Nicotinamide Adenine Dinucleotide Phosphate Cytochrome Research Articles

NADPH–cytochrome P450 reductase knockdown decreases the response to precocene I in the migratory locust Locusta migratoria

Precocene I is a juvenile hormone antagonist that needs to be activated via oxidative biotransformation catalyzed by cytochrome P450 (CYP). NADPH–cytochrome P450 reductase (CPR) supplies CYP with electrons in the oxidation–reduction process; however, its functional role in the activation of precocene I remains unexplored. Here, the representative characteristics of CPRs were analyzed in the CPR gene of Locusta migratoria (LmCPR), the result of model docking indicated that the hydrogen bonds were formed between reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and NADPH-, FAD-, FMN-domains of LmCPR, respectively. Treating the fourth-instar nymphs with precocene I decreased the juvenile hormone titers of nymphs to 0.55-fold of that in acetone-treated controls, and extended the interval time between fourth- and fifth-instar nymphs. 68.75% of the treated fourth-instar nymphs developed into precocious adults in the fifth-instar. LmCPR knockdown decreased the response to precocene I in the nymphs, the occurrence rate of precocious adults induced by precocene I treatment reduced by 23.11%. Therefore, LmCPR may be involved in the activation of precocene I in L. migratoria. In addition, we generated an active recombinant LmCPR protein using a prokaryotic expression system, its activity in reducing cytochrome c was 33.13 ± 11.50 nmol CytCred/min/μg protein. This study lays the foundation for further research on the role of LmCPR in precocene I activation.

Proteomic changes of CD4+/CD25+/forkhead box p3+ regulatory T cells in a 30-day rat model of sepsis survival.

Sepsis is defined as life threatening organ dysfunction arising from a dysregulated host response to infection. The outcomes of sepsis include early mortality, delayed mortality and recovery, and depend on the inflammatory response. Previous studies have demonstrated that regulatory T cells (Tregs) are important in determining the outcome of sepsis, as their suppressive function serves a role in maintaining immune homeostasis. However, Treg-mediated immunosuppression during the course of sepsis remains unclear and little is known about the survival of patients following diagnosis. Studying the survivors of sepsis may explain the mechanisms of natural recovery. Therefore, a 30-day rat model of sepsis survival was established in the current study. Cluster of differentiation CD4+/CD25+/forkhead box p3+ Tregs were isolated from the blood and spleens of rats undergoing cecal ligation and puncture or sham surgery, using flow cytometry. Proteomic analysis was performed using nano high-performance liquid chromatography-mass spectrometry. Several different biological pathways associated with uncommon differentially-expressed proteins were identified in the blood and spleen survivor and sham groups. Extracellular-regulated kinase/mitogen-activated protein kinase, as well as integrin and actin cytoskeletal pathway elements, including Ras-related protein 1b, talin 1 and filamin A, were associated with Tregs in the blood. Pathway elements associated with cell cycle regulators in the B-cell translocation gene family of proteins, tumor necrosis factor receptor superfamily member 4, Hippo signaling, P70-S6 kinase 1, phosphatidylinositol 3-kinase/protein kinase B signaling and 1,25-dihydroxyvitamin D3 biosynthesis were associated with Tregs from the spleen including phosphatase 2A activator regulatory factor 4, histone arginine methyltransferase, CD4, major histocompatibility complex class I antigens, 14-3-3 protein θ and nicotinamide adenine dinucleotide phosphate cytochrome P450 reductase. These results explain the mechanism by which Tregs naturally recover and indicates that Tregs in the blood and spleen vary. Differentially-expressed proteins serving a role in these pathways provide additional insight for the identification of new targets for the diagnosis and treatment of sepsis.

Involvement of NADPH oxidase and glutathione in zinc-induced dopaminergic neurodegeneration in rats: Similarity with paraquat neurotoxicity

An association between excessive zinc (Zn) accumulation in brain and incidences of Parkinson's disease (PD) has been shown in several epidemiological and experimental investigations. The involvement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and glutathione (GSH) in the pathogenesis of PD has also been proposed in a few studies. Despite the implicated role of oxidative stress in PD, the entire mechanism of Zn-induced dopaminergic neurodegeneration has not yet been clearly understood. The present study aimed to investigate the involvement of NADPH oxidase and GSH in Zn-induced dopaminergic neurodegeneration and also to assess its similarity with paraquat (PQ)-induced rat model of PD. Male Wistar rats were treated either with Zn (20mg/kg; i.p.) or PQ (5mg/kg; i.p.) in the presence and absence of NADPH oxidase inhibitor, apocynin (10mg/kg; i.p.) and a GSH precursor, N-acetyl cysteine (NAC; 200mg/kg; i.p.) either alone or in combination along with the respective controls. Apocynin and/or NAC pre-treatment significantly alleviated Zn- and PQ-induced changes in neurobehavioral deficits, number of dopaminergic neurons and contents of the striatal dopamine and its metabolites. Apocynin and/or NAC also mitigated Zn- and PQ-induced alterations in oxidative stress, NADPH oxidase activation and cytochrome c release, caspases-9 and -3 activation and CD11b expression. The results obtained thus suggest that Zn induces oxidative stress via the activation of NADPH oxidase and depletion of GSH, which in turn activate the apoptotic machinery leading to dopaminergic neurodegeneration similar to PQ.

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.

A Professional and Personal Odyssey

As I began to write this article reflecting on my professional career in biomedical research, many memories of people, activities, situations, and experiences were swirling in my head. Because of the extraordinary support that I have received from my family, they are never far from my thoughts. I believe that sharing a few of these memories will be helpful for those who find themselves in similar situations or periods of professional development. This is not meant to be a comprehensive review of the fields to which I have had the privilege of contributing but a personal stroll down a path in which I have found extraordinary pleasure and from which I have derived a sense of accomplishment.

Effect of cell density and epidermal growth factor on the inducible expression of CYP3A and CYP1A genes in human hepatocytes in primary culture.

The influence of cell density and epidermal growth factor (EGF) on the expression and inducibility of cytochrome P450 (CYP) genes of the CYP3A and CYP1A families in adult human hepatocytes in primary culture has been evaluated. Only when cultured at subconfluence and in the presence of EGF did hepatocytes exhibit a proliferative response, assessed by measuring DNA synthesis and cyclin A accumulation. In the absence of EGF, the accumulation of CYP3A4 and CYP1A2 messenger RNAs (mRNAs) in response to their respective inducers (rifampicin and dioxin) was dramatically decreased in subconfluent culture with respect to confluent cultures. The presence of EGF only slightly decreased the accumulation of these mRNAs in both confluent and subconfluent cultures. The accumulation of CYP2D6 and CYP2E1 proteins, which are constitutively expressed in confluent cultures, and the production of fibrinogen and apolipoprotein (Apo) B100 exhibited similar behavior, while nicotinamide adenine dinucleotide phosphate cytochrome c reductase activity was affected neither by cell density nor by EGF. In contrast, the accumulation of CYP1A1 mRNA in response to dioxin was similar in confluent and subconfluent cultures, irrespective of the presence of EGF. Interestingly, CYP3A7, a gene that is preferentially expressed in the fetal liver, was expressed constitutively neither in confluent nor in subconfluent cultures, irrespective of the presence of EGF. It is concluded that the loss of cell-cell contacts rather than the proliferative status of cells per se is responsible for the dramatic decrease in the expression of CYP genes, normally expressed in the adult human liver.

Effects of piperonyl butoxide and β‐naphthoflavone on cytochrome P4501A expression and activity in Atlantic salmon (Salmo salar L.)

AbstractThe effects of piperonyl butoxide (PBO) and β‐naphthoflavone (BNF) on cytochrome P4501A (CYP1A) expression and activity in juvenile Atlantic salmon (Salmo salar L.) with regard to time and temperature was investigated. The time exposure study was performed at 8 °C, and the results show that PBO, although acutely functioning as a CYP1A inhibitor, is able to induce CYP1A expression in salmon liver. Both PBO and BNF give highest induction of CYP1A mRNA 48 h after intraperitoneal injection (five‐ and 14‐fold, respectively). The mRNA levels induced by PBO and BNF were sustained during the 8 d of the experiment (four‐ and 11‐fold, respectively). The CYP1A protein content measured by enzyme‐linked immunosorbent assay demonstrated the highest induction by PBO 8 d after exposure (eightfold) and by BNF 4 d after exposure (11‐fold). Activity of CYP1A measured by ethoxyresorufin‐O‐deethylase (EROD) demonstrated inhibition after PBO treatment the first 24 h after exposure, followed by threefold induction from 48 h and to the end of the experiment (8 d). β‐Naphthoflavone strongly induced EROD activity, with the highest levels occurring 4 d after treatment (56‐fold) and 8 d after treatment (22‐fold). In the temperature study, the results demonstrated temperature compensation, as salmon acclimated to 7°C for 3 weeks had a significantly higher EROD activity than those acclimated to 11 and 15°C. This was not reflected in significantly higher levels of reduced nicotinamide adenine dinucleotide phosphate cytochrome c reductase activity or CYP1A protein. The inductive properties of PBO and BNF on CYP1A expression was also demonstrated in primary cultures of salmon hepatocytes.

Cerebral metabolism of imipramine and a purified flavin-containing monooxygenase from human brain

Flavin-containing monooxygenase (FMO), previously reported both from hepatic and extrahepatic tissues, including brain, catalyze the oxidation of certain xenobiotics and drugs that contain a nucleophilic heteroatom. Psychoactive drugs, including the antidepressant imipramine, are substrates for the brain FMO. Since FMO-mediated metabolism of these drugs might contribute to local pharmacodynamic modulation within the human brain, the metabolism of imipramine by human brain FMO was studied in further detail. In the present study, the FMO activity was determined in human brain microsomes by estimating the actual amount of imipramine N-oxide formed. It was then compared with the corresponding activity measured using substrate (imipramine)-stimulated rates of nicotinamide adenine dinucleotide phosphate (NADPH) oxidation, which was significantly higher than the activity estimated as the amount of N-oxide assayed using high-pressure liquid chromatography (HPLC). The brain FMO activity was measurable only in the presence of detergents (sodium cholate or Lubrol PX) or in microsomes that were freeze-thawed several times. The activity was inhibited by an antibody to rabbit pulmonary FMO, but an antiserum to the rat liver NADPH cytochrome P-450 reductase had no effect indicating that cytochrome P-450 was not involved in the above metabolic pathway. The optimum pH for N-oxidation of imipramine was found to be 8.5; thermolability experiments indicated that the FMO activity was completely lost only after the incubation of brain microsomes at 45 degrees C for 20 minutes. An FMO purified to apparent homogeneity from a human brain had a molecular weight of 71,000 Da. The purified enzyme cross-reacted with the antibody to rabbit pulmonary FMO and efficiently catalyzed the metabolism of imipramine to its N-oxide. The human brain clearly contains an active FMO system, and it is conceivable that such enzymes are significantly involved in the local metabolism and modulation of pharmacological and/or toxic effects of certain xenobiotics, including psychoactive drugs.

The Ontogeny of Key Endoplasmic Reticulum Proteins in Human Embryonic and Fetal Red Blood Cells

Recently, using immunohistochemical methods, we surprisingly found that endoplasmic reticulum glucose-6-phosphatase is present in human embryonic and fetal red blood cells (RBCs) but not in adult RBCs. The fact that an endoplasmic reticulum enzyme, whose major site of expression in adults is the liver, is present in human embryonic and fetal RBCs, particularly nucleated cells, indicated that it would be sensible to determine whether these cells also contain other endoplasmic reticulum enzyme systems normally found in adult liver. Therefore, we have studied the expression of other endoplasmic reticulum proteins and found that human embryonic and fetal RBC precursors contain other protein components of the glucose-6-phosphatase system, ie, the phosphate and glucose transport proteins as well as other enzymes (eg, uridine diphosphate-glucuronosyltransferases, cytochrome P450 isozymes, nicotinamide adenine dinucleotide phosphate cytochrome P450 oxidoreductase, and prostaglandin H synthase). In addition, we also found the predominantly cytosolic markers 15-hydroxyprostaglandin dehydrogenase, prostaglandins PGE2 and 13,14-dihydro-15-keto-PGE2. The expression of key enzymes that control glucose production, detoxification of endobiotics and xenobiotics, and the regulation of prostaglandin levels in embryonic and early fetal RBCs means that these cells may have an important role in protecting the developing conceptus before it establishes an efficient circulation and before all tissues fully express their normal complement of these enzymes.

Cytochrome P450 and associated monooxygenase activities in the rat and human spinal cord: induction, immunological characterization and immunocytochemical localization.

We have discovered cytochrome P450 and associated monooxygenase activities in microsomes prepared from spinal cord tissues from rats and a human. Cytochrome P450 levels and nicotinamide adenine dinucleotide phosphate cytochrome c reductase activities in microsomes from rat spinal cord were similar to those observed from the whole brain. However, certain monooxygenase activities were significantly lower in the rat spinal cord microsomes as compared to the corresponding activities observed in the whole brain. Cytochrome P450-mediated monooxygenase activities were also detectable in microsomes prepared from human spinal cord. Immunoblot analyses of rat and human spinal cord microsomes using antisera to various forms of hepatic cytochrome P450 namely (2B1 + 2B2), 1A1, 1A2 and 2E1 revealed the presence of immunologically similar forms. The spinal cord microsomes also cross-reacted with the antiserum to the phenobarbital-inducible form of rat brain cytochrome P450. Immunocytochemical stain was predominant in the gray horns of the rat spinal cord. At the cervical level, lamina 1 and 2 representing the substantia gelatinosa were intensely stained. In the ventral horns, lamina 7, 8 and 9 containing the large motor neurons were strongly labelled, while small neurons revealed variable staining. In the white matter, the glial cells were stained but the axons remained non-reactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Antipyrine elimination and hepatic microsomal enzyme activity in patients with liver disease

Induction of hepatic monooxygenases reflected by 7-ethoxycoumarin O-deethylase has been proposed to be associated with the initiation of liver damage. This study investigated a possible correlation between 7-ethoxycoumarin O-deethylase, reduced nicotinamide adenine dinucleotide phosphate cytochrome c reductase and benzypyrene hydroxylase activity in liver biopsy specimens of 31 patients with liver disease and antipyrine elimination, an in vivo parameter of hepatic monooxygenase activity. No correlation was found between the enzyme activities and antipyrine clearance or half-life. When microsomal enzyme activities were compared with the formation rate of 4-hydroxyantipyrine, 3-methylhydroxyantipyrine, and norantipyrine, a correlation was found only between benzo[alpha]pyrene hydroxylase and 3-methylhydroxyantipyrine (r = 0.89; p less than 0.0005). There was also a correlation between 7-ethoxycoumarin O-deethylase and reduced nicotinamide adenine dinucleotide phosphate cytochrome c reductase (0.56; p less than 0.05). Our data suggest that antipyrine elimination is not related to 7-ethoxycoumarin O-deethylase activity in liver disease. However, the formation rate of antipyrine metabolites, rather than antipyrine half-life and clearance, may correlate with the activity of certain microsomal enzymes.

肝ミクロソームの酸化的薬物代謝酵素(チトクロムP-450)系に対する合成ステロイド剤・Danazolの阻害作用

The inhibitory effect of danazol, a synthetic androgen, on the hydroxylations of testosterone as a model for endogenous steroids and the dealkylations of aminopyrine and 7-ethoxycoumarin as models for xenobiotics in mouse hepatic microsomes in vitro was studied. Danazol inhibited these enzyme activities in a dose-dependent fashion. The inhibition constants (Ki) of danazol for these enzyme activities were 1-4 orders of magnitude lower than those of cimetidine, while there was a great difference among the inhibitory potencies of danazol for each enzyme activity. Addition of danazol to microsomal preparation resulted in a reverse type I difference spectrum and the spectrophotometric analysis revealed that danazol had a high affinity for cytochrome P-450 with dissociation constants (Ks) of 0.9 and 4.2 microM, which were 2 orders of magnitude lower than those of cimetidine. On the other hand, danazol did not significantly affect reduced nicotinamide adenine dinucleotide phosphate cytochrome c reductase activity and levels of cytochrome P-450 in the microsomes. These results suggest that danazol is a highly potent inhibitor for several cytochrome P-450-mediated metabolisms of testosterone and xenobiotics in mouse hepatic microsomes.

Dual granule localization of the dormant NADPH oxidase and cytochrome b559 in human neutrophils

The subcellular localization of the microbicidal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and associated b-cytochrome was investigated in human neutrophils. In unperturbed neutrophils 85% of b-cytochrome and the major part of membrane-bound components of the NADPH oxidase co-sedimented with markers for specific granules and gelatinase. Using cytochrome b559 as a marker for membrane-bound components of the NADPH oxidase in quantitative studies we observed that, of the remaining 15%, the vast majority co-sedimented with latent alkaline phosphatase, a marker for a newly identified mobilizable intracellular compartment. Only a small fraction co-localized with the plasma membranes. Azurophil granules contained a protease activity which rapidly inactivated the NADPH oxidase components present in other membranes. Stimulation of the neutrophils with formyl-methionyl-leucyl-phenyl-alanine and leukotriene B4 which caused minimal degranulation of specific granules, resulted in translocation of b-cytochrome to the plasma membrane, concomitant with incorporation of alkaline phosphatase into the plasma membrane.

Influence of phenobarbital dosage, dietary crude protein level and interval to measurement on hepatic monoxygenase activity in ewes.

Hepatic monoxygenases metabolize steroids that in turn influence reproduction. Experiments were conducted to establish the effect of dose of phenobarbital and level of dietary protein on hepatic monoxygenase. In Exp. 1, ewes were given either 0, .5, 1, 2 or 3 g phenobarbital (PB) daily for 4 to 8 d. After 48 h after last dose of PB, cytochrome P-450 was higher (P less than .01) in all ewes given PB than in controls and was higher (P less than .01) in ewes given PB for 8 d than in ewes treated for 4 d. In Exp. 2, either 0 or 1 g of PB was given orally for 8 d. Liver samples were collected 1, 4, 7 or 10 d after last treatment. Both cytochrome P-450 (P less than .01) and nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome c reductase (P less than .06) were higher in ewes receiving PB than in controls. Cytochrome P-450 was twice as high in treated ewes as in control ewes on the 8th d of treatment, but concentrations returned to control levels 10 d after last treatment. Microsomal protein and cytochrome b5 were not affected by PB (P greater than .10). Ewes in Exp. 3 receiving a diet containing 14.8% crude protein for 10 d had higher levels of cytochrome P-450 (P less than .01) than ewes fed 4.7% crude protein. Protein did not affect microsomal protein or NADPH cytochrome c reductase. These data suggest that a relationship among PB, dietary protein and monoxygenases exists and provide information that will lead to a better understanding of the relationship between diet and reproduction.

Effects of antimycotics on hepatic steroid metabolism.

When male ddY mice were treated with consecutive doses of 10 and 100 mg/kg of miconazole (MCZ) or ketoconazole (KCZ), imidazole-containing antimycotics, once a day for 3 d, a dose-dependent shortening of pentobarbital sleeping time was observed for MCZ, while no change in the sleeping time was observed for KCZ. Even at a low dose (10 mg/kg), MCZ significantly increased cytochrome P-450 content and reduced nicotinamide adenine dinucleotide phosphate cytochrome c reductase activity. Simultaneously, hydroxylase activities of testosterone as a model of endogenous steroids, and aminopyrine N-demethylase and 7-ethoxycoumarin O-deethylase activities were increased, while KCZ lacked inducing properties even at a high dose (100 mg/kg). The change in hepatic oxidative metabolism of cortisol (F) in a patient before, during and after treatment with progressively increasing doses of 2-10 mg/kg/d of MCZ for 14 d was examined by monitoring urinary 6 beta-hydroxycortisol (6 beta-OHF), an oxidative metabolite of F. The ratio of 6 beta-OHF to F in 24-h urine decreased by 15% from the original level on day 1, and then it began to increase on day 7 to reach 2.4 times the original level on day 14. These results suggest that MCZ, but not KCZ, has inducing activity for hepatic cytochrome P-450-dependent oxidative metabolism of steroids and xenobiotics, in addition to its known inhibitory activity.

Effects of ozagrel (OKY-046), a thromboxane synthase inhibitor, on oxidative drug-metabolizing enzymes in mouse hepatic microsomes.

The inhibitory effects of ozagrel (OZA), an imidazole derivative and a specific thromboxane synthase inhibitor, on a monooxygenase system in mouse hepatic microsomes were studied. Pentobarbital sleeping time was significantly prolonged by i.p. administration of a single dose of 100 mg/kg of OZA, and the potency of OZA for the prolongation of sleeping time was similar to that of cimetidine. In vitro, OZA inhibited aminopyrine N-demethylase, aniline hydroxylase and testosterone 6 beta- and 7 alpha-hydroxylase activities in hepatic microsomes with inhibition constants (Ki) of 0.19-3.72 mM. The potency and the mode of inhibition of OZA for these enzyme activities were similar to those of cimetidine, while no inhibitory effect of OZA on testosterone 16 alpha-hydroxylase activity was found. A spectrophotometric study revealed that the imidazole moiety of OZA binds to cytochrome P-450 and has little effect on reduced nicotinamide adenine dinucleotide phosphate cytochrome c reductase activity. These results indicated that OZA is an inhibitor of some cytochrome P-450-mediated drug metabolism in hepatic microsomes.

Cytochromes P-450scc, P-450(17)alpha, adrenodoxin, and reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase in bovine follicles and corpora lutea. Changes in specific contents during the ovarian cycle.

To investigate the basis for the pattern of ovarian steroid production during the bovine estrous cycle, the tissue concentrations of major steroidogenic enzymes, 17 alpha-hydroxylase cytochrome P-450 and cholesterol side-chain cleavage cytochrome P-450 (cytochrome P-450scc), and their respective electron donors, NADPH-cytochrome P-450 reductase and adrenodoxin, were estimated and compared with those of the nonsteroidogenic enzymes, cytochrome c oxidase and F1-ATPase. The levels of these enzymes were estimated in medium sized (9-11 mm) and large (14-18 mm) follicles after removal of follicular fluid by centrifugation, and corpora lutea from the early, early-mid late-mid, and late stages of the luteal phase (n = 5 per group). The specific contents of all enzymes and electron donors were determined by immunoblot analysis, except for cytochrome c oxidase, which was quantified by determination of specific activity. The specific (per microgram of tissue homogenate protein) and total (per follicle or corpus luteum) tissue contents of 17 alpha-hydroxylase cytochrome P-450 increased 0.5- and 5-fold respectively from medium sized to large follicles, but then decreased to undetectable levels in corpora lutea in the early luteal phase, and remained undetectable throughout the luteal phase. In contrast, the specific content of NADPH-cytochrome P-450 reductase was similar between follicles and corpora lutea. The specific contents of cytochrome P-450scc, adrenodoxin and cytochrome c oxidase in follicles were similar to those of corpora lutea of the early luteal phase. However, by the early-mid luteal phase the specific contents of luteal cytochrome P-450scc (490 +/- 46 vs. 5709 +/- 982 cpm/micrograms protein) and adrenodoxin (44 +/- 15 vs. 705 +/- 229 cpm/micrograms protein) were increased, by 12- and 15-fold, respectively (P less than 0.05). In contrast, cytochrome c oxidase activity (29.1 +/- 10.1 vs. 108.6 +/- 20.7 nmol/mg tissue protein X min) and the specific content of F1-ATPase increased only 3- to 4-fold reflective of an increase in numbers of mitochondria. The levels of these enzymes remained elevated until the late luteal phase when they declined markedly. It is concluded that the induction of synthesis of P-450scc and adrenodoxin after ovulation is specific and does not merely reflect biogenesis of mitochondria during luteinization. Moreover the changes in the types of steroids produced by the ovarian compartments throughout the estrous cycle are a reflection of changes in the tissue content of steroidogenic enzymes.

Ascorbic acid potentiates the substrate-specific inhibition of mixed-function oxidation and the stimulation of NADPH oxidation caused by paraquat.

Paraquat inhibits the in vitro hepatic microsomal metabolism of both ethylmorphine and aniline. Inclusion of ascorbate with paraquat in the incubations did not alter the paraquat effect on ethylmorphine N-demethylase activity but potentiated the inhibition of aniline p-hydroxylase activity. Ascorbate alone was without effect on the metabolism of either substrate. Paraquat stimulated the hepatic microsomal oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) equally in the absence of mixed-function oxidase (MFO) substrates or in the presence of ethylmorphine; in the presence of aniline the rate of NADPH oxidation was significantly greater. Also, in the presence of aniline, ascorbate potentiated the paraquat-induced NADPH oxidation, while it was ineffective with paraquat on NADPH oxidation in the presence of ethylmorphine or in the absence of substrates for the microsomal MFO system. The potentiated inhibition of aniline metabolism, concomitant with the potentiated stimulation of NADPH oxidation, was consistent whether liver microsomal fractions were prepared from control rats or from animals induced with phenobarbital. Investigation of possible influences on NADPH cytochrome c reductase activity was precluded by the rapid nonenzymatic reduction of cytochrome c by ascorbate. The paraquat-ascorbate redox couple would not reduce cytochrome P-450. These data suggest that a paraquat interaction with the active microsomal MFO enzyme system plays a role in the depletion of cellular NADPH stores that occurs after paraquat administration in vivo. This mechanism may play a significant role in the development of paraquat toxicity and in the potentiated toxicity observed with ascorbate and paraquat.

Hepatic monooxygenase activities in subjects with a genetic defect in drug oxidation

Microsomes were prepared from surgical wedge biopsy specimens of the livers of 49 patients and from intact liver lobes of 9 kidney transplant donors with the objective of defining interindividual differences in the content and activities of six monooxygenase activities and their relationship to a common genetic defect in drug metabolism known as “debrisoquine polymorphism.” Sixty-one patients were phenotyped in vivo with respect to their urinary metabolic ratio of debrisoquine to 4-hydroxydebrisoquine. Forty-seven patients were found to be extensive metabolizers, 12 patients were arbitrarily classified as “intermediate” metabolizers, and only 2 patients were poor metabolizers. The formation of the 4-hydroxy metabolite from debrisoquine in hepatic microsomes from extensive metaboizers was 1.33 ± 0.437 nmol · mg protein−1 · 15 min−1 (mean ± SD, n = 9). Microsomes of the 2 poor metabolizers formed 0.13 and 0.18 nmol 4-hydroxydebrisoquine · mg−1 · 15 min−1. Microsomes of intermediate metabolizers produced 0.464 ± 0.115 (n = 5) of 4-hydroxydebrisoquine and were distinguished from extensive and poor metabolizers. There was no correlation between the capacity for debrisoquine hydroxylation and the total concentration of microsomal cytochrome P450, cytochrome b5, or the activities of nicotinamide adenine dinucleotide phosphate-cytochrome c reductase, aminopyrine-N-demethylase, aryl hydrocarbon hydroxylase, ethoxycoumarin-O-deethylase, aldrin epoxidase, and 2-biphenylhydroxylase. These studies indicate that genetically defective in vivo metabolism of debrisoquine is caused by a deficiency of a monooxygenase reaction in liver microsomes. Moreover, the findings suggest that direct measurement of the debrisoquine oxidation deficiency may allow the identification of heterozygous carriers of the defect. This conclusion remains to be verified by pedigree analysis.

Effect of Cholestasis Produced by Bile Duct Ligation on Hepatic Heme and Hemoprotein Metabolism in Rats

Cholestasis produced by bile duct ligation has been associated with decreased concentrations of hepatic microsomal cytochrome P450 and decreased hepatic microsomal oxidative drug metabolism. Bile duct ligation producing cholestasis results in a marked increase in hepatic microsomal heme oxygenase activity, with corresponding decreases in hepatic microsomal cytochrome P450 concentration, reduced form of nicotinamide adenine dinucleotide phosphate-cytochrome c reductase activity, and hepatic delta-aminolevulinic acid synthetase activity. As sham-operated rats also demonstrate a less prolonged decrease in cytochrome P450 concentration and reduced form of nicotinamide adenine dinucleotide phosphate-cytochrome c reductase activity, the metabolic effects of surgery and anesthesia must also be involved in these alterations in microsomal oxidative drug metabolism. The relative rate of hepatic cytochrome P450 synthesis and of degradation are both decreased after bile duct ligation. These data suggest that decreased hepatic microsomal cytochrome P450 concentrations in cholestasis are partly the result of decreased cytochrome P450 synthesis. Increased levels of heme oxygenase activity are not related to increased cytochrome P450 turnover, but may instead reflect enlargement and increased catabolism of a free heme pool resulting from decreased hemoprotein (cytochrome P450) synthesis.