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Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive

Research Article

volume 60 issue 10 (october 2026) : 1830-1836,   Doi: 10.18805/IJAR.B-4453

Alterations in Biochemical Markers of Liver and Kidney Function and Oxidative Stress Indices on Sub-acute Exposure of Enrofloxacin in Albino Rats

P
Prateek Mishra1,*
V
Vidhi Gautam2
R
R.K. Sharma2
A
Anushri Tiwari2
1Department of Veterinary Pharmacology and Toxicology, Nanaji Deshmukh Veterinary Science University, Jabalpur-482 002, Madhya Pradesh, India.
2Department of Veterinary Pathology, Nanaji Deshmukh Veterinary Science University, Jabalpur-482 002, Madhya Pradesh, India.
Cite article:- Mishra Prateek, Gautam Vidhi, Sharma R.K., Tiwari Anushri (2026). Alterations in Biochemical Markers of Liver and Kidney Function and Oxidative Stress Indices on Sub-acute Exposure of Enrofloxacin in Albino Rats . Indian Journal of Animal Research. 60(10): 1830-1836. doi: 10.18805/IJAR.B-4453.

ABSTRACT

Background: The present study was performed to evaluate Alterations in biochemical markers of liver and kidney function and oxidative stress indices on sub-acute exposure of enrofloxacin in albino rats. 

Methods: Albino rat weighing 200-300 gm were treated with enrofloxacin orally with the help of gavage needle and devided into three groups each group consist six rats. The rats of group I were served as control. However, rats of group II and III were treated with enrofloxacin @ 5 mg/kg b.wt., enrofloxacin @ 10 mg/kg b.wt. respectively. All the groups received medication orally, once daily for 28 days and blood samples were collected on the 28 day of experiment by the help of capillary tube and processed by laboratory methods. 

Result: Enrofloxacin altered the functions of liver and kidney as indicated by the alteration in the biochemical markers of liver and kidney function. Enrofloxacin significantly enhanced the concentration of biochemical markers of liver function viz. ALT, AST, GGT, ALP, albumin and bilirubin as compared to control. The concentration of biochemical markers of kidney function viz. BUN and creatinine was significantly enhanced after enrofloxacin administration as compared to control. Enrofloxacin administration induced oxidative stress in rats as indicated by significant decrease in concentration of catalase, superoxide dismutase and reduced glutathione in blood as compared to control. However, lipid peroxidation was significantly enhanced as indicated by increased level of MDA in blood as compared to control Thus, enrofloxacin have ability to altered the biochemical markers of liver and kidney and also have potency to induced oxidative stress in albino rats after the sub-acute exposure for 28 days. 

INTRODUCTION

Enrofloxacin, a 6-fluoroquinolone, is one of the most widely used antimicrobials in veterinary practice. It is a bactericidal drug with a broad spectrum antibacterial activity against a range of bacteria, including the strains resistant to other antimicrobial agents. The mechanism of action of enrofloxacin consists of inhibition of bacterial DNA gyrase, which plays basic role in the process of DNA replication, leading to inhibition of synthesis of bacterial proteins. The metabolism of enrofloxacin takes place in the liver through the N-oxidation, N-dealkylation and de-ethylation processes. Enrofloxacin is mainly excreted through urine and bile (Vancutsem et al., 1990).

Fluoroquinolone antibiotics may cause adverse effects such as chondrotoxicity, renal damage, retinal damage, dys-glycemia, cardiac arrhythmia and even tendon rupture. These side effects of fluoroquinolones are associated with reduced collagen synthesis and induced oxidative stress (Elbe et al., 2015). Induction of oxidative stress has been reported with the administration of ofloxacin, ciprofloxacin, levofloxacin, gatifloxacin and enrofloxacin (Ibrahim and Yarsan, 2011). There is evidence that free radical formation plays an important role in fluoroquinolone induced cartilage defect and photo toxicity (Gurbay et al., 2006).

Keeping the following facts in mind, the present study was conducted to determine the oxidative stress induced by sub-acute exposure of enrofloxacin in albino rats.

MATERIAL AND METHODS

Experimental animals
 
The study was conducted on healthy albino rats of 6-7 wk age, weighing around 150-200 gm and carried out in Department of Veterinary Pharmacology and Toxicology, (NDVSU), Jabalpur in year 2020. The experiment was approved by the Institutional Animal Ethical Committee (IAEC) of College of Veterinary Science and Animal Husbandry, Nanaji Deshmukh Veterinary Science University (NDVSU), Jabalpur.
 
Experimental design
 
Eighteen rats were randomly divided into three groups with six rats in each group. The oxidative stress induced by sub-acute exposure of enrofloxacin was evaluated in the groups of rats. The experiment was conducted for 28 days.
 
Design of experiment


Collection of blood sample
 
Blood was collected on day 0 and day 28 from the retro-orbital plexus with the help of capillary tube as described by Archer and Riley (1981). Blood was collected in heparinised vials and used for biochemical and oxidative stress parameter study.
 
Biochemical studies
 
Plasma was separated from heparinised blood samples and refrigerated at 4°C for biochemical studies. The following biochemical markers of liver and kidney function were estimated by using Semi-auto analyzer with respective commercially available kits of ERBA, manufactured by Transasia Bio-Medicals Ltd., Daman.
1. Aspartate aminotransferase (AST) (IU/L)
2. Alanine transaminase (ALT) (IU/L)
3. Alkaline phosphatase (ALP) (IU/L)
4. Bilirubin (mg/dl)
5. GGT-Gamma glutamyl transpeptidase (U/L)
6. Albumin (g/dl)
7. Creatinine (mg/dl)
8. Blood urea nitrogen (BUN) (mg/dl)
 
Assessment of oxidative stress indices
 
After blood collection the samples were centrifuged at 2000 rpm for 15 min to separate plasma. The layer of white blood cells above the packed erythrocytes was discarded. Erythrocyte pellet was washed three times with 0.15 M NaCl, diluted (33 per cent) in phosphate buffer saline (mM: NaCl, 136.9, KCl, 2.68; KH2·PO4, 1.47; and Na2·HPO4, 6.62; pH 7.4) and kept at 4°C until further analysis. The 33 per cent packed erythrocytes were used for the estimation of LPO, GSH, Glutathione reductase, Catalase and Superoxide dismutase activity by using Helios double beam spectrophotometer. LPO and GSH were measured on the day of blood collection (Prins and Loos, 1969).
 
Lipid peroxidation
 
Membrane peroxidative damage in erythrocytes was
determined in terms of malondialdehyde (MDA) production
 
as method suggested by Rehman (1984).
 
Reagents
 
1) Trichloroacetic acid (TCA) (10 per cent solution): 10 gm of TCA was dissolved in distilled water and the volume was made up to 100 ml with distilled water.
2) Thiobarbituric acid (TBA) (0.67 per cent solution): 0.67 gm of TBA in 100 ml of distilled water and warm up for dissolving TBA.
 
Procedure
 
One ml of 33 per cent of erythrocyte was incubated at 37°C for 2 hrs. To each sample, 1 ml of 10 per cent w/v TCA was added. After thorough mixing, the reaction mixture was centrifuged at 2000 rpm for 10 minutes. Then 1 ml of 0.67 per cent w/v of TBA was added in 1 ml of supernatant and kept in boiling water bath for 10 minutes. Reaction was cooled and diluted with 1ml of distilled water. Blank was made by adding all the reagents except the packed erythrocyte. The absorbance of these samples was read at wavelength 535 nm.
 
Calculation
 
Calculation was done by using molar extinction coefficient (EC) of MDA-TBA complex at 535 nm, i.e., 1.56× 108 /M/cm. The amount of LPO expressed as nano mole (nM) of MDA formed per ml of packed RBCs.
 
 
OD = Optical density
DF = Dilution factor
 
Reduced glutathione (GSH)
 
GSH was estimated by the 5, 5’ dithiobis (2- nitrobenzoic acid) (DNTB) method suggested by Prins and Loos (1969).
 
Reagents
 
1) H2SO4- 0.08 N in distilled water.
2) Tungustate solution, containing 0.3 M Na2WO4 and 0.1 Methylene diamine tetra acetic acid.
3) TRIS buffer, pH 8.0, a solution containing 1M tris hydroxymethyl amino methane and adjusted with HCI to pH 8.0.
4) DNTB reagent, a solution containing 0.14 M NaCl, 0.009M Na2HPO4, 0.000013 M, NaH2PO4 and 40 mg/100 mI DNTB (prepared fresh).
 
Procedure
 
RBCs pack (0.2 ml) (33 per cent dilution in PBS) was added to 4 ml of 0.08 N H2SO4 and mixed carefully. After 10 minutes of standing at room temperature, 0.5 ml of Tungustate solution was added to clear haemolysate. The tube was stoppered and mixture was shaken vigorously for 5 minutes. The stopper was removed and suspension was allowed to stand for 5 minutes in order to avoid bubble formation on top of the supernatant. The suspension was then centrifuged for 15 minutes at 2000 rpm at room temperature. After centrifugation, 2 ml of supernatant was added to 2.5 ml of tris-buffer, 0.2 ml of DNTB reagent was added and mixed well. Within a minute, absorbance was measured at 412 nm against blank by using Spectrophotometer in which 2 ml of distilled water was substituted for supernatant.
 
Calculation
 
Calculation was done by using the following formula and results were expressed as micromole (µM) of GSH per ml of packed RBCs.
 
 
 
Superoxide dismutase
 
Superoxide dismutase (SOD) activity was determined in the blood supernatant according to the method of Madesh and Balasubramanian (1998). A calorimetric assay involving generation of superoxide by pyrogallol auto-oxidation and the inhibition of superoxide dependent reduction of tetrazolium dye (MTT) [3-(4-5 dimethyl thiazol- 2-4) 2, 5-diphenyl tetrazolium bromide]. One unit of SOD was that which caused a 50 percent inhibition of reduction rate of MTT.
 
Reagent
 
1) Sample: 1:20 haemolysate
2) Phosphate buffer saline
3) MTT(1.25 mM) fresh 12.61 mg/10 ml
4) Pyrogallol(100 mM) fresh 12.61 mg/100 ml distilled water
5) DMSO (Dimethyl sulfoxide)
 
Procedure 
 
In a set of tubes 0.65 ml PBS (pH 7.4) was taken. To this 30 ìl (MTT) was added. Then, 75 μl of pyrogallol was added to both the tubes. This mixture was incubated for 5 min at room temperature and the reaction was stopped by adding 0.75 ml of dimethyl sulfoxide and finally 10 μl of sample was added to second tube to find out the percentage of reduction of MTT formation. The absorbance was read at 570 nm and the activity has been expressed as Unit. One unit of SOD was defined as the micrograms of haemoglobin causing 50 per cent inhibition in the MTT reduction.
 
Catalase
 
Catalase activity in erythrocytes was assayed by the spectrophotometric method of Aebi (1983).
 
Reagents
 
1) Phosphate buffer saline
2) Haemolysate (10 per cent)
3) Hydrogen peroxide (10 mM)
 
Procedure
 
Two ml of phosphate buffer (50 mM, pH 7.0) and 10 μl hemolysate (10 per cent) were taken in a cuvette. Reaction was started by adding 1 ml H2O2 (10 mM) and the absorbance was recorded at every 10 sec for 1 min at 240 nm against water blank. The activity of catalase has been expressed as mmol H2O2 utilized/min/mg haemoglobin.
 
Statistical analysis
 
Means and standard error were obtained as per standard procedure. Parameters were analyzed by using the method of complete randomized design with seven treatments allotted to groups of six animals each. The difference between treatments was tested statistically for their significance (Snedecor and Cochran, 1994).

RESULTS AND DISCUSSION

Biochemical studies
 
In the present research work, biochemical study was carried out to study the alterations in biochemical markers of liver and kidney function on sub-acute exposure of enrofloxacin in albino rats.
 
Biochemical markers of liver function
 
The mean values of biochemical markers of liver function viz. Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transferase (GGT), Alkaline phosphatase (ALP), albumin and bilirubin in rats treated with enrofloxacin have been presented in Table (01).

Table 1: Effect of enrofloxacin on biochemical markers of liver function after sub- acute exposure in albino rats.


 
ALT (SGPT) and AST (SGOT)
 
The concentration of ALT in control was 79.75 ± 7.50 IU/L. Enrofloxacin, significantly increased the concentration of ALT and this increase was 91.38 ± 0.22 IU/L at the dose rate of 5 mg/kg b.wt. orally for 28 days. In higher doses i.e.10 mg/kg b.wt. orally for 28 days, enrofloxacin further increased the concentration of ALT to 106.32 ± 0.10. The concentration of AST in control was 159.98 ± 6.27 IU/L. Enrofloxacin, significantly increased the concentration of AST and this increase was 177.33 ± 0.49 IU/L at the dose rate of 5 mg/kg b.wt., orally for 28 days. In higher doses i.e.10 mg/kg b.wt., orally for 28 days, enrofloxacin further increased the concentration of AST to 180.53 ± 0.09 IU/L. In the present investigation, elevated level of serum ALT and AST after subacute exposure of enrofloxacin are in agreement with the result of Guo et al., (2018). ALT and AST are liver enzyme markers used as an indicator of enrofloxacin induced hepatic changes in albino rats (Jiang et al., 2014). Corum et al., (2014) suggested that enrofloxacin administration in rats significantly increased levels of liver enzymes AST and ALT after sub- acute treatment.
 
GGT (Gamma glutamyl transferase)
 
The concentration of GGT (Gamma glutamyl transferase) in control was 7.07 ± 0.03 IU/L. Enrofloxacin, significantly increased the concentration of GGT and this increase was 7.40 ± 0.04 IU/L after the subacute exposure of enrofloxacin at the dose rate of 5 mg/kg b.wt., orally for 28 days. In higher doses i.e.10 mg/kg b. wt., orally for 28 days enrofloxacin further increased the concentration of GGT to 7.70 ± 0.05 IU/L. Serum GGT is sensitive, highly specific and indicative enzyme for liver function. Sureshkumar et al., (2013) suggested that enrofloxacin after the sub- acute administration increased the level of enzyme GGT in serum.  Ellakany et al., (2007) found the elevation in serum GGT level after the administration of enrofloxacin in chickens.
 
ALP (Alkaline phosphatase)
 
The concentration of alkaline phosphatase in control was 449.50±2.54. Enrofloxacin significantly increased the concentration of ALP and this increase was 532.50 ± 0.96 IU/L after the subacute exposure of enrofloxacin at the dose rate of 5 mg/kg b.wt. orally for 28 days. In higher doses i.e.10 mg/kg b.wt. orally for 28 days, enrofloxacin further increased the concentration of ALP to 545.17 ± 3.71 IU/L. Alkaline phosphatase is used to detect liver disease and is also indicative enzyme for liver function. Elkholy et al., (2009) showed that administration of enrofloxacin @ 10 mg /kg body weight daily for five successive days caused elevation of serum ALP when given orally. Davoren and Mainstone (1993) and Moustafa et al., (1998) showed that oral administration of enrofloxacin for 15 consecutive days caused elevation in serum ALP level in dogs.
 
Albumin and bilirubin
 
The concentration of albumin in control was 4.05 ± 0.03. Enrofloxacin, significantly increased the concentration of albumin and this increase was 4.33 ± 0.03 g/dl after the administration of enrofloxacin at the dose rate of 5 mg/kg b.wt., orally for 28 days. In higher doses i.e.10 mg/kg b.wt., orally for 28 days, enrofloxacin further increased the concentration of albumin to 4.50 ± 0.05 g/dl. The concentration of Bilirubin in control was 0.107± 0.003 mg/dl. Enrofloxacin, significantly increased the concentration of bilirubin and this increase was 0.127± 0.003 mg/dl after the administration of enrofloxacin at the dose rate of 5 mg/kg b.wt. orally for 28 days. In higher doses i.e.10 mg/kg b.wt. orally for 28 days, enrofloxacin further increased the concentration of bilirubin to 0.137± 0.002 mg/dl. Kock et al., (1987) also suggested that enrofloxacin, after the sub- acute administration increased the level of albumin and bilirubin. Kaneko et al., (1997) and Jain (1986) also reported the elevation in serum albumin and bilirubin level after the administration of enrofloxacin. Enrofloxacin induced oxidative stress contributes to liver injury and elevates concentration of albumin and bilirubin.
 
Biochemical markers of kidney function
 
The mean values of biochemical markers of kidney function viz. blood urea nitrogen and creatinine in rats treated with enrofloxacin have been presented in (Table 2).

Table 2: Effect enrofloxacin on biochemical markers of kidney function after sub-acute exposure in albino rats.


 
Creatinine and blood urea nitrogen
 
The concentration of creatinine in control was 0.640±0.021 mg/dl Enrofloxacin, significantly increased the concentration of creatinine and this increase was 0.847 ± 0.013 mg/dl after the administration of enrofloxacin at the dose rate of 5 mg/kg b.wt. In higher doses i.e.10 mg/kg b.wt enrofloxacin further increased the concentration of creatinine to 1.052±0.023 mg/dl. The concentration of blood urea nitrogen in control was 14.35± 0.23 mg/dl. Enrofloxacin, at the dose rate of 5 mg/kg b.wt., orally for 28 days did not affect the BUN level in serum significantly and the mean value was 14.62± 0.03. However, enrofloxacin in higher doses i.e.10 mg/kg b.wt., orally for 28 days, significantly increased the concentration of BUN to 23.84±0.02 mg/dl. Creatinin and blood urea nitrogen are potent indicators of renal impairment. In this study enrofloxacin administration significantly increased the concentration of creatinine and BUN in serum as compared to control. Khan and Rampal (2014) indicated that sub-acute exposure of enrofloxacin increased the concentration of creatinine and BUN level. Sureshkumar et al., (2013) mentioned enrofloxacin induced changes in biomarkers of kidney function and found increased concentration of creatinine and blood urea nitrogen.
 
Oxidative stress indices
 
The mean values of oxidative stress indices viz. lipid peroxidation (MDA), Superoxide dismutase (SOD), reduced glutathione (GSH) and catalase in rats treated with enrofloxacin have been presented in (Table 3).

Table 3: Effect of enrofloxacin in oxidative stress indices after sub- acute exposure in albino rat.


 
Lipid peroidation (MDA)
 
Lipid peroxidation was calculated in terms of nM MDA/gm of blood on day 28 of experiment in albino rats. The concentration of LPO in control was 4.63±0.02 nM MDA/gm of blood, however, enrofloxacin at dose of 5 mg/kg b.wt and 10 mg/kg b.wt. significantly increased the concentration of LPO and the mean values were 5.20 ±0.03 and 5.78±0.03 nM MDA /gm of blood, respectively. MDA is the end product of lipid peroxidation, due to oxidation of polyunsaturated fatty acids by free radicals. The increase in its concentrations in the kidney tissue revealed an enhanced lipid peroxidation leading to tissue damage as well as the failure of the antioxidants to prevent excessive production of free radicals (Karadeniz et al., 2008). Dhuley, (1999) observed that enrofloxacin after administration, increased concentration of malondialdehyde in blood. In the present study, the concentration of LPO was increased significantly in enrofloxacin treated group as compared to control group.  Sarban et al.  (2005) reported an increase in MDA levels in the plasma associated with enrofloxacin administration as indicated by the onset of lipid hydroperoxide.
 
SOD (Superoxide dismutase)
 
Superoxide dismutase was calculated in terms of U/g of Hb on day 28 of experiment in albino rats. The concentration of SOD in control was 1.22±0.01 U/g of Hb, however enrofloxacin at dose of 5 mg/kg b.wt and 10 mg/kg b.wt. significantly decreased the concentration of SOD and the mean values were 0.88±0.01 and 0.84±0.02 U/g respectively. SOD activity contributes to increasing the level of superoxide radicals, thus increased oxidative stress. Suresh et al., (2013) indicated that enrofloxacin administration reduced superoxide dismutase (SOD). Sakaguchi and Furusawa (2006) indicated that enrofloxacin administration caused formation of reactive oxygen species and reduced the level of superoxide dismutase. The findings of present study are in agreement with the findings of Suresh et al., (2013) and Sakaguchi and Furusawa (2006).
 
GSH (Reduced glutathione)
 
Reduced glutathione was calculated in terms of µmol /ml of blood on day 28 of experiment in albino rats. The concentration of GSH in control was 340.67±0.21 µmol /ml of blood, however, enrofloxacin at dose of 5 mg/kg b.wt and 10 mg/kg b.wt. Significantly decreased the concentration of GSH and the mean values were 332.57±0.24 and 330.42±0.44 µmol /ml of blood respectively. Glutathione peroxidase reduces H2O2 to H2O by oxidizing reduced glutathione. A decrease in glutathione peroxidase would increase the concentration of H2O2, thus signaling an increase in further oxidative stress. The decreased renal antioxidant capacity due to excessive ROS production could aggravate oxidative damage in rats. GSH is an essential compound for maintaining cellular integrity and metabolism. It has been proposed that antioxidants maintain the concentration of reduced GSH. Cerreras et al.  (2005) suggested that metabolism of enrofloxacin residues, generate free radicals and contribute to an increase in oxidative stress, promoting inhibition of cellular enzymes by the reduction in glutathione peroxidase (GSHPx). In agreement with that GSH concentration was significantly reduced after enrofloxacin treatment compared to control group in present study. Keskin et al., (2005) and Gurbay et al., (2006) indicated that administration of enrofloxacin in rats produced reactive oxygen species and increased oxidative stress. In agreement with that GSH concentration was significantly lowered after enrofloxacin treatment compared to control group in present study.
 
Catalase
 
Catalase was calculated in terms of µmol H2O2 decomposed/min/gm Hb on day 28 of experiment in albino rats. The concentration of CAT in control was 235.08±0.38 µmol H2O2 decomposed. However, enrofloxacin at dose of 5 mg/kg b.wt and 10 mg/kg b.wt. significantly decreased the concentration of CAT and the mean values were 157.83±0.48 and 131.57±0.20 µmol H2O2 decomposed, respectively. Decrease in catalase activity would increase H2O2 concentration in the cell, leading to increased oxidative stress (Chelikani et al., 2004). Yazar and Tras (2001) suggested that metabolism of enrofloxacin residues, generate free radicals and contribute to an increase in oxidative stress, promoting inhibition of cellular enzymes by the reduction in catalase (CAT). The findings of present study are in agreement with Yazar and Tras, as in the present research, CAT concentration was significantly lower in enrofloxacin treated group as compared to control group.

​

Plate 1: ERBA EM-200 Auto-analyzer



Plate2: Spectrophometer.

CONCLUSION

Enrofloxacin at the doses of 5 mg/kg body wt. and 10 mg/kg body wt., administered orally, daily for 28 days in albino rats induced the liver impairment as indicated by increased concentration of biochemical markers of liver function viz. ALT, AST, GGT, ALP, albumin and bilirubin and at the doses of 5 mg/kg body wt. and 10 mg/kg body wt., orally, daily for 28 days in albino rats induced the renal impairment as indicated by increased concentration of biochemical markers of kidney function viz. BUN and creatinine. Enrofloxacin at the doses of 5 mg/kg body wt. and 10 mg/kg body wt., orally, daily for 28 days in albino rats induced oxidative stress as indicated by altered oxidative stress indices viz. catalase, superoxide dismutase, reduced glutathione and lipid peroxidation in blood.

CONFLICT OF INTEREST

All authors declare that they have no conflict of interest.

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    Research Article

    Alterations in Biochemical Markers of Liver and Kidney Function and Oxidative Stress Indices on Sub-acute Exposure of Enrofloxacin in Albino Rats

    P
    Prateek Mishra1,*
    V
    Vidhi Gautam2
    R
    R.K. Sharma2
    A
    Anushri Tiwari2
    1Department of Veterinary Pharmacology and Toxicology, Nanaji Deshmukh Veterinary Science University, Jabalpur-482 002, Madhya Pradesh, India.
    2Department of Veterinary Pathology, Nanaji Deshmukh Veterinary Science University, Jabalpur-482 002, Madhya Pradesh, India.
    Cite article:- Mishra Prateek, Gautam Vidhi, Sharma R.K., Tiwari Anushri (2026). Alterations in Biochemical Markers of Liver and Kidney Function and Oxidative Stress Indices on Sub-acute Exposure of Enrofloxacin in Albino Rats . Indian Journal of Animal Research. 60(10): 1830-1836. doi: 10.18805/IJAR.B-4453.

    ABSTRACT

    Background: The present study was performed to evaluate Alterations in biochemical markers of liver and kidney function and oxidative stress indices on sub-acute exposure of enrofloxacin in albino rats. 

    Methods: Albino rat weighing 200-300 gm were treated with enrofloxacin orally with the help of gavage needle and devided into three groups each group consist six rats. The rats of group I were served as control. However, rats of group II and III were treated with enrofloxacin @ 5 mg/kg b.wt., enrofloxacin @ 10 mg/kg b.wt. respectively. All the groups received medication orally, once daily for 28 days and blood samples were collected on the 28 day of experiment by the help of capillary tube and processed by laboratory methods. 

    Result: Enrofloxacin altered the functions of liver and kidney as indicated by the alteration in the biochemical markers of liver and kidney function. Enrofloxacin significantly enhanced the concentration of biochemical markers of liver function viz. ALT, AST, GGT, ALP, albumin and bilirubin as compared to control. The concentration of biochemical markers of kidney function viz. BUN and creatinine was significantly enhanced after enrofloxacin administration as compared to control. Enrofloxacin administration induced oxidative stress in rats as indicated by significant decrease in concentration of catalase, superoxide dismutase and reduced glutathione in blood as compared to control. However, lipid peroxidation was significantly enhanced as indicated by increased level of MDA in blood as compared to control Thus, enrofloxacin have ability to altered the biochemical markers of liver and kidney and also have potency to induced oxidative stress in albino rats after the sub-acute exposure for 28 days. 

    INTRODUCTION

    Enrofloxacin, a 6-fluoroquinolone, is one of the most widely used antimicrobials in veterinary practice. It is a bactericidal drug with a broad spectrum antibacterial activity against a range of bacteria, including the strains resistant to other antimicrobial agents. The mechanism of action of enrofloxacin consists of inhibition of bacterial DNA gyrase, which plays basic role in the process of DNA replication, leading to inhibition of synthesis of bacterial proteins. The metabolism of enrofloxacin takes place in the liver through the N-oxidation, N-dealkylation and de-ethylation processes. Enrofloxacin is mainly excreted through urine and bile (Vancutsem et al., 1990).

    Fluoroquinolone antibiotics may cause adverse effects such as chondrotoxicity, renal damage, retinal damage, dys-glycemia, cardiac arrhythmia and even tendon rupture. These side effects of fluoroquinolones are associated with reduced collagen synthesis and induced oxidative stress (Elbe et al., 2015). Induction of oxidative stress has been reported with the administration of ofloxacin, ciprofloxacin, levofloxacin, gatifloxacin and enrofloxacin (Ibrahim and Yarsan, 2011). There is evidence that free radical formation plays an important role in fluoroquinolone induced cartilage defect and photo toxicity (Gurbay et al., 2006).

    Keeping the following facts in mind, the present study was conducted to determine the oxidative stress induced by sub-acute exposure of enrofloxacin in albino rats.

    MATERIAL AND METHODS

    Experimental animals
     
    The study was conducted on healthy albino rats of 6-7 wk age, weighing around 150-200 gm and carried out in Department of Veterinary Pharmacology and Toxicology, (NDVSU), Jabalpur in year 2020. The experiment was approved by the Institutional Animal Ethical Committee (IAEC) of College of Veterinary Science and Animal Husbandry, Nanaji Deshmukh Veterinary Science University (NDVSU), Jabalpur.
     
    Experimental design
     
    Eighteen rats were randomly divided into three groups with six rats in each group. The oxidative stress induced by sub-acute exposure of enrofloxacin was evaluated in the groups of rats. The experiment was conducted for 28 days.
     
    Design of experiment


    Collection of blood sample
     
    Blood was collected on day 0 and day 28 from the retro-orbital plexus with the help of capillary tube as described by Archer and Riley (1981). Blood was collected in heparinised vials and used for biochemical and oxidative stress parameter study.
     
    Biochemical studies
     
    Plasma was separated from heparinised blood samples and refrigerated at 4°C for biochemical studies. The following biochemical markers of liver and kidney function were estimated by using Semi-auto analyzer with respective commercially available kits of ERBA, manufactured by Transasia Bio-Medicals Ltd., Daman.
    1. Aspartate aminotransferase (AST) (IU/L)
    2. Alanine transaminase (ALT) (IU/L)
    3. Alkaline phosphatase (ALP) (IU/L)
    4. Bilirubin (mg/dl)
    5. GGT-Gamma glutamyl transpeptidase (U/L)
    6. Albumin (g/dl)
    7. Creatinine (mg/dl)
    8. Blood urea nitrogen (BUN) (mg/dl)
     
    Assessment of oxidative stress indices
     
    After blood collection the samples were centrifuged at 2000 rpm for 15 min to separate plasma. The layer of white blood cells above the packed erythrocytes was discarded. Erythrocyte pellet was washed three times with 0.15 M NaCl, diluted (33 per cent) in phosphate buffer saline (mM: NaCl, 136.9, KCl, 2.68; KH2·PO4, 1.47; and Na2·HPO4, 6.62; pH 7.4) and kept at 4°C until further analysis. The 33 per cent packed erythrocytes were used for the estimation of LPO, GSH, Glutathione reductase, Catalase and Superoxide dismutase activity by using Helios double beam spectrophotometer. LPO and GSH were measured on the day of blood collection (Prins and Loos, 1969).
     
    Lipid peroxidation
     
    Membrane peroxidative damage in erythrocytes was
    determined in terms of malondialdehyde (MDA) production
     
    as method suggested by Rehman (1984).
     
    Reagents
     
    1) Trichloroacetic acid (TCA) (10 per cent solution): 10 gm of TCA was dissolved in distilled water and the volume was made up to 100 ml with distilled water.
    2) Thiobarbituric acid (TBA) (0.67 per cent solution): 0.67 gm of TBA in 100 ml of distilled water and warm up for dissolving TBA.
     
    Procedure
     
    One ml of 33 per cent of erythrocyte was incubated at 37°C for 2 hrs. To each sample, 1 ml of 10 per cent w/v TCA was added. After thorough mixing, the reaction mixture was centrifuged at 2000 rpm for 10 minutes. Then 1 ml of 0.67 per cent w/v of TBA was added in 1 ml of supernatant and kept in boiling water bath for 10 minutes. Reaction was cooled and diluted with 1ml of distilled water. Blank was made by adding all the reagents except the packed erythrocyte. The absorbance of these samples was read at wavelength 535 nm.
     
    Calculation
     
    Calculation was done by using molar extinction coefficient (EC) of MDA-TBA complex at 535 nm, i.e., 1.56× 108 /M/cm. The amount of LPO expressed as nano mole (nM) of MDA formed per ml of packed RBCs.
     
     
    OD = Optical density
    DF = Dilution factor
     
    Reduced glutathione (GSH)
     
    GSH was estimated by the 5, 5’ dithiobis (2- nitrobenzoic acid) (DNTB) method suggested by Prins and Loos (1969).
     
    Reagents
     
    1) H2SO4- 0.08 N in distilled water.
    2) Tungustate solution, containing 0.3 M Na2WO4 and 0.1 Methylene diamine tetra acetic acid.
    3) TRIS buffer, pH 8.0, a solution containing 1M tris hydroxymethyl amino methane and adjusted with HCI to pH 8.0.
    4) DNTB reagent, a solution containing 0.14 M NaCl, 0.009M Na2HPO4, 0.000013 M, NaH2PO4 and 40 mg/100 mI DNTB (prepared fresh).
     
    Procedure
     
    RBCs pack (0.2 ml) (33 per cent dilution in PBS) was added to 4 ml of 0.08 N H2SO4 and mixed carefully. After 10 minutes of standing at room temperature, 0.5 ml of Tungustate solution was added to clear haemolysate. The tube was stoppered and mixture was shaken vigorously for 5 minutes. The stopper was removed and suspension was allowed to stand for 5 minutes in order to avoid bubble formation on top of the supernatant. The suspension was then centrifuged for 15 minutes at 2000 rpm at room temperature. After centrifugation, 2 ml of supernatant was added to 2.5 ml of tris-buffer, 0.2 ml of DNTB reagent was added and mixed well. Within a minute, absorbance was measured at 412 nm against blank by using Spectrophotometer in which 2 ml of distilled water was substituted for supernatant.
     
    Calculation
     
    Calculation was done by using the following formula and results were expressed as micromole (µM) of GSH per ml of packed RBCs.
     
     
     
    Superoxide dismutase
     
    Superoxide dismutase (SOD) activity was determined in the blood supernatant according to the method of Madesh and Balasubramanian (1998). A calorimetric assay involving generation of superoxide by pyrogallol auto-oxidation and the inhibition of superoxide dependent reduction of tetrazolium dye (MTT) [3-(4-5 dimethyl thiazol- 2-4) 2, 5-diphenyl tetrazolium bromide]. One unit of SOD was that which caused a 50 percent inhibition of reduction rate of MTT.
     
    Reagent
     
    1) Sample: 1:20 haemolysate
    2) Phosphate buffer saline
    3) MTT(1.25 mM) fresh 12.61 mg/10 ml
    4) Pyrogallol(100 mM) fresh 12.61 mg/100 ml distilled water
    5) DMSO (Dimethyl sulfoxide)
     
    Procedure 
     
    In a set of tubes 0.65 ml PBS (pH 7.4) was taken. To this 30 ìl (MTT) was added. Then, 75 μl of pyrogallol was added to both the tubes. This mixture was incubated for 5 min at room temperature and the reaction was stopped by adding 0.75 ml of dimethyl sulfoxide and finally 10 μl of sample was added to second tube to find out the percentage of reduction of MTT formation. The absorbance was read at 570 nm and the activity has been expressed as Unit. One unit of SOD was defined as the micrograms of haemoglobin causing 50 per cent inhibition in the MTT reduction.
     
    Catalase
     
    Catalase activity in erythrocytes was assayed by the spectrophotometric method of Aebi (1983).
     
    Reagents
     
    1) Phosphate buffer saline
    2) Haemolysate (10 per cent)
    3) Hydrogen peroxide (10 mM)
     
    Procedure
     
    Two ml of phosphate buffer (50 mM, pH 7.0) and 10 μl hemolysate (10 per cent) were taken in a cuvette. Reaction was started by adding 1 ml H2O2 (10 mM) and the absorbance was recorded at every 10 sec for 1 min at 240 nm against water blank. The activity of catalase has been expressed as mmol H2O2 utilized/min/mg haemoglobin.
     
    Statistical analysis
     
    Means and standard error were obtained as per standard procedure. Parameters were analyzed by using the method of complete randomized design with seven treatments allotted to groups of six animals each. The difference between treatments was tested statistically for their significance (Snedecor and Cochran, 1994).

    RESULTS AND DISCUSSION

    Biochemical studies
     
    In the present research work, biochemical study was carried out to study the alterations in biochemical markers of liver and kidney function on sub-acute exposure of enrofloxacin in albino rats.
     
    Biochemical markers of liver function
     
    The mean values of biochemical markers of liver function viz. Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transferase (GGT), Alkaline phosphatase (ALP), albumin and bilirubin in rats treated with enrofloxacin have been presented in Table (01).

    Table 1: Effect of enrofloxacin on biochemical markers of liver function after sub- acute exposure in albino rats.


     
    ALT (SGPT) and AST (SGOT)
     
    The concentration of ALT in control was 79.75 ± 7.50 IU/L. Enrofloxacin, significantly increased the concentration of ALT and this increase was 91.38 ± 0.22 IU/L at the dose rate of 5 mg/kg b.wt. orally for 28 days. In higher doses i.e.10 mg/kg b.wt. orally for 28 days, enrofloxacin further increased the concentration of ALT to 106.32 ± 0.10. The concentration of AST in control was 159.98 ± 6.27 IU/L. Enrofloxacin, significantly increased the concentration of AST and this increase was 177.33 ± 0.49 IU/L at the dose rate of 5 mg/kg b.wt., orally for 28 days. In higher doses i.e.10 mg/kg b.wt., orally for 28 days, enrofloxacin further increased the concentration of AST to 180.53 ± 0.09 IU/L. In the present investigation, elevated level of serum ALT and AST after subacute exposure of enrofloxacin are in agreement with the result of Guo et al., (2018). ALT and AST are liver enzyme markers used as an indicator of enrofloxacin induced hepatic changes in albino rats (Jiang et al., 2014). Corum et al., (2014) suggested that enrofloxacin administration in rats significantly increased levels of liver enzymes AST and ALT after sub- acute treatment.
     
    GGT (Gamma glutamyl transferase)
     
    The concentration of GGT (Gamma glutamyl transferase) in control was 7.07 ± 0.03 IU/L. Enrofloxacin, significantly increased the concentration of GGT and this increase was 7.40 ± 0.04 IU/L after the subacute exposure of enrofloxacin at the dose rate of 5 mg/kg b.wt., orally for 28 days. In higher doses i.e.10 mg/kg b. wt., orally for 28 days enrofloxacin further increased the concentration of GGT to 7.70 ± 0.05 IU/L. Serum GGT is sensitive, highly specific and indicative enzyme for liver function. Sureshkumar et al., (2013) suggested that enrofloxacin after the sub- acute administration increased the level of enzyme GGT in serum.  Ellakany et al., (2007) found the elevation in serum GGT level after the administration of enrofloxacin in chickens.
     
    ALP (Alkaline phosphatase)
     
    The concentration of alkaline phosphatase in control was 449.50±2.54. Enrofloxacin significantly increased the concentration of ALP and this increase was 532.50 ± 0.96 IU/L after the subacute exposure of enrofloxacin at the dose rate of 5 mg/kg b.wt. orally for 28 days. In higher doses i.e.10 mg/kg b.wt. orally for 28 days, enrofloxacin further increased the concentration of ALP to 545.17 ± 3.71 IU/L. Alkaline phosphatase is used to detect liver disease and is also indicative enzyme for liver function. Elkholy et al., (2009) showed that administration of enrofloxacin @ 10 mg /kg body weight daily for five successive days caused elevation of serum ALP when given orally. Davoren and Mainstone (1993) and Moustafa et al., (1998) showed that oral administration of enrofloxacin for 15 consecutive days caused elevation in serum ALP level in dogs.
     
    Albumin and bilirubin
     
    The concentration of albumin in control was 4.05 ± 0.03. Enrofloxacin, significantly increased the concentration of albumin and this increase was 4.33 ± 0.03 g/dl after the administration of enrofloxacin at the dose rate of 5 mg/kg b.wt., orally for 28 days. In higher doses i.e.10 mg/kg b.wt., orally for 28 days, enrofloxacin further increased the concentration of albumin to 4.50 ± 0.05 g/dl. The concentration of Bilirubin in control was 0.107± 0.003 mg/dl. Enrofloxacin, significantly increased the concentration of bilirubin and this increase was 0.127± 0.003 mg/dl after the administration of enrofloxacin at the dose rate of 5 mg/kg b.wt. orally for 28 days. In higher doses i.e.10 mg/kg b.wt. orally for 28 days, enrofloxacin further increased the concentration of bilirubin to 0.137± 0.002 mg/dl. Kock et al., (1987) also suggested that enrofloxacin, after the sub- acute administration increased the level of albumin and bilirubin. Kaneko et al., (1997) and Jain (1986) also reported the elevation in serum albumin and bilirubin level after the administration of enrofloxacin. Enrofloxacin induced oxidative stress contributes to liver injury and elevates concentration of albumin and bilirubin.
     
    Biochemical markers of kidney function
     
    The mean values of biochemical markers of kidney function viz. blood urea nitrogen and creatinine in rats treated with enrofloxacin have been presented in (Table 2).

    Table 2: Effect enrofloxacin on biochemical markers of kidney function after sub-acute exposure in albino rats.


     
    Creatinine and blood urea nitrogen
     
    The concentration of creatinine in control was 0.640±0.021 mg/dl Enrofloxacin, significantly increased the concentration of creatinine and this increase was 0.847 ± 0.013 mg/dl after the administration of enrofloxacin at the dose rate of 5 mg/kg b.wt. In higher doses i.e.10 mg/kg b.wt enrofloxacin further increased the concentration of creatinine to 1.052±0.023 mg/dl. The concentration of blood urea nitrogen in control was 14.35± 0.23 mg/dl. Enrofloxacin, at the dose rate of 5 mg/kg b.wt., orally for 28 days did not affect the BUN level in serum significantly and the mean value was 14.62± 0.03. However, enrofloxacin in higher doses i.e.10 mg/kg b.wt., orally for 28 days, significantly increased the concentration of BUN to 23.84±0.02 mg/dl. Creatinin and blood urea nitrogen are potent indicators of renal impairment. In this study enrofloxacin administration significantly increased the concentration of creatinine and BUN in serum as compared to control. Khan and Rampal (2014) indicated that sub-acute exposure of enrofloxacin increased the concentration of creatinine and BUN level. Sureshkumar et al., (2013) mentioned enrofloxacin induced changes in biomarkers of kidney function and found increased concentration of creatinine and blood urea nitrogen.
     
    Oxidative stress indices
     
    The mean values of oxidative stress indices viz. lipid peroxidation (MDA), Superoxide dismutase (SOD), reduced glutathione (GSH) and catalase in rats treated with enrofloxacin have been presented in (Table 3).

    Table 3: Effect of enrofloxacin in oxidative stress indices after sub- acute exposure in albino rat.


     
    Lipid peroidation (MDA)
     
    Lipid peroxidation was calculated in terms of nM MDA/gm of blood on day 28 of experiment in albino rats. The concentration of LPO in control was 4.63±0.02 nM MDA/gm of blood, however, enrofloxacin at dose of 5 mg/kg b.wt and 10 mg/kg b.wt. significantly increased the concentration of LPO and the mean values were 5.20 ±0.03 and 5.78±0.03 nM MDA /gm of blood, respectively. MDA is the end product of lipid peroxidation, due to oxidation of polyunsaturated fatty acids by free radicals. The increase in its concentrations in the kidney tissue revealed an enhanced lipid peroxidation leading to tissue damage as well as the failure of the antioxidants to prevent excessive production of free radicals (Karadeniz et al., 2008). Dhuley, (1999) observed that enrofloxacin after administration, increased concentration of malondialdehyde in blood. In the present study, the concentration of LPO was increased significantly in enrofloxacin treated group as compared to control group.  Sarban et al.  (2005) reported an increase in MDA levels in the plasma associated with enrofloxacin administration as indicated by the onset of lipid hydroperoxide.
     
    SOD (Superoxide dismutase)
     
    Superoxide dismutase was calculated in terms of U/g of Hb on day 28 of experiment in albino rats. The concentration of SOD in control was 1.22±0.01 U/g of Hb, however enrofloxacin at dose of 5 mg/kg b.wt and 10 mg/kg b.wt. significantly decreased the concentration of SOD and the mean values were 0.88±0.01 and 0.84±0.02 U/g respectively. SOD activity contributes to increasing the level of superoxide radicals, thus increased oxidative stress. Suresh et al., (2013) indicated that enrofloxacin administration reduced superoxide dismutase (SOD). Sakaguchi and Furusawa (2006) indicated that enrofloxacin administration caused formation of reactive oxygen species and reduced the level of superoxide dismutase. The findings of present study are in agreement with the findings of Suresh et al., (2013) and Sakaguchi and Furusawa (2006).
     
    GSH (Reduced glutathione)
     
    Reduced glutathione was calculated in terms of µmol /ml of blood on day 28 of experiment in albino rats. The concentration of GSH in control was 340.67±0.21 µmol /ml of blood, however, enrofloxacin at dose of 5 mg/kg b.wt and 10 mg/kg b.wt. Significantly decreased the concentration of GSH and the mean values were 332.57±0.24 and 330.42±0.44 µmol /ml of blood respectively. Glutathione peroxidase reduces H2O2 to H2O by oxidizing reduced glutathione. A decrease in glutathione peroxidase would increase the concentration of H2O2, thus signaling an increase in further oxidative stress. The decreased renal antioxidant capacity due to excessive ROS production could aggravate oxidative damage in rats. GSH is an essential compound for maintaining cellular integrity and metabolism. It has been proposed that antioxidants maintain the concentration of reduced GSH. Cerreras et al.  (2005) suggested that metabolism of enrofloxacin residues, generate free radicals and contribute to an increase in oxidative stress, promoting inhibition of cellular enzymes by the reduction in glutathione peroxidase (GSHPx). In agreement with that GSH concentration was significantly reduced after enrofloxacin treatment compared to control group in present study. Keskin et al., (2005) and Gurbay et al., (2006) indicated that administration of enrofloxacin in rats produced reactive oxygen species and increased oxidative stress. In agreement with that GSH concentration was significantly lowered after enrofloxacin treatment compared to control group in present study.
     
    Catalase
     
    Catalase was calculated in terms of µmol H2O2 decomposed/min/gm Hb on day 28 of experiment in albino rats. The concentration of CAT in control was 235.08±0.38 µmol H2O2 decomposed. However, enrofloxacin at dose of 5 mg/kg b.wt and 10 mg/kg b.wt. significantly decreased the concentration of CAT and the mean values were 157.83±0.48 and 131.57±0.20 µmol H2O2 decomposed, respectively. Decrease in catalase activity would increase H2O2 concentration in the cell, leading to increased oxidative stress (Chelikani et al., 2004). Yazar and Tras (2001) suggested that metabolism of enrofloxacin residues, generate free radicals and contribute to an increase in oxidative stress, promoting inhibition of cellular enzymes by the reduction in catalase (CAT). The findings of present study are in agreement with Yazar and Tras, as in the present research, CAT concentration was significantly lower in enrofloxacin treated group as compared to control group.

    ​

    Plate 1: ERBA EM-200 Auto-analyzer



    Plate2: Spectrophometer.

    CONCLUSION

    Enrofloxacin at the doses of 5 mg/kg body wt. and 10 mg/kg body wt., administered orally, daily for 28 days in albino rats induced the liver impairment as indicated by increased concentration of biochemical markers of liver function viz. ALT, AST, GGT, ALP, albumin and bilirubin and at the doses of 5 mg/kg body wt. and 10 mg/kg body wt., orally, daily for 28 days in albino rats induced the renal impairment as indicated by increased concentration of biochemical markers of kidney function viz. BUN and creatinine. Enrofloxacin at the doses of 5 mg/kg body wt. and 10 mg/kg body wt., orally, daily for 28 days in albino rats induced oxidative stress as indicated by altered oxidative stress indices viz. catalase, superoxide dismutase, reduced glutathione and lipid peroxidation in blood.

    CONFLICT OF INTEREST

    All authors declare that they have no conflict of interest.

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