Biomedical subjects

P J O'Brien

Publications and source records attributed to P J O'Brien.

At least 55 records · Page 3Linked to original sources

PubMed1999

Oxygen activation during peroxidase catalysed metabolism of flavones or flavanones.

Flavonoids containing phenol B rings, e.g. naringenin, naringin, hesperetin and apigenin, formed prooxidant metabolites that oxidised NADH upon oxidation by peroxidase/H2O2. Extensive oxygen uptake occurred which was proportional to the NADH oxidised and was increased up to twofold by superoxide dismutase. Only catalytic amounts of flavonoids and H2O2 were required indicating a redox cycling mechanism that activates oxygen and generates H2O2. NADH also prevented the oxidative destruction of flavonoids by peroxidase/H2O2 until the NADH was depleted. These results suggest that prooxidant phenoxyl radicals formed by these flavonoids cooxidise NADH to form NAD radicals which then activated oxygen. Similar oxygen activation mechanisms by other phenoxyl radicals have been implicated in the initiation of atherosclerosis and carcinogenesis by xenobiotic phenolic metabolites. This is the first time that a group of flavonoids have been identified as prooxidants independent of transition metal catalysed autoxidation reactions.

Catalysis↗

PubMed1999

Role of the cellular redox state in modulating acute ethanol toxicity in isolated hepatocytes.

OBJECTIVES: To propose a mechanism for ethanol induced hepatocytotoxicity. DESIGN AND METHODS: Hepatocytotoxicity was determined at various concentrations of oxygen and agents involved in NADH metabolism. RESULTS: At 1% O2, hepatocytes were nearly 8-fold more susceptible to ethanol than at 95% O2 (carbogen). Cytotoxicity at 1% O2 was enhanced in the presence of glycolytic substrates that generate NADH (e.g., sorbitol or xylitol), and prevented by glycolytic substrates that reoxidise NADH (e.g., fructose or dihydroxyacetone). Susceptibility to ethanol correlated with the cytosolic redox state (lactate; pyruvate ratio). Cytotoxicity also correlated with reactive oxygen species (ROS) formation. Cytotoxicity was averted by ROS scavengers or the ferric chelator desferoxamine but was increased by hydroxylamine, a catalase inhibitor, or by prior glutathione depletion. Ethanol induced cytotoxicity was also decreased by inhibitors of alcohol/aldehyde dehydrogenases or CYP2E1, an alcohol inducible cytochrome P450. CONCLUSIONS: A cytotoxic mechanism was proposed where the sustained increase in NADH levels, resulting from ethanol metabolism, maintains CYP2E1 in a more reduced state that increases ROS formation.

Alcohol Dehydrogenase↗

PubMed1999

The effects of heme-binding proteins on the peroxidative and catalatic activities of hemin.

The plasma proteins hemopexin (Hx) and albumin (Alb) are known to bind heme with high and medium affinity, respectively. To study how this binding modifies heme catalytic reactivity, the effects of Hx, human serum Alb (HSA), and bovine serum Alb (BSA) on the peroxidase- and catalaselike activities of hemin were investigated. These hemin activities were found to be inhibited by 50 to 60% with either HSA or BSA, and by 80 to 90% with Hx. The heme complexes with Hx or Alb (1:1 = protein:heme) therefore had a much lower reactivity toward H2O2 and Cum-OOH than the nonprotein heme. A kinetic analysis suggested that binding to Hx or Alb inhibited the primary activation of heme by H2O2, the step common for both peroxidase- and catalaselike activities of hemin. It is thought that by complexing heme, the Hx and Alb can prevent the toxic effects of extracellular heme in blood plasma.

Animals↗

PubMed1999

Catalytic promiscuity and the evolution of new enzymatic activities.

Several contemporary enzymes catalyze alternative reactions distinct from their normal biological reactions. In some cases the alternative reaction is similar to a reaction that is efficiently catalyzed by an evolutionary related enzyme. Alternative activities could have played an important role in the diversification of enzymes by providing a duplicated gene a head start towards being captured by adaptive evolution.

Bacteria↗

PubMed1999

Glutathione-dependent generation of reactive oxygen species by the peroxidase-catalyzed redox cycling of flavonoids.

Catalytic concentrations of apigenin (a flavone containing a phenol B ring) and naringin or naringenin (flavanones containing a phenol B ring) caused extensive GSH oxidation at a physiological pH in the presence of peroxidase. Only catalytic H2O2 concentrations were required, indicating a redox cycling mechanism that generated H2O2 was involved. Extensive oxygen uptake ensued, the extent of which was proportional to the extent of GSH oxidation to GSSG and was markedly increased by superoxide dismutase. These results suggest that prooxidant phenoxyl radicals formed by these flavonoids co-oxidized GSH to form thiyl radicals which activated oxygen. GSH also prevented the peroxidase-catalyzed oxidative destruction of these flavonoids which suggests that phenoxyl radicals initiated the oxidative destruction. This is the first time that a group of flavonoids have been identified as prooxidants independent of autoxidation reactions catalyzed by the transition metal ions Fe3+, Fe2+, Mn2+, and Cu2+.

Catalysis↗

PubMed1999

Early fixation of the vertically unstable pelvis: the role of iliosacral screw fixation of the posterior lesion.

OBJECTIVES: To evaluate the effectiveness of the use of iliosacral screw fixation in the management of the vertically unstable pelvis. STUDY DESIGN: Retrospective analysis with clinical follow-up of patients to assess functional outcome. METHODS: Thirty-eight vertically unstable pelvic fractures were treated using iliosacral screw fixation. Anterior fixation was by means of plating in sixteen pelves and by external fixation in fifteen pelves. Four pelves had no anterior fixation. Complications were recorded and radiographs were analyzed to classify fractures and identify screw misplacement and malunion. Twenty-six patients had a functional evaluation. RESULTS: Five patients (13 percent) suffered a pulmonary embolus in the early postoperative period, one of which was fatal, a hospital mortality of 2.6 percent. Screw misplacement occurred in five patients but there were no adverse sequelae. In thirty-four cases with radiographic follow-up, malunion was noted in fifteen cases (44 percent). A lower rate of malunion (36 percent) was noted with internal fixation of the anterior lesion. Of twenty-six patients with long-term follow-up, only four (15 percent) had no pain. Sacroiliac fusion for pain was performed in three patients (11 percent). Twelve patients (46 percent) returned to their preinjury occupation, six patients (23 percent) changed occupation, and nine patients (30 percent) had not yet returned to work by last follow-up. CONCLUSIONS: Iliosacral screw fixation is a useful method of fixation in the vertically unstable pelvis but needs to be augmented by rigid anterior fixation to minimize malunion.

Adolescent↗

PubMed1998

Catecholic iron complexes as cytoprotective superoxide scavengers against hypoxia:reoxygenation injury in isolated hepatocytes.

Reactive oxygen species including superoxide radicals (O2-.) have been implicated in the pathogenesis of radiotherapy, ischemia-reperfusion injury, aging, and inflammatory diseases. In the present work, 2:1 catecholic iron complexes were found to be more effective than uncomplexed catechols at protecting hepatocytes against hypoxia:reoxygenation cell injury. They also decreased markedly the level of reactive oxygen species formed before cytotoxicity ensued. Furthermore, these catecholic iron complexes were also more effective than uncomplexed catechols at scavenging superoxide radicals generated both enzymatically and nonenzymatically. The superoxide radical scavenging activity of catecholic iron complexes seemed to correlate with the redox potential of catechols. These results suggest that cytoprotection by catechols may involve an initial chelation with iron to form a complex that is a much more effective superoxide radical scavenger than the catechol itself.

Animals↗

PubMed1998

Hepatocyte-catalysed detoxification of cyanide by L- and D-cysteine.

The hepatocyte metabolic pathways involved in the detoxification of cyanide by cysteine have been investigated in vitro using hepatocytes isolated from Sprague-Dawley rats. Cyanide toxicity towards isolated hepatocytes could be prevented by the addition of L- or D-cysteine, cystine, or the cysteine metabolites thiosulfate and mercaptopyruvate, which markedly increased thiocyanate formation. Prior depletion of hepatocyte GSH markedly increased thiosulfate formation from L- or D-cysteine without affecting thiocyanate formation from L- or D-cysteine. This suggested that the major metabolic pathway for thiocyanate formation did not involve thiosulfate. Mercaptopyruvate was a more likely metabolic intermediate, as thiocyanate formation from L-cysteine but not thiosulfate was inhibited markedly by aminooxyacetate, a cysteine aminotransferase inhibitor, and propargylglycine, a gamma-cystathionase inhibitor. Furthermore, propargylglycine prevented L-cysteine cytoprotection against cyanide toxicity. Thiocyanate formation from D-cysteine likely also involved mercaptopyruvate, as thiocyanate formation from D-cysteine but not L-cysteine was inhibited by benzoate, an inhibitor of D-amino acid oxidase. Furthermore, benzoate prevented D-cysteine cytoprotection against cyanide toxicity. Cystine may also be an intermediate, as hepatocyte thiocyanate formation from added L-cysteine was inhibited when L-cysteine autoxidation was prevented with the copper chelator bathocuproine disulfonate. Furthermore, thiocyanate formation by rat liver homogenates with L-cystine was far more rapid than that with L-cysteine. Hepatocyte thiocyanate metabolic intermediates of beta-mercaptopyruvate and thiocystine were proposed for L-cysteine, and beta-mercaptopyruvate was proposed for D-cysteine.

Animals↗

PubMed1998

Usefulness of transesophageal echocardiography in predicting mortality and morbidity in stroke patients without clinically known cardiac sources of embolus.

This study tested the hypothesis that stroke patients without a cardiac source of embolism suspected by clinical examination can be risk stratified by transesophageal echocardiography. Forty ischemic stroke patients without atrial fibrillation, prosthetic valves, ejection fraction < 20%, or recent myocardial infarction underwent multiplane transesophageal echocardiography: 24 (designated high risk) had > or = 1 of the following: left heart thrombus, vegetation, mass or spontaneous echo contrast, mobile ascending aortic or arch debris, patent foramen ovale, atrial septal defect or aneurysm, mitral annular calcification, mitral valve thickening, prolapse or mitral valve strands. End points were death, recurrent stroke, transient ischemic attack, myocardial infarction or peripheral embolism. Thirty-eight patients (95%) (23 high, 15 low risk) were followed for 14 +/- 8 months: 9 (24%) died of vascular causes including 4 who had a cardiac cause of death and 5 who had fatal strokes. Eight had recurrent strokes (4 nonfatal) and 1 nonfatal myocardial infarction occurred. Cardiovascular survival was predicted by transesophageal echocardiography: survival rates were 92% (low risk) and 63% (high risk) at 24 months (p = 0.036). Left atrial enlargement was independently associated with death from stroke (fatal stroke occurred in 25% of those with atrial enlargement compared to 8% of those with normal atrial dimension, p < or = 0.03), as was left atrial spontaneous echo contrast (50% died vs 9% without contrast, p < or = 0.03). Left ventricular hypertrophy and aortic atherosclerosis were both associated with the risk of recurrent stroke (30% of patients with ventricular hypertrophy had recurrent stroke compared to 10% with normal wall thickness (p < or = 0.05); 30% with aortic atherosclerosis had a recurrent stroke compared to none with a normal aorta (p < or = 0.05). Thus, transesophageal echocardiography clearly identifies patients at a high risk for cardiovascular mortality and morbidity after stroke despite an unsuspected source of embolism by clinical examination.

Aged↗

PubMed1998

The glutathione dependence of inorganic sulfate formation from L- or D-cysteine in isolated rat hepatocytes.

The GSH dependence of the metabolic pathways involved in the conversion of cysteine to sulfate in intact cells has been investigated. It was found that hepatocyte-catalysed sulfate formation from added L-cysteine did not occur if hepatocyte GSH was depleted beforehand, but was restored when GSH levels recovered. Furthermore, sulfate formation did not recover in GSH-depleted hepatocytes if GSH synthesis was prevented with buthionine sulfoximine. Thiosulfate formation was, however, markedly enhanced in GSH-depleted hepatocytes. These results suggest that thiosulfate is an intermediate in the formation of inorganic sulfate from L-cysteine and that GSH was required for the conversion of thiosulfate to inorganic sulfate. Much less sulfate was formed if the cysteine was replaced with cysteinesulfinate. Furthermore, sulfate formation from L-cysteine was markedly inhibited by the addition of the transaminase inhibitor DL-cycloserine or the gamma-cystathionase inhibitor DL-propargylglycine. The major routes of sulfate formation from L-cysteine therefore seems to involve pathways that do not involve L-cysteinesulfinate. Similar amounts of sulfate were formed from D-cysteine as L-cysteine. Thiosulfate instead of sulfate was also formed in GSH-depleted hepatocytes. However, sulfate formation from D-cysteine differed from L-cysteine in that it was inhibited by the D-aminoacid oxidase inhibitor sodium benzoate and was not affected by transaminase or gamma-cystathionase inhibitors. These results suggest that thiosulfate is an intermediate in sulfate formation from D-cysteine and involves the oxidation of D-cysteine by D-amino acid oxidase to form beta-mercaptopyruvate.

Alkynes↗

PubMed1997

The prevention of ferric nitrilotriacetate-induced nephro- and hepatotoxicity by methylenedioxybenzene antioxidants.

Previously it was shown that methylenedioxybenzenes (MDBs), particularly isosafrole, were highly effective at preventing CCl4-induced liver necrosis in vivo (Z.S. Zhao, P.J. O'Brien, The prevention of CCl4-induced liver necrosis in mice by naturally occurring methylenedioxybenzenes, Toxicol. Appl. Pharmacol., 140 (1996) 411-421), probably as a result of forming metabolic intermediate complexes with cytochrome P450. In the following it was shown that pretreatment of mice with isosafrole also completely prevented ferric nitrilotriacetate (FeNTA)-induced renal necrosis and lipid peroxidation, even though metabolic activation by cytochrome P450 is not involved. The naturally occurring or synthetic MDBs that prevented CCl4 hepatotoxicity also prevented hepatocyte lipid peroxidation. induced by FeNTA, but other cytochrome P450 inhibitors were ineffective. These compounds, in decreasing order of antioxidant effectiveness, were sesamol, 4-t-butyl-methylenedioxybenzene, isosafrole, piperonyl butoxide and 4-bromo-methylenedioxybenzene and safrole, whereas, benzodioxole, 3,4-(methylenedioxy)-toluene and 1,2-(methylenedioxy)-4-nitrobenzene were ineffective. Pre-incubating the hepatocytes with P450 inhibitors decreased the protective effects of isosafrole, suggesting that the catecholic metabolites of MDBs were responsible for the antioxidant activity. A greater inhibition of FeNTA-induced lipid peroxidation by catecholic metabolites was observed. Since cytochrome P450 did not participate in FeNTA-induced hepatocyte or microsomal lipid peroxidation, it is likely that the antioxidant properties of MDBs or their catecholic metabolites also contribute to their in vivo protection against CCl4 or FeNTA-induced hepato- or nephrotoxicity.

Animals↗

PubMed1997

Targeting of HIV- and SIV-infected cells by CD4-chemokine receptor pseudotypes.

Retroviral vectors containing CD4 and an appropriate chemokine receptor were evaluated for the ability to transduce cells infected with human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). These CD4-chemokine receptor pseudotypes were able to target HIV- and SIV-infected cell lines and monocyte-derived macrophages in a manner that corresponded to the specificity of the viral envelope glycoprotein for its CD4-chemokine receptor complex. This approach could offer a way to deliver antiviral genes directly to HIV-infected cells in vivo and could provide an additional treatment strategy in conjunction with existing antiviral therapies.

Animals↗

PubMed1997

Rapid and specific efflux of glutathione before hepatocyte injury induced by hypoxia.

Hypoxia caused the efflux of glutathione (GSH) from hepatocytes before membrane lysis occurred. Dithiothreitol (DTT), a thiol reductant, greatly increased the hypoxia induced GSH efflux as well as the subsequent membrane lysis. The NADH generating nutrients sorbitol and beta-hydroxybutyrate as well as ethanol also enhanced hepatocyte GSH efflux and cell injury, whereas on the other hand NADH oxidising metabolic intermediates, e.g., acetoacetate or the artificial electron acceptor methylene blue, partly prevented GSH efflux and membrane lysis. Hypoxia induced GSH efflux and cytotoxicity were also prevented by oxypurinol, a xanthine oxidase inhibitor, as well as by the polyphenolic antioxidant quercetin, suggesting that reactive oxygen species contributed to the GSH efflux and cell lysis. The above results suggest that reductive stress caused by hypoxia activates the redox sensitive sinusoidal GSH transporter that is likely responsible for the GSH efflux before membrane lysis ensues.

Animals↗

PubMed1997

Phenylenediamine induced hepatocyte cytotoxicity redox. Cycling mediated oxidative stress without oxygen activation.

Muscle necrosis induced by various phenylenediamine derivatives has been correlated with their autoxidation rate. However, a more detailed investigation of the cytotoxic mechanism using a model system of isolated hepatocytes and 2,3,5,6-tetramethylphenylenediamine (DD) shows little oxygen activation as indicated by the absence of cyanide resistant respiration, lipid peroxidation and lack of cytoprotection by iron chelators, superoxide dismutase mimics and xanthine oxidase inhibitors. Cytotoxicity was however attributed to oxidative stress as GSH was not only rapidly oxidized to GSSG but mixed protein disulfide formation also occurred. Furthermore, the disulfide reductant dithiothreitol added some time after DD restored protein thiols and prevented further cytotoxicity. This oxidative stress was attributed to a futile two electron redox cycle involving oxidation of DD to the corresponding diimine by the mitochondrial electron transport chain and rereduction by DT diaphorase. Evidence suggesting this was that both diimine accumulation and the ensuing cytotoxicity were markedly increased by inactivating hepatocyte DT diaphorase but were prevented by a subtoxic concentration of the mitochondrial respiratory inhibitor cyanide. Furthermore, addition of NADH generating substrates such as lactate, sorbitol, xylitol or ethanol prevented DD induced GSH oxidation and cytotoxicity. This suggests that DD undergoes intracellular redox cycling without oxygen activation until the hepatocyte is unable to maintain redox homeostasis and mixed protein disulfide cytotoxicity ensues.

Animals↗

PubMed1997

Hydrogen peroxide supports human and rat cytochrome P450 1A2-catalyzed 2-amino-3-methylimidazo[4,5-f]quinoline bioactivation to mutagenic metabolites: significance of cytochrome P450 peroxygenase.

We show that the naturally occurring hydroperoxide hydrogen peroxide is highly effective in supporting the cytochrome P450 1A2 peroxygenase-catalyzed metabolic activation of the heterocyclic aromatic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to genotoxic metabolites. Mutagenicity was assessed by the Ames assay with Salmonella typhimurium strain YG1012 and an activation system consisting of hydroperoxides plus either 3-methylcholanthrene-induced rat liver microsomes (rP4501A) or human P450 1A2-containing microsomes (hP4501A2). The mutagenic response was dependent on the concentration of microsomal protein, IQ, and hydroperoxides. The addition of hydrogen peroxide or tert-butyl hydroperoxide to rP4501A greatly enhanced the yield of histidine prototrophic (His+) revertants. This increase was inhibited, in a concentration-dependent manner, by alpha-naphthoflavone, a P450 1A inhibitor. Hydrogen peroxide was the most effective peroxygenase cofactor, particularly with hP4501A2 (K(m) = 0.1 mM). The hydroperoxide-supported activation of IQ produced reactive intermediates which bound to 2'-deoxyguanosine; LC/MS analysis of the adducts revealed the same major (protonated) adduct at m/z = 464.4 as previously reported for the DNA adduct formed (in vivo or in vitro) by the mixed function-catalyzed bioactivation system. None of the peroxidase-catalyzed IQ metabolites (nitro-, azo-, or azoxy-IQ) were detected. In conclusion, hydrogen peroxide in the physiological/pathological concentration range may be able to support the metabolic activation of arylamines to genotoxic products through the cytochrome P450 peroxygenase pathway.

Animals↗

PubMed1997

Compensatory up-regulation of cardiac SR Ca2+-pump by heat-shock counteracts SR Ca2+-channel activation by ischemia/reperfusion.

We tested the hypothesis that heat-shock protected myocardial Ca2+-cycling by sarcoplasmic reticulum from ischemia and reperfusion (I/R) injury. Twenty-four hours after increasing body temperature to 42 degrees C for 15 min, rat hearts were isolated, Langendorff-perfused, and subjected to 30 min ischemia then 30 min reperfusion. Left ventricles were homogenized and their ionized Ca2+ concentration monitored with indo- during Ca2+-uptake in the presence and absence of the Ca2+-release channel (CRC) modulator ryanodine. Tissue content of heat-shock protein 72 (HSP 72) was analyzed. Exposure to I/R resulted in a 37% enhancement of CRC activity but no effect on Ca2+-pumping activity, resulting in 25% decreased net Ca2+-uptake activity. Pre-exposure to heat-shock resulted in a 10-fold increase in HSP 72, and a 25% enhancement of maximal Ca2+-pumping activity which counteracted the effect of I/R on CRC and net Ca2+-uptake activities. This protection of SR Ca2+-cycling was associated with partial protection of myocardial physiological performance. Net Ca2+-uptake activity was correlated with the left ventricular developed pressure and its rate of change. We conclude that one of the mechanisms by which heat-shock protects myocardium from I/R injury is to upregulate SR Ca2+-pumping activity to counteract the enhanced SR Ca2+-release produced by I/R.

Animals↗

PubMed1997

Rapid, simple and sensitive microassay for skeletal muscle homogenates in the functional assessment of the Ca-release channel of sarcoplasmic reticulum: application to diagnosis of susceptibility to malignant hyperthermia.

A microassay is demonstrated for functional characterization of the Ca(2+)-release channel (CRC) of sarcoplasmic reticulum (SR) of skeletal muscle using swine with susceptibility to malignant hyperthermia (MH). Diluted muscle homogenates, indo-1 and ratiometric dual-emission spectrofluorometry are used to monitor Ca(2+)-lowering activity in real-time in the presence and absence of ryanodine at exposures that open and close the CRC. Reactions are initiated with 50 microM CaCl2 to raise ionized Ca2+ concentration near 1 microM and MgATP to activate the Ca(2+)-ATPase pump. Oxalate is included to precipitate Ca2+ within the SR. The assay requires less than 30 mg muscle, which may be cryopreserved, and is completed within 20 min of thawing the tissue. Maximum SR Ca(2+)-ATPase pumping and CRC activities, degree of CRC activation, and Ca(2+)-buffering capacity can be determined. Using this assay we studied muscle from MH-susceptible swine and demonstrated that whereas maximal Ca(2+)-ATPase pumping and CRC activities are normal, the CRC activity after addition of a bolus of Ca2+ is 50% greater in heterozygotes and 100% greater in homozygotes for the MH mutation. Hypersensitivity to CRC agonists, such as caffeine, and an associated hyposensitivity to CRC antagonists such as Mg2+ is also demonstrated. Genotypes for the MH mutation site can be discriminated from each other by determining Ca(2+)-lowering activities and the effect of ryanodine on them.

Animals↗

PubMed1997

Knee pain after tibial nailing.

OBJECTIVE: To determine the incidence and natural history of knee pain following tibial nailing. DESIGN: A retrospective analysis of patients treated by tibial nailing evaluating a consecutive series of patients with isolated tibial shaft fractures. SETTING: A level one trauma center in Vancouver, British Columbia. PATIENTS: A group of 107 consecutive patients with 110 tibial fractures treated by interlocking tibial nailing. INTERVENTION: Patients were contacted and interviewed by the authors. Clinical records and radiographs were analysed. MAIN OUTCOME MEASUREMENTS: Incidence of knee pain; time of onset; relationship of nail position on radiographs to knee pain; relationship to knee pain to site of nail insertion; response to nail removal. RESULTS: At a mean follow-up period of thirty-two months (12-58 months), sixty-one (57%) patients (63 of 110 knees) had developed anterior knee pain. There was no correlation between nail protrusion and knee pain. Insertion of the nail through the patella tendon was associated with a higher incidence of knee pain compared to the paratendon site of nail insertion (77% and 50% respectively). Of patients with knee pain, 80% (49/61) required nail removal. At a mean duration of 16 months following nail removal, pain was completely relieved in 22 patients and partially relieved in 17. In the remaining 10 patients, there was no improvement. CONCLUSIONS: Based on this data, we would recommend a parapatellar tendon incision for nail insertion, and nail removal for those patients with a painful knee. The causes of knee pain after tibial nailing are multi-factorial and require further study.

Adolescent↗