under peer review2 19 group at the 9a position of the aglycone ring. the resulting dibasic...

*Corresponding author: Email: [email protected]; Phone: +2348068525502

Research paper1

Influence of Azithromycin Treatment on Hepatic2

Lipid Peroxidation and Antioxidant Defence3

Systems of Rats4

5Olayinka Ebenezer Tunde* and Ore Ayokanmi6

Biochemistry Unit, Department of Chemical Sciences, P.M.B. 1066, Ajayi Crowther University, Oyo, Nigeria.789

ABSTRACT1011

Aim: Azithromycin is a semisynthetic macrolide antibiotic commonly indicated for use in middle ear,upper and lower respiratory tract infections, bronchitis, and community-acquired pneumonia with awell-established safety and efficacy profile. The antibiotic is usually well tolerated; however, it hasbeen associated with rare cases of progressive cholestatic hepatitis and fulminant hepatic failure. Thisstudy was therefore designed to examine the effect of two preparations of azithromycin; Zithromax250® (5.6 mg/kg body weight, b.w.) and Zithromax 500® (11.2 mg/ kg b.w.) on some hepatic and renalfunction indices, oxidative stress and antioxidant defense system in rats.

Study Design: Toxicological, Histological and Biochemical study.

Place and Duration of study: Biochemistry Unit, Department of Chemical Sciences, Faculty ofNatural Sciences, Ajayi Crowther University, Oyo, Nigeria between March 2012 and April 2012.

Methodology: Thirty rats (Wistar strain) weighing between 180 – 200 g were randomly assigned intothree treatment groups of ten animals each; Group A (Control) did not receive any drug, Group Breceived Zithromax 250® (5.6 mg/ kg b.w) and Group C received Zithromax 500® (11.2mg/ kg b.w)twice daily for seven days.

Results: Urea, Creatinine and Bilirubin levels were significantly (p=0.05) elevated in the plasma of therats that received Zithromax 250® and Zithromax 500® by 20.0% and 35.0%, 78.0% and 130.0%, and14.0% and 47.4% respectively when compared to the control. Activities of some marker enzymes;aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) andGamma-glutamyl transferase (GGT) were also significantly increased (p=0.05) in the plasma oftreated animals by 19.0% and 36.6%, 27.0% and 36.4%, 16.0% and 23.5%, and 43.9% and 80.5%respectively. Also, there was a significant increase (p=0.05) in plasma Triglycerides, Total cholesterol,HDL- and LDL-cholesterol in the treated animals by 20% and 31.2%, 19% and 35.4%, 24% and74.3%, and 33% and 35.2% respectively. Furthermore, Zithromax 250® and Zithromax 500®

significantly(p=0.05) reduced the levels of hepatic Ascorbic acid, Glutathione (GSH) and activities ofGlutathione-S-transferase (GST) by 35%, 66.3% and 34% and 56.1%, 45% and 50%, respectively. Inaddition, there was a significant decrease in the activities of hepatic Catalase (CAT), and Superoxidedismutase (SOD) by 67.4% and 43.0%, and 43% and 50% respectively in the two treated group.These were accompanied by a significant increase (p=0.05) in hepatic lipid peroxidation (MDA) by81% and 91.6% respectively in the treated groups. The histology of the liver revealed sinusoidal andportal congestion and a mild periportal cellular infiltration by mononuclear cells, by Zithromax 500®.Likewise, kidney histopathology revealed severe cortical congestion and hemorrhage and few tubulescontaining protein casts in the treated group.

Conclusion: In conclusion, the administration of Zithromax 250® and Zithromax 500® induced markedrenal and liver damage, oxidative stress and altered the antioxidant status in rats.

12Keywords: Azithromycin, liver and renal damage, oxidative stress, antioxidants13

1415

1. INTRODUCTION16Azithromycin (9-deoxo-9a-aza-9a-methyl-9a-homoerythromycin, Fig. 1), an erythromycin17semisynthetic derivative is formed by inserting a methyl-substituted nitrogen in place of the carbonyl18

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group at the 9a position of the aglycone ring. The resulting dibasic 15-membered ring macrolide19derivative is more appropriately referred to as an ‘‘azalide.’’ This structural modification gives20azithromycin several distinct advantages over erythromycin: makes the compound more stable in21acid, significantly increases the serum half-life, improved pharmacokinetic profiles, gastrointestinal22tolerability, oral bioavailability and tissue penetration, and results in increased activity against gram-23negative organisms when compared with erythromycin [1]. Its extensive coverage against all the24major respiratory pathogens, excellent safety profiles and desirable tissue distribution into the25infection sites make azithromycin the drug of choice for the treatment of respiratory tract infections [2].26Azithromycin is frequently prescribed for the treatment of middle ear and upper respiratory tract27infections, bronchitis, and community-acquired pneumonia [3].28

Macrolide antibacterial agents including azithromycin are lipophilic and are extensively distributed in29body fluids and tissues. They exert their antibacterial effects by reversibly binding to the 50s subunit30of the bacterial ribosome. This interaction inhibits RNA-dependent protein synthesis by preventing31transpeptidation and translocation reactions [4]. The advanced macrolide – azithromycin is a32structural analogue of erythromycin that has similar mechanism of action.33

34

Fig. 1: Azithromycin35

Certain macrolide antibiotics have been documented to be hepatotoxic in animals [5] and human36subjects in clinical studies [6, 7, 8]. Macrolide antibiotics are also known to alter the physiological37redox homeostasis leading to oxidative stress [9, 10], and lipid peroxidation. Cells require oxygen for38certain metabolic processes including the metabolism of xenobiotic and in the process certain39destructive species referred to as reactive oxygen species (ROS) are generated [11]. The cell40protects itself from these ROSs, by the actions of certain low molecular weight substances such as41glutathione, vitamin A, E, and C (the non enzymic antioxidants) and enzymic antioxidants such as42superoxide dismutase (SOD), catalase (CAT) etc.43

The effects of azithromycin on liver damage has been well documented in clinical studies [6, 7, 12, 8],44however, information regarding the hepatotoxic and renal effects of azithromycin as well as its effect45on the antioxidants status in nonclinical studies is still lacking. Therefore, the objective of this study46was to investigate the effect of azithromycin on the markers of renal and hepatic toxicity, hepatic47redox status and lipid peroxidation in rats.48

492. MATERIAL AND METHODS50

512.1 Chemicals and Reagent52

Azithromycin (Zithromax® 250 mg and 500 mg tablets) was a product of Pfizer, New York,53Glutathione, 1-Chloro, 2, 4-dinitrobenzene (CDNB), 5, 5-dithio bis-2-nitrobenzoic acid (DTNB),54epinephrine, and hydrogen peroxide (H2O2) were all purchased from Sigma Chemical Company55(London, UK). Assay kits for alanine transferase (ALT), aspartate aminotransferase (AST), alkaline56phosphatase (ALP), gamma glutamyl transpeptidase (GGT), Urea, Creatinine, total Bilirubin, Total57cholesterol, triglycerides (TG), high density lipoprotein (HDL) cholesterol, and low density lipoprotein58(LDL) cholesterol were obtained from Randox® laboratories ltd (Antrim, UK). All other reagents used59were of analytical grade.60

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2.2 Animals and Treatments63

2.2.1 Animals64

In bred 5-6 week-old male rats (Wistar strain) weighing 200±20g were used in this study. The rats65were bred and housed in the animal house of the Department of Chemical sciences, Ajayi Crowther66University, Oyo, Nigeria. They were kept in wire meshed cages at room temperature and under67controlled light cycle (12-hr light: dark).They were fed with commercial rat chow (Ladokun feeds,68Ibadan, Nigeria) and supplied water ad libitum. All experiments were carried out in the Biochemistry69laboratory of Ajayi Crowther University, Oyo, Nigeria and conducted without anaesthesia and protocol70conforms to the guidelines of the National Institute of Health [13] for laboratory animal care and use.71

2.2.2 Treatments72

Thirty (30) healthy male albino rats (Wistar strain) were randomly divided into three groups of 10 rats73each. Group I (Control) received physiological saline, while groups II and III were administered74Zithromax 250® (5.6 kg/ b.w) and Zithromax 500® (11.2 kg/ b.w) respectively as presented in Table 1.75The treatments were administered in divided doses, twice daily for seven days. The rats were76sacrificed 24hrs after the last treatment.77

Table 1: Experimental Design78

Experimental Groups Treatments

Group I (n=10) Control (Normal saline)

Group II (n=10) Zithromax 250® (5.6mg/kg.b.w)

Group III (n=10) Zithromax 500® (11.2mg/kg.b.w)

79

2.3 Collection of blood samples for plasma preparation80

Blood was collected from the inferior vena cava of heart of the animals into heparinized tubes, and the81rats were sacrificed by cervical dislocation. Plasma was prepared by centrifuging blood samples for82ten minutes at 3000 x g in an Eppendorf (UK) bench centrifuge. The clear supernatant was used for83the estimation of plasma lipid profiles and enzymes.84

2.4 Preparation of cytosolic fractions85

The liver, excised from rat, blotted of blood stains, rinsed in 1.15%KCl was homogenized in 4 volumes86of ice-cold 0.01 M potassium phosphate buffer, (pH 7.4). The homogenates were centrifuged at8712,500 g for 15 min at 4°C and the supernatants, termed the post-mitochondrial fractions (PMF) were88aliquoted and used for enzyme assays.89

2.5 Renal and liver functions test90

Plasma Creatinine, urea and bilirubin determination was done using Randox® diagnostic kits. Methods91for Creatinine assays are based on colorimetric alkaline picrate methods Jaffe [14] with Creatinine-92picrate complex measured at 492nm. The urea determination method was based on the Fenton93reaction Tietz et al. [15] with the Diazine chromogen formed absorbing strongly at 540nm. The94dimethy sulphoxide method by Tietz, et al. [16] was used for bilirubin determination. The dimethyl95sulphoxide form a coloured compound with maximum absorption at 550nm.96

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2.6 Determination of plasma AST, ALT, ALP and GGT activities99

Plasma AST, ALT, ALP and GGT activities were determined using Randox® diagnostic kits.100Determination of AST and ALT activities were based on the principle described by Reltman and101Frankel [17]. AST was measured by monitoring the concentration of oxaloacetate hydrazone formed102with 2,4-dinitrophenylhydrazine at 546nm and ALT was measured by monitoring the concentration of103pyruvate hydrazone formed with 2,4-dinitrophenylhydrazine at 546 nm. ALP was determined in104accordance with the principles of Tietz [15]. The p-nitrophenol formed by the hydrolysis of p-105Nitrophenyl phosphate confers yellowish colour on the reaction mixture and its intensity can be106monitored at 405nm to give a measure of enzyme activity. GGT activity was measured based on a107modification of the method described by Theodorsen et al. [18] using Randox diagnostic kits.108

2.7 Determination of plasma lipid profiles109

The plasma total cholesterol, HDL- cholesterol, LDL- cholesterol and triglycerides were determined110using Randox® diagnostic kits and the determination were based on CHOD-PAD enzymatic111colorimetric method of Trinder [19]. Cholesterol in the presence of Cholesterol Oxidase and112Peroxidase enzymes produced 4-(p)-benzoquinone-monoiminophenazone whose absorbance is read113at 546 nm. Low-density lipoprotein (LDL and VLDL) and Chylomicron fractions are precipitated114quantitatively by the addition of phosphotungstic acid in the presence of magnesium ions. After115centrifugation, the cholesterol concentration in the HDL (High density lipoprotein) fraction which116remains in the supernatant is determined enzymatically. The Triglycerides are determined after117enzymatic hydrolysis with lipases. The indicator is a quinoneimine formed from hydrogen peroxide, 4-118aminophenazone and 4-chlorophenol under the catalytic influence.119

2.8 Non-enzymatic antioxidants and lipid peroxidation Assay120

Hepatic vitamin C was determined chemically according to the method of Erel et al. [20] using dinitro121phenyl hydrazine (DNPH), while hepatic glutathione was determined according to the method of122Jollow et al. [21]. The chromophoric product resulting from the reaction of Ellman’s reagent with the123reduced glutathione, 2-nitro-5-thiobenzoic acid possesses a molar absorption at 412 nm which was124read in a spectrophotometer. Reduced GSH is proportional to the absorbance at 412 nm. The extent125of lipid peroxidation (LPO) was estimated by the method of Vashney and Kale [22], the method126involved the reaction between malondialdehyde (MDA; product of LPO) and thiobarbituric acid to yield127a stable pink chromophore with maximum absorption at 532 nm.128

2.10 Antioxidant enzymes assay129

The procedure of Misra and Fridovich [23] as described by Magwere et al. [24] was used for the130determination of hepatic superoxide dismutase (SOD) activity by measuring the inhibition of auto-131oxidation of epinephrine at pH 10.2 and 300C. Hepatic catalase activity was determined according to132the method of Asru [25] by measuring the reduction of dichromate in acetic acid to chromic acetate at133570 nm. Hepatic Glutathione S-transferase (GST) activity was determined by the method described134by Habig et al. [26] using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate.135

2.11 Protein determination136

Protein determination of plasma and all fractions was estimated by the method of Lowry et al. [27]137using bovine serum albumin as standard.138

2.12 Histopathological studies139

The method of Baker and Silverton [28] was employed for the processing of liver for histopathological140studies.141

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2.13 Statistical analysis144

Results were expressed as mean of 10 replicates ± SD. Data obtained were subjected to one-way145Analysis of Variance (ANOVA) and complemented with Student’s t-test using Stat Pac Statistical146Software. Statement of statistical significance was based on p<0.05.147

1483. RESULTS AND DISCUSSION149

1503.1 Effect of Azithromycin on the levels of plasma Creatinine, Urea and Bilirubin in151rats152Table 2 shows the effects of Azithromycin treatment on plasma Creatinine, Urea and Bilirubin level.153Azithromycin (as Zithromax 250mg® and Zithromax 500mg®) treatment significantly(p=0.05) increased154the plasma Creatinine, Urea and Bilirubin levels in the rats by 78% and 130%, 21% and 35%, and15514% and 47.4%, respectively when compared with control.156

1573.2 Effect of Azithromycin on the levels of plasma enzymes in rats158

159Effects of Azithromycin (Zithromax 250mg® and Zithromax 500mg®) treatment on Plasma Alkaline160Phosphatase (ALP), Alanine aminoransferase (ALT), Aspartate aminotransferase (AST) and γ-161Glutamyl transpeptidase (GGT) activities in rats were shown in Table 3. Zithromax 250mg® and162Zithromax 500mg® treatments significantly(p=0.05) increased the plasma ALP, ALT, AST and GGT163activities in the rats by 16% and 127.4%, 20% and 36.4%, 20% and 36.6% and 43% and 80.5%164respectively when compared with control.165

1663.3 Effect of Azithromycin on the levels of plasma lipid profiles in rats167

168The effects of Azithromycin (Zithromax 250mg® and Zithromax 500mg®) treatment on Plasma Lipid169Profiles in rats are presented in Table 4. Zithromax 250mg® and Zithromax 500mg® treatment170significantly increased the plasma Total Cholesterol, HDL- and LDL- Cholesterol concentrations in the171rats by 19% and 35.44%; 24% and 74.32%; and 33% and 35.19% respectively when compared172with control (p=0.05). Similarly, plasma Triglyceride level was also significantly increased in the173treated group by 20% and 31.2% respectively when compared with the control (p=0.05).174

1753.4 Effect of Azithromycin on the levels of hepatic antioxidant enzymes in rats176

177Table 5 shows the effects of Azithromycin (Zithromax 250mg® and Zithromax 500mg®) treatment on178Hepatic Superoxide dismutase (SOD) and Catalase (CAT) activities in rats. Zithromax 250mg® and179Zithromax 500mg® treatment significantly decrease the SOD and CAT activities in the liver of rats by18027% and 50%; and 30% and 50% respectively when compared with control (p=0.05). Hepatic181glutathione-S-transferase (GST) activity was also significantly reduced following treatment with182Zithromax 250mg® and Zithromax 500mg® by 45% and 56.1% respectively (Fig. 4).183

1843.5 Effect of Azithromycin on the levels of hepatic non-enzymatic antioxidant and lipid185peroxidation in rats186

187The hepatic Vitamin C concentration is shown in Fig 2 following treatment with Azithromycin188(Zithromax 250mg® and Zithromax 500mg®). The Vitamin C level was significantly decreased by 35%189and 66.3% respectively in the treated groups when compared with the control (p=0.05). Also, the GSH190level was significantly decreased by 34% and 67.4% respectively in the treated groups when191compared with the control (Fig.3). Furthermore, hepatic lipid peroxidation (MDA) level was192significantly increased by 81% and 91.7% in the treated groups respectively when compared with the193control (Fig.5).194

1953.6 Effect of Azithromycin on the histopathology of kidney and liver of rats196

197Plates 1 and 2 are photomicrographs showing the effect of azithromycin on the liver and kidney198respectively. The Zithromax 250 mg resulted in mild periportal cellular infiltration by mononuclear cells199

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(Plate 1B). A sinusoidal and portal congestion with mild periportal cellular infiltration by mononuclear200cells was observed in the Zithromax 500mg group (Plate 1C). In the kidney, Zithromax 250 mg,201produces protein casts in the tubular lumen (Plate 2B), while, severe cortical congestion and202haemorrhage, and few tubules containing protein casts was observed in the Zithromax 500mg group203(Plate 2C).204

205206

Table 2: Effect of Azithromycin on Plasma Creatinine, Urea and Bilirubin levels in rats.207208

TREATMENT CREATININE(mg/dl)

UREA(mg/dl)

TOTAL BILIRUBIN(mg/dl)

Control 0.14+0.01 46.8+0.83 0.57+0.09Zithromax 250mg® 0.25+0.01 (78.5%)* 56.4+1.5 (20.5%)* 0.65+0.01 (13.4%)*Zithromax 500mg® 0.32+0.01 (130%)* 63.2+1.9 (35%)* 0.85+0.03 (47.4%)*

209The values are the Means + SD (range) for ten rats in each group; *significantly different from the210

control p=0.05; Values in parenthesis represent percentage (%) increase.211212

Table 3: Effects of Azithromycin on Plasma Alkaline Phosphatase (ALP), Alanine213Aminotransferase (ALT), Aspartate Aminotransferase (AST) and -Glutamyl transpeptidase214(GGT) activities in rats.215

216TREATMENT ALP (U/l) ALT (U/l) AST (U/l) GGT (U/l)

Control 103.2+1.9 52.2+3.1 61.2+3.6 8.2+0.8Zithromax 250mg® 120+2.9 (16.2%)* 66.2+2.2 (26.8%)* 73.4+1.6 (19.9%)* 11.8+0.8 (43.9%)*Zithromax 500mg® 127.4+1.5 (23.5%)* 71.2+1.9 (36.4%)* 83.6+3.1 (36.6%)* 14.8+0.8 (80.5%)*



Table 4: Effects of Azithromycin on Plasma Lipid Profiles in rats.221222

TREATMENT TOTAL-cholesterol

(mg/dl)

HDL-cholesterol(mg/dl)

LDL-cholesterol(mg/dl)

TRIGLYCERIDE(mg/dl)

Control 41.2+1.9 14.8+0.8 21.6+1.5 43.6+2.6

Zithromax 250mg® 49.2+1.4 (19.4%)* 18.4+0.5 (24.3%)* 28.8+1.3 (33.3%)* 52.6+2.0 (20.6%)*

Zithromax 500mg® 55.8+1.8 (35.4%)* 25.8+2.1 (74.3%)* 29.2+1.6 (35.2%)* 57.2+4.1 (31.2%)*223

The values are the Means + SD (range) for ten rats in each group; *significantly different from the224control p=0.05; Values in parenthesis represent percentage (%) increase.225

226Table 5: Effects of Azithromycin on Hepatic Superoxide dismutase (SOD) and Catalase227activities in rats.228

229TREATMENT SOD Activity (units) CATALASE Activity

(μmole H2O2 consumed/min)Control 8+0.7 0.36+0.02

Zithromax 250mg® 7.4+1.1 (7.5%)* 0.26+0.01 (28%)*Zithromax 500mg® 4+0.7 (50%)* 0.18+0.02 (50%)*


control p=0.05; Values in parenthesis represent percentage (%) decrease. 1 unit of232SOD activity was given as the amount of SOD necessary to cause 50% inhibition of the oxidation of233

adrenaline to adrenochrome during 1 minute.234

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UREA(mg/dl)












(mg/dl)



TRIGLYCERIDE(mg/dl)

Control 41.2+1.9 14.8+0.8 21.6+1.5 43.6+2.6

Zithromax 250mg® 49.2+1.4 (19.4%)* 18.4+0.5 (24.3%)* 28.8+1.3 (33.3%)* 52.6+2.0 (20.6%)*

Zithromax 500mg® 55.8+1.8 (35.4%)* 25.8+2.1 (74.3%)* 29.2+1.6 (35.2%)* 57.2+4.1 (31.2%)*224









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UREA(mg/dl)












(mg/dl)



TRIGLYCERIDE(mg/dl)

Control 41.2+1.9 14.8+0.8 21.6+1.5 43.6+2.6

Zithromax 250mg® 49.2+1.4 (19.4%)* 18.4+0.5 (24.3%)* 28.8+1.3 (33.3%)* 52.6+2.0 (20.6%)*

Zithromax 500mg® 55.8+1.8 (35.4%)* 25.8+2.1 (74.3%)* 29.2+1.6 (35.2%)* 57.2+4.1 (31.2%)*225









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235

Figure 2: Influence of Azithromycin on hepatic Vitamin C concentration in Rats.236

The values are the Means + SD (range) for ten rats in each group *significantly different from the237control, p=0.05.238

239

240

Figure 3: Influence of Azithromycin on Hepatic reduced Glutathione (GSH) level in Rats.241


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Figure 4: Influence of Azithromycin on Hepatic Glutathione-S-transferase (GST) activity in246Rats.247


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Figure 5: Influence of Azithromycin on Hepatic Lipid peroxidation (MDA) level in Rats.251


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Plate 1: Effect of Azithromycin on histology of the Liver cell. A - Control: No visible lesions seen;257B - 250 mg Azithromycin: There is mild periportal cellular infiltration by mononuclear cells. C - 500mg258

Azithromycin: There is sinusoidal and portal congestion and there is also mild periportal cellular259infiltration by mononuclear cells.260

261

262

Plate 2: Effect of Azithromycin on histology of the Kidney cell A - Control: No visible lesions263seen; B - 250 mg Azithromycin: There are protein casts in the tubular lumen; C - 500mg Azithromycin:264There is severe cortical congestion and haemorrhage. There are also few tubules containing protein265

casts.266

267

A B

C

A B

C

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3.7 Discussion268

Azithromycin (Zithromax(R)) is an azalide, a subclass of macrolide antibiotics, structurally modified269from erythromycin. Its efficacy and potential to inhibit microorganisms had led to its increased use. It270is well-known that Azithromycin is widely used to treat or prevent certain bacterial infections, most271often those causing middle ear infections, strep throat, pneumonia, typhoid, and sinusitis. However,272an increasing number of evidence indicates that it has risk of hepatotoxicity and cardio-toxicity as273adverse effect [8, 29].274

Oxidative stress mediated by reactive oxygen species (ROS) has been implicated as a common link275between chronic liver damage and hepatic fibrosis [30]. The human body contains an array of276antioxidant defense mechanisms (non-enzymatic and enzymatic antioxidants) to remove harmful277ROS as soon as they are formed and to prevent their deleterious effects [31]. The non-enzymatic278antioxidants include vitamins E and C, and reduced glutathione (GSH), while the enzymatic279antioxidants include superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase280(GSHPx).281

In the present study, the effect of two preparations of azithromycin; Zithromax 250® (5.6mg/kg.b.w)282and Zithromax 500® (11.2 mg/kg.b.w) on markers of renal and hepatic toxicity, lipid profile and283antioxidant status was investigated in rats. We observed that the two preparations of Azithromycin284induce renal and liver failure in the rats. This was obvious from the renal and liver function test as285plasma concentration of creatinine, urea and bilirubin increased significantly in the treated groups,286suggesting impairment of renal and liver function. Urea and creatinine are metabolic waste products287that are freely filtered by the glomeruli of the kidneys [32] and their serum/plasma concentrations are288commonly used to screen for renal or cardiovascular diseases [33, 34]. Elevation of the plasma levels289of creatinine and urea is an indication of abnormal renal function [35]. Increases in the plasma290bilirubin concentration are generally the consequence of bile accumulation as a result of impairment291to intrahepatic or extrahepatic bile flow, increased production associated with accelerated erythrocyte292destruction, or altered bilirubin metabolism [36, 37, 38].293

An integrated evaluation of data from the plasma levels of ALT, AST, ALP and GGT has been shown294to be crucial in the identification of hepatobiliary injury [39]. Hepatotoxic drugs has been shown to295cause damage to the liver cell membranes which makes marker enzymes like AST, ALT and ALP296leak into serum and show increased activities [40]. Hepatic causes of increased serum ALT and AST297activities are known to include hepatocellular necrosis, injury, or regenerative or reparative activity298[41, 42, 38]. Plasma/serum ALP elevation has been attributed to increased osteoblastic activity and299hepatobiliary diseases [43].The elevated level of aminotransferases and phosphatases observed in300the 5.6 and 11.2 mg/kg dose of Zithromax® treated animals is highly indicative of hepatic toxicity [44,30145].302

The hepatotoxic effects of azithromycin appear to progress through generation of free radicals and303ROSs as evident in reduction in the levels of enzymic and non-enzymic antioxidants. In the present304investigation, we observed a significant reduction in the activities of SOD, CAT and GST in the liver of305azithromycin- treated animals. The significantly decreased activity of the ROS scavenging enzyme,306SOD and CAT, by exposure to azithromycin, conforms to previous report on macrolide antibiotics [46].307This may be due to the damaging effects of free radicals possibly generated by the action of the drug.308

The antioxidants enzymes CAT and SOD represent the primary intracellular antioxidants defence309mechanism against oxidative stress [20]. Catalase is a tetrameric hemoprotein present in the liver310cells and erythrocytes at high concentration [47]. It is generally accepted that H2O2 can be detoxified311by catalase which removes it when present at high concentration. Catalase is known to be inhibited312by ROS such as superoxides anion which converts it to ferroxy and ferryl states that are inactive313forms of enzymes [48]. SOD catalyzes the disputation of superoxide radicals (O2

-) to hydrogen314peroxide (H2O2) and molecular oxygen (O2). SOD therefore protects catalase against inhibition by315

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superoxide anion. Thus the balance of this enzyme system may be essential to get rid of superoxide316anion and peroxides generated in subcellular compartments of the liver. The antioxidant enzymes,317Superoxide dismutase and Catalase constitute a mutually supportive team of defense against reactive318oxygen species.319

Glutathione-S-Transferase (GST) is a family of multifunctional isozymes found in all eukaryotes,320catalyzing both glutathione dependent conjugation and reduction reactions [49]. One main function of321GST is to catalyze the biotransformation of xenobiotics, including drugs detoxification in the322mercapturic acid pathway, leading to the elimination of toxic compounds [50], and also acting as an323antioxidant enzyme [51]. In this study, GST activities were inhibited in animals treated with the two324preparations of Azithromycin. This observation, therefore, suggests that these drugs may alter the325expression and activities of antioxidant enzymes as a result of toxic metabolites generated during326their biotransformation.327

The level of reduced GSH is a measure of non-enzymic antioxidant and cellular redox status of cells328in higher animals [52]. In addition to vitamin A and β-carotene, Ascorbic acid (Vitamin C) is known to329represent the first line of antioxidant defence [53, 54] and this vitamin is likely to be most susceptible330to free radical oxidation. Ascorbate is a good free radical scavenger due to its chemical properties331[55]. Studies have shown that the redox state of intracellular vitamin C is controlled by the intracellular332level of GSH [56]. Our results showed that Azithromycin treatment decreased the overall redox333status in the liver as indicated by a significant decrease in the level of hepatic GSH and Vitamin C.334The observed decrease in GSH and Vitamin C levels further confirmed the formation of reactive335oxygen species or toxic metabolites from the two preparation of Azithromycin.336

The thiobarbituric acid assay is a satisfactory means of assessing lipid peroxidation (LPO) through337assay of MDA in biological samples. The elevated level of malondialdehyde (MDA) in the liver of338Azithromycin treated rat is a clear manifestation of excessive formation of free radicals and activation339of lipid peroxidation systems. The level of MDA is a good indicator of the degree of lipid peroxidation340and tissue damage. The accumulation of LPO products observed in this study agrees with previous341studies on certain macrolide antibiotics [57, 58]. The significant increase in the level of lipid342peroxidation may be related to the reduction in the enzymic and non enzymic antioxidant systems,343leading to accelerated oxidation of lipids. Lipid peroxidation has been shown to cause a significant344alteration in the structural integrity and functions of cell membranes [59].345

Lipid profile analysis revealed a significant increase in the plasma level of total cholesterol, LDL-346cholesterol, HDL-cholesterol and triglycerides by the two preparations of Azithromycin. It has been347suggested that cholesterol is a general indicator of the level of lipid in the circulation [60] and together348with polyunsaturated fatty acids (PUFA) are the main components of LDL. PUFA is the substrate349required for MDA formation and the amount of peroxidised lipid formed may be related both to the350amount of substrate and to the level of lipid peroxidation (LPO). Oxidation of LDL-cholesterol is known351to result in the depletion of lipoprotein antioxidants and subsequent accumulation of cholesterol esters352[59].353

In the present investigation, we observed that azithromycin induced renal and hepatic toxicity in rats354as explained by the photomicrograph of rat kidney and liver sections. The histological alterations355observed in the liver of azithromycin - treated rats characterized by sinusoidal and portal congestion,356and mild periportal cellular infiltration by mononuclear cells coupled with the presence of protein casts357in the kidney tubular lumen as well as severe cortical congestion and haemorrhage is an indication of358disruption of cellular architecture. All these abnormalities might have resulted due to the formation of359highly reactive radicals because of oxidative threat caused by the drug which disrupted normal360cellular functioning of the liver and the kidney.361

362

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4. CONCLUSION363

In summary, azithromycin induced renal and hepatic damage in rats and its mechanism of toxicity364appears to proceed through the generation of free radicals or depletion of the antioxidant systems.365

COMPETING INTERESTS366

We declared that there are no competing interests.367

AUTHORS’ CONTRIBUTIONS368

This research work was carried out in collaboration between the two authors – the experimental369designed, the study, the statistical analysis, Interpretation of biochemical findings and preparation of370journal manuscript.371

CONSENT372

Not applicable.373

ETHICAL APPROVAL374

Not applicable.375

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