1. Genetic ToxicologyDr R B Cope BVSc BSc(Hon 1) PhD cGLPCP DABT ERT

2. Quick Review of RelevantBasic Biology 3. Definitions Genotoxic: Non-genotoxic: Direct-acting mutagen: Indirect-acting mutagen: Pro-mutagen: Proximate mutagen: 4. Definitions Ultimate mutagen: Alkylating agent: Radiomimetic: Non-radiomimetic: DNA macrolesion DNA microlesion 5. Direct-acting mutagens By definition, these are highly DNA reactive agents thatdo not require prior metabolism for direct DNAinteractions; Generally very reactive chemicals, often with relativelyshort environmental half-lives; Tend to be highly reactive electrophiles; Typically act at the site of first contact; 6. Direct-acting mutagens Chemical stability, transport, membrane permeabilitygenerally determine the degree of mutagenicityassociated with these agents; Direct-acting mutagens are generally mutagenic atmultiple tissue sites and in essentially all species; 7. Direct-acting mutagens Classical examples: Nitrogen and sulfur mustards; Methyl methane sulfonate; Propane sulfone, Ethyleneimine Beta propiolactone Dimethylsulfate 8. Classical Structural Alerts for Reactive, Electrophilic Direct-Acting Mutagens Cl-CH2-O-CH2-Cl HaloethersEpoxides Strained lactonesSulfonatesEnals 9. Mechanisms of Chemical Interactionwith DNA: Electrophiles The majority of genotoxic chemicals thatdirectluy interact with DNA are eitherelectrophiles or are metabolized toelectrophiles; Electrophiles are positively charged speciesthat are attracted to an electron richcenter i.e. a nucleophile; 10. Mechanisms of Chemical Interactionwith DNA: Electrophiles An electrophile (literally electron-lover) is areagent attracted to electrons thatparticipates in a chemical reaction byaccepting an electron pair in order to bondto a nucleophile; Most electrophiles are positively charged,have an atom that carries a partial positivecharge, or have an atom that does nothave an octet of electrons; 11. Mechanisms of Chemical Interactionwith DNA: Electrophiles Specific areas of DNA behaves like a nucleophile; DNA nucleophilic centers: Ring nitrogens of the DNA bases are generally themost reactive centers; Ring oxygens of the DNA bases are also reactivecenters; S: groups in DNA are also targets Critically, the N7 of guanine (most reactive); the N3 ofadenine and the O6 of guanine are the most commonsites 12. Nucleophilic Centers of DNA Bases 13. Mechanisms of Chemical Interactionwith DNA: Electrophiles Electrophilic damage may also occur to thephosphodiester backbone of DNA: 14. Mechanisms of Chemical Interactionwith DNA: Electrophiles Different electrophiles display differentpreferences for the various DNA nucleophilicsites and different spectra of damage; One of the intensively researched concepts isthat the DNA damage spectrum for differentelectrophiles could be used as a marker ofenvironmental exposure: has not workedbecause of the variable ability of DNA to repairdifferent types of DNA damage plus the oftentissue-specific nature of mutation spectra. 15. Mechanisms of Chemical Interactionwith DNA: Oxidization Oxidation of DNA bases is a normalbackground biological event associated withaerobic metabolism and other cell reactions; Important endogenous oxidizing agents are:H2O2, superoxide anion, nitric oxide species, lipidperoxides, hydroxyl radicals, Fenton reactionproducts; 16. Mechanisms of Chemical Interactionwith DNA: Oxidization Endogenous oxidative damage to DNA occursat a rate of about 120 occurrences per cell perhour in NORMAL cells! In general, oxidative damage to DNA isrepaired with high fidelity at a maximal rate ofabout 105 base pairs per hour; Critically, there are a number of antioxidantpathways that limit the amount ofendogenously oxidative species that areproduced under normal circumstances; 17. Mechanisms of Chemical Interactionwith DNA: Oxidization Oxidative damage to DNA occurs most readilyat guanine residues due to the high oxidationpotential of this base relative to cytosine,thymine, and adenine; Most common oxidized DNA adduct is 8-hydroxyguanine followed by thymine glycol. 18. Mechanisms of Chemical Interactionwith DNA: Hydrolysis - deamination,depurination, and depyrimidination Deamination of cytosine: Spontaneous deamination is the hydrolysisreaction of cytosine into uracil, Corrected for by the removal of by uracil-DNAglycosylase, generating an abasic (AP) site; The AP site is then corrected by base excisionrepair; 19. Mechanisms of Chemical Interactionwith DNA: Hydrolysis -deamination, depurination, anddepyrimidination 5-methylcytosine: Spontaneous deamination of5-methylcytosine results in thymine; This is the most common single nucleotidemutation. In DNA, this reaction can be correctedby the enzyme thymine-DNA glycosylase; 20. Mechanisms of Chemical Interactionwith DNA: Hydrolysis - deamination,depurination, and depyrimidination Guanine: Deamination of guanine results in theformation of xanthine. Xanthine, in a manner analogous to the enoltautomer of guanine, selectively base pairs withthymine instead of cytosine. This results in a post-replicative transition mutation, where the originalG-C base pair transforms into an A-T base pair.Correction of this mutation involves the use ofalkyladenine glycosylase during base excisionrepair; 21. Mechanisms of Chemical Interactionwith DNA: Hydrolysis - deamination,depurination, and depyrimidination Adenine: Deamination of adenine results in theformation of hypoxanthine. Hypoxanthine, in a manner analogous to theimine tautomer of adenine, selectively base pairswith cytosine instead of thymine. This results in apost-replicative transition mutation, where theoriginal A-T base pair transforms into a G-C basepair; 22. Mechanisms of Chemical Interactionwith DNA: Hydrolysis -deamination, depurination, anddepyrimidination Depurination is a chemical reaction of purinedeoxyribonucleosides, deoxyadenosine anddeoxyguanosine, in which the -N-glycosidic bond ishydrolytically cleaved releasing a nucleic base,adenine or guanine, respectively. Deoxyribonucleosides and their derivatives aresubstantially more prone to depurination than theircorresponding ribonucleoside counterparts. Loss of pyrimidine bases (Cytosine and Thymine)occurs by a similar mechanism, but at asubstantially lower rate. 23. Mechanisms of Chemical Interactionwith DNA: Hydrolysis - deamination,depurination, and depyrimidination When depurination occurs with DNA, it leads to theformation of apurinic site and results in an alterationof the structure; As many as 5,000 purines are lost this way each dayin a typical human cell; [In cells, one of the main causes of depurination isthe presence of endogenous metabolitesundergoing chemical reactions; 24. Mechanisms of Chemical Interactionwith DNA: Hydrolysis - deamination,depurination, and depyrimidination Apurinic sites in double-stranded DNA are efficientlyrepaired by portions of the base excision repair(BER) pathway; Depurinated bases in single-stranded DNAundergoing replication can lead to mutations,because in the absence of information from thecomplementary strand, BER can add an incorrectbase at the apurinic site, resulting in either atransition or transversion mutation; Depurination is known to play a major role in cancerinitiation. 25. Mechanisms of Chemical Interactionwith DNA: Intercalation Intercalation occurs when ligands of anappropriate size and chemical nature fitthemselves in between base pairs of DNA; Intercalating agents are generallypolycyclic, aromatic, and planar, and goodDNA stains; Important examples: Ethidium bromide (DNA stain); Anticancer agents:proflavine, daunorubicin, doxorubicin, dactinomycin, thalidomide. 26. Molecular structure of ethidium intercalated between two pairs of adenine-uracil base pairs. 27. Mechanisms of Chemical Interactionwith DNA: Intercalation In order for an intercalator to fit between base pairs,the DNA must dynamically open a space betweenits base pairs by unwinding; The amount of unwinding depends on the specificagent; This unwinding induces local structural changes tothe DNA strand resulting in functional changes:inhibition of transcription and replication and DNArepair processes, Intercalating agents are commonly potentmutagen. 28. Mechanisms of Chemical Interactionwith DNA: Intercalating agents Important examples: 8,9 epoxide of aflatoxin B1; Acridine dyes. 29. Mechanisms of Chemical Interactionwith DNA: DNA cross linking Crosslinks occur when exogenous or endogenousagents react with two different positions in the DNA; Crosslinks can occur in the same DNA strand(intrastrand crosslink) or between opposite strands(interstrand crosslink); Crosslinks between DNA and protein can occur; 30. Mechanisms of Chemical Interactionwith DNA: DNA cross linking Crosslinks impair DNA replication if the crosslink is notrepaired; Mechanisms: Bifunctional alkylating agents (e.g. methylenedimethanesulphonate, sulphur mustard, methylmethanesulphonate): mostly act on adjacent N7-guanine bases; Cisplatin: 1,2-intrastrand d(GpG) adducts (via N7-guanine bases); 31. Mechanisms of Chemical Interactionwith DNA: DNA cross linking Mechanisms: Nitrous acid is formed in the stomach fromdietary nitrites (meat prreservatives): formsinterstrand DNA crosslinks at the aminogroupof N2 of guanine at CG sequences; Malondialdehyde from lipid peroxidation:forms etheno adduct-derived interstrandcrosslinks; 32. Mechanisms of Chemical Interactionwith DNA: DNA cross linking Mechanisms: Psoralens: photoactivated by UVA formcovalent adducts with thymine, one type ofwhich is an intrastrand crosslinking reactiontargets TA sequences intercalating in DNAand linking one base of the DNA with the onebelow it. Psoralen adducts cause replicationarrest and is used in the treatment of psoriasisand vitiligo; 33. Mechanisms of Chemical Interactionwith DNA: DNA cross linking Mechanisms: Aldehydes such as acrolein andcrotonaldehyde found in tobacco smoke orautomotive exhaust can form DNAinterstrand crosslinks in DNA. Formaldehyde (HCHO) induces protein-DNAand protein-protein crosslinks; 34. Mechanisms of Chemical Interactionwith DNA: DNA single strand breaks Single strand breaks are an extremely commonphenomenon thousands of incidents per cellper day; Mechanisms of production: Free radical attack (notably with radiation-induced DNA damage); DNA alykylation (i.e. electrophylic attack); Many of the mechanisms are poorlyunderstood; 35. Mechanisms of Chemical Interactionwith DNA: DNA double strand breaks Particularly hazardous to the cell because they canlead to genome rearrangements: regarded as themost dangerous of DNA lesions; Mechanisms are poorly understood, howeveroxidization and alkylating agents are able toproduce these DNA lesions; DSBs are induced by a number of differentmechanisms, including exposure to ionizingradiation, radiomimetic drugs, collapse ofreplication forks when the replication machineryencounters single-stranded breaks (SSBs) in thetemplate DNA, 36. Micro-DNA Lesions: Small damage with BIG outcomes. 37. The Ability to Chemically InteractWith DNA is Not Enough For a mutation to occur: Exposure of DNA generally must occur at the righttime in the cell cycle; A DNA lesion must be chemically produced; The DNA lesion must not be so gross as to preventDNA replication and/or produce cell death; 38. The Ability to Chemically InteractWith DNA is Not Enough For a mutation to occur: The lesion must persist in the DNA long enough for atleast 1 cell division (i.e. the fixing of the mutationwithin the cell genome; often referred to as theexpression time in genetox assays); The DNA lesion must not trigger the G1/S checkpoint(i.e. apoptosis or senescence; more on this in thecarcinogenesis sildes); The DNA lesion must not trigger the intra-S phasecheckpoint (if it does, repair is the likely outcome); 39. The Cell Cycle and Mutation:The vulnerability of the S phase. Small mutations are most likely to occur whenthe DNA is being copied i.e. S phase; Reason for this is that the DNA double helix isunwound and the 2 strands are separated single DNA strands are particularly vulnerable tochemical attack; 40. (A) Nucleoside triphosphates serve as a substrate for DNA polymerase, according to themechanism shown on the top strand. Each nucleoside triphosphate is made up of threephosphates (represented here by yellow spheres), a deoxyribose sugar (beige rectangle)and one of four bases (differently colored cylinders). The three phosphates are joined toeach other by high-energy bonds, and the cleavage of these bonds during thepolymerization reaction releases the free energy needed to drive the incorporation ofeach nucleotide into the growing DNA chain. The reaction shown on the bottom strand,which would cause DNA chain growth in the 3 to 5 chemical direction, does not occur innature. (B) DNA polymerases catalyse chain growth only in the 5 to 3 chemical direction,but both new daughter strands grow at the fork. The leading strand grows continuously,whereas the lagging strand is synthesized by a DNA polymerase through the backstitchingmechanism illustrated. Thus, both strands are produced by DNA synthesis in the 5 to 3direction. 41. Proteins at the Y-shaped DNA replication fork: These proteins are illustrated schematically in panel a of the figure below, but in reality, thefork is folded in three dimensions, producing a structure resembling that of the diagram in the inset b. Focusing on the schematic illustrationin a, two DNA polymerase molecules are active at the fork at any one time. One moves continuously to produce the new daughter DNAmolecule on the leading strand, whereas the other produces a long series of short Okazaki DNA fragments on the lagging strand. Bothpolymerases are anchored to their template by polymerase accessory proteins, in the form of a sliding clamp and a clamp loader. A DNAhelicase, powered by ATP hydrolysis, propels itself rapidly along one of the template DNA strands (here the lagging strand), forcing openthe DNA helix ahead of the replication fork. The helicase exposes the bases of the DNA helix for the leading-strand polymerase to copy.DNA topoisomerase enzymes facilitate DNA helix unwinding. In addition to the template, DNA polymerases need a pre-existing DNA orRNA chain end (a primer) onto which to add each nucleotide. For this reason, the lagging strand polymerase requires the action of a DNAprimase enzyme before it can start each Okazaki fragment. The primase produces a very short RNA molecule (an RNA primer) at the 58end of each Okazaki fragment onto which the DNA polymerase adds nucleotides. Finally, the single-stranded regions of DNA at the forkare covered by multiple copies of a single-strand DNA-binding protein, which hold the DNA template strands open with their basesexposed. In the folded fork structure shown in the inset, the lagging-strand DNA polymerase remains tied to the leading-strand DNApolymerase. This allows the lagging-strand polymerase to remain at the fork after it finishes the synthesis of each Okazaki fragment. As aresult, this polymerase can be used over and over again to synthesize the large number of Okazaki fragments that are needed to producea new DNA chain on the lagging strand. In addition to the above group of core proteins, other proteins (not shown) are needed for DNAreplication. These include a set of initiator proteins to begin each new replication fork at a replication origin, an RNAseH enzyme to removethe RNA primers from the Okazaki fragments, and a DNA ligase to seal the adjacent Okazaki fragments together to form a continuous DNAstrand. 42. The Cell Cycle and Mutation:The S phase checkpoint. The S-phase checkpoint is a surveillancemechanism, mediated by the protein kinases ATRand Chk2 in human cells; Responds to to DNA damage by co-ordinating aglobal cellular response necessary to maintaingenome integrity; A key aspect of this response is the stabilization ofDNA replication forks, which is critical for cellsurvival; A defective checkpoint causes irreversiblereplication-fork collapse and leads to genomicinstability, a hallmark of cancer cells. 43. If DNA replication is blocked (e.g. by a DNA adduct) ssDNA regions at stalledforks continue to grow because MCM (minichromosome maintenance complex)helicase continues DNA unwinding, although uncoupled from DNA synthesis.The ssDNA binds RPA (replication protein A), which triggers the activation of thecheckpoint response. This process is initiated by the recruitment of the Mec1/ATRsensor to RPA-coated ssDNA at stalled forks by its regulatory subunit, Ddc2 (ATRIPin human cells). Mec1 then phosphorylates Mrc1 (the homologue of humanClaspin), a mediator that transduces the signal from Mec1 to the effector kinaseRad53, which becomes phosphorylated and activated. 44. The Cell Cycle and Mutation:The S phase checkpoints. The S-phase checkpoint response co-ordinates DNAreplication, DNA repair and cell-cycle progression andregulates processes such as firing of replication origins,stabilization of DNA replication forks in response to DNAdamage or replicative stress, resumption of stalled DNAreplication forks, transcriptional induction of DNAdamage response genes, choice of the repair pathwayand inhibition of mitosis until replication is completed; The S-phase checkpoint is required for cellular viability inDNA damage or replicative-stress conditions 45. Types of Mutations: Point mutations. Point mutation = single base substitution = thereplacement of a single base nucleotide with anothernucleotide of the genetic material (either DNA or RNA); Point mutations most commonly occur during S phase(i.e. DNA replication); The term also includes insertions or deletions of a singlebase pair which will result in a frame shift mutation; Classifications: By type: deletion, transition, insertion, or transversion; By the effect on function: nonsense, missense and silent; 46. Types of Mutations: Point mutations. Transversions (beta mutations): The substitution of a purine for a pyrimidine or vice versa; Can only be repaired by a spontaneous reversion; Transitions produce large chemical changes to DNAstructure; thus the consequences of this change tend to bemore drastic than those of transitions. Transversions are classically caused by ionizing radiation andalkylating agents. 47. Types of Mutations: Point mutations. Transitions (alpha mutations): A point mutation that changes a purine nucleotide to anotherpurine (A G) or a pyrimidine nucleotide to another pyrimidine (C T); Approximately two out of three single nucleotide polymorphisms(SNPs) are transitions; Transitions can be caused by oxidative deamination andtautomerization; Although there are twice as many possible transversions, transitionsappear more often in genomes, possibly due to the molecularmechanisms that generate them; 5-Methylcytosine is more prone to transition than unmethylatedcytosine, due to spontaneous deamination. 48. Types of Mutations: Point mutations. Nonsense mutations are point mutations in a sequence of DNAthat results in a premature stop codon, or a nonsense codon inthe transcribed mRNA, and in a truncated, incomplete, andusually nonfunctional protein product; 49. Types of Mutations: Point mutations. Missense mutations are point mutations in which a singlenucleotide is changed, resulting in a codon that codes for adifferent amino acid i.e. a non-synonymous change (mutationsthat change an amino acid to a stop codon are considerednonsense mutations, rather than missense mutations). There are2 possible outcomes: Conservative mutations: Result in an amino acid change.However, the properties of the amino acid remain the same(e.g., hydrophobic, hydrophilic, etc). At times, a change to oneamino acid in the protein is not detrimental to the organism as awhole. Most proteins can withstand one or two point mutationsbefore their functioning changes; Non-conservative mutations: Result in an amino acid change thathas different properties than the wild type. The protein may lose itsfunction, which can result in a disease in the organism. 50. Types of Mutations:Frame shift mutations. Silent mutations: Code for the same amino acid. A silent mutation has no effect on the functioning of the protein; A single nucleotide can change, but the new codon specifiesthe same amino acid, resulting in an non-mutated protein; This type of change is also called synonymous change, since theold and new codon code for the same amino acid; This is possible because 64 codons specify only 20 amino acids; Different codons can lead to differential protein expressionlevels. 51. Types of Mutations: Point mutations. A frame shift mutation (also called a framing error or a readingframe shift) is a genetic mutation caused by indels (insertions ordeletions) of a number of nucleotides that is not evenly divisible bythree from a DNA sequence; Remember that it takes 3 DNA and RNA nucleotides to code for aspecific amino acid in a protein i.e. the reading frames consist of3 nucleic acid residues; Thus insertion or deletion of a DNA nucleotide can change thereading frame (i.e. the grouping 3s of the codons), resulting in acompletely different translation from the original;The earlier in thesequence the deletion or insertion occurs, the more altered theprotein produced is. 52. Types of Mutations: Point mutations. Frame shift mutations; The earlier in the sequence the deletion or insertionoccurs, the greater the effect on protein structure. This isbecause all of the reading frames after the insertion ordeletion will be altered i.e. earlier in the relevant DNA codingsequence the error is, the bigger the overall change to thedownstream amino acid composition of the protein; 53. Types of Mutations: Point mutations. Frame shift mutations; Frameshift mutations will also alter the first stop codon ("UAA","UGA" or "UAG") encountered in the sequence. Thepolypeptide being created could be abnormally short orabnormally long, and will most likely not be functional; Frameshift mutations frequently result in severe geneticdiseases 54. DNA Repair Mechanisms. High level overview only; The self-repair of DNA is unique amongst biological molecules; Major types of mammalian DNA repair: Base excision repair; Nucleotide excision repair; Mismatch repair; Recombinational repair. 55. DNA Repair Mechanisms. DNA repair capacity varies by mechanism, tissue, organ,individual and species; Not all DNA adduct types are equal: some are more easilyrepaired than others and some types cannot be repaired; Because of the above 2 points, the use of the number of DNAadducts per cell is NOT a reliable predictor of genetic hazardUNLESS there is very detailed information regarding the kineticsof DNA adduct removal in the specific target tissue and targetspecies! 56. DNA Repair Mechanisms. In general terms, DNA repair are saturatable mechanisms i.e.there is maximal threshold for the amount of DNA that can berepaired within a given set of parameters; All DNA repair mechanisms have the ability to detect and repairDNA, and if the DNA repair is successful, the impact of theoriginal DNA damage on the animal is reduced or eliminated; 57. DNA Repair Mechanisms: In general terms, when low levels of DNA damage occur (i.e.below saturation for repair), error-free (high fidelity) repairoccurs. When there are large amounts of DNA damage (i.e.above the saturation for repair), error prone DNA repairpredominates; However: there are big species, sex, age, tissue, and organdifferences; The type of DNA adduct has a significant influence overwhich type of repair predominates; 58. DNA Repair Mechanisms: Intrinsic variability within human populations There is a very large variability to repair DNA damage between individuals: up to 65% of average rate in populations without inherited DNA repair defects (e.g. XP); People with XP have DNA repair capacity of ~1-2% of normal; There are differences in DNA repair capacity between rodents and humans: you cannot directly extrapolate results unless you accurate kinetics across the relevant species; 59. DNA Repair Mechanisms: Mechanisms where only one strand is damaged When only one of the two strands of a double helixhas a defect (i.e. only one of the 2 strands has adamaged/missing nucleotide), the other strand canbe used as a template to guide the correction of thedamaged strand; These types of DNA repair are called excision repair:remove the damaged nucleotide and replace it withan undamaged nucleotide complementary to thatfound in the undamaged DNA strand 60. DNA Repair Mechanisms: Base excision repair Base excision repair: Repairs damage to a single base caused by oxidation,alkylation, hydrolysis, or deamination; The damaged base is removed by a DNA glycosylase; The DNA base is then recognized by an enzyme calledAP endonuclease, which cuts the phosphodiesterbond; The missing part is then resynthesized by a DNApolymerase, and a DNA ligase performs the final nick-sealing step; 61. Base Excision Repair 62. BER: Short Patch 63. BER: Long Patch 64. DNA Repair Mechanisms: Base excision repair Base excision repair: BER is the major repair pathway involved in the removalof non-bulky damaged nucleotides; In general BER is a high fidelity process i.e. not errorprone; BER protects both nuclear and mitochondrial DNA; Heritable defects in BER (particularly DNA polymerase)are associated with cancer. 65. DNA Repair Mechanisms:Nucleotide excision repair Nucleotide excision repair (NER), which recognizesbulky, helix-distorting lesions; A specialized form of NER known as transcription-coupled repair deploys NER enzymes to genes thatare being actively transcribed; 66. 1) 3 protein complexesare involved in DNA-damage recognition:XPA, XPC-HR23 and RPA.2) These proteins recruitsTranscription factor II(TFIIH) that incorporatetwo helicases: XPB andXPD that unwinds a 30 bpDNA fragment aroundthe DNA damage.3) After DNA unwinding,damaged-DNA strand isexcised by XPG and theXPF-ERCC1 complex at 3and 5 sites respectively.4) After excision,damaged-DNA strand isremoved and replacedby re-synthesizing thetemplate complementaryDNA strand bypolymerase complex (Pol.E/D, replication protein A(RPA) and replicationfactor C). 67. DNA Repair Mechanisms:Nucleotide excision repair Mismatch repair (MMR), which corrects errors of DNAreplication and recombination that result inmispaired (but undamaged) nucleotides; MMR functions primarily as a proof reader followingDNA replication 68. Mismatch Repair 69. DNA Repair Mechanisms:Nucleotide excision repair Mismatch repair (MMR), which corrects errors of DNAreplication and recombination that result inmispaired (but undamaged) nucleotides; MMR functions primarily as a proof reader followingDNA replication 70. Macro DNA Damage: Macro DNA damage = damage to chromosomes =clastenogenesis e.g. single strand breaks, doublestrand breaks, sister chromatid exchange, non-homologous end joining, changes in ploidy; Abnormal chromosome number = aneuploidy; Increased chromosome number = polyploidy; 71. Macro DNA Damage: Sister chromatid exchange Exchange of genetic material between two identicalsister chromatids or between chromosomes with identicalmutations; Primarily occurs during S phase; Four to five sister chromatid exchange/chromosomepair/mitosis is within the normal range, 14-100 exchangesis not normal and presents a danger to the organis; Mediated by the homologous end-joining mechanism foDNA; 72. Macro DNA Damage: Sister chromatid exchange (equal cross over) SCEs can be induced by various genotoxic treatmentsthat result in double DNA strand breaks, suggesting thatSCEs reflect a DNA repair process i.e. they are measure ofDNA damage; This process is considered to be conservative and error-free, since no information is generally altered duringreciprocal interchange by homologous recombination. Most forms of DNA damage induce chromatid exchangeupon replication fork collapse: Holliday model; Occurs during prophase I of meiosis (pachytene) in aprocess called synapsis. 73. Macro DNA Damage:Other potential outcomes ofDNA double strand breaks SCE (equal cross over) is the least harmful outcomeof a DSB; Other potentially catestrophic outcomes include dueto misrepair of DSBs include: Inversions; Interstitial deletions; Terminal deletions; Translocations; Unequal crossovers. 74. Macro DNA Damage: Other potential outcomes of DNA double strand breaks Inversions: Inversions thatinvolve thecentromere arecalled pericentricinversions; Those that do notinvolve thecentromere arecalled paracentricinversions; Inversions potentiallyhave massive effectson gene function. 75. Macro DNA Damage:Other potential outcomes ofDNA double strand breaks Interstitial deletions: Causes include thefollowing: Lossesfrom translocation;chromosomalcrossovers within achromosomalinversion; unequalcrossing over;breaking withoutrejoining 76. Macro DNA Damage:Other potential outcomes ofDNA double strand breaks 77. Macro DNA Damage: Other potential outcomes of DNA double strand breaks Translocation = a chromosome abnormality caused by rearrangement of parts between nonhomologous chromosomes; Gene fusion may be created when the translocation joinstwo otherwise separated genes: very important incarcinogenesis as it may place the coding region of arelatively inactive gene with normally very active genepromoter region inappropriate upregulation of a gene(alternatively gene silencing may occur); 78. Macro DNA Damage:Other potential outcomes ofDNA double strand breaks Translocation: There are two main types, reciprocal (also known as non- Robertsonian) and non-reciprocal (Robertsonian); Also, translocations can be balanced (in an even exchangeof material with no genetic information extra or missing, andideally full functionality) or unbalanced (where the exchangeof chromosome material is unequal resulting in extra ormissing genes). 79. Macro DNA Damage: Other potential outcomes ofDNA double strand breaks Reciprocal translocations: Reciprocal translocations are usually an exchange ofmaterial between nonhomologous chromosomes; Estimates of incidence range from about 1 in 500 humannewborns; Such translocations are usually harmless and may be foundthrough prenatal diagnosis; However, carriers of balanced reciprocal translocations haveincreased risks of creating gametes with unbalancedchromosome translocations leading to miscarriages orchildren with abnormalities. Most balanced translocation carriers are healthy and do nothave any symptoms. 80. Macro DNA Damage: Other potentialoutcomes of DNA double strand breaks Nonreciprocal (Robertsonian) translocations: This type of rearrangement involves two acrocentricchromosomes that fuse near the centromere region with lossof the short arms The resulting karyotype in humans leaves only 45chromosomes since two chromosomes have fused together 81. Macro DNA Damage: Other potentialoutcomes of DNA double strand breaks Nonreciprocal (Robertsonian) translocations: This has no direct effect on the phenotype since the onlygenes on the short arms of acrocentrics are common to all ofthem and are present in variable copy number (nucleolarorganiser genes). Robertsonian translocations have beenseen involving all combinations of acrocentric chromosomes.The most common translocation in humans involveschromosomes 13 and 14 and is seen in about 0.97 / 1000newborns. Carriers of Robertsonian translocations are not associatedwith any phenotypic abnormalities, but there is a risk ofunbalanced gametes which lead to miscarriages orabnormal offspring. 82. Macro DNA Damage: Other potential outcomes of DNA double strand breaks Balanced translocations: no genetic material is lost 83. Macro DNA Damage: Other potential outcomes of DNA double strand breaks Unbalanced translocation: genetic material is lost from onechromosome but gained by another. This means that theprogeny will either have missing genetic material or extragenetic material 84. Macro DNA Damage: Other potential outcomes of DNA double strand breaks Acentric fragments: a segment of a chromosomethat lacks a centromere; 85. Macro DNA Damage:Other potential outcomes ofDNA double strand breaks Acentric fragments: Because acentric fragments lack a centromere, the cannot attachto the mitotic spindle i.e. acentric fragments are not evenlydistributed to the daughter cells in cell division (mitosis and meiosis).As a result one of the daughters will lack the acentric fragment; Lack of the acentric fragment in one of the daughter cellsmay have deleterious consequences, depending on thefunction of the DNA in this region of the chromosome; In the case of a gamete, it will be fatal if essential DNA iscontained in that DNA segment; In the case of a diploid cell, the daughter cell lacking theacetric fragment will show expression of any recessive genesfound in the homologous chromosome. 86. Acentric fragments are lost from the nuclei of cells followingmitosis or meiosis. They form a micronucleus. 87. Macro DNA Damage: Changes in chromosome number Changes to chromosome number can result from: Nonreciprocal (Robertsonian) translocations; Errors in chromosomal segregation i.e. a wholechromosome is left behind during anaphase ofmitosis or meiosis; Damage to the mitotic spindle: e.g. griseofulvin,pcalitaxel, colecemid, vinblastine; 88. Macro DNA Damage: Changes inchromosome number Kinetocore: The protein structure on chromatids where the spindle fibers attach during cell division to pull sister chromatids apart; The kinetochore forms in eukaryotes, assembles on the centromere and links the chromosome to microtubule polymers from the mitotic spindle during mitosis and meiosis; Kinetochores start, control and supervise the striking movements of chromosomes during cell division; 89. Macro DNA Damage: Changes inchromosome number Kinetocore: The protein structure on chromatids where the spindle fibers attach during cell division to pull sister chromatids apart; The kinetochore forms in eukaryotes, assembles on the centromere and links the chromosome to microtubule polymers from the mitotic spindle during mitosis and meiosis; Kinetochores start, control and supervise the striking movements of chromosomes during cell division; 90. Macro DNA Damage: Changes in chromosome number Kinetocore: Kinetochores are critical in initiating/avoiding thespindle checkpoint 91. Macro DNA Damage: Changes inchromosome number The spindle checkpoint: The spindle checkpoint (= spindle assembly checkpoint = mitoticcheckpoint), is a cellular mechanism responsible for detection of: Correct assembly of the mitotic spindle Attachment of all chromosomes to the mitotic spindle in a bipolar manner Congression of all chromosomes at the metaphase plate. When just one chromosome (for any reason) remains lagging duringcongression, the spindle checkpoint machinery generates a delayin cell cycle progression: the cell is arrested, allowing time for repairmechanisms to solve the detected problem. After some time, if theproblem has not been solved, the cell will be targeted for apoptosis(programmed cell death), a safety mechanism to avoid thegeneration of aneuploidy, a situation which generally has dramaticconsequences for the organism. 92. Macro DNA Damage: Changes in chromosome number Micronuclei can also contain whole chromosomes thatwere improperly attached to the mitotic spindle duringanaphase: these are detectable by staining themicronuclei for kinetocores; There are at least major causes of micronuclei: The formation of chromosome fragments that lack acentromere (and thus a kinetocore cannot attach o themitotic spindle); The loss of a whole chromosome which has failed to attachto the mitotic spindle or has broken off the mitotic spindle.This effect is produced by spindle agents; Extrachromosomal double minutes. 93. Macro DNA Damage: Changes inchromosome number So what the heck is a double minute you ask (no it is not 120seconds): Double minutes are small fragments of extrachromosomal DNA,which have been observed in a large number of human tumors; They are a manifestation of gene amplification during thedevelopment of tumors, which give the cells selective advantagesfor growth and survival; They frequently harbor amplified oncogenes and genes involved indrug resistance; Double minutes, like actual chromosomes, are composed ofchromatin and replicate in the nucleus of the cell during celldivision; Unlike typical chromosomes, they are composed of circularfragments of DNA, up to only a few million base pairs in size andcontain no centromere or telomere. 94. Micronucleusfluorescently labeledfor kinetocores 95. Mechanisms of micronuclei formation. (A)Aneugenic agents prevent the formation ofthe spindle apparatus during mitosis. The use ofthese agents generates micronuclei as aconsequence of whole chromosomes laggingbehind at anaphase. These chromosomes areleft out of the cell nucleus at the end of mitosis;The DNA in the micronuclei could balance thenuclear DNA and result in a completegenome, or be additional to the cellsgenome. (B) Clastogenic agents induce micronuclei bybreaking the double helix of DNA, therebyforming acentric fragments. These fragmentsare incapable of adhering to the spindle fibresand integrating in the daughter nuclei, andare thus left behind during mitosis.(C) Micronuclei can also contain highlyamplified gene sequences, derived fromextrachromosomal double minutes (DM)(yellow dots indicate the presence of a DM).(D(I)) Torsion between the two centromeres ofa dicentric chromosome would give rise to theformation of an anaphase bridge that isfrequently resolved by breakage. The bridgebreakage often results in the formation ofacentric fragments that are not included inany of the daughter cell nuclei and form oneor more micronuclei at the end of mitosis. (D(II)) It has also been described that, instead ofbreaking, dicentric chromosomes involved inanaphase bridges are sometimes detachedfrom the two centrosomes, left behind atanaphase and sequestered into micronuclei. 96. US EPA Testing Requirements & TiersTier Test Types Assessment FunctionRapid, low cost screening, Ames (bacterial reverse mutation)typically for agents where In vitro mammalian cell mutation (e.g.human exposure is low. Hazard Identification 1 mouse lymphoma TK)If all results are negative, In vitro chromosome aberration orgenerally no further micronucleus assaytesting is required In vivo testing: at least one or more of: in Required if there is vivo micronucleus, comet assay, in vivosignificant human 2 DNA binding, in vivo unscheduled DNA exposure or +ve results in synthesis, transgenic mouse models Tier 1Required if +ve results in In vivo tests in germ cells (i.e. dominantTier 2. Provides a basis for 3 lethal, germ cell micronucleus, germ cellhazard assessment of DNA binding, germ cell USD,germ line effectsProvides a quantitative Quantitative in vivo tests for germ cellassessment of germ cell mutation (specific locus test, visible or 4 biochemical markers [mouse spot],mutations for use inquantitative risk heritable translocation test in mice)assessments 97. Germ Cell Versus Somatic Mutation:Female germ cell The timing of exposure is critically important: Mutation is most likely to occur during the S phase(during DNA replication) i.e. mitosis and meiosis; 98. Germ Cell Versus Somatic Mutation: Female germ cell The timing of exposure is critically important: In humans (and most mammals), this means: During mitosis of oogonium during fetal development (between weeks 4 and 30 in humans; between days 14.5 and 18.5 in the rat and between days 10.5-12.5 in mice); There are no further S phases until after fertilization and the formation of a zygote; Remember: DNA repair occurs in oocytes! 99. Germ Cell Versus Somatic Mutation: Female germ cell 100. Germ Cell Versus Somatic Mutation: Female germ cell Consequences in terms of germ cell mutation: The critical timing for female germ cell mutation inmammals occurs at the prenatal stage ofdevelopment! Ooctyes are RESISTANT to mutation by non-radiomimetic chemicals (i.e. chemicals that do notproduce chromosome or chromatid breaks); Oocytes are susceptible to radiation andradiomimetic chemicals; 101. Germ Cell Versus Somatic Mutation: Male germ cells Same basic principle holds true: S phase of celldivision processes is most susceptible tomutagenesis; S phase in spermatogenesis occurs duringspermatogonial stem cell stage of development; Unlike in females, spermatogonial stem cellmitosis occurs throughout the life time of males! Late spermatids and spermatozoa lack DNArepair also susceptible to unrepaired DNAdamage! 102. Germ Cell Versus Somatic Mutation Overall, males are generally regarded as beingat greater risk of generating germ cell mutationsthan females because of the continuous, life-time replication of spermatogonial stem cells(where as oogonium are only generated in largenumbers during prenatal development infemales). 103. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Fundamental principles: These assays are for micro-DNA damage; Mutant bacterial test strains are created/selected (typicallyloose the capacity for synthesis of an amino acid) so theyrequire some form of additional supplementation (usually anamino acid) in order to grow these are called auxotrophs(auxotrophy is the inability of an organism to synthesize aparticular organic compound required for its growth); 104. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Fundamental principles: The mutation required to produce the auxotrophic strain isgenerally either a point mutation or a frame shift mutation i.e.a small DNA sequence change. 105. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Fundamental principles: In order for an auxotroph to grow in medium where thesupplement is NOT present, they must undergo a reversemutation in order to regain the capacity to synthesize theessential substance for growth; Once the bacteria have undergone a reverse mutaiton, theyare able to grow in media that DO NOT contain thesupplement (typically an amino acid; These reverse mutations are typically point mutations orframe shifts. 106. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Fundamental principles: Many test strains have features that make them moresensitive for the detection of mutations: Specific responsive DNA sequences at the reversion sites; Increased cell permeability to large molecules Lack of DNA repair or enhancement of error-prone DNA repair; 107. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Advantages: Fast; Cheap; Quick screening for micro-DNA damage; Extensive database/library of the effects of a very diversearray of chemicals; Generally has acceptable false positive/false negative levels(i.e. good sensitivity, specificity, precision and predictivevalue) Although many compounds that are positive in this test aremammalian carcinogens, the correlation is not absolute. It isdependent on chemical class and there are carcinogensthat are notdetected by this test because they act throughother, non-genotoxic mechanisms or mechanismsabsent inbacterial cells. 108. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Although many compounds that are positive in this test aremammalian carcinogens, the correlation is not absolute. It isdependent on chemical class and there are carcinogens thatare not detected by this test because they act through other,non-genotoxic mechanisms or mechanismsabsent in bacterialcells. 109. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Disadvantages: Utilizes prokaryotic cells, which differ from mammalian cells in such factors as uptake, metabolism, chromosome structure and DNA repair processes; Tests conducted in vitro generally require the use of an exogenous source of metabolic activation. In vitro metabolic activation systems cannot mimic entirely the mammalian in vivo conditions; The test does not provide direct information on themutagenic and carcinogenic potency of a substance inmammals. 110. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Limitations: Difficult to use with substances that are potent bacteriocidesor bacteriostats (i.e. substances that block mitosis); Culture media are hydrophilic i.e. requires specializedtechniques for highly lipophilic substances (e.g. petroleumdistillates); Special techniques are required for gases, vapors orsubstances that evaporate at 37OC; 111. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Metabolic activation systems: The objective is to replicate at least some of the majorbiotransformation pathways in vitro; Classical system is liver S9 fraction: Rats are dosed with Arochlor 1254 (a PCB mixture that acts as a potent inducer of liver CYP and UGT enzymes via the AhR pathway) livers are homogenized centrifuged at 9000 g for 20 minutes supernatant is collected; S9 contains cytosol and microsomes (= smooth endoplasmic reticulum): microsomes component contains cytochrome P450 isoforms (phase I metabolism);cytosolic portion contains the major part of the activities of transferases (phase II metabolism) 112. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Metabolic activation systems: Classical system is liver S9 fraction: A NADPH-regenerating system or NADPH solution is required to supply the energy demand of the CYP enzymes (powers the CYP cycle); For the catalytic activity of phase II enzymes, addition of exogenous cofactors is necessary: UDPGA and alamethicin for UGT; acetyl CoA, DTT, and acetyl CoA regenerating g system for NAT; PAPS for ST; and GT for GST; 113. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Bacteria and strains required for OECD 471: Salmonella typhimurium TA1535 rfa+ uvrB+ hisG46: S. typhimurium TA1537 rfa+ uvrB+ hisC3076; S. typhimurium TA98 rfa+ uvrB+ hisD3052 pKM101; S. typhimurium TA100 rfa+ uvrB+ hisG46 pKM101; Escherichia coli WP2 trp+ uvrA; In order to detect cross-linking mutagens it may be preferable toinclude TA102 or to add a DNA repair-proficient strain of E.coli [e.g.E.coli WP2 or E.coli WP2 (pKM101)] 114. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Bacteria and strains required for OECD 471:So what does this allmean? His genotype: location of the deletion mutation in the histidineoperon (operon is a functioning unit of genomic DNA containing acluster of genes under the control of a single regulatory signal orpromoter. Net result is that the bacterial carrying this mutationcannot synthesize histidine and are auxotrophs); rfa mutation: A mutation (rfa) in all strains that leads to a defectivelipopolysaccharide (LPS) layer that coats the bacterial surface,making the bacteria more permeable to bulky chemicals; uvrB and uvrA mutations: The uvrB deletion mutation eliminates theaccurate excision repair mechanism, thereby allowing more DNAlesions to be repaired by the error-prone DNA repairmechanism.The deletion through the biotin gene makes the bacteria biotindependent. 115. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Bacteria and strains required for OECD 471:So what does this allmean? Plasmid pKM101: present in strains TA1535 and TA1538 resulting inthe corresponding isogenic strains TA100, TA98, TA97, TA102 andTA104. Plasmid pKM101 enhances chemical and UV-inducedmutagenesis via an increase in the recombination DNA repairpathway. The plasmid confers ampicillin resistance, which is aconvenient marker to detect the presence of the plasmid; Insertion of the mutation hisG428 on the multi-copy plasmid pAQlwhich was introduced in strain TA102 with the aim of amplifying thenumber of target sites. To enhance the ability of this strain to detectDNA crosslinking agents, the uvrB gene was retained making thebacterium DNA repair proficient 116. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Bacteria and strains required for OECD 471: So what does this allmean? trp+: Trp operon is that codes for the production of tryptophan. trp+E. coli are auxotrophs for tryptophan; Note: all strains except S. typhimurium TA102 are also biotindependent (i.e. are both histidine and biotin auxotrophs). 117. E. Coli Wp2 uvrA: 118. Notes: A trace of histidine (ortryptophandepending on theauxotroph) + biotinare incorporated toallow for 1 or 2 roundsof cell replication inorder to fix anymutations present; The plateincorporation methodis reputed to increasethe assay sensitivityand to allow thetesting of suspensionsas well as solutions oftest article 119. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Modifications to the standard plate incorporation assay: The preincubation assay: the tester strains are exposed to thechemical for a short time (20 to 30min) in a small volume (0.5ml) ofeither buffer or S-9 mix, prior to plating on glucose agar minimalmedium (GM agar) supplemented with a trace amount of histidine.With few exceptions it is believed that this assay is more sensitivethan the plate incorporation assay, because short-lived mutagenicmetabolites may have a better chance reacting with the testerstrains in the small volume of preincubation mixture, and theeffective concentration of S-9 mix in the preincubation volume ishigher than that on the plate. 120. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Modifications to the standard plate incorporation assay: The desiccator assay for liquids and gases: the use of a closedchamber is recommended for testing highly volatile chemicals andgases; The Kado Salmonella microsuspension assay for testing samples ofsmall volumes; Testing chemicals in a reduced oxygen atmosphere: anaerobicenvironments, such as anaerobic chambers, have been used tostudy mutagenicity of chemicals and fecal samples under reducedoxygen levels. 121. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Modifications to the standard plate incorporation assay: Fluctuation method: The fluctuation method is performed entirely in liquidculture and is scored by counting the number of wells that turn yellow frompurple in a 96-well microplate. If bacteria are able to revert back tometabolic competence they will continue to replicate and turn the liquidmedia acid. By including a pH indicator in the media, the frequency ofmutation is counted as the number of wells out of 96 which have changedcolor. The fluctuation method is comparable to the traditional pour plate methodin terms of sensitivity and accuracy, however, it does have a number ofadvantages, namely, allowing for the testing of higher concentrations ofsample (up to 75% v/v), increasing the sensitivity and extending itsapplication to low-level environmental mutagens.[20] The fluctuation method also has a simple colorimetric endpoint; countingthe number of positive wells out of a possible 96 wells is much less timeconsuming than counting individual colonies on an agar plate. 122. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Strain checks: Histidine dependence (his): streak a loopful of the culture across aGM agar plate supplemented with an excess of biotin. Because allthe Salmonella strains are histidine dependent, there should be nogrowth on the plates. Biotin dependence (bio): streak a loopful of the culture across aGM agar plate supplemented with an excess of histidine. Thereshould be no growth on the plate except for strain TA102 which isbiotin independent. Biotin and histidine dependence (bio, his): streak a loopful of theculture across a GM agar plate supplemented with an excess ofbiotin and histidine. Growth should be observed with all strains. 123. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Strain checks: rfa marker: streak a loopful of the culture across a GM agar platesupplemented with an excess of biotin and histidine. Apply 10 l ofa sterile 0.1% crystal violet solution. All Salmonella strains shouldshow a zone of growth inhibition (crystal violet is a relatively largebacteriocidal molecule [mw = 407.979] which cannot penetratethe bacteria if a normal cell wall is present); Presence of plasmid pKM101 (ampicilline resistance): apply in thecenter of a plate 10 l of ampicilline solution. Streak a loopful of thepKM101-carrying Salmonella culture across an agar platesupplemented with an excess of histidine and biotin. Growth shouldbe observed. 124. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Strain checks: Spontaneous mutant frequency: use the standard plateincorporation assay procedure without the inclusion of a solvent fordetermining the spontaneous mutant frequency (negative control)of each of the tester strains. When the spontaneous control valuesfall outside an acceptable range the genetic integrity of the strain isconsidered compromised, and a new culture should be isolated. 125. Classical Assays for Genotoxicity:Bacterial reverse mutation assays Controls: Must have: suitable positive control for non-metabolic activationand metabolic activation + solvent/vehicle negative control; 126. For tests with metabolic activation: 127. Classical Assays for Genotoxicity: Bacterial reverse mutation assays Evaluation of results: Surviving populations: usually a 2 3-log range of doses are usedand the highest of these doses is selected to show some degree ofbacterial toxicity (background clearing or reduction in the numberof spontaneous mutants); Dose-response phenomena: a mutagen should display a cleardose-related increase in revertant colonies (can be influenced bypoor dose range selection); 128. Classical Assays for Genotoxicity: Bacterial reverse mutation assays Evaluation of results: Generally speaking a mutagen will produce a positive doseresponse over at least 3 different concentrations with the hiighestincrease in revertants being 2 3 times that of the level ofspontaneous revertants in the negative control plates; Pattern: TA-1535 and TA-100 are derived from the same parentalstrain, thus commonly the responses of these two strains should benearly identical; The results (particularly the pattern of reversion) should berepeatable and consistent; Specific gene sequencing may be of use in some cases. 129. Classical Assays for Genotoxicity: Mouse lymphoma L5178Y tk+/- forward mutation assay (OECD 476) Basic principle of the assay: Cells deficient in thymidine kinase (TK) due to the mutationTK+/- TK-/- are resistant to the cytotoxic effects of thepyrimidine analogue trifluorothymidine (TFT); TFT is converted to TFT-monophosphate by thymidine kinase TFT-triphosphate (TFT-TP) is incorporated into DNA, resultingin cytocidal effects. 130. Classical Assays for Genotoxicity: Mouse lymphoma L5178Y tk+/- forward mutation assay (OECD 476) Basic principle of the assay: Thymidine kinase proficient cells are sensitive to TFT, whichcauses the inhibition of cellular metabolism and halts furthercell division; Thus mutant cells are able to proliferate in the presence ofTFT, whereas normal cells, which contain thymidine kinase,are not. 131. Classical Assays for Genotoxicity: Mouse lymphoma L5178Y tk+/- forward mutation assay (OECD 476) Basic principle of the assay: The TK mutations have no effect on the growth of cells innormal media because normal DNA synthesis does notinvolve the TK pathway; The L5178Y cells also have point mutations in both p53alleles,. Because p53 is an important protein in the DNAdamage response in the cell, the L5178Y cell line isinadequate in its response to DNA damage, which isarguably important for enhanced assay sensitivity for thedetection of genotoxic compounds. 132. Classical Assays for Genotoxicity: Mouse lymphoma L5178Y tk+/- forward mutation assay (OECD 476) Basic principle of the assay: Assay is usually conducted with and without S9 metabolicactivation; Controls: Vehicle/solvent negative control; Positive controls: methylmethanesulfonate for studies withoutmetabolic activation; cyclophosphamide, benzo(a)pyreneand 3-methylcholanthrene for tests with metabolicactivation; 133. Classical Assays for Genotoxicity: Mouse lymphoma L5178Y tk+/- forward mutation assay (OECD 476) Dose range: Selected so that the test doses span the range from 0% to80% reduction in cell growth with or without S9 activation. 134. Classical Assays for Genotoxicity: Mouse lymphoma L5178Y tk+/- forward mutation assay (OECD 476) Assay acceptance criteria: The average absolute cloning efficiency must be in the range of 65 120% (ability of single cells to form a new colony); The average increase in the vehicle control cell populationshould be 8 -32 fold over 2 days; Background forward mutation frequency should beacceptable; 135. Classical Assays for Genotoxicity: Mouse lymphoma L5178Y tk+/- forward mutation assay (OECD 476) Assay acceptance criteria: The frequency of forward mutation in the positive controls must be consistent with historical data for the lab; The assay must include applied concentrations that reach 5mg/mL or 10 mM (which ever is the lower) for test articles thatcause little or no cytotoxicity; The assay must include applied concentrations that reducethe relative cell growth by approximately 20%; OR Reach a concentration that exceeds the solubility limit. 136. Classical Assays for Genotoxicity: Mouse lymphoma L5178Y tk+/- forward mutation assay (OECD 476) Interpretation: Cell colony size on agar: large mutant colonies have few genetic changes other than the tk+/- tk-/- forward mutation; small mutant colonies generally represent more extensive genetic modification of the cells; A mutant frequency of at least 2 times that in the negativecontrol is suggestive of a positive control (induction ofmutation is an additive process and not a multiple overbackground the global evaluation factor should be used); 137. Classical Assays for Genotoxicity: Mouse lymphoma L5178Y tk+/- forward mutation assay (OECD 476) Interpretation: Global evaluation factor = the mean of each vehicle control mutant frequency distribution + 1 SD across multiple test labs; The GEF for the agar assay method = 90; The GEF for 96 well plate assays = 126; A positive assay is one where the mutant frequency for thetest article equals or exceeds the GEF PLUS there is astatistically demonstrable dose trend present; A negative assay is one where the mutant frequency for thetest article is below the GEF PLUS there is no statisticallydemonstrable dose trend present 138. Classical Assays for Genotoxicity: Mouse lymphoma L5178Y tk+/- forward mutation assay (OECD 476) Interpretation: If only one criterion is met, additional studies are needed to clarify the test outcome 139. Classical Assays for Genotoxicity: Other mammalian cell forward mutation tests(OECD 476) The HRPT assay: Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) isa transferase that catalyzes conversion of hypoxanthine toinosine monophosphate and guanine to guanosinemonophosphate; Test cell lines are HRPT+/- i.e. have a single functional HRPTallele that is lost with mutation; Multiple different mammalian cell lines with this genotypeare available: V79 Chinese hamster cells, AS52 Chinesehamster cells, Chinese hamster ovary cells (CHO) and humanTK6 human lymphoblastoid cells. 140. Classical Assays for Genotoxicity:Other mammalian cell forwardmutation tests(OECD 476) The HRPT assay: Basic principles: HRPT converts 6-thioguanine (TG) or 8-azaguanine (AG) to non-toxic metabolites; Cells that have HRPT function are able to survive andreplicate in the presence of TG or AG; Those cells that lack HRPT function due to a forward mutationdie in the presence of TG or AG; The assay is conducted and assessed in a manner that issimilar to the TK assay. 141. Classical Assays for Genotoxicity:Other mammalian cell forwardmutation tests(OECD 476) The XHRPT assay: Transgene of xanthineguanine phosphoribosyl transferase(XHRPT); Works on the same principle as the HRPT assay; XHRPT is located on autosomal chromosomes where as HRPTis located on the X chromosome. 142. Classical Assays for Genotoxicity: Other mammalian cell forward mutation tests(OECD 476) The TK, HPRT and XPRT mutation tests detect different spectra ofgenetic events. The autosomal location of TK and XPRT mayallow the detection of genetic events (e.g. large deletions) notdetected at the HPRT locus on X-chromosomes. 143. Classical Assays for Genotoxicity:In vivo mammalian forward mutationassays in transgenic animals. The Big Blue mouse assay: The genome of these mice has been manipulated suchthat every cell contains, stably integrated into theDNA, multiple tandem copies of a bacterial lac I repressorgene; lf the mice are exposed to mutagens, there is a smallprobability that a mutation will occur somewhere alongthe inserted sequence; Any mutation will lead to an inactive lac I gene and lacrepressor protein, meaning the gene (lacZ) for beta-galactosidase will no longer be repressed; 144. Classical Assays for Genotoxicity:In vivo mammalian forward mutationassays in transgenic animals. The Big Blue mouse assay: The DNA is extracted from the tissues of the treated mouse the vector is isolated and used to make functionalbacteriophages E. coli cells are mixed with thebacteriophage and spread on a solid culture medium thebacteriophages infect and destroy ("lyze") the E. coli cells --?this causes clear circular zones, called plaques, to appear ina "lawn" of bacteria Before they die, cells that have beeninfected by bacteriophages carrying a mutated lac I willproduce beta-galactosidase This reacts with a substrate inthe culture medium turning it blue Count both colorlessand blue plaques The number of blue plaques divided bythe total number of plaques gives the mutation frequency. 145. Classical Assays for Genotoxicity:In vivo mammalian forward mutationassays in transgenic animals. The Big Blue mouse assay: Bacteriophages with non-mutated genes produce colorlessplaques because no beta-galactosidase is synthesized; 146. Classical Assays for Genotoxicity:In vivo mammalian forward mutationassays in transgenic animals. The muta-mouse assay operates in a manner similar to theBig Blue except it uses a LacZ gene 147. Classical Assays for Genotoxicity: In vivo mammalian forward mutation assays in transgenic animals. In vivo forward mutation assays offer very substantialadvantages: The complete array of metabolic processes are present; Tissue specific metabolism is taken into account rather than just the over simplified metabolism that occurs with S9; Tissue and even cell-type specific mutation rates can be measured. This includes germ cell (spermatogonial) mutations in males!; Takes into account toxicokinetic and toxicodynamic differences between different organs, tissues and cell types; In general, tissue specific mutation frequencies generallymatch the distribution of mutation in live animals! 148. Classical Assays for Genotoxicity: In vitro chromosomal aberration assay (OECD 473) Basic principle of the assay: Typically Chinese hamster ovary cells strain CHO-WBL ATCCCCL61 are used (fibroblastic cell line); Chromosomal number of 21 with a low frequency of spontaneous mutations; Alternatively human peripheral blood lymphocytes that havebeen stimulated to divide using a mitogen (PHA) are used; 149. Classical Assays for Genotoxicity: In vitro chromosomal aberration assay (OECD 473) Basic principle of the assay: Cell cultures are exposed to the test substance both withand without metabolic activation At predetermined intervals after exposure of cell cultures tothe test substance, they are treated with a metaphase-arresting substance (e.g. Colcemid or colchicine),harvested, stained and metaphase Cells are analysed microscopically for the presence ofchromosome aberrations. 150. Classical Assays for Genotoxicity: In vitro chromosomal aberration assay (OECD 473) Basic principle of the assay: Cell cultures are exposed to the test substance both withand without metabolic activation At predetermined intervals after exposure of cell cultures tothe test substance, they are treated with a metaphase-arresting substance (e.g. Colcemid orcolchicine), harvested, stained and metaphase Cells are analysed microscopically for the presence ofchromosome aberrations. 151. Classical Assays for Genotoxicity: In vitro chromosomal aberration assay (OECD 473) Measurement of results: This assay does NOT determine aneuploidy (i.e. increased ordecreased number of chromosomes; this is because ofartifacts associated with the cell analysis preparation): onlycells with a normal chromosome number are analysed; The types of chromosomal aberrations that are detectedare: Simple breaks; Complex exchanges; Gaps; Dicentric chromosomes; Ring chromosomes. 152. Classical Assays for Genotoxicity: In vitro chromosomal aberration assay (OECD 473) Advantages: Accurate identification of all the different chromosomemutation types; Possible co-detection of mitotic indices; No full automatic but interactive scoring possible; Disadvantages: High false positive rate; Labor intensive and time consuming; Heavily dependent on operator/reader skill. 153. Classical Assays for Genotoxicity: In vivo bone marrow chromosomal aberration assay (OECD 475) Advantages over the in vitro method: Takes into account in vivo TK and TD to a certain extent; Can test different routes of exposure; Full in vivo metabolism (but with some limitations, particularlygiven that the tissue analyzed is bone marrow); 154. Classical Assays for Genotoxicity:In vivo bone marrow chromosomalaberration assay (OECD 475) Disadvantages over the in vitro method: Exposure of the bone marrow must occur in order for the testto be valid this may need to be demonstrated by a TKstudy; If there is evidence that the test substance, or a reactivemetabolite, will not reach the targettissue, it is notappropriate to use this test; 155. Classical Assays for Genotoxicity:In vivo bone marrow chromosomalaberration assay (OECD 475) Disadvantages over the in vitro method: If metabolism to an ultimate mutagen is required, then theblood and tissue T must be long enough for bone marrowexposure to occur unless local metabolism in the bonemarrow occurs; Risk of excessive toxicity producing distorted results: doseranging studies are often needed. 156. Classical Assays for Genotoxicity: In vivo bone marrow chromosomal aberration assay (OECD 475) Principle of the assay: Animals (typically rats or Chinese hamsters) are exposed tothe test substance by an appropriate route of exposure andare sacrificed at appropriate times after treatment; Prior to sacrifice, animals are treated with a metaphase-arresting agent (e.g., colchicine or Colcemid); Chromosome preparations are then made from the bonemarrow cells and stained, and metaphase cells are analysedfor chromosome aberrations. 157. Classical Assays for Genotoxicity: In vivo spermatogonial chromosomal aberration assay (OECD 483) Assay is similar in principle to OECD 475; Important difference is that it tests for GERM CELLchromosomal damage; If there is evidence that the test substance, or a reactivemetabolite, will not reach the target tissue, it is notappropriate to use this assay. 158. Classical Assays for Genotoxicity:Unscheduled DNA Synthesis OECD428 This is essentially a test of DNA repair that occursoutside of the normal period of DNA synthesis in cells; Measures DNA synthesis in cells which are not in the Sphase of the cell cycle; The assay measures global genomic nucleotideexcision repair (NER); 159. Classical Assays for Genotoxicity:Unscheduled DNA Synthesis OECD428 The classical OECD assay measures DNA synthesis bymeasuring the incorporation of 3H-thymidine or BrDUinto DNA; More modern techniques use specific DNA dyes andflow cytometry: faster, more accurate, providesmore information; 160. Classical Assays for Genotoxicity: Unscheduled DNA Synthesis OECD428 The test requirements are very broad: Can be done in vitro or in vivo; Primary cell cultures or established cell lines can be used; Test is performed with or without metabolic activation; In order to discriminate between UDS and normal semi-conservative DNA replication, cell replication is inhibited orminimized using an arginine-deficient medium, low serumcontent, or by hydroxyurea in the culture medium; For flow cytometric methods, inhibition of the cell cycle is notrequired. 161. Classical Assays for Genotoxicity: Unscheduled DNA Synthesis OECD428 Given that the assay measures nucleotide excision repair, it iscritical that: At least some of the DNA lesions produced by the test articleare repairable by nucleotide excision repair; The test cell line/animal line is capable of relatively normalnucleotide excision repair 162. Classical Assays for Genotoxicity: Mammalian Erythrocyte Micronucleus Test OECD 474 Basic principle of the assay: It is an assay of macro-chromosomal damage (chromosomefragments lacking a kinetocore/centromere) OR damage tothe mitotic spindle (whole chromosomes chromosomefragments containing a kinetocore); The assay detects lagging chromosome fragments orlagging chromosomes; 163. Classical Assays for Genotoxicity: Mammalian Erythrocyte Micronucleus Test OECD 474 Basic principle of the assay: Takes advantage of the fact that when bone marrowerythroblast develops into a polychromatic erythrocyte thecell nucleus is extruded from the cell however DNAcontaining micronuclei are not extruded from the cell; Micronuclei can be detected by cell staining and flowcytometry. The lack of a normal cell nucleus makes thedetection of micronuclei much easier. 164. Classical Assays for Genotoxicity: Mammalian Erythrocyte Micronucleus Test OECD 474 This assay is regarded as the highest tier assay of the commonlyconducted genetic toxicology assays for chemicals and otheragents; 165. Classical Assays for Genotoxicity: Mammalian Erythrocyte Micronucleus Test OECD 474 Important features: Classically outbred rats and mice are used in preference toinbred strains in order to reduce the likelihood of strainspecific responses (classically CD-1 [ICR] BR mice and/orSprague-Dawley CD [SD] IGS BR rats); Animals are usually 8 -10 weeks old at the start of the study; Various routes of administration, including parenteral routes,are available. Unless there are specific reasons not to, thestudy route of exposure should match those of the likelyhuman routes of exposure; Parenteral routes (IV, IP) are used if absorption is poor or ifthere are other technical constraints regarding dosing. 166. Classical Assays for Genotoxicity: Mammalian Erythrocyte Micronucleus Test OECD 474 Important features: The IV route of exposure has a particular advantage in that itguarantees exposure of the bone marrow; If non-IV routes of exposure are used, there must be sufficientTK data that demonstrates that bone marrow exposureoccurs or is likely to occur; If the test article is a pro-mutagen (i.e. requires metabolicactivation), then there must be reasonable certainty thatappropriate metabolic activation can occur in the bonemarrow OR the active metabolites have a chemical half-lifethat is long enough for them to reach the bone marrow! 167. Classical Assays for Genotoxicity: Mammalian Erythrocyte Micronucleus Test OECD 474 Important features: Ideally, 2 positive controls should be used: Cyclophosphamide: requires metabolic activation; A substance that does not require metabolic activation (e.g. ethyl methanesulfonate) 168. Classical Assays for Genotoxicity: Mammalian Erythrocyte Micronucleus Test OECD 474 Positive controls: Can be administered by a different route than the testarticle; Can be administered as a single dose; Negative controls: typically a solvent or vehicle control 169. Classical Assays for Genotoxicity: Mammalian Erythrocyte Micronucleus Test OECD 474 No standard treatment schedule (i.e. 1, 2, or more treatmentsat 24 h intervals) can be recommended. The samples fromextended dose regimens are acceptable as long as a positiveeffect has been demonstrated for this study or, for a negativestudy, as long as toxicity has been demonstrated or the limitdose has been used, and dosing continued until the time ofsampling. Test substances may also be administered as a splitdose, i.e., two treatments on the same day separated by nomore than a few hours, to facilitate administering a largevolume of material. 170. Classical Assays for Genotoxicity: Mammalian Erythrocyte Micronucleus Test OECD 474 The test can be performed in 2 ways: Animals are treated with the test substance once. Samples ofbone marrow are taken at least twice, starting not earlier than24 hours after treatment, but not extending beyond 48 hoursafter treatment with appropriate interval(s) between samples.The use of sampling times earlier than 24 hours after treatmentshould be justified. Samples of peripheral blood are taken atleast twice, starting not earlier than 36 hours after treatment,with appropriate intervals following the first sample, but notextending beyond 72 hours. When a positive response isrecognized at one sampling time, additional sampling is notrequired. 171. Classical Assays for Genotoxicity: Mammalian Erythrocyte Micronucleus Test OECD 474 The test can be performed in 2 ways: If 2 or more daily treatments are used (e.g. two or moretreatments at 24 hour intervals), samples should be collectedonce between 18 and 24 hours following the final treatment forthe bone marrow and once between 36 and 48 hours followingthe final treatment for the peripheral blood (12). 172. Classical Assays for Genotoxicity: Mammalian Erythrocyte Micronucleus Test OECD 474 Dose rates: If a range finding study is performed because there are no suitabledata available, it should be performed in the same laboratory,using the same species, strain, sex, and treatment regimen to beused in the main study (13). If there is toxicity, three dose levels areused for the first sampling time. These dose levels should cover arange from the maximum to little or no toxicity. At the latersampling time only the highest dose needs to be used. The highestdose is defined as the dose producing signs of toxicity such thathigher dose levels, based on the same dosing regimen, would beexpected to produce lethality. Toxicity can take any form. A commonly used measure is areduction in the ratio of polychromatic to normochromaticerythrocytes (what does this say about the bone marrow?); 173. Classical Assays for Genotoxicity: Mammalian Erythrocyte Micronucleus Test OECD 474 Dose rates: Substances with specific biological activities at low non-toxic doses(such as hormones and mitogens) may be exceptions to the dose-setting criteria and should be evaluated on a case-by-case basis.The highest dose may also be defined as a dose that producessome indication of toxicity of the bone marrow (e.g. a reduction inthe proportion of immature erythrocytes among total erythrocytesin the bone marrow or peripheral blood). In general, a maximum limit dose of 2 g/kg is used for substanceswith low toxicity 174. Classical Assays for Genotoxicity: Mammalian Erythrocyte Micronucleus Test OECD 474 Sexes: In general, if there is data that indicates that there are no sexdifferences in toxicity, only males are used; If there is data indicating sex differences, BOTH sexes MUSTbe used. 175. Classical Assays for Genotoxicity:Mammalian Erythrocyte MicronucleusTest OECD 474 Assay acceptance criteria: % micronucleated PCE in the negative control group must liewithin the historical normal range for the lab and must bebelow 0.4%; Must be a statistically significant increase in the %micronucleated PCE relative to the negative controls in thepositive control group and this increase should be consistedwith historical data for the lab; Either the limit dose is reached or at least some non-leathaltoxicity must be present in the highest test article dose 176. Classical Assays for Genotoxicity:Mammalian Erythrocyte MicronucleusTest OECD 474 What constitutes a positive response? A statistically significant increase in micronucleatedPCE in at least one of the test article doses relative tothe negative controls; OR A statistically significant dose response relationship ispresent; The results make biological sense. 177. Classical Assays for Genotoxicity:Rare Assays Mouse Spot Test (OECD 484) Now extremely difficult to perform because the appropriatemouse strains are no longer available (lost); It is a high tier assay in terms of risk assessment/hazardclassification; Often now replaced by Big Blue or Mutamouse An in vivo test in mice in which developing embryos areexposed to a test agent. The target cells are melanoblasts; The target genes are those which control the pigmentationof the coat hairs; 178. Classical Assays for Genotoxicity: Rare Assays Mouse Spot Test (OECD 484) The developing embryos are heterozygous for a number of thesecoat colour genes; A mutation in, or loss of (by a variety of genetic events), thedominant allele of such a gene in a melanoblast results in theexpression of the recessive phenotype in its descendantcells, constituting a spot of changed colour in the coat of theresulting mouse. The number of offspring with these spots, mutations, are scored andtheir frequency is compared with that among offspring resultingfrom embryos treated with the solvent only. The mouse spot testdetects presumed somatic mutations in foetal cells. 179. Figure 2Defects in melanocyte development cause white spotting, while thestem cell defect results hair graying.(A) A Ednrbs-l/Ednrbs-l mouse demonstrating extensive piebald spotting. (B) AKitW-2J/+ mouse demonstrating a white head blaze, and small dorsal spot on theback. (C) A Sox10Lacz/+ mouse exhibiting the characteristic white belly spot. (D)A Mitfvit/vit mouse (upper) exhibits gradual hair graying. A lower mouse is age-matched control. Adapted from the WEB site of the European Society forPigment Cell Research (ESPCR), Color genes: http://www.espcr.org/micemut/. 180. Classical Assays for Genotoxicity:Rare Assays Mouse Heritable Translocation Assay (OECD 485) This is a particularly useful assay because it is an in vivomeasure of germ cell macro-chromosomal damage; Currently there is no commonly used in vivo equivalent to thisassay; There are 2 forms of the assay: Detection of changes in fertility of the F1 progeny of exposed animals; Cytogenetic examination of the F1 progeny of exposed animals; 181. Classical Assays for Genotoxicity: Rare Assays Mouse Heritable Translocation Assay (OECD 485) The assay primarily measures the frequency of structuralchromosome assays that do not result in a loss ofgenetic material i.e. reciprocal translocations; Reciprocal translocations = fetal survival + nomalformations; Non-reciprocal translocations usually mean fetal death; 182. Classical Assays for Genotoxicity: Rare Assays Mouse Heritable Translocation Assay (OECD 485) Reciprocal translocation, however, result in a loss of fertility inthe F1 generation due to the production of unbalancedchromosomes in the gametes (i.e. unbalancedtranslocations); 183. Classical Assays for Genotoxicity:Rare Assays Insect and yeast OECD assays: rarely if ever used. You willoccasionally come across them in historical data sets.Treat them with caution! 184. Other Common Genotoxicity Assays: The comet assay = Single Cell Electrophoresis Assay; Simple, reliable and particularly useful!; Well accepted assay; Can also be used as a very simple, but effective andaccurate assay of DNA repair; 185. Other Common Genotoxicity Assays: The comet assay Principle: Measures DNA strand breaks (i.e. clastenogenesis); Can be done in vitro or in vivo; Can be used for specific tissues or even specific cells; Cells embedded in agarose on a microscope slide are lysed withdetergent and high salt to form nucleoids containing supercoiledloops of DNA linked to the nuclear matrix; Electrophoresis at high pH results in structures resembling comets,observed by fluorescence microscopy; The intensity of the comet tail relative to the head reflects thenumber of DNA breaks 186. A Few Slides of Gratuitous SilynessAfter 228 power point slides on genetic toxicology, I figured youwere entitled.. 187. Before Genetic Tox After Genetic Tox 188. ?