Molecular/Toxicology Genotoxicity Flashcards
What are primary tools in toxicology?
Chemistry, biochemistry, and molecular biology.
What tools does chemistry have to offer?
Analytical methods for toxicants and their metabolites.
What tools does biochemistry have to offer?
methods for investigating metabolism and
modes of toxic action.
What tools does molecular biology have to offer?
methods for investigating
the roles of genes and gene expression in
toxicology.
Who and when was the DNA structure discovered?
Watson and Crick in 1953
What occurred in the 1960s?
The definition of the genetic code.
Molecular toxicology is what?
restricted to the events involving DNA, RNA, Proteomics, and Metabolomics.
What is the goal of molecular toxicology?
define underlying molecular mechanisms of toxicity.
What do molecular techniques help you understand?
the mechanisms and responses of an organism to a toxic insult.
What areas does genetic toxicology work with?
Cytotoxicity, mutations, cytogenetic abberations, DNA damage and repair, and carcinogenesis.
What are the areas that molecular toxicology encompass?
Genetic toxicology, Epigenetics, bioinformatics, Toxicogenomics, Proteomics, metabolomics, gene expression, and gene function.
What are the 3 main areas of molecular toxicology needed to know for class?
Genetic tox (mutations and cytogenetic aberrations), toxicogenomics, and gene expression.
What is genetic toxicology?
Study and characterization of physical and chemical agents that affect the hereditary material of living organisms.
What are hereditary materials?
DNA and RNA
What is the primary goal of a genetic toxicologist?
Detect and understand the properties of agents that induce deleterious effects in genetic elements at doses below those that induce physiological toxicity.
How did the field of genetic toxicology originate?
With the work of HJ muller and C Auerbach.
Who was HJ Muller?
Radiation mutagenesis
Who was C. Auerbach?
Chemical mutagenesis.
What occurred to Muller in 1927?
He discovered ionizing radiation, specifically X-rays, induced mutations in drosophila
Who publicized the adverse implications of inappropriate use of medical x-rays?
HJ Muller in 1930
What were X-rays being used for in 1930s?
To xrays your feet inside the shoe to be sure it fit well.
In 1946 What did Charlotte Auerbach discover?
chemicals could induce mutations in Drosophila.
What type of chemicals did C. Aurbach investigations used?
Mustard Gas use in WW2
C. Auerbach was one of the first scientist to consider what?
Chemical mutagens could induce genetic damage in humans.
What did the two major advances in the 1970s provide clearer understanding to?
mechanisms of mutagenesis and the potential role of mutagens in carcinogenesis.
What did James and Elizabeth Miller demonstrate?
Chemical carcinogens could react to form stable covalent derivatives with DNA, RNA, Proteins.
Formation of derivatives required metabolisms of what?
Who discovered this?
The chemical to primary and subsequent metabolites.
James and Elizabeth Muller.
What did James and Elizabeth Muller establish?
Requirement of metabolism for some carcinogens.
What did Bruce Ames and his colleagues design?
An assay with Salmonella Tryphimurium to detect chemically induced reverse mutations.
The Ames Assay was to use to detect chemically induced reverse mutations where?
At the histidine locus.
What role does the Ames Assay play?
An important role in hazard identification.
Ames assay includes several bacteria strains that contain what?
A specific mutation in the histidine locus.
Tester strains are unable to grow in the absence of what?
Histidine.
Mutations must be reverted to normal functions why?
to be detected in assay (histidine independence)
What does TA1535 detect?
base pair substitutions
What does TA1538 detect?
-1 base pair frameshifts
What does TA1537 detect?
+1 base pair framshifts.
What is the experimental procedure for the Ames Assay?
Bacteria are treated with a test chemical.
Plated onto a histidine-deficient growth medium.
Colonies that develop are scored as mutants.
How can Ames assay also be conducted using S9?
(exogenous metabolizing
system) to detect mutagens that require metabolic
activation to DNA-reactive intermediate.
What type of approaches do we use now?
Omic approaches including
microarrays and whole genome sequencing.
What type of advances were made in mid 70s and mid 80s?
~200 short-term genotoxicity and
mutagenicity assays developed
Mutation induction, DNA damage,
DNA repair, cytotoxicity
What are three types of genetic alterations induced by a toxicant?
Mutagenesis, clastogenesis, Aneuploidy.
What is mutagenesis?
Loss, addition, or alteration of a small number of base pairs.
What is Clastogenesis?
Loss, addition, or rearrangement of parts of chromosomes.
What is Aneuploidy?
Acquisition or loss of a complete chromosome.
Base pair substitutions and modifications is an example of what?
Gene mutations: mutagenesis
Frameshift mutations (deletions/insertions) is an example of what?
Mutagenesis
Changes in the number of sets of chromosomes is an example of what?
Clastogenesis and aneuploidy
Changes in the number of chromosomes is what?
Clastogenesis and aneuploidy
Changes in the structure of the chromosomes (deletion, duplication, and translocation) is an example of what?
Clastogenesis and aneuploidy
Substituting one base for another is what?
Gene mutations: remember A-T and G-C
What can cause erroneous base pairing?
Incorporation of a mutagen into the DNA molecule.
Which is worse: BP sub or Frameshift?
Framshift
What is a frameshift mutation?
Base pair addition or deletion.
What agents may intercalate into the DNA?
Ethidium bromide and acridine orange.
Agents that intercalate into the DNA may result in what?
Frameshift mutation.
In frameshift mutation what occurs to the order of the genetic code?
It is altered and reading from the base code is shifted.
What is seriously affected by a frameshift mutation?
Transcription of information.
What is polyploidy chromosomal damage?
Changes in the number of sets of chromosomes.
What is aneuploidy chromosomal damage?
changes in the number of chromosomes.
What are types of changes within the chromosome structure?
deletion, duplication, and translocation.
What are genetic toxicology assays developed to screen?
environmental products for carcinogens.
Genetic toxicology assays are developed to identify what?
putative carcinogens in body fluids.
Genetic toxicology assays are developed to define what?
mechanisms of activation of chemical carcinogens and describing their reactions with DNA.
Genetic toxicology assays are developed to understand what?
the process of anti-mutagenicity and anticarcinogenicity.
Genetic toxicology assays are developed to undergo what type of research?
basic and molecular mutation.
Genetic toxicology assays are developed to predict what?
carcinogenicity in regulatory standards for testing environmental chemicals.
What are the ideal characteristics of a genotoxicity assay?
sensitivity and repoducibility and to be able to detect the entire mutagenic spectrum.
What does it mean when a genotoxicity assay has ideal sensitivity and reproducibility?
ability to detect a small mutational effect above the spontaneous mutation rate.
done in any given laboratory.
What does it mean when a genotoxicity assay has ideal capacity to detect the entire mutagenic spectrum?
Ability to detect changes in chromosomes number and induction of chromosomes aberrations and rearrangements. –> ability to detect single gene mutations and DNA damage.
What are two assays used to detect gene mutations?
Ames assay and gene mutation assays in cell culture lines.
How does the ames assay detect mutations?
Bacterial assay- Various strains to detect specific mutations
Mainly hazard identification
How does the Gene mutation assay in cell culture lines detect mutations?
Target certain loci for specific mutations
What is an example of a gene mutation assay in cell culture lines?
Mouse lymphoma assay
(MLA) that targets the thymidine kinase
(TK) as selection marker
What type of assays are used to detect chromosomal aberrations?
chromosomal/cytogenetic assays.
What do chromosomal/cytogenetic assays assess?
metaphase spreads
How do the chromosomal/cytogenetic assays assess metaphase spreads?
by using new technology called Fluorescent in situ hybridization
(FISH)
What does FISH stand for?
Fluorescent in situ hybridization.
What is FISH?
it assays various chromosomal aberrations.
Where can FISH be done?
cell culture, plants, animals, and humans.
What is genomic DNA isolation used for?
for evaluating genomic DNA
What is genomic DNA isolation?
series of steps to purify DNA from cell.
What is needed for gel electrophoresis?
determine DNA size, genomic DNA assessing degradation, and cut DNA with restriction enzymes.
What is a southern blot?
analysis of specific DNA sequences.
What do southern blots do?
determine if gene is present, copy number of gene, and genotype of genetically modified animals.
What does PCR stand for?
polymerase chain reaction
What does PCR do?
amplifies targeted DNA sequences using specially designed primers.
PCR can make billions of copies of what?
target sequence of DNA in a few hrs.
When was PCR first invented?
1984
What is PCR now used for?
cloning, mutagenesis, and analysis of gene expression.
What is RNA isolation in gene expression assays?
extra caution to prevent degredation of RNA, snapshot of gene expression.
What is northern blot?
Measures the amount and size of RNAs transcribed and estimates their abundance.
What are methods for the Northern blot?
runs in agarose denaturing gel, transfer to membrane, probe specific DNA sequence, and probe detection.
Why do many assays utilize stable cDNA synthesized from RNA sequence?
because a double strand is stronger than a single strand.
What does quantitative PCR do?
Quantify target gene relative to a reference gene.
What does toxicogenomics stand for?
genome wide analysis in toxicology.
What does genomics mean?
the study of genes and their functions.
What does transcriptomics mean?
The study of mRNA
What does Proteomics?
The study of proteins.
What does metabolomics mean?
The study of molecules involved in cellular metabolism.
What does glycomics mean?
The study of cellular carbohydrates
What does lipomics mean?
The study of cellular lipids.
What does ionomics mean?
All mineral nutrients and trace elements of an organism.
What includes transcriptomics, proteomics, and metabolomics?
Toxicogenomics.
Transcriptomics compares what?
the genes expressed (mRNA) in an organism that have been exposed to a drug, chemical, or a toxin to those of unexposed organisms.
What is the goal of transcriptomics?
to identify patterns of gene expressions related to specific chemicals or chemical classes to be used as endpoints in assessing toxicity.
What is the end result of microarray transcriptome analysis?
differentially expressed gene list related to specific chemical.
Why is microarray transcriptome analysis good?
if you don’t know molecule targets initially.
Why evaluate for genetic damage?
lethal mutations, cancer, developmental abnormalities.
