Document eYak4Ba0nEyyKYRVDxmaM69e

Toxicology, 46 (1987) 2 9 -4 2 ..................... Elsevier Scientific Publishers Ireland Ltd. AROCLOR 1254 AS A 2,3,7,8-TETRACHLORODIBENZOp-DIOXIN ANTAGONIST: EFFECTS ON ENZYME INDUCTION AND IMMUNOTOXICITY R. BANNISTER, D. DAVIS, T. ZACHAREWSKI. I. TIZARD and S. SAFE Departments o f Physiology and Pharmacology and Microbiology and ~ Veterinary Medicine, Texas A A M University, College Station, T X 77843 (C/.SJU (Received March 10th. 1987) (Accepted April 24th. 1987) of An international journal concerned w ith the effects o f chem icals on living system s 783743 GENP 010932 ' '*' of chemical on living syitsms i ' ',''i- ' r Toxicology is a journal for the publication of original scientific papers on the biological effects arising from the adm inistration of chemical com pounds, principally to animals, tissues or cells, but alto to man. Such com pounds include industrial chemicals and residues, chemical contam inants, consum er products, drugs, metals, pesticides, food additives, cosmetics, and additives to animal feeding stuffs. Preference.will be given to investigations dealing with the mechanisms of action of toxic agents. Papers describing m olecular interactions w ith cellular and genetic pro cesses w ill be w elcom ed. Quantitative toxicological studies w ill be published -that are o f relevance to risk assessment and regulatory management of exposure hazards and safety evaluation. This applEea.particularJjy to, carcinogenicity, mutagenicity, em bryotoxicity and related areas, as well as to alternatives to the use of anim als in toxicological experimentation. Epidem iological studies bearing toxicological significance to man fall-within; the scope of the journal. The Editors w ould also welcome the subm ission o f concise and pertinent review o n currant issues in toxicology. \ " T, MANAGING EDITORS: H.P. WITSCHl, OakRldge; TN,U.S.A.; ^ EDITORIAL BOARD K J. NETTER, Marburg* F .R &*>- -*w " --V *.*. * * ' ? f ,.. .' ". F. Barglund. Stockholm R. Hsndsrson. Albuquerque, NM W.O. Bemdt. Omette. NE J.B. Hook. Phlladslphie. PA- J.W. Bridges, Guildford F A : da le Ig M *. Arm- Arbor, Ml A.B. Buckpitt, Owls, CA . - Rilrona^flasM rchTrtsogla ParteMC A. Castro, Buenos Aires W.G. Charisn, Ontario ,. . ^ mN. Chamotf, Research TrienePwfc, NC J.W. Denial. L ittle Seddow*. ~ G. Dithaimer. S tra ti *** .. . O. Ecoblchon, Montreal J.RlK;Kkinaoh,rwTroAkiyModsn`*.WT7^* i.T^;^7*" " K-Ko|HmtTWtyte ,V/ ft. kjsinriKBtiiAk^if^'- T Lsfis.'EastMIUstonvM jV , v , . ttXr^oomiQftam,CT ;-,;r ;;.+1 PjS. Ellee. Redbridge - v ^ ^ ^ y ^ J L filC a a m d a ^ P lra ra in y . ^ EX. Fowlkes. C ln d n ittd ,OH-j S. G am ttlnl. Milan --V.,,'..'.-.--''vv'C ' -V-W" PJ. HakUnen. Qncinntattii,,Off?; S. Harrison, Birm i ' , AJEJd.MclJervtuxxkxr^/r-^'v^;.v'. r ,. ~: P. Milas, Morgantown, W V G J . Mulder, Leiden R. Neal. 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Va , 1, ^ " 1 - . . ,* Authorisation to photocopy Item * fo r {ntarrtd or psrsonaf usi ^ th k ln ta m d wpacMM Lsa o f maetfte d ie n ti; is granted by Elsevier Scientific'Publishers Ireland. Ltd.- fo r librane* and other u ii r r rsgHtarad wfdv the~CCpyrlQht Clsarana. Cantar (CCCl .Transactional Reoonlng Servics, prirvldsd that tha basa faa o f S09JBG p sr copvvpIus iX O l par paga E&peid dlfeefty.to CCC, 27 Congress St^SW m , MA 01070,0300-483X/87/S03XO + .00. 7i'!*' ' r\ GENP 010933 783744 Tox Else AE A> IM R.I Dep Vet (Ret (Act su: cyt last II : anc fea teil pot 4-Ij mai littl due mie pmt TCI ago Arc 2,3, of ; cree Thii Add Abbi rafie poly< activ tetra 0300 U Print Toxicology, 46 (1987) 29--42 Elsevier Scientific Publishers Ireland Ltd. AROCLOR 1254 AS A 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN ANTAGONIST: EFFECTS ON ENZYME INDUCTION AND IMMUNOTOXICITY R. BANNISTER, D. DAVIS, T. ZACHAREWSKI, I. TIZARD and S. SAFE Departments o f Physiology and Pharmacology and Microbiology and Parasitology, College of VeUrinary Medicine, Texas A A M University, College Station, TX 77843 (U.S.A.) (Received March 10th, 1987) (Accepted April 24th, 1987) SUMMARY 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and Aroclor 1254 induced the cytochrome iM 50 dependent monooxygenases, aryl hydrocarbon hydroxy lase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma H-4II E cells and C57BL/6J mice. It has been proposed that both Aroclor 1254 and 2,3,7,8-TCDD induce these enzymes via a common mechanism which features initial binding to the aryl hydrocarbon (Ah) cytosolic receptor pro tein. The major difference between these compounds was the relative potency (i.e. 2,3,7,8-TCDD > Aroclor 1254). Cotreatment of ra t hepatoma H4-II E cells or C57BL/6J mice with a dose of 2,3,7,8-TCDD which submaximally induces AHH and EROD and a dose of Aroclor 1254 which exhibited little or no induction activity resulted in significant antagonism of the in duction effects of 2,3,7,8-TCDD. For example, cotreatment of C57BL/6J mice with 2,3,7,8-TCDD (15 nmol/kg) and Aroclor 1254 (25, 75 and 150 /mol/kg) resulted in up to 23% antagonism of AHH induction by 2,3,7,8TCDD. Moreover, cotreatment with a higher dose of the 2,3,7,8-TCDD agonist (30 or 50 nmol/kg) partially reversed some of the antagonism by Aroclor 1254. In vivo antagonism was observed only at Aroclor 1254/ 2,3,7,8-TCDD molar ratios of 1667:1, 5000:1 and 10 000:1. Administration of 2,3,7,8-TCDD (3.72 nmol/kg) to C57BL/6J mice resulted in a 76% de crease in the splenic plaque forming cell response to sheep red blood cells. This T-cell mediated immunotoxic effect of 2,3,7,8-TCDD segregates with Address ail correspondence to: S. Sale. Abbreviations: Ah, aryl hydrocarbon: AHH, aryl hydrocarbon hydroxylase; EROD, ethoxyresorufin O-deethylase; HCBP, hexachlorobiphenyl; PCBs, polychlorinated biphenyls; PCDDs, polychlorinated dibenzo>p-dioxiiis; PCDFs, polychlorinated dibenzofurans; SARs. structure activity relationships: 2,3,7,8-TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxixi; TCDF, 2,3,7,8tetrachlorodibenzofuran. 0300-483X/87/303.50 1987 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland 29 GENP 010934 7837^5 tionships (SARs) have recently been reported for several structurally diverse PCDDs, PCDFs and PCBs [9--11,20]. Comparable SARs were observed for cytosolic receptor binding, the in vitro induction of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD), the in vivo induction of these same monooxygenase enzymes, body weight loss and thymic atrophy (in the rat). Previous studies with agonists for the estrogen receptor have demon strated that some compounds which exhibit moderately high estrogen receptor binding affinities but low estrogenic activities (e.g. estriol and some triphenylethylene analogs) can antagonize the effects of estradiol [21,22]. Similarly, recent studies in our laboratory have shown th at both 1,3,6,8- and 2,4,6,8-tetrachlorodibenzofuran (TCDF) exhibit relatively high cytosolic receptor binding affinities (1.25 x 10'7 M and 1.5 X 10"3 M, respectively) but are only weakly active as inducers of AHH and EROD in ra t hepatoma H-4-II E cells in culture [23]. Cotreatment of the cells with 2,3,7,8-TCDD and either of the 2 PCDF isomers demonstrated that both compounds antagonized the induction of the monooxygenases by 2,3,7,8-TCDD. Previous studies [8] have reported that the commercial PCB, Aroclor 1254 also exhibited moderate binding affinity to the cytosolic receptor protein (6.6 X 10~* M) but was a weak agonist for most receptor-mediated respon ses in vivo and in vitro [24]. This paper reports the activity of Aroclor 1254 as a 2,3,7,8-TCDD antagonist in ra t hepatoma H-4-II E cells and in C57BL/ 6 mice. MATERIALS AND METHODS Chemicals 2,3,7,8-TCDD and ethoxyresorufin were synthesized in this laboratory to greater than 99% purity as determined by gas chromatography [10]; Aroclor 1254 was a gift from Dr. 0 . Hutzinger; NADPH and NADH were pur chased from the United States Biochemical Corporation (Cleveland, OH). Benzo[a]pyrene was purchased from Sigma Chemical Co.; DNA-grade hydroxylapatite was purchased from Bio-Rad Laboratories (Richmond, CA). All other ehamirala were of the highest quality commercially available. pH]2,3,7,8-TCDD was synthesized in the laboratory by the chlorination of l,6pH 2]dibenzop-dioxin followed by chlorination and HPLC purification of the product (> 98% pure) [5], In vivo enzyme induction studies Male C57BL/6J, 3--4 weeks of age, were obtained from the Jackson Laboratories, Bar Harbor, ME. The mice were housed in plastic cages with hardwood bedding, allowed free access to Teklab Laboratory Rodent Chow, (Teklab, Madison, WI) and water and maintained on a diurnal 12-h light/12h dark cycle. Aroclor 1254 or 2,3,7,8-TCDD were dissolved in com oil and administered by intraperitoneal injection (10 ml/kg). The C57BL/6J mice treated with Aroclor 1254 and/or 2,3,7,8-TCDD were sacrificed after 72 h. 31 - GETSiP 010936 783747 Corn oil (10 ml/kg) served as a vehicle control for all studies. Livers were perfused via the hepatic portal vein with ice cold saline containing 0.1 mM EDTA, weighed and placed in beakers containing homogenization buffer. Livers were washed and then homogenized using a Potter-Elvehjem homogenizer in 4 volumes of 0.25 M sucrose with 0.1 mM EDTA. A microsomal pellet was prepared by centrifuging the homogenate at 10 000 g for 20 min and then centrifuging the resultant supernatant at 100 000 g for 1 h. The pellet was resuspended in homogenization buffer and stored at - 80 C until used for the enzyme assays. Ethoxyresorufin o-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH) were determined as previously described [25,26]. Protein concentrations were determined [27] using bovine serum albumin as a protein standard. All data are expressed as means S.D. Sig nificance was determined by a two-way ANOVA and Dunnet's t-test [28]. In vitro enzyme induction Rat hepatoma H-4-II E cells were grown in m inim um essential medium without ribo- or deoxyribonucleotides but with L-glutamine. The medium was supplemented with 10% fetal calf serum, 10% calf serum, gentamycin sulfate (50 pg/ml) and Fungizone (2.5 pg/ml) and cultures were maintained in a humidified 5% CO, atmosphere at 37 C. A t confluency stock cultures were trypsinized and seeded in 25-cm2 Corning culture flasks at a density of 0.6 x 10 cells/flask (Day 1). On Day 2, the spent medium was aspirated off and replaced with fresh medium containing the test compound in DMSO to a final DMSO concentration of 0.5%. Dose-response induction studies with at least 6 different concentrations of each inducer were carried out. On Day 3, the cultures reached confluency and were harvested. Cultures were washed 3 times with PBS (pH 7.4) and scraped off with a rubber policeman in Tris-sucrose (0.05--0.02 M) buffer (pH 8.0). The cell suspensions were centrifuged and resuspended in buffer. After protein determination [27], the cell suspensions were adjusted to a final protein concentration of 1 mg/ml and whole cell suspensions were assayed for enzyme activity. EROD and AHH activities were measured fiuorimetrically [25,26] and the EC50 values determined as previously described [9--11]. Receptor binding assays Hepatic cytosol ~ 3 --5 mg/ml) was incubated with different concentrations of pH]2,3,7,8-TCDD and Arodor 1254 (100, 500 and 667 nM) for 2 h at 20 C. Each assay was performed in duplicate. A hydroxylapatite slurry (Bio Rad Lab; 3 parts gel in 5 parts phosphate or HEDG buffer) was freshly pre pared. The phosphate buffer was 50 mM Tris--HC1, 1 mM KH2P 0 4. After incubation, 200 pi aliquot of the cytosol was transferred to a second tube containing *250 pi of the hydroxylapatite slurry. The tubes were incubated on ice for 30 min with shaking every 10 min. The mixture was then resus pended in 2 ml phosphate buffer and centrifuged at 800 g for 2 min. The hydroxylapatite pellets were washed 3 additional times with phosphate buffer (2.0 ml buffer) and then resuspended in 1.0 ml absolute ethanoL The 32 GENP 010937 783748 hydroxylapatite-ethanol solution was vortexed and transferred to scintilla tion vials and any remaining hydroxylapatite was washed into the vial with an additional 1 ml of ethanol; 10 ml of scintillation cocktail was added and samples were counted for radioactivity. Saturation binding of pH]2,3,7,8TCDD with C57BL/6J hepatic cytosolic receptor protein was determined in the presence of different concentrations of the competitive ligand, Aroclor 1254 and the results are summarized in Fig. 2. Plaque forming cell (PFC) assay The method employed was the "Cunningham" variant of the Jerne PFC as say [29,30]. C57BL/6J mice at 8 weeks were purchased from the Jackson Laboratories, Bar Harbor, ME. Upon arrival the animals were allowed to acclimate for 5--7 days before treatment. On day 1 the animals were in jected (i.p.) with the appropriate chemical as described for the enzyme induction studies (Aroclor 1254 and/or 2,3,7,8-TCDD) and sheep red blood ells (SRBC, 4 x 108 cells) were injected after 5 days. The animals were sac rificed by cervical dislocation 10 days after treatment with the chemicals and the spleens were removed and placed individually in 60-mm petri dishes containing 10 ml of cold Eagle's MEM media supplemented with Hank's salts (KC Biological). Each spleen was then teased through a 70-pan nylon screen (Spectrum Medical, Inc.) which was saturated with 2 ml of the Eagle's media. The screens were washed of remaining cells by the addition of 8 ml of media. The cell solution was then spun at 220 g for 10 min and the pellets resuspended in 2 ml of fresh media. Appropriate dilutions of the suspensions were then made with 0.1 ml of a dilution mixed with 0.2 ml of a 20% SEBO solution and 0.1 ml of a 1--5 guinea pig serum dilution. Through capillary action, microliter aliquots of the mixtures were placed in "Cunningham" slide chambers and incubated for 60 min at 37 C. Viable cell counts were determined by Eosin Y staining. RESULTS 2,3,7,8-TCDD evoked a dose-dependent induction of AHH and EROD in rat hepatoma H-4-I1 E cells at concentrations of 10"12--10"* M. A t higher dose levels (> 10"8 M), 2,3,7,8-TCDD was toxic to the cells and this was evi denced by a dose-dependent decrease in the AHH induction maximum with increasing concentrations of 2,3,7,8-TCDD (Fig. 1). The dose-dependent in crease and subsequent `decrease of AHH and EROD induction has previously been reported for other halogenated aromatics [24]. Moreover, for some weak agonists such as Aroclor 1254, cellular toxicity is noted at con centrations which are insufficient to elicit maximum induction of the cytochrome fMSO-dependent monooxygenases (i.e. see Fig. 1). Cotreatment of the rat hepatoma cells with a non-toxic concentration of Aroclor 1254 33 GENP 010938 783749 Fig. 1. Dose response induction of AHH (pmol/mg protein/min) in rat hepatoma H-4-II E cello by Aroclor 1254 (O) and 2.3,7.8-TCDD ( x ). Cellular toxicity by Aroclor 1254 and 2,3,7,8-TCDD waa observed at concentrations > 10*M and 10" M, respectively. (1 x 10"7 M) and a concentration of 2,3,7,8-TCDD which elicited a submaximal AHH and GEOD induction response (1 x 10"9 M) resulted in the an tagonism of the induction activity of 2,3,7,8-TCDD by Aroclor 1254 (Table 1). Cotreatment of the cells with the same concentration of Aroclor 1254 (1 x 10'7 M) and a higher concentration of 2,3,7,8-TCDD (2.25 X 10~* M) par- TABLE I EFFECTS OF 2,3,7,8-TCDD, AROCLOR 1254 AND 2,3,7,8-TCDD PLUS AROCLOR 1254 (COTREATED AS INDUCERS OF AHH AND EROD IN RAT HEPATOMA H-4-II E CELLS IN CULTURE Inducer Solvent (control) Aroclor 1254 2,3,7,8-TCDD 2,3,7,8-TCDD Aroclor 1254 plus 2,3,7,8-TCDD Aroclor 1254 plus 2,3,7,8-TCDD Concen tration (M) _ 1 x 10- 1 x 10-* 2.25 X 10- 1 X 10- 1 X io-* 1 X 10- 2.25 X 10"* ERODb 0 0 74.0 20.1 77.9 19.9 46.8 3.99" 65.6 2.21 AHHb 0 0 155.9 14.6 179.0 19.7 105.7 11.7" 117.0 7.36 S i gnificantly lower (P < 0.01) than observed after treatment with 2,3,7,8-TCDD (1.0 x 10~* M) alone. ^pmol/mg protein/min. 34 a z c G // / Fig. pres: the ; in th tivel abse of p coeff prep tiall ana pati Aro acts T clor mal< ER( 2,3/ thyr of A kg : GENP 010939 783750 ^. Fig. 2. Double reciprocal plat analysis of the saturation binding of [3HJ2.3,7.8-TCDD in the presence of different concentrations of Arodor 1254 (100, 500 and 667 nM). The y intercepts of the double-reciprocal plots of the saturation binding of [3H]2,3,7,8-TCDD with the Ah receptor in the presence of 100, 500 and 667 nM Arodor 1254 were 0.0091, 0.0068 and 0.0092. respec tively. The linear correlation coefficients were 0.9925, 0.9984 and 0.9995, respectively. In the absence of Arodor 1254, the y intercept of the double*reriprocal plot of the saturation binding of [3H]2,3,7,S-TCDD with the Ah receptor was 0.0074 and the correlation linear correlation coefficient was 0,9899 (data not shown). The incubations were carried out using hepatic cytosol preparation (3--5 mg/ml) and a range of [3H]2,3,7,8-TCDD concentrations (from 0.8 to 11 nM). tially overcame the antagonism by Aroclor 1254, Double reciprocal plot analysis of the saturation binding curves of [3H]2,3,7,8-TCDD and rat he patic cytosolic receptor in the presence of different concentrations of Aroclor 1254 is summarized in Fig. 2 'and demonstrates th at Aroclor 1254 acts as a competitive antagonist for the receptor-2,3,7,8-TCDD interaction. Table II summarizes the dose-response effects of 2,3,7,8-TCDD and Aro clor 1254 as inducers of hepatic microsomal AHH and EROD in immature male C57BL/6J mice. Maximal induction of AHH (4.06 nmol/mg/min) and EROD (4.86 nmol/mg/min) were observed at a dose level of 50 nmol/kg of 2,3,7,8-TCDD. No overt toxicity (e.g. body weight loss, hepatotoxidty or thymic atrophy) was observed at this dose level. In contrast, administration of Aroclor 1254 to C57BL/6J mice at doses of 5, 25, 75, 150 and 400 jimol/ kg significantly induced EROD only at the highest dose of Aroclor 1254 35 GENP 010940 783751 TABLE l i THE DOSE-RESPONSE EFFECTS OF 2,3,7,8-TCDD AND AROCLOR 1254 AS INDUCERS OF HEPATIC MICROSOMAL AHH AND EROD IN MALE C57BL/6J MICE In = 5) Compound Cora dii 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD 2,3,7,8-TCDD Aroclor 1254 Aroclor 1254 Aroclor 1254 Aroclor 1254 Aroclor 1254 Dose (fimol/kg) __ 0.001 0.003 0.010 0.015 0.03 0.05 5 25 75 150 400 ERODb 0.06 0.004 0.33 0.08* 1.39 0.54* 3.31 0.50* 3.96 0.16* 4.19 * 0.30* 4.86 0.61* 0.06 0.006 0.05 0.03 0.06 0.01 0.09 i 0.04 0.25 0.05* AHHb 0.21 0.08 0.64 0.16* 1.67 0.71* 3.36 0.07* 3.56 0.43* 3.67 0.30* 4.06 0.37* 0.18 0.06 0.41 0.06* 0.39 0.06* 0.40 0.08* 0.55 0.07* `Significantly different (P < 0.01) from the corn oil controls, ^nmol/mg protein/min. TABLE III COTREATMENT OF C57BL/6J MICE WITH AROCLOR 1254 AND 2,3,7,8-TCDD: EFFECTS ON AHH AND EROD INDUCTION (n = 5) Inducers Dose (pmol/lcg) ERODc AHHC Aroclor 1254 plus 2,3,7,8-TCDD Aroclor 1254 plus 2,3,7,8-TCDD Aroclor 1254 plus 2,3,7,8-TCDD Aroclor 1254 plus 2,3,7,8-TCDD Aroclor 1254 plus 2,3,7,8-TCDD Aroclor 1254 plus 2,3,7,8-TCDD Aroclor 1254 plus 2,3,7,8-TCDD `5 0.015 25 0.015 75 0.015 150 0.015 400 0.015 25 0.03 25 0.06 3.79 0.58 3.05 * 0.47* 3.09 0.34* 3.22 0.07b 3.96 0.45 4.19 0.39 4.93 0.25 3.34 0.42 2.84 0.33* 2.88 0.21* 3.09 A 0.27* 3.55 0.18 3.78 0.25 4.10 0.20 Significantly lower "IP < 0.01) btP < 0.05) than induction values observed after treatment with 2,3,7,8-TCDD (15 nmol/kg) alone; the induction values for 2,3,7,8-TCDD (0.015, 0.03 and 0.5 fdnol/kg) are shown in Table II. cnmol/mg protein/min. 36 E N P 010941 783752 VJM and marginally increased AHH at doses above 25 pmol/kg. Table III sum* marizes the effects of coadministration of Aroclor 1254 (5, 25, 75, 150 or 400 pmol/kg) with a dose of 2,3,7,8-TCDD (15 nxnol/kg) which elicited a submaximal AHH and EROD induction response. Cotreatment of 2,3,7,8TCDD with 25 or 75 pmol/kg of Aroclor significantly inhibited the induction of AHH and EROD by 2,3,7,8-TCDD. Coadministration of Aroclor 1254 (25 pmol/kg) -with higher doses of 2,3,7,8-TCDD (30 and 50 nmol/kg) reversed the antagonistic effects of Aroclor 1254. Table IV summarizes the effects of 2,3,7,8-TCDD and Aroclor 1254 on the T-cell dependent plaque-forming cell (PFC) assay after exposure of TABLE IV EFFECTS OF 2,3,7,8-TCDD, AROCLOR 1254 AND 2,3,7,8-TCDD PLUS AROCLOR 1254 ON THE PLAQUE FORMING CELL ASSAY IN MICE PRETREATED WITH SHEEP RED BLOOD CELLS Treatment (dose) PFC/apleen X 10 PFC/109 viable spleen cells % Control (PFC/spleen x 10s) Com oil* 1.61 * 0.17 562 a 76 100 2,3,7,8-TCDD (3.72 mnol/kg)b (11.2 nmol/kg) 0.38 A 0.03e 0.20 0.03c 156 40c 62 12 24 12 Aroclor 1254 (5 pmol/kg) (15 fimol/kg)b (75 pmol/lcg) (150 pmol/lcg) 2,3,7,8-TCDD (3.72 nmol/kg) plus Aroclor 1254 (5 pmoi/kg) 1.57 0.11 1.60 * 0.87 1.57 A 0.23 1.20 A 0.22 1.70 A 0.30 561 140 465 114 594 A 35 394 A 35 491 A 62 98 100 98 75 106 2,3,7,8-TCDD (3.72 nmol/kg) plus Aroclor 1254 (15 jimol/kg)b 1.49 A 0.08 480 A 48 93 2,3,7,8-TCDD (3.72 nmol/kg) plus Aroclor 1254 (75 imal/kg) 1.66 * 0.28 558 A 52 103 2,3,7,8-TCDD (3.72 nmol/kg) plus Aroclor 1254 (150 pmol/kg) 0.92 A 0.08* 372 A 81c 57 2,3,7,8-TCDD (11.2 nmol/kg) plus Aroclor 1254 (75 pmol/kg) 0.58 A 0.10* *11 B innU . *>8animala; all other 4 animals/group. Significantly different IP < 0.01) than the com oil controls. 170 A 10c 36 37 GENP 010942 783753 C57BL/6J mice to sheep red blood cells. Administration of 2,3,7,8-TCDD (3.72 and 11.2 nmol/kg) resulted in only 24 and 12% PFCs/106 spleen cells respectively, compared to the com oil-treated animals. In contrast, adminis tration of Aroclor 1254 at dose levels of 5, 15, and 75 pmol/kg did not significantly impair the immune response although administration of a higher dose of Aroclor 1254 (150 pmol/kg) did evoke some decrease in the plaque forming cell response. Cotreatment of the mice with 2,3,7,8-TCDD (3.72 nmol/kg) and 5, 15, or 75 pmol/kg Aroclor 1254 resulted in significant protection of these nimnls from the immunotoxic effects of 2,3,7,8-TCDD (Table IV). Significant antagonism of 2,3,7,8-TCDD was also observed in animals cotreated with the highest dose of .Aroclor 1254 (150 pmol/kg) which was also immunotoxic in this assay when administered alone. Coadministra tion of a higher dose of 2,3,7,8-TCDD (11.2 nmol/kg) with Arcolor 1254 (75 pmol/kg) reversed some of the antagonistic effects of the commercial PCB mixture. DISCUSSION This study describes the in vitro and in vivo interactive effects of 2,3,7,8TCDD and Aroclor 1254, 2 compounds which individually elicit a number of common biologic and toxic effects in diverse animal species. For example, both of these halogenated aryl hydrocarbons induce the same monooxygen ase enzymes (e.g. AHH and EROD) and their associated cytochrome P-450 isozymes, cause body weight loss, hepatotoxicity and porphyria, thymic atrophy and immunotoxirity [1--3,24]. In addition, 2,3,7,8-TCDD and Aro clor 1254 competitively bind to the Ah or 2,3,7,8-TCDD receptor protein [8]. These results support the proposed common mechanism of action for both compounds; however, it is apparent from this study and others th at the major differences between Aroclor 1254 and 2,3,7,8-TCDD are their toxic and biologic potencies. The results summarized in Tables I and II clearly illustrate the quantitative differences between 2,3,7,8-TCDD and Aroclor 1254 as inducers of AHH and EROD. In ra t hepatoma cells, the ECm for 2,3,7,8-TCDD as an inducer of AHH and EROD was 9.78 x 10"11 and 1.90 x 10~to M respectively whereas the ECM for Aroclor 1254 as an inducer of these enzymes was > 10~* M. Moreover in C57BL/6J mice, 2,3,7,8TCDD was > 50 000 times more potent than Aroclor 1254 as an inducer of hepatic microsomal AHH and EROD. 2,3,7,8-TCDD and Aroclor 1254 and several PCB congeners are immunotoxic and inhibit the plaque forming an tibody response in C57BL/6J mice challenged with sheep erythrocytes [13-- 17,31]. The EDU for inhibition of this response by Aroclor 1254 in C57BL/ 6N mice was between 250 and 500 mg/kg [31] whereas the EDMfor 2,3,7,8TCDD was <1.2 pg/kg [14]. Previous in vitro studies have reported th at 1,3,6,8- and 2,4,6,8tetrachlorodibenzofuran antagonize the induction of AHH and EROD in rat hepatoma H-4-II E cells in culture [23]. Like Aroclor 1254, both of these iso mers exhibit moderate competitive receptor binding affinities and are weak 38 783754 inducers of AHH and EROD in the cell culture system. Table I summarizes the interactive effects of 2,3,7,8-TCDD and Aroclor 1254 in the ra t hepa toma H-4-II E system and at Aroclor 1254/2,3,7,8-TCDD concentration ratios of 100:1 there was a significant inhibition of the induction activity of 2,3,7,8-TCDD by Aroclor 1254. Moreover at a higher dose of the agonist (2.25 x 10~* M) some of the inhibitory effects of Aroclor 1254 were over come. A comparable interaction study was carried out in C57BL/6J mice. The mice were cotreated with a dose of 2,3,7,8-TCDD which elicited a submaximal induction response for hepatic microsomal AHH and EROD (15 nmol/kg) and one of several doses of Aroclor 1254. The results showed the Aroclor significantly reduced the induction of AHH and EROD by 2,3,7,8TCDD (15 nmol/kg) only at doses of 25, 75 and 150 pmol/kg. The role of Aroclor 1254 as a 2,3,7,8-TCDD antagonist was further supported by the effects of cotreating the animal with the most effective "inhibitory" dose of Aroclor 1254 (25 pimol/kg) and higher doses of 2,3,7,8-TCDD (30 and 50 nmol/kg) (Table III). These results showed that at higher dose levels of the agonist the partial antagonist activity of Aroclor 1254 was decreased com plementing the results obtained in the in vitro studies. It was also apparent that Aroclor 1254 was a more effective partial antagonist of the induction responses by 2,3,7,8-TCDD in cell culture. Significant antagonism of AHH was observed at Aroclor 1254/2,3,7,8-TCDD ratios of 100:1 in cell cultures whereas antagonism was only observed in the mice at ratios between 1667:1 and 10 000:1. Several reports have shown that 2,3,7,8-TCDD and related compounds are immunotoxic and impair many T-cell mediated functions [13--17,31-- 34]. Moreover the T-cell dependent plaque forming cell response to sheep red blood cells is one of the most sensitive indicators of exposure to toxic halogenated aryl hydrocarbons and studies in genetically inbred mice have provided strong evidence that this response is Ah receptor-mediated. Table IV summarizes the immunosuppressive effects of 2,3,7,8-TCDD and Aroclor 1254 in C57BL/6J mice using the plaque forming cell assay response to sheep red blood cells. 2,3,7,8-TCDD was highly immunotoxic whereas doses of 5, 15 and 75 pmol/kg of Aroclor 1254 were inactive in this assay system. This latter result was not surprising since Aroclor 1254 was previously reported to be inactive at doses of < 125 mg/kg (382 pmol/kg) [31]. Cotreat ment of the mice with 2,3,7,8-TCDD (3.72 nmol/kg) and different doses of Aroclor 1254 (5, 15 or 75 pmol/kg) resulted in complete protection from the immunotoxic effects of 2,3,7,8-TCDD by Aroclor 1254 (5, 15 or 75 pmol/kg). Moreover, the antagonistic effects of Aroclor 1254 (75 pmol/kg) can be par tially overcome by a higher dose of the 2,3,7,8-TCDD agonist (11.2 nmol/ kg). The interactive effects of 2,3,7,8-TCDD, a potent Ah receptor agonist, and Aroclor 1254 suggest that this commercial PCB mixture can antagonize at least 2 Ah receptor mediated responses, namely immunotoxicity and the induction of cytochrome *450-dependent monooxygenases (AHH and EROD). These effects are dependent on the dose of both the antagonist and 39 GENP 010944 783755 agonist and their respective antagonist/agonist ratios. For antagonism of AHH and EROD induction by 2,3,7,8-TCDD in C57BL/6J mice, an Aroclor 1254/2,3,7,8-TCDD ratio of 1667:1 gave the m axim um antagonism (23%) of the EROD induction response. In contrast* Aroclor 1254/2,3,7,8-TCDD ratios from 1340:1 to 20 160:1 resulted in complete protection from the immunotoxic effect of 2,3,7,8-TCDD (3.72 nmoi/kg). The results demonstrate that the "window of antagonism" for Aroclor 1254 is clearly dependent not only on the absolute and relative doses of Aroclor 1254 and 2,3,7,8-TCDD but on the specific receptor-mediated response which is being measured. However, it was observed that for Aroclor 1254/2,3,7,8-TCDD ratios which result in antagonism by Aroclor 1254 of AHH and EROD induction (in vivo and in vitro) and immunotoxidty, an increased dose of 2,3,7,8-TCDD over came some of the partial antagonism by Aroclor 1254. These results, coupled with the double-reciprocal plot analysis of the in vitro binding data (Fig. 2) suggest that the antagonist activity of Aroclor 1254 is related to the competitive inhibition of 2,3,7,8-TCDD binding to the Ah receptor and is not due to cellular toxicity caused by Aroclor 1254. A previous study has reported that 2,3-dimethyI-5-t-butyI-lf3-benzodioxole (DBBD) inhibits EROD induction by 3-methylcholanthrene [35]. However, it is unlikely that the mechanism of antagonism for this com pound involves competitive inhibition of receptor binding since DBBD exhibits a low binding affinity for the Ah receptor [36]. A more recent study suggested that the activity of DBBD may be associated with down regula tion of the Ah receptor [37]. A recent paper [38] reported that l-amino-3,7,8trichlorodibenzo-p-dioxin was also an effective antagonist for 2,3,7,8-TCDD induced myelotoxicity (in vivo) and enzyme induction (in vitro). They also proposed that this compound acts as a competitive antagonist and their results complement the data reported in this study which demonstrates that Aroclor 1254 antagonizes 2,3,7,8-TCDD-mediated enzyme induction and immunotoxidty in C57BL/6J mice. It is interesting to note that the relatively high ratios of Arodor 1254/ 2,3,7,8-TCDD which result in antagonist activity are comparable to the ratios of PCBs/PCDFs or PCBs/PCDDs which have been identified in the analysis of human tissues and environmental samples [39,40]. It is conceiv able that environmental or acddental exposure to these mixtures within certain exposure levels may result in some protection from the highly toxic PCDDs and PCDFs by PCBs and this possibility is currently being investi gated in our laboratory. ACKNOWLEDGEMENTS The finantial assistance of the National Institutes of Health (ES-03843) and the Texas Agricultural Experiment Station is gratefully acknowledged. The experimental assistance of Ms. B. 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Chemosphere, 15 (1986) 1585. 41 T. Sawyer and S. Safe, PCB isomers and congeners: induction of aryl hydrocarbon hydrox ylase and ethoxyreeorufin O-deethylase enzyme activities in rat hepatoma cells. Toxicol. Lett.. 13 (1982) 87. 42 GENP 010947 783758 -- Review author: y Animal Ex -- It is ex an ethic Address -- Author and cor Summery The art Key words A list c script. I ducinq activity Figures -- Line dri provide -- Half-tor Colour to the e Tobies -- should t References -- should t Referen referenc -- In the r< 1. A. G< fluor 2. A. Fr 3. T.A. Comt -- Female -- For jou Lancer. M anuscript To avoid th 1 A ft pap 2 The be cc 3 The (i) tc () e 4 A cc TOX shoo 5 EacfThi: be ac Acterxiaem Adverti: MU US UK: T.< 22S 181