EJJecfs o/StreJs on Marine organisms (Gray, J. S. & Christiansen, M. E., eds.), pp. 527-543, J. Wiley and Sons Ltd, Chichester and New York Batel, R., Bihari, N. & Zahn. R. K. (1988) C‘omp. Riochem. I’hysiol. 90C, 435-438 Waters, L. C. & Nix, C. E. (1988) I’eAtic, Hiochem. I’hysiol. 30, 214-227 Kirchin, M. A. (1988) Cyrochromc /’-4SO o/ the common mrt.s.sel, Mytiiris edrilis L: purtiul purijicztion and charucterizution, Ph.D. thesis, University of Surrey, U.K. Gilewicz, M., Guillaume, J. C., Carles, D.. Leveau, M. & Bertrand, J . C. ( 1984) Mar. Biol. 80, 155- 159 M. Wilber, personal communication Kaipainen, P., Koivusaari, U. & Long, M. (1985) C’omp. Riochem. I’hysiol. SIC, 293-296 Stegeman, J. J.(1985)Mur.Bio/. 89,21-30 Bihari. N., Hatel, R., Kurelec, B. & Zahn, R. K. ( 1984) Sci. 7utul Environ. 35,41-51 Livingstone, D. R. & Farrar, S. V. ( 1 984) Sci. Total Environ. 39, 209-235 Takimoto, Y., Ohshima, M. & Miyamoto, J. ( I 987) Ecoroxicol. Environ. Suf 13, 118-125 Smith, S. L. & Mitchell, M. J . ( I 988) Biochem. Biophys. Hex C‘ommun. 154,559-563
19 62. Soumoff, C. ’& Skinner, D. M. ( 1988) Cbmp. Biochem. I’hy,siol. 93C, 139- 144 63. Singer, S. C. & Lee, R. F. ( 1 977) Biol. Bull. 153, 377-386 64. Fries, C. R. & Lee, R. F. ( 1984) Mar. Biol. 79, 187- 193 65. Conner, J. W. & Lee, R. F. ( 1 982) in Cytochrorne 1’4.50 Biochemistry, Biophysics and Environrnenral Implications (Laitinen, M. & Hanninen, O., eds.), pp. 245-249, Academic Press, New York 66. Quattrochi, L. C. & Lee, R. F. ( 1984) C’omp. Riochrm. 1’hy.siol. 7 9 c , 171-176 67. James,M.0.(1984) Mar. Envir. Kes. 14, 1 - 1 I 68. Spry, J. A., Livingstone, D. R., Wiseman, A,, Gibson, G. G. & Goldfarb, P. S.( 1989) Hiochem. Soc. Truns. 18, 10 13- I 0 14 69. Singer, S. C., March, P. E., Jr, Gonsoulin, F. & Lee, R. F. ( 1 980) Chmp. Biochern. I’hysiol. 65C, 129- I 34 70. Walters, J. M., Cain, R. B., Higgins, 1. J. & Corner, E. D. S. ( 1 979) J . Mar. Riol. Ass. U.K. 59,553-563 71. Lindstrom-Seppa, P. & Hanninen, 0. (1986) Arch. Toxicol. (Suppl. Y), 374-377
Received 1 I August 1989
Structure, function and regulation of cytochrome P-450forms in fish JOHN J. STEGEMAN,* BRUCE R. WOODIN and ROXANNA M. SMOLOWITZ Biology Ilepurrment, Woods Hole Oceunogruphic Institution, Woods Hole, M A 02543, U.S.A.
450 proteins involved in xenobiotic metabolism in fish, drawing heavily on a more detailed review . We also present preliminary results of new studies on important aspects of relationships among cytochrome 1’-450 forms in fish.
Introdir ct iori
Cytochrome ”-450 plays key roles in determining the biological action including toxicity of pollutant chemicals, drugs and therapeutic agents, and many chemical carcinogens, catalysing the activation and inactivation of these compounds. The extent to which these functions occur in different individuals or species exposed to various compounds will depend to a large degree on the complement of different cytochrome ”-450 proteins present, their catalytic functions, and their regulation. The diverse structure and function of mammalian cytochrome f - 4 5 0 proteins are well known from studies with purified cytochromes ”-450, and with monoclonal and polyclonal antibodies and cDNA probes to these forms 111. These mammalian forms are well represented in a recently developed nomenclature based on gene sequence information for cytochrome I-’-450 forms, which describes a gene superfamily and organizes all sequences known at the time into P450 gene families and subfamilies 12 I. Research on mammalian cytochrome P-450 continues to dominate the literature, but there is a growing recognition of its biological significance in other animals, and of our need to know the diversity and biochemistry of cytochrome P-450 proteins in these groups. The 20000 species of fish extant ,represent about one-half of the known vertebrate species. The fishes present extraordinary diversity, inhabiting virtually all of the world’s aquatic environments. They also represent a significant source of protein for humans. The study of cytochrome ”-450 forms in fish thus acquires importance from evolutionary and toxicological standpoints. In this paper, we summarize information on cytochrome P*To whom correspondence should be addressed. Abbreviations used: EROD, ethoxyresororufin 0-de-ethylase; AHH, aryl hydrocarbon hydroxylase; PAH, polynuclear aromatic hydrocarbon; BNF, P-naphthoflavone; MAb, monoclonal antibody; PB, phenobarbital.
Cytochrome 1’-450jorms in fish ’The general properties of total microsomal cytochrome 1’450 in liver and other organs of various fish species have been described in detail in earlier reviews [ 5 , 6 ] .As in mammals, the diversity of catalytic functions of fish liver microsoma1 cytochrome 1’-450 is extensive. Reactions include epoxidation, hydroxylation, dealkylation, S-oxidation, Noxidation and most other types of reactions ascribed to cytochrome 1’-450 in mammalian systems. Many of the commonly used substrates for evaluating mono-oxygenase or mixed-function oxidase reactions are, in mammalian systems, linked to a single form or a family of cytochrome P450. A multiplicity of cytochrome P-450 forms in fish can be inferred by comparing patterns of specific catalytic functions in fish microsomal systems with the patterns of activities observed in mammals. Some of these activities, such as ethoxyresorufin 0-de-ethylase (EROD) activity and testosterone 6P-hydroxylase activity have been known to be under different regulation 171, clearly indicating the presence of multiple catalysts. Based on physicochemical and catalytic properties, distinct forms of hepatic cytochrome 1’-450 have been identified in several fish species. Five cytochrome P-450 fractions have been obtained from the marine teleost scup (Stenotomus chrysops) ,four from cod (Gudus morhuo) ,and as many as nine from the freshwater species, rainbow trout (Sulmo gairdnerii) [lo].Many of the forms identified in these species have been considered in a recent reviews 14, 111. Antibodies have been prepared to a number of these cytochromes P-450, and cross-reacting proteins have been identified in other teleosts by immunoblot analysis. Relationships between most of the teleost forms thus far described are not yet known. However, as previously summarized [ 1 11, a close relationship has been demonstrated between cytochromes P450E from scup, P450c from cod,
BIOCHEMICAL SOCIETY TRANSACTIONS
20 and P450LM4 from rainbow trout. These proteins have similar spectral properties (Fez -CO absorbance maxima at 447 nm) and are the catalysts for aryl hydrocarbon hydroxylase (AHH) and EROD activities. These forms are all strongly inhibited by a-naphthoflavone. Western-blot analysis has demonstrated that these proteins are also the primary forms induced in these species by polynuclear aromatic hydrocarbon (PAH) and p-naphthoflavone (BNF). More recently, a reciprocal analysis with antibodies to each of these purified forms has demonstrated an antigenic relationship among them [ 121. The molecular and catalytic properties of the teleost A H H and EROD catalysts are very similar to those of the PAH-inducible P450 1Al (see ) enzymes in mammals. Consistent with this, antibodies to these teleost proteins also recognize the PAH- or BNF-inducible cytochrome f-450 forms in mammalian species. A relationship between scup P450E and rat P450c was first demonstrated using monoclonal antibodies (MAb) to these proteins. MAb 1-12-3 to scup P450E, and MAb C4 and C6 to rat P450c, each recognized the other protein [ 13, 141. Further analysis with antibodies to various cytochromes P-450 has confirmed relationships between hydrocarbon-inducible cytochromes 1’-450 throughout the vertebrata . Recently, a cDNA for a teleost BNF-inducible cytochrome P-450, trout P450LM4b, also called P,450, has been cloned and sequenced [ 151. The sequence similarity between the trout P,450 coding regions and the same regions of mammalian P450 IA forms is great enough to conclude that the trout P,450 is in fact a teleost representative of P450 1Al. +
Teleost cytochrome 1’450 IA I sequence comparison
The amino acid sequences of teleost cytochrome P,450 (P450 I A l ) forms show an intriguing degree of similarity for a limited segment of the N-terminal region. The N-terminal amino acid sequence reported for scup P450E ,and that inferred from trout P,450 [ 151, have only one of the first 10 residues different. Recently we obtained (unpublished work) additional sequence data for the N-terminus of scup P450E; the same high degree of similarity appeared in the first 25 residues of the two teleost proteins (Table 1).By comparison, this region of the mammalian P450 1Al representatives shows a like degree of similarity only between the two rodent species, while rabbits and rats differ substantially (Table 1). The sequence similarity evident in these proteins, even in this limited portion of the molecules, is higher than might be expected based on the phylogenetic relationship of the species from which they were obtained. The teleost orders Salmoniformes (trout) and Perciformes (scup) have diverged Table I . Sequence similarity in N-terminal 25 residues of cytochrome 1’450 IAl forms Comparison to:
Rat Mouse Rabbit Human
Rat Rat Rat Rat
84 64 68
in the geological record at least as far back as 70 mybp. The mammalian orders Lagomorpha (rabbits) and Rodentia (rodents) appear to have diverged at about the same time, of the order of > 60 million years ago. Highly similar proteins in distantly related species suggest the action of evolutionary pressures to conserve these proteins. The N-terminal region of cytochromes P-450 includes a sequence apparently involved in insertion of the polypeptide into the membrane. It is possible that the similarity in scup and trout N-terminal regions reflects some feature related to similarities in poikilotherm endoplasmic reticulum membranes, different from those in mammals. Xenobiotic induction of teleost cytochrome P450 IA I
The functional and structural similarities evident in the proteins indicate that the induction of P450 I A l in teleost and mammalian species might occur by similar mechanisms. Measurement of mRNA by in vitro translation and/or Northern-blot analysis, and of protein by Western-blot analysis, has revealed temporal relationships with the mRNA for P450E or P,450 increasing before increases in the levels of P450E protein occur [16, 171. This is consistent with the operation of a receptor-mediated system in teleosts similar to that found in mammals. However, definitive evidence for the nature of such receptor in fish is lacking, and the structural requirements for the range of inducers not yet defined. In addition to induction in liver, P450E is induced in a number of extrahepatic organs of fish. lmmunoblot analysis of scup gill, kidney, gut and heart with MAb 1-12-3 shows a pronounced induction of P450E by BNF in each of these organs. The induction in heart is particularly strong, with the P450E content increasing from less than 0.05 to 0.5 nmol/ mg of protein of cardiac microsomes. Furthermore, the induction in extrahepatic organs has been found to occur in selected cell types, identified using MAb 1-12-3 to scup P450E. In some organs there is a restricted localization. Thus, the pillar cells (endothelial cells) of gill and the endothelial cells in the heart are the primary sites of P450E induction in those organs. Additional cell types are involved in some other organs (Table 2). The biological features controlling the induction in different cell types are yet to be established. Furthermore, whether modulators of P450 IA 1 expression in liver, such as steroids or temperature, also affect induction in these other organs is unknown. Phenobarbital induction
A major distinction between fish and mammalian cytochromes P 4 5 0 that continues to attract attention is the lack of any clear-cut induction by phenobarbital (PB) or pheno-
Table 2. Cellular characterization of cytochrome 1’4SOE induced in scup
Similarity ( O h )
*Data on mammalian N-terminal residues are from Black & Coon [ 181and from Nelson & Strobel [ 191. Trout data are from [ 1 51. ?Direct sequencing of P450E from scup was accomplished using an Applied Biosystems automatic sequencer, according to the methods outlined by ABI. The first 10 residues were identical to those previously determined by manual Edman degradation .
Induction verified by immunoblot
Gill Heart Gut Brain
+ + +-
Cell types where P450E is induced Hepatocytes, ductular epithelium, endothelium Tubular and ductal epithelium, endothelium Pillar cells and epithelium Endocardium and endothelium Mucosal epithelium and endothelium Endothelium
*Formalin-fixed, paraffin-embedded sections stained with MAB 1-1 2-3 to P450E and peroxidase-labelled second antibodies, as
described in .
D EV EL0 PM E N T 0F M I X ED- F U N CTI0N 0X 1DAS E S Table 3 . I’B-induction of anti-scup 1’4SOH cross-reacrive protein in ral liver micrusomes
Supported by N.I.H. grants ES-04220 and CA-44306. Contribution No. 7 165 of the Woods Hole Oceanographic Institution.
Western blotting was done as in , with anti-scup P450B. Data are expressed as densitometric area per unit of microsomal protein. Equivalent amounts of microsomal protein were applied to the gels. Rats (Sprague-Dawley) had been treated with 8 0 mg o f phenobarbital/kg and rnicrosorncs prepared on dxy 3.
1. Ortiz de Montellano, P. R. ( 1986) in C’yloc~hrome1’450. .Strirc,lure, Mec~harii.sm.ririrl B i o c ~ h e r n i ~Plenum t ~ ~ , Press, New York 2. Nebert, D. W.. Adesnik, M., Coon. M. J., Estabrook, R. W.. Gonzalez. F. J., Guengerich, F. P.. Gunsalus, 1. C., Johnson, E. F., Kemper. B.. Levin, W.. Phillips. I. R.,Sato, R. & Water-
rnan,M.R.(19X7)DNA6, 1-11 3. Cohen. D. M. ( 1979) /’roc. C’ulif:Ac~rd..Sci. 17, 34 1-346 4. Stegernan,J.J.(1989)Xenobioticu 19, 1093-1 110 5. Bend, J. R. di James, M. 0. ( I 978) in Hiochemicul and H i o physicul I’er.spec~ives in Murine Biology (Malins, D. C. di Sargent, J. R.. eds.), pp. 125- 188, Academic Press, New York 6 . Stegernan, J. J. ( I 9 8 I ) in I’olyc:,.c~lic~ tfydrocurbons and C’cinwr (Gelboin, H. G. & Ts’O, P. 0. P., eds.), pp. 1-60, Academic
barbital-type inducers in teleosts. T h e lack of PB-type induction in some groups (fish, reptilia, amphibia) must b e due to either a lack of structural genejs) coding for PB-inducible forms, o r t o the lack of a regulatory mechanism like that acting to control expression of the related genes in mammals. T h e mechanism of PB induction has yet to b e described. However, we recently obtained evidence indicating the identity of a fish cytochrome 1’-450 related to a major PBinducible form in mammals. We have detected single proteins in untreated fish liver microsomes that strongly cross-react with antibodies to mammalian PB-inducible P450 IlBl (rat P450b or PB4) (J. Stegeman, B. Woodin & D. Waxman, unpublished work). Antibodies to scup P450B [ 131 strongly recognize an identical band in these same species. T h e antiscup P 4 5 0 B also detected the same bands in PB-induced rats as seen by anti-P450 PB-4 (Table 3). Antibodies to the scup and rat proteins also recognized the antigens, scup P450B and rat P 4 5 0 PB-4 (IIBl),purified from these species. Defining relationships between these anti-P450 IIB 1 cross-reacting proteins in fish and the mammalian PB-inducible proteins, should help to focus our efforts to understand the lack of PB-response in fish, and could aid in identifying thc mechanism of PB induction. Further defining the spectrum of cytochromes 1’-450 in teleosts will enhance their utility as model systems in toxicology or carcinogenesis, as well as providing a basis for predicting susceptibility to environmental chemical effects in aquatic systems.
Appl. I’hormac,ol. 94, 246-253 I X . Black, S. di Coon, M. (1986) in (yiorhrome /’-450. Sirrrc?rm~, Mechunism, and Niocliemistry (Ortiz dc Montellano, P. R., ed.), pp. 16 1-2 16. Plenum Press, New York 19. Nelson, D. R. & Strobel, H. W. (1987) Mol. Riol. Evol. 4, 572-593 20. Smolowitz. R. M.. Moore. M. J. di Sicgeman. J. J. ( 1 9x9) blur. Environ. Hex in the press.
We are grateful to our collaborators. particularly M. Kochersperger, Applied Biosystems, for contributions t o this research.
Received I I August 19x9
Press, New York 7. Stegeman. J. J. & Woodin, B. A. (1984) Mar. Environ. H c ~ s .14, 422-425 8. Klotz. A,, Stegeman, J . J. & Walsh, C. (1983) Arch. RiocArm. Biophys. 226,578-592 9. Goksoyr, A . ( 1985) Hiochim. Biophys. ACIU840,409-417 10. Miranda, C. L.. Wang, J.-L., Henderson, M. C. & Buhler. D. R. (1989) Arch. Hiochem. Biuphys. 268, 227-238 I 1. Stegeman, J. J. & Kloepper-Sam. P. J. ( 1987) Environ. Heulth I’erspect. 17, X7-95 12. Goksoyr, A.. Andcrsson, T., Buhler. D. R.. Stegeman, J. J.. Williams. D. E. & Forlin, L. ( I Y X Y j I ~ i o d i c w I’liurmucd. . in the
press 3. Klotz, A,, Siegeman, J., Woodin, B., Snowberger, E., Thomas, P. & Walsh, C. ( 19x6) Arch. Biochem. Hiophyv. 249, 326-338 4. Park, S., Miller, H., Klotz, A,. Kloepper-Sams, P., Stegernan, J. & Gelboin. H. ( 1986) Arch. Hiochem. Hiop/iy.s. 249, 339-350 5. Heilmann. L. J.. Sheen, Y.-Y., Bigelow. S. W. & Nebert, 0. W. ( 1988) IINA 7,379-387 6 . Kloepper-Sams. P. J. & Stegeman. J. J. ( 1 989) Arch. Hiochcw1. Biophys. 268,525-535
7. Haasch, M. L., Kleinow, K. M. & Lech, J. J. ( 1 988) Toxic,ol.
Molecular genetics of the human cytochrome P-450system C. R O L A N D WOLF,*? J O H N S. MILES,* A L A N G O U G H $ and NIGEL K. SPURR$ ’Iinperiul C’uricer Kesrurch Fiitid? Labomtory o f Muleciclur I’licirtiitri~ok~~~. Uiriwrsitv Ilepcrrttneiit of 1~ioc~hemi.stty Ilirgh Kobsoti ~iril[iiirg,(ieorgc Syittcrr., Ediithirrgh Et I8 YXD, U.K. oird $ Iiriyeriul Ciiircer Kesetrrch Frrnd, Ilirmuir Genetic Kcsoirrces Uiiit. C‘lure Ilull Lahorutories, Nluriche Luiie. Soirtlr Miinitis, ~’olter.s k i r , Ilerts. EN6 .?LK, U.K.
Iritrocliiction Adaption t o chemicals in the environment is a major factor in the survival and evolution o f living species and, as ii consequence, all living organisms have developed mechanisms t o combat the cytotoxic effects o f environmental chemicals. T h e systems which have evolved are characteristic for each organism and their evolution has undoubtcdly depended o n many factors, including the living environment, source of nutrient. etc. Yl‘o whom correspondence should be addressed.
O n the basis o f the above, it would be highly surprising if the detoxication capacity of different mammalian species, for example, rats and man would bc the same, and diffcrcnccs in the drug-metabolizing enzymes, such as the cytochromes 1’4 5 0 o r glutathione transferaseh arc expected. This obviously has important implications for the extrapolation of toxicological data obtaincd in small mammals t o man, and in many cases will explain significant differenccs between small mammals in their susceptibility to certain chemical toxins and carcinogens. It is, therefore, of central importance to identify where the similaritics and differences in drug-metabolizing enzymes between small mammals and man lie. T h e cytochrome 1’-450-dcpcndcnt mono-oxygenasc system is of central importance in determining our response to foreign chemicals. These proteins arc intimately involvcd in both the deactivation of chemical toxins as well as in the metabolism of certain compounds t o toxic. mutagenic and carcinogenic products. Cytochromes 1’-450 are encoded by several multigcnc familics with each protein exhibiting a unique spectrum of activities [ 1-81 (Table 1). T h e major tissue of drug metabolism is the liver and the individual vari-