A proteomics study of chemically induced cirrhosis in rat liver revealed the mechanism of thioacelamide hepatotoxicity 3

For example, after 3 weeks of treatment, most of the experimental rats had scores of 2s to 3s at ten different fields being analyzed Table 3-1 as compared to the scores of 0s and 1s for

Chapter 3

Results

3.1 Histo-pathology of rat liver tissues

Generally, with increasing duration of TAA treatment, the fibrous content in the livers increased as compared to the controls For example, after 3 weeks of treatment, most of the experimental rats had scores of 2s to 3s at ten different fields being analyzed (Table 3-1) as compared to the scores of 0s and 1s for the corresponding controls This shows that with only 3 weeks of TAA treatment, the rat livers had developed increased deposition of collagen, but septa were still absent or incomplete in the liver samples (Figure 3-1)

However, after 6 weeks (Table 3-2 and Figure 3-2) and 10 weeks (Table 3-3 and Figure 3-3) of treatment, most of the ten fields examined in the treated livers had maximum scores of 4 This means that most of the liver at this stage had developed full- blown cirrhosis, with increase of collagen and formation of complete septa, some even with obvious nodularity (marked with “n”), as observed in Figure 3-2 and 3-3 Collectively, these results show that our approach had successfully generated fibrosis and cirrhosis in the rat livers The details of the histo-pathological slides and scores are illustrated from Table 3-1 and Figure 3-1 onwards

Table 3-1 Histo-pathological scores at ten different fields for rats treated with TAA for 3

weeks

Rat Serial No Histo-pathological scores at ten different fields for rats

after 3 weeks of TAA treatment

Figure 3-1 Tissue sections (x20) of rat livers stained with Mason Trichrome stain for

3 week control

3 week experimental

Table 3-2 Histo-pathological scores at ten different fields for rats treated with TAA for 6

weeks

Rat Serial No Histo-pathological scores at ten different fields for rats

after 6 weeks of TAA treatment

Figure 3-2 Tissue sections (x20) of rat livers stained with Mason Trichrome stain

6 week control

6 week experimental

n

Table 3-3 Histo-pathological scores at ten different fields for rats treated with TAA for

10 weeks

Rat Serial No Histo-pathological scores at ten different fields for rats

after 10 weeks of TAA treatment

10 week control

10 week experimental

n

We performed student’s t test to confirm the differences At 3 weeks, the mean fibrosis score of the control animals was 0.483 The corresponding score for the experimental animals was 2.7, which was significantly higher than its control (p <0.05)

At 6 weeks, the livers of the experimental animals were cirrhotic on macro- and microscopic examination The fibrosis score was 3.95 and was significantly higher that its control, 0.533 (p < 0.05) Similarly, 10 weeks experimental animals had gross cirrhosis The mean fibrosis score was 3.98 versus 0.25 in controls (p < 0.05) After 3 weeks of TAA treatment, there was evidence of increased fibrosis but no evidence of cirrhosis However, definite cirrhotic nodules were obvious in the 6 and 10 weeks experimental rat livers (Figures 3-2 and 3-3)

In fibrotic and cirrhotic livers, total collagen is increased up to eight fold, mainly

because of collagen type I deposits (Rojkind et al., 1982) and the primary source of

collagen is the perisinusoidal stellate cells Following fibrosis and cell deaths, hepatocellular regeneration occurs with the thickening of liver cell-plates The parenchyma expands against the constraining fibrous septa and tends to take up a spherical shape, forming nodules Hence, in cirrhosis, the nodularity of the liver is mostly the result of fibrosis dissecting the parenchyma in small uniform acinar or subacinar nodules in micronodular types and in lobular and plurilobular large non- uniform nodules in macronodular forms Regenerative nodules develop in the midst of scars but are a late phenomenon

3.2 2-DE of TAA-treated rat liver tissues

In generating the 2-DE gels, six pairs of control and treated samples were used for each time point with triplicate gels per sample Firstly, the “best gels” were visually analyzed for differentially expressed protein spots The best gel was the representative gel chosen from the triplicate gels based on good image resolution and spot sharpness and

it contained the highest number of spots To avoid ambiguity, only spots that were prominently different in the 2-DE gels were chosen Subsequently, representative gels of all the controls were collectively analyzed for consistent spots This process was repeated for the best gels of the treated samples Then protein spots that were consistent

in at least four out of six pairs were chosen and labeled on a “master gel” This methodology is illustrated in figures 3-4 and 3-5

ONLY CONSISTENT REGIONS ARE SELECTED A*

Figure 3-4 depicts the first part of our approach for image analysis First, the best gel from each sample was chosen

They were represented by A*, B* and 1*, 2* Then A* was compared visually with 1* for differentially expressed spots This was repeated for B* and 2* These comparisons were based on the following criteria: i.) Spots were of reasonable sizes and intensities ii.) Only spots that were present/absent or very high in contrast to very low intensity and to avoid ambiguity, faint or small spots were excluded Spots that were over-expressed in any one gel were labeled on that gel itself Finally, gels from the same group i.e A* and B* were compared so that differentially expressed spots that were reproducible were detected and labeled on a “Master gel”

PDQuest® software analysis

Sample A: 3 of the best duplicate gels

MASTER GEL FOR TREATED GROUP –

AS A GUIDE

Sample B: 3 of the best duplicate gels

Sample 1: 3 of the best duplicate gels

MASTER GEL FOR CONTROL GROUP –

AS A GUIDE

Sample 2: 3 of the best duplicate gels

Figure 3-5 depicts the second part of our approach for image analysis First, with the help of a “Master gel” from visual

analysis (Figure 3-4), the reproducibility of differentially expressed spots of 3 of the best gels for each sample was confirmed This was performed with PDQuest®, software for image analysis, which collectively analyzed the intensities of all the selected spots at the same time thus providing statistically-significant spot data

3.2.1 Visual analysis results

Our preliminary visual analysis yielded several regions of interest that was

further analyzed with PQQuest® later These regions are displayed in each representative

gels as the following images

3.2.1.1 Representative 2-D gels for 3 week control samples

3.2.1.2 Representative 2-D gels for TAA-treated samples (3-week)

3.2.1.3 Representative 2-D gels for 6 week control samples

3.2.1.4 Representative 2-D gels for TAA-treated samples (6-week)

Figure 3-9 shows a best experimental gel from the 6 week group

Differentially expressed protein spots of interest are circled At the right are

molecular weight markers labeled in kDa unit

3.2.1.5 Representative 2-D gels for 10 week control samples

3.2.1.6 Representative 2-D gels for TAA-treated samples (10-week)

kDa

3.2.2 PDQuest® software analysis

A further analysis of the selected spots by PDQuest® yielded normalized data called spot quantity that reflects the total intensity of a defined spot This information was calculated with the following formula:

Spot height is the peak of the Gaussian representation of the spot which is measured in optical densities (O.D.s) σx is the standard deviation of the Gaussian distribution of the spot in the direction of the x axis, and σy is the standard deviation in the direction of the y axis

These data can provide quantitative data for each spot within a group of gels and thus level of up- or down-regulation can be estimated based on the ratio of two groups of gels These data are represented as folds of changes in Table 3-4

3.3 Identification of protein spots

To identify the differentially expressed protein spots, these spots were excised and trypsinized before being analyzed by mass spectrometry The methods are described

in Chapter 2

Spot quantity = Spot height * π * σx * σy

3.3.1 Excision of spots of interest from CONTROL gels

A total of 41 spots were excised for identification These spots were regulated in the experimental animals as illustrated in Figure 3-12

down-3.3.2 Excision of spots of interest from EXPERIMENTAL gels

A total of 24 spots were excised for identification These spots were regulated in the experimental animals as illustrated in Figure 3-13

up-3.3.3 Protein spots excised from CONTROL gels

Figure 3-12 depicts a representative CONTROL gel with spots of interest labeled with numbers

These spots were subsequently excised, trypsinzied and analyzed with mass spectrometry Table 3-4 shows a list of protein identities generated from these spots

3.3.4 Protein spots excised from EXPERIMENTAL gels

Figure 3-13 depicts a representative EXPERIMENTAL gel with spots of interest labeled with

numbers These spots were subsequently excised, trypsinzied and analyzed with mass spectrometry Table 3-4 shows a list of protein identities generated from these spots

3.4 Compiled list of differentially-expressed proteins

Table 3-4 shows a compiled list of protein spots that underwent quantitative changes after TAA treatment The superscript (a.) denotes the accession numbers of proteins derived from Swiss-Prot and NCBI non-redundant databases (*) while (b.) protein coverage was calculated based on the percentage of amino acids residues covered

in a particular protein by the matched peptides (c.) Values for experimental pI and molecular weight were derived from gel images with software written in-house Finally (d.) is the ratios of differential expression derived from the normalized average optical densities, provided by PD-Quest® A “+” sign in this table indicates the up-regulation of the corresponding protein in the TAA-treated samples while a “-” sign denotes down- regulation in the TAA-treated samples “#” represents subcellular localization of protein

as predicted with PSORT (http://psort.nibb.ac.jp/) Hollow (C) indicates that the spot was present as an overloaded, hollow spot in the control gels but not in the experimental gels The reverse is true for Hollow (E) While “SAT” indicates that the spot was saturated To ensure the confidence of the “fit”, MOWSE score was assigned for each possible protein identified The MOWSE score reported by MS-Fit was based on the

scoring system described by Pappin et al (Pappin et al, 1993).

Theoretical MW/pI/

Protein Coverage b / Sequence Covered

Experimental

MW x 10 -3 /pI c / subcellular localization

3-week Diff exp d

6-week Diff exp d

10-week Diff exp d

31517/ 8.4

38%

44-56 (K)NSSVGLIQLNRPK(A) 107-115 (R)TFQDCYSGK(F) 116-125 (K)FLSHWDHITR(I) 158-178 (K)AQFGQPEILLGTIPGAGGTQR(L) 186-197 (K)SLAMEMVLTGDR(I)

212-228 (K)IFPVETLVEEAIQCAEK(I) 242-257 (K)ESVNAAFEMTLTEGNK(L) 242-260 (K)ESVNAAFEMTLTEGNKLEK(K) 262-272 (K)LFYSTFATDDR(R)

Table 3-4 Differentially-expressed proteins

No Accession No a /

Protein Name

MOWSE/

Matched Peptides/

Theoretical MW/pI/

Protein Coverage b / Sequence Covered

Experimental

MW x 10 -3 /pI c / subcellular localization

3-week Diff exp d

6-week Diff exp d

10-week Diff exp d

186-197 (K)SLAMEMVLTGDR(I) 1Met-ox 212-228 (K)IFPVETLVEEAIQCAEK(I) 242-260 (K)ESVNAAFEMTLTEGNKLEK(K) 262-272 (K)LFYSTFATDDR(R)

5 P14141/ Carbonic anhydrase III (RAT)

31.0/ 7.1 Cytoplasmic

No Significant Difference

Hollow/

1963.7+68.8

Hollow/ 1152.2+72.8

6 P14141/ Carbonic anhydrase III (RAT)

No Accession No a /

Protein Name

MOWSE/

Matched Peptides/

Theoretical MW/pI/

Protein Coverage b / Sequence Covered

Experimental

MW x 10 -3 /pI c / subcellular localization

3-week Diff exp d

6-week Diff exp d

10-week Diff exp d

269-291 (K)IVAPELYIAVGISGAIQHLAGMK (D) 302-321 (K)DPEAPIFQVADYGIVADLFK (V) 322-331 (K)VVPEMTEILK (K)

172-183 (R)LKLPAVVTADLR(L) 174-183 (K)LPAVVTADLR(L) 219-230 (K)VSVISVEEPPQR(S)

31.2/ 7.6 Mitochondrial matrix

No Significant Difference

Hollow/

2178.9+71.2

Hollow/ 816.4+85.2

No Accession No a /

Protein Name

MOWSE/

Matched Peptides/

Theoretical MW/pI/

Protein Coverage b / Sequence Covered

Experimental

MW x 10 -3 /pI c / subcellular localization

3-week Diff exp d

6-week Diff exp d

10-week Diff exp d

153-165 (R)IEQYNATQPLQQK(V) 177-193 (R)EELFQLFGYGEVVFVSK(D) 198-209 (K)HLGFRSAGEALK(G) 1PO4 250-270 (R)VVDTLGAGDTFNASVIFSLSK(G) 271-279 (K)GNSMQEALR(F)

271-279 (K)GNSMQEALR(F) 1Met-ox 280-289 (R)FGCQVAGK(K)

34.3/ 6.17 Endoplasmic

36.9/ 9.08 Mitochondrial matrix

36.9/ 9.47 Mitochondrial matrix

No Accession No a /

Protein Name

MOWSE/

Matched Peptides/

Theoretical MW/pI/

Protein Coverage b / Sequence Covered

Experimental

MW x 10 -3 /pI c / subcellular localization

3-week Diff exp d

6-week Diff exp d

10-week Diff exp d

297-315 (K)TPILLGSVAHQIYRMMCSK(G)

1Met-ox 320-330 (K)KDFSSVFQYLR(E) 321-330 (K)DFSSVFQYLR(E)

No Significant Difference

No significant

No Accession No a /

Protein Name

MOWSE/

Matched Peptides/

Theoretical MW/pI/

Protein Coverage b / Sequence Covered

Experimental

MW x 10 -3 /pI c / subcellular localization

3-week Diff exp d

6-week Diff exp d

10-week Diff exp d

22.2/ 5.88 cytoplasmic (PSORT)

113-127 (R)FSTVAGESGSADTVR(D) 113-130 (R)FSTVAGESGSADTVRDPR(G) 136-156 (K)FYTEDGNWDLVGNNTPIFFIR(D) 211-221 (R)HMNGYGSHTFK(L) 1Met-ox 251-263 (R)LAQEDPDYGLR(D)

288-301 (K)EAETFPFNPFDLTK(V) 355-363 (R)LFAYPDTHR(H) 366-382 (R)LGPNYLQIPVNCPYRAR(V) 2PO4 1Cys-am

383-388 (R)VANYQR(D) 432-444 (R)FNSANEDNVTQVR(T) 445-449 (R)TFYTK(V) 1PO4 481-492 (K)NFTDVHPDYGAR(I)

60.6/ 6.99

Hollow/

No Accession No a /

Protein Name

MOWSE/

Matched Peptides/

Theoretical MW/pI/

Protein Coverage b / Sequence Covered

Experimental

MW x 10 -3 /pI c / subcellular localization

3-week Diff exp d

6-week Diff exp d

10-week Diff exp d

432-444 (R)FNSANEDNVTQVR(T) 469-476 (K)DAQLFIQR(K) 481-492 (K)NFTDVHPDYGAR(V) 493-506 (R)VQALLDQYNSQKPK(N)

60.6/ 7.12 Peroxisomal

No Significant Difference

Hollow/

1531.4+59.8

Hollow/ 513.8+156.4

244-252 (K)NLPVEEAGR(L) 253-263 (R)LAQEDPDYGLR(D) 355-363 (R)LFAYPDTHR(H) 355-364 (R)LFAYPDTHRHR(L) 1PO4 366-380 (R)LGPNYLQIPVNCPYR(A) 383-388 (R)VANYQR(D)

432-444 (R)FNSANEDNVTQVR(T) 450-456 (K)VLNEEER(K)

No Accession No a /

Protein Name

MOWSE/

Matched Peptides/

Theoretical MW/pI/

Protein Coverage b / Sequence Covered

Experimental

MW x 10 -3 /pI c / subcellular localization

3-week Diff exp d

6-week Diff exp d

10-week Diff exp d

244-252 (K)NLPVEEAGR(L) 253-263 (R)LAQEDPDYGLR(D) 355-363 (R)LFAYPDTHR(H) 355-365 (R)LFAYPDTHRHR(L) 1PO4 366-380 (R)LGPNYLQIPVNCPYR(A) 383-388 (R)VANYQR(D)

432-444 (R)FNSANEDNVTQVR(T) 450-456 (K)VLNEEER(K)

469-476 (K)DAQLFIQR(K) 481-492 (K)NFTDVHPDYGAR(V) 493-506 (R)VQALLDQYNSQKPK(N)

27.1/ 6.89

No Accession No a /

Protein Name

MOWSE/

Matched Peptides/

Theoretical MW/pI/

Protein Coverage b / Sequence Covered

Experimental

MW x 10 -3 /pI c / subcellular localization

3-week Diff exp d

6-week Diff exp d

10-week Diff exp d

156-164 (R)NDISWNFEK (F) 165-175 (K)FLVGPDGVPVR (R)

35.6/ 6.22

534.7+39.4/ SAT