To Örebro University

BACKGROUND AND OBJECTIVES: The pesticide metabolite p,p'-DDE has been associated with left ventricular (LV) mass and known risk factors for LV hypertrophy in humans and in experimental models. We hypothesized that the associations of p,p'-DDE with LV hypertrophy risk factors, namely elevated glucose, adiposity and hypertension, mediate the association of p,p'-DDE with LV mass.

METHODS: p,p'-DDE was measured in plasma from 70-year-old subjects (n = 988) of the Prospective Study of the Vasculature in Uppsala Seniors (PIVUS). When these subjects were 70-, 75- and 80- years old, LV characteristics were measured by echocardiography, while fasting glucose, body mass index (BMI) and blood pressure were assessed with standard clinical techniques.

RESULTS: We found that p,p'-DDE levels were associated with increased fasting glucose, BMI, hypertension and LV mass in separate models adjusted for sex. Structural equation modeling revealed that the association between p,p'-DDE and LV mass was almost entirely mediated by BMI (70%), and also by hypertension (19%).

CONCLUSION: The obesogenic effect of p,p'-DDE is a major determinant responsible for the association of p,p'-DDE with LV mass.

Background and objective: During recent years, some persistent organic pollutants (POPs) have been linked to atherosclerosis. One group of POPs, the poly- and perfluoroalkyl substances (PFASs) have not been investigated with regard to atherosclerotic plaques.

Methods: Carotid artery atherosclerosis was assessed by ultrasound in 1016 subjects aged 70 years in the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study. Eight PFASs were detected in >75% of participants' plasma by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS).

Results: No significant linear associations were observed between the PFASs and intima-media thickness (IMT), or the echogenicity in the intima-media complex (IM-GSM, a marker of lipid infiltration in the artery) when men and women were analyzed together. Neither was occurrence of carotid plaques related to PFASs levels. However, highly significant interactions were observed between some PFASs and sex regarding both IM-GSM and plaque prevalence. Perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), and perfluoroundecanoic acid (PFUnDA), were all related to IM-GSM in a positive fashion in women (p=0.002-0.003), while these relationships were negative in men. The levels of PFUnDA were significantly related to carotid plaque in women (OR 1.59, 95%CI 1.03-2.43, p=0.03), but not in men (OR 0.93, 95%CI 0.62-1.42, p=0.75).

Conclusions: In this cross-sectional study, a pronounced gender difference was observed regarding associations between some PFASs, especially the long-chain PFUnDA, and markers of atherosclerosis, with more pronounced relationships found in women. These findings suggest a sex-specific role for PFASs in atherosclerosis.

During an investigation of the potential associated with coupling packed column supercritical fluid chromatography (pSFC) to mass spectrometry for the analysis of Dechlorane Plus and related compounds, it was found that negative ion atmospheric pressure chemical ionization (APCI) was a promising ionization technique. In the course of maximizing the responses associated with the target analytes, it proved useful to examine some aspects of the complex nature and reactivity of the corona discharge plasma generated to explain the observed ionization products. Various dopants/reagents were screened for both APCI and atmospheric pressure photoionization (APPI) in negative ion mode and mechanisms of ionization involving superoxide were elucidated based on the results obtained. Superoxide formation was found to be temperature dependent and directly related to the intensity of the ion cluster [M-Cl+O](-) obtained for the target DP analytes. Furthermore, triethylamine was identified as a reagent capable of suppressing unwanted side reactions during the ionization process and maximizing response associated with the analytes of interest. The applicability of pSFC-APCI/MS for the separation and detection of Dechlorane Plus and related compounds was demonstrated by analyzing Lake Ontario sediment and comparing the results with values reported in the scientific literature.

Currently used analytical techniques for halogenated aromatic environmental contaminants such as polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and biphenyls (PCBs), also known as legacy persistent organic pollutants, are based on gas chromatographic separation of target analytes and detection by mass spectrometry. The coupling of packed column supercritical fluid chromatography (SFC) to atmospheric pressure ionization mass spectrometry (API/MS) could allow for the concurrent analysis of thermally labile and legacy halogenated environmental contaminants if ionization can be sufficiently optimized. The evaluation of positive ion atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) as well as possible charge transfer dopants for the generation of molecular ion isotopomeric clusters of halogenated environmental contaminants with minimal fragmentation has been completed. Using the investigated parameters, positive ion APPI was found to be the more sensitive technique. Of the aromatic and cycloalkane dopants investigated, only fluorobenzene and trifluorotoluene were found to be effective dopants for the halogenated aromatic target analytes (PCDDs, PCDFs, and PCBs). Experiments involving deuterated dopants confirmed that reactive species generated by cycloalkanes were quenched by the SFC eluent rendering them unusable in conjunction with the investigated separation technique. Alternatively, aromatic dopants were found to be less susceptible to quenching by the SFC eluent and fluorobenzene was determined to be the most effective charge transfer dopant for PCDDs, PCDFs, and PCBs. To demonstrate the applicability of the optimized ionization conditions, SFC-API/MS has been used for the concurrent analysis of legacy halogenated aromatic environmental contaminants (PCDDs, PCDFs, and PCBs) and thermally labile analytes (alpha, beta, and gamma isomers of hexabromocyclododecane).

The application of phosphorus based flame retardants as replacements for commonly used halogenated flame retardants has been gaining interest due to the possibility that these compounds may have a less significant impact on human and environmental health. Unfortunately, little is known about the chemical compositions of many of the technical products (which often are mixtures) and a single separation technique for concurrent analysis of these types of compounds has not been identified. This paper reports the results of an investigation into the constituents of three halogen free organophosphate flame retardants (OPFRs), resorcinol bis(diphenyl phosphate) (RDBPP), bisphenol A bis(diphenyl phosphate) (BPA-BDPP), and 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO). The major components of commercial samples of RDBPP and BPA-BDPP were isolated by preparative TLC and characterized by NMR. A commercial sample of DOPO was found to be essentially pure, but its analysis is complicated by the fact that it can exist in ring-open and ring-closed forms. With the structures of the components confirmed by NMR, multiple analytical separation techniques (gas chromatography (GC), liquid chromatography (LC), and packed column supercritical fluid chromatography (pSFC)) were investigated for the analysis of these three technical products. Packed column supercritical fluid chromatography allows the separation of the components of all three OPFRs, including the two forms of DOPO, in a single run.

Objectives: Since the metabolism of the organochlorine pesticide dichlorodiphenyltrichloroethane (DDT) is not fully known in humans, we evaluated if circulating levels of a major breakdown product of DDT, p,p'-DDE, were related to genome-wide genetic and methylation variation in a population-based sample.

Methods: In the population-based Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study (1016 subjects all aged 70), circulating levels of p, p'-DDE were analyzed by high-resolution chromatography coupled to high-resolution mass spectrometry (HRGC/HRMS). Genetic variants were genotyped and imputed (1000 Genomes reference, March 2012 release). Methylation sites were assayed using the Illumina HumanMethylation450 array in whole blood. A genome-wide association study (GWAS) approach was applied.

Results: Evidence for genome-wide significant association with p,p'-DDE levels was observed only for a locus at chromosome 19 corresponding to the CYP2B6 gene (lead SNP rs7260538). Subjects being homozygote for the G allele showed a median level of 472 ng/g lipid, while the corresponding level for those being homozygote for the T allelewas 192 ng/g lipid (p= 1.5x10(-31)). An analysis conditioned on the lead SNP disclosed a distinct signal in the same gene (rs7255374, position chr19: 41520351; p= 2.2 x 10(-8)). A whole-genome methylation analysis showed one significant relationship vs. p,p'-DDE levels (p= 6.2 x 10(-9)) located 7 kb downstreamthe CYP2B6 gene (cg27089200, position chr19: 41531976). This CpG-sitewas also related to the lead SNP (p = 3.8 x 10(-35)), but mediated only 4% of the effect of the lead SNP on p, p'-DDE levels.

Conclusion: Circulating levels of p, p'-DDE were related to genetic variation in the CYP2B6 gene in the general elderly population. DNA methylation in this gene is not closely linked to the p, p'-DDE levels.

Metabolic syndrome and mitochondrial dysfunction have been linked to elevated serum levels of persistent organic pollutants (POPs). However, it is not clear which specific POPs contribute to aryl hydrocarbon receptor (AhR)-dependent bioactivity or inhibit mitochondrial function in human subjects. Here, we measured the cumulative bioactivity of AhR ligand mixture (AhR bioactivity) and the effects on mitochondrial function (ATP concentration) in recombinant Hepa1c1c7 cells incubated with raw serum samples obtained from 911 elderly subjects in the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) cohort. Plasma concentrations of 30 POPs and plastic chemicals have previously been determined in the same PIVUS subjects. Linear regression analysis demonstrated that total toxic equivalence (TEQ) values and polychlorinated biphenyls (PCBs) were significantly correlated with AhR bioactivity (positively) and ATP concentration (negatively). Serum AhR bioactivities were positively associated with some PCBs, regardless of their dioxin-like properties, but only dioxin-like PCBs stimulated AhR bioactivity. By contrast, PCBs mediated a reduction in ATP content independently of their dioxin-like properties. This study suggests that AhR bioactivity and ATP concentrations in serum-treated cells may be valuable surrogate biomarkers of POP exposure and could be useful for the estimation of the effects of POPs on human health.

Aim: It has previously been reported that the environmental contaminants perfluoroalkyl substances (PFASs) are linked to atherosclerosis in cross-sectional studies. Since cross-sectional studies could be subject to reverse causation, we here analyzed if the longitudinal changes in PFASs during a 10 years follow-up were related to the change in intima-media thickness (IMT) during the same period.

Methods: In the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study,1,016 individuals were investigated at age 70. 826 of those were reinvestigated at age 75 and 606 at age 80 years. Eight different PFASs and carotid artery intima-media thickness (IMT, ultrasound) were measured at the three time-points.

Results: IMT increased 0.058 mm during the 10-year period (p<0.0001). Following adjustment for baseline values of PFASs (age 70) and sex, the changes in plasma levels of 6 of the 8 measured PFASs were significantly related to the change in IMT over the 10-year follow-up period (p<0.0062 using Bonferroni correction for 8 tests). Further adjustment for traditional CV risk factors (HDL and LDL-cholesterol, smoking, systolic blood pressure, statin use, fasting glucose and serum triglycerides) did only affect these relationships marginally.

Conclusions: The change in plasma levels of several PFASs during 10 years was related the increase in IMT seen during the same period, giving further evidence that PFASs might interfere with the atherosclerotic process.

BACKGROUND: In 2012, drinking water contaminated with per- and polyfluoroalkyl substances (PFASs), foremost perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic acid (PFHxS) at levels over 20ng/L and 40ng/L, respectively, was confirmed in Uppsala, Sweden.

OBJECTIVES: We assessed how a longitudinally sampled cohort's temporal trend in PFAS plasma concentration was influenced by their residential location and determined the plausible association or disparity between the PFASs detected in the drinking water and the trend in the study cohort.

METHODS: The Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) cohort provided plasma samples three times from 2001 to 2014. Individuals maintaining the same zip code throughout the study (n = 399) were divided into a reference (no known PFAS exposure), low, intermediate and high exposure area depending on the proportion of contaminated drinking water received. Eight PFASs detected in the majority (75%) of the cohort's plasma samples were evaluated for significant changes in temporal PFAS concentrations using a random effects (mixed) model.

RESULTS: PFHxS plasma concentrations continued to significantly increase in individuals living in areas receiving the largest percentage of contaminated drinking water (p < 0.0001), while PFOS showed an overall decrease. The temporal trend of other PFAS plasma concentrations did not show an association to the quality of drinking water received.

CONCLUSIONS: The distribution of contaminated drinking water had a direct effect on the trend in PFHxS plasma levels among the different exposure groups, resulting in increased concentrations over time, especially in the intermediate and high exposure areas. PFOS and the remaining PFASs did not show the same relationship, suggesting other sources of exposure influenced these PFAS plasma trends.