A. Bast

Doxorubicin-induced cardiotoxicity monitored by ECG in freely moving mice A new model to test potential protectors

Abstract In laboratory animals, histology is most commonly used to study doxorubicin-induced cardiotoxicity. However, for monitoring during treatment, large numbers of animals are needed. Recently we developed a new method to measure ECG values in freely moving mice by telemetry. With this model we investigated the effect of chronic doxorubicin administration on the ECG of freely moving BALB/c mice and the efficacy of ICRF-187 as a protective agent. The ST interval significantly widened from 15.0±1.5 to 56.8±11.8 ms in week 10 (7 weekly doses of 4 mg/kg doxorubicin given i.v. plus 3 weeks of observation). The ECG of the control animals did not change during the entire study. After sacrifice the hearts of doxorubicin-treated animals were enlarged and the atria were hypertrophic. As this schedule exerted more toxicity than needed to investigate protective agents, the protection of ICRF-187 was determined using a dose schedule with lower general toxicity (6 weekly doses of 4 mg/kg doxorubicin given i.v. plus 2 weeks of observation). On this schedule, the animals’ hearts appeared normal after sacrifice and ICRF-187 (50 mg/kg given i.p. 1 h before doxorubicin) provided almost full protection. These data were confirmed by histology. The results indicate that this new model is very sensitive and enables monitoring of the development of cardiotoxicity with time. These findings result in a model that allows the testing of protectors against doxorubicin-induced cardiotoxicity as demonstrated by the protection provided by ICRF-187.

Monohydroxyethylrutoside as protector against chronic doxorubicin‐induced cardiotoxicity

1 The clinical use of the antitumour agent, doxorubicin, is largely limited by the development of a cumulative dose‐related cardiotoxicity. This toxicity is generally believed to be caused by the formation of oxygen free radicals. In earlier studies it was established that flavonoids, naturally occurring antioxidants, can provide some degree of protection. In this study we investigated whether 7‐monohydroxyethylrutoside (monoHER), a powerful antioxidative flavonoid with extremely low toxicity, can provide protection to an extent comparable to the clinically successful Cardioxane (ICRF‐187). 2 Balb/c mice of 20–25 g were equipped i.p. with a telemeter to measure ECG. They were given 6 i.v. doses of doxorubicin (4 mg kg −1) at weekly intervals. ICRF‐187 (50 mg kg−1) or monoHER (500 mg kg−1) were administered i.p. 1 h before doxorubicin administration. In the 2 monoHER groups the treatment continued with either 1 or 4 additional injections per week. A saline and monoHER treated group served as controls. After these 6 weeks, they were observed for another 2 weeks. 3 At the end of this study (week 8) the ST interval had increased by 16.7 ±2.7 ms (mean ± s.e.mean) in doxorubicin‐treated mice. At that time, the ST interval had increased by only 1.8 ±0.9 ms in ICRF‐187 co‐mediated mice and in monoHER co‐medicated mice by only 1.7 ±0.8 and 5.1 ± 1.7 ms (5‐ and 2‐day schedule, respectively, all P< 0.001 relative to doxorubicin and not significantly different from control). The ECG of the control animals did not change during the entire study. The QRS complex did not change in either group. 4 It can be concluded that monoHER protects against doxorubicin‐induced cardiotoxicity and merits further evaluation in this respect.

Alpha-tocopheryl phosphate is a novel apoptotic agent

Alpha-tocopheryl succinate (TOS) is a well-known potent and selective apoptotic agent. This apoptotic activity has been ascribed to its detergent-like property which is also shared by the structurally related compound, alpha-tocopheryl phosphate (TOP). TOP meets the structural requirements that have been described for the apoptotic activity of TO esters, i.e. the combination of three structural, one functional, one signalling and one hydrophobic domain. In this study, we have investigated the effect of TOP on the osteosarcoma cell line MG-63 using TOS as a reference compound. As compared with TOS, TOP showed a higher proliferative and apoptosis inducing activity on the MG-63 cancer cell line. The cytotoxic effect of TOP and TOS seems to be due to the effect of the intact compounds, since only a minor conversion into alpha-tocopheryl (TO) could be detected. EPR experiments showed that TOS and TOP reduced membrane fluidity, whereas TO had no effect. In addition, induction of erythrocyte hemolysis by TOP depended on the pH. These results suggest that the detergent-like activity of these compounds might be involved in their biological effect. Due to the potent biological activities, TOP might be clinically useful.

Effect of dimethyl sulfoxide (DMSO) on the electrocardiogram (ECG) in freely moving male balb/c mice

1. Since dimethyl sulfoxide (DMSO) is a solvent which is often used for drugs in animal studies, we investigated the effect of a daily administration of DMSO on the telemetrically obtained electrocardiogram (ECG) in freely moving male Balb/c mice. 2. During treatment with 4.5 ml 100% DMSO/kg i.p. 5 days per week during 3 weeks, DMSO caused substantial cardiotoxicity. The ST-interval increased significantly after 1 week by 2.2 +/- 1.3 msec and also the ECG wave form changed completely in time. 3. During treatment with 4.5 ml 50% DMSO/kg i.p. 5 days per week during 3 weeks, no significant difference was observed compared with the control animals. 4. During the entire study the maximal heart rate and body weight remained constant in all treated groups. 5. The data indicate that DMSO can not be used in a 100% concentration to dissolve compounds that are tested for protection against the cardiotoxicity of cytostatics.

Identification of the metabolites of the antioxidant flavonoid 7-mono-O-(β-hydroxyethyl)-rutoside in mice.

The clinical use of the anticancer drug doxorubicin is limited by severe cardiotoxicity. In mice, the semisynthetic antioxidant flavonoid 7-mono-O-(β-hydroxyethyl)-rutoside (monoHER) has been successfully used as a protector against doxorubicin-induced cardiotoxicity. However, most monoHER has already been cleared from the body at the time that doxorubicin concentrations are still high. This result suggests that not only the parent compound monoHER itself but also monoHER metabolites could be responsible for the observed cardioprotective effects in mice. Therefore, in the present study, we investigated the metabolism of monoHER in mice. Mice were administered 500 mg/kg monoHER intraperitoneally. At different time points after monoHER administration, bile was collected and analyzed for the presence of monoHER metabolites. The formed metabolites were identified by liquid chromatography-diode array detection-time of flight-mass spectrometry. Thirteen different metabolites were identified. The observed routes of monoHER metabolism are methylation, glucuronidation, oxidation of its hydroxyethyl group, GSH conjugation, and hydrolysis of its disaccharide. In line with other flavonoids, methylated monoHER and the monoHER glucosides are expected to have relatively high cellular uptake and low clearance from the body. Therefore, these metabolites might contribute to the observed protection of monoHER against doxorubicin-induced cardiotoxicity.

Stability of monoHER in an aqueous formulation for i.v. administration.

MonoHER is a semisynthetic flavonoid used successfully in modulating the cardiotoxic effect of doxorubicin but not its antitumor activity. The oral bioavailability of monoHER is <1%. Therefore, it should be prepared as an i.v. formulation for use in clinical trials. The solubility of monoHER in water is highly pH dependent. At pH</=8.3 the drug precipitates 4 h after preparation. DMSO was tested for enhancing the solubility of monoHER in aqueous solutions. In all DMSO-based aqueous solutions monoHER recrystalized again at pH<8.3 and room temperature within 4 h after preparation. Moreover, the stability of monoHER was lower in a DMSO stock solution than after dilution with an aqueous solution. The stability of monoHER was tested in alkaline solutions (pH 8.3 and 9.5) using an HPLC-DAD procedure to detect all possible degradation products within 10 min after injection. Minor degradation occurred to monoHER in alkaline solutions when exposed to daylight or 1% H(2)O(2). MonoHER intensively degraded when exposed to a high temperature (80 degrees C). The stability of monoHER was almost the same in saline or 5% glucose when kept at room temperature and an alkaline pH of 8.3 and 9.5. Under shelf-life conditions the stability of monoHER in 5% glucose (pH 8.4), decreased with about 10% during 48 h after preparation.

Changes in neuroreceptor function of tracheal smooth muscle following acute ozone exposure of guinea pigs

We studied the effect of in vivo ozone inhalation (3 ppm, 2 h) on neuroreceptor function in guinea pig tracheal smooth muscle in vitro and the role of the epithelial layer in this process. Changes in smooth muscle tension after stimulation of the muscarinic- and beta-adrenergic receptor were recorded isometrically and stained tracheal tissue sections were histologically evaluated for changes in the epithelial and smooth muscle layer. Ozone exposure resulted in an increase in maximal contraction following stimulation of the muscarinic receptor, whereas pD2 values remained unchanged. After stimulation of the beta-adrenergic receptor no increase in maximal relaxation but only an increase in pD2 value was observed after correction for differences in precontraction level in control- and ozone-exposed situations. Mechanical removal of the epithelial layer resulted in a slight increase of the maximal contraction level after stimulation with methacholine in the control situation, whereas exposure to ozone resulted in a strong decrease of the maximal contraction level under these conditions. Histological stainings showed a slight and focal influx of neutrophilic granulocytes in the epithelial layer, submucosal layer and airway lumen after exposure to ozone. These data support the idea that ozone is able to increase the maximal degree of airway narrowing upon muscarinergic stimulation, i.e. a hyperreactivity response. The results also suggest that functionally altered epithelium plays an important role in the process of ozone-induced hyperreactivity, possibly linked with an early inflammatory response.

Fully automated determination of a new anthracycline N-l-leucyldoxorubicin and six metabolites in plasma by high-performance liquid chromatography with on-line sample handling

N-l-Leucyldoxorubicin (Leu-Dox) was developed as a prodrug of doxorubicin (Dox) in order to diminish the cardiotoxic side-effect associated with repeated anthracycline treatment. To study the pharmacokinetics of Leu-Dox, Dox and other metabolites a sensitive and selective assay was needed. Leu-Dox and six of its known metabolites were extracted from plasma using an in-line reversed-phase precolumn (40-50 microns C8 particles). The trapped analytes were subsequently flushed to the analytical column (3 microns C18) using 0.5 ml of phosphate buffer (pH 3.5)-acetonitrile (2:1, v/v), which also served as the isocratic mobile phase. Within 12 min, a clean baseline-resolved chromatogram is obtained by fluorescence detection. Recoveries were almost quantitative and highly reproducible, with standard deviations less than or equal to 5.4% and less than or equal to 2.7% at spiked concentrations of 10 and 100 nM. Using 300 microliters of plasma, detection limits ranged from 0.3 to 0.8 nM at a signal-to-noise ratio of 3. The calibration curves were linear from 1 to 300 nM (r2 greater than or equal to 0.999) for each of the seven compounds. The between-day accuracy was in the range 91-99% and 99-105% at 10 and 100 nM, respectively, with standard deviations of 1-4%. Application of the assay to the analysis of plasma from patients after administration of Leu-Dox proved successful.

The effect of ozone exposure on the release of eicosanoids in guinea-pig BAL fluid in relation to cellular damage and inflammation

The observed effects after ozone exposure strongly depend on ozone concentration and exposure time. We hypothesized that depending on the O3 exposure protocol, mainly either an oxidant damage or an inflammation will determine the O3 toxicity. We compared two different ozone exposure protocols: an acute exposure (3 ppm 2 h) for studying the oxidant damage and an exposure (1 ppm 12 h) where an inflammatory component is also probably involved. We measured LDH activity and protein and albumin exudation as markers for cellular damage. After the acute exposure an increase in LDH activity was measured and after exposure to 1 ppm ozone for 12 h the exudation of protein and albumin was also enhanced. The histological examinations showed a neutrophilic inflammatory response only after exposure to 1 ppm ozone for 12 h. The acute exposure protocol resulted in an increased release of PGE2, PGD2, PGF2α and 6-ketoPGF1α whereas exposure to 1 ppm ozone for 12 h led to an additional release of LTB4. No effects were measured on the release of TxB2 and LTC4/D4/E4. These changed amounts of eicosanoids will probably contribute to the ozone-induced lung function changes.

Acute exposure to ozone does not influence neuroreceptor density and sensitivity in guinea pig lung

The effects of acute exposure of guinea pigs to 3 ppm of ozone for 2 h on the receptor density and sensitivity of the muscarinergic-, the histaminergic- and the beta-adrenergic receptor systems were studied, in order to provide more insight in the complex mechanisms underlying the well known ozone-induced changes in receptor functionality. The exposure to ozone did not change either the total amount of receptors present in lung tissue, nor the receptor sensitivity of the systems studied. Although no effects were observed, this does not yet fully exclude the receptor system for being a target of ozone exposure. The receptor function can be changed after exposure to ozone, e.g., the coupling with the G-protein can be influenced. Furthermore, the G-protein itself may have been altered or changes can occur at lower levels in the receptor signal transmission route leading to functional changes after stimulation of the receptor with an agonist.