Human group II phospholipase A2. Characterization of monoclonal antibodies and immunochemical quantitation of the protein in synovial fluid.

Murine monoclonal and rabbit polyclonal antibodies were generated against human group II phospholipase A2 (PLA2) in order to study the role of this enzyme in inflammatory disease, the source of its synthesis, and the interaction of PLA2 with its substrate. Monoclonal antibody PLA187 exhibits potent inhibitory activity toward human PLA2 using autoclaved E. coli membranes as the substrate. Three other monoclonal antibodies (PLA184, PLA185, and PLA186) also inhibit enzyme activity, but with about 50-fold less potency. Based on the results of double-antibody competition experiments and enzyme inhibition profiles, PLA184 and PLA185 appear to recognize the same epitope. Monoclonal antibody PLA186 recognizes an epitope which is spatially distinct from that recognized by PLA184/185. The results also suggest that the epitope recognized by PLA187 may overlap with both epitopes recognized by PLA186 and PLA184/185. A double-antibody sandwich ELISA was developed using a combination of PLA185 and rabbit polyclonal antibody against PLA2. The ELISA provides a sensitive and quantitative method for monitoring specifically group II PLA2 in various biological sources, independent of factors which may affect enzyme activity. We have utilized this assay to quantitate PLA2 levels in synovial fluid from the joints of individuals with rheumatoid arthritis as well as from non-arthritic joints. Our results indicate that elevated levels of group II PLA2 in synovial fluid are not necessarily associated with arthritis.

A simple, non-chromatographic purification procedure for monoclonal antibodies. Isolation of monoclonal antibodies against cytochrome P450 isozymes.

A simple, non-chromatographic purification procedure for monoclonal antibodies from mouse ascites fluid is described. This procedure, which is rapid, inexpensive, and has high capacity involves the precipitation of contaminating proteins with caprylic acid followed by precipitation of immunoglobulin using ammonium sulfate. This two-step procedure is shown to be effective for the purification of various immunoglobulins including IgG1, IgG2a and IgG2b. In the present report, more than 30 monoclonal antibodies directed against cytochrome P450 isozymes have been purified by this method and characterized.

PCBs: structure-function relationships and mechanism of action.

Numerous reports have illustrated the versatility of polychlorinated biphenyls (PCBs) and related halogenated aromatics as inducers of drug-metabolizing enzymes and the activity of individual compounds are remarkably dependent on structure. The most active PCB congeners, 3,4,4',5-tetra-, 3,3',4,4'-tetra-, 3,3',4,4',5-penta- and 3,3',4,4',5,5'-hexachlorobiphenyl, are substituted at both para and at two or more meta positions. The four coplanar PCBs resembled 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) in their mode of induction of the hepatic drug-metabolizing enzymes. These compounds induced rat hepatic microsomal benzo(a)pyrene hydroxylase (aryl hydrocarbon hydroxylase, AHH) and cytochromes P-450a, P-450c and P-450d. 3,4,4',5-Tetrachlorobiphenyl, the least active coplanar PCB, also induced dimethylaminoantipyrine N-demethylase and cytochromes P-450b+e and resembled Aroclor 1254 as an inducer of the mixed-function oxidase system. Like Aroclor 1254, all the mono-ortho- and at least eight di-ortho-chloro analogs of the coplanar PCBs exhibited a "mixed-type" induction pattern and induced microsomal AHH, dimethylaminoantipyrine NM-demethylase and cytochromes P-450a-P-450e. Quantitative structure-activity relationships (QSARs) within this series of PCBs were determined by comparing their AHH induction potencies (EC50) in rat hepatoma H-4-II-E cells and their binding affinities (ED50) for the 2,3,7,8-TCDD cytosolic receptor protein. The results showed that there was an excellent correlation between AHH induction potencies and receptor binding avidities of these compounds and the order of activity was coplanar PCBs (3,3',4,4' -tetra-, 3,3',4,4',5-penta- and 3,3',4,4',5,5'-hexachlorobiphenyls) greater than 3,4,4',5-tetrachlorobiphenyl approximately mono-ortho coplanar PCBs greater than di-ortho coplanar PCBs. It was also apparent that the relative toxicities of this group of PCBs paralleled their biological potencies. The coplanar and mono-ortho coplanar PCBs also exhibit differential effects in the inbred C57BL/6J and DBA/2J mice. These compounds induce AHH and cause thymic atrophy in the former "responsive" mice whereas at comparable or higher doses none of these effects are observed in the nonresponsive DBD/2J mice. Since the responsiveness of these two mice strains is due to the presence of the Ah receptor protein in the C57BL/6J mice and its relatively low concentration in the DBA/2J mice, the results for the PCB cogeners support the proposed receptor-mediated mechanism of action.

Induction of two immunochemically related rat liver cytochrome P-450 isozymes, cytochromes P-450c and P-450d, by structurally diverse xenobiotics.

We have previously shown that purified rat liver cytochromes P-450c and P-450d share some but not all immunochemical determinants (Reik, L. M., Levin, W., Ryan, D.E., and Thomas, P.E. (1982) J. Biol. Chem. 257, 3950-3957). Antibody to cytochrome P-450d cross-reacts with cytochrome P-450c to form an immunoprecipitin band in the Ouchterlony test, but no detectable immunoprecipitin ring is formed in a radial immunodiffusion assay. However, the addition of purified cytochrome P-450c to purified cytochrome P-450d in the radial immunodiffusion assay alters the cytochrome P-450d standard curve. Appropriate corrections have been made for the interference of cytochrome P-450c in the immunoquantitation of cytochrome P-450d. Twelve structurally diverse xenobiotics have been examined for their capacity to modulate the levels of cytochrome P-450d, as well as cytochromes P-450a, P-450b, and P-450c, in rat liver microsomes. Five compounds (isosafrole, 3-methylcholanthrene, beta-naphthoflavone, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and phenothiazine) and the polychlorinated biphenyl mixture Aroclor 1254 are potent in vivo inducers of cytochrome P-450d (0.44-0.89 nmol of cytochrome P-450d/mg of microsomal protein). Control rats have low levels of this microsomal hemoprotein (0.04-0.05 nmol of cytochrome P-450d/mg of microsomal protein). Isosafrole induces cytochrome P-450d to a greater extent than cytochrome P-450c, Aroclor 1254 induces both hemoproteins to similar extents, and the remaining four compounds preferentially induce cytochrome P-450c relative to cytochrome P-450d. All of these structurally diverse compounds induce cytochromes P-450d and P-450c, suggesting that the inducibility of these cytochrome P-450 isozymes, but not cytochromes P-450a and P-450b, is linked.

Characterization of nine monoclonal antibodies against rat hepatic cytochrome P-450c. Delineation of at least five spatially distinct epitopes.

Spleen cells from a BALB/cByJ mouse previously immunized with purified rat liver microsomal cytochrome P-450c were fused with myeloma cells (P3X63Ag8.653) and 10 hybridoma clones secreting antibody against cytochrome P-450c were selected for characterization. The monoclonal antibodies (C1-C10) were purified from mouse ascites fluid and nine were determined to be distinct immunoglobulins. C6 was an IgG2b, whereas the rest were of the IgG1 subclass. A competitive enzyme-linked immunoassay was used to show that the antibodies were directed against at least five spatially distinct epitopes on cytochrome P-450c. Additional evidence for the recognition of distinct epitopes was provided by Ouchterlony immunoprecipitation of cytochrome P-450c with mixtures of appropriate monoclonal antibodies. Differences in antibody reactivity provided evidence for a sixth overlapping epitope that was recognized by two antibodies (C4 and C6). Three monoclonal antibodies to the same epitope on cytochrome P-450c, (CD2, CD3, and CD5) cross-reacted strongly with cytochrome P-450d, another isozyme induced by 3-methylcholanthrene treatment of rats. The antibodies that did not cross-react with cytochrome P-450d contained kappa light chains, whereas the three cross-reacting antibodies contained lambda light chains. None of the monoclonal antibodies cross-reacted with purified cytochromes P-450a, P-450b, P-450e, P-450f, P-450g, or P-450h or any other cytochrome P-450 in "Western blots" of liver microsomes from untreated or 3-methylcholanthrene-treated rats. C8 was a potent inhibitor of metabolism catalyzed by cytochrome P-450c in a reconstituted system as well as microsomes from 3-methylcholanthrene-treated rats. This antibody effected maximal inhibition of catalytic activity at an approximately 0.5:1 molar ratio of IgG to cytochrome P-450c, i.e. one antibody-binding site per epitope on cytochrome P-450c.

Purification, characterization and regulation of five rat hepatic microsomal cytochrome P-450 isozymes.

1. Five hepatic microsomal cytochrome P-450 isozymes (cytochromes P-450a, P-450b, P-450c, P-450d, P-450e) have been purified to apparent homogeneity from immature male rats treated with various xenobiotics. 2. The unique electrophoretic properties, substrate specificities and spectral characteristics of these haemoproteins have been compared and contrasted. 3. Structural studies of these cytochrome P-450 isozymes have included NH2-terminal amino acid sequence analyses, as well as electrophoretic profiles of limited proteolytic digests and cyanogen bromide fragments of the haemoproteins. 4. Specific antibodies have been prepared against four of the isozymes and used to evaluate immunochemical relationships among these cytochrome P-450s by Ouchterlony double-diffusion analyses. 5. The levels of some of these cytochrome P-450 isozymes have been quantified immunologically in hepatic microsomal preparations from untreated rats and following induction by phenobarbital, 3-methylcholanthrene or Aroclor 1254. 6. Antibodies directed against cytochromes P-450a and P-450b have been used to establish the presence of more than one 7 alpha- and 16 alpha-testosterone hydroxylase in rat hepatic microsomes. The relative contributions of cytochromes P-450c and P-450d to the overall microsomal metabolism of benzo[a]pyrene have been evaluated using antibodies to these haemoproteins.

Regulation of three forms of cytochrome P-450 and epoxide hydrolase in rat liver microsomes. Effects of age, sex, and induction.

A procedure for the preparation of monospecific antibody directed against rat liver microsomal cytochrome P-45-a is described. This antibody, together with monospecific antibodies to cytochromes P-450b and P-450c, has been used to show that these three forms of cytochrome P-450 are distinct and share no common antigenic determinants. These antibodies (a) give single immunoprecipitin bands with detergent-solubilized microsomes; (b) do not cross-react with the purified heterologous antigens in Ouchterlony double diffusion analyses; (c) have no effect on catalytic activity of the heterologous antigens but completely inhibit the enzymatic activity of the homologous antigens; and (d) remove only the homologous antigen from detergent-solubilized microsomes when covalently bound to a solid support. With radial immunodiffusion assay, we have quantitated these three forms of cytochrome P-450 in liver microsomes after treatment of rats with seven different inducers of cytochrome P-450. The levels of these cytochrome P-450 isozymes vary independently and are also regulated by the age and sex of the animal. The antibodies have also been used to assess the contribution of cytochromes P-450a, P-450b, and P-450c in the metabolism of xenobiotics by rat liver microsomes. A large proportion of benzo(a)pyrene metabolism and testosterone 16 alpha-hydroxylation in microsomes from untreated rats is not catalyzed by cytochromes P-450a, P-450b, and P-450c. Epoxide hydrolase, another microsomal enzyme involved in the metabolism of xenobiotics, was also quantitated by radial immunodiffusion after prior treatment of rats with microsomal enzyme inducers. The inductions of epoxide hydrolase varies independently of the induction of cytochromes P-450a, P-450b, and P-450c.

Monoclonal antibodies distinguish among isozymes of the cytochrome P-450b subfamily.

Polyclonal antibody has been shown previously to react identically with cytochromes P-450b and P-450e purified from Long Evans rats and a strain variant of cytochrome P-450b purified from Holtzman rats (P-450bH). In the present study, an array of 12 different monoclonal antibodies produced against cytochrome P-450b has been used to distinguish among these closely related phenobarbital-inducible rat hepatic cytochromes P-450. In immunoblots and enzyme-linked immunosorbent assays, 10 monoclonal antibodies bind to cytochromes P-450b, P-450e, and P-450bH; one monoclonal antibody (B50) recognizes cytochromes P-450b and P-450bH but not cytochrome P-450e; and one monoclonal antibody (B51) is specific for cytochrome P-450b. In addition, one monoclonal antibody (BEF29) reacts strongly with cytochrome P-450f, and another antibody (BEA33) reacts weakly with cytochrome P-450a. No cross-reactions with cytochromes P-450c, P-450d, and P-450g-P-450j were detected with any of the monoclonal antibodies in these assays. Six spatially distinct epitopes on cytochrome P-450b were identified, and differences in antibody reactivity provided evidence for three additional overlapping epitopes. Several monoclonal antibodies are potent inhibitors of testosterone and benzphetamine metabolism supported by cytochrome P-450b in a reconstituted system. B50 and BE52 do not inhibit metabolism of the two substrates by microsomes from untreated rats, but inhibit benzphetamine N-demethylation and testosterone metabolism to 16 alpha- and 16 beta-hydroxytestosterone as well as androstenedione formation 67-94% by microsomes from phenobarbital-treated rats. No other pathways of testosterone metabolism are inhibited by these monoclonal antibodies. The differential inhibition of microsomal metabolism of benzphetamine and testosterone by these monoclonal antibodies is a reflection of the content and inducibility of cytochromes P-450b and P-450e as well as other cytochrome P-450 isozymes.

Monoclonal antibodies of differentiating specificities as probes of cytochrome P450h (2C11).

A panel of 30 monoclonal antibodies against rat hepatic microsomal cytochrome P450h (2C11) has been produced, purified, and characterized. A broad range of reactivities was observed when 13 purified rat cytochrome P450 isozymes were tested for epitope relatedness in a noncompetitive enzyme-linked immunosorbent assay or on immunoblots. Several antibodies were antigen-specific, others reacted with additional members of the 2C subfamily, and other monoclonal antibodies recognized cytochromes P450 from the 2E, 2B, 2A, and 1A subfamilies. Cytochromes P450p (3A1) and P4501 (3A2) did not react with any of the antibodies. A minimum of seven spatially distinct epitopes on cytochrome P450h were defined by the panel of antibodies. Immunoblot analysis of rat microsomes illustrated the male specificity of cytochrome P450h expression which extended to extrahepatic tissues including kidney and lung. A survey of various species by immunoblot analysis with several antibodies revealed little if any epitope relatedness among microsomal proteins from rats, mice, rabbits, hamsters, squirrel monkeys, guinea pigs, or humans. All of the antibodies were screened as potential inhibitors of cytochrome P450h-mediated testosterone hydroxylation in a reconstituted system. Although most of the antibodies were noninhibitory, greater than 70% inhibition of 2 alpha- and 16 alpha-hydroxylation of testosterone was observed with selected antibodies. These inhibitory antibodies gave similar results when benzphetamine N-demethylation was evaluated in the reconstituted system. The inhibitory antibodies were then used to assess the role of cytochrome P450h in microsomal benzphetamine N-demethylation, since this isozyme exhibits high catalytic activity for this substrate. Only 20-25% inhibition of benzphetamine metabolism was attained in microsomal preparations from adult male rats, and the antibodies did not influence the microsomal catalytic activity of immature males or females or adult females. Thus, despite the high level of expression of cytochrome P450h in microsomes from adult male rats and the high catalytic activity of the purified protein for benzphetamine, this isozyme contributes only a small portion of the metabolism of this substrate in microsomes.

Polyclonal and monoclonal antibodies as probes of rat hepatic cytochrome P-450 isozymes.

Cytochrome P-450 is the terminal oxidase of an electron transport system that is responsible for the oxidative metabolism of a large variety of endogenous and exogenous compounds. This broad substrate selectivity is caused by multiple isozymes of cytochrome P-450 and the wide substrate selectivity of many of these isozymes. We have isolated 11 isozymes of cytochrome P-450 from the livers of rats (cytochromes P-450a-P-450k). We have found both polyclonal and monoclonal antibodies increasingly useful to distinguish among these isozymes and to quantitate enzyme levels in liver microsomal preparations where as many as 15 or more cytochrome P-450 isozymes are present. Several of these isozymes show considerable immunochemical relatedness to each other, and operationally they can be grouped into families of immunochemically related isozymes that include cytochromes P-450b and P-450e in one family, cytochromes P-450c and P-450d in another, and cytochromes P-450f-P-450i, and P-450k in a third family. Immunoquantitation of some of these isozymes has revealed dramatic increases of over 50-fold in the levels of certain of these isozymes when exogenous compounds are administered to rats.

Use of monoclonal antibody probes against rat hepatic cytochromes P-450c and P-450d to detect immunochemically related isozymes in liver microsomes from different species.

Nine distinct monoclonal antibodies raised against purified rat liver cytochrome P-450c react with six different epitopes on the antigen, and one of these epitopes is shared by cytochrome P-450d. None of these monoclonal antibodies recognize seven other purified rat liver isozymes (cytochromes P-450a, b, and e-i) or other proteins in the cytochrome P-450 region of "Western blots" of liver microsomes. Each of the monoclonal antibodies was used to probe "Western blots" of liver microsomes from untreated, or 3-methylcholanthrene-, or isosafrole-treated animals to determine if laboratory animals other than rats possess isozymes immunochemically related to cytochromes P-450c and P-450d. Two protein-staining bands immunorelated to cytochromes P-450c and P-450d were observed in all animals treated with 3-methylcholanthrene (rabbit, hamster, guinea pig, and C57BL/6J mouse) except the DBA/2J mouse, where no polypeptide immunorelated to cytochrome P-450c was detected. The conservation of the number of rat cytochrome P-450c epitopes among these species varied from as few as two (guinea pig) to as many as five epitopes (C57BL/6J mouse and rabbit). The relative mobility in sodium dodecyl sulfate-gels of polypeptides immunorelated to cytochromes P-450c and P-450d was similar in all species examined except the guinea pig, where the polypeptide related to cytochrome P-450c had a smaller Mr than cytochrome P-450d. With the use of both monoclonal and polyclonal antibodies, we were able to establish that purified rabbit cytochromes P-450 LM4 and P-450 LM6 are immunorelated to rat cytochromes P-450d and P-450c, respectively.

Hexachlorobenzene and substituted pentachlorobenzenes (X-C6Cl5) as inducers of hepatic cytochrome P-450-dependent mono-oxygenases.

Hexachlorobenzene (HCB) and 2,3,4,4',5-pentachlorobiphenyl induced a similar spectrum of cytochrome-P-450-dependent mono-oxygenase activities in the rat, including 4-dimethylaminoantipyrine N-demethylase, aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD). Levels of individual cytochrome P-450 isozymes and various mono-oxygenase activities in liver microsomes from rats treated with substituted pentachlorobenzene (X-C6Cl5) and 4'-substituted-2,3,4,5-tetrachlorobiphenyl (X-C12 H5Cl4) analogues demonstrated the remarkable effects of substituent structure on induction activities. The chloro- and bromopentachlorobenzenes induced hepatic microsomal 4-dimethylaminoantipyrine N-demethylase, AHH and EROD; the iodo-, fluoro-, acetamino- and nitropentachlorobenzene analogues together with pentachlorobenzene weakly induced both AHH and EROD (approximately 2-fold or less); and the remaining substituted pentachlorobenzenes tested (X = CH3, OCH3 and OH) were relatively inactive as inducers of microsomal mono-oxygenases. Substituent effects were observed for the induction of liver microsomal cytochromes P-450a, P-450b + e, P-450c, P-450d and total cytochrome P-450 by the X-C6Cl5 and X-C12H5Cl4 analogues. The chloro- and bromopentachlorobenzene analogues in both series induced total cytochrome P-450 and cytochromes P-450a to P-450d, whereas the hydroxy-, methyl- and methoxy-substituted analogues in both series were relatively inactive as inducers of cytochrome P-450. Iodo-, fluoro- and nitropentachlorobenzene were weak 3-methylcholanthrene-type inducers and, in contrast to the corresponding biphenyl analogues, had little or no effect on the induction of cytochromes P-450a, P-450c and P-450d.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of two members of the steroid-inducible cytochrome P450 subfamily (3A) in rats.

Monoclonal antibodies have been successfully isolated which are isozyme-specific for cytochrome P450p (3A1) or P4501 (3A2), two members of the steroid-inducible cytochrome P450 subfamily exhibiting 89% amino acid sequence homology, and these antibodies show less than 5% cross-reaction with 11 other cytochromes P450 (P450a-P450k). A library of 28 purified monoclonal antibodies was established and characterized as to epitope specificity. Appropriate antibodies were selected and utilized to investigate the regulation of expressed cytochrome P450p and P4501 proteins as a function of age, sex, and treatment of rats with various inducing agents. Cytochrome P450p is not detectable in hepatic microsomes from untreated immature or adult male and female rats. Following dexamethasone treatment, expression of cytochrome P450p is observed in all groups with the levels reaching 30-37% of total microsomal cytochrome P450. Administration of other inducers such as pregnenolone 16 alpha-carbonitrile also yield enhanced levels of cytochrome P450p. Measurable amounts of constitutive cytochrome P4501 were detected in hepatic microsomes from immature and adult males as well as immature females but not in adult females. Cytochrome P4501 expression is inducible by dexamethasone in immature rats of both sexes and adult males, although dexamethasone is more effective as an inducer of cytochrome P450p than cytochrome P4501. Hence, not only is cytochrome P4501 protein expressed in immature animals of both sexes, it is also inducible in both sexes. These studies show that constitutive expression and induction of steroid-inducible cytochrome P450s may vary as a function of age.

Bispecific humanized anti-IL-2 receptor alpha beta antibodies inhibitory for both IL-2- and IL-15-mediated proliferation.

Humanized anti-Tac (HAT) and Mik beta1 (HuMik beta 1) Abs directed at IL-2R alpha and IL-2R beta, respectively, inhibit IL-2 binding and biological activity and together act synergistically in vitro. The Abs have been used successfully in primate models of allograft rejection, graft-vs-host disease, and autoimmunity. We produced bifunctional humanized anti-IL-2R alpha beta Abs (BF-IgG) to combine the specificity of the two Abs into one entity by fusing HAT-producing NSO cells and HuMik beta 1-producing Sp2/0 cells. BF-IgG was purified using protein G-Sepharose affinity chromatography, followed by IL-2R alpha and IL-2R beta affinity chromatography and hydrophobic interaction chromatography. BF-IgG exhibited both anti-IL-2R alpha and anti-IL-2R beta specificities in binding assays. While the Ab binds the IL-2R with intermediate affinity (Kd = 2.82 nM), it does not inhibit IL-15 binding to its high affinity IL-15R. In Kit225/K6 (IL-2R alpha beta gamma+) cells, BF-IgG was 10-fold more potent than a HAT/HuMik beta 1 equimolar mixture in blocking IL-2-induced proliferation and, unexpectedly, was at least 65-fold more active than the mixture in blocking IL-15-induced proliferation. This dual inhibitory activity may be due to cross-linking of the IL-2R alpha and IL-2R beta, thus blocking IL-2 binding and possibly impeding the association of IL-2R beta with IL-15R. BF-IgG has potent immunosuppressant activities against both IL-2- and IL-15-mediated responses, and this antagonist could be more efficacious than HAT and/or HuMik beta 1 for the treatment of autoimmunity and the prevention of allograft rejection.

Differential time course of induction of rat liver microsomal cytochrome P-450 isozymes and epoxide hydrolase by Aroclor 1254.

The time course of induction of rat liver microsomal cytochromes P-450a, P-450b + P-450e, P-450c, and P-450d and epoxide hydrolase has been determined in immature male rats administered a single large dose [1500 mumol (500 mg)/kg body wt] of the polychlorinated biphenyl mixture Aroclor 1254. Differential regulation of these xenobiotic-metabolizing enzymes was indicated by their characteristic patterns of induction. The rate of induction of cytochrome P-450a and epoxide hydrolase was relatively slow, and steady-state levels of these enzymes were maintained from approximately Days 9 to 15 after Aroclor 1254 treatment. In contrast, cytochrome P-450c was maximally induced 2 days after Aroclor 1254 treatment and remained at a constant level through Day 15. Steady-state levels of cytochrome P-450d, beginning 1 week after Aroclor 1254 treatment, were preceded by a fairly rapid rate of induction and possibly by a small decline from maximal levels observed around Days 4 to 5. Like those of the other cytochrome P-450 isozymes and epoxide hydrolase, the levels of cytochromes P-450b + P-450e were constant from Day 9 to 15 after Aroclor 1254 treatment. However, an unexpected but reproducible decline (approximately 25%) in total cytochrome P-450 content observed between Days 4 and 9 after Aroclor 1254 treatment principally reflected a dramatic and totally unanticipated decrease (approximately 45%) in the level of cytochromes P-450b + P-450e. This transient decline in the level of cytochromes P-450b + P-450e was not due to an unusual effect of a mixture of polychlorinated biphenyls, since identical results were obtained with two individual congeners, namely 2,3,4,5,4'-penta- and 2,3,4,5,3',4'-hexachlorobiphenyl, that induced the same isozymes as Aroclor 1254. In contrast, when rats were treated with 2,4,5,2',4',5'-hexachlorobiphenyl, which induces cytochromes P-450a and P-450b + P-450e and epoxide hydrolase but not cytochromes P-450c or P-450d, maximal levels of cytochromes P-450b + P-450e were attained on Day 4 and no decrease was observed over the next 11 days. These results suggest that there may be an interaction in the regulation of induction of certain individual cytochrome P-450 isozymes.

Effects of three recombinant human leukocyte interferons on drug metabolism in mice.

Three recombinant human leukocyte interferons (IFLrA, IFLrD, and a hybrid IFLrA/D) that differ markedly in their antiviral activity in murine L cells were examined for their effects on hepatic microsomal drug metabolism in adult female CD-1 mice. When administered for 1 or 3 consecutive days, IFLrA/D, which exhibited the highest antiviral activity in murine L cells, caused a dose-dependent decrease in cytochrome P-450 content and in the rate of metabolism in vitro of benzo[a]pyrene, hexobarbital, 7-ethoxycoumarin, benzphetamine, and zoxazolamine. The concentration of cytochrome b and the activity of NADPH-cytochrome c reductase were also depressed when IFLrA/D was administered for 3 days. Similar but somewhat smaller changes were observed following treatment of mice with IFLrD, which possessed approximately 1% of the antiviral activity of IFLrA/D in murine L cells. In contrast, IFLrA, which was essentially devoid of antiviral activity in the mouse cell line, failed to depress cytochrome P-450 levels and in vitro drug metabolism activity in a consistent or dose-dependent manner. Cytochrome P-450 content and the in vitro rate of metabolism of benzphetamine and zoxazolamine were maximally depressed 8-24 hr after a single intraperitoneal injection of 1.5 micrograms of interferon per mouse; at this time the interferons were no longer detectable in serum. Near-normal levels of cytochrome P-450 and in vitro drug metabolism activity were restored by 48 hr after a single injection of interferon. Treatment of mice with 1.5 micrograms of IFLrA/D once daily for 3 days prolonged hexobarbital sleeping time but not zoxazolamine paralysis time, whereas neither of these was influenced by treatment with IFLrA or D. The results indicate that an interferon-dependent process reduces the level of microsomal cytochrome P-450 in liver and potentiates the pharmacological actions of certain drugs in mice.