Total Cytochrome P450 Research Articles

CYP-mediated drug metabolism in the brain impacts drug response.

The functional role of cytochrome P450 (CYP) enzymes in the brain is an exciting and evolving field of research. CYPs are present and active in the brain, with heterogeneous patterns of expression, activity, and sensitivity to modulation across cell types, regions, and species. Despite total brain CYP expression being a fraction of hepatic CYP expression, the expanding literature of in vitro and in vivo experiments has provided evidence that brain CYPs can impact acute and chronic drug response, susceptibility to damage by neurotoxins, and are associated with altered personality, behaviour, and risk of neurological disease. They may also play a role in endogenous neurotransmitter and neurosteroid homeostasis. This review goes through the characterization of brain CYPs across species, the patterns of susceptibility of brain CYPs to exogenous induction, and recent preclinical evidence of the potential role of brain CYPs in vivo (e.g. CYP2D), along with the development of experiment paradigms that allow modulation of brain CYP activity without affecting CYP activity in the liver. Understanding brain CYP function, and changes therein, may provide unique strategies for the development of CNS-acting therapeutics metabolized locally in the brain, as well as therapeutics to target brain CYPs directly.

Relationship between metabolic enzyme activities and bioaccumulation kinetics of PAHs in zebrafish (Danio rerio)

Many studies have investigated bioaccumulation and metabolism of polycyclic aromatic hydrocarbons (PAHs) in aquatic organisms. However, lack of studies investigated both processes simultaneously, and the interaction between these two processes is less understood so far. This study investigated the bioaccumulation kinetics of PAHs and metabolic enzyme activities, including total cytochrome P450 (CYPs) and total superoxide dismutase (T-SOD), in zebrafish. Mature zebrafish were exposed to the mixture of phenanthrene and anthracene under constant concentration maintained by passive dosing systems for 16days. The results showed that PAH concentrations in zebrafish experienced a peak value after exposure for 1.5days, and then decreased gradually. The bioaccumulation equilibrium was achieved after exposure for 12days. Both of the uptake rate constants (ku) and the elimination rate constants (ke) decreased after the peak value. The variation of PAH concentrations and metabolic enzyme activities in zebrafish had an interactive relationship. The activities of CYPs and T-SOD increased initially with the increase of PAH concentrations, but decreased to the lowest state when PAH concentrations reached the peak value. When the bioaccumulation equilibrium of PAHs was achieved, CYPs and T-SOD activities also reached the steady state. In general, CYPs and T-SOD activities were activated after exposure to PAHs. The decrease of PAH concentrations in zebrafish after the peak value may be attributed to the great drop of ku and the variation of CYPs activities. This study suggests that an interactive relationship exists between bioaccumulation kinetics of PAHs and metabolic enzyme activities in aquatic organisms.

Metabolism of inhaled methylethylketone in rats

Methylethylketone (MEK) is widely used in industry, often in combination with other compounds. Although nontoxic, it can make other chemicals harmful. This study investigates the fate of MEK in rat blood, brain and urine as well as its hepatic metabolism following inhalation over 1 month (at 20, 200 or 1400 ppm). MEK did not significantly accumulate in the organism: blood concentrations were similar after six-hour or 1-month inhalation periods, and brain concentrations only increased slightly after 1 month’s exposure. Urinary excretion, based on the major metabolites, 2,3-butanediols (± and meso forms), accounted for less than 2.4% of the amount inhaled. 2-Butanol, 3-hydroxy-2-butanone and MEK itself were only detectable in urine in the highest concentration conditions investigated, when metabolic saturation occurred. Although MEK exposure did not alter the total cytochrome P450 concentration, it induced activation of both CYP1A2 and CYP2E1 enzymes. In addition, the liver glutathione concentration (reduced and oxidized forms) decreased, as did glutathione S-transferase (GST) activity (at exposure levels over 200 ppm). These metabolic data could be useful for pharmacokinetic model development and/or verification and suggest the ability of MEK to influence the metabolism (and potentiate the toxicity) of other substances.

CRISPR knockout rat cytochrome P450 3A1/2 model for advancing drug metabolism and pharmacokinetics research

Cytochrome P450 (CYP) 3A accounts for nearly 30% of the total CYP enzymes in the human liver and participates in the metabolism of over 50% of clinical drugs. Moreover, CYP3A plays an important role in chemical metabolism, toxicity, and carcinogenicity. New animal models are needed to investigate CYP3A functions, especially for drug metabolism. In this report, Cyp3a1/2 double knockout (KO) rats were generated by CRISPR-Cas9 technology, and then were characterized for viability and physiological status. The Cyp3a1/2 double KO rats were viable and fertile, and had no obvious physiological abnormities. Compared with the wild-type (WT) rat, Cyp3a1/2 expression was completely absent in the liver of the KO rat. In vitro and in vivo metabolic studies of the CYP3A1/2 substrates indicated that CYP3A1/2 was functionally inactive in double KO rats. The Cyp3a1/2 double KO rat model was successfully generated and characterized. The Cyp3a1/2 KO rats are a novel rodent animal model that will be a powerful tool for the study of the physiological and pharmacological roles of CYP3A, especially in drug and chemical metabolism in vivo.

The effects of metronidazole on Cytochrome P450 Activity and Expression in rats after acute exposure to high altitude of 4300 m

BackgroundThe purpose is to observe the changes of CYP450 enzyme activity expression as well as the physiological and pathological states of Wistar rats given metronidazole drug intervention after acute exposure to high altitude of 4300m. Methods and resultsThirty healthy adult male Wistar rats of average weight 200 ± 20 g were randomly assigned into three groups of 10 rats per group as follows: Group A (55m), Group B (4300m) and Group C (metronidazole intervention, 4300m). After three days, the main blood gas levels were detected and the liver tissue pathological slices were observed. The enzymatic activity of CYP1A2 and 3A1 as well as the total protein, total CYP450, cytochrome b5 and the protein expression of CYP450 isoforms CYP1A2 and 3A1 were detected. Compared with Group A and Group B, the expression of total CYP450 and CYP1A2 was significantly reduced (P<0.01or P<0.05), the enzymatic activity of CYP1A2 and CYP3A1 were significantly reduced (P<0.05, P<0.01), respectively and CYP3A1 had no significant changes (P>0.05). Compared with Group B and Group C the enzymatic activity of CYP1A2 had no significant changes (P>0.050), the enzymatic activity of CYP3A1 was significantly reduced (P<0.01). The total cytochrome p450, CYP1A2 and cytochrome b5 were significantly reduced (P<0.01 and P<0.05), respectively and CYP3A1 had no significant changes (P>0.05). Pathological observation showed that rats in Group A were normal with no significant pathological changes in their livers; rats in Group B suffered from liver injuries and edema with a few of them caught inflammatory cell infiltration; rats in Group C caught liver cell edema, inflammatory diseases and lobular vena cava expansion. ConclusionsAcute exposure to high altitude CYP450 isoform expression and the enzymatic activity were significantly reduced, both the physiology and pathology of rats were substantially impaired, and which maybe resulted from the hypoxia and drug intervention interrelated effected in plateau filed test.

Improvement of the chemical inhibition phenotyping assay by cross-reactivity correction.

The fraction of an absorbed drug metabolized by the different hepatic cytochrome P450 (CYP) enzymes, relative to total hepatic CYP metabolism (fmCYP), can be estimated by measuring the inhibitory effects of presumably selective CYP inhibitors on the intrinsic metabolic clearance of a drug using human liver microsomes. However, the chemical inhibition data are often affected by cross-reactivities of the chemical inhibitors used in this assay. To overcome this drawback, the cross-reactivities exhibited by six chemical inhibitors (furafylline, montelukast, sulfaphenazole, ticlopidine, quinidine and ketoconazole) were quantified using specific CYP enzyme marker reactions. The determined cross-reactivities were used to correct the in vitro fmCYPs of nine marketed drugs. The corrected values were compared with reference data obtained by physiologically based pharmacokinetics simulation using the software SimCYP. Uncorrected in vitro fmCYPs of the nine drugs showed poor linear correlation with their reference data (R2=0.443). Correction by factoring in inhibitor cross-reactivities significantly improved the correlation (R2=0.736). Correcting in vitro chemical inhibition results for cross-reactivities appear to offer a straightforward and easily adoptable approach to provide improved fmCYP data for a drug.

The neuroprotective action of naringenin on oseltamivir (Tamiflu) treated male rats

The aim of this study is to evaluate the protective action of naringenin (a flavonoid) on the brain functions of oseltamivir treated rats. 24 rats were divided into 4 groups as follows: control, naringenin treated rats (Nar, 50mg/kgbwt/day), oseltamivir treated rats (Tam, 0.75mg/kgbwt twice daily) and naringenin+oseltamivir treated rats (Nar+Tam). All the drugs were received via oral gavage for five days. The animals on the 5th day were trained in Y maze. Then, on the 6th day, rats were decapitated and the brain was excised for determination of total antioxidant capacity (TAC), total oxidant capacity (TOC), total nitric oxide (TNO), Ca ATPase, total cytochrome P450 (CYP450) contents and brain fatty acid binding proteins FABP7. The results showed a significant increase in the TOC, TNO and CYP450 in Tam treated rats while a significant decrease was noticed in TAC, Ca ATPase and FABP7 in the same group in comparison with the control. Nar+Tam treated rats exhibited a significant decrease in TOC, TNO and CYP450 and a significant increase in TAC, Ca ATPase and FABP7 in comparison with Tam treated rats. An improvement in Y maze behavior and all the investigated parameters was noticed in Nar+Tam treated rats as compared with the oseltamivir treated rats. The results suggest that Nar has a neurophysiological and behavioral protective effect on oseltamivir side effects on the brain functions.

Hepatic expression of cytochrome P450 in Zucker diabetic fatty rats

In this study, the hepatic expression of cytochrome P450 (CYP) enzymes, including CYP1A1/2, 2B1, 2C11, 2E1, 3A1/2, and 4A, was investigated in 5-week-old (insulinresistant state) and 11-week-old (diabetic) Zucker diabetic fatty (ZDF) rats. Serum glucose and glycated hemoglobin levels were increased in 11-week-old ZDF rats, but not in 5-weekold ZDF rats. Hyperinsulinemia was observed in both age groups. The microsomal protein, total CYP, CYP reductase, CYP1A1/2, and CYP3A1 levels did not differ between 5- and 11-week-old ZDF rats and their respective control rats, while CYP4A was up-regulated in both groups. Hepatic levels of cytochrome b5, CYP2B1, CYP2C11, CYP2E1, and CYP3A2 were decreased in 5-week-old ZDF rats, but not in 11-week-old ZDF rats. Similarly, pentoxyresorufin O-depentylase, testosterone 2α- and 16α-hydroxylase, chlorzoxazone 6- hydroxylase, and midazolam 1′- and 4-hydroxylase activities were decreased only in 5-weekold ZDF rats. Based on these results, the 5-week-old ZDF rats exhibited down-regulation of the major CYP enzymes. These results suggest that hepatic expression of CYP enzymes may be dysregulated during development in ZDF rats. With the exception of CYP2B1 and CYP4A, the hepatic levels and activities of CYP were comparable between 11-week-old ZDF and control rats, suggesting that xenobiotic metabolism is normally regulated in the early diabetic state.

Cytochrome P450 Activity in Ex Vivo Cornea Models and a Human Cornea Construct

The pharmacokinetic behaviors of novel ophthalmic drugs are often preliminarily investigated in preclinical studies using ex vivo animal cornea or corneal cell culture models. During transcorneal passage, topically applied drugs may be affected by drug metabolizing enzymes. The knowledge regarding the functional expression of metabolic enzymes in corneal tissue is marginal; thus, the aim of this study was to investigate cytochrome P450 activity in an organotypic three-dimensional human cornea construct and to compare it with porcine and rabbit corneas, which are commonly used ex vivo cornea models. The total cytochrome P450 activity was determined by measuring the transformation of 7-ethoxycoumarin. Furthermore, the expression of the cytochrome P450 enzyme 2D6 (CYP2D6) was investigated at the protein level using immunohistochemistry and western blotting. CYP2D6 activity measurements were performed using a d-luciferin-based assay. In summary, similar levels of the total cytochrome P450 activity were identified in all 3 cornea models. The protein expression of CYP2D6 was confirmed in the human cornea construct and porcine cornea, whereas the signals in the rabbit cornea were weak. The analysis of the CYP2D6 activity indicated similar values for the human cornea construct and porcine cornea; however, a distinctly lower activity was observed in the rabbit cornea.

Antidiabetic and antioxidant effects of saponarin from Gypsophila trichotoma on streptozotocin-induced diabetic normotensive and hypertensive rats

BackgroundDiabetes and hypertension are diseases that often coexist, which increases the risk of chronic organ damages and cardiovascular complications. PurposeTo evaluate the effects of saponarin, isolated from Gypsophila trichotoma Wend, on blood pressure, glycemia, body weight, and liver biochemical parameters related to oxidative stress in diabetic normotensive Wistar Kyoto rats (NTR) and spontaneously hypertensive rats (SHR). MethodsDiabetes was induced by administration of streptozotocin (40mg/kg, i.p.). The following biochemical parameters: reduced glutathione (GSH), malondialdehyde (MDA), total cytochrome P450, aniline hydroxylase (AH) activity, as well as the activities of antioxidant enzymes such as glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) were measured in the livers of euthanized rats. ResultsSaponarin exerted slight antihypertensive activity in non-diabetic SHR, judged by 19% (p<0.05) decrease of the initial blood pressure. However, such effect was not observed in streptozotocin-induced diabetic SHR (SHR-D). Streptozotocin-induced diabetes was evidenced by 78% (p<0.05) and by 171% (p<0.05) increase in blood glucose level in NTR and SHR, respectively. In non-diabetic SHR the initial MDA quantity was by 36% (p<0.05) higher and the initial GSH levels were by 28% (p<0.05) lower in comparison to non-diabetic NTR. Significant decrease in the activities of GPx, GR, and GST was measured in the livers of all diabetic rats. Treatment with saponarin ameliorated the above mentioned liver parameters in both diabetic strains, however its effects were less pronounced in the diabetic SHR group. ConclusionTaken together our data indicate that diabetes and hypertension in combination are more difficult to be modulated by saponarin.

Evaluation of cytochrome P450 2C9 activity in normal, healthy, adult Western Indian population by both phenotyping and genotyping.

Objectives:Cytochrome P450 2C9 (CYP2C9) is a member of cytochrome P450 (CYP) family that accounts for nearly 18% of the total CYP protein content in the human liver microsomes and catalyzes almost 15–20% of the drugs. Considering the paucity of data on the polymorphisms of CYP2C9 in Western Indian population, the present study was conducted to evaluate the prevalence of CYP2C9 polymorphisms (*1, *2 and *3) and correlate it with the activity using flurbiprofen (FLB) as a probe drug.Materials and Methods:A 100 mg FLB capsule was administered to 298 healthy adult participants. Venous blood samples were analyzed at 2 h postdose for the estimation of FLB and 4-hydroxy FLB. Metabolic ratio (MR) was calculated to determine the extent of poor metabolizer (PM) and rapid metabolizer status using probit plot. Genotyping of CYP2C9 polymorphism was performed using polymerase chain reaction-restriction fragment length polymorphism technique.Results:Of the total 298 participants, phenotype was assessable in 288 and genotype was performed in 289 participants. The median (range) MR of the study population was 6.6 (1.65–66.05). Five participants were found to be PMs by phenotype. Of the total 289 participants, 209 (72.3%) (66.7, 77.2) had CYP2C9*1/*1, 25 (8.7%) (5.8, 12.7) with CYP2C9*1/*2, 55 (19%) (14.8, 24.1) had CYP2C9*1/*3, 3 (1%) (0.3, 3.3) had CYP2C9*2/*3 genotype. A significant association between phenotype and genotype was observed.Conclusion:To conclude, the present study found significant association of CYP2C9 activity by both phenotype and genotype and these findings have to be corroborated in different kinds of patients.

The ameliorating effects of vitamin E on hepatic antioxidant system and xenobiotic-metabolizing enzymes in fenvalerate-exposed iodine-deficient rats

This study investigated the effects of vitamin E (VE) on hepatic antioxidant system and drug-metabolizing enzymes in fenvalerate (FEN)-exposed iodine-deficient (ID) Wistar rats. ID was produced by perchlorate containing drinking water. VE was introduced by a loading dose of 100 mg/kg/d, i.g. for the first three days in the last week of feeding period; then with a single maintenance dose of 40 mg/kg on the 4th day. During last week, FEN groups (F) received 100 mg/kg/d, i.p. FEN. VE alone did not significantly affect thyroid hormones and antioxidant parameters; however, significantly increased total cytochrome P450 (38%) and cytochrome b5 levels (36%). In all ID groups, plasma thyroid-stimulating hormone (TSH) levels increased markedly, but remained at control level in vitamin E plus FEN receiving iodine-deficient group (IDVF) group. Glutathione peroxidase activity showed marked increases in F (19%) and FEN-exposed iodine-deficient group (IDF, 48%) groups. FEN treatment significantly increased total cytochrome P450 (28%) and thiobarbituric acid reactive substance levels (36%), as well as 7-ethoxyresorufin O-deethylase (120%), 7-penthoxyresorufin O-deethylase (139%) and glutathione S-transferase (15%) activities and decreased total glutathione concentrations (28%) versus control. Overall results suggest that vitamin E has ameliorating effects on the measured parameters in ID and/or FEN exposure.

Experimental protoporphyria: effect of bile acids on liver damage induced by griseofulvin.

The effect of bile acids administration to an experimental mice model of Protoporphyria produced by griseofulvin (Gris) was investigated. The aim was to assess whether porphyrin excretion could be accelerated by bile acids treatment in an attempt to diminish liver damage induced by Gris. Liver damage markers, heme metabolism, and oxidative stress parameters were analyzed in mice treated with Gris and deoxycholic (DXA), dehydrocholic (DHA), chenodeoxycholic, or ursodeoxycholic (URSO). The administration of Gris alone increased the activities of glutathione reductase (GRed), superoxide dismutase (SOD), alkaline phosphatase (AP), gamma glutamyl transpeptidase (GGT), and glutathione-S-transferase (GST), as well as total porphyrins, glutathione (GSH), and cytochrome P450 (CYP) levels in liver. Among the bile acids studied, DXA and DHA increased PROTO IX excretion, DXA also abolished the action of Gris, reducing lipid peroxidation and hepatic GSH and CYP levels, and the activities of GGT, AP, SOD, and GST returned to control values. However, porphyrin accumulation was not prevented by URSO; instead this bile acid reduced ALA-S and the antioxidant defense enzymes system activities. In conclusion, we postulate that DXA acid would be more effective to prevent liver damage induced by Gris.

Berberine protects liver from ethanol-induced oxidative stress and steatosis in mice

Alcohol consumption is customary in many cultures and it is a common human behavior worldwide. Binge ethanol and chronic alcohol consumption, two usual drinking patterns of human beings, produce a state of oxidative stress in liver and disturb the liver function. However, a safe and effective therapy for alcoholic liver disease in humans is still elusive. This study identified the natural product berberine as a potential agent for treating or preventing ethanol-induced liver injury. We demonstrated that berberine attenuated oxidative stress resulted from binge drinking in liver by reducing hepatic lipid peroxidation, glutathione exhaust and mitochondrial oxidative damage. Furthermore, berberine also prevented the oxidative stress and macrosteatosis in response to chronic ethanol exposure in mice. Either the total cytochrome P450 2E1 or the mitochondria-located cytochrome P450 2E1, which is implicated in ethanol-mediated oxidative stress, was suppressed by berberine. On the other hand, berberine significantly blunted the lipid accumulation in liver due to chronic alcohol consumption, at least partially, through restoring peroxisome proliferator-activated receptor α/peroxisome proliferator-activated receptor-gamma Co-activator-1α and hepatocyte nuclear factor 4α/microsomal triglyceride transfer protein pathways. These findings suggested that berberine could serve as a potential agent for preventing or treating human alcoholic liver disease.

Human recombinant cytochrome P450 enzymes display distinct hydrogen peroxide generating activities during substrate independent NADPH oxidase reactions.

Microsomal enzymes generate H2O2 in the presence of NADPH. In this reaction, referred to as "oxidase" activity, H2O2 is generated directly or indirectly via the formation of superoxide anion. In the presence of redox active transition metals, H2O2 can form highly toxic hydroxyl radicals and, depending on the "oxidase" activity of individual cytochrome P450 isoenzymes, this can compromise cellular functioning and contribute to tissue injury. In the present studies, we compared the initial rates of H2O2 generating activity of microsomal preparations containing various human recombinant cytochromes P450s. In the absence of cytochrome P450s the human recombinant NADPH cytochrome P450 reductase (CPR) generated low, but detectable amounts of H2O2 (∼0.04 nmol H2O2/min/100 units of reductase). Significantly greater activity was detected in preparations containing individual cytochrome P450s coexpressed with CPR (from 6.0 nmol H2O2/min/nmol P450 to 0.2 nmol/min/nmol P450); CYP1A1 was the most active, followed by CYP2D6, CYP3A4, CYP2E1, CYP4A11, CYP1A2, and CYP2C subfamily enzymes. H2O2 generating activity of the cytochrome P450s was independent of the ratio of CYP/CPR. Thus, similar H2O2 generating activity was noted with the same cytochrome P450s (CYP3A4, CYP2E1, and CYP2C9) expressed at or near the ratio of CYP/CPR in human liver microsomes (5-7), and when CPR was present in excess (CYP/CPR = 0.2-0.3). Because CYP3A4/5/7 represent up to 40% of total cytochrome P450 in the liver, these data indicate that these enzymes are the major source of H2O2 in human liver microsomes.

Role of Kupffer cells in ischemic injury in alcoholic fatty liver

BackgroundThis study was designed to evaluate the role of Kupffer cells (KCs) in hepatic drug metabolizing dysfunction after hepatic ischemia–reperfusion (IR) in alcoholic fatty liver. Materials and methodsRats were fed the Lieber-DeCarli diet for 5 wk to develop alcoholic fatty liver, then were subjected to 90 min of hepatic ischemia and 5 h of reperfusion. For ablation of KCs, rats were pretreated with gadolinium chloride (GdCl3) 48 and 24 h before the IR procedure. ResultsAfter the IR procedure, ethanol diet (ED)-fed rats had higher serum aminotransferase activity compared with the control diet-fed rats. These changes were attenuated by GdCl3. The ED-fed rats exhibited increased hepatic microsomal total cytochrome P450 (CYP) content and nicotinamide adenine dinucleotide phosphate-CYP reductase and CYP1A1, 1A2, 2B1, and 2E1 isozyme activity. After hepatic IR, these increases were reduced to lower levels than observed in the sham group, except CYP2E1 activity. Increases in CYP2E1 activity and its expression were augmented after hepatic IR in ED-fed animals, but were attenuated by GdCl3. Finally, toll-like receptor 4 and myeloid differentiation primary response gene 88 protein expression, nuclear translocation of nuclear factor-κB and activator protein 1, and levels of proinflammatory mediators were further increased in ED-fed animals compared with control diet-fed animals after IR. These increases were attenuated by GdCl3. ConclusionsWe suggest that KCs contribute to hepatic drug metabolizing dysfunction during hepatic IR in alcoholic fatty liver via the toll-like receptors 4-mediated inflammatory response.

Effects of age increase on hepatic expression and activity of cytochrome P450 in male C57BL/6 mice.

Effects of aging on hepatic expression and activity of cytochrome P450 (CYP) isoforms were investigated in male mice aged 2, 6, 18, and 30months. Microsomal protein, total CYP, cytochrome b5 and NADPH-dependent cytochrome P450 reductase contents in liver were fully expressed in young (2-month-old) mice. Neither Cyp1a1 nor Cyp2c was detected in any aged mice. And Cyp1a2 was maximally expressed at 2months and decreased with age. Hepatic levels of Cyp2b10 and Cyp3a11 were decreased in 30-month-old mice. Hepatic Cyp2e1 levels were constantly maintained from 2-month to 30-month old mice. Hepatic activities of ethoxyresorufin-O-deethylase and methoxyresorufin-O-demethylase were gradually decreased after 6months. The 30-month-old mice exhibited the lowest activity of midazolam 1'-hydroxylase. Pentoxyresorufin-O-depenthylase activity was decreased in 30-month-old mice, but not statistically significant. There were no significant differences in hepatic activities of chlorzoxazone 6-hydroxylase and midazolam 4-hydroxylase. The present study shows that increasing age, especially 30-month-old mice, leads to decrease in expression and activity of hepatic CYP isoforms, suggesting that aging mice exhibit poor hepatic drug-metabolizing capacity.

Ascorbic acid deficiency decreases hepatic cytochrome P-450, especially CYP2B1/2B2, and simultaneously induces heme oxygenase-1 gene expression in scurvy-prone ODS rats.

The mechanisms underlying the decrease in hepatic cytochrome P-450 (CYP) content in ascorbic acid deficiency was investigated in scurvy-prone ODS rats. First, male ODS rats were fed a diet containing sufficient ascorbic acid (control) or a diet without ascorbic acid (deficient) for 18 days, with or without the intraperitoneal injection of phenobarbital. Ascorbic acid deficiency decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial cytochrome oxidase (COX) complex IV subunit I protein, and simultaneously increased heme oxygenase-1 protein in microsomes and mitochondria. Next, heme oxygenase-1 inducers, that is lipopolysaccharide and hemin, were administered to phenobaribital-treated ODS rats fed sufficient ascorbic acid. The administration of these inducers decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial COX complex IV subunit I protein. These results suggested that the stimulation of hepatic heme oxygenase-1 expression by ascorbic acid deficiency caused the decrease in CYP content in liver.

The revised human liver cytochrome P450 "Pie": absolute protein quantification of CYP4F and CYP3A enzymes using targeted quantitative proteomics.

The CYP4F subfamily of enzymes has been identified recently to be involved in the metabolism of endogenous compounds (arachidonic acid and leukotriene B4), nutrients (vitamins K1 and E), and xenobiotics (pafuramidine and fingolimod). CYP4F2 and CYP4F3B are reported to be expressed in the human liver. However, absolute concentrations of these enzymes in human liver microsomes (HLMs) and their interindividual variability have yet to be determined because of the lack of specific antibodies. Here, an liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based targeted quantitative proteomic approach was employed to determine the absolute protein concentrations of CYP4F2 and CYP4F3B compared with CYP3A in two panels of HLMs (n = 31). As a result, the human hepatic cytochrome P450 (P450) "pie" has been revised to include the contribution of CYP4F enzymes, which amounts to 15% of the total hepatic cytochrome P450 enzymes. CYP4F3B displayed low interindividual variability (3.3-fold) in the HLM panels whereas CYP4F2 displayed large variability (21-fold). However, CYP4F2 variability decreased to 3.4-fold if the two donors with the lowest expression were excluded. In contrast, CYP3A exhibited 29-fold interindividual variability in the same HLM panels. The proposed marker reaction for CYP4F enzymes pafuramidine/DB289 M1 formation did not correlate with CYP4F protein content, suggesting alternate metabolic pathways for DB289 M1 formation in HLMs. In conclusion, CYP4F enzymes are highly expressed in the human liver and their physiologic and pharmacologic roles warrant further investigation.

Identification of metabolic pathways and enzyme systems involved in the in vitro human hepatic metabolism of dronedarone, a potent new oral antiarrhythmic drug

The in vitro metabolism of dronedarone and its major metabolites has been studied in human liver microsomes and cryopreserved hepatocytes in primary culture through the use of specific or total cytochrome P450 (CYP) and monoamine oxidase (MAO) inhibitors. The identification of the main metabolites and enzymes participating in their metabolism was also elucidated by using rhCYP, rhMAO, flavin monooxygenases (rhFMO) and UDP-glucuronosyltransferases (rhUGT) and liquid chromatography/tandem mass spectrometry (LC/MS-MS) analysis. Dronedarone was extensively metabolized in human hepatocytes with a metabolic clearance being almost completely inhibited (98 ± 2%) by 1-aminobenzotriazole. Ketoconazole also inhibited dronedarone metabolism by 89 ± 7%, demonstrating the crucial role of CYP3A in its metabolism. CYP3A isoforms mostly contributed to N-debutylation while hydroxylation on the butyl-benzofuran moiety was catalyzed by CYP2D6. However, hydroxylation on the dibutylamine moiety did not appear to be CYP-dependent. N-debutyl-dronedarone was less rapidly metabolized than dronedarone, the major metabolic pathway being catalyzed by MAO-A to form propanoic acid-dronedarone and phenol-dronedarone. Propanoic acid-dronedarone was metabolized at a similar rate to that of N-debutyl-dronedarone and was predominantly hydroxylated by CYP2C8 and CYP1A1. Phenol-dronedarone was extensively glucuronidated while C-dealkyl-dronedarone was metabolized at a slow rate. The evaluation of the systemic clearance of each metabolic process together with the identification of both the major metabolites and predominant enzyme systems and isoforms involved in the formation and subsequent metabolism of these metabolites has enhanced the overall understanding of metabolism of dronedarone in humans.