Monoclonal antibodies to cytochromeP-450 isozymes

TIPS - January 1983 m

Current awareness series Key developments

in pharmacology

Monoclonal antibodies to cytochrome P-450 isozymes Liver microsomal cytochrome P-450 was originally thought to be a single protein species with an extraordinarily broad substrate specificity. In the 20 years or more since the demonstration of the pivotal role of this remarkable cytochrome in the metabolism of xenobiotics, this original idea has been replaced by the realization that many P-450 isozymes exist. The existence of these multiple isozymes, which have varying substrate specificities and whose levels can be independently modulated in response to challenge with xenobiotic chemicals, ensures that a broad spectrum of foreign compounds can be metabolized while the deleterious effects of unwanted oxidation of various hormones or protoxins will be minimized. In a recent study 1, S. S. Parket al. utilized hybridoma technology to produce some potentially very useful tools for the investigation of structural and catalytic differences among P-450 isozymes. Monoclonal antibodies can be produced by fusing antibody-producing B lymphocytes with myeloma cells, which can be maintained indefinitely in animal hosts or in culture. When the resulting hybrids are cloned, cells are obtained that produce a single, highly specific antibody in virtually unlimited quantities. Since these antibodies are specific to a single antigenic determinant, consisting of a small number of amino acids, they can be extremely valuable in discriminating small structural differences between two related proteins or isozymes. The authors have produced monoclonal antibodies to two P-450 isozymes: P-450LM2, a phenobarbital-inducible form isolated from rabbit liver microsomes, and P-450LM4, a form induced by 5,6benzoflavone and polycyclic aromatic hydrocarbons, also isolated from rabbit liver microsomes. Seven clones were obtained that produced antibodies to P-450LM2, and which could be grouped into three sub-

setS: those producing antibody that inhibited LM2-catalyzed aryl hydrocarbon hydroxylase activity; those producing antibody that precipitated LM2 but did not inhibit it, and those producing antibody that bound to, but did not inhibit or precipitate LM2. They obtained 4 clones that produced antibody to P-450 LM4; 3 produced lgG antibodies, and one produced lgM antibodies. All of the IgG antibodies bound to LM4 and inhibited LM4-catalyzed aryl hydrocarbon hydroxylase activity, but none precipitated pure LM4 measurable by Ouchterlony double diffusion analysis. The IgM antibody bound to LM4, but was not tested as an inhibitor of aryl hydrocarbon hydroxylase activity. The cross-reactivity of the two groups of antibodies was tested; antibodies to LM2 did not precipitate or inhibit LM4, and antibodies to LM4 did not precipitate or inhibit LM2. These are two somewhat restrictive measures of antibody-antigen interaction, especially with monoclonal antibodies which do not readily form precipitatable 'lattices' with the antigen. A radiometric binding test, which the authors employed to measure reactivity to the intended antigen but did not use to measure cross-reactivity with the other isozyme might have revealed cross-reactivity, and by inference structural homology, not shown by the influence of the antibody on enzyme activity alone. It was also shown that those antibodies which inhibited LM2-catalyzed aryl hydrocarbon hydroxylase activity inhibited the production by LM2 of all identifiable benzo(a)pyrene metabolites as well as the O-de-ethylation of ethoxycoumarin by LM2. The use of monoclonal antibodies for the investigation of P-450-1inked monooxygenase systems promises to answer many questions which may not be approachable by other techniques. For example, it may eventually be possible to

prepare antibodies specific for substrate or sixth ligand binding sites, or for sites of interaction of P-450 with P-450 reductase or cytochrome bs. The ability to block or deactivate one of these sites without perturbing the others could be useful in studying mechanistic questions. Of more immediate interest, however, may be the applicability of monoclonal antibodies to delineation of the degree of structural homology among various P-450 isozymes. These techniques should eventually allow assessment of the interrelation of catalytically similar P-450s obtained from different organs, individuals, or species, as well as structural relationships of catalytically dissimilar P-450s obtained from the same tissue. They may also provide a means of settling the current controversy over the number and characteristics of P-450 isozymes induced by polycyclic aromatic hydrocarbonsz3 Admittedly a great deal of work will be necessary before monoclonal antibodies can be applied to questions such as those outlined above. What this paper and other similar studies4 5 show is the feasibility of production of these valuable tools. It provokes many exciting questions. THOMAS M GUENTHNER Department of Pharmacology. Umverstty of Illinois Medwal Center. 835 S Wolcott Av , P.O. Bo.t 6998. Chwago, Illinois 6068(I, U ~.A

Reading

list

I park. S. S , Cha. S -J , Mdler. H . Persson. A

V ,

Coon, M. J and Gelbom, H V (1982) Mol. Pharmacol. 21,248-258 2 Thomas, P E, Rok, L M. Ryan, D E and Levm, W (1981)./ Bud Chem. 256, 1044-1052 3 Chen. Y T, Lang, M A , Jensen. N M , Neglshi, M , Tukey, R. H , Sidransky,E., Guenthner,T M and Neben, D. W (1982)Eur J. Rtochem. 122, 361-368 4 park, S S., Persson. A V, Coon, M J and Gelboin, H. V (1980)FEBS l,ert 116,231-235 5 Boobis, A. R., Slade, M, B , Stem, C, Lewis, K. M and Davies, D S. (1981) l.tfe .¢;ct. 29, 1,443-1448

FJscvcr B~edJcal Press 1981 0165 - 6147181/(J4X~l- 0(XX]I$OI 00