Sulfhydryl Homeostasis Research Articles

Paraptosis: a non-classical paradigm of cell death for cancer therapy.

Due to the sustained proliferative potential of cancer cells, inducing cell death is a potential strategy for cancer therapy. Paraptosis is a mode of cell death characterized by endoplasmic reticulum (ER) and/or mitochondrial swelling and cytoplasmic vacuolization, which is less investigated. Considerable evidence shows that paraptosis can be triggered by various chemical compounds, particularly in cancer cells, thus highlighting the potential application of this non-classical mode of cell death in cancer therapy. Despite these findings, there remain significant gaps in our understanding of the role of paraptosis in cancer. In this review, we summarize the current knowledge on chemical compound-induced paraptosis. The ER and mitochondria are the two major responding organelles in chemical compound-induced paraptosis, which can be triggered by the reduction of protein degradation, disruption of sulfhydryl homeostasis, overload of mitochondrial Ca2+, and increased generation of reactive oxygen species. We also discuss the stumbling blocks to the development of this field and the direction for further research. The rational use of paraptosis might help us develop a new paradigm for cancer therapy.

Plumbagin induces paraptosis in cancer cells by disrupting the sulfhydryl homeostasis and proteasomal function

Plumbagin (PLB) is an active secondary metabolite extracted from the roots of Plumbago rosea. In this study, we report that plumbagin effectively induces paraptosis by triggering extensive cytoplasmic vacuolation followed by cell death in triple negative breast cancer cells (MDA-MB-231), cervical cancer cells (HeLa) and non-small lung cancer cells (A549) but not in normal lung fibroblast cells (WI-38). The vacuoles originated from the dilation of the endoplasmic reticulum (ER) and were found to be empty. The cell death induced by plumbagin was neither apoptotic nor autophagic. Plumbagin induced ER stress mainly by inhibiting the chymotrypsin-like activity of 26S proteasome as also evident from the accumulation of polyubiquitinated proteins. The vacuolation and cell death were found to be independent of reactive oxygen species generation but was effectively inhibited by thiol antioxidant suggesting that plumbagin could modify the sulfur homeostasis in the cellular milieu. Plumbagin also resulted in a decrease in mitochondrial membrane potential eventually decreasing the ATP production. This is the first study to show that Plumbagin induces paraptosis through proteasome inhibition and disruption of sulfhydryl homeostasis and thus further opens up the lead molecule to potential therapeutic strategies for apoptosis-resistant cancers.

Paraptosis in the anti-cancer arsenal of natural products.

Given the problems with malignant cancer cells showing innate and acquired resistance to apoptosis, we need alternative means to induce cell death in cancer. Paraptosis is a type of programmed cell death that is characterized by dilation of the endoplasmic reticulum (ER) and/or mitochondria. Although relatively little is known regarding the molecular basis of paraptosis, the underlying mechanism clearly differs from that of apoptosis. Recent studies have shown that various natural products, including curcumin, celastrol, 15d-PGJ2, ophiobolin A, and paclitaxel, demonstrate anti-cancer effects by inducing the paraptosis-associated cell death, which was commonly characterized by vacuolation derived from the ER. Perturbation of cellular proteostasis due to proteasomal inhibition and disruption of sulfhydryl homeostasis, generation of reactive oxygen species, and/or imbalanced homeostasis of ions (e.g., Ca(2+) and K(+)) appear to contribute to the accumulation of misfolded protein and proteotoxicity in this process. Given the pathophysiological importance of paraptosis and the debate regarding the importance of apoptosis in solid tumor, we need to collect the available knowledge regarding paraptosis and suggest future directions in the field. Here, we review the morphological and biochemical features of paraptosis, the natural products that induce paraptosis-associated cell death, their proposed mechanisms, and the significance of paraptosis as a potential anti-cancer strategy. Such work and future clarifications should enable the development of new strategies for preventing cancer and/or combating malignant cancer.

Proteome-wide identification and quantification of S-glutathionylation targets in mouse liver.

Protein S-glutathionylation is a reversible post-translational modification regulating sulfhydryl homeostasis. However, little is known about the proteins and pathways regulated by S-glutathionylation in whole organisms and current approaches lack the sensitivity to examine this modification under basal conditions. We now report the quantification and identification of S-glutathionylated proteins from animal tissue, using a highly sensitive methodology combining high-accuracy proteomics with tandem mass tagging to provide precise, extensive coverage of S-glutathionylated targets in mouse liver. Critically, we show significant enrichment of S-glutathionylated mitochondrial and Krebs cycle proteins, identifying that S-glutathionylation is heavily involved in energy metabolism processes invivo. Furthermore, using mice nulled for GST Pi (GSTP) we address the potential for S-glutathionylation to be mediated enzymatically. The data demonstrate the impact of S-glutathionylation in cellular homeostasis, particularly in relation to energy regulation and is of significant interest for those wishing to examine S-glutathionylation in an animal model.

Sulfhydryl-mediated redox signaling in inflammation: role in neurodegenerative diseases.

Posttranslational modifications of cysteine sulfhydryl (-SH) moieties, e.g., S-nitrosylation, S-glutathionylation, or S-sulfuration, play an important role in cellular response to oxidative stress. Reversible cysteine modifications alter protein function and can play a critical role in redox signal transduction. Perturbation of sulfhydryl homeostasis is a hallmark of many diseases, including neurodegenerative disorders. Besides direct oxidative stress within the neurons, inflammation of the central nervous system as well as the periphery is implicated also in the development and progression of neurodegeneration. Therefore, perturbation of redox regulation of key inflammatory mediators is an important component of neurodegenerative diseases. Many proteins involved in inflammation have been shown to undergo S-nitrosylation (-SNO) and/or S-glutathionylation (-SSG) with functional consequences. The mechanistic and functional relationships between these two modifications have yet to be thoroughly investigated. While protein-SNO intermediates in some cases may signal independently of protein-SSG intermediates, the relatively unstable nature of protein-SNO derivatives in the presence of GSH suggests that protein-SNO formation in many cases may serve as a precursor for protein-SSG modifications. In this review, we describe the cysteine modifications of specific inflammation-mediating proteins and their relationship to inflammatory responses such as cytokine and chemokine production. In particular, we consider evidence for sequential protein-SNO→protein-SSG modifications of these proteins. We conclude that cysteine modifications of critical regulatory proteins are likely to play a central role in the onset and progression of neuroinflammatory diseases and thus should be studied thoroughly in this context.

A comparative study of genotoxicity and oxidative stress before and after using lemon balm and cinnamon in subjects exposed to Nickel Welding Fumes

By producing reactive oxygen and nitrogen species, metal-induced toxicity and carcinogenicity alter DNA bases, increase lipid peroxidation, and change calcium and sulfhydryl homeostasis. The purpose of the study was to investigate putative effects of Lemon balm and Cinnamomum zeylanicum on the blood markers of welding workers. We measured nickel and chromium levels, biochemical parameters, blood oxidative stress markers, total antioxidant capacity (TAC), Myeloperoxidase (MPO), lipid peroxidation (LPO), and DNA damage. The study was conducted on 55 male workers who worked in an industrial subjects exposed to Nickel Welding Fumes. The participants were administered Lemon balm and Cinnamon extract infusion 1.5 (0.12 extract) and 0.25(0.013 extract) g/100 mL, respectively, they have drunken twice a day for 30 days at 7:00 AM and 2:00 PM every day. In order to analyze the achieved data, paired t-test and Pearson correlation coefficient have been used. After using the extract infusion, a significant increase revealed in TAC (p= 0.007). Also, administration of infusion decreased DNA damage but it was not statistically significant. After administration of infusion, a decrease in LPO and MPO were observed (p= 0.014, p=0.000 respectively). Also there is positive correlation between ALP and Ni with 8-oH-dG and also, between Ni and TAC. The results indicate that using infusion causes to raise in a TAC and reduce in DNA damage.

Protective effects of the thioredoxin and glutaredoxin systems in dopamine-induced cell death

Although the etiology of sporadic Parkinson disease (PD) is unknown, it is well established that oxidative stress plays an important role in the pathogenic mechanism. The thioredoxin (Trx) and glutaredoxin (Grx) systems are two central systems upholding the sulfhydryl homeostasis by reducing disulfides and mixed disulfides within the cell and thereby protecting against oxidative stress. By examining the expression of redox proteins in human postmortem PD brains, we found the levels of Trx1 and thioredoxin reductase 1 (TrxR1) to be significantly decreased. The human neuroblastoma cell line SH-SY5Y and the nematode Caenorhabditis elegans were used as model systems to explore the potential protective effects of the redox proteins against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity. 6-OHDA is highly prone to oxidation, resulting in the formation of the quinone of 6-OHDA, a highly reactive species and powerful neurotoxin. Treatment of human cells with 6-OHDA resulted in an increased expression of Trx1, TrxR1, Grx1, and Grx2, and small interfering RNA for these genes significantly increased the cytotoxic effects exerted by the 6-OHDA neurotoxin. Evaluation of the dopaminergic neurons in C. elegans revealed that nematodes lacking trxr-1 were significantly more sensitive to 6-OHDA, with significantly increased neuronal degradation. Importantly, both the Trx and the Grx systems were also found to directly mediate reduction of the 6-OHDA-quinone in vitro and thus render its cytotoxic effects. In conclusion, our results suggest that the two redox systems are important for neuronal survival in dopamine-induced cell death.

A review of metal‐catalyzed molecular damage: protection by melatonin

Metal exposure is associated with several toxic effects; herein, we review the toxicity mechanisms of cadmium, mercury, arsenic, lead, aluminum, chromium, iron, copper, nickel, cobalt, vanadium, and molybdenum as these processes relate to free radical generation. Free radicals can be generated in cells due to a wide variety of exogenous and endogenous processes, causing modifications in DNA bases, enhancing lipid peroxidation, and altering calcium and sulfhydryl homeostasis. Melatonin, an ubiquitous and pleiotropic molecule, exerts efficient protection against oxidative stress and ameliorates oxidative/nitrosative damage by a variety of mechanisms. Also, melatonin has a chelating property which may contribute in reducing metal-induced toxicity as we postulate here. The aim of this review was to highlight the protective role of melatonin in counteracting metal-induced free radical generation. Understanding the physicochemical insights of melatonin related to the free radical scavenging activity and the stimulation of antioxidative enzymes is of critical importance for the development of novel therapeutic strategies against the toxic action of these metals.

Lead exposure exacerbates cardiovascular risk.

Cardiovascular disease (CVD) is the leading cause of mortality all over the world. However, majority of CVDs are caused by risk factors that can be controlled, treated or modified, such as hypertension, cholesterol, overweight/obesity, tobacco use, sedentary lifestyles and diabetes. Various environmental toxicants like Lead (Pb) are also important preventable causes of variation in CVDs as reported in different human populations [1]. Although important measures have already been implemented in a number of countries to reduce environmental Pb exposure which includes the use of unleaded gasoline, Pb free paints, solder of canned foods, and glazed ceramics used for storage and preparation of food, it is still a major environmental health hazard in specific communities and targeted high-risk populations [2]. Pb intoxication has been found to be associated with hypertension and promotes atherosclerosis in experimental animals [2, 3]. Hypertensive effects of Pb has also been documented in workers chronically exposed to high Pb levels [4]. Several epidemiological and clinical studies have found a link between chronic Pb exposure and elevated blood pressure [1]. Although a dose–response relationship between Pb exposure and blood pressure has also been reported however shape of the dose response relationship is not completely elucidated, especially at low levels of exposure. It is still not clear what should be the lowest level of Pb exposure that shall not be associated with Pb toxicity and its impact on blood pressure, although in the available studies there seems to be no evidence of a threshold effect [5]. The precise mechanisms explaining a hypertensive effect of low chronic exposure to environmental Pb are unknown. Muntner et al. [6] has reported an inverse association between estimated glomerular filtration rate and blood Pb level at Pb concentration below 5 μg/dl in general population studies, indicating that Pb-induced reductions in renal function could play a major role in hypertension. Enhanced oxidative stress, down-regulation of nitric oxide, stimulation of the renin-angiotensin system and soluble guanylate cyclase are among other potential mechanisms which could result in increased vascular tone and peripheral vascular resistance [5]. Metal-mediated formation of free radicals may enhance lipid peroxidation and changes in calcium and sulfhydryl homeostasis. Pb by promoting the production of reactive oxygen species, triggers a cycle of oxidative stress and inflammation in target tissues. Depletion of sulfhydryl reserves in cells provides an indirect mechanism for redox-inactive metals induced oxidative stress. These findings suggest a possible involvement of oxidative stress in the pathophysiology of Pb toxicity. Although oxidative stress is one of the important factor in the pathophysiology of Pb toxicity, it is not clear whether these alterations are the cause of the oxidative damage or a consequence of it. Increased expression and production of inflammatory markers in association with Pb exposure have also been reported in humans [2]. Most of the data available have been drawn from experimental studies and there are few cohort studies that evaluate the association of Pb with clinical cardiovascular outcomes in general population settings. The National Health and Nutrition Examination Surveys (NHANES) II and III showed a statistically significant increase in cardiovascular mortality with increasing blood Pb levels, although there was an overall decline in Pb levels in the US adults. Similarly the British Regional Heart study showed positive but non-significant association of coronary heart disease or stroke with elevated Pb levels. However, the unresolved issue is the impact of uncontrolled confounding and measurement error on the relative risk estimates in studies of Pb and clinical cardiovascular end points. Similarly the validity of occupational studies on Pb and cardiovascular mortality is limited by different methodological problems like bias of observations in occupationally exposed workers, which cannot be extrapolated to general population [5]. Various factors, both conventional and novel risk factors have been shown to be responsible for the morbidity and mortality of patients with cardiovascular diseases. No attention has so far been given to environmental toxicants and other trace elements such as Pb, which can exacerbate the disease condition. A prospective pro-atherogenic effect, even if modest compared to other conventional risk factors would have a significant impact in sensitive population groups. In view of this it becomes important to explore the precise role and mechanism of Pb in development of atherosclerosis and CVD.

Manumycin A inhibits triple-negative breast cancer growth through LC3-mediated cytoplasmic vacuolation death

Therapy resistance can be attributed to acquisition of anti-apoptotic mechanisms by the cancer cells. Therefore, developing approaches that trigger non-apoptotic cell death in cancer cells to compensate for apoptosis resistance will help to treat cancer effectively. Triple-negative breast cancers (TNBC) are among the most aggressive and therapy resistant to breast tumors. Here we report that manumycin A (Man A), an inhibitor of farnesyl protein transferase, reduces cancer cell viability through induction of non-apoptotic, non-autophagic cytoplasmic vacuolation death in TNBC cells. Man A persistently induced cytoplasmic vacuolation and cell death through the expression of microtubule-associated protein 1 light chain 3 (LC3) and p62 proteins along with endoplasmic reticulum (ER) stress markers, Bip and CHOP, and accumulation of ubiquitinated proteins. As inhibitors of apoptosis and autophagy failed to block cytoplasmic vacuolation and its associated protein expression or cell death, it appears that these processes are not involved in the death induced by Man A. Ability of thiol antioxidant, NAC in blocking Man A-induced vacuolation, death and its related protein expression suggests that sulfhydryl homeostasis may be the target of Man A. Surprisingly, normal human mammary epithelial cells failed to undergo cytoplasmic vacuolation and cell death, and grew normally in presence of Man A. In conjunction with its in vitro effects, Man A also reduced tumor burden in vivo in xenograft models that showed extensive cytoplasmic vacuoles and condensed nuclei with remarkable increase in the vacuolation-associated protein expression together with increase of p21, p27, PTEN and decrease of pAkt. Interestingly, Man A-mediated upregulation of p21, p27 and PTEN and downregulation of pAkt and tumor growth suppression were also mimicked by LC3 knockdown in MDA-MB-231 cells. Overall, these results suggest novel therapeutic actions by Man A through the induction of non-apoptotic and non-autophagic cytoplasmic vacuolation death by probably affecting ER stress, LC3 and p62 pathways in TNBC but not in normal mammary epithelial cells.

Mechanisms of Altered Redox Regulation in Neurodegenerative Diseases—Focus on S-Glutathionylation

Neurodegenerative diseases are characterized by progressive loss of neurons. A common feature is oxidative stress, which arises when reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) exceed amounts required for normal redox signaling. An imbalance in ROS/RNS alters functionality of cysteines and perturbs thiol-disulfide homeostasis. Many cysteine modifications may occur, but reversible protein mixed disulfides with glutathione (GSH) likely represents the common steady-state derivative due to cellular abundance of GSH and ready conversion of cysteine-sulfenic acid and S-nitrosocysteine precursors to S-glutathionylcysteine disulfides. Thus, S-glutathionylation acts in redox signal transduction and serves as a protective mechanism against irreversible cysteine oxidation. Reversal of protein-S-glutathionylation is catalyzed specifically by glutaredoxin which thereby plays a critical role in cellular regulation. This review highlights the role of oxidative modification of proteins, notably S-glutathionylation, and alterations in thiol homeostatic enzyme activities in neurodegenerative diseases, providing insights for therapeutic intervention. Recent studies show that dysregulation of redox signaling and sulfhydryl homeostasis likely contributes to onset/progression of neurodegeneration. Oxidative stress alters the thiol-disulfide status of key proteins that regulate the balance between cell survival and cell death. Much of the current information about redox modification of key enzymes and signaling intermediates has been gleaned from studies focused on oxidative stress situations other than the neurodegenerative diseases. The findings in other contexts are expected to apply to understanding neurodegenerative mechanisms. Identification of selectively glutathionylated proteins in a quantitative fashion will provide new insights about neuropathological consequences of this oxidative protein modification.

Levodopa Activates Apoptosis Signaling Kinase 1 (ASK1) and Promotes Apoptosis in a Neuronal Model: Implications for the Treatment of Parkinson's Disease

Oxidative stress is implicated in the etiology of Parkinson's disease (PD), the second most common neurodegenerative disease. PD is treated with chronic administration of l-3,4-dihydroxyphenylalanine (levodopa, L-DOPA), and typically, increasing doses are used during progression of the disease. Paradoxically, L-DOPA is a pro-oxidant and induces cell death in cellular models of PD through disruption of sulfhydryl homeostasis involving loss of the thiol-disulfide oxidoreductase functions of the glutaredoxin (Grx1) and thioredoxin (Trx1) enzyme systems [Sabens, E. A., Distler, A. M., and Mieyal, J. J. (2010) Biochemistry 49 (12), 2715-2724]. Considering this loss of both Grx1 and Trx1 activities upon L-DOPA treatment, we sought to elucidate the mechanism(s) of L-DOPA-induced apoptosis. In other contexts, both the NFκB (nuclear factor κB) pathway and the ASK1 (apoptosis signaling kinase 1) pathway have been shown to be regulated by both Grx1 and Trx1, and both pathways have been implicated in cell death signaling in model systems of PD. Moreover, mixed lineage kinase (MLK) has been considered as a potential therapeutic target for PD. Using SHSY5Y cells as model dopaminergic neurons, we found that NFκB activity was not altered by L-DOPA treatment, and the selective MLK inhibitor (CEP-1347) did not protect the cells from L-DOPA. In contrast, ASK1 was activated with L-DOPA treatment as indicated by phosphorylation of its downstream mitogen-activated protein kinases (MAPK), p38 and JNK. Chemical inhibition of either p38 or JNK provided protection from L-DOPA-induced apoptosis. Moreover, direct knockdown of ASK1 protected from L-DOPA-induced neuronal cell death. These results identify ASK1 as the main pro-apoptotic pathway activated in response to L-DOPA treatment, implicating it as a potential target for adjunct therapy in PD.

Levodopa Deactivates Enzymes That Regulate Thiol−Disulfide Homeostasis and Promotes Neuronal Cell Death: Implications for Therapy of Parkinson’s Disease

Parkinson's disease (PD), characterized by dopaminergic neuronal loss, is attributed to oxidative stress, diminished glutathione (GSH) levels, mitochondrial dysfunction, and protein aggregation. Treatment of PD involves chronic administration of Levodopa (l-DOPA) which is a pro-oxidant and may disrupt sulfhydryl homeostasis. The goal of these studies is to elucidate the effects of l-DOPA on thiol homeostasis in a model akin to PD, i.e., immortalized dopaminergic neurons (SHSY5Y cells) with diminished GSH content. These neurons exhibit hypersensitivity to l-DOPA-induced cell death, which is attributable to concomitant inhibition of the intracellular thiol disulfide oxidoreductase enzymes. Glutaredoxin (Grx) was deactivated in a dose-dependent fashion, but its content was unaffected. Glutathione disulfide (GSSG) reductase (GR) activity was not altered. Selective knockdown of Grx resulted in an increased level of apoptosis, documenting the role of the Grx system in neuronal survival. l-DOPA treatments also led to decreased activities of thioredoxin (Trx) and thioredoxin reductase (TR), concomitant with diminution of their cellular contents. Selective chemical inhibition of TR activity led to an increased level of apoptosis, documenting the Trx system's contribution to neuronal viability. To investigate the mechanism of inhibition at the molecular level, we treated the each isolated enzyme with oxidized l-DOPA. GR, Trx, and TR activities were little affected. However, Grx was inactivated in a time- and concentration-dependent fashion indicative of irreversible adduction of dopaquinone to its nucleophilic active-site Cys-22, consistent with the intracellular loss of Grx activity but not Grx protein content after l-DOPA treatment. Overall l-DOPA is shown to impair the collaborative contributions of the Grx and Trx systems to neuron survival.

A novel role for MAP1 LC3 in nonautophagic cytoplasmic vacuolation death of cancer cells

Thiol reactive cyclopentenone prostaglandin, 15-deoxy-Δ12, 14-Prostaglandin J2, induced a novel, non-apoptotic and Map1 LC3 dependent but non-autophagic form of cell death in colon, breast and prostate cancer cell lines, characterized by extensive cytoplasmic vacuolation with dilatation of endoplasmic reticulum. Disruption of sulfhydryl homeostasis, which resulted in ER stress, accumulation of ubiquitinated proteins and subsequent ER dilation, contributed to PPARγ independent cell death by 15d-PGJ2. Absence of intracellular organelles in these vacuoles, shown by Electron Microscopy and unique fragmentation of Lamin B, suggested this form of cell death to be different from autophagy and apoptosis. Cell death induced by 15d-PGJ2 is prevented by cycloheximide and actinomycin D, suggesting a requirement of new protein synthesis for death with cytoplasmic vacuolation. Here, we report for the first time that upregulation and processing of autophagy marker LC3 is an important event in non-autophagic cytoplasmic vacuolation and cell death. Notably, knockdown of LC3 conferred significant protection against 15d-PGJ2 induced cytoplasmic vacuolation and cell death suggesting a novel role of LC3 in a death process other than autophagy.

Mechanistic and Kinetic Details of Catalysis of Thiol-Disulfide Exchange by Glutaredoxins and Potential Mechanisms of Regulation

Glutaredoxins are small, heat-stable proteins that exhibit a characteristic thioredoxin fold and a CXXC/S active-site motif. A variety of glutathione (GSH)-dependent catalytic activities have been attributed to the glutaredoxins, including reduction of ribonucleotide reductase, arsenate, and dehydroascorbate; assembly of iron sulfur cluster complexes; and protein glutathionylation and deglutathionylation. Catalysis of reversible protein glutathionylation by glutaredoxins has been implicated in regulation of redox signal transduction and sulfhydryl homeostasis in numerous contexts in health and disease. This forum review is presented in two parts. Part I is focused primarily on the mechanism of the deglutathionylation reaction catalyzed by prototypical dithiol glutaredoxins, especially human Grx1 and Grx2. Grx-catalyzed protein deglutathionylation proceeds by a nucleophilic, double-displacement mechanism in which rate enhancement is attributed to special reactivity of the low pK(a) cysteine at its active site, and to increased nucleophilicity of the second substrate, GSH. Glutaredoxins (and Grx domains) have been identified in most organisms, and many exhibit deglutathionylation or other activities or both. Further characterization according to glutathionyl selectivity, physiological substrates, and intracellular roles may lead to subclassification of this family of enzymes. Part II presents potential mechanisms for in vivo regulation of Grx activity, providing avenues for future studies.

What is the Functional Significance of the Unique Location of Glutaredoxin 1 (GRx1) in the Intermembrane Space of Mitochondria?

Glutaredoxins (GRx) catalyze reversible protein glutathionylation. They are implicated in sulfhydryl homeostasis and regulation of redox signal transduction, controlling various cellular processes like DNA synthesis, defense against oxidative stress, apoptosis signaling, and DNA-binding of transcription factors. Two isoforms of GRx are well characterized in mammals: GRx1, the "cytosolic" form, and GRx2, the "mitochondrial" form. Here we report documentation of GRx1 in mitochondria, localized exclusively in the intermembrane space and segregated from GRx2, localized exclusively in the mitochondrial matrix. We hypothesize that GRx1 and GRx2 in their unique locations regulate different functions of the mitochondria via reversible S-glutathionylation.

Metals, Toxicity and Oxidative Stress

Metal-induced toxicity and carcinogenicity, with an emphasis on the generation and role of reactive oxygen and nitrogen species, is reviewed. Metal-mediated formation of free radicals causes various modifications to DNA bases, enhanced lipid peroxidation, and altered calcium and sulfhydryl homeostasis. Lipid peroxides, formed by the attack of radicals on polyunsaturated fatty acid residues of phospholipids, can further react with redox metals finally producing mutagenic and carcinogenic malondialdehyde, 4-hydroxynonenal and other exocyclic DNA adducts (etheno and/or propano adducts). Whilst iron (Fe), copper (Cu), chromium (Cr), vanadium (V) and cobalt (Co) undergo redox-cycling reactions, for a second group of metals, mercury (Hg), cadmium (Cd) and nickel (Ni), the primary route for their toxicity is depletion of glutathione and bonding to sulfhydryl groups of proteins. Arsenic (As) is thought to bind directly to critical thiols, however, other mechanisms, involving formation of hydrogen peroxide under physiological conditions, have been proposed. The unifying factor in determining toxicity and carcinogenicity for all these metals is the generation of reactive oxygen and nitrogen species. Common mechanisms involving the Fenton reaction, generation of the superoxide radical and the hydroxyl radical appear to be involved for iron, copper, chromium, vanadium and cobalt primarily associated with mitochondria, microsomes and peroxisomes. However, a recent discovery that the upper limit of "free pools" of copper is far less than a single atom per cell casts serious doubt on the in vivo role of copper in Fenton-like generation of free radicals. Nitric oxide (NO) seems to be involved in arsenite-induced DNA damage and pyrimidine excision inhibition. Various studies have confirmed that metals activate signalling pathways and the carcinogenic effect of metals has been related to activation of mainly redox-sensitive transcription factors, involving NF-kappaB, AP-1 and p53. Antioxidants (both enzymatic and non-enzymatic) provide protection against deleterious metal-mediated free radical attacks. Vitamin E and melatonin can prevent the majority of metal-mediated (iron, copper, cadmium) damage both in vitro systems and in metal-loaded animals. Toxicity studies involving chromium have shown that the protective effect of vitamin E against lipid peroxidation may be associated rather with the level of non-enzymatic antioxidants than the activity of enzymatic antioxidants. However, a very recent epidemiological study has shown that a daily intake of vitamin E of more than 400 IU increases the risk of death and should be avoided. While previous studies have proposed a deleterious pro-oxidant effect of vitamin C (ascorbate) in the presence of iron (or copper), recent results have shown that even in the presence of redox-active iron (or copper) and hydrogen peroxide, ascorbate acts as an antioxidant that prevents lipid peroxidation and does not promote protein oxidation in humans in vitro. Experimental results have also shown a link between vanadium and oxidative stress in the etiology of diabetes. The impact of zinc (Zn) on the immune system, the ability of zinc to act as an antioxidant in order to reduce oxidative stress and the neuroprotective and neurodegenerative role of zinc (and copper) in the etiology of Alzheimer's disease is also discussed. This review summarizes recent findings in the metal-induced formation of free radicals and the role of oxidative stress in the carcinogenicity and toxicity of metals.

Human brain thioltransferase: constitutive expression and localization by fluorescence in situ hybridization

Thioltransferase (glutaredoxin) is a member of the family of thiol-disulfide oxido-reductases that maintain the sulfhydryl homeostasis in cells by catalyzing thiol-disulfide interchange reactions. One of the major consequences of oxidative stress in brain is formation of protein-glutathione mixed disulfide (through oxidation of protein thiols) which can be reversed by thioltransferase during recovery of brain from oxidative stress. Here we have visualized the location of thioltransferase in brain regions from seven human tissues obtained at autopsy. Constitutively expressed thioltransferase activity was detectable in all human brains examined although inter-individual variations were seen. The enzyme activity was significantly higher in hippocampus and cerebellum as compared to other regions. Constitutive expression of thioltransferase mRNA was detectable by Northern blot analysis. Localization of thioltransferase mRNA by fluorescence in situ hybridization revealed its presence predominantly in neurons in the cerebral cortex, Purkinje and granule cell layers of the cerebellum, granule cell layer of the dentate gyrus and in the pyramidal neurons of CA1, CA2 and CA3 subfields of hippocampus. These discrete neuronal concentrations of thioltransferase would be consistent with an essential role in modulating recovery of protein thiols from mixed disulfides formed during oxidative stress.

Acute Cadmium Exposure Inactivates Thioltransferase (Glutaredoxin), Inhibits Intracellular Reduction of Protein-glutathionyl-mixed Disulfides, and Initiates Apoptosis

Oxidative stress broadly impacts cells, initiating regulatory pathways as well as apoptosis and necrosis. A key molecular event is protein S-glutathionylation, and thioltransferase (glutaredoxin) is a specific and efficient catalyst of protein-SSG reduction. In this study 30-min exposure of H9 and Jurkat cells to cadmium inhibited intracellular protein-SSG reduction, and this correlated with inhibition of the thioltransferase system, consistent with thioltransferase being the primary intracellular catalyst of deglutathionylation. The thioredoxin system contributed very little to total deglutathionylase activity. Thioltransferase and GSSG reductase in situ displayed similar dose-response curves (50% inhibition near 10 micrometer cadmium in extracellular buffer). Acute cadmium exposure also initiated apoptosis, with H9 cells being more sensitive than Jurkat. Moreover, transfection with antisense thioltransferase cDNA was incompatible with cell survival. Collectively, these data suggest that thioltransferase has a vital role in sulfhydryl homeostasis and cell survival. In separate experiments, cadmium inhibited the isolated component enzymes of the thioltransferase and thioredoxin systems, consistent with the vicinal dithiol nature of their active sites: thioltransferase (IC(50) approximately 1 micrometer), GSSG reductase (IC(50) approximately 1 micrometer), thioredoxin (IC(50) approximately 8 micrometer), thioredoxin reductase (IC(50) approximately 0.2 micrometer). Disruption of the vicinal dithiol on thioltransferase (via oxidation to C22-SS-C25; or C25S mutation) protected against cadmium, consistent with a dithiol chelation mechanism of inactivation.

Rat brain thioltransferase: regional distribution, immunological characterization, and localization by fluorescent in situ hybridization.

Thioltransferase (TTase) is a member of the family of thiol-disulfide oxidoreductases that are involved in the maintenance of sulfhydryl homeostasis in cells by catalyzing thiol-disulfide interchange reactions. One of the major consequences of oxidative stress in brain is the formation of protein-glutathione mixed disulfides (through oxidation of protein thiols), which can be reversed by TTase during the recovery of brain from oxidative stress. We therefore examined the presence of TTase in brain regions from rat. In the rat, TTase activity in the whole brain was comparable with the corresponding activity in liver, but significantly higher in hippocampus. The enzyme activity was significantly lower in striatum and cerebellum compared with activity in whole brain. Rat brain TTase shared immunological similarity with the human red blood cell enzyme, but not with the pig liver enzyme. The constitutive expression of the mRNA to TTase was demonstrable by northern blotting. Localization of the TTase mRNA in rat brain by fluorescent in situ hybridization showed the presence of high amounts of mRNA in the olfactory bulb, cortex, and hippocampus and its predominant localization in the neurons. TTase mRNA was also present in Purkinje cells in the cerebellum, in giant reticular neurons in the midbrain, and in the striatal and thalamic neurons. This study demonstrates the constitutive presence of a functional TTase system in brain and delineates the regional and cellular localization of the enzyme in rat brain.