Protein expression profiling of glutathione S-transferase pi null mice as a strategy to identify potential markers of resistance to paracetamol-induced toxicity in the liver (original) (raw)
Related papers
1989
The role of the glutathione peroxidase/reductase (GSH-Px/GSSG-Rd) enzyme system in protection from paracetamol toxicity was investigated in isolated mouse hepatocytes in primary culture. The effect of inhibitors of these enzymes on the toxicity of paracetamol and on t-butylhydroperoxide (r-BOOH), used as a positive control, was determined. 1,3-Bis(chloroethyl)-1-nitrosourea (BCNU) was used to inhibit GSSG-Rd, and goldthioglucose (GTG) used to inhibit GSH-Px. Both these inhibitors increased cell membrane damage in response to oxidative stress initiated by t-BOOH. However, they also increased the susceptibility of hepatocytes to paracetarnol toxicity, indicating that a component of paracetamol's toxic effect involves formation of species that are detoxified by the GSH-Px/GSSG-Rd enzymes. To further examine the role of these enzymes, age-related differences in their activity were exploited. Hepatocytes from two-week-old mice were less susceptible to both j-BOOH and paracetamol toxicity than were those from adult mice. This corresponds to higher activity of cytosolic GSH-Px/ GSSG-Rd in this age group. However, after inhibition of GSSG-Rd with BCNU, hepatocytes from these postnatal mice were more susceptible to paracetamol toxicity. This suggests that the higher activity of GSH-Px/GSSG-Rd in hepatocytes from two-week-old mice is responsible for their reduced susceptibility to paracetamol toxicity. The data indicate that the GSH-Px/GSSG-Rd enzymes contribute to protection from paracetamol toxicity and suggest that formation of peroxides contributes to this drug's hepatotoxic effects.
PLoS ONE, 2011
Background: Glutathione S-transferases (GSTs) metabolize drugs and xenobiotics. Yet despite high protein sequence homology, expression of p-class GSTs, the most abundant of the enzymes, varies significantly between species. In mouse liver, hepatocytes exhibit high mGstp expression, while in human liver, hepatocytes contain little or no hGSTP1 mRNA or hGSTP1 protein. p-class GSTs are known to be critical determinants of liver responses to drugs and toxins: when treated with high doses of acetaminophen, mGstp1/2+/+ mice suffer marked liver damage, while mGstp1/22/2 mice escape liver injury. Methodology/Principal Findings: To more faithfully model the contribution of p-class GSTs to human liver toxicology, we introduced hGSTP1, with its exons, introns, and flanking sequences, into the germline of mice carrying disrupted mGstp genes. In the resultant hGSTP1+mGstp1/22/2 strain, p-class GSTs were regulated differently than in wild-type mice. In the liver, enzyme expression was restricted to bile duct cells, Kupffer cells, macrophages, and endothelial cells, reminiscent of human liver, while in the prostate, enzyme production was limited to basal epithelial cells, reminiscent of human prostate. The human patterns of hGSTP1 transgene regulation were accompanied by human patterns of DNA methylation, with bisulfite genomic sequencing revealing establishment of an unmethylated CpG island sequence encompassing the gene promoter. Unlike wild-type or mGstp1/22/2 mice, when hGSTP1+mGstp1/22/2 mice were overdosed with acetaminophen, liver tissues showed limited centrilobular necrosis, suggesting that p-class GSTs may be critical determinants of toxininduced hepatocyte injury even when not expressed by hepatocytes. Conclusions: By recapitulating human p-class GST expression, hGSTP1+mGstp1/22/2 mice may better model human drug and xenobiotic toxicology.
PROTEOMICS, 2008
Chemically reactive metabolites (CRMs) are thought to be responsible for a number of adverse drug reactions through modification of critical proteins. Methods that defined the chemistry of protein modification at an early stage would provide invaluable tools for drug safety assessment. Here, human GST pi (GSTP) was exploited as a model target protein to determine the chemical, biochemical and functional consequences of exposure to the hepatotoxic CRM of paracetamol (APAP), N-acetyl-p-benzoquinoneimine (NAPQI). Site-specific, dose-dependent modification of Cys47 in native and His-tagged GSTP was revealed by MS, and correlated with inhibition of glutathione (GSH) conjugating activity. In addition, the adaptation of iTRAQ labelling technology to define precisely the quantitative relationship between covalent modification and protein function is described. Multiple reaction monitoring (MRM)-MS of GSTP allowed high sensitivity detection of modified peptides at physiological levels of exposure. Finally, a bioengineered mutant cytochrome P450 with a broad spectrum of substrate specificities was used in an in vitro reaction system to bioactivate APAP: in this model, GSTP trapped the CRM and exhibited both reduced enzyme activity and site-specific modification of the protein. These studies provide the foundation for the development of novel test systems to predict the toxicological potential of CRMs produced by new therapeutic agents.
Role of α Class Glutathione S-Transferases as Antioxidant Enzymes in Rodent Tissues
Toxicology and Applied Pharmacology, 2002
Glutathione S-transferases (GST) are multifunctional proteins. α class GSTs are known to catalyze glutathione peroxidase reactions, in addition to their major activity, i.e., conjugation of electrophiles to glutathione. In the present work, the contribution of rat and mouse α class GSTs to glutathione-dependent reduction of phospholipid hydroperoxides has been studied., Results of these studies indicate that the α class GST fraction, which consists of three isoforms, has glutathione peroxidase activity toward phospholipid hydroperoxides residing in biological membranes, without the need of prior phospholipase C action. Immunotitration studies using antibodies specific to the α class GSTs, GSTA1-1, GSTA2-2, and GSTA3-3, indicate that these GST isozymes account for approximately half of the glutathione peroxidase activity toward phospholipid hydroperoxides present in the 28,000g supernatant fractions of rat and mouse liver extracts. GSTs contribute proportionally lesser fraction of this activity in other tissues in which α class GSTs are less prevalent. In mice, the contribution of α class GSTs to the overall glutathione peroxidase activity is indistinguishable in wild-type mice and knockout mice lacking the major selenoenzyme, glutathione peroxidase 1, an enzyme that does not act on intact phospholipid hydroperoxides. These results are consistent with our previous studies on human α class GSTs (Yang, et al. J. Biol. Chem. 276, 19220–19230, 2001) and demonstrate that α class GSTs are of physiological importance, not only in the conjugative detoxification of electrophiles, but are also an essential component of cellular antioxidant defense mechanisms.
Archives of Biochemistry and Biophysics, 1999
In order to elucidate the protective role of glutathione S-transferases (GSTs) against oxidative stress, we have investigated the kinetic properties of the human ␣-class GSTs, hGSTA1-1 and hGSTA2-2, toward physiologically relevant hydroperoxides and have studied the role of these enzymes in glutathione (GSH)-dependent reduction of these hydroperoxides in human liver. We have cloned hGSTA1-1 and hGSTA2-2 from a human lung cDNA library and expressed both in Escherichia coli. Both isozymes had remarkably high peroxidase activity toward fatty acid hydroperoxides, phospholipid hydroperoxides, and cumene hydroperoxide. In general, the activity of hGSTA2-2 was higher than that of hGSTA1-1 toward these substrates. For example, the catalytic efficiency (k cat /K m) of hGSTA1-1 for phosphatidylcholine (PC) hydroperoxide and phosphatidylethanolamine (PE) hydroperoxide was found to be 181.3 and 199.6 s ؊1 mM ؊1 , respectively, while the catalytic efficiency of hGSTA2-2 for PC-hydroperoxide and PE-hydroperoxide was 317.5 and 353 s ؊1 mM ؊1 , respectively. Immunotitration studies with human liver extracts showed that the antibodies against human ␣-class GSTs immunoprecipitated about 55 and 75% of glutathione peroxidase (GPx) activity of human liver toward PC-hydroperoxide and cumene hydroperoxide, respectively. GPx activity was not immunoprecipitated by the same antibodies from human erythrocyte hemolysates. These results show that the ␣-class GSTs contribute a major portion of GPx activity toward lipid hydroperoxides in human liver. Our results also suggest that GSTs may be involved in the reduction of 5-hydroperoxyeicosatetrae-noic acid, an important intermediate in the 5-lipoxygenase pathway.
Selective expression of the three classes of glutathione S-transferase isoenzymes in mouse tissues
Toxicology and Applied Pharmacology, 1990
The suitability of mouse as an animal model for studying the glutathione Stransferase (GST)-mediated detoxification mechanisms has been studied by analyzing the expression of the OL, /J, and T classes of glutathione S-transferase isoenzymes in mouse brain, heart, kidney, spleen, liver, and muscle. Individual isoenzymes from each of these tissues have been purified, characterized. and classified into the three known classes of GST. These studies demonstrate that GST isoenzymes are variably expressed in different mouse tissues, suggesting that their expression is tissue specific. A major isoenzyme, belonging to the r class, with a pZ value in the range of 8.6-9.1 and an approximate subunit M, value of 22,500 was detected in each tissue investigated in this study. A variable number of p class isoenzymes with subunit M, values of 26,500 were expressed in all mouse tissues studied, except spleen and muscle. Only liver and kidney showed the expression of an 01 class isoenzyme, each having a basic pZ value and subunit I%& of approximately 25,000. Another minor acidic 01 class isoenzyme, also with a subunit M, value of 25,000, was detected in liver, kidney, and brain. While multiple GST isoenzymes were detected in all other tissues studied, only spleen showed the presence of a single isoenzyme, which belonged to the rr class. These results reveal considerable differences in the GST isoenzyme composition of mouse tissues as compared to rat and human tissues. However, several apparent similarities in mouse and human tissues exist, suggesting that the mouse model can be used to analyze the GST-mediated detoxification mechanisms in humans. o 1990 Academic Press, Inc.
Glutathione-S-transferase (GST) is a family of enzymes involved in the detoxification of toxic and carcinogenic compounds. In the present study, the effect of thioacetamide (TA), a hepatotoxic and hepatocarcinogenic compound, on the activity and expression of GST of Wistar female rats was tested. Animals were treated with a single dose of TA (250 mg/kg) for 12, 24, 48 and 72 hours. GST activity toward the broad substrate 1-chloro-2,4-dinitrobenzene was enhanced by TA. The protein level of the GST classes alpha and mu as well as the mRNA level of several GST subunits were also positively affected by the TA treatment. Female Wistar rats of the same age but from two other different colonies had their GST activity either inhibited or not affected by TA. The basal mRNA level of class alpha and class mu GST was also tested in female Wistar rats obtained from five different sources. Differences in the basal level of class alpha mRNA were observed in rats from at least three different sources, while class mu mRNA level was distinct in two groups of animals.
Increased resistance to acetaminophen hepatotoxicity in mice lacking glutathione S -transferase Pi
Proceedings of the National Academy of Sciences, 2000
Overdose of acetaminophen, a widely used analgesic drug, can result in severe hepatotoxicity and is often fatal. This toxic reaction is associated with metabolic activation by the P450 system to form a quinoneimine metabolite, N -acetyl- p -benzoquinoneimine (NAPQI), which covalently binds to proteins and other macromolecules to cause cellular damage. At low doses, NAPQI is efficiently detoxified, principally by conjugation with glutathione, a reaction catalyzed in part by the glutathione S -transferases (GST), such as GST Pi. To assess the role of GST in acetaminophen hepatotoxicity, we examined acetaminophen metabolism and liver damage in mice nulled for GstP ( GstP1/P2 (−/−) ). Contrary to our expectations, instead of being more sensitive, GstP null mice were highly resistant to the hepatotoxic effects of this compound. No significant differences between wild-type ( GstP1/P2 (+/+) ) mice and GstP1/P2 (−/−) nulls in either the rate or route of metabolism, particularly to glutathio...
Glutathione S-transferase (GST) is a superfamily enzyme which plays a major role in detoxification of xenobiotic compounds by catalyzing the conjugation of xenobiotic to cellular glutathione (GSH). GST-P is an important class of GSTs which is expressed during the early stage of life and during developmental stages. Its activity is relatively high during embryogenesis and immediately after birth and diminished in normal adult rat liver. To investigate the effects of hepatotoxic agents such as acetaminophen (APAP) and aflatoxin B1 (AFB1) on liver GST-P in rats during postnatal age, suckling rats age (14±2 days old) were divided into groups (n=5) and treated with both APAP (250 or 450 mg/kg B.W) and AFB1 (3 mg/kg B.W). Livers were removed at different time intervals (2, 6, 12, 18 and 24 h) and processed for GST and GST-P activity at protein and mRNA levels (RT-PCR). Administration of a single high dose of AFB1 (3 mg/kg BW) and APAP (450 mg/kg BW) to weanling rats caused a significant (P< 0.05) induction in total GST activity in developing rats. Based on the Western blotting technique and GST-P specific mRNA amplification by RT-PCR, the GST-pi protein level and its expression were not affected by APAP or AFB1. Despite the inducible effects of AFB1 and APAP on liver total GST activity, GST-P remained unaffected in response to the drugs at protein and mRNA levels.
Toxicology in Vitro, 2002
Previous studies from this laboratory indicated that glutathione (GSH) conjugate formation with acetaminophen (APAP) is remarkably induced in liver of weanling rats in response to a single overdose of the drug administered intraperitoneally (ip). Increased APAP-GSH conjugation has been attributed to inducible glutathione S-transferases (GSTs) in dividing hepatocytes. In order to verify this finding, an in vitro reconstitution assay containing liver microsomes (source of cytochrome P-450) and cytosolic fractions (source of GST) from livers and kidneys of adult and weanling rats has been established. In vitro incubation of the reaction mixture was followed by solvent extraction, enzymatic digestion and HPLC analysis of the conjugate. Under controlled conditions, in vitro, the rate of APAP-GSH conjugation reflected the GST activity of cytosolic sample added to incubation system. The activity of cytosolic GST in catalyzing this reaction was measured using cytosols prepared from various tissue sources, particularly from animals pretreated with dietary butylated hydroxylanisole (BHA). The extent of APAP-GSH conjugate formation mediated by cytosols varied in this order: BHA-treated adult liver >BHA-treated weanling liver > control adult liver >control weanling liver> BHA-adult kidney >control adult kidney > BHA weanling kidney > control weanling kidney. In contrast to findings obtained from in vivo experiments, the rate of GST-dependent APAP conjugate formation with GSH in vitro is not induced in the presence of exogenous drug. #