Eva Kvasničková

Iron is not involved in oxidative stress-mediated cytotoxicity of doxorubicin and bleomycin

The anticancer drugs doxorubicin and bleomycin are well‐known for their oxidative stress‐mediated side effects in heart and lung, respectively. It is frequently suggested that iron is involved in doxorubicin and bleomycin toxicity. We set out to elucidate whether iron chelation prevents the oxidative stress‐mediated toxicity of doxorubicin and bleomycin and whether it affects their antiproliferative/proapoptotic effects.

New iron chelators in anthracycline-induced cardiotoxicity

The use of anthracycline anticancer drugs is limited by a cumulative, dose-dependent cardiac toxicity. Iron chelation has long been considered as a promising strategy to limit this unfavorable side effect, either by restoring the disturbed cellular iron homeostasis or by removing redox-active iron, which may promote anthracycline-induced oxidative stress. Aroylhydrazone lipophilic iron chelators have shown promising results in the rabbit model of daunorubicin-induced cardiomyopathy as well as in cellular models. The lack of interference with the antiproliferative effects of the anthracyclines also favors their use in clinical settings. The dose, however, should be carefully titrated to prevent iron depletion, which apparently also applies for other strong iron chelators. We have shown that a mere ability of a compound to chelate iron is not the sole determinant of a good cardioprotector and the protective potential does not directly correlate with the ability of the chelators to prevent hydroxyl radical formation. These findings, however, do not weaken the role of iron in doxorubicin cardiotoxicity as such, they rather appeal for further investigations into the molecular mechanisms how anthracyclines interact with iron and how iron chelation may interfere with these processes.

Effect of ivermectin on activities of cytochrome P450 isoenzymes in mouflon (Ovis musimon) and fallow deer (Dama dama).

Ivermectin is an antiparasitic drug widely used in veterinary and human medicine. We have found earlier that repeated treatments of rats with high doses of this drug led to significant increase of cytochrome P450-dependent 7-methoxyresorufin O-demethylase (MROD) and 7-ethoxyresorufin O-deethylase (EROD) activities in hepatic microsomes. In the present study, the effects of ivermectin on cytochrome P450 (CYP) activities were investigated in mouflon (Ovis musimon) and fallow deer (Dama dama). This study was conducted also to point out general lack of information on both basal levels of CYP enzymes and their inducibilities by veterinary drugs in wild ruminants. Liver microsomes were prepared from control animals, mouflons, after single or repeated (six doses in six consecutive days) treatments with therapeutic doses of ivermectin (0.5 mg kg(-1) of body weight), and fallow deer exposed to repeated doses of ivermectin under the same conditions. Alkyloxyresorufins, testosterone and chlorzoxazone were used as the specific substrate probes of activities of the CYP isoenzymes. A single therapeutic dose of ivermectin significantly induced (300-400% of the control group) the activities of all alkyloxyresorufin dealkylases tested in mouflon liver microsomes. Repeated doses of ivermectin also caused an increase of these activities, but due to fair inter-individual differences, this increase was not significant. The administration of ivermectin led to an induction (170-210% of the control) of the testosterone 6beta- and 16alpha-hydroxylase activities in mouflon liver but no significant modulation of chlorzoxazone hydroxylase (CZXOH) activity was found in mouflon liver. CYP-dependent activities in hepatic microsomes were generally higher in fallow deer than in mouflons. However, with the exception of slight increase in the 7-benzyloxyresorufin O-dealkylase (BROD) activities, no significant modulation of the other activities was observed. The induction of CYP3A-like isoenzyme was confirmed by immunoblotting only in the microsomes from mouflons administered with repeated doses of ivermectin; however, no significant increase of CYP1A isoenzymes was observed due to a weak cross-reactivity of anti-rat CYP1A1/2 polyclonal antibodies used in the study. The results indicate that ivermectin should be considered as an inducer of several cytochrome P450 isoenzymes, including CYP1A, 2B and 3A subfamilies, in mouflons. The comparison of induction effect of ivermectin in rat, mouflon and fallow deer also demonstrates the inter-species differences in inducibility of CYP enzymes.

High-performance liquid chromatography study of stereospecific microsomal enzymes catalysing the reduction of a potential cytostatic drug, oracin: Interspecies comparison

One of the main metabolites of oracin (I) ¿6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo-5,6-dihydro-11H-indeno[1,2- c] isoquinoline¿, a potential cytostatic drug, is 11-dihydrooracin (II) ¿(+),(-)-6-[2-(2-hydroxyethyl)aminoethyl]-5-oxo-11-hydroxy-5,6-dihydro-1 1H- indeno[1,2-c]isoquinoline¿, a metabolite formed by the reduction of oracins pro-chiral centre on C 11. This metabolite has been found in all laboratory species in vitro and in vivo and it constitutes the main metabolite in man. The stereospecificity of reducing enzymes participating in the oracin biotransformation pathway was investigated using microsomal preparations from standard laboratory animals. Enzyme stereospecificity has been defined as preferential formation by the enzyme of the (+) or (-) stereoisomer of II. Significant interspecies differences were observed in the stereospecificity of the respective biotransformation enzymes. HPLC quantitative determinations of both enantiomers were performed using a Chiralcel OD-R column as chiral stationary phase with excellent resolution and stability.

Chromatographic characterization of in vitro metabolites of 5-[2-(N,N-dimethylamino)ethoxy]-7-oxo-7H-benzo-[c]fluorene

Detection reactions and RF values in thin-layer chromatography on silica gel were studied for the antineoplastic drug Ih (benfluron) and related substances. On incubation of Ih with homogenate fractions of mammalian livers the N-oxide Ii and 5-[2-(N,N-dimethylamino)ethoxy]-7-hydroxy-7 H-benzo[c]fluorene (IIh) were established as products, and 5-[2-(N-methylamino)ethoxy]-7-oxo-7 H-benzo[c]fluorene (Ig), 5-[2-(N-methylamino)ethoxy]-7-hydroxy-7 H-benzo[c]fluorene (IIg) and a phenolic product of benfluron (IV) were tentatively identified.

Sex differences in stereospecificity of oracin reductases in rat in vitro and in vivo.

In vitro and in vivo experiments to investigate possible stereospecific aspects of oracin reduction in relation to rat gender have been conducted. Incubation of oracin with rat microsomes, cytosol, and hepatocytes in the presence of various coenzymes and under aerobic or anaerobic conditions provided evidence for sex differences in the formation of 11-dihydrooracin (DHO) enantiomers. The greatest sex differences were seen in hepatocytes where females showed higher stereospecificity of the reductases than males. While female biotransformation enzymes preferentially generated approximately 82% of (+)-DHO, male enzymes gave only rise to 63% of (+)-DHO. Males displayed higher stereospecificity than females in the microsomal fraction. However, in the cytosolic fraction females exhibited higher stereospecificity than males. Similarly, in in vivo studies, the ratio of (+)- and (-)-DHO in faeces and urine gave no indication of the significant differences between the male and female rat. Enzyme stereospecificity has been defined as preferential formation of the (+)- or (-)-stereoisomer of 11-DHO by the respective enzyme. HPLC quantitative determinations of both enantiomers were performed using a Chiralcel OD-R column as the chiral stationary phase with excellent resolution and stability.

Effect of substituents on microsomal reduction of benzo(c)fluorene N-oxides

The potential benzo(c)fluorene antineoplastic agent benfluron (B) displays high activity against a broad spectrum of experimental tumours in vitro and in vivo. In order to suppress some of its undesirable properties, its structure has been modified. Benfluron N-oxide (B N-oxide) is one of benfluron derivatives tested. The main metabolic pathway of B N-oxide is its reduction to tertiary amine B. A key role of cytochrome P4502B and P4502E1 in B N-oxide reduction has been proposed in the rat. Surprisingly, B N-oxide is reduced also in the presence of oxygen although all other N-oxides undergo reduction only under anaerobic conditions. With the aim to determine the influence of the N-oxide chemical structure and its redox potential on reductase affinity, activity and oxygen sensitivity five relative benzo(c)fluorene N-oxides were prepared. A correlation between the redox potential measured and the non-enzymatic reduction ability of the substrate was found, but no effect of the redox potential on reductase activity was observed. Microsomal reductases display a high affinity to B N-oxide (apparent K(m) congruent with0. 2 mM). A modification of the side-chain or nitrogen substituents has led to only a little change in apparent K(m) values, but a methoxy group substitution on the benzo(c)fluorene moiety induced a significant K(m) increase (ten-fold). Based on kinetic study results, the scheme of mechanism of cytochrome P450 mediated benzo(c)fluorene N-oxides reduction have been proposed. All benzo(c)fluorene N-oxides under study were able to be reduced in the presence of oxygen. Changes in the B N-oxide structure caused an extent of anaerobic conditions preference. The relationship between the benzo(c)fluorene N-oxide structure and the profile of metabolites in microsomal incubation was studied and important differences in the formation of individual N-oxide metabolites were found.

A comparison between stereospecificity of oracin reduction and stereoselectivity of oxidation of 11-dihydrooracin enantiomers in vitro in rat and guinea pig

11-dihydrooracin (DHO) arises from the potential cytostatic drug oracin through the metabolic conversion of its prochiral centre (C11). The participation of reduction enzymes on production of DHO enantiomer under various incubation conditions were tested in rat and guinea pig microsomal and cytosolic fractions. Interesting differences in stereospecificity of oracin reduction enzymes were found. Reduction stereospecificity was further studied on rat and guinea pig isolated hepatocytes. The enantiomers were detected in rat and guinea pig hepatocytes in the (+)/(-) ratio 63/37 and 32/68 respectively. As the differences in the amounts of DHO enantiomers can be caused not only by stereospecificity of oracin reduction but also by subsequent conversion of the enantiomer, stereoselectivity of DHO oxidation to oracin was investigated. Synthetically prepared pure (+)- and (-)-DHO were incubated with rat or guinea pig microsomes and cytosol and with various coenzymes under aerobic or anaerobic conditions. Significant oxidation of DHO to oracin was observed in rat microsomes. This oxidation depends on NADPH and O2 and is stereoselective for (+)-DHO. The formation of oracin in the guinea pig was greater in cytosol than microsomes, but no significant preference for a particular DHO enantiomer was found.

Elimination of Benfluron and its Metabolites in the Faeces and Urine of Rats

The study of the biotransformation of the potential cytostatic benfluron has been continued. The elimination of benfluron and of nine of its metabolites whose structure had been established, mainly on the basis of the comparison of their IR, MS and NMR spectra with those of standards, was studied. After oral administration of 500 mg.kg-1 to rats, the amounts of these substances in the faeces and urine were followed up by high-performance liquid chromatography for five days. Striking qualitative and quantitative differences were observed in the elimination of benfluron and its metabolites by both routes.

Inhibition study of rabbit liver cytosolic reductases involved in daunorubicin toxication

Anthracycline cardiotoxicity represents the most unfavorable side effect of these highly efficient anticancer drugs. Several biotransformation enzymes have been described to contribute to their cardiotoxicity. Besides the activities of CYP450 isoforms which lead to the generation of reactive oxygen species (ROS), the cytosolic reductases have attracted attention nowadays. The reductases known to metabolize anthracyclines to C13-hydroxyanthracyclines are carbonyl reductase (CR, 1.1.1.184) and the aldo-keto reductases (AKR1C2, 1.3.1.20; AKR1A1, 1.1.1.2). Their participation in the formation of the toxic C13-hydroxymetabolite has been investigated in rabbit using diagnostic inhibitors of CR and AKR1C2. The kinetics and the type of reductase inhibition exerted by the two inhibitors have been described and it was found that CR was the main daunorubicin reductase at both optimal and physiological pH with the kinetic parameters for daunorubicin reduction of and The IC50 values for quercitrin and flufenamic acid were 5.45 ± 1.37 μM and 3.68 ± 1.58 μM, respectively. The inhibition was uncompetitive for both inhibitors and irreversible in the case of flufenamic acid.