Transcript SOS/umu-Test as A Tool for Genotoxicity Assessment

SOS/umu-Test
as
A Tool for Genotoxicity Assessment
By 劉文堅
Advisor: 袁如馨
防災與水環境研究中心
Why umu-test ?
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A low cost experiment assay: no high cost chemicals
and complicated apparatus is needed in conducting
the experiment.
High-throughput: umu-test enable high amount of
sample to be tested at a time.
Short time requirement: umu-test utilizes bacteria
which has short life cycle.
High sensitivity: accuracy of umu-test has been
proven
Theory of umu-test
Mutagen
exposure
Sos response
umuC-LacZ fused protein
• ONPG is used as color indicator
β-galactosidase
ONPG
• As soon as the bacteria senses genotoxicity
existance, SOS response is transmitted to
produce umuC-LacZ fused protein
• The protein will produce β-galactosidase
Enzyme
production
(colourless)
• Firstly, Salmonella typhimuium
TA1535/pKK1002 will be exposed to
genotoxicants
o-nitrophenyl
(Yellow-colored)
Absorbance
measurement
• ONPG (colorless) will be converted into onitrophenyl (yellow color) by the existance
of β-galactosidase
• Absorbance measurement is done under
spectrophotometer
• The amount of β-galactosidase correlates to
the level of toxicity
• Induction ratio obtained by calculation
IR calculation
Genotoxicity test of 4-NQO
14
1600
-galactosidase activity
Induction ratio
1400
12
10
1200
1000
8
800
6
600
4
Induction ratio
-galactosidase activity (unit)
1800
400
2
200
10
5
2.
5
5
1.
2
25
0.
6
12
5
0.
3
0.
1
0.
0
56
25
0
78
15
0
4-NQO (mg/L)
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The recent conducted toxicity test result on 4-NQO (well known
genotoxicants) indicates the ability of umu test in toxic assessment.
The Dashed line represent concentration of 4-NQO at 2-fold greater than
the control response, which tells us every black point (IR point) that is
higher than the dashed line are considered to be genotoxic.
Future Study
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Application of umu-test on various toxic samples in
the environment.
Continuous cooperation with the inventor of the
umu-test to increase the efficiency of the test by
experimenting on different strains of Salmonella.
Comparison of the genotoxicity result by umu-test
with those by Comet assay, to establish the database
of potential genotoxicants from industrial wastewater.