ABSTRACT: The microsomal protein cytochrome b5 , which is located in the membrane of the endoplasmic reticulum, has been shown to modulate many reactions catalyzed by cytochrome P450 (CYP) enzymes. We investigated the influence of exposure to the anticancer drug ellipticine and to two environmental carcinogens, benzo[a]pyrene (BaP) and 1-phenylazo-2-naphthol (Sudan I), on the expression of cytochrome b5 in livers of rats, both at the mRNA and protein levels. We also studied the effects of these compounds on their own metabolism and the formation of DNA adducts generated by their activation metabolite(s) in vitro. The relative amounts of cytochrome b5 mRNA, measured by real-time polymerase chain reaction analysis, were induced by the test compounds up to 11.7-fold in rat livers. Western blotting using antibodies raised against cytochrome b5 showed that protein expression was induced by up to sevenfold in livers of treated rats. Microsomes isolated from livers of exposed rats catalyzed the oxidation of ellipticine, BaP, and Sudan I and the formation of DNA adducts generated by their reactive metabolite(s) more effectively than hepatic microsomes isolated from control rats. All test compounds are known to induce CYP1A1. This induction is one of the reasons responsible for increased oxidation of these xenobiotics by microsomes. However, induction of cytochrome b5 can also contribute to their enhanced metabolism.

• Keywords
• DNA, Drug research, Enzymes, High pressure liquid chromatography,
• Publication type
• Journal Article MeSH

Sudan I [1-(phenylazo)-2-hydroxynaphthalene, C.I. Solvent Yellow 14, CAS No: 842-07-9] is used as the compound employed in chemical industry and to color materials such as hydrocarbon solvents, oils, fats, waxes, plastics, printing inks, shoe and floor polishes and gasoline. Such a wide used could result in a considerable human exposure. Sudan I is known to cause developments of tumors in the liver or urinary bladder in rats, mice, and rabbits, and is considered a possible weak human carcinogen and mutagen. This carcinogen is also a potent contact allergen and sensitizer. Here, we compare the data concerning the Sudan I oxidative metabolism catalyzed by cytochrome P450 (CYP) and peroxidase enzymes, which has been investigated in our laboratory during the last two decades. These two types of enzymes are responsible both for Sudan I detoxication and activation. Among the Sudan I metabolites, C-hydroxylated derivatives and a dimer of Sudan I are suggested to be the detoxication metabolites formed by CYPs and peroxidases, respectively. Metabolic activation of Sudan I by both types of enzymes leads to formation of reactive species (the benzenediazonium ion by CYP and Sudan I radicals by peroxidase) that bind to DNA and RNA, generating covalent adducts in vitro and in vivo. Whereas the structure of the major adduct formed by the benzenediazonium ion in DNA has already been identified to be the 8-(phenylazo)guanine adduct, the structures of adducts formed by peroxidase, have not been characterized as yet. Biological significance of the DNA adducts of Sudan I activated with CYP and peroxidase enzymes and further aims of investigations in this field are discussed in this study.

• Keywords
• Sudan I, carcinogenic azo dye, cytochrome P450, oxidative activation, peroxidase,
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• Journal Article MeSH

The efficiencies of NADPH-dependent phenol hydroxylase (EC 1.14.13.7) and catechol 1,2-dioxygenase (EC.1.13.11.1) in biodegradation of phenol in the cytosolic fraction isolated from yeast Candida tropicalis were investigated. Enzymatic activities of both NADPH-dependent phenol hydroxylase and catechol 1,2-dioxygenase were detected in the cytosolic fraction of C. tropicalis grown on medium containing phenol. Using the procedure consisting of chromatography on DEAE-Sepharose, fractionation by polyethylene glycol 6000 and gel permeation chromatography on Sepharose 4B the enzyme responsible for phenol hydroxylation in cytosol, NADPH-dependent phenol hydroxylase, was isolated from the cytosolic fraction of C. tropicalis close to homogeneity. However, fractionation with polyethylene glycol 6000 lead to a decrease in catechol 1,2-dioxygenase activity. Therefore, another procedure was tested to purify this enzyme. Gel permeation chromatography of proteins of the eluate obtained by chromatography on a DEAE-Sepharose column was utilized to separate phenol hydroxylase and catechol 1,2-dioxygenase. Among gel permeation chromatography on columns of Sephadex G-100, Sephacryl S-300 and Sepharose 4B tested for their efficiencies to isolate phenol hydroxylase and catechol 1,2-dioxygenase, that on Sephacryl S-300 was found to be suitable for such a procedure. Nevertheless, even this chromatographic method did not lead to obtain catechol 1,2-dioxygenase in sufficient amounts and purity for its further characterization. The data demonstrate the progress in resolving the enzymes responsible for the first two steps of phenol degradation by the C. tropicalis strain.

• Keywords
• Candida tropicalis, NADPH-dependent phenol hydroxylase, biodegradation, catechol-1,2-dioxygenase, environmental pollutants, phenol, yeast,
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• Journal Article MeSH

N-(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 that N-(2-methoxyphenyl)hydroxylamine is metabolized by microsomes of both species mainly to o-aminophenol and a parent carcinogen, o-anisidine, whereas 2-methoxynitrosobenzene (o-nitrosoanisole) is formed as a minor metabolite. Another N-(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) in N-(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, catalyze N-(2-methoxyphenyl)hydroxylamine conversion to form both its reductive metabolite, o-anisidine, and o-aminophenol. CYP2E1 is the most efficient enzyme catalyzing conversion of N-(2-methoxyphenyl)hydroxylamine to o-aminophenol.

• Keywords
• N-(2-methoxyphenyl)hydroxylamine, cytochrome P450, metabolism, o-anisidine, oxidation,
• Publication type
• Journal Article MeSH

Cytochrome P450 (CYP) is a heme protein oxidizing various xenobiotics, as well as endogenous substrates. Understanding which CYP enzymes are involved in metabolic activation and/or detoxication of different compounds is important in the assessment of an individual's susceptibility to the toxic action of these substances. Therefore, investigation which of several in vitro experimental models are appropriate to mimic metabolism of xenobiotics in organisms is the major challenge for research of many laboratories. The aim of this study was to evaluate the efficiency of different in vitro systems containing individual enzymes of the mixed-function monooxygenase system to oxidize two model substrates of CYP3A enzymes, exogenous and endogenous compounds, α-naphtoflavone (α-NF) and testosterone, respectively. Several different enzymatic systems containing CYP3A enzymes were utilized in the study: (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 b(5) (Supersomes™), (iv) membranes isolated from of Escherichia coli, containing recombinant human CYP3A4 and cytochrome b(5), and (v) purified human CYP3A4 or rabbit CYP3A6 reconstituted with NADPH:CYP reductase with or without cytochrome b(5) in liposomes. The most efficient systems oxidizing both compounds were Supersomes™ containing human CYP3A4 and cytochrome b(5). The results presented in this study demonstrate the suitability of the supersomal CYP3A4 systems for studies investigating oxidation of testosterone and α-NF in vitro.

• Keywords
• cytochrome P450 3A, metabolism, α-naphthoflavone,
• Publication type
• Journal Article MeSH

About biological affecting of flavonoids on animal organisms is known less,thus we selected flavonoids, flavanones and flavones, and their glycosides, which wereexamined as potential inducers of cytochrome(s) P450 when administrated by gavages intoexperimental male rats. The study was focused on induction of CYP1A1, the majorcytochrome P450 involved in carcinogen activation. The data obtained demonstrate thenecessity of taking into account not only ability of flavonoids to bind to Ah receptor(induction factor) but also to concentrate on their distribution and metabolism (includingcolon microflora) in the body. After that we examined certain flavonoids as potential inducers of cytochrome P450, we wanted to suggest and optimize suitable electrochemical technique for determination of selected flavonoids (quercetin, quercitrin, rutin, chrysin and diosmin) in body liquids. For these purposes, we selected square wave voltannetry using carbon paste electrode. Primarily we aimed on investigation of their basic electrochemical behaviour. After that we have optimized frequency, step potential and supporting electrolyte. Based on the results obtained, we selected the most suitable conditions for determination of the flavonoids as follows: frequency 180 Hz, step potential 1.95 mV/s and phosphate buffer of pH 7 as supporting electrolyte. Detection limits (3 S/N) of the flavonoids were from units to tens of nM except diosmin, where the limit were higher than μM. In addition, we attempted to suggest a sensor for analysis of flavonoids in urine. It clearly follows from the results obtained that flavonoids can be analysed in the presence of animal urine, because urine did not influence much the signals of flavonoids (recoveries of the signals were about 90 %).

• Keywords
• antioxidant, cancer, carbon paste electrode, cytochrome P450, flavonoids, square wave voltammetry, western blot analysis,
• Publication type
• Journal Article MeSH