Molecular Biology of Liver Tumors Following Chemical Exposure Robert C. Sills, DVM, PhD Diplomate, ACVP

1. Molecular Biology of Liver Tumors Following Chemical ExposureRobert C. Sills, DVM, PhDDiplomate, ACVP

2. Overview • Molecular Pathology Review • Molecular Pathology Case Studies • Oxazepam (Mice) • Hepatocellular neoplasms • Hepatoblastomas • Riddelliine (Rats and Mice) • Liver hemangiosarcomas • Research Team

3. Neoplastic Liver LesionsB6C3F1 Mouse Hepatocellular Adenoma Hepatocellular Carcinoma Hepatoblastoma Hemangiosarcoma

4. Molecular Pathology Review

5. Major Genes Involved in Carcinogenesis Proto-oncogenes Tumor suppressor genes • Involved in cellular growth and differentiation • Must be activated in cancer • Point mutation • Chromosomal translocation • Gene amplification • Negative growth regulator • Must be inactivated or lost in cancer • Point mutation • Loss of gene or chromosome • Methylation

6. Case Study in Mice OxazepamNon-genotoxic

7. Oxazepam • Central nervous system depressant • Prescribed widely for treatment of anxiety • Metabolite of benzodiazepines (valium) • Valium has been prescribed at an annual rate of greater than 25 million times in US

8. Incidences of Neoplasams and Nonneoplastic Lesions of the Liverof Female B6C3F1 Mice in the 2-Year Feed Study of Oxazepam Dose (ppm) 0 125 2,500 5,000 2-Year Study Liver (Number of rats examined) 50 50 50 50 Centrilobular Hypertophy 0 2(1.5) 11**(2.5) 29**(2.9) Hepatocellular Adenoma 25 35* 35* 36* Hepatocellular Carcinoma 9 5 49** 44** Hepatoblastomas 0 1 8** 8** *P<0.05 **P<0.01

9. Approach for Evaluating the Mechanism of Carcinogenesis B6C3F1 Mouse Oxazepam Tissue Cellular Molecular ? Liver Centrilobular Hypertrophy Cytochrome p450 Hepatocellular Neoplasms Hepatoblastomas Bucher et al, 1994 Fund. Appl. Tox, 23, 280,1994 Griffen et al, 1995 Tox. Let. 76, 251,1995

10. OXAZEPAM METABOLITES OXAZEPAM CANCER GENES Cytochrome P450Oxidative Damage

11. Isoprostane (oxidative damage marker) in Livers ofB6C3F1 Mice Following 6-months Oxazepam Exposure 70 60 (n=3) 50 40 (n=3) 30 20 10 0 Control Oxazepam Tomer, Devereaux, NIEHS, 2000

12. Cytochrome P450Oxidative Damage OXAZEPAM METABOLITES OXAZEPAM H-ras proto-oncogene Hepatocellular Tumors Hepatoblastomas

13. Ras Genes and Cancer • Commonly mutated in both animal and human tumors • Contributes to understanding of pathogenesis of cancer • Links between chemical exposure and signature mutations in cancer • Robust spontaneous data base for comparison with chemically induced tumors

14. Ras Signal Transduction Pathway Growth factor Growth factor receptor GTP GDP Inactivation by Hydrolysis of GTP Activation of MAP kinase pathway BLOCKED IN MUTANT RAS Activation of transcription Cell cycle progression Robbins and Cotran Pathologic Basis of Disease, 2005

15. H-ras Proto-Oncogene Evaluation

16. H-ras Mutations in Hepatocellular Adenomas and Carcinomas from Oxazepam Treated Mice Treatment Tumor Incidence Tumors with Mutations Control, 0 p.p.m. Oxazepam 125 p.p.m. Oxazepam 2500 p.p.m. Oxazepam 5000 p.p.m. 11/20 (55%) 13/37 (35%) 2/25 (8%) 0/21 (0%) 28/50 (56%) 36/50 (72%) 50/50 (100%) 47/50 (94%) Historical Control - H-ras mutations, 80/126 (63%) Hepatoblastomas - No H-ras Mutations

17. Revisit Hypothesis:Assessment of Mutations in Cancer Genes b-catenin Gene Oxazepam Exposure Liver Tumors Induction of Cytochrome p450 b–Catenin Mutations Oxygen Radicals DNA Damage Genetic Alterations in Cancer Genes

18. Assessment of Mutations in Cancer Genes from Oxazepam Induced Liver Tumors • b-Catenin gene • First cancer gene where mutations identified in both mouse and human hepatocellular neoplasms • Mutations of b-catenin also a major factor in colon cancer and melanomas • Hot spot for mutations: Codons 32-45 De La Coste, et al., PNAS, 95: 8847-8851, 1998

19. b-catenin ProteinCancer APC Cancer Cell Normal Cell Adapted, Science 281: 1439, 1998

20. Assessment of Mutations in Cancer Genes from Oxazepam Induced Liver Tumors • b-Catenin gene • Determine the mutation frequency and pattern of b-catenin mutations in spontaneous and oxazepam induced hepatocellular neoplasms and hepatoblastomas • Determine if the b-catenin protein accumulated in oxazepam induced liver tumors

21. Strategy for Evaluating DNA from Tumors for Mutations

22. Single-Strand Conformational Analysis (SSCA)

23. SSCA Analysisb-catenin Mutations N 1 2 3 4 5 6 7 8 9 10 Devereux, T.R, Sills, R.C., Barrett, J.C et al., Oncogene, 18: 4726-4733, 1999

24. G G T T A A C C Codon 41 C C A C C A/G Normal Mutation Direct Sequencingb-catenin Mutations

25. Mutation Frequency of b-Cateninin Hepatocellular Tumors of B6C3F1 Treatment Mutation Frequency Control 2/22 (9%) Oxazepam 18/42 (41%) a ap<0.001 when comparing mutation frequency to controls Devereaux, Sills, Barret et al., Oncogene 18, 4726, 1999

26. Examples of b-Catenin Mutations in B6C3F1Mouse Hepatocelluar Neoplasms Tumor Group Codon Mutation Bases Control 32 GAT to GCT A to C 33 TCT to TTT C to T Oxazepam 32 GAT to GGT A to G 32 GAT to GTT A to T 32 GAT to CAT G to C 32 GAT to AAT G to A 33 TCT to TAT C to A 33 TCT to TTT C to T 34 GGA to AGA G to A 34 GGA to GTA G to T 34 GGA to GAA G to A 41 ACC to ATC C to T Devereux, Sills, Barrett et al., Oncogene 18, 4726-33, 1999

27. Assessment of Mutations • Increase in point mutations at guanine bases following oxazepam exposure is consistent with the theory that oxygen radicals contributed to their formation as these genetic lesions can arise from oxidative damage • Mutations can occur as the result of oxidative damage to guanine residues resulting in the production of 8-oxoguanine

28. Oxazepam Hepatoblastomasb-Catenin Protein Expression

29. Immunohistochemical Detectionb-catenin ProteinB6C3F1 Mouse Cancer cells Hepatoblastoma Science, 281, 1439, 1998 Anna, Sills, Devereux et al. Cancer Res., 60, 2864, 2000

30. b-catenin Mutations in Hepatoblastomas From B6C3F1 Mice Treated with Oxazepam Tumor Group Codon mutation (amino acid) Frequency Codon 32 GAT to GGT (Asp to Gly) + Del. Codons 5-8 Del. Codons 5-7 Del. Codons 36-48 Del. Codons 23-49 Codon 34 GGA to GTA (Gly to Val) Del. Codons 5-13 Del. Codons 16-36 Del. Codons 21-43 Oxazepam 1 2 3 4 5 6 7 8 8/8 (100%) Anna, C.H., Sills, R.C., Devereux et al., Cancer Res., 60, 2864-2868, 2000

31. Western Blot Analysis of Proteins Associated with b-Catenin Mutations and Cancer Methylene Chloride Oxazepam 1 2 3 4 5 6 7 8 9 10 b-Catenin N – – + + + + + + + Mutation Cyclin D1 4 1 0 0 0 1 15 18 30 43 C-Myc Actin N A C C C C C A A C Anna, CH., Ida, M., Sills, R.C., Devereux, T.R., Tox. Appl. Pharmacology, 190: 135-145, 2003

32. Summary Oxazepam Tissue Cellular Molecular Hepatocellular Neoplasms Hepatoblastomas Liver Endogenous Source of Superoxide Anion Radicals Centrilobular Hypertrophy Cytochrome p450 DNA Damage b-catenin gene • b-catenin protein • Cyclin D1

33. Case Study in Rats and MiceHemangiosarcomas RiddelliineGenotoxic

34. Riddelliine • Belongs to a class of pyrrolizidine alkaloids • Isolated from plants of the genera Crotalaria, Amsinckia, and Senecio • Plants may contaminate human food sources, and intact plants and their seeds may contaminate commercial grain

35. Strategy for Examining Molecular Mechanisms of Liver Hemangiosarcomas in F344 Rats and B6C3F1Mice • Mechanistic Studies (NCTR) • Identification of activated riddelliine metabolites • 32P-postlabeling/HPLC method for identification of riddelliine-derived DNA adducts • Detection and quantification of adducts in livers of F344/N rats orally gavaged with riddelliine for 3 or 6 months • Molecular Studies (NIEHS) • Identification of K-ras/p53 mutations in hemangiosarcomas in B6C3F1 mice • Relevance of Mechanistic Studies to Humans

36. Metabolism of Riddelliine to Activated Metabolite

37. Metabolism of Riddelliine to Activated Metabolite

38. Total HPLC DHR-derived DNA Adducts in Liver DNA of Rats Fed Riddelliine for 3 and 6 Months

39. Relationships Between Administered Dose, AdductLevels, and Hemangiosarcoma Incidence of Rats

40. Tumor Incidence in Rats and Mice Rats Mice Cho, M.W., Chan, P. et al., Cancer Letters, 193: 119-125, 2003

41. Molecular LevelAssessment of Mutations in Cancer Genes Hypothesis Riddelliine Exposure Liver Hemangiosarcomas Induction of Cytochrome p450 P53 Mutations Ras Mutations Dehydroretronecine (DHR) Metabolite DNA DHR-Derived Adducts – Cancer Genes

42. Rationale for Evaluating Cancer Genes in Hemangiosarcomas of Mice • Vinyl chloride induce similar tumors in humans and rats • Vinyl chloride genotoxic intermediates DNA etheno adducts • N2-ethenoguanine: K-ras G A transitions • N6-etheno adduct: p53 A T transversions

43. Mutation Analysis of K-ras Oncogene Hemangiosarcomas B6C3F1 Mice K-ras Codon 12 Mutation Frequency 0/13a GGT (Normal Sequence) Spontaneous Hemangiosarcomas 7/12 GGT GTT (Mutation) Riddelliine Hemangiosarcomas aVarious Hemangiosarcomas in control B6C3F1 mice in NTP studies

44. Direct SequencingK-ras Mutations G G T T A A C C K-ras C G G Codon 13 T G/T G T G G Codon 12 Normal Mutation

45. p53 Gene is a tumor suppressor gene which causes G1 and G2 arrest, promotes apoptosis and loss of function causes genomic instability • Mutated p53 gene increased half life of protein detected by immunohistochemistry • Most commonly mutated tumor suppressor gene in human cancer S Cell Cycle p53 G2 G1 M Apoptosis p53 Gene Nature Reviews, Genetics 2001

46. P53 Protein ExpressionRiddelliineHemangiosarcomas

47. Relevance of Mechanistic Data to Humans • Do human liver microsomes metabolize riddelliine? • Do human liver microsomes with DNA and riddelliine form DHR-derived DNA adducts?

48. Metabolism of Riddelliine to Activated Metabolite

49. Human Liver Microsomal Metabolism 0.7 DHR 0.6 N-oxide 0.5 0.4 0.3 0.2 0.1 0 MR FR M1 M2 M3 M4 F1 F2 F3 F4 Rat Human Human

50. 32P-Postlabeling/HPLC Analysis of DHR-Derived Adducts Formed from Metabolism of Riddelliine by Liver Microsomes The liver microsomes were from female F344/N rats or male or female humans.