- Email: [email protected]
in vitro assay
Chemosphere, Voi.23, Nos.ll-12, Printed in Great Britain
pp 1855-1868,
1991
0 ~ 5 - 6 5 3 5 / 9 1 $3.00 + 0.00 Pergamon Press plc
Carcinogenic and co-carcinogenic potential of 2,3,7,8-tetrachlorodibenzo-p-dioxin in a ho,qt-med~ted in vivo/in ~-o way • • a b Th. M u s a , A. E s m a e l l l , B. S o h l a t t e r ~ , H. H s ~ m m , , ~ " and P. Chandra*
• M o l e c u l a r Biology (ZBC), University Medical School, T h e o d o r S t e r n - K a i 7, D-6000 F r a n k f u r t 71, FRG a Umweltbundcsamt, Bismarckplatz 1, W-1000 Berlin 33, F R O b Universitit T~bingen, Institut for Organische Chemic Auf der MorgensteUe 18, W-7400 T0bingen 1, F R O ABSTRACT In an in v i v o / i n v i t r o a s s a y s y s t e m (Massa e t al., 1990) we have d e t e c t e d t h e carcinogenic activity o f 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - p - d i o x i n CrCDD). The carcinogenic p o t e n t i a l m e a s u r e d in t h i s syst e m is c o n c e n t r a t i o n - d e p e n d e n t . E x p e r i m e n t s w i t h o t h e r carcinogenic c o m p o u n d s have revealed t h a t TCDD at low d o s e s c a n act as c o - c a r c i n o g e n . At h i g h e r c o n c e n t r a t i o n s TCDD induces T N F - a production.
INTRODUCTION TCDD elicits a wide s p e c t r u m o f biochemical a n d p a t h o p h y s i o l o g i c a l e f f e c t s which are m e d i a t e d by its capacity t o induce aryl h y r o c a r b o n h y d r o x y l a s e (AHH) (Gonzales e t a l , 1984; Israel and Whitlock,1984). The p r e s e n t c o n c e p t of its m o l e c u l a r m o d e of action as s u m m a r i z e d r e c e n t l y by W h i t l o c k (Whitlock, 1990) involves t h e a c t i v a t i o n o f CYFIA! gene coding for c y t o c h r o m e P450IAI The activation o f t h i s gene d e p e n d s o n t h e i n t e r a c t i o n b e t w e e n TCDD and its specific r e c e p t o r w h i c h is a DNA binding p r o t e i n . The DNA r e c o g n i t i o n site f o r t h e dioxin Ah r e c e p t o r c o m p l e x h a s b e e n r e p o r t e d by Denison (Denison e t al., 1986). A l t h o u g h t h e e x a c t m e c h a n i s m by which t h e r e c e p t o r - e n h a n c e r i n t e r a c t i o n a c t i v a t e s t h e t r a n s c r i p t i o n o f t h e CYP1A1 gene is n o t yet clear, it h a s b e e n p r o p o s e d t h a t o t h e r c e l l u l a r p r o t e i n s c o n t r i b u t e which m o d u l a t e t h e i n t e r a c t i o n b e t w e e n l i g a n d - r e c e p t o r c o m p l e x and t h e e n h a n c e r (VChitlock, 1990). TCDD h a s b e e n s h o w n t o b e carcinogenic in mice a n d r a t s in long chronic feeding s t u d i e s (Van Miller e t al., 1977; Kociba e t al., 1978; T o t h e t al., 1978; Kociba e t al., 1979) and t o p o s s e s s a t u m o r p r o m o t i n g e f f e c t in m o u s e skin (Di Giovanni e t al., 1977; Berry e t al., 1978. Kouri e t al., 1978; Kociba e t al., 1979). However, unlike o t h e r carcinogenic c o m p o u n d s o f t h e series o f polyc h l o r i n a t e d h y d r o c a r b o n s , TCDD s h o w e d n o c o v a l e n t binding t o r a t liver protein, r i b o s o m e s ,
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1856
R N A o r DNA u n d e r in vivo c o n d i t i o n s ( P o l a n d a n d Glover. 1979). M o r e o v e r in r i v e a n d in vitrt, s t u d i e s h a v e f a i l e d t o p r o d u c e any c o n c l u s i v e e v i d e n t s a b o u t its m u t a g e n i c i t y ( N e b e r t e t al., 1976; G i l b e r t e t al.. 1980: G e i g e r a n d Neal. 1981). F o r t h i s r e a s o n it w a s i n e r e s t i n g f o r u.,. 1c~ e v a l u a t e t h e c a r c i n o g e n i c p o t e n t i a l o f T C D D in t h e h o s t - m e d i a t e d
in r i v e / i n vitro a s s a y w i t h
p e r i t o n e a l m u r i n e m a c r o p h a g e s f o r t h e d e t e c t i o n o f c a r c i n o g e n i c c h e m i c a l s w h i c h h a s rect~ntI~ b e e n e s t a b l i s h e d in o u r laboratory, ( M a s s a e t al,. 1990),
MATERIAL AND METHODS H o s t m e d i a t e d in v i v o / i n v i t r o a s s a y All a n i m a l s w e r e 8 w e e k - . o l d m a l e m i c e o f t h e i n b r e d NMR1 s t r a i n w e i g h i n g a p p r o x i m a t e l y 30g. They were obtained from the central Breeding Laboratories of the IIniversity of Frankfurt and were maintained under specific pathogen free conditions. They had free access to standard diet ( A l t r o m i n ) a n d w a t e r . All c h e m i c a l s w e r e r e a g e n t g r a d e a n d w e r e d i s s o l v e d f o r e a c h e x p e r i m e n t i m m e d i a t e l y b e f o r e u s e . At day 0, 125tlg l i p o p o l y s a c c h a r i d (Sigma, LPS, E. coil, s e r o t y p e no. 0127:B8). d i s s o l v e d in l m l p h o s p h a t e b u f f e r e d s a l i n e (PBS). w a s a s c e p t i c a l l y a d m i n i s t e r e d t o e a c h m o u s e i n t r a p e r i t o n e a l l y . S u b s t a n c e s t o be e x a m i n e d w e r e d i s s o l v e d o r e m u l s i f i e d in Iml PBS c o n t a i n i n g 100rig 1 2 - O - t e t r a d e c a n o y l p h o r b o l - 1 3 - a c e t a t e ,
(TPA, Sigma, P-8139) o r w e r e
d i s s o l v e d in 0 . 2 m l e m u l s i o n o f 30~$ D M S O a n d 70X p e a n u t o i l (TCDD a n d TBrDD) e m u l s i f i e d in 0 . 8 m l PBS a n d 1 0 0 n g T P A . T h i s c o c k t a i l w a s t h e n a d m i n i s t e r e d Control
animals were
given either
at day 4 i n t r a p e r i t o n e a l l y .
PBS c o n t a i n i n g 100ng TPA. o r
PBS a l o n e
or
0.2ml
c,f
30X D M S O / 7 0 X p e a n u t o i l in 0 . 8 m l PBS. M a c r o p h a g e s w e r e c o l l e c t e d by r e p e a t e d p e r i t o n e a l lavag~, f o u r d a y s latt, r. T h e a p p r o x i m a t e yield o f m a c r o p h a g e s p e r m o u s e w a s 2 - 4 × 106. T h e s u s p e n d e d p e r i t o n e a l
macrophages were
c e n t r i f u g e d at 600 x g f o r 10 rain., r e s u s p e n d e d a n d w a s h e d t w o t i m e s u s i n g 5 m l o f a cell c u l t u r e m e d i u m ( u p - m e d i u m ) , c o n t a i n i n g 2 / 3 H a n k ' s 199 ( S e r o m e d ) w i t h 10X f o e t a l c a l f s e r u m (FCS) a n d IX penicillin / s t r e p t o m y c i n
a n d 1/3 c o n d i t i o n e d H a n k ' s m e d i u m (CSF). T h e p r o d u c t i o n ~ f
c o n d i t i o n e d H a n k ' s m e d i u m is d e s c r i b e d b e l o w . One half of the resuspended (one b o t t l e / m o u s e .
cells (2.5ml/mouse)
was plated into a sterile culture bottle
S 0 m l , 2Scm 2. N u n c . No. 163371). s u p p l e m e n t e d by t h e s a m e v o l u m e o f u p
m e d i u m . T h e b o t t l e s w e r e i n c u b a t e d at 3 7 ° C in an a t m o s p h e r e c o n t a i n i n g 5X COo_. N o n a d h e r i n g c e l l s w e r e r e m o v e d 24 h o u r s l a t e r by c h a n g i n g t h e m e d i u m . M e t a b o l i c a l l y acidified m e d i u m w a s r e p l a c e d by c o n d i t i o n e d m e d i u m w i t h i n t h e f i r s t week. T h e n acidified m e d i u m w a s r e p l a c e d by H a n k ' s 199 m e d i u m c o n t a i n i n g 10X FCS a n d 1X p e n i c i l l i n / s t r e p t o m y c i n The second half of the suspension was transfered
(1/1).
i n t o s o f t a g a r as f o l l o w s : O n e 24 wel!
p l a t e (Greiner, No. 662160) w a s u s e d f o r e a c h m o u s e . F i r s t 0 , 2 m l u n d e r l a y e r w a s p i p e t t e d i n t o e a c h well. A f t e r i t s s o l i d i f i c a t i o n 0.2ml u p p e r l a y e r w i t h t h e p e r i t o n e a l m a c r o p h a g e s at a c o n c e n t r a t i o n 2 - 4 x l0 s c e l l s / r o t
w e r e a d d e d . T h e p l a t e s w e r e t h e n i n c u b a t e d at 37°C, in a w a t e r
s a t u r e d a t m o s p h e r e c o n t a i n i n g 5X CO~. 24 h o u r s l a t e r 0 . 2 m l c o n d i t i o n e d m e d i u m w a s a d d e d p e r well. 5 t o 6 d a y s l a t e r t h e g r o w t h o f cell c o l o n i e s w a s e v a l u a t e d . U n d e r l a y e r : 0.6X a g a r fDifco, No. 0140-01): 59.4g H a n k ' s 199 (IX s t r e p t o m y c i n e / p e n i c i l l i n e ) : 20.0X f e t a l c a l f s e r u m : 20.0Z c o n ditioned medium(contains
CSF). U p p e r l a y e r : S0.0X u p - m e d i u m
p h a g e s ) ; 29.4~ 11ank's 199 (IX s t r e p t o m y c i n fetal calf serum.
(containing peritoneal
macro-
/ p e n i c i l l i n h 0.6~ a g a r (Difco. No. 0140 01): 20.(IX
Ig57
Production of conditioned m e d i u m M o u s e fibroblasts L-929 ceils were g r o w n in Hank's 199 m e d i u m containing 10% F C S and I% streptomycin, /penicillin. 2 x 106 cells (of 1 x 10s ceIl/ml) were given into a culture bottle (Nunc, 260mi, 80cm2}. They were cultured at 37°C after being equilibrated with a 5X C O 2 in air t w o days beyond the time reaching confluency. Supernatants of these cultures were collected. After centrifugation at 2000 x g for I0 minutes the supernatant was filtered through a m e m brane filter (Millipore, No. S L G V 025BS). This filtrate was used as conditioned m e d i u m containing C SF.
Evaluation of the test: The t r a n s f o r m i n g potential of s u b s t a n c e s was c h a r a c t e r i z e d as d e s c r i b e d e l s e w h e r e ( M a s s a e t al.. 1990}: briefly, m i c r o s c o p i c a l l y d i s t i n g u i s h a b l e c l o n e sizes w e r e divided into 10 c l a s s e s (C0-C9). The f r e q u e n c i e s o f c l o n e sizes o f a d e f i n e d c l a s s w e r e d e t e r m i n e d for e a c h 2 4 - w e l l plate. T h e y w e r e r e l a t e d t o t h e cell n u m b e r a n d were r e p r e s e n t e d as indicated in Table 1-3. T h e m i c r o s c o p i c a l l y d e t e r m i n e d f r e q u e n c y o f t h e c l o n e size o f each c l a s s w a s multiplied w i t h a f a c t o r c o n s i d e r i n g t h e s i g n i f i c a n c e o f t h e c l o n e size. The r e s u l t i n g p r o d u c t s o f c l a s s e s C 0 - C 9 were s u m m e d u p f o r e a c h 24-well p l a t e ( r e s u l t o f one animal) s e p a r a t e l y . The m e d i a n o f e a c h e x p e r i m e n t a l g r o u p c o n s i s t i n g o f 5 t o 6 a n i m a l s and r e p r e s e n t i n g S t o 6 24-well p l a t e s desig-nares the t r a n s f r o m i n g potency of the respective substance.
T N F - ~ ELISA Resident peritoneal macrophages for the first control group without any injection were recovered from 8 week old male N M R l - m i c e by repeated Washing of the peritoneal cavity with PBS. This peritoneal macrophages were washed 3 × and suspended to I x 1 0 6 P e M O per ml H a n k ' s 199 c o n t a i n i n g 10% FCS a n d 1% s t r e p t o m y c i n e / p e n i c i l l i n e . 4 ml o f s u s p e n s i o n w a s p l a t e d i n t o a s t e r i l e c u l t u r e b o t t l e (Nunc. S0ml, 2Scm2). A f t e r 2h i n c u b a t i o n at 37°C and 5% CO 2 n o n ~ a d h e r e n t cells were r e m o v e d by t w o r i g o r o u s r i n s e s . Second c o n t r o l g r o u p w a s injected 0.2ml o f 30% DMSO/70% p e a n u t o i l e m u l s i f i e d in 0.8ml PBS. E x p e r i m e n t a l g r o u p w a s injected w i t h t h e s a m e cocktail b u t c o n t a i n e d various c o n c e n t r a t i o n s o f TCDD, i n t r a p e r i t o n e a l l y . 14 days p o s t a d m i n i s t r a t i o n t h e m a c r o p h a g e s w e r e h a r v e s t e d by r e p e a t e d w a s h i n g o f t h e p e r i t o n e a l cavity a n d p l a t e d into c u l t u r e b o t t l e s at s a m e c o n c e n t r a t i o n as d e s c r i b e d f o r f i r s t c o n t r o l group. A f t e r t h e initial 2h a d h e r e n c e period, t h e m o n o l a y e r s w e r e ow.rlaid with 3.6ml s e r u m - f r e e H a n k ' s 199 m e d i u m c o n t a i n i n g 1% s t r e p t o m y c i n /penicillin
a n d 20ttg LPS f o r TNF-~ induction.
All m a c r o p h a g e p o p u l a t i o n s were >95% m o n o n u c l e a r p h a g o c y t e s as d e t e r m i n e d by s t a i n i n g , m o r p h o l o g i c and p h a g o c y t i c indices. C e l l - f r e e s u p e r n a t a n t s were c o l l e c t e d a f t e r 18h i n c u b a t i o n 0 . 4 m l o f 10-fold c o n c e n t r a t e d c o a t i n g p u f f e r (1.6g NaCO3+3,0g NaHCO 3 in 100ml aqua d e s t . . pH 9,6) f o r t h e e n z y m e - l i n k e d i m m u n o s o r b e n t a s s a y (ELISA) was added t o e a c h 3.6ml s u p e r n a ~ r a n t . 170,tl w a s p l a t e d i n t o each well o f f | a t
b o t t o m m i c r o w e l l m o d u l e s (Nunc. micro well
m o d u l e , no. 4-69949). Each p l a t e carried its own internal s t a n d a r d . The s t a n d a r d c o n s i s t s ¢~f a log 2 d i l u t i o n o f r e c o m b i n a n t routine t u m o r n e c r o s i s f a c t o r alfa ( E r n s t - B o e h r i n g e r - l n s t i t u t f u r Arzneimittelforschung, recombinant murine tumor necrosis facto alfa, E. cull-derived, no. 4296-17) in coating buffer. After incubation over night at 4 ° C and repeated washing with buffer
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(PBS + 0.0SZ T w e e n 20(Serva, T w e e n 20 p o l y o x y e t h y t e n e s o r b i t a n m o n o l a u r e a t e , no. 37470) + bovine s e r u m a l b u m i n e (BSA) I%) and b l o c k i n g lh w i t h PBS • IZ BSA rabbit polyctonal antibodies t o m u r i n e TNF-~ ( E r n s t - B o e h r i n g e r - l n s t i t u t f u r A r z n e i m i t t e l f o r s c h u n g , rabbit polyclonal anti bodies t o routine T N F - a , no. 70S9-65) were added in a c o n c e n t r a t i o n o f 1/1S00 and i n c u b a t e d 2h at 37°C. Biotinylated g o a t a n t i - r a b b i t a n t i b o d i e s (Medac, no. 6600) were added at a c o n c e n t r a t i o n o f 1/1000 f o r l h at 37°C. A f t e r w a s h i n g , it w a s i n c u b a t e d w i t h h o r s e r a d i s h p e r o x i d a s e (Vector, no. A2004) 0.Sh at
37°C (Sml
PBS + IZBSA + S~l h o r s e r a d i s h peroxidase) and incuba-
t e d 0.Sh w i t h o r t h o p h e n y l diamin h y d r o c h l o r i d e (OPD) (Stag OPD ÷ 0.Sml m e t h a n o l + 49.5ml a q u a d e s t . * Spl H202 ) at r o o m t e m p e r a t u r e . A b s o r p t i o n was d e t e r m i n e d u s i n g a micro ELISA autoreader.
RESULTS In t h e h o s t - m e d i a t e d in v i v o / i n vitro a s s a y w i t h peritoneal m a c r o p h a g e s . TCDD revealed a cellt r a n s f o r m i n g p o t e n t i a l t h a t s h o w e d a d o s e - d e p e n d e n t r e s p o n s e (table 1). T h e h i g h e s t c o n c e n t r a t i o n o f TCDD u s e d w a s 10~ of" LDs0 (LDs0 in mice: 125pg per kg) t o minimize t h e a c u t e t o x i c i t y as m u c h as p o s s i b l e . T h e c e l l - t r a n s f o r m i n g p o t e n t i a l o f TCDD at 0 . 3 9 ~ g / k g ( 7 . 9 n g / m o u s e ) was 1.3 w h i c h i n c r e a s e d t o 8.7S at 12.S~g/kg ( 2 5 0 n g / m o u s e ) . M a c r o p h a g e s t r e a t e d w i t h t h e cocktail c o n t a i n i n g p e a n u t oil, DMSO, PBS a n d TPA s h o w e d no t r a n s f o r m i n g p o t e n t i a l in t h i s s y s t e m . The c o - c a r c i n o g e n i c activity o f TCDD is i l l u s t r a t e d in t a b l e 2. In earlier work ( M a s s a e t al., 1990}, we have s h o w n , t h a t d i p h e n y l h y d a n t o i n at c o n c e n t r a t i o n s h i g h e r t h a n I S 0 ~ g / m o u s e h a s a c e l l - t r a n s f o r m i n g e f f e c t in o u r m a c r o p h a g e a s s a y s y s t e m . To s t u d y t h e c o - c a r c i n o g e n i c activity o f TCDD we have o m i t t e d TPA f r o m t h e cocktail. U n d e r t h e s e c o n d i t i o n s , TCDD at 0 . 3 9 ~ g / k g e x h i b i t s a very weak c e l l t r a n s f o r m i n g p o t e n t i a l o f 0.S. P h e n y t o i n at 1 0 0 p g / m o u s e s h o w e d no c e l l - t r a n s f o r m i n g p o t e n t i a l . However, t h e c o - a d m i n i s t r a t i o n o f TCDD leads t o t h e p o t e n t i a t i o n o f t h e o n c o g e n i c p o t e n t i a l o f p h e n y t o i n t o 2.9.
The co-carcinogenic activity of T C D D in table 3, Administration of T C D D
is dependent on the treatment schedule, as illustrated
t w o days prior to phenytoin, or two days after the pheny-
toin treatment has no effect on the cell-transforming potential of phenytoin. T C D D
admini-
stered at the same time as phenytoin leads to the highest potentiation of the carcinogenic potential of phenytoin.
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Table 1. Frequencies of the clone sizes in the host-mediated in vivo/in vitro assay with peritoneal routine macrophages after administration of various amounts of T C D D
together with TPA.
Control/' carcinogenic substance
No.of Frequency of clone sizea plate Unspecific Specific
Transforming potential of various concentrations
0.2mi +0.8ml +100ng i,p, per
DMSO/P.oil PBS TPA NMRI-mouse
1 2 3 4 5 6
10 4.0 3.35.0 --4.2--
1.3 1,3
0.0
0,2ml OMSO/P.oil +250ngTCDD +0.Sml PBS +100ng TPA i.p, per NMRI-mouse
1 2 3 4 5 6
5.0 22 76 8.8 47 25
0.5 6.0 19 3.0 15 9.0
0.5 3.0 3.3 0.3 6.3 5.0
8.75
0.2ml DMSO/P.oil +15.6ngTCDD +0.Sml PBS +100ng I-PA i.p. per NMRI-mouse
1 2 3 4 5 6
63 10 125 110 33
18 4,3 32 54 19
8.0 1.7 0.8 0.8 2.3 5.8 25 2.0 1.0 1.0 17 1.0
4.1
0.2ml +7.8ng +O.Sml +100ng i.p per
1 2 3 4 5 6
140 90 100 150 75 85
32 4.2 74 6.0 4.0 16
1.3 1.0
1.3
DMSO/P.oil TCDD PBS TPA NMRI-mouse
0.5 1.5 2.0 1.3 1.0 1.0 0 . 5 -
-
2.3 1.8 0.5 0.3 0 . 8 - -
--
2.7 1.2 1.6
aThe clone sizes according to cell n u m b e r (S-9, 10-14 ....)I00) and their frequencies are depicted for calculation of the transforming potential, as given under Material and Methods, or according to results published earlier (Massa et ai.,1990). S-9, all clones with S cells/clone to 9 cells/ clone were allocated to the class 5-9. S a m e procedure for classes C2 {10-14) to C9 (>I00l. bTransformlng potential of T C D D expressed as median of each experimental group.
Table 2. Co-carcinogenic effect of T C D D
with phenytoin.
Control/ carcinogenic substance
No.of Frequency of clone,size j plate Unspecific Specific
Transforming potential of TCDD and phenytoin b
0,2ml DMSO/P.oil +7.8ng TCDD +O.Sml PBS i.0. per NMRI-mouse
1 2 3 4 5
200 140 150 66 83
4.0 4,0 25 60 4.2
0.5
0.2ml +0.8ml +100pg i.p. per
DMSO/P.oil PBS phenytoin NMRI-mouse
1 2 3 4 5
166 133 160 150 30
3.3 2.3 33 40 --
2.0 0.7 0.7
0.2ml DMSO/P.oil +7,Sng TCDD +0.Sml PBS +100pg phenytoin i.p per NMRI-mouse
1 2 3 4 5
60 66 70 50 170
12 6.6 13 13 50
1.4 1.2 0.8 0.4 0.6 0.2 0.7 0.3 1.0 0.3 1.0 0.3 0.31.0 0.3 0 . 3 0.3 0 . 3 -
a.
0,5 1.0 0.3 2.7
0.0
bFo r description, see legend of table I.
2.9
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Table 3. EffeCt of application schedule on the co-carcinogenic potential of TCDD. Control/ carcinogenic substance
4. 1 ~ 0.2ml +7.Sng +0.8ml +lOOpg
No.of plate
Frequency o f clor~size a Unspecific
Specific
5-9
10-I#,
15-19 ~-L:u, 25-L~ 30-4~ 50-8g 70-;9 >100
50 2.0
35 5.0 3.5 2.5 0.8 -0.4
5.0 33
2.0 1.0 6.7 5.0 2.3 1.7 1.7 1.0 1.0
DMSO/P.oil TCDD PBS phenytoin
1 2 3 4 5
100 30 5.050 100
O.2ml D M S O / P . o i l +O.~ml PBS +lOOu.q phenytoin
1 2
60
A, Day
2. Day
Transforming potential o f TCDD/ phenytoin b 3.9
3.0 1.0
0.3
3
3.0 3 . 0 1.0 8 . 0 LO
0.3 . . . . . . . .
PBS
5
125 2 5
2.0
~ . 2~/#hl D M S O / P . o i l + , . e n a p-r~D +O .Sml 4: ~ +8,~ml ht ~4sSO/P'°"
1 2
2005.0
0.7
3 4
+lOOIA8 phenytoin
5
3002 5 1.7 2 2 1.5
0.8 0.5 . . . . . . . .
++ :
0.5
Table 4 . Co-carcinogenic effect of T O D D Control/ carcinogenic substance
No.of plate
compared with tumor promotor TPA.
Frequency o f cloresize a Unspecific
Specific
5-£
tS-~ 20-25 25-~ 30-$~ 50-6g 70-~ >~0
~-~
lml PBS 1 i.D. per N M R I - m o u s e 2 3 4 5
lml PBS 1 +100ng TPA 2 i.p. per N M R I - m o u s e 3
Transforming potential o f phenytoin,TPA ph./TPA, ph./TCDD b 0,0
5.0--
0.0 25
2.5
.
.
.
.
2.5 --
.
..............
4 5
4.0 . . . . . . . . . . . . tO --
0.2ml +0.8ml +100pg i.p. per
DMSO/P,oil PBS phenytoin NMRI-mouse
1 2 3 4 5
21 2 . 9 2.0 0.7 3.3 0.7 5.5-
0.2ml +0.8ml +100ng +100#g i.p per
DMSO/P.oil PBS TPA phenytoin NMRI-mouse
1 2 3 4 5
233 111 25 t50 55
0.2ml +7.8ng +0.Sml +100lzg i.p per
DMSO/P.oil TCDD PBS phenytoin NMRI-mouse
1 2 3 4 5
100 5 0 30 2.0
17 18 5.3 12 2.8
0.0
0.3 0.3 0,3 .
.
.
.
.
.
.
.
3.3 2.7 0.6 . . . . . . . . 1.0 --0.6
10
-
35 5.0 3.5 2.5 0.8 0.4
3.9
5.0 50 5.0 100 3 3
2.0 1.0 . . . . . . 6.7 5.0 2.3 1.7 1.7 1.0 1.0
a.b Experimental details are described in material and m e t h o d s , and under t a b l e 1.
1861
O u r n e x t a p p r o a c h w a s t o d i s t i n g u i s h b e t w e e n t h e c o - c a r c i n o g e n l c e f f e c t o f TCDD and t h e t u m o r - p r o m o t l n g activities o f TPA. As f o l l o w s f r o m t a b l e 4, n e i t h e r TFA ( 1 0 0 n g = 2 7 0 p m o l / m o u s e , MTPA = 364.44), n o r p h e n y t o i n (100pg/mouse) h a s any cell t r a n s f o r m i n g p o t e n t i a l a t d o s e s u s e d in t h e c o n t r o l e x p e r i m e n t s . The c o - a d m l n i s t r a t i o n o f TPA a n d p h e n y t o i n e x h i b i t a weak o n c o g e n l c p o t e n t i a l o f 1.01 however, t h e c o - a d m l n l s t r a t i o n o f TCDD(7.Sng=24pmol/mouse, MTCDD = 321.69) a n d p h e n y t o i n p o t e n t i a t e s t h e c e l l - t r a n s f o r m i n g p o t e n t i a l t o 3.9. The t e s t s y s t e m d e v e l o p e d in o u r l a b o r a t o r y h a s b e e n u s e d t o s t u d y t h e s t r u c t u r e a c t i v i t y r e l a t i o n s h i p in r e s p o n s e t o t h e i r cell t r a n s f o r m i n g potential. W e t h e r e f o r e have c o m p a r e d t h e cell t r a n s f o r m i n g p o t e n t i a l o f TCDD w i t h i t s b r o m o analog 2 , 3 , 7 , 8 - t e t r a b r o m o d i b e n z o - p - d i o x i n (TBrDD}. As f o l l o w s f r o m t a b l e S, t h e cell t r a n s f o r m i n g p o t e n t i a l o f TCDD is 7 t i m e s m o r e t h a n t h a t o f TBrDD a t t h e same m o l a r c o n c e n t r a t i o n and u n d e r identical e x p e r i m e n t a l c o n d i t i o n s . It is i n t e r e s t i n g t h a t we c o u l d o b t a i n a p e r m a n e n t cell line f r o m p e r i t o n e a l m a c r o p h a g e s o f mice t r e a t e d w i t h TBrDD. The biochemical a n d serological c h a r a c t e r i z a t i o n o f t h i s cell line will be i n t e r e s t i n g t o i n v e s t i g a t e t h e m e c h a n i s m s o f c a r c i n o g e n e s i s by TCDD, TBrDD and similar c o m p o u n d s . Table S. Cell-transforming potential of T C D D Control/ carcinogenic substance
No.of Frectuency of clonesize a plate Unspecific Specific s-~ 10-~
0.2ml +0.Smt +lOOng i.p. per
DMSO/P.oil PBS TPA NMRI-mouse
and TBrDD.
1 2 3 4 5
~-~ ~-~ 25-2g 30~ 5o.~g 7o..~ > ~
Transforming potential of phenytoin,TPA ph,/TPA, b ph./TCDD 0.0
14 5.0 8.0 3.0 7.0-7.2
0.2ml DMSO/P.oil +125ng TCDD = 0.39nMol +0.Sml PBS +lOOng TPA
1 2 3 4 5
280 90 190 80
3.0 6.0 3 . 0 -
90 15 210 68
4.0 2.0 11.0 6.0 7.0 1.0 1.0 --
0.2mi DMSO/P.oil +195ng TBrDD = 0.39nMot +O.8ml PBS +lO0ng TPA
1 2 3 4 5
25060
10.00.3 0.3 0.3
300 75 11 1 55 18
5.0 1.0 1.0
2.0 0 . 6 -
-1.0
a'bExperimental details are described in material and methods, and under table I.
O n e of the toxic effects of T C D D
reported in a number of investigations is weightloss (Holco-
m b et al., 19881 Pltlss et al., 1988; Hebert et al., 1990). Cachexia has also been noted in cancer patients. Oliff et al. (1987) have recently correlated cachexia in tumor bearing animals with the secretion of TNF-~. In these experiments tumors were induced in nude mice with C H O cells which had an integrated genetic construct of TNF-~. Cachexia was noted in mice injected with this particular construct C H O / T N F - 2 0 and a m u c h smaller effect was observed in animals injected with C H O
cells without the construct. This motivated us to look for the effect of T C D D
on T N F - ~ secretion in our cell system.
1862
A s f o l l o w s f r o m t a b l e 6 T C D D a t c o n c e n t r a t i o n s a b o v e l $ ~ g / k g is a b l e t o i n d u c e T N F - ~ s e c r e t i o n in a c o n c e n t r a t i o n d e p e n d e n t m a n n e r . C o n c e n t r a t i o n s
lower than this were unable to in-
d u c e T N F - ~ . I n t h e s e e x p e r i m e n t s T C D D w a s a p p l i e d in a s i n g l e d o s e ; h o w e v e r t h e s a m e a m o u n t o f T C D D ( $ 0 ~ g / k g ) e x t r a c t e d in 3 d o s e s g a v e t h e s a m e r e s u l t s ( l o w e r p a n e l , t a b l e 6).
Table 6. Induction of tumor necrosis factor • by T C D D .
Control/substance
day of i n t r m p e r i toneal injection b 1 S 9
TNP-~ • Units
ng
75pg/kg T C D D
X
141 + 6.4
II.7Sa + 0.S
50{~glkg T C D D
X
88
*-2.3
7.33 +0.2
2$tLg/kg T C D D
X
39
+-1.7
3.25
l$~g/kg T C D D
X
0
0
l,Stlg/kg T C D D
X
0
0
control (DMSOIPoil/PBS)
X
0
0
3-16.6ttg/kg= S 0 ~ g / k g T C D D
X
X
X
94
control (DMSO/Poil/PBS)
X
X
X
0
+-3.6
+-0.15
7.80
+0.3
control(DMSO/Poil/PBS)
CFA d
X
359 +-15.8 30.0
+-1.3
75~g/kg T C D D
CFA
X
396 +-12.2 33.0
+1.0
• T N F - ~ per 106 peritoneal macrophages and per ml medium. Serumfree m e d i u m samples were assayed for T N F - ~ activity by T N F - a ELISA at day 14 .as described under material and methods. I ng of TNF-~ is equivalent with 12 units. 1 unit of activity results in 50g lysis of fibroblasts using the standard L929 cytotoxity assay. bTCDD
was dissolved in 0.2ml emulsion of 30% D M S O
and 70% peanutoil emulsified in 0.8mi
PBS. This cocktail was then administered at day I, 5 or/and 9 intraperitoneally (marked with X). Control animals were given 0.2ml of 30% D M S O I T O Z
peanutoil in 0.8ml PBS.
°Each experiment contained 15 animals for each substance and concentration divided into three independent experimental groups. Each experimental group was analyzed 8-fold seperately. l~ach value depicts the m e a n of 24 experimental measurements with standard deviation. d 0.Sml complete Freunds adjuvant per m o u s e
DISCUSSION The environmental contaminant T C D D and related compounds are exogenous agents with carcinogenic potential in experimental animals. T C D D has been s h o w n to induce hepatic, pulmonary and skin tumors in long term feeding experiments (Van Miller et al., 1977:Kociba et al.. 1978a and 1978b; Toth et al., 1978; NTP, 1980). In particular, a number of recent studies have revealed that T C D D is a very potent promotor of hepatocarclnogenesis in female rats (Pitot et al., 1980; lqodstrSm and Ahlborg, 1989; FlodstrSm et al., 1990). Although T C D D is a potent carcinogen its m o d e of action is unknown. It has bvv~n shown that T C D D does not act as mltogen in bacterial systems (Gilbert et al. 1980: Geiger and Neal, 1981). nor does T C D D
s h o w co-
valent interaction with nucleic acids (Kondorosi et al., 1973; Poland and Glover, 1979): however, marginal effects on the incidence of the chromosome, aberration in vivo have been reported
1863
{Green a n d M o r e l a n d , 1975; Loprieno e t al., 1982). T h e s e s t u d i e s lead t o t h e c o n c l u s i o n t h a t TCDD h a s n o n e , o r very l i t t l e g e n o t o x i c activity w h i c h m a k e s it d i f f i c u l t t o explain its c a r c i n o genic e f f e c t s . The r e s u l t s p r e s e n t e d h e r e s h o w clearly t h a t TCDD h a s a h i g h c e l l - t r a n s f o r m i n g p o t e n t i a l i n o u r h o s t - m e d i a t e d in v i v o / i n v i t r o a s s a y w i t h p e r i t o n e a l routine m a c r o p h a g e s . So far, we k n o w o f t w o r e p o r t s w h i c h have b e e n able t o s h o w t h e m u t a g e n i c activity o f TCDD in animal s y s t e m s . Hay (1983) r e p o r t e d t h e m a t a g e n l c activity o f TCDD in a b a b y h a m s t e r kidney cell t r a n s f o r m a t i o n assay, a n d Rogers e t al. {1982) have r e p o r t e d t h a t TCDD is a direct a c t i n g m u t a g e n f o r L5178y cells. In t h e i r s t u d y , t r e a t m e n t w i t h a high {0.02 - 0 . 5 ~ g / m l ) c o n c e n t r a t i o n of TCDD p r o d u c e d dose d e p e n d e n t i n c r e a s e s in m e t h o t r e x a t e r e s i s t a n c e o f cells. A l t h o u g h t h e a u t h o r s i m p l i c a t e d t h i s finding as a direct e f f e c t o f TCDD a c t i n g as a f r a m e - s h i f t m u t a g e n ; however, it c a n n o t b e e x c l u d e d t h a t t h e m e t h o t r e x a t e r e s i s t a n c e c o u l d be a r e s u l t o f t h e a m p l i f i c a t i o n o f t h e germ f o r d t h y d r o f o l a t e r e d u c t a s e . In a n o t h e r study, A b e r n e t h y e t al. (1985) have s t u d i e d t h e e f f e c t o f TCDD in a cell t r a n s f o r m a t i o n a s s a y s y s t e m involving C 3 H / 1 0 T I / 2 m o u s e e m b r y o f i b r o b l a s t s . T h e s e a u t h o r s failed t o o b serve any t r a n s o r m i n g activity o f TCDD with, o r w i t h o u t TPA. However c o n t i n o u s t r e a t m e n t o f t h e s e c e l l s w i t h low c o n c e n t r a t i o n s o f TCDD ((= 4pM) e n h a n c e d f o c u s p r o d u c t i o n in c u l t u r e s pretreated with N-methyl-N'-nitroN-nitrosoguanidine. From this study the authors conclude that p r o m o t i o n ol~ t r a n s f o r m a t i o n is t h e p r e d o m i n a n t e f f e c t o f TCDD in t h e s e cells. F r o m o u r r e s u l t s it is evident t h a t t h e cell t r a n s f o r m i n g activity o f TCDD is c o n c e n t r a t i o n dep e n d e n t . W e c o u l d a l s o e s t a b l i s h t h e c o - c a r c i n o g e n l c activity o f TCDD in t h i s s y s t e m at c o n c e n t r a t i o n s a t w h i c h TCDD h a s n o cell t r a n s f o r m i n g activity. W e d e s i g n a t e t h i s e f f e c t as a c o carcinogenic activity b e c a u s e t h e p r o m o t o r activity is applicable f o r t h o s e s u b s t a n c e s w h i c h t h e m s e l v e s have n o carcinogenic activity at any c o n c e n t r a t i o n . The s e c o n d p a r a m e t e r w h i c h also d i s t i n g u i s h e s TCDD f r o m a c l a s s i c p r o m o t o r like TPA is t h e a p p l i c a t i o n schedule. The t u m o r p r o m o t i n g e f f e c t o f TPA is s e q u e n t i a l in t h e sense, t h a t t h e initiating c a r c i n o g e n h a s t o be applied f i r s t , f o l l o w e d by TPA. In o u r s t u d i e s w i t h TCDD, t h e c o - c a r c i n o g e n i c e f f e c t o f TCDD was o b s e r v e d only u n d e r c o n d i t i o n s w h e n TCDD a n d p h e n y t o i n were a d m i n s t e r e d s i m u l t a n e o u s l y . F u r t h e r m o r e , we o b s e r v e d a s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p in t h e carcinogenic p o t e n t i a l o f TCDD a n d its s t r u c t u r e a n a l o g TBrDD. The b r o m o derivative h a d a m u c h l o w e r cell t r a n s f o r m i n g p o t e n t i a l t h a n TCDD. I n t e r e s t i n g l y , we have n o w b e e n ale t o e s t a b l i s h a m a c r o p h a g e cell line f r o m mice t r e a t e d w i t h TBrDD. The b i o c h e m i c a l a n d serological c h a r a c t e r i z a t i o n o f t h i s cell line will b e i m p o r t a n t t o e l u c i d a t e t h e m e c h a n i s m o f cell t r a n s f o r m a t i o n by TCDD. Recent s t u d i e s by Durra e t al. {1988) indicate t h a t TCDD c a n m o d u l a t e t h e e x p r e s s i o n o f g e n e s o t h e r t h a n r e s p o n s i b l e f o r A h - r e c e p t o r in p r o d u c i n g oncogenic e f f e c t s . They have b e e n able t o c h a r a c t e r i z e a 2.1-kb long mRNA w h i c h e n c o d e s t h e e n z y m e aldehyde d e h y d r o g e n a s e {aldehyde: NADP + o x l d o r e d u c t a s e , EC 1.2.1.5.) in llver o f r a t s t r e a t e d w i t h TCDD. The i n t e r e s t i n g a s p e c t o f t h i s i n v e s t i g a t i o n is t h e o b s e r v a t i o n t h a t t h i s 2.1-kb mRNA was p r e s e n t in primary r a t liver h e p a t o m a s f r o m u n t r e a t e d rats. This o b s e r v a t i o n s u g g e s t s t h a t TCDD c a n elicit oncogenic e v e n t s by s o m e r e g u l a t o r y p r o c e s s e s decoupled from the Ah-receptor. The e f f e c t o f TCDD o n T N F - a s e c r e t i o n h a s b e e n r e c e n t l y r e p o r t e d by Clark e t al. (1990) u s i n g TCDD r e s p o n s i v e a n d n o n r e s p o n s i v e mice. T h e s e a u t h o r s have d o c u m e n t e d an increasing T N F - a p r o d u c t i o n by TCDD; whereby, t h e e f f e c t was m o r e p r o n o u n c e d in t h e TCDD r e s p o n s i v e mice a n d m o r e o v e r , it was c o n c e n t r a t i o n d e p e n d e n t . The m i n i m u m d o s e n e e d e d f o r a s i n g i f i c a n t rise f o r T N F - a p r o d u c t i o n w a s 10~g/kg or m o r e ; t h e m a x i m u m i n c r e a s e o f T N F - a was r e p o r t e d at 500~g TCDD/kR body weight. The f a c t t h a t t h e i n d u c t i o n o f T N F - a was dose d e p e n d e n t in TCDD r e s p o n s i v e mice h a s lead t h e s e a u t h o r s t o p o s t u l a t e t h a t T N F - a p r o d u c t i o n is m e d i a t e d via t h e A h - r e c e p t o r . In o u r e x p e r i m e n t s we c o u l d o b s e r v e t h e i n d u c t i o n o f T N F - a p r o d u c t i o n only at
1864
doses about I5~g/kg body weight. The induction of TNF-~ by T C D D is dose dependent. It is interesting to note, that the cell transforming potential of T C D D was m a x i m u m at 12.5tlg/kg body weight (2S0ng/mouse). Since at this concentration w e did not observe any T N F - a production, w e believe that the cell transforming activity of T C D D tion. W e have investigated the effect of T C D D
is not regulated by T N F - a produc-
on T N F - a production and its consequence on the
body weight {unpublished data}. W e could observe a loss of body weight which was related to T C D D concentration and the amount of T N F - a production. The highest cachexia effect was observed in mice treated with 75~g/kg T C D D
and this group exhibited the highest level of TNF-~.
$UMk/ARY W e have developed a host-medlated assay system for the detection of the transforming action of chemical carcinogens on peritoneal macrophages (Massa et al., 1990). Directly as well as indirectly acting carcinogenic substances administered intraperitoneally to NMRI mice could be examined in this way. Resident m a ~ o p h a g e s were recovered by peritoneal lavage from treated and untreated mice and were cultured in soft agar. After 5-6 days normal and transformed cells could he distinguished. Statistical analysis comparing cells from 2,3,7,8-tetrachlorodibenzo-pdioxin{TCDD)-treated animals with those from control mice proved that the test is positive at least on a significance level of S• using the t-test. T C D D
revealed a cell-transforming potential
that showed a dose-dependent response in this host-mediated assay. The co-carcinogenic activity of T C D D
was established ~a experiments with diphenylhydantoin. L o w doses ofdiphenylhy-
dantoin which did not exhibit any t x a r m f ~
pot~tial in our system gained a high oncogenic
potential by the simultaneous administration of low doses of T C D D which, as well had no transforming potentiaL W e
have compared the cell transforming potential of T C D D
analog TBrDD. The Cell transforming potential of T C D D
with its b r o m o
is 7 times more than that of TBrDD.
Using monospecific antibodies to tumor necrosis factor~ (TNF-~} wehave found that T C D D stimulates the secretion of TNF-~. The experimental data reported here lead to the conclusion that T C D D has a carcinogenic as well as a co-carcinogenic activity and has the property to induce TNF-a.
l~y words: 2,3,7,8-tetrachlordibenzo-p-dioxin
- 2,3,7,8-tetrabromodibenzo-p-dioxin - Dlphenyl-
hydantoin - T u m o r necrosis factor a - T N F - ~ ELISA - Host mediated in vivo/in vitro assay
lu-~--wl~llks~mts. This work was supported financially by Umweltbundesamt, Berlin (Grant, no. 106 03 098). W e are grateful to Dr. G.R Adolf (Ernst Boehringer-lnstitut fur Arzneimittelforschung, Vienna} for the gifts of recombinant routine TN]F-~ and rabbit polyclonal antibodies to murine TNF-a. It is a pleasure to acknowledge the skilled technical assistance of Mrs. Anja Lerch.
1865
Abernethy DJ, Greenlee F, Huband JC, Boreiko CJ (1985) 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) promotes the transformation of C3H/10T1/2 cells. Carcinogenesis 6(4):651-653 Berry DL, Di Giovanni J, Juchau M, Brachen WM, Gleason GL, Slaga TJ (1978) Lack of tumor-promoting ability of certain environmental chemicals in tow-stage mouse skin tumorigenesis assay. Res. Commun. Chem. Pathol. Pharmacol 20:101-107 Clark G, Tritscher A, Rucier G, Taylor M (1990) Dose-dependent increase in tumor necrosis factor-alpha production in TCDD exposed mice is Ahreceptor dependent. 10th International Meeting Dioxin 90, Short papers, Eco-lnforma Press, Bayreuth, Volume 1:77-80 Oenison MS, Harper PA, Okey AB (1986) Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Codistiribution of unoccupied receptor with cytosolic marker enzymes during fractionation of mouse liver, rat liver, and cultured Hepa lclc7 cells. Eur. J. Biochem. 155:223-29. DI Giovanni J., Viaje A., Berry DL, Slaga TJ, Juchau MR (1977) Tumorinitiating ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and Aroclor1254 in two-stage system of mouse skin carcinogenesis. Bull. Environ. Contain. Txicol. 18:552-557 Dunn TJ, Lindahl R, Pitot HC (1988) Differential gene expression in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD).Noncoordinate regulation of a TCDD-induced aldehyde dehydrogenase and cytochrome P-450c in the rat. The Journal of Biological Chemistry 263(22): 10878-10886 Flodstrbm S and Ahlborg UG (1989) Tumor promoting effects of 2,3,7,8-tetrachtorodibenzo-p-dioxin (TCDD) - effects of exposure duration administration schedule and type of dict. Chemosphere, 19:779-783 Flodstr~m S, Busk L, Ahlborg UG (1990) Enhancement of altered hepatic loci development by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) effects of vitamin A supplementation and the type of dict. 10th international Meeting Dioxin 90, Bayreuth, sept. 12-15 1990, volume 1, 173-176 Geiger LE, Neal RA {1981) Mutagenicity testing of 2,3,7,8-tetrachlorodibenzo-p-dioxin in histidine auxotrophs of Salmonella typhimurium. Toxicol. Appl. Pharmacol. 59(1):125-129 Gilbert P, Saint-Ruf G, Poncelet F, Mercier M (1980) Genetic effects of chlorinated anilines and azobenzenes on Salmonella typhimurium. Arch Environ. Contain. Toxicol. 9(5):533-541
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Gonzalez FJ, Tukey RH, Nebert DW (1984) Strucktural gene products of the Ah locus. Transcriptional regulation of cytochrome P~-450 and P3-450 mRNA levels by 3-methylcholanthrene. Mol. Pharmacol. 26:117-2t Green S, Moreland FS (1975) Cytogenetic evaluation of several dioxins in the rat. Toxicol. Appl. Pharmacol. 33:161 Hay A (1983) The mutagenic properties of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Abstracts of Americ&n Chemical Society National Meeting 23(2):14 Hebert CD, Harris MW, Elwell MR, Birnbaum LS (1990) Relative toxicity and tumor-promoting ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and i,2,3,4,7,8-hexachlorodibenzofuran (HCDF) in hairless mice. Toxicology and applied Pharmacology, 102:362-377 Holcomb M, Cheng Y, Safe S (t988) Biologic and toxic effects of polychlorinated dibenzo-p-dioxin and dibenzofuran congeners in the guinea pig. Quantitative structure-activity relationships. Ann chemical Pharmacology, 37(8):1535-1539 israel DI, Whitlock JP Jr. (1984) Regulation of cytochrome PC450 gene transcription by 2,3,7,8-tetrachlorodibenzo-p-dioxin in wildtype and variant mouse hepatoma cells. J. Biol. Chem. 259:5400-2 Kociba RJ, Keyes DG, Beyer JE, Carreon RM, Wade CE, Dittenber DA, Kalnins RP, Frauson LE, Park CN, Barnard SD, Hummel RA, Humiston CG (1978a) Results of a two-year chronic toxicity and oncogenicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats. Toxicol. Appl. Pharmacol. 46:279-303 Kociba RJ, Keyes DG, Beyer JE, Carreon RM (1978b) Toxicologic studies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCOD) in rats. Toxicol. Occup. Med. 4:281-287 Kociba RJ, Keyes DG, Beyer JE, Carreon RM, Gehring PJ (1979) Long term toxicologic Studies of 2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals. Ann. N.Y. Acad. Sci. 320:397-404 Kondorosi A, Fedoresak I, Solymosy F, Ehrenberg L, Osterman-Golkar S (1973) Inactivation of QBRNA by electrophiles. Murat. Res. 17:149-161 Kouri RE, Rude TH, Joglekar R, Dansette PM, Jerina DM, Atlas SA, Owens IS, Nebert DW (1978) 2,3,7,8-tetrachlorodibenzo-p-dioxin as cocarcinogen causing 3-methylcholanthrene-initiated subcutaneous tumors in mice genetically "nonresponsive" at Ah locus. Can. Res. 38:2777-2783
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Loprieno N, Skrana I, Rusciano D, Lascialfari D, Lari T (1982) In vivo cytogenetic studies on mice and rats exposed to tetrachlorodibenzo-p-dioxin (TCDD). In: Chlorinated dioxins and related compounds, impact on the environment. Proceedings of a workshop held at the Istitutio Superiore die Sanita, Rome, Italy, 22-24 Oct. 1980. Ed.: Hutzinger O. et al., Pergamon Press, New York, 419-428 Massa Th, Gerber T, Pfaffenholz V, Chandra A, Schlatterer B, Chandra P (1990) A host mediated in vivo/in vitro assay with peritoneal murine macrophages for the detection of carcinogenic chemicals. J. Cancer Res. Clin. Oncol. Volume 116, no 4:357-364 Nebert D, Thorgiersson S, Felton J (1976) Genetic differences in mutagenesis, carcinogenesis and drug toxicity. In: de Serros F, Fotets J, Bend J, Philpot R (eds) In vitro metabolic activation in mutagenesis testing. Elsevier/North Holland Biomedical Press, Amsterdam. pp 105-124. NTP (1980) National toxicology program. Bioassay of 2,3,7,8-tetrachlorodibenzo-p-dioxin for possible carcinogenicity (garage studie). Carcinogenesis testing prgram, National Cancer Institute (NIH), Bethesda, MD; NTP, Research Triangle Park, NC. U.S. Department of Health and Human Services Publication no. 82-1765 Oliff A, Defeo-Jones D, Boyer M, Martinez D, Kiefer D, Vuocolo G, Wolfe A, Socher SH (1987) Tumors secreting human TNF/Cachectin induce cachexia in mice. Cell, 50:555-563 Pitot HC, Goldsworthy T, Campbell HA, Poland A (1980) Quantitative evaluation of the promotion by 2,3,7,8-tetrachlorodibenzo-p-dioxin of hepatocarcinogenesis from diethylnitrosamine. Cancer Res. 40:3616-3620 PIUss N, Poiger H, Hohbach C, Suter M, Schlatter C (1988) Subchronic toxicity of 2,3,4,7,8-Pentachlorodibenzofuran (PeCDF) in rats. Chemosphere, 17(6): 1099-1110 Poland A, Glover E (1979) An estimate of the maximum in vivo covalent Binding of 2,3,7,8-tetrachlorodibeno-p-dioxin to rat liver protein, ribosomal RNA, and DNA. Cancer Research 39:3341-3344 Rogers AM, Andersen ME, Back KC (1982) Mutagenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin and perfluoro-n-decanoic acid in L5178Y mouse lymphoma cells. Mutat. Res. 105:445-449 Toth K, Sugar J, Somfai-Relle S, Bence J (1978) Carcinogenic bioassay of the herbic;ide 2,4,5-trichlorophenoxyethanol (TCPE) with different 2,3,7,8tetrachlorodibenzo-p-dioxin (Dioxin) content in Swiss mice. Prog. Biochem. Pharmacol., 14:82-93
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Van Miller JP, Lalich JJ, Allen JR (1977) Increased incidence of neoplasms in rats exposed to low levels of 2,3,7,8-tetrachlorodibenzo-pdioxin. Chemosphere 6:537-544 Whitlock JP Jr (1990) Genetic and molecular aspects of 2,3,7,8-tetrachlorodibenzo-p-dioxin action. Annu. Rev. Pharmacol. Toxicol. 30:251-77
pp 1855-1868,
1991
0 ~ 5 - 6 5 3 5 / 9 1 $3.00 + 0.00 Pergamon Press plc
Carcinogenic and co-carcinogenic potential of 2,3,7,8-tetrachlorodibenzo-p-dioxin in a ho,qt-med~ted in vivo/in ~-o way • • a b Th. M u s a , A. E s m a e l l l , B. S o h l a t t e r ~ , H. H s ~ m m , , ~ " and P. Chandra*
• M o l e c u l a r Biology (ZBC), University Medical School, T h e o d o r S t e r n - K a i 7, D-6000 F r a n k f u r t 71, FRG a Umweltbundcsamt, Bismarckplatz 1, W-1000 Berlin 33, F R O b Universitit T~bingen, Institut for Organische Chemic Auf der MorgensteUe 18, W-7400 T0bingen 1, F R O ABSTRACT In an in v i v o / i n v i t r o a s s a y s y s t e m (Massa e t al., 1990) we have d e t e c t e d t h e carcinogenic activity o f 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - p - d i o x i n CrCDD). The carcinogenic p o t e n t i a l m e a s u r e d in t h i s syst e m is c o n c e n t r a t i o n - d e p e n d e n t . E x p e r i m e n t s w i t h o t h e r carcinogenic c o m p o u n d s have revealed t h a t TCDD at low d o s e s c a n act as c o - c a r c i n o g e n . At h i g h e r c o n c e n t r a t i o n s TCDD induces T N F - a production.
INTRODUCTION TCDD elicits a wide s p e c t r u m o f biochemical a n d p a t h o p h y s i o l o g i c a l e f f e c t s which are m e d i a t e d by its capacity t o induce aryl h y r o c a r b o n h y d r o x y l a s e (AHH) (Gonzales e t a l , 1984; Israel and Whitlock,1984). The p r e s e n t c o n c e p t of its m o l e c u l a r m o d e of action as s u m m a r i z e d r e c e n t l y by W h i t l o c k (Whitlock, 1990) involves t h e a c t i v a t i o n o f CYFIA! gene coding for c y t o c h r o m e P450IAI The activation o f t h i s gene d e p e n d s o n t h e i n t e r a c t i o n b e t w e e n TCDD and its specific r e c e p t o r w h i c h is a DNA binding p r o t e i n . The DNA r e c o g n i t i o n site f o r t h e dioxin Ah r e c e p t o r c o m p l e x h a s b e e n r e p o r t e d by Denison (Denison e t al., 1986). A l t h o u g h t h e e x a c t m e c h a n i s m by which t h e r e c e p t o r - e n h a n c e r i n t e r a c t i o n a c t i v a t e s t h e t r a n s c r i p t i o n o f t h e CYP1A1 gene is n o t yet clear, it h a s b e e n p r o p o s e d t h a t o t h e r c e l l u l a r p r o t e i n s c o n t r i b u t e which m o d u l a t e t h e i n t e r a c t i o n b e t w e e n l i g a n d - r e c e p t o r c o m p l e x and t h e e n h a n c e r (VChitlock, 1990). TCDD h a s b e e n s h o w n t o b e carcinogenic in mice a n d r a t s in long chronic feeding s t u d i e s (Van Miller e t al., 1977; Kociba e t al., 1978; T o t h e t al., 1978; Kociba e t al., 1979) and t o p o s s e s s a t u m o r p r o m o t i n g e f f e c t in m o u s e skin (Di Giovanni e t al., 1977; Berry e t al., 1978. Kouri e t al., 1978; Kociba e t al., 1979). However, unlike o t h e r carcinogenic c o m p o u n d s o f t h e series o f polyc h l o r i n a t e d h y d r o c a r b o n s , TCDD s h o w e d n o c o v a l e n t binding t o r a t liver protein, r i b o s o m e s ,
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R N A o r DNA u n d e r in vivo c o n d i t i o n s ( P o l a n d a n d Glover. 1979). M o r e o v e r in r i v e a n d in vitrt, s t u d i e s h a v e f a i l e d t o p r o d u c e any c o n c l u s i v e e v i d e n t s a b o u t its m u t a g e n i c i t y ( N e b e r t e t al., 1976; G i l b e r t e t al.. 1980: G e i g e r a n d Neal. 1981). F o r t h i s r e a s o n it w a s i n e r e s t i n g f o r u.,. 1c~ e v a l u a t e t h e c a r c i n o g e n i c p o t e n t i a l o f T C D D in t h e h o s t - m e d i a t e d
in r i v e / i n vitro a s s a y w i t h
p e r i t o n e a l m u r i n e m a c r o p h a g e s f o r t h e d e t e c t i o n o f c a r c i n o g e n i c c h e m i c a l s w h i c h h a s rect~ntI~ b e e n e s t a b l i s h e d in o u r laboratory, ( M a s s a e t al,. 1990),
MATERIAL AND METHODS H o s t m e d i a t e d in v i v o / i n v i t r o a s s a y All a n i m a l s w e r e 8 w e e k - . o l d m a l e m i c e o f t h e i n b r e d NMR1 s t r a i n w e i g h i n g a p p r o x i m a t e l y 30g. They were obtained from the central Breeding Laboratories of the IIniversity of Frankfurt and were maintained under specific pathogen free conditions. They had free access to standard diet ( A l t r o m i n ) a n d w a t e r . All c h e m i c a l s w e r e r e a g e n t g r a d e a n d w e r e d i s s o l v e d f o r e a c h e x p e r i m e n t i m m e d i a t e l y b e f o r e u s e . At day 0, 125tlg l i p o p o l y s a c c h a r i d (Sigma, LPS, E. coil, s e r o t y p e no. 0127:B8). d i s s o l v e d in l m l p h o s p h a t e b u f f e r e d s a l i n e (PBS). w a s a s c e p t i c a l l y a d m i n i s t e r e d t o e a c h m o u s e i n t r a p e r i t o n e a l l y . S u b s t a n c e s t o be e x a m i n e d w e r e d i s s o l v e d o r e m u l s i f i e d in Iml PBS c o n t a i n i n g 100rig 1 2 - O - t e t r a d e c a n o y l p h o r b o l - 1 3 - a c e t a t e ,
(TPA, Sigma, P-8139) o r w e r e
d i s s o l v e d in 0 . 2 m l e m u l s i o n o f 30~$ D M S O a n d 70X p e a n u t o i l (TCDD a n d TBrDD) e m u l s i f i e d in 0 . 8 m l PBS a n d 1 0 0 n g T P A . T h i s c o c k t a i l w a s t h e n a d m i n i s t e r e d Control
animals were
given either
at day 4 i n t r a p e r i t o n e a l l y .
PBS c o n t a i n i n g 100ng TPA. o r
PBS a l o n e
or
0.2ml
c,f
30X D M S O / 7 0 X p e a n u t o i l in 0 . 8 m l PBS. M a c r o p h a g e s w e r e c o l l e c t e d by r e p e a t e d p e r i t o n e a l lavag~, f o u r d a y s latt, r. T h e a p p r o x i m a t e yield o f m a c r o p h a g e s p e r m o u s e w a s 2 - 4 × 106. T h e s u s p e n d e d p e r i t o n e a l
macrophages were
c e n t r i f u g e d at 600 x g f o r 10 rain., r e s u s p e n d e d a n d w a s h e d t w o t i m e s u s i n g 5 m l o f a cell c u l t u r e m e d i u m ( u p - m e d i u m ) , c o n t a i n i n g 2 / 3 H a n k ' s 199 ( S e r o m e d ) w i t h 10X f o e t a l c a l f s e r u m (FCS) a n d IX penicillin / s t r e p t o m y c i n
a n d 1/3 c o n d i t i o n e d H a n k ' s m e d i u m (CSF). T h e p r o d u c t i o n ~ f
c o n d i t i o n e d H a n k ' s m e d i u m is d e s c r i b e d b e l o w . One half of the resuspended (one b o t t l e / m o u s e .
cells (2.5ml/mouse)
was plated into a sterile culture bottle
S 0 m l , 2Scm 2. N u n c . No. 163371). s u p p l e m e n t e d by t h e s a m e v o l u m e o f u p
m e d i u m . T h e b o t t l e s w e r e i n c u b a t e d at 3 7 ° C in an a t m o s p h e r e c o n t a i n i n g 5X COo_. N o n a d h e r i n g c e l l s w e r e r e m o v e d 24 h o u r s l a t e r by c h a n g i n g t h e m e d i u m . M e t a b o l i c a l l y acidified m e d i u m w a s r e p l a c e d by c o n d i t i o n e d m e d i u m w i t h i n t h e f i r s t week. T h e n acidified m e d i u m w a s r e p l a c e d by H a n k ' s 199 m e d i u m c o n t a i n i n g 10X FCS a n d 1X p e n i c i l l i n / s t r e p t o m y c i n The second half of the suspension was transfered
(1/1).
i n t o s o f t a g a r as f o l l o w s : O n e 24 wel!
p l a t e (Greiner, No. 662160) w a s u s e d f o r e a c h m o u s e . F i r s t 0 , 2 m l u n d e r l a y e r w a s p i p e t t e d i n t o e a c h well. A f t e r i t s s o l i d i f i c a t i o n 0.2ml u p p e r l a y e r w i t h t h e p e r i t o n e a l m a c r o p h a g e s at a c o n c e n t r a t i o n 2 - 4 x l0 s c e l l s / r o t
w e r e a d d e d . T h e p l a t e s w e r e t h e n i n c u b a t e d at 37°C, in a w a t e r
s a t u r e d a t m o s p h e r e c o n t a i n i n g 5X CO~. 24 h o u r s l a t e r 0 . 2 m l c o n d i t i o n e d m e d i u m w a s a d d e d p e r well. 5 t o 6 d a y s l a t e r t h e g r o w t h o f cell c o l o n i e s w a s e v a l u a t e d . U n d e r l a y e r : 0.6X a g a r fDifco, No. 0140-01): 59.4g H a n k ' s 199 (IX s t r e p t o m y c i n e / p e n i c i l l i n e ) : 20.0X f e t a l c a l f s e r u m : 20.0Z c o n ditioned medium(contains
CSF). U p p e r l a y e r : S0.0X u p - m e d i u m
p h a g e s ) ; 29.4~ 11ank's 199 (IX s t r e p t o m y c i n fetal calf serum.
(containing peritoneal
macro-
/ p e n i c i l l i n h 0.6~ a g a r (Difco. No. 0140 01): 20.(IX
Ig57
Production of conditioned m e d i u m M o u s e fibroblasts L-929 ceils were g r o w n in Hank's 199 m e d i u m containing 10% F C S and I% streptomycin, /penicillin. 2 x 106 cells (of 1 x 10s ceIl/ml) were given into a culture bottle (Nunc, 260mi, 80cm2}. They were cultured at 37°C after being equilibrated with a 5X C O 2 in air t w o days beyond the time reaching confluency. Supernatants of these cultures were collected. After centrifugation at 2000 x g for I0 minutes the supernatant was filtered through a m e m brane filter (Millipore, No. S L G V 025BS). This filtrate was used as conditioned m e d i u m containing C SF.
Evaluation of the test: The t r a n s f o r m i n g potential of s u b s t a n c e s was c h a r a c t e r i z e d as d e s c r i b e d e l s e w h e r e ( M a s s a e t al.. 1990}: briefly, m i c r o s c o p i c a l l y d i s t i n g u i s h a b l e c l o n e sizes w e r e divided into 10 c l a s s e s (C0-C9). The f r e q u e n c i e s o f c l o n e sizes o f a d e f i n e d c l a s s w e r e d e t e r m i n e d for e a c h 2 4 - w e l l plate. T h e y w e r e r e l a t e d t o t h e cell n u m b e r a n d were r e p r e s e n t e d as indicated in Table 1-3. T h e m i c r o s c o p i c a l l y d e t e r m i n e d f r e q u e n c y o f t h e c l o n e size o f each c l a s s w a s multiplied w i t h a f a c t o r c o n s i d e r i n g t h e s i g n i f i c a n c e o f t h e c l o n e size. The r e s u l t i n g p r o d u c t s o f c l a s s e s C 0 - C 9 were s u m m e d u p f o r e a c h 24-well p l a t e ( r e s u l t o f one animal) s e p a r a t e l y . The m e d i a n o f e a c h e x p e r i m e n t a l g r o u p c o n s i s t i n g o f 5 t o 6 a n i m a l s and r e p r e s e n t i n g S t o 6 24-well p l a t e s desig-nares the t r a n s f r o m i n g potency of the respective substance.
T N F - ~ ELISA Resident peritoneal macrophages for the first control group without any injection were recovered from 8 week old male N M R l - m i c e by repeated Washing of the peritoneal cavity with PBS. This peritoneal macrophages were washed 3 × and suspended to I x 1 0 6 P e M O per ml H a n k ' s 199 c o n t a i n i n g 10% FCS a n d 1% s t r e p t o m y c i n e / p e n i c i l l i n e . 4 ml o f s u s p e n s i o n w a s p l a t e d i n t o a s t e r i l e c u l t u r e b o t t l e (Nunc. S0ml, 2Scm2). A f t e r 2h i n c u b a t i o n at 37°C and 5% CO 2 n o n ~ a d h e r e n t cells were r e m o v e d by t w o r i g o r o u s r i n s e s . Second c o n t r o l g r o u p w a s injected 0.2ml o f 30% DMSO/70% p e a n u t o i l e m u l s i f i e d in 0.8ml PBS. E x p e r i m e n t a l g r o u p w a s injected w i t h t h e s a m e cocktail b u t c o n t a i n e d various c o n c e n t r a t i o n s o f TCDD, i n t r a p e r i t o n e a l l y . 14 days p o s t a d m i n i s t r a t i o n t h e m a c r o p h a g e s w e r e h a r v e s t e d by r e p e a t e d w a s h i n g o f t h e p e r i t o n e a l cavity a n d p l a t e d into c u l t u r e b o t t l e s at s a m e c o n c e n t r a t i o n as d e s c r i b e d f o r f i r s t c o n t r o l group. A f t e r t h e initial 2h a d h e r e n c e period, t h e m o n o l a y e r s w e r e ow.rlaid with 3.6ml s e r u m - f r e e H a n k ' s 199 m e d i u m c o n t a i n i n g 1% s t r e p t o m y c i n /penicillin
a n d 20ttg LPS f o r TNF-~ induction.
All m a c r o p h a g e p o p u l a t i o n s were >95% m o n o n u c l e a r p h a g o c y t e s as d e t e r m i n e d by s t a i n i n g , m o r p h o l o g i c and p h a g o c y t i c indices. C e l l - f r e e s u p e r n a t a n t s were c o l l e c t e d a f t e r 18h i n c u b a t i o n 0 . 4 m l o f 10-fold c o n c e n t r a t e d c o a t i n g p u f f e r (1.6g NaCO3+3,0g NaHCO 3 in 100ml aqua d e s t . . pH 9,6) f o r t h e e n z y m e - l i n k e d i m m u n o s o r b e n t a s s a y (ELISA) was added t o e a c h 3.6ml s u p e r n a ~ r a n t . 170,tl w a s p l a t e d i n t o each well o f f | a t
b o t t o m m i c r o w e l l m o d u l e s (Nunc. micro well
m o d u l e , no. 4-69949). Each p l a t e carried its own internal s t a n d a r d . The s t a n d a r d c o n s i s t s ¢~f a log 2 d i l u t i o n o f r e c o m b i n a n t routine t u m o r n e c r o s i s f a c t o r alfa ( E r n s t - B o e h r i n g e r - l n s t i t u t f u r Arzneimittelforschung, recombinant murine tumor necrosis facto alfa, E. cull-derived, no. 4296-17) in coating buffer. After incubation over night at 4 ° C and repeated washing with buffer
1858
(PBS + 0.0SZ T w e e n 20(Serva, T w e e n 20 p o l y o x y e t h y t e n e s o r b i t a n m o n o l a u r e a t e , no. 37470) + bovine s e r u m a l b u m i n e (BSA) I%) and b l o c k i n g lh w i t h PBS • IZ BSA rabbit polyctonal antibodies t o m u r i n e TNF-~ ( E r n s t - B o e h r i n g e r - l n s t i t u t f u r A r z n e i m i t t e l f o r s c h u n g , rabbit polyclonal anti bodies t o routine T N F - a , no. 70S9-65) were added in a c o n c e n t r a t i o n o f 1/1S00 and i n c u b a t e d 2h at 37°C. Biotinylated g o a t a n t i - r a b b i t a n t i b o d i e s (Medac, no. 6600) were added at a c o n c e n t r a t i o n o f 1/1000 f o r l h at 37°C. A f t e r w a s h i n g , it w a s i n c u b a t e d w i t h h o r s e r a d i s h p e r o x i d a s e (Vector, no. A2004) 0.Sh at
37°C (Sml
PBS + IZBSA + S~l h o r s e r a d i s h peroxidase) and incuba-
t e d 0.Sh w i t h o r t h o p h e n y l diamin h y d r o c h l o r i d e (OPD) (Stag OPD ÷ 0.Sml m e t h a n o l + 49.5ml a q u a d e s t . * Spl H202 ) at r o o m t e m p e r a t u r e . A b s o r p t i o n was d e t e r m i n e d u s i n g a micro ELISA autoreader.
RESULTS In t h e h o s t - m e d i a t e d in v i v o / i n vitro a s s a y w i t h peritoneal m a c r o p h a g e s . TCDD revealed a cellt r a n s f o r m i n g p o t e n t i a l t h a t s h o w e d a d o s e - d e p e n d e n t r e s p o n s e (table 1). T h e h i g h e s t c o n c e n t r a t i o n o f TCDD u s e d w a s 10~ of" LDs0 (LDs0 in mice: 125pg per kg) t o minimize t h e a c u t e t o x i c i t y as m u c h as p o s s i b l e . T h e c e l l - t r a n s f o r m i n g p o t e n t i a l o f TCDD at 0 . 3 9 ~ g / k g ( 7 . 9 n g / m o u s e ) was 1.3 w h i c h i n c r e a s e d t o 8.7S at 12.S~g/kg ( 2 5 0 n g / m o u s e ) . M a c r o p h a g e s t r e a t e d w i t h t h e cocktail c o n t a i n i n g p e a n u t oil, DMSO, PBS a n d TPA s h o w e d no t r a n s f o r m i n g p o t e n t i a l in t h i s s y s t e m . The c o - c a r c i n o g e n i c activity o f TCDD is i l l u s t r a t e d in t a b l e 2. In earlier work ( M a s s a e t al., 1990}, we have s h o w n , t h a t d i p h e n y l h y d a n t o i n at c o n c e n t r a t i o n s h i g h e r t h a n I S 0 ~ g / m o u s e h a s a c e l l - t r a n s f o r m i n g e f f e c t in o u r m a c r o p h a g e a s s a y s y s t e m . To s t u d y t h e c o - c a r c i n o g e n i c activity o f TCDD we have o m i t t e d TPA f r o m t h e cocktail. U n d e r t h e s e c o n d i t i o n s , TCDD at 0 . 3 9 ~ g / k g e x h i b i t s a very weak c e l l t r a n s f o r m i n g p o t e n t i a l o f 0.S. P h e n y t o i n at 1 0 0 p g / m o u s e s h o w e d no c e l l - t r a n s f o r m i n g p o t e n t i a l . However, t h e c o - a d m i n i s t r a t i o n o f TCDD leads t o t h e p o t e n t i a t i o n o f t h e o n c o g e n i c p o t e n t i a l o f p h e n y t o i n t o 2.9.
The co-carcinogenic activity of T C D D in table 3, Administration of T C D D
is dependent on the treatment schedule, as illustrated
t w o days prior to phenytoin, or two days after the pheny-
toin treatment has no effect on the cell-transforming potential of phenytoin. T C D D
admini-
stered at the same time as phenytoin leads to the highest potentiation of the carcinogenic potential of phenytoin.
1859
Table 1. Frequencies of the clone sizes in the host-mediated in vivo/in vitro assay with peritoneal routine macrophages after administration of various amounts of T C D D
together with TPA.
Control/' carcinogenic substance
No.of Frequency of clone sizea plate Unspecific Specific
Transforming potential of various concentrations
0.2mi +0.8ml +100ng i,p, per
DMSO/P.oil PBS TPA NMRI-mouse
1 2 3 4 5 6
10 4.0 3.35.0 --4.2--
1.3 1,3
0.0
0,2ml OMSO/P.oil +250ngTCDD +0.Sml PBS +100ng TPA i.p, per NMRI-mouse
1 2 3 4 5 6
5.0 22 76 8.8 47 25
0.5 6.0 19 3.0 15 9.0
0.5 3.0 3.3 0.3 6.3 5.0
8.75
0.2ml DMSO/P.oil +15.6ngTCDD +0.Sml PBS +100ng I-PA i.p. per NMRI-mouse
1 2 3 4 5 6
63 10 125 110 33
18 4,3 32 54 19
8.0 1.7 0.8 0.8 2.3 5.8 25 2.0 1.0 1.0 17 1.0
4.1
0.2ml +7.8ng +O.Sml +100ng i.p per
1 2 3 4 5 6
140 90 100 150 75 85
32 4.2 74 6.0 4.0 16
1.3 1.0
1.3
DMSO/P.oil TCDD PBS TPA NMRI-mouse
0.5 1.5 2.0 1.3 1.0 1.0 0 . 5 -
-
2.3 1.8 0.5 0.3 0 . 8 - -
--
2.7 1.2 1.6
aThe clone sizes according to cell n u m b e r (S-9, 10-14 ....)I00) and their frequencies are depicted for calculation of the transforming potential, as given under Material and Methods, or according to results published earlier (Massa et ai.,1990). S-9, all clones with S cells/clone to 9 cells/ clone were allocated to the class 5-9. S a m e procedure for classes C2 {10-14) to C9 (>I00l. bTransformlng potential of T C D D expressed as median of each experimental group.
Table 2. Co-carcinogenic effect of T C D D
with phenytoin.
Control/ carcinogenic substance
No.of Frequency of clone,size j plate Unspecific Specific
Transforming potential of TCDD and phenytoin b
0,2ml DMSO/P.oil +7.8ng TCDD +O.Sml PBS i.0. per NMRI-mouse
1 2 3 4 5
200 140 150 66 83
4.0 4,0 25 60 4.2
0.5
0.2ml +0.8ml +100pg i.p. per
DMSO/P.oil PBS phenytoin NMRI-mouse
1 2 3 4 5
166 133 160 150 30
3.3 2.3 33 40 --
2.0 0.7 0.7
0.2ml DMSO/P.oil +7,Sng TCDD +0.Sml PBS +100pg phenytoin i.p per NMRI-mouse
1 2 3 4 5
60 66 70 50 170
12 6.6 13 13 50
1.4 1.2 0.8 0.4 0.6 0.2 0.7 0.3 1.0 0.3 1.0 0.3 0.31.0 0.3 0 . 3 0.3 0 . 3 -
a.
0,5 1.0 0.3 2.7
0.0
bFo r description, see legend of table I.
2.9
1860
Table 3. EffeCt of application schedule on the co-carcinogenic potential of TCDD. Control/ carcinogenic substance
4. 1 ~ 0.2ml +7.Sng +0.8ml +lOOpg
No.of plate
Frequency o f clor~size a Unspecific
Specific
5-9
10-I#,
15-19 ~-L:u, 25-L~ 30-4~ 50-8g 70-;9 >100
50 2.0
35 5.0 3.5 2.5 0.8 -0.4
5.0 33
2.0 1.0 6.7 5.0 2.3 1.7 1.7 1.0 1.0
DMSO/P.oil TCDD PBS phenytoin
1 2 3 4 5
100 30 5.050 100
O.2ml D M S O / P . o i l +O.~ml PBS +lOOu.q phenytoin
1 2
60
A, Day
2. Day
Transforming potential o f TCDD/ phenytoin b 3.9
3.0 1.0
0.3
3
3.0 3 . 0 1.0 8 . 0 LO
0.3 . . . . . . . .
PBS
5
125 2 5
2.0
~ . 2~/#hl D M S O / P . o i l + , . e n a p-r~D +O .Sml 4: ~ +8,~ml ht ~4sSO/P'°"
1 2
2005.0
0.7
3 4
+lOOIA8 phenytoin
5
3002 5 1.7 2 2 1.5
0.8 0.5 . . . . . . . .
++ :
0.5
Table 4 . Co-carcinogenic effect of T O D D Control/ carcinogenic substance
No.of plate
compared with tumor promotor TPA.
Frequency o f cloresize a Unspecific
Specific
5-£
tS-~ 20-25 25-~ 30-$~ 50-6g 70-~ >~0
~-~
lml PBS 1 i.D. per N M R I - m o u s e 2 3 4 5
lml PBS 1 +100ng TPA 2 i.p. per N M R I - m o u s e 3
Transforming potential o f phenytoin,TPA ph./TPA, ph./TCDD b 0,0
5.0--
0.0 25
2.5
.
.
.
.
2.5 --
.
..............
4 5
4.0 . . . . . . . . . . . . tO --
0.2ml +0.8ml +100pg i.p. per
DMSO/P,oil PBS phenytoin NMRI-mouse
1 2 3 4 5
21 2 . 9 2.0 0.7 3.3 0.7 5.5-
0.2ml +0.8ml +100ng +100#g i.p per
DMSO/P.oil PBS TPA phenytoin NMRI-mouse
1 2 3 4 5
233 111 25 t50 55
0.2ml +7.8ng +0.Sml +100lzg i.p per
DMSO/P.oil TCDD PBS phenytoin NMRI-mouse
1 2 3 4 5
100 5 0 30 2.0
17 18 5.3 12 2.8
0.0
0.3 0.3 0,3 .
.
.
.
.
.
.
.
3.3 2.7 0.6 . . . . . . . . 1.0 --0.6
10
-
35 5.0 3.5 2.5 0.8 0.4
3.9
5.0 50 5.0 100 3 3
2.0 1.0 . . . . . . 6.7 5.0 2.3 1.7 1.7 1.0 1.0
a.b Experimental details are described in material and m e t h o d s , and under t a b l e 1.
1861
O u r n e x t a p p r o a c h w a s t o d i s t i n g u i s h b e t w e e n t h e c o - c a r c i n o g e n l c e f f e c t o f TCDD and t h e t u m o r - p r o m o t l n g activities o f TPA. As f o l l o w s f r o m t a b l e 4, n e i t h e r TFA ( 1 0 0 n g = 2 7 0 p m o l / m o u s e , MTPA = 364.44), n o r p h e n y t o i n (100pg/mouse) h a s any cell t r a n s f o r m i n g p o t e n t i a l a t d o s e s u s e d in t h e c o n t r o l e x p e r i m e n t s . The c o - a d m l n i s t r a t i o n o f TPA a n d p h e n y t o i n e x h i b i t a weak o n c o g e n l c p o t e n t i a l o f 1.01 however, t h e c o - a d m l n l s t r a t i o n o f TCDD(7.Sng=24pmol/mouse, MTCDD = 321.69) a n d p h e n y t o i n p o t e n t i a t e s t h e c e l l - t r a n s f o r m i n g p o t e n t i a l t o 3.9. The t e s t s y s t e m d e v e l o p e d in o u r l a b o r a t o r y h a s b e e n u s e d t o s t u d y t h e s t r u c t u r e a c t i v i t y r e l a t i o n s h i p in r e s p o n s e t o t h e i r cell t r a n s f o r m i n g potential. W e t h e r e f o r e have c o m p a r e d t h e cell t r a n s f o r m i n g p o t e n t i a l o f TCDD w i t h i t s b r o m o analog 2 , 3 , 7 , 8 - t e t r a b r o m o d i b e n z o - p - d i o x i n (TBrDD}. As f o l l o w s f r o m t a b l e S, t h e cell t r a n s f o r m i n g p o t e n t i a l o f TCDD is 7 t i m e s m o r e t h a n t h a t o f TBrDD a t t h e same m o l a r c o n c e n t r a t i o n and u n d e r identical e x p e r i m e n t a l c o n d i t i o n s . It is i n t e r e s t i n g t h a t we c o u l d o b t a i n a p e r m a n e n t cell line f r o m p e r i t o n e a l m a c r o p h a g e s o f mice t r e a t e d w i t h TBrDD. The biochemical a n d serological c h a r a c t e r i z a t i o n o f t h i s cell line will be i n t e r e s t i n g t o i n v e s t i g a t e t h e m e c h a n i s m s o f c a r c i n o g e n e s i s by TCDD, TBrDD and similar c o m p o u n d s . Table S. Cell-transforming potential of T C D D Control/ carcinogenic substance
No.of Frectuency of clonesize a plate Unspecific Specific s-~ 10-~
0.2ml +0.Smt +lOOng i.p. per
DMSO/P.oil PBS TPA NMRI-mouse
and TBrDD.
1 2 3 4 5
~-~ ~-~ 25-2g 30~ 5o.~g 7o..~ > ~
Transforming potential of phenytoin,TPA ph,/TPA, b ph./TCDD 0.0
14 5.0 8.0 3.0 7.0-7.2
0.2ml DMSO/P.oil +125ng TCDD = 0.39nMol +0.Sml PBS +lOOng TPA
1 2 3 4 5
280 90 190 80
3.0 6.0 3 . 0 -
90 15 210 68
4.0 2.0 11.0 6.0 7.0 1.0 1.0 --
0.2mi DMSO/P.oil +195ng TBrDD = 0.39nMot +O.8ml PBS +lO0ng TPA
1 2 3 4 5
25060
10.00.3 0.3 0.3
300 75 11 1 55 18
5.0 1.0 1.0
2.0 0 . 6 -
-1.0
a'bExperimental details are described in material and methods, and under table I.
O n e of the toxic effects of T C D D
reported in a number of investigations is weightloss (Holco-
m b et al., 19881 Pltlss et al., 1988; Hebert et al., 1990). Cachexia has also been noted in cancer patients. Oliff et al. (1987) have recently correlated cachexia in tumor bearing animals with the secretion of TNF-~. In these experiments tumors were induced in nude mice with C H O cells which had an integrated genetic construct of TNF-~. Cachexia was noted in mice injected with this particular construct C H O / T N F - 2 0 and a m u c h smaller effect was observed in animals injected with C H O
cells without the construct. This motivated us to look for the effect of T C D D
on T N F - ~ secretion in our cell system.
1862
A s f o l l o w s f r o m t a b l e 6 T C D D a t c o n c e n t r a t i o n s a b o v e l $ ~ g / k g is a b l e t o i n d u c e T N F - ~ s e c r e t i o n in a c o n c e n t r a t i o n d e p e n d e n t m a n n e r . C o n c e n t r a t i o n s
lower than this were unable to in-
d u c e T N F - ~ . I n t h e s e e x p e r i m e n t s T C D D w a s a p p l i e d in a s i n g l e d o s e ; h o w e v e r t h e s a m e a m o u n t o f T C D D ( $ 0 ~ g / k g ) e x t r a c t e d in 3 d o s e s g a v e t h e s a m e r e s u l t s ( l o w e r p a n e l , t a b l e 6).
Table 6. Induction of tumor necrosis factor • by T C D D .
Control/substance
day of i n t r m p e r i toneal injection b 1 S 9
TNP-~ • Units
ng
75pg/kg T C D D
X
141 + 6.4
II.7Sa + 0.S
50{~glkg T C D D
X
88
*-2.3
7.33 +0.2
2$tLg/kg T C D D
X
39
+-1.7
3.25
l$~g/kg T C D D
X
0
0
l,Stlg/kg T C D D
X
0
0
control (DMSOIPoil/PBS)
X
0
0
3-16.6ttg/kg= S 0 ~ g / k g T C D D
X
X
X
94
control (DMSO/Poil/PBS)
X
X
X
0
+-3.6
+-0.15
7.80
+0.3
control(DMSO/Poil/PBS)
CFA d
X
359 +-15.8 30.0
+-1.3
75~g/kg T C D D
CFA
X
396 +-12.2 33.0
+1.0
• T N F - ~ per 106 peritoneal macrophages and per ml medium. Serumfree m e d i u m samples were assayed for T N F - ~ activity by T N F - a ELISA at day 14 .as described under material and methods. I ng of TNF-~ is equivalent with 12 units. 1 unit of activity results in 50g lysis of fibroblasts using the standard L929 cytotoxity assay. bTCDD
was dissolved in 0.2ml emulsion of 30% D M S O
and 70% peanutoil emulsified in 0.8mi
PBS. This cocktail was then administered at day I, 5 or/and 9 intraperitoneally (marked with X). Control animals were given 0.2ml of 30% D M S O I T O Z
peanutoil in 0.8ml PBS.
°Each experiment contained 15 animals for each substance and concentration divided into three independent experimental groups. Each experimental group was analyzed 8-fold seperately. l~ach value depicts the m e a n of 24 experimental measurements with standard deviation. d 0.Sml complete Freunds adjuvant per m o u s e
DISCUSSION The environmental contaminant T C D D and related compounds are exogenous agents with carcinogenic potential in experimental animals. T C D D has been s h o w n to induce hepatic, pulmonary and skin tumors in long term feeding experiments (Van Miller et al., 1977:Kociba et al.. 1978a and 1978b; Toth et al., 1978; NTP, 1980). In particular, a number of recent studies have revealed that T C D D is a very potent promotor of hepatocarclnogenesis in female rats (Pitot et al., 1980; lqodstrSm and Ahlborg, 1989; FlodstrSm et al., 1990). Although T C D D is a potent carcinogen its m o d e of action is unknown. It has bvv~n shown that T C D D does not act as mltogen in bacterial systems (Gilbert et al. 1980: Geiger and Neal, 1981). nor does T C D D
s h o w co-
valent interaction with nucleic acids (Kondorosi et al., 1973; Poland and Glover, 1979): however, marginal effects on the incidence of the chromosome, aberration in vivo have been reported
1863
{Green a n d M o r e l a n d , 1975; Loprieno e t al., 1982). T h e s e s t u d i e s lead t o t h e c o n c l u s i o n t h a t TCDD h a s n o n e , o r very l i t t l e g e n o t o x i c activity w h i c h m a k e s it d i f f i c u l t t o explain its c a r c i n o genic e f f e c t s . The r e s u l t s p r e s e n t e d h e r e s h o w clearly t h a t TCDD h a s a h i g h c e l l - t r a n s f o r m i n g p o t e n t i a l i n o u r h o s t - m e d i a t e d in v i v o / i n v i t r o a s s a y w i t h p e r i t o n e a l routine m a c r o p h a g e s . So far, we k n o w o f t w o r e p o r t s w h i c h have b e e n able t o s h o w t h e m u t a g e n i c activity o f TCDD in animal s y s t e m s . Hay (1983) r e p o r t e d t h e m a t a g e n l c activity o f TCDD in a b a b y h a m s t e r kidney cell t r a n s f o r m a t i o n assay, a n d Rogers e t al. {1982) have r e p o r t e d t h a t TCDD is a direct a c t i n g m u t a g e n f o r L5178y cells. In t h e i r s t u d y , t r e a t m e n t w i t h a high {0.02 - 0 . 5 ~ g / m l ) c o n c e n t r a t i o n of TCDD p r o d u c e d dose d e p e n d e n t i n c r e a s e s in m e t h o t r e x a t e r e s i s t a n c e o f cells. A l t h o u g h t h e a u t h o r s i m p l i c a t e d t h i s finding as a direct e f f e c t o f TCDD a c t i n g as a f r a m e - s h i f t m u t a g e n ; however, it c a n n o t b e e x c l u d e d t h a t t h e m e t h o t r e x a t e r e s i s t a n c e c o u l d be a r e s u l t o f t h e a m p l i f i c a t i o n o f t h e germ f o r d t h y d r o f o l a t e r e d u c t a s e . In a n o t h e r study, A b e r n e t h y e t al. (1985) have s t u d i e d t h e e f f e c t o f TCDD in a cell t r a n s f o r m a t i o n a s s a y s y s t e m involving C 3 H / 1 0 T I / 2 m o u s e e m b r y o f i b r o b l a s t s . T h e s e a u t h o r s failed t o o b serve any t r a n s o r m i n g activity o f TCDD with, o r w i t h o u t TPA. However c o n t i n o u s t r e a t m e n t o f t h e s e c e l l s w i t h low c o n c e n t r a t i o n s o f TCDD ((= 4pM) e n h a n c e d f o c u s p r o d u c t i o n in c u l t u r e s pretreated with N-methyl-N'-nitroN-nitrosoguanidine. From this study the authors conclude that p r o m o t i o n ol~ t r a n s f o r m a t i o n is t h e p r e d o m i n a n t e f f e c t o f TCDD in t h e s e cells. F r o m o u r r e s u l t s it is evident t h a t t h e cell t r a n s f o r m i n g activity o f TCDD is c o n c e n t r a t i o n dep e n d e n t . W e c o u l d a l s o e s t a b l i s h t h e c o - c a r c i n o g e n l c activity o f TCDD in t h i s s y s t e m at c o n c e n t r a t i o n s a t w h i c h TCDD h a s n o cell t r a n s f o r m i n g activity. W e d e s i g n a t e t h i s e f f e c t as a c o carcinogenic activity b e c a u s e t h e p r o m o t o r activity is applicable f o r t h o s e s u b s t a n c e s w h i c h t h e m s e l v e s have n o carcinogenic activity at any c o n c e n t r a t i o n . The s e c o n d p a r a m e t e r w h i c h also d i s t i n g u i s h e s TCDD f r o m a c l a s s i c p r o m o t o r like TPA is t h e a p p l i c a t i o n schedule. The t u m o r p r o m o t i n g e f f e c t o f TPA is s e q u e n t i a l in t h e sense, t h a t t h e initiating c a r c i n o g e n h a s t o be applied f i r s t , f o l l o w e d by TPA. In o u r s t u d i e s w i t h TCDD, t h e c o - c a r c i n o g e n i c e f f e c t o f TCDD was o b s e r v e d only u n d e r c o n d i t i o n s w h e n TCDD a n d p h e n y t o i n were a d m i n s t e r e d s i m u l t a n e o u s l y . F u r t h e r m o r e , we o b s e r v e d a s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p in t h e carcinogenic p o t e n t i a l o f TCDD a n d its s t r u c t u r e a n a l o g TBrDD. The b r o m o derivative h a d a m u c h l o w e r cell t r a n s f o r m i n g p o t e n t i a l t h a n TCDD. I n t e r e s t i n g l y , we have n o w b e e n ale t o e s t a b l i s h a m a c r o p h a g e cell line f r o m mice t r e a t e d w i t h TBrDD. The b i o c h e m i c a l a n d serological c h a r a c t e r i z a t i o n o f t h i s cell line will b e i m p o r t a n t t o e l u c i d a t e t h e m e c h a n i s m o f cell t r a n s f o r m a t i o n by TCDD. Recent s t u d i e s by Durra e t al. {1988) indicate t h a t TCDD c a n m o d u l a t e t h e e x p r e s s i o n o f g e n e s o t h e r t h a n r e s p o n s i b l e f o r A h - r e c e p t o r in p r o d u c i n g oncogenic e f f e c t s . They have b e e n able t o c h a r a c t e r i z e a 2.1-kb long mRNA w h i c h e n c o d e s t h e e n z y m e aldehyde d e h y d r o g e n a s e {aldehyde: NADP + o x l d o r e d u c t a s e , EC 1.2.1.5.) in llver o f r a t s t r e a t e d w i t h TCDD. The i n t e r e s t i n g a s p e c t o f t h i s i n v e s t i g a t i o n is t h e o b s e r v a t i o n t h a t t h i s 2.1-kb mRNA was p r e s e n t in primary r a t liver h e p a t o m a s f r o m u n t r e a t e d rats. This o b s e r v a t i o n s u g g e s t s t h a t TCDD c a n elicit oncogenic e v e n t s by s o m e r e g u l a t o r y p r o c e s s e s decoupled from the Ah-receptor. The e f f e c t o f TCDD o n T N F - a s e c r e t i o n h a s b e e n r e c e n t l y r e p o r t e d by Clark e t al. (1990) u s i n g TCDD r e s p o n s i v e a n d n o n r e s p o n s i v e mice. T h e s e a u t h o r s have d o c u m e n t e d an increasing T N F - a p r o d u c t i o n by TCDD; whereby, t h e e f f e c t was m o r e p r o n o u n c e d in t h e TCDD r e s p o n s i v e mice a n d m o r e o v e r , it was c o n c e n t r a t i o n d e p e n d e n t . The m i n i m u m d o s e n e e d e d f o r a s i n g i f i c a n t rise f o r T N F - a p r o d u c t i o n w a s 10~g/kg or m o r e ; t h e m a x i m u m i n c r e a s e o f T N F - a was r e p o r t e d at 500~g TCDD/kR body weight. The f a c t t h a t t h e i n d u c t i o n o f T N F - a was dose d e p e n d e n t in TCDD r e s p o n s i v e mice h a s lead t h e s e a u t h o r s t o p o s t u l a t e t h a t T N F - a p r o d u c t i o n is m e d i a t e d via t h e A h - r e c e p t o r . In o u r e x p e r i m e n t s we c o u l d o b s e r v e t h e i n d u c t i o n o f T N F - a p r o d u c t i o n only at
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doses about I5~g/kg body weight. The induction of TNF-~ by T C D D is dose dependent. It is interesting to note, that the cell transforming potential of T C D D was m a x i m u m at 12.5tlg/kg body weight (2S0ng/mouse). Since at this concentration w e did not observe any T N F - a production, w e believe that the cell transforming activity of T C D D tion. W e have investigated the effect of T C D D
is not regulated by T N F - a produc-
on T N F - a production and its consequence on the
body weight {unpublished data}. W e could observe a loss of body weight which was related to T C D D concentration and the amount of T N F - a production. The highest cachexia effect was observed in mice treated with 75~g/kg T C D D
and this group exhibited the highest level of TNF-~.
$UMk/ARY W e have developed a host-medlated assay system for the detection of the transforming action of chemical carcinogens on peritoneal macrophages (Massa et al., 1990). Directly as well as indirectly acting carcinogenic substances administered intraperitoneally to NMRI mice could be examined in this way. Resident m a ~ o p h a g e s were recovered by peritoneal lavage from treated and untreated mice and were cultured in soft agar. After 5-6 days normal and transformed cells could he distinguished. Statistical analysis comparing cells from 2,3,7,8-tetrachlorodibenzo-pdioxin{TCDD)-treated animals with those from control mice proved that the test is positive at least on a significance level of S• using the t-test. T C D D
revealed a cell-transforming potential
that showed a dose-dependent response in this host-mediated assay. The co-carcinogenic activity of T C D D
was established ~a experiments with diphenylhydantoin. L o w doses ofdiphenylhy-
dantoin which did not exhibit any t x a r m f ~
pot~tial in our system gained a high oncogenic
potential by the simultaneous administration of low doses of T C D D which, as well had no transforming potentiaL W e
have compared the cell transforming potential of T C D D
analog TBrDD. The Cell transforming potential of T C D D
with its b r o m o
is 7 times more than that of TBrDD.
Using monospecific antibodies to tumor necrosis factor~ (TNF-~} wehave found that T C D D stimulates the secretion of TNF-~. The experimental data reported here lead to the conclusion that T C D D has a carcinogenic as well as a co-carcinogenic activity and has the property to induce TNF-a.
l~y words: 2,3,7,8-tetrachlordibenzo-p-dioxin
- 2,3,7,8-tetrabromodibenzo-p-dioxin - Dlphenyl-
hydantoin - T u m o r necrosis factor a - T N F - ~ ELISA - Host mediated in vivo/in vitro assay
lu-~--wl~llks~mts. This work was supported financially by Umweltbundesamt, Berlin (Grant, no. 106 03 098). W e are grateful to Dr. G.R Adolf (Ernst Boehringer-lnstitut fur Arzneimittelforschung, Vienna} for the gifts of recombinant routine TN]F-~ and rabbit polyclonal antibodies to murine TNF-a. It is a pleasure to acknowledge the skilled technical assistance of Mrs. Anja Lerch.
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Abernethy DJ, Greenlee F, Huband JC, Boreiko CJ (1985) 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) promotes the transformation of C3H/10T1/2 cells. Carcinogenesis 6(4):651-653 Berry DL, Di Giovanni J, Juchau M, Brachen WM, Gleason GL, Slaga TJ (1978) Lack of tumor-promoting ability of certain environmental chemicals in tow-stage mouse skin tumorigenesis assay. Res. Commun. Chem. Pathol. Pharmacol 20:101-107 Clark G, Tritscher A, Rucier G, Taylor M (1990) Dose-dependent increase in tumor necrosis factor-alpha production in TCDD exposed mice is Ahreceptor dependent. 10th International Meeting Dioxin 90, Short papers, Eco-lnforma Press, Bayreuth, Volume 1:77-80 Oenison MS, Harper PA, Okey AB (1986) Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Codistiribution of unoccupied receptor with cytosolic marker enzymes during fractionation of mouse liver, rat liver, and cultured Hepa lclc7 cells. Eur. J. Biochem. 155:223-29. DI Giovanni J., Viaje A., Berry DL, Slaga TJ, Juchau MR (1977) Tumorinitiating ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and Aroclor1254 in two-stage system of mouse skin carcinogenesis. Bull. Environ. Contain. Txicol. 18:552-557 Dunn TJ, Lindahl R, Pitot HC (1988) Differential gene expression in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD).Noncoordinate regulation of a TCDD-induced aldehyde dehydrogenase and cytochrome P-450c in the rat. The Journal of Biological Chemistry 263(22): 10878-10886 Flodstrbm S and Ahlborg UG (1989) Tumor promoting effects of 2,3,7,8-tetrachtorodibenzo-p-dioxin (TCDD) - effects of exposure duration administration schedule and type of dict. Chemosphere, 19:779-783 Flodstr~m S, Busk L, Ahlborg UG (1990) Enhancement of altered hepatic loci development by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) effects of vitamin A supplementation and the type of dict. 10th international Meeting Dioxin 90, Bayreuth, sept. 12-15 1990, volume 1, 173-176 Geiger LE, Neal RA {1981) Mutagenicity testing of 2,3,7,8-tetrachlorodibenzo-p-dioxin in histidine auxotrophs of Salmonella typhimurium. Toxicol. Appl. Pharmacol. 59(1):125-129 Gilbert P, Saint-Ruf G, Poncelet F, Mercier M (1980) Genetic effects of chlorinated anilines and azobenzenes on Salmonella typhimurium. Arch Environ. Contain. Toxicol. 9(5):533-541
1866
Gonzalez FJ, Tukey RH, Nebert DW (1984) Strucktural gene products of the Ah locus. Transcriptional regulation of cytochrome P~-450 and P3-450 mRNA levels by 3-methylcholanthrene. Mol. Pharmacol. 26:117-2t Green S, Moreland FS (1975) Cytogenetic evaluation of several dioxins in the rat. Toxicol. Appl. Pharmacol. 33:161 Hay A (1983) The mutagenic properties of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Abstracts of Americ&n Chemical Society National Meeting 23(2):14 Hebert CD, Harris MW, Elwell MR, Birnbaum LS (1990) Relative toxicity and tumor-promoting ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and i,2,3,4,7,8-hexachlorodibenzofuran (HCDF) in hairless mice. Toxicology and applied Pharmacology, 102:362-377 Holcomb M, Cheng Y, Safe S (t988) Biologic and toxic effects of polychlorinated dibenzo-p-dioxin and dibenzofuran congeners in the guinea pig. Quantitative structure-activity relationships. Ann chemical Pharmacology, 37(8):1535-1539 israel DI, Whitlock JP Jr. (1984) Regulation of cytochrome PC450 gene transcription by 2,3,7,8-tetrachlorodibenzo-p-dioxin in wildtype and variant mouse hepatoma cells. J. Biol. Chem. 259:5400-2 Kociba RJ, Keyes DG, Beyer JE, Carreon RM, Wade CE, Dittenber DA, Kalnins RP, Frauson LE, Park CN, Barnard SD, Hummel RA, Humiston CG (1978a) Results of a two-year chronic toxicity and oncogenicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats. Toxicol. Appl. Pharmacol. 46:279-303 Kociba RJ, Keyes DG, Beyer JE, Carreon RM (1978b) Toxicologic studies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCOD) in rats. Toxicol. Occup. Med. 4:281-287 Kociba RJ, Keyes DG, Beyer JE, Carreon RM, Gehring PJ (1979) Long term toxicologic Studies of 2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals. Ann. N.Y. Acad. Sci. 320:397-404 Kondorosi A, Fedoresak I, Solymosy F, Ehrenberg L, Osterman-Golkar S (1973) Inactivation of QBRNA by electrophiles. Murat. Res. 17:149-161 Kouri RE, Rude TH, Joglekar R, Dansette PM, Jerina DM, Atlas SA, Owens IS, Nebert DW (1978) 2,3,7,8-tetrachlorodibenzo-p-dioxin as cocarcinogen causing 3-methylcholanthrene-initiated subcutaneous tumors in mice genetically "nonresponsive" at Ah locus. Can. Res. 38:2777-2783
1867
Loprieno N, Skrana I, Rusciano D, Lascialfari D, Lari T (1982) In vivo cytogenetic studies on mice and rats exposed to tetrachlorodibenzo-p-dioxin (TCDD). In: Chlorinated dioxins and related compounds, impact on the environment. Proceedings of a workshop held at the Istitutio Superiore die Sanita, Rome, Italy, 22-24 Oct. 1980. Ed.: Hutzinger O. et al., Pergamon Press, New York, 419-428 Massa Th, Gerber T, Pfaffenholz V, Chandra A, Schlatterer B, Chandra P (1990) A host mediated in vivo/in vitro assay with peritoneal murine macrophages for the detection of carcinogenic chemicals. J. Cancer Res. Clin. Oncol. Volume 116, no 4:357-364 Nebert D, Thorgiersson S, Felton J (1976) Genetic differences in mutagenesis, carcinogenesis and drug toxicity. In: de Serros F, Fotets J, Bend J, Philpot R (eds) In vitro metabolic activation in mutagenesis testing. Elsevier/North Holland Biomedical Press, Amsterdam. pp 105-124. NTP (1980) National toxicology program. Bioassay of 2,3,7,8-tetrachlorodibenzo-p-dioxin for possible carcinogenicity (garage studie). Carcinogenesis testing prgram, National Cancer Institute (NIH), Bethesda, MD; NTP, Research Triangle Park, NC. U.S. Department of Health and Human Services Publication no. 82-1765 Oliff A, Defeo-Jones D, Boyer M, Martinez D, Kiefer D, Vuocolo G, Wolfe A, Socher SH (1987) Tumors secreting human TNF/Cachectin induce cachexia in mice. Cell, 50:555-563 Pitot HC, Goldsworthy T, Campbell HA, Poland A (1980) Quantitative evaluation of the promotion by 2,3,7,8-tetrachlorodibenzo-p-dioxin of hepatocarcinogenesis from diethylnitrosamine. Cancer Res. 40:3616-3620 PIUss N, Poiger H, Hohbach C, Suter M, Schlatter C (1988) Subchronic toxicity of 2,3,4,7,8-Pentachlorodibenzofuran (PeCDF) in rats. Chemosphere, 17(6): 1099-1110 Poland A, Glover E (1979) An estimate of the maximum in vivo covalent Binding of 2,3,7,8-tetrachlorodibeno-p-dioxin to rat liver protein, ribosomal RNA, and DNA. Cancer Research 39:3341-3344 Rogers AM, Andersen ME, Back KC (1982) Mutagenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin and perfluoro-n-decanoic acid in L5178Y mouse lymphoma cells. Mutat. Res. 105:445-449 Toth K, Sugar J, Somfai-Relle S, Bence J (1978) Carcinogenic bioassay of the herbic;ide 2,4,5-trichlorophenoxyethanol (TCPE) with different 2,3,7,8tetrachlorodibenzo-p-dioxin (Dioxin) content in Swiss mice. Prog. Biochem. Pharmacol., 14:82-93
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Van Miller JP, Lalich JJ, Allen JR (1977) Increased incidence of neoplasms in rats exposed to low levels of 2,3,7,8-tetrachlorodibenzo-pdioxin. Chemosphere 6:537-544 Whitlock JP Jr (1990) Genetic and molecular aspects of 2,3,7,8-tetrachlorodibenzo-p-dioxin action. Annu. Rev. Pharmacol. Toxicol. 30:251-77