logo assessment of cytotoxicity and acute toxcity of selected endocrine disrupting compounds...
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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
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.
DEFINITIONENDOCRINE 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
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-Nonylphenol
•
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 (EC50) that causes 50% decrease in bioluminescence.
EXPERIMENTAL METHODS - MICROTOX
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015
EXPERIMENTAL METHODS – MTT ASSAY
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015
RESULTS – MICROTOX ASSAY
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015
-100
0
100
200
300
400
500
600
700
800
900
-7.2
794.2
115.1 108.1 89.2 76 61.2 46.8 40.25 2.2
EC50 VALUES FOR SELECTED XENOBIOTICS
Series1
EC
50 [
umol
/dm
3]
RESULTS – MTT ASSAY
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015
175.2 87.6 43.8 21.9 4.4 2.20%
20%
40%
60%
80%
100%
120%
140%
160% 3h BPA
4tOP
BADGE
DES
Dioctyl phthalate
Diethyl phthalate
4NP
BPA bis (2-dihy-droxypropyl) ether
BPA bis (3-chloro-2-hydroxypropyl) ether
Xenobiotic concentration in test well [umol/dm3]
% C
ontr
ol
175.2 87.6 43.8 21.9 4.4 2.20%
20%
40%
60%
80%
100%
120%
140% 6hBPA
4tOP
BADGE
DES
Dioctyl phthalate
Diethyl phthalate
4NP
BPA bis(2,3-dihy-droxypropyl) ether
BPA (3-chloro-2-hydroxypropyl) ether
Xenobiotic concentration in test well [umol/dm3]
% C
ontr
ol
175.2 87.6 43.8 21.9 4.4 2.20%
20%
40%
60%
80%
100%
120%
140%24h BPA
4tOP
BADGE
DES
Dioctyl phthalate
Diethyl phthalate
4NP
BPA bis(2,3-dihy-droxypropyl) ether
BPA (3-chloro-2-hydroxypropyl) ether
Xenobiotic concentration in test well [umol/dm3]
%C
ontr
ol
175.2 87.6 43.8 21.9 4.4 2.20%
20%
40%
60%
80%
100%
120% 72h BPA
4tOP
BADGE
DES
Dioctyl phthalate
Diethyl phthalate
4NP
BPA bis (2,3-di-hydroxypropyl) ether
BPA (3-chloro-2-hydroxypropyl) ether
Xnobiotic concentration in test well [umol/dm3]
% C
ontr
ol
RESULTS – MTT ASSAY
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques. Valencia, September 2015
BPA bis (
2,3-d
ihydr
oxyp
ropy
l) eth
er
Diethy
l phth
alate
BPA
BPA bis (
3-ch
loro-
2-hy
drox
ypro
pyl)
ether
DES
BADGE
Dioctyl
phtha
late
4-no
nylfe
nol
4-ter
t-okty
lfeno
l0
500
1000
1500
2000
2500
3000
3500
4000CUMULATIVE CHART OF BIOLOGICAL EFFECT
72h 24h
6h 3h
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