Biochemical study and gene expression of Glutathione-S-Transferase (GST)in induced asthma in Rat

Introduction

Glutathione S-transferases (GST) is a family of enzymes that play an of detoxification enzymes.  All eukaryotic specie has multiple GSTisoenzyme that are bound by cytosolic and membranes (1). The role of glutathione S-transferases as a defense against potentially toxic compounds. Glutathione-S-transferases (GSTs) are enzymes that catalyze nucleophilic attacks by glutathione on compounds containing electrophilic centers (2). GST is present as soluble and membrane bound forms and it’s found in highest concentration in the liver,kidney, lung and intestine. Human GSTs are divided into three main families: cytosolic, mitochondrial, and microsomal. cytosolic GSTs are subdivided into seven distinct classes designated ,named as Alpha (α), Mu (μ), Pi (π), Sigma (σ), Theta (θ), Zeta (ξ), Omega (ω) and  Kappa (κ) ⁽⁵⁾.  Three classes Alpha, mu and pi are involved in drug metabolism and detoxification (3, 4). Four additional classes of this super family, called Beta (β), Delta (δ), Phi (φ), and Tau (τ) are represented in bacteria, insects and plants ⁽⁶⁾. In glutathione redox cycle cycle, most of the enzymes catalyze the conjugation of reduced Glutathione (GSH) with compounds that include an electrophilic center through the formation of a thioether bond between the sulphur atom of GSH and the substrate ⁽⁷⁾.

Asthma is characterized by chronic airway inflammation and oxidative stress in the lungs has been implicated in its pathogenesis. Most of ROS and other free radicals are generated as by-products of normal cellular metabolic activities.Firstly in the mitochondria or during the enzymatic oxidation. ROS have long been recognized as important mediators of inflammation and cell death. Oxygen free radicals are highly reactive molecules result in oxidative damage of biological molecules such as, lipids, and DNA and proteins and alter their functions⁽⁸⁾. The shift in the balance between oxidants and antioxidants in favor of oxidants is termed oxidative stress. These are countered by enzymatic and non-enzymatic antioxidants, including dietary antioxidants, such as vitamins C, glutathione (GSH), phenolic compounds, alkaloids, non-protein amino acids and a-tocopherols⁽⁹⁾. A family of enzymes, the glutathione-S-transferases(GSTs), has the general function of conjugating glutathione with electrophilic substances that are capable of generating free radicals, thus leading to detoxification of their effects. Genetic polymorphisms associated with reduced activity of GSTs are therefore of interest in the study of disease susceptibility. Two common deletion polymorphisms of GSTM1 and GSTT1 genes have been associated with asthma⁽¹⁰⁾

Experimental design

Sixty malesratsused in this study,the average weight of rats were (140 ± 10) gm and (8-9) week age were divided into four groups. Every group contained 15 rats. First group received distilled water daily for three weeks then injected with (0.5ml) normal saline one dose on 1^st, 3rd, 5th day as a control. Second  group  received distilled water  for three weeks  then was injected subcutaneous  with three doses of 100µg  of  egg  albumin on 1^st,3^rd,5^th day . Third  group induced  asthma by injected  of 100µg  of egg   albumin on 1^st,3^rd,5^th day and then  received  local propolis  for three weeks  at doses 200mg /kg.The last group received local propolis at doses 200mg /kg for three weeks, then injected subcutaneous with three doses of 100µg of egg albumin on 1^st, 3rd, 5th day.

Materials and methods

Biochemical Tests

A–Determination of Serum Reduced Glutathione (GSH) level⁽¹¹⁾

The disulfide of 5, 5-Dithiobis (2-nitrobenzoic acid) (DTNB) is readily reduced by sulfahydryl group of GSH to an intensely yellow compound .That reduced Compound measured absorbance   at 412 nm and is directly proportional to the GSH concentration, by using UV-VIS Spectrophotometer(APLEPD-303 UV).

B–Determination of Serum Nitric Oxide Level⁽¹²⁾

A reaction between nitrite and sulfanilamide and N-(1-naphthyl) ethylene diamine produced a pink colored complex which is measured by UV-VIS Spectrophotometer(APLEPD-303 UV)at 540 nm wave length.

C-Assament activity of serum GlutathioneS-Transferase(GST) ⁽¹³⁾

The activity was determined by using 1-chloro-2, 4-dinitrobenzene (CDNB) as Substrate,The conjugation of (CDNB) with GSH proceeds by nucleophilic aromatic substitution of chlorine by thiol group of GSH, producing a dinitrophenylthioether and chloride ion, measured absorbance at 340nm by using UV-Visible spectrophotometer(SHIMADZU UV-1650PC).

Enzyme partially purified–2

A. Purification of Glutathione-s-Transferase(GST) from Rat liver.

All steps were performed at 4 ^oC unless stated otherwise.

Step 1: Preparation of Cytosol Fractions

Rat liver (2 g) was cut into small pieces and homogenized in 10 mM ice-cold Tris-acid pH 8.0 in a blender. The homogenate was filtrated through 2 layers of gauze to remove connective tissue.    The homogenate was centrifuged at 5,000 rpm for one hour. The resulting supernatant was then centrifuged for 30 minute. The cytosol fraction was obtained, and stored at -20 Cᵒ ⁽¹³⁾.

Step 2: Ammonium Sulfate Fractionation

Protein was precipitated using ammonium sulfate (NH4)₂SO₄ saturation (50%) or protein salting out ⁽¹⁴⁾. Solid ammonium sulfate (1.45) gm per (5 ml) supernatant was added with stirring to the enzyme extract and the mixture was stirred until all of the ammonium sulfate had dissolved; the suspension was centrifuged for (30) min. The protein in precipitate and supernatant are determined using the modified Lowry method ⁽¹⁵⁾, and the GST is determined in each fraction ⁽¹³⁾

Step 3: Dialysis

Dialysis was made using a semipermeable cellophane dialysis membrane with M.wt. cut off (<10000) dalton.The dialysis containing the suspension in (Step 2) was dialyzed against 0.015M phosphate buffer, pH 6.25.The solution was stirred with a magnetic stirrer overnight at (4) oC. The buffer was changed twice during dialysis ⁽¹⁶⁾. The protein of the dialyzed enzyme was estimated by modified Lowry method ⁽¹⁵⁾.

Step 4: Gel Filtration Chromatography

This step was done in column dimension of 2 × 100 cm containing gel using Sephadex G-75 to high (80 cm) ⁽¹⁶⁾. 0.5 ml of the protein, which was obtained in (Step 3), was applied on the top of a bed Sephadex G-75, followed by phosphate buffer solution. Elution of protein was carried out at a flow rate (52.3) ml / hour.

Step 5: Freeze-Dryer (Lyophilization) Technique

The enzyme fraction which was obtained from gel filtration was lyophilized to obtain a powder of a concentrated protein. The enzyme was kept in a deep freeze at (-20) C⁰ in a tight sample tube to be used in further investigations.

B- Kinetic properties of purified glutathione-S-transferase enzyme

Different concentration of CDNB (2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 mM) was reacted with 0.001 gm/ 1 ml of purified enzyme in 1 ml phosphate buffer, and then read the absorbance of reaction at 340 nm every minute for 10 minute. K_m and V_max were calculated by the line Weaver-Burk plot equation.

Gene expression of Glutathione-S-Transferrase (GST)

Primers

The primers were used in this study, GAPDH gene primers used as Housekeeping gene, GSTM1 gene and GAPDH geneprimers used as target genes. These primers were designed by using NCBI- Gene Bank data base and Primer 3 design online, the primers used in quantification of gene expression using qRT-PCR techniques based SYBER Green DNA binding dye, and supplied from (Bioneer, Korea) company.

Table1: GSTM 1 Primer, GAPDH Primer

 

Quantitative Reverse Transcription Real-Time PCR

Quantitative Reveres Transcription Real-Time PCR technique was performed for estimation of relative quantification (gene expression analysis). This technique was done according to method described by ⁽¹⁷⁾.

Results and discussion

Biochemical Analysis

A- Glutathione (GSH) level

The  asthmatic  rats (G2)  showed significant decline  in the mean of serum GSH level as  compared with serum samples in control group(G1)(P <0.05) .The oral administration of EEP and then induced asthma in (G3)  caused  a significant elevation in GSH as compared with results found in asthmatic group(G2) (P <0.05).Animal’ sadministration with EEP before induced asthma in (G4) showed almost the same concentration in GSH level as compared with control group as shown table (2).

Table2: Comparison between the control group (G1) and the three treatments on both alone to indicate any treatments more effect of GSH

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