Potent Protection against Aflatoxin-Induced Tumorigenesis throughInduction of Nrf2-Regulated Pathways by the Triterpenoid1-[2-Cyano-3-,12-Dioxooleana-1,9(11)-Dien-28-Oyl]ImidazoleMelinda S. Yates,1Mi-Kyoung Kwak,1Patricia A. Egner,1John D. Groopman,1Sridevi Bodreddigari,2Thomas R. Sutter,2Karen J. Baumgartner,3B.D. Roebuck,3Karen T. Liby,3Mark M. Yore,3Tadashi Honda,4Gordon W. Gribble,4Michael B. Sporn,3and Thomas W. Kensler11Johns Hopkins University, Baltimore, Maryland;2University of Memphis, Memphis, Tennessee; and3Dartmouth Medical School and4Dartmouth College, Hanover, New HampshireAbstractSynthetic triterpenoid analogues of oleanolic acid are potentinducers of the phase 2 response as well as inhibitors ofinflammation. We show that the triterpenoid, 1-[2-cyano-3-,1 2-dioxoole ana-1,9(1 1)-dien-28-oyl]imidazole (CDDO-Im),is a highly potent chemopreventive agent that inhibitsaflatoxin-induced tumorigenesis in rat liver. The chemo-preventive potency of CDDO-Im was evaluated by measuringinhibition of fo rmation of putative preneoplastic lesions(glutathione S-transferase P positive foci) in the liver of ratsexposed to aflatoxin B1. CDDO-Im produces an 85% reductionin the hepatic focal burden of preneoplastic lesions at 1 Mmol/kg body weight and a >99% reduction at 100 Mmol/kg bodyweight. CDDO-Im treatment reduces levels of aflatoxin-DNAadducts by f40% to 90% over the range of 1 to 100 Mmol/kgbody weight. Additionally, changes in mRNA levels of genesinvolved in aflatoxin metabolism were measured in rat liverfollowing a single dose of CDDO-Im. GSTA2, GSTA5, AFAR, andEPHX1 transcripts are elevated 6 hours following a 1 Mmol/kgbody weight dose of CDDO-Im. Microarray analysis using wild-type and Nrf2 knockout mice confirms that many phase 2 andantioxidant genes are induced in an Nrf2-dependent mannerin mouse liver following treatment with CDDO-Im. Thus, low-micromole doses of CDDO-Im induce cytoprotective genes,inhibit DNA adduct formation, and dramatically block hepatictumorigenesis. As a point of reference, oltipraz, an establishedmodulator of aflatoxin metabolism in humans, is 100-foldweaker than CDDO-Im in this rat antitumorigenesis model.The unparalleled potency of CDDO-Im in vivo highlights thechemopreventive promise of targeting Nrf2 pathways withtriterpenoids. (Cancer Res 2006; 66(4): 2488-94)IntroductionInduction of phase 2 enzymes is an effective mechanism ofprotection against carcinogenesis, mutagenesis, and other formsof toxicity mediated by carcinogens. Phase 2 enzymes, such asglutathione S-transferases (GST) and UDP-glucuronosyl trans-ferases, are involved in the metabolism of carcinogens andfunction to facilitate their elimination. The protective effects ofphase 2 enzyme induction have been shown in a wide variety ofmodels. For example, oltipraz and related dithiolethiones arepotent phase 2 inducers that protect against acute toxicity causedby acetaminophen, allyl alcohol, and aflatoxin in mice, hamsters,and rats, respectively. Dithiolethiones are also chemoprotectivein animal models of chemically induced carcinogenesis targetingliver, lung, colon, small intestine, forestomach, bladder, mammaryglands, and skin (1). Dithiolethiones are especially ef fective atinhibit ing aflatoxin-induced hepatocarcinogenesis in rats (2).Furthermore, the feasibility and potential importance of phase 2enzyme induction has been confirmed in humans. Several studieshave shown that oltipraz modulates phase 2 enzymes in humans(3). GST activity is doubled in peripheral lymphocytes after dosingwith 125 mg oltipraz (4). Increased GST activity in peripheralmononuclear cells and colon mucosa biopsies following treatmentwith oltipraz has also been reported (5). Clinical trials of oltiprazhave been conducted in residents of Qidong, People’s Republic ofChina, who are at increased risk for development of hepatocellularcarcinoma, in part due to consumption of aflatoxin-contaminatedfoods, such as corn and peanuts. Oltipraz increased phase 2conjugation of the ultimate carcinogenic species, aflatoxin-8,9-oxide, yielding higher rates of excretion of aflatoxin-mercapturicacid in urine (6). Increased formation of the aflatoxin-mercapturicacid results from GST conjugation of the epoxide and is inverselyassociated with levels of aflatoxin DNA adducts formed in liver andexcreted into urine.The mechanisms that result in protection by dithiolethiones andother classes of phase 2 inducers are under investigation. TheKeap1-Nrf2 signaling pathway seems to play a central role inthe constitutive and inducible expression of many phase 2 genes,including GSTs (7). Inducers may interact with critical cysteines inKeap1 through oxidoreduction or alkylation, allowing the tran-scription factor Nrf2 to escape proteosomal degradation and toaccumulate in the nucleus. In turn, Nrf2 binds as heterodimerswith 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-disruptedmice have revealed that Nrf2 regulates the inducible expressionof multiple catego ries of genes, inclu ding an tioxidative/anti-inflammator y genes, molecular chaperones/stress response genes,proteasome subunit genes, as well as carcinogen-metabolizingenzymes (8). The multiple components of such a broad-basedadaptive response allow for protection against electrophile andoxidant stresses, both of which are components of carcinogenesis.Nrf2-deficient mice are greatly predisposed to chemically inducedDNA damage and exhibit higher susceptibility toward cancerNote: Results from this study were presented at the AACR International Conferenceon Frontiers in Cancer Prevention Research in Baltimore, Maryland, 2005.Requests for reprints: Thomas W. Kensler, Department of Environmental HealthSciences, 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: [email protected] American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-3823Cancer Res 2006; 66: (4). February 15, 20062488www.aacrjournals.orgResearch Articledevelopment in several models of chemical carcinogenesis (9).Moreover, Nrf2-disrupted mice are refractory to the protectiveeffects of inducers, such as oltipraz, highlighting the importance ofthe Keap1-Nrf2-ARE signaling pathway as a molecular target forprevention.Oleanolic acid is a naturally occurring triterpenoid. Several of