BACKGROUND: Oxidative stress plays a crucial role in the pathogenesis of many skeletal diseases by inducing osteocyte death. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of various antioxidant gene expressions through antioxidant response element (ARE) against cellular oxidative stress and can be induced by various stimulants, including the phytochemicals methysticin (MET) and L-sulforaphane (SFN). This study aimed to establish an osteocyte in vitro model to investigate the pharmacological effects of MET and SFN on the Nrf2/ARE pathway.
METHODS: MLO-Y4 murine osteocytes and the stably transduced MLO-Y4-SIN-lenti-ARE reporter gene cell line were used. MET and SFN were used as Nrf2 inducers. The cytotoxicity of MET, SFN, and hydrogen peroxide (H2O2) was evaluated using the CytoTox-Glo™ Assay. Time- and dose-dependent ARE induction was examined by Monoluciferase Assay. The mRNA and protein expressions of Nrf2 target markers, such as heme-oxygenase 1 (Ho-1), NADPH quinone dehydrogenase 1 (Nqo1), and thioredoxin reductase 1 (Txnrd1), were detected by RT-qPCR, Western Blot, and immunofluorescence staining, respectively. Osteogenesis markers, osteopontin, and osteocalcin were compared with and without treatment by immunofluorescence staining.
RESULTS: The experimental data showed that MET and SFN induced ARE activity in a time- and dose-dependent manner and increased the mRNA and protein expression of antioxidant markers compared to vehicle-treated controls. The protein expression of osteopontin and osteocalcin in the samples treated with SFN were significantly higher than without treatment, and the number of cell death treated with SFN was significantly lower than without treatment under H2O2-induced stress conditions.
CONCLUSIONS: Nrf2 inducers MET and SFN increased the mRNA expression of antioxidant genes through the Nrf2/ARE pathway in osteocytes. Notably, SFN increased the protein expression of osteocyte-associated osteogenic markers and suppressed cell death under H2O2-induced stress condition. Thus, Nrf2 stimulators can exert stress-relieving and osteogenic effects on osteocytes.

Gold(III) complexes with 1,7- and 4,7-phenanthroline ligands, [AuCl3(1,7-phen-κN7)] (1) and [AuCl3(4,7-phen-κN4)] (2) were synthesized and structurally characterized by spectroscopic (NMR, IR and UV-vis) and single-crystal X-ray diffraction techniques. In these complexes, 1,7- and 4,7-phenanthrolines are monodentatedly coordinated to the Au(III) ion through the N7 and N4 nitrogen atoms, respectively. In comparison to the clinically relevant anti-angiogenic compounds auranofin and sunitinib, gold(III)-phenanthroline complexes showed from 1.5- to 20-fold higher anti-angiogenic potential, and 13- and 118-fold lower toxicity. Among the tested compounds, complex 1 was the most potent and may be an excellent anti-angiogenic drug candidate, since it showed strong anti-angiogenic activity in zebrafish embryos achieving IC50 value (concentration resulting in an anti-angiogenic phenotype at 50% of embryos) of 2.89μM, while had low toxicity with LC50 value (the concentration inducing the lethal effect of 50% embryos) of 128μM. Molecular docking study revealed that both complexes and ligands could suppress angiogenesis targeting the multiple major regulators of angiogenesis, such as the vascular endothelial growth factor receptor (VEGFR-2), the matrix metalloproteases (MMP-2 and MMP-9), and thioredoxin reductase (TrxR1), where the complexes showed higher binding affinity in comparison to ligands, and particularly to auranofin, but comparable to sunitinib, an anti-angiogenic drug of clinical relevance.

Oxidative stress plays a role in UV-induced melanoma, which may arise from melanocytic nevi. We investigated whether oral administration of the antioxidant N-acetylcysteine (NAC) could protect nevi from oxidative stress in vivo in the setting of acute UV exposure. The minimal erythemal dose (MED) was determined for 100 patients at increased risk for melanoma. Patients were randomized to receive a single dose (1,200 mg) of NAC or placebo, in double-blind fashion, and then one nevus was irradiated (1-2 MED) using a solar simulator. One day later, the MED was redetermined and the irradiated nevus and a control unirradiated nevus were removed for histologic analysis and examination of biomarkers of NAC metabolism and UV-induced oxidative stress. Increased expression of 8-oxoguanine, thioredoxin reductase-1, and γ-glutamylcysteine synthase modifier subunit were consistently seen in UV-treated compared with unirradiated nevi. However, no significant differences were observed in these UV-induced changes or in the pre- and postintervention MED between those patients receiving NAC versus placebo. Similarly, no significant differences were observed in UV-induced changes between subjects with germline wild-type versus loss-of-function mutations in the melanocortin-1 receptor. Nevi showed similar changes of UV-induced oxidative stress in an open-label post-trial study in 10 patients who received NAC 3 hours before nevus irradiation. Thus, a single oral dose of NAC did not effectively protect nevi from UV-induced oxidative stress under the conditions examined. Cancer Prev Res; 10(1); 36-44. ©2016 AACR.

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.

Selenium (Se) is an essential trace element in human nutrition, but its role in certain health conditions, particularly among Se sufficient populations, is controversial. A genome-wide association study (GWAS) of blood Se concentrations previously identified a locus at 5q14 near BHMT. We performed a GW meta-analysis of toenail Se concentrations, which reflect a longer duration of exposure than blood Se concentrations, including 4162 European descendants from four US cohorts. Toenail Se was measured using neutron activation analysis. We identified a GW-significant locus at 5q14 (P < 1 × 10(-16)), the same locus identified in the published GWAS of blood Se based on independent cohorts. The lead single-nucleotide polymorphism (SNP) explained ∼1% of the variance in toenail Se concentrations. Using GW-summary statistics from both toenail and blood Se, we observed statistical evidence of polygenic overlap (P < 0.001) and meta-analysis of results from studies of either trait (n = 9639) yielded a second GW-significant locus at 21q22.3, harboring CBS (P < 4 × 10(-8)). Proteins encoded by genes at 5q14 and 21q22.3 function in homocysteine (Hcy) metabolism, and index SNPs for each have previously been associated with betaine and Hcy levels in GWAS. Our findings show evidence of a genetic link between Se and Hcy pathways, both involved in cardiometabolic disease.

BACKGROUND: The thioredoxin system maintains redox balance through the action of thioredoxin and thioredoxin reductase. Thioredoxin regulates the activity of various substrates, including those that function to counteract cellular oxidative stress. These include the peroxiredoxins, methionine sulfoxide reductase A and specific transcription factors. Of particular relevance is Redox Factor-1, which in turn activates other redox-regulated transcription factors.
METHODS: Experimentally defined transcription factor binding sites in the human thioredoxin and thioredoxin reductase gene promoters together with promoters of the major thioredoxin system substrates involved in regulating cellular redox status are discussed. An in silico approach was used to identify potential putative binding sites for these transcription factors in all of these promoters.
CONCLUSIONS: Our analysis reveals that many redox gene promoters contain the same transcription factor binding sites. Several of these transcription factors are in turn redox regulated. The ARE is present in several of these promoters and is bound by Nrf2 during various oxidative stress stimuli to upregulate gene expression. Other transcription factors also bind to these promoters during the same oxidative stress stimuli, with this redundancy supporting the importance of the antioxidant response. Putative transcription factor sites were identified in silico, which in combination with specific regulatory knowledge for that gene promoter may inform future experiments.
CONCLUSIONS: Redox proteins are involved in many cellular signalling pathways and aberrant expression can lead to disease or other pathological conditions. Therefore understanding how their expression is regulated is relevant for developing therapeutic agents that target these pathways.