Toxic effects
•
•
•
•
•
•
Acute / chronic
Reversible / irreversible
Immediate / delayed
Idiosyncratic - hypersensitivity
Local / systemic
Target organs
Toxicological Screening
Molecular assays
• DNA damage (eg AP sites)
• DNA binding (eg adducts)
• Altered gene expression (Gene
arrays)
• Altered protein synthesis (Proteomics)
DNA Binding
Gene array
Protein 2D gel
Toxicological Screening
Bioassays
•
•
•
•
•
Cell Toxicity Assays (Short Term)
Cell Genotoxicity Assays (Short Term)
Tissue culture (Primary cells, cell lines)
Isolated perfused organs
Whole Animal Studies (Medium - Long
Term)
Cell Toxicity Assays
•
•
•
•
•
Trypan Blue Exclusion
Neutral Red Uptake
ATP Production
MTT assay
Assays for apoptosis (TUNEL)
In vitro short-term tests for
genotoxicity
Tests for mutagenesis/carcinogenesis
Bacterial mutagenesis e.g. Ames assay
Mammalian mutagenesis
e.g. Mouse Lymphoma L 5178Y, V79
DNA repair e.g. Sister Chromatid Exchange (SCE)
Chromosome damage, Cytogenetics
e.g. Chromosome Aberrations (CA),
Strand Breakage
Cell transformation e.g. BALB/3T3, C3H/10T 1/2
Tissue culture
Isolated perfused liver
Isolated
perfused
lung
Bioassays
in vivo
Tests for initiators
Skin Tumors in Mice (Sencar)
Pulmonary Tumors in Mice (Strain A)
Tests for promoters
Skin Tumors in Mice (Sencar)
Pulmonary Tumors in Mice (Strain A)
Altered Foci in Rodent Liver
Breast Cancer in Female Rats (Huggins model)
NTP Cancer Bioassays
Bioassays
in vivo
Experimental Design
Generally two or more species, equal numbers of each gender
Short-term: Acute
24-48 h
Sub-acute
Sub-chronic 10% of life-span, 90 days
Observe: Food consumption, body weight gain, general
appearance, behavior
At sacrifice: Organ weights, gross pathology,microscopic
examination of major organs, clinical laboratory
tests on blood and urine. Other studies if indicated
by observations, clinical chemistry or gross lesions.
Determine MTD for long-term studies.
Bioassays
Long-term: Chronic
Entire Life-span
Observe: Food consumption, body weight gain, behavior
Mortality and morbidity, Cancer
At sacrifice: Organ weights, gross pathology,
histopathology. More detailed assessment of tumors
To define the nature of the toxicity, carcinogencity,
NOAEL
Biomarkers
Biomarkers of Exposure
Metabolites (urine, saliva, blood)
DNA adducts (peripheral blood
lymphocytes)
Protein adducts (blood)
Biomarkers of Effect
Chromosomal aberrations
Mutations