Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold

Abstract

In experimental animals and humans, aflatoxin B1 (AFB1) is a potent hepatic toxin and carcinogen. The synthetic oleanane triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), a powerful activator of Keap1-Nrf2 signaling, protects against AFB1-induced toxicity and preneoplastic lesion formation (GST-P-positive foci). This study assessed and mechanistically characterized the chemoprotective efficacy of CDDO-Im against AFB1-induced hepatocellular carcinoma (HCC). A lifetime cancer bioassay was undertaken in F344 rats dosed with AFB1 (200 μg/kg rat/day) for four weeks and receiving either vehicle or CDDO-Im (three times weekly), one week before and throughout the exposure period. Weekly, 24-hour urine samples were collected for analysis of AFB1 metabolites. In a subset of rats, livers were analyzed for GST-P foci. The comparative response of a toxicogenomic RNA expression signature for AFB1 was examined. CDDO-Im completely protected (0/20) against AFB1-induced liver cancer compared with a 96% incidence (22/23) observed in the AFB1 group. With CDDO-Im treatment, integrated level of urinary AFB1-N(7)-guanine was significantly reduced (66%) and aflatoxin-N-acetylcysteine, a detoxication product, was consistently elevated (300%) after the first AFB1 dose. In AFB1-treated rats, the hepatic burden of GST-P-positive foci increased substantially (0%-13.8%) over the four weeks, but was largely absent with CDDO-Im intervention. The toxicogenomic RNA expression signature characteristic of AFB1 was absent in the AFB1 + CDDO-Im-treated rats. The remarkable efficacy of CDDO-Im as an anticarcinogen is established even in the face of a significant aflatoxin adduct burden. Consequently, the absence of cancer requires a concept of a threshold for DNA damage for cancer development.

Fig. 1

Schematic of dosing protocol. Time of treatment with (*) 30 μmol CDDO-Im, p.o., (▼) 200 μg AFB₁/kg body weight, p.o., and (U) collection of 24 hr urine sample.

Fig. 2

Growth rates of rats treated with (●) AFB₁ or (○) AFB₁ + CDDO-Im. Insert, mean body weight around the period of dosing (bar). An asterisk indicates significant difference by treatment group, p < 0.05. Standard errors were smaller than symbols unless indicated.

Fig. 3

Kaplan Meier curves for proportion of rats free of hepatocellular carcinoma after receiving (●) AFB₁ or (○) AFB₁ + CDDO-Im.

Fig. 4

Urinary and hepatic levels of aflatoxin-DNA adducts and mercapturic acid. (A) Urinary excretion of aflatoxin-N7-guanine during dosing phase in rats maintained on lifetime bioassay (bars). Circles indicate the mean dose of AFB₁ administered as calculated at weekly intervals during the dosing period. (■, ●) AFB₁ or (□, ○) AFB₁ + CDDO-Im. (B) Urinary excretion of aflatoxin-N-acetylcysteine (mercapturic acid) during dosing phase in rats maintained on lifetime bioassay. (■) AFB₁ or (□) AFB₁ + CDDO-Im. Values are mean ± SE (n=10).

Fig. 5

Hepatic levels of aflatoxin-N⁷-guanine (N7) receiving ()AFB₁ or () AFB₁ + CDDO-Im and 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxyaflatoxin B₁ (FAPyr) receiving () AFB₁ or () AFB₁ + CDDO-Im in DNA isolated 24 hr after the most recent dose of AFB₁ over a 1 to 4 week dosing period. Values are mean ± SE (n=3-7).

Fig. 6

Effect of aflatoxin alone and with CDDO-Im intervention on levels of transcript signatures of aflatoxin-induced hepatocarcinogenesis at day 28 of dosing protocol. RFC, relative fold change. Values are mean ± SE (n=3). * Differs from concurrent untreated rats (no AFB₁), p < 0.05). ≠ Differs from AFB₁, p < 0.05. Lower right: RFC in AFB₁ group plotted versus AFB₁ + CDDO-Im group. Proximity of the gene name to 1 on the Y-axis indicates near return of gene expression levels to control (no AFB₁ treatment) levels.