Biomedical subjects
S D Aust
Publications and source records attributed to S D Aust.
Ischemia, resuscitation, and reperfusion: mechanisms of tissue injury and prospects for protection.
Since its introduction in 1960, CPR has evolved into a complex program involving not only the medical community but also the lay public. Currently, program activities include instruction of the lay public in basic life support techniques, development and deployment of emergency medical systems, recommendations for drug protocols for advanced cardiac life support and, most recently, introduction of new methods for tissue protection following resuscitation. After 25 years of experience, we are beginning to understand the pathophysiology of tissue ischemia during cardiac arrest and the interventions required to improve chances of survival and quality of life of the cardiac arrest victim. Recent data in the literature suggest that modification of certain interventions in the resuscitation program may be needed. The poor neurologic outcomes with prolonged standard CPR show that it is not protective after 4 to 6 minutes of cardiac arrest. Modifications to this technique, including SVC-CPR or IAC-CPR, have not been shown to increase resuscitability or hospital discharge rates. Human studies of open-chest cardiac massage are needed to evaluate this option. Defibrillation is the definitive treatment for ventricular fibrillation. Greater emphasis should be placed on the earliest possible delivery of this treatment modality. Computerized defibrillators may provide greater and earlier access to defibrillation in the homes of patients at high risk of ventricular fibrillation. They may also be applicable by untrained public service personnel (police and firemen), individuals in geographically inaccessible areas (aircraft), or emergency medical technicians in rural areas where skill retention is a significant problem. Calcium has no proved benefit in cardiac resuscitation. There is biochemical evidence that it may be harmful in brain resuscitation. Its use in resuscitation should be discontinued. The dose of epinephrine currently advocated in the ACLS protocols may be inadequate to increase aortic diastolic pressure and coronary and cerebral perfusion pressures and thus aid resuscitation. Animal studies indicate that substantial increases in the current dosage are needed to achieve these effects. Human studies are needed to verify these results. A role for calcium antagonists in the treatment of postarrest encephalopathy has been demonstrated in animals and is currently undergoing clinical trials. Iron-dependent lipid peroxidative cell membrane injury may be important in the pathogenesis of postarrest encephalopathy. Animal studies suggest that the iron chelator deferoxamine may have a significant therapeutic role in the treatment of postarrest encephalopathy.
Free radicals and environmental toxins.
Some chemicals that contaminate our environment exert their toxic effects by virtue of their ability to form free radicals. In the absence of sufficient quenching reactions, these reactive radicals can attack biomolecules, resulting in their oxidative degradation. Biological membranes which contain polyunsaturated fatty acids are most susceptible to oxidative degradation (lipid peroxidation), although oxidation of DNA may have more severe biological consequences. Free radicals species can be generated by at least two mechanisms in vivo. The first, of which carbon tetrachloride (CCl4) is the classic example, is the biotransformation of the chemical to a free radical species. Metabolism of CCl4 to the trichloromethyl radical by the hepatic mixed-function oxidase system results in the initiation of lipid peroxidation, protein-lipid cross linkages, and trichloromethyl adducts with DNA, protein, and lipid. The second mechanism for forming free radicals involves their reduction to less stable free radical intermediates which are oxidized by molecular oxygen to give superoxide (O2-.). In the presence of transition metals, such as iron, O2-. can be converted to other oxygen radical species, such as the hydroxyl radical (.OH), an extremely powerful oxidant capable of cleaving DNA, oxidizing protein, and initiating lipid peroxidation. Under many conditions, lipid peroxidation appears not to be initiated by .OH, but rather by an iron-oxygen complex. Regardless of the identity of the initiating species, transition metals are required for most of the deleterious reactions of oxygen. Superoxide and certain organic radicals have been found to release iron from ferritin.
Myocardial tissue iron delocalization and evidence for lipid peroxidation after two hours of ischemia.
Ischemic tissue injury has been proposed to be in part due to oxygen-radical-mediated lipid peroxidation. In vitro studies of such reactions show that they are thermodynamically unfavorable unless catalyzed by transitional metals such as iron in low molecular weight species (LMWS iron), ie, the iron-ADP complex. This study tests for iron delocalization into a LMWS pool during myocardial ischemia and for increased tissue malondialdehyde (MDA), a product of lipid peroxidation. Anesthesia was induced in eight dogs (weighing 20 to 30 kg) with ketamine and maintained by ventilation with 1% halothane. The left anterior descending coronary artery was ligated in four animals, and the circumflex coronary artery was ligated in the other four. Two hours after ligation, the animals were sacrificed by a central venous injection of KCl. Tissue samples were immediately taken from the ischemic zone and from the corresponding nonischemic zone. MDA was determined by the thiobarbituric acid assay. LMWS iron was determined on a tissue ultrafiltrate by the o-phenanthroline assay. Statistical data analysis used the matched-pair two-tailed t test. LMWS iron was 18.3 nM/100 mg in ischemic tissue versus 13.1 nM/100 mg in nonischemic tissue (t = 4.14; P less than .01). MDA was 0.91 nM/100 mg in ischemic tissue versus 0.83 nM/100 mg in nonischemic tissue (t = 7.27; P less than .005). We conclude that there is a significant increase in tissue LMWS iron and in MDA after two hours of regional myocardial ischemia. This iron might be the catalyst for maturation of tissue injury during reperfusion as observed by other investigators.(ABSTRACT TRUNCATED AT 250 WORDS)
Importance of the polyunsaturated fatty acid to vitamin E ratio in the resistance of rat lung microsomes to lipid peroxidation.
Rat lung microsomes and liposomes made from isolated lung microsomal lipids were found to be much more resistant to lipid peroxidation than those from liver in both enzymatic and nonenzymatic systems. The polyunsaturated fatty acid (PUFA) content of isolated lung microsomal lipids was 28% of total fatty acids, while liver was 54%. The vitamin E (alpha-tocopherol) content of isolated lung microsomal lipids was 2.13 nmol/mumol lipid phosphate and that of liver was 0.43. Individually, neither the lower PUFA content nor higher vitamin E levels could account for the resistance of lung microsomal lipids to peroxidation. Distearoyl-L-alpha-phosphatidylcholine and/or alpha-tocopherol were added to liver microsomal lipids to achieve different PUFA to vitamin E ratios at PUFA contents of 28% or 54%, and the resulting liposomes were subjected to an NADPH-dependent lipid peroxidation system utilizing cytochrome P450 reductase, EDTA-Fe+3, and ADP-Fe+3. Liposomes having PUFA to vitamin E ratios less than approximately 250 nmol PUFA/nmol vitamin E were resistant to peroxidation, whereas lipid peroxidation, as evidenced by malondialdehyde production, occurred in liposomes having higher ratios. When lipid peroxidation occurred, 40%-60% of the liposomal vitamin E was irreversibly oxidized. Irreversible oxidation did not occur in the absence of lipid peroxidation. These studies indicated that the low PUFA to vitamin E ratio in lung microsomes and isolated microsomal lipids was sufficient to account for the observed resistance to lipid peroxidation.
Transferrin-dependent lipid peroxidation.
The potential for iron bound to transferrin to be released and promote the peroxidation of phospholipid liposomes was investigated using ADP as a low molecular weight chelator and superoxide generated by the xanthine/xanthine oxidase system as the reducing agent. Lipid peroxidation in this system was dependent upon transferrin as the source of iron; increasing the transferrin concentration resulted in increased rates of lipid peroxidation. Increasing the xanthine oxidase activity also caused increased rates of peroxidation. Catalase stimulated rates of peroxidation at all xanthine oxidase activities tested. Conditions resulting in the most rapid release of iron from transferrin (low pH, high ADP) did not promote the greatest rates of lipid peroxidation, indicating that at neutral pH, rates of lipid peroxidation may be limited by the availability of iron. It is concluded that transferrin is not a likely source of iron for catalysis of deleterious biological oxidations such as lipid peroxidation in vivo.
Oxidation of persistent environmental pollutants by a white rot fungus.
The white rot fungus Phanerochaete chrysosporium degraded DDT [1,1,-bis(4-chlorophenyl)-2,2,2-trichloroethane], 3,4,3',4'-tetrachlorobiphenyl, 2,4,5,2',-4',5'-hexachlorobiphenyl, 2,3,7,8-tetrachlorodibenzo-p-dioxin, lindane (1,2,3,4,5,6-hexachlorocylohexane), and benzo[a]pyrene to carbon dioxide. Model studies, based on the use of DDT, suggest that the ability of Phanerochaete chrysosporium to metabolize these compounds is dependent on the extracellular lignin-degrading enzyme system of this fungus.
Toxicity of 3,4,5,3',4',5'-hexabrominated biphenyl and 3,4,3',4'-tetrabrominated biphenyl.
Immature male rats were given a single equimolar dose (21.3 mumol/kg body wt) of 3,4,5,3',4',5'-hexabromobiphenyl (HBB) or 3,4,3',4'-tetrabromobiphenyl (TBB) and terminated at various times up to 14 days after treatment. Hepatic microsomal aryl hydrocarbon hydroxylase (AHH) activity for the TBB treatment group was maximal at Day 2 and then steadily decreased, whereas this activity was induced in 1 day and remained high for the HBB treatment group. Tissue concentrations of HBB appeared to be unchanged over time whereas tissue concentrations of TBB decreased in a biphasic manner. Rates of in vitro metabolism of TBB with hepatic microsomes from TBB-treated animals showed a similar time-course relationship to AHH induction. HBB caused moderate to severe hepatic changes while TBB-treated rats had only mild hepatic changes. The relative binding of TBB by the hepatic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was about 10 times that of HBB. The results suggest that even though the receptor-binding affinities imply that TBB should be more toxic than HBB, it is less toxic than HBB because it is metabolized. Studies with the chlorinated analogs of TBB and HBB suggested that PCB behave similarly. These results also suggest that receptor binding and AHH induction do not accurately reflect toxicity for polyhalogenated aromatic hydrocarbons which are metabolized, presumably because continued occupation of the receptor and persistent induction of some enzyme activity are required for toxicity.
Studies on the structure-activity relationships for the metabolism of polybrominated biphenyls by rat liver microsomes.
The in vitro metabolism of polybrominated biphenyl (PBB) congeners by cytochrome P-450-dependent monooxygenases was investigated using hepatic microsomes isolated from immature male rats pretreated with 3-methylcholanthrene (MC) or phenobarbital (PB). MC pretreatment increased the NADPH-dependent microsomal metabolism of pure PBB congeners which possessed adjacent nonhalogenated ortho and meta carbons on at least one ring. 4,4'-Dibromobiphenyl (-DBB) was metabolized at the fastest rate, followed by 3,4,4'-tribromobiphenyl, 3,4,3',4'-tetrabromobiphenyl (-TBB), 2,3,3',4'-TBB, 2,5,3',4'-TBB, and 2,4,2',5'-TBB in decreasing order. It appeared that further bromination prevented metabolism since 2,4,5,3',4'-pentabromobiphenyl (-PBB), 2,3,4,2',4',5'-hexabromobiphenyl (-HBB), and 2,3,4,5,3'.4'-HBB were not metabolized although they possess adjacent nonhalogenated ortho and meta carbons. PB pretreatment increased in vitro rat hepatic microsomal metabolism of PBB congeners which possessed adjacent nonhalogenated meta and para carbons on at least one ring. 2,2'-DBB was metabolized at the fastest rate, followed by 2,4,2',5'-TBB, 2,5,2',5'-TBB, 2,3,3',4'-TBB, 2,5,3',4'-TBB, and 2,4,5,2',5'-PBB in decreasing order. The results suggest that the rates of metabolism of PBB congeners are dependent upon the positions of bromine and the form of cytochrome P-450 induced. In vitro rates of metabolism of 3,4,3',4'-TBB using hepatic microsomes isolated from rats pretreated with either 3,4,5,3',4',5'-HBB or 3,4,3',4'-TBB were also investigated. There was good correlation between the rates of 3,4,3',4'-TBB metabolism, induction of microsomal ethoxyresorufin-O-deethylase activity, and specific content of MC-inducible cytochrome P-450 (P-450 beta NF-B). The results suggest that the isozyme P-450 beta NF-B is responsible for the metabolism of 3,4,3',4'-TBB.
Ferritin and superoxide-dependent lipid peroxidation.
Ferritin was found to promote the peroxidation of phospholipid liposomes, as evidenced by malondialdehyde formation, when incubated with xanthine oxidase, xanthine, and ADP. Activity was inhibited by superoxide dismutase but markedly stimulated by the addition of catalase. Xanthine oxidase-dependent iron release from ferritin, measured spectrophotometrically using the ferrous iron chelator 2,2'-dipyridyl, was also inhibited by superoxide dismutase, suggesting that superoxide can mediate the reductive release of iron from ferritin. Potassium superoxide in crown ether also promoted superoxide dismutase-inhibitable release of iron from ferritin. Catalase had little effect on the rate of iron release from ferritin; thus hydrogen peroxide appears to inhibit lipid peroxidation by preventing the formation of an initiating species rather than by inhibiting iron release from ferritin. EPR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide was used to observe free radical production in this system. Addition of ferritin to the xanthine oxidase system resulted in loss of the superoxide spin trap adduct suggesting an interaction between superoxide and ferritin. The resultant spectrum was that of a hydroxyl radical spin trap adduct which was abolished by the addition of catalase. These data suggest that ferritin may function in vivo as a source of iron for promotion of superoxide-dependent lipid peroxidation. Stimulation of lipid peroxidation but inhibition of hydroxyl radical formation by catalase suggests that, in this system, initiation is not via an iron-catalyzed Haber-Weiss reaction.
Characterization of the photolysis of 2,4,5,2',4',5'-hexabromobiphenyl.
2,4,5,2',4',5'-Hexabromobiphenyl (2,4,5-HBB) was irradiated with ultraviolet light in hexane with stirring, and photolysis was monitored by gas chromatography (GC). 2,4,5-HBB decomposed at an average rate of 0.66 +/- 0.02 mumol/min and the reaction appeared zero order from 0.159 to 1.59 mM 2,4,5-HBB. Several polybrominated biphenyl (PBB) congeners were identified as photoproducts of 2,4,5-HBB. 2,4,5,2',5'-Pentabromobiphenyl (-PBB), formed by para debromination, accumulated at a higher rate than did 2,4,5,3',4'-PBB, formed by ortho debromination. 2,4,5,2',4'-PBB was formed by meta debromination. 3,4,3',4'-Tetrabromobiphenyl (-TBB) was found as a secondary photoproduct, formed by ortho debromination of 2,4,5,3',4'-PBB. 2,5,2',5'-TBB and 2,4,2',5'-TBB were formed by debromination of 2,4,5,2',5'-PBB para and meta, respectively. 2,5,3',4'-TBB could be formed by either ortho debromination of 2,4,5,2',5'-PBB or para debromination of 2,4,5,3',4'-PBB. Rates of degradation and accumulation of the penta- and tetrabrominated biphenyls were also studied. The ultraviolet spectra of the 2,4,5-HBB photolysis mixture, as well as the purified components, were studied and are also reported.
Photolysis products of 2,4,5,2',4',5'-hexabromobiphenyl: hepatic microsomal enzyme induction and toxicity in Sprague-Dawley rats.
The irradiation of 2,4,5,2',4',5'-hexabromobiphenyl (2,4,5-HBB) by ultraviolet light created a mixture of lower brominated polybrominated biphenyl (PBB) congeners. Three photoproducts, 2,4,5,3',4'-pentabromobiphenyl (-PBB), 2,4,5,2',5'-PBB, and 3,4,3',4'-tetrabromobiphenyl (3,4-TBB), as well as 2,4,5-HBB and the photolyzed 2,4,5-HBB mixture, were administered to rats as a single ip injection (90 mg/kg, except 3,4-TBB, which was given at 2 mg/kg) 2 weeks before sacrifice. All treatments except 3,4-TBB induced NADPH-cytochrome P-450 reductase and aminopyrine-N-demethylase activities while all treatments except 2,4,5-HBB induced ethoxyresorufin-O-deethylase and UDP-glucuronosyltransferase activities. Thymus to body weight and spleen to body weight ratios were unchanged compared to controls for all treatments whereas an increase in the liver weights was observed for all treatment groups. Histologic examination revealed that the photolyzed 2,4,5-HBB mixture caused moderate to severe hepatocyte enlargement. Results of tissue analysis for the pure PBB congeners indicated that 2,4,5,2',5'-PBB and 3,4-TBB were metabolized in vivo and this was confirmed by in vitro metabolism studies. The results revealed that the photolyzed 2,4,5-HBB mixture caused a mixed-type induction of hepatic drug-metabolizing enzymes. This is most likely due to the effect of 2,4,5-HBB and toxic congeners formed during the irradiation of 2,4,5-HBB. 2,4,5,3',4'-PBB, which is toxic and apparently not metabolized, is believed to be the major congener contributing to the increased toxicity of the photolyzed 2,4,5-HBB mixture since 3,4-TBB was metabolized and appeared not to be as potent as inducer of aryl hydrocarbon hydroxylase activity.
Post resuscitation iron delocalization and malondialdehyde production in the brain following prolonged cardiac arrest.
Assays for brain tissue malondialdehyde (MDA) and low molecular weight chelated (LMWC) iron were used to examine samples of the cerebral cortex obtained from dogs 2 h after resuscitation from a 15-min cardiac arrest. The effect of post-resuscitation treatment with lidoflazine and/or desferrioxamine was similarly examined. Non-ischemic brain samples had LMWC iron levels (in nmol/100 mg tissue) of 12.32 + 2.60 and MDA levels (in nmol/100 mg tissue) of 8.46 + 1.35. Animals subjected to cardiac arrest and resuscitation and standard intensive care (SIC) had LMWC iron levels of 37.04 + 4.58 (p less than .01 against non-ischemic controls) and MDA levels of 12.24 + 1.9 (p less than .05 against non-ischemic controls). All treatment interventions significantly reduced the LMWC iron (p less than .05), but only treatment with desferrioxamine alone significantly reduced MDA (p less than .05), although a trend toward reduction of the MDA was also evident in animals treated with both desferrioxamine and lidoflazine. LMWC iron levels are increased in the post-ischemic brain, and this increase may be related to lipid peroxidation in the brain following resuscitation from cardiac arrest. These changes are probably pathologic and are amenable to pharmacologic intervention.
Role of metals in oxygen radical reactions.
Partially-reduced forms of dioxygen or "oxy-radicals" (superoxide, O2-/HO2; hydrogen peroxide, H2O2; hydroxyl radical X OH) and oxidants of comparable reactivity are implicated in an increasing number of physiological, toxicological, and pathological states. Transition metal catalysis is recognized as being integral to the generation and the reactions of these activated oxygen species. Factors such as pH and chelation govern the reactivity of the transition metals with dioxygen and "oxy-radicals" and therefore influence the apparent mechanisms by which oxidative damage to phospholipids, DNA, and other biomolecules is initiated. In biological systems the concentrations of redox-active transition metals capable of catalyzing these reactions appears to be relatively low. However, under certain conditions metal storage and transport proteins (ferritin, transferrin, ceruloplasmin, etc.) may furnish additional redox active metals.
Paraquat and ferritin-dependent lipid peroxidation.
A lipid peroxidation system consisting of phospholipid liposomes, paraquat, ADP, and NADPH-cytochrome P450 reductase was constituted using ferritin as the sole source of iron. Lipid peroxidation was completely inhibited by superoxide dismutase, essentially not affected by mannitol, but markedly stimulated by catalase. Similar effects of these scavenging agents were observed in incubations void of ADP. These data suggest that O2-, produced by the redox cycling of paraquat, can release iron from ferritin and thereby promote lipid peroxidation. The effects of catalase and mannitol suggest that the initiation of peroxidation, in either the presence or absence of ADP, is not significantly dependent upon the hydroxyl radical produced via an iron-catalyzed Haber-Weiss reaction.
Rat liver microsomal NADPH-dependent release of iron from ferritin and lipid peroxidation.
Microsomes prepared by the usual method of differential centrifugation were found to contain ferritin, superoxide dismutase (SOD), and catalase which could be removed by chromatography on Sepharose CL-2B. Addition of purified rat liver ferritin to chromatographed microsomes resulted in a significant stimulation of NADPH-dependent lipid peroxidation which was inhibited by exogenously added SOD. Iron release from ferritin by these microsomes was also inhibited by SOD. Ferritin did not promote NADPH-dependent microsomal lipid peroxidation when added to microsomes isolated in the usual manner, presumably due to the endogenous SOD present in the microsomes. Accordingly, only very low rates of iron release from ferritin were observed with these microsomes. Paraquat (PQ), which generates superoxide O2-. via redox cycling, greatly stimulated iron release from ferritin and lipid peroxidation in chromatographed microsomes. Paraquat had no effect on iron release from ferritin or lipid peroxidation in microsomes. which were not chromatographed unless they were first treated with CN- to inhibit endogenous SOD. These studies indicate that the majority of microsomal iron is contained within ferritin and that following release by O2-. this iron serves to promote the peroxidation of microsomal lipids.
Postischemic tissue injury by iron-mediated free radical lipid peroxidation.
Cell damage initiated during ischemia matures during reperfusion. Mechanisms involved during reperfusion include the effects of arachidonic acid and its oxidative products prostaglandins and leukotrienes, reperfusion tissue calcium overloading, and damage to membranes by lipid peroxidation. Lipid peroxidation occurs by oxygen radical mechanisms that require a metal with more than one ionic state (transitional metal) for catalysis. We have shown that cellular iron is delocalized from the large molecules where it is normally stored to smaller chemical species during postischemic reperfusion. Postischemic lipid peroxidation is inhibited by the iron chelator deferoxamine. Intervention in the reperfusion injury of membranes by chelation of transitional metals is a new and promising therapeutic possibility for protection of the heart and brain.
Cardiac arrest and resuscitation: brain iron delocalization during reperfusion.
We hypothesize that brain injury from cardiac arrest occurs during reperfusion and is in part mediated by iron-dependent lipid peroxidation. We conducted a study to examine the time course of brain iron delocalization and lipid peroxidation in an animal model of cardiac arrest and resuscitation. Assays for brain tissue iron in low-molecular-weight species (LMWS iron) used the o-phenanthroline test on an ultrafiltered (molecular weight less than 30,000) tissue sample; malondialdehyde (MDA), a product of lipid peroxidation, in brain tissue was assayed by the thiobarbituric acid test (TBA). Samples of the parietal cortex from 11 nonischemic control dogs (Group 1) had LMWS iron levels of 9.6 +/- 4.9 nmol/100 mg tissue and MDA levels of 7.7 +/- 2.0 nmol/100 mg tissue. Samples from the parietal cortex taken from five dogs after 15 minutes of cardiac arrest (Group 2) had LMWS iron levels of 9.3 +/- 3.1 nmol/100 mg tissue and MDA levels of 6.1 +/- 1.0 nmol/100 mg tissue. Samples from the parietal cortex taken from five dogs after 45 minutes of cardiac arrest (Group 3) had LMWS iron levels of 6.7 +/- 3.3 nmol/100 mg tissue and MDA levels of 5.6 +/- 0.4 nmol/100 mg tissue. There was no significant difference among the three groups for either LMWS iron or MDA. Five dogs were subjected to 15 minutes of cardiac arrest and definitive resuscitation by internal cardiac massage and defibrillation (Group 4). Following resuscitation the chest was closed and the dogs were given intensive care for two hours.(ABSTRACT TRUNCATED AT 250 WORDS)