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

PRINCIPLE OF THE AMES ASSAY Histidine is an amino acid essential for cell growth “Normal” wild-type bacteria make their own histidine: No His Ames strains have defects in the genes that code for histidine synthesis No His No His Mutations may reverse the defects (reversion) + Chemical + His Revertants

Tissue culture

Isolated perfused liver

Isolated perfused lung

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:Acute24-48 h Sub-acute Sub-chronic10% 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:ChronicEntire 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 Bioassays

Biomarkers Biomarkers of Exposure Metabolites (urine, saliva, blood) DNA adducts (peripheral blood lymphocytes) Protein adducts (blood) Biomarkers of Effect Chromosomal aberrations Mutations