Research Article Potent Protection against Aflatoxin Induced Tumorigenesis through Induction of Nrf2 Regulated Pathways by the Triterpenoid 1 2 Cyano 3 12 Dioxooleana 1 9 11 Dien 28 Oyl Imidazole 1 1 1 1 Melinda S Yates Mi Kyoung Kwak Patricia A Egner John D Groopman Sridevi Bodreddigari 2 3 3 3 3 Thomas R Sutter Karen J Baumgartner B D Roebuck Karen T Liby Mark M Yore 4 4 3 1 Tadashi Honda Gordon W Gribble Michael B Sporn and Thomas W Kensler 2 1 Johns Hopkins University Baltimore Maryland 2University of Memphis Memphis Tennessee and 3Dartmouth Medical School and Dartmouth College Hanover New Hampshire 4 Abstract Synthetic triterpenoid analogues of oleanolic acid are potent inducers of the phase 2 response as well as inhibitors of inflammation We show that the triterpenoid 1 2 cyano3 12 dioxooleana 1 9 11 dien 28 oyl imidazole CDDO Im is a highly potent chemopreventive agent that inhibits aflatoxin induced tumorigenesis in rat liver The chemopreventive potency of CDDO Im was evaluated by measuring inhibition of formation of putative preneoplastic lesions glutathione S transferase P positive foci in the liver of rats exposed to aflatoxin B1 CDDO Im produces an 85 reduction in the hepatic focal burden of preneoplastic lesions at 1 Mmol kg body weight and a 99 reduction at 100 Mmol kg body weight CDDO Im treatment reduces levels of aflatoxin DNA adducts by f40 to 90 over the range of 1 to 100 Mmol kg body weight Additionally changes in mRNA levels of genes involved in aflatoxin metabolism were measured in rat liver following a single dose of CDDO Im GSTA2 GSTA5 AFAR and EPHX1 transcripts are elevated 6 hours following a 1 Mmol kg body weight dose of CDDO Im Microarray analysis using wildtype and Nrf2 knockout mice confirms that many phase 2 and antioxidant genes are induced in an Nrf2 dependent manner in mouse liver following treatment with CDDO Im Thus lowmicromole doses of CDDO Im induce cytoprotective genes inhibit DNA adduct formation and dramatically block hepatic tumorigenesis As a point of reference oltipraz an established modulator of aflatoxin metabolism in humans is 100 fold weaker than CDDO Im in this rat antitumorigenesis model The unparalleled potency of CDDO Im in vivo highlights the chemopreventive promise of targeting Nrf2 pathways with triterpenoids Cancer Res 2006 66 4 2488 94 Introduction Induction of phase 2 enzymes is an effective mechanism of protection against carcinogenesis mutagenesis and other forms of toxicity mediated by carcinogens Phase 2 enzymes such as glutathione S transferases GST and UDP glucuronosyl transferases are involved in the metabolism of carcinogens and Note Results from this study were presented at the AACR International Conference on Frontiers in Cancer Prevention Research in Baltimore Maryland 2005 Requests for reprints Thomas W Kensler Department of Environmental Health Sciences Johns Hopkins University Bloomberg School of Public Health Room E7541 615 North Wolfe Street Baltimore MD 21205 Phone 410 955 4712 Fax 410 955 0116 E mail tkensler jhsph edu I2006 American Association for Cancer Research doi 10 1158 0008 5472 CAN 05 3823 Cancer Res 2006 66 4 February 15 2006 function to facilitate their elimination The protective effects of phase 2 enzyme induction have been shown in a wide variety of models For example oltipraz and related dithiolethiones are potent phase 2 inducers that protect against acute toxicity caused by acetaminophen allyl alcohol and aflatoxin in mice hamsters and rats respectively Dithiolethiones are also chemoprotective in animal models of chemically induced carcinogenesis targeting liver lung colon small intestine forestomach bladder mammary glands and skin 1 Dithiolethiones are especially effective at inhibiting aflatoxin induced hepatocarcinogenesis in rats 2 Furthermore the feasibility and potential importance of phase 2 enzyme induction has been confirmed in humans Several studies have shown that oltipraz modulates phase 2 enzymes in humans 3 GST activity is doubled in peripheral lymphocytes after dosing with 125 mg oltipraz 4 Increased GST activity in peripheral mononuclear cells and colon mucosa biopsies following treatment with oltipraz has also been reported 5 Clinical trials of oltipraz have been conducted in residents of Qidong People s Republic of China who are at increased risk for development of hepatocellular carcinoma in part due to consumption of aflatoxin contaminated foods such as corn and peanuts Oltipraz increased phase 2 conjugation of the ultimate carcinogenic species aflatoxin 8 9oxide yielding higher rates of excretion of aflatoxin mercapturic acid in urine 6 Increased formation of the aflatoxin mercapturic acid results from GST conjugation of the epoxide and is inversely associated with levels of aflatoxin DNA adducts formed in liver and excreted into urine The mechanisms that result in protection by dithiolethiones and other classes of phase 2 inducers are under investigation The Keap1 Nrf2 signaling pathway seems to play a central role in the constitutive and inducible expression of many phase 2 genes including GSTs 7 Inducers may interact with critical cysteines in Keap1 through oxidoreduction or alkylation allowing the transcription factor Nrf2 to escape proteosomal degradation and to accumulate in the nucleus In turn Nrf2 binds as heterodimers with small Maf proteins to the antioxidant response elements ARE found in the promoter regions of many phase 2 genes Comparative genomic studies in wild type and Nrf2 disrupted mice have revealed that Nrf2 regulates the inducible expression of multiple categories of genes including antioxidative antiinflammatory genes molecular chaperones stress response genes proteasome subunit genes as well as carcinogen metabolizing enzymes 8 The multiple components of such a broad based adaptive response allow for protection against electrophile and oxidant stresses both of which are components of carcinogenesis Nrf2 deficient mice are greatly predisposed to chemically induced DNA damage and exhibit higher susceptibility toward cancer 2488 www aacrjournals org Protection against Tumorigenesis by CDDO Im development in several models of chemical carcinogenesis 9 Moreover Nrf2 disrupted mice are refractory to the protective effects of inducers such as oltipraz highlighting the importance of the Keap1 Nrf2 ARE signaling pathway as a molecular target for prevention Oleanolic acid is a naturally occurring triterpenoid Several of its synthetic analogues have