Induction of a cytochrome P-450-dependent fatty acid monooxygenase in Bacillus megaterium by a barbiturate analog, 1-[2-phenylbutyryl]-3-methylurea

Summary In previous publications from our laboratory, we reported that a soluble, cytochrome P-450-dependent fatty acid monooxygenase from Bacillus megaterium ATCC 14581 can be induced by phenobarbital and a variety of other barbiturates. The tested barbiturates showed an excellent correlation between increasing lipophilicity and increasing inducer potency (Kim BH, Fulco AJ; Biochem Biophys Res Commun 116: 843–850, 1983). The only exception proved to be mephobarbital (N-methylphenobarbital) which, although more lipophilic than phenobarbital, is not an inducer of fatty acid monooxygenase activity. We have now found that 1-[2-phenylbutyryl]-3-methylurea (PBMU), an acylurea that can be derived from mephobarbital by hydrolytic cleavage of the barbiturate ring, is an excellent inducer of this activity. Paradoxically, the addition of mephobarbital to the bacterial growth medium containing PBMU significantly enhances the apparent potency of the acylurea to induce fatty acid monooxygenase activity as measured in cell-free extracts. When cell-free extracts of cells grown separately in PBMU or mephobarbital are mixed no enhancement of activity is seen. This finding suggests that the effect of mephobarbital is to somehow increase the efficiency of PBMU as an inducer of the P-450-dependent fatty acid monooxygenase rather than to induce an activator of this enzyme or a rate-limiting component of the monooxygenase system. Finally, both mephobarbital and PBMU induce the synthesis of total cytochrome P-450 in B. megaterium although PBMU is a much more potent P-450 inducer. For cytochrome P-450 induction, however, there is no synergistic or even additive effect when mephobarbital and PBMU are used together in the bacterial growth medium.Abbreviations PBMU 1-[2-phenylbutyryl]-3-methylurea - M.P. melting point     

The importance of serum lipoproteins in the cytolytic action of 7 beta-hydroxycholesterol on cultured hepatoma cells

A soluble cytochrome P-450-dependent fatty acid monooxygenase activity obtained from Bacillus megaterium ATCC 14581 can be induced by at least 13 different barbiturates. In general, the potency of these compounds as inducers increases with their increasing lipophilicity. We have now shown that at least 4 of these barbiturates (phenobarbital, secobarbital, pentobarbital and methohexital) seem to induce the same active cytochrome P-450-containing enzyme by a non-substrate type mechanism. The partially purified enzymes obtained from cultures induced with each of the 4 barbiturates tested were all of similar molecular size (Mr = 130,000 +/- 10,000) and had similar turnover numbers (1400-1800 +/- 300) with either palmitoleate or myristate as substrates. None of the tested barbiturates served as substrates, activators or inhibitors of any of the monooxygenase preparations, nor did they appear to interact in any way with the monooxygenase enzyme or the P-450 component.     

Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium

A unique cytochrome P-450-dependent fatty acid monooxygenase from Bacillus megaterium ATCC 14581 is strongly induced by phenobarbital (Narhi, L. O., and Fulco, A. J. (1982) J. Biol. Chem. 257, 2147-2150) and many other barbiturates (Kim, B.-H., and Fulco, A. J. (1983) Biochem. Biophys. Res. Commun. 116, 843-850). This monooxygenase has now been purified to homogeneity from pentobarbital-induced bacteria as a single polypeptide with a molecular weight of 119,000 +/- 5,000 daltons. In the presence of NADPH and O2, it can catalyze the oxygenation of long chain fatty acids without the aid of any other protein. The enzyme has a catalytic center activity of 4,600 nmol of fatty acid oxygenated per nmol of P-450 (the highest activity yet reported for a P-450-dependent monooxygenase) and also functions as a highly active cytochrome c reductase in the presence of NADPH. The purified holoenzyme is a soluble protein containing 40 mol % hydrophobic amino acid residues and 1 mol each of FAD and FMN/mol of heme. It is isolated and purified in the low spin form but is converted to the high spin form in the presence of long chain fatty acids. The enzyme, which catalyzes the omega-2 hydroxylation of saturated fatty acids and the hydroxylation and epoxidation of unsaturated fatty acids has its highest affinity (Km = 2 +/- 1 microM) for the C15 and C16 chain lengths.     

Nonsubstrate induction of a soluble bacterial cytochrome P-450 monooxygenase by phenobarbital and its analogs

A soluble, cytochrome P-450-dependent fatty acid hydroxylase--epoxidase complex from Bacillus megaterium ATCC 14581 can be induced more than 100-fold by the addition of phenobarbital or one of its analogs (hexobarbital) to the growth medium. These barbiturate inducers are apparently not substrates for the enzyme nor do they activate the monooxygenase in the cell-free system. The induction efficiency of both phenobarbital and hexobarbital can be significantly increased with respect to monooxygenase activity by autoclaving the inducer in the growth medium rather than by adding it to the medium after autoclaving. Turnover numbers of about 3 000 nmoles of substrate oxygenated per min per nmole of P-450 were obtained in crude cell-free preparations obtained from maximally induced cultures. Our data indicate that products formed by heating phenobarbital or hexobarbital in the growth medium are significantly better inducers of monooxygenase activity than are the unaltered drugs.     

Acylureas: A new class of barbiturate-like bacterial cytochrome P-450 inducers

The soluble, cytochrome P-450-dependent fatty acid monooxygenase of Bacillus megaterium ATCC 14581 is induced by phenobarbital and at least twelve other barbiturates (Kim, B.-H. and Fulco, A.J. 91983) Biochem. Biophys. Res. Commun. 116, 843–850). We have since found that the inducer potency of phenobarbital and of six other of these barbiturates was enhanced by adding them to growth medium prior to sterilization by autoclaving. A similar ‘activation’ was effected simply by autoclaving these barbiturates in distilled water at pH 8.0. When the hydrolytic products resulting from such treatment of phenobarbital were identified and screened for inducer activity, the major product, 2-phenulbutyrylurea, was found to be 3–5-times more potent than phenobarbital itself. The racemic mixture, (±-)-2-phenylbutyryluera was somewhat more active as an inducer than was either of the enantiomers (±) or (?) tested singly. Of the other hydrolytic products of phenobarbital, only 2-phenylbutyramide had significant inducer activity (about the same as phenobarbital). Among other ureides tested, tow monosubstituted acetylureas (phenylacetylurea and dodecanoylurea) were inactive as inducers, but six of seven disubstituted acetylureas were better inducers than 2-phenylbutyrylurea.     

Purification and characterization of pentobarbital-induced cytochrome P-450BM-1 from Bacillus megaterium ATCC 14581

When Bacillus megaterium ATCC 14581 is grown in the presence of barbiturates, a cytochrome P-450-dependent fatty acid monooxygenase (Mr 120000) is induced (Kim, B.-H. and Fulco, A.J. (1983) Biochem. Biophys. Res. Commun. 116, 843-850). Gel filtration chromatography of a crude monooxygenase preparation from pentobarbital-induced B. megaterium indicated that not all of the induced cytochrome P-450 present in the extract was accounted for by this high-molecular-weight component. Further purification revealed the presence of two additional but smaller cytochrome P-450 species. The minor component, designated cytochrome P-450BM-2, had a molecular mass of about 46 kDa, but has not yet been completely purified or further characterized. The major component, designated cytochrome P-450BM-1, was obtained in pure form, exhibited fatty acid monooxygenase activity in the presence of iodosylbenzenediacetate, and has been extensively characterized. Its Mr of 38000 makes it the smallest cytochrome P-450 yet purified to homogeneity. Although it is a soluble protein, a complete amino acid analysis indicated that it contains 42% hydrophobic residues. By the dansyl chloride procedure the NH2-terminal amino acid is proline; the penultimate NH2-terminal residue is alanine. The absolute absorption spectra of cytochrome P-450BM-1 show maxima in the same general regions as do P-450 cytochromes from mammalian or other bacterial sources, but they differ in detail. The oxidized form of P-450BM-1 has absorption maxima at 414, 533 and 567 nm, while the reduced form has peaks at 410 and 540 nm. The absorption maxima for the CO-reduced form of P-450BM-1 are found at 415, 448 and 550 nm. Antisera from rabbits immunized with pure P-450BM-1 strongly reacted with and precipitated this P-450, but showed no detectable affinity for either the 46 kDa P-450 or the 120 kDa fatty acid monooxygenase.     

Occurrence of a barbiturate-inducible catalytically self-sufficient 119,000 dalton cytochrome P-450 monooxygenase in bacilli

In a recent publication (Narhi, L.O. and Fulco, A.J.[1986] J. Biol. Chem. 261, 7160-7169) we described the characterization of a catalytically self-sufficient 119,000 Dalton cytochrome P-450 fatty acid monooxygenase (P-450BM-3) induced by barbiturates in Bacillus megaterium ATCC 14581. We have now examined cell-free preparations from 12 distinct strains of B. megaterium and from one or two strains each of B. alvei, B. brevis, B. cereus, B. licheniformis, B. macerans, B. pumilis and B. subtilis for the presence of this inducible enzyme. Using Western blot analyses in combination with assays for fatty acid hydroxylase activity and cytochrome P-450, we were able to show that 11 of the 12 B. megaterium strains contained not only a strongly pentobarbital-inducible fatty acid monooxygenase identical to or polymorphic with P-450BM-3 but also significant levels of two smaller P-450 cytochromes that were the same as or similar to cytochromes P-450BM-1 and P-450BM-2 originally found in ATCC 14581. Unlike the 119,000 Dalton P-450, however, the two smaller P-450s were generally easily detectable in cultures grown to stationary phase in the absence of barbiturates and, with some exceptions, were not strongly induced by pentobarbital. None of the non-megaterium species of Bacillus tested exhibited significant levels of either fatty acid monooxygenase activity or cytochrome P-450. The one strain of B. megaterium that lacked inducible P-450BM-3 was also negative for BM-1 and BM-2. However, this strain (ATCC 13368) did contain a small but significant level of another P-450 cytochrome that others have identified as the oxygenase component of a steroid 15-beta-hydroxylase system. Our evidence suggests that the BM series of P-450 cytochromes is encoded by chromosomal (rather than by plasmid) DNA.     

Phenobarbital induces cytochrome P-450- and cytochrome P-448-dependent monooxygenases in rat hepatoma cells

The induction by phenobarbital (PB) of aldrin epoxidase (AE) and aryl hydrocarbon hydroxylase (AHH), markers of cytochrome P-450- and cytochrome P-448-dependent monooxygenases, was studied in cell lines derived from Reuber H35 rat hepatoma which differ widely in their degree of differentiation. The following results were obtained: (1) PB induced AE 2-6-fold and AHH 2-4-fold in the differentiated clones, Fao, 2sFou, and C2Rev7 during an exposure period of 72 h. The barbiturate increased AHH but not AE in the dedifferentiated clone H5, the poorly differentiated line H4IIEC3/T, and in the well differentiated line H4IIEC3/G-. (2) Continuous presence of the barbiturate was required for maintaining the induction of the two monooxygenase activities in C2Rev7 cells. (3) Maximum induction of AE was observed at a PB concentration of 1.5-3.0 mM. (4) The effects of 7,8-benzoflavone on AHH-activities induced by phenobarbital in C2Rev7 and H5 cells suggested that they are mediated by cytochrome P-450- and cytochrome P-448-dependent monooxygenase forms, respectively. Thus, the flavonoid had only a slight inhibitory effect on PB-induced AHH in C2Rev7 cells, but strongly inhibited PB-induced AHH in H5 cells. The induction of AE and of 7,8-benzoflavone-inhibitable AHH in 2sFou cells indicated that PB is capable of inducing cytochromes P-450 and cytochrome P-448 in the same cell.     

Induction of cytochrome P-450 forms in liver microsomes of rats in the early neonatal period after administration of phenobarbital and 3-methylcholanthrene

The activity of cytochrome P-450 dependent monooxygenase system from rat liver microsomes after induction by phenobarbital and 3-methylcholantrene in early neonatal period (3-16 days after birth) was studied. It was found that the total amount of cytochrome P-450 increases after injection of these inducers in neonatal rats of all age groups. In parallel, in the case of 3-methylcholantrene induction the benz(a)pyrene hydroxylase and 7-ethoxyresorufin deethylase activities increase; phenobarbital induction causes a rise in the benzphetamine-N-demethylase and benz(a)pyrene hydroxylase activities. Immunochemical analysis involving the use of antibodies specifically directed against cytochrome P-450 of adult rats revealed that the level of cytochrome P-450 in the case of 3-methylcholantrene induction increases from 5 to 50%, whereas that of cytochrome P-450 upon phenobarbital induction increases from 5 to 40% in liver microsomes of 3- and 16-day-old rats. The mode of inhibition of various substrates metabolism by antibodies in neonatal rat microsomes suggests that the 3-methylcholantrene-induced cytochrome P-448, like in adult rats, participates in the hydroxylation of benz(a)pyrene and O-deethylation of 7-etoxyresorufin. The participation of phenobarbital-induced cytochrome P-450 in the metabolism of benzphetamine and aldrin in neonatal rats is much lower than in the adult ones. The metabolism of benz(a)pyrene in phenobarbital-induced neonatal rat microsomes in all age groups is not inhibited by antibodies. The age-dependent differences in inhibition of metabolism and the increase in the benz(a)pyrene hydroxylase activity in phenobarbital-induced rats suggest that the spectrum of inducible forms of cytochrome P-450 in neonatal rats differ from that in adult animals.     

Mechanism of inhibiting the cytochrome P-450-dependent monooxygenase system in liver with fluorocarbons]

The inducer of the liver monooxygenase system perfluorodecalin added to microsomes as a submicron emulsion forms an enzyme-substrate complex with cytochrome P-450. The K(app) values for the perfluorodecalin binding to cytochrome P-450 in microsomes isolated from the livers of control and phenobarbital-treated rats are 5 x 10(-5) M and 2.3 x 10(-6) M, respectively. Perfluorodecalin competitively inhibits the binding of substrates to cytochrome P-450 and decreases the rates of monooxygenase reactions. Perfluorodecalin extrusion from the active center of cytochrome P-450 occurs when an excess of perfluorocarbons non-interacting with cytochrome P-450 is added to microsomes. There is a significant vagueness in the rates of various monooxygenase reactions because of simultaneous induction and inhibition of monooxygenase enzymes after perfluorodecalin administration to rats. The data obtained are consistent with the hypothesis that constitutive forms of cytochrome P-450 are primary receptors for xenobiotic-inducers of phenobarbital-type cytochrome P-450 isoforms.     

Cloning of the gene encoding a catalytically self-sufficient cytochrome P-450 fatty acid monooxygenase induced by barbiturates in Bacillus megaterium and its functional expression and regulation in heterologous (Escherichia coli) and homologous (Bacillus megaterium) hosts

In a previous publication (Narhi, L. O., and Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169) we described the characterization of a 119,000-dalton P-450 cytochrome that is strongly induced by barbiturates in Bacillus megaterium. In the presence of NADPH and O2, this single polypeptide can catalyze the hydroxylation of long-chain fatty acids without the aid of any other protein. The gene encoding this unique monooxygenase (cytochrome P-450BM-3) has now been cloned by an immunochemical screening technique. The Escherichia coli clone harboring the recombinant plasmid produces a 119,000-dalton protein that appears to be electrophoretically and immunochemically identical to the B. megaterium enzyme and contains the same N-terminal amino acid sequence. The recombinant DNA product also exhibits the characteristic cytochrome P-450 spectrum and is fully functional as a fatty acid monooxygenase. In E. coli, the synthesis of P-450BM-3 is directed by its own promoter included in the DNA insert and proceeds constitutively at a very high rate but is not stimulated by pentobarbital. However, when the cloned P-450BM-3 gene, either intact or in a truncated form, is introduced back into B. megaterium via an E. coli/Bacillus subtilis shuttle vector, its expression is constitutively repressed but is induced by pentobarbital. This finding demonstrates that the regulatory region of the P-450BM-3 gene that responds to barbiturates is included in the cloned DNA. The evidence also indicates that pentobarbital cannot directly act on the gene to cause induction but presumably interacts with another component such as a repressor molecule that is present in B. megaterium but is absent in the E. coli clone.     

Specificity in the activation and inhibition by flavonoids of benzo[a]pyrene hydroxylation by cytochrome P-450 isozymes from rabbit liver microsomes

The effect of flavone and 7,8-benzoflavone on the metabolism of benzo[a]pyrene to fluorescent phenols by five cytochrome P-450 isozymes obtained from rabbit liver microsomes was determined. Benzo[a]pyrene metabolism was stimulated more than 5-fold by the addition of 600 microM flavone to a reconstituted monooxygenase system consisting of NADPH, cytochrome P-450 reductase, dilauroylphosphatidylcholine, and cytochrome P-450LM3c or cytochrome P-450LM4. In contrast, an inhibitory effect of flavone on benzo[a]pyrene metabolism was observed when cytochrome P-450LM2, cytochrome P-450LM3b, or cytochrome P-450LM6 was used in the reconstituted system. 7,8-Benzoflavone (50-100 microM) stimulated benzo[a]pyrene metabolism by the reconstituted monooxygenase system about 10-fold when cytochrome P-450LM3c was used, but benzo[a]pyrene hydroxylation was strongly inhibited when 7,8-benzoflavone was added to the cytochrome P-450LM6-dependent system. Smaller effects of 7,8-benzoflavone were observed on the metabolism of benzo[a]pyrene by the cytochrome P-450LM2-, cytochrome P-450LM3b-, and cytochrome P-450LM4-dependent monooxygenase systems. These results demonstrate that the activating and inhibiting effects of flavone and 7,8-benzoflavone on benzo[a]pyrene metabolism depend on the type of cytochrome P-450 used in the reconstituted monooxygenase system.     

The rabbit pulmonary monooxygenase system. Immunochemical and biochemical characterization of enzyme components.

The enzymatic components of the rabbit pulmonary monooxygenase system, cytochromes P-450I and P-450II and NADPH-cytochrome P-450 reductase, are immunochemically distinct proteins. In pulmonary microsomes, the N-demethylation of benzphetamine, amino-pyrine, and ethylmorphine, and the O-deethylation of 7-ethoxycoumarin are dependent only on cytochrome P-450I, and the hydroxylation of coumarin is apparently catalyzed by both cytochromes. Cytochrome P-450II is immunochemically distinct from the major forms of hepatic cytochrome P-450 induced by phenobarbital or 3-methylcholanthrene, whereas cytochrome P-450I is indistinguishable from the former on the basis of physical and catalytic as well as immunochemical characteristics. Pulmonary and hepatic NADPH-cytochrome P-450 reductases also have identical physical, catalytic, and immunochemical properties. The lack of response of the lung monooxygenase system to phenobarbital, therefore, is apparently not due to an inability of the lung to synthesize the enzymes induced by phenobarbital in the liver. The relatively high proportion of cytochrome P-450I in the lung appears to be responsible for the higher rates (per nmol of P-450) of N-demethylation that have been observed in rabbit pulmonary as compared to hepatic microsomal fractions.     

Directed modification of the structure of cytochrome P-450 substrates as a method of generating new inducers of the microsomal monooxygenase system

Using the previously obtained data on the substrate-type induction of monooxygenase by xenobiotics of phenobarbital type, the method of conversion of typical substrates for cytochrome P-450 into inducers of biosynthesis of this enzymatic system by blocking in the substrate molecule of the position subjected to oxidative conversion in the enzyme active center was tested. The introduction of the methyl group in the omega-1 position of amobarbital, of Cl- into positions 2 and 4 of biphenyl and the substitution of methyl groups for the isopropyl groups in the 4-N(CH3)2 position of aminopyrine provides for marked induction of these derivatives of cytochrome P-450 and some monooxygenase activities.     

Cytochrome P-450-dependent H2O2 production demonstrated in vivo. Influence of phenobarbital and allylisopropylacetamide

By administration of allylisopropylacetamide, an inhibitor of cytochrome P-450, we demonstrated that cytochrome P-450 is involved in the production of H2O2 during aminopyrine metabolism and phenobarbital induction in both the unanaesthetized guinea pig and rat. In the guinea pig we also found evidence for the existence of a basal cytochrome P-450-dependent H2O2 production, i.e. in the absence of exogenous substrate. Catalase participates in the decomposition of H2O2 produced in the endoplasmic reticulum where cytochrome P-450 is localized.     

Induction of cytochrome P-450 isozymes by mirex and chlordecone

The effect of the insecticides, mirex and chordecone (Kepone), on the cytochrome P-450 monooxygenase system in C57BL/6N mouse liver microsomes was studied. Mice were treated intraperitoneally with low (6 mg/kg) and high (30 mg/kg) doses of mirex and chlordecone in corn oil for 2 days. For comparison, mice were also treated with either phenobarbital (PB) or 3-methylcholanthrene (3-MC). All treatments significantly increased the hepatic microsomal P-450 content over that of controls. Benzphetamine N-demethylase, ethoxyresorufin O-deethylase, benzo[a]pyrene hydroxylase, and acetanilide hydroxylase activities were also determined. Mirex and chlordecone resembled phenobarbital with respect to the induction of monooxygenase activities. Immunoquantitation with antibodies to purified P-450 IIB1 (Pb-induced P-450) and P-450 IA1 (3-MC-induced P-450) indicated that mirex and chlordecone induced P-450 IIB1 in a dose-dependent manner. The high dose of mirex also induced a small amount of a protein cross reacting with the antibody to IA1. The induction of this isozyme did not, however, contribute significantly to the monooxygenase activities measured.     

Microsomal ethanol-oxidizing system in Euglena gracilis. Similarities between Euglena and mammalian cell systems.

1. ADH activity of Euglena grown with 50 mM ethanol decreased, but MEOS activity increased with a corresponding increase in the total amount of cytochrome P-450. 2. Phenobarbital treatment increased the total amount of cytochrome P-450. 3. CO and KCN, cytochrome P-450 ligands, diminished acetaldehyde formed from ethanol oxidation by MEOS. 4. The amounts of NAD(P)H cytochrome c reductases and cytochrome b5 type, components of microsomal monooxygenase reaction, have been spectrophotometrically measured. 5. NAD(P)H cytochrome c reductases activities were induced by phenobarbital. 6. DMSO, an inhibitor of rabbit MEOS, inhibited O2 consumption (11-20%) by Euglena grown with an ethanol, but not a lactate medium. 7. These studies indicate the presence of cytochrome P-450-dependent MEOS in Euglena similar to that in the mammalian hepatic cell.     

Suppression of the inductive effects of phenobarbital and 3-methylcholanthrene on ascorbic acid synthesis and hepatic cytochrome P-450-linked monooxygenase systems by the interferon inducers, poly rI.rC and tilorone.

The interferon inducing agents, poly rI·rC and tilorone, cause a marked depression of hepatic cytochrome P-450-linked monooxygenase systems. Ascorbate synthesis and hepatic monnoxygenase systems are induced by phenobarbital and 3-methylcholanthrene. Poly rI·rC and tilorone suppressed the induction of ascorbate synthesis, P-450 and monooxygenase activity (ethylmorphine N-demethylase and benzo[a]pyrene hydroxylase) by phenobarbital. 3-Methylcholanthrene-induced ascorbate synthesis was suppressed by poly rI·rC, but equivocal results were obtained with tilorone. Induction of P-450 by 3-methylcholanthrene was suppressed completely by poly rI·rC or tilorone, but that of benzo[a]pyrene hydroxylase was lowered by only 40%, thus demonstrating the selective depressive action of interferon inducing agents on different species of P-450.     

Comparison of the inducing effect of triphenyldioxan, bis-(dichloropyridyloxy)benzene and phenobarbital on the liver monooxygenase

The induction by triphenyldioxane (TPD) of cytochrome P-450 in rat liver microsomes was studied. It was demonstrated that TPD injection in a single dose (10 mg/kg of body mass) is associated with a marked induction of cytochromes P-450 b/e (cytochrome PB-forms) in rat liver microsomes and a significant increase in the benzphetamine-N-demethylase activity typical of cytochrome P-450b. In other words, TPD is a potent inducer of PB-type, the inducing effect being attained by an injection of a single dose of TPD which is by one order of magnitude less than that of phenobarbital. It can be assumed that this compound shows a high affinity for the hypothetical receptor responsible for cytochrome P-450b synthesis. It was shown also that TPD does not induce the monooxygenase system of mouse liver, whereas 1,4-bis[2-(dichloropyridyloxy)]benzene (DPB) is a potent inducer of PB-type in mice, being fairly ineffective in rats. Hence, the species-specific effect of TPD and DPB appears to be opposite.     

A dual assay for the specific screening of 3-methylcholanthrene- and phenobarbital-like chemical inducers of cytochrome P-450 monooxygenases

We present and evaluate a dual assay, the CYPIA (Cytochrome P-450 induction assay) for the detection and the simultaneous identification of chemicals belonging either to the 3-methylcholanthrene or phenobarbital classes of cytochrome P-450 monooxygenase inducers. These inducers play an important role in the mutagenic activation of chemical compounds as well as in many pharmacological and toxicological events and therefore should be screened by drug and chemical designers. After treatment of male rats or mice by chemicals, the liver preparations (S9) have been used in the Salmonella typhimurium test, to activate either ethidium bromide or cyclophosphamide into mutagenic metabolites. These transformations are specifically catalyzed by cytochrome P-450-dependent monooxygenases induced by 3-methylcholanthrene-like and phenobarbital-like chemical inducers, respectively, The mutagenicity data were strikingly correlated with other methods (production of [³H]benzo[a]pyrene bay-region metabolites, benzphetamine demethylase activity, immunological double-diffusion analysis). Compared to the latter methods, the CYPIA, based on a single and widespread technology, introduces an interesting simplification, and improves the specificity and the sensitivity of the responses.