Comparative Genome Analysis and Global Phylogeny of the Toxin Variant Clostridium difficile PCR Ribotype 017 Reveals the Evolution of Two Independent Sublineages.

The diarrheal pathogen Clostridium difficile consists of at least six distinct evolutionary lineages. The RT017 lineage is anomalous, as strains only express toxin B, compared to strains from other lineages that produce toxins A and B and, occasionally, binary toxin. Historically, RT017 initially was reported in Asia but now has been reported worldwide. We used whole-genome sequencing and phylogenetic analysis to investigate the patterns of global spread and population structure of 277 RT017 isolates from animal and human origins from six continents, isolated between 1990 and 2013. We reveal two distinct evenly split sublineages (SL1 and SL2) of C. difficile RT017 that contain multiple independent clonal expansions. All 24 animal isolates were contained within SL1 along with human isolates, suggesting potential transmission between animals and humans. Genetic analyses revealed an overrepresentation of antibiotic resistance genes. Phylogeographic analyses show a North American origin for RT017, as has been found for the recently emerged epidemic RT027 lineage. Despite having only one toxin, RT017 strains have evolved in parallel from at least two independent sources and can readily transmit between continents.

In vitro and in vivo therapeutics of ß-thujaplicin on LPS-induced inflammation in macrophages and septic shock in mice.

ß-thujaplicin, an active constituent from Chamaecyparis obtusa, has been shown to have acaricidal and antimicrobial effects. Very few studies have focused on the potential of the anti-inflammatory effect of ß-thujaplicin. Moreover, its capability of inhibiting inflammatory mediators e.g. TNF-a gene transcription, nitric oxide (NO) and prostaglandin E2, remains unknown. Besides those molecular mechanisms behind the anti-inflammatory effect of ß-thujaplicin, solid proof of its effectiveness in vivo has not yet been studied. In our study, in vitro effects of ß thujaplicin were verified on RAW 264.7 macrophages which were stimulated by LPS. Indomethacin was used as a positive control. The inducible NO production after stimulation was measured by Griess reagent. PGE2, IL-6 and TNF-α were measured by ELISA methods. Protein expressions of iNOS, COX2, and NF-κB were evaluated by Western blotting. Septic ICR mice were administered 20 mg/kg of LPS and then the mortality rate was monitored. Within the concentration range which was devoid of cytotoxicty, ß-thujaplicin exhibited a clear dose-dependent inhibition on LPS-induced NO production. Furthermore, ß-thujaplicin inhibited LPS-induced PGE2, IL-6, and TNF-α production as well as iNOS, COX2, and NF- κB protein expression more substantially potent than indomethacin. In agreement with the in vitro study, ß-thujaplicin was shown to be effective in vivo for inhibiting LPS-induced NO and TNF-α production and a significant decrease in mortality rate of mice suffering from septic shock was observed. This study demonstrates the potential of ß-thujaplicin in treatment of inflammation and sepsis. These effects occur through an efficient blockage of TNF-α and iNOS production. ß-thujaplicin efficacy is comparable to that of indomethacin thus it can be a substitution but bear less depletion of PGE2, making this compound very promising in clinical applications. ß-thujaplicin, an active constituent from Chamaecyparis obtusa, has been shown to have acaricidal and antimicrobial effects. Very few studies have focused on the potential of the anti-inflammatory effect of ß-thujaplicin. Moreover, its capability of inhibiting inflammatory mediators e.g. TNF-alpha gene transcription, nitric oxide (NO) and prostaglandin E2, remains unknown. Besides those molecular mechanisms behind the anti-inflammatory effect of ß-thujaplicin, solid proof of its effectiveness in vivo has not yet been studied. In our study, in vitro effects of ß-thujaplicin were verified on RAW 264.7 macrophages which were stimulated by LPS. Indomethacin was used as a positive control. The inducible NO production after stimulation was measured by Griess reagent. PGE2, IL-6 and TNF-alpha were measured by ELISA methods. Protein expressions of iNOS, COX2, and NF-kB were evaluated by Western blotting. Septic ICR mice were administered 20 mg/kg of LPS and then the mortality rate was monitored. Within the concentration range which was devoid of cytotoxicty, ß-thujaplicin exhibited a clear dose-dependent inhibition on LPS-induced NO production. Furthermore, ß-thujaplicin inhibited LPS-induced PGE2, IL-6, and TNF-alpha production as well as iNOS, COX2, and NF-kB protein expression more substantially potent than indomethacin. In agreement with the in vitro study, ß-thujaplicin was shown to be effective in vivo for inhibiting LPS-induced NO and TNF-alpha production and a significant decrease in mortality rate of mice suffering from septic shock was observed. This study demonstrates the potential of ß-thujaplicin in treatment of inflammation and sepsis. These effects occur through an efficient blockage of TNF-alpha and iNOS production. ß-thujaplicin efficacy is comparable to that of indomethacin thus it can be a substitution but bear less depletion of PGE2, making this compound very promising in clinical applications.

Cingulate circuits are associated with escalation of heroin use and naloxone-induced increases in heroin self-administration.

Opioid use disorder (OUD) is defined as a compulsion to seek and take opioids, loss of control over intake and the development of a negative emotional state when access to opioids is denied. Using functional magnetic resonance imaging (fMRI) data in a rat model of OUD, we demonstrate that the escalation of heroin self-administration (SA) and the increased heroin SA following an injection of an opioid receptor antagonist (naloxone) are associated with changes in distinct brain circuits, centered on the cingulate cortex (Cg). Here, SA escalation score was negatively associated with changes in resting state functional connectivity (rsFC) between the Cg and the dorsal striatum. Conversely, increased heroin SA following naloxone injection, was associated with increased connectivity between the Cg and the extended amygdala and hypothalamus. Naloxone-induced increased SA was also positively associated with changes in the amplitude of low frequency fluctuations within the Cg, a measure of spontaneous neuronal activity. Characterizing the distinct brain circuit and behavior changes associated with different facets of addiction increases our understanding of OUD and may provide insight into addiction prevention and treatment.

Toll-like receptor 2 deficiency improves insulin sensitivity and hepatic insulin signalling in the mouse.

AIMS/HYPOTHESIS: Substantial evidence suggests a link between elevated inflammation and development of insulin resistance. Toll-like receptor 2 (TLR2) recognises a large number of lipid-containing molecules and transduces inflammatory signalling in a variety of cell types, including insulin-responsive cells. Considering the contribution of the fatty acid composition in TLR2-depedent signalling, we hypothesised that the inflammatory signals transduced by TLR2 contribute to insulin resistance. METHODS: Mice deficient in TLR2 were used to investigate the in vivo roles of TLR2 in initiating and maintaining inflammation-associated insulin resistance and energy homeostasis. RESULTS: We first recapitulated the observation with elevated expression of TLR2 and inflammatory cytokines in white adipose tissue and liver of ob/ob mice. Aged or high-fat-fed TLR2-deficient mice were protected from obesity and adipocyte hypertrophy compared with wild-type mice. Moreover, mice lacking TLR2 exhibited improved glucose tolerance and insulin sensitivity regardless of feeding them regular chow or a high-fat diet. This is accompanied by reductions in expression of inflammatory cytokines and activation of extracellular signal-regulated kinase (ERK) in a liver-specific manner. The attenuated hepatic inflammatory cytokine expression and related signalling are correlated with increased insulin action specifically in the liver in TLR2-deficient mice, reflected by increased insulin-stimulated protein kinase B (Akt) phosphorylation and IRS1 tyrosine phosphorylation and increased insulin-suppressed hepatocyte glucose production. CONCLUSIONS/INTERPRETATION: The absence of TLR2 attenuates local inflammatory cytokine expression and related signalling and increases insulin action specifically in the liver. Thus, our work has identified TLR2 as a key mediator of hepatic inflammation-related signalling and insulin resistance.

Narrowband ultraviolet B phototherapy for patients with refractory uraemic pruritus: a randomized controlled trial.

BACKGROUND: Pruritus is very common in uraemic patients, but the treatment remains challenging. Studies regarding narrowband ultraviolet B (NB-UVB) phototherapy for uraemic pruritus are rare. OBJECTIVES: To investigate whether or not NB-UVB phototherapy is an effective treatment for uraemic pruritus. METHODS: We conducted a single-blind, randomized, controlled trial for patients with refractory uraemic pruritus. The treatment group received NB-UVB phototherapy three times per week for 6 weeks. The dose of NB-UVB started from 210 mJ cm(-2) and was increased by 10% each time. The control group received time-matched exposures to long-wave UVA radiation. A visual analogue scale (VAS) score was evaluated weekly for pruritus intensity for 12 weeks. The characteristics of pruritus were also assessed by a questionnaire at baseline and after 6 weeks of phototherapy. RESULTS: Both the NB-UVB and control groups had significant and comparable improvement in the pruritus intensity VAS scores during the period of phototherapy and follow-up. Compared with the control group, the NB-UVB group showed a significant improvement in the involved body surface area affected by pruritus (P = 0·006), but not in sleep quality. More detailed regression and estimating analysis revealed that the patients in the NB-UVB group had lower pruritus intensity scores at week 6, week 10 and week 12. This may indicate a beneficial difference at certain time points, but the effect seems marginal. CONCLUSIONS: NB-UVB phototherapy does not show a significant effect in reducing pruritus intensity compared with a control group for refractory uraemic pruritus. Further studies are warranted.

Exposure to traffic exhausts and oxidative DNA damage.

AIMS: To assess the relations between exposure to traffic exhausts and indicators of oxidative DNA damage among highway toll station workers. METHODS: Cross-sectional study of 47 female highway toll station workers exposed to traffic exhausts and 27 female office workers as a reference group. Exposure assessment was based on average and cumulative traffic density and a biomarker of exposure, urinary 1-hydroxypyrene-glucuronide (1-OHPG). Urinary 8-hydroxydeoxyguanosine (8-OHdG) was used as a biomarker of oxidative DNA damage. Plasma nitric oxide (NO) was measured as an indicator of oxidative stress related to traffic exhaust exposure. RESULTS: The mean concentration of urinary 8-OHdG was substantially higher among the exposed non-smokers (13.6 microg/g creatinine) compared with the reference non-smokers (7.3 microg/g creatinine; difference 6.3, 95% CI 3.0 to 9.6). The mean concentration of NO among the exposed (48.0 micromol/l) was also higher compared with the reference non-smokers (37.6 micromol/l; difference 10.4, 95% CI -0.4 to 21.2). In linear regression adjusting for confounding, a change in log(8-OHdG) was statistically significantly related to a unit change in log(1-OHPG) (beta = 0.372, 95% CI 0.081 to 0.663). CONCLUSIONS: Results indicate that exposure to traffic exhausts increases oxidative DNA damage. Urinary 8-OHdG is a promising biomarker of traffic exhaust induced oxidative stress.

Elevated extracellular glutamate levels increased the formation of hydroxyl radical in the striatum of anesthetized rat.

Results from various in vitro experiments have indicated that excitotoxicity and oxidative stress are two interrelated major mechanisms in causing neuronal damage in brain disorders such as cerebral ischemia/reperfusion. Thus, we have conducted experiments to investigate whether in the striatum of anesthetized rats the elevated brain extracellular concentrations of glutamate could increase the formation of hydroxyl radical. Elevation of glutamate levels and trapping of hydroxyl radical were accomplished by perfusing Ringer solutions containing both glutamate and salicylic acid through microdialysis probes implanted in rat striatum. The formation of hydroxyl radical was quantitated as the increased amounts of 2,3 and 2,5 dihydroxybenzoic acid (DHBA) which were the hydroxylative products of salicylic acid. Eluted microdialysates were directly injected onto high performance liquid chromatography (HPLC) with electrochemical detector via an on-line automatic injector. This method was authenticated by in vitro studies employing Fenton-type hydroxyl radicals generation system. Our results indicated that elevated glutamate concentrations (15 mM, 1.5 mM, and 150 microM glutamate in perfusing solutions) would significantly increased both the concentrations of 2,3 and 2,5 DHBA. In conclusion, we have obtained direct evidence showing that the elevated glutamate concentrations in brain extracellular space would increase the formation of hydroxyl radical, and these results implied that oxidative stress occurring in brain disorders might be induced by excitotoxicity.

In vivo evidence of hydroxyl radical formation induced by elevation of extracellular glutamate after cerebral ischemia in the cortex of anesthetized rats.

The in vivo interrelation between excitotoxicity and oxidative stress following cerebral ischemia in the cortex of anesthetized rats was investigated. Cerebral ischemia was induced by ligation of the bilateral common carotid arteries and the unilateral middle cerebral artery. Microdialysis perfusion with on-line high-performance liquid chromatography was used to monitor the hydroxyl radical levels. Extracellular hydroxyl radical levels were quantitated as the increased formation of 2.3 and 2.5 dihydroxybenzoic acid (DHBA), the hydroxylative products of salicylic acid contained in the microdialysis perfusion solutions. Elevated cortex extracellular glutamate content, resulting from the cerebral ischemia, caused an increase in the formation of hydroxyl radicals. Exogenous perfusion of authentic glutamate solutions through implanted microdialysis probes also resulted in increased hydroxyl radical formation in the cortex. The 2.3 and 2.5 DHBA levels remained elevated for an entire 80-min ischemic period. These results suggest that, after cerebral ischemia, increased oxidative stress did occur in anesthetized rats, and the oxidative stress may result from increased excitotoxicity.

The roles of reactive oxygen species and endogenous opioid peptides in ischemia-induced arrhythmia of isolated rat hearts.

Although the formation of oxygen-derived free radicals (or reactive oxygen species; ROS) and the release of endogenous opioid peptides (EOP) have been independently reported to be the major arrhythmogenic factors in ischemic hearts, possible relations between these two factors have seldom been investigated. Thus, we studied whether the ROS and EOP were related in the progression of ischemia-induced arrhythmias. Isolated rat hearts perfused in the Langendorff mode were treated with dynorphin A1-13 (kappa EOP receptor agonist), and/or allopurinol (xanthine oxidase inhibitor), before the onset of ischemia induced by ligating the left coronary arteries. Ischemic period lasted for 30 min, during which cardiac rhythms were recorded. At the end of ischemia, hearts were analyzed for the glutathione and ascorbate levels. Allopurinol (100 nmoles/heart) was effective in reducing the severity of arrhythmia (arrhythmia score: Mean +/- SEM 3.00 +/- 0.80 for allopurinol, 5.75 +/- 0.41 for placebo, p < 0.01), while dynorphin (10 micrograms/heart) potentiated the arrhythmia (6.71 +/- 0.52, p < 0.05 vs. placebo). Coadministration of allopurinol and dynorphin was capable of reducing arrhythmia (5.57 +/- 0.65) when compared with the administration of dynorphin alone (6.71 +/- 0.52, p < 0.05). Tissue oxidative stress was evaluated by the concentrations of glutathione (GSH) and ascorbate. Allopurinol did not significantly elevate tissue GSH concentrations (1.46 +/- 0.05 mumoles/g wet wt) in ischemic hearts, while dynorphin alone significantly decreased the GSH concentrations (0.96 +/- 0.08, p < 0.05) when compared with the placebo (1.32 +/- 0.03). The dynorphin-induced GSH decrease cannot be reversed by coadministration with allopurinol (0.90 +/- 0.104). Allopurinol significantly elevated tissue ascorbate levels (0.16 +/- 0.01) when compared with placebo (0.10 +/- 0.01, p < 0.05). Interestingly, dynorphin alone also elevated the tissue ascorbate concentrations (0.16 +/- 0.02). Coadministration of allopurinol and dynorphin further spiked the ascorbate levels (0.28 +/- 0.05, p < 0.01). In conclusion, the results suggested that ischemia-induced arrhythmia mechanisms might involve the formation of superoxide and other ROS, which were probably generated from the release of EOP (or EOP/EOP receptor interactions). Superoxide, the formation of which can be inhibited by allopurinol that exerted antiarrhythmic effect, was probably scavenged by ascorbate in myocardial ischemia. The ROS resulting from EOP/EOP receptor interactions were probably scavenged by glutathione system. Elevated ascorbate levels in dynorphin-treated hearts might result from the compensatory synthesis induced by decreased glutathione levels.

The involvement of glutamate in recall of the conditioned NK cell response.

The molecular mechanisms responsible for the conditioned enhancement of natural killer (NK) cell activity were investigated. The primary goal of the study was to examine the roles of glutamate and gamma-aminobutyric acid (GABA) in recall of the conditioned NK cell response. Both neurochemical blocking assay and high performance liquid chromatography (HPLC) technique were used in the study. Results from the neurochemical blocking assay demonstrated that glutamate but not GABA was required in recall of the conditioned NK cell response. NMDA but not the kainate/AMPA receptors, are believed to be involved. The levels of glutamate that were released and/or taken up also appeared to be critical in that interruption of glutamate release and/or uptake blocked the conditioned NK cell response. Results from the HPLC analysis, however, did not show any significant difference in the glutamate content between the conditioned and control brains.

Effect of diethylmaleate on liver extracellular glutathione levels before and after global liver ischemia in anesthetized rats.

Glutathione (GSH), present in a high concentration in the liver, serves important protective functions. We investigated the effect of lowered tissue GSH content, accomplished by diethylmaleate (DEM) administration, on liver extracellular GSH levels before and after global ischemia in anesthetized rats. Liver extracellular GSH levels were determined by microdialysis perfusion and an on-line high performance liquid chromatography system. Global liver ischemia was induced by ligation of the hepatic pedicles including the hepatic artery, portal vein, and bile duct. DEM (4 mmol/kg) significantly lowered both the liver tissue GSH levels (1.36 +/- 0.26 micromol/g wet wt vs 9.50 +/- 0.55 micromol/g wet wt for the untreated) and the liver extracellular GSH levels (4.3 +/- 2.4 microM vs 25.2 +/- 8.7 microM for the untreated). Global liver ischemia induced a dramatic increase in the liver extracellular GSH level. Although the liver tissue GSH level was lowered following DEM treatment, DEM administration did not affect significantly ischemia-induced elevation of extracellular GSH (when presented as fold increase relative to basal value). In conclusion, DEM showed a direct effect on liver extracellular GSH content in anesthetized rats. However, DEM treatment did not affect the relative release of GSH following global liver ischemia.

A comparison on the emission of polycyclic aromatic hydrocarbons and their corresponding carcinogenic potencies from a vehicle engine using leaded and lead-free gasoline.

Our objective in this study was to assess the effect of using two kinds of lead-free gasoline [including 92-lead-free gasoline (92-LFG) and 95-lead-free gasoline (95-LFG), rated according to their octane levels] to replace the use of premium leaded gasoline (PLG) on the emissions of polycyclic aromatic hydrocarbons (PAHs) and their corresponding benzo[a]pyrene equivalent (BaP(eq)) amounts from the gasoline-powered engine. The results show that the three gasoline fuels originally contained similar total PAHs and total BaP(eq) contents; however, we found significant differences in the engine exhausts in both contents. The above results suggest that PAHs originally contained in the gasoline fuel did not affect the PAH emissions in the engine exhausts. The emission factors of both total PAHs and total BaP(eq) obtained from the three gasoline fuels shared the same trend: 95-LFG > PLG > 92-LFG. The above result suggests that when PLG was replaced by 95-LFG, the emissions would increase in both total PAHs and total BaP(eq), but when replaced by 92-LFG would lead to the decreased emissions of both contents. By taking emission factors and their corresponding annual gasoline consumption rates into account, we found that both total PAH and total BaP(eq) emissions increased from 1994 to 1999. However, the annual increasing rates in total BaP(eq) emissions were slightly higher than the corresponding increasing rates in total PAHs.

Circadian variations in plasma and erythrocyte concentrations of glutamate, glutamine, and alanine in men on a diet without and with added monosodium glutamate.

Variations in plasma and erythrocyte concentrations of glutamate, glutamine, and alanine during the day were studied in 10 healthy men fed ordinary Taiwanese meals, first without and, 1 week later, with monosodium glutamate (MSG) added. MSG at a level of 15, 40, and 45 mg/kg (total, 100 mg/kg/d) was added, respectively, to the breakfast, lunch, and dinner meals. Heparinized blood samples were collected over 24 hours with 1- to 3-hour intervals. In both trials, plasma glutamate concentrations increased significantly after lunch and dinner. Although the circadian variations of plasma glutamate were small (between 32 and 53 micromol/L), the levels nevertheless varied significantly as a function of the time of day in both trials. Considering that the dietary intake of glutamate was high when MSG was added, the low plasma glutamate concentration over 24 hours indicates that glutamate is actively metabolized. On the other hand, the concentrations of erythrocyte glutamate (507 to 631 micromol/L) and glutamine (427 to 613 micromol/L) did not show a significant postprandial increase or circadian variation. Nevertheless, the concentration of plasma glutamine (539 to 657 micromol/L) varied significantly as a function of time in both trials. The plasma concentration of alanine (274 to 494 micromol/L) increased significantly after each meal and decreased significantly from 2:00 to 5:00 AM in both trials. Both plasma and erythrocyte alanine concentrations varied significantly as a function of time. These results show that the substantial amount of MSG intake had no apparent effect on the circadian variation profiles of blood glutamate, glutamine, and alanine.

Lowered brain glutathione by diethylmaleate decreased the glutamate release induced by cerebral ischemia in anesthetized rats.

The effect of lowered brain glutathione content on the glutamate release following cerebral ischemia was investigated. Diethylmaleate (4 mmol/kg, i.p.), a commonly used chemical reagent for tissue glutathione depletion, significantly reduced the ischemia-induced glutamate release. The release of another excitatory amino acid aspartate was not affected by the diethylmaleate administration. These results suggested that part of the elevated glutamate content induced by ischemia might result from the cellular GSH.

Elevated extracellular glutamate concentrations increased malondialdehyde production in anesthetized rat brain cortex.

Oxidative stress is believed to be involved in the damaging mechanism of excitotoxic insult. Thus, we investigated the effect of elevated extracellular glutamate levels on malondialdehyde production, a common index of lipid peroxidation, in anesthetized rat brain cortex. Elevation of extracellular glutamate levels was achieved either by exogenously perfusing glutamate solutions, or by perfusing L-trans-pyrrolidine-2,4-dicarboxylate (PDC), a competitive inhibitor of glutamate uptake transporter, through an implanted microdialysis probe. Malondialdehyde levels in the microdialysates, which were reacted with thiobarbituric acid, were analyzed by a high performance liquid chromatography system equipped with a fluorescence detector. Perfusion of glutamate (1.5 and 15 mM) resulted in dose-dependent increases in extracellular malondialdehyde production (as high as a 6-fold increase in malondialdehyde production following perfusion of 15 mM glutamate solution). PDC (3.14 and 31.4 mM), not only significantly increased the extracellular glutamate levels in a dose-dependent manner, but also dramatically increased malondialdehyde production (as high as 20-fold increase). These results suggest that excitotoxicity induces oxidative stress in anesthetized rat brain cortex, as evidenced by the glutamate-induced increase in malondialdehyde production.

Effects of isoflavones containing soy protein isolate compared with fish protein on serum lipids and susceptibility of low density lipoprotein and liver lipids to in vitro oxidation in hamsters.

The effects of dietary soy protein isolate (SPI), ethanol-extracted SPI (E-SPI) low in isoflavones, and fish protein (FP) on the concentration of blood lipids and the susceptibility of low density lipoprotein (LDL) to copper-induced oxidation were compared in male golden Syrian hamsters fed a moderate hypercholesterolemic semi-purified diet for 10 weeks. SPI, E-SPI, and FP were incorporated into the isonitrogenous experimental diets as protein sources. The SPI group exhibited significantly lower serum total cholesterol concentration compared with the E-SPI group (P < 0.05) and the FP group (P < 0.01). Both the SPI and E-SPI groups showed lower LDL cholesterol (P < 0.001 and P < 0.05, respectively) and less LDL apolipoprotein B (P < 0.01) compared with the FP group. The distribution pattern of serum lipoprotein cholesterol fractions of the SPI and E-SPI groups were similar to each other, but different from that of the FP group. The lysine/arginine ratio of the three diets was significantly correlated with serum total cholesterol concentration (r = 0.462, P = 0.023). The resistance of LDL to copper-induced oxidation was greater in the SPI group than in the E-SPI and FP groups as assessed by the lower concentrations of thiobarbituric acid-reactive substances (TBARS) and the longer lag time required for the formation of conjugated dienes (P < 0.01). Livers of hamsters fed the FP diet had a higher amount of TBARS than those of hamsters fed SPI (P < 0.01) and E-SPI (P < 0.05) diets. The SPI diet showed sparing effects on alpha-tocopherol contents in both serum and liver. It seems likely that soy isoflavones protect the circulating and membrane lipids by sparing alpha-tocopherol and endogenous antioxidants.

Alpha-tocopherol acetate significantly suppressed the increase in heart interstitial 8-hydroxydeoxyguanosine following myocardial ischemia and reperfusion in anesthetized rats.

The effect of alpha-tocopherol acetate, an aqueous form of alpha-tocopherol, on the increase in heart interstitial 8-hydroxydeoxyguanosine (8-OH-dG) levels following myocardial ischemia/reperfusion was investigated. A microdialysis probe was implanted in the left ventricular interstitial space of anesthetized rat hearts. Myocardial ischemia was induced by ligating the left anterior descending coronary artery. Levels of 8-OH-dG in microdialysates were analyzed via an on-line high-performance liquid chromatography system equipped with an electrochemical detector. The 8-OH-dG levels significantly increased (maximum 3.6-fold of increase relative to basal value) during the 60-min reperfusion stage following a 20 min ischemia. Administration of alpha-tocopherol acetate (20 mg/kg, intravenous, bolus) at 3 min prior to onset of reperfusion, significantly suppressed the reperfusion-induced increase in 8-OH-dG levels. These results suggested that one of the possible protective effect of alpha-tocopherol acetate was to reduce oxidative DNA damage during in myocardial ischemia and reperfusion.

Increased formation of interstitial hydroxyl radical following myocardial ischemia: possible relationship to endogenous opioid peptides.

The effects of myocardial ischemia and reperfusion on interstitial hydroxyl radical production, in the left ventricular myocardium of anesthetized cats, were investigated. Ringer's solution containing salicylic acid was perfused through an implanted microdialysis probe. Hydroxyl radical production was evaluated as the 2,3 and 2,5 dihydroxybenzoic acid (DHBA) concentrations in the microdialysates by an on-line high performance liquid chromatography system. Myocardial ischemia for 60 min, induced by ligation of the left anterior descending coronary artery, significantly increased both 2,3 and 2,5 DHBA levels when compared with the sham-operated cats. Naloxone (1 mg/kg, bolus, intravenous), an endogenous opioid peptide receptor antagonist, significantly suppressed the ischemia-induced production of hydroxyl radicals. Myocardial ischemia also induced cardiac arrhythmia. Naloxone reduced the severity of ischemia-induced arrhythmia, as observed by a significantly lower arrhythmia score (1.4 +/- 0.2 vs. 4.6 +/- 0.4 for control), and by diminished incidence of ventricular tachycardia (0/7 vs. 8/8 for control) and ventricular fibrillation (0/7 vs. 3/8 for control). Furthermore, perfusion of dynorphin (0.25 microgram, 2.5 micrograms and 25 micrograms), an endogenous opioid peptide receptor agonist, increased hydroxyl radical production. Our results suggest that, in anesthetized cats, myocardial ischemia can induce production of interstitial hydroxyl radical in left ventricular myocardium, and this production may involve the actions of released endogenous opioid peptides on their receptors.