MATERIALS AND METHODS

Experimental Animals Ethics Committee of Istanbul University, Cerrahpasa Faculty of Medicine approved the following study. About 80 healthy adult male Sprague Dawley rats, weighing 250-300 g at the start of experiment were used to conduct this study.

Drugs: Locally purchased formulation of Cipro and Doxo were used, Ciproxin Flacon (400 mg; Bayer, Turkey) and Adriamycin Ampoule (10 mg/5 mL; Deva Pharmaceuticals, Turkey), respectively.

Dose schedules: Rats were randomly divided into two main groups i.e., acute and chronic and 10 subgroups, having 8 animals in each to receive the same cumulative doses of Doxo but in different schedules. The doses and schedules are given as follows.

Rats were given different doses (6 and 15 mg kg^-1) of Doxo alone and in combination with Cipro (20 mg kg^-1; daily) as single intraperitonial injection (i.p.). Multiple i.p. injections of Doxo (1 and 2.5 mg kg^-1 twice a week, cumulative dose: 6 and 15 mg kg^-1, respectively) alone and along with Cipro (20 mg kg^-1, i.p. daily) were given to rats for the duration of three weeks. Whereas, control group rats received serum physiological solution.

Plasma analysis: Blood samples were collected on the third day of last injection following acute and chronic treatment protocol. Plasma cTnI levels were determined using a commercially available Enzyme-Linked Immuno Sorbent Assays (ELISA) kits (East Biopharm, China).

General toxicity: General toxicity was estimated by measuring the survival percentage and by considering the relative organ weight. At the end of chronic and acute dose protocols, rats were sacrificed by cervical dislocation; heart, liver, testes, spleen and kidneys were removed quickly after visual inspection and their weights were recorded. Organ weight was expressed as relative organ weight, i.e., as follows:

Statistical analysis: All values are presented as Mean±SE. Statistical significance (p<0.05) was determined by one-way ANOVA followed by post hoc Tukeys test (Graph Pad Prism 4.0). Whereas, two-way ANOVA was carried out to study the interaction between Doxo and Cipro groups.

RESULTS

General toxicity
Survival percentage: Rats treated with chronic high dose of Doxo alone (2.5 mg kg^-1) showed 50% mortality rate, which was significantly higher than that of control, Doxo (1 mg kg^-1) and Doxo+Cipro (1 +20 mg kg^-1) treated rats (p<0.05, Fig. 1). Whereas, mortality rate was 38% in rats treated with Doxo+Cipro (2.5 +20 mg kg^-1) in comparison to control. No mortality was observed in rats of chronic Doxo (1 mg kg^-1) and Doxo+Cipro (1+20 mg kg^-1). The rats of acute group under low and high doses (6 and 15 mg kg^-1 or with Cipro 20 mg kg^-1) therapy maintained 100% survival.

Relative organ weight: The relative heart weight of rats treated with Doxo 2.5 mg kg^-1 showed significant reduction than that of control (34. 6%). Whereas, 56% of reduction was restored by the addition of Cipro (20 mg kg^-1) to this group (p<0.0001). There was no significant difference between the other groups (Fig. 2).

The relative liver weight of rats treated with Doxo 1 mg kg^-1 showed significant increase of 34.6% with respect to control (p<0.01). However, Doxo+Cipro (2.5+20 mg kg^-1) in comparison to Doxo 1 mg kg^-1 showed a significantly minor liver atrophy (22%, Fig. 3).

Fig. 1:	Percentage survival (Kaplan-Meier Plot) of rats treated with multiple i.p. injection of saline, Doxo (1 or 2.5 mg kg-1) alone and in combination with Cipro (20 mg kg-1 daily) for the duration of 3 weeks and till the end of experiment. Dotted lines representing SE. *p<0.05 vs. Control, Doxo (2.5 mg kg-1), Doxo+Cipro (2.5+20 mg kg-1)

All the treated group except Doxo+Cipro (1+20 mg kg^-1) showed a significant reduction of 76-80% in relative spleen weight than that of control (p<0.0001, Fig. 4).

A significant dose dependent increase (26%) in relative kidney weight was observed in Doxo (2.5 mg kg^-1) group (p<0.0001). While, it was restored to control values when Cipro (20 mg kg^-1) was added to this group (Fig. 5).

Relative testes weight of rats treated with Doxo (2.5 mg kg^-1) showed a significant reduction of (50%) (p<0.0001). However, the addition of Cipro (20 mg kg^-1) to this group brought the levels back to normal (Fig. 6).

Whereas, rats under the acute schedule of Doxo or Doxo+Cipro groups did not show significant variations in organ weights.

Cardiac troponin-I (cTnI) plasma concentration: Extend of cardiac injury induced by Doxo-Cipro following acute and chronic dose protocols was determined by estimating the plasma cTnI levels of treated rats. The rats given saline solution showed 1.317±0.12 ng mL^-1 cTnI levels during the experiment. The administration of Doxo (1 or 2.5 mg kg^-1) alone and along with Cipro (20 mg kg^-1) showed a significant increase (p<0.0001) in cTnI levels in comparison to control (Fig. 7).

Fig. 2:	Effect of Doxo alone and along with Cipro on relative heart weight of Sprague Dawley rats. Values are the Means±S.E, bars having similar letters do not differ significanly (p<0.0001)

Fig. 3:	Effect of Doxo alone and along with Cipro on relative liver weight of Sprague Dawley rats. Values are the Means±S.E, bars having similar letters do not differ significanly (p<0.01)

Fig. 4:	Effect of Doxo alone and along with Cipro on relative spleen weight of Sprague Dawley rats. Values are the Means±S.E, bars having similar letters do not differ significanly (p<0.0001)

Fig. 5:	Effect of Doxo alone and along with Cipro on relative kidney weight of Sprague Dawley rats. Values are the Means±S.E, bars having similar letters do not differ significanly (p<0.0001)

Fig. 6:	Effect of Doxo alone and along with Cipro on relative testes weight of Sprague Dawley rats. Values are the Means±S.E, bars having similar letters do not differ significanly (p<0.0001)

The highest concentration of cTnI was found in rats treated with high dose of Doxo (2.5 mg kg^-1) alone followed by Doxo+Cipro (2.5+20 mg kg^-1) (4.405±0.07464, 4.335±0.1913 ng mL^-1, respectively).

Similar trend was observed in the plasma cTnI concentration of rats that followed acute protocol. Single i.p. injection of low or high doses of Doxo (6 or 15 mg kg^-1) alone and in combination with Cipro (20 mg kg^-1) showed significant increase in cTnI plasma levels than that of control (p<0.0001, Fig. 7). The highest increase in plasma cTnI concentration was observed in rats given high dose of Doxo (15 mg kg^-1) alone (3.921±0.2018 ng mL^-1) closely followed by Doxo+Cipro (15+20 mg kg^-1), (3.925±0.2836 ng mL^-1).

The significance was at par, when plasma cTnI levels of rats treated with chronic low dose Doxo (1 mg kg^-1, cumulative 6 mg kg^-1) was compared with acute low dose Doxo (6 mg kg^-1) alone or along with Cipro (Fig. 8). Similarly, chronic high dose Doxo (2.5 mg kg^-1: Cumulative 15 mg kg^-1) and acute high dose Doxo (15 mg kg^-1) groups, either alone or in combination with Cipro were found to be statistically similar in terms of cTnI plasma levels (Fig. 9).

Fig. 7:	cTnI plasma levels of Doxo alone and along with Cipro following chronic and acute dose protocols. Values are the Means±S.E, bars with similar letters do not differ significanly (p<0.0001)

Fig. 8:	Cardiac troponin-I (cTnI) plasma concentration of rats administered with acute Doxo alone and along with Cipro. Values are the Means±S.E, bars with similar letters do not differ significanly (p<0.05)

DISCUSSION

A dose dependent cumulative cardio-toxicity of Doxo always remains a major clinical concern and led to the development of several animal models of chronic and acute cardio-toxicity (Bertazzoli et al., 2014; Herman et al., 2000).

Fig. 9:	Cardiac troponin-I (cTnI) plasma concentration of rats treated chronically with Doxo alone and along with Cipro. Values are the Means±S.E, bars with similar letters do not differ significanly (p<0.05)

Cipro is a well-known broad spectrum antibiotic that has been successfully used in daily clinical practice as well as in oncology as a supportive therapy for immuno-compromised patients. So, the hypothesis behind this experiment was to study the interaction of Cipro with Doxo and to assess its cardio-protective or cardio-toxic effects, following different dose protocols in Sprague Dawley rats.

The use of plasma levels of cardiac troponin-I (cTnI) has been well-established in clinical practice to measure cardio-toxicity in cancer patients (Lipshultz et al., 2004; Urbanova et al., 2009). It has also been used as biomarker to identify the extent of cardiac injury in preclinical studies (Bertinchant et al., 2003; Herman et al., 1998). The current report showed an increase in cTnI levels at each dose of Doxo alone or in combination with Cipro followed either acute or chronic dose schedule. However, the maximum cTnI concentration was observed in rats treated with high doses of Doxo alone (2.5 and 15 mg kg^-1 in chronic and acute dose schedules, respectively) and in combination with Cipro (2.5+20 mg kg^-1 and 15+20 mg kg^-1). The probable cause of increase in cTnI levels is damage to myocardium which could be due to anemic hypoxia resulting due to alteration in erythropoiesis (Wallace et al., 2004). A dose dependent increase in cardiac lesions was also reported in Fischer rats treated with Doxo 1-2 mg kg^-1 upto 10-14 weeks (Mettler et al., 1977) and spontaneously hypertensive Wister-Kyoto rats treated with cumulative dose of 12 mg kg^-1 (Herman et al., 1985). Literature indicated that myocardial injury increases with increase in cumulative dose, as observed in Sprague Dawley rats treated with the cumulative dose of 10, 13.5 and 18 mg kg^-1 and in rabbits administered with 11, 14, 16 mg kg^-1 of Doxo (Solcia et al., 1981) and same is confirmed by our study.

On the other hand, literature showed that Cipro causes cardiotoxicity by initiating the oxidative stress in the heart, nitric oxide is thought to play a key role in this toxicity in juvenile rats (Saracoglu et al., 2009). Alternative hypotheses can be the involvement of prostaglandins (Das, 1981), histamine (Bristow et al., 1983) and C-13 anthracycline metabolites (Olson et al., 1988) as mediators of cardiac toxicity. However, in the current study Cipro did not cause any significant change in plasma cTnI levels, but our previous reports indicated a significant increase in Doxo plasma concentrations after the concomitant use (Shahzadi and Yazici, 2014). This shows that there is a pharmacokinetic interaction but this interaction did not cause any significant effects on plasma cTnI concentration. It can be speculated that the non-significant increase in cTnI plasma levels of Cipro treated group in comparison to Doxo group can be due to the use of minimum effective dose of Cipro. The minimum effective dose was selected to reduce the other possible toxicities induced by Cipro. However, further investigation is needed to evaluate the exact role of Cipro in myocardial injury.

Doxo is administered systemically so it may harm the healthy tissues as well, leading to unfavorable effects to tissues, which ultimately, reduces the quality of life in cancer patients. In this study, Doxo alone and in combination with Cipro following different dose schedule in Sprague Dawley rats was tested to examine their effects on different body organs. Results indicated that the rats treated with high dose of Doxo (2.5 mg kg^-1) alone under chronic dose schedule leads to atrophy of rats heart (Fig. 2), that can be attributed to apoptosis and necrosis of heart tissues which is usually observed when cardiac cells are exposed to Doxo (Zhu et al., 2009). The loss of cardiac structure and deteriorated function usually observed in long-term treatment with Doxo (Migrino et al., 2008; Shakir and Rasul, 2009) which is in accordance with the findings of the current study recorded under chronic model. Whereas, the results of the following study also showed that Cipro helps in the restoration of heart cells by decreasing the Doxo induced necrosis.

Relative liver weight showed non-significant differences in comparison to control while interestingly a slight increase in liver weight was observed in Doxo 1 mg kg^-1 treated rats in comparison to Doxo+Cipro (2.5+20 mg kg^-1) (Fig. 3). The slight increase in liver weight at low dose Doxo is not clear, however, the possibility of hyperlipidemia cannot be ruled out.

All the treatment groups showed decrease in relative spleen weight except Doxo+Cipro (1+20 mg kg^-1) when compared with control. Drastic decrease in relative spleen weight was observed in the rats treated with high dose of Doxo alone (2.5 mg kg^-1 following chronic schedule) which can be correlated with changes in hematological parameters (Herman et al., 1985). Hematological changes such as low RBC, Hb and HCT counts was observed in patients suffering from breast cancer (Panis et al., 2012) as well seen in hypertensive and Wister-Kyoto rats treated with 12 mg kg^-1 cumulative dose of Doxo (Herman et al., 1985). Literature also showed bone marrow depression in patient undergoing chemotherapy with Doxo (Carvalho et al., 2009; Vici et al., 2011).

Relative weight of kidney was increased in rats treated with Doxo which is in accordance to earlier findings of Sharkey et al. (2013), who reported that this increase was the result of glomerular damage, as observed in patients undergoing Doxo treatment (Bardi et al., 2007). In present study, application of Doxo along with Cipro at all doses improved the kidney weight of rats; literature also confirmed that Cipro does not exert nephrotoxicity in rats (Baykal et al., 2005).

Testicular atrophy was observed in rats treated with chronic Doxo alone both with low and high doses (1 and 2.5 mg kg^-1). The effect was severe at chronic high dose of Doxo. These findings are in accordance with previous findings which suggest that Doxo treated rats showed testicular toxicities (Xin et al., 2012). It is evident from the literature that chemotherapeutic agents also affects the non-target tissues like testes (Da Cunha et al., 1983; Suter et al., 1997) and Doxo inhibits spermatogenesis (Howell and Shalet, 2005; Yeh et al., 2007) and finally leads to infertility. The underlying mechanism for Doxo induced testicular toxicity is yet not clear but recent findings strongly suggest the oxidative stress from lipid peroxidation (Atessahin et al., 2006; Yeh et al., 2007) and cellular apoptosis (Shinoda et al., 1999) as major reasons. The reason regarding the improvement in relative testes weight in rats treated with Cipro+Doxo in present study is not clear and needs further investigation. The increased toxic effects can be subjected to pharmacokinetic interaction. Our previous study showed an increase in plasma Doxo concentration when Doxo was combined with Cipro (Shahzadi and Yazici, 2014).

Whereas, interestingly organ weights showed non-significant differences in acute model which was contrasting to the previous findings (Minotti et al., 2004; Pereira et al., 2011; Weiss, 1992) who reported that acute model caused hypertrophy of heart. In addition to this, it is also reported that a single injection of 10 mg kg^-1 Doxo initiated decrease in heart mass followed by a restoration to control values (Lushnikova et al., 2004) which was not observed in our acute model that can be due to low sample size or may be the use of different traits of rats. As there are genetic differences which can alter the metabolism and vulnerability to toxic agents (Kacew, 1996; Mas et al., 2000). So, further investigation is required for the better interpretation of results in acute and chronic model of Doxo along with Cipro, because both of these drugs are commonly prescribed in chemotherapy and can lead to unseen toxicity.

CONCLUSION

Findings of this study confirm that Doxo showed dose dependent cardio-toxicity and Cipro did not show any significant change in cTnI levels. Whereas, in general Cipro played an important role in improvement of organs weight. This combination needs further investigation in terms of cardio-toxicity so that this combination can be safely prescribed in clinical practice to the patients undergoing chemotherapy with Doxo and need Cipro for being immune-compromised or to treat other complications.

Abberivations

ACKNOWLEDGEMENT

The present study was supported by Bilimsel Arastirma Projeleri Birimi (BAP), Istanbul University grant 29224.