Antioxidants and selenocompounds inhibit 3,5-dimethylaminophenol toxicity to human urothelial cells.

Exposure to alkyl anilines may lead to bladder cancer, which is the second most frequent cancer of the urogenital tract. 3,5-dimethylaniline is highly used in industry. Studies on its primary metabolite 3,5-dimethylaminophenol (3,5-DMAP) showed that this compound causes oxidative stress, changes antioxidant enzyme activities, and leads to death of different mammalian cells. However, there is no in vitro study to show the direct effects of 3,5-DMAP on human bladder and urothelial cells. Selenocompounds are suggested to decrease oxidative stress caused by some chemicals, and selenium supplementation was shown to reduce the risk of bladder cancer. The main aim of this study was to investigate whether selenocompounds organic selenomethionine (SM, 10 µmol/L) or inorganic sodium selenite (SS, 30 nmol/L) could reduce oxidative stress, DNA damage, and apoptosis in UROtsa cells exposed to 3,5-DMAP. 3,5-DMAP caused a dose-dependent increase in intracellular generation of reactive oxygen species, and its dose of 50 µmol/L caused lipid peroxidation, protein oxidation, and changes in antioxidant enzyme activities in different cellular fractions. The comet assay also showed single-strand DNA breaks induced by the 3,5-DMAP dose of 50 µmol/L, but no changes in double-strand DNA breaks. Apoptosis was also triggered. Both selenocompounds provided partial protection against the cellular toxicity of 3,5-DMAP. Low selenium status along with exposure to alkyl anilines can be a major factor in the development of bladder cancer. More mechanistic studies are needed to specify the role of selenium in bladder cancer.

Untargeted Proteomics and Systems-Based Mechanistic Investigation of Artesunate in Human Bronchial Epithelial Cells.

The antimalarial drug artesunate is a semisynthetic derivative of artemisinin, the principal active component of a medicinal plant Artemisia annua. It is hypothesized to attenuate allergic asthma via inhibition of multiple signaling pathways. We used a comprehensive approach to elucidate the mechanism of action of artesunate by designing a novel biotinylated dihydroartemisinin (BDHA) to identify cellular protein targets of this anti-inflammatory drug. By adopting an untargeted proteomics approach, we demonstrated that artesunate may exert its protective anti-inflammatory effects via direct interaction with multiple proteins, most importantly with a number of mitochondrial enzymes related to glucose and energy metabolism, along with mRNA and gene expression, ribosomal regulation, stress responses, and structural proteins. In addition, the modulatory effects of artesunate on various cellular transcription factors were investigated using a transcription factor array, which revealed that artesunate can simultaneously modulate multiple nuclear transcription factors related to several major pro- and anti-inflammatory signaling cascades in human bronchial epithelial cells. Artesunate significantly enhanced nuclear levels of nuclear factor erythroid-2-related factor 2 (Nrf2), a key promoter of antioxidant mechanisms, which is inhibited by the Kelch-like ECH-associated protein 1 (Keap1). Our results demonstrate that, like other electrophilic Nrf2 regulators, artesunate activates this system via direct molecular interaction/modification of Keap1, freeing Nrf2 for transcriptional activity. Altogether, the molecular interactions and modulation of nuclear transcription factors provide invaluable insights into the broad pharmacological actions of artesunate in inflammatory lung diseases and related inflammatory disorders.

Cytoplasmic and nuclear toxicity of 3,5-dimethylaminophenol and potential protection by selenocompounds.

Most common alkylanilines in the environment are 2,6-dimethylaniline (2,6-DMA), 3,5-dimethylaniline (3,5-DMA), and 3-ethylaniline (3-EA). 3,5-Dimethylaminophenol (3,5-DMAP), a metabolite of 3,5-DMA, is of particular interest, as it is potentially genotoxic. Supplementation with organic or inorganic forms of selenium (Se) may reduce toxicity following exposure to a wide variety of environmental chemicals. This study was designed to evaluate the protective effects of sodium selenite (SS) and selenomethionine (SM) at varying time points of supplementation (24 h and 72 h) against the cytotoxicity, reactive oxygen species (ROS) production, and genotoxicity of 3,5-DMAP in CHO AS52 cells. 3,5-DMAP caused dose-dependent increase of cytotoxicity, ROS production and genotoxicity, and generated free radicals in the nuclei. Thioredoxin reductase (TrxR), catalase and glutathione reductase activities, and glutathione levels were significantly lower while lipid peroxidation and protein oxidation levels were higher after 3,5-DMAP treatment in both cytoplasm and the nucleus vs. control. After 24 h, both SS and SM provided protection in antioxidant/oxidant status of the 3,5-DMAP-treated cells; however other than supplying higher glutathione peroxidase and TrxR activities, 72 h supplementation did not provide advanced improvement. Selenocompounds may be beneficial against cytotoxic and genotoxic potential of 3,5-DMAP and might protect both nucleus and cytoplasm following exposure to alkylanilines.

Endogenously produced nitric oxide mitigates sensitivity of melanoma cells to cisplatin.

Melanoma patients experience inferior survival after biochemotherapy when their tumors contain numerous cells expressing the inducible isoform of NO synthase (iNOS) and elevated levels of nitrotyrosine, a product derived from NO. Although several lines of evidence suggest that NO promotes tumor growth and increases resistance to chemotherapy, it is unclear how it shapes these outcomes. Here we demonstrate that modulation of NO-mediated S-nitrosation of cellular proteins is strongly associated with the pattern of response to the anticancer agent cisplatin in human melanoma cells in vitro. Cells were shown to express iNOS constitutively, and to generate sustained nanomolar levels of NO intracellularly. Inhibition of NO synthesis or scavenging of NO enhanced cisplatin-induced apoptotic cell death. Additionally, pharmacologic agents disrupting S-nitrosation markedly increased cisplatin toxicity, whereas treatments favoring stabilization of S-nitrosothiols (SNOs) decreased its cytotoxic potency. Activity of the proapoptotic enzyme caspase-3 was higher in cells treated with a combination of cisplatin and chemicals that decreased NO/SNOs, whereas lower activity resulted from cisplatin combined with stabilization of SNOs. Constitutive protein S-nitrosation in cells was detected by analysis with biotin switch and reduction/chemiluminescence techniques. Moreover, intracellular NO concentration increased significantly in cells that survived cisplatin treatment, resulting in augmented S-nitrosation of caspase-3 and prolyl-hydroxylase-2, the enzyme responsible for targeting the prosurvival transcription factor hypoxia-inducible factor-1α for proteasomal degradation. Because activities of these enzymes are inhibited by S-nitrosation, our data thus indicate that modulation of intrinsic intracellular NO levels substantially affects cisplatin toxicity in melanoma cells. The underlying mechanisms may thus represent potential targets for adjuvant strategies to improve the efficacy of chemotherapy.