Genistein induction of human sulfotransferases in HepG2 and Caco-2 cells.

Sulfotransferases are phase II drug-metabolizing enzymes. While the induction of sulfotransferases by hormones and endogenous molecules is relatively well known, induction by xenobiotics is not well studied. Isoflavones are naturally occurring phyto-oestrogens, mainly existing in soy food products. They have been described as health-promoting, disease-preventing dietary supplements and as agents with cancer-preventive activities. Recently, isoflavones have been reported to interact with nuclear receptors, including those that are known to mediate the induction of drug-metabolizing enzymes. In the present investigation, the isoflavone genistein was shown to be a xenobiotic inducer of human sulfotransferases in transformed human liver cells (HepG2) and colon carcinoma cells (Caco-2). Enzymatic activity assay, Western blot, and real-time reverse transcription-polymerase chain reaction (RT-PCR) results demonstrated that genistein significantly induced protein and mRNA expression of human simple phenol sulfotransferase (hSULT1A1) and human dehydroepiandrosterone sulfotransferase (hSULT2A1) in HepG2 and Caco-2 cells. The induction was time-dependent and dose-dependent. Western blot results agreed well with real-time RT-PCR results, suggesting that induction occurred at the gene transcription level. This isoflavone is the first nutritionally related phyto-oestrogen shown to induce human sulfotransferases in HepG2 and Caco-2 cells.

Sulfation of the isoflavones genistein and daidzein in human and rat liver and gastrointestinal tract.

Phytoestrogens, in particular the isoflavone aglycones genistein and daidzein, are thought to be the bioactive components of soy. Like estrogens, isoflavones can be sulfur-conjugated. However, although isoflavones in the serum are found largely in the form of glucuronide and sulfur conjugates following soy consumption, little is known regarding the relative contributions of sulfotransferases in the liver and small intestine to isoflavone sulfation. Since the sulfates may be deconjugated in target tissues, circulating isoflavone sulfates may act as a source of tissue aglycones. In the current study genistein and daidzein sulfotransferase activities were measured in cytosol from human and rat liver and gastrointestinal tract. Isoflavone sulfation in the human gastrointestinal (GI) tract was correlated with activities towards substrates for previously characterized human sulfotransferases. Western blots of human cytosols were also conducted using antisera towards human sulfotransferases SULT1E1 and SULT2A1. Whereas rat liver was almost fourfold more active than small intestine in sulfation of genistein, in the human, activities in the two tissues were comparable. In contrast, intestinal sulfation of daidzein was comparable to hepatic sulfation in the rat and significantly greater in the human. Genistein and daidzein sulfation occurred throughout the human GI tract, but with a different distribution and different interindividual variability. Whereas genistein sulfation in the human GI tract correlated significantly with sulfation of the prototypical human phenolic sulfotransferase SULT1A family substrate 2-naphthol (r2 = 0.71), daidzein sulfotransferase activity did not correlate with activities towards any prototypical sulfotransferase substrate or with genistein sulfation. Our results suggest that metabolism in the human GI tract has an important role in the generation of potentially bioactive isoflavone sulfates and a major role for the human phenolic sulfotransferase SULT1A family in metabolism of genistein in the gut. However, human intestinal daidzein sulfation appears to be catalyzed by a separate enzyme.

Arginine residues in the active site of human phenol sulfotransferase (SULT1A1).

Cytosolic sulfotransferases (STs) catalyze the sulfation of hydroxyl containing compounds. Human phenol sulfotransferase (SULT1A1) is the major human ST that catalyzes the sulfation of simple phenols. Because of its broad substrate specificity and lack of endogenous substrates, the biological function of SULT1A1 is believed to be an important detoxification enzyme. In this report, amino acid modification, computer structure modeling, and site-directed mutagenesis were used for studies of Arg residues in the active site of SULT1A1. The Arg-specific modification reagent, 2,3-butanedione, inactivated SULT1A1 in an efficient, time- and concentration-dependent manner, suggesting Arg residues play an important role in the catalytic activity of SULT1A1. According to the computer model, Arg78, Arg130, and Arg257 may be important for SULT1A1 catalytic activity. Site-directed mutagenesis results demonstrated that the positive charge on Arg78 is not critical for SULT1A1 because R78A is still active. In contrast, a negative charge at this position, R78E, completely inactivated SULT1A1. Arg78 is in close proximity to the site of sulfuryl group transfer. Arg257 is located very close to the 3'-phosphate in adenosine 3'-phosphate 5'-phosphosulfate (PAPS). Site-directed mutagenesis demonstrated that Arg257 is critical for SULT1A1: both R257A and R257E are inactive. Although Arg130 is also located very close to the 3'-phosphate of PAPS, R130A and R130E are still active, suggesting that Arg130 is not a critical residue for the catalytic activity of SULT1A1. Computer modeling suggests that the ionic interaction between the positive charge on Arg257, and the negative charge on 3'-phosphate is the primary force stabilizing the specific binding of PAPS.