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© 2006 American Society for Nutrition J. Nutr. 136:2785-2790, November 2006

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Dietary Genistein Stimulates Anion Secretion Across Female Murine Intestine1

Layla Al-Nakkash2,*, Lane L. Clarke4,5, George E. Rottinghaus6, Yinchieh J. Chen6, Kim Cooper3 and Leona J. Rubin4,7,8

2 Department of Physiology and 3 Department of Biomedical Sciences, Midwestern University, Glendale, AZ 85308; 4 Department of Biomedical Sciences of the College of Veterinary Medicine, 5 Dalton Cardiovascular Research Center, 6 Veterinary Medical Diagnostic Laboratory of the College of Veterinary Medicine, 7 Department of Medical Pharmacology and Physiology of the College of Medicine, and 8 Center for Gender Physiology and Environmental Adaptation, University of Missouri, Columbia, MO

* To whom correspondence should be addressed. E-mail: lalnak{at}midwestern.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Genistein, a naturally occurring isoflavone, augments in vitro epithelial anion transport via activation of the cystic fibrosis transmembrane conductance regulator chloride channel. In this study, we examined whether chronic dietary exposure to 600 mg/kg genistein (600 G) for 1 mo would stimulate anion secretion across wild-type (Wt, normal) murine intestine. Anion secretion was assessed in freshly excised segments of murine jejuna by measuring short circuit current (Isc) and comparing with jejunal segments from mice fed 0 mg/kg genistein (0 G). Basal and forskolin-stimulated anion secretions were augmented (P < 0.05) in female but not in male mice fed 600 G, compared with their counterparts fed 0 G. Serum genistein concentrations were greater in both female and male mice fed 600 G (~3.5–6.9 µmol/L) than those fed 0 G (~100 nmol/L). Anion substitution experiments and bumetanide-sensitivity demonstrated that chloride was the major anion mediating the increased secretion. A smaller bicarbonate component was not augmented by consumption of the genistein diet. These data indicate that chronic exposure to dietary genistein stimulates a sex-dependent increase in basal and forskolin-stimulated chloride secretion across murine intestine.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Genistein, a naturally occurring isoflavonic phytoestrogen, is found in high concentrations in soy products, legumes, and grains (1). Epidemiologic studies indicate multiple health benefits for humans consuming soy-based foods, including reduced risk of cancers (2–5), reduced incidence of coronary artery disease (6,7), reduced cholesterol levels (8), and reduced blood pressure (9). The cellular targets and mechanisms underlying the multiple health benefits of soy products are unclear.

Additionally, in cell culture systems, we and others have shown that genistein stimulates the epithelial chloride channel, wild-type (Wt)9 cystic fibrosis transmembrane conductance regulator protein (CFTR) (10). More recent studies have shown that genistein increases the open probability of the most common cystic fibrosis (CF) disease-associated mutation, {Delta}F508-CFTR, to levels comparable to those seen in Wt-CFTR (11,12). These data suggest that genistein may have therapeutic potential for treating CF. Moreover, the effectiveness of genistein to improve {Delta}F508-CFTR in in vitro cell systems with a 50% effective concentration of 5 µmol/L (13,14) is within the physiological range attainable by dietary modifications. Genistein is readily absorbed across the intestines and can readily reach micromolar concentrations in serum (15). Intestinal whole-animal absorption of genistein was evaluated in rats (16), indicating that genistein is highly bioavailable. Mice consuming 750 mg/L genistein generate plasma genistein concentrations of ~2 µmol/L (17). These levels are comparable to a soy milk diet in humans that also results in plasma genistein concentrations of ~2 µmol/L (18).

Acute bilateral application of genistein to Wt murine distal colon (19) increased short circuit current (Isc), which is indicative of increased anion secretion. Additionally, acute application of genistein increased current and conductance in depolarized colonic mucosa from normal and CF mice (20). The flavonol quercetin also increased chloride (and likely bicarbonate) secretion in rat small and large intestine (21).

To our knowledge, to date there are no studies assessing the effect of chronic dietary genistein on intestinal function in mice. Data presented here describe the effect of chronic (1 mo) dietary supplementation with genistein on small intestine epithelial anion secretion in mice. We hypothesized that chronic exposure to elevated levels of dietary genistein would generate an increase in transepithelial chloride secretion in intestinal epithelia via augmentation of CFTR channel activity.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Mice. Wild type (Wt) C57B1/6J mice (female and male) were purchased from Jackson Laboratory at 7 wk of age and were housed in an animal care facility with 12:12-h light:dark cycle, 5/cage to 2–3 mo of age. Mice consumed food and water ad libitum. Body weight was measured weekly during the diet study, and general health was monitored biweekly. Mice were randomly assigned to a genistein-containing (600 G) or genistein-free (0 G) diet for a period of 4 wk. Mice were killed by asphyxiation using 100% CO2, which was followed by surgical thoracotomy to induce pneumothorax. Animal care and treatments were conducted in accordance with established guidelines and all protocols were approved by the Research and Animal Care and Use Committee of Midwestern University.

    Diets. Casein-based diets were prepared by Dr. R. S. MacDonald (Department of Nutrition, Iowa State University) and contained either 600 G or 0 G (Table 1). Importantly, diets contained equivalent amounts of protein (20.3 g), carbohydrate (66 g), and fat (5 g) and had an estimated energy content of 16.28 kJ/g. Diets were formulated as a powder and mice were allowed to eat ad libitum. Although food intake was not monitored, all mice in this study continued to gain weight.


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  		TABLE 1 Composition of experimental diets

 
    Serum genistein and estradiol. At the time the mice were killed, blood samples were obtained by heart puncture, serum was separated by centrifugation (2000 x g; 20 min), and samples were stored at –80°C. Serum samples were analyzed for genistein concentration by HPLC, using a modification of the methodology by Franke et al. (22). Values are means of duplicate serum samples. Serum samples were analyzed for estradiol concentrations by radioimmunoassay as described previously (23,24). 125I-labeled estradiol and estradiol antibody were obtained from ICN Biochemicals. Samples were run in a single assay following extraction with ethyl acetate:chloroform (80:20, Fisher Scientific). Sensitivity for the assays was 0.5 pg for estradiol and the intra- and interassay CV were ~3 and 11%, respectively.

    Bioelectric measurement of intestinal secretion. Via an abdominal incision, ~5 cm of mid-jejunum was removed and placed in ice-cold oxygenated Krebs bicarbonate ringer (KBR). Each mouse yielded 3–4 jejunum pieces, isolated as previously described (19,25–27). Jejunum sections mounted in the Ussing chambers had 0.238 cm2 exposed surface area. Transepithelial Isc (reported as µA/cm2) was measured via an automatic voltage clamp (VCC-600, Physiologic Instruments) and the experimental conditions and methods were previously described (28). Intestinal tissue pieces were constantly maintained in 1 µmol/L indomethacin (to minimize tissue exposure to endogenously generated prostanoids resulting from manipulation and mounting of the tissue) (29). Glucose (10 mmol/L) was added to the serosal KBR bath and mannitol (10 mmol/L) substituted for glucose in the mucosal KBR bath to avoid an inward current due to Na+-coupled glucose transport (28). Once mounted, the serosal side was exposed to tetrodotoxin (0.1 µmol/L) to minimize variations in intrinsic intestine neural tone (30). Intrinsic neural tone limits the absorptive capacity of the murine mucosa, and neural block is denoted by a decrease in Isc.

    Experimental protocols. Tissues were exposed to KBR for 20 min and steady-state basal Isc measured at that time. cAMP-dependent anion secretion was assessed by bilateral application of 10µmol/L forskolin (at 20 min) and steady-state forskolin response was taken at 60 min. Addition of bumetanide (100 µmol/L, serosal), a Na+/K+/2Cl–cotransporter, indicates the chloride secretory component. At the end of each experiment, glucose (10 mmol/L, mucosal) was added to stimulate Na+-coupled glucose transport to assess tissue viability (denoted by >10% increase in Isc). Tissues that failed to respond to glucose within this parameter were discarded. On separate intestinal segments we determined the Na+-coupled glucose transport, as assessed by the phloridzin-sensitive Isc (phloridzin inhibits the Na+-glucose symporter) in the presence of mucosal glucose, with mannitol added to the serosal side to maintain osmolarity. Most experiments were performed in the presence of KBR (chloride/bicarbonate present). To determine the contribution of chloride secretion to the genistein-stimulated basal and forskolin-stimulated Isc, chloride-free KBR (bicarbonate present) was utilized. To test for the presence of a bicarbonate secretory component, tissues were bathed in chloride/bicarbonate-free KBR. KBR contained the following (mmol/L): 115 NaCl, 25 NaHCO3, 5 KCl, 1.2 MgCl2 and 1.2 CaCl2 mmol/L, pH 7.4. Chloride-free KBR contained the following: 0.4 KH2PO4, 2.4 K2HPO4, 115 NaGluconate, 25 NaHCO3, 2.4 hemicalcium, 1.2 MgSO4, and 3 CaSO4, pH 7.4. Chloride-bicarbonate-free KBR contained the following (mmol/L): 115 NaGluconate, 5 KGluconate, 25 NaTES, 3 CaSO4, 2.4 hemicalcium, and 1.2 MgSO4, pH 7.4.

    Chemicals. Forskolin was purchased from Calbiochem and stored as 20 mmol/L stock in dimethyl sulfoxide (DMSO) at –20°C. All other chemicals were obtained from Sigma Chemical.

    Statistics. Values are means ± SEM. Numbers in parentheses represent numbers of tissues used from individual mice. Analysis was computed using a mixed 2-way ANOVA to compare time (repeated measure) and diet (independent measure) or using a mixed 2-way ANOVA pairwise comparison with Bonferroni multiple comparison test.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Physical characteristics of the mice. During the 4-wk study diet, both groups maintained a steady increase in weight. Weight (males and females from both diet groups) at the start of the diet study was 22.43 ± 0.44 g (n = 45) and at the end of the diet study was 25.44 ± 0.56 g (n = 45).

Serum genistein concentration was negligible in female (107.17 ± 19.42 nmol/L, n = 8) and male (104.97 ± 8.59 nmol/L, n = 5) mice fed the 0 G diet. In those fed the 600 G diet, the serum genistein concentration was greater (P < 0.05) in both females (7731.36 ± 3831.74 nmol/L, n = 6; range = 1110.18, 3580.84, 3054.66, 20958.57, 1538.16, 16145.89 nmol/L) and males (3974.77 ± 2141.54 nmol/L, n = 5; range = 6517.19, 737.09, 1231.28, 1057.66, 10330.59 nmol/L). The serum estradiol concentration did not differ between female mice fed the 600 G diet (33.41 ± 6.98 pmol/L, n = 5) and the 0 G diet (40.38 ± 5.14 pmol/L, n = 5).

    Ussing chamber bioelectric measurements. Basal Isc increased in female mice fed 600 G for 4 wk compared with those fed 0 G (P = 0.0021, Fig. 1A). Interestingly, we found that jejuna removed from male mice fed 600 G for 4 wk did not exhibit an increased basal anion secretion (Fig. 1B) and basal Isc was comparable in jejuna removed from male mice fed either 600 G or 0 G. Furthermore, females fed 600 G had a higher basal Isc than males fed 600 G (P = 0.0023, Fig. 1C). Bilateral application of 10 µmol/L forskolin increased Isc in all mice and diet groups (female and male), which peaked and then remained elevated. However, the steady-state forskolin-stimulated Isc was greater in female mice fed 600 G than in those fed 0 G (P = 0.0007, Fig. 1A). In contrast, in male mice, the steady-state forskolin-stimulated Isc was unchanged by the 600 G diet, compared with the 0 G diet (Fig. 1B). Females fed the 600 G diet generated greater forskolin-stimulated Isc than males fed the same diet (P = 0.00084, Fig. 1C).


Figure 1
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  		Figure 1  Basal and cAMP-stimulated Isc in intestine from female (A) and male (B) mice fed 0 G and 600 G diets for 4 wk. Additions: forskolin (10 µmol/L, bilateral); bumetanide (100 µmol/L, serosal); glucose (1 mmol/L, mucosal). Values are means ± SEM, n = 5–11. * Different from corresponding 0 G values, P < 0.05. Basal (left) and forskolin-stimulated Isc (right) in intestines from female and male mice fed the 600 G diet. * Different from males, P < 0.05.

 
Subsequent treatment with bumetanide (100 µmol/L, serosal) decreased the forskolin-stimulated Isc in both diet groups. The bumetanide-sensitive component for female mice fed 600 G (–60.93 ± 9.45 µA/cm2) was greater (P = 0.0012) than that in female mice fed 0 G (–31.32 ± 5.38 µA/cm2), suggesting a dietary genistein-induced chloride secretory component. Na+-coupled glucose transport did not differ between males and females fed the genistein-free diet (61.58 ± 26.57 µA/cm2, n = 7, and 68.25 ± 17.90 µA/cm2, n = 7, respectively). Female mice fed 600 G generated a glucose-stimulated Isc (compared with basal levels, P = 0.004), whereas males did not (Fig. 2).


Figure 2
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  		Figure 2  Glucose-stimulated phloridzin-sensitive Isc in intestine from female and male mice fed the 600 G diet. Additions: glucose (10 mmol/L mucosal); phloridzin (100 µmol/L mucosal). Values are means ± SEM, n = 10 (females) or 3 (males). * Different from 20 min, P < 0.05.

 
Bathing jejuna from female mice fed 600 G in bilateral chloride-free KBR did not change the magnitude of the basal Isc (Fig. 3A), but reduced the steady-state forskolin-stimulated Isc by 61% (P = 0.005). The residual current in chloride-free KBR, which was insensitive to bumetanide (Fig. 3A), was likely due to the secretion of an alternate anion, such as bicarbonate. Basal Isc was further reduced in the chloride/bicarbonate-free KBR (P = 0.045, Fig. 3A). In addition, the steady-state forskolin-stimulated Isc was also reduced (P = 0.0002) in a chloride/bicarbonate-free ringer (an 80% decrease from that obtained in the chloride-containing KBR) in chloride/bicarbonate-free KBR (Fig. 3A).


Figure 3
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  		Figure 3  Effect of bilateral chloride-free and chloride/bicarbonate-free KBR on the basal and forskolin-stimulated Isc in intestine of female mice fed 600 G (A) or 0 G (B) diets. Additions: forskolin (10 µmol/L, bilateral), bumetanide (100 µmol/L, serosal) and glucose (10 mmol/L, mucosal). Values are means ± SEM. In A, chloride-containing KBR, n = 5; chloride-free KBR, n = 5; chloride/bicrabonate-free KBR, n = 6 tissues from 2 mice. In B, chloride-containing KBR, n = 3; chloride-free KBR, n = 3; chloride/bicarbonate-free KBR, n = 8 tissues from 3 mice. # Different from chloride/bicarbonate-free at that time, P < 0.05. * Different from chloride-free at that time, P < 0.05.

 
To better assess whether the genistein in the genistein-containing diet selectively increased chloride secretion in female mice, comparable experiments were performed on jejuna from female mice fed the 0 G diet (Fig. 3B). For those female mice fed 0 G, basal Isc did not differ in chloride-free KBRor chloride/bicarbonate-free KBR when compared with chloride-containing KBR. Chloride removal or chloride/bicarbonate removal also did not change the steady-state forskolin-stimulated Isc in chloride-containing KBR. These data suggest that the increased Isc observed in female mice is due to the 600 G diet.


   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
To our knowledge, this study provides the first evidence that chronic consumption of dietary genistein (600 G) increases both basal and cAMP-stimulated Isc in freshly isolated jejuna segments removed from female mice, but not males. This increased Isc is comprised mainly of chloride secretion based on the following evidence: 1) bumetanide reduced the forskolin-stimulated Isc and this bumetanide-sensitive component was significantly increased in female genistein-fed mice compared with the genistein-free group; 2) female mice fed genistein exhibited increased basal Isc and forskolin-stimulated Isc that was reduced in chloride-free KBR (from our studies it appears that only the chloride secretory component was elevated with the genistein-fed females); and 3) some anion secretion persisted in jejuna removed from female mice even in the presence of chloride-free KBR. Because the small intestine does secrete bicarbonate (31), this remaining anion secretion was likely due to bicarbonate secretion. Removal of both bicarbonate and chloride on Isc in jejuna from female mice generated further reductions in basal and forskolin-stimulated Isc, which is indicative of a bicarbonate component. Accordingly, this bicarbonate component was of a similar magnitude (~40 µA/cm2) in female mice fed either 600 G or 0 G, which suggested that increased dietary genistein does not increase the bicarbonate-mediated anion secretion.

In the normal murine intestine, chloride secretory responses to forskolin have long been detected, and the major route for the chloride exit across the apical membrane is via the CFTR chloride channel (32–34). Nonetheless, other potential pathways exist for chloride to exit across the apical membrane of intestinal epithelia. Recently, apical chloride secretory pathways (ClC-2 and/or ClC-4) were identified (35,36); however, there is little information regarding the effects of genistein on these chloride channels. Based on the large body of evidence in the literature regarding genistein's action in vitro on CFTR (10,13,14), we predicted that at least one pathway mediating genistein's action in the jejuna epithelia would be on the CFTR chloride channel.

Flavonoids (such as genistein) are found naturally in soy and plants and are digested in an average daily diet. Soy-rich diets have been shown to generate micromolar serum genistein concentrations (37). In fact, 1 µmol/L serum genistein concentrations can be obtained in rats consuming a diet containing 750 µg genistein/g (38). More recently, Bhandari et al. (39) showed a relation between dietary genistein and serum levels in mice (1000 mg/kg dietary genistein generated ~1.5 µmol/L serum genistein and 500 mg/kg dietary genistein generated ~0.5 µmol/L serum genistein) after 4 wk of consuming the study diets. In comparison, our female and male mice fed 600 G had serum genistein levels of 7.73 ± 4.038 µmol/L and 4.18 ± 2.25 µmol/L, respectively. Thus, based on research evidence that µmol/L levels of genistein are required to stimulate CFTR channel activity, our data attests that regular continued consumption of genistein has a viable potential to maintain serum levels in a low (<5) µmol/L range. Large variations in serum genistein levels obtained from mice fed 600 G could be due to uncontrolled eating behaviors by the mice (the mice in this study were not fasted prior to experimentation). Furthermore, clearance rates of serum genistein may be different between females and males; in fact, evidence suggests that female mice have greater circulating concentrations than males after consuming identical genistein-rich diets (40,41).

Our observation, that intestinal epithelial anion secretion was increased only in female mice, and not males, following chronic dietary genistein exposure, was intriguing. Several potential mechanisms could account for the sex-dependent difference in genistein's action on intestinal secretion. First, it is presently unknown whether levels of CFTR expression in male and female murine intestinal epithelia are equivalent under normal dietary conditions (less CFTR expression in males vs. females could account for this difference in chloride secretion). It is also unclear whether dietary genistein alters trafficking of CFTR to the epithelial plasma membrane. Such regulation of CFTR trafficking in intact epithelial tissue has been shown in the Spiny Dogfish rectal gland (42) and, more recently, in a cell culture system (43). Future studies are required to examine levels of CFTR expression. Second, genistein is capable of binding to estrogen receptors ER{alpha} and ERß (44,45), both of which are found in the intestine (46,47). Thus, sex-dependent differences in intestinal epithelial function could be predicted if dietary genistein were to act via an ER-mediated pathway, and/or if there were differences in the ER types or numbers present in male and female murine intestine.

Interestingly, Singh et al. (48) reported that 17ß-estradiol caused a rapid and reversible inhibition of forskolin-stimulated chloride secretion across T84 epithelial cell monolayers (Ki = 9 µmol/L). The difference in response by 17ß-estradiol on forskolin-stimulated chloride secretion in T84 monolayers and our observations with genistein could be due to: 1) they used a concentration of forskolin (10 µmol/L) which generally elicits a maximal CFTR-mediated current (49) that can not be further potentiated, 2) it is possible that 17ß-estradiol [like genistein (50)] has a biphasic stimulatory and inhibitory effect, and 3) the cell systems used are different and therefore involve alternative pathways in either system. Additionally, genistein has been shown to upregulate expression of genes through the estrogen response element in an intestinal epithelial cell line (51). Whether dietary genistein modifies gene expression remains to be seen.

Third, in serum pharmacokinetic studies, male and female mice (C57BL6), given either identical subcutaneous administration of genistein (1–5 d) or fed genistein–containing diets (28 d), generated differences in maximal concentrations of total genistein. Females had greater circulating levels than males (40,41). Thus, reduced circulating levels of genistein in males may account for the sex-dependent differences we observed. This may provide some of the mechanistic rationale for the reduced basal and forskolin-stimulated Isc response exhibited by males fed the genistein-rich diet. Lastly, there is evidence to suggest that estradiol activates cAMP-dependent protein kinase activity in rat colon (52). Because genistein is structurally similar to estradiol and binds to estrogen receptors, it is conceivable that genistein could potentially also activate the cAMP-dependent protein kinase pathway, which would thereby augment CFTR activity in the intestinal epithelia.

Although the mechanism(s) underlying the genistein-mediated increased anion secretion in the intestinal epithelium are currently unclear, this study provides, to our knowledge, the first evidence that increased consumption of dietary genistein, a naturally occurring isoflavone, increases both basal and cAMP-stimulated intestinal anion secretion in female mice.


   ACKNOWLEDGMENTS
 
The authors thank Dr. A. W. Jones, Dr. V. Huxley, and Dr. T.-C. Hwang (Department of Medical Pharmacology and Physiology, Center for Gender Research and Environmental Adaptation and Dalton Cardiovascular Research Center) for helpful discussions. In addition, we thank Dr. R. MacDonald (Iowa State University, Department of Nutrition) for formulating the casein-based diets.


   FOOTNOTES
 
1 Funded by the NIH (HL07094, to the Department of Medical Pharmacology and Physiology, University of Missouri-Columbia), a COR research grant from the College of Veterinary Medicine (L.A.) and NASA. Back

9 Abbreviations used: 600 G, 600 mg/kg genistein; 0 G, 0 mg/kg genistein; Isc, short circuit current; CFTR, cystic fibrosis transmembrane conductance regulatory protein; CF, cystic fibrosis; Wt, wild-type; KBR, Krebs bicarbonate ringer. Back

Manuscript received 28 February 2006. Initial review completed 28 March 2006. Revision accepted 22 August 2006.


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 Materials and Methods
 Results
 Discussion
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