Glucuronidation, oxidative metabolism, and bioactivation of enterolactone in rhesus monkeys

https://doi.org/10.1016/j.abb.2004.06.023Get rights and content

Abstract

Enterolactone (ENL) and enterodiol (END) are mammalian lignans derived from the plant lignans matairesionol (MAT), secoisolariciresinol (SECO), and other dietary precursors. ENL was found to undergo extensive glucuronidation with rhesus liver microsomes to form O-glucuronides at both phenolic hydroxy groups. In addition to glucuronidation, ENL was found to be a good substrate for oxidative metabolism. The major products had a m/z of 313 or 295 by LC–MS analysis in negative ion mode and were determined to be products of monohydroxylation of ENL. The m/z 295 products were the result of a dehydration of the m/z 313 in the MS source. Addition of N-acetylcysteine (NAC) to the NADPH incubations resulted in a decrease of at least 2 major monohydroxylated products and the formation of a major and several minor new products with a m/z of 474. The major adduct was isolated, purified for NMR, and confirmed to be the NAC adduct of the ENL catechol. Incubations of ENL with liver microsomes containing UDPGA, NADPH, and NAC resulted in the formation of ENL-glucuronides; no NAC adducts were detected by LC–MS. Incubations of ENL with human and rhesus hepatocytes resulted in several metabolites. The major metabolites in hepatocytes were the glucuronic acid conjugates; minor amounts of the sulfate conjugate(s) and monohydroxylated products were also detected by LC–MS. Glutathione or other thiol adducts were not detected in hepatocytes.

Conclusion. The high efficiency and specificity for the glucuronidation of ENL decrease its potential toxicity via CYP450 bioactivation.

Section snippets

Materials

UDPGA, bis-tris-propane, magnesium chloride (MgCl2), alamethicin, ammonium acetate (NH4OAc), N-acteyl-l-cysteine (NAC), NADPH, and d-saccharic acid 1,4-lactone were obtained from Sigma (St. Louis, MO). Microsomes expressing human UGT isoforms were purchased from BD Biosciences (San Jose, CA). BCA protein assay kit was obtained from Pierce (Rockford, IL). ENL was obtained from Fluka Chemicals (Buchs, Switzerland). Potassium phosphate monobasic (KH2PO4), acetonitrile (ACN), and glacial acetic

Results

The in vitro glucuronidation of ENL (Table 1) was examined in liver microsomes from female rhesus monkey along with microsomes containing 3 expressed rhesus UGTs (UGT1A01, UGT2B9∗2, and UGT2B33). The in vitro intrinsic clearance (Vmax/Km) value, by way of glucuronidation for ENL in liver microsomes was 660 μl/min/mg. To determine the major UGT isoform involved in the glucuronidation of ENL in rhesus, the Km values in recombinant rhesus UGT isoforms were compared to those obtained from rhesus

Discussion

This paper describes the in vitro metabolism of ENL in rhesus monkey (Fig. 8) and human liver preparations, along with the structural identification of a metabolite generated from a reactive intermediate. A comparison of the kinetic parameters (Km) for glucuronidation of ENL with the rhesus UGT1A01, UGT2B9∗2, and UGT2B33 to those in rhesus liver microsomes suggests that UGT2B9∗2 and/or UGT2B33 are possibly involved in the glucuronidation of ENL. Additional data from incubations of ENL with

Acknowledgment

The authors wish to thank Jenny Stevens for her input into the final preparation of the manuscript.

References (41)

  • L.Q. Wang

    J. Chromatogr. B Anal. Technol. Biomed. Life Sci.

    (2002)
  • E. Bowey et al.

    Food Chem. Toxicol.

    (2003)
  • K.E. Bach Knudsen et al.

    J. Nutr.

    (2003)
  • H. Adlercreutz et al.

    J. Steroid. Biochem. Mol. Biol.

    (1992)
  • H. Adlercreutz

    Lancet Oncol.

    (2002)
  • H. Adlercreutz et al.

    Anal. Biochem.

    (1998)
  • H. Adlercreutz et al.

    J. Steroid Biochem. Mol. Biol.

    (1995)
  • J.W. Lampe

    J. Nutr.

    (2003)
  • W. Yue et al.

    J. Steroid Biochem. Mol. Biol.

    (2003)
  • R. Ramanathan et al.

    J. Am. Soc. Mass Spectrom.

    (1998)
  • E. Jacobs et al.

    J. Steroid Biochem. Mol. Biol.

    (1999)
  • B. Dean et al.

    Arch. Biochem. Biophys.

    (2004)
  • B. Dean et al.

    Arch. Biochem. Biophys.

    (2002)
  • C.P.a.t.E.C. Committee on Toxicity of Chemicals in Food,...
  • L.U. Thompson et al.

    Nutr. Cancer

    (1991)
  • S.R. Stitch et al.

    Nature

    (1980)
  • L.H. Xie et al.

    Chem. Pharm. Bull. (Tokyo)

    (2003)
  • W.R. Phipps et al.

    J. Clin. Endocrinol. Metab.

    (1993)
  • M.E. Burow et al.

    J. Clin. Endocrinol. Metab.

    (2001)
  • M. Axelson et al.

    Nature

    (1982)
  • Cited by (30)

    • Pharmacokinetic modeling: Prediction and evaluation of route dependent dosimetry of bisphenol A in monkeys with extrapolation to humans

      2011, Toxicology and Applied Pharmacology
      Citation Excerpt :

      The choice of animal models for comparing BPA pharmacokinetics to humans is important. The adult monkey has been identified as the best animal model for the study of steroid glucuronidation based on: (1) the high degree of concordance between adult humans and monkeys (but not dog, bovine, swine, rat, or mouse) for glucuronidation of steroid hormones; (2) the UGT isoforms expressed in hepatic, extra-hepatic (including GI tract), and steroid target tissues of monkeys and humans; (3) the closer gene homology between monkey and human UGT 1A and 1B isoforms than with rat or rabbit; (4) the many similarities in substrate specificity (Barbier and Bélanger, 2003; Dean et al., 2004; Stevens et al., 1993) in that many of the human UGT 1A and 2B isoforms catalyzing glucuronidation of BPA (i.e., UGT 2B15, 2B7, 1A9, 1A8, 1A1; Doerge et al., 2010) are homologous with those important in steroid hormone metabolism in monkeys (Barbier and Bélanger, 2003); and (5) similarities in oral absorption and bioavailability between monkeys and humans (Chiou and Buehler, 2002). Finally, the general value of the developing non-human primate model for developmental toxicology studies includes the similar reproductive physiology and endocrinology, the similar progression of stages of embryo–fetal development, similar organogenesis, and minor morphological and developmental differences in placentae (Chellman et al., 2009), and a similar pattern of bilirubin glucuronidation and transport that develops immediately after birth in term newborn rhesus monkeys and humans (Gartner et al., 1977).

    • Oat Phenolics: Biochemistry and Biological Functionality

      2011, Oats: Chemistry and Technology: Second Edition
    • Multi-functional sample preparation procedure for measuring phytoestrogens in milk, cereals, and baby-food by liquid-chromatography tandem mass spectrometry with subsequent determination of their estrogenic activity using transcriptomic assay

      2009, Analytica Chimica Acta
      Citation Excerpt :

      Indeed, phytoestrogens are known to occur in vegetal as precursors, i.e. associated with glucoside, acetyl and/or malonyl moieties (Fig. 3), which have very poor or no biological activity. Conversely, they are mainly present as biologically active aglycone forms in animal fluids and food products of animal origin, due to metabolism reactions inducing the hydrolysis of precursors [55–58]. As a consequence, our expected sample preparation procedure dedicated to phytoestrogens in vegetal-based material had to include an efficient hydrolysis step, in order to finally measure the total aglycone forms resulting from the cleavage of all precursors initially present in the sample.

    • Uptake and metabolism of enterolactone and enterodiol by human colon epithelial cells

      2005, Archives of Biochemistry and Biophysics
      Citation Excerpt :

      Although loss of lignan sulfates due to protein binding has been suggested as a risk when analyzing plasma [20], this is unlikely to explain our data, because we have used the whole cell medium sample with only clean-up by centrifugation. Thus, our data agree with the above mentioned in vivo data and also with a recent study where enterolactone glucuronides and smaller amounts of sulfates were produced by rhesus monkey and human hepatocytes [23]. However, they are in contrast with two earlier publications on breast and liver cancer cell lines, where the main enterolactone conjugates were monosulfates (78% for HepG2 liver cells and 91% for MCF-7 breast cells).

    • Small-Volume and High-Sensitivity NMR Probes

      2005, Annual Reports on NMR Spectroscopy
      Citation Excerpt :

      Applications of 3 mm probe technology during 2004 were largely oriented toward small-sample structure characterization. Dean and colleagues168 reported a study of the glucuronidation, oxidative metabolism, and bioactivation of enterolactone in the rhesus monkey that relied on the sensitivity of 3 mm probe capabilities. Vorsa and co-workers169 reported the characterization of an extensive series of flavonols found in cranberry (Vaccinium macrocarpon) powder.

    View all citing articles on Scopus
    View full text