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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Supplementary Material
  8. Acknowledgements
  9. References

Objective:

We and others have shown relationships between circulating levels of persistent organic pollutants (POPs) and different measures of obesity in both cross-sectional and prospective studies. Since viscerally located fat seems to be the most harmful type, we investigated whether plasma POP levels were more closely related to visceral adipose tissue (VAT) than to subcutaneous adipose tissue (SAT).

Design and Methods:

Thousand hundred and sixteen subjects aged 70 years were investigated in the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study; 23 POPs were analyzed using high-resolution gas chromatography/high-resolution mass spectrometry. Abdominal magnetic resonance imaging, measuring VAT and SAT, respectively, was performed in a representative subsample of 287 subjects.

Results:

The less chlorinated polychlorinated biphenyl (PCB) congeners (105 and 118), and the pesticides dichlorodiphenyldichloroethylene (DDE), hexachlorobenzene (HCB), and trans-nonachlordane (TNC) were positively related to both VAT and SAT, whereas the more highly chlorinated PCBs (153, 156, 157, 169, 170, 180, 194, 206, and 209) were inversely related to both VAT and SAT. PCB189 was related to the VAT/SAT ratio in an inverted U-shaped manner (P = 0.0008).

Conclusions:

In conclusion, the results were in accordance with our previous studies using waist circumference and fat mass as obesity measure. However, the novel finding that PCB189 was related to the VAT/SAT ratio deserves further investigation since exposure to this PCB congener, which has previously been linked to diabetes development, might thereby play a role in the distribution of abdominal adipose tissue.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Supplementary Material
  8. Acknowledgements
  9. References

Persistent organic pollutants (POPs) include for example organochlorine (OC) pesticides such as dichlorodiphenyltrichloroethane (DDT), industrial chemicals such as polychlorinated biphenyls (PCBs) and polybrominated flame retardants including polybrominated diphenylethers (PBDEs), and dioxins. As the umbrella term implies, POPs degrade slowly and therefore persist for many years in the environment and in living organisms. Moreover, POPs accumulate in fatty tissues of humans and wildlife and bioaccumulate, i.e., body burdens are higher at higher levels in the food chain. Due to both their persistency and their widespread use, POPs such as DDT and PCBs are still found at considerable levels in biological matrices such as human tissues and breast milk (1) although their use was banned in most of the Western world over 30 years ago due to their toxicity.

Many POPs, including OC pesticides and PCBs, are endocrine-disrupting compounds and have gained increasing attention in this respect in recent years, as endocrine-disrupting compounds are emerging as a possible contributing explanatory factor to the obesity and diabetes epidemics (2-6). The global prevalence of overweight and obesity, defined as BMI ≥25 and BMI ≥30, respectively, has risen dramatically in high- as well as in low-income countries over the past few decades and is predicted to increase further in the future (7,8). In 2008, 1.5 billion adults worldwide were overweight, and 500 million of these were obese (9).

We and others have previously shown, in both cross-sectional and prospective studies, that POP plasma levels are associated with different measures of obesity (4,10-15). Within the framework of the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study, comprising 1,016 women and men aged 70 years, we have shown that increased plasma concentrations of OC pesticides, dioxin, and less chlorinated PCBs are positively related to obesity, whereas plasma concentrations of higher chlorinated PCBs are negatively related to obesity (13,15).

In the present cross-sectional study, we investigated the relationships between plasma levels of 21 POPs with areas of visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT), measured in a transversal abdominal section by magnetic resonance imaging (MRI), in a representative subset of the PIVUS cohort. Our results further expand the results from Lee et al. (13) and Rönn et al. (15), where the PIVUS cohort was examined using waist circumference and fat mass determined by dual-energy X-ray absorptiometry, respectively. Since viscerally located fat seems to be the most harmful type in terms of increased risk for cardiovascular disease and/or diabetes (16,17), we evaluated the hypothesis that POP plasma levels were related to the distribution of abdominal adipose tissue as determined by abdominal MRI. Our primary outcome variable was thus the VAT/SAT ratio, providing a quantitative measure of the abdominal fat distribution, but data for VAT and SAT are also given separately.

Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Supplementary Material
  8. Acknowledgements
  9. References

Subjects

Eligible for the study were all subjects aged 70 living in the community of Uppsala, Sweden. The subjects were randomly chosen from the register of community living. A total of 1,016 subjects participated, giving a participation rate of 50.1%. The study was approved by the ethics committee at Uppsala University.

All subjects were investigated in the morning after an overnight fast. No medication or smoking was allowed after midnight. The subjects were asked to answer a questionnaire about their medical history, education level, exercise habits, smoking habits, and regular medication. Education level was divided into three groups: <9 years, 9-12 years, and >12 years. Exercise habits were divided into four groups: <2 times light exercise (no sweat) per week, ≥2 times light exercise per week, 1-2 times heavy exercise (sweat) per week, and >2 times heavy exercise per week. Venous blood samples were collected and stored at −70 °C until analysis. Lipid determination was performed at the laboratory of the University Hospital in Uppsala, Sweden, using a standard laboratory technique and was performed on an Architect C 8000 analyzer (Abbott Laboratories, Abbott Park, IL) and reported using SI units. Some basic characteristics of subjects are given in Table 1 and further in Supplementary Table S1 online.

Table 1. Mean and s.d. as well as median and 25th and 75th percentile for the investigated variables
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Chemical analysis

POP levels were analyzed in stored plasma samples according to Salihovic et al. (18). A total of 23 POPs were measured: 16 PCBs; five OC pesticides: p,p′-dichlorodiphenyldichloroethylene (p,p′-DDE), hexachlorobenzene (HCB), cis-chlordane, trans-chlordane, and trans-nonachlordane (TNC); octachlorinated dibenzo-p-dioxin (OCDD); and one polybrominated diphenyl ether (BDE47). Briefly, samples were prepared using solid-phase extraction and analyzed using high-resolution gas chromatography coupled to high-resolution mass spectrometry. Among the 23 POPs measured, the detection rate was above 95.5% for all POPs except OCDD (80.6%) and BDE47 (72.2%). Two OC pesticides, cis-chlordane and trans-chlordane, with detection rates of <10% in the study population were not included in the final statistical analyses. Plasma concentrations were lipid adjusted and are expressed as ng/g lipid (shown in Table 1).

Abdominal MRI

MRI of the abdominal region was performed in 287 randomly selected subjects from the PIVUS cohort. VAT and SAT areas (cm2) were manually segmented from a single 10 mm thick axial steady-state-free precession slice as previously described (19). Based on repeated measurements of VAT and SAT in 22 subjects, the reproducibility/coefficients of variation were found to be 5.9 and 3.4%, respectively.

Statistical analysis

All variables were evaluated regarding non-normality, and variables with a skewed distribution, such as plasma triglycerides and POP concentrations, were ln-transformed. Differences between POP evels in three BMI groups (normal weight BMI <25 kg/m2, overweight 25-30 kg/m2, and obesity >30 kg/m2) were evaluated by ANOVA following adjustment for gender, education level, exercise, and smoking. In separate linear regression models for each environmental contaminant, the contaminant levels (as ln-transformed continuous variables) were related to VAT and SAT measurements by abdominal MRI. In each model, we adjusted for gender, education level, exercise, and smoking. POP levels were also divided into quintiles to evaluate potential nonlinear relationships, using both a linear and a quadratic term.

For all POPs and all outcomes, interactions between POP levels and gender were evaluated. Since no such interactions were observed, no stratification on gender was performed.

STATA 11 (Stata, College Station, TX) was used for calculations. For the primary outcome variable, the VAT/SAT ratio, a Bonferroni correction for the number of POPs was applied to determine the α-level for statistical significance (0.05/21 = 0.00238).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Supplementary Material
  8. Acknowledgements
  9. References

POPs vs. overweight and obesity

Plasma levels of the pentachlorinated PCB congeners 105 and 118, as well as of the OC pesticides DDE and TNC, were increased in overweight and obese subjects compared to normal weight subjects (Table 2). In contrast, most of the more highly chlorinated (substituted with 6-10 chlorine atoms) PCBs (PCB153, 156, 157, 169, 170, 180, 194, 206, and 209); exhibited reduced plasma levels in overweight and obese subjects (Table 2). No significant relationships were identified for OCDD or BDE47.

Table 2. Medians and 25th and 75th percentiles of persistent organic pollutants in normal weight, overweight, and obese subjects
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POPs vs. VAT and SAT by MRI

Similar to the relations described above, plasma levels of the less chlorinated (substituted with 4-5 chlorine atoms) PCB congeners 74, 105, and 118, together with all three OC pesticides included in the results analyses were found positively related to VAT and SAT areas measured by MRI (Table 3). In addition, levels of the more highly chlorinated PCBs 156, 157, 169, 170, 180, 189, 194, 206, and 209 were again negatively related to VAT, SAT, or both (Table 3). No significant relationships were identified for OCDD or BDE47.

Table 3. Linear regression analysis between persistent organic pollutants and VAT, SAT and the VAT/SAT ratio
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POPs vs. the VAT/SAT ratio by MRI

Of the investigated POPs, PCB169 and PCB189 tended to be related to the VAT/SAT ratio, a marker of the relative deposition of visceral and subcutaneous fat (Table 3), but none of these relationships was significant following adjustment for multiple testing of this primary outcome variable.

Nonlinear relationships

Based on quintile analysis, nonlinear relationships were found for PCB74, with maximum VAT in the fourth quintile; and for PCB194, with maximum VAT seen already in the second quintile (Supplementary Table S2 online). No significant nonlinear relationships were found between SAT and any of the analyzed POPs (Supplementary Table S3 online). Regarding the VAT/SAT ratio, nonlinear relationships were found for the four hepta- and octachlorinated PCBs 170, 180, 189, and 194 (Supplementary Table S4 online), with PCB189 levels showing the strongest association, with maximum VAT/SAT ratio in the third quintile (Pquadratic = 0.0008). This relationship remained significant following adjustment for multiple testing of this primary outcome variable.

In secondary analyses, we added information on total caloric intake (mean 1,889 (SD 505) kcal/day) and percent fat intake (32% (SD 4.8%)) (data available in a random 86% of the population by 7-day food item recordings) as additional confounders in the analysis. This did however only marginally affect the major results previously described.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Supplementary Material
  8. Acknowledgements
  9. References

The present cross-sectional study showed that plasma levels of OC pesticides and PCB congeners with no more than five chlorine substitutions were positively related to abdominal VAT and SAT, whereas PCB congeners substituted with six or more chlorine atoms exhibited negative relations. Moreover, we found a significant inverted U-shaped relationship for PCB189 and the VAT/SAT ratio, indicating that PCB189 might play a role in where abdominal adipose tissue is deposited. Our results are in agreement with the previous PIVUS studies using waist circumference (970 subjects) and dual-energy X-ray absorptiometry (890 subjects) as obesity indexes (13,15). However, the use of abdominal MRI, or abdominal computed tomography scanning as an alternative, permits the important distinction between abdominal visceral and subcutaneous fat and thereby expands and specifies our understanding of the role of POPs in obesity.

Our results, moreover, agree well with the findings by Dhooge et al. where plasma levels of the hexa- and heptachlorinated marker PCBs 138, 153, and 180 were negatively related to BMI in adults, whereas DDE, HCB, and the pentachlorinated PCB118 showed positive relationships (10). Dirinck et al. also found negative relationships between plasma levels of PCBs 153, 170, and 180 and BMI, but did not observe significant relationships between DDE and BMI (11). Elobeid et al. found positive associations between plasma levels of OCDD and both BMI and waist circumference, and negative associations between TNC and BMI (12), which is in contrast to our results. It is important to remember, however, that all three studies mentioned above investigated relationships in subjects with a much broader age span than in the PIVUS cohort. As younger subjects are included, any effects of direct POP exposure during later periods in life may be confounded with effects resulting from pre- and/or perinatal exposure according to the theory on the developmental origin of adult disease (6). While the relative homogeneity of our subjects entails limitations as to the applicability of our results to the general population, it also permits the detection of subtle effects and isolates the effects of postnatal exposure. Most of the POPs were both introduced and banned during the life of the PIVUS subjects, i.e., they have been subjected to high environmental POP levels without having been exposed during fetal life or through breastfeeding.

Our main result, the relationship between PCB189 and the VAT/SAT ratio, was found to be nonmonotonic in that the highest VAT/SAT ratio was found in the third, and not the fifth, quintile. In fact, the VAT/SAT ratio was lower in the fifth quintile compared to the third. Similar nonmonotonic relationships have been described previously regarding POP exposure and obesity measures, as well as the metabolic syndrome and diabetes (14), although the exact molecular mechanisms behind this phenomenon are unknown. Linearity between concentration of a ligand and occupancy of the corresponding receptor is generally seen only up to the concentration by which 10% of the receptors are occupied. At higher concentrations, a nonlinear ligand-receptor occupancy relationship is generally not seen (20,21). Sometimes even downregulation of receptors are seen at high concentrations (22). Thus, based on experimental knowledge on ligand-receptor occupancy kinetics, nonlinear relationships do not seem to be unrealistic, but it is unclear whether this is the mechanism behind the particular findings in the present study.

We found that the relationships between obesity indexes and PCBs with low or high degree of chlorination were of opposite signs. This might be due to differences in effects of PCB congeners, but is more likely due to differences in kinetic properties such as the half-lives of the congeners. A recent review on the kinetics of different POPs showed half-lives ranging from a couple of years for the less chlorinated PCBs, to >20 years for some of the highly chlorinated PCBs (23). In a model developed by Wolff and coworkers (24), an inverse relationship between fat mass and circulating POPs will be found following the major exposure, because the major part of the POPs are stored in fat tissue. However, given no further exposure and a constant fat mass, after some 2-3 half-lives of the compounds this negative relationship turns into a positive relationship due to the elimination of the POPs. Thus, for the highly chlorinated compounds, the time during which a negative relationship between fat mass and circulating PCBs is seen will be considerably longer than for the less chlorinated PCBs. It might be that the time from the exposure peak in the 1970s to the measurements in the present study ∼30 years later could produce relationships between obesity indexes and PCBs with low or high degree of chlorination with opposite signs. This picture is further complicated by recurrent exposure and the increase in fat mass usually seen with aging.

Another important feature to consider is that different fat depots might contribute unequally to the concentrations of POPs measured in the circulation. In a recent study by Yu et al. (25), POP concentrations differed substantially between different fat depots. Thus, it might well be that the elimination rates from different adipose tissues differ and thereby the contributions of VAT and SAT to the measured circulating concentrations would differ and explain some of the findings in the present study.

The large number of compounds in this study permits a thorough comparison of effects of different POPs. However, the correspondingly large number of statistical tests increases the risk for false positive results. For our primary outcome, the VAT/SAT ratio, we applied the Bonferroni multiple testing correction. Furthermore, we obtained similar nonlinear relationships for three other highly chlorinated PCBs, so although PCB189 was the only PCB below the Bonferroni corrected P value threshold, low-dose effects were also seen for PCB170, PCB180, and PCB194 with P values in the 0.05-0.01 range for quadratic trend, supporting the view that the PCB189 finding is not only a chance finding. Additional associations found were regarded as supplementary and therefore not subjected to correction, so these results must therefore be taken with caution until reproduced by others.

In conclusion, we confirmed that plasma levels of OC pesticides and PCBs are associated with both VAT and SAT in an elderly population. In addition, the appliance of abdominal MRI technology adds the dimension of the site of abdominal adipose tissue deposition through the VAT/SAT ratio, which showed a significant inverted U-shaped relationship for PCB189. Exposure to PCB189 might thereby play a role in where adipose tissue is deposited. This result deserves further attention, especially because high levels of highly chlorinated PCB congeners have previously been linked to the development of diabetes (14,26).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Supplementary Material
  8. Acknowledgements
  9. References

This study was financially supported by the Swedish Research Council (V.R.) and the Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning (Formas).

Table  . 
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References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Supplementary Material
  8. Acknowledgements
  9. References
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