Stabilization of microbial cytochrome P-450 activity by creation of station-phase conditions in a continuously operated immobilized-cell reactor.

Bacillus megaterium (ATCC 13368) exhibits cytochrome P-450 monooxygenase activity (referred to herein as Cyt P-450 meg) catalyzing 15 beta-steroid hydroxylation. This activity belongs to the widespread ferredoxin reductase-ferredoxin-Cyt P-450 type of monooxygenases, providing a representative model system for this type of activity. The level of Cyt P-450 meg activity reaches its maximum in the cells during the stationary phase of the growth curve and is not affected by Cyt P-450 inducers. Here we present the development of an approach for stabilizing the Cyt P-450 meg system so that it performs continuous steroid hydroxylation and will be a model system for Cyt P-450-based detoxification. It is based on cell immobilization and simulation of stationary-phase conditions in a continuously operated fluidized-bed bioreactor. The combination of an appropriate immobilization technique, operational conditions, and medium composition provided a stabilized cell environment resulting in "freezing" of a physiological steady-state analog under stationary phase conditions, allowing stable performance of continuous hydroxylation for several weeks. It is suggested that this approach may be extended for use with other environmentally induced enzymatic activities.     

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.     

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.     

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.     

Isolation and characterization of cytochrome P-450meg.

The cytochrome P-450-dependent steroid 15 beta-hydroxylase system from Bacillus megaterium has been resolved into three components, 1) a NADPH-specific, FMN-containing flavoprotein reductase, molecular weight 55-60 000; 2) an iron-sulfur protein, molecular weight 13,000 and 3) cytochrome P-450meg, molecular weight 52,000. The cytochrome component has been purified to homogeneity, as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing in polyacrylamide gel, and its amino acid composition has been determined. Cytochrome P-450meg has a pI of 4.9, a Stokes radius of 27 A and a sedimentation constant of 3.3 S. Electron paramagnetic resonance and optical spectra are typical of a low-spin cytochrome P-450. The fluorescence spectrum is indicative of a tryptophane residue in a relatively non-polar environment. In recombination experiments, the electron flow was shown to proceed from the reductase via the iron-sulfur protein to the cytochrome. It is also possible to exchange the different components of the mitochondrial 11 beta-hydroxylase system from bovine adrenals for corresponding components in B. megaterium. Substrate specificity studies indicate that only steroids with a 3-oxo-delta 4-configuration are hydroxylated by the B. megaterium hydroxylase system. When oxidizing agents were used, hydroxylation occurred both in positions 15 alpha and 15 beta. Further substrate specificity studies have shown that aniline and imipramine can function as substrates for the bacterial system.     

cDNA-directed expression of rat testosterone 7 alpha-hydroxylase using the modified vaccinia virus, T7-RNA-polymerase system and evidence for 6 alpha-hydroxylation and delta 6-testosterone formation

The modified vaccinia virus, T7-RNA-polymerase cDNA-expression system was used to express rat cytochrome P-450a. Various parameters such as host-cell type and density, and duration of infection were tested to optimize the level of expression of cytochrome P-450a enzyme activity. Cytochrome P-450a expressed from the cDNA sequence was exclusively incorporated into the membrane-containing portions of the cell lysates, as expected from its normal association in the liver endoplasmic reticulum. The enzyme displayed a carbon-monoxide-reduced-cytochrome-P-450a difference spectrum with a Soret maximum of 450 nm. Activity measurements revealed that cytochrome P-450a produced three metabolites of testosterone; 7 alpha-hydroxytestosterone and 6 alpha-hydroxytestosterone and delta 6-testosterone at a ratio of about 38:1:1. Under the appropriate conditions, the vaccinia-virus, T7-RNA-polymerase system produces high levels of a single form of cytochrome P-450 in cells that are virtually devoid of endogenous cytochrome P-450. Analysis of the cytochrome P-450 in its natural membrane-bound state, as opposed to artificial-lipid reconstitution studies of purified enzymes, allows accurate and confident measurements of substrate specificities.     

Characterization of recombinant Bacillus megaterium cytochrome P-450 BM-3 and its two functional domains

Bacillus megaterium cytochrome P-450BM-3 and its two functional domains, the heme and flavin domains, have been purified and characterized using an Escherichia coli expression system. Recombinant P-450BM-3 behaves both spectrally and enzymatically the same as the enzyme produced from the natural host, B. megaterium, and another E. coli system recently described (Bouddupalli, S. S., Estabrook, R. W., and Peterson, J. A. (1990) J. Biol. Chem. 265, 4233-4239). Reduction of the flavins in P-450BM-3 domain with NADPH appears to be very similar to microsomal P-450 reductases where two reducing equivalents are consumed to fully reduce the FMN while the FAD is converted to the semiquinone in an one electron reduction. NADPH reduction of the heme occurs only in the presence of substrate suggesting, by analogy with the cytochrome P-450CAM system, a possible increase in iron redox potential of the heme upon substrate binding which facilitates electron transfer from the flavins to the heme. The flavin domain retains a high level of cytochrome c reductase activity and also reacts with NADPH to give a 3-electron reduced product. The heme domain retains the ability to bind substrate and generates the characteristic 450-nm absorption band upon reduction in the presence of CO. The heme domain has been crystallized and a preliminary set of x-ray diffraction data obtained.     

Rat pulmonary microsomal cytochrome P-450 enzymes involved in the activation of procarcinogens.

Rat lung microsomal cytochrome P-450 (P-450) enzymes have been characterized with regard to their catalytic specificities towards activation of several procarcinogens to genotoxic metabolites in Salmonella typhimurium TA1535/pSK1002. We first examined the roles of rat liver microsomal P-450 enzymes in the activation of benzo[a]pyrene and its 7,8-diol enantiomers to genotoxic products, and found that P-450 1A1 is a major catalyst for the activation of these potential procarcinogens in rat livers. Using lung microsomes isolated from rats treated with various P-450 inducers we obtained evidence that at least three P-450 enzymes are involved in the activation of several procarcinogens. Immunoinhibition studies support the view that benzo[a]pyrene and its 7,8-diol derivatives, other dihydrodiol derivatives of polycyclic aromatic hydrocarbons, and 3-amino-1-methyl-5H-pyrido[4,3-b]indole are activated to genotoxins mainly by rat P-450 1A1, which is inducible in rat lungs by 5,6-benzoflavone and the polychlorinated biphenyl mixture Aroclor 1254. Activation of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline and 2-amino-3-methylimidazo[4,5-f]quinoline may be catalyzed by another P-450 enzyme because the activities were not induced by treatment with 5,6-benzoflavone or Aroclor 1254. The observation that both activities were inhibited by antibodies raised against P-450 1A2 and by 7,8-benzoflavone suggests a role for an enzyme of P-450 1A family, probably P-450 1A2, in rat lung microsomes. The activation of aflatoxin B1 and sterigmatocystin appears to be catalyzed by other P-450 enzyme(s) rather than the P-450 1A family as judged by the different responses of activities to the P-450 inducers and the specific antibodies in rat lung microsomes. Interestingly, lung microsomal activation of several procarcinogens was found to be suppressed in rats treated with isosafrole and pregnenolone 16 alpha-carbonitrile. Thus, the results support the roles of different P-450 enzymes in the activation of procarcinogens in rat lung microsomes.     

Aryl hydrocarbons as inducers of cytochrome P-450 in Acinetobacter calcoaceticus

Summary The inducibility of cytochrome P-450 in Acinetobacter calcoaceticus by some compounds known as typical inducers of hepatic cytochromes P-450 was investigated. Besides biphenyl also indene and phenanthrene are inducers, whereas compounds of the so-called phenobarbital type are not. Biphenyl appears to be the most effective inducer with regard to the yield of cytochrome P-450/mg of cell protein. By addition of the compounds in the vapour phase an induction of the protein by naphthalene could be demonstrated. The results are indicative of the existence of bacterial cytochromes P-450 that resemble hepatic cytochromes.     

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.     

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.     

Characterization of a cytochrome P-450-dependent steroid hydroxylase system present in Bacillus megaterium.

Cell-free extracts from sonically disrupted Bacillus megaterium ATCC 13368 hydroxylated a variety of 3-oxo-delta4-steroids in position 15beta in the presence of NADPH and O2. Ring A-reduced, aromatic and 3beta-hydroxy-delta5-steroids did not serve as substrates for the 15beta-hydroxylase system. Using ion exchange chromatography on DEAE-cellulose and gel filtration on Ultrogel ACA-54 it was possible to resolve the hydroxylase system into three proteins: a strictly NADPH-dependent FMN-containing (megaredoxin reductase), an iron-sulfur protein (megaredoxin), and cytochrome P-450 (P-450meg). The activity of the 15beta-hydroxylase system was fully reconstituted upon combination of these three proteins and addition of NADPH. Megaredoxin had an apparent sulfur to iron ration of 0.98 and showed g-signals at 1.90, 1.93, and 2.06 when analyzed by electron paramagnetic reso0 times and the preparation contained 1 to 2 nmol of cytochrome P-450 per mg of protein. This preparation of cytochrome P-450meg sedimented as a homogeneous zone on sucrose gradients with a sedimentation coefficient of 3.3 S and contained 0.94 nmol of heme per nmol of cytochrome P-450. The oxidized form of cytochrome P-450meg showed absolute absorption maxima at 416, 528, and 565 nm whereas the reduced form showed maxima at 411 and 542 nm. The following scheme is suggested for the electron transport in the 15beta-hydroxylase system in B. megaterium: NADPH leads to megaredoxin reductase leads to megaredoxin leads to cytochrome P-450meg.     

Genetic differences in response to pulmonary cytochrome P-450 inducers and oxygen toxicity

The effects of cytochrome P-450 inducers on O2 toxicity were studied in mice. We first examined three cytochrome P-450 inducers, which differ by their specific tissue affinity: phenobarbital sodium (PB), essentially active in the liver, and 3-methylcholanthrene (3-MC) and beta-naphthoflavone (BNF), which are also active in the lung. Both BNF and 3-MC increased the survival rate and significantly decreased pulmonary edema (pulmonary water and wet-to-dry weight ratio) in C57BL/6J mice exposed to hyperoxia (O2 greater than or equal to 95%), whereas PB had no protective effect. In the second part of this study, we compared the action of BNF in two strains of mice. In one (C57BL/6J), cytochrome P-450 can be induced by aromatic hydrocarbons, whereas in the other (DBA/2J) cytochrome P-450 is not inducible by these compounds. Protection against O2 toxicity was assessed in terms of lethality and pulmonary edema and of lung lipid peroxidation (assessed by measuring malondialdehyde). BNF only protected against O2 toxicity in the inducible strain. This protective effect of BNF on O2 toxicity in C57BL/6J mice was associated mainly with a large increase in the components of the cytochrome P-450 system (cytochrome P-450 and cytochrome b5) in the lung. The activity of pulmonary superoxide dismutase was also slightly increased, but the enhancement was not statistically significant. In contrast, in DBA/2J mice neither the components of the cytochrome P-450 system nor the activity of superoxide dismutase showed any increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Musk xylene is a novel specific inducer of cytochrome P-450IA2.

The effect of musk xylene on contents of both cytochrome P-450IA1 and cytochrome P-450IA2 in rat liver was investigated using Western blotting analysis. Rats were treated i.p. for five consecutive days with either 50, 100 or 200 mg musk xylene/kg body weight. Musk xylene increased both total cytochrome P-450 and cytochrome b5 contents in rat liver microsomes. Musk xylene induced cytochrome P-450IA2 (384 pmol/mg protein) strongly and preferentially and the ratio of cytochrome P450IA2/P-450IA1 was about 12 at the lowest dose tested. Musk xylene also induced the cytochrome P-450IA1 dose-dependently, but these extents were very small (32-174 pmol/mg protein). These results suggest that musk xylene may be a more specific inducer for cytochrome P-450IA2 than any other inducers reported.     

The effect of pyridine and pyridine-N-oxide on the monooxygenase system of rat liver microsomes]

Effects of pyridine and pyridine-N-oxide on the monooxygenase system of rat liver microsomes have been studied. Pyridine (200 mg/kg) increased total cytochrome P-450 content and activated metabolism of some specific substrates 24 hours after injection. There was an increase in the degree of p-nitrophenol and chlorzoxazone hydroxylation due to increasing ethanol-induced cytochrome P-450IIE1 content. Pyridine was also able to induce cytochrome P-450IIB1 in rat microsomes; this reaction was accompanied by acceleration of 7-pentoxyresorufin 0-dealkylation. Cytochrome P-450IA1 appearance in liver microsomes was associated with increasing content of cytochrome P-450IA2. Dealkylation rates for specific substrates (7-ethoxyresorufin and 7-methoxyresorufin) were also increased. Similar to pyridine, pyridine-7-oxide induced cytochromes P-450IIE1, P-450IIB1/B2, and P-450IA1/A2, resulting in activation of specific substrate metabolism. Hence, pyridine and its derivative pyridine-N-oxide can be regarded as effective inducers of cytochrome P-450.     

Characteristics of a cytochrome P-450-dependent fatty acid omega-2 hydroxylase from bacillus megaterium.

The fatty acid (omega-2) hydroxylase from Bacillus megaterium ATCC 14581 was examined with respect to some general enzymatic properties attributed to an intact complex isolated in a partially purified state. Hydroxylase specific activity was found to increase with increasing protein concentration in a manner consistent with a reversible association of the components in the complex. There was a substantial kinetic lag phase for palmitate hydroxylation which was abolished by a substrate preincubation in the absence of NADPH. The substrate bound and presumably activated the hydroxylase complex without the formation of a substrate-derived intermediated. The oxidation of NADPH and the hydroxylation of palmitate were found to occur in a one to one molar ration, independent of the protein concentration. Finally, a cytochrome P-450 component of the complex was identified on the basis of its CO-binding difference spectrum. It appears, that this cytochrome P-450 component is not identical to P-450 meg of the steroid hydroxylase system of B. megaterium ATCC 13368, since progesterone, an active substrate for the latter, is not hydroxylated by the preparation from B. megaterium ATCC 14581.     

Regulation of cytochrome P-450j, a high-affinity N-nitrosodimethylamine demethylase, in rat hepatic microsomes

Polyclonal antibodies were produced in rabbits against purified cytochrome P-450j isolated from isoniazid-treated adult male rats. The monospecificity of immunoadsorbed antibody to cytochrome P-450j was demonstrated by Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays, and immunoblots. Immunoquantitation results indicated that rat liver microsomal cytochrome P-450j content decreases between 3 and 6 weeks of age in both the male and female animal. Several xenobiotics, such as Aroclor 1254, mirex, and 3-methylcholanthrene, repressed cytochrome P-450j levels when administered to male rats. Isoniazid, dimethyl sulfoxide, pyrazole, 4-methylpyrazole, and ethanol were inducers of cytochrome P-450j in rat liver although these compounds showed different inducing potencies. Microsomes from adult male rats with chemically induced diabetes also contained elevated levels of cytochrome P-450j compared to untreated animals. Cytochrome P-450j levels were measurable in kidney, whereas this isozyme was barely detectable in lung, ovaries, and testes; however, extrahepatic cytochrome P-450j was inducible by isoniazid. Approximately 80-90% of microsomal N-nitrosodimethylamine demethylation was inhibited by antibody to cytochrome P-450j whether the microsomes were isolated from untreated rats or animals administered inducers or repressors of cytochrome P-450j. The residual catalytic activity resistant to antibody inhibition may be a reflection of the inaccessibility of a certain amount of cytochrome P-450j due to interference by NADPH-cytochrome P-450 reductase based on results obtained with the reconstituted system. There was a good correlation (r2 = 0.87) between cytochrome P-450j content and N-nitrosodimethylamine demethylase activity in microsomes from rats of different ages and treated with various xenobiotics. The evidence presented indicates that cytochrome P-450j is the primary, and perhaps sole, microsomal catalyst of N-nitrosodimethylamine demethylation at substrate concentrations relevant to hepatocarcinogenesis induced by N-nitrosodimethylamine.     

Modification of enzymatic activity and difference spectra of cytochrome P-450 from various sources by cholesterol side chain cleavage inhibitors

Several groups of compounds were tested for their ability to inhibit cholesterol side chain cleavage and induce spectral change in cytochrome P-450 from bovine corpus luteum, bovine adrenal cortex, and human placental mitochondria. The substances tested include: steroids, pyridines, glutarimides, anilines and imidazoles. Good correlation was found between spectral change and enzymatic inhibition, especially in the corpus luteum which has only a single P-450-linked steroid hydroxylase. The cholesterol side chain cleavage enzyme systems from each of the three sources appear to have similar affinities for the inhibitors, which adds further support to the concept that these cytochrome P-450s are functionally identical.