Gene expression of apoptosis-related genes,stress protein and antioxidant enzymes in hemocytes of white shrimp Litopenaeus vannamei under nitrite stress

Apoptotic cell ratio and mRNA expression of caspase-3, cathepsin B (CTSB), heat shock protein 70 (HSP70), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx) and thioredoxin (TRx) in hemocytes of white shrimp Litopenaeus vannamei exposed to nitrite-N (20 mg/L) was investigated at different stress time (0, 4, 8, 12, 24, 48 and 72 h). The apoptotic cell ratio and mRNA expression level of CTSB were significantly increased in shrimp exposed to nitrite-N for 48 and 72 h. Caspase-3 mRNA expression level significantly increased by 766.50% and 1811.16% for 24 and 48 h exposure, respectively. HSP70 expression level significantly increased at 8 and 72 h exposure. MnSOD mRNA expression in hemocytes up-regulated at 8 and 48 h, while CAT mRNA expression level increased at 24 and 48 h. GPx expression showed a trend that increased first and then decreased. Significant increases of GPx expression were observed at 8 and 12 h exposure. Expression level of TRx reached its highest level after 48 h exposure. These results suggest that nitrite exposure induces expression of apoptosis-related genes in hemocytes, and subsequently caused hemocyte apoptosis. Meanwhile, expression levels of HSP70 and antioxidant enzymes up-regulated to protect the hemocyte against nitrite stress.     

Mitochondrial matrix P53 sensitizes cells to oxidative stress

A mitochondrial matrix-specific p53 construct (termed p53-290) in HepG2 cells was utilized to determine the impact of p53 in the mitochondrial matrix following oxidative stress. H₂O₂ exposure reduced cellular proliferation similarly in both p53-290 and vector cells, and p53-290 cells demonstrating decreased cell viability at 1 mM H₂O₂ (~ 85% viable). Mitochondrial DNA (mtDNA) abundance was decreased in a dose-dependent manner in p53-290 cells while no change was observed in vector cells. Oximetric analysis revealed reduced maximal respiration and reserve capacity in p53-290 cells. Our results demonstrate that mitochondrial matrix p53 sensitizes cells to oxidative stress by reducing mtDNA abundance and mitochondrial function.     

Design,synthesis and biological evaluation of novel 3,4,5-trisubstituted aminothiophenes as inhibitors of p53–MDM2 interaction. Part 2

Five series of novel 3,4,5-trisubstituted aminothiophene derivatives and analogs were designed and synthesized based on our previous studies. All target compounds were evaluated for their p53–MDM2 binding inhibitory activities and anti-proliferation activities against A549 and PC3 tumor cell lines. Twelve compounds displayed comparable p53–MDM2 binding inhibitory activities to that of Nutlin-3. Among them, compound 7a exhibited marked binding affinity (IC₅₀ = 0.086 μM). In addition, most target compounds showed potent anti-proliferation activities with IC₅₀ values at low micromolar level. A good selective profile for wild-type p53 expression cell line was also observed. Molecular docking analysis was performed as well to predict possible binding modes of target compounds with MDM2.     

Association of p53 and MDM2 polymorphisms with risk of human papillomavirus (HPV)-related esophageal squamous cell carcinoma (ESCC)

PurposeThough polymorphisms of the tumor suppressor gene p53 have been extensively investigated in numerous tumors, particularly tumors associated with human papillomavirus (HPV) infection. However, the results remain controversial. Our previous study showed that HPV serostatus is not an independent risk factor for esophageal squamous cell carcinoma (ESCC) in nonsmokers and nondrinkers. Given the roles of p53 and HPV E6 as well as MDM2 oncoproteins in p53 degradation, we validated the association of p53 and MDM2 polymorphisms with ESCC risk stratified by HPV16 sero-status.MethodsSingle nucleotide polymorphisms of p53 Arg72Pro (rs1042522) and MDM2 (rs937283) in 307 ESCC patients and 311 healthy controls were genotyped. The presence or absence of HPV16 in serum was measured by enzyme-linked immunosorbent assay. Multivariable logistic regression analysis was used to evaluate the possible associations of p53 and MDM2 polymorphisms with ESCC risk stratified by HPV16 sero-status.ResultsPatients carrying p53 Arg/Arg or Arg/Pro had a higher risk of esophageal SCC (P < 0.001, Odds ratio [OR] 4.98, 95% confidential interval [CI] 3.46–7.17), however, not found in MDM2 rs937283. The risk of esophageal SCC increased significantly among patients carrying p53 Arg/Arg, or Arg/Pro and HPV16-seropositivity (P < 0.001, OR 9.33, 95% CI 5.44–16.0), but not for MDM2 rs937283. The risk of esophageal SCC was further elevated among patients carrying Arg/Arg or Arg/Pro and HPV16-seropositivity who were smokers (P < 0.001, OR 27.05, 95% CI 11.06–66.16) or drinkers (P < 0.001, OR 13.20, 95% CI 5.74–30.38).ConclusionHPV16 seropositivity synergized with p53 Arg/Arg or Arg/Pro and increased ESCC risk, especially in smokers or drinkers.     

Interactive effects of elevated temperature and CO2 levels on metabolism and oxidative stress in two common marine bivalves (Crassostrea virginica and Mercenaria mercenaria)

Marine bivalves such as the hard shell clams Mercenaria mercenaria and eastern oysters Crassostrea virginica are affected by multiple stressors, including fluctuations in temperature and CO₂ levels in estuaries, and these stresses are expected to be exacerbated by ongoing global climate change. Hypercapnia (elevated CO₂ levels) and temperature stress can affect survival, growth and development of marine bivalves, but the cellular mechanisms of these effects are not yet fully understood. In this study, we investigated whether oxidative stress is implicated in cellular responses to elevated temperature and CO₂ levels in marine bivalves. We measured the whole-organism standard metabolic rate (SMR), total antioxidant capacity (TAOC), and levels of oxidative stress biomarkers in the muscle tissues of clams and oysters exposed to different temperatures (22 and 27 °C) and CO₂ levels (the present day conditions of ~ 400 ppm CO₂ and 800 ppm CO₂ predicted by a consensus business-as-usual IPCC emission scenario for the year 2100). SMR was significantly higher and the antioxidant capacity was lower in oysters than in clams. Aerobic metabolism was largely temperature-independent in these two species in the studied temperature range (22–27 °C). However, the combined exposure to elevated temperature and hypercapnia led to elevated SMR in clams indicating elevated costs of basal maintenance. No persistent oxidative stress signal (measured by the levels of protein carbonyls, and protein conjugates with malondialdehyde and 4-hydroxynonenal) was observed during the long-term exposure to moderate warming (+ 5 °C) and hypercapnia (~ 800 ppm CO₂). This indicates that long-term exposure to moderately elevated CO₂ and temperature minimally affects the cellular redox status in these bivalve species and that the earlier observed negative physiological effects of elevated CO₂ and temperature must be explained by other cellular mechanisms.     

MDM2 promoter polymorphism is associated with increased susceptibility to hepatocellular carcinoma in Turkish population

Background: The mouse double minute 2 (MDM2) gene represents one of the central nodes in the p53 pathway. A naturally occurring T/G single nucleotide polymorphism (SNP) in the intronic promoter of MDM2, SNP309 (rs2279744), was shown to influence MDM2 expression and p53 activity. SNP in the promoter region of MDM2 gene has recently been shown to be associated with accelerated tumor formation in both hereditary and sporadic cancers in humans. In this study, we aim to evaluate the association of SNP309 with the risk of hepatocellular carcinoma (HCC) development among Turkish population. Methods: MDM2 SNP309 polymorphism was investigated in 110 confirmed subjects with HCC and 110 cancer-free control subjects matched on age, gender, smoking and alcohol consumption by using a polymerase chain reaction-restriction fragment length polymorphism assay. Results: The allele frequencies of case subjects (T, 0.48; G, 0.52) were significantly different from those of control subjects (T, 0.65; G, 0.35) (p = 0.003). The proportion of GG genotype of the SNP309 in patients with HCC (26%) was significantly higher than that in patients without HCC (14%). We observed that compared with the TT genotype, the genotypes containing G allele [TG (OR, 2.19; 95% CI, 1.18–4.07; p = 0.013) or GG (OR, 3.63; 95% CI, 1.65–8.00; p = 0.001)] were associated with significant increased susceptibility to HCC. Conclusion: Our findings suggest that the MDM2 promoter SNP309 G allele is associated with presence of HCC in Turkish population.     

Multi-organ toxicological impact of fungicide propiconazole on biochemical and histological profile of freshwater fish Channa punctata Bloch

Fungicides are a pesticide that particularly kills or destroy fungi responsible for several diseases associated to humans and other living organisms. Assessment of toxic effects and mechanisms of fungicide action is important because humans and domesticated animals get exposed to these pesticides through a wide variety of applications. Several fungicides are being used at the large scale for the crop protection from the fungal invasion. Propiconazole (PCZ), a trazole-containing fungicide, is widely used in China and various Asian countries for food crop protection which made it easily to exposed to the aquatic system. Long term usage of PCZ may contaminate the water bodies, but its toxicity to aquatic organisms is not well studied. In this study, freshwater fish, Channa punctata Bloch was exposed to different sub-lethal concentrations of the fungicide, PCZ (0.5 and 5 ppm) for a period of 96 h. Various biochemical assays and histological alterations were measured to determine the organ toxicity caused by PCZ exposure particularly in liver, kidney and gills of the fishes. Compared to the control group, fish exposed to PCZ (96 h) showed marked dose dependent toxicity. The levels of lipid peroxidation (LPO) and protein carbonyls (PC), oxidative stress biomarker of liver, kidney and gills in the experimental group were significantly higher (P < 0.05 and P < 0.001) compared to the control group. Levels of reduced glutathione (GSH) and non-protein thiols (NP-SH) decreased significantly (P <0.05–0.001) in all analyzed intoxicated organs of the PCZ exposed fishes. Activity of glutathione-S-transferase (GST), glutathione peroxidase (GPx) and catalase (CAT) in fungicide treated groups was significantly lowered (P < 0.05–0.001). In addition, histopathological examination in the organs showed significant changes like atrophy of primary and secondary gill lamellae, infiltrations, inflammation, hepatocyte degeneration, vacuolization and necrotic kidney. Thus, PCZ exposure altered the oxidative stress homeostasis and brought about histopathological changes which may serve as potential biomarkers of the PCZ toxicity in the laboratory set-up for potential risk assessment.     

HIV proteins (gp120 and Tat) and methamphetamine in oxidative stress-induced damage in the brain: Potential role of the thiol antioxidant N-acetylcysteine amide

An increased risk of HIV-1 associated dementia (HAD) has been observed in patients abusing methamphetamine (METH). Since both HIV viral proteins (gp120, Tat) and METH induce oxidative stress, drug abusing patients are at a greater risk of oxidative stress-induced damage. The objective of this study was to determine if N-acetylcysteine amide (NACA) protects the blood brain barrier (BBB) from oxidative stress-induced damage in animals exposed to gp120, Tat and METH. To study this, CD-1 mice pre-treated with NACA/saline, received injections of gp120, Tat, gp120 + Tat or saline for 5 days, followed by three injections of METH/saline on the fifth day, and sacrificed 24 h after the final injection. Various oxidative stress parameters were measured, and animals treated with gp120 + Tat + Meth were found to be the most challenged group, as indicated by their GSH and MDA levels. Treatment with NACA significantly rescued the animals from oxidative stress. Further, NACA-treated animals had significantly higher expression of TJ proteins and BBB permeability as compared to the group treated with gp120 + Tat + METH alone, indicating that NACA can protect the BBB from oxidative stress-induced damage in gp120, Tat and METH exposed animals, and thus could be a viable therapeutic option for patients with HAD.     

Genotoxicity and oxidative stress in fish after a short-term exposure to silver nanoparticles

This study examined the effects of waterborne silver nanoparticles (AgNPs) on juvenile fish Piaractus mesopotamicus (“pacú”), and analyzed toxicological endpoints such as metal burdens, oxidative stress and genotoxicity in a short-term assay. Fish were individually exposed to 0 (control), 2.5, 10, and 25 μg AgNPs/L. After 24 h, silver accumulation was greater in the brain than the liver and gills at all silver concentrations. Fish exposed to higher AgNPs concentrations showed major alterations in oxidative stress markers. An increase in lipid peroxidation (LPO) levels was observed in the liver of fish exposed to 10 μg AgNPs/L with no changes in the antioxidant enzymes activities. In the case of the 25 μg AgNPs/L treatment, a hepatic activation of the enzymatic antioxidant defense occurred, and LPO levels resulted unaltered. On the other hand, the brain presented the highest LPO levels at both 10 and 25 μg AgNPs/L exposures. The AgNPs toxicity was also evidenced by the DNA damage in fish erythrocytes at higher concentrations. Summarizing, a short exposure to sublethal concentrations of AgNPs is enough to generate deleterious effects on fish, including DNA damage.     

Yokukansan,a traditional Japanese herbal medicine,alleviates the emotional abnormality induced by maladaptation to stress in mice

The aim of the present study was to examine the effect of yokukansan, a traditional Japanese herbal medicine that is composed of Atractylodis lanceae Rhizoma, Poria, Cnidii Rhizoma, Uncariae Uncis cum Ramulus, Angelicae Radix, Bupleuri Radix and Glycyrrhizae Radix, on the emotional abnormality induced by maladaptation to stress in mice. Mice were exposed to repeated restraint stress for 60 or 240 min/day for 14 days. From the 3rd day of stress exposure, mice were given yokukansan orally (p.o.) or the 5-HT_1A receptor agonist flesinoxan intraperitoneally (i.p.) immediately after the daily exposure to restraint stress. After the final exposure to restraint stress, the emotionality of mice was evaluated using an automatic hole-board apparatus. A single exposure to restraint stress for 60 min induced a decrease in head-dipping behavior in the hole-board test. This emotional stress response disappeared in mice that had been exposed to repeated restraint stress for 60 min/day for 14 days, which confirmed the development of stress adaptation. In contrast, mice that were exposed to restraint stress for 240 min/day for 14 days did not develop this stress adaptation, and still showed a decrease in head-dipping behavior. The decreased emotionality observed in stress-maladaptive mice was significantly recovered by chronic treatment with yokukansan (1000 mg/kg, p.o.) as well as flesinoxan (0.25 and 0.5 mg/kg, i.p.) immediately after daily exposure to stress. These findings suggest that yokukansan may have a beneficial effect on stress adaptation and alleviate the emotional abnormality under conditions of excessive stress.     

Mangiferin attenuates oxidative stress induced renal cell damage through activation of PI3K induced Akt and Nrf-2 mediated signaling pathways

BackgroundMangiferin is a polyphenolic xanthonoid with remarkable antioxidant activity. Oxidative stress plays the key role in tert-butyl hydroperoxide (tBHP) induced renal cell damage. In this scenario, we consider mangiferin, as a safe agent in tBHP induced renal cell death and rationalize its action systematically, in normal human kidney epithelial cells (NKE).MethodsNKE cells were exposed to 20 µM mangiferin for 2 h followed by 50 µM tBHP for 18 h. The effect on endogenous ROS production, antioxidant status (antioxidant enzymes and thiols), mitochondrial membrane potential, apoptotic signaling molecules, PI3K mediated signaling cascades and cell cycle progression were examined using various biochemical assays, FACS and immunoblot analyses.ResultstBHP exposure damaged the NKE cells and decreased its viability. It also elevated the intracellular ROS and other oxidative stress-related biomarkers within the cells. However, mangiferin dose dependently, exhibited significant protection against this oxidative cellular damage. Mangiferin inhibited tBHP induced activation of different pro-apoptotic signals and thus protected the renal cells against mitochondrial permeabilization. Further, mangiferin enhanced the expression of cell proliferative signaling cascade molecules, Cyclin d1, NFκB and antioxidant molecules HO-1, SOD2, by PI3K/Akt dependent pathway. However, the inhibitor of PI3K abolished mangiferin's protective activity.ConclusionsResults show Mangiferin maintains the intracellular anti-oxidant status, induces the expression of PI3K and its downstream molecules and shields NKE cells against the tBHP induced cytotoxicity.General significanceMangiferin can be indicated as a therapeutic agent in oxidative stress-mediated renal toxicity. This protective action of mangiferin primarily attributes to its potent antioxidant and antiapoptotic nature.     

Effect of thermal stress on metabolic and oxidative stress biomarkers of Hoplosternum littorale (Teleostei,Callichthyidae)

The present study aimed to investigate in Hoplosternum littorale (Hancock, 1828) the effects of different water temperatures (10 °C, 25 °C-control group- and 33 °C) on physiologic and metabolic traits following acute (1 day) and chronic (21 days) exposures. We analyzed several biomarker responses in order to achieve a comprehensive survey of fish physiology and metabolism under the effect of this natural stressor. We measured morphological indices, biochemical and hematological parameters as well as oxidative stress markers. To evaluate energy consumption, muscle and hepatic total lipid, protein and glycogen concentrations were also quantified. Extreme temperatures exposures clearly resulted in metabolic adjustments, being liver energy reserves and plasma metabolites the most sensitive parameters detecting those changes. We observed reduced hepatosomatic index after acute and chronic exposure to 33 °C while glycogen levels decreased at both temperatures and time of exposure tested. Additionally, acute and chronic exposures to 10 °C increased liver lipid content and plasma triglycerides. Total protein concentration was higher in liver and lower in plasma after chronic exposures to 10 °C and 33 °C. Acute exposition at both temperatures caused significant changes in antioxidant enzymes tested in the different tissues without oxidative damage to lipids. Antioxidant defenses in fish failed to protect them when they were exposed for 21 days to 10 °C, promoting higher lipid peroxidation in liver, kidney and gills. According to multivariate analysis, oxidative stress and metabolic biomarkers clearly differentiated fish exposed chronically to 10 °C. Taken together, these results demonstrated that cold exposure was more stressful for H. littorale than heat stress. However, this species could cope with variations in temperature, allowing physiological processes and biochemical reactions to proceed efficiently at different temperatures and times of exposure. Our study showed the ability of H. littorale to resist a wide range of environmental temperatures and contributes for the understanding of how this species is adapted to environments with highly variable physicochemical conditions.     

Transcriptional responses of neonatal mouse lung to hyperoxia by Nrf2 status

Hyperoxia exposure can inhibit alveolar growth in the neonatal lung through induction of p21/p53 pathways and is a risk factor for the development of bronchopulmonary dysplasia (BPD) in preterm infants. We previously found that activation of nuclear factor erythroid 2 p45-related factor (Nrf2) improved survival in neonatal mice exposed to hyperoxia likely due to increased expression of anti-oxidant response genes. It is not known however, whether hyperoxic induced Nrf2 activation attenuates the growth impairment caused by hyperoxia in neonatal lung. To determine if Nrf2 activation modulates cell cycle regulatory pathway genes associated with growth arrest we examined the gene expression in the lungs of Nrf2^−/− and Nrf2^+/+ neonatal mice at one and 3 days of hyperoxia exposure.MethodsMicroarray analysis was performed in neonatal Nrf2^+/+ and Nrf2^−/− lungs exposed to one and 3 days of hyperoxia. Sulforaphane, an inducer of Nrf2 was given to timed pregnant mice to determine if in utero exposure attenuated p21 and IL-6 gene expression in wildtype neonatal mice exposed to hyperoxia.ResultsCell cycle regulatory genes were induced in Nrf2^−/− lung at 1 day of hyperoxia. At 3 days of hyperoxia, induction of cell cycle regulatory genes was similar in Nrf2^+/+ and Nrf2^−/− lungs, despite higher inflammatory gene expression in Nrf2^−/− lung.Conclusionp21/p53 pathways gene expression was not attenuated by Nrf2 activation in neonatal lung. In utero SUL did not attenuate p21 expression in wildtype neonatal lung exposed to hyperoxia. These findings suggest that although Nrf2 activation induces expression of anti-oxidant genes, it does not attenuate alveolar growth arrest caused by exposure to hyperoxia.     

A rapid in vivo zebrafish model to elucidate oxidative stress-mediated PCB126-induced apoptosis and developmental toxicity

Dioxin-like 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) is one of the most potent and widespread environmental pollutants. Although PCB126-induced toxicity is related to the aryl hydrocarbon receptor pathway, there is still no study that has constructed an in vivo visual model to clarify the role of the Nrf2/ARE signaling pathway in the oxidative stress mechanism of PCB126-induced toxicity. In the present study, an in vivo zebrafish model of nrf2a fused to enhanced green fluorescent protein (nrf2a-eGFP) was constructed. The zebrafish embryos microinjected with nrf2a-eGFP (72 h postfertilization) were exposed to various concentrations of PCB126 (0, 25, 50, 100, 200 μg/L) or 30 mM N-acetylcysteine (NAC)+200 μg/L PCB126. After 72 h exposure, PCB126 significantly increased the malformation rates and induced eGFP expression in a dose-dependent manner in several zebrafish tissue types. The distribution of eGFP fluorescence coincided with developmental deformity sites. NAC pretreatment effectively counteracted PCB126-induced developmental toxicity including heart rate, pericardial edema, and body length. The highest PCB126 dose, 200 μg/L, produced marked apoptosis in the eye, gill, and trunk detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. At 48 and 72 h exposure, 200 μg/L PCB126 affected glutathione metabolism as evidenced by decreased glutathione and increased glutathione disulfide concentrations, indicative of oxidative stress. These effects were also counteracted by NAC pretreatment. Furthermore, the Nrf2-regulated genes gclc, gpx, gstp1, and hmox1 were significantly induced at 24, 48, and 72 h at the highest PCB126 exposures but not in the NAC-pretreated group. In addition, a significant increase in ROS generation was detected in zebrafish larvae at 72 h PCB126 exposure, which might offer a link for future mechanistic studies. Collectively, these data suggest that PCB126-induced developmental toxicity and apoptosis in the nrf2a-eGFP-injected zebrafish model are due to oxidative stress mediated by disruption to glutathione metabolism and changes in Nrf2-regulated gene expression.     

Developmental fluoxetine exposure increases behavioral despair and alters epigenetic regulation of the hippocampal BDNF gene in adult female offspring

A growing number of infants are exposed to selective serotonin reuptake inhibitor (SSRI) medications during the perinatal period. Perinatal exposure to SSRI medications alter neuroplasticity and increase depressive- and anxiety-related behaviors, particularly in male offspring as little work has been done in female offspring to date. The long-term effects of SSRI on development can also differ with previous exposure to prenatal stress, a model of maternal depression. Because of the limited work done on the role of developmental SSRI exposure on neurobehavioral outcomes in female offspring, the aim of the present study was to investigate how developmental fluoxetine exposure affects anxiety and depression-like behavior, as well as the regulation of hippocampal brain-derived neurotrophic factor (BDNF) signaling in the hippocampus of adult female offspring. To do this female Sprague–Dawley rat offspring were exposed to prenatal stress and fluoxetine via the dam, for a total of four groups of female offspring: 1) No Stress + Vehicle, 2) No Stress + Fluoxetine, 3) Prenatal Stress + Vehicle, and 4) Prenatal Stress + Fluoxetine. Primary results show that, in adult female offspring, developmental SSRI exposure significantly increases behavioral despair measures on the forced swim test, decreases hippocampal BDNF exon IV mRNA levels, and increases levels of the repressive histone 3 lysine 27 tri-methylated mark at the corresponding promoter. There was also a significant negative correlation between hippocampal BDNF exon IV mRNA levels and immobility in the forced swim test. No effects of prenatal stress or developmental fluoxetine exposure were seen on tests of anxiety-like behavior. This research provides important evidence for the long-term programming effects of early-life exposure to SSRIs on female offspring, particularily with regard to affect–related behaviors and their underlying molecular mechanisms.