Microsomal benzo(a)pyrene hydroxylase in Aspergillus ochraceus TS: Assay and characterization of the enzyme system

Microsomal preparations of $\text{Aspergillus ochraceus}$ TS oxidised benzo(a)pyrene very efficiently in the presence of NADPH and O₂ and exhibits a pH optimum of 8.0–8.2. The hydroxylation is also effected in presence of NaI04. Hydroxylation was inhibited by metyrapone, SKF-525A, PCMB, imidazole, carbon monoxide and flavone but not by cyanide, azide and antimycin A indicating thereby the involvement of cytochrome P-450 in this reaction. Inhibition by cytochrome C is consistant with the participation of NADPH-cytochrome C reductase in this hydroxylation. Reduced microsomes and its solubilized preparation, when treated with carbon monoxide, showed absorption maxima at 453 and 449 respectively. Different classical inducers of cytochrome P-450 induce the benzo(a)pyrene hydroxylase activity to varying degree and as such suggests the existence of multiple forms of cytochrome P-450 in this fungus.     

Presence of cytochrome P-450- and cytochrome P-448-dependent monooxygenase functions in hepatoma cell lines

Cell lines derived from Reuber H-4-II-E hepatoma cells and their hybrids that differ in the expression of liver-specific functions are shown to contain different forms of monooxygenases. According to 1) the specificity toward the substrates benzo(a)pyrene, aldrin and chenodexycholic acid, 2) the kinetics of the epoxidation of aldrin, 3) the response to inducers, such as benz(a)anthracene and dexamethasone, and 4) the $\text{in}$$\text{vitro}$ modifier 7,8-benzoflavone, the monooxygenases predominating in differentiated cell lines belong to the cytochrome P-450-dependent enzyme(s), those in the less differentiated lines belong to the cytochrome P-448-dependent form(s).     

Multiple forms of cytochrome P-450: resolution and purification of rabbit liver aryl hydrocarbon hydroxylase.

We describe the resolution and partial purification of two minor forms of cytochrome P-450 from liver microsomes of rabbits treated with 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin. Both forms have different electrophoretic mobilities when compared to the major form of cytochrome P-450 isolated from this source. The two cytochromes show different activities with several substrates. One form is very active in the hydroxylation of benzo($\text{a}$)pyrene when reconstituted with highly purified NADPH-cytochrome P-450 reductase.     

Solubilization and partial purification of cytochrome P-450 from rat lung microsomes

Cytochrome P-450 from rat lung microsomes has been solubilized and purified 8-fold by using affinity chromatography on an ω-amino-$\text{n}$-octyl derivative of Sepharose 4B. The purified fraction was free of cytochrome $\text{b}$₅ and NADPH-cytochrome $\text{c}$ reductase and showed spectral characteristics similar to those of lung microsomal cytochrome P-450. When combined with NADPH-cytochrome $\text{c}$ reductase partially purified from liver microsomes, the cytochrome P-450 fraction supported the hydroxylation of benzo (α)pyrene and the activity was proportional to the content of the hemoprotein. No absolute requirement for phosphatidylcholine was found.     

Liver microsomal DT diaphorase: noninvolvement in hydroxylation of benzo(a)pyrene.

A highly purified reconstituted system isolated from the microsomes of 3-methylcholanthrene-treated rats consisting of cytochrome P-448, NADPH-cytochrome $\text{c}$ reductase and synthetic dilauroyl phosphatidylcholine had no DT diaphorase activity, but hydroxylated benzo[a]pyrene at a faster rate than microsomes from 3-methylcholanthrene-treated rats. DT diaphorase purified from liver microsomes of 3-methylcholanthrene-treated rats when added to this reconstituted system did not stimulate or inhibit benzo[a]pyrene hydroxylation, nor could it replace or NADPH-cytochrome $\text{c}$ reductase in supporting the reaction. We therefore conclude that microsomal DT diaphorase is not involved in microsomal hydroxylation of benzo[a]pyrene to its phenolic products despite the observation that both DT diaphorase activity and the hydroxylation of benzo[a]pyrene are induced by 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin     

Benzo(a)pyrene hydroxylase from Saccharomyces cerevisiae. Substrate binding, spectral and kinetic data

Saccharomyces cerevisiae, brewer's yeast, produces a microsomal benzo(a)pyrene hydroxylase when grown at high glucose concentrations of which the haemoprotein, cytochrome P-450 (RH, reduced-flavoprotein:oxygen oxidoreductase (RH-hydroxylating) EC 1.14.14.1) is a component. We report here kinetic data derived from Lineweaver-Burk plots of benzo(a)pyrene hydroxylation. The Michaelis constant was decreased by growth of the yeast in the presence of benzo(a)pyrene showing the induction of a form of the enzyme more specific for this compound. NADPH or cumene hydroperoxide could be used as cofactors by this enzyme, although with different Km and V values for benzo(a)pyrene. A solubilised and a solubilised, immobilised enzyme preparation were capable of benzo(a)pyrene hydroxylation, using cumene hydroperoxide but not NADPH as the cofactor. Benzo(a)pyrene was found to produce a modified type I spectral change with yeast and rat liver microsomes. The interaction of benzo(a)pyrene with cytochrome P-450 was investigated further by means of an equilibrium gel filtration technique. There appeared to be 20 binding sites per mol ofcytochrome P-450 for benz(a)pyrene, in both yeast and rat liver microsomes.     

Purification and characterization of the cytochrome P-448 component of a benzo(a)pyrene hydroxylase from Saccharomyces cerevisiae

Cytochrome P-448, a type of cytochrome P-450, from brewer's yeast (Saccharomyces cerevisiae) grown under conditions of glucose repression was isolated and purified. Triton X-100 in very low concentration proved to be very effective in stabilizing P-448 in the microsomal fraction and later prevented its conversion to cytochrome P-420 through solubilization with various ionic and nonionic detergents. Highest yields were obtained with 1% sodium cholate, in the presence of 0.1% Triton X-100 and reduced glutathione. A novel combination of hydrophobic adsorption and other chromatographic techniques was used for the purification of cytochrome P-448. These involve the use of amino octyl-Sepharose 4B, instead of the low-yielding aminohexyl derivative, followed by the fast-running hydroxyapatite-cellulose column. Finally, the use of DEAE-Sephacel was found to increase greatly the purity of the cytochrome P-448 obtained. The molecular weight of this preparation was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (M_r, 55,500). Using the known molar extinction coefficient of the carbon monoxide-difference spectrum the estimate of degree of purity of cytochrome P-448 obtained by this purification procedure was between 88 and 97%. Electrophoresis also showed that this preparation was completely homogeneous and assays showed that it was also completely free of cytochrome b_s, cytochrome c reductase and cytochrome P-420. Purified cytochrome P-448 reconstituted with cytochrome P-450 (cytochrome c) reductase, isolated from yeast, showed 10-fold higher aryl hydrocarbon hydroxylase activity with benzo[a]pyrene as a substrate than the corresponding microsomal fraction enzyme. Kinetics of benzo[a]pyrene hydroxylation were determined: K_m (33 μm) was comparable with that reported for purified hepatic cytochrome P-448. The number of binding sites of microsomal and purified cytochromes P-450 (from liver of phenobarbital-induced rats) and yeast cytochrome P-448 with benzo[a]pyrene has been determined using and equilibrium gel filtration method. There is one binding site in each case (contrast with six sites for microsomal enzymes). The Scatchard plot gives number of binding sites, apparent association constants (K), and the equivalent dissociation constants (K_s). Comparison is made with spectral dissociation constants for these enzymes and benzo[a]pyrene. Thus the proportion bound, dissociation constant (K_s), and stoichiometry of rat liver (phenobarbital induced) and yeast cytochrome P-448 with benzo[a]pyrene were compared with corresponding values for microsomal fractions of both systems. Purified enzymes had higher K_s values in both cases, and the proportion of enzyme that bound benzo[a]pyrene was high (53%) for liver and this value is 100% for purified enzyme from yeast, which is the same as the value obtained for the microsomal enzyme from yeast.     

The degradation of different forms of cytochrome P-450 in vivo by fluroxene and allyl-iso-propylacetamide.

Treatment of uninduced, phenobarbital and 3-methylcholanthrene induced rats with fluroxene and allyl-$\text{iso}$-propylacetamide decreased hepatic microsomal cytochrome P-450 and equivalently decreased microsomal heme, aniline binding and $\text{p}$-nitroanisole demethylase. In contrast, ethylmorpnine demethylase, benzpyrene-3-hydroxylase and ethoxyresofurin deethylase were not in all cases decreased in proportion to the loss of cytochrome P-450. After phenobarbital induction fluroxene and allyl-$\text{iso}$-propylacetamide degrade multiple forms of cytochrome P-450, but degrade in the greatest amounts the form(s) of cytochrome P-450 inducible by phenobarbital. After 3-methylcholanthrene induction fluroxene preferentially degrades cytochrome P-448, while allyl-$\text{iso}$-propylacetamide is relatively specific for the form(s) of cytochrome P-450 inducible by phenobarbital.     

Evidence for cytosolic benzo(a)pyrene carrier proteins which function in cytochrome P450 oxidation in rat liver

Solutions of cytosolic proteins from rat liver contain benzo(a)pyrene solubilizing activity capable of serving as a carrier between solid state benzo(a)pyrene and microsomal cytochrome P₄₅₀. Fractionation of benzo(a)pyrene-saturated cytosolic proteins on a Sephadex G-100 column or by sucrose density gradients produced benzo(a)pyrene peaks of about 46,000 daltons and a very high molecular weight material. The protein-bound benzo(a)pyrene obtained in both peaks was oxidized rapidly by microsomes in the presence of NADPH, indicating that the benzo(a)pyrene carrier activity is capable of presenting the substrate to the cytochrome P₄₅₀. Liver cytosolic proteins from rats receiving intraperitoneal injection of [¹⁴C] benzo(a)pyrene was chromatographed on a column of Sephadex G-75. Radioactivity eluted at the same positions of the chromatogram as did the carrier activities described above. These results indicate that these benzo(a)pyrene carrier proteins may have an $\text{in}\text{vivo}$ role in the metabolism of benzo(a)pyrene.     

Cytochrome P-450 inducibility by ethanol and 7-ethoxycoumarin O-deethylation in S.cerevisiae

The level of cytochrome P-450 and some enzymatic activity cytochrome P-450 dependent in a diploid strain (D7) of $\text{S.cerevisiae}$ are affected by the substrate supporting growth and its concentration and, in particular, by the growth phase of the culture. For these reasons we tested the hypothesis that the induction of the monooxygenase system in the D7 strain when grown in high concentration of glucose depended on one product of glycolysis, ethanol. There was a strict correlation between the level of cytochrome P-450 and the ethanol concentration. Moreover we developed a sensitive test measuring the ethoxycoumarin O-deethylation in order to detect the enzymatic activity cytochrome P-450 dependent in whole yeast cells, in different growth conditions.     

The effect of enzyme induction on the irreversible binding of ethynyloestradiol to guinea pig liver microsomes

The effect of pretreatment with phenobarbitone, rifampicin, β-naphthoflavone, antipyrine and spironolactone on the irreversible binding of ethynyloestradiol to guinea pig liver microsomes has been examined and the corresponding changes in microsomal P-450 content and cytochrome $\text{c}$ reductase activity measured. Rifampicin produced the greatest increase (220%) in irreversible binding while phenobarbitone produced the greatest increase in both microsomal P-450 content (172%) and cytochrome $\text{c}$ reductase activity (210%). There was no correlation of irreversible binding with either microsomal P-450 content or with cytochrome $\text{c}$ reductase activity.     

Effects of phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls on sex-specific forms of cytochrome P-450 in liver microsomes of rats

The effects of treatment with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls (PCB) on the amounts of sex-specific forms of cytochrome P-450, namely P-450-male and P-450-female, in male and female rats were studied. Although treatment with phenobarbital, 3-methylcholanthrene or PCB markedly increased the total amount of hepatic cytochrome P-450, P-450-male and P-450-female were rather decreased or not significantly changed. Thus, the percentages of P-450-male and P-450-female in the total cytochrome P-450 were decreased in liver microsomes from the treated rats. The increases in specific cytochrome P-450, such as P-448-H, P-448-L, and P-450I-c accounted for the increase in the total amount of cytochrome P-450 in the treated rats. The treatment with phenobarbital or PCB increased the activities of testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase and aminopyrine N-demethylase more markedly in female rats than in male rats. Similarly, the treatment with 3-methylcholanthrene increased benzo(a)pyrene hydroxylase more markedly in female rats. Therefore, the sex-differences in testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase, and aminopyrine N-demethylase activities became smaller after the drug treatment. These results indicate that sex-specific P-450-male and P-450-female were unaffected, or even depressed by the agents in some cases.     

Metabolism of (−)-trans-(3R,4R)-dihydroxy-3,4-dihydrochrysene to diol epoxides by liver microsomes

Metabolism of biosynthetic (?)-trans-(3R,4R)-dihydroxy-3,4-dihydrochrysene by liver microsomes from control, phenobarbital-treated and 3-methylcholanthrene-treated rats was investigated. Although previous studies of the metabolism of related benzo[a]pyrene and benzo[e]pyrene dihydrodiols which also prefer the diaxial conformation had indicated that diol epoxides were minor metabolites, the diastereomeric chrysene 3,4-diol-1,2-epoxides-$\text{1}$ and $\text{?2}$ were major metabolites (66–90%). All three types of microsomes metabolized the chrysene 3,4-dihydrodiol at low but essentially similar rates (0.5–0.7 nmol substrate/nmol cytochrome P-450/min).     

Hydrodynamic properties of monomeric cytochromes P-450LM2 and P-450LM4 in n-octylglucoside solution

Sedimentation equilibrium and sedimentation velocity measurements were carried out on cytochrome P-450_LM2 from phenobarbital-treated rabbit liver and on cytochrome P-450_LM4 from 5,6-benzoflavone-treated rabbit liver in the presence of the nonionic detergent $\text{1-O-}\text{n}\text{-}\text{octyl}\text{-β-}\text{D}\text{-}\text{glucopyranoside}$. P-450_LM2 was monomeric with a molecular weight of 48,800 and a Stokes radius of 3.1 nm in 7 g/l detergent and P-450_LM4 was monomeric with a molecular weight of 49,800 and a Stokes radius of 2.6 nm at 5 g/l detergent. Both particles were spherical in shape under these conditions. Neither cytochrome was irreversibly denatured at these detergent concentrations as indicated by the ability to form substantial amounts (>60%) of the CO adduct with an absorption maximum at 451 nm (P-450_LM2) or 448 nm (P-450_LM4) when diluted into detergent-free buffer containing CO and sodium dithionite.     

Purification of a substrate complex of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-treated rabbits.

Cytochrome P-450 was purified as a 3-methylcholanthrene complex from liver microsomes of 3-methylcholanthrene-treated rabbits to a specific content of 17 to 18 nmoles per mg of protein with a yield of about 10 %. The purified protein gave only a single protein band on sodium dodecylsulfate-urea-poly-acrylamide gel electrophoresis, and its apparent molecular weight was estimated to be about 54,000, a value which is higher than that for cytochrome P-450 from phenobarbital-treated rabbits by about 4,000. The reconstituted system containing the purified cytochrome and NADPH-cytochrome $\text{c}$ reductase was active in NADPH-dependent hydroxylation of benzo[α]pyrene.     

Immunochemical study on the participation of cytochrome b5 in drug oxidation reactions of mouse liver microsomes.

Highly purified divalent and monovalent antibodies against cytochrome $\text{b}{}_5$, anti-$\text{b}{}_5$ immunoglobulin G (IG) and anti-$\text{b}{}_5$ Fab', were used in elucidating the role of this cytochrome in the drug-oxidizing enzyme system of mouse liver microsomes. Anti-$\text{b}{}_5$ IG strongly inhibited not only NADH-supported but also NADPH-supported oxidation of 7-ethoxycoumarin and benzo(a)pyrene, but had no inhibitory action on the oxidation of aniline. Anti-$\text{b}{}_5$ Fab' also inhibited NADH-supported and NADPH-supported benzo(a)pyrene hydroxylation. These observations indicate an essential role of cytochrome $\text{b}{}_5$ in the transfer of electrons not only from NADH but also from NADPH to cytochrome P-450 in the microsomal oxidation of some drugs, but not of aniline.     

Benzo(a)pyrene metabolism by purified forms of rabbit liver microsomal cytochrome P-450, cytochrome b5 and epoxide hydrase in reconstituted phospholipid vesicles

The roles of rabbit liver cytochrome b₅, epoxide hydrase and various forms of cytochrome P-450 in the NADPH-dependent metabolism of benzo(a)pyrene were examined. After incorporation of the purified enzymes into phospholipid vesicles, using the cholate gel filtration technique, the various types of cytochrome P-450 did exhibit different stereospecificities in the oxygenation of the substrate. Cytochrome P-450_LM2 was found to efficiently convert benzo(a)pyrene in the presence of epoxide hydrase to 4,5-dihydroxy-4,5-dihydrobenzo(a)pyrene whereas cytochrome P-450_LM4 primarily participated in the formation of 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene. By contrast, benzo(a)pyrene was not metabolized by cytochrome P-450_LM3. Cytochrome b₅ enhanced cytochrome P-450_LM2-catalyzed oxygenations 5-fold, whereas cytochrome P-450_LM4-dependent oxygenations proceeded at a 3 times higher rate when cytochrome b₅ was present in the membrane.     

Specificity in the interaction of phospholipids and fatty acids with vesicle reconstituted cytochrome P-450. A spin label study

The association of fatty acids, androstane, phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid with purified and phospholipid-vesicle reconstituted cytochrome $\text{P-450}$ was studied by spin labeling. Spin-labeled fatty acids were found to be motionally restricted by cytochrome $\text{P-450}$ in both phospholipid vesicles and in microsomes to a much greater extent than spin-labeled phospholipids. The equilibrium of spin-labeled fatty acid between the bulk membrane lipid and the protein interface could be shifted towards an increased amount in the bulk phospholipid phase by the addition of oleic acid or lysophosphatidylcholine, but not by sodium cholate. Microsomes from different animals showed a variable extent of motional restriction of fatty acids, independent of pretreatment of the animals with phenobarbital or β-naphthoflavone, of cytochrome $\text{P-450}$ content, of the presence of type I and type II substrates for cytochrome $\text{P-450}$. These differences are attributed to the presence of varying amounts of lipid breakdown products in the microsomal membrane such as lysolipids or fatty acids which compete with the externally added spin-labeled fatty acids, or with spin-labeled androstane for the binding to cytochrome $\text{P-450}$. The negative charge of the fatty acid was found to be involved in its association with the protein. Cytochrome $\text{P-450}$ was shown to interact only with a few spin-labeled phospholipid molecules in such a way that the motional restriction of the spin acyl chains can be detected by electron paramagnetic resonance ($\text{τ}{}_{\mathrm{<mtext>R</mtext>}}\text{> 10}{}^{\mathrm{?8}}\text{s}$). The number of associated lipid molecules per protein probably is too small to form a complete shell around the protein. This lipid-protein interaction could be destroyed by the addition of sodium cholate, in contrast to the fatty acid-protein interaction.     

Induction of hepatic microsomal propranolol N-desisopropylase activity by 3-methylcholanthrene and sudan III

Metabolism of propranolol in liver microsomes was markedly induced in rats and $\text{C57BL}\text{6J}$ mice treated with 3-methylcholanthrene (3-MC) or sudan III, inducers of cytochrome P-448. 7,8 Benzoflavone inhibited propranolol metabolism in microsomes from treated rats. 3-MC did not induce propranolol metabolism in genetically nonresponsive $\text{DBA}\text{2}$ mice. High-performance liquid chromatographical analysis of propranolol metabolites revealed a 6-fold increase in propranolol N-desisopropylase activities in liver microsomes from sudan III- or 3-methylcholanthrene-treated rats. It is concluded that propranolol N-desisopropylation is predominantly catalyzed by cytochrome P-448.     

Benzo(a)pyrene pretreatment of Drosophila simulans mutant strain results in the induction of aberrant isoform of cytochrome P-450 with increased capacity to metabolize benzo(a)pyrene]

The basal level of benzo(a)pyrene monooxygenase, epoxide hydrolase and glutathione S-transferase activity as well as the content of cytochrome P-450 were found the same in both compared benzo(a)pyrene (BP) sensitive D. simulans strain 364yv and BP-resistant wild one (Turku). Phenobarbital pretreatment resulted in the same increase level of these enzyme activities in both strains. BP-pretreatment of 364yv flies decreased the amount of the cytochrome P-450 but raised up the turnover of BP per molecule of cytochrome P-450. SDS-polyacrylamide gel electrophoresis of the microsomal proteins from BP-pretreated 364yv flies (but not from Turku) showed an increased hemoprotein content in the 56000 band. The relationship between BP-sensitivity of the strain 364yv and BP-induced aberrant isoform of the cytochrome P-450 has been discussed.