1. The National Institute of Environmental Health Sciences The National Institutes of Health The Department of Health and Human Services NTP The National Toxicology Program The Department of Health and Human Services Molecular Biology of Liver Tumors Following Chemical Exposure Robert C. Sills, DVM, PhD Diplomate, 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 Lesions B6C3F1 Mouse Hepatocellular Carcinoma Hemangiosarcoma Hepatocellular Adenoma Hepatoblastoma

4. Molecular Pathology Review

5. Major Genes Involved in Carcinogenesis Tumor suppressor genesProto-oncogenes  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 Oxazepam Non-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 Liver of Female B6C3F 1 Mice in the 2-Year Feed Study of Oxazepam Dose (ppm)01252,5005,000 2-Year Study Liver (Number of rats examined)50505050 Centrilobular Hypertophy02(1.5)11**(2.5)29**(2.9) Hepatocellular Adenoma2535*35*36* Hepatocellular Carcinoma9549**44** Hepatoblastomas 018**8** *P<0.05 **P<0.01

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

10. Cytochrome P450 Oxidative Damage OXAZEPAM METABOLITES CANCER GENES

11. Isoprostane (oxidative damage marker) in Livers of B6C3F1 Mice Following 6-months Oxazepam Exposure 0 10 20 30 40 50 60 70 (n=3) ControlOxazepam Tomer, Devereaux, NIEHS, 2000

12. Cytochrome P450 Oxidative Damage OXAZEPAM METABOLITES 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 Robbins and Cotran Pathologic Basis of Disease, 2005 Activation of MAP kinase pathway BLOCKED IN MUTANT RAS Inactivation by Hydrolysis of GTP GDP GTP Activation of transcription Cell cycle progression Growth factor Growth factor receptor

15. H-ras Proto-Oncogene Evaluation

16. H-ras Mutations in Hepatocellular Adenomas and Carcinomas from Oxazepam Treated Mice Treatment 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%) Tumors with Mutations Tumor Incidence Historical Control - H-ras mutations, 80/126 (63%) Hepatoblastomas - No H-ras Mutations

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

18. Assessment of Mutations in Cancer Genes from Oxazepam Induced Liver Tumors   -Catenin gene First cancer gene where mutations identified in both mouse and human hepatocellular neoplasms Mutations of  -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.  -catenin Protein Cancer Adapted, Science 281: 1439, 1998 APC Normal Cell Cancer Cell

20. Assessment of Mutations in Cancer Genes from Oxazepam Induced Liver Tumors   -Catenin gene Determine the mutation frequency and pattern of  -catenin mutations in spontaneous and oxazepam induced hepatocellular neoplasms and hepatoblastomas Determine if the  -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 Analysis  -catenin Mutations N Devereux, T.R, Sills, R.C., Barrett, J.C et al., Oncogene, 18: 4726-4733, 1999 1 2 3 4 5 6 7 8 9 10

24. Direct Sequencing  -catenin Mutations A C G T A C G T Normal Mutation CCACCA C A/G Codon 41

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

26. Examples of  -Catenin Mutations in B6C3F 1 Mouse Hepatocelluar Neoplasms Devereux, Sills, Barrett et al., Oncogene 18, 4726-33, 1999 Tumor GroupCodonMutationBases Control32GAT to GCTA to C 33 TCT to TTT C to T Oxazepam32GAT to GGTA to G 32GAT to GTTA to T 32GAT to CATG to C 32GAT to AATG to A 33TCT to TATC to A 33TCT to TTTC to T 34GGA to AGAG to A 34GGA to GTAG to T 34GGA to GAAG to A 41ACC to ATCC to T

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 Hepatoblastomas  -Catenin Protein Expression

29. Immunohistochemical Detection  -catenin Protein B6C3F1 Mouse Anna, Sills, Devereux et al. Cancer Res., 60, 2864, 2000 Science, 281, 1439, 1998 Cancer cells Hepatoblastoma

30.  -catenin Mutations in Hepatoblastomas From B6C3F1 Mice Treated with Oxazepam Tumor Group Oxazepam 1 2 3 4 5 6 7 8 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 8/8 (100%) Frequency Codon mutation (amino acid) Anna, C.H., Sills, R.C., Devereux et al., Cancer Res., 60, 2864-2868, 2000

31. Western Blot Analysis of Proteins Associated with  -Catenin Mutations and Cancer Cyclin D1 C-Myc Actin N – ACCCCCAAC N–+++++++ 21345678910 Methylene Chloride Oxazepam Mutation  -Catenin 41000115183043 Anna, CH., Ida, M., Sills, R.C., Devereux, T.R., Tox. Appl. Pharmacology, 190: 135-145, 2003

32. Summary Oxazepam Tissue Cellular Molecular Liver Centrilobular Hypertrophy Cytochrome p450 Endogenous Source of Superoxide Anion Radicals DNA Damage  -catenin gene  -catenin protein Cyclin D 1 Hepatocellular Neoplasms Hepatoblastomas

33. Case Study in Rats and Mice Hemangiosarcomas Riddelliine Genotoxic

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 32 P-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

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

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

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

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

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 Spontaneous Hemangiosarcomas Riddelliine Hemangiosarcomas 7/12 0/13 a Mutation Frequency K-ras Codon 12 GGT (Normal Sequence) a Various Hemangiosarcomas in control B6C3F1 mice in NTP studies GGT GTT (Mutation)

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

45. p53 Gene p53 G1 S G2 M Cell Cycle Apoptosis Nature Reviews, Genetics 2001  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

46. P53 Protein Expression Riddelliine Hemangiosarcomas

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 0.1 0.2 0.3 0.4 0.5 0.6 0.7 MRFRM1M2M3M4F1F2F3F4 DHR N-oxide Rat Human

50. 32 P-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.

51. Summary  Riddelliine induces liver hemangiosarcomas through a genotoxic mechanism.  Riddelliine-derived DNA adducts are dose-dependent and persistent, and responsible for liver hemangiosarcoma induction.  The greater DNA adduct levels, K-ras and p53 mutations in endothelial cells correlate with riddelliine-induced liver hemangiosarcomas in rats and mice.

52. Research Team