Tissue-Specific Distribution of Genistein, Daidzein and Bisphenol

Food Sci. Technol. Res., ++ (,), +21ῌ+3-, ,**/

Tissue-speciﬁc Distribution of Genistein, Daidzein and Bisphenol A in Male Sprague-Dawley Rats after Intragastric Administration

+ῌ + + +ῌῌ Shin YASUDA , Po-Sheng WU , Masaaki OKABE , Hirofumi TACHIBANA+ and Koji YAMADA

+ Laboratory of Food Chemistry, Division of Applied Biological Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, Japan

Received December 0, ,**.; Accepted March ,-, ,**/

A mixture of genistein, daidzein and bisphenol A was intragastrically given to male Sprague-Dawley rats. The tissue distribution of these compounds and equol, a metabolite of daidzein, was examined by high-performance liquid chromatography after administration. In the serum, liver and kidney, genistein, daidzein and bisphenol A were clearly detected at 0 to ,. h. Equol was detected at * h and levels subsequently dropped and then increased again, reaching the initial level at ,. h. In the mesenteric lymph node (MLN), thymus and spleen, genistein and daidzein were detected at 0 to ,. h. Bisphenol A and equol in the MLN was detectable at 0 to ,. hand,. h, respectively. In the testicle, epididymis, spermatheca and prostate, higher levels of genistein and daidzein were detected around 0 h. Bisphenol A was detected in the epididymis at 0 to +, h. These results suggest that the distribution of these chemicals and their tissue a$nities in rats vary from tissue to tissue.

Keywords: genistein, daidzein, bisphenol A, equol

Introduction These reports imply that isoflavones in foodstu#s and Soybean has been known to be a healthy foodstu# in bisphenol A from chemical products can be easily ab- Asia for centuries and its benefits have more recently sorbed into our bodies and can exert a biological e#ect become widely known in the West. Isoflavones, a class of similar to that of estrogen. Although these compounds phytoestrogens, are the major phytochemicals present in are metabolized into a conjugate form in the body, they soybean. The health-related and clinical benefits of isofla- are also thought to act in target tissues in the free form vones have been reported (Munro et al., ,**-). The pro- (Setchell, +332). However, it is not entirely clear how posed physiological functions of isoflavones include bind- these compounds are metabolized or what they act upon. ing to estrogen receptors (Han et al., ,**,), radical Therefore, it is important to determine the target tissues scavenging activities (Patel et al., ,**+), and antipro- of these estrogenic compounds. In order to investigate liferative e#ects on cancer cells (Traganos et al., +33,). the location of these compounds in the body for further Genistein and daidzein are isoflavone aglycons in soybean study, we examined the tissue distribution of genistein, seeds (Kudou et al., +33+). Equol is the main product daidzein, and bisphenol A in rats after intragastric admin- from daidzein, and the metabolism of equol has been istration. Besides these three compounds, the amount of reported to depend on intestinal gut flora in humans equol was also analyzed as a metabolite using a high- (Setchell and Adlercreutz, +332). Its antioxidant activity performance liquid chromatography (HPLC) system with and a$nity for estrogen receptors have also been a coulometric array detector. The level of change in reported (Setchell et al., ,**,). each chemical within ,. hours of administration was also Bisphenol A is an industrial chemical that is principally investigated in a variety of tissues, such as drug metabolic- used as a monomer in the manufacturing of poly- excretive, immune and reproductive tissues in rats. carbonate plastics and epoxy resins (Krishnan et al., +33-). It is present in the coatings of food and beverage contain- Materials and Methods ers (Brotons et al., +33/; Hoyle and Budway, +331)aswell Materials Daidzein and genistein were purchased as in dental sealants and fillings (Imai, +333). Bisphenol from Fujicco Co. (Kyoto, Japan), and equol from Fluka A has been reported to accelerate puberty in female rats Chemie AG (Buchs, Switzerland). Bisphenol A was ob- (Ashby and Tinwell, +332). This chemical has been tained from Sigma Chemical Co. (St. Louis, MO, USA). designated a xenoestrogen because of its estrogenic e#ect. HPLC-grade acetonitrile and distilled water were pur- ῌ To whom correspondence should be addressed. chased from Kanto Chemical Co. (Tokyo, Japan) and / E-mail: [email protected] methanol from Nacalai Tesque (Kyoto, Japan). Type H- ῌῌ To whom inquiries about the paper should be addressed. b-glucuronidase from Helix pomatia was obtained from E-mail: [email protected] Sigma. Ethanol, sodium acetate and acetic acid were 188 S. YASUDA et al. purchased from Nacalai Tesque. Experimental animals A mixture of genistein, daidzein and bisphenol A (.* mg/ml each) was prepared in sa%ow- er oil (Rinoru Oil Mills Co., Nagoya, Japan). Eight-week- old male Sprague-Dawley (SD) rats (Seac Yoshitomi, Fukuoka, Japan), approximately ,/*ῌ,2* g/body, were fed a nonpurified diet (type MF, Oriental Yeast, Tokyo, Japan) and drinking water for five days after arrival. The animals were then individually housed in four groups of four rats each in a room controlled at ,*῏. The rats were denied food for +* h, and then + ml of the above mixture was intragastrically administered under light diethyl ether anesthesia. Without administration (* h) or at 0, +,,or,. h after administration, these animals were killed by withdrawing blood from the abdominal aorta under anesthesia. After blood collection, tissues were excised immediately, and stored at ῌ-*῏ until analysis. All animals were kept in accordance with the Guidelines on Animal Experiments from the Faculty of Agriculture and Graduate Course of Kyushu University, and Law No. +*/ and Notification No. 0 of the Japanese government. Analysis of estrogenic compounds Genistein, daidzein, Fig. +. Tissue-distribution of isoflavones, bisphenol A and bisphenol A and equol were measured by HPLC analysis equol after intragastric administration in rats. The data after b-glucuronidase treatment as described (Yasuda et shown illustrates the total-amount of genistein (filled), ,**. +/* al., ). Briefly, ml of rat serum was treated with or daidzein (hatched), bisphenol A (unfilled) and equol (dotted). without ,0.1 kU/ml of b-glucuronidase for -* min at -1῏; Rats were intragastrically administered a mixture containing if any of the samples contained a conjugate-form of the .* mg each of genistein, daidzein and bisphenol A. Rats test compounds, they were enzymatically hydrolyzed. were killed 0 h later and the rat serum (A) or each tissue The estrogenic compounds in these samples were extra- homogenate (B) was applied to HPLC after ,0.1 kU/ml of cted three times with ethanol and the residues after evap- b-glucuronidase treatment and extraction. The data shown oration were resolved in mobile solution. Each tissue are mean values from four test rats. These compounds was homogenized in physiological saline at *., g/ml on were not found in the renal, epididymal and brown adipose tissues. MLN, mesenteric lymph node. ice, and +/* ml of the homogenate was resolved in mobile solution after the enzyme treatment and evaporation. The compounds were separated by HPLC using an SCL-+* A system controller (Shimadzu, Kyoto, Japan) equipped from four di#erent individuals. with two LC-+*AD pumps (Shimadzu) and a Mightysil RP- +2 (L) GP (particle size / mm, +/*῎..0 mm, Kanto Chemical) Results column in a CTO-+*Asup column oven (Shimadzu) at -*῏. Estrogenic compound levels in various tissues Levels of A gradient elution, set at a flow rate of *.0 mL/min, was estrogenic compounds as total-amount values (free plus used to separate each compound. The mobile solution conjugate form) were assessed in various tissues 0 h after consisted of two eluents: solution A (an 2*: ,* mixture of intragastric administration (Fig. +). Genistein, daidzein, a /* mM sodium acetate bu#er (pH ..2) and methanol) and bisphenol A and equol levels were detectable in the serum B(a.*: .*: ,* mixture of a /* mM sodium acetate bu#er at +++2, ++.-, -// and 0. ng/ml, respectively (Fig. +A). (pH ..2), methanol and acetonitrile). The initial condi- Each tissue contained various amounts of each estrogenic tions were ,*ῌ of solution B for +* min, a linear increase compound; indeed, each tissue demonstrated a specific to +**ῌ B over ,/ min, and a +* minholdat+**ῌ B. A /* ml preference for a compound (Fig. +B). The liver, kidney portion of each sample was subjected to HPLC. Using an and mesenteric lymph node (MLN), particularly, con- eight-channel coulometric electrode array detector (model tained over ,*** ng/g total, and the spleen contained the /0** Coul Array, ESA, Chelmsford, MA, USA), these es- least. The ratio of each estrogenic compound was shown trogenic compounds were simultaneously measured. De- to be tissue-dependent and did not correspond to the tection of each compound was performed using peak composition of the administered mixture. Genistein and height at 0** mV and the electrode oxidative potentials daidzein were detectable in all tested tissues, but ranged from -/* to 1** mV with /* mV increments. De- bisphenol A was detectable only in the serum, liver, tection limits of genistein, daidzein, bisphenol A and kidney, MLN and epididymis. Equol was detectable in equol were -*, +/, +1 and +/ ng/ml in the serum, and +.2, only the serum and liver at this time point. These es- 1/, 2. and 11 ng/g in the tissues, respectively. Data was trogenic compounds were not detected in the epididymal, represented as mean values or mean῍standard deviation renal and interscapular brown adipose tissues. Tissue-distribution of Isoflavones and Bisphenol A 189

Fig. ,. Time-dependent changes in isoflavone, bisphenol A and equol levels in rat drug metabolic-excretive tissues after intragastric administration. Data are shown as meansῌSD (n῍.) of total-amount (filled) or free-form (unfilled) for each compound. Rats were intragastrically administered a mixture containing genistein, daidzein and bisphenol A. Rats were killed at *, 0, +, or ,. h after administration, and the serum or tissue homogenate treated with or without b-glucuronidase was applied to HPLC.

Estrogenic compound levels in the drug metabolic- detected in the liver and kidney at * h, and decreased at 0 excretive tissues In the serum, liver, and kidney, which to +, h post administration. In the liver, the free-form are representative drug metabolic-excretive tissues, time- amounts of genistein and daidzein were found to be .3 dependent changes in the level of each estrogenic com- and 0/ῌ of the total, respectively. Likewise, bisphenol A pound were determined after administration of the mix- amounted to ,*ῌ of the total-amount at 0 to ,. h. In the ture (Fig. ,). Levels were analyzed as total-amount or kidney, the free-form of genistein and daidzein amounted free-form amount. In the serum, maximum levels of the to .* and -0ῌ of the total-amount, respectively. total-amount of genistein and daidzein were found after Estrogenic compound levels in immune tissues In the the first 0 h, but bisphenol A reached the highest level at MLN, thymus and spleen, which represent the immune +, h and this level was sustained until ,. h post adminis- tissues, time-dependent changes in the level of each es- tration. Equol was detected at * h; the level then drop- trogenic compound were determined after administration ped and increased again until the initial level was reached (Fig. -). In the MLN, the maximum level for the total- at ,. h. Except for equol, serum amounts of the free-form amount of genistein and daidzein was found after the first for each compound represented less than 2ῌ of the total- 0 h. The free-form amounts represented 12 and 1.ῌ of amount at each time point. In the liver and kidney, the the total-amount at each time point between 0 and ,. h. maximum level of the total-amount for genistein and The highest level of the total-amount of bisphenol A was daidzein was demonstrated at the first 0 h, but bisphenol found at 0 h and sustained until +, h. The free-form of A showed its highest level around +, hr. Equol was bisphenol A amounted to 0+ῌ of the total-amount, and 190 S. YASUDA et al.

Fig. -. Time-dependent changes in isoflavone, bisphenol A and equol levels in the immune tissues of rats after intragastric administration. Data are shown as meansῌSD (n῍.) of total-amount (filled) or free-form (unfilled) of each compound. Rats were intragastrically administered a mixture containing genistein, daidzein and bisphenol A. Rats were killed at *, 0, +, or ,. h after administration, and the serum or tissue homogenate treated with or without b-glucuronidase was applied to HPLC. MLN, mesenteric lymph node.

this level was sustained from 0 to ,. h. Equol was total-amount at each time point between * and ,. hinthe detected only at ,. h post administration, and 1/ῌ was in testicle, and about 1,ῌ and /.ῌ in the epididymis, respec- the free-form. In the thymus, the maximum total-amount tively. In the epididymis, total-amount levels of bisphenol of genistein and daidzein was found at the first 0 or +, h, A were temporarily detected at 0 to +, h, wholly in the with 2/ and 03ῌ, respectively, in the free-form. In the conjugated form. In the spermatheca and prostate, the spleen, the maximum level of the total-amount of maximum levels of the total-amounts of genistein and genistein and daidzein was found at the first 0 h, with 21 daidzein were found to be at the first 0 h. The free-form and 1.ῌ, respectively, in the free-form. of genistein and daidzein amounted to 12 and /2ῌ of the Estrogenic compound levels in the reproductive tissues In total-amount in the spermatheca, and 1. and /3ῌ in the the testicle, epididymis, spermatheca, and prostate, which prostate, respectively. represent the reproductive tissues, time-dependent changes in the level of each estrogenic compound were deter- Discussion mined after administration (Fig. .). In the testicle and Soybean isoflavones, bisphenol A and equol have been epididymis, the total-amount of genistein was initially recognized as estrogenic compounds. It has been detected at * h, and the maximum level was found at the revealed that soybean isoflavones in foodstu#s and first 0 h. The maximum level of the total-amount of bisphenol A from chemical products can be absorbed into daidzein was found to be at the first 0 h. The free-form of our bodies and exert a biological e#ect similar to endoge- genistein and daidzein amounted to 00 and 0.ῌ of the nous estrogen. However, it is not entirely clear how Tissue-distribution of Isoflavones and Bisphenol A 191

Fig. .. Time-dependent changes in isoflavone, bisphenol A and equol levels in rat reproductive tissues after intragastric administration. Data are shown as meansῌSD (n῍.) of total-amount (filled) or free-form (unfilled) of each compound. Rats were intragastrically administered a mixture containing genistein, daidzein and bisphenol A. Rats were killed at *, 0, +, or ,. h after administration, and the serum or tissue homogenate treated with or without b-glucuronidase was applied to HPLC. these compounds are metabolized or where they act. In that these compounds mainly exist as glucuronide or this study, we tried to determine the localization of these sulfate conjugates in the human body (Setchell, +332; compounds, including a metabolite, equol, in their target Thomas et al., ,**+; Ikezuki et al., ,**,). The estrogenic tissues. In a preliminary feeding experiment, we noticed compound levels detected in this study were evaluated as that administration of an isoflavone compound at ,- mg/ total-amount (free plus conjugate-form) or free-form body/day for three weeks resulted in levels too low to be amount in samples with and without b-glucuronidase detected in a variety of tissues. Therefore, the mixture treatment. administered to rats in this study contained .* mg each of Total levels of each administered compound detected in genistein, daidzein and bisphenol A. It has been reported the serum or tissue homogenates revealed that they had 192 S. YASUDA et al. di#erent tissue-specific distributions (Fig. +). Equol was late in these tissues due to intake of commercial pellets. also detected in the serum and liver, although it was not Indeed, the commercially available pellet we used con- administered. The total-amount level of each estrogenic tained 3/ mg/g of genistein and /3 mg/g of daidzein. compound taken in summation was found to be highest in Estrogenic compounds have been shown to have preven- the liver and kidney and lowest in the spleen at this time tive e#ects on prostate carcinoma cells (Rice et al., ,**,), point. Also, no compound was detected in the adipose and they have been detected in human prostatic fluid tissues. It has been suggested that there are specific (Morton et al., +331) and in the rat prostate (Chang et al., absorption, metabolism and excretion rates for each ,***). These reproductive tissues may be target tissues isoflavone (Piskula, ,***; Munro et al., ,**-) and bisphenol where the estrogenic compounds can function. A (Zalko et al., ,**-; Domoradzki et al., ,**.). The data In conclusion, the data presented in this report repre- obtained suggested that each compound has a tissue- sent a systematic tissue distribution for estrogenic com- specific a$nity. Furthermore, the tissues tested were pounds including genistein, daidzein, bisphenol A and split into three classes: drug metabolic-excretive, immune, equol after oral administration. More work is warranted and reproductive. Each estrogenic compound was evalu- in order to fully elucidate not only the distribution but ated as the total-amount and free-form amount in tissue also the action mechanism of the potential role of these samples at *, 0, +,,and,. h after administration. compounds. These data may be useful in focusing fur- Estrogenic compound levels were examined in the ther research on target tissues where estrogenic com- serum, liver, and kidney (Fig. ,). In rodent and human pounds can act or be metabolized. serum, isoflavones are known to exist mainly (up to 3*ῌ) in their glucuronide-conjugate forms, and some of them Acknowledgments The authors are indebted to Dr. Masao are aglycons (Holder et al., +333). The data obtained Yamasaki for critical reading of the manuscript. We thank Dr. showed that most genistein, daidzein, bisphenol A and Perry Seto for proofreading the manuscript. equol exists in conjugate-form in the serum. Drug metabolism and detoxification mainly takes place in the References Ashby, J. and Tinwell, H. (+332). Uterotropic activity of bisphenol liver and kidney. It has been reported that isoflavones A in the immature rat. Environ. Health Perspect., +*0, 1+3ῌ1,*. are conjugated by liver-specific glucuronosyltransferase Brotons, J., Olea-Serrano, M., Villalobos, M., Pedraza, V. and Olea, (Doerge et al., ,***) and sulfotransferase (Pai et al, ,**+), N. (+33/). Xenoestrogens released from lacquer coating in food and that glucuronosyltransferase is also highly expressed cans. Environ. Health Perspect., +*-, 0*2ῌ0+,. in kidney cells (Yokota et al., +331). Urinary isoflavones, Brouwers, E., L’ homme, R., Al-Maharik, N., Lapcik, O., Hampl, R., equol and bisphenol A have already been reported to Wahala, K., Mikola, H. and Adlercreutz, H. 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