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1 J. Fd Hyg. Sfety 21(2), (2006) p, v v, v v, p, p q In Vitro x ü zá½ Á Á xá û Á Áy Á 1 Á Á w wœ w œ w w wp w Oestrogenic Activity of Prbens In Vitro Estrogen Assys Sung-Hoon Lee, Sun-Jung Kim, Jung-Rn Prk, Eun-Hye Jo, Nm-Shik Ahn, Joon-Suk Prk, Je-Woong Hwng, Ji-Youn Jung 1, Yong-Soon Lee, nd Kyung-Sun Kng %FQBSUNFOUPG7FUFSJOBSZ1VCMJD)FBMUI$PMMFHFPG7FUFSJOBSZ.FEJDJOF4FPVM/BUJPOBM6OJWFSTJUZ4FPVM,PSFB %FQBSUNFOUPG$PNQBOJPOBOE-BCPSBUPSZ"OJNBM4DJFODF,POHKV/BUJPOBM6OJWFSTJUZ$IVOHOBN,PSFB 3FDFJWFE.BZ"DDFQUFE+VOF ABSTRACT The use of underrm nd body cre cosmetics with oestrogenic chemicl excipients (prticulrly the prbens) nd the hypothesized ssocition with brest cncer incidence, prticulrly in women. It is noted tht the type of cosmetic product is irrelevnt (e.g. ntiperspirnt/deodornt versus body lotion, moisturizers or sprys versus crems) nd ttention must focus on issues of ctul exposure to chemicls through continued derml ppliction of body cre products nd the endocrine/hormonl ctivity nd toxicity of the chemicls in the formultions. To evlute the estrogenic ctivities of prbens such s ethylprben, butylprben, propylprben, isobutylprben nd isopropylprben, we used recombinnt yests contining the humn estrogen receptor [Scchromyces cerevisie ER + LYS 8127], humn brest cncer MCF-7 cell lines nd humn estrogen receptor α nd β. In E-screen ssys, isopropylprben is the most estrogenic prben, nd in ER competition ssy, isobutylprben is the most estrogenic prben. We evluted isopropylprben ws most ctive in the recombinnt yest ssy, followed by propylprben, ethylprben, isobutylprben nd butylprben. Results from this study demonstrte tht prbens re observed in humn endocrine system. Therefore, we hve shown tht the prbens is induced the estrogenic ctivities similr to 17β-estrdiol nd Bisphenol-A. Key words: ethylprben, butylprben, propylprben, isobutylprben, isopropylprben, estrogenic effect, endocrine disruptor q q w l k» pq, v vq, pq, p q š, ý, w, ý w, kj y t ƒ š yw 1,2). w ƒ w ù ù, tw s q ƒ w HPLC mw y w q ü s ü w 3). w, q y p Author to whom correspondence should be ddressed. w ww w q ü w w w 2,4~6). q 1990 û y w w ƒ œ» w 7,8). w r w š v w y t ƒ yw p, š ƒ sƒw» w z ƒ w š w 9). FDA w, 77% y t 99%ƒ q w wš, 0.32% pq v vq 0.07% pq q q w y t š w. ƒ t 1% w/w q w» w š š š 100

2 0FTUSPHFOJD"DUJWJUZPG1BSBCFOT*O7JUSP&TUSPHFO"TTBZT )., w, w 1~1 6mg/Kg q wš, 2 w 4~6 mg/kg w š š 11)., t š x ƒ 1% ƒ w wš. t 2004 EFSA ( w t ) š t ƒ, w, ƒœ t w w z, p-hydroxybenzotes sƒ tw. t ƒ, w, ƒœ t w w z t q w w, w q 1994 t w z(scf) sƒw p, p, v v q sww ADI 0~10 mg/kg bw w 10). z» x x mw x ADI ADI yw, v vq ADI w NOAEL yw ADI «šw š š w. w x 10 mg/ Kg/dy w š 10). q w w pq ü ùkú ƒ š q 12) x q in vitro l w x mw q ü sƒw. x pq, pq, v vq, pq, v vq (Sigm-ldrich) š [2,3,6,7-H3]estrdiol (Sigm-ldrich)( w E2) Bisphenol-A ( w BPA) (Sigm-ldrich). x MCF-7 s w q E-Screening x 5% Fetl Bovine Serum (FBS)(Gibco) 1% PSN ntibiotic mixture (Gibco)ƒ ƒ Phenol red-free D- medi œ w s MCF-7 s 5% CO 2, 37 o C j l(snyo) w. x MCF-7 s 6-well culture plte (Nunc) wš, e 5% dextrn coted chrcol-stripped FBS (Hyclone) 0.3% PSN ntibiotic mixtureƒ sw D- medi y w w š e, 5% dextrn coted chrcol-stripped FBS (Hyclone) 1% PSN ntibiotic mixture w w 3 w.» x sw w w š, e 3, 0.1N NOH (Sigm) ew DNA content spectrophotometer (Beckmn) w OD 260 nm d w 13). q ER w x x E2 w ERα ERβ w x RBA (reltive binding ffinities) d w» w w. ER w x x ü w d w» w EPA «šw ERα ERβ competition binding ssy kit (Pnver) w «š x w xw. x ƒ q 10 M l 4 w M¾ ƒ 10 w š E2 10 M l 4 6 w 2 10 M¾ ƒ 10.» xÿ-rÿ» (Tecn) w ƒ w r Ÿ (Polriztion) w š 100% 10 5 M E2 rÿ wš Origin6.0 m v w IC 50 w 14). ER sl β-glctosidseƒ z w x w q p sƒ Scchromyces cerevisie ER+ LYS 8127 (YER) z, shking incubtor (300 rpm) k. z ƒƒ CUP1 metllothionine promoter her sww vector sl l estrogen response element, β-gl xw. S. cerevisie ER+ LYS g/ml yest nitrogen bse, 2% dextrose, 30 µg/ml L-lysine-HCl, 35 µ/ml L- histidine-hcl sww wš, Tble 1. Prolifertive effect of prbens Compound C mx RPP b RPE c 17β-estrdiol % butyl prben % propyl prben % ethyl prben % isobutyl prben % isopropyl prben % bisphenol-a % C mx is the concentrtion of the test compound giving mximl prolifertion RPP b (reltive prolifertive potency) is the rtio of the Cmx of the test compound to tht of 17β-estrdiol. RPE c (reltive prolifertive effect) is the rtio of mximl cell yield of the test compound to tht of 17β-estrdiol, expressed s percentge.

3 102 4VOH)PPO-FFFUBM 20% glycerol ƒw -80 o C w w. z ƒ w w w z 500 um CuSo 4 ƒw, 50 ml p w. ƒƒ ew shking incubtor 18 w z, w w w z 96-well plte 100 ul w. ƒ well Z buffer 100ul wš 20 z microplte ELISA reder w 420 nm 590 nm w d w 4,5,13,15). m SAS w ANOVA z, Dunnett't test 5% w. š MCF-7 s w q E-Screening x MCF-7 s x ƒ q E2 BPA ew 3 z d (Tble 1). E2 ( ) w 10 M 2.5 s z š BPA 10 M 2.2 s 8 z. 5 q pq, v vq, pq, v vq E2 w 10,000 û s š pq Fig. 1. Prolifertion of MCF-7 prolifertion treted with E2 (17β-estrdiol) (A), BPA (B),ethylprben (C), propylprben (D), butylprben (E), isobutylprnben (F), isopropylprben (G). Cell number fter tret ment of cells for 3 dys with the indicted concentrtions of chemicls is expressed s fold induction reltive to tht of cells treted with vehicle (0.1% ethnol) lone (1.0). E2 of (B)~(G) is expressed s fold induction vlue t E2 10-9M. ech point is men of three determintions nd significnt differences s compred with control : * P<0.05.

4 0FTUSPHFOJD"DUJWJUZPG1BSBCFOT*O7JUSP&TUSPHFO"TTBZT 103 Fig. 2. Estrogenic ctivity of E2 (17β-estrdiol) (A), BPA (B), ethylprben (C), propylprben (D), butylprben (E), isobutyl - prnben (F), isopropylprben (G) in Yest recombinnt ssy. Ech bsorbnce vlue is detected fter tretment of yest with the indicted concentrtions of chemicls is expressed s bsorbnce vlue t 420 nm. E2 of (B)~(G) is expressed bsorbnce vlue t E2 10-9M. ech point is men of three determintions nd significnt differences s compred with control : * P< ,000 û s. w pq, v vq, pq, v vq 10 4 M pq 10 M Cmx ùkþš q 4 ƒ ƒ E2 w z (RPE) v vq > pq >v vq > pq > p q (Fig 1). q ER w x ER α β w x x ü w d w inhibitory concentrtion 50% (IC 50 ) E2 w ERα ERβ w x RBA(reltive binding ffinities) d w (Tble 2). ERα ERβ E2 IC 50 ƒƒ M, M š q IC M~ M. q ƒ ERα w E2 w (RBA) pq > pq >v vq = v vq > pq ùkûš ERβ w RBA pq > pq > v vq àv vq > pq. w pq E2 w

5 104 4VOH)PPO-FFFUBM Tble 2. Reltive binding ffinity of prbens to humn ERα nd ERβ E2 100 (ERα), 200 (ERβ)ƒ û y w š BPA ERα 3 ƒ w 16). Compound IC 50 ERα RBA b IC 50 ERβ RBA b 17β-estrdiol butyl prben propyl prben ethyl prben isobutyl prben isopropyl prben bisphenol-a Ech vlue ws the men of triplicte determintions in more three experiments. IC 50 is the concentrtion of the compound tht inhibits binding of 17β-estrdiol to ER by 50% b RBA ws clculted by the eqution (IC 50 of 17β-estrdiol/IC 50 of competitor) 100 ER sl β-glctosidseƒ z w x w q p sƒ ER w z VIT promoter reporter (luc or CAT) ww q ER ww xw ü w. E2 ( ) w 10 M 7 p š BPA 10 M 6 p 7 Tble 3. Estrogenic ctivity of prbens in the Yest Screen Compound EC 50 C mx b 17β-estrdiol butyl prben propyl prben ethyl prben isobutyl prben isopropyl prben bisphenol-a Ech vlue ws the men of triplicte determintions. EC 50 is the concentrtion of the test compound giving hlf-mximl estrogenic ctivity. b C mx is the concentrtion of the test compuond giving mximl estrogenic ctivity.. 10 M E2 w š 5 q v vq 10 5 M, pq, pq, v vq 10 4 M, pq 10 M Cmx 3 kùü. q ƒ ƒ E2 w p pq > pq > pq >v vq > v vq ùkû. l (KFDA EDS-311)., 2006 w w w (KRF-005- E00076). ER sl β-glctosidseƒ z w x w q ü 17) w. x x w E2 BPA w q p w. E2 10 M ƒ y š, BPA 10 M p 7 ƒ. q v vq 10 M 10 M¾ xw 3, p ƒw, 10 M ƒ w p. w E2 w, 10 M 7 10 M¾ v vq E2 ü 3. v vq w ù q 10 M 4 10 M v vq 5 10 M 3 10 M pq 4 p. w MCF-7 s s ERαƒ w p p ER w s w. q x ü BPA ü w w p BPA y 1000 š E2 w MCF-7 s w. 72 BPA E2 w MCF-7 s w z DNA d w, E2 10 M 2.5 s w, BPA 10-8M 2.2 s w. pq

6 0FTUSPHFOJD"DUJWJUZPG1BSBCFOT*O7JUSP&TUSPHFO"TTBZT M 10 4 M¾ MCF-7 s ƒ gš, 10 4 M w s 2.2 w w p. v vq, pq, pq v vq 10 5 M 2 s š, v v q RPE 104%, ü. wr, q ER w q x ü w d w» w ERα ERβ competition binding ssy kit w xw. x E2 w q ERα ERβ w x RBA(reltive binding ffinities) d w. q ERα w x. E2 IC š, pq w RBA 0.952ƒ. w x BPA RBA w, pq 3 ƒ ü 16). q ERβ w x. ERα ƒ pq M IC50 ƒ RBA q ERα β w w š E2 w w ü ww w e w š. q pq ww w ü BPA j ƒ š ùkû. in vitro q p d w š w yw ¼ ù k» ƒ q p w ùkû 2,18)., t ù y t yw w ü j w e ƒ š q. š x 1. Rstogi S.C., Schouten A., de Kruijf N. nd Weijlnd J.W. : Contents of methyl-, ethyl-, propyl-, butyl- nd benzylprben in cosmetic products. Contct Dermtitis 32(1), (1995) 2. T. Okubo., Y. Yokoym., K. Kno. nd I. Kno : ERdependent estrogenic ctivity of prbens ssessed by prolifertion of humn brest cncer MCF-7 cells nd expression of ERα nd ERβ nd PR. Food nd Chemicl Toxicology 39, (2001) 3. Drbre P.D., Aljrrh A., Miller W.R., Coldhm N.G., Suer M.J. nd Pope G.S. : Concentrtions of prbens in humn brest tumours. J. Appl. Toxicol. 24(1), 5-13 (2004) 4. Routledge., J. Prker., J. Odum., J. Ashby. nd J. P. Sumpter. Some lkyl hydroxy benzote preservtives (Prbens) re estrogenic. Toxicology nd Applied Phrmcology 153, 12?19 (1998) 5. Nishihr., J. Nishikw., T. Knym., F. Dkeym., K. Sito., M. Imgw., S. Tktori., Y. Kitgw., S. Hori nd H. Utsumi : Estrogenic ctivities of 517 chemicls by yest twohybrid ssy. Journl of Helth Science 46, 282?298 (2000) 6. Andersen : Finl report on the sfety ssessment of isobutylprben nd isopropylprben. Journl of the Americn College of Toxicology 14, 364?372 (1995) 7. S. Oishi : Effects of propyl prben on the mle reproductive system. Food nd Chemicl Toxicology 40, (2002) 8. Song B.L., Li H.Y. nd Peng D.R. : in vitro spermicidl ctivity of prbens ginst humn spermtozo. Contrception 39(3), (1989) 9. Hrvey P.W. nd Drbre P. : Endocrine disrupters nd humn helth: could oestrogenic chemicls in body cre cosmetics dversely ffect brest cncer incidence in women? J. Appl. Toxicol. 24(3), (2004) 10. Soni M.G. nd Tylor S.L. : Greenberg N.A., Burdock G.A. Evlution of the helth spects of methyl prben: review of the published literture. Food nd Chemicl Toxicology 40(10), (2002) 11. Elder R.L. : Finl Report on the sfety ssessment of methylprben, ethyl -prben, propylprben, nd butyl - proben. J. Am. Coll. Toxicol. 3, (1984) 12. Kng K.S., CHE J.H., oung RYU D.Y.,, Kim T.W., LI G.X. nd LEE Y.S. Decresed Sperm Number nd Motile Activity on the F1 Offspring Mternlly Exposed to Butyl p- Hydroxybenzoic Acid (Butyl Prben). J. Vet Med Sci 64, (2002) 13. Prk, J.S., Lee, B.J., Kng, K.S., Ti, J.H., Cho, J.J., Cho, M.H., Inoue T. nd Lee, Y.S. : Hormonl effects of severl chemicls in recombinnt yest, MCF-7 cells nd uterotrophic ssys in mice. J. Microbiol. Biotechnol 10, (2000) 14. Bolger R., Wiese T.E., Ervin K., Nestich S. nd Checovich W. : Rpid screening of environmentl chemicls for estrogen receptor binding cpcity. Environ. Helth Perspect 106(9), (1998) 15. Edwin J. Routledge., J. Prker., J. Odum., J. Ashby., nd J. P. Sumpter. : Some Alkyl Hydroxy Benzote Preservtives (Prbens) Are Estrogenic. Toxicology And Applied Phrmcology 153, (1998) 16. Hn D.H., Denison M.S., Tchibn H. nd Ymd K. Reltionship between estrogen receptor-binding nd estrogenic ctivities of environmentl estrogens nd suppression by

7 106 4VOH)PPO-FFFUBM flvonoids. Biosci. Biotechnol. Biochem. 66(7), (2002) 17. Kng K.S., Cho S.D. nd Lee Y.S. : Additive estrogenic ctivities of the binry mixtures of four estrogenic chemicls in recombinnt yest expressing humn estrogen receptor. J. Vet. Sci. 3(1), 1-5 (2002) 18. Routledge E. J. nd Sumpter, J.p. : Structurl fetures of lkylphenolic chemicls ssocited with estrogenic ctivity. J. Biol. Chem. 272, (1997)