Lycopene Ameliorate the Gentamicin Induced Nephrotoxicity in White Mice Model

Aminoglycoside antibiotics are broadly utilized in the treatment of gram-negative bacterial infections (Chen and Kaye 2011). These anti-biotics have the potential for causing of nephrotoxicity and it has assessed that practically 25% of patients who get positive clinical effects may encounter nephrotoxicity. Gentamicin preferably accumulates in the proximal renal tubules in cortex due to membrane transporters of proteins and cations in kidney (Selby et al., 2009) and gives rise to vascular and tubular damages. Accumulated gentamycin activates reactive oxygen species markers like, ROS, lipid peroxidase (LPO) and serum creatinine. Moreover it was stated that gentamicin toxicity increase the pro-inflammatory cytokines (Priyamvada et al., 2008). Oxidative stress produced by gentamicin leads to activation of caspase mediated apoptosis (Sanchez-Gonzalez et al., 2011; El-Bahr, 2015).

Lycopene is a lipid soluble carotenoid pigment with the chemical formula C40H56. This compound is responsible for the red color of many fruits such as tomato, grapes and water-melon with the highest concentration in tomatoes (Sigurdson et al., 2017). Lycopene was reported to have antioxidant effect due to the nature of its structure. Lycopene can keep many diseases away from the human body such as cancer, hypertension and others (Wildman 2016; Liu, 2017). The antioxidant effect of lycopene was estimated to be ten folds higher as compared to other antioxidants and may reaches up to 100 folds than that for  vitamin E in diseases like cancer and  in in-vitro studies, hence recommended as daily nutrient supplement (Ascenso et al., 2013,). Long time ago, lycopene was used in traditional medicine in treatment of nephrotoxicity and hepatotoxicity as an antioxidant (Sadek et al., 2015; Iranshahy et al., 2018). Moreover it was also reported to suppress significantly proinflammatory cytokines production in in-vitro studies (Shyu et al., 2008). Results of in in-vitro studies recorded that Lycopene inhibits the expression of inducible nitic oxide synthase gene which consequently inhibit the production of NO induced by bacterial lipopolysaccharide (LPS) (Rafi et al., 2007 and Saedisomeolia et al., 2009). Proinflammatory cytokines such as IL-6 and tumor necrosis factor alpha (TNF-α) expression had also been reported to be inhibited by lycopene (Liu and Chen, 2016). Cadmium induced nephrotoxicity was recorded to be reduced by lycopene (Sharma and Vijaya, 2015) and adjusted the level of urea, creatinine, Lipid peroxidation and malondialdehyde (MDA) level. According to the high antioxidant effect and protective effect of lycopene and due to the rareness of studies regarding the antibiotic gentamicin, the aim of this study is to clarify the renal protective effect lycopene after challenge by gentamicin.

Chemicals
 
Gentamicin and lycopene were purchased from Sigma Aldrich Company. All chemical those used in the biochemical analysis were purchased from Human Diagnostic Worldwide (Max-Planck-Ring 21, Wiessbaden, Germany). Antibodies for different cytokines were obtained from Biosource, USA.
 
Animals
 
In this study 32 adult male mice weighting around 25-30 g were used. Animals were obtained from Animal facility, Department of Biological Sciences, College of Science, King Faisal University. Mice were acclimatizedd in the laboratory environment of controlled temperature, light/dark cycle for two weeks before the experiment. Mice used in this experiment were supplied by standard pellet diet. Water and food were supplied three times a week. Mice care was in compliance with the guidelines of animal ethical committee of KFU.
 
Experimental protocol
 
Mice were divided into four groups (8 mice each). Group I: control group received a daily dose of corn oil for three weeks. Group II: gentamicin group received gentamicin 80 mg/kg of animal body weight for three weeks. Group III: treated group received both gentamicin and lycopene (4mg/kg and 80mg/kg), respectively for three weeks. Group IV: lycopene group received 4 mg/kg for three weeks. At the end of the experiment, animals were anesthetized and serum were collected for biochemical investigations. Kidney were collected and divided into two portions one for histopathological study and the other for the biochemical and molecular analysis. 
 
Biochemical analysis
 
Different marker for kidney, creatinine and serum urea were investigated.  Cytokines (IL-6, TNF-α, IL-4 and IL-10) were investigated using enzyme-linked immunosorbent assay kit (Biosource, USA) according to manufacturer protocol. Oxidative stress markers (MDA, GPx and SOD) were investigated in kidney homogenate according to the company protocol. Serum glucose was also investigated.
 
Semi-quantitative PCR
 
Total RNA from kidney tissue was extracted and mRNA purified.  The obtained mRNA was reverse transcripted into cDNA using oligo-dt12-28 primer (GIBCO-Invitrogen, USA). cDNA stored until use in -20^oC. Primers for cytokines (TNF-α, IL-6, IL-4 and IL-10) sequences as shown in (Table 1) with annealing temperature 54, 50, 52, 51 respectively. The expected product was 438bp, 336bp, 318 and 482.


 
Histopathological studies
 
Kidney samples those taken for histopathological investigation subjected to routine hematoxylin and eosin processing. Stained slides were examined under normal light microscope.
 
Statistical analysis
 
All one way analysis of variance (ANOVA) was used to analyze all numerical data. The analysis was performed throughout mean, standard deviation, maximum and minimum range. Significant data were considered at (P value≤0.05).

Gentamicin is an antibiotic induces a nephrotoxicity in a great percent of the patient those receive it (Abdel-Raheem et al., 2009). The nephrotoxicity induced by gentamicin due to it accumulation selectively in proximal tubule cells leading to loss of cell integrity and finally cell degeneration and apoptosis occur (Liu et al., 2016 and Veljkovic et al., 2016). Results of kidney markers in mice serum gave an indication for nephrotoxicity where the level of both marker, urea and creatinine were recorded a highly significant increase (P≤0.05) in the serum of mice challenged with gentamicin (Fig 1). These results were supported and in agree with many previous studies such as (Bapaydin et al., 2017, Katary and Salahuddin, 2017). Creatinine and urea low clearance rate from the blood indicate the impaired kidney function (Calderon et al., 2010). Mice treated with lycopene showed a significant decrease in serum level of both kidney markers (urea and creatinine) (Fig 1).



The oxidative status of the kidney was determined by estimation of oxidative enzymes markers. Injection of gentamicin cause a significant decrease (P£0.05) of GPx and SOD in kidney tissue homogenate and increase MDA level (Fig 2). Gentamicin cause an oxidative stress (Chashmi, 2017 and Mosaad et al., 2016). This may be due to production of free radicals by enhancing the production of mitochondrial hydrogen peroxide and other ions those leads to peroxidation of membrane lipid and finally disturbance of osmotic balance and cell degeneration. Decreasing of SOD, GPx by gentamicin injection also reported to indicate antioxidants defense which related to glomerular filtration impairment (El-Kashef et al., 2015 and Manikandan et al., 2011) leading to increasing level of serum glucose (Fig 2). Results of this study show a repairing of SOD, GPx and MDA (Fig 2) where it inhibit the production of reactive oxygen species (ROS). Lycopene is characterized by giving an electron ability which scavenge most of the free radicals induced by gentamicin. This is an agreement with Daniel et al., 2015. Results indicate that lycopene is a great antioxidant among the whole carotenoids where the lycopene treated group results usually more or less near to the normal group.

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Results also showed that mice challenged with gentamicin showed a significant (P≤0.05) increase in the level of cytokines (TNF-α, IL-6, IL-4 and IL-10) Fig (3). Treatment of mice with lycopene at the same time results in highly significant (P≤0.05) decrease of both types of cytokines (pro-inflammatory and anti-inflammatory). Mice those received lycopene only showed a serum cytokines (both types) and glucose more or less near to that of normal group (Fig 3). Serum cytokines estimation of this work reveal a significant increase (P≤0.05) in all investigated cytokines in the gentamicin challenged mice (Fig 3). The increase of the inflammatory mediators indicate that gentamicin affect the inflammatory oxidative pathway. Inflammatory cytokines had been reported to play an important role in nephrotoxicity (Kirbas et al., 2015). Using lycopene treatment significantly decreases the level of both types cytokines (Fig 3). These results were confirmed by semi-quantitative PCR where the expression rate of the TNF, IL-6, IL-4 and IL-10 were changed due to treatment with lycopene (Fig 4). Results obtained by this study were supported with previous one where it was reported that lycopene can alleviate the inflammatory status of the kidney (Oguz et al., 2015).

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The oxidative status of the kidney tissue induced by gentamicin and its attenuation by lycopene were confirmed by histopathological studies in this work. Stained slides showed a protective effect of lycopene on the kidney parenchyma from damage by oxidation the finally reduces nephrotoxicity (Fig 5,6). Tubular cells necrosis and tubular dilation is also reduced in lycopene treated group (Fig 5,6). Infiltration of the kidney tissue by several leucocytes indicate the inflammatory status of the kidney (Fig 5,6).

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Results of this work conclude that lycopene is very powerful antioxidant that have a protective effect against the renal toxicity induced by Gentamicin. It is also indicates that lycopene alleviate the inflammatory and the oxidative stress status of the kidney cells and protect the integrity of the cell membrane of the proximal convoluted kidney tubule. So the author support using of lycopene as a protective for kidney diseases in the form of food supplementation.

The author acknowledge the deanship of scientific Research at King Faisal University for the financial support under Nasher Track (Grant No. 186341).

The author declare that there is no conflict of interests regarding the publication of this article.