LOGO ASSESSMENT OF CYTOTOXICITY AND ACUTE TOXCITY OF SELECTED ENDOCRINE DISRUPTING COMPOUNDS COMMONLY PRESENT IN FOOD PRODUCTS Katarzyna Owczarek, Błażej Kudłak, Zofia Mazerska, Jacek Namieśnik Gdańsk University of Technology Chemical Faculty Department of Analytical Chemistry 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015

EDCs are exogenous substances which, by disrupting the proper functioning of the endocrine system, cause undesired health effects on the organism exposed to them or on their offspring. DEFINITION ENDOCRINE DISRUPTING COMPOUNDS

MECHANISMS OF ENDOCRINE DISRUPTION  Modify hormone synthesis pathways  Disrupt hormone excretion mechanisms  Disrupt cell/tissue hormone transport pathways  Bind to receptors  Disrupt hormone degradation pathways  Mimic the functioning of endogenous hormones  Antagonism with synthesis of natural hormones or their metabolism  Change level or activity of the hormonal receptors 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015

6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015 EDC PRESENT IN THE ENVIRONMENT EXAMPLES Polycyclic aromatic hydrocarbons (PAHs) Polichlorinated biphenyls (PCBs) Phthalates Chloroorganic pesticides Alkylphenols Bisphenols

SOURCES OF EDCs IN FOOD PRODUCTS 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015 Animal products Vegetable products Grain products Food additives SOURCES OF EDCs IN HUMAN DIET NATURAL ARTIFFICIAL EDCs transfered from food packaging

THE AIM OF THE RESEARCH The aim of this work was to assess the cytotoxicity and acute toxicity of selected xenobiotics that commonly contaminate both food and environment using bioanalytical methods. 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015

EDCs SELECTED FOR THE RESEARCH Bisphenol A BADGE and its derivatives 4-t-octylphenol Diethyl and dioctyl phthalates 4-Nonylpheno l Diethylstilbestrol

6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015 BIOASSAYS USED FOR TOXICITY ASSESMENT MTT CELL VIABILITY ASSAY Based on the measurement of mitochondrial metabolic activity by the detection of reduction of the soluble yellow MTT tetrazolium salt to purple MTT formazan by the action of mitochondrial dehydrogenase. BIOASSAYS MICROTOX ® ASSAY Based on bioluminescence of seawater bacteria Vibrio fischeri. It is used to evaluate acute toxicity of the sample which is estimated by decrease in bioluminescence intensity. The designated parameter is effective concentration (EC 50 ) that causes 50% decrease in bioluminescence.

E XPERIMENTAL METHODS - MICROTOX 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015 ABSORBANCE READING AFTER EXPOSURE ADDITION OF THE XENOBIOTICS MODEL SOLUTIONS TO THE BACTERIA SUSPENSION ABSORBANCE READING BEFORE EXPOSURE PREPARATION OF DILUTION SERIES Diluent - 2% NaCl PREPARATION OF STOCK SOLUTION OF THE SELECTED XENOBIOTICS Preparig the stock solutions in absolute ethanol Preparing dilutions wth the final EtOH concentration 2 % ADJUSTMENT OF THE OSMOLARITY 2% NaCl PREPARATION OF BACTERIAL SUSPENSION Mixing the lyophilized bacteria with reconstitution solution

EXPERIMENTAL METHODS – MTT ASSAY 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015 ABSORBANCE READING λ=550 nmTecan Infinite ® 200 PRO microplate reader DISSOLUTION of FORMAZAN CRYSTALS IN75µL DMSO Mixing ADDITION OF 50 µL MTT SOLUTION 4-hours incubation ADDITION OF FRESH GROWTH MEDIUM (200 uL) AFTER INCUBATION CELLS INCUBATION WITH XENOBIOTICS 3, 6, 24, 72 hours ADDITION OF XENOBIOTIC SOLUTIONS TO CELL SUSPENSION Stock solutions – 4mg of pure substane per 1 mL of ethanol absolute Dil ution of stock solutions in growth medium to concentrations 0,5- 40 µg/mL INCUBATION 24 hours, 37 o C PLATING MCF-7 CELLS ON 96-WELL PLATE Cells density - 8x10 3 cells/well

RESULTS – MICROTOX ASSAY 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015

RESULTS – MTT ASSAY 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015

RESULTS – MTT ASSAY 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015

CONCLUSIONS  The results obtained in two toxicity bioassays do not seem to be correlated, although in both cases 4-nonylphenol and 4-tert- octylphenol show the highest toxicity  The lack of correlation may be the result of the differences in test organisms and the principle of the assays  Hormesis phenomenon observed in MTT test for bisphenol A and diethyl phthalate after short incubation period may indicate an estrogenic activity of these chemicals 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015

LOGO 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015