TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

104,367-374

(1990)

SHORT COMMUNICATION Interaction

of Carbon Tetrachloride with ,&Naphthoflavone-Mediated Cytochrome P450 Induction in Winter Flounder (Pseudopleuronectes americanus)’

Interaction of Carbon Tetrachloride with @-Naphthoflavone-Mediated Cytochrome P450 Induction in Winter Flounder (Pseudopleuronectes americanus). KLEINOW, K. M., DROY, B. F., BUHLER, D. R., AND WILLIAMS, D. E. (1990). Toxicol. Appl. Pharmacol. 104, 367-374. The interaction between /3-naphthoflavone induction (BNF, 100 mg/kg) and carbon tetrachloride (CC&; 1 ml/kg) hepatotoxicity was examined in the flounder. Treatment groups composed of control, BNF, Ccl,, and BNF/CC& were compared in terms of cytochrome P450 isozyme content (LM4,,; LMz), catalytic activity, isozyme distribution, SGGT-SGPT levels, and pathology. CCI, administration resulted in significant reductions in both the constitutive P450 (LM,) and the BNF-inducible isozyme (LM& as well as elevations in SGPT and SCOT levels. The decline in LM4b isozyme content was reflected by stoichiometric decreases in ethoxyresorufin-Odeethylase activities. BNF/CCIJ coadministration was protective in part against CCL, hepatotoxicity. lmmunohistochemistry indicated that LMdb was diffusely distributed throughout the liver. These interactions have demonstrated a multiple P450 isozyme involvement, the protective nature of BNF against CCL, hepatotoxicity in the flounder, the ability to maintain an inductive response in face of Ccl, coadministration, and the diffuse distributional pattern of LM4s in the flounder l&r. 0 1990 Academic press, 1nc.

Cytochrome P450-dependent activity in fish with CC& in mammals for the suicide dehas been shown to be induced by exposure to struction of P450 b,e, the major PB-inducible low levels of environmental pollutants forms (Noguchi et al., 1982) and cytochrome (Payne, 1976; Melancon et al., 1987). ThisreP450 et, the ethanol-inducible form (English sponse to environmental xenobiotics has fo- and Anders, 1985) as well as for the destruccused attention upon P450 induction as a tion of P-450d, one of the 3-methylcholanpossible biological monitoring tool. Exposure threne-inducible isozymes (Sesardic et al., of fish to polluted waters, however, presents 1989). Unlike mammals which are responsive to not only inducing agents to the fish but also, in certain areas, hepatotoxic agents (National PB and polycyclic-aromatic hydrocarbon Research Council, 1980). (PAH)-like inducers, evidence for P450 inCarbon tetrachloride (CCL), a known duction in fish has come largely with PAHs. aquatic pollutant and hepatotoxin, has been Studies in trout have identified two major widely studied in experimental mammalian P450 isozymes’ including a PAH-inducible toxicology in regard to its interaction with in- form (LM&, which appears to be the homoducers and effect upon cytochrome P450. logue to the major /I-naphthoflavone (BNF)Early studies have shown CCL treatment to induced form in other vertebrates and a condecrease activity (Head et al., 198 1) and con- stitutive noninducible P450 (LMP). Although tent (Sasame et al., 1968) of phenobarbital the constitutive trout P450 (LM,) does not (PB)-induced P450 while leaving activities as- appear to be inducible, it has been shown to sociated with 3-methylcholanthrene-induced * The terminology for trout cytochrome P45Os LMdb P450 largely unaffected. Evidence now exists and LM2 is based upon relative mobility on SDS-PAGE gels. Although this nomenclature system is patterned after that of rabbit cytochrome P45Os no structural or functional similarity is implied.

’ Presented in part at the 27th Meeting of the Society of Toxicology, Dallas, TX, February 1988. 367

0041008X/90

$3.00

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

368

SHORT COMMUNICATION

be effective in activating aflatoxin B, (AFB,) to the carcinogen AFB,-2,3 epoxide (Williams and Buhler, 1983). In this regard, trout LM2 is similar to PB-inducible P450 isozymes in the rat (Yoshizawa et al., 1982). Major differences in the localization and magnitude of CC& hepatotoxicity exist between mammals and fish. While hepatic centrilobular toxicoses with CCL correlate with the distribution of PB-inducible P450 in mammals (Baron et al., 1978) diffuse focal necrosis is a more common lesion in fish (Gingerich, 1982). The relationship between P450 isozyme distribution and Ccl,-induced liver necrosis has not been addressed in fish species. A preliminary study was undertaken to characterize the influence of Ccl, upon cytochrome P450 isozymes and the effect of P450-inducing agents (i.e., BNF) on CCL-induced hepatotoxicity in the winter flounder. Of particular interest was the effect of CC14cytochrome P450 isozyme interactions upon the reliability of induction as an environmental monitoring method. Dosage and sampling regimes were based upon optimization with time and response of glutamic pyruvic transaminase elaboration (Statham et al., 1978) and BNF induction (Vodicnik et al., 1984). MATERIALS

AND

METHODS

Rabbit anti-trout LM4, IgG and LMI IgG were prepared as previously described (Williams and Buhler, 1984). The trout LM.,t, antibody has been shown to inhibit the liver microsomal benzo[a]pyrene-hydroxylase activity catalyzed by the winter flounder as well as to display immunochemical cross-reactivity with P450 isozymes sharing similar activities in other fish species (Williams, d a/., 1986). Goat anti-rabbit colloidal gold conjugates, silver enhancement, and SDS-PAGE supphes were obtained from Bio-Rad Laboratories (Richmond, CA). Resorufin and BNF, were obtained from Aldrich Chemica’Co., Inc. (Milwaukee, WI). ‘I-Ethoxyresorufm was obtained from Molecular Probes (Eugene, OR). Peroxklase-antiperoxidase complex was obtained from D&K0 @anta Batbara, CA). All other chemicals were of the highest purity oommerciaIly available.

200 3 El (I)

150

8 2

100

(0 k3

50

%5

0 CON:ROL

&

Cc3L4

m&CL4

FIG. 1. Serum aspartate aminotransferase (ASTSGOT) levels and serum glutamic pyruvic transaminase (ALT-SGPT) levels for IIounder in (1) control; (2) BNF; (3) CCL; and (4) CCl,/BNF treatments. Error bars represent means + SD. N = 3 for each treatment.

Mount Desert Island, Maine, were housed for acclimation and experimental purposes under natural photoperiods in running seawater (13.0-152°C). Flounder were administered inducer and/or hepatotoxin alone or in combination to provide four treatment groups of three animals each: (I) control; (2) inducer; (3) hepatotoxin; and (4) inducer/hepatotoxin in combination. The inducer (BNF; 100 mg/kg) and hepatotoxin (CC&; 1 ml/ kg) were administered to appropriate groups by ip injection and by gavage, respectively. Compounds were administered to appropriate treatment groups 1 (CCL,) and 5 (BNF) days prior to sample collection. Following blood collection, the animals were killed and the livers excised. Livers were divided for subsequent histological, immunohistochemical, and microsome preparation. Microsomal preparation and enzyme assays. Serum aspartate aminotransferase and glutamic pyruvic transaminase activities, utilized as indices of liver damage, were determined calorimetrically according to a modification (90-min incubation) of the method of Reitman and Frankel(1957). Hepatic microsomes were prepared by the differential ultracentrifugation procedure described by Elcombe and Lech (1979). EthoxyresorulinO-deethylase activities were measured spectroIluorometrically using the techniques of Burke and Mayer (1974).

SDS PAGE, western blots ati immunostaining. Microsomes were subjected to SDSPAGE eIectrophoresis (Laemmli, 1970). Stacking and resolving gels (0.75 mm) contained total acrylamide concentrations of 4 and IO%, respectively. The gels were run under a constant voltage setting of 200 V. After electropboresis the gels were transblotted at 100 V onto nitroceIIulose. The nitmcelIulose transfer was fixed by heat at 7o’C for 30 min. Al& fixation, the blots were immunosta&ed utilizing a BioRad Immun-Blot Assay Kit with &her rabbit anti-trout P450 Ll& or LMz primary IgG am&o&es and goat anti-r&

369

SHORT COMMUNICATION TABLE 1 CYTOCHROME P450 ISOZYME CONTENT AND MON~XYGENASE IN HEPATIC

Treatmen@ Corn oil control PNF @NF/CClb

FROM

ACTIVITY

FLOUNDER

L&L

LM2

(pmol/mg)

WWmg)

EROD’ (pmol/min/mg)

6.2 + 0.3 6.7 + 0.5 2.0 + 0.2* 3.7 + 1.1

139.5 f 1198.0 2 14.1 + 700.5 f

19.8 73.3 2.9 52.2

CCh

MICROSOMES

-t 10.1 f 25.0* + 5.0* -+ 7.0*

11.0 648.0* 24.3* 338.1*

a Corn oil was administered ip to all treatment groups; BNF treatment was 100 mg/kg ip; and CC& was at 1 ml/kg by gavage. ’ Each entry represents means -+ SD of three animals. ’ Ethoxyresorufin-O-deethylation. * Significantly different from controls: p < 0.05 t test and two-way ANOVA.

bit gold conjugate. Gold conjugate enhancement was then performed utilizing a modification of the silver staining technique of Danscher and Norgaard (1983). The resulting blots were quantified by densitometry. The microsomal P450 (LM4,, or LMz) content was calculated using a graded series of the corresponding purified trout isozyme for a standard curve. Morphological analysis. Representative regions of each liver were dehydrated, followed by an overnight infiltration in 100% catalyzed glycol methacrylate (GMA) monomer. Tissues were then embedded in GMA, processed by routine methods, and stained with Mayer’s hematoxylin and eosin (Preece, 1972). Zmmunohistochemistry. Fresh liver sections (2 X 4 X 4 mm) were frozen in isopentane at liquid nitrogen temperatures and freeze-dried under vacuum at -40°C overnight for immunohistochemistry. Tissues were vacuum-infiltrated with GMA monomer, allowed to polymerize overnight, and sectioned. Primary rabbit antitrout P450 antibody and a swine antibody to the rabbit immunoglobulins followed by the rabbit horseradish peroxidase-antiperoxidase (PAP) complex were used in the peroxidase-antiperoxidase immunostaining technique. The slides, following the immunostaining, were incubated with 3,3’-diaminobenzidine tetrahydrochloride for 5 min, rinsed with water, counterstained with Mayer’s hematoxylin (2 min), blued in ammonia water, and prepared for viewing. Statistical comparisons between groups for all quantitative assayswere performed using the t test and two-way ANOVA at p G 0.05.

RESULTS

AND

DISCUSSION

When BNF was coadministered with CC& there was an apparent protective effect

against CC& hepatotoxicity as evidenced by reduced SGPT/SGOT levels (Fig. 1). CCL resulted in significant reductions in hepatic EROD activity as well as in LMdb and LM2 content (Table 1). These toxic effects of CC& were in part ameliorated by BNF coadministration. Immunochemical staining for LMai, was demonstrated as an increased granular appearance for BNF and BNF/CC1, treatments (Fig. 2). In both cases LMdb was distributed in a homogeneous, apparently unorganized staining pattern. Evidence of cellular damage for the CCL, treatment was demonstrated microscopically (H&E) in some animals as diminished cellulardefinition, nuclear size alterations, and cellular vacuolation (Fig. 3). CC4 administered to flounder under the conditions described resulted in the reduction of both the major PAH-inducible and constitutive P450 isozymes. The mechanism for the isozyme destruction is unknown. It is possible that both isozymes may be involved in the suicide substrate metabolism of CCL,. Alternatively the wholesale loss of P450 (LMdb and LMJ may result from a chaotrophic action of CC& (Moody et al., 1986), or by the diffusive action of reactive intermediates. Recent studies in mammals have demonstrated the destruction of the PAH-inducible isozyme (P45Od) with CCL adminis-

SHORT COMMUNICATION

370

3~.

2. Immunohistochemical

localization of LMdb in flounder liver sections. Treatments: (A) control:

(B)CCL,; (C) BNF: and (D) BNF/CCl+ The diffuse granular staining demonstrates homogeneous distributiol ‘1of P450 LM4b with BNF and BNF/CC& treatments (arrows) (PAP-LM,,;

450x).

SHORT

COMMUNICATION

FIG. 2-Continued

371

372

SHORT

FIG 3. Light photomicrograph resulted in areas with diminished (Hand E X450).

COMMUNICATION

of liver sections cellular definition

from flounder. (a) control: and nuclear size alterations

(b) Ccl, (1 ml/kg). CCL in some animals (a ITOW)

373

SHORT COMMUNICATION

tration (Sesardic et al., 1989). The findings presented here in the flounder further support the involvement of PAH-inducible isozymes in CCL, hepatotoxicity. Administration of CCL, to BNF-pretreated flounder altered but did not significantly abolish the inductive response of LMkb content or EROD activity in the time frame of the study. Maintenance of the inductive effect of BNF with CC& coadministration could be related to the time of sampling or stoichiometric considerations between the induced LM4,, isozyme and the toxicologically significant Ccl4 species. The protective nature of BNF as noted by serum enzymes and LM2 levels suggest that a more plausible explanation may be the induction of biotransformation enzymes responsible for the production of nontoxic CC& metabolites. PAHs have been shown to be active in the induction of both phase I and phase II enzymes in other species (Jefcoate, 1983). Alterations in LMdb content with the various treatments were nearly stoichiometric with the corresponding EROD activities. These results reflect the high specificity of EROD for PAH-inducible P450. Importantly, these results also suggest that activity was not affected independently from the P450 isozyme content. The reductions in EROD activity with CC& administration were most probably related to destruction of P450. Although BNF and BNF/CCh treatment groups demonstrated hepatic immunochemical staining with LMdb antibody, the control and Ccl, treatments lacked definitive staining with the peroxidase-antiperoxidase method utilized. These results were probably related to the relative isozyme content and the method sensitivity. Immunological staining of livers from BNF-treated animals demonstrated a uniform LM4,-P450 distribution throughout the liver parenchyma. These results in the flounder, like those reported for the scup and rainbow trout (Miller et al., 1988), demonstrate that the BNF-inducible isozyme was not regionally distributed.

Mechanistically, these studies have demonstrated (I) that CC& toxicity in the flounder affected both the LMb and LMz P-450 isozymes, (2) that BNF was protective against Ccl, hepatotoxicity, (3) that an inductive response could be maintained in the face of CC14 administration, and (4) that P450 LM4,, was uniformly distributed in the liver of the flounder. While the data presented suggest that P450 induction, even in the presence of a hepatotoxin, may have utility as an environmental monitoring tool, the conditions of dose, period of exposure, and sequence of hepatotoxin-inducer exposure can greatly influence the hepatotoxin/inducer interaction. It is conceivable that at some point P450 induction and hepatotoxin-induced reductions would lead to no demonstrable inductive effect. Furthermore, hepatotoxins other than CCL, may elicit differing profiles of interaction. ACKNOWLEDGMENTS We thank Mona Lemoyne and Cheryl Crowder for their valuable assistance. This work was supported by the Lucille P. Markey Charitable Trust and the Mount Desert Island Biological Laboratory-Marine and Freshwater Biomedical Sciences Specialized Research Center Grant EHS 1 P30 ES03828-02.

REFERENCES BARON, J., REDICK, J. A., AND GUENGERICH, F. P. (1978). Immunohistochemical localization of cytochromes P-450 in rat liver. Life Sci. 23,2627-2632. BURKE, M. D., AND MAYER, R. T. (1974). Ethoxyresorufin: Direct fluorimetric assayof a microsomal Odealkylation which is preferentially inducible by 3-methyl-cholanthrene. DrugMetab. Dispos. 2,583-588. DANSCHER, G., AND NORGAARD, J. 0. R. (1983). Light microscopic visualization of colloidal gold on resinembedded tissue. J. Histochem. Cytochem. 31, I3941398.

ELCOMBE,C.R.,ANDLECH, J.J.(1979).Inductionand characterization of hemo-protein(s) P-450 and monooxygenase in rainbow trout (Salmo gairdnen]. Toxicol. Appl. Pharmacol. 49,437-450. ENGLISH, J. C., AND ANDERS, M. W.(1985). Evidence for metabolism of N-nitrosodimethylamine and car-

374

SHORT COMMUNICATION

bon tetrachloride by a common isozyme of cytochrome P-450. Drug Metab. Dispos. 13,449-452. GINGERICH, W. H. (1982). Hepatic toxicology of fishes. In Aquatic Toxicology (L. J. Weber, Ed.), pp. 55-105. Raven Press. New York. HEAD, B., MOODY, D. E.. Woo, C. H., AND SMIJCKLER, E. A. (198 I). Alterations of specific forms of cytochrome P-450 in rat liver during acute carbon tetrachloride intoxication. Toxicol. Appl. Pharmacol. 61, 286-295. JEFCOATE, C. R. ( 1983). Integration of xenobiotic metabolism in carcinogen activation and detoxication. In Biological Basis QfDetoxication (J. Caldwell and W. B. Jakoby. Eds.). pp. 31-76. Academic Press, New York. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680-685. MELANCON, M. J., YEO. S.. AND LECH, J. J. (1987). Hepatic monooxygenase activity in carp and bullheads exposed to river water. Environ. Toxicof. Chem. 6, 127-135. MILLER, M. R., HINTON, D. E., BLAIR, J. J.. AND STEGEMAN, J. J. (1988). Immunohistochemical localization of cytochrome P-450E in liver, gill and heart of scup (Stenotomus chrysops) and rainbow trout (Salmo gairdneri). Mar. Environ. Res. 24, 37-39. MOODY, D. E., HEAD, B., Woo, C. H., JAMES. J. L., AND SMUCKLER, E. A. (1986). NADPH-dependent and independent loss of cytochrome P-450 in control and phenobarbital-induced rat hepatic microsomes incubated with carbon tetrachloride. Exp. Mol. Pathol. 44, 318-328. National Research Council (I 980). Drinking Water and Health Vol. 3, National Academy of Sciences. Washington, DC. NOCXICHI, T.. FONG, K. L., LAI, E. K., ALEXANDER, S. S., KING, M. M., OLSON, L., POYER, J. L., AND MCCAY, P. B. (1982). Specificity of a phenobarbital-induced cytochrome P-450 for metabolism of carbon tetrachloride to the trichloromethyl radical. Biochem. Phurmacol. 31,6 15-624. PAYNE, J. F. (1976). Field evaluation of benzopyrene hydroxylase induction as a monitor for marine pollution. Science 191,945-946. PREECE,A. (1972). A Manualfor Histologic Technicians. 3rd ed., Little, Brown, Boston. REITMAN, S.. AND FRANKEL, S. (1957). A calorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Amer. J. Clin. Pathol. 28,56-63.

SASAME, H. A.. CASTRO, J. A., AND GILLEI-~E, J. R. (1968). Studies on the destruction of liver microsomal cytochrome P-450 by carbon tetrachloride administration. Biochem. Pharmacol. 17, 1759- 1768. SESARDIC, D., RICH, K. J., EDWARDS, R. J., DAVIES, D. S., AND BOOBIS, A. R. (1989). Selective destruction of cytochrome P-450d and associated monooxygenase activity by carbon tetrachloride in the rat. Xenobiotica 19,795-S 11. STATHAM, C. N., CROFT, W. A., AND LECH. J. J. (1978). Uptake, distribution and effects of carbon tetrachloride in rainbow trout. (Salmogairdneri) Toxicol. Appl. Pharmacol. 45,13 1- 140. VODICNIK, M. J., RAU. L. A., AND LECH. J. J. (1984). The effect of monooxygenase inducing agents on the incorporation of [‘%]methionine into hepatic microsomal protein of rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 79C, 21 L-276. WILLIAMS, D. E., AND BUHLER, D. R. (I 983). Purified form of cytochrome P-450 from rainbow trout with high activity toward conversion of aflatoxin B, to aflatoxin Br-2.3-epoxide. Cancer Res. 43,4752-47X. WILLIAMS, D. E., AND BUHLER, D. R. (1984). Benzo[a]pyrene-hydroxylase catalyzed by purified isozymes of cytochrome P-450 from /3-naphthoflavone-fed rainbow trout. Biochem. Pharmacol. 33,3743-3753. WILLIAMS, D. E., MASTERS, B. S. S., LECH, J. J., AND BUHLER, D. R. (1986). Sex differences in cytochrome P-450 isozyme composition and activity in kidney microsomes of mature rainbow trout. Biochem. Pharmacol. 35,20 17-2023. YOSHIZAWA, H., UCHIMARU, R.. KAMATAKI, T., KATO, R., AND UENO, Y. (1982). Metabolism and activation of aflatoxin Br by reconstituted cytochrome P-450 system of rat liver. Cancer Res. 42, I 120-I 124. KEVIN M. KLEINO~*.~ BRAD F. DROY* DONALD R. BUHLER~ DAVID E. WrLLIAMst * Department of Veterinary Physiology Pharmacology and Toxicology, School Q/’ Veterinary Medicine Louisiana State University Baton Rouge, Louisiana 70803 t Marine Fresh water Biomedical Sciences Specialized Center for Research Oregon State University “. Corvallis, Oregon 97331 Received August 9, I989 ’ To whom correspondence should be addressed.