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Background: Exposure of skin to long-wave UV radiation (UVA) increases the cellular levels of reactive oxygen species (ROS), which have been linked to apoptosis induction through the damage of lipids, proteins, and nucleic acids. Peroxiredoxin I (Prx I) is one of a family of antioxidant proteins that plays a protective role against oxidative damage; however the role of Prx I in UVA-induced damage remains to be clarified., Objective: Here we investigated the protective role of Prx I against UVA-induced changes using mouse embryonic fibroblasts (MEFs) derived from Prx I homozygous knockout (Prx I (-/-)) mice., Methods: Prx I (-/-) and wild-type (Prx I (+/+)) MEFs were subjected to UVA irradiation, and the resulting apoptosis was analyzed using flow cytometry, quantitative real-time PCR, and western blotting., Results: Prx I (-/-) MEFs showed enhanced sensitivity to UVA treatment, exhibiting increased apoptosis and ROS production compared to Prx I (+/+) MEFs. Consistent with the increase in apoptosis, p53 expression was significantly higher, while Bcl-2, Bcl-xL, and Nrf2 expressions were all lower in Prx I (-/-) versus (+/+) MEFs. The UVA-induced inflammatory response was upregulated in Prx I (-/-) MEFs, as indicated by increased expressions of IκB, TNFα, and IL-6. Evidence was presented indicating that Prx I impacts these pathways by modifying critical signaling intermediates including p53, IκB, and Nrf2., Conclusion: Our results indicate that Prx I plays a protective role against UVA-induced oxidative damage by controlling ROS accumulation. Both the UVA-induced apoptotic and inflammatory signals were found to be modulated by Prx I., (Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Expression of antioxidant enzymes is regulated by transcription factor NF-E2-related factor (Nrf2) and induced by oxidative stress. Reactive oxygen species contribute to the formation of several types of cochlear injuries, including age-related hearing loss and gentamicin ototoxicity. In this study, we examined the roles of Nrf2 in age-related hearing loss and gentamicin ototoxicity by measuring auditory brainstem response thresholds in Nrf2-knockout mice. Although Nrf2-knockout mice maintained normal auditory thresholds at 3 months of age, their hearing ability was significantly more impaired than that of age-matched wild-type mice at 6 and 11 months of age. Additionally, the numbers of hair cells and spiral ganglion cells were remarkably reduced in Nrf2-knockout mice at 11 months of age. To examine the importance of Nrf2 in protecting against gentamicin-induced ototoxicity, 3-day-old mouse organ of Corti explants were cultured with gentamicin. Hair cell loss caused by gentamicin treatment was enhanced in the Nrf2-deficient tissues. Furthermore, the expressions of some Nrf2-target genes were activated by gentamicin treatment in wild-type mice but not in Nrf2-knockout mice. The present findings indicate that Nrf2 protects the inner ear against age-related hearing injuries and gentamicin ototoxicity by up-regulating antioxidant enzymes and detoxifying proteins., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The anticancer agent cis-diamminedichloroplatinum (cisplatin) is a first-line chemotherapeutic agent for oral cancer. Cell exposure to cisplatin is associated with increased oxidative stress and post-translational changes in components of apoptosis pathways, including p38 Mitogen-activated protein kinase (MAPK), c-Jun-NH2-kinase (JNK), and extracellular signal-regulated kinase (ERK). Peroxiredoxin (Prx) I is an oxidative stress-inducible protein expressed in many tissues and important for reducing reactive oxygen species in vivo; however, whether Prx I helps protect cells from cisplatin injury is unknown. In this report, we examined the effects of Prx I on cell sensitivity to cisplatin-induced apoptosis. Mouse embryo fibroblasts (MEFs) derived from Prx I-deficient mice showed increased cisplatin-induced apoptosis compared with wild-type MEFs. Cisplatin treatment also led to increased activation of p38 MAPK and JNK, and reduced ERK phosphorylation in Prx I-deficient MEFs compared with wild-type MEFs. Furthermore, JNK- and ERK-specific inhibitors protected the Prx I-deficient MEFs from cisplatin-induced apoptosis, but Prx I-deficient MEFs remained more sensitive than wild-type MEFs when treated with a p38 MAPK-specific inhibitor. These findings indicate that Prx I modulates the cisplatin-evoked activation of MAPKs that lead to apoptosis, and Prx I may thus represent a useful target as a protective therapy against cisplatin cytotoxicity.

The transcription factor Nrf2 is a key regulator for hepatic induction of detoxifying enzymes, antioxidative stress genes and Mrp efflux transporters. We aimed to investigate whether Nrf2 activation counteracts liver injury associated with cholestasis. The role of Nrf2 activation in counteracting cholestatic liver injury was studied using a bile duct-ligation (BDL) model of Keap1 gene-knockdown (Keap1-kd) mice that represent the sustained activation of Nrf2 in the liver. Upon Nrf2 activation, Keap1-kd mice showed large increases in Mrp efflux transporters, detoxifying enzymes and antioxidative stress genes in the livers. After BDL, the number of hepatic parenchymal necrosis and the reactive oxygen species content were significantly smaller in the livers of the Keap1-kd mice than in those of the WT mice. Moreover, the increase in serum bilirubin levels was attenuated in the Keap1-kd mice. In conclusion, the results suggest a hepatoprotective role of sustained Nrf2 activation against liver injury associated with cholestasis.

Much of the cell injury caused by ultraviolet A (UVA) irradiation is associated with oxidative stress. Quercetin is a major natural polyphenol that is known to protect cells from UVA-induced damage. Here, we investigated the molecular mechanism of this protection. Quercetin pretreatment strongly suppressed UVA-induced apoptosis in human keratinocyte HaCaT cells, markedly increased protein levels of the transcription factor Nrf2, induced the expression of antioxidative genes, and dramatically reduced the production of reactive oxygen species following UVA irradiation. Importantly, these beneficial effects were greatly attenuated by downregulating Nrf2 expression. Thus, quercetin protects cells from UVA damage mainly by elevating intracellular antioxidative activity via the enhanced accumulation of a transcription factor for antioxidant genes, Nrf2.

We previously demonstrated that Nrf2 regulates oxidized LDL-mediated CD36 expression in macrophages. The current study aimed to determine the mechanism of Nrf2-mediated macrophage CD36 induction. Treatment with the Nrf2 activator diethylmaleate, but not PPARgamma specific ligands, caused marked upregulation of CD36 in mouse macrophage RAW264.7 cells. Similarly, Nrf2 activators induced CD36 expression in bone marrow-derived macrophages in a Nrf2-dependent manner. Induced expression of the three alternative first exons of mouse CD36, deemed 1A, 1B, and 1C, occurred upon Nrf2 activation with exon1A mainly contributing to the CD36 expression. Four antioxidant response elements (AREs) lie within close proximity to these three exons, and chromatin immunoprecipitation assays demonstrated that two AREs upstream of exon1A, the distal 1A-ARE1, and the proximal 1A-ARE2, were Nrf2-responsive. Luciferase reporter assays conclusively demonstrated that 1A-ARE2 is the critical regulatory element for the Nrf2-mediated gene expression. Thus Nrf2 directly regulates CD36 gene expression by binding to 1A-ARE2.

Peroxiredoxin I (Prx I) is a key cytoplasmic peroxidase that reduces intracellular hydroperoxides in concert with thioredoxin. To study the role of tissue Prx I in protection from oxidative stress, we generated Prx I-/- mice by gene trapping. We then evaluated the acute-phase tissue damage caused by ferric-nitrilotriacetate (Fe-NTA). Increases in serum aspartate aminotransferase and alanine aminotransferase levels were significantly greater in Prx I-/- than wild-type mice, 4 and 12 h after the injection of Fe-NTA. Using real-time EPR imaging, we examined the reduction of the stable paramagnetic nitroxyl radical 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl in vivo, and found that the half-life of this spin probe in the liver and kidney was significantly prolonged in the Prx I-/- mice. These results demonstrate that Prx I-/- mice have less reducing activity and are more susceptible to the damage mediated by reactive oxygen species in vivo than wild-type mice.

Sub-lethal levels of arsenic induce upregulation of stress proteins. We here report for the first time that inorganic arsenic activates the transcription factor Nrf2, which controls the expression of oxidative stress-induced proteins. Treatment of cultured MC3T3-E1 osteoblasts with arsenite or arsenate induced increase of Nrf2, followed by transcriptional activation of target genes encoding HO-1, Prx I, and A170. We found that arsenate (200-800 micro M) only slightly increased the normal 60kDa A170 protein but markedly increased higher molecular mass forms of A170 (HMM-A170) that appeared as smeared bands. Arsenate also markedly increased ubiquitin-conjugated cellular proteins, suggesting that HMM-A170 was one of the poly-ubiquitinated proteins. Arsenite (50-100 micro M) also induced accumulation of HMM-A170 and ubiquitin-conjugated proteins. These results provide the first direct evidence that toxic arsenics impair the normal function of A170. Our findings provide a potential diagnostic tool for monitoring biotoxicity in cells and tissues in response to arsenic compounds.