Substudy 1

Toxicological analyses of PFCs/POPs; their time trend levels and impact on steroid hormone and dioxin receptor functions, cellular stress and possible counteraction by trace elements.

Time trend levels:
POPs/PFCs, trace elements and metals will be assessed in blood from the ABCB and PFCs in blood from DNBC. Trace elements and metals (including up to 53 elements e.g. Pb, Hg, Cd, Cu, Zn, Mn, As, Se, Ni, Cr, Fe etc.) and 17 PFCs, including 11 PFCAs (C4 to C14), 5 perfluoroalkyl-sulfonates (PFASs; C4 to C10) and 1 sulfonamide (perflurooctane-sulfonamide, PFOSA), will be determined at the National Environmental Research Institute (NERI). Other POPs including 14 PCBs and 10 OCPs will be analyzed at Le Centre de Toxichologie, Quebec, Canada. The POP/PFC analyses for GRBC will be measured as for the 2 Danish birth cohorts.

The POP data of the MISA samples will be obtained from NILU (Norwegian Institute for Air Research) Tromsoe, Norway. For the CHBC POP levels will be determined at the Nankai University, China.

All measurements will be performed under the Quality and assurance Controls.

Steroid hormone and dioxin receptor functions:
To support the traditional chemical exposure assessment of single POPs and PFCs, ex vivo cellular assays will be used to determine the effect biomarkers as the integrated biological effects of the serum POP/PFC mixtures on the estrogen- (ER), androgen- (AR) or aryl hydrocarbon- (AhR) receptors.
Serum extraction for ER and AR: A Solid Phase Extraction (SPE) followed by High Performance Liquid Chromatography (HPLC) fractionation will be performed to obtain the serum fraction containing the total mixture of bioaccumulated lipophilic POPs and PFCs.
Serum extraction for AhR: To obtain the lipophilic POP fraction, serum will be extracted using SPE and clean-up on multiple silica and florisil columns.
Receptor effect biomarkers: The serum extracts containing the total serum POP/PFC mixtures are applied for (xeno)hormone- and dioxin-like activity measurement using receptor mediated luciferease reporter gene bioassays.

Status of oxidative stress:
Endogenous hormones (estradiol, testosterone), total antioxidant status (TAS), the ratio of glutathione (GSH) to oxidized glutathione (GSSG) and DNA damage will be determined on ABCB samples using commercially available kits.

a) Time trends of POPs/PFCs will be determined for the following periods: 1996-2002 (DNBC) and 2008-2015(ABCB) while the cellular effect biomarkers on the function of hormone and dioxin receptors and the status for cellular oxidative stress and DNA damage will be determined for the period 2011-2014 (ABCB). The time trends of PFCs in ABCB maternal blood at 12th week of gestation from nulliparous women will be determination in 1600 samples (250/year 2008-2010; 200/year 2011-2014; 50 2015), the other POPs, trace elements, metals, status of oxidative stress, and PFCs in cord blood will be determined in 200 samples (each 50/year in 2011-2014).
b) The obtained data in a) (chemical measurements, effect biomarkers and oxidative stress parameters) will be used to evaluate possible associations between POP/PFC exposures and fetal and infant outcomes such as birth weight, infant growth, head circumference, and gestational age. PFCs measured during pregnancy and in cord blood will be used to estimate the mother to child PFC ratio.

Scientific team:
Responsible leader
Professor, director Eva C. Bonefeld-Jørgensen, Centre for Arctic Health, Department of Public Health, Aarhus University
Associated Professor Manhai Long, Centre for Arctic Health, Department of Public Health, Aarhus University
Post Doc Mandana Ghisari, Centre for Arctic Health, Department of Public Health, Aarhus University
PhD Tox student Christian Bjerregaard-Olesen, Centre for Arctic Health, Department of Public Health, Aarhus University
Technician Duy Dang, Centre for Arctic Health, Department of Public Health, Aarhus University
Professor Tine Brink Henriksen, The Aarhus Birth Cohort Biobank, Perinatal Epidemiology Research unit, Department of Pediatrics, Aarhus University Hospital, Skejby
Senior Scientist Rossana Bossi, National Environmental Research Institute (NERI), Department of Atmospheric Environment, Aarhus University
Senior Scientist Gert Asmund, National Environmental Research Institute (NERI), Department of Arctic Environment, Aarhus University