Biomarkers of Lead-Induced Toxicity in Channel Catfish and Human Liver Carcinoma (HepG2) Cells

1
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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Background and Rationale

• 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

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Background and Rationale

• 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

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Toxicological Profile of Lead Poisoning in
Children

• --------------------------------------------------
  --------------------------------------------------
  -
• 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
• --------------------------------------------------
  --------------------------------------------------
  -

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Background and Rationale

• 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.

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Why Channel Catfish ?

• 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

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Research Objectives and Approach

• 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

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Research Objectives and Approach

• 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

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Preliminary Studies

• Conductivity
• Total dissolved solids

• Constants
• 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

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Water Quality
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Acute Toxicity of Lead to Channel Catfish 48
hrs Exposure
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Preliminary Findings

• 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

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Cytotoxicity of Lead Nitrate to HepG2 Cells 48
hrs Exposure
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Stress Gene Promoter/Response Element-CAT Fusion
Constructs

• --------------------------------------------------
  --------------------------------------------------
  -------------
• 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
• --------------------------------------------------
  --------------------------------------------------
  -------------

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Deliverables

• 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