Complete Protection against Aflatoxin B 1 -induced Liver Cancer with a Triterpenoid: DNA Adduct Dosimetry and Genotoxic Threshold

Transcription

1 Complete Protection against Aflatoxin B 1 -induced Liver Cancer with a Triterpenoid: DNA Adduct Dosimetry and Genotoxic Threshold Bill D. Roebuck 1, John D. Groopman 2, and Thomas W. Kensler 2,3 1 Department of Pharmacology and Toxicology, The Geisel School of Medicine at Dartmouth, Hanover, New Hampshire 2 Department of Environmental Health Sciences, Bloomberg School of Public Health, Baltimore, Maryland 3 Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania

2 Natalie M. Johnson, Patricia A. Egner, Victoria K. Baxter, Michael B. Sporn, Ryan S. Wible, Thomas R. Sutter, John D. Groopman, Thomas W. Kensler, and Bill D. Roebuck Complete Protection against Aflatoxin B 1 -Induced Liver Cancer with a Triterpenoid: DNA Adduct Dosimetry, Molecular Signature, and Genotoxicity Threshold Cancer Prevention Research 7: , 2014 Baltimore, 1983 Collaborative efforts for nearly 4 decades Groopman Kensler Roebuck

3 General Background of Aflatoxins as Acute Hepatotoxin and Hepatocellular Carcinogen

4 H Aflatoxins H CH 3 Aflatoxin B 1 (AFB 1 ) Discovered in UK ~1960 in moldy, toxic animal feed Lethal protein component to poultry feed ( Turkey X disease ) Chemical and biological properties Highly fluorescent, heat stable Multiple forms - aflatoxin B 1 most toxic/carcinogenic Liver carcinogen in virtually all animals tested Acute toxicant and carcinogen for humans Frequent contaminant of improperly stored food crops Produced by strains of mold Aspergillus flavus (A.flavus toxin = Aflatoxin ) Spores globally distributed in soil Mold grows on food crops not properly dried Maize (corn) Groundnuts (peanuts)

5 AFB 1 as Hepatocellular Carcinogen Sensitive µg/kg/day Rainbow Trout Pekin Duck Fischer Rat Sprague-Dawley Rat Zebrafish, Guppy, Medaka Intermediate Brook Trout, Tree Shrew Hamster Squirrel Monkey? [Human]? Rhesus, African Green, & Cynomolgus monkey Chicken Mouse Amphibians Resistant mg/kg/day Channel Catfish, Salmon

6 Hepatic Carcinogenesis: Aflatoxins Aflaxtoxins-induced liver cancer in rats: 1963 Rainbow trout (Salmo gairdneri) sensitive Cheap & easy to use large numbers Mouse resistant to aflatoxin Activates AFB 1, but detoxifies efficiently Fischer (F344 strain) very sensitive Carcinogenic response: males > females Hepatocellular carcinomas - 1 to 2 years 40 rats/arm: large & expensive experiment

7 Worldwide Morbidity and Mortality for Liver Cancer World Cancer Report 2014, IARC

8 Worldwide Morbidity and Mortality for Liver Cancer World Cancer Report 2014, IARC

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10 Incidence of Hepatocellular Carcinoma in USA Altekruse et al. Am. J. Gastro., 2014

11 Highly Reproducible and Quantitative Animal Model for Aflatoxin B 1 -induced Hepatocellular Carcinogenesis

12 H H CH 3 aflatoxin DNA adducts preneoplastic lesions liver cancer & metabolites HURS WEEKS MNTHS

13 Aflatoxin forms several different DNA adducts AFB1 AFB 1 AFB 1 Hydrolysis HPLC pmol/mg DNA H 2 N A HN 5 N B C D AFB N NH E CH AFB -FAPY F 1 H DIL I Frac tion Number AFB1-N7-Gua H 2 N HN N AFB N NH CH

14 Search for the Most Mutagenic Lesion 8 9 P 450 CH 3 Aflatoxin B1 (AFB1) CH 3 AFB1-8,9-xide AFB AFB N N HN CH HN H 2 N N NH H 2 N N NH DNA H 3 C H HN N H 2 N N N CH 3 P P 3 AFB1-N7-Gua 3 P P 3 FAPY-Major 3 P P 3 FAPY-Minor AFB1-N7 Guanine H Smela et al., 2002 ther FAPY Rotamers 3 P AP Site P 3

15 Multistage Carcinogenesis Sequential steps 1. Initiation Rapid, DNA damage, mutations 2. Post-initiation events (promotion) ver relatively long time 3. Progression Late event

16 Quantitative Predictive Rat Model Rat: F344 or Sprague-Dawley 100 to 110 g at initial dose of AFB 1 Physiologically cannot vomit Caging: suspended wire mesh Diet: AIN 76A or AIN 93 series Dietary acclimation Foci analysis: weeks post-initiation Initially, 16 weeks Currently, 5 weeks

17 Quantitative Model (cont.) Putative preneoplastic lesions enzymatic positive foci gamma glutamyl transpeptidase positive adenosine triphosphatase deficiency glucose-6-phosphatase deficiency 3 H-thymidine labeling Glutathione S-transferase (GST-P foci) A fetal enzyme liver and placental form Immunohistochemical assay Acetone fixed tissue & paraffin imbedded

18 AFB 1 Carcinogenesis in Young Rat = 25 μg AFB 1 Autopsy Autopsy FCI CANCERS Time (weeks)

19 Weeks following initiation Fixed liver in paraffin H & E stain GST-P stained foci

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22 Microscopic view of two-dimensional section of liver Morphometric Transformation Mean Focal Diameter Number Foci/cm 3 Focal Volume %

23 Quantitative microscopymorphometry bserved (2-D) Tissue area Number of transected foci Area of transected focus Focal classes Calculated (3-D) Foci / unit volume Foci / liver Mean focal diameter Volume % of liver occupied by foci (tumor burden)

24 Two examples showing general utility of quantitative morphometric analysis of AFB 1 -induced foci for understanding chemical carcinogenesis and chemoprevention

25 Schistosoma Schsitosoma mansoni mansoni

26 Aflatoxin mercapturic acid (urine) Aflatoxin M 1 (urine) CYP1A2 GSTs H H CH 3 CYPs 1A2 3A4 H H H 2 N HN N H N N H H CH 3 DNA Aflatoxin B 1 Aflatoxin-8,9-epoxide AP site CH 3 DNA H 2 N HN N H N N H H CH 3 Aflatoxin N 7 -guanine (urine) other metabolites Aflatoxin albumin adduct (serum)

27 AFB 1 Carcinogenesis in Young Rats 25 μg AFB 1 Autopsy Autopsy FCI CANCERS Time (weeks)

28 Inhibition of Hepatocellular Carcinogenesis with 10 doses of AFB 1 Focal Incidence Focal Volume % (mean ± SE) HCC (%) Aflatoxin 10/10 36 ± 17 5/45 (11) Aflatoxin plus oltipraz 4/10 1 ± 1 0 Roebuck et al., 1991

29 ltipraz Reduces Aflatoxin-DNA Adduct Formation in Rat Liver

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31 Aflatoxin mercapturic acid (urine) Aflatoxin M 1 (urine) CYP1A2 GSTs H H CH 3 CYPs 1A2 3A4 H H H 2 N HN N H N N H H CH 3 DNA Aflatoxin B 1 Aflatoxin-8,9-epoxide AP site CH 3 DNA H 2 N HN N H N N H H CH 3 Aflatoxin N 7 -guanine (urine) other metabolites Aflatoxin albumin adduct (serum)

32 ltipraz intervention: foci, adenomas and adenocarcinomas Treatment Incidence (%) Number of rats No lesions Foci Adenomas HCC No intervention 41 1 (2.4) 4 (9.8) 2 (4.9) 34 (82.9) Intervention 40 4 (10) 13 (32.5) 4 (10) 19 (47.5)* *P<0.05, Fisher s Exact Test

33 ltipraz intervention: foci, adenomas and adenocarcinomas Treatment Incidence (%) Number of rats No lesions Foci Adenomas HCC No intervention 41 1 (2.4) 4 (9.8) 2 (4.9) 34 (82.9) Intervention 40 4 (10) 13 (32.5) 4 (10) 19 (47.5)* *P<0.05, Fisher s Exact Test Significant decrease in HCC Shift toward less advanced lesions

34 Chemoprevention by Reduction and Delay Effect of ltipraz on Aflatoxin-induced HCC in Rats Proportion Free of HCC Control ltipraz Extent of Protection 22% delay in onset: p = % reduction in incidence: p = WEEKS Kensler et al., CEBP. 6: , 1997

35 Reduction in Hepatic AFB-DNA Adduct Levels UNDERESTIMATES Antitumorigenic Efficacy of ltipraz Kensler et al, CRT, 1999

36 Reduction in Hepatic AFB-DNA Adduct Levels UNDERESTIMATES Antitumorigenic Efficacy of ltipraz 90% Foci Burden 50% DNA adducts Kensler et al, CRT, 1999

37 Triterpenoids Synthetic analogs of oleanolic acid with anti-inflammatory and antitumorigenic activity Inhibit growth, induce cell cycle arrest, and induce apoptosis in breast cancer cell lines 1 Inhibit tumor growth in melanoma and leukemia mouse models 2 Potent inducers of phase 2 enzymes in vitro 3 Functions in part through Nrf2 signaling in vitro 3,4 NC H CDD-Imidazolide (TP235) N N 1 Lapillonne et al. Cancer Res Place et al. Clin Cancer Res Dinkova-Kostova et al. PNAS Liby et al. Cancer Res 2005

38 Protocol for Evaluation of CDD-Im as an Inhibitor of Aflatoxin-Induced Tumorigenesis in Male F344 Rats * * * * * * * * * Weeks of experiment * 1, 3, 10, 30, or 100 µmol CDD-Im/kg body weight by gavage 25 µg aflatoxin B 1 per rat by gavage 6 h after CDD-Im

39 CDD-Im Inhibits Aflatoxin-DNA Adduct Formation in Rat Liver Aflatoxin-N 7 -guanine (pmol/mg DNA) ** P < * P < 0.02 x ± SE (n=4) 0 Vehicle CDD-Im (µmol/kg body weight)

40 Volume Percent GST-P Positive Foci CDD-Im Inhibits Aflatoxin-Induced Tumorigenesis in Rat Liver Yates et al, Cancer Res, 2006 AFB + vehicle no AFB 100 X CDD-Im Roebuck et al ltipraz Dose of Hepatoprotectant per dose (µmol/kg body weight)

41 Paper for Today s Webinar Natalie M. Johnson, Patricia A. Egner, Victoria K. Baxter, Michael B. Sporn, Ryan S. Wible, Thomas R. Sutter, John D. Groopman, Thomas W. Kensler, and Bill D. Roebuck Complete Protection against Aflatoxin B 1 -Induced Liver Cancer with a Triterpenoid: DNA Adduct Dosimetry, Molecular Signature, and Genotoxicity Threshold Cancer Prevention Research 7: , 2014

42 Protocol for Induction of HCC with AFB 1 in Rats A Urine AFB 1 CDD-Im U U U U U U * * * * * * * * * * * * * * * Bi-weekly 12 Weeks of Age Lifetime N=43 B Sacrifice (N=6/timepoint) * CDD-Im (30µmol/kg) AFB 1 (200µg/kg)

43 Complete Protection Against Aflatoxin-Hepatocarcinogenesis In a Lifetime Bioassay by the Nrf2 Inducer CDD-Im in Rats Percent Free of Hepatocellular Carcinoma AFB 1 22/ Weeks of Age AFB 1 + CDD-Im 0/20 Johnson et al. CAPR (2014)

44 g 6.3x Solid

45 f 6.3x cystic

46 Comparison of foci: AFB 1 vs AFB 1 +CDD-Im Treatment Age at death (weeks) Foci number bserved Mean Diameter, mm (range) AFB ( ) AFB 1 + CDD-Im ( ) ( )

47 Protocol for Induction of HCC with AFB1 in Rats Urine AFB 1 CDD-im U U U U U U * * * * * * * * * * * * * * * Bi-weekly 12 B Weeks of Age Lifetime N=43 Sacrifice (N=6/timepoint)

48 Table 1. Hepatic burden of GST-P Foci Treatment Days of Number Focal volume % AFB 1 of rats Mean (range) None 0 a 3 0 AFB (0-0.04) ( ) ( ) ( ) AFB1 + CDD-Im a Evaluated at day ( ) (0-0.02)

49 There are lots of aflatoxin-dna adducts in rats that never develop liver cancer! THERE MUST BE A GENTXICITY THRESHLD Aflatoxin-N7-guanine Excretion (pmol/mg creatinine) [ ] AFB 1 AFB 1 + CDD-Im * * * * * Days of AFB 1 Treatment Average Dose of AFB1 (µg/day) [ ] URINE

50 There are lots of aflatoxin-dna adducts in rats that never develop liver cancer! THERE MUST BE A GENTXICITY THRESHLD Hepatic Aflatoxin-N7-guanine (pmol/mg DNA) [ ] not done N7 FAPyr * * * * * * * * Hepatic Aflatoxin-FAPyr (pmol/mg DNA) [ ] Days of AFB 1 Treatment LIVER

51 CNCLUSINS DNA adduct formation presumed NECESSARY for carcinogenesis by genotoxic carcinogens (e.g., aflatoxin) Although DNA adducts may lead to mutations, an adduct is not equivalent to a mutation (and not all mutagens are carcinogens) Substantial aflatoxin-dna damage is NT SUFFICIENT for development of liver cancer Carcinogenesis is a multi-step, chronic process and adduct burden likely only accounts for a fraction of disease risk prediction Prevention of tumor development can occur with achievable reduction in DNA adduct levels. While a carcinogen-dna adduct (genotoxic) threshold is demonstrable, and biologically plausible, it may not be actionable.