Biomarkers of lead induced toxicity in channel catfish and human liver carcinoma hepg2 cells
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Biomarkers of Lead-Induced Toxicity in Channel Catfish and Human Liver Carcinoma (HepG2) Cells. Paul B. Tchounwou Environmental Toxicology Research Laboratory, Jackson State University, Jackson, MS, USA Environmental Technology Consortium Washington, D.C. , March 10-11, 2003.

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Biomarkers of lead induced toxicity in channel catfish and human liver carcinoma hepg2 cells
Biomarkers of Lead-Induced Toxicity in Channel Catfish and Human Liver Carcinoma (HepG2) Cells

Paul B. Tchounwou

Environmental Toxicology Research Laboratory,

Jackson State University,

Jackson, MS, USA

Environmental Technology Consortium

Washington, D.C. , March 10-11, 2003


Background and rationale
Background and Rationale Human Liver Carcinoma (HepG2) Cells

  • Lead is a non-essential element that exhibits a high degree of toxicity to humans

  • It is highly toxic to children, and affects virtually every system of the body

  • It can damage a child kidneys, and central nervous system, and cause anemia

  • At very high levels, lead can cause coma, convulsions and death

  • Even low levels of lead are harmful


Background and rationale1
Background and Rationale Human Liver Carcinoma (HepG2) Cells

  • Levels as low as 10 ug/dL of blood have been associated with:

    • decreased intelligence

    • behavior problems

    • reduced physical stature and growth, and

    • impaired hearing

  • A child is estimated to lose 2 IQ points for each 10 ug/dL increase in blood lead level

    • Most evidence of harmful effects is found in children whose blood lead levels exceed 10 ug/dL


  • Toxicological profile of lead poisoning in children
    Toxicological Profile of Lead Poisoning in Children Human Liver Carcinoma (HepG2) Cells

    -----------------------------------------------------------------------------------------------------

    Blood Lead Level Adverse Toxicological

    (ug/dL) Effects

    -----------------------------------------------------------------------------------------------------

    10 Reduced IQ, hearing, growth, behavior problems

    20 Impaired nerve function

    30 Reduced Vitamin D metabolism

    42 Damage to blood forming system

    55 Severe stomach cramps

    70 Severe anemia

    80 Kidney damage

    90 Severe brain damage

    130 Death

    -----------------------------------------------------------------------------------------------------


    Background and rationale2
    Background and Rationale Human Liver Carcinoma (HepG2) Cells

    • Most studies with lead have focused on its effects on organ systems such as the nervous system, the red blood cells, and the kidneys which are considered the primary targets of toxicity.

    • However, little is known about its adverse effects on the liver and other tissues. The literature is also scarce regarding the molecular mechanisms of lead-induced toxicity in mammalian systems.


    Why channel catfish
    Why Channel Catfish ? Human Liver Carcinoma (HepG2) Cells

    • Aquatic organisms (fish/crawfish) are important sources of food, and income for several residents

    • Mississippi is No. 1 producer of catfish in the nation

    • Contaminated fish may constitute a significant route of human exposure to lead thru the food chain


    Research objectives and approach
    Research Objectives and Approach Human Liver Carcinoma (HepG2) Cells

    • To assess the acute and chronic toxicities of lead to channel catfish (ictalurus punctatus)

      • Static renewal bioassays with fish exposed to lead nitrate (acute)

      • Flow-through system bioassays with fish exposed to lead nitrate (chronic)

  • To determine the cytotoxicity of lead

    • MTT assay for cell viability with transformed human hepatocytes exposed to lead nitrate


  • Research objectives and approach1
    Research Objectives and Approach Human Liver Carcinoma (HepG2) Cells

    • To predict the molecular mechanisms of lead-induced toxicity

      • Gene Profile (CAT-Tox) to assess the transcriptional activation of stress genes

      • Microarray analysis for large scale gene expression

  • To identify the potential biomarkers of exposure, sensitivity and effect associated with lead exposure

    • Western Blot and densitometric analyses

    • Histopathological examinations


  • Preliminary studies

    Constants: Human Liver Carcinoma (HepG2) Cells

    Volume of Water (1.2L)

    Renewal of Medium (every 24hrs)

    Time of exposure (96hrs)

    Number fish (4)

    No Feeding or Aeration

    Water Quality:

    pH

    Dissolved oxygen

    Temperature

    Conductivity

    Total dissolved solids

    Preliminary Studies


    Water quality
    Water Quality Human Liver Carcinoma (HepG2) Cells



    Preliminary findings
    Preliminary Findings Exposure

    • The concentration of lead has a direct effect on the mortality rate of channel catfish.

    • The toxicity of lead is dose- and time-dependent.

    • Lead is acutely toxic to channel catfish


    Cytotoxicity of lead nitrate to hepg 2 cells 48 hrs exposure
    Cytotoxicity of Lead Nitrate to HepG Exposure2 Cells – 48 hrs Exposure


    Stress gene promoter response element cat fusion constructs
    Stress Gene Promoter/Response Element-CAT Fusion Constructs Exposure

    -----------------------------------------------------------------------------------------------------------------

    Promoter Biologic Function

    -----------------------------------------------------------------------------------------------------------------

    CYP1A1 Cytochrome-P450 1A1 Phase I biotransformation enzyme

    GST Ya Glutathion-s-transferase Phase II biotransformation enzyme

    XRE Xenobiotic Resp. Elt. Binding site for Ah-receptor

    CRE cAMP Response Elt. Binding site for the CREB protein

    RARE Retinoic Acid RE Binding site for RA

    HMTIIA Metallothionein Sequestration of heavy metals

    HSP70 Heat Shock Protein Cytoplasmic protein chaperone

    GRP78 Glucose-Regulated P ER protein chaperone

    GADD45/153 GA & DNA Damage P Cell cycle regulation

    FOS c-fos Member of AP-1 TF complex

    NFkBRE Nuclear Factor Binding site to the NFkB TF

    p53RE Tumor Suppressor P Binding site for the p53 TF

    -----------------------------------------------------------------------------------------------------------------


    Deliverables
    Deliverables Exposure

    • Education and workforce development

      • Strengthen curriculum in the areas of environmental toxicology and risk assessment

      • Educate and train under-represented students in the areas of environmental toxicology and risk assessment

  • Contribution to science / Research

    • Review and update the toxicological profile of lead

    • Identify and elucidate the mechanisms of lead-induced toxicity

  • Scientific publications ( 3 in 3 years)

  • Scientific presentations ( 6 in 3 years)

  • Faculty development in the area of environmental toxicology and risk assessment

    • Seminars / workshops / conferences


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