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jus, *nd Tort, , tlo fljJS , H tk l *? z> -i m tab ., 38. oM n J* r 2 - *1. 8, -9-512. -w ' . J. Pham. -- il. - ,Eur. J. *; - i." Abstracts. . . L e if 84-^94. rittfn f Clin.. t M ?; 'C' * -. r; ' h3 * . iv; ft - i n d u c e d p y jtn e .M T m r c iiin e n ia ^ 0 1 1 6 ^ 0 ^ :^ 3 ,7 ,8 - T e tra c W o rta ^ IB e tliy ls iilb e s to l a n d B e n z o (a )p y re iie > , *,'t **i'v*.'*f* ... ,* J. H . D E A N , M .L L U S T E R . G . A . B O O R M A N . K . C H A E . L . D . L A U E R . UL i f i f . L U B K E L . D . L A W S O N a n d :' . 4 r i S L & , W I L S O N National T a c a b ^ ^ m ' ^ ' u b i o a i r fB a rlm m In a l Q ta lta r . National hbtftots c^EcyjrixBasntaf Hodp h Sciences. SHL P.O. Bax. 12233. . / Rdw arehTttag^ Q rnliaa 277U&`t/SA iSTSACT ' *K . 7 V ^"'' ' P ^ ' " K te s tin g ,b a tte r y v i l i b e d e s c r i b e d f p r asscstfing" a lte r a tio n * in .immunological f u n c tio n in d h o s t r e s i s t a n c e fo llo w in g e x p o s u re .o f B^C^Pi mice to TCDD, DES and 3P. TCDD i s ' a m ajor c o n ta m in a n t.o f t h e d e f o l i a n t 2 ,4 ,5 .T (a g e n t o r a n g e ); DES Ls a p o te n t s y n th e tic e s tro g e n u se d th e r a p e u ti c a lly to p rev e n t menopausal symptoms and p o s t - c o i t a l i a p l a n t a d o n ^ and as".a /.v e ig b t prom otsne i n c a t t l e ; and BP i s th e p rin c ip a l carcin o g en in c ig a re tte ;s m o k e - a n d .c o a l-ta r. Mice exposed p e r in a ta lly to TCDD. C5j l u g /k g ) - e x h l b ite d -iark ed ; th'yxaia. a tro p h y and d e p re sse d m ito g e n ic resp o n se s R asistends to L i* t* r ia BsmaifytGgmfaj- -graa^-oagatlTS endotoxin and, tra n sp la n ta b le syngeneic tumor- c e l l s ' veret:Tikertd<B'.impaired.--'The mice Appeared -to have a s p e c if ic T - c e ll d e f e c t a s s o d s ta d w ith ; e V re e d p c b r-fo r TCDD p r e s e n t a t h ig h le v e ls in thym ic ti s s u d . Mice, ex p o sed to.DES a t . ; 8 , 0 , j2 .0 and 0 . 2 mgl/kg o f body y a ig h t f o r 5 days exhibited severe thyaM .attophj& fieoos^enied. by .d ep ressio n of the follow ing params t e r s : a n tib o d y PFC re s p o n s e t o 5KBC and LPS; ' d e la y e d . c u ta n e o u s h y p e r s e n s i t i v i t y ; ly m p h o p rp life ra tiv e resp o n se to-T~: end B-ce11 m ito g ea i a n d .a lio a n tig e n ; bone marrow c e l l u l a r i c y ; and stem an d -g ran u lo cy term acro p h ag a p ro g e n ito r c e l l p r o l i f e r a tio n . Macrophages were a c tiv a te d r e la tiv e to phag o cy tic, p r o lif e r a tiv e and tu a o ric id al responaas w ith evidence o f suppressor c a ll a c tiv ity . Host resistan ce param eters w ere lik e w is e im p a ire d . T hus, DES ex p o su re r s s u t t e d I n RES s ti m u la tio n , severely depressed im ounological-end h o st re a lsta n c a fu n ctio n , due in p art to macrophage s u p p r e s s o r c e l l s e n d .p o s s i b ly .a T - c e l l d e f e c t.- - F i n a ll y , BP was dosed a t 400 , 200 and 50'm g /k g o f b o d y rv eig fat over 10 d a y s . BP exp o sed m ice demons tr a c e d d ep re ssed e n tib o d y PFC re sp o n se s;-' ly ap h o p ro l i f e r s t i v e re sp o n se s to m ito g en s; and depressed numbers o f bona marrow m acrophage-granulocyte p ro g e n ito rs . Macrophage fun ctio n and h o st re s is ta n c e to- A rte ria l and tiaeor c e lls were u n affec ted . These data demoncrate the. s e n s itiv ity o f th e l i t i n a response fo r d e te c tin g s e le c tiv e or 'tion-specific chem ical to x ic ity and support the hypothesis th a t c e rta in carcinogens m ay.operate v ia im u h o lo g ic e l mechanisms. INTRODUCTIOH During th e p e s t d e c a d e , th e itmaune sy stem h a a b een examined in e x p e rim e n ta l an im als for its fu n ctio n al in te g rity fallow ing exposure to a v a rie ty of chemicals of e n v ir onmental concern. C e rta in chem icals have been found to produce a lte r a tio n s in r^'cV ' >% M* ' ' ;`;^V*\ *,' -C''r *.'. ',' l :; * , 1< ' , `'.' Vf '' - . J i r r a l l s ; ' 1979b',e).>: L lktW lT ^t5i5h*klration have been obscrved `La c e r ta in c-v-:. population of Indlv lg u ala exposed t o chem icals through accid en tal o r occupational-^ .fcgp&aure.^ A lt bough,-a. f cvchegdcla-,m ay b e .jia la c tiv a ly to x ic to a p a r tic u la r 1 u b -T-v . population of lymphoid c e ll o f th e /jre tic u lo e o d o ch e lia l yaten (RES), such . thymocyta, i n cho ca se o f c h e ^ ^ ii^ c & h y d rp c m rb o n ; 2,3,7,6-tetracfalorodibenJ7pT & i' diaxin.(TCDD) (L u ste r and.co-yp r j198 0 b ) , i t l a our. hyp o th esis th a t exam ination of Lamina fu n c tlo ti.,ia D oat caaai^jidda. a n . c tr a la v a 1 o f a n a I c lv lc y fo r d e fin in g ; c e l l u l a r t o x i c i t y . . T his .Lac r a is e d s e n s i t i v i t y o fj t h a . immune response fo r d e te c tin g celj.ulac; to x ic ity re a u lta from th e .ra p id p rp lifaratlo n ..an d dynastic nature of Ttva^- ? c e l l u l a r components o f th e -inmine. sy stem .This concept is supported by human-ch'em^~- . i c a l . t o x i c i t y s t u d i e s i a th e f o n t o f c l i n i c a l chem otherapy in w hich c e l l s o f t e S ''*' a r e th e m oat f r e q u e n t t a r g e t o rg a n f o r c h e m ic a l in ju r y .. The f u n c t i o n a l . i n t e g r i t y - ''< of .CHI o r th e q u a n t i t a t i o n o f bona marrow, c l e p c n ts fo llo w in g chem otherapy i s . freq u en tly determ ined, to d e fin e th e 'le v e l o f ;to x ic ity and to re g u la te dosage. There are se v e ra l conaide r a t Iona y^eu, d esig n in g a comprehensive iessunotoxicity , assessm ent (p ro to c o l) w ith ch em icals.d ,,en v iro n m en tal concern, as fo llo w s: - (1) The planned ro u te of exposure should^adoic aa .clo sely as p o ssib le the a c tu a l en v iro n -'-m ental exposure ( i . c . o r a l , p a r e n te r a l , in h a la tio n , e tc .) ;- (2) The dosage employed should, somewhat exceed -the- r e a l . l i f Ie...expoaure l e v e l s w ith c o n s id e r a t io n o f body .V* w eig h t to s u r f a c e r a p i o ; (1). .The d o sa g e e le c te d sh o u ld .b e below th o s e w h ich , produce o v e r t t o x ic e f f e c t s - `( i . e . v n e g a tiv e w e ig h t g a in o r g ro ss a l t e r a t i o n s 'i n liv e r enryme le v e ls ) ; and (4 ).:th e a g e -o f th e anim al s e le c te d fo r exposure should be c o n s id e re d c a r e f u l l y s in c e some c h e m ic a ls ( e . g . TCDO) h a v e - a lr e a d y been demon s t r a t e d to have more p o te n t i 'usaunol o g i c s 1 a l t e r a t i o n s vheti a d m in is te re d d u rin g -v p r e - a n d /o r p o s tn a ta L l i f e v h iL e ^ th e immune sy stem i s u n d erg o in g m a tu r a tio n . The p r o to ty p e c h e m ic a ls s e l e c t e d 'f o r 'i m u n o t o x i c i t y a s se ss m e n t by o u r la b o ra to ry '.'-'. re p re s e n t th re e broad c la s s e s o f ...chemical^ .th a t .are n o t .only c a rc in o g e n ic b u t'o f ;'' .UQAtQIF mrirnntunfal __.3\a aa ' inrliwl* hal Ao*nar*if. arrtfM^i j* KwImrarKrtnd _-**** specific s p e c ific a lly ti-iv (a )p y re n e (BP).. ,. . . ' ; . *"- >v : * *- . : TCDD i s th e p rim a ry c h l o r i n a t e d rfib e n x o - p -d lo x in c o n ta m in a n t prod u ced d u rin g t h e '"' s y n th e s is o f 2 , 4 ,5 - t r i c h l o r o p h e n o x y a e e t a t a . (2 ,4 ,5 -.T ) , th e l a t t e r b e in g one of -the' **. m ajor to x ic c o n s titu e n ts o f th e d e f o lia n t'.a g e n t orange. (Courtney and co-w orker a,*' 1979). TCDO h as been found to a l t e r th e ivaunocom petence o f r a t s , m ice, guinea*" pigs and monkeys as w ell as a l t e r r e s is ta n c e to b a c te r ia l, p p ra'sic e and sy n g en eic' tumor c e l l c h a lle n g e in m ice ( s e e re v ie w by Vos, 1977; F a ith , L u s te r and Vos, 1979)'. The s tu d y w ith TCDD d e s c r ib e d h e r e r e p r e s e n t s h i g h l i g h t s from a d e t a i l e d p r e /p o a c - - '-U- n a t a l e x p o su re s tu d y in BCs Fi n i c e (L u ster; and c o -w o rk e rs, 1980). S tu d ie s -o n th e ', p re se n c e ot a s p e c i f i c re c e p to r,lo r.T C D D i n thymic- t i s s u e w ere s l i d co n d u c te d . .."r~r<- D i e t h y l s t i l b e a t r o l , I s . a n o n - s t e r o i d a l e a tr o g e n i n i t i a l l y s y n th e s is e d by Dobbs :and a s so c ia te s (1938) d u rin g a search fo r s y n th e tic estro g e n s. Since i t s discovery i t has been used th e ra p e u tic a lly as.a n -# a tro g e n replacem ent to co n tro l menopausal symptoms, su p p ress l a c t a t i o n , . p re v e n t th re a te n e d a b o rtio n s , and p rev en t p o s tc o ita l im plantacion ("m orning a f t e r p ill" ) ., aa w ell as to tr e a t b re a s t and p r o s ta tic ca n ce r p a t i e n t s (s e e re v ie w by H o lle r and F is h , 1974).* DES has a ls o been e x te n s iv e ly used a s a grow th prom ocant in c a t t l e , sheep and p o u ltr y (s e e rev iew by McHartin and co -w o rk e rs, 1978). I t was im p lic a te d as a p o te n tia l ca rcin o g e n or c o c a rc in o g e n in humans when H e r b s t, U lf e ld e ra n d and P o sk an zer (1971) o b serv ed t h a t 7 of 8 g i r l s w ith a very ra re c l e a r - c e ll adenocarcinom a of the vagina were the r e a u l t of h ig h * r i k p re g n a n c ie a in w hich th e m others r e c e iv e d DES to p re v e n t a b o r tio n . These o b s e rv a tio n s have been ex ten d ed and confirm ed in o v er 302 c a se s o f v ag in al as w ell as c e r v ic a l adenocarcinom a (H erbst and co-w orkers, 1977). V. I l $ V 'f fes'mwf*l ru in ..;; patiooal tu b -... as the nso-p- -... . . . .amin a tio n fining detscting . o the an chea- -- of RES .cegrty-. ia <* ci cy . (I) The... nv ir n - . : employed* body .eh . i in r : should i demonripg oratory but of irbooi beino 1 o t. che. piiae* r>w7 ?Ce/po*t- s on che atei* : - '* * bb* v e ry i-Cr usai to o lt a l atlc 1u t ' by fati o t ry *4 th a t -tfftba fea sts ?3 iWr- ' T * * t * & ! * *'*i * P f " * - 1 # L.-. .i - ... -/M-JAesasmtt'.' f lanuno to x ic ity -` ^ v i^ eS f - .; --R ecen tly DES-'haa beeQ*:hcm'. v i ' o n rc s ll- ta a d t a c a d - l j a k u h l t t * ) ^ ' .` r e l a t i v e l y potent ' inmunos upp re s a iv a H i " 1-- "* 1578, I980;:' X n ll* ^ ,r ^ 5 M b ) o r'b d lc -e x p o su ra (Boorman a n d .co -v o rk e L u s te r end c o - v o r k e r s ^ l^ S O a ; '" Dean* a n d co -w o rk ers', 1 9 8 0 ). S t u d i e s * ' ehig r e p o t t hLghL ig h t 'c ^ \ f o d i t i g s v i t h ' DES; ' . - >' . . ..................-r i-; " '* * * -** P o ly c y c lic , a r o m a tle h y d r c V rt h j.b f ly h ic h ' b s a r o ( a ) p y ren e U t m a jo r c oq ere- formed b y incomp le te^cocba* tio ^ -o ffo * i i l fu e ls end tobacco aa b y -p ro d u c t* , o f c o a l c o n v e c tio n 1' p r o c a s s e * . ' They a rie'a c te d * upon by a r y l i h ^ r c V r /,:. h y d ro x y la s e . to m etab...o..l...i.c...a..s..?..V......t..b...:.p...o...t.e...n..t.. c a rc in o g e n p o t e n t i a l . The a i g n t f I c k n c m ^ ?4 : ^ f . ^ ' dbhe^tip.ic.caa--t:taarcr Le nLongoegne~n-nindduucceedd:-.lInra&afutniaook'kuppp_rc_s_siion l a th_e._t_u_m__o r;ig e n:ic p oit.e n~tla T^ o f .lk t 1*`i:'.:'* c a rc in o g e n , ia n o t - y e t f u l l y u n d e rs to o d ,- H alngreena. and a s s o c i a t e s (1952),:p b ' e ^ f '^ * j3 ^ & In s c u d ie s r e p o r te d here-,- i j u l t 'B'Ci'Ti'ace v e r e ' exposed cu ta n e o u s in je c tio n s * - fo llo w e d b y an- e v a lu a tio n , o f in s u n o l o g ic a l param eters.- ' ' MATERIALS AND METHODS ~ TCDD, DES and BP E xposure ; P r e /p o o tn a ta l TCDDe x p o s u re v as `a c c o m p lish e d a s p r e v io u s ly d e s c r ib e d L u s te r and c o -w o rk e rs (fS B O ).' TCDD' v a* s y n th e s iz e d by th e LEC a t SIEHS'-'at - g r e a t e r ;' chan 992 p u r i t y . ' V ir g in fem ale C57BI/6 m ice w ere m ated w ith m ale C3H ic e -:a n d ; " preg n an cy v a s d e te rm in e d b y ''t h e `p re s e n c e o f a v a g in a l p lu g . M other* .v'e re -a d m ih --^ . . / . ; V v ! ' L scered TCDD d is s o l v e d i n c o rn o i l by g av sg e a t day*T4 o f g e s t a t i o n snd-;sg^.a.*biiw*. VV^`. days l , 7 and 14 f o l lo v in g b i r t h a t d o sa g e s o f 1 .0 , 5 . 0 . and 1 5 .0 u g /k g "}iyL:;~ ' w eig h t. - Mice u sed f o r c o n tr o ls re c e iv e d co rn o i l . lam m o lo g ic a l as se ss m e n t .vas. t- br; in itia te d 7 days s f e e r veaning shd h o st re s is ta n c e param eters were examined 6-8 :\'-r _ weeks a f t e r w ean in g .* ' -*.*- D i e t h y l s c i l b e s t r o l ' (DES) was o b ta in e d from Sigma C h em ical`Company (S c . L o u is , H O ).. S ix to e i g h t week o ld fem ale BCFi m ice w ere i n j e c t e d s u b c u ta n e o u s ly (sc)- w ith . 0 .2 , 2 and 8 mg/kg body w e ig h t *o DES d is s o lv e d i n 0 .1 ml o f . c o r n o i l f o r f iv e . _ co n sec u tiv e d a y s . C ontrol. icaV receiv ed corn o i l alo n e . ' Im m unological a n d a o s t ., 2 '^ r e s i s t a n c e s t u d i e s v e r c cp e r f o r a e d 3 -5 d a y s fo llo w in g th e l a s t d o s e . ' 'C 'V B e n ro (s)p y re n e (BP) wms lilc e v ls a p u rc h a se d from Sigma C hem ical Com pany.-*S i r t o ; ' w eig h t week o ld fem ale BCiFt m ica ''v e r a d m in is te r e d BP d is s o lv e d in c o rn o i l by subcutaneous I n je c tio n to a t o t a l dose o f 50, 200 and 400 mg/kg body w eig h t; b y a r-a .j.,, , . v 14-day p erio d . Three to fiv a 'd a y s a f te r the la s t treatm en t dosage Insnunoiogicil.v' and h o st r e s is ta n c e param eters were ev a lu a te d . Comprehensive E v alu atio n of Inm unological and Host R e sistan c e P aram eters The a s s a y p a n e l employed In th e I n s u n o lo g ic a l, p a t h o to x ic o lo g ic a l and h o s t r e s i s tance e v a lu a tio n o f ia o u n o to x ic ity has p re v io u sly been d e s c rib e d (Dean and co w o rk e rs, 1979b). The m ethods u t i l i s e d have lik e w is e been d e s c r ib e d in d e t a i l c (L uccer and CO-WOrfcC*, lp8phc;^';.r*^nvwtfh^aen d rcAor-wwbarfcera, I960; Dciq And c o -w o rk er!'^ , .` J979c) .* ' lf;, " *' '" '* '-<<- ^ TCDD R e ce p to r Amt t y .. ' *" * * - -', * .v, . l a a t y p i c a l a s s a y , 3 ` l / . o f a lOOiOOOlX-g s u p e rn a ta n t from t i s s u e h a v in g a p r o t e i n - c o n c e n tr a tio n o f ' a p p r o x im a te ly 0 .'S/iiig/ml. i n 20 -mM T rie HCi b u f f e r c o n ta in in g 0 .2 M KC1 end I mM EDTA (pH 7 . 4 ) # ,Va j.n q ^ A te d :w ith 2K 4Q f dp o f l - N - ( 5 - l 2 i I - i o d o v e l e r - saido)- 3 ,7 ,8-trichlorodibtutarp-dlio^a^J(A 1* I-d io x ia), plus a 300-fold ex cel of u n i abe le d TCDD f o r 2 h o u r i afc. 0*C -.i'afA-^ s h a k e r b a th . The unbound 12SI - d i o r i a va reeovqd. b y * a c tiv a te d c h a r c o a l p e rfo riie d by- in c u b a tin g 0 .8 mL a l i q u o t o f th e in c u b -- a tio n m ixture w ith 0 ,4 1 o f c h a tc o a l/d e x tr a n suspension, c e n trifu g in g a t 300 x g f o r 10 m in u te s , a n d - c o u n tin g 0 . 6 `ml tlA u p e rn A ta n t in a gamma c o u n te r . Each a s s a y " was p erform ed i n d u p l i c a t e . - S p e c i f i c .b i n d in g i s th e d if f e r e n c e betw een 1211 - d io x in 1 bound in th e ab sen c e o f any- unlabsled..yCDD and * * * I-d io x in bound in th e p rese n ce * o f e x c e ss u n la b e le d TCDD,' i .; -.X'1~ TX*-' - .. / > S ta tis tic a l A nalysis !- . - S'jUy The M a n n -^ itn e y U t e s t o ^ C h i ^ i u a r e i x e s t was employed tc a s s e s s the s ig n ific a n c e of treatm ent e f f e c ts .' -D ose-responss tteo d 's'w ere determ ined by Jo n c k h ee re 's t e s t ; D ifferences' betw een m itogen responses-'yere determ ined'by S tu d e n t's t - t e s t . - '' -4. l " y. 1 ^wr. e.su.wl. ts - ; . * * / .?****'xy i;* .* T sb le 1 su m s a riz e * t h e ` ffe e t O t `d i s j s ic a l e x p o su re on body and s e l e c t e d o rg an w eights. A sig n ifican t-d ecrease'-m ^b o d y .w eig h t gain vsa observed in th e S ug/kg .TCDD d osage g ro u p . Thymus v eig lit'W U -jth y m n a-to 'b o d y w e ig h t r a t i o s were a ls o -.sig n ifican tly '.red u ced in tfiiad o sag f^g ro u p ,-.. H istopathological ev alu atio n of th e' . thymis rev e ale d se v e re th y m is;atro p h y w ith d e p le tio n of lymphocytes from c o r t i c a l regions in the 5 pg/kg d o s a g e ' ` *- *7 *- # . '.T able. 1 a l s o s u o s a r i x e s b o d y -;S i^ ,j(e ie C t* d 'o rg a n w eights- from DES exp o sed m ic e . Ko . e v id e n c e Of ^ o v a r t to s ^ 'c it ^ .w ^ ^ - ^ s e r w s d ^ a t any o f th e DES d o s e s . DES in d u ced a 1* s lig h t in crease in body w eight ` ighr^be expected, w hile producing a dram atic d e c re a s e in thym is w e ig h ts w hich ra n g e d ;fro m 50 to 752 d ep en d in g on d o s e . On h i s t o p a t h o l o g i c a l e v a l u a t i o n , m a r k ^ * a l t e r a t i o n s o f thymus s i r e c h a r a c t e r iz e d by marked d e p l e ti o n o f c o r t i c a l lym phocyte- a s a f u n c tio n o f DES d o se was o b s e rv e d . S pleen and l i v e r w e ig h ts w ere it^creased V BP p roduced a d ose re s p o n s e tr e n d (p < 0 .0 1 ) .tow ards body and thymus w eig h t lo s s which was not s t a t i s t i c a l l y s i g n i f ic a n t a t any o f th e .c o n c e n tr a tio n * ` te s te d (T a b le 1 ) . O ther o rg a n w e ig h ts w ere u n a lte r e d . ..H is to p e e c o lo g ie s ^evaluation o f o th e r lymphoid organs re v e a le d no marked p a th o lo g y ; " ^ \- V-" Jv ` -'v ~ The e v a lu a tio n o f bone marrow c e llu la S rity , sp le e n c o lo n ie s (CFU-S) in x - i r r a d i a t e d r e c ip ie n ts , and th e number of bone marrow c e l l s form ing m acrophage-granulocyte c o lo n ie s (CFU-CH) i s s u m a r i z s d . i n T ab le 2 . Bone marrow c e l l u l a r ic y and th e number o f c e l l s fo rm in g CFU-S c o l o n i e s / S * 4 0 * c e l l s was s i g n i f i c a n t l y d e p re s s e d in th e h igh TCDD dosage g ro u p . CFU-Qi c o lo n ie s w ere s i g n i f i c a n t l y red u c ed in th e h ig h dosage g ro u p . The e f f e c t o f DES e x p o s u re on bone marrow c e l l u l a r i t y and CFU-s i s a ls o sum aarixed in T able 2. The o b serv ed h y p o c e llu l a r ity o f th e bone marrow was s i g n i f i c a n t ( p < 0 .0 1 ) and lik e w is e d o te --r e l e t e d . The number o f bone marrow CFU-CH as m easured by i n v i t r o c u ltu r e s e x h ib ite d a d o se-d ep en d en t r e d u c tio n . The numbers of h em ato p o ietic p le u rip o te n t stem c e l l s (CFU-S'*) were lik e w ise dem onstrated to have a s ig n ific a n t (p < 0 .0 1 ) dose-dependent red u ctio n . r N 3 A *m1 iJ OP. n fft.<DP. 3C H| s c \*At HV *Oi n-1 n Zf i-- f --n o n O. i3c d-O1 < a*tn -p a* xn m p Ml 0Pp0f- PAap*-Pagc nAnHrAPr>- A " i* IA P- it (V M (t Ik Vi 6 rN* tnwtr 13 AC 83 ri*<*r-t* ? *nw1 cOr K.S 3aA. AON n ** t? xo* BBc . 0*, fHnCi* 8Na-K** Hi ^ A ,O, rt n rt n A 8 Oaf A p XAT Ck o A H VI 0 O A 4 >1 Hi O rrtt* . G A D " 1C A A 01 Are 'rei rre 3HHMn*** ss n'vnil r *a1 n Nk n (Hii drtI* IM MiM*nrNt*prt* XT H rt* A re g. w A i Htro SAn OHXritg-N>KNM n gi rt P A<' > Or*i <M o raUt *- A A* HA* rt X D P 8. 8 i Art fry.. /y ** ; ' d'* C heaicel Dose TABLE 1 E f f e c t o f TCDD, DES end BP on Body end S e le c te d Orgen W eights iy; > .Oi.-I Body W eight Thymus w eig h t Thymui/body w eig h t S p leen `.w eight L iv e r w e ig h t-.- R ig h t ld d n e y (g) Cog) r e tio (M0>) (mg) (mg) w eight1 (g) TCDD TCDD TCDD 0 I Vi/kg 5 Mg/kg 13.6* 12.6 10.9C 61 58 36b 4.5 4.6 3.3b 84 840 HD 82 846 HD 70b 7l4b * HD!, DES 0 DES 0. 2 DES 2.0 DES 8 .0 Dose resp o n se 20.8 21.6 22.8 22.1 US 4$ 22c 14C 12c o.o; 2.3 l.0 c 0.6C 0.5C <0.01 120 140 160b 205c <0.01 . 1063 ' ' 294b J666c 207GC <0.0r,, .. 133.. 146. 135; I39: I? i BP 0. BP 50 BP 200 BP ` 400 Dose resp o n se 21.2 20.9 20.2 19.6 <0.05 63.3 3.0 59.4 2.9 57.0 ' 2.8 4 48.4 .. - 2.4 * -io.oi / * .' `ns- 97 97 . 92 . .`joo *' *. ; : .,'iis - _____ :______v ,h - 1050 040 .. 1050 1150 .NS 14^' :S; l l i . 138;: : W 15% - ;v s --Heen velue of 7-8 m ice/group; b -- e ig n if ic e n tly d if f e r e n t from c o n tro l e t p < 0 .0 5 ; ',c -- e ig n i f i c e n t l y - d l f f l i t from c o n tro l e t p < 0.01 ; NS -- Not s i g n if ic e n tly d i f f e r e n t ; ND -- Not done. *:i v WAff\i ` ' - -r, . .' i; jv .v, 'vw *t.'i-;V.'v' f-' *-1,--<*Tr** -s-;? cr^. ;-fc"rsiv*-, r* 'V - v*V* " Kntrorti `i. " t-t *.*< T r * ^ ' .V ' 1 \( ,t l * * 9 * *t '' TABLE 2 E f f e c t of TCDD, DES end BP on Bone Merrow C e l l u l a r i t y , P ro g e n ito r C e ll C o lo n ie s, end T -C e ll D ependent (SRBC) and In d ep en d en t (LPS) A ntibody P laque Forming C e lls ..fe"'"- *r * ! V* r i - ' 4'1 :f *Jr/ \r i 'r* >il1 ii<1 -n -*> Chemical Doce N ucleated c e lia / femur x 10-4 CFU-S/ 5 x 10"' c e ll* CFU-CM/ 10s c e lla SRBC PFC/ IO1 s p le e n c e l l a LPS PFC/ : y 10? sp leen call* o r Hk t i t e r (lo g 2) TCDD TCDD TCDD 0 1 yg/fcg 5 17.7 . 18.6 (4XO* u . e (l6X4)b 10.3 < *29.6 ND . 36 .8 7 .0 - (32Xfjb A 26.0 ' ND 1- ' I8XO v (1 2X4):'>- ; ; m\ ; no.; ' " ;> t '.'HD n -'.ND - , NO * S . DES 0 DE5 0 .2 a g /k g DES 2 .0 DES 8 .0 Dose resp o n se BP 0 BP 50 mg/kg BP 200 BP 600 Dose resp o n se ` - 19.1 15.7 13.7 12.9 <0.01 (18X4) (28X4)b (32XOc 19.8 22 .8 (15X+) 21 .1 (6XO 26.3 <23XOb <0.05 6.8 - Sii. 5 6 .6 (3X4) 40.9 (25X O b 6 .0 . (12XO ; 34.6 . (36XOc 4 . (4JX4)C 33.5 (38X4)c < 0.0r V <0.01 : ioo3 1039 (4XO -742 (26XOb. 775 (23X+)b . :: <0.01, . 0.6 ' 108 '8 . 5 (20X4)6 92 8 .5 (20X+)C 89 5 .7_.(46X t)c .,64 <0.01^ <t).01 (15X4) ( 1 8X4)b (41XOc 1510 1133 (25X4) 984 (35X4)6- 378 (75X4)C T < o .o r- `` 6': 1 6.00 5.67 3.67 <0.01 729 3B9 256 99 <0.01 < 10X 4) 05X 4)5 <>! *- -.-*c. (47X+)C (65X 0 c. (86X4) *v a -- Mean v a lu e of 6-8 mice /g ro iip .'w ith X change in p a r e n th e s is ; b -- S i g n i f i c a n t l y d i f f e r e n t from c o n tr o l a t p < 0.05; c -- S ig n ific a n tly d if f e r e n t from c o n tro l a t p < 0 .0 l; d -- Ile m a g g lu tin in /c ite r (lo g 2 ). ? B m :" mm* i. \\ ~''Aj m sa n an e o f iB ounocoxiclty fmA\ rmtmm\k?** d o a a g a - la v a ls ,- w h ile Cha n u o ta r o f CFU-GM c o lo n ia s v as red u c ed sc Che 200 and 400 mg/fcg l e v e l . V- i .*' ----- - , The JgM a n tib o d y p la q u e fo rm in g c a l l (PFC) ra sp o n a a co sheep red blood c a l l * (SRBC) was- s i g n i f i c a n t l y reduced* ( p < 0 .0 5 ) ac Che 2 and 8 mg/kg d o ta g e of DES (T ab le 2) And P f C t.p e r CocaL s p le e n v a r a ' l i k e w it a red u ced o v e r 40Z. The tecum a n tib o d y ' t i t e r - co th e T-celL in d e p e n d e n t a n t ig e n E. a o l i t lip o p o ly s a c c h a r id e (LFS) v a t s i m i l a r l y . " re d u c e d , up to 45Z in Che h ig h e x p o s u re g ro u p . The n o a t s t r i k i n g e f f e c t o b se rv e d i n ' chc; e v a lu a c io n o f BP v a s che d e p re sse d . PFC-.y re s p o n s e to b o th T.-detoendent and T -io d e p n d e n t s t i m u l i . The r e s p o n s e .to SRBC-vas.-V:- d e p re sse d f r o 2S.tr o W Z a n d t h e re s p o n s e to LFS v aa d e p re s s e d from 47 co 86Z, _ depending on th e tre a tm e n t group examined.* Uv. . . `,V.< A ssessm ent o f :th e ly o p h o _ p ro lifa ra c iv a ~(LP) re s p o n s e o f s p le e n c e l l s frota TCDD exposed w e a n lin g s ,~to m ito g e n s , r e v e a le d s i g n i t x c a n t i v d e p re s s e d p r o l i f e r a t i v e TBsponTca co th e T - c e l l m ito g en s PHA and Con A, w hich v e r c d o se -d e p e n d e n t, bu t n o t - TO tA n - c e i i m ito g en PS T able 5 ) . <--------------------* :\* 1 " 7* T able 3 a ls o su sm a rlz c s th e e f f e c t s o f DES a d m in is t r a tio n on i n v i t r o s p le n ic LP r e s p o n s e s . LP r e s p o n s e s to sh e T -ly m p h o cy te ito g e o s FHA and Con A w ere s e v e r e ly depressed in tre a te d n ic e . S u p p ressio n of th e se responses ranged from approxim ately 30Z in .the 0 .2 n g /k g d o sag e group to 70Z i n th e h ig h e s t d o sag e g ro u p . A dose re sp o n se r e f f e c t was n o t se en , in th e re s p o n s e to th e 8 -c e lL m ito g en LFS. - The LPS resp o n se was augmented i n th e 0 .2 and 2 .0 u g /k g dosage groups w ith a 291 d e c re a s e in th e 8 mg/kg dosage g ro u p , w hich was n o t s t a t i s t i c a l l y s i g n i f i c a n t when com pared . to c o n t r o l s . .S u p p r e s s io n o f th e m ixed le u k o c y te c u l t u r e (KLC) re sp o n se s o c c u rre d in m ice from th e 2 .0 and 8 .0 mg/kg DCS d o sag e ( d a ta n o t show n). *" * .As - LP re s p o n s e s to th e T - c e l l ito g e n s FHA an d Con A w ere ( p r e s s e d in BP c r e a te d m ice up to 60Z and 54Z r e s p e c t i v e l y (T a b le 3).. The B - c e ll re s p o n s e co LPS was enhanced a t che, lo w est dose and d e p re sse d 54Z a t th e h ig h est^ d o se . A ll m itogens e x h ib ite d dose-dependent depression. r i. Hacrophage numbers and fu n c tio n vas examined in mice exposed to a l l 't h r e e ch em icals. F u n c tio n was evaluaC ed u s in g a s s a y s f o r SRBC p h a g o c y to s is , leu k em ia t a r g e t c e l l (HBL-2) c y t o s t a s i s , m a cro p h ag e -m ed ia ted stumor c e l l c y c o ly s is and s u p p r e s s o r c e l l a c t i v i t y . A nim als expo sed to TCDD and BP e x h ib ite d norm al H i.f u n c t i o n and num bers. In c o n t r a s t , m ice exposed to DES e x h i b ite d an enhanced number o f p e r i t o n e a l Hi (Table 4) which e x h ib ite d enhanced p h ag o cy tic, c y to s ta tic and c y to ly tic p o te n tia l in d ic a tiv e of Hi a c tiv a tio n (Table 4 ). In an a tte m p t to u n d ersta n d th e m echanism o f TCDD-induced T - c e ll d e f ic ie n c y , m ice w ere exam ined f o r th e p re s e n c e o f a p u t a t i v e r e c e p to r f o r TCDD in th e thymus and o th e r t i s s u e s . T hese s t u d i e s (T a b le 5) r e v e a le d a r e c e p to r f o r TCDD p r e s e n t a t h ig h c o n c e n tr a tio n s in thymus (114 fm/mg o f p r o t e i n ) , Co a l e s s e r e x t e n t in l i v e r and p re s e n t a t o n ly a v ery low c o n c e n tra tio n in h e a r t m u scle. C om petition e x p e ri ments dem onstrated the r e la tiv e s p e c if ic ity of th is re c e p to r (Table 5 ). Thus, the r e c e p to r fo r TCDD in thymus p ro b a b ly h e lp s a c c o u n t f o r th e s e l e c t i v e t o x i c i t y o f the chem ical fo r thymus-dependent lym phocytes. Hoc t s u s c e p t i b i l i t y to ch a l le-nge w ith 1.2 * 10* v ia b le L i s t e r i a (LD20) and sy n g e n eic PYB6 tumor c e l l s (TD20) in TCDD ex p o sed mice is summarized in T able 6 . in c re a s e d m o r ta lity to L. m onocytogenes and tumor ta k e fre q u en c y fo llo w in g FYB6 c h a lle n g e was o b se rv e d in TCDD ex p osed mice even a t Che lo w est l e v e l o f TCDD ex am in ed . E ndotoxin hyper s e n s i t i v i t y was n o t p e r f o ^ .-TABLE 3 , E f f e c t of TCDD, DES and. BP on--the ^y o tp b o p ro liferativ * Response* o f Hice J v ` -V Chemical Do** C o n tro l Msan CPM x 10" *-TdR I n c o r p o r i t i on (X ch an g e) POL Con A LPS TCDD -r " TCDD TCDD 0 I Ug/kg 5 DES 0 DES - 0 .2 mg/kg DES 2 .0 DES 8 .0 Dose resp o n se BP 0 BF 50 * mg/leg BP 200 BP 400 Dose resp o n se 1.0 1.1 . 0.6 , . 0 .9; ;; 0 .8 1.2 1.0 NS 0.5 0.5 0.5". 0 .3 ' <0.05 31.4*. T. 40.3 35.9, (I4X t) 41.2 14.9 (S2X4)C : 25.7 2 7 .5 ` ' .?'*** v 4 2 3 .4 - 17.3 <37X4)> 16.1 12.2 (56X4)C 12.1 " 4 .8 (83X4)c 8.3 <0.01 <0.01 (2X+) (36X4)b (31X4) (48X4)c (65X4)C ' 77.7 71.3 52.8 31.3 <0.01 (8X4) (32X4)C (60X4)c 82.0 87.3 (6X+) 54.7 (33X4)b 3 7 .? <54X4)5 <0.01 3 .8 ' 4.6 (21Xt) 3.1 (18X4) 8.2 13.6 (65X t)b 10.0 (22Xt) 5 .8 (29X4) NS 16.9 22.4 16.8 7.7 <0.05 (3 2 2 t)b (54Z4)c a -- M e a n 'v a lu e 'o f 6 -8 n ic e /g ro u p ;, b -- S i g n i f i c a n t l y d i f f e r e n t from c o n t r o l a t p < 0 .0 5 c -- S ig n ific a n tly d if f e r e n t from c o n tro l a t p < 0 .0 1 . V TABLE 4 E f f e c t o f DES E xp o su re on P e r ito n e a l M acrophage F u n c tio n Dosage (mg/kg) T o ta l number o f peritoneal c e lls x 10" */mouse Z 5BBC phagocytosis re la tiv e to control 0 g.2 2 8 Dose resp o n se 2.76* 3.50b 4.4QC s .6 2 c <0.01 1 * ;' . 92X 155Xb 150Xb <0.05 X Macrophage c y to sta sis (20:1) 1 28X 86 Xc 90 Xc <0.01 X Net C ytolysis (10:1) - -2Z 2 5 .2Xb 2 4 .7Xb <0.05 a -- Hean v a lu e o f 8 n ic e /g ro u p ; b -- A ctual value* v e re s i g n i f i c a n t l y d i f f e r e n t iro n c o n tro l a t p < 0 .0 5 ; c -- A ctual v alu es .were s ig n if ic a n tly d if f e r e n t from c o n tro l a t p < 0 .0 1 . TABLE 5 C o n c e n tra tio n o f TCDD R e c e p to r i n C y to so l o f Mouse T is s u e T issu e c y to s o l TCDD R e c e p to r (fa o l/m g p r o t e i n ) 2 , 3 , 7 ,6-TCDO 2 ,3,7,8-T C D F * I -N E ,- 3 , 7 ,8-TRICDD* Thymus L iv e r H eart 114 73 94 58 10 ND 56 30 ND a -- Compounds th a t com pete f o r s p e c i f i c b in d in g o f l J i I - d i o x i n in c y to s o l f r a c t i o n ; ND -- Not d o n e. l*. o*f1t hff-thh o Lon M<rMLMcflrMN 1 OO w"X-h- r* p 1 * * *1 i t-1 an tDA o Lon* OUA.'J OD*^ s H** wn iVUHMfOl' (X/) COo rs> --OvCB tM4O- MK-*o> /oCMns* ' .cr, KW--0N4"0 wM--4K> ' fc M1 (ft AotEB*r . < oMl .: *n ' 1: TABLE 6 E f f e c t o f TCDD, DES and BP E xposure on H ost R e s is ta n c e P a ra m e te rs Chemical TCDD TCDD TCDD DES . DES DES DES BP BP BP BP Dose PYB6 Tumor C e lls L is te ria Challenge Ho. tu o o rs/N o . c h a lle n g e d , H o .d e a th s /ty o . c h a lle n g e d . (Z tum ors) j (X m o r t a l i t y ) . 0 1 lig/kg 5 0 0 .2 mg/kg 2.0 8 .0 0 50 ng /k g 200 400 4/18 6/10. 4/9 2/10 3/10 8/10 9/10 0/10 1/10 0/10 .2/10 (22Z) (60X)b (44X) (20X) (30Z) (80X)b (90X)b COX) C10X> (OX) <20X> .... 7/25 6/15 -. . M /15 : 2/10 ! 10/10 10/10 a/8 0/10 1/10 1 / to 0/10 (28X) (40X) (73X)C . (20X) (I00X )b <l00X).b ' I00X)* (OX) (1X) (I0Z) (I0X) Endotoxin Challenge Ho. deatha/H o., c h a lle n g e d ^ '; . (I m ortality) * <? : ND ND HD /10 10 6/9 - 9/10 4/9 J V ? (67X)b ` (90X)b (44X) '.i" /(! X J' 'j 1/10 C10I). 3 /10 (30X) 7/10 , (70X)* 2/10 C20X) }jr a -- S ig n i f i c a n t l y 'd i f f e r e n t from c o n tro l a t p < 0.05 by C h i-sq u are a n a ly s is - o r S tu d e n t's T - t e s t ; b -- S ig n if ic a n tly d if f e r e n t from c o n tro l a t p < 0 .0 l by C h i-sq u a re a n a ly s is or S tu d e n t's T - t e s t . o --tft -Ft ; * I, l X-.r Y ... ,* ... ... > v; ;" . . -f -it ... * -v r--.\Y-, :?: . -*; .. ' if? .. '-t " r-Y Y ;1., i '*V< v?.y i-i'/Y ,.. .j.ip.iiR fnnm H C T iivm m ppinv -Jr-M -*'' hi **`12*A'S* . wW*-'.^ A^ 'n U u tA v V ..... .... i i j r i v 46 . ' \ vH'. .. . -,.V -J f.v *> - a. *'rf.t`,j >.v. ;\ }. . , Tab Is. 6 "a l o ' aujm safiids h f f c t ^ o f ji* i^ ix p 6 * u r ' in -a d u lt fem ale BC|Ft mic:. on' the jfre^iuviY of.P T B i tu m o r* !-fc ^ c v i^ ia ^ ^ le v e l (5 x 10*) o f cumo^'. , ^^3iO S ^of 'tu ao r t ^ i o c c ^ r d ^ f^ L c rv iiig "nor i t^ c H ir f i ^ i ^ t n ^ b d c ' adm iniitared- 8 .0 o r 2 .0 mg/kg ofV; ' DES. * Ttia'm aan la te n c y in 'd a y * to 'tu m o r " d a ta c tio n wax* n o t s i g n i f i c a n t l y a l t e r e d in DES expcxad m lce. aor^ wea th (Bean; tu o o r v o li e ( d a ta n o t show n). L ikew ise', T ab le 6 . s u o s i 'i^ i''c h a L le n g e 'a a d 'iD o r c ility ,* tu d ie x ; w ith L i s t e r i a i n m ice f o llo w in g DES- .exposuriT. '. M ice from, a l l th r e e DES'-tr e a tm e n t groups, d em o n strate d a s i g n i f i c a n t o r ta lit y v * ip < O .O I)."to a i i j r t a i ^ ; c K a l l e n g e d o se o f org an ism s c h a t k i l l e d o n ly 20Z / .. of_ th\COli-iol t r e a t e d c o n t r o l s ': ' i i i e ` m ean'la te n c y to d e a th in d ay s was lik e w is e ' . r e d u c e d .- ia /th e DES'"t r e a t e d >`g ro u p s ,w ith a n ' a v e rag e la te n c y o f from 4 .8 to 5 .4 days a r co$pa*d to . 8 . 0 ' d ay s'.in ~ th e ..c o n tr o l g ro u p . . .t . ''*"." '" '- 7,, .. ; DES exposed an tm alaTw r e r a i i 3 f s i g n i f i c a n t l y * ( p < 0 .0 1 ) o re s e n s i t i v e to a c h a lle n g e ` d o se o il g ra m -n e g a tiv e e n d o to x in ' (E* c c l i ) th a n were C o n tro l m ice (T a b le 6) w ith a 67.-90X. m o r t a l i t y ' f re q u e n c y , k d o s e -re s p o n s e p a t t e r n was n o t o b se rv e d s in c e th e highest/D E S 'dose ^ 8 .0 E^/kg) 'failed '^ tb 'd em o n strate s ig n ific a n tly in creased endo to x in jaojrt a l i ty.'^ / ' ...... BP e x p o s u re f s i l e d to s l t e f b o 'st r e s i s t a n c e 't o tumor c e l l o r '- L ic te r ia c h a lle n g e , '* *a lth- o\ uitg,#h*^i3nci r e a s e d m o4*r t a;`lLi tky*'>w,-as oLba*s."e, riv.*e.-d*->fo*llo *w in g en d o to x in, c h a lle n g e (T ab le 6 ) . i"-V ..V 'V h ' t: ^ ^ J ; , D i f e c o s s i 6 N ' ' ^ The* n a t i o n a l T o x ic o lo g y `Program (NTP) i s exam ining a v a r i e t y o f in v iv o and in v i t r o a p p ro a c h e s a s w e ll as th e a p p l i c a t i o n o f new r e s e a r c h d i s c i p l i n e s * t o th e field 'o .,td x ic o lo g y fo r the e v a lu a tio n s 'o f chem icals of environm ental concern. .Issu n o lo g y r e p r e s e n t s o n e 'o f th e d i s c i p l i nes t h a t t he NTP i s em ploy in g to exam ine possible ta rg e t organ to x ic ity as w ell as generalized to n e rty a t the celL u iarT h d bo l e e u l a r l e v e l . F u rth e rm o re , Che e f f e c t s o f ch em ic a ls otT T ioft"d e fe n c e to j , - ta c t t r i a l * v i r a l ; ftm gsl and p a r a a ir te d ise a se s as w en as the s u rv e illa n c e and Z' - ^ e ^ r o c t i o n b f tw o p la s rifd a lly 't r a h s f a m s d c e l l s are* s im u lta n e o u s ly b e in g e v a l u a t e d / r I t i* o u r /g oa l";i t h i i i k S f t o _Js y s c e m a tic a lly 'a p p ly th e d i s c i p l i n e s o f immunology and ' " m icrobiology in a com prehensive a rin et'to ''stu d y im m unotoxicity and a lte re d h o st ` -. ^_.. r e s i s t a a c k aa v e T ll.'a s /to /d e f in e a p a n e l o f s e n s i t i v e * s a y * to s c r e e n f o r c h e m ie a l- V i u d u c e d / c b a n g e s in IcooiaolO gical srxl h o s t r e s is ta n c e p zram eters. E x ten siv e ' s tu d ie s/c o n d u c te d ; at'M U X S 'have .s u g g e s te d 'th a t the ro u te of exposure as w ell as th e tim e 'o f . e x p o tire /re la tiv e to th e m a tu ra tio n s ! developm ent o f th e isxoune'system . should be o f paramount" c o n s id e r a tio n 'in th e d esig n o f e v a lu a tio n p ro to c o ls . S ev eral s t u d i e s h a v e d e m o n stra te d t h a t p r e - 'a n d 'p o s t n a t a l e x p o su re to c e r t a i n c h e m ic a ls ( e . g . TCDD o r DES) d u r in g th e * m a tu ra tio n o f th e th y m u s-d e p e n d e n t`e le m e n ts o f th e isaune system o fte n produces' m ore`profound issaunological e f f e c ts than a d u lt exposure (see review by F a ith and co w o rk ers)'o r produce L ong-lasting e ffec ts* th ro u g h a d u lt life ^ O C A lla n d . ` 1980b)-; * Th u s.- th e f o cu s' o f th e d i s c i p l i n e o f imauriology on th e v*?' -- e v a lu a tio n ''o f c h e a l c t l 't o x i c i t i e s "m ay'prov id e b e n e tic i a l in fo rm a tio n to a c m t a t e 'b e t t e r Human r i s k asaeli& tfiC T ' `. The e v a lu a tio n o f th e "pro to ty p e chem icals h ig h lig h te d h ere s u b s ta n tia te th ese b e lie f s reg ard in g the im portance of ev a lu a tin g im uno lo g ic a l param eters. Profound r e d u c tio n i n thymus w e ig h ts w ere o b se rv e d fo llo w in g p r e / p o s t n a t a l e x p o s u re to TCDD and fo llo w in g a d u l t ex p o su re to DES a t e x p o su re le v e ls w hich w ere n o t o v e r t l y to x ic by c l a s s ic a l, to x ic o lo g ic a l p a ra m e te rs. A s l i g h t d e c re a se in body w eight g ain o c c u rre d a t th e 5 yg/lcg TCDD doaage g ro u p , b u t n o t th e 1 u g /k g g ro u p , where norm al liv e r w eights and serum c h e m is trie s were found. These d a ta s u b s ta n tia te p rev ious r e p o r ta o f s e l e c t i v e thymus a tro p h y fo llo w in g TCllu ex p o su re . R e c e n tly , t a r i s te d t D ^ E T i r m y r e p o r te d th e p re se n c e o f a r e c e p to r Cor TCDD in thymus t i s s u e and l i v e r which was r e c e n tly confirm ed by Chae o f th is la b o ra to ry and re p o rte d h e r e . The p re se n c e o t h i s r e c e p to r may e x p la in th e s e l e c t i v e t o x i c i t y o f TCDD Cor th y m o c y te s. I ! jF | ale on f tumor ' -g/kg o ft> V . ( alterad in ' lis a , Tabi 6. in s DES If leene ed o n ly 20X m i< Likew ise ' 13 5 .4 day* a challenge 1 6) w ith a " ,inc the (sed en d o - jialLenge, (Table 6) . d in to the ncern. a -examine l l u l a r and jto [nee and evaluated." o lo g y aneT* ed host* r cheaicalive ' w ell a x ,7"' une system.. I*. Several " em icals* of the u lt exposure ugh a d u lt ?n the fa c ilita te :he*e | Profound -e to TCDD rely toxic gain re nom al previous ar Is ted c and liv e r The thym ocytes, "v". irvp?' *-r`f i r X e v t 1 ^1 .A ssessm en t o f Zsnsimot o x i c i t y a lth o u g h ,thc e x a c t lo c a ti o n o f t h i s r e c e p to r s t i l l a w a its d e f i n i t i o n . Sjgrdjfcant ly. reduced thymus w e ig h ts .o c c u r r e d .in s l l DES do sag e groups Thymus, h i a to lpgy-^taVaj ` Q a e v ^ S ly ic p h o id d e p t * t ; i o a / ,o & t r t l c a l ire g io n * i<n" ftp s':-a Wa---r--T-'-a-*---:--r-n--iwr r-F V r tF if fis-rkl~- , i?. T flt aid-rjot produc sL guificani'thym ui- w eight lo s s ' o r H isto lo g ic a l lte re tip n ^ w tic h ' nay suggest p rim arily a B ^cell d e fe c t ra th e r than T -c e ll a lte r a tio n s . ' V ... . . .\ .* ` pnpe n i r r n v TM 1IuL arl ry nj pr o g e n it o r c a l l (CFU-S and CFU-GHl S tudija 'r a i T ^ |V v ' genneeira l h y p o c e l l u l a r i t y T:Oo llTo w in g BP t r e a tm e n t. f o llo w In. th in e. g c a TC se toi&f. anTCdDDDBSth e e x^fpioyspuorce e- ual lnudl hyper a rlty * ce is l Tul pro a ritv baEX ^ ^ ^ -V-% 'to a d i r e c t t o x ic e f f e c t bn th e p l e u r a l - p o t e n t i a l stem c e l l s in c e CFU-S'y a i u e s i ^ '---' were d e p r e s s e d , w h ile CFU-GM c o lo n ie s (g ra n u lo c y te /m a c to p h a g e p r o g e n i t o r ^ , in c re a se d '. In c o n t r a s t , i n DES t r e a t e d m ic e, bone marrow CFU-Oi m lo n ie i'- w a ta if e ^ .f ^ ;/,; d e p re sse d and a t th e h ig h e s t DES d o se CFU-S c e l l s w ere a l s o d e p r e s s e d . . cau sed in c r e a s e d BH c e l ^ u l a r i t y accom panied by d e p re s s e d CFU-S and C F U -t ^ ^ a l e # > * ` 'V :<* ` T his o b s e rv a tio n a w a its f u r t h 'e r d e f i n i t i o n . T hus, BH c e l l u l a r i cy and prc^ltirv:'rv. ; >4> c e ll c lo n in g ca p a c ity ; ap p ear, to r e p r e s e n t s e n s itiv e e n d p o in ts f o r i-snun o to r i c i t y e v a lu a tio n e s p e c i a l l y s in c e - 'iym phoid e le m e n ta a r e a so u rc e o f p r o lif e r e to ^ Y in d jV i n h ib i to r y f a c t o r s f o r BM h e m o s ta s is ( H e tc a lf , 1978) and a r e b e lie v e d t o 'p r o v i d e r -. im portant re g u la to ry c ir c u ii* whose a l te r a t io n s a re probably r e f le c te d in c e l l u l a r i t y and p r o g e n i t o r `c e l l l e v e l s . ' DES tre a tm e n t produced, a m o d erate b u t s i g n i f i c a n t r e d u c tio n in th e PFC r e s p o n s e '.to SRBC and a n tib o d y t i t e r to LPS. S in c e DES in d u c e s M0 s u p p r e s s o r c e i l s (L u s te r; an d cow orkers, 1980) th e d e p re s s e d re s p o n s e s c o u ld r e s u l t from a l t e r e d H0 f u n c tio n o r a d i r e c t a c tio n o f . th e s u p p r e s s o r c e l l s on th e AB p ro d u c in g c e l l s . In c o n t r a s t , BP produced a d ra m a tic d e p r e s s io n i n IgM a n tib o d y FFCs to th e a n tig e n s SRBC".i75Z+) and LPS (6X +). T his o b s e r v a tio n e x te n d s th e r e c e n t o b s e rv a tio n s o f Urso and G engorian (I9 6 0 ) and e a r l i e r r e p o r ts ' by o th e r s t h a t BP and o 'th e r p o ly c y c lic a r o m atic h y d ro ca rb o n s d e p re s s th e a n tib o d y re s p o n s e to SRBCs ( S tje r n s w a r d , 1965,-: 1966; B all, 1970). ^. s f Exam ination of fu n c tio n a l resp o n ses of lym phocytes to T - and B -ce ll mitogens^,arc b e lie v e d , to r e p r e s e n t b o th an e s tim a tio n o f .T, B and n u l l c e l l le v e l s a s v o l l '.a a m onitor the fu n c tio n a l in te g r ity o f th e se p o p u la tio n s (bean, 1979a). R ecenC'-studies in ca n ce r p a t i e n t s have r e v e a le d th e im p o rta n ce o f th e LF a s s a y in th e imauno l o g i c a l s ta g in g o f can cer p a tie n ts r e l a t i v e ro s u r v iv a l and re c u rre n c e of tumor (Cannon-and cow orkers, 1980). The s ig n ific a n c e o f c h e m ic a l-in d u c e d 'a lte r a tio n s in f u n c tio n a l. LP re sp o n se s and th e lo n g -te rm co n seq u en ce o r su c a im p a irm en ts a w a its further" 'd e f i n i t i o n , _,, S tu d ie s w ith TCDD ex p o sed w e a n lin g s 's u g g e s te d more s e v e re ly , d e p re s s e d lan - c e l l f u n c ti o n s . H are d e t a i l e d s t u d i e s ( L u s te r and co -w orkers' ,'^T? 80a) rev ealed chat th e d epressed resp o n se s to T -c e ll m itogens was due to an i n t r i n s i c d e fe c t and not re la te d to su p p resso r c e l l a c t i v i t y sin c e th e se c e i l s f a ile d , to tran sfer depressed responses in co -cu ltu re. The e x p re s s io n o f s u p p r e s s o r a c t i v i t y from a d h e r e n t c e l l s in DES c r e a te d mice may be r e f l e c t e d in enhanced m acrophage c y t o * ta x i s o c c u r r in g i n TIES exp o sed mic*e.-.`;\Y* Recent studies dem onstrated th a t in d u ctio n of adherent suppressor c e ll a c tiv ity -' re s u lts from the s tim u la tio n o f c y c lic n u c le o tid e s by p rosT aglandins which s tr o gens have been shown Co o f te n mimic (Webb and Ja m ie so n , 1976; Weissman and co w orkers, 1975). Phren (1963) observed chat to p ic a l a p p lic a tio n of p o ly c y c lic arom atic hydrocarbons prolonged tr a n s p la n ta tio n r e je c tio n of a l l o g r a f t s . The ro u te of exposure or dose of BP employed may e x p la in th e f a i l u r e o f BP to s i g n i f i c a n t l y a l t e r h o s t r e s i s t a n c e in our s tu d y , sin c e BP seemed to a l t e r p r im a r il y B - c e ll re s p o n s e s ( i . e . a n tib o d y form ation), but not T -cell responses. We observed in c re a s e d m o r t a l i t y f o llo w in g Lift te ri.n r h a l l e n gft in TCDD and DES exposed mi r e BP d id n o t a l t e r L i s t e r i a s u s c e p t i b i l i t y . The in c re a s e d m o r ta lity c **SiJSti& iiid ` 46 J . H*. Dean a t a t . o b serv ed f o llo w in g DES was . a s s o c ia t e d w ith -a; s i g n i f i c a n t in c r e a s e in Che number o f v ia b le L i a ta ria l a th e sp le en s e n d ;liy e r i. a t 4 -day*. (Dean and co-w orker*. I960), a tic?* when T -c e l i*iaaaixrity ie^cbo^gfay^C ^^ecfaxpT^ * *e d ^ (T rip a th y en d Mackane*s,->4969)',y * b i t b a c t e r i a l . cotm ca v e r e e i M ila r 'to r f i o h c r o i m ic a 's c day 1, a c in e when Hfl a r e th o u g h t to e x e r t t h e i r g r e a t e s t e f f e c t .' T h e s e 'd a ta s u g g e s t- th a t th e in c r e a s e d __ X i a t e r t a - s u s c e p t i b i l i t y found f o llo w in g TCPD and DES ex p o su re r e s u l t * ir o n a T-oeXL d e f e c ts In p revious stu d i.es Thigpen- aod>co-vorfcer7T i s T r a erved in creased ; M o r ta lity fo llo w in g S a lm o n e lla c h a lle n g e - I n TCDD exposed B ic e , a f a c t which some f * e l co u ld be r e l a t e d t o 1th e in c re a s e d s e n s i t i v i t y o f th e s e m ice to g ra a rn e g a tiv e endotoxin. Likew ise, K eller (I9 6 0 ):found th a t sy n th e tic estrogens increased phagocytosis, but th a t enhanced*phagocytosis did not p ro te c t ag a in st m icrobial i n f e c t i o n . In c o n t r a s t , 'N ic o l a n d .co w o rk ers (1964) found c h a t OES s tim u la te d p h ag o cy to sis and prolonged c u r v i r a l 'o f Swiss mice a g a in s t Pnetanococcus type' I and P a s te u r e lla s e p tio a . Immunity to-.ft*m&oaoaous i s th o u g h t to be T -c e ll independent 'w h ic h may p a r t i a l l y e x p l a in th e p r e v io u s r e s u l t s so d th e p o s s i b le c o n f l i c t w ith ' d s t a r e p o r te d h e r e . THU; th e iamaino l o g i c a l d e f e c t w hich r e s u l t s in in c re a s e d s u s c e p t i b i l i t y to L i s t a r t a follow ing-T U D Q otc u t s e x p o su re .s t r o n g l y im p lic a te s a T ceLL d e f e c t w hich ifl 'BUppOgCfea py f u n c ti o n a l a s s a y s o f T - c e l l s . lutaor s u s c e p t i b i l i t y s t u d i e s ' fo llo w in g c h a lle n g e w ith 5 * tO1'V ia b le sy n g e n eic PYB6 tumor c e l l s r e v e a le d t h a t n o n - t r e a t e d - a d u l t BCaF i -mice r e s i s t e d tumor fo rm a tio n w ith only a 10-201 frequency o f tum or--form ation. '-In c o n tr a s t, mice exposed to TCDD i n u te r o o r DES a s a d u l t s had a 'tu m o r fre q u e n c y o f u p * to 60Z f o llo w in g TCDD and up -to 90Z f o llo w in g DES e x p o su re .- - I n 11 c a s e s th e tum ors w ere p r o g r e s s iv e . and r e s u l t e d i n d e a th . BP d id n o t a f f e c t th e fre q u e n c y o f tumor ta k e s fo llo w in g challenge in th is'm o d e l. The mecha*ni.sbm' by w hich th e u n im aun.ixed h o s t c a n r e s-i. s t curior o-r a l l o g r a f t- c h a lle n ge i s b e lie v e d to in v o lv e "imaune s u r v e illa n c e " aecKaniams r e q u ir in g immunocompetent 'hjflgH -Be p en d e n t lym phocytes _(T - c e l I s 1 end m acrophages IB u r n e t. 1970). o th fCDB / f and pks have been shown tco a l t e r T - c e l i im m unocoopetence in t h i s r e p o r t . // P rev io u s s tu d ie s (C ass, 1964) in b o th m ale and' fem ale C3H.mice dem onstrated a s i g n i f i c a n t l y in c r e a s e d d o s e - ^ e la te d - 'in c id a n c e -of masmax7 <-tumors fo llo w in g DES e x p o s u re . The in c id e n c e o f mansaary-*tumors f o llo w in g DES was enhanced o v er DES alone by r a d ia tio n exposure (S e g a lb o ff and K ax field ,^ 1971), a n o th e r ag en t which - a lte r s T -c e ll fu n c tio n . In a l a t e r r e p o r t. Case end co-w orkers (1974) dem onstrated a re q u ire m e n t f o r im irine mammary tum or "v iru s (HKTV) f o r -th e m atinary tumor in d u c tiv e e f f e c ts - o f DES.--'*-'- ' --r-'w*. *- - - ; .-.i-.-- . BP ex p o su re f a i l e d to a l t e r s u s c e p t i b i l i t y to L i s t e r i a w hich i s c o n s is c e n t w ith prim arily a B -cell d efect. S e n s itiv ity to g ram -negative endo to x in was a p p a re n tly in c re a se d fo llo w in g exposure to a l l th r e e c h e ts ic a ls . T hese d a t a s u g g e s t t h a t - t h e d e t o x i f i c a t i o n o f LPS i s r e l a t e d to a n * i n t a c t Md p o p u la tio n -end p o s s i b ly aven r e q u i r i n g B - c e lls . The d a ta p rese n ted here d em o n strate -th e s e n s itiv ity of the imsune system and the assay panel proposed for d etec tin g c e llu la r to x ic ity . ./^Iteratio n of T -ccll function appeared to c o r r e l a t e w ith in c re a s e d s u s c e p t i b i l i t y to b a c t e r i a l and tumor c e l l 1"ft-*-, S tu d ie s to c o r r e l a t e b e tw e e n -a s a a y s o f lym phocyte f u n c tio n and h o s t resistan ce are cu rren tly in progress. REFERENCES B a ll, J.K . (1970) Im sunosuppression and ca rc in o g e n e sis: c o n tra s tin g e ffe c ts w ith 7 ,1 2"d i th y lb e n r(a )a n C h ra c in e , b e n t(a )p y re n e , and 3 -m e th y lc h o la n th rc n e . J. Nat. 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