Chemometers to Determine the Environmental and Human Exposome by Mixtures of Pollutants

EXPOSO-METER - Characterizing the Environmental and Human Exposome of Mixtures of Pollutants at High Trophic Levels

Motivation of this study

Human life is full of chemicals: They support the provision of cure, clothes, shelter, transportation and even nourishment of the world population. But can they also represent a risk for humans? Many of these chemicals can be taken up into the human body. Often, they are transformed and excreted directly, but some chemicals persist over longer time periods or accumulate in living organisms. Since there are many thousands of chemicals in the environment and products humans use on a daily basis, there may be complex mixtures around, and consequently also in the human body. The investigators start a new project at the Helmholtz Centre for Environmental Research (UFZ) in Leipzig, Germany. It is called EXPOSO-METER and attempts to make these chemicals measurable in an easy and comparable way, to better characterize this potential hazard. With the valuable contribution of the participants and collaborators, the investigators will be able to describe and compare the situation for similar groups of study participants in Central Europe, North America and Asia. The aim is to characterize the chemicals and see which potential effects their presence may have. In the project, the investigators will also compare human samples to material from the environment, e.g., seal and fox samples, to improve the understanding of the occurrence and fate of chemicals in the environment and in the human body.

Determination of sample size

The research project EXPOSO-METER does not aim for a large cohort study but for a proof-of-concept pilot study that will form the basis for future work. Therefore, two approaches were followed to determine the sample size:

1. Based on a literature review of similar studies, a closely matching example from a high-level journal was identified. It worked with 16 individuals in total from two German cities (different tissues, in total 88 samples). The work in EXPOSO-METER will by far exceed this number of study participants. Furthermore, three global regions are expected to show explicit large-scale differences due to distinct lifestyle and exposure conditions in the U.S., Europe and South Korea.
2. Cochran's formula [n= (Z^2 p(1-p))/e^2] was applied for sample size calculation: with a 90 percent confidence level (Z=1.645), maximum variability (50 percent, i.e., p=0.5) and +/-10 percent precision (e=0.1), it renders a minimum sample size of 68 participants.

As a combined result, the minimum sample size is therefore 50 study participants per region, with 100 study participants being ideal per region.

Sample collection

The following distribution of study participants is targeted: 25 female and 25 male participants of 30-40 years, and 5 female and 5 male participants of each of these age groups: 20-30, 40-50, 50-60, 60-70, 70-80 years. The following samples are collected: (1) 2-20 g of excized subcutaneous lipid from the lower abdomen, placed in a glass vial labelled with a unique sample identification number (ID) and stored at -20 °C; (2) 5 mL of blood using a tube filled with the complexation agent ethylenediaminetetraacetic acid (EDTA) and labelled with the ID and stored at -20 °C. In addition, the study participants are asked to fill the participants' questionnaire to be labelled with the ID. Once complete, the sample set is shipped to the UFZ laboratories.

Sample processing at UFZ

Lipid tissue: Samples will be thawed, homogenized and its lipid content determined. (a) Passive equilibrium sampling: To the sample, silicone thin-film samplers are added, which are medical grade silicone sheets of a suitable size and thickness, precleaned using Soxhlet extraction with ethyl acetate. After approx. 48 h (required for equilibration) the silicone is removed, its surface cleaned thoroughly and the chemicals from the silicone are extracted with ethyl acetate. (b) Total extraction: A subsample of the lipid tissue will be extracted directly with different mixtures of solvents. The solvent from both approaches is evaporated, the samples are cleaned via a range of columns, and the chemicals are analyzed by gas/liquid chromatography (GC/LC) coupled to high-resolution mass spectrometry (HRMS) or tandem mass spectrometry (MS/MS). In total, 150 + 400 chemicals will be screened for using the two techniques. For quality assurance/quality control, stable isotope-labelled internal standard compounds are used. The results will be set into context across regions, and data from the questionnaires will be used to support data interpretation.

Blood: The blood samples will be submitted to an extraction procedure currently under development, similar to the approaches described above, the extracts are cleaned, concentrated and analyzed by GC/LC-HRMS (or MS/MS), as described above.

Possible extensions

As a first option to further extend this work, the investigators aim to explore non-targeted screening of the broad mixtures of chemicals that are quantified in a targeted mode at UFZ. Likely there are many more chemicals, yet unidentified, that would be of interest to study, either at UFZ, via the EXPOSO-METER collaboration or via other related projects. For other projects, written consent will be sought before starting the work.

As a second option, the investigators would be interested in adding to the chemical profiling described above by exploring the application of bioanalytical tools (bioassays) to characterize the mixture effects elicited by the complex array of chemicals present in the human samples. This could either be done at UFZ, via the EXPOSO-METER collaboration or via other future projects. For other projects, written consent will be sought before starting the work.

Inclusion Criteria:

• willingness to contribute to the EXPOSO-METER research
• cover geographical patterns
• cover all sexes and age groups to derive patterns

Exclusion Criteria:

• none