Inability of desferrioxamine to limit tissue injury in the ischaemic and reperfused rabbit heart.

The role of iron-catalysed hydroxyl radical production in development of myocardial injury was examined in an in vivo rabbit heart with regional ischaemia (45 min) and reperfusion (3 h). Open-chest anaesthetised rabbits were assigned to control (saline) or desferrioxamine-treated [30 mg/kg subcutaneously (s.c.) plus 1, 10, 20, or 100 mg/kg/h intravenously (i.v.) starting 15 min before ischaemia] groups. Haemodynamics and ECG were monitored throughout. Following reperfusion, hearts were excised and perfused in vitro with buffer. The artery was religated, and fluorescent particles were injected to delineate the ischaemic zone. Tetrazolium staining was used to define necrosis. Planimetry was performed on photographed heart slices for calculation of the size of the hearts and of the ischaemic and infarcted zones. Infarct size (as a percentage of ischaemic zone size) in control hearts was 51.2 +/- 5.8% (n = 10) and in the groups treated with desferrioxamine 1, 10, 20, and 100 mg/kg/h it was 53.1 +/- 11.7% (n = 8), 45.3 +/- 6.3% (n = 9), 65.1 +/- 10.1% (n = 6), and 52.5 +/- 9.4% (n = 7), respectively (p = NS vs. control in each instance). In addition, no antiarrhythmic effects were observed at any dose. Thus, iron-catalysed hydroxyl radical damage may not play a role in the pathogenesis of tissue injury under these conditions.

Metabolism of metronidazole and antipyrine in isolated rat hepatocytes. Influence of sex and enzyme induction and inhibition.

The metabolism of metronidazole and antipyrine was investigated in freshly isolated hepatocytes from 7 male and 6 female control Wistar rats, 8 males and 5 females pretreated with phenobarbital (PB) and 3 males pretreated with 3-methylcholanthrene (MC). Pretreatment with PB increased the intrinsic clearance (CLi = Vmax/Km) of metronidazole to its acetic acid (MAA) and hydroxy metabolite (HM) 7- and 2.8-fold in the males and 3.2- and 3.0-fold in the females, whereas MC treatment increased the values 9- and 10-fold, respectively (P less than 0.05). The CLi of metronidazole to HM and its glucuronide conjugate was higher in the control and PB treated male than in the corresponding female groups, whereas the rank order was reversed for sulphate formation (P less than 0.05). SKF 525A was a more potent inhibitor of MAA formation than of HM formation, except in the PB treated male group. Pretreatment with MC increased the inhibitory potency of alpha-naphthoflavone and antipyrine toward MAA and HM formation. In male rats PB treatment increased the CLi of antipyrine to 3-hydroxymethyl-(HMAP), nor-(NORAP) and 4-hydroxyantipyrine (OHAP) 2.5-, 2.1- and 4.5-fold, respectively (P less than 0.05). Pretreatment with MC in male and with PB in female rats had no significant effect on antipyrine metabolism. SKF 525A was a more potent inhibitor of HMAP and OHAP formation than of NORAP formation. Treatment with MC increased the inhibitory potency of alpha-naphthoflavone toward the formation of all antipyrine metabolites. Metronidazole increased the formation rate of HMAP, but inhibited the formation of NORAP and OHAP, particularly the latter. The results suggest that the formation of MAA, HM, HMAP, NORAP and OHAP from metronidazole and antipyrine is catalyzed by different cytochrome P-450 isozymes, which may be supplemented or substituted by PB or MC induced species. The involved P-450 isozymes have more or less overlapping substrate and product specificity. Metronidazole appears to be a sensitive probe for detection and identification of PB and MC type induction.

Dusts causing pneumoconiosis generate .OH and produce hemolysis by acting as Fenton catalysts.

Silicates causing pneumoconiosis function as Fenton catalysts to generate hydroxyl radicals (.OH) when incubated with hydrogen peroxide and a reducing substance. In contrast, silicates which do not cause pneumoconiosis demonstrate no Fenton activity. Catalytic activity is decreased by pretreatment of silicates with the iron chelators deferoxamine or transferrin. Hemolysis from silicates is decreased by interventions which remove superoxide anion or hydrogen peroxide from the medium, or by pretreatment of dusts with iron chelators. Thus, asbestos and nonfibrous silicates may cause pneumoconiosis through a common oxidant mechanism by catalyzing production of toxic .OH radicals in the lung.

Oxidant-induced DNA damage of target cells.

In this study we examined the leukocytic oxidant species that induce oxidant damage of DNA in whole cells. H2O2 added extracellularly in micromolar concentrations (10-100 microM) induced DNA strand breaks in various target cells. The sensitivity of a specific target cell was inversely correlated to its catalase content and the rate of removal of H2O2 by the target cell. Oxidant species produced by xanthine oxidase/purine or phorbol myristate acetate-stimulated monocytes induced DNA breakage of target cells in proportion to the amount of H2O2 generated. These DNA strand breaks were prevented by extracellular catalase, but not by superoxide dismutase. Cytotoxic doses of HOCl, added to target cells, did not induce DNA strand breakage, and myeloperoxidase added extracellularly in the presence of an H2O2-generating system, prevented the formation of DNA strand breaks in proportion to its H2O2 degrading capacity. The studies also indicated that H2O2 formed hydroxyl radical (.OH) intracellularly, which appeared to be the most likely free radical responsible for DNA damage: .OH was detected in cells exposed to H2O2; the DNA base, deoxyguanosine, was hydroxylated in cells exposed to H2O2; and intracellular iron was essential for induction of DNA strand breaks.

Differential formation of hydroxyl radicals by adriamycin in sensitive and resistant MCF-7 human breast tumor cells: implications for the mechanism of action.

Adriamycin-stimulated formation of .OH in sensitive and resistant subline of human breast tumor cells (MCF-7) has been examined by electron spin resonance spectroscopy. It was shown that adriamycin significantly stimulated the formation of .OH spin adducts [5,5-dimethyl-1-pyrroline N-oxide (DMPO)-OH] in the sensitive cells but not in the resistant cells. By use of spin-broadening techniques and inhibition of .OH with high molecular weight poly(ethylene glycol), which does not enter intact cells, it was shown that 60-65% of adriamycin-induced .OH were located extracellularly and were metal ion dependent since they were decreased in the presence of desferal. Furthermore, superoxide dismutase and catalase, enzymes that detoxify superoxide and hydrogen peroxide, also significantly inhibited adriamycin-induced .OH formation and protected against the cytotoxicity of adriamycin. The differential .OH formation in these two cell lines is not due to diminished activities of flavin-dependent activating enzymes nor decreased accumulation of the drug in the cells but appears to be related to enhanced activities of detoxifying enzymes, particularly, glutathione peroxidases in the resistant cells.

Myeloperoxidase as an effective inhibitor of hydroxyl radical production. Implications for the oxidative reactions of neutrophils.

Hydroxyl radicals have been generated from hydrogen peroxide and superoxide (produced with xanthine oxidase), and an iron (EDTA) catalyst, and detected with deoxyribose, or in some cases with benzoate or alpha-keto-gamma-methiolbutyric acid. Purified myeloperoxidase, and neutrophils stimulated with fMet-Leu-Phe and cytochalasin B, strongly inhibited this hydroxyl radical production in a concentration-dependent manner. Supernatants from stimulated cells also inhibited, and inhibition by cells or supernatant was prevented by azide. There was much less inhibition by myeloperoxidase-deficient neutrophils. Inhibition thus was due to myeloperoxidase released by the cells. With neutrophils stimulated with phorbol myristate acetate, which release very little myeloperoxidase, hydroxyl radical production was enhanced due to the additional superoxide produced by the cells. It is concluded that under conditions where neutrophils release myeloperoxidase as well as superoxide and hydrogen peroxide, breakdown of hydrogen peroxide by myeloperoxidase would make conditions unfavorable for hydroxyl radical production.

Free radical damage to cultured porcine aortic endothelial cells and lung fibroblasts: modulation by culture conditions.

Culture conditions modulating cell damage from xanthine plus xanthine oxidase-derived partially reduced oxygen species were studied. Porcine thoracic aorta endothelial cells and porcine lung fibroblasts were maintained in monolayer culture. Cells were prelabeled with 51Cr before xanthine plus xanthine oxidase exposure. Endothelial cells showed 30 to 100% more lysis than fibroblasts and thus seemed more sensitive to this oxidant stress. The effect of cell culture age, as indicated by population doubling level (PDL), was examined. Response of low PDL endothelial cells and fibroblasts subjected to oxidant stress was compared with the response of PDL 15 cells. Both low PDL endothelial cells and fibroblasts responded differently to the lytic effect of xanthine oxidase-derived free radicals than did higher PDL cells. Specific activities of the antioxidant enzymes catalase, manganese superoxide dismutase, copper-zinc superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase were measured in both low and high PDL fibroblasts and endothelial cells. Antioxidant enzyme specific activities could only partially explain the differences in response to oxidant stress between fibroblasts and endothelial cells and between low and high PDL cells. Cell culture medium composition modulated the rate of production, and relative proportions of xanthine plus xanthine oxidase-derived partially reduced species of oxygen, i.e. superoxide, hydrogen peroxide, and hydroxyl radical. Serum content of medium was important in modulating free radical generation; superoxide production rates decreased 32%, H2O2 became undetectable, and hydroxyl radical generation decreased 54% in the presence of 10% serum. The medium protein and iron content also modulated free radical generation. The data suggest that cell culture media constituents, cell type, and cell culture age greatly affect in vitro response of cells subjected to oxidant stress.

Prevention of granulocyte-mediated oxidant lung injury in rats by a hydroxyl radical scavenger, dimethylthiourea.

Toxic, partially reduced metabolites of oxygen (toxic oxygen radicals) are increasingly implicated in acute leukocyte-mediated tissue injury. To further probe the roles of oxygen radicals in acute lung edema, I studied the effects of a recently described and very potent oxygen radical scavenger, dimethylthiourea (DMTU) (Fox, R. B., R. N. Harada, R. M. Tate, and J. E. Repine, 1983, J. Appl. Physiol., 55:1456-1459) on polymorphonuclear leukocyte (PMN) oxidant function and on two types of lung injury mediated by oxygen radicals and PMN. DMTU (10 mM) blocked 79% of hydroxyl radical (OH) production by PMN in vitro without interfering with other PMN functions, such as O-2 production, myeloperoxidase activity, chemotaxis, degranulation, or aggregation. When isolated rat lung preparations were perfused with PMN activated to produce OH, lung weights were increased from 2.3 +/- 0.2 to 11.2 +/- 0.8 g. DMTU (10 mM) prevented 70% of these increases (lung weights, 5.0 +/- 1.1 g, P less than 0.005). Finally, when intact rats were exposed to 100% O2 for 66 h, lung weight:body weight ratios were increased from 5.78 +/- 0.33 to 8.87 +/- 0.16 g. DMTU (500 mg/kg) prevented 83% of this hyperoxia-induced lung edema in vivo (lung:body weight ratios, 6.05 +/- 0.21, P less than 0.001). Pharmacokinetic studies showed that DMTU diffused effectively into lung interstitial fluids and had a relatively long half-life (25-35 h) in the circulation. Because a variety of oxygen radicals, such as superoxide (O-2), hydrogen peroxide (H2O2), or OH are produced by PMN, there is usually some uncertainty about which one is responsible for injury. However, in these studies, DMTU did not scavenge O-2 and scavenged H2O2 only very slowly while scavenging OH very effectively. Therefore, DMTU may be useful in the investigation of the roles of oxygen radicals, especially OH, in acute granulocyte-mediated tissue injury.

The role of iron chelates in hydroxyl radical production by rat liver microsomes, NADPH-cytochrome P-450 reductase and xanthine oxidase.

Uninduced rat liver microsomes and NADPH-Cytochrome P-450 reductase, purified from phenobarbital-treated rats, catalyzed an NADPH-dependent oxidation of hydroxyl radical scavenging agents. This oxidation was not stimulated by the addition of ferric ammonium sulfate, ferric citrate, or ferric-adenine nucleotide (AMP, ADP, ATP) chelates. Striking stimulation was observed when ferric-EDTA or ferric-diethylenetriamine pentaacetic acid (DTPA) was added. The iron-EDTA and iron-DTPA chelates, but not unchelated iron, iron-citrate or iron-nucleotide chelates, stimulated the oxidation of NADPH by the reductase in the absence as well as in the presence of phenobarbital-inducible cytochrome P-450. Thus, the iron chelates which promoted NADPH oxidation by the reductase were the only chelates which stimulated oxidation of hydroxyl radical scavengers by reductase and microsomes. The oxidation of aminopyrine, a typical drug substrate, was slightly stimulated by the addition of iron-EDTA or iron-DTPA to the microsomes. Catalase inhibited potently the oxidation of scavengers under all conditions, suggesting that H2O2 was the precursor of the hydroxyl radical in these systems. Very high amounts of superoxide dismutase had little effect on the iron-EDTA-stimulated rate of scavenger oxidation, whereas the iron-DTPA-stimulated rate was inhibited by 30 or 50% in microsomes or reductase, respectively. This suggests that the iron-EDTA and iron-DTPA chelates can be reduced directly by the reductase to the ferrous chelates, which subsequently interact with H2O2 in a Fenton-type reaction. Results with the reductase and microsomal systems should be contrasted with results found when the oxidation of hypoxanthine by xanthine oxidase was utilized to catalyze the production of hydroxyl radicals. In the xanthine oxidase system, ferric-ATP and -DTPA stimulated oxidation of scavengers by six- to eightfold, while ferric-EDTA stimulated 25-fold. Ferric-desferrioxamine consistently was inhibitory. Superoxide dismutase produced 79 to 86% inhibition in the absence or presence of iron, indicating an iron-catalyzed Haber-Weiss-type of reaction was responsible for oxidation of scavengers by the xanthine oxidase system. These results indicate that the ability of iron to promote hydroxyl radical production and the role that superoxide plays as a reductant of iron depends on the nature of the system as well as the chelating agent employed.

Role of hydroxyl radical in polymorphonuclear leukocyte-mediated bactericidal activity.

Among the antimicrobial systems of polymorphonuclear leukocytes (PMNs) are those that are oxygen-dependent. Recent interest has been directed at the hydroxyl radical(OH.), a highly reactive reduction product of oxygen, as a possible mediator of microbicidal activity. The role of OH. in PMN microbicidal activity is discussed in relation to the observations that (1) an enzymic OH. generating system consisting of xanthine oxidase plus acetaldehyde is bactericidal, (2) phagocytosing PMNs convert substrates like methional and 5,5-dimethyl pyrroline-1-oxide to products suggestive of OH.-mediated reactions and (3) PMN-mediated microbicidal activity is partially inhibited by scavengers of OH.

Indicator media for microorganisms degrading chlorinated pesticides.

A bromocresol purple liquid indicator medium and an eosin-methylene blue agar have been developed for the demonstration and isolation of microorganisms able to degrade the chlorinated herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D). Plates of the eosin-methylene blue agar indicate individual 2,4-D-degrading bacterial colonies. Both indicator systems show the production of acid, presumably hydrochloric, during degradation of the 2,4-D in the media. Concentrations of 2,4-D required to give an acid reaction in media with varying concentrations of yeast extract were determined; the production of about 0.24 mmol of hydrochloric acid seems necessary to counteract the buffering effect of 100 mg of metabolized yeast extract. Acid production from the herbicide, 4-chloro-2-methylphenoxyacetic acid(MCPA), which in the salt form could yield only small amounts of hydrochloric acid, was inconsistent. The two indicator media should be useful in investigations of the microbial degradation of other acid-yielding halogenated pesticides.