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1 J. Fd Hyg. Safety 20(1), (2005) Ÿ w y, w s z w Á½ xá Á Á yá v w t œw Antioxidative, Antimutagenic and Cytotoxic Effects of the Mineral Water Seung-shi Ham, Soo-hyun Kim, Seon-young Moon, Mi-Sun Jeon, Deog-Hwan Oh and Cheng-Bi Cui 4DIPPMPG#JPUFDIOPMPHZBOE#JPFOHJOFFSJOH,BOHXPO/BUJPOBM6OJWFSTJUZ$IVODIPO,PSFB ABSTRACT This study was performed to observe the components, antioxidative, antimutagenic and cytotoxic effects of the mineral water using AOAC method, DPPH free radical donating method, Ames test and SRB assay. Mineral water contained eleven kinds of minerals among the total seventeen components and sodium and potassium ion were main components. Mineral water showed electron donating activities (175.9 µg). The inhibition rate of mineral water (200 µg/plate) in the Sallmonella typhimurium TA98 strain showed 54% against the mutagenesis induced by Trp-P-1. In addition, same concentration of mineral water the Sallmonella typhimurium TA100 strain showed highest 67%, 65.8% and 63% inhibition against B(a)P, 4NQO and Trp-P-1, respectively. The cytotoxic effects of mineral water against the cell lines with Human lung carcinoma (A549), Human breast adenocarcinoma (MCF-7), Human stomach adenocarcinoma (AGS) and Human cervical adenocarcinoma (Hela) were inhibited with the increase of the mineral water. The treatment of 50 µg/well of mineral water showed cytotoxicities of 66%, 47.6%, 37.7% and 45.6% against A549, MCF-7, AGS and Hela. Key words: Antimutagenicity, cytotoxicity, mineral water w w w» w».», m,, w wš ƒ wwš y w» w š» swwš ƒ s wwš. w» O 2, N 2, CO 2, H 2 S, CH 4 H 2 ƒ y» He, Ne, Ar, Kr, Xe š Rnƒ. w w 1) l 25 o C w w ³ ù l e w wù. w w x w ƒ w k wš Ÿ w w ƒ y mw sƒw v w š. Ÿ w ùp, e, Author to whom correspondence should be addressed. e,, p, p p, ³,, w» š. ù,,, y w» e w ùkû. p» w» w ƒ ùp (Na) e (K) s w s» w w w. (Mg) w w g l» x y w» w. e (Ca) w w öe e w šx». ³ (SiO 2 ) ³ e y w g. š p(li) v w w w w p w ùe w Ÿ k. (Fe) w w» s z y w. (Zn) w v v š ù ƒ. p p(sr) 99%ƒ x 53

2 54 4FVOHTIJ)BNFUBM e š. p p e q w. Ÿ w. Ÿ l e w wù f j p v w Ÿ w 1) š Ÿ yw sƒ, w 2) Ÿ s k w 3) Ÿ w x w ƒ w k. Ÿ y» w sƒ ƒ ³ w w š. x w Ÿ w 943 m(3yœ) w millipore filter(0.45 µm) w z þ š w x w. s v w RPMI 1640 Dulbecco's Modified Eagle Medium(DMEM), Hepes buffer(gibco), Fetal bovine serum(fbs), Trypsin-EDTA Gibco (U.S.A.) l w. š w x v w 4-Nitroquinoline-1-oxide (4NQO) N-methyl-N'- nitro-n-nitrosoguanidine(mnng) Sigmaz l w 3-amino-1,4-dimethyl-5H-pyrido- (4,3-b)indole(Trp-P-1), benzo(a)pyrene(b(a)p), L-histidine dimethyl sulfoxide(dmso) z p w. s x³ x w s s s A549(Lung carcinoma, Human), s MCF-7(Breast adenocarcinoma, pleural effusion, Human), s AGS(stomach adenocarcinoma, Human), ª s HeLa (Cervical adenocarcinoma, Human) š s 293(transformedprimary human embryonal kidney) Korea Cell Line Bank(KCLB) l w w Salmonella typhimurium LT-2 histidine (auxotroph) TA98, TA100, Califonia w B.N. Ames l œ. Ÿ» AOAC 10) w. œ (electron donating ability) w w y y Ÿ Choi 11) w œ w w y y d w. 4 ml M 1,1-diphenyl-2-picrylhydrazyl solution(in methanol) 1 ml ƒw z, 30 ew z 517 nm Ÿ d w. œ ƒ x 3z w RC 50 ƒ Ÿ 1/2 j t w, œ (standard curve) mw w. s d s w s ù d w SRB(sulforhodamine B) assay 12) w. 10% fetal bovine serum ƒƒ s s s(a549), s(mcf-7), s (AGS), ª s(hela), s(293) w w RPMI 1640 DMEM cells/ml 100 µl ƒ well ƒw 24 (37 o C, 5% CO 2 )w z 12.5, 25, 37.5, 50 µg/ well 100 µl ƒw 48 w. z aspirator wš þ w 10%(w/v) TCA 100 µl ƒw z 1 4C ew z o x. plate» k z 1% (v/v) acetic acid 0.4%(w/v) SRB 100 µl ƒw 30 g. ww SRB w» w 1%(v/ v) acetic acid w.» plate 10 mm Tris buffer 100 µl z 540 nm microplate reader Ÿ d w. w x(antimutagenicity test) Ames test w preincubation 13) w x w š x w 4NQO, MNNG B(a)P Trp-P- 1 w. ³ k glass cap tube

3 "OUJPYJEBUJWF"OUJNVUBHFOJDBOE$ZUPUPYJD&GGFDUTPGUIF.JOFSBM8BUFS 55 50, 100, 150, 200 µg/plate ƒwš ƒƒ 50 µl ƒw. 10% S-9 mix 250 µl ƒƒ ƒw.» k Salmonella typhimurium ³ 100 µl w z 0.2 M sodium phosphate buffer ƒw vƒ 700 µlƒ w. 37 C 20 k w o histidine/biotin ƒ top agar(45 C) o 2 ml ƒw yww z w minimal glucose agar plate wš sqš y g 37 C 48 o w (his+ revertant colony) d w w q w. ƒ x mw w w y y w (inhibition, %) ùkü. 3z x w s³ e ùkü. Inhibition(%) = [(M S 1 )/(M S 0 )] 100, M: w, S 0 :, S 1 : ƒw š Ÿ Ÿ w 943 m(3 yœ) l w w ƒ?» w Table 1 17.» 11 ƒ» ùp e w p. š, û, k ƒ y. Table 2. Antioxidant effect of Kyung-san mineral water Sample RC 50 * (µg) mineral water 1Ü(0.1%) Ü(0.2%) Ü(0.4%) Ü(0.6%) Ü(0.8%) Ü(1.0%) BHT** 12.4 α-tocopherol 14.6 *Amount required for 50% inhibition of DPPH after 30 min. **Dibutyl hydroxy toluene w y y Ÿ w w w yz ƒ ³ w» w Ÿ w DPPH free radical y d w w y y sƒw. x Table 2 Ÿ w y z 10 (1.0%) RC µg α-tocopherol(rc 50 =14.6 µg) w w y y ùkü. w z Ÿ y mw» w S. typhimurium TA98 ³ w 4NQO B(α)P Trp-P-1 y ³ w. x Fig. 1 ƒ y ùkü 200 µg/plate ƒ Trp-P-1 w 54.2% ƒ y ùkü B(α)P 43.1% y ùkü. š 4NQO w 40% û y ùkü. Table 1. Analysis of mineral water Item Hole NO Hole NO Item Depth(m) 694 1, Free CO Temperature( o C) Cl ph SO 4 8,487 6, Total solid 8,084 8, F 5.2 ND 1.4 Na 2,320 1,502 1,982 SiO K Fe Ca Zn Mg Cu ND ND Sr Pb ND ND Li 21.0 ND 16.1 H 2 S ND ND ND HCO Mn CO 2 ND ND ND Al (unit : %)

4 56 4FVOHTIJ)BNFUBM Fig. 1. The Antimutagenic effects of mineral water against each mutagen on Salmonella typhimurium TA98. Fig. 3. Inhibitory effects of growth of human cancer cells in adding the mineral water. Fig. 2. The Antimutagenic effects of mineral water against each mutagen on Salmonella typhimurium TA100. wr, S. typhimurium TA100 ³ w 4NQO MNNG, B(α)P Trp-P-1 y xw S. typhimurium TA98 ³ ƒ ƒ y ùkü 200 ml/plate ƒ ƒƒ 65.8%, 58.6%, 67.0% 63.1% MNNG w š 60% y ùkü. w y r ù Ÿ y ùkü sƒ (Fig. 2). s z Fig. 4. Inhibitory effects of growth of mineral water on human transfomed primary embryonal kindney (293). x ƒ s w Ÿ s ³ w» w s A549, Hela, AGS MCF-7 s 293 w SRB assay ww. Ÿ w A549, Hela, AGS MCF-7 s w wz mw Fig. 3 Ÿ 50 µg/well ƒ ƒƒ 66%, 45.6%, 37.7% 47.6% z ùkü. w z ùkù Trp-P-1, B(α)P, MNNG 4NQO w z w y ù kü. w s 293 w Ÿ s z 20% w û Ÿ ƒ s w û z ùký (Fig. 4).

5 "OUJPYJEBUJWF"OUJNVUBHFOJDBOE$ZUPUPYJD&GGFDUTPGUIF.JOFSBM8BUFS 57 q w. w 943 m(3 yœ) l w Ÿ Á» w 17» 11 ƒ» ùp e w p. š û k ƒ y. š w y y, w s x w y y w y ùkü. MNNG, 4NQO, Trp-P-1 B(α)P w w x S. typhimurium TA98 w Trp-P µg/plate ƒ 54% y ùkü. w S. typhimurium TA100 ³ 4NQO MNNG ƒƒ 65.8% 58.6% y B(α)P Trp-P-1 w ƒƒ 67% 63% y ùkü. š Ÿ w y ³ w» w s z 50 µg/well ƒ A549, Hela, AGS MCF-7 w ƒƒ 66%, 45.6%, 37.7% 47.6% z ùkü. w s 293 w s z 20% w û Ÿ ƒ s w û z ùký. š x 1., wy yw, q Ÿ, pp (1996).s 2. Kim JH, Choi YW, Chung TK : Rapid simultaneous determination of anions in mineral water by single column ion chromatography. Kor J Chemical Society 39, (1995). 3. Nam SH : A study on a classification technique of natural mineral waters by its constitution and physico-chemical properties. Kor J Env Hlth Soc, 14(1), (1988). 4. Ko IS, Kim DH : Investigation on trace elements distribution in hot an cold mineral spring waters in the south Korea. Bull K H Pharma Sci, 12, (1984). 5. AOAC. Offcial methods of analysis(15th ed.). Arlington, VA : Association of Official Analytical Chemists (1990). 6. Choi JS, Park JH, Kim HG, Young HS, Mun SI : Screening for antioxidant activity of plants and marune algae and its active principles from Prunus daviana. Kor J Pharmacol 24, (1993). 7. Scudiero DA, Shoemarker RH, Paul KD, Monks A, Tiemey S, Nofziger TH, Currens MJ, Seniff D, Boyd MR : Evaluation of a solube tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res 48, (1988). 8. Yahagi T, Nagao M, Seino Y, MSTSUSHIMA T, Sugimura T, Okada M : Mutagenicites of N-nitrosoamines on Salmonella. Mutation Res 48, (1997).