(Micronucleus Assay and Chorioallantoic Membrane Assay)
Toxicity Testing • Toxicology testing, safety assessment -conducted to determine the degree to which a substance can damage a living or non-living organisms - Identify hazards and risk factors that have the potential to cause harm (hazard identification) Toxicity Testing Categorized by: • Method of application • In Vitro • In Vivo • Chronicity • Acute • Subchronic • Chronic • On Route of administration of the toxicant • Oral • Dermal • Inhalation • Injection (IV, IM, IP, SC) Toxicity Testing The major difference between repeated dose and subchronic toxicity studies is the duration: repeated dose toxicity studies are conducted over a duration of 28 days, and subchronic toxicity studies are carried out over 90 days. limited exposure of an animal to a substance (acute toxicity) as well as repeated, long-term exposure (chronic toxicity). Substances are also tested for more specific endpoints such as cytotoxicity (ability to damage cells), mutagenicity (ability to cause changes in genetic material), carcinogenicity (ability to cause cancer), and teratogenicity (ability to cause birth defects).Identify hazards and risk factors that have the potential to cause harm (hazard identification) I. Chemical and Physical Properties • Probable contaminants from synthesis as well as intermediates and waste products from synthetic process II. Exposure and Environmental Fate A. Degradation studies: hydrolysis, photodegradation, etc. B. Degradation in soil, water and under various conditions C. Mobility and dissipation in soil, water, and air D. Accumulation in plants, aquatic animals, wild terrestial animals, food plants, and animals III. In vivo Tests A. Acute 1. LD50 and LC50- oral dermal or inhaled 2. Eye irritation 3. Dermal irritation 4. Dermal sensitization B. Subchronic 1.30- to 90-day feeding 2. 30- to 90-day dermal or irritation exposure C. Chronic/ Reproduction 1. Chronic Feeding (including oncogenicity tests) 2. Teratogenicity 3. Reproduction (multi-generation) III. In vivo Tests D. Special Tests 1. Neurotoxicity 2. Potentiation 3. Metabolism 4. Pharmacodynamics 5. Behavior IV. In Vitro Tests A. Mutagenicity (Ames Test) – Prokaryote - Salmonella typhimurium reverse mutation assay B. Genotoxicity MICRONUCLEUS ASSAY INTRODUCTION Micronucleus Test – In vivo toxicity study developed by Schmid and co- workers in 1975.
– Used to screen compounds for clastogenic
(chromosome-breaking) and aneugenic (loss of whole chromosome activity). • Micronuclei are extranuclei bioamarker of a chromosome damage • One of the standard cytogenetic tools implemented to assess micronuclei formation (signifying chromosomal damage) subsequent to exposure to genotoxic/cytotoxic agents. • Mutagenic and genotoxic property of the test substance. Figure 1: Illustration on formation of micronuclei from damaged chromosomes (Journal of Radiation and Cancer Research) Score binucleated cells with main nuclei that are separate and of approximately equal size, main nuclei that touch and even overlap as long a nuclear boundaries are able to be distinguished, and main nuclei that are linked by nucleoplasmic bridges Do not score: trinucleated, quadranucleated, or multinucleated cells, or cells where main nuclei are undergoing apoptosis (because MN may be gone already or may be caused by apoptotic process)
The combination of the micronucleus assay with fluorescence in situ
hybridization (FISH) Micronucleus ( Howell-Jolly bodies) – chromosomal breakage or spindle damage during metaphase/anaphase. – Typically round with as diameter of about 1/20 to 1/5 of the erythrocytes. – Some are almost almond-shaped. Principle: Genetic material replicates and divides equally between two daughter cells. – Micronuclei are found in a variety of different marrow cells (myeloblast, myelocytes, erythroblasts, erythrocytes) – Majority are found in polychromatic (immature) erythrocytes – Giemsa stain: Immature: blue Mature: pinkish, orange CHORIOALLANTOIC MEMBRANE ASSAY • Screening assay for potential pro-and anti-angiogenic agents • An evaluation tool for angiogenesis. • Assessment of angiogenesis is an indirect assessment of the carcinogenicity of a drug sample • Angiogenesis is the formation of new blood vessels and essential in wound healing and placental formation during pregnancy. It arises in tumors and cancers and may feed and sustain them and allow cell proliferation and migration. In terms of cancer, tumor angiogenesis is the consequence of an angiogenic imbalance in which pro-angiogenic factors predominate over anti-angiogenic factors. • Alternative versatile, cost effective and ethically unobjectionable in vivomodel for many applications • List of feasible applications: • Angiogenesis (induction, inhibition) • Tumour cell behaviour (angiogenesis, proliferation, apoptosis, invasion, migration, metastasis) • Short term tissue culture (tumour samples, small pieces of bone, liver, skin etc) • Toxicity assay (HET-CAM) • Testing Biomaterials and drugs UNTREATED CELECOXIB