The role of free radicals in asbestos-induced diseases.

Asbestos exposure causes pulmonary fibrosis and malignant neoplasms by mechanisms that remain uncertain. In this review, we explore the evidence supporting the hypothesis that free radicals and other reactive oxygen species (ROS) are an important mechanism by which asbestos mediates tissue damage. There appears to be at least two principal mechanisms by which asbestos can induce ROS production; one operates in cell-free systems and the other involves mediation by phagocytic cells. Asbestos and other synthetic mineral fibers can generate free radicals in cell-free systems containing atmospheric oxygen. In particular, the hydroxyl radical often appears to be involved, and the iron content of the fibers has an important role in the generation of this reactive radical. However, asbestos also appears to catalyze electron transfer reactions that do not require iron. Iron chelators either inhibit or augment asbestos-catalyzed generation of the hydroxyl radical and/or pathological changes, depending on the chelator and the nature of the asbestos sample used. The second principal mechanism for asbestos-induced ROS generation involves the activation of phagocytic cells. A variety of mineral fibers have been shown to augment the release of reactive oxygen intermediates from phagocytic cells such as neutrophils and alveolar macrophages. The molecular mechanisms involved are unclear but may involve incomplete phagocytosis with subsequent oxidant release, stimulation of the phospholipase C pathway, and/or IgG-fragment receptor activation. Reactive oxygen species are important mediators of asbestos-induced toxicity to a number of pulmonary cells including alveolar macrophages, epithelial cells, mesothelial cells, and endothelial cells. Reactive oxygen species may contribute to the well-known synergistic effects of asbestos and cigarette smoke on the lung, and the reasons for this synergy are discussed. We conclude that there is strong evidence supporting the premise that reactive oxygen species and/or free radicals contribute to asbestos-induced and cigarette smoke/asbestos-induced lung injury and that strategies aimed at reducing the oxidant stress on pulmonary cells may attenuate the deleterious effects of asbestos.     

Characterization of genetic instability in radiation- and benzene-induced murine acute leukemia.

This study, using the CBA/Ca mouse as a model, compares genetic lesions associated with radiation- and benzene-induced acute leukemias. Specific types of leukemia included in the analyses are radiation-induced acute myeloid leukemia (ML), and benzene-induced lymphoblastic leukemias, lymphomas, or mix-lineage leukemias. These leukemias have histopathological characteristics similar to those seen in human acute leukemias. G-band cytogenetic analysis showed that specific deletions involving regions D-E of one copy of mouse chromosome 2 [del(2)(D-E)] were frequently associated in both radiation- and benzene-induced acute leukemias. In addition, translocations of chr2(D-E) were also observed in some cases. These results suggest an important role of chr2 (D-E) deletions and translocations in the development of radiation- and benzene-induced murine acute leukemias. Fluorescence in situ hybridization with DNA probes specific for 2(D-E), constructed in our laboratory by means of chromosomal microdissection and PCR amplification, also demonstrate 2(D-E) deletions and/or translocations in these leukemic cells. Aneuploidy of chromosomes 3, 15, 16, and Y were also frequently detected in benzene-induced leukemic cells with or without lesions on chr2. These cytogenetic findings support the previous observations that metabolites of benzene lead to spindle-fiber disruption or abnormal cytokinesis in exposed animals. In summary, genetic instabilities observed in leukemic cells isolated from mice that had developed leukemia after exposure to radiation or benzene are syntenic with those frequently detected in patients with myelodysplastic syndrome, acute ML, and acute lymphoblastic leukemia. Thus, the CBA/Ca mouse has several characteristics that make it an excellent model for the study of radiation or benzene leukemogenesis in humans.     

The role of free radicals in cold injuries

Cold injury is a tissue trauma produced by exposure to freezing temperatures and even brief exposure to a severely cold and windy environment. Rewarming of frozen tissue is associated with blood reperfusion and the simultaneous generation of free oxygen radicals. In this review is discussed the current understanding of the mechanism of action of free oxygen radicals as related to cold injury during rewarming. Decreased energy stores during ischaemia lead to the accumulation of adenine nucleotides and liberation of free fatty acids due to the breakdown of lipid membranes. On rewarming, free fatty acids are metabolized via cyclo-oxygenase and adenine nucleotides are metabolized via the xanthine oxidase pathway. These may be the source of free oxygen radicals. Leukocytes may also play a major role in the pathogenesis of cold injury. Oxygen radical scavengers, such as superoxide dismutase and catalase, may help to reduce the cold induced injury but their action is limited due to the inability readily to cross the plasma membrane. Lipid soluble antioxidants are likely to be more effective scavengers because of their presence in membranes where peroxidative reactions can be arrested.     

The role of free radicals in the pathophysiology of muscular dystrophy.

Null mutation of any one of several members of the dystrophin protein complex can cause progressive, and possibly fatal, muscle wasting. Although these muscular dystrophies arise from mutation of a single gene that is expressed primarily in muscle, the resulting pathology is complex and multisystemic, which shows a broader disruption of homeostasis than would be predicted by deletion of a single-gene product. Before the identification of the deficient proteins that underlie muscular dystrophies, such as Duchenne muscular dystrophy (DMD), oxidative stress was proposed as a major cause of the disease. Now, current knowledge supports the likelihood that interactions between the primary genetic defect and disruptions in the normal production of free radicals contribute to the pathophysiology of muscular dystrophies. In this review, we focus on the pathophysiology that results from dystrophin deficiency in humans with DMD and the mdx mouse model of DMD. Current evidence indicates three general routes through which free radical production can be disrupted in dystrophin deficiency to contribute to the ensuing pathology. First, constitutive differences in free radical production can disrupt signaling processes in muscle and other tissues and thereby exacerbate pathology. Second, tissue responses to the presence of pathology can cause a shift in free radical production that can promote cellular injury and dysfunction. Finally, behavioral differences in the affected individual can cause further changes in the production and stoichiometry of free radicals and thereby contribute to disease. Unfortunately, the complexity of the free radical-mediated processes that are perturbed in complex pathologies such as DMD will make it difficult to develop therapeutic approaches founded on systemic administration of antioxidants. More mechanistic knowledge of the specific disruptions of free radicals that underlie major features of muscular dystrophy is needed to develop more targeted and successful therapeutic approaches.     

The role of free radicals in cerebral hypoxia and ischemia

This review focuses on the effects that ischemia and hypoxia have on the cerebral cortex and the cerebellum during different periods of life. The acute interruption or reduction of cerebral blood flow, that can be induced by several factors and clinical pathologies, reduces available oxygen to the nervous system and this causes either focal or global brain damage, with characteristic biochemical and molecular alterations that can result in permanent or transitory neurological sequelae or even death. Under these circumstances, an increase in the activity of different isoforms of nitric oxide synthase occurs and nitric oxide is produced. This excess of nitric oxide reacts with cellular proteins yielding nitrotyrosine, thus contributing to cerebral damage. This phenomenon has been studied at different stages of perinatal and postnatal development, including aging animals. Both the duration and the intensity of the ischemic injury were evaluated. In all cases there is overproduction of nitric oxide in ischemia, which may represent an effort to reestablish normal blood flow. Unfortunately, in many cases this response becomes excessive and it triggers a cascade of free-radical reactions, leading to modifications of cerebral plasticity and overt injury.     

Physico-chemical modeling of the role of free radicals in photodynamic therapy. IV. Quantitative aspects of photodynamic effects on free radicals generated in cell cultures

Production and the mechanism of the interactions of free radicals generated by stimulated macrophages in the presence of luminol and a free radical inhibitor was investigated to determine the possibility of using luminol-dependent chemiluminescence for studying photodynamic effects in biology. Earlier measurements have been revisited and additional experiments performed indicating that oxidation products of luminol neither inhibit the in vitro formation of radicals nor quench CL. Simulation based on the mechanism suggested revealed that the likely value for the rate constant of the primary step between luminol and superoxide anion radicals producing luminol radicals is 5x10(2)-1x10(3) M-1s-1. It has been established that the ratio of the concentration of radicals generated by the biological system to that formed by oxidation of luminol exceeds 10(3); that is, the contribution of the latter is negligible and the system is appropriate to measure quantitatively the effect of excited photosensitizers on free radicals.     

The role of oxygen free radicals in the development of chronic renal failure.

This study examined whether there is increased production of oxygen free radicals during chronic renal failure. Rats subjected to 3/4 nephrectomy and sham operated controls were killed after 3 weeks. Lipid extracts of plasma and renal tissue were examined by HPLC and kidney specimens were also analyzed by EPR spectroscopy. The redox capacity of blood was assessed using nitroblue tetrazolium and plasma ascorbate levels were measured with HPLC. There was no detectable renal production of oxygen free radicals in rats with chronic renal failure. Kidney parenchymal content of other oxidants and the oxidant:reductant ratio were similar in control and uremic animals. The plasma redox capacity and ascorbate levels were elevated in uremic rats. We conclude that early in the course of chronic renal failure, there is not excessive production of oxygen free radicals. There is accumulation of reductants, primarily ascorbate, in the plasma of uremic animals.     

Exercise-induced brachial artery vasodilation: role of free radicals

Originally thought of as simply damaging or toxic "accidents" of in vivo chemistry, free radicals are becoming increasingly recognized as redox signaling molecules implicit in cellular homeostasis. Indeed, at the vascular level, it is plausible that oxidative stress plays a regulatory role in normal vascular function. Using electron paramagnetic resonance (EPR) spectroscopy, we sought to document the ability of an oral antioxidant cocktail (vitamins C, E, and alpha-lipoic acid) to reduce circulating free radicals, and we employed Doppler ultrasound to examine the consequence of an antioxidant-mediated reduction in oxidative stress on exercise-induced vasodilation. A total of 25 young (18-31 yr) healthy male subjects partook in these studies. EPR spectroscopy revealed a reduction in circulating free radicals following antioxidant administration at rest ( approximately 98%) and as a consequence of exercise ( approximately 85%). Plasma total antioxidant capacity and vitamin C both increased following the ingestion of the antioxidant cocktail, whereas vitamin E levels were not influenced by the ingestion of the antioxidants. Brachial artery vasodilation during submaximal forearm handgrip exercise was greater with the placebo (7.4 +/- 1.8%) than with the antioxidant cocktail (2.3 +/- 0.7%). These data document the efficacy of an oral antioxidant cocktail in reducing free radicals and suggest that, in a healthy state, the aggressive disruption of the delicate balance between pro- and antioxidant forces can negatively impact vascular function. These findings implicate an exercise-induced reliance upon pro-oxidant-stimulated vasodilation, thereby revealing an important and positive vascular role for free radicals.     

Physicochemical modeling of the role of free radicals in photodynamic therapy

A new kinetic approach is suggested and experimentally supported for quantification of the spin-trapping procedure. Accordingly, the concentration of the spin adduct formed in the interaction between the spin-trap DMPO and various free radicals (cyanopropylperoxy, cumylperoxy, phenylethylperoxy, and hydroperoxy radicals) generated by the initiated oxidation of the parent molecules is followed by kinetic ESR spectrometry. The initial sections of the corresponding kinetics are linear indicating that during this period the consumption of the adduct is negligible and thus the rate of accumulation (W(A)) approximates the rate of formation (W(f)): W(A) approximately W(f) = k(ST)[Rad(*)][DMPO], supported also by the fact that the rate of initiation of oxidation equals W(A) at high [DMPO]. In addition, the circulatory experimental apparatus enables calculation of the rate of molecular decomposition of the adduct by stopping circulation (W(f) becomes negligible) and following the decrease of the ESR signal. Corresponding rate constants are summarized.     

Sensor-based measurements of the role and interactions of free radicals in cellular systems

Direct real-time electrochemical measurements have offered new insight into the importance of free radical interplay in a number of cell culture and in vivo models of neurodegenerative processes. This review highlights investigations carried out in this laboratory of real-time superoxide and nitric oxide free radical generation, and presents evidence of complex inter-relationships between these species. These include: a novel function for astrocytic nitric oxide synthase in controlling neuronal nitric oxide availability; and the demonstration that extracellular superoxide flux can lead to the generation of NO by glial cells. The possible consequences of these interactions are discussed.     

Stimulation of microsomal lipid peroxidation by iron and cysteine. Characterization and the role of free radicals.

The stimulation of microsomal lipid peroxidation by FeSO4 and cysteine has been investigated. Although both FeSO4 and cysteine alone promoted an increase in malonaldehyde production, when these agents were added together to microsomes the resultant level of malonaldehyde was greater than the sum of the amounts formed by these pro-oxidants when acting individually. A further indication of an interaction between FeSO4 and cysteine was shown by the inhibitory action of chelating agents. Stimulation of peroxidation was shown to be independent of microsomal protein, including cytochrome P-450. The system has been characterized for the effects of cysteine, Fe2+ and O2 concentrations, pH, temperature and antioxidants. The results indicate that the high level of peroxidation attained with this system, its non-enzymic character and the involvement of hydroxyl radicals make it particularly useful for the investigation of the action of antioxidants. Furthermore it may also be a model of way in which decompartmentalized, delocalized or 'free' iron initiates peroxidation in vivo.     

Cat's claw inhibits TNFalpha production and scavenges free radicals: role in cytoprotection

Cat's claw (Uncaria tomentosa) is a medicinal plant from the Amazon River basin that is widely used for inflammatory disorders and was previously described as an inhibitor of NF-kappaB. Cat's claw was prepared as a decoction (water extraction) of micropulverized bark with and without concentration by freeze-drying. Murine macrophages (RAW 264.7 cells) were used in cytotoxicity assays (trypan blue exclusion) in response to the free radical 1, 1-diphenyl-2-picrilhydrazyl (DPPH, 0.3 microM) and ultraviolet light (UV) light. TNFalpha production was induced by lipopolysaccharide (LPS 0.5 microg/ml). Cat's claw was an effective scavenger of DPPH; the EC(50) value for freeze-dried concentrates was significantly less than micropulverized (18 vs. 150 microg/ml, p <.05). Cat's claw (10 microg/ml freeze-dried) was fully protective against DPPH and UV irradiation-induced cytotoxicity. LPS increased TNFalpha media levels from 3 to 97 ng/ml. Cat's claw suppressed TNFalpha production by approximately 65-85% (p <.01) but at concentrations considerably lower than its antioxidant activity: freeze-dried EC(50) = 1.2 ng/ml, micropulverized EC(50) = 28 ng/ml. In conclusion, cat's claw is an effective antioxidant, but perhaps more importantly a remarkably potent inhibitor of TNFalpha production. The primary mechanism for cat's claw anti-inflammatory actions appears to be immunomodulation via suppression of TNFalpha synthesis.     

Oxygen free radicals play a role in cellular differentiation: An hypothesis

Evidence from a variety of sources supports the view that oxygen free radicals play a role in cellular differentiation. It is postulated that cellular differentiation is accompanied by changes in the redox state of cells. Differentiated cells have a relatively more prooxidizing or less reducing intracellular environment than the undifferentiated or dedifferentiated cells. Changes in the redox balance during differentiation appear to be due to an increase in the rate of O₂⁻ generation. Differentiated cells, in general, exhibit higher rates of cyanide-resistant respiration, cyanide-insensitive SOD activity, and peroxide concentration and lower levels of GSH as compared to undifferentiated cells. The effects of free radicals on cellular differentiation may be mediated by the consequent changes in ionic composition.