Thioredoxin reductase-1 levels are associated with NRF2 pathway activation and tumor recurrence in non-small cell lung cancer.

Activating mutations in the KEAP1/NRF2 pathway characterize a subset of non-small cell lung cancer (NSCLC) associated with chemoresistance and poor prognosis. We herein evaluated the relationship between 64 oxidative stress-related genes and overall survival data from 35 lung cancer datasets. Thioredoxin reductase-1 (TXNRD1) stood out as the most significant predictor of poor outcome. In a cohort of NSCLC patients, high TXNRD1 protein levels correlated with shorter disease-free survival and distal metastasis-free survival post-surgery, including a subset of individuals treated with platinum-based adjuvant chemotherapy. Bioinformatics analysis revealed that NSCLC tumors harboring genetic alterations in the NRF2 pathway (KEAP1, NFE2L2 and CUL3 mutations, and NFE2L2 amplification) overexpress TXNRD1, while no association with EGFR, KRAS, TP53 and PIK3CA mutations was found. In addition, nuclear accumulation of NRF2 overlapped with upregulated TXNRD1 protein in NSCLC tumors. Functional cell assays and gene dependency analysis revealed that NRF2, but not TXNRD1, has a pivotal role in KEAP1 mutant cells' survival. KEAP1 mutants overexpress TXNRD1 and are less susceptible to the cytotoxic effects of the TXNRD1 inhibitor auranofin when compared to wild-type cell lines. Inhibition of NRF2 with siRNA or ML-385, and glutathione depletion with buthionine-sulfoximine, sensitized KEAP1 mutant A549 cells to auranofin. NRF2 knockdown and GSH depletion also augmented cisplatin cytotoxicity in A549 cells, whereas auranofin had no effect. In summary, these findings suggest that TXNRD1 is not a key determinant of malignant phenotypes in KEAP1 mutant cells, although this protein can be a surrogate marker of NRF2 pathway activation, predicting tumor recurrence and possibly other aggressive phenotypes associated with NRF2 hyperactivation in NSCLC.

Role of the Enzymatic Environment in the Reactivity of the AuIII-C

The action mechanism of anticancer gold(III) complexes is a multi-step process and depends on their redox stability. First, the gold(III) complex undergoes a ligand exchange reaction in the presence of cellular thiols, such as those available in the active site of the enzyme TrxR, and then, the AuIII → AuI reduction occurs. Most experimental and theoretical studies describe these processes under chemical conditions without considering the enzyme structure effect. In the present study, molecular models are proposed for the [AuIII(C^N^C)(SHCys-R)]+ adduct, with the [AuIII(C^N^C)]+ moiety bonded to the Cys498 residue in the C-terminal arm of the TrxR. This one represents the product of the first ligand exchange reaction. Overall, our results suggest that the exchange of the auxiliary ligand (for instance, Cl- to S-R) plays a primary role in increasing the reduction potential, with the enzyme structure having a small effect. The parent compound [AuIII(C^N^C)Cl] has E° = -1.20 V, which enlarges to -0.72 V for [AuIII(C^N^C)CH3SH]+ and to -0.65 V for the largest model studied, Au-trx. In addition to the effect of the enzyme structure on the redox stability, we also analyze the Au transfer to the enzyme using a small peptide model (a tetramer). This reaction is dependent on the Cys497 protonation state. Thermodynamics and kinetic analysis suggests that the C^N^C ligand substitution by Cys497 is an exergonic process, with an energy barrier estimated at 20.2 kcal mol-1. The complete transfer of the Au ion to the enzyme's active site would lead to a total loss of enzyme activity, generating oxidative damage and, consequently, cancer cell death.

Accessing the transcriptional status of selenoproteins in skin cancer-derived cell lines.

BACKGROUND: Selenoproteins are selenocysteine (Sec)-containing proteins that exhibit numerous physiological functions, mainly antioxidative activities. Studies have suggested that several human selenoproteins play an important role in tumor initiation and progression, including melanoma. METHODS: Using RNA-seq data set from Sequence Reads Archive (SRA) experiments published at the National Center for Biotechnology Information (NCBI), we determined and compared the transcriptional levels of the 25 selenoproteins-coding sequences found in 16 human-derived melanoma cell lines and compared to four melanocyte controls. RESULTS: 15 selenoprotein-coding genes were found to be expressed in melanoma and normal melanocyte cells, and their mRNA levels varied among the cell lines. All melanoma cells analyzed with BRAF or NRAS mutations presented upregulated levels of SELENOI, TXNRD1, and SELENOT transcripts and downregulated levels of SELENOW and SELENON transcripts in comparison with melanocytes controls. Moreover, SELENOW, SELENON, SELENOI, TXNRD1, and SELENOT-coding transcripts were affected when BRAF-mutated A375 cells were treated with CPI203, A771726 or Vorinostat drugs. CONCLUSION: Our results indicate that melanoma cells can modify, in a different manner, the selenoprotein transcript levels, as a possible mechanism to control tumor progression. We suggest that the usage of diet and supplements containing selenium should be carefully used for patients with melanoma.

Association between TXNRD1 polymorphisms and anti-tuberculosis drug-induced hepatotoxicity in a prospective study.

Anti-tuberculosis drug-induced hepatotoxicity (ATDH) is a serious adverse reaction to anti-tuberculosis (TB) treatment. Thioredoxin reductase 1 (TXNRD1), encoded by the TXNRD1 gene, is an important enzyme involved in oxidant challenge. TXNRD1 plays a key role in regulating cell growth and transformation, and protects cells against oxidative damage. We investigated the association between TXNRD1 polymorphisms and ATDH susceptibility. In this prospective study, 280 newly diagnosed TB patients were followed-up for 3 months after beginning anti-TB therapy. Tag single-nucleotide polymorphisms (tag-SNPs) of TXNRD1 were selected using Haploview 4.2 based on the HapMap database of the Chinese Han in Beijing (CHB) panel. Genotyping was performed using the MassARRAY platform. Of the 280 patients enrolled in this study, 33 were lost to follow-up, 24 had ATDH, and 223 were free from ATDH. After adjusting for sex, age, smoking status, and body mass index, there were no significant differences in the allele and genotype frequency distributions of TXNRD1 SNPs between the ATDH and non-ATDH groups (all P > 0.05). The haplotype analysis showed that haplotype TCAGCC was associated with an increased risk of ATDH susceptibility [P = 0.024, OR (95%CI) = 6.273 (1.023-38.485)]. Further stratified analyses showed that the haplotype TCAGCC was associated with ATDH susceptibility in female subjects [P = 0.036, OR (95%CI) = 5.711 (0.917-35.560)] and non-smokers [P = 0.029, OR (95%CI) = 6.008 (0.971-37.158)]. Our results suggest that TXNRD1 variants may favor ATDH susceptibility in females and non-smokers. Further studies are required to verify this association.

Synthesis and biological evaluation of new nitrogen-containing diselenides.

The antioxidant properties of organoselenium compounds have been extensively investigated with the aim of developing new drugs, since oxidative stress is responsible for a variety of chronic human diseases. Herein, we reported the synthesis of new nitrogen-containing diselenides by a simple and efficient synthetic route. The products were obtained in good to excellent yields and their identification and characterization were achieved by NMR and HRMS techniques. The new derivatives may represent promising structures with different biological activities, which can act against oxidative stress through diverse mechanisms of action. The glutathione peroxidase-like assay (GPx-like activity) of the new synthesized compounds indicated that they reduced H2O2 to water at the expense of PhSH. The best results were obtained with diselenide 2b, which was 9 times more active than the standard organoselenium drug ebselen and, in contrast, this compound was not reduced by hepatic TrxR. All of the new compounds inhibited Fe(II)-induced TBARS.

Acute exposure to low lead levels and its implications on the activity and expression of cytosolic thioredoxin reductase in the kidney.

Renal thioredoxin reductase-1 (TrxR-1) activity is stimulated at lead doses lower than that necessary to inhibit δ-aminolevulinate dehydratase activity (δ-ALA-D), which is a classical early biomarker of lead effects. Thus, we hypothesized that the activity of TrxR-1 could be a more sensitive early indicator of lead effects than is δ-ALA-D. To evaluate this hypothesis, we assessed the blood and renal TrxR-1 activity and its gene expression along with biomarkers of oxidative damage, antioxidant enzyme activities and biomarkers of lead exposure in rats acutely exposed to lead. A histopathological analysis was performed to verify renal damage. The increase in renal TrxR-1 activity paralleled the increase in the blood and renal lead levels at 6, 24 and 48 hr after the exposure to 25 mg/kg lead acetate (p < 0.05), whereas its expression was increased 24 and 48 hr after exposure. These effects were not accompanied by oxidative or tissue damage in the kidneys. Blood TrxR-1 activity was not affected by lead exposure (up to 25 mg/kg). Erythrocyte δ-ALA-D activity was inhibited 6 hr after the exposure to 25 mg/kg lead acetate (p < 0.05) but recovered thereafter. Renal δ-ALA-D activity decreased 24 and 48 hr after the exposure to 25 mg/kg lead acetate. There were no changes in any parameters at lead acetate doses <25 mg/kg. Our results indicate that blood TrxR-1 activity is not a suitable indicator of lead effects. In contrast, the increase in renal TrxR-1 expression and activity is implicated in the early events of lead exposure, most likely as a protective cellular mechanism against lead toxicity.

Intracerebral injection of oil cyst content of human craniopharyngioma (oil machinery fluid) as a toxic model in the rat brain.

Craniopharyngiomas (CPs) are benign epithelial cystic tumors of the sellar and suprasellar region with a high survival rate and high recurrence in children. CPs contain dense oily fluid, but little is known yet about this content and its contribution to tissue damage and tumoral growth. In this study, we developed a simple experimental model produced by intracortical injection to rats of the cyst fluid content collected from human CPs to explore its possible contribution to brain tissue damage. The cyst fluid of the CPs ("oil machinery fluid") was collected during surgical removal, briefly preserved and further tested in rats through intracortical infusion. The group receiving "oil machinery fluid" presented increased reactive oxygen species formation, oxidative damage to lipids and reactive gliosis accompanied by augmented immunoreactivity to peroxiredoxin and thioredoxin reductase 1 at 15, 30 and 45 days post-injection. Other markers of inflammation and cell damage were stimulated at all post-lesion days tested. There was also a body weight gain. The persistence of tissue damage and oxidative stress suggests that "oil machinery fluid" exerts progressive alterations similar to those observed in patients with CPs, supporting the concept that some components of cyst fluid may contribute to brain tissue damage in these patients.

Evaluation of in vitro antioxidant effect of new mono and diselenides.

This study was designed to examine the antioxidant activity in vitro of novel mono- and diselenide compounds. We compared whether the formation of p-methyl-selenol from compounds 1-phenyl-3-(p-tolylselanyl)propan-2-amine (C1) and 1,2-dip-tolyldiselenide (C4) and o-methoxy-selenol from compounds 1-(2-methoxyphenylselanyl)-3-phenylpropan-2-amine (C2) and 1,2-bis(2-methoxyphenyl)diselenide (C3) may be involved in their antioxidant effects. The compounds were tested against Fe(II) and sodium nitroprusside (SNP)-induced lipid peroxidation in rat brain and liver homogenates. Likewise, the antioxidant capacity of the compounds was assessed by their ability to decolorize the DPPH radical as well as the Fe(II) chelating assay through the reduction of molybdenum(VI) (Mo6+) to molybdenum(V) (Mo5+). This colorimetric assay was also used to quantify thiol peroxidase (GPx) and oxidase activity and thioredoxin reductase (TrxR) activity. The results showed that the novel selenide compounds inhibit the thiobarbituric acid reactive species (TBARS) induced by different pro-oxidants, but the monoselenides effects were significant only at concentrations higher than the concentrations of the diselenides. Similarly, the total antioxidant activity was higher in the diselenides. Moreover, GPx and TrxR activity was only observed for the diselenides, which indicates that these compounds are more stable selenol molecules than monoselenides.

Disubstituted diaryl diselenides as potential atheroprotective compounds: Involvement of TrxR and GPx-like systems.

Oxidative modifications of low-density lipoproteins (LDLs) have a determinant role in atherogenesis and the study of agents that can modulate LDL oxidation is of pharmacological and therapeutic significance. Therefore, the aim of this study was to evaluate the antioxidant effect of the disubstituted diaryl diselenides, p-methoxyl-diphenyl diselenide (p-CH(3)O-C(6)H(4)Se)(2) (DM) and p-chloro-diphenyl diselenide (p-Cl-C(6)H(4)Se)(2) (DC), on Cu(2+)-induced LDL oxidation. Both compounds caused a dose-dependent inhibition of human serum and isolated LDL oxidation evidenced by the increasing of the lag phase of lipid peroxidation and decreased the lipid oxidation rate (V(max)). The protein moieties from isolated LDL were also protected from Cu(2+)-induced oxidation. Moreover, the disubstituted diaryl diselenides efficiently decreased the oxidized LDL (ox-LDL) induced foam cell formation in J774A.1 macrophage cells. Mechanistically, we have demonstrated that the antioxidant and antiatherogenic effects of DM and DC are related to formation of their selenol intermediates (RSeH) either by a direct reaction with endogenous thiols (GPx-like activity) or via their reduction by TrxR (using NADPH as electron donor). Considering the powerful effect of DM and DC against LDL-induced toxicity, they could be considered for developing of new therapeutic approaches to preventing and treating atherosclerosis and cardiovascular diseases.

Imbalance in superoxide dismutase/thioredoxin reductase activities in hypercholesterolemic subjects: relationship with low density lipoprotein oxidation.

BACKGROUND: There is a relationship among hypercholesterolemia, oxidative stress and inflammation in the atherogenesis. Thus, the objective of the present study was to assess paraoxonase (PON1), superoxide dismutase (SOD) and thioredoxin reductase (TrxR-1) activities and their relationship with lipids, oxidative stress and inflammation in subjects with different low density lipoprotein-cholesterol (LDL) levels. METHODS: Serum lipids, highly sensitive C-reactive protein (hs-CRP), lipid and protein oxidation, oxidized LDL (LDLox) and LDLox autoantibodies (LDLoxAB) levels and enzymes activities were measured in a total of 116 subjects that were divided into the following groups according to their LDL levels: low-LDL group (LDL < 100 mg/dL, n = 23), intermediate-LDL group (LDL 100-160 mg/dL, n = 50) and high-LDL group (LDL > 160 mg/dL, n = 43). RESULTS: The LDLox and hs-CRP levels increased in the high-LDL group (2.7- and 3.7- fold, respectively), whereas the intermediate and high-LDL groups had higher LDLoxAB (2.2- and 3.1-fold) when compared to low-LDL group (p < 0.05). Similarly, SOD activity, the atherogenic index (AI) and protein oxidation were also higher in the intermediate (1.3-, 1.3- and 1.2-fold) and high-LDL (1.6-, 2.3- and 1.6-fold) groups when compared to the low-LDL group (p < 0.05). Lipid oxidation and SOD/TrxR-1 ratio increased only in the high-LDL group (1.3- and 1.6-fold) when compared to the low-LDL group (p < 0.05). The SOD/TrxR-1 ratio was positively correlated to TBARS (r = 0.23, p < 0.05), LDLox (r = 0.18, p < 0.05), LDLoxAB (r = 0.21, p < 0.05), LDL (r = 0.19, p < 0.05) and AI (r = 0.22, p < 0.05). PON1 and TrxR-1 activities were similar among groups. CONCLUSIONS: Some oxidative events initiate when LDL levels are clinically acceptable. Moreover, hypercholesterolemic patients have an imbalance in SOD and TrxR-1 activities that is positively associated to LDL oxidation.

Low 8-oxo-7,8-dihydro-2'-deoxyguanosine levels and influence of genetic background in an Andean population exposed to high levels of arsenic.

BACKGROUND: Arsenic (As) causes oxidative stress through generation of reactive oxygen species. 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a sensitive marker of oxidative DNA damage, has been associated with As exposure in some studies, but not in others, possibly due to population-specific genetic factors. OBJECTIVES: To evaluate the association between As and 8-oxodG in urine in a population with a low urinary monomethylated As (%MMA) and high dimethylated As (%DMA), as well as the genetic impact on (a) 8-oxodG concentrations and (b) the association between As and 8-oxodG. MATERIALS AND METHODS: Women (N=108) in the Argentinean Andes were interviewed and urine was analyzed for arsenic metabolites (ICPMS) and 8-oxodG (LC-MS/MS). Twenty-seven polymorphisms in genes related to oxidative stress and one in As(+III)methyltransferase (AS3MT) were studied. RESULTS: Median concentration of 8-oxodG was 4.7 nmol/L (adjusted for specific weight; range 1.6-13, corresponding to 1.7 microg/g creatinine, range 0.57-4.8) and of total urinary As metabolites (U-As) 290 microg/L (range 94-720; 380 microg/g creatinine, range 140-1100). Concentrations of 8-oxodG were positively associated with %MMA (strongest association, p=0.013), and weakly associated with U-As (positively) and %DMA (negatively). These associations were strengthened when taking ethnicity into account, possibly reflecting genetic differences in As metabolism and genes regulating oxidative stress and DNA maintenance. A genetic influence on 8-oxodG concentrations was seen for polymorphisms in apurinic/apyrimidinic endonuclease 1 (APEX1), DNA-methyltransferases 1 and 3b (DNMT1, DNMT3B), thioredoxin reductase 1 (TXNRD1) and 2 (TXNRD2) and glutaredoxin (GLRX). CONCLUSION: Despite high As exposure, the concentrations of 8-oxodG in this population were low compared with other As-exposed populations studied. The strongest association was found for %MMA, stressing that some inconsistencies between As and 8-oxodG partly depend on population variations in As metabolism. We found evidence of genetic impact on 8-oxodG concentrations.

Effect of lead acetate on cytosolic thioredoxin reductase activity and oxidative stress parameters in rat kidneys.

Oxidative stress has been suggested to be an important molecular mechanism of toxic effects of lead in the kidney. Thioredoxin reductase-1 is a selenoprotein involved in many cellular redox processes. This study evaluated the effect of acute and chronic exposure intraperitoneally to lead acetate on thioredoxin reductase-1 activity and on other oxidative stress parameters in the rat kidney, as well as on indicators of renal function commonly used to assess lead poisoning. Acute exposure to 25 mg/kg lead acetate increased superoxide dismutase and thioredoxin reductase-1 activity (after 6, 24 and 48 hr), while exposure to 50 mg/kg lead acetate increased catalase activity (after 48 hr) and inhibited delta-aminolevulinate dehydratase activity (after 6, 24 and 48 hr) in the kidney (P < 0.05). Chronic exposure (30 days) to 5 mg/kg lead acetate inhibited delta-aminolevulinate dehydratase and increased glutathione S-transferase, non-protein thiol groups, catalase, thioredoxin reductase-1 and uric acid plasma levels, while exposure to 25 mg/kg lead acetate reduced body weight and delta-aminolevulinate dehydratase, but increased glutathione S-transferase, non-protein thiol groups and uric acid plasma levels (P < 0.05). No changes were observed in thiobarbituric acid reactive substances, glutathione peroxidase, creatinine or inorganic phosphate levels after either acute or chronic exposure. Our results suggest that thioredoxin reductase-1 may be an early indicator of acute exposure to low lead doses.