Following a presentation of basic principles of carcinogenesis with emphasis on liver cancer development and intrinsic susceptibility factors, examples of specific rat and mouse animal models of hepatocarcinogenesis are provided.

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Liver Carcinogenesis and Use of Rat and Mouse Models

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1. Liver Carcinogenesis and Use of Rat and Mouse Models R.R. Maronpot maronpot@me.com
2. Global Importance of Liver Cancer • 6th most common cancer type worldwide – 3rd most common cause of cancer death worldwide • 748,300 new liver cancer cases in 1980 – 695,900 liver cancer related deaths – 70-80 % hepatocellular carcinoma – Highest incidence in Asia and sub-Saharan Africa
3. Liver 57 % Lung 22 % Kidney 22 % Mammary gland 14 % Hematopoeitic 13 % Forestomach 12 % Thyroid 10 % Vascular System 9 % Evidence of carcinogenic activity (n=290 out of ~600 NTP rat & mouse carcinogenicity studies)
4. Unlike the situation with human hepatocellular carcinoma, rodent hepatocellular carcinoma development is usually not secondary to infections (with some exceptions).
5. Outline • Carcinogenesis overview – Multistage process – Lesion progression • Rodent hepatocarcinogenesis • Animal models of hepatocarcinogenesis • Other – Tumor regression – Non-heptocellular liver neoplasia – Cell proliferation
6. Overview of Carcinogenesis • Complex disease with multiple causes • Influenced by multiple intrinsic and extrinsic factors • Multistep progressive process at the genetic and phenotypic level
7. Causes of Cancer • Infection (viruses and parasites) • Genes and gene mutations • Enhanced cell proliferation • Chemicals • Hormones • Radiation • Diet and life style • Sunlight
8. ure 6. FIGURE 7. Intrinsic and extrinsic factors modulating specific gene expression and its effect on tissue phenotype and function. GENE PHENOTYPIC & FUNCTIONAL EFFECTS Intrinsic Modulating Factors • Metabolism • Receptors • Differentiation receptors • Signal transduction • DNA repair • Cell-cell communication • Growth factors • Cytokines • Angiogenesis • Inflammation • Chemical agents • Physical agents • Viruses • Radiation • Diet • Life style • Bacteria • Parasites Extrinsic Modulating FactorsRNA Protein Effect on Cell Effect on Tissue Effect on Organism Modulating Factors
9. Modulating Factorsre 6. FIGURE 7. Intrinsic and extrinsic factors modulating specific gene expression and its effect on tissue phenotype and function. GENE PHENOTYPIC & FUNCTIONAL EFFECTS Intrinsic Modulating Factors • Metabolism • Receptors • Differentiation receptors • Signal transduction • DNA repair • Cell-cell communication • Growth factors • Cytokines • Angiogenesis • Inflammation • Chemical agents • Physical agents • Viruses • Radiation • Diet • Life style • Bacteria • Parasites Extrinsic Modulating FactorsRNA Protein Effect on Cell Effect on Tissue Effect on Organism Cellproliferation
10. Overview of Carcinogenesis DNA Repair
11. Metaplasia Physiological hyperplasia Dysplasia Anaplasia
12. Outline • Carcinogenesis overview – Multistage process – Lesion progression • Rodent hepatocarcinogenesis • Animal models of hepatocarcinogenesis • Other – Tumor regression – Non-heptocellular liver neoplasia – Cell proliferation
13. – Hepatocellular – Hepatoblastoma – Cholangial – Hemangial Rodent Liver Cancer
14. Drinkwater & Bennett, 1991 Lung Colon Liver Skin 1.0 0.8 0.6 0.4 0.2 0 Relative Susceptibility of Inbred Mouse Strains to Chemically Induced Carcinogenesis
15. Figure 1. Serum ALT levels 24 hours after dosing with APAP (300mg/kg) or vehicle (0.5% methylcellulose). From I. Rusyn, University of North Carolina
16. Sex Differences in Liver Positive 2-Year Bioassays • 54 Liver positive rat bioassays – 13 (24%) in males – 8 (15%) in females – 33 (61%) in both sexes • 120 Liver positive mouse bioassays – 14 (12%) in males – 37 (31%) in females – 69 (57%) in both sexes
17. Age-related lesions (Male B6C3F1 mouse) Mice with lesions (%) Age (mos) Focus Adenoma Carcinoma < 12 0 0 0 12-18 12 17 5 18-24 31 32 18 24- 30 21 46 34 30-36 28 63 34 Harada, et al. In: Pathology of the Mouse, Maronpot; Ed. 1999
18. Age-related lesions (F344 rats) lesion (%) Age (mos) Focus Adenoma Carcinoma 6 50 % 12 80 % > 12 100 % 1% 1% Eustis, et al. In: Pathology of the Fisher rat, Boorman, et al.; Ed. 1990
19. Susceptible Intermediate Resistant Fischer F344 Sprague Dawley Copenhagen Donryu Wistar DRH August Brown Norway Marshall Wistar-Kyoto Wood, et al. 2002. Carcinogenesis 23(1):1-9. -foci don’t progress; no diff apoptosis rates; polygenic, modifier genes; role of oval cell. Rat strain sensitivity
20. Male Mouse Liver Tumors King-Herbert & Thayer – 2006
21. Relative susceptibilities of selected strains to liver tumor induction High susceptibility Intermediate susceptibility Relatively resistant C3H C57BR/cdJ BALB/c CBA FVB C57BL/6 B6C3F1 SM/J C57BL/10 DBA/2 (infant model) P/J 129 Tif:MAGf CE/J DBA/2 (> 5 weeks old) C3H x CBA LP SWR CBA x C57BL/10 AKR/J A C3H x A/J CD-1 IF DBA/2 x CE/J NMRI RF LP x 129 A x C57BL/6 LP x DBA/2 C57BL x A LP x C57BL/10 A x C57BL/10 129 x DBA/2 C57BL/6 x BALB/c
22. Genetic loci implicated in mouse hepatocarcinogenesis Locus identifier Mouse chromosome Hcs7 1 Hcf2 1 Hcs4 2 Hcs5 5 Hcs1 7 Hcs2 8 Hcr2 10 Hcs3 12 Hcf1 17 Hcs6 19 Hcs = Hepatocarcinogen sensitivity Hcr = Hepatocarcinogen resistance Hcf = Hepatocarcinogenesis female
23. Overview of Carcinogenesis DNA Repair
24. Multistage Hepatocarcinogenesis normal focus of altered hepatocytes hepatocellular adenoma hepatocellular carcinoma H-ras activation altered Brca1 altered TGFa Cathepsins Osteopontin Goliath MIG MHC class II B-catenin apoptosis c-fos cyr61
25. Progression • Foci of cellular alteration – Earliest proliferative lesions – Initially increase in number and then decrease • Adenomas – Some arise within foci – Increase in prevalence before carcinomas – Some remain and some progress to carcinomas • Carcinomas – Some arise within adenomas – Increase in prevalence after emergence of adenomas – Rate of increased prevalence similar to that of adenomas
26. Metaplasia Physiological hyperplasia Dysplasia Anaplasia Focus of cellular alteration Hepatocellular adenoma Hepatocellular carcinoma
27. Initiated cell? Focus of cellular alteration Hepatocellular adenoma Hepatocellular carcinoma Carcinoma arising in an adenoma Adenoma arising in a focus
28. Carcinoma arising in an Adenoma
29. Carcinoma arising in an Adenoma
30. Defining Diagnostic Criteria • What is hyperplasia versus neoplasia in the broad context of toxicologic pathology – There is a range of change – Diagnoses determined by training, published literature, and experience – The greater the experience, the broader the ranges of non-neoplastic and benign NORMAL PATHOLOGICAL HYPERPLASIA AND PRENEOPLASIA ADENOMA CARCINOMA
31. NORMAL PATHOLOGICAL HYPERPLASIA AND PRENEOPLASIA ADENOMA CARCINOMA
32. Outline • Carcinogenesis overview – Multistage process – Lesion progression • Rodent hepatocarcinogenesis • Animal models of hepatocarcinogenesis • Other – Tumor regression – Non-heptocellular liver neoplasia – Cell proliferation
33. Rodent Models of Hepatocarcinogenesis • Variety of models used to study factors influencing development of hepatocellular carcinoma (HCC) – Pathogenesis of HCC – Metastasis – Identification of key pathways – Identification of key mediators – Identification of new treatment modalities
34. Rodent Models for Liver Tumor Induction • Conventional bioassays – CD-1, B6C3F1, NMRI, C57BL/10 • Single/multiple doses to adult rats • Neonatal mouse model • Initiation-promotion models – Necrogenic dose of initiator – Initiator after partial hepatectomy – Neonatal initiation • Genetically engineered models
35. Mouse Models of Hepatocarcinogenesis • Xenograft models – HCC lines in SCID mice • Orthotopic models • Transgenic GEM models – Viral: HBV, HCV – Cell cycle related: p53 KO + liver specific factors – C-myc; c-myc+E2F-1; c-myc+TGFa; SV40 T Ag – Telomere dysfunction models – Pathway-specific models: Wnt/b-catenin; IGF2; HGF • Chemically induced models: choline deficiency; DEN, 2-AAF, Vinyl carbamate
36. Chemically Induced Models • Neonatal mouse model • Solt-Farber rat model • Medium-term rat liver focus model Based on Initiation and Promotion Protocols
37. Overview of Carcinogenesis DNA Repair
38. Chemically Induced Models • Neonatal mouse model • Solt-Farber rat model • Medium-term rat liver focus model
39. Chemically Induced Models • Neonatal mouse model – IP injection of 15-day old mice with DEN or VC* – Endpoints – basophilic foci (G-6-P’ase negative), adenomas, carcinomas – Foci in less than 18 weeks; adenomas in less than 40 weeks, carcinomas in less than 56 weeks • Solt-Farber rat model • Medium-term rat liver focus model * Genotoxic agents; not necrogenic
40. Vinyl Carbamate (VC) Studies Newborn Mouse Model • Single intraperitoneal dose of vinyl carbamate at day 15 • 0.03 and 0.15 -M VC/ gram body weight • No further treatment • Periodic sacrifice of mice over a 24-30 month period
41. Foci 0.15 -M 0.03 -M Control 0.15 μM 0.03 μM Control Males
42. Hepatocellular Adenoma 0.15 -M 0.03 -M Control 0.15 μM 0.03 μM Control
43. Hepatocellular Carcinoma 0.15 -M 0.03 -M Control 0.15 μM 0.03 μM Control
44. Male Data
45. Foci Adenoma Carcinoma B6C3F1 C3H C57BL/6 Male Data
46. Age-specific Tumor Response Strain Differences
47. Sex Differences in Liver Tumor Response Female Male Male Female
48. Chemically Induced Models • Neonatal mouse model • Solt-Farber rat model • Medium-term rat liver focus model
49. Solt-Farber 1976 Model Presence of basophilic foci + _ _ _ Basal diet Basal diet plus 0.02% AAF DEN DEN DEN SALINE PH PH PH SH 1 week 1 week 1 week 1 week
50. Chemically Induced Models • Neonatal mouse model • Solt-Farber rat model • Medium-term rat liver focus model – Necrogenic dose of DEN at 6 weeks – Treatment with test substance 2 weeks later – Partial hepatectomy 1 week later – Treat with test substance for 5 more weeks – Quantitate PGS-T foci – (Note: Doesn’t work for peroxisome proliferators)
51. Medium Term Rat Liver Focus Model
52. Medium Term Rat Liver Focus Model
53. promotioninitiation progression
54. GSTP/PGST
55. Medium-Term Multi-organ Model • Dose sequentially with DEN, MNU, and DHPN • Then given test agent for 14 weeks with sacrifice at 18 weeks – DEN – liver – DHPN – thyroid, lung, kidney, urinary bladder, lung – MNU – thyroid, urinary bladder, hematopoietic • Dose sequentially with DHPN, EHEN, and DMAB • Then given test agent for 16 weeks • DHPN – thyroid, lung, kidney, urinary bladder, lung – EHEN – kidney – DMAB – prostate
56. Outline • Carcinogenesis overview – Multistage process – Lesion progression • Rodent hepatocarcinogenesis • Animal models of hepatocarcinogenesis • Other – Tumor regression – Non-heptocellular liver neoplasia – Cell proliferation
57. Malarkey, et al. 1995. Carcinogenesis 16:2617-
58. Malarkey, et al. 1995. Carcinogenesis 16:2617-
59. Tumor regression – Hepatocellular tumors – Neuroblastoma – Fibrosarcoma – Germinoma – Renal cell carcinoma – Lung cancer – Malignant melanoma – Lymphoma – Mouse mammary tumors
60. – Chlordane – Phenobarbital – Nafenopin – Clofibrate • Peroxisome Proliferator WY-14,643 Tumor regression non-genotoxic hepatocarcinogens
61. – Hepatocellular – Hepatoblastoma – Cholangial – Hemangial Rodent Liver Cancer
62. Cholangioma Cholangiocarcinoma Oval cell proliferation Cholangiofibrosis Cholangiofibroma ? ?
63. Generalized Neoplasia • Hemangioma/Hemangiosarcoma • Histiocytic Sarcoma • Lymphoma • Mononuclear Cell Leukemia • Erythroleukemia • Myelodysplasia