E. Monti

Protective effect of the nitroxide tempol against the cardiotoxicity of adriamycin

Nitroxides are cell permeable, stable radicals that have been shown to exert antioxidant effects in several experimental models. In the present study, the ability of the piperidine nitroxide TEMPOL to prevent the acute cardiac toxicity of Adriamycin (ADR), which depends on oxygen-derived free radical generation, was assessed in isolated rat hearts. The results obtained show that TEMPOL (2.5 mM) significantly reduces the contractile impairment as well as the lipid peroxidation observed in rat heart preparations perfused with 100 micrograms/ml of ADR for 60 min. Both direct interaction with free radicals and decrease of Fe(II) availability (by stable oxidation and/or by chelation) seem to contribute to the cardioprotective effect to TEMPOL. HPLC and EPR studies of the subcellular distribution of TEMPOL indicate that substantial amounts of the nitroxide localize to the mitochondrial and microsomal fractions, in an ordered environment possibly corresponding to the interface between membrane and aqueous compartments.

Antiproliferative effect of the piperidine nitroxide TEMPOL on neoplastic and nonneoplastic mammalian cell lines.

The stable nitroxide 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) is widely used as a probe in biophysical studies and as an antioxidant in several experimental models. The potential cytotoxic effects of TEMPOL were tested on a panel of human and rodent cell lines, and the nitroxide proved to be significantly more effective in inhibiting the growth of neoplastic than nonneoplastic cell lines after a 96-h exposure. More detailed studies on MCF-7/WT cells indicate that at least 24 h are necessary for TEMPOL to induce irreversible cell damage, which seems to be related to the reactivity of the nitroxyl group. This observation, together with the antagonistic effect of N-acetylcysteine, suggests an involvement of free radical-mediated processes. Cell cycle studies indicate a biphasic effect of TEMPOL, with a short-term accumulation of the cells in the G1 phase and a later increase in G2/M phase; the pattern of DNA fragmentation observed in TEMPOL-treated cells points to an apoptotic mode of cell death. In conclusion, our data suggest that, while the possible cytotoxic effects of TEMPOL should not be overlooked when using this compound as a biophysical probe or antioxidant, these same properties could be exploited as a novel approach to cancer chemotherapy, especially in tumor cells exhibiting unfavorable characteristics, such as a multidrug-resistant phenotype or loss of hormone receptors.

Protective activity of the spin trap tert-bytyl-α-phenyl nitrone (PBN) in reperfused rat heart

Abstract The aim of this work was to ascettain whether free radicals play a causal role in the injury occurring in myocardial ischemia and reperfusion. To this purpose we observed whether spin-trapping compounds protect the heart when used at a concentration capable of reacting with free radicals. The lipophilic spin trap α-phenyl-t-butyl nitrone (PBN) was used because it is taken up by the myocites. Isolated Langendorff rat hearts were subjected to ischemia according to two schemes: “Model A” = 30 min zero-flow ischemia followed by 30 min reperfusion; “Model B” = 60 min of low-flow ischemia (10% of the individual value; N 2 saturated) followed by 30 min reperfusion. Treated groups received in addition 5.0 m m PBN which was supplied continuously. The following parameters were measured throughout the experiment: contractile performance (RPP); coronary flow (CF); CPK; phosphocreatine (PCr), ATP, inorganic phosphate ( P i ), intracellular pH (pH i ). The pathology obtained by “Model A” is more severe than that of Model B, and partly irreversible. During the ischemic phase in “Model A”, contractility, PCr and ATP dropped to near zero; during initial reflow CPK rose about 13-fold and P i rose 2.5-fold, while pH i decreased to 6.1. During reperfusion, a partial recovery of PCr, P i and pH i was observed, while RPP and ATP did not increase; PBN treatment improved significantly PCr and CPK, while the other parameters were unaffected. During ischemia, “Model B” hearts showed a drop of contractility to near zero, of PCr to 35%, of ATP to 50%; CPK rose 7-fold and P i 1.5-fold; pH i was not modified. During reperfusion, all parameters recovered in part, with exception of P i . PBN developed a marked protective activity on all tested parameters, which gained a nearly normal value. The results of the present investigations show that the lipophilic spin trap PBN partly protects the heart from the ischemia/reperfusion injury, thus confirming that free radicals play a causal role in this pathology; the continuous loading of the tissue with the drug can be an important factor for obtaining the protective effect.

Subcellular distribution of two spin trapping agents in rat heart : possible explanation for their different protective effects against doxorubicin-induced cardiotoxicity

Previous investigations, performed on isolated rat atria, showed that the lipophylic spin-trapping agent N-tert-butyl- alpha-phenylnitrone (PBN) is able to prevent the acute cardiotoxic effects produced by doxorubicin (DXR), whereas the hydrophylic compound 5,5-dimethyl-pyrroline-N-oxide (DMPO) is inactive. The present study was designed to ascertain whether differences in the pharmacological effects of the two spin traps are related to their different subcellular distribution. Langendorff rat hearts were perfused for 60 minutes with [14C]-DXR and either PBN or DMPO. The subcellular mapping of the three compounds was performed by measuring DXR by liquid scintillation counting, PBN by GC/MS, and DMPO by HPLC in the following isolated fractions: nuclei, mitochondria, sarcoplasmic reticulum, sarcolemma, cytosol. DMPO was shown to accumulate in the cytosolic compartment; both PBM and DXR are taken up by nuclei and mitochondria, while only trace amounts of DXR were detected in the sarcoplasmic reticulum. These results suggest that mitochondrial (and not sarcoplasmic) enzymes are mainly involved in DXR-induced free radical production, which is thought to cause the acute cardiotoxic effects of DXR. An involvement of DXR-induced free radical generation in the nuclear compartment seems unlikely in the short-term in vitro effects observed with the experimental model adopted for these studies, although it may play a role in the delayed pathology.

Effect of ICRF-187 pretreatment against doxorubicin-induced delayed cardiotoxicity in the rat

Doxorubicin (DXR), administered iv in rats at the weekly dose of 3 mg/kg for 5 weeks, significantly impaired body weight gain and induced irreversible ECG alterations, mainly consisting of a progressive prolongation of ST and QT intervals. Five weeks after the last DXR administration, the contractile performance of atria isolated from treated animals was significantly reduced. At the same time, relevant morphologic lesions, consisting of myocyte vacuolization and myofibrillar loss, were also present in the myocardium of the same rats. The study showed that ICRF-187, administered ip at a dose of 125 mg/kg, significantly prevented body weight loss. QT and ST prolongation, and the decreased contractile force induced by DXR. In addition, ICRF-187 caused a significant reduction in incidence and severity of myocardial lesions. The cardioprotective effect of ICRF-187 is not mediated by a modification in DXR pharmacokinetics in heart, since the drug was actually found to increase DXR uptake in myocardial cells.

Protective effects of spin-trapping agents on adriamycin-induced cardiotoxicity in isolated rat atria

Adriamycin (ADR) is known to exert a severe negative inotropic effect on isolated myocardial preparations; a role for free radical generation has been hypothesized. Spin-trapping of free radicals has been extensively exploited in ESR studies, both in cell-free systems and in intact tissues. The interaction between spin-traps and free radicals should in principle stop the reaction cascade leading to cellular damage. Based on this hypothesis, the possible cardioprotective action of three spin-trapping agents, 5,5-dimethyl-l-pyrroline-N-oxide (DMPO), N-tert-butyl-alpha-phenylnitrone (PBN) and alpha-(4-pyridyl 1-oxide) N-tert-butylnitrone (POBN), was tested on isolated rat atria incubated in the presence of ADR; maximal non-cardiotoxic concentrations were used (50, 10 and 50 mM respectively) in order to achieve a maximal spin-trapping effect. A varying degree of protection was observed with the three compounds, directly correlated to their hydrophobicity, as assessed by chloroform/water partition coefficients. It is proposed that ADR-induced free radical generation is responsible for the acute cardiotoxic effects of the drug; this seems to be a site-specific mechanism restricted to one or more hydrophobic cellular compartment/s, since only lipophilic spin-trapping agents are able to prevent the development of the negative inotropic effect of ADR.

EFFECT OF GLUTATHIONE AND N- ACETYLCYSTEINE ON IN VITRO AND IN VIVO CARDIAC TOXICITY OF DOXORUBICIN

The effects of two sulfhydryl compounds, glutathione (GSH) and N-acetylcysteine (NAC), on the cardiotoxicity of doxorubicin (DXR) were tested on in vitro and in vivo models. DXR was administered to rats as 4 weekly i.v. doses of 3 mg/kg. GSH (1.5 mmoles/kg), given i.v. 10 min before and 1 hr after DXR, was found to prevent the development of the delayed cardiotoxic effects of DXR, as assessed by electrocardiographic and mechanical parameters, as well as by histological examination of left ventricular preparations. In contrast, equimolar oral doses of NAC (1 hr before and 2 hrs after DXR) were found to be ineffective. Both GSH and NAC prevented the negative inotropic effect produced by DXR on isolated rat atria. A good correlation exists between the cardioprotective effects of the two agents and their ability to enhance the non-protein sulfhydryl group content of the myocardium. Differences observed in vivo between GSH and NAC might be accounted for by pharmacokinetic factors.

Relationship between doxorubicin-induced ECG changes and myocardial alterations in rats.

The aim of the present study was to evaluate the dose- and time-dependence of the effect displayed by doxorubicin (DXR) on the electrocardiogram (ECG) and to establish the relationship between structural alterations of the myocardium and ECG changes in rats administered DXR, at a dose of 1.5 or 3.0 mg/kg, every 3 days for a total of three administrations. The most interesting findings consisted of a dose-dependent, but reversible prolongation of the QRS complex, and in a dose-dependent and progressive irreversible increase in QaT and, in particular, in SaT duration. Furthermore, animals treated with the higher DXR dose showed a slight increase in serum K+ concentration and a significant decrease in serum Ca2+ levels. A good correlation was found between the morphologic score indicating the degree of observed tissue damage and SaT prolongation. These results therefore support the usefulness of measuring this ECG parameter for monitoring the development of DXR-induced cardiotoxicity in rats.

Myocardial contractility and heart pharmacokinetics of adriamycin following a single administration in rat

SummaryA single administration of adriamycin (DXR) 6.0 mg/kg i. v. to rats brings about a biphasic impairment of the maximal myocardial contractile performance, measured as dF/dt of ex vivo isolated atria incubated in the presence of calcium concentrations varying up to 12 mM. The initial impairment of the contractile performance peaks 1 week after DXR administration and recovers within 3 weeks (acute phase of cardiotoxicity). After this time and up to the end of the observation period (8 weeks after treatment), delayed cardiotoxicity occurs, showing a progressive and irreversible impairment of the contractile performance of the atria. This behaviour parallels the previously shown ECG and morphological abnormalities. Tissue determinations of DXR showed that the drug is present in myocardium during the acute phase of cardiotoxicity, while the metabolite adriamycinol is not detectable 1 week after DXR administration. These data show that the presence of DXR and/or metabolites in heart muscle is not necessary for the delayed form of cardiotoxicity to become apparent and suggest that this form of cardiotoxicity is related to a mechanism different from that involved in acute cardiotoxicity.

Effect of piribedil and one of its metabolites on the concentration of homovanillic acid in the rat brain

Abstract Piribedil and its metabolite (pyrimidyl-2′)-1-(dihydroxy-3′,4′-benzyl)-4-piperazine dichlorhydrate (PdHBP), like apomorphine, decrease the level of HVa in the rat striatum. The effect appears rapidly and it lasts for about 2 hr. Piribedil antagonizes the rise of striatal HVA elicited by chlorpromazine, haloperidol and fenfluramine. Piribedil, PdHBP and apomorphine did not counteract the increase of striatal HVA induced by d-amphetamine.