Biological sampling

In the EPHOR project, the relation between the working-life exposome (occupational as well as non-occupational factors) and non-communicable diseases will be studied. This includes the targeted and agnostic common biomonitoring, omics and bioassays on traditional biological matrices in addition to method development for non-invasive monitoring. Blood samples will be used for metals, cotinine, white blood cell count, cytokines, hydroxydeoxyguanosine, mitochondrial DNA copy number, telomere length, GWAS, epigenomics, targeted RNA expression, pyrosequencing, cell-free DNA methylation, proteomics and fluorescent oxidation products (FlOPs).

Exposure to metals can result in oxidative damage. Therefore, Pb and As and possible other metals (e.g. Cd, Hg, Pt, Fe, Mn) will be measured in blood. Measuring cotinine gives an insight into the smoking behavior of the participants and will be done for samples from WP6 (asthma/COPD short-term study) and WP7 (shift workers). Furthermore, epigenome-wide association studies (EWAS) help elucidate associations between altered DNA methylation and health outcomes as well as environmental impacts on DNA methylation which is integral to the EPHOR study (WP6 and WP7). DNA methylation will additionally be studied using bisulfite Pyrosequencing.

It is a technique based upon the “sequencing by synthesis” approach. It provides quantitative and highly reproducible methylation data at single-base resolution, and it requires relatively low quantities of DNA. This technique will be applied for samples from WP6 and 7. In addition, efficiently identifying circulatory proteins may provide a more mechanistic overview associated disease phenotypes, and at the same time help in development of predictive biomarkers (WP6). In this context another cell free marker, the cell-free DNA (cfDNA) in blood plasma is emerging as a powerful tool for disease diagnostics and biomarker development. And therefore, epigenetic marks found on cfDNA, such as DNA methylation, can be used as biomarkers for exposure and disease and will be validated in the present study (WP6). Moreover, 8-hydroxydeoxyguanosine (8-OHdG) will be used for measurement of oxidative damage. Blood samples from participants in WP6 and 7 cohorts will serve as matrix for 8-OHdG quantification.

In addition, also FlOPs will be evaluated. FlOPs are a global marker of oxidation processes, including protein and DNA oxidation and lipid peroxidation are of growing interest in epidemiology. This biomarker of damage has been found to be associated with chronic diseases including asthma. Furthermore, relative mitochondrial DNA copy number (mtDNAcn) and relative telomere length (rTL) will both serve as biomarkers for senescence/DNA damage. In addition, mtDNAcn will be used for the assessment of oxidative stress, whereas rTL will be used for the assessment of biological ageing in relation to occupational exposure. mtDNAcn and rTL will be assessed in cases and controls from WP6 and shift workers from WP7. Moreover, digital droplet (dd)PCR provides high-precision, absolute quantification of nucleic acid target sequences in a wide-range of applications including analysis of gene expression, microRNA analysis and genomic alterations such as copy number variations (CNV). ddPCR will be used to assess biomarkers for occupational exposure to shift work and other exposures i.e. stress, etc. (WP6 and 7).

Finally, investigations of the normal sleep–wake cycle showed that the production of pro-inflammatory cytokines exhibit peaks during early nocturnal sleep whereas the anti-inflammatory cytokine activity peaks during daytime wakefulness. Consequently, mistimed sleep and the associated alterations of circadian rhythms (like in a night shift work) are suggested to lead to disturbed immune responses which might contribute to the increased risk for infection, autoimmune diseases, cardiovascular and metabolic disorders, and cancer. A panel of 30 cytokines, chemokines and growth factors will be measured in plasma samples to assess changes in their profile in night shift workers compared to day shift workers. Concentrations will be measured at multiple time-points for several days based on feasibility in all contributing cohorts.

Certain other parameters (e.g. IgE, CC16, YKL40) that have an added benefit for the study could be measured in the samples that will be collected during the project. Urine can serve as a non-invasive matrix for the assessment of internal exposure to metals and polycyclic aromatic hydrocarbons (PAHs). Cadmium, arsenic and PAHs are known to be carcinogenic to humans. Therefore, it is important to measure the internal exposure. Metal as well as PAH analysis will be performed on samples from the asthma/COPD study (WP6) and the working-life exposome study in shift workers (WP7). Steroid hormones (including glucocorticoids, androgens and progestogens) play important roles in key human functions. Their concentrations might be altered by night shift. Melatonin is a hormone mainly released by the pineal gland at nighttime.

The main role of melatonin is the regulation of the sleep-wake cycle, although it plays additional roles such as antioxidant, body weight regulation or reproduction. Saliva is a well-established matrix for melatonin determination. Quantification of salivary melatonin is especially useful for capturing sharp changes in the pineal activity. Saliva will be used as a non-invasive matrix to measure steroid profile concentrations and melatonin concentrations in shift workers from WP7. Concentration will be measured at multiple timepoints. Exhaled breath analysis will be performed for both WP6, as part of the asthma/COPD short-term study, and WP7, as part of the working-life exposome study in shift workers. The volatile compounds on breath will be measured and identified by high resolution GC-MS. A panel of volatile compounds on breath related to mechanisms such as oxidative stress and inflammation makes up the core of the analysis, while discovery analysis will be performed on the collected data to identify new potential compounds which correlate occupational exposures. Exhaled breath analysis will be performed for both WP6, as part of the asthma/COPD short-term study, and WP7, as part of the working-life exposome study in shift workers. Non-volatile breath targets captured can be analyzed using LC-MS and/or ELISA.

A panel of volatile and non-volatile compounds on breath related to mechanisms such as oxidative stress and inflammation makes up the core of the analysis, while discovery analysis will be performed on the collected data to identify new potential compounds which correlate occupational exposures. In WP6, cfDNA methylation and proteomics will be studied in exhaled breath condensate samples from WP6. By using exhaled breath condensate, an alternative matrix for blood is evaluated.