Biomedical subjects

Terrence J Monks

Publications and source records attributed to Terrence J Monks.

24 records · Page 2Linked to original sources

PubMed2003

An integrated approach to identifying chemically induced posttranslational modifications using comparative MALDI-MS and targeted HPLC-ESI-MS/MS.

Identification of multiple and novel posttranslational modifications remains a major challenge in proteomics. The present approach uses comparative analysis by matrix-assisted laser/desorption ionization (MALDI) MS of proteolytic digests from control and treated proteins to target differences due to modifications, without initial assumption as to type or residue localization. Differences between modified and unmodified digest MS spectra highlight peptides of interest for subsequent tandem mass spectrometry (MS/MS) analysis. Targeted HPLC-electrospray ionization (ESI)-MS/MS is then used to fragment peptides, and manual de novo sequencing is used to determine the amino acid sequence and type of modification. This strategy for identifying posttranslational modifications in an unbiased manner is particularly useful for finding modifications produced by exogenous chemicals. Successful characterization of chemically induced posttranslational modifications and novel chemical adducts is given as an example of the use of this strategy. Histone H4 from butyrate-treated LLC-PK1 cells is separated on a gel into bands representing different overall charge state. Bands are analyzed by comparative MALDI-MS and LC-MS/MS to identify the sites of methylation and acetylation. Previous attempts to identify chemically adducted proteins in vivo have been unsuccessful in part due to a lack of understanding of the final adduct form. Cytochrome c is adducted in vitro with benzoquinone, an electrophilic metabolite of benzene capable of interacting with nucleophilic sites within proteins. De novo sequencing identifies a novel cyclized diquinone adduct species as the major reaction product, targeting Lys and His residues at two specific locations on the protein surface. This unpredicted reaction product is characterized using our unbiased methods for detection and demonstrates the important influence of protein structure on chemical adduction.

Acetylation↗

PubMed2003

Reduced constitutive 8-oxoguanine-DNA glycosylase expression and impaired induction following oxidative DNA damage in the tuberin deficient Eker rat.

The Tsc-2 tumor suppressor gene encodes the protein tuberin, a multi-functional protein with sequence homology to the GTPase activating protein (GAP) for Rap1. Mutations in the Tsc-2 gene are associated with the development of renal tumors. The Eker rat (Tsc-2(EK/+)) bears a mutation in one allele of the Tsc-2 gene, which predisposes these animals to renal cancer. Treatment of wild-type (Tsc-2(+/+)) and mutant (Tsc-2(EK/+)) Eker rats with 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ; 7.5 micro mol/kg. i.v.), a potent redox active and nephrotoxic metabolite of hydroquinone increases the incidence of renal tumors only in animals carrying the mutant Tsc-2(EK/+) allele. We now show that the constitutive expression of 8-oxoguanine-DNA glycosylase (OGG1) in Tsc-2(EK/+) rats is three-fold lower than in wild-type Tsc-2(+/+) rats. Moreover, treatment of wild-type and mutant Eker rats with TGHQ greatly increases 8-oxo-deoxyguanosine (8-oxo-dG) levels within the outer stripe of the outer medulla. Tsc-2(EK/+) rats, with lower constitutive renal OGG1 expression, experience substantially higher levels of 8-oxo-dG than do wild type Tsc-2(+/+) rats. Interestingly, whereas OGG1 expression was rapidly (4 h) induced in Tsc-2(+/+) rats following exposure to TGHQ, it was significantly reduced in Tsc-2(EK/+) rats. The combination of the higher constitutive expression of OGG1 in Tsc-2(+/+) rats, and its rapid induction in response to TGHQ treatment, coupled to the initial decrease in OGG1 expression in Tsc-2(EK/+) rats, results in Tsc-2(EK/+) OGG1 protein levels just 5% of those seen in Tsc-2(+/+) rats 8 h after treatment. Coincidentally, 8-oxo-dG levels in Tsc-2(+/+) rats 8 h after treatment with TGHQ are just 5% of those that occur in Tsc-2(EK/+) rats. The results indicate that the Tsc-2 gene influences constitutive OGG1 expression and the ability of OGG1 to respond to an oxidative stress, consistent with the proposal that Tsc-2 is an acute-phase response gene. In keeping with this view, acute TGHQ-induced cytotoxicity was greater in Tsc-2(EK/+) rats than in Tsc-2(+/+) rats. The mechanism(s) coupling tuberin expression to the regulation of OGG1 are not known and are under investigation.

Animals↗

PubMed2002

Role of metabolites in MDMA (ecstasy)-induced nephrotoxicity: an in vitro study using rat and human renal proximal tubular cells.

The metabolism of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) has recently been implicated in the mechanisms underlying ecstasy-induced neurotoxicity and hepatotoxicity. However, its potential role in ecstasy-induced kidney toxicity has yet to be investigated. Thus, primary cultures of rat and human renal proximal tubular cells (PTCs) were used to investigate the cytotoxicity induced by MDMA and its metabolites methylenedioxyamphetamine (MDA), alpha-methyldopamine (alpha-MeDA), and the glutathione (GSH) conjugates 5-(glutathion- S-yl)-alpha-MeDA and 2,5- bis(glutathion- S-yl)-alpha-MeDA. Cell viability was evaluated using the mitochondrial MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. MDMA and MDA were not found to be toxic to either rat or human PTCs at any concentration tested (100-800 micro M). In contrast, 800 micro M alpha-MeDA caused 60% and 40% cell death in rat and human PTCs, respectively. Conjugation of alpha-MeDA with GSH resulted in the formation of even more potent nephrotoxicants. Thus, exposure of rat and human PTC monolayers to 400 micro M 5-(glutathion- S-yl)-alpha-MeDA caused approximately 80% and 70% cell death, respectively. 5-(Glutathion- S-yl)-alpha-MeDA (400 micro M) was more toxic than 2,5- bis(glutathion- S-yl)-alpha-MeDA to rat renal PTCs but equally potent in human renal PTCs. Pre-incubation of rat PTCs with either acivicin, an inhibitor of gamma-glutamyl transpeptidase (gamma-GT), or bestatin, an inhibitor of aminopeptidase M, resulted in increased toxicity of 5-(glutathion- S-yl)-alpha-MeDA but had no effect on 2,5- bis(glutathion- S-yl)-alpha-MeDA-mediated cytotoxicity. The present data provide evidence that metabolism is required for the expression of MDMA-induced renal toxicity in vitro. In addition, metabolism of 5-(glutathion- S-yl)-alpha-MeDA by gamma-GT and aminopeptidase M to the corresponding cystein- S-yl-glycine and/or cystein- S-yl conjugates is likely to be associated with detoxication of this compound. Thus, it appears that toxicity induced by thioether metabolites of ecstasy at the apical membrane of renal proximal tubular cells is the result of extracellular events, presumably redox cycling.

Animals↗

PubMed2002

Mitogen-activated protein kinases contribute to reactive oxygen species-induced cell death in renal proximal tubule epithelial cells.

Extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNK/SAPK), and p38 mitogen-activated protein kinase (MAPK) were all rapidly activated in a ROS-dependent manner during 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ)-mediated oxidative stress and oncotic cell death in renal proximal tubule epithelial cells (LLC-PK1). TGHQ-induced phosphorylation of ERK1/2 and JNK MAPKs required epidermal growth factor receptor (EGFR) activation, whereas p38 MAPK activation was EGFR independent. In contrast to their established roles in cell survival, TGHQ-activated ERK1/2 and p38 MAPK (but not JNK) appear to contribute to cell death, since inhibition of ERK1/2 or p38 MAPKs with PD098059 or SB202190 respectively, attenuated TGHQ-mediated cell death. TGHQ increased AP-1 and NFkappaB DNA-binding activity, but whereas pharmacological inhibition of ERK1/2 or p38 MAPKs attenuated AP-1 DNA binding activity, it potentiated TGHQ-mediated NFkappaB activation. Consistent with a role for NFkappaB activation in the cytoprotective response to ROS in renal epithelial cells, an anti-NFkappaB peptide SN50 suppressed the protective effects of ERK inhibition (PD098059 treatment). The data provide evidence that the activation of MAPKs by ROS in renal epithelial cells plays an important role in oncotic cell death, and NF-kB is involved in the cytoprotective effects of PD098059.

Animals↗

PubMed2002

Cell proliferation is insufficient, but loss of tuberin is necessary, for chemically induced nephrocarcinogenicity.

Although 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ; 2.5 micromol/kg ip) markedly increased cell proliferation within the outer stripe of the outer medulla (OSOM) of the kidney in both wild-type (Tsc2(+/+)) and mutant Eker rats (Tsc2(EK/+)), only TGHQ-treated Tsc2(EK/+) rats developed renal tumors, indicating that cell proliferation per se was not sufficient for tumor development. Tuberin expression was initially induced within the OSOM after TGHQ treatment but was lost within TGHQ-induced renal tumors. High extracellular signal-regulated kinase (ERK) activity occurred in the OSOM of Tsc2(EK/+) rats at 4 mo and in TGHQ-induced renal tumors. Cyclin D1 was also highly expressed in TGHQ-induced renal tumors. Reexpression of Tsc2 in tuberin-negative cells decreased ERK activity, consistent with the growth-suppressive effects of this tumor suppressor gene. Thus 1) stimulation of cell proliferation after toxicant insult is insufficient for tumor formation; 2) tuberin induction after acute tissue injury suggests that Tsc2 is an acute-phase response gene, limiting the proliferative response after injury; and 3) loss of Tsc2 gene function is associated with cell cycle deregulation.

Animals↗

PubMed2002

The metabolism and toxicity of quinones, quinonimines, quinone methides, and quinone-thioethers.

Quinones are ubiquitous in nature and constitute an important class of naturally occurring compounds found in plants, fungi and bacteria. Human exposure to quinones therefore occurs via the diet, but also clinically or via airborne pollutants. For example, the quinones of polycyclic aromatic hydrocarbons are prevalent as environmental contaminants and provide a major source of current human exposure to quinones. The inevitable human exposure to quinones, and the inherent reactivity of quinones, has stimulated substantial research on the chemistry and toxicology of these compounds. From a toxicological perspective, quinones possess two principal chemical properties that confer their reactivity in biological systems. Quinones are oxidants and electrophiles, and the relative contribution of these properties to quinone toxicity is influenced by chemical structure, in particular substituent effects. Modification to the quinone nucleus also influences quinone metabolism. This review will therefore focus on the differences in structure and metabolism of quinones, and how such differences influence quinone toxicology. Specific examples will be discussed to illustrate the diverse manner by which quinones interact with biological systems to initiate and propagate a toxic response.

Alkylation↗