Krzysztof Gwoździński

Oxidative stress induced in rat liver by anticancer drugs doxorubicin, paclitaxel and docetaxel

PURPOSE Oxidative stress generated by anticancer drugs in non-targeted tissues, is considered as a significant factor responsible for their severe side effects, e.g. cardiotoxicity, neurotoxicity and hepatotoxicity. Lack of data on the effect of concurrent administration of commonly used anticancer drugs: doxorubicin (DOX), paclitaxel (PTX) and docetaxel (DTX) on normal tissue, prompted us to examine the markers of oxidative stress in the liver of rats treated with these drugs. MATERIAL/METHODS Male Wistar rats of average weight 200 g were injected intraperitoneally (i.p.) with 10 mg/kg of body weight (b.w.) of DOX, PTX and DTX. The drugs were given alone or in combinations DOX+taxane. The activities of superoxide dismutase (SOD), catalase (CAT), low molecular weight and total thiols and thiobarbituric acid-reactive substances (TBARS) were estimated. RESULTS Combination of two drugs generated greater changes than single agents. Concurrent administration of DOX and PTX increased SOD activity and TBARS, decreased the amount of low molecular weight and total thiols, but did not cause any changes in the activity of catalase. Combination of DOX and DTX induced similar changes except for the activity of catalase, which decreased after the treatment. Of the three drugs only DTX significantly decreased the activity of SOD. However, both taxanes increased the activity of catalase. Although a decrease in concentration of -SH groups, depletion of glutathione and an increase of TBARS were observed after treatment with single drugs, the changes were not statistically significant. CONCLUSION Concurrent administration of DOX and taxane induced enhanced oxidative stress in comparison to single drugs, which suggests their synergistic prooxidant mode of action in liver.

Effect of aspirin on conformation and dynamics of membrane proteins in platelets and erythrocytes.

The effect of the chemical modifications induced by aspirin (acetylsalicylic acid), acetyl chloride or salicylate on platelet membranes and erythrocyte ghosts has been investigated by means of fluorescence quenching and ESR spectroscopy in relation to our earlier findings of acetylation-induced reduction of platelet and erythrocyte membrane lipid fluidity. Only aspirin was found to induce disorders in the lipid-protein matrix and membrane protein conformation. The apparent distance separating the membrane tryptophan and bound 1-anilino-8-naphthalenesulphonate (ANS) molecules was decreased after aspirin action in both platelet and erythrocyte membranes. This resulted in a significant increase in the maximum energy transfer efficiency. The decrease in the ratio of the amplitudes of low-field peaks of weakly to strongly immobilized fractions of maleimide spin label (4-maleimido-2,2,6-6-tetramethylpiperidine-1-oxyl) and the rise in the relative rotational correlation time of iodoacetamide spin label [4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-1-oxyl] indicate that aspirin effectively immobilizes membrane proteins in the plane of the lipid bilayer, whereas neither acetyl chloride or salicylate gave rise to detectable effects. We conclude that aspirin-induced alterations in membrane protein structure induce a reorganization of lipid assembly as well as rearrangements in the membrane protein pattern, and consequently alterations in lipid-protein interactions. Thus, the interaction of aspirin with platelet and erythrocyte membranes may induce local conformational changes in membranes, which are discussed in connection with impairment of platelet function. A new mode of protein chemical modification by aspirin is suggested which involves the generation of reactive salicylic residue during the fast degradation of aspirin under physiological conditions.

The comparison of the effects of heavy metal ions on the antioxidant enzyme activities in human and fish dicentrarchus labrax erythrocytes

1. The effect of the increasing concentrations of CuSO4 and HgCl2 (0.01-0.3 mmol/l) on erythrocyte haemolysis and the activities of peroxide metabolism enzymes: superoxide dismutase, catalase, peroxidase and glutathione peroxidase was investigated in human erythrocytes and the nucleated red blood cells of marine fish (Dicentrarchus labrax). 2. The results show that both heavy metal ions had only little effect on haemolysis and antioxidant enzyme activities in human erythrocytes; in contrast the effect of heavy metals on fish erythrocytes was statistically significant when compared to control values. 3. Copper was found to have more pronounced effect than mercury on the erythrocytes of Dicentrarchus labrax; otherwise there were no significant differences between the toxic effects of both ions on human erythrocytes. 4. We suggest that the mechanism of copper-induced haemolysis may be different from that of mercuric ion in the erythrocytes of Dicentrarchus labrax.

The effects of in vivo and in vitro non-enzymatic glycosylation and glycoxidation on physico-chemical properties of haemoglobin in control and diabetic patients

The erythrocyte deformability, which is related to erythrocyte internal viscosity, was suggested to depend upon the physico-chemical properties of haemoglobin. In the present study we employed ESR spectroscopy on order to explore further the extent to which the in vivo or in vitro glycation and/or glycoxidation might affect haemoglobin structure on conformation. We revealed that under both in vivo and in vitro conditions the attachment of glucose induced a mobilization of thiol groups in the selected domains of haemoglobin molecules ( the increased h+1/h0 parameter of maleimide spin label, MSL; 0.277 +/- 0.021 in diabetics vs 0.338 +/- 0.017 in controls, n = 12, P < 0.0001). The relative rotational correlation time (tau c) of two spin labels, TEMPONE and TEMPAMINE, respectively, in erythrocyte insides (5.22 +/- 0.42 in diabetics, n = 21 vs 4.79 +/- 0.38, n = 16 in controls, P < 0.005) and in the solutions of in vitro glycated haemoglobin, were increased. Neither oxidation nor crosslinking of thiol groups was evidenced in glycated and/or oxidized haemoglobin. In addition, erythrocyte deformability was found to be reduced in type 2 diabetic patients (6.71 +/- 1.08, n = 28 vs 7.31 +/- 0.96, n = 21, P < 0.015). In conclusion, these observations suggest that: the attachment of glucose to haemoglobin might have decreased the mobility of the Lys-adjacent Cys residues, thus leading to the increased h+1/h0 parameter of MSL. Such structural changes in haemoglobin owing to non-enzymatic glycosylation may contribute to the increased viscosity of haemoglobin solutions (r = 0.497, P < 0.0035) and the enhanced internal viscosity of diabetic erythrocytes (r = 0.503, P < 0.003). We argue that such changes in haemoglobin, and consequently in red blood cells, might contribute to the handicapped oxygen release under tissue hypoxia in the diabetic state.

Melittin-induced alterations in dynamic properties of human red blood cell membranes.

The interaction of bee venom melittin with erythrocyte membrane ghosts has been investigated by means of fluorescence quenching of membrane tryptophan residues, fluorescence polarization and ESR spectroscopy. It has been revealed that melittin induces the disorders in lipid-protein matrix both in the hydrophobic core of bilayer and at the polar/non-polar interface of melittin complexed with erythrocyte membranes. The peptide has been found to act most efficiently at the concentration of the order of 10(-10) mol/mg membrane protein. The apparent distance separating the membrane tryptophan and bound 1-anilino-8-naphthalenesulphonate (ANS) molecules is decreased upon melittin binding, which results in a significant increase of the maximum energy transfer efficiency. Significant changes in the fluorescence anisotropy of both 1,6-diphenyl-1,3,5-hexatriene and 1-anilino-8-naphthalenesulphonate bound to erythrocyte ghosts, which have been observed in the presence of melittin and crude venom, indicate membrane lipid bilayer rigidization. The effect of crude honey bee venom has been found to be of similar magnitude as the effect of pure melittin at the concentration of 10(-10) mol/mg membrane protein. Using two lipophilic spin labels, methyl 5-doxylpalmitate and 16-doxylstearic acid, we found that melittin at its increasing concentrations induces a well marked rigidization in the deeper regions of lipid bilayer, whereas the effect of rigidization near the membrane surface maximizes at the melittin concentration of 10(-10) mol/mg (10(-4) mol melittin per mole of membrane phospholipid). The decrease in the ratio hw/hs of maleimide and the rise in relative rotational correlation time (tau c) of iodacetamid spin label, indicate that melittin effectively immobilizes membrane proteins in the plane of the lipid bilayer. We conclude that melittin-induced rigidization of the lipid bilayer may induce a reorganization of lipid assemblies as well as the rearrangements in membrane protein pattern and consequently the alterations in lipid-protein interactions. Thus, the interaction of melittin with erythrocyte membranes is supposed to produce local conformational changes in membranes, which are discussed in the connection with their significance during the synergistic action of melittin and phospholipase of bee venom on red blood cells.

Quercetin attenuates oxidative stress in the blood plasma of rats bearing DMBA-induced mammary cancer and treated with a combination of doxorubicin and docetaxel.

The development of side-effects during doxorubicin-docetaxel (DOX-DTX) chemotherapy is considered as related to generation of oxidative stress by DOX. The addition of docetaxel potentiates this effect. Thus, antioxidants are assumed as a promising remedy for neutralizing deteriorating effects of reactive oxygen species (ROS) in pathological conditions and polyphenolic antioxidants are suitable candidates for such a therapeutic approach. We evaluated the ability of quercetin to attenuate oxidative stress developed during the process of DMBA carcinogenesis and DOX-DTX chemotherapy in the blood plasma of rats bearing mammary tumors. We have found that quercetin significantly improved the plasma nonenzymatic antioxidant capacity (NEAC) and reduced lipid peroxidation, which suggest the beneficial effect of flavonoid. The inclusion of quercetin to the DOX-DTX chemotherapy was also advantageous. A considerable decrease of carbonyls and lipid peroxidation products (TBARS) and improvement of the endogenous antioxidant defense system (an increase of NEAC, thiols and SOD activity) were observed compared to rats treated with DOX-DTX chemotherapy. These results suggest that quercetin could protect blood plasma constituents against oxidative damage evoked by DOX and DTX.

Response of Daphnia's antioxidant system to spatial heterogeneity in Cyanobacteria concentrations in a lowland reservoir.

Many species and clones of Daphnia inhabit ecosystems with permanent algal blooms, and they can develop tolerance to cyanobacterial toxins. In the current study, we examined the spatial differences in the response of Daphnia longispina to the toxic Microcystis aeruginosa in a lowland eutrophic dam reservoir between June (before blooms) and September (during blooms). The reservoir showed a distinct spatial pattern in cyanobacteria abundance resulting from the wind direction: the station closest to the dam was characterised by persistently high Microcystis biomass, whereas the upstream stations had a significantly lower biomass of Microcystis. Microcystin concentrations were closely correlated with the cyanobacteria abundance (r = 0.93). The density of daphniids did not differ among the stations. The main objective of this study was to investigate how the distribution of toxic Microcystis blooms affects the antioxidant system of Daphnia. We examined catalase (CAT) activity, the level of the low molecular weight antioxidant glutathione (GSH), glutathione S-transferase (GST) activity and oxidative stress parameters, such as lipid peroxidation (LPO). We found that the higher the abundance (and toxicity) of the cyanobacteria, the lower the values of the antioxidant parameters. The CAT activity and LPO level were always significantly lower at the station with the highest M. aeruginosa biomass, which indicated the low oxidative stress of D. longispina at the site with the potentially high toxic thread. However, the low concentration of GSH and the highest activity of GST indicated the occurrence of detoxification processes at this site. These results demonstrate that daphniids that have coexisted with a high biomass of toxic cyanobacteria have effective mechanisms that protect them against the toxic effects of microcystins. We also conclude that Daphnias resistance capacity to Microcystis toxins may differ within an ecosystem, depending on the blooms spatial distribution.

The flavonoid quercetin: Possible solution for anthracycline-induced cardiotoxicity and multidrug resistance

Anthracycline chemotherapy is often used in the treatment of various malignancies. Its application, however, encounters several limitations due to development of serious side effects, mainly cardiotoxicity and may be ineffective due to multidrug resistance (MDR). Many different compounds have been evaluated as poorly effective in the protection against anthracycline side effects and in the prevention from MDR. Thus, continuous investigational efforts are necessary to find valuable protectants and the flavonoid quercetin (Q) seems to be a promising candidate. It is present in relatively high amounts in a human diet and the lack of its toxicity, including genotoxicity has been confirmed. The structure of Q favours its high antioxidant activity, the potential to inhibit the activity of oxidative enzymes and to interact with membrane transporter proteins responsible for development of MDR, e.g. P-glycoprotein. Furthermore, Q can influence cellular signalling and gene expression, and thus, alter response to exogenous genotoxicants and oxidative stress in normal cells. It accounts for its chemopreventive and anticancer properties. Overall, these properties might indicate the possibility of application of Q as cardioprotectant during anthracycline chemotherapy. Moreover, numerous biological properties displayed by Q might possibly result in the reversal of MDR in tumour cells and improve the efficacy of chemotherapy. However, these beneficial effects towards anthracycline-induced complications of chemotherapy have to be further explored and confirmed both in animal and clinical studies. Concurrently, investigations aimed at improvement of the bioavailability of Q and further elucidation of its metabolism after application in combination with anthracyclines are needed.

Anti-tumor potential of nitroxyl derivative Pirolin in the DMBA-induced rat mammary carcinoma model: A comparison with quercetin

BACKGROUND Considering the role of oxidative stress in carcinogenesis, we investigated the effect of synthetic antioxidant Pirolin (3-carbamoyl-2,2,5,5-tetramethylpyrroline-1-oxyl) on breast cancer progression. Since the anticancer drugs may cause cardiotoxicity due to oxidative stress in the heart muscle, we also evaluated Pirolin performance in heart tissue and compared its effect with that of the natural dietary flavonoid quercetin. METHODS Sprague-Dawley rats were administered with 7,12-dimethylbenz(a)anthracene (DMBA) and then treated ip with an antioxidant (each at a dose of 10mg/kg b.w.) for 14 days. The histopathology of tumors, their size and multiplicity were assesed. The effect of antioxidants on heart tissue was evaluated by the oxidative stress markers and poly (ADP-ribose) polymerase 1 (PARP 1) cleavage. RESULTS The median number of tumors and their volume, at the end of the study, were considerably smaller in both antioxidant-treated groups. We found a better antioxidative performance of quercetin in the heart, since a restoration of the GSH pool and decreased amount of hydroperoxides were observed. Antioxidants did not prevent cardiomyocytes from apoptosis. CONCLUSION The attenuation of tumor progression by Pirolin was comparable with the action of quercetin. No negative changes were observed in the heart of animals after Pirolin treatment. Thus, its use in targeting deregulated redox pathways should be further studied.

Antioxidant enzyme activities and lipid peroxidation in Mytilus galloprovincialis from the French Mediterranean coast

Antioxidant enzyme activities and lipid peroxidation in Mytilus galloprovincialis from the French Mediterranean coast In the present work we have studied some of the indicators of oxidative damage of the digestive gland tissue of two populations of mussels Mytilus galloprovincialis: native mussels cultured in an aquaculture farm and contaminated mussels collected from the polluted marine area of the French Mediterranean coast located nearby Tamaris and La Seyne-sur-mer - small towns in close proximity to Toulon.