Glycosylation of bisphenol A by freshwater microalgae

Glycosylation of bisphenol A by freshwater microalgae

Nobuyoshi Nakajima a,*,Tetsuya Teramoto b ,Fumie Kasai a ,Tomoharu Sano c ,Masanori Tamaoki a ,Mitsuko Aono a ,Akihiro Kubo a ,Hiroshi Kamada b ,

Yoshitaka Azumi d ,Hikaru Saji a

a

Environmental Biology Division,National Institute for Environmental Studies,Onogawa 16-2,Tsukuba,Ibaraki 305-8506,Japan

b

Graduate School of Biosystem Science,University of Tsukuba,Japan

c

Laboratory for Intellectual Fundamentals for Environmental Studies,National Institute for Environmental Studies,Japan

d

School of Science,Kanagawa University,Japan

Received 22July 2006;received in revised form 23May 2007;accepted 26May 2007

Available online 16July 2007

Abstract

The endocrine disruptor bisphenol A (BPA,4,40-isopropylidenediphenol)is used to manufacture polycarbonate plastic and epoxy resin linings of food and beverage cans,and the residues from these products are then sometimes discharged into rivers and lakes in waste leachates.However,the fate of BPA in the environment has not yet been thoroughly elucidated.Considering the e?ect of BPA on aquatic organisms,it is important that we estimate the concentration of BPA and its metabolites in the aquatic environment,but there are few data on the metabolites of BPA.Here,we focused on freshwater microalgae as organisms that contribute to the biodegradation or bio-transformation of BPA in aquatic environments.When we added BPA to cultures of eight species of freshwater microalgae,a reduction in the concentration of BPA in the culture medium was observed in all cultures.

BPA was metabolized to BPA glycosides by Pseudokirchneriella subcapitata ,Scenedesmus acutus ,Scenedesmus quadricauda ,and Coelastrum reticulatum ,and these metabolites were then released into the culture medium.The metabolite from P.subcapitata ,S.acutus ,and C.reticulatum was identi?ed by FAB-MS and 1H-NMR as bisphenol A-mono-O -b -D -glucopyranoside (BPAGlc),and another metabolite,from S.quadricauda ,was identi?ed as bisphenol A-mono-O -b -D -galactopyranoside (BPAGal).These results demonstrate that freshwater microalgae that inhabit universal environments can metabolize BPA to its glycosides.Because BPA glycosides accumu-late in plants and algae,and may be digested to BPA by b -glycosidase in animal intestines,more attention should be given to levels of BPA glycosides in the environment to estimate the ecological impact of discharged BPA.ó2007Elsevier Ltd.All rights reserved.

Keywords:Metabolite;Glucoside;Galactoside;Endocrine disruptors

1.Introduction

We are exposed to numerous chemical substances,some of which are called endocrine disrupting compounds (EDCs).EDCs are often manufactured as components of insecticides,herbicides,and plastics and then discharged to the environment.They are detected in surface water,ground water supplies or sewage e?uent worldwide (Sta-ples et al.,1998;Bolz et al.,2001;Yamamoto et al.,2001;

Kolpin et al.,2002;Petrovic et al.,2004).Exogenous appli-cation of EDCs interferes with normal hormone activities by acting as agonist or antagonists (Sonnenschein and Soto,1998).Exposure to EDCs may result in adverse e?ects on reproduction and development in wildlife (Guil-lette et al.,1995;Fry,1995;Baatrup and Junge,2001;Hill et al.,2002;Stoker et al.,2003;Lee et al.,2006).

Bisphenol A [BPA,2,2-(4,4-dihydroxydiphenyl)pro-pane],one of the most commonly produced EDCs,has been used as a material in polycarbonate plastic and in epoxy resin linings of food and beverage cans (Staples et al.,1998).Global production of BPA was more than

0045-6535/$-see front matter ó2007Elsevier Ltd.All rights reserved.doi:10.1016/j.chemosphere.2007.05.088

*

Corresponding author.Tel./fax:+81298502490.E-mail address:naka-320@nies.go.jp (N.Nakajima).

https://www.360docs.net/doc/ba2884817.html,/locate/chemosphere

Chemosphere 69(2007)

934–941

2214000tons in2003(Burridge,2003).It has been demon-strated that BPA causes apoptotic cell death in swordtail (Xiphophorus helleri)testes(Kwak et al.,2001)and increased mortality of medaka(Oryzias latipes)eggs(Shi-oda and Wakabayashi,2000).It also a?ected growth of the aquatic midge(Hahn et al.,2002;Watts et al.,2003; Lee et al.,2006),altered the development of the reproduc-tive organs of caiman(Caiman latirostris)and induced abnormal development of Xenopus laevis and freshwater sponges(Hill et al.,2002;Stoker et al.,2003;Sone et al., 2004).Considering the e?ect of BPA on wildlife,it is important that we estimate the concentrations of BPA and its metabolites in the aquatic environment.Since BPA has been observed in waste land?ll leachates,rivers, seas,and soils(Staples et al.,1998;Bolz et al.,2001; Yamamoto et al.,2001;Kolpin et al.,2002;Yamauchi et al.,2003)much attention was given on biodegradation and biotransformation of BPA.In case of bacteria and basidiomycetes,BPA is broken down to4-hydroxybenzoic acid and4-hydroxyacetophenone by oxidative cleavage reaction catalyzed by speci?c peroxidase or dioxygenase. (Spivack et al.,1994;Hirano et al.,2000;Ike et al.,2000; Kang and Kondo,2002;Kang et al.,2004;Sasaki et al., 2005;Zhang et al.,2007).It has been reported that animal cells and plant cells could transform BPA to its glucuronide and glycoside,respectively(Kanank and Sullivan,1966; Nakajima et al.,2002,2004;Noureddin et al.,2004).In addition,plant cells are able to oxidize BPA to its hydroxy-lated form and cleave to monophenols.(Hamada et al., 2002;Chai et al.,2003).

Microalgae,as a base of the food chain are responsible for nearly a half of global net primary production(Field et al.,2007).While the actions of not only bacteria but also microalgae should be considered in the fate of BPA in the aquatic environment,literatures that described about metabolism or biotransformation of BPA by microalgae are limited.(Hirooka et al.,2005;Peng et al.,2006).Here, we identify metabolites of BPA by4species of microalgae that are universal inhabitants of freshwater bodies such as rivers and lakes.

2.Materials and methods

2.1.Chemicals and microalgae

BPA(99.0%)was obtained from Tokyo Kasei Kogyo (Tokyo,Japan).A stock solution of BPA(1mg mlà1) was prepared in40%(v/v)dimethyl sulfoxide.Eight species of microalgae,Pseudokirchneriella subcapitata(NIES-35), Scenedesmus acutus(NIES-94),Scenedesmus quadricauda (NIES-96),Micractinium pusillum(NIES-151),Coelastrum reticulatum(NIES-245),Carteria cerasiformis(NIES-424), Gonium pectorale(NIES-468),and Cyanophora paradoxa (NIES-763),were purchased from the Microbial Culture Collection at the National Institute for Environmental Studies(MCC-NIES),Tsukuba,Japan.2.2.Culture conditions

Microalgae were grown in500-ml?asks,each contain-ing300ml of the liquid medium recommended by MCC-NIES(Kasai et al.,2004).Media inoculated with algae were cultivated in a temperature-controlled room at 25°C,with continuous illumination at a photon?ux den-sity of40l mol mà2sà1(white?uorescent tubes;FL 40SW,Osram,Yokohama,Japan).Under these condi-tions,most of the algal cultures reached the late log phase or the stationary phase in20–30days.

2.3.Measurement of dry weights of microalgae

Samples with di?erent cell concentrations were pre-pared.After measurement of the optical densities(OD) of the cultures by a spectrophotometer at a wavelength of680nm(DU640,Beckman Coulter,Inc,Fullerton, CA,USA),the cells were collected on a glass micro-?ber ?lter(GF/C47-mm?,Whatman,Middlesex,UK).The?l-ters were dried at70°C for24h,and the dry weight of the algae was determined with a digital balance.The linear regression between dry weight(mg mlà1),de?ned as‘‘y’’, and OD680were obtained as the following expressions and coe?cients of correlation(r2):P.subcapitata,y= 0.345·OD680à0.0047(r2=0.9996);S.acutus,y=0.4·OD680à0.0076(r2=0.9998);S.quadricauda,y= 0.8255·OD680à0.0185(r2=0.9967);M.pusillum,y= 0.4503·OD680à0.0094(r2=0.9978); C.reticulatum, y=0.5607·OD680à0.0027(r2=0.9991);C.cerasiformis, y=0.9103·OD680à0.0102(r2=0.9935);G.pecto-rale,y=0.4485·OD680à0.0067(r2=0.9982);and C. paradoxa,y=0.459·OD680à0.0123(r2=0.9981).

2.4.Growth inhibition tests

Growth inhibition tests were performed in test tubes with screw caps(16·150mm);each tube contained5ml of algal culture in the late-log phase.After BPA had been added to each culture at2,5and10mg là1,the samples were cultivated for8days.For the control,the same vol-ume of40%dimethyl sulfoxide was added to the culture. At each sampling day(0,1,2,4,and8),inhibition of algal growth was evaluated from the dry weight calculated as described above.

2.5.Analysis of BPA in algal cultures

Two milligrams per liter of BPA was added to each algal culture in the late-log phase,and culture was then contin-ued for8days.For the control,liquid medium without inoculation was supplemented with the same concentration of BPA.The reduction of BPA in each culture was ana-lyzed by HPLC at0,12,4,and8days.Aliquots(300l l) of culture were centrifuged(14338g,4°C,10min)to remove the cells and insoluble materials.Fifty microliters of the supernatant was then injected into the HPLC system

N.Nakajima et al./Chemosphere69(2007)934–941935

equipped with UV–VIS detector(degasser,DGU-12A; pump,LC-10ATVP;detector,SPD-10AVP;column oven, CTO-10AVP;recorder,C-R8A;Shimadzu,Kyoto,Japan). The mobile phase was50%methanol(methanol/distilled water,v/v)with a?ow rate at1ml minà1,the column was a C-18(150·3.9mm;Symmetry,Nihon Waters, Tokyo,Japan),and the oven temperature was40°C.The absorbance at a wavelength of217nm was monitored.

2.6.Measurement of radiolabeled BPA in algal cells

Algal cultures were exposed to2mg là1of BPA con-taining7.4kBq of propyl-2[14C]bisphenol A(Moravek Biochemicals,Brea,CA,USA).On each sampling day (0,2,4,and8),aliquots(300l l)of algal cultures were centrifuged(14338g,4°C,30s)and then10l l of the supernatant was mixed with3ml of Aquasol-2(Perkin Elmer Japan,Yokohama,Japan).To measure the radio-activity in the cells,aliquots of the cultures were?ltered on0.45-l m membranes(MF-Millipore membrane?lter ?25mm,Nihon Millipore,Tokyo,Japan)and washed with culture medium;then the radioactivities of the?lters as well as the supernatants were determined by liquid scintillation counter(Tri-Carb,Packard,Groningen, Netherlands).

2.7.Experiments on BPA disappearance in algal cultures

Cells were cultured in test tubes with screw caps (16·150mm),and then5ml of algal culture in the station-ary phase was exposed to10mg là1of BPA.These subcul-tures were kept for10days in a temperature-controlled room at25°C with continuous illumination at a photon ?ux density of4l mol mà2sà1(white?uorescent tubes; FL40SW,Osram,Yokohama,Japan).For the control, liquid medium without algal cells was supplemented with 10mg là1of BPA.The concentration of BPA in each cul-ture was analyzed by HPLC at0,4,7,and10days.Ali-quots(600l l)of culture were centrifuged(14338g,4°C, 10min),and then50l l of each supernatant was analyzed by HPLC system as described above.

2.8.Isolation and identi?cation of metabolites of BPA by microalgae

The algal cultures were exposed to10mg là1of BPA for 10days,and the cultures were concentrated and then se-parated by HPLC as described above.The peaks of the metabolites were collected and concentrated by evapora-tion,and then the molecular weights of the metabolites were analyzed by FAB-MS with a glycerol matrix(JMS-700GC-MS,JEOL,Tokyo,Japan).The metabolites were dissolved in deuterium oxide(D2O)and their structure analyzed by1H-NMR(JNM-ALPHA500,JEOL,Tokyo, Japan).The1H-NMR signals were annotated with the sig-nals of4-nitrophenyl-b-D-galactopyranoside and4-nitro-phenyl-b-D-mannopyranoside.

In order to identify hexose moiety,a few micrograms of the metabolite by S.quadricauda were digested for2h using the following enzyme solutions(100l l).(1)a-gluco-sidase(2mU),potassium acetate(57mM,pH6.8,37°C);

(2)b-glucosidase(2mU),sodium acetate(80mM,pH 5.0,37°C);(3)a-galactosidase(2mU),potassium phos-phate(80mM,pH 6.5,25°C);(4)b-galactosidase (2mU),sodium phosphate(80mM,pH7.3,37°C),MgCl2 (30mM),2-mercaptoethanol(45mM);(5)a-mannosidase (2mU),sodium citrate(30mM,pH4.5,37°C);(6)b-man-nosidase(2mU),sodium acetate(10mM,pH4.0,25°C). All enzymes were purchased from Sigma(Saint Louis, MO,USA).Each reaction product was then analyzed by HPLC as described above,with50%aqueous methanol as a solvent.

3.Results

3.1.E?ects of BPA on growth of microalgae

BPA at up to5mg là1had no e?ect on the algal growth. BPA at10mg là1inhibited the growth of P.subcapitata but did not a?ect the other species(data not shown).Therefore, we chose2–5mg là1for the subsequent experiments.

3.2.Reduction in BPA concentration by microalgae

Substantial reductions in the BPA concentrations in the culture media were observed after8days’incubation with the algae(Fig.1a and b).The degree of reduction on a dry weight basis after8days were11,16,5,3,5,7,1, and2l g(mg dry weight)à1for P.subcapitata,S.acutus, S.quadricauda,M.pusillum,C.reticulatum,C.cerasifor-mis,G.pectorale,and C.paradoxa,respectively(Fig.2). In the absence of cells there was no reduction(data not shown).To determine whether the reduction in BPA con-centration was merely a consequence of its accumulation in the algal cells,radiolabeled BPA was added to the cul-tures and the radioactivity in the cells was measured. BPA accumulated to levels of2,0.5,1,1,1,2,1and3l g (mg dry weight)à1in the cells of P.subcapitata,S.acutus, S.quadricauda,M.pusillum,C.reticulatum,C.cerasifor-mis,G.pectorale,and C.paradoxa,respectively(Fig.2). In the cells of P.subcapitata,S.acutus,S.quadricauda, M.pusillum,C.reticulatum,and C.cerasiformis the degree of reduction in the level of BPA in the culture was higher than the degree of accumulation in the cells.These results suggest that these species metabolize BPA and release the metabolites into the media.We then tried to con?rm the release of metabolites by HPLC analysis.

3.3.Isolation and identi?cation of metabolites

by microalgae

We exposed algal culture to10mg là1of BPA and the metabolites released from the algal cells into the culture media were analyzed by HPLC after10days.Some peaks

936N.Nakajima et al./Chemosphere69(2007)934–941

with retention times di?erent from those of BPA were detected in their cultures.In cultures of these species,with the exception of M.pusillum ,some metabolites were detected by HPLC analysis.A major metabolite in the C.cerasiformis culture was detected at approximately 2min of retention time by HPLC analysis,but this metabolite could not be identi?ed (data not shown).Whereas the retention time of the major metabolites in the cultures of P.subcapitata ,S.acutus ,and C.reticulatum was approxi-mately 4.0min,that in the S.quadricauda culture was approximately 3.8min (Fig.3).The major peak obviously increased with the concomitant decrease in BPA concentra-tion (Fig.3).These metabolites were isolated by repeated HPLC.The metabolites in the P.subcapitata ,S.acutus ,

S.quadricauda ,and C.reticulatum cultures were analyzed

by FAB-MS,and their molecular weights were determined to be 390.This corresponded to that of the BPA glycoside reported by Nakajima et al.(2002).To determine the struc-tures of the metabolites,they were analyzed by 1H-NMR.The NMR spectrum of the metabolites in the cultures of P.subcapitata ,S.acutus ,and C.reticulatum corresponded to that of BPA-mono-O -b -D -glucopyranoside (Nakajima et al.,2002)(Fig.4a).On the other hand,the signals (d 3.82–d 4.99)from the metabolite in the S.quadricauda culture were clearly di?erent from those of b -D -glucopyr-anoside.We named this metabolite ‘X’(Fig.4b).

3.4.Identi?cation of hexose in the metabolite in the S.quadricauda culture

To estimate the structure of the hexose moiety,X was treated with several hexosidases.Both b -galactosidase and b -mannosidase could digest X to BPA,but BPA was not detected when X was treated with the other enzymes (a -glucosidase,b -glucosidase,a -galactosidase,or a -mannosi-dase,data not shown).This indicated that the hexose might be b -D -galactose or b -D -mannose.To further distinguish between these two,the NMR spectrum of X was compared with those of 4-nitrophenyl-b -D -galactopyranoside and 4-nitrophenyl-b -D -mannopyranoside.On the 1H-NMR spec-trum of 4-nitrophenyl-b -D -galactopyranoside,the signal of the anomeric proton appeared as doublet at 5.19ppm (J =7.7Hz),and the signal of 40-H was shifted to lower mag-netic ?eld at 4.03ppm because of equatorial position.In case of the mannopyranoside,anomeric proton signal was shown at 5.48ppm as singlet,and the signal of 20-H was observed at 4.22ppm by shield e?ect (Fig.5a and b).On the NMR spectrum of X.the anomeric proton signal appeared as doublet at 4.99ppm (J =7.3Hz),and the 1H signal which shifted to lower magnetic ?eld was 40-H (3.98ppm,Fig.5c).These results indicated that the hexose of X was b -D -galactose.Therefore,metabolite X was iden-ti?ed as BPA-mono-O -b -D -galactopyranoside (Fig.5c).

concentration of BPA their cultures was HPLC at 0,Vertical bars standard deviations P.subcapitata ;S.acutus ;quadricauda ;;pusillum ;cerasiformis ;pectorale ;and C.paradoxa .

N.Nakajima et al./Chemosphere 69(2007)934–941937

4.Discussion

We studied whether freshwater microalgae contribute to the biodegradation or biotransformation of BPA in aquatic environments.As experimental materials we used eight species universally inhabiting freshwater environments. Reduction in the BPA concentration in cultures of these algae was caused by BPA accumulation in the cells and its transformation to BPA glycosides and unknown metab-olites.In two species(G.pectorale and C.paradoxa)the degrees of BPA reduction in whole culture were only1 and2l g(mg dry weight)à1of algae,respectively,and both of these concentrations roughly corresponded to those of the BPA incorporated into the algal cells(Fig.2).These results suggest that both species merely accumulated BPA.On the other hand,for other species(P.subcapitata, S.acutus,S.quadricauda,M.pusillum,C.reticulatum,and C.cerasiformis)the levels of reduction of BPA were, respectively,5.5,32,5,3,5,and3.5times the levels of BPA incorporated in the algal cells(Fig.2).The radioactiv-

ity in the algal cells was measured at0,2,4,and8days,but

it?uctuated little after2days(data not shown).This result

suggests that the incorporation of radioactivity into algal

cells was saturated at2days,suggesting that these species

not only accumulated BPA but also discharged or metabo-

lized it.Major metabolites from the cultures of P.subcap-

itata,S.acutus,S.quadricauda,and C.reticulatum were

detected at approximately3.8or4.0min by HPLC analysis

(Fig.3);these metabolites were released into the culture

media.These compounds were identi?ed by FAB-MS

and1H-NMR.One was BPA-mono-O-b-D-glucopyrano-

side from the P.subcapitata,S.acutus,and C.reticulatum

cultures(Fig.4a),and the other was BPA-mono-O-b-D-galactopyranoside from the S.quadricauda culture (Fig.5).These results indicate that these algal species can

transform BPA to BPA glycosides.In these species the

degree of reduction in the level of BPA in the culture was

higher than the degree of accumulation in the cells.

938N.Nakajima et al./Chemosphere69(2007)934–941

Biotransformation of BPA to BPA glycosides appears to be responsible for the di?erence between reduction and accu-mulation of BPA in these species.

What is the biological signi?cance of glycosylation of BPA by microalgae?In the plant kingdom,glycosylation may be a detoxi?cation reaction to hydroxylated com-pounds such as phenolic compounds (Pridham,1964).We have found that cultured tobacco cells and seedlings can detoxify BPA by glycosylation (Nakajima et al.,2002).The cultured cells rapidly absorbed BPA,trans-formed it to BPAGlc,and then released the BPAGlc into the culture medium.These successive reactions closely resemble the algal reactions revealed here.We also previously reported that tobacco cells successively meta-bolize BPAGlc to BPA-di-O -b -D -glucopyranoside and BPA-mono-O -b -D -gentiobioside (Nakajima et al.,2004).Although we could not isolate them,substances with the same retention times as these metabolites were

detected

Fig.4.NMR spectra of metabolites of BPA.Chemical structures attributable to the signals are indicated.(a)Spectrum of a metabolite from P.subcapitata ,S.acutus ,and C.reticulatum identi?ed it as BPA-mono-O -b -D -glucopyranoside.(b)Spectrum of a metabolite from S.quadricauda

.

Fig.5.NMR spectra of sugar moiety of 4-nitrophenyl-b -D -galactopyranoside (a);4-nitrophenyl-b -D -mannopyranoside (b);and X (c).Chemical structure of each compound is indicated.

N.Nakajima et al./Chemosphere 69(2007)934–941939

in our algal cultures(data not shown),suggesting that microalgae may also be able to transform BPA glycosides to other glycosides.

Glycosylation of BPA is catalyzed by phenol glucosyl transferase in higher plants(Pridham,1964;Kreuz et al., 1996).This enzyme catalyzes the transfer of glucose moiety from UDP-glucose to the hydroxyl group of phenol com-pounds.Microalgae might also transform BPA to glyco-sides by the action of glycosyl transferase.Because glycosyl transferases utilize sugar nucleotides as their sub-strates,it is easily explained that not only UDP-glucose but also UDP-galactose were used in BPA glycosylation as observed in S.quadricauda.It is well known that UDP-glucose4-epimerase catalyzes mutual transformation between UDP-glucose and UDP-galactose(Leloir,1951). On the other hand,epimerization of glycosylated phenol is scarcely reported.Therefore,we can postulate that the formation of BPAGlc and BPAGal was catalyzed by di?er-ent glycosyl transferases.

How much estrogenic activity do BPA glycosides have? Our previous research demonstrated that estrogenic activ-ity of BPAGlc was one-third that of BPA estimated by ELISA-based estrogen receptor competitive binding assay and was under the detectable level by yeast two-hybrid assay(Morohoshi et al.,2003).Estrogenic activity is depen-dent on binding ability to hormonal receptors.Therefore,it is easy to postulate that BPAGal also have lower estrogenic activity because chemical structure of BPAGal is nearly the same to BPAGlc Our result suggests that microalgae metabolize BPA to non or less-toxic forms.Because many phenolic compounds are synthesized in plants and utilized in residues of amino acids,the cell wall,and pigments,mic-roalgae probably utilize glycosylation for detoxi?cation of phenolic compounds such as BPA.

It should be noted that all the microalgae that could metabolize BPA to glycosides were green algae.This result is interesting from an evolutionary point of view.We pre-viously demonstrated that most tracheophytes have BPA glucosyltransferase activity in their leaves(Nakajima et al.,2005),indicating that this reaction may be conserved in the plant kingdom.On the other hand,in the mammal liver UDP-glucuronic acid is used to conjugate BPA with glucuronic acid to yield BPA-b-glucuronide,which is excreted into the urine(Kanank and Sullivan,1966).These results indicate that another but similar reaction has evolved in the animal kingdom.

Besides glycosylation,what reactions are used to metab-olize BPA in microalgae?In the case of bacteria and basidiomycetes BPA was degraded primarily to4-hydroxy-benzoic acid and4-hydroxyacetophenone by cleavage of bisphenol to monophenols(Spivack et al.,1994;Hirano et al.,2000;Ike et al.,2000;Kang and Kondo,2002;Kang et al.,2004;Sasaki et al.,2005;Zhang et al.,2007).In higher plants,monophenols were also reported as metabolites of BPA in addition to monohydroxyl BPA and BPA-glyco-sides(Hamada et al.,2002;Chai et al.,2003).Oxidative enzymes such as speci?c monooxygenases,dioxygenases or peroxidases are thought to participate in the cleavage reaction.In case of green algae,Hirooka et al.(2005) reported that Chlorella fusca transformed BPA to mono-hydroxyl BPA but they could not detect monophenols in the metabolites.In this study,we isolated BPA-glycosides but could not identify cleavage products of BPA,suggesting that green algae so far investigated may not have BPA-oxidizing enzymes or their expression levels may be too low to degrade su?cient amount of BPA for detection.

Finally,what is the signi?cance of glycosylation of BPA in environmental toxicology?Because the estrogenic activ-ity of BPA is greatly decreased by the biotransformation, the toxicity of BPA is probably mitigated in the aquatic environment.However,BPAGlc accumulates in plant leaves(Nakajima et al.,2002;Noureddin et al.,2004)and may be digested to BPA by b-glucosidase in the animal intestine,suggesting that BPAGlc could be the source of BPA in a bioaccumulated form even if BPAGlc itself is non-toxic.Therefore,attention should be given to the level of BPAGlc in the environment if we are to estimate the eco-logical impact of discharged BPA.

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