AQP3-Dependent PI3K/Akt Modulation in Breast Cancer Cells.

Aquaporin 3 (AQP3) is a peroxiporin, a membrane protein that channels hydrogen peroxide in addition to water and glycerol. AQP3 expression also correlates with tumor progression and malignancy and is, therefore, a potential target in breast cancer therapy. In addition, epithelial growth factor receptor (EGFR) plays an important role in breast cancer. Therefore, we investigated whether disruption of the lipid raft harboring EGFR could affect AQP3 expression, and conversely, whether AQP3 silencing would affect the EGFR/phosphoinositide-3-kinase (PI3K)/Protein kinase B (PKB or Akt) signaling pathway in breast cancer cell lines with different malignant capacities. We evaluated H2O2 uptake, cell migratory capacity, and expression of PI3K, pAkt/Akt in three breast cancer cell lines, MCF7, SkBr3, and SUM159PT, and in the nontumorigenic breast epithelial cell line MCF10A. Our results show different responses between the tested cell lines, especially when compared to the nontumorigenic cell line. Neither lipid raft disruption nor EGF stimuli had an effect on PI3K/Akt pathway in MCF10A cell line. AQP3-silencing in SkBr3 and SUM159PT showed that AQP3 can modulate PI3K/Akt activation in these cells. Interestingly, SUM159PT cells increase nuclear factor-E2-related factor 2 (NRF2) in response to lipid raft disruption and EGF stimuli, suggesting an oxidative-dependent response to these treatments. These results suggest that in breast cancer cell lines, AQP3 is not directly related to PI3K/Akt pathway but rather in a cell-line-dependent manner.

Angiotensin II type 1 receptor is involved in flow-induced vasomotor responses of isolated middle cerebral arteries: role of oxidative stress.

This study aimed to determine the mechanosensing role of angiotensin II type 1 receptor (AT1R) in flow-induced dilation (FID) and oxidative stress production in middle cerebral arteries (MCA) of Sprague-Dawley rats. Eleven-week old, healthy male Sprague-Dawley rats on a standard diet were given the AT1R blocker losartan (1 mg/mL) in drinking water (losartan group) or tap water (control group) ad libitum for 7 days. Blockade of AT1R attenuated FID and acetylcholine-induced dilation was compared with control group. Nitric oxide (NO) synthase inhibitor Nω-nitro-l-arginine methyl ester (l-NAME) and cyclooxygenase inhibitor indomethacin (Indo) significantly reduced FID in control group. The attenuated FID in losartan group was further reduced by Indo only at Δ100 mmHg, whereas l-NAME had no effect. In losartan group, Tempol (a superoxide scavenger) restored dilatation, whereas Tempol + l-NAME together significantly reduced FID compared with restored dilatation with Tempol alone. Direct fluorescence measurements of NO and reactive oxygen species (ROS) production in MCA, in no-flow conditions revealed significantly reduced vascular NO levels with AT1R blockade compared with control group, whereas in flow condition increased the NO and ROS production in losartan group and had no effect in the control group. In losartan group, Tempol decreased ROS production in both no-flow and flow conditions. AT1R blockade elicited increased serum concentrations of ANG II, 8-iso-PGF2α, and TBARS, and decreased antioxidant enzyme activity (SOD and CAT). These results suggest that in small isolated cerebral arteries: 1) AT1 receptor maintains dilations in physiological conditions; 2) AT1R blockade leads to increased vascular and systemic oxidative stress, which underlies impaired FID.NEW & NOTEWORTHY The AT1R blockade impaired the endothelium-dependent, both flow- and acetylcholine-induced dilations of MCA by decreasing vascular NO production and increasing the level of vascular and systemic oxidative stress, whereas it mildly influenced the vascular wall inflammatory phenotype, but had no effect on the systemic inflammatory response. Our data provide functional and molecular evidence for an important role of AT1 receptor activation in physiological conditions, suggesting that AT1 receptors have multiple biological functions.

Chronic High Fat Diet Intake Impairs Hepatic Metabolic Parameters in Ovariectomized Sirt3 KO Mice.

High fat diet (HFD) is an important factor in the development of metabolic diseases, with liver as metabolic center being highly exposed to its influence. However, the effect of HFD-induced metabolic stress with respect to ovary hormone depletion and sirtuin 3 (Sirt3) is not clear. Here we investigated the effect of Sirt3 in liver of ovariectomized and sham female mice upon 10 weeks of feeding with standard-fat diet (SFD) or HFD. Liver was examined by Folch, gas chromatography and lipid hydroperoxide analysis, histology and oil red staining, RT-PCR, Western blot, antioxidative enzyme and oxygen consumption analyses. In SFD-fed WT mice, ovariectomy increased Sirt3 and fatty acids synthesis, maintained mitochondrial function, and decreased levels of lipid hydroperoxides. Combination of ovariectomy and Sirt3 depletion reduced pparα, Scd-1 ratio, MUFA proportions, CII-driven respiration, and increased lipid damage. HFD compromised CII-driven respiration and activated peroxisomal ROS scavenging enzyme catalase in sham mice, whereas in combination with ovariectomy and Sirt3 depletion, increased body weight gain, expression of NAFLD- and oxidative stress-inducing genes, and impaired response of antioxidative system. Overall, this study provides evidence that protection against harmful effects of HFD in female mice is attributed to the combined effect of female sex hormones and Sirt3, thus contributing to preclinical research on possible sex-related therapeutic agents for metabolic syndrome and associated diseases.

Arachidonic Acid Metabolites of CYP450 Enzymes and HIF-1α Modulate Endothelium-Dependent Vasorelaxation in Sprague-Dawley Rats under Acute and Intermittent Hyperbaric Oxygenation.

Acetylcholine-induced vasorelaxation (AChIR) and responses to reduced pO2 (hypoxia-induced relaxation (HIR), 0% O2) were assessed in vitro in aortic rings of healthy male Sprague-Dawley rats (N = 252) under hyperbaric (HBO2) protocols. The studied groups consisted of the CTRL group (untreated); the A-HBO2 group (single HBO2; 120 min of 100% O2 at 2.0 bars); the 24H-HBO2 group (examined 24 h after single exposure) and the 4D-HBO2 group (four consecutive days of single HBO2). AChIR, sensitivity to ACh and iNOS expression were decreased in the A-HBO2 group. HIR was prostanoid- and epoxyeicosatrienoic acid (EET)-mediated. HIF-1α expression was increased in the 24H-HBO2 and 4D-HBO2 groups. LW6 (HIF-1α inhibitor) decreased HIR in the 24H-HBO2 group. HBO2 affected the expression of COX-1 and COX-2. CYP2c11 expression was elevated in the 24H-HBO2 and 4D-HBO2 groups. Concentrations of arachidonic acid (AA) metabolites 14(15)-DiHET, 11(12)-DiHET and 8(9)-DiHET were increased in A-HBO2 and 24H-HBO2. An increased concentration of 8(9)-EET was observed in the A-HBO2 and 24h-HBO2 groups vs. the CTRL and 4D-HBO2 groups, and an increased concentration of 5(6)-DiHET was observed in the 24H-HBO2 group vs. the 4D-HBO2 group. The 20-HETE concentration was increased in the A-HBO2 group. All were determined by LC-MS/MS of the aorta. The results show that AChIR in all groups is mostly NO-dependent. HIR is undoubtedly mediated by the CYP450 enzymes' metabolites of AA, whereas HIF-1α contributes to restored HIR. Vasoconstrictor metabolites of CYP450 enzymes contribute to attenuated AChIR and HIR in A-HBO2.

Cytotoxicity of gamma irradiated aflatoxin B1 and ochratoxin A.

Toxicity of gamma irradiated mycotoxins aflatoxin B1 (AFB1) and ochratoxin A (OTA) was investigated in vitro. AFB1 and OTA stock solutions (50 mM, in methanol) were gamma irradiated (5 and 10 kGy) and non-irradiated and irradiated mycotoxins solutions were tested for cytotoxicity on Pk15, HepG2 and SH-SY5Y cell lines (MTT assay, 1-500 µM concentration range; 24 h exposure). Degradation of mycotoxin molecules was examined by liquid chromatography tandem mass spectrometry (HPLC-MS/MS). AFB1 and OTA radiolytic products were less toxic than the parent mycotoxins to all of the tested cell lines. Gamma irradiation even at 5 kGy had effect on AFB1 and OTA molecules however, this effect was dependent on chemical structure of mycotoxin. Since gamma irradiation at low dose reduced initial level of both mycotoxins, and gamma irradiated mycotoxins had lower toxicity in comparison to non-irradiated mycotoxins, it can be concluded that gamma irradiation could be used as decontamination method.

High salt intake shifts the mechanisms of flow-induced dilation in the middle cerebral arteries of Sprague-Dawley rats.

The goal of the present study was to examine the effect of 1 wk of high salt (HS) intake and the role of oxidative stress in changing the mechanisms of flow-induced dilation (FID) in isolated pressurized middle cerebral arteries of male Sprague-Dawley rats ( n = 15-16 rats/group). Reduced FID in the HS group was restored by intake of the superoxide scavenger tempol (HS + tempol in vivo group). The nitric oxide (NO) synthase inhibitor Nω-nitro-l-arginine methyl ester, cyclooxygenase inhibitor indomethacin, and selective inhibitor of microsomal cytochrome P-450 epoxidase activity N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide significantly reduced FID in the low salt diet-fed group, whereas FID in the HS group was mediated by NO only. Cyclooxygenase-2 mRNA (but not protein) expression was decreased in the HS and HS + tempol in vivo groups. Hypoxia-inducible factor-1α and VEGF protein levels were increased in the HS group but decreased in the HS + tempol in vivo group. Assessment by direct fluorescence of middle cerebral arteries under flow revealed significantly reduced vascular NO levels and increased superoxide/reactive oxygen species levels in the HS group. These results suggest that HS intake impairs FID and changes FID mechanisms to entirely NO dependent, in contrast to the low-salt diet-fed group, where FID is NO, prostanoid, and epoxyeicosatrienoic acid dependent. These changes were accompanied by increased lipid peroxidation products in the plasma of HS diet-fed rats, increased vascular superoxide/reactive oxygen species levels, and decreased NO levels, together with increased expression of hypoxia-inducible factor-1α and VEGF. NEW & NOTEWORTHY High-salt (HS) diet changes the mechanisms of flow-induced dilation in rat middle cerebral arteries from a combination of nitric oxide-, prostanoid-, and epoxyeicosatrienoic acid-dependent mechanisms to, albeit reduced, a solely nitric oxide-dependent dilation. In vivo reactive oxygen species scavenging restores flow-induced dilation in HS diet-fed rats and ameliorates HS-induced increases in the transcription factor hypoxia-inducible factor-1α and expression of its downstream target genes.

Yeast aquaporin regulation by 4-hydroxynonenal is implicated in oxidative stress response.

Reactive oxygen species, especially hydrogen peroxide (H2 O2 ), contribute to functional molecular impairment and cellular damage, but also are necessary in normal cellular metabolism, and in low doses play stimulatory role in cell proliferation and stress resistance. In parallel, reactive aldehydes such as 4-hydroxynonenal (HNE), are lipid peroxidation breakdown products which also contribute to regulation of numerous cellular processes. Recently, channeling of H2 O2 by some mammalian aquaporin isoforms has been reported and suggested to contribute to aquaporin involvement in cancer malignancies, although the mechanism by which these membrane water channels are implicated in oxidative stress is not clear. In this study, two yeast models with increased levels of membrane polyunsaturated fatty acids (PUFAs) and aquaporin AQY1 overexpression, respectively, were used to evaluate their interplay in cell's oxidative status. In particular, the aim of the study was to investigate if HNE accumulation could affect aquaporin function with an outcome in oxidative stress response. The data showed that induction of aquaporin expression by PUFAs results in increased water permeability in yeast membranes and that AQY1 activity is impaired by HNE. Moreover, AQY1 expression increases cellular sensitivity to oxidative stress by facilitating H2 O2 influx. On the other hand, AQY1 expression has no influence on the cellular antioxidant GSH levels and catalase activity. These results strongly suggest that aquaporins are important players in oxidative stress response and could contribute to regulation of cellular processes by regulation of H2 O2 influx. © 2017 IUBMB Life, 69(5):355-362, 2017.

AQP3 and AQP5 Modulation in Response to Prolonged Oxidative Stress in Breast Cancer Cell Lines.

Aquaporins are membrane pores regulating the transport of water, glycerol, and other small molecules across membranes. Among 13 human aquaporins, six have been shown to transport H2O2 and are therefore called peroxiporins. Peroxiporins are implicated in cancer development and progression, partly due to their involvement in H2O2 transport. Oxidative stress is linked to breast cancer development but is also a mechanism of action for conventional chemotherapy. The aim of this study is to investigate the effects of prolonged oxidative stress on Aquaporin 3 (AQP3), Aquaporin 5 (AQP5), and signaling pathways in breast cancer cell lines of different malignancies alongside a non-tumorigenic breast cell line. The prolonged oxidative stress caused responses in viability only in the cancer cell lines, while it affected cell migration in the MCF7 cell line. Changes in the localization of NRF2, a transcription factor involved in oxidative stress response, were observed only in the cancer cell lines, and no effects were recorded on its downstream target proteins. Moreover, the prolonged oxidative stress caused changes in AQP3 and AQP5 expression only in the cancer cell lines, in contrast to their non-malignant counterparts. These results suggest peroxiporins are potential therapeutic targets in cancer treatment. However, further research is needed to elucidate their role in the modulation of therapy response, highlighting the importance of research on this topic.

Shewanella spp. from wastewater treatment plant-affected environment: isolation and characterization.

Bacteria from the genus Shewanella are inhabitants of marine and freshwater ecosystems, recognized fish spoilage bacteria, but less known as fish disease agents. Shewanella spp. isolated from fish living in waters close to effluents of a wastewater treatment plant (WWTP) were not previously characterized. We have tested Shewanella isolates from WWTP-affected waters and related fish. Genotypic characterization identified most strains as S. baltica and S. oneidensis. In order to investigate the sensibility and accuracy of their MALDI-TOF MS identification, they were grown on two culture media enriched by various NaCl concentrations, incubated at different temperatures and duration. We analyzed their antimicrobial susceptibility on a panel of antimicrobial drugs and capacity for biofilm production. With a view to demonstrate their capacity to produce fatty acids, we assessed the impact of different culture media on their lipid profile. We performed zebrafish embryotoxicity tests to simulate the environmental infection of the earliest life stages in S. baltica-contaminated waters. The best MALDI-TOF MS identification scores were for strains cultivated on TSA for 24 h at 22 °C and with supplementation of 1.5% NaCl. Less than 17% of isolates demonstrated antimicrobial resistance. Most isolates were weak biofilm producers. Strain-to-strain variation of MIC and MBC was low. The major fatty acids were C15:0, C16:0, C16:1, C17:1, and iC15:0. Exposure of Danio rerio to different S. baltica concentrations induced severe effects on zebrafish development: decreased heartbeat rate, locomotor activity, and melanin pigmentation. S. baltica passed through chorionic pores of zebrafish.

NanoUPLC-QTOF-MS/MS Determination of Major Rosuvastatin Degradation Products Generated by Gamma Radiation in Aqueous Solution.

Rosuvastatin, a member of the statin family of drugs, is used to regulate high cholesterol levels in the human body. Moreover, rosuvastatin and other statins demonstrate a protective role against free radical-induced oxidative stress. Our research aimed to investigate the end-products of free radical-induced degradation of rosuvastatin. To induce the radical degradation, an aqueous solution of rosuvastatin was irradiated using different doses of gamma radiation (50-1000 Gy) under oxidative conditions. Rosuvastatin and related degradation products were separated on nanoC18 column under gradient elution, and identification was carried out on hyphenated nanoUPLC and nanoESI-QTOF mass spectrometer system. Elemental composition analysis using highly accurate mass measurements together with isotope fitting algorithm identified nine major degradation products. This is the first study of gamma radiation-induced degradation of rosuvastatin, where chemical structures, MS/MS fragmentation pathways and formation mechanisms of the resulting degradation products are detailly described. The presented results contribute to the understanding of the degradation pathway of rosuvastatin and possibly other statins under gamma radiation conditions.

The influence of antioxidants in the thiyl radical induced lipid peroxidation and geometrical isomerization in micelles of linoleic acid.

The biomimetic model of micelles of linoleic acid containing 2-mercaptoethanol and the antioxidant was examined under gamma irradiation up to 400 Gy in aerobic or deoxygenated conditions where thiyl radicals are the main reactive species. Lipid peroxidation was retarded by ascorbic acid and α-tocopherol, whereas this process was strongly inhibited by resveratrol as effectively as the ascorbic acid/α-tocopherol mixture. Furthermore, antioxidants have a much stronger inhibitory effect on the peroxidation in the presence of 2-mercaptoethanol, and at the same time show protective properties of the double bond, decreasing the cis-trans isomerization. Under anaerobic conditions, cis-trans isomerization occurred and antioxidants efficiency increased along the series: resveratrol < α-tocopherol < ascorbic acid. This result is explained taking into account the double bond localization in the hydrophobic core of the micelle and the need of co-localization of the antioxidant in order to get an anti-isomerizing activity and protection of the natural lipid geometry.