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.     

Mechanism of interaction between cytochromes P-450 RLM5 and b5: evidence for an electrostatic mechanism involving cytochrome b5 heme propionate groups

The role of cytochrome b5 heme propionate groups in the functional interactions between cytochromes P-450 RLM5 and b5 has been investigated by comparing the capacity of RLM5 to interact with both native b5 and a b5 derivative in which the native heme was replaced with ferric protoporphyrin IX dimethyl ester (DME-b5). Both forms of b5 interacted with RLM5 causing an increase in the RLM5 spin state from 28 to 68% high-spin RLM5 at saturation, as judged using uv-visible spectrophotometry. However, DME-b5 exhibited a 7-fold weaker affinity for RLM5. The apparent dissociation constant (Kd) for the interaction between RLM5 and b5 was also shown to be a strong function of ionic strength, in a manner consistent with the involvement of electrostatic attraction in complex formation. Reconstitution of b5 into an RLM5-dependent monooxygenase system stimulated the p-nitroanisole demethylase rate about 25-fold and 7-ethoxycoumarin deethylase about 6-fold. DME-b5, however, produced only 30% of the stimulation of RLM5-dependent turnover of p-nitroanisole observed at equivalent concentrations of native b5 without a change in Km. With 7-ethoxycoumarin, turnover was 50% diminished. The diminished capacity of DME-b5 to stimulate RLM5-dependent substrate turnover was shown not to be due to impairment of electron flow between NADPH-cytochrome P-450 reductase and DME-b5, since the Km of reductase for DME-b5 is 2.5-fold lower, and the Vmax is actually increased, but rather to an impairment of some aspect of functional interaction between the DME-b5 and RLM5. The data show that complex formation between cytochrome P-450 and b5 involves electrostatic attraction mediated in part by cytochrome b5 heme propionate groups.     

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.     

Induction and catalytic activities of cytochromes P-450b/e and P-450c in the liver microsomes of neonatal rats

The activities of cytochrome P-450-dependent monooxygenases has been investigated in the liver microsomes of newborn rats (3-16 days after birth) induced with PB or 3-MC. It has been shown that the induction by PB and 3-MC results in the increase of both the total amount of cytochrome P-450 as determined by the CO-reduced spectrum and the amount of induced forms P-450b/e and P-450c respectively. In the course of induction of the specific forms of cytochrome P-450 BP-hydroxylase and 7-ER-O-deethylase activities increased at 3-MC-induction, while BPh-N-demethylase and BP-hydroxylase increased at PB-induction. Analysis of inhibition of monooxygenase reactions with antibodies has showed that only P-450c was involved in metabolism of BP and 7-ER. Participation of P-450b/e in BPh N-demethylation was notably lower in the neonates in comparison to the adult rats. In the one-week-old rats induced with 3-MC a considerable rate of BP hydroxylation and 7-ER O-deethylation (2-4.5 nmol of product min-1 mg-1) has been observed despite a small amount of P-450 (0.02-0.1 nmol/mg of protein). This fact shows the higher catalytic activity of this cytochrome P-450 in the neonates compared to similar characteristics of P-450c in the 3-MC-induced microsomes. Metabolism of BP in the PB-microsomes of the neonatal rats was inhibited neither by anti-P-450b/e nor anti-P-450c in contrast to the adults, where this reaction was inhibited by antibodies against P-450b/e.     

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.     

Immunolocalization of cytochromes P-450olf1 and P-450olf2 in rat olfactory mucosa

Previously, we described two olfactory-specific cytochromes P-450: rat cytochrome P-450olf1 (IIG1), identified by cDNA cloning, and bovine cytochrome P-450olf2 (IIA), identified by peptide microsequencing of a transmembranal polypeptide (p52). Here we describe the preparation of polyclonal antisera against peptide sequences of these proteins and their use in the immunolocalization of cytochromes P-450olf1 and P-450olf2 in rat olfactory mucosa. Immunoreactivities related to both enzymes are found in the subepithelial Bowman's glands of olfactory mucosa. Practically no immunoreactivity was found in other rat tissues, including liver, lung, kidney and respiratory mucosa. In addition, double-labeling experiments demonstrated that cytochromes P-450olf1 and P-450olf2 are present in the same population of Bowman's glands. The olfactory-specific localization of cytochromes P-450olf1 and P-450olf2 is consistent with a role for these enzymes in the modification or clearance of odorants from the chemosensory tissue.     

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.     

Lipid peroxidation and the degradation of cytochrome P-450 heme

The enzyme content and functional capacities of mesophyll chloroplasts from Atriplex spongiosa and maize have been investigated. Accompanying evidence from graded sequential blending of leaves confirmed that mesophyll cells contain all of the leaf pyruvate, P_i dikinase, and PEP carboxylase activities and a major part of the adenylate kinase and pyrophosphatase. 3-Phosphoglycerate kinase, NADP glyceraldehyde-3-P-dehydrogenase, and triose-P isomerase activities were about equally distributed between mesophyll and bundle sheath cells but other Calvin cycle enzymes were very largely or solely located in bundle sheath cells. In A. spongiosa extracts of predominantly mesophyll origin the proportion of the released pyruvate, P_i dikinase, adenylate kinase, pyrophosphatase, 3-phosphoglycerate kinase, and NADP glyceraldehyde-3-P dehydrogenase retained in pelleted chloroplasts was similar but varied between 30 and 80% in different preparations. The proportion of these enzymes and NADP malate dehydrogenase recovered in maize chloroplast preparations varied between 15 and 35%. Washed chloroplasts retained most of the activity of these enzymes but ribulose diphosphate carboxylase and other Calvin cycle enzyme activities were undetectable. Among the evidence for the integrity of these chloroplasts was their capacity for light-dependent conversion of pyruvate to phosphoenolpyruvate and O₂ evolution when 3-phosphoglycerate or oxaloacetate were added. These results support our previous conclusions about the function of mesophyll chloroplasts in C₄-pathway photosynthesis and clearly demonstrate that they lack Calvin cycle activity.     

Rat liver cytochrome P-450b, P-420b, and P-420c are degraded to biliverdin by heme oxygenase

In this report we provide data, for the first time, demonstrating the conversion of the heme moiety of certain cytochrome P-450 and P-420 preparations, to biliverdin, catalyzed by heme oxygenase. We have used purified preparations of cytochromes P-450c, P-450b, P-450/P-420c, or P-450/P-420b as substrates in a heme oxygenase assay system reconstituted with heme oxygenase isoforms, HO-2 or HO-1, NADPH-cytochrome c (P-450) reductase, biliverdin reductase, NADPH, and Emulgen 911. With cytochrome P-450b or P-450/P-420b preparations, a near quantitative conversion of degraded heme to bile pigments was observed. In the case of cytochrome P-450/P-420c approximately 70% of the degraded heme was accounted for as bilirubin but only cytochrome P-420c was appreciably degraded. The role of heme oxygenase in this reaction was supported by the following observations: (i) bilirubin formation was not observed when heme oxygenase was omitted from the assay system; (ii) the rate of degradation of the heme moiety was at least threefold greater with heme oxygenase and NADPH-cytochrome c (P-450) reductase than that observed with reductase alone; and (iii) the presence of Zn- or Sn-protoporphyrins (2 microM), known competitive inhibitors of heme oxygenase, resulted in 70-90% inhibition of bilirubin formation.     

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.