1 15 1 https://www.nal.usda.gov/exhibits/speccoll/files/original/2a1438290323f998babe3b6e753443a5.pdf 09154f08eff60cd54691dceac6b97c49 PDF Text Text Item ID Number Author °2434 D Andersen, Melvin E. Corporate Author RBDOrt/ArtldeTltlB The Toxicity of Perfluoro-n-decanoic Acid and 2,3,7,8Tetrachlorodibenzo-p-dioxin in L5178Y Mouse Lymphoma 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 �. tfc si. O'f 19 •.. • \. ',:';'.,. . ,-..,7 ^Mtfl^i l.t frK-.-!'».'! ."Si*? ".< X -V '1 Vii THE TOXICITY-OF PtRFLUORO-N.DECAKOIC ACID r1 IS178Y MOUSS: LYMPHOWA CELLS) i IW £. GEORGK TOXiCOLO'iY BRA NCH WCkOli'OWGICAL ASSOC/A TES s>21 RIV&R ROAD KETHSSDA. MAX 1'i^ KEtMETH&BACK, PH.D. • £.;^:^iS^t^i?^« - • ; - • • • ••-;.••.•.'•.•; • ; • • .: *,*-^/j • . ,;., ' • • » • • " . " • : ; . . - ' • • ' • . - . . ' . . '';c-";»j5,l �*• NOTICES V»'li. f, U3 C'lVcrjH.-n-ut iji'»ivvi!iy». £p-\'ifi«Nll. ; <,T;i<. Or n\lli'f l'«t'« Jt)'<: '..'.i:(! f'.-j .-i!).1: ;.Ur!i > •? i.'':''.i('! tH.JTi )• ci.:"n:i:v!y ivlfiti-ii (rovernir;'. :;< ;.'"(vc«r''ra'-ril i-jy,'>'(i(i ••«, less* <;«•>%••„« !iir-i»:! ;J.-;-»;4'.y ..M •.: i :u> tv".|'--:i.".«r-:'it; ii'-r arty oi>H/rnli«;i whutsti'.vi-r. nn- f»rm;>;:.: '.';!, l'uv,-i:!iCid. or in JT.V w«y supplied the sai;! drawing, ept'cifzctiiionb, or i>th«?r data. r> nt't -to U- rt'gaf.)-i bv impiiaili'iiv or cth»rwi«'j. as w «ny manner J'u:snj.in[f thft hoMt-r «">r t«»iy o;'«t r pinrK'in <.r corpttt attntt, « c *nnv«yinp «ny rijjhts or pcrmissbr. to mmiufActare, «?«, or sull.ntiy patcntvd invcivtion Uiut »uuy i« :.: way l»o i alatnd t\icre<o. . 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: . Notional Tcnhiuc !M8f/ Povt Hwyr.1 Road p.fieW, Virginia 22161 . Ft-dcnil Govcrnmifnt sacncits and iholr contractors te»;i tiered witK Dt!i«ir.-j TcchnJca! Information M.-r i:Vtould (Urc-ct rcqut-stn far copi<>» of thir- report to: . r.jft! Trfiinicallnformation Center Cameron &tntion Al««andrin. Virginia TECHMSCAL REVUSvV A!*O APPROVAL AF/C'J.IL-TR-82-.IO �<" A T ) . I N C** T M t S f* I-T.AI) INSTKWTIONS MM n|.'K COMI'I.I T I N I V T O K M :>i C t t ' l l Nl'f, C « T A t o d miM'.itV"*"" REPORT DOCUMENTATION PAGE * 4 t-l.'iioo c 1 I T U T f r/tlf .'.«rr,flHr' Technical Kcport The T o x i c i t y of I'orf luoro-n-di*caiti>ic Acid and 2,3,7,B-Tctr.ichlorodibenzo-p-dioxin in L51V8Y Mouse l.yiophoiiia^ Cells ___ . * « ~ ~ ....... I C O ' i t n » C T OH CHM.T NUMflt"«(»l H. E. Anderson. A. M. Rogers*, M. E. George and K. C. Biick** . . . « *-}K« U N I T NUUUCnS AKAMKL, Toxic ll.izards D i v i s i o n , AMD, AFSC, Wrip.ht-Pattcrson AFB, Ohio 4 54 3-3 6 ntI'O»7 D A T E ' c o * i « ' O L L i r . » r t c c tAMtc MARCH 1903 p»oe ^' A T K M L N T <»f tt.it 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 20814 AFAMRL Primary Investigator: Dr. M. E. Andersen, AFAMRL/THB, (513) 255-5150. t KCV WORDS fCootlm/. «i i.v.». «n» «l r Perfluoro-n-decanoic Acid Toxicology L5178Y Mouse Lymphoma Cells Cell Culture V. Krythrocyte Fragility 2,3,7,8-Tctrachlorodibento-p-dioxin •Polyfluorinated acids <C*«**(«H«« «n «»*•«•• «lrf* If nc«*«B*rr •• VPerfluoro-n-decanoic acid (PFDAj causes toxic sequelae in vivo very siailar to those caused by 2,3.7,8-tetraehlorodibenco-p-dioxin (ICDD). The toxicity of these two compounds, several other polyfluorinated fatty acids, and corresponding liydrogenated fatty acids have been studied in vitro in L5176Y mouse lymphoraa cells. Below concentrations which cause cell lysis f>500 i*g/tal)t PFDA did not affect .suspension growth. After 24 hr treatment with concentrations between I and 100 flg/ml treated cells no longer grew into clones when plated in semi-soft agar. This impairment of clone-forming ability was reveiciole att»r growth of % OD ,'?:"„ 1473 «,T,OHor. ICCu*<TV CL*t»iriC*TiOM or »AGC <***• O �iccuniVY CLASiiFiCATibM or THIS CAsef!""" o«r< K«U». treated cells in fresh medium for 36 hr. Pert luoi'o-n-octanoic acid did not impair clon«i- Co fining ability at any concentration; anil neither did the straight-chain hydroRi-n.it «?d Catty acid Analogs. All polyfluorinalotl acids tested (either p*r( luorinatc'J or w-hydro-analogs) wilh chain length 9 or greater caused ir.ipauinunt. of clone-forming ability aftur treatment with concentrations 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 TCDD concentrations of 0.01 iig/ml or greater, placed ccllts formed clones with altered morphology. These clones were less discrete, lacking a clearly defined boundary. The effect on clone morphology required 36 hr treatment of cells with TCDD in (suspension and was reversible following 48 hr growth in fresh medium. Cell division time in suspension was 10-12 hrs and wan unaffected by I'FDA or TCDIKiyln vivo PFUA treatment altered erythrocyte fragility in rats. . It is suggested that the toxicity of I'FDA and TCDD in vivo and in 1.517BY cells in vitro may be due to an ability of these chemicals to interfere with normal structure and/or function of biological membranes^. *ecu«iT» Ck*ttiriCATio« or •"• �PRtFACK This research was performed in the I'.icchemical Toxicology Branch, Toxic lUir.ards Division, Air Force Aerospace Medical Research Laboratory from January 1980 through December 1981. It was performed in support of Task 2312V1, "Toxicological Mechafiisms of Air Force Cheraicols and Materials;" Work Unit 2312V118, "Effects of Air Force Propel Iants and Chemicals on Metabolic Mechanisms." Portions of this work were presented nt the 21st Annual Meeting of the Society of Toxicology, Boston, Massachusetts, 22-26 February 1982. Accession For KTIS CRAfcl DIIC TAB Unftnuc'inced JustificationBy_ .. .. JDintri'-.if-:i/ Avr»U 'V I'.tv Ccdos .'.•••• . ...:/or |Dlct ' r> :! .. I I �ItiTKOUUCTION I'er lluorinated f.'tlty Jcids, \>r;ri luorinated sulfonic acids, nud appropriate derivatives ari' used cc-..aut!rci<il ly in numerous applications which take advar.caf.o of their exceptional surf.'ictant properties and extreme chemical and thermal stability (Cuenthncr and Victor, 1962). Most commercially important 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 and Long, 1980). Loss is known of the tox.icity of longer chain analogs. In an abstract Andersen ct al. (1981) described the acute toxicity of perfluoro-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 than PFOA and its toxicity differed both quantitatively and qualitatively from that of the shorter chain analog. Tox.ic signs and target organs for PFDA were 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/kg and rats intubated with 90 rog/kg lost nearly 50% of their initial body weight before dying two to three weeks after intubation. As docs TCDD, PFDA caused severe thyoic atrophy. As part of a comparison of the biological effects of PFOA and TCDD, we have evaluated the toxicity of those chemicals in Fuvural isolated cell systems. In part, this paper describes effects of various poiyfluorinated fatty acids, hydrogonared fatty acids, and TCDD on growth characteristics of L5178Y 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 was used because T-lyaphocytes appear to be targets of I'FDA and TCDD toxicity in rodents. This conclusion was based on the narked thynic cortical atrophy noted in animals treated with either of these chemicals. L5178Y cells are commonly used for mutagenicity testing and the mutagcnic potential of these chemicals in L5178Y cells is reported elsewhere (Rogers et al., 1982). In addition, United results of osmotic fragility studies of erythrocytes from rats treated with PFDA are described in an attempt to relate altered osmotic fragilities to the effects of PFDA on L5178Y cells. MATERIALS AND METHODS LS178Y Mouse Lymphoroa Cells; L5178Y. cells were originally obtained from Dr. C. F. Arlett. MRC, Cell Mutation Unit, Brighton, England. They were routinely screened for raycoplasna contamination. The routine methods for maintenance of L5178Y cells and the soft agar cloning technique were as described elsewhere (Cole and Arlett, 1976), except that McCoy's 5A medium (supplemented with penicillin, streptomycin, sodium pyruvate, and IOZ horse scrum) was used instead of Fischer's medium. For toxicity experiment*, L5178Y cells were treated for 24 hrs with doses of PFDA ranging from 0.01 ug/ml to I mg/ml, or for 48 hrs with doses of TCDD ranging from 0.001 tig/ml to 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 20Z hone serum. Cells were plated for growth in soft agar, and plates were examined 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 commercially. Thc> latt<?r two compounds contain a single hydrogen at the omega position. I'erfluoro-n-dodccanoic acid*1 (71Z CnF23C02ll; 3% CiQF2iC02H; 22 CgF^C^H; remainder unidentified, nonfunctional fluorocarbon) and TCDD were gifts5. For L5178Y studios, TCDD was dissolved in acetone and the fatty acids and fluorinated analogs were dissolved in dimethylsulfoxide except pcrfluorinatod dodecanoic was also dissolved in acetone. Osmotic Fragility: Male Fischer 344 rats (200-300 g) were treated ip with SO mg PFDA/kg, I'ropylene glycolruater (50:50 v/v) was used as diluent with a final dosing volume of 2 ml/kg. Treated and diluent control rats in groups of four to five were killed at various times after injection. Blood was drawn from the inferior vena cava after opening the abdomen of anesthetized rats and erythrocytes harvested by centrifugation. Osmotic fragility was determined as described in Dacie and Lewis (1963). Curves were constructed for heraolysis at 10 saline concentrations between 0.25 and 0.852. Data presented are percent hcmolysis at a single intermediate saline concentration, 0.42. RESULTS Fatty Acids; PFDA had little effect on 1,5178Y suspension growth at concentrations 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 cells nor debris were visible in suspensions at these concentrations. In comparison to the dose-response curve for suspension growth, the curve for cloneforming ability was shifted some 2.5 log units to the left: the EDjo-24 hr for impairing clone-forming ability was approximately 3 x 10"*• ug/nl. To our knowledge, this ability - dissociating the markers of suspension growth and clone-forming ability in these transformed cells - has not been reported for any other chemical. Perfluorinated-n-dodeca.noic and ll-H-eicosafluoro-nundecanoic acid caused a similar displacement of the two dose response curves (Table 1). On the other hand, PFOA which was slightly less toxic to cells in suspension than was PFDA did not show the differential toxicity with respect to suspension and clonal growth. The u-H-hexadecafluoro-nnonanoic acid displaced the dose response curves for suspension and clone forming ability, but the displacement was less than that seen with PFDA (Table 1), With hydrogenated fatty acid analogs from Cg to GU, toxicity was equal both in suspension and in agar (Table 2). 1 Aldrich Chemical Company, Milwaukee, WI 53233. 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. 2 �O.O ^ A fl* to VI 3 3 A 3OO • • 3C ** VI nc o— r* VI VI 1 O *"* rro «. o* —* -» 3 O r» <^ O •*» <•» fc -« J I 2T A O O OOO •* n <A v> **• A n c §£ 9 JrJ 10 3 5 » a. -i VI 3 a I/I O « * | ?5 o - 1 — OOO5CO OIOB 1 ODIOI0 tfi 'Q.'txwr<J** 00 vO u» ^ g 3 -n o O • t OKXOV. VI — 01 S I 1 ; I-H+I+ !+•!•» | + + 'x 1 —us <ji »«-%i | * 2 1 1 OOO ' —o*** 7* r* • T O ni ^^ 1 1 CQODOJ-sl 1 t fMINJPJvn c I/I a ^ o ! 1 OOO— \oo 00 •"^ o 3 ~* a •i l§I OOCTl 3 § O "** 1 3C *»—•• * 3 VI £S5i gg no i r» 5" 7e> o r* »* 3 A*< • 2? ve 3" --0 3 -»i 50 *~ A VI n3 i O O f O o W -J^ H | ., !" T OJ _^ — Ul S- — t* \' ta o a- oa 7re O i , ^ O o 3 3 r» vi rVI -«• U> ciTlS 0 T ^ T 3 -!« 00 O* vi 3" O O — o re r»re e -•» ct — re r> n — — ftt VI —-— £1 | ^ **— \ •>< . «* 0- ° .£ VQ ^ .5 w m 5 T / O > •• O r* — r> 3 O ^. T O C o o a.v> 3 n t V* •** 1 |O - * ' ^— "^" — a 3-3 vi A & ••• ^ *r""""^ "O r*r «-* (£ <^ ~t — ' o o r» n *i> — . -*»o c 3 3 3T3 wT"2.m *^ 3 C* D O W» •n rt» T m CLQ» 1 a n O rn z -^ ° ^ > 5 / / *+* / / /^ 0 ^ a rn r.— -*^*^" ^ ~*^ O o *"T3 O • ^-33- r-*^ > 1 ! t ^ 5 / H f o -i y (9 ^ 3 <->e- — n 4 ° / 3 O ft1 VI O A "* •* ^ V vi c — —o o rt> Q -S J> O 33 -s» r- on ^ o O» rt 1 i/> —• r* ft> fl> 3 •e n O 06 , O V> T O tte T'oa CX — ^> •* O O O.3 1 3 O 0 ••• 1 fi 3 U3 O 3 •< a -tvre^T — to 13 VI — Growth as Percent of Control VI r* O >< — 3 VI ?o. — B.3 Tl CXA "1 n t**l 3 O. ST o^ „ •S — A O 3"S £+, 5 A" 3 r* — -Iff O. O g er-n-o ^-^ o t £ »o 3 — V>< C 0> "3 *£ -••N* o •c X. r- -H 0 —£ 3 0 M O O | & 2 IS S.? — ^ o^31 o -o 3 2, O 1 i-l -""5.1 —.»<-» !§ C over's " T C. •*.*'* c i/i 1 I r> o s O 3 Perfluoro n-decanoi Acid 3- 9X I ft 3 fik >0 A O 3 f) lO — .0. 0> 1 D 3 VI ~ B a n ft o< 1 ? «*. o C 1 ** O< vTvi 3 O A n §s T ^ C n <* — — o tt *• c 0? 3 A *^"»Brt» CL« 3 >S3, 3 VI 3 1 f^ ^3 40 1 i0 0 VI 3 -0 VI M XI iJSSSSS 3 A f* V uoisuad O 3 o* c f\ ' O -•- O -*.-*' —O 3 O3 — ^ *• < C I/* Iff *% th "t to OT m •^ t/> — O *• ^ X •jy O 3^ �Table ?. Hi" f.itoct of VArujnr. Fatty Acids c.n Growth of L5178Y Kou«- l.yi^hoiM Ctlls in Suspension *nU on llioif Clone forn.irq Ability in bciM-Soft Agar Dose Non.uioic Acid_ SUMIOIISion Aijar Hocanoic Acid SUspenstbn AjjoF Uiutecanoic Acin" SuVp*''CTo'«''"VaT (liy/ml) (% Control) (X Control) (X Control) 0.01 0.1 1 10 50 100 500 1000 85* 82 92 90 85 72 33, b - 98 98 90 94 98 8B 26 — 106 94 100 84 75 63, «b b « 96 94 93 90 8? 63 - 95 85 75 8? 82 4? —b — 96 93 96 93 86 44 --*> a Numbers in both columns arc growth as percent of growth'of control cells. ° These concentrations dissolved cells in suspension. Titac Co E f f e c t and Reversibility: Cell division time for L5178Y cells in suspension under growth conditions used in this study was 10 to 12 hours. An experiment was performed to see if cells required a period of treatment with PFDA before diminished clone-forming ability could be observed. Cells wen; grown in suspension containing 0.5 ug PFDA/ml and removed at various times for plating to observe loss of clone-forming ability (Fig. 2a). There was a lag of 8 hr before any appreciable e f f e c t was observed and the time of treatment required to reduce plating e f f i c i e n c y to SOX of control was about 12 h r , i.e., one cell generation. Cells were also grown for 24 hr in the presence of 0.5 Ug of PFDA/ml harvested by centrifugation and washed in fresh growth medium. These treated cells were resuspended for growth in fresh, uncontaminated medium and aliquots withdrawn after various times for plating (Fig* 2b). The decreased plating efficiency was reversible, but recovery was more prolonged than the time required to induce the diminished clone-forming ability. The time of growth in freth medium necessary to restore 502 plating efficiency was nearly 36 hr, or about three cell generations. Cell division time of L5178Y cells in suspension was unaffected by pretreatment with 0.5 ug PFDA/nl. Piox in: In L5178Y cells TCDD did not dissociate growth in suspension from growth in soft agar at any concentration tested, up to 0.5 Ug/ml. However, the morphology of the clones obtained after treating cells in suspension with concentrations of TCDD between 0.01 and O.S pg/ml, was markedly d i f f e r e n t from controls (Fig. 3). Instead of the well-circumscribed, circular clusters of control clones, those clones formed after dicxintreatment were less-discrete and lacked a well-defined border. After growing cells in suspension for 46 hr in the presence of 0.003 ug TCDD/ml, �TtME TO RESPOr4SE/TIME TO RECOVERY OF L5178Y CELLS TREATED WITH PFOA. 10O-X B e O 60- « u 60 k. o Q. £ 4 °i o O 20- "3 c o o 24 24 48 96 Time (hr.) Figure 2. Time course of impairment and recovery of cloning ability in LS178Y cel'is treated with 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 to percent of untreated cells which give rise to clones. B: Time to recovery: cells were treated in suspension vith 0.5 jug PFOA/ml for ?A hr, and harvested by centrifugation. Aliquots were 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 clones formed 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 dioxin required to produce alterations affecting 50% of the formed clones when cells were initially maintained in suspension with dioxin for 48 hr before plating, was about 0.01 pg/ml, i.e., about 3 x lO'^M. In time-course experiments analogous to those in Fig. 2, but conducted with 0.01 vS dioxin/ml, the time of treatment in suspension required to produce 502 of maximum response in altering clone morphology was about 36 hr. A time to recovery of normal growth characteristics was also estimated for cells grown initially for 48 hr in the presence of 0.01 Ug TCDD/ml. The time of growth in fresh medium required to give a SOX recura to normal clonal morphology was about 48 hr. As noted with PFDA, effects on clone growth were reversible, but recovery and expression times for the effects were longer with TCDD than with PFDA. Red Blood Cell Fragility; Rat red blood cells were obtained from rats killed at various times after ip injection of 50 mg PFDA/kg. There was increased resistance to hemolysis after treatment with PFDA (Fig. 4) and the time course of decreased fragility was similar to the time course of weight loss in treated rats (Andersen et al., 1981). .6 �Control clones derived from cnlls treated in suspension . - -viv.. .,s H, with OMSO. ••'/.& B. Clones derived from cells treat.td in suspension for 48 hr with 0.25 u<j TCDO/ml with UMSO as diluent. ,»• '• 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 cultured cell types and found no toxicity in any of these mammalian cell lines at treatment concentrations of up to 10~^M and contact times of up to two weeks. Markers for toxicity included (1) alterations in the morphology of cells or the cell cultures, (2) percentage viable cells, and (3) growth rate. Among the 23 cell lines were five lyraphoid cell types derived from thyraic cortex - three were murine and two were virally transformed human leukocytes. All these cell types were tested for growth in suspension and cell viability by trypan blue exclusion. Beatty'et al. (1975) found that TCDD had no effect on growth or morphology of normal human lymphocytes in suspension. Our results are similar to the extent 'that TCDD did not affect growth or cell viability of LS178Y cells in suspension. The altered growth characteristics observed in this paper are more subtle and only apparent when cells are grown in semi-soft agar, where they are constrained to gro»r in close proximity to each other. The concentration dependence of the effect with TCDD is such that a 48 hr treatment with 0.01 pg/ml (i.e., about 3 x 10~8H TCDD) causes the effect in most of the treated cells. This concentration is reasonable for physiological significance stnce the mouse is about 300 ug TCDD/kg, or about 1 Pmoles/kg (McConnell et al., 1978). 7 . '•'* �lOOi • S (3 60 t»> * w (rt " 60 O T T i T';'T '.;] • "55 j O tr. i- 0) •5 "0 X "o E * T 1 '• a* vf* fj X L T ~t 20 . ••' ^ :.; - '** • j*l i< ? '•u.f § O pii •'•'.'1 ' . .''i ;'. i :' '.! ' if- .,',, S'> ..4 '•'.'.1 '••&$ ;-•.. / /!.? 0 DA Y 2 DA Y 3 DA^' 14 ^•^ DAVrso Figure 4. Relative osmotic fra^ilitiei of red blood cells from rats injected ip with 60 m9 PrOA/kg and killed at various tiiw>i after injection. Data are iwan and standard deviation (n « 4-5). From the overall curves with 10 salt concentrations, the concentration 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 had 505 iiemolysis at 0.45, 0.44, 0.45, and Q.43X. respectively. Alterations in clone morphology seen after TCDD are striking, but estimations of concentration dependence are e s s e n t i a l l y q u a l i t a t i v e , i.e., the percentage of abnormal clones is estimated by inspection and making a distinction between normal and slightly abnormal clones is d i f f i c u l t . We have maintained a restrictive d e f i n i t i o n of what constitutes an abnormal clone and scoring was done solely by Dr. A. M. Rogers. For these reasons, the estimated £050 Cor the effects with TCDD are probably high. More quantitative determinations of these TCDD dose response curves await determination of the biochemical basis of the altered morphology and methods to unequivocally identify altered clonal units. With PFDA, results are readily quantified since treated cells no longer proliferate in semi-soft agar. The EDjQ - 24 hr for the loss of clone-forming abilicy was 0.3 Pg/ml (i.e., about 6 x 10~'a); this contrasts to a single dose 1,1)59 in mice of about 100-150 ing PFDA/kg or 0.2-0.3 mnoles/kg (Andersen et a!., 1981; Van Rafelghem and Andersen, unpublished experiments, 1981). With the polyfluorinated acids examined, this toxicity is present with acids of chain length greater than 8. The differences in single cell toxicity between the fluorinated octanoic and decanoic acids are striking, but consistent with the d i f f e r e n t acute toxicity reported for these two acids in rats. The hydrogenated f a t t y acids are without d i f f e r e n t i a l e f f e c t on 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 ) . Tin- mol.'Cul.ir l);u;i<. nf ih<- i<::p:i!ily i s tiiiknown. Subtle change:; t:iay li.'tvv orciinvd .' r t-tll mi-nbrant.'!. to i n h i b i t j-iowth of cells vh<M) maintained in close contact. In t h i s regard, tin- i.'S.-v'U io fragility ritsultti surest a biological Kii/inb.-.'we i.iore t os isitnnt to liyi-u^snot i t insult. Increased resistance can be- due to a variety of causv.s, on.- ol vliich is altered wi'.mbrmu.1 composition (Kuipor ot a 1 . , 1971). Prcl iroinjiry uLudit-s in our laboratory have- now shown that i-rythrocyt'-s Irora PF!M-tr«aU:«i .Mur.iaU also show increased nwmbranQ f l u i d i t y (M. Cc-orj-o ,iiul M. K. Anderson, uti[uii)li.sh(-i1 studies,' 19B2), and that thv total fatty acid coinposi lion' iii the liv«r l i p i d pool 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?.). While if.dirc'ct, these results sup. nest:' composit ional <'ind functional altei'at. ions in membranes subsequent to S'KDA oxposurt in tiic rat in vivo. There is no unifying hypothesis explaining i he toxic ity of TCDD and materials causing; similar toxic effects, i.e., certain polyhalogenatcd biphenyls (Sleight et al., 1981; Biocca et al., 1961) and long-chain pert 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, are toxic due to effects on cc.ll weir.branes rosui t in,; in inicr fen-nice v.'ith cell-eel! or eel l-n-.nliator interactions. These effrcts could either be direct or c;ediatc(l by interference 1 with same undogonuus horr.-.or.al control of t:ionibr;uie cocTiposit ion/ function. Toxicity would r.ot be a result ol cell necrosis or grossly visible organellar alterations, but from wore subtle structural alterations of biomembranes ar.ur laboratory. The cell line used for this research was so-called TK */+ with regard to the gene locus for the enr.ycne thyuidine kinoae (TK) . Our stock of these cells, brought to Dayton from England by Dr. Rogers, was destroyed during a malfunction of the deep freeze storage uint. .'We have not observed differential effects on suspension and clonal growth with L5178Y TK*/~ cells a cell line more commonly used in mutation research and, therefore, much more readily available. It appears that future work on this phenomenon will have to be restricted to the TK*/* cells. REFERENCES Andersen, M.E., Baskin, C., and Rogers, A.M.,.1981, "The acute toxicity of perfluoro-n-decanoic acid: Similarities with 2,3,7 ,8-tetrachJorodibenzodioxin," The Toxicologist, 1, 16. Beatty, P.W., Lemback, K.J., Holscher, M.A., and Neal, R.A., 1975, "Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on mammalian cells in tissue cultures," Toxicol. Appl. Pharmacol., 31, 309-312. �''•' •>*•.•!•. M, • ''"I' 1 -'. ' » • ' • • , i.:>:««-. K . , H s K i i n . - y . . I . H . , .-UK! M.'i«n-, -I. A . , . '"I- > :c i t > * • • ! ••••l«Tt-'il :.. 'n.','.- -I r i <;.'i) tn'»:.lflil«i'«'l'i jiiu-ny t isor.oi si i n l!'<.- iaoit*<«, <\<l, , J . ,nt:l A r l . . - t t . < : . K . , l ' i / f i . " K . i h y l me! ti.iit.-sul [.:,;>:-.,n •• i.iu( ;iK.i'n»>*i s w i i l i I.M?;'.Y :..i>ur.i- I yir,pti.ti:ia cells. A roiiij.ai i *..>» w i i h ouabaMi, t liiu.-u.-.tuiH' , iind o ." Mucii.iojj ii<-_s . , J4 , SUJ-i>/(i. Da •,•»«.'. J.V. and Lou-jt:, S.M., rrAj.-tii.i ' ; ^ . , !..i-::ion, 19C3. ; . r i : l i t l . , ('.(). .liui l.on^, J . M . . I'^riO, "Aniq.al l o x i c i t y studies w i t h .im:r..)oitjm (-0! lli-oroix t.-iiii>;a"." A.TIIM-. i r.t! . I v j . C<iionth:x>r , K . A . an;i V i c t o r , M . L . , IS'K., "Surface a c t i v e u.<i(.or i j Is f r o w J>IT i luorociirboxyt i v ami j'or 1 1 uoro.su I t c n i c .'iciils," I n d u s t r i a l and En t -4»<H;rin>' {••••.ri. I'rorf. Kos. i ! i v - . •• i6b"U>*. " * Km:ts<ni. J.C. and Poland, A., 19fiO. "? ,3,7 .S-t F a i l u r r r to doraonstra:*; l o x i c i t y in t w o n t y - t h r e o c u l t u r e d c e l l types. Ton i col. l' • V«. 3/7-383.' , P . J . C . , 1.ivn«f, A . , and .*•<..• \ o r s t o i n . N . , 1971, "t iianj-.es in l i p i d conpoi, i t i o n ".nd iisc.itic f r a g i l i t y i>( orythrucytoi. of hamster induc«sd t-y Stoat i'K[.«>sur..-," H.io--:!.j:5. iij,i-];iws. AtM « . 2'«.^. 300-3»'i. McConnt-ll. K . K . , Moc-ri-. .I.A.. Has-uai.. J . K . . and H a r r i s . M . W . . I V / B . "Tintir.r.j.iarnt ivc c o x i c i t y o( ch 5<>t inatca dii^neo-p-dioxinr- jn oic<' and piini-a pitfs." T'\K_J£i>l. Aji|-l . jM|»arr.i.M.-ul. , 44 , )J5-J5f>. 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 lycphoroa US178Y)," Biochora. Ph.arciacol. )0, 3375-3377. ' ^ OUon, C.T.. Anderson. M . E . , Ccor^i', M.E., Van Rafelghem, M . J . , and Hack, A.M.. 1982, "The toxicology of perf luorodocanoic acid in rodents," presented «t th« Thirt«cnth Annual Conference on Environmental Toxicology, Dayton, Ohio. Rogers, A.M.. Andersen, U.K.. and Back. K.C.. W82, "Hut*R«nici ty of 2.3.7.8totrachlorodibcnzo-p-dxoxin and per f luoro-n-d«*canoic acid in rtiouto lyciphoma cells," Mutation Res. lOS, Sloip.l\t, S.C., Render, J . A . , AVoso, B.T.. Aust , S.D.. and Nachrcinec. R . . 1931, "Comparative toxtcopatholony of fire-matter BP-6, 2 ,2,4,4' ,5,5'-hexabrnmobiphenyl (HBB) and 3.3' ,4,4* ,5.5'-HBB a f t e r 10 and 30 days of d i e t a r y a d m i n i s t r a t i o n to rats," The ToxicoloRt itt I, 12. 10 � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young. For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 096 Folder The folder containing the original item. 2434 Series The series number of the original item. Series V Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Andersen, Melvin E. Marilyn E. George Date A point or period of time associated with an event in the lifecycle of the resource 1983-03-01 Title A name given to the resource The Toxicity of Perfluoro-n-decanoic Acid and 2,3,7,8-Tetrachlorodibenzo-p-dioxin in L5178Y Mouse Lymphoma Cells