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Protective role of granulocyte colony-stimulating factor against adriamycin induced cardiac, renal and hepatic toxicities
Toxicology Letters 187 (2009) 40–44
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Protective role of granulocyte colony-stimulating factor against adriamycin induced cardiac, renal and hepatic toxicities Xu-Wei Hou a,∗ , Yu Jiang b , Li-Fang Wang c , Hai-Ying Xu a , Hong-Min Lin d , Xiu-Ying He a , Jian-Jun He d , Sheng Zhang d a
Department of Cardiology, Hangzhou First Municipal Hospital and Hangzhou Hospital, Nanjing Medical University, Huan-sha Road 26, Hangzhou 310006, China Department of Cardiology, Jiangxi Provincial Corps Hospital, Chinese People’s Armed Police Force, Nanchang 330030, China Department of Basic Medicine, Zhejiang Medical College, Hangzhou 310053, China d Department of Internal Medicine, Baodiao Hospital, Zoucheng City 273511, China b c
a r t i c l e
i n f o
Article history: Received 18 November 2008 Received in revised form 20 January 2009 Accepted 21 January 2009 Available online 3 February 2009 Keywords: Granulocyte colony-stimulating factor Adriamycin Toxicity Peroxidative alterations
a b s t r a c t Adriamycin (ADR) causes dose-dependant toxicities in heart, liver and kidneys via inducing the peroxidative alterations in organ tissues. Recent studies showed that the granulocyte colony-stimulating factor (G-CSF) exerts beneficial effects on heart, liver and kidney injuries induced by different pathological conditions. We hypothesize that G-CSF have a protective effect on ADR induced cardiac, renal and hepatic toxicities by inhibiting the peroxidative alterations in organ tissues. Wistar rats were randomly divided into control, ADR, ADR + phosphate buffered saline (PBS) and ADR + G-CSF group (n = 16 in each group). ADR was administered intraperitoneally every other day at the dose of 2.5 g/kg each time per rat (total six times of injection during 2 weeks). Rats in the ADR + GCSF group were injected subcutaneously with G-CSF at the dose of 50 g/(kg day) (for 8 consecutive days). After 8 weeks, the serum and urine biochemistry variables were determined. The malondialdehyde (MDA) level and the glutathione (GSH) content in the heart, the liver and the kidney tissues were measured. ADR caused significant cardiac, renal and hepatic toxicities indicated by the serum and urine biochemistry variables. The tissue MDA level in the heart, kidney and liver in rats treated with ADR were markedly elevated, while the GSH content in these tissues were significantly reduced. G-CSF administration palliated the cardiac, renal and hepatic toxicities. Notably, G-CSF induced significant reduction of MDA level and increase of GSH content in the heart, kidney and liver tissues. This study suggests that G-CSF play an overall protective effect on ADR-induced toxicities in heart, liver and kidneys and the inhibition of tissue peroxidative alterations might contribute to this beneficial effect. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Adriamycin (ADR) is an anti-neoplastic agent used in the treatment of a variety of human neoplasms. However, its clinical use is severely restricted by dose-dependant toxicity in various tissues, including the heart, the liver and the kidneys. ADR-induced cardiotoxicity is characterized by progressive ventricular dilatation and congestive heart failure with a poor prognosis (DeAtley et al., 1999; Cini Neri et al., 1991; Sazuka et al., 1989a,b; Praga et al., 1979; Singal and Iliskovic, 1998; Kakadekar et al., 1997). Experimental studies in animals showed that ADR caused a renal toxicity and produced progressive glomerular injuries (Barbey et al., 1989; Manabe et al., 2001; Malarkodi et al., 2003; Deepa and Varalakshmi, 2005). ADR-induced hepatotoxicity was reported as well (You et al., 2007;
∗ Corresponding author. Tel.: +86 571 87065701x20681; fax: +86 571 87065701x10324. E-mail address: [email protected] (X.-W. Hou). 0378-4274/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2009.01.025
Gokcimen et al., 2007; Goeptar et al., 1993; Bertolatus et al., 1991). These adverse effects of ADR inhibit its clinical use. The exact mechanism of ADR-induced toxicity remains unclear. Some researchers proposed that ADR-induced toxicity is most likely mediated by the formation of an iron–anthracycline complex that generates reactive free radicals (ROS), which in turn, causes diverse oxidative damage on critical cellular components and membrane lipids in the plasma membranes and mitochondria (DeGraff et al., 1994; Lee et al., 1991; Sazuka et al., 1989a,b). This view is well supported by the fact that antioxidants prevent the ADR-induced toxicity in experimental animals as well as in human (Mohamed et al., 2000; Cole et al., 2006). The granulocyte colony-stimulating factor (G-CSF) is a growth factor that promotes the neutrophil and bone marrow stem cells to the peripheral circulation. Neutrophil can generate superoxide and other forms of ROSs. Recent studies showed G-CSF exerts beneficial effects on heart, liver and kidney injuries induced by a variety of pathological conditions. In the setting of the ADR-induced cardiotoxicity, G-CSF treatment protects the cardiomyocyte ultra-
X.-W. Hou et al. / Toxicology Letters 187 (2009) 40–44
structure, inhibits the myocyte apoptosis and enhances the heart function (Tomita et al., 2004; Hou et al., 2006). G-CSF administration increases the hepatic sinusoidal perfusion and enhances the hepatocyte proliferation, thus contributing to liver tissue regeneration after liver injury or liver resection (Sidler et al., 2008; Spahr et al., 2008). Animal studies showed that G-CSF mobilization accelerates the improvement of renal function and prevents the renal tubular injury induced by cisplatin and facilitates the vascular regeneration in mouse kidneys after an ischemia/reperfusion injury (Iwasaki et al., 2005; Akihama et al., 2007). Until now, the effect of G-CSF on ADR-induced oxidative stress and peroxidative alterations are poorly understood. We hypothesize that G-CSF might inhibit the excessive generation of oxidative stress, thus providing an overall protective role in the heart, liver and kidney toxicities induced by ADR administration in rats.
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3. Results 3.1. Blood and urine biochemical variables Table 1 showed the blood and urine biochemical variables in all rats 8 weeks after G-CSF and PBS administration. These biochemical variables are commonly used for the assessment of heart, liver and kidney damages. ADR treatment induced significant increases in the CK-MB, LDH, Cr, BUN, ALT and AST serum levels as well as the 24-h urine albumin level compared to those in the control group. This means that ADR administration caused systemic organ toxicities in rats. In rats from ADR + G-CSF group, the serum CK-MB, LDH, Cr, BUN, ALT, AST levels and the 24H urine albumin level were significantly lowered 8 weeks after the G-CSF administration, when significant differences were seen compared with those in rats from ADR and ADR + PBS groups.
2. Materials and methods 2.1. Animal preparation and samples collection
3.2. Tissue MDA level and GSH content
Adult male Wistar rats (body weight 226 ± 15.5 g) were randomly divided into four groups: control, ADR, ADR + phosphate buffered saline (PBS) and ADR + G-CSF group (n = 16 in each group). ADR was injected intraperitoneally at the dose of 2.5 g/kg each time per rat. The injections were performed every other day during 2 weeks (eight times of injections in all). All rats except the control rats received ADR administration. After 2 weeks of wash-off period, the rats in the ADR + G-CSF group were injected subcutaneously with the G-CSF at the dose of 50 g/(kg day) per rat for 8 consecutive days. Similarly, rats in ADR + PBS group were injected with PBS. After 8 weeks of observation, all rats were anesthetized with ether and the blood samples were collected by heart puncture. The heart, the liver and the kidneys were removed rapidly, perfused with cold saline solution and used for the analysis as described below. The study was approved by the Ethics Committee of Nanjing Medical University.
Fig. 1 shows the tissue MDA levels in the heart, the liver and the kidneys during a time course of 8 weeks. ADR administration induced dramatic, constant increases of MDA production in these tissue samples. A temporary, but dramatic elevation of MDA level was observed in each tissue sample 1 week after G-CSF administration, after which the MDA levels begun to decline constantly. Eight weeks later, the tissue MDA levels in rats from the ADR + GCSF group reached their minimums, with a significant difference compared to those of ADR and ADR + PBS groups, respectively. Fig. 2 shows the GSH content in the heart, the liver and the kidney tissue samples. Compared with that of controls, the GSH content in ADR treated rats decreased dramatically. The G-CSF treatment induced a constant increase of GSH content in three organs. This trend lasted for 8 weeks. After 8 weeks, the GSH content in the heart, the liver and the kidney tissues in ADR + G-CSF group were markedly elevated, with a significant difference compared to those of ADR and ADR + PBS groups, respectively.
2.2. Blood and urine biochemical variables Blood samples were coagulated at room temperature for 30 min followed by centrifugation for the extraction of the serum. The serum creatine kinase MB (CK-MB) levels were assayed using the CK-MB test kit (Bayer Diagnostics). CK-MB activity was measured in freshly separated serum according to the manufacturer’s instructions. The serum lactate dehydrogenase (LDH) activity was estimated using a commercially available kit using an UV–vis spectrophotometer. The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were determined according to manufacturer’s instructions. The serum creatinine (Cr) and blood urea nitrogen (BUN) level was measured by Jaffa’s method with a creatinine test kit and by the urease indophenol method with a nitrogen B test kit. The 24 h urine was collected 1 day before sacrifice for albumin analysis. The albumin level was measured by a bromo-cresol green method using an A/GB test kit. 2.3. Tissue malondialdehyde (MDA) measurement The MDA level in the heart, liver and kidney samples was determined using the thiobarbiturate reaction. 0.5 mL of 0.5% butylated hydroxytoluene was added to 2 mL of tissue homogenate to prevent lipid autoxidation. After tissue protein precipitation and centrifugation, 1 mL of 0.67% thiobarbiturate–water solution was added to the supernatant and boiled for 60 min. After cooling, the optical density was assayed at a wavelength of 530 nm. We used 1,1,3,3-tetraethoxypropane as the standard. The MDA level was expressed as nmol/g tissue. 2.4. Tissue glutathione (GSH) measurement The GSH content of the heart, the liver and the kidney samples was determined. Fifty microliters of tissue extract diluted 10-fold in 100 mM NaHP2O4 and 5 mM ethylene–diaminetetra–acetic acid buffer at pH 7.5 was added to 50 L of glutathione oxidoreductase (5 U/50 L) and 50 L of 2.5 mM 5,5 -dithiobis-(2nitrobenzoic acid). The reaction was initiated by the addition of 50 L of 1.2 mM nicotinamide adenine dinucleotide phosphate in buffer. The rate of color change at 410 nm, which is proportional to the amount of total GSH in the samples, was monitored using a plate reader. The results are expressed as mol/mg protein. 2.5. Statistics All data were expressed as mean ± S.E.M. Statistical significance of differences was analyzed by the one-way ANOVA with PASS 13.0 and a P value <0.05 was considered as statistically significant.
4. Discussion This study, for the first time, evaluated the effect of G-CSF on the ADR-induced toxicities in heart, liver and kidneys and its influence on the tissue peroxidative alterations in these organs. In accordance with previous studies, we observed that ADR induced overall cardiac, renal and hepatic toxicities in rats. The G-CSF administration significantly reduced the CK-MB, LDH, Cr, BUN, ALT, AST level and the 24-h urine albumin level, suggesting that G-CSF provides overall protective effects in heart, liver and kidney. In this study, the tissue peroxidative alterations in the three organs were markedly inhibited by G-CSF, as the MDA level was significantly reduced and the GSH content was dramatically increased. We propose that the inhibition of peroxidative alterations might contribute to the beneficial effect of G-CSF in reducing ADR-induced cardiac, renal and hepatic toxicities. In a physiological condition, intracellular ROS acts as a second messenger to a variety of growth factors, including G-CSF, which induces the activation of down stream signaling molecules (Zhu et al., 2006). However, when the ROS is excessively produced, for instance, in the setting of ADR administration, it becomes deleterious and causes damage on cellular components and membrane lipids in the plasma membranes and mitochondria. The lipid peroxidation occurs in cellular components and membrane lipids when the ROS levels are elevated, leading to the release of reactive aldehydes, such as MDA. The effect of G-CSF on the peroxidative alterations was explored previously. Kitagawa et al. reported that G-CSF enhanced superox-
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Table 1 Biochemical variables in all rats. Group Control ADR ADR + PBS ADR + G-CSF * # +
CK-MB (U/L) 78.3 1770.6 1879.6 599.9
± ± ± ±
11.2 113.6* 143.7* 155.8+ , *
LDH (U/L) 119.6 2100.4 2231.9 681.7
± ± ± ±
BUN (mmol/L) 43.8 234.7* 176.9* 154.4+ , *
4.6 21.5 23.6 14.5
± ± ± ±
1.9 9.6* 11.8* 3.2+ , #
Cr (mol/L) 43.7 223.6 243.9 134.6
± ± ± ±
11.6 67.8* 112.4* 77.3+ , #
Urine albumin (mg) 4.6 23.6 25.6 14.2
± ± ± ±
1.9 9.4* 11.2* 7.8+ , *
AST (U/L) 37.6 376.8 376.9 134.5
± ± ± ±
12.5 45.6* 63.6* 49.2+ , #
ALT (U/L) 34.7 228.8 312.7 165.3
± ± ± ±
7.4 68.4* 56.2* 56.1+ , *
P < 0.01 vs. controls. P < 0.05 vs. ADR + PBS. P < 0.01 vs. ADR + PBS.
ide release in human granulocytes stimulated by the chemotactic peptide (Kitagawa et al., 1987). Zhu et al. reported that GCSF induced ROS production in both a time-dependent and dose-dependent manner in G-CSF receptor-transfected murine Ba/F3 cells and 32D cells through Lyn-PI3K-Akt pathway (Zhu et al., 2006). In aplastic anemia patients, Ohsaka et al. found that intravenous administration of another growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), enhanced the FMLP-induced O2− release in neutrophil (Ohsaka et al., 1992). However, the controversial results were reported as well. A recent study showed that G-CSF had a strong inhibitory effect (about 60% inhibition) on the production of ROS induced by cis-
platin in blood platelets, suggesting that G-CSF has a protective effect against the excessive oxidative stress (Olas et al., 2000). Ozbirecikli et al. found that GM-CSF administration decreased the 1–2 dimethylhydrazine-induced alterations in superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in rat colon cancer (Ozbirecikli et al., 2007). In an irradiation induced skin injury rat model, Kilic et al. reported that GM-CSF significantly reduced the lipid peroxidation and enhanced GSH content in the injured skin (Kilic et al., 2000). As the protocols of these studies, for example, the cell or tissue type, the time and methods for peroxidase assessment and the experimental conditions are quite different, we therefore argue that these differences might account for the disagreement of the above
Fig. 1. The tissue MDA levels in the heart the liver and the kidneys during 8 weeks of time course. ADR administration induced constant increases of MDA production in all samples. A temporary, but dramatic elevation of MDA level was observed in each sample 1 week after G-CSF administration, after which the MDA levels begun to decline constantly and reached their minimums in ADR + G-CSF group 8 weeks later with a significant difference compared to those of ADR and ADR + PBS groups, respectively.
X.-W. Hou et al. / Toxicology Letters 187 (2009) 40–44
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Fig. 2. The GSH content in the heart, the liver and the kidney tissue samples. Compared with that of controls the GSH content in ADR-treated rats decreased dramatically. During 8 weeks, the G-CSF treatment induced a constant increase of GSH content in all organs. After 8 weeks, the GSH content in the heart, the liver and the kidney tissues in ADR + G-CSF group were markedly elevated, with a significant difference compared to those of ADR and ADR + PBS groups, respectively.
studies. This implies that the effect of G-CSF on generation of ROS and lipid peroxidase is very complex. In the present study, we observed that G-CSF administration induced a temporal, but dramatic elevation of MDA levels in heart, liver and kidney tissues. This phenomenon lasted only 1 week, after which the MDA levels declined constantly. This suggests a timedependent dual effect of G-CSF on ADR-induced MDA level in organ tissues. This dual effect phenomenon of G-CSF was reported by Park et al. They reported that G-CSF exerts dual opposing effects on endothelial cells in that it induces an inflammatory reaction leading to CRP production; meanwhile it has direct beneficial effects protecting endothelial cells from the deleterious effects of CRP through activation of Akt/eNOS pathway, leading to an increase in NO production (Park et al., 2008). As a pleiotropic cytokine, G-CSF activates neutrophil to generate superoxide and other forms of ROS (Balazovich et al., 1991). Therefore, we postulate that G-CSF administration induce a temporal inflammation in organ tissues, where the neutrophil are recruited and activated, leading to the elevation of ROS generation. This caused the temporal elevation of MDA levels in organ tissues. As previous studies showed that endogenously generated NO is a powerful scavenger of excessive ROS (Wartenberg et al., 2003),
we postulate this inflammatory reaction was rapidly neutralized by other beneficial effects of G-CSF, e.g. enhancing the endogenous generation of NO or improve the impaired endothelial function, which the mechanism may includes: (1) via activating the Akt/eNOS pathway, leading to an increase in endogenous NO production (Park et al., 2008). (2) Via enhancing the release of the anti-inflammatory (e.g. interleukin 10, IL-10) and anti-oxidant cytokine (e.g. CRP, tumour necrosis factor-alpha and TNF-alpha) in tissue, thus shifting the balance between the pro- and anti-inflammatory cytokines towards a more favorable anti-inflammatory net effect. A recent clinical study conducted by Ikononidis and Papadimitriou et al. found a greater increase of IL-10 and reduction of TNF-alpha/IL10 after the G-CSF treatment compared to placebo. In their study, although CRP and TNF-alpha were higher, TNF-alpha/IL-10 was markedly lowered by G-CSF treatment compared to placebo, leading to improvement in endothelial function (Ikononidis et al., 2008) 3. By increasing the mobilization of endothelial progenitor cells (EPCs). The mobilized EPCs have a favorable effect on endothelium by substituting damaged endothelial cells and reducing atherosclerotic plaque formation (Hill et al., 2003). We have limitations in this study. Firstly, we did not assess the other forms of lipid peroxidation, e.g. the concentrations of O(2)(−),
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H(2)O(2), catalase and so on. Secondly, we did not evaluate the tissue NO level and the Akt/eNOS pathway activity, as well as the proand anti-inflammatory cytokines, e.g. IL 10, CRP, TNF-alpha, which would be helpful to understand the mechanism of G-CSF. Thirdly, we did not have a group receiving PBS only in order to determine whether the sole PBS administration induce a organ toxicity in rat or influence the ADR-induced toxicity, thus, a potential bias effect might occur on our results. In conclusion, in this study we observed that G-CSF palliated ADR-induced toxicities in heart, liver and kidneys. Also, G-CSF significantly reduced the MDA level and increased the GSH content in these organ tissues. This result suggests that G-CSF play a protective effect on ADR-induced adriamycin induced cardiac, renal and hepatic toxicities and the inhibition of tissue peroxidative alterations might contribute to this beneficial effect. Conflict of interest statement There is no potential conflict of interest for any of the contributing authors. Acknowledgements This work was supported by a Project Grant from Nanjing Medical University (No. 2004B163). We thank Mrs. Karen Dini Haast at Zhejiang University for her help in correcting this manuscript. References Akihama, S., Sato, K., Satoh, S., et al., 2007. Bone marrow-derived cells mobilized by granulocyte-colony stimulating factor facilitate vascular regeneration in mouse kidney after ischemia/reperfusion injury. Tohoku J. Exp. Med. 213 (December (4)), 341–349. Balazovich, K.J., Almeida, H.I., Boxer, L.A., 1991. Recombinant human G-CSF and GM-CSF prime human neutrophils for superoxide production through different signal transduction mechanisms. J. Lab. Clin. Med. 118 (December (6)), 576–584. Barbey, M.M., Fels, L.M., Soose, M., et al., 1989. Adriamycin affects glomerular renal function: evidence for the involvement of oxygen radicals. Free Radic. Res. Commun. 7 (3–6), 195–203. Bertolatus, J.A., Klinzman, D., Bronsema, D.A., et al., 1991. Evaluation of the role of reactive oxygen species in doxorubicin hydrochloride nephrosis. J. Lab. Clin. Med. 118 (November (5)), 435–445. Cini Neri, G., Neri, B., Bandinelli, M., et al., 1991. Anthracycline cardiotoxicity: in vivo and in vitro effects on biochemical parameters and heart ultrastructure of the rat. Oncology 48, 327–333. Cole, M.P., Chaiswing, L., Oberley, T.D., et al., 2006. The protective roles of nitric oxide and superoxide dismutase in adriamycin-induced cardiotoxicity. Cardiovasc. Res. 69 (January (1)), 186–197. DeAtley, S.M., Aksenov, M.Y., Aksenova, M.V., et al., 1999. Adriamycin-induced changes of creatine kinase activity in vivo and in cardiomyocyte culture. Toxicology 134, 51–62. Deepa, P.R., Varalakshmi, P., 2005. Biochemical evaluation of the inflammatory changes in cardiac, hepatic and renal tissues of adriamycin-administered rats and the modulatory role of exogenous heparin-derivative treatment. Chem. Biol. Interact. 156 (October (2–3)), 93–100. DeGraff, W., Hahn, S.M., Mitchell, J.B., et al., 1994. Free radical modes of cytotoxicity of adriamycin and streptonigrin. Biochem. Pharmacol. 48 (October (7)), 1427–1435. Goeptar, A.R., Te Koppele, J.M., et al., 1993. Cytochrome P450 2B1-mediated oneelectron reduction of adriamycin: a study with rat liver microsomes and purified enzymes. Mol. Pharmacol. 44 (December (6)), 1267–1277. Gokcimen, A., Cim, A., Tola, H.T., et al., 2007. Protective effect of N-acetylcysteine, caffeic acid and vitamin E on doxorubicin hepatotoxicity. Hum. Exp. Toxicol. 26 (June (6)), 519–525. Hill, J.M., Zalos, G., Halcox, J.P., Schenke, W.H., Waclawiw, M.A., Quyyumi, A.A., Finkel, T., 2003. Circulating endothelial progenitor cells, vascular function and cardiovascular risk. N. Engl. J. Med. 348, 593–600.
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Protective role of granulocyte colony-stimulating factor against adriamycin induced cardiac, renal and hepatic toxicities Xu-Wei Hou a,∗ , Yu Jiang b , Li-Fang Wang c , Hai-Ying Xu a , Hong-Min Lin d , Xiu-Ying He a , Jian-Jun He d , Sheng Zhang d a
Department of Cardiology, Hangzhou First Municipal Hospital and Hangzhou Hospital, Nanjing Medical University, Huan-sha Road 26, Hangzhou 310006, China Department of Cardiology, Jiangxi Provincial Corps Hospital, Chinese People’s Armed Police Force, Nanchang 330030, China Department of Basic Medicine, Zhejiang Medical College, Hangzhou 310053, China d Department of Internal Medicine, Baodiao Hospital, Zoucheng City 273511, China b c
a r t i c l e
i n f o
Article history: Received 18 November 2008 Received in revised form 20 January 2009 Accepted 21 January 2009 Available online 3 February 2009 Keywords: Granulocyte colony-stimulating factor Adriamycin Toxicity Peroxidative alterations
a b s t r a c t Adriamycin (ADR) causes dose-dependant toxicities in heart, liver and kidneys via inducing the peroxidative alterations in organ tissues. Recent studies showed that the granulocyte colony-stimulating factor (G-CSF) exerts beneficial effects on heart, liver and kidney injuries induced by different pathological conditions. We hypothesize that G-CSF have a protective effect on ADR induced cardiac, renal and hepatic toxicities by inhibiting the peroxidative alterations in organ tissues. Wistar rats were randomly divided into control, ADR, ADR + phosphate buffered saline (PBS) and ADR + G-CSF group (n = 16 in each group). ADR was administered intraperitoneally every other day at the dose of 2.5 g/kg each time per rat (total six times of injection during 2 weeks). Rats in the ADR + GCSF group were injected subcutaneously with G-CSF at the dose of 50 g/(kg day) (for 8 consecutive days). After 8 weeks, the serum and urine biochemistry variables were determined. The malondialdehyde (MDA) level and the glutathione (GSH) content in the heart, the liver and the kidney tissues were measured. ADR caused significant cardiac, renal and hepatic toxicities indicated by the serum and urine biochemistry variables. The tissue MDA level in the heart, kidney and liver in rats treated with ADR were markedly elevated, while the GSH content in these tissues were significantly reduced. G-CSF administration palliated the cardiac, renal and hepatic toxicities. Notably, G-CSF induced significant reduction of MDA level and increase of GSH content in the heart, kidney and liver tissues. This study suggests that G-CSF play an overall protective effect on ADR-induced toxicities in heart, liver and kidneys and the inhibition of tissue peroxidative alterations might contribute to this beneficial effect. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Adriamycin (ADR) is an anti-neoplastic agent used in the treatment of a variety of human neoplasms. However, its clinical use is severely restricted by dose-dependant toxicity in various tissues, including the heart, the liver and the kidneys. ADR-induced cardiotoxicity is characterized by progressive ventricular dilatation and congestive heart failure with a poor prognosis (DeAtley et al., 1999; Cini Neri et al., 1991; Sazuka et al., 1989a,b; Praga et al., 1979; Singal and Iliskovic, 1998; Kakadekar et al., 1997). Experimental studies in animals showed that ADR caused a renal toxicity and produced progressive glomerular injuries (Barbey et al., 1989; Manabe et al., 2001; Malarkodi et al., 2003; Deepa and Varalakshmi, 2005). ADR-induced hepatotoxicity was reported as well (You et al., 2007;
∗ Corresponding author. Tel.: +86 571 87065701x20681; fax: +86 571 87065701x10324. E-mail address: [email protected] (X.-W. Hou). 0378-4274/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2009.01.025
Gokcimen et al., 2007; Goeptar et al., 1993; Bertolatus et al., 1991). These adverse effects of ADR inhibit its clinical use. The exact mechanism of ADR-induced toxicity remains unclear. Some researchers proposed that ADR-induced toxicity is most likely mediated by the formation of an iron–anthracycline complex that generates reactive free radicals (ROS), which in turn, causes diverse oxidative damage on critical cellular components and membrane lipids in the plasma membranes and mitochondria (DeGraff et al., 1994; Lee et al., 1991; Sazuka et al., 1989a,b). This view is well supported by the fact that antioxidants prevent the ADR-induced toxicity in experimental animals as well as in human (Mohamed et al., 2000; Cole et al., 2006). The granulocyte colony-stimulating factor (G-CSF) is a growth factor that promotes the neutrophil and bone marrow stem cells to the peripheral circulation. Neutrophil can generate superoxide and other forms of ROSs. Recent studies showed G-CSF exerts beneficial effects on heart, liver and kidney injuries induced by a variety of pathological conditions. In the setting of the ADR-induced cardiotoxicity, G-CSF treatment protects the cardiomyocyte ultra-
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structure, inhibits the myocyte apoptosis and enhances the heart function (Tomita et al., 2004; Hou et al., 2006). G-CSF administration increases the hepatic sinusoidal perfusion and enhances the hepatocyte proliferation, thus contributing to liver tissue regeneration after liver injury or liver resection (Sidler et al., 2008; Spahr et al., 2008). Animal studies showed that G-CSF mobilization accelerates the improvement of renal function and prevents the renal tubular injury induced by cisplatin and facilitates the vascular regeneration in mouse kidneys after an ischemia/reperfusion injury (Iwasaki et al., 2005; Akihama et al., 2007). Until now, the effect of G-CSF on ADR-induced oxidative stress and peroxidative alterations are poorly understood. We hypothesize that G-CSF might inhibit the excessive generation of oxidative stress, thus providing an overall protective role in the heart, liver and kidney toxicities induced by ADR administration in rats.
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3. Results 3.1. Blood and urine biochemical variables Table 1 showed the blood and urine biochemical variables in all rats 8 weeks after G-CSF and PBS administration. These biochemical variables are commonly used for the assessment of heart, liver and kidney damages. ADR treatment induced significant increases in the CK-MB, LDH, Cr, BUN, ALT and AST serum levels as well as the 24-h urine albumin level compared to those in the control group. This means that ADR administration caused systemic organ toxicities in rats. In rats from ADR + G-CSF group, the serum CK-MB, LDH, Cr, BUN, ALT, AST levels and the 24H urine albumin level were significantly lowered 8 weeks after the G-CSF administration, when significant differences were seen compared with those in rats from ADR and ADR + PBS groups.
2. Materials and methods 2.1. Animal preparation and samples collection
3.2. Tissue MDA level and GSH content
Adult male Wistar rats (body weight 226 ± 15.5 g) were randomly divided into four groups: control, ADR, ADR + phosphate buffered saline (PBS) and ADR + G-CSF group (n = 16 in each group). ADR was injected intraperitoneally at the dose of 2.5 g/kg each time per rat. The injections were performed every other day during 2 weeks (eight times of injections in all). All rats except the control rats received ADR administration. After 2 weeks of wash-off period, the rats in the ADR + G-CSF group were injected subcutaneously with the G-CSF at the dose of 50 g/(kg day) per rat for 8 consecutive days. Similarly, rats in ADR + PBS group were injected with PBS. After 8 weeks of observation, all rats were anesthetized with ether and the blood samples were collected by heart puncture. The heart, the liver and the kidneys were removed rapidly, perfused with cold saline solution and used for the analysis as described below. The study was approved by the Ethics Committee of Nanjing Medical University.
Fig. 1 shows the tissue MDA levels in the heart, the liver and the kidneys during a time course of 8 weeks. ADR administration induced dramatic, constant increases of MDA production in these tissue samples. A temporary, but dramatic elevation of MDA level was observed in each tissue sample 1 week after G-CSF administration, after which the MDA levels begun to decline constantly. Eight weeks later, the tissue MDA levels in rats from the ADR + GCSF group reached their minimums, with a significant difference compared to those of ADR and ADR + PBS groups, respectively. Fig. 2 shows the GSH content in the heart, the liver and the kidney tissue samples. Compared with that of controls, the GSH content in ADR treated rats decreased dramatically. The G-CSF treatment induced a constant increase of GSH content in three organs. This trend lasted for 8 weeks. After 8 weeks, the GSH content in the heart, the liver and the kidney tissues in ADR + G-CSF group were markedly elevated, with a significant difference compared to those of ADR and ADR + PBS groups, respectively.
2.2. Blood and urine biochemical variables Blood samples were coagulated at room temperature for 30 min followed by centrifugation for the extraction of the serum. The serum creatine kinase MB (CK-MB) levels were assayed using the CK-MB test kit (Bayer Diagnostics). CK-MB activity was measured in freshly separated serum according to the manufacturer’s instructions. The serum lactate dehydrogenase (LDH) activity was estimated using a commercially available kit using an UV–vis spectrophotometer. The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were determined according to manufacturer’s instructions. The serum creatinine (Cr) and blood urea nitrogen (BUN) level was measured by Jaffa’s method with a creatinine test kit and by the urease indophenol method with a nitrogen B test kit. The 24 h urine was collected 1 day before sacrifice for albumin analysis. The albumin level was measured by a bromo-cresol green method using an A/GB test kit. 2.3. Tissue malondialdehyde (MDA) measurement The MDA level in the heart, liver and kidney samples was determined using the thiobarbiturate reaction. 0.5 mL of 0.5% butylated hydroxytoluene was added to 2 mL of tissue homogenate to prevent lipid autoxidation. After tissue protein precipitation and centrifugation, 1 mL of 0.67% thiobarbiturate–water solution was added to the supernatant and boiled for 60 min. After cooling, the optical density was assayed at a wavelength of 530 nm. We used 1,1,3,3-tetraethoxypropane as the standard. The MDA level was expressed as nmol/g tissue. 2.4. Tissue glutathione (GSH) measurement The GSH content of the heart, the liver and the kidney samples was determined. Fifty microliters of tissue extract diluted 10-fold in 100 mM NaHP2O4 and 5 mM ethylene–diaminetetra–acetic acid buffer at pH 7.5 was added to 50 L of glutathione oxidoreductase (5 U/50 L) and 50 L of 2.5 mM 5,5 -dithiobis-(2nitrobenzoic acid). The reaction was initiated by the addition of 50 L of 1.2 mM nicotinamide adenine dinucleotide phosphate in buffer. The rate of color change at 410 nm, which is proportional to the amount of total GSH in the samples, was monitored using a plate reader. The results are expressed as mol/mg protein. 2.5. Statistics All data were expressed as mean ± S.E.M. Statistical significance of differences was analyzed by the one-way ANOVA with PASS 13.0 and a P value <0.05 was considered as statistically significant.
4. Discussion This study, for the first time, evaluated the effect of G-CSF on the ADR-induced toxicities in heart, liver and kidneys and its influence on the tissue peroxidative alterations in these organs. In accordance with previous studies, we observed that ADR induced overall cardiac, renal and hepatic toxicities in rats. The G-CSF administration significantly reduced the CK-MB, LDH, Cr, BUN, ALT, AST level and the 24-h urine albumin level, suggesting that G-CSF provides overall protective effects in heart, liver and kidney. In this study, the tissue peroxidative alterations in the three organs were markedly inhibited by G-CSF, as the MDA level was significantly reduced and the GSH content was dramatically increased. We propose that the inhibition of peroxidative alterations might contribute to the beneficial effect of G-CSF in reducing ADR-induced cardiac, renal and hepatic toxicities. In a physiological condition, intracellular ROS acts as a second messenger to a variety of growth factors, including G-CSF, which induces the activation of down stream signaling molecules (Zhu et al., 2006). However, when the ROS is excessively produced, for instance, in the setting of ADR administration, it becomes deleterious and causes damage on cellular components and membrane lipids in the plasma membranes and mitochondria. The lipid peroxidation occurs in cellular components and membrane lipids when the ROS levels are elevated, leading to the release of reactive aldehydes, such as MDA. The effect of G-CSF on the peroxidative alterations was explored previously. Kitagawa et al. reported that G-CSF enhanced superox-
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Table 1 Biochemical variables in all rats. Group Control ADR ADR + PBS ADR + G-CSF * # +
CK-MB (U/L) 78.3 1770.6 1879.6 599.9
± ± ± ±
11.2 113.6* 143.7* 155.8+ , *
LDH (U/L) 119.6 2100.4 2231.9 681.7
± ± ± ±
BUN (mmol/L) 43.8 234.7* 176.9* 154.4+ , *
4.6 21.5 23.6 14.5
± ± ± ±
1.9 9.6* 11.8* 3.2+ , #
Cr (mol/L) 43.7 223.6 243.9 134.6
± ± ± ±
11.6 67.8* 112.4* 77.3+ , #
Urine albumin (mg) 4.6 23.6 25.6 14.2
± ± ± ±
1.9 9.4* 11.2* 7.8+ , *
AST (U/L) 37.6 376.8 376.9 134.5
± ± ± ±
12.5 45.6* 63.6* 49.2+ , #
ALT (U/L) 34.7 228.8 312.7 165.3
± ± ± ±
7.4 68.4* 56.2* 56.1+ , *
P < 0.01 vs. controls. P < 0.05 vs. ADR + PBS. P < 0.01 vs. ADR + PBS.
ide release in human granulocytes stimulated by the chemotactic peptide (Kitagawa et al., 1987). Zhu et al. reported that GCSF induced ROS production in both a time-dependent and dose-dependent manner in G-CSF receptor-transfected murine Ba/F3 cells and 32D cells through Lyn-PI3K-Akt pathway (Zhu et al., 2006). In aplastic anemia patients, Ohsaka et al. found that intravenous administration of another growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), enhanced the FMLP-induced O2− release in neutrophil (Ohsaka et al., 1992). However, the controversial results were reported as well. A recent study showed that G-CSF had a strong inhibitory effect (about 60% inhibition) on the production of ROS induced by cis-
platin in blood platelets, suggesting that G-CSF has a protective effect against the excessive oxidative stress (Olas et al., 2000). Ozbirecikli et al. found that GM-CSF administration decreased the 1–2 dimethylhydrazine-induced alterations in superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in rat colon cancer (Ozbirecikli et al., 2007). In an irradiation induced skin injury rat model, Kilic et al. reported that GM-CSF significantly reduced the lipid peroxidation and enhanced GSH content in the injured skin (Kilic et al., 2000). As the protocols of these studies, for example, the cell or tissue type, the time and methods for peroxidase assessment and the experimental conditions are quite different, we therefore argue that these differences might account for the disagreement of the above
Fig. 1. The tissue MDA levels in the heart the liver and the kidneys during 8 weeks of time course. ADR administration induced constant increases of MDA production in all samples. A temporary, but dramatic elevation of MDA level was observed in each sample 1 week after G-CSF administration, after which the MDA levels begun to decline constantly and reached their minimums in ADR + G-CSF group 8 weeks later with a significant difference compared to those of ADR and ADR + PBS groups, respectively.
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Fig. 2. The GSH content in the heart, the liver and the kidney tissue samples. Compared with that of controls the GSH content in ADR-treated rats decreased dramatically. During 8 weeks, the G-CSF treatment induced a constant increase of GSH content in all organs. After 8 weeks, the GSH content in the heart, the liver and the kidney tissues in ADR + G-CSF group were markedly elevated, with a significant difference compared to those of ADR and ADR + PBS groups, respectively.
studies. This implies that the effect of G-CSF on generation of ROS and lipid peroxidase is very complex. In the present study, we observed that G-CSF administration induced a temporal, but dramatic elevation of MDA levels in heart, liver and kidney tissues. This phenomenon lasted only 1 week, after which the MDA levels declined constantly. This suggests a timedependent dual effect of G-CSF on ADR-induced MDA level in organ tissues. This dual effect phenomenon of G-CSF was reported by Park et al. They reported that G-CSF exerts dual opposing effects on endothelial cells in that it induces an inflammatory reaction leading to CRP production; meanwhile it has direct beneficial effects protecting endothelial cells from the deleterious effects of CRP through activation of Akt/eNOS pathway, leading to an increase in NO production (Park et al., 2008). As a pleiotropic cytokine, G-CSF activates neutrophil to generate superoxide and other forms of ROS (Balazovich et al., 1991). Therefore, we postulate that G-CSF administration induce a temporal inflammation in organ tissues, where the neutrophil are recruited and activated, leading to the elevation of ROS generation. This caused the temporal elevation of MDA levels in organ tissues. As previous studies showed that endogenously generated NO is a powerful scavenger of excessive ROS (Wartenberg et al., 2003),
we postulate this inflammatory reaction was rapidly neutralized by other beneficial effects of G-CSF, e.g. enhancing the endogenous generation of NO or improve the impaired endothelial function, which the mechanism may includes: (1) via activating the Akt/eNOS pathway, leading to an increase in endogenous NO production (Park et al., 2008). (2) Via enhancing the release of the anti-inflammatory (e.g. interleukin 10, IL-10) and anti-oxidant cytokine (e.g. CRP, tumour necrosis factor-alpha and TNF-alpha) in tissue, thus shifting the balance between the pro- and anti-inflammatory cytokines towards a more favorable anti-inflammatory net effect. A recent clinical study conducted by Ikononidis and Papadimitriou et al. found a greater increase of IL-10 and reduction of TNF-alpha/IL10 after the G-CSF treatment compared to placebo. In their study, although CRP and TNF-alpha were higher, TNF-alpha/IL-10 was markedly lowered by G-CSF treatment compared to placebo, leading to improvement in endothelial function (Ikononidis et al., 2008) 3. By increasing the mobilization of endothelial progenitor cells (EPCs). The mobilized EPCs have a favorable effect on endothelium by substituting damaged endothelial cells and reducing atherosclerotic plaque formation (Hill et al., 2003). We have limitations in this study. Firstly, we did not assess the other forms of lipid peroxidation, e.g. the concentrations of O(2)(−),
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H(2)O(2), catalase and so on. Secondly, we did not evaluate the tissue NO level and the Akt/eNOS pathway activity, as well as the proand anti-inflammatory cytokines, e.g. IL 10, CRP, TNF-alpha, which would be helpful to understand the mechanism of G-CSF. Thirdly, we did not have a group receiving PBS only in order to determine whether the sole PBS administration induce a organ toxicity in rat or influence the ADR-induced toxicity, thus, a potential bias effect might occur on our results. In conclusion, in this study we observed that G-CSF palliated ADR-induced toxicities in heart, liver and kidneys. Also, G-CSF significantly reduced the MDA level and increased the GSH content in these organ tissues. This result suggests that G-CSF play a protective effect on ADR-induced adriamycin induced cardiac, renal and hepatic toxicities and the inhibition of tissue peroxidative alterations might contribute to this beneficial effect. Conflict of interest statement There is no potential conflict of interest for any of the contributing authors. Acknowledgements This work was supported by a Project Grant from Nanjing Medical University (No. 2004B163). We thank Mrs. Karen Dini Haast at Zhejiang University for her help in correcting this manuscript. References Akihama, S., Sato, K., Satoh, S., et al., 2007. Bone marrow-derived cells mobilized by granulocyte-colony stimulating factor facilitate vascular regeneration in mouse kidney after ischemia/reperfusion injury. Tohoku J. Exp. Med. 213 (December (4)), 341–349. Balazovich, K.J., Almeida, H.I., Boxer, L.A., 1991. Recombinant human G-CSF and GM-CSF prime human neutrophils for superoxide production through different signal transduction mechanisms. J. Lab. Clin. Med. 118 (December (6)), 576–584. Barbey, M.M., Fels, L.M., Soose, M., et al., 1989. Adriamycin affects glomerular renal function: evidence for the involvement of oxygen radicals. Free Radic. Res. Commun. 7 (3–6), 195–203. Bertolatus, J.A., Klinzman, D., Bronsema, D.A., et al., 1991. Evaluation of the role of reactive oxygen species in doxorubicin hydrochloride nephrosis. J. Lab. Clin. Med. 118 (November (5)), 435–445. Cini Neri, G., Neri, B., Bandinelli, M., et al., 1991. Anthracycline cardiotoxicity: in vivo and in vitro effects on biochemical parameters and heart ultrastructure of the rat. Oncology 48, 327–333. Cole, M.P., Chaiswing, L., Oberley, T.D., et al., 2006. The protective roles of nitric oxide and superoxide dismutase in adriamycin-induced cardiotoxicity. Cardiovasc. Res. 69 (January (1)), 186–197. DeAtley, S.M., Aksenov, M.Y., Aksenova, M.V., et al., 1999. Adriamycin-induced changes of creatine kinase activity in vivo and in cardiomyocyte culture. Toxicology 134, 51–62. Deepa, P.R., Varalakshmi, P., 2005. Biochemical evaluation of the inflammatory changes in cardiac, hepatic and renal tissues of adriamycin-administered rats and the modulatory role of exogenous heparin-derivative treatment. Chem. Biol. Interact. 156 (October (2–3)), 93–100. DeGraff, W., Hahn, S.M., Mitchell, J.B., et al., 1994. Free radical modes of cytotoxicity of adriamycin and streptonigrin. Biochem. Pharmacol. 48 (October (7)), 1427–1435. Goeptar, A.R., Te Koppele, J.M., et al., 1993. Cytochrome P450 2B1-mediated oneelectron reduction of adriamycin: a study with rat liver microsomes and purified enzymes. Mol. Pharmacol. 44 (December (6)), 1267–1277. Gokcimen, A., Cim, A., Tola, H.T., et al., 2007. Protective effect of N-acetylcysteine, caffeic acid and vitamin E on doxorubicin hepatotoxicity. Hum. Exp. Toxicol. 26 (June (6)), 519–525. Hill, J.M., Zalos, G., Halcox, J.P., Schenke, W.H., Waclawiw, M.A., Quyyumi, A.A., Finkel, T., 2003. Circulating endothelial progenitor cells, vascular function and cardiovascular risk. N. Engl. J. Med. 348, 593–600.
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