Theophylline metabolism by rat liver microsomal monooxygenases

Theophylline metabolism has been studied in a reconstituted monooxygenase system with purified forms of cytochrome P-450: P-450a, P-450b, P-450d and P-450k as well as in liver microsomes of control and 3-methylcholanthrene-induced rats. Cytochrome P-450 isoforms, P-450a and P-450b, had no effect on theophylline metabolism, whereas forms P-450d and P-450k induced the synthesis of 1.3-dimethyluric acid (1.3-DMA) at the rates of 900 and 330 pmol/min/nmol of protein, respectively. The catalytic activity of these isoforms was fully inhibited by homologous monospecific antibodies. P-450c catalyzed the formation of a nonidentified metabolite. In microsomes of control animals antibodies specifically directed to cytochrome P-450k suppressed the rate of 1.3-DMA synthesis by 73%, whereas antibodies specifically raised against P-450c+d--by 11%. In microsomes of methylcholanthrene-induced animals the rate of 1.3-DMA synthesis was increased two-fold. This activity was inhibited by 61% by antibodies to cytochrome P-450k and by 18% by anti-P-450c+d antibodies.     

Biochemical bases of ftorafur-potentiating effects of perfluorodecalin, inducer of cytochrome P-450 dependent microsomal monooxygenases]

Activity of cytochrome P-450-dependent monooxygenase system is significantly lower in hepatoma H-2-73 and Lewis lung carcinoma-bearing mice as compared to that in control animals. An inhibition of the cytochrome P-450 system was observed after the injection of ftorafur. Treatment of the mice with perfluorodecalin markedly increased the antineoplastic activity of ftorafur determined by a loss of the leucocytes and of the weight hepatoma H-2-73- and Lewis lung carcinoma resistant to ftorafur alone.     

Phenobarbital-type induction of cytochrome P-450 in liver microsomes by perfluorodecalin

Cytochrome P-450 induction in hepatic microsomes after injections of rats with a fluorocarbon emulsion containing perfluorodecalin was studied in comparison with phenobarbital and methylcholanthrene type inductions. It was shown that perfluorodecalin injection as well as the phenobarbital one cause an increase in the cytochrome P-450 content, NADPH-cytochrome c reductase activity, the rates of benzphetamine N-demethylation and aldrin epoxidation in the microsomes. Using the Ouchterlony double immunodiffusion test with antibodies against cytochrome P-450b, an immunological identity of cytochrome P-450 isoforms during perfluorodecalin and phenobarbital inductions was shown. Upon "rocket" immunoelectrophoresis the recovery of cytochrome P-450 which is immunologically indistinguishable from cytochrome P-450b was approximately 72% in perfluorodecalin-induced microsomes. The activity of benzphetamine demethylase and aldrin epoxidase was inhibited by antibodies against cytochrome P-450b. These results suggest that in rat hepatic microsomes perfluorodecalin induces the cytochrome P-450 isoform whose immunological properties and substrate specificity correspond to those of phenobarbital-type cytochrome P-450.     

Age-dependent induction and repression of rat liver microsomal hydroxylase systems by estradiol

The activities of the hepatic microsomal 2α-, 2β-, 18- and 7α-hydroxylase systems active on 5α-[4-^14C] androstane-3α,17β-diol were studied in male and female rats which had been castrated and spayed at 14, 24, 35 and 45 days of age, treated for 5 days with 500 μg of estradiol benzoate per kg body weight and killed 6 days after gonadectomy. The hepatic microsomal 15β-hydroxylase enzyme system active on 5α-[1,2-³H] androstane-3α, 17β-diol 3,17-disulphate was also measured in these animals as well as in an additional group of animals that were gonadectomized at 56 days of age, treated with estradiol benzoate and killed at 62 days of age. The hydroxylase systems active on the free steroid substrate were all relatively unresponsive towards enstradiol in the developing rat, in contrast to the strong effects on hepatic hydroxylase activities previously noted following treatment of adult male rats with estradiol. On the other hand, the 15β-hydroxylase system active on disulphurylated 5α-androstane-3α, 17β-diol was inducible in both male and female rats of 41 and 51 days of age and in male rats of 61 days of age.     

Induction of cytochrome P-450 in rat liver microsomes by perfluorodecalin

Perfluorodecalin was incorporated into phospholipid liposomes and injected intraperitoneally in various dozes. The maximal cytochrome P-450 induction is reached 48 hours after perfluorodecalin injection. Cytochrome P-450 content increases 4 times after perfluorodecalin injection in dose of 0.6 ml/kg in homogenate, and 6 times after perfluorodecalin injection in a dose of 0.4 ml/kg in microsomes. Phenobarbital and perfluorodecalin induce several cytochrome P-450 isozymes and cause the appearance of a new isozyme with mass 56 kD absent in microsomes of intact CBA mice. Perfluorodecalin induction strongly increased the rate of NADPH-dependent aminopyrine nN-demethylation (6-7 times per mg of microsomal protein and 1.5 times per nmol cytochrome P-450). The rate of NADPH-dependent hydroxylation of aniline was not affected by perfluorodecalin induction.     

Nuclear ADP-ribosyl transferase activity correlates with induction of P-450 monooxygenases by phenobarbital in rat liver microsomes

The effect of phenobarbital treatment on the nuclear ADP-ribosyl transferase activity has been studied in parallel with microsomal cytochrome P-450 concentration and related mono-oxygenase activities, in rat liver. A marked activation of the ADP-ribosyl transferase was observed 24 h after phenobarbital administration. The chronological study performed between 0-6 days after phenobarbital treatment showed a sharp increase in this nuclear enzyme activity, to approximately equal to 270% of the control value produced in 48 h. The administration of 5'-methylnicotinamide in vivo, an inhibitor of ADP-ribosyl transferase activity in vitro, produced a decrease both of the induction of liver microsomal cytochrome P-450 mono-oxygenases and nuclear ADP-ribosyl transferase activity. The role of nuclear ADP-ribosyl transferase in the adaptative response of the liver cell to phenobarbital is discussed.     

Comparison of microsomal and solubilized monooxygenases from rat and rabbit by proton magnetic relaxation.

The paper presents results of a comparative study of the haem environment, by proton magnetic relaxation, in P-450 and P-448 monooxygenases from rat and rabbit, induced by phenobarbital and 3-methylcholanthrene, in both species. It was established that the method yields information on the accessibility of the haem iron for solvent molecules (protons), both in microsomes and in solubilized samples of various degrees of purification, i.e. association. The state of micelles in the solutions does not alter the haem iron accessibility. A slight difference was found for the microsomes suspended in a phosphate vs. pyrophosphate buffer, but this is without any consequence with regard to the species and form differences. The correlation time for the highly purified LM2 fraction of rabbit P-450 could not be determined more precisely than before for a sample of lower purity, because the relaxation rates are frequency independent. The correlation time for the rat P-448 monooxygenase was determined by dispersion measurements to be (4.1 +/- 0.4) x 10(-11) s. It was found that the PMRx behaviours of rabbit and rat monooxygenases are more alike in microsomes than in the partially purified solubilized form. The solubilization produces a pronounced alteration of the PMRx temperature dependence only for the rat 3-MC induced monooxygenase P-448. For the P-450 form the haem iron becomes less accessible on solubilization, both for the rabbit and the rat liver monooxygenases, whereas in case of rat liver P-448 the accessibility is considerably enhanced on solubilization. There is a substantial structural specificity of the haem environments from the two animal species, the one from rat being tighter. The reduced, NO-bound rabbit liver monooxygenase was studied also, but the results are not yet conclusive, except the fact that the unpaired spin from NO is thoroughly shielded from the solvent compared with the haem iron from the original sample. The following series of increased haem-iron accessibility emerges from the PMRx studies known so far: rat (P-448) less than rabbit (P-448) less than rat (P-450) less than rabbit (P-450) in microsomes, and rabbit (P-448, with 3-MC bound?) less than Pseudomonas putida (P-450) rat less than (P-448), less than rat (P-450) less than rabbit (P-450) from solubilized samples. For the latter, it appears that increased enzymic specificity goes along with a closing of the haem cleft.     

The stimulation of rat liver microsomal CDP-diacylglycerol formation by guanosine triphosphate

GTP has been found to markedly enhance the formation of CDP-diacylglycerol in rat liver microsomes. Neither GDP, GMP nor the nonhydrolyzable analogues of GTP increased the synthesis of the liponucleotide. The GTP stimulation of phosphatidate cytidylyltransferase activity is inhibited by EDTA and NaF. GTP enhances the activity of the enzyme in a concentration-, time-, and temperature-dependent manner and preincubation of rat liver microsomes with GTP produces a persistently activated phosphatidate cytidylyltransferase. GTP reduces the Km for phosphatidic acid, but has no effect on either the Km for CTP or the Vmax of the reaction. GTP, by stimulating the activity of the phosphatidate cytidylyltransferase, enhances the formation of phosphatidylinositol from CTP, phosphatidic acid, and inositol. Evidence is presented suggesting that the mechanism by which GTP stimulates the activity of the phosphatidate cytidylyltransferase involves a covalent modification of the enzyme itself or a protein intimately associated with the phosphatidate cytidylyltransferase.     

Effects of phenobarbitone and short-term exposure to heat on microsomal cytochrome P-450 and associated monooxygenases in rat liver

Short-term exposure of the control and phenobarbitone-treated rats to high ambient temperature caused a different response of the hepatic microsomal cytochrome P-450-dependent monooxygenase system participating in the oxidation of aniline, aminopyrine and p-nitroanisole. The highest differences of the enzyme activities were observed in rats exposed to 28 degrees C, as compared with animals exposed to 21 degrees C or 37 degrees C.     

The induction of sister chromatid exchanges by cyclophosphamide in the presence of differently induced microsomal fractions of rat liver.

The induction of sister chromatid exchanges can be monitored by a test that incorporates the factor of metabolic activation in a simple manner. The results with cyclophosphamide show that in this test the induction of the metabolizing enzymes of the rat liver homogenates used is very important. 3-Methylcholanthrene induces little if any extra conversion of cyclophosphamide to SCE-inducing metabolites, compared with no induction. Aroclor 1254 and phenobarbital however, were very good inducers. The difference found between the liver homogenates concerning SCE induction corresponded with the differences in cyclophosphamide metabolism, which was measured as the decrease in NADPH induced by cyclophosphamide.     

Multiple forms of liver microsomal flavin-containing monooxygenases: complete covalent structure of form 2

Hepatic flavin-containing monooxygenases catalyze NADPH-dependent oxygenation of a wide variety of drugs that possess a nucleophilic heteroatom. Two forms of these enzymes (form 1 and 2) have been isolated from rabbit liver microsomes and partially characterized (Ozols, J., 1989, Biochem. Biophys. Res. Commun. 163, 49-55). The complete amino acid sequence of form 2 is presented here. Sequence determination was achieved by pulsed liquid-phase and solid-phase sequencing of 40 peptides generated by chemical and enzymatic cleavages, including CNBr cleavage of tryptophanyl residues. Form 2 monooxygenase contains 533 amino acid residues and has a molecular weight of 60,089. The COOH terminus of this enzyme is very hydrophobic and presumably functions to anchor the protein to the membrane. Form 2 is readily degraded, since a form lacking residues 1 to 278 and a form without the COOH-terminal segment were also isolated from solubilized membrane preparations. The amino acid sequence of form 2 is 52% identical to that of form 1 and shows 55% identity to the sequence of rabbit lung monooxygenase derived from the cDNA data. The putative FAD and NADP binding segments around residues 9 and 190 are conserved in all three forms. Three variable segments can also be identified in these isoforms. These are residues 308 to 321, residues 408 to 421, and the membrane binding domain, residues 505 to 533. A comparison of the presently limited amino acid sequence data of flavin-containing monooxygenases (FMOs) implies that a particular FMO in different mammalian species may be very similar, but isozymes within a species may exhibit more extensive variability with respect to homology and catalytic activity. This study documents the structural diversity of a second hepatic FMO from rabbit liver and establishes this class of drug-metabolizing enzymes as a family of related proteins.     

Metabolism of melphalan by rat liver microsomal glutathione S-transferase

One of the major problems in the treatment of human cancer is the phenomenon of drug resistance. Increased glutathione (gamma-glutamylcysteinylglycine, GSH) conjugation (inactivation) due to elevated level of cytosolic glutathione S-transferase (GST) is believed to be an important mechanism in tumor cell resistance. However, the potential involvement of microsomal GST in the establishment of acquired drug resistance (ADR) remains uncertain. In our experiments, a combination of liquid chromatography/electrospray ionization/mass spectrometry (LC/ESI/MS) was employed for structural characterization of the resulting conjugates between GSH and melphalan, one of the alkylating agents. The spontaneous reaction of 1mM melphalan with 5mM GSH at 37 degrees C in aqueous phosphate buffer for 1h gave primarily the monoglutathionyl and diglutathionyl melphalan derivatives, with small amounts of mono- and dihydroxy melphalan derivatives. We demonstrated that rat liver microsomal GST presented a strong catalytic effect on the reaction as determined by the increase of monoglutathionyl and diglutathionyl melphalan derivatives and the decrease of melphalan. We showed that microsomal GST was activated by melphalan in a concentration- and time-dependent manner. Microsomal GST which was stimulated approximately 1.5-fold with melphalan had a stronger catalytic effect. Thus microsomal GST may play a potential role in the metabolism of melphalan in biological membranes, and in the development of ADR.     

Metabolism of chlorambucil by rat liver microsomal glutathione S-transferase

Clinical efficacy of alkylating anticancer drugs, such as chlorambucil (4-[p-[bis [2-chloroethyl] amino] phenyl]-butanoic acid; CHB), is often limited by the emergence of drug resistant tumor cells. Increased glutathione (gamma-glutamylcysteinylglycine; GSH) conjugation (inactivation) of alkylating anticancer drugs due to overexpression of cytosolic glutathione S-transferase (GST) is believed to be an important mechanism in tumor cell resistance to alkylating agents. However, the potential involvement of microsomal GST in the establishment of acquired drug resistance (ADR) to CHB remains uncertain. In our experiments, a combination of lipid chromatography/electrospray ionization mass spectrometry (LC/ESI/MS) was employed for structural characterization of the resulting conjugates between CHB and GSH. The spontaneous reaction of 1mM CHB with 5 mM GSH at 37 degrees C in aqueous phosphate buffer for 1 h gave primarily the monoglutathionyl derivative, 4-[p-[N-2-chloroethyl, N-2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG) and the diglutathionyl derivative, 4-[p-[2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG2) with small amounts of the hydroxy-derivative, 4-[p-[N-2-S-glutathionylethyl, N-2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBSGOH), 4-[p-[bis[2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBOH2), 4-[p-[N-2-chloroethyl, N-2-S-hydroxyethyl]amino]phenyl]-butanoic acid (CHBOH). We demonstrated that rat liver microsomal GST presented a strong catalytic effect on these reactions as determined by the increase of CHBSG2, CHBSGOH and CHBSG and the decrease of CHB. We showed that microsomal GST was activated by CHB in a concentration and time dependent manner. Microsomal GST which was stimulated approximately two-fold with CHB had a stronger catalytic effect. Thus, microsomal GST may play a potential role in the metabolism of CHB in biological membranes, and in the development of ADR.     

Ring hydroxylation of di-t-butylhydroxytoluene by rat liver microsomal preparations

3,5-Di-t-butylhydroxytoluene (compound I) was converted into 4-hydroperoxy-4-methyl-2,6-di-t-butylcyclohexa-2,5-dienone (compound II), 4-hydroxy-4-methyl-2,6-di-t-butylcyclohexa-2,5-dienone (compound III) and 2,6-di-t-butyl-4-hydroxymethylphenol (compound IV) by rat liver microsomal preparations in the presence of NADPH and air. The oxidation of compound (I) by m-chloroperbenzoic acid also produced the same compounds. These results suggest that hydroperoxide can be an intermediate in aromatic hydroxylation and that biological oxygenations resemble per-acid reactions.     

Acetylenic amines as inhibitors of microsomal monooxygenases

A new class of compounds: acetylenic amines, possessing structural similarity with the known inhibitor SKF-525A, and their saturated analogues, has been studied for its effect on the microsomal cytochrome P-450-dependent monooxygenases (MM). Significant differences in sensitivity of different substrate oxidation reactions in experiments with the mouse liver MM were observed. It was shown that the acetylenic amines investigated 13-30 times exceeded their saturated analogues as to the ability to inhibit aminopyrine and benzo[a]pyrene oxidation, and differed but slightly from their analogues with respect to p-nitroanisole and paraoxon oxidation. Benzo[a]pyrene hydroxylase of the house-fly abdomens was less sensitive to the compound investigated than that of the mouse liver, however, in contrast to it exhibited selectively to the diphenyl derivatives and not to the monophenyl ones.