Sigma XI Research Rresentation

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  • 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 bodys 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 incorporat