Methods in Toxicological Research [1] Genotoxicity and Carcinogenecity

1. Methods in Toxicological Research [1] Genotoxicity and Carcinogenecity 1.1 Carcinogenesis, oncogenes and DNA repair mechanisms 1.2 DNA adducts, DNA damage and Comet assay 1.3 Genotoxicity, mutagenesis and Carcinogenesis testing Prof. K.M. Chan, Dept. of Biochemistry and Environmental Science Program, CUHK. Tel: 3163-4420; email: kingchan@cuhk.edu.hk

2. 1.1 Carcinogenesis, oncogenes and DNA repair mechanisms Basic concepts • Causes of cancer: radiation, chemical carcinogens, viruses, aging plus the above factor(s). • Natural cancers result from the interaction of multiple events over time. • Chronic exposure of initiator and promoter of chemicals. • All involved oncogenes and anti-oncogenes (recessive oncogenes) products. 1.1.1 Carcinogenesis

3. Cancer is a disease related to aberrant gene expression • Oncogenes are altered forms of genes that also occur in unaltered form as proto-oncogenes in normal cells. • They are known as oncogenes as they trigger tumor or cancer developments. • Anti-oncogene or recessive oncogenes refer to those genes normally depress cellular proliferations or tumor development, when they got mutated, cancer began to develop. 1.1.1 Carcinogenesis

4. Transformation Experiments • Transfection experiments adding tumor cell DNA conferred cancer traits. • DNA extracted from mouse cells after chemical transformation was put back in normal fibroblasts. • Select some transformed cells to be injected into mouse. • Mouse developed tumor. Adapted from Bishop and Hanafusa, 1996. Proto-oncogenes in normal and neoplastic cells. In: Scientific American Molecular Oncology. Sci. Amer. Inc., New York, pp61-83. 1.1.1 Carcinogenesis

5. 1.1.2 Cancer: cell proliferates out of control • Mutated growth factor works inappropriately. • Mutated receptorsmight turn cells on even without incoming signals. • Mutated signal proteins can turn genes on without normal triggers. • Mutated transcription factors that turn on genes without external triggers or stimulants. 1.1.1 Carcinogenesis

6. Normal pathway of signal transduction leading to gene expression to control cell division could be triggered when those involved genes got mutated. cytoplasm 1. Growth factor 3. Signal Transducer Anti-oncogenes are usually gene suppressors, suppressing gene transcription 2. Cell surface receptor Cell Division 4. Transcription factor nucleus 1.1.1 Carcinogenesis

7. Tumours (tumors): • Benign: pose no or little risk to its host. • Malignant: become life-threatening because of its invasiveness and their power to metastasize (move away and migrate to invade other normal organ and cells). • Neoplasia: relatively autonomous growth of malignant cells in the host. Sarcoma derived from mesodermic cells such as leukemia, carcinomas derived from endoderm or ectoderm. • Solid tumor. 1.1.1 Carcinogenesis

8. Tumor cell characteristics: • Increase cell division rate • Cannot differentiate • Multiple stages and progression may take a long time (months to years) • Different triggers work at different stages inducing different oncogenes or antioncogenes, forming a complex etiology of cancer 1.1.1 Carcinogenesis

9. Progression of cancer and events leading to neoplasia (Harris 1987) 1.1.1 Carcinogenesis

10. 1.1.2 Oncogenes • Bishop and Varmus (1989) got their Nobel for the discovery of “src” gene carried in Rous sarcoma virus (RSV). • Src is a tyrosine kinase. It phosphorylates surface receptor for fibronectin to lose cell attachment causing malignancy development. • Oncogenes were found to have their cellular counterparts called proto-oncogenes in normal cells. 1.1.2 Oncogenes

11. Changing Proto-oncogenes into oncogenes (by comparing normal and cancer cells) • DNA sequence substitution (point mutation) • DNA sequence deletion • Translocation of gene segment, especially promote or enhancer insertion • Gene amplification (increase of numbers of gene copies) 1.1.2 Oncogenes

12. DNA sequence substitution • Point mutations • C-Ha-ras, c-Ki-ras, c-N-ras; c means cellular = proto-oncogene • 12th, 13th, 61st codons are hot spots for point mutations • Causing colon carcinoma, lung carcinoma, etc. 1.1.2 Oncogenes

13. Activation of Protooncogenes via point mutation • Comparing gene sequences of protooncogenes and oncogenes, several hot spots have point mutations found in some oncogenes. • P21 ras is a very good example. • A substantial number of human tumors (10-15%) contained activated ras. • It is a membrane bound protein with GTPase activity, transducing signals from growth factor receptors. • E.g. Nitrosomethylurea and dimethylbenz(a)anthracene (DMBA) were found to induce H-ras mutations. 1.1.2 Oncogenes

14. Transforming ras genes. From Bos et al. (1998) 1.1.2 Oncogenes

15. Formation of o6-methylguanine DNA • Nitrosomethylurea is an alkylating agent known to induce the formation of o6-methylguanine in DNA. • G – A transition observed at the 12th codon of H-ras. • DMBA (7,12-dimethylbenz[a]anthracene)induced A – T transversion at codon 61 at H-ras. 1.1.2 Oncogenes

16. 1.1.2 Oncogenes

17. Oncogenes are also amplified in some tumors: • C-myc, 20X, leukemia and lung carcinoma • C-erbB, 30X, epi-dermoid carcinoma • C-K-ras, 4-20X in colon carcinoma cell-line, 30-60X in adrenal-cortical carcinoma line. 1.1.2 Oncogenes

18. Tumor Suppressor Genes • Oncogene acts at dominant manner. • Anti-oncogenes act as recessive manner because they are tumour suppressor. • They normally inhibit neoplastic transformation. • Because they are recessive genes, some if not all cases are heritable. 1.1.2 Oncogenes

19. Retinoblastoma Gene • 1 per 20,000 births. • fetal retinal layer has neoplastic cells. • high risk to get cancer somewhere as they get older. • heritable form had lymphocytes with chromsome 13 broken. • In sporadic cases, abnormal pair of ch13 found in tumour. Adapted from Lewin, 1995. Gene V. 1.1.2 Oncogenes

20. 1.1.2 Oncogenes

21. p53 • Small cell lung carcinoma, breast carcinoma and osteosarcoma • 17p chromosome • DNA binding protein • Controlling signals for cell cycle development, it’s a transcription regulator. 1.1.2 Oncogenes

22. Loss of mutation repair capability K-ras p53 1.1.2 Oncogenes Development of Colon Cancer Molecular events from normal bowel mucosa to adenomatous polyp and finally invasive cancer(adapted from Bishop and Weinberg, 1996. Molecular Oncology, Scientific American, Inc. pp187).

23. Proliferating Cell Nuclear Antigen, PCNA • Cyclin to control cell division (not an onco-protein • Induced by p53 • Interact with p21 • Control DNA replication (w/o p53) • If non functional or abnormal could trigger apoptosis • Markers for carcinogenesis 1.1.2 Oncogenes

24. 1.1.3 DNA repair mechanism • Radiation or chemicals cause DNA damage. • Xeroderma pigmentosum (XP) in humans related to UV exposures. • XP patients develop benign and malignant skin tumors. • UV induces such deleterious processes including mutagensis, carcinogenesis, and cell-death. • Aging accompanies with malfunction of protective DNA repair mechanism prone to be easier to have cancers. 1.1.3 DNA Repair

25. Adapted from Watson, 1994. Molecular Biology of the Genes. 1.1.3 DNA Repair

26. SOS Responses in E. coli • The SOS regulon uses Lex A protein as a repressor. • However, Rec A gene is not entirely repressed by Lex A. • When DNA damage happened, DNA replication is halted, single-stranded (ss) DNA exposed to bind with Rec A protein. • Rec A-ssDNA can inactivate Lex A. • As the Lex A repressor is inactivated, the SOS genes including Rec A become activated to repair the DNA in E. coli. 1.1.3 DNA Repair

27. The SOS Regulon Adapted from Lehninger 2003 Principles of Biochemistry. 1.1.3 DNA Repair

28. Genes induced as part of the SOS response Adapted from Lehninger Principles of Biochemistry. 1.1.3 DNA Repair

29. DNA Repair Systems in E. coli • Mismatch repair • Base-excision repair • Nucleotide-excision repair • Direct repair The repair systems in eukaryotic cells are much more complicated. From the XP patients, scientists have identified many genes in human involved in DNA repair. 1.1.3 DNA Repair

30. Mismatch Repair Adapted from Lehninger Principles of Biochemistry. 1.1.3 DNA Repair

31. Base Excision Repair Adapted from Lehninger Principles of Biochemistry. 1.1.3 DNA Repair

32. Nucleotide Excision Repair 1.1.3 DNA Repair Adapted from Lehninger Principles of Biochemistry.

33. DNA repair systems in E. coli Adapted from Lehninger Principles of Biochemistry. 1.1.3 DNA Repair

34. Major Pathways of DNA Damage Repair. Direct Repair 1.1.3 DNA Repair

35. 1.2 DNA Adducts, Damage and Comet Assay • E.g. Aflatoxin B1-guanine adduct, benzo(a)pyrene-purine adducts. • These chemicals are transformed by Phase I enzymes to become active in attacking DNA at specific sites. • Adducts can form links with adjacent DNA base on the same strand, thus no base-pairing formed with opposite strand. • Base substitution occur (AFB1 induces only transversion) resulting in point mutation, as the adducts can bypass the repair mechanisms. 1.2 DNA Adducts, Damage and Comet Assay

36. Detection of DNA adducts • Methods for DNA-carcinogen adduct analysis include: • 32P post-labeling assay. • Immunoassays for specific adducts. • Fluorescence assay. • Mass spectrometry detection methods. • Urinalysis of alkylpurine to study alkylation damage of DNA. • Comet Assay. 1.2.1 DNA Adducts

37. Post-labeling Analysis DNA samples possibly contain carcinogen-adducted DNA Cleavage into nucleotides by micrococcal endonuclease and spleen exonuclease Adapted from Klaassen, C.D. 1996. Cassarett & Doull’s Toxicology. The Basic Science of Poisons. 5th ed., McGraw-Hill Books Inc. Ap + Gp+ Tp+ Cp + m5Cp + Xp + Yp……… adducts Normal nucleotides T4 Polynucleotide Kinase (PNK) + γ 32PATP *pAp + *pGp+ *pTp+ *pCp + *pm5Cp + *pXp + *pYp…... Removal of normal nucleotides by PE1-cellulose or reverse phase HPLC *pXp + *pYp…... 1.2.1 DNA Adducts Separation and detection of adducts, e.g. PEI-cellulose TLC Autoradiography

38. Autoradiography of Sole liver DNA treated with BaP. Adapted from Varanasi et al., 1989. 1.2.1 DNA Adducts

39. Mass Spectrometry (GC-MS) and Urinalysis • Urine samples were tested with GC-MS. • Spontaneous depurination occur to yield two unusual classes of DNA adducts: N-7-alkylguanines and N-3-alkyladenines. • Glycosylases excision occur and within days or 24 hours, adducts can be detected in urine samples. Patients’ samples could be detected for adducts on HPLC columns. • Urinalysis is a popular non-invasive method, and HPLC or immunoassay have been developed for the above two adducts. 1.2.1 DNA Adducts

40. Sensitivities of some DNA and hemoglobin adduct analyses 1.2.1 DNA Adducts Hemoglobin adducts are useful but not sensitive enough for bio-monitoring.

41. 1.2.2 DNA damage and Comet Assay • Observe nuclear DNA with single cell gel electrophoresis (SCGE) and fluorochrome-staining method • Prepare blood cells or cells to be analyzed • Put in agarose and run alkali electrophoresis in a small chamber • View individual cell and its nucleus after DNA staining with SYBR green or ethidium bromide • Damaged DNA become comet-shaped • Count numbers of damaged nuclei (% damaged) • May study tail length to nuclei radius ration and classify the degree of damages

42. 1 3 2 Comet Assay

43. Comet Assay 4

44. Limitations and validity of comet assay • It is a quantitative assay provide easy and rapid analysis of DNA integrity • Complicated with 5 classes: intact, with tail, tail = nucleus, tail > nucleus, tail only (nucleus damged totally). However, • DNA released from nuclei could be related to poor sample preparation and live sample is preferred • Cannot measure DNA repair • Cannot confirm if the chemical tested is directly or indirectly related to the damage • Common for environmental monitoring, e.g. in mussel hemolymph, in vivo or in vitro assay of animal cells. 1.2.2 DNA damage and Comet Assay

45. 1.3 Genotoxicity, mutagenicity and Carcinogenicity Tests • Mutagenicity testings: Ames tests, Mutatox, Sister Chromatid Exchange, mammalian cell test. To identify if a chemical could cause mutation. • Called genotoxicity tests to be exact. • Carcinogenicity testings: chemical screening, rat liver altered foci assay, whole animal tests over 6 month and 2 years. To identify if a chemical could cause cancer in vivo. • Direct testing of chemicals for their mechanisms of mutagenicities and genotoxicities with target proteins or enzymes and specific oncogene mutations studied.

46. 1.3.1 GENOTOXIC &MUTAGENICITY TESTING Modified from Ecobichon, D.J. (1992) The Basic of Toxicity Testing. CRC Press. Chapter 6. Mutagenesis-Carcinogenesis. pp127. (Table 3).

47. 1.3.1 GENOTOXIC &MUTAGENICITY TESTING Ames Test

48. 1.3.1 GENOTOXIC &MUTAGENICITY TESTING Ames 1973 developed a rapid screening method based on mutation of Salmonella typhimurium. The mutant strains used in the Ames Tests are histidine defective (unable to synthesize histidine). Back mutation make them able to survive on plates without histidine. TA1535 is used for base-pair substitutions TA153F and TA1538 are used for frameshift mutations Adapted from Lowy, D.R. 1996 The Causes of Cancer. In: American Scientific Molecular Oncology. Sci. Amer., Inc., New York, pp41-59.

49. ICH (Int’l. Conf. on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use) Test Battery. • Bacteria reverse mutation assay (Salmonella and Escherichia tester strains) • In vitro chromosome aberration test • Mouse lymphoma assay • Rodent micronucleus assay 1.3.1 GENOTOXIC &MUTAGENICITY TESTING

50. Syrian Hamster Embryo (SHE) cell transformation assay Normal cells • Results correlate well with rodent carcinogenicity testing. • Morphological tests for 24 h, 7 days or both. • Seed 105 /cm cells w/o feeder layer for 24 h or 3 days, followed by chemical exposure for 3 days, or 7-10 days • Study transformed foci as preserved in fixative and stain 1.3.1 GENOTOXIC &MUTAGENICITY TESTING Transformed cells Short term in vitro assays