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  • Author: Markéta Martínková x
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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

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, Karel Naiman, Markéta Martínková, Václav Martínek, Martina Svobodová, Heinz Schmeiser and Eva Frei

Genotoxic mechanisms for the carcinogenicity of the environmental pollutants and carcinogens o-anisidine and 2-nitroanisole follow from adducts generated by their metabolite N-(2-methoxyphenyl)hydroxylamine with deoxyguanosine in DNA

An aromatic amine, o-anisidine (2-methoxyaniline) and its oxidative counterpart, 2-nitroanisole (2-methoxynitrobenzene), are the industrial and environmental pollutants causing tumors of the urinary bladder in rats and mice. Both carcinogens are activated to the same proximate carcinogenic metabolite, N-(2-methoxyphenyl)hydroxylamine, which spontaneously decomposes to nitrenium and/or carbenium ions responsible for formation of deoxyguanosine adducts in DNA in vitro and in vivo. In other words, generation of N-(2-methoxyphenyl)hydroxylamine is responsible for the genotoxic mechanisms of the o-anisidine and 2-nitroanisole carcinogenicity. Analogous enzymes of human and rat livers are capable of activating these carcinogens. Namely, human and rat cytochorme P450 2E1 is the major enzyme oxidizing o-anisidine to N-(2-methoxyphenyl)hydroxylamine, while xanthine oxidase of both species reduces 2-nitroanisole to this metabolite. Likewise, O-demethylation of 2-nitroanisole, which is the detoxication pathway of its metabolism, is also catalyzed by the same human and rat enzyme, cytochorme P450 2E1. The results demonstrate that the rat is a suitable animal model mimicking the fate of both carcinogens in humans and suggest that both compounds are potential carcinogens also for humans.

Open access

Karel Naiman, Helena Dračínská, Martin Dračínský, Markéta Martínková, Václav Martínek, Petr Hodek, Martin Štícha, Eva Frei and Marie Stiborová

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

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.