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Barbora Mrázová, Markéta Martínková, Václav Martínek, Eva Frei and Marie Stiborová

Optimalization of preparation of apocytochrome b5 utilizing apo-myoglobin

Cytochrome b5 (cyt b5), a component of endoplasmic reticulum membrane, plays a role in modulation of enzymatic activity of some cytochrome P450 (CYP) enzymes. The effect of apo-cytochrome b5 on this enzymatic system has not been investigated in details, because preparation of cyt b5 as a pure protein failed in many laboratories. In order to prepare the native apo-cytochrome b5 in a large scale we utilized a protein with higher affinity toward the heme; the apo-myoglobin from the equine skeletal muscle. In the first step, we extracted heme moiety from the native myoglobin by butanone extraction. Than the effect of pH on spontaneous heme release from both proteins was investigated: purified rabbit cyt b5 as well as equine skeletal muscle myoglobin. The prepared apo-myoglobin was incubated with the cyt b5 and heme transfer was monitored as a shift of absorption maximum from 413 to 409 nm in pH varying between 3-6 (10 mM KH2PO4, pH 3-6). Here, we obtained 43 mg of the equine skeletal muscle apo-myoglobin (43% yield). The optimal pH range for heme transfer from cyt b5 into apo-myoglobin was between 4.2 and 5. Native apo-cytochrome b5 was successfully prepared using procedure described here.

Open access

Marie Stiborová, Jitka Poljaková, Tomáš Eckschlager, Rene Kizek and Eva Frei

DNA and histone deacetylases as targets for neuroblastoma treatment

Neuroblastoma, a tumor of the peripheral sympathetic nervous system, is the most frequent solid extra cranial tumor in children and is a major cause of death from neoplasia in infancy. Still little improvement in therapeutic options has been made, requiring a need for the development of new therapies. In our laboratory, we address still unsettled questions, which of mechanisms of action of DNA-damaging drugs both currently use for treatment of human neuroblastomas (doxorubicin, cis-platin, cyclophosphamide and etoposide) and another anticancer agent decreasing growth of neuroblastomas in vitro, ellipticine, are predominant mechanism(s) responsible for their antitumor action in neuroblastoma cell lines in vitro. Because hypoxia frequently occurs in tumors and strongly correlates with advanced disease and poor outcome caused by chemoresistance, the effects of hypoxia on efficiencies and mechanisms of actions of these drugs in neuroblastomas are also investigated. Since the epigenetic structure of DNA and its lesions play a role in the origin of human neuroblastomas, pharmaceutical manipulation of the epigenome may offer other treatment options also for neuroblastomas. Therefore, the effects of histone deacetylase inhibitors on growth of neuroblastoma and combination of these compounds with doxorubicin, cis-platin, etoposide and ellipticine as well as mechanisms of such effects in human neuroblastona cell lines in vitro are also investigated. Such a study will increase our knowledge to explain the proper function of these drugs on the molecular level, which should be utilized for the development of new therapies for neuroblastomas.

Open access

Lenka Vilímková, Jan Páca, Veronika Kremláčková, Jan Páca and Marie Stiborová

Isolation of cytoplasmic NADPH-dependent phenol hydroxylase and catechol-1,2-dioxygenase from Candida tropicalis yeast

The efficiencies of NADPH-dependent phenol hydroxylase (EC and catechol 1,2-dioxygenase (EC. 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.

Open access

Lucie Bořek-Dohalská, Petr Hodek, Jiří Hudeček and Marie Stiborová

Experimental approaches to evaluate activities of cytochromes P450 3A

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 b5 (Supersomes™), (iv) membranes isolated from of Escherichia coli, containing recombinant human CYP3A4 and cytochrome b5, and (v) purified human CYP3A4 or rabbit CYP3A6 reconstituted with NADPH:CYP reductase with or without cytochrome b5 in liposomes. The most efficient systems oxidizing both compounds were Supersomes™ containing human CYP3A4 and cytochrome b5. 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.

Open access

Jitka Křížková, Kamila Burdová, Marie Stiborová, Vladimír Křen and Petr Hodek

The effects of selected flavonoids on cytochromes P450 in rat liver and small intestine

In recent years, the consumption and use of dietary supplements containing concentrated phytochemicals (e.g. flavonoids) increased dramatically. Flavonoids, as foreign compounds (xenobiotics), have great potential to modulate the activity of cytochrome P450s (CYPs), xenobiotic-metabolizing enzymes involved in the activation and detoxification of food and environmental carcinogens. Thus, the aim of this study was to investigate the effects of model glycosylated and deglycosylated flavonoids on CYPs in rat liver and small intestine, as the two main organs responsible for xenobiotic metabolism, after p.o. administration by gastric gavages. The effects of two glycosylated flavonoids (isoquercitrin, rutin) and their aglycone (quercetin) on CYPs were determined using Western blotting technique and specific activity assays with alkyl-resorufin derivatives. In liver microsomes, a considerable increase of all the measured marker activities (EROD, MROD, PROD) was observed only after isoquercitrin treatment. To evaluate the effects of flavonoids on CYPs along small intestine, the tissue was dissected into proximal (near pylorus), middle and distal parts. Of all the tested compounds, isoquercitrin was the most efficient CYP inducer, namely in the middle part of small intestine. Obtained data demonstrate the different effects of flavonoid glycosides and aglycone on CYP expression in rat liver and small intestine. Since these phytochemicals are xenobiotics, and thus they can increase the human risk of cancer development, their consumption in large quantities should be carefully considered.

Open access

Marie Stiborová, Jiří Hudeček, Eva Frei and Heinz Schmeiser

Contribution of biotransformation enzymes to the development of renal injury and urothelial cancer caused by aristolochic acid: urgent questions, difficult answers

Ingestion of aristolochic acid (AA) is associated with the development of aristolochic acid nephropathy, which is characterized by chronic renal failure, tubulointerstitial fibrosis and urothelial cancer. AA may also cause a similar type of kidney fibrosis with malignant transformation of the urothelium, the Balkan endemic nephropathy. Understanding which enzymes are involved in AA activation and/or detoxication is important in the assessment of a susceptibility to this carcinogen. The most important human enzymes activating AA by simple nitroreduction in vitro are hepatic and renal cytosolic NAD(P)H:quinone oxidoreductase, hepatic microsomal cytochrome P450 1A2 and renal microsomal NADPH:cytcohrome P450 reductase, besides cyclooxygenase, which is highly expressed in urothelial tissue. Despite extensive research, contribution of most of these enzymes to the development of these diseases is still unknown. Hepatic cytochromes P450 were found to detoxicate AA in mice, and thereby protect the kidney from injury. However, which of cytochromes P450 are the most important in this process both in animal models and in humans have not been entirely resolved as yet. In addition, the relative contribution of enzymes found to activate AA to species responsible for induction of urothelial cancer in humans remains still to be resolved.

Open access

Jitka Poljaková, Tomáš Eckschlager, Jana Hřebačková, Jan Hraběta and Marie Stiborová

The comparison of cytotoxicity of the anticancer drugs doxorubicin and ellipticine to human neuroblastoma cells

Ellipticine is an antineoplastic agent, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of covalent DNA adducts mediated by cytochromes P450 and peroxidases. Here, the cytotoxicity of ellipticine to human neuroblastoma derived cell lines IMR-32 and UKF-NB-4 was investigated. Treatment of neuroblastoma cells with ellipticine was compared with that of these cancer cells with doxorubicin. The toxicity of ellipticine was essentially the same as that of doxorubicin to UKF-NB-4 cells, but doxorubicin is much more effective to inhibit the growth of the IMR-32 cell line than ellipticine. Hypoxic conditions used for the cell cultivation resulted in a decrease in ellipticine and/or doxorubicin toxicity to IMR-32 and UKF-NB-4 neuroblastoma cells.

Open access

Martina Svobodová, Helena Dračínská, Markéta Martínková, Jiří Hudeček, Petr Hodek, Eva Frei and Marie Stiborová

Oxidation of carcinogenic 2-nitroanisole by rat cytochromes P450 - similarity between human and rat enzymes

2-Nitroanisole (2-NA) is an important industrial pollutant and a potent carcinogen for rodents. Understanding which cytochrome P450 (CYP) enzymes are involved in its metabolism are important to assess an individual's susceptibility to this environmental carcinogen. The aim of this study was to evaluate the efficiency of rat hepatic CYPs to oxidize 2-NA, to examine the metabolites formed during such an oxidation, and to compare such efficiencies of rat CYPs with those of human. 2-NA is oxidized by rat hepatic microsomes to 2-nitrophenol (2-NP) as the major metabolite, and to 2,6-dihydroxynitrobenzene (2,6-DNB) and 2,5-dihydroxynitrobenzene (2,5-DNB) as the minor products. All these metabolites are suggested as detoxication products. Using hepatic microsomes of rats pre-treated with specific CYP inducers and microsomes from Baculovirus transfected insect cells expressing recombinant rat and human CYP enzymes we found that rat recombinant CYP2E1, 2D2, 2B2, 2C6 and 1A1, as well as orthologous human CYP enzymes are the most efficient enzymes metabolizing 2-NA. However, human CYP1A1 oxidize 2-NA with a higher efficiency than the enzyme of rats. The results show the participation of orthologous CYPs in 2-NA oxidation by both species and underline the suitability of rat species as a model to evaluate human susceptibility to 2-NA.

Open access

Marie Stiborová, Jitka Poljaková, Eva Martínková, Lucie Bořek-Dohalská, Tomáš Eckschlager, Rene Kizek and Eva Frei

Ellipticine cytotoxicity to cancer cell lines - a comparative study

Ellipticine is a potent antineoplastic agent exhibiting multiple mechanisms of action. This anticancer agent should be considered a pro-drug, whose pharmacological efficiency and/or genotoxic side effects are dependent on its cytochrome P450 (CYP)- and/or peroxidase-mediated activation to species forming covalent DNA adducts. Ellipticine can also act as an inhibitor or inducer of biotransformation enzymes, thereby modulating its own metabolism leading to its genotoxic and pharmacological effects. Here, a comparison of the toxicity of ellipticine to human breast adenocarcinoma MCF-7 cells, leukemia HL-60 and CCRF-CEM cells, neuroblastoma IMR-32, UKF-NB-3 and UKF-NB-4 cells and U87MG glioblastoma cells and mechanisms of its action to these cells were evaluated. Treatment of all cells tested with ellipticine resulted in inhibition of cell growth and proliferation. This effect was associated with formation of two covalent ellipticine-derived DNA adducts, identical to those formed by 13-hydroxy- and 12-hydroxyellipticine, the ellipticine metabolites generated by CYP and peroxidase enzymes, in MCF-7, HL-60, CCRF-CEM, UKF-NB-3, UKF-NB-4 and U87MG cells, but not in neuroblastoma UKF-NB-3 cells. Therefore, DNA adduct formation in most cancer cell lines tested in this comparative study might be the predominant cause of their sensitivity to ellipticine treatment, whereas other mechanisms of ellipticine action also contribute to its cytotoxicity to neuroblastoma UKF-NB-3 cells.

Open access

Dagmar Aimová, Jitka Poljaková, Věra Kotrbová, Michaela Moserová, Eva Frei, Volker Arlt and Marie Stiborová

Ellipticine and benzo(a)pyrene increase their own metabolic activation via modulation of expression and enzymatic activity of cytochromes P450 1A1 and 1A2

Two compounds known to covalently bind to DNA after their activation with cytochromes P450 (CYPs), carcinogenic benzo(a)pyrene (BaP) and an antineoplastic agent ellipticine, were investigated for their potential to induce CYP and NADPH:CYP reductase (POR) enzymes in rodent livers, the main target organ for DNA adduct formation. Two animal models were used in the study: (i) rats as animals mimicking the fate of ellipticine in humans and (ii) mice, especially wild-type (WT) and hepatic POR null (HRN™) mouse lines. Ellipticine and BaP induce expression of CYP1A enzymes in livers of experimental models, which leads to increase in their enzymatic activity. In addition, both compounds are capable of generating DNA adducts, predominantly in livers of studied organisms. As determined by 32P postlabelling analysis, levels of ellipticine-derived DNA adducts formed in vivo in the livers of HRN™ mice were reduced (by up to 65%) relative to levels in WT mice, indicating that POR mediated CYP enzyme activity is important for the activation of ellipticine. In contrast to these results, 6.4 fold higher DNA binding of BaP was observed in the livers of HRN™ mice than in WT mice. This finding suggests a detoxication role of CYP1A in BaP metabolism in vivo. In in vitro experiments, DNA adduct formation in calf thymus DNA was up to 25 fold higher in incubations of ellipticine or BaP with microsomes from pretreated animals than with controls. This stimulation effect was attributed to induction of CYP1A1/2 enzymes, which are responsible for oxidative activation of both compounds to the metabolites generating major DNA adducts in vitro. Taken together, these results demonstrate that by inducing CYP1A1/2, ellipticine and BaP modulate their own enzymatic metabolic activation and detoxication, thereby modulating their either pharmacological (ellipticine) and/or genotoxic potential (both compounds).