A proteomics study of chemically induced cirrhosis in rat liver revealed the mechanism of thioacelamide hepatotoxicity 2

2.4.1 Preparation of lysis buffer Lysis buffer is prepared as the following: Table 2-2 Composition of lysis buffer for 2-D gels Buffer components Amount/Volume added Concentration... B

Chapter 2 Materials and Methods

Ethylenediaminetetraacetic acid (EDTA)

Phenylmethyl sulfonyl fluoride (PMSF)

Thioacetamide (TAA)

Trifluoroacetic acid (TFA)

All common laboratory chemicals

Bio-Rad Laboratories (U.S.A.)

40% acrylamide/ bis solution in 37.5: 1 ratio

Ammonium persulfate (APS)

Dithiothreitol (DTT)

EDTA disodium dihydrate

Fluka Chemie (Switzerland)

Modified porcine trypsin

PIERCE (Rockford, Bedford, MA, USA)

Coomassie® Plus Protein Assay

2.2 Chemical induction of liver fibrosis

2.2.1 Experimental animals

8-9 weeks old male Wistar-Furth rats were purchased from Sembawang Animal Centre, Singapore They were housed separately in an animal room on a 12 h light-dark cycle with free access to food and water These rats were kept for a week for acclimatization prior to thioacetamide (TAA) administration

To prepare a 10% TAA solution, 10 g of TAA was weighed and dissolved in 100

ml of autoclaved de-ionized water The container was wrapped up with aluminum foil and stored at 4ºC TAA crystals reformed during storage so just before injection, TAA was redissolved by stirring with a magnetic stirrer

TAA was administered to the rats via intraperitoneal injection Each rat was weighed before being injected with 300 mg/kg of TAA using the formula below: if the rat weighs N kg, then 10% TAA to inject is 3 x N ml Each experimental rat was injected for 3 times per week, using a 27G 1/2 needle with at least a day’s rest in between injections The rats were divided into 3 groups; each group was treated for 3, 6 and 10 weeks respectively The paired controlled animal received no treatment

2.2.4 Sacrifice of animals

After the final injection, the rats were left untreated for a week to clear the circulating TAA from their systems before sacrifice During sacrifice, the rats were anesthetized with ether in a hood before surgery

2.2.5 Surgical removal of liver tissue

During sacrifice, a consistent lobe of liver was surgically removed, sliced into small pieces, stored in 1 ml cryo-vials and immediately snapped-frozen in liquid nitrogen Then, the tissues were transferred to -152ºC freezer for storage

Following fixation, the liver was embedded in paraffin and sectioned 4-µm sections were mounted on slides and stained with Masson Trichrome stain The tissue sections were examined blind by a trained liver pathologist Each section was graded under light microscopy according to the amount of positively stained collagen The degree of liver fibrosis was graded according to a modified scoring system below as

reported by Ruwart et al (Ruwart et al, 1989)

sections with little or no nodularity, each field was examined at x20 magnifications Each animal would have a total minimum score of 0 or maximum score of 4

Table 2-1 The scoring scheme used in the assessment of liver histopathology Score Description

3 Definite increase with incomplete septa (those septa which do not

interconnect with each other so as to divide the parenchyma into separate fragments)

4 Definite increase with complete septa but thin septa (those septa

which interconnect with each other so as to divide the parenchyma into separate fragments)

2.4 Preparation of liver tissue lysate for 2DE

Liver tissues were kept in liquid nitrogen upon retrieval from the freezer They were ground with a pestle and mortar in liquid nitrogen until finely powdered For safety purpose, the pounding was performed in a class II cell culture hood with full laminar flow ventilation Subsequently, lysis buffer consisting of 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 40 mM tris(hydroxymethyl aminomethane (Tris), 1 mM PMSF (Sigma), 0.5 mg/ml DNase, and 0.5 mg/ml RNase was then added to the powder and the grinding continued until the buffer mixed well with the tissue in powder form

Finally, the mixture was left to thaw Tissue lysate was then carefully taken up with a pipette into an Eppendorf tube and subjected to ultracentrifuge for 1 h at 100, 000

x g The resulting supernatant was retrieved and kept in -80ºC deep freezer

2.4.1 Preparation of lysis buffer

Lysis buffer is prepared as the following:

Table 2-2 Composition of lysis buffer for 2-D gels

Buffer components Amount/Volume added Concentration

2.6 Two-dimensional gel electrophoresis (2-DE)

2.6.1 First dimension - isoelectric focusing (IEF)

2.6.1.1 Rehydration of dehydrated IPG strips

For rehydration, precast 18 cm long, pH 3-10 non-linear, dry immobilized pH gradient strips (IPGs) were immersed overnight in rehydration buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 20 mM DTT and 5% IPG buffer (pH 3-10)

This process was first performed by pipetting 340 µL of the rehydration buffer into the ceramic strip holders Then, the protective plastic layer of the IPG gel was removed and the IPG gel was placed onto the strip holder with the gel-coated surface facing down, so that the gel surface was in contact with the rehydration buffer To prevent uneven rehydration, trapped air bubbles were smoothened carefully with a clean forceps This is followed by covering the gel with a layer of paraffin oil and a plastic cover so as to prevent exposure of the rehydration buffer to air This is important because crystallation of the highly concentrated urea might occur due to evaporation of the buffer Rehydration should take at least 10 h and it is usually performed overnight Rehydration is an important step because it ensures that the dehydrated strips were rehydrated so that they are able to adsorb solubilized protein samples during isoelectric focusing (IEF)

2.6.1.2 Preparation of rehydration buffer

Rehydration buffer was prepared according to the Composition below Aliquots

of 1 ml each were transferred to an Eppendorf tube and stored at -20°C

Buffer components Amount/Volume added Concentration

Table 2-3 Composition of rehydration buffer for rehydration of IPG strips

2.6.1.3 Isoelectric focusing (IEF)

All IEF experiments were performed with Amersham Pharmacia IPGphor machines IPG gel rehydrated overnight was first rinsed by being immersed into a clean cylinder of purified water The strip was then blotted dry with a piece of clean filter paper and transferred to a universal strip holder with the gel-coated surface facing up This is followed by placing a sample loading cup carefully near the acidic end of the IPG gel 20

µl of rehydration buffer was carefully pipetted into the loading cup to test for any leakages, and was discarded later after ensuring that the cup sat properly in the strip holder

100 µg of protein sample in lysis buffer was made up to 20 µl with rehydration buffer and was then pipetted carefully into the empty loading cup Two pieces of filter paper wetted with reversed osmosis water were placed at both the acidic and basic ends

of the strips These two pieces of wet papers were then covered with electrodes A layer

of paraffin oil was laid on the gel strip to prevent evaporation of buffer due to high voltages This was followed by placing the plastic cover on top of the setup Finally, IEF was conducted with the following steps:

Table 2-4 Voltage gradient applied for isoelectric focusing

Steps Voltages (V) Mode Volt-Hours (V-hr)

Upon completion, the IPG strips were recovered, kept in a Pyrex glass tube and stored immediately in a -80°C

2.6.2 Second dimension

2.6.2.1 Equilibration of IPG strips with SDS equilibration buffer

Before being transferred to the second dimensional SDS gel, it is necessary to equilibrate the IPG strips in 2 steps The first step involved reduction of the disulphide bonds Gel strips were placed in an equilibration buffer consisting of 6 M urea, 30% glycerol, 2% SDS, 50 mM Tris-HCl (pH 6.8) and 1% (w/v) DTT and shaken slowly on a rocker for 15 minutes This equilibration step is required because it reduced the disulphide bonds and conditions the IPG strips with the SDS buffer system required for the second dimensional separation

This was followed by the alkylation step whereby the equilibration buffer was discarded and replenished with a fresh batch of equilibration buffer made of 6 M urea, 30% glycerol, 2% SDS, 50 mM Tris-HCl (pH 8.8), and 2.5 % (w/v) iodoacetamide (IAA) This step allowed the reduced free thiol groups of cysteines to be alkylated so as

to prevent reoxidation This additional step replaced the reductant with iodoacetamide

2.6.2.2 Preparation of SDS equilibration buffers

The equilibration buffer was prepared as follows:

Table 2-5 Composition of buffer used for equilibration of IPG strips

Stock Solutions pH 6.8 buffer

Amount/volume

pH 8.8 buffer Amount/volume

Final Concentration

2.6.2.3 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SDS-PAGE is a rapid method for protein separation, quantification, and characterization SDS, being an ionic detergent, binds to proteins at a uniform negative charge density and denatures them Thus, SDS-bound polypeptide is separated in the gel according to its molecular weight alone SDS-PAGE with the tris-glycine system (Laemlli, 1970) was chosen as the second dimension for its unsurpassed resolution and ease of use 10% T SDS-PAGE gels were cast as the following composition:

Table 2-6 Composition of SDS-PAGE used for second dimensional separation

Stock solution Volume (ml)

40% acrylamide/ bis solution in 37.5: 1 ratio 50.0

2.6.2.4 Preparation of sample loading buffer

Protein samples need to be solubilized and denatured before SDS-PAGE This was accomplished by dissolving protein samples in the sample loading buffer 2x sample loading buffer was used for liquid sample and was prepared as the table below Aliquots

of 1 ml each were transferred to Eppendorf tubes and stored in freezer at -20°C

Table 2-7 Composition of sample loading buffer for SDS-PAGE

2.6.2.5 Preparation of electrode buffer

Tris-glycine buffer was the electrode buffer used in our SDS-PAGE experiments

It was prepared as follows:

Table 2-8 Composition of electrode buffer used for SDS-PAGE

Ingredients Amount/Volume

2.6.2.6 Molecular weight standards

Broad range standard molecular weight markers (Bio-Rad) used included rabbit

skeletal muscle myosin (200 000 Da), E Coli beta galactosidase (116, 250 Da), rabbit

muscle phosphorylase b (97 000 Da), bovine serum albumin (66 200 Da), hen egg white ovalbumin (45 000 Da), bovine carbonic anhydrase (31 000 Da), soybean trypsin inhibitor (21 500 Da) and hen egg white lysozyme (14 400 Da)

2.6.2.7 Treatment of protein molecular weight markers

Molecular weight markers were mixed with 2x SDS sample loading buffer at a ratio of 1:1 and then heated at 95°C for 5 min prior to application About 5 µl of this mixture was dispensed carefully onto a piece paper application strip before being transferred to the second dimension

2.6.2.8 Transfer of IPG strip to the second dimension

After SDS equilibration, the IPG strip was rinsed in purified water and blotted dry before being transferred to the second dimension by gently laying the strip on top of a SDS-PAGE gel Protein molecular weight markers loaded on a piece of application strip was inserted at one end of the strip This was followed by gently dispensing 0.75% agarose to seal the IPG strip and the paper strip to the second dimension

2.6.2.9 SDS-PAGE running conditions

The second dimensional gel was performed using a Bio-rad Protean IIxi gel set with the power pack set at a constant current of 30 mA per gel and the system was cooled

to 10°C with a Pharmacia LKB – MultiTemp II cool water circulator

2.7 Image visualization and analysis

To detect and visualize any protein spots, the 2-D gels were stained with silver After gel electrophoresis, the transparent gels were recovered and immediately kept in fixing solution prior to staining to visualize protein spots Silver staining was performed

according to Shenchenko et al (Shenchenko et al., 1996) Silver stain can detect up to

nanogram of proteins and hence is suitable for staining 2-D gels

2-D gels were first immersed in fixing solution for 30 min before washing with 50% methanol in water for another 30 min Then, the gels were washed again with water for 60 min followed by sensitization with 0.02% sodium thiosulphate for 2 min After the gels were rinsed with water twice for 2 min each, they were incubated with chilled 0.1% w/v silver nitrate for 40 min at 4°C After discarding the silver nitrate and rinsing with 2 changes of water for 1 min each, the gels were developed in 0.04% formalin (35% formaldehyde in water) in 2% sodium bicarbonate When the desired image intensity was attained, the developing solution was discarded and the gel incubated with 1.46% EDTA disodium dihydrate for 10 min to stop the development The silver stain procedure was completed with 3 rinses of water for 5 min each

2.7.2 Preparation of silver stain reagents

Reagents for silver stain were prepared as follows:

Table 2-9 Composition of reagents used in silver stain

Solutions Ingredients Composition

Developing Solution formalin 0.04%

2.7.3 Image acquisition and analysis

Stained gels were scanned using a Molecular Dynamics Personal Densitometer

SI (Sunnyvale, CA, USA) at 1200 dots per inch (dpi) Images were saved and archived as gel format for subsequent image analysis

Analysis of 2-D gel images for the detection of differentially expressed proteins was performed in 2 steps First, a visual inspection of the representative gels (controls compared to the treated samples) was performed to identify protein spots that were markedly different in their expression levels This process is also called the “eye-balling” approach Then, a more detailed analysis of all the triplicates gels were performed using the image analysis software, PDQuest (Bio-Rad)

2.7.3.1 Eye-balling approach

In this analysis, visual inspection of the representative gels was performed The representative gel is the “best gel” chosen from the triplicate gels to represent a liver sample It was chosen based on good image resolution, spot sharpness and highest number of spots For comparison, the best gel of a control sample was paired up with its respective treated counterpart to identify the proteins spot with markedly different intensities

To avoid ambiguity, only spots that were present/absent or prominently different

in intensities were chosen Subsequently, all the control representative gels were collectively analyzed for spots that were consistent among the same controls This process was repeated for all the best gels of the treated samples Labeled spots that were present at least in 4 out of 6 pairs of the representative gels are chosen and labeled on a

“Master Gel”