Development and Validation of Nutrition and Food Safety Educational Material for Fish Processors in Nigeria.

INTRODUCTION: fish can be an affordable and accessible animal-source food in many Low- and Middle-Income Countries (LMIC). BACKGROUND: Traditional fish processing methods pose a risk of exposing fish to various contaminants that may reduce their nutritional benefit. In addition, a lack of literacy may increase women fish processors' vulnerability to malnutrition and foodborne diseases. OBJECTIVE: The overall aim of the project was to educate women and youth fish processors in Delta State, Nigeria about the benefit of fish in the human diet and to develop low literacy tools to help them better market their products. The objective of this study was to describe the development and validation of a low-literacy flipbook designed to teach women fish processors about nutrition and food safety. METHOD: developing and validating instructional material requires understanding the population, high-quality and relevant graphics, and the involvement of relevant experts to conduct the content validation using the Content Validity Index (CVI) and the index value translated with the Modified Kappa Index (k). RESULT: The Item-level Content Validity Index (I-CVI) value of all domains evaluated at the initial stage was 0.83 and the Scale-level Content Validity Index (S-CVI) was 0.90. At the final stage, the material was validated with CVI 0.983 by four experts and satisfied the expected minimum CVI value for this study (CVI ≥ 0.83, p-value = 0.05). The overall evaluation of the newly developed and validated flipbook was "excellent". CONCLUSIONS: the developed material was found to be appropriate for training fish processors in Nigeria in nutrition and food safety and could be modified for a population of fish processors in other LMICs.

Identification of oncogenic signatures in the inflammatory colon of C57BL/6 mice fed a high-fat diet.

Adoption of an obesogenic diet such as a high-fat diet (HFD) results in obesity, bacterial dysbiosis, chronic inflammation, and cancer. Gut bacteria and their metabolites are recognized by interleukin-1 (IL-1R)/toll-like receptors (TLRs) which are essential to maintain intestinal homeostasis. Moreover, host extracellular microRNAs (miRNAs) can alter bacterial growth in the colon. Characterization of the underlying mechanisms may lead to identifying fecal oncogenic signatures reflecting colonic health. We hypothesize that an HFD accelerates the inflammatory process and modulates IL-1R/TLR pathways, gut microbiome, and disease-related miRNA in the colon. In this study, 4-week-old C57BL/6 mice were fed a modified AIN93G diet (AIN, 16% energy fat) or an HFD (45% energy fat) for 15 weeks. In addition to increased body weight and body fat composition, the concentrations of plasma interleukin 6 (IL-6), inflammatory cell infiltration, ß-catenin, and cell proliferation marker (Ki67) in the colon were elevated > 68% in the HFD group compared to the AIN group. Using a PCR array analysis, we identified 14 out of 84 genes with a ≥ 24% decrease in mRNA content related to IL-1R and TLR pathways in colonic epithelial cells in mice fed an HFD compared to the AIN. Furthermore, the content of Alistipes bacteria, the Firmicutes/Bacteroidetes ratio, microRNA-29a, and deoxycholic and lithocholic acids (secondary bile acids with oncogenic potential) were 55% greater in the feces of the HFD group compared to the AIN group. Collectively, this composite, a multimodal profile may represent a unique HFD-induced fecal signature for colonic inflammation and cancer in C57BL/6 mice.

Changes in the Fecal Metabolome Accompany an Increase in Aberrant Crypt Foci in the Colon of C57BL/6 Mice Fed with a High-Fat Diet.

High-fat diet (HFD)-induced obesity is a risk factor for colon cancer. Our previous data show that compared to an AIN-93 diet (AIN), a HFD promotes azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) formation and microbial dysbiosis in C57BL/6 mice. To explore the underlying metabolic basis, we hypothesize that AOM treatment triggers a different fecal metabolomic profile in C57BL/6 mice fed the HFD or the AIN. We found that 65 of 196 identified metabolites were significantly different among the four groups of mice (AIN, AIN + AOM, HFD, and HFD + AOM). A sparse partial least squares discriminant analysis (sPLSDA) showed that concentrations of nine fecal lipid metabolites were increased in the HFD + AOM compared to the HFD, which played a key role in overall metabolome group separation. These nine fecal lipid metabolite concentrations were positively associated with the number of colonic ACF, the cell proliferation of Ki67 proteins, and the abundance of dysbiotic bacteria. These data suggest that the process of AOM-induced ACF formation may increase selective fecal lipid concentrations in mice fed with a HFD but not an AIN. Collectively, the accumulation of these critical fecal lipid species may alter the overall metabolome during tumorigenesis in the colon.

Food Insecurity and Depression among US Adults: NHANES 2005-2016.

A growing body of evidence suggests that food insecurity is associated with adverse mental health outcomes such as depression and anxiety. In this study, the relationship between food insecurity and depression was examined using data from the 2005−2016 National Health and Nutrition Examination Survey (NHANES). Food insecurity was assessed with the 18-item United States Food Security Survey Module with zero affirmative responses indicating high food security, 1 or 2 affirmative responses indicating marginal food security, and ≥3 affirmative responses indicating food insecurity. Depression was assessed with the Patient Health Questionnaire-9 with scores ≥10 indicating depression. Data were analyzed from 28,448 adult participants aged 20 or older. Food insecurity was present in 19.2% of the sample population (n = 5452). Food security status was significantly associated with gender, race, education level, marital status, smoking status, and BMI (Rao-Scott chi-square, p < 0.05). Fully food secure and very low food security adults experienced depression at a rate of 5.1% and 25.8%, respectively (Rao-Scott chi-square, p < 0.0001). Participants with very low food security had a significantly greater odds of depression than food secure adults, OR = 3.50 (95% CI: 2.98, 4.12). These findings suggest that food insecurity is a significant risk factors for depression in US adults over 20 years of age. To address this issue in our citizenry, police initiatives and public health interventions addressing both food access and mental health should be prioritized.

Vulnerability of Triple-Negative Breast Cancer to Saponin Formosanin C-Induced Ferroptosis.

Targeting ferritin via autophagy (ferritinophagy) to induce ferroptosis, an iron- and reactive oxygen species (ROS)-dependent cell death, provides novel strategies for cancer therapy. Using a ferroptosis-specific inhibitor and iron chelator, the vulnerability of triple-negative breast cancer (TNBC) MDA-MB-231 cells to ferroptosis was identified and compared to that of luminal A MCF-7 cells. Saponin formosanin C (FC) was revealed as a potent ferroptosis inducer characterized by superior induction in cytosolic and lipid ROS formation as well as GPX4 depletion in MDA-MB-231 cells. The FC-induced ferroptosis was paralleled by downregulation of ferroportin and xCT expressions. Immunoprecipitation and electron microscopy demonstrated the involvement of ferritinophagy in FC-treated MDA-MB-231 cells. The association of FC with ferroptosis was strengthened by the results that observed an enriched pathway with differentially expressed genes from FC-treated cells. FC sensitized cisplatin-induced ferroptosis in MDA-MB-231 cells. Through integrated analysis of differentially expressed genes and pathways using the METABRIC patients' database, we confirmed that autophagy and ferroptosis were discrepant between TNBC and luminal A and that TNBC was hypersensitive to ferroptosis. Our data suggest a therapeutic strategy by ferroptosis against TNBC, an aggressive subtype with a poor prognosis.

Identification of Selenoprotein H Isoforms and Impact of Selenoprotein H Overexpression on Protein But Not mRNA Levels of 2 Other Selenoproteins in 293T Cells.

BACKGROUND: Selenoprotein H (SELONOH), a member of the thioredoxin-like family proteins, is prioritized to degradation in selenium (Se) insufficiency. Recent studies implicate protective roles of SELENOH in oxidative stress, cellular senescence, and intestinal tumorigenesis. Although the nonselenoprotein H0YE28 is suggested as shortened SELENOH according to genomic and proteomic data repositories, this variant has not been verified biochemically. OBJECTIVES: We sought to identify SELENOH isoforms and explore the impact of Se flux on selenoprotein expression in SELENOH-overexpressing cells. METHODS: A vector expressing a FLAG (the DYKDDDDK sequence) tag on the N-terminal end of wild-type SELENOH was constructed and transiently transfected into 293T cells incubated with graded concentrations of Na2SeO3 (0-200 nM). Cells were subjected to immunoprecipitation, LC-MS/MS protein analysis, immunoblotting, qRT-PCR, and senescence assays. Data were analyzed by 1-way or 2-way ANOVA. RESULTS: Results of anti-FLAG immunoblotting showed that FLAG-SELENOH transfection increased (3.7-fold; P < 0.05) protein levels of the long, but not the short, SELENOH variants in the presence of Na2SeO3 (100 nM). By contrast, SELENOH mRNA levels were increased by 53-fold upon FLAG-SELENOH transfection but were comparable with or without supplemental Se (100 nM). LC-MS/MS analyses of anti-FLAG immunoprecipitates designated both anti-FLAG bands as SELENOH and co-identified three 60S ribosomal and 9 other proteins. Overexpression of FLAG-SELENOH 1) reduced glutathione peroxidase 1 and thioredoxin reductase 1 expression at the protein rather than the mRNA level in the absence but not presence of supplemental Se (100 nM; P < 0.05); 2) increased mRNA levels of 3 heat shock proteins (HSP27, HSP70-1A, and HSP70-1B; P < 0.05); and 3) reduced senescence induced by H2O2 (20 µM, 4 hours; P < 0.05). CONCLUSIONS: These cellular studies demonstrate a Se-independent, shortened SELENOH variant and suggest competition of overexpressed FLAG-SELENOH with 2 other selenoproteins for the expression at the protein but not the mRNA level in Se insufficiency.

Dietary Selenium Requirement for the Prevention of Glucose Intolerance and Insulin Resistance in Middle-Aged Mice.

BACKGROUND: Although dietary selenium (Se) deficiency or excess induces type 2 diabetes-like symptoms in mice, suboptimal body Se status usually causes no symptoms but may promote age-related decline in overall health. OBJECTIVES: We sought to determine the dietary Se requirement for protection against type 2 diabetes-like symptoms in mice. METHODS: Thirty mature (aged 4 mo) male C57BL/6J mice were fed a Se-deficient torula yeast AIN-93M diet supplemented with Na2SeO4 in graded concentrations totaling 0.01 (basal), 0.04, 0.07, 0.10, and 0.13 (control) mg Se/kg for 4 mo (n = 6) until they were middle-aged (8 mo). Droplets of whole blood were used to determine glucose tolerance and insulin sensitivity in the mice from ages 5 to 8 mo. Postmortem serum, liver, and skeletal muscle were collected to assay for selenoprotein expression and markers of glucose metabolism. Data were analyzed by 1-way ANCOVA with or without random effects for time-repeated measurements using live mice or postmortem samples, respectively. RESULTS: Compared with control, the consumption of basal diet increased (P < 0.05) fasting serum insulin (95% CI: 52%, 182%) and leptin (95% CI: 103%, 118%) concentrations in middle-aged mice. Dietary Se insufficiency decreased (P < 0.05) 1) glucose tolerance (13-79%) and insulin sensitivity (15-65%) at ≤0.10 mg Se/kg; 2) baseline thymoma viral proto-oncogene phosphorylation on S473 (27-54%) and T308 (22-46%) at ≤0.10 and ≤0.07 mg Se/kg, respectively, in the muscle but not the liver; and 3) serum glutathione peroxidase 3 (51-83%), liver and muscle glutathione peroxidase 1 (32-84%), serum and liver selenoprotein P (28-42%), and liver and muscle selenoprotein H (39-48%) and selenoprotein W (16-73%) protein concentrations at ≤0.04, ≤0.10, ≤0.07, and ≤0.10 mg Se/kg, respectively. CONCLUSIONS: Mice fed diets containing ≤0.10 mg Se/kg display impaired glucose tolerance and insulin sensitivity, suggesting increased susceptibility to type 2 diabetes by suboptimal Se status at levels ≤23% of nutritional needs.

Superior inhibitory efficacy of butyrate over propionate and acetate against human colon cancer cell proliferation via cell cycle arrest and apoptosis: linking dietary fiber to cancer prevention.

Intake of dietary fiber may protect against colon cancer. The anticancer property is associated with an increased production of short chain fatty acids (SCFAs), including acetate, propionate and butyrate, during dietary fiber fermentation in the colon. However, the mechanisms remain to be determined. We hypothesized that butyrate exhibits a stronger inhibitory potential against colon cancer cell proliferation compared with acetate and propionate. We determined the half maximal inhibitory concentrations (IC50) of SCFAs in HCT116 human colon cancer cell proliferation by examining cell growth curves. At 24- and 48-hour time points, IC50 (mmol/L) concentrations of acetate, propionate, and butyrate were [66.0 and 29.0], [9.2 and 3.6], and [2.5 and 1.3], respectively. Consistent with the greater anti-proliferative effect, butyrate exhibits >3-fold stronger potential for inducing cell cycle arrest at the G2 phase with a drop in S-phase fraction (including c-Myc/p21 signaling) and apoptosis when compared with acetate and propionate. Subsequently, we focused on the effect of butyrate on apoptotic gene expression. Using a PCR array analysis, we identified 17 pro-apoptotic genes, 6 anti-apoptotic genes, and 4 cellular mediator genes with >1-fold increase or decrease in mRNA levels out of 93 apoptosis related genes in butyrate-treated HCT116 cells when compared with untreated HCT116 cells. These genes were mainly involved in the TNF, NFκB, CARD, and BCL-2 regulated pathways. Taken together, our data indicate a greater inhibitory efficacy of butyrate over propionate and acetate against human colon cancer cell proliferation via cell cycle arrest and apoptosis.

Selenoprotein T Promotes Proliferation and G1-to-S Transition in SK-N-SH Cells: Implications in Parkinson's Disease.

BACKGROUND: Selenium is prioritized to the brain mainly for selenoprotein expression. Selenoprotein T (SELENOT) protects dopaminergic, postmitotic neurons in a mouse model of Parkinson's disease (PD). OBJECTIVE: We hypothesized a proliferative role of SELENOT in neural cells. METHODS: To assess SELENOT status in PD, sedated male C57BL/6 mice at 10-12 wk of age were injected with 6-hydroxydopamine in neurons, and human peripheral blood mononuclear cells were isolated from 9 healthy subjects (56% men, 68-y-old) and 11 subjects with PD (64% men, 63-y-old). Dopaminergic neural progenitor-like SK-N-SH cells with transient SELENOT overexpression or knockdown were maintained in the presence or absence of the antioxidant N-acetyl-l-cysteine and the calcium channel blocker nimodipine. Cell cycle, proliferation, and signaling parameters were determined by immunoblotting, qPCR, and flow cytometry. RESULTS: SELENOT mRNA abundance was increased (P < 0.05) in SK-N-SH cells treated with 1-methyl-4-phenylpyridinium iodide (3.5-fold) and peripheral blood mononuclear cells from PD patients (1.6-fold). Likewise, SELENOT was expressed in tyrosine hydroxylase-positive dopaminergic neurons of 6-hydroxydopamine-injected mice. Knockdown of SELENOT in SK-N-SH cells suppressed (54%; P < 0.05) 5-ethynyl-2'-deoxyuridine incorporation but induced (17-47%; P < 0.05) annexin V-positive cells, CASPASE-3 cleavage, and G1/S cell cycle arrest. SELENOT knockdown and overexpression increased (88-120%; P < 0.05) and reduced (37-42%; P < 0.05) both forkhead box O3 and p27, but reduced (51%; P < 0.05) and increased (1.2-fold; P < 0.05) cyclin-dependent kinase 4 protein abundance, respectively. These protein changes were diminished by nimodipine or N-acetyl-l-cysteine treatment (24 h) at steady-state levels. While the N-acetyl-l-cysteine treatment did not influence the reduction in the amount of calcium (13%; P < 0.05) by SELENOT knockdown, the nimodipine treatment reversed the decreased amount of reactive oxygen species (33%; P < 0.05) by SELENOT overexpression. CONCLUSIONS: These cellular and mouse data link SELENOT to neural proliferation, expanding our understanding of selenium protection in PD.

The Thioredoxin-Like Family of Selenoproteins: Implications in Aging and Age-Related Degeneration.

The thioredoxin-like (Rdx) family proteins contain four selenoproteins (selenoprotein H, SELENOH; selenoprotein T, SELENOT; selenoprotein V, SELENOV; selenoprotein W, SELENOW) and a nonselenoprotein Rdx12. They share a CxxU or a CxxC (C, cysteine; x, any amino acid; U, selenocysteine) motif and a stretch of eGxFEI(V) sequence. From the evolutionary perspective, SELENOW and SELENOV are clustered together and SELENOH and SELENOT are in another branch. Selenoproteins in the Rdx family exhibit tissue- and organelle-specific distribution and are differentially influenced in response to selenium deficiency. While SELENOH is nucleus-exclusive, SELENOT resides mainly in endoplasmic reticulum and SELENOW in cytosol. SELENOV is expressed essentially only in the testes with unknown cellular localization. SELENOH and SELENOW are more sensitive than SELENOT and SELENOV to selenium deficiency. While physiological functions of the Rdx family of selenoproteins are not fully understand, results from animal models demonstrated that (1) brain-specific SELENOT knockout mice are susceptible to 1-methyl-4-phenylpyridinium-induced Parkinson's disease in association with redox imbalance and (2) adult zebrafishes with heterozygous SELENOH knockout are prone to dimethylbenzanthracene-induced tumorigenesis together with increased DNA damage and oxidative stress. Further animal and human studies are needed to fully understand physiological roles of the Rdx family of selenoproteins in redox regulation, genome maintenance, aging, and age-related degeneration.

Butyrate Inhibits Cancerous HCT116 Colon Cell Proliferation but to a Lesser Extent in Noncancerous NCM460 Colon Cells.

Butyrate, an intestinal microbiota metabolite of dietary fiber, exhibits chemoprevention effects on colon cancer development. However, the mechanistic action of butyrate remains to be determined. We hypothesize that butyrate inhibits cancerous cell proliferation but to a lesser extent in noncancerous cells through regulating apoptosis and cellular-signaling pathways. We tested this hypothesis by exposing cancerous HCT116 or non-cancerous NCM460 colon cells to physiologically relevant doses of butyrate. Cellular responses to butyrate were characterized by Western analysis, fluorescent microscopy, acetylation, and DNA fragmentation analyses. Butyrate inhibited cell proliferation, and led to an induction of apoptosis, genomic DNA fragmentation in HCT116 cells, but to a lesser extent in NCM460 cells. Although butyrate increased H3 histone deacetylation and p21 tumor suppressor expression in both cell types, p21 protein level was greater with intense expression around the nuclei in HCT116 cells when compared with that in NCM460 cells. Furthermore, butyrate treatment increased the phosphorylation of extracellular-regulated kinase 1/2 (p-ERK1/2), a survival signal, in NCM460 cells while it decreased p-ERK1/2 in HCT116 cells. Taken together, the activation of survival signaling in NCM460 cells and apoptotic potential in HCT116 cells may confer the increased sensitivity of cancerous colon cells to butyrate in comparison with noncancerous colon cells.

Loss of Selenium-Binding Protein 1 Decreases Sensitivity to Clastogens and Intracellular Selenium Content in HeLa Cells.

Selenium-binding protein 1 (SBP1) is not a selenoprotein but structurally binds selenium. Loss of SBP1 during carcinogenesis usually predicts poor prognosis. Because genome instability is a hallmark of cancer, we hypothesize that SBP1 sequesters cellular selenium and sensitizes cancer cells to DNA-damaging agents. To test this hypothesis, we knocked down SBP1 expression in HeLa cervical cancer cells by employing a short hairpin RNA (shRNA) approach. Reduced sensitivity to hydrogen peroxide, paraquat and camptothecin, reactive oxygen species content, and intracellular retention of selenium after selenomethionine treatment were observed in SBP1 shRNA HeLa cells. Results from Western analyses showed that treatment of HeLa cells with selenomethionine resulted in increased SBP1 protein expression in a dose-dependent manner. Knockdown of SBP1 rendered HeLa cells increased expression of glutathione peroxidase-1 but not glutathione peroxidase-4 protein levels and accelerated migration from a wound. Altogether, SBP1 retains supplemental selenium and sensitizes HeLa cancer cells to clastogens, suggesting a new cancer treatment strategy by sequestering selenium through SBP1.

Redox-sensitive MAPK and Notch3 regulate fibroblast differentiation and activation: a dual role of ERK1/2.

Myofibroblastic transformation, characterized by upregulation of α-smooth muscle actin in response to profibrotic agents such as TGF-ß1, is considered as a major event leading to fibrosis. The mechanistic basis linking myofibroblast differentiation to idiopathic pulmonary fibrosis and the disease treatment remain elusive. In this study, we studied roles of MAPK, Notch, and reactive oxygen species (ROS) during the differentiation of IMR-90 lung fibroblasts at basal level and induced by TGF-ß1. Our results demonstrated that ROS-dependent activation of p38, JNK1/2 and Notch3 promoted basal and TGF-ß1-induced differentiation and expression of extracellular matrix proteins. In stark contrast, ERK1/2 was suppressed by ROS and exhibited an inhibitory effect on the differentiation but showed a weak promotion on the expression of extracellular matrix proteins. TGF-ß1-induced Notch3 expression depended on p38 and JNK1/2. Interestingly, Notch3 was also downstream of ERK1/2, suggesting a complex role of ERK1/2 in lung function. Our results suggest a novel ROS-mediated shift of dominance from the inhibitory ERK1/2 to the stimulatory p38, JNK1/2 and Notch3 during the pathological progression of IPF. Thus, targeting ERK1/2 signaling for activation and p38, JNK1/2 and Notch3 for inhibition may be of clinical potential against lung fibrosis.

N-acetylcysteine negatively regulates Notch3 and its malignant signaling.

Notch3 receptor is expressed in a variety of cancers and the excised active intracellular domain (N3ICD) initiates its signaling cascade. N-acetylcysteine (NAC) as an antioxidant has been implicated in cancer prevention and therapy. In this study, we demonstrated a negative regulation of Notch3 by NAC in cancer cells. HeLa cells treated with NAC exhibited a time- and concentration-dependent decrease in Notch3 levels and its downstream effectors Hes1 and HRT1 in a manner independent of f-secretase or glutathione. In contrast, NAC did not affect protein levels of Notch1, the full length Notch3 precursor, or ectopically expressed N3ICD. Although SOD, catalase and NAC suppressed reactive oxygen species in HeLa cells, the first two antioxidants did not impact on Notch3 levels. While the mRNA expression of Notch3 was not altered by NAC, functional inhibition of lysosome, but not proteasome, blocked the NAC-dependent reduction of Notch3 levels. Furthermore, results from Notch3 silencing and N3ICD overexpression demonstrated that NAC prevented malignant phenotypes through down-regulation of Notch3 protein in multiple cancer cells. In summary, NAC reduces Notch3 levels through lysosome-dependent protein degradation, thereby negatively regulates Notch3 malignant signaling in cancer cells. These results implicate a novel NAC treatment in sensitizing Notch3-expressing tumors.

Nuclear selenoproteins and genome maintenance.

Selenium is an essential metalloid required for the expression of selenoproteins. While cells are constantly challenged by clastogens of endogenous and exogenous origins, genome integrity is maintained by direct repair of DNA damage, redox balance, and epigenetic regulation. To date, only five selenoproteins are experimentally demonstrated to reside in nucleus, exclusively or partially, including selenoprotein H, methionine-R-sulfoxide reductase 1, glutathione peroxidase-4, thioredoxin reductase-1, and thioredoxin glutathione reductase. All these five selenoproteins have demonstrated or potential roles in redox regulation and genome maintenance. Selenoprotein H is known to transactivate the expression of a couple of genes against oxidative stress. The thioredoxin reductase-1b isoform delivers estrogen receptor-α and -ß to the nucleus. Nuclear glutathione peroxidase-4 epigenetically and globally inhibits gene expression through the maintenance of chromatin compactness in testes. Continued studies on how these and additional nuclear selenoproteins regulate genome stability will have profound impact on advancing our understanding in selenium regulation of optimal health. © 2015 IUBMB Life, 68(1):5-12, 2016.

Paradoxical Roles of Antioxidant Enzymes: Basic Mechanisms and Health Implications.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated from aerobic metabolism, as a result of accidental electron leakage as well as regulated enzymatic processes. Because ROS/RNS can induce oxidative injury and act in redox signaling, enzymes metabolizing them will inherently promote either health or disease, depending on the physiological context. It is thus misleading to consider conventionally called antioxidant enzymes to be largely, if not exclusively, health protective. Because such a notion is nonetheless common, we herein attempt to rationalize why this simplistic view should be avoided. First we give an updated summary of physiological phenotypes triggered in mouse models of overexpression or knockout of major antioxidant enzymes. Subsequently, we focus on a series of striking cases that demonstrate "paradoxical" outcomes, i.e., increased fitness upon deletion of antioxidant enzymes or disease triggered by their overexpression. We elaborate mechanisms by which these phenotypes are mediated via chemical, biological, and metabolic interactions of the antioxidant enzymes with their substrates, downstream events, and cellular context. Furthermore, we propose that novel treatments of antioxidant enzyme-related human diseases may be enabled by deliberate targeting of dual roles of the pertaining enzymes. We also discuss the potential of "antioxidant" nutrients and phytochemicals, via regulating the expression or function of antioxidant enzymes, in preventing, treating, or aggravating chronic diseases. We conclude that "paradoxical" roles of antioxidant enzymes in physiology, health, and disease derive from sophisticated molecular mechanisms of redox biology and metabolic homeostasis. Simply viewing antioxidant enzymes as always being beneficial is not only conceptually misleading but also clinically hazardous if such notions underpin medical treatment protocols based on modulation of redox pathways.

Selenoprotein H suppresses cellular senescence through genome maintenance and redox regulation.

Oxidative stress and persistent DNA damage response contribute to cellular senescence, a degeneration process critically involving ataxia telangiectasia-mutated (ATM) and p53. Selenoprotein H (SelH), a nuclear selenoprotein, is proposed to carry redox and transactivation domains. To determine the role of SelH in genome maintenance, shRNA knockdown was employed in human normal and immortalized cell lines. SelH shRNA MRC-5 diploid fibroblasts under ambient O2 displayed a distinct profile of senescence including ß-galactosidase expression, autofluorescence, growth inhibition, and ATM pathway activation. Such senescence phenotypes were alleviated in the presence of ATM kinase inhibitors, by p53 shRNA knockdown, or by maintaining the cells under 3% O2. During the course of 5-day recovery, the induction of phospho-ATM on Ser-1981 and γH2AX by H2O2 treatment (20 µm) subsided in scrambled shRNA but exacerbated in SelH shRNA MRC-5 cells. Results from clonogenic assays demonstrated hypersensitivity of SelH shRNA HeLa cells to paraquat and H2O2, but not to hydroxyurea, neocarzinostatin, or camptothecin. While SelH mRNA expression was induced by H2O2 treatment, SelH-GFP did not mobilize to sites of oxidative DNA damage. The glutathione level was lower in SelH shRNA than scrambled shRNA HeLa cells, and the H2O2-induced cell death was rescued in the presence of N-acetylcysteine, a glutathione precursor. Altogether, SelH protects against cellular senescence to oxidative stress through a genome maintenance pathway involving ATM and p53.

Discovery of systematic responses and potential biomarkers induced by ochratoxin A using metabolomics.

Ochratoxin A (OTA) is known to be nephrotoxic and hepatotoxic in rodents when exposed orally. To understand the systematic responses to OTA exposure, GC-MS- and (1)H-NMR-based metabolomic techniques together with histopathological assessments were applied to analyse the urine and plasma of OTA-exposed rats. It was found that OTA exposure caused significant elevation of amino acids (alanine, glycine, leucine etc.), pentose (ribose, glucitol, xylitol etc.) and nucleic acid metabolites (pseudouridine, adenosine, uridine). Moreover, myo-inositol, trimethylamine-oxide (TMAO), pseudouridine and leucine were identified as potential biomarkers for OTA toxicity. The primary pathways included the pentose phosphate pathway (PPP), the Krebs cycle (TCA), the creatine pathway and gluconeogenesis. The activated PPP was attributed to the high requirements for nicotinamide adenine dinucleotide phosphate (NADPH), which is involved in OTA metabolism through cytochrome P450. The elevated gluconeogenesis and TCA suggest that energy metabolism was involved. The up-regulated synthesis of creatinine reveals the elevated catabolism of proteins. These findings provide an overview of systematic responses to OTA exposure and metabolomic insight into the toxicological mechanism of OTA.

Methylseleninic acid sensitizes Notch3-activated OVCA429 ovarian cancer cells to carboplatin.

Ovarian cancer, the deadliest of gynecologic cancers, is usually not diagnosed until advanced stages. Although carboplatin has been popular for treating ovarian cancer for decades, patients eventually develop resistance to this platinum-containing drug. Expression of neurogenic locus notch homolog 3 (Notch3) is associated with chemoresistance and poor overall survival in ovarian cancer patients. Overexpression of NICD3 (the constitutively active form of Notch3) in OVCA429 ovarian cancer cells (OVCA429/NICD3) renders them resistance to carboplatin treatment compared to OVCA429/pCEG cells expressing an empty vector. We have previously shown that methylseleninic acid (MSeA) induces oxidative stress and activates ataxia-telangiectasia mutated and DNA-dependent protein kinase in cancer cells. Here we tested the hypothesis that MSeA and carboplatin exerted a synthetic lethal effect on OVCA429/NICD3 cells. Co-treatment with MSeA synergistically sensitized OVCA429/NICD3 but not OVCA429/pCEG cells to the killing by carboplatin. This synergism was associated with a cell cycle exit at the G2/M phase and the induction of NICD3 target gene HES1. Treatment of N-acetyl cysteine or inhibitors of the above two kinases did not directly impact on the synergism in OVCA429/NICD3 cells. Taken together, these results suggest that the efficacy of carboplatin in the treatment of high grade ovarian carcinoma can be enhanced by a combinational therapy with MSeA.