Item ID Number °2434 D

Author Andersen, Melvin E.

Corporate Author

RBDOrt/ArtldeTltlB The Toxicity of Perfluoro-n-decanoic Acid and 2,3,7,8-Tetrachlorodibenzo-p-dioxin in L5178Y MouseLymphoma Cells

Journal/Book Title

Year 1983

Month/Day March

Color D

Number of Images 14

DBSCriptOn NOteS AFAMRL-7A-82-50

Thursday, November 01, 2001 Page 2434 of 3007

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THE TOXICITY-OF PtRFLUORO-N.DECAKOIC ACIDr 1

IS178Y MOUSS: LYMPHOWA CELLS) i

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WCkOli'OWGICAL ASSOC/A TESs>21 RIV&R ROADKETHSSDA. MAX 1'i

KEtMETH&BACK, PH.D.

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I'l#aas tlfl not request copio* of thiB rej>ort frorn Air Force A<*rc«spa« M(;dic;.l Itoturcli Lc« !>«».» tor y.Additional copiva may bi purchased from: .

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Ft-dcnil Govcrnmifnt sacncits and iholr contractors te»;i tiered witK Dt!i«ir.-j TcchnJca! InformationM.-r i:Vtould (Urc-ct rcqut-stn far copi<>» of thir- report to: .

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TECHMSCAL REVUSvV A!*O APPROVAL

AF/C'J.IL-TR-82-.IO

<" AT) . IN C** TMtS f*

REPORT DOCUMENTATION PAGE

* 1 I TUT f r/tlf .'.«rr,flHr'

The T o x i c i t y of I'orf luoro-n-di*caiti>ic Acid and2,3,7,B-Tctr . ichlorodibenzo-p-dioxin in L51V8YMouse l.yiophoiiia^ Cells ___

* ~ ....... ~. «H. E. Anderson. A. M. Rogers*, M. E. Georgeand K. C. Biick**

AKAMKL, Toxic ll.izards Division, AMD, AFSC,Wrip.ht-Pattcrson AFB, Ohio 4 54 3-3

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Technical Kcport

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. . .« *-}K« U N I T NUUUCnS

6'ntI'O»7 DATE

MARCH 1903p»oe

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Approvotl for public release; distribution unlimited.

II. iUPPLCMCNTAHV NOTCS

* Microbiological Associates, 5221 Rtv««r Road. Bcthesda, MO 20816** Department of Preventive Medicine and Biometrics, UniCorned Services

University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814AFAMRL Primary Investigator: Dr. M. E. Andersen, AFAMRL/THB, (513) 255-5150.t KCV WORDS fCootlm/. «i i.v.». «n» «l rPerfluoro-n-decanoic AcidToxicologyL5178Y Mouse Lymphoma CellsCell CultureKrythrocyte Fragility

2,3,7,8-Tctrachlorodibento-p-dioxin•Polyfluorinated acids

V.

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•• VPerfluoro-n-decanoic acid (PFDAj causes toxic sequelae in vivo very siailarto those caused by 2,3.7,8-tetraehlorodibenco-p-dioxin (ICDD). The toxicity ofthese two compounds, several other polyfluorinated fatty acids, and correspondingliydrogenated fatty acids have been studied in vitro in L5176Y mouse lymphoraacells. Below concentrations which cause cell lysis f>500 i*g/tal)t PFDA did notaffect .suspension growth. After 24 hr treatment with concentrations between Iand 100 flg/ml treated cells no longer grew into clones when plated in semi-softagar. This impairment of clone-forming ability was reveiciole att»r growth of %

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treated cells in fresh medium for 36 hr. Pert luoi'o-n-octanoic acid did notimpair clon«i- Co fining ability at any concentration; anil neither did thestraight-chain hydroRi-n.it «?d Catty acid Analogs. All polyfluorinalotl acidstested (either p*r( luorinatc'J or w-hydro-analogs) wilh chain length 9 orgreater caused ir.ipauinunt. of clone-forming ability aftur treatment withconcentrations that were non-toxic in suspension. TCD1) (highest dose, 0.5'(fg/ml) had no effect on growth in suspension^ After A8 hr treatment with TCDDconcentrations of 0.01 iig/ml or greater, placed ccllts formed clones withaltered morphology. These clones were less discrete, lacking a clearlydefined boundary. The effect on clone morphology required 36 hr treatment ofcells with TCDD in (suspension and was reversible following 48 hr growth infresh medium. Cell division time in suspension was 10-12 hrs and wanunaffected by I'FDA or TCDIKiyln vivo PFUA treatment altered erythrocytefragility in rats. . It is suggested that the toxicity of I'FDA and TCDD in vivoand in 1.517BY cells in vitro may be due to an ability of these chemicals tointerfere with normal structure and/or function of biological membranes^.

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PRtFACK

This research was performed in the I'.icchemical Toxicology Branch, ToxiclUir.ards Division, Air Force Aerospace Medical Research Laboratory fromJanuary 1980 through December 1981. It was performed in support of Task2312V1, "Toxicological Mechafiisms of Air Force Cheraicols and Materials;"Work Unit 2312V118, "Effects of Air Force Propel Iants and Chemicals onMetabolic Mechanisms." Portions of this work were presented nt the 21stAnnual Meeting of the Society of Toxicology, Boston, Massachusetts, 22-26February 1982.

Accession ForKTIS CRAfclDIIC TABUnftnuc'incedJustification-

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ItiTKOUUCTION

I'er lluorinated f.'tlty Jcids, \>r;ri luorinated sulfonic acids, nud appro-priate derivatives ari' used cc-..aut!rci<il ly in numerous applications which takeadvar.caf.o of their exceptional surf.'ictant properties and extreme chemicaland thermal stability (Cuenthncr and Victor, 1962). Most commerciallyimportant derivative!* are based on perfluoroalkyl chain lengths of 5 to 1.The acucc and subchronie toxicity of ammonium per£luoro-n-octanoatc (PFOA)has beo.n described in detail in both rats and rhesus monkeys (Griffith andLong, 1980). Loss is known of the tox.icity of longer chain analogs.

In an abstract Andersen ct al. (1981) described the acute toxicity ofperfluoro-n-decanoic acid (I'FDA; nonadeca£luoro-n-decanoic acid; CiQF^O^U)in a variety of rodent species. Thin acid was significantly more toxic thanPFOA and its toxicity differed both quantitatively and qualitatively fromthat of the shorter chain analog. Tox.ic signs and target organs for PFDAwere similar to those seen with 2,3,7,8-tetrachlorodibenso-p-dioxin (TCDU).The single dose oral LD<JQ - 30 days of PFDA in male rats was about 30 rag/kgand rats intubated with 90 rog/kg lost nearly 50% of their initial bodyweight before dying two to three weeks after intubation. As docs TCDD, PFDAcaused severe thyoic atrophy.

As part of a comparison of the biological effects of PFOA and TCDD, wehave evaluated the toxicity of those chemicals in Fuvural isolated cellsystems. In part, this paper describes effects of various poiyfluorinatedfatty acids, hydrogonared fatty acids, and TCDD on growth characteristics ofL5178Y incuse lyirphoma Cislls, a T-cell derived lymphona (Muller et al., 1981),which grows both in suspension and in semi-soCt .ipar. A T-cell lyctphoma wasused because T-lyaphocytes appear to be targets of I'FDA and TCDD toxicity inrodents. This conclusion was based on the narked thynic cortical atrophynoted in animals treated with either of these chemicals. L5178Y cells arecommonly used for mutagenicity testing and the mutagcnic potential of thesechemicals in L5178Y cells is reported elsewhere (Rogers et al., 1982). Inaddition, United results of osmotic fragility studies of erythrocytes fromrats treated with PFDA are described in an attempt to relate altered osmoticfragilities to the effects of PFDA on L5178Y cells.

MATERIALS AND METHODS

LS178Y Mouse Lymphoroa Cells; L5178Y. cells were originally obtained fromDr. C. F. Arlett. MRC, Cell Mutation Unit, Brighton, England. They wereroutinely screened for raycoplasna contamination. The routine methods formaintenance of L5178Y cells and the soft agar cloning technique were asdescribed elsewhere (Cole and Arlett, 1976), except that McCoy's 5A medium(supplemented with penicillin, streptomycin, sodium pyruvate, and IOZ horsescrum) was used instead of Fischer's medium. For toxicity experiment*,L5178Y cells were treated for 24 hrs with doses of PFDA ranging from 0.01ug/ml to I mg/ml, or for 48 hrs with doses of TCDD ranging from 0.001 tig/mlto 0.50 iig/ml. At the end of the treatment period, cells were centrifuged,washed in McCoy's 5A medium and reeuspcnded in McCoy's 5A containing 20Zhone serum. Cells were plated for growth in soft agar, and plates wereexamined for clones after 9-10 d«ys incubation in a humidified CC>2

incubator. Horse serum was obtained from Gifoco-Uiocult. Penicillin,str<>|icoiriycin, and sodium pyruvate wore obtained from Sigma.

Ch'.'mica 1 c: Fatty acids (ail > 99% pure)', pcrfluoro-n-decmioic acid (per(luoro-n-octanoic acid (>96£)*, ll-H cicosafluoro-n-undecanoic acid(97-99%)2, and 9-H hexadecafluoro-n-nonanoic acid* were obtained com-mercially. Thc> latt<?r two compounds contain a single hydrogen at the omegaposition. I'erfluoro-n-dodccanoic acid*1 (71Z CnF23C02ll; 3% CiQF2iC02H; 22CgF^C^H; remainder unidentified, nonfunctional fluorocarbon) and TCDD weregifts5. For L5178Y studios, TCDD was dissolved in acetone and the fattyacids and fluorinated analogs were dissolved in dimethylsulfoxide exceptpcrfluorinatod dodecanoic was also dissolved in acetone.

Osmotic Fragility: Male Fischer 344 rats (200-300 g) were treated ip withSO mg PFDA/kg, I'ropylene glycolruater (50:50 v/v) was used as diluent witha final dosing volume of 2 ml/kg. Treated and diluent control rats ingroups of four to five were killed at various times after injection. Bloodwas drawn from the inferior vena cava after opening the abdomen of anesthe-tized rats and erythrocytes harvested by centrifugation. Osmotic fragilitywas determined as described in Dacie and Lewis (1963). Curves were con-structed for heraolysis at 10 saline concentrations between 0.25 and 0.852.Data presented are percent hcmolysis at a single intermediate saline concen-tration, 0.42.

RESULTS

Fatty Acids; PFDA had little effect on 1,5178Y suspension growth at concen-trations below 100 ug/ml (Fig. I). At concentrations of 500 Pg/ral or above,cells were dissolved by the surfactant action of the acid and neither cellsnor debris were visible in suspensions at these concentrations. In compari-son to the dose-response curve for suspension growth, the curve for clone-forming ability was shifted some 2.5 log units to the left: the EDjo-24 hrfor impairing clone-forming ability was approximately 3 x 10"*• ug/nl. Toour knowledge, this ability - dissociating the markers of suspension growthand clone-forming ability in these transformed cells - has not been reportedfor any other chemical. Perfluorinated-n-dodeca.noic and ll-H-eicosafluoro-n-undecanoic acid caused a similar displacement of the two dose responsecurves (Table 1). On the other hand, PFOA which was slightly less toxic tocells in suspension than was PFDA did not show the differential toxicitywith respect to suspension and clonal growth. The u-H-hexadecafluoro-n-nonanoic acid displaced the dose response curves for suspension and cloneforming ability, but the displacement was less than that seen with PFDA(Table 1), With hydrogenated fatty acid analogs from Cg to GU, toxicitywas equal both in suspension and in agar (Table 2).

1 Aldrich Chemical Company, Milwaukee, WI 53233.2 PCR Research Chemicals, Inc., Gainesville, FL 32602.3 Alfred Bader Library of Rare Chemicals, Division of Aldrich Chemical

Company, Milwaukee, WI 53233.* Commercial Chemicals Division 3M, 3M Center, St Paul, MN 55144.5 Dow Chemical USA, Midland, MI 46460.

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Table ?.

Hi" f . i toct of VArujnr. Fatty Acids c.n Growth of L5178Y Kou«- l.yi hoiM Ctllsin Suspension *nU on llioif Clone forn.irq Ability in bciM-Soft Agar

Non.uioic Acid_ Hocanoic Acid Uiutecanoic Acin"Dose SUMIOIISion Aijar SUspenstbn AjjoF SuVp*''CTo'«''"VaT

(liy/ml) (% Control) (X Control) (X Control)

0.01 85* 98 106 96 95 960.1 82 98 94 94 85 93

1 92 90 100 93 75 9610 90 94 84 90 8? 9350 85 98 75 8? 82 86

100 72 8B 63, 63 4? 44500 33, 26 «b - —b

1000 -b — «b — --*>

a Numbers in both columns arc growth as percent of growth'of controlcells.

° These concentrations dissolved cells in suspension.

Titac Co E f f e c t and Revers ib i l i ty : Cell division time for L5178Y cells insuspension under growth conditions used in this study was 10 to 12 hours.An experiment was performed to see if cells required a period of treatmentwith PFDA before diminished clone-forming ability could be observed. Cellswen; grown in suspension containing 0.5 ug PFDA/ml and removed at varioustimes for pla t ing to observe loss of clone-forming abil i ty (Fig. 2a). Therewas a lag of 8 hr before any appreciable e f fec t was observed and the time oftreatment required to reduce plating ef f ic iency to SOX of control was about12 hr , i.e., one cell generation.

Cells were also grown for 24 hr in the presence of 0.5 Ug of PFDA/mlharvested by centrifugation and washed in fresh growth medium. Thesetreated cells were resuspended for growth in fresh, uncontaminated mediumand aliquots withdrawn after various times for plating (Fig* 2b). Thedecreased plating efficiency was reversible, but recovery was more prolongedthan the time required to induce the diminished clone-forming ability. Thetime of growth in freth medium necessary to restore 502 plating efficiencywas nearly 36 hr, or about three cell generations. Cell division time ofL5178Y cells in suspension was unaffected by pretreatment with 0.5 ugPFDA/nl.

Piox in: In L5178Y cells TCDD did not dissociate growth in suspension fromgrowth in soft agar at any concentration tested, up to 0.5 Ug/ml. However,the morphology of the clones obtained after treating cells in suspensionwith concentrations of TCDD between 0.01 and O.S pg/ml, was markedlyd i f fe ren t from controls (Fig. 3). Instead of the well-circumscribed,circular clusters of control clones, those clones formed after dicxin-treatment were less-discrete and lacked a well-defined border. Aftergrowing cells in suspension for 46 hr in the presence of 0.003 ug TCDD/ml,

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Time (hr.)Figure 2. Time course of impairment and recovery of cloning ability in LS178Y cel'is treatedwith PFDA in suspension. A: Time to effect: cells were grown for various lengths of time(x-axis) in suspension in a medium containing 0.5 tig PFOA/ml and plated in semi-soft agar.Growth is expressed as percent of plated cells forming clones after treatment relative topercent of untreated cells which give rise to clones. B: Time to recovery: cells weretreated in suspension vith 0.5 jug PFOA/ml for ?A hr, and harvested by centrifugation. Aliquotswere removed and grown in fresh, uncontawinated medium for various lengths of time (it-axis).Cells were then plated to observe recover./ of the ability to form clones.

all clones formed after plating were normal; at 0.01 vg/ml, most clonesformed were abnormal; and by O.S tig/ml, all clones had altered morphology.By inspection of the plates, the £050, that is the concentration of dioxinrequired to produce alterations affecting 50% of the formed clones whencells were initially maintained in suspension with dioxin for 48 hr beforeplating, was about 0.01 pg/ml, i.e., about 3 x lO'^M.

In time-course experiments analogous to those in Fig. 2, but conductedwith 0.01 vS dioxin/ml, the time of treatment in suspension required toproduce 502 of maximum response in altering clone morphology was about 36hr. A time to recovery of normal growth characteristics was also estimatedfor cells grown initially for 48 hr in the presence of 0.01 Ug TCDD/ml. Thetime of growth in fresh medium required to give a SOX recura to normalclonal morphology was about 48 hr. As noted with PFDA, effects on clonegrowth were reversible, but recovery and expression times for the effectswere longer with TCDD than with PFDA.

Red Blood Cell Fragility; Rat red blood cells were obtained from ratskilled at various times after ip injection of 50 mg PFDA/kg. There wasincreased resistance to hemolysis after treatment with PFDA (Fig. 4) and thetime course of decreased fragility was similar to the time course of weightloss in treated rats (Andersen et al., 1981).

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B. Clones derived from cellstreat.td in suspension for48 hr with 0.25 u<j TCDO/mlwith UMSO as diluent.

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Figure 3. Altered clone morphology after treating 15178V cells in suspension with TCOO.

DISCUSSION

Knutson and Poland (1980) studied the effects of TCDD on 23 culturedcell types and found no toxicity in any of these mammalian cell lines attreatment concentrations of up to 10~ M and contact times of up to twoweeks. Markers for toxicity included (1) alterations in the morphology ofcells or the cell cultures, (2) percentage viable cells, and (3) growthrate. Among the 23 cell lines were five lyraphoid cell types derived fromthyraic cortex - three were murine and two were virally transformed humanleukocytes. All these cell types were tested for growth in suspension andcell viability by trypan blue exclusion. Beatty'et al. (1975) found thatTCDD had no effect on growth or morphology of normal human lymphocytes insuspension. Our results are similar to the extent 'that TCDD did not affectgrowth or cell viability of LS178Y cells in suspension. The altered growthcharacteristics observed in this paper are more subtle and only apparentwhen cells are grown in semi-soft agar, where they are constrained to gro»rin close proximity to each other. The concentration dependence of theeffect with TCDD is such that a 48 hr treatment with 0.01 pg/ml (i.e., about3 x 10~8H TCDD) causes the effect in most of the treated cells. Thisconcentration is reasonable for physiological significance stnce the mouse

is about 300 ug TCDD/kg, or about 1 Pmoles/kg (McConnell et al., 1978).7

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Figure 4. Relative osmotic fra^ilitiei of red blood cells from rats injected ip with60 m9 PrOA/kg and killed at various tiiw>i after injection. Data are iwan and standarddeviation (n « 4-5). From the overall curves with 10 salt concentrations, the concen-tration at which SOX horool/sis occurred was 0.43, 0.38, 0.34, and 0.43^, rcsperMvely,in treated rats at 2, 6, lo. sod 30 days. Control groupi it these sampling t ', s had505 iiemolysis at 0.45, 0.44, 0.45, and Q.43X. respectively.

Alterations in clone morphology seen after TCDD are striking, butest imations of concentration dependence are essential ly qual i ta t ive, i.e.,the percentage of abnormal clones is estimated by inspection and making adistinction between normal and slightly abnormal clones is diff icul t . Wehave maintained a restrictive definit ion of what constitutes an abnormalclone and scoring was done solely by Dr. A. M. Rogers. For these reasons,the estimated £050 Cor the effects with TCDD are probably high. Morequantitative determinations of these TCDD dose response curves awaitdetermination of the biochemical basis of the altered morphology and methodsto unequivocally identify altered clonal units.

With PFDA, results are readily quantified since treated cells no longerproliferate in semi-soft agar. The EDjQ - 24 hr for the loss ofclone-forming abilicy was 0.3 Pg/ml (i.e., about 6 x 10~'a); this contraststo a single dose 1,1)59 in mice of about 100-150 ing PFDA/kg or 0.2-0.3mnoles/kg (Andersen et a!., 1981; Van Rafelghem and Andersen, unpublishedexperiments, 1981).

With the polyfluorinated acids examined, this toxicity is present withacids of chain length greater than 8. The differences in single celltoxicity between the fluorinated octanoic and decanoic acids are striking,but consistent with the d i f ferent acute toxicity reported for these twoacids in rats. The hydrogenated fa t ty acids are without differential effecton clone-forming ability of L5178Y cells. In terms of cell lysis, expressed

8

as t.oxicity in sii.-.iicnsicri , livlro^.^n.-ii ri! .nut ; ,••! y i 1 u<u i;i.iti-d f a t t y acidsabciilt » '(U ' J't'l «.'llt (IriyK'S I ."•istl '.> ) .

Tin- mol.'Cul.ir l);u;i<. nf ih< - i<::p:< i r:::t'(il <•'. c lonc-l'ui ini.V; ;.!>i!ily i stiiiknown. Subtle change:; t:iay li.'tvv orciinvd .' r t-tll mi-nbrant.'!. to inhibit j-iowthof cells vh<M) maintained in close contact. In this regard, tin- i.'S.-v'U iofragility ritsultti surest a biological Kii/inb.-.'we i.iore t os isitnnt to liyi-u^snot i tinsult. Increased resistance can be- due to a variety of causv.s, on.- ol vliichis altered wi'.mbrmu.1 composition (Kuipor ot a 1 . , 1971). Prcl iroinjiry uLudit-s inour laboratory have- now shown that i-rythrocyt'-s Irora PF!M-tr«aU:«i .Mur.iaU alsoshow increased nwmbranQ f l u i d i t y (M. Cc-orj-o ,iiul M. K. Anderson, uti[uii)li.sh(-i1studies,' 19B2), and that thv total fatty acid coinposi lion' iii the liv«r l i p i dpool in these r.its shows a drar.uitic shift ^ownrd incn-asin;; unsaiur.'ition,I'Spec.ially in the Rtcaric to olexc acid ratio (Ulsoii cc al., I'.'lv?.). Whileif.dirc'ct, these results sup. nest:' composit ional <'ind functional altei'at. ions inmembranes subsequent to S'KDA oxposurt in tiic rat in vivo.

There is no unifying hypothesis explaining i he toxic ity of TCDD andmaterials causing; similar toxic effects, i.e., certain polyhalogenatcdbiphenyls (Sleight et al., 1981; Biocca et al., 1961) and long-chainpert loor inated fatty acids of chain length y or abox'e (Anderson -?t al.,1981). It may b>.» that these various chemicals, including I'FDA and TCDD, aretoxic due to effects on cc.ll weir.branes rosui t in,; in inicr fen-nice v.'ithcell-eel! or eel l-n-.nliator interactions. These effrcts could either be director c;ediatc(l by interference1 with same undogonuus horr.-.or.al control of t:ionibr;uiecocTiposit ion/ function. Toxicity would r.ot be a result ol cell necrosis orgrossly visible organellar alterations, but from wore subtle structuralalterations of biomembranes ar.<i attendant disc urbances in intercellularcommunication. This hypothesis is und-.-r active investigation in i>urlaboratory.

The cell line used for this research was so-called TK */+ with regard tothe gene locus for the enr.ycne thyuidine kinoae (TK) . Our stock of thesecells, brought to Dayton from England by Dr. Rogers, was destroyed during amalfunction of the deep freeze storage uint. .'We have not observeddifferential effects on suspension and clonal growth with L5178Y TK*/~ cells -a cell line more commonly used in mutation research and, therefore, much morereadily available. It appears that future work on this phenomenon will haveto be restricted to the TK*/* cells.

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Mti Her. W.E.C. . 7.ahn. I'.K.. M a i d t i o f . A. . Schrodt-r, II. C., and l:r.frawa, H. ,19.SI, "Bestatin, a stir.ulator of polyuon-.c asccxbly in T-ccll lycphoroaUS178Y)," Biochora. Ph.arciacol. )0, 3375-3377. ' ^

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