Apoptotic Gene Expression in Endosulfan Exposed IBD Infected Birds

Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, Bareilly; Department of Pharmacology, College of Veterinary Science and Animal Husbandry, Pandit Deen Dayal Upadhyay Veterinary and Animal Sciences University, Mathura, India; Avian Disease Section, Indian Veterinary Research Institute, Izatnagar, Bareilly; Central Instrumentation Facility, Indian Veterinary Research Institute, Izatnagar, Bareilly; Division of Animal Genetics, Indian Veterinary Research Institute, Izatnagar, Bareilly; Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar, Bareilly, *Corresponding author: deep_biotek@yahoo.com


INTRODUCTION
Endosulfan is a member of the cyclodiene group of organochlorine pesticides and is used worldwide in agriculture.The cyclodiene insecticides are among the most toxic and environmentally persistent insecticides known so far.Endosulfan is a highly toxic pesticide, and it belongs to toxicity class 1 in the Environmental Protection Agency (EPA) (ExToxNet, 2000).Endosulfan is also a known environmental endocrine disrupter (Rose et al., 1999).Organochlorine insecticides are now harshly regulated in the United States but are still in production and are used extensively in developing countries because of their effectiveness and inexpensiveness.Along with picrotoxinbinding, cyclodienes inhibit Na+, K+-ATPase, but more importantly inhibit Ca2+, Mg2+-ATPase.This leads to an accumulation of intracellular free calcium ions, which promotes the release of neurotransmitters and subsequent depolarization of adjacent neurons.Endosulfan is stable in the environment and, because of its lipophilicy, can be stored in fat tissue and released later during sickness or starvation.Endosulfan has genotoxic effects on HeoG2 cells (Yuquan et al., 2000) and has been
IBDV is a double stranded RNA virus belonging to the Avibirnavirus genus in the family Birnaviridae causes an acute disease known as Gumboro disease.This disease is characterized by high mortality and morbidity.Chickens infected with IBDV at <3 weeks of age usually have little or no clinical signs but they are immunosuppressed.IBDV can induce apoptosis in chicken peripheral blood lymphocytes (Vasconcelos andLam 1994, Jain et al., 2013), indicating that it may play a crucial role in the depletion of lymphoid cells, in apoptosis and immunosuppression.Previous studies have shown chicken embryo fibroblasts exhibited morphological and biochemical features of apoptosis when infected in vitro with IBDV (Tham et al., 1995, Kumar 2009).Infection of chicken embryos and young chickens with IBDV induced contagious disease of young chickens (Vasconcelos and Lam 1995).
There are several types of viruses pesticides contribute to oxidative stress and apoptosis.Reports also confirm the ability of endosulfan and IBDV to induce apotosis independently.But no report confirms the toxic effects of endosulfan inducing apoptosis, heat stress and other related genes of apoptotic functions in IBDV infection.The main purpose of the present research was to investigate the side effects of sub acute exposure of endosulfan in environmentally realistic conditions on IBD infected chickens, and secondarily to analyze the potential pathways involved in endosulfan-induced apoptosis in IBDV infected birds.

Animals
Day old chicks were procured from Central Avian Research Institute, Izatnagar, India.The chicks were acclimatized for seven days and maintained under controlled conditions of temperature and light (12/12-h light/dark cycle).Food and water were provided ad-libitum.After the period of acclimatization the chicks were divided into three groups (Control (Group-1), IBD infected birds (Group-2) and endosulfan intoxicated IBD infected birds (Group-3) comprising seven birds in each group.

Preparation of Pesticides
Endosulfan was purchased from Sigma Aldrich Ltd. and was mixed in feed with a rate of 30 ppm (30 mg/kg feed).The endosulfan-mixed feed were prepared just before use and fed to chicks ad-libitum.

Treatments
All the chicks were infected with the IBD IM+ strain by ocular route with 10 3.0 TCID 50 of virus on day 7. Same day chicks were exposed to 30 ppm of endosulfan-mixed feed for up to 28 days.The dose of the endosulfan in the present study was chosen based on the study of Aggarwal et al., (2003).

Sample Collection
After the exposure period of 21 days, chicks were sacrificed.Heparinized blood, bursa and the liver were collected aseptically and processed under standard protocol of RNeasy® Minikit of Qiagen for the gene expression study with single step real-time PCR and DNA content analysis using PI dye using FACS (Fluorescence-activated cell sorting) technique.

Apoptosis Detection (Flowcytometric Method Using Propidium Iodide Dye)
The experimental birds were sacrificed and bursa was collected.Bursa was minced finely with scissors, and minced tissue was washed several times in PBS.Bursal cell suspension obtained from control and IBDV infected birds were layered on Histopaque and centrifuged at 1500 r.p.m. for 10 minute.The cells at the interphase was collected, washed twice in Phosphate buffer saline and suspended in 500 µl of propidium iodide hypotonic buffer and incubated at 37 C for 15 Minuit in the dark.The PI fluorescence was measured through FL-2 filter in Becton Dickenson Flowcytometer (NJ, USA).

RESULTS AND DISCUSSION
The FACS analysis using Propedium Iodide dye showed increase in the percentage of apoptotic cells in bursal cells harvested from endosulfan fed IBDV infected birds from that of IBD alone infected and control birds.In endosulfan intoxicated IBDV infected birds the proportion of cells with a lower DNA content increased considerably, than the IBDV alone infected and control birds, indicating the profound induction of apoptosis.
Previous studies revealed that endosulfan induces oxidative stress (Dorval et al., 2003Sohn et al., 2004, Ayub et al., 2003, Dorts et al., 2009, Ballesteros et al., 2009, El-Shenawy 2010).Previous studies have indicated that certain chemical exposures can result in the alteration of secondary messengers, such as free radicals or ROS, and these alterations have been linked to the induction of apoptosis in immune cells (Corcoran et al., 1994).Free radicals influence gene expression, regulate cellular responses to cytokines, as well as proliferative events of a cell.All these events have also been implicated as possible triggering mechanisms of apoptosis.Oxidative stress may trigger the mitochondrial pathway.Increased permeability of the mitochondrial membrane during apoptosis triggers the release of cytochrome c from mitochondria in the apoptotic process.The release of cytochrome c into the cytoplasm is a critical step, which is followed by the activation of different caspases like caspase-9 and caspase-3 (Shen et al., 2004).In the present study there is significant down regulation of mitochondrial pathway genes like MCL-1 and BCL-XL was observed in both the endosulfan intoxicated IBDV infected birds (0.36±0.23 and 0.33±0.008respectively) (Figure 1) and IBDV alone infected birds and (0.11±0.0012 and 0.008±0.0012respectively) in comparison to control birds.Down regulation in of mitochondrial pathway genes were significantly more profound in the IBDV alone infected birds.This finding suggests though the roles of intrinsic pathway genes in apoptosis of hepatocytes in IBDV infected endosulfan intoxicated birds are there but it is of less magnitude in endosulfan intoxicated IBDV infected birds.This resulted in several morphological and biochemical changes leading to apoptosis of the cell (Hengartner 2000).Mitochondrial (Intrinsic) pathway was shown to get activated at different time point of virus life cycle in IBDV induced apoptosis (Kumar et al., 2010).Anti-apoptotic genes like BCL-2 and BCL-Xl acts as inhibitors of cell death and the suppression of apoptosis.BCL-2 can neutralize one another's function, suggesting that their relative concentration in the given cell may act as a rheostat for the suicide program.BCL-2 is localized on the cytoplasmic face of the mitochondrial outer membrane, endoplasmic reticulum and nuclear envelope and counter balance the oxidative damage done to these compartments and affect their behavior (Hockenberry 1995, Antonsson et al., 1997, Yang and Korsmeyer 1999).
Mitochondrial DNA is also heavily damaged by ROS at the bases.Transcription factors such as NF-kB, p53 and AP-1 are also sensitive to redox changes in mammalian cells, primarily through redox regulation of their DNA binding regions (Morel and Barouki 1999;Ho and Bray 1999;Du et al., 1999;Williams and Smith 1993;Li et al., 2013;Li et al., 2014).NF-kB is a transcription factor that has a crucial role in regulation of cell survival, protecting cells from apoptosis under most circumstances and accelerating apoptosis in others.NF-kB is activated via degradation of the inhibitor of NF-kB (IkB.In the present study, gene expression studies by real-time PCR revealed the mRNA level expression of apoptogenic genes Caspases-3 and NF-kB were found positively regulated in the endosulfan intoxicated IBDV infected birds (1.33±0.22 and 3.99±1.2respectively) and down regulated in IBD alone infected birds ( 0.93±0.0014and 0.10±0.0029respectively) in comparison to control birds.The result suggests that the NF-kB pathway and the executioner caspase of extrinsic/intrinsic pathway, caspase-3 plays the pivotal role in endosulfan induced apoptosis in IBDV infected birds.Endosulfan induces uncoupling of oxidative phosphorylation.Oxidative stress occurs under conditions when oxygen radical production is greater than the detoxification capacity of the cell (Kowaltowski andVercesi 1999, Halliwell andGutteridge 1989).Oxidative stress occurs when redox homeostasis within the cell is altered.Injury to cells occurs only when the ROS overwhelm the biochemical defenses of the cell (Sies 1986, Horton and Fairhurst 1987, Sies 1985).Reactive oxygen species, (in particular, hydroxyl radicals) can react with all biological macromolecules, i.e. lipids, proteins, nucleic acids and carbohydrates.The initial reaction generates a second radical, which reacts with a second macromolecule and oxidative damage in membrane phopholipids, pose a constant threat to cellular integrity and function.The lipid radicals generated during the early encounter with an oxidant add molecular oxygen to produce lipid dioxyl radical (Jaescheke 1995).
It appears that oxidative stress acts as a pleiotropic modulator in both these pathways.Indeed, ROS have been described as second messengers for several growth factors and cytokines.It has been shown that the transcription factors such as NF-kB and AP-1, which is stimulated by ROS, could mediate apoptotic gene expression.Under conditions of mild oxidative stress, cell cycle-related genes are repressed to increase the lengthening of G1-phase.Excess ROS production causes GSH depletion and oxidative stress.Oxidative stress leads to disruption of the mitochondrial transmembrane potential and causes release of cytochrome C and other apoptosis related proteins to cytosol and finally leads to apoptosis.In the present study increased apoptotic DNA content may be due to endosulfan-induced ROS generation and oxidative damage to the cells (Kannan et al., 2000).IBDV induces apoptosis through activation of caspases (Kumar et al., 2009).Genes like hemeoxigenase-1 (0.16±0.07),Cyclin-D1 (0.58±0.44) and HSP-70 (0.42± 0.26) were negatively regulated as compared to control birds suggest that cells prosurvival machinery is not selected till this stage of intoxication.There was significant down regulation in FAS-L (0.32±0.16) gene in endosulfan exposed IBD infected birds as compared to control and IBD infected birds revealed that the activation of Caspase-3 and increased mRNA level expression might have followed the intrinsic pathway of apoptosis by activating Caspase-8.Apoptotic pathway genes CARD, RIPIC, FAS-L, Caspase-10 down regulation shows there less significance in combined apoptosis induced by IBDV infection and sub acute endosulfan intoxication.
The gene expression, in general, is modulated by both physiological and environmental stimuli.The results of the study confirm that the experimental exposure of IBDV infected birds with endosulfan affect mitochondrial pathway, Caspase-3 (extrinsic/intrinsic pathway) and NF-κB (oxidative stress-induced) pathways of apoptosis in the liver.The study shows that the infectious disease IBD in conjugation with the environmental pesticides pollutants like endosulfan, may lead to grave prognosis of the disease and may cause more economic losses.

Figure 1 :
Figure 1: Real-time RT-PCR analysis of mRNA level of genes associated with apoptotic pathways; MCL 1; Caspase 8; Caspase 3; Fas l; Cyclin D1; Caspase 10; NFkB; Heme Oxygenase 1 gene; Heat Shock Protein 70 gene; RIPK 2; Caspase Recruitment Domain gene; BCL XL; BCL 2; Data shown are mean±SEM of [log2 (RQ) value].RQ value, which was calculated as described in material & methods, is an indicative of mRNA expression level for each gene at different time interval (d pi).a, b, c : means within each gene that are not denoted with common letter are different p < 0.05