Document gbOmMjM6BMQpD8vgazxoameKG

Toxicology Letters, 38 (1987) 299-306 Elsevier '. 299 T X L 01844 AROCLOR 1254 AS AN ANTAGONIST OF THE TERATOGENICITY OF 2,3,7,8-TETRACHLORODIBENZO-p-DlOXIN (Aroclor 1254; 2,3,7,8-TCDD; antagonism) J.M. HAAKE*. S. SAFE*, K. MAYURA6 and T.D. PHILLIPS6 'Department o f Physiology and Pharmacology, 6Department o f Public Health, College o f Veterinary Medicine, Texas A&.M University, College Station, TX 77843 (U S.A.) (Received 17 April 1987) (Revision received 30 May 1987) (Accepted 6 June 1987) SUMMARY Administration of 2,3.7,8-teirachlorodibenzo-p-dioxin (TCDD. 20 *tg/kg) to pregnant C57BL/6J mice (on day 10) resulted in 62ff7o fetuses with cleft palate per litter without any observable maternal toxicity. In contrast, Aroclor 1254 administered at a dose of 750 unol/kg was not teratogenic. Cotreatment of the pregnant mice with both Aroclor 1254 (244 mg/kg) and 2.3.7,8-TCDD (20 jig/kg) resulted in an S ^ o incidence of cleft palate per litter. In contrast. Aroclor 1254 did not afford any protection from the teratogenicity of dexamethasone in C57BL/6J mice. Previous studies have shown that Aroclor 1254 can act as a partial antagonist of the microsomal enzyme induction and immunotoxic effects of 2,3,7,8-TCDD in C57BL/6J mice and this paper demonstrates that the commercial polychlorinated biphenyl mixture also antagonizes 2,3.7,8-TCDD-mediated teratogenicity in this strain of mice. INTRODUCTION Polychlorinated biphenyls (PCBs) are widely used industrial compounds which have been identified in almost every component of the global ecosystem including fish, wildlife and human adipose tissue, blood and milk [1-4], During the past few years, the more toxic halogenated aryl hydrocarbons, namely the polychlorinated Address for correspondence: S. Safe, Department of Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843, U.S.A. Abbreviations: Ah, aryl hydrocarbon; AHH, aryl hydrocarbon hydroxylase: PCBs. polychlorinated biphenyls; PCDDs. polychlorinated dibenzo-/?-dioxins; PCDFs, polychlorinated dibenzofurans; TCDD. tetrachlorodibenzo-p-dioxin. 0378-4274/87/S 03.50 1987 Elsevier Science Publishers B.V. (Biomedical Division) GENP 010948 783759 inn diben/olurans ( I'C DI s) and dibenzo-p-dioxins (I'C D D s) liavc also been identified in I he cnviroiinicnl |5-8| and in human issue including m ilk samples |9 I 6|. Since I he PCDFs and I'CDDs arc industrial or combuslion bypioducls ||7 - I 9 |, lie levels o f llicsc compounds are usually > IO '-fo ld lower in most environmental samples. Moreover, high resolution analyses o f the human m ilk and adipose tissue have con firmed the presence o f 2,3,7,8-tclraclilorodibenzo-/>-dioxin ( I COD), the most toxic halogenated aryl hydrocarbon and several other toxic 2,3,7,8-subsliiulcd I' CDDs and I' CDFs |9 -I6 |. The toxicity and mechanism o f action o f halogenated aryl hydrocarbons including I' CBs, PCDDs and PCDFs have been extensively investigated |20-26|. I aboralory animal studies show that these compounds elicit a number o f common toxic responses including: body weight loss, thym ic atrophy and im m uuotoxicity, teratogenicity and reproductive problems, hepatoloxicity and porphyria, car cinogenicity, effects on the endocrine system and dermal toxicity. A ll o f these responses are not evoked in any single animal species; however, body weight loss and thym ic atrophy are the signs o f toxicity most frequently observed in laboratory animals. Most incidents involving environmental or accidental human exposure to the toxic I'CDDs and I' CDFs arc associated w ith concomitant exposme to higher levels o f I'CBs and these arc generally reflected in human tissue levels in which the I' C B s /l' CDDs, PCDFs ratios arc >1000:1. For example in the ^ usho and Yu Cheng poisoning in Japan and Taiwan respectively, several thousand individuals were poisoned w ith rice oil contaminated with PCBs and PCDFs |27.28). The ratios o i PCBs/PCDFs in the contaminated oil and the blood ol exposed individuals were generally in the order o f 184-667:1 and 15(H)-1890:1 respectively |27|. Since the in teractive effects o f concurrent exposure to halogenated aryl hydiocarbons is not well understood, this study reports teratogenicity o f combined exposure to 2.3.7.8-1C D D and Aroclor 1254, a commercial PC B form ulation. MAI LRIAI.S AND METHODS Chemicals 2 ,3 ,7 ,8 -lC D D was prepared in our laboratory and the purity was > 99tt/o as determined by gas-liquid and high-pressure liquid chrom atographic analysis. Dexamclhasonc was purchased from Sigma Chemical C o., St. I.ouis, MO. A roclor 1254 was a generous gift o f M r. B. C h ittim , W ellington Environmental Consultants, Ouelph, O n t., Canada. A nim al treatment M ature male C57BL./6J mice and virgin female C 57B I./6N mice (20 25 g) were purchased Irom Harlan Sprague-Dawley, Indianapolis, IN and maintained on food and water ad libitum at the Texas A A M University Laboratory A nim al Resources 783760 .101 and Research facility. At the end o f a 7-day acclimation period, females were mated with the males overnight. The follow ing morning, females were checked for the presence ol vaginal plugs. I he presence o f a vaginal plug was denoted as day 0 o f pregnancy, females with vaginal plugs were weighed and marked. Pregnancies were confirmed by measuring weight gain. I lie dams were treated by oral gavage with either coin oil (10 m l/kg ), A roclor 1254 (244 m g/kg), 2,3,7,8-TC DD (20 fig/kg), A ro d o r I 2M (244 m g/kg) followed by 2,3,7,8-TC DD (20 tg/kg), dexamclhasone (90 m g/kg). or A roclor 1254 (244 m g/kg) followed by dexamthasone (90 m g/kg). A ll these compounds or mixtures were administered in corn oil (10 m l/k g ) by oral gavage. A roclor 1254 alone was administered on day 9 o f pregnancy; corn oil and 2.3.7.R I t 'D I ) were given on day 10 and dexamethasone on day 13. I he animals receiving 2 chemicals were given A roclor 1254 on the day prior to either 2,3,7,8I t 1)1) or dexamethasone, i.e. A roclor 1254 on day 9 and 2,3.7,8-TC D I) on day 10 or Aroclor 1254 on day 12 and dexamethasone on day 13 o f pregnancy. C ontrols were either untreated or treated with corn oil (10 m l/k g ). On day 17, the females were killed with C O j or by cervical dislocation. The weights o f the dams and their respective liver weights were recorded. The uterine horns were exposed and the number o f resorptions, dead and live fetuses were counted. Live fetuses were rem ov ed from die uterus, weighed and examined for gross abnorm alities. The fetuses were fixed in B onin's solution for at least I week and examined for internal soft tissue anomalies as previously described (29,30|. Statistical differences were determined using an analysis o f variance procedure (A N O V A ). RF.SIJI IS AND DISCUSSION Table I summarizes the effects o f 2,3,7,8-TCDD, A ro clo r 1254, 2,3,7,8-TC DD plus A roclor 1254, dexamethasone and dexamethasone plus A roclor 1254 on C 57BI./6J females and their fetuses. No apparent signs o f maternal toxicity were observed in any o f the treatment groups. The percentages o f fetuses with hydrone phrosis per litter for the treatment groups were: untreated (1.3 4.3); corn o il (5.7 9.9), A roclor 1254 (33.4 18.0); 2,3,7,8-TC DD (87.8 20.4); A roclor 1254 + 2.3.7.8- TC D D (96.0 11.0); dexamethasone (3.2 7.3); and A roclor 1254 + dexamethasone (3.7 5.5). The dose o f A ro clo r 1254 (224 m g/kg) caused con siderable hydronephrosis and. not surprisingly, did not antagonize the effects o f 2.3.7.8- T C D D . Hydronephrosis is a more sensitive indicator o f exposure to toxic halogcnalcd aryl hydrocarbons |3I,38,39) and at a dose level o f 244 m g/kg, A ro clo r 1254 acts as an agonist for this response. At a dose o f 20 jig /k g , 2,3,7,8-TC D D caus ed 61.8% fetal cleft palate per litter whereas A roclor 1254 (244 m g/kg) did not cause any cleft palate in the litters. In contrast, hydronephrosis was observed in animals exposed to 2,3,7,8-TCDD or A roclor 1254 (data not shown). Cotreatmcnl o f the mice with a teratogenic dose o f 2,3,7,8-TC DD (20 n g /kg ) plus A ro clo r 1254 (244 m g/kg) resulted in only 8.2% fetal cleft palate per litter which was significantly GENP 010949 85.8 102 n CT w. o rr-'li s3 o -C 1/ cS ~ H n H O fl oo Uu. S & O VO V fc *1 oo fl fl oo II O NO ZS * co V O o oo oo H >o o o ^ O h* fl fl fl O- oo -t r- o */ 9 11.2 z 9.3 88.8 * 9.3 'Significantly different from corn oil-treated group at P<0.05. " 'Values with such signs are significantly different from each other at /><0.05. (90 mg/kg)___________ 9 0.64* : 0.21 12.50 * 2.23' J 7.78 z 9.3 plus dxamthasone cw 5 I? 3? -- d *> ff fl fl oo s o r r o V V \ ?! w --^ Z= oM on fN ff fl fl O' r- g -- w- Q $ rn r i -- fl fl fl w- p 1 1 .5 9 1 4 .4 7 oo or i r fl fl fl o NO N^l 1-- o r- O ^ T*f1 ff<-*> ^^ M- S _** <^r U ^ Ci <> rX 7 ^ E oo^O ^^ h. rO< X <M 3-- ~ Ci 0 wE O^ Ui l XV <-*t t" E 0C0 r"wi _o N, 4 .% 783761 m decreased from (lie teratogenic effects observed with 2,3,7,8-TC DD alone. These results contrast with previous studies on the interactive effects o f individual PCBs and 2,3,7,8-TC DD as teratogens (311. For example, the teratogenicity o f 2.3.7.8- T C D D was unaffected by cotreatment with 2 ,2 ',4 ,4 ',5 ,5 '-h e x a c h lo ro b iphenyl whereas 2,3,3' ,4,4' ,5-hexachlorobiphenyl potentiated the teratogenicity o f 2.3.7.8- T C D D . Previous reports have shown (hat 2,3,3' ,4,4' ,5-hexachlorobiphenyl evokes several typical 2,3,7,R-TCDD-like responses such as the induction o f cytochrome P-450t and related ntonooxygenascs, body weight loss, thym ic atrophy and im m unotoxicity (32-36|. Moreover, this PCB congener binds with moderate a f fin ity to the Ah receptor protein (37|. In contrast, 2 ,2 ',4 ,4 ',5,5'-hexachlorobiphcnyl docs not elicit Ah receptor-mediated effects in laboratory animals 120,33). Subsequent studies on the interactive effects o f 2,3,7,8-TC DD and 2 ,3 ,3 ',4 ,4 ',5hexachlorobiphenyl as teratogens suggest that the interactive effects are additive 138) and recent work shows that the interactive effects o f 2,3,7,8-TC DD and 2,3,7,8 tetrachlorodibenzofuran (TC D F) as teratogens are also additive (39). A lthough A roclor 1254 is a complex m ixture o f PCB isomers and congeners, this mixture, like 2,3,3' ,4,4' ,5-hexachlorobiphenyl, also elicits a broad spectrum o f Ah receptor-mediated responses including im m unotoxicity, thym ic atrophy, body weight loss, porphyria and the induction o f cytochrome P-450c and related monooxygenases and exhibits moderate a ffin ity fo r the Ah receptor protein (20,32-37). In fact, both A roclor 1254 and 2,3,3',4,4',5-hexachlorobiphenyl are weak Ah receptor agonists compared to 2,3,7,8-TC DD and their competitive binding a f finities for the hepatic Ah receptor are comparable (37|. One m ajor difference be tween 2 ,3 ,3 ',4 ,4 ',5-hexachlorobiphenyl and A ro clo r 1254 is the relative potency o f the former compound. For example, the in vivo E D 50 values for body weight loss, thymic atrophy and hepatic microsomal aryl hydrocarbon hydroxylase in the rat for 2 ,3 ,3 ',4 ,4 ' ,5 ' hexachlorobiphenyl are 180, 180 and 7 ^m o l/k g |36|; the corre sponding values for A roclor 1254 are at least 10 times higher (40). Moreover, A roclor 1254 exhibits a competitive Ah receptor binding a ffin ity only 500 times lower than 2,3,7,8-TCDD whereas the toxicity and enzyme induction activity o f the latter compound is at least 10' times greater in the rat |4 I|. A lthough a direct com parison o f the teratogenic potency o f A roclor 1254 and 2,3,7,8-TC DD has not been made, the ED 50 for teratogenicity (cleft palate) in this study was < 20 jrg/kg whereas the F.Di0 for Kanechlor 500 in the ddY strain o f mice was >2000 /im o l/k g (42). We therefore propose that A ro clo r 1254 may act as a partial 2,3,7,8-TC DD antagonist by competing for the Ah receptor and thus lowering the cellular levels o f (he Ah receptor available for interaction w ith the potent agonist, 2,3,7,8-TCDI>. F.vidcncc which supports this hypothesis include the follow ing: (a) colreatment o f C 57BL/6J mice with A roclor 1254 and 2,3,7,8-TC D D at A ro clo r 1254/2,3,7,8 IC D D ratios >1000:1 showed that A roclor 1254 can significantly antagonize the in duction o f 2 Ah receptor-mediated responses, namely the induction o f aryl hydrocarbon hydroxylase (A H H ) and the plaque-form ing ceil response to sheep GENP 010950 304 erythrocytes [43]; (b) Aroclor 1254 also antagonizes the induction o r A H II by 2,3,7,8-TCDD in rat liepaloma H-4-II E cells in culture [43]; (c) double-reciprocal plot analysis o f the Ah rcceplor-2,3,7,8-TCDD binding isotherms in the presence o r dirrerent concentrations o f Aroclor 1254 gave data which are consistent with com petitive inhibition o r 2,3,7,8-TCDD-receptor binding by A roclor 1254 [43]; (d) Aroclor 1254 docs not act as a non-specific teratogen antagonist since Aroclor 1254 docs not antagonize the teratogenicity o r dexamelhasonc, a synthetic glucocorticoid horm one analog which acts through initial binding with the glucocorticoid receptor (see Table I). These results further extend the spectrum or 2,3,7,8-TCDD-mcdialed responses which can be antagonized by A roclor 1254. M oreover since hum an environm ental exposure to the toxic PCDD and PC D F congeners occurs in concert with sim ultaneous exposure and bioaccumulalion o f PCB$ [1-4,9-16], it is possible that the non-toxic environmental PCB levels may arford some measure o f protection from these toxins since the environmental ratios o f PCB/loxic PCDDs + PCDFs [7-9,12-15] are comparable to ratios in which A roclor 1254 exhibits antagonistic ac tivity (this paper; [43)). Current studies in o u r laboratory are focused on further delineating the mechanism o f the interactive effects o f commercial PCBs and in dividual PCB congeners with the toxic 2,3,7,8-5ubstiluted PCDDs and PCDFs. ACKNOWLEDGEMENTS Financial assistance was provided by the National Institute o f Health (ES-03843), the Texas Agricultural Experiment Station and the Chester J. Reed Endowment. REFERENCES t R .W . 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Safe, Aroclor 1254 as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist: effects on enzyme induction and immunotoxicity. Toxicology, in press. ENP 010952 783763