▪ Abstract This review describes the three mammalian glutathione transferase (GST) families, namely cytosolic, mitochondrial, and microsomal GST, the latter now designated MAPEG. Besides detoxifying electrophilic xenobiotics, such as chemical carcinogens, environmental pollutants, and antitumor agents, these transferases inactivate endogenous α,β-unsaturated aldehydes, quinones, epoxides, and hydroperoxides formed as secondary metabolites during oxidative stress. These enzymes are also intimately involved in the biosynthesis of leukotrienes, prostaglandins, testosterone, and progesterone, as well as the degradation of tyrosine. Among their substrates, GSTs conjugate the signaling molecules 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) and 4-hydroxynonenal with glutathione, and consequently they antagonize expression of genes trans-activated by the peroxisome proliferator-activated receptor γ (PPARγ) and nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). Through metabolism of 15d-PGJ₂, GST may enhance gene expression driven by nuclear factor-κB (NF-κB). Cytosolic human GST exhibit genetic polymorphisms and this variation can increase susceptibility to carcinogenesis and inflammatory disease. Polymorphisms in human MAPEG are associated with alterations in lung function and increased risk of myocardial infarction and stroke. Targeted disruption of murine genes has demonstrated that cytosolic GST isoenzymes are broadly cytoprotective, whereas MAPEG proteins have proinflammatory activities. Furthermore, knockout of mouse GSTA4 and GSTZ1 leads to overexpression of transferases in the Alpha, Mu, and Pi classes, an observation suggesting they are part of an adaptive mechanism that responds to endogenous chemical cues such as 4-hydroxynonenal and tyrosine degradation products. Consistent with this hypothesis, the promoters of cytosolic GST and MAPEG genes contain antioxidant response elements through which they are transcriptionally activated during exposure to Michael reaction acceptors and oxidative stress.

The glutathione S-transferases (GST) represent a major group of detoxification enzymes. All eukaryotic species possess multiple cytosolic and membrane-bound GST isoenzymes, each of which displays distinct catalytic as well as noncatalytic binding properties: the cytosolic enzymes are encoded by at least five distantly related gene families (designated class alpha, mu, pi, sigma, and theta GST), whereas the membrane-bound enzymes, microsomal GST and leukotriene C, synthetase, are encoded by single genes and both have arisen separately from the soluble GST. Evidence suggests that the level of expression of GST is a crucial factor in determining the sensitivity of cells to a broad spectrum of toxic chemicals. In this article the biochemical functions of GST are described to show how individual isoenzymes contribute to resistance to carcinogens, antitumor drugs, environmental pollutants, and products of oxidative stress. A description of the mechanisms of transcriptional and posttranscriptional regulation of GST isoenzymes is provided to allow identification of factors that may modulate resistance to specific noxious chemicals. The most abundant mammalian GST are the class alpha, mu, and pi enzymes and their regulation has been studied in detail. The biological control of these families is complex as they exhibit sex-, age-, tissue-, species-, and tumor-specific patterns of expression. In addition, GST are regulated by a structurally diverse range of xenobiotics and, to date, at least 100 chemicals have been identified that induce GST; a significant number of these chemical inducers occur naturally and, as they are found as nonnutrient components in vegetables and citrus fruits, it is apparent that humans are likely to be exposed regularly to such compounds. Many inducers, but not all, effect transcriptional activation of GST genes through either the antioxidant-responsive element (ARE), the xenobiotic-responsive element (XRE), the GST P enhancer l(GPE), or the glucocorticoid-responsive element (GRE). Barbiturates may transcriptionally activate GST through a Barbie box element. The involvement of the Ah-receptor, Maf, Nrl, Jun, Fos, and NF-κB in GST induction is discussed. Many of the compounds that induce GST are themselves substrates for these enzymes, or are metabolized (by cytochrome P-450 monooxygenases) to compounds that can serve as GST substrates, suggesting that GST induction represents part of an adaptive response mechanism to chemical stress caused by electrophiles. It also appears probable that GST are regulated in vivoby reactive oxygen species (ROS), because not only are some of the most potent inducers capable of generating free radicals by redox-cycling, but H₂O₂has been shown to induce GST in plant and mammalian cells: induction of GST by ROS would appear to represent an adaptive response as these enzymes detoxify some of the toxic carbonyl-, peroxide-, and epoxide-containing metabolites produced within the cell by oxidative stress. Class alpha, mu, and pi GST isoenzymes are overexpressed in rat hepatic preneoplastic nodules and the increased levels of these enzymes are believed to contribute to the multidrug-resistant phenotype observed in these lesions. The majority of human tumors and human tumor cell lines express significant amounts of class pi GST. Cell lines selected in vitrofor resistance to anticancer drugs frequently overexpress class pi GST, although overexpression of class alpha and mu isoenzymes is also often observed. The mechanisms responsible for overexpression of GST include transcriptional activation, stabilization of either mRNA or protein, and gene amplification. In humans, marked interindividual differences exist in the expression of class alpha, mu, and theta GST. The molecular basis for the variation in class alpha GST is not known. Absence of certain class mu and theta GST can be attributed to deletion of the GSTMIgene in 50% of the population and deletion of the GSTTIgene in 16% of the population. The biological consequences of failure to express hGSTMI or hGSTTI protein can include susceptibility to bladder, colon, skin, and possibly lung cancer.

The p62/SQSTM1 (sequestosome 1) protein, which acts as a cargo receptor for autophagic degradation of ubiquitinated targets, is up-regulated by various stressors. Induction of the p62 gene by oxidative stress is mediated by NF-E2-related factor 2 (NRF2) and, at the same time, p62 protein contributes to the activation of NRF2, but hitherto the mechanisms involved were not known. Herein, we have mapped an antioxidant response element (ARE) in the p62 promoter that is responsible for its induction by oxidative stress via NRF2. Chromatin immunoprecipitation and gel mobility-shift assays verified that NRF2 binds to this cis-element in vivo and in vitro. Also, p62 docks directly onto the Kelch-repeat domain of Kelch-like ECH-associated protein 1 (KEAP1), via a motif designated the KEAP1 interacting region (KIR), thereby blocking binding between KEAP1 and NRF2 that leads to ubiquitylation and degradation of the transcription factor. The KIR motif in p62 is located immediately C-terminal to the LC3-interacting region (LIR) and resembles the ETGE motif utilized by NRF2 for its interaction with KEAP1. KIR is required for p62 to stabilize NRF2, and inhibition of KEAP1 by p62 occurs from a cytoplasmic location within the cell. The LIR and KIR motifs cannot be engaged simultaneously by LC3 and KEAP1, but because p62 is polymeric the interaction between KEAP1 and p62 leads to accumulation of KEAP1 in p62 bodies, which is followed by autophagic degradation of KEAP1. Our data explain how p62 contributes to activation of NRF2 target genes in response to oxidative stress through creating a positive feedback loop.

Increases in the intracellular levels of reactive oxygen species (ROS), frequently referred to as oxidative stress, respresents a potentially toxic insult which if not counteracted will lead to membrane dysfunction, DNA damage and inactivation of proteins. Chronic oxidative stress has numerous pathological consequences including cancer, arthritis and neurodegenerative disease. Glutathione-associated metabolism is a major mechanism for cellular protection against agents which generate oxidative stress. It is becoming increasingly apparent that the glutathione tripeptide is central to a complex multifaceted detoxification system, where there is substantial inter-dependence between separate component members. Glutathione participates in detoxification at several different levels, and may scavenge free radicals, reduce peroxides or be conjugated with electrophilic compounds. Thus, glutathione provides the cell with multiple defences not only against ROS but also against their toxic products. This article discusses how glutathione biosynthesis, glutathione peroxidases, glutathione S-transferases and glutathione S-conjugate efflux pumps function in an integrated fashion to allow cellular adaption to oxidative stress. Co-ordination of this response is achieved, at least in part, through the antioxidant responsive element (ARE) which is found in the promoters of many of the genes that are inducible by oxidative and chemical stress. Transcriptional activation through this enhancer appears to be mediated by basic leucine zipper transcription factors such as Nrf and small Maf proteins. The nature of the intracellular sensor(s) for ROS and thiol-active chemicals which induce genes through the ARE is described. Gene activation through the ARE appears to account for the enhanced antioxidant and detoxification capacity of normal cells effected by many cancer chemopreventive agents. In certain instances it may also account for acquired resistance of tumours to cancer chemotherapeutic drugs. It is therefore clear that determining the mechanisms involved in regulation of ARE-driven gene expression has enormous medical implications.