Genistein and other soya isoflavones are potent ligands for transthyretin in serum and cerebrospinal fluid

British Journal of Nutrition (2006), 95, 1171–1176 q The Authors 2006 DOI: 10.1079/BJN20061779 Genistein and other soya isoflavones are potent ligands for transthyretin in serum and cerebrospinal fluid Branislav Radović*, Birgit Mentrup and Josef Köhrle Institut für Experimentelle Endokrinologie und Endokrinologisches Forschungszentrum (EnForCé), Charité Universitätsmedizin Berlin, Schumannstrasse 20-21, 10117, Berlin, Germany (Received 15 July 2005 – Revised 24 January 2006 – Accepted 10 February 2006) Consumption of soya-based nutrients is increasing in modern society because of their potentially protective effects against chronic diseases. Soya products are also heavily advertised as alternative drugs for relief from symptoms of the menopause and for hormone replacement therapy. However, because of their oestrogenic activity, negative effects of isoflavones have been postulated. Therefore, we analysed influences of soya isoflavones, major soya constituents with endocrine activity, on thyroxine (T4) binding to its distribution proteins. Serum binding of 125I-labelled L -T4 was analysed in the absence or presence of increasing concentrations of soya isoflavones using non-denaturing PAGE for analysis. Complete displacement of [125I]T4 binding to transthyretin (TTR) was observed in human serum incubated with genistein at concentrations . 10 mM ; interference started at .0·1 mM . Glycitein showed decreased and daidzein the lowest displacement potency. [125I]T4 was displaced to albumin in rat and to T4-binding globulin in human serum. Soya isoflavones also obstruct [125I]T4 binding to TTR in human cerebrospinal fluid (CSF). The inhibitory effect was confirmed in direct binding assays using purified TTR with 50 % inhibitory concentration values of 0·07 mM for genistein, 0·2 mM for glycitein and 1·8 mM for daidzein. The present study underlined a potent competition of soya isoflavones for T4 binding to TTR in serum and CSF. Isoflavones might alter free thyroid hormone concentrations resulting in altered tissue availability and metabolism. As a consequence of this interference, one could expect a disturbance in the feedback regulation of hormonal networks, including the pituitary – thyroid– periphery axis during development and in adult organisms. Thyroxine: Transthyretin: Genistein: Isoflavones: Cerebrospinal fluid Several experimental and clinical studies have shown that consumption of soya and soya food products have beneficial effects on human health. Particularly, studies on soya isoflavones revealed their possible protective role against different forms of cancer, osteoporosis, CVD and renal disease (Tham et al. 1998; Messina, 1999; Anderson et al. 1999; Lissin & Cooke, 2000; Jin & MacDonald, 2002; Cross et al. 2004; Duncan et al. 2005). Their structural similarities to 17b-oestradiol, and ability to preferentially bind to oestrogen receptor b (50 % inhibitory concentration (IC50) values of 8·4 nM for genistein and 100 nM for daidzein) and sex hormone-binding globulin (Kuiper et al. 1998; Dixon & Ferreira, 2002; Doerge & Sheehan, 2002), identified these soya isoflavones as acting as potential selective oestrogen receptor modulators, this being the reason for their wide but controversial use in relieving postmenopausal symptoms in women. Despite the numerous beneficial effects of soya isoflavones, epidemiological and experimental data also exist showing an adverse effect on human health. Soya isoflavones exhibit oestrogen activity but, administered during development, can cause adverse oestrogen effects in experimental animals (Dixon & Ferreira, 2002). The main isoflavone genistein also inhibits tyrosine kinase (IC50 about 150 mM ) and other protein kinases by acting as a competitive inhibitor of ATP binding at higher doses than needed for oestrogen receptor binding (Akiyama et al. 1987). Recently it was found that mice neonatally exposed to genistein develop uterine cancer later in their life, reminiscent of certain effects of oestrogen analogues, such as diethylstilbestrol (Newbold et al. 2001). The negative effects of soya on the pituitary –thyroid axis are also well described in human subjects and animals. The studies on rats revealed associations between goitrogenesis and soya consumption, and the protective effect of adequate dietary iodide intake (Block et al. 1961; Nordsiek, 1962; Konijn et al. 1972; Kay et al. 1988). Hypothyroidism and goitre were also observed in infants consuming soya formula (van Wyk et al. 1959; Hydovitz, 1960; Shepard et al. 1960; Ripp, 1961; Pinchera et al. 1965; Labib et al. 1989; Chorazy et al. 1995; Jabbar et al. 1997); however, goitre was reversed after switching to cows’ milk or iodine supplementation. There are at least three different levels at which soya isoflavones can interact with the thyroid hormone system: at the thyroid gland; in metabolism (with feedback mechanisms); with thyroid hormone transport proteins (Köhrle, 2000). Genistein and daidzein were identified as potent inhibitors of thyroid peroxidase, a key enzyme in thyroid hormone synthesis, in vitro and in vivo (Divi et al. 1997; Chang & Doerge, 2000; Doerge & Sheehan, 2002). They block both Abbreviations: CSF, cerebrospinal fluid; IC50, 50 % inhibitory concentration; T4, thyroxine; TBG, thyroxine-binding globulin; TTR, transthyretin. * Corresponding author: Dr Branislav Radovic, fax þ49 30450524922, email branislav.radovic@charite.de Downloaded from https://www.cambridge.org/core. IP address: 54.70.40.11, on 04 Sep 2018 at 18:02:08, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms . https://doi.org/10.1079/BJN20061779 1172 B. Radović et al. (Young et al. 1982). The sera and CSF samples (10 ml) were incubated in 1·5 ml Eppendorf tubes for 30 min at room temperature with 10 ml [125I]T4 (about 740 Bq) diluted in 0·02 M- phosphate buffer (pH 9) in the absence or presence of increasing concentrations of soya isoflavones (0·1 –100 mmol/l). Samples (60 ml) of the incubated mixture were loaded on nondenaturing PAGE gels and run for 14 h at 50 V in a tri(hydroxymethyl)-aminomethane-glycine native running buffer (pH 8·4). The temperature was maintained at 68C by the Bio-Rad Protean II xi cooling electrophoresis chamber (Bio-Rad Laboratories, Hercules, CA, USA). Gels were sealed in a plastic transparent bag and exposed to phosphoimager plates overnight, before scanning. The distribution of radiolabelled T4 to individual binding proteins was analysed and quantified by a Cyclone storage phosphor screen (Packard Instrument Company Inc., Meriden, CT, USA). iodination of tyrosine residues on thyroglobulin and the coupling of two iodinated tyrosine molecules to yield iodothyronines. Daidzein and genistein also affect the metabolism of thyroid hormones and iodide re-utilisation in the human thyroid by inhibition of sulfotransferase enzymes (Ebmeier & Anderson, 2004). In the healthy adult rat, thyroid hormones are transported to target tissues primarily bound to transthyretin (TTR), the major serum distributor protein for thyroid hormone in rodents (Young et al. 1982). This is in contrast to human serum, where thyroxine-binding globulin (TBG) binds thyroxine (T4) with highest affinity. Flavonoids are strongly and preferentially bound to TTR in most species, including man, but show no or only minor competition with thyroid hormone for binding to TBG, or to serum albumin (Köhrle et al. 1989). In the present in vitro study, we report the influence of soya isoflavones (Fig. 1) on thyroid hormone binding and distribution, in particular on the binding of T4 to its distributor proteins in human and rat serum as well as in human cerebrospinal fluid (CSF). In vitro thyroxine–transthyretin competition-binding studies The analysis of the capacity of soya isoflavones to compete with T4 binding to purified human TTR was performed as described previously (Somack et al. 1982), with slight modifications (Auf’mkolk et al. 1986). The assay mixture was a 0·1 M -tri(hydroxymethyl)-aminomethane-HCl buffer (pH 8·0) containing 0·1 M- NaCl and 1 mM -EDTA, purified human TTR (2·5 mg/ ml ¼ 23 nmol/l), 125I-labelled L -T4 (610 Bq/tube, about 50 000 cpm) and competitors (soya isoflavones) with increasing concentrations (0·001–10 mmol/l), in a total volume of 100 ml. Control incubations contained 1 % dimethylsulfoxide, which was the solvent, instead of the competitor. The incubation mixtures were allowed to reach binding equilibrium at room temperature for 30 min, and incubation was stopped by adding 0·5 ml ice-cold dextran-coated charcoal. TTR-bound and free [125I]T4 were separated after 10 min of incubation at 48C by 10 min centrifugation at 3000 g. The decanted supernatant fraction was counted in an LKB Wallac 1277 g counter (Wallac, Milton Keynes, Bucks, UK). Unspecific binding, determined by adding L -T4 (10 mmol/l), was subtracted to obtain specific binding data. All analyses were performed with data from at least three different experiments performed in duplicate. Calculation of binding parameters was performed with GraphPad Prism version 4 for Windows (GraphPad Software Inc., San Diego, CA, USA). Materials and methods Chemicals and materials 125 I-labelled L -T4 (specific activity 4·99–6·10 MBq/mg) was purchased from Perkin Elmer (Billerica, MA, USA). 3,30 ,5,50 -Tetraiodo-L -T4 was kindly provided from Henning Berlin (Germany). Human purified TTR and TBG were prepared by Vivian Cody (Hauptman-Woodward Medical Research Institute, Buffalo, NY, USA). The soya isoflavone genistein was purchased from Sigma-Aldrich (Germany) and isoflavones daidzein and glycitein were kindly provided by Sabine Kulling (Karlsruhe, Germany). The Wistar rat serum pool was kindly provided by Franziska Götz (Institute of Experimental Endocrinology, Charité, Berlin, Germany) and the human serum pool and CSF pool from Lutz Schomburg and Ulrich Schweizer (Institute of Experimental Endocrinology, Charité, Berlin, Germany), respectively. Analysis of thyroxine binding to serum proteins Binding of 125I-labelled T4 to serum and CSF proteins was assessed by non-denaturing PAGE, as previously described (A) O (B) I HO NH2 I OH O I HO I OH (C) H3 C O OH OH (D) O O OH O O HO O HO O Fig. 1. Structural formulas of thyroxine (A) and soya isoflavones used in the study: genistein (B); glycitein (C); daidzein (D). Downloaded from https://www.cambridge.org/core. IP address: 54.70.40.11, on 04 Sep 2018 at 18:02:08, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms . https://doi.org/10.1079/BJN20061779 Genistein – a potent ligand for transthyretin Results 1173 TTR presented in Fig. 2 (A) (lane 3) was slightly shifted compared with native TTR, either because it was partially ‘denatured’ or devoid of retinol-binding protein after purification. Thebindingof[125I]T4 toTTRinthepresenceofsaturatingconcentrations of soya isoflavones is presented in Fig. 3. The sigmoidal dose–response curves describe the relationships between the isoflavones’ concentration and [125I]T4 bound to human purified TTR.Genisteinwasthestrongestcompetitor,showingpractically the same displacement as the unlabelled L -T4 used as a control in each experiment (IC50 ¼ 0·07 and 0·08 mM , respectively). Glycitein inhibited [125I]T4 binding to TTR with about four times less potency (IC50 ¼ 0·2 mM ) and daidzein was the weakest competitor for binding to TTR (IC50 ¼ 1·8 mM ). Scatchard analysis of the representative binding data yielded the following dissociation constants: T4 (65 (SD 19) nM ); genistein (59 (SD 13) nM ); glycitein (71 (SD 22) nM ); daidzein (131 (SD 109) nM ). Increasing concentrations of soya isoflavones added to human and rat serum and human CSF progressively inhibited the binding of 125I-labelled L -T4 to TTR. Native PAGE images presented in Fig. 2 show a representative example of the most potent competitor, genistein. Complete inhibition of [125I]T4 binding to TTR was achieved at a concentration of . 10 mmol genistein/l in human serum with interference starting at . 0·1 mM concentrations (Fig. 2 (A)). The labelled hormone was displaced from TTR to TBG and albumin. The displacement potency decreased with the decrease in concentration of the competitor. Fig. 2 (B) shows the effect of genistein on [125I]T4 binding to TTR in human CSF, which contains no TBG or albumin. Complete and marked displacement was observed at concentrations of 100 and 10 mmol/l respectively, leading to an increased amount of free [125I]T4 (lanes 5 and 6). Genistein added to pooled rat serum markedly inhibited the [125I]T4 binding to TTR at the concentrations of 100 and 10 mmol/l (Fig. 2 (C)). The labelled hormone was displaced from TTR to albumin. The other soya isoflavones, glycitein and daidzein, influence the [125I]T4 binding to serum and CSF proteins in the same manner, but with lower potency (data not shown). Purified Discussion The results presented in the present in vitro study clearly demonstrate the inhibitory effect of soya isoflavones on the binding of T4 to the serum and CSF thyroid hormone transport (A) TBG ALB TTR Free[125I]T4 1 2 3 4 5 6 7 8 (B) TTR Free[125I]T4 1 2 3 4 5 6 7 8 (C) ALB TTR 4 5 6 7 Fig. 2. Representative autoradiographs of [125I]thyroxine ([125I]T4) bound to serum and cerebrospinal fluid (CSF) thyroid hormone-binding proteins. Effect of the isoflavone genistein added in vitro on binding of [125I]T4 to binding proteins in (A) human serum, (B) human CSF and (C) rat serum. In (A) the lanes are: lane 1, free [125I]T4; lane 2, purified human thyroxine-binding globulin (TBG); lane 3, purified human transthyretin (TTR); lane 4, control human serum; lane 5, human serum þ100 mM -genistein; lane 6, human serum þ10 mM -genistein; lane 7, human serum þ1 mM -genistein; lane 8, human serum þ0·1 mM -genistein. In (B) the lanes are: lane 1, free [125I]T4; lane 2, purified human TBG; lane 3, purified human TTR; lane 4, control human CSF; lane 5, CSF þ 100 mM -genistein; lane 6, CSF þ 10 mM -genistein; lane 7, CSF þ1 mM -genistein; lane 8, CSF þ0·1 mM -genistein. In (C) the lanes are: lane 4, control rat serum; lane 5, rat serum þ100 mM -genistein; lane 6, rat serum þ10 mM -genistein; lane 7, rat serum þ1 mM -genistein. ALB, albumin. Downloaded from https://www.cambridge.org/core. IP address: 54.70.40.11, on 04 Sep 2018 at 18:02:08, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms . https://doi.org/10.1079/BJN20061779 1174 B. Radović et al. [125I]T4 bound (%) 100 75 50 25 0 0·001 0·01 0·1 1 10 Concentration (µM) 100 Fig. 3. Dose–response displacement of [125I]thyroxine ([125I]T4; A) from transthyretin (TTR) by soya isoflavones genistein (W), glycitein (D) and daidzein (L). Data points are mean values of at least three measurements in duplicate, with standard deviations represented by vertical bars. [125I]T4 – TTR binding data were normalised for each experiment to span the range from 0 to 100 %. Values for 50 % inhibitory concentration (mM) were: T4, 0·08; genistein, 0·07; glycitein, 0·2; daidzein 1·8. protein, TTR. As no competition was observed for the binding of [125I]T4 to albumin and TBG (data not shown), the addition of soya isoflavones to rat and human serum resulted in the displacement of [125I]T4 from TTR to these transport proteins. Displacement also occurred in human CSF, but because of the absence of other specific binding proteins for T4, the free [125I]T4 fraction was evidently increased. Genistein was the strongest competitor, showing binding affinity comparable with that of unlabelled T4. Glycitein and daidzein exhibited lower competition potency. Selective binding of genistein and daidzein to TTR in plasma determined by an antibody capture–HPLC method, as recently published by Green et al. (2005), is in agreement with the present data. The results presented in the present study are highly relevant both biologically and medically because the obtained IC50 concentrations are in the range of published soya isoflavone plasma concentrations. Doerge & Sheehan (2002) reported that adults eating typical Asian diets have blood concentrations of 0·1 –1·2 mmol total soya isoflavones/l. Three adult volunteers had concentrations of 0·5 –0·9 mmol/l after eating soya nutritional supplements and even 2–7 mmol/l concentrations of soya isoflavones were measured in the serum of seven infants eating soya infant formula. Manach et al. (2005) reviewed ninety-seven polyphenol bioavailability studies and showed similar results; for example, concentrations of 1·74 and 1·33 mmol genistein/l were measured in plasma 6 h after intake of soya milk and tofu, respectively. As these values are far above IC50 values obtained in vitro for genistein (0·07 mmol/l) and even glycitein (0·2 mmol/l), displacement of T4 from TTR could be equally possible in vivo. Altered binding of T4 to TTR, associated with altered free thyroid hormone levels, might be possible and might exert physiological effects. Watanabe et al. (2000) reported altered T4 and triiodothyronine values in young premenopausal women, after administration of physiological doses of isoflavones. Similar doses of soya isoflavones caused only modest hormonal effects in postmenopausal women (Duncan et al. 1999). Selective binding of (iso)flavonoids to TTR might also indicate a role of TTR in distribution and targeting of soya isoflavones to steroid-regulated tissues, an effect independent of T4 competition. As reported by Cassidy et al. (1994) and Watanabe et al. (2000), physiological doses of isoflavones can cause changes in sex hormone production and perturb menstruation. The recently published data (Jefferson et al. 2005) indicate a potential deleterious role of isoflavones during pregnancy, which is of particular concern. The authors reported that neonatal exposure to genistein at environmentally relevant doses caused abnormal oestrous cycles, altered ovarian function, early reproductive senescence, and subfertility in mice. Whether TTR itself or altered thyroid hormone economy as suggested by the present observations contribute to alterations of the reproductive axis reported by Jefferson et al. (2005) remains to be studied. Competitive in vitro binding with TTR in CSF revealed a clear increase in free T4 concentration because there is no other alternative specific thyroid hormone carrier able to bind displaced T4. TBG is not present in CSF (Davidsson et al. 2001; Matsumoto et al. 2003) and the amount of albumin which has the lowest affinity to bind T4 (Tabachnick & Giorgio, 1964) is too low compared with TTR to bind displaced [125I]T4. Chanoine et al. (1992) also reported the transient increase in serum and CSF free T4 concentration after administering low and high doses of synthetic flavonoid EMD21388, respectively. However, the occupation of the sole hormone distributor protein and evident disturbance in binding properties postulate an effect on brain metabolism, if it is known that thyroid hormones are intimately involved in the regulation of the central nervous system. Experimental studies show that the central nervous system has strict requirements for thyroid hormones; in the brain, the concentrations of both T4 and the more active metabolite triiodothyronine tend to be kept within a narrow range even in the presence of extreme fluctuations of circulating T4 level (Dratman et al. 1983). This fact additionally underlines the importance of TTR binding in CSF considering that TTR binds both T4 and triiodothyronine (although having different affinities) (Cody, 2002), which are present in equimolar concentration in the brain in contrast to the serum. However, further in vivo studies are required to clarify the physiological and molecular implications of disturbed T4 binding, particularly on the central nervous system. The negative influence of soya isoflavones on thyroid hormone synthesis by means of blocking thyroid peroxidase has been well described in vitro and in vivo. Numerous studies on rats and human subjects raise concerns on anti-thyroid effects, including goitre formation, especially in infants consuming soya formula (van Wyk et al. 1959; Hydovitz, 1960; Shepard et al. 1960; Ripp, 1961; Pinchera et al. 1965; Kay et al. 1988; Labib et al. 1989; Ishizuki et al. 1991; Chorazy et al. 1995; Jabbar et al. 1997; Ikeda et al. 2000). There are also opposite reports (Klein, 1998; Merritt & Jenks, 2004) indicating that dietary isoflavones in soya infant formulas do not adversely affect human health. Besides the inhibitory effects of flavonoids on thyroid peroxidase, iodine deficiency is a very important risk factor for thyroid dysfunction and goitre development in both man and rats. An adequate iodine supply is absolutely advantageous for preventing the goitrogenic effects of soya isoflavones, especially in the relatively high-risk group of patients with congenital hypothyroidism solely dependent on exogenous thyroid hormone supply, or in patients with transient hypothyroidism after thyroidectomy. The present data contribute to unveiling the mechanism of action of soya isoflavones in goitrogenesis and disturbance Downloaded from https://www.cambridge.org/core. IP address: 54.70.40.11, on 04 Sep 2018 at 18:02:08, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms . https://doi.org/10.1079/BJN20061779 Genistein – a potent ligand for transthyretin of the thyroid hormone homeostasis, while emphasising the role of binding and distributor proteins, particularly TTR. TTR is the main thyroid hormone transport protein in rodents, but, in man, despite the 20-fold higher concentration in serum relative to that of TBG, it plays a lesser role in iodothyronine transport (Woeber & Ingbar, 1968). Only a minor fraction of T4 is bound to serum albumin in both man and rats, despite the very high binding capacity (Köhrle et al. 1989). In vitro and in vivo competitive-binding studies have revealed up to now that flavonoids interfere with T4 binding, not with all thyroid hormone distribution proteins, but preferentially with TTR (Köhrle, 2000). TTR is a highly conserved tetrameric protein with two binding domains and three pairs of halogen-binding pockets in each of them (Cody, 2002). At physiological conditions, only one T4 binding domain is occupied, since the negative cooperativity in binding to the second domain decreases the binding affinity of a second hormone molecule (Ferguson et al. 1975). Structural data for the human TTR –T4 complex has revealed that T4 binds in a ‘forward’ mode with its phenolic OH group buried deep within the binding channel, while the synthetic flavone EMD21388 binds to human TTR in a manner different from T4 (Cody, 2002). After 12 h incubation with EMD21388, binding occurred only in one domain in a ‘forward’ mode, while 24 h incubation data showed forward binding in both domains with the bromoflavone bound deeper in the channel than T4. After 48 h incubation EMD21388 binds in both a ‘forward’ mode and a ‘reverse’ mode. Ciszak et al. (1992) reported the similar binding manner for a bromoaurone analogue. The fact that soya isoflavones tested in the present study have a similar structure to synthetic bromoflavone means that a similar binding manner could be expected. Therefore, soya isoflavones could also exhibit alternative binding orientations, which may explain such strong binding affinities for TTR (Auf’mkolk et al. 1986). Structure–activity correlation in the present study revealed the soya isoflavone genistein as the strongest binding competitor, indicating the same binding potency as L -T4. Obviously, the 5-OH group is important for binding, most probably by occupying one of the three halogen-binding pockets, while the 7-OH group in the meta-position occupies the other. In addition, the ability of the 5-OH group of genistein to form an intramolecular hydrogen bond with the 4-keto group leading to a pseudo aromatic ring (Chen et al. 1995) could also participate in increased binding to TTR compared with other soya isoflavones. The reason for the ten-fold increase in binding potency of glycitein compared with daidzein should be searched for in the 6-methoxy group which is absent in daidzein. According to Cody (2002), the presence of the 3methyl of EMD21388 appears crucial for effective binding and stabilisation of the TTR tetramer. The important van der Waal’s interactions may also be formed between the 6-methoxy group of daidzein and adequate amino acids in the binding pocket. However, the further crystallographic analyses of TTR –ligand co-crystal complexes are required to clarify these structure –activity relationships. Naturally occurring chalcones, including phloretin, aurones and flavonoids, exhibit a clear concentration-dependent displacement of T4 from TTR binding in vitro with IC50 values in the range of 0·1– 50 mM (Köhrle, 2000). The present data contribute to competition-binding studies made thus far, therefore revealing the isoflavone genistein as the strongest naturally occurring T4 competitor. Further in vivo studies will clarify 1175 the expected disturbance in the feedback regulation of hormonal networks, including the pituitary –thyroid –periphery axis and will shed more light on the role of TTR in this feedback circuit. Acknowledgements We thank Ms Manuela Topp and Ms Anita Kinne for perfect technical assistance and Dr Lutz Schomburg for useful discussion. The present study was supported by the German Research Council (Deutsche Forschungsgemeinschaft), no. Ko 922/12-1. 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