Microsomal Cytochrome P450 Reductase Research Articles

Effects of Thioacetamide—Induced Cirrhosis, after a Drug Washout Period, on the Michaelis–Menten Kinetics of Major Cytochrome P450 Enzymes in the Rat Liver Microsomes

PurposeThioacetamide (TAA) is used to induce liver cirrhosis in rodents. However, the drug itself directly inhibits cytochrome P450 (CYP450) enzymes. The purpose of this study was to determine the effects of TAA‐induced liver cirrhosis, after a washout period to eliminate TAA, on the Michaelis‐Menten (MM) kinetics and protein contents of the major CYP450 enzymes CYP2D, CYP2E1, and CYP3A in rat liver microsomes.MethodsAdult, male Sprague‐Dawley rats were treated for 10 weeks with either TAA (200 mg/kg ip, 3 days per week) or vehicle (n = 7/each group). After a 10‐day washout period, animals were euthanized, and the liver was collected after cardiac perfusion with cold saline. Liver microsomes (MC) were prepared by differential centrifugation. The activities of the liver microsomal CYP2D (dextromethorphan O‐demethylation), CYP2E1 (chlorzoxazone 6‐hydroxylation) and CYP3A (midazolam 1′‐hydroxylation) were estimated by measurement of the metabolites by LC‐MS/MS. The microsomal CYP450 reductase (CPR) activity was analyzed spectrophotometry. Furthermore, the protein expressions of the isoenzymes were determined using Western blot analysis. The MM kinetic parameters maximum velocity (Vmax) and MM constant (Km) were determined using nonlinear regression analysis. Unpaired t‐test was used for statistical analysis of data, with a p value of < 0.05 as significant.ResultsWhereas the MM kinetics of dextromethorphan demethylation (CYP2D) were best fit by a two‐enzyme equation, those of chlorzoxazone hydroxylation (CYP2E1) and midazolam 1′‐hydroxylation (CYP3A) were best fit by a single‐enzyme equation. Cirrhosis resulted in significant reductions in the Vmax values for all the tested microsomal activities. The microsomal high‐ and low‐capacity Vmax values for CYP2D demethylase activity were significantly reduced by 74% (p value < 0.0001) and 51% (p < 0.05), respectively, in the TAA group. This was associated with a significant (p < 0.001) reduction (71%) in the protein contents of CYP2D in the TAA group. However, Km values were not affected by cirrhosis. For CYP2E1, cirrhosis caused a significant reduction (52%) in the Vmax value (p < 0.0001) for the chlorzoxazone hydroxylation activity. However, the Km values and protein contents of CYP2E1 remained unchanged. The Vmax and Km values of microsomal CYP3A hydroxylase activity, resulting in 1′‐hydroxylation of midazolam, were significantly reduced by 71% (p < 0.001) and 46% (p < 0.05), respectively, which was associated with a 30% reduction (p < 0.05) in the protein contents of CYP3A. Cirrhotic animals also showed a 31% decline (p < 0.05) in the microsomal CPR activity.ConclusionsTAA‐induced cirrhosis in rats resulted in a decreased liver microsomal activity of CPR and major CYP450 enzymes (CYP2D, CYP2E1, and CYP3A). Except for CYP2E1, the decreased isoenzyme activities were also associated with decreases in the protein concentrations of the isoenzymes. However, the cirrhosis‐induced decrease in the CYP2E1 activity, which was in the absence of a change in its protein levels, is possibly due to a decrease in the microsomal CPR activity.Support or Funding InformationFunding: Chapman University School of Pharmacy

Inhibition of NADPH–cytochrome P450 reductase by tannic acid in rat liver microsomes and primary hepatocytes: Methodological artifacts and application to ischemia–reperfusion injury

Tannic acid (TA) inhibits nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) activity, which is measured by reduction of cytochrome c, in rat liver microsomes (RLMs). In the current study, we noticed that TA directly reduces cytochrome c in the absence of microsomes, thus confounding the CPR activity assay. A method is presented that measures CPR activity in the presence of TA by subtracting the cytochrome c reduction in the absence of NADPH (TA effect) from that in the presence of NADPH (TA plus CPR effect). The method was used to determine the inhibitory effect of TA in RLMs, recombinant CPR enzyme, and primary hepatocytes. Additionally, application of TA in a study of role of CPR in a primary rat hepatocyte model of ischemia-reperfusion (IR) was investigated. TA showed concentration-dependent, complete inhibition of CPR with half maximal inhibitory concentration (IC(50) ) values of 58.2 μM in RLMs and 54.6 and 275 μM in primary rat hepatocytes in the absence and presence of serum in the medium, respectively. Additionally, inhibition of CPR by TA was associated with a significant reduction in reactive oxygen species and cell death after IR injury. These data may be useful in future studies using TA as an inhibitor of CPR in microsomes and primary hepatocytes.

Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species

Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and β-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from β-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury.

Exploring the Electron Transfer Properties of Neuronal Nitric-oxide Synthase by Reversal of the FMN Redox Potential

In nitric-oxide synthase (NOS) the FMN can exist as the fully oxidized (ox), the one-electron reduced semiquinone (sq), or the two-electron fully reduced hydroquinone (hq). In NOS and microsomal cytochrome P450 reductase the sq/hq redox potential is lower than that of the ox/sq couple, and hence it is the hq form of FMN that delivers electrons to the heme. Like NOS, cytochrome P450BM3 has the FAD/FMN reductase fused to the C-terminal end of the heme domain, but in P450BM3 the ox/sq and sq/hq redox couples are reversed, so it is the sq that transfers electrons to the heme. This difference is due to an extra Gly residue found in the FMN binding loop in NOS compared with P450BM3. We have deleted residue Gly-810 from the FMN binding loop in neuronal NOS (nNOS) to give Delta G810 so that the shorter binding loop mimics that in cytochrome P450BM3. As expected, the ox/sq redox potential now is lower than the sq/hq couple. Delta G810 exhibits lower NO synthase activity but normal levels of cytochrome c reductase activity. However, unlike the wild-type enzyme, the cytochrome c reductase activity of Delta G810 is insensitive to calmodulin binding. In addition, calmodulin binding to Delta G810 does not result in a large increase in FMN fluorescence as in wild-type nNOS. These results indicate that the FMN domain in Delta G810 is locked in a unique conformation that is no longer sensitive to calmodulin binding and resembles the "on" output state of the calmodulin-bound wild-type nNOS with respect to the cytochrome c reduction activity.

Role of a novel dual flavin reductase (NR1) and an associated histidine triad protein (DCS-1) in menadione-induced cytotoxicity

Microsomal cytochrome P450 reductase catalyzes the one-electron transfer from NADPH via FAD and FMN to various electron acceptors, such as cytochrome P450s or to some anti-cancer quinone drugs. This results in generation of free radicals and toxic oxygen metabolites, which can contribute to the cytotoxicity of these compounds. Recently, a cytosolic NADPH-dependent flavin reductase, NR1, has been described which is highly homologous to the microsomal cytochrome P450 reductase. In this study, we show that over-expression of NR1 in human embryonic kidney cells enhances the cytotoxic action of the model quinone, menadione. Furthermore, we show that a novel human histidine triad protein DCS-1, which is expressed together with NR1 in many tissues, can significantly reduce menadione-induced cytotoxicity in these cells. We also show that DCS-1 binds NF1 and directly modulates its activity. These results suggest that NR1 may play a role in carcinogenicity and cell death associated with one-electron reductions.

NAD(P)H:quinone oxidoreductase-1-dependent and -independent cytotoxicity of potent quinone Cdc25 phosphatase inhibitors.

Cdc25 dual-specificity phosphatases coordinate cell cycle progression and cellular signaling. Consequently, Cdc25 inhibitors represent potential anticancer agents. We evaluated >10,000 compounds for inhibition of human Cdc25 phosphatases and identified many potent and selective inhibitors, which all contained a quinone. Bioreductive enzymes frequently detoxify or activate quinones. Therefore, we evaluated the effect of NAD(P)H:quinone oxidoreductase-1 (NQO1) and reductase-rich microsomes on the activity of three quinone-containing Cdc25 inhibitors: 2-(2-hydroxyethylsulfanyl)-3-methyl-1,4-naphthoquinone (Cpd 5, compound 5; NSC 672121), 2,3-bis-(2-hydroxyethylsulfanyl)-1,4-naphthoquinone (NSC 95397), and 6-chloro-7-(2-morpholin-4-yl-ethylamino)quinoline-5,8-dione (NSC 663284). Each inhibitor was reduced by human NQO1 (K(m) of 0.3-0.5 microM) but none by microsomes. Compounds were evaluated with six cancer cell lines containing different amounts of NQO1: HT-29 (1056 nmol/mg/min), HCT116 (660 nmol/mg/min), sublines HCT116-R30A (28 nmol/mg/min) and HCT-116R30A/NQ5 (934 nmol/mg/min), MDA-MB-231/Q2 (null NQO1), and subline MDA-MB-231/Q6 (124 nmol/mg/min) but containing similar amounts of microsomal cytochrome P450 reductase and cytochrome b(5) reductase. Growth inhibition and G2/M arrest by Cpd 5 was proportional to NQO1 levels, requiring 4- to 5-fold more Cpd 5 to inhibit HCT-116 or HCT-116R30A/NQ5 compared with HCT-116R30A. In contrast, in all tested cell lines irrespective of NQO1 level, growth inhibition and G2/M arrest by NSC 95375 and NSC 663284 were similar (average IC(50) of 1.3 +/- 0.3 and 2.6 +/- 0.4 microM, respectively). NSC 95375 and NSC 663284 also caused similar Cdk1 hyperphosphorylation, indicating similar Cdc25 inhibition. However, lower Cpd 5 concentrations were needed to produce Cdk1 hyperphosphorylation in sublines with minimal NQO1. Thus, NQO1 detoxified Cpd 5, probably by reducing it to a less active hydroquinone, whereas NSC 95397- and NSC 663284-generated cytotoxicity was unaffected by NQO1.

Cyclosporin A-induced free radical generation is not mediated by cytochrome P-450.

1. Reactive oxygen species (ROS) have been proposed to play a role in the side effects of the immunosuppressive drug cyclosporin A (CsA). 2. The aim of this study was to investigate whether cytochrome P-450 (CYP) dependent metabolism of CsA could be responsible for ROS generation since it has been suggested that CsA may influence the CYP system to produce ROS. 3. We show that CsA (1 -- 10 microM) generated antioxidant-inhibitable ROS in rat aortic smooth muscle cells (RASMC) using the fluorescent probe 2,7-dichlorofluorescin diacetate. 4. Using cytochrome c as substrate, we show that CsA (10 microM) did not inhibit NADPH cytochrome P-450 reductase in microsomes prepared from rat liver, kidney or RASMC. 5. CsA (10 microM) did not uncouple the electron flow from NADPH via NADPH cytochrome P-450 reductase to the CYP enzymes because CsA did not inhibit the metabolism of substrates selective for several CYP enzymes that do not metabolize CsA in rat liver microsomes. 6. CsA (10 microM) did not generate more radicals in CYP 3A4 expressing immortalized human liver epithelial cells (T5-3A4 cells) than in control cells that do not express CYP 3A4. 7. Neither diphenylene iodonium nor the CYP 3A inhibitor ketoconazole were able to block ROS formation in rat aortic smooth muscle or T5-3A4 cells. 8. These results demonstrate that CYP enzymes do not contribute to CsA-induced ROS formation and that CsA neither inhibits NADPH cytochrome P-450 reductase nor the electron transfer to the CYP enzymes.

Purification of Soluble Cytochrome b5 as a Component of the Reductive Activation of Porcine Methionine Synthase

In mammals, methionine synthase plays a central role in the detoxification of the rogue metabolite homocysteine. It catalyzes a transmethylation reaction in which a methyl group is transferred from methyltetrahydrofolate to homocysteine to generate tetrahydrofolate and methionine. The vitamin B12 cofactor cobalamin plays a direct role in this reaction by alternately accepting and donating the methyl group that is in transit from one substrate (methyltetrahydrofolate) to another (homocysteine). The reactivity of the cofactor intermediate cob(I)alamin renders the enzyme susceptible to oxidative damage. The oxidized enzyme may be returned to the catalytic turnover cycle via a reductive methylation reaction that requires S-adenosylmethionine as a methyl group donor, and a source of electrons. In this study, we have characterized an NADPH-dependent pathway for the reductive activation of porcine methionine synthase. Two proteins are required for the transfer of electrons from NADPH, one of which is microsomal and the other cytoplasmic. The cytoplasmic protein has been purified to homogeneity and is soluble cytochrome b5. It supports methionine synthase activity in the presence of NADPH and the microsomal component in a saturable manner. In addition, purified microsomal cytochrome P450 reductase and soluble cytochrome b5 reconstitute the activity of the porcine methionine synthase. Identification of soluble cytochrome b5 as a member of the reductive activation system for methionine synthase describes a function for this protein in non-erythrocyte cells. In erythrocytes, soluble cytochrome b5 functions in methemoglobin reduction. In addition, it identifies an additional locus in which genetic polymorphisms may play a role in the etiology of hyperhomocysteinemia, which is correlated with cardiovascular diseases.

NO news is good news.

The recent determination of the crystal structure of microsomal cytochrome P450 reductase, a diflavin protein that shuttles electrons from NADPH to the P450 heme, represents a significant advance towards the understanding of cytochromes P450. A similar advance was made in a related enzyme system, nitric oxide synthase (NOS). The crystal structure of the NOS heme domain reveals a very different architecture to that observed in P450s and offers significant insight into the production of nitric oxide, one of nature's most important regulatory molecules.

Generation of a free radical from calphostin C by microsomal cytochrome P450 reductase in rat and human liver.

Calphostin C (cal C) is a potent and specific protein kinase C inhibitor that is thought to be photoactivated to generate a reactive perylenesemiquinone free radical intermediate. In this electron spin resonance spectrometry study, we show, for the first time, an enzymatic light-independent mechanism of cal C free radical formation. Such a free radical was generated in liver microsomes from rat and humans, and its formation was dependent on the presence of NADPH, functional microsomes, and protein and cal C concentrations. Inhibitor and purified enzyme studies showed that cytochrome P450 reductase is responsible for the cal C free radical observed, which is probably a perylenesemiquinone.

Biotransformation enzyme activities in the olfactory organ of rainbow trout (Oncorhynchus mykiss). Immunocytochemical localization of cytochrome P4501A1 and its induction by β-naphthoflavone

Olfaction is a crucial function in most fish species, but little is known about biotransformation enzymes in the olfactory organ. This study demonstrates that biotransformation enzymes usually found in the rainbow trout liver, are present in the olfactory organ as well. While microsomal cytochrome P450 reductase, p-nitrophenol hydroxylase and cytosolic glutathioneS-transferase presented similar levels in both the olfactory organ and the liver, microsomal 7-ethoxyresorufinO-deethylase (EROD), 7-ethoxycoumarinO-deethylase, and 7-pentoxyresorufinO-deethylase were much lower in the olfactory organ (77-, 35-, 200-times respectively). Furthermore, microsomes from the olfactory organ were able to perform testosterone hydroxylation only in the 16α-position while testosterone was hydroxylated in the 16β-position by liver microsomes. Using polyclonal antibodies raised against perch cytochrome P4501A1, the immunoreactive protein was shown to be strongly expressed in various cellular types forming the nonsensory epithelium. Some immunostaining was also reported in the nonsensory cellular elements constituting the sensory epithelium, while olfactory receptor cells failed to show cytochrome P4501A1-immunoreactivity. Finally, the exposure of rainbow trout to waterborne β-naphthoflavone (0.1 μg ml(-1)) for 2 or 4 days resulted in a higher induction of EROD activity in the olfactory organ compared to the liver. The presence of biotransformation enzymes in the olfactory organ of rainbow trout addresses the question of their involvement in the detoxication/toxication of pollutants as well as in the olfactory function.

Effects of acetylsalicylic acid on parameters related to peroxisome proliferation in mouse liver

Male C57 BL/6 mice were exposed to 1.0% (w/w) acetylsalicylic acid (ASA) in their diet for 10 days and effects related to peroxisome proliferation were subsequently examined. A 2.2-fold increase in mitochondrial protein content was obtained. The activities of the peroxisomal enzymes, lauroyl-CoA oxidase, palmitoyl-CoA oxidation and catalase, were enhanced 4.5-, 4.0- and 2.1-fold, respectively. There was a dramatic increase (9.1-fold) in microsomal cytochrome P450 IVA-catalysed activity, a 1.6-fold induction of total microsomal P450 content and a 2-fold induction of microsomal cytochrome P450 reductase activity (measured as NADPH-cytochrome c reductase). Catalase activity in the cytosol was induced 5.2-fold and DT-diaphorase activity was increased 3.5- and 3.2-fold in the cytosol and mitochondria, respectively. There was a significant increase in the susceptibility of microsomes to lipid peroxidation. Smaller increases in superoxide dismutase, glutathione transferase and glutathione peroxidase activities were also observed. The possible relevance of these effects to the pharmacology of ASA is discussed.

Susceptibility of channel catfish (Ictalurus punctatus) and brown bullhead (Ameriurus nebulosus) to oxidative stress: a comparative study

Many pollutants in aquatic systems have been shown to exert toxic effects related to oxidative stress. Biochemical parameters of xenobiotic metabolism and oxidative stress were examined in two benthic fish species, channel catfish and brown bullhead, which differ in their apparent susceptibility to pollutant-mediated neoplasia. These parameters included enzyme activities and production of reactive oxygen species by hepatic subcellular fractions. Hepatic ethoxyresorufin O-deethylase (EROD) activity was markedly higher in catfish than in bullhead. This interspecific difference was also observed following induction of EROD activities by β-naphthoflavone (βNF). Conversely, hepatic microsomal cytochrome P-450 reductase (P-450R) activity was higher in bullhead and was unaffected in both species by βNF treatment. Hepatic catalase, DT diaphorase, glutathione reductase, glutathione S-transferase and total glutathione content were significantly higher in catfish as well. Hepatic glutathione peroxidase activity was significantly higher in bullhead. No significant interspecific difference was observed in hepatic superoxide dismutase activity. Cytosolic rates of menadione-mediated superoxide anion and hydrogen peroxide production were higher in pooled catfish liver samples as compared to bullhead. Conversely, microsomal rates of superoxide and hydrogen peroxide production were higher in bullhead, consistent with relative P-450R activities in the two species. Catfish liver homogenate was more effective than bullhead homogenate at inhibiting iron/ ascorbate-mediated production of 8-hydroxydeoxyguanosine (8-OHdG) in vitro.

Expression, purification, and properties of the flavoprotein domain of cytochrome P-450BM-3. Evidence for the importance of the amino-terminal region for FMN binding.

Comparison of the amino acid sequences of several microsomal cytochrome P-450 reductases to the flavoprotein domain (BMR) of cytochrome P-450BM-3 has revealed that this class of flavoproteins contains evolutionarily conserved regions that are important for their interaction with nucleotide substrates and cofactors. In order to understand the properties of BMR, the region encoding this protein, beginning at residue Lys-472 of cytochrome P-450BM-3, was subcloned and expressed in Escherichia coli. The recombinant protein (more than 50% of host-soluble proteins) was purified to homogeneity using conventional purification procedures. BMR (Mr 66,000) showed typical flavoenzyme absorbance spectra, contained FAD and FMN in a stoichiometry of 1:1, and catalyzed reduction of several artificial electron acceptors with rates comparable to those of the microsomal NADPH-cytochrome P-450 oxidoreductase. Limited trypsinolysis of BMR, under non-denaturing conditions, revealed that the protease removed the NH2-terminal 122 residues. This region was postulated to contain amino acids that are important for FMN binding (Porter, T. D. (1991) Trends Biochem. Sci. 16, 154-158). Consistent with this hypothesis, the major tryptic product of BMR (BMR-52, Mr 52,000) contained only FAD, in an equimolar ratio to the protein. Also, like the FMN-depleted microsomal NADPH-cytochrome P-450 oxidoreductase (Kurzban, G. P., Howarth, J., Palmer, G., and Strobel, H. W. (1990) J. Biol. Chem. 265, 12272-12279), BMR-52 was active for only catalyzing ferricyanide reduction. These data provide strong experimental evidence for a discrete multidomain structure of BMR, as proposed for the membrane-bound reductases, with an amino-terminal FMN binding region and carboxyl-terminal FAD- and NADPH binding regions. Thus, BMR strongly resembles the microsomal cytochrome P-450 reductase and offers an opportunity to better understand the structure-function relationships of this class of flavoproteins.

The cloning of cholesterol 7 α-hydroxylase: Will molecular techniques help us understand the physiology of bile acid synthesis?

The rate-limiting step in bile acid biosynthesis is catalyzed by the microsomal cytochrome P-450 cholesterol 7 α-hydroxylase. The expression of this enzyme is subject to feedback regulation by sterols and is thought to be coordinately regulated with enzymes in the cholesterol supply pathways, including the low-density lipoprotein receptor and 3-hydroxy-3-methylglutarylcoenzyme A reductase and synthase. Here we report the purification of rat microsomal P-450 cholesterol 7 α-hydroxylase and the determination of a partial amino acid sequence. Oligonucleotides derived from peptide sequence were used to clone a full-length cDNA encoding microsomal P-450 cholesterol 7 α-hydroxylase. DNA sequence analysis of the cDNA revealed a microsomal P-450 cholesterol 7 α-hydroxylase protein of 503 amino acids with a predicted molecular weight of 56,890, which represents a novel family of cytochrome P-450 enzymes. Transfection of a microsomal P-450 cholesterol 7 α-hydroxylase cDNA into simian COS cells resulted in the synthesis of a functional enzyme whose activity was stimulated in vitro by the addition of rat microsomal cytochrome P-450 reductase protein. RNA blot hybridization experiments indicated that the messenger RNA for microsomal P-450 cholesterol 7 α-hydroxylase is found only in the liver. The levels of this messenger RNA increased when bile acids were depleted by dietary cholestyramine and decreased when bile acids were consumed. Dietary cholesterol led to an increase in microsomal P-450 cholesterol 7 α-hydroxylase messenger RNA levels. The enzymatic activity of microsomal P-450 cholesterol 7 α-hydroxylase paralleled the observed changes in messenger RNA levels. These results suggest that bile acids and sterols are able to alter the transcription of the microsomal P-450 cholesterol 7 α-hydroxylase gene and that this control explains the previously observed feedback regulation of bile acid synthesis. A complete cDNA encoding cholesterol 7 α-monooxygenase (EC 1.14 13.17), which had been isolated from rat liver cDNA libraries by using specific antibodies to the enzyme (Noshiro M, Nishimoto M, and Okuda K. FEBS Lett 1989;257:97–100) was totally sequenced. The cDNA contained a 1,509 bb open-reading frame encoding 503 amino acid residues (Mr = 56,880) and an unusually long 3′-untranslated region rich in AT sequence in the total length of 3,545 bp. The predicted amino acid sequence displays less than 30% similarity to other sequenced cytochrome P-450s, indicating that the 7 α-hydroxylase constitutes a novel family of cytochrome P-450. The AT-rich region often contained ATTTA motifs, 5′-AAT-3′ or 5′-TAA-3′ trinucleotides reported to be involved in rapidly degrading messenger RNA. Using the specific antibodies and the cDNA as probes, a diurnal variation of the levels of the three factors (i.e., enzyme protein, messenger RNA and enzyme activity) was studied on rat livers prepared at various times of the day. In normal animals, all three factors exhibited maximum level at 10 PM and minimum at 10 AM. No significant sexual difference was observed. Cholestyramine feeding increased all three factors at 10 AM close to the maximum levels of the normal rats but did not show a significant increase at 10 PM. On the contrary, starvation markedly decreased all three factors either at 10 AM or at 10 PM, while still maintaining the diurnal variation. A good correlation of the levels of messenger RNA to the enzyme activities and the protein levels demonstrates that pretranslational regulation is most likely a mechanism for the circadian rhythm of 7 α-hydroxylase. The marked diurnal fluctuation of the amount of protein and the level of messenger RNA also indicates their rapid turnover. The short half-life of messenger RNA could be correlated with the structure of the 3′-untranslated region of the messenger RNA characteristic of rapidly degrading messenger RNA (i.e., abundance of motif, AUUUA and existence of 5′-AAU-3′ or 5′-UAA-3′ trinucleotides in single-stranded regions of the secondary structure).

Cloning and regulation of cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis.

The rate-limiting step in bile acid biosynthesis is catalyzed by the microsomal cytochrome P-450 cholesterol 7 alpha-hydroxylase (7 alpha-hydroxylase). The expression of this enzyme is subject to feedback regulation by sterols and is thought to be coordinately regulated with enzymes in the cholesterol supply pathways, including the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase and synthase. Here we report the purification of rat 7 alpha-hydroxylase and the determination of a partial amino acid sequence. Oligonucleotides derived from peptide sequence were used to clone a full-length cDNA encoding 7 alpha-hydroxylase. DNA sequence analysis of the cDNA revealed a 7 alpha-hydroxylase protein of 503 amino acids with a predicted molecular weight of 56,890 which represents a novel family of cytochrome P-450 enzymes. Transfection of a 7 alpha-hydroxylase cDNA into simian COS cells resulted in the synthesis of a functional enzyme whose activity was stimulated in vitro by the addition of rat microsomal cytochrome P-450 reductase protein. RNA blot hybridization experiments indicated that the mRNA for 7 alpha-hydroxylase is found only in the liver. The levels of this mRNA increased when bile acids were depleted by dietary cholestyramine and decreased when bile acids were consumed. Dietary cholesterol led to an increase in 7 alpha-hydroxylase mRNA levels. The enzymatic activity of 7 alpha-hydroxylase paralleled the observed changes in mRNA levels. These results suggest that bile acids and sterols are able to alter the transcription of the 7 alpha-hydroxylase gene and that this control explains the previously observed feedback regulation of bile acid synthesis.

Purification and characterization of human placental aromatase cytochrome P-450.

Aromatase cytochrome P-450, which catalyzes the conversion of androgens to estrogens, was purified from human placental microsomes. The enzyme was extracted with sodium cholate, fractionated by ammonium sulfate precipitation, and subjected to column chromatography in the presence of its substrate, androstenedione, and the nonionic detergent, Nonidet P-40. The preparation exhibits a single major band when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a specific content of 11.5 nmol of P-450/mg of protein. The purified enzyme displays spectroscopic properties typical of the ferric and ferrous forms of cytochrome P-450. Full enzymatic activity can be reconstituted with rabbit liver microsomal cytochrome P-450 reductase and Nonidet P-40. Purified aromatase cytochrome P-450 displays catalytic characteristics similar to the enzyme in intact microsomes in the aromatization of androstenedione, 19-hydroxyandrostenedione and 19-oxoandrostenedione. Testosterone and 16 alpha-hydroxytestosterone are aromatized at maximal rates similar to androstenedione, and all substrates exhibit relative affinities corresponding to those observed in microsomes. We have raised rabbit antibodies to the purified enzyme which show considerable specificity and sensitivity on immunoblots.

Oxy radical formation during redox cycling of the bleomycin-iron (III) complex by NADPH-cytochrome P-450 reductase

Bleomycin was aerobically incubated with FeCl 3, NADPH, isolated rat-liver microsomal cytochrome P-450 reductase and methional. The conversion of methional to ethene, which indicates oxy radicals, was determined. Ethene formation depended on oxygen, NADPH, FeCl 3 and the enzyme. About equimolar concentrations of bleomycin and FeCl 3 resulted in optimal ethene formation. Dimethyl sulfoxide, mannitol, glycerol, glutathione and glutathione disulfide inhibited ethene formation. These results indicate that oxy radicals are formed after reduction of the bleomycin-Fe-complex by NADPH-cytochrome P-450 reductase.

The drug and carcinogen metabolism system of rat colon microsomes

The hydroxylation of N- and O-methyl drugs and polycyclic hydrocarbons has been demonstrated in microsomes prepared from colon mucosal cells. The hydroxylation of the drugs benzphetamine, ethylmorphine, p-nitroanisole, and p-nitrophenetole by colon microsomes is inducible two- to fourfold by pretreatment with phenobarbital/hydrocortisone. Colon microsomal benzo[α]pyrene hydroxylation is inducible 35-fold by pretreatment with β-naphthoflavone. Phenobarbital/hydrocortisone pretreatment also induces a fourfold increase in the specific content of colon microsomal cytochrome P-450, while β-naphthoflavone pretreatment causes a shift in the reduced CO difference spectrum peak to 448 nm and an eightfold increase in the specific content of this cytochrome. SKF 525-A inhibits the hydroxylation of the drug benzphetamine by colon microsomes or liver microsomes by 77% at a concentration of 2.0 m m. 7,8-Benzoflavone, on the other hand, inhibits the hydroxylation of the polycyclic hydrocarbon benzo[α]pyrene by colon microsomes by 76% and by liver microsomes by 44% at a concentration of 10 μ m. Carbon monoxide, an inhibitor of oxygen interaction with cytochromes P-450 and P-448, inhibits benzphetamine hydroxylation and benzpyrene hydroxylation by colon microsomes 30 and 51%, respectively, at an oxygen to carbon monoxide ratio of 1:10. The K m values of colon microsomal cytochrome P-450 reductase for the artificial electron acceptors cytochrome c, dichloroindophenol, and ferricyanide (10–77 μ m) are in agreement with those for purified rat liver cytochrome P-450 reductase. These data support the conclusions that hydroxylation of drugs and polycyclic hydrocarbons is catalyzed by colon mucosal microsomes and that the hydroxylation activity is attributable to a cytochrome P-450-dependent drug metabolism system similar to that found in liver microsomes.

Studies on the role of protein synthesis in cell injury by toxic agents: I. Effect of cycloheximide administration on several factors modulating carbon tetrachloride-induced liver necrosis

Cycloheximide pretreatment (1 mg/kg, ip) slightly attenuated the hepatic necrosis 24 hr after CCl 4 administration and this attenuation was more obvious at 72 hr. A dose of cycloheximide, 2 mg/kg, evoked a marked protective effect even at 24 hr. Cycloheximide administration (1 mg/kg) significantly reduced the body temperature in CCl 4-treated rats but did not change the CCl 4 concentrations in the liver. Prior treatment of the rats with cycloheximide decreased the amount of binding of 14CCl 4 to liver microsomal lipids and reduced CCl 4-induced lipid peroxidation at 1 hr but not at 6 hr after administration of the hepatotoxin. Cycloheximide did not alter the pentobarbital sleeping time of the rats nor did it interact with microsomes to give spectral changes. Cycloheximide did not alter microsomal cytochrome P-450 reductase activity. Administration of cycloheximide prior to CCl 4 did not prevent the CCl 4-induced decrease in cytochrome P-450 or glucose 6-phosphatase activity caused by the hepatotoxin. However it did prevent the CCl 4-induced polysome breakdown and it did attenuate the severity of the lesions caused by CCl 4 at the ultrastructural level.