M. Machala ( ) · K.Nezveda Veterinary Research Institute, 62132 Brno, Czech Republic A. Irizar · A.Bu-Abbas ·C. Ioannides Molecular Toxicology Group, University of Surrey, Guildford, Surrey,GU2 5XH, UK Arch Toxicol (1996) 71: 57—63 ( Springer-Verlag 1996 METABOLIC ACTIVATION/INACTIVATION Miroslav Machala · Karel Nezveda · Amaia Irizar Ali Bu-Abbas · Costas Ioannides Expression and inducibility of cytochrome P450 proteins in the liver of chick embryo Received: 6 February 1996/Accepted: 3 May 1996 AbstractThe expression andinducibilityof cyto- chrome P450 proteins in the liver of chick embryo were investigated using substrate probes and/or immunolo- gically using polyclonal antibodies to the mammalian isoforms.Antibodies to CYP1A1 recognised a single protein which wasinducible by structurally diverse chemicals, including aminocompounds, and was paral- lelled by increases in the O-dealkylations of ethoxy- and methoxyresorufin and in the bioactivation of Glu- P-1. When probed with antibodies to CYP2E1 an im- munoreacting protein was revealed which was induced by phenobarbital but not acetone; a second protein became apparent following the treatment with pheno- barbital. The increase in apoprotein levels was accom- panied by similar increasesin p-nitrophenolhy- droxylase.Antibodiesto CYP2C11 recognised two immunorelated proteins, of which one was inducible by phenobarbital. The same inducer, but not dex- amethasone, enhanced the N-demethylation of eryth- romycin but antibodies to rat CYP3A1 failed to detect any proteins. Finally, lauric acid hydroxylation was not detectable in chick embryo and, moreover,no im- munoreacting band was visible following probing of microsomes with anti-CYP4A1. It is concluded that proteins immunorelated to the mammalian CYP1 and CYP2 families are expressed in chick embryo but the regulation of the latter family in the embryo by exogenous chemicals differs markedly from that estab- lished for mammals. Key words Cytochromes P450 · Enzyme induction · Chick embryo Introduction The cytochromeP450-dependentmixed-function oxidases comprises a ubiquitous enzyme system the presenceof which has been demonstrated in both prokaryotic and eukaryotic cells. It is characterized by a very broad substrate specificity — the consequence existing as a number of proteins assigned to families and subfamilies, each protein displaying its own sub- strate specificity (Guengerich 1993). Cytochromes P45 can catalyse the metabolism of structurally diverse xenobiotics as well as of critical endogenous substrate such as hormones, vitamins and fatty acids. Families one (CYP1),two (CYP2) and three (CYP3) are largely involved in the metabolism ofxenobioticswhich may additionally induce them. CYP4 is also an in- ducible family, but its main catalytic function is in the metabolism of fatty acids and prostaglandins (Bain et al.1985). Chicken hepatic cytochromes P450 have not attrac- ted the same attention as their rodentorthologues. Proteins belonging to the CYP1A subfamily have been described in theliver of chick embryo and adult chicken by a number of workers, and their inducibility by polycyclic aromatic hydrocarbons and planar poly- chlorinated biphenyls has been established (Sinclair et al. 1989; Hokama et al. 1988; Brunstro¨ m and Ander- sson 1988; Nakai et al. 1992; Machala et al. 1996). Tw phenobarbital-inducible forms, termed CYP2H1 and CYP2H2, probably belonging to the CYP2B subfam- ily, have been demonstrated in the liver of chickens an chicken embryos (Hansen and May 1989; Sinclair et al 1990).However,these chicken proteins have limited structural similarity to the mammalian forms, but dis- play N-demethylation activity towards aminopyrine and benzphetamine; CYP2H1 can also p-hydroxylate nitrophenol(Sinclair et al. 1990).A cytochrome P450 form,inducible by acetone and displaying high p-nitrophenol hydroxylase activity, thus presumably