Metabolism of Carcinogenic Azo Dye Sudan I by Rat, Rabbit, Minipig and Human Hepatic Microsomes

2002 ◽  
Vol 67 (12) ◽  
pp. 1883-1898 ◽  
Author(s):  
Václav Martínek ◽  
Marie Stiborová
Keyword(s):  
Cytochromes P450 ◽  
Human Health Risk ◽  
Enzymatic System ◽  
Cytochrome P450 1A1 ◽  
Predominant Role ◽  
Hepatic Microsomes ◽  
Sudan I ◽  
Microsomal Enzymatic System

We investigated the ability of hepatic microsomal samples from different species including human to metabolize rodent carcinogen Sudan I (C.I. Solvent Yellow 14, 1-(phenylazo)-2-naphthol). A comparison between experimental animals and the human microsomal enzymatic system is essential for the extrapolation of animal carcinogenicity data to assess human health risk. Major metabolites produced from Sudan I by microsomes of all species were C-hydroxylated derivatives identified as 1-[(4-hydroxyphenyl)azo]-2-naphthol and 1-(phenylazo)naphthalene-2,6-diol. Additional minor C-hydroxylated products of Sudan I oxidation were 1-[(4-hydroxyphenyl)azo]naphthalene-2,6-diol and 1-[(3,4-dihydroxyphenyl)- azo]-2-naphthol. Human microsomes generated the pattern of Sudan I metabolites reproducing that formed by hepatic microsomes of rats. While microsomes of rabbit and minipig favored the production of the metabolite hydroxylated in position 6 of the naphthol ring of the Sudan I molecule, those of human and rat predominantly produced 1-[(4-hydroxyphenyl)azo]-2-naphthol. Therefore, rat microsomes are a suitable in vitro system mimicking the metabolism of Sudan I in humans. To define the role of specific cytochromes P450 in the Sudan I metabolism by rat microsomes, we investigated the modulation of Sudan I oxidation by specific inducers and selective inhibitors of these enzymes. The results suggest that cytochromes P450 1A1 and 3A are responsible for Sudan I metabolism by rat microsomes. Using purified enzymes, their ability to oxidize Sudan I was confirmed. The data clearly demonstrate the predominant role of cytochrome P450 1A1 in the Sudan I metabolism and suggest a carcinogenic potency of this rodent carcinogen for humans.

Effect of Ginkgo biloba extract on rat hepatic microsomal CYP1A activity: role of ginkgolides, bilobalide, and flavonols

2004 ◽  
Vol 82 (1) ◽  
pp. 57-64 ◽  
Author(s):  
I fan Kuo ◽  
Jie Chen ◽  
Thomas K.H Chang
Keyword(s):  
Ginkgo Biloba ◽  
Ginkgo Biloba Extract ◽  
Inhibitory Potency ◽  
Hepatic Microsomes ◽  
Cyp1a Activity ◽  
Ginkgo Biloba Extracts ◽  
The Individual ◽  
Vitro Effect

The present study investigated the in vitro effect of Ginkgo biloba extracts and some of the individual constituents (ginkgolides, bilobalide, and flavonols such as kaempferol, quercetin, isorhamnetin, and their glycosides) on CYP1A-mediated 7-ethoxyresorufin O-dealkylation in hepatic microsomes isolated from rats induced with β-naphthoflavone. G. biloba extract competitively inhibited CYP1A activity, with an apparent Ki value of 1.6 ± 0.4 µg/mL (mean ± SE). At the concentrations present in the G. biloba extracts, ginkgolides A, B, C, and J and bilobalide did not affect CYP1A activity, whereas kaempferol (IC50 = 0.006 ± 0.001 µg/mL, mean ± SE), isorhamnetin (0.007 ± 0.001 µg/mL), and quercetin (0.050 ± 0.003 µg/mL) decreased this activity. The monoglycosides (1 and 10 µg/mL) and diglycosides (10 µg/mL) of kaempferol and quercetin but not those of isorhamnetin also inhibited CYP1A activity. The order of inhibitory potency was kaempferol ~ isorhamnetin > quercetin, and for each of these flavonols the order of potency was aglycone >> monoglycoside > diglycoside. In summary, G. biloba extract competitively inhibited rat hepatic microsomal CYP1A activity, but the effect was not due to ginkgolides A, B, C, or J, bilobalide, kaempferol, quercetin, isorhamnetin, or the respective flavonol monoglycosides or diglycosides.Key words: bilobalide, CYP1A, cytochrome P450, Ginkgo biloba, ginkgolide, flavonol.

The Possible Role of Hydroxylation in the Detoxification of Atrazine in Mature Vetiver (Chrysopogon zizanioides Nash) Grown in Hydroponics

2005 ◽  
Vol 60 (5-6) ◽  
pp. 427-434 ◽  
Author(s):  
Sylvie Marcacci ◽  
Muriel Raveton ◽  
Patrick Ravanel ◽  
Jean-Paul Schwitzguébel
Keyword(s):  
Cytochromes P450 ◽  
Resistance Mechanism ◽  
Uv Spectra ◽  
Chrysopogon Zizanioides ◽  
Retention Factors ◽  
Glutathione S Transferases ◽  
Layer Chromatography ◽  
Major Metabolic Pathway

The resistance mechanism of vetiver (Chrysopogon zizanioides) to atrazine was investigated to evaluate its potential for phytoremediation of environment contaminated with the herbicide. Plants known to metabolise atrazine rely on hydroxylation mediated by benzoxazinones, conjugation catalyzed by glutathione-S-transferases and dealkylation probably mediated by cytochromes P450. All three possibilities were explored in mature vetiver grown in hydroponics during this research project. Here we report on the chemical role of benzoxazinones in the transformation of atrazine.Fresh vetiver roots and leaves were cut to extract and study their content in benzoxazinones known to hydroxylate atrazine, such as 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)- one (DIBOA), 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) and their mono- and di-glucosylated forms. Identification of benzoxazinones was performed by thin layer chromatography (TLC) and comparison of retention factors (Rf) and UV spectra with standards: although some products exhibited the same Rf as standards, UV spectra were different. Furthermore, in vitro hydroxylation of atrazine could not be detected in the presence of vetiver extracts. Finally, vetiver organs exposed to [14C]-atrazine did not produce any significant amount of hydroxylated products, such as hydroxyatrazine (HATR), hydroxydeethylatrazine (HDEA), and hydroxy-deisopropylatrazine (HDIA). Altogether, these metabolic features suggest that hydroxylation was not a major metabolic pathway of atrazine in vetiver.

scholarly journals Cytochrome P450-mediated metabolism of N-(2-methoxyphenyl)-hydroxylamine, a human metabolite of the environmental pollutants and carcinogens o-anisidine and o-nitroanisole

2008 ◽  
Vol 1 (3-4) ◽  
pp. 218-224 ◽  
Author(s):  
Karel Naiman ◽  
Helena Dračínská ◽  
Martin Dračínský ◽  
Markéta Martínková ◽  
Václav Martínek ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Cytochromes P450 ◽  
Environmental Pollutants ◽  
Human Metabolite ◽  
Hepatic Microsomes ◽  
Minor Metabolite ◽  
Reactive Compound ◽  
A Minor

Cytochrome P450-mediated metabolism ofN-(2-methoxyphenyl)-hydroxylamine, a human metabolite of the environmental pollutants and carcinogenso-anisidine ando-nitroanisoleN-(2-methoxyphenyl)hydroxylamine is a human metabolite of the industrial and environmental pollutants and bladder carcinogens 2-methoxyaniline (o-anisidine) and 2-methoxynitrobenzene (o-nitroanisole). Here, we investigated the ability of hepatic microsomes from rat and rabbit to metabolize this reactive compound. We found thatN-(2-methoxyphenyl)hydroxylamine is metabolized by microsomes of both species mainly too-aminophenol and a parent carcinogen,o-anisidine, whereas 2-methoxynitrosobenzene (o-nitrosoanisole) is formed as a minor metabolite. AnotherN-(2-methoxyphenyl)hydroxylamine metabolite, the exact structure of which has not been identified as yet, was generated by hepatic microsomes of rabbits, but its formation by those of rats was negligible. To evaluate the role of rat hepatic microsomal cytochromes P450 (CYP) inN-(2-methoxyphenyl)hydroxylamine metabolism, we investigated the modulation of its metabolism by specific inducers of these enzymes. The results of this study show that rat hepatic CYPs of a 1A subfamily and, to a lesser extent those of a 2B subfamily, catalyzeN-(2-methoxyphenyl)hydroxylamine conversion to form both its reductive metabolite,o-anisidine, ando-aminophenol. CYP2E1 is the most efficient enzyme catalyzing conversion ofN-(2-methoxyphenyl)hydroxylamine too-aminophenol.

scholarly journals Distinct Roles for Two CYP226 Family Cytochromes P450 in Abietane Diterpenoid Catabolism by Burkholderia xenovorans LB400

2007 ◽  
Vol 190 (5) ◽  
pp. 1575-1583 ◽  
Author(s):  
Daryl J. Smith ◽  
Marianna A. Patrauchan ◽  
Christine Florizone ◽  
Lindsay D. Eltis ◽  
William W. Mohn
Keyword(s):  
Cytochromes P450 ◽  
Degradation Pathway ◽  
Dehydroabietic Acid ◽  
Burkholderia Xenovorans ◽  
Substrate Removal ◽  
Burkholderia Xenovorans Lb400 ◽  
Abietane Diterpenoids ◽  
Insertion Mutants

ABSTRACT The 80-kb dit cluster of Burkholderia xenovorans LB400 encodes the catabolism of abietane diterpenoids. This cluster includes ditQ and ditU, predicted to encode cytochromes P450 (P450s) belonging to the poorly characterized CYP226A subfamily. Using proteomics, we identified 16 dit-encoded proteins that were significantly more abundant in LB400 cells grown on dehydroabietic acid (DhA) or abietic acid (AbA) than in succinate-grown cells. A key difference in the catabolism of DhA and AbA lies in the differential expression of the P450s; DitU was detected only in the AbA-grown cells, whereas DitQ was expressed both during growth on DhA and during growth on AbA. Analyses of insertion mutants showed that ditQ was required for growth on DhA, ditU was required for growth on AbA, and neither gene was required for growth on the central intermediate, 7-oxo-DhA. In cell suspension assays, patterns of substrate removal and metabolite accumulation confirmed the role of DitU in AbA transformation and the role of DitQ in DhA transformation. Spectral assays revealed that DitQ binds both DhA (dissociation constant, 0.98 ± 0.01 μM) and palustric acid. Finally, DitQ transformed DhA to 7-hydroxy-DhA in vitro. These results demonstrate the distinct roles of the P450s DitQ and DitU in the transformation of DhA and AbA, respectively, to 7-oxo-DhA in a convergent degradation pathway.

scholarly journals Nitric oxide is a mediator of the decrease in cytochrome P450-dependent metabolism caused by immunostimulants

1993 ◽  
Vol 90 (23) ◽  
pp. 11147-11151 ◽  
Author(s):  
O G Khatsenko ◽  
S S Gross ◽  
A B Rifkind ◽  
J R Vane
Keyword(s):  
Nitric Oxide ◽  
Cytochrome P450 ◽  
Nitric Oxide Synthase ◽  
Cytochromes P450 ◽  
Hepatic Metabolism ◽  
Spectral Studies ◽  
Rat Liver Microsomes ◽  
Hepatic Microsomes ◽  
Oxide Synthase

Bacterial lipopolysaccharide (LPS) and a diverse array of other immunostimulants and cytokines suppress the metabolism of endogenous and exogenous substances by reducing activity of the hepatic cytochrome P450 mixed-function oxidase system. Although this effect of immunostimulants was first described almost 40 yr ago, the mechanism is obscure. Immunostimulants are now known to cause NO overproduction by cells via induction of nitric oxide synthase. We have investigated whether NO overproduction is involved in suppressing hepatic metabolism by LPS. In vitro treatment of hepatic microsomes with NO, produced by chemical decomposition of 3-morpholinosydnonimine or by nitric oxide synthase, substantially suppressed cytochrome P450-dependent oxygenation reactions. This effect of NO was seen with hepatic microsomes prepared from two species (rat and chicken) and after exposure to chemicals that induce distinct molecular isoforms of cytochromes P450 (beta-naphthoflavone, 3-methylcholanthrene, and phenobarbital). Spectral studies indicate that NO reacts in vitro with both Fe(2+)- and Fe(3+)-hemes in microsomal cytochromes P450. In vivo, LPS diminished the phenobarbital-induced dealkylation of 7-pentoxyresorufin by rat liver microsomes and reduced the apparent P450 content as measured by CO binding. These LPS effects were associated with induction of NO synthesis; LPS-induced NO synthesis showed a strong positive correlation with the severity of cytochrome P450 inhibition. The decrease in both hepatic microsomal P450 activity and CO binding caused by LPS was largely prevented by the selective NO synthase inhibitor N omega-nitro-L-arginine methyl ester. Our findings implicate NO over-production as a major factor mediating the suppression of hepatic metabolism by immunostimulants such as LPS.

Cytochrome P-450 from Tulip Bulbs (Tulipa fosteriana L.) Oxidizes an Azo Dye Sudan I (1-Phenylazo-2-hydroxynaphthalene, Solvent Yellow 14) in vitro

1996 ◽  
Vol 61 (11) ◽  
pp. 1689-1696
Author(s):  
Marie Stiborová ◽  
Hana Hansíková
Keyword(s):  
Azo Dye ◽  
Microsomal Fraction ◽  
Point Of View ◽  
Transport Chain ◽  
Sudan I ◽  
Tulip Bulbs ◽  
Metabolism Of Xenobiotics ◽  
Cytochrome P 450

The microsomal fraction from tulip bulbs (Tulipa fosteriana L.) contains cytochrome P-450 enzymes catalyzing the NADPH-dependent oxidation of the xenobiotic substrate, an azo dye Sudan I (1-phenylazo-2-hydroxynaphthalene, Solvent Yellow 14). C-Hydroxy derivatives [1-(4-hydroxyphenylazo)-2-hydroxynaphthalene, 1-phenylazo-2,6-dihydroxynaphthalene, 1-(4-hydroxyphenylazo)-2,6-dihydroxynaphthalene] and the benzenediazonium ion are the products of the Sudan I oxidation. The oxidation of Sudan I has also been assessed in a reconstituted electron-transport chain with the isolated cytochrome P-450, isolated plant NADPH-cytochrome P-450 reductase and phospholipid. The results are discussed from the point of view of the role of cytochromes P-450 in the metabolism of xenobiotics in plants.

Cytochrome P450 3A activities and their modulation by α-naphthoflavone in vitro are dictated by the efficiencies of model experimental systems

2010 ◽  
Vol 75 (2) ◽  
pp. 201-220 ◽  
Author(s):  
Lucie Bořek-Dohalská ◽  
Marie Stiborová
Keyword(s):  
Cytochrome B ◽  
Cytochromes P450 ◽  
Cytochrome P450 3A ◽  
Experimental Conditions ◽  
Experimental Systems ◽  
Hepatic Microsomes ◽  
Enzymatic Systems ◽  
Testosterone Hydroxylation ◽  
Hydroxylated Metabolite

The knowledge on efficiencies of different in vitro systems containing cytochromes P450 (CYP) of a 3A subfamily is crucial to screen potential substrates of these CYPs. We evaluated and compared efficiencies of several in vitro CYP3A enzymatic systems to oxidize the model substrates, α-NF and testosterone, under the standardized experimental conditions. Five CYP3A systems were tested: (i) human hepatic microsomes rich in CYP3A4, (ii) hepatic microsomes of rabbits treated with a CYP3A6 inducer, rifampicine, (iii) microsomes of Baculovirus transfected insect cells containing recombinant human CYP3A4 and NADPH:CYP reductase with or without cytochrome b5 (SupersomesTM), (iv) membranes isolated from Escherichia coli, containing recombinant human CYP3A4, NADPH:CYP reductase and cytochrome b5, and (v) human CYP3A4 or rabbit CYP3A6 reconstituted with NADPH:CYP reductase with or without cytochrome b5 in liposomes. All systems oxidize testosterone to its 6β-hydroxylated metabolite and α-NF to trans-7,8-dihydrodiol and 5,6-epoxide. The most efficient systems oxidizing both compounds were CYP3A4-SupersomesTM containing cytochrome b5, followed by human hepatic microsomes. This finding suggests these systems to be suitable for general evaluating a variety of compounds as potential substrates of CYP3A4. The lowest efficiencies to oxidize α-NF and testosterone were found for CYP3A4 expressed in membranes of E. coli, and for reconstituted CYP3A4 or CYP3A6. Utilizing the tested enzymatic systems, we also explain here the discrepancies, which showed previously the controversial effects of α-NF on CYP3A-mediated reactions. We demonstrate that inhibition or stimulation of the CYP3A-mediated testosterone hydroxylation by α-NF is dictated by efficiencies of individual enzymatic systems to oxidize the CYP3A substrates.

scholarly journals Regulation of aryl hydrocarbon receptor activity in porcine cumulus–oocyte complexes in physiological and toxicological conditions: the role of follicular fluid

Reproduction ◽  
2007 ◽  
Vol 133 (5) ◽  
pp. 887-897 ◽  
Author(s):  
Daniela Nestler ◽  
Michaela Risch ◽  
Bernd Fischer ◽  
Paola Pocar
Keyword(s):  
Follicular Fluid ◽  
Cumulus Cells ◽  
Cell Types ◽  
Protective Role ◽  
Cytochrome P450 1A1 ◽  
Aryl Hydrocarbon ◽  
Arylhydrocarbon Receptor ◽  
Aryl Hydrocarbon Receptor Activity

The arylhydrocarbon receptor (AhR) mediates the adverse effects of dioxin-like compounds. However, it has also been reported that the AhR may exert a role in ovarian physiology. In the present study, porcine cumulus–oocyte complexes (COCs) were matured in vitro in the presence of 10% follicular fluid. Expression of AhR and its partner, AhR nuclear translocator occurs in immature COCs. After in vitro maturation (IVM), an up-regulation of AhR and cytochrome P450 1A1 (CYP1A1; the main AhR-target gene) was observed. To explore the role of the AhR during IVM, we exposed the COCs to 50 μM β-napthoflavone (βNF). The treatment induced a marked up-regulation of CYP1A1 mRNA, indicating both constitutive and inducible AhR activity. However, in contrast to what was observed in other cell types, no sign of toxicity was observed in COCs. To investigate if components of porcine follicular fluid may exert a protective role against AhR ligands, we exposed porcine COCs to βNF, in the absence of follicular fluid. In these conditions, a marked decrease in the percentage of matured oocytes, concomitant with an increase in oocyte degeneration, was observed. Furthermore, βNF increased apoptosis in cumulus cells in the absence of follicular fluid, whereas βNF has no effects when COCs were treated in the presence of porcine follicular fluid (pFF). In conclusion, these results suggest the presence of unknown endogenous AhR-ligand(s) during porcine IVM and that a dysregulation of this mechanism may result in ovotoxicity by inducing apoptosis in cumulus cells. However, this phenomenon is interrupted by the presence of follicular fluid, indicating a putative protective role for follicular fluid components against exogenous insults.

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