Dichlorodiphenyldichloroethylene (DDE) is a persistent synthetic organic pollutant detected throughout the world in sediments, in the food chain, and in human tissues. Its precursor dichlorodiphenyltrichloroethane (DDT) is still being produced and used today, particularly for indoor spraying against malaria.
DDE at very high levels suppresses both innate and acquired immunity in animal models.1-4 In humans, DDE at environmental background levels (Table 1) has been shown to suppress activity of T lymphocyte cells after stimulation (decrease in IL2, IFμ, TNFα, and concanavalin A).5-8 In addition, counts of lymphocytes and immunolobulins have been related to DDE levels.5-10 However, the clinical relevance of these findings is unknown. In children, a moderate increase in risk of acute infections during the first year of life with increasing levels of DDE has been reported in Inuit communities,11 in Japan,12 and in Taiwan,13 as well as in Swedish children exposed to levels lower than the Inuit.14 However, the effects of DDE in these studies could not be separated from those of polychlorinated biphenyls (PCBs). PCBs have been related to several immunologic biomarkers in adolescents from Belgium,15 with a smaller thymus size in neonates from Slovakia,16 and with changes in the immune profile and prevalence of otitis media and chickenpox in children from the Netherlands.17,18 Similar immunologic effects have been found with other organochlorine compounds, such as hexachlorobenzene (HCB).19
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TABLE 1: Human Studies on Immune Markers and DDE
Only 1 study has been able to isolate the immunologic effects of DDE from those of PCBs or other organochlorine compounds in humans. This prospective study found early-life exposure to DDE to be associated with childhood asthma at the ages of 4 years20 and 6 years21 in the birth cohort of Menorca, Spain. Lower respiratory tract infections during infancy may contribute to the development of asthma.22
Our aim is to study, in a new birth cohort recruited from the year 2004, the association between prenatal levels of DDE and lower respiratory tract infection in infancy. In addition, we aim to isolate effects of DDE from those of other common organochlorine compounds found in human tissues, such as PCBs and HCB.
METHODS
Study Population
A general population-based birth cohort was established in the city of Sabadell (Catalonia, Spain) as part of the INMA—Infancia y Medio Ambiente (Environment and Childhood) Project.23 Between July 2004 and July 2006, 657 pregnant women who visited the public-health center of Sabadell for a first prenatal visit in the first trimester were recruited. This center serves most of the pregnant women (at least 80%) in the city. Approximately 50% of those who met the eligibility criteria (age >16 years, intention to deliver in the city, and no problems of communication) agreed to participate. Among the 657 recruited women, 584 (89%) had organochlorine compounds assayed and completed the child's 6-month visit; 538 (82%) also completed the 14-month visit. These mother-infant pairs were included in this study. Those who completed the 6-month follow-up (n = 584) and those who did not (n = 73) differed for maternal education (27% of participants had only primary school level vs. 42% of nonparticipants) and maternal asthma (7% vs. 19%). Given a different pattern of organochlorine compound levels and covariates in native (n = 520) and immigrant mothers (n = 64), all analyses were stratified by migration status. Due to the small number of immigrant mothers, the present paper presents only the 520 mother-infant pairs from native mothers (results from the immigrant population are in the eAppendix [https://links.lww.com/EDE/A402]).
The study protocol was approved by the ethics committee of the Institut Municipal d'Asistencia Sanitaria (CEIC-IMAS), Barcelona. Maternal informed consent and parental informed consent for the offspring were obtained.
Outcome Assessment
The assessment of lower respiratory tract infection was based on reports by the mother in interviewer-led questionnaires administered during the 6 and 14 month visits. Occurrence of a lower respiratory tract infection was defined as a positive answer to the question “Has a doctor told you that your son/daughter has had a chest infection?” A second question referred to a specific doctor diagnosis of bronchitis, bronchiolitis, or pneumonia. Agreement between the 2 questions was 92%.
Covariate Assessment
Information on covariates was obtained through questionnaires administered during the first and third trimesters of pregnancy. These covariates included maternal education, socioeconomic background (based on maternal occupation classified by International Standard Classification of Occupations [ISCO88 code]), marital status, maternal health, and obstetric history, parity, medication use, alcohol and smoking habits, anthropometric measures, and dietary intake. Information on breast-feeding was collected in the interview at 6 and 14 months. Information on postnatal smoking habits was collected in the 14-month interview. In serum extracted during the first trimester of pregnancy, levels of total IgE (an objective surrogate of maternal atopy) were determined using electrochemiluminescence, at the Laboratori de Referencia de Catalunya.
Exposure Assessment
Maternal blood was drawn during the first trimester of pregnancy, and serum was stored in glass tubes at −20°C. Levels of organochlorine compounds (HCB, β-hexachlorocyclohexane (β-HCH), p,p′-DDT, p,p′-DDE, and PCB congeners 28, 118, 138, 153, and 180) were analyzed in these serum samples using methods described elsewhere.24 The limit of detection was 0.071 ng/mL. Values with nondetectable levels were set at a value of half the detection limit. All exposures are expressed on a lipid basis using the method described elsewhere.25 Correlation between results adjusted and not adjusted for lipid levels were 0.973 for DDE and 0.954 for PCBs.
Statistical Analysis
We used 4 definitions of lower respiratory tract infection according to the report of any chest infection in specific reporting periods: lower respiratory tract infection in the first 6 months; lower respiratory tract infection between 6 and 14 months; intermittent lower respiratory tract infection (any chest infection in 1 of the 2 periods); and recurrent lower respiratory tract infection (chest infection in both periods).
We presented analyses using lipid-adjusted organochlorine compounds to facilitate comparison with other studies, and because the coefficients obtained without lipid adjustment did not appreciably change from those obtained after lipid adjustment (change less than 0.005). The organochlorine compound levels were analyzed as categorical variables using tertiles as cut-offs, and also as continuous variables. For PCBs, only coefficients of the continuous variables are presented because generalized additive modeling did not show departure from linearity (Pgain of linearity >0.4 in all cases). When treated as continuous variables, organochlorine compounds were log-transformed to approach normal distributions.
Poisson regression models were used to estimate relative risks for the association between lower respiratory tract infection and DDE levels. We present results after adjusting for any variable with a P < 0.2 for the bivariate association with the outcome. The following cofactors were evaluated: maternal age, social class, education, smoking during pregnancy and the postnatal period, parity, allergy (defined by the report of food or drug allergy, allergic rhinitis, or atopic dermatitis), asthma, total IgE, weight, child sex, birth weight, and predominant breast-feeding (exclusive breast-feeding with the possible addition of orange juice or water).
RESULTS
About 20% of the babies of native mothers had a lower respiratory tract infection the first half-year of life, and about 30% did between the months 6 and 14 of life (Table 2). Six percent of children had been diagnosed with bronchiolitis during the first half year of life, whereas during months 6-14 most diagnosed infections were bronchitis. Thirteen percent of babies had a recurrent lower respiratory tract infection in both periods. The corresponding frequencies among infants born to immigrant mothers were similar (eTable 1 [https://links.lww.com/EDE/A402]).
TABLE 2: Description of Reported Doctor Diagnosis of Lower Respiratory Tract Infections in Infants, the Sabadell Birth Cohort (n = 520)
DDE was the organochlorine compound with the highest concentrations (Table 3). DDE was quantifiable in almost all mothers, but DDT was not detectable in any sample. DDE levels were much higher in immigrant mothers than in mothers born in Spain, whereas for other organochlorine compounds the patterns varied (eTable2 [https://links.lww.com/EDE/A402]). The pollutant with the second-highest concentration was HCB. Among the PCBs, congener 153 was the most common.
TABLE 3: Organochlorine Levels (ng/g Lipid at 25th, 50th, and 75th Percentiles) at First Trimester of Pregnancy, the Sabadell Birth Cohort (n = 520)
Crude organochlorine compound levels were higher among mothers of children with lower respiratory tract infection than in those without (Table 4). Maternal age at delivery, social class, education, smoking, parity, allergy, total IgE, sex of the child, and breast-feeding were related to lower respiratory tract infection.
TABLE 4: Organochlorine Levels and Covariates by Lower Respiratory Tract Infections Status in Infants
These covariables showed different patterns of association with the specific organochlorines (Table 5). Although DDE increased with lower social class, PCB concentrations decreased with lower social class, poorer education, parity, and total IgE levels. All organochlorine compounds increased with maternal age and decreased with prenatal smoking. No differences were seen for gender of the child. Women who breast-fed longer had lower levels of organochlorine compounds, except PCBs, although differences were typically rather small. HCB and βHCH were also associated with education and parity.
TABLE 5: Geometric Mean of Organochlorine Levels (ng/g Lipid) by Maternal and Child Characteristics
DDE was the only organochlorine compound that showed a strong adjusted association with lower respiratory tract infection. In particular, there was an association, even after adjusting for PCBs, with lower respiratory tract infection occurring during the first 6 months of life (for the second tertile compared with the first, and for continuous DDE), during the period between 6 and 14 months (for the second and third tertile), as well as for recurrent lower respiratory tract infection (for the second and third tertile, and as a continuous variables) (Table 6). Increases in relative risks were similar in the second and third tertiles (compared with the first) in all DDE models. PCBs did not show an association in the 1-pollutant models (data not shown). In the 2-pollutant models (Table 6), relative risks for DDE showed very little change in comparison with the 1-pollutant models, whereas the risks with PCB were not elevated. Inclusion of HCB and β-HCH in the models did not change the coefficients of DDE or PCBs obtained in the 2-pollutant models (not shown). Correlation coefficients between DDE and other organochlorides ranged from 0.25 to 0.39, and the coefficients between PCBs and the other organochloride compounds were from 0.38 to 0.49. The associations for HCB and β-HCH in the 1- and 2-pollutant models were much weaker than those observed for DDE. Adjustment for other variables included in Table 3 did not change the coefficients. The association between DDE and lower respiratory tract infection was not modified after stratification by breast-feeding or maternal asthma or allergy (all P values for interaction >0.25). Among children born to immigrant women, the relative risks with increasing levels of DDE, but were modest, with broad confidence intervals (eTable 3 [https://links.lww.com/EDE/A402]).
TABLE 6: Adjusted Associations Between pp′DDE (in Categories Defined by Tertiles or Continuous) and Lower Respiratory Tract Infections: 1- and 2-Pollutant Models
DISCUSSION
Maternal DDE levels were associated with early-life lower respiratory tract infection, at 6 months and 14 months of age, even after adjusting for PCBs or other organochlorine compounds, whereas PCBs were not associated with lower respiratory tract infections. Adjustment for other risk factors did not change the results. The fact that maternal DDE levels were associated with lower respiratory tract infection both in children who were breast-fed and those who were formula-fed, suggests that most of the effect occurred during prenatal life. Thus, alteration in the developing immune system after exposure to DDE during prenatal life could result in long-term impairment and increased risk of lower respiratory tract infection. The exact mechanism, however, is unknown. The lack of a linear dose-response pattern, with similar effect size for the second and third tertile of DDE exposure, is difficult to explain from a biologic perspective.
Several experiments in animals have shown that DDE induces changes in immunity that may lead to an increased susceptibility to viral infections.1-4 In humans some studies have assessed the association of DDE with general immunologic markers at levels moderately higher than the present study (Table 1). All of these studies are cross-sectional and relatively small, and they did not adjust for PCBs. In persons living near a waste dump in North Carolina, those with higher plasma DDE levels had lower mitogen-induced lymphoproliferative activity and increased total lymphocytes and IgA levels.5 In a small study in farmers also in the United States, DDE levels were associated with an increase of interleukin-4 (which, in addition to down-regulating T helper 1 (TH1)-type cytokines, influences natural killer responsible of viral response).6 Another study of US farmers showed that IgG levels (involved in secondary response to infections) decreased with increasing DDE levels, and autoantibodies increased.9 In German school children, DDE levels were associated with an increase of IgE as well as in total lymphocytes.10 A small study in pregnant women from Australia showed a depression of T helper cell Type 1 cytokines.8 Overall, these studies are not conclusive about the specific immunologic pathways, although several of them suggest the occurrence of a depression of T helper cell Type 1 after cell stimulation, which is responsible for response to viral infections. Two studies in children, one with higher and one with lower levels of DDE than the present study, have found an increase of lower respiratory tract infection with increasing DDE levels, although effects could not be isolated from those of other organochlorine compounds.11,14
Furthermore, a birth cohort study from Menorca (Spain) found an association between prenatal exposure to DDE and asthma.20,21 Several prospective studies have established a link with asthma development during the first decade of life for hospitalized lower respiratory tract infection22 and outpatient lower respiratory tract infection.26 Continued follow-up of existing birth cohorts is needed to find out if the observed association between DDE and lower respiratory tract infection during first years of life is followed by an association between DDE and asthma at school age, when the asthma phenotypes are clinically detectable.
We found different levels of DDE between native and immigrant mothers, and no detectable levels of DDT. DDE is a highly lipophilic metabolite formed from DDT. DDT was found during periods of active use of DDT.27 DDT has been banned in Europe for decades before this study, which explains the undetectable levels of DDT. DDE, however, is more persistent than its parent compound and is still ingested directly through foods containing animal fat.28 The DDE levels we observe probably originate from dietary exposures. The distinct exposure patterns in immigrant women may be explained by a different dietary pattern, although recent direct exposure to DDT in their country of origin is also a possibility. The fact that PCB levels were higher among mothers born in Spain also points towards a different dietary pattern. By contrast, we found an association of DDE with lower respiratory tract infection in both groups (P for interaction >0.40), whatever the origin of DDE, which reinforces the evidence for an immunologic effect of DDE.
PCBs, whose industrial use has also been forbidden for decades, are less common than DDE for both mothers born in Spain and those born elsewhere, although in the latter group the ratio of DDE to PCBs was higher. Adjustment for PCBs hardly changed the associations between DDE and lower respiratory tract infection, whereas PCBs were not associated with lower respiratory tract infection. Similarly, other organochlorine compounds did not explain the effect found for DDE.
Several risk factors have been related to lower respiratory tract infection in infants from similar cohorts in Europe: parity, male sex, maternal allergy, breast-feeding, and low social class.29 Maternal smoking has also been found to be associated with lower respiratory tract infection, but its inter-relationships with other factors such as breast-feeding30 or genetic susceptibility31 are complex. The associations observed in this study followed the direction and size expected, except for smoking. One explanation could be that 46% of smokers reported having quit before week 12 of pregnancy, and those who continued reduced by half the number of cigarettes (to an average of 5 per day). Many of the women who smoked throughout the entire pregnancy continued to smoke up to 14 months after delivery, and although 84% of them reported never smoking in front of the child, it is impossible to disentangle the pre- and postnatal smoking effects. Detailed research on the effects of smoking and inter-relations with other factors is required to understand the absence of an effect of smoking on lower respiratory tract infection in this cohort.
A major limitation of our study is the lack of serology or culture to confirm the lower respiratory tract infection diagnosis. However, we used repeated items to define lower respiratory tract infection from repeated questionnaires at different time periods, and our results were consistent whichever definition criteria was selected. A second limitation is nonresponse. Differences between participants and nonparticipants were minimal for most variables (social class, smoking, parity, maternal age, allergy, BMI, sex, birth weight, breast-feeding); differences for maternal education and asthma were greater, but their inclusion in the models did not change the associations. The strengths of the study were its prospective nature, the consistency of the findings using repeated questionnaires and repeated definitions, and the ability to adjust for PCBs (which were important because of the relatively poor correlation among the organochlorine compounds).
Overall, the immunologic suppression effect of DDE in experimental studies with DDE could explain the association observed between DDE and lower respiratory tract infection independently of PCBs and other organochlorine compounds. Lower respiratory tract infection can be a source of substantial morbidity in infancy as well as a possible risk factor for subsequent childhood asthma, and DDE is a common xenobiotic in human tissues. This study reinforces the hypothesis that exposures during prenatal life could have a critical effect on the development of the immune and respiratory systems.
ACKNOWLEDGMENTS
We are grateful to Silvia Fochs, Anna Sànchez, Maribel López, and Nuria Pey for their assistance in contacting the families and administering the questionnaires. We particularly thank all the participants for their generous collaboration. A full roster of the INMA-Sabadell Study Investigators can be found at http://www.proyectoinma.org/cohorts/sabadell/membres-sabadell.html.
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