Download assessment of cytotoxicity and acute toxcity of

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