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

2. 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

3. 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

4. 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 -----------------------------------------------------------------------------------------------------

5. 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.

6. 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

7. 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

8. 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

9. 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 Conductivity Total dissolved solids Preliminary Studies

10. Water Quality

11. Acute Toxicity of Lead to Channel Catfish – 48 hrs Exposure

12. 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

13. Cytotoxicity of Lead Nitrate to HepG2 Cells – 48 hrs Exposure

14. 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 -----------------------------------------------------------------------------------------------------------------

15. 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