Cardiotoxicity Induced by Intratracheal Instillation of Diesel Exhaust Particles in Mice, and the Protective Effects of Carnosol: Suppression of Inflammation and Oxidative and Nitrosative Stress via Modulation of NF-κb/MAPKs Signaling Pathways.

BACKGROUND/AIMS: Inhaled particulate air pollution is associated with cardiotoxicity with underlying mechanisms including oxidative stress and inflammation. Carnosol, commonly found in rosemary and sage, is known to possess a broad range of therapeutic properties such as antioxidant, anti-inflammatory and antiapoptotic. However, its cardioprotective effects on diesel exhaust particles (DEPs)-induced toxicity have not been studied yet. Hence, we evaluated the potential ameliorative effects of carnosol on DEPs-induced heart toxicity in mice, and the underlying mechanisms involved. METHODS: Mice were intratracheally instilled with DEPs (1 mg/kg) or saline, and 1 hour prior to instillation they were given intraperitoneally either carnosol (20 mg/kg) or saline. Twenty-four hours after the DEPs instillation, multiple parameters were evaluated in the heart by enzyme-linked immunosorbent assay, colorimetric assay, Comet assay and Western blot technique. RESULTS: Carnosol has significantly reduced the elevation in the plasma levels of lactate hydrogenase and brain natriuretic peptide induced by DEPs. Likewise, the augmented cardiac levels of proinflammatory cytokines, lipid peroxidation, and total nitric oxide in DEPs-treated groups were significantly normalized with the treatment of carnosol. Moreover, carnosol has markedly reduced the heart mitochondrial dysfunction, as well as DNA damage and apoptosis of mice treated with DEPs. Similarly, carnosol significantly reduced the elevated expressions of phosphorylated nuclear factor-кB (NF-кB) and mitogen-activated protein kinases (MAPKs) in the hearts. Furthermore, the treatment with carnosol has restored the decrease in the expression of sirtuin-1 in the hearts of mice exposed to DEPs. CONCLUSION: Carnosol significantly attenuated DEP-induced cardiotoxicity in mice by suppressing inflammation, oxidative stress, DNA damage, and apoptosis, at least partly via mechanisms involving sirtuin-1 activation and the inhibition of NF-кB and MAPKs activation.

Effects of Waterpipe Smoke Exposure on Experimentally Induced Chronic Kidney Disease in Mice.

Tobacco smoking is an independent risk factor in the onset of kidney disease. To date, there have been no reports on the influence of waterpipe smoke (WPS) in experimentally induced chronic kidney disease (CKD) models. We studied the effects and mechanisms of actions of WPS on a mouse model of adenine-induced CKD. Mice fed either a normal diet, or an adenine-added diet and were exposed to either air or WPS (30 min/day and 5 days/week) for four consecutive weeks. Plasma creatinine, urea and indoxyl sulfate increased and creatinine clearance decreased in adenine + WPS versus either WPS or adenine + saline groups. The urinary concentrations of kidney injury molecule-1 and adiponectin and the activities of neutrophil gelatinase-associated lipocalin and N-acetyl-ß-D-glucosaminidase were augmented in adenine + WPS compared with either adenine + air or WPS groups. In the kidney tissue, several markers of oxidative stress and inflammation were higher in adenine + WPS than in either adenine + air or WPS groups. Compared with the controls, WPS inhalation in mice with CKD increased DNA damage, and urinary concentration of 8-hydroxy-2-deoxyguanosine. Furthermore, the expressions of nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPKs) (ERK and p38) were elevated in the kidneys of adenine + WPS group, compared with the controls. Likewise, the kidneys of adenine + WPS group revealed more marked histological tubular injury, chronic inflammation and interstitial fibrosis. In conclusion, WPS inhalation aggravates kidney injury, oxidative stress, inflammation, DNA damage and fibrosis in mice with adenine-induced CKD, indicating that WPS exposure intensifies CKD. These effects were associated with a mechanism involving NF-κB, ERK and p38 activations.

Comparative Evaluation of the Effects of Amorphous Silica Nanoparticles on the Erythrocytes of Wistar Normotensive and Spontaneously Hypertensive Rats.

Silica nanoparticles (SiNPs) are one of the most widely used nanomaterials. SiNPs can encounter erythrocytes and hypertension is strongly linked to abnormalities in the functional and structural characteristics of erythrocytes. As little is known about the combinatorial effect of SiNP-hypertension interactions on erythrocytes, the aim of this work was to study the effects triggered by hypertension on SiNPs induced hemolysis and the pathophysiological mechanism underlying it. We compared the interaction of amorphous 50 nm SiNPs at various concentrations (0.2, 1, 5 and 25 µg/mL) with erythrocytes of normotensive (NT) and hypertensive (HT) rats in vitro. Following incubation of the erythrocytes, SiNPs induced significant and dose-dependent increase in hemolysis. Transmission electron microscopy revealed erythrocyte deformity in addition to SiNPs taken up by erythrocytes. The erythrocyte susceptibility to lipid peroxidation was significantly increased. The concentration of reduced glutathione, and activities of superoxide dismutase, and catalase were significantly increased. SiNPs significantly increased intracellular Ca2+. Likewise, the concentration of the cellular protein annexin V and calpain activity was enhanced by SiNPs. Concerningly, all the tested parameters were significantly enhanced in erythrocytes from HT rats compared to NT rats. Our results collectively demonstrate that hypertension can potentially exacerbate the in vitro effect induced by SiNPs.

Impact of Intratracheal Administration of Polyethylene Glycol-Coated Silver Nanoparticles on the Heart of Normotensive and Hypertensive Mice.

Silver nanoparticles are widely used in various industrial and biomedical applications; however, little is known about their potential cardiotoxicity after pulmonary exposure, particularly in hypertensive subjects. We assessed the cardiotoxicity of polyethylene glycol (PEG)-coated AgNPs in hypertensive (HT) mice. Saline (control) or PEG-AgNPs (0.5 mg/kg) were intratracheally (i.t.) instilled four times (on days 7, 14, 21, and 28 post-angiotensin II or vehicle [saline] infusion). On day 29, various cardiovascular parameters were evaluated. Systolic blood pressure and heart rate were higher in PEG-AgNPs-treated HT mice than in saline-treated HT or PEG-AgNPs-treated normotensive mice. The heart histology of PEG-AgNPs-treated HT mice had comparatively larger cardiomyocyte damage with fibrosis and inflammatory cells when compared with saline-treated HT mice. Similarly, the relative heart weight and the activities of lactate dehydrogenase and creatine kinase-MB and the concentration of brain natriuretic peptide concentration were significantly augmented in heart homogenates of HT mice treated with PEG-AgNPs compared with HT mice treated with saline or normotensive animals exposed to PEG-AgNPs. Similarly, the concentrations of endothelin-1, P-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 in heart homogenates were significantly higher than in the other two groups when HT mice were exposed to PEG-AgNPs. Markers of inflammation and oxidative and nitrosative stress were significantly elevated in heart homogenates of HT mice given PEG-AgNPs compared with HT mice treated with saline or normotensive animals exposed to PEG-AgNPs. The hearts of HT mice exposed to PEG-AgNPs had significantly increased DNA damage than those of HT mice treated with saline or normotensive mice treated with AgNPs. In conclusion, the cardiac injury caused by PEG-AgNPs was aggravated in hypertensive mice. The cardiotoxicity of PEG-AgNPs in HT mice highlights the importance of an in-depth assessment of their toxicity before using them in clinical settings, particularly in patients with pre-existing cardiovascular diseases.

Comparative Study on the Chronic Vascular Responses Induced by Regular Versus Occasional Waterpipe Smoke Inhalation in Mice.

BACKGROUND/AIMS: Waterpipe smoke (WPS) is the second most prevalent form of smoking in the world. There are ample evidences about the vascular alterations caused by regular WPS (Reg-WPS). Nonetheless, comparison of the chronic vascular response induced by regular versus occasional WPS (Occ-WPS) exposure is very scarce. METHODS: We investigated, in BALB/c mice, the effects of Occ-WPS (30 minutes/day, 1 day/week) versus Reg-WPS (30 minutes/day, 5 days/week) for 6 months on thrombogenicity and platelet aggregation in vivo and in vitro. Moreover, various markers of endothelial integrity, inflammation and oxidative stress were assessed by enzyme-linked immunosorbent assay and colorimetric assay. Control mice were exposed to air. RESULTS: Our results showed that either Occ-WPS or Reg-WPS exposure shortened the thrombotic time in pial microvessels in vivo. Moreover, in pial venules, this effect was more marked in Reg-WPS group (-47%) compared with Occ-WPS (-34%). Similarly, exposure to either Occ-WPS or Reg-WPS reduced the prothrombin time and activated partial thromboplastin time. Platelet count was increased only in Reg-WPS exposure. Exposure to either Occ-WPS or Reg-WPS induced platelet aggregation in vitro. In addition, there was a statistically significant difference between Occ-WPS and Reg-WPS groups in platelet count and aggregation. Plasma concentration of tissue factor (+98%), P-selectin (+14%) and E-selectin (+16%) were significantly increased in Occ-WPS group compared with air exposed group. Likewise, compared with air group Reg-WPS caused an increase in concentration of tissue factor (+193%), P-selectin (+21%) and E-selectin (+42%). Nevertheless, only Reg-WPS induced a decrease (-38%) in the plasma concentration of tissue plasminogen activator. Notably, our results showed a statistically significant difference between Occ-WPS and Reg-WPS groups in the concentration of tissue factor. Erythrocyte numbers, hemoglobin concentration, hematocrit and lactate dehydrogenase activity were augmented only in Reg-WPS group compared with either control or Occ-WPS groups. Likewise, only Reg-WPS induced an increase in proinflammatory cytokines, tumor necrosis factor-α and interleukin-1ß compared with either control or Occ-WPS groups. However, markers of oxidative stress including 8-isoprostane and total antioxidants were enhanced in both Occ-WPS and Reg-WPS compared with control group. CONCLUSION: Our data confirm the vascular toxicity of the chronic Reg-WPS exposure and shows that even occasional chronic exposure to WPS caused thrombosis, platelet aggregation, endothelial alterations and oxidative stress. The latter findings are an additional cause of concern about the long-term toxicity of occasional waterpipe smoking.

Effect of smoking cessation on chronic waterpipe smoke inhalation-induced airway hyperresponsiveness, inflammation, and oxidative stress.

Waterpipe smoking (WPS) prevalence is increasing globally. Clinical and laboratory investigations reported that WPS triggers impairment of pulmonary function, inflammation, and oxidative stress. However, little is known if smoking cessation (SC) would reverse the adverse pulmonary effects induced by WPS. Therefore, we evaluated the impact of WPS inhalation for 3 mo followed by 3 mo of SC (air exposure) compared with those exposed for either 3 or 6 mo to WPS or air (control) in C57BL/6 mice. To this end, various physiological, biochemical, and histological endpoints were evaluated in the lung tissue. Exposure to WPS caused focal areas of dilated alveolar spaces and foci of widening of interalveolar spaces with peribronchiolar moderate mixed inflammatory cells consisting of lymphocytes, macrophages, and neutrophil polymorphs. The latter effects were mitigated by SC. Likewise, SC reversed the increase of airway resistance and reduced the increase in the levels of myeloperoxidase, matrix metalloproteinase 9, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß in lung tissue induced by WPS. In addition, SC attenuated the increase of oxidative stress markers including 8-isoprostane, glutathione, and catalase induced by WPS. Similarly, DNA damage, apoptosis, and the expression of NF-κB in the lung induced by WPS inhalation were alleviated by CS. In conclusion, our data demonstrated, for the first time, to our knowledge, that SC-mitigated WPS inhalation induced an increase in airway resistance, inflammation, oxidative stress, DNA injury, and apoptosis, illustrating the benefits of SC on lung physiology.

Exacerbation of Coagulation and Cardiac Injury in Rats with Cisplatin-Induced Nephrotoxicity Following Intratracheal Instillation of Cerium Oxide Nanoparticles.

BACKGROUND/AIMS: Exposure to particulate air pollution is associated with increased cardiovascular morbidity and mortality. These effects are particularly aggravated in patients with pre-existing kidney diseases. Cerium oxide nanoparticles (CNPs), used as diesel fuel additives, are emitted in vehicle exhaust and affect humans when inhaled. However, thrombotic and cardiac injury resulting from pulmonary exposure to CNPs in experimental acute kidney injury (AKI) is not fully understood. The objective of the present study was to evaluate the thrombotic and cardiac injury effects of CNPs in a rat model of AKI. METHODS: AKI was induced in rats by a single intraperitoneal injection of cisplatin (CDDP, 6 mg/kg). Six days after injection, rats were intratracheally (i.t.) instilled with either CNPs (1 mg/kg) or saline (control), and various cardiovascular variables and markers of inflammation, oxidative stress and DNA injury were assessed by enzyme linked immunosorbent assay, colorimetric assay, single-cell gel electrophoresis assay and immunohistochemistry, the following day. RESULTS: Compared with individual CDDP or CNPs treatments, the combined CDDP + CNPs treatment elevated significantly the coagulation function, relative heart weight, and troponin I, lactate dehydrogenase, interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and total nitric oxide levels in the plasma. In heart homogenates, the combination of CDDP and CNPs induced a significant increase in IL-6, TNFα, catalase, and glutathione. Furthermore, significantly more DNA damage was observed in this group than in the CDDP or CNPs groups. Immunohistochemical analysis of the heart revealed that expression of nuclear factor erythroid-derived 2-like 2 (Nrf2) and glutathione peroxidase by cardiac myocytes and endothelial cells was increased in the CDDP + CNPs group more than in either CDDP or CNPs group. CONCLUSION: I.t. administration of CNPs in rats with AKI exacerbated systemic inflammation, oxidative stress, and coagulation events. It also aggravated cardiac inflammation, DNA damage, and Nrf2 expression.

Waterpipe Tobacco Smoke Inhalation Triggers Thrombogenicity, Cardiac Inflammation and Oxidative Stress in Mice: Effects of Flavouring.

The consumption of water-pipe smoking (WPS) has been promoted by the use of flavoured tobacco. However, little is known about the impact of flavouring on the cardiovascular toxicity induced by WPS inhalation. Here, we compared the cardiovascular effects and underlying mechanism of actions of plain (P) (unflavoured) versus apple-flavoured (AF) WPS (30 minutes/day, 5 days/week for 1 month) in mice. Control mice were exposed to air. Both P- and AF-WPS inhalation induced an increase in systolic blood pressure, thrombogenicity and plasma concentration of fibrinogen and von Willebrand factor. In heart homogenates, AF-WPS inhalation caused an increase of 8-isoprostane and a decrease in the levels of reduced glutathione (GSH) and superoxide dismutase (SOD). Nevertheless, P-WPS decreased only the activity of SOD. The concentrations of tumour necrosis factor α and interleukin 1ß were increased only in heart homogenates of mice exposed to AF-WPS. Although both P- and AF-WPS increased the concentration of troponin I in heart homogenates and induced DNA damage, the concentration of cleaved caspase 3 was only increased in mice exposed to AF-WPS. Immunohistochemical analysis of the hearts showed that both P- and AF- WPS inhalation decreased the expression of SOD. Moreover, the expression of nuclear factor erythroid-derived 2-like 2 at nuclear level in the heart was higher in both AF-WPS and P-WPS compared with control group, and the effect observed in AF-WPS group was more significant than that seen in P-WPS group. Likewise, the concentration of heme oxygenase-1 was significantly increased in both P-WPS and AF-WPS groups compared with control group, and the effect seen in AF-group was higher than that observed in P-WPS group. In conclusion, our findings showed that both P- and AF-WPS induce thrombogenicity and cardiac injury, and that this toxicity is potentiated by the presence of flavouring.

Impact of Pulmonary Exposure to Cerium Oxide Nanoparticles on Experimental Acute Kidney Injury.

BACKGROUND/AIMS: Cerium oxide nanoparticles (CeO2 NPs) are released from diesel engines that use cerium compounds as a catalytic agent to decrease the diesel exhaust particles, leading to human exposure by inhalation to CeO2 NPs. We have recently demonstrated that pulmonary exposure to CeO2 NPs induces lung inflammation, thrombosis, and oxidative stress in various organs including kidneys. It is well known that particulate air pollution effects are greater in patients with renal diseases. The aim of this study is to investigate the effects of pulmonary exposure to CeO2 NPs in a rat model of acute kidney injury (AKI). METHODS: AKI was induced in rats by a single intraperitoneal injection of cisplatin (CP, 6 mg/kg). Six days later, the rats were intratracheally (i.t.) instilled with either CeO2 NPs (1 mg/kg) or saline (control), and various renal and pulmonary endpoints were assessed 24 h afterward using histological, colorimetric assay, enzyme-linked immunosorbent assay and Comet assay techniques. RESULTS: CP alone decreased body weight, and increased water intake, urine volume and relative kidney weight. CP also increased the plasma concentrations urea and creatinine, and decreased creatinine clearance. In the kidneys, CP significantly increased renal injury molecule-1, interleukin-6 (IL-6), tumor necrosis factor α (TNFα) and glutathione concentrations, and caused renal tubular necrosis, and DNA injury assessed by Comet assay. All these actions were significantly aggravated in rats given both CP and CeO2 NPs. Histopathological changes in lungs of CeO2 NPs-treated rats included marked interstitial cell infiltration and congestion. These were aggravated by the combination of CP + CeO2 NPs. Moreover, this combination exacerbated the increase in the concentrations of TNFα and IL-6, and the decrease in the activity of pulmonary catalase and total nitric oxide concentration, and lung DNA damage. CONCLUSION: We conclude that the presence of CeO2 NPs in the lung exacerbated the renal and lung effects of CP-induced AKI.

Thrombosis and systemic and cardiac oxidative stress and DNA damage induced by pulmonary exposure to diesel exhaust particles and the effect of nootkatone thereon.

Adverse cardiovascular effects of particulate air pollution persist even at lower concentrations than those of the current air quality limit. Therefore, identification of safe and effective measures against particle-induced cardiovascular toxicity is needed. Nootkatone is a sesquiterpenoid in grapefruit with diverse bioactivities including anti-inflammatory and antioxidant effects. However, its protective effect on the cardiovascular injury induced by diesel exhaust particles (DEPs) has not been studied before. We assessed the possible protective effect of nootkatone (90 mg/kg) administered by gavage 1 h before intratracheal instillation of DEPs (30 µg/mouse). Twenty-four hours after the intratracheal administration of DEPs, various thrombotic and cardiac parameters were assessed. Nootkatone inhibited the prothrombotic effect induced by DEPs in pial arterioles and venules in vivo and platelet aggregation in whole blood in vitro. Also, nootkatone prevented the shortening of activated partial thromboplastin time and prothrombin time induced by DEPs. Nootkatone inhibited the increase of plasma concentration of fibrinogen, plasminogen activator inhibitor-1, interleukin-6, and lipid peroxidation induced by DEPs. Immunohistochemically, hearts showed an analogous increase in glutathione and nuclear factor erythroid-derived 2-like 2 expression by cardiac myocytes and endothelial cells after DEP exposure, and these effects were enhanced in mice treated with nootkatone + DEPs. Likewise, heme oxygenase-1 was increased in mice treated with nootkatone + DEPs compared with those treated with DEPs or nootkatone + saline. The DNA damage caused by DEPs was prevented by nootkatoone pretreatment. In conclusion, nootkatoone alleviates DEP-induced thrombogenicity and systemic and cardiac oxidative stress and DNA damage, at least partly, through nuclear factor erythroid-derived 2-like 2 and heme oxygenase-1 activation. NEW & NOTEWORTHY Nootkatoone, a sesquiterpenoid found in grapefruit, alleviates the thrombogenicity and systemic and cardiac oxidative stress and DNA damage in mice exposed to diesel exhaust particles. Nootkatone-induced boosting of nuclear factor erythroid-derived 2-like 2 and heme oxygenase-1 levels in the heart of mice exposed to diesel exhaust particles suggests that its protective effect is, at least partly, mediated through nuclear factor erythroid-derived 2-like 2 and heme oxygenase-1 activation.

The in Vitro Effect of Polyvinylpyrrolidone and Citrate Coated Silver Nanoparticles on Erythrocytic Oxidative Damage and Eryptosis.

BACKGROUND/AIMS: Silver nanoparticles (AgNPs) are increasingly used as antimicrobial agents and drug carriers in various biomedical fields. AgNPs can encounter erythrocytes either directly following intravenous injection, or indirectly via translocation from the site of administration. However, information regarding the pathophysiological effects and possible mechanism of action of AgNPs on the erythrocytes are still inadequately studied. Thus, the aim of our study was to investigate the mechanism underlying the effect of coating and concentration of AgNPs on mouse erythrocytes in vitro. METHODS: We studied the interaction of polyvinylpyrrolidone (PVP) and citrate (CT) coated AgNPs (10 nm) at various concentrations (2.5, 10, 40 µg/ml) with mouse erythrocytes in vitro using various techniques including transmission electron microscopy (TEM), hemolysis, and colorimetric measurement of markers of oxidative stress comprising malondialdehyde (MDA), reduced glutathione (GSH), and catalase (CAT). Intracellular calcium (Ca2+) was determined using Fura 2AM fluorescence. Annexin V was quantified using ELISA and the caspase 3 was determined both flurometrically and by western blot technique. RESULTS: Following incubation of the erythrocytes with AgNPs, both PVP- and CT- AgNPs induced significant and dose - dependent increase in hemolysis. TEM revealed that both PVP- and CT- AgNPs were taken up by erythrocytes. The erythrocyte susceptibility to lipid peroxidation measured by MDA was significantly increased in both PVP-and CT- AgNPs. The concentration of GSH and CAT activity were significantly decreased by both types of AgNPs. Additionally, PVP- and CT- AgNPs significantly increased intracellular Ca2+ in a dose -dependent manner. Likewise, the concentration of the cellular protein annexin V was significantly and dose - dependently enhanced by both types of AgNPs. Furthermore, PVP- and CT- AgNPs induced significant increase in calpain activity in incubated erythrocytes. CONCLUSION: We conclude that both PVP- and CT- AgNPs causes hemolysis, and are taken up by erythrocytes. Moreover, we demonstrated that AgNPs induces oxidative stress and eryptosis. These findings provide evidence for the potential pathophysiological effect of PVP-and CT- AgNPs on erythrocyte physiology.

Chronic exposure to water-pipe smoke induces cardiovascular dysfunction in mice.

Water-pipe tobacco smoking is becoming prevalent in all over the world including Western countries. There are limited data on the cardiovascular effects of water-pipe smoke (WPS), in particular following chronic exposure. Here, we assessed the chronic cardiovascular effects of nose-only WPS exposure in C57BL/6 mice. The duration of the session was 30 minutes/day, 5 days/week for 6 consecutive months. Control mice were exposed to air. WPS significantly increased systolic blood pressure. The relative heart weight and plasma concentrations of troponin-I and B-type natriuretic peptide were increased in mice exposed to WPS. Arterial blood gas analysis showed that WPS caused a significant decrease in [Formula: see text] and an increase in [Formula: see text] WPS significantly shortened the thrombotic occlusion time in pial arterioles and venules and increased the number of circulating platelet. Cardiac lipid peroxidation, measured as thiobarbituric acid-reactive substances, was significantly increased, while superoxide dismutase activity, total nitric oxide activity, and glutathione concentration were reduced by WPS exposure. Likewise, immunohistochemical analysis of the heart revealed an increase in the expression of inducible nitric oxide synthase and cytochrome c by cardiomyocytes of WPS-exposed mice. Moreover, hearts of WPS-exposed mice showed the presence of focal interstitial fibrosis. WPS exposure significantly increased heart DNA damage assessed by Comet assay. We conclude that chronic nose-only exposure to WPS impairs cardiovascular homeostasis. Our findings provide evidence that long-term exposure to WPS is harmful to the cardiovascular system and supports interventions to control the spread of WPS, particularly amid youths.NEW & NOTEWORTHY No data are available on the chronic cardiovascular effects of water-pipe smoke (WPS). Our findings provide experimental evidence that chronic exposure to WPS increased blood pressure, relative heart weight, troponin I, and B-type natriuretic peptide in plasma and induced hypoxemia, hypercapnia, and thrombosis. Moreover, WPS caused cardiac oxidative stress, DNA damage, and fibrosis.

Water-Pipe Smoke Exposure-Induced Circulatory Disturbances in Mice, and the Influence of Betaine Supplementation Thereon.

BACKGROUND/AIMS: It has been shown, both experimentally and clinically, that water-pipe smoke (WPS) exposure adversely affects the cardiovascular system (CVS) through the generation of oxidative stress and inflammation. Betaine, a naturally occurring compound in common foods, has antioxidant and anti-inflammatory actions. However, its potential to mitigate the adverse effect of WPS on the CVS has never been reported before. This is the subject of this study in mice. METHODS: Mice were exposed daily for 30 min to either normal air (control), or to WPS for two consecutive weeks. Betaine was administered daily by gavage at a dose of 10mg/kg, 1h before either air or WPS exposure. RESULTS: Betaine mitigated the in vivo prothrombotic effect of WPS in pial arterioles and venules. Moreover, it reversed the WPS-induced decrease in circulating platelets. Likewise, betaine alleviated platelet aggregation in vitro, and the shortening of activated partial thromboplastin time and prothrombin time induced by WPS. Betaine reduced the increase of plasminogen activator inhibitor-1 and fibrinogen concentrations in plasma induced by WPS. Betaine also diminished the WPS-induced increase of plasma concentrations of interleukin 6 and tumor necrosis factor α, and attenuated the increase of lipid peroxidation and superoxide dismutase. Immunohistochemical analysis of the heart revealed an increase in the expression of inducible nitric oxide synthase and cytochrome C by cardiomyocytes of the WPS-exposed mice. These effects were averted by betaine. CONCLUSION: Our findings suggest that betaine treatment significantly mitigated WPS-induced hypercoagulability, and inflammation, as well as systemic and cardiac oxidative stress.

Chronic Exposure to Water-Pipe Smoke Induces Alveolar Enlargement, DNA Damage and Impairment of Lung Function.

BACKGROUND/AIM: Epidemiological evidence indicates that water-pipe smoking (WPS) adversely affects the respiratory system. However, the mechanisms underlying its effects are not well understood. Recent experimental studies reported the occurrence of lung inflammation and oxidative stress following acute and subacute exposure to WPS. Here, we wanted to verify the extent of inflammation and oxidative stress in mice chronically-exposed to WPS and to evaluate, for the first time, its effect on alveolar injury and DNA damage and their association with impairment of lung function. METHODS: Mice were nose-only exposed to mainstream WPS (30 min/day; 5 days/week for 6 consecutive months). Control mice were exposed using the same protocol to atmospheric air only. At the end of the exposure period, several respiratory parameters were assessed. RESULTS: In bronchoalveolar lavage fluid, WPS increased neutrophil and lymphocyte numbers, lactate dehydrogenase, myeloperoxidase and matrix metallopeptidase 9 activities, as well as several proinflammatory cytokines. In lung tissue, lipid peroxidation, reactive oxygen species, superoxide dismutase activity and reduced glutathione were all increased by WPS exposure. Along with oxidative stress, WPS exposure significantly increased lung DNA damage index. Histologically the lungs of WPS-exposed mice had foci of mixed inflammatory cells infiltration in the interalveolar interstitium which consisted of neutrophils, lymphocytes and macrophages. Interestingly, we found dilated alveolar spaces and alveolar ducts with damaged interalveolar septae, and impairment of lung function following WPS exposure. CONCLUSION: We show the persistence of lung inflammation and oxidative stress in mice chronically-exposed to WPS and demonstrate, for the first time, the occurrence of DNA damage and enlargement of alveolar spaces and ducts associated with impairment of lung function. Our findings provide novel mechanistic elucidation for the long-term effects of WPS on the respiratory system.

Prolonged Pulmonary Exposure to Diesel Exhaust Particles Exacerbates Renal Oxidative Stress, Inflammation and DNA Damage in Mice with Adenine-Induced Chronic Renal Failure.

BACKGROUND/AIMS: Epidemiological evidence indicates that patients with chronic kidney diseases have increased susceptibility to adverse outcomes related to long-term exposure to particulate air pollution. However, mechanisms underlying these effects are not fully understood. METHODS: Presently, we assessed the effect of prolonged exposure to diesel exhaust particles (DEP) on chronic renal failure induced by adenine (0.25% w/w in feed for 4 weeks), which is known to involve inflammation and oxidative stress. DEP (0.5m/kg) was intratracheally (i.t.) instilled every 4th day for 4 weeks (7 i.t. instillation). Four days following the last exposure to either DEP or saline (control), various renal endpoints were measured. RESULTS: While body weight was decreased, kidney weight increased in DEP+adenine versus saline+adenine or DEP. Water intake, urine volume, relative kidney weight were significantly increased in adenine+DEP versus DEP and adenine+saline versus saline. Plasma creatinine and urea increased and creatinine clearance decreased in adenine+DEP versus DEP and adenine+saline versus saline. Tumor necrosis factor α, lipid peroxidation and reactive oxygen species were significantly increased in adenine+DEP compared with either DEP or adenine+saline. The antioxidant calase was significantly decreased in adenine+DEP compared with either adenine+saline or DEP. Notably, renal DNA damage was significantly potentiated in adenine+DEP compared with either adenine+saline or DEP. Similarly, systolic blood pressure was increased in adenine+DEP versus adenine+saline or DEP, and in DEP versus saline. Histological evaluation revealed more collagen deposition, higher number of necrotic cell counts and dilated tubules, cast formation and collapsing glomeruli in adenine+DEP versus adenine+saline or DEP. CONCLUSION: Prolonged pulmonary exposure to diesel exhaust particles worsen renal oxidative stress, inflammation and DNA damage in mice with adenine-induced chronic renal failure. Our data provide biological plausibility that air pollution aggravates chronic renal failure.

Ultrasmall superparamagnetic iron oxide nanoparticles acutely promote thrombosis and cardiac oxidative stress and DNA damage in mice.

BACKGROUND: Ultrasmall superparamagnetic iron oxide nanoparticles (USPIO) are being developed for several biomedical applications including drug delivery and imaging. However, little is known about their possible adverse effects on thrombosis and cardiac oxidative and DNA damage. METHODS: Presently, we investigated the acute (1 h) effect of intravenously (i.v.) administered USPIO in mice (0.4, 2 and 10 µg/kg). Diesel exhaust particles (DEP; 400 µg/kg) were used as positive control. RESULTS: USPIO induced a prothrombotic effect in pial arterioles and venules in vivo and increased the plasma plasminogen activator inhibitor-1 (PAI-1). Both thrombogenicity and PAI-1 concentration were increased by DEP. The direct addition of USPIO (0.008, 0.04 and 0.2 µg/ml) to untreated mouse blood dose-dependently induced in vitro platelet aggregation. USPIO caused a shortening of activated partial thromboplastin time (aPTT) and prothrombin time (PT). Similarly, DEP administration (1 µg/ml) triggered platelet aggregation in vitro in whole blood. DEP also reduced PT and aPTT. The plasma levels of creatine phosphokinase-MB isoenzyme (CK-MB), lactate dehydrogenase (LDH) and troponin-I were increased by USPIO. DEP induced a significant increase of CK-MB, LDH and troponin I levels in plasma. The cardiac levels of markers of oxidative stress including lipid peroxidation, reactive oxygen species and superoxide dismutase activity were increased by USPIO. Moreover, USPIO caused DNA damage in the heart. Likewise, DEP increased the markers of oxidative stress and induced DNA damage in the heart. CONCLUSION: We conclude that acute i.v. administration of USPIO caused thrombosis and cardiac oxidative stress and DNA damage. These findings provide novel insight into the pathophysiological effects of USPIO on cardiovascular homeostasis, and highlight the need for a thorough evaluation of their toxicity.

Short-term nose-only water-pipe (shisha) smoking exposure accelerates coagulation and causes cardiac inflammation and oxidative stress in mice.

BACKGROUND/AIM: Water-pipe smoking (WPS) has acquired worldwide popularity, and is disseminating particularly rapidly in Europe and North America. However, little is known about the short-term cardiovascular effects of WPS. METHODS: Presently, we assessed the short-term cardiovascular effects of nose-only exposure to mainstream WPS in BALB/c mice for 30 min/day for 5 consecutive days. Control mice were exposed to air. At the end of the exposure period, several cardiovascular endpoints were measured. RESULTS: WPS did not affect the number of leukocytes and the plasma concentrations of C-reactive protein, tumor necrosis factor α (TNFα) and interleukin-6 (IL-6). Likewise, plasma levels of lipid peroxidation (LPO), reduced glutathione (GSH) and catalase were not affected by WPS. By contrast, WPS aggravated in vivo thrombosis by shortening the thrombotic occlusion time in pial arterioles and venules. The number of circulating platelets was reduced by WPS suggesting the occurrence of platelet aggregation in vivo. Elevated concentrations of fibrinogen and plasminogen activator inhibitor-1 were seen after the exposure to WPS. Blood samples taken from mice exposed to WPS and exposed to adenosine diphosphate showed more platelet aggregation. The heart concentrations of IL-6 and TNFα were augmented by WPS. Likewise, heart levels of LPO, reactive oxygen species and the antioxidants catalase and GSH were increased by WPS. However, the systolic blood pressure and heart rate were not affected by WPS. CONCLUSION: It can be concluded that short-term exposure to WPS exerts procoagulatory effects and induce cardiac inflammation and oxidative stress. At the time point investigated, there was no evidence for blood inflammation or oxidative stress.

Reproductive toxicity to male mice of nose only exposure to water- pipe smoke.

BACKGROUND/AIMS: Water-pipe smoking (WPS) is popular in the Middle East and is starting to gain popularity in several Western countries as well. It is widely and erroneously perceived to be less harmful than other forms of tobacco use. The reproductive adverse effects of cigarette smoking have been studied before with conflicting results, but data on the possible adverse reproductive effects of WPS are lacking. Here, we assessed the effects of nose-only exposure to mainstream WPS generated by commercially available honey-flavored "moasel" tobacco in mice. METHODS: The duration of the session was 30 min/day for one month. Control mice were exposed to air. Twenty-four h after the last exposure, mice were killed and the testes and plasma removed for analysis. In testicular homogenates total protein, alkaline phosphatase activity, several indices of oxidative damage and Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) were quantified. The plasma concentrations of leptin, testosterone, estrogen and luteinizing hormone (LH) were also measured. Histological analysis of testes and lungs was also conducted. RESULTS: WPS caused statistically significant decreases in the plasma concentrations of leptin, testosterone, and LH, and in the concentrations of total protein and the antioxidant indices measured. A statistically non-significant decrease in VEGFR2 protein in the WPS--exposed mice compared to the control mice was also found. The body and testicular weights of mice exposed to WPS, as well as their testicular alkaline phosphatase activity and light microscopic histology, and plasma estrogen concentration were all not significantly affected by WPS. CONCLUSION: Further studies on the functional implications of these findings in mice exposed to WPS for longer durations are warranted.

Diesel Exhaust Particles Induce Impairment of Vascular and Cardiac Homeostasis in Mice: Ameliorative Effect of Emodin.

BACKGROUND/AIM: There is strong epidemiological and clinical evidence that components of the cardiovascular system are adversely affected by particulate air pollutants through the generation of inflammation and oxidative stress. Emodin (1,3,8-trihydroxy-6- methylanthraquinone), which is commonly found in the roots of rhubarb plant, has strong antioxidant and anti-inflammatory effects. However, its possible protective effect on the cardiovascular effect of particulate air pollutants has never been reported before. METHODS: We tested, in Tuck-Ordinary mice, the possible ameliorative effect of emodin on the acute (24h) cardiovascular effects of diesel exhaust particles (DEP, 1 mg/kg) or saline (control). Emodin (4 mg/kg) was administered intraperitoneally 1h before and 7h after pulmonary exposure to DEP. Twenty four h following DEP exposure, several cardiovascular endpoints were assessed. RESULTS: Emodin significantly prevented the increase of leukocyte (n=8, P<0.001) and erythrocyte (n=8, P<0.01) numbers caused by DEP. Likewise, emodin abrogated DEP-induced increase of heart tissue levels of interleukin 1ß (n=8, P<0.01) and tumour necrosis factor α (n=8, P<0.05), and significantly mitigated the change of the activities of antioxidant enzymes superoxide dismutase (n=8, P<0.001) and glutathione reductase (n=8, P<0.05). Emodin abolished the in vivo prothrombotic effect of DEP in pial arterioles (n=6, P<0.01) and venules (n=6, P<0.001). Similarly, emodin prevented platelet aggregation in vitro in whole blood (n=4-5, P<0.01), and the shortening of activated partial thromboplastin time (n=4, P<0.001) and prothrombin time (n=4, P<0.01) caused by DEP. CONCLUSION: We conclude that emodin treatment has consistently protected against DEP-induced impairment of vascular and cardiac homeostasis in mice. Our study provides experimental evidence that the use of functional food such as emodin, pending further studies, can be considered a useful agent and may have the potential to protect or mitigate the cardiovascular detrimental effects observed in people living in cities with high concentrations of particulate air pollution.

Pancreatic effects of diesel exhaust particles in mice with type 1 diabetes mellitus.

BACKGROUND/AIMS: Epidemiologically, diabetics are more prone to the adverse health effects of particulate air pollution than healthy individuals. We recently demonstrated an increased cardiovascular and respiratory susceptibility to diesel exhaust particles (DEP) in mice with type 1 diabetes. However, the pancreatic effects of DEP in healthy and diabetic mice are unknown. METHODS: Presently, we evaluated the pancreatic impact of DEP in healthy mice, and mice with streptozotocin-induced type 1 diabetes. Four weeks following induction of diabetes, mice were intratracheally instilled (i.t.) with either DEP (0.4 mg/kg) or saline, and several histological and biochemical endpoints were measured 24 h thereafter. RESULTS: Neither the histology nor the stain for apoptosis in the pancreatic islets and exocrine glands were affected by DEP. In diabetic mice exposed to saline, the islet cells showed cellular vacuolation and apoptotic islet cells (71.6 ± 2.6%). In diabetic mice exposed to DEP, a more marked decrease in the size and number of islet cells with cellular vacuolation along with a significant increase of apoptotic islet cells (79.1 ± 1.7 %, P<0.05) were observed. In diabetic mice, DEP increased significantly pancreatic amylase activity and markers of oxidative stress including 8-isoprostane, superoxide dismutase and reduced glutathione compared with either diabetic mice exposed to saline or non-diabetic mice exposed to DEP. Staining for inducible nitric oxide synthase (iNOS) in healthy mice exposed to either saline or DEP showed no staining in either pancreatic islets cells or acini. In saline-treated diabetic mice, a mild cytoplasmic staining for iNOS in some pancreatic islet cells was observed. Notably, in diabetic mice exposed to DEP, a marked cytoplasmic staining for iNOS in most pancreatic islet cells and some acinar cells was seen. CONCLUSION: We conclude that DEP caused detrimental effects on the pancreas of diabetic mice, and that oxidative stress is responsible, at least partially, for the observed effects.