UNLABELLED: 1. Aristolochic acid I (AAI) is the predominant component in plant extract of Aristolochia genus that is involved in development of aristolochic acid nephropathy, Balkan endemic nephropathy and urothelial cancer. The diseases do not develop in all individuals exposed to AAI and patients exhibit different clinical outcomes. Differences in the activities of enzymes catalyzing the metabolism of AAI might be one of the reasons for this individual susceptibility. 2. Understanding which human enzymes are involved in reductive activation of AAI generating AAI-DNA adducts, and/or its detoxication to the O-demethylated metabolite, aristolochic acid Ia (AAIa), is necessary in the assessment of the susceptibility to this compound. 3. This review summarizes the results of the latest studies utilizing genetically engineered mouse models to identify which human and rodent enzymes catalyze the reductive activation of AAI to AAI-DNA adducts and its oxidative detoxication to AAIa in vivo. 4. The use of hepatic cytochrome P450 (Cyp) reductase null (HRN) mice, in which NADPH:Cyp oxidoreductase (Por) is deleted in hepatocytes, Cyp1a1((-/-)), Cyp1a2((-/-)) single-knockout, Cyp1a1/1a2((-/-)) double-knockout and CYP1A-humanized mice revealed that mouse and human CYP1A1 and 1A2, besides mouse NAD(P)H: quinone oxidoreductase, were involved in the activation of AAI but CYP1A1 and 1A2 also oxidatively detoxified AAI.

• MeSH
• adukty DNA metabolismus   MeSH
• cytochrom P-450 CYP1A1 genetika   metabolismus   MeSH
• cytochrom P-450 CYP1A2 genetika   metabolismus   MeSH
• enzymy genetika   metabolismus   MeSH
• inhibitory enzymů farmakologie   MeSH
• karcinogeny metabolismus   farmakokinetika   MeSH
• kyseliny aristolochové metabolismus   farmakokinetika   toxicita   MeSH
• lidé MeSH
• metabolická inaktivace * MeSH
• myši knockoutované * MeSH
• myši MeSH
• NAD(P)H dehydrogenasa (chinon) antagonisté a inhibitory   MeSH
• NADPH-cytochrom c-reduktasa genetika   metabolismus   MeSH
• nemoci ledvin chemicky indukované   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

BACKGROUND: The enzyme NADPH-P450 oxidoreductase (POR) is the main electron donor to all microsomal CYPs. The possible contribution of common POR variants to inter- and intra-individual variability in drug metabolism is of great pharmacogenetic interest. AIM: To search for POR polymorphic alleles and estimate their frequencies in a Jewish population. MATERIALS & METHODS: We analyzed the POR gene in 301 Ashkenazi and Moroccan Jews. RESULTS: A total of 30 POR SNPs were identified, nine in the noncoding regions and 21 in the protein-coding regions (ten synonymous, 11 missense). Six of these missense variants are previously undescribed (S102P, V164M, V191M, D344N, E398A and D648N). CONCLUSION: The data collected in this study on missense POR SNPs, interpreted in light of the crystallographic structure of human POR, indicate that some POR missense variants may be potential biomarkers for future POR pharmacogenetic screening.

• MeSH
• farmakogenetika MeSH
• frekvence genu MeSH
• genetické markery MeSH
• haplotypy MeSH
• jednonukleotidový polymorfismus MeSH
• lidé MeSH
• missense mutace MeSH
• molekulární modely MeSH
• NADPH-cytochrom c-reduktasa chemie   genetika   MeSH
• sekvenční analýza DNA MeSH
• vazebná nerovnováha MeSH
• židé genetika   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Geografické názvy
• Izrael MeSH
• Maroko MeSH

Aristolochic acid (AA) causes aristolochic acid nephropathy, Balkan endemic nephropathy, and their urothelial malignancies. To identify enzymes involved in the metabolism of aristolochic acid I (AAI), the major toxic component of AA we used HRN (hepatic cytochrome P450 [Cyp] reductase null) mice, in which NADPH:Cyp oxidoreductase (Por) is deleted in hepatocytes. AAI was demethylated by hepatic Cyps in vitro to 8-hydroxy-aristolochic acid I (AAIa), indicating that less AAI is distributed to extrahepatic organs in wild-type (WT) mice. Indeed, AAI-DNA-adduct levels were significantly higher in organs of HRN mice, having low hepatic AAI demethylation capacity, than in WT mice. Absence of AAI demethylation in HRN mouse liver was confirmed in vitro; hepatic microsomes from WT, but not from HRN mice, oxidized AAI to AAIa. To define the role of hepatic Cyps in AAI demethylation, modulation of AAIa formation by CYP inducers was investigated. We conclude that AAI demethylation is attributable mainly to Cyp1a1/2. The higher AAI-DNA adduct levels in HRN than WT mice were the result of the lack of hepatic AAI demethylation concomitant with a higher activity of cytosolic NAD(P)H:quinone oxidoreductase (Nqo1), which activates AAI. Mouse hepatic Cyp1a1/2 also activated AAI to DNA adducts under hypoxic conditions in vitro, but in renal microsomes, Por and Cyp3a are more important than Cyp1a for AAI-DNA adduct formation. We propose that AAI activation and detoxication in mice are dictated mainly by AAI binding affinity to Cyp1a1/2 or Nqo1, by their turnover, and by the balance between oxidation and reduction of AAI by Cyp1a.

• MeSH
• adukty DNA MeSH
• biotransformace MeSH
• cytochrom P-450 CYP1A1 metabolismus   MeSH
• cytochrom P-450 CYP1A2 metabolismus   MeSH
• játra enzymologie   metabolismus   MeSH
• karcinogeny farmakokinetika   MeSH
• krysa rodu rattus MeSH
• kyseliny aristolochové farmakokinetika   MeSH
• lidé MeSH
• metylace MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• NADPH-cytochrom c-reduktasa genetika   metabolismus   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Ellipticine is an antineoplastic agent, which forms covalent DNA adducts mediated by cytochromes P450 (CYP) and peroxidases. We evaluated the role of hepatic versus extra-hepatic metabolism of ellipticine, using the HRN (Hepatic Cytochrome P450 Reductase Null) mouse model, in which cytochrome P450 oxidoreductase (POR) is deleted in hepatocytes, resulting in the loss of essentially all hepatic CYP function. HRN and wild-type (WT) mice were treated i.p. with 1 and 10 mg/kg body weight of ellipticine. Multiple ellipticine-DNA adducts detected by (32)P-postlabelling were observed in organs from both mouse strains. Highest total DNA binding levels were found in liver, followed by lung, kidney, urinary bladder, colon and spleen. Ellipticine-DNA adduct levels in the liver of HRN mice were up to 65% lower relative to WT mice, confirming the importance of CYP enzymes for the activation of ellipticine in livers, recently shown in vitro with human and rat hepatic microsomes. When hepatic microsomes of both mouse strains were incubated with ellipticine, ellipticine-DNA adduct levels with WT microsomes were up to 2.9-fold higher than with those from HRN mice. The ratios of ellipticine-DNA adducts in extra-hepatic organs between HRN and WT mice of up to 4.7 suggest that these organs can activate ellipticine and that more ellipticine is available in the circulation. These results and the DNA adduct patterns found in vitro and in vivo demonstrate that both CYP1A or 3A and peroxidases participate in activation of ellipticine to reactive species forming DNA adducts in the mouse model used in this study.

• MeSH
• adukty DNA analýza   metabolismus   MeSH
• cytochrom P-450 CYP1A1 metabolismus   MeSH
• cytochrom P-450 CYP3A metabolismus   MeSH
• DNA metabolismus   účinky léků   MeSH
• elipticiny metabolismus   toxicita   MeSH
• hepatocyty enzymologie   účinky léků   MeSH
• injekce intraperitoneální MeSH
• izotopové značení metody   MeSH
• jaterní mikrozomy enzymologie   účinky léků   MeSH
• játra enzymologie   účinky léků   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• NADPH-cytochrom c-reduktasa genetika   metabolismus   nedostatek   MeSH
• protinádorové látky metabolismus   toxicita   MeSH
• radioizotopy fosforu MeSH
• umlčování genů MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH

Antley-Bixlerův syndrom (ABS) je vzácné kongenitální onemocnění charakterizované četnými kraniofaciálními, skeletálními a v některých případech i urogenitálními abnormalitami vyplývajícími z porušené steroidogeneze. Donedávna jedinou známou genetickou příčinou u sporadických případů ABS byly dominantní mutace v genu pro receptor pro fibroblastový růstový faktor 2 (FGFR2). Současný výzkum však velmi překvapivě odhalil, že za projevy Antley-Bixlerova syndromu spojeného s poruchami steroidogeneze a urogenitálními anomáliemi stojí mutace v genu POR kódujícím enzym NADPH-cytochrom P450 oxidoreduktázu (CYPOR). CYPOR je flavoprotein, obsahuje dva flaviny, flavin adenin dinukleotid (FAD) a flavin mononukleotid (FMN) a také sekvenci pro vazbu NADPH. Enzym je monomer organizován do čtyř domén. Sekvence 25 aminokyselin v N-terminální části proteinu zodpovídá za mikrozomální lokalizaci proteinu. CYPOR je hlavním elektronovým dárcem pro mikrozomálně lokalizované cytochromy P450, které se účastní metabolismu xenobiotik a biosyntézy steroidních hormonů. Mutace v genu POR vedou k zdánlivé insuficienci některých P450 enzymů. Asociace CYPOR s ABS odhalila nové skutečnosti ohledně tohoto onemocnění. Dle současných výsledků se zdá, že pacienti se skeletálními anomáliemi charakteristickými pro ABS, kteří však nesou mutaci v genu POR a mají porušenou steroidogenezi, představují nové onemocnění, POR deficienci.

Antley-Bixler syndrome (ABS) is a rare congenital disorder characterized by numerous craniofacial, skeletal and, in some cases, urogenital abnormalities resulting from disordered steroidogenesis. Known genetic causes in sporadic cases of ABS include dominant mutations in the fibroblast growth factor 2 receptor gene (FGFR2). Recent research shows surprisingly that symptoms of Antley-Bixler syndrome, combined with disordered steroidogenesis and urogenital anomalies, are caused by mutations in the POR gene that encodes NADPH-cytochrome P450 oxidoreductase (CYPOR). CYPOR is a four domaincontaining monomeric flavoprotein that contains two flavins, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), a binding site for NADPH, and the N-terminal sequence of 25 amino acids which determines the microsomal localization of the protein. CYPOR is the electron donor to microsomally localized cytochromes P450 that participate in xenobiotic metabolism and steroidogenesis. Mutations in the POR gene lead to apparent diminished activity of some P450 enzymes. Association of CYPOR with ABS discloses new facts about this disease and recent research shows that patients with ABS-like skeletal anomalies, but with mutations in the POR gene and disordered steroidogenesis, represent a new disorder called POR deficiency.

• MeSH
• financování organizované MeSH
• kraniofaciální abnormality diagnóza   etiologie   genetika   MeSH
• muskuloskeletální abnormality diagnóza   etiologie   genetika   MeSH
• NADPH-cytochrom c-reduktasa genetika   MeSH
• Southernův blotting metody   využití   MeSH
• urogenitální abnormality diagnóza   etiologie   genetika   MeSH
• vrozené poruchy metabolismu steroidů diagnóza   etiologie   genetika   MeSH

Ingestion of aristolochic acid (AA) is associated with the development of AA-nephropathy and Balkan endemic nephropathy, which are characterized by chronic renal failure, tubulointerstitial fibrosis, and urothelial cancer. Understanding which enzymes are involved in AA activation and/or detoxification is important in assessing susceptibility to AA. Xiao et al. demonstrate that hepatic cytochrome P450s in mice detoxicate AA and thereby protect kidney from injury. The relative contribution of enzymes activating AA to induce urothelial cancer in humans remains to be resolved.

• MeSH
• játra enzymologie   MeSH
• karcinogeny metabolismus   toxicita   MeSH
• kyseliny aristolochové metabolismus   toxicita   MeSH
• metabolická inaktivace MeSH
• myši MeSH
• NADPH-cytochrom c-reduktasa genetika   metabolismus   MeSH
• nemoci ledvin enzymologie   chemicky indukované   MeSH
• urologické nádory enzymologie   chemicky indukované   MeSH
• urotel MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• komentáře MeSH

Cryptosporidium parvum contains a unique fusion protein pyruvate:NADP+ oxidoreductase (CpPNO) that is composed of two distinct, conserved domains, an N-terminal pyruvate:ferredoxin oxidoreductase (PFO) and a C-terminal cytochrome P450 reductase (CPR). Unlike a similar fusion protein that localizes to the mitochondrion of the photosynthetic protist Euglena gracilis, CpPNO lacks an N-terminal mitochondrial targeting sequence. Using two distinct polyclonal antibodies raised against CpPFO and one polyclonal antibody against CpCPR, Western blot analysis has shown that sporozoites of C. parvum express the entire CpPNO fusion protein. Furthermore, confocal immunofluorescence and transmission electron microscopy confirm that CpPNO is localized within the cytosol rather than the relict mitochondrion of C. parvum. The distribution of this protein is not, however, strictly confined to the cytosol. CpPNO also appears to localize posteriorly within the crystalloid body.

• MeSH
• Cryptosporidium parvum cytologie   enzymologie   genetika   MeSH
• cytosol enzymologie   MeSH
• Euglena gracilis cytologie   enzymologie   MeSH
• financování organizované MeSH
• fluorescenční mikroskopie MeSH
• ketonoxidoreduktasy analýza   genetika   imunologie   MeSH
• konfokální mikroskopie MeSH
• NADPH-cytochrom c-reduktasa analýza   genetika   imunologie   MeSH
• organely enzymologie   MeSH
• protozoální proteiny analýza   MeSH
• pyruvátsynthasa analýza   genetika   imunologie   MeSH
• sporozoiti cytologie   enzymologie   MeSH
• transmisní elektronová mikroskopie MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH

The development of bicistronic systems for coexpression of recombinant human cytochrome P450 enzymes (P450s) with their redox partner, NADPH-cytochrome P450 reductase (NPR), has enabled P450 activity to be reconstituted within bacterial cells. During expression of recombinant P450 2E1 and some other forms, we observed the formation of a blue pigment in bacterial cultures. The pigment was extracted from cultures and shown to comigrate with standard indigo on TLC. UV-visible spectroscopy and mass spectrometric analysis provided further support for identification of the pigment as indigo. Indigo is known to form following the spontaneous oxidation of 3-hydroxyindole. Accordingly, we speculated that indole, formed as a breakdown product of tryptophan in bacteria, was hydroxylated by the P450 system, leading to indigo formation. Bacterial membranes containing recombinant P450 2E1 and human NPR were incubated in vitro with indole and shown to catalyze formation of a blue pigment in a time- and cofactor-dependent manner. These studies suggest potential applications of mammalian P450 enzymes in industrial indigo production or in the development of novel colorimetric assays based on indole hydroxylation. Copyright 1999 Academic Press.

• MeSH
• biologické pigmenty * metabolismus   MeSH
• cytochrom P-450 CYP2E1 genetika   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• geny MeSH
• indigotindisulfonát sodný MeSH
• indoly * metabolismus   MeSH
• lidé MeSH
• NADPH-cytochrom c-reduktasa genetika   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• systém (enzymů) cytochromů P-450 genetika   metabolismus   MeSH
• techniky in vitro MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH