Sigma XI Research Rresentation
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Transcript of Sigma XI Research Rresentation
THE PROTECTIVE EFFECT OF GLUTATHIONE,
alpha- LIPOIC ACID, AND N-ACETYL-L-
CYSTEINE AGAINST ACRYLAMIDE AND
ACETAMIDE IN Danio rerio EMBRYOS
By Diya Jayram
Purpose
Toxins pose a serious threat to the environment and
our health
This study explores the effectiveness of the
antioxidants, glutathione, alpha-Lipoic acid, and
N-acetyl-L-cysteine
What are toxins?
Harmful agents in the environment.
Normally, the body works in order to remove these toxins.
When toxins pass the body’s capacity to remove them, they start to build up and are stored.
People are exposed to numerous toxins in their daily lives.
Acrylamide and its Dangers (ACR)
One of the foremost health problems
Formed when asparagine is heated to high
temperatures in the presence of certain sugars
People exposed to its toxicity on a daily basis.
Probable human carcinogen (B2)
Acetamide and its dangers (A)
Solvent, plasticizer, and a penetrating agent
Workers in the plastics and chemical industries
Carcinogen, may be a genotoxin, and can be a
reproductive toxin.
Causes irritation in eyes, skin, and mucous membranes
Combustible
Gives off toxic fumes in a fire
Neurotoxicity and its dangers
Is an effect of toxicity
Is toxicity in the nervous system
Causes brain damage, memory loss, limb weakness, vision loss,
headaches, cognitive issues, and behavioral problems.
Neurons are at greatest risk
Oligodendrocytes, astrocytes, and microglia are next in the
order of risk
Oxidative Stress and its Dangers
Is an effect of toxicity
An imbalance between the synthesis of free radicals and the
ability to counter harmful effects through neutralization
One can reduce oxidative stress through the use of antioxidants.
(Lobo, Patil, Phatak, & Chandra, 2010).
Antioxidants prevent the loss of free radicals and inhibit oxidation
by donating a free electron without destabilizing themselves.
The antioxidants used in this experiment are glutathione,
alpha-Lipoic acid, and N-acetyl-L-cysteine.
Glutathione (G)
Very important antioxidant found in plants, animals, fungi, and
bacteria.
Capable of rebalancing free radicals and antioxidant system.
Naturally made by the liver
It is soluble in water
Glutathione depletion results in apoptosis
alpha-Lipoic Acid (ALA)
An antioxidant derived from yeast, liver, broccoli, potatoes,
and more.
Soluble in water and ethanol.
Treats diabetes and its nerve related symptoms.
Prevents cell damage in body and restores certain vitamins.
Protects the brain from damage, for instance, from
neurotoxicity caused by acrylamide toxicity.
N-acetyl-L-cysteine (NAC)
Mucolyic agent that reduces the viscosity of mucous
secretions
Antidote for acetaminophen overdose and has a anti-
apoptotic effect, thus, may prevent cancer.
Inhibits viral stimulation by reactive oxgen species
(ROS) in patient with HIV.
Cu/Zn-Superoxide Dismutase (SOD1)
Antioxidant enzyme
Keeps cells safe from metabolic waste
Converts oxygen to harmless water
Pivotal role in oxidative stress
Subdues effects of ROS
Danio rerio Embryos
Genetic similarity to humans
We share 70% of our genes with zebrafish
Optical clarity
Easy to introduce mutations
These characteristics make the zebrafish embryo the perfect correlative and predictive model for assessing toxicity
Taken by IGTRCN
Hypotheses1. If the embryos are exposed to glutathione, alpha-Lipoic acid, or N-acetyl-L-cysteine after being
exposed to toxins, then there will be no protective effect.
2. If the embryos are exposed to glutathione after being exposed to acrylamide, then there will be
a high protection efficiency due to its capability of rebalancing free radicals.
3. If the embryos are exposed to glutathione after being exposed to acetamide, then there will be a
high protection efficiency due to its capability of rebalancing free radicals.
4. If the embryos are exposed to alpha-Lipoic acid after being exposed to acrylamide, then there
will be a high protection efficiency due to its capability of neutralizing free radicals.
5. If the embryos are exposed to alpha-Lipoic acid after being exposed to acetamide, then there
will be a high protection efficiency due to its capability of neutralizing free radicals.
6. If the embryos are exposed to N-acetyl-L-cysteine after being exposed to acrylamide, then there
will be a high protection efficiency due to anti-apoptotic effect.
7. If the embryos are exposed to N-acetyl-L-cysteine after being exposed to acetamide then there
will be a high protection efficiency due to anti-apoptotic effect.
Procedure
All embryos were kept in groups of 50 embryos/50 ml of embryo medium.
There was 5 petri dishes per group.
To measure the amount of SOD activity, I performed the SOD1 Assay. To conduct this assay:
Created proteins samples using a motor-and-pestle to lyse and a centrifuge to homogenize the tissue
Absorbance of the samples and standards were measured using a spectrophotometer and compared to a standard curve to visualize the upregulation and downregulation of the antioxidant enzyme.
Procedures Performed Organized By hpf (hours post
fertilization)
48 hpf 72 hpf 96 hpf
Exposed to
a toxin
Exposed to
an
antioxidant
N/A
Mortality Counts X X X
Heartbeats X X
Eye Width X X
Body Length X X
Protein Sample
Made
X
Layout of Conditions
Embryos
Embryos
Embryos Embryos
EmbryosEmbryosEmbryos
Embryos
A/ACR
G
G
NACNAC
ALA
ALA
A/ACR
1
65
432
87
A/ACR
A/ACR
Positive control Positive control Positive control
Negative control
Positive control
Abbreviation Table
Alpha-Lipoic acid ALA
N-acetyl-L-cysteine NAC
Acrylamide ACR
Average of 5
trials
ALA was the only
ineffective
antioxidant
against
acrylamide. Every
measurement
above 2 shows
high mortality.
NAC was the only
ineffective
antioxidant. It was
ineffective alone
and against
acrylamide
toxicity. The
healthy ranges
are shown above.
Abbreviation Table
Alpha-Lipoic acid ALA
N-acetyl-L-cysteine NAC
Acrylamide ACR
Average of 5
trials
In terms of growth, glutathione was ineffective alone and against acrylamide toxicity; ALA was
ineffective against acetamide toxicity. In terms of body length, NAC and glutathione was
ineffective against acetamide toxicity; and ALA was ineffective against acrylamide toxicity. The
healthy ranges are shown above.
Abbreviation Table
Alpha-Lipoic acid ALA
N-acetyl-L-cysteine NAC
Acrylamide ACR
Average of 5
trials
Alpha-Lipoic acid
was ineffective
against
acrylamide
toxicity. Every
measurement
above 2 shows
high mortality.
Abbreviation Table
Alpha-Lipoic acid ALA
N-acetyl-L-cysteine NAC
Acrylamide ACR
Average of 5
trials
Abbreviation Table
Alpha-Lipoic acid ALA
N-acetyl-L-cysteine NAC
Acrylamide ACR
N-acetyl-L-
cysteine was
ineffective alone
and against
acrylamide.
Healthy range is
shown
Average of 5
trials
Standard Curve Equation: y =5.2099x+0.8943; R^2=0.2289
Glutathione was innefective alone, against acetamide, and against
acrylamide. N-acetyl-L-cysteine was only ineffective against
acrylamide.
ConclusionIn conclusion, the phenotypical results were
that glutathione was effective alone and
against acetamide toxicity; N-acetyl-L-
cysteine was only effective against
acetamide toxicity; and alpha-Lipoic acid
was the most effective alone and against
acrylamide toxicity. The molecular results
were that glutathione was ineffective alone
and against all toxins; and, N-acetyl-L-
cysteine was ineffective against acrylamide.
The phenotypical and molecular
measurements illustrates that alpha-Lipoic
acid and glutathione can be used as a daily
supplement and that N-acetyl-L-cysteine
can only be used against certain toxins.
Application
Toxicity causes most predominant health problems, such as cancer.
75-80% of cancers caused by environmental carcinogens
This study can be applied to:
Lessening toxicity
Stabilizing heartbeats
Stabilizing growth
Prevents cancer and other serious diseases
Stabilizing enzyme activity
Lessening oxidative stress and neurotoxicity
Limitations
Does not apply to all toxins and antioxidants
Zebrafish embryos have a high mortality rate,
therefore, mortality counts could have been skewed
This limitation was overcome by measuring mortality at
48 hpf for precise and accurate measurements.
Future Research
Research could be expanded to incorporate more
environmental carcinogens and antioxidants
Toxins such as benzene and arsenic
Antioxidants such as beta-carotene and selenium
Acknowledgements
I would like to acknowledge:
Mrs. Leya Joykutty, Mr. Kepa Oyarbide, and Dr. Sood for their support and mentoring
Dr. Issac Skromne, professor at University of Miami, for providing me with the zebrafish embryos
Dr. Bingham, professor at Barry University, for providing me with the zebrafisn embryo