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Background: It is evaluated that a few million individuals worldwide are experiencing Arsenic (As) harmfulness coming about because of anthropogenic discharges. There is likewise proof to propose that As can affect the peripheral, as well as, the central nervous system (CNS). On the contrary, thymoquinone (TQ), a biologically active ingredient of Nigella sativa has exhibited numerous neuro-pharmacological traits since ancient times.Hypothesis/purpose: In the present study, the neuroprotective efficacy of TQ was explored by primarily studying its antioxidant and anti-apoptotic potential against Arsenic trioxide (As2O3) induced toxicity in SH-SY5Y human neuroblastoma cell lines.Study Design: For experimentation, cells were seeded in 96 well tissue culture plates and kept undisturbed for 24 h to attain proper adhesion. After 75-80% confluence, cells were pretreated with 10 µM and 20 µM thymoquinone (TQ) for 1 h After adding 2 µM As, cells were set aside for incubation for 24 h without changing the medium.Methods: The mitigatory effects of TQ with particular reference to cell viability and cytotoxicity, the generation of reactive oxygen species, DNA damage, and mitochondrial dynamics were studied.Results: Pretreatment of SH-SY5Y cells with TQ (10 and 20 μM) for an hour and subsequent exposure to 2 μM As2O3 protected the SH-SY5Y cells against the neuro-damaging effects of the latter. Also, the SH-SY5Y cells were better preserved with increased viability, repaired DNA, less free radical generation and balanced transmembrane potential than those exposed to As2O3 alone. TQ pretreatment also inhibited As2O3-induced exacerbation in protein levels of BAX and PARP-1 and restored the loss of Bcl2 levels.Conclusion: The findings of this study suggest that TQ may prevent neurotoxicity and As2O3-induced apoptosis and cytotoxicity. It is, therefore, worth studying further for its potential to reduce the risks of arsenic-related neurological implications. [ABSTRACT FROM AUTHOR]

Background: Licochalconce (LC) H is an artificial compound in the course of synthesizing LCC in 2013. So far, few studies on the effects of LCH have been found in the literature. Despite progress in treatment modalities for oral cancer, the cure from cancer has still limitations.Purpose: The effects of LCH were investigated on human oral squamous cell carcinoma (OSCC) cells to elucidate its mechanisms.Study Design: We explored the mechanism of action of LCH by which it could have effects on JAK2/STAT3 signaling pathway.Methods: To confirm LCH anti-cancer effect, analyzed were MTT assay, DAPI staining, soft agar, kinase assay, molecular docking simulation, flow cytometry and Western blotting analysis.Results: According to docking and molecular dynamics simulations, the predicted pose of the complex LCH and JAK2 seems reasonable and LCH is strongly bound to active JAK2 with opened activation loop. The LCH inhibitor is surrounded by specific ATP-binding pocket in which it is stabilized by forming hydrogen bonds and hydrophobic interactions. It is shown that LCH plays as a competitive inhibitor in an active state of JAK2. LCH caused a dose-dependent decrease in phosphorylation of JAK2 and STAT3. More interestingly, LCH suppressed JAK2 kinase activity in vitro by its direct binding to the JAK2. LCH significantly inhibited the JAK2/STAT3 signaling pathway, causing the down-regulation of target genes such as Bcl-2, survivin, cyclin D1, p21 and p27. In addition, LCH inhibited cell proliferation and colony formation of OSCC cells in a dose- and time-dependent manner, as well as induction of cell apoptosis through extrinsic and intrinsic pathway. The induction of apoptosis in OSCC cells by LCH was evident in the increased production of ROS, loss of mitochondrial membrane potential, release of cyto c, variation of apoptotic proteins and activation of caspase cascade.Conclusion: LCH not only induces apoptosis in OSCC cells through the JAK/STAT3 signaling pathway but also inhibits cell growth. It is proposed that LCH has a promising use for the chemotherapeutic agent of oral cancer. [ABSTRACT FROM AUTHOR]

Mitochondria are functionally versatile organelles. In addition to their conventional role of meeting the cell's energy requirements, mitochondria also actively regulate innate immune responses against infectious and sterile insults. Components of mitochondria, when released or exposed in response to dysfunction or damage, can be directly recognized by receptors of the innate immune system and trigger an immune response. In addition, despite initiation that may be independent from mitochondria, numerous innate immune responses are still subject to mitochondrial regulation as discrete steps of their signaling cascades occur on mitochondria or require mitochondrial components. Finally, mitochondrial metabolites and the metabolic state of the mitochondria within an innate immune cell modulate the precise immune response and shape the direction and character of that cell's response to stimuli. Together, these pathways result in a nuanced and very specific regulation of innate immune responses by mitochondria. [ABSTRACT FROM AUTHOR]

Accumulation of advanced glycation end products (AGEs) in tissues and serum plays important roles in diabetes-associated complications. Therefore, the identification of antiglycating compounds is attracting considerable interest. In this study, the structural changes associated with the glycation of superoxide dismutase (SOD) and its protection by thymoquinone (TQ) have been investigated by biophysical techniques. Incubation of SOD with glucose, methylglyoxal (MG) or both at 37 ̊C resulted in progressive hyperchromicity at 280 nm, intrinsic fluorescence quenching at 310 nm, decrease in negative ellipticity at 208 nm, AGE-specific fluorescence enhancement in the wavelength range 400–480 nm and Thioflavin T (ThT) fluorescence enhancement at 480 nm (fibrillar state enhancement). Therefore, glycation by glucose or MG induced both tertiary and secondary structural changes in SOD and formation of AGEs and fibrils. The changes were more and faster with MG than with glucose since MG is a stronger glycating agent than glucose. TQ offered protection against glucose or MG-induced glycation of SOD as observed by a reduction in the structural changes, formation of AGEs and fibrils. Thus, TQ can be used for reducing diabetic complications many of which are due to protein glycation. [ABSTRACT FROM AUTHOR]

• Thermoregulation is a necessary component of daily life. • COVID-19 causes thermal dysregulation and increases core body temperature. • Individuals with multiple sclerosis (MS) are sensitive to increases in core body temperature. • Being physically active triggers adaptations that alleviate COVID-19-induced thermal dysregulation. • Engaging in regular physical activity before and after infection with COVID-19 attenuates worsening clinical signs and reduces recovery time in patients with MS. Thermoregulation is a homeostatic mechanism that is disrupted in some neurological diseases. Patients with multiple sclerosis (MS) are susceptible to increases in body temperature, especially with more severe neurological signs. This condition can become intolerable when these patients suffer febrile infections such as coronavirus disease-2019 (COVID-19). We review the mechanisms of hyperthermia in patients with MS, and they may encounter when infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Finally, the thermoregulatory role and relevant adaptation to regular physical exercise are summarized. [ABSTRACT FROM AUTHOR]

Abstract: Melastatin-like transient receptor potential 2 (TRPM2) channel is a redox sensitive Ca2+-permeable cation channel that can be gated by H2O2 binding to the channel''s enzymatic Nudix domain. Since the mechanisms that lead to TRPM2 action in response to H2O2 are not understood, we examined the effects of various antioxidants on H2O2-induced TRPM2 cation channel currents in transfected Chinese hamster ovary (CHO) cells. The CHO cells were transfected with cDNA coding for TRPM2. Membrane currents were measured with the conventional whole cell patch-clamp technique. The intracellular solution contained ethylenediamine tetraacetic acid (EDTA) as a chelator for Ca2+ and heavy metal ions instead of ethylene glycol tetraacetic acid (EGTA). Moreover, we chose an intracellular Ca2+ concentration calculated to be in the range of 1 μM. H2O2 (10 mM) was added extracellularly to the bath chamber. With these conditions, we were able to evoke TRPM2 currents consistently with H2O2. We next tested whether vitamins C and E or glutathione (GSH) would prevent or attenuate the induction of TRPM2 currents by H2O2 when applied extracellularly or intracellularly. Unexpectedly, administration of these antioxidants did not inhibit activation of TRPM2 by H2O2. In conclusion, TRPM2 channels were constitutively activated by H2O2 although we could not detect any inhibitory effect of the antioxidants on H2O2-induced TRPM2 cation channel currents in CHO cells. [Copyright &y& Elsevier]

Abstract: TNF-α, GSH depletion and CYP2E1 are factors that play an important role in alcoholic liver disease. Activation of NF-κB prevents hepatocyte damage caused by TNF-α. This work describes the effect of NF-κB inhibition on toxicities caused by GSH depletion or arachidonic acid (AA) treatment in liver cells, and evaluates the possible influence of CYP2E1 overexpression. Cells were exposed to the NF-κB inhibitor BAY11-7082, in the absence or presence of l-buthionine sulfoximine (BSO) to block GSH synthesis. BSO toxicity was higher in CYP2E1-expressing E47 HepG2 cells compared to control cells; the incubation with BAY11-7082 potentiated BSO toxicity in both cell lines comparably. Several other agents which suppress activation of NF-κB increased BSO toxicity in E47 cells. NF-κB inhibition, however, did not sensitize E47 cells to AA toxicity. Suppressing activity of NF-κB also potentiated BSO, but not AA toxicity, in isolated rat hepatocytes. BAY11-7082 plus BSO induced a greater p38 MAPK activation as compared to BAY11-7082 or BSO alone, and a p38 MAPK inhibitor protected against the synergistic toxicity. In summary, inhibition of NF-κB sensitizes liver cells to toxicity linked to GSH depletion, probably accelerating the processes of thiol homeostasis deregulation and induction of apoptosis through a mechanism mediated by p38 MAPK. [Copyright &y& Elsevier]

Abstract: Schizandrae chinensis, a traditional Chinese medicine herb, has been used to treat hepatitis B disease in Chinese hospital clinic. We have isolated two bioactive compounds, deoxyschizandrin and γ-schizandrin, from S. chinensis. In the present, we reported that deoxyschizandrin and γ-schizandrin could induce apoptosis in human promyelocytic leukemia cells (HL-60), as characterized by DNA fragmentation and poly (ADP) ribose polymerase (PARP) cleavage. Further molecular analysis showed that deoxyschizandrin and γ-schizandrin caused the loss of mitochondrial membrane potential (ΔΨm), cytochrome c release from mitochondrion to cytosol, truncation of Bid protein, and activation of caspase-3 and -9. However, they did not increase the intracellular level of reactive oxygen species (ROS). Antioxidants such as N-acetyl cysteine (NAC) and catalase did not block the apoptosis induced by deoxyschizandrin or γ-schizandrin. These findings suggest that deoxyschizandrin and γ-schizandrin-induced apoptosis in HL-60 cells involved ROS-independent mitochondrial dysfunction pathway. [Copyright &y& Elsevier]

Abstract: Reactive oxygen species (ROS) are implicated in 6-hydroxydopamine (6-OHDA) injury to catecholaminergic neurons; however, the mechanism(s) are unclear. In addition to ROS generated during autoxidation, 6-OHDA may initiate secondary cellular sources of ROS that contribute to toxicity. Using a neuronal cell line, we found that catalytic metalloporphyrin antioxidants conferred protection if added 1 h after exposure to 6-OHDA, whereas the hydrogen peroxide scavenger catalase failed to protect if added more than 15 min after 6-OHDA. There was a temporal correspondence between loss of protection and loss of the ability of the antioxidant to inhibit 6-OHDA-induced ERK phosphorylation. Time course studies of aconitase inactivation, an indicator of intracellular superoxide, and MitoSOX red, a mitochondria targeted ROS indicator, demonstrate early intracellular ROS followed by a delayed phase of mitochondrial ROS production, associated with phosphorylation of a mitochondrial pool of ERK. Furthermore, on initiation of mitochondrial ROS and ERK activation, 6-OHDA-injured cells became refractory to rescue by metalloporphyrin antioxidants. Together with previous studies showing that inhibition of the ERK pathway confers protection from 6-OHDA toxicity, and that phosphorylated ERK accumulates in mitochondria of degenerating human Parkinson''s disease neurons, these studies implicate mitochondrial ERK activation in Parkinsonian oxidative neuronal injury. [Copyright &y& Elsevier]

Abstract: To protect tissues from damaging effects of reactive oxygen species (ROS), organisms possess enzymatic and non-enzymatic antioxidant systems. Cytosolic-enzyme catalase (CAT) is a component of the antioxidant defence system that reduces hydrogen peroxide (H2O2) to water (H2O). The aim of this study was to assess the variation of antioxidant enzyme CAT activity in brain, kidney and liver of adult male mice according to tissue-specific and temporal patterns within a 24-h period (12:12 L/D). The CAT activity was assayed at 4-h intervals. The Cosinor test programme was used to detect and confirm the best corresponding rhythm. In liver, the circadian rhythm of CAT was associated with ultradian components. The prominent circadian rhythm (with a period τ =24 h) showed a peak located at the middle of the dark phase, more precisely ≅ 17 HALO (Hours After Light Onset). In kidney, only a circadian rhythm of CAT was validated with a peak time located at ≅ 17 HALO. However, in brain, the time pattern of CAT activity showed two peak times at ≅ 1 and ≅ 17 HALO, illustrating the existence of an ultradian rhythm (with a period τ =12 h). The results showed significant organ differences with the highest activity in liver, compared with kidney (−89%) and brain (−98%). This might be related to several factors such as their respective physiological function, the risk of exposure to oxidative damage and the balance between synthesis and degradation of proteins during “normal metabolism”. Moreover, CAT activity revealed differences in time-related changes across a 24-h period that were more obvious in peak levels between the three tissues. [Copyright &y& Elsevier]

Abstract: The DNA aggregation was found in aloe-emodin-induced H460 cell apoptosis in this study. Aloe-emodin (40μM)-induced DNA single strand breaks were observed by comet assay. Aloe-emodin induced decreases in the mRNA of DNA repair enzymes such as hMTH1, hOGG1 and APE. Although the activity of the radical-scavenging enzyme SOD was enhanced by aloe-emodin, the effects of aloe-emodin on H460 cell apoptosis were suspected to result from the prooxidant. These results suggest that aloe-emodin induced DNA damage through generation of reactive oxygen species in human lung carcinoma cells. [Copyright &y& Elsevier]

Abstract: Hypericum perforatum is a medicinal plant species containing many polyphenolic compounds, namely flavonoids and phenolic acids. Since polyphenolic compounds have a high antioxidant potential, in this study we evaluated the effect of H. perforatum in an animal model of acute inflammation, carrageenan-induced pleurisy. We report here that H. perforatum extract (given at 30 mg/kg orally, bolus prior to carrageenan) exerts potent anti-inflammatory effects in an animal model of acute inflammation. Injection of carrageenan into the pleural cavity of mice elicited an acute inflammatory response characterized by fluid accumulation in the pleural cavity which contained a large number of neutrophils (PMNs) as well as an infiltration of PMNs in lung tissues and subsequent lipid peroxidation (as determined by thiobarbituric acid-reactant substance measurement) and increased production of tumor necrosis factor-α, (TNF-α) and interleukin-1β (IL-1 β). All parameters of inflammation were attenuated by H. perforatum extract. Furthermore, carrageenan induced an upregulation of the expression of adhesion molecules ICAM-1, as well as an increase in the amounts of nitrotyrosine and poly(ADP-ribose) (PAR), as determined by immunohistochemical analysis of lung tissues. The degree of staining for the ICAM-1, nitrotyrosine, and PAR was significantly reduced by H. perforatum extract. Additionally, we demonstrate that these inflammatory events were associated with the activation of nuclear factor-κB (NF-κB) and signal transducer and activator transcription-3 (STAT-31) activation in the lung. NF-κB and STAT-3 activation were significantly inhibited by H. perforatum extract treatment. Taken together, our results indicate that prevention of the activation of NF-κB and STAT-3 by H. perforatum extract reduces the development of acute inflammation. [Copyright &y& Elsevier]

Summary: Betel quid (BQ) chewing is popular in Taiwan, India, and many southeast-Asian countries. BQ chewing has strong association with the risk of oral leukoplakia (OL), oral submucous fibrosis (OSF), and oral cancer (OC). BQ components exhibit genotoxicity and may alter the structure of DNA, proteins and lipids, resulting in production of antigenicity. BQ ingredients are also shown to induce keratinocyte inflammation by stimulating the production of prostaglandins, TNF-α, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in keratinocytes. These events may provoke tissue inflammation, early cell-mediated immunity (CMI), and immune surveillance in BQ chewers. However, BQ components also directly affect the functional activities of immunocompotent cells, and moreover tumor cells may hypo-respond to the CMI via diverse mechanisms such as induction of apoptosis of lymphocytes, induction of production of suppressor T cells, downregulation of MHC molecules in tumor cells, etc. Clinically, an alteration in lymphocyte subsets, a decrease in total number of lymphocytes, and a reduction in functional activities of CMI have been observed in isolated peripheral blood mononuclear cells (PBMC) and tumor infiltrated lymphocytes (TIL) in patients with OSF, OL or OC. Adaptation of tumor cells to immune system may promote clonal selection of resistant tumor cells, leading to immune tolerance. Future studies on effects of BQ components on CMI and humoral immunity in vitro and in vivo can be helpful for chemoprevention of BQ-related oral mucosal diseases. To elucidate how virus infection, tobacco, alcohol and BQ consumption, and other environmental exposure affect the immune status of patients with oral premalignant lesions or OC will help us to understand the immunopathogenesis of OC and to develop immunotherapeutic strategies for OC. [Copyright &y& Elsevier]

Abstract: Previously, we reported that a 1-butanol soluble extract of the edible red alga Palmaria palmata, known as dulse, exhibited hydroxyl and stable free radical scavenging activity as well as inhibition of lipid peroxidation, attributed to the reducing activity and polyphenol content of the dulse extract. In the present study, we evaluated the antioxidant and antiproliferative activities of two grades of dulse harvested from Canadian Maritime locations differing in UV radiation exposure (i.e. west versus east coasts of Grand Manan Island, New Brunswick). The 1-butanol soluble extract from Grade 1 dulse (reduced UV-exposure) exhibited lower reducing activity versus Grade 2 dulse (greater UV exposure) reflecting a lower requirement for endogenous antioxidant protection. Grade 1 and 2 dulse extracts both inhibited (p ⩽0.03) AAPH-induced lipid peroxidation, but had no effect on AMVN-induced lipid peroxidation, demonstrating the aqueous nature of the antioxidants involved. The Grade 1 and 2 dulse extract inhibition (p <0.05) of HeLa cell proliferation was dose-dependent over 0.5–5.0mg/mL and maximal at 48 and 72h incubation. The antiproliferative effects of the Grade 1 and 2 dulse extracts in the present study likely reflect the bioactivity of the polyphenol content of these extracts. [Copyright &y& Elsevier]

Abstract: Erythrocytes are a convenient model to understand the membrane oxidative damage induced by various xenobiotic-prooxidants. In this investigation, we have examined the potency of Fenvalerate (FEN) and its metabolite, p-chlorophenyl isovaleric acid (p-CPIA) to induce oxidative stress response in rat erythrocytes in vitro in terms of lipid peroxidation and effects on selected antioxidant enzymes. Susceptibility of erythrocytes to FEN exposure was further investigated in terms of morphological alterations by scanning electron microscopy and protein damage by gel electrophoresis of erythrocyte ghosts. Following in vitro exposure, FEN caused a significant induction of oxidative damage in erythrocytes at concentrations beyond 0.1mM as evidenced by increased thiobarbituric acid reactive substances (TBARS) levels. The response was both concentration and time dependent. At higher concentrations, significant decreases in the activities of vital antioxidant enzymes viz., catalase, superoxide dismutase, glutathione transferase and glutathione reductase were also discernible clearly suggesting the potency of both, parent compound and its metabolite to induce oxidative stress in erythrocytes. Scanning electron micrographs of erythrocytes following FEN exposure at higher concentrations revealed various degrees of distortion in shape and ruptured membranes. Furthermore, gel electrophoresis studies revealed consistent and significant aggregation of only band 3 protein in erythrocyte membranes exposed to either FEN or p-CPIA at higher concentrations. These in vitro findings show that FEN and its metabolite have the propensity to cause significant oxidative damage in rat erythrocytes, which is associated with marked damage to membrane proteins. These data suggest that both structural and functional perturbations may ensue in erythrocytes following exposure to FEN at higher concentrations under in vivo situations. [Copyright &y& Elsevier]

Abstract: Using flow cytometry, we quantitatively evaluated anti-tumor-promoting activity of rice bran extracts by measuring inhibition of Epstein-Barr virus early-antigen activation (EBV-EA) induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). This assay measures anti-tumor-promoting activity and cytotoxicity of target substances using the same batch of cells. Using this short-term procedure, we have determined the anti-tumor-promoting activity of 70% ethanol–water extracts of bran (outer layer) from seeds of five pigmented rice cultivars: Jumlalocal-1, DZ 78, Elwee, LK1-3-6-12-1-1, and LK1A-2-12-1-1. The results show that, compared to an extract from the non-pigmented white cooking rice variety Chuchung, the extracts from the pigmented varieties strongly inhibited phorbol ester-induced tumor promotion in marmoset lymphoblastoid cells B95-8 in vitro. These findings may facilitate development and use of new health-promoting rice varieties. [Copyright &y& Elsevier]

Abstract: Purpose: This study was performed to examine the effect of hypercholesterolemia on inducible nitric oxide synthase (NOS-2) expression and oxidative tissue injury in an experimental rat model of elevated IOP. Methods: Wistar rats were maintained on either regular chow or a high-cholesterol diet for 24 weeks. Intraocular pressure (IOP) was elevated in hypercholesterolemic rats by unilaterally cauterizing three episcleral vessels. Rats were divided into four experimental groups as follows; hypercholesterolemia, hypercholesterolemia+elevated IOP, elevated IOP and control. NOS-2 distribution, lipid peroxidation and retinal nerve fiber layer (RNFL) thickness was evaluated in all experimental groups at the end of 24 weeks. Results: Light microscopic evaluation of retinas in hypercholesterolemic rats revealed breaks and discontinuation in focal areas in the outer nuclear layer (ONL). NOS-2 positive staining was observed throughout the outer plexiform layer (OPL), inner plexiform layer (IPL) and ganglion cell layer (GCL) in rats with elevated IOP and/or hypercholesterolemia. Calculated values of RNFL thickness in hypercholesterolemic rats were significantly higher than those in the control and elevated IOP group. Vitreous malondialdehyde (MDA) levels detected in elevated IOP (3.51±0.31nmol/mg protein) and hypercholesterolemia+elevated IOP (5.14±1.28nmol/mg protein) groups were significantly higher than those detected in hypercholesterolemic (1.92±1.43nmol/mg protein) and control (1.89±0.24nmol/mg protein) groups. Conclusion: The presented data confirms hypercholesterolemia as a risk factor in the development of glaucomatous optic neuropathy (GON) and suggests that increased circulating cholesterol may exacerbate disease progression by inducing NOS-2 expression and elevating oxidant tissue injury. [Copyright &y& Elsevier]

Abstract: This study examined the effects of the organochlorine (OC) insecticides chlordane, o,p′-DDT, dieldrin, endosulfan, kepone, methoxychlor, and toxaphene on human HepG2 cytochrome P450 (1A-EROD and 2B-PROD) activities and glutathione (GSH) levels. Cells were exposed for 24h at high concentrations (1, 5 or 10mM) and for 48h at lower concentrations ranging from 0.01 to 1mM to evaluate dose responses. Our results show that after 48h all but dieldrin significantly induced both P4501A and 2B. P4502B responses were greater at all exposure concentrations and times. Mixed responses in GSH levels were observed. All OCs except dieldrin and MXC significantly depleted GSH after 24h. At 48h, chlordane, endosulfan and toxaphene significantly increased GSH at low levels and decreased GSH at high levels, while kepone and methoxychlor produced significant declines in GSH at all concentrations. These results support findings of OC insecticides inducing CYP1A, 2B in rats, with CYP2B responses more important. GSH levels declined when P4502B activity was significantly elevated and were significantly increased in the absence of significant P450 activity, suggesting that GSH levels influence the catalytic activity of the cytochrome P450s and the cytochrome P450s influence the cell’s ability to regulate GSH. [Copyright &y& Elsevier]

Neopterin is well established as a reliable marker in HIV-1 infection. Neopterin concentrations measured in urine or serum indicate sensitively the course and progression of the disease as well as efficacy of anti-retroviral therapy. The main trigger for neopterin production is Th1-type cytokine interferon-γ. During acute HIV-1 infection, enhanced formation of neopterin occurs already at a very early time point, before antibody seroconversion takes place. After this stage, neopterin concentrations in serum and urine closely correlate with virus load in the circulation of HIV-1-infected patients. Data provide evidence for an important role of immune activation and Th1-type cytokine interferon-γ in the pathogenesis of HIV-1 infection.This review subsumes the importance of neopterin as a marker in HIV-1 infection. Further evidence is increasing, that neopterin derivatives might modulate immune response by interfering with the cellular redox balance, activating redox-sensitive transcription factors, or inducing apoptosis in specific cell types. The possible impact of neopterin derivatives and of other biochemical pathways induced by interferon-γ such as indoleamine 2,3-dioxygenase in chronic diseases like HIV-1 infection is discussed. [Copyright &y& Elsevier]

Abstract: Several novel antioxidant-iron chelators bearing 8-hydroxyoxyquinoline moiety were synthesized, and various properties related to their iron chelation, and neuroprotective action were investigated. All the chelators exhibited strong iron(III) chelating and high antioxidant properties. Chelator 9 (HLA20), having good permeability into K562 cells and moderate selective MAO-B inhibitory activity (IC50 110μM), displayed the hightest protective effects against differentiated P19 cell death induced by 6-hydroxydopamine. EPR studies suggested that Chelator 9 also act as radical scavenger to directly scavenge hydroxyl radical. [Copyright &y& Elsevier]

Reactive oxygen species (ROS) can be generated following cell stimulation and function as intracellular signaling molecules. To determine signaling components involved in ROS induction, human U937 blood cells grown in 10% serum were exposed to serum-free media. It was previously reported that serum withdrawal (SW) killed cells by elevating cellular ROS levels. This study showed that SW activates phosphoinositide 3-kinase (PI3K). PI3K activation was evident after the ROS levels began increasing, and an antioxidant blockade of this increase resulted in PI3K activation suppression. Interestingly, the inhibition of PI3K activity/activation using either its specific inhibitor or dominant-negative mutant attenuated the subsequent additional increase in the ROS levels. These results suggest that SW-induced ROS activate PI3K, which in turn promotes the process leading to ROS accumulation. The present study also revealed that both ROS and PI3K support SW-induced cell death by activating stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). Overall, it appears that SW triggers a positive mutual interaction between ROS and PI3K, which amplifies signals required for the induction of an SAPK-dependent death pathway. [Copyright &y& Elsevier]

Abstract: In the following, we have compared the cytotoxic mechanisms of the chromate CrO42- and arsenite AsO2-. Chromate (Cr (VI)) cytotoxicity was associated with reactive oxygen species (ROS) formation, lipid peroxidation and loss of mitochondrial membrane potential, which were prevented by catalase, antioxidants and ROS scavengers. Hepatocyte glutathione was also rapidly oxidized. Chromate reduction was inhibited in glutathione depleted hepatocytes, and glutathione depleted hepatocytes were also much more resistant to chromate induced cytotoxicity, ROS formation and lipid peroxidation. This suggests that chromate is reductively activated by glutathione. Chromate cytotoxicity also involved lysosomal injury and protease activation, which were prevented by lysosomotropic agents, endocytosis inhibitors, protease inhibitors and ROS scavengers. On the other hand, arsenite cytotoxicity was associated with much less oxidative stress, and lysosomal damage did not occur. However, arsenite cytotoxicity was also associated with loss of mitochondrial membrane potential, which in contrast to chromate cytotoxicity was inhibited by the ATP generators fructose, xylitol and glutamine. Arsenite induced cytotoxicity, mitochondrial membrane potential decline and also ROS formation were significantly increased by inactivating hepatocyte methionine synthase or hepatocyte methyl transferase. However, methyl donors such as betaine, methionine or folic acid prevented arsenite but not chromate cytotoxicity, and this suggests that arsenite is detoxified by reductive methylation. In conclusion, chromate induced cytotoxicity could be attributed to oxidative stress and lysosomal damage, whereas arsenite induced cytotoxicity could be attributed to mitochondrial toxicity and ATP depletion. [Copyright &y& Elsevier]

Abstract: Polyphenols are ubiquitous substances in the diet. Their anti-oxidative, anti-inflammatory and anti-viral effects are of interest for human health, and polyphenols such as luteolin are used at high concentrations in food supplements. The aim of this project was to determine the intrinsic effects of luteolin in H4IIE rat hepatoma cells. Luteolin is relatively toxic, cell death was caused via induction of apoptosis as detected by DNA-ladder formation, by nuclear fragmentation and activation of apoptotic enzymes (caspase-2, -3/7, -9 and -8/10). Luteolin (250μM, 24h) increased the caspase-3/7 activity four-fold and the caspase-9 activity six-fold. In a time course experiment caspase-9 is activated after 6h, while caspase-2 and -3/7 are activated after 12h. After 24h, caspase-8/10 also displays activation. We found a concentration-dependent increase in malondialdehyde release suggesting a prooxidative effect of luteolin. Furthermore, we analysed DNA strand break formation by luteolin and found a distinct increase of DNA strand breaks after incubation for 3h with 100μM luteolin, a concentration which induces oligonucleosomal DNA cleavage at 24h. In conclusion, the sequence of events is compatible with the assumption that luteolin triggers the mitochondrial pathway of apoptosis, probably by inducing DNA damage. [Copyright &y& Elsevier]

Abstract: Troglitazone, a thiazolidinedione containing compound, was widely used to treat non-insulin dependent-diabetes. Unfortunately, troglitazone was associated with a sporadic liver toxicity that led to a cessation of its use clinically. Here we show that troglitazone induces a rapid and dose-dependent drop of mitochondrial membrane potential in liver HepG2 cells. The decrease in mitochondrial membrane potential induced by 100μM troglitazone was completed after 5min and similar in magnitude to that caused by carbonyl cyanide m-chloro phenylhydrazone. The troglitazone-induced loss of mitochondrial membrane potential preceded changes in cell permeability and cell count. In addition, troglitazone-induced a rise of intracellular calcium, subsequent to the drop in mitochondrial membrane potential, which was blocked by EGTA and the Na+/Ca2+ exchange inhibitor bepridil. Finally, application of 100μM troglitazone for 24h to HepG2 cells resulted in activation of caspase 3. The results of this study shed light on the molecular mechanisms by which troglitazone can cause cytotoxicity. [Copyright &y& Elsevier]

Abstract: The spectrophotometric method with 2,4-dinitrophenylhydrazine (DNPH) and the alkaline single-cell gel electrophoresis (comet assay) were used to assess the possibility of three polyphenolic compounds (tannic, ellagic and gallic acid) to induce the changes in proteins and DNA. B14 Chinese hamster cells were exposed to various concentrations of used polyphenols (1–60μM), hydrogen peroxide (10 and 40μM) copper ions (50μM), and the effect of phenols alone and in combination with Cu2+ ions and H2O2 was determined. The results reveal that polyphenolic acids in higher concentrations than 1μM contribute to the changes in the tested molecules and cause protein and DNA damage. The combined treatment of polyphenols with Cu2+ ions and with H2O2 demonstrates that the lowest dose of phenols (1μM) can act as an antioxidant agent against used chemicals. The DNA repair process was also studied. The data obtained in this experiment demonstrate that the most effective DNA repair occurs 1h after the removal of phenolic compounds. The used methods and obtained results provide additional information about the potential use of these phenols not only as antioxidants but also as pro-oxidants in biological systems. [Copyright &y& Elsevier]

To determine whether the Orf17 (NtpA) protein of Escherichia coli, a MutT-type enzyme, functions as a hydrolyzing enzyme for a damaged deoxyribonucleotide, we purified the recombinant Orf17 protein and incubated it with oxidized deoxyribonucleotides. Of the deoxyribonucleoside 5′-triphosphates tested, 8-hydroxy-2′-deoxyadenosine 5′-triphosphate was hydrolyzed by this protein. Unexpectedly, the Orf17 protein degraded 8-hydroxy-2′-deoxyadenosine 5′-diphosphate 2.3-fold more efficiently than the corresponding triphosphate. Thus, this protein is the first MutT-type enzyme that hydrolyzes both the triphosphate and diphosphate derivatives of a deoxyribonucleoside, with similar efficiencies. These results suggest that the Orf17 protein may be involved in the hydrolysis of oxidized dATP and dADP. [Copyright &y& Elsevier]

The Fanconi anemia (FA) proteins overlap with those of homologous recombination through FANCD1/BRCA2, but the biochemical functions of other FA proteins are largely unknown. By constructing and characterizing a null fancg mutant (KO40) of hamster CHO cells, we show that FancG protects cells against a broad spectrum of genotoxic agents. KO40 is consistently hypersensitive to both alkylating agents that produce monoadducts and those that produce interstrand crosslinks. KO40 cells were no more sensitive to mitomycin C (3×) and diepoxybutane (2×) than to 6-thioguanine (5×), ethylnitrosourea (3×), or methyl methanesulfonate (MMS) (3×). These results contrast with the pattern of selective sensitivity to DNA crosslinking agents seen historically with cell lines from FA patients. The hypersensitivity of KO40 to MMS was not associated with a higher level of initial DNA single-strand breaks; nor was there a defect in removing MNU-induced methyl groups from DNA. Both control and MMS-treated synchronized G1-phase KO40 cells progressed through S phase at a normal rate but showed a lengthening of G2 phase compared with wild type. MMS-treated and untreated early S-phase KO40 cells had increased levels of Rad51 foci compared with wild type. Asynchronous KO40 treated with ionizing radiation (IR) exhibited a normal Rad51 focus response, consistent with KO40 having only slight sensitivity to killing by IR. The plating efficiency and doubling time of KO40 cells were nearly normal, and they showed no increase in spontaneous chromosomal aberrations or sister chromatid exchanges. Collectively, our results do not support a role for FancG during DNA replication that deals specifically with processing DNA crosslinks. Nor do they suggest that the main function of the FA protein “pathway” is to promote efficient homologous recombination. We propose that the primary function of FA proteins is to maintain chromosomal continuity by stabilizing replication forks that encounter nicks, gaps, or replication-blocking lesions. [Copyright &y& Elsevier]

Abstract: Background: It has been proposed that chlorella extracts have antioxidative and anti-inflammatory effects. Methods: RAW 264.7 murine macrophage cell line was preincubated with various concentrations (0–100 μg/ml) of chlorella dichloromethane extract (CDE) and stimulated with lipopolysaccharide (LPS) to induce oxidative stress and inflammation. Results: Treatments of CDE reduced thiobarbituric acid-reactive substances (TBARS) accumulation, enhancing glutathione level and activities of antioxidative enzymes including superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-px), and glutathione reductase in LPS-stimulated macrophages than LPS-only treated cells. Nitric oxide (NO) production was significantly suppressed in a dose-dependent manner (p<0.05) with an IC50 of 30.5 μg/ml. Treatment of CDE at 50 μg/ml suppressed NO production to 6% of LPS-control. Treatment with CDE suppressed the levels of inducible nitric oxide synthase (iNOS) protein and mRNA expressions. The specific DNA binding activities of nuclear factor kappa B (NFκB) on nuclear extracts from CDE treatments were significantly suppressed with an IC50 of 62.7 μg/ml in a dose-dependent manner. Conclusions: CDE ameliorates NO production and iNOS expression through the down-regulation of NFκB activity, which may be mediated by attenuated oxidative stress in RAW 264.7 macrophages. [Copyright &y& Elsevier]

Excessive exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues including the breast. 4-Hydroxyequilenin (4-OHEN), a major catechol metabolite of equine estrogens present in Premarin®, an estrogen replacement formulation, has been shown to induce apoptosis and DNA damage in human breast cancer cells. It also has the potential to be a tumor initiator or promoter and complete carcinogen. To further understand the effects and mechanisms of equine catechol estrogen metabolite 4-OHEN action in vitro, human non-tumorigenic mammary epithelial MCF 10A cell line was used to study the toxic effects of 4-OHEN. In this study, we observed that 4-OHEN caused dose-dependent increases in apoptosis and DNA damage as measured by the DAPI nuclear screening assay and the Comet assay, respectively. Interestingly, cells treated with 100nM 4-OHEN biweekly for 4 weeks became resistant to cisplatin-induced apoptosis. The resistance to apoptosis of the 100nM 4-OHEN-treated cells was through multiple regulatory mechanisms. Compared to the DMSO-treated cells, the 100nM 4-OHEN-treated cells had higher GSH levels and total SOD activity, and a stronger GSH response after cisplatin treatment. Expression levels of several genes involved in cell growth, DNA repair, and apoptosis were either up- or down-regulated. These data indicate that long-term low-level equine estrogen metabolite exposure could induce DNA damage and initiate cells to become resistant to apoptosis. [Copyright &y& Elsevier]

Abstract: Photodynamic therapy (PDT) is recently developed as an effective treatment for malignant disease. In PDT, the photosensitizer eradicates tumour by induction of apoptosis. In this study, we investigated the mechanistic actions of a recently developed second generation photosensitizer, Zn–BC–AM, on nasopharyngeal carcinoma (NPC) cells. Zn–BC–AM was found to localize in the mitochondria, endoplasmic reticulum (ER), and golgi body. Photoactivation of Zn–BC–AM loaded NPC cells resulted in a rapid collapse of mitochondrial membrane potential (Δψm) (15min), followed by the release of cytochrome c (1h), and activation of caspases-9 and -3 (4h). Expression of ER chaperones Bip/Grp78 and Grp94, and ER resident lectin-like chaperone calnexin (CNX) was also enhanced in PDT-stressed NPC cells. Caspase-12, an important caspase involved in ER stress-induced apoptosis, was also activated. Inhibition of Ca2+ uptake into mitochondria by ruthenium red (RR) or loading the cells with EGTA–AM, an agent that buffers intracellular Ca2+ released from ER, resulted in a significant reduction of Zn–BC–AM PDT-induced cell death. These observations suggest that both ER and mitochondria are the subcellular targets of Zn–BC–AM. Effective activation of ER- and mitochondria-mediated apoptotic pathways is responsible for Zn–BC–AM PDT-induced NPC cell death. [Copyright &y& Elsevier]

Abstract: A comparison of Cd2+ and Ca2+ effects on in vitro rat liver mitochondria function and a further study of their interaction were conducted. Similarity and distinction in action of rotenone, oligomycin, N-ethylmaleimide, dithiothreitol, catalase, dibucaine, ruthenium red, cyclosporin A (CsA), and ADP on Cd2+ and/or Ca2+-induced mitochondrial dysfunction were revealed. We found that rotenone exerted a strong protective action both against Ca2+ and Cd2+-produced mitochondrial membrane permeabilization (MMP). In contrast to Ca2+, catalase and dibucaine did not influence on main Cd2+ effects. In NH4NO3 medium N-ethylmaleimide (NEM) at low concentrations increased markedly Cd2+-produced swelling of non-energized mitochondria, whereas it exhibited a partial reversal effect following energization. In sucrose medium low [NEM] did not change Cd2+-produced mitochondrial swelling. High [NEM] promoted synergistic increase of the Cd2+-produced swelling in NH4NO3 medium; all above effects were reversed (and prevented) by dithiothreitol, DTT. We shown also that when exogenous Ca2+ and Pi were simultaneously present in NH4NO3 medium, DTT reversed only partially Cd2+-produced swelling of succinate plus rotenone-energized mitochondria, while DTT recovery action was complete when either Ca2+ or Pi were separately administered to the Cd2+-treated mitochondria. Besides, DTT added following a low Cd2+ pulse in KCl medium containing exogenous Ca2+ induced a substantial enhancing of sustained Cd2+ stimulation of mitochondrial basal respiration and the stimulation was CsA-sensitive, while the activation promoted by low [Cd2+] alone was totally eliminated by DTT supplement. We observed the similar respiratory activation earlier when high concentrations of Cd2+ in the absence of added Ca2+ were used but it was completely CsA-insensitive. A possible involvement of respiratory chain components, namely complex I (P-site) and complex III (S-site) in Cd2+and/or Ca2+-produced MMP was discussed. [Copyright &y& Elsevier]

Redox-sensitive cysteine residues sense and transduce changes in cellular redox status caused by the generation of reactive oxygen species and the presence of oxidised thiols. Oxidation of such cysteines is converted into signals that control cell regulatory pathways and induction of gene expression. A variety of proteins, including transcription factors, molecular chaperones and protein tyrosine phosphatases, are regulated via redox processes. Common mechanisms underlie the sensitivity of cysteines to redox, such as proximity to polar and charged groups, and signal transduction is exerted via conformational changes that are conferred by the formation of disulfide and cyclic sulfenamide covalent bonds, and sulfenic and sulfonic acids. [Copyright &y& Elsevier]

Plant small GTPases belonging to the Rop, Arf, and Rab families are regulators of vesicle trafficking. Rop GTPases regulate actin dynamics and modulate H2O2 production in polar cell growth and pathogen defence. A candidate Rop GDP to Rop GTP exchange factor (RopGEF) SPIKE1 is involved in the morphogenesis of leaf epidermal cells. The ArfGEF GNOM regulates the endosomal recycling of the PIN proteins, which are involved in polar auxin transport. Intracellular localisation of small GTPases and functional studies using dominant mutant versions of Arf and Rab GTPases are defining novel plant-specific membrane compartments, especially those that participate in endosomal vesicle trafficking. [Copyright &y& Elsevier]

It is widely acknowledged that the function of the original uncoupling protein, UCP1, is uncoupling of substrate oxidation from ATP synthesis, and that its physiological purpose is thermogenesis. The mechanisms and physiological functions of the novel uncoupling proteins, identified within the past seven years, are as yet poorly understood. These novel uncoupling proteins are part of a large family comprising approximately 35 mitochondrial anion carrier proteins. UCP2 and UCP3 appear to function in reactive oxygen species handling and/or in fatty acid metabolism; uncoupling might occur secondarily. There is little information on UCP4 and UCP5 (BMCP1), and phylogenetic analyses indicate that they are further removed from UCP1 than mitochondrial anion carrier proteins, and have distinct functions. [Copyright &y& Elsevier]

The present study aimed at comparing in vitro the apoptogenic properties of metallic cobalt (Co), tungsten carbide (WC) and tungsten carbide–cobalt (WC–Co) in conditions known to cause genotoxicity. Human peripheral blood mononucleated cells were incubated with 2.0–6.0μg/ml of Co alone or mixed with WC particles and 33.3–100.0μg/ml WC alone for up to 24h. Under these culture conditions the majority (60%) of the cobalt metal particles were almost immediately solubilised in the culture medium, while WC remained under the form of particles that were progressively phagocytosed by monocytes. Apoptosis was assessed by Annexin-V staining, flow cytometry and analysis of DNA fragmentation by ELISA. Metallic Co-particles induced apoptosis in vitro. Furthermore, although so far considered as biologically inert, WC particles also induced apoptosis. When compared with its individual components WC–Co displayed an additive apoptotic effect in the DNA fragmentation assay. Apoptosis induced by WC particles was found largely dependent on caspase-9 activation and occurred presumably in monocytes, while that induced by Co involved both caspase-9 and -8 activation. The data suggest that apoptosis induced by the tested WC–Co mixture results from the additive effects of WC apoptosis induced in monocytes and Co-specific apoptosis in both monocytes and lymphocytes. The apoptogenic properties of these metals may be important in the mechanism of lung pathologies induced by the cobalt-containing particles. [Copyright &y& Elsevier]

Mitochondria play an important role in the integration and transmission of cell death signals, activating caspases and other cell death execution events by releasing apoptogenic proteins from the intermembrane space. The BCL-2 family of proteins localize (or can be targeted) to mitochondria and regulate the permeability of the mitochondrial outer membrane to these apoptotic factors. Recent evidence suggests that multiple mechanisms may regulate the release of mitochondrial factors, some of which depend on the action of caspases. [Copyright &y& Elsevier]

The present study was carried out to investigate the effects of copper (Cu) intake on lipid profile, oxidative stress and tissue damage in normal and in diabetic condition. Since diabetes mellitus is a situation of high-risk susceptibility to toxic compounds, we examined potential early markers of Cu excess in diabetic animals. Male Wistar rats, at 60-days-old were divided into six groups of eight rats each. The control(C) received saline from gastric tube, the no-diabetic(Cu-10), treated with 10mg/kg of Cu(Cu++–CuSO4, gastric tube), no-diabetic with Cu-60mg/kg(Cu-60), diabetic(D), diabetic low-Cu(DCu-10) and diabetic high-Cu(DCu-60). Diabetes was induced by an ip injection of streptozotocin (60mg/kg). After 30days of treatments, no changes were observed in serum lactate dehydrogenase, alanine transaminase and alkaline phosphatase, indicating no adverse effects on cardiac and hepatic tissues. D-rats had glucose intolerance and dyslipidemic profile. Cholesterol and LDL-cholesterol were higher in Cu-60 and DCu-60 than in C, Cu-10 and D and DCu-10 groups respectively. Cu-60 rats had higher lipid hydroperoxide (HP) and lower superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) serum activities than C and Cu-10 rats. LH was increased and GSH-Px was decreased, while no alterations were observed in SOD and catalase in serum of DCu-60 animals. DCu-60 rats had increased urinary glucose, creatinine and albumin. In conclusion, Cu intake at high concentration induced adverse effects on lipid profile, associated with oxidative stress and diminished activities of antioxidant enzymes. Diabetic animals were more susceptible to copper toxicity. High Cu intake induced dyslipidemic profile, oxidative stress and kidney dysfunction in diabetic condition. Copper renal toxicity was associated with oxidative stress and reduction at least, one of the antioxidant enzymes. [Copyright &y& Elsevier]

Abstract: The ubiquitin proteasome system (UPS) is the major protein quality control system in eukaryotic cells. Many neurodegenerative diseases are characterized by aggregates and inclusions of aberrant proteins, implying a sub-optimal functioning or defective UPS. The last few years have seen increasing evidence for the involvement of the UPS in neurodegenerative disorders, including Alzheimer''s disease (AD). Notably, decreases in proteasome activity were detected in several cortical areas in AD patients. In addition, proteins that accumulate in the classical hallmarks of AD were linked to UPS function. This review specifically discusses the involvement of the UPS in AD pathogenesis. First, a detailed overview of the UPS is presented, after which AD pathology and its relation to the UPS is discussed. [Copyright &y& Elsevier]

Apoptosis is a highly regulated cellular suicide program crucial for metazoan development. However, dysfunction of apoptosis also leads to several diseases. Yeast undergoes apoptosis after application of acetic acid, sugar- or salt-stress, plant antifungal peptides, or hydrogen peroxide. Oxygen radicals seem to be key elements of apoptotic execution, conserved during evolution. Furthermore, several yeast orthologues of central metazoan apoptotic regulators have been identified, such as a caspase and a caspase-regulating serine protease. In addition, physiological occurrence of cell death has been detected during aging and mating in yeast. The finding of apoptosis in yeast, other fungi and parasites is not only of great medical relevance but will also help to understand some of the still unknown molecular mechanisms at the core of apoptotic execution. [Copyright &y& Elsevier]

Clusterin (CLU) plays numerous roles in mammalian cells after stress. A review of the recent literature strongly suggests potential roles for CLU proteins in low dose ionizing radiation (IR)-inducible adaptive responses, bystander effects, and delayed death and genomic instability. Its most striking and evident feature is the inducibility of the CLU promoter after low, as well as high, doses of IR. Two major forms of CLU, secreted (sCLU) and nuclear (nCLU), possess opposite functions in cellular responses to IR: sCLU is cytoprotective, whereas nCLU (a byproduct of alternative splicing) is a pro-death factor. Recent studies from our laboratory and others demonstrated that down-regulation of sCLU by specific siRNA increased cytotoxic responses to chemotherapy and IR. sCLU was induced after low non-toxic doses of IR (0.02–0.5Gy) in human cultured cells and in mice in vivo. The low dose inducibility of this survival protein suggests a possible role for sCLU in radiation adaptive responses, characterized by increased cell radioresistance after exposure to low adapting IR doses. Although it is still unclear whether the adaptive response is beneficial or not to cells, survival of damaged cells after IR may lead to genomic instability in the descendants of surviving cells. Recent studies indicate a link between sCLU accumulation and cancer incidence, as well as aging, supporting involvement of the protein in the development of genomic instability. Secreted after IR, sCLU may also alter intracellular communication due to its ability to bind cell surface receptors, such as the TGF-β receptors (types I and II). This interference with signaling pathways may contribute to IR-induced bystander effects. We hypothesize that activation of the TGF-β signaling pathway, which often occurs after IR exposure, can in turn activate the CLU promoter. TGF-β and IR-inducible de novo synthesized sCLU may then bind the TGF-β receptors and suppress downstream growth arrest signaling. This complicated negative feedback regulation most certainly depends on the cellular microenvironment, but undoubtedly represents a potential link between IR-induced adaptive responses, genomic instability and bystander effects. Further elucidation of clusterin protein functions in IR responses are clearly warranted. [Copyright &y& Elsevier]

Abstract: Background:: We aimed to investigate the possible association between Mn-SOD polymorphism in the mitochondrial targeting sequence and asthma. Methods:: Alanine or valine polymorphism in the signal peptide of Mn-SOD gene was evaluated using a primer pair to amplify a 107-bp fragment followed by digestion with NgoM IV. Results:: No significant difference in genotype frequencies was found between patients and controls. Conclusion:: These results suggest no major modifying role for the Mn-SOD gene polymorphism in patients with asthma. [Copyright &y& Elsevier]

Ultraweak luminescence (UL) changes and sister-chromatid exchanges (SCEs) induced by Al toxicity were investigated in root tip cells of barley (Hordeum vulgare L.). A low positive correlation between relative UL rates and root relative elongation rates (RERs) was observed in 30 barley cultivars. During 1.5–24h exposure to 20μM Al solution, the UL value in cv Humai 16 (Al-tolerant) was higher than that in cv 2000-2 (Al-sensitive), suggesting that UL is a possible mechanism by which Al-tolerant cultivars avoid reactive oxygen species (ROS) toxicity. Upon raising Al levels (>50μM), the increase for UL in Al-tolerant and sensitive cultivars possibly involved cell death. A significant increase in the SCE rate was observed in cv 2000-2 but not in cv Humai 16 at 20μM Al level, suggesting that mechanisms of Al-tolerance might block the occurrence of Al-induced SCEs. With raising Al levels (0–640μM), SCE rates first increased and then decreased in the two cultivars, but their Al levels inducing the highest SCE rate were 10μM in cv 2000-2 and 320μM in cv Humai 16. In cv 2000-2, SCEs reached saturation status after 4h treatment with 10μM Al, suggesting that SCEs can be considered as a sensitive indicator for Al phytotoxicity. Ascorbic acid (ASA) pretreatment could significantly inhibit Al-induced SCE occurrence, indicating that Al indirectly induces SCEs via Al-triggered ROS formation. To our knowledge, this is the first report demonstrating that Al can induce UL changes and SCEs in root tip cells, and that may serve as possible indicators and thereby provide new insights into the mechanisms of Al toxicity and tolerance in barley. [Copyright &y& Elsevier]

The in vitro effects of resveratrol (RES) on apoptotic pathway in human chronic myeloid (K562) and acute lymphoblastic (HSB-2) leukemia cells were investigated. RES treatment of both cell types significantly and irreversibly inhibited their growth, associated with extensive apoptosis and increase in hypodiploid cells. Cell cycle analysis showed accumulation in G1 phase in HSB-2 drug exposed cells, while only K562-treated cells exhibited a marked accumulation in S phase with a concomitant decrease in G1 and G2/M at 24h. Moreover, RES caused internucleosomal DNA fragmentation, even if K562 cells were found less sensitive to the drug, as compared to HSB-2 cells, which also reacted earlier to the treatment. RES-induced apoptosis was associated with an increase of Bax expression and a marked release of cytochrome c from mitochondria. Interestingly, K562 cells exhibited a basal content of glutathione 10-fold that of HSB-2 cells, which increased after 24–48h RES exposure, together with increment of glutathione reductase and peroxidase activities. However, the major resistance to apoptosis of K562 cells cannot be attributed to their higher pool of reducing power, since neither the inhibition of glutathione synthesis by buthionine sulphoximine nor glutathione depletion by diethylmaleate, sensitized these cells. In addition, glutathione enrichment of HSB-2 cells by N-acetylcysteine did not prevent the apoptotic effects of RES. Our data indicate that RES commitment to apoptosis in both cell lines is independent from the intracellular content of glutathione, while it is associated with either the enhanced expression of Bax and cytochrome c release. [Copyright &y& Elsevier]

Low doses, chronic exposure to mercurial organic compounds is a worldwide health concern and could be pathogenetically relevant as co-factor in several neurodegenerative diseases. In this in vitro study we wanted to further improve our knowledge on the mechanisms of toxicity of methylmercury hydroxide (MeHgOH) in the unprimed PC12 cell line. Cell viability, mitochondrial function, redox state, and cell morphology were recorded at different time points to sequence the events leading to cell death. The lowest cytotoxic concentration and EC50 were 0.3 and 1.3μM, respectively. 5μM MeHgOH was fatal for 80% of the cell population after 24h; within 1h it caused glutathione (GSH) depletion and a partial dissipation of Δψm. At this concentration, reactive oxygen species (ROS) generation was only slight and delayed. After 6h more than 50% of ATP was available and caspase 3 was active. Time-lapse confocal microscopy showed that only a fraction of the cells completed apoptosis while others turned toward necrosis (necrapoptosis). Pre-incubation with N-acetylcysteine (NAC) and GSH but not Cyclosporin A rescued over 80% of the cells.These results provide experimental evidence that, in this cell model, MeHgOH triggers cell death via a primary depletion of GSH but in the absence of ROS overproduction. [Copyright &y& Elsevier]

Ascorbic acid is necessary for optimal insulin secretion from pancreatic islets. We evaluated ascorbate recycling and whether it is impaired by increased glucose metabolism in the rat β-cell line INS-1. INS-1 cells, engineered with the potential for overexpression of glucokinase under the control of a tetracycline-inducible gene expression system, took up and reduced dehydroascorbic acid to ascorbate in a concentration-dependent manner that was optimal in the presence of physiologic d-glucose concentrations. Ascorbate uptake did not affect intracellular GSH concentrations. Whereas depletion of GSH in culture to levels about 25% of normal also did not affect the ability of the cells to reduce dehydroascorbic acid, more severe acute GSH depletion to less than 10% of normal levels did impair dehydroascorbic acid reduction. Culture of inducible cells in 11.8 mM d-glucose and doxycycline for 48 h enhanced glucokinase activity, increased glucose utilization, abolished d-glucose-dependent insulin secretion, and increased generation of reactive oxygen species. The latter may have contributed to subsequent decreases in the ability of the cells both to maintain intracellular ascorbate and to recycle it from dehydroascorbic acid. Cultured β cells have a high capacity to recycle ascorbate, but this is sensitive to oxidant stress generated by increased glucose metabolism due to culture in high glucose concentrations and increased glucokinase expression. Impaired ascorbate recycling as a result of increased glucose metabolism may have implications for the role of ascorbate in insulin secretion in diabetes mellitus and may partially explain glucose toxicity in β cells. [Copyright &y& Elsevier]