Development of New Genoproteimic Diagnostic Tools for the Toxilogical Assessment of Endocrine Disruptors in Food - ENDIF

Institution CRP Gabriel Lippmann
Partenaire(s) : CRP-Santé , Laboratoire National de Santé
Du : 01/02/2006
Au : 31/01/2009
Budget : 983 437,00€
Contact(s) : Hoffmann Lucien

Summary

Pollutants, originating either from industrial sources such as PCB’s (polychlorinated biphenyls) or from agricultural practices, mainly pesticides (e.g. atrazine) can act as endocrine disrupting compounds (EDC). These molecules can exhibit hormonal activity and therefore perturb physiological functions in humans, resulting e.g. in fertility-problems. These xenobiotics can be very persistent in the environment and accumulate in the food-chain. In addition to these man-made compounds, a couple of natural originating compounds, such as mycotoxins, may also act as EDC.

The objective of the present investigation was to both quantify PCB’s, zearalenone, atrazine and degradation products (DEA, DIA) in food and water in Luxembourg, and to study their effects on human cells by proteomic means. Fish and seafood products (n=90) were investigated on their PCB content (13 congeners) by GC-MS analysis. Water sources (n=70), tap water (n=34), and bottled water (n=25) were investigated for pesticides by HPLC-MS/MS analysis, as was zearalenone in wheat from field samples (n=17). In parallel, human MCF-7 cells were exposed to PCB-153 and atrazine at 100-500 ppb over 24h. Proteins were then isolated, separated into membrane and cytosol fractions, and identified by 2-D DIGE/SDS-PAGE and MALDI-TOF.

Atrazine and degradation products were detectable in most water sources (range 0-202 ng/L), bottled water (0-21 ng/L), and tap water (0-111 ng/L), however, levels remained below EU guidelines. Zearalenone levels up to 208 mg/kg from wheat collected from Luxembourgish fields were detected. Highest PCB-concentrations were found in fish, especially eel (ca. 450 mg/kg for PCB-153), with PCB-153 and 138 being the major PCB’s. A variety of additional foods including meat, milk, and dairy products showed only very low contamination with PCBs. A total of 49 proteins from the cytosol and 40 from the membrane were identified as being differentially expressed.

In general, Atrazine tended to result in underexpression, PCB-exposure in overexpression, effecting a variety of proteins partaking in the cell cytoskeleton integrity, DNA stability, and potentially spermatogenesis. Isolating for these sub- cellular compartments allowed for detecting differences among the low abundant proteins. Verification of a number of these differentially expressed proteins by western blotting techniques is ongoing. Despite PCB and atrazine banishment, both remain detectable in the environment, albeit in concentrations not surpassing EU-guidelines. In fish, especially eel future monitoring of PCB concentration seems warranted. Likewise, future studies on the effects of PCBs, especially their mixtures, on human cells are needed.

Refereed Scientific Publications

• Lasserre, J.P., Fack, F., Planchon, S., Revets, A., Renaut, J., Gutleb, A., Hoffmann, L, Bohn, T. Effects of the endocrine disruptors atrazine and PCB 153 on the protein expression of MCF-7 human cells (2009). Journal of Proteome Research 8, 5485-96.

Other Publications

• Not applicable