Sigma XI Research Rresentation

26
THE PROTECTIVE EFFECT OF GLUTATHIONE, alpha- LIPOIC ACID, AND N-ACETYL-L- CYSTEINE AGAINST ACRYLAMIDE AND ACETAMIDE IN Danio rerio EMBRYOS By Diya Jayram

Transcript of Sigma XI Research Rresentation

Page 1: 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

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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

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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.

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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)

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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

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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

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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.

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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

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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.

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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.

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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

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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

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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.

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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

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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

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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.

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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

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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

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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

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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

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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.

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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.

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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

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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.

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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

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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