Haematological and Histopathological Effects of Mosquito Coil Smoke on Kidney-An Experimental study on Albino Rat

Masooma Syed, Sheikh Tousia, Kamaldeep Singh Balowria, Nusrat Jabeen, Nafis Ahmad Faruqi, Arshi Beg Senior Resident, Department of Anatomy, J.N.M.C, A.M.U, Aligarh, MD, Department of Anatomy, MD, Department of Anatomy, Medical officer.PHC, Bhalla Doda, Associate Professor, Post graduate Department of Anatomy, GMC Jammu, Professor, Department of Anatomy, Department Of Anatomy,J.N.M.C, A.M.U,Aligarh, 6 Resident, Department of Oncopathology, Tata Memorial Hospital, Mumbai.


Introduction
Mosquitoes have long been identified as the main vectors of many human and animal diseases like West Nile fever, malaria, dengue, etc. The latest estimates from WHO put new cases of malaria worldwide at 214 million in the year 2015 with an estimated 4,38,000 deaths. According to NVBDCP, there were around 0.67 million cases of malaria, with 84 million related deaths in the year 2017 till the month of September in India. [1] Mosquito coils are the most preferred anti-mosquito products in low-income communities because they are cheap and readily available and are burned indoors as a common practice in many households of South America, Africa and Asian countries like India. The most common active ingredients in mosquito coils, which comprise about 0.3-0.4% of the coil mass are pyrethroids that are effective against many genera of mosquitoes including Aedes, [2] Anopheles and Mansonia. The first pyrethroid (fenvalerate) was commercialized in 1978. [3] At present, the class of pyrethroids includes 42 active ingredients, differing in chemical structure or in relative stereoisomer composition The health implications of burning one mosquito coil is equivalent to the release of the same amount of particulate matter as burning 75 to 137 cigarettes, and emitting formaldehyde equivalent to 51 cigarettes. [4] Pyrethroids have been documented to cause clinical symptoms such as increased urination, jerky movements, ataxia, incoordination, staggering gait, dizziness, altered blood biochemistry, hepatotoxicity, haematoxicity and neurotoxicity. [5] Research also indicates substantial alteration in various organs of different animals exposed to chronic inhalation of mosquito coils smoke. Changes have been reported as early as 7 days. [6] Previous report on histopathological assessments of the kidney tissues of the rats exposed to mosquito coil smoke showed severe multifocal congestion, cystic dilatation in the medulla; proteinaceous casts within ducts, interstitial mononuclear cellular infiltration and widespread fibrosis. [6] It has also been reported that mosquito coil burning leads to increased level of blood urea and creatinine levels. [7] In the present study we intend to do a detailed investigation into the histopathological alteration in kidneys and ISSN (0): 2455-5274; ISSN (P): 2617-5207 hematological-indices (serum urea, creatinine, sodium and potassium)in rats after prolonged and variable exposure to mosquito coil smoke. As substantial data is not available on the effects of mosquito coils containing d-trans allethrin, therefore the present study will provide information of great social impact.

Subjects and Methods
The present study was carried out on 30 albino rats of Wistar strain of both sexes. The rats were procured from the "Central Animal House" of Government Medical College, Jammu. The clearance for use of animals was obtained from "Institutional Ethics Committee" of Government Medical College, Jammu The animals were housed in propylene cages of dimensions 44x28.6x30 cm3, which were placed in a room of size 10x12 sq. ft. with cross ventilation and facility of fan, where they were acclimatized for two weeks under standard laboratory condition (12 hour light and 12 hour darkness, temperature at 25±1℃), with ad libitum access to food and water.

Exposure of the rats
The animals were exposed to mosquito coil smoke for eight hours a day (average period of time that humans sleeps in a day) using mosquito repellent coil brand containing 0.1% w/w of d-trans allethrin. The mosquito coil was placed at a distance of 30 cm from the cage Group A Unexposed to coil smoke. Group B Exposed for 4 weeks Group C Exposed for 6 weeks Group D Exposed for 8 weeks Group E Exposed for 10 weeks

Collection of Blood
At the end of exposure period blood was collected from each rat using the retro-orbital procedure. The tip of the capillary tube was placed at the medial canthus of the eye under the nictitating membrane. As soon as the sinus was punctured, blood entered the tube by capillary action. 1 ml was collected in a simple vial and sent for kidney function tests, which included, serum creatinine, urea, sodium and potassium levels. Serum urea was estimated by GLDH-urease method. [8] Serum creatinine was estimated by alkaline picrate method. [9] Serum sodium and potassium levels where estimated using respective Accucare Diagnostic Kit (LAB-CARE Diagnostic India Pvt. Ltd., Mumbai ). The results were analysed using one way ANOVA test.

Dissection of the kidneys
Each rat was placed in a beaker containing chloroform soaked cotton . A lid was placed over the beaker for about 5-6 minutes to sacrifice the rats. The sacrificed rats were laid supine on the dissecting tray and pinned down at fore-and hind-feet in stretched out position. A midline incision was made in abdominal wall and skin was retracted to open the abdominal cavity. The intestines were displaced to one side to visualise the kidney of opposite sides. The renal vessels and ureters were ligated and cut and the kidneys removed. Tissue of 3mm size was cut from kidney by using sharp blade and placed in 10% formalin solution of, the following composition. [10] I. Formalin : 100m II. Tap water : 900m III. Sodium chloride : 8.5gms The tissue samples were embedded into paraffin wax and sectioned into 8 um thick sections using a microtome.
Haematoxylin and eosin was used to stain the tissue sections.  Serum sodium and serum potassium showed no significant change in all experimental groups even after exposure to coil smoke for 10 weeks. [ Table 5 and 6]

Haematological Indices
We found an increased level of Serum urea and creatinine in all experimental groups compared to the control. A statistically significantly increase (p<0.001) was however only observed in Groups C,D, and E which were exposed to mosquito coil smoke for 6,8 and 10 weeks respectively [Tables 3 and 4]

Histopathological findings Normal Rat Kidney (Group A)
The proximal convoluted tubules were more numerous in number and were lined by a single layer of low columnar or pyramidal cells which had round nuclei and granular cytoplasm staining deeply with eosin. The cell boundaries of lining epithelium were not clearly defined and the apical surfaces of the cells showed brush border that almost filled the lumen. The nuclei were euchromatic and central in position. The distal convoluted tubules were less numerous in number and they were lined by cuboidal cells that lacked a brush border and contained lightly stained cytoplasm with central euchromatic nucleus. In the medulla the thick descending and ascending segments of the loops of were lined by similar cells as the distal convoluted tubules. The collecting tubules and ducts of Bellini were lined by cuboidal or columnar epithelium with clear, lightly stained cytoplasm and distinct cell outlines. They had larger lumina and were regular in shape and lacked brush border. The thin segments of loops of Henle were lined by simple squamous epithelium with central bulging nuclei. [ Figure 1]

Group B
The kidneys of the group B rats showed no change in architecture of cortex and medulla. In the cortex, the renal corpuscles appeared normal, with mild congestion of the glomeruli. The interstitium showed mild congestion,focal haemorrhage and focal chronic inflammatory infiltrate. The medulla also showed focal vascular congestion. The tubules appeared normal with normal lining epithelium and empty lumina. The interstitium of the medulla showed mild vascular congestion and haemorrhage with focal chronic inflammatory infiltrate. [ Figure 2]

Group C
The renal corpuscles in the cortex showed moderate congestion of the glomerular tuft of capillaries. Most of the proximal convoluted tubules showed slight dilatation. Their lumina were filled with eosinophilic proteinaceous material. The distal convoluted tubules also appeared dilated with eosinophilic proteinaceous material filling the lumina. The tubules showed swelling of the lining epithelial cells with ballooning degeneration and fragmentation. The interstitium showed moderate congestion and focal chronic inflammatory infiltrate. In the medulla blood vessels were moderately congested. All the tubules showed proteinaceous material in the lumina. The interstitium also showed focal chronic inflammatory infiltrate, vascular congestion and haemorrhage.

Group D
In the cortex there was marked congestion of the glomerular capillaries. The proximal convoluted tubules appeared dilated with eosinophilic proteinaceous material in the lumina of approximately half of the tubules. Half of the tubules were normal appearing with empty lumina. The distal convoluted tubules also showed dilatation with eosinophilic material filling some lumina only, the rest of the tubules were normal appearing with empty lumina. The interstitium showed focal congestion, haemorrhage with focal chronic inflammatory cell collection in the interstitium. The medulla showed focal vascular congestion. Half of the tubules in the medulla were filled with eosinophilic hyaline material and the other half of tubules appeared normal. The interstitium of the medulla also showed focal vascular congestion, haemorrhage and chronic inflammatory infiltrate.[ Figure 4]

Group E
In the cortex there was marked congestion and infiltration of the glomerular capillaries. The interstitium also showed signs of marked congestion and infiltration.[ Figure 5]

Discussion
Agarwal et al. [7] conducted a study on 15 rats for a period of 4 weeks. They demonstrated an increase in serum urea levels from 16.03±1.33 mg/dl (control group) to 18.16±0.42 mg/dl (4 weeks exposure) and the serum creatinine levels from 0.48±0.03 mg/dl to 0.63±0.04 mg/dl (4 weeks exposure). The results demonstrated a significant increase in both the values, which is in line with results of the present study. Significant increase in the serum creatinine levels (p<0.05) was also reported by Ugwu et al. [11] and Ogbonnia et al. [12] in experimental groups of rats exposed to different brands of mosquito coils for a period of 31 days, which supports our result of increase in serum creatinine in group B i.e. 4 weeks of exposure. Ibiam et al. [13] conducted a study on rats using two different brands of mosquito coil for a period of 4 weeks. Their results indicated a significant increase in serum creatinine in both exposed groups, which is again similar to our finding in the present study for Group B rats. According to a study conducted by Okine et al. [14] chronic exposure of mosquito coil smoke for 6 weeks showed no significant increase in serum creatinine levels. The study is in discordance to our study. Siddique et al. [15] in their study on Wistar rats exposed for 4 weeks reported haemorrhage, congestion of blood vessels, cellular infiltration and fibrosis in the interstitium of kidneys. These findings are in concordance with the results obtained in our study for group B that was exposed for a period of 4 weeks. Further Agarwal et al. [16] also reported marked symptoms of renal tissue impairment in rats exposed to mosquito coil smoke for 4 weeks, which included interstitial infiltration that was in favour of our study. They also reported glomerular degeneration and tubular degeneration that was also observed in our study. Idowu et al. [17] reported no morphological changes in exposed rats till 8 weeks when they reported mild interstitial congestion. Full congestion was reported at around 16 weeks of exposure. The study is in discordance with our study as we observed congestion as early as 4 weeks and maximum congestion was reported in the exposed animals at 10 weeks Ayorinde et al. [18] reported no change in the histology of kidneys. of exposed animals and the same was also observed by Okine et al. [14] which is in discordance with our study. Urea is formed by deamination of amino acids in the liver, and then it is transported by blood to the kidneys where it is excreted with urine. [19] Elevation of serum urea observed in the present study in response to pesticides exposure may be explained by: 1) impairment in its synthesis as a result of impaired hepatic function, 2) disturbance in protein metabolism and 3) decrease in the filtration rate of the kidney. [20] Creatinine is a waste product that is normally filtered from the blood and excreted with urine. Increase in creatinine level in response to pesticide exposure indicates renal disease and may be a result of impaired glomerular function. [22,23]

Conclusion
The coil smoke can be toxic for humans as well and general masses should be made aware about the possible hazards of the over-use of mosquito coils. Adequate measures should be taken to ensure minimal exposure to coil smoke during domestic use.