Comparative In Vitro Toxicity
Gabrielle
1,2
Maldonado ,
Mark
1,3
Wilson
1
2
Emerging Scholars Environmental Health Science Academy, Chalmette High School
3 Department of Global Environmental Health Sciences, Tulane University School of Public Health and Tropical Medicine
Abstract
In the Emerging Scholars program at Tulane University, I with the help of my mentor used different assays to measure cytotoxicity. We used ethanol concentrations based on alcohol blood
concentrations known to affect humans. Different concentrations of ethanol were then applied to Hep G2 liver cells to see how they responded. We observed how they reacted by applying five
different assays: colony, MTT, alamarBlue®, SRB, and Trypan Blue Exclusion. We then raised the doses reasoning that the liver inside a human body cleans a percentage of the alcohol from the blood
and the remaining percentage that remains is the blood alcohol concentration levels. After all the experiments were commenced, we compared the results from the different assays to determine
sensitivity and ease of use.
Introduction
Methods
Colony Assay
Cytotoxicity is defined as being toxic to cells. In a lab
there are several different ways to assess
cytotoxicity. Setting up an experiment to see which
assay works the best for cytotoxicity is important
because it’ll help determine which assay to use later
for future experiments.
To assess cytotoxicity, there first has to be a target
and a toxicant. In this case, it was liver cells and
ethanol respectively. Liver cells were used because
they are the ones that break up alcohol in the human
body. Experiments were set up with two exposure
time points, a one hour and overnight exposure, and
with concentrations of ethanol based on
concentrations already known to affect humans.
MTT Assay
Materials
Petri dishes
Medium
Ethanol
Hep G2 cells
Crystal Violet
•
•
•
•
•
1)
2)
3)
4)
5)
6)
7)
8)
One drink has 0.5 US fl oz (15 ml) alcohol by volume
Incubate for 2-4 hours
Put in
spectrophotometer
Over-expression
Emotional swings
Anger or sadness
Boisterousness
Decreased libido
And visualize results:
More cells= more purple
Results
Concentration
Reasoning
Depth perception
Peripheral vision
Glare recovery






Reflexes
Reaction time
Gross motor control
Staggering
Slurred speech
Temporary erectile
dysfunction
Possibility of temporary
alcohol poisoning
Drinks
1
2
3
4
0.20–0.29



Severe motor
impairment
Loss of consciousness
Memory blackout
140
104
0.30–0.39


Severe central nervous system
depression
Unconsciousness
Possibility of death




Bladder function
Breathing
Dysequilibrium
Heart rate
5
0.40–0.50
>0.50



General lack of behavior
Unconsciousness
Possibility of death
High risk of poisoning
Possibility of death
Breathing
Heart rate
Positional Alcohol
Nystagmus
40 kg
45 kg
55 kg
64 kg
73 kg
82 kg
91 kg
100 kg 109 kg
100 lb 120 lb 140 lb 160 lb 180 lb 200 lb 220 lb 240 lb
80
–
0.04
0.03
0.03
0.02
0.02
0.02
0.02
0.02
0.05
0.05
0.04
0.03
0.03
0.03
0.02
0.02
0.02
–
0.08
0.06
0.05
0.05
0.04
0.04
0.03
0.03
0.10
0.09
0.08
0.07
0.06
0.05
0.05
0.04
0.04
–
0.11
0.09
0.08
0.07
0.06
0.06
0.05
0.05
0.15
0.14
0.11
0.10
0.09
0.08
0.07
0.06
0.06
–
0.15
0.12
0.11
0.09
0.08
0.08
0.07
0.06
0.20
0.18
0.15
0.13
0.11
0.10
0.09
0.08
0.08
1 hour
Linear (overnight)
60
Linear (1 hour)
y = -130.1x + 112.12
R² = 0.3375
1 hr
96
overnight
y = 104.03e-0.303x
R² = 0.8348
94
Expon. (1 hr)
Expon. (overnight)
90
20
88
0
0
0.1
0.2
0.3
0.4
0.5
0.6
y = 102.17e-0.291x
R² = 0.8241
86
0
% Etoh
alamarBlue® Cell Viability
–
0.19
0.16
0.13
0.12
0.11
0.09
0.09
0.08
0.25
0.23
0.19
0.16
0.14
0.13
0.11
0.10
0.09
0.1
0.2
0.3
% Etoh
0.4
0.5
0.6
120.00
120%
120
100%
100.00
80%
80.00
–
0.30
0.23
0.27
0.19
0.23
0.16
0.19
0.14
0.17
0.13
0.15
0.11
0.14
0.10
0.12
0.09
0.11
1 hour
60
Expon. (overnight)
Expon. (1 hour)
7
–
0.26
0.22
0.19
0.16
0.15
0.13
0.12
0.11
0.35
0.32
0.27
0.23
0.20
0.18
0.16
0.14
0.13
overnight
60%
1 hr
Linear (overnight)
40%
Linear (1 hr)
40
y = -2.4402x + 0.979
R² = 0.7302
20%
8
–
0.30
0.25
0.21
0.19
0.17
0.15
0.14
0.13
0.40
0.36
0.30
0.26
0.23
0.20
0.18
0.17
0.15
0
0
10
–
0.34
0.28
0.24
0.21
0.19
0.17
0.15
0.14
0.45
0.41
0.34
0.29
0.26
0.23
0.20
0.19
0.17
–
0.38
0.31
0.27
0.23
0.21
0.19
0.17
0.16
0.51
0.45
0.38
0.32
0.28
0.25
0.23
0.21
0.19
overnight
60.00
1 hour
Expon. (overnight)
40.00
Expon. (1 hour)
y = 18.903e-0.036x
R² = 0.0513
20.00
y = -3.1949x + 0.7523
R² = 0.5146
y = 47.565e-0.058x
R² = 0.4411
10
20
0%
y = 61.778e-0.046x
R² = 0.6001
30
40
0%
10%
20%
30%
40%
50%
% Etoh
50
% Etoh
Subtract approximately 0.01 every 40 minutes after drinking.
BAC Charts from Virginia Tech
Acknowledgments
Colony Assay
•
•
•
•
•
Based on cell proliferation
Uses petri dishes
Uses the most materials
Easy to contaminate
Takes ≈ 2 weeks
MTT Assay
• Based on working mitochondrial functions
• Purple crystals are formed in healthy cells
• The cells are then lysed, so it’s a one way
trip
• The crystals undergo solubilization
• The more healthy cells there are the more purple the dye
gets
• Takes a day
alamarBlue® Assay
• Based on the conversion of resazurin to resorufin
• Resazurin becomes fluorescent due to the reductive
reactions of active cells
• More fluorescence equals more living cells
• Takes about 5 hours to do
• The cells can be used again
0.00
0.00
10.00
•
•
•
•
•
Based on protein binding of SRB dye
The binding fixes the cells onto the well plates
Doesn’t differentiate between dead and live cells
At high doses of ethanol can fix dead cells and skew data
Takes 3 days to do
Trypan Blue Exclusion Assay
SRB Cell Viability
Trypan Blue Cell Viability
overnight
Conclusions
SRB Assay
92
• A hybridizing of effects as described at Alcohol's Effects from Virginia Techand Federal Aviation Regulation (CFR) 91.17: Alcohol and
Flying (hosted onFlightPhysical.com)
•
5)
6)
98
overnight
y = -44.79x + 88.431
R² = 0.9901
80
9



4)
102
20

3)
100
6
Stupor
Loss of understanding
Impaired sensations
Possibility of falling unconscious
2)
Method
Set up 96 well
plate
Treat the cells
with ethanol
Trypsinize to get
cells off plate
Stain w/ trypan
blue
Put in centrifuge
Count w/
hemocytometer
MTT Assay Cell Viability
% Viability
Subtle effects that can
be detected with special
tests
1)
at 585
106
40





excitation at 548 & emission
Materials
96 well plates
Medium
Hep G2 cell
Ethanol
SRB
Method
Set up 96 well plate
Treat with ethanol
Fix cells
Stain cells
Wash cells
Read plate
Cell Viability
0.10–0.19





4) Put in fluorescence scanner:
•
•
•
•
•
•
100
90 lb
0.06–0.09
3) Add dye
Materials
96 well plates
Medium
Hep G2 cells
Ethanol
centrifuge
Trypan Blue
100
% viabilty
0.010–0.029 Average individual appears normal
Blunted feelings
Disinhibition
Extroversion
2) Treat with ethanol
Impairment

Mild euphoria
Relaxation
Joyousness
Talkativeness
Decreased inhibition
1)
2)
3)
4)
5)
6)
120
Body weight


0.030–0.059 


•
•
•
•
•
1) Set up 96 well plate
Add MTT
% viability
Behavior
Materials
96 well plates
Medium
Hep G2 cells
Ethanol
alamarBlue®
Method
Approximate blood alcohol percentage (by vol.) [4]
Male
Female
BAC (% by vol.)
SRB Assay
160
% Viability
Progressive effects of
•
•
•
•
•
Treat with ethanol
Colony Assay Cell Viability
alcohol[2]
alamarBlue® Assay
Materials
• 96 well plates
• Medium
• Hep G2 cells
• Ethanol
• MTT
Method
1) Set up 96 well plate
Method
Set up petri dishes
2)
Treat with ethanol
Change the medium
3)
Put in incubator and wait ≈ 2 weeks
4)
Take off medium
5)
Wash with PBS
Stain with crystal violet
6)
Count cell colonies
Trypan Blue Assay
20.00
y = 37.588e-0.071x
R² = 0.4107
30.00
% Etoh
40.00
50.00
•
•
•
•
•
•
Based on cell membrane structure
Trypan blue is added and is absorbed by dead cells
A lot of counting is required
There are several steps in which cells can be lost
It isn’t practical for continuous lab work
The data isn’t very good as many cells were lost in the
process
• Takes a day to do
In Summary:
• alamarBlue® was the least time intensive and agreed well
with other metrics of cytotoxicity, and the cells were still
viable.
• The difference between in vivo and in vitro is worth
mentioning.
- In vitro testing is not always the best representation of
what goes on in the body (in vivo)
• Other testing should be done to see if our results are cell
type dependent
I would like to thank Dr. Wickliffe for the use of his lab. I would also like to thank Mrs. Perrault for bringing me into this program. I thank my family for supporting me as always. Finally, I thank the Ph. D students on the 21st floor for giving me nudges in the right direction. This work was supported
www.postersession.com
by the Gulf Region Outreach Program (GRHOP) which is funded from the Deepwater Horizon Medical Benefits Class Action Settlement approved by the U.S. District Court in New Orleans on January 11, 2013.