Plumbagin and juglone induce caspase-3-dependent apoptosis involving the mitochondria through ROS generation in human peripheral blood lymphocytes

The phytochemicals plumbagin and juglone have recently been gaining importance because of their various pharmacological activities. In this study, these compounds are shown to induce concentration- and time-dependent toxicity in human peripheral blood lymphocytes via the apoptotic pathway. Flow cytometry data revealed the occurrence of about 28% early apoptotic cells after 6 h exposure to 10 μM plumbagin and 35% late apoptotic cells and about 43% sub-G1 population after 24 h. The cytotoxic effect of plumbagin was at least twofold higher than that of juglone as evidenced by the IC₅₀ value for cytotoxicity. Characteristic apoptotic features such as chromatin condensation and apoptotic body formation were observed through TEM, and membrane blebbing and cell surface smoothening were seen in SEM studies. Generation of ROS was evidenced through the HPLC analysis of superoxide-specific 2-OH-E+ formation. In addition, a decrease in GSH levels parallel to ROS production was observed. Reversal of apoptosis in both NAC- and Tempol-pretreated cells indicates the involvement of both ROS generation and GSH depletion in plumbagin- and juglone-induced apoptosis. The mechanistic pathway involves a decrease in MMP; alterations in the levels of Bcl-2, Bax, and cytosolic cytochrome c; and PARP-1 cleavage subsequent to caspase-3 activation.     

Pregnenolone protects the PC-12 cell line against amyloid beta peptide toxicity but its sulfate ester does not

Pregnenolone (P), the main precursor of the steroids, and its sulfate ester, pregnenolone sulfate (PS), are the major neurosteroids produced in the neural tissue. Many neuroendocrinological studies stressed the neuroprotective role of neurosteroids although it has been suggested that the inhibition of P and PS synthesis can delay neuronal cell death. The potential roles of P and PS in vital neuronal functions and in amyloid beta peptide (Aβ) toxicity are not clearly identified. This work aims to investigate the effects of P and PS on cell viability and Aβ peptide toxicity in a concentration and exposure time-dependent manner in rat PC-12 cells. The cells were treated with 20 μM Aβ peptide 25-35 and variable concentrations of P and PS ranging from 0.5 μM to 100 μM. To examine the effects of steroid treatment on Aβ peptide toxicity, 0.5 μM (low) and 50 μM (high) neurosteroids were used. The cell viability and lactate dehydrogenase release of cells were evaluated after 24, 48 and 72 h. Morphological changes of cells were also examined. The treatment with higher than 1 μM concentrations of P and PS significantly decreased the cell viability comparing to untreated cells. At lower concentrations, P and PS had no toxic actions until 72 h. The Aβ treatment resulted in a significant decrease in cell viability comparing to untreated cells. P showed a dose-dependent protective effect against Aβ peptide in PC-12 cells. But its sulfate ester did not have the same effect on Aβ peptide toxicity, even it significantly decreased cell viability in Aβ-treated cells. Consequently, the discrepant effects of P and PS on Aβ peptide toxicity may provide insight on the pathogenesis of Alzheimer’s disease.     

Antidepressants attenuate the dexamethasone-induced decrease in viability and proliferation of human neuroblastoma SH-SY5Y cells: A involvement of extracellular regulated kinase (ERK1/2)

Excessive glucocorticoid levels in depressed patients have been associated with atrophic changes in some brain regions, but only few studies suggest that some antidepressants can interfere with deleterious effect of glucocorticoids on neuronal cells. The aim of the present study was to examine the effect of dexamethasone (DEX), a synthetic glucocorticoid and some antidepressants from different chemical groups (imipramine, desipramine, amitriptyline, citalopram, fluoxetine, reboxetine and tianeptine) on SH-SY5Y cells cultured in the medium containing steroid-free serum. DEX in concentrations from 1 to 100 μM did not change LDH release but exposure to 10 μM and 100 μM DEX for 24, 48 and 72 h caused a significant reduction in cell viability and proliferation as confirmed by MTT reduction and BrdU ELISA assays, respectively. Twenty four-hour incubation of cells with antidepressants (0.05–10 μM) and DEX (10 μM) showed that imipramine, amitriptyline, desipramine, citalopram and fluoxetine at concentrations from 0.1 up to 1 μM, reboxetine (0.1 μM) and tianeptine (0.05 μM) prevented the DEX-induced decreases in cell viability and proliferation rate. The protective effects of antidepressants were ameliorated by inhibitors of MAPK/ERK1/2, but not PI3-K/Akt pathway as shown for imipramine, fluoxetine and reboxetine. Moreover, Western blot analysis showed the decrease in the activated form of ERK1/2 (p-ERK) after DEX treatment and this effect was inhibited by imipramine. Thus, the reduction in SH-SY5Y cell viability caused by DEX appears to be related to its antiproliferative activity and some antidepressant drugs in low concentrations attenuate this effect by mechanism which involves the activation of MAPK/ERK1/2 pathway.     

Promising effects of the 4HPR-BSO combination in neuroblastoma monolayers and spheroids

To enhance the efficacy of fenretinide (4HPR)-induced reactive oxygen species (ROS) in neuroblastoma, 4HPR was combined with buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, in neuroblastoma cell lines and spheroids, the latter being a three-dimensional tumor model. 4HPR exposure (2.5-10 μM, 24 h) resulted in ROS induction (114-633%) and increased GSH levels (68-120%). A GSH depletion of 80% of basal levels was observed in the presence of BSO (25-100 μM, 24 h). The 4HPR-BSO combination resulted in slightly increased ROS levels (1.1- to 1.3-fold) accompanied by an increase in cytotoxicity (110-150%) compared to 4HPR treatment alone. A correlation was observed between the ROS-inducing capacity of each cell line and the increase in cytotoxicity induced by 4HPR-BSO compared to 4HPR. No significant correlation between baseline antioxidant levels and sensitivity to 4HPR or BSO was observed. In spheroids, 4HPR-BSO induced a strong synergistic growth retardation and induction of apoptosis. Our data show that BSO increased the cytotoxic effects of 4HPR in neuroblastoma monolayers and spheroids in ROS-producing cell lines. This indicates that the 4HPR-BSO combination might be a promising new strategy in the treatment of neuroblastoma.     

Discovery of 1,2,4-thiadiazolidine-3,5-dione analogs that exhibit unusual and selective rapid cell death kinetics against acute myelogenous leukemia cells in culture

4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) was previously identified as an antileukemic agent exhibiting no evident toxicity toward normal hematopoietic cells. An SAR study has been carried out to examine the effect of varying the C-2 and C-4- substituents on the thiadiazolidinone ring of TDZD-8 on antileukemic activity. These studies resulted in the identification of more druglike analogs that exhibited comparable potency to TDZD-8 in killing acute myelogenous leukemia (AML) cells in culture. Surprisingly, the cell death kinetics induced by several of these novel analogs on MV-411 cells were extremely fast, with commitment to death occurring within 30 min. At a concentration of 10 μM, 3f (LD₅₀ = 3.5 μM) completely eradicated cell viability of MV-411 cells within 2 h, while analog 3e (LD₅₀ = 2.0 μM) decimated cell viability within 30 min at a concentration of 10 μM and effectively abolished cell viability at 5 μM within 1-2 h.     

Sensitive voltammetric determination of thymol in essential oil of Carum copticum seeds using boron-doped diamond electrode

Essential oil of Carum copticum seeds, obtained from a local shop, was extracted and content of thymol was analyzed using square-wave voltammetry at boron-doped diamond electrode. The effect of various parameters, such as pH of supporting electrolyte and square-wave voltammetric parameters (modulation amplitude and frequency), was examined. In Britton–Robinson buffer solution (pH 4), thymol provided a single and oval-shaped irreversible oxidation peak at +1.13 V versus silver/silver chloride potassium electrode (3 M). Under optimal experimental conditions, a plot of peak height against concentration of thymol was found to be linear over the range of 4 to 100 μM consisting of two linear ranges: from 4 to 20 μM (R² = 0.9964) and from 20 to 100 μM (R² = 0.9993). The effect of potential interferences such as p-cymene and γ-terpinene (major components in essential oil of C. copticum seeds) was evaluated. Thus, the proposed method displays a sufficient selectivity toward thymol with a detection limit of 3.9 μM, and it was successfully applied for the determination of thymol in essential oil of C. copticum seeds. The Prussian blue method was used for validation of the proposed electroanalytical method.     

Effect of pitavastatin on apolipoprotein A-I production in HepG2 cell

There are few reports describing the mechanism of HDL-elevating action of HMG-CoA reductase inhibitors (statins). As it is considered that the key step of HDL production is the secretion of apolipoprotein A-I (apoA-I), we investigated the effect of statins on apoA-I synthesis and secretion by HepG2 cell to elucidate the mechanism of the action. Each statin induced apoA-I expression (mRNA and protein) dose-dependently: the rank order of the apoA-I induction pitavastatin (3 μM) > simvastatin (10 μM) > atorvastatin (50 μM). The induction of apoA-I by statins disappeared with addition of mevalonate, which indicates that the effect is HMG-CoA reductase inhibition-dependent. Based on HMG-CoA reductase inhibition, pitavastatin-induced apoA-I more efficiently than simvastatin and atorvastatin. Further study revealed that pitavastatin increased ABCA1 mRNA in HMG-CoA reductase-dependent manner and that Rho and Rho kinase inhibitor (C3T and Y27632) increased apoA-I production in the HepG2 cells. These results suggest that pitavastatin efficiently increases apoA-I in the culture medium of HepG2 cells by promoting apoA-I production through inhibition of HMG-CoA reductase and suppression of Rho activity and by protecting apoA-I from catabolism through ABCA1 induction and lipidation of apoA-I.     

The improving effect of reduced glutathione on boar sperm cryotolerance is related with the intrinsic ejaculate freezability

Reduced glutathione (GSH) improves boar sperm cryosurvival and fertilising ability when added to freezing extenders. Poor freezability ejaculates (PFE) are known to present lower resistance than good freezability ejaculates (GFE) to cryopreservation procedures. So far, no study has evaluated whether the ability of GSH to counteract the cryopreservation-induced injuries depends on ejaculate freezability (i.e. GFE vs. PFE). For this reason, thirty boar ejaculates were divided into three equal volume fractions and cryopreserved with or without GSH at a final concentration of either 2 or 5 mM in freezing media. Before and after freeze–thawing, sperm quality was evaluated through analysis of viability, motility, integrity of outer acrosome membrane, ROS levels, integrity of nucleoprotein structure, and DNA fragmentation. Ejaculates were classified into two groups (GFE or PFE) according to their post-thaw sperm motility and viability assessments in negative control (GSH 0 mM), after running cluster analyses. Values of each sperm parameter were then compared between treatments (GSH 0 mM, GSH 2 mM, GSH 5 mM) and freezability groups (GFE, PFE). In the case of GFE, GSH significantly improved boar sperm cryotolerance, without differences between 2 and 5 mM. In contrast, PFE freezability was significantly increased when supplemented with 5 mM GSH, but not when supplemented with 2 mM GSH. In conclusion, PFE need a higher concentration of GSH than GFE to improve their cryotolerance.     

First report of an anti-tumor, anti-fungal, anti-yeast and anti-bacterial hemolysin from Albizia lebbeck seeds

A monomeric 5.5-kDa protein with hemolytic activity toward rabbit erythrocytes was isolated from seeds of Albizia lebbeck by using a protocol that involved ion-exchange chromatography on Q-Sepharose and SP-Sepharose, hydrophobic interaction chromatography on Phenyl-Sepharose, and gel filtration on Superdex 75. It was unadsorbed on both Q-Sepharose and SP-Sepharose, but adsorbed on Phenyl-Sepharose. Its hemolytic activity was fully preserved in the pH range 0-14 and in the temperature range 0-100 °C, and unaffected in the presence of a variety of metal ions and carbohydrates. The hemolysin reduced viability of murine splenocytes and inhibited proliferation of MCF-7 breast cancer cells and HepG2 hepatoma cells with an IC₅₀ of 0.21, 0.97, and 1.37 μM, respectively. It impeded mycelial growth in the fungi Rhizoctonia solani with an IC₅₀ of 39 μM but there was no effect on a variety of other filamentous fungi, including Fusarium oxysporum, Helminthosporium maydis, Valsa mali and Mycosphaerella arachidicola. Lebbeckalysin inhibited growth of Escherichia coli with an IC₅₀ of 0.52 μM.     

N-acetylcysteine, coenzyme Q10 and superoxide dismutase mimetic prevent mitochondrial cell dysfunction and cell death induced by d-galactosamine in primary culture of human hepatocytes

d-Galactosamine (d-GalN) induces reactive oxygen species (ROS) generation and cell death in cultured hepatocytes. The aim of the study was to evaluate the cytoprotective properties of N-acetylcysteine (NAC), coenzyme Q₁₀ (Q₁₀) and the superoxide dismutase (SOD) mimetic against the mitochondrial dysfunction and cell death in d-GalN-treated hepatocytes. Hepatocytes were isolated from liver resections. NAC (0.5 mM), Q₁₀ (30 μM) or MnTBAP (Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (1 mg/mL) were co-administered with d-GalN (40 mM) in hepatocytes. Cell death, oxidative stress, mitochondrial transmembrane potential (MTP), ATP, mitochondrial oxidized/reduced glutathione (GSH) and Q₁₀ ratios, electronic transport chain (ETC) activity, and nuclear- and mitochondria-encoded expression of complex I subunits were determined in hepatocytes. d-GalN induced a transient increase of mitochondrial hyperpolarization and oxidative stress, followed by an increase of oxidized/reduced GSH and Q₁₀ ratios, mitochondrial dysfunction and cell death in hepatocytes. The cytoprotective properties of NAC supplementation were related to a reduction of ROS generation and oxidized/reduced GSH and Q₁₀ ratios, and a recovery of mitochondrial complexes I + III and II + III activities and cellular ATP content. The co-administration of Q₁₀ or MnTBAP recovered oxidized/reduced GSH ratio, and reduced ROS generation, ETC dysfunction and cell death induced by d-GalN. The cytoprotective properties of studied antioxidants were related to an increase of the protein expression of nuclear- and mitochondrial-encoded subunits of complex I. In conclusion, the co-administration of NAC, Q₁₀ and MnTBAP enhanced the expression of complex I subunits, and reduced ROS production, oxidized/reduced GSH ratio, mitochondrial dysfunction and cell death induced by d-GalN in cultured hepatocytes.     

Dopamine but not l-dopa stimulates neural glutathione metabolism. Potential implications for Parkinson’s and other dopamine deficiency states

Dopamine is produced first by hydroxylalation of l-tyrosine to l-dihydroxyphenylalanine (l-dopa) and subsequently by the decarboxylation of l-dopa to dopamine catalysed by the enzymes tyrosine hydroxylase and aromatic l-amino acid decarboxylase (AADC) respectively. Reduced glutathione (GSH) acts as a major cellular antioxidant. We have investigated the role of dopamine in the control of GSH homeostasis in brain cells. The SH-SY5Y human neuroblastoma cell line was found to increase intracellular GSH levels in response to 50 μM dopamine treatment. Similarly the 1321N1 human astrocytoma cell line was found to increase GSH release in response to 50 μM dopamine. The same concentration of l-dopa was also found to increase intracellular GSH in SH-SY5Y cells, however when AADC was inhibited this affect was abolished. Furthermore 1321N1 cells which were found to have almost undetectable levels of AADC activity did not increase GSH release in response to 50 μM l-dopa. These results suggest that at these concentrations dopamine has the potential to act as a signal for the upregulation of GSH synthesis within neuronal-like cells and for the increased trafficking of GSH from astrocytes to neurons. This effect could potentially relate to the activation of antioxidant response elements leading to the induction of phase II detoxifying enzymes including those involved in GSH synthesis and release. The inability of l-dopa to produce a similar effect when AADC was inhibited or when AADC activity was absent indicates that these effects are relatively specific to dopamine. Additionally dopamine but not l-dopa treatment led in an increase in complex I activity of the respiratory chain in SH-SY5Y cells which may be related to the effect of dopamine on GSH levels.     

Protective Effect of Saffron Extract and Crocin on Reactive Oxygen Species-Mediated High Glucose-Induced Toxicity in PC12 Cells

Diabetic neuropathy is one of the most frequent complications of diabetes. Despite some studies, the exact mechanism of glucose neurotoxicity has not been fully elucidated. Increased reactive oxygen species (ROS) has proposed as a possible mechanism. Crocus sativus L. (saffron) has been known as a source of antioxidants. Therefore, neuroprotective effect of saffron extract, its active component crocin and γ-glutamylcysteinylglycine (GSH) was studied in glucose-induced neurotoxicity, using PC12 cells as a suitable in vitro model of diabetic neuropathy. Cell viability was quantitated by MTT assay. ROS was measured using DCF-DA by flow cytometry analysis. The result showed that glucose (13.5 and 27 mg/ml) reduced the cell viability of PC12 cells after 4 days. Saffron extract (5 and 25 mg/ml), crocin (10 and 50 μM) and GSH (10 μM) could decrease this toxicity. Glucose toxicity was consistent with increased ROS production which reduced by saffron, crocin and GSH pretreatment. These results suggest saffron and its carotenoid crocin could be potentially useful in diabetic neuropathy treatment.     

Involvement of catalase in the apoptotic mechanism induced by apigenin in HepG2 human hepatoma cells

Apigenin has been reported to inhibit proliferation of cancer cells; however, the mechanism underlying its action is not completely understood. Here, we evaluated the effects of apigenin on the levels of expression and activity of antioxidant enzymes, and the involvement of ROS in the mechanism of cell death induced by apigenin in HepG2 human hepatoma cells. Upon treatment with apigenin, HepG2 cells displayed a reduction in cell viability in a dose- and time-dependent manner, and some morphological changes. In addition, apigenin treatment induced ROS generation and significantly decreased the mRNA levels and activity of catalase and levels of intracellular GSH. On the other hand, apigenin treatment did not alter the expression or activity levels of other antioxidant enzymes. Addition of exogenous catalase significantly reduced the effects of apigenin on HepG2 cell death. We also demonstrated that HepG2 cells are more sensitive to apigenin-mediated cell death than are primary cultures of mouse hepatocytes, suggesting a differential toxic effect of this agent in tumor cells. Our results suggest that apigenin-induced apoptosis in HepG2 cells may be mediated by a H₂O₂-dependent pathway via reduction of the antioxidant defenses.     

Protective effects of sulfated chitooligosaccharides against hydrogen peroxide-induced damage in MIN6 cells

Sulfated chitooligosaccharides (COS-S) with different degrees of substitution (DS) were obtained by the chlorosulfuric acid/pyridine method. Protective effects of COS-S against hydrogen peroxide (H₂O₂)-induced damage were investigated in pancreatic β-cells MIN6 cell line. The cell viability, morphology, insulin contents, malondialdehyde (MDA) inhibition, lactate dehydrogenase (LDH) release and the levels of antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidise (GPx) were evaluated under oxidative damage by 150 μM H₂O₂ for 6 h. COS-S did not show any harmful or inhibitory effect on cell growth at concentrations ranging from 0.1 to 0.5 mg/ml. While COS-S could enhance the cell viability, decrease the production of ROS, and reduce the MDA level as well as LDH level in oxidative damaged β-cells by being an antioxidant. The underlining mechanisms of protective effects of COS-S are partly due to the enhancement of antioxidant enzyme activity and inhibition of intracellular ROS production, along with suppressing MIN6 cell apoptosis subsequent to the amelioration of ROS. Moreover, increased DS might contribute to the defense mechanisms against H₂O₂-induced oxidative damage in MIN6 cells. These results indicated that the antioxidant properties of COS-S hold great potential for the oxidative diseases treatment, and the sulfate content of polysaccharides made great role in regulating antioxidant activities.     

Mouse recombinant leptin protects human hepatoma HepG2 against apoptosis,TNF-α response and oxidative stress induced by the hepatotoxin–ethanol

Obesity is a risk factor for hepatocellular carcinoma (HCC) complicated with alcoholic liver disease (ALD) and cryptogenic cirrhosis. Leptin is a 16-kDa antiobesity hormone secreted mainly by adipocytes. The role of leptin on alcohol-mediated effects in cell line is yet to be unraveled. Therefore, we investigated the effect of leptin against ethanol-elicited cytoxicity in human hepatoma cell lines (HepG2). HepG2 cells were treated with leptin (31.2 nM), ethanol (500 mM), ethanol + leptin and untreated cells served as control. 48 h after treatment, cell viability, apoptosis, TNF-α secretory response and oxidative damage were analysed. Our results suggest that leptin at a concentration of 31.2 nM prevents ethanol elicited cytotoxicity as evidenced by MTT and trypan blue dye exclusion assay. Leptin also inhibited ethanol-induced apoptosis, which was confirmed by [³H] thymidine uptake and cell cycle analysis using propidium iodide (PI) staining. Further, simultaneous leptin treatment along with ethanol showed protection against ethanol mediated cellular damage as indicated by significantly decreased levels of reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) and significantly increased levels of reactive nitrogen species (RNS), reduced glutathione (GSH) and elevated activities of superoxide dismutase (SOD) and catalase (CAT). In addition, leptin downregulated the secretion of tumor necrosis factor-α (TNF-α) by ethanol-induced HepG2 cells. Our results demonstrate that simultaneous leptin treatment along with ethanol could be useful in preventing the damage produced by ethanol, which might be of therapeutic interest.     

Trans-10, cis-12 conjugated linoleic acid induced cell death in human colon cancer cells through reactive oxygen species-mediated ER stress

Dietary conjugated linoleic acids (CLA) are fatty acid isomers with anticancer activities produced naturally in ruminants or from vegetable oil processing. The anticancer effects of CLA differ upon the cancer origin and the CLA isomers. In this study, we carried out to precise the effects of CLA isomers, c9,t11 and t10,c12 CLA, on mechanisms of cell death induction in colon cancer cells. We first showed that only t10,c12 CLA treatment (25 and 50 μM) for 72 h triggered apoptosis in colon cancer cells without affecting viability of normal-derived colon epithelial cells. Exposure of colon cancer cells to t10,c12 CLA activated ER stress characterized by induction of eIF2α phoshorylation, splicing of Xbp1 mRNA and CHOP expression. Furthermore, we evidenced that inhibition of CHOP expression and JNK signaling decreased t10,c12 CLA-mediated cancer cell death. Finally, we showed that CHOP induction by t10,c12 CLA was dependent on ROS production and that the anti-oxidant N-acetyl-cysteine reduced CHOP induction-dependent cell death. These results highlight that t10,c12 CLA exerts its cytotoxic effect through ROS generation and a subsequent ER stress-dependent apoptosis in colon cancer cells.     

Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography–electrospray ionization–tandem mass spectrometry

A wide variety of sulfur metabolites play important roles in plant functions. We have developed a precise and sensitive method for the simultaneous measurement of several sulfur metabolites based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) and ³⁴S metabolic labeling of sulfur-containing metabolites in Arabidopsis thaliana seedlings. However, some sulfur metabolites were unstable during the extraction procedure. Our proposed method does not allow for the detection of the important sulfur metabolite homocysteine because of its instability during sample extraction. Stable isotope-labeled sulfur metabolites of A. thaliana shoot were extracted and utilized as internal standards for quantification of sulfur metabolites with LC–MS/MS using S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), glutathione (GSH), and glutathione disulfide (GSSG) as example metabolites. These metabolites were detected using electrospray ionization in positive mode. Standard curves were linear (r² > 0.99) over a range of concentrations (SAM 0.01–2.0 μM, SAH 0.002–0.10 μM, Met 0.05–4.0 μM, GSH 0.17–20.0 μM, GSSG 0.07–20.0 μM), with limits of detection for SAM, SAH, Met, GSH, and GSSG of 0.83, 0.67, 10, 0.56, and 1.1 nM, respectively; and the within-run and between-run coefficients of variation based on quality control samples were less than 8%.     

Nox2-derived ROS in PPARγ signaling and cell-cycle progression of lung alveolar epithelial cells

Reactive oxygen species (ROS) play important roles in peroxisome proliferator-activated receptor γ (PPARγ) signaling and cell-cycle regulation. However, the PPARγ redox-signaling pathways in lung alveolar epithelial cells remain unclear. In this study, we investigated the in vivo and in vitro effects of PPARγ activation on the levels of lung ROS production and cell-cycle progression using C57BL/6J wild-type and Nox2 knockout mice (n = 10) after intraperitoneal injection of a selective PPARγ agonist (GW1929, 5 mg/kg body wt, daily) for 14 days. Compared to vehicle-treated mice, GW1929 increased significantly the levels of ROS production in wild-type lungs, and this was accompanied by significant up-regulation of PPARγ, Nox2, PCNA, and cyclin D1 and phosphorylation of ERK1/2 and p38MAPK. These effects were absent in Nox2 knockout mice. In cultured alveolar epithelial cells, GW1929 (5 μM for 24 h) increased ROS production and promoted cell-cycle progression from G0/G1 into S and G2/M phases, and these effects were abolished by (1) adding a PPARγ antagonist (BADGE, 1 μM), (2) knockdown of PPARγ using siRNA, or (3) knockout of Nox2. In conclusion, PPARγ activation through Nox2-derived ROS promotes cell-cycle progression in normal mouse lungs and in cultured normal alveolar epithelial cells.     

Equine spermatozoa stored in the epididymis for up to 96 h at 4 °C can be successfully cryopreserved and maintain their fertilization capacity

After injury or death of a valuable male, recovery of epididymal spermatozoa may be the last chance to ensure preservation of its genetic material. The objective of this research was to study the effect of sperm storage, at 4 °C up to 96 h, in the epididymides obtained from castrated horses and its effect on different functional sperm parameters. Aims were to study the effect of (1) sperm storage on viability and chromatin condensation; (2) pre-incubation of recovered epididymal sperm in the freezing extender, prior cryopreservation, on viability and chromatin condensation; and (3) freezing–thawing on viability, chromatin condensation, ROS generation, protein tyrosine phosphorylation and heterologous fertilization rate (ICSI and IVF using bovine oocytes) of sperm recovered from the epididymis up to 96 h post castration. The average volume (720 ± 159 μL) and the concentration (6.5 ± 0.4 × 10⁹ spermatozoa/mL) of sperm recovered from the epididymis were not affected by storage. Sperm viability after refrigeration at 4 °C for up to72 h was similar (P < 0.01). The effect of sperm dilution in the freezing media showed similar values up to 48 h, while viability was preserved up to 72 h (P < 0.01). Cryopreserved spermatozoa show similar viability between different storage times. Chromatin condensation was not affected by storage time; however, incubation for 30 min in freezing medium and freezing–thawing process induced an increase in the chromatin decondensation. ROS generation was not affected by storage up to 96 h. Epididymal storage did not affect sperm protein tyrosine phosphorylation patterns; although the pattern of phosphorylation changed to strong staining of the equatorial segment when the sperm where capacitated in sperm–TALP. Finally, successful and similar pronuclear formation (analyzed by ICSI) and in vitro penetration (evaluated with bovine zone free oocyte) was observed using cryopreserved sperm obtained from prolong epididymal storage at 4 °C. In conclusion, cryopreservation of epididymal stallion sperm stored for up to 72 h in the epididymis at 4 °C, maintain both viability and ability to fertilize in vitro.