Differential role of reactive oxygen intermediates in photofrin-I- and photofrin-II-mediated photoenhancement of lipid peroxidation in epidermal microsomal membranes.

Photoradiation therapy with porphyrins and light offers an alternative approach to the management of certain types of cancer. The mechanism of tissue destruction mediated by this modality is poorly understood. In this study, epidermal microsomes incubated in vitro with Photofrin-I (Pf-I) and Photofrin-II (Pf-II) followed by exposure to radiation (approximately 400 nm) resulted in increased (180%) NADPH-supported (enzymatic) as well as ADP/iron-supported (140%) (nonenzymatic) lipid peroxidative damage as measured by malondialdehyde formation. Lipid peroxidation by Pf-I and Pf-II was found to be differentially affected by quenchers of singlet oxygen (2,5-dimethylfuran, histidine, beta-carotene, ascorbic acid, and sodium azide), superoxide anion (superoxide dismutase), and the hydroxyl radical (sodium benzoate, mannitol, and ethanol). Catalase, a quencher of hydrogen peroxide, afforded significant protection only against Pf-II-enhanced lipid peroxidative damage while it had little effect against the Pf-I-mediated reaction. Deuterium oxide, which is known to increase the half-life of singlet oxygen, was found to enhance Pf-I-mediated lipid peroxidation but produced insignificant effects upon Pf-II-mediated photosensitization. Our results indicate that Pf-I and Pf-II, which are employed for the photodynamic therapy of malignant tumors, evoke membrane damage by generating different reactive oxygen species. The Pf-I-mediated photodestruction mainly involves a type II mechanism via singlet oxygen formation, whereas Pf-II-mediated photodestruction preferentially involves a type I mechanism by generating superoxide anions and hydroxyl radicals. Our data indicate that tumor necrosis evoked by porphyrins and light is likely due to the generation of reactive oxygen species.

Free radicals, immunoglobulins and complement as mediators of inflammation.

There is evidence for both oxygen-centred free radicals and products of complement activation acting as mediators of inflammation. Evidence for the generation and reaction of free radicals at sites of inflammation can only be indirect and circumstantial due to their very transient nature. Evidence for complement activation in several inflammatory conditions, including rheumatoid arthritis is strong. These mediator classes individually possess a range of potential proinflammatory activities. Their effects may be linked through the formation of immune complexes and the activation of polymorphonuclear leukocytes. Their actions will also be linked with and modulated by the activities of other mediators mentioned only briefly in this chapter. The relative importance of the different mediators in any particular inflammatory condition is difficult to ascertain. The importance of free radicals and complement will be better understood when drugs specifically and unequivocally aimed at their control are identified. This potential for therapeutic advances in the treatment of inflammatory disorders has yet to be realized.

Evidence suggesting a role for hydroxyl radical in passive Heymann nephritis in rats.

We examined the effect of scavengers of reactive oxygen metabolites on proteinuria in the passive Heymann nephritis model of membranous nephropathy. Passive Heymann nephritis was induced by a single intravenous injection of anti-Fx1A IgG in a dose of 10 mg/100 g body weight. Superoxide dismutase, a scavenger of superoxide or catalase which destroys hydrogen peroxide, did not affect the proteinuria. In contrast, dimethylthiourea (DMTU, 500 mg/kg followed by 125 mg/kg ip twice a day), a scavenger of hydroxyl radical, markedly reduced the proteinuria (day 5: anti-Fx1A 53 +/- 13, n = 18; anti-Fx1A + DMTU, 21 +/- 6 mg/24 h, n = 15, P less than 0.001). Experiments with 125I-labeled anti-Fx1A antibody demonstrated that DMTU did not affect the amount of antibody deposited in the kidney. Semiquantitative estimation of IgG and complement deposition in the kidney showed no differences between the DMTU-treated and control rats. A second hydroxyl radical scavenger, sodium benzoate (150 mg/kg ip twice a day), also resulted in marked reduction in proteinuria (day 5: anti-Fx1A 56 +/- 7, n = 9; anti-Fx1A + benzoate, 14 +/- 4 mg/24 h, n = 8, P less than 0.01). Because of the participation of iron in biological systems to generate hydroxyl radical, we also examined the effect of deferoxamine (DFO, 35 mg/day), an iron chelator, on the anti-Fx1A-induced proteinuria. There was a significant reduction in proteinuria in rats treated concurrently with DFO (day 5: anti-Fx1A 67 +/- 13, n = 15; anti-Fx1A + DFO, 29 +/- 4 mg/24 h, n = 15, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for HCO3- conductance pathways in nutrient membrane of bullfrog antrum.

The effect of changing the nutrient HCO3- concentration on potential difference (PD) and resistance in bullfrog antrum bathing in CI- media was determined. Changes in HCO3- concentration were from 25 mM to several lower concentrations and back to 25 mM. A plot of /delta PD/ versus log [HCO3-] gave a linear relation for changes of HCO3- concentration from 25 down to 3.1 mM and back to 25 mM but deviated to some extent for changes to 1.6 mM. In these experiments, changes from higher to lower HCO3- concentrations gave a less rapid initial PD response than those in the reverse direction. This result eliminated H+ conductance pathways as being predominant. Experiments were done in which in the first part changes were made in nutrient solution from 5 percent CO2 and 25 mM HCO3- to 0.6 percent CO2 and 3 mM HCO3- and in the second part the same changes with a simultaneous changes of secretory solution from 5 percent to 10 percent CO2. The magnitude of PD decrease was greater by 4.5 mV in the second part. This result indicated that HCO3- conductance pathways rather than OH- conductance pathways are predominated . There was no evidence of HCO3-, OH-, and H+ conductance pathways in secretory membranes.

Lipid peroxidation and acute lung injury after thermal trauma to skin. Evidence of a role for hydroxyl radical.

The authors have previously shown that thermal injury to the skin of rats results in the development of acute lung injury that is susceptible to systemic treatment of animals with catalase and dependent on the presence of neutrophils. The current studies have been expanded for exploration of the nature of the neutrophil-derived oxygen products responsible for the lung injury and have also focused on evidence of the appearance of products of lipid peroxidation (conjugated dienes). With respect to the former, treatment of rats with iron chelators (deferoxamine mesylate, 2,3-dihydroxybenzoic acid), with scavengers of hydroxyl radical (dimethyl sulfoxide, dimethyl thiourea, sodium benzoate), or with vitamin E affords a significant degree of protection from acute lung injury as assessed by changes in lung vascular permeability and by morphologic parameters. These data suggest that lung vascular injury after thermal trauma of the skin is related to the generation by neutrophils of the hydroxyl radical. Conjugated dienes have been demonstrated to appear sequentially both in the burned skin (at 1/4 hour) and in the lungs (at 2 hours), as well as in the plasma (with peaks at 1/2 and at 3 hours) after thermal injury. The appearance of the conjugated dienes in plasma at the two intervals of time is greatly diminished if animals are pretreated with the iron chelator deferoxamine, with catalase, or with scavengers of hydroxyl radical. Furthermore, the appearance of conjugated dienes in plasma at 30 minutes and 3 hours is significantly diminished if animals are depleted of neutrophils, complement-depleted, or the burned skin is excised immediately after thermal injury. These data indicate a linkage between thermal trauma of skin, secondary injury of lung, and appearance in plasma and tissues of products of lipid peroxidation.

Role of oxygen-derived free radicals in hemorrhagic shock-induced gastric lesions in the rat.

This study was designed to determine whether oxygen-derived free radicals play a role in the pathogenesis of gastric lesions produced by hypotensive ischemia in the rat. To achieve this goal, allopurinol, an inhibitor of xanthine oxidase (the enzyme responsible for the formation of superoxide radicals); superoxide dismutase, a scavenger of superoxide radicals (O2-); and dimethyl sulfoxide, a scavenger of hydroxyl radicals (OH) were used. In the anesthetized rat, HCl (0.1 N) was instilled into the pylorus-ligated stomach, and the rat was bled to reduce the blood pressure to less than 30 mmHg. The blood pressure was maintained at less than 30 mmHg for 20 min and then the shed blood was retransfused. Twenty minutes after the retransfusion the rat was killed, the stomach was removed, and the area of gastric mucosal lesions was measured. Both allopurinol and superoxide dismutase, but not dimethyl sulfoxide, significantly protected against hemorrhagic shock-induced gastric lesions. These findings suggest that oxygen-derived free radicals, particularly O2-, play an important role in the formation of gastric lesions produced by ischemia plus HCl.

Natural killer cell-mediated lysis involves an hydroxyl radical-dependent step.

The role of oxygen radicals in lysis of K562 target cells by human natural killer (NK) cells was determined by addition of scavengers of these free radicals. Lysis was greatly reduced under hypoxic conditions. Superoxide dismutase and cytochrome c, scavengers of superoxide anions, and catalase and scavengers of hypochlorite had no effect on lysis. Of 15 hydroxyl radical scavengers tested, 13 inhibited lysis. These were not toxic, because cell morphology and spontaneous chromium release were not affected and preculture with scavengers was not inhibitory. These scavengers differed widely in structure, but degree of inhibition of lysis correlated with their rate constants (k) for reaction with hydroxyl radical (k vs log inhibitor concentration required to decrease lysis by 50%: r = -0.9202, p less than 0.001), showing that inhibition was due to inactivation of the hydroxyl radical. Target cell binding was not reduced at concentrations that inhibited lysis. Inhibitors of the lipoxygenase pathway also decreased lysis, suggesting this pathway to be the source of hydroxyl radicals. In view of the reported requirements for hydroxyl radical-mediated lipid peroxidation for optimal secretory activity in a number of cell types, it appears that the generation of hydroxyl radicals by NK cells is required for delivery of cytotoxic factors.

The hydroxyl-hydrogen ion concentration ratio during hypothermia.

Fundamental physicochemical characteristics of the acid-base related constituents of extracellular and intracellular fluid spaces of vertebrates in relation to changes in temperature have been reviewed. Emphasis has been placed upon the dissociation constant of water, the solubility constant of CO2, the dissociation constant of histidine imidazole, the hydroxyl-hydrogen ion ratio, the protein charge state and the alpha-imidazole regulation concept. Because pN and pKIm change in parallel when temperature varies, the OH/H ratio and the alpha-imidazole value for any sample of blood or plasma held anaerobically in vitro are invariant with changing temperature, since a constant CO2 content is maintained. Thus, when blood or plasma cools, pH increases and PCO2 decreases, but relative alkalinity and the protein charge state remain constant. These responses are solely the consequence of physical constants, that is, equilibrium constants and gas solubility, changing with temperature. In vivo, the set of PCO2 is established in each poikilothermic species by its normal ventilatory pattern designed to maintain constant CO2 content. Regulation in vivo in poikilotherms consists of adjustments of ventilation per unit metabolism (VA/VCO2) appropriate to every temperature. When the ventilatory and renal mechanisms of human beings are suppressed by anesthesia and hypothermia, their extracellular and intracellular responses mimic those of poikilotherms. Clinical management of hypothermia in humans requires ventilatory control using oxygen-augmented room air without added CO2 monitored by pH measurements of arterial blood warmed anaerobically to 37 degrees C. Finally, the need for new techniques to measure intracellular pH as temperature is lowered and some areas for further investigation are suggested.