Genotoxicity - toxicology slide 4

taught by Dr. Ramez Labib; 1.5hrs

what is the general objective of genetic toxicology?

• Identification of materials with the potential to induce genetic damages in humans

• provide support for the evaluation and interpretation of carcinogenicity and other toxicological results

• provide support for regulatory decisions

• alert producers and users of potential hazard

Mutagen

a substance that under the correct metabolic and cellular conditions has the ability to alter a cell’s genetic information (DNA) in a heritable manner

Carcinogen

a substance that under the correct metabolic and cellular conditions has the ability to transform a normal cell to a neoplastic cell (cancer)

how does a genotoxic agent effect DNA?

it can go down 2 paths

1. microlesions - mutation type effect

   1. error-free repair

      1. no effect

   2. Mis-repair or misreplication

      1. point mutation

2. macrolesions - change in structure of chromosome(s)

   chromosome rearrangement or deletion 

Outcome of Genetic Damage

• mutation in somatic cells

  • Cancer

  • Aging

• Mutation in Germinal Cells

  • Genetic Diseases

what does this diagram represent?

The Balance of DNA Damage and Repair

what are 3 major challenges to the integrity of the DNA molecule in vivo?

Lesions inflected by:

endogenous and environmental DNA-damaging agents and replication errors which have escaped the editing process during DNA synthesis 

Exogenous sources

• UV (sunlight)

• Pollution (hydrocarbons

• Smoking

• Foodstuffs

• Radiotherapy

  • Ionizing Radiation

  • X-rays

• Chemotherapy (Alkylating agents)

  • Cisplatin

  • Mitomycin C

  • Cyclophosphamide

  • Psoralen

  • Melphalan

Endogenous Sources

• Oxidative damage by free radicals

  • oxygen metabolism

• Replicative errors

• Spontaneous alternations in DNA

• Alkylating agents (Malondialdehyde)

What are the common types of DNA Damage and Spontaneous Alterations 

• Exogenous Sources

  • Radiotherapy

  • Chemotherapy

• Endogenous Sources

*look at pic for visual representations 

transition

the substitution of an amino acid to another of the same amino acid (Val - Val)

transversion

the substitution of an amino acid to a different amino acid (Val-Pro)

Types of mutations - description 

• Point mutation (missense, nonsense, silent) - Change of base pair

• Deletion mutation - removal of one or more bases

• Insertion mutation - insertion of one or more bases

• Frameshift mutation - deletion or insertion of a number of bases that cannot be divided by 3

• Inversion mutation - inversion of a sequence of bases (may cause frame shift)

what are these mutations caused by?

• Point mutation (missense, nonsense, silent) - Replication mistake, repair mistake, chemically altered base that mis-pairs 

• Deletion mutation - intercalating chemicals, DNA polymerase slips mobile genetic elements 

• Insertion mutation - intercalating chemicals, mobile genetic elements

• Frameshift mutation - intercalating chemicals, mobile genetic elements

• Inversion mutation - mobile genetic elements 

Consequences of Point Mutations and Small Deletions

• missense mutation

  • substitution of 1 amino acids for another. Most common type of mutation since most codons make an amino acid. usually (no always) relatively minor — often have no phenotype

• nonsense mutation

  • single base change that generates a new stop codon causing truncation of protein synthesis. Often nonfunctional since many amino acids can be affected

what is human cancer linked to and is there any hope for cancer?

• 80% of human cancer is linked to chemicals

• Primary event in cancer is DNA damage

The good news: DNA is the only macromolecule which can be repaired 

;..;.;

What is some insight/info on DNA Repair?

• All these others can be replaced, but DNA must be preserved

• Importance of maintaining the integrity of the DNA is Highlighted by the Investment of Energy in DNA Repair

• Less than 1/1000 Lesions become a mutation

• Diverse DNA repair systems reflect diversity of DNA damage

• Multiple systems recognize common lesions

• Most DNA repair mechanisms rely on the complementary information in the DNA

what the mechanisms of DNA Repair?

• Direct reversal repair: MGMT - O6-methylguanine DNA methyltransferase 

• Excision repair:

  • base excision repair (BER)

  • nucleotide excision repair(NER)

  • mismatch repair

what kind of repair is base excision repair and what does it do in basic terms?

Excision repair; fixes abnormal bases (uracil, hypoxanthine, alkylated bases)

what does (BER) stand for?

base excision repair

what kind of repair is nucleotide excision repair and what does it do in basic terms?

Excision repair; fixes large structural changes and helix distortions (pyrimidine dimers, bulky base adducts)

what does (NER) stand for?

nucleotide excision repair

what kind of repair is mismatch repair and what does it do in basic terms?

Excision repair; fixes mismatches

how does MGMT Direct Reversal Repair work? (google)

DNA repair pathway where the O6-methylguanine-DNA methyltransferase (MGMT) enzyme directly reverses damage by transferring an alkyl group, such as a methyl group, from the damaged O6 position of guanine to its own cysteine residue

*look at pic for visual representation of mechanism

what does Base Excision Repair do? (list more detail)

• damaged bases are removed by free bases

• primarily responsible for removal or oxidative and alkylation damage

*most genes in this pathway are essential for it to work

*thought to have a important role in aging

what does Nucleotide Excision Repair do? (list more detail)

• damaged bases are removed by oligonucleotides

• primarily responsible for removal of UV-induced damage and bulky adducts

• also removes ~ 20% of oxidative damage

*deficient in human disorders

what are Regulations for Mutagenicity Testing?

• ICH - International Council for Harmonization (followed by FDA) 

  • protocols that need to be followed to determine whether ur chemical(drug) is genotoxic

• US EPA(environmental protection agency) - responsible for registration of new chemicals or pesticides

• EU Dangerous Substance Legislation

• Japan regulations

• Redbook - guidelines for food regulations that need to be tested (followed by FDA)

ICH requires a S2B Genotoxicity Testing Battery, what does that entail?

a series of different tests that need to be run (going up in tiers) that lets u test to see if ur drug is carcinogenic

*all of these are required for submission of a new drug by the FDA

ICH S2B Genotoxicity Testing Battery tests (in order)

1. Ames test - testing for mutations in bacteria

   1. must be done on 2 different strains 

      1. Salmonella and E.coli

2. Mammalian cell evaluation for mutation or chromosomal damage

   1. In vitro cytogenetics (CHO, CHL, HPBL)

   2. In vitro mouse lymphoma (TK ± assay)

3. In vivo evaluation for chromosomal damage

   1. Micronucleus assay (mouse or rat)

   2. Rat bone marrow chromosome aberration assay

US EPA Mutagenicity Testing Requirements

a series of different tests that need to be run (going up in tiers) that lets u test to see if ur pesticide is carcinogenic

*all of these are required for submission of a new pesticide by the EPA

US EPA Mutagenicity Testing Requirements tests (in order)

• Ames test

• Mouse Lymphoma TK +/- gene mutation assay

  • OR, u can do the CHO/HGPRT gene mutation assay AND in vitro chromosomal aberration assay if u want to do a different cell line (this is in place of mouse lymphoma)

• Rodent micronucleus test 

EU New Chemical Mutagenicity Testing

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Japan Regulations on Mutagenicity Testing

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