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Inhaled xenobiotics such as tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone are mainly metabolized by phase I oxidase cytochrome P450, family 2, subfamily A, polypeptide 13 (CYP2A13), phase II conjugate UDP glucuronosyltransferase 2 family, polypeptide B17 (UGT2B17), and phase III transporter ATP-binding cassette, subfamily B (MDR/TAP), member 1 (ABCB1), with genetic polymorphisms implicated in lung cancer. Their genetic interaction and pulmonary expression regulation are largely unknown. We analyzed joint association for CYP2A13 and ABCB1 polymorphisms in 2 independent lung cancer case populations (669 and 566 patients) and 1 common control population (749 subjects), and characterized the transacting function of the lung development-related transcription factor forkhead box A2 (FOXA2). We undertook FOXA2 overexpression and down-regulation in lung epithelial cell lines, analyzed functional impact on the transactivation ofCYP2A13,UGT2B17, andABCB1, and measured correlation for their expressions in lung tissues. We found a substantial reduction in cancer risk (OR 0.39; 95% CI 0.25-0.61; Pinteraction = 0.029) associated with combined genotypes for CYP2A13 R257C and a functionary regulatory variant in the cis element of ABCB1 synergistically targeted by GATA binding protein 6 and FOXA2. Genetic manipulation of FOXA2 consistently influenced its binding to and transactivation of the promoters of CYP2A13, UGT2B17, and ABCB1, whose mRNA and protein expressions were all consistently correlated with those of FOXA2 in both tumorous and normal lung tissues. We therefore establish FOXA2 as a core transcriptional modulator for pulmonary xenobiotic metabolic pathways and uncover an etiologically relevant interaction between CYP2A13 and ABCB1, furthering our understanding of expression and function of the xenobiotic metabolism system. [ABSTRACT FROM AUTHOR]

Mitochondria, the major source of cellular energy in the form of ATP, respond to changes in substrate availability and bioenergetic demands by employing rapid, short-term, metabolic adaptation mechanisms, such as phosphorylation-dependent protein regulation. In mammalian cells, an intramitochondrial CO2-adenylyl cyclase (AC)-cyclic AMP (cAMP)-protein kinase A (PKA) pathway regulates aerobic energy production. One target of this pathway involves phosphorylation of cytochrome c oxidase (COX) subunit 4-iso-form 1 (COX4i1), which modulates COX allosteric regulation by ATP. However, the role of the CO2-sAC-cAMP-PKA signalosome in regulating COX activity and mitochondrial metabolism and its evolutionary conservation remain to be fully established. We show that in Saccharomyces cerevisiae, normoxic COX activity measured in the presence of ATP is 55% lower than in the presence of ADP. Moreover, the adenylyl cyclase Cyrl activity is present in mitochondria, and it contributes to the ATP-mediated regulation of COX through the normoxic subunit Cox5a, homologue of human COX4i1, in a bicarbonate-sensitive manner. Furthermore, we have identified 2 phosphorylation targets in Cox5a (T65 and S43) that modulate its allosteric regulation by ATP. These residues are not conserved in the Cox5b-containing hypoxic enzyme, which is not regulated by ATP. We conclude that across evolution, a CO2-sAC-cAMP-PKA axis regulates normoxic COX activity. [ABSTRACT FROM AUTHOR]

Cytochrome P450 (CYP) 4A and 4F enzymes metabolize arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). Although CYP4A-derived 20-HETE is known to have prohypertensive and proangiogenic properties, the effects of CYP4F-derived metabolites are not well characterized. To investigate the role of CYP4F2 in vascular disease, we generated mice with endothelial expression of human CYP4F2 (Tie2-CYP4F2-Tr). LC/MS/MS analysis revealed 2-fold increases in 20-HETE levels in tissues and endothelial cells (ECs), relative to wild-type (WT) controls. Tie2-CYP4F2-Tr ECs demonstrated increases in growth (267.1±33.4 vs. 205.0±13% at 48 h) and tube formation (7.7±1.1 vs. 1.6±0.5 tubes/field) that were 20-HETE dependent and associated with up-regulation of prooxidant NADPH oxidase and proangiogenic VEGF. Increases in VEGF and NADPH oxidase levels were abrogated by inhibitors of NADPH oxidase and MAPK, respectively, suggesting the possibility of crosstalk between pathways. Interestingly, IL-6 levels in Tie2-CYP4F2-Tr mice (18.6±2.7 vs. 7.9±2.7 pg/ml) were up-regulated via NADPH oxidase- and 20-HETE-dependent mechanisms. Although Tie2-CYP4F2-Tr aortas displayed increased vasoconstriction, vasorelaxation and blood pressure were unchanged. Our findings indicate that human CYP4F2 significantly increases 20-HETE production, CYP4F2-derived 20-HETE mediates EC proliferation and angiogenesis via VEGF- and NADPH oxidase-dependent manners, and the Tie2-CYP4F2-Tr mouse is a novel model for examining the pathophysiological effects of CYP4F2-derived 20-HETE in the vasculature. [ABSTRACT FROM AUTHOR]

The biosynthesis of steroid hormones, essential to the stwvival of all mammals, is dependent on the activity of 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3βBHSD). 3βHSD activity is, in turn, influenced by cytochrome-b5 (Cyt-b5). However, the mechanism through which this occurs is unknown. In this study, we investigated this mechanism by evaluating the influence of Cyt-b5 on the dehydrogenase and isomerase activities of 3βHSD. Capra hircus 3IBHSD was overexpressed in SF-9 cells, using a baculovirus expression system, and purified. Substrate and cofactor kinetics were determined spectrophotometrically in the presence and absence of purified Ovis aries liver Cyt-b5. Nonspecific enzyme activity was evaluated by zero-enzyme, -substrate, and -cofactor blanks. Fusion proteins, 3βHSD-eCFP, and Cyt-b5-eYFP were subsequently coexpressed in COS-1 cells and analyzed for FRET. A CFP-YFP fusion protein served as positive control, while coexpression of 3βHSD- eCFP and cytochrome P450 17α-hydroxylase/17,20 lyase-eYFP (CYP17AI-eYFP) served as negative control. Results showed Cyt-b5 to decrease the Km,NAD+ value of 3βHSD ≈3.5-fold while increasing the Vmax,app of the dehydrogenase reaction ≈17%. FRET analysis showed COS-1 cells coexpressing 3βHSD-eCFP and Cyt-b5-eYFP to exhibit a FRET signal ≈9-fold greater than that of the negative control. These results indicate that Cyt-b5 augments 3βHSD activity v/a an allosteric mechanism by increasing the affinity of the enzyme toward NAD+. [ABSTRACT FROM AUTHOR]

Cerebral amyloid angiopathy (CAA) is an age-associated condition and a common finding in Alzheimer's disease in which amyloid-β (Aβ) vascular deposits are featured in >80% of the cases. Familial Aβ variants bearing substitutions at positions 21-23 are primarily associated with CAA, although they manifest with strikingly different clinical phenotypes: cerebral hemorrhage or dementia. The recently reported Piedmont L34V Aβ mutant, located outside the hot spot 21-23, shows a similar hemorrhagic phenotype, albeit less aggressive than the widely studied Dutch E22Q variant. We monitored the apoptotic events occurring after stimulation of human brain microvascular endothelial and smooth muscle cells with nonfibrillar structures of both variants and wild-type Aβ40. Induction of analogous caspase-mediated mitochondrial pathways was elicited by all peptides, although within different time frames and intensity. Activated pathways were susceptible to pharmacological modulation either through direct inhibition of mitochondrial cytochrome c release or by the action of pan- and pathway-specific caspase inhibitors, giving a clear indication of the independent or synergistic engagement of both extrinsic and intrinsic mechanisms. Structural analyses of the Aβ peptides showed that apoptosis preceded fibril formation, correlating with the presence of oligomers and/or protofibrils. The data support the notion that rare genetic mutations constitute unique paradigms to understand the molecular pathogenesis of CAA. [ABSTRACT FROM AUTHOR]

Activation of the phagocyte NADPH oxidase (phox) requires the association of cytosolic proteins (p67-phox, p47-phox, p40-phox, and Rac1/2) with the membrane cytochrome b558, leading to a hemoprotein conformation change. To clarify this mechanism, the phagocyte NADPH oxidase complex was isolated through cytochrome b558 purification after three chromatographic steps. The purified neutrophil complex was constitutively active in the absence of an amphiphile agent with a maximum turnover (125 mol 02- ·s-1·mol heme b-1), indicating that cytochrome b558 has been activated by cytosolic proteins and is in an "open conformation," able to transfer a maximum rate of electrons. In contrast, the phox complex prepared with B lymphocyte cytosol shows a lower constitutive turnover (∼50 mol O2- ·s-1·mol heme b-1). Analysis of phox complex components by Western blot and mass spectrometry showed the presence of cytosolic factors (especially p67-phox) and structural proteins (moesin, ezrin). To investigate the difference in activity of phox complexes, we evaluated the effect of MRP8 and MRP14, specifically expressed in neutrophils, on the activity of the B lymphocyte complex. MRPs induce the switch between the partially and the fully "open" cytochrome b558 conformation. Moreover, their effect was independent of p67-phox. Data point out two potential cytochrome b558 activation states. [ABSTRACT FROM AUTHOR]

Presents abstracts of studies on enzyme structure and function. "Characterization and Mutation of Rat Cytochrome P450C24A1 (CYP24A1) From the Vitamin D Pathway," by Andrew Annalora, Katya Bobrovnikova-Marjon, Andrzej Pastuszyn, Mark Chiu, Sandra Graham and John Omdahl; "Stabilization of Protein Kinase C by Phosphorylation," by Jaime Marach; "Structural Basis for K-Ras Farnesylation," by Bret Beyer and Hee-Won Park; "Benzyl Isothiocyanate Binds at a Discrete Site Within the Xenobiotic Substrate Region of Glutathione S-Transferase pi," by Luis A. Ralat and Roberta F. Colman; "Cloning, Expression and Characterization of the Mitochondrial Creatine Kinase," by Sona Sona and William Ellington.

Several lines of evidence indicate that mitochondria-mediated reactive oxygen species (ROS) generation is a major source of oxidative stress in the cell. Release of cytochrome c from mitochondria is a central event in apoptosis induction and appears to be mediated by ROS. Dissociation of cytochrome c from the IMM, where it is bound to cardiolipin, represents a necessary first step for cytochrome c release. In the present study, the role of ROS and cardiolipin in the release of cytochrome c from rat liver mitochondria was investigated. ROS were produced by mitochondria oxidizing succinate in the nonphosphorylating state. Cytochrome c was quantitated by a new, very sensitive and rapid reverse-phase HPLC method. We found that succinate-supported ROS production resulted in a release of cytochrome c from mitochondria and a parallel loss of cardiolipin content. These effects were directly and significantly correlated and also abolished by ADP, which prevents succinate-mediated ROS production. The ROS-induced cytochrome c release was independent from MPT and appears to involve VDAC. It is suggested that mitochondrial-induced ROS production promotes cytochrome c release from mitochondria by a two-steps process, consisting of the dissociation of this protein from cardiolipin, followed by permeabilization of the outer membrane, probably by interaction with VDAC. The data may help clarify the molecular mechanism underlying the release of cytochrome c from the mitochondria to the cytosol and the role of ROS and cardiolipin in this release.--Petrosillo, G., Ruggiero, F. M., Paradies, G. Role of reactive oxygen species and cardiolipin in the release of cytochrome c from mitochondria. [ABSTRACT FROM AUTHOR]

This article discusses a study on the effects of gossypol on Bax/Bak-independent activation of apoptosis and cytochrome c release. The researchers screened out compounds that can activate caspase-dependent apoptosis in Bak/Bax double knockout. The study found that gossypol induced cytochrome c release in a mitochondrial permeability transition pore independent manner in Bax/Bak cells.

Presents a summary of a research on the role of inductible isoform of heme oxygenase (HO) on mitochondria heme content and metabolism. Expression of HO-1 protein in microsomes and mitochondria during Western blot and immune-electron microscopy; Association of the induction of mitochondrial HO-1 with a decrease of mitochondrial heme content and protein expression of cytochrome oxygenase subunit I.

Presents a summary of a research on the oxidative stabilization of iso-1-cytochrome c by redox-inspired protein engineering. Occurrence of cytochrome c oxidative inactivation; Modulation of the partition of the oxidizing equivalents toward the exogenous substrate by site-directed mutagenesis; Preservation of the macroscopic stability properties of the mutant cytochrome c variants.

Presents findings of a study on the role of basal endogenous sphingosine kinase-1 (SK1) in cell regulation. Effect of SK1 on the cell cycle profile of MCF-7 breast cancer cells; Cause of caspase activation, cytochrome release and Bax oligomerization; Effect of SK1 loss on the mass levels of ceramide.

Presents abstracts of studies on biotransformation and drug transport, presented at the Experimental Biology/International Union of Physiological Sciences 2005 congress held in San Diego, California. "Ontogeny of Brain Mdr1a Expression: Implications for Cyclosporine A Neurotoxicity in Young Children," by Philip Acott, David Worth et al.; "Regulation of the Multidrug Resistance-Associated Protein 3 by Bile Acids," by Tanya Christina McCarthy, Xiufeng Li and Christopher J. Sinal; "Differences in the Inhibition of Cytochromes P450 3A4 and 3A5 by Metabolite-Inhibitor Complex Forming Drugs," by Jason Boer, Kyle Allen et al.

Focuses on a study which characterized the genomic structure of mouse cytochromes P450 (CYP) 3a13 in order to identify the molecular basis of CYP3a regulation. Way in which CYP have been demonstrated; Responsibility of the CYP3A subfamily; Analysis of the catalytic ability of expressed CYP3a13 toward clinically active drugs.

We previously reported that the prototypical liver X receptor (LXR) agonists, 24(S),25-epoxycholesterol, an endogenous oxysterol, and T0901317, a synthetic ligand, also induce rodent CYP3A expression in primary cultured hepatocytes as a consequence of pregnane X receptor (PXR) activation, albeit at somewhat higher concentrations than are necessary to activate LXR. In this study, we examined the effects of these two agents on CYP3A4 mRNA content in primary cultured human hepatocytes and on human PXR activity, using a transactivation assay in HepG2 cells. Treatment of primary cultured human hepatocytes with 1 µM or 10 nM T0901317 increased CYP3A4 content ∼50-fold (n=3 preparations) or ∼10-fold (n=2 preparations), respectively. By comparison, treatment with 10 or 30 µM 24(S),25-epoxycholesterol did not increase CYP3A4 mRNA content more than ∼2-fold in any preparation. T0901317 potently activated PXR-responsive reporter expression with an EC50 of ∼17 nM, and the maximal increase produced at 1 µM T0901317 (31-fold) was approximately the same as that produced by treatment with 50 pM rifampicin (28-fold). Treatment with 24(S),25-epoxycholesterol at concentrations up to 30 µM had no effect on PXR activity. These results demonstrate that T0901317 is a potent human PXR activator and CYP3A4 inducer, while 24(S), 25-epoxycholesterol is inactive in this regard. [ABSTRACT FROM AUTHOR]

To elucidate the mechanism by which cytochrome b5 (cyt b5) affects catalysis by cytochrome P450 (P450) 2B4, the kinetics of product formation by oxyferrous cyt P450 2B4 were compared in the presence of cyt b5 and cyt P450 reductase (CPR) under single turnover conditions. In the presence of cyt b5, P450 catalyzes the oxidation of cyclohexane monophasically (k=29 ± 4.5 s-1), forming 58 pmoles of cyclohexanol/nmole P450. In contrast, in the presence of CPR, P450 catalyzes the oxidation of cyclohexane ≅ 10-fold slower (k= 2.5 ± 0.35 s-1) and less efficiently, generating 21 pmoles of cyclohexanol/nmole P450. The ten-fold different catalytic rate constants of cyt b5 and CPR enabled us to investigate whether cyt b5 or CPR catalyzed the oxidation of cyclohexane when both were present. Increasing the cyt b5 diminished the amount of cyclohexanol produced by CPR. Under steady-state conditions, increasing the concentration of cyt b5 progressively inhibits NADPH consumption while product formation increases at cyt b5: P450 ratios ≦ 1 and decreases at cyt b5: P450 ratios ≧ 1. These data are consistent with the hypothesis that cyt b5 binds to P450 and prevents CPR from reducing ferric P450 and initiating catalysis. Present experiments, previous mutagenesis studies, and model complexes suggest that cyt b5 and CPR complete for a binding site on cyt P450 2B4. [ABSTRACT FROM AUTHOR]

Presents abstracts of studies on RNA presented at the Experimental Biology/International Union of Physiological Sciences 2005 Congress in San Diego, California. "Understanding the Role of Phosphorylation in the Nonsense Mediated mRNA Decay Surveillance Mechanism," by Ivan Joel Cajigas, et al; "Differential Tissue Expression of the Vitamin D Regulating Cytochromes CYP224A1 and CYP27B1 Hydroxylase in Pigs During Different Regulatory States," by Charlotte Mobarak, et al; "In Vitro Analysis of RNA Helicases Involved in Ribosome Biogenesis," by Chieyu Lin, et al.

Presents information on several studies on cytochrome p450. Characterization of cytochrome p450; Analysis of heme-oxygen enzymology; Information on the pharmacogenomics of cytochrome p450.

The present study is to investigate the protective effect of dandelion hot water extract (DWE) on liver injury induced by carbon tetrachloride (CCl4) in Sprague-Dawley rats. Dandelion samples were extracted by hot water and lyophilized. The freeze-dried DWE was dissolved in third distilled water and administered to rats by gavage at 500mg/kg, 2g/kg respectively for 7 consecutive days. The acute liver injury was induced by carbon tetrachloride (500ul/kg. p.o.) on the eighth day and 24 hours later all rats are sacrificed. The DWE supplement significantly decreased the serum alanine and aspartate aminotransferase (ALT and AST) activity. Comparing with the CCl4 intoxicated group, the contents of glutathione peroxidase, glutathione reductase and superoxide dismutase increased dose--dependent manner, and mRNA and protein expression levels of cytochrome p450 2E1 significantly decreased in the dandelion administered group. These results indicate that DWE has protective effect on acute liver inflammation induced by CCl4 in rats. [ABSTRACT FROM AUTHOR]

This study was designed to evaluate the detoxification effect of Chlorella vulgaris extract in carbon tetrachloride-induced hepatotoxic Sprague-Dawley rats. Hepatic toxification was evident by hepatic ballooning degeneration, fatty metamorphosis, necrosis, multinuclear cells, and fibrotic changes in histopathological study. Elevated levels of serum aminotransferase and lipids were also observed in these rats. The Chlorella vulgaris extract restored levels of the biochemical markers of the phase I and II detoxification, and hepatic microsomal CYP2B1mRNA with a manner of dose response (p<0.05). We concluded that the Chlorella vulgaris extract is effective in detoxifying carbon tetrachloride-induced hepatotoxicity. Moreover, the effects seem to be relevant to increases in CYP2B1mRNA, cytochrome P450, NADPH-cytochrome P450 reductase, glutathione and glutathione-S-transferase. [ABSTRACT FROM AUTHOR]

Cytochrome b5 reductase (cb5r), a member of the FNR family of flavoprotein transhydrogenases, catalyzes the NADH-dependent reduction of cytochrome b5 (cb5). The putative nucleotide sequence for the D. melanogaster enzyme was used to clone and generate a histidine-tagged form of the soluble, catalytic domain of cb5r. The recombinant protein was purified to homogeneity and exhibited absorption and CD spectra comparable to the wild type rat cb5r diaphorase domain, suggesting similar protein folding and flavin incorporation. Oxidation-reduction potentials in both the absence (-275mV) and presence (-195mV) of NAD+ were comparable to values previously obtained for the corresponding rat domain (-268 mV and -190 mV). Thermal stability studies revealed that the fly domain exhibited decreased thermal stability (49oC) as compared to that of the rat protein (55oC). Initial-rate kinetic studies showed a decreased turnover rate in the fly diaphorase domain, with kcat's of 283s-1 and 268s-1 for the NADH:FR and NADH:BR activities respectively, as compared to values of 800s-1 and 600s-1 for the rat domain. Km's for NADH and cb5 were comparable to that of wild type rat cb5r. These results provide direct comparison of an enzyme from two divergent phylogenetic sources using identical expression systems and indicate a species-based difference in the structural, spectroscopic, kinetic and thermodynamic properties of the enzyme. [ABSTRACT FROM AUTHOR]

Recessive congenital methemoglobinemia is characterised by a deficiency of NADH-cytochrome b5 reductase and exhibits two phenotypes. Both types are associated with cyanosis, type I is benign while type II is accompanied by severe neurological defects. The primary function of cb5r is reduction of methemoglobin. With the development of a heterologous expression system it is possible to characterise novel mutations in terms of their enzymatic function and protein stability. Sequencing the DIA1 gene from patient with apparent type I RCM revealed a heterozygous deletion of D255 in exon 9. In addition, a homozygous C to A change was found in exon 4 predicting an amino acid change of proline to histidine at codon 92. Thus one allele carries a double mutation. Previously, a similar mutation 3 codons away, P95H, was detected in an individual with type II RCM. Characterising the P92H and D255del variants, indicated that the P92H mutation did not dramatically affect the function of cb5r but decreased the thermostability of the protein. In contrast, the P92H and D255- double mutant exhibited decreased enzyme activity, lower affinity towards the NADH co-factor and reduced thermostability. The previously described P95H mutation also exhibited decreased protein stability but not complete loss of function. Consequently, the pathophysiology of RCM appears to be influenced by the residual activity of the individual cb5r variants. [ABSTRACT FROM AUTHOR]

Cytochrome b5 reductase (cb5r), a member of the FNR family of flavoprotein transhydrogenases, catalyzes the NADH-dependent reduction of cytochrome b5. Multiple sequence alignments revealed a conserved "GxGxxP" motif in this family involved in binding reduced pyridine nucleotides. X-ray crystallography shows that this motif is important in binding the adenosine moiety of NADH. To investigate the roles of these residues, a series of alanine variants were generated, recombinantly expressed in E. coli and purified to homogeneity. Each mutant protein incorporated FAD in a 1:1 cofactor/protein stoichiometry and exhibited absorption and CD spectra that were identical to those of wild-type cb5r, indicating both correct protein folding and similar flavin environments. Oxidation/reduction potentials for the FAD/FADH2 couple (n=2) were comparable to wild-type protein (E° = -272 mV). All six mutants showed decreased NADH:FR activities, on the order of WT > I183A > P185A > T181A > G182A > G180A > T184A with T184A retaining only 18% of the wild-type activity. The affinity for NADH was increased in the G180A and T184A variants, with all others showing decreased affinity on the order of G182A > T184A > WT > I183A > P185A > T181A > G180A with the Km NADH for G180A 11-fold greater than that of the wild type. Both Ks H4NAD and Ks NADH values confirmed that the GxGxxP mutants compromised both NADH and NAD+ binding affinities. [ABSTRACT FROM AUTHOR]

In addition to their effects on lipid metabolism and glucose homeostasis, peroxisome proliferators-activated receptor-alpha (PPAR-α) also plays a role in the regulation of vascular tone and blood pressure. In the current study, we assessed the efficacy of the PPAR-α agonist, fenofibrate on blood pressure and kidney function in type II diabetic rats. 26-week-old male Zucker diabetic fatty rats (ZDF, GmiCrl-fa/fa, blood glucose: 525±30 mg/dl) were fed with fenofibrate (100 mg/kg/day) for 5 weeks. Compared to their lean littermates (129±1 mmHg), ZDF rats have higher systolic blood pressure (SBP) (148±4 mmHg). Fenofibrate administration significantly lowered SBP to 129±4 mmHg in ZDF rats, whereas it had no effect on SBP in Zucker lean rats. Renal cortical protein levels of cyclin D1 and CYP2C23 were assessed by Western blot. Expression of renal cyclin D1 protein was upregulated in ZDF rats compared to lean controls and fenofibrate significantly decreased cyclin D1 in kidney cortex of ZDF rats. In contrast, fenofibrate treatment markedly increased the main renal epoxygenase isoform, CYP2C23 protein by 80% in ZDF rats. Interestingly, these fenofibrate effects were observed in the absence of changes in blood glucose, insulin or lipid levels. Taken together, our results demonstrate that PPAR-α agonist fenofibrate decreases arterial blood pressure, which is associated with its upregulation of renal epoxygenase CYP2C23 and downregulation of cyclin D1 protein expression in Zucker diabetic fatty rats. [ABSTRACT FROM AUTHOR]

CYP2A13 is the most efficient P450 in the metabolic activation of NNK. The aim of this study was to determine the extent of variation in the levels of CYP2A13 expression in human lung microsomes, and to identify any correlation between CYP2A13 expression and lung microsomal NNK metabolism. The expression of CYP2A13 protein was examined in adult human lung microsomes, following affinity purification, using an immunoblotting method that allows resolution of CYP2A6 and CYP2A13. CYP2A6 protein was detected in most lung biopsies examined, but the expression of CYP2A13 protein was undetected in many lung biopsy samples that were positive for CYP2A6 protein detection. Quantitative RNA-PCR analysis confirmed that the variable expression of CYP2A6 and CYP2A13 proteins was consistent with variations in the levels of the corresponding mRNAs in the same tissue samples. We also found that lung microsomal samples with detectable CYP2A13 protein had higher rates of NNK metabolic activation than those with no detectable CYP2A13 had, regardless of whether CYP2A6 was detected. These results indicate that there are large interindividual variations in CYP2A13 expression in the lung, and that a higher expression of CYP2A13 protein is associated with higher activities in human lung microsomal NNK metabolic activation, which might lead to a greater susceptibility to smoking-related lung cancer. [ABSTRACT FROM AUTHOR]

In this study, MJ-29(6-pyrrolidinyl-2-(2-substituted phenyl)-4-quinazolinones derivatives) was examined for its growth inhibition effect on U937 human leukemia cell line. After 24 hours treatment of MJ-29,PI incorporation was applied to determine the proliferation rate. A dose and time-dependent decrease in cell proliferation was observed. The cytotoxic effect of MJ-29 on normal cells (HUVEC and peripheral mononuclear cells) was lower than that on U937 cells of treatment. Cell cycle analysis of U937 cells showed that MJ-29 induced significant G2/M arrest and apoptosis. Immuno-fluorescence microscopy and in vitro tubulin assembly assays indicated that MJ-29 binding to tubulin and disrupts microtubule organization. We found that MJ-29 up-regulated the protein levels of p161-CDK1,Chk1,Chk2,p21 and increased cyclin B and CDK1 kinase activity. MJ-29-treated U937 cells presented typical apoptosis by DAPI staining, increase of Annexin V binding, internucleosomal DNA cleavage. In addition, release of cytosolic cytochrome c¡BPro-caspase-9¡BApaf-1 and AIF, down-regulation of Bcl-2, Bcl-xL¡B xIAP¡BclAP-1¡BclAP-2 and up-regulation of phospho-Bcl-2¡BBax¡BBad¡BBak were detected by western blotting. Caspases activity assay and Sub-G1 nuclei analysis of MJ-29-treated U937 indicated that the MJ-29 induced apoptosis was mainly mediated by activation of caspase 9 and 3. Our results indicated that the ability of MJ-29 to inhibit cell proliferation may be mediated by both the induction of mitotic arrest and apoptosis. µ [ABSTRACT FROM AUTHOR]

Preceding studies have shown that cardiac cytochrome c oxidase (CCO) deficiency occurs in the offspring of Cu-deficient rats on postnatal days (PND) 15 and 21. In order to determine if the CCO deficiency resulted from low prenatal Cu intake rather than from low postnatal Cu intake, pups from dams fed AIN93G diet containing 1 mg Cu/kg (CuD dams) were cross fostered to dams fed diet containing 6 mg Cu/kg (CuA dams) and vice versa on PND 1. Pups that remained with their birth dams served as controls. All dams began dietary treatment with either CuD or CuA diets 3 weeks before conception and remained on their respective diets until PND 21. On PND 21, CCO activities (U/mg protein, mean±SEM) in isolated cardiac mitochondria were 3.25±0.06 in pups of CuA dams (N=26), 3.30±0.11 in pups of CuA dams cross fostered to CuD dams (N=8), 2.52±0.10 in pups of CuD dams cross fostered to CuA dams (N=10), and 1.81±0.07 in pups of CuD dams (N=18). CCO activity was lower (P<0.05, Tukey's test) in pups of CuDdams cross fostered to CuA dams compared to pups of CuA dams or pups of CuA dams cross fostered to CuD dams, but higher (P<0.05) compared to pups of CuD dams. Differences in heart Cu concentrations corresponded to the differences in CCO activity. These findings indicate that Cu intake during pregnancy has a greater influence than postnatal Cu intake on cardiac CCO in the first generation. [ABSTRACT FROM AUTHOR]

Introduction: The use of smokeless tobacco (ST) is popular in USA, Europe and South East Asian countries. Apart from oral cancer, the molecular mechanisms involved in other ST-related health effects are unknown. Hence, the effects of chronic use of aqueous extract of 'gutkha' (AEGT) on cytochrome p450 (cyp) mediated genotoxic damage in liver, lung, and kidney of male Wistar rats were studied. Materials and Methods: Animal Experiments were conducted after Institutes Animal Ethical Clearance. The lyophilized AEGT was orally administered to animals at a low-dose (96 mg/kg body weight/day) for 2 and 32 weeks, and at a high-dose (960 mg/kg body weight/day) for 2 weeks. The immunohistochemical expression of cyp1A1, 1A2, 2E1 and GST-μ; myeloperoxidase (MPO) and caspase-3 activity and DNA fragmentation in above organs and plasma TNF-α, protein carbonyls and blood micronuclei formation were measured. Results: Low-dose of AEGT administration for 32 weeks differentially expressed the lung, liver and kidney cyp1A1, 1A2, 2E1 and GST-μ. There was significant increase (p<0.05) in MPO, caspase-3 activity and DNA fragmentation. The extent of this increase was less due to high-dose of AEGT for 2 weeks. High-dose and low-dose AEGT for 2 and 32 weeks significantly enhanced plasma TNF-α by 7% and 23%, protein carbonyls by 4% and 12% respectively. Micronuclei formation in whole blood was increased by 14% in long term group. Conclusion: Chronic use of AEGT induces cytochrome p450's, which mediates genotoxic damage in various organs of rats. The differential damage by AEGT may be due to variable sensitivities and functions carried out by these organs. These changes may be responsible for AEGT-induced pathogenesis. Supported by PGIMER, Chandigarh, India. [ABSTRACT FROM AUTHOR]

Cytochrome P-450 (P450)enzymes play an important role during the drug metabolism due to it catalyzes a great amoung of reactions. However, their use for, in vitro assays has several limitations, the main one is the use of the cofactor (NADPH). In this work the catalytic activity of P450 using NADP+ reduced with silica hydride was evaluated. The results obtained showed that the reduction of NADP+ with silica hydride was concentration and time-dependent. In addition, P450 mantained its activity when NADP+ and silica hydride were added during the reaction, however its activity was less than when NADPH was employed. It is due to the fact that silica hydride is able to reduce not only to NADP+ but also to iron atom from P450, avoiding or diminishing the aniline affinity by the active site. The reduction of the iron atom with silica hydride was corroborated by electronic paramagnetic resonance (EPR). On the other hand, when NADP+ was reduced with silica hydride before being added to the reaction, the P450 activity was better than when NADPH was used. Therefore, it is worthnoting that the use of NADP+ reduced with silica hydride before to be added to the reaction could be a good system for studying biotransformation reactions. [ABSTRACT FROM AUTHOR]

Schering 66712 (SCH66712) is a known mechanism-based inhibitor of human cytochrome P450 2D6. Spectral binding assays with purified recombinant protein indicate that SCH66712 displays classical Type I binding with ferric P450 2D6 with KS of 0.39± 0.10 µM. Incubation of SCH66712 with purified 2D6 and NADPH resulted in a loss of P450 heme-CO difference spectrum. Pyridine hemochrome assays also showed loss of heme over time. SDS-PAGE analysis of inactivation assays with human liver microsomes, recombinant microsomes or reconstituted purified 2D6 demonstrated that 3H- and 14C-labeled SCH66712 were irreversibly bound to the apoprotein in SCH66712 inactivated samples and that the association was NADPH dependent. ESI-LC-MS analysis of the SCH66712 inactivated samples showed that the mass of the inactivated 2D6 apoprotein increased by approximately 380 Da (SCH66712, 338 Da). [ABSTRACT FROM AUTHOR]

Despite the breadth of studies on complexes of the mitochondrial electron transport chain, molecular details of the tendency of these complexes to assemble into supermolecular respirasomes are unclear. Previously we established that in mild detergent extracts of mitochondria from Saccharomyces cerevisiae lacking cardiolipin (crd1 null strains), cytochrome bc1 complex (Complex III) and cytochrome c oxidase (Complex IV) do not form a tetrameric (III2IV2) supermolecular complex as observed in extracts from wild type mitochondria (J. Biol. Chem. 277: 43553-56). This result correlates with the cytochrome c pool-like kinetic behavior in electron transfer between Complexes III and IV in intact mitochondria lacking cardiolipin as compared to their respirasome-like behavior in wild type mitochondria (J. Biol. Chem. 280: 29403-08). In this study classical biochemical techniques were applied to purify the supercomplex (III2IV2) in the active state. Negative stain electron microscopy was used to investigate the structural organization of the supercomplex. Image processing was performed combining tomographic and single particle analysis. These studies revealed the organization of the supercomplex to be a central dimer of III flanked at opposite sides with monomers of IV (IV-III2-IV), suggesting an overall symmetry of the C2 type. The first two authors contributed equally. [ABSTRACT FROM AUTHOR]

CYP2A13.1 has high efficiencies in metabolically activating tobacco-specific nitrosamines. CYP2A13.2 has two sequence variations, Arg25Gln and Arg257Cys. We had previously found that the Arg257Cys change leads to decreases in CYP2A13 activity. In this study, we compared the activities of heterologously expressed CYP2A13.1 and CYP2A13.2 proteins using a number of known CYP2A13.1 substrates, including the tobacco-specific procarcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Our results indicate that CYP2A13.2 is only slightly less active than CYP2A13.1 with all substrates tested. We also found that the *2 allele is associated with a slightly lower allelic expression than that of the *1 allele, and that this decrease in expression may be due to a deletion in the 5′-flanking region of CYP2A13*2. Consistent with the differing allelic expression of the *1 and *2 alleles, total CYP2A13 mRNA levels in *1/*2 lung samples appeared to be lower than those in *1/*1 samples, although large inter-sample variations made it difficult to demonstrate statistical significance of the difference. These findings suggest that the reported association of the *2 allele with decreased incidences of lung adenocarcinoma in smokers may not be solely due to a decrease in CYP2A13 function; other genetic variations in linkage disequilibrium with the *2 allele might also be involved. [ABSTRACT FROM AUTHOR]

CYP2G1 is a cytochrome P450 monooxygenase expressed uniquely in the olfactory mucosa (OM). The lateral nasal gland (LNG), the largest anterior gland in the nasal cavity, is an important organ for the production of odorant binding proteins. Cyp2g1-null mouse was recently found to have an increased resistance to acetaminophen (AP) cytotoxicity in the LNG, even though CYP2G1 is not expressed in that organ. The aim of this study is to identify mechanisms that link CYP2G1 expression in the OM to AP toxicity in the LNG. We found that the increased resistance of LNG to AP toxicity was accompanied by reduced AP levels, and AP-induced decreases in non-protein thiol (NPT) levels, in the LNG. However, in vitro metabolic activation of AP by LNG microsomes was found previously to be unaltered in the Cyp2g1-null mouse. Subsequent microarray analysis identified 18 genes that are differentially expressed in the LNG of Cyp2g1-null and wild-type mice. Among the up-regulated are three genes encoding secretory carrier proteins, including prostaglandin D2 synthase (L-PGDS), androgen binding protein A (Abpa), and androgen binding protein B (Abpb). We propose that the increased expression of these proteins in the LNG of the Cyp2g1-null mice contributes to the reduction in target tissue AP levels and increased resistance to AP toxicity. Furthermore, since the expression of both L-PGDS and Abp is regulated by steroid hormones, which are potential Cyp2G1 substrates in the OM, we propose that the loss of CYP2GI in the OM leads to changes in steroid hormone levels in the LNG, thereby altering the expression of secreted carrier proteins in the LNG. Studies designed to test these hypotheses are underway. [ABSTRACT FROM AUTHOR]

2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MelQx) is one of the most abundant mutagens formed during high temperature cooking of proteinaceous foods. Nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells were constructed with stable transfection of human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. Cells transfected with NAT2*4 had significantly higher levels of SMZ N-acetyltransferase (p < 0.0001) and N-hydroxy-MelQx O-acetyltransferase (p = 0.0093) catalytic activity than cells transfected with NAT2*5B. Only cells transfected with both CYP1A1 and NAT2*4 showed concentration-dependent cytotoxicity and mutagenesis, dG-C8-MelQx DNA adduct levels were dose-dependent in each cell line and in the order: untransfected < transfected with CYP1A1 < transfected with CYP1A1 & NAT2*5B < transfected with CYP1A1 & NAT2*4. At each concentration of MelQx tested, (0.75 µM, 1.5 µM and 3.0 µM), MelQx DNA adduct levels and mutants were significantly higher (p < 0.001) in CYP1A1/NAT2*4 than CYP1A1/NAT2*SB cells. These results strongly support activation of MelQx by CYP1A1 and a robust effect of human NAT2 genetic polymorphism on MelQx --induced mutagenesis and DNA adduct levels. Partially supported by USPHS grant CA034627 from the National Cancer Institute. [ABSTRACT FROM AUTHOR]

We analyzed a time course of dexamethasone (DEX)-induction at PND25 and PND60 in male and female rats fed soy protein isolate (SPI) or casein (CAS) based AIN93G diets throughout development to examine molecular mechanisms underlying increased CYP3A expression and inducibility after SPI-feeding. At PND25, SPI-feeding increased CYP3A1 and CYP3A2 mRNA expression (p < 0.05) only in male liver but increased CYP3A expression (p < 0.05) in both sexes at PND60. After 50 mg/kg DEX, CYP3A1 mRNA expression was induced >200-fold in SPI-fed males and females peaking 17 h post-injection but only 100-150-fold in CAS-fed animals (p < 0.05). A similar increase in DEX-inducibility was observed in CYP3A1 at PND60. CYP3A2 mRNA was also induced more in SPI-fed rats, but DEX-induction was only 2-4-fold. Measurement of steady-state HnRNA for CYP3A1 using RT-PCR with intron-specific primers showed no difference with diet and only 3-4-fold induction peaking at 8 h post-DEX: Quantitation of newly synthesized CYP3A1 RNA transcripts by nuclear run-on analysis using biotinylated nucleotides followed by avidin-bead precipitation and real-time RT-PCR demonstrated dramatic induction 8 h post-DEX, but no effects of diet on constitutive or induced levels of transcription. These data suggest that primary transcripts of CYP3A have a short half life relative to its mature mRNA and that while DEX increases transcription, increased mRNA expression and inducibility following SPI-feeding involves posttranscriptional effects on RNA processing. [ABSTRACT FROM AUTHOR]

Fucoidan is a sulfated polysaccharide from the brown alga, Fucus vesiculous and has been reported to inhibit tumor angiogenesis. The present study examined fucoidan-mediated apoptosis in the HT-29 human colon cancer cell line. Fucoidan substantially decreased viable HT-29 cell numbers and increased apoptotic cell numbers in a dose-dependent manner (0 - 20 µg/mL). Western blot analyses of total cell lysates revealed that fucoidan increased the levels of cleaved caspase-8, -9, -7, and -3, and poly(ADP-ribose) polymerase (PARP). Fucoidan increased the levels of the proapoptotic protein Bak and truncated Bid. In addition, fucoidan increased the translocation of cytochrome c and Smac/Diablo from the mitochondria to the cytoplasm. The levels of X-linked inhibitor of apoptosis protein (XIAP) and survivin were decreased by treatment of fucoidan. The caspase-8 inhibitor Z-IETD-FMK attenuated the fucoidan-induced activation of caspases-8 and -3, increase in tBid, PARP cleavage, and apoptosis. We have demonstrated that the induction of apoptosis by fucoidan in HT-29 human colon cancer cells may be mediated via the activation of the caspase-8 pathway leading to changes in mitochondrial events, which are associated with cytochrome c and Smac/Diablo release and subsequent activation of the caspase-9/ caspase-3 cascade [ABSTRACT FROM AUTHOR]

Despite the breadth of studies on complexes of the mitochondrial electron transport chain, molecular details of the tendency of these complexes to assemble into supermolecular respirasomes are unclear. Previously we established that in mild detergent extracts of mitochondria from Saccharomyces cerevisiae lacking cardiolipin (crd1 null strains), cytochrome bc1 complex (Complex III) and cytochrome c oxidase (Complex IV) do not form a tetrameric (III2IV2) supermolecular complex as observed in extracts from wild type mitochondria (J. Biol. Chem. 277: 43553-56). This result correlates with the cytochrome c pool-like kinetic behavior in electron transfer between Complexes III and IV in intact mitochondria lacking cardiolipin as compared to their respirasome-like behavior in wild type mitochondria (J. Biol. Chem. 280: 29403-08). In this study classical biochemical techniques were applied to purify the supercomplex (III2IV2) in the active state. Negative stain electron microscopy was used to investigate the structural organization of the supercomplex. Image processing was performed combining tomographic and single particle analysis. These studies revealed the organization of the supercomplex to be a central dimer of III flanked at opposite sides with monomers of IV (IV-III2-IV), suggesting an overall symmetry of the C2 type. The first two authors contributed equally. [ABSTRACT FROM AUTHOR]

Presents an abstract of the study "Cytochrome P450 Enzymes and Their Many Roles: The William C. Rose Award Lecture," by Frederick Peter Guengerich, presented at the Experimental Biology/International Union of Physiological Sciences 2005 congress held in San Diego, California. Characteristics of the enzymes; Information on other studies on the enzymes.