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Methods Conclusions

Comparative In Vitro ToxicityGabrielle Maldonado1,2, Mark Wilson1,3

1 Emerging Scholars Environmental Health Science Academy, 2 Chalmette High School3 Department of Global Environmental Health Sciences, Tulane University School of Public Health and Tropical Medicine

Introduction

Results

Acknowledgments

AbstractIn 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.

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.

Progressive effects of alcohol[2]

BAC (% by

vol.)Behavior Impairment

0.010–

0.029

Average individual appears normal

Subtle effects that can be detected with special tests

0.030–

0.059

Mild euphoria Relaxation Joyousness Talkativeness Decreased inhibition

Concentration

0.06–0.09

Blunted feelingsDisinhibitionExtroversion

ReasoningDepth perceptionPeripheral visionGlare recovery

0.10–0.19

Over-expression Emotional swings Anger or sadness Boisterousness Decreased libido

Reflexes Reaction time Gross motor control Staggering Slurred speech Temporary 

erectile dysfunction Possibility of

temporary alcohol poisoning

0.20–0.29

Stupor Loss of understanding Impaired sensations Possibility of falling

unconscious

Severe motor impairment

Loss of consciousness

Memory blackout

0.30–0.39

Severe central nervous system depression

Unconsciousness Possibility of death

Bladder function Breathing Dysequilibrium Heart rate

0.40–0.50

General lack of behavior Unconsciousness Possibility of death

Breathing Heart rate Positional Alcohol N

ystagmus

>0.50High risk of poisoningPossibility of death

• 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)

•  BAC Charts from Virginia Tech

Male

Fema

le

Approximate blood alcohol percentage (by vol.)[4]

One drink has 0.5 US fl oz (15 ml) alcohol by volume

Drink

s

Body weight

40 kg 45 kg 55 kg 64 kg 73 kg 82 kg 91 kg100 k

g

109 k

g

90 lb 100 lb 120 lb 140 lb 160 lb 180 lb 200 lb 220 lb 240 lb

1–

0.05

0.04

0.05

0.03

0.04

0.03

0.03

0.02

0.03

0.02

0.03

0.02

0.02

0.02

0.02

0.02

0.02

2–

0.10

0.08

0.09

0.06

0.08

0.05

0.07

0.05

0.06

0.04

0.05

0.04

0.05

0.03

0.04

0.03

0.04

3–

0.15

0.11

0.14

0.09

0.11

0.08

0.10

0.07

0.09

0.06

0.08

0.06

0.07

0.05

0.06

0.05

0.06

4–

0.20

0.15

0.18

0.12

0.15

0.11

0.13

0.09

0.11

0.08

0.10

0.08

0.09

0.07

0.08

0.06

0.08

5–

0.25

0.19

0.23

0.16

0.19

0.13

0.16

0.12

0.14

0.11

0.13

0.09

0.11

0.09

0.10

0.08

0.09

6–

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

7–

0.35

0.26

0.32

0.22

0.27

0.19

0.23

0.16

0.20

0.15

0.18

0.13

0.16

0.12

0.14

0.11

0.13

8–

0.40

0.30

0.36

0.25

0.30

0.21

0.26

0.19

0.23

0.17

0.20

0.15

0.18

0.14

0.17

0.13

0.15

9–

0.45

0.34

0.41

0.28

0.34

0.24

0.29

0.21

0.26

0.19

0.23

0.17

0.20

0.15

0.19

0.14

0.17

10–

0.51

0.38

0.45

0.31

0.38

0.27

0.32

0.23

0.28

0.21

0.25

0.19

0.23

0.17

0.21

0.16

0.19

Subtract approximately 0.01 every 40 minutes after drinking.

0 0.1 0.2 0.3 0.4 0.5 0.675

80

85

90

95

100

105

110

f(x) = 102.169753827522 exp( − 0.291014024014734 x )R² = 0.824092408742528

f(x) = 104.032203859436 exp( − 0.303235103598819 x )R² = 0.834840298943853

MTT Assay Cell Viability

1 hrExponential (1 hr)overnightExponential (overnight)

% Etoh

% V

iabilit

y

0 0.1 0.2 0.3 0.4 0.5 0.60

20

40

60

80

100

120

140

160

f(x) = − 130.100606204321 x + 112.120841296586R² = 0.33745462196037

f(x) = − 44.7900054144063 x + 88.4312391315204R² = 0.99010974202512

Colony Assay Cell Viability

overnightLinear (overnight)1 hourLinear (1 hour)Linear (1 hour)Linear (1 hour)

% Etoh

% v

iabi

lty

Colony Assay Materials

• Petri dishes • Medium • Ethanol• Hep G2 cells• Crystal Violet

Method1) Set up petri dishes2) Treat with ethanol3) Change the medium4) Put in incubator and wait ≈ 2 weeks5) Take off medium6) Wash with PBS7) Stain with crystal violet8) Count cell colonies

Colony Assay• Based on cell proliferation• Uses petri dishes• Uses the most materials• Easy to contaminate• Takes ≈ 2 weeksMTT 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 dayalamarBlue® 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 againSRB Assay• 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 doTrypan Blue Exclusion Assay• 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

MTT Assay Materials

• 96 well plates• Medium• Hep G2 cells• Ethanol• MTT

Method1) Set up 96 well plate

2) Treat with ethanol

3) Add MTT

4) Incubate for 2-4 hours

5) Put in spectrophotometer

6) And visualize results: More cells= more purple

alamarBlue® Assay

Materials• 96 well plates• Medium• Hep G2 cells• Ethanol• alamarBlue®

Method

1) Set up 96 well plate

2) Treat with ethanol

3) Add dye

4) Put in fluorescence scanner:

excitation at 548 & emission

at 585

0.00 10.00 20.00 30.00 40.00 50.000.00

20.00

40.00

60.00

80.00

100.00

120.00

f(x) = 37.5877821099938 exp( − 0.0707299894722076 x )R² = 0.410664197003987

f(x) = 18.9029836584435 exp( − 0.035983972041381 x )R² = 0.0512558349361063

SRB Cell Viability

overnightExponential (overnight)1 hourExponential (1 hour)

% Etoh

Cell V

iabilit

y

0 5 10 15 20 25 30 35 40 45 500

20

40

60

80

100

120

f(x) = 61.7779537639857 exp( − 0.0462612948640661 x )R² = 0.600061681087446

f(x) = 47.5653239248289 exp( − 0.0582948234964829 x )R² = 0.44110380564507

alamarBlue® Cell Viability

overnightExponential (overnight)1 hourExponential (1 hour)

% Etoh

% V

iabilit

y

SRB AssayTrypan Blue Assay

Materials• 96 well plates• Medium• Hep G2 cell• Ethanol• SRB

Method

1) Set up 96 well plate

2) Treat with ethanol

3) Fix cells

4) Stain cells

5) Wash cells

6) Read plate

Materials• 96 well plates • Medium• Hep G2 cells• Ethanol• centrifuge• Trypan Blue

Method1) Set up 96 well

plate2) Treat the cells

with ethanol3) Trypsinize to get

cells off plate4) Stain w/ trypan

blue5) Put in centrifuge6) Count w/

hemocytometer

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 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.

0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50%0%

20%

40%

60%

80%

100%

120%

f(x) = − 2.44021995471436 x + 0.979001566836973R² = 0.730183932149749

f(x) = − 3.1948505309207 x + 0.752251503696564R² = 0.51457567811568

Trypan Blue Cell Viability

overnightLinear (overnight)1 hrLinear (1 hr)

% Etoh

% v

iabilit

y