in vitro assay

Chemosphere, Vol.25, Nos.7-10, pp 1085-1090, 1992 Printed in Great Britain

0045-6535/92 $5.00 + 0.00 Pergamon Press Ltd.

Carcinogenic and c0-carcin0genicpotentialof 2,3,7,8-tetrachl0r0dlbenz0di0xinin a

h0st-mediatedInvlv0/invitroassay "r.M--.~, A. ~-mMttd, H. rortmeyer 2 , e. ScSdatterer 8, H. Hagen~-~'¶ r. Chandm'Z iLaboratorlum fiirMolekularbiologie (ZBC}, KlinLkum der Johann-Wolf gang Goethe Hniverslt~it, D-6000 Frankfurt/Maln 71, ]:RG 2Tierversuchsanlage der Johann-Wolfgang Goethe Hniversitiit Frankfurt aHmweltbundesamt, D-1000 Berlin *Instltut flit Organlsche Chemic der Universitiit Ttibingen ~To w h o m correspondence should be sent

Abstract: In an in vivo/in vitro assay system (Massa et al., 1990) we have detected the carcinogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin {TCDD). The carcinogenic potential measured in this system is concentration-dependent. Experiments with other carcinogenic compounds have revealed that T C D D at low doses can act as co-carcinogen. Cells transformed by TBrDD, the bromo-derivatlve of dioxin, induced tumors in nude mouse.

Introduction: T C D D elicits a wide spectrum of biochemical and pathophysiological effects which axe mediated by its capacity to induce aryl hyrocarbon hydroxylase (AHH) (Gonzales et al., 1984; Israel and WhitIock,1984). T C D D has been s h o w n to be carcinogenic in mice and rats in long chronic feeding studies (Van Miller et al., I977: Kociba et al.,1978; Toth et al.,1978: Kociba et al., 1979} and to possess a tumor promoting effect in m o u s e skin (Di Giovanni et al., 1977: Berry et al.. 1978: Kouri et al., 1978; Kociba et al., 19791. However. unlike other carcinogenic compounds of the series of polychlorinated hydrocarbons, T C D D showed no covalent binding to rat liver protein, ribosomes, R N A or D N A under in vivo conditions (Poland and Glover, 19791. Moreover in vivo and in vitro studies have failed to produce any conclusive evidence about its mutagenicity (Nebert et al., 1976: Gilbert et al..1980; Geiger and Neal, 1981}. For this reason, it was interesting for us to evaluate the carcinogenic potential of T C D D in the host-mediated in vivo/in vitro assay (which has recently been established in our laboratory (Massa et al., 19901) with peritoneal murine macrophages for the detection of carcinogenic chemicals.

Material and methoda: W e have detected the carcinogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in a host mediated in vivo/in vitro assay system as previously described by Massa et al.,1990. Briefly, animals were 8 week-old male mice of the inbred N M R I strain weighing approximately 30g. At' day 0, 12S~g lipopolysaccharide (LPS} dissolved in Iml phosphate buffered saline (PBS),was asceptically administered to each m o u s e intraperitoneally. Substances to be examined were dissolved or emulsified in Iml PBS containing 100ng 12-O-tetradecanoylphorbol-13-acetate, (TPA} or were dissolved in 0.2ml emulsion of 30Z D M S O and 70Z peanutoil emulsified in 0.8ml PBS containing 100ng TPA. This cocktail was then administered at day 4 intraperitoneal]y. Macrophages were collected by repeated peritoneal lavage four days later and were resuspended in a special cell culture m e d i u m Cup-medium} containing cell-stimulating-factor (CSF}. One half of the resuspended cells (2,5ml/mouse} was given into a sterile culture bottle (one bottle/ mouse}. The second half of the suspension was transfered into soft agar and was incubated at 37°C. in a water saturated atmosphere containing 5Z C O 2. 5 to 6 days later the growth of cell colonies was evaluated. The transforming potential of substances was characterized as described elsewhere (Massa et al., 1990L briefly, microscopically distinguishable clone sizes were divided into I0 classes (C0-C9). The frequencies of clone sizes of a defined class were determined for each 24-well plate. They were related to the cell number and were represented as indicated in Table I-5. The microscopically determined frequency of the c l o n e size o f e a c h 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. T h e 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 w e r e s u m m e d u p f o r e a c h 24 w e l l p l a t e ( r e s u l t o f o n e animal} s e p a r a t e l y . T h e 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 a n d r e p r e s e n t i n g 5 t o 6 24 well plates designates the transfroming potency of the respective substance. 1085

1086

Results end discussion: In the host-mediated in vivo/in vitro assay with peritoneal macrophages, T C D D revealed a cell-transforming potential that showed a dose-dependent response {table I). The highest concentration of T C D D Table I. Erequencies of the clone sizes in the host-mediated in vivo/in vitro assay after administration of various amounts of T C D D to(~ether with TPA. FreQuency of clone size a

Control/ carcinogenic substance

No.of plate

0.2ml +0.8rot +lO0ng i.p. per

1 2 3 4 5 6 1 2 3 4 5 6

6.0 22 ,76 8.8 4,7 25

0.2ml DMSO/P.oil +15.6ngTCDD +0.8ml PBS +100ng TPA i.D. Ioer NMRI-mouse

l 2 3 4 5 6

0.2ml +7.8ng +0.Sml +lO0ng i o per

1 2 3 4 5 6

DMSQ/P.oil PBS TPA NMRI-mouse

0.2ml OMSO/P.oil +250ngTCDD +0.8ml PBS +100ng TPA i.p. per NMRI-mouse

OMSO/P.oil TCDD PBS TPA NMRI-mouse

Unspecific

Specific

~.-9 ~

1~-~9 20-2¢25-~ 30-¢9 50-~ 70-9g >100

Transformino potential of " various concentrations of TCDD b

10 4.0 3.36.0 --

1.3 1.3

O.O

0.5 6.0 79 3.0 75 9.0

0.5 3.0 3.3 0.3 6.3 5.0

8.7b

63 10 125 ~70 33

78 4.3 32 5419

8.0 1.7 0.8 0.8 2.3 5.8 25 2.0 1.0 1.0 17 1.0

4.1

I40 90 100 750 75 85

32 4.2 74 6.0 4.0 t6

1._t 1.0

1.3

4.2--

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 t o cell n u m b e r (5-% 10-14 .... >100) and t h e i r frequencies are p r e s e n t e d for calculation o f the t r a n s f o r m i n g potential according t o results published earlier (Massa et a1.,1990). 5-9, all clones with S c e l l s / c l o n e t o 9 c e l l s / c l o n e were allocated t o the class 5-9. Same procedure for c l a s s e s C2 (I0-14) t o C9 (>100). b T r a n s f o r m i n g potential o f TCDD e x p r e s s e d as median o f each experimental group.

Table 2. Co-carcinogenic e f f e c t o f TCDD with phenytoin. Controi/ carcinogenic substance

No.of plate

Frequency of clone sizea Unspecific

Specific

5-9

10-!~.

15-1g 20-25 25-29 30-4g 50-6g 70-99 >100

4.0 4.0 25 60 4.2

0.5

0.2ml DMSO/P.oil +7.Sng TCDD +O.8ml PBS i.p. per NMRI-mouse

1 2 3 4 5

200 740 750 66 83

0.2mi +0.8mi +100Bg i.p. oer

DrvlSO/R.oii PBS phenytoin NMRI-mouse

1 2 3 4 5

f66 3.3 ;33 2.3 T60 33 150 40 30 --

0.2ml +7.8rig +0.Sml +lOOBg i.o per

OMSO/P.oil TCDD PB5 phenytoin NMRI-mouse

] 2 3 4 5

60 66 .70 50 170

i2 6.6 ~3 13 50

Transforming •otential of ~-CDD and, phenytoin D 0.5

i.0 0.3 2.7

0.0 2.0 0.7 0.7 1.4 ] . 2 0 i m 0.4 0.6 0.2 0/7 0,3 1.00.3 1.0 0.3 0.31.0 0.3 0.3 0.3 0.3 -

a,b For description, see legend o f table 1

2.9

1087

used was 10Z of LDs0 (LDs0 in mice: 12S#g per kg) to minimize the acute toxicity as m u c h as possible. The cell-transform~.g potential of T C D D at 0.39vg/kg (7.9ng/mouse) was 1.3 (for calculation see material and methods} which increased to 8.7S at 12.S~glkg (2S0nglmouse). Macrophages treated with the cocktail containing peanut oil,D M S O , PBS and T P A showed no transforming potential in this system. The co-carcinogenic activity of T C D D is illustrated in table 2. In earlier work (Massa et al.,1990). we have shown, that diphenylhydantoin at concentrations higher than IS0~g/mouse has a ceil-transforming effect in our macrophage assay system. Phenytoin gives reasons for being carcinogenic to humans or to animals is according to the classificationof the IARC (1987 B). To study the co-carcinogenic activity of T C D D w e Treble 3. Effect of application schedule on the co-carcinogenic potential of T C D D . Controt/ No.of Frequency of cione size Transforming carcinogenic substance

plate

4.Tag 0.2ml +7.8ng +0.8ml +lO0l~g

i 2 3 4 5

100 50 30 2.0 5.050 5.0 100 33

1 2 3 4 5

60 3,0 3.0 !.0

3.0 1.0

8.0

LO

0.3

125 25

2.0

200 6,0

0.7

DMS©/P.oil TCDD PSS phenytoin

2 .Tag O.2ml DMSO/P.oil tO 8ml PBS ~1~O#9 phemytoir~ d'(~TL~ I DMSO/P.oil +7.Snq TCDD ~O.8m'q PBS

2xTag .2rim DMSO,'P.oil 1 4.Ta~l O.2mi OMSO/P.oii +{(~6#g phenytoin

3

4 5

Unspecific

Specific

5-9

15-19 20-2#, 25-29 30-¢9 50-5g 70-99 >100

10-1~.

35 5 0 3.5 2.5 0.8 0.4

s,~bsta~,ce

3.9

2.0 1.0 6.7 5.0 2,3 1.7 1.7 1.0 1.0 0.3

0.5

3 0 0 --

25 22

1.7 1.5

0.8 0.5

Table 4. Co-carcinogenic effect of T C D D Controi/ carcinogenic

compared with tumor promotor TPA.

No.of Frequency of cione size plate

Transforming potential of

Unspecific Specific

5-0

lml PBS 1 i.D. 0er NMRI-mouse 2 3 4 5 lml PBS 1 +100ng TPA 2 i.D. per NMRI-mouse 3 4 5

~otential of TCDD/ phenytoin

~-~

15-~g ~

~h~n,t~

~-2g 30-~9 50-6g 70-~ >100

bh./TCDD

0.0 5.0--

0.0 25

2.5

2.5 -

4.0-10 --

0.2ml +0.8ml +100#g i.p. 0er

OMSO/P.oil PBS Dhenytoin NMRI-mouse

l 2 3 4 5

21 2.9 2.0 0.7 3.3 0.7 5.5-

0.3 0.3 0.3

0.0

0.2rap +0.8ml +100ng +100#g i.o per

DMSO/P.oit PBS TPA phenytoin NMRI-mouse

1 2 3 4 5

233 111 25 150 56

3.3 2.7 0.6

1.0

0.2ml +7.8rig +0.8ml +100#g i.p per

OMSO/P.oil TCDD PBS phenvtoin NMRI-mouse

1 2 3 4 5

1OO -gO 30 2.0 5.0 _50 5.0 100 33

17 78 6.3 12 2.8

1.0 -0.6 35 08 . . 2.0 6.7

5.0 . . 10 5.0

3.b 2.b 0.4 . 2.3 1.7 1.7 10

39

1.0

1088

have o m i t t e d TPA f r o m t h e cocktail. H n d e r t h e s e c o n d i t i o n s , T C D D at 0 . 3 % g / k g e x h i b i t s a very weak cell t r a n s f o r m i n g p o t e n t i a l o f 0.5. P h e n y t o i n at 1 0 0 u 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 . H o w e v e r , t h e c o - a d m i n i s t r a t i o n o f TCDD leads to 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 c o - c a r c i n o g e n i c activity o f TCDD is d e p e n d e n t on t h e t r e a t m e n t s c h e d u l e , as i l l u s t r a t e d in t a b l e 3. A d m i n i s t r a t i o n o f TCDD t w o days prior t o p h e n y t o i n , or t w o days a f t e r t h e p h e n y t o i n t r e a t m e n t h a s no e f f e c t on 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 p h e n y t o i n . T C D D a d m i n i s t e r e d at t h e s a m e t i m e as p h e n y t o i n leads t o t h e h i g h e s t p o t e n t i a t i o n o f t h e carcinogenic p o t e n t i a l o f p h e n y t o i n . O u r n e x t a p p r o a c h was 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 i c e f f e c t of TCDD and t h e t u m o r - p r o m o t i 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 TPA (100ng=270 p m o l / m o u s e . MTPA= 354.44). n o r p h e n y t o i n ( 1 0 0 g g / m o u s e ) 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 at d o s e s used in t h e c o n t r o l experi m e n t s . The c o - a d m i n i s t r a t i o n of TPA and p h e n y t o i n e x h i b i t s a weak o n c o g e n i c p o t e n t i a l o f 1.0; however, t h e c o - a d m i n i s t r a t i o n o f TCDD(7.Snf~=24pmol/mouse, MTCDD = 321.69) and p h e n y t o i n !aotentiates 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 to 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 been u s e d to s t u d y t h e s t r u c t u r e activity 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 !aotential. We 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 tial o f TCDD w i t h its b r o m o a n a l o g 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 5, t h e ceil t r a n s f o r m i n g p o t e n t i a l of TCDD is 7 t i m e s m o r e t h a n t h a t o f TBrDD at t h e s a m e 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 . F r o m l o n g - t e r m c u l t u r e d c e l l s g r o w n as m o n o l a y e r s , we have s u c c e e d e d in e s t a b l i s h i n g a p e r m a n e n t cell link f r o m peritoneal m a c r o p h a g e s o f mice t r e a t e d with TBrDD. T h e s e c e l l s were t e s t e d for t h e i r t u m o r i Figure 1

T u m o r i g e n i c p o t e n t i a l o f cells t r a n s f o r m e d by TBrDD in a t h y m i c n u / n u m o u s e .

1 x 106 c u l t u r e d c e l l s were injected s u b c u t a n e o u s l y on t w o sites. The figure s h o w s t h e g r o w t h of t u m o r s a f t e r 3 weeks o f t r e a t m e n t

1089

S. Cell-transforming potential of T C D D and TBrDD. Control/ No.of Frequency of clone size carcinogenic plate substance Unspecific Specific

Table

5-g

0.2ml +0.Smt +100ng i.p. per

DMSO/P.oil PBS TPA NMRI-mouse

1 2 3 4 5

~-~

~-~ ~0-u 2~-2n t0-40 s0-sn 70-~ >100

Transforming potential of phenytoin TPA ph./TPA.' ph./TCDD 0.0

14 5.0 8.0 3.0 7.0-

0.2ml DMSQ/P.oil +125ng TCDD = 0.39nMol +0.Sml PBS +100n,q TPA

1 2 3 4 5

280 90 190 80 90 15 210 68

410

0.2ml DMSO/P.oil +195ng TBrDD = 0.39nMol +0.8mt PBS +100ng TPA

1 2 3 4 5

250 60

10.00.3 0.3 0.3

300 75 11 1 55 18

1.0 1.0

3.0

72

6.0 3.0

2.0 0.6

--

-

2 i0

11,0 6.0 7.0 1.0 1.0 1.0

5.0

genic p o t e n t i a l in athymic n u / n u mice. Figure 1 s h o w s t h e d e v e l o p m e n t o f t u m o r s in a n u / n u m o u s e inj e c t e d w i t h t h e s e cells. The t u m o r s appeared on b o t h t w o iniected sites, 3 weeks a f t e r the s u b c u t a n e o u s injection o f t h e s e cells (1 x 106cells per site). One o f t h e t o x i c e f f e c t s o f TCDD r e p o r t e d in a n u m b e r o f investigations is w e i g h t l o s s (Holcomb e t al., 1988; H e b e r t e t al., 1990). Cachexia has also been n o t e d in cancer patients. Oliff e t al. (1987) have r e c e n t l y c o r r e l a t e d cachexia in t u m o r bearing animals with t h e s e c r e t i o n o f TNF-~. This motivated us t o look for t h e e f f e c t o f TCDD on TNF-u s e c r e t i o n in our cell s y s t e m (Table 6). Even a induction o f TNF-~ by TCDD whithin a period o f l 4 days d o e s n ' t prevents the growth o f large cell colonies as we have s h o w n in t a b l e s l-S. Table 6. Induction of tumor necrosis factor ~ by TCDD. day o f intraperiTNF-~ • toneal injection b Control/substance Hnits 1

5

9

ng c

7Sl~g/kg TCDD S0~g/kg TCDD 2SLzg/kg TCDD IS~zg/kg TCDD l,S~g/kg TCDD control (DMSO/Poil/PBS)

X X X X X

141 +-6.4 88 +-2.3 39 ±1.7 0 0

II.7S +-0.S 7.33 +-0.2 3.25 + 0.1S 0 0

X

0

0

3*16.6~g/kg= S0~g/kgTCDD c o n t r o l {DMSO/Poil/PBS)

X X

X X

X X

94 0

+-3.6

7,80

+-0.3

aTNF-~ per 106peritoneal macrophages and per ml medium. Serumfree medium samples were assayed for TNF-~ activity by TNF-~ ELISA at day 14. 1 ng of TNF-~ is equivalent with 12 units. I unit of activity results in S0g lysis of fibroblasts using the standard L929 cytotoxity assay. b T C D D was dissolved in 0.2ml emulsion of 30Z D M S O and 70% peanutoil emulsified in 0.8ml PBS. This cocktail was then administered at day I, S or/and 9 intraperitoneaIIy (marked with X). Control animals were given 0.2ml of 30% D M S O / 7 0 % peanutoil in 0,8ml PBS, aEach experiment contained 15 animals for each substance and concentration divided into three independent experimental groups. Each experimental group was analyzed 8-fold seperately. Each value depicts the mean of 24 experimental measurements with standard deviation.

1090

][~tflll'l~ll~lll: 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. Pathcl. Pharmacol 20:101-107 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 fiver, 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-tetrachlorodibenzop-dioxin (TCDD) and Aroclor1254 in two-stage system of mouse skin carcinogenesis. Bull. Environ. Contain. Txicot. 18:552-557 Geiger LE, Neal RA (1981) Mutagenicity testing of 2,3,7,8-tetrachlorodibenzo-p-dioxin in histidine auxotrophs of Salmonella typhimur]um. 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 Gonzatez FJ, Tukey RH, Nebert DW (1984) Strucktural gene products of the Ah locus. Transcriptional regulation of cytochrome P1-450 and P2-450 mRNA levels by 3-methylcholanthrene. Mol. Pharmacol. 26:117-21 Hebert CD, Harris IvlW, Elwetl MR, Birnbaum LS (1990) Relative tuxicity arid tumur-promot;ng ability of 2,3,7,8-tetracHorodibenzo-p-dioxin (TCDD), and 1,2,3,4,7,8-hexachlorodibenzofuran (HCDF) in hairless mice. Toxicology and app!ied Pharmacology, 102:362-377 Holcomb M, Cheng Y, Safe S (1988) Biologic and toxic effects of polychiorinated dibenzo-D-dioxin and dibenzofuran congeners in the guinea pig. Quantitative structure-activity relationships. Ann. chemical Pharmaco!ogy, 37(8):1535-1539 IARC 1987 B IARC monographs on the evaluation of carconogenic risks to humans overall evaluations of carcinogenictiy: an updating of IARC monographs from Volumes 1 to 42,Lyon, supol7 Israel DI, Whitlock JP Jr. (1984) Regulation of cytochrome P1-450 gene transcription by 2,3,7,8-tetrachlorodibenzo-p-dioxin in wild-type 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 (TCDD) in rats. Toxicol. Occup. Med. 4:28!-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 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 causiqg 3-methylcholanthrene-init]ated subcutaneous tumors in mice genetically "nonresponsive" at Ah locus. Can. Res. 38:2777-2783 Massa Th, Gerber T, Pfaffenholz V, Chandra A, Schlatterer B, Chandra P (1990) A host mediated in vivo/in vitro assay with peritoneal routine macrophages for the detection of carcinogenic chemicals. J. Cancer Res. Clin. Oneol. 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, Folets J, Bend J, Philpot R (eds) In vitro metabolic activat!on in mutagenesis testing. E!sevier/ North Holland Biomedical Press, Amsterdam. pp 105-124. 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, 5 0 : 5 5 5 - 5 6 3 Poland A, Glover E (1979) An estimate of the maximum in vivo covalent Binding of 2,3,7,8-tetrachlorodibeno-pdioxin to rat liver protein, ribosomal RNA, and DNA. Cancer Research 3 9 : 3 3 4 ! - 3 3 4 4 Toth K, Sugar J, Somfai-Relle S, Bence J (1978) Carcinogenic bioassav of the herbicide 2,4,5-trichiorophenoxyethanol {TCPE) with different 2,3,7,8-tetrachlorodibenzo-p-dioxin (Dioxin) content in Swiss mice. Prcg. Biochem. Pharmacol., 1 4 : 8 2 - 9 3 Van Miller JP, Lalich JJ, Allen JR (1977) Increased incidence of neoplasms in rats exposed to low levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Chemosphere 6:537-544 Whitlock JP Jr (1990) Genetic and molecular aspects of 2,3,7,8-tetrachlorodibenzo-p-dioxin action. Annu Rev Pharrnacol. Toxicol. 30:251-77