Induction and regulation of cytochrome P450 K-5 (lauric acid hydroxylase) in rat renal microsomes by starvation

The effects of starvation on rat renal cytochrome P-450s were studied. The content of spectrally measured cytochrome P-450 in the renal microsomes of male rats increased 2-fold with 72 h starvation, but cytochrome b5 and NADPH-cytochrome P-450 reductase were not induced. 7-Ethoxycoumarin O-dealkylation and aniline hydroxylation activities of the renal microsomes of control male rats were very low but were induced 2.5-3-fold by 72 h starvation. Aminopyrine N-demethylation and lauric acid hydroxylation activities were induced 1.5-2-fold by 72 h starvation. The changes in catalytic activities suggested that the contents of individual cytochrome P-450s in the renal microsomes were altered by starvation. The contents of some cytochrome P-450s were measured by Western blotting. P450 DM (P450IIE1), a typical form of cytochrome P-450 induced by starvation in rat liver, was barely detected in rat kidney and was induced 2-fold by 72 h starvation. P450 K-5, a typical renal cytochrome P-450 and lauric acid hydroxylase, accounted for 81% of the spectrally measured cytochrome P-450 in the renal microsomes of control male rats and was induced 2-fold by 72 h starvation. P450 K-5 was not induced in rat kidney by treatment with chemicals such as acetone or clofibrate. The renal microsomes of male rats contained 6-times as much P450 K-5 as those of female rats. These results suggest that P450 K-5 is regulated by an endocrine factor.     

Characterization of three cytochrome P450s purified from renal microsomes of untreated male rats and comparison with human renal cytochrome P450

Three renal cytochrome P450s (P450 K-2, K-4, and K-5) were purified from renal microsomes of untreated male rats. Also, the human renal cytochrome P450 (P450 HK) was partially purified from renal microsomes and its properties were compared with those of the rat renal cytochrome P450s. The molecular weight of P450 K-2, K-4, and K-5 was 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The absolute spectrum of the oxidized forms indicated that they had the low-spin state of heme, and the CO-reduced spectral maxima of P450 K-2, K-4, and K-5 were at 449, 451, and 452 nm, respectively. NH2-terminal sequence analysis of P450 K-2, K-4, and K-5 showed that these forms were different from hepatic cytochrome P450s purified previously. P450 K-2, K-4, and K-5 catalyzed the O-dealkylation of 7-ethoxycoumarin but were not efficient in the hydroxylation of testosterone. Aminopyrine was metabolized by P450 K-2 and K-4 but not by P450 K-5. Lauric acid was metabolized efficiently by all of these forms in the presence of cytochrome b5. The regiospecificity of these forms toward lauric acid was different. P450 K-2 hydroxylated lauric acid only at the (omega-1)-position, not at the omega-position. P450 K-4 and K-5 hydroxylated lauric acid at both the omega- and (omega-1)-positions. The ratios of omega/(omega-1)-hydroxylation activity of P450 K-4 and K-5 were 2.5 and 7.8, respectively. Human P450 HK was purified 220-fold and its specific content was 2.0 nmol/mg of protein. The Soret maxima of P450 HK were at 418 nm for the oxidized form, 416 nm for the reduced form, and 450 nm for the CO-reduced form. P450 HK catalyzed the O-dealkylation of 7-ethoxycoumarin but was not efficient in aminopyrine N-demethylation or testosterone hydroxylation. P450 HK had high lauric acid omega- and (omega-1)-hydroxylation activities in the presence of cytochrome b5, especially omega-hydroxylation. These properties resembled those of P450 K-5 most closely. Anti-P450 K-5 antibody cross-reacted with P450 HK as well as P450 K-5 and only one band was stained on immunostained Western blotting for partially purified P450 HK. The molecular weight of P450 HK was 52,000 on Western blotting.     

A novel cytochrome P450 3A isoenzyme in rat intestinal microsomes

PCR with several pairs of primers facilitates screening for new isoenzymes among highly homologous cytochrome P450s (CYPs). Combinations of two pairs of primers, which amplify N- and C-terminal coding sequences of either CYP3A1/CYP3A23 or CYP3A2 detected the presence of a previously unrecognized CYP3A in enterocyte microsomes isolated from rats. PCR, Northern blot, and immunoblotting with specific antibodies indicated that this isoenzyme is clearly distinguishable from CYP3A1, 3A23 or 3A2. Sequencing of a 285 bp coding fragment of this gene revealed 97% similarity with rat olfactory CYP3A9 (P450olf3).     

Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of a large number of endogenous compounds and the majority of ingested environmental chemicals, leading to their elimination and often to their metabolic activation to toxic products. This enzyme system therefore provides our primary defense against xenobiotics and is a major determinant in the therapeutic efficacy of pharmacological agents. To evaluate the importance of hepatic P450s in normal homeostasis, drug pharmacology, and chemical toxicity, we have conditionally deleted the essential electron transfer protein, NADH:ferrihemoprotein reductase (EC, cytochrome P450 reductase, CPR) in the liver, resulting in essentially complete ablation of hepatic microsomal P450 activity. Hepatic CPR-null mice could no longer break down cholesterol because of their inability to produce bile acids, and whereas hepatic lipid levels were significantly increased, circulating levels of cholesterol and triglycerides were severely reduced. Loss of hepatic P450 activity resulted in a 5-fold increase in P450 protein, indicating the existence of a negative feedback pathway regulating P450 expression. Profound changes in the in vivo metabolism of pentobarbital and acetaminophen indicated that extrahepatic metabolism does not play a major role in the disposition of these compounds. Hepatic CPR-null mice developed normally and were able to breed, indicating that hepatic microsomal P450-mediated steroid hormone metabolism is not essential for fertility, demonstrating that a major evolutionary role for hepatic P450s is to protect mammals from their environment.     

Alpha-tocopherol regulation of hepatic cytochrome P450s and ABC transporters in rats

To test the hypothesis that supra-elevated hepatic alpha-tocopherol concentrations would up-regulate mechanisms that result in increased hepatic alpha-tocopherol metabolism and excretion, rats received daily subcutaneous alpha-tocopherol injections (10 mg/100 g body wt) and then were sacrificed on Day 0 or 12 h following their previous injection on Days 3, 6, 9, 12, 15, and 18. Liver alpha-tocopherol concentrations increased from 12 +/- 1 nmol/g (mean +/- SE) to 819 +/- 74 (Day 3), decreased at Day 9 (486 +/- 67), and continued to decrease through Day 18 (338 +/- 37). alpha-Tocopherol metabolites and their intermediates increased and decreased similarly to alpha-tocopherol albeit at lower concentrations. There were no changes in known vitamin E regulatory proteins, i.e., hepatic alpha-tocopherol transfer protein or cytochrome P450 (CYP) 4F. In contrast, both CYP3A and CYP2B, key xenobiotic metabolizing enzymes, doubled by Day 6 and remained elevated, while P450 reductase increased more slowly. Consistent with the decrease in liver alpha-tocopherol concentrations, a protein involved in biliary xenobiotic excretion, p-glycoprotein, increased at Day 9, doubling by Day 15. Thus hepatic alpha-tocopherol concentrations altered hepatic proteins involved in metabolism and disposition of xenobiotic agents.     

Metabolism of chlorobenzene with hepatic microsomes and solubilized cytochrome P-450 systems

Chlorobenzene is converted to mixture of o-, m-, and p-chlorophenols in perfused rat livers, and in the following cell-free hepatic preparations from rat: postmitochondrial supernatant, microsomes, and reconstituted soluble hemoprotein systems. The percentage of m-chlorophenol steadily decreases with increasing resolution of the hemoprotein-monoxygenase system. Prior treatment of rats with 3-methylcholanthrene results in a large increase in rate of formation of the ortho isomer in all hepatic systems. Prior treatment with phenobarbital results in moderate increases in rates of formation of all three chlorophenols. Formation of the three chlorophenols is inhibited to similar extents by carbon monoxide, metyrapone, and high concentrations of glutathione. SKF-525a and 7,8-benzoflavone inhibit formation of o- and p-chlorophenols to a greater extent than that of the meta isomer; with microsomal preparations NADH greatly potentiates NADPH-dependent formation of p-chlorophenol, moderately potentiates formation of m-chlorophenol and has little effect on formation of o-chlorophenol. Dihydrodiols are not significant metabolites of Chlorobenzene with the soluble hemoprotein systems. These results, in concert with changes in proportions of phenolic metabolites seen during resolution of hepatic systems from the intact cell of the perfused liver to the soluble hemoprotein, are at least consonant with the hypothesis that chlorophenol production from Chlorobenzene is catalyzed by three different enzymes, two of which form arene oxide intermediates and one of which catalyzes a direct formation of a phenol. Thus, o- and p-chlorophenols result, respectively, on isomerization of intermediate 3- and 4-chlorobenzene oxides, while formation of m-chlorophenol would appear to occur via a direct oxidative pathway. In vitro conjugation of the arene oxide with glutathione or hydration is not a significant pathway. Assay of chlorophenols and dihydrodiols of Chlorobenzene was by high-pressure liquid chromatography.     

Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay

The major phenobarbital-inducible form of cytochrome P-450 (cytochrome P-450 PB) was purified to homogeneity from rat liver microsomes and rabbit antibodies prepared against the purified enzyme. Using these antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytochrome P-450 PB in microsomes which was sensitive at the nanogram level. The content of cytochrome P-450 PB was determined in hepatic microsomes from rats treated with various xenobiotics. Phenobarbital and Aroclor 1254 pretreatments resulted in several-fold increases in immunoreactive cytochrome P-450 PB over control levels. ELISA measurements of cytochrome P-450 PB were also carried out over a 48-h time course of phenobarbital induction in liver microsomes. Significant increases over control levels were seen at 16 h and beyond. Measurements of ELISA-detectable cytochrome P-450 PB were made in microsomes following the administration of CCl4 to phenobarbital-pretreated rats. Immunoreactive cytochrome P-450 PB was observed to decrease less rapidly than the spectrally detectable enzyme in the microsomal membranes. Inhibition of heme synthesis was carried out by the administration of 3-amino-1,2,4-triazole (AT) to rats. Concomitant pretreatment with phenobarbital and AT resulted in levels of ELISA-detectable cytochrome P-450 PB which were significantly increased over control levels, while spectrally detectable levels of total holoenzyme remained unchanged. These results support the idea that this cytochrome P-450 may exist, at least partly, in the microsomal membrane in an inactive or apoprotein form.     

Regulation of cytochrome P-450j, a high-affinity N-nitrosodimethylamine demethylase, in rat hepatic microsomes

Polyclonal antibodies were produced in rabbits against purified cytochrome P-450j isolated from isoniazid-treated adult male rats. The monospecificity of immunoadsorbed antibody to cytochrome P-450j was demonstrated by Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays, and immunoblots. Immunoquantitation results indicated that rat liver microsomal cytochrome P-450j content decreases between 3 and 6 weeks of age in both the male and female animal. Several xenobiotics, such as Aroclor 1254, mirex, and 3-methylcholanthrene, repressed cytochrome P-450j levels when administered to male rats. Isoniazid, dimethyl sulfoxide, pyrazole, 4-methylpyrazole, and ethanol were inducers of cytochrome P-450j in rat liver although these compounds showed different inducing potencies. Microsomes from adult male rats with chemically induced diabetes also contained elevated levels of cytochrome P-450j compared to untreated animals. Cytochrome P-450j levels were measurable in kidney, whereas this isozyme was barely detectable in lung, ovaries, and testes; however, extrahepatic cytochrome P-450j was inducible by isoniazid. Approximately 80-90% of microsomal N-nitrosodimethylamine demethylation was inhibited by antibody to cytochrome P-450j whether the microsomes were isolated from untreated rats or animals administered inducers or repressors of cytochrome P-450j. The residual catalytic activity resistant to antibody inhibition may be a reflection of the inaccessibility of a certain amount of cytochrome P-450j due to interference by NADPH-cytochrome P-450 reductase based on results obtained with the reconstituted system. There was a good correlation (r2 = 0.87) between cytochrome P-450j content and N-nitrosodimethylamine demethylase activity in microsomes from rats of different ages and treated with various xenobiotics. The evidence presented indicates that cytochrome P-450j is the primary, and perhaps sole, microsomal catalyst of N-nitrosodimethylamine demethylation at substrate concentrations relevant to hepatocarcinogenesis induced by N-nitrosodimethylamine.     

A world of cytochrome P450s

The world we live in is a biosphere influenced by all organisms who inhabit it. It is also an ecology of genes, with some having rather startling effects. The premise put forth in this issue is cytochrome P450 is a significant player in the world around us. Life and the Earth itself would be visibly different and diminished without cytochrome P450s. The contributions to this issue range from evolution on the billion year scale to the colour of roses, from Darwin to Rachel Carson; all as seen through the lens of cytochrome P450.     

Interactions of cytochrome P450s with their ligands

Cytochrome P450s (CYPs) are heme-containing monooxygenases that contribute to an enormous range of enzymatic function including biosynthetic and detoxification roles. This review summarizes recent studies concerning interactions of CYPs with ligands including substrates, inhibitors, and diatomic heme-ligating molecules. These studies highlight the complexity in the relationship between the heme spin state and active site occupancy, the roles of water in directing protein–ligand and ligand–heme interactions, and the details of interactions between heme and gaseous diatomic CYP ligands. Both kinetic and thermodynamic aspects of ligand binding are considered.     

Purification and characterization of cytochrome P450 2E2 from hepatic microsomes of neonatal rabbits

The alcohol-inducible P450 2E subfamily in the rabbit has two known members that differ in only 16 amino acid residues scattered throughout the polypeptide chain. P450 2E1 has been thoroughly characterized, and is known to have diverse inducers and substrates. Little is known, however, about the properties of P450 2E2, since efforts to isolate this isozyme from adult rabbits have been unsuccessful. In the present study, 2E2 was purified to electrophoretic homogeneity from liver microsomes of neonatal rabbits with the use of 4-methylpyrazole as a stabilizing agent. The purified cytochrome was identified as 2E2 by NH2-terminal amino acid sequence analysis as well as by immunoblot analysis with three different antibodies to 2E1. Purified 2E2, in contrast to 2E1, is predominantly low-spin in the presence of 20% glycerol, but is in a mixed high- and low-spin state as the concentration of glycerol is decreased. The catalytic properties of purified 2E1 and 2E2 were compared in the reconstituted system with a variety of substrates, including alcohols, ethers, nitrosamines, and aromatic compounds. Differences between the two enzymes in catalytic activity and in the interaction with cytochrome b5 were observed with some but not all of the substrates tested. Purified 2E1 and 2E2 both consume molecular oxygen relatively rapidly during NADPH oxidation in the absence of an added substrate, and stoichiometric determinations indicated that only about 20% of the O2 was reduced to H2O2, with the remainder apparently undergoing four-electron reduction to water.     

Heterogeneity of cytochrome P-450 in rat testis microsomes.

Cytochrome P-450 appears to be a component of the steroid-coverting enzymes, 17alpha-hydroxylase and 17,20-lyase, which catalyze sequential steps in sex hormone synthesis. Further evidence indicates that the steroid substrates of these enzymes bind to cytochrome P-450 during catalysis. The present report deals with the problem of whether a single form of cytochrome P-450 mediates both enzyme reactions or whether two enzymes are involved. Both activities are competitively inhibited by a number of the same inhibitors. Because K1 values of competitive inhibitors are dissociated constants, and thus a property of the cytochrome, different magnitudes of K1, determined for the same inhibitor with each enzyme, are consistent with the participation of more than one form of cytochrome P-450. Differences in the K1 values were found to be statistically significant and varied from 3- to 10-fold. Two competitive inhibitors retarded velocities with one reaction but not the other. In addition, the enzyme activities were markedly different in their sensitivity to carbon monoxide inhibition. The conclusion based on these two lines of evidence is that separate enzymes and different forms of cytochrome P-450 are involved in each reaction.     

Highly purified cytochrome P-448 and P=450 from rat liver microsomes.

Cytochrome P-448 from 3-methylcholanthrene-treated rats has been purified to a specific content of greater than 20 nmoles/mg protein, and cytochrome P-450 from phenobarbital-treated rats to greater than 17 nmoles/mg protein. Both cytochromes are catalytically active when reconstituted with lipid and NADPH-cytochrome c reductase and exhibit differential substrate specificities for benzphetamine and benzo[a]pyrene. Cytochrome P-448 has a minimum molecular weight of approximately 53,000, and cytochrome P-450, 48,000 by SDS polyacrylamide gel electrophoresis.     

Cytochromes P450 involved with benzene metabolism in hepatic and pulmonary microsomes

Benzene is an occupational hazard and environmental toxicant found in cigarette smoke, gasoline, and the chemical industry. The major health concern associated with benzene exposure is leukemia. The toxic effects of benzene are dependent on its metabolism by the cytochrome P450 enzyme system. Previous research has identified CYP2E1 as the primary P450 isozyme responsible for benzene metabolism at low concentrations, whereas CYP2B1 is involved at higher concentrations. Our studies using microsomal preparations from human, mouse, and rat indicate that CYP2E1 is the P450 isozyme primarily responsible for benzene metabolism in lung and in liver. CYP2B isozymes have little involvement in benzene metabolism by either lung or liver. Our results also indicate that isozymes of the CYP2F subfamily may play a role in benzene metabolism by lung.     

Improved cloning and expression of cytochrome P450s and cytochrome P450 reductase in yeast

Combination of the pYeDP60 yeast expression system with a modified version of the improved uracil-excision (USER) cloning technique provides a new powerful tool for high-throughput expression of eukaryotic cytochrome P450s. The vector presented is designed to obtain an optimal 5' untranslated sequence region for yeast (Kozak consensus sequence), and has been tested to produce active P450s and NADPH-cytochrome P450 oxidoreductase (CPR) after 5' end silent codon optimization of the cDNA sequences. Expression of two plant cytochrome P450s, Sorghum bicolor CYP79A1 and CYP71E1, and S. bicolor CPR2 using the modified pYeDP60 vector in all three cases produced high amounts of active protein. High-throughput functional expression of cytochrome P450s have long been a troublesome task due to the workload involved in cloning of each individual P450 into a suitable expression vector. The redesigned yeast P450 expression vector (pYeDP60u) offers major improvements in cloning efficiency, speed, fidelity, and simplicity. The modified version of the USER cloning system provides great potential for further development of other yeast vectors, transforming these into powerful high-throughput expression vectors.     

Activities of human recombinant cytochrome P450 isoforms and human hepatic microsomes for the hydroxylation ofAlternaria toxins

TheAlternaria toxins alternariol (AOH), alternariol-9-methyl ether (AME), altenuene (ALT) and isoaltenuene (iALT) undergo extensive oxidative metabolism, but the cytochrome P450 (CYP) isoforms responsible for the reported hydroxylation reactions are yet unknown. In the present study, the activities of twelve human CYP isoforms for the hydroxylation of AOH, AME, ALT and iALT at different positions have been determined. The most active monooxygenase for AOH and AME was CYP1A1, and lower activities were observed for CYP1A2, 2C19 and 3A4. Hydroxylation at C-2 of AOH and AME was the preferred reaction of most isoforms. For ALT and iALT, CYP2C19 had the highest activity, followed by 2C9 and 2D6. The dominating metabolite of all active isoforms was the 8-hydroxylated ALT and iALT. The activities of the CYP isoforms are consistent with the pattern of metabolites of theAlternaria toxins obtained with pooled human hepatic microsomes. Based on the activities of the CYP isoforms, a significant extrahepatic hydroxylation must be expectede.g. in the lung and esophagus for AOH and AME, and in the intestine and ovaries for ALT and iALT. As all major hydroxylation products are catechols, the extrahepatic metabolism ofAlternaria toxins may be of toxicological relevance. Presented at the 30^th Mycotoxin Workshop, Utrecht, Netheriands, April 28–30. 2008. Financial support: State of Baden-Württemberg (Research Program “Mycotoxins” as part of the Research Initiative “Food and Health”)     

Induction of renal cytochrome P-450 in hepatic microsomes of diabetic rats

We purified two forms of cytochrome P-450 which was induced in hepatic microsomes of diabetic male rates treated with streptozotocin. One of these corresponded to P-450j. The other form, designated P450 DM-2, had a minimum molecular weight 53000 and a CO-reduced absorption maximum at 452 nm. The P450 DM-2 efficiently catalyzed the omega- and (omega-1)-hydroxylation of lauric acid, but was not efficient in metabolizing aminopyrine, 7-ethoxycoumarin, aniline, N-nitrosodimethylamine, or testosterone. The NH2-terminal sequence of P450 DM-2 was identical to that of P450 K-5, the major renal cytochrome P-450. Both forms gave very similar electrophoretic patterns of proteolytic digests. P450 DM-2 and P450 K-5 are closely related forms.     

Metabolons involving plant cytochrome P450s

Arranging biological processes into “compartments” is a key feature of all eukaryotic cells. Through this mechanism, cells can drastically increase metabolic efficiency and manage complex cellular processes more efficiently, saving space and energy. Compartmentation at the molecular level is mediated by metabolons. A metabolon is an ordered protein complex of sequential metabolic enzymes and associated cellular structural elements. The sub-cellular organization of enzymes involved in the synthesis and storage of plant natural products appears to involve the anchoring of metabolons by cytochrome P450 monooxygenases (P450s) to specific domains of the endoplasmic reticulum (ER) membrane. This review focuses on the current evidence supporting the organization of metabolons around P450s on the surface of the ER. We␣outline direct and indirect experimental data that describes P450 enzymes in the phenylpropanoid, flavonoid, cyanogenic glucoside, and other biosynthetic pathways. We also discuss the limitations and future directions of metabolon research and the potential for application to metabolic engineering endeavors.