Biochemical and histological alterations induced by the smoke of allethrin based mosquito coil on mice model

Mosquito coil is the slow-burning structure made mainly of insecticides along with inert materials such as wood floor, coconut shell powder, starch etc. Upon burning, MC emits smokes, containing single or multiple insecticides, which creates a defending environment and protects the subject from several mosquito borne lethal diseases including malaria, filaria and dengue. Pyrethroids and Pyrethrins are the main active ingredients of MC. Among the several types of pyrethroid, allethrin especially d-transallethrin (belong to the type I pyrethroids) is being used in almost all brands of mosquito coil in South Asian territory. Allethrin is a type of neurotoxin, acting both on the peripheral and central nervous systems by modifying the kinetics of voltage-sensitive sodium channel, resulting in increasing of sodium permeability across the channel and paralysis of insect’s organs [1]. On the other hand pyrethroids have well been reported to induce oxidative stress and alter antioxidant level in different organ systems of rodent animal [2, 3].

MC smoke is considered as a potent air pollutant in indoor environment. One MC, upon burning, produces such amount of particulate matter equivalent to that of 75–137 cigarettes [4]. Along with insecticides, MC smoke also contains sub-micrometer particles, volatile, semi-volatile organic compounds and gaseous pollutants such as polyaromatic hydrocarbons [5, 6]. After using the MC in a room, it was observed that the maximum concentration of allethrin (0.0120 ppm) prevails till 30–35 min followed by a gradual decline in 6 h [7]. During overnight burning, subjects at close proximity are exposed to sub-micrometer particles, metal fumes, free radicals and vapors from smoke which ultimately reaches the alveolar region of lung and leading to irritation of the upper respiratory tract [6]. Chronic inhalation of MC smoke has been reported to correlate with asthma and persistent wheeze in human [8] along with focal declination and metaplasia in tracheal epithelium in rodents [5, 9]. Prolong exposure and subsequent entry of allethrin along with other MC derived particulate matters into red blood cells lead to the alteration of blood biochemical parameters including hemoglobin percentages [10, 11]. Exposure of pyrethrin based MC smoke has also been reported to considerable increase in white blood cell (WBC), especially basophil and lymphocyte in rat [12]. This noxious synthetic chemical is proven to be responsible for cellular, tissue and organs injury due to exerting its action on the membrane phospholipids, resulting membrane fluidity and tissue damage [11, 13].

The toxic smoke, emitted from MC is also reported to be responsible for inducing chromosomal aberrations in pulmonary alveolar macrophages and bone marrow [14, 15]. Recently, Madhubabu et al., (2012) have shown that MC smoke induces elevated level of reactive oxygen species (ROS) as well as over-expression of stress responsive gene, p53 [16]. Oxidative stress (DNA damage mediated) induced up-regulation of p53 is considered as the critical and early event of mitochondria mediated apoptosis [17]. Therefore, it could be postulated that MC smoke would induce apoptotic mediated tissue damage in subject with prolong exposure.

Subtropical climate, Poor drainage system and dense population are triggering the mosquito pressure day by day in tropical region including Bangladesh. As a cheap and instant remedy, middle and lower income groups are regularly using MC. Thus, a huge number of people are undesirably exposing to toxic fine particles and free radicals present in the smoke of MC and are subjecting themselves to various health complications regularly [18]. Therefore, this study was aimed to analyze the patterns of damages in the histo-architecture of lung and liver tissues as well as the alteration of biochemical parameters of blood in mice model inhaled with the mosquito coil smoke.

MC contains pyrethrin based insecticide but still widely being used in the tropical and subtropical region including Bangladesh as a common approach to protect mosquito from entering into home. MC generally has higher acceptance over liquid vaporizer due to its lower cost and ease of use. Upon burning, MC releases over 60 organic compounds, fine and ultrafine particles and heavy metals [5]. The smoke is composed of two phases: gas and particle phase [26]. During overnight burning, the smoke of particle phase containing carbon particles, heavy metals, aldehydes ultimately reaches peripheral region of lung and leading to progressive cellular injury and destruction of mucus membrane [9].

The present study demonstrates that critical biochemical parameters of blood were altered significantly in MC smoke inhaled animals. The activity of transaminases (ALT and AST) was increased significantly (at P < 0.05) in smoke inhaled animals (Table 1). Elevated activity of these hepatic enzymes is strongly correlated with hepatic injury [11]. It is either due to the direct effects of allethrin on the membrane phospholipids of hepatocytes and/or indirect effects caused due to the by-products derived from pyrethroid metabolism. Allethrin is membrane active substance, which acts upon membrane phospholipids and increases membrane fluidity [13] and leading to leak out cellular enzymes in extracellular matrix eventually increasing transaminases activity in blood. Elevated transaminase level has also been reported previously by Karthikeyan et al., (2006) in mice, exposed to mosquito repellent mat vapor [27].

The exposure of MC smoke resulted dose dependent depletion of BUN and total protein activity in all inhaled animals. This finding is also supported by a number of observations which demonstrated that kidney functions were not negatively altered in MC smoke inhaled rat [5, 11]. Similarly, the MC smoke has previously been reported to reduce serum total protein activity both in rodent animal and human subject [27, 28]. Higher protein degradation with subsequent reduction of total protein activity of blood in smoke inhaled subject has also been confirmed by marked increase of plasma free amino acid levels [28].

The elevated activity of some parameters of lipid profile in smoke inhaled mice in current study (Table 1) is not accord with the findings conducted by Narendra et al.,(2008), which demonstrates decreased plasma CHOL and LDL activity in human subjects, who inhaled the smoke of allethrine based MC regularly for long period of time [28]. Elevated LDL activity is considered to be the risk factor for cardiac condition, because it transports cholesterol from the liver to peripheral tissues. However, it is the first study that documented the raised cholesterol level in rodent animal treated with allethin based MC smoke via inhalation.

The oxidative damage, induced by ROS is the hallmark of cellular stress which modulates a number of signaling pathways including carcinogenesis, apoptosis, necrosis and inflammatory pathway. Free radical and volatile organic compounds present in the smoke of MC is considered as the potent contributor of oxidative DNA damage and tissue injury. Under these stress conditions, the histological organization of pulmonary and hepatic tissues becomes affected which led to severe organ injury. Histological study of lung tissue demonstrates extensive destruction of the alveolar septa and bronchial epithelial wall resulting in loss of alveoli as well as capillary surface area. Thus, the current study shows the clear evidence of severe emphysema as a result of long time inhalation of allethrin based MC smoke (Fig. 1E-F). The key event of the development of emphysema is the imbalance between proteases and anti-proteases, which is well established [29]. The ROS, induced by MC smoke, interferes with the antioxidant defense system [16], which would triggers inflammatory response in damage sites [30]. Furthermore, over expression of p53 in MC induced animals [16] is also considered as the potent contributor of pulmonary and hepatic inflammation. Under stress condition, p53 has been reported to activate pro-inflammatory cytokines at the site of stress induced pulmonary tissue in cigarette smoke inhaled rat [31]. However, the recruited inflammatory cells along with proteolytic enzymes act upon the anti-proteases (collagen and elastin) in alveolar region and inactivate them which in turn results in the destruction of alveolar septa and eventually develop emphysema [29]. Additional evidence has also confirmed the involvement of neutrophil in the degradation of elastin fibers with subsequent development of emphysema [32].

Emphysema in smoke inhaled animals is also accompanied by extensive thickening of alveolar septa and bronchial epithelial wall, which indicate hyperplasia (over tissue growth). Co-existence of emphysema and hyperplasia in pulmonary tissue was also observed in mice expressing TNF-α at elevated level [33]. Therefore, it can be postulated that current pulmonary injury would also due to the up-regulation of TNF-α in some extent. Because, elevated TNF-α level has been reported to deplete the total cellular glutathione (antioxidant enzyme) level in both in vitro and in vivo model [34, 35]. However, over tissue growth (TAS and TBW in Fig. 1D) in the current findings would also be due to extensive proliferation and deposition of two primary connective tissue components i,e. elastin and collagen in emphysematous areas [36]. Accumulation of inflammatory cells probably induces deposition of extracellular matrix in the sub epithelium region and contributes in thickening of bronchial epithelial wall [37]. The structure of epithelial lining of bronchi is converted from pseudo-stratified columnar ciliated epithelium into thickened constricted bronchi (CB in fig-1C and 1B), which might limit air passing through the bronchi to the peripheral region of lung- major clinical feature in patient with COPD [38]. The activation of fibroblasts by inflammatory cells and with subsequent thickening of bronchial epithelial wall and alveolar septa are considered as airway resistance and airway remodeling against ongoing destruction of alveolar septa [37].

The co-existence of necrotic and apoptotic mediated liver damage is the novel finding of this study (AC and NC in Fig. 2E-F). Necrotic characteristics were mainly found in higher duration inhaling groups (3 h and 2 h), probably due to allethrin induced toxicity on cell membranes. Necrotic cells death characteristically affects large fields of tissue (NC in figure- 2F) rather than a single cell and induce to release cytoplasmic contents into the surrounding cellular environment which leads to trigger inflammatory cells to be accumulated (NC in Fig. 2E-F).

Histological observation of hepatic tissue also reveals degeneration of hepatocytes in such a way, affecting single cell or small area of tissue without recruiting inflammatory cells: the critical feature of apoptotic cells (AC in Fig. 2E) [39]. Some hepatocytes of 1 h inhaled mice shows condensation of cytoplasm and chromatin- the key event of apoptosis mediated cell death (CCA in fig-2C). However, it is the first time study that we have observed apoptotic mediated cell death induced by the smoke of allethrin based MC. Oxidative DNA damage with subsequent up-regulation of p53 is the perquisite of mitochondria mediated apoptosis [40]. Several studies earlier reported that ROS induced DNA damage and up-regulation of p53 in MC smoke inhaled rat [15, 16]. Although, the mechanism and cellular features of apoptosis and necrosis are different, but both the phenomenon share a common a biochemical network described as “apoptosis-necrosis continuum” [41]. The interplay between apoptosis and necrosis may take place at the same time in the same tissue depending upon the types and degrees of death stimuli [42], which would also be supportive to the current dose dependent co-existence of apoptosis and necrosis in smoke inhaled tissues.

This study has some limitations relating to the conclusion of the results. Here, we stained the respiratory and hepatic tissue only with hematoxiline-eosin, though other staining technique such as DAPI staining could be done to observe apoptotic cells more precisely. In this study, we could not check the function of respiratory system such as X-ray analysis respiratory system due to lack of proper facilities. In spite of some confines, these findings would help to select proper medication for diseases induced by the mosquito coil smoke, and this study gives the proposal to modify the chemical structure of the insecticide for avoiding unexpected effects on human body.

In this study, we found the biochemical and histological effects of the mosquito coil smoke using mice model. Biochemically, inhalation of the mosquito coil smoke altered dose dependently in all of the blood parameters tested. In histological approach, the progressive destruction of lung tissue and sequential necrotic degeneration of liver tissue were newly characterized as toxicological phenomena of the mosquito coil smoke.