Mutagenesis, Vol. 17, No. 6, 551-579,
November 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Abstracts of the United Kingdom Environmental Mutagen Society 25th Anniversary Meeting, June 30 to July 3, University of Plymouth, UK
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1. The enemy within: Endogenous DNA lesions as substrates for DNA repair |
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 Top 1. The enemy within:... 2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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T. Lindahl, D.E. Barnes, A. Dulic, H. Nilsen, P. Robins, B. Sedgwick, S. Trewick
Clare Hall Laboratories, Cancer Research UK London Research Institute, South Mimms, Herts EN6 3LD, UK
Chromatin is susceptible both to environmental mutagens and to many endogenously generated compounds. Intracellular DNA damage can occur by hydrolysis and oxidation as well as by reaction with lipid peroxidation products, hydroxylated estrogens, endogenously generated alkylating agents, and amino acid degradation products. Such potentially mutagenic and carcinogenic events are counteracted by DNA repair. The major repair pathways have been elucidated, but there are still some surprises when the correction mechanisms for unusual DNA lesions are investigated. A novel mechanism of DNA repair involves direct reversal of base alkylation damage by oxidative DNA demethylation employing iron-dependent free-radical chemistry.
Variable DNA repair due to single nucleotide polymorphisms in relevant genes may turn out to be a key factor to explain variations in individual susceptibility within a population. Transgenic mice with specific gene knockouts provide unique tools to evaluate the importance of DNA repair for suppression of carcinogenesis and aging. We have constructed KO mice defective in either the uracil-DNA glycosylase (UNG) or 8-oxoG-DNA glycosylase (OGG1). The abnormal accumulation of DNA damage in vivo and other phenotypic traits occur in such mice. Crosses with other repair-defective mice have been performed to enhance biological effects and eliminate back-up repair functions. Such experiments further clarify the physiological roles of these DNA repair enzymes, and interactions between different repair pathways.
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2. Molecular mechanisms of UV-induced mutations as revealed by the study of human cells expressing DNA polymerase 
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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Alain Sarasin, Patricia Kannouche*, Alan R. Lehmann* and Anne Stary
Laboratory of Genetic Instability and Cancer, UPR 2169 CNRS, 94801 Villejuif, France and *Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, UK.
Solar UV induces lesions at bipyrimidine sites in genomic DNA. These photoproducts are normally removed or tolerated by error-free processes. However lesions that escape these error-free mechanisms can give rise to mutations. Thus cells from patients with xeroderma pigmentosum (XP), a rare autosomal, recessive human syndrome characterized by early and numerous skin lesions and cutaneous tumours on sun-exposed body sites, are hypermutable after UV-irradiation due to a defect in Nucleotide Excision Repair (NER). Cells from XP variant (XPV) patients are also UV-hypermutable even though NER is normal. XPV cells are deficient in DNA polymerase , which is able to carry out `error free' bypass of cyclobutane pyrimidine dimers. It is likely that in the absence of pol in XPV cells, other recently discovered polymerases are able to bypass the DNA lesions, but with lower efficiency and lower fidelity. The high rate of misincorporation by these polymerases can explain the cancer-proneness of the XP variant patients. DNA polymerases involved in translesion synthesis in human cells appear to be constitutively expressed, in contrast to what has been found in bacteria. We have used UV-irradiated shuttle vectors to determine the mutagenic characteristics of DNA polymerases implicated in translesion synthesis in normal cells, XPV cells or XPV cells complemented with the wild type pol gene. In normal cells, the bypass by pol of UV-induced DNA lesions containing Cs appears to be more error prone than those containing Ts. In contrast, in XPV cells, deficient in pol , the proportion of mutations at lesions containing Ts is increased, suggesting that pol bypasses lesions containing Ts in a relatively error-free manner. UV-irradiation of host cells prior to transfection with unirradiated shuttle vectors increases the mutation frequency substantially in normal, XPV and corrected XPV cells to similar extents. This demonstrates that mutagenic polymerases are activated after UV-irradiation and produce mutations in undamaged DNA. The similar increase in normal and XP-V cells suggests that pol is unlikely to be responsible for this increase.
The results obtained with shuttle vectors correlate pretty well with previous studies analysing the type and distribution of mutations in key genes modified in human skin cancers such the p53 gene.
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3. Mechanisms of carcinogenicity/chemotherapy by O6-methylguanine |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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Geoffrey P. Margison1, Andrew C. Povey2, Mauro F. Santibáñez Koref3
1Cancer Research UK Carcinogenesis Group, Paterson Institute for Cancer Research, Manchester M20 4BX, UK, 2Centre for Occupational and Environmental Health, University of Manchester, Manchester M13 9PL, UK, 3Max Delbrück
Centre for Molecular Medicine, Robert Rössle Str. 10, Berlin 13 092, Germany
Alkylating agents are a structurally diverse group of compounds that cause a wide range of biological effects including cell death, mutation, and cancer. DNA damaged by these agents contains widely different amounts of 12 alkylated purines/pyrimidines and two phosphotriester isomers but the biological effects appear to be mediated predominantly by attack at the O6-position of guanine. DNA extracted from various normal human tissues contains detectable levels of O6-methylguanine, the source of which has not been defined. Given that this lesion can generate point mutations and can initiate mismatch-repair mediated DNA recombination following DNA replication, it seems worthwhile to consider the possible contribution of these events to the aetiology of human cancer. In terms of cancer treatment, certain cancer chemotherapeutic agents exploit the cytotoxicity of O6-methylguanine. However, repair of this damage by O6-alkylguanine-DNA alkyltransferase is often very efficient in resistant tumour tissues, but inefficient in tissues that manifest toxic side effects. This has given rise to strategies for repair modulation to enhance chemotherapeutic efficacy.
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4. Characterisation of the Fanconi anaemia gene pathway using Chinese hamster cell mutants |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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James B. Wilson1, Mark A. Johnson1, Alan D. D'Andrea2, Nigel J. Jones1
1School of Biological Sciences, Donnan Laboratories, University of Liverpool, Liverpool, L69 7ZD, UK. 2Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA
Fanconi anaemia (FA) is a chromosome instability syndrome that exhibits mutagen-sensitivity (especially to DNA cross-linkers) and has a high predisposition to cancer, particularly acute myeloid leukaemia. Mutations in at least eight genes can give rise to the disorder. Five of the proteins (FANCA, C, E, F and G) assemble in a multi-protein nuclear complex that, with BRCA1, is required for activation of a sixth, FANCD2. During S-phase, or following DNA damage, FANCD2 is monoubiquitinated and is targeted to BRCA1/ RAD51 nuclear foci. We are studying several Chinese hamster (CH) mutants that are phenotypically similar to FA cells, including two newly isolated diepoxybutane-sensitive mutants. Neither mutant expresses the activated isoform of FANCD2, indicating a defect in the FA gene pathway. Additionally, immunoprecipitation (IP) western-blotting analysis shows that the FA protein complex fails to form in one of the two mutants. This mutant also exhibits extreme cellular and chromosomal sensitivity to other cross-linking agents including MMC. Mutant NM3 has been shown to be defective in the FANCG/XRCC9 gene. Transfection of the human FANCG/XRCC9 cDNA restores the FA protein complex and expression of activated FANCD2 in NM3 and also corrects its sensitivity to MMC, DEB, bleomycin and MMS/EMS. These data indicate that cellular resistance to these diverse agents requires FANCG, and that the FA pathway, via activation of FANCD2, is involved in maintaining genomic stability in response, not only to interstrand cross-links, but also other types of DNA damage (Carcinogenesis, 22, 1939–1946). We are screening other CH mutants for their involvement in the FA gene pathway by assaying them for the expression of the two FANCD2 isoforms. CH cell lines defective in FA genes are likely to prove important in characterising the FA pathway, its role in maintaining genomic stability and its interactions with other proteins/pathways including BRCA1 and RAD51-mediated homology-directed DNA repair.
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5. Variation in DNA repair capacity in normal humans |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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A.R. Collins, V. Harrington
Rowett Research Institute, Aberdeen, UK.
Much effort has been made to provide an accurate estimate of the background level of base oxidation in the DNA of normal human cells. Many published results were artificially high because of oxidation of guanine during sample preparation. It now seems that the true level is no more than one 8-oxoguanine per 106 guanines. This low level is maintained as a dynamic steady state; input of damage is limited by antioxidant defences, and the damage that does occur is removed by cellular DNA repair. Variation in DNA repair capacity can result from genetic polymorphisms, of which several are known. It is also possible that repair is modulated by environmental factors. There are different approaches to measuring repair rates as a biomarker in population studies. Cells can be incubated after treatment with DNA damaging agent and the amount of damage remaining monitored with time. Freshly isolated lymphocytes seem to be poor at repair of H2O2-induced damage, perhaps because they suffer further input of damage from atmospheric oxygen during the incubation. In an alternative in vitro repair assay based on the comet assay, cultured cells containing oxidised bases are embedded in agarose and lysed; they provide the substrate for repair enzymes in a simple extract prepared from the lymphocytes under test. The extract and substrate are incubated, and the incision rate estimated from the rate of accumulation of DNA breaks. This assay is specific for base excision repair (Ogg1). Differences between individuals are maintained when the same subjects are sampled again. We have found a stimulation of this repair activity by dietary supplementation, and are currently investigating repair in a population occupationally exposed to asbestos fibres. (Work supported by EC contract no. QLK4-1999-01629.)
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6. Genetic Effects Of Environmental Contaminants: From Molecules To Populations |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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John W. Bickham
Department of Wildlife and Fisheries Sciences, Texas A&M University, College Station TX, USA 77843-2258
Environmental contaminants cause a cascade of effects in wildlife from the molecular level to the population level. Genotoxic chemicals affect DNA directly by forming bulky adducts which can lead to somatic or heritable mutations. Somatic mutations cause cellular damage that is ultimately expressed as a diseased condition. Such stress can reduce viability, survivability, and reproductive success. Heritable effects, such as deleterious germ-line mutations, also produce these effects. Subsequently the genetic makeup of populations might be altered by the reduction of genetic variability, the increase of deleterious alleles, or the fixation of low-frequency alleles as the population becomes adapted to new environmental conditions. Most of these effects are likely to be sublethal, but the implications for the long-term survival of populations inhabiting impacted areas can be profound. Population genetic effects also provide a challenge to the ecotoxicologist using biomarkers to document pollution because contaminated and reference populations are usually assumed to be identical. Selection for survivorship in contaminated environments, as well as immigration into contaminated sites that act as ecological sinks, can confuse the interpretation of biomarker responses. Studies show that population genetic data, used in combination with biomarkers, achieve a more complete understanding of contaminant effects in wildlife populations.
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7. From ethics to genetics: protecting the environment with respect to ionizing radiation. |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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R.J. Pentreath
Environmental Systems Science Centre, The University of Reading, Whiteknights, Reading RG6 6AL, UK.
Much is known about the biological effects of radiation, and the theory and practice of human radiation protection has been developed in a systematic way. But there has been no parallel attempt to develop a systematic approach to protection of the environment. This situation is now changing and it has many facets, including the ethical issues surrounding the question of why and how the environment should be protected. These range from strictly anthropocentric to what have been termed biocentric, and ecocentric, points of view. Similarly, the needs of environmental management in general have been considered, from environmental exploitation, pollution control, and the needs of conservation and habitat protection, all of which are often reflected in some form of legislative agreement. One of the major features of the successful development of the system to protect humans has been the concept of Reference Man. It has therefore been proposed that a system based on the concept of a number of Reference Fauna and Flora might be used to form the basis of a system for environmental protection. This would allow the development of a more common approach to estimates of radiation exposure, dosimetry, and the systematic characterisation of different forms of radiation effects. Such effects, at the level of DNA damage, are probably very similar for all plant and animal cells, but their expression at tissue, organ, and whole-body levels is markedly different. Of relevance to environmental protection, however, are broad categories of effect, such as early mortality, reduced reproductive success, and scorable cytogenetic effects. These, in turn, may have important consequences for estimating and evaluating the impact of industrial practices, waste disposal, and the risks of accidents on the interpreted needs of conservation, habitat protection, and the maintenance of biodiversity – often depending, of course, on one's ethical point of view.
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8. Ecogenotoxicological evaluation of tritium (3H) on marine invertebrates |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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J.A. Hagger, M.H. Depledge, A.N. Jha
School of Biological Sciences, Plymouth Environmental Research Centre, University of Plymouth, Plymouth PL4 8AA, UK
The marine environment is often the ultimate recipient of radioactive contaminants. Tritium (3H), a soft beta emitter, is discharged in relatively large quantities all over the world, including the UK. Despite attempts to reduce the discharges of radionuclides, the amounts are likely to increase due to a perceived need for more nuclear power (to reduce emission of greenhouse gases) and to advance naval submarine technology. Although considered to be one of the least hazardous isotopes to humans, discharge of tritium is of concern given the fact that it is rapidly dispersed in the environment and taken up by organisms. Despite growing scientific and public concern, there has been however very limited study to evaluate the impact of this highly important radionuclide on aquatic biota. An integrated study was carried out to evaluate the potential effects of tritium (0.37–370 k Bq/ml or 0.01-10 µCi/ml) at various levels of biological organisation. The study evaluated the genotoxic (chromosomal aberrations and sister chromatid exchanges), cytotoxic (proliferative rate index), developmental and survival effects in embryo-larval stages of two ecologically relevant marine invertebrates, Mytilus edulis (blue mussel) and Platynereis dumerilii (ragworm). Depending upon the exposure scenario, dose received by the embryo-larval stages of these organisms was calculated to be in the range of 0.007–23.6 mGy. The study revealed mussels to be more sensitive than the worms for all the end points evaluated. The effects at chromosomal and cellular levels following exposure to this concentration/dose range were correlated with abnormality and mortality of the growing embryo-larvae. The increase in the mortality for mussels was found to be significantly correlated to both cytotoxic and genotoxic effects whereas for the worms an increase in abnormal embryo-larvae correlated to genotoxic parameters. The study emphasises the need of evaluating the biological effects of exposure to environmentally realistic levels of radionuclides in the natural biota.
9. Taking mutation testing into extreme environments.
David R. Dixon and Linda R.J. Dixon
Southampton Oceanography Centre, Waterfront Campus, European Way, Southampton SO14 3ZH, United Kingdom
Apart from their laboratory application, mutation-testing procedures have been used extensively in field studies to address questions relating to environmental quality and the effects of pollution on various types of wildlife. Given its widespread use as a sink for the disposal of chemicals and radionuclides, the marine environment has featured extensively in this type of study. Apart from its importance as a home for a wide variety of organisms, a large proportion of the world's human population lives close to or derives its food from estuarine or marine sources. However, unlike standard mutation testing procedures, which are aimed at a single species, environmental studies have to address both human interests and those of a wide variety of other species. Unfortunately, the complexities of many test procedures limit their application outside the laboratory environment. During our deep-sea studies, the opportunity arose to investigate the special adaptations that allow members of the hydrothermal-vent fauna to survive and reproduce in what is arguably one of the most contaminated environments on the face of the planet. However, much of this research needed to be performed while on board ship, which is not a suitable platform for carrying out electrophoresis and certain other types of laboratory analysis. In an attempt to overcome these practical difficulties, tissue samples (from the Atlantic vent mussel Bathymodiolus azoricus) were first preserved in a relatively non-toxic buffer, which retained the DNA integrity (5% SDS, 250mM EDTA, PH 8, and 50 mM Tris, pH 8), and then subjected to neutral gel electrophoresis (GE) once back in a terrestrial laboratory. (Note alkaline GE was less informative due to a high incidence of alkali-labile sites.) The neutral GE assay gave promising results with a significant increase in DNA damage being recorded when animals were: a) initially collected from their high pressure environment (80 and 170 bars) and b) subsequently exposed to a range of mutagens including a pro-oxidant (H2O2). Conversely, a reduction in DNA damage was observed when: c) animals were kept at 1 bar for a few days and d) following their removal from the toxicant. These findings suggest that DNA repair, which is particularly evident in younger individuals, plays an important part in animal survival in this extreme deep-sea environment. Apart from this specific application to hydrothermal-vent studies, the GE assay offers access to a wide range of other, lesser-known species, both aquatic and terrestrial, which have previously proved inaccessible to mutation testing.
This study forms part of the research programme of the EU-funded VENTOX project (EVK3CT1999-00003).
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10. Effects of Exposure to 2,4-D or TCDD on Gonadal Differentiation in the Soft-shell Clam, Mya arenaria. |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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R.A. Butler1, R.J. Van Beneden1,2, George R. Gardner3
1School of Marine Sciences, University of Maine, Orono, ME 04469, USA. 2Department of Biochemistry, Microbiology and Molecular Biology, University of Maine, Orono, ME 04469, USA. 3Environmental Protection Agency, Narragansett, RI 02882, USA
The vertebrate aryl hydrocarbon receptor (AHR) mediates the toxic response to TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) and other halogenated aromatic hydrocarbons (HAHs). Populations of soft-shell clams (Mya arenaria) from eastern Maine have historically exhibited a high prevalence of gonadal tumours of unknown aetiology. One hypothesis is that development of these tumours results from HAH exposure and is mediated through the AHR. To address this, adult clams from a feral population in eastern Maine with a zero prevalence of gonadal tumours were exposed in the laboratory to either the herbicide 2,4-D (2,4-diphenoxyacetic acid) or TCDD (+/– diethylnitrosamine, DEN). Clams were exposed under static conditions for 48 hrs and returned to flow-through tanks for 120 hr recovery between exposures and during post-treatment. Exposure Protocol I: control; 2 pulses of 100 ppm DEN; 2 of 10 pptr TCDD; and 2 of DEN plus 2 of TCDD. Exposure II: control; five pulses to 5 or 10 ppm 2,4-D. Individuals were sacrificed at six months post exposure. Histological analysis did not indicate tumour formation. Both 2,4-D and TCDD inhibited gametogenesis to an extent that gender was indeterminate. Exposure to 2,4-D resulted in a significant dose-dependent increase in individuals with undifferentiated gonads, males appearing more susceptible to treatment than females. TCDD (+/– DEN) inhibited gamete development to a greater extent in males than females. Expression of the cell cycle regulator proteins p53 and AHR was investigated. Levels of p53 protein were significantly lower in animals exposed to 2,4-D, but not in those exposed to TCDD. Female clams exposed to TCDD exhibited significantly lower levels of AHR protein than controls. Expression of AHR protein appeared to correlate with eggs; none was detected in male or undifferentiated gonads. Studies are underway to further investigate the molecular basis of interference with gametic differentiation.
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11. Toxicogenomics in non-model organisms: use of custom microarrays and subtractive hybridisation to study differential gene expression in the European flounder |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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J.K. Chipman, T.D. Williams, S.D. Minchin, B. Lyons, K. Gensberg
The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
There is increasing interest in gaining information on gene expression changes to supplement measures of genetic toxicity in relation to carcinogen hazard identification. Whereas this is relatively tractable in model organisms for which adequate sequence information and other databases are available, the situation is somewhat more difficult when studying genotoxicity in organisms of environmental relevance. Since analysis of p53 and ras mutations has failed to characterise hepatic tumours in the European flounder, gene expression profiles to indicate pollutant impact are particularly required. We have initiated studies by designing degenerate primers for a range of genes of toxicological interest and amplified and sequenced gene fragments from flounder ovary and liver cDNA and genomic DNA. Seventy-four of these Expressed Sequence Tags (ESTs) were identified by homology, whereas thirty had novel sequences. PCR products from these clones were used to produce a cDNA microarray. In a pilot study, mRNA was prepared from the liver of flounder (pooled from ten female fish) from the relatively polluted Tyne estuary (known to have elevated PAH, metal and xenoestrogen concentrations) and the relatively unpolluted Alde estuary. cDNA was labelled with Cy5-dCTP or Cy3-dCTP and hybridised to the array. Genes that were upregulated (1.5 to 5-fold) in polluted samples included NADP-menadione oxidoreductase (NMO), a zona-pellucida related transcript (ZPC), delta aminolevulinic acid synthase (
-ALAS), glycoprotein 36B (GP36B), metallothionein and three novel transcripts. Downregulated (<0.66-fold) transcripts included 
-fibrinogen, 
2HS-glycoprotein and an unidentified transcript containing a zinc finger motif. The mean coefficient of variation for each gene was 11%. Subtractive suppressive hybridisations between liver cDNA of flounder isolated from the Alde and Tyne have, to date, provided ~250 clones. Initial data show that these are largely novel sequences, several, however have been identified by homology and are currently under investigation as potential novel biomarkers. Now that the strategy for this feral species has been taken to proof of concept, these additional genes will be added to the microarray for further analysis of flounder from laboratory and field exposures. Additional subtractive work is currently identifying differentially expressed genes following laboratory exposures including that to the genotoxic agent benzo(a)pyrene. Through a recent EU collaboration, a more comprehensive EST array is being produced from a normalised flounder liver library to allow an `open' system of profiling of responses to be achieved.
This work was funded by the NERC with support from CEFAS
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12. Mutagenicity, rodent carcinogenicity and endocrine disruption: standard protocols for established assays versus the need to deploy new assays. |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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John Ashby
Syngenta Central Toxicology Laboratory, Alderley Park, Cheshire
Two influences are currently making it difficult to evaluate the toxicity of chemicals. The first is uncertainty regarding `gold standards'; the second is the conflict intrinsic to the competing needs for standard regulatory test protocols and the need for rapid deployment of newly developed assays. Discussion of these complicating influences is the best way of reducing their impact. The gold standard problem is illustrated by the following questions - should cytogenetic assays or accelerated transgenic rodent cancer bioassay models be judged in their own right, or in relation to their correlation with 2-year rodent carcinogenicity bioassay data? The assay protocol problem is illustrated by current anguishing over a standard test protocol for the 50-year old rodent uterotrophic assay when a more pressing need is for the integration of newer molecular techniques into screening for endocrine disruptors.
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13. Multicolour-FISH in two and three dimensions |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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Michael R. Speicher
Institut für Humangenetik, Technische Universität München, München, Germany
Recent developments in molecular cytogenetics open new avenues to study mechanisms of interaction of chemicals with chromosomes. Chemicals may induce both numerical and structural aberrations and in addition chromosomal instability. For a detailed analysis, sophisticated approaches at single cell resolution are needed. Multicolour-FISH allows new opportunities to analyse the genome in 2-dimensions, e.g. on metaphase spreads, or in 3-dimensions, e.g. in interphase nuclei. For twenty-four colour karyotyping, we use 7-fluorochrome multiplex-FISH (M-FISH). Advantages include that karyotyping can be automated and that the resolution for the detection of small interchromosomal rearrangements is unprecedented as compared to the classical banding technologies. M-FISH has been applied to study the effects of exposure to different agents, such as bulky-adduct-forming agents or methylating agents, on chromosomal stability.
For other applications it may be necessary to analyse the genome at the cellular level within the natural tissue context. Therefore, we developed a multicolour deconvolution technique for three-dimensional (3D) microscopy, which generates 3D data by optically sectioning the specimen. Deconvolution refers to a computational method used to reduce out-of-focus fluorescence in 3D microscopy images. Images are captured using an epifluorescence microscope equipped with a motorized table to collect a stack of images at defined levels in z-direction. After deconvolution, 3D-reconstruction algorithms are applied. Multiple regions within the genome can now be analysed simultaneously within biological specimen with a thickness of up to 30 µm.
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14. Genome stability in Fanconi anaemia: telomere studies and molecular biology of FANCD2 |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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J. Surrallés, M. Bogliolo, O. Cabré, E. Callén, V. Castillo, A. Creus, R. Marcos
Group of Mutagenesis, Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
Fanconi anaemia (FA) is a rare genetic disease characterized by chromosome instability, congenital malformations, progressive bone marrow failure and cancer susceptibility. The genetics of FA is highly heterogeneous with at least 8 different genes involved (FANCA, B, C, D1, D2, E, F and G), all of them except FANCB and FANCD1 have been cloned. FA proteins A, C, E, F, and G assemble in a nuclear complex that is required for the activation, via monoubiquitination, of FANCD2. The recently cloned FANCD2 gene is thought to be a key player in the FA pathway since FANCD2 binds to BRCA1 in nuclear foci and is phosphorylated by ATM in response to genetic damage. Thus, the two tumour supressor pathways, AT and FA, converge on FANCD2. We have cloned and sequenced* the cDNA of the Drosophila melanogaster homologue to hFANCD2 to show that although dFANCD2 is only partially similar in sequence to hFANCD2, the residues of ATM dependent phosphorylation and ACEFG-complex dependent monoubiquitination are highly conserved during evolution, indicating an essential role of these post-translational modifications in the FA pathway. We also observed that FANCD2 relocates to the site of damage in locally UV-irradiated nuclei in a progressive and persistent manner and forms nuclear foci only in S-phase cells. Since telomeres are related to chromosome stability, haematopoiesis and carcinogenesis and, in addition, FANCD2 binds to mouse autosomal telomeres in diplonema, we have studied telomere biology in FA. Our Q-FISH experiments showed that telomeres in FA patients shorten faster than in age-matched controls due to both replication-mediated shortening in bone marrow stem cells and accumulation of deleterious breaks at telomeres of differentiated T-cells. Consistent with shorter telomeres, we observed a 10-fold increase in end-fusions in FA. Immunohistochemistry studies in FA cell lines and corrected counterparts by retrovirus-mediated gene transfer of FANCA and FANCD2 showed that a functional FA pathway is not required for telomere binding of TRF2, the major telomere-binding protein preventing chromosome end-fusions. Finally, laser confocal microscopy analysis of FANCD2 and TRF2 suggest that, unlike NBS1, FANCD2 does not bind to telomeres in S-phase cells indicating that FANCD2 plays no role at telomeres during replication.
(*gene bank accession number AJ459772)
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15. What do human micronuclei contain? |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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H. Norppa, G. Falck
Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Topeliuksenkatu 41 a A, FIN-00250 Helsinki, Finland
As micronuclei (MN) derive from both chromosome breakage and chromosomes lagging behind in anaphase, the MN assay can be used to show both clastogenic and aneugenic effects. The distinction between the two phenomena is important, since the exposure studied often induces only one type of MN. This particularly concerns the use of MN as a biomarker of human genotoxic exposure and effects, where differences in MN frequencies between the exposed subjects and referents are expected to be small. A specific analysis of the right type of MN may considerably increase the sensitivity of detecting the exposure effect. MN harbouring whole chromosomes can be distinguished from those harbouring chromosomal fragments by the presence of a kinetochore or a centromere, using kinetochore antibodies or centromeric fluorescence in situ hybridization (FISH). The proportion of centromere-positive MN increases with age, which has primarily been attributed to an age-dependent micronucleation of the X and Y chromosomes. There is little evidence for a preferential inclusion of other human chromosomes in MN. The X chromosome tends to lag behind in anaphase, ending up in MN more frequently than autosomes. X laggards often appear to contain both sister chromatids, but the ratio of double and single chromosomes in MN is unknown. An open question is also whether micronucleation equally occurs for chromosome-type and chromatid-type fragments or terminal and interstitial fragments. MN contents have been suggested to be modified by, e.g., genotoxic agents, age, sex, cell culture, and the use of cytochalasin B. Understanding the mechanistic origin of MN is essential for the proper use of this cytogenetic endpoint in biomarker studies, genotoxicity testing, and risk assessment.
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16. Application of molecular cytogenetics: Identification of an upstream regulator of HNF-4 expression by mapping the translocation breakpoint in a family with diabetes.
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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S. Ellard1, A.L. Gloyn1, M. Shepherd 1R.T. Howell2, E.M. Parry3, A. Jefferson4, E.R. Levy4, A.T. Hattersley1
1Department of Diabetes and Vascular Medicine, University of Exeter, 2Regional Cytogenetics Centre, Southmead Hospital, Bristol, 3School of Biological Sciences, University of Wales, Swansea, 4The Wellcome Trust Centre for Human Genetics, University of Oxford
Monogenic human disorders have been used as paradigms for complex genetic disease and as tools for establishing important insights into mechanisms of gene regulation and transcriptional control. Maturity-onset diabetes of the young (MODY) is a monogenic dominantly inherited form of diabetes that is characterised by defective insulin secretion from the pancreatic beta-cells. Mutations in six different genes have been identified which result in this condition. Although a wide variety of mutation types have been identified, including missense, nonsense, frameshift and splice site mutations, there have been no reports of a chromosome deletion or translocation resulting in MODY. We report a pedigree where MODY co-segregates with a balanced translocation [karyotype 46, XX t(3;20) (p21.2;q12)]. The chromosome 20 breakpoint is within the region of one of the known MODY genes, HNF-4 (MODY1), 20q12. Fluorescence in situ hybridisation (FISH) analysis demonstrated that the breakpoint does not disrupt the coding region of this gene, but lies upstream of the conventional promoter (P1) and downstream of the recently described, alternate distal pancreatic promoter(P2) and HNF-4. We therefore report the first case of MODY due to a chromosome abnormality and provide evidence to confirm the crucial role of an upstream regulator of HNF-4 gene expression.
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17. An Analysis of the in vitro aneugenic activity of Bisphenol A. |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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Elizabeth M. Parry, Chiara Corso, Emma Quick, George Johnson, James M. Parry
Centre for Molecular Genetics and Toxicology, School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea, SA2 8PP, UK.
Bisphenol A (BP-A) is a monomer widely used as a raw material in the production of plastic products including polycarbonate drinks containers. A number of studies have demonstrated that BP-A has oestrogenic activity and can be classified as an environmental endocrine disruptor (Krishnan et al 1993).
We have undertaken studies in cultured human and hamster cells to evaluate the potential of BP-A to induce aneuploidy and to determine the mechanisms which may lead to aneugenic activity. In an in vitro binucleate micronucleus assay (Fenech 2000) in metabolically competent MCL-5 human lymphoblastoid cells, BP-A was a potent inducer of micronuclei (up to 10x at 30 µg/ml). The involvement of an aneugenic mechanism in the induction of micronuclei was indicated by the increase in the proportion of micronuclei containing whole chromosomes revealed by staining with kinetochore antibodies. This aneugenic activity of BP-A was confirmed by the analysis of the distribution of chromosome 7 and 20 in the nuclei of binucleate cells which showed a dose dependent increase in the non-disjunction of these two chromosomes.
To investigate the mechanism of the aneugenic activity of BP-A we undertook an analysis of the morphology of dividing cell in Chinese hamster V79 cells using staining with brilliant blue R (for spindle structure), Safranin O (for chromosome behaviour) and specific antibodies of ß and tubulins (for spindle and polar structures). These results demonstrated that BP-A induces dose dependent modifications of microtubular organising centres leading to multipolar cell divisions. Chromosome segregation in multipolar cells may result in significant levels of aneuploidy in BP-A treated cultures.
References
Krishnan A, et al 1993. Endocrinology 132: 2279–2286.
Fenech M 2000. Mutation Res 455: 81–95.
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18. The detection of p53 mutations in pre-malignant tissues; a biomarker for cancer risk. |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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Gareth Jenkins
Swansea Clinical School/School of Biological Sciences, University of Wales Swansea, SA28PP.
It is well known that point mutations arising in tumour suppressor genes and/or proto-oncogenes are early events in cancer progression. Indeed these point mutations often drive tumour formation. If detected early enough in cancer progression, these mutations can potentially predict cancer progression rates in individuals. In addition, this mutation data could also shed light on the causative mutagen exposure, due to the fact that mutagens induce characteristic mutation patterns (fingerprints). However, whilst it is relatively easy to detect late stage mutations in tumour tissues, it is extremely difficult to detect them in pre-malignant tissues. This is a consequence of the early mutations being present in a few cells surrounded by an excess of non-mutated cells. Most molecular techniques which are employed to detect tumour mutations (DNA sequencing, SSCP, DGGE etc) are not sensitive enough to detect mutations in tissues where less than 10% of the cells are mutated. Hence our search for methods capable of detecting very rare mutational events, such as those present in pre-malignant tissues. One methodology developed by us, namely Restriction Site Mutation (RSM), has been particularly useful in detecting rare p53 mutations. RSM was initially employed in our group to detect mutation fingerprints in a variety of animal models/in vitro systems. These studies validated the RSM methodology and determined mutation fingerprints for several model mutagens. Recent work has extended the application of RSM to detecting rare p53 mutations in pre-malignant oesophageal and gastric tissue. Our aim in these studies has been to identify pre-malignant patients possessing the highest risk of cancer progression. In addition, by comparison with previously obtained mutation fingerprints, causative mutagen exposures can be suggested.
Reference
Jenkins, GJS, et al (1999). Mutagenesis 14, 439–448.
Jenkins, GJS, et al (2001). Mutation Research 498, 135–144.
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19. Paradigm changes in radiation genetics: possible implications for genetic toxicology |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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Bryn A. Bridges
Genome Damage and Stability Centre, University of Sussex, Science Park Road, Falmer, Brighton, BN1 9RQ
Recent work on germ line effects in the irradiated male mouse has indicated that repeat DNA sequences, of which there are many, do not always behave in accordance with the paradigms that we have previously accepted (reviewed by Bridges (2001). A proportion of repeat sequences are spontaneously unstable and a comparable degree of instability is also manifested after irradiation. It may also be seen in the descendents of the irradiated cells following fertilisation. Since we know the numbers and types of radiation lesions and since these are more or less randomly distributed in the DNA, it follows that the mutations in these sequences must outnumber the radiation lesions that they sustain by two orders of magnitude. Thus there is a new paradigm, namely that mutations may arise at regions other than the site of damage and that there is some sort of amplification process that extends the effect of DNA damage to undamaged regions of DNA. There are implications of this for (i) mutation spectrum studies, (ii) dose response curves, and (iii) monitoring of exposed individuals and populations (since these markers are extremely responsive to DNA damage). There are also unresolved questions concerning the validity of the mouse model for humans and whether these mutations have any implications for the health of offspring resulting from fertilisation by sperm from an irradiated male (including effects manifest in utero or at birth). In principle there is no reason why this new paradigm and its consequences should not also apply to DNA damage caused by chemicals, although there is at present almost no relevant experimental evidence.
Reference
Bridges, B A (2001) Radiation Research 156, 631–641.
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20. Metabolic activation of the suspected human carcinogen 3-nitrobenzanthrone by human acetyltransferases and human sulfotransferase |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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V.M. Arlt1, H. Glatt2, E. Muckel2, U. Pabel2, B. Sorg3, H.H. Schmeiser3, D.H. Phillips1
1Section of Molecular Carcinogenesis, Institute of Cancer Research, Sutton, UK. 2German Institute of Human Nutrition, Potsdam, Germany. 3German Cancer Research Center, Heidelberg, Germany.
3-Nitrobenzanthrone (3-NBA) is an extremely potent mutagen and suspected human carcinogen that has been identified in diesel exhaust and in airborne particulate matter, and it has been shown to form multiple DNA adducts in vitro and in vivo in rats. In order to investigate whether human N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) contribute to the metabolic activation of 3-NBA we used a panel of newly constructed Chinese hamster V79MZ derived cell lines expressing human NAT1, NAT2 or human SULT1A1, respectively, as well as TA1538-derived Salmonella typhimurium strains expressing human NAT1 (DJ400) or NAT2 (DJ460). The formation of 3-NBA-derived DNA adducts was analysed by 32P-postlabelling after exposing V79 cells to 0.01 µM 3-NBA or 0.1 µM N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA), a potential metabolite of 3-NBA. Up to 4 major and 2 minor adducts were detectable, the major ones being identical to those detected previously in DNA from rats treated with 3-NBA in vivo. Comparison of DNA binding between different V79MZ derived cells revealed that human NAT2 and, to a lesser extent, human NAT1 and human SULT1A1, contribute to the genotoxic potential of 3-NBA and N-Ac-N-OH-ABA to form DNA adducts. However, the extent of DNA binding by 3-NBA was higher at a 10-fold lower dose than by N-Ac-N-OH-ABA, suggesting that N-Ac-N-OH-ABA is not a major intermediate in the formation of 3-NBA-derived adducts. 3-NBA showed a 6-fold and 2.7-fold higher mutagenic activity in Salmonella strains expressing human NAT2 and human NAT1, respectively, whereas N-Ac-N-OH-ABA was only weakly mutagenic in Salmonella DJ460 expressing human NAT2. Our results indicate that O-acetylation and O-sulfonation by human NATs and SULTs may contribute significantly to the high mutagenic and genotoxic potential of 3-NBA. Moreover, the as-yet-unidentified four major 3-NBA-derived adducts may be DNA adducts without an N-acetyl group.
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21. An adaptation of single cell gel electrophoresis that enables the differentiation of comets derived from apoptotic, necrotic or viable cells |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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N. Morley1, A. Rapp2, H. Dittmar2, A. Curnow1, L. Salter1, D. Gould1 & O. Greulich2
1Cornwall Dermatology Research Project, G14 PHLS, Penventinnie Lane, Treliske, Truro, Cornwall, TR1 3LQ, UK. 2Institute for Molecular Biotechnology, Department of the Single Cell and Molecular Techniques, Postfach 100813, D-07708 Jena, Germany.
Single cell gel electrophoresis (SCGE) otherwise known as the `comet assay' because of the comet-like appearance of the DNA visualised during the assay, is a popular method of assessing DNA damage induced by environmental genotoxins at the single cell level. The state of the cell from which the comet has derived is generally assumed to be viable, although a necrotic or apoptotic cell may produce a highly damaged comet. This could result in an increased level of DNA damage thus inflating the level of DNA damage induced by the compound being tested. A method of distinguishing different types of cell (apoptotic, necrotic or viable) is therefore required.
We have developed an adaptation to the SCGE technique that, for the first time, enables the visualisation of comets derived from viable cells or from cells in various stages of apoptosis and necrosis. In our system, HeLa cells were grown on clear (window) microscope slides. The cells were exposed to a range of insults and then stained with annexin V, calcein blue and propidium iodide and viewed using a fluorescence microscope. The membranes of apoptotic cells appeared green, the cytosol of viable cells fluoresced blue and the DNA of necrotic cells stained red. At this point an image was captured and compared with that produced at exactly the same position after SCGE had been performed. This enabled the differentiation of comets derived from apoptotic, necrotic or viable cells.
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22. Development of model mammalian systems for the study of mechanisms of chromatid breaks. |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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Peter E. Bryant, Graham Armstrong and Lindsey Gray.
Cancer Biology Group, School of Biology, University of St Andrews, St Andrews KY16 9TS,
Chromatid breaks are not only a marker of individual radiosensitivity, but are also linked with susceptibility to breast and other types of cancers (1-3). For example, around 40% of sporadic breast cancer cases show elevated chromatid radiosensitivity, compared with only 6% of normal individuals, indicating the presence of low-penetrance susceptibility genes that also confer chromatid radiosensitivity (1,4,5). Recent modelling (6) and experiments (7) in our laboratories lead us to conclude that chromatid breaks are not simply expanded DNA double-strand breaks (dsb), but result from chromatin rearrangements involving megabase domains. Some 10-20% of chromatid breaks are known to result from inter-chromatid rearrangements (6-8). We predict that the remaining 80% of chromatid breaks will result from intra-chromatid rearrangements, leading to an inversion adjacent to the chromatid break. To study the mechanism of conversion of dsb into visible chromatid breaks we developed a number of Chinese hamster cell lines, each containing unique dsb site (I-SceI endonuclease recognition site) and inducible endogenous expression of the I-SceI gene. A plasmid vector (pCMVI-SceI, containing the 18 bp recognition site, constructed in our laboratories) was transfected into Chinese hamster (CHOK1) cells using Effectine and selected in G418. One of several transfectant clones (CHO10) was then further transfected with two plasmids encoding for inducible endogenous expression of I-SceI endonuclease under the control of the ecdysone analogue ponasterone-A. The positions of chromatid breaks are first established using DAPI staining, and then we use a FISH probe (fluorescence-tagged pCMVI-SceI) in the induced cells to highlight the specific dsb site and to seek evidence of inversions adjacent to break sites. Treatment of cells with ponasterone-A yielded an increasing frequency of chromatid breaks on the specific chromosome with time. Break frequency reached a plateau after about 8 h following addtion of ponasterone-A. Using FISH, preliminary data support the rearrangement model of chromatid breaks.
References
Scott, D. et al. (1994) Lancet, 344, 1444.
Terzoudi, G.I. et al. (2000) Int. J. Radiat. Biol. 76, 607–615.
Baria, K. et al (2001) British Journal of Cancer. 84, 892–8966.
Riches, A.C. et al. (2001) British Journal of Cancer. 85, 1157–61.
Roberts, S.A. et al. (1999) American Journal of Human Genetics. 65, 784–794.
Bryant, P.E. (1998) Int. J. Radiat. Biol. 73, 243–251.
Rogers-Bald, M. et al. (2000) Int. J. Radiat. Biol. 76, 23–29.
Harvey, A.N. et al. (1997). Int. J. Radiat. Biol. 71, 21–28.
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23. DNA damage - bad fat, good fat. |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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Neil Beeharry, Jillian E. Lowe, Alma Rosales Hernandez, Flavia Fucassi, Peter J. Cragg, Michael H.L. Green, Irene C. Green
School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Moulsecoomb, Brighton BN2 4GJ, UK
Polynsaturated fats with a -C=C-C-C=C- site are the main target for lipid peroxidation and subsequent formation of mutagenic metabolites, but it is diets high in saturated fats which are most strongly associated with adverse health effects. We find that the common saturated fatty acid, palmitic acid (chain length : unsaturation; 16:0), is a potent inducer of DNA damage in an insulin-secreting cell line, and in primary human fibroblasts. Damage is prevented by two different antioxidants, -lipoic acid and 3,3'-methoxysalenMn(III) (EUK134), which also partly prevent palmitic acid-induced apoptosis and growth inhibition. It might be expected that a combined treatment with a saturated and polyunsaturated fatty acid would be especially harmful, since it would both generate oxidative stress, and provide a substrate for lipid peroxidation. Instead, we find that palmitic acid-induced DNA damage is prevented by the polyunsaturated fatty acid, linoleic acid (18:2, cis-9,cis-12-octadecadienoic acid), which is therefore acting as a protective agent against oxidative stress, rather than as a source of oxidative stress-generated mutagenic metabolites. This dual action of polyunsaturated fats may account for some of the complexities in linking dietary fat intake to cancer.
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24. Cellular glutathione status and p53 protein expression: Biomarkers for the risk assessment of betel-nut chewers |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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K. Kumpawat, A. Chatterjee
Genetics Laboratory, Department of Zoology, North-Eastern Hill University, Shillong-793022, India.
Betel-nut chewing related oral mucosal lesions are potential hazards to a large population worldwide. It was reported that arecoline (a potent alkaloid in betel-nut) administered orally to mice induced DNA damage and delay in cell kinetics which were reduced by N-acetylcysteine (a precursor of glutathione). Therefore, the aim of this study was to monitor DNA damage in raw betel nut (RBN) chewers from North-Eastern India, with respect to their endogenous glutathione (GSH) status. Blood was collected from donors who were categorised as non, moderate and heavy chewers based on the amount of RBN consumption. Chromosomal aberrations (CAs), sister chromatid exchanges (SCEs) and cell kinetics were analysed in lymphocytes in vitro. From the isolated lymphocytes total GSH was measured and total protein was extracted from the lymphocytes by using RIPA (Radioimmunoprecipitation assay) buffer for determination of p53 protein level by immunoblotting techniques. SCEs and the delay in cell kinetics were significantly increased in heavy chewers, while the increase in Cas was marginal. The level of total GSH was reduced significantly in heavy chewers and a lesser depletion was observed in moderate chewers. The expression of p53 protein was significantly enhanced in heavy chewers, which could be due to induction of DNA damage by the RBN ingredients. The data show a significant delay in cell kinetics in heavy chewers, which could be related to higher expression of p53 protein. It is evident that increased p53 level could arrest cell cycle progression and allow the damaged DNA to be repaired. GSH serves as a major endogenous cellular defense against toxic effects of xenobiotics and GSH depletion may lead to significant sensitization. This depletion of GSH in heavy chewers might make the cells more vulnerable to DNA damage by genotoxic RBN-ingredients. Therefore, the GSH status and p53 protein level could become intermediate biomarkers for the risk assessment of RBN chewers.
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25. The alkaline comet assay as a predictive test of bladder cancer cell radiosensitivity and chemosensitivity. |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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Manar A.L. Moneef1, Karen J. Bowman1, R. Paul Symonds1, Roger C. Kockelbergh2, William P. Steward1, George D.D. Jones1
1Department of Oncology, University of Leicester, Leicester, UK. 2Department of Urology, Leicester General Hospital, Leicester, UK.
In the UK the two main treatment options for invasive bladder cancer are radiotherapy (RT) or cystectomy. However, ~50% of patients undergoing RT fail to respond. If tumour cell radiosensitivity could be predicted in advance, it may be possible to significantly improve tumour control rate by selecting for immediate RT those patients whose tumours are radiation sensitive; additionally, patients who would benefit from initial surgery would be identified earlier. The alkaline comet assay (ACA) is a highly sensitive method for the measurement of single strand breaks (SSBs) and alkali labile sites, and can readily detect levels of this damage induced by clinical radiation doses. In the present study, using a panel of human bladder tumour cell lines of differing radiosensitivities, the relationship of cell survival to the induction and repair of DNA SSBs and alkali-labile sites was assessed via clonogenic assay and ACA, respectively. We have found that initial levels of damage maintain the same rank order as cell survival, with greater levels of damage noted in the radiosensitive cell lines. Furthermore, the extent of repair and level of residual damage also tends to correlate with cell survival. Finally, cells isolated from human bladder cancer biopsies also reveal a range of radio-responses, as determined by ACA, at clinically relevant doses. Other studies, using a version of ACA developed to assess DNA-DNA inter-strand cross-links, have examined the effects of the common chemotherapeutic agents mitomycin C and cis-platinum on bladder cancer cells. As for the radiation studies, the levels of cross-links maintain the same rank order as survival, with higher levels of cross-links being observed in the chemosensitive cell lines. Overall, our studies demonstrate ACA to be a good predictor of bladder cancer cell radiosensitivity and chemosensitivity.
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26. The Role of DNA Damage in the Development of Barrett's Oesophagus and Oesophageal Adenocarcinoma |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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C.P. Wild, J.R. Olliver, L.J. Hardie, G.W.B. Clark1, S. Dexter, D. Chalmers2.
Molecular Epidemiology Unit and 1Department of Surgery, School of Medicine, University of Leeds, 2Gastroenterology Unit, Leeds General Infirmary, Leeds, LS2 9JT, UK.
The strongest risk factor for oesophageal adenocarcinoma (ADC) is the presence of the precursor condition, Barrett's oesophagus (BO), where the squamous epithelium is replaced by a metaplastic columnar epithelium, usually in response to gastro-oesophageal reflux (GOR). Molecular changes that occur during the development of ADC may prove useful markers to assess the progression from BO to ADC (Bani-Hani et al 2000; JNCI 92; 1316-1321). These molecular changes could result from DNA damage caused by GOR and therefore we examined whether GOR is associated with an elevation in DNA damage in biopsies from BO patients and controls. For each BO patient, biopsies were collected from normal squamous oesophageal, gastric and Barrett's mucosa. Control patients had normal squamous oesophageal tissue sampled. Reflux history, medication, smoking and alcohol intake were recorded. DNA damage (strand breaks, SBs) was analysed using the alkaline `Comet' assay. Preliminary findings indicate a significantly raised (p<0.01) level of SBs in BO tissue (27.4% {23.3–30.0%}) (median % tail DNA {1st–3rd quartile, N=19) when compared with matched squamous mucosa (17.1 %{13.7–22.5%}) from the same patient. Out of 19 Barrett's patients, 17 showed higher levels of DNA damage in BO tissue compared to squamous mucosa. In contrast there was no significant difference (p>0.05) in the level of SBs when comparing BO (27.4% {23.3–30.0%}) and gastric mucosa (25.0 %{18.5–29.4%}. Somewhat surprisingly, levels of SBs in the squamous tissue of control patients (26.1 %{20.5–29.6%}) were higher than those in the squamous tissue of BO patients (17.1 %{13.7–22.5%}). There was no association between DNA damage levels and age, sex, Helicobactor pylori infection, prescription of NSAIDs, alcohol or smoking but the type of proton pump inhibitor (PPI) prescribed to BO patients appeared to significantly influence DNA damage levels.
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27. A proposed approach for genotoxicity risk assessment |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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K.L. Dearfield1, M.C. Cimino 2, N.E. McCarroll3, I. Mauer3, L.R. Valcovic1
U.S. Environmental Protection Agency (EPA), 1Office of Research and Development, 2Office of Pollution Prevention and Toxics, 3Office of Pesticide Programs, Washington, DC 20460, USA
Regulatory agencies evaluate environmental risks via a risk assessment process that involves the analysis of a substantial body of scientific data. To provide consistency, agencies develop guidelines for generation of data and for assessment of toxicity and exposure. Recent advances in genetic toxicity (mutagenicity) testing and in risk assessment approaches are prompting a renewed effort to harmonize genotoxicity risk assessment across the world. Genotoxicity risk assessment historically focused on transmissible germ cell genetic risk.
However, somatic cell genetic risk has also been a major risk consideration, particularly in support of carcinogenicity assessments. We propose an approach in which genotoxicity assessments for germ cell and somatic cell risks are developed within the risk assessment paradigm used by EPA. A classification scheme for agents is based on any inherent genotoxicity present, dose-responses observed in the data, and a preliminary exposure analysis. The classification leads to an initial level of concern for genotoxic risk to humans. A total risk characterization is performed using all other relevant toxicity data with the genotoxicity data and a comprehensive exposure evaluation. This characterization is ultimately applied to generate a final level of concern for genotoxic risk to humans. The final level of concern and characterized genotoxicity risk assessment are communicated to decision makers for possible regulatory action(s) and to the public.
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28. The Role of UKEMS in the Development of Testing Guidelines |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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David J. Kirkland
Covance Laboratories Ltd., Otley Road, Harrogate HG3 1PY, England.
Twenty years ago UKEMS established a sub-committee to determine the minimal professional criteria that should be achieved to comply with mutagenicity testing requirements in the United Kingdom. Recommendations on the conduct of basic and supplementary tests were published respectively in 1983 and 1984. Despite their local distribution, these recommendations had impact around the world. Further guidelines for statistical evaluation of mutagenicity test data, and revisions to the first 2 volumes, followed. By the early 1990s the mood was for international harmonisation rather than national or regional isolation. The processes by which UKEMS had achieved its testing recommendations in the 1980s and early 1990s were successfully employed in the International Workshops for Genotoxicity Testing, of which 3 have now been held, and made a significant impact on OECD guidelines and ICH guidance.
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29. Antigenotoxic Properties of Selenium Compounds on Potassium Dichromate |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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Eduardo Cemeli, Joanna Carder, Diana Anderson
Department of Biomedical Sciences, University of Bradford, Richmond Road, Bradford, West Yorkshire, BD7 1DP
Heavy metals are found in practically all living organisms. They are essential for nutrition. Non-essential metals are found as contaminants of food–stuffs and are ingested daily. Thus in the human diet, there is the presence of both essential and toxic metals. Selenium is an environmental metal that occurs ubiquitously and is produced throughout the world for various industrial activities. Selenium has been shown to have anticarcinogenic effects and preventive effects in clinical and epidemiological studies. Selenium supplements can inhibit chemically-induced tumours. Selenium has been considered by IARC not to be carcinogenic for man but from the viewpoint of genotoxicity, selenium has not been adequately studied. In contrast, hexavalent chromium is classified as a known respiratory carcinogen producing DNA damage through free oxygen radicals. In the present study, therefore, we examined the effects of three selenium compounds, sodium selenate, sodium selenite and selenious acid on potassium dichromate in the Ames test using strain TA102, and in the Comet assay in human lymphocytes. In the Ames test, it was shown that potassium dichromate produced a highly mutagenic response, whilst the three selenium compounds did not. In combination, sodium selenate reduced the response of potassium dichromate, but sodium selenite and selenious acid had no effect. In the Comet assay, all selenium compounds produced an increased response and so did potassium dichromate. In combination with selenium, however, only sodium selenate reduced the response of potassium dichromate, whereas sodium selenite exacerbated it and selenious acid had no effect. Thus, sodium selenate reduces effects of potassium dichromate in both assay systems. In conclusion, the results of this study confirm the antigenotoxic effects of sodium selenate.
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30. Establishment of a multiple-endpoint genotoxicity test system based on human cells |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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M. Honma, A. Hakura, K.F. Miura, T. Morita, S. Nakayama, H. Oka, S. Sato, Y. Sugiki, Y. Yamashita, A. Wakata, S. Wakuri, M. Hayashi
(The collaborative study group for the genotoxicity tests based on human cells, Mammalian Mutagenesis Study Group of the Environmental Mutagen Society of Japan)
Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
A number of genotoxicity tests has been developed and validated over the years. It is difficult to evaluate their capacity and to compare their results mutually, however, because each genotoxicity test differs in its biological system (prokaryotic, eukaryotic, in vitro, and in vivo) and in its test protocol (concentration, treatment period, etc). In order to establish a multiple-endpoint genotoxicity test system based on human cells including DNA damage, gene mutation, and chromosome aberration, we conducted a collaborative study involving 25 laboratories under the cooperation of JEMS/MMS. Using human lymphoblastoid cell lines TK6 and WTK-1, which originate from the same progenitor cell, but WTK-1 is p53-mutant cell line, we evaluated the genotoxicity of some chemicals by the Comet assay (COM), TK-gene mutation assay (TK), and micronucleus test (MN) according to a definite protocol. We examined 14 chemicals; 3 were mutagens and 11 were clastogens that were reported to be negative in a bacterial reverse mutation assay. Two mutagens, MNNG and 4NQO, clearly induced TK and MN in both cells. On the other hand, some clastogens tended to yield TK-positive response in WTK-1 rather than TK6. This may be associated with the different p53 status. The correspondence of results among COM, TK, and MN are now being investigated. These studies hold promise for the establishment of a humanized genotoxicity test system as well as for the elucidation of mechanisms of mutation and chromosome aberration after DNA damage.
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31. Karyotypic analysis of a rat egr-1/GFP transgenic model and chromosomal localization of the transgene. |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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C. Corso1, J.P. Slade2, D.A. Carter2, J.M. Parry1
1University of Wales Swansea, Centre for Molecular Genetic and Toxicology, Swansea, UK. 2School of Biosciences, Cardiff University, UK
The production of transgenic rodents through the illegitimate recombination of pronuclear-injected DNA can result in insertional disruption of host genes. Insertional mutants have formed both important models of mammalian development and routes to the molecular cloning of previously undefined loci. The development and application of transgenic models is increasingly important for research in mutagenesis and carcinogenesis. Using a promoter-reporter construct (egr-1/NGF1-A-d4EGFP) designed to delineate the spatial and temporal activity of the egr-1 promoter, multiple lines of transgenic rats were produced. One line, of intermediate copy-number, displayed an overt, and heritable phenotype. Although transgenic models are very well characterized from a phenotypic point of view there is a paucity of data regarding their karyotypic status. Classic cytogenetic analysis was employed to investigate the karyotypic stability of bone marrow cells of the transgenic rat model. Fluorescence In Situ Hybridization was used to localize the transgene (TG) at a chromosomal level and to detect the number of gene insertions throughout the genome. Our results show chromosomal instability due to a variable chromosomal modal number and the presence of chromosome fragments demonstrating mosaics within the bone marrow cells. FISH has demonstrated the presence of positive signals on the q arm of chromosome 3 in 100% of the cells. However, most of the cells appear to be monosomic for the chromosome 3 and show FISH positive signals at different chromosomal sites, mainly at the periphery of small chromosome or chromosome fragments. These findings might suggest that the TG insertion has induced amplification of the TG itself and or/breakage at the insertion site. In view of these findings, further experiments need to be carried out to estimate whether the overt phenotype of the rats is due to the genomic instability consequent to the insertion rather than the disruption of a single gene. Our results indicate the need for comprehensive analysis of the genome of transgenic models.
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32. Chromosome aberrations in patients with hip replacement |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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A.T. Doherty, R. Nesbitt, R.T. Howell, C.P. Case
Bristol Implant Research Centre. University of Bristol Department of Orthopeadic Surgery, Southmead Hospital. Bristol. U.K. BS10 5NB.
Joint replacement surgery has been an enormous success. However 20% of joint replacements fail within 20 years and require revision surgery. Some studies have suggested that prolonged exposure to metal debris following joint replacement might predispose to development of malignancy. We have previously shown an increase of chromosome aberrations in bone marrow adjacent to a worn prosthesis.
The level of chromosome aberrations in peripheral blood lymphocytes (PBL) from patients with failed hip replacements was investigated. In addition, the ability of wear debris worn from the hip joint to induce aberrant cell divisions was studied in vitro.
PBL have been taken from cohorts of patients undergoing revision of total hip arthroplasty (THR) and primary THR. Fluorescent in situ hybridisation (FISH) has been used to determine cumulative genetic damage by painting chromosomes 1, 2 & 3 using three colours in order to detect stable chromosome translocations from 600 cells per patient (n=100). The Cytochalasin-B blocked micronucleus assay was performed to determine in vitro mutagenicity of wear debris extracted from metal stained tissue adjacent to the worn prosthesis. Blood samples from the patients involved were also analysed for the levels of chromium, cobalt and nickel by ICP-MS (Inductively Coupled Plasma Mass Spectrometry) to determine any link between chromosome aberrations and metal levels.
The data collected have indicated that the level of chromosome translocations in revision patients is double that observed in primary patients and significantly higher than the general population. In Vitro a dose-related increase in aberrations, have been observed particularly from wear debris from titanium alloy prostheses.
There was an increase of aneuploidy in peripheral blood lymphocytes in patients at revision surgery of worn titanium alloy prostheses. In vitro we have induced aneuploidy with Ti alloy wear debris.
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33. Is aristolochic acid a risk factor for Balkan endemic nephropathy-associated urothelial cancer? |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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V.M. Arlt1, D. Ferluga2, M. Stiborova3, A. Pfohl-Leszkowicz4, M. Vukelic5, S. Ceovic1, H.H. Schmeiser6, J.P. Cosyns7
1Section of Molecular Carcinogenesis, Institute of Cancer Research, Sutton, SM2 5NG, UK. 2University of Ljubljana, Slovenia. 3Charles University, Prague, The Czech Republic. 4Ecole Nationale Supérieure Agronomique de Toulouse, France. 5General Hospital, Slavonski Brod, Croatia. 6German Cancer Research Center, Heidelberg, Germany. 7Université Catholique de Louvain Medical School, Brussels, Belgium.
Aristolochic acid nephropathy (AAN) is a unique type of rapidly progressive interstitial fibrosis, associated with urothelial cancer and related to the prolonged intake of Chinese herbal remedies (Aristolochia species) containing nephrotoxic and carcinogenic aristolochic acid (AA) (Arlt et al., 2002a). On both clinical and morphological grounds, AAN is very similar to another fibrosing nephropathy, the Balkan endemic nephropathy (BEN), including the association with urothelial tumours. It has been suggested that the mycotoxin ochratoxin A is associated with BEN, but also AA was considered as a possible causal factor in BEN. In this pilot study we assessed AA exposure in endemic areas for BEN (Arlt et al., 2002b). Using the 32P-postlabelling method we analysed renal tissue from three female farmers who lived in endemic areas and in two of whom an upper urinary tract cancer had developed, for AA-DNA adducts. In two of these patients of whom one had a urothelial tumour the major adenosine adduct of aristolochic acid I, 7-(deoxyadenosin-N6-yl)-aristolactam I (dA-AAI), the most abundant AA-DNA adduct found in AAN patients, was observed and identified by cochromatographic analyses on TLC and HPLC. Adduct levels ranged from 5.6 to 17.1 adducts per 109 nucleotides. Our data clearly demonstrate that people living in endemic areas for BEN have been exposed to AA. Thus, AA should be considered as a potential risk factor in the development of urothelial cancer in endemic areas. The epidemiology of AAN and AAN-associated urothelial malignancies might provide a clue to urothelial malignancies in endemic areas. Whether AA plays a role also in BEN remains to be further assessed in patients with unequivocal diagnosis of BEN and in patients with other nephropathies but living in endemic areas for BEN.
References
Arlt et al.(2002a) Mutagenesis, 17, 265-277.
Arlt et al.(2002b) Int. J. Cancer, in press.
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34. G:C A:T mutations in the liver lacI gene of Big Blue® rats fed leucomalachite green
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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M. G. Manjanatha, S.D. Shelton, M. Bishop, J.S. Shaddock, V. Dobrovolsky, R.H. Heflich, S.J. Culp.
Food and Drug Administration, National Center for Toxicological Research, Jefferson, AR 72079 USA.
Leucomalachite green (LMG) is the major metabolite of malachite green, a triphenylmethane dye that has been used widely as an antifungal agent in the fish industry. Concern over the use of malachite green is due to the potential for consumer exposure, suggestive evidence of tumour promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. Previously, we reported significant increases in DNA adducts and lacI mutant frequency in the livers of female Big Blue (BB) rats fed LMG for up to 32 weeks [ Culp et al., Mutation Res. (in press)]. Although these results suggested that LMG is a mutagen, preliminary results from a two-year carcinogenicity study indicate that LMG is not carcinogenic in female rats. In this study, we have further characterized the mutagenicity of LMG in BB rats. Bone marrow and splenic lymphocytes were examined for induction of micronuclei and Hprt mutants, respectively, after 4, 16, and 32 weeks of LMG feeding. No significant increases in the frequency of micronuclei or Hprt mutants were observed for any of the doses or time points assayed. Molecular analysis of liver lacI mutants from treated rats revealed that 31% (17/55) were clonal in origin and that the majority (32/38) of the independent mutations were basepair substitutions. The predominant type of mutation was G:CA:T transition (47%) and the majority of these involved CpG sites of the lacI gene. When corrected for clonality, lacI mutation frequency in treated rats was not significantly different from the control. Further, the lacI mutational spectrum in treated rats was similar to that found for control rats. Taken together, these data indicate that LMG is neither a mutagen nor a carcinogen in female rats and that the increase in lacI mutant frequency observed in the liver of treated rats may be due to expansion of spontaneous lacI mutations.
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35. A study on the origin of the two hairs cells in the wing Spot test of Drosophila melanogaster |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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R. El Hamss1, M. Idaomar1, A. Muñoz Serrano2, A. Alonso-Moraga 2
93 1Université Abdelmalek Essaâdi, Unité de Biologie Cellulaire et Moléculaire (BCM) BP 2121, 002 Tétouan, Morocco. 2Universidad de Córdoba, Departamento de Genética, San Alberto Magno s/n, 14071 Córdoba, Spain.
In order to determine the origin of cells with two hairs `false mwh' in Somatic Mutation And Recombination Test (SMART) in the wing of Drosophila melanogaster, three different crosses carrying markers mwh and flr3on the left arm of the chromosome 3 were set up. The larvae from these crosses differ with respect to their bioactivation capacity. Three-day-old larvae were treated with urethane in parallel of a water control. Wings from emerging flies were scored for the occurrence of spontaneous and induced spots. Parallel to `true spots'(single mwh and flr spots, twin spots), false mwh were also scored.
Our results, with water control, show an increased frequency of false mwh proportionally with the bioactivation level. The same result, but in much higher frequencies was obtained with urethane. Also, this phenomenon is dose-dependent. The balancer-heterozygous wings mwh flr+/TM3, show a higher frequency of two cell hairs than the marker-heterozygous mwh flr+/mwh+ flr wings. Furthermore, a scoring of wings from a wild strain of our laboratory indicates the presence of cells with two hairs. From our data, we can conclude that the two hairs cells in Drosophila melanogaster is a phenotype whose expression is influenced not only by the mwh gene but also by other genes with two-hairs manifestations sensitive to mutagens and influenced by the metabolism level.
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36. p53-dependent nucleotide excision repair of biologically significant levels of DNA adducts in human cells |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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D.R. Lloyd1, P.C. Hanawalt2
1Research School of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ; 2Department of Biological Sciences, Stanford University, Stanford, CA94305-5020, USA.
We have developed an experimental approach for studying the global nucleotide excision repair (NER) of DNA adducts at biologically significant levels in human cells. By exploiting the sensitivity of the 32P-postlabelling assay, a technique commonly used for the detection of DNA adducts in human tissues, it has been possible to monitor cellular excision repair of such lesions at levels comparable to those documented in certain human populations as a result of environmental exposure. We have used this highly sensitive experimental approach to investigate the efficiency of NER of carcinogen-DNA adducts in human cells in which the expression of the p53 tumour suppressor protein can be regulated. The NER of the major N2-guanine adduct formed by benzo(a)pyrene diol epoxide (BPDE) was found to be p53-dependent. Similarly, the removal of the 4 major adducts formed by benzo(g)chrysene diol epoxide (BCDE) was more efficient in the presence of p53; the removal of BCDE-adenine adducts was slightly more p53-dependent than corresponding guanine adducts. The requirement of p53 for efficient NER was most notable when both BPDE and BCDE adducts were present at low levels (around 1 adduct per 107 nucleotides). Since these adduct levels are comparable with those found in biopsy tissues from environmentally-exposed individuals, e.g. smokers, the results from these studies are particularly significant in terms of human carcinogenesis related to environmental exposure. The manner in which p53 controls the efficiency of NER at these biologically significant levels of DNA adducts, and its possible role in the regulation of lesion recognition proteins, is currently under investigation.
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37. Synergistic mutagenicity of benzo[a]pyrene diol epoxide and UV radiation in the supF gene |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450...41. Bone marrow micronucleus...42. Comet assay in...43. Xenobiotic diacyglycerols:...45. Fluorescence in situ...46. Factors Responsible for...47. Two-stage cell...48. A method to...49. In vitro Genotoxicity...50. The effects of...51. Free radical activity...52. Application of the...53. ILSI HESI Structure-Activity...54. Evaluation of gene...55. Is MutaTMMouse insensitive...56. Biophotonic imaging of...57. Assessment of DNA...58. The Influence of...59. Gene-expression analysis by...60. An investigation into...61. Bisphenol-A induces multiple...62. Genotoxicity study of...64. Antimutagenic effect study...65. Effect of p53-dosage...66. Chromosomal radiosensitivity...67. Validation Studies on...68. The comet assay...69. Elucidation of the...70. Bile salt and...71. Evaluation of the...72. An in vitro...73. Arsenate increases...74. Detection of genetic...75. Distribution of breakpoints...76. The responses of...77. Sensitivity of different...78. The Lack of...79. Comparisons of repair...80. Induction of Micronuclei...81. Genotoxicity studies on...82. Development of an...83. Molecular epidemiological...84. Evaluation of the... |
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K.I.E. McLuckie1, M. Gaskell1, G.C.K. Roberts2, P.B. Farmer1, E.A. Martin3, G.D.D. Jones4, M.N. Routledge5
1Cancer Biomarkers and Prevention Group, The Biocentre, University Road, Leicester, LE1 7RH. 2Biological NMR Centre, University of Leicester, Leicester, LE1 9HN. 3Genetic Toxicology, AstraZeneca, Alderley Park, Macclesfield, SK10 4TG. 4Dept. of Oncology, Hodgkin Building, University of Leicester, Leicester, LE1 9HN (5) Molecular Epidemiology Unit, University of Leeds, Leeds, LS2 9JT.
Exposure of DNA to environmental mutagens frequently occurs in the form of a complex mixture of agents. The effects on DNA of combined exposures may be different to exposure from individual agents.
We have previously shown that UV irradiation of benzo[a]pyrene diol epoxide (BPDE) treated DNA induces a synergistic increase in mutation frequency compared to either treatment alone (Routledge, et al, 2001). As a continuation of this work, pSP189 plasmid DNA was treated with BPDE before and after treatment with UVC radiation, as well as with UVC or BPDE alone. The level of DNA damage by BPDE was determined to be between 0.2 and 2.2 adducts per 104 nucleotides using 32P-postlabelling. The mutation frequency in the supF gene increased with all treatments in comparison to control (plasmid treated with solvent only). Separate treatment with BPDE or UVC independently increased the mutation frequency by up to 40-fold or 86-fold over control, respectively. Treatment with BPDE followed by UVC increased mutation frequency by 656-fold over control. Treatment with UVC followed by BPDE increased mutation frequency by 60-fold, compared to control. The types of mutations varied for the different treatments. BPDE alone gave mainly GCTA transversions (65% of all mutations) and UVC alone gave mainly GCAT transitions (78% of all mutations). Combined treatments preferentially induced transition substitution mutations, of which the GCAT mutation was prevalent (59% and 63% for BPDE first and UVC first, respectively). The distribution of mutations and position of hotspots, in the supF gene, differed depending on which treatment was administered first.
These results suggest that the large increase in mutation frequency of the BPDE followed by UVC treatment is due to photoactivation of BPDE adducts rather than interaction between UV and BPDE adducts on the same plasmid molecule.
Reference
Routledge, M. N., et al. (2001) Carcinogenesis 22, 1231–1238.
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38. Cells cultured at low density have increased frequencies of micronuclei |
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Top1. The enemy within:...2. Molecular mechanisms of...3. Mechanisms of...4. Characterisation of the...5. Variation in DNA...6. Genetic Effects Of...7. From ethics to...8. Ecogenotoxicological...10. Effects of Exposure...11. Toxicogenomics in non-model...12. Mutagenicity, rodent...13. Multicolour-FISH in two...14. Genome stability in...15. What do human...16. Application of molecular...17. An Analysis of...18. The detection of...19. Paradigm changes in...20. Metabolic activation of...21. An adaptation of...22. Development of model...23. DNA damage -...24. Cellular glutathione status...25. The alkaline comet...26. The Role of...27. A proposed approach...28. The Role of...29. Antigenotoxic Properties of...30. Establishment of a...31. Karyotypic analysis of...32. Chromosome aberrations in...33. Is aristolochic acid...34. G:C->A:T mutations in...35. A study on...36. p53-dependent nucleotide...37. Synergistic mutagenicity of...38. Cells cultured at...39. In vitro comet...40. Human Cytochrome P450... |
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