Adverse influence of β-methylcholanthrene on detoxification function of chick embryo brain glutathione S-transferases and degenerative changes of brain

© 2018 Sreenivasulu Dasari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercialShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). *Corresponding Author Sreenivasulu Dasari, Postdoctoral Fellow, Department of Biochemistry, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh, India. E-mail: dasarisreenivaasulu @ gmail.com Adverse influence of β-methylcholanthrene on detoxification function of chick embryo brain glutathione S-transferases and degenerative changes of brain


INTRODUCTION
Glutathione S-transferase (GST: EC 2.5.1.18)is an enzyme that can detoxify reactive metabolites like drugs and environmental chemicals, so that GST is regarded as toxicologically crucial enzymes (Arakawa et al., 2013).GSTs are exhibiting multiple reactions with a multitude of endogenous and exogenous substrates (Salinas and Wong, 1999).GST super gene family is an important cellular defence enzyme against endogenous and exogenous toxic chemical compounds; some of them have carcinogenic potential (Ates et al., 1995;Strange and Fryer, 1999).GSTs are contributing in the phase II biotransformation of xenobiotics and carcinogens (Voso et al., 2008).In a review, Hayes and Pulford (1995) said that the GSTs are evolved to protect cell against reactive oxygen metabolites.Li et al. (2013) observed that the GST isoforms concrete relationship with oxidative stress.GST is an oxidative stress marker, it may play protective role when free radicals are produced (Acharjee and Mahanta, 2014).GSTs can detoxify wide range hazardous substances by transferase activity and GST associated peroxidase activity (Dasari et al., 2017a).The presence of GST activity in wild birds appeared in 1968.Usually, birds are extremely mobile; it is difficult to exclude them from polluted areas.They eat contaminated food and drink contaminated water by the agricultural chemical formulas, industrial chemical pollutants etc.In birds, the GSTs which play key role to detoxify toxic agents, they act as biomarker to assess the health condition of birds (Dasari et al., 2017b).These chemical pollutants show direct effect on brain.Chick embryo brain GST (CB I & CB II ) protein biomarkers can protect the developing brain from environmental toxic chemical agents (Dasari et al., 2017c).
β-methylcholanthrene (MC) is one of the polycyclic aromatic hydrocarbon, experimental tumour promoting carcinogenic agent in biochemical research.Throughout the world, the urban environments are filled with PAH mainly by fossil fuel combustion (Perera et al., 2014).Heating systems, vehicle motors and the inadequate combustion of solid and fuel oils are usually the common sources of PAH, which consider as main reason for air pollution (O'Donnell et al., 2006).Carcinogenic, mutagenic and immunosuppressive effects of PAHs are well established (Armstrong et al., 2004).PAHs are light sensitive, resistance to heat, corrosion resistance and many exhibit physiological actions (Akyuz and Cabuk, 2010).PAHs are used as intermediaries in pharmaceuticals, agricultural products, photographic products, thermosetting plastics, lubricating materials and many other chemical products (Kaminski et al., 2008).PAHs are composed of two or more benzene rings bonded as linear, cluster or angular arrangements (Di Toro et al., 2000).Usually vegetable oils are contaminated by PAHs during technological processes that are direct fire drying, in connection to this that the combustion products come into contact with the oil seeds including oil (Speer et al., 1990).Lannerö et al. (2008) said that the tobacco smoke contains more than forty PAHs which are known or suspected carcinogens.PAHs are un-metabolized toxic compounds; some of them can bind to cellular proteins and DNA (Armstrong et al., 2004).Whatever concentrations of drugs and chemicals are usually safe to well-developed brain are more vulnerable to the developing brain (Bal-Price et al., 2010).Tilson (2000) was suggested that the non-availability of blood brain barrier (BBB) in the developing brain is one of the reasons for easy susceptibility to drugs and chemicals.That the lipophilic nature of PAH facilitate to cross placenta as well as immature blood brain barrier (BBB) and thereby target the developing brain (Liu et al., 2011;Nielsen et al., 2011).PAHs are interacting with brain tissues (Tayebati et al., 2009).
The exposure of PAHs to developing brain is very common in case of maternal cigarette smoking during pregnancy (Rodgman et al., 2000;Polanska et al., 2009).It was studied that the maternal cigarette smoking as well as alcohol consumption during pregnancy causes poor intelligence quotient (Batty et al., 2006), behavioural problems (Thapar et al., 2003) and psychiatric disorders (Grandjean and Landrigan, 2006) in offspring.Singh et al. (2013) was concluded that at early stage of neuronal development is metabolically very active than mature cells but more susceptible to PAH, so that MC may impair developing brain function.That the aryl hydrocarbon receptor (AHR) is the crucial mediator of MC toxicity which translocate from cytoplasm into nucleus (Omiecinski et al., 2010).The present study was aimed to evaluate the adverse influence of β-methylcholanthrene (MC) on detoxification function of chick embryo brain glutathione S-transferases including degenerative changes in developing brain.

Maintenance of eggs and route of chemical administration
Fertilized Babcock strain chicken eggs were obtained from poultry department, Sri Venkateswara Veterinary University, Tirupati.Eggs weighing about 50 grams were allowed for incubation at 37°C, maintained 60% humidity in incubator, and rotated them daily twice.For chemical administration, a hole was made at the blunt end of the egg on the shell where chemical was injected with a micro syringe though air sac.The inner shell membrane of egg is in direct contact with chorio-allantoic membrane.Therefore this route of administration allowed faster uptake of the chemical substance than yolk injections.During this process the survival embryos were tested using candler light.The dead eggs were discarded and survived eggs were selected for the experiments.

Administration schedule of MC
24 hours interval: On fifteenth day embryos were treated with MC in coconut oil through air sac for three days with an interval of 24 hours to a total concentration of 6 ppm at rate of 2 ppm per 10 µl of each dose.Control embryos were maintained by vehicle only.
48 hours interval: On twelfth day embryos were treated with MC in coconut oil through air sac for three days with an interval of 48 hours to a total concentration of 6 ppm at rate of 2 ppm per 10 µl of each dose.Control embryos were maintained by vehicle only.

Tissue collection and sample preparation
On 18 th day, control and MC treated embryos were collected and brain tissue was collected by pressing small head with forceps, washed them with cold 50 mM Tris HCL buffer (pH 8.0) containing 1 mM EDTA, to remove excess blood and body fluids, some of collected tissue was immediately fixed for histological study and remaining tissues was preserved at −20°C for further experimentation.
Brain tissues were slightly thawed, minced with scissors and then prepared twenty per cent homogenate with 50 mM Tris HCL buffer (pH 8.0) containing 0.25 M sucrose and PMSF by using potter Elvijhem homogenizer which is already in an ice jacket.The homogenate was subjected to filter through cheese cloth in order to remove floating lipid materials.The resultant homogenate was then centrifuged at 4°C two times by 45 minutes at 10,000 rpm.The resultant supernatant was known as cytosolic GSTs fraction and it was used for GST activity and specific activity assays.Usually, fresh tissues were used for all assays.

GST activity assay
Brain GST activity was assayed by the method of Habig et al. (1974).The reaction mixture consists 1 ml of 0.3 M phosphate buffer (pH 6.5), 30 mM CDNB 100 µl, 30 mM GSH 100 µl and enzyme 100 µl.This reaction mixture was made up to 3 ml with distilled water.An increase in absorbance was measured at 340 nm by using spectrophotometer.One unit of GST activity was defined either as formation one µ mole of 2,4 dinitrophenol-glutathione conjugate per minute or one µ mole of substrate consumed per minute.

Estimation of protein
Protein concentration was determined in both control and treated cytosolic fractions as well as in affinity purified GST sample by the method of Lowry et al. (1951).

Antisera production
That the affinity purified GST protein was used for immunization of Rabbits (Newzealand White Male, 3 months old) to produce antibodies after SDS PAGE analysis as described by Laemmli (1970).Rabbit was obtained from Dept. of physiology, Sri Venkateswara Veterinary University, Tirupati.Blood was collected from rabbit and serum was stored as control for further analysis.That the rabbit hair was removed at 4-6 sites and then 1 ml of 200 mg of affinity purified GST protein was emulsified with an equal volume of Freund's complete adjuvant and that emulsified mixture was injected subcutaneously to the rabbit at 4 to 6 sites.The booster doses were given with an interval of a week for about four to five times.Titer of antibodies was tested before the fifth dose of immunization.The last dose was given with an incomplete adjuvant.One week after the last injection, the rabbit was bled and the serum was centrifuged at 6,000 rpm by Remi centrifuge for 30 minutes at 4°C and that the collected supernatant was considered as antisera of chick embryos brain GSTs.

Electro-blotting
According to Towbin et al. (1979), electro-blotting was carried out by using Mini Blot Module (Vertical Gel System-EC 120, USA) and Nitro cellulose (NC) membrane.SDS-PAGE gel was placed for 30 min in Towbin buffer i.e. 25 mM Tris HCL (pH 8.8), 192 mM glycine and 20% methanol.The NC membrane was washed several times with distilled water and then with Towbin transfer buffer until it was equilibrated and precaution was taken that the membrane should not become dry.The blotting stack was assembled on the top of stainless steel grid cathode located in the trough of the frame stand of the Mini Blot Module, to which a small amount of transfer buffer was added.
After the assembly, and then filled with the transfer buffer (about 2.5 Lit).The red lead was connected to the anode and the black lead to the cathode, and the proteins transfer was allowed to move anions to the direction of anode.The transfer process was performed at 4°C for overnight using a constant voltage of 35 V.The NC membrane was removed and placed in the Ponceau-S stain to check the transferred of protein bands.

Immunostaining of NC membrane
Immunostaining was carried out according to the instruction manual provided with the goat anti-rabbit IgG secondary antibodies and all of the incubations were performed in a minimum of 5 ml of solutions in each step with continuous shaking at room temperature.
The electro-blotted NC membrane was incubated in the blocking solution (5% non-fat dry milk powder) in transfer buffer saline (TBS) for 30 minutes.After that, the NC membrane was incubated with chick embryo brain anti GST primary antibodies (1:2000 dilutions) in the blocking solution for 30 minutes.After hybridization with primary anti bodies, NC membrane was then washed five times with TBS (5 min each) and incubated with the secondary antibody (goat anti rabbit IgG-ALP conjugate) with the 1:5000 dilution in TBS for 5 hours.After that the membrane was washed three times (5 min each) with TBS and then the ALP conjugate color developing solution (BCIP/NBT) was added.The specific protein bands started to appear after 10-30 min.Finally, the membranes were carefully dried and the images were obtained by using a scanner connected to the computer.

Histopathological studies
According to Humason (1972), brain tissue histological examination was carried out.Brain tissues were collected from both control and treated embryos and washed them gently with physiological saline i.e. 0.9% NaCl, in order to remove blood and fat debris adheres to them.The tissues were allowed for fixation in 10% formalin until processing and then the tissues were washed under running tap water to remove the fixative.After dehydration of tissue by a graded series of alcohol the tissues allowed clear by using methyl benzoate and subjected to embed in paraffin wax.The tissue sections were cut with 6 µ thickness and then allowed for staining with haematoxylin and eosin (H & E).That the sections were mounted with Canada balsam, after complete dehydration and clear.Finally, the sections were observed under light microscope.

Statistical analysis
That all the data which related to this study and documented results were calculated from three experiments and presented as the mean ± standard deviation (SD).Student t-test was used in this study to identify the MC administered brain samples differed from the mean for the respective vehicle controls.That the differences between an experimental groups at the level of p < 0.05 were regarded as significant

Adverse effect of MC on brain GSTs
As shown in Table 2 and Figure 1A & 1B, MC treated brain sample by 24 hours interval, with the substrate CDNB, GST specific activity was increased to 2.7 fold and 4.49 fold in response to 2 ppm and 4 ppm MC respectively, but decreased to 2.53 fold in response to 6 ppm MC than 4.25 fold of control.With the substrates BSP and pNBC, GST specific activities were increased to 1.52 fold & 1.15 fold as well as 2.5 fold and 2.19 fold in response to 2 ppm and 4 ppm MC, respectively, but decreased to 1.42 fold and 0.64 fold, in response to 6 ppm MC, respectively, than 2.13 fold and 1.55 fold of control, respectively.With the substrate EPNP, GST specific activity was increased to 1.26 fold and 2.51 fold in response 2 ppm and 4 ppm MC, respectively, but decreased to 0.56 fold in response to 6 ppm MC than 1.45 fold of control.With the substrate p-NPA, GST specific activity was increased to 2.6 fold and 4.56 fold in response to 2 ppm and 4 ppm MC, but decreased to 2.13 fold in response to 6 ppm MC than 3.25 fold of control.With the substrate CHP, GST specific activity was increased to 1.01 fold and 1.46 fold in response to 2 ppm and 4 ppm MC, respectively, but decreased to 1.29 fold in response to 6 ppm MC than 2.3 fold of control.The documented values are average of three separate experiments of three samples.Mean ± standard deviation (SD).Student test (a = p < 0.05 is regarded as significant specific activity of GST).Doubling the dose 2 ppm MC per day for chick embryos by 24 hours interval.1 unit of GST activity is defined as 1 μ mole of GSH conjugate formed/min/mg protein (CDNB, BSP, pNBC, EPNP, pNPA), 1 n mole of NADPH oxidised/min/mg protein (CHP).As shown in Table 3 and Figure 2A & 2B, MC treated brain sample by 48 hours interval, with the substrate CDNB, GSTs specific activity was increased to 1.59 fold and 2.89 fold in response to 2 ppm and 4 ppm MC, respectively, but decreased to 4.62 fold in response to 6 ppm MC than 6.56 fold of control.With the substrates BSP and p-NBC, GST specific activities were increased to 1.66 fold & 1.15 fold as well as 3.73 fold and 2.07 fold in response to 2 ppm and 4 ppm MC, respectively, but decreased to 2.09 fold and 0.97 fold in response to 6 ppm MC, respectively than 3.12 fold and 1.94 fold of control, respectively.With the substrate EPNP, GST specific activity was increased 0.98 fold and 1.96 folds in response 2 ppm and 4 ppm MC, respectively, but decreased to 0.81 fold in response to 6 ppm MC than 1.83 fold of control.With the substrate p-NPA, GST specific activity was increased to 1.38 fold and 3.34 fold in response to 2 ppm and 4 ppm MC, respectively, but decreased to 3.21 fold in response to 6 ppm MC than 4.85 fold of control.With the substrate CHP, GST specific activity was increased to 1.31 fold and 2.68 fold in response to 2 ppm and 4 ppm MC, respectively, but decreased to 2.09 fold in response to 6 ppm MC than 3.3 fold of control.The documented values are average of three separate experiments of three samples.Mean ± standard deviation (SD).Student test (a = p < 0.05 is regarded as significant specific activity of GST).Doubling the dose 2 ppm MC per day for chick embryos by 48 hours interval.1 unit of GST activity is defined as 1 μ mole of GSH conjugate formed/min/mg protein (CDNB, BSP, pNBC, EPNP, pNPA), 1 n mole of NADPH oxidised/min/mg protein (CHP).

Western blot analysis
As shown in Figure 3, in twenty four hours interval, GST expression levels were significantly increased in response to 2 ppm MC and 4 ppm MC, but GST expression levels were decreased in response to 6 ppm MC.Similarly, in forty eight hours interval, GST expression levels were significantly increased in response to 2 ppm MC and 4 ppm MC, but GST expression levels were decreased in response to 6 ppm.Initially, GST expression was activated when MC entered in to cell, but excess infiltration of MC lead to deactivation of GST expression.

Developing chick embryo brain injury by MC
As shown in Figure 4, control cerebellum molecular layer, granular layer; Purkinje cell layer with in granular layer and white matter between two granular layers were noticed.As shown in Figure 5 & 6, 6ppm MC administered section were injured in both intervals.In 24 hours interval, 6 ppm MC administered brain section cerebellar layers degeneration, expansion of molecular layer towards granular layer and vacuolation were noticed as shown in Figure 5.In 48 hours interval, 6 ppm MC administered brain section severe cerebral haemorrhages and vacuolation was noticed as shown Figure 6.
In the present study, expressed brain GST isoenzymes by the administration of MC were screened by using model substrates such as CDNB, BSP, pNBC, EPNP, pNPA and CHP as shown in   ppm MC than 2.13 fold and 1.55 fold of control for 24 intervals, respectively, as well as 3.12 fold and 1.94 fold of control for 48 hours interval, respectively.As shown in Table 2 & 3 As shown in Hence forth the specific activity was decreased to 1.29 fold by 24 hours interval and 2.09 fold by 48 hours interval in response to 6 ppm MC respectively, than 2.3 fold and 3.3 fold of control, in both intervals, respectively.
Mannervik and Jensson (1982) said that the mu (μ) GST catalyse the GSH conjugation with BSP as well as CDNB.Mu (μ) class GST has highest specific activity with the substrate CDNB (Contreras-Vergara et al., 2007).Hayes and Pulford (1995) said that, mu (μ) GST has shown significant activity with pNBC and EPNP.As shown in Table 2 & 3 Mannervik et al. (1988) said that, a substrate of specific class GST can show activity with other class GST.Mu (μ) GST activity was enhanced with pNPA and EPNP than genetic variants (Kurtovic et al., 2007).Wild type alpha (α) GST activity was increased with pNPA and CHP (Zhang et al., 2012).Mclellan et al. (1992) and Mitchell et al. (1997) were said that the alpha (α) and mu (μ) GSTs are present in brain and other organs.(1997), Dasari et al. (2016).Mannervik et al. (1985) said that the alpha (α) class GST activity towards CHP, represents the selenium independent glutathione peroxidase activity.Chang et al. (1990), said that the physiological role of non-selinium GPx activity is associated with the GSTs is very interesting.Among GST enzymes that the alpha (α) can actively protect cells from lipid hydroperoxides which generated by oxidative stress (Spector et al., 2000).That the turkey alpha GSTs which have highest catalytic activity towards CDNB and CHP (Kim et al., 2011). Yang et al. (2001) said that the alpha GST can perform large part of peroxidase activity and this kind of activity is necessary part of defense mechanism of cell.As shown in Table 2 &  As shown in Table 2 it was observed that the GST activity was increased by 24 hours interval and 48 hours interval, in response to 2 ppm MC and 4 ppm MC, respectively.This may be due to increased process of detoxification.As in Figure 3, immunoblot with primary antibody that specific to chick embryo brain GSTs, showed band pattern similar to purified GST band pattern (CB I and CB II , our published work) and it was observed that the increased levels of GST expression in response to 2 ppm MC and 4 ppm MC, it may be due to increased process of detoxification.As shown in Table 2 & 3, Figure 1A & 1B, Figure 2A & 2B, it was observed that the GST activity decreased in both intervals, in response to 6 ppm.This may be due to suppression of GST by excess infiltration of MC.Similarly as shown in Figure 3, it was observed that the decreased levels of GST expression in response to 6 ppm MC in both intervals, this may be due to suppression of GST expression by MC.GST activity increased when expression of GST increased hence detoxification process increases, but GST activity decreased when expression of GST supressed because of excess accumulation toxic chemical agents (Dasari et al., 2018).In this study, we observed the similar results.Through histopathology study, as shown in Figure 4, in chick embryo control cerebellum section has three intact layers.But MC treated cerebellum sections were injured severely as shown in Figure 5 & 6, in both intervals.PAHs are shows moderate to high acute toxicity to birds and aquatic beings (Abdel-Shafy et al., 2016).PAHs interact with brain tissue (Tayebati et al., 2009).GST specific isoform variation can help in deeper view of histopathological conditions (Acharjee and Mahanta, 2014).Significant level expression of alpha GST is a signal for more complex damage (Giannini et al., 2001).Mu GSTs play a key role in neuroprotection (Dagnino-Subiabre et al., 2000).As shown in the Figure 5

CONCLUSION
GST isoenzymes expressed by the administration of MC, showed significant specific activity with the model substrates like BSP, p-NBC, p-NPA and CHP including CDNB.GSTs specific activity with substrates BSP and pNBC showed the existence of μ GST; with substrate p-NPA showed the existence of α and μ GSTs.GSTs specific activity with the substrate CHP showed the existence of GST associated peroxidase activity.Continues infiltration of MC into brain may supress the expression GST and ultimately made brain injury.Key conclusion, both specific activity and western blot studies proved that MC is a potent neurotoxic chemical compound, that supress chick embryo brain CB I and CB II ) GSTs and made brain degeneration.

CONFLICT OF INTEREST
Authors do not have any potential conflict of interest.

Fig. 1 :
Fig. 1: (A) Expression levels of brain GSTs in response to MC by 24 hours interval.(B) MC administered brain GSTs specific activity with model substrates by 24 hours interval.

Fig. 2 :
Fig. 2: (A) Expression levels of brain GSTs in response to MC by 48 hours interval.(B) MC administered brain GSTs specific activity with model substrates by 48 hours interval.

Fig. 3 :
Fig. 3: Western blot of chick embryo brain GSTs expressed by MC.As shown in Table 2 & 3, Figure 1A & 1B, Figure 2A & 2B, with the substrate CDNB, GST specific activity was increased to 2.7 fold & 4.49 fold by 24 hours interval and 1.59 fold & 2.89 fold by 48 hours interval, in response to 2 ppm MC and 4 ppm MC.Henceforth, the specific activity was decreased to 2.53 fold by 24 hours interval and 4.62 fold by 48 hours interval, in response to 6 ppm MC than 4.25 fold and 6.56 fold of control in both intervals, respectively.As shown in Table 2 & 3, Figure 1A & 1B, Figure 2A & 2B, with the substrates BSP and pNBC, GST specific activities were increased to 1.52 fold & 1.15 fold as well as 2.5 fold & 2.19 fold by 24 hours interval and 1.66 fold & 1.15 fold as well as 3.73 fold & 2.07 fold by 48 hours interval in response to 2 ppm MC and 4 ppm MC, respectively.Henceforth, the specific activity was decreased to 1.42 fold & 0.64 fold by 24 hours interval and 2.09 fold & 0.97 fold by 48 hours interval, in response to 6
, Figure 1A & 1B, Figure 2A & 2B, with the substrate EPNP, GST specific activity was increased to 1.26 fold & 2.51 fold by 24 hours interval and 0.98 fold & 1.96 fold by 48 hours interval, in response 2 ppm MC and 4 ppm MC, respectively.Henceforth, the specific activity was decreased to 0.56 fold by 24 hours interval and 0.81 fold by 48 hours interval, in response to 6 ppm MC than 1.45 fold and 1.83 fold of control in both intervals, respectively.As shown in Table 2 & 3, Figure 1A & 1B, Figure 2A & 2B, with the substrate pNPA, GST specific activity was increased to 2.6 fold & 4.56 fold by 24 hours interval and 1.38 fold & 3.34 fold by 48 hours interval, in response to 2 ppm MC and 4 ppm MC.Henceforth, the specific activity was decreased to 2.13 fold by 24 hours interval and 3.21 fold by 48 hours interval in response to 6 ppm MC, respectively than 3.25 fold and 4.85 fold of control in both intervals, respectively.

Table 1 :
GST specific activity studies procedures summary.

Table 2 :
Effect of MC on chick embryo brain GSTs by 24 hours interval.

Table 3 :
Effect of MC on chick embryo brain GSTs by 48 hours interval.
Table 2 & 3, Figure 1A & 1B, Figure 2A & 2B, with the substrate CHP, GST specific activity was increased 1.01 fold & 1.46 fold by 24 hours interval and 1.31 fold & 2.68 fold by 48 hours interval in response to 2 ppm MC and 4 ppm MC.