Epidemiología, Instituto de Salud Carlos III, Government ofSpain, the Regional Government of Andalucía — Spain (SAS 01/264,grant numbers P09-CTS-5488 Project of Excellence, and SAS PI-0675-2010), the Spanish Ministry of Science and Innovation (Ramon y CajalProgram-MFFC), the EU Commission (CONTAMED FP7-ENV-212502),Instituto de Salud Carlos III (FIS PI11/0610), and the Granada Researchof Excellence Initiative on BioHealth “GREIB” from the University ofGranada (CEB-005). None of the authors has any current or potentialcompeting financial interests. The results would not have beenachieved without the selfless collaboration of the staff from Santa Anaand San Cecilio Hospitals and the patients who took part in the study.

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