Interaction of 1-ethynylpyrene and hepatic CYPIA1 from rainbow trout

.g Chemico-BiologicalInteractions 93 (1994) 1-10

Interaction of 1-ethynylpyrene and hepatic CYPIA1 from rainbow trout P . C . L e e *a'b'c, A . K . D a s m a h a p t r a a'c aDivision of Gastroenterology, Department of Pediatrics, bDepartment of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI. 53226, USA CMarine and Freshwater Biomedical Research Core Center, University of Wisconsin Milwaukee, Milwaukee, W1. 53201, USA

Received 23 August 1993; revision received 6 October 1993; accepted 8 October 1993

Abstract 1-Ethynylpyrene (EP) inhibits microsomal 7-ethoxyresorufin O-deethylase (EROD) activities from the liver of both trout and rat. Trout hepatocytes treated with EP had increased microsomal EROD activity and a concomitant increase in CYPIAI protein as measured by Western blot analysis. EP at 10 -9-10 -6 M did not interfere with B-naphthoflavone (BNF) induction of CYPIA1 in trout hepatocytes. In contrast, treatment with a-naphthoflavone (ANF), another inhibitor of CYPIA1, resulted in a significant inhibition of the basal (at 10 -6 M ANF) and BNF induced (at 10-s M and higher concentrations of ANF) level of EROD activity. ANF also did not significantly induce CYPIAI protein but led to attenuation of the induction of CYPIA1 proteins by BNF in trout hepatocyte cultures. Thus, in trout hepatocytes, EP acts differently from ANF in the modulation of CYPIA1. Keywords: Trout hepatocyte; Ethynylpyrene; Cytochrome P450; a-Naphthoflavone;/~-Naphthoflavone

1. Introduction The synthetic compound 1-ethynylpyrene (EP) is a potent, mechanistic based, inhibitor of mammalian microsomal cytochrome P450 dependent hydroxylation of benza[a]pyrene (BP) in vitro [1]. EP administration to rats, however, did not cause * Corresponding author, Department of Pediatrics, Medical College of Wisconsin, MACC Fund Research Center, 8701 Watertown Plank Road, Milwaukee, Wl 53226, USA. 0009-2797/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0009-2797(93)03238-P

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P.C. Lee, ,4.K. Dasmahaptra/Chem.-Biol. Interact. 93 (1994) 1-10

significant changes in the cytochrome P450 content nor in diminution of (BP) hydroxylation activity in the liver microsome [1]. EP inhibits the formation of DNA adducts of dimethylbenza[a]anthracene (MBA) and BP in mouse skin and reduces the incidence of skin tumor initiation by MBA and BP, presumably by inhibiting the metabolism of MBA and BP by cytochrome P450-dependent monooxygenases [2,3]. Fish liver contains cytochrome P450 monooxygenase that is in many ways similar to its mammalian counterparts [4]. Rainbow trout CYPIAI cDNA has been cloned and shown to encode a protein, with 522 amino acid residues, that shows considerable homology to mammalian CYPIAI [5]. Furthermore, antisera to rat p450c (CYPIAI) cross-react with rainbow trout CYPIAI indicating immunological similarity between the CYPIA1 of the two species [6]. In spite of the basic similarities, more stringent amino acid comparison shows recognizable differences. Since the 10 amino acid residues in the amino terminal region in rainbow trout CYPIA1 are different from that of the rat [6], it is possible that differences in other parts of the molecule also exist. These differences would confer subtle differences in the function and regulation of the trout and mammalian hepatic CYPIA1. 1-Ethynylpyrene has been very useful in the study of the mechanism of carcinogenesis and regulation of CYPIAI in mammals [1-3,7,8]. With the increasing interest in the use of fish as models for the study of carcinogenesis and drug metabolism [9-11], it is important to see ifEP also affects CYPIAI in fish such that it can be used in similar studies. In the present study we characterized the interaction of EP, a defined suicide inhibitor for the mammalian CYPIA1, and the CYPIAI from rainbow trout liver by in vitro inhibition assays. We also investigated and contrasted the effects of EP and ANF (ot-naphthoflavone) on the induction of CYPIAI by/3-naphthoflavone (BNF) using rainbow trout primary hepatocyte culture. 2. Materials and methods 2.1. Chemicals Except otherwise stated, all chemicals were from Sigma Chemical Company (St. Louis, MO). Calf serum and medium M199 were from GIBCO (Long Island, NY); rabbit anti-trout CYPIA1 serum (LM4B) was kindly provided by Drs John Lech and Mary Haasch, Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI. l-Ethynylpyrene was a gift from Dr William Alworth, Department of Chemistry, Tulane University, New Orleans, LA. Nitrocellulose membrane and horseradish peroxidase conjugated goat anti-rabbit IgG were from BioRad (Richmond, CA). 2.2. Methods Microsomes from the livers of rats and rainbow trout, treated either with BNF or vehicle (dimethylsulfoxide, DMSO), were prepared by differential centrifugation as described previously [12]. Protein concentration was determined by the Bradford method [13] using the BioRad® reagent (BioRad Laboratories, Richmond, CA) with BSA as the standard. 7-Ethoxyresorufin O-deethylase (EROD) activity in micro-

P. C Lee. ,4,K. Dasma~taptra/ Ckem.~Biol, Interact~ 93, (1994)- 1.- 10

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1-EP concentrations (uM) Fig. 1, Dose dependence of EP inhibition of trout and rat hepatic microsomal EROD activities at 22°C and 37°C, EP at concentrations indicated was added at the beginning of the assay and allowed to remain for the duration of the assay. Three separate microsomal fractions from 3 different liver tissues were used for the assay. Each assay was performed in duplicate and the results averaged. For comparison, EROD activities were normalized to that of the corresponding control values (i.e. value obtained from the assay tubes with no EP added; this was arbitrarily assigned as 100%). I"1, hepatic microsomal fractions from untreated animals; I , microsomal fractions from BNF-treated animals. Mean ± S.D.

3

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P.C. Lee, A.K. Dasmahaptra / Chem.-Biol. Interact. 93 (1994) 1-10

somes was determined spectrofluorometrically following the method of Burke and Mayer [14] using an Aminco SPF spectrofluorometer as previously described [15]. In EP inhibition studies, EP was added at the beginning of the assay. Rainbow trout hepatocytes were isolated and cultured as previously reported [15]. Cell viability was evaluated by trypan blue dye exclusion. Freshly isolated hepatocytes (20 x 106) in 20 ml of culture medium were seeded onto a 100-mm diameter plastic culture dishes (Falcon) precoated with rainbow trout skin extract (7.6 t~g protein/cm2). Culture dishes were incubated at 16°C in air. Hepatocytes were allowed to attach to culture dishes for 24 h before the start of experimental treatments./3Naphthoflavone, a-naphthoflavone, and EP at various concentrations and combinations as specified were then added to the culture medium. Control cultures received the same amount of the vehicle (DMSO) only. Reagents alone or in mixtures were added simultaneously and cells were harvested 24 h after the initial exposure. Microsomal fractions were prepared from the hepatocytes and their EROD activities and CYPIA1 concentrations were determined. Western blots for the determination of CYPIA1 protein were carried out as described previously [12] using antisera LM4B which is specific for the trout CYPIA1. 2.3. Statistics Results are reported as mean 4- S.D. For multiple sample comparison, ANOVA was used and difference between two means was then evaluated by Student's t-test with P < 0.05 considered as significant.

3. Results Co-incubating EP at various concentrations with the substrate ethoxyresorufin affected the microsomal EROD activities (Fig. 1). At 37°C, the physiological temperature for the rat enzyme, slight inhibition was seen at 10 -7 and 10 -6 M concentra-

Table 1 Microsomal EROD activity from trout hepatocytes cultured in DMSO (dimethylsulfoxide - - control), or BNF (/3-naphthoflavone) in the presence and absence of EP (I-ethynylpyrene) EP concentration (M)

EROD activity (pmol/min/mg protein) DMSO

0 10 -9 10 -8 10 -7 10 -6

8.9 20.7 17.0 21.9 16.5

± ± ± ± ±

BNF (10 -s M) 1.1 0.6* 2.7* 2.7* 2.6*

22.2 25.2 25.0 27.4 15.6

± ± ± ± ±

4.2 2.9 8.4 6.2 3.2

Values are mean 4- S.D. from 3 separate hepatocyte cultures. BNF and EP were added at 24 h and cells were harvested 48 h after plating. *Values significantly different from corresponding values without exposure to EP with P _< 0.05.

P.C. Lee, A,K. Dasmahaptra/ Chem.-Biol. Interact. 93 (1994) 1-10

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Table 2 Microsomal E R O D activities from trout hepatocytes cultured in DMSO (control), or BNF in the presence and absence of A N F (c~-naphthoflavone) A N F (M)

E R O D activities (pmol/min/mg protein)

0 10 -9 10 -8 10 -7 10 -6

DMSO

BNF (10 -s M)

14.6 ± 4.2 13.9 ± 3.9 11.7 ± 4.2 9.8 ± 3.6 6.9 ± 1.8"

42.8 38.1 28.3 28.8 18.0

± ± ± ± ±

5.6 4.4 7.1" 8.8* 1.5"

Values are mean • S.D. from 3 separate hepatocyte cultures. BNF and A N F were added at 24 h and cells were harvested 48 h after plating. • Values significantly different from corresponding values without exposure to A N F with P < 0.05.

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Fig. 2. Western blots of microsomal fractions from cultured hepatocytes in control,/~-naphthoflavone (BNF) and I-ethynylpyrene (EP)-treated at various concentrations (10-9-10 -6 M). Upper panel: BNF alone or BNF and EP were added at 24 h and cells were harvested at 48 h after plating. Lower panel: BNF alone or EP alone was added at 24 h and cells were harvested at 48 h after plating. Equal amount of microsomal protein (10 t~g each) was applied to each lane. Blots were developed with rabbit anti-trout CYPIA (LM4B) serum. For each treatment, 2 lanes are shown, with each derived from cultured hepatocytes from a different fish.

P.C. Lee, A.K. Dasrnahaptra/ Chem.-Biol. Interact. 93 (1994) 1-10

tions of EP. At 10 -5 M of EP the inhibition was pronounced and only 30-40% of the original activities remained. EP inhibits EROD activities in microsomes from both the control and BNF-stimulated rats. The trout microsome showed no EROD activity at 37°C. EROD activity of the trout microsome was therefore determined at 22°C, which is a more physiological temperature for the trout. At 22°C, significant inhibition was evident at 10 -6 M of EP (20-30% of control). At 10 -5 M, EP inhibition reached 70-80%. Rat microsomes evaluated at 22°C for comparison showed less inhibition by EP at similar concentrations. To evaluate the modulating effect of EP on microsomal CYPIA1 in trout liver, hepatocyte cultures were treated with varying concentrations of EP in the absence and presence of BNF. EP alone led to increases in EROD activity (Table 1). EP did not inhibit the induction of microsomal EROD activities by BNF. For comparison, ANF, a known antagonist of CYPIA1 was tested in the same system in the presence and absence of BNF (Table 2). Incubation of hepatocytes with ANF alone at the concentrations of 10 -9 t o 10 -7 M had no effect on the EROD activity of the subsequently isolated microsomal fractions. When the concentration of ANF was increased to 10 -6 M, the microsomal fraction isolated from these treated hepatocytes showed a decrease in EROD activity. When ANF was

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Fig. 3. Western blots of microsomal fraction from cultured hepatocytes in control, /3-naphthoflavone (BNF) and o~-naphthoflavone (ANF)-treated at various concentrations (10-9-10-6M). Upper panel: BNF alone or BNF and A N F were added at 24 h and cells were harvested at 48 h after plating. Lower panel: BNF alone or A N F alone was added at 24 h and cells were harvested at 48 h after plating. Other conditions were as described in legend to Fig. 2 except the lower panel shows only 1 lane for each treatment group.

P.C. Lee;~.a:K. Oasma~,~tra/ Che,r,:-Bio/,J,,teract: 9.~ C~994) t-tO given together with BNF to trout hepatocytes, the microsomal fraction obtained subsequently from these cells showed lower EROD activities compared with that treated with BNF alone. Significantly reduced levels were reached at ANF concentrations of 10 -8 M and higher. Since EP is a potent inhibitor of CYPIA 1 enzyme activity, measurement of EROD activities may not be representative of the CYPIA 1 protein concentrations. Western blot assays were done to evaluate the effect of EP on CYPIAI expression. Fig. 2 indicates that EP at the concentrations used (10-9-10 -6 M), has no effect on the induction of CYPIA 1 by BNF. Further, EP by itself is a good inducer of CYPIA 1 and does not seem to show any dose repsonse over the range of concentrations used (10-9-10 -6 M). At a concentration of 10 -8 M, EP induced CYPIAI to a similar extent as an equivalent amount of BNF. Fig. 3 shows a Western blot analysis of CYPIA1 protein concentrations in hepatocytes exposed to BNF and/or ANF. While BNF treatment led to increases of CYPIA1 over the basal (untreated) level, ANF even at 10 -6 M had minimal effect on the level of CYPIAI in the same hepatocyte cultures, Fig. 3 also shows that ANF when added together with BNF attenuated the induction of CYPIAI by BNF. 4. Discussion

Fish has become a practical and useful model for the study of carcinogenesis and drug metabolism [9-11]. Knowledge of the fish cytochrome P450 system, however, lags behind that of the mammalian counterparts. A better understanding of fish cytochrome P450 is needed to provide the basis for better interpretation of the result of carcinogenesis and xenobiotic metabolism studies in these species. 1-Ethynylpyrene, a benzo[a]pyrene analogue, has been used for characterizing different metabolic activation pathways catalysed by different cytochrome monooxygenases. The specific inhibitory effect of EP on mammalian CYPIAI makes it an ideal experimental probe for the investigation of CYPIAI function in different species. The present results indicate that the trout microsomal EROD activity is also inhibited by EP. Compared with rat microsomes, the dose response curves suggest that the trout microsomal hepatic EROD activity is at least as sensitive to EP as the rat hepatic microsomes. Since EP inhibition of EROD activity was equally effective against microsome fractions from tissues with and without prior stimulation with BNF and BNF is known to be a specific inducer of CYPIAI in trout liver, EP is thus an effective inhibitor for CYPIAI from both fish and mammalian species. The present study also shows that exposure of trout hepatocytes to EP increases the EROD activity in microsomes subsequently isolated from the EP-treated cells. These results suggest that EP might act as an inducer of CYPIAI in trout hepatocytes. In contrast, in rats treated with EP, there is only a slight increase in BP hydroxylation (a step catalysed by the CYPIAI enzyme) [1]. The difference observed might be because of the high dose of EP ( - 2 × 10 -4 M) used in the rat study. Such a dose might leave enough residual EP in the subsequently isolated microsomal fraction to inhibit, at least partially, the catalytic activity of CYPIA1. In mouse skin

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Interact. 93 (1994) 1-10

epidermis, EP treatment in vivo inhibited the binding of 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene to DNA. This is in agreement with the interpretation that EP inhibits CYPIA1 activity thus preventing the conversion of these procarcinogens to their active metabolites and lower their overall binding to DNA [2]. Since measurements of CYPIA1 protein were not presented in these studies direct comparison to our results is not possible. There is a difference between the response of trout hepatocytes and rat liver or mouse skin to EP. EP induction of CYPIA1 in trout hepatoctyes was further confirmed by the increase in CYPIA1 protein as measured by Western blot analysis. 1-Ethynylpyrene when given together with BNF at optimal concentration to trout hepatocytes neither increased nor decreased the EROD activities or CYPIA 1 protein, suggesting both EP and BNF act through similar mechanisms in the induction of CYPIA1. Our results also showed that the behavior of ANF, a known CYPIAI inhibitor, on trout hepatocytes is quite different from that of EP. At a dose of 10 -8 M and higher, A N F led to a decrease in measurable EROD activities in the microsomes from treated trout hepatocytes. It reached the significant level only at 10 -6 M of ANF. In this regard, our observation is different from that reported for human keratinocytes in which ANF, even at 10 -4 M, has no effect on the basal level of P450IAI mRNA [8]. In rat hepatoma cells, however, A N F at 10 -5 M caused a dramatic increase in P450IA1 mRNA [12]. At concentrations of 10 -8 M and higher, ANF also inhibited the induction of EROD activity by BNF. The inhibitory effect of A N F in the BNF induction of CYPIA1 in the trout hepatocyte parallels that seen in human keratinocytes where A N F inhibited the induction of CYPIA1 by both BNF and T C D F (2,3,7,8-tetrachlorodibenzofuran) [81 and in rat hepatoma cells and mouse lymphocytes where ANF inhibited the induction of CYPIA1 by TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) at 10-7-10-5 M [16,17]. The difference in the behavior of A N F and EP as observed in the trout hepatocyte system is most likely due to the difference in their modes of action. In mammals, ANF is a very weak inducer and only induces CYPIA1 at very high concentrations ( > 10 -5 M) [16]. ANF however is a very potent inhibitor in that even at concentrations of 10 -7 M, it competitively inhibits the binding of arylhydrocarbon (e.g. TCDD) to the Ah (arylhydrocarbon) receptor [16,18,19]. The same mechanism is probably operating in the trout hepatocyte such that ANF competes with BNF for Ah receptor binding and inhibits the induction of CYPIAI by BNF. EP, in contrast, is itself a substrate of CYPIA1 and only inhibits the action of CYPIAI following its own metabolism [1]. EP is also an inducer in that it induces CYPIAI. At present, we can only speculate as to the mechanism of EP induction of CYPIAI. It is probably similar to that of BNF, i.e. by binding to the Ah receptor, since we do not see an additive effect when EP and BNF are presented to the hepatocyte simultaneously. Competition studies between EP and BNF or arylhydrocarbons in the binding to Ah receptors and subsequently to the XRE (xenobiotic response element) would provide some needed answers. In summary, we have shown that EP acts on fish hepatocyte microsomes but differs from that of ANF. EP like ANF can be a useful probe for the study of CYPIA1 function in fish.

P. C. Lee, A,K. Dasmahaptra / Chem..Biol. Interact. 93 (1994) 1-10

Acknowledgement This work was supported in part by NIH grant No. ES04184. We thank Mr Kris Kosteretz for his technical help in the preparation of trout hepatocytes and Dr S.L. Werlin in the preparation of the manuscript.

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J.A. Blank, A.N. Tucker, J. Sweatlock, T.A. Gasiewicy and M.I. Luster, a-Naphthoflavone antagonism of 2,3,7,8-tetracholorodibenzo-p-dioxin induced murine lymphocyte ethoxyresorutin O-deethylase activity and immunosuppression, Mol. Pharmacol., 32 (1987) 168-172. 18 M. Merchant, L. Arellano and S. Safe, The mechanism of action of o-naphthoflavone as an inhibitor of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYPIAI gene expression, Arch. Biochem. Biophys., 281 (1990) 84-88. I9 M. Merchant, V. Krishnan and S. Safe, Mechanism of action of o-naphthoflavone as an Ah receptor antagonist in MCF-7 human breast cancer cells, Toxicol. Appl. Pharmacol., 120 (1993) 179-185. 17