Introduction

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

A growing number of industrial chemicals has been reported to act as endocrine disrupters in mammals and other animals (Hoyer, 2001). One of the prominent environmental hormones, bisphenol A³ (2,2-bis[4-hydroxyphenyl]propane), has demonstrated estrogenic activity having adverse effects on the reproductive system (Kim et al., 2001; Chen et al., 2002). Bisphenol A is widely used in the manufacture of epoxy, polycarbonate, and polyester-styrene resins (National Toxicology Program, 1982), and traces of the compound can be easily leached from food containers and tableware made of such plastics and can be taken up by human beings through eating and drinking (Brotons et al., 1995; vom Saal et al., 1998). In vitro, bisphenol A has stimulated cell proliferation as well as the induction of progesterone receptors on MCF-7 human breast cancer cells (Krishnan et al., 1993). In the reproductive tract of female rats, a single high dose of the compound (37.5-150 mg/kg) was reported to induce cell differentiation, and c-fos proto-oncogene expression (Steinmetz et al., 1998). Exposing pregnant CF-1 mice for 7 days to bisphenol A (2.4 mg/kg) evoked early puberty of the female offspring, significantly hastening vaginal opening and onset of estrous (Howdeshell et al., 1999).

Bisphenol A introduced orally into the body must pass through the intestine and liver before arriving at the reproductive organs, where irreversible damage may be inflicted. To elucidate the mechanism governing the detrimental effects on the target organs, it is essential to clarify metabolism and disposition of the compound during its journey through the gastrointestinal tract. Previously, we found that bisphenol A in rat liver is extensively glucuronidated by UGT2B1, an isoform of UDP-glucuronosyltransferase (Yokota et al., 1999). Additional study showed that the compound is glucuronidated in rat perfused liver and is excreted exclusively into the bile (Inoue et al., 2001). In the intestine, however, which provides the foremost barrier against ingested toxicants, behavior of the compound has not been delineated.

In experiments applying 1-naphthol to rat-everted intestine, glucuronidation activity toward the phenolic compound was evident, and the resultant glucuronide was expelled from the enteric mucosal cells into the lumen (Inoue et al., 1999). These observations, together with our hepatic findings to date, led us to conduct the present work using everted intestine to determine the fate of bisphenol A that enters the intestine of the rat.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

Chemicals. Bisphenol A was purchased from Kanto Chemical Co. (Tokyo, Japan); high-performance liquid chromatography (HPLC) grade acetonitrile from Labscan Ltd. (Dublin, Ireland); -glucuronidase (type B-1; from bovine liver) from Sigma-Aldrich (St. Louis, MO). Bisphenol A glucuronide purified from the bile after rat liver perfusion with 7.5 µmole bisphenol A (Inoue et al., 2001) was quantified by HPLC by using the difference between -glucuronidase-treated sample and untreated sample, and was used as a standard.

Animals. Male Sprague-Dawley rats (8-weeks old; 300-340 g) were used in all experiments. The rats were housed under standard conditions and given food and water ad libitum. The animals were handled according to the Laboratory Animal Control Guidelines of Rakuno Gakuen University, which is based on the Guide for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health.

Preparation of Everted Intestine. Krebs Ringer's bicarbonate buffer (135.0 mM Na⁺, 5.0 mM K⁺, 2.5 mM Ca²⁺, 1.2 mM Mg²⁺, 122.4 mM Cl, 25.0 mM HCO³, 10.0 mM glucose) was used in all experiments. The buffer solution was aerated by 95% O₂ + 5% CO₂ and the pH was adjusted to 7.4. The rats were euthanized by decapitation, and the jejunum, ileum, and colon were flushed with cold Krebs Ringer's buffer. The bowels of the animals were excised and prepared according to a modification of the segmentation and eversion method described previously (Inoue et al., 1999). Briefly, with the exception of the duodenum, the excised small intestine was lavaged and divided into four sections of equal length as quickly as possible. The distal portion of each section was excised and trimmed to 10 cm and designated I, II, III, and IV in distal order, with segment I being from the jejunum and segment IV from the distal ileum. In the same manner, the colon was excised, washed, and trimmed to a final segment of 10 cm taken from the distal end.

HPLC Analysis of Reaction Products. The mucosal and serosal samples were filtered by a disposable disk filter (HLC-DISK3) from Kanto Chemical Co. and stored at 80°C until analysis. The samples were analyzed by an HPLC system (Tosoh, Tokyo, Japan) based on the method described previously (Yokota et al., 1999). Briefly, the samples were eluted with a solution of acetonitrile/H₂O/acetic acid (37:63:0.1 v/v/v) on a constant flow rate at 1 ml/min. The eluted samples were analyzed with UV 222-nm detection using TSK-gel ODS-80Ts-reversed phase column (4.6 × 250-mm; Tosoh Tokyo, Japan). The results were recorded with C-R6A integrator from Shimadzu (Tokyo, Japan).

-Glucuronidase Reaction. After being filtered, the various mucosal and serosal buffer solutions were allowed to react with the -glucuronidase (Inoue et al., 2001), and the products were analyzed by HPLC to verify whether the metabolite was the glucuronide. Hardly any sulfatase activity of the -glucuronidase was detected by HPLC under the same conditions as those for -naphthyl sulfate, indicating that the metabolite was in fact the glucuronide.

Statistical Analysis. All data were presented as the mean ± S.E., and the means were compared by use of analysis of variance, with the p value of 0.05 as the level of significance.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

High-Performance Liquid Chromatography of Buffer Solution. Elution profiles obtained by HPLC are shown in Fig. 1. In mucosal buffer solution from the intestinal segments treated with 50 µM bisphenol A, three peaks (numbered as 1, 2, and 3) were observed. With increasing incubation time, the peak eluted at ~5.6 min (peak 1) increased gradually, and the last peak (peak 3) decreased (Fig. 1, A-C). In serosal buffer solution, a modest peak (peak 3) of the substrate appeared at ~19 min (Fig. 1D). In the solution containing -glucuronidase, which cleaves the glucuronide, unconjugated bisphenol A appeared (Fig. 1E; peak 3), signaling that the first peak (peak 1) represented the bisphenol-A glucuronide. An unidentified peak eluted at ~6.8 min (peak 2) did not change dynamically with incubation time.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   HPLC chromatograms of mucosal and serosal buffer solutions derived from rat everted intestine treated with bisphenol A (50 µM). Samples here are from the ileum, segment IV (see Fig. 1). Mucosal buffer solution sampled at 20-min incubation (A), 40 min (B), and 60 min (C); serosal buffer solution at 60-min incubation (D); serosal buffer solution treated with -glucuronidase (E). Peak 1, bisphenol-A glucuronide increased with incubation time; peak 2, unknown peak; and peak 3, unconjugated bisphenol A.

Bisphenol A Absorption and Transport. On each addition of bisphenol A (10, 50, or 100 µM) to the mucosal side, bisphenol A concentration in the mucosal fluid decreased with incubation. As shown in Table 1, the rate of bisphenol A disappearance from the mucosal compartment was notable on a high dose of bisphenol A (100 µM). Although the distal intestine showed the greatest extent of bisphenol A disappearance, the data were not significant among the five segments of the intestine (p > 0.052).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Absorption of unconjugated bisphenol A in the individual serosal fluid of rat everted intestine and colon. The jejunum, ileum, and colon were segmented into lengths of 10 cm each, and bisphenol A was added to the mucosal buffer solution of each segment in concentrations of 10 µM (--), 50 µM (--), and 100 µM (--). Segments I, II, III, and IV are from the jejunum and ileum in distal order.

Bisphenol A Glucuronidation. Bisphenol-A glucuronide expelled into the mucosal side as well as that transported into the serosal side increased with the incubation period (Fig. 3). In contrast to the excretion of the glucuronide to the mucosal side, an ~10-min time lag was observed in the transport of glucuronide into the serosal side, suggesting that the intestinal wall may have interfered with the diffusion of the glucuronide in the everted intestine experimental system. Although both the excretion and transport of the glucuronide increased in relation to the administrative dose of bisphenol A, the amounts appeared to reach plateau with a high dose (100 µM) of the compound.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Glucuronidation of bisphenol A in rat everted intestine. A, the resulting glucuronide excreted to the mucosal side; B, the resulting glucuronide transported to the serosal side. Symbols are the same as those of Fig. 2.

View larger version (44K): [in this window] [in a new window]   Fig. 4.   Bisphenol-A glucuronidation and excretion to the mucosal side and transport to the serosal side within 60-min incubation in relation to the dose applied. Bisphenol A was added to the mucosal buffer solution of each segment in concentrations of 10 µM (), 50 µM (), and 100 µM (). I, II, III, and IV indicate the intestinal sites in distal order from the ligament of Trietz.

Fate of Bisphenol A at 60-min Postapplication. In the small intestine at 60-min postapplication of low-dose bisphenol A (10 µM), the absorbed substrate was almost completely glucuronidated, and the resulting glucuronide was expelled into the mucosal and serosal buffer solutions (Fig. 5). In contrast, the amount of bisphenol A that eluded detection increased on a high dose of bisphenol A (100 µM). The amount remaining unaccounted for (i.e., of unknown fate) was especially prominent in the colon.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Absorption and glucuronidation of bisphenol A at 60-min incubation in rat everted intestine in relation to the dose applied. Concentrations (10, 50, and 100 µM) of bisphenol A added into the mucosal buffer solution are depicted horizontally. Total decrease of mucosal bisphenol A during 60-min incubation is depicted in the sum total of the fractional column in each intestinal segment. Bisphenol A glucuronide secreted to the mucosal side (), bisphenol A glucuronide transported to the serosal side (), bisphenol A transported to serosal side () bisphenol A of unknown fate (). BPA, bisphenol A; BPA-GA, bisphenol A glucuronide; M, secreted into mucosal side; S, transported into serosal side.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

Results of this study affirm that, in the intestine of Sprague-Dawley rats exposed to bisphenol A, 1) most of the compound absorbed by the intestine is glucuronidated within the intestinal wall; 2) the resulting glucuronide is eliminated preferentially into the mucosal side in the small intestine and into the serosal side in the colon; and 3) on a high-dose exposure to bisphenol A, the relative absorption of unconjugated bisphenol A increases dramatically.

Bisphenol A Glucuronidation during Absorption. As suggested by the present results, the proximal intestine is seen as playing a highly protective role against ingested bisphenol A. Bisphenol A in the lumen of the rat intestine was highly glucuronidated during its passage through the intestine, with most of the compound excreted to the mucosal side as glucuronide, which is low in estrogenic activity (Matthews et al., 2001). This was particularly evident for the proximal jejunum, where mucosal excretion of the glucuronide greatly exceeded serosal excretion. Thus, it appears that the proximal jejunum defends the body against potential adverse effects of orally introduced bisphenol A by limiting entry of the free compound into the blood stream and by curtailing exposure to the middle and distal parts of the intestine. In line with these results, low exposure has been reported in association with oral intake of bisphenol A (Pottenger et al., 2000). Comparing the concentration-time profiles for bisphenol A in the blood of F344 rats exposed intraperitoneally and those exposed orally to the compound, Pottenger et al. (2000) found that oral administration results in a low exposure to unconjugated bisphenol A. In the light of our present findings, the diminution of exposure to unconjugated bisphenol A on oral administration may be ascribed to high glucuronidation of the compound in the proximal intestine, which is the foremost barrier to damage from oral administration.

Excretion of the Resulting Glucuronide. Whereas in the intestine the bisphenol A glucuronide was excreted into the mucosal side, the direction of elimination was reversed in the colon, where transport was into the serosal side. Recently, ATP-dependent transporters have been described as mediating the transport of the glucuronide across the cell membrane (Oude Elferink et al., 1995). In rat liver a member of the ATP-binding cassette family, namely, multidrug resistance associated protein (MRP), is reported to be capable of mediating transmembrane excretion of a wide range of amphiphathic compounds, including bilirubin-, estrogen- and xenobiotic-glucuronide (Yamazaki et al., 1996). In the rat intestine, MRP2, localized in the apical domain of the enterocyte, is distributed in the proximal intestine (Mottino et al., 2001) and MRP3, localized in the basolateral domain, is distributed mainly in the ileum and colon (Rost et al., 2002). Intriguingly, the apical and basolateral directions of bisphenol A glucuronide excretion in our study parallels the distribution patterns of MRP2 and MRP3, respectively. Thus the supposition may be made that the elimination direction of bisphenol A glucuronide is governed by the distribution of an organic anion transporter system such as MRP.

Appearance of Serosal Bisphenol A in the Colon. As the lumen was the site into which large amounts of the bisphenol A glucuronide were eliminated in our study, presumably the excreted glucuronide would flow with the luminal contents into the distal intestine. In the colon, most likely the glucuronide would be deconjugated by lumen bacterial -glucuronidase, an enzyme known to generate toxic and carcinogenic substances (Reddy et al., 1992). Deconjugation by lumen bacterial -glucuronidase is known to be involved in the reactivation of an antitumor chemical derived from Irinotecan (Kaneda and Yokokura, 1990). Furthermore, a deglucuronidated Irinotecan derivative (SN-38 glucuronide) is reabsorbed in the distal intestine, where it damages the mucosa (Takasuna et al., 1996). In the light of these reports, the notable absorption and transport of unconjugated bisphenol A to the serosal side in the rat colon in our study suggests that the deconjugated bisphenol A is eventually reabsorbed by the colon. This proposition concurs with Upmeier et al. (2000), who recently, in a toxicokinetic study of bisphenol A in female DA/Han rats, have shown the possibility of enterohepatic recirculation and protracted absorption of bisphenol A from the intestinal tract. These findings bear out that, for ingested bisphenol A, the metabolism and disposition of the compound in the intestinal tract play a pivotal role in mediating the degree of toxic damage by the compound.

	Conclusion

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

Because the intestine absorbs environmental estrogens introduced orally into the body, it is important to trace the fate of such compounds before inflow into the bloodstream. Evidence is accruing that most bisphenol A that is ingested is glucuronidated during its absorption by the small intestine and by its passage through the liver (Inoue et al., 2001). The present study has established that bisphenol A is excreted into the intestinal lumen as a glucuronide, bearing out that the gastrointestinal tract is a strategic pathway against invasion of bisphenol A at target organs such as the gonads and the brain. Further work is warranted to determine the fate of the enteroluminal glucuronide in its complete pathway before excretion.