<p>Effects of TR1 and TR2 knock down on 2-DG treated CaSki cells. (A & C) CaSki and SiHa cells were transfected with control and TR1, TR2 and TR1+TR2 siRNA and after 24h, treated with 20mM 2-DG, 48h and CFA was performed. *p<0.01 Control + DG vs TR1/TR2+DG, *p<0.01 Control vs TR1/2 siRNA. (B & D) CaSki and SiHa cells were transfected with scrambled and TR1 and TR2 siRNA and protein levels were determined by Western blot.</p>

Energy-intensive kidney reabsorption processes essential for normal whole-body function are maintained by tubular epithelial cell metabolism. Tubular metabolism changes markedly following acute kidney injury (AKI), but which changes are adaptive versus maladaptive remain poorly understood. In publicly available data sets, we noticed a consistent downregulation of the mitochondrial pyruvate carrier (MPC) after AKI, which we experimentally confirmed. To test the functional consequences of MPC downregulation, we generated novel tubular epithelial cell-specific Mpc1 knockout (MPC TubKO) mice. 13C-glucose tracing, steady-state metabolomic profiling, and enzymatic activity assays revealed that MPC TubKO coordinately increased activities of the pentose phosphate pathway and the glutathione and thioredoxin oxidant defense systems. Following rhabdomyolysis-induced AKI, MPC TubKO decreased markers of kidney injury and oxidative damage and strikingly increased survival. Our findings suggest that decreased mitochondrial pyruvate uptake is a central adaptive response following AKI and raise the possibility of therapeutically modulating the MPC to attenuate AKI severity.

Role of anti-oxidant NAC on 2-DG, BSO and AUR induced cell viability. (A-B) Cells were seeded and treated with 2-DG and BSO for 24h followed by 5hrs of 10mM NAC with replacement of 2-DG, BSO and AUR for another 24h. Cell viability was determined by Alamar blue. Statistical Analysis: (A) BA vs NAC+BA, p<0.05, N=3 (B) BA vs NAC+BA and DBA vs NAC+DBA, p<0.05, N=3.

<p>Effects of TR1 and TR2 knock down on 2-DG treated CaSki cells. (A & C) CaSki and SiHa cells were transfected with control and TR1, TR2 and TR1+TR2 siRNA and after 24h, treated with 20mM 2-DG, 48h and CFA was performed. *p<0.01 Control + DG vs TR1/TR2+DG, *p<0.01 Control vs TR1/2 siRNA. (B & D) CaSki and SiHa cells were transfected with scrambled and TR1 and TR2 siRNA and protein levels were determined by Western blot.</p>

<p>Effect of catalase over expression in the clonogenic survival of 2-DG, BSO and AUR treated cells. (A-B) Cells were seeded and transduced with Ad CAT and Ad empty viruses and CFA was done as described in the materials and methods section. (C) CaSki cells were treated with 20mM 2-DG, 1mM BSO and 100nM AUR for 24h along with or without CQ (60μM) and cell viability was determined by Alamar Blue. Statistical Analysis: (A) *p < 0.05 Ad-empty vs Ad-CAT BSO (B) p < 0.07 Ad-empty vs Ad-CAT for DG, BSO and BA. (C) ***DBA vs DBA+CQ, p<0.05, N=3.</p>

The toxicity of many "inert" ingredients of pesticide formulations, such as safeners, is poorly characterized, despite evidence that humans may be exposed to these chemicals. Analysis of ToxCast data for dichloroacetamide safeners with the ToxPi tool identified benoxacor as the safener with the highest potential for toxicity, especially liver toxicity. Benoxacor was subsequently administered to mice via oral gavage for 3 days at concentrations of 0, 0.5, 5, and 50 mg/kg bodyweight (b.w.). Bodyweight-adjusted liver and testes weights were significantly increased in the 50 mg/kg b.w. group. There were no overt pathologies in either the liver or the intestine. 16S rRNA analysis of the cecal microbiome revealed no effects of benoxacor on alpha- or beta-diversity; however, changes were observed in the abundance of certain bacteria. RNAseq analysis identified 163 hepatic genes affected by benoxacor exposure. Benoxacor exposure expressed a gene regulation profile similar to dichloroacetic acid and the fungicide sedaxane. Metabolomic analysis identified 9 serum and 15 liver metabolites that were affected by benoxacor exposure, changes that were not significant after correcting for multiple comparisons. The activity of antioxidant enzymes was not altered by benoxacor exposure. In vitro metabolism studies with liver microsomes and cytosol from male mice demonstrated that benoxacor is enantioselectively metabolized by cytochrome P450 enzymes, carboxylesterases, and glutathione S-transferases. These findings suggest that the minor toxic effects of benoxacor may be due to its rapid metabolism to toxic metabolites, such as dichloroacetic acid. This result challenges the assumption that inert ingredients of pesticide formulations are safe.

Cystic fibrosis-related diabetes (CFRD) is one the most common comorbidities in cystic fibrosis (CF). Pancreatic oxidative stress has been postulated in the pathogenesis of CFRD, but no studies have been done to show an association. The main obstacle is the lack of suitable animal models and no immediate availability of pancreas tissue in humans. In the CF porcine model, we found increased pancreatic total glutathione (GSH), glutathione disulfide (GSSG), 3-nitrotyrosine- and 4-hydroxynonenal-modified proteins, and decreased copper zinc superoxide dismutase (CuZnSOD) activity, all indicative of oxidative stress. CF pig pancreas demonstrated increased DHE oxidation (as a surrogate marker of superoxide) in situ compared to non-CF and this was inhibited by a SOD-mimetic (GC4401). Catalase and glutathione peroxidase activities were not different between CF and non-CF pancreas. Isolated CF pig islets had significantly increased DHE oxidation, peroxide production, reduced insulin secretion in response to high glucose and diminished secretory index compared to non-CF islets. Acute treatment with apocynin or an SOD mimetic failed to restore insulin secretion. These results are consistent with the hypothesis that CF pig pancreas is under significant oxidative stress as a result of increased O2●− and peroxides combined with reduced antioxidant defenses against reactive oxygen species (ROS). We speculate that insulin secretory defects in CF may be due to oxidative stress.

Ketogenic diets (KD) are high in fat and low in carbohydrates, forcing cells to utilize mitochondrial fatty acid oxidation for energy production. Since cancer cells demonstrate increased mitochondrial oxidative stress relative to normal cells, we hypothesized that a KD may selectively enhance metabolic oxidative stress in head and neck cancer cells, sensitizing them to radiation and platinum-based chemotherapy without causing increased toxicity in surrounding normal tissues. This hypothesis was tested in preclinical murine xenografts and in a phase 1 clinical trial (NCT01975766). In this study, mice bearing human head and neck cancer xenografts (FaDu) were fed either standard mouse chow or KetoCal® KD (90% fat, 8% carbohydrate, 2% protein) and exposed to ionizing radiation. Tumors were harvested from mice to test for glutathione, a biomarker of oxidative stress. In parallel, patients with locally advanced head and neck cancer were enrolled in a phase 1 clinical trial where they consumed KD and received radiation with concurrent platinum-based chemotherapy. Subjects consumed KetoCal KD via percutaneous endoscopic gastrostomy (PEG) tube and were also allowed to orally consume water, sugar-free drinks, and foods approved by a dietitian. Oxidative stress markers including protein carbonyls and total glutathione were assessed in patient blood samples both pre-KD and while consuming the KD. Mice bearing FaDu xenografts that received radiation and KD demonstrated a slight improvement in tumor growth rate and survival compared to mice that received radiation alone; however a variation in responses was seen dependent on the fatty acid composition of the diet. In the phase 1 clinical trial, a total of twelve patients were enrolled in the study. Four patients completed five weeks of the KD as per protocol (with variance in compliance). Eight patients did not tolerate the diet with concurrent radiation and platinum-chemotherapy (5 were patient decision and 3 were removed from study due to toxicity). The median number of days consuming a KD in patients who did not complete the study was 5.5 (range: 2-8 days). Reasons for discontinuation included "stress of diet compliance" (1 patient), grade 2 nausea (3 patients), and grade 3 fatigue (1 patient). Three patients were removed from the trial due to dose-limiting toxicities including: grade 4 hyperuricemia (2 patients) and grade 3 acute pancreatitis (1 patient). Median weight loss was 2.95% for the KD-tolerant group and 7.92% for patients who did not tolerate the diet. In conclusion, the ketogenic diet shows promise as a treatment combined with radiation in preclinical mouse head and neck cancer xenografts. A phase 1 clinical trial evaluating the safety and tolerability of KD demonstrated difficulty with diet compliance when combined with standard-of-care radiation therapy and cisplatin chemotherapy.

The purpose of this study was to determine if radiation (RT)-resistant cervical cancers are dependent upon glutamine metabolism driven by activation of the PI3K pathway and test whether PI3K pathway mutation predicts radiosensitization by inhibition of glutamine metabolism. Cervical cancer cell lines with and without PI3K pathway mutations, including SiHa and SiHa PTEN-/- cells engineered by CRISPR/Cas9, were used for mechanistic studies performed in vitro in the presence and absence of glutamine starvation and the glutaminase inhibitor, telaglenastat (CB-839). These studies included cell survival, proliferation, quantification of oxidative stress parameters, metabolic tracing with stable isotope-labeled substrates, metabolic rescue, and combination studies with L-buthionine sulfoximine (BSO), auranofin (AUR), and RT. In vivo studies of telaglenastat ± RT were performed using CaSki and SiHa xenografts grown in immune-compromised mice. PI3K-activated cervical cancer cells were selectively sensitive to glutamine deprivation through a mechanism that included thiol-mediated oxidative stress. Telaglenastat treatment decreased total glutathione pools, increased the percent glutathione disulfide, and caused clonogenic cell killing that was reversed by treatment with the thiol antioxidant, N-acetylcysteine. Telaglenastat also sensitized cells to killing by glutathione depletion with BSO, thioredoxin reductase inhibition with AUR, and RT. Glutamine-dependent PI3K-activated cervical cancer xenografts were sensitive to telaglenastat monotherapy, and telaglenastat selectively radiosensitized cervical cancer cells in vitro and in vivo These novel preclinical data support the utility of telaglenastat for glutamine-dependent radioresistant cervical cancers and demonstrate that PI3K pathway mutations may be used as a predictive biomarker for telaglenastat sensitivity.

Cancer cells, including melanoma, often metastasize regionally through lymphatics before metastasizing systemically through the blood; however, the reason for this is unclear. We discovered that melanoma cells in lymph experience less oxidative stress and form more metastases than melanoma cells in blood. Immunocompromised mice with patient-derived melanomas and immunocompetent mice with mouse melanomas had more melanoma cells per microliter of tumor-draining lymph than tumor-draining blood. Cells metastasizing through blood, but not lymph, became dependent on the ferroptosis inhibitor GPX4. Cells pre-treated with chemical ferroptosis inhibitors formed more metastases than untreated cells after intravenous, but not intralymphatic, injection. We observed multiple differences between lymph fluid and blood plasma that may contribute to decreased oxidative stress and ferroptosis in lymph, including higher levels of glutathione and oleic acid, and less free iron, in lymph. Oleic acid protected melanoma cells from ferroptosis in an Acsl3-dependent manner and increased their capacity to form metastatic tumors. Melanoma cells from lymph nodes were more resistant to ferroptosis and formed more metastases after intravenous injection than melanoma cells from subcutaneous tumors. Exposure to the lymphatic environment thus protects melanoma cells from ferroptosis and increases their ability to survive during subsequent metastasis through the blood. Citation Format: Sean J. Morrison. Lymph protects metastasizing melanoma cells from ferroptosis [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr IA20.

Despite dramatic improvements in outcomes arising from the introduction of targeted therapies and immunotherapies, metastatic melanoma is a highly resistant form of cancer with 5 year survival rates of <35%. Drug resistance is frequently reported to be associated with changes in oxidative metabolism that lead to malignancy that is non-responsive to current treatments. The current report demonstrates that triphenylphosphonium(TPP)-based lipophilic cations can be utilized to induce cytotoxicity in pre-clinical models of malignant melanoma by disrupting mitochondrial metabolism. In vitro experiments demonstrated that TPP-derivatives modified with aliphatic side chains accumulated in melanoma cell mitochondria; disrupted mitochondrial metabolism; led to increases in steady-state levels of reactive oxygen species; decreased total glutathione; increased the fraction of glutathione disulfide; and caused cell killing by a thiol-dependent process that could be rescued by N-acetylcysteine. Furthermore, TPP-derivative-induced melanoma toxicity was enhanced by glutathione depletion (using buthionine sulfoximine) as well as inhibition of thioredoxin reductase (using auranofin). In addition, there was a structure-activity relationship between the aliphatic side-chain length of TPP-derivatives (5-16 carbons), where longer carbon chains increased melanoma cell metabolic disruption and cell killing. In vivo bio-distribution experiments showed that intratumoral administration of a C14-TPP-derivative (12-carbon aliphatic chain), using a slow-release thermosensitive hydrogel as a delivery vehicle, localized the drug at the melanoma tumor site. There, it was observed to persist and decrease the growth rate of melanoma tumors. These results demonstrate that TPP-derivatives selectively induce thiol-dependent metabolic oxidative stress and cell killing in malignant melanoma and support the hypothesis that a hydrogel-based TPP-derivative delivery system could represent a therapeutic drug-delivery strategy for melanoma.

Maternal stress during pregnancy is associated with increased risk of psychiatric disorders in offspring, but embryonic brain mechanisms disrupted by prenatal stress are not fully understood. Our lab has shown that prenatal stress delays inhibitory neural progenitor migration. Here, we investigated redox dysregulation as a mechanism for embryonic cortical interneuron migration delay, utilizing direct manipulation of pro- and antioxidants and a mouse model of maternal repetitive restraint stress starting on embryonic day 12. Time-lapse, live-imaging of migrating GAD67GFP+ interneurons showed that normal tangential migration of inhibitory progenitor cells was disrupted by the pro-oxidant, hydrogen peroxide. Interneuron migration was also delayed by in utero intracerebroventricular rotenone. Prenatal stress altered glutathione levels and induced changes in activity of antioxidant enzymes and expression of redox-related genes in the embryonic forebrain. Assessment of dihydroethidium (DHE) fluorescence after prenatal stress in ganglionic eminence (GE), the source of migrating interneurons, showed increased levels of DHE oxidation. Maternal antioxidants (N-acetylcysteine and astaxanthin) normalized DHE oxidation levels in GE and ameliorated the migration delay caused by prenatal stress. Through convergent redox manipula-tions, delayed interneuron migration after prenatal stress was found to critically involve redox dysregulation. Redox biology during prenatal periods may be a target for protecting brain development.

Superoxide dismutase 1 (SOD1) is the principal cytoplasmic superoxide dismutase in humans and plays a major role in redox potential regulation. It catalyses the transformation of the superoxide anion (O2•-) into hydrogen peroxide. Heterozygous variants in SOD1 are a common cause of familial amyotrophic lateral sclerosis. In this study we describe the homozygous truncating variant c.335dupG (p.C112Wfs*11) in SOD1 that leads to total absence of enzyme activity. The resulting phenotype is severe and marked by progressive loss of motor abilities, tetraspasticity with predominance in the lower extremities, mild cerebellar atrophy, and hyperekplexia-like symptoms. Heterozygous carriers have a markedly reduced enzyme activity when compared to wild-type controls but show no overt neurologic phenotype. These results are in contrast with the previously proposed theory that a loss of function is the underlying mechanism in SOD1-related motor neuron disease and should be considered before application of previously proposed SOD1 silencing as a treatment option for amyotrophic lateral sclerosis.

Therapies for lung cancer patients initially elicit desirable responses, but the presence of hypoxia and drug resistant cells within tumors ultimately lead to treatment failure. Disulfiram (DSF) is an FDA approved, copper chelating agent that can target oxidative metabolic frailties in cancer vs. normal cells and be repurposed as an adjuvant to cancer therapy. Clonogenic survival assays showed that DSF (50-150 nM) combined with physiological levels of Cu (15 mu M CuSO4) was selectively toxic to H292 NSCLC cells vs. normal human bronchial epithelial cells (HBEC). Furthermore, cancer cell toxicity was exacerbated at 1% O-2, relative to 4 or 21% O-2. This selective toxicity of DSF/Cu was associated with differential Cu ionophore capabilities. DSF/Cu treatment caused a> 20-fold increase in cellular Cu in NSCLCs, with nearly two-fold higher Cu present in NSCLCs vs. HBECs and in cancer cells at 1% O-2 vs. 21% O-2. DSF toxicity was shown to be dependent on the retention of Cu as well as oxidative stress mechanisms, including the production of superoxide, peroxide, lipid peroxidation, and mitochondrial damage. DSF was also shown to selectively (relative to HBECs) enhance radiation and chemotherapy-induced NSCLC killing and reduce radiation and chemotherapy resistance in hypoxia. Finally, DSF decreased xenograft tumor growth in vivo when combined with radiation and carboplatin. These results support the hypothesis that DSF could be a promising adjuvant to enhance cancer therapy based on its apparent ability to selectively target fundamental differences in cancer cell oxidative metabolism.

OBJECTIVE:Reduction of oxidized methionines is emerging as a major protein repair pathway. The lack of methionine sulfoxide reductase A (MsrA) exacerbates cardiovascular disease phenotypes driven by increased oxidative stress. However, the role of MsrA on maintaining cellular homeostasis in the absence of excessive oxidative stress is less well understood.METHODS AND RESULTS:Constitutive genetic deletion of MsrA increased formation of p62-containing protein aggregates, activated autophagy, and decreased a marker of apoptosis in vascular smooth muscle cells (VSMC). The association of Keap1 with p62 was augmented in MsrA-/- VSMC. Keap1 targets the transcription factor Nrf2, which regulates antioxidant genes, for proteasomal degradation. However, in MsrA-/- VSMC, the association of Nrf2 with Keap1 was diminished. Whereas Nrf2 mRNA levels were not decreased in MsrA-/- VSMC, we detected decreased ubiquitination of Nrf2 and a corresponding increase in total Nrf2 protein in the absence of biochemical markers of oxidative stress. Moreover, nuclear-localized Nrf2 was increased under MsrA deficiency, resulting in upregulation of Nrf2-dependent transcriptional activity. Consequently, transcription, protein levels and enzymatic activity of glutamate-cysteine ligase and glutathione reductase were greatly augmented in MsrA-/- VSMC.SUMMARY:Our findings demonstrate that reversal of methionine oxidation is required for maintenance of cellular homeostasis in the absence of increased oxidative stress. These data provide the first link between autophagy and activation of Nrf2 in the setting of MsrA deletion.

Abstract Purpose: A ketogenic diet (KD) is a high fat, low carbohydrate diet that forces cells to utilize mitochondrial fatty acid oxidation for energy production. It has been previously shown that cancer cells have defects in mitochondrial oxidative metabolism resulting in increased oxidative stress relative to non-malignant cells. Because radiation and platinum based chemotherapy also induce oxidative damage, we hypothesized that a KD may enhance radiation and platinum chemotherapy efficacy in head and neck cancer. The KD's ability to enhance radiation and chemotherapy efficacy were assessed in murine head and neck cancer xenograft models. The safety and tolerability of a KD in combination with standard of care radiation and platinum-based chemotherapy in head and neck cancer patients was assessed in a phase I clinical trial. Methods: Mice bearing FaDu human head and neck cancer xenografts were fed either standard mouse chow or commercially prepared KD and treated with varying combination of cetuximab (CTX), cisplatin, and/or radiation (IR) and tumor growth was measured via calipers. Tumor homogenates and mice serum were harvested to test for markers including glutathione, 4HNE, PCNA, and IL6. Based on this study and other previously published pre-clinical studies, a phase I clinical trial in locally advanced head and neck cancer combining standard radiation and chemotherapy while consuming a KD was initiated. Subjects consumed a KD by PEG or nasogastric-tube and were also allowed to orally consume water, sugar-free drinks approved by the dietician, and approved snacks and meals approved by the dietician. Oxidative stress markers including protein carbonyl and glutathione were assessed. Results: Mice bearing xenografts treated with KD + IR + CTX had significantly increased survival relative to mice treated with IR + CTX. Collected tumor homogenates in KD + IR + CTX treated mice demonstrated increased 4HNE modified proteins relative to tumor homogenates from IR +CTX mice. In the phase I clinical trial, a total of twelve subjects were enrolled in the study. Four subjects completed five weeks of the KD as per protocol. Median follow-up from completion of RT was 13.1 months (range 8.0-26.7 months). Of the eight subjects that did not tolerate the diet, median number of days subjects stayed on diet was 5.5 (range: 2-8 days). Reasons for discontinuation included “stress of diet compliance” (1), grade 2 nausea (3), and grade 3 fatigue (1). Three subjects were removed for dose limiting toxicities: grade 4 hyperuricemia (2) and grade 3 acute pancreatitis (1). Median weight loss was 2.95% for the KD group and 7.92% for subjects that did not tolerate the diet. Subject serums demonstrated increased oxidative stress as they continued the diet. Conclusions: KD shows promise as adjuvant treatment in mouse xenograft model of head and neck cancer but in our small study there was difficulty with compliance and tolerability when combined with standard of care radiation and chemotherapy. Citation Format: Daniel MA, Carryn Anderson, Samuel N. Rodmann, Kayla Follmer, Visarut Buranasudja, Kellie Bodeker, Jessica R. Parkhurst, Michael McCormack, Andrew Davis, Douglas R. Spitz, John Buatti, Bryan G. Allen, Melissa A. Fath. Ketogenic diet with concurrent chemoradiation in head and neck squamous cell carcinoma: Pre-clinical and phase I trial results [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-252.

Abstract Highly glycolytic cervical cancers largely resist treatment by cisplatin and coadministered pelvic irradiation as the present standard of care. In this study, we investigated the effects of inhibiting glycolysis and thiol redox metabolism to evaluate them as alternate treatment strategies in these cancers. In a panel of multiple cervical cancer cell lines, we evaluated sensitivity to inhibition of glycolysis (2-deoxyglucose, 2-DG) with or without simultaneous inhibition of glutathione and thioredoxin metabolism (BSO/AUR). Intracellular levels of total and oxidized glutathione, thioredoxin reductase activity, and indirect measures of intracellular reactive oxygen species were compared before and after treatment. Highly radioresistant cells were the most sensitive to 2-DG, whereas intermediate radioresistant cells were sensitive to 2-DG plus BSO/AUR. In response to 2-DG/BSO/AUR treatment, we observed increased levels of intracellular oxidized glutathione, redox-sensitive dye oxidation, and decreased glucose utilization via multiple metabolic pathways including the tricarboxylic acid cycle. 2-DG/BSO/AUR treatment delayed the growth of tumors composed of intermediate radioresistant cells and effectively radiosensitized these tumors at clinically relevant radiation doses both in vitro and in vivo. Overall, our results support inhibition of glycolysis and intracellular redox metabolism as an effective alternative drug strategy for the treatment of highly glycolytic and radioresistant cervical cancers. Significance: This study suggests a simple metabolic approach to strike at an apparent Achilles' heel in highly glycolytic, radioresistant forms of cervical cancers, possibly with broader applications in cancer therapy. Cancer Res; 78(6); 1392–403. ©2018 AACR.

Selenium plays an essential role in redox biology and redox toxicology. It is found at the active site of redox enzymes such as, glutathione peroxidases (GPx) and thioredoxin reductases (TrxR). Cell culture media is typically deficient in selenium; this deficiency could limit seleno-enzyme expression and activity, compromising the translation of data from in vitro experiments to in vivo settings. The level of selenium in cell culture media should always be a consideration. However, any supplementation must be within a range to overcome deficiency and yet not induce toxicity. The optimal level of supplementation has not been well characterized. To determine optimal levels of Se-supplementation of cell culture media we introduced varying levels of sodium selenite (Na2SeO3) or seleno-L-methionine (SLM). We established, that the window to avoid potential toxicity can be widened by using SLM instead of selenite. We assessed the expression and activity of GPx1 and GPx4 to determine the level of optimal selenium supplementation for a variety of cells. Supplementing cell culture media containing 10% FBS with 100 – 300 nM SLM, we were able to maximize GPx1 and GPx4 enzyme expression and activity. Interestingly, data from a clinical trial at The University of Iowa indicates that supplementing Se in humans does not increase GPx1 activity in blood. Subjects received ~30x the RDA (55 mg d-1) of Se as SLM daily for more than 7 mo. The level of total Se in blood increased about 50-fold. However, the activity of GPx1 in blood remained constant. Our cell culture data show the importance of supplementing cell culture media with Se to insure full activity of these enzymes. This simple maneuver will increase the rigor and reproducibility of cell culture experiments and may lead to better translation of data addressing basic redox biology.

Oxidative stress plays a fundamental role in abdominal aortic aneurysm (AAA) formation. Activated polymorphonuclear leukocytes (or neutrophils) are associated with AAA and express myeloperoxidase (MPO), which promotes inflammation, matrix degradation, and other pathological features of AAA, including enhanced oxidative stress through generation of reactive oxygen species. Both plasma and aortic MPO levels are elevated in patients with AAA, but the role of MPO in AAA pathogenesis has, heretofore, never been investigated. Here, we show that MPO gene deletion attenuates AAA formation in two animal models: ANG II infusion in apolipoprotein E-deficient mice and elastase perfusion in C57BL/6 mice. Oral administration of taurine [1% or 4% (wt/vol) in drinking water], an amino acid known to react rapidly with MPO-generated oxidants like hypochlorous acid, also prevented AAA formation in the ANG II and elastase models as well as the CaCl2 application model of AAA formation while reducing aortic peroxidase activity and aortic protein-bound dityrosine levels, an oxidative cross link formed by MPO. Both MPO gene deletion and taurine supplementation blunted aortic macrophage accumulation, elastin fragmentation, and matrix metalloproteinase activation, key features of AAA pathogenesis. Moreover, MPO gene deletion and taurine administration significantly attenuated the induction of serum amyloid A, which promotes ANG II-induced AAAs. These data implicate MPO in AAA pathogenesis and suggest that studies exploring whether taurine can serve as a potential therapeutic for the prevention or treatment of AAA in patients merit consideration.NEW & NOTEWORTHY Neutrophils are abundant in abdominal aortic aneurysm (AAA), and myeloperoxidase (MPO), prominently expressed in neutrophils, is associated with AAA in humans. This study demonstrates that MPO gene deletion or supplementation with the natural product taurine, which can scavenge MPO-generated oxidants, can prevent AAA formation, suggesting an attractive potential therapeutic strategy for AAA.