Untreated Rat Liver Microsomes Research Articles

Differential expression and function of three closely related phenobarbital-inducible cytochrome P-450 isozymes in untreated rat liver

The levels of expression of cytochromes P-450b and P-450e (both inducible by phenobarbital (PB) and differing by only 14 of 491 amino acids) in liver microsomes from untreated male rats were separately quantitated by Western blotting with a polyclonal antibody raised against P-450b that is equally effective against P-450e (anti P-450 b e ). A protein with mobility identical to P-450e was detected in all microsomal samples. Microsomes from uninduced livers of individual male rats from five different strains exhibited only minor interstrain and interindividual variability in the expression of P-450e (17 ± 5 pmol P-450e/mg microsomal protein) with the exception of the Brown Norway strain (8.5 ± 0.5 pmol P-450e/mg). Expression of P-450b varied widely from undetectable levels (<2 pmol/mg) in most Sprague-Dawley rats to about 50% of P-450e levels in Fischer and Brown Norway strains. Anti P-450 b e inhibited total metabolism of 7,12-dimethylbenz[ a]anthracene (DMBA) by uninduced microsomes, to an extent dependent on rat strain (15–30%), predominantly through inhibition of formation of 12-hydroxymethyl-7-methyl BA (12HOMMBA) (65–85%), the major metabolite of purified P-450e. A specific activity for P-450e-dependent DMBA metabolism was calculated from four sets of microsomes where the P-450b content was either undetectable or very low (0.7–1.0 nmol/nmol P-450e/min −1). Comparable calculated activities were, however, obtained from other untreated rat liver microsomes where P-450b levels were significant. Polymorphism in P-450b was detected but did not affect total P-450b expression or the sensitivity of DMBA metabolism to anti P-450 b e . A fourth band of greater mobility than P-450b (apparent M r < 50,000), was also recognized by anti P-450 b e . The intensity of this band did not vary among individual rats or among the different strains and therefore did not correlate with the sensitivity of microsomal DMBA metabolism to anti P-450 b e . A monoclonal antibody (MAb) against P-450b (2-66-3) recognized P-450's b, b 2, and e on Western blots but did not react with this higher mobility band. MAb 2-66-3 and two other MAbs produced against P-450b inhibited 12-methylhydroxylation of DMBA by untreated rat liver microsomes to the same extent as anti P-450 b e . Following PB induction, P-450b was induced to about double the level of P-450e in most rat strains examined. This study demonstrates that cytochrome P-450e is selectively expressed in livers of untreated rats and plays a substantial role in the metabolism of DMBA, despite its relatively low level of expression (⩽3% of the total spectrally detectable P-450).

Incorporation of cytochrome b5 into rat liver microsomal membranes : Impairment of cytochrome P-450-dependent mixed function oxidase activity

Cytochrome b 5 was purified to electrophoretic homogeneity from the liver microsomes of untreated rats and reincorporated into liver microsomes from phenobarbital-treated rats, resulting in an approximate three-fold enrichment of the cytochrome b 5 specific content (1.5 nmol haemoprotein · mg −1 protein). Our results have shown that the N-demethylation of benzphetamine was progressively inhibited in cytochrome b 5-fortified microsomal preparations. Using stopped flow, visible difference spectrophotometry, the NADPH-driven reduction kinetics of cytochrome P-450 were examined in the modified microsomes over the first few seconds of reaction. Increasing the amount of incorporated cytochrome b 5 resulted in a progressive inhibition of the initial, fast phase reduction rate constant of microsomal cytochrome P-450, both in the absence and presence of the type I substrate benzphetamine. Although the initial rate of NADPH-driven cytochrome b 5 reduction was the same for both native and cytochrome b 5-fortified microsomes, the extent of cytochrome b 5 reduction was greater in the fortified microsomes. If cytochrome b 5 has a positive role to play in cytochrome P-450-dependent mixed function oxidase activity either as an effector or in electron transfer or both, the former haemoprotein must be already present in sufficient concentrations in the native microsomes.

The predominant role of S-oxidation in rat liver metabolism of thiaarenes

Thiaarenes are metabolized by liver microsomes of untreated rats predominantly to sulfones and sulfoxides. After pretreatment of rats with monooxygenase inducers, ring oxidation of thiaarenes is also observed. In case of benzo[ b]naphtho[2,3- d]thiophene the formation of a p-quinone takes place. Rat liver microsomal metabolism of the thiaarenes tested as substrates did not resemble that of the polycyclic aromatic hydrocarbon (PAH) isosters at all.

Assay of mannose-6-phosphatase in untreated and detergent-disrupted rat-liver microsomes for assessment of integrity of microsomal preparations

An accurate, precise, and convenient procedure was developed for measurement of the latency of the low-Km mannose-6-phosphatase activity for the purpose of assessment of the membrane permeability barrier in microsomes. This approach is based on previous work of Arion et al. [J. Biol. Chem. (1976) 251, 4901-4907] and consists of measurement of mannose-6-phosphatase activity in the untreated microsomal fraction and in the corresponding microsomes that are fully disrupted in order to eliminate the membrane permeability barrier. Complete disruption of rat liver microsomes was achieved by incubation for 60 min at 0 degree C in the presence of 4 mM zwitterionic detergent 3-[(3-cholamido-propyl)dimethyl-ammonio]-2-hydroxy-1-propane sulphonate (Chapso). That the microsomal membrane permeability barrier was eliminated under those conditions was suggested by the fact that the enzyme activation (up to 50-fold) produced by this pretreatment was at least as large as the effect of any other previously reported disruptive procedure. Disruption of the microsomes by Chapso or by ultrasonication markedly enhanced the thermolability of the mannose-6-phosphatase activity. In addition, exposure of the microsomes to high concentrations of Chapso produced enzyme inactivation that could be partially reversed by dilution of the detergent prior to assaying the enzymic activity. Investigation of these enzyme inactivation phenomena under various incubation conditions for disruption of the microsomes by Chapso and for subsequent assay of mannose-6-phosphatase activity in the presence of Chapso enabled us to define conditions under which instability of the enzyme was undetectable. Using these optimized procedures for disruption of microsomes and assay of hexose-6-P phosphohydrolase, we found that the low-Km mannose-6-phosphatase activity of untreated rat liver microsomes consistently was less than 5% of the total enzyme activity in the fully disrupted microsomes. Accurate and precise assay of the structural latency of mannose-6-phosphatase in membrane preparations must be performed under well-controlled conditions, with special attention to the marked thermolability of the enzyme in the presence of detergent, and is a prerequisite for using this approach for the purpose of assessing intactness of microsomal preparations.

Stereoselective metabolism of 9-methyl-, 9-hydroxymethyl- and 9,10-dimethylanthracenes: absolute configurations and optical purities of trans-dihydrodiol metabolites.

Stereoselective metabolism of 9-methylanthracene (9-M-A), 9-hydroxymethylanthracene (9-OHM-A) and 9,10-dimethylanthracene (9,10-DM-A) by liver microsomes from untreated rats, and from rats pretreated with either 3-methylcholanthrene or phenobarbital was studied. The metabolites were separated by h.p.l.c. and characterized by analysis of the u.v.-visible, mass and n.m.r. spectral data and compared to published spectral data. The identified trans-dihydrodiol metabolites were 9-M-A trans-1,2- and 3,4-dihydrodiols, 9-OHM-A trans-1,2- and 3,4-dihydrodiols, and 9,10-DM-A trans-1,2-dihydrodiol. The absolute configuration and optical (enantiomeric) purity of the trans-dihydrodiol metabolites were determined. In order to assist in determining the absolute configuration of these trans-dihydrodiols, metabolism of 9-bromoanthracene by liver microsomes of rats pretreated with 3-methylcholanthrene was performed and its trans-1,2- and 3,4-dihydrodiol metabolites were determined to both possess an R,R absolute configuration. Absolute configurations of the trans-dihydrodiol metabolites of 9-methylanthracene, 9-hydroxymethylanthracene and 9,10-dimethylanthracene were then determined by comparison of their CD spectra with those of anthracene 1R,2R-dihydrodiol or 9-bromoanthracene 1R,2R-dihydrodiol. The optical purities of the trans-dihydrodiol metabolites were determined by analysis of the chiral stationary phase h.p.l.c. profiles of the dihydrodiols or their corresponding tetrahydrodiol derivatives. The results indicated that the R,R enantiomers were the predominant products and that liver microsomes of rats pretreated with 3-methylcholanthrene exhibited much higher stereoselectivity than the liver microsomes of untreated rats or rats pretreated with phenobarbital in the formation of trans-dihydrodiols from anthracene, 9-methylanthracene, 9-hydroxymethylanthracene and 9,10-dimethylanthracene. The methyl and hydroxymethyl substituents slightly decreased the stereoselectivity of the trans-dihydrodiol formation.

Metabolism of 1-nitrobenzo[a]pyrene by rat liver microsomes to potent mutagenic metabolites.

1-,3- and 6-Nitrobenzo[a]pyrene (nitro-BaP), which are prototypes of nitro polycyclic aromatic hydrocarbons (nitro-PAHs) derived from a carcinogenic parent PAH, benzo[a]pyrene, are environmental contaminants and potent bacterial mutagens. In this study, the aerobic and hypoxic metabolism of 1-nitro-BaP by rat liver microsomes was studied. Aerobic metabolism of 1-nitro-BaP yielded 1-nitro-BaP trans-7,8- and 9,10-dihydrodiol, while metabolism under hypoxic conditions yielded 1-amino-BaP. The metabolites formed from aerobic metabolism of 1-nitro-BaP and 1-nitro-BaP trans-9,10-dihydrodiol by liver microsomes of untreated rats and rats pretreated with 3-methylcholanthrene and phenobarbital were quantified. Comparison of these results with those obtained with BaP and BaP trans-9,10-dihydrodiol indicates that nitro substitution at the 1-position of BaP markedly affects the regioselectivity of the P-450-containing enzymes. 1-Nitro-BaP and the three metabolites were potent mutagens in Salmonella typhimurium TA98, both in the absence and in the presence of an exogenous metabolic activation system (S9). The direct and S9-mediated mutagenicities of 1-nitro-BaP and the two dihydrodiols were decreased in the nitroreductase-deficient strain TA98NR, while TA98/1,8-DNP6, an O-acetylase-deficient strain, was less sensitive to the two dihydrodiols, both with and without S9, and 1-nitro-BaP with S9. 1-Amino-BaP was equally mutagenic in all three tester strains. These observations indicate that: the metabolism of 1-nitro-BaP involves several pathways leading to mutagenic activation; the major activation pathways of 1-nitro-BaP involve nitroreduction; nitroreduction followed by O-acetylation is the major activation pathway of 1-nitro-BaP trans-7,8- and 9,10-dihydrodiol; and 1-amino-BaP is a potent direct-acting mutagen.

Formation and disposition of nitrosochloramphenicol in rat liver

It has been suggested that in the chloramphenicol-induced aplastic anemia nitrosochloramphenicol may be involved as a toxic intermediate. We found that aminochloramphenicol, which reportedly is formed from chloramphenicol by intestinal bacteria, is N-oxygenated by liver microsomes of untreated rats with apparent K m = 0.4 mM and V max = 0.28 nmole/min/mg protein. These values are in close agreement with those reported for aniline N-oxygenation. Reductive reactions, however, eliminate the N-oxygenation products at markedly higher rates. As judged from hemoglobin-free singlepass liver perfusion experiments, N-hydroxy-chloramphenicol is reduced at rates faster than 300 nmole/min/g liver wet, and nitrosochloramphenicol is eliminated at rates faster than 1.5 μmole/min/g liver. At least two NADPH- and two NADH-dependent cytosolic enzymes are responsible for nitrosochloramphenicol reduction. Determination of the kinetic parameters of these enzymes by stop-flow analysis revealed the contribution of enzymes, one of it being alcohol dehydrogenase, with Michaelis constants in the micromolar range. Despite this high reducing capacity, about 10% of nitrosochloramphenicol reacted with GSH under formation of glutathionesulfinamidochloramphenicol and GSSG released from the liver into bile and venous effluent. At high nitrosochloramphenicol load these reactions led to glutathione depletion of the liver, caused membrane damage, and impaired bile production. At low nitrosochloramphenicol load, i.e. below 0.5 μmole/min/g, no relevant nitrosochloramphenicol passed the liver. These data together with the previously reported reactions of nitrosochloramphenicol within human blood suggest that nitrosochloramphenicol, if formed at all in the intestine or liver, is rather unlikely to be transferred to the critical target.

Regulation of cytochrome P-450. Immunochemical quantitation of eight isozymes in liver microsomes of rats treated with polybrominated biphenyl congeners.

Specific immunochemical techniques were used to quantitate the levels of eight isozymes of cytochrome P-450 (P-450) and epoxide hydrolase in liver microsomes of untreated rats and rats treated with phenobarbital, 3-methylcholanthrene, a mixture of these two compounds, nine individual polybrominated biphenyl (PBB) congeners, and a commercial mixture of PBBs. Levels of two 3-methylcholanthrene-inducible P-450s (designated P-450 beta NF-B and P-450 beta NF/ISF-G) varied over two orders of magnitude and were highly correlated. The levels of four phenobarbital-inducible P-450s (designated P-450PB-B, P-450PB-C, P-450PB-D, and P-450PB/PCN-E) were all correlated to each other. The level of one form, P-450UT-A, which was present at substantial levels in untreated rats, was inversely correlated to the levels of P-450 beta NF-B and P-450 beta NF/ISF-G. Among the PBB congeners which were examined, the presence of bromine at carbons o to the biphenyl bridge favored the induction of P-450PB-B, P-450PB-C, P-450PG-D, and P-450PB/PCN-E but did not necessarily eliminate the ability to induce P-450 beta NF-B and P-450 beta NF-ISF-G. PBB congeners with 2,2'-dibromo substitution induced P-450 beta NF-B and P-450 beta NF/ISF-G if one of the biphenyl rings contained bromines at positions 2,3, and 4. The induction of P-450UT-F was found to occur to a small extent with three of the compounds and is not readily explained in terms of structure-activity relationships. Although correlations were found among levels of some of the forms of P-450, several important exceptions were noted in relative levels of the individual enzymes. While the correlative data are useful in predicting induction patterns, all eight forms of P-450 appear to be independently regulated to some extent.

Purification and characterization of liver microsomal cytochromes p-450: electrophoretic, spectral, catalytic, and immunochemical properties and inducibility of eight isozymes isolated from rats treated with phenobarbital or beta-naphthoflavone.

Eight different forms of cytochrome P-450 (P-450) were purified to electrophoretic homogeneity by a common procedure from liver microsomes of rats treated with phenobarbital or beta-naphthoflavone. Antibodies were prepared to seven of these forms in rabbits. The eight P-450s were distinguished by spectral properties of the ferric, ferrous, and ferrous carbonyl forms, apparent monomeric molecular weights, peptide mapping, immunological reactivity as discerned by double-diffusion immunoprecipitin analysis and crossed immunoelectrophoresis, and catalytic activities toward the substrates acetanilide, aminopyrine, aniline, benzo[a]-pyrene, d-benzphetamine, N,N-dimethylnitrosamine, 7-ethoxycoumarin, 7-ethoxyresorufin, ethylmorphine, p-nitroanisole, testosterone, and (R)- and (S)-warfarin. Crossed sodium dodecyl sulfate-polyacrylamide gel immunoelectrophoresis was used to estimate the levels of each of the eight forms of P-450 present in the liver microsomes of untreated rats and rats treated with phenobarbital, 5,6-benzoflavone, pregnenolone-16 alpha-carbonitrile, isosafrole, or the polychlorinated biphenyl mixture Aroclor 1254. In each situation, the sum of the levels of these eight P-450s was at least as high as the spectrally determined P-450 content. The results clearly demonstrate that individual forms of P-450 can be induced by different compounds and that a single compound can lower the level of one form of P-450 while inducing one or more other forms of P-450. Catalytic activities toward each of the substrates observed with microsomal preparations are compared to rates predicted on the basis of the content of each of the eight P-450s. These studies provide a basis for further studies on the regulation of individual P-450s, the physical properties of the different P-450s, and the metabolic consequences of changes in the forms of P-450 in rat liver models.

Purification and characterization of two constitutive forms of rat liver microsomal cytochrome P-450.

Two constitutive forms of cytochrome P-450 isozymes, designated RLM3 and RLM5, have been purified from untreated rat liver microsomes. RLM3 and RLM5 have minimum molecular weights of 50,000 and 51,000, respectively, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Absolute oxidized spectra indicate that RLM3 is essentially all in the low spin state, whereas RLM5 contains some high spin component. The CO-reduced difference spectral maximum of RLM3 is at 449 nm and RLM5 is at 451 nm. RLM3 preferentially hydroxylates testosterone at 6 beta and 7 alpha positions and forms an even higher amount of an as yet unidentified polar metabolite, whereas RLM5 hydroxylates testosterone preferentially at 2 beta and 16 alpha positions, and exhibits 6 beta-hydroxylase activity similar to RLM3. RLM5 metabolizes aminopyrine, ethylmorphine, and benzphetamine all faster than does RLM3. When RLM3 and RLM5 were digested with proteolytic enzymes, peptide maps showed that these two cytochrome P-450 isozymes have different primary structures. Cyanogen bromide digestion breaks both isozymes into fragments too small to compare on polyacrylamide gels. Amino acid analyses indicate that RLM3 and RLM5 have very similar but not identical compositions. NH2-terminal residues were determined by Edman degradation and high pressure liquid chromatography separation of phenylthiohydantoin-derivatives. The first four residues of both enzymes are identical, NH2-Met-Asp-Pro-Val, but the fifth residue of RLM5 is -Leu-while that of RLM3 is -Val-. These sequences differ dramatically from all other P-450 isozymes reported to date.

The metabolism of pyrene by rat liver microsomes and the influence of various mono-oxygenase inducers.

1. Pyrene metabolite g.l.c. profiles were recorded and metabolites identified by mass spectrometry. 2. Pyrene is metabolized by liver microsomes of untreated rats to 1-hydroxypyrene, 4,5-dihydroxy-4,5-dihydropyrene, two different diphenols and a triol, tentatively identified as 1,4,5-trihydroxy-4,5-dihydropyrene. 3. Pretreatment with phenobarbital or polychlorinated biphenyls favours oxidation at the K-region, whereas cytochrome P-448 inducers stimulate oxidation at the non-K-region of pyrene. 4. 1-Hydroxypyrene does not inhibit pyrene oxidation. 5. Pyrene diphenols are formed by secondary oxidation of 1-hydroxypyrene. 6. Triols are formed from dihydrodiols by secondary oxidation.

Selective induction of rat liver microsomal cytochrome P-448 by 3,4,5,3',4'-pentachlorobiphenyl and its effect on liver microsomal drug metabolism.

Pretreatment of rats with 3, 4, 5, 3', 4'-pentachlorobiphenyl (PenCB) at a single i.p. dose of 10 mg/kg caused a marked increase of cytochrome P-448 (P448) in liver microsomes. The liver microsomes of PenCB-treated rats (PenCB-microsomes) showed similar but somewhat different properties from those of 3-methylcholanthrene (MC)-treated rats (MC-microsomes) with respect to its CO difference spectrum, electrophoretic pattern on sodium dodecyl sulfate (SDS)-polyacrylamide gel and immunochemical reactivity with the antibodies prepared against the hemoproteins from phenobarbital (PB)- or β-naphthoflavone (BNF)-treated rat liver microsomes. Furthermore, PenCB pretreatment decreased not only demethylation of aminopyrine and codeine, but also hydroxylation of aniline, to which PenCB-microsomes showed lower affinity than MC-microsomes. Trace amounts of PenCB inhibited the metabolism of aniline and aminopyrine by MC-microsomes but not by untreated rat liver microsomes. On SDS-polyacrylamide gel electrophoretograms the ratio of cytochrome P450 (P450) to P448 in PenCB-microsomes was much smaller than that in MC-microsomes. These results may suggest that a part of the differences between the catalytic properties of PenCB- and MC-microsomes is due to the inhibitory effect of residual PenCB in PenCB-microsomes or/and the marked change of a relative ratio of P450 to P448 in PenCB-microsomes. A possibility that PenCB may induce a different P448 from that induced by MC is also discussed.

Purification and properties of cytochrome P-450 obtained from liver microsomes of untreated rats by lauric acid affinity chromatography.

Purification and properties of cytochrome P-450 obtained from liver microsomes of untreated rats by lauric acid affinity chromatography.

Investigation of the Component Reactions of Oxidative Sterol Demethylation: ROLE OF AN ENDOGENOUS MICROSOMAL SOURCE OF REDUCING EQUIVALENTS

Abstract Incubation of 50 mg of rat liver microsomal protein with either 25 µg of crude snake venom (Crotalus adamanteus) or 1 µg of purified phospholipase A results in essentially complete loss of NAD+-dependent methyl sterol demethylase activity (R-CH3 → R-H + CO2). However, the snake venom-treated microsomes contain all of the known component enzymes of demethylase and microsomal electron carriers. Methyl sterol demethylase activity is restored either by the addition of supernatant fraction from snake venom treatment or by the addition of reduced pyridine nucleotides. Thus, the source of reducing equivalents for the multistep mixed function oxidase of demethylase has been examined extensively. Evidence for an endogenous, NAD+-dependent cytochrome b5-reducing system has been found in untreated rat liver microsomes. Snake venom treatment removes the endogenous reducing system from microsomes. Concomitant loss of the NAD+-dependent methyl sterol demethylase and the reducing system is observed. Each enzymic activity is restored by the addition of supernatant fraction, which is obtained from the snake venom treatment. The presence of the endogenous reducing system and the known dependence of the mixed function oxidase upon a source of reducing equivalents explains the paradox observed earlier that fully active methyl sterol demethylase of rat liver microsomes is observed in the absence of reduced pyridine nucleotide when NAD+ is the only added cofactor. The rate of generation of reducing equivalents by the endogenous NAD+-dependent system is slow, but the rate is substantially faster than the rate of methyl sterol oxidase, the mixed function oxidase of demethylase. The endogenous reducing system appears to obtain reducing equivalents via a microsomal alcohol dehydrogenase which is readily released by the snake venom treatment Both commercial horse liver alcohol dehydrogenase and isolated rat liver alcohol dehydrogenase restore the NAD+-dependent methyl sterol demethylase activity that is lost upon snake venom treatment; the rat liver enzyme is approximately 10 times as efficacious.