Two-Dimensional Electrophoretic Analysis of Compartment- Specific Hepatic Protein Charge Modification Induced by Thioacetamide Exposure in Rats

Witzmann, Frank A.; Fultz, Carla D.; Mangipudy, Raja S.; Mehendale, Harihara M.

doi:10.1006/faat.1996.0083

Cited by 29 publications

(9 citation statements)

References 20 publications

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“…This putative charge train phenomenon has also been observed among proteins targeted by other reactive metabolites [e.g., acetaminophen (34) and thioacetamide (35)], but the origins of this phenomenon are unclear. Because reactive metabolites can modify ionizable protein nucleophiles such as lysine, histidine, and even carboxylate groups, it is theoretically possible that adduction could shift the apparent pI of the targeted protein.…”

Section: Resultsmentioning

confidence: 84%

“…However, because the fractional adduction of target proteins by chemically reactive metabolic intermediates is on average quite low (e13%; see Table 1), it seems most unlikely that the charge trains observed here result from adduction of ionizable groups by reactive metabolites. Furthermore, a number of apparently "normal" proteins are also known to show charge trains on 2DE; examples include liver fatty acid binding protein (36), protein disulfide isomerase (35,37,38), Grp78 (39), and a plant lectin protein viscumin (40).…”

Section: Resultsmentioning

confidence: 99%

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A Proteomic Analysis of Bromobenzene Reactive Metabolite Targets in Rat Liver Cytosol in Vivo

Koen

Gogichaeva

Alterman

et al. 2007

Chem. Res. Toxicol.

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Metabolic activation and protein covalent binding are early and apparently obligatory events in the cytotoxicity of many simple organic chemicals including drugs and natural products. Although much has been learned about the chemistry of reactive metabolite formation and reactivity toward protein nucleophiles, progress in identifying specific protein targets for reactive metabolites of various protoxins has been much slower. We previously reported nine microsomal and three cytosolic proteins as targets for reactive metabolites of bromobenzene in rat liver. These results, and contemporary work by others, indicate that protein covalent binding is not totally random in cells. Moreover, as protein targets for other protoxins were identified, little commonality of target proteins became apparent. In the present work, we used two-dimensional gel electrophoresis to separate liver cytosolic proteins from rats treated with 14C-bromobenzene; 110 of the 836 observed spots contained measurable radioactivity that varied over a 600-fold range of adduct density. Of these 110 spots, in-gel digestion coupled with mass spectrometry identified apparently single proteins in 57 spots. A few other spots clearly contained more than one identifiable protein, and in several cases, the same protein was identified in several spots having different apparent molecular masses and/or pI. Altogether, 33 unique new protein targets for bromobenzene metabolites were identified and compared to those known for acetaminophen, naphthalene, butylated hydroxytoluene, benzene, thiobenzamide, and halothane via a target protein database available at http://tpdb.medchem.ku.edu:8080/protein_database/. With increasing numbers of target proteins becoming known, more commonality in targeting by reactive metabolites from diverse chemical agents may be seen. Such commonality may help to separate toxicologically significant covalent binding events from a background of covalent binding that is toxicologically inconsequential.

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Section: Resultsmentioning

confidence: 84%

Section: Resultsmentioning

confidence: 99%

A Proteomic Analysis of Bromobenzene Reactive Metabolite Targets in Rat Liver Cytosol in Vivo

Koen

Gogichaeva

Alterman

et al. 2007

Chem. Res. Toxicol.

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“…The expression of GRP78 is activated, and intracelluar levels of protein are increased, by cellular stresses including glucose starvation or treatment with inhibitors of protein glycosylation, dithiothreitol, metals such as Pb 2+ , or alkylating agents such as iodoacetamide, but (unlike other heat shock proteins) not by high temperature. GRP78 arises from a single-copy gene but is often observed as a set of 3-5 charge variants on 2D electro-phoresis (17,18); these forms are thought to be related to the autophosphorylation and/or ADP-ribosylation which GRP78 is known to undergo. Chemical stresses which induce GRP78 synthesis render cells more tolerant to insults such as calcium ion overload, oxidative stress, and alkylating agents, whereas preventing GRP78 synthesis sensitizes cells to these stresses (19).…”

Section: Resultsmentioning

confidence: 99%

Identification of Seven Proteins in the Endoplasmic Reticulum as Targets for Reactive Metabolites of Bromobenzene

Koen

Hanzlik

2002

Chem. Res. Toxicol.

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The hepatotoxicity of bromobenzene is strongly correlated with the covalent binding of chemically reactive metabolites to cellular proteins, but up to now relatively few hepatic protein targets of these reactive metabolites have been identified. To identify additional hepatic protein targets we injected an hepatotoxic dose of [14C]bromobenzene to phenobarbital-pretreated male Sprague-Dawley rats ip. After 4 h, their livers were removed and homogenized, and the homogenates fractionated by differential ultracentrifugation. The highest specific radiolabeling (6.1 nmol equiv 14C/mg of protein) was observed in a particulate fraction (P25) sedimented at 25000g from a 6000g supernatant fraction. Proteins in this fraction were separated by two-dimensional electrophoresis and, after transblotting, analyzed for radioactivity by phosphorimaging. More than 20 radiolabeled protein spots were observed in the blots. For 17 of these spots, peptide mass maps were obtained using in-gel digestion with trypsin, followed by MALDI-TOF mass spectrometric analysis of the resulting peptide mixtures. By searching genomic databases, the 17 sets of MS-derived peptide masses were found to match predicted tryptic fragments of just 7 proteins. Spots 1-4 matched with 78 kDa glucose regulated protein (GRP78), protein disulfide isomerase isozyme A1 (PDIA1), endoplasmic reticulum protein ERp29, and PDIA6, respectively. Spots 5 and 6, 7-11, and 12-17 presented as apparent "charge trains" of spots, each of which gave peptide mixtures closely similar to those of other spots within the train. The proteins present in these sets of spots were identified as transthyretin, serum albumin precursor and PDIA3, respectively. The possible relationship of the adduction of these proteins to the toxicological outcome is discussed.

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“…This result is similar to that obtained in a previous study by Witzmann et al . [45] These molecular chaperones can regulate protein turnover and assembly and protect cells from harmful conditions, including oxidative stress [46]. Coates et al .…”

Section: Discussionmentioning

confidence: 99%

Biological Responses to Perfluorododecanoic Acid Exposure in Rat Kidneys as Determined by Integrated Proteomic and Metabonomic Studies

et al. 2011

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BackgroundPerfluorododecanoic acid (PFDoA) is a perfluorinated carboxylic chemical (PFC) that has broad applications and distribution in the environment. While many studies have focused on hepatotoxicity, immunotoxicity, and reproductive toxicity of PFCAs, few have investigated renal toxicity.Methodology/Principal FindingsHere, we used comparative proteomic and metabonomic technologies to provide a global perspective on renal response to PFDoA. Male rats were exposed to 0, 0.05, 0.2, and 0.5 mg/kg/day of PFDoA for 110 days. After 2-D DIGE and MALDI TOF/TOF analysis, 79 differentially expressed proteins between the control and the PFDoA treated rats (0.2 and 0.5 mg-dosed groups) were successfully identified. These proteins were mainly involved in amino acid metabolism, the tricarboxylic acid cycle, gluconeogenesis, glycolysis, electron transport, and stress response. Nuclear magnetic resonance-based metabonomic analysis showed an increase in pyruvate, lactate, acetate, choline, and a variety of amino acids in the highest dose group. Furthermore, the profiles of free amino acids in the PFDoA treated groups were investigated quantitatively by high-coverage quantitative iTRAQ-LC MS/MS, which showed levels of sarcosine, asparagine, histidine, 1-methylhistidine, Ile, Leu, Val, Trp, Tyr, Phe, Cys, and Met increased markedly in the 0.5 mg dosed group, while homocitrulline, α-aminoadipic acid, β-alanine, and cystathionine decreased.Conclusion/SignificanceThese observations provide evidence that disorders in glucose and amino acid metabolism may contribute to PFDoA nephrotoxicity. Additionally, α2u globulin may play an important role in protecting the kidneys from PFDoA toxicity.

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Two-Dimensional Electrophoretic Analysis of Compartment- Specific Hepatic Protein Charge Modification Induced by Thioacetamide Exposure in Rats

Cited by 29 publications

References 20 publications

A Proteomic Analysis of Bromobenzene Reactive Metabolite Targets in Rat Liver Cytosol in Vivo

A Proteomic Analysis of Bromobenzene Reactive Metabolite Targets in Rat Liver Cytosol in Vivo

Identification of Seven Proteins in the Endoplasmic Reticulum as Targets for Reactive Metabolites of Bromobenzene

Biological Responses to Perfluorododecanoic Acid Exposure in Rat Kidneys as Determined by Integrated Proteomic and Metabonomic Studies

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