A photo-activated, protein-based, NO/H 2O 2 generating system with tumoricidal activity composed of the nitric oxide derivative of apo-metallothionein (thionein-NO) and glucose oxidase

The S-nitroso derivative of apo-metallothionein (thionein) was prepared by transnitrosation with S-nitroglutathione. The thionein-NO thus formed has an absorption maximum at 334 nm. Light-induced NO release from thionein-NO was demonstrated by flash
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  "i" ....... A.~D RtOIIIt~I~'Y i1= ~11 ,ul .,, E LS EV l E R Journal of Photochemistry and Photohiology B: Biology 41 I ItS7 ) 249--254 A photo-activated, protein-based, NO/H.,O., ge,lerating system with tumoricidal activity composed of the nitric oxide derivative of apo-metallothionein thionein-NO) and glucose oxidase ~ Marie Tannous , Natalie Labbe ~, Robert W. Redmond b Bulent Mutus ~'* Departmelzt oJ'Chemi.~'tr and Biot'hemi,~try, I'niver.wty oJ' Wmd.w~r. Windxor. Ont.. NgB 3P4. Canada h tVelhnan l.ahmatories ,?fPhotomedicine. Department a[Dennamlogy. Harrard Medical S,'h,,t,L ,~ht.~sachu~ett.v General Hov, itaL B,,.~u,t..tlA 112114. USA Received 23 July 1997; accepted 3 November 1997 Abstract The S-nitroso derivalive of apo-metallothionein thionein ) was prepared by transnitrosation with S-nitrosoglutathione. The thitmein-NO thus h)rmed has an absorption maximum at 334 rim. Light-induced NO release from thionein-NO was demonstrated by flash photolysis. This system produces peroxynitrite at neutral pH as evidenced by nitrotyrosine formation. The c2~toloxic po ential of this protein-ha,veal, ight- activated NO/H,O2 generating system was demonstrated by exposing human colon adenocarcinoma cells (SW 948) in culture ~o thionein- NO and glucose exidase in the presence and absence of light. The cell density ot the samples. 72 h subsequent to receiving 1 h of light exposure, decreased by ~ 98cb, relative to controls. In comparison, cell density of the samples that were incubated in the pre~nce of catala,~ and did not meceive light treatment, decreased by only ~ 22% after 72 h. © 1997 Elsevier Science S.A. Keyword.w Metallolhioncin: Nitric ~xidc, Glucose oxidase: Pcroxynitril¢: Hydrogen peroxide I Introduction Recent evidence indicates that the cytoxic activity of NO is amplilied by ~ 2.5 [ 1 ] to eight-lold 12 ] in the presence of hydrogen peroxide. The exact mechanism of toxicity is unknown but is thought to involve a strong oxidant tbrmed from a combination of trace metals/NO/hydrogen peroxide. NO when produced in excess is cytoxic [31. In the cellular milieu, the toxic effects are augmented by its reaction with superoxide ( O:'- ) yielding peroxynitrite ( ONOO - ). Peroxynitrite ( OONO- ) is formed by the reaction of nitric oxide (NO) and superoxide anion (O_,'-) [41. OONO- once protonated, rapidly decomposes to H + and NO3-, through an intermediate complex possessing characteristics of both nitrogen dio:dde (NO_,') and hydroxyl radical (OH') I51. In proteins, peroxynitrite decomposition most com- monly leads to the nitration of Tyr and Phe side chains as * Corresponding author. Fel.: + 1-519-25342 32: fax: + 1-519-973 7098: e-mail: J The technology described n this manuscript s the subject of a previsional US patent application iled November 1997. I01 I-1344//$17.00 ~ i997 El.~vier Science S.A. All tights re,fred P IS I01 I- 1344( 97)00115-2 well as ~he oxidation of Trp [61. In fact, tyrosine nit~ttion of proteins is used as evidence for the in vivo detection of OONO- [ 71. OONO- formation has been detected in acti- vated macrophages and other cells associated with eucaryotic defense mechanisms which can generate both NO attd super- oxide in large amounts [ 8-101. Reaction of NO with thiols like glutathione (GSH) resuit~ in stable S-nitrosothiolates (RS-NO). We have ob~rved that S-nitrosothiols are photolabile. Irradiation with either 545 or 350 nm light leads to the release of NO [ I I, 121. Metallothioneins (MTs) are ubiquitous low molecular weight (6000-7000 D) mehai-binding proteins 1131. MTs are ideal candidates as stable protein NO carriers as they are rich in Cys residues: 20 oft,.: total 60 amino acids comprising human MTs are cysteines [ 141. It. this study, we have used the enzyme glucose oxidase, which catalyzes the conversion of glucose to glcconic acid plus H_.O2, and the S-nitroso derivative of apo-metallo- thionein (thionein-NO), as the NO carrier. Thionein, with its 20 Cys thiols, can carry 20 times more NO in comparison to small molecular weight S-nJtrosothiols like S-nitrosogluta-  25(1 M. Tannous et aL /Journal ~ Photochemistry and Photobiology B: Biology 41 1997) 249-254 tnione. Thionein-NO is stable to transnitrosation by physio- logically relevant thiols. Furthermore. the photosensitivity of the S-nitroso bond means that this peroxynitrite generating system can be photo-regulated. The cytoxicity of thi~ system has b'~en demonstrated against SW 948 cells in or, lit, re. 2. Materials and methods 2. I. Cell culture The human colon adenocarcinoma cell line SW 948 was pufcha~d ffoi-a ihe American Type Culture Collection (ATCC CCL 237, batch F-12139). and maintained in 90% Leibovitz's L 15 medium (Gibco) plus 10% heat-inactivated fetal bovine .serum. in a CO_,-free incubator with atmospheric air at 37°C. Viability was determined by the ability of the cells to exclude trypan blue dye (0.2 vol.C~ final concentration ). 2.2. MT purification Rabbits were injected subcutaneously with ZnCI2 ( 100 p.g/kg body weight) daily for 7 days in order to induce MT. Rabbit liver ( 50 g) was homogenized in 80 ml of cold ( 5°C ) Buffer A (20 mM Tris-CI. 250 mM glucose, 100 mM ~mercaptoethanol, pH 9.3). The cytosolic fraction ( ~50 ml L obtained by centdfugation, was chromatographed on a column of Sephade;, G-75 ( 5 cmx 50 cm ) equilibrated with Buffer B (Buffer A without glucose). The low molecular weight fraction with characteristic red-orange fluorescence was collected and applied to a column of DEAE-cellulose (2.5 cm×5 cm) which was then washed with 50 column volume,; of Buffer B. The crude MT was eluted with an NaCI gradient ( 0-0.25 M ). The MT was further purified on a Bio- Logic system (Bio-Rad) on a Merc LiChroprep RP- 18 col- umn with a linear gradient (0.1 vol.% trifluoroacetic acid { TFA)--60% vol./vol, acetonitrile/0. I vol.% TFA). ~ arified rabbit liver was also purchased from Sigma ( St. Louis. MO). 2.3. T-NO synthesis MT ( ~ 20 mg in 5.0 ml Buffer B) was then incubated overnight with dithiothreitol (DTI') (50 rag) at 5°C. This mixture was incubated with 50 ml of TFA in order to remove the metal ions from MT. The small molecular weight com- pounds were removed by chromatography on a column ( 1.5 cm × 20 cm) of Sephadex G-25 ( superfine ) equilibrated with 0. 1% TFA. Tne concentration of thionein was determined using its molar absorption coefficient at 214 nm (48 200 M - L m ' ). The free thiol concentration in the thionein was determined by Ellman's reagent [ 15 ]. The T-NO was formed by incu- bating the thionein solution (in 100 mM Tris-CI, pH 8.0), with 100-fold molar excess (over free thiol) of S-nitroso- glutathione (GSNO) for 30 rain at room temperature. The excess GSNO was removed by chromatography on a column ( 1.5 cm X 20cm ) of Sephadex G-25 (superfine) equilibrated with I00 mM Tris-CI, pH 8.0. T-NO was protected from light by aluminum foil. The molar extinction of thionein NO was estimated by Eq. ( I ), where the absorbance of T-NO ( 334 nm ) was divided by the product of free thiols/T ( deter- mined by Ellman's reagent and the extinction coefficient of T (214 nm) ) and IT-NOI ( [T-NOI = [T] ×dil factor in G-25 chromatography). A3.o, nm mol SH ,',,~' 7 x IT- NO] The molar absorption of thionein S-NO estimated by this procedure was 1820 M ~ cm- ' at 334 nm. 2.4. Measurement oJphotolydc NO release fronl T-NO by flash photo vsis The laser flash photolysis apparatus was essentially as described by Krieg et al. and Avelinc et al. [ 16,171. The excitation source was the frequency-tripled output ofa Quan- tel YG660A Nd/YAG laser at 335 (pulse duration of l0 ns, 620 p.I/pulse) (Continuum, Santa Clara CA). A solution containing T-NO ( 1.2 p,M) plus oxyhemoglobin ( l0 p.M) in sodium phosphate ( 100 raM, pH 7.4l was exposed to five 3.3 mJ pulses. The change in absorbance (425 and 405 rim), subsequent to each pulse, was monitored as a function of time. 2 5 Measurement t~photolylic NO release of T-NO by direct inetlsllrenlelll till till NO, analyzer T-NO ( 1.2 p,M, total volume 1.0 ml ) was placed in a glass screwcap test tube ( 15 ml ) fitted with a rubber septum which was equilibrated with N2 gas. The beadspace gas was with- drawn with a gas-tight syringe (25 ml) and directly injected into a chemilumnescent NO analyzer (Thermo Environmen- tal Instruments model 42). The gas withdrawn was replaced with N2. 2.6. Measnrenlent of peroxynitrite formalion by monitoring nitration of tyrosine at neutral pH Four 1.0 ml samples of L- 15 medium containing BSA ( 100 Ixg ). T-NO ( I 0 p,M ) and glucose oxidase (GOD) ( 62 p,M ) were made. To two of these samples I mg of catalase was added. Two samples, one with catalase and one without, were exposed to light from an overhead projector for I h while the other two (+catalase) were protected from light. 20 p,I of each sample were treated with an equal volume of sample application buffer (0.017 M Tris, I% SDS, 20% glycerol, 4 saturated bromophenol blue and 10% 2-mercaptoethanol vol./vol, at pH 6.8). The mixture was boiled for l0 rain. 20 [~1 of the reduced denatured sample were applied to 10%  M. Tannous et aL / Journal of Photochemistry and Phom'~ioh~g.v B: Biology 41 ¢ 1097j 249-254 251 SDS--polyacrylamide gels for electrophoresis. Resolved pro- teir, bands were transferred electrophoretically onto a nitro- cellulose support medium at 50 V for 30 rain using a transfer buffer of 25 mM "Iris base/192 mM glycine/20% methanol. The blots were blocked overnight with 50 mM Tris base. pH 7.4 containing 0.5 M sodium chloride (TBS) and 2% poly- vinylpyrrolidone. After washing with TBS containing 0.05% Tween-20, pH 7.5 (TI'BS), the blots were incubated for I h with anti-nitrotyrosine (rabbit monoclonal lgG) diluted 1:400 in TI'BS. The excess antiserum was removed by 4 x 5 rain washings with TrBS. The blots were then treated with a i:3000 dilution of anti-rabbit-lgG conjugated to alkaline phosphatase in "ITB~q for I h. After two washes in TI'BS, one wash in TBS, bromochloroindolyl phosphate/nitroblue tetra- zolium substrate was added to visualize the primary/second- ar) antibody complexes. After color development the blots were washed with distilled water and dried between paper towels. 2. Z Cell viabili~. Monolayer cultures: SW 948 cells were grown in Leibov- itz's L- 5 medium, 90%; fetal bovine serum, 10%; antibiotic- free. The cells were detached using 0.25% trypsin. 0.03% EDTA for 15 rain. resuspended in media and centrifuged twice. They were then transferred to four sets of 12-well plates at a final concentration of I 210 000 cells/ml, and exposed to one of the following treatments, using (+) camlase (I mg/ml) in each treatment (in triplicate, total volume 1.0 ml): treatment I: T-NO ( 10 ItM); ~,tt:,,mcm 2: T-NG ( 10 p.M), GOD (62 p,M); treatment 3: GOD, 62 p,M; treat- ment 4 (control): cells in 1.0 ml of medium. Two sets were covered with aluminum foil, the other two sets were exposed to light (10 cm above lnFocus® overhead projector) for 60 min, both placed in a 37°C incubator. The dark and the light-exposed samples were counted with a hemocytometer at I and 72 h after exposure to the various treatments in the presence of Trypan Blue. 3. Results and discussion The UV-Vis absorption spectra of T (thionein) and T-NO are presented in Fig. I. T is spectrally transparent in the wavelength region 330--500 nm [181. The small peak observed ~ 285 nm with T (Fig. I, open circles) is thought to result from incomplete removal of the metal ions bound to metallothionein. The formation of the NO derivative of T (Fig. I, filled circles) is confirmed from the characteristic S-nitroso absorbance at 334 nm [ 191. The molar absorption coefficient (at 334 nm) for thionein S-NO was estimated to be 1820 M-~ cm-~. This value is approximately two-fold larger than S-NO absorption coefficients observed for small molecular weight thiols [201 and approx, two-fold smaller than that reported for the NO derivative of bovine serum albumin [ 211. '<11.5 m 311 410 511 ~ll (-,-) Fig. I. UV-Vis spe~:tm of thionein and thioncin-NO: hioncin ( 32 I.LM Iopen circlc.,O, hionein-NO derivati'.'e (32 F.M) (filled cir¢le~) in O.I M sodium phosphate, pH 7.4. The photolability of the S-NO bond of T-NO is demon- strated in Fig. 2. 1.0 ml aliquots ofT-NO { 15 p,M) in phos- phate buffer (0.1 M, pH 7.4) were added to two matched cuvettes (semi-micro). One cuvctte was wrapped in alumi- :~um foil in order to protect it from light. Both cuvettes were then placed l0 cm above an overhead projector ( lnFocus® ). UV-Vis spectra were recorded at various time intervals fol- lowing light exposure. The spectrum of the ,,+ample protected from light was unchanged after I h (Fig. 2, open triangles it=O) and filled circles (t= I h) ). The absorption peak of the photo-irradiated sample decreased by ~ 56% after 15 rain of exposure (Fig. 2, filled triangles) and was nearly totally abolished after I h of exposure ( Fig. 2, open diamonds). The stability of T-NO to transnitromtion (Eq. (2)) by thiols was te+~.ed b~' RS-NO + R'S - --+ ,S- + R'S---NO (2) adding GSH, L-Cys or/3-mercaptoethanol (fl-ME) ( I mM ) to T-NO ( 22.3 itM, 18.9 NO/tool ). Since the S-NO extinc- 0,0 ~-- +. .... -_ ...... ,3100 ~ 400 4SO SOl rim) Fig. 2. UV-Vis spcctPa f thionein-NO as a fimetion f the time of pho~oir- radiation. hionein-NO 15 l.tM) pro~ected rom light at t=0, open Lri- angles- t= 1 h. filled circles); thionein-NO ( 15 p.M) expo.~d to light li~" various ime ntervals: = 15 rain ( filled riangle+,+ . t = I h ( open diamonds) in 0.1 M +sodium phosphate, pH 7.4.  252 M. Tamums et al. / Jourmd t~f Photochemistty and Photohio~ogv B: Biology 41 1997) 249-254 tion coefficient ofT-NO (334 nm. 1820 M - e cm - ~ is larger than those of GSNO (960 M - ~ cm - ~ . L-Cys-NO ( 870 M - cm- ~ and ,8--Me-NO ( 772 M - ~ cm - ~ I 17 i the transnitro- sation rate constant can therefore be estimated by monitoring the decrease in the thionein-S-NO absorbance at 334 nm as a function of time. The second order rate constants were estimated from the pseudo-first order treatment of ',he tran- snitrosation data. The largest k2 (0.163 + 0.0 56 M - ~ s - ~ was obtained with GSH. whereas L-Cys yielded the smallest (0.000686+0.000034 M-e s-e). The non-physiological thiol, ft--ME resulted in an intermediate rate constant (0.0340_+ 0.00481 M - ~ s- r ). These rate constants will yield very slow reaction rates with physiologically relevant con- centrations of the reactants. Serum [ GSH 1 is ~ I 0 I-tM 122 I. if 10 p,M of GSH and T-NO are left to react. 50% of the T-NO-bound NO would be lost to transnitrosation in 7.09 days. In order for T-NO to function in this system, it must yield NO upon photolysis, in previous studies, we have shown that al:hough small molecular weight S-nitrosothiols such as GSNO do yield NO upon photolysis, the S-NO derivative of the protein, bovine serum albumin does not [ 121. It wa,,, therefore crucial to test whether NO could be generated by T-NO photolysis. It is well documented that NO (and not NO * or NO-) reacts with oxyhemoglobin ( Fe-" *. A ... 425 nm ) converting it to methemoglobin (Fe ~'. '~m~ 408 rim) I23-251. In this study T-NO ( 1.2 p,M) was photolyzed by 355 nm light in the presence of oxyhemoglobin ( 10 pM ) in a flash photolysis instrument. The absorbance changes subsequent to the nan- osecond flash were monitored at 425 and 408 nm (Fig. 3). The liberation of NO was confirmed as the absorbance increased at 408 nm and a concomitant decrease was observed at 425 nm at the same rate (Fig. 3). Irradiation of oxyhe- moglobin alone gave rise to no transient absorption changes at either wavelength ~ not shown). In order to further confirm photolytic NO release, a sample of T-NO ( 1.2 p,M, total volume 1.0 ml ) was placed in a glass screwcap test tube ( 15 ml) fitted with a rubber septum. The head space gas was withdrawn with ~; gas-tight syringe 25 ml and directly injected into a chemilumnescent NO analyzer. There was no NO detected when the sample was protected from light. 1-..'awever, upon exposure of the sample to light fiom an overhead projector for I h. the head space gas contained ~ 4 ixM NO. In the next step of these studies we tested the cytoxic potential of producing NO and H_~Oz molecules by a protein- based means in the extracellular milieu. In these cell culture experiments, glucose oxidase was chosen as an enzyme- based method for the production of H,O_~ . This enzyme catalyzes the conversion of glucose to gluconic acid plus H_~O_, 1241. This enzyme can also produce H_~O, from D-galacto~. albeit at 0.5% of the rate observed for glucose 125], This is significant as the media for the cells used (human colo~ adenocarcinoma cells SW 948) contains D-galactose ~,nd not D-glucose. The other component for o. t'o~ . ,; | ***' ~,T.¢ nll~q ~A ~ ~ .~.o~ ~ A -oo0~ -Oar s i a * ooooo ooo~ o nnlo o O01s 0 OO2O 11 me w..) Fig. 3. Flash photolysis evidence for the photo-generation of NO from T-NO. Transient absorption changes were measured at 408 (circles) and 425 ( riangles ) nm li~llo,.','ing irect excitation at 355 nm ofT-NO ( 1.2 ItM ) in the presence of oxyhemoglobin ( 10 p.M) in sodium phosphate 0.1 M, pH 7.4. ONOO- production. NO can be produced photochemically by photolysis of T-NO. In these experiments, which are summarized in Fig. 4, the ce '~ were exposed to four different treatments: I, T-NO: 2, T-NO + GOD, 3. GOD: 4. control ( cells in I ml of media ). all ( + ) catalase. One compt-.te set was exposed to light for (60 min) by placing the 12 well plates on an overhead pro- jector. The other set was kept in the dark. The effectiveness of T-NO and GOD in combination is evident after I h in the irradiated set, where the cell viability decreases by ~ 76% without catalase and by 2% with catalase. This value increases to 98% (no catalase) and to 65% (catalase) in 72 h. in contrast, in samples receiving GOD, T-NO, ::L:cata- lase and no light the cell viability it decreased by only 56% ( no catalase) and 22c;~ (catalase) in 72 h indicating that H _,O_, is an essential component of this cytoxic system. However, irradiated T-NO was also quite effective. 67 and 91% kill being observed after I and 72 h. respectively. This is thought to result from T-NO derived NO interacting with endoge- neous, intraccllular HzO_,. which cannot be removed by the extracellular application of catalase. For clarity the actual % values obtained after the various treatments are given in Table I. Farias-Eisner etal. 12 ] have demonstrated that NO/H-,Oz tor.icity is mediated through metal ions ( traces ofFe ~ ÷/Fe -~ + ). These authors suggest that NO can reduce trace Fe 3 ÷ by Eq. (3). Fe-" + thus produced can yield "OH via Eq. (4). NO+Fe 3~ --,1Fe~ +/-NO~Fe-~ +/-NO ÷ ] (3) Fe -~ ~ +H _, O, --*Fe 3 + +HO- +'OH (4) They also suggest two possible routes for ONOO- produc- tion ( Eqs. ( 5 )-( 7 ) ) via the reaction of the products of Eqs. (3) and (-1). [ Fc3 +.NO,~Fe-" +_NO + I + H_, O_,-,Fe-~ ÷ ONOO- + 2H ~- (5) • OH+H20,-*H20+'OOH (6)  M. Tamum.~ et aL /Jounial o[Photochvmixto and Phorohioh~g~ B: Biology 41 t 1~#~7; 24~,L254 253 r L+C lhr L F:g. 4. The cytoxic effect of T-NO and gluco~,e oxidam to human cohm ademx'arcinuma cells in culture SW 948 con ,,iabilit,,. I and 72 h al~er cxpor, re tu the following treatments: I. T-NO t 60 aM ): 2. T-NO t 60 la.M ) plus (;OD ( 62 itM ): 3. (;O13 162 p.M )" -I. coronal, cells in grov, h medium ahme. either in the dark no catalase (D). treated with catala,,e ( D + C I or subsequent to t~) sin o1" expo,,urc to light no catala,.e t L I. treated ,.,.+ith calala~,e I I. + C I. Viabilit.x is expressed as a ";;- of control cell density ( i.e. treatment 41 at each time i't~fint. Table I Actual ¢,; values obtained after the various treatments Treatment Cell viabi itv ( +3 ) I h light I h dark 72 h hght 72 h dark T-NO 33 t,'0 9 7O T-NO + catalase 76 89 19 81 T-NO + GOD 24. 76 2 44 T-NO + GOD + catalase 98 t~0 35 78 GOD 88 75 3t,J 91 GOD + catalase IIH) 86 77 94 Control IIX) I X) I X) ] X) Control + catala~,e I In 1 x) I in I x) NO+'OOH-,ONOO + H + (7) When mM NO (g) was introduced to trace H202 and Fe salts ( 0.4 p.M )+ trace amounts of NO~ -. the stable decomposition product of ONOO-. were detected. In the present study, the cell culture medium ( Leibov:,:z's L-15) is composed of 2 to 140 mM inorganic salts and no metal chelators, as a result, trace Fe ,on contamination is expected. We therefore wanted to check for ONGO- pro- duction under the condition:+ employed in the cell culture experiments. Nitrotyrosine has been widely used as a marker of peroxynitrite formation. In these experiments. BSA sam- ples in L-15 medium plus T-NO and GOD with or without catalase were irradiated for I h or kept in the dark. The samples were electrophoresed, transferred onto nitre.cellulose and probed with anti-nitrotyrosine (rabbit) primary antibod- ies followed by anti-rabbit IgG conjugated to alkaline phos- phatase. The results are presented in Fig. 5. A single nitrated band at the correct molecular mass for BSA was detected when the sample was treated with light for I h in the absence of catalase ( lane 2 ). On the other hand, samples treated with light and catalase ( lane 3 ) or dark ( lane 4 ) show no evidence of nitrotyrosylated protein These results indicate that some peroxynitrite is generated when NO is photolytically release in the presence of the enzymatically generated H,02. How- ever. the question of whether ONOO- is responsible for the observed cytoxicity cannot he answered at this time. Both metallothionein [ 261 and GOD [ 27 [ ha~,e previously been conjugated to antibodies. The cy~otoxic potential dem- onstrated here by the combination of T-NO and GOD in solution could be increased many fold upon conjugation of T-NO and GOD to antibodies raised against specific cancer cell antigens. In summary, we have demonstrated that metallothionein. once stripped from its metal ions. can serve as a potent NO carrier. Thionein can carry up to 18 mol of NO/mol via S-nitrosation of its 20 constituent Cys thiols and thus serve as a multiple source of NO (c.f. GSNO. which produces only one NO per molecule). T-NO is stable to transnitro~tioa by the ubiquitous intra- and extracellular thiol, glutathione. The S-nitrosothiol bond of T-NO is photolabile and yields NO upon photolysis. This property can he utilized for phot(x:hem- ical release of NO from T-NO. We have shown that ONOO can be generated upon photolysis of S-nitrosothiols in the presence of H202. We were able to demonstrate that the combination of T-NO with GOD in the pre.~nce of visible light can decreases cell viability of human colon adeno- carcinoma cells by 98% within 72 h. We suggest that the effectiveness of this technique may bc enhanced by the co-
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