Cytotoxic and xenoestrogenic effects via biotransformation of trans-anethole on isolated rat hepatocytes and cultured MCF-7 human breast cancer cells

Abstract

The metabolism and action of trans-anethole (anethole) and the estrogen-like activity of the compound and its metabolites were studied in freshly isolated rat hepatocytes and cultured MCF-7 human breast cancer cells, respectively. The incubation of hepatocytes with anethole (0.25–2.0 mM) caused a concentration- and time-dependent cell death accompanied by losses of cellular ATP and adenine nucleotide pools. Anethole at a weakly toxic level (0.5 mM) was metabolized to 4-methoxycinnamic acid (4MCA), 4-hydroxy-1-propenylbenzene (4OHPB), and the monosulfate conjugate of 4OHPB; the levels of 4OHPB sulfate and 4MCA reached approximately 20 and 200 μM within 2 hr, respectively, whereas that of free unconjugated 4OHPB was less than approximately 0.5 μM. At a moderately toxic concentration (1.0 mM), unconjugated 4OHPB reached approximately 10 μM, followed by abrupt loss of 3′-phosphoadenosine 5′-phosphosulphate (PAPS). Based on cell viability and adenine nucleotide levels, 4OHPB was more toxic than anethole and 4MCA. The addition of 2,6-dichloro-4-nitrophenol (50 μM), an inhibitor of sulfotransferase, enhanced the anethole-induced cytotoxicity associated with losses of ATP, PAPS, and 4OHPB sulfate, and symmetrically increased the unconjugated 4OHPB concentration. 4OHPB as well as diethylstilbestrol (DES) and bisphenol A (BPA), which are known xenoestrogenic compounds, competitively displaced 17β-estradiol bound to the estrogen receptor α in a concentration-dependent manner; ic 50 values of these compounds were approximately 1×10 −5, 1×10 −8 and 5×10 −5 M, respectively. 4OHPB also caused a concentration (10 −8 to 10 −6 M)-dependent proliferation of MCF-7 cells, whereas neither anethole nor 4MCA (10 −9 to 10 −5 M) affected cell proliferation. However, at higher concentrations (>10 −4 M), 4OHPB rather than anethole and 4MCA was cytotoxic. These results suggest that the biotransformation of anethole induces a cytotoxic effect at higher concentrations in rat hepatocytes and an estrogenic effect at lower concentrations in MCF-7 cells based on the concentrations of the hydroxylated intermediate, 4OHPB.