Elevated branched-chain amino acid promotes atherosclerosis progression by enhancing mitochondrial-to-nuclear H2O2-disulfide HMGB1 in macrophages.

As the essential amino acids, branched-chain amino acid (BCAA) from diets is indispensable for health. BCAA supplementation is often recommended for patients with consumptive diseases or healthy people who exercise regularly. Latest studies and ours reported that elevated BCAA level was positively correlated with metabolic syndrome, diabetes, thrombosis and heart failure. However, the adverse effect of BCAA in atherosclerosis (AS) and its underlying mechanism remain unknown. Here, we found elevated plasma BCAA level was an independent risk factor for CHD patients by a human cohort study. By employing the HCD-fed ApoE-/- mice of AS model, ingestion of BCAA significantly increased plaque volume, instability and inflammation in AS. Elevated BCAA due to high dietary BCAA intake or BCAA catabolic defects promoted AS progression. Furthermore, BCAA catabolic defects were found in the monocytes of patients with CHD and abdominal macrophages in AS mice. Improvement of BCAA catabolism in macrophages alleviated AS burden in mice. The protein screening assay revealed HMGB1 as a potential molecular target of BCAA in activating proinflammatory macrophages. Excessive BCAA induced the formation and secretion of disulfide HMGB1 as well as subsequent inflammatory cascade of macrophages in a mitochondrial-nuclear H2O2 dependent manner. Scavenging nuclear H2O2 by overexpression of nucleus-targeting catalase (nCAT) effectively inhibited BCAA-induced inflammation in macrophages. All of the results above illustrate that elevated BCAA promotes AS progression by inducing redox-regulated HMGB1 translocation and further proinflammatory macrophage activation. Our findings provide novel insights into the role of animo acids as the daily dietary nutrients in AS development, and also suggest that restricting excessive dietary BCAA consuming and promoting BCAA catabolism may serve as promising strategies to alleviate and prevent AS and its subsequent CHD.

Mitochondria-derived H2O2 triggers liver regeneration via FoxO3a signaling pathway after partial hepatectomy in mice.

Reactive oxygen species (ROS) can induce oxidative injury and are generally regarded as toxic byproducts, although they are increasingly recognized for their signaling functions. Increased ROS often accompanies liver regeneration (LR) after liver injuries, however, their role in LR and the underlying mechanism remains unclear. Here, by employing a mouse LR model of partial hepatectomy (PHx), we found that PHx induced rapid increases of mitochondrial hydrogen peroxide (H2O2) and intracellular H2O2 at an early stage, using a mitochondria-specific probe. Scavenging mitochondrial H2O2 in mice with liver-specific overexpression of mitochondria-targeted catalase (mCAT) decreased intracellular H2O2 and compromised LR, while NADPH oxidases (NOXs) inhibition did not affect intracellular H2O2 or LR, indicating that mitochondria-derived H2O2 played an essential role in LR after PHx. Furthermore, pharmacological activation of FoxO3a impaired the H2O2-triggered LR, while liver-specific knockdown of FoxO3a by CRISPR-Cas9 technology almost abolished the inhibition of LR by overexpression of mCAT, demonstrating that FoxO3a signaling pathway mediated mitochondria-derived H2O2 triggered LR after PHx. Our findings uncover the beneficial roles of mitochondrial H2O2 and the redox-regulated underlying mechanisms during LR, which shed light on potential therapeutic interventions for LR-related liver injury. Importantly, these findings also indicate that improper antioxidative intervention might impair LR and delay the recovery of LR-related diseases in clinics.

Exercise and dietary intervention ameliorate high-fat diet-induced NAFLD and liver aging by inducing lipophagy.

Exercise and dietary intervention are currently available strategies to treat nonalcoholic fatty liver disease (NAFLD), while the underlying mechanism remains controversial. Emerging evidence shows that lipophagy is involved in the inhibition of the lipid droplets accumulation. However, it is still unclear if exercise and dietary intervention improve NAFLD through regulating lipophagy, and how exercise of skeletal muscle can modulate lipid metabolism in liver. Moreover, NAFLD is associated with aging, and little is known about the effect of lipid accumulation on aging process. Here in vivo and in vitro models, we found that exercise and dietary intervention reduced lipid droplets formation, decreased hepatic triglyceride in the liver induced by high-fat diet. Exercise and dietary intervention enhanced the lipophagy by activating AMPK/ULK1 and inhibiting Akt/mTOR/ULK1 pathways respectively. Furthermore, exercise stimulated FGF21 production in the muscle, followed by secretion to the circulation to promote the lipophagy in the liver via an AMPK-dependent pathway. Importantly, for the first time, we demonstrated that lipid accumulation exacerbated liver aging, which was ameliorated by exercise and dietary intervention through inducing lipophagy. Our findings suggested a new mechanism of exercise and dietary intervention to improve NAFLD through promoting lipophagy. The study also provided evidence to support that muscle exercise is beneficial to other metabolic organs such as liver. The FGF21-mediated AMPK dependent lipophagy might be a potential drug target for NAFLD and aging caused by lipid metabolic dysfunction.

Hydrogen peroxide modulates the proliferation/quiescence switch in the liver during embryonic development and posthepatectomy regeneration.

AIMS: The liver undergoes marked changes in the rate of proliferation during normal development and regeneration through the coordinated activity of numerous signaling pathways. Little is known, however, about the events that act upstream of these signaling pathways. Here, we explore the modulatory effects of hydrogen peroxide (H2O2) on these pathways in the context of liver development and regeneration. RESULTS: We show that H2O2 production during liver development and after partial hepatectomy is tightly regulated in time by specific H2O2-producing and scavenging proteins and dose dependently triggers two distinct pathways. Sustained elevated H2O2 levels are required for the activation of ERK signaling and trigger a shift from quiescence to proliferation. Contrastingly, sustained decreased H2O2 levels are required for the activation of p38 signaling and trigger a shift from proliferation to quiescence. Both events impact the cyclin D and Rb pathways and are involved in liver development and regeneration. Pharmacological lowering of H2O2 levels reduces the extent of fetal hepatocyte proliferation and delays the onset of liver regeneration. Chemical augmentation of H2O2 levels in adult hepatocytes triggers proliferation and delays the termination of liver regeneration. INNOVATION: Our results challenge the traditional view of H2O2 as a deleterious stressor in response to liver damage and identify a novel role of endogenous H2O2 in liver development and regeneration. CONCLUSIONS: Endogenous H2O2 production is tightly regulated during liver development and regeneration. H2O2 constitutes an important trigger for the proliferation and quiescence transition in hepatocytes via the concentration-dependent activation of the ERK or p38 pathway.

Saponins from Aralia taibaiensis attenuate D-galactose-induced aging in rats by activating FOXO3a and Nrf2 pathways.

Reactive oxygen species (ROS) are closely related to the aging process. In our previous studies, we found that the saponins from Aralia taibaiensis have potent antioxidant activity, suggesting the potential protective activity on the aging. However, the protective effect of the saponins and the possible underlying molecular mechanism remain unknown. In the present study, we employed a D-galactose-induced aging rat model to investigate the protective effect of the saponins. We found that D-galactose treatment induced obvious aging-related changes such as the decreased thymus and spleen coefficients, the increased advanced glycation end products (AGEs) level, senescence-associated ß-galactosidase (SAß-gal) activity, and malondialdehyde (MDA) level. Further results showed that Forkhead box O3a (FOXO3a), nuclear factor-erythroid 2-related factor 2 (Nrf2), and their targeted antioxidants such as superoxide dismutase 2 (SOD2), catalase (CAT), glutathione reductase (GR), glutathione (GSH), glutamate-cysteine ligase (GCL), and heme oxygenase 1 (HO-1) were all inhibited in the aging rats induced by D-galactose treatment. Saponins supplementation showed effective protection on these changes. These results demonstrate that saponins from Aralia taibaiensis attenuate the D-galactose-induced rat aging. By activating FOXO3a and Nrf2 pathways, saponins increase their downstream multiple antioxidants expression and function, at least in part contributing to the protection on the D-galactose-induced aging in rats.

Ischemic neurons activate astrocytes to disrupt endothelial barrier via increasing VEGF expression.

Blood-brain barrier (BBB) disruption occurring within the first few hours of ischemic stroke onset is closely associated with hemorrhagic transformation following thrombolytic therapy. However, the mechanism of this acute BBB disruption remains unclear. In the neurovascular unit, neurons do not have direct contact with the endothelial barrier; however, they are highly sensitive and vulnerable to ischemic injury, and may act as the initiator for disrupting BBB when cerebral ischemia occurs. Herein, we employed oxygen-glucose deprivation (OGD) and an in vitro BBB system consisting of brain microvascular cells and astrocytes to test this hypothesis. Neurons (CATH.a cells) were exposed to OGD for 3-h before co-culturing with endothelial monolayer (bEnd 3 cells), or endothelial cells plus astrocytes (C8-D1A cells). Incubation of OGD-treated neurons with endothelial monolayer alone did not increase endothelial permeability. However, when astrocytes were present, the endothelial permeability was significantly increased, which was accompanied by loss of occludin and claudin-5 proteins as well as increased vascular endothelial growth factor (VEGF) secretion into the conditioned medium. Importantly, all these changes were abolished when VEGF was knocked down in astrocytes by siRNA. Our findings suggest that ischemic neurons activate astrocytes to increase VEGF production, which in turn induces endothelial barrier disruption.

NADPH oxidase-mitochondria axis-derived ROS mediate arsenite-induced HIF-1α stabilization by inhibiting prolyl hydroxylases activity.

Arsenic exposure has been shown to induce hypoxia inducible factor 1α (HIF-1α) accumulation, however the underlying mechanism remains unknown. In the present study, we tested the hypothesis that arsenic exposure triggered the interaction between NADPH oxidase and mitochondria to promote reactive oxygen species (ROS) production, which inactivate prolyl hydroxylases (PHDs) activity, leading to the stabilization of HIF-1α protein. Exposure of human immortalized liver cell line HL-7702 cells to arsenite induced HIF-1α accumulation in a dose-dependent manner, which was abolished by SOD mimetic MnTMPyP. Inhibition of NADPH oxidase with diphenyleneiodonium chloride (DPI) or inhibition of mitochondrial respiratory chain with rotenone significantly blocked arsenite-induced ROS production, and the mitochondria appeared to be the major source of ROS production. Arsenite treatment inhibited HIF-1α hydroxylation by prolyl hydroxylases (PHDs) and increased HIF-1α stabilization, but did not affect HIF-1α mRNA expression and Akt activation. Supplementation of ascorbate or Fe(II) completely abolished arsenite-induced PHDs inhibition and HIF-1α stabilization. In conclusion, these results define a unique mechanism of HIF-1α accumulation following arsenic exposure, that is, arsenic activates NADPH oxidase-mitochondria axis to produce ROS, which deplete intracellular ascorbate and Fe(II) to inactivate PHDs, leading to HIF-1α stabilization.

Mechanism of acute lung injury due to phosgene exposition and its protection by cafeic acid phenethyl ester in the rat.

The mechanism of phosgene-induced acute lung injury (ALI) remains unclear and it is still lack of effective treatments. Previous study indicated that oxidative stress was involved in phosgene-induced ALI. Caffeic acid phenethyl ester (CAPE) has been proved to be an anti-inflammatory agent and a potent free radical scavenger. The purpose of this study was to investigate the protective effects of CAPE on phosgene-induced ALI and identify the mechanism, in which oxidative stress and inflammation were involved. The phosgene was used to induce ALI in rats. The results showed that after phosgene exposure, total protein content in BALF was not significantly changed. The increase of MDA level and SOD activity induced by phosgene was significantly reduced by CAPE administration, and the decrease of GSH level in BALF and lung were significantly reversed by CAPE. CAPE also partially blocked the translocation of NF-κB p65 to the nucleus, but it had little effect on the phosphorylation of p38 MAPK. In conclusion, CAPE showed protective effects on lung against phosgene-induced ALI, which may be related with a combination of the antioxidant and anti-inflammatory functions of CAPE.

Ethyl pyruvate protects rats from phosgene-induced pulmonary edema by inhibiting cyclooxygenase2 and inducible nitric oxide synthase expression.

Phosgene is a poorly water-soluble gas penetrating the lower respiratory tract which can induce acute lung injury characterized by a latent phase of fatal pulmonary edema. Pulmonary edema caused by phosgene is believed to be a consequence of oxidative stress and inflammatory responses. Ethyl pyruvate (EP) has been demonstrated to have anti-inflammatory and anti-oxidative properties in vivo and in vitro. The potential therapeutic role of EP in phosgene-induced pulmonary edema has not been addressed so far. In the present study, we aim to investigate the protective effects of EP on phosgene-induced pulmonary edema and the underlying mechanisms. Rats were administered with EP (40 mg kg(-1)) and RAW264.7 cells were also incubated with it (0, 2, 5 or 10 µm) immediately after phosgene (400 ppm, 1 min) or air exposure. Wet-to-dry lung weight ratio (W:D ratio), nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production, cyclooxygenase2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, and mitogen-activated protein kinases activities (MAPKs) were measured. Our results showed that EP treatment attenuated phosgene-induced pulmonary edema and decreased the level of NO and PGE(2) dose-dependently. Furthermore, EP significantly reduced COX-2 expression, iNOS expression and MAPK activation induced by phosgene. Moreover, specific inhibitors of MAPKs reduced COX-2 and iNOS expression induced by phosgene. These findings suggested that EP has a protective role against phosgene-induced pulmonary edema, which is mediated in part by inhibiting MAPK activation and subsequently down-regulating COX-2 and iNOS expression as well as decreasing the production of NO and PGE(2).

Poly(ADP-ribose) polymerase-1 inhibition by arsenite promotes the survival of cells with unrepaired DNA lesions induced by UV exposure.

Human arsenic exposure is associated with increased risk of skin cancer, and arsenite greatly enhances ultraviolet (UV)-induced skin tumors in a mouse model of carcinogenesis. Inhibition of DNA repair is one proposed mechanism for the observed cocarcinogenicity. We have previously demonstrated that low concentrations of arsenite inhibit poly(ADP-ribose) polymerase (PARP)-1, thus interfering with DNA repair process triggered by UV radiation. Because overactivation of PARP-1 often leads to apoptotic cell death, and unrepaired DNA lesions promote genomic instability and carcinogenesis, we hypothesized that inhibition of PARP-1 by arsenic may promote the survival of potentially "initiated carcinogenic cells," i.e., cells with unrepaired DNA lesions. In the present study, we tested this hypothesis on UV-challenged HaCat cells. Cells were pretreated with 2µM arsenite for 24 h before UV exposure. Outcome parameters included apoptotic death rate, PARP-1 activation, apoptotic molecules, and retention of DNA lesions. UV exposure induced PARP-1 activation and associated poly(ADP-ribose) production, apoptosis-inducing factor release, cytochrome C release, and caspases activation, which led to apoptotic death in HaCat cells. Pretreatment with 2µM arsenite significantly inhibited UV-induced cell death as well as the associated molecular events. Notably, knockdown of PARP-1 with small interfering RNA completely abolished the antagonism of arsenite. Furthermore, arsenite pretreatment led to long-term retention of UV-induced cyclobutane pyrimidine dimers. Together, these results suggest that low concentration of arsenite reduces UV-induced apoptosis via inhibiting PARP-1, thus promoting the survival of cells with unrepaired DNA lesions, which may be an important mechanism underlying arsenic cocarcinogenic action.

Protective effect of the peroxisome proliferator-activated receptor (PPAR)-γ, ligand rosiglitazone on tert-butyl hydroperoxide-induced QZG cell injury.

Tert-butyl hydroperoxide (t-BHP) can induce cell injury by forming free radical intermediates. Peroxisome proliferator-activated receptor (PPAR)-γ is a ligand-activated transcription factor belonging to nuclear hormone receptor superfamily, and is involved in oxidative stress response. Thiazolidinedione rosiglitazone is a potent PPARγ agonist. The main aim of this study was to investigate the protective effect of rosiglitazone on QZG cells from t-BHP-induced toxicity. MTT assay showed that t-BHP treatment resulted in decreased cell viability in a concentration dependent manner. Under 400 µM t-BHP treatment, QZG cell displayed significant loss of viability and dramatic morphological changes characterized by changing in shape from triangle to spherical, disappearance of cell cilia, swollen mitochondrial and typical apoptotic alteration such as condensation of chromatin, and appearance of crescent under light microscopy and electronic microscopy, respectively. Flow cytometry analysis indicated that 30.90±1.70% QZG cells were undergoing apoptosis compared to that of the control cells (2.80±0.85%, P<0.05). There was substantial population of the cells undergoing necrosis (28.5.%). 25 µM rosiglitazone treatment inhibited the t-BHP-induced cell toxicity significantly by restoring the cell viability, reducing cell population undergone apoptosis to normal level (3.5%) and ameliorating t-BHP-induced pathological changes. Real-time RT-PCR results showed that 400 µM t-BHP caused dramatic down-regulation of PPARγ expression in QZG cells, whereas combining treatment with 25 µM rosiglitazone resistant to PPARγ expression to normal level partially. Overall, our results indicate that rosiglitazone has protective effect against t-BHP-induced QZG cell injury. The protective effect of rosiglitazone is involved in its regulation on the function of PPARγ.

Pentoxifylline inhibits intercellular adhesion molecule-1 (ICAM-1) and lung injury in experimental phosgene-exposure rats.

Phosgene inhalation results in acute lung injury (ALI) mostly, pulmonary edema and even acute respiratory distress syndrome, but there is no specific antidote. Inflammatory cells play an important role in the ALI caused by phosgene. Intercellular adhesion molecule-1 (ICAM-1) is a critical factor for inflammatory organ injury. We hypothesized that pentoxifylline (PTX), an inhibitor of leukocyte activation, would have a protective effect on experimental phosgene-induced lung injury rats by inhibiting ICAM-1. To prove this hypothesis, we used rat models of phosgene (400 ppm x 1 min)-induced injury to investigate: (1) the time course of lung injury (control 1, 3, 6, 12, 24, and 48 h group), including pathological changes in hematoxylin and eosin staining and transmission electron microscope, myeloperoxidase (MPO) activity by colorimetric method and ICAM-1 protein level detected by western blot, (2) At 3 h after phosgene exposure, protective effects of different dosages of PTX (50 mg/kg and 100 mg/kg) administration were evaluated by MPO activity, ICAM-1 differential expression and WBC count in bronchoalveolar lavage fluid. The results showed that inflammatory cells emerged out of lung blood vessels at 3 h after phosgene exposure. The MPO activity of lung tissue increased significantly from 3 to 48 h after phosgene exposure (P < 0.05) and ICAM-1 expression presented a similar change, especially at 3 h and 24 h (P < 0.05). After pretreatment and treatment with PTX (100 mg/kg), significant protective effects were shown (P < 0.05). These data supported our hypothesis that PTX reduced phosgene-induced lung injury, possibly by inhibiting ICAM-1 differential expression.

Normobaric hyperoxia reduces the neurovascular complications associated with delayed tissue plasminogen activator treatment in a rat model of focal cerebral ischemia.

BACKGROUND AND PURPOSE: A major limitation of tissue plasminogen activator (tPA) thrombolysis for ischemic stroke is the narrow time window for safe and effective therapy. Delayed tPA thrombolysis increases the risk of cerebral hemorrhage and mortality, which, in part, is related to neurovascular proteolysis mediated by matrix metalloproteinases (MMPs). We recently showed that normobaric hyperoxia treatment reduces MMP-9 expression and blood-brain barrier disruption in the ischemic brain. Therefore, we hypothesized that normobaric hyperoxia could increase the safety of delayed tPA thrombolysis in stroke. METHODS: Male Sprague-Dawley rats were exposed to normobaric hyperoxia (95% O(2)) or normoxia (21% O(2)) during 5-hour filament occlusion of the middle cerebral artery followed by 19-hour reperfusion. Thirty minutes before reperfusion, saline or tPA was continuously infused to rats over 1 hour. Outcome parameters were neurological score, mortality rate, brain edema, hemorrhage volume, and MMP-9. Hemorrhage was quantified with a hemoglobin spectrophotometry method. Edema was evaluated as hemispheric enlargement. MMP-9 was measured by gelatin zymography. RESULTS: In normoxic rats, delayed tPA treatment at 4.5 hours after stroke onset resulted in high mortality, more severe neurological deficits, increased hemorrhage volumes, and augmented MMP-9 induction compared with saline. Rats treated with combined normobaric hyperoxia and tPA showed significantly reduced tPA-associated mortality, brain edema, hemorrhage, and MMP-9 augmentation as compared with tPA alone. CONCLUSIONS: Our results suggest that early normobaric hyperoxia treatment may represent an important strategy to increase the safety of delayed tPA thrombolysis in ischemic stroke.

Normobaric hyperoxia attenuates early blood-brain barrier disruption by inhibiting MMP-9-mediated occludin degradation in focal cerebral ischemia.

Early blood-brain barrier (BBB) disruption resulting from excessive neurovascular proteolysis by matrix metalloproteinases (MMPs) is closely associated with hemorrhagic transformation events in ischemic stroke. We have shown that normobaric hyperoxia (NBO) treatment reduces MMP-9 increase in the ischemic brain. The aim of this study was to determine whether NBO could attenuate MMP-9-mediated early BBB disruption following ischemic stroke. Rats were exposed to NBO (95% O(2)) or normoxia (30% O(2)) during 90-min middle cerebral artery occlusion, followed by 3-hour reperfusion. NBO-treated rats showed a significant reduction in Evan's blue extravasation in the ischemic hemisphere compared with normoxic rats. Topographically, Evan's blue leakage was mainly seen in the subcortical regions including the striatum, which was accompanied by increased gelatinolytic activity and reduced immunostaining for tight-junction protein, occludin. Increased gelatinolytic activities and occludin protein loss were also observed in isolated ischemic microvessels. Gel gelatin zymography identified that MMP-9 was the main enzymatic source in the cerebral microvessels. Incubation of brain slices or isolated microvessels with purified MMP-9 revealed specific degradation of occludin. Inhibition of MMP-9 by NBO or MMP-inhibitor, BB1101, significantly reduced occludin protein loss in ischemic microvessels. These results suggest that NBO attenuates early BBB disruption, and inhibition of MMP-9-mediated occludin degradation is an important mechanism for this protection.

Correlation between sPLA2-IIA and phosgene-induced rat acute lung injury.

Secreted phospholipase A(2) of group IIA (sPLA(2)-IIA) has been involved in a variety of inflammatory diseases, including acute lung injury. However, the specific role of sPLA(2)-IIA in phosgene-induced acute lung injury remains unidentified. The aim of the present study was to investigate the correlation between sPLA(2)-IIA activity and the severity of phosgene-induced acute lung injury. Adult male rats were randomly exposed to either normal room air (control group) or a concentration of 400 ppm phosgene (phosgene-exposed group) for there are 5 phosgene-exposed groups altogether. For the time points of 1, 3, 6, 12 and 24 h post-exposure, one phosgene-exposed group was sacrificed at each time point. The severity of acute lung injury was assessed by Pa(O2)/F(IO2) ratio, wet-to-dry lung-weight ratio, and bronchoalveolar lavage (BAL) fluid protein concentration. sPLA(2)-IIA activity in BAL fluid markedly increased between 1 h and 12 h after phosgene exposure, and reached its highest level at 6 h. Moreover, the trend of this elevation correlated well with the severity of lung injury. These results indicate that sPLA(2)-IIA probably participates in phosgene-induced acute lung injury.

Inhibition of poly(ADP-ribose) polymerase-1 by arsenite interferes with repair of oxidative DNA damage.

Arsenic enhances skin tumor formation when combined with other carcinogens, including UV radiation (UVR). In this study we report that low micromolar concentrations of arsenite synergistically increases UVR-induced oxidative DNA damage in human keratinocytes as detected by 8-hydroxyl-2'-deoxyguanine (8-OHdG) formation. Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in base excision repair, a process that repairs 8-OHdG lesions. Arsenite suppresses UVR-induced PARP-1 activation in a concentration-dependent manner. Inhibition of PARP-1 activity by 3-aminobenzamide or small interfering RNA silencing of PARP-1 expression significantly increases UVR-induced 8-OHdG formation, suggesting that inhibition of PARP-1 activity by arsenite contributes to oxidative DNA damage. PARP-1 is a zinc finger protein, and mass spectrometry analysis reveals that arsenite can occupy a synthetic apopeptide representing the first zinc finger of PARP-1 (PARPzf). When the PARPzf peptide is preincubated with Zn(II) followed by incubation with increasing concentrations of arsenite, the ZnPARPzf signal is decreased while the AsPARPzf signal intensity is increased as a function of arsenite dose, suggesting a competition between zinc and arsenite for the same binding site. Addition of Zn(II) abolished arsenite enhancement of UVR-stimulated 8-OHdG generation and restored PARP-1 activity. Our findings demonstrate that arsenite inhibits oxidative DNA damage repair and suggest that interaction of arsenite with the PARP-1 zinc finger domain contributes to the inhibition of PARP-1 activity by arsenite. Arsenite inhibition of poly(ADP-ribosyl)ation is one likely mechanism for the reported co-carcinogenic activities of arsenic in UVR-induced skin carcinogenesis.

Dual actions involved in arsenite-induced oxidative DNA damage.

Arsenic is a recognized human carcinogen, but the mechanism of carcinogenesis is not well understood. Oxidative stress and inhibition of DNA damage repair have been postulated as potential carcinogenic actions of arsenic. The present study tests the hypothesis that arsenite not only induces oxidative stress but also inhibits the activity of the DNA base excision repair protein, poly(ADP-ribose) polymerase-1 (PARP-1), leading to exacerbation of the oxidative DNA damage induced by arsenic. HaCat cells were treated with arsenite for 24 h before measuring 8-hydroxyl-2'-deoxyguanosine (8-OHdG), PARP-1 activity, and reactive oxygen species (ROS). Zinc supplementation and PARP-1 siRNA were used to increase or decrease, respectively, the PARP-1 protein's physiological function. At high concentrations (10 microM or higher), arsenite greatly induced oxidative DNA damage, as indicated by 8-OHdG formation. At lower concentrations (1 microM), arsenite did not produce detectable 8-OHdG, but was still able to effectively inhibit PARP-1 activity. Zinc supplementation reduced the formation of 8-OHdG, restored the PARP-1 activity inhibited by arsenite, but did not decrease ROS production. SiRNA knockdown of PARP-1 did not affect the 8-OHdG level induced by arsenic, while it greatly increased the 8-OHdG level produced by hydrogen peroxide indicating that PARP-1 is a molecular target of arsenite. Our findings demonstrate that in addition to inducing oxidative stress at higher concentrations, arsenite can also inhibit the function of a key DNA repair protein, PARP-1, even at very low concentrations, thus exacerbating the overall oxidative DNA damage produced by arsenite, and potentially, by other oxidants as well.

Alterations of Fc gamma receptor I and Toll-like receptor 4 mediate the antiinflammatory actions of microglia and astrocytes after adrenaline-induced blood-brain barrier opening in rats.

Blood-brain barrier (BBB) opening occurs under many physiological and pathological conditions. BBB opening will lead to the leakage of large circulating molecules into the brain parenchyma. These invasive molecules will induce immune responses. Microglia and astrocytes are the two major cell types responsible for immune responses in the brain, and Fc gamma receptor I (FcgammaRI) and Toll-like receptor 4 (TLR4) are the two important receptors mediating these processes. Data suggest that activation of the FcgammaRI pathway mediates antiinflammatory processes, whereas activation of TLR4 pathway leads to proinflammatory activities. In the present study, we tested the hypothesis that BBB opening could lead to alterations in FcgammaRI and TLR4 pathways in microglia and astrocytes, thus limiting excessive inflammation in the brain. The transient BBB opening was induced by adrenaline injection through a caudal vein in Sprague-Dawley rats. We found that the FcgammaRI pathway was significantly activated in both microglia and astrocytes, as exhibited by the up-regulation of FcgammaRI and its key downstream molecule Syk, as well as the increased production of the effector cytokines, interleukin (IL)-10 and IL-4. Interestingly, after transient BBB opening, TLR4 expression was also increased. However, the expression of MyD88, the central adapter of the TLR4 pathway, was significantly inhibited, with decreased production of the effector cytokines IL-12a and IL-1beta. These results indicate that, after transient BBB opening, FcgammaRI-mediated antiinflammatory processes were activated, whereas TLR4-mediated proinflammatory activities were inhibited in microglia and astrocytes. This may represent an important neuroprotective mechanism of microglia and astrocytes that limits excessive inflammation after BBB opening.

Low concentration of arsenite exacerbates UVR-induced DNA strand breaks by inhibiting PARP-1 activity.

Epidemiological studies have associated arsenic exposure with many types of human cancers. Arsenic has also been shown to act as a co-carcinogen even at low concentrations. However, the precise mechanism of its co-carcinogenic action is unknown. Recent studies indicate that arsenic can interfere with DNA-repair processes. Poly(ADP-ribose) polymerase (PARP)-1 is a zinc-finger DNA-repair protein, which can promptly sense DNA strand breaks and initiate DNA-repair pathways. In the present study, we tested the hypothesis that low concentrations of arsenic could inhibit PAPR-1 activity and so exacerbate levels of ultraviolet radiation (UVR)-induced DNA strand breaks. HaCat cells were treated with arsenite and/or UVR, and then DNA strand breaks were assessed by comet assay. Low concentrations of arsenite (

Time course for expression of VEGF and its receptor and regulator levels of contraction and relaxation in increased vascular permeability of lung induced by phosgene.

Acute lung injury (ALI) induced by phosgene increases risk of serious edema and mortality. Increased permeability of the microvascular endothelium is implicated in the progression of ALI, but the processing interaction and time course activity of the vascular regulators in exudation are still not understood. The main aim of this study was to investigate the time course and potential role for vascular endothelial growth factor (VEGF), its receptors, and some vascular function regulators related to increased vascular permeability of lung induced by phosgene. Sprague Dawley rats were randomly divided into seven groups according to time post phosgene exposure (control, and 1, 3, 6, 12, 24, and 48 h groups). Lung tissue was removed to evaluate VEGF isoforms, fms-like tyrosine kinase receptor 1 (Flt-1), and kinase insert domain containing region (KDR/Flk-1) by reverse-transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Blood samples were collected for measurement of plasma endothelin-1 (ET-1) and nitric oxide (NO) level. The results showed that the mRNA and protein expression profile of the VEGF system after phosgene exposure was time dependent. The VEGF system expression in lung tissue was related closely to the level of ET-1 and NO. In conclusion, increased permeability of the lung microvascular endothelium induced by phosgene was primarily a result of differential expression of VEGF and its receptors, and was related to the level of ET-1 and NO. The results suggest that the cooperation of VEGF system, ET-1, and NO plays a critical role, and all those parameters emerge as time dependent in the early phase of the permeability process induced by phosgene exposure.