Protective mechanisms of atorvastatin against doxorubicin-induced hepato-renal toxicity

Biomedicine & Pharmacotherapy xxx (2013) xxx–xxx

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BIOPHA-3323; No. of Pages 10

Original article

Protective mechanisms of atorvastatin against doxorubicin-inducedhepato-renal toxicity

Mohamed A. El-Moselhy a,1, Azza A.K. El-Sheikh b,*,1

a Department of Pharmacology and Toxicology, Faculty of Pharmacy, Minia University, 61511 Minia, Egyptb Department of Pharmacology, Faculty of Medicine, Minia University, 61511 Minia, Egypt

A R T I C L E I N F O

Article history:

Received 16 August 2013

Accepted 24 September 2013

Keywords:

Atorvastatin

Doxorubicin

Oxidative stress

Abbreviations:

ALT, Alanine transaminase

AST, Aspartate transaminase

Ator, Atorvastatin

Bax, Bcl-2-associated X protein

BUN, Blood urea nitrogen

DOX, Doxorubicin

eNOS, Endothelial nitric oxide synthase

GSH, Reduced glutathione

MDA, Malondialdehyde

NF-kB, Nuclear factor-kB

NO, Nitric oxide

TNF-a, Tumor necrosis factor-a

A B S T R A C T

To investigate the mechanisms by which the anticancer drug doxorubicin (DOX)-induced hepato-renal

damage could be prevented by the cholesterol-lowering statin, atorvastatin (Ator), Ator (10 mg/kg) was

administered orally for 10 days, and, in independent rat groups, DOX hepato-renal toxicity was induced

via a single i.p. dose of 15 mg/kg at day 5 of experiment, with or without Ator. DOX caused deterioration

in hepato-renal function, as it significantly increased blood urea nitrogen (BUN), creatinine, alanine

transaminase (ALT) and aspartate transaminase (AST) compared to control, with distortion in normal

renal and hepatic histology. Pretreatment with Ator preserved kidney and liver function and histology.

DOX caused oxidative stress as indicated by significant decrease in reduced glutathione (GSH) level and

catalase activity with increase in malondialdehyde (MDA) compared to control. Combined DOX/Ator

significantly reversed these values compared to DOX in both kidney and liver. DOX caused nitrosative

stress, as it increased tissue nitric oxide compared to control. Concomitant DOX/Ator treatment

decreased NO in kidney and liver. Furthermore, DOX caused inflammatory effects indicated by up-

regulation of hepato-renal nuclear factor-kB (NF-kB) expression and increment of tumor necrosis factor-

a (TNF-a) tissue concentration, with down-regulation of endothelial nitric oxide synthase (eNOS). DOX

also caused apoptotic effect, as it up-regulated the apoptotic marker, Bcl-2-associated X protein (Bax),

expression in liver and kidney. Using Ator with DOX reversed hepato-renal inflammatory and apoptotic

marker expression. These findings suggest Ator as a protective adjuvant against DOX toxicity, via

antioxidant, anti-nitrosative, anti-inflammatory and anti-apoptotic mechanisms.

� 2013 Elsevier Masson SAS. All rights reserved.

Available online at

ScienceDirectwww.sciencedirect.com

1. Introduction

Doxorubicin (DOX), also known as adriamycin, was isolated inthe 1960s from a species of actinobacteria called Streptomyces

peucetius [1]. Since then, DOX has been successfully used as one ofthe first-line anticancer drugs against solid and hematologicalmalignancies [2]. DOX exerts its pharmacological anticanceractions through preferentially targeting and intercalating withthe DNA of rapidly dividing tumor cells, causing cell cycle blockagein the G2 phase [3]. Disappointingly, DOX use has been constraineddue to its multi-organ toxic effects, including its effects on the liver

* Corresponding author. Tel.: +2 0100 6756740.

E-mail address: [email protected] (Azza A.K. El-Sheikh).1 El-Moselhy M.A. and El-Sheikh A.A.K. contributed equally to this work in

designing the research, performing the research, contributing in data analysis and

writing the paper.

Please cite this article in press as: El-Moselhy MA, El-Sheikh AAK. Prohepato-renal toxicity. Biomed Pharmacother (2013), http://dx.doi.or

0753-3322/$ – see front matter � 2013 Elsevier Masson SAS. All rights reserved.

http://dx.doi.org/10.1016/j.biopha.2013.09.001

[4] and kidney [5]; the main drug detoxifying excretory organs inthe body. The molecular mechanisms underlying DOX-inducedtoxicity is multi-factorial and, to date, not fully characterized. Still,the most acceptable theory attributes initiation of such toxicity tooxidative stress [6]. Other factors contributing to organ toxicityincludes DOX generation of inflammatory cascade [7], and,eventually, programmed cellular death, apoptosis [8]. We haverecently studied the mechanisms of nephrological toxic effects ofDOX [5], emphasizing the role of oxidative stress and apoptosis.Other studies also focused on antioxidants as possible preventiveadjuvant drugs to overcome DOX toxicity [9–11], but so far, noeffective and clinically applicable treatment is yet discovered toprevent DOX-induced hepato-renal damage.

Atorvastatin (Ator) is a well-tolerated cholesterol-loweringstatin that acts through inhibiting 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase, essential for cholesterolproduction via the mevalonate pathway [12]. Previous reportssuggested that statins may improve DOX-induced cardiotoxicity in

tective mechanisms of atorvastatin against doxorubicin-inducedg/10.1016/j.biopha.2013.09.001


mailto:[email protected]



M.A. El-Moselhy, A.A.K. El-Sheikh / Biomedicine & Pharmacotherapy xxx (2013) xxx–xxx2

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laboratory animal models [13,14] as well as in humans [15].However, the protective effect of statins on DOX-induced toxicityin the liver [16,17] and kidney [18,19] is still controversial.

The present study was designed to investigate the possibleprotective effects of Ator against DOX-induced toxicity in ratkidney and liver, in addition to exploring the role of inflammatorymediators, tumor necrosis factor (TNF)-a and nuclear factor-kB(NF-kB), as well as the apoptotic marker, Bcl-2-associated Xprotein (Bax) as possible mechanisms.

2. Materials and methods

2.1. Chemicals

Atorvastatin was kindly provided by Eipico (Egypt). DOX HCl10 mg was purchased from Pharmacia Italia (SPA, Italy). Kits forexamining blood urea nitrogen (BUN), creatinine, alanine transa-minase (ALT), aspartate transaminase (AST), reduced glutathione(GSH) and catalase were purchased from Biodiagnostic (Egypt).TNF-a enzyme-linked immuno sorbent assay (ELISA) kit waspurchased from WKEA-Med supplies Corp. (China). Triology wassupplied by Cell Marque (USA). Power Stain Poly HRP DAB Kitwas purchased from Genemed Biotechnologies (USA). The ready touse rabbit polyclonal antibody NF-kB/p65 was purchased fromLabvision Corp. (USA). Concentrated rabbit polyclonal Bax andendothelial nitric oxide synthase (eNOS) antibodies were pur-chased from Biorbyt Ltd. (UK).

2.2. Experimental design

Forty adult male Wistar rats of 190–240 g weight werepurchased from the National Research Centre, Giza, Egypt.Throughout the experiments, rats were housed in the standardanimal facility, 3 or 4 animals/cage. Tap water and laboratory chowwere supplied ad libitum. Before the start of experiments, animalswere left to acclimatize for 2 weeks. After acclimatization period,animals were divided into 4 groups: control group (n = 7),atorvastatin-treated group (n = 7) receiving single daily oral doseof 10 mg/kg/day atorvastatin by gastric gavage for 10 days [20],DOX-treated group (n = 13) receiving single i.p. dose of 15 mg/kgDOX at day 5 of the experiment [5] and DOX/atorvastatin-treatedgroup (n = 13) receiving both DOX and atorvastatin treatments aspreviously indicated. Total rat body weights were recorded beforethe start and at the end of the 10-day experiment.

2.3. Sample preparation and evaluation of kidney

and liver function

After 5 days of DOX injection, rats were sacrificed by cervicaldislocation. Venous blood samples were collected from the jugularvein, centrifuged at 5000 rpm for 15 min, and serum was collectedand stored at �80 8C till used. Using colorimetric diagnostic kitsaccording to the manufacturer’s instructions, assessment of renalfunction and nephrotoxicity was done by determination of BUNand serum creatinine, whereas of liver function and hepatotoxicityby serum ALT and AST. Both kidneys and liver were rapidly excisedand weighed. Kidney and liver sections were fixed in 10% formalinand embedded in paraffin for histopathological and immunohis-tochemical examinations. The rest of the kidney and liver tissueswas snap-frozen in liquid nitrogen and kept at �80 8C. To preparetissue homogenate, kidneys and livers were homogenized (Glas-Col homogenizer) and a 20% w/v homogenate was prepared in ice-cold phosphate buffer (0.01 M, pH 7.4). The homogenate wascentrifuged at 3000 rpm for 20 min and the supernatant was thendivided over several containers to avoid sample thawing andrefreezing, and was kept at �80 8C till used.


2.4. Analysis of oxidative stress markers in kidney and

liver homogenate

Biochemical oxidative stress markers were determined in renaland hepatic tissue homogenate, including evaluating GSH con-centration, catalase activity and lipid peroxide content. A spectro-photometric kit was used for assessment of GSH. In brief, themethod is based on that the sulfhydryl component of GSH reactswith 5,5-dithio-bis-2-nitrobenzoic acid (Ellman’s reagent) produ-cing 5-thio-2-nitrobenzoic acid having a yellow color, that wasmeasured colorimetrically at 405 nm (Beckman DU-64 UV/VISspectrophotometer). Results were expressed as mmol/g tissue.Assessment of catalase antioxidant enzymatic activity wasdetermined in tissue homogenate from the rate of decompositionof H2O2 at 510 nm by colorimetric kit. The results were expressedas unit/g tissue. Tissue content of lipid peroxides was determinedby biochemical assessment of thiobarbituric acid reacting sub-stance through spectrophotometric measurement of color at535 nm, using 1,1,3,3-tetramethoxypropane as standard. Theresults were expressed as equivalents of malondialdehyde(MDA) in tissue homogenate in nmol/g tissue [21].

2.5. Assessment of nitrosative stress marker and TNF-a in kidney

and liver homogenate

For the assessment of nitrosative stress, the stable oxidationend products of nitric oxide (NO), nitrite and nitrate was used as anindex of NO production, as NO has a half-life of only a few seconds,being readily oxidized to nitrite then to nitrate. The method usedwas based on Griess reaction [22] that depends on the spectro-photometric measurement of total nitrites at 540 nm after theconversion of nitrate to nitrite by copperized cadmium granules,using nitric acid as a standard. Results were expressed as nmol/100 mg tissue. TNF-a was determined according to ELISA kitmanufacturer’s instructions. TNF-a was assessed in 10 mL ofkidney or liver homogenate using the supplied 96-wells ELISAplate. The plate was read using ELISA plate reader at 450 nm.

2.6. Histopathological and immunohistochemical examination

The specimens from the kidneys and liver were collected andfixed in 10% buffered neutral formalin solution, dehydrated ingradual ethanol (70–100%), cleared in xylene, and embedded inparaffin. Five-mm thick paraffin sections were prepared and thenroutinely stained with hematoxylin and eosin (H&E) dyes [23].Stained slides were microscopically analyzed using light micro-scopy (Olympus CX41). For immunohistochemical staining, sec-tions were cut into 4 m then fixed at 65 8C for 1 hour. Triology thatcombines the three pretreatment steps: deparaffinization, rehy-dration and antigen unmasking, was used to enhance standardi-zation of the pretreatment step and produce more consistentresults. Slides were placed in a coplin jar containing 200 mL oftriology working solution at 120 8C for 15 min, after whichpressure was released and slides were allowed to cool for 30 min.Sections were then washed tris buffer saline. Quenchingendogenous peroxidase activity was performed by immersingslides in 3% hydrogen peroxide for 10 min. The rabbit polyclonalNF-kB antibody was employed as it is (ready to use), while rabbitpolyclonal Bax and eNOS were diluted according to theirmanufacturer’s specification at 1:500 and 1:1000, respectively.After applying the antibodies, slides were incubated overnight at4 8C. Poly HRP enzyme conjugate was applied for 20 minutes, afterwhich DAB chromogen was applied for 2 min. After rinsing DAB,counterstaining with Mayer Hematoxylin and cover slipping wereperformed as the final steps before slides were examined underthe light microscope.



Table 1Effect of atorvastatin (Ator) on change (D) of total body weight (wt), organ/total wt

ratios, and kidney and liver function tests in doxorubicin (DOX)-induced toxicity.

Control Ator DOX DOX/Ator

D Body wt (%) 100 � 4 99 � 5 78 � 2a 91 � 4b

Kidney/wt ratio 5.2 � 0.6 5.3 � 0.8 7.1 � 0.9a 5.9 � 0.5b,c

Liver/wt ratio 189.1 � 28.4 195.6 � 23.6 352.8 � 44.4a 249.2 � 39.2b

BUN (mg/dL) 6.3 � 0.6 6.6 � 0.7 7.9 � 0.8a 6.9 � 0.5b,c

Creatinine (mg/dL) 1.05 � 0.27 0.99 � 0.13 2.91 � 0.83a 1.52 � 0.62b,c

ALT (U/dL) 38.4 � 4.3 42.3 � 5.2 63.3 � 11.9a 50.1 � 8.1b

AST (U/dL) 131.2 � 20.7 145.9 � 15.6 183.9 � 42.1a 140.5 � 14.6b,c

Kidney/wt and liver/wt are ratios of weight of respective organ/total body

weight � 1000 ratio. BUN: blood urea nitrogen; ALT: alanine transaminase; AST:

aspartate transaminase. Values are representation of 6–11 observations as

means � SEM. Results are considered significantly different when P < 0.05.a Significant difference compared to control.b Significant difference compared to DOX group.c No significant difference compared to control.

M.A. El-Moselhy, A.A.K. El-Sheikh / Biomedicine & Pharmacotherapy xxx (2013) xxx–xxx 3

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2.7. Statistical analysis

The data was analyzed by one-way ANOVA followed by DunnettMultiple Comparison Test. The values are represented as mean-s � SEM. All statistical analysis was done using GraphPad Prism(GraphPad Prism software, 2011). The differences were consideredsignificant when the calculated P value is less than 0.05.

3. Results

3.1. Effect of Ator on change in total body weight, organ/body weight

ratio and hepato-renal function in DOX-induced toxicity

Total body weight was measured at the beginning and at theend of the experiment. The percent of change of body weightbetween the initial and final weights was significantly lower inDOX-treated group compared to control (Table 1). Administrationof Ator with DOX significantly improved the percent of change oftotal body weight compared to DOX sole therapy. In addition, theratio between the weight of the kidney and the final body weightwas significantly higher compared to control, which wassignificantly reversed in the DOX group pre-treated with Ator.Similarly, DOX caused significant increase in liver/total bodyweight ratio compared to control, which was reversed bypretreatment with Ator. Interestingly, Ator pretreatment affectedthe kidney/weight ratio more than the liver/weight ratio, as theformer decreased to levels not significantly different from control.Markers of renal function, BUN and creatinine, as well as markersof hepatic function, AST and ALT, were significantly elevated inDOX-treated group compared to control. On the other hand, DOX/Ator group showed significantly better renal and hepatic markerscompared to DOX group.

3.2. Effect of Ator on tissue oxidative, nitrosative and inflammatory

markers DOX-treated rats

GSH concentration, catalase activity and MDA level weredetermined as markers of oxidative stress, whereas nitrite/nitrateratio was assessed as an indicator of nitrosative stress, and TNF-aas an inflammatory marker. DOX-treated group showed significantdecrease in GSH concentration and catalase activity comparedwith untreated control in both kidney and liver (Table 2).Concomitant treatment of DOX with Ator increased renal andhepatic GSH and catalase values to levels statistically higher fromDOX-treated group. On the other hand, renal and hepatic MDA, NOand TNF-a levels increased in DOX-treated group compared tocontrol. This increase was reversed by pretreatment with Ator in

Table 2Effect of atorvastatin (Ator) on reduced glutathione (GSH), catalase, malondialdehyde (M

and liver exposed to doxorubicin (DOX).

Kidney

Control Ator DOX D

GSH

(mmol/g.tissue protein)

12.9 � 1.9 13.2 � 0.9 6.7 � 0.8a

Catalase

(U/g.tissue protein)

9.8 � 1.1 10.3 � 0.8 6.2 � 1.9a

MDA

(nmol/g.tissue protein)

38.3 � 3.3 36.9 � 4.2 62.9 � 9.9a

NO

(nmol/0.1 g tissue protein)

89.3 � 5.5 86.3 � 4.2 130.9 � 12.2a

TNF-a(ng/g.tissue protein)

91.9 � 8.2 88.3 � 10.7 179.2 � 21.8a 1

Values are represented as means � SEM of 6–11 observations.a Significant difference compared to control.b Significant difference compared to DOX.c No significant difference compared to control. Significant difference is reported wh


DOX/Ator group which showed significantly lower levels of theseparameters compared to DOX alone. Interestingly, by comparingsignificance levels, Ator seems to improve oxidative, nitrosativeand inflammatory markers in the kidney better than the liver, as itreversed these markers in the kidney to levels comparable to thatof the untreated control group.

3.3. Effect of Ator on renal and hepatic histopathology in

DOX-treated rats

Kidney histopathological examination revealed that controlgroup had normal glomeruli and renal tubules (Fig. 1; left panel).Ator-treated group was almost normal with only mild localizedcloudy swelling, and vacuolation in the renal epithelia. DOX-treated group, on the other hand, showed severe congestion inrenal blood vessels and glomeruli, with multifocal areas ofcoagulative necrosis. Glomerular Bowman’s spaces showeddilatation with hyaline casts in some renal tubules. Focal necroticareas were observed infiltrated with round cells of mostlylymphocytes and macrophages. Severe cloudy swelling, vacuolarand hydropic degeneration were detected. Treatment with Atortogether with DOX alleviated the severity of the lesions describedin DOX-treated group, as the DOX/Ator group showed normal renaltubules with only mild vacuolated glomerulus and few hyalinecasts. Little cloudy swelling and hydropic degeneration wereobserved.

Liver histopathological examination revealed normal hepaticstructure in untreated controls, with normal lobular architecture,

DA), nitric oxide (NO) and tumor necrosis factor-alpha (TNF-a) levels in rat kidney

Liver

OX/Ator Control Ator DOX DOX/Ator

11.9 � 0.4b,c 21.9 � 3.8 20.8 � 3.8 12.3 � 2.5a 17.2 � 1.8b

8.8 � 0.9b,c 10.5 � 0.6 9.9 � 0.5 7.1 � 0.7a 8.9 � 0.4b

42.9 � 8.7b,c 26.8 � 6.3 31.1 � 4.7 48.3 � 6.9a 37.5 � 7.3b

94.9 � 8.8b,c 78.7 � 9.1 81.3 � 4.6 139.3 � 12.4a 94.9 � 10.4b

11.3 � 17.9b,c 50.8 � 8.8 54.4 � 9.6 134.7 � 26.8a 87.2 � 22.2b

en P < 0.05.



Fig. 1. Effect of atorvastatin (Ator) on hepato-renal histopathology in doxorubicin (DOX)-treated rats. A photomicrograph of a section in rat kidney (left panel) and liver

(right panel). Left panel: (A) kidney of control group showing normal renal structure, (B) kidney of Ator-treated group showing mild cloudy swelling (arrow) and

vacuolation in the renal epithelia (arrowheads), (C) kidney of DOX-treated group showing congested glomeruli (arrows) besides necrotic (arrowhead) and degenerated

renal tubules with the insert showing necrotic renal tubules (N) and interstitial aggregation of round cells (arrows), (D) kidney of group receiving both DOX and Ator

showing hyaline cast inside the lumen of the renal tubule (arrow). Right panel: (E) liver of control group showing normal hepatic structure, (F) liver of Ator-treated group

showing congested blood vessel (arrow), (G) liver of DOX-treated group showing portal area with hyperplasia in the lining epithelia of the bile duct, fibrous connective

tissue proliferation and round cells infiltration (arrow) besides congested blood vessel (arrowhead), (H) liver of group treated with both DOX and Ator showing

hyalinization in the wall of portal blood vessels and mild fibrous connective tissue in the portal tract (arrowhead). (H&E, 100�; bar 100 mm).


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central vein and radiating hepatic cords (Fig. 1; right panel). Liverof Ator-treated group seemed almost normal, with only mildlycongested portal blood vessels and little hyperplasia in the liningepithelium of the bile ducts. Treatment with DOX alone causedinfarction with large irregular areas of coagulative necrosisinvaded and surrounded with erythrocytes and leukocytes. Focal


interstitial and portal aggregations of lymphocytes in addition tohydropic degeneration and fatty changes were also detected. Atortreatment together with DOX decreased the severity of the lesions,as hepatic cells showed only mild hydropic degeneration and fattychange, with some apoptotic bodies and hyalinization in the wallof the blood vessels. Scoring the histological alterations confirmed



Table 3Effect of atorvastatin (Ator) on severity of histopathological lesions in doxorubicin

(DOX)-induced nephrotoxicity in rats.


Tubular degeneration 0 � +++ ++

Tubular necrosis 0 0 ++ +

Leukocyte infiltrates 0 0 ++ �Glomerular damage 0 0 ++ �Hyaline casts 0 � ++ +

Score level 0 was considered normal while � is considered borderline. Scores+, ++,

and +++ are mild, moderate, and severe levels, revealing less than 25, 50, and 75%

histopathological alterations of total fields examined, respectively. Score represents

values obtained from tissue sections of 6 animals of each group, 5 fields/section (�400).

Table 4Effect of atorvastatin (Ator) on severity of histopathological lesions in doxorubicin

(DOX)-induced hepatotoxicity in rats.


Degeneration 0 + +++ +

Necrosis and apoptosis 0 0 N+++ A++

Thrombosis 0 0 ++ 0

Biliary hyperplasia 0 + ++ �Leukocyte infiltrates 0 0 +++ �

Score level 0 was considered normal while � is considered borderline. Scores+, ++,

and +++ are mild, moderate, and severe levels, revealing less than 25, 50, and 75%

histopathological alterations of total fields examined, respectively. Score represents

values obtained from tissue sections of 6 animals of each group, 5 fields/section. N:

necrosis; A: apoptosis (�400).


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that Ator co-therapy with DOX improved the pathological findingsand reverted signs of architectural distortion compared to DOXalone in both kidney (Table 3) and liver (Table 4).

3.4. Effect of Ator on renal and hepatic eNOS expression

in DOX-treated rats

Immunohistochemical staining by eNOS was performed inkidney and liver (Fig. 2; left and right panels, respectively). Controlrat kidney revealed eNOS expression in the endothelial layer of theglomeruli, peri-tubular capillaries and tubular epithelial cells(Fig. 2A). Comparable expression was observed in Ator-treatedgroup (Fig. 2B). On the other hand, DOX treatment causedsignificant decrease in eNOS expression (Fig. 2C), which wasreversed in DOX/Ator group (Fig. 2D). Liver of control and Ator ratgroup revealed expression of eNOS in the portal and sinusoidalvascular endothelia as well as in hepatocytes (Fig. 2F and G,respectively). DOX-treated group, however, showed significantdecrease in eNOS expression (Fig. 2H), which was reversed in DOX/Ator group (Fig. 2I). Semiquantitative analysis, performed tocalculate the degree of significance of protein expression, showedthat percent of immunopositive cells in kidney and liver (Fig. 2Eand J, respectively) was 38.8 � 2.1 and 35.1 � 1.9 in control groupand significantly decreased to reach 1.2 � 0.3 and 1.4 � 0.2 in DOX-treated group. After combined Ator treatment, percent of immuno-positive cells significantly increased compared to DOX alone to reach17.4 � 1.1 and 20.6 � 2.2 (P < 0.05).

3.5. Effect of Ator on renal and hepatic NF-kB expression

in DOX-treated rats

As a confirmative marker of inflammation, immunostainingwith NF-kB was performed in rat kidney (Fig. 3; left panel) andliver (Fig. 3; right panel). Kidneys from control (Fig. 3A) and Ator-treated (Fig. 3B) groups showed mild cytoplasmic expression ofNF-kB, indicating that NF-kB was present in its inactive form. Inkidneys of rats treated with DOX alone, NF-kB was observed to behighly expressed in renal nuclei (Fig. 3C), indicating nuclear


translocation, up-regulation and activation of NF-kB. Ator treat-ment together with DOX caused less staining of NF-kB in renalnuclei (Fig. 3D). In liver sections from control and Ator-treatedgroups, NF-kB expression was mildly expressed in the cytoplasmof hepatocytes (Fig. 3F and G, respectively). Following DOXtreatment, NF-kB immunoreactivity was highly expressed in thenucleus (Fig. 3H). A marked decrease in NF-kB staining wasobserved in the nucleus of combined DOX and Ator therapy, withmild cytoplasmic staining (Fig. 3I). Semiquantitative analysis wasperformed for scoring renal sections for assessment of nuclearlocalization of NF-kB in the kidney and liver (Fig. 3E and J,respectively). Percent of nuclei positive for NF-kB was about 30folds higher in kidneys of DOX group compared to that of control(29.9 � 2.6 vs 2.8 � 0.4), whereas Ator combined with DOXsignificantly reverted these values (14.8 � 2.1) to levels significantfrom DOX alone (P < 0.05). In the liver, more than 40% of all nucleiwere positive for NF-kB in sections obtained from the rats subjectedto DOX alone compared to less than 1% in those from control.Compared to DOX-treated group, rats receiving DOX/Ator showedmarked reduction in such percent to around 14%.

3.6. Effect of Ator on renal and hepatic Bax expression

in DOX-treated rats

Immunohistochemical localization of Bax was performed andserved as an apoptotic marker in kidney (Fig. 4; left panel) and liver(Fig. 4; right panel). In the kidneys of control group, Bax expressionwas minimal (Fig. 4A), which was insignificantly different fromAtor-treated group (Fig. 4B). Obvious marked Bax staining,however, was observed in DOX-treated group (Fig. 4C), whichwas more prominent in the proximal tubular epithelial cells. InDOX/Ator-treated group, significantly less Bax expression wasobserved compared to the group receiving DOX alone (Fig. 4D).Semiquantitative analysis was performed to calculate the sig-nificance of change in protein expression in the kidney (Fig. 4E) andliver (Fig. 4J). Percent of immunopositive cells in the kidney was2.1 � 0.4 in control, significantly increased in sole DOX therapy to45.8 � 2.6 and reversed to only 9.8 � 0.7 in DOX/Ator group. In theliver, percent of immunopositive cells was 2.7 � 0.3, 5.5 � 0.9,40.5 � 1.7, and 9.5 � 2.1 in control, Ator-, DOX-, and DOX/Ator-treated groups, respectively.

4. Discussion

Due to the importance of DOX in treatment of various types ofmalignancies, many hypotheses have been proposed for themechanisms underlying DOX toxicity and measures to circumventit. Unfortunately, the vast majority of reported studies weredirected to DOX cardiotoxic effects, with very few aiming at itshepato-renal damage. Here, we proved that Ator, the most widelyprescribed lipid-lowering statin globally [24], confers protectionagainst DOX-induced hepato-renal damage. Ator was previouslyreported to confer protection against DOX-induced testicular- andcardiotoxicity in mice [25]. In addition, prophylactic use of Atorwas effective in protecting against DOX-induced cardiomyopathyin humans [26]. Moreover, Ator had shown enhancement ofaccumulation of DOX in tumor cells [27], as well as independentanti-tumor effects [28]. Collective Ator cardio-protection and anti-tumor activity, together with its hepato-renal protection againstDOX-induced toxicity proved in the present study, may draw theattention to Ator as possible adjuvant in DOX chemotherapy.

In the present study, pretreatment with Ator in a dose of 10 mg/kg/day for 10 days improved kidney and liver functions, as well ashistopathological changes that have been altered due to DOXtherapy. Beneficial effects of Ator on renal function tests have beenpreviously reported in humans [29]. In contrast, effects of Ator on



Fig. 2. Effect of atorvastatin (Ator) on endothelial nitric oxide synthase (eNOS) immunohistochemical staining of doxorubicin (DOX)-treated and untreated rat kidney and

liver. Localization of eNOS immunoreactivity (�100) in the kidney (left panel) and liver (right panel) of (A and F) control group, (B and G) Ator-treated group, (C and H) DOX-

treated group and (D and I) concomitant DOX with Ator-treated group, respectively. E and J show semiquantitative analysis of eNOS immunohistochemical staining results in

kidney and liver, respectively. Values are represented as means � SE of number of eNOS immunopositive cells in sections of 6 animals of each group, 5 fields/section. a: significant

difference compared with control, b: significant difference compared from DOX group. Significant difference is reported when P < 0.05.


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Please cite this article in press as: El-Moselhy MA, El-Sheikh AAK. Protective mechanisms of atorvastatin against doxorubicin-inducedhepato-renal toxicity. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.09.001


Fig. 3. Effect of atorvastatin (Ator) on nuclear factor-kappa B (NF-kB) immunohistochemical staining of doxorubicin (DOX)-treated and untreated rat kidney and liver.

Localization of NF-kB immunoreactivity (�100) in the kidney (left panel) and liver (right panel) of (A and F) control group, (B and G) Ator-treated group, (C and H) DOX-

treated group and (D and I) concomitant DOX with Ator-treated group, respectively. E and J show semiquantitative analysis of NF-kB immunohistochemical staining results in

kidney and liver, respectively. Values are represented as means � SE of number of NF-kB immunopositive cells in sections of 6 animals of each group, 5 fields/section. a: significant

difference compared with control, b: significant difference compared from DOX group. Significant difference is reported when P < 0.05.


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Fig. 4. Effect of atorvastatin (Ator) on Bcl-2-associated X protein (Bax) immunohistochemical staining of doxorubicin (DOX)-treated and untreated rat kidney and liver.

Localization of Bax immunoreactivity (�100) in the kidney (left panel) and liver (right panel) of (A and F) control group, (B and G) Ator-treated group, (C and H) DOX-treated

group and (D and I) concomitant DOX with Ator-treated group, respectively. E and J show semiquantitative analysis of Bax immunohistochemical staining results in kidney

and liver, respectively. Values are represented as means � SE of number of Bax immunopositive cells in sections of 6 animals of each group, 5 fields/section. a: significant difference

compared with control, b: significant difference compared from DOX group. Significant difference is reported when P < 0.05.


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liver function enzymes, ALT and AST, have been controversial.While some studies reported that Ator caused an increase in ALTand AST [30], others reported no effect [31] or even decrease [32] inthe level of these enzymes. The reason is probably the differentdosage regimens of Ator, as, indeed, Ator in a dose of 80 mg/kg/dayfor 4 weeks caused hepatotoxicity in rats, whereas rats receiving20 mg/kg/day for the same duration had no hepatotoxic effects[33].

In the present study, DOX enhanced oxidative stress markers inkidney and liver, as it decreased tissue GSH level and catalaseactivity, while elevating the content of lipid peroxidation products.Ator succeeded in reverting DOX-induced hepato-renal oxidativestress markers, in concurrent with previous studies reporting Atorbeneficial effects on such markers in other models of liver andkidney damage [20,32,34]. DOX-induced oxidative stress has, forlong, been incriminated in initiating its multi-organ toxicity, as itundergoes bio-reductive activation by redox-cycling via its uniquechemical structure that favors free radical formation [35]. Despitethat earlier studies have suggested that the anticancer efficacy ofDOX is related to its pro-oxidant properties, several more recentstudies have shown the opposite and now it is acceptable thatoxidative stress is not a major contributor in DOX anticanceractivity [1]. Enhancement of endogenous antioxidant capacity viaup-regulating antioxidant enzymes, thus, does not affect DOXchemotherapeutic efficacy [36].

In the current study, DOX increased nitrosative stress as evidentby increasing hepato-renal tissue NO level. Pretreatment with Atorreversed DOX-induced nitrosative stress via decreasing tissue NOin both organs. NO is a free radical gas with dual action, that mightmediate cytoprotection or cytotoxicity [11]. Most studies sug-gested that NO generation, through up-regulation of inducibleform of NOS (iNOS), is one of the main mechanisms of DOX-induced cytotoxicity [9,37–39]. Still, others suggested that DOXdown-regulates iNOS [40], and decreases NO [41]. Studies evensuggested that DOX-induced toxicity is due to NO insufficiency ordys-regulation, which could be corrected by adjuvant inorganicnitrate [42]. Moreover, some studies suggested formulations ofDOX containing NO-releasing groups, as nitrooxy-DOX [43].

In the present study, DOX down-regulated eNOS expression inliver and kidney, while Ator pretreatment reversed DOX effect oneNOS. Our results are in concurrent with previous studies that havesuggested up-regulation of eNOS as a possible protectivemechanism of Ator [44–47]. Nevertheless, the effect of DOX oneNOS is still not conclusive. In previous studies, DOX showedvariable effects, ranging from up-regulating eNOS in the kidney[11] to inhibiting eNOS activity in the endothelium [48]. A studyshowed that the unselective NOS inhibitor, nitro-L-argininemethyl ester, increased DOX-induced mortality [49], suggestingthe inhibition of NOS as one of DOX-induced toxic mechanisms. Astudy employing iNOS knockout and eNOS transgenic micehighlighted the complexity of NOS physiology and reported thatvarious NOS isozymes have differential roles in DOX-inducedtoxicity in the heart [50]. Similar studies are required to elucidatethe role of NOS isoforms in DOX-induced toxicity in the liver andkidney.

In the present study, DOX-induced inflammatory markers as itincreased TNF-a tissue concentration and up-regulated NF-kB, aswell as increased the pro-apoptotic protein, Bax, expression in bothkidney and liver, while concomitant treatment with Ator reversedthese findings. Ator has been previously reported to possess anti-inflammatory properties via inhibiting NF-kB pathway in the liver,as in ischemia/reperfusion [47] and fructose-fed [51] models.Similarly, Ator-mediated down-regulation of renal NF-kB wasreported in gentamicin-induced nephrotoxicity [20]. It has beensuggested that TNF-a down-regulates eNOS expression [52],which is in concurrence with our results. Interestingly, Ator was


reported to possess tumor-specific pro-apoptotic effects, as itinduces Bax in lymphoma [53] and leukemic cells [54] leading totheir death, while in non-tumor cells, Ator had no effect on Baxexpression [55] and, in some studies, even down-regulated Bax[56], indicating its anti-apoptotic effect.

The sequence of events leading to DOX-induced multi-organdamage is complex, but is most probably initiated by DOXredox-cycling [57]. During this process, DOX gains an electronand is reduced into DOX radical. This reaction is catalyzed byseveral endogenous enzymes, including catalase [35] andeNOS [58]. Re-oxygenation then occurs and DOX returns to itsoriginal form, generating superoxide free radicals, hydrogenperoxide and toxic hydroxyl radical [59]. The resultant radicalsfurther promote oxidative stress through altering redox status of keyproteins, as GSH that can become oxidized forming disulfide [60]. Freeradicals also stimulate TNF-a, that would further activate multiplesignaling pathways, including NF-kB inflammatory pathway [61]and the pro-apoptotic protein, Bax [62]. According to our recentreview on the crosstalk between free radical formation, nitrosativestress, inflammatory process and apoptosis [63], these pathways aremore complex and intermingling, which requires more in-depthmolecular studies to elucidate their overall role in the protective effectof Ator.

5. Conclusion

We confirm that Ator, besides its cholesterol-lowering benefits,possesses manifold properties, as antioxidant, anti-nitrosative, anti-inflammatory and anti-apoptotic, which can ameliorate DOX-induced hepato-renal toxicity by intervening with several steps ofDOX-induced toxic vicious circuit. Our results, thus, suggest Ator aspotentially successful adjuvant therapy for DOX chemotherapy thatmay widen the therapeutic window of DOX as an anticancer drug.

Disclosure of interest

The authors declare that they have no conflicts of interestconcerning this article.

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Protective mechanisms of atorvastatin against doxorubicin-induced hepato-renal toxicity

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