Phase II metabolism of the soy isoflavones genistein and daidzein in humans, rats and mice: a cross-species and sex comparison

Abstract

Soy isoflavones (IF) are in the focus of biomedical research since more than two decades. To assess their bioactivity, IF are investigated in rats and mice as a model. As the biological activity of IF is affected by their biotransformation, our aim was to comprehensively compare the conjugative and microbial metabolism of daidzein and genistein in adult humans, rats and mice of both sexes. One identical soy extract and a validated LC–MS method were used for all studies. We detected considerable differences between the three species. In rats and mice, sex-specific differences were observed in addition. The major plasma phase II metabolites in humans were the 7-sulfo-4′-glucuronides (39–49 %) and, in case of genistein, also the diglucuronide (34 %), whereas in mice monosulfates (33–41 %) and monoglucuronides (30–40 %) predominated. In male rats the disulfates (23–62 %) and 7-sulfo-4′-glucuronides (19–54 %) were predominant, while in female rats the 7-glucuronides (81–93 %) exhibited highest concentrations. The portion of aglycones was low in humans (0.5–1.3 %) and rats (0.5–3.1 %) but comparatively high in mice (3.1–26.0 %), especially in the case of daidzein. Furthermore, substantial differences were observed between daidzein and genistein metabolism. In contrast to humans, all rats and mice were equol producer, independent of their sex. In conclusion, there are marked differences between humans, rats and mice in the profile of major metabolites following IF phase II metabolism. These differences may contribute to resolve inconsistencies in results concerning the bioactivity of IF and should be considered when applying findings of animal studies to humans, e.g., for risk assessment.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2

Abbreviations

DAI:

Daidzein

DG:

Diglucuronide

DS:

Disulfate

GEN:

Genistein

G:

Glucuronide

IF:

Isoflavones

LOD:

Limit of detection

LOQ:

Limit of quantification

S:

Sulfate

SD:

Standard deviation

References

  1. Alekel DL, Van Loan MD, Koehler KJ, Hanson LN, Stewart JW, Hanson KB, Kurzer MS, Peterson CT (2010) The Soy Isoflavones for Reducing Bone Loss (SIRBL) Study: a 3-y randomized controlled trial in postmenopausal women. Am J Clin Nutr 91:218–230

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. Allred CD, Allred KF, Ju YH, Goeppinger TS, Doerge DR, Helferich WG (2004) Soy processing influences growth of estrogen-dependent breast cancer tumors. Carcinogenesis 25:1649–1657

    CAS  Article  PubMed  Google Scholar 

  3. Allred CD, Twaddle NC, Allred KF, Goeppinger TS, Churchwell MI, Ju YH, Helferich WG, Doerge DR (2005) Soy processing affects metabolism and disposition of dietary isoflavones in ovariectomized Balb/c mice. J Agric Food Chem 53:8542–8550

    CAS  Article  PubMed  Google Scholar 

  4. Andrade JE, Ju YH, Baker C, Doerge DR, Helferich WG (2015) Long-term exposure to dietary sources of genistein induces estrogen-independence in the human breast cancer (MCF-7) xenograft model. Mol Nutr Food Res 59:413–423

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Bayer T, Colnot T, Dekant W (2001) Disposition and biotransformation of the estrogenic isoflavone daidzein in rats. Toxicol Sci 62:205–211

    CAS  Article  PubMed  Google Scholar 

  6. Blei T, Soukup ST, Schmalbach K, Pudenz M, Möller FJ, Egert B, Wörtz N, Kurrat A, Müller D, Vollmer G, Gerhäuser C, Lehmann L, Kulling SE, Diel P (2015) Dose-dependent effects of isoflavone exposure during early lifetime on the rat mammary gland: studies on estrogen sensitivity, isoflavone metabolism, and DNA methylation. Mol Nutr Food Res 59:270–283

    CAS  Article  PubMed  Google Scholar 

  7. Bolca S, Urpi-Sarda M, Blondeel P, Roche N, Vanhaecke L, Possemiers S, Al-Maharik N, Botting N, De Keukeleire D, Bracke M, Heyerick A, Manach C, Depypere H (2010) Disposition of soy isoflavones in normal human breast tissue. Am J Clin Nutr 91:976–984

    CAS  Article  PubMed  Google Scholar 

  8. Casanova M, You L, Gaido KW, Archibeque-Engle S, Janszen DB, Heck HA (1999) Developmental effects of dietary phytoestrogens in Sprague–Dawley rats and interactions of genistein and daidzein with rat estrogen receptors alpha and beta in vitro. Toxicol Sci 51:236–244

    CAS  Article  PubMed  Google Scholar 

  9. Chen M, Rao Y, Zheng Y, Wei S, Li Y, Guo T, Yin P (2014) Association between soy isoflavone intake and breast cancer risk for pre- and post-menopausal women: a meta-analysis of epidemiological studies. PLoS ONE 9:e89288

    Article  PubMed  PubMed Central  Google Scholar 

  10. Coward L, Smith M, Kirk M, Barnes S (1998) Chemical modification of isoflavones in soyfoods during cooking and processing. Am J Clin Nutr 68:1486S–1491S

    CAS  PubMed  Google Scholar 

  11. Doerge DR, Chang HC, Churchwell MI, Holder CL (2000) Analysis of soy isoflavone conjugation in vitro and in human blood using liquid chromatography-mass spectrometry. Drug Metab Dispos 28:298–307

    CAS  PubMed  Google Scholar 

  12. Dwivedi C, Downie AA, Webb TE (1987) Net glucuronidation in different rat strains: importance of microsomal beta-glucuronidase. FASEB J 1:303–307

    CAS  PubMed  Google Scholar 

  13. Grosser G, Döring B, Ugele B, Geyer J, Kulling SE, Soukup ST (2014) Transport of the soy isoflavone daidzein and its conjugative metabolites by the carriers SOAT, NTCP, OAT4, and OATP2B1. Arch Toxicol. doi:10.1007/s00204-014-1379-3

    PubMed  Google Scholar 

  14. Gu L, House SE, Prior RL, Fang N, Ronis MJJ, Clarkson TB, Wilson ME, Badger TM (2006) Metabolic phenotype of isoflavones differ among female rats, pigs, monkeys, and women. J Nutr 136:1215–1221

    CAS  PubMed  Google Scholar 

  15. Hakkak R, Korourian S, Shelnutt SR, Lensing S, Ronis MJJ, Badger TM (2000) Diets containing whey proteins or soy protein isolate protect against 7,12-dimethylbenz(a)anthracene-induced mammary tumors in female rats. Cancer Epidemiol Biomark Prev 9:113–117

    CAS  Google Scholar 

  16. Holder CL, Churchwell MI, Doerge DR (1999) Quantification of soy isoflavones, genistein and daidzein, and conjugates in rat blood using LC/ES-MS. J Agric Food Chem 47:3764–3770

    CAS  Article  PubMed  Google Scholar 

  17. Hosoda K, Furuta T, Ishii K (2011) Metabolism and disposition of isoflavone conjugated metabolites in humans after ingestion of kinako. Drug Metab Dispos 39:1762–1767

    CAS  Article  PubMed  Google Scholar 

  18. Ishii J, Hosoda K, Furuta T (2013) In: Preedy VR (ed) Isoflavones: chemistry analysis function and effects. Royal Society of Chemistry, London

    Google Scholar 

  19. Islam MA, Bekele R, vanden Berg JHJ, Kuswanti Y, Thapa O, Soltani S, van Leeuwen FXR, Rietjens IMCM, Murk AJ (2015) Deconjugation of soy isoflavone glucuronides needed for estrogenic activity. Toxicol In Vitro. doi:10.1016/j.tiv.2015.01.013

    PubMed  Google Scholar 

  20. Jin Z, MacDonald RS (2002) Soy isoflavones increase latency of spontaneous mammary tumors in mice. J Nutr 132:3186–3190

    CAS  PubMed  Google Scholar 

  21. Ju YH, Fultz J, Allred KF, Doerge DR, Helferich WG (2006) Effects of dietary daidzein and its metabolite, equol, at physiological concentrations on the growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in ovariectomized athymic mice. Carcinogenesis 27:856–863

    CAS  Article  PubMed  Google Scholar 

  22. Khaodhiar L, Ricciotti HA, Li L, Pan W, Schickel M, Zhou J, Blackburn GL (2008) Daidzein-rich isoflavone aglycones are potentially effective in reducing hot flashes in menopausal women. Menopause 15:125–132

    PubMed  PubMed Central  Google Scholar 

  23. Kinjo J, Tsuchihashi R, Morito K, Hirose T, Aomori T, Nagao T, Okabe H, Nohara T, Masamune Y (2004) Interactions of phytoestrogens with estrogen receptors a and b (III). Estrogenic activities of soy isoflavone aglycones and their metabolites isolated from human urine. Biol Pharm Bull 27:185–188

    CAS  Article  PubMed  Google Scholar 

  24. Kurrat A, Blei T, Kluxen FM, Mueller DR, Piechotta M, Soukup ST, Kulling SE, Diel P (2015) Lifelong exposure to dietary isoflavones reduces risk of obesity in ovariectomized Wistar rats. Mol Nutr Food Res. doi:10.1002/mnfr.201500240

    Google Scholar 

  25. Lamartiniere CA, Cotroneo MS, Fritz WA, Wang J, Mentor-Marcel R, Elgavish A (2002) Genistein chemoprevention: timing and mechanisms of action in murine mammary and prostate. J Nutr 132:552S–558S

    PubMed  Google Scholar 

  26. Liu L, Klaassen CD (1996a) Ontogeny and hormonal basis of female-dominant rat hepatic sulfotransferases. J Pharmacol Exp Ther 279:386–391

    CAS  PubMed  Google Scholar 

  27. Liu L, Klaassen CD (1996b) Ontogeny and hormonal basis of male-dominant rat hepatic sulfotransferases. Mol Pharmacol 50:565–572

    CAS  PubMed  Google Scholar 

  28. Manach C, Donovan JL (2004) Pharmacokinetics and metabolism of dietary flavonoids in humans. Free Radic Res 38:771–785

    CAS  Article  PubMed  Google Scholar 

  29. Mentor-Marcel R, Lamartiniere CA, Eltoum I-E, Greenberg NM, Elgavish A (2001) Genistein in the diet reduces the incidence of poorly differentiated prostatic adenocarcinoma in transgenic mice (TRAMP). Cancer Res 61:6777–6782

    CAS  PubMed  Google Scholar 

  30. Molzberger A, Soukup S, Kulling S, Diel P (2013) Proliferative and estrogenic sensitivity of the mammary gland are modulated by isoflavones during distinct periods of adolescence. Arch Toxicol 87:1129–1140

    CAS  Article  PubMed  Google Scholar 

  31. Mortensen A, Kulling SE, Schwartz H, Rowland I, Ruefer CE, Rimbach G, Cassidy A, Magee P, Millar J, Hall WL, Birkved FK, Sorensen IK, Sontag G (2009) Analytical and compositional aspects of isoflavones in food and their biological effects. Mol Nutr Food Res 53:S266–S309

    Article  PubMed  Google Scholar 

  32. Owens W, Ashby J, Odum J, Onyon L (2003) The OECD program to validate the rat uterotrophic bioassay. Phase 2: dietary phytoestrogen analyses. Environ Health Perspect 111:1559–1567

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Penza M, Montani C, Romani A, Vignolini P, Ciana P, Maggi A, Pampaloni B, Caimi L, Di Lorenzo D (2007) Genistein accumulates in body depots and is mobilized during fasting, reaching estrogenic levels in serum that counter the hormonal actions of estradiol and organochlorines. Toxicol Sci 97:299–307

    CAS  Article  PubMed  Google Scholar 

  34. Perez-Vizcaino F, Duarte J, Santos-Buelga C (2012) The flavonoid paradox: conjugation and deconjugation as key steps for the biological activity of flavonoids. J Sci Food Agric 92:1822–1825

    CAS  Article  PubMed  Google Scholar 

  35. Pritchett JJ, Kuester RK, Sipes IG (2002) Metabolism of bisphenol A in primary cultured hepatocytes from mice, rats, and humans. Drug Metab Dispos 30:1180–1185

    CAS  Article  PubMed  Google Scholar 

  36. Pugazhendhi D, Watson KA, Mills S, Botting N, Pope GS, Darbre PD (2008) Effect of sulphation on the oestrogen agonist activity of the phytoestrogens genistein and daidzein in MCF-7 human breast cancer cells. J Endocrinol 197:503–515

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. Ronis MJ, Little JM, Barone GW, Chen G, Radominska-Pandya A, Badger TM (2006) Sulfation of the isoflavones genistein and daidzein in human and rat liver and gastrointestinal tract. J Med Food 9:348–355

    CAS  Article  PubMed  Google Scholar 

  38. Schwartz H, Sontag G, Plumb J (2009) Inventory of phytoestrogen databases. Food Chem 113:736–747

    CAS  Article  Google Scholar 

  39. Setchell KD, Brown NM, Zhao X, Lindley SL, Heubi JE, King EC, Messina MJ (2011) Soy isoflavone phase II metabolism differs between rodents and humans: implications for the effect on breast cancer risk. Am J Clin Nutr 94:1284–1294

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Shelnutt SR, Cimino CO, Wiggins PA, Ronis MJ, Badger TM (2002) Pharmacokinetics of the glucuronide and sulfate conjugates of genistein and daidzein in men and women after consumption of a soy beverage. Am J Clin Nutr 76:588–594

    CAS  PubMed  Google Scholar 

  41. Soucy NV, Parkinson HD, Sochaski MA, Borghoff SJ (2006) Kinetics of genistein and its conjugated metabolites in pregnant Sprague-Dawley rats following single and repeated genistein administration. Toxicol Sci 90:230–240

    CAS  Article  PubMed  Google Scholar 

  42. Soukup ST, Al-Maharik N, Botting N, Kulling SE (2014) Quantification of soy isoflavones and their conjugative metabolites in plasma and urine: an automated and validated UHPLC-MS/MS method for use in large-scale studies. Anal Bioanal Chem 406:6007–6020

    CAS  Article  PubMed  Google Scholar 

  43. Walaszek Z, Hanausek-Walaszek M, Webb TE (1986) Dietary glucarate-mediated reduction of sensitivity of murine strains to chemical carcinogenesis. Cancer Lett 33:25–32

    CAS  Article  PubMed  Google Scholar 

  44. Wangen KE, Duncan AM, Xu X, Kurzer MS (2001) Soy isoflavones improve plasma lipids in normocholesterolemic and mildly hypercholesterolemic postmenopausal women. Am J Clin Nutr 73:225–231

    CAS  PubMed  Google Scholar 

  45. Wenzel E, Somoza V (2005) Metabolism and bioavailability of trans-resveratrol. Mol Nutr Food Res 49:472–481

    CAS  Article  PubMed  Google Scholar 

  46. Wu AH, Yu MC, Tseng CC, Pike MC (2008) Epidemiology of soy exposures and breast cancer risk. Br J Cancer 98:9–14

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. Yang Z, Zhu W, Gao S, Xu H, Wu B, Kulkarni K, Singh R, Tang L, Hu M (2010) Simultaneous determination of genistein and its four phase II metabolites in blood by a sensitive and robust UPLC–MS/MS method: application to an oral bioavailability study of genistein in mice. J Pharm Biomed Anal 53:81–89

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. Yang Z, Kulkarni K, Zhu W, Hu M (2012) Bioavailability and Pharmacokinetics of Genistein: mechanistic Studies on its ADME. Anti-Cancer Agents Med Chem 12:1264–1280

    CAS  Article  Google Scholar 

  49. Yeh S-L, Lin Y-C, Lin Y-L, Li C-C, Chuang C-H (2015) Comparing the metabolism of quercetin in rats, mice and gerbils. Eur J Nutr. doi:10.1007/s00394-015-0862-9

    Google Scholar 

  50. Yu C, Shin YG, Chow A, Li Y, Kosmeder JW, Lee YS, Hirschelman WH, Pezzuto JM, Mehta RG, van Breemen RB (2002) Human, rat, and mouse metabolism of resveratrol. Pharm Res 19:1907–1914

    CAS  Article  PubMed  Google Scholar 

  51. Zhang Y, Song TT, Cunnick JE, Murphy PA, Hendrich S (1999) Daidzein and genistein glucuronides in vitro are weakly estrogenic and activate human natural killer cells at nutritionally relevant concentrations. J Nutr 129:399–405

    CAS  PubMed  Google Scholar 

  52. Zhang Y, Hendrich S, Murphy PA (2003) Glucuronides are the main isoflavone metabolites in women. J Nutr 133:399–404

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was funded by the German Research Foundation (DFG), grants KU 1079/9-1, DI 716/12-2 and VO 410/12-1. The project is part of the collaborative research project entitled IsoCross “Isoflavones: Cross-species comparison on metabolism, estrogen sensitivity, epigenetics, and carcinogenesis”.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Sabine E. Kulling.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the State Medical Chamber Baden-Württemberg, Stuttgart, Germany, and the ethics commission of the German Sport University Cologne, Cologne, Germany as well as with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. All guidelines of the Institutional Animal Care and Use Committee regulated by the German federal law for animal welfare were followed.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 351 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Soukup, S.T., Helppi, J., Müller, D.R. et al. Phase II metabolism of the soy isoflavones genistein and daidzein in humans, rats and mice: a cross-species and sex comparison. Arch Toxicol 90, 1335–1347 (2016). https://doi.org/10.1007/s00204-016-1663-5

Download citation

Keywords

  • Isoflavones
  • Glucuronides
  • Sulfates
  • Human
  • Mouse
  • Rat