Glutathione Peroxidase

Ferroptosis is a form of nonapoptotic cell death for which key regulators remain unknown. We sought a common mediator for the lethality of 12 ferroptosisinducing small molecules. We used targeted metabolomic profiling to discover that depletion of glutathione causes inactivation of glutathione peroxidases (GPXs) in response to one class of compounds and a chemoproteomics strategy to discover that GPX4 is directly inhibited by a second class of compounds. GPX4 overexpression and knockdown modulated the lethality of 12 ferroptosis inducers, but not of 11 compounds with other lethal mechanisms. In addition, two representative ferroptosis inducers prevented tumor growth in xenograft mouse tumor models. Sensitivity profiling in 177 cancer cell lines revealed that diffuse large B cell lymphomas and renal cell carcinomas are particularly susceptible to GPX4-regulated ferroptosis. Thus, GPX4 is an essential regulator of ferroptotic cancer cell death.

This study investigates the effects of acute exposure to organophosphate insecticide malathion (250mg/kg, i.p.) and/or ZnCl2 (5mg/kg, i.p.), with the following parameters: lipid peroxidation and the activity of acetylcholinesterase (AChE), glutathione reductase (GR), glutathione S-transferase (GST), glutathione peroxidase (GPx), glucose-6-phosphate dehydrogenase (G6PDH), and the levels of total glutathione (GSH-t) in the hippocampus and cerebral cortex of female rats. Malathion exposure

10. Methods used to house and feed male C57BL/6 mice, a commonly used model in aging research with an average life-span of ϳ30 months, were recently described [T. D. Pugh, T. D. Oberley, R. Weindruch, Cancer Res. 59, 642 (1999)]. 11. Total RNA was extracted from frozen tissue by using TRIZOL reagent (Life Technologies). Polyadenylate [poly(A) ϩ ] RNA was purified from the total RNA with oligo-dT-linked Oligotex resin (Qiagen). One microgram of poly(A) ϩ RNA was converted into doublestranded cDNA (ds-cDNA) by using SuperScript Choice System (Life Technologies) with an oligo-dT primer containing a T7 RNA polymerase promoter (Genset). After second-strand synthesis, the reaction mixture was extracted with phenol-chloroformisoamyl alcohol, and ds-cDNA was recovered by ethanol precipitation. In vitro transcription was performed by using a T7 Megascript Kit (Ambion) with 1.5 l of ds-cDNA template in the presence of a mixture of unlabeled ATP, CTP, GTP, and UTP and biotin-labeled CTP and UTP [bio-11-CTP and bio-16-UTP (Enzo)]. Biotin-labeled cRNA was purified by using an RNeasy affinity column (Qiagen), and fragmented randomly to sizes ranging from 35 to 200 bases by incubating at 94°C for 35 min. The hybridization solutions contained 100 mM MES, 1 M Na ϩ , 20 mM EDTA, and 0.01% Tween 20. The final concentration of fragmented cRNA was 0.05 g/l in the hybridization solutions. After hybridization, the hybridization solutions were removed and the gene chips were washed and stained with streptavidinphycoerythrin. DNA chips were read at a resolution of 6 m with a Hewlett-Packard GeneArray Scanner. 12. Detailed protocols for data analysis of Affymetrix microarrays and extensive documentation of the sensitivity and quantitative aspects of the method have been described [D. J. Lockhart, Nature Biotechnol. 14, 1675 (1996)]. Briefly, each gene is represented by the use of ϳ20 perfectly matched (PM) and mismatched (MM) control probes. The MM probes act as specificity controls that allow the direct subtraction of both background and cross-hybridization signals. The number of instances in which the PM hybridization signal is larger than the MM signal is computed along with the average of the logarithm of the PM:MM ratio (after background subtraction) for each probe set. These values are used to make a matrix-based decision concerning the presence or absence of an RNA molecule. Positive average signal intensities after background subtraction were observed for over 4000 genes for all samples. To determine the quantitative RNA abundance, the average of the differences representing PM minus MM for each gene-specific probe family is calculated, after discarding the maximum, the minimum, and any outliers beyond 3 SDs. Averages of pairwise comparisons were made between animals with Affymetrix software. To determine the effect of age, each 5-month-old mouse (n ϭ 3) was compared to each 30-month-old (n ϭ 3) mouse, generating a total of nine pairwise comparisons. To determine the effect of diet, 30-month-old CR-fed (n ϭ 3) and 30-month-old control-fed (n ϭ 3) animals were similarly compared. Pearson correlation coeficients were calculated between individual animals in the same age/diet groups. No correlation coeficient between

A systematic study on the in vitro interactions of 7-20 nm spherical silver nanoparticles (SNP) with HT-1080 and A431 cells was undertaken as a part of an on-going program in our laboratory to develop a topical antimicrobial agent for the treatment of burn wound infections.

For the past 40 years or so, oxidative stress has been increasingly recognized as a contributing factor in aging and in various forms of pathophysiology generally associated with aging. Our view of oxidative stress has been largely ''superoxide-centric'', as we focused on the pathological sources of this oxygen-derived free radical and the types of molecular havoc it can wreak, as well as on the protection provided by the antioxidant enzymes, especially the superoxide dismutases, catalases, and glutathione peroxidases. In the last decade our view of oxidative stress has broadened considerably, and it is now often seen as an imbalance that has its origins in our genes, and the ways in which gene expression is regulated. At the center of this new focus is the transcription factor called nuclear factor (erythroid-derived 2)-like 2, or Nrf2. Nrf2 is referred to as the ''master regulator'' of the antioxidant response, modulating the expression of hundreds of genes, including not only the familiar antioxidant enzymes, but large numbers of genes that control seemingly disparate processes such as immune and inflammatory responses, tissue remodeling and fibrosis, carcinogenesis and metastasis, and even cognitive dysfunction and addictive behavior. Thus, the dysregulation of Nrf2-regulated genes provides a logical explanation for the connections, both direct and indirect, between observable oxidative stress and perhaps 200 human diseases involving these various physiological processes, each reflecting a network involving many gene products. The evolutionary self-association of these many genes under the common control of Nrf2 suggests that the immune and inflammatory systems may present the largest demand for increased antioxidant protection, apart from constitutive oxidative stress resulting from mitochondrial oxygen consumption for metabolic purposes. Gene expression microarray data on human primary vascular endothelial cells and on the SK-N-MC human neuroblastoma-derived cell line have been obtained in response to the dietary supplement Protandim, a potent composition of highly synergistic phytochemical Nrf2 activators. Pathway analysis of results shows significant modulation by Protandim of pathways involving not only antioxidant enzymes, but of those related to colon cancer, cardiovascular disease, and Alzheimer disease.