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David Kirkland
<ul><li>Could a +ve  in vitro  result be explained by: </li></ul><ul><ul><li>Production of reactive oxygen species by reac...
DMEM  F-10  F-12  RPMI  RPMI  McCoys  Williams Hepes  5A  E  M of H 2 O 2  produced -  +  -  +  -  +  -  +  -  +  -  +  -...
% cells with CA (solid line) Relative cell count (%) (dashed line) Incubation time (hr) of EGCG in medium before stopping ...
<ul><li>K m  for most biochemical reactions is <100  μ M yet top concentration for testing is 10 mM </li></ul><ul><ul><li>...
Chemical Previous LEC New LEC Allyl isovalerate 2.81 mM in  MLA +ve at 0.55 mM in CA (3 hr +S9) Benzofuran 1.27 mM in MLA ...
<ul><li>Many different measures of cytotoxicity can be used and are acceptable under OECD </li></ul><ul><ul><li>They may n...
% cells with CA Survival measure relative to control Conc. of drug(  g/ml) 50% toxicity
<ul><li>COLIPA trial has looked at relative increase in cell count (RICC) or relative population doubling (RPD) with 6 con...
Negative Positive Positive Equivocal *** p≥0.001
<ul><li>Bacterial-specific metabolites </li></ul><ul><li>Nitroreduction </li></ul><ul><li>Rat liver S9 </li></ul><ul><li>I...
<ul><li>Sodium azide is converted to mutagenic form (azidoalanine) by O-acetylserine(thio)lyase in bacteria </li></ul><ul>...
<ul><li>Bacteria are very efficient at nitroreduction </li></ul><ul><li>Mammalian nitroreductase is oxygen-labile </li></u...
<ul><li>Rat may produce metabolites not produced by humans </li></ul><ul><li>“ Induction” of rat livers with Aroclor or ph...
<ul><li>Quercetin is clearly genotoxic  in vitro </li></ul><ul><ul><li>formation of active oxygen by redox cycling </li></...
<ul><li>Interaction with non-DNA targets </li></ul><ul><li>DNA-reactive chemicals/metabolites at high concentrations, but ...
<ul><li>Non-relevant  mechanisms for +ves  in vitro  would not be expected to occur in animals  in vivo , or only under ce...
<ul><li>Enzyme induction </li></ul><ul><li>Imbalance of DNA precursors </li></ul><ul><li>Energy depletion </li></ul><ul><l...
<ul><li>Inhibitors of DNA gyrase can also cause other changes both in bacteria and mammalian cells </li></ul><ul><ul><li>I...
<ul><li>Deoxyribonucleotide pool imbalances  aberrant DNA replication/repair  multitude of genetic effects  -   mutation, ...
<ul><li>Paracetamol (acetaminophen) inhibits ribonucleotide reductase, but genotoxicity probably due to NAPQI metabolite <...
<ul><li>DNA-damage by various chemicals, or enhancement of others, accompanied by glutathione depletion  </li></ul><ul><ul...
<ul><li>Multi-fibre nature of mitotic spindle means it can function effectively even when experiencing some damage </li></...
<ul><li>MN that contain whole chromosomes (that failed to attach to spindle) will contain centromeres </li></ul><ul><li>Ca...
<ul><li>If no convincing experimental evidence of threshold or non-relevant mechanism, evidence of absence of direct DNA-m...
<ul><li>Concept acknowledges human exposure threshold (including those of unknown toxicity) below which no significant hum...
<ul><li>Gold database contained genotoxic and non-genotoxic carcinogens </li></ul><ul><li>ILSI Europe Task Group (Kroes  e...
<ul><li>MN are usually measured in bone marrow cells </li></ul><ul><li>Erythrocyte precursor cells at the time of their la...
In vivo  micronucleus assay 6-8 h 18-22 h Erythroblasts Immature erythrocytes Mature erythrocytes Bone marrow Peripheral b...
<ul><li>Acute study most common for bone marrow </li></ul><ul><ul><li>1 administration with 2 sampling times (24 & 48 hr) ...
<ul><li>Micronuclei can also be measured in young reticulocytes (CD71 expressing) in peripheral blood </li></ul><ul><li>Be...
Peripheral Blood MN Methodology Inside the Cytometer FITC PI 488nm laser NCE  (No Fluorescence) Reticulocyte  (Green) MN-R...
EMS - 3 hr  Peripheral Blood MN Methodology Data Output
<ul><li>MN can also be measured in liver </li></ul><ul><li>Surgery (partial hepatectomy) is needed to make cells divide </...
 
<ul><li>Howell-Jolly bodies are small nuclear remnants that have the colour of a pyknotic nucleus & contain DNA </li></ul>...
<ul><li>Administration of EPO </li></ul><ul><li>Hypoxia leads to secretion of EPO </li></ul><ul><ul><li>iron deficiency </...
MN % mg/kg
<ul><li>In MN test at 200 mg/kg, body temperature dropped from 37 o C to 24 o C within 6 hr </li></ul><ul><li>Temperature ...
MN/1000 PCE mg/kg Drug X
<ul><li>Most widely used is the single cell gel electrophoresis (Comet) assay </li></ul><ul><ul><li>Measures DNA damage as...
<ul><li>Cells mixed with agarose </li></ul><ul><li>Spread on microscope slides </li></ul><ul><li>Lysis </li></ul><ul><li>U...
<ul><li>Unwinding DNA at different pH expresses different types of DNA damage as strand breaks (SBs), resulting in fragmen...
Data Collected by Image Analysis <ul><li>Length of DNA migration   </li></ul><ul><li>(smallest detectable DNA)   </li></ul...
Cells with DNA migration Cells without DNA migration
<ul><li>Correlation of  in vivo  Comet assay results with rodent carcinogenicity very high </li></ul><ul><ul><li>Particula...
<ul><li>Mice have been engineered to carry a bacterial  lacI  or  lacZ  transgene on a lambda bacteriophage vector </li></...
<ul><li>MUTATION IN TRANSGENES </li></ul>Treat mice take tissues isolate DNA package colour selection ( LacI ) positive se...
Cohesive end BamH1 BamH1 BamH1 BamH1 XHol BamH1 EcoR1 EcoR1 EcoR1 EcoR1 BamH1 Cohesive end LacZ 0 50 kb
<ul><li>The lambda phage will lyse the sensitive bacteria and form plaques </li></ul><ul><li>By growing the bacteria on ag...
<ul><li>Muta TM Mouse  (lacZ ) and Big Blue ( lacI ) most widely used </li></ul><ul><li>Treatment period is usually 28 day...
<ul><li>Is there a credible mechanistic/metabolic reason why +ve results  in vitro  are expected to have threshold, or onl...
<ul><li>CRITERIA FOR POSITIVES </li></ul><ul><li>Statistical significance at 1 or more dose points </li></ul><ul><ul><li>s...
<ul><li>I hope I have been able to show that  decision making with genotoxicity data is not easy </li></ul><ul><li>There a...
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Health Canada Genetic Tox Lecture Part 2

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This is Part 2 of a presentation on Genetic Toxicology that was given by Dr. David Kirkland to scientific staff at Health Canada in Nov. 2010. Part 1 is availabile here in ppt and as a webinar at the LinkedIn DABT CE group link

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Health Canada Genetic Tox Lecture Part 2

  1. 1. David Kirkland
  2. 2. <ul><li>Could a +ve in vitro result be explained by: </li></ul><ul><ul><li>Production of reactive oxygen species by reaction between test substance and culture medium? * [*next slide] </li></ul></ul><ul><ul><li>Degradation of test chemical in medium? </li></ul></ul><ul><ul><li>Confounding effects of apoptosis or necrosis? </li></ul></ul><ul><ul><li>Possible effects on kinases? </li></ul></ul><ul><ul><li>Topoisomerase II or DNA synthesis inhibition? </li></ul></ul><ul><ul><li>Interference by stray UV light? </li></ul></ul><ul><ul><li>Nucleotide pool imbalance? </li></ul></ul><ul><ul><li>Metabolic overload? </li></ul></ul>
  3. 3. DMEM F-10 F-12 RPMI RPMI McCoys Williams Hepes 5A E  M of H 2 O 2 produced - + - + - + - + - + - + - + EGCG Sig. clastogenic dose of H 2 O 2
  4. 4. % cells with CA (solid line) Relative cell count (%) (dashed line) Incubation time (hr) of EGCG in medium before stopping reaction McCoy’s 5A F-10
  5. 5. <ul><li>K m for most biochemical reactions is <100 μ M yet top concentration for testing is 10 mM </li></ul><ul><ul><li>ICH will probably agree 1 mM acceptable for pharms but will this be accepted for other chemicals? </li></ul></ul><ul><li>Recent review (Parry et al) identified 24 rodent carcs –ve in Ames, only +ve in mammalian cells at >1 mM </li></ul><ul><ul><li>Some are non-genotoxic carcs so not expected to be detected </li></ul></ul><ul><ul><li>9 other chemicals re-tested, and either negative up to cytotoxic concs or 10 mM, or positive at 2 mM (200 μ g/ml) and below </li></ul></ul><ul><li>Proposal for new upper limit of 1 mM or 500 μ g/ml, whichever is the higher </li></ul>
  6. 6. Chemical Previous LEC New LEC Allyl isovalerate 2.81 mM in MLA +ve at 0.55 mM in CA (3 hr +S9) Benzofuran 1.27 mM in MLA -ve at in MLA up to 1 mM (64-99% toxicity ). Increased MF at 2 mM but almost 100% toxicity Caffeic acid 1.11 mM in MLA +ve 0.4 mM in MLA (24 hr –S9) and 1 mM in CA (20 hr –S9) Chlorobenzene 1.11 mM in MLA +ve at 0.6 mM in MLA (3 hr +S9) Daminozide 13.75 mM in CA; 11.25 mM in MLA -ve up to 10 mM (CA & MLA) Furan 1.47 mM in CA +ve at 0.8 mM in MLA (3 hr +S9) and at 4 mM in CA (3 hr +S9) Methylolacrylamide 2.94 mM in CA +ve at 2 mM in CA (20 hr –S9) and MLA (24 hr –S9) Toluene 2.44 mM in MLA -ve up to toxic doses (10% RTG) in MLA (not tested in CA) Monuron 6.54 mM in CA -ve in CA up to 4.25 mM which induced >50% toxicity (- and + S9)
  7. 7. <ul><li>Many different measures of cytotoxicity can be used and are acceptable under OECD </li></ul><ul><ul><li>They may not all give the same conc for 50% toxicity </li></ul></ul><ul><ul><li>Greenwood et al (2004) showed several non-DNA reactive clastogens were -ve if 50% toxic conc chosen by population doubling instead of cell counts </li></ul></ul><ul><ul><li>Further evidence of this from COLIPA trial at Covance using in vitro MN test on 19 “false positive” chemicals </li></ul></ul><ul><li>A rigorous comparison (collaborative trial) of different measures of cytotoxicity is needed </li></ul><ul><ul><li>Cell counts, population doubling, replication index, RTG, ATP levels, LDH leakage, Alamar Blue etc. </li></ul></ul><ul><ul><li>Rodent cells, human lymphocytes, TK6 etc. </li></ul></ul><ul><ul><li>Need measures other than mitotic index for lymphocytes </li></ul></ul>
  8. 8. % cells with CA Survival measure relative to control Conc. of drug(  g/ml) 50% toxicity
  9. 9. <ul><li>COLIPA trial has looked at relative increase in cell count (RICC) or relative population doubling (RPD) with 6 confirmed false +ves </li></ul><ul><ul><li>Relative cell count (RCC) picked higher concs for 50% toxicity than RICC or RPD and therefore more prone to give false positive results with non-carcinogens </li></ul></ul><ul><li>Recent collaborative trial on in vitro MN test showed that range of 14 “real +ves” all detected as +ve if RICC or RPD used </li></ul><ul><ul><li>No false -ves </li></ul></ul>
  10. 10. Negative Positive Positive Equivocal *** p≥0.001
  11. 11. <ul><li>Bacterial-specific metabolites </li></ul><ul><li>Nitroreduction </li></ul><ul><li>Rat liver S9 </li></ul><ul><li>In vitro specific effects </li></ul><ul><li>In addition compounds may disrupt non-DNA targets (e.g. spindle) and exhibit a threshold </li></ul>
  12. 12. <ul><li>Sodium azide is converted to mutagenic form (azidoalanine) by O-acetylserine(thio)lyase in bacteria </li></ul><ul><li>Mammalian cells express enzyme but azidoalanine is not formed </li></ul><ul><ul><li>different sub-type of enzyme? </li></ul></ul><ul><ul><li>is azidoalanine rapidly converted to non-mutagenic intermediate? </li></ul></ul><ul><li>Sodium azide is not mutagenic for mammalian cells </li></ul>
  13. 13. <ul><li>Bacteria are very efficient at nitroreduction </li></ul><ul><li>Mammalian nitroreductase is oxygen-labile </li></ul><ul><ul><li>where intestinal bacteria may not be exposed, there may be no risk to humans </li></ul></ul><ul><li>Metronidazole is bacterial mutagen (via nitroreduction) but -ve in mammalian cells </li></ul><ul><ul><li>mammalian metabolism is via side chain (NO 2 intact) </li></ul></ul><ul><ul><li>lung tumours not consistent with bacterial nitroreduction </li></ul></ul><ul><ul><li>is reduced metronidazole unable to pass from the bacteria into surrounding mammalian tissue? </li></ul></ul>
  14. 14. <ul><li>Rat may produce metabolites not produced by humans </li></ul><ul><li>“ Induction” of rat livers with Aroclor or phenobarbital/ ß-naphthoflavone results mainly in elevation of Phase I (activation) enzymes rather then Phase II (detoxification) enzymes </li></ul><ul><ul><li>CYP1A and 2B enzymes at 40x levels found in normal liver </li></ul></ul><ul><ul><li>In normal testing we do not add cofactors such as glutathione, acetyl CoA (for acetylation), PAPS (for sulphation), or UDPGA (for glucuronidation) for detoxification pathways </li></ul></ul><ul><li>Understanding relative importance of Phase I & II metabolism can determine whether +ve results may have threshold of detoxification which may not be exceeded under normal human exposure </li></ul>
  15. 15. <ul><li>Quercetin is clearly genotoxic in vitro </li></ul><ul><ul><li>formation of active oxygen by redox cycling </li></ul></ul><ul><li>All in vivo genotoxicity studies are -ve </li></ul><ul><li>10 carcinogenicity studies are -ve </li></ul><ul><li>Humans take 50 mg/day in food </li></ul><ul><li>In vivo quercetin is rapidly methylated via catechol-O-methyltransferase, and redox cycling is negligible </li></ul>
  16. 16. <ul><li>Interaction with non-DNA targets </li></ul><ul><li>DNA-reactive chemicals/metabolites at high concentrations, but which, at low concs, are effectively conjugated and unable to form adducts </li></ul><ul><li>Repair mechanisms that enable cells to tolerate low levels of potentially mutagenic DNA adducts without biological consequences </li></ul><ul><ul><li>Difficult to demonstrate and quantify, but some DNA-reactive carcinogens have been shown to exhibit thresholds </li></ul></ul>
  17. 17. <ul><li>Non-relevant mechanisms for +ves in vitro would not be expected to occur in animals in vivo , or only under certain conditions </li></ul><ul><li>Chemicals inducing DNA damage via a threshold mechanism can produce +ve results in vivo if a high enough exposure can be achieved </li></ul><ul><li>Safety evaluation of a threshold genotoxin is therefore quantitative rather than qualitative , and more difficult to manage without animal data </li></ul>
  18. 18. <ul><li>Enzyme induction </li></ul><ul><li>Imbalance of DNA precursors </li></ul><ul><li>Energy depletion </li></ul><ul><li>Production of active oxygen species </li></ul><ul><li>Lipid peroxidation </li></ul><ul><li>Sulphydryl depletion </li></ul><ul><li>Nuclease release from lysosomes </li></ul><ul><li>Inhibition of protein synthesis </li></ul><ul><li>Protein denaturation </li></ul><ul><li>Ionic imbalance </li></ul><ul><li>High osmolality </li></ul><ul><li>Low pH </li></ul>* High cytotoxicity was not identified at this time
  19. 19. <ul><li>Inhibitors of DNA gyrase can also cause other changes both in bacteria and mammalian cells </li></ul><ul><ul><li>Inhibitors of topo I or II do not bind to DNA but seem to increase no. of strand breaks or prevent their resealing </li></ul></ul><ul><ul><li>Activity can produce point mutations, but predominantly chrom abs, recombination and aneuploidy </li></ul></ul><ul><li>Chemicals primarily inhibiting mammalian topo enzymes (m-AMSA, etoposide) will be genotoxic at human exposures </li></ul><ul><ul><li>Critical to determine whether primary or secondary effect & concs at which topo inhibition occurs </li></ul></ul>
  20. 20. <ul><li>Deoxyribonucleotide pool imbalances aberrant DNA replication/repair multitude of genetic effects - mutation, DNA breaks, chrom abs and MN </li></ul><ul><li>Should lead to genotoxicity without DNA interaction - should be a threshold </li></ul><ul><li>Methotrexate, other antifolate drugs, hydroxyurea and FUdR disrupt nucleotide triphosphate levels </li></ul><ul><ul><li>Can effects be reversed by restoring nucleotide pools balance? </li></ul></ul><ul><ul><ul><li>Chrom abs induced by excessive thymidine are reduced by simultaneous addition of deoxycytidine </li></ul></ul></ul><ul><ul><li>If genotoxic response only partially reversed, 2nd (non-threshold) mechanism may be involved? </li></ul></ul>
  21. 21. <ul><li>Paracetamol (acetaminophen) inhibits ribonucleotide reductase, but genotoxicity probably due to NAPQI metabolite </li></ul><ul><ul><li>NAPQI conjugates to glutathione </li></ul></ul><ul><ul><li>If conjugation saturated, chom abs & DNA damage induced  threshold of conjugation </li></ul></ul><ul><li>Other situations may be similar </li></ul><ul><ul><li>Determine conjugation threshold and absence of DNA adducts and genotoxicity below this level </li></ul></ul><ul><ul><li>Compare threshold with human exposures and employ conservative safety factors </li></ul></ul>
  22. 22. <ul><li>DNA-damage by various chemicals, or enhancement of others, accompanied by glutathione depletion </li></ul><ul><ul><li>Acrolein, chloropropanones, some acrylate esters, 1,3-butadiene, isobutene, various metals, dichlorvos, diethylmaleate </li></ul></ul><ul><li>Obtain supporting evidence of threshold </li></ul><ul><ul><li>Look for different responses in different cell types with different constitutive levels of glutathione </li></ul></ul><ul><ul><li>Add glutathione to treated cultures to see how the genotoxic response is modified or eliminated </li></ul></ul>
  23. 23. <ul><li>Multi-fibre nature of mitotic spindle means it can function effectively even when experiencing some damage </li></ul><ul><li>Widely accepted that spindle inhibitors exhibit threshold (non-DNA target) </li></ul><ul><ul><li>Identify induction of polyploidy or centromere +ve micronuclei </li></ul></ul><ul><ul><li>Non-disjunction between daughter nuclei is most sensitive measure of threshold (Elhajouji, Marshall, Zijno) </li></ul></ul>
  24. 24. <ul><li>MN that contain whole chromosomes (that failed to attach to spindle) will contain centromeres </li></ul><ul><li>Can look for these using fluorescently-tagged DNA probe against 1 or all (pan) centromeres </li></ul><ul><li>Induction of predominantly centromere +ve MN indicates aneuploidy mode of action </li></ul><ul><ul><li>Will have a threshold because results from damage to spindle and not to DNA </li></ul></ul><ul><ul><li>Important in risk assessment and establishing safe levels of exposure </li></ul></ul>
  25. 25.
  26. 26.
  27. 27. <ul><li>If no convincing experimental evidence of threshold or non-relevant mechanism, evidence of absence of direct DNA-mediated effect can help </li></ul><ul><ul><li>Should be no DNA adducts if chemical interacts with non-DNA target </li></ul></ul><ul><ul><li>Can 14 C-labelled chemical bind to DNA of cells/bacteria at concs. that are genotoxic? </li></ul></ul><ul><ul><ul><li>24 hr treatments with “hot” chemical may not be possible </li></ul></ul></ul><ul><ul><li>32 P-postlabelling is more sensitive, but selection of solvents/conditions affected by type of adduct (but if no adduct expected????) </li></ul></ul><ul><ul><ul><li>How convincing is absence of spots? </li></ul></ul></ul>
  28. 28. <ul><li>Concept acknowledges human exposure threshold (including those of unknown toxicity) below which no significant human health risk exists </li></ul><ul><li>Based on analysis of carcinogenic potencies initially of 343 (and subsequently >700) chemicals from Gold et al database (CPDB) </li></ul><ul><li>FDA threshold of regulation for food contact materials determined that, for most carcinogens, lifetime exposure to 1.5 μ g/day would increase cancer risk by 1 in 10 6 </li></ul>
  29. 29. <ul><li>Gold database contained genotoxic and non-genotoxic carcinogens </li></ul><ul><li>ILSI Europe Task Group (Kroes et al , 2004) determined that for genotoxic chemicals (high potency carcinogens, structural alerts), TTC would be 0.15 μ g/day for lifetime risk of 1 in 10 6 </li></ul><ul><li>For drug substances, providing health benefits, argued that lifetime cancer risk of 1 in 10 5 would be acceptable, therefore TTC of 1.5 μ g/day proposed for genotoxic impurities </li></ul>
  30. 30. <ul><li>MN are usually measured in bone marrow cells </li></ul><ul><li>Erythrocyte precursor cells at the time of their last division are the target </li></ul><ul><li>When the nucleus is expelled (to form an erythrocyte) any chromosome fragments or non-incorporated whole chromosomes are left behind and appear as micronuclei </li></ul><ul><li>MN can also be measured in reticulocytes in peripheral blood </li></ul><ul><li>Centromeric probes can be used (aneuploidy) </li></ul>
  31. 31. In vivo micronucleus assay 6-8 h 18-22 h Erythroblasts Immature erythrocytes Mature erythrocytes Bone marrow Peripheral blood Clastogen
  32. 32.
  33. 33.
  34. 34. <ul><li>Acute study most common for bone marrow </li></ul><ul><ul><li>1 administration with 2 sampling times (24 & 48 hr) or 2 administrations with 1 sampling time (24 hr) </li></ul></ul><ul><li>5 animals/sex per dose group </li></ul><ul><li>3 dose levels if toxic (or 1 if non-toxic) </li></ul><ul><li>2000 polychromatic erythrocytes (PCE) per animal scored for MN </li></ul><ul><li>Ratio of PCE to normochromatic erythrocytes gives measure of bone marrow toxicity </li></ul><ul><li>Measure concentration of chemical in plasma </li></ul>
  35. 35. <ul><li>Micronuclei can also be measured in young reticulocytes (CD71 expressing) in peripheral blood </li></ul><ul><li>Because spleen of many species removes micronucleated erythrocytes, need to score larger numbers of cells to detect a +ve effect </li></ul><ul><ul><li>Successfully done for mice, rats, dogs & humans </li></ul></ul><ul><li>This can be done by flow cytometry where 20,000 to 2 million cells per sample can be scored </li></ul><ul><li>Animals can be bled at different intervals (don’t have to sacrifice as for bone marrow – non-invasive) and pre-dosing bleeds provide additional control data </li></ul>
  36. 36. Peripheral Blood MN Methodology Inside the Cytometer FITC PI 488nm laser NCE (No Fluorescence) Reticulocyte (Green) MN-Reticulocyte (Green + Red) Platelet (Yellow) Key PE
  37. 37. EMS - 3 hr Peripheral Blood MN Methodology Data Output
  38. 38. <ul><li>MN can also be measured in liver </li></ul><ul><li>Surgery (partial hepatectomy) is needed to make cells divide </li></ul><ul><li>Alternative recommendation is to test in young rats (before liver is fully grown) but metabolism is also not mature </li></ul><ul><ul><li>Results not relevant for adult rats may be obtained </li></ul></ul><ul><li>Techniques also being developed to measure MN in skin, colon, testes etc. </li></ul>
  39. 40. <ul><li>Howell-Jolly bodies are small nuclear remnants that have the colour of a pyknotic nucleus & contain DNA </li></ul><ul><ul><li>Spherical in shape; usually only 1/cell but may be numerous </li></ul></ul><ul><li>Usually removed by the spleen (present in blood after splenectomy & in hyposplenic state, in haemolytic anaemia, megaloblastic anaemia) </li></ul><ul><li>During normal erythrocyte maturation they arise from nuclear fragmentation (karyorrhexis) or incomplete expulsion of the nucleus </li></ul><ul><li>Stimulate erythropoiesis and increased karyorrhexis will lead to a transient increase in MN </li></ul><ul><ul><li>erythropoiesis is a response to secretion of erythropoietin (EPO) by the kidney </li></ul></ul>
  40. 41. <ul><li>Administration of EPO </li></ul><ul><li>Hypoxia leads to secretion of EPO </li></ul><ul><ul><li>iron deficiency </li></ul></ul><ul><ul><li>thyroid dysfunction </li></ul></ul><ul><ul><li>thyroid, adrenocortical and human growth hormones </li></ul></ul><ul><ul><li>hypothermia? - reduced O 2 uptake by Hb? </li></ul></ul><ul><ul><li>hyperthermia? - cancer patients on hyperthermic therapy show increased EPO </li></ul></ul>
  41. 42. MN % mg/kg
  42. 43. <ul><li>In MN test at 200 mg/kg, body temperature dropped from 37 o C to 24 o C within 6 hr </li></ul><ul><li>Temperature was still as low as 29 o C at 48 hr </li></ul><ul><li>10 o C decreases in temperature for several hours, induced by reserpine & chlorpromazine, have resulted in 2-3-fold increases in MN at late sampling times </li></ul><ul><li>Hypothermia rescue study therefore performed with CNS Drug X </li></ul>
  43. 44. MN/1000 PCE mg/kg Drug X
  44. 45. <ul><li>Most widely used is the single cell gel electrophoresis (Comet) assay </li></ul><ul><ul><li>Measures DNA damage as it migrates from lysed cells in an electrical field (electrophoresis) - the more damaged the DNA, the more of it and the further it migrates </li></ul></ul><ul><ul><li>Any cell culture, or any tissue from which single cell suspensions can be made can be used </li></ul></ul><ul><li>Cells do not have to be dividing </li></ul><ul><li>By using alkaline conditions any lesions in DNA are converted to strand breaks </li></ul>
  45. 46. <ul><li>Cells mixed with agarose </li></ul><ul><li>Spread on microscope slides </li></ul><ul><li>Lysis </li></ul><ul><li>Unwinding (pH): expresses different types of DNA damage as strand breaks – next slides </li></ul><ul><li>Electrophoresis </li></ul><ul><li>Neutralization </li></ul><ul><li>Image analysis: Automated, Manual </li></ul>
  46. 47. <ul><li>Unwinding DNA at different pH expresses different types of DNA damage as strand breaks (SBs), resulting in fragments of DNA. </li></ul><ul><ul><li>7-8 - Double Strand Breaks, Crosslinks </li></ul></ul><ul><ul><li>12.1 - Strand Breaks, Excision-repair Sites, Crosslinks </li></ul></ul><ul><ul><li>>13 - Strand Breaks, Alkali-labile Sites, Excision-repair Sites, Crosslinks </li></ul></ul><ul><li>These fragments of DNA are then subjected to electrophoresis and the DNA migration pattern analysed. </li></ul>More SBs Smaller DNA fragments Increased DNA migration
  47. 48. Data Collected by Image Analysis <ul><li>Length of DNA migration </li></ul><ul><li>(smallest detectable DNA) </li></ul><ul><li>% Migrated DNA </li></ul><ul><li>(amount of migrating DNA) </li></ul><ul><li>Olive Tail Moment </li></ul><ul><li>(migrated DNA x tail length) </li></ul>Head Tail Length of DNA migration
  48. 49. Cells with DNA migration Cells without DNA migration
  49. 50. <ul><li>Correlation of in vivo Comet assay results with rodent carcinogenicity very high </li></ul><ul><ul><li>Particularly useful with compounds negative in bone marrow MN study (Kirkland & Speit, 2008) </li></ul></ul><ul><li>Unclear whether DNA strand breaks can result from cytotoxic effects </li></ul><ul><li>Most published results with in vivo Comet assay from 1 lab, therefore consistency and ability to “travel” not well established </li></ul><ul><li>Collaborative trials on-going to try to establish OECD guideline </li></ul>
  50. 51. <ul><li>Mice have been engineered to carry a bacterial lacI or lacZ transgene on a lambda bacteriophage vector </li></ul><ul><li>Multiple copies of the transgene exist in each cell </li></ul><ul><li>The transgene is neutral and confers no advantage or disadvantage to the mouse </li></ul><ul><li>After treatment of the mice, the DNA is extracted, “packaged” into viable lambda bacteriophage, which are assayed on sensitive bacteria where mutations in the transgene can be detected </li></ul>
  51. 52. <ul><li>MUTATION IN TRANSGENES </li></ul>Treat mice take tissues isolate DNA package colour selection ( LacI ) positive selection ( LacZ ) (cut at cos sites:1 lambda vector per phage) 1 lacZ or lacI gene
  52. 53. Cohesive end BamH1 BamH1 BamH1 BamH1 XHol BamH1 EcoR1 EcoR1 EcoR1 EcoR1 BamH1 Cohesive end LacZ 0 50 kb
  53. 54. <ul><li>The lambda phage will lyse the sensitive bacteria and form plaques </li></ul><ul><li>By growing the bacteria on agar containing a particular substrate for the ß-galactosidase enzyme, mutant plaques can form a colour whereas wild-type plaques are colourless </li></ul><ul><li>For the lacZ system a positive selection system has been devised which means wild-type phage do not form plaques (reduces size of assay) </li></ul>
  54. 55.
  55. 56. <ul><li>Muta TM Mouse (lacZ ) and Big Blue ( lacI ) most widely used </li></ul><ul><li>Treatment period is usually 28 days (followed by further 3-day expression period) to accommodate slow-acting mutagens </li></ul><ul><ul><li>Mutations from fast-acting mutagens plateau </li></ul></ul><ul><li>At least 3 dose levels and 5 animals/sex/group </li></ul><ul><li>Mutations can be detected in any tissue, therefore useful for site-of-contact mutagens (skin, GI tract, lungs) </li></ul><ul><li>Limitation - not sensitive to chemicals inducing large DNA deletions (delete through cos sites; will not package) </li></ul><ul><ul><li>Gpt -delta or spi mutation models (see papers by Nohmi et al ) are able to detect deletion mutations </li></ul></ul>
  56. 57. <ul><li>Is there a credible mechanistic/metabolic reason why +ve results in vitro are expected to have threshold, or only occur in conditions to be irrelevant for human exposure? </li></ul><ul><li>Are there -ve results from 2 appropriate in vivo tests? </li></ul><ul><ul><li>need toxicokinetic data to relate exposure to the genotoxic concentrations in vitro and to human exposure </li></ul></ul><ul><li>Are exposure (or safety) margins acceptable? </li></ul><ul><ul><li>human plasma C max compared with highest no-effect concentration (NOEC) in +ve in vitro test(s) </li></ul></ul><ul><ul><li>human exposure (area under conc. curve, AUC) compared with AUC in -ve in vivo tests </li></ul></ul>
  57. 58. <ul><li>CRITERIA FOR POSITIVES </li></ul><ul><li>Statistical significance at 1 or more dose points </li></ul><ul><ul><li>sensitive e.g. p<0.01 for 1.4-fold increase in Ames mutants (Dunnett's test) </li></ul></ul><ul><li>Fold increase over control </li></ul><ul><ul><li>conservative e.g. get significant dose responses without breaching pre-set level </li></ul></ul><ul><li>Frequencies exceed historical control range </li></ul><ul><ul><li>good for low frequency events (chromosomal aberrations, micronuclei), not so for Ames or tk mutations </li></ul></ul><ul><li>Frequencies exceed pre-set level </li></ul><ul><ul><li>Global evaluation factor used for mouse lymphoma assay </li></ul></ul><ul><li>Dose response </li></ul><ul><ul><li>probably most relevant in terms of biological effect </li></ul></ul><ul><li>Reproducibility </li></ul><ul><ul><li>would be required for publication in peer-reviewed journal </li></ul></ul>
  58. 59. <ul><li>I hope I have been able to show that decision making with genotoxicity data is not easy </li></ul><ul><li>There are many factors involving study design and potential for artefacts that need to be considered in order to understand whether a test result truly indicates lack of, or potential for, human hazard </li></ul><ul><li>Thank you for your attention </li></ul>

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