Attenuation Of Methotrexate-induced Kidney Toxicity In Rat Model By Antioxidants

Cancer is a major burden of disease worldwide. Cancer treatment involves the use of chemotherapy which employs a wide group of drugs that have cytotoxic effects which preferentially, but not exclusively, target the rapidly dividing cancer cells (Cholawara et al., 2011). Chemotherapeutic agents include methotrexate which is used either alone or in a combination with other agents. Also, it is used in the treatment of rheumatoid arthritis and other inflammatory conditions (Crostein 1996). In cancer cells, methotrexate competitively inhibits dihydrofolate reductase an enzyme required for tetrahydrofolate synthesis. The resultant effect of MTX inhibits the synthesis of DNA, RNA, thymidylates and proteins (Ravi et al., 2002). Although the use of MTX has been beneficial to most patients, however, it can cause significant toxicity, including kidney injury in 2%–12% of patients. Nephrotoxicity results from crystallization of methotrexate in the renal tubular lumen, leading to tubular toxicity. Renal toxicity leads to impaired methotrexate clearance and prolonged exposure to toxic concentrations (Howard et al., 2016). Studies in animal model of MTX administered rats reported oxidative and nitrosative stresses and intensive degenerative changes (Abdel-Daim, 2017).

Melatonin (N-acetyl-5-methoxytryptamine) an endogenous neurohormone is an evolutionary conserved indole amine synthesized from tryptophan that is mainly secreted by the pineal gland (Gurpinar et al., 2012). It has antioxidant (Galano et al.,2011), anti-flammatory activities and immunomodulatory effect (Kireev et al ,2008). It stimulates anti-oxidants production against oxygen-and nitrogen-based reactive molecules (Sudnikovich et al., 2007) in exogenous administration. Also, Melatonin can modulate a variety of neural, endocrine, and immure functions (Toma et al., 2007). ). Also, MT can protect against a wide variety of processes and agents involved in damage tissue via free radical mechanisms. (Reites et al., 2003)

N-acetyl cysteine is a sulfhydryl-containing compound rapidly absorbed into various tissues following on oral dose (Stockley RA., 2008). NAC is an effective scavenger of free radicals as it interacts with ROS such as OH and H2O2 as well as a major contributor to maintenance of the cellular GSH status and can minimize the oxidative effect of ROS through correcting or preventing GSH depletion (Atkuri et al., 2007). As a drug, NAC represents perhaps the ideal xenobiotic, capable of directly entering endogenous biochemical processes as a result of its own metabolism (Sahin and Alatas, 2013).The biological activity of NAC is attributed to the sulfhydryl group, while its acetyl-substituted amino group affords its protection against oxidative and metabolic processes (Bonanomi L et al., 1980). It serves as an antidote for acetaminophen poisoning. It is administered orally and intravenously for the treatment of paracetamol poisoning (Green et al., 2013).

Alpha-lipoic acid (ALA), a dithiol compound derived from octanoic acid, which plays an essential role in mitochondrial dehydrogenase reactions. It acts as a cofactor in the multi-enzyme complexes and is an essential substance in energy production via the citric acid cycle (Hagen et al., 1999). It is a unique and potent antioxidant that delivers antioxidant activity in both fat- and water-soluble medium. It has an antioxidant effect in both its oxidized (LA) and reduced (DHLA [dihydrolipoic acid]) forms (Goraca et al., 2011). DHLA is capable of exerting an antioxidant effect directly by donating electrons to a pro-oxidant or an oxidized molecule (Scholich et al., 1989). ALA has been documented to have positive effects on a wide variety of clinical conditions, which is completely consistent with its antioxidant and anti-inflammatory effect (Jung et al., 2012; Kaya-Dagistanli et al., 2013). Therefore, this study examine the effect of Alpha-lipoic acid, melatonin and n-acetylcysteine against rat model of methotrexate-induced kidney damage.

Materials and Methods

Drugs/Chemicals

Melatonin, Alpha –lipoic acid and n-acetyl cysteine used for this study were manufacture by Puritan’s Pride Premium, Mason Vitamins Inc while the methotrexate was manufactured by Biochem Pharmaceutical industries limited.

Dose selection

Doses used in this study are 20mg/kg of MTX, (Endorgan et al., 2015), NAC 10mg/k of NAC (Adikwu and Bokolo, 2017), 10mg/kg of MT (Adikwu et al., 2015), and 10mg/kg of ALA (Adikwu et al., 2017)

Animals

Adult albino rats (of both sexes) with an average weight of 164 ± 5g used for this study were obtained from the animal house of the department of Pharmacology and Toxicology Niger Delta University, Wilberforce Island, Bayelsa state. The animals were housed in cages (6 per cage) and allowed to acclimatize for one week in a well -ventilated room, maintained at a room temperature of 28±2 oC, under natural lighting condition. They were fed with standard rodent chew and given tap water ad libitum.

Experimental procedure

The sixty rats were grouped into 4 (A-D). Groups A and C contained six rats each whereas groups C and D contained 24 rats each which were subdivided in sub-group B1-B4 and D1-D4 of six rats each. Rats in group A were treated with water intraperitoneally (ip) while those in group B (B1-B4) were daily administered with10mg/kg of NAC, 10mg/kg of MT , 10mg/kg ALA and a combination of MT and ALA ip for 5 days . Rats in group C were daily administered with 20 mg/kg MTX ip on the 5th day. Rats in group D (D1-D4) were administered with NAC, MT, ALA and a combination of MT and ALA ip for five days. On the fifth day all the animals in group D were administered with 20 mg/kg of MTX ip after which the animals were sacrificed on the 6th day after an overnight fast and blood samples were collected and kidney harvested.

Biochemical analysis

Blood samples were then centrifuged at 2000 rpm for 10 min in a centrifuge to separate serum samples from the cells. Serum urea, creatinine, uric acid, total protein, and albumin levels were determined in the serum by routine colorimetric methods using standard laboratory test kits (RANDOX Laboratories Ltd., Crumlin, UK). The liver and kidney samples taken were washed in saline in an ice bath and homogenized in the ratio 1:10 (w:v) with ice-cold 150 mM KC1 and evaluated for oxidative stress marker. Kidney malondialdehyde (MDA) was assayed as reported by Buege and Aust whereas total glutathione (GSH) was evaluated as reported by Sedlak and Lindsay 1968. Superoxide dismutase (SOD) was measured as reported by Misra and Fridovich 1972 whereas catalase (CAT) was analysed using the method of Aebi (1984). Glutathione peroxidase (GPx) activity assayed according to Rotruck et al. 1973 whereas protein content was determined according to Lowry et al.1951

Histopathology

The sections of the kidney of rats were taken and gently rinsed with physiological saline solution (0.9% NaCl). They were fixed in 75 ml of saturated aqueous picric acid, 25 ml of 40% formaldehyde and glacial acetic acid) for 24 hours. The kidney tissues were processed and embedded in paraffin wax. Sections of 5µ thickness were cu using microtome. The sections were stained with Harris hematoxylin and counter stained with eosin, dissolved in 95% alcohol. After dehydration and cleaning, the sections were mounted and photographed