Association of endocrine active environmental compounds with body mass index and weight loss following bariatric surgery

The objective of this study was to study associations of a wide range of halogenated biphenyls, dibenzo-p-dioxins, dibenzofurans, and diphenylethers with body mass index (BMI) and evaluate changes in their concentration following bariatric surgery ​ . Methods Subcutaneous fat, visceral fat, and liver tissue samples were collected from 106 patients undergoing Roux-en-Y gastric bypass surgery for weight loss or patients who were undergoing abdominal surgery for non-bariatric reasons. We measured concentrations of an extensive panel of chlorinated and brominated biphenyls, dioxins, and furans, and brominated diphenylethers in the samples. We conducted linear regression to examine associations with BMI, adjusting for age and gender. Changes in concentration for indicator chemicals were evaluated in samples collected following bariatric surgery in a small sub-population. Results After BMI and in a of after surgery and loss.


Abstract Introduction
The objective of this study was to study associations of a wide range of halogenated biphenyls, dibenzo-p-dioxins, dibenzofurans, and diphenylethers with body mass index (BMI) and evaluate changes in their concentration following bariatric surgery .

Methods
Subcutaneous fat, visceral fat, and liver tissue samples were collected from 106 patients undergoing Roux-en-Y gastric bypass surgery for weight loss or patients who were undergoing abdominal surgery for non-bariatric reasons. We measured concentrations of an extensive panel of chlorinated and brominated biphenyls, dioxins, and furans, and brominated diphenylethers in the samples. We conducted linear regression to examine associations with BMI, adjusting for age and gender. Changes in concentration for indicator chemicals were evaluated in samples collected following bariatric surgery in a small sub-population.

Results
After adjustments for age and gender and correction for multiple testing seven ortho-chlorinated biphenyls, one non-ortho-chlorinated biphenyl, four PCDD/F's and one ortho-brominated biphenyl were associated with BMI. The strongest associations between BMI and lipid-adjusted concentrations were seen with PCB-105 in subcutaneous fat (beta=16.838 P-val=1.45E-06) PCB-126 in visceral fat (beta=15.067 P-val=7.72E-06) and PCB-118 (beta=14.101 P-val=2.66E-05) in liver. The concentrations of sum PCBs, chlorinated TEQ's and brominated compounds increased significantly with weight loss in subcutaneous fat in a group of ten individuals resampled up to five years after bariatric surgery and substantial weight loss.

Conclusion Introduction
Halogenated biphenyls, dibenzo-p-dioxins, dibenzofurans, and diphenylethers are chemicals that can interfere with endocrine systems and are implicated in the pathogenesis of obesity, cancer, birth defects, and other developmental disorders (1)(2)(3)(4)(5). There are increasing concerns that some of these chemicals can interfere with regulatory processes involved in lipid metabolism and in the control of adipocyte function resulting in excess weight and obesity (6,7). While some of these chemicals are of natural origin, the majority are synthetic chemicals, which have been released by human activities into the environment. Among the chemicals in this broad category are polychlorinated biphenyls (PCBs) and structurally related polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), the brominated dioxins (PBDDs), furans (PBDFs), and biphenyls (PBBs), diphenyl ethers (PBDEs) (8,9). The patterns of distribution of these chemicals in the body have been studied in numerous species (10). These chemicals are highly lipophilic and tend to distribute in the body primarily on the basis of the lipid content of the tissue, with data indicating that in general, the concentration of these compounds in lipid throughout the body is usually very similar (8).
PCDD/Fs, PBDD/Fs, and coplanar PCBs are also found in liver tissue as they bind to a specific protein, cytochrome p4501a2, which is present in the liver and can be induced by dioxin activation of the aryl hydrocarbon receptor (11,12). As a result, hepatic concentrations of these compounds can significantly exceed the concentrations expected on the basis of the lipid content of the liver.
Concentrations of many of these environmental chemicals have been monitored in the blood in the US population through the US National Health and Nutrition Examination Survey (NHANES), however, to our knowledge, there have been no systematic biomonitoring studies for chlorinated dioxins in UK populations and there are no UK studies looking at the association of environmental chemicals with BMI and obesity. Furthermore, since the concentrations of these compounds vary with the lipid content in the body, there are concerns that their concentrations might increase in the body following rapid weight lipid loss following procedures such as bariatric surgery (13)(14)(15). There are limited data looking at the changes in the body concentrations of these environmental chemicals following bariatric surgery and no data looking at the changes of these in liver tissue following weight loss (16,17). For example, Jansen A et.al. (18) analyzed 17 different polyfluoroalkylated substances (PFASs) to assess the effect of weight loss before and one year after bariatric surgery, and showed that the the plasma levels of all PFASs decreased upto 4-34%. Another study (19) examined 17 persistent organic pollutants (POPs) and 13 PFAA at baseline and following bariatric surgery and showed that there was a significant increase in POP levels in response obesity surgery.
The prevalence of obesity is increasing in the western world and genetic factors explain only a small proportion of the heritability of BMI/obesity (20,21). It is therefore important to investigate the association of known potentially endocrine active compounds with BMI. The objectives of this study were 1) to look at the association of selected persistent lipophilic environmental chemicals with BMI and 2) compare the concentration of these compounds in the body before and after bariatric surgery in a subset of the study population.

Study population and sample collection
The study population consisted of patients undergoing Roux-en-Y gastric bypass surgery for weight loss or patients who were undergoing abdominal surgery for non-bariatric reasons

Sample analysis
The method (23) used for the preparation, extraction and analysis of samples is comprehensively validated, formally accredited and forms part of the modular CEN method EN16215:2012. In brief, samples were fortified with 13C-labelled analogues of target compounds and exhaustively extracted using mixed organic solvents. PBDEs and ortho-substituted PCBs/PBBs were separated from non-ortho substituted PCBs/PBBs, PCDD/Fs and PBDD/Fs by fractionation on activated carbon. The two fractions were further purified using adsorption chromatography on alumina. Analytical measurement was carried out using high-resolution gas chromatography-high resolution mass spectrometry (HRGC-HRMS) for all analytes apart from the ortho-substituted PCBs which were analysed by high-resolution gas chromatography-unit resolution mass spectrometry (HRGC-LRMS). For example, in some cases, particularly for liver samples, the collected sample was of insufficient mass to allow analysis for all of the target chemical groups. In some cases the analytical results were flagged as "indicative" and were inconsistent with other data collected for the same compounds, and the decision was made to exclude those analytical results from the statistical analyses. Finally, for brominated dioxins and furans, 17 out of 21 contributing to toxic equivalency (TEQ) were measured due to the lack of available standards.
Specifically, octa-brominated dioxin and furan were not measured and only one hepta-brominated (a hepta-brominated furan) was measured. Although these have low toxic equivalency factor (TEF) values they are likely to contribute to total brominated TEQ, so it is likely that the brominated TEQ values will be somewhat underestimated.

Statistical methods
Summary statistics for each measured congener in each tissue (detection frequency, geometric mean, geometric standard deviation, minimum and maximum) were generated in R 3.5.0.
Several studies have shown a significant association of age with distribution of dioxin chemicals in the population. A simple bivariate correlation which will be confounded by age, is therefore not useful and can be misleading. Hence, we assessed the association of these environmental chemicals using regression analysis adjusting for both age and gender. Linear regression analysis implemented in R was used to test the association of BMI with the log10-transformed concentration of each compound, adjusted for age and gender. Bonferroni correction was used to identify a threshold for statistical significance (0.05/number of tests) to account for multiple testing. Since the environmental chemicals showed a high degree of correlation in subcutaneous tissues, visceral fat and liver we used a conservative Bonferroni correction of 0.05/63 where 63 is the number of chemicals measured across all the tissues.
Chlorinated PCBs and PCDD/Fs were analysed in baseline and follow-up samples from 10 participants. Sensitivity analysis was performed by 1) imputing missing data with random forest algorithm imputation in R and 2) by using raw values on environmental chemicals (rather than log-transformed).
To assess changes in analyte concentrations following weight loss, changes in the sum of three persistent indicator PCBs (138, 153, and 180) and in lower bound estimates of chlorinated ED were examined. Baseline and follow-up fat samples were analysed for selected brominated compounds in 10 individuals. BDE 153 was selected as a marker to examine changes in persistent brominated compound concentrations in these two participants.
Shapiro-Wilk tests were used to compare the median concentrations of the analytes before and after the bariatric surgery.

Changes in the concentrations selected compounds following a bariatric surgery
Follow-up subcutaneous fat biopsies of sufficient volume to allow analysis were collected from 10 bariatric surgery group participants. The biopsies were collected following an average of 2.8 years after initial surgery (range 1 to 4.8 years) and participants lost an average of 35.3% of their pre-surgery body weight. Fat mass was estimated at the baseline surgery time point and at the follow-up sample time using a prediction formula based on age, gender, and BMI (24). Based on these estimates, the participants lost an average of 48.6% of their body fat (range 38.1 to 60.6%). BDE-153 was also measured in two participants before and after surgery and the mean concentrations increased 2.5 μg/kg lipid to 4.04 μg/kg lipid.

Discussion
This is the first study in the UK population looking at the association of a suite of lipophilic, persistent chlorinated and brominated biphenyls, dioxins, furans, and diphenylethers with BMI as well as changes in their concentration following bariatric surgery and weight loss.
We show that several ortho-chlorinated biphenyls and non-ortho-chlorinated biphenyls and PCDD/F's are associated with BMI and that the concentrations of these environmental chemicals in fat tissues increase following bariatric surgery.
Our study showed that BMI was positively associated with selected polychlorinated biphenyls PCBs and structurally related polychlorinated dibenzo-p-dioxins dibenzofurans (PCDD/Fs) whereas BMI was not generally associated with the brominated dioxins (PBDDs), furans (PBDFs), and biphenyls (PBBs), diphenyl ethers (PBDEs) after correction for multiple testing. The positive association between concentrations of many of these compounds and age has been well documented in the literature; however, studies looking at the association with BMI with environmental chemicals have been equivocal (25). Since the increase in BMI also correlates with an increase in body lipids the concentrations of environmental chemicals in the body can increase or decrease depending upon the time course of environmental exposure to these compounds, acquisition of body fat, and lipophilic properties of these compounds. As a result, it is difficult to establish a causal relationship between these compounds and BMI. A study by Dirinck et al.(26)  With the increasing prevalence of obesity in the general population in many countries, evaluation of if, and how much, dioxin levels are associated with obesity becomes relevant to risk assessment for these compounds. There are various sources of these endocrine active chemicals, and according to some studies, exposure to chlorinated POPs by the general population continues, mostly through consumption of fatty foods of animal origin. Thus a high intake of animal source food can lead to higher levels of endocrine active chemicals leading to higher BMI.
The associations between BMI and environmental chemicals during the developmental period and childhood can be explored by prospective cohort studies looking at the exposure to environmental chemicals at birth and follow-up weight in the study participants; however, such studies have been limited. In a Belgian prospective cohort study (27) in the paediatric population, cord blood PCB ( congeners 118, 138, 153, 170, and 180) concentrations were associated with increased follow-up BMI. Another study (28) showed that female foetuses with the highest transplacental PCB exposures were heavier for their heights than other girls (at age 11 years) who were not exposed, however, these associations were only significant in caucasian participants. However, other studies have shown either negative (29) or null associations between PCBs and BMI (30).
Animal studies have also provided some evidence showing associations with obesity. A study by Ruzzin et al.(31) showed that male rats fed a high-fat diet of crude salmon oil (HFC) containing high levels of persistent organic pollutants (POPs) or refined salmon oil (HFR) with significantly lower environmental chemicals levels showed increased abdominal obesity, and elevated levels of diacylglycerol, triacylglycerol, and total cholesterol in HFC rats compared with the HFR rats. HFC-fed rats developed insulin resistance, impaired lipid and glucose metabolism, and hepatosteatosis, confirming that chronic exposure to environmental chemicals severely impairs insulin sensitivity and contributes to abdominal obesity in male rats.
A further issue relates to the impact of weight reduction upon the concentration and amount of dioxins in the body. There is the possibility that significant weight reduction could cause release of dioxin from adipose tissue depots, as the adipose tissue is lost, and that the concentrations in blood and adipose tissue of dioxin and other toxic lipophilic chemicals could increase if the dioxin remains in the body as adipose tissue is lost, potentially increasing the internal exposure level for toxicologically relevant tissues or receptors. There is evidence that weight loss is associated with an increase in PCBs and other highly lipophilic In this study, we also show that estimated concentrations of selected compounds increased in remaining fat depots following weight loss induced by bariatric surgery. Our findings are in agreement with an earlier study (16,32) which showed that the serum PCB concentrations Our study had several limitations. Since this is a cross-sectional study, we are not able to establish a causal link between environmental chemicals and BMI. It is possible that the association of the environmental chemicals with BMI simply reflects the excess accumulation of lipophilic environmental chemicals in participants with high BMI and high concentration of body lipids. Furthermore, in this cross-sectional study, we are not able to adjust for the timecourse of lifelong environmental exposure to these chemicals, which are important determinants of their concentration in the body. Also, the scope of this study was limited to investigating the association of these endocrine-active chemicals with BMI and changes in their concentration following bariatric surgery. Further studies are needed to investigate if the accumulation of these chemicals following bariatric surgery increases the likelihood of endocrine or hormonal dysfunction in the long term, though in the short term the beneficial effects of bariatric surgery would be more evident. All the participants from this study were recruited, from Hull and East Riding of Yorkshire, which is a highly stable population epidemiologically; therefore, different environmental exposure is less likely than in a mobile population, but a detailed migratory history was not obtained to confirm this.
However, this study provides first paired liver and adipose tissue samples from adults for a range of environmental chemicals, including some with no previous human liver tissue data.
We also show that if an environmental chemical is associated with BMI, this association is likely to be consistent across subcutaneous fat, visceral fat and in liver fat and likely is a consequence of high correlation levels across these depots. We also provide evidence of environmental chemicals re-distribution following weight loss, which has implications for patients experiencing significant weight loss, whether following surgery, dietary restriction, or secondary to illness. In summary, we show an association between BMI and various polychlorinated biphenyls PCBs and structurally related polychlorinated dibenzo-p-dioxins dibenzofurans (PCDD/Fs) and increased subcutaneous fat concentrations following weight loss. Further molecular, genetic and animal studies are needed to elucidate potential causal associations between these compounds and obesity..

Data availability Statement
The data that support the findings of this study are available from the corresponding author uponreasonable request.