Pretreatment with Tamoxifen does not Change Acute Epirubicin Cardiotoxicity in Rats

The use of anthracycline antibiotics as anticancer agents is limited by a cumulative dose-related cardiotoxicity (Wojtacki et al. 2000). Tamoxifen, a synthetic non-steroidal antioestrogen, in addition to its application in treatment and prevention of breast cancer, is also a modulator of multidrug resistance and an antioxidant (Perumal et al. 2005). Combination of epirubicin and tamoxifen has been used in breast cancer (Pico et al. 2004) and lung cancer therapy (Chen et al. 2000). Furthermore, high-dose tamoxifen have been used in phase I trials in patients with advanced malignancies (Perez et al. 2003). Clinical studies have not reported any increased cardiotoxicity with the combination that outweighs beneficial effects (Wils et al. 1999; Fargeot et al. 2004; Pico et al. 2004). Due to its multiple properties, tamoxifen can reverse resistance to anthracyclines (Lucci et al. 1999), but the possibility of increased cardiotoxicity of anthracyclines may represent a clinical problem. In this preliminary study the combination was examined in an isolated rat heart model. Animals and treatments. Male Wistar rats (Møllegaard Breeding Laboratory, Ejby, Denmark), weighing 150 g were housed in grid-bottom metal wire cages in a room maintained at a 12 hr light/dark cycle and a temperature of 20–22 °. The animals were acclimatized for 14 days before start of experiments, and had free access to a standard rat diet (Bekay Feeds, B & K Universal, UK) and water during this period and during the following treatment period. Rats were separated in two groups (tamoxifen and control) and received either tamoxifen (12 mg/ml) or vehicle 1,2-propandiol (control group) by a gastric tube each morning for 9 days. The volume administered was based on the actual weight of the rat and a dosage of 40 mg/kg. The rats were weighed at the start of experiments, and at regular intervals during treatment. All experiments were approved by The Norwegian Animal Research Authority and conducted according to the European Convention for the Protection of Vertebrates used for scientific purposes. Cardiotoxicity model. The rats were anaesthetised with an intraperitonial injection of pentobarbital 50 mg/kg after 9 days of treatment, and hearts were excised for isolated heart perfusion after an intravenous injection of 500 IU of heparin. A Langendorff constant volume perfusion isolated rat heart model described in detail previously were used to analyze the cardiotoxicity of epirubicin (Schjøtt et al. 1996a & b). A water-filled latex balloon mmHg was placed in the left ventricle and connected to a pressure transducer for the recording of left ventricular developed pressure (LVDP), heart rate and secondarily derived contractility indices (dP/dt). The balloon was inflated to achieve a level of left ventricular end diastolic pressure (LVEDP) of 4–8 mmHg during stabilization. The balloon volume was then held constant for the duration of the experiment. A second pressure transducer was connected to a side arm of the aortic cannula for the recording of aortic pressure, an index of coronary vascular resistance during constant volume perfusion. An epicardial electrocardiogram (ECG) was recorded throughout the experimental period by two electrodes attached directly to the heart. The ECGs were analyzed to determine the incidence of ventricular fibrillation. The heart was considered to be in ventricular fibrillation if an irregular undulating baseline was seen on the ECG. The heart was considered to be in sinus rhythm if normal sinus complexes occurring in a regular rhythm were apparent on the ECG. Pressure signals and ECG were amplified (Quadbridge and Bio Amp, AD Instruments, London, UK), and processed by a Power Lab system (AD Instruments, London, UK). The perfusion medium was a modified, oxygenated (95% O2 and 5% CO2) Krebs-Henseleit bicarbonate buffer containing (in mmol/l): 118.5 NaCl, 25.0 NaHCO3, 11.1 glucose, 4.7 KCl, 1.2 KH2PO4, 1.2 CaCl2, 1.2 MgSO4. Hearts were frozen in liquid nitrogen at the end of experiments for biochemical analysis. Myocardial content of epirubicin, tamoxifen and metabolites was analysed by high performance liquid chromatography (HPLC) techniques described in detail previously (Schjøtt et al. 1996a; Kisanga et al. 2003). Experimental protocol and statistical analysis. Hearts were allowed to stabilize for 20 min. with constant volume flow conditions (12 ml/min.). Hearts which failed to achieve a LVDP of 70 mmHg or developed serious arrhythmias in this period were excluded from the experiments. The final number of rats in each group was 6. Experiments started by a 5 min. period where baseline values were obtained (baseline period). Epirubicin (2 mg/ml at a rate 0.1 ml min.) was infused by a side arm of the aortic cannula for 20 min. (epirubicin period) followed by a 10 min. wash-out phase (recovery period). Statistical analysis was performed using SPSS for Windows (Release 11.0.1. 2001. Chicago: SPSS Inc). A repeated measurements analysis was used for comparing physiological data and weight, and a Students t-test for biochemical data. Data are presented as mean±S.E.M. P-values of less than 0.05 were considered significant. Results. There were no differences between the groups concerning baseline values, except that tamoxifen caused a significant (P<0.05) reduction in weight compared to the control group (table 1). The effect of tamoxifen on weight in rats has been reported previously (Darvari & Boroujerdi 2005), and is associated with reduced food and water consumption. Infusion of epirubicin depressed LVDP and secondarily derived contractility indices to about 40–60% of baseline in both groups while the effect on heart rate was negible. Left ventricular developed pressure heart rate product (LVDP×heart rate) were 48±8% for vehicle and 50±4% for tamoxifen at the end the epirubicin infusion. The corresponding values at the end of a 10 min. recovery period were 55±5% and 66±7% (fig. 1). During infusion of epirubicin and recovery, the expected increase and subsequent decrease in LVEDP and aortic pressure was observed. No significant differences were observed in physiological or electrophysiological parameters during epirubicin infusion or recovery between the groups. Cardiac content of tamoxifen and metabolites was significantly increased after feeding, but myocardial content of epirubicin were similar in the two groups at the end of experiments (table 1). Left ventricular developed pressure heart rate product (LVDP×HR) during baseline, epirubicin and recovery period. Discussion. The relevance of the present model for clinical cardiac toxicity may be argued. However, the acute and chronic toxicity of anthracyclines seems to share the same mechanisms, with chronic toxicity arising from repeated episodes of acute toxicity with cumulative damage as a result (Pelikan et al. 1986). Furthermore, the isolated rat heart model has been suggested as a valuable tool for screening toxicity of drug combinations (Pouna et al. 1996). The effect of epirubicin on physiological parameters were similar to results observed in two previous studies (Schjøtt et al. 1996a & b), indicating a good reliability of the toxicological model. Importantly, the levels of epirubicin in the perfusate in the model (Schjøtt et al. 1996a) are comparable to Cmax values reported after high-dose epirubicin in man (Basser et al. 1994). The haemodynamic effects of anthracyclines reflect alterations in systolic and diastolic properties (Joyeux et al. 2001). Moreover, they involve generation of free radicals, lipid peroxidation and impaired calcium control (Fogli et al. 2004; Minotti et al. 2004). Doxorubicin has been shown to induce serious arrhythmias in a comparable model with different experimental design (Joyeux et al. 2001), but we observed no serious arrhythmias during or after epirubicin infusion. This discrepancy could be due to use of a anthracycline with different pharmacokinetic and cardiotoxic properties (Ramanathan-Girish & Boroujerdi 2001). In agreement with previous results in rats we observed that high tissue levels of tamoxifen and metabolites were reached after short treatment duration (Kisanga et al. 2003). Tissue distribution and metabolism of tamoxifen in rats resembles that in man, and heart levels of tamoxifen and metabolites in our results were comparable to those observed in human tissues after clinical doses (Lien et al. 1991). Inhibition of lipid peroxidation in rat liver microsomes by 4-hydroxytamoxifen and tamoxifen has been reported (Wiseman 1994). Furthemore, enhanced expression of MnSOD, the primary antioxidant enzyme in heart mitochondria, has been demonstrated in mice after tamoxifen treatment (Daosukho et al. 2005). In a trial including 64 postmenopausal women a decreased concentration of malondialdehyde, an end-product of lipid peroxidation, and remarkably increased levels of enzymatic and non-enzymatic antioxidants were demonstrated after 3 and 6 months of tamoxifen (20 mg/day) (Thangaraju et al. 1994). Thus, cardioprotection of tamoxifen in the clinic could be related to an enhanced antioxidant effect. The lack of any significant interaction between tamoxifen and epirubicin in our results is unclear, but tamoxifen in a dose of 1 mg/kg for 11 days is reported to inhibit p-glycoprotein (Darvari & Boroujerdi 2005) in rat hearts. A hypothesis is that enhanced antioxidant status (protective effect) is balanced by a change in myocardial distribution of epirubicin (damaging effect). We conclude that pretreatment with tamoxifen does not change the acute cardiotoxicity of epirubicin in this model, and the present results support previous clinical observations (Fargeot et al. 2004).