S-transferase Isozymes Research Articles

Toxicogenomics of bromobenzene hepatotoxicity: a combined transcriptomics and proteomics approach

Toxicogenomics is a novel approach integrating the expression analysis of thousands of genes (transcriptomics) or proteins (proteomics) with classical methods in toxicology. Effects at the molecular level are related to pathophysiological changes of the organisms, enabling detailed comparison of mechanisms and early detection and prediction of toxicity. This report addresses the value of the combined use of transcriptomics and proteomics technologies in toxicology. Acute hepatotoxicity was induced in rats by bromobenzene administration resulting in depleted glutathione levels and reduced average body weights, 24 hr after dosage. These physiological symptoms coincided with many changes of hepatic mRNA and protein content. Gene induction confirmed involvement of glutathione- S-transferase isozymes and epoxide hydrolase in bromobenzene metabolism and identified many genes possibly relevant in bromobenzene toxicity. Observed glutathione depletion coincided with induction of the key enzyme in glutathione biosynthesis, γ-glutamylcysteine synthetase. Oxidative stress was apparent from strong upregulation of heme oxygenase, peroxiredoxin 1 and other genes. Bromobenzene-induced protein degradation was suggested from two-dimensional gel electrophoresis, upregulated mRNA levels for proteasome subunits and lysosomal cathepsin L, whereas also genes were upregulated with a role in protein synthesis. Both protein and gene expression profiles from treated rats were clearly distinct from controls as shown by principal component analysis, and several proteins found to significantly change upon bromobenzene treatment were identified by mass spectrometry. A modest overlap in results from proteomics and transcriptomics was found. This work indicates that transcriptomics and proteomics technologies are complementary to each other and provide new possibilities in molecular toxicology.

Specificity of murine glutathione S-transferase isozymes in the glutathione conjugation of (-)-anti- and (+)-syn-stereoisomers of benzo[g]chrysene 11,12-diol 13,14-epoxide.

Specificities of murine glutathione (GSH) S-transferase (GST) isozymes mGSTA1-1, mGSTA2-2, mGSTA3-3 and mGSTA4-4 (alpha class), mGSTP1-1 (pi class) and mGSTM1-1 (mu class) for GSH conjugation of (-)-anti- and (+)-syn-stereoisomers of benzo[g]chrysene 11, 12-diol 13,14-epoxide (B[g]CDE), the activated metabolites of the environmental pollutant benzo[g]chrysene (B[g]C), have been determined. When GST activity was determined as a function of varying (-)-anti- or (+)-syn-B[g]CDE concentration (10-320 microM) at a fixed saturating concentration of GSH (2 mM), each isozyme obeyed Michaelis-Menten kinetics. mGSTA1-1 was significantly more efficient than other murine GSTs in the GSH conjugation of not only (-)-anti-stereoisomer but also (+)-syn-B[g]CDE. For example, the catalytic efficiency (k(cat)/K(m)) of mGSTA1-1 towards (-)-anti-B[g]CDE was approximately 2.3- to 16.6-fold higher compared with other murine GSTs. Likewise, mGSTA1-1 was approximately 2.7-, 6.7-, 4.4- and 12.4-fold more efficient than mGSTA2-2, mGSTA3-3, mGSTP1-1 and mGSTM1-1, respectively, in catalyzing the GSH conjugation of (+)-syn-B[g]CDE. Interestingly, mGSTA4-4, which also belongs to class alpha, was virtually inactive towards both stereoisomers of B[g]CDE. The results of the present study indicate that murine GSTs, especially alpha class isozymes, significantly differ in their ability to detoxify B[g]CDE stereoisomers and that mGSTA1-1 plays a major role in the detoxification of both (-)-anti- and (+)-syn-B[g]CDE, which among four B[g]CDE stereoisomers are formed from the carcinogen B[g]C as major DNA binding metabolites.

Molecular analysis and mapping of two genes encoding maize glutathione S-transferases (GST I and GST II).

Maize glutathione S-transferase (GST) isozymes are encoded by a gene family comprising at least five genes, three of which (Gst I, II and III) have recently been isolated and sequenced. The enzymes are active as homo or heterodimers and exhibit intraspecific polymorphism including a "null" variant for the two major isoforms expressed in roots. Northern blot analyses performed on total root RNA from "null" and "plus" genotypes, using Gst I- and Gst II-specific probes, indicated that the Gst I gene controls the expression of the two major GST isoforms expressed in roots. Gst I and Gst II were mapped by RFLP analysis using an F2 population of 149 individuals previously characterized. Gst I was localized on the long arm of chromosome 8, while two putative Gst II loci were mapped to chromosome 8 (70 cM from Gst I) and 10, respectively.

P-220 Enhancing effects of ursodeoxycholic acid (UDCA) on glutathione S-transferase (GST) enzyme activities and isozyme concentrations in mouse liver cytosol

P-220 Enhancing effects of ursodeoxycholic acid (UDCA) on glutathione S-transferase (GST) enzyme activities and isozyme concentrations in mouse liver cytosol

Effects of altered calcium homeostasis on the expression of glutathione S- transferase isozymes in primary cultured rat hepatocytes

The effects of altered Ca 2+ homeostasis on glutathione S-transferase (GST) isozyme expression in cultured primary rat hepatocytes were examined. Isolated hepatocytes were cultured on Vitrogen substratum in serum-free modified Chee's essential medium and treated with Ca 2+ ionophore A23187 at 120 hr post-plating. GST activity increased slightly, albeit significantly, in a concentration-dependent manner in A23187-treated hepatocytes relative to untreated controls. Western blot analysis using GST class α and μ specific antibodies showed an ∼1.6-and 1.5-fold increase in the class α, Ya and Yc subunits, respectively, whereas no significant increase (∼1.2-fold) in class μ GST expression was observed following A23187 treatment. Northern blot analysis revealed an ∼5-fold increase in GST class α and an ∼7-fold increase in class μ GST mRNA levels in ionophore-treated hepatocytes compared to untreated cells. Results of the Western and Northern blot analyses of the ionophore-treated hepatocytes were compared with those obtained for tert-butyl hydroperoxide-treated cells. Inununoblot analysis showed a significant increase in the expression of GST class α, Ya and Yc subunits, ∼1.8- and 1.7-fold, respectively, for tert-butyl hydroperoxide-treated hepatocytes as compared to controls, with little or no increase in class μ GSTs. Northern blot analysis showed ∼3- and 2-fold increases, respectively, in class α and μ GST mRNA levels, following the tert-butyl hydroperoxide treatment. The results of the present investigation show that alterations in Ca 2+ homeostasis produced by either Ca 2+ ionophore A23187 or tert-butyl hydroperoxide treatment of hepatocytes enhanced the expression of GST isozymes in primary cultured rat hepatocytes.

A single step method for the separation of rat liver cytosolic glutathione S-transferase isozymes.

A simple, single step chromatographic method was developed to separate the liver cytosolic glutathione S-transferase (GSH-S-t) isozymes from each other and from the bulk of the cytosolic protein. Five peaks of GSH-S-t activity, tested with 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate, were eluted. By comparison of the activities with CDNB and the other substrate 3,4-dichloronitrobenzene (DCNB) the five peaks could be identified as GSH-S-t isozymes C, B, A, and AA, being GSH-S-t isozyme C eluted in two different peaks. The method was used to detect a decrease of specific GSH-S-t isozymes in the cytosol of rats intoxicated with carbon tetrachloride, as compared with control rats.