Effect of n -hexane extract of Nigella sativa on gentamicin-induced nephrotoxicity in rats

The present study investigated whether the administration of the n -hexane extract of the Nigella sativa Linn. (kalajira) ameliorates gentamicin-induced nephrotoxicity in rats. Gentamicin (100 mg/kg/day for 7 days) was administered and nephrotoxicity was evaluated biochemically (significantly decreased reduced glutathione in renal cortex and significantly increased serum creatinine and serum urea) and histologically (moderate degree of proximal tubular damage). The n -hexane extract of N. sativa (5 mL/kg/day) was administered as pre-, post- and concomitant treatment for 7 days in the nephrotoxic rats. Statistically significant amelioration in all the biochemical parameters supported by significantly improved renal cortical histology was observed in the n -hexane extract of N. sativa treated nephrotoxic rats, which was more evident in the post-treatment group than the pre- treatment and the concomitantly-treated group. It is suggested that some ingredients contained in the n -hexane extract of N. sativa effected in ameliorating the signs of nephrotoxicity and that the specific active principle of the n -hexane extract of N. sativa responsible for this amelioration if obtained, would be more useful.


Introduction
Gentamicin is an important aminoglycoside antibiotic commonly used in treating life-threatening gram negative infections (Ali, 1995).However its usefulness is limited by signs of nephrotoxicity, which may occur in 13-30% of treated patients (Mathew, 1992).Lipid peroxidation may occur in the course of gentamicin administration (Ramsammy et al., 1985), giving rise to free radicals (Yang et al., 1995), which are highly toxic to tissue (Feldman et al., 1982).Oxidation and necrosis by apoptosis may occur.
Antioxidants have been shown to ameliorate signs of gentamicin-induced nephrotoxicity (Ali and Mousa, 2001).A recent study suggested that N. sativa oil (Ali, 2004) given prophylactically was able to alleviate gentamicin nephrotoxicity in rats.N. sativa (kalajira) occupies a unique position among the herbal products of Southeast Asia as a natural remedy for a number of illnesses.Its anti-bacterial, hypolipidaemic, anti-diabetic and anti-hypertensive properties have been reported (Ara, 1999;Saha, 2004;Uddin, 2002;Rashid et al., 1987).The seeds or compounds isolated have been found to be useful in a number of models of nephrotoxicity.The phytochemical, pharmacological and toxicological properties of N. sativa have recently been reviewed (Ali and Blunden, 2003).Attempts were made to obtain agents that can ameliorate or potentiate the nephrotoxicity of gentamicin (Ali, 2003;Mingeot-Leclercq and Tulkens, 1999).Among these agents, extract of medicinal plants like garlic (Pedraza-Chaverri et al., 2000), and N. sativa oil (Ali, 2004) have been reported to possess properties to ameliorate gentamicin-induced nephrotoxicity.One common feature of the herbal agents is that they all have anti-oxidant properties (Prasad et al., 1996;Burits and Bucar, 2000).A potential therapeutic approach to ameliorate gentamicin-induced renal damage would have very important clinical consequence (Mengeot-Leclercq and Tulkens, 1999).
In the present work, we have attempted to test and compare the possible pre-, post-and concomitant action of n-hexane extract of N. sativa on gentamicin-induced nephrotoxicity in rats.

Chemicals and reagents
Gentamicin (80 mg/mL) was obtained from the Essential Drug Company Ltd. (Bangladesh).N. sativa was purchased from the local market.Chemicals and reagents of estimation of serum creatinine and serum urea were obtained from Human GmbH (Germany).Reduced glutathione was purchased from Loba Cheme (India).5,5-dithiobis-2-nitrobenzoic acid (DTNB) was from Sigma Chemicals (USA).

n-Hexane extract of N. sativa
The seeds of N. sativa (800 g) were soaked in 3 liters of n -hexane for 48-72 hours.They were filtered and the filtrate was concentrated in a rotary evaporator.The concentrate was freeze-dried, and a concentrated dark brown oily n-hexane extract of N. sativa was obtained.

Animals
Adult male rats aged between 8-12 weeks, weighing 200 -230 g were obtained from the animal house of Bangabandhu Sheikh Mujib Medical University.Normal rat-feed, water ad libitum was provided under 12 hours light-dark schedule at room temperature (24-28°C).Rats were divided into 12 groups, 6 rats in each control group and 10 rats in each experimental group.

Experimental design
Rats were treated with distilled water (2.5 mL/kg/day orally), gentamicin (100 mg/kg/day by subcutaneous injection), soybean oil (5 mL/kg/day orally) or extract of N. sativa (5 mL/kg/ day orally) for 7 days followed by none or above concentration of distilled water, gentamicin, soybean oil or extract of N. sativa for another 7 days.Rats were sacrificed on either day 8 th or 15 th . 2 mL of blood was collected in clean test tubes and centrifuged (4000 x g for 5 min).The serum obtained was stored at 0-4°C for estimation of creatinine and urea concentrations.The kidneys were excised, blotted on a filter paper and weighed in pairs.The cortex was dissected out and a portion was placed in formalin for subsequent histological processing.Another portion of the renal cortex was processed for biochemical estimation of reduced glutathion (GSH) concentration.

Biochemical measurements
GSH concentration was measured in homogenates of the renal cortex spectrophotometically (Sedlak and Lindsay, 1968).Serum creatinine concentration was measured by Jaffè reaction (Spencer, 1986).Serum urea concentration was measured by enzymatic colorimetric method (Fawcett and Scott, 1960).

Histological procedure
Small portion of cortex of the representative kidneys were fixed in 10% formalin, dehydrate in graded alcohol and embedded in paraffin wax, sectioned at 5 μm thickness and stained with Hematoxylin and Eosin (H & E) for light microscopic examination.Renal proximal tubular damage was assessed on the basis of arbitrary score (Teixeira et al., 1982) as follows: 0 for no cell necrosis; 1 for mild, usually single cell necrosis in sparse tubules; 2 for moderate, sparse tubules showing more than one cell involvement; 3 for marked, tubules in almost every power field exhibiting total necrosis; and 4 for massive, total necrosis.

Protein analysis
Protein content was determined by Biuret method (Weichselbaum et al., 1946)

Statistical analysis
The results obtained from the experiments are represented as mean ± SEM of the number of samples.Data were analyzed by Student's unpaired 't' test, and significant difference between means ± SEM of the different groups were estimated using one-way analysis of variance (ANOVA) followed by Student's unpaired 't' test.

Results
The groups of rats injected subcutaneously with gentamicin for 7 days, sacrificed on day 8 and 15 had serum creatinine and serum urea concentrations significantly (p<0.001)increased while the renal cortical reduced glutathione concentrations of these groups of rats were significantly reduced (p<0.001)compared to those in the control rats.This would suggest that these rats were made model for nephrotoxicity (Table I).This assumption was supported by histological observation of the H & E stained transverse sections through the renal cortex, which suggested massive damage to the proximal tubules (score 3) of these groups of rats (Figure 1B).The transverse sections through the renal cortex of the vehicle control groups of rats showed almost identical architecture with no damage to proximal tubules (Figure 1A).The n-hexane extract of N. sativa treated rats differed from the vehicle control rats by an elevated (p<0.001)concentration of renal cortical reduced glutathione, while the other biochemical parameters were identical (no statistical difference could be obtained) almost to those of the control rats, and the histology did not differ from those of the control groups of rats.Treatment of nephrotoxic rats with the n-hexane extract of N. sativa as pre-, postand concomitantly mitigated the increases in serum creatinine and urea, and the decreases in reduced glutathione (GSH) while compared to those of the gentamicin-treated group sacrificed on day 15 (Table I).But none of the parameters in any of the groups were identical or closer to those of the control groups (Table I).However, the post-treatment group (gentamicin followed by n-hexane extract of N. sativa) demonstrated significant reduction in serum creatinine and serum urea concentrations (p<0.001) and also a significant elevation (p<0.001) of renal cortical glutathione concentrations compared to those observed in the pre treatment group (n-hexane extract of N. sativa followed by gentamicin) and the concomitantly treated (gentamicin and n-hexane extract of N. sativa concomitantly treated).Histological picture obtained in the post treatment was also apparently improved (score 1).However, the histological score obtained in both the pre treatment and concomitantly treated group were score 2. The post-treatment, therefore, remained as the most alleviated treatment group from toxic damage of gentamicin (Figure 1C).

Discussion
In the present study, nephrotoxicity induced by gentamicin was evidenced by depletion of renal cortical GSH and increases in serum creatinine and serum urea concentrations.This was supported by proximal These observations were similar to those of Ali (2004) who reported similar biochemical and histological changes suggestive of nephrotoxicity.The n-hexane extract of N. sativa possess strong anti-oxidant properties (Badary et al., 2000;Burtis and Bucar, 2000) that is why they were used in the present study to expect that the action of the toxic free radicals (Feldman et al., 1982) in the course of gentamicin administration, causing oxidative damage to the renal cortex would be antagonized.Reports about similar ameliorating action of anti-oxidants upon gentamicin nephrotoxicity is available (Ali and Mousa, 2001;Naidu et al., 2000;Ali, 2004).We have used three treatment groups to ameliorate gentamicin nephrotoxocity e.g., the n-hexane extract of N. sativa followed by gentamicin (pretreatment), gentamicin followed by n-hexane extract of N. sativa (post treatment) and lastly, concomitant administration of gentamicin and the n-hexane extract of N. sativa (concomitant treatment).The results of biochemical and histologycal observations indicate that the groups of rat which received gentamicin followed by n-hexane extract of N. sativa better overcome the toxic actions when compared to the other two groups.
Gentamicin treatment in rats gives rise to free radicals (Walker and Shah, 1988;Yang et al., 1995) that induces oxidative damage at the cellular level of the renal cortex (Feldman et al., 1982).Agents with anti-oxidant action could antagonize the depletion of the reduced glutathione (Sandhya and Varalakshmi, 1997;Pedraza-Chaverri et al., 2000;Ali, 2004).Probably the antioxidant action of the n-hexane extract of N. sativa prevents the oxidation of renal tissue.The cellular GSH content was therefore not depleted.

Conclusion
The present study suggests that the n-hexane extract of N. sativa was able to produce considerable alleviation from the nephrotoxic action of gentamicin in the adult male rats.The anti-oxidant action perhaps better exerted in presence of oxidative damage and prior supply of anti-oxidant may not protect the tissue to the expected degree in absence of free radicals.The mechanism of alleviation and the specific dose and ingredients of the n-hexane extract of N. sativa for complete nephroprotection remains to be identified.

Figure 1 :
Figure 1: Representative photograph of sections of renal cortex under light-microscope of rats treated with either distilled water (A), gentamicin (100 mg/kg/day for 7 days (B) and gentamicin plus extract of N. sativa (5 mL/kg/day for next 7 days) (C).H & E 400x

Table I Effects of gentamicin and n-hexane extract (N. sativa) on biochemical parameters and histology
tubular histology suggestive of gross tubular damage.