Katarina Johansson

Microsomal Glutathione Transferase 1 Protects Against Toxicity Induced by Silica Nanoparticles but Not by Zinc Oxide Nanoparticles

Microsomal glutathione transferase 1 (MGST1) is an antioxidant enzyme located predominantly in the mitochondrial outer membrane and endoplasmic reticulum and has been shown to protect cells from lipid peroxidation induced by a variety of cytostatic drugs and pro-oxidant stimuli. We hypothesized that MGST1 may also protect against nanomaterial-induced cytotoxicity through a specific effect on lipid peroxidation. We evaluated the induction of cytotoxicity and oxidative stress by TiO2, CeO2, SiO2, and ZnO in the human MCF-7 cell line with or without overexpression of MGST1. SiO2 and ZnO nanoparticles caused dose- and time-dependent toxicity, whereas no obvious cytotoxic effects were induced by nanoparticles of TiO2 and CeO2. We also noted pronounced cytotoxicity for three out of four additional SiO2 nanoparticles tested. Overexpression of MGST1 reversed the cytotoxicity of the main SiO2 nanoparticles tested and for one of the supplementary SiO2 nanoparticles but did not protect cells against ZnO-induced cytotoxic effects. The data point toward a role of lipid peroxidation in SiO2 nanoparticle-induced cell death. For ZnO nanoparticles, rapid dissolution was observed, and the subsequent interaction of Zn2+ with cellular targets is likely to contribute to the cytotoxic effects. A direct inhibition of MGST1 by Zn2+ could provide a possible explanation for the lack of protection against ZnO nanoparticles in this model. Our data also showed that SiO2 nanoparticle-induced cytotoxicity is mitigated in the presence of serum, potentially through masking of reactive surface groups by serum proteins, whereas ZnO nanoparticles were cytotoxic both in the presence and in the absence of serum.

A novel persulfide detection method reveals protein persulfide- and polysulfide-reducing functions of thioredoxin and glutathione systems.

Protein Persulfide Detection Protocol reveals vital roles for thioredoxin and glutathione systems in maintaining sulfane sulfur homeostasis in cells and in vivo. Hydrogen sulfide signaling involves persulfide formation at specific protein Cys residues. However, overcoming current methodological challenges in persulfide detection and elucidation of Cys regeneration mechanisms from persulfides are prerequisites for constructing a bona fide signaling model. We here establish a novel, highly specific protein persulfide detection protocol, ProPerDP, with which we quantify 1.52 ± 0.6 and 11.6 ± 6.9 μg/mg protein steady-state protein persulfide concentrations in human embryonic kidney 293 (HEK293) cells and mouse liver, respectively. Upon treatment with polysulfides, HEK293 and A549 cells exhibited increased protein persulfidation. Deletion of the sulfide-producing cystathionine-γ-lyase or cystathionine-β-synthase enzymes in yeast diminished protein persulfide levels, thereby corroborating their involvement in protein persulfidation processes. We here establish that thioredoxin (Trx) and glutathione (GSH) systems can independently catalyze reductions of inorganic polysulfides and protein persulfides. Increased endogenous persulfide levels and protein persulfidation following polysulfide treatment in thioredoxin reductase-1 (TrxR1) or thioredoxin-related protein of 14 kDa (TRP14) knockdown HEK293 cells indicated that these enzymes constitute a potent regeneration system of Cys residues from persulfides in a cellular context. Furthermore, TrxR1-deficient cells were less viable upon treatment with toxic amounts of polysulfides compared to control cells. Emphasizing the dominant role of cytosolic disulfide reduction systems in maintaining sulfane sulfur homeostasis in vivo, protein persulfide levels were markedly elevated in mouse livers where hepatocytes lack both TrxR1 and glutathione reductase (TR/GR-null). The different persulfide patterns observed in wild-type, GR-null, and TR/GR-null livers suggest distinct roles for the Trx and GSH systems in regulating subsets of protein persulfides and thereby fine-tuning sulfide signaling pathways.

Multiple roles of microsomal glutathione transferase 1 in cellular protection: A mechanistic study

The aim of this study was to investigate the involvement of membrane-bound microsomal glutathione transferase 1 (MGST1) in cellular resistance against oxidative stress as well as its mechanism of protection. MGST1 is ubiquitously expressed and predominantly located in the endoplasmic reticulum and outer mitochondrial membrane. Utilizing MCF7 cells overexpressing MGST1 we show significant protection against agents that are known to induce lipid peroxidation (e.g., cumene hydroperoxide and tert-butylhydroperoxide) and an end-product of lipid peroxidation (e.g., 4-hydroxy-2-nonenal). Furthermore, our results demonstrate that MGST1 protection can be enhanced by vitamin E when toxicity depends on oxidative stress, but not when direct alkylation is the dominant mechanism. Mitochondria in MGST1-overexpressing cells were shown to be protected from oxidative insult as measured by calcium loading capacity and respiration. MGST1 induces cellular resistance against cisplatin. Here we used vitamin E to elucidate whether oxidative stress caused by cisplatin is significant for cell toxicity. The results indicate that oxidative stress and induction of lipid peroxidation are not the most prominent toxic mechanism of cisplatin in our cell system. We thus conclude that MGST1 protects cells (and mitochondria) by both conjugation and glutathione peroxidase functions. A new protective mechanism against cisplatin is also indicated.

Thioredoxin-related protein of 14 kDa is an efficient L-cystine reductase and S-denitrosylase.

Significance Several functions in cells require reductive processes, i.e., the enzymatic catalysis of a transfer of electrons to specific cellular substrates. One major reductive system in the cytosol of human cells depends upon thioredoxin 1 (Trx1), which in turn is kept reduced by thioredoxin reductase 1 (TrxR1) using NADPH. In the present study it is shown that another protein in addition to Trx1, called thioredoxin-related protein of 14 kDa (TRP14), is highly efficient together with TrxR1 in catalyzing reduction of L-cystine or nitric oxide-derivatized cysteine residues. It is also shown that TRP14, in contrast to Trx1, is resistant to inactivation by hydrogen peroxide. These findings reveal that several TrxR1-dependent functions in cells may not be propelled solely by Trx1, but instead relate to activities of TRP14. Thioredoxin-related protein of 14 kDa (TRP14, also called TXNDC17 for thioredoxin domain containing 17, or TXNL5 for thioredoxin-like 5) is an evolutionarily well-conserved member of the thioredoxin (Trx)-fold protein family that lacks activity with classical Trx1 substrates. However, we discovered here that human TRP14 has a high enzymatic activity in reduction of l-cystine, where the catalytic efficiency (2,217 min−1⋅µM−1) coupled to Trx reductase 1 (TrxR1) using NADPH was fivefold higher compared with Trx1 (418 min−1⋅µM−1). Moreover, the l-cystine reduction with TRP14 was in contrast to that of Trx1 fully maintained in the presence of a protein disulfide substrate of Trx1 such as insulin, suggesting that TRP14 is a more dedicated l-cystine reductase compared with Trx1. We also found that TRP14 is an efficient S-denitrosylase with similar efficiency as Trx1 in catalyzing TrxR1-dependent denitrosylation of S-nitrosylated glutathione or of HEK293 cell-derived S-nitrosoproteins. Consequently, nitrosylated and thereby inactivated caspase 3 or cathepsin B could be reactivated through either Trx1- or TRP14-catalyzed denitrosylation reactions. TRP14 was also, in contrast to Trx1, completely resistant to inactivation by high concentrations of hydrogen peroxide. The oxidoreductase activities of TRP14 thereby complement those of Trx1 and must therefore be considered for the full understanding of enzymatic control of cellular thiols and nitrosothiols.

Microsomal glutathione transferase 1: mechanism and functional roles.

Microsomal glutathione transferase 1 (MGST1) belongs to a superfamily named MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism). This family is represented in all life forms, except archae. Of the six human members, three are specialized in the synthesis of leukotrienes and prostaglandin E, whereas the others (MGST1–3) have potential roles in drug metabolism. MGST1 has a well-established role in the conjugation of electrophiles and oxidative stress protection, whereas MGST2 and 3 have been less studied. Here, we review the recent advances regarding the structure, mechanism, and functional roles of MGST1. Emerging data show that the enzyme is overexpressed in certain tumors and support a role for the enzyme in protecting cells from cytostatic drugs.

Changes in the central projection pattern of vibrissae innervating primary sensory neurons after peripheral nerve injury in the rat

The central representation of a normal vibrissa nerve and the corresponding nerve after transection and regeneration of the infraorbital nerve has been studied by the use of transganglionic transport of horseradish peroxidase in the adult rat. The normal vibrissa nerve terminated in a well-defined area within nucleus caudalis and C1 dorsal horn. In contrast, the regenerated vibrissa nerve showed a widespread central termination pattern indicating a pronounced loss of somatotopic organization. These changes in somatotopic organization could contribute to an inability to correctly localize a sensory stimulus; this is a common clinical finding after peripheral nerve injury and regeneration.

Characterization of new potential anticancer drugs designed to overcome glutathione transferase mediated resistance.

Resistance against anticancer drugs remains a serious obstacle in cancer treatment. Here we used novel strategies to target microsomal glutathione transferase 1 (MGST1) and glutathione transferase pi (GSTP) that are often overexpressed in tumors and confer resistance against a number of cytostatic drugs, including cisplatin and doxorubicin (DOX). By synthetically combining cisplatin with a GST inhibitor, ethacrynic acid, to form ethacraplatin, it was previously shown that cytosolic GST inhibition was improved and that cells became more sensitive to cisplatin. Here we show that ethacraplatin is easily taken up by the cells and can reverse cisplatin resistance in MGST1 overexpressing MCF7 cells. A second and novel strategy to overcome GST mediated resistance involves using GST releasable cytostatic drugs. Here we synthesized two derivatives of DOX, 2,4-dinitrobenzenesulfonyl doxorubicin (DNS-DOX) and 4-mononitrobenzenesulfonyl doxorubicin (MNS-DOX) and showed that they are substrates for MGST1 and GSTP (releasing DOX). MGST1 overexpressing cells are resistant to DOX. The resistance is partially reversed by DNS-DOX. Interestingly, the less reactive MNS-DOX was more cytotoxic to cells overexpressing MGST1 than control cells. It would appear that, by controlling the reactivity of the prodrug, and thereby the DOX release rate, selective toxicity to MGST1 overexpressing cells can be achieved. In the case of V79 cells, DOX resistance proportional to GSTP expression levels was noted. In this case, not only was drug resistance eliminated by DNS-DOX but a striking GSTP-dependent increase in toxicity was observed in the clonogenic assay. In summary, MGST1 and GSTP resistance to cytostatic drugs can be overcome and cytotoxicity can be enhanced in GST overexpressing cells.

HER2-Positive Tumors Imaged Within 1 Hour Using a Site-Specifically 11C-Labeled Sel-Tagged Affibody Molecule

A rapid, reliable method for distinguishing tumors or metastases that overexpress human epidermal growth factor receptor 2 (HER2) from those that do not is highly desired for individualizing therapy and predicting prognoses. In vivo imaging methods are available but not yet in clinical practice; new methodologies improving speed, sensitivity, and specificity are required. Methods: A HER2-binding Affibody molecule, ZHER2:342, was recombinantly fused with a C-terminal selenocysteine-containing tetrapeptide Sel-tag, allowing site-specific labeling with either 11C or 68Ga, followed by biodistribution studies with small-animal PET. Dosimetry data for the 2 radiotracers were compared. Imaging of HER2-expressing human tumor xenografts was performed using the 11C-labeled Affibody molecule. Results: Both the 11C- and 68Ga-labeled tracers initially cleared rapidly from the blood, followed by a slower decrease to 4–5 percentage injected dose per gram of tissue at 1 h. Final retention in the kidneys was much lower (>5-fold) for the 11C-labeled protein, and its overall absorbed dose was considerably lower. 11C-ZHER2:342 showed excellent tumor-targeting capability, with almost 10 percentage injected dose per gram of tissue in HER2-expressing tumors within 1 h. Specificity was demonstrated by preblocking binding sites with excess ligand, yielding significantly reduced radiotracer uptake (P = 0.002), comparable to uptake in tumors with low HER2 expression. Conclusion: To our knowledge, the Sel-tagging technique is the first that enables site-specific 11C-radiolabeling of proteins. Here we present the finding that, in a favorable combination between radionuclide half-life and in vivo pharmacokinetics of the Affibody molecules, 11C-labeled Sel-tagged ZHER2:342 can successfully be used for rapid and repeated PET studies of HER2 expression in tumors.

Association of interleukin 8 with myocardial infarction: Results from the Stockholm Heart Epidemiology Program

BACKGROUND Interleukin 8 (IL8) has been contradictorily associated with the risk of myocardial infarction (MI). AIM To investigate the association of IL8 serum levels with the risk of MI and the association of the IL8 (IL8) and IL8 receptors (CXCR1 and CXCR2) genetic variants with IL8 levels and MI risk in a large case control study, the Stockholm Heart Epidemiology Program. METHODS AND RESULTS IL8 levels (pg/mL) were divided into quartiles and the MI risk was calculated by logistic regression and expressed as odds ratio (OR) and 95% CI. Two IL8 SNPs (rs4073A/T, rs2227306C/T) and three SNPs tagging CXCR1 and CXCR2 (rs4674258C/T, rs1008563C/T, rs6723449T/C) were analyzed for association with IL8 levels and with MI risk. Multivariate adjusted ORs for MI risk by IL8 levels in the highest quartiles indicated reduced point estimates in both women (OR 0.37; 95% CI 0.2-0.8) and men when compared to the lowest quartile. In female cases, IL8 levels decreased progressively in the six months after MI (p=0.03). IL8, CXCR1 and CXCR2 genetic variants were not associated with IL8 levels. In men, the T allele at the IL8 SNP rs4073 was associated with a slight increase in the MI risk under an additive and a recessive model of inheritance. CONCLUSIONS IL8 serum levels were associated with a reduced occurrence of MI among women, whereas IL8 was associated with a slightly increased risk among men, possibly through different mechanisms. These data suggest that the biological effects of IL8 on MI risk may vary over time and warrant further cohort studies with repetitive IL8 measurements.

Characterization of a new fluorogenic substrate for microsomal glutathione transferase 1

A new thiol-reactive electrophilic, disubstituted rhodamine-based fluorogenic probe (bis-2,4-dinitrobenzenesulfonyl rhodamine [BDR]) with very high quantum yield was synthesized and described recently [A. Shibata et al., Bioorg. Med. Chem. Lett. 18 (2008) 2246-2249]. Because hydrophobic electrophiles are often conjugated by glutathione transferases, the BDR or monosubstituted rhodamine derivatives (2,4-dinitrobenzenesulfonyl rhodamine [DR]) were tested with microsomal glutathione transferase 1 (MGST1) and shown to function as substrates. The kinetic parameters for purified enzyme and DR were k(cat)=0.075+/-0.005 s(-1) and K(m)=21+/-3 microM (k(cat)/K(m)=3.6 x 10(3)+/-5.6 x 10(2)M(-1)s(-1)), giving a rate enhancement of 10(6) compared with the nonenzymatic reaction. In cells overexpressing MGST1, the addition of BDR caused a time-dependent increase of fluorescence compared with control cells. Preincubating the cells with a thiol reagent (N-ethylmaleimide) abolished the fluorescent signal. By using DR, we could determine the MGST1 activity in whole cell extracts with high sensitivity. In addition, the activity could be increased by thiol reagents (a hallmark of MGST1). Thus, we have identified a new fluorogenic substrate for MGST1 that will be a useful tool in the study of this enzyme and related enzymes.