Cancer chemopreventive activity and metabolism of isoliquiritigenin, a compound found in licorice

Abstract

Isoliquiritigenin (2',4',4-trihydroxychalcone; ILG), a chalcone found in licorice root and many other plants, has shown potential chemopreventive activity through induction of phase II enzymes such as quinone reductase-1 in murine hepatoma cells. In this study, the in vivo metabolism of ILG was investigated in rats. In addition, ILG glucuronides and ILG-glutathione adducts were observed in human hepatocytes and in livers from rats treated with ILG. ILG glucuronides were detected in both plasma and rat liver tissues. In addition, in a full-term cancer chemoprevention study conducted with 7,12-dimethylbenz(a)anthracene-treated female Sprague-Dawley rats, dietary administration of ILG slightly increased tumor latency but had a negative effect on the incidence of mammary tumors starting at approximately 65 days after 7,12-dimethylbenz(a)anthracene administration. Further, no significant induction of phase II enzymes was found in mammary glands, which is consistent with the low level of ILG observed in these tissues. However, ILG significantly induced quinone reductase-1 activity in the colon, and glutathione as well as glutathione S-transferase in the liver. Analysis of mRNA expression in tissues of rats treated with ILG supported these findings. These results suggest that ILG should be tested for chemopreventive efficacy in nonmammary models of cancer.

Figure 1

ILG induces GSH in H4IIE cells. Cells were treated with 10–160 µM ILG, 10 µM 4'-bromoflavone (4'BF), or DMSO (0.5% final concentration) as control (C) for 24 h and then analyzed for GSH. Results are shown as fold-induction relative to the level observed in the control. Results are the means of three determinations ± SD. * Significantly different from control values (p < 0.001).

Figure 2

A) Negative ion electrospray selected reaction monitoring (SRM) LC-MS-MS chromatograms showing detection of phase I metabolites M1–M7 of ILG after incubation with rat liver microsomes in the presence of NADPH. B) Mass chromatograms of ILG phase II glucuronide conjugates formed by rat liver microsomes in the presence of UDPGA. Metabolites MG3, MG4, and MG5 correspond to 4-glucuronosylisoliquiritigenin, 2′-glucuronosylisoliquiritigenin, and 4′-glucuronosylisoliquiritigenin, respectively, and MG1 and MG2 are monoglucuronides of liquiritigenin (see structures in Figure 3). C) Computer-reconstructed selected ion chromatograms of ILG glucuronides and sulfate conjugates detected in rat plasma using high resolution LC-MS with negative ion electrospray. Although different HPLC systems were used for parts B and C, the same five glucuronide conjugates were detected in rats as were observed with in vitro systems.

Figure 3

A) Phase I metabolites of ILG formed during incubation with rat liver microsomes and NADPH. Based on accurate mass measurements, HPLC retention times, MS/MS analyses, and comparison to data reported by Guo et al. (29), the structures of metabolites M1, M2, M3, M4, M5, M6, and M7 were assigned as liquiritigenin, 7,8,4′-trihydroxychalcone, sulfuretin, 7,3′,4′-trihydroxychalcone, davidigenin, trans-6,4′-dihydroxyaurone, and cis-6,4′-dihydroxyaurone, respectively. B) Structures of ILG glucuronide conjugates formed by rat liver microsomes in the presence of UDPGA.

Figure 4

(A and B) Positive ion electrospray LC-MS-MS chromatograms showing the detection of two abundant GSH adducts, GSH1 and GSH2, in lysates of human hepatocytes that had been incubated with ILG. Precursor ion scanning was used to detect ions that fragmented to form the characteristic GSH product ion of m/z 308. (C) Negative ion electrospray MS-MS with collision-induced dissociation and SRM was used to detect GSH1 in rat liver following administration of ILG. The structures of GSH1 and GSH 2 were determined by comparison with synthetic standards, and the fragmentation patterns are based on high resolution product ion tandem mass spectrometry with accurate mass measurement.

Figure 5

Effect of dietary ILG on percent incidence in rats of observable mammary tumors (A), number of tumors (B), and body weight (C). Female Sprague-Dawley rats were given a single i.g. dose of 7,12-dimethylbenz(a)anthracene (DMBA) on day 0. ILG was included in the rat chow from 7 days prior to DMBA administration (−7) to the end of the study. The rat treatment groups were as follows: ◆ DMBA in sesame oil; ■ DMBA and 7.5 g/kg diet of ILG; ▲ DMBA and 10.0 g/kg diet of ILG; × 10.0 g/kg diet of ILG.

Figure 6

Effect of dietary ILG on QR-1 induction (A), GST induction (B), and GSH levels (C) in rat liver (■), colon (□) and mammary gland (). Induction was calculated by comparing group 2 (DMBA and 7.5 g/kg diet of ILG) and group 3 (DMBA and 10.0 g/kg diet of ILG) with group 1 (DMBA in sesame oil), and group 4 (10.0 g/kg diet of ILG) with group 5 (basal diet). Treatment groups were significantly different (p<0.05) from the DMBA only control group 1 (*) or from the basal diet control group 5 (**) with n = 6.