Vulnerability of Three Days Old Chick Embryos to Permethrin Induced Toxicities

| Agricultural and domestic use of permethrin, a pyrethroid insecticide, has led to intoxication of varying intensities in non target organisms including human. In present study, teratogenicity and embryotoxicity of permethrin was tested in developing chick(Gallus domesticus). Different concentrations of permethrin (0, 5, 10 and 20ppm) were prepared in sterilized distilled water. These doses were injected into eggs on 3rd day while recovery of embryos was done on 7th day of incubation. Disrupted embryos with microcephly, hydrocephaly, short neck, micromelia, amelia, micrognathia, agnathia, cataract, ectopiacordis, omphalocoel, axis distortion, anencephaly, anophthalmia, microphthalmia, phocomelia with reduced body weight and crownrump length in all dose groups were obtained. Adverse histological changes appeared in the form of disrupted and malformed visceral organs, vertebral bone and spina bifida as compared to control. The findings of this study clearly indicate that permethrin is potentially toxic to developing chicks, especially the highest concentrations used in the study. Article History Received: November 11, 2017 Revised: April 04, 2018 Accepted: April 14, 2017 Published: May 15, 2018 Authors’ Contributions SA conceived the concept, supervised and executed the research. FZ, NA and YN performed the experimental work. CA did statistical analysis and wrote the manuscript.


Introduction
S everal disorders like cancers, infertility, and above all, birth defects are on the role of indiscriminate use of pesticides (Mostafalou and Abdollahi, 2017). Permethrin, a pyrethroid insecticide is widely used domestically and agriculturally for vector control (Khan et al., 2012;Liѐnard et al., 2013). It is used as anti-lice (Clark et al., 2015) as well as house fly and cockroach control (Zhu et al., 2016). Prenatal exposure to permthrin in human is found to be associated with activation of immune activity in cord blood (Neta et al., 2011). Human body has always been at expense of the drug through skin, dermal, inhalation, and oral path (Nakamura et al., 2007;Saieva et al., 2004;Wei et al., 2013). Non-dietary exposure is reported as most important among children (Zartarian et al., 2012). The *Corresponding author Dr. Shagufta Andleeb, drshagufta@ue.edu.pk, s_andleeb@yahoo.com drastic effects of multiple concentrations of permethrin to non-target organism have been documented in various studies. It is well known for inducing mitochondrial functional impairment, altered immune system and oxidative stress in adult rats (Falcioni et al., 2010;Nasuti et al., 2008). While, early life exposure of rats showed an increase in proinflammatory cytokines and changes in heart cells, liver and many other long term defects in adult age (Carloni et al., 2012;Fedeli et al., 2013;Gabbianelli et al., 2013;Nasuti et al., 2014). A wide range of concentrations of permethrin (0.013-70ppm) is announced as gonadotoxic (Issam et al., 2011;Mostafa et al., 2016;Yuan et al., 2010;Zhang et al., 2008), while the other (2-75ppm) as neurotoxic (Farag et al., 2006;Imanishi et al., 2013;Nasuti et al., 2013) for rat as well as mice. Mutagenic potential has been triggered inhuman lymphocytes (in vitro) by 50-200ppm of permethrin (Barrueco et al., 1994). Even topical exposure of 5% permethrin in infants for the treatment of scabies has led to the metabolic acidosis (Goksugur et al., 2015). Shagufta Andleeb* 1 , Fariha Zahid 1 , Noor-ul-Ain 1 , Yousra Nisar 1 , Chaman Ara 2 Vulnerability of Three Days Old Chick Embryos to Permethrin Induced Toxicities 1 Eighty fertilized eggs of Gallus domesticus were divided equally into four groups. Considering LD50 (676ppm) announced by Anwar et al. (2004), three groups were given different concentrations of permethrin i.e. 5, 10 and 20ppm prepared in distilled water.While, fourth group (C) was normal control (given 0ppm of permethrin). On 3 rd day of incubation at 37±0.5˚C, a small surface area of the eggshell was cleaned and sterilized with help of cotton and ethanol to make a small window. Following application of 0.1 ml of each dose of permethrin, window was sealed by a drop of molten paraffin wax. Later on, eggs were incubated over a layer of cotton cushionat 37±0.5˚C at some distance from each other. Ventilators of the incubator were kept on for aeration. A water-filled beaker was kept inside to provide moisture. Eggs were rotated twice daily for proper development of the embryos. Following recovery on day 7 and subsequent fixation in Bouin's fluid, embryos were analyzed for different parameters like wet body weight, crown rump length and morphological anomalies. Histological study of embryos was done through serial sectioning (using paraffin wax) and hematoxylin and eosin staining. The slides were observed for histological study using microscope SWIFT (M4000-D) and photo-micrographed with the help of digital camera BESTSCOPE (BUC2-500C).
Ethical parameters set by ethical Committee University of the Punjab were observed during the study. The data are expressed as mean ± SEM. Statistical analysis is performed by one-way analysis of variance (ANOVA)-Tukey's multiple comparison test using Prism Graphpad 5.01 Software (San Diego, CA) to establish significant differences (p < 0.05) among groups.

Results
Embryos recovered from control group were well developed with normal body weight, size and organs. The average bodyweight, CR length of treated groups decreased significantly (p<0.05) than control (Table I). Embryos receiving 0ppm of permethrin were obtained with normal growth, while, those treated with 5 ppm of permethrin showed, hydrocephaly, micromelia, micrognathia, cataract, distorted axis (Figure 1a and b), amelia, short neck, microcephly, agnathia (absence of beak), ectopiacordis, and omphalocoel. The other dose (10ppm) induced microcephaly, ectopiacordis, short neck, microphthalmia, and phocomelia in developing embryos, while embryos treated with highest dose (20ppm) were adversely affected showing various anomalies like amelia, microphthalmia, ectopiacordis, hydrocephaly, agnathia and omphalocoel (Figure 1c).   Among these malformations, hydrocephaly, agnathia (absence of beak), amelia, cataract, microphthalmia and ectopiacordis appeared in most of the embryos (Table  II). Histological preparations of embryos from different dose groups revealed adverse effects of insecticide on de-velopment of various body parts. Spina bifida, disrupted centrum of vertebral bone and malformed visceral organs were observed in highest dose group (Figure 2).

Discussion
Reduced body weight and CR length, severe morphological as well as skeletal malformations, and disrupted viscera (Figure 1 and 2) in present study indicate the intense teratogenic potential of even lower concentrations of permethrin. Microphthalmia, agnathia and ectopiacordis appeared as the most prevalent malformation in all embryos (Table II). A comparable study in chick embryo came up with almost similar abnormalities including microphthalmia, axis distortion, spina bifida, hydrocephaly, exencephaly, ectopiacordis, gastroschisis, meningocephalocoel, with increased incidences of micromelia and micrognathia, reduced body weight and CR length in exposure toeven lower doses (0.625, 1.25, 2.5 and 5ppm) of permethrin as compared to control (Andleeb et al., 2014). Similar kind of abnormalities, including axial and appendicular skeletal structures have been observed in exposure to cypermethrin, a pyrethroid, in chick embryo too (Uggini et al., 2012). Fetal exposure to insecticides is found to induce abnormal organogenesis including skeleton (Michal et al., 1993). Permethrin may affect neurite outgrowth through interference with intracellular calcium regulation (Fergusonand Audesirk, 1990). In a study, pyrethroid exposure has been found to be associated negatively to neural and mental development of infants (Xue et al., 2013).
In another study, higher concentrations of permethrin (50, 100 and 200 ppm) have notable effects on various enzymes, glucose, glycogen, total protein and lipids, cholesterol, uric acid, DNA and RNA contents of kidney of developing chick. In addition to these pronounced enzymatic and molecular changes, tubular necrosis, small sized and distorted glomeruli with blood cell infiltration, damaged endothelium of Bowman's capsule and epithelium of glomeruli were also encountered (Anwar, 2003a). Necrosis, pyknosis, increased sinusoidal spaces along with blood cell infiltration in 16 day old as well as newly hatched young chicks were documented on receiving the drug on day zero of incubation. Such findings indicate that pre-developmental exposure is quite lethal for developing liver (Anwar, 2003b;Anwar et al., 2004;Anwar and Shakoori, 2010).
Permethrin is also a proven nephrotoxic and hepatotoxic in rat (Patrick-Iwuanyanwu and Charles, 2014). Significant decrease in weight of brain was observed in chick embryos as compared to heart and liver when treated with 5ppm permethrin (Alhifi, 2010). This teratogenic attribute of permethrin is may be by interfering the functioning of detoxifying enzymes (Ezeji et al., 2012).
Insufficient comprehensive study on toxicity of permethrin in chick embryo, entertains probable assumptions for chick embryo regarding histopathological toxicities induced by permethrin, accomplished by peeping into toxic effects induced in other animals. Testicular damage, decrease in testis weight and serum testosterone concentrations in mice have been manifested owing to 25, 50 and 100ppm of permethrin ( Jin et al., 2012). Disrupted architecture, diminished count of mature sperm cells, wider luminal diameter, reduced interstitial spaces in testis (Omotoso et al., 2014), and necrosis in addition to apoptosis in kidney has been characterized in rats in consequence of treatment with permethrin (Guvenc et al., 2013). Dose-dependent effects have been reported in splenic as well as thymic cellularity among mice (Prater et al., 2002). Such deformities and toxicities are debatable for humans too. Various biomarkers of permethrin like trans-and cis-3-(2,2-dichlorovinyl)-2, 2-dimethylcyclopropane-l-carboxylic acid metabolites (trans-DCCA, cis-DCCA), and 3-phenoxybenzoic acid (3-PBA) have been detected in urine even through inhalation (Ferland et al., 2015) and once inside the blood circulation, it may cross the placenta and target various organs of fetus (Borkhardt et al., 2003). Moreover, hardly fifty percent is eliminated after 84hrs of oral administration of very small amount , and may cause neurodegeneration leading to Par-kinson's disease (Nasuti et al., 2017).
Various mechanisms of toxocities of pyrethroids have been presented (Ray and Fry, 2006;Soderlund, 2012). Oxidative stress is the mechanism of action of many developmental toxicants (Hansen, 2006) including pyrethroids (Zepeda-Arce et al., 2017) and embryo is the most vulnerable to oxidative stress (Dennery, 2007). Moreover, electron transfer, unfolded protein response, apoptosis leading to disrupted endocrine functioning and central nervous system, DNA attack, enzyme inhibition, interference with hormonal action, proteins, and mitochondria, and unavailability of ATP, GTP, and detoxifying enzymes are other possible mechanisms exploited by insecticides as well as pesticides to disrupt the normal development (Kovacic and Somanathan, 2006;Kupsco and Schlenk, 2015;Ngoula et al., 2012;Zhang et al., 2017). Unfolded protein response (UPR) mediators and signaling proteins respond efficiently to toxicants and also play important role in prenatal development (Cornejo et al., 2013). Endoplasmic reticulum stress due to UPR is well known to induce apoptosis in cells in response to reactive oxygen species (ROS) generated by toxicants (Gong et al., 2017).

Conclusions
It is concluded from the present study that permethrin is highly toxic and teratogenic, as it has been found to cause severe skeletal and visceral abnormalities in chick embryo. Therefore, misuse of the insecticide and associated health risks is challenging for the government and health department to educate the workers involved in its formulation and handling, and create awareness among household as well as agricultural users regarding dose application and precautionary measurements.