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
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Complete Protection against Aflatoxin B1-induced Liver Cancer
with a Triterpenoid: DNA Adduct Dosimetry and Genotoxic Threshold
Bill D. Roebuck1, John D. Groopman2, and Thomas W. Kensler2,3
1Department of Pharmacology and Toxicology, The Geisel School of Medicine at Dartmouth,Hanover, New Hampshire
2Department of Environmental Health Sciences, Bloomberg School of Public Health,Baltimore, Maryland
3Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania
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 B1-Induced Liver Cancer with a Triterpenoid: DNA Adduct Dosimetry,
Molecular Signature, and Genotoxicity Threshold
Cancer Prevention Research 7:658-665, 2014
Baltimore, 1983
Collaborative efforts for nearly 4 decades
Groopman
Kensler
Roebuck
General Background of Aflatoxins as
Acute Hepatotoxin and Hepatocellular Carcinogen
Aflatoxins
• 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 B1 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
O
O
O
O
O
H
H
OCH3
Aflatoxin B1 (AFB1)
• Maize (corn)• Groundnuts
(peanuts)
AFB1 as Hepatocellular CarcinogenSensitive µg/kg/day Rainbow Trout
• Significant decrease in HCC• Shift toward less advanced lesions
WEEKS70 75 80 85 90 95
Prop
ortio
n Fr
ee o
f HCC
0.2
0.4
0.6
0.8
1.0
OltiprazControl
Extent of Protection22% delay in onset: p = 0.00242% reduction in incidence: p = 0.001
Chemoprevention by Reduction and DelayEffect of Oltipraz on Aflatoxin-induced HCC in
Rats
Kensler et al., CEBP . 6:603-610, 1997
Reduction in Hepatic AFB-DNA Adduct LevelsUNDERESTIMATES
Antitumorigenic Efficacy of Oltipraz
Kensler et al, CRT, 1999
Reduction in Hepatic AFB-DNA Adduct LevelsUNDERESTIMATES
Antitumorigenic Efficacy of Oltipraz
Kensler et al, CRT, 1999
50%↓ DNA adducts
90%↓ Foci Burden
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
CDDO-Imidazolide(TP235)
1Lapillonne et al. Cancer Res 20032Place et al. Clin Cancer Res 20033Dinkova-Kostova et al. PNAS 20054Liby et al. Cancer Res 2005
O
NCO
N
N
H
O
* 1, 3, 10, 30, or 100 µmol CDDO-Im/kg body weight by gavage
25 µg aflatoxin B1 per rat by gavage 6 h after CDDO-Im
Protocol for Evaluation of CDDO-Im as an Inhibitor of Aflatoxin-Induced Tumorigenesis in Male F344 Rats
Weeks of experiment0 1 2 3 7 8
* * * * * * * * *
CDDO-Im Inhibits Aflatoxin-DNA Adduct Formation in Rat Liver
CDDO-Im (µmol/kg body weight)
Afla
toxi
n-N
7 -gu
anin
e(p
mol
/mg
DN
A)
0
1
2
3
4
5
6
Vehicle 1 3 10 30 100
∗
∗∗
∗∗ ∗∗
∗∗
** P < 0.001* P < 0.02x ± SE (n=4)
Dose of Hepatoprotectant per dose(µmol/kg body weight)
0 1 10 100 1000
Vol
ume
Per
cent
GST
-P P
osit
ive
Foci
0.001
0.01
0.1
1 AFB + vehicle
no AFB
CDDO-Im
CDDO-Im Inhibits Aflatoxin-Induced Tumorigenesis in Rat Liver
Roebuck et al. 2003
100X
Yates et al, Cancer Res, 2006
Oltipraz
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 B1-Induced Liver Cancer with a Triterpenoid: DNA Adduct Dosimetry,
Molecular Signature, and Genotoxicity Threshold Cancer Prevention Research 7:658-665, 2014
Paper for Today’s Webinar
Weeks of Age
5 6 7 8 9 10 12Bi-weekly
U U U U U U
* * * * * * * * * * * * * * *
Protocol for Induction of HCC with AFB1 in Rats
UrineAFB1CDDO-Im Lifetime
N=43
Sacrifice (N=6/timepoint)
A
B
* CDDO-Im (30µmol/kg)AFB1 (200µg/kg)
Weeks of Age20 40 60 80 100
Perc
ent F
ree
ofH
epat
ocel
lula
r Car
cino
ma
0
20
40
60
80
100
22/23
AFB1 + CDDO-Im 0/20
AFB1
Complete Protection Against Aflatoxin-HepatocarcinogenesisIn a Lifetime Bioassay by the Nrf2 Inducer CDDO-Im in Rats
Johnson et al. CAPR (2014)
122-926g 6.3x Solid
122-926f 6.3x cystic
Comparison of foci: AFB1 vs AFB1+CDDO-Im
Treatment
Age at death
(weeks)Foci
number
Observed Mean Diameter, mm
(range)
AFB1 35 5 1.30 (1.92-0.85)
AFB1 + CDDO-Im 93 9 0.43 (0.93-0.15)
95 2 1.12 (1.82-0.42)
111 1 0.32
12 0.53
Weeks of Age
5 6 7 8 9 10 12Bi-weekly
U U U U U U
* * * * * * * * * * * * * * *
Protocol for Induction of HCC with AFB1 in Rats
UrineAFB1CDDO-im Lifetime
N=43
Sacrifice (N=6/timepoint)
B
Table 1. Hepatic burden of GST-P FociTreatment Days of