A proteomics study of chemically induced cirrhosis in rat liver revealed the mechanism of thioacelamide hepatotoxicity 4

Chapter Discussion Chapter 4.1 General discussion Oral administration and intraperitoneal injection of TAA are both established methods in the generation of fibrosis and cirrhosis models of animals, particularly in the rats We adopted the latter, which is achieved by injecting 200-300 mg/kg of TAA to the rats for a span of to months (Chiijiwa et al., 1994; Zhao et al., 2002) TAA was chosen for our studies mainly because TAA-induced nodules in rats are more prominent than that of those induced by other chemicals and the histology of the TAA model was more akin to human liver cirrhosis (Li et al., 2002) TAA is a thiono-sulfur containing compound that is both necrogenic (Landon et al., 1986) and carcinogenic (Kizer et al., 1985) to the liver In 1984, TAA toxicity was first associated with thioacetamide S-oxide (TSO), which is derived from the biotransformation of TAA by the microsomal FAD-monooxygense (FMO) system (Chieli et al., 1984) Recently, certain isoforms of cytochrome P450 such as CYP2E1 and CYP2B (Lee et al., 2003) were reported as also being involved in the metabolic activation of TAA to its toxic metabolites During the biotransformation of TAA, both flavin-containing monooxygenase (FMO) and cytochrome P450 reduce dioxygen to superoxide anion, which is in turn, catalyzed (Ekström et al., 1989) to form hydrogen peroxide, an important reactive oxygen species (ROS) that is held responsible for cellular oxidative stress Therefore, it is not surprising to find that biotransformation of TAA is accompanied by liver injury associated with oxidative damage, as evidenced by the presence of glutathione depletion 147 Chapter (Sanz et al., 2002), an increase in malondialdehyde (MDA) (So et al., 2002) and the disappearance of tetraploid hepatocytes (Díez-Fernández et al., 1993) Nevertheless, the detailed biochemical mechanisms underlying this hepatotoxic process of TAA remain largely unknown In our attempt to unravel the pathogenesis of TAA-induced liver cirrhosis in rat liver and also to identify potential biomarkers for early detection of liver fibrosis, our 2DE-MS approach had successfully identified 59 protein spots which displayed differential expression upon TAA administration, some of which could be correlated with the chronicity of TAA administration Subsequently, these proteins were categorized according to their common functions and properties As a result, we obtained four functional clusters as shown in Table 4-1 We attempted to deduce the basis of TAAinduced toxicity related to fibrosis These deductions were then collaborated and supported by evidences available in the literature The inter-relationship of the four clusters of proteins is presented as Fig 4-11 148 Chapter Cluster Energy Metabolism Protein Long chain acyl-CoA dehydrogenase (↓) Electron transfer flavoprotein α subunit (↓) Electron transfer flavoprotein α subunit (↓) Enoyl CoA hydratase (5 isoforms) (↓) Functions β-oxidation Cluster Iron and Heme Related Protein Ferritin light chain (↓) Functions iron storage Cluster Carbonyl Metabolizing Protein Aldose reductase (↑) Functions detoxify aldehydes β-oxidation Albumin (↓) heme binding Aldose reductase-like protein (↑) detoxify aldehydes β-oxidation Haptoglobin (↓) heme binding Aldehyde dehydrogenase (↑) Hemopexin (↑) heme binding 3-α-hydroxysteroid dehydrogenase (↓) anti-oxidant, binding to lipid and DNA hydroxyperoxides hemoprotein Malate dehyrogenase (2 isoforms) (↓) Krebs Cycle Glycine N-methyl transferase (↓) methionine breakdown glutathione Stransferase Pi (↑) anti-oxidant, binding to H2O2 hemoprotein sulfite oxidase (↓) GPx selenium dependent (↓) Functions detoxification Catalase (↓) Protein detoxify aldehydes β-oxidation & branched chain amino acid breakdown Cluster Glutathione Related Table 4-1 Differentially expressed proteins with common properties and functions were categorized to four clusters Modes of regulation are included where (↓) signifies down-regulation while (↑) signifies up-regulation 149 Chapter 4.2 Cluster 1: energy metabolism Proteins categorized as the first cluster are involved in fatty acid β-oxidation, the Krebs cycle, breakdown of branched chain amino acids such as isoleucine and valine as well as breakdown of methionine 4.2.1 Fatty acid catabolism As shown in Table 3-1 in Chapter 3, long chain acyl-CoA dehydrogenase (LCAD) was down-regulated at least two folds in the treated rat liver samples from the third week of TAA treatment Fatty acids are broken down through the β-oxidation of fatty acyl-CoA (LCAD), which is a flavoenzyme responsible for the formation of a trans - α, β double bond in fatty acyl-CoA through dehydrogenation, the first committed step of fatty acid breakdown This reaction involves the removal of a proton at Cα of fatty acylCoA and transfer of a hydride ion equivalent from Cβ to FAD prosthetic group of LCAD, forming FADH2 Subsequently, electron transferring flavoprotein (ETF) transfers an electron pair from the FADH2 prosthetic group to the flavo-iron-sulfur protein ETF: ubiquinone oxidoreductase and is followed by reactions in the electron transport chain in the mitochondria Interestingly, both α and β subunits of ETF were also found to be downregulated as well since the third week of TAA treatment 150 Chapter Following the dehydrogenation of the fatty acyl-CoA to form an enoyl-CoA, enoyl-CoA hydratase catalyzes the stereo-specific addition of H2O to its substrate’s trans-α, β double bond to form 3-L-Hydroxyacyl-CoA, a secondary alcohol Coincidentally, the second enzyme in the β-oxidation pathway, short chain enoyl-CoA hydratase (3 isoforms) was down-regulated A detailed description of this pathway and the differentially expressed proteins are illustrated in Fig 4-1 151 Chapter Fatty acyl Co-A FAD ETF LCAD ETFox Electron transport chain FADH2 trans- -Enoyl-CoA H2O Enoyl-CoA hydratase 3-L-Hydroxyacyl-CoA NAD+ 3-L-hydroxyacylCoA dehydrogenase NADH + H+ β-Ketoacyl-CoA CoASH β-Ketoacyl-CoA thiolase Fatty acyl-CoA (2 C shorter) + Acetyl-CoA or Fatty acyl-CoA (2 C shorter) + Succinyl-CoA (from βoxidation of odd chain fatty acids derived from diet Figure 4-1 shows the fatty acid β-oxidation pathway Proteins labeled in red were downregulated as found in our 2DE-MS experiments It is interesting to note that the enzymes involved in the first two steps of fatty acid β-oxidation are down-regulated 152 Chapter 4.2.1.1 Cluster 1: Hypothesis derived from observations It has been well documented in the literature that the first step of fatty acyl-CoA breakdown involving acyl-CoA dehydrogenases and electron transfer flavoproteins (ETF) are crucial for survival This has been exemplified in the deficiency of acyl-CoA dehydrogenases in sudden infant death syndrome (SIDS) and Jamaican vomiting sickness, both of which can lead to death SIDS involves the Lys 304 → Glu mutation in medium chain acyl-CoA dehydrogenases (MCAD) while the second involves consumption of the poisonous ackee fruit that contain hypoglycin A, which was converted to methylenecyclopropylacetyl-CoA (MCPA-CoA), a mechanism inhibitor of acyl-CoA dehydrogenases Our results show that enzymes involved in fatty acid metabolism are downregulated Decrease in β-oxidation might lower ATP production It was also suspected that a decreased β-oxidation may lead to accumulation of fatty acids in the affected livers (Grimbert et al., 1993) (Fig 4.2) 153 Chapter TAA Administration Decrease in β-Oxidation of Lipid? Accumulation of Fatty Acids? Figure 4-2 Suppression of fatty acid β-oxidation by TAA, as implicated by the 2DE-MS data, may result in the accumulation of fatty acids in the liver, causing fatty change or steatohepatitis 154 Chapter 4.2.1.2 Supporting evidence from scientific literature This hypothesis turned out to be supported by published results Firstly, a paper by Müller and Dargel (Müller et al., 1984) showed rat livers chronically treated with TAA exhibited a decrease in fatty acid oxidation They also showed that the structural and functional alterations of mitochondria from chronically treated rats remained unaffected for at least two weeks after cessation of TAA application Besides, several early reports have also recorded fatty change in liver as a result of TAA toxicity (Smith et al., 1968; Vorbrodt et al., 1966) For instance, Zimmermann (Zimmerman, 1976) reported that TAA is moderately steatogenic while Fernandez (Fernandez et al., 1997) found that the nodular cirrhosis of TAA-treated rat livers developed increased collagen content and lipid accumulation This hypothesis and its accompanying verifications are illustrated in detail as Fig 4-3 Collectively, our results are in concert with the biochemical and toxicological observations pertaining to TAA-administered livers These results indicate that the downregulation of enzymes involved in β-oxidation might be the underlying cause for the observed TAA-induced decrease in β-oxidation and development of steatohepatitis 155 Chapter the most reactive free radicals known and they have the ability to react with a wide range of cellular constituents such as amino acids, pyrimidine residues of DNA and membrane lipids to initiate a chain of free radical reactions Interestingly, catalase and glutathione peroxidase (GPx) were both found to be down-regulated in our TAA-treated rat livers, as shown in Table 3-1 We believe that such a down-regulation is associated with TAA-induced oxidative stress since it had been shown (Van Remmen et al., 1996) that the decline in catalase and GPx expression in cultured hepatocytes was correlated with a decrease in the reduced glutathione to oxidized glutathione (GSH/GSSG) ratio and an increase in lipid peroxidation Both catalase and GPx are responsible for the breakdown of H2O2 Therefore, when these two enzymes become depleted, as is our case, intracellular H2O2 should accumulate, thus enhancing the Fenton and Haber-Weiss reactions Such a phenomenon may provide a plausible, albeit partial explanation for the increased oxidative stress, liver injury and the ensuing liver fibrosis in TAA hepatotoxicity as reported in the literature 167 Chapter T-FLC ↓ I *Free Iron? *Hemosiderin? II + OH + OH- + O2Free Radicals *H2O2 OXIDATIVE STRESS *FentonHaber-Weiss Chemistry Figure 4-9 Iron can be released from ferritin by reactive oxygen species (ROS) (Biemond P et al., 1988) Ferritin was also reported as degraded, with the release of iron, by autophagic vacuolar apparatus in macrophages during inflammation [Sakaida et al., 1990] Free iron is dangerous as it can act as a catalyst in the Fenton (Crichton et al., 2002) and Haber-Weiss (Haber et al., 1934) reactions These reactions involve hydrogen peroxide (H2O2), and thus can potentiate oxygen toxicity by the generation of a wide range of free radical species, including hydroxyl radicals (OH.) 168 Chapter 4.3.4 Summary for Cluster In Cluster 2, we showed that the declined biosynthesis of heme and cytochrome P450 in TAA-treated rat liver, as reported in several toxicological studies, could be attributed to the depletion of succinyl-CoA, as derived from Cluster Depletion of succinyl-CoA can affect ALA-synthase (Bonkowsky et al., 1977), the first and also the rate-limiting step in heme biosynthesis In fact, TAA had been shown to inhibit ALAsynthase (Matsuura et al., 1983) As a result, heme biosynthesis decreased Since heme is a positive regulator of cytochrome P450 mRNA transcription, decrease in the level of heme also spells the decline in the level of cytochrome P450 Secondly, we showed that free iron is a highly probable product of ferritin degradation, an outcome of TAA-induced oxidative stress Coupled with the decrease in catalase and GPx, accumulated H2O2 and free iron can potentially cause massive oxidative damage to liver as a result of Fenton and Haber-Weiss reactions These are illustrated in Fig 4-10 169 Chapter Succinyl-CoA *Heme Biosynthesis *cyt P450 Catalase (↓) T-FLC (↓) *Free Iron? *Hemosiderin? *H2O2 accumulates *Fenton-Haber-Weiss Chemistry OH + OH- + O 2- Figure 4-10 As a result of succinyl-CoA depletion, heme biosysnthesis and cytochrome P450 decreased Besides, a down-regulation of tissue ferritin light chain and catalase was also observed The release of free iron from T-FLC can result in Fenton-Haber Weiss chemistry and consequently, oxidative damage The down-regulation of catalase, on the other hand, can result in the accumulation of hydrogen peroxide, an agent for oxidative stress 170 Chapter 4.4 Cluster 3: metabolism of carbonyl compounds A group of interesting proteins in which all of them save one were up-regulated was found This group of proteins metabolizes carbonyl compounds such as aldoses and aldehydes These unique proteins were classified under Cluster In this cluster, aldose reductase (AR) and aldose reductase-like protein (rARLP) were up-regulated in the TAAtreated rat livers (Table 3-1) Both of them belong to the aldose reductases (AKR1B) family under the aldo-keto reductase (AKR) superfamily In addition, aldehyde dehydrogenase was also found to be up-regulated AR, a cytoplasmic NADPH-dependent enzyme that converts glucose to sorbitol, is also involved in the detoxification of harmful aldehydes (Maser, 1995) that are generated during oxidative stress such as in rat hepatoma (Takahasi et al., 1995) as a result of lipid peroxidation More specifically, AR was shown to detoxify 4- hydroxynonenal (HNE) (Srivastava et al., 2002; Spycher et al., 1996), one of the most potent aldehyde products of lipid peroxidation (Daniel et al., 1987) Hence, it is not surprising to find that over-expression of AR is also found to be induced by malondialdehyde (MDA) and HNE (Koh et al., 2000; Rittner et al., 1999), both of which are reactive products of membrane lipid peroxidation Meanwhile, rARLP was first found to be over-expressed in rat hepatomas induced by nitroso compounds as reported by Zeindl-Eberhart et al (Zeindl-Eberhart et al., 1994, 2001, 1997) In 1998, Cao and co-workers (Cao et al., 1998) described a human homolog of rARLP as being present in HCC rARLP shares a 70-90% sequence 171 Chapter identity with AR (Zeindl-Eberhart et al., 2001) and was assumed to act as a functionally active embryonic AR (Zeindl-Eberhart et al., 1997) It was suggested that rARLP could potentially serve as a marker enzyme in early hepatic neoplasia (Zeindl-Eberhart et al., 1997) Our results showed that it was indeed up-regulated consistently in our TAAtreated sample after weeks of treatment (Fig 3-18) At this stage, the histology of the rat livers appeared abnormal but was not cirrhotic The absence of cirrhotic nodules in the weeks samples can be clearly observed in our histology slides (Fig 3-1) Four isoforms of aldehyde dehydrogenases (ALDH) were up-regulated in our liver samples They are considered as general detoxifying enzymes which can eliminate toxic biogenic and xenobiotic aldehydes (Harrington et al., 1987; Jakoby et al., 1990), thus removing the highly reactive aldehyde functional groups In our 2-DE results, ALDH 1A1 was found to be up-regulated It belongs to the class ALDH which is constitutive and inducible in the liver cytoplasm of rat (Lindahl, 1992) Small aliphatic aldehydes such as acetaldehyde, propionaldehyde and MDA (Crow et al., 1974; Greenfield et al., 1977; Deitrich et al., 1966; Tottmar et al., 1973) as well as 4hydroxyalkenals such as HNE are excellent substrates for class ALDH (Lindahl, 1984) In summary, the up-regulation of these aldehyde-metabolizing enzymes, as observed in our 2-DE results, is a strong indication of the presence of increased lipid peroxides in TAA treated rats This is consistent with the findings that showed TAA administration results in oxidative damage associated liver injury 172 Chapter Lipid Peroxides Aldose Reductase (↑) Aldose Reductase-like (↑) Aldehyde dehydrogenase (↑) Figure 4-11 shows enzymes (aldose reductases and aldehyde dehydrogenase) that catalyze and detoxify lipid peroxides were up-regulated in the livers of the rats after administration of TAA 173 Chapter 4.5 Cluster 4: Glutathione-related proteins Five isoforms of GST-Pi (7-7) which were strongly up-regulated upon TAA administration (Table 3-1) were found Two isoforms (pIs 6.12 and 6.20) in particular, were up-regulated in a time-dependent manner, being most prominent at the tenth week treatment stage (Fig 3-19) This observation is in accord with a paper by Tsuchida (Tsuchida et al., 1992) who described that GST-Pi (7-7) was strongly expressed not only in hepatic foci and hepatoma, but also in initiated cells at the very early stages of chemical hepatocarcinogenesis Besides being capable of conjugating glutathione (GSH) to electrophilic compounds derived from the biotransformation of xenobiotics, GST-Pi (7-7) is capable of acting as a selenium-independent GPx towards lipid peroxides (Meyer et al., 1985) and DNA hydroxyperoxides (Tan et al., 1986) On the other hand, we detected the down-regulation of isoforms of seleniumdependent glutathione peroxidase (GPx) GPx removes H2O2 by coupling its reduction to H2O with oxidation of reduced glutathione (GSH) Besides H2O2, GPx also acts on other peroxide such as fatty acid hydroxyperoxides and synthetic hydroxyperoxides It has been well documented that ROS can down-regulate GPx (Fujii et al., 1999) In addition, decreased GPx activity was detected in rodents treated with compounds that induce GST activity (Hayes et al., 1991, 1999; McLellan et al., 1994) and this finding is consistent with our proteomic studies 174 Chapter GST-P (7-7) (↑) Lipid Peroxides and DNA hydroxyperoxides Selenium dependent GPx (↓) H2O2 Figure 4-12 shows that glutathione transferases that catalyze and detoxify lipid peroxides and DNA hydroxyperoxides were up-regulated in the livers of the treated rats after administration of TAA, indicating the presence of oxidative stress On the other hand, selenium-dependent glutathione peroxidase was down-regulated 175 Chapter 4.6 Conclusion: Integration of Clusters 1-4 Finally, the four clusters are integrated to form a bigger picture (Fig 4-12) First, TAA administration resulted in the decrease of three primary metabolic pathways that are involved in the production of succinyl-CoA (Cluster 1) As a result, heme biosynthesis was reduced and this led to a reduction in cytochrome P450 Besides, free iron was released from ferritin by reactive oxygen species, which are the products of TAA biotransformation Free iron could initiate Fenton and Haber-Weiss reactions in the presence of H2O2 to potentiate oxidative damage (Cluster 2) Oxidative damage resulted in lipid peroxidation and the production of highly reactive aldehydes That could explain our observation of an increased expression of carbonyl-metabolizing enzymes (aldose reductase and aldehyde dehydrogenase) that could react with these toxic aldehydes to detoxify them (Cluster 3) Besides, glutathione transferase 7-7, capable of reacting with lipid hydroxyperoxides, was also up-regulated (Cluster 4) In contrast, selenium- dependent glutathione peroxidase and catalase were down-regulated Both enzymes are responsible for the breakdown of H2O2 When these two enzymes become depleted, intracellular H2O2 should accumulate, thus enhancing the Fenton and Haber-Weiss reactions and resulting in more severe oxidative damage In conclusion, a detailed analysis of the global protein expression profiles of the TAA-treated rat liver proteome at different time points had enabled us to derive relevant proteomics data associated with TAA hepatotoxicity However, 2DE-MS data can only provide information about protein abundance and possible modifications, but not functional relationship between biological molecules such as proteins In this thesis, we 176 Chapter have shown for the first time that proteomics data can be readily correlated and integrated with the biochemical, clinical and physiological observations associated with TAAinduced liver toxicity and fibrosis into an “overview model” This model can now provide us and other investigators the framework for the further experimental verification of the molecular basis of TAA-induced hepatotoxicity 177 Chapter β-Oxidation 1.) LCAD 2.) enoyl-CoA hydratase 3.) ETF Cluster ROS Cluster Ile & Val breakdown enoyl-CoA hydratase Met breakdown 1.) GNMT Malate DeH Figure 4-13 An intergration of the clusters of proteins differentially-expressed gives a bigger picture of the effects of TAA on rat livers GPx selenium dependent Kreb Cycle GST-P (7-7) *Succinyl-CoA *Heme Biosynthesis Lipid Peroxides Aldose Reductase Sulfte Oxidase Cyt *P450 Macrophages ? Aldose Reductase-like protein Lipid Peroxidation Catalase T-FLC Aldehyde Dehydrogenase 3-α-hydroxysteroid dehydrogenase *Free Iron? *Hemosiderin? + *H2O2 accumulates Cluster *Fenton-Haber-Weiss Chemistry OH + OH- + O2- Cluster Activation of Hepatic Stellate Cells 178 Chapter 4.7 Future work Although 2DE-MS proteomics approach was able to provide data in the differential expression of proteins in TAA-induced liver cirrhosis in rat livers, thus suggesting some important points regarding its mechanism, I think that there are certain aspects that need further investigations Besides, there are also some experimental aspects that can be addressed in order to obtain better results First, in this experiment, the whole liver tissue is used for protein profiling instead of individual liver cell types Hepatic stellate cells undergo proliferation upon activation, therefore, the over-expression of STAP and other HSC-specific proteins might not reflect the actual level of protein over-expression but the increase in the level of protein might be due to cellular proliferation Hence, we think that it would be better if individual cell types can be isolated and then had their proteome characterized Until now, the most promising method is laser capture- micro-dissection (LCM), but the cost and time taken could be prohibitive Secondly, we realized that one of the disadvantages of 2DE is the inability to detect low-abundance proteins This is clearly reflected in our data where most of the differentially expressed proteins are metabolic enzymes that are of high level of expression There is no low-abundance proteins involved in cellular signaling detected, for instance I think that it would be more convincing if a parallel experiment using ICAT and LC-MS/MS could be performed Finally, Western-blot can also be done to verify the differential level of protein expression However, the cost for experimental 179 Chapter setup such as LC-MS/MS and raising/purchasing different antibodies are a major concern Third, we hypothesized that the depletion of succinyl-CoA could be the underlying reason behind the development of a fatty liver Although supported by proteomics data and other non-proteomics studies, we think that it is crucial to establish a biochemical assay to examine the level of succinyl-CoA in the livers treated with TAA to verify the hypothesis At the same time, another assay to investigate the relationship of succinyl-CoA and heme biosynthesis also needs to be set up in order to provide solid experimental data for the hypothesis Fourth, since free iron was proposed as one of the damaging agent in TAAinduced liver cirrhosis, it is preferable to measure the level of free iron in the cirrhotic rat livers so as to confirm that free iron level is indeed increased in the diseased livers Finally, we had identified novel proteins which were differentially expressed in TAA-treated livers They are HSCO protein, RIKEN cDNA 0610012D14 and “Similar to RIKEN cDNA 2310016A09” Further characterization of their biological function and structure would be interesting Besides, they can serve as potential biomarkers for liver cirrhosis in rat models However, validation studies are required before they can be used as biomarkers These studies will include: i.) looking into the presence of these proteins in other cirrhosis models to find out if these proteins are present in all cirrhosis and at the same time if they are specific to a class of drugs ii.) find out if these proteins are present 180 Chapter in humans as well and finally iii.) examine the site of expression of these proteins if they are secreted to body fluids 181 ... found that in vivo TAA administration to male rat had been demonstrated to inhibit δ-aminolevulinic acid (ALA) synthetase (Matsuura et al., 1983) In the same paper, Matsuura and co-workers also... vacuolar apparatus in macrophages during inflammation (Sakaida et al., 1990) Since oxidative stress and inflammation are both implicated to be associated with TAA hepatotoxicity, the release of. .. regulated in our 2-D gels, catalyzes the transfer of water to a fatty acid, can also play a major role in the metabolic breakdown of branched chain amino acids such as valine and isoleucine The