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The interactions of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 6-methyl-1,3,8-trichlorodibenzofuran in chick embryo hepatocytes
Toxic'. in Vitro Vol. 6, No. 4, pp. 373-380, 1992 Printed in Great Britain. All rights reserved
0887-2333/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
THE INTERACTIONS OF 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN A N D 6-METHYL-1,3,8-TRICHLORODIBENZOFURAN IN CHICK EMBRYO HEPATOCYTES C. YAO and S. SAVE* Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4466, USA (Received 11 October 1991; revisions received 11 December 1991)
A~tract--Treatment of chick embryo hepatocytes in ovo and in culture with 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) resulted in a dose-dependent induction of microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities. Significant induction was observed in the hepatocytes at TCDD doses as low as 10-" mol/egg (in ovo) and 10-~°M (in culture). In contrast, 6-methyl-l,3,8-trichlorodibenzofuran (MCDF) was a relatively weak inducer of these activities and only 10-20% of the induction responses observed for TCDD were elicited by MCDF at doses of 10 -6 mol/egg o r 1 0 - 7 M in culture. Co-treatment of the chick embryo hepatocytes with TCDD (10 ~0mol/egg in ovo; 10-~°M in culture) and different concentrations of MCDF (10 -6 to 10 Smol/egg in ovo and 10-7 and 10 -s M in culture) resulted in minimal inhibition of TCDD-induced enzyme activities in ovo and a 37 to 50% inhibition in culture. The partial antagonist activity of MCDF in the chick embryo hepatocytes in culture paralleled the interactive effects previously reported in rodent liver and transformed rodent cell lines. TCDD (10 -7 to 10 -I° M) also caused an accumulation of hepta- and octacarboxyporphyrins in chick embryo hepatocytes (10 -7 to 10 -9 M); however, MCDF (10 -6 and 10-5 M) elicited similar responses and MCDF did not significantly decrease the TCDD-induced porphyrogenic response in these cells. These results suggest that chick embryo hepatocytes in culture will serve as a useful model for investigating TCDD-induced gene transcription and the effects and mechanism of action of antagonists.
INTRODUCTION
2,3,7,8-Tetrachlorodibenzo-p-dioxin ( T C D D ) has been used as a prototype to investigate the effects and mechanism of action of the toxic halogenated aryl hydrocarbons. T C D D and related compounds elicit a broad spectrum of toxic and biochemical responses including a wasting syndrome, thymic atrophy, hepatotoxicity and porphyria, endocrine effects, developmental and reproductive toxicity, dermal-toxicity, and the induction of both phase I and II drugmetabolizing enzymes (Poland and Knutson, 1982; Safe, 1986; Whitlock, 1986 and 1987). The effects caused by T C D D and related compounds are dependent on a number of factors including the age, sex, species and strain of the test animal. The molecular mechanism of TCDD-induced cytochrome P4501A1 (CYP1A1) gene expression has been extensively investigated as a model for under*To whom all correspondence should be addressed. AHH = aryl hydrocarbon hydroxylase; ALA = 6-aminolaevulinic acid; BSA = bovine serum albumin; oL-DTT = DL-dithiothreitol; DMSO = dimethyl sulphoxide; EDTA=ethylenediaminetetraacetic acid; E R O D = ethoxyresorufin O-deethylase; HBSS = Hanks' balanced salt solution; MCDF = 6-methyl-l,3,8trichlorodibenzofuran; TCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDF = 2,3,7,8-tetrachlorodibenzofuran.
Abbreviations:
standing the other TCDD-induced toxic and biochemical responses. The results support the initial binding of T C D D to a cytosolic (aryl hydrocarbon, Ah) receptor that accumulates in the nucleus of target cells and acts as a transcriptional enhancer (reviewed in Whitlock, 1986 and 1987). Recent studies have identified several compounds, including substituted dibenzofurans and dibenzo-pdioxins, polychlorinated biphenyls and ~t-naphthoflavone, that inhibit several TCDD-induced responses (Astroff et al., 1988; Bannister et al., 1987 and 1989; Blank et al., 1987; Davis and Safe, 1990; Harris et al., 1989; Keyes et al., 1986; Luster et al., 1986; Merchant et al., 1990; Yao and Safe, 1989). One of these compounds, 6-methyl- 1,3,8-trichlorodibenzofuran ( M C D F ) , partially inhibited several TCDD-induced responses including immunotoxicity, teratogenicity, porphyria and C Y P 1 A I induction (Astroff et al., 1988; Bannister et al., 1989; Yao and Safe, 1989). In contrast, like T C D D , M C D F was moderately active as an anti-oestrogen in the female rat uterus and in human breast cancer cells in culture (Astroff and Safe, 1988). Thus, M C D F exhibited both partial agonist and partial antagonist activities, and this behaviour is similar to that of the clinically used antioestrogen tamoxifen, which elicits both oestrogen and anti-oestrogen responses in laboratory animals (Jordan and Koch, 1989; Jordan and Murphy, 1990). 373
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C. YAO and S. SAFE
This study reports the interactive effects of M C D F and TCDD in chick embryo hepatocytes in ovo and in culture. The chick embryos are Ah-responsive and are a useful model for investigating the mechanisms of at least two responses, namely the halogenated aromatic hydrocarbon-induced CYP1AI gene expression and porphyria (Denison et al., 1986; Lambrecht et al., 1988a,b; Rifkind et al., 1984; Sassa et al., 1984; Yao et al., 1990).
MATERIALS A N D M E T H O D S
Chemicals and biochemicals
TCDD and M C D F have previously been prepared in this laboratory ( > 9 8 % purity). Benzo[a]pyrene, DL-dithiothreitol (oL-DTT), N-2-hydroxythylpiperazine-N'-2-ethanesulphonic acid (HEPES), bovine serum albumin (BSA), Tris, ammonium chloride, ethylenediaminetetraacetic acid (EDTA, disodium salt), 6-aminolaevulinic acid (ALA) and Folin-Ciocalteu's phenol reagent were purchased from Sigma Chemical Co. (St Louis, MO, USA). Trypsin, insulin, 3,3',5-triiodo-e-thyronine (sodium salt), collagenase and hyaluronidase of tissue culture grade were also purchased from Sigma Chemical Co. Penicillin and streptomycin sulphate were purchased from Pfizer Co. (New York. NY, USA) and Eli Lilly and Co. (Indianapolis, IN, USA), respectively. Williams' medium E was purchased from either Hazelton (Lenaxa, KS, USA) or Gibco (Grand Island, NY, USA). Antiseptic Zephiran chloride was obtained from Winthrop Laboratories (New York, NY, USA). N A D P H and N A D H were purchased from United States Biochemical Corp. (Cleveland, OH, USA). Resorufin was purchased from Eastman Kodak Co. (Rochester, NY, USA) and ethoxyresorufin was prepared in this laboratory. Porphyrin standards were purchased from Porphyrin Products (Logan, UT, USA). Isolation o f chick embryo hepatocytes Incubation of eggs. Primary cultures of hepatocytes from 16- to 18-day-old white leghorn chick embryos were prepared and maintained in Williams' medium E as described by Sinclair et al. (1982). Eggs were incubated at 37°C (85% humidity) for 16 to 18 days in a Petersime model no. 5 incubator (Gettysberg, OH, USA) with rotation every hour. Preparation o f hepatocytes and culture conditions.
Incubated eggs were candled, marked, and sterilized with antiseptic Zephiran chloride before the embryos were removed. The livers were dissected from the embryos, pooled, and washed twice in Hanks' balanced salt solution (HBSS) without calcium and magnesium. Washed livers were minced into small pieces (about 2 mm 2) and digested for 30 min with 0.066% trypsin in HBSS containing penicillin and streptomycin. Contaminating red blood cells were lysed and removed as described by Sassa and Kappas
(1977). The resulting hepatocytes were resuspended in 200 volumes of serum-free Williams' medium E (WEHI) containing insulin (1 # g/ml), dexamethasone (0.3/~g/ml) and triiodothyonine (1/~g/ml) as described by Sinclair et al. (1979). Cells were cultured in an incubator at 37°C with a humidified air:CO2 (95 : 5) atmosphere. Treatments and enzyme induction. After incubation for 24 hr, the medium was changed to serum-free Williams medium E containing dexamethasone and triiodothyronine (WEH) but no insulin. Cells were cultured in 25-cm 2 flasks. The test compounds in dimethyl sulphoxide (DMSO) were added to the culture media ( < 0 . 4 % DMSO in all experiments), and the induction of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD) activities were determined after 19 hr as described (Yao et al., 1990) using fluorescence assays (Nebert and Gelboin, 1968; Pohl and Fouts, 1980). 3 hr before harvesting the cells, ALA was added to the media at 150/~M (final concentration). At the end of incubation, the cells were rinsed twice with phosphate buffered saline (0.90% NaCI, pH 7.4), scraped from the flasks, resuspended in Tris-sucrose buffer and assayed for enzyme activity. Protein concentrations were determined by the method of Lowry et al. (1951) using BSA as a standard. Porphyria studies
For porphyria studies, cells were treated with the test chemicals for 24 hr. 6 hr before harvesting the cells, ALA (150/~M) was added to the media. The cells were removed from the culture flask in the culture media, sonicated for 5 sec and 2 ml of the sonicate was added to an equal volume of acetoneconcentrated HC1 (97.5:2.5, v/v). The mixture was vortexed for 30sec and centrifuged at 1000g for 10 min at 4°C. Aliquots (40-50/d) of the supernatant were injected into a Beckman (Berkeley, CA, USA) HPLC equipped with two pumps and model 420 controller. Porphyrin peaks were quantified by interpolation of the peak heights with the peak heights from standard curves. The chromatographic conditions of Woods et al. (1984) and Ford et al. (1981) were adopted. These included two solvents, 0.05 M-phosphate-methanol buffer (pH 3.5) designated as solvent A and HPLC-grade methanol designated as solvent B. A flow rate of 1.0 ml/min was used. The elution gradient used was as follows (ratios are for B to A): 1:9 at time 0; 1 : 1 at the end of 3 min; 7 : 3 at the end of 10 min; 99 : 1 from 15 to 23 min. The HPLC column was allowed to re-equilibrate at a B to A ratio of 1:9 for 10min before the next injection. The individual porphyrins were detected by an interfaced LDC Fluoromonitor model 1311 (Riviera Beach, FL, USA) with the excitation filter set at 410nm and an emission filter with a cut-off at 600 nm. The signal was quantified simultaneously by a Hewlett Packard HP 3390A integrator (Avondale, PA, USA) directly connected to the detector.
TCDD/MCDF interactions in chick embryo hepatocytes Ah-receptor determination
After treatment o f the cells with 1 nM-[3H]TCDD for 2 hr, the cultured chick embryo hepatocytes were scraped from a 150-cm 2 flask and washed with phosphate buffered saline. The washed cell pellet was resuspended in 1.5 ml H E D buffer (25 mM-HEPES, 1.5 mM-EDTA and 1 mM-DL-DTT, pH 7.6) and then transferred to a 15-ml W h e a t o n homogenizing tube. Cells were homogenized by five full-burst strokes o f a teflon pestle. The h o m o g e n a t e was transferred to a 15-ml disposable polypropylene tube with an additional 1.5ml H E G D M ( H E D + I 0 % glycerol (v/v) + 20 mM-sodium molybdate). The homogenate was then centrifuged at 1000g for 10 min at 4°C. The resultant pellet was washed twice as described above with 10 ml H E G D M . The washed pellet was resuspended in 3 ml H E G D M buffer containing 0.5 M-KCI (pH 8.5) and homogenized by 12 full-burst strokes o f a teflon pestle. The h o m o g e n a t e was transferred to a 5-ml polycarbonate Beckman centrifuge tube and allowed to stand at 4°C for 1 hr. A non-specific binding baseline was obtained by incubating the cells with [3H]TCDD and a 200-fold excess o f 2,3,7,8tetrachlorodibenzofuran (TCDF). U n b o u n d and loosely b o u n d [3]TCDD was removed by adding the samples to dextran-coated charcoal [0.2 mg charc o a l : 0 . 0 2 m g dextran per 1 ml nuclear extract pelleted from H E G D ( H E D + 10% glycerol) buffer]. The dextran-coated charcoal was resuspended on a vortex mixer and the sample was incubated for 15 min at 4°C; the dextran-coated charcoal was then removed by centrifugation at 4000g for 10min at 2°C. Aliquots (300/~1) o f sample were layered onto linear sucrose gradients (5 ~ 25%) prepared in H E G buffer containing 0.4 M-KC1. Gradients were centrifuged at 2°C for 2.5 hr at 404,000 g. After centrifugation, 30 four-drop fractions were collected from each gradient and radioactivity in each fraction was determined by liquid scintillation counting. The procedure was repeated three times and the sedimentation coefficient was determined as a mean + SD.
375
Table 1. Dose-responseeffectsof TCDD as an inducer of AHH and EROD activities in chick embryo hepatocytesin ot,o TCDD conch (mol/egg)
AHH activity (pmol/mg/min)t
EROD activity (pmol/mg/min)t
0 (Control)~ 85.3 +- 7.6 90 + 8.0 TCDD (10 N) 1350 + 65.3** 513 +- 19.4"* TCDD (10 10) 1700 +- 56.6** 784 +- 33.0** TCDD (10 9) 1730 +-62.9** 1010 + 57.8** TCDD (10 s) 1840 +-98.8** 1140 +-70.6** TCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin AHH = aryl hydrocarbon hydroxylase EROD= ethoxyresorufin O-deethylase tEnzyme activities were measured after 48 hr of treatment. Results are expressed as means+ SD. Values marked with asterisks differ significantly (Student's t-test) from the corresponding value for the control group (**P < 0.01). :~Dimethyl sulphoxide(<0.4%). porphyria assays are expressed as means + SD and at least three determinations were carried out for each treatment group. RESULTS Table 1 summarizes the d o s e - r e s p o n s e effects o f T C D D on microsomal A H H and E R O D activities in chick embryo hepatocytes in ovo 48 hr after treatment. T C D D caused a dose-dependent induction o f both A H H and E R O D activities. T C D D was more effective as an inducer o f A H H activity than as an inducer o f E R O D activity in these cells. In chick embryo hepatocytes in culture, 19 hr after treatment T C D D caused significant induction of A H H and E R O D activities at all concentrations (10 -10, 10 -9, 10 -8, 10 -7 and 10-6M) and A H H activity was more readily induced than E R O D activity (Table 2). The induced A H H and E R O D activities both peaked at a T C D D concentration o f 10-9M. Moreover, at higher (>I0-9M) concentrations of T C D D , there was a dose-related reduction in A H H and E R O D activities that was due to cytotoxicity. Table 3 summarizes the interactive effects of T C D D with M C D F on the hepatic microsomal A H H and E R O D activities in chick embryo hepatocytes in ovo 48 hr after treatment. M C D F alone at concentrations o f 10 6, 10 7 and 10 8M significantly
In ovo treatment
After incubation o f the eggs at 37°C for 17 days, a hole (about 2 m m 2) was made with an electric engraver in the egg shell directly over the air sac. Test c o m p o u n d s in D M S O or appropriate solvent controls were injected onto the inner shell m e m b r a n e with a l-ml sterile syringe, and the hole was sealed with melted wax. After the treated eggs had been incubated for a further 48 hr, the embryos were assayed for hepatic microsomal enzyme activities as described above. At least six eggs were used per treatment group and the results are expressed as means + SD. Statistics
The differences between different treatments were determined using Student's t-test. The results from the chick embryo hepatocyte induction and
Table 2. Dos~response effectsof TCDD as an inducer of AHH and EROD activities in chick embryo hepatocytesin culture TCDD AHH activity~" EROD activityt concn (M) ( p m o l / m g / m i n ) (pmol/mg/min) 0 (Control):~ 10 l0 10 9 10 8 10 7 10-6
0 494+ 12.7"* 606 + 32.7** 334 + 70.5** 132+- 16.2"* 65.7 +- 7.9**
11.3 + 0.5 146+-2.7"* 248 +- 15.1"* 114 +- 22.6** 35.6+-4.0** 18.5 +- 1.6"*
TCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin AHH = aryl hydrocarbon hydroxylase EROD= ethoxyresorufinO-deethylase tEnzyme activitiesweremeasured after 19 hr of incubation (hepatocytes were treated with TCDD for 16 hr, then 150,UM-h-aminolaevulinic acid was added for a further 3-hr incubation). Some cytoxicity was observedin the cells treated with 10 7 or 10 6MTCDD. :~Dimethyl sulphoxide (<0.4%). Values are expressed as means_ SD, and those marked with asterisks differ significantly(Student's t-test) from the corresponding value for the control group (**P < 0.01).
376
C. YAO and S. SAFE Table 3. The interactive effects of TCDD EROD activities in chick Treatment (concm mol/egg) None (control)§ TCDD(10 10) MCDF (10.6) MCDF (10 7) MCDF(10 8) TCDD(10 ~0)+MCDF(10 6) TCDD (10 10) + MCDF (10 7) TCDD(10 10)+MCDF(10 8)
and MCDF on the induction of AHH and embryo hepatocytes in ovo AHH activityll EROD activityll (pmol/mg/min) (pmol/mg/min) 39.5 ± 9.7 0 2351+83.0"* 1530±108"* 417 _+5.0** 77.6 ± 5.7 234 ± 27.2** 14.0 ± 3.0 210_+ 11.6"* 0 2100- 131"*t 1270 _+70.8"'2 2470 _+ 103"* 1550 + 152 2530±193"* 1470±75.1
TCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin MCDF = 6-methyl-1,3,8-trichlorodibenzofuran AHH = aryl hydrocarbon hydroxylase EROD = ethoxyresorufin O-deethylase IIEnzyme activities were measured after 48 br of treatment. §Dimethyl sulphoxide (<0.4%). Values are means + SD, and those marked with superscripts differ significantly (Student's t-test) as follows: l,. the control group: **P < 0.01; v. the TCDD-treated group, t P <0.05; :~P < 0.01. i n d u c e d A H H activity. A t I0 6 a n d 10-7M, E R O D activity was also induced. T C D D (10 l°M) significantly i n c r e a s e d b o t h e n z y m e activities, a n d the increases were m u c h higher t h a n t h o s e o b s e r v e d at any o f the d o s e s o f M C D F . In the c o - t r e a t m e n t studies, M C D F at I 0 - 6 M significantly a n t a g o n i z e d the i n d u c t i o n o f A H H a n d E R O D activities c a u s e d by T C D D , w h e r e a s at lower c o n c e n t r a t i o n s (10 -7 a n d 10-SM) M C D F did n o t significantly alter these e n z y m e activities. In chick e m b r y o h e p a t o c y t e s in culture, 1 9 h r after t r e a t m e n t M C D F alone c a u s e d s o m e induction o f A H H a n d E R O D activities c o m p a r e d with c o n t r o l cells; T C D D at 10-1°M c a u s e d a significant increase in b o t h e n z y m e activities to levels m u c h g r e a t e r t h a n with M C D F a l o n e (Table 4). In the c o - t r e a t m e n t studies, M C D F at c o n c e n t r a t i o n s o f 10 -7 a n d 10 8 M significantly a n t a g o n i z e d the i n d u c t i o n o f A H H a n d E R O D activities c a u s e d by T C D D (10-10 M). T h e a n t a g o n i s m o f the i n d u c t i o n o f A H H a n d E R O D activities by M C D F was o b s e r v e d at T C D D : M C D F m o l a r ratios o f 1:1000 a n d 1:100, respectively. Fig. 1 illustrates the sucrose d e n s i t y g r a d i e n t profile o f n u c l e a r extracts f r o m chick e m b r y o h e p a t o c y t e s t r e a t e d with 1 nM[ 3 H ] T C D D for 2 h r in the p r e s e n c e o r a b s e n c e o f a 200-fold excess o f 2 , 3 , 7 , 8 - T C D F . T h e s e d i m e n t a t i o n
coefficient for the specifically b o u n d p e a k was 6.2 + 0.4 S. T a b l e 5 s u m m a r i z e s the c o n c e n t r a t i o n - d e p e n d e n t effects o f T C D D o n p o r p h y r i n levels in chick e m b r y o h e p a t o c y t e s in culture after 24 h r o f e x p o s u r e . A t all c o n c e n t r a t i o n s , TCDD caused significant elevation in u r o p o r p h y r i n [(COOH)8] a n d h e p t a c a r b o x y p o r p h y r i n [ ( c o o n ) 7 ] levels in a d o s e - d e p e n d e n t m a n n e r with a m a x i m a l 3.7-, 5.5-, 9.5- a n d 11.6-fold increase in u r o p o r p h y r i n levels a n d a m a x i m a l 4-, 4.8-, 6.5- a n d 7.3-fold increase in h e p t a c a r b o x y p o r p h y r i n levels at c o n c e n t r a t i o n s o f 10 -9, 10 8, 10 7 a n d 10-6M, respectively. A 3.9-, 5.1-, 7.8- a n d 9.2fold increase in ( C O O H ) 8 + ( C O O H ) 7 porphyrin levels was o b s e r v e d in cells t r e a t e d with T C D D c o n c e n t r a t i o n s o f 10 -9, 10 -8, 10 -7 a n d 10-6M, respectively. T h e r e were n o significant differences b e t w e e n any t r e a t m e n t g r o u p s (including the c o n t r o l g r o u p ) in the levels o f total p o r p h y r i n s . L o w levels o f the ( C O O H ) s + ( c o o n ) 7 p o r p h y r i n s were o b s e r v e d in the c o n t r o l g r o u p b u t the total p o r p h y r i n levels were similar to t h o s e in the T C D D t r e a t e d cells. In chick e m b r y o h e p a t o c y t e s in culture, 24 hr of t r e a t m e n t with M C D F a l o n e c a u s e d a significant increase in p o r p h y r i n levels with a 3.6- a n d 5.3-fold increase in u r o p o r p h y r i n , a 36- a n d 47.2-fold increase
Table 4. The interactive effects of TCDD and MCDF on the induction of AHH and EROD activities in chick embryo bepatocytes in culture Treatment AHH activity3" EROD activityt (concn, M) (pmol/mg/min) (pmol/mg/min) None (control)§ 40.5 ± 3.1 11.5 + 1.0 TCDD (10 to) 583 ± 13.6"* 249 ± 21.0"* MCDF(10 7) 98.2_+ 18.0"* 13.0_+ 1.4" MCDF (10 s) 59.7 + 5.0** 10.7 ± 2.1 * TCDD (10-to) + MCDF (10 7) 289 + 22.3"*~: 175 ± 9.5**5 TCDD (10 ~0) + MCDF (10 s ) 369 ± 16.5"*~ 193 + 11.3"*:~ TCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin MCDF = 6-methyl-1,3,8-trichlorodibenzofuran AHH = aryl hydrocarbon hydroxylase EROD = ethoxyresorufin O-deethylase tEnzyme activities were measured after 19 hr of incubation (hepatocytes were treated with the test chemicals or mixtures for 16 hr and then 150/~r,i-f-aminolaevulinic acid was added for a further 3-hr incubation). §Dimethyl sulphoxide (<0.4%). Values are means _+SD, and those marked with superscripts differ significantly (Student's t-test) as follows: v. the control group, *P < 0.05; **P < 0.01; v. the TCDD-treated group, ~P < 0.01.
TCDD/MCDF interactions in chick embryo hepatocytes
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Fig. 1. Sucrose density gradient elution profile of nuclear extracts from chick embryo hepatocytes treated with 1 nM-[3H]TCDD (ll) or l nM-[3H]TCDD plus a 200-fold excess of 2,3,7,8-TCDF (O). The sedimentation coefficient for the specifically-bound peak (i.e. total-non-specific binding) was 6.2 __+0.4 S (three determinations) and the level of the nuclear Ah-receptor complex was 37 fmol/mg protein. The experimental details are summarized in Materials and Methods. in heptacarboxyporphyrin, and a 3.3- and 4.8-fold increase in (COOH)8 + (COOH)7 porphyrin levels at concentrations o f 10 -6 and 10-SM, respectively (Table 6). T C D D (10-SM) caused significant increases in all observed porphyrin levels [4.5-, 41.2and 3.9-fold increase for (COOH)8, (COOH)7 and (COOH)8 + (COOH)7 porphyrin levels, respectively]. The TCl~D-induced increase in porphyrin levels was greater than that observed for M C D F at 10 -6 M. C o - t r e a t m e n t o f the cells with T C D D (10 -8 M) and M C D F (10 -6 M) resulted in higher levels o f (COOH)8, ( c o o n ) 7 and (COOH) s + (COOH)7 porphyrins than those observed after treatment with T C D D (10 -8 M) or M C D F (10 -6 M) alone. The porphyrin patterns in the cells co-treated with T C D D and M C D F were comparable with the results observed after treatment with T C D D alone. The co-treatment studies with various concentrations o f M C D F and T C D D resuited in a non-additive elevation o f porphyrin levels,
and it was a p p a r e n t that M C D F was a moderately active porphyrinogen in chick embryo hepatocytes in culture. In a second study on the interactive effects o f T C D D (10 -8 and 10 -7 M) and M C D F (10 -5 M) on porphyrin levels in chick embryo hepatocytes in culture (Table 7), M C D F (10 -5 M) alone caused 1.7-, 9.5- and 3.6-fold increases in (COOH)8, (COOH)7 and ( c o o n ) 8 + (COOH)7 porphyrin levels, respectively. T C D D caused significant increases in all observed porphyrin levels with 3.1- and 4.7-fold increases for (COOH)8, 15.6- and 20.5-fold increases for (COOH)7, and 6.2and 8.6-fold increases in the ( C O O H ) s + ( C O O H ) 7 porphyrin levels at concentrations o f 10 -8 M and 10 -7 M, respectively. Co-treatment o f the cells with T C D D (10 -8 M) and M C D F (10-SM) resulted in (COOH)8, (COOH)7 and ( C O O H ) 8 + ( C O O H ) 7 porphyrin levels that were higher than those observed after treatment with
Table 5. Dose-responseeffectsof TCDD on porphyrin levels in chick embryo hepatocytesin culture Porphyrin levels (pmol/dish)t:~ TCDD (M) 0 (control)~ ]0 -9 10-8 10-7 10-6
concn
(COOH)8 90.5 + 17.0(3.9) 333 + 23.2**(18.6) 495 + 21.2"* (31.6) 864 _.+77.1"* (41.7) 1050 ___35.7**(44.6)
(COOH)7 121 _+ 11.7(5.3) 483 _+30.2**(26.9) 584 ± 31.7**(37.3) 789 _+44.0** (38.1) 885 + 107"*(37.5)
(COOH)8 + (COOH)7 211 _+28.3 (9.2) 816 _+52.5**(45.5) 1080 + 52.5**(68.9) 1650 ± 117"* (79.8) 1940 +_ 139"* (82.1)
Total 2280 + 74.7 1790 + 69.0 1570 + 29.7 2070 + 104 2360 + 175
TCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin tPorphyrin levelsweredeterminedafter 24 hr of incubation (hepatocytesweretreated with the test chemicalsfor 16 hr, then 150 ~ M-t~-aminolaevulinic acid was added for a further 8-hr incubation). :~Values in parentheses are as a percentageof total porphyrin. §Dimetbyl sulphoxide ( < 0.4%) Values are means + SD and those marked with asterisks differ significantly(Student's t-test) from the correspondingvalue for the control group (**P < 0.01).
378
C. YAO a n d S. SAFE Table 6. The interactive effects of TCDD and M C D F on porphyrin levels in chick embryo hepatocytes in culture Porphyrin levels (pmol/dish)'t:~
Treatment (concn, M) None (control)§ T C D D (10 5) M C D F ( 1 0 6) M C D F (10 5) TCDD(10 S)+MCDF(10 TCDD(10 S)+MCDF(10
5) 5)
(COOH) 8
(COOH)7
(COOH) 8 h- (COOH) 7
83.5 + 46.3 (3.3) 383 + 127" (22.7) 297+41.5'*(11.8) 437 ± 42.0"* (24.8) 424±43.4"*(27.1) 524±69.1"*(31.2)
16.9 ± 19.2 (7.8) 696 ± 184"* (41.2) 610±42.3"*(24.2) 800 ± 56.1"* (45.5) 767±66.8**(49.0) 847±87.l**(50.4)
278 ± 47.1 (11.1) 1080 _+ 310"* (63.9) 906+81.8"*(36) 1330±92.8"*(70.3) 1190± 106"*(76.1) 1370± 152"*(81.6)
Total 2500 ± 864 1690 ± 75.3 2520_+ 138"* 1760_+152"* 1570+ 106 1680± 150
T C D D = 2,3,7,8-tetrachlorodibenzo-p-dioxin M C D F = 6-methyl-l,3,8-trichlorodibenzofuran tPorphyrin levels were determined after 24 hr of incubation (hepatocytes were treated with the test chemicals for 16 hr, then 150/~ M-6-aminolaevulinic acid was added for a further 8-hr incubation). ++Values in parentheses are as a percentage of total porphyrin. §Dimethyl sulphoxide ( < 0.4%). Values are means ± SD, and those marked with asterisks differ significantly (Student's t-test) from the corresponding values for the control group (*P <0.05; **P < 0.01).
Table 7. The interactive effects of T C D D and M C D F on porphyrin levels in chick embryo hepatocytes in culture Porphyrin levels (pmol/dish)% Treatment (concn, M)
(COOH)~
None (control)§ T C D D ( 1 0 8) T C D D ( 1 0 7) M C D F (10 5) TCDD(10 8)+MCDF(10 TCDD(10 7)+MCDF(10
5) 5)
132 ± 12.6 (5.1) 4 0 7 ± 148"*(26.8) 6 2 5 + 152"*(33.1) 220 ± 69.0* (12.2) 469±80.1"*(26.0) 685±60.5"*(30.9)
(COOH) 7 43.4 _+ 87.2 (1.7) 673±241"*(44.3) 882±183"*(46.8) 414 ± 84.2** (22.9) 734±130"*(45.7) 968±257"(43.7)
(COOH)8 + (COOH) 7 176 ± 94.6 (6.8) 1080±386"*(71.1) 1510±332"*(79.9) 634 ± 143"* (35.1) 1200±206"*(71.7) 1650±317"*(74.6)
Total 2610 ± 60.1 1520±638" 1890±327 1810 ± 752** 1760±317' 2220_+388
T C D D = 2,3,7,8-tetrachlorodibenzo-p-dioxin M C D F = 6-methyl-l,3,8-trichlorodibenzofuran fPorphyrin levels were determined after 24 hr of incubation (hepatocytes were treated with the test chemicals for 16 hr, then 150 p M6-aminolaevulinic acid was added for a further 8-hr incubation). ~.Values in parentheses are as a percentage of total porphyrin. §Dimethyl sulphoxide (<0.4%). Values are means _+ SD, and those marked with asterisks differ significantly (Student's t-test) from the corresponding values for the control group (*P <0.05; **P < 0.01).
T C D D (10 -8 M) or M C D F (10 -5 M) alone. Comparable results were observed after co-treatment of the cells with 10 7M-TCDD and 10-SM-MCDF. The results obtained for the cells treated with M C D F plus T C D D (Tables 6 and 7) are approximately additive if the porphyrin values for the control cells are subtracted from those obtained for the different treatment groups. DISCUSSION
The results summarized in Tables 1 and 2 demonstrate that T C D D caused a concentration-dependent induction of hepatic microsomal A H H and E R O D activities in chick embryo hepatocytes in o v o and in culture. Maximum induction of both enzyme activities was observed at concentrations as low as 10 9M and 10 -8 mol/egg for hepatocytes in culture and in ovo, respectively, and significant induction was observed in o v o at a concentration of 10- ~ mol/egg. In the cultured cells there was a decrease in the induced enzyme activities at concentrations greater than 10 9M, and this was probably due to cytotoxicity. These data demonstrate that the chick embryo hepatocytes were highly Ah-responsive, and this was consistent with the identification of the nuclear Ah-receptor in the cultured hepatocytes (Fig. 1). The sedimentation coefficient for this receptor was 6.2_+ 0.4S and the level of the Ah-receptor was 37 fmol/mg protein. These values are similar to those
obtained from many other Ah-responsive cell lines (Safe, 1988). The effects of M C D F and TCDD-induced A H H and E R O D activities was also determined in chick embryo hepatocytes in o v o and in culture. On the basis of the results obtained for the enzyme induction activity of T C D D alone (Table 1), a dose of 10 -~° mol/egg was chosen for the interactive studies. Treatment of the eggs with M C D F alone showed that this compound was significantly less active than T C D D as an inducer of A H H and E R O D activity. In the co-treatment studies, M C D F significantly inhibited the induction of A H H and EROD activity by TCDD. However, this partial antagonism was observed only at the highest dose of M C D F (10-6mol/egg), and the induction activity in the co-treated eggs (TCDD plus MCDF) was only 20% lower than that observed in the eggs treated with T C D D (10-J°mol/egg) alone. In contrast, co-treatment of the cultured hepatocytes with 10 ~0M-TCDD and M C D F resulted in a 50 and 37% decrease in A H H induction, at 10 7 and 10 8 M-MCDF, respectively. Interactions of similar magnitude have been observed in rat hepatic microsomes and in rat hepatoma H-4-II E cells (Astroff e t al., 1988). Chick embryo hepatocytes in culture can be used to determine the effects of T C D D antagonists on two responses mediated by the Ah-receptor, namely the induction of A H H and EROD activities and porphyria. The latter response is characterized by the
TCDD/MCDF interactions in chick embryo hepatocytes accumulation of (COOH) 7 and (COOH)8 porphyrins. It was apparent that T C D D concentrations greater t h a n 10-6M were required for the accumulation of maximum levels of the (COOH)~ + (COOH)7 porphyrins (Tables 5-7). In contrast, maximum induction of A H H activity by T C D D was observed at concentrations of 10 9M (Table 2). Similar results have been observed in C57BL/6 mice, in which the doses of T C D D that are required to cause porphyria are higher than those required for enzyme induction (Yao and Safe, 1989). M C D F only weakly induced A H H and E R O D activity in chick embryo hepatocytes at concentrations of 10 -7 or 10-SM (Table 4), whereas at a 10 6M, M C D F caused significant accumulation of (COOH)8 and (COOH)7 porphyrins in chick embryo hepatocytes (Table 6). The results in Tables 5-7 show that M C D F is only 100 to 1000 times less potent than T C D D as a porphyrinogen. In contrast, the T C D D : M C D F potency ratios were more than l 0 4 t o 10 5 for most other Ah-receptor-mediated toxic responses, including porphyrinogenicity in C57BL/6 mice (Astroff et al., 1988; Bannister et al., 1989; Harris et al., 1989; Yao and Safe, 1989). The apparent inversion of the expected relative potencies of M C D F as a monooxygenase enzyme inducer and as a porphyrinogen was unexpected, and the reasons for these potency differences are unknown. However, because of the unexpectedly high porphyrogenicity of M C D F in cultured chick embryo hepatocytes, no antagonism of T C D D (10 8 and 10 7M)-induced porphyria was observed and it is unlikely that antagonism would be observed at lower concentrations of TCDD. Thus, chick embryo hepatocytes show promise as useful models for investigating the molecular mechanisms associated with partial antagonism of T C D D induced C Y P I A 1 gene expression. Current studies are focused on the development of new antagonists that can also be used to probe the mechanisms associated with the development of porphyria in chick embryo hepatocytes. Acknowledgements--This work was supported by the National Institutes of Health (ES03843 and ES03554) and the Texas Agricultural Experiment Station. S. Safe is a Burroughs Wellcome Toxicology Scholar. REFERENCES
Astroff B. and Safe S. (1988) Comparative antiestrogenic activities of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 6methyl-l,3,8-trichlorodibenzofuran in the female rat. Toxicology and Applied Pharmacology 95, 435-443. Astroff B., Zacharewski T., Safe S., Arlotto M. P., Parkinson A., Thomas P. and Levin W. (1988) 6-Methyl1,3,8-trichlorodibenzofuran as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist: inhibition of the induction of rat cytochrome P-450 isozymes and related monooxygenase activities. Molecular Pharmacology 33, 231 236. Bannister R., Biegel L., Davis D., Astroff B. and Safe S. (1989) 6-Methyl-l,3,8-trichlorodibenzofuran (MCDF) as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist in C57BL/6 mice. Toxicology 54, 139-150.
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Bannister R., Davis D., Zacharewski T., Tizard I. and Safe S. (1987) Aroclor 1254 as a 2,3,7,8-tetrachlorodibenzo-pdioxin antagonist: effects on enzyme induction and immunotoxicity. Toxicology 46, 29-42. Blank J. A., Tucker A. N., Sweatlock J., Gasiewicz T. A. and Luster M. I. (1987) ~-Naphthoflavone antagonism of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced murine lymphocyte ethoxyresorufin O-deethylase activity and immunosuppression. Molecular Pharmacology 32, 168-172. Davis D. and Safe S. (1990) Immunosuppressive activities of polychlorinated biphenyls in C57BL/6 mice: structure-activity relationships as Ah receptor agonists and partial antagonists. Toxicology 63, 97-111. Denison M. S., Okey A. B., Hamilton J. W., Bloom S. E. and Wilkinson C. S. (1986) Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin: ontogeny in chick embryo liver. Journal of Biochemical Toxicology 1, 39-49. Ford R. E., Ou C. N. and Ellefson R. D. (1981) Liquid chromatographic analysis for urinary porphyrins. Clinical Chemistry 27, 397-401. Harris M., Zacharewski T., Astroff A. and Safe S. (1989) Partial antagonism of 2,3,7,8-tetrachlorodibenzo-pdioxin-mediated induction of aryl hydrocarbon hydroxylase by a 6-methyl-l,3,8-trichlordibenzofuran: mechanistic studies. Molecular Pharmacology 35, 729 735. Jordan V. C. and Koch R. (1989) Regulation of prolactin synthesis in vitro by estrogenic and antiestrogenic derivatives of estradiol and estrone. Endocrinology 124, 1717--1726. Jordan V. C. and Murphy C. S. (1990) Endocrine pharmacology of antiestrogens. Endocrine Review 11, 578-610. Keyes B., Piskorska-Pliszczynska J. and Safe S. (1986) Polychlorinated dibenzofurans as 2,3,7,8-TCDD antagonists in vitro inhibition of monooxygenase induction. Toxicology Letters 31, 151 158. Lambrecht R. W., Sinclair P. R., Bement W. J. and Sinclair J. F. (1988b) Uroporphyrin accumulation in cultured chick embryo hepatocytes: comparison of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3,Y,4,4'-tetrachlorobiphenyl. Toxicology and Applied Pharmacology 96, 507 516. Lambrecht R. W., Sinclair P. R., Bement W. J., Sinclair J. F., Carpenter H. M., Buhler D. R., Urquart A. J. and Elder G. H. (1988a) Hepatic uroporphyrin accumulation and uroporphyrinogen decarboxylase activity in cultured chick-embryo hepatocytes and in Japanese quail (Coturnix coturnix japonica) and mice treated with polyhalogenated aromatic compounds. Biochemical Journal 253, 131 138. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265-275. Luster M. I., Hong L. I., Osborne R., Blank J. A., Clark G., Silver M. T., Boorman G. A. and Greenley W. F. (1986) l-Amino-3,7,8-trichlorodibenzo-p-dioxin: a specific antagonist for TCDD-induced myelotoxicity. Biochemical and Biophysical Research Communications 139, 747-756. Merchant M., Arellano L. and Safe S. (1990) The mechanism of action of c~-naphthoflavone as an inhibitor of 2,3,7,8-tetrachlorodibenzo-p-dioxin induced CYP1A1 gene expression. Archives of Biochemistry and Biophysics 281, 84-89. Nebert D. W. and Gelboin H. V. (1968) Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture: assay and properties of induced enzyme. Journal oJ Biological Chemistry 242, 6242-6249. Pohl R. J. and Fouts R. J. (1980) A rapid method for assaying the metabolism of 7-ethoxyresorufin by microsomal subcellular fractions. Analytical Biochemistry 107, 150-155.
Poland A. and Knutson J. C. (1982) 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons. Examination of the mechanism of toxicity.
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Annual Review of Pharmacology and Toxicology 22, 517-554. Rifkind A. B., Firpo A. and Alonso D. R. (1984) Coordinate induction of cytochrome P-448-mediated mixed function oxidase and histopathologic changes produced acutely in chick embryo liver by polychlorinated biphenyl congeners. Toxicology and Applied Pharmacology 72, 343 354. Safe S. H. (1986) Comparative toxicology and mechanism of action of polychlorinated dibenzo-p-dioxins and dibenzofurans. Annual Review of Pharmacology and Toxicology 26, 371-399. Safe S. H. (1988) The aryl hydrocarbon receptor. ISI Atlas of Science. Pharmacology 2, 78 83. Sassa S., Deverneuil H. and Kappas A. (1984) Inhibition of uroporphyrinogen decarboxytase activity in polyhalogenated aromatic hydrocarbon poisoning. In Biological Mechanism of Dioxin Action. Edited by A. Poland and R. D. Kimbrough, Banbury Report No. 18. pp. 215-224. Cold Spring Harbor Press, Cold Spring Harbor, NY. Sassa S. and Kappas A. (1977) Induction of 6-aminolevulinate synthase and porphyrins in cultured liver cells maintained in chemically defined medium. Journal of Biological Chemistry 252, 2428-2436. Sinclair J. F., Sinclair P. R. and Bonkowsky H. L. (1979) Hormonal requirements for the induction of cytochrome
P450 in hepatocytes cultured in a serum-free medium. Biochemical and Biophysical Research Communications 86, 710-717. Sinclair J. F., Sinclair P. R., Healey J. F., Smith E. L. and Bonkowsky H. L. (1982) Decrease in hepatic cytochrome P-450 by cobalt. Evidence for a role of cobalt protoporphyrin. Biochemical Journal 204, 103 109. Whitlock J. P. (1986) The regulation of cytochrome P-450 gene expression. Annual Review of Pharmacology and Toxicology 26, 333 369. Whitlock J. P. (1987) The regulation of gene expression of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Pharmacology Reviews 39, 147 161. Woods J. S., Eaton D. L. and Lukens C. B. (1984) Studies on porphyrin metabolism in the kidney: effects of trace metals and glutathione on renal uroporphyrinogen decarboxylase. Molecular Pharmacology 26, 336 341. Yao C., Panigrahy B. and Safe S. (1990) Utilization of cultured chick embryo hepatocytes as in vitro bioassays for polychlorinated biphenyls (PCBs): quantitative structure-induction relationships. Chemosphere 21, 1007 1016. Yao C. and Safe S. (1989) 2,3,7,8-Tetrachlorodibenzo-pdioxin-induced porphyria in genetically inbred mice: partial antagonism and mechanistic studies. Toxicology and Applied Pharmacology 100, 208 216.
0887-2333/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
THE INTERACTIONS OF 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN A N D 6-METHYL-1,3,8-TRICHLORODIBENZOFURAN IN CHICK EMBRYO HEPATOCYTES C. YAO and S. SAVE* Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4466, USA (Received 11 October 1991; revisions received 11 December 1991)
A~tract--Treatment of chick embryo hepatocytes in ovo and in culture with 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) resulted in a dose-dependent induction of microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities. Significant induction was observed in the hepatocytes at TCDD doses as low as 10-" mol/egg (in ovo) and 10-~°M (in culture). In contrast, 6-methyl-l,3,8-trichlorodibenzofuran (MCDF) was a relatively weak inducer of these activities and only 10-20% of the induction responses observed for TCDD were elicited by MCDF at doses of 10 -6 mol/egg o r 1 0 - 7 M in culture. Co-treatment of the chick embryo hepatocytes with TCDD (10 ~0mol/egg in ovo; 10-~°M in culture) and different concentrations of MCDF (10 -6 to 10 Smol/egg in ovo and 10-7 and 10 -s M in culture) resulted in minimal inhibition of TCDD-induced enzyme activities in ovo and a 37 to 50% inhibition in culture. The partial antagonist activity of MCDF in the chick embryo hepatocytes in culture paralleled the interactive effects previously reported in rodent liver and transformed rodent cell lines. TCDD (10 -7 to 10 -I° M) also caused an accumulation of hepta- and octacarboxyporphyrins in chick embryo hepatocytes (10 -7 to 10 -9 M); however, MCDF (10 -6 and 10-5 M) elicited similar responses and MCDF did not significantly decrease the TCDD-induced porphyrogenic response in these cells. These results suggest that chick embryo hepatocytes in culture will serve as a useful model for investigating TCDD-induced gene transcription and the effects and mechanism of action of antagonists.
INTRODUCTION
2,3,7,8-Tetrachlorodibenzo-p-dioxin ( T C D D ) has been used as a prototype to investigate the effects and mechanism of action of the toxic halogenated aryl hydrocarbons. T C D D and related compounds elicit a broad spectrum of toxic and biochemical responses including a wasting syndrome, thymic atrophy, hepatotoxicity and porphyria, endocrine effects, developmental and reproductive toxicity, dermal-toxicity, and the induction of both phase I and II drugmetabolizing enzymes (Poland and Knutson, 1982; Safe, 1986; Whitlock, 1986 and 1987). The effects caused by T C D D and related compounds are dependent on a number of factors including the age, sex, species and strain of the test animal. The molecular mechanism of TCDD-induced cytochrome P4501A1 (CYP1A1) gene expression has been extensively investigated as a model for under*To whom all correspondence should be addressed. AHH = aryl hydrocarbon hydroxylase; ALA = 6-aminolaevulinic acid; BSA = bovine serum albumin; oL-DTT = DL-dithiothreitol; DMSO = dimethyl sulphoxide; EDTA=ethylenediaminetetraacetic acid; E R O D = ethoxyresorufin O-deethylase; HBSS = Hanks' balanced salt solution; MCDF = 6-methyl-l,3,8trichlorodibenzofuran; TCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDF = 2,3,7,8-tetrachlorodibenzofuran.
Abbreviations:
standing the other TCDD-induced toxic and biochemical responses. The results support the initial binding of T C D D to a cytosolic (aryl hydrocarbon, Ah) receptor that accumulates in the nucleus of target cells and acts as a transcriptional enhancer (reviewed in Whitlock, 1986 and 1987). Recent studies have identified several compounds, including substituted dibenzofurans and dibenzo-pdioxins, polychlorinated biphenyls and ~t-naphthoflavone, that inhibit several TCDD-induced responses (Astroff et al., 1988; Bannister et al., 1987 and 1989; Blank et al., 1987; Davis and Safe, 1990; Harris et al., 1989; Keyes et al., 1986; Luster et al., 1986; Merchant et al., 1990; Yao and Safe, 1989). One of these compounds, 6-methyl- 1,3,8-trichlorodibenzofuran ( M C D F ) , partially inhibited several TCDD-induced responses including immunotoxicity, teratogenicity, porphyria and C Y P 1 A I induction (Astroff et al., 1988; Bannister et al., 1989; Yao and Safe, 1989). In contrast, like T C D D , M C D F was moderately active as an anti-oestrogen in the female rat uterus and in human breast cancer cells in culture (Astroff and Safe, 1988). Thus, M C D F exhibited both partial agonist and partial antagonist activities, and this behaviour is similar to that of the clinically used antioestrogen tamoxifen, which elicits both oestrogen and anti-oestrogen responses in laboratory animals (Jordan and Koch, 1989; Jordan and Murphy, 1990). 373
374
C. YAO and S. SAFE
This study reports the interactive effects of M C D F and TCDD in chick embryo hepatocytes in ovo and in culture. The chick embryos are Ah-responsive and are a useful model for investigating the mechanisms of at least two responses, namely the halogenated aromatic hydrocarbon-induced CYP1AI gene expression and porphyria (Denison et al., 1986; Lambrecht et al., 1988a,b; Rifkind et al., 1984; Sassa et al., 1984; Yao et al., 1990).
MATERIALS A N D M E T H O D S
Chemicals and biochemicals
TCDD and M C D F have previously been prepared in this laboratory ( > 9 8 % purity). Benzo[a]pyrene, DL-dithiothreitol (oL-DTT), N-2-hydroxythylpiperazine-N'-2-ethanesulphonic acid (HEPES), bovine serum albumin (BSA), Tris, ammonium chloride, ethylenediaminetetraacetic acid (EDTA, disodium salt), 6-aminolaevulinic acid (ALA) and Folin-Ciocalteu's phenol reagent were purchased from Sigma Chemical Co. (St Louis, MO, USA). Trypsin, insulin, 3,3',5-triiodo-e-thyronine (sodium salt), collagenase and hyaluronidase of tissue culture grade were also purchased from Sigma Chemical Co. Penicillin and streptomycin sulphate were purchased from Pfizer Co. (New York. NY, USA) and Eli Lilly and Co. (Indianapolis, IN, USA), respectively. Williams' medium E was purchased from either Hazelton (Lenaxa, KS, USA) or Gibco (Grand Island, NY, USA). Antiseptic Zephiran chloride was obtained from Winthrop Laboratories (New York, NY, USA). N A D P H and N A D H were purchased from United States Biochemical Corp. (Cleveland, OH, USA). Resorufin was purchased from Eastman Kodak Co. (Rochester, NY, USA) and ethoxyresorufin was prepared in this laboratory. Porphyrin standards were purchased from Porphyrin Products (Logan, UT, USA). Isolation o f chick embryo hepatocytes Incubation of eggs. Primary cultures of hepatocytes from 16- to 18-day-old white leghorn chick embryos were prepared and maintained in Williams' medium E as described by Sinclair et al. (1982). Eggs were incubated at 37°C (85% humidity) for 16 to 18 days in a Petersime model no. 5 incubator (Gettysberg, OH, USA) with rotation every hour. Preparation o f hepatocytes and culture conditions.
Incubated eggs were candled, marked, and sterilized with antiseptic Zephiran chloride before the embryos were removed. The livers were dissected from the embryos, pooled, and washed twice in Hanks' balanced salt solution (HBSS) without calcium and magnesium. Washed livers were minced into small pieces (about 2 mm 2) and digested for 30 min with 0.066% trypsin in HBSS containing penicillin and streptomycin. Contaminating red blood cells were lysed and removed as described by Sassa and Kappas
(1977). The resulting hepatocytes were resuspended in 200 volumes of serum-free Williams' medium E (WEHI) containing insulin (1 # g/ml), dexamethasone (0.3/~g/ml) and triiodothyonine (1/~g/ml) as described by Sinclair et al. (1979). Cells were cultured in an incubator at 37°C with a humidified air:CO2 (95 : 5) atmosphere. Treatments and enzyme induction. After incubation for 24 hr, the medium was changed to serum-free Williams medium E containing dexamethasone and triiodothyronine (WEH) but no insulin. Cells were cultured in 25-cm 2 flasks. The test compounds in dimethyl sulphoxide (DMSO) were added to the culture media ( < 0 . 4 % DMSO in all experiments), and the induction of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD) activities were determined after 19 hr as described (Yao et al., 1990) using fluorescence assays (Nebert and Gelboin, 1968; Pohl and Fouts, 1980). 3 hr before harvesting the cells, ALA was added to the media at 150/~M (final concentration). At the end of incubation, the cells were rinsed twice with phosphate buffered saline (0.90% NaCI, pH 7.4), scraped from the flasks, resuspended in Tris-sucrose buffer and assayed for enzyme activity. Protein concentrations were determined by the method of Lowry et al. (1951) using BSA as a standard. Porphyria studies
For porphyria studies, cells were treated with the test chemicals for 24 hr. 6 hr before harvesting the cells, ALA (150/~M) was added to the media. The cells were removed from the culture flask in the culture media, sonicated for 5 sec and 2 ml of the sonicate was added to an equal volume of acetoneconcentrated HC1 (97.5:2.5, v/v). The mixture was vortexed for 30sec and centrifuged at 1000g for 10 min at 4°C. Aliquots (40-50/d) of the supernatant were injected into a Beckman (Berkeley, CA, USA) HPLC equipped with two pumps and model 420 controller. Porphyrin peaks were quantified by interpolation of the peak heights with the peak heights from standard curves. The chromatographic conditions of Woods et al. (1984) and Ford et al. (1981) were adopted. These included two solvents, 0.05 M-phosphate-methanol buffer (pH 3.5) designated as solvent A and HPLC-grade methanol designated as solvent B. A flow rate of 1.0 ml/min was used. The elution gradient used was as follows (ratios are for B to A): 1:9 at time 0; 1 : 1 at the end of 3 min; 7 : 3 at the end of 10 min; 99 : 1 from 15 to 23 min. The HPLC column was allowed to re-equilibrate at a B to A ratio of 1:9 for 10min before the next injection. The individual porphyrins were detected by an interfaced LDC Fluoromonitor model 1311 (Riviera Beach, FL, USA) with the excitation filter set at 410nm and an emission filter with a cut-off at 600 nm. The signal was quantified simultaneously by a Hewlett Packard HP 3390A integrator (Avondale, PA, USA) directly connected to the detector.
TCDD/MCDF interactions in chick embryo hepatocytes Ah-receptor determination
After treatment o f the cells with 1 nM-[3H]TCDD for 2 hr, the cultured chick embryo hepatocytes were scraped from a 150-cm 2 flask and washed with phosphate buffered saline. The washed cell pellet was resuspended in 1.5 ml H E D buffer (25 mM-HEPES, 1.5 mM-EDTA and 1 mM-DL-DTT, pH 7.6) and then transferred to a 15-ml W h e a t o n homogenizing tube. Cells were homogenized by five full-burst strokes o f a teflon pestle. The h o m o g e n a t e was transferred to a 15-ml disposable polypropylene tube with an additional 1.5ml H E G D M ( H E D + I 0 % glycerol (v/v) + 20 mM-sodium molybdate). The homogenate was then centrifuged at 1000g for 10 min at 4°C. The resultant pellet was washed twice as described above with 10 ml H E G D M . The washed pellet was resuspended in 3 ml H E G D M buffer containing 0.5 M-KCI (pH 8.5) and homogenized by 12 full-burst strokes o f a teflon pestle. The h o m o g e n a t e was transferred to a 5-ml polycarbonate Beckman centrifuge tube and allowed to stand at 4°C for 1 hr. A non-specific binding baseline was obtained by incubating the cells with [3H]TCDD and a 200-fold excess o f 2,3,7,8tetrachlorodibenzofuran (TCDF). U n b o u n d and loosely b o u n d [3]TCDD was removed by adding the samples to dextran-coated charcoal [0.2 mg charc o a l : 0 . 0 2 m g dextran per 1 ml nuclear extract pelleted from H E G D ( H E D + 10% glycerol) buffer]. The dextran-coated charcoal was resuspended on a vortex mixer and the sample was incubated for 15 min at 4°C; the dextran-coated charcoal was then removed by centrifugation at 4000g for 10min at 2°C. Aliquots (300/~1) o f sample were layered onto linear sucrose gradients (5 ~ 25%) prepared in H E G buffer containing 0.4 M-KC1. Gradients were centrifuged at 2°C for 2.5 hr at 404,000 g. After centrifugation, 30 four-drop fractions were collected from each gradient and radioactivity in each fraction was determined by liquid scintillation counting. The procedure was repeated three times and the sedimentation coefficient was determined as a mean + SD.
375
Table 1. Dose-responseeffectsof TCDD as an inducer of AHH and EROD activities in chick embryo hepatocytesin ot,o TCDD conch (mol/egg)
AHH activity (pmol/mg/min)t
EROD activity (pmol/mg/min)t
0 (Control)~ 85.3 +- 7.6 90 + 8.0 TCDD (10 N) 1350 + 65.3** 513 +- 19.4"* TCDD (10 10) 1700 +- 56.6** 784 +- 33.0** TCDD (10 9) 1730 +-62.9** 1010 + 57.8** TCDD (10 s) 1840 +-98.8** 1140 +-70.6** TCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin AHH = aryl hydrocarbon hydroxylase EROD= ethoxyresorufin O-deethylase tEnzyme activities were measured after 48 hr of treatment. Results are expressed as means+ SD. Values marked with asterisks differ significantly (Student's t-test) from the corresponding value for the control group (**P < 0.01). :~Dimethyl sulphoxide(<0.4%). porphyria assays are expressed as means + SD and at least three determinations were carried out for each treatment group. RESULTS Table 1 summarizes the d o s e - r e s p o n s e effects o f T C D D on microsomal A H H and E R O D activities in chick embryo hepatocytes in ovo 48 hr after treatment. T C D D caused a dose-dependent induction o f both A H H and E R O D activities. T C D D was more effective as an inducer o f A H H activity than as an inducer o f E R O D activity in these cells. In chick embryo hepatocytes in culture, 19 hr after treatment T C D D caused significant induction of A H H and E R O D activities at all concentrations (10 -10, 10 -9, 10 -8, 10 -7 and 10-6M) and A H H activity was more readily induced than E R O D activity (Table 2). The induced A H H and E R O D activities both peaked at a T C D D concentration o f 10-9M. Moreover, at higher (>I0-9M) concentrations of T C D D , there was a dose-related reduction in A H H and E R O D activities that was due to cytotoxicity. Table 3 summarizes the interactive effects of T C D D with M C D F on the hepatic microsomal A H H and E R O D activities in chick embryo hepatocytes in ovo 48 hr after treatment. M C D F alone at concentrations o f 10 6, 10 7 and 10 8M significantly
In ovo treatment
After incubation o f the eggs at 37°C for 17 days, a hole (about 2 m m 2) was made with an electric engraver in the egg shell directly over the air sac. Test c o m p o u n d s in D M S O or appropriate solvent controls were injected onto the inner shell m e m b r a n e with a l-ml sterile syringe, and the hole was sealed with melted wax. After the treated eggs had been incubated for a further 48 hr, the embryos were assayed for hepatic microsomal enzyme activities as described above. At least six eggs were used per treatment group and the results are expressed as means + SD. Statistics
The differences between different treatments were determined using Student's t-test. The results from the chick embryo hepatocyte induction and
Table 2. Dos~response effectsof TCDD as an inducer of AHH and EROD activities in chick embryo hepatocytesin culture TCDD AHH activity~" EROD activityt concn (M) ( p m o l / m g / m i n ) (pmol/mg/min) 0 (Control):~ 10 l0 10 9 10 8 10 7 10-6
0 494+ 12.7"* 606 + 32.7** 334 + 70.5** 132+- 16.2"* 65.7 +- 7.9**
11.3 + 0.5 146+-2.7"* 248 +- 15.1"* 114 +- 22.6** 35.6+-4.0** 18.5 +- 1.6"*
TCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin AHH = aryl hydrocarbon hydroxylase EROD= ethoxyresorufinO-deethylase tEnzyme activitiesweremeasured after 19 hr of incubation (hepatocytes were treated with TCDD for 16 hr, then 150,UM-h-aminolaevulinic acid was added for a further 3-hr incubation). Some cytoxicity was observedin the cells treated with 10 7 or 10 6MTCDD. :~Dimethyl sulphoxide (<0.4%). Values are expressed as means_ SD, and those marked with asterisks differ significantly(Student's t-test) from the corresponding value for the control group (**P < 0.01).
376
C. YAO and S. SAFE Table 3. The interactive effects of TCDD EROD activities in chick Treatment (concm mol/egg) None (control)§ TCDD(10 10) MCDF (10.6) MCDF (10 7) MCDF(10 8) TCDD(10 ~0)+MCDF(10 6) TCDD (10 10) + MCDF (10 7) TCDD(10 10)+MCDF(10 8)
and MCDF on the induction of AHH and embryo hepatocytes in ovo AHH activityll EROD activityll (pmol/mg/min) (pmol/mg/min) 39.5 ± 9.7 0 2351+83.0"* 1530±108"* 417 _+5.0** 77.6 ± 5.7 234 ± 27.2** 14.0 ± 3.0 210_+ 11.6"* 0 2100- 131"*t 1270 _+70.8"'2 2470 _+ 103"* 1550 + 152 2530±193"* 1470±75.1
TCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin MCDF = 6-methyl-1,3,8-trichlorodibenzofuran AHH = aryl hydrocarbon hydroxylase EROD = ethoxyresorufin O-deethylase IIEnzyme activities were measured after 48 br of treatment. §Dimethyl sulphoxide (<0.4%). Values are means + SD, and those marked with superscripts differ significantly (Student's t-test) as follows: l,. the control group: **P < 0.01; v. the TCDD-treated group, t P <0.05; :~P < 0.01. i n d u c e d A H H activity. A t I0 6 a n d 10-7M, E R O D activity was also induced. T C D D (10 l°M) significantly i n c r e a s e d b o t h e n z y m e activities, a n d the increases were m u c h higher t h a n t h o s e o b s e r v e d at any o f the d o s e s o f M C D F . In the c o - t r e a t m e n t studies, M C D F at I 0 - 6 M significantly a n t a g o n i z e d the i n d u c t i o n o f A H H a n d E R O D activities c a u s e d by T C D D , w h e r e a s at lower c o n c e n t r a t i o n s (10 -7 a n d 10-SM) M C D F did n o t significantly alter these e n z y m e activities. In chick e m b r y o h e p a t o c y t e s in culture, 1 9 h r after t r e a t m e n t M C D F alone c a u s e d s o m e induction o f A H H a n d E R O D activities c o m p a r e d with c o n t r o l cells; T C D D at 10-1°M c a u s e d a significant increase in b o t h e n z y m e activities to levels m u c h g r e a t e r t h a n with M C D F a l o n e (Table 4). In the c o - t r e a t m e n t studies, M C D F at c o n c e n t r a t i o n s o f 10 -7 a n d 10 8 M significantly a n t a g o n i z e d the i n d u c t i o n o f A H H a n d E R O D activities c a u s e d by T C D D (10-10 M). T h e a n t a g o n i s m o f the i n d u c t i o n o f A H H a n d E R O D activities by M C D F was o b s e r v e d at T C D D : M C D F m o l a r ratios o f 1:1000 a n d 1:100, respectively. Fig. 1 illustrates the sucrose d e n s i t y g r a d i e n t profile o f n u c l e a r extracts f r o m chick e m b r y o h e p a t o c y t e s t r e a t e d with 1 nM[ 3 H ] T C D D for 2 h r in the p r e s e n c e o r a b s e n c e o f a 200-fold excess o f 2 , 3 , 7 , 8 - T C D F . T h e s e d i m e n t a t i o n
coefficient for the specifically b o u n d p e a k was 6.2 + 0.4 S. T a b l e 5 s u m m a r i z e s the c o n c e n t r a t i o n - d e p e n d e n t effects o f T C D D o n p o r p h y r i n levels in chick e m b r y o h e p a t o c y t e s in culture after 24 h r o f e x p o s u r e . A t all c o n c e n t r a t i o n s , TCDD caused significant elevation in u r o p o r p h y r i n [(COOH)8] a n d h e p t a c a r b o x y p o r p h y r i n [ ( c o o n ) 7 ] levels in a d o s e - d e p e n d e n t m a n n e r with a m a x i m a l 3.7-, 5.5-, 9.5- a n d 11.6-fold increase in u r o p o r p h y r i n levels a n d a m a x i m a l 4-, 4.8-, 6.5- a n d 7.3-fold increase in h e p t a c a r b o x y p o r p h y r i n levels at c o n c e n t r a t i o n s o f 10 -9, 10 8, 10 7 a n d 10-6M, respectively. A 3.9-, 5.1-, 7.8- a n d 9.2fold increase in ( C O O H ) 8 + ( C O O H ) 7 porphyrin levels was o b s e r v e d in cells t r e a t e d with T C D D c o n c e n t r a t i o n s o f 10 -9, 10 -8, 10 -7 a n d 10-6M, respectively. T h e r e were n o significant differences b e t w e e n any t r e a t m e n t g r o u p s (including the c o n t r o l g r o u p ) in the levels o f total p o r p h y r i n s . L o w levels o f the ( C O O H ) s + ( c o o n ) 7 p o r p h y r i n s were o b s e r v e d in the c o n t r o l g r o u p b u t the total p o r p h y r i n levels were similar to t h o s e in the T C D D t r e a t e d cells. In chick e m b r y o h e p a t o c y t e s in culture, 24 hr of t r e a t m e n t with M C D F a l o n e c a u s e d a significant increase in p o r p h y r i n levels with a 3.6- a n d 5.3-fold increase in u r o p o r p h y r i n , a 36- a n d 47.2-fold increase
Table 4. The interactive effects of TCDD and MCDF on the induction of AHH and EROD activities in chick embryo bepatocytes in culture Treatment AHH activity3" EROD activityt (concn, M) (pmol/mg/min) (pmol/mg/min) None (control)§ 40.5 ± 3.1 11.5 + 1.0 TCDD (10 to) 583 ± 13.6"* 249 ± 21.0"* MCDF(10 7) 98.2_+ 18.0"* 13.0_+ 1.4" MCDF (10 s) 59.7 + 5.0** 10.7 ± 2.1 * TCDD (10-to) + MCDF (10 7) 289 + 22.3"*~: 175 ± 9.5**5 TCDD (10 ~0) + MCDF (10 s ) 369 ± 16.5"*~ 193 + 11.3"*:~ TCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin MCDF = 6-methyl-1,3,8-trichlorodibenzofuran AHH = aryl hydrocarbon hydroxylase EROD = ethoxyresorufin O-deethylase tEnzyme activities were measured after 19 hr of incubation (hepatocytes were treated with the test chemicals or mixtures for 16 hr and then 150/~r,i-f-aminolaevulinic acid was added for a further 3-hr incubation). §Dimethyl sulphoxide (<0.4%). Values are means _+SD, and those marked with superscripts differ significantly (Student's t-test) as follows: v. the control group, *P < 0.05; **P < 0.01; v. the TCDD-treated group, ~P < 0.01.
TCDD/MCDF interactions in chick embryo hepatocytes
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Fig. 1. Sucrose density gradient elution profile of nuclear extracts from chick embryo hepatocytes treated with 1 nM-[3H]TCDD (ll) or l nM-[3H]TCDD plus a 200-fold excess of 2,3,7,8-TCDF (O). The sedimentation coefficient for the specifically-bound peak (i.e. total-non-specific binding) was 6.2 __+0.4 S (three determinations) and the level of the nuclear Ah-receptor complex was 37 fmol/mg protein. The experimental details are summarized in Materials and Methods. in heptacarboxyporphyrin, and a 3.3- and 4.8-fold increase in (COOH)8 + (COOH)7 porphyrin levels at concentrations o f 10 -6 and 10-SM, respectively (Table 6). T C D D (10-SM) caused significant increases in all observed porphyrin levels [4.5-, 41.2and 3.9-fold increase for (COOH)8, (COOH)7 and (COOH)8 + (COOH)7 porphyrin levels, respectively]. The TCl~D-induced increase in porphyrin levels was greater than that observed for M C D F at 10 -6 M. C o - t r e a t m e n t o f the cells with T C D D (10 -8 M) and M C D F (10 -6 M) resulted in higher levels o f (COOH)8, ( c o o n ) 7 and (COOH) s + (COOH)7 porphyrins than those observed after treatment with T C D D (10 -8 M) or M C D F (10 -6 M) alone. The porphyrin patterns in the cells co-treated with T C D D and M C D F were comparable with the results observed after treatment with T C D D alone. The co-treatment studies with various concentrations o f M C D F and T C D D resuited in a non-additive elevation o f porphyrin levels,
and it was a p p a r e n t that M C D F was a moderately active porphyrinogen in chick embryo hepatocytes in culture. In a second study on the interactive effects o f T C D D (10 -8 and 10 -7 M) and M C D F (10 -5 M) on porphyrin levels in chick embryo hepatocytes in culture (Table 7), M C D F (10 -5 M) alone caused 1.7-, 9.5- and 3.6-fold increases in (COOH)8, (COOH)7 and ( c o o n ) 8 + (COOH)7 porphyrin levels, respectively. T C D D caused significant increases in all observed porphyrin levels with 3.1- and 4.7-fold increases for (COOH)8, 15.6- and 20.5-fold increases for (COOH)7, and 6.2and 8.6-fold increases in the ( C O O H ) s + ( C O O H ) 7 porphyrin levels at concentrations o f 10 -8 M and 10 -7 M, respectively. Co-treatment o f the cells with T C D D (10 -8 M) and M C D F (10-SM) resulted in (COOH)8, (COOH)7 and ( C O O H ) 8 + ( C O O H ) 7 porphyrin levels that were higher than those observed after treatment with
Table 5. Dose-responseeffectsof TCDD on porphyrin levels in chick embryo hepatocytesin culture Porphyrin levels (pmol/dish)t:~ TCDD (M) 0 (control)~ ]0 -9 10-8 10-7 10-6
concn
(COOH)8 90.5 + 17.0(3.9) 333 + 23.2**(18.6) 495 + 21.2"* (31.6) 864 _.+77.1"* (41.7) 1050 ___35.7**(44.6)
(COOH)7 121 _+ 11.7(5.3) 483 _+30.2**(26.9) 584 ± 31.7**(37.3) 789 _+44.0** (38.1) 885 + 107"*(37.5)
(COOH)8 + (COOH)7 211 _+28.3 (9.2) 816 _+52.5**(45.5) 1080 + 52.5**(68.9) 1650 ± 117"* (79.8) 1940 +_ 139"* (82.1)
Total 2280 + 74.7 1790 + 69.0 1570 + 29.7 2070 + 104 2360 + 175
TCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin tPorphyrin levelsweredeterminedafter 24 hr of incubation (hepatocytesweretreated with the test chemicalsfor 16 hr, then 150 ~ M-t~-aminolaevulinic acid was added for a further 8-hr incubation). :~Values in parentheses are as a percentageof total porphyrin. §Dimetbyl sulphoxide ( < 0.4%) Values are means + SD and those marked with asterisks differ significantly(Student's t-test) from the correspondingvalue for the control group (**P < 0.01).
378
C. YAO a n d S. SAFE Table 6. The interactive effects of TCDD and M C D F on porphyrin levels in chick embryo hepatocytes in culture Porphyrin levels (pmol/dish)'t:~
Treatment (concn, M) None (control)§ T C D D (10 5) M C D F ( 1 0 6) M C D F (10 5) TCDD(10 S)+MCDF(10 TCDD(10 S)+MCDF(10
5) 5)
(COOH) 8
(COOH)7
(COOH) 8 h- (COOH) 7
83.5 + 46.3 (3.3) 383 + 127" (22.7) 297+41.5'*(11.8) 437 ± 42.0"* (24.8) 424±43.4"*(27.1) 524±69.1"*(31.2)
16.9 ± 19.2 (7.8) 696 ± 184"* (41.2) 610±42.3"*(24.2) 800 ± 56.1"* (45.5) 767±66.8**(49.0) 847±87.l**(50.4)
278 ± 47.1 (11.1) 1080 _+ 310"* (63.9) 906+81.8"*(36) 1330±92.8"*(70.3) 1190± 106"*(76.1) 1370± 152"*(81.6)
Total 2500 ± 864 1690 ± 75.3 2520_+ 138"* 1760_+152"* 1570+ 106 1680± 150
T C D D = 2,3,7,8-tetrachlorodibenzo-p-dioxin M C D F = 6-methyl-l,3,8-trichlorodibenzofuran tPorphyrin levels were determined after 24 hr of incubation (hepatocytes were treated with the test chemicals for 16 hr, then 150/~ M-6-aminolaevulinic acid was added for a further 8-hr incubation). ++Values in parentheses are as a percentage of total porphyrin. §Dimethyl sulphoxide ( < 0.4%). Values are means ± SD, and those marked with asterisks differ significantly (Student's t-test) from the corresponding values for the control group (*P <0.05; **P < 0.01).
Table 7. The interactive effects of T C D D and M C D F on porphyrin levels in chick embryo hepatocytes in culture Porphyrin levels (pmol/dish)% Treatment (concn, M)
(COOH)~
None (control)§ T C D D ( 1 0 8) T C D D ( 1 0 7) M C D F (10 5) TCDD(10 8)+MCDF(10 TCDD(10 7)+MCDF(10
5) 5)
132 ± 12.6 (5.1) 4 0 7 ± 148"*(26.8) 6 2 5 + 152"*(33.1) 220 ± 69.0* (12.2) 469±80.1"*(26.0) 685±60.5"*(30.9)
(COOH) 7 43.4 _+ 87.2 (1.7) 673±241"*(44.3) 882±183"*(46.8) 414 ± 84.2** (22.9) 734±130"*(45.7) 968±257"(43.7)
(COOH)8 + (COOH) 7 176 ± 94.6 (6.8) 1080±386"*(71.1) 1510±332"*(79.9) 634 ± 143"* (35.1) 1200±206"*(71.7) 1650±317"*(74.6)
Total 2610 ± 60.1 1520±638" 1890±327 1810 ± 752** 1760±317' 2220_+388
T C D D = 2,3,7,8-tetrachlorodibenzo-p-dioxin M C D F = 6-methyl-l,3,8-trichlorodibenzofuran fPorphyrin levels were determined after 24 hr of incubation (hepatocytes were treated with the test chemicals for 16 hr, then 150 p M6-aminolaevulinic acid was added for a further 8-hr incubation). ~.Values in parentheses are as a percentage of total porphyrin. §Dimethyl sulphoxide (<0.4%). Values are means _+ SD, and those marked with asterisks differ significantly (Student's t-test) from the corresponding values for the control group (*P <0.05; **P < 0.01).
T C D D (10 -8 M) or M C D F (10 -5 M) alone. Comparable results were observed after co-treatment of the cells with 10 7M-TCDD and 10-SM-MCDF. The results obtained for the cells treated with M C D F plus T C D D (Tables 6 and 7) are approximately additive if the porphyrin values for the control cells are subtracted from those obtained for the different treatment groups. DISCUSSION
The results summarized in Tables 1 and 2 demonstrate that T C D D caused a concentration-dependent induction of hepatic microsomal A H H and E R O D activities in chick embryo hepatocytes in o v o and in culture. Maximum induction of both enzyme activities was observed at concentrations as low as 10 9M and 10 -8 mol/egg for hepatocytes in culture and in ovo, respectively, and significant induction was observed in o v o at a concentration of 10- ~ mol/egg. In the cultured cells there was a decrease in the induced enzyme activities at concentrations greater than 10 9M, and this was probably due to cytotoxicity. These data demonstrate that the chick embryo hepatocytes were highly Ah-responsive, and this was consistent with the identification of the nuclear Ah-receptor in the cultured hepatocytes (Fig. 1). The sedimentation coefficient for this receptor was 6.2_+ 0.4S and the level of the Ah-receptor was 37 fmol/mg protein. These values are similar to those
obtained from many other Ah-responsive cell lines (Safe, 1988). The effects of M C D F and TCDD-induced A H H and E R O D activities was also determined in chick embryo hepatocytes in o v o and in culture. On the basis of the results obtained for the enzyme induction activity of T C D D alone (Table 1), a dose of 10 -~° mol/egg was chosen for the interactive studies. Treatment of the eggs with M C D F alone showed that this compound was significantly less active than T C D D as an inducer of A H H and E R O D activity. In the co-treatment studies, M C D F significantly inhibited the induction of A H H and EROD activity by TCDD. However, this partial antagonism was observed only at the highest dose of M C D F (10-6mol/egg), and the induction activity in the co-treated eggs (TCDD plus MCDF) was only 20% lower than that observed in the eggs treated with T C D D (10-J°mol/egg) alone. In contrast, co-treatment of the cultured hepatocytes with 10 ~0M-TCDD and M C D F resulted in a 50 and 37% decrease in A H H induction, at 10 7 and 10 8 M-MCDF, respectively. Interactions of similar magnitude have been observed in rat hepatic microsomes and in rat hepatoma H-4-II E cells (Astroff e t al., 1988). Chick embryo hepatocytes in culture can be used to determine the effects of T C D D antagonists on two responses mediated by the Ah-receptor, namely the induction of A H H and EROD activities and porphyria. The latter response is characterized by the
TCDD/MCDF interactions in chick embryo hepatocytes accumulation of (COOH) 7 and (COOH)8 porphyrins. It was apparent that T C D D concentrations greater t h a n 10-6M were required for the accumulation of maximum levels of the (COOH)~ + (COOH)7 porphyrins (Tables 5-7). In contrast, maximum induction of A H H activity by T C D D was observed at concentrations of 10 9M (Table 2). Similar results have been observed in C57BL/6 mice, in which the doses of T C D D that are required to cause porphyria are higher than those required for enzyme induction (Yao and Safe, 1989). M C D F only weakly induced A H H and E R O D activity in chick embryo hepatocytes at concentrations of 10 -7 or 10-SM (Table 4), whereas at a 10 6M, M C D F caused significant accumulation of (COOH)8 and (COOH)7 porphyrins in chick embryo hepatocytes (Table 6). The results in Tables 5-7 show that M C D F is only 100 to 1000 times less potent than T C D D as a porphyrinogen. In contrast, the T C D D : M C D F potency ratios were more than l 0 4 t o 10 5 for most other Ah-receptor-mediated toxic responses, including porphyrinogenicity in C57BL/6 mice (Astroff et al., 1988; Bannister et al., 1989; Harris et al., 1989; Yao and Safe, 1989). The apparent inversion of the expected relative potencies of M C D F as a monooxygenase enzyme inducer and as a porphyrinogen was unexpected, and the reasons for these potency differences are unknown. However, because of the unexpectedly high porphyrogenicity of M C D F in cultured chick embryo hepatocytes, no antagonism of T C D D (10 8 and 10 7M)-induced porphyria was observed and it is unlikely that antagonism would be observed at lower concentrations of TCDD. Thus, chick embryo hepatocytes show promise as useful models for investigating the molecular mechanisms associated with partial antagonism of T C D D induced C Y P I A 1 gene expression. Current studies are focused on the development of new antagonists that can also be used to probe the mechanisms associated with the development of porphyria in chick embryo hepatocytes. Acknowledgements--This work was supported by the National Institutes of Health (ES03843 and ES03554) and the Texas Agricultural Experiment Station. S. Safe is a Burroughs Wellcome Toxicology Scholar. REFERENCES
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