Fundamental and Applied Toxicology
Regular ArticleTwo-Dimensional Electrophoretic Analysis of Compartment- Specific Hepatic Protein Charge Modification Induced by Thioacetamide Exposure in Rats
Abstract
Thioacetamide (TA) is a well-known hepatotoxicant. It has been reported that an obligate intermediate of TA binds to proteins with the formation of acetylimidolysine derivatives that are responsible for TA-induced hepatotoxic effects. TA has also been reported to cause chemically induced cell death via both apoptosis and necrosis. The objective of this study was 2-fold: first, to investigate the effect of TA exposure on protein charge modifications in the rat liver and second, to study the role of these molecular correlates in the regulation of cell death. Male Sprague–Dawley rats (200–225 g, 7–8 weeks old) were divided into four major groups and treated intraperitoneally with a 12-fold dose range of TA (50, 150, 300, and 600 mg TA/kg) dissolved in water. Using whole liver extracts, alterations in the hepatic protein pattern following treatment with the 12-fold dose range of TA were studied using high-resolution, two-dimensional polyacrylamide gel electrophoresis and computerized image analysis. The results indicate that charge modification was clearly evident as early as 2 hr with the lowest dose of 50 mg TA/kg. At this dose and time endoplasmic reticulum proteins, calreticulin, grp78, and ER60 exhibited acidic charge variants. The effect of TA became more prominent with dose and time. Generally the elevation of charge modification indices (CMI) by TA appeared to reach a peak between 4 and 6 hr and then while CMI either leveled off or declined in the lower two doses of 50 and 150 mg TA/kg, it continued to remain elevated with the higher doses of 300 and 600 mg TA/kg. This dichotomy in the elevation of CMI is in close correspondence to the pattern of cell death observed with a similar dose range of TA, where lower doses (50 and 150 mg TA/kg) predominantly cause cell death via apoptosis while higher doses cause cell death via necrosis. Delayed charge modification was observed with the cytosolic hsc70s with the 300 and 600 mg TA/kg treatments, indicating that the reactive metabolite(s) slowly leak out into the cytosol from the endoplasmic reticulum. There were no alterations in the mitochondrial proteins hsp60 and grp75, suggesting that TA has no effect on the mitochondrion, its effects primarily being confined to the endoplasmic reticulum. The concept of looking at these proteins as biomarkers of tissue injury has validity. These changes may be indicators of bioactivation and adduct formation and also may be signaling events in the regulation of the mode of cell death.
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The toxic effect of thioacetamide on rat liver in vitro
2010, Toxicology in VitroThioacetamide (TAA) is a hepatotoxin frequently used for experimental purposes which produces centrilobular necrosis after a single dose administration. In spite of the fact that oxidative stress seems to play a very important role in the mechanism of TAA-induced injury, the effect of TAA on hepatocytes in primary culture with respect to the influence on mitochondria has yet to be verified.
Hepatocytes were incubated for 24 h in a medium containing TAA (0–70 mmol/l). Glutathione content (GSH/GSSG), reactive oxygen species and malondialdehyde formation were assessed as markers of cell redox state. Toxicity was determined by lactate dehydrogenase leakage and WST-1 assay. The functional capacity of hepatocytes was evaluated from albumin and urea production. Mitochondrial metabolism was assessed by measuring mitochondrial membrane potential and oxygen consumption.
Our results show that a profound decrease in the GSH level in hepatocytes precedes a sharp rise in endogenous ROS production. ROS production correlates with an increase in lipoperoxidation. Mitochondria are affected by TAA secondarily as a consequence of oxidative stress. Oxidation of the NADH-dependent substrates of respiratory Complex I is significantly more sensitive to the toxic action of TAA than oxidation of the flavoprotein-dependent substrate of Complex II. Mitochondria can also maintain their membrane potential better when they utilize succinate as a respiratory substrate. It appears that GSH should be depleted below a certain critical level in order to cause a marked increase in lipid peroxidation. Mitochondrial injury can then occur and cell death develops.
Thioacetamide
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Proteomic profiling of intestinal prechylomicron transport vesicle (PCTV)-associated proteins in an animal model of insulin resistance (94 char)
2010, Journal of ProteomicsIntestinal overproduction of apolipoprotein B (apoB)-48-containing chylomicrons is increasingly recognized as an underlying factor in metabolic dyslipidemia commonly observed in insulin-resistant states. Enhanced chylomicron assembly and secretion has been documented in animal models of insulin resistance, but the underlying mechanistic factors are unknown. Chylomicron assembly occurs through a series of complex vesicular interactions involving prechylomicron transport vesicles (PCTVs), which transport lipids from the endoplasmic reticulum (ER) to the Golgi. We report proteomic profiles of PCTVs isolated from the enteric ER in the small intestine of the fructose-fed hamster, an established model of diet-induced insulin resistance. Using 2D gel electrophoresis and tandem mass spectrometry, PCTVs were characterized and proteomic profiles of PCTV-associated proteins from insulin-resistant and control enterocytes were developed, with the intention of identifying proteins involved in insulin signaling attenuation and lipoprotein overproduction. A number of PCTV-associated proteins were found to be differentially expressed including microsomal triglyceride transfer protein (MTP), apoB-48, Sar1 and VAMP7. We postulate that altered expression of Sar1 and MTP may contribute to increased chylomicron assembly in the fructose-fed hamster. These findings have increased our understanding of the intracellular assembly and transport of nascent chylomicrons and potential cellular factors responsible for lipoprotein overproduction in insulin-resistant states.
Evaluation of cultured, precision-cut rat liver slices as a model to study drug-induced liver apoptosis
2005, ToxicologyThe precision-cut liver slice culture model has been used widely to investigate drug metabolism and drug-induced necrosis. However, apoptosis, a key mediator of liver toxicity remains to be studied in this model. We evaluated apoptosis induced by thioacetamide (TAA) in rat liver slices, and in livers taken from TAA-treated rats as a control. Rat liver slices were treated with 50, 75 and 100 mM of TAA for 15 h. Histopathological examination of the liver slices revealed specific centrilobular localization of apoptotic hepatocytes at 75 mM but randomly distributed at 100 mM. Apoptosis in centrilobular hepatocytes was confirmed by appearance of cleavage products of caspase-3 and DNA fragmentation studied by TUNEL method. A concentration-dependent release of cytochrome c was observed in the slices, suggesting a role for mitochondria in the apoptosis triggered by TAA. The in vitro results were compared to the data obtained in male Sprague-Dawley rats given a single ip injection of 40 mg/kg TAA and sacrificed 1, 2, 3 and 6 h after dosing. Histopathological analyses showed specific centrilobular localization of apoptosis after 6 h treatment. Caspase-3 activation, DNA fragmentation and cytochrome c release were also observed in the liver of rats treated with TAA. Overall these data indicated that precision-cut liver slices provide a valuable in vitro system to study drug-induced liver apoptosis.
Differential signatures of protein expression in marmoset liver and thymus induced by single-dose TCDD treatment
2005, Toxicology2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an ubiquitously distributed environmental pollutant. Health effects have been studied intensively, but low-dose effects are quite complex and not yet fully understood. In many studies, the immune system was identified as the most sensitive target. Here, we demonstrate changes of protein expression in liver and thymus of male marmosets (Callithrix jacchus) which were subjected to a single dose of a subcutaneous injection of 100 ng/kg body weight TCDD. Histopathological examination revealed myocardial fibrosis, but there were no significant findings in pathology and histopathology of liver and thymus. In order to detect more subtle treatment-related changes, we performed a comparative proteomic investigation of liver and thymus using a 2-D gel electrophoresis based proteomics approach. Fluorescence labeling and automated image analysis was used to enhance sensitivity and reproducibility. In both organs, distinct changes of protein expression were detected which were more pronounced in thymus, where the pattern of deregulated proteins could be clearly related to immune responses. In the thymus of treated animals, several toxicologically relevant factors were increased, including chaperones, glycerol-3-phosphate dehydrogenase, and adseverin. Among others, vimentin, Ca-dependent protease and protein disulfide isomerase were downregulated. In the liver, transferrins, lamin A and HSP70 were upregulated, whereas thymidine phosphorylase (synonyms: endothelial cell growth factor, PD-ECGF, gliostatin) was significantly reduced. Comparative analysis of deregulated proteins in both organs revealed a pattern of related functions, which fits well into the existing knowledge of the toxic processes and mechanisms underlying TCDD-mediated toxicity.
The use of toxicokinetic and toxicodynamic data in risk assessment: An international perspective
2002, Science of the Total EnvironmentCitation Excerpt :Hence, proteome analysis as a basis for functional genomics (Jornvall and Jolles, 2000) has obviously gained interest because it permits the qualitative and quantitative ascertainment of a broad spectrum of proteins which can be related to specific functions in cell physiology. First examples in using this approach are given by studies from Anderson et al. (1995) on oltipraz, Witzmann et al. (1996) on thioacetamide, Aicher et al. (1998) cyclosporin A and Edvardsson et al. (1999) on the peroxisome proliferator, WY 14643. At present, the methodology of gene profiling has been used to demonstrate interaction of substrate with cytochrome P-450s, thus providing early information on possible metabolic pathways, which at least for drug candidates, is important information.
Risk assessment of chemicals is a process which is usually based on data derived from animal testing in which the exposure of animals results in toxicological effects. By extrapolation, the dose/exposure in humans, which will not result in toxicological effects (‘safe dose’, ‘safe exposure’), is estimated. Traditional approaches use ‘safety factors’ or ‘uncertainty factors’ to extrapolate from animal to man and from the ‘mean’ subject to the general population, including sensitive subgroups. Traditionally, a default factor of 10 has been used to account for interspecies variation. It is proposed that this factor be subdivided into a subfactor to address the toxicokinetic aspects and a second subfactor for the toxicodynamic aspects. Likewise, a default factor of 10 with subfactors is proposed to account for the intraspecies variability. In the framework of the International Program on Chemical Safety's (IPCS) project on the Harmonization of Approaches to the Assessment of Risk from Exposure to Chemicals, an activity has been initiated to provide guidance to risk assessors on the use of quantitative chemical specific data to account for interspecies variation and interindividual variability in risk assessment. To address the toxicokinetic aspects, the active species, the relevant internal exposure and the adequate metrics must be considered. Data quality and availability, in vitro or in vivo, the route of administration and the relevant dose level are relevant information for interspecies extrapolation. The availability of experimental data, including the relevance of the population studied, the number of subjects and/or samples obtained in the relevant group allow one to estimate the population distribution, e.g. difference between central tendency and given percentiles. In a similar fashion, the toxicodynamic data must be addressed. In addition to the identification of the active chemical species, the relevant endpoint must be determined. In extrapolation from animal to man, in most of the cases, the definitive endpoint (e.g. anemia) is lacking. It can be substituted by in vitro data (e.g. in vitro hemolysis) if it is a key event and relevant for the toxicity in animal as well as in humans. In extrapolating from animal to man, the dose–effect relationship plays an important role. To account for the toxicodynamic variability in the human population, similar aspects have to be taken into consideration, which have been discussed for toxicokinetics. The IPCS document is available at the IPCS website and risk assessors are invited to use the framework and report back their experience with it to enable revision and improvement.