Oxidative Stressors Research Articles

Oxidative stress biomarkers in newborn calves: Comparison among artificial insemination, in vitro fertilization and cloning

Oxidative stress occurs when there is greater than optimal production of reactive oxygen species (ROS) or an antioxidant system failure. Calves produced using in vitro fertilization (IVF) or cloning (CA) have greater mortality rates, with greater incidence of respiratory diseases, which could be explained by the deleterious outcomes from oxidative stress. Calves were studied that were produced using: artificial insemination (AI; n = 20), in vitro fertilization (IVF; n = 15) or cloning (CA; n = 15). Blood samples were collected at 6, 12, 24 and 48 h subsequent to the time of birth. The cloned calves had greater ROS production from lipid peroxidation, with greater thiobarbituric acid reactive substances. This factor was associated with a lesser amount of superoxide dismutase in the CA. Calves produced using IVF had a greater activity of catalase and glutathione peroxidase, either due to greater production of hydrogen peroxide or greater efficiency of enzymatic response of these neonates. Calves produced using AI had greater concentrations ​​of reduced thiol groups. These associated factors may indicate there is greater oxidative stress in calves produced by IVF and cloning than with use of AI, however in these calves there was an effective response to these oxidative stressors within 48 h subsequent to birth. Hence, calves produced using IVF and by cloning have greater ROS production when compared to calves produced using AI. The calves produced using IVF, however, had a greater enzymatic activity or were more efficient in adapting to ROS when compared to calves produced by cloning.

A Method for Deciphering Major Drivers of Bacterial Iron Stress Response in the Presence of Oxidative Stressors.

This paper describes a method for deciphering major drivers of bacterial stress response using an empirically informed computational approach. We develop a working model of iron flux regulation and concomitant oxidative stress response in Escherichia coli. The integrated model is used to investigate the temporal effects of iron and hydrogen peroxide stress on bacterial growth and metabolism. We employ a sensitivity analysis platform and, using various measures, probe for major mechanistic drivers of the bacterial response to iron stress.

Quantitative Estimation of Oxidative Stress in Cancer Tissue Cells Through Gene Expression Data Analyses.

Quantitative assessment of the intracellular oxidative stress level is a very important problem since it is the basis for elucidation of the fundamental causes of metabolic changes in diseased human cells, particularly cancer. However, the problem proves to be very challenging to solve in vivo because of the complex nature of the problem. Here a computational method is presented for predicting the quantitative level of the intracellular oxidative stress in cancer tissue cells. The basic premise of the predictor is that the genomic mutation level is strongly associated with the intracellular oxidative stress level. Based on this, a statistical analysis is conducted to identify a set of enzyme-encoding genes, whose combined expression levels can well explain the mutation rates in individual cancer tissues in the TCGA database. We have assessed the validity of the predictor by assessing it against genes that are known to have anti-oxidative functions for specific types of oxidative stressors. Then the applications of the predictor are conducted to illustrate its utility.

The mitochondrial peroxiredoxin displays distinct roles in different developmental stages of African trypanosomes

Hydroperoxide reduction in African trypanosomes relies on 2-Cys-peroxiredoxins (Prxs) and glutathione peroxidase-type enzymes (Pxs) which both obtain their reducing equivalents from the trypanothione/tryparedoxin couple and thus act as tryparedoxin peroxidases. While the cytosolic forms of the peroxidases are essential, the mitochondrial mPrx and Px III appear dispensable in bloodstream Trypanosoma brucei. This led to the suggestion that in this developmental stage which is characterized by a mitochondrion that lacks an active respiratory chain, only one of the two peroxidases might be required. Here we show that bloodstream cells in which the Px III gene is deleted and mPrx is down-regulated by RNA interference, proliferate as the parental cells indicating that both mitochondrial peroxidases are dispensable. However, when we raised the culture temperature to 39 °C, mPrx-depleted cells died indicating that under conditions mimicking a fever situation in the mammalian host, the protein becomes essential. In contrast, depletion of mPrx in insect stage procyclic T. brucei causes a proliferation defect under standard conditions at 27 °C, in the absence of any stress. In the absence of mPrx, a tryparedoxin-coupled roGFP2 biosensor expressed in the mitochondrial matrix is unable to respond to antimycin A treatment. Thus mPrx reduces mitochondrial H2O2 with the generation of trypanothione disulfide and acts as peroxidase. However, mPrx-depleted procyclic cells neither display any alteration in the cytosolic or mitochondrial trypanothione redox state nor increased sensitivity towards exogenous oxidative stressors suggesting that the peroxidase activity is not the crucial physiological function. After prolonged mPrx-depletion, the cells almost stop proliferation and display a highly elongated shape and diminished MitoTracker Red staining. In contrast to the situation in the mammalian bloodstream T. brucei and Leishmania, mPrx appears to play a constitutive role for the morphology, mitochondrial function and proliferation of the insect stage of African trypanosomes.

Clinical Manifestations of Hereditary Leiomyomatosis and Renal Cell Carcinoma: a Case Report

Introduction: Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a rare genetic disorder etiologically stemming from mutations in the FH gene. This disorder has been reported in 300 families worldwide. We were presented with the following case as part of the Rare Disease Interdepartmental Science Case Competition (RISCC) in order to successfully diagnose the patient and examine the pathophysiology, socioeconomics, and treatment strategy of this disorder.Case Presentation: A 27-year-old woman presented to the emergency room with a two-week history of hematuria. Her medical history included a single benign skin lesion. She did not report a family history of cancer but stated that her mother underwent a hysterectomy for unknown reasons.Clinical Investigations: An abdominal ultrasound revealed an isoechoic nodular formation on the right kidney, and CT scans confirmed the presence of a 7-cm renal tumour and extensive leiomyomas in the patient’s uterus. Genetic testing revealed a mutation in the fumarate hydratase (FH) gene, leading to the diagnosis of hereditary leiomyomatosis and renal cell carcinoma.Pathophysiology: HLRCC is a condition caused by an autosomal dominant germline mutation in the FH gene, characterized by skin lesions, uterine leiomyomas, and aggressive renal cell carcinoma. FH acts as a tumour suppressor gene and encodes the fumarase protein. HLRCC arises from a variety of missense mutations in the FH gene that prevent the formation of a full fumarase tetramer. The disease is likely related to the dosage of the gene, rather than the site of mutation. Consequently, there is no known genotype/phenotype correlation. Fumarate inhibits HIF-α hydroxylases, so loss of the FH gene causes fumarate accumulation within cells and prevents the degradation of HIF-α. This induces a state of pseudohypoxia and leads to upregulation of genes involved in cell growth and tumorigenesis. Fumarate accumulation also modulates the Keap1-Nrf2 pathway by inducing activation of Nrf2 and allows for the protection of cancer cells from oxidative and electrophilic stressors. Furthermore, deficiency of fumarase prevents oxidative phosphorylation, thus promoting aerobic glycolysis, the most common method of metabolism used by cancer cells.Socioeconomics: The risk of infertility and developing an aggressive cancer makes it essential to have frequent and thorough screening procedures early in life for children with a family history of HLRCC. Medical costs, infertility risk, and frequent hospital visits breed anxiety and stress for patients and family members. Fortunately, organizations like HLRCC Family Alliance exist to provide accessible information and represent a welcoming support community.Treatment Strategy: To treat the patient’s renal tumour, we suggest a radical nephrectomy with wide margins due to the aggressive nature of renal cell carcinoma. Additional imaging should be performed to identify and excise any metastatic tumours. The patient should also consult a gynecologist regarding her extensive uterine leiomyomas and will likely undergo a hysterectomy or myomectomy. A full dermatological exam should be performed to locate additional cutaneous leiomyomas. We also strongly suggest that family members undergo genetic testing. Lastly, we recommend the patient connect with support groups such as HLRCC Family Alliance to receive continued social support following her diagnosis and throughout her life.

Loci associated with conception rate in crossbred beef heifers.

The inability of beef cattle to maintain full term pregnancies has become an economic concern for the beef industry. Herd management and nutritional improvements have alleviated environmental impacts on embryonic and fetal loss, yet additional gains can be made through genomic selection. The objectives of this study were to identify loci and gene-sets in crossbred beef heifers associated with the number of services required to become pregnant (TBRD) and heifer conception rate at first service (HCR1). Heifers (n = 709) from a commercial beef operation underwent one round of artificial insemination, before exposure to bulls for natural service for 50 days. Pregnancy and time of conception was determined by ultrasound 35 days after the breeding season. Heifers were genotyped using the GeneSeek (Lincoln, NE) Bovine GGP50K BeadChip prior to genome-wide association analyses (GWAA) conducted using an EIGENSTRAT-like model to identify loci associated (P < 1 × 10−5) with TBRD and HCR1. One locus was associated (P = 8.97 × 10−6) with TBRD on BTA19 and included the positional candidate gene ASIC2, which is differentially expressed in the endometrium of fertility classified heifers, and the positional candidate gene, SPACA3. Gene-set enrichment analyses using SNP (GSEA-SNP) data, was performed and identified one gene-set, oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen as enriched (NES = 3.15) with TBRD and contained nine leading edge genes that contributed to the enrichment of the gene set. The enriched gene-set is involved in catalyzing oxidation-reduction reactions, which have been associated with oxidative stressors impacting pregnancy success. No loci were associated nor gene-sets enriched with HCR1. Identification of loci, positional candidate genes, gene-sets and leading edge genes enriched for fertility facilitate genomic selection that allows producers to select for reproductively superior cattle, reduce costs associated with infertility, and increase percent calf crop.

Dietary nanoencapsulated quercetin homeostated transcription of redox-status orchestrating genes in zebrafish (Danio rerio) exposed to silver nanoparticles

The present study assessed the protective effect of chitosan-nanoencapsulated quercetin (Qu-ChiNPs) against oxidative stress caused by silver nanoparticles (AgNPs). To this end, the transcription of prime genes regulating hepatic Keap1-Nrf2 pathway as well as downstream antioxidant enzymes were monitored prior to and after oxidative stress by AgNPs. Zebrafish (Danio rerio; n = 225) was assigned into five experimental groups based on feeding with diets supplemented with different additives as follows: negative and positive control groups, without additive; ChiNPs, 400 mg nanochitosan per kg diet; Quercetin, 400 mg free quercetin per kg diet; and Qu-ChiNPs, 400 mg Qu-ChiNPs per kg diet. At the end of the feeding trial (40 days), the experimental groups, except the negative control, were exposed to sublethal concentration of AgNPs (0.15 mg L−1) for 96h. Before exposure to AgNPs, free quercetin-treated diet significantly upregulated Keap1, Nrf2, Cat, SOD, GPx, and GST genes in the liver tissue when compared with the control diet, whereas Qu-Chi.NPs downregulated their transcription to the lowest levels. After exposure to AgNPs, all genes exhibited different responses in the AgNPs-exposed groups. The highest transcription of Nrf2, Cat, SOD, GPx, and GST was observed in the positive group, with being upregulated about 8, 10, 8, 8, and 7 times, respectively, when compared to the respective ones in the negative control. However, Keap1 showed a reverse response with being transcripted 12 times lower. The quercetin treatments, especially Qu-Chi.NPs, significantly reduced the transcription of Nrf2, Cat, SOD, GPx, and GST genes, yet enhanced Keap1 expression. Qu-Chi.NPs reduced the expression of Nrf2, SOD, Cat, GPx, and GST about 11, 10, 15, 10, and 10 times, respectively, yet increased that of Keap1 about 12 times. Taken together, nanoencapsulation can improve the antioxidant efficacy of quercetin against AgNPs toxicity and might reduce involvement of the cellular antioxidant system through tuning redox status. More broadly, it would be interesting to assess the effects of Qu-Chi.NPs against other metallic and organic oxidative stressors or pollutants.

Donor sex, age and ethnicity impact stored red blood cell antioxidant metabolism through mechanisms in part explained by glucose 6-phosphate dehydrogenase levels and activity.

Red blood cell (RBC) storage in the blood bank promotes the progressive accumulation of metabolic alterations that may ultimately impact the erythrocyte capacity to cope with oxidant stressors. However, the metabolic underpinnings of the capacity of RBC to resist oxidant stress and the potential impact of donor biology on this phenotype are not known. Within the framework of the REDS-III RBC-Omics study, RBC from 8,502 healthy blood donors were stored for 42 days and tested for their propensity to hemolyse following oxidant stress. A subset of extreme hemolysers donated a second unit of blood, which was stored for 10, 23, and 42 days and profiled again for oxidative hemolysis and metabolomics (599 samples). Alterations of RBC energy and redox homeostasis were noted in donors with high oxidative hemolysis. RBC from females, donors over 60 years old, donors of Asian/South Asian race-ethnicity, and RBC stored in additive solution- 3 were each independently characterized by improved antioxidant metabolism compared to, respectively, males, donors under 30 years old, Hispanic and African American race ethnicity donors, and RBC stored in additive solution-1. Merging metabolomics data with results from an independent genome-wide association study on the same cohort, we identified metabolic markers of hemolysis and glucose 6-phosphate dehydrogenasedeficiency, which were associated with extremes in oxidative hemolysis and dysregulation in nicotinamide adenine dinucleotide phosphate and glutathione- dependent detoxification pathways of oxidized lipids. Donor sex, age, ethnicity, additive solution and glucose 6-phosphate dehydrogenase status impact the metabolism of the stored erythrocyte and its susceptibility to hemolysis following oxidative insults.

AMPK is Activated During Sleep Deprivation in Caenorhabditis elegans Ryan Schuck, Edwin Li, Matthew D. Nelson Saint Joseph’s University, Department of Biology, Philadelphia, PA 19131

Sleep conserves energy and allows for its reallocation towards biosynthetic pathways, suggesting that one of sleep’s functions is cellular restoration. During times of sickness, cells are damaged and sleep is heightened, supporting an additional immune function for sleep. In some cases, sleep is directly induced by signaling mechanisms of the immune response. Work in model systems, such as Caenorhabditis elegans and Drosophila melanogaster, have identified a protective sleep state called stress‐induced sleep (SIS), which resembles sleep during sickness. SIS occurs following exposure to environmental stressors such as noxious temperatures, osmotic imbalances, physical damage and UV irradiation. While the signaling molecules regulating SIS have begun to be elucidated, the mechanisms linking sleep to cellular restoration are still unknown. A master regulator of nutrient/energy sensing, adenosine monophosphate‐activated protein kinase (AMPK), which is highly conserved from invertebrates to mammals, is a candidate for such a link. In mammalian cells, AMPK is activated by low levels of ATP and oxidative stressors, the latter occurring through the overproduction of reactive oxygen species. In both cases, activation of AMPK requires phosphorylation of a threonine residue (T172). We used CRISPR to make an AMPK mutant in C. elegans in which the predicted, homologous T172 site is changed to an alanine, to determine if this amino acid is conserved functionally. We find that these mutants live significantly shorter lives than wild type animals following exposure to paraquat, a known inducer of oxidative stress. Moreover, these animals provide us with an important control for testing our broader hypothesis that AMPK links sleep to cellular repair during SIS. To test this, we conducted a temporal western blot analysis, assessing AMPK’s phosphorylation levels during SIS in both wild type animals and those that are genetically sleep deprived. We find that AMPK is not significantly activated during times of normal SIS, however, mutant animals that lack sleep‐promoting neuropeptides, display increased AMPK activation 24 hours after stress exposure. These mutants also display reduced longevity in response to oxidative stress. We hypothesize that sleep deprivation promotes an increase in AMPK phosphorylation in an attempt to provide cellular protection/repair during SIS.Support or Funding InformationNSF Award #1845020 (PI Nelson)

Serum deprivation primes cancer cells to adapt and survive oxidative stress

Inadequate nutrient intake, a survival benchmark in cancer cells, leads to oxidative stress (OS) disrupting homeostasis, activating signaling, and altering metabolism. As reactive oxygen species (ROS) increase, oxidative stressors activate mechanisms allowing cancer cells to adapt and survive. Our data suggests that nutrient‐deprived cancer cells tolerate OS. Moreover, a quiescent phenotype along with nuclear factor‐kappaB (NF‐κB) signaling may synergistically protect tumor cells from OS induced by nutrient deprivation. In DU145 prostate cancer cell line, we observed that serum‐deprived cells maintained viability after exposure to H2O2 and transitioned to a quiescent phenotype via up‐regulated quiescent markers. Additionally, we transiently silenced NF‐κB (RelA/p65) via siRNA, and demonstrated the percentage of total apoptosis in DU145 cells significantly increased in serum‐deprived cells exposed to H2O2. Furthermore, we observed that inhibition of quiescence significantly reduced NF‐κB‐mediated cell survival in response to OS using two quiescence inhibitors targeting Mirk/Dyrk1b kinase (quiescence inducer), AZ191 and NCGC00185981‐05/ML195; concomitantly, a distinct NF‐κB nuclear localization was not observed. It is unclear how prostate cancer cells adapt and survive oxidative stress; however, we hypothesize nutrient deprivation primes cancer cells for OS cell survival, concurrent with a transition to a quiescent phenotype and NF‐κB signaling.Support or Funding InformationThese studies were supported by the NIH/NIGMS/RISE #5R25GM060414‐15 and NIH/NIHMD/RCMI #5G12MD007590.

Neuroprotective and neurotoxic outcomes of androgens and estrogens in an oxidative stress environment

BackgroundThe role of sex hormones on cellular function is unclear. Studies show androgens and estrogens are protective in the CNS, whereas other studies found no effects or damaging effects. Furthermore, sex differences have been observed in multiple oxidative stress-associated CNS disorders, such as Alzheimer’s disease, depression, and Parkinson’s disease. The goal of this study is to examine the relationship between sex hormones (i.e., androgens and estrogens) and oxidative stress on cell viability.MethodsN27 and PC12 neuronal and C6 glial phenotypic cell lines were used. N27 cells are female rat derived, whereas PC12 cells and C6 cells are male rat derived. These cells express estrogen receptors and the membrane-associated androgen receptor variant, AR45, but not the full-length androgen receptor. N27, PC12, and C6 cells were exposed to sex hormones either before or after an oxidative stressor to examine neuroprotective and neurotoxic properties, respectively. Estrogen receptor and androgen receptor inhibitors were used to determine the mechanisms mediating hormone-oxidative stress interactions on cell viability. Since the presence of AR45 in the human brain tissue was unknown, we examined the postmortem brain tissue from men and women for AR45 protein expression.ResultsNeither androgens nor estrogens were protective against subsequent oxidative stress insults in glial cells. However, these hormones exhibited neuroprotective properties in neuronal N27 and PC12 cells via the estrogen receptor. Interestingly, a window of opportunity exists for sex hormone neuroprotection, wherein temporary hormone deprivation blocked neuroprotection by sex hormones. However, if sex hormones are applied following an oxidative stressor, they exacerbated oxidative stress-induced cell loss in neuronal and glial cells.ConclusionsSex hormone action on cell viability is dependent on the cellular environment. In healthy neuronal cells, sex hormones are protective against oxidative stress insults via the estrogen receptor, regardless of sex chromosome complement (XX, XY). However, in unhealthy (e.g., high oxidative stress) cells, sex hormones exacerbated oxidative stress-induced cell loss, regardless of cell type or sex chromosome complement. The non-genomic AR45 receptor, which is present in humans, mediated androgen’s damaging effects, but it is unknown which receptor mediated estrogen’s damaging effects. These differential effects of sex hormones that are dependent on the cellular environment, receptor profile, and cell type may mediate the observed sex differences in oxidative stress-associated CNS disorders.

Cryptic prophages in a blaNDM-1-bearing plasmid increase bacterial survival against high NaCl concentration, high and low temperatures, and oxidative and immunological stressors.

In this study, we investigated the effect of cryptic prophage regions in a blaNDM-1-bearing plasmid, which was identified in a patient from South Korea, on the survival of bacteria against adverse environmental conditions. First, we conjugated the intact plasmid and plasmids with deleted cryptic prophages into Escherichia coli DH5α. The E. coli transconjugants carrying the plasmid with intact cryptic prophages showed increased survival during treatment with a high concentration of NaCl, high and low temperatures, an oxidative stressor (H2O2), and an immunological stressor (human serum). By contrast, the transconjugants carrying the plasmid with a single-cryptic prophage knockout did not show any change in survival rates. mRNA expression analyses revealed that the genes encoding sigma factor proteins were highly upregulated by the tested stressors and affected the expression of various proteins (antioxidant, cell osmosis-related, heat shock, cold shock, and universal stress proteins) associated with the specific defense against each stress. These findings indicate that a bacterial strain carrying a plasmid with intact carbapenemase gene and cryptic prophage regions exhibited an increased resistance against simulated environmental stresses, and cryptic prophages in the plasmid might contribute to this enhanced stress resistance. Our study indicated that the coselection of antibiotic resistance and resistance to other stresses may help bacteria to increase survival rates against adverse environments and disseminate.

Alda-1, an activator of ALDH2, ameliorates Achilles tendinopathy in cellular and mouse models

Achilles tendinopathy has a high re-injury rate and poor prognosis. Development of effective therapy for Achilles tendinopathy is important. Excessive accumulation of ROS and resulting oxidative stress are believed to cause tendinopathy. Overproduction of hydrogen peroxide (H2O2), the most common ROS, could lead to the tendinopathy by causing oxidative damage, activation of endoplasmic reticulum (ER) stress and apoptotic death of tenocytes. Activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) is expected to alleviate oxidative stress and ER stress. Alda-1 is a selective and potent activator of ALDH2. In this study, we examined the cytoprotective benefit of Alda-1, an activator of ALDH2, on H2O2-induced Achilles tendinopathy in cellular and mouse models. We prepared cellular and mouse models of Achilles tendinopathy by treating cultured Achilles tenocytes and Achilles tendons with oxidative stressor H2O2. Subsequently, we studied the protective benefit of Alda-1 on H2O2-induced Achilles tendinopathy. Alda-1 pretreatment attenuated H2O2-induced cell death of cultured Achilles tenocytes. Treatment of Alda-1 prevented H2O2-induced oxidative stress and depolarization of mitochondrial membrane potential in tenocytes. Application of Alda-1 attenuated H2O2-triggered mitochondria- and ER stress-mediated apoptotic cascades in cultured tenocytes. Alda-1 treatment ameliorated the severity of H2O2-induced Achilles tendinopathy in vivo by preventing H2O2-induced pathological histological features of Achilles tendons, apoptotic death of Achilles tenocytes and upregulated expression of inflammatory cytokines IL-1β and TNF-α. Our results provide the evidence that ALDH2 activator Alda-1 ameliorates H2O2-induced Achilles tendinopathy. Alda-1 could be used for preventing and treating Achilles tendinopathy.

Oxidative Stress Induction Is a Rational Strategy to Enhance the Productivity of Antrodia cinnamomea Fermentations for the Antioxidant Secondary Metabolite Antrodin C.

Antioxidant metabolites contribute to alleviating oxidative stress caused by reactive oxygen species (ROS) in microorganisms. We utilized oxidative stressors such as hydrogen peroxide supplementation to increase the yield of the bioactive secondary metabolite antioxidant antrodin C in submerged fermentations of the medicinal mushroom Antrodia cinnamomea. Changes in the superoxide dismutase and catalase activities of the cells indicate that ROS are critical to promote antrodin C biosynthesis, while the ROS production inhibitor diphenyleneiodonium cancels the productivity-enhancing effects of H2O2. Transcriptomic analysis suggests that key enzymes in the mitochondrial electron transport chain are repressed during oxidative stress, leading to ROS accumulation and triggering the biosynthesis of antioxidants such as antrodin C. Accordingly, rotenone, an inhibitor of the electron transport chain complex I, mimics the antrodin C productivity-enhancing effects of H2O2. Delineating the steps connecting oxidative stress with increased antrodin C biosynthesis will facilitate the fine-tuning of strategies for rational fermentation process improvement.

Phenol metabolism of two cultivars of durum wheat (Triticum durum Desf.) as affected by ozone and flooding stress

AbstractTo compare the phenolic responses under oxidative stressors, plants of two Italian cultivars of durum wheat (Claudio and Mongibello) were (a) exposed to ozone (O3) (80 ppb, 5 hr/day for 70 consecutive days), with the aim to investigate the changes of phenolic compound contents in their leaves, or (b) flooded (seven consecutive days). Plants showed O3‐induced visible injury, but their photosynthetic performance was not affected by the pollutant. Specifically, Claudio showed a higher O3 tolerance than Mongibello. The major value of the present study is undoubtedly the pioneering investigation of phenolic metabolism of durum wheat under O3. We identified 12 foliar phenolic compounds in all leaf samples (i.e. controls, exposed to O3 and flooded): ten phenolic acids, a flavanol (catechin hydrate) and a phenolic aldehyde (syringaldehyde). Overall, O3 exposure resulted in accumulations of phenolic compounds, especially in Claudio. These responses can be likely considered a fine‐regulated repair process that equipped Claudio stressed plants with an antioxidant system capable of scavenging oxidative stress. Different phenolic variations were found in flooded plants, suggesting that phenolic response to environmental constraints is stress specific. Our study confirms that investigations and characterization of specific phenolic profiles of crop cultivars under oxidative stress may be helpful in breeding programmes.

Evaluation of Antioxidant Activity of Spice-Derived Phytochemicals Using Zebrafish.

Various dietary phytochemicals seem to display antioxidant activity through the NF-E2-related factor 2 (Nrf2) pathway. However, few studies have demonstrated its antioxidant effect and Nrf2 dependency at the animal level. We constructed a zebrafish-based assay system to analyze the in vivo antioxidant activity of phytochemicals and examined the activity of 10 phytochemicals derived from spices, using this system as a pilot study. Hydrogen peroxide and arsenite were used as oxidative stressors, and Nrf2 dependency was genetically analyzed using an Nrf2-mutant zebrafish line. The activities of curcumin, diallyl trisulfide and quercetin were involved in the reduction of hydrogen peroxide toxicity, while those of cinnamaldehyde, isoeugenol and 6-(methylsulfinyl)hexyl isothiocyanate were involved in the reduction of arsenite toxicity. The antioxidant activities of these phytochemicals were all Nrf2 dependent, with the exception of cinnamaldehyde, which showed strong antioxidant effects even in Nrf2-mutant zebrafish. In summary, we succeeded in constructing an assay system to evaluate the in vivo antioxidant activity of various phytochemicals using zebrafish larvae. Using this system, we found that each spice-derived phytochemical has its own specific property and mechanism of antioxidant action.

Abstract TP478: Mechanisms of Brain Endothelial Erythrophagocytosis: Insights Into the Pathogenesis of Cerebral Microbleeds

Background: Cerebral microbleeds, MRI signatures of cerebral microhemorrhage, are thought to arise from brain microvessel disruption. Our prior work suggests an alternate origin for some microbleeds, emphasizing brain endothelial erythrophagocytosis (BEE) producing microhemorrhage-like lesions (“pseudo-microbleeds”) in absence of vascular disruption (Frontiers Cell Neurosci 9-18; PMID: 30237761). We demonstrated an erythrophagocytic phenotype of brain microvascular endothelium for oxidatively-stressed red blood cells (RBC), and migration of iron-rich hemoglobin across the brain endothelium. However, cellular mechanisms underlying BEE and hemoglobin passage across brain endothelium are not defined. Herein we investigated a) role of oxidative stress and RBC phosphatidylserine (PS) exposure in BEE and b) intracellular endosomal trafficking of RBC within brain endothelial cells. Methods: Murine brain endothelial cells (bEnd.3 cells) were incubated with 2x10 6 mouse RBC treated with 3mM tert-butylhydroperoxide (t-BHP, an oxidative stressor) or sterile PBS (control) for 48h at 37°C, in the presence of 7.5 μg annexin V to cloak PS, or vitamin C (15μM-1500μM) to reduce reactive oxygen species (ROS). BEE was evaluated by hematoxylin &amp; eosin stain and diaminofluorene assay for hemoglobin. RBC ROS levels, PS exposure, and cell viability were measured. bEnd.3 cells were immunostained to visualize RBC in early and late endosomes, the latter being the presumed site of RBC degradation post-erythrophagocytosis. Results: tBHP induced both ROS production and PS exposure in RBC. There was a 9-fold increase (p&lt;0.001) in BEE of t-BHP-RBC compared with control. Vitamin C reduced RBC ROS levels (70%, p&lt;0.001) and PS exposure (27%, p&lt;0.001), while annexin V blocked RBC PS exposure (65%, p&lt;0.001). BEE was significantly attenuated by annexin V (63%, p&lt;0.001) and vitamin C (39%, p&lt;0.001). No change in bEnd.3 viability was observed and t-BHP-RBC localized to both early and late endosomes. Conclusions: These data demonstrate the importance of RBC PS exposure in BEE, and describe the intracellular trafficking of RBC in brain endothelial cells. These findings provide insights into the development of microhemorrhage-like lesions and cerebral microbleeds.

Cytoprotective effects of berry anthocyanins against induced oxidative stress and inflammation in primary human diabetic aortic endothelial cells

Type 2 diabetes is associated with oxidative stress and low-grade inflammation resulting in endothelial dysfunction (ED). This study determined to explore the protective effects of berry-derived anthocyanins (AC) with potent antioxidant and anti-inflammatory activities in human diabetic endothelial cells upon oxidative and inflammatory stressors. Cultured healthy human aortic endothelial cells (HAEC) and diabetic human aortic endothelial cells (D-HAEC) exposed to oxidative stress by hydrogen peroxide (H2O2, 75 μM) and lipopolysaccharide (LPS, 1 μg/mL) as an inflammatory inducer before treatment with AC (50 μl/ml). The results from cytotoxicity assays showed that AC had no significant effects in cell viability (P-value < 0.0001), and exposure to H2O2 75 μM had a less toxic effect (P-value < 0.05). Although, AC significantly decreased H2O2-induced cytotoxicity and oxidative stress in both HAEC and D-HAEC cell lines (P-value < 0.0001), no positive impact of AC was found on the GSSG/GSH ratios (P-value < 0.05). Exposure to the LPS increased the production of IL-6 in both HAEC and D-HAEC cell lines (P-value < 0.0001), whereas AC treatment reduced LPS‐induced IL‐6 production in both cell lines with a more robust impact on D-HAEC (P-value < 0.0001). While LPS increased inflammasome assembling and caspase-1 activation, AC treatment inhibited caspase-1 activation in D-HAEC (P ≤ 0.05). This study indicated that berry anthocyanins reduced oxidative stress and inflammation via the inhibition of the NF-ƙB signaling pathway, which contributes to mitigating the diabetes-induced up-regulation of NF-ƙB.

Plant-Derived Isoquinoline Alkaloids that Target Ergosterol Biosynthesis Discovered by Using a Novel Antifungal Screening Tool

Background: The ergosterol pathway is a prime antifungal target as it is required for fungal survival, yet is not involved in human homeostasis. Methods to study the ergosterol pathway, however, are often time-consuming. The minimum inhibitory concentration (MIC) assay is a simple research tool that determines the lowest concentration at which a novel antimicrobial is active in vitro with limited scope to determine the mechanism of action for a drug. Methods: In this study, we show that by adding hydrogen peroxide, an oxidative stressor, or glutathione (GSH), an antioxidant, to modify a commonly performed MIC assay allowed us to screen selectively for new antifungal drugs that target ergosterol biosynthesis in fungi. A human pathogen and dermatophyte,Microsporum gypseum, was used as a test organism. When exposed to ergosterol targeting drugs, the hydrogen peroxide treatment significantly decreased fungal survival by reducing ergosterol in the cell wall, whereas GSH increased survival of M. gypseum. Further, by performing a series of experiments with M. gypseum and Trichophyton rubrum, it was determined that the oxidative stress from hydrogen peroxide causes cell death at different developmental stages based on fungal species. Findings: These findings allow us to describe a simple, high-throughput method for simultaneously screening new antifungal drugs for activity and effects on the ergosterol pathway. Interpretation: By using this tool, two isoquinoline alkaloids were discovered to be potent inhibitors of ergosterol biosynthesis in vitro by reducing the amount of ergosterol without affecting the expression of 1,3-β-glucan. Both compounds also significantly reduced the severity of acanthosis, hyperkeratosis, spongiosis and dermal edema in vivo. Funding Statement: This work was supported by Faculty Research Grant, Hong Kong Baptist University (FRG 2/17-18/103), the Hong Kong Baptist University (HKBU) Interdisciplinary Research Matching Scheme (RC-IRMS/15-16/02),NSFC funding 31670360, 81973293, U1702286 and Natural Science Foundation of SZU 860-000002110131. Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: Female guinea pigs were used for the in vivo antifungal study, which was approved by the laboratory animal ethics committee of Shenzhen University, China [Approval number: SYXK (Yue) 2014-0140].

Novel oral multifunctional antioxidant prevents noise-induced hearing loss and hair cell loss

Oxidative stress is a major contributor to noise-induced hearing loss, the most common cause of hearing loss among military personnel and young adults. HK-2 is a potent, orally-active, multifunctional, redox-modulating drug that has been shown to protect against a wide range of neurological disorders with no observed side effects. HK-2 protected cochlear HEI-OC1 cells against various forms of experimentally-induced oxidative stressors similar to those observed during and after intense noise exposure. The mechanisms by which HK-2 protects cells is twofold, first by its ability to reduce oxidative stress generated by free radicals, and second, by its ability to complex biologically active transition metals such as Fe+2, thus reducing their availability to participate in the Fenton reaction where highly toxic hydroxyl radicals are generated. For the rat in vivo studies, HK-2 provided significant protection against noise-induced hearing loss and hair cell loss. Noise-induced hearing loss was induced by an 8–16 kHz octave band noises presented for 8 h/d for 21 days at an intensity of 95 dB SPL. In the Prevention study, HK-2 was administered orally beginning 5 days before the start of the noise and ending 10 days after the noise. Treatment with HK-2 dose-dependently reduced the amount of noise-induced hearing impairment, reflected in the cochlear compound action potential, and noise-induced hair cell loss. In a subsequent Rescue experiment in which HK-2 was administered for 10 days starting after the noise was turned off, HK-2 also significantly reduced the amount of hearing impairment, but the effect size was substantially less than in the Prevention studies. HK-2 alone did not adversely affect HEI-OC1 cell viability, nor did it cause any adverse changes in rat body weight, behavior, cochlear function or hair cell integrity. Thus, HK-2 is a novel, safe, orally-deliverable and highly effective otoprotective compound with considerable potential for preventing hearing loss from noise and other hearing disorders linked to excessive oxidative stress.