, Volume 167, Issue 1, pp 46–53 | Cite as

The soya isoflavone content of rat diet can increase anxiety and stress hormone release in the male rat

  • David E. HartleyEmail author
  • Jessica E. Edwards
  • Claire E. Spiller
  • Nazmul Alom
  • Sonia Tucci
  • Pallab Seth
  • Mary L. Forsling
  • Sandra E. File
Original Investigation



Most commercial rodent diets are formulated with soya protein and therefore contain soya isoflavones. Isoflavones form one of the main classes of phytoestrogens and have been found to exert both oestrogenic and anti-oestrogenic effects on the central nervous system. The effects have not been limited to reproductive behaviour, but include effects on learning and anxiety and actions on the hypothalamo-pituitary axis. It is therefore possible that the soya content of diet could have significant effects on brain and behaviour and be an important source of between-laboratory variability.


To determine whether behaviour in two animal tests of anxiety, and stress hormone production, would differ between rats that were fed a diet which was free of soya isoflavones and other phytoestrogens (iso-free) and those that were fed a diet which contained 150 μg/g of the isoflavones genistein and daidzein (iso-150). This controlled diet has an isoflavone concentration similar to that in the maintenance diet routinely used in our institution.


Male rats were randomly allocated to the iso-free and iso-150 diets and their body weights and food and water consumption were recorded for 14 days. They were then maintained on the same diets, but housed singly for 4 days, before testing in the social interaction and elevated plus-maze tests of anxiety. Corticosterone concentrations in both dietary groups were determined under basal conditions and after the stress of the two tests of anxiety. Vasopressin and oxytocin concentrations were determined after brief handling stress.


The groups did not differ in food or water intake, body weight or oxytocin concentrations. Compared with the rats fed the iso-free diet, the rats fed the iso-150 diet spent significantly less time in active social interaction and made a significantly lower percentage of entries onto the open arms of the plus-maze, indicating anxiogenic effects in both animal tests. The groups did not differ in their basal corticosterone concentrations, but the iso-150 group had significantly elevated stress-induced corticosterone concentrations. Stress-induced plasma vasopressin concentrations were also significantly elevated in the iso-150 diet group compared with the iso-free rats.


Major changes in behavioural measures of anxiety and in stress hormones can result from the soya isoflavone content of rat diet. These changes are as striking as those seen following drug administration and could form an important source of variation between laboratories.


Phytoestrogen Anxiety Social interaction Elevated plus-maze Corticosterone Vasopressin Oxytocin 



These experiments were supported by a grant from the Dunhill Medical Trust. We are indebted to Dr. Tobin of Harlan Teklad UK Ltd. for the detailed calculations of the isoflavone content of our diet.


  1. Almstrup K, Fernandez MF, Petersen JH, Olea N, Skakkebaek NE, Leffers H (2002) Dual effects of phytoestrogens result in U-shaped dose–response curves. Environ Health Perspect 110:743–748PubMedGoogle Scholar
  2. Andrews N, File SE (1993) Handling history of rats modifies behavioural effects of drugs in the elevated plus-maze test of anxiety. Eur J Pharmacol 235:109–112PubMedGoogle Scholar
  3. Appenrodt E, Schnabel R, Schwarzberg H (1998) Vasopressin administration modulates anxiety-related behavior in rats. Physiol Behav 64:543–547CrossRefPubMedGoogle Scholar
  4. Baldwin HA, File SE (1989) Flumazenil prevents the development of chlordiazepoxide withdrawal in rats tested in the social interaction test of anxiety. Psychopharmacology 97:424–426Google Scholar
  5. Barlow SM, Knight AF, Sullivan FM (1979) Plasma corticosterone responses to stress following chronic oral administration of diazepam in the rat. J Pharm Pharmacol 31:23–26PubMedGoogle Scholar
  6. Boettger-Tong H, Murthy L, Chiappetta C, Kirkland JL, Goodwin B, Adlercreutz H, Stancel GM, Makela S (1998) A case of a laboratory animal feed with high estrogenic activity and its impact on in vivo responses to exogenously administered estrogens. Environ Health Perspect 106:369–373Google Scholar
  7. Brimble MJ, Balment RJ, Smith CP, Windle RJ, Forsling ML (1991) Influence of oxytocin on sodium excretion in the anaesthetised Brattleboro rat. J Endocrinol 129:49–54PubMedGoogle Scholar
  8. Brown NM, Setchell KD (2001) Animal models impacted by phytoestrogens in commercial chow: implications for pathways influenced by hormones. Lab Invest 81:735–747PubMedGoogle Scholar
  9. Carney JA, Walker BL (1973) Mode of killing and plasma corticosterone concentrations in the rat. Lab Anim Sci 23:675–676PubMedGoogle Scholar
  10. Costall B, Jones BJ, Kelly ME, Naylor RJ, Tomkins DM (1989) Exploration of mice in a black and white test box: validation as a model of anxiety. Pharmacol Biochem Behav 32:777–785PubMedGoogle Scholar
  11. Costall B, Kelly ME, Onaivi ES, Naylor RJ (1990) The effect of ketotifen in rodent models of anxiety and on the behavioural consequences of withdrawing from treatment with drugs of abuse. Naunyn Schmiedebergs Arch Pharmacol 341:547–551PubMedGoogle Scholar
  12. Cotroneo MS, Wang J, Fritz WA, Eltoum IE, Lamartiniere CA (2002) Genistein action in the prepubertal mammary gland in a chemoprevention model. Carcinogenesis 23:1467–1474CrossRefPubMedGoogle Scholar
  13. De Boer SF, Van der Gugten J, Slangen JL (1990) Brain benzodiazepine receptor-mediated effects on plasma catecholamine and corticosterone concentrations in rats. Brain Res Bull 24:843–847PubMedGoogle Scholar
  14. De Boer SF, Slangen JL, Van der Gugten J (1991) Effects of buspirone and chlordiazepoxide on plasma catecholamine and corticosterone levels in stressed and nonstressed rats. Pharmacol Biochem Behav 38:299–308PubMedGoogle Scholar
  15. Degen GH, Janning P, Diel P, Bolt HM (2002) Estrogenic isoflavones in rodent diets. Toxicol Lett 128:145–157PubMedGoogle Scholar
  16. Dunn J, Scheving L (1971) Plasma corticosterone levels in rats killed sequentially at the "trough" or "peak" of the adrenocortical cycle. J Endocrinol 49:347–348PubMedGoogle Scholar
  17. Dunn AJ, File SE (1987) Corticotropin-releasing factor has an anxiogenic action in the social interaction test. Horm Behav 21:193–202PubMedGoogle Scholar
  18. Fernandes C, Arnot MI, Irvine EE, Bateson AN, Martin IL, File SE (1999) The effect of treatment regimen on the development of tolerance to the sedative and anxiolytic effects of diazepam. Psychopharmacology 145:251–259CrossRefPubMedGoogle Scholar
  19. File SE (1979) Effects of ACTH4-10 in the social interaction test of anxiety. Brain Res 171:157–160PubMedGoogle Scholar
  20. File SE (1980) The use of social interaction as a method for detecting anxiolytic activity of chlordiazepoxide like drugs. J Neurosci Methods 2:219–238PubMedGoogle Scholar
  21. File SE (1984) The validation of animal tests of anxiety—pharmacological implications. Pol J Pharmacol Pharm 36:505–512PubMedGoogle Scholar
  22. File SE (1988) The benzodiazepine receptor and its role in anxiety. Br J Psychiatry 152:599–600PubMedGoogle Scholar
  23. File SE (1994) Chronic exposure to noise modifies the anxiogenic response, but not the hypoactivity, detected on withdrawal from chronic ethanol treatment. Psychopharmacology 116:369–372Google Scholar
  24. File SE, Andrews N (1991) Low but not high doses of buspirone reduce the anxiogenic effects of diazepam withdrawal. Psychopharmacology 105:578–582Google Scholar
  25. File SE, Andrews N (1993) Benzodiazepine withdrawal: behavioural pharmacology and neurochemical changes. Biochem Soc Symp 59:97–106PubMedGoogle Scholar
  26. File SE, Hyde JR (1978) Can social interaction be used to measure anxiety? Br J Pharmacol 62:19–24Google Scholar
  27. File SE, Lister RG (1983) Interactions of ethyl-beta-carboline-3-carboxylate and Ro 15–1788 with CGS 8216 in an animal model of anxiety. Neurosci Lett 39:91–94PubMedGoogle Scholar
  28. File SE, Peet LA (1980) The sensitivity of the rat corticosterone response to environmental manipulations and to chronic chlordiazepoxide treatment. Physiol Behav 25:753–758PubMedGoogle Scholar
  29. File SE, Pellow S (1984) The anxiogenic action of Ro 15–1788 is reversed by chronic, but not by acute, treatment with chlordiazepoxide. Brain Res 310:154–156PubMedGoogle Scholar
  30. File SE, Pellow S (1985) Chlordiazepoxide enhances the anxiogenic action of CGS 8216 in the social interaction test: evidence for benzodiazepine withdrawal? Pharmacol Biochem Behav 23:33–36CrossRefPubMedGoogle Scholar
  31. File SE, Seth P (2003) A review of 25 years of the social interaction test. Eur J Pharmacol 463:35–53Google Scholar
  32. File SE, Vellucci SV (1978) Studies on the role of ACTH and of 5-HT in anxiety, using an animal model. J Pharm Pharmacol 30:105–110Google Scholar
  33. File SE, Vellucci SV, Wendlandt S (1979) Corticosterone—an anxiogenic or an anxiolytic agent? J Pharm Pharmacol 31:300–305PubMedGoogle Scholar
  34. File SE, Lister RG, Nutt DJ (1982) The anxiogenic action of benzodiazepine antagonists. Neuropharmacology 21:1033–1037CrossRefPubMedGoogle Scholar
  35. File SE, Pellow S, Braestrup C (1985) Effects of the β-carboline, FG 7142, in the social interaction test of anxiety and the holeboard: correlations between behaviour and plasma concentrations. Pharmacol Biochem Behav 22:941–944PubMedGoogle Scholar
  36. File SE, Mabbutt PS, Walker JH (1988) Comparison of adaptive responses in familiar and novel environments: modulatory factors. Ann NY Acad Sci 525:69–79PubMedGoogle Scholar
  37. File SE, Baldwin HA, Hitchcott PK (1989) Flumazenil but not nitrendipine reverses the increased anxiety during ethanol withdrawal in the rat. Psychopharmacology 98:262–264Google Scholar
  38. File SE, Zharkovsky A, Hitchcott PK (1992) Effects of nitrendipine, chlordiazepoxide, flumazenil and baclofen on the increased anxiety resulting from alcohol withdrawal. Prog Neuropsychopharmacol Biol Psychiatry 16:87–93PubMedGoogle Scholar
  39. File SE, Andrews N, al-Farhan M (1993) Anxiogenic responses of rats on withdrawal from chronic ethanol treatment: effects of tianeptine. Alcohol Alcohol 28:281–286PubMedGoogle Scholar
  40. File SE, Zangrossi H Jr, Sanders FL, Mabbutt PS (1994) Raised corticosterone in the rat after exposure to the elevated plus-maze. Psychopharmacology 113:543–546PubMedGoogle Scholar
  41. File SE, Kenny PJ, Ouagazzal AM (1998) Bimodal modulation by nicotine of anxiety in the social interaction test: role of the dorsal hippocampus. Behav Neurosci 112:1423–1429Google Scholar
  42. Forsling ML, Peysner K (1988) Pituitary and plasma vasopressin concentrations and fluid balance throughout the oestrous cycle of the rat. J Endocrinol 117:397–402PubMedGoogle Scholar
  43. Forsling ML, Stromberg P, Akerlund M (1982) Effect of ovarian steroids on vasopressin secretion. J Endocrinol 95:147–151PubMedGoogle Scholar
  44. Gibbs DM (1984) Dissociation of oxytocin, vasopressin and corticotropin secretion during different types of stress. Life Sci 35:487–491CrossRefPubMedGoogle Scholar
  45. Haas DA, George SR (1988) Single or repeated mild stress increases synthesis and release of hypothalamic corticotropin-releasing factor. Brain Res 461:230–237PubMedGoogle Scholar
  46. Harbuz MS, Lightman SL (1992) Stress and the hypothalamo-pituitary-adrenal axis: acute, chronic and immunological activation. J Endocrinol 134:327–339PubMedGoogle Scholar
  47. Hatton GI (1990) Emerging concepts of structure-function dynamics in adult brain: the hypothalamo-neurohypophysial system. Prog Neurobiol 34:437–504PubMedGoogle Scholar
  48. Hrabovszky E, Kallo I, Hajszan T, Shughrue PJ, Merchenthaler I, Liposits Z (1998) Expression of estrogen receptor-beta messenger ribonucleic acid in oxytocin and vasopressin neurons of the rat supraoptic and paraventricular nuclei. Endocrinology 139:2600–2604PubMedGoogle Scholar
  49. Husain K, Manger WM, Rock TW, Weiss RJ, Weiss RA, Birkner J, Dufton S, Hart C, Frantz AG (1975) Plasma vasopressin (VP) in rats: effect of stressful stimuli. Clin Res 13:573AGoogle Scholar
  50. Kampov-Polevoy AB, Matthews DB, Gause L, Morrow AL, Overstreet DH (2000) P rats develop physical dependence on alcohol via voluntary drinking: changes in seizure thresholds, anxiety, and patterns of alcohol drinking. Alcohol Clin Exp Res 24:278–284Google Scholar
  51. Kao YC, Zhou C, Sherman M, Laughton CA, Chen S (1998) Molecular basis of the inhibition of human aromatase (estrogen synthetase) by flavone and isoflavone phytoestrogens: a site-directed mutagenesis study. Environ Health Perspect 106:85–92Google Scholar
  52. Kawashima K, Inoue T, Tsutsumi N, Endo H (1996) Effect of KCA-098 on the function of osteoblast-like cells and the formation of TRAP-positive multinucleated cells in a mouse bone marrow cell population. Biochem Pharmacol 51:133–139CrossRefPubMedGoogle Scholar
  53. Kelestimur H, Leach RM, Ward JP, Forsling ML (1997) Vasopressin and oxytocin release during prolonged environmental hypoxia in the rat. Thorax 52:84–88PubMedGoogle Scholar
  54. Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, van der Burg B, Gustafsson JA (1998a) Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 139:4252–4263PubMedGoogle Scholar
  55. Kuiper GG, Shughrue PJ, Merchenthaler I, Gustafsson JA (1998b) The estrogen receptor beta subtype: a novel mediator of estrogen action in neuroendocrine systems. Front Neuroendocrinol 19:253-286CrossRefPubMedGoogle Scholar
  56. Laflamme N, Nappi RE, Drolet G, Labrie C, Rivest S (1998) Expression and neuropeptidergic characterization of estrogen receptors (ERalpha and ERbeta) throughout the rat brain: anatomical evidence of distinct roles of each subtype. J Neurobiol 36:357–378CrossRefPubMedGoogle Scholar
  57. Lahti RA, Barsuhn C (1974) The effect of minor tranquilizers on stress-induced increases in rat plasma corticosteroids. Psychopharmacologia 35:215–220PubMedGoogle Scholar
  58. Le Bail JC, Varnat F, Nicolas JC, Habrioux G (1998) Estrogenic and antiproliferative activities on MCF-7 human breast cancer cells by flavonoids. Cancer Lett 130:209–216CrossRefPubMedGoogle Scholar
  59. Le Fur G, Guilloux F, Mitrani N, Mizoule J, Uzan A (1979) Relationships between plasma corticosteroids and benzodiazepines in stress. J Pharmacol Exp Ther 211:305–308PubMedGoogle Scholar
  60. Lephart ED, Thompson JM, Setchell KD, Adlercreutz H, Weber KS (2000) Phytoestrogens decrease brain calcium-binding proteins but do not alter hypothalamic androgen metabolizing enzymes in adult male rats. Brain Res 859:123–131CrossRefPubMedGoogle Scholar
  61. Lephart ED, West TW, Weber KS, Rhees RW, Setchell KD, Adlercreutz H, Lund TD (2002) Neurobehavioral effects of dietary soy phytoestrogens. Neurotoxicol Teratol 24:5–16CrossRefPubMedGoogle Scholar
  62. Lund TD, Lephart ED (2001) Dietary soy phytoestrogens produce anxiolytic effects in the elevated plus maze. Brain Res 913:180–184CrossRefPubMedGoogle Scholar
  63. Lund TD, West TW, Tian LY, Bu LH, Simmons DL, Setchell KD, Adlercreutz H, Lephart ED (2001) Visual spatial memory is enhanced in female rats (but inhibited in males) by dietary soy phytoestrogens. Biomed Central Neurosci 2:20CrossRefGoogle Scholar
  64. Makela S, Poutanen M, Lehtimaki J, Kostian ML, Santti R, Vihko R (1995) Estrogen-specific 17 beta-hydroxysteroid oxidoreductase type 1 (E.C. as a possible target for the action of phytoestrogens. Proc Soc Exp Biol Med 208:51–59PubMedGoogle Scholar
  65. Pan Y, Anthony M, Watson S, Clarkson TB (2000) Soy phytoestrogens improve radial arm maze performance in ovariectomized retired breeder rats and do not attenuate benefits of 17beta-estradiol treatment. Menopause 7:230–235PubMedGoogle Scholar
  66. Patisaul HB, Dindo M, Whitten PL, Young LJ (2001) Soy isoflavone supplements antagonize reproductive behavior and estrogen receptor alpha- and beta-dependent gene expression in the brain. Endocrinology 142:2946–2952PubMedGoogle Scholar
  67. Pellow S, File SE (1986) Anxiolytic and anxiogenic drug effects on exploratory activity in an elevated plus-maze: a novel test of anxiety in the rat. Pharmacol Biochem Behav 24:525–529Google Scholar
  68. Pellow S, Chopin P, File SE, Briley MJ (1985) Validation of open:closed arm entries in an elevated plus maze as a measure of anxiety in the rat. Neurosci Methods 14:149–167PubMedGoogle Scholar
  69. Pericic D, Pivac N (1996) Effects of diazepam on conflict behaviour and on plasma corticosterone levels in male and female rats. Naunyn Schmiedebergs Arch Pharmacol 353:369–376PubMedGoogle Scholar
  70. Peysner K, Forsling ML (1990) Effect of ovariectomy and treatment with ovarian steroids on vasopressin release and fluid balance in the rat. J Endocrinol 124:277–284PubMedGoogle Scholar
  71. Piskula MK, Yamakoshi J, Iwai Y (1999) Daidzein and genistein but not their glucosides are absorbed from the rat stomach. FEBS Lett 447:287–291CrossRefPubMedGoogle Scholar
  72. Rodgers RJ, Haller J, Holmes A, Halasz J, Walton TJ, Brain PF (1999) Corticosterone response to the plus-maze: high correlation with risk assessment in rats and mice. Physiol Behav 68:47–53CrossRefPubMedGoogle Scholar
  73. Roy BN, Reid RL, Van Vugt DA (1999) The effects of estrogen and progesterone on corticotropin-releasing hormone and arginine vasopressin messenger ribonucleic acid levels in the paraventricular nucleus and supraoptic nucleus of the rhesus monkey. Endocrinology 140:2191–2198PubMedGoogle Scholar
  74. Sajdyk TJ, Schober DA, Gehlert DR, Shekhar A (1999) Role of corticotropin-releasing factor and urocortin within the basolateral amygdala of rats in anxiety and panic responses. Behav Brain Res 100:207–215PubMedGoogle Scholar
  75. Santell RC, Chang YC, Nair MG, Helferich WG (1997) Dietary genistein exerts estrogenic effects upon the uterus, mammary gland and the hypothalamic/pituitary axis in rats. J Nutr 127:263–269PubMedGoogle Scholar
  76. Spina G, Merlo-Pich E, Akwa Y, Balducci C, Basso M, Zorrilla P, Britton T, Rivier J, Vale W, Koob F (2002) Time-dependent induction of anxiogenic-like effects after central infusion of urocortin or corticotropin-releasing factor in the rat. Psychopharmacology 160:113–121CrossRefPubMedGoogle Scholar
  77. To CT, Anheuer ZE, Bagdy G (1999) Effects of acute and chronic fluoxetine treatment of CRH-induced anxiety. Neuroreport 10:553–555PubMedGoogle Scholar
  78. Weber KS, Setchell KD, Lephart ED (2001) Maternal and perinatal brain aromatase: effects of dietary soy phytoestrogens. Brain Res Dev Brain Res 126:217–221CrossRefPubMedGoogle Scholar
  79. Whitten PL, Patisaul HB, Young LJ (2002) Neurobehavioral actions of coumestrol and related isoflavonoids in rodents. Neurotoxicol Teratol 24:47–54CrossRefPubMedGoogle Scholar
  80. Windle RJ, Shanks N, Lightman SL, Ingram CD (1997) Central oxytocin administration reduces stress-induced corticosterone release and anxiety behavior in rats. Endocrinology 138:2829–2834PubMedGoogle Scholar
  81. Wotjak CT, Kubota M, Liebsch G, Montkowski A, Holsboer F, Neumann I, Landgraf R (1996) Release of vasopressin within the rat paraventricular nucleus in response to emotional stress: a novel mechanism of regulating adrenocorticotropic hormone secretion? J Neurosci 16:7725–7732PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • David E. Hartley
    • 1
    Email author
  • Jessica E. Edwards
    • 1
  • Claire E. Spiller
    • 1
  • Nazmul Alom
    • 1
  • Sonia Tucci
    • 1
  • Pallab Seth
    • 1
  • Mary L. Forsling
    • 1
  • Sandra E. File
    • 1
  1. 1.Psychopharmacology Research Unit, Centre for NeuroscienceHodgkin Building, Kings College London, Guy's CampusLondonUK

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