Rodent Liver Tumors: NCI/NTP Historic Perspective
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1. Rodent Liver Tumors: NCI/NTP Historic Perspective Bob Maronpot, Raleigh, NC STP Annual Meeting – 2023
2. Rodent Liver Tumors: NCI/NTP Historic Perspective Bob Maronpot, Raleigh, NC STP Annual Meeting – 2023 Acknowledgements: Dave Malarkey & Arun Pandiri • A little bit of NCI/NTP rodent bioassay history • NTP liver tumor data • Liver tumor images • Current NTP safety assessment perspective
3. 1700’s 1950 1960 1970 1980 1990 2000 2011 • Bernardino Ramazzini – 1713 • Breast cancer in nuns • John Hill – 1761 • Snuff & oral/nasal cancer • Percival Pott – 1775 • Scrotal cancer • Elmslie -1866 (Kashmir) • Epithelioma of abdomen & thighs • Rehn – 1895 • Bladder cancer in aniline dye workers • Yamagiwa & Ichikawa – 1918 • Tar & soot on rabbit ears 1775 – Scrotal cancer in chimney sweeps. Cancer was attributed to the tar and soot in the chimneys. This is an early and famous example of occupational cancer in humans. Rodent Bioassay Timeline
4. 1700’s 1950 1960 1970 1980 1990 2000 2011 • Bernardino Ramazzini – 1713 • Breast cancer in nuns • John Hill – 1761 • Snuff & oral/nasal cancer • Percival Pott – 1775 • Scrotal cancer • Elmslie -1866 (Kashmir) • Epithelioma of abdomen & thighs • Rehn – 1895 • Bladder cancer in aniline dye workers • Yamagiwa & Ichikawa – 1918 • Tar & soot on rabbit ears Rodent Bioassay Timeline
5. 1700’s 1950 1960 1970 1980 1990 2000 2011 • Murphy & Sturm – 1925 • Lung tumors in tar-painted mice • Cook et al. – 1932 • Cancer induction by PAHs • Sasaki & Yoshida – 1935 • o-amidoazotoluene diet and liver cancer; effects of dose on latacency and use of stop studies • Berenblum – 1941 • Concept of co-carcinogenesis • Initiation, promotion, progression • Magee & Barnes – 1956 • Nitrosamines & liver cancer in rats • Bernardino Ramazzini – 1713 • Breast cancer in nuns • John Hill – 1761 • Snuff & oral/nasal cancer • Percival Pott – 1775 • Scrotal cancer • Elmslie -1866 (Kashmir) • Epithelioma of abdomen & thighs • Rehn – 1895 • Bladder cancer in aniline dye workers • Yamagiwa & Ichikawa – 1918 • Tar & soot on rabbit ears Rodent Bioassay Timeline
6. 1700’s 1950 1960 1970 1980 1990 2000 2010 • Bernardino Ramazzini – 1713 • John Hill – 1761 • Percival Pott – 1775 • Elmslie -1866 • Jonathon Hutchinson – 1888 • Rehn – 1895 • Yamagiwa & Ichikawa – 1918 • Murphy & Sturm – 1925 • Cook et al. – 1932 • Sasaki & Yoshida – 1935 • Berenblum – 1941 • Magee & Barnes – 1956 • Realization that chemicals, environmental factors, and aspects of lifestyle cause cancer
7. Concept of the rodent bioassay & its establishment by the National Cancer Insititute (NCI) •1962 – First contracted bioassay •1969 – Innes et al*., study published •20,000 mice; 127 different chemicals; 18-mo studies •Selection of B6C3F1 mouse •1971 – National Cancer Act •Decision made to standardize bioassay testing •~1975 – Inbred F344 rat selected •Small size, vigor & survival, disease resistance *Innes et al., JNCI 42(6): 1104-1114 (1969)
8. Thou shalt use standardize tests
9. Thou shalt use two species
10. Thou shalt use the MTD & 1/2 MTD
11. 1700’s 1950 1960 1970 1980 1990 2000 2010 NCI NTP CANCER BIOASSAY TIMELINE The NCI cancer bioassay 50 Male and 50 female F344 rats 50 Male and 50 female B6C3F1 mice Maximum tolerated dose & 1/2 MTD Test duration of 18 months or 2 years Pathology evaluation
12. 1700’s 1950 1960 1970 1980 1990 2000 2010 NCI NTP Input from National and International Organizations Refining of the Bioassay • Standardization of bioassay – Originally designed for screening • Extensive pathology with peer review* – Standardization of diagnostic nomenclature • Statistical evaluation standardized • Historical control database • Search for alternative models *Maronpot & Boorman (1982) Toxicol Pathol 10(2): 71-78
13. 1700’s 1950 1960 1970 1980 1990 2000 2010 NCI NTP Limitations of the bioassay Alternative models & ancillary approaches • Resource intensive • Not validated • Inherent insensitivity for detecting weak or moderate carcinogens • Single chemical exposure vs “real world” • Not sure if an agent has carcinogenic potential under actual human exposure conditions • Debate regarding relevance • Rodent-specific mechanisms • High doses • Strain A mouse • Two-stage & neonatal models • Humanized mice • Ito medium-term model • Transgenic models • Local subcutaneous injection • Medaka & guppy models • Genotoxicity batteries
14. # Mouse (%), n=490* Rat (%), n=490* 1 Liver (27.1) Liver (10.6) 2 Lung (8.8) Kidney, tubular cell (9.2) 3 Forestomach (4.7) Mammary gland (5.9) 4 Hematopoietic system (4.5) Lung (4.6) 5 Harderian gland Thyroid gland, follicular cell (2.7) Thyroid gland, follicular cell (4.5) 6 Kidney, tubular cell (2.5) Forestomach (4.3) 7 Vascular System (Unspecified) (2.3) Urinary bladder (4.1) 8 Mammary gland (2.2) Skin (3.8) 9 Ovary (2) Hematopoietic system (3.7) 10 Skin (1.6) Adrenal medulla Oral cavity Zymbal gland (3.5) Target organs of chemical-induced carcinogenicity *n=490 studies where the same chemical was tested in both F344 rats and B6C3F1 mice Courtesy of A. Pandiri 2020
15. Historical control incidences of liver tumors in rats (F344/N) and mice (B6C3F1) Background liver tumor incidence Tumor type Male Mouse % (Range%) Female Mouse % (Range%) Male Rat % (Range%) Female Rat % (Range%) Hepatocellular Adenoma 54.91 (34-78) 25.68 (10-67) 1.43 (0-6) 0.86 (0-4) Hepatocellular Carcinoma 30 (16-50) 12.93 (4-20) 0.57 (0-4) 0.14 (0-2) Hepatoblastoma 3.27 (0-8) 0.55 (0-2) 0 0 Combined 71.82 (62-84) 34.43 (16-73) 2 (0-6) 1 (0-4) Mouse, n=550; Rat, n=700 Courtesy of A. Pandiri 2020
16. Liver 57 % Lung 22 % Kidney 22 % Mammary gland 14 % Hematopoietic 13 % Forestomach 12 % Thyroid 10 % Vascular System 9 % Frequency of tissue response in 290 cancer- positive NTP mouse and/or rat bioassays Data courtesy of D. Malarkey 2022
17. • 30% (146) of 490 NTP studies had an hepatocellular tumor response in rats and/or mice* • Species dependence: mouse – 95/146 (65%), rat – 14/146 (9.6%), or both species 37/146 (25.3%) Liver tumor incidences based on 490 studies Liver tumors N=146/490* Mouse Male n (%) Mouse Female n (%) Rat Male n (%) Rat Female n (%) Nodule 0 1 (0.6) 10 (6.8) 6 (4.1) Hepatocellular Adenoma 6 (4.1) 14 (9.5) 1 (0.6) 5 (3.4) Hepatocellular carcinoma 64 (43.8) 88 (60.2) 31 (21.2) 30 (20.5) Hepatoblastoma 20 (13.7) 13 (8.9) 0 (0) 1 (0.6) Combined 90 (61.6) 116 (79.5) 42 (28.8) 42 (28.8) * 490 studies with same chemical tested in both rats and mice Data courtesy of A. Pandiri 2022
18. Hepatocellular Adenomas and Carcinomas
19. Hepatic Foci of Cellular Alteration
20. Hepatocellular Adenoma Untreated Male B6C3F1 Mouse
21. Hepatocellular Adenoma
22. Hepatocellular Carcinoma
23. Hepatocellular Carcinoma
24. Progression of Proliferative Liver Lesions Basophilic Focus Hepatocellular adenoma Metastatic carcinoma Hepatocellular carcinoma
25. Carcinoma Arising in Adenoma
26. Hepatoblastoma Turusov et al., Tox Path 30(5):580-591 (2002) (63/140 studies had hepatoblastoma) (Evaluated 500 hepatoblastomas)
27. Cholangioma Sprague Dawley Male
28. Cystic Cholangioma Male F344 Rat Control
29. Cholangiocarcinoma B6C3F1 Untreated male
30. Hepatocholangiocarcinoma Hepatocholangioma Treated Female Sprague Dawley Treated Male F344
31. Hepatocholangiocarcinoma with intestinal metaplasia
32. Other types of liver tumors Hemangiosarcoma Histiocytic sarcoma Stellate cell tumor Lymphoma
33. What have we learned from the conventional bioassay with respect to liver tumors?
34. Malarkey, DE, Hoenerhoff, MJ, and Maronpot, RR. 2018. Carcinogenesis: Manifestations and Mechanisms in Fundamentals of Toxicologic Pathology, 3rd Edition, Wallig, MA, Haschek, WM, Rousseaux, CG, Bolon, B, and Mahler, BW, Editors, Academic Press, San Diego. Pp 83-104.
35. 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
36. There were and still are some strong opinions about the significance of rodent bioassays
38. 1700’s 1950 1960 1970 1980 1990 2000 2010 NCI NTP 2022 • Nuclear receptor activation • CAR/PXR, AhR, PPAR-a • Cytotoxicity and regenerative hyperplasia • Endocrine modifiers • Epigenetic modifiers • Mitogen/tumor promoter • Inflammation • Oxidative stress • Hormonal perturbation • Immunosuppression • Suppression of apoptosis Mechanisms associated with bioassay tumor responses
39. Contemporary NTP efforts • Core set of mechanistic assays • DNA repair & reactivity • Receptor-mediated assays • Intercellular communication • Enzyme induction • Cell cycle perturbations • Endocrine disruption • Effects on methylation • Oxidative stress • Immunosuppression • Other contemporary investigative approaches • NEGCARC (Genotoxicity, endocrine, histopathology) for pharmaceuticals • Tox 21 & high throughput screening assays • Genomics, proteomics, metabonomics • Mutations in cancer genes • Structure activity relationships • Epigenetic changes • Adverse outcome pathway/MOA Throughput QSAR Relationships Cell culture and Genetox assays Organoids, metabolically competent Lower order model organisms Rodent models Human relevance
40. Prechronic liver lesions as predictors of liver carcinogenicity* B6C3F1 Mouse • 25 of the 27 (92%) liver tumor positive studies were correctly identified in 90-day studies based on combination of liver hypertrophy, cytomegaly, necrosis and increased liver weight (p < 0.001) • 18 false positives F344 Rat • 7 of 11 (64%) liver tumor positive studies were correctly identified in 90-day studies based on combination of liver hypertrophy, cytomegaly, and necrosis (p<0.01) • 16 false positives *Based on examination of 83 B6C3F1 and 87 F344 90-day studies with corresponding 2-year studies Allen et al., Toxicologic Pathology 32:393-401 (2004)