Activity of key enzymes of heme metabolism and cytochrome P-450 content in the rat liver in experimental rhabdomyolysis and hemolytic anemia

The 5-aminolevulinate synthase, heme oxygenase, tryptophan-2,3-dioxygenase activities, the content of total heme and cytochrome P-450 content in the rat liver and absorption spectrum of blood serum in Soret region under glycerol model of rhabdomiolisis and hemolytic anemia caused by single phenylhydrazine injection have been investigated. The glycerol injection caused a considerable accumulation of heme-containing products in the serum and the increase of the total heme content, holoenzyme, total activity and heme saturation of tryptophan-2,3-dioxygenase, as well as the increase of the 5-aminolevulinate synthase and heme oxygenase activities in the liver during the first hours of its action and the decrease of cytochrome P-450 content in 24 h. Administration of phenylhydrazine lead to the increasing of hemolysis products content in blood serum too, although it was less expressed. The phenylhydrazine injection caused the increase of activities of 5-aminolevulinate synthase, holoenzyme, total activity and heme saturation of tryptophan-2,3-dioxygenase, as well as decrease of cytochrome P-450 content in the rat liver in 2 h. The increase of the total heme content and heme oxygenase activity has been observed in 24 h. The effect of heme arrival from the blood stream, as well as a direct influence of glycerol and phenylhydrazine on the investigated parameters are discussed.     

Immunohistochemical localizations of cytochromes P-450 in rat liver

Antibodies produced against two forms of cytochrome P-450, PB-B and MC-B, which were purified to apparent homogeneity from hepatic microsomes of rats pretreated with phenobarbital and 3-methylcholanthrene, respectively, have been employed to localize these hemoproteins immunohistochemically at the light microscopic level in the livers of untreated rats. Using these antibodies in an unlabeled antibody peroxidase-antiperoxidase technique, immunohistochemical staining for the cytochromes P-450 was detected in parenchymal cells throughout the liver lobule. The patterns of immunohistochemical staining intensity observed with the two antibodies, however, were quite different. Exposure of liver sections to the antibody to cytochrome P-450 PB-B resulted in intense immunostaining within the centrilobular regions but produced staining of considerably weaker intensity in the peripheral regions of the lobule. In contrast to these observations, the antibody to cytochrome P-450 MC-B yielded a more uniform pattern of immunohistochemical staining, with the intensity of staining being only slightly greater in the centrilobular regions. The results of this immunohistochemical study thus demonstrate that different patterns of distribution exist for different forms of cytochrome P-450 within the liver lobule and that the greatest concentration of cytochrome P-450 occurs within the centrilobular regions of the liver.     

Immunoquantitation of cytochrome b5 and methylcholanthrene-induced cytochromes P-450

The enzyme-linked immunosorbent assay (ELISA) has been investigated for its ability to quantitate hydrophobic proteins like cytochromes b5 and P-450 at the subnanogram level. Issues encountered that have broad significance not only for ELISA, but for other qualitative and quantitative immunoassays as well, include the effects of detergent, the discriminatory capacity of ELISA, and the method for determining an assay's selectivity.     

Tissue-specific expression of rat mRNAs homologous to cytochromes P-450b and P-450e.

The tissue-specific expression of cytochrome P-450b and P-450e mRNAs was examined with synthetic 18-mer oligomer probes in the liver, lung, kidney, and testis of control and inducer pretreated adult rats. RNAs homologous to the P-450e probe were detected in trace amounts in control and 3-methylcholanthrene (MC) induced livers and at high levels in livers from phenobarbital (PB) induced animals. P-450e mRNA levels were below detection limits in the other tissues examined, regardless of pretreatment. In contrast, mRNAs homologous to the P-450b oligomer were detected at low levels in control and inducer pretreated lung and testis, and at high levels in PB induced liver. No P-450b mRNAs were detected in these assays in RNA isolates from the kidney or from control or MC pretreated liver. Solution hybridization data indicated that the rat lung contained 9-12%, and the testis, 6-9%, respectively, of the levels of P-450b mRNA measured in the PB induced liver. Results from oligo(dT)-cellulose and poly(U)-affinity experiments indicated that the hepatic mRNAs for P-450b and P-450e were present predominantly in the bound, polyadenylated fraction, whereas the homologous lung and testes P-450b mRNAs predominated in the flow-thru fractions.     

Partial purification and properties of cytochromes P-450 and P-448 from rat liver microsomes

Cytochromes P-450 and P-448 in rat liver microsomes were solubilized with sodium cholate and were partially purified. The preparations contained 5.0–5.5 nmoles of cytochrome P-450 or P-448 per mg of protein; contamination with cytochrome P-420 and cytochrome b₅, was less than 10% of the total heme content. The absolute spectra of Cytochromes P-450 and P-448 differed only slightly; both hemoproteins had a Soret peak at 418–419 nm in the oxidized absolute spectra and at 448 and 450 nm in the reduced plus CO absolute spectra. Both hemoproteins showed typical type I (benzphetamine) and type II (aniline) binding spectra but differed in their binding of hexobarbital (another type I substrate). The total phospholipid content of the preparation (per mg protein) has been reduced by approximately 90% relative to microsomes and the hemoprotein has been purified 20–25 fold with respect to phospholipid. The partially purified hemoprotein fractions, after combination with a reductase and lipid fraction, were capable of oxidizing a variety of substrates inluding drugs, steroids, and chemical carcinogens.     

Phenobarbital induction of rat liver cytochromes P-450b and P-450e. Quantitation of specific RNAs by hybridization to synthetic oligodeoxyribonucleotide probes

Cytochromes P-450b and P-450e are extremely homologous and immunochemically indistinguishable proteins that are coordinately induced by phenobarbital in rat liver. To assess the effect of phenobarbital on mRNA levels for each of these hemoproteins we performed solution hybridization and Northern blot experiments with synthetic oligodeoxynucleotide probes of defined sequence. Our data demonstrate that phenobarbital administration to rats resulted in marked increases in levels of hepatic mRNA for both cytochrome P-450b and cytochrome P-450e, with a 4- to 5-fold greater accumulation of P-450b mRNA vis à vis P-450e mRNA. The level of hepatic mRNA increased from less than 3 molecules/cell of each mRNA in untreated rats, to 630 and 130 molecules/cell for P-450b and P-450e, respectively, in phenobarbital-treated rats. Data obtained in Northern blot hybridization experiments demonstrated that the size of the mRNAs for each protein were identical, being approximately 1800 bases in length.     

Activity of 5-aminolevulinate synthase in rat liver during degradation of cytochrome P-450 caused by administration of cadmium chloride

The 5-aminolevulinate synthase, tryptophan-2,3-dioxygenase activities and cytochrome P-450 content in the rat liver was studied in different terms after CdCl2 administration and after administration of metal salt against a background of 2-hours action of alpha-tocopherol. The lowering of activity of 5-aminolevulinate synthase in 2 h with the consequent increase of the enzyme activity in 6 h and 24 h was detected. The holoenzyme activity and heme saturation of tryptophan-2,3-dioxygenase increased 6 h after CdCl2 administration. The holoenzyme activity and the total activity of tryptophan-2,3-dioxygenase rised in 24 h. The level of cytochrome P-450 lowered. Preliminary administration of alpha-tocopherol prevented changes of studied parameters 24 h after CdCl2 administration. The relationship between decrease of cytochrome P-450 level and 5-aminolevulinate synthase activation are discussed.     

Isoenzyme-specific phosphorylation of cytochromes P-450 and other drug metabolizing enzymes

A series of fourteen cytochrome P-450 isoenzymes was treated with three different protein kinases and found to divide into isoenzymes phosphorylated by both the cyclic AMP-dependent kinase and the calcium-phospholipid-dependent kinase (P-450 PB 3a and PB 2e), by none of these kinases (P-450 PB 1b, MC 1b, UT 1, and thromboxane synthase), and by either the cyclic AMP-dependent kinase (P-450 LM 2, PB 2d, and PB 3b) or the calcium-phospholipid-dependent kinase (P-450 PB 1a, PB 2a, MC 1a, LM 3c, and LM 4). Other components of the monooxygenase system, cytochrome P-450 reductase, cytochrome b5, cytochrome b5 reductase as well as microsomal epoxide hydrolase, were poor substrates for the kinases employed. On the other hand, glutathione transferases 1-2 and 4-4, but not 3-3, were relatively good substrates for the calcium-phospholipid-dependent kinase.     

Coordinate induction of multiple mRNAs specific for rat liver phenobarbital-inducible cytochromes P-450

Utilizing two-dimensional gel electrophoresis, the polypeptide composition of a purified microsomal cytochrome P-450 preparation isolated from phenobarbital-treated Long-Evans rats obtained from Charles River Laboratories has been examined. The purified protein consists of three polypeptides with nearly identical subunit molecular weights (approximately 52,000) but differing in net charge. These three polypeptides can be detected in liver microsomes isolated from phenobarbital-treated rats by immunoblot analysis but are virtually absent in microsomes isolated from untreated rats. All three polypeptides appear to be products of distinct mRNAs since they can be immunoprecipitated from rabbit reticulocyte lysates programmed with poly(A+)-RNA isolated from phenobarbital-treated rats. The amount of functional mRNA specific for the P-450 polypeptides increases dramatically in response to an acute administration of phenobarbital; however, in untreated rats the amount of functional mRNA was below the level of detection by the translational assay. These data are consistent with the very low level of the phenobarbital-inducible cytochromes P-450 in liver microsomes isolated from untreated rats. Finally, the data indicate that all three cytochrome P-450 mRNAs increase rapidly in response to phenobarbital administration and are regulated coordinately.     

Variations in immunoreactivity for phenobarbital- and 3-methylcholanthrene-inducible cytochromes P-450, and NADPH-cytochrome P-450 reductase in rat liver over twenty-four hours

Summary To reveal distribution patterns of phenobarbital-and 3-methylcholanthrene-inducible cytochromes P-450 (PB and MC) and NADPH-cytochrome P-450 reductase (P-450red) within the liver acinus of untreated rats, and their variations over 24 h, hepatic samples were examined by immunohistochemistry and image-analyzer at evenly spaced six time points over 24 h. When examined in semi-thin sections obtained from Epon-embedded, freeze-dried, and paraformaldehyde vapor-phase fixed materials, the immunoreactivity for these enzymes showed different distribution patterns within the liver acinus. Immunodeposits for PB were predominantly distributed in perivenous hepatocytes, whereas those for MC and P-450red were slightly more intense in periportal hepatocytes at each time point. The immunoreactivity for PB and MC in both perivenous and periportal hepatocytes increased during the dark period, peaking early in the light period. These variations coincide well with our previous morphometric results (Uchiyama and Asari, 1984); the volume and surface densities of rough endoplasmic reticulum (rER) in hepatocytes increased during the dark period. On the other hand, weak fluctuation was demonstrated in the immunoreactivity for P-450red in hepatocytes of both zones. These results suggest that PB and MC are retained in rER rather than smooth endoplasmic reticulum (sER) of hepatocytes obtained from untreated rats. These enzymes in sER may be short in their half-life spans.     

Zonal distribution of cytochromes P-450 and related enzymes of bovine adrenal cortex--quantitative assay of concentrations and total contents

The zona glomerulosa, zona fasciculata, zona reticularis, and medulla were separated from bovine adrenal glands and cytochromes P-450 and related enzymes in each zone were investigated immunochemically by Western blotting using antisera from chickens or rabbits against cytochromes P-450scc, P-450(11)beta, P-450s21, and b5, NADH-cytochrome b5 reductase, NADPH-cytochrome P-450 reductase, NADPH-adrenodoxin reductase, and adrenodoxin. Concentrations of cytochrome P-450(11)beta, NADPH-cytochrome P-450 reductase, and cytochrome b5 per milligram of protein of homogenate were higher in the zona glomerulosa than in the other zones; the levels of the other components were higher in the zona fasciculata. The total enzyme content of all components was the highest in the zona fasciculata. The amount of adrenodoxin was about 10 times that of NADPH-adrenodoxin reductase in each zone.     

Factors involved in the regulation of the levels and activities of rat liver cytochromes P-450

Methods have been developed for the purification of eight rat liver microsomal cytochrome P-450 (P-450) isoenzymes. Another rat P-450, responsible for the metabolism of the genetic polymorphism prototype debrisoquine, has also been partially purified from rat liver. Six P-450s have been purified to electrophoretic homogeneity from human liver preparations. The rat and human P-450s can be quantified in crude samples using 'immunoblotting' methods coupled with peroxidase visualization. A study on the effects of a family of polybrominated biphenyl congeners led to the conclusion that the levels of all of the rat P-450s considered above are under some degree of independent regulation. In monolayer culture, different P-450s show different stabilities and levels of several are selectively regulated by various media components. Studies with the eight isolated rat P-450s indicate that the iron spin state, oxidation-reduction potential (Fe3+/Fe2+ couple), and catalytic activity towards substrates are not related to each other. The major function of phospholipid in reconstituted P-450/NADPH-P-450 reductase systems is the facilitation of formation of a complex of the two proteins. Studies on the regioselective hydroxylation of warfarin have been used to develop an order of binding affinity of the different P-450s for NADPH-P-450 reductase.     

Studies on the molecular organization of cytochromes P-450 and b5 in the microsomal membrane.

1. The relative orientations of the heme groups of cytochromes P-450 and b5 in the microsomal membrane have been studied by the technique of electron paramagnetic resonance. The results show that the heme plane of cytochrome P-450 lies in the same plane as the membrane surface, whereas the cytochrome b5 heme plane has a random orientation. 2. No significant broadening or change in relaxation properties of the gz component of low spin cytochrome P-450 occurred when cytochrome b5 was reduced by redox poising. It is concluded that there is little or no paramagnetic coupling between the heme groups of the two species. 3. The results favor a model in which no tight complex between cytochromes P-450 and b5 is present, the species being independent and interacting only by random molecular collisions or via other intermediate species.     

Oxidation-reduction properties of rat liver cytochromes P-450 and NADPH-cytochrome p-450 reductase related to catalysis in reconstituted systems

A series of equilibrium and kinetic measurements involving the oxidation-reduction properties of purified rat liver NADPH-cytochrome P-450 reductase and eight different purified rat liver cytochromes P-450 (P-450s) were carried out. Apparent spin states of P-450 iron were determined in the absence and presence of a number of known substrates by using second-derivative and conventional near-UV absorbance spectroscopy. Many of the substrates examined did not produce significant changes in the apparent iron spin state, even when binding could be demonstrated with equilibrium dialysis. Further, the spin state was not correlated to catalytic activity of the P-450s in reconstituted enzyme systems. The oxidation-reduction potentials were determined for the ferric/ferrous couples of each of the eight P-450s in the presence and absence of known substrates, as well as other proteins suspected of altering the potentials. The midpoint potential (Em,7) ranged from -350 to -289 mV for the P-450s under these conditions. In some cases Em,7 was raised with the addition of substrates, but the extent of the increase was no greater than +33 mV. The Em,7 of one P-450 (P-450 beta NF/ISF-G) was not changed significantly when the fraction of high-spin iron varied between 11 and 67%. Steady-state spectral studies provided evidence for the accumulation of an oxygenated ferrous intermediate (or a derived product) of one P-450 (P-450PB-B) in the presence of a substrate, cyclohexane. Studies on the donation of electrons from cytochrome b5 and a series of dyes to this complex suggest that it has an effective Em,7 (for reduction) of approximately +50 mV. In studies with one of the P-450s, steady-state spectral studies indicated that the three-electron-reduced form of NADPH-P-450 reductase accumulates, consistent with the view that this form of the reductase is involved in the reduction of P-450 from the ferric to the ferrous state.     

Age-related changes in the iron spin state of testosterone-binding rat liver microsomal cytochromes P-450

The aging process is accompanied by decreased drug metabolism as well as lower levels of sex hormones such as testosterone. We examined the age-dependence of liver microsomal cytochromes P-450 from young (3 months) and old (24 months) male rats by absorption and ESR spectroscopy. Spectral perturbations by testosterone were used to identify testosterone-specific P-450 forms. Absorption difference spectra indicated that testosterone induced a greater conversion of P-450 to the high spin form in young rats than in old rats. ESR signals corresponding to total low spin P-450 were of higher intensity in the young rats and were increased by testosterone. Testosterone also interconverted one low spin P-450 species to another. These results demonstrate age-related differences in the types and amounts of testosterone-specific P-450's in rats.