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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 ENDOCRINE DISRUPTING COMPOUNDS DEFINITION 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. 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015 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 EDC PRESENT IN THE ENVIRONMENT Polycyclic aromatic hydrocarbons (PAHs) Polichlorinated biphenyls (PCBs) Alkylphenols EXAMPLES Bisphenols Phthalates Chloroorganic pesticides 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015 SOURCES OF EDCs IN FOOD PRODUCTS SOURCES OF EDCs IN HUMAN DIET NATURAL Animal products Vegetable products ARTIFFICIAL EDCs transfered from food packaging Food additives Grain products 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015 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 Diethyl and dioctyl phthalates • • 4-t-octylphenol 4-Nonylphenol • Diethylstilbestrol 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015 BIOASSAYS USED FOR TOXICITY ASSESMENT BIOASSAYS 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 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 (EC50) that causes 50% decrease in bioluminescence. . 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015 EXPERIMENTAL METHODS MICROTOX PREPARATION OF BACTERIAL SUSPENSION Mixing the lyophilized bacteria with reconstitution solution ADJUSTMENT OF THE OSMOLARITY 2% NaCl PREPARATION OF STOCK SOLUTION OF THE SELECTED XENOBIOTICS Preparing dilutions wth the final EtOH concentration 2 % Preparig the stock solutions in absolute ethanol PREPARATION OF DILUTION SERIES Diluent - 2% NaCl ABSORBANCE READING BEFORE EXPOSURE ADDITION OF THE XENOBIOTICS MODEL SOLUTIONS TO THE BACTERIA SUSPENSION ABSORBANCE READING AFTER EXPOSURE 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015 EXPERIMENTAL METHODS – MTT ASSAY PLATING MCF-7 CELLS ON 96-WELL PLATE Cells density - 8x103 cells/well INCUBATION 24 hours, 37oC ADDITION OF XENOBIOTIC SOLUTIONS TO CELL SUSPENSION Stock solutions – 4mg of pure substane per 1 mL of ethanol absolute Dilution of stock solutions in growth medium to concentrations 0,540 µg/mL CELLS INCUBATION WITH XENOBIOTICS 3, 6, 24, 72 hours ADDITION OF FRESH GROWTH MEDIUM (200 uL) AFTER INCUBATION ADDITION OF 50 µL MTT SOLUTION 4-hours incubation DISSOLUTION of FORMAZAN CRYSTALS IN75µL DMSO Mixing λ=550 nm ABSORBANCE READING Tecan Infinite® 200 PRO microplate reader 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015 RESULTS – MICROTOX ASSAY 900 EC50 VALUES FOR SELECTED XENOBIOTICS 794.2 800 700 EC50 [umol/dm3] 600 500 400 300 200 115.1 100 108.1 89.2 76 61.2 46.8 40.2 5 2.2 0 -7.2 -100 Dioctyl phthalate BPA bis (2,3-dihydroxypropyl) ether BPA (2,3-dihydroxypropyl) glycidyl ether Diethyl phthalate BADGE 4tOP BPA (3-chloro-2-hydroxypropyl)(2,3-dihydroxypropyl) ether BPA BPA bis(3-chloro-2-hydroxypropyl) ether 4NP DES 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015 RESULTS – MTT ASSAY BPA 3h 160% BPA 6h 140% 4tOP 4tOP 140% 120% 100% % Control % Control 120% DES 100% 80% Dioctyl phthalate 60% Diethyl phthalate 4NP Dioctyl phthalate 60% 175.2 87.6 43.8 21.9 4.4 2.2 175.2 87.6 BPA bis (3-chloro-2hydroxypropyl) ether BPA 24h 140% 4tOP 120% BADGE 100% DES 4NP 0% BPA bis (2dihydroxypropyl) ether Xenobiotic concentration in test well [umol/dm 3] Diethyl phthalate 20% 20% 0% DES 80% 40% 40% 43.8 21.9 4.4 2.2 Xenobiotic concentration in test well [umol/dm 3] 72h 120% Dioctyl phthalate 60% Diethyl phthalate 40% 4NP 20% 0% 175.2 87.6 43.8 21.9 4.4 2.2 Xenobiotic concentration in test well [umol/dm3] BPA bis(2,3dihydroxypropyl) ether BPA (3-chloro-2hydroxypropyl) ether BPA (3-chloro-2hydroxypropyl) ether BPA 100% 80% BPA bis(2,3dihydroxypropyl) ether 4tOP % Control %Control BADGE BADGE BADGE 80% DES 60% Dioctyl phthalate 40% Diethyl phthalate 20% 4NP BPA bis (2,3dihydroxypropyl) ether 0% 175.2 87.6 43.8 21.9 4.4 2.2 Xnobiotic concentration in test well [umol/dm 3] BPA (3-chloro-2hydroxypropyl) ether 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015 RESULTS – MTT ASSAY 4000 CUMULATIVE CHART OF BIOLOGICAL EFFECT 3500 72h 24h 6h 3h 3000 2500 2000 1500 1000 500 0 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 4tert-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