Amphotericin B enhances the synthesis and release of the immunosuppressive agent gliotoxin from the pulmonary pathogen Aspergillus fumigatus

Jou nal o Medical Mic obiology (2004), 53, 1–7 DOI 10.1099/jmm.0.45626-0 45626 &2004 SGM P in ed in G ea B i ain 1 Co espondence Ke in Ka anagh [email p o ec ed] Recei ed 5 Feb ua y 2004 Accep ed 29 Ap il 2004 Ampho e icin B enhances he syn hesis and elease o he immunosupp essi e agen glio oxin om he pulmona y pa hogen Aspe gillus umiga us Eme P. Ree es, Thomas Mu phy, Paul Daly and Ke in Ka anagh Medical Mycology Uni , Na ional Ins i u e o Cellula Bio echnology, Depa men o Biology, Na ional Uni e si y o I eland Maynoo h, Co. Kilda e, I eland Exposu e o he pulmona y pa hogen Aspe gillus umiga us o ampho e icin B al e s memb ane pe meabili y as indica ed by he escape o amino acids and p o ein om he mycelium. Ampho e icin B exposu e o pe iods o 2–4 h also leads o inc eased elease o he immunosupp essi e agen glio oxin in o he su ounding cul u e medium. Examina ion o he in acellula glio oxin concen a ion ollowing exposu e o ampho e icin B indica ed ele a ed le els wi hin he hyphae as well as in he cul u e medium – an e ec which was also e iden upon exposu e o A. umiga us o DMSO. These esul s indica e ha in pa allel wi h he abili y o ampho e icin B o ac as a ungis a ic agen i can also induce he syn hesis o glio oxin and acili a e i s elease by inc easing he pe meabili y o he ungal cell memb ane. Inc eased syn hesis o glio oxin may esul om he commencemen o seconda y me abolism in he p esence o ampho e icin B. The abili y o ampho e icin B o enhance he syn hesis and elease o glio oxin may exace ba e he e ec s o he oxin and acili a e ungal in asion o pulmona y issue. INTRODUCTION The ungus Aspe gillus umiga us is a pulmona y pa hogen capable o inducing disease in hose wi h p e-exis ing pulmona y mal unc ion (e.g. as hma, cys ic ib osis), disease (e.g. ube culosis, lung cance ) o unde going immuno- supp essi e he apy p io o o gan ansplan a ion (Denning, 1996a; F ase , 1993; Daly & Ka anagh, 2001). Th ee o ms o aspe gillosis a e ecognized clinically: sap o- phy ic, alle gic and in asi e. In asi e aspe gillosis (IA) is he mos se ious o m o disease as i in ol es he in asion o iable issue and may p oduce a mo ali y a e o 80–95 % (Denning, 1996b, 1998). IA has eme ged as an impo an disease in ecen decades due o he use o agg essi e immunosupp essi e he apy causing p olonged neu openia in he ea men o cance and leukaemia (Daly & Ka anagh, 2001). Despi e agg essi e an i- ungal chemo he apy, dea h due o IA usually esul s 7–14 days pos -diagnosis (Denning, 1996b). A. umiga us p oduces a ange o seconda y me aboli es such as glio oxin, hel olic acid and umagillin which may acili a e i s g ow h and pe sis ence in he lung (Ami ani e al., 1995a; Hogan e al., 1996). Ex ac s ob ained om spu um sols o pa ien s wi h aspe gillosis damage human espi a o y epi he- lial cells (Ami ani e al., 1995a). Subsequen analysis con- i med ha glio oxin de i ed om A. umiga us was he oxic agen and ha hel olic acid was also capable o comple e cilios asis and epi helial cell dis up ion (Ami ani e al., 1995b). Glio oxin (C13H14N2O4S2, molecula mass 326.4) is an epipoly hiodioxopipe azine (Wa ing & Bea e , 1996) which displays immunosupp essi e p ope ies in i o (Su on e al., 1994). Glio oxin is capable o inhibi ing mac ophage unc- ion and adhe ence o plas ic su aces (Be ou e al., 2002; Eichne e al., 1986) and may al e he immune esponse o Aspe gillus as i can induce apop o ic cell dea h in mac o- phages (Wa ing, 1990), cells o he spleen (B ai hwai e e al., 1987) and he immune sys em (Su on e al., 1994). The immunosupp essi e p ope ies o glio oxin ha e been e al- ua ed o supp essing he whole body immune esponse p io o o gan ansplan a ion (Su on e al., 1995). Glio oxin is also capable o inducing cell dea h in a mu ine ib oblas ic cell line (Pi a, 1994). Glio oxin has been de ec ed in samples om animals (Richa d & DeBey, 1995; Richa d e al., 1996) and humans (Shah e al., 1995) whe e i may acili a e ungal pe sis ence and coloniza ion o issue. In addi ion, glio oxin has been implica ed in he des uc ion o lung pa enchyma in IA (Su on e al., 1996) and he pene a ion o blood essels in angio-in asi e aspe gillosis (F ase , 1993) Con en ional he apy o he con ol o aspe gillosis elies upon he use o he polyene, ampho e icin B (Ellis, 2002) and he azoles, i aconazole and luconazole (Canu o & Rode o, 2002). Ampho e icin B displays ungis a ic ac i i y and unc ions by o ming ape u es in he cell memb ane, by A icle numbe = 45626 Doc opic is: pa h i Abb e ia ion: IA: In asi e aspe gillosis. complexing he memb ane s e ol, e gos e ol (Abu-Salah, 1996). Each po e consis s o an annulus o eigh ampho- e icin B molecules linked hyd ophobically o e gos e ol. The hyd oxyl esidues o he polyene d ug ace inwa ds o gi e an e ec i e po e diame e o 0.4–1.0 nm. Ampho e icin B– e gos e ol po es allow he exi o in acellula cons i uen s (e.g. ions and small molecula mass compounds) and he en y o hyd ogen ions leading o inc eased cy oplasmic acidi y (Cohen, 1998). While ampho e icin B can induce he o ma ion o ape u es in he ungal cell memb ane, he e ec may no be a al o he cell and ‘ eco e y’ may be possible (Liao e al., 1999). I has been es ablished ha while a speci ic concen a ion o ampho e icin B may inhibi eplica ion, as indica ed by lack o ungal g ow h, speci ic cellula unc ions (e.g. ans-memb ane po en ial, in acellula enzyme ac i i y and memb ane in eg i y) s ill ope a e and, o e ime, he ungus may esume he abili y o g ow and di ide (Liao e al., 1999). An al e na i e explana ion o he mode o ac ion o ampho- e icin B has ecen ly been p oposed and sugges s ha , in addi ion o he o ma ion o po es in he ungal cell memb ane, ampho e icin B also al e s he pe meabili y o he phospholipid bilaye by changing he memb ane phase, i.e. inc easing he luidi y o he bilaye (Venegas e al. 2003). Howe e , he dominan e ec o ampho e icin B appea s o be he o ma ion o po es in he memb ane by complexing e gos e ol. The abili y o ampho e icin B o induce he elease o in acellula cons i uen s in Candida albicans ia memb ane ape u es has been es ablished p e iously (Ghosh & Ghosh, 1963) and aises he possibili y o he elease o low molecula mass oxins om pa hogenic ungi. Consequen ly, he aim o his s udy was o es ablish whe he exposu e o A. umiga us o ampho e icin B could lead o he elease o a low molecula mass oxin such as glio oxin; a p ocess which could ha e he po en ial o exace ba e he immunosupp essi e and in asi e abili ies o A. umiga us. MATERIAL AND METHODS A. umiga us cul u e condi ions. A. umiga us ATCC 26933 (ob- ained om he Ame ican Type Cul u e Collec ion) was used in his s udy. Aspe gillus cul u es we e g own in minimal essen ial medium Eagle (MEM) (Sigma Ald ich) o RPMI medium supplemen ed wi h 5 % ( / ) e al cal se um (Sigma Ald ich) a 37 8C and 200 .p.m., o up o 4 days. S ocks we e main ained on mal ex ac aga (MEA) (Oxoid). E ec o ampho e icin B on g ow h o A. umiga us .MEA pla es con aining spo ula ing Aspe gillus colonies we e washed wi h 10 ml 0.1 % ( / ) Tween 80 (Me ck) in PBS (pH 7.2) (Sigma Ald ich) o isola e conidia. Conidia we e washed wice in s e ile PBS, cen i uged (1500 g, 5 min in a Beckman GS-6 cen i uge) and coun ed using an haemocy ome e . Flasks con aining MEM (25 ml) we e inocula ed wi h 13105Aspe gillus conidia o gi e a densi y o 4 3103ml1and incuba ed a 37 8C and 200 .p.m. Cul u es we e supplemen ed wi h ampho e icin B a inal concen a ions o 0.04, 0.08, 0.16 and 0.32 ìgml 1. Flasks we e emo ed a each ime poin and he con en s il e ed h ough a Wha man No. 1 il e in a Bu ¨chne unnel and ai - d ied. A g ow h cu e was cons uc ed o d y ungal biomass e sus incuba ion ime. E alua ion o memb ane leakage. Se en y- wo-hou -old cul u es (25 ml) o A. umiga us we e ha es ed by cen i uga ion (2056 g,20 min), washed h ee imes in PBS and he hyphal mass esuspended in 25 ml PBS. Ampho e icin B (0.04, 0.08, 0.16 o 0.32 ìgml 1) o DMSO (Sigma Ald ich) (0.5 % / ) was added and he cul u es incuba ed a 37 8C and 200 .p.m. o a u he 1, 2 o 4 h. A each ime poin a cul u e was emo ed and he con en s il e ed h ough a Wha man No. 1 il e . The il a e was passed h ough a 0.45 ìM sy inge il e (Sa o ius) and ee amino acids o p o ein we e measu ed as desc ibed below. Amino acid concen a ion was de e mined by he ninhyd in colo i- me ic me hod and is exp essed in e ms o aspa ic acid and glu amic acid, which we e used as s anda ds. A ninhyd in (Sigma Ald ich) solu ion (200 ìl; s ock: 0.35 g in 100 ml e hanol) was added o each sample (1 ml) and hea ed o 95 8C o 4 min. A e cooling o oom empe a u e in an ice ba h, he abso bance a 570 nm was eco ded on a spec opho ome e (Beckman DU 640). To de e mine he quan i y o p o ein eleased om he hyphal mass, samples we e assayed using he B ad o d eagen (Bio-Rad), wi h BSA (Sigma Ald ich) as s anda d. Ex ac ion o glio oxin om A. umiga us cul u e il a e. Hyphae o Aspe gillus we e emo ed om he MEM cul u e medium o RPMI medium by il a ion and an equal olume (25 ml) o chlo o o m (Hype Sol ; BDH) was added o he il a es. Following con inual mixing o 30 min, he chlo o o m ac ion was collec ed and e apo- a ed o d yness in a Bu ¨chi (B inkmann Ins umen s; Wes bu y, NY) o o e apo a o . D ied ex ac s we e dissol ed in 250 ìl me hanol (Hype Sol , BDH) and s o ed a 70 8C un il assayed. Ex ac ion o glio oxin om hyphae o A. umiga us .The ex ac ion o in acellula glio oxin om hyphae o A. umiga us was pe o med as ollows: hyphae we e eco e ed om MEM cul u e medium (50 ml) o RPMI medium (50 ml) by il a ion. Hyphae we e washed in PBS and g ound o a ine powde unde liquid N2using a p e-chilled pes le and mo a . The g ound hyphae we e esuspended in 10 ml 6 M HCl. Chlo o o m (50 ml) was added and he mix u e s i ed a oom empe a u e o 30 min. The sample was pou ed in o a sepa a ion unnel and he glio oxin ex ac ed in he lowe chlo o o m laye . Following chlo o o m e apo a ion, he d ied ex ac s we e dissol ed in 250 ìl me hanol and le els o glio oxin quan i ied by Re e sed Phase- HPLC. Quan i ica ion o glio oxin by HPLC. Glio oxin was de ec ed by Re e sed Phase-HPLC (Spec a-Physics). The mobile phase was 34.9% ( / ) ace oni ile (Hype Sol , BDH), 0.1 % ( / ) i luo oace ic acid (Sigma Ald ich) and 65 % ( / ) deionized-dis illed wa e . Glio oxin ex ac (20 ìl) was injec ed on o a C18 Hewle Packa d column. A s anda d cu e o peak a ea e sus glio oxin concen a ion was cons uc ed using glio oxin s anda ds (50, 100 and 200 ng ml1) dissol ed in me hanol (Sigma Ald ich). S a is ical analysis. All assays we e pe o med on h ee independen occasions. Resul s p esen ed a e he mean s anda d e o . S a is ical analysis we e pe o med using S uden ’s wo ailed - es wi h alues o P,0.05 conside ed s a is ically signi ican . E. P. Ree es and o he s 2 Jou nal o Medical Mic obiology 53 RESULTS The e ec o ampho e icin B on he g ow h o A. umiga us Cul u e medium was inocula ed wi h conidia o A. umiga us a an ini ial densi y o 4 3103ml1and incuba ed as desc ibed. An un ea ed cul u e and ou cul u es supple- men ed wi h di e en concen a ions o ampho e icin B we e used. The esul s (Fig. 1) indica e ha while he concen a ions o ampho e icin B employed he e ac as ungis a ic agen s o e he i s 24 h, he a e o g ow h o he cul u es supplemen ed wi h 0.04 and 0.08 ìg ampho- e icin B ml1is app oxima ely he same as he con ol o e he 24–72 h pe iod. Following 96 h incuba ion, he ea ed cul u es eached a mass o app oxima ely 65–70 % o ha o he con ol. This expe imen demons a ed ha he concen- a ions o ampho e icin B chosen o subsequen wo k we e ungis a ic a he han ungicidal, as used clinically (Liao e al., 1999). To e i y ha he ampho e icin B concen a ions employed in subsequen expe imen s we e ungis a ic a he han ungicidal, 48 h cul u es o A. umiga us [hyphal mass 127.9 mg (d y weigh )] g own in MEM cul u e medium we e supplemen ed wi h ampho e icin B a inal concen a- ions o 0.16 o 0.32 ìgml 1and g own o a u he 24 h. Hyphae we e ha es ed, washed wi h PBS and esuspended in esh medium o 24 h in he absence o ampho e icin B. The hyphal mass o he cul u e supplemen ed wi h 0.16 ìg ampho e icin B ml1had inc eased o 155.4 mg (d y weigh ) while ha o he cul u e supplemen ed wi h ampho e icin B a a concen a ion o 0.32 ìgml 1was 111.6 mg (d y weigh ). This inding indica es ha ampho e icin B a 0.16 ìgml 1does no p e en subsequen g ow h o he cul u e, and ha he 0.32 ìgml 1ampho e icin B ea men does no cause wide-scale hyphal cell dea h, as he d y weigh was s ill 87 % o he un ea ed con ol. The e ec o ampho e icin B on elease o in acellula cons i uen s om A. umiga us I has been es ablished p e iously ha ampho e icin B can induce he elease o in acellula cons i uen s om pa ho- genic ungi (Ghosh & Ghosh, 1963; Cohen, 1998). We sough o es ablish whe he his occu ed in A. umiga us and o de e mine whe he i con ibu ed o he enhanced elease o low molecula mass compounds. Se en y- wo-hou -old cul u es o A. umiga us we e exposed o ampho e icin B a inal concen a ions o 0.04, 0.08, 0.16 o 0.32 ìgml 1o 0.5 % ( / ) DMSO o 1, 2 o 4 h and he elease o amino acids and p o ein was moni o ed. DMSO was employed he e as a posi i e con ol since i is known o al e he pe meabili y o he cell memb ane (Yu & Quinn, 1998). The esul s show ha exposu e o 0.16 o 0.32 ìg ampho e icin B ml1o 0.5 % DMSO o pe iods o 2 o 4 h lead o he elease o ele a ed le els o amino acids compa ed o he ele an con ols (Fig. 2). In he case o exposu e o A. umiga us o 0.32 ìg ampho e icin B ml1 o 4 h, 2.741 0.069 ìg amino acid (mg hyphae)1was eleased. Hyphal mass [mg (25 ml cul u e)–1] Time (h) 0 5 10 15 20 25 30 35 40 45 024487296 Fig. 1. E ec o ampho e icin B on he g ow ho A. umiga us .Cul u e medium was supplemen ed wi h ampho e icin B p io o inocula ion wi h A. umiga us conidia. d, Con ol; s,0 .4; j,0 .08; h,0 .16; n, 0.32 ìg ampho e icin B ml1. The hyphal mass was de e mined a 24 h in e als. Ampho e icin B (µg ml–1) Amino acids [∝ g (mg hyphae)–1] 0 0·5 1 1·5 2 2·5 3 Con 0·04 0·08 0·16 0·32 DMSO Con 0·04 0·08 0·16 0·32 DMSO Con 0·04 0·08 0·16 0.32 DMSO 1 h 2 h 4 h ** * * ** * * * Fig. 2. E ec o ampho e icin B on elease o amino acids om hyphae o A. umiga us . Cul u es o A. umiga us we e supplemen ed wi h ampho e icin B and he elease o amino acids was assessed using he ninhyd in assay. DMSO was used as a posi i e con ol a a concen a ion o 0.5 % ( / ). *, S a is ically signi ican ( P ,0.05) di e ence compa ed o he ele an con ol. Ampho e icin-B-media ed elease o glio oxin om A. umiga us h p://jmm.sgmjou nals.o g 3 Enhanced p o ein elease om A. umiga us was also e iden a e exposu e o ampho e icin B o 2 o 4 h (Fig. 3). In his case he g ea es elease occu ed when 0.32 ìg ampho e icin Bml 1was employed o 4 h (35.57 2.56). Release o glio oxin om A. umiga us exposed o ampho e icin B Ampho e icin B can induce he elease o amino acids and p o ein om cul u es o A. umiga us o e a ela i ely sho ime pe iod (Figs 2 and 3). Since glio oxin is a low molecula mass dipep ide i could escape om A. umiga us in he same manne as amino acids and p o eins. Cul u es o A. umiga us which had been g own o a pe iod o 72 h o a hyphal mass o 29 mg we e supplemen ed wi h ampho e icin B a inal concen a ions o 0.04, 0.08, 0.16 o 0.32 ìgml 1and he amoun o glio oxin in he cul u e il a es was assessed a a ious ime poin s a e he addi ion. A e 1 h, he con ol cul u e demons a ed a glio oxin concen a ion o 210.336.1 ng (mg hyphae)1(Fig. 4). Those cul u es supplemen ed wi h 0.16 o 0.32 ìg ampho e icin B ml1 showed ele a ed le els o glio oxin in he cul u e supe - na an s, pa icula ly a e 2 and 4 h. The highes concen a- ion o glio oxin [506.624.7 ng (mg hyphae)1] was de ec ed in he cul u e supplemen ed wi h 0.32 ìg ampho- e icin B ml1a e 2 h incuba ion (Fig. 4). E ec o DMSO on glio oxin elease by A. umiga us Ampho e icin B unc ions by binding e gos e ol in he ungal cell memb ane and c ea ing po es h ough which in acellu- la cons i uen s may escape (Abu-Salah, 1996). I was pos ula ed ha he ele a ed le els o glio oxin obse ed in ampho e icin-B-supplemen ed cul u es could be due o he inc eased pe meabili y o he ungal cell memb ane. To es his hypo hesis 72-h-old cul u es o A. umiga us we e supplemen ed wi h DMSO, which also has he abili y o al e he pe meabili y o cell memb anes, bu in a di e en manne o ampho e icin B (Yu & Quinn, 1998). The esul s demon- s a ed a signi ican inc ease in glio oxin in he cul u e medium a e 2 h in hose cul u es supplemen ed wi h 0.5 % DMSO (Fig. 5). Following 4 h exposu e o DMSO, he glio oxin concen a ion in he cul u e medium supple- men ed wi h 0.5 % DMSO eached a alue o 431.14 23.1 ng (mg hyphae)1. Examina ion o in acellula and ex acellula concen a ion o glio oxin ollowing exposu e o A. umiga us o ampho e icin B o DMSO The ele a ed le els o glio oxin in ampho e icin-B- o DMSO-supplemen ed cul u es may be due o inc eased memb ane pe meabili y. Al e na i ely, he wo agen s could exe a ungis a ic e ec and his migh , in u n, lead o he inc eased syn hesis o seconda y me aboli es such as glio ox- in. To asce ain whe he his was he case he glio oxin concen a ion in supplemen ed medium and in he hyphae o A. umiga us was de e mined. In his case cul u es we e g own o 48 h and hen supplemen ed wi h he ele an concen a ion o ampho e icin B o DMSO o a u he 24 h. The esul s e ealed enhanced le els o glio oxin in he hyphae as well as in he cul u e medium o hose cul u es supplemen ed wi h ampho e icin B o DMSO (Fig. 6). The in a-hyphal glio oxin concen a ion in he cul u e Ampho e icin B (µg ml–1) P o ein [∝ g (mg hyphae)–1] Con 0·04 0·08 0·16 0·32 DMSO Con 0·04 0·08 0·16 0·32 DMSO Con 0·04 0·08 0·16 0·32 DMSO 0 5 10 15 20 25 30 35 40 45 1 h 2 h 4 h * * ** * * * ** * * * * * * Fig. 3. P o ein elease om A. umiga us ollowing exposu e o ampho e icin B. Cul u es we e supplemen ed wi h ampho e icin B and he elease o p o ein om hyphae was measu ed. DMSO was used as a posi i e con ol a a concen a ion o 0.5 % ( / ). *, S a is ically signi ican ( P ,0.05) di e ence compa ed o he ele- an con ol. Ampho e icin B (µg ml–1) Glio oxin [ng (mg hyphae) –1 ] 1 h 2 h 4 h 0 100 200 300 400 500 600 Con 0·04 0·08 0·16 0·32 Con 0·04 0·08 0·16 0·32 Con 0·04 0·08 0·16 0·32 * * * * *** Fig. 4. Release o glio oxin om A. umiga us cul u es supplemen ed wi h ampho e icin B. The concen a ion o glio oxin in ampho e icin-B- supplemen ed cul u es was assessed by HPLC analysis. *, S a is ically signi ican ( P ,0.05) di e ence compa ed o he ele an con ol. E. P. Ree es and o he s 4 Jou nal o Medical Mic obiology 53 supplemen ed wi h 0.16 ìg ampho e icin B ml1was 732.1 37.9ngmg 1compa ed o he con ol which showed a glio oxin concen a ion o 88.64.9ngmg 1. Simila ly, he glio oxin concen a ion in he MEM cul u e medium was 273.511.9ngml 1compa ed o 146.14.5ngml 1in he con ol. In he case o he cul u es supplemen ed wi h 0.5 % ( / ) DMSO, he glio oxin concen a ion in he cul u e medium was 227 12.1ngml 1(con ol: 146.114.5 ng ml1) while ha in he hyphae was ound o be 405.520.2ngmg 1compa ed o 88.64.9ngmg 1in he ele an con ol. In he case o A. umiga us g own in RPMI medium (Fig. 6) he ex acellula glio oxin concen a- ion was 39.47 1.2ngml 1in he con ol whe eas i was 205.19 8.2ngml 1when he cul u e was supplemen ed wi h ampho e icin B a a inal concen a ion o 0.16 ìgml 1. The in acellula glio oxin concen a ion inc eased om 3.63 0.13 ng (mg hyphae)1in he con ol o 41.4 0.74 ng (mg hyphae)1in he ampho e icin-B- ea ed cul- u e. Al hough di e en le els o glio oxin we e p oduced by A. umiga us in MEM and RPMI cul u e media, he abili y o ampho e icin B o inc ease he in acellula and ex acellula glio oxin le els was obse ed in cul u es g own in bo h media. DISCUSSION In ec ion by A. umiga us is a se ious h ea o he heal h o pa ien s immunocomp omised as a esul o disease, pul- mona y mal unc ion o medical he apy (Daly & Ka anagh, 2001) and, in he case o IA, may ha e a mo ali y a e o 85– 90 % (Denning, 1996b). Pa o he abili y o A. umiga us o colonize he lung is i s capaci y o supp ess he local immune esponse (Hogan e al., 1996). This can be achie ed h ough he ac ion o oxins and enzymes, and he abili y o glio oxin o acili a e coloniza ion is well cha ac e ized (Su on e al., 1995 & 1996). Glio oxin may supp ess he unc ion o pulmona y mac ophages and also a ec he gene al immune esponse o pa hogens by des oying cells o he spleen and he immune sys em (Su on e al., 1994). One o he p incipal d ugs used o comba aspe gillosis is he polyene ampho e icin B, which c ea es po es in he ungal cell memb ane h ough which in acellula cons i uen s escape (Abu-Salah, 1996; Cohen, 1998). I was pos ula ed ha such po es migh also se e o allow glio oxin o escape om he hyphae o A. umiga us, which could exace ba e glio oxin-media ed damage o pulmona y issue and he supp ession o he immune esponse in he in ec ed pa ien . As a consequence we sough o de e mine he esponse o hyphae o A. umiga us o ampho e icin B in e ms o he syn hesis and elease o glio oxin. Using ungis a ic concen a ions o ampho e icin B (Fig. 1) i was es ablished ha exposu e o cul u es o A. umiga us o ampho e icin B inc eased he elease o amino acids (Fig. 2) and p o ein (Fig. 3) 2–4 h a e addi ion. These inding sugges ed ha ampho e icin B was inducing he elease o in acellula cons i uen s om he ungal hyphae, possibly h ough he c ea ion o po es in he cell memb ane. Ele a ed 1 h 4 h 2 h Glio oxin [ng (mg hyphae) –1 ] 0 50 100 150 200 250 300 350 400 450 500 DMSO (%) Con 0·25 0·5 1·0 Con 0·25 0·5 1·0 Con 0·25 0·5 1·0 * * * * * * * Fig. 5. E ec o DMSO on elease o glio oxin om A. umiga us . Cul u es o A. umiga us we e supplemen ed wi h DMSO and he elease o glio oxin o e a 4 h pe iod was assessed by HPLC analysis. *, S a is ically signi ican ( P ,0.05) di e ence compa ed o he ele an con ol. 0 50 100 150 200 250 300 MEM AmpB (0·08µg ml–1) MEM Con ol MEM AmpB (0·16µg ml–1) MEM DMSO (0·5%) 0 100 200 300 400 500 600 700 800 900 RPMI AmpB (0·16µg ml–1) RPMI Con ol * * * * + + + + Ex acellula glio oxin [ng (ml cul u e)–1] In acellula glio oxin [ng (mg hyphae)–1] Fig. 6. In a- and ex acellula concen a ion o glio oxin in cul u es supplemen ed wi h ampho e icin B o DMSO. Cul u es o A. umiga us we e supplemen ed wi h ampho e icin B (0.08 o 0.16 ìgml 1)o DMSO (0.5 % / ). Ex acellula (open ba s) and in acellula ( illed ba s) glio oxin was ex ac ed and quan i ied by HPLC. S a is ically signi ican di e ences ( P ,0.05) compa ed o he ele an con ols a e indica ed by * o +. Ampho e icin-B-media ed elease o glio oxin om A. umiga us h p://jmm.sgmjou nals.o g 5 le els o glio oxin we e also de ec ed in media o cul u es supplemen ed wi h ampho e icin B (Fig. 4) o DMSO (Fig. 5). DMSO is commonly used as a sol en in biological science and can al e cell pe meabili y by a ec ing he s uc u e and in e ac ion o lipids in memb anes (Yu & Quinn, 1998; Go deliy e al., 1998) which may explain he elease o amino acids, p o ein and glio oxin obse ed he e. Gi en he p e iously es ablished abili y o ampho e icin B o induce he elease o in acellula cons i uen s om C. albicans (Ghosh & Ghosh, 1963), i appea ed ha his mechanism migh also be ope a ing he e o acili a e he elease o glio oxin. Howe e , when he in acellula con- cen a ion o glio oxin was asce ained i was ound o be ele a ed, along wi h aised le els de ec ed in he cul u e supe na an . In he case o MEM cul u es supplemen ed wi h 0.08 o 0.16 ìg ampho e icin B ml1, he in acellula concen a ion o glio oxin ose om 88.64.9ngmg 1 o 217.38 18.8ngmg 1and 732.137.9ngmg 1, espec- i ely (Fig. 6). This e ec was also appa en in RPMI-g own cul u es o A. umiga us. The inc eased concen a ion o glio oxin in cul u e supe - na an may be explained by he well es ablished abili y o ampho e icin B o inc ease he pe meabili y o he ungal cell memb ane ia he c ea ion o po es (Ghosh & Ghosh, 1963; Cohen, 1998). Howe e , he ele a ed le el o in acellula glio oxin could also con ibu e o he highe le els de ec ed in he cul u e. The abili y o ampho e icin B o inc ease he syn hesis o glio oxin may be media ed h ough i s ungi- s a ic abili ies. When applied o cul u es o C. albicans, ampho e icin B hal s eplica ion bu he cells emain iable (Liao e al., 1999) and he ungus may eco e he abili y o di ide in he absence o he polyene. Hal ing he cell’s g ow h may o ce he p oduc ion o seconda y me aboli es such as glio oxin. In e ec ampho e icin B may ha e wo dis inc e ec s on he ungal cell: (i) The ungis a ic e ec may hal eplica ion, lea ing he cell ali e bu capable o syn hesizing seconda y me aboli es such as glio oxin; (ii) The abili y o he polyene o c ea e ape u es in he ungal cell memb ane allows he escape o small molecula mass compounds, including glio oxin. The consequence o ampho e icin B exposu e a he cellula le el would be ele a ed syn hesis and elease, ia memb ane ape u es, o glio oxin. Al hough DMSO has a di e en p ima y e ec on cell memb anes (Yu & Quinn, 1998), i also induces ele a ed syn hesis and elease o glio oxin. In he case o exposu e o A. umiga us o DMSO, he ele a ed le els o glio oxin in he supe na an may be due o he inc eased pe meabili y o he ungal cell memb ane. The leakage o me aboli es may hinde he cell’s abili y o di ide, empo a ily allowing i syn hesize seconda y me aboli es such as glio oxin. The wo k p esen ed he e indica es ha when exposed o ampho e icin B in i o A. umiga us p oduces and sec e es he immunosupp essi e agen glio oxin a an ele a ed a e. Ampho e icin B he apy has many undesi able side e ec s (Cohen, 1998) and new o mula ions a e designed o minimize he oxici y owa ds hos issue (Ha sel & Bola d, 1996). Apa om i s di ec e ec on he hos , ampho e icin B may also induce he syn hesis and elease o seconda y me aboli es by A. umiga us which may con ibu e o g ea e pulmona y damage and dec eased immune unc ion bo h locally and sys emically. While he wo k p esen ed he e examined he esponse o A. umiga us o ampho e icin B in an in i o model, in i o e alua ion o his phenomenon may be equi ed o ully assess i s clinical signi icance. Recen wo k has indica ed ha ungi can su i e exposu e o ela i ely high le els o ampho e icin B, main ain ce ain cellula unc ions and po en ially esume eplica ion (Liao e al., 1999). This s udy has es ablished ha ampho e icin B may induce he syn hesis and elease o glio oxin om A. umiga us. This phenomenon has he po en ial o con ibu e o ele a ed le els o pulmona y damage (Eichne e al., 1986; Su on e al., 1996) and immunosupp ession (Su on e al., 1994, 1995) associa ed wi h his oxin. ACKNOWLEDGEMENTS This wo k was conduc ed independen ly o any pha maceu ical company wi h in e es s in an i- ungal d ug de elopmen o ma ke ing. This wo k was suppo ed by unding om he Highe Educa ion Au ho i y h ough PTRLI 3. REFERENCES Abu-Salah, K. M. (1996). Ampho e icin B: an upda e. B i J Biomed Sci 53, 122–133. Ami ani, R., Mu ayama, T., Nawada, R., Lee, W. J., Niimi, A., Suzuki, K., Tanaka, E. & Kuze, F. (1995a). 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