Oral Exposure to Nitrocellulose Thinner Solvent Induces Nephrotoxicity in Male Albino Wistar Rats

INTRODUCTION

Environmental pollution and health hazards are known to be associated with the use of various industrial solvents including hexane, toluene, benzene, among others. Generally, different types of chemical solvents are used by different industries for different purposes. Some of these solvents may have very toxic effects in humans and other species of organisms. Nitocellulose thinner is one of the known solvents with various use in different industries. This solvent is generally used in furniture, paint and automobile spray painting industries. It may also be used in mixtures with other solvents in domestic or industrial products. Nitrocellulose thinner is an important industrial solvent containing different organic chemical substances, including ethylbenzene or toluene and butyl acetate. These chemical substances are known to constitute chemical pollutants in the environments where they are used. Typically, it has been reported that these chemical agents, among others, have been detected in household and workplace air (WHO, 1996, 2005).

The kidney is known to be responsible for the maintenance of the constant extracellular environment through its involvement in the excretion of such catabolites as urea, creatinine, uric acid and regulation of water and electrolyte balance. Abnormal concentration of these catabolites and some electrolytes in the plasma or serum therefore serve as a clear indication of renal function impairment (Gidado et al., 2001; Nwankwo et al., 2006; Haddy et al., 2006; Crook, 2007; Zanna et al., 2008). Impairment of the renal functions may be caused by several diseased conditions and exposure to certain reactive or toxic metabolites, i.e., nephrotoxic substances (Chatterjea and Shinde, 2002; Jimoh and Odutuga, 2004; Crook, 2007). For instance, certain mixtures of hydrocarbon, gasoline vapours, lead, insecticides, pesticides and other chemical solvents have been reported to pose some degrees of adverse effects on the functional integrity of the renal tissues in humans and experimental animals (Wang et al., 2002; Dioka et al., 2004; Boogaard et al., 2005; Adeniran et al., 2006; Hernandez-Serrato et al., 2006; Patil et al., 2007; Uboh et al., 2008, 2009, 2011; Salawu et al., 2009). Also, exposure to lead from automobile exhaust is reported to be a risk factor for nephrotoxicity among traffic policemen (Mortada et al., 2001). Renal function impairment manifests in a variety of different clinical presentations, some of which may be asymptomatic. The renal function impairment with asymptomatic presentations can only be detected by routine laboratory examinations. Azotaemia, a clinical condition associated with renal function impairment, is one of such presentations that can rightly be detected by laboratory findings. The condition is characterized by elevated levels of serum creatinine, urea and BUN (Cotran et al., 1999). A persistently increased serum creatinine is reported to be one of the risk factors for chronic kidney disease, which may results in renal failure (Mortada et al., 2001).

The kidney functions may therefore be assessed from the level of some electrolytes (such as K⁺ Na⁺, Cl^- and HCO^3-), metabolites (such as creatinine, uric acid, urea and blood urea nitrogen) in the plasma and the histological analysis of the ultrastructural status of the renal tissues (Nwankwo et al., 2006; Atangwho et al., 2007; Crook, 2007; Uboh et al., 2008, 2009, 2011). Nephrotoxic response, resulting in renal tissue dysfunction, has been reported to be characterized by the distortions in the architectural integrity of the glomerular tubules. This may be evidenced by lesions on different segments of the nephron, including exacerbation of hyaline droplet formation as the primary change, development of granular casts as a sequelae and finally chronic nephrosis as a consequence of granular cast formation obstructing the nephron. Renal dysfunction of any kind may affect all or parts of the nephron to some extent, impairing either the glomerular or tubular functions. Generally, the net effect of renal dysfunction on the plasma and urine depends on the proportion of the glomeruli to tubules that are affected and on the number of nephrons involved. In this present study, the changes in some serum renal function indices and histopathology of the renal tissue, hence, nephrotoxicity, associated with oral exposure to nitrocellulose thinner were assessed in male albino Wistar rats.

MATERIALS AND METHODS

Chemicals and reagents: Nitrocellulose thinner (POLYGARD^®, MISWA CHEMICALS LTD, England), Randox, Dialab diagnostic, CRESCENT diagnostic and AXIOM Gestllschaft for Dignostica and Biochemica mBH reagent kits were used for the biochemical and electrolyte assays. All the other chemicals used in this study were of high grade of purity commercially available.

Animal handling and experimental design: Twenty four mature male albino Wistar rats, weighing between 120 to 150 g were obtained from Biochemistry Department Experimental Research Animal House of the University of Calabar, Calabar, Nigeria. They were fed with a standard laboratory diet and tap water. Illumination was 12 h light/dark cycle and room temperature was 25±2°C. The animals were divided into four groups, [i.e., one control (I) and three experimental groups (II, III and IV)], which consisted of six apparently normal albino Wistar rats per group. The experimental groups II, III and IV were exposed daily to 10, 20 and 30 mg kg^-1 body weight, respectively of nitrocellulose thinner by oral administration for 30 days, while the control group was given normal saline. In this study, all the animal experimentations were carried out following the guidelines for the care and use of laboratory animals obtained from the Institutional Animal Ethics Committee. This research work carried out between September and October, 2011.

Collection and handling of blood serum for analyses: Twenty-four hours after last exposure, the animals were anaesthetized with chloroform vapour and dissected. Whole blood from each animal was collected by cardiac puncture into well labelled non-heparinized sample tubes and allowed to clot for 3 h in iced water. The serum was separated from the clots after centrifuging at 10,000 rpm for 5 min into well-labelled plain sample bottles and used for assays. The kidneys were also collected for histopathological examinations.

Biochemical assays
Serum protein: The concentration of total serum protein was assayed spectrophotometrically by Biuret method using Randox reagent kits (Randox Laboratory Ltd, United Kingdom, BT29 4QY).

Serum urea and blood urea nitrogen: The concentration of urea in serum was estimated by the endpoint colorimetric method using Dialab reagent kits (Searcy et al., 1967). In this method, urease enzyme hydrolyses urea to ammonia and carbon dioxide. The ammonia so formed reacts with alkaline hypochloride and sodium salicylate in the presence of sodium nitroprusside to form a coloured chromophore which was measured with DREL 3000 HACH (England) model spectrophotometer.

Serum uric acid level: The concentration of uric acid in the serum of the experimental animal was estimated by enzymatic colorimetric test using CRESCENT diagnostic kit (Saudi Arabia) according to the procedure given in the kit protocol.

Serum creatinine: The concentration of serum creatinine was assayed based on the reaction of creatinine with an alkaline solution of sodium pirate to form a red complex. The red coloured complex which is proportional to the concentration of creatinine in the sample was measured spectrophotometrically.

Serum calcium level: The level of calcium in the serum samples of both the test and control animals was measured using AXIOM Gestllschaft for Dignostica and Biochemica mBH kit (Deutschland).

Serum sodium: Serum sodium concentration was estimated using Mg-Uranylacetate reaction method described in Dialab diagnostic kits (Trinder, 1957). Sodium in serum is precipitated with Mg-Uranylacetate, the remaining uranyl ions form a yellow-brown complex with thioglycolic acid. The difference between reagent blank analyses is proportional to the sodium chloride.

Serum chloride: The level of chloride in serum was determined using mercuric thiocyanate reaction method described in Dialab diagnostic kits (Tietz, 1976). Chloride ions in the sample react with mercuric thiocyanate displacing the thiocyanate ions. The displaced thiocyanate ions react with ferric ions producing a coloured complex.

Serum bicarbonae ion: Serm bicarbonate ion (HCO^3-) concentrations were determined as described by Tietz et al. (1999), using Randox reagent kit.

Histopatholoigical examination: For light microscopic examination, liver and kidney tissues from each groups were fixed with 10% buffered formalin, embedded with paraffin. After routine processing, paraffin sections of each tissue were cut into 4 μm thickness and stained with haematoxylin and eosin.

Statistical analysis: All the data obtained for the biochemical assay results were presented as Mean±SEM. These results were analyzed using the Statistical Package for Social Sciences (SPSS for windows, version 17.0). Comparison were made between and within the experimental groups using one-way Analysis of Variance (ANOVA), followed by pair wise comparison between each test and control groups using Student’s t-test. Values of less than 0.05 (i.e., p≤0.05) were regarded as statistically significant.