Radomír Hrdina

In vitro analysis of iron chelating activity of flavonoids

Flavonoids have been demonstrated to possess miscellaneous health benefits which are, at least partly, associated with iron chelation. In this in vitro study, 26 flavonoids from different subclasses were analyzed for their iron chelating activity and stability of the formed complexes in four patho/physiologically relevant pH conditions (4.5, 5.5, 6.8, and 7.5) and compared with clinically used iron chelator deferoxamine. The study demonstrated that the most effective iron binding site of flavonoids represents 6,7-dihydroxy structure. This site is incorporated in baicalein structure which formed, similarly to deferoxamine, the complexes with iron in the stoichiometry 1:1 and was not inferior in all tested pH to deferoxamine. The 3-hydroxy-4-keto conformation together with 2,3-double bond and the catecholic B ring were associated with a substantial iron chelation although the latter did not play an essential role at more acidic conditions. In agreement, quercetin and myricetin possessing all three structural requirements were similarly active to baicalein or deferoxamine at the neutral conditions, but were clearly less active in lower pH. The 5-hydroxy-4-keto site was less efficient and the complexes of iron in this site were not stable at the acidic conditions. Isolated keto, hydroxyl, methoxyl groups or an ortho methoxy-hydroxy groups were not associated with iron chelation at all.

The role of reactive oxygen and nitrogen species in cellular iron metabolism.

The catalytic role of iron in the Haber–Weiss chemistry, which results in propagation of damaging reactive oxygen species (ROS), is well established. In this review, we attempt to summarize the recent evidence showing the reverse: That reactive oxygen and nitrogen species can significantly affect iron metabolism. Their interaction with iron-regulatory proteins (IRPs) seems to be one of the essential mechanisms of influencing iron homeostasis. Iron depletion is known to provoke normal iron uptake via IRPs, superoxide and hydrogen peroxide are supposed to cause unnecessary iron uptake by similar mechanism. Furthermore, ROS are able to release iron from iron-containing molecules. On the contrary, nitric oxide (NO) appears to be involved in cellular defense against the iron-mediated ROS generation probably mainly by inducing iron removal from cells. In addition, NO may attenuate the effect of superoxide by mutual reaction, although the reaction product—peroxynitrite—is capable to produce highly reactive hydroxyl radicals.

In vitro interactions of coumarins with iron

Coumarins are a large group of natural substances with diverse pharmacological properties that may predetermine some of them for the prevention and/or treatment of cardiovascular diseases and also other pathologies. Free iron participates in the production of reactive oxygen species (ROS) and plays an important role in the pathogenesis of cardiovascular diseases. Therefore, chelation of iron may attenuate some ROS consequences, but on the other hand, reduction of ferric ions to ferrous ones is unfavourable and leads to intensification of ROS production. In this study, we have examined the interaction of iron with coumarins which has been rarely analyzed. A series of naturally occurring and chemically synthesized 4-methylcoumarins were analyzed for their ferrous and total iron-chelating properties and compared with standard iron chelator deferoxamine. The iron chelation activity was assessed by a simple spectrophotometric approach based on the specific indicator for ferrous ions--ferrozine. The methodology was also extended for the measurement of total iron. Among the tested coumarins, ortho-dihydroxyderivatives were the most potent iron chelators and 7,8-dihydroxy-4-methylcoumarin even reached the efficiency of deferoxamine in neutral pH. However, these ortho-dihydroxycoumarins did not bind iron firmly in acidic conditions (e.g., in acute myocardial infarction) and, moreover, they reduced ferric ions that could lead to intensification of the Fenton chemistry. Other tested coumarins did not substantially chelate iron with the exception of ortho-diacetoxycoumarins. Conclusively, the use of iron-chelating coumarins in acidic conditions may be disadvantageous in contrast to neutral conditions.

Rabbit model for in vivo study of anthracycline‐induced heart failure and for the evaluation of protective agents

Cardiac toxicity associated with chronic administration of anthracycline (ANT) antibiotics represents a serious complication of their use in anticancer chemotherapy, but can also serve as a useful experimental model of cardiomyopathy and congestive heart failure.

Iron reduction potentiates hydroxyl radical formation only in flavonols

Flavonoids, substantial components of the human diet, are generally considered to be beneficial. However, they may possess possible pro-oxidative effects, which could be based on their reducing potential. The aims of this study were to evaluate the ability of 26 flavonoids to reduce ferric ions at relevant pH conditions and to find a possible relationship with potentiation of hydroxyl radical production. A substantial ferric ions reduction was achieved under acidic conditions, particularly by flavonols and flavanols with the catecholic ring B. Apparently corresponding bell-shaped curves displaying the pro-oxidant effect of flavonols quercetin and kaempferol on iron-based Fenton reaction were documented. Several flavonoids were efficient antioxidants at very low concentrations but rather inefficient or pro-oxidative at higher concentrations. Flavonols, morin and rutin were progressively pro-oxidant, while 7-hydroxyflavone and hesperetin were the only flavonoids with dose-dependent inhibition of hydroxyl radical production. Conclusively, administration of flavonoids may lead to unpredictable consequences with few exceptions.

Comparative study of chronic toxic effects of daunorubicin and doxorubicin in rabbits

This study compares the chronic toxicity of two anthracyclines–daunorubicin and doxorubicin, commonly used for induction of anthracycline cardiomyopathy in the rabbit model. Such a comparative study has not been published until now. Both drugs were administered intravenously to male Chinchilla rabbits in doses at 3 mg/ kg (50 mg/m2) once weekly for 10 weeks. Selected biochemical, haematological and cardiovascular parameters and body weights were regularly monitored; additionally, a histological evaluation of heart, kidney and liver was performed at the end of the experiment. In the daunorubicin group, there were marked signs of the progressive development of heart failure, like the significant increases of the pre-ejection period/left ventricular ejection time index values (up to 134%)–and histological changes within the myocardium were also observed. On the other hand, the 10-week doxorubicin administration did not cause these changes that are typical for heart injury. Haematotoxicity, manifested particularly by aplastic anaemia, was apparent in both the experimental groups. Significant body weight loss (by 45.2%) and high premature mortality (100% versus 36.4%) reflected a greater general toxicity, especially nephrotoxicity of doxorubicin in comparison with daunorubicin. Further studies are necessary to find a possible explanation for these findings.

In vitro evaluation of copper-chelating properties of flavonoids

Copper is an essential trace element involved in plenty of redox reactions in living systems, however, unbound copper ions cause damage to various biomolecules via excessive generation of reactive oxygen species. Flavonoids, ubiquitous plant secondary metabolites, possess complex effects on human health and chelation of transient metal ions is one of their proposed mechanisms of action. In this in vitro study, 26 flavonoids from various subclasses were screened for their interactions with both copper oxidation states at four (patho)physiologically relevant pH conditions (4.5, 5.5, 6.8 and 7.5) by two spectrophotometric approaches and compared with the clinically used copper chelator trientine. In a slightly competitive environment, the majority of flavonoids were able to chelate cupric ions, however, under more competitive conditions, only flavones and flavonols were able to chelate both cupric and cuprous ions. Apparently, the 2,3-double bond was essential for stable copper chelation. The most efficient copper chelation sites were the 3-hydroxy-4-keto group in flavonols and the 5,6,7-trihydroxyl group in flavones. On the other hand, the 3′,4′-dihydroxyl group was associated only with a weak activity. 3-Hydroxyflavone, kaempferol and partly baicalein were even more potent than trientine in the acidic environment, however, none of the tested flavonoids was able to surpass it at physiological pH or slightly acidic conditions. In conclusion, flavonoids possessing appropriate structural characteristics were efficient copper chelators and some of them were even more potent than trientine under acidic conditions.

Lack of cardiotoxicity of a new antineoplastic agent, a synthetic derivative of indenoisochinoline: comparison with daunorubicin in rabbits

Abstract The effect of repeated i.v. administration (once weekly, 12 administrations) of a new antineoplastic agent, Oracin (6-[2-(2-hydroxyethyl)aminoethyl]5,11-dioxo-5,6-dihydro-11H-indeno [1,2-c]-isochinoline hydrochloride, 10 mg/kg) and daunorubicin (3 mg/ kg) were investigated in rabbits in vivo. The criterion of occurrence of cardiotoxicity was compared with a control group of animals. Noninvasive polygraphic records were used to evaluate the function of the heart. The morphological changes of the heart were evaluated after the death of animals. There were no significant changes found in the ratio of the pre-ejection period/left ventricular ejection time (PEP : LVET ratio) after administration of Oracin (values between 0.3080 and 0.3310) or in the control group (values between 0.3425 and 0.3885). The administration of daunorubicin induced a significant, progressive increase in the PEP : LVET ratio (0.3775–0.9473), which was significantly different both from the Oracin-treated and the control group of animals. Histological examination of the hearts from the control group revealed normal structure of the myocardium including minute changes (dispersed cardiomyocytes with intensively eosinophilic cytoplasm, and several single cells with degenerated myofibrils) similar to the normal changes in muscle tissue. A very similar scenario was found in the Oracin group with the exception of one case where a slightly higher number of degenerated and necrotic cells was occurring. Administration of daunorubicin resulted in severe dispersed damage of the myocardium (myocytolysis with subsequent interstitial fibrosis), the changes being markedly different from those of the Oracin treatment and the control group. On the basis of our results it is possible to conclude that the administration of Oracin (10 mg/kg i.v.) did not induce signs of cardiotoxicity in rabbits in vivo.

Cardiac biomarkers in a model of acute catecholamine cardiotoxicity

Coronary heart disease and in particular its most serious form — acute myocardial infarction (AMI) — represents the most common cause of mortality in developed countries. Better prognosis may be achieved by understanding the etiopathogenetic mechanisms of AMI. Therefore, a catecholamine model of myocardial injury, which has appeared to be very similar to AMI in human in some aspect, was used. Male Wistar:Han rats were randomly divided into two groups: control group (saline) and isoprenaline group (ISO; synthetic catecholamine, 100 mg.kg— 1 subcutaneously [s.c.]). After 24 hours, functional parameters were measured, biochemical markers in the blood and metals content in the heart tissue were analysed and histological examination was performed. ISO caused marked myocardial injury that was associated with myocardial calcium overload. Close correlation between myocardial impairment (i.e. serum TnT, stroke volume index and wet ventricles weight) and the levels of myocardial calcium was observed. Direct reactive oxygen species (ROS) involvement was documented only by non-significant increase in malonyldialdehyde 24 hours after ISO injury. Moreover, myocardial element analysis revealed no significant changes as for the content of zinc and iron while selenium and copper increased in the ISO group although it reached statistical significance only for the latter.

Novel method for rapid copper chelation assessment confirmed low affinity of D-penicillamine for copper in comparison with trientine and 8-hydroxyquinolines

Copper is an essential trace element involved in many physiological processes. Since disorder of copper homeostasis is observed in various pathologies, copper chelators may represent a promising therapeutic tool. This study was aimed at: 1) formation of an in vitro methodology for screening of copper chelators, and 2) detailed analysis of the interaction of copper with clinically used D-penicillamine (D-PEN), triethylenetetramine (trientine), experimentally tested 8-hydroxyquinolines, and the disodium salt of EDTA as a standard chelator. Methodology based on bathocuproinedisulfonic acid disodium salt (BCS), usable at (patho)physiologically relevant pHs (4.5-7.5), enabled assessment of both cuprous and cupric ions chelation and comparison of the relative affinities of the tested compounds for copper. In the case of potent chelators, the stoichiometry could be estimated too. Clioquinol, chloroxine and EDTA formed very stable complexes with Cu(+)/Cu(2+) at all tested pHs, while copper complexes with trientine were stable only under neutral or slightly acidic conditions. Non-substituted 8-hydroxyquinoline was a less efficient copper chelator, but still unequivocally more potent than D-PEN. Both 8-hydroxyquinoline and D-PEN chelation potencies, similarly to that of trientine, were pH-dependent and decreased with pH. Moreover, only D-PEN was able to reduce cupric ions. Conclusively, BCS assay represents a rapid, simple and precise method for copper chelation measurement. In addition, lower binding affinity of D-PEN compared with 8-hydroxyquinolines and trientine was demonstrated.