Genetic Toxicology.

What are the characteristics of the abbreviated standard assay

- Typically use TA98 & TA100 -Can substitute or add strains as needed -with or without S9 -8 doses -2X replicates per dose

transformed cells in a she assay express what

- random orientation, 3-D stacking of cells - increased nuclear to cytoplasmic ratio - cells frequently more basophilic than normal - criss-crossing of cells at perimeter and interior

IF you dont have bone marrow toxicity how can you demonstrate target tissue exposure

1) Measure blood plasma levels following treatment 2) directly measure drug material in bone marrow 3) Autoradiography assemssements of tissue eposure

What are the two possible outcomes of genetic damage?

1) You can have a mutation in somatic cells that causes either aging or cancer 2) Or you can have a mutation is germinal cells which causes genetic diseases

What are the three major challenges to the integrity of the DNA in vivo?

1) are lesions inflicted by endogenous and environmental DNA-damaging agents (endogenous and environmental is what you want to know 3) Replication errors Endogenous agents, Exogenous like UV light, and replication errors

You have 100 transformed colonies in the she assay and you have 200 total colonies, what is the transformed frequency

100/200= 50 percent.

How many tester strains does the abbreviated standard assay usually use

2

How accurate is the SHE assay

89 percent

What is a structural aberration

: a change in chromosome structure detectable by microscopic examination of the metaphase stage of cell division, observed as deletions and fragments, intrachanges or interchanges.

What is the difference between a carcinogen and a mutagen

A mutagen alters DNA in a heritable manner while a carcinogen transforms a normal cell to a neoplastic cell

What is a mutagen?

A substance that under the coreect metabolic and cellular conditions has the ability to alter a cells DNA in a heritable manner.

What is the UDS assay

A tier 2 assay that is a short-term genotoxicity assay that allows identification of substances that induce DNA repair in liver cells of treated animals

What are microlesions

A type of lesion that occurs when a chemical reacts with DNA and it creates a mutation.

What are the two types of typical screening assays

Abbreviated standard assay and the Spot test assay

Base excision repair has an important role in

Aging

What is the difference between Mouse lymphoma assay along with cho/hgprt compared to the ames tes

Ames is not a mammallian system. The CHo/HGPRT and mouse lymphomas are mammalian systems. The mouse lymphoma and the HGPRT/CHO assays cause a mutation and quantitate it while the ames test reverses a natural mutation and quantitates it.

In ICH, testing what is the protocol you have to follow as in what testing must be done

Ames test, then you have to do in vitro and you have the option of in vivo if the company says you need to do that.

What is the list of testing the EPA has to do?

Ames, In vivo mouse lymphoma, rodent micronucleus test.

Why do we add S9 in the ames assay?

Bacteria lacks metabolic capacity so we add s9 and it is a blend of cytochrome p450. And this is good because procarcinogens and promutagens need to be metabolized to become a true carcinogen or mutagen.

This type of repair fixes abnormal bases in DNA

Base excision repair

What are the two types of excision repairs

Base excision repair and nucleotide excision repair.

What is the difference between base excision repair and nucelotide excision repair in rescpect to what they are responsible in removing

Base excision-- removes oxidative and alkylation damage. Nucleotide excision-- Removes UV-induced damage and bulky adducts and 20 percent of oxidative damage

What is the difference between the types of mutations that are detectable by MLA or CHO

Both can detect point mutations and allele loss, but only MLA can detect multi locus mutations, chromosome alterations, aneuploidy.

What is a substance that under the coreect metabolic and cellular conditions has the ability to transform a normal cell to a neoplastic cell?

Carcinogen.

What is a chromosome type abberation

Causes structural chromosome damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site.

80 percent of human cancer is linked to

Chemicals

This a type of chromosomal aberration that causes structural chromosome damage expressed as breakage of single chromatids or breakage and reunion between chromatids.

Chromatid type aberration

This is a type of abberation Causes structural chromosome damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site.

Chromosome type aberration

In ICH and OECD guidelines, for a positive result how do you confirm it

Clear positives dont need one

The purpose of this type of assay is to detect more chromosomal effects. To detect the potential of a test article or its metabolites to induce damage to chromosomes.

Cytogenic assay

What is the only macromolecule that can be repaird?

DNA

This detects the clastogenic potential of a test material

Definitive assay

Tis type of mutation removes on or more bases

Deletion

What does the in vivo micronucleus test detect?

Detects chromosome fragments or whole chromosomes excluded from the nuclei at cell division in erythrocytes.

What does the mouse lymphoma assay detect?

Detects mutations at the thymidine kinase locus of mouse lymphoma cells. It can also detect multi locus mutations, chromosome alterations and anueloidy

This type of repair mechanism where the damaged area or lesion is repaired directly by specialised proteins in our body

Direct reversal repair

What are the mechanisms of DNA repair

Direct reversal repair. Excision repair Mismatch repair

What is the primary thing that happens to usually cause cancer

Dna Damage

What are the two different types of microlesions

Error-free repairs can occur so youre good or it can be a problem in repair leading to a mutation

This type of mutation causes a deletion or insertion of a number of bases that cannot be divided by three

Frameshift

Imbalance between repair mechanisms and exposure to chemical could lead to what

Genetic instability and cause cancer, etc.

This assay detects mutations at the Hypoxanthine-guanine phosphoribosyl transferase locus of the chinese hamster ovary cells

HGPRT/CHO

In regards to mammalian cell forward mutation assays, what are the two types

HGRPT and mouse lymphoma

What are the general objectives of genetic toxicology

Identify materials that could induce mutations in DNA in humans 2) Provide reasoning and support to examine an unknown molecule 3) Provide support for regulatroy reasons 4) Avoid potential hazards and alert manufacturers.

When is a test article evaluated and verified as positive in ICH and OECD?

If it causes Dose-related increase in mean revertants with or without S9 over two increasing concentrations

When do negative results in ICH and OECD not get repeated.

If you can justify them they dont have to be repeated but if you can tjustify them it needs to be repeated

What is the difference between base excision repair and nucleotide excision repair in respect to damaged bases

In base excision repair, damaged bases are removed as free bases and in nucleotide excision repair, damaged bases are removed as oligonucleotides.

The purpose of this test is to identify substances that cause cytogenic damage which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes.

In vivo micronucleus test

This test Detects chromosome fragments or whole chromosomes excluded from the nuclei at cell division in erythrocytes

In vivo micronucleus test

What are the two assay methods of the ames test?

Incorportation and preincubation Incorporation is more traditional while preincubation is more sensitive.

An increase in the frequency of micronucleated polychromatic erythrocytes in animal is an indication of

Induced chromosome damage

This type of mutation inserts on or more bases

Insertion

What are transversions

Interchanges of purine for pyramidine bases, which involve exchange of one ring or two ring sructures.

What are Transitions

Interchanges of two ring purines or of one ring pyramideine.

This type of mutation causes an inversion of a sequence of bases

Inversion

What is a macrolesion

It is another type of lesion that occurs when a genotoxic agent affects DNA and it causes a chromosomal effect like rearrangement or deletion

Why is the HGPRT locus so important?

It results in the gene to produce the enzyme responsible for putting guanine into the DNA. So you want to see if the test particle is causing mutation in the gene producing the enzyme. If this hapens, then the CHO cells are not goign to be able to incorporate guanine in the DNA.

What is the way testing is allotted to the European union ?

Larger volume you use, the more tests you have to do

Waht are the cons to the abbreviated standard assay

Limited strains can be used

Genotoxic agent or chemicals when they react with DNA, they are able to induce two types of lesions, what are these lesions

Microlesions and macrolesions.

What is the difference between the micronucleus assay and the metaphase analysis in vivo assays based on what they use

Micronucleus uses rat or mouse bone marrow erythrocytes or mouse peripheral blood erythrocytes. Metaphase uses rat or mouse bone marrow or germ cells

This type of repair fixes mismatches

Mismatch repair

This is a type of point mutation that causes substitution of 1 amino acid for another

Missense

What is the most common types of mutations

Missense

When peripheral blood is used, which organism do you use, mouse or rat

Mouse

What is a substance that under the correct metabolic and cellular conditions has the ability to alter a cells DNA in a heritable manner.

Mutagen

How can you accelerate a deamination

Nitrites

What are the limitations of an in vivo study

Not appropriate if the compound is not systemically absorbed like aluminum based antacids

What are the cons of the ames test?

Not mammal system Can not detect epigenetic cawrcinogems Not 100 percent accurate

This type of repair fixes large structural changes and helix distortions

Nucleotide excision repair

In human disorder, which type of repair is usually absent

Nucleotide excision repair.

Aneupolidy is what kind of chromosomal aberration

Numerical

When would a SHE assay be issued

On a case by case basis

What are some endogenous agents

Oxidative damage by free radicals, replicative errors, alkylating agents, spontaneous alterations of DNA

This type of mutation changes a base pair

Point mutation

What are the five main types of mutations

Point mutations deletion mutation insertion frameshift inversion

What does the CHO/HGPRT assay detect

Point mutations and allele loss.

What are the pros of the ames test?

Predicts carcinogens to a high degree 80-85 percent . It is a short term assay Very cheap Versatile

What is the objectives of the modified ames test

Predicts resuts of full- GLP assay reduces sample amount requirement Accelerates turnover time Reduces cost

What are the pros to the abbreviated standard assay

Quantitative results and its fully comparable to a standard assay

This serves to select doses for the definitive portion of the study.

Range finding micronucleus assay

What is used for the metaphase analysis of in vivo cytogenetic assays?

Rat or mouse bone marroe or germ cells

What is used for the micronucleus in vivo mouse assays

Rat or mouse bone marrow red blood cells Mouse peripheral blood erythrocytes

In vivo assays usually use what animals

Rats or mice

: a change in chromosome structure detectable by microscopic examination of the metaphase stage of cell division, observed as deletions and fragments, intrachanges or interchanges.

Structural aberration of chromosomes

What are the advantages of the in vivo cytogenetic assays?

The test article goes through endogenous metabolic activation and it gets detoxified. You can mimic routes of human exposure and evaulate it, it is performed in somatic and germ cells and more relevant to human risk assessment. You also dont have to add S9 for metabolic activation.

How many test article dose levels are evaluated for structural and numerical aberrations in an in vitro chromosome aberration study

Three

What are tier 2 assays used for

To answer specific questions

For both the CHO/HGPRT and mouse lymphoma assays, what is the purpose

To evaluate the genotoxic potential of the test article bases on quantitation of forward mutation

T/F SHE assay has a high frequency of false positives

True

This is a short-term genotoxicity assay that allows identification of substances that induce DNA repair in liver cells of treated animals

UDS

What are two types of tier 2 assays used

UDS (unscheduled DNA synthesis) Assy Syrian Hamster Embryo (SHE) Assay

what are some exogenous agents

UV, Smoking, X-rays

In ICH and OECD guidelines, how do you clarify equivocal results?

Using modified condition

What is a numerical aberration

a change in the number of chromosomes from the normal number characteristic of the cells utilised

What does deamination do?

a cytosine will be changed to uracil and as a result you see a T translated in further replications and you dont want a T. You want a G.

What is a carcinogen

a substance that under the coreect metabolic and cellular conditions has the ability to transform a normal cell to a neoplastic cell?

What is the purpose of the ames test?

evaluate a chemical's genotoxicity by measuring its ability to induce reverse mutations at selected loci of several bacterial strains Youre starting off with mutated bacteria and adding test particle to bacteria to see if that chemical will result in a mutation that reverses the exisiting mutation.

What types of cells are used in UDS assay

hepatocytes

When wont the preincubution method of the ames assay not work?

if the test particle is too toxic

What is a direct reversal repair

mechanism of repair where the damaged area or lesion is repaired directly by specialised proteins in our body

This is a type of mutation that causes a single base change that generated a new stop codon causing truncation of protein synthesis.

nonsense

a change in the number of chromosomes from the normal number characteristic of the cells utilised

numerical aberration

What is a chromatid type aberration

structural chromosome damage expressed as breakage of single chromatids or breakage and reunion between chromatids.

What are the criteria for a valid test in ICH and OECD

tester strain genotypes are verified spontaneous revertant frequencies tester strain titers positive control values Must have 3 nontoxic levels Must demonstrate txicity or mutagenecity at top dose or have tested up to 5 mg.plate