This document discusses various types of mutagenicity tests, including molecular, gene, and chromosomal level tests. It describes three important mutagenicity tests in detail: the Ames test, HPRT gene test, and mouse micronucleus test. The Ames test uses bacteria to identify mutagenic chemicals. The HPRT test detects mutations in the HPRT gene of mammalian cells. The mouse micronucleus test examines mouse bone marrow for evidence of chromosomal damage and mutation. These three tests are commonly used to screen chemicals for potential mutagenicity and carcinogenicity.
Genotoxicity refers to the ability of substances to damage genetic material like DNA. Genotoxicity testing aims to identify substances that may induce DNA damage through in vitro and in vivo assays. The Ames test is a widely used bacterial reverse mutation assay to assess mutagenicity. Mammalian cell assays like the micronucleus test and comet assay are also employed to study chromosomal damage and DNA breakage in vivo. Standard test batteries from organizations like OECD provide guidelines for various assays to thoroughly evaluate genotoxic potential of new chemicals and drugs. Genotoxic agents can cause cancer, mutations and birth defects by interacting with and damaging DNA.
genotoxicity describes the property of chemical agents that damages the genetic information within a cell causing mutations, which may lead to cancer. While genotoxicity is often confused with mutagenicity, all mutagens are genotoxic, whereas not all genotoxic substances are mutagenic
The micronucleus assay is a test used to detect potential genotoxic compounds. It works by identifying micronuclei, which form during cell division from chromosome fragments or whole chromosomes damaged by genotoxins. The assay has regulatory approval and can be conducted in vitro using cell cultures or in vivo using rodents. Cells or animals are exposed to test compounds, cell division is blocked, and cells are analyzed microscopically for the presence of micronuclei to determine if the compound caused genetic damage. The micronucleus assay is a simple, reliable, and reproducible method for toxicological testing.
Good Laboratory Practice(GLP) by Kashikant YadavKashikant Yadav
Good Laboratory Practice (GLP) regulations were created by the FDA in 1978 to ensure quality and integrity of safety data from non-clinical health and environmental safety studies. GLP provides a framework for the organizational processes and conditions under which these studies are planned, performed, monitored, recorded, reported and archived. It aims to make sure data submitted to regulatory authorities is a true reflection of study results. Key aspects of GLP include requirements for facilities, equipment, test systems, personnel, standard operating procedures, study plans and reports. The overall goal of GLP is to promote quality test data for regulatory decision making.
This document discusses long term toxicity studies to assess carcinogenicity. It defines carcinogens and describes the process of carcinogenesis in three stages - initiation, promotion, and progression. Carcinogens can be genotoxic, directly damaging DNA, or non-genotoxic, inducing cancer through other mechanisms. A variety of in vivo and in vitro test systems are used to evaluate carcinogenic potential, from short term mutagenicity tests to long term chronic bioassays in animals. Factors that contribute to chemical carcinogenesis in humans include lifestyle, environmental and occupational exposures, medical treatments, and infectious agents.
Toxicokinetics describes how the body handles toxicants over time through absorption, distribution, metabolism and excretion (ADME). It is important in drug development to generate kinetic data for toxicity assessment, check safety ratios, and set safe dose levels in clinical trials. Toxicokinetic evaluation helps reduce animal testing, understand inter-individual differences in responses, and has applications in screening anticancer drugs, cell-based assays, and other areas of research.
Toxicokinetics is the study of how the body affects a toxic substance over time through absorption, distribution, metabolism, and excretion. Toxicokinetic studies help explain toxicity results by quantifying exposure levels in animals and relating them to dose levels and time. Such studies are important for interpreting toxicity findings, designing further studies, and assessing the relevance of results to human safety. Key objectives include describing systemic exposure levels in toxicity studies and relating them to toxic effects.
Genotoxicity refers to the ability of substances to damage genetic material like DNA. Genotoxicity testing aims to identify substances that may induce DNA damage through in vitro and in vivo assays. The Ames test is a widely used bacterial reverse mutation assay to assess mutagenicity. Mammalian cell assays like the micronucleus test and comet assay are also employed to study chromosomal damage and DNA breakage in vivo. Standard test batteries from organizations like OECD provide guidelines for various assays to thoroughly evaluate genotoxic potential of new chemicals and drugs. Genotoxic agents can cause cancer, mutations and birth defects by interacting with and damaging DNA.
genotoxicity describes the property of chemical agents that damages the genetic information within a cell causing mutations, which may lead to cancer. While genotoxicity is often confused with mutagenicity, all mutagens are genotoxic, whereas not all genotoxic substances are mutagenic
The micronucleus assay is a test used to detect potential genotoxic compounds. It works by identifying micronuclei, which form during cell division from chromosome fragments or whole chromosomes damaged by genotoxins. The assay has regulatory approval and can be conducted in vitro using cell cultures or in vivo using rodents. Cells or animals are exposed to test compounds, cell division is blocked, and cells are analyzed microscopically for the presence of micronuclei to determine if the compound caused genetic damage. The micronucleus assay is a simple, reliable, and reproducible method for toxicological testing.
Good Laboratory Practice(GLP) by Kashikant YadavKashikant Yadav
Good Laboratory Practice (GLP) regulations were created by the FDA in 1978 to ensure quality and integrity of safety data from non-clinical health and environmental safety studies. GLP provides a framework for the organizational processes and conditions under which these studies are planned, performed, monitored, recorded, reported and archived. It aims to make sure data submitted to regulatory authorities is a true reflection of study results. Key aspects of GLP include requirements for facilities, equipment, test systems, personnel, standard operating procedures, study plans and reports. The overall goal of GLP is to promote quality test data for regulatory decision making.
This document discusses long term toxicity studies to assess carcinogenicity. It defines carcinogens and describes the process of carcinogenesis in three stages - initiation, promotion, and progression. Carcinogens can be genotoxic, directly damaging DNA, or non-genotoxic, inducing cancer through other mechanisms. A variety of in vivo and in vitro test systems are used to evaluate carcinogenic potential, from short term mutagenicity tests to long term chronic bioassays in animals. Factors that contribute to chemical carcinogenesis in humans include lifestyle, environmental and occupational exposures, medical treatments, and infectious agents.
Toxicokinetics describes how the body handles toxicants over time through absorption, distribution, metabolism and excretion (ADME). It is important in drug development to generate kinetic data for toxicity assessment, check safety ratios, and set safe dose levels in clinical trials. Toxicokinetic evaluation helps reduce animal testing, understand inter-individual differences in responses, and has applications in screening anticancer drugs, cell-based assays, and other areas of research.
Toxicokinetics is the study of how the body affects a toxic substance over time through absorption, distribution, metabolism, and excretion. Toxicokinetic studies help explain toxicity results by quantifying exposure levels in animals and relating them to dose levels and time. Such studies are important for interpreting toxicity findings, designing further studies, and assessing the relevance of results to human safety. Key objectives include describing systemic exposure levels in toxicity studies and relating them to toxic effects.
Target identification in drug discoverySwati Kumari
The document discusses target identification in drug discovery. It begins by defining a target and explaining that target identification is the first step in drug discovery. It then discusses various approaches to target identification, including direct biochemical methods, genetic interaction methods, and computational inference methods. The document also discusses characteristics of drug targets and how drugs interact with targets at the molecular level. It provides examples of tools that can be used for target identification and validation, such as microarrays, antisense technology, and proteomics. In summary, the document outlines the process of target identification in drug discovery and various methods that can be used to identify and validate potential drug targets.
This document discusses genetic toxicology and genotoxicity testing. It defines genetic toxicology as the study of agents that can damage DNA and chromosomes. It also defines genotoxicity tests as in vitro and in vivo tests to detect compounds that induce genetic damage. The document outlines the importance of genotoxicity testing and describes some standard tests used, including the Ames test, chromosome aberration test, and micronucleus test. It provides details on procedures for these common genotoxicity assays and discusses recommendations for interpreting genotoxicity test results.
This document discusses various methods for testing the mutagenicity of chemicals, including both prokaryotic and eukaryotic cell systems. It describes the Ames test which uses Salmonella bacteria to identify mutagens, as well as other prokaryotic methods like the host-mediated assay and coliform assay. Eukaryotic methods discussed include the Saccharomyces forward mutation assay, mammalian cell tests, and in vivo assays like the micronucleus test and dominant lethal assay. The document provides details on the procedures and principles of many of these important mutagenicity testing methods.
This document discusses various cell culture techniques. It covers topics such as types of cell culture (primary, secondary, cell lines), culturing cells (adherent vs suspension), cell isolation techniques, subculturing cells, cryopreservation, and assays to measure cell viability. A variety of cell culture equipment is also described, including incubators, microscopes, cell counters, refrigerators/freezers, and liquid nitrogen storage. Finally, the basic components and types of cell culture media are outlined.
PRINCIPLES AND APPLICATIONS OF CELL VIABILITY ASSAY (MTT ASSAY)Durgadevi Ganesan
The document discusses the principles and applications of MTT cell viability assays. The MTT assay is a colorimetric assay that measures the reduction of MTT by mitochondrial succinate dehydrogenase in metabolically active cells to form an insoluble purple formazan product. The amount of formazan produced is directly proportional to the number of viable cells and can be used to measure cell proliferation, viability, and cytotoxicity in response to drugs or other factors. The MTT assay is inexpensive, rapid, quantitative, and reproducible, making it well-suited for applications like cytotoxicity testing, drug screening, and measuring responses to growth factors.
Fundamentals Of Genetic Toxicology In The Pharmaceutical Industry Sept 2010TigerTox
Historical and current perspectives on genetic toxicology, with commentary and slides on assay predictivity and shortcomings, regulatory guidance, and high-throughput screens to enhance preclinical drug safety.
This presentation gives the brief idea of the various guidelines carried out to study the genetic damage to cells when there is a discover of new active molecule.
Polymorphism affecting drug metabolismDeepak Kumar
Genetic polymorphisms can affect how individuals metabolize and respond to drugs. Variations in genes encoding drug-metabolizing enzymes like CYP450 isoforms can result in poor, intermediate, extensive, or ultra-rapid metabolizer phenotypes. This impacts how effectively an individual metabolizes and eliminates drugs from the body. The effects of inhibitors and inducers on drug metabolism also differ depending on a person's metabolizer phenotype. Understanding these genetic factors is important for predicting drug responses and interactions between a drug and other substances in an individual.
In vitro toxicity testing methods are used to identify potentially hazardous chemicals and confirm lack of toxicity in new substances like drugs and food additives. Cell culture methods screen for toxicity by assessing cell morphology, viability, growth and specialized functions. Both qualitative tests like extraction and direct contact methods, and quantitative assays measuring metabolic activity and membrane integrity are used. While 2D cell cultures have limitations, 3D tissue models better maintain native cell morphology and functions, improving predictions for drug toxicity testing and reducing animal testing and clinical trial failures.
There are three main types of cell culture: primary cell culture, where cells are directly separated from tissue and grown; secondary cell culture, where primary cells are subcultured and transferred to new vessels; and cell lines, which can be either finite, with a limited lifespan, or continuous, capable of indefinite growth. Primary cell culture can involve adherent cells that attach to surfaces or suspension cells that float freely. Secondary culture involves detaching and transferring adherent cells to new vessels using enzymatic methods.
HPLC - High Performance Liquid ChromatographyDivya Basuti
The document discusses High Performance Liquid Chromatography (HPLC). It explains that HPLC is a type of liquid chromatography that uses pumps to force the mobile phase through a column packed with porous particles or beads under high pressure. This allows for effective separation of mixtures as the components elute from the column at different rates depending on their interactions with the stationary phase. The document provides details on the typical components of an HPLC system including the solvent delivery system, pumps, injector, columns, detectors, and data processing unit.
This document provides information on gel electrophoresis techniques. It defines electrophoresis as a method to separate macromolecules like DNA, RNA, and proteins based on size and charge using an electrical current applied through a gel. It describes the principles, types of gels used (agarose or polyacrylamide), sample preparation, running procedures, and staining steps for separating DNA, RNA, and proteins. Protocols for agarose gel electrophoresis of DNA, formaldehyde gel electrophoresis of RNA, and SDS-PAGE gel electrophoresis of proteins are outlined in detail.
High throughput screening (HTS) is a process used in drug discovery to quickly test large numbers of chemical compounds and biological agents for biological activity against a disease state or condition of interest. The goal of HTS is to identify "hits" or "leads" that show desired activity at low concentrations and have a new chemical structure. Cell-based assays are an important type of HTS that uses live cells to more accurately model biological systems and provide information on bioavailability, cytotoxicity, and effects on biochemical pathways. Key elements of cell-based assays include a cellular component, a target molecule to detect cellular responses, and instrumentation to conduct and analyze the assay.
Cell death, also known as programmed cell death, occurs through various pathways including apoptosis, autophagy, and necrosis. Apoptosis, or programmed cell death, involves two main pathways - the intrinsic pathway which is triggered by cellular stress and the extrinsic pathway which is triggered by death ligands binding to cell surface death receptors. Both pathways activate caspases that break down cellular components leading to cell death. Autophagy is the natural and regulated mechanism by which cells degrade and recycle unnecessary or dysfunctional cellular components through the formation of autophagosomes and lysosomal degradation. Necrosis is unregulated cell death caused by external factors like infection, trauma or ischemia and results in the premature death of cells and tissue damage
Equipments used , types of culture and media, subculturing, secondary culture, finite & continuous cell lines, cryopreservation and applications of cell culture
Genetic toxicology involves assessing the effects of physical and chemical agents on DNA and genetic processes in living cells. It examines the health impacts of genetic alterations in somatic and germ cells, mechanisms that induce alterations like DNA damage and repair, and formation of gene mutations. A variety of assays are used to detect genetic alterations, with goals of identifying mutagenic chemicals and repair mechanisms. These assays examine DNA damage, mutations in nonmammalian and mammalian models, and chromosomal aberrations. Germ cell mutagenesis is also evaluated through assays measuring gene mutations and chromosomal alterations.
This document describes a glucose uptake assay to analyze glucose transport activity in differentiated 3T3 L1 cells. The assay involves treating starved cells with insulin or plant extracts, then exposing the cells to a radioactive cocktail containing tagged glucose. Uptake of the tagged glucose is measured using liquid scintillation counting to analyze the effect of treatments on glucose uptake activity.
Cell viability and proliferation assays measure aspects of cellular health and function, such as membrane integrity, metabolic activity, and DNA synthesis. Common assays include MTT, which measures mitochondrial activity; ATP assays, which measure ATP concentration as a marker of viability; Sulforhodamine B, which binds cellular proteins to measure biomass; and propidium iodide staining, which detects compromised membranes. These assays are useful for screening drug toxicity and effects on cell growth.
Genotoxicity_studies M pharmacy Pharmacology.pptxAyodhya Paradhe
This document discusses various genotoxicity studies and guidelines. It introduces the concept of genotoxicity and describes several important tests used in genotoxicity assessment, including the Ames test, in vitro mammalian cell micronucleus test, in vivo mammalian erythrocyte micronucleus test, in vitro mammalian chromosomal aberration test, and in vivo mammalian bone marrow chromosome aberration test. It provides details on the principles, procedures, and reporting of results for these key genotoxicity tests.
Target identification in drug discoverySwati Kumari
The document discusses target identification in drug discovery. It begins by defining a target and explaining that target identification is the first step in drug discovery. It then discusses various approaches to target identification, including direct biochemical methods, genetic interaction methods, and computational inference methods. The document also discusses characteristics of drug targets and how drugs interact with targets at the molecular level. It provides examples of tools that can be used for target identification and validation, such as microarrays, antisense technology, and proteomics. In summary, the document outlines the process of target identification in drug discovery and various methods that can be used to identify and validate potential drug targets.
This document discusses genetic toxicology and genotoxicity testing. It defines genetic toxicology as the study of agents that can damage DNA and chromosomes. It also defines genotoxicity tests as in vitro and in vivo tests to detect compounds that induce genetic damage. The document outlines the importance of genotoxicity testing and describes some standard tests used, including the Ames test, chromosome aberration test, and micronucleus test. It provides details on procedures for these common genotoxicity assays and discusses recommendations for interpreting genotoxicity test results.
This document discusses various methods for testing the mutagenicity of chemicals, including both prokaryotic and eukaryotic cell systems. It describes the Ames test which uses Salmonella bacteria to identify mutagens, as well as other prokaryotic methods like the host-mediated assay and coliform assay. Eukaryotic methods discussed include the Saccharomyces forward mutation assay, mammalian cell tests, and in vivo assays like the micronucleus test and dominant lethal assay. The document provides details on the procedures and principles of many of these important mutagenicity testing methods.
This document discusses various cell culture techniques. It covers topics such as types of cell culture (primary, secondary, cell lines), culturing cells (adherent vs suspension), cell isolation techniques, subculturing cells, cryopreservation, and assays to measure cell viability. A variety of cell culture equipment is also described, including incubators, microscopes, cell counters, refrigerators/freezers, and liquid nitrogen storage. Finally, the basic components and types of cell culture media are outlined.
PRINCIPLES AND APPLICATIONS OF CELL VIABILITY ASSAY (MTT ASSAY)Durgadevi Ganesan
The document discusses the principles and applications of MTT cell viability assays. The MTT assay is a colorimetric assay that measures the reduction of MTT by mitochondrial succinate dehydrogenase in metabolically active cells to form an insoluble purple formazan product. The amount of formazan produced is directly proportional to the number of viable cells and can be used to measure cell proliferation, viability, and cytotoxicity in response to drugs or other factors. The MTT assay is inexpensive, rapid, quantitative, and reproducible, making it well-suited for applications like cytotoxicity testing, drug screening, and measuring responses to growth factors.
Fundamentals Of Genetic Toxicology In The Pharmaceutical Industry Sept 2010TigerTox
Historical and current perspectives on genetic toxicology, with commentary and slides on assay predictivity and shortcomings, regulatory guidance, and high-throughput screens to enhance preclinical drug safety.
This presentation gives the brief idea of the various guidelines carried out to study the genetic damage to cells when there is a discover of new active molecule.
Polymorphism affecting drug metabolismDeepak Kumar
Genetic polymorphisms can affect how individuals metabolize and respond to drugs. Variations in genes encoding drug-metabolizing enzymes like CYP450 isoforms can result in poor, intermediate, extensive, or ultra-rapid metabolizer phenotypes. This impacts how effectively an individual metabolizes and eliminates drugs from the body. The effects of inhibitors and inducers on drug metabolism also differ depending on a person's metabolizer phenotype. Understanding these genetic factors is important for predicting drug responses and interactions between a drug and other substances in an individual.
In vitro toxicity testing methods are used to identify potentially hazardous chemicals and confirm lack of toxicity in new substances like drugs and food additives. Cell culture methods screen for toxicity by assessing cell morphology, viability, growth and specialized functions. Both qualitative tests like extraction and direct contact methods, and quantitative assays measuring metabolic activity and membrane integrity are used. While 2D cell cultures have limitations, 3D tissue models better maintain native cell morphology and functions, improving predictions for drug toxicity testing and reducing animal testing and clinical trial failures.
There are three main types of cell culture: primary cell culture, where cells are directly separated from tissue and grown; secondary cell culture, where primary cells are subcultured and transferred to new vessels; and cell lines, which can be either finite, with a limited lifespan, or continuous, capable of indefinite growth. Primary cell culture can involve adherent cells that attach to surfaces or suspension cells that float freely. Secondary culture involves detaching and transferring adherent cells to new vessels using enzymatic methods.
HPLC - High Performance Liquid ChromatographyDivya Basuti
The document discusses High Performance Liquid Chromatography (HPLC). It explains that HPLC is a type of liquid chromatography that uses pumps to force the mobile phase through a column packed with porous particles or beads under high pressure. This allows for effective separation of mixtures as the components elute from the column at different rates depending on their interactions with the stationary phase. The document provides details on the typical components of an HPLC system including the solvent delivery system, pumps, injector, columns, detectors, and data processing unit.
This document provides information on gel electrophoresis techniques. It defines electrophoresis as a method to separate macromolecules like DNA, RNA, and proteins based on size and charge using an electrical current applied through a gel. It describes the principles, types of gels used (agarose or polyacrylamide), sample preparation, running procedures, and staining steps for separating DNA, RNA, and proteins. Protocols for agarose gel electrophoresis of DNA, formaldehyde gel electrophoresis of RNA, and SDS-PAGE gel electrophoresis of proteins are outlined in detail.
High throughput screening (HTS) is a process used in drug discovery to quickly test large numbers of chemical compounds and biological agents for biological activity against a disease state or condition of interest. The goal of HTS is to identify "hits" or "leads" that show desired activity at low concentrations and have a new chemical structure. Cell-based assays are an important type of HTS that uses live cells to more accurately model biological systems and provide information on bioavailability, cytotoxicity, and effects on biochemical pathways. Key elements of cell-based assays include a cellular component, a target molecule to detect cellular responses, and instrumentation to conduct and analyze the assay.
Cell death, also known as programmed cell death, occurs through various pathways including apoptosis, autophagy, and necrosis. Apoptosis, or programmed cell death, involves two main pathways - the intrinsic pathway which is triggered by cellular stress and the extrinsic pathway which is triggered by death ligands binding to cell surface death receptors. Both pathways activate caspases that break down cellular components leading to cell death. Autophagy is the natural and regulated mechanism by which cells degrade and recycle unnecessary or dysfunctional cellular components through the formation of autophagosomes and lysosomal degradation. Necrosis is unregulated cell death caused by external factors like infection, trauma or ischemia and results in the premature death of cells and tissue damage
Equipments used , types of culture and media, subculturing, secondary culture, finite & continuous cell lines, cryopreservation and applications of cell culture
Genetic toxicology involves assessing the effects of physical and chemical agents on DNA and genetic processes in living cells. It examines the health impacts of genetic alterations in somatic and germ cells, mechanisms that induce alterations like DNA damage and repair, and formation of gene mutations. A variety of assays are used to detect genetic alterations, with goals of identifying mutagenic chemicals and repair mechanisms. These assays examine DNA damage, mutations in nonmammalian and mammalian models, and chromosomal aberrations. Germ cell mutagenesis is also evaluated through assays measuring gene mutations and chromosomal alterations.
This document describes a glucose uptake assay to analyze glucose transport activity in differentiated 3T3 L1 cells. The assay involves treating starved cells with insulin or plant extracts, then exposing the cells to a radioactive cocktail containing tagged glucose. Uptake of the tagged glucose is measured using liquid scintillation counting to analyze the effect of treatments on glucose uptake activity.
Cell viability and proliferation assays measure aspects of cellular health and function, such as membrane integrity, metabolic activity, and DNA synthesis. Common assays include MTT, which measures mitochondrial activity; ATP assays, which measure ATP concentration as a marker of viability; Sulforhodamine B, which binds cellular proteins to measure biomass; and propidium iodide staining, which detects compromised membranes. These assays are useful for screening drug toxicity and effects on cell growth.
Genotoxicity_studies M pharmacy Pharmacology.pptxAyodhya Paradhe
This document discusses various genotoxicity studies and guidelines. It introduces the concept of genotoxicity and describes several important tests used in genotoxicity assessment, including the Ames test, in vitro mammalian cell micronucleus test, in vivo mammalian erythrocyte micronucleus test, in vitro mammalian chromosomal aberration test, and in vivo mammalian bone marrow chromosome aberration test. It provides details on the principles, procedures, and reporting of results for these key genotoxicity tests.
This is Part 1 of a presentation on Genetic Toxicology that was given by Dr. David Kirkland to scientific staff at Health Canada in Sept. 2010. Part 2 is availabile in ppt
TOXICOLOGY STUDY IS VERY ESSENTIAL FOR DRUG DISCOVERY. INTERNATIONAL COUNCIL FOR HARMONIZATION (ICH) HAS IMPLEMENTED SOME BASIC RULES AND REGULATION REGARDING THE TOXICITY STUDY ON ANIMAL DURING PRE-CLINICAL TRIAL, WHICH IS A PART OF DRUG DISCOVERY PROCESS. HERE SOME OF THE BASIC TEST ARE DISCUSSED ALONG WITH SOME BASIC CONCEPTS OF GENETICS. HOPE THIS WILL HELP THE STUDENTS TO UNDERSTAND THE TOPIC.
The Ames test is a bacterial mutagenicity test that uses strains of Salmonella typhimurium bacteria to determine if a chemical is mutagenic. It is based on the principle of reverse mutation, where auxotrophic mutant bacteria that cannot synthesize the amino acid histidine are exposed to a test chemical. If the chemical is mutagenic, it can induce a reverse mutation in the histidine gene, allowing the bacteria to grow in a histidine-deficient medium. The test involves mixing the chemical with rat liver enzymes before incubating it with the bacterial strains, and counting the number of colonies formed, with more colonies indicating higher mutagenicity. The Ames test is a fast, inexpensive way to screen
Shital Magar presented on in vitro genotoxicity testing based on OECD guidelines. The presentation covered the objectives of genotoxicity testing, introduction to genotoxicity, history, and details of key in vitro tests including bacterial reverse mutation assay, mammalian cell gene mutation tests, mammalian chromosomal aberration test, and mammalian cell micronucleus test. Parameters and limitations of each test were discussed along with examples of software used to analyze genotoxicity results.
The Ames test is a bacterial mutagenicity assay used to identify potential carcinogens. It uses strains of Salmonella typhimurium bacteria that are unable to synthesize the amino acid histidine and contain mutations in DNA repair systems. When these bacteria are exposed to a mutagen, some bacteria will experience a reverse mutation that allows them to synthesize histidine. These revertant bacteria will form colonies on a histidine-deficient growth medium. The Ames test is a quick and inexpensive method to screen chemicals compared to animal carcinogenicity studies. It has helped identify many carcinogens and led to the regulation of harmful substances. However, it also has limitations as bacteria differ from human metabolism and some known carcinogens are
Principles of cell viability assays by surendra.pptxSurendra Chowdary
1.DYE EXCLUSION ASSAYS:
Dye exclusion assays are the simplest methods that are based on utilization of different dyes such as trypan blue, eosin, congo red, and erythrosine B, which are excluded by the living cells, but not by dead cells.
For these assays, although staining procedure is quite straightforward, experimental procedure may be time-consuming in case of large sample sizes.
a. Trypan blue stain assay:
Trypan blue stain assay has initially been developed in 1975 to measure viable cell count and is still used as a confirmatory test for measuring changes in viable cell number caused by a drug or toxin.
Trypan blue stain, a large negatively charged molecule, is one of the simplest assays that are used to determine the number of viable cells in a cell suspension.
Principle:
The principle of this assay is that living cells have intact cell membranes that exclude the trypan blue stain, whereas dead cells do not.
Cell suspension is mixed with the trypan blue stain and examined visually under light microscopy to determine whether cells include or exclude the stain.
A viable cell will have a clear cytoplasm, whereas a nonviable cell will have a blue cytoplasm.
Reagent preparation:
To perform the trypan blue stain assay, 0.4% trypan blue stain and phosphate- buffered saline (PBS) or serum-free medium are obtained.
Trypan blue stain should be stored in dark and filtered after prolonged storage.
As trypan blue stain binds to serum proteins and causing misleading results, serum-free medium should be used to obtain reliable results.
Assay Protocol:
The cell suspension to be tested is centrifuged at 100 g for 5 min.
The supernatant is discarded and the pellet is resuspended in 1-ml PBS solution or serum-free medium.
Then, one portion of this cell suspension is mixed with one portion of trypan blue stain.
The mixture is allowed to stay at room temperature for 3 min. It is important to note that the cells should be counted within 3–5 min of mixing with trypan blue, as longer incubation periods will lead to cell death and hence reduced viability counts.
Following the incubation, a drop of the mixture is applied to a hemocytometer, which is placed on the stage of a binocular microscope.
Viable cells will remain unstained, and nonviable cells will stain, in the hemocytometer and these cells are counted separately.
.
Calculation:
After counting viable and nonviable cells, the total number of viable cells per milliliter of aliquot is determined by multiplying the total number of viable cells by 2, which is the dilution factor for trypan blue.
Similarly, total number of cells per milliliter of aliquot is determined by addition of number of viable and nonviable cells and multiplying it by 2.
Then, the percentage of viable cells is calculated using the following equation.
% Viable cells = Total number of viable cells per milliliter of aliquot × 100.
Total number of cells per milliliter of aliquot
2.COLORIMETRIC ASSAYS:
Colorimetric assays
This document discusses various methods for detecting mutations in microorganisms. It describes that mutations can be detected if they cause an altered phenotype. It then explains several techniques for detecting mutations including replica plating, resistance selection, substrate utilization, and the Ames test. Replica plating involves transferring bacterial colonies from a "master plate" to plates with different growth conditions to identify mutants unable to grow in certain conditions. Resistance selection and substrate utilization detect mutants able to grow in the presence of antibiotics or use alternative carbon sources. The Ames test uses Salmonella strains to identify whether chemicals cause mutations detectable as increased reversion to histidine prototrophy.
This document provides an overview of genetic toxicity testing guidelines. It discusses the history and aims of toxicity studies. Various in vitro and in vivo genetic toxicology tests are described, including tests for gene mutation, chromosomal abnormalities, and primary DNA damage. Key tests covered include the mammalian erythrocyte micronucleus test, mammalian bone marrow chromosomal aberration test, rodent dominant lethal assay, and mouse heritable translocation assay. The principles, procedures, and parameters of these tests are summarized. References on genetic toxicology guidance documents and studies are also provided.
The document discusses mutagenicity and carcinogenicity of environmental factors. It defines key terms like mutagen, mutagenicity, carcinogen and carcinogenicity. It describes various types of mutagens and carcinogens. It also summarizes different methods to test for mutagenicity and carcinogenicity, including tests on the molecular, gene and chromosomal level like the Ames test, comet assay and micronucleus test. The document provides an overview of the process of carcinogenesis and challenges in evaluating human carcinogenicity.
The document describes screening methods for new anticancer drugs. It discusses how cancer arises from genetic mutations and different cancer types. Current treatments include chemotherapy, surgery and radiation. There is a need for more selective anticancer agents due to drug resistance and side effects. Various in vitro and in vivo screening assays are described to test compounds for cytotoxicity against cancer cells and tumors in animal models. The goal is to develop more effective and safer anticancer drugs.
In vitro toxicity testing methods can more cost-effectively identify potentially hazardous chemicals compared to animal testing. Cell culture models are commonly used to screen for general toxicity through estimation of cell viability, morphology, and growth. Both qualitative and quantitative cytotoxicity assays are employed, such as membrane integrity assays measuring LDH leakage and viability assays measuring mitochondrial or lysosomal activity via dyes like MTT or neutral red. While 2D cell cultures have limitations, 3D tissue models better maintain native cell morphology and functionality for improved prediction of drug toxicity and efficacy compared to animal models.
In vitro toxicity testing methods can more cost-effectively identify potentially hazardous chemicals compared to animal testing. Common techniques include extracting chemicals from test materials and monitoring their effects on cultured cells through microscopy and assays measuring cell viability, morphology, and metabolism. While 2D cell cultures are widely used, 3D cultures better mimic in vivo conditions and may improve predictions of drug toxicity to potentially reduce clinical trial failures. A variety of quantitative assays exist to complement qualitative observations, with the MTT, neutral red, calcein, and LDH assays among the most frequently employed.
In vitro toxicity testing methods can more cost-effectively identify potentially hazardous chemicals compared to animal testing. Common techniques include extracting chemicals from test materials and monitoring their effects on cultured cells through microscopy and assays measuring cell viability, morphology, and metabolism. While 2D cell cultures are widely used, 3D cultures better mimic in vivo conditions and may improve predictions of drug toxicity to potentially reduce clinical trial failures. A variety of quantitative assays exist to complement qualitative observations, with the MTT, neutral red, calcein, and LDH assays among the most frequently employed.
Principles & Applications of cell viability assays (MTT Assays)VidyaNani
This document discusses cell viability assays, specifically focusing on principles and applications of MTT assays. It defines cell viability and describes various types of cell viability assays including dye exclusion, colorimetric, fluorometric, luminometric, and flow cytometric assays. The document provides details on the MTT assay, including its principle, protocol, and applications for measuring cell proliferation, cytotoxicity, and metabolic activity. The MTT assay is a common colorimetric assay that measures the reduction of yellow MTT by mitochondrial dehydrogenases in viable cells to produce purple formazan crystals.
The document discusses alternatives to animal toxicity testing for phototoxicity and carcinogenicity evaluation. It summarizes the 3T3 neutral red uptake photo toxicity test used to assess phototoxic potential in vitro. It also describes tests to detect genotoxic carcinogens like the Ames assay and mouse lymphoma assay, and non-genotoxic carcinogens using the Syrian hamster embryo cell transformation assay which can identify carcinogens through morphological transformation of cells.
This document summarizes research presented on isolating and characterizing human fetal liver stem cells. The key steps involved isolating stem cells from human fetal liver tissue using markers like AFP, CK18, and albumin. The isolated stem cells were then characterized using techniques like cell counting, viability assays, immunocytochemistry, MTT assays, and PCR to analyze RNA expression and confirm the presence of stem cell markers. The overall aim was to isolate fetal liver stem cells and characterize their properties for potential applications in areas like blood transfusions, organ transplantation, and disease research.
1. This study investigated the prevalence of integrons and antimicrobial resistance genes in 110 clinical isolates of Enterobacter species collected from hospitals in Tehran, Iran between 2012-2013.
2. The study found that 45 isolates (41%) contained integrons, with class 1 integrons being most common. Integron-positive isolates showed higher resistance to antibiotics like augmentin, trimethoprim-sulfamethoxazole, and cefoxitin.
3. Ten integron-positive isolates were found to be ESBL producers. Common resistance genes identified included blaTEM (20%), blaCTX-M-1 (15.6%), and genes encoding aminoglycoside
Macroeconomics- Movie Location
This will be used as part of your Personal Professional Portfolio once graded.
Objective:
Prepare a presentation or a paper using research, basic comparative analysis, data organization and application of economic information. You will make an informed assessment of an economic climate outside of the United States to accomplish an entertainment industry objective.
How to Add Chatter in the odoo 17 ERP ModuleCeline George
In Odoo, the chatter is like a chat tool that helps you work together on records. You can leave notes and track things, making it easier to talk with your team and partners. Inside chatter, all communication history, activity, and changes will be displayed.
Thinking of getting a dog? Be aware that breeds like Pit Bulls, Rottweilers, and German Shepherds can be loyal and dangerous. Proper training and socialization are crucial to preventing aggressive behaviors. Ensure safety by understanding their needs and always supervising interactions. Stay safe, and enjoy your furry friends!
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A Survey of Techniques for Maximizing LLM Performance.pptx
Mutagenicity, Carcinogenicity, Genotoxicity Tests
1. S. Archana
(Reg no. 22701502)
I MSc Biochemistry
V.V.Vanniapermual college for
women, Virudhunagar.
2. contents
Introduction
Types of mutagenicity tests
Molecular level
Gene level
Chromosomal level
Three important tests
AMES Test
HPRT Test
Mouse Micronucleus Test
3. Introduction
The purpose of mutagenicity testing is to
identify substances that can cause genetic
alterations in somatic and/or germ cells and use
this information in regulatory decisions.
Mutagenicity testing is the first step to screen the
chemicals for their potential to be a pesticide,
food additive, or drug.
The most widely used mutation test is Ames test,
developed by Ames, which is performed in different
strains of Salmonella typhimurium and in Escherichia
coli.
4. The most widely used mutation test is Ames test,
developed by Ames, which is performed in different
strains of Salmonella typhimurium and in Escherichia
coli.
5. Types of mutagenicity tests
It can be separated on the basis of
1)Molecular level
2)gene level
3) chromosomal level
1)Molecular level:
Comet assay –
The comet assay (single-cell gel electrophoresis) is a
simple method for measuring deoxyribonucleic acid (DNA)
strand breaks in eukaryotic cells.
Cells embedded in agarose on a microscope slide are
lysed with detergent and high salt to form nucleoids containing
supercoiled loops of DNA linked to the nuclear matrix.
6.
7. 2) Gene level test
TK Gene Mutation test :
The in vitro mammalian cell gene mutation
test (OECD 490), also referred to as the mouse
lymphoma assay, is used to detect a spectrum of
genetic events denoting gene mutations induced by
chemical substances in the cell lines that measure
mutation at thymidine kinase (TK).
8.
9. In vivo pig gene mutation assay:
The Pig-a gene mutation assay has emerged
as a valuable tool for quantifying in vivo and in
vitro mutational events. The Pig-a locus is located at
the X-chromosome, giving the advantage that one
inactivated allele can give rise to a mutated phenotype,
detectable by multicolour flow cytometry.
10.
11. 3)Chromosomal level test
In Vivo Micronucleus Assay:
The micronucleus test (MNT) is used to determine if a
compound is genotoxic by evaluating the presence of micronuclei.
Micronuclei may contain chromosome fragments produced from
DNA breakage (clastogens) or whole chromosomes produced by
disruption of the mitotic apparatus (aneugens).
SCE assay:
It is a powerful technique to visually detect the physical
exchange of DNA between sister chromatids. SCEs could result
as a consequence of DNA damage repair by homologous
recombination (HR) during DNA replication.
13. Important tests
The three most commonly conducted assays are:
1)The Ames Salmonella typhimurium reverse mutation
assay,
2)The Chinese hamster ovary (CHO) hypoxanthine-
guanine phosphoribosyltransferase (HGPRT) in vitro
cytogenetics assay, and
3)The mouse micronucleus test .
14.
15. AMES TEST
Ames test it is a biological assay to assess the
mutagenic potential of chemical compounds. It
utilizes bacteria to test whether a given chemical can
cause mutations in the DNA of the test organism. The
test was developed by Bruce N. Ames in 1970s to
determine if a chemical at hand is a mutagen.
Objective
To determine the mutagenic activity of
chemicals by observing whether they cause mutations
in sample bacteria.
16. Principle
Ames test uses several strains of bacteria (Salmonella, E.coli) that
carry a particular mutation.
Point mutations are made in the histidine (Salmonella
typhimurium) or the tryptophan (Escherichia coli) operon,
rendering the bacteria incapable of producing the corresponding
amino acid.
These mutations result in his- or trp- organisms that cannot
grow unless histidine or tryptophan is supplied.
But culturing His- Salmonella is in a media containing certain
chemicals, causes mutation in histidine encoding gene, such
that they regain the ability to synthesize histidine (His+). This is
to say that when a mutagenic event occurs, base substitutions or
frameshifts within the gene can cause a reversion to amino acid
prototrophy. This is the reverse mutation.
These reverted bacteria will then grow in histidine- or
tryptophan-deficient media, respectively.
17. A sample’s mutagenic potential is assessed by exposing
amino acid-requiring organisms to varying
concentrations of chemical and selecting for the
reversion event. Media lacking the specific amino acid
are used for this selection which allow only those cells
that have undergone the reversion to histidine /
tryptophan prototrophy to survive and grow. If the test
sample causes this reversion, it is a mutagen.
18. Method:
I ) Isolate an auxotrophic strain of Salmonella Typhimurium for
histidine. (ie. His-ve)
II) Prepare a test suspension of his-ve Salmonella Typhimurium in a
plain buffer with test chemical (eg. 2-aminofluorene). Also add a
small amount of histidine.
Note: small amount of histidine is required so bacteria starts growing.
Once histidine is depleted only those bacteria mutated to gain the
ability to synthesize histidine form colonies.
III) Also prepare a control suspension of His-
ve Salmonella Typhimurium but without test chemicals.
IV) Incubate the suspensions at 37°C for 20 minutes
V) Prepare the two agar plate and spread the suspension on agar plate.
VI) Incubate the plates at 37°C for 48 hours.
VII) After48 hours count the number of colonies in each plate.
19.
20. Result Interpretation
The mutagenicity of chemicals is proportional to number of
colonies observed.
If there is a large number of colonies on the test plate in
comparison to control, then such chemical are said to be
mutagens.
Very few numbers of colonies can be seen on control plate also.
This may be due to spontaneous point mutation on hisidine
encoding gene.
Uses
1)While Ames test is used to identify the revert mutations
which are present in strains, it can also be used to detect the
mutagenicity of environmental samples such as drugs, dyes,
reagents, cosmetics, waste water, pesticides and other substances
which are easily solubilized in a liquid suspension.
2)AMES test are used in pharmacy research before a
compound given to a animal.
21. 3)Smoker Urine test for mutagenicity is possible by the
use of AMES test.
4)Primary DNAdamage test can be done through
AMES test.
5)This test is highly sensible for testing the
mutagnicity of water and solid soil sample from the
nuclear.
Merits:
1)Simple, rapid and robust bacterial assay.
2)Ease and low cost of the test make it invaluable for
screening substances in our environment for possible
carcinogenicity.
3)Ames test can detects suitable mutants in large
population of bacteria with high sensitivity.
22. limitation
1)Some substances that cause cancer in laboratory
animals (dioxin, for example) do not give a positive
Ames test (and vice-versa)
2)Ames assay consists of Salmonella
typhimurium strains and so it is not a perfect model
for human.
3)Some sample shows false postive test. That is
scattered large population microbes integates only
spontaneous mutation not mutation due to the
compound taken as test.Aggregated population of
microbes only positive for test compound.
23. HPRT GENE TEST
In Vitro Mammalian Cell Gene Mutation Test
(HPRT Gene)
The in vitro mammalian cell gene mutation test is used
to detect mutations of the hypoxanthine-guanine
phosphoribosyltransferase (HPRT) gene in Chinese
hamster ovary (CHO) or lung (V79) fibroblasts.
Screening and Regulatory Support
1)Cultures are incubated with several concentrations of the
test compound for three to four hours in the presence
and absence of metabolic activation (S9).
24. 2) Cells are subcultured for seven to eight days after
treatment to allow expression of the mutant
phenotype, and then plated in media with and
without 6-thioguanine (TG) to select for mutants
and determine cloning efficiency.
3) A positive outcome is characterized by a
statistically significant, dose-dependent increase
in mutant frequency that exceeds historical
negative control limits.
25. When To Perform
Screening
Reduced volume and/or abbreviated formats available
REACH requirement
As part of Annex VIII testing
Mutagenicity
To confirm presumed mutagenic activity arising from
limited/small genetic damage (e.g., positive Ames or
large colony MLA)
To assess mutagenicity when the Ames assay may not be
appropriate (e.g., antibiotics, nanomaterials)
26.
27. The Mouse Micronucleus Test
Screening and Regulatory Support
Male and/or female rats or mice are treated with the test compound at
three dose levels, usually two or three times at 24-hour intervals.
Approximately 24 hours after the last dose, bone marrow or peripheral
blood is collected to determine the frequency of micronucleated
polychromatic erythrocytes (MN-PCEs) or micronucleated
reticulocytes (MN-RETs), respectively.
A positive outcome is characterized by a statistically significant, dose-
dependent increase in MN-PCEs or MN-RETs that exceeds historical
control limits.
Can be combined with standard toxicology tests, the comet assay and
the Pig-a assay.
Administration routes include: oral, intravenous, infusion and
inhalation
28. When to Perform
Screening
Abbreviated formats available or can be added to non-GLP
tolerability studies
IND-enabling
As part of the ICH S2(R1) standard battery (Option 1 or 2)
REACH requirement
To follow up a positive result in any of the Annex VII or VIII
genotoxicity tests