Breast cancer resistance protein (ABCG2) determines distribution of genistein phase II metabolites: reevaluation of the roles of ABCG2 in the disposition of genistein

Abstract

It was recently proposed that the improved oral bioavailability of genistein aglycone and conjugates in Bcrp1(-/-) mice is mainly due to increased intestinal absorption of aglycone and subsequent elevated exposure to conjugation enzymes. Here we tested this proposed mechanism and found that intestinal absorption of genistein aglycone did not increase in Bcrp1(-/-) mice compared with wild-type mice using an in situ mouse intestinal perfusion model and that inhibition of breast cancer resistance protein (BCRP) in Caco-2 cells also did not significantly increase permeability or intracellular concentration of aglycone. Separately, we showed that 5- to 10-fold increases in exposures of conjugates and somewhat lower fold increases (<2-fold) in exposures of aglycone were apparent after both oral and intraperitoneal administration in Bcrp1(-/-) mice. In contrast, the intestinal and biliary excretion of genistein conjugates significantly decreased in Bcrp1(-/-) mice without corresponding changes in aglycone excretion. Likewise, inhibition of BCRP functions in Caco-2 cells altered polarized excretion of genistein conjugates by increasing their basolateral excretion. We further found that genistein glucuronides could be hydrolyzed back to genistein, whereas sulfates were stable in blood. Because genistein glucuronidation rates were 110% (liver) and 50% (colon) higher and genistein sulfation rates were 40% (liver) and 42% (colon) lower in Bcrp1(-/-) mice, the changes in genistein exposures are not mainly due to changes in enzyme activities. In conclusion, improved bioavailability of genistein and increased plasma area under the curve of its conjugates in Bcrp1(-/-) mice is due to altered distribution of genistein conjugates to the systemic circulation.

Fig. 1.

The blood concentrations of genistein (A), G-7-G (B), G-4′-G (C), G-4′-S (D), and G-7-S (E) after oral administration of genistein at both 2 and 20 mg/kg in wild-type and BCRP knockout mice. The blood concentrations of G-7-G and G-4′-G were below the detection limit in wild-type mice for the 2 mg/kg group and 24-h samples of BCRP(−/−) mice. Data are presented as means ± S.D; n = 5.

Fig. 2.

The blood concentrations of genistein (A), G-7-G (B), G-4′-S (C), and G-7-S (D) after intraperitoneal administration of genistein at 20 mg/kg in wild-type FVB and Bcrp1(−/−) mice (n = 5). The blood concentrations of G-7-G and G-7-S were below the detection limit in FVB mice at 24 h. Data are presented as means ± S.D.

Fig. 3.

The intestinal absorption of genistein aglycone in wild-type FVB and Bcrp1(−/−) mice (n = 4). Data are presented as means ± S.D. The perfusion containing 10 μM genistein was performed at a flow rate of 0.191 ml/min. SI, small intestine.

Fig. 4.

The apical excretion of G-7-G (A), G-4′-G (B), G-4′-S (C), and G-7-S (D) in Caco-2 cells in the presence or absence of KO143. Two concentrations of genistein (2 and 10 μM) and 5 μM KO143 were used as inhibitor. Data are presented as means ± S.D; n = 3. *, p < 0.05.

Fig. 5.

The basolateral excretion of G-7-G (A), G-4′-G (B), G-4′-S (C), and G-7-S (D) and the transport of genistein (E) from the apical to basolateral side in Caco-2 cells in the presence or absence of KO143. Two concentrations of genistein (2 and 10 μM) were used, and 5 μM KO143 were used as inhibitor. Data are presented as means ± S.D; n = 3. *, p < 0.05.

Fig. 6.

Deconjugation of genistein glucuronides and sulfates in fresh blood at 37°C in a shaking water bath for up to 4 h (n = 3). Genistein glucuronide (combination of G-7-G and G-4′-G; 3 μM) and genistein (3.8 μM) were incubated in fresh blood taken from Bcrp1(−/−) rats for genistein glucuronides hydrolysis. Genistein sulfates [combination of G-7-S and G-4′-S (0.8 μM) and aglycone (2.2 μM)] were incubated in the same condition as for genistein sulfate hydrolysis. SI, small intestine.

Fig. 7.

The genistein glucuronidation rate (A) and sulfation rate (B) in liver, small intestine and colon S9 fractions prepared from FVB and BCRP(−/−) mice. Genistein (10 μM) was used. Data are presented as means ± S.D; n = 4. *, p < 0.05.

Fig. 8.

Schematic representation showing the absorption of genistein aglycone and distribution of its conjugates in intestine, bile and blood (or plasma). This is a schematic presentation and as such is an approximate representation of the major enzymes, and efflux transporters involved, and not exhaustive. Green triangle with P, genistein; magenta shape with G, genistein glucuronides; magenta diamond with s, genistein sulfates; arrows, efflux transporters for genistein conjugates transport; large green hollow arrows, proposed role of Bcrp; brown filled arrows, proposed role of other efflux transporters; small green solid arrows: aglycone transport via passive diffusion. Arrow size indicates the importance (or size) of its role in genistein conjugate transport. MRP, represented by large solid blue arrows, represents the MRP efflux transporters localized to the basolateral side. Currently, only one MRP transporter (i.e., MRP3) was shown to be involved in the basolateral efflux of conjugates (Kitamura et al., 2010), but other related MRP efflux transporters may also function. Note: Genistein has the same transport pathway in small intestine and colon. To keep the figure simple, some of the enzyme and transporter icons were omitted in the colon compartment. In the colon compartment, efflux transporters were represented by simple magenta solid arrows without identifying the actual efflux transporters, which are expected to be identical to those in the small intestine (although the expression levels may be different).