Effects of dihydrolipoic acid (DHLA), α-lipoic acid. N-acetyl cysteine and ascorbate on xenobiotic-mediated methaemoglobin formation in human erythrocytes in vitro

α-Lipoic acid, dihydrolipoic acid (DHLA), N-acetyl cysteine and ascorbate were compared with methylene blue for their ability to attenuate and/or reduce methaemoglobin formation induced by sodium nitrite, 4-aminophenol and dapsone hydroxylamine in human erythrocytes. Neither α-lipoic acid, DHLA, N-acetyl cysteine nor ascorbate had any significant effects on methaemoglobin formed by nitrite, either from pre-treatment, simultaneous addition or post 30 min addition of the agents up to the 60 min time point, although N-acetyl cysteine did reduce methaemoglobin formation at 120 min (P<0.05). In all three treatment groups at 30, 60 and 120 min, there were no significant effects mediated by DHLA or N-acetyl cysteine on 4-aminophenol (1 mM)-mediated haemoglobin oxidation. Ascorbate caused marked significant reductions in 4-aminophenol methaemoglobin in all treatment groups at 30–120 min except at 30 min in the simultaneous addition group (P<0.0001). Neither α-lipoic acid, nor N-acetyl cysteine showed any effects on hydroxylamine-mediated methaemoglobin formation at 30 and 60 in all treatment groups. In contrast, DHLA significantly reduced hydroxylamine-mediated methaemoglobin formation at all three time points after pre-incubation and simultaneous addition (P<0.001), while ascorbate was ineffective. Compared with methylene blue, which was effective in reducing methaemoglobin formation by all three toxins (P<0.01), ascorbate was only highly effective against 4-aminophenol mediated methaemoglobin, whilst the DHLA-mediated attenuation of dapsone hydroxylamine-mediated methaemoglobin formation indicates a possible clinical application in high-dose dapsone therapy.     

Circulating levels of advanced glycation end products (AGE) and interleukin-6 (IL-6) are independent determinants of serum asymmetric dimethylarginine (ADMA) levels in patients with septic shock

There is a growing body of evidence that nitric oxide (NO) excess plays a central role in the pathogenesis of hypotension and organ failure in patients with septic shock. In addition, recently, asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase, has been shown to contribute to the regulation of vascular tone via modulation of NO generation in vivo. However, the kinetics and regulation of serum levels of ADMA in patients with septic shock are largely unknown. Since high mobility group box 1 (HMGB1)-receptor for advanced end products (RAGE) axis is supposed to be involved in the lethality in septic shock, we examined the correlations among serum levels of ADMA, endotoxin, interleukin-6 (IL-6), soluble form of RAGE (sRAGE) and RAGE ligands such as HMGB1 and advanced glycation end products (AGE) in septic shock patients. Fifteen septic shock patients (10 males and 15 females, mean age: 70.1 ± 8.5 years) and fifteen age- and sex-matched healthy volunteers were included in this study. The criteria of the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference were used for diagnosis of septic shock. All the subjects underwent a complete history and physical examination, determination of blood chemistries, including serum levels of ADMA, endotoxin, IL-6, HMGB1, AGE and sRAGE. Linear and multiple stepwise regression analysis were performed for the determinants of serum levels of ADMA. Serum levels of ADMA were significantly higher than those in healthy volunteers (0.98 ± 0.21 nmol/mL vs. 0.30 ± 0.05 nmol/mL, p < 0.0001). In univariate analysis, creatinine (p < 0.005), endotoxin (p < 0.001), IL-6 (p < 0.001), HMGB1 (p < 0.001), AGE (p < 0.001) and sRAGE (p < 0.001) were significantly associated with serum ADMA levels. After performing multivariate stepwise regression analyses, IL-6 (p = 0.001), AGE (p = 0.002) and creatinine (p = 0.013) still remained significant independently. The present study is the first demonstration that ADMA levels were significantly elevated in patients with septic shock and that serum IL-6, AGE and creatinine levels were independent determinants of ADMA in these patients. Given the harmful effects of NO excess in septic shock, ADMA levels may be increased as a counter-system against inflammation and oxidative stress in this life-threatening disorder.     

Biomarkers of nucleic acid oxidation, polymorphism in, and expression of, hOGG1 gene in styrene-exposed workers

This study investigated nucleic acid oxidation associated with styrene exposure, mRNA expression levels of hOGG1 gene and the role of the genetic polymorphism Ser326Cys of human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) in 60 styrene-exposed workers and 50 unexposed clerks. Biomarkers of exposure (styrene in blood, mandelic and phenylglyoxylic acids and 4-vinylphenol in urine) and urinary biomarkers of nucleic acid oxidation, namely 8-oxo-7,8-dihydro-2′-deoxyguanosine (U-8-oxodGuo), 8-oxo-7,8-dihydroguanosine (U-8-oxoGuo) and 8-oxo-7,8-dihydroguanine (U-8-oxoGua) were determined by liquid chromatography–tandem mass spectrometry. The levels of 8-oxodGuo adduct and 2′-deoxyguanosine (dGuo) were measured by HPLC in DNA from white blood cells (WBC). Genomic DNA and RNA from blood samples were used to characterize the Ser326Cys polymorphism and the mRNA expression levels of the hOGG1 gene, respectively, by PCR-based methods. Exposed workers showed lower values of 8-oxodGuo/10⁵ dGuo ratio in WBC-DNA but higher concentrations of U-8-oxoGuo compared to controls (p = 0.002 and p = 0.008, respectively, t-test for independent samples). In the whole group, all urinary biomarkers of nucleic acid oxidation correlated with both the sum of mandelic and phenylglyoxylic acids (rho > 0.33, p < 0.0001) and 4-vinylphenol (rho > 0.29, p < 0.001), whereas 8-oxodGuo/10⁵ dGuo in WBC showed a negative correlation with exposure parameters (rho < −0.24, p < 0.02). Subjects bearing the hOGG1 Ser/Ser genotype showed lower values of 8-oxodGuo/10⁵ dGuo in WBC than those with at least one variant Cys allele (0.34 ± 0.16 vs 0.45 ± 0.21, p = 0.008). In the subgroup of hOGG1 Ser/Ser subjects, laminators showed lower levels of WBC 8-oxodGuo/10⁵ dGuo ratio and significantly higher concentrations of U-8-oxoGua than controls (p = 0.07 and p = 0.01, respectively, t-test for independent samples). Interestingly, workers showed higher levels of hOGG1 expression compared to controls (p < 0.0005). Styrene exposure seems to be associated with oxidation damage to nucleic acids, particularly to RNA and with an induction of the BER system.     

Renal effects and vascular reactivity induced by Tityus serrulatus venom.

Tityus serrulatus, popularly known as yellow scorpion, is one of the most studied scorpion species in South America and its venom has supplied some highly active molecules. The effects of T. serrulatus venom upon the renal physiology in human showed increased renal parameters, urea and creatinine. However, in perfused rat kidney the effects were not tested until now. Isolated kidneys from Wistar rats, weighing 240-280 g, were perfused with Krebs-Henseleit solution containing 6% (g weight) of previously dialysed bovine serum albumin. The effects of T. serrulatus venom were studied on the perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), sodium tubular transport (%TNa+), potassium tubular transport (%TK+) and chloride tubular transport (%TCl-). Tityus serrulatus venom (TsV; 10 microg/mL) was added to the system 30 min after the beginning of each experiment (n=6). This 30 min period was used as an internal control. The mesenteric bed was perfused with Krebs solution kept warm at 37 degrees C by a constant flow (4 mL/min), while the variable perfusion pressure was measured by means of a pressure transducer. The direct vascular effects of TsV (10 microg/mL/min; n=6), infused at a constant rate (0.1 mL/min), were examined and compared to the infusion of the vehicle alone at the same rate. TsV increased PP (PP30'=127.8+/-0.69 vs PP60'=154.2+/-14 mmHg*, *p<0.05) and RVR (RVR30'=6.29+/-0.25 vs RVR60'=8.03+/-0.82 mmHg/mLg(-1)min(-1)*, *p<0.05), decreased GFR (GFR30'=0.58+/-0.02 vs GFR60'=0.46+/-0.01mLg(-1)min(-1)*, *p<0.05) and UF (UF30'=0.135+/-0.001 vs UF60'=0.114+/-0.003mLg(-1)min(-1)*, *p<0.05). Tubular transport was not affected during the whole experimental period (120 min). On the other hand, the infusion of TsV (10 microg/mL/min) increased the basal perfusion pressure of isolated arteriolar mesenteric bed (basal pressure: 74.17+/-3.42 vs TsV 151.8+/-17.82 mmHg*, *p<0.05). TsV affects renal haemodynamics probably by a direct vasoconstrictor action leading to decreased renal flow.     

Correlates of arterial stiffness in an ageing population: Role of asymmetric dimethylarginine

A number of previous investigators have demonstrated that arterial augmentation index (AIx), a measure of apparent arterial stiffness, reflects in part vascular endothelial function, and that AIx is modulated by nitric oxide (NO) responses. We evaluated AIx in a population of 253 ageing subjects (mean age 63.4 ± 6 (standard deviation, SD) years) and its relationship to (i) plasma levels of asymmetric dimethylarginine (ADMA), a marker and mediator of vascular endothelial dysfunction and (ii) the ratio of ADMA to its non-metabolised enantiomer symmetric dimethylarginine (SDMA), an inverse index of ADMA metabolic clearance. Evaluation was performed by univariate followed by multivariate analyses.On multivariate analyses, both ADMA (β = 0.16, p = 0.01) and ADMA:SDMA (β = 0.21, p < 0.001) ratio were significant direct correlates of AIx. Other significant correlates of AIx on multivariate analysis were: use of angiotensin-converting enzyme inhibitors/angiotensin-receptor blockers (ACEi/ARB) (β = −0.24, p = 0.004), smoking history (β = 0.15, p = 0.007), male gender (β = −0.38, p < 0.001), creatinine clearance (CrCL) (β = −0.25, p < 0.001), and history of hypertension (β = 0.17, p = 0.04).We conclude that (1) endothelial dysfunction engendered by impairment of NO synthesis may represent the basis for increased arterial stiffness in ageing individuals and (2) the fundamental biochemical anomaly may be impairment of ADMA clearance. These pathophysiological factors are likely to be relevant to optimize therapy to ameliorate disorders of arterial compliance in the ageing population.     

Performance-enhancing and thermoregulatory effects of intracerebroventricular dopamine in running rats

To assess the role of central dopamine on metabolic rate, heat balance and running performance, 2.0 µL of 5 × 10^− 3 M dopamine solution (DA) or 0.15 M NaCl (SAL) was intracerebroventricularly injected in Wistar rats 1 min before running on a motor-driven treadmill, according to a graded exercise protocol, until fatigue. Oxygen consumption (VO₂) and body temperature (T_b) were recorded at rest, during exercise, and after 30 min of recovery. DA induced a marked increase in workload (~ 45%, p < 0.05). At fatigue point, DA-injected rats attained ~29% higher maximum oxygen consumption (VO_2max) and ~0.75 °C higher T_b than SAL-injected rats. Despite the higher VO_2max and T_b attained during exercise, DA-treated rats reached VO₂ basal values within the same recovery period and dissipated heat ~33% faster than SAL-treated rats (p < 0.05). The mechanical efficiency loss rate was ~40% lower in DA than in SAL-treated rats (p < 0.05), however, the heat storage was ~35% higher in the DA group (p < 0.05). Our results demonstrate that increased DA availability in the brain has a performance-enhancing effect, which is mediated by improvements in the tolerance to heat storage and increases in the metabolic rate induced by graded exercise. These data provide further evidence that central activation of dopaminergic pathways plays an important role in exercise performance.     

Differential Effects of a Dual Orexin Receptor Antagonist (SB-649868) and Zolpidem on Sleep Initiation and Consolidation,SWS, REM Sleep,and EEG Power Spectra in a Model of Situational Insomnia

Orexins have a role in sleep regulation, and orexin receptor antagonists are under development for the treatment of insomnia. We conducted a randomised, double-blind, placebo-controlled, four-period crossover study to investigate the effect of single doses of the dual orexin receptor antagonist SB-649868 (10 or 30 mg) and a positive control zolpidem (10 mg), an allosteric modulator of GABA_A receptors. Objective and subjective sleep parameters and next-day performance were assessed in 51 healthy male volunteers in a traffic noise model of situational insomnia. Compared with placebo, SB-649868 10 and 30 mg increased total sleep time (TST) by 17 and 31 min (p<0.001), whereas after zolpidem TST was increased by 11.0 min (p=0.012). Wake after sleep onset was reduced significantly by 14.7 min for the SB–6489698 30 mg dose (p<0.001). Latency to persistent sleep was significantly reduced after both doses of SB–6489698 (p=0.003), but not after zolpidem. Slow wave sleep (SWS) and electroencephalogram (EEG) power spectra in non-REM sleep were not affected by either dose of SB-640868, whereas SWS (p< 0.001) and low delta activity (<=1.0 Hz) were increased, and 2.25–11.0 Hz activity decreased after zolpidem. REM sleep duration was increased after SB-649868 30 mg (p=0.002) and reduced after zolpidem (p=0.049). Latency to REM sleep was reduced by 20.1 (p=0.034) and 34.0 min (p<0.001) after 10 and 30 mg of SB-649868. Sleep-onset REM episodes were observed. SB-649868 was well tolerated. This dual orexin receptor antagonist exerts hypnotic activity, with effects on sleep structure and the EEG that are different from those of zolpidem.     

Characterization of a double‐hit murine model of acute respiratory distress syndrome

The aim of the present study was to characterize a murine model of acute respiratory distress syndrome (ARDS) abiding by the Berlin definition of human ARDS and guidelines for animal models of ARDS. To this end, C57BL/6NCrl mice were challenged with lipopolysaccharide (LPS; 15 mg/kg, i.p.) followed 18 h later by injection of oleic acid (OA; 0.12 mL/kg, i.v.). Controls received saline injection at both time points. Haemodynamics were monitored continuously. Arterial blood gas analyses were performed just before and every 30 min after OA challenge. Ninety minutes after OA challenge, the chest of mice was scanned using micro‐computed tomography (CT). Cytokine concentrations were measured in plasma samples. Lungs were harvested 90 min after OA challenge for histology, immunohistochemistry, lung weight measurements and tissue cytokine detection. A histological lung injury score was determined. Eighteen hours after LPS challenge, mice exhibited a severe systemic inflammatory response syndrome. Oxygenation declined significantly after OA injections (P_ao ₂/F_io ₂ 283 ± 73 and 256 ± 71 mmHg at 60 and 90 min, respectively; P < 0.001). Bilateral patchy infiltrates were present on the micro‐CT scans. Histology revealed parenchymal damage with accumulation of polymorphonuclear neutrophils, intra‐alveolar proteinacous debris and few hyaline membranes. The lung wet : dry ratio indicated damage to the alveolar capillary membrane. Cytokine patterns evidenced a severe local and systemic inflammatory state in plasma and lung tissue. In conclusion, the described two‐hit model of ARDS shows a pathological picture of ARDS closely mimicking human ARDS according to the Berlin definition and may facilitate interpretation of prospective experimental results.     

Assessment of biochemical and hematological parameters in rats injected with Tityus serrulatus scorpion venom

The aim of this work was to evaluate the hematological changes induced by Tityus serrulatus venom (TsV). Blood of Wistar rats was collected 0.5, 2, 6 and 24 h after i.p. injection of TsV (0.5 mg/kg) or saline (controls). Two additional groups were injected with 0.67 mg/kg and 0.25 mg/kg of TsV and the blood was collected after 0.5 and 2 h, respectively. The results showed an increase on hematocrit (Ht), red blood cells (RBC) count, hemoglobin concentration (Hb), albumin and total protein, mainly 2–6 h after envenoming. Increase in serum activities of amylase, creatine kinase and aspartate aminotransferase were also observed, indicating tecidual damages. Hyperglycemia was observed at all times analyzed, as a consequence of catecholamine release. No significant changes were detected in the urea, [Na⁺] and [Ca²⁺], but an increase of [Mg²⁺], [K⁺] and conductivity was observed. TsV induced a reduction of erythrocytes osmotic fragility as consequence of dehydration and increase in plasma electrolytes concentration, as evidenced by its higher conductivity. This study demonstrated that TsV is able to induce severe hematological changes, that appear within the first hours after envenoming, justifying the seeking of medical attention as soon as possible to avoid worsening of clinical symptoms.     

Does Dietary Deoxynivalenol Modulate the Acute Phase Reaction in Endotoxaemic Pigs?—Lessons from Clinical Signs,White Blood Cell Counts,and TNF-Alpha

We studied the interaction between deoxynivalenol (DON)-feeding and a subsequent pre- and post-hepatic immune stimulus with the hypothesis that the liver differently mediates the acute phase reaction (APR) in pigs. Barrows (n = 44) were divided into a DON-(4.59 mg DON/kg feed) and a control-diet group, surgically equipped with permanent catheters pre- (V. portae hepatis) and post-hepatic (V. jugularis interna) and infused either with 0.9% NaCl or LPS (7.5 µg/kg BW). Thus, combination of diet (CON vs. DON) and infusion (CON vs. LPS, jugular vs. portal) created six groups: CON_CON_jug.-CON_por., CON_CON_jug.-LPS_por., CON_LPS_jug.-CON_por., DON_CON_jug.-CON_por., DON_CON_jug.-LPS_por., DON_LPS_jug.-CON_por.. Blood samples were taken at −30, 15, 30, 45, 60, 75, 90, 120, 150, 180 min relative to infusion and analyzed for leukocytes and TNF-alpha. Concurrently, clinical signs were scored and body temperature measured during the same period. LPS as such induced a dramatic rise in TNF-alpha (p < 0.001), hyperthermia (p < 0.01), and severe leukopenia (p < 0.001). In CON-fed pigs, an earlier return to physiological base levels was observed for the clinical complex, starting at 120 min post infusionem (p < 0.05) and persisting until 180 min. DON_LPS_jug.-CON_por. resulted in a lower temperature rise (p = 0.08) compared to CON_LPS_jug.-CON_por.. In conclusion, APR resulting from a post-hepatic immune stimulus was altered by chronic DON-feeding.     

Safety assessment of a modified acetolactate synthase protein (GM-HRA) used as a selectable marker in genetically modified soybeans

Acetolactate synthase (ALS) enzymes have been isolated from numerous organisms including soybeans (Glycine max; GM-ALS) and catalyze the first common step in biosynthesis of branched chain amino acids. Expression of an ALS protein (GM-HRA) with two amino acid changes relative to native GM-ALS protein in genetically modified soybeans confers tolerance to herbicidal active ingredients and can be used as a selectable transformation marker. The safety assessment of the GM-HRA protein is discussed. Bioinformatics comparison of the amino acid sequence did not identify similarities to known allergenic or toxic proteins. In vitro studies demonstrated rapid degradation in simulated gastric fluid (<30 s) and intestinal fluid (<1 min). The enzymatic activity was completely inactivated at 50 °C for 15 min demonstrating heat lability. The protein expressed in planta is not glycosylated and genetically modified soybeans expressing the GM-HRA protein produced similar protein/allergen profiles as its non-transgenic parental isoline. No adverse effects were observed in mice following acute oral exposure at a dose of at least 436 mg/kg of body weight or in a 28-day repeated dose dietary toxicity study at doses up to 1247 mg/kg of body weight/day. The results demonstrate GM-HRA protein safety when used in agricultural biotechnology.     

Time Course Analysis of the Effects of Botulinum Neurotoxin Type A on Pain and Vasomotor Responses Evoked by Glutamate Injection into Human Temporalis Muscles

The effect of botulinum neurotoxin type A (BoNTA) on glutamate-evoked temporalis muscle pain and vasomotor responses was investigated in healthy men and women over a 60 day time course. Subjects participated in a pre-BoNTA session where their responses to injection of glutamate (1 M, 0.2 mL) and saline (0.2 mL) into the temporalis muscles were assessed. On Day 1, BoNTA (5 U) was injected into one temporalis muscle and saline into the contralateral temporalis muscle, in a randomized order. Subjects then received intramuscular injections of glutamate (1 M, 0.2 mL) into the left and right temporalis muscles at 3 h and subsequently 7, 30 and 60 days post-injection of BoNTA. Pain intensity, pain area, and neurogenic inflammation (skin temperature and skin blood perfusion) were recorded. Prior to BoNTA treatment, glutamate evoked significantly greater pain and vasomotor reactions (P < 0.001) than saline. BoNTA significantly reduced glutamate-evoked pain intensity (P < 0.05), pain area (P < 0.01), skin blood perfusion (P < 0.05), and skin temperature (P < 0.001). The inhibitory effect of BoNTA was present at 3 h after injection, peaked after 7 days and returned to baseline by 60 days. Findings from the present study demonstrated a rapid action of BoNTA on glutamate-evoked pain and neurogenic inflammation, which is in line with animal studies.     

Race differences in factors relating to smoking initiation

To investigate race differences in retrospectively-reported early smoking experiences, we studied African-American (n = 48) and Caucasian (n = 155) current smokers who participated in a study designed to identify phenotypic and genotypic factors associated with smoking. Compared with Caucasian smokers, African-American smokers were less educated (mean ± s.e.m.: 13.3 ± 0.25 vs. 14.3 ± 0.16; p < .01), had higher BMI (28.9 ± 1.06 vs. 26.7 ± 0.52; p < .05), and smoked significantly fewer cigarettes/day (14.1 ± 1.00 vs. 18.4 ± 0.74; p < .01). Ninety percent of African-American smokers consumed menthol cigarettes, as opposed to 25% of Caucasian smokers. African-American smokers were significantly older than Caucasian smokers upon initial smoking experimentation (17.4 ± 1.1 vs. 14.7 ± 0.3; p < .05) and onset of regular smoking (19.7 ± 0.9 vs. 17.4 ± 0.4; p < .05). African-American smokers were significantly more likely than Caucasian smokers to endorse global pleasurable sensations (48% vs. 30%; p < .05), “pleasurable rush or buzz” (62% vs. 43%; p < .05), and “relaxing” (45% vs. 27%; p < .05) as early experiences with smoking, whereas Caucasian smokers were marginally more likely to report dizziness and difficulty inhaling (61% vs. 45%; p < .10 and 48% vs. 31%; p < .10, respectively). Caucasian smokers were significantly more likely to endorse friends (6.9 ± 0.2 vs. 4.8 ± 0.4; p < .0001) and “perk me up” (4.2 ± 0.3 vs. 3.1 ± 0.4; p < .05) and marginally more likely to endorse buzz (4.2 ± 0.2 vs. 3.4 ± 0.5; p < .10) as reasons for starting to smoke. Further research is needed to determine the relative contributions of genetic, developmental, and socio-cultural factors to these findings.     

Mechanisms of p,p′-DDE-induced apoptosis in human peripheral blood mononuclear cells

p,p′-Dichlorodiphenyldichloroethylene (DDE), the most stable metabolite of organochlorine insecticide p,p′-dichlorodiphenyltrichloroethane (DDT), has been detected in human populations living in malaria-endemic areas of México where this insecticide was used. DDE induces apoptosis in peripheral blood mononuclear cells (PMBC); however, the molecular mechanism of cell death induced by this compound is poorly understood. In the present study, PBMC isolated from healthy individuals (not exposed to DDE) were incubated in the presence of increasing concentrations of p,p′-DDE (0–80 μg/ml) over time. When PBMC were treated with low p,p′-DDE concentration (10 μg/ml) an antioxidant response and biomarkers of inflammation were induced, indicating a pro-inflammatory state. Moreover, when PBMC were treated with high p,p′-DDE concentration (80 μg/ml) several apoptotic biochemical events were triggered, such as activation of caspase-8, Bid, caspase-9 and caspase-3, as well as degradation of PARP and ubiquitination. The results described in this study show a possible inflammatory condition and the involvement of both extrinsic and intrinsic pathways in the induction of apoptosis in DDE-treated PBMC.     

Cypermethrin-induced histopathological and biochemical changes in Nile tilapia (Oreochromis niloticus), and the protective and recuperative effect of ascorbic acid

The objective of this study was to investigate the effects of ascorbic acid on the toxicity of cypermethrin's on histopathological lesions in tissues and protein, glycogen levels in Oreochromis niloticus. Nile tilapia was exposed to 0.22 and 0.44 μg/l cypermethrin + control diet, 0.22 and 0.44 μg/l cypermethrin + ascorbic acid supplemented diet for 20 days. The fish were allowed recuperation period of 15 days in pesticide-free water and fed with ascorbic acid suplementation diet. In light microscopic investigation, histopathological lesions were observed in the gill, liver and kidney. The severity of lesions accreted depending on increased pesticide concentration and control diet. Some of the lesions were reversible or at least were less pronounced after recuperation period. Protein levels decreased in some groups after treatment period according to control groups (p < 0.05). The highest depletions in liver, muscle and gill protein levels were found in 0.44 μg/l cypermethrin + ascorbic acid supplemented diet group (62.23%), in 0.22 μg/l cypermethrin + control diet group (53.12%) and in 0.44 μg/l cypermethrin + control diet group (61.87%) after 10 days, respectively. These levels increased at the end of the recuperation period. The highest depletion in liver glycogen levels was found in 0.22 μg/l cypermethrin + control diet group (50.50%) after 10 days (p < 0.05). At the end of recuperation period, there was no difference between the groups (except 0.22 μg/l cypermethrin + ascorbic acid supplemented diet group) and controls. The decrease of muscle glycogen, except 0.22 μg/l cypermethrin + ascorbic acid supplemented diet group, was recorded at the end of 10 and 20 days. In the recuperation period, an increase was observed at all groups. These results revealed that the histopathology, protein and glycogen can work as good indicators of stress of a toxicant on fish. Ascorbic acid serves fish as an antitoxic agent against pesticide toxicity.     

Pharmacological analysis of the neutrophil migration induced by D. rostrata lectin: Involvement of cytokines and nitric oxide

In the present study, we investigated the involvement of resident cell and inflammatory mediators in the neutrophil migration induced by chemotactic activity of a glucose/mannose-specific lectin isolated from Dioclea rostrata seeds (DrosL). Rats were injected i.p. with DrosL (125–1000 μg/cavity), and at 2–96 h thereafter the leukocyte counts in peritoneal fluid were determined. DrosL-induced a dose-dependent neutrophil migration accumulation, which reached maximal response at 24 h after injection and declines thereafter. The carbohydrate ligand nearly abolished the neutrophil influx. Pre-treatment of peritoneal cavities with thioglycolate which increases peritoneal macrophage numbers, enhanced neutrophil migration induced by DrosL by 303%. However, the reduction of peritoneal mast cell numbers by treatment of the cavities with compound 48/80 did not modify DrosL-induced neutrophil migration. The injection into peritoneal cavities of supernatants from macrophage cultures stimulated with DrosL (125, 250 and 500 μg/ml) induced neutrophil migration. In addition, DrosL treatment induced cytokines (TNF-α, IL-1β and CINC-1) and NO release into the peritoneal cavity of rats. Finally, neutrophil chemotaxis assay in vitro showed that the lectin (15 and 31 μg/ml) induced neutrophil chemotaxis by even 180%. In conclusion, neutrophil migration induced by D. rostrata lectin occurs by way of the release of NO and cytokines such as IL-1β, TNF-α and CINC-1.     

Lactococcus lactis subsp. cremoris FC alleviates symptoms of colitis induced by dextran sulfate sodium in mice

Probiotics have been used to treat human gastrointestinal inflammations including inflammatory bowel disease (IBD). However, the exact mechanisms by which probiotics act to protect against intestinal inflammation have yet to be fully elucidated. The aim of this study was to evaluate anti-inflammatory effects of Lactococcus lactis subsp. cremoris FC using in vivo and in vitro inflammation models. Colitis was induced in C57BL/6 mice by administration of 3% dextran sulfate sodium to drinking water. In the cellular level assessment, a gut inflammation model with the co-culture system consisting Caco-2 cells and RAW264.7 cells stimulated by LPS was used. Administration of L. lactis subsp. cremoris FC significantly ameliorated shortening of colon length and histological score of the colon in DSS-induce colitis mice. In addition, the treatment of L. lactis subsp. cremoris FC improved the aberrant mRNA expression in inflamed tissue near to control level through notable suppression of TNF-α (P < 0.05), IFN-γ (P < 0.05), IL-6, iNOS, and MIP-2 mRNA expression. In addition, in a gut inflammation model, treatment with L. lactis subsp. cremoris FC resulted in significant down-regulation of IL-8 mRNA expression in Caco-2 cells and inhibition of NF-κB nuclear translocation in RAW264.7 cells. Our findings indicate that administration of L. lactis subsp. cremoris FC improves negative effects of DSS-induced colitis in mice through the inhibition of inflammatory cell infiltration.     

Hypotensive effects of diltiazem to normals and essential hypertensives

Summary The hypotensive effect of acute and long-term, intravenous and oral administration of the calcium antagonist, diltiazem, was investigated in 8 normotensive volunteers and 55 patients with essential hypertension. Diltiazem i.v. infusion of 45 mg/h (0.5 mg/min, then 1.0 mg/min, each for 30 min rapidly decreased both blood pressure (BP) from 164±22/98±8 to 144±15/86±9 mmHg (mean±SD) and total peripheral resistance from 32.6±8.4 to 25.3±5.4 mmHg/l/min (p<0.001), and increased stroke volume from 58.2±9.5 to 64.2±8.6 ml/beat (p<0.05). It altered neither heart rate nor cardiac output in the hypertensives (n=10). Oral diltiazem 60 mg rapidly decreased BP from 155±10/103±6 to 142±12/90±8 mmHg after 3 hours (p<0.01/p<0.001) in hypertensives (n=8), but not in normotensives (n=8). Diltiazem 90 mg p.o. decreased BP from 157±15/102±9 to 129±13/83±8 mmHg (p<0.01) in hypertensives (n=15), and reduced the heart rate from 71±8 to 65±8 beats/min (p<0.01). The drug did not change plasma renin activity either in normotensives or hypertensives. The fall in diastolic BP was correlated with the plasma diltiazem concentration (r=0.910, n=6, p<0.05). Long-term treatment with diltiazem 30mg t.d.s. decreased BP from 163±12/104±8 to 145±9/88±9 mmHg (p<0.001, n=13), and 60mg t.d.s. decreased BP from 169±15/102±6 to 148±13/87±8 mmHg (p<0.001, n=8), and significantly reduced the heart rate (p<0.01) in hypertensives. Thus, the hypotensive action of diltiazem, which is due to arterial dilatation, is effective, either on intravenous or oral administration, during acute and long-term treatment of essential hypertension.     

Effect of lidocaine administration at the nucleus locus coeruleus level on lateral hypothalamus-induced antinociception in the rat

Several lines of evidence have shown that stimulation or inactivation of lateral hypothalamus (LH) produces antinociception. In this study, we assessed the role of nucleus locus coeruleus (LC) in antinociceptive response induced by LH stimulation or inactivation in the rat. The cholinergic agonist carbachol (125 nmol/0.5 μl saline) or lidocaine (2%; 0.5 μl) was unilaterally microinjected into the LH with the LC inactivation concurrently. Antinociceptive responses were obtained by tail-flick test and represented as maximal possible effect (MPE) at 5, 10, 15, 20, 30 and 60 min after drug administration. The results showed that microinjection of carbachol into the LH significantly induced antinociception at 5 and 10 min (p < 0.001). This effect was significantly blocked by microinjection of lidocaine into the LC. On the other hand, microinjection of lidocaine into LH-induced antinociception at 5 (p < 0.01) and 10 (p < 0.05) min after administration. However, inactivation of the LC following the LH inactivation increased MPE at 5 min after injection. These findings support the conclusion that antinociception produced by LH stimulation or inactivation involves two separate mechanisms. It seems that analgesic response induced by LH stimulation is mediated in part by the subsequent activation of spinally projecting noradrenergic neurons in the LC cell group.