Role of Iron in the Cellular Effects of Asbestos.

Research to understand how the physical characteristics of asbestos fibers impact the pathological effects has intensified in the past 10 years. The role that iron, intrinsic or acquired, may play has been the subject of many of these investigations. Asbestos catalyzes many of the same reactions that iron does, including lipid peroxidation and DNA damage. At physiological pH, mobilization of iron from the fibers by a low-molecular-weight chelator greatly enhances these reactions. Iron is also mobilized from asbestos fibers in human lung epithelial (A549) cells after the fibers are endocytized. The iron appears to move from a low-molecular-weight pool to nonferritin proteins and ferritin. The amount of iron in the low-molecular-weight pool was directly correlated with the toxicity of the asbestos. Glutathione (CSH) levels in these cells were dramatically reduced after asbestos treatment as a result of CSH efflux. This was not related to the iron associated with the fibers, but appeared to be due in some way to the silicate structure. The presence of the mobilized iron in the cells depleted of CSH creates a very oxidizing environment. Asbestos treatment of cells resulted in DNA oxidation, as assessed by formation of 8-hydroxy-2'-deoxyguanosine (8-oxo-dCJ. The DNA oxidation was dependent upon the iron associated with the fibers and the enzymatic generation of (•) NO by the cells. The induction of the inducible form of nitric oxide synthase (iNOS), responsible for the production of "NO, was dependent upon the presence of iron from the fibers and the decrease in CSH. One without the other did not lead to induction of iNOS. Asbestos was mutagenic in hgprt-gpt(+) V79 Chinese hamster lung cells. The mutagenicity was dependent upon iron, intrinsic or acquired. Addition of (•) NO synergistically increased the mutagenicity of the asbestos, suggesting that in cells that respond to asbestos by making (•) NO, the response will be enhanced. This also suggests that the responses will be enhanced in the presence of activated macrophages producing (•) NO or in the presence of cigarette smoke, because of the presence of (•) NO and iron chelators in the smoke.

Differential toxicities of cyclophosphamide and its glutathione metabolites to A549 cells.

Cyclophosphamide (CP), a widely used antineoplastic agent, is metabolized to species responsible for both the therapeutic and toxic effects of this drug. Acrolein is believed to be the primary toxic metabolite. This alpha,beta-unsaturated aldehyde reacts rapidly with glutathione (GSH) and can then be further metabolized to the mercapturic acid derivatives. The toxicities of the acrolein-glutathione adduct, 3-oxopropyl glutathione (oxoPrGSH) and the acrolein mercapturic acid derivatives S-3 oxopropyl N-acetylcysteine (oxoPrMCA) and S-3 hydroxypropyl N-acetylcysteine (hydroxyPrMCA) have not been fully tested. OxoPrMCA, hydroxyPrMCA and oxoPrGSH were synthesized. The toxicities of these compounds, along with those of CP and acrolein, were assessed by measuring their effects on the growth of human type II A549 lung carcinoma cells using the alamarBlue assay. Each compound was incubated with A549 cells under serum-free conditions for 2 hr, followed by 94 hr more growth in the presence of fresh medium with serum. A 50% reduction in cell growth 72 hr after treatment was achieved with 83 muM oxoPrMCA or 4 muM acrolein. No significant toxicity was seen with hydroxyPrMCA (10 mM) or oxoPrGSH (5 mm). CP (5 mM) also had no effect on the growth of A549 cells under these conditions. This latter finding is consistent with previous evidence that CP requires metabolic activation to exert its toxicity. When present during xenobiotic exposure, GSH (2 mm) almost completely protected against the growth inhibition caused by 1 mM oxoPrMCA or 10 mum acrolein. N-Acetylcysteine (1 mM) also prevented the toxicity caused by 1 mM oxoPrMCA and provided significant protection against the growth inhibition induced by 10 muM acrolein. These data support the concept that toxicity from oxoPrMCA may be due to the release of acrolein.

Iron mobilization from crocidolite asbestos by human lung carcinoma cells.

Neutron-activated crocidolite, containing 55Fe and 59Fe, was used to determine whether iron was mobilized from crocidolite phagocytized by cultured human lung carcinoma cells (A549 cells). Cells were treated with neutron-activated crocidolite in medium at pH 6.8 or 7.4 for 24 h. The mobilization of iron into two subcellular fractions, 10,000g supernatant (total iron) or < 10,000 MW [low-molecular-weight (LMW)] was monitored using scintillation counting. Iron was mobilized from crocidolite at a rate similar to that observed in vitro when citrate was incubated with crocidolite for 24 h at pH 7.4, but the amount mobilized was greater when cells were cultured at pH 6.8 than at 7.4. Iron mobilization was not due to the medium nor did it appear to be due to differences in the amount of crocidolite phagocytized. At the highest concentration of crocidolite used for treatment at pH 7.4 (4.5 micrograms/cm2), a total of 3600 pmol iron/10(6) cells was mobilized of which 54 pmol/10(6) cells was in a LMW fraction. After estimation of the volume of the cells, this was calculated to be equivalent to an intracellular concentration of 1.4 mM iron of which 22 microM was in the LMW fraction. Cell survival decreased linearly as the iron mobilized into the LMW fraction increased, independent of the pH of the culture medium being used. These results suggest that iron mobilization from crocidolite into a LMW fraction may represent "iron overload" in cells which have phagocytized the fibers and may be responsible for crocidolite-dependent cytotoxicity and possibly other crocidolite-dependent biological effects.

Reduction of glutathione disulfide and the maintenance of reducing equivalents in hypoxic hearts after the infusion of diamide.

A tissue's response to an oxidative stress is related to its capacity to supply reducing equivalents and may be affected by energy levels. The ability of intact rat heart tissue to supply NADPH and reduce glutathione disulfide (GSSG) produced by diamide was determined under normoxic or hypoxic conditions with and without glycolytic energy production. Cardiac ATP and phosphocreatine (PCr) levels remained relatively constant (approximately 20 nmol/mg dry weight) during a 60 min perfusion with oxygenated Krebs-Henseleit buffer containing glucose. Levels of ATP and PCr were depleted 85-92% following 60 min of hypoxia. A 5 min infusion of 800 microM diamide, after 60 min of normoxia or hypoxia, oxidized 70-80% of cardiac glutathione (GSH), but had no effect on total glutathione. After a subsequent 25 min diamidefree perfusion, 75-85% of the GSSG formed was reduced in both normoxic and hypoxic hearts. The removal of glucose, or the inhibition of glycolysis with 2-deoxy-D-glucose, did not affect GSSG reduction. Cardiac NADH levels were increased from 0.05 to 0.5 nmol/mg dry weight after 60 min hypoxia in hearts perfused with or without glucose. A 5 min infusion of diamide in hypoxic hearts slightly decreased NADH levels, but there was no further change after a subsequent 25 min diamide-free period. Inhibition of glutathione reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea prevented GSSG reduction, showing NADPH was required. However, NADPH levels were not affected by hypoxia or diamide infusion and remained constant at 0.2 nmol/mg dry weight in hearts perfused with or without glucose. Inhibition of glycolysis with 2-deoxy-D-glucose also did not affect NADPH levels. These results demonstrate that hypoxia did not affect the ability of oxidatively stressed, intact heart tissue to supply NADPH for the reduction of GSSG. In addition, GSSG reduction was independent of energy levels and appeared to be unaffected by glucose availability. NADH may be involved in maintaining NADPH levels through interconversion pathways.

Cellular reducing equivalents and oxidative stress.

Energy has been proposed to play a role in the ability of cells and tissues to defend against oxidative stress, even though the ultimate antioxidant capacity of a tissue is determined by the supply of reducing equivalents. The pathways involved in supplying reducing equivalents in response to an oxidative stress remain unclear, particularly if competing reactions such as ATP synthesis are active. Glutathione (GSH), a major component of cellular antioxidant systems, is maintained in the reduced form by glutathione reductase. Although this enzyme is specific for NADPH, the ability of intact cells, isolated mitochondria (which are a major source of free radicals and contain antioxidant systems independent of the rest of the cell), and whole tissues to supply reducing equivalents and maintain normal levels of GSH appears to involve NADH. This article reviews available data regarding the source and pathways by which reducing equivalents are made available to reduce exogenous oxidants, and suggests energy is not a factor. An improved understanding of the mechanism by which reducing equivalents are supplied by tissues to respond to an oxidative stress may direct future research toward designing strategies for augmenting the ability of tissues to defend themselves against oxidative stress induced by reperfusion or xenobiotics.

Iron associated with asbestos bodies is responsible for the formation of single strand breaks in phi X174 RFI DNA.

The ability of amosite cored asbestos bodies isolated from human lungs to catalyse damage to phi X174 RFI DNA in vitro was measured and compared with that of uncoated amosite fibres with a similar distribution of length. Asbestos bodies (5000 bodies) suspended for 30 minutes in 50 mM NaCl containing 0.5 micrograms phi X174 RFI DNA, pH 7.5, did not catalyse detectable amounts of DNA single strand breaks. Addition of the reducing agent ascorbate (1 mM), however, resulted in single strand breaks in 10% of the DNA. Asbestos bodies in the presence of a low molecular weight chelator (1 mM) and ascorbate catalysed the formation of single strand breaks in 21% of the DNA with citrate or 77% with ethylenediamine tetra-acetic acid (EDTA), suggesting that mobilisation of iron may increase damage to DNA. Preincubation for 24 hours with desferrioxamine B, which binds iron (Fe (III)) and renders it redox inactive, completely inhibited the reactivity of asbestos bodies with DNA, strongly suggesting that iron was responsible. Amosite fibres (5000 fibres/reaction), with a similar length distribution to that of the asbestos bodies, did not catalyse detectable amounts of single strand breaks in DNA under identical reaction conditions. The results of the present study strongly suggest that iron deposits on the amosite core asbestos bodies were responsible for the formation of DNA single strand breaks in vitro. Mobilisation of iron by chelators seemed to enhance the reactivity of asbestos bodies with DNA. It has been postulated that the in vivo deposition of the coat material on to fibres may be an attempt by the lung defenses to isolate the fibre from the lung surface and thus offer a protective mechanism from physical irritation. These results suggest, however, that the iron that is deposited on asbestos fibres in vivo may be reactive, potentially increasing the damage to biomolecules, such as DNA, above that of the uncoated fibres.

Iron mobilization from crocidolite asbestos greatly enhances crocidolite-dependent formation of DNA single-strand breaks in phi X174 RFI DNA.

The ability of the iron associated with asbestos to catalyze damage to phi X174 RFI DNA was determined and compared with iron mobilized from asbestos. Asbestos (1 mg/ml) suspended for 30 min in 50 mM NaCl containing 0.5 micrograms phi X174 RFI DNA, pH 7.5, did not catalyze detectable amounts of DNA single-strand breaks (SSB). However, addition of ascorbate (1 mM) resulted in 19, 26, 7 or 8% DNA with SSB for crocidolite, amosite, chrysotile or tremolite respectively. The percentage of DNA with SSB induced by each form of asbestos was directly related to its iron content. Inclusion of desferrioxamine B, which binds Fe(III) rendering it redox inactive, completely inhibited asbestos-dependent formation of DNA SSB, suggesting that iron was responsible for catalyzing the formation of DNA SSB. Mobilization of Fe(II) from crocidolite by citrate, EDTA or nitrilotriacetate (1 mM) in the absence of ascorbate resulted in 15, 33 or 63% DNA with SSB respectively. This activity was completely inhibited by compounds considered to be .OH scavengers, i.e. mannitol, 5,5-dimethyl-1-pyrroline N-oxide or salicylate (100 mM). Preincubation of crocidolite with citrate (1 mM) for 24 h resulted in mobilization of 52 microM iron and increased ascorbate-dependent induction of DNA SSB compared with crocidolite that was preincubated without citrate. Iron mobilized by citrate was entirely responsible for crocidolite-dependent formation of DNA SSB as evidenced by complete inhibition with desferrioxamine B. Therefore, the results of the present study strongly suggest that iron was responsible for asbestos-dependent generation of oxygen radicals, which resulted in the formation of DNA SSB. Mobilization of iron by chelators, followed by redox cycling, greatly enhanced crocidolite-dependent formation of DNA SSB. Thus, mobilization of iron in vivo by low mol. wt chelators may lead to the increased production of reactive oxygen species resulting in damage to biomolecules, such as DNA.

Iron-catalyzed reactions may be responsible for the biochemical and biological effects of asbestos.

The most carcinogenic forms of asbestos contain iron to levels as high as 36% by weight and catalyze many of the same biochemical reactions that freshly prepared solutions of iron do, i.e. oxygen consumption, generation of reactive oxygen species, lipid peroxidation and DNA damage. The participation of iron from asbestos in these reactions has been demonstrated using the iron chelator desferrioxamine B which inhibits iron-catalyzed reactions. Iron appears to be redox active on the asbestos fiber, but chelation and subsequent iron mobilization from asbestos by a variety of chelators, e.g. citrate, EDTA or nitrilotriacetate, makes the iron more redox active resulting in greater oxygen consumption and production of oxygen radicals in the presence of reducing agents. Iron also appears to be important for some of the asbestos-dependent biological effects on tissues or cells in culture, such as phagocytosis, cytotoxicity, lipid peroxidation and DNA damage. Therefore, redox cycling of iron to generate oxygen radicals at the surface of the fiber and/or in solution, as mobilized, low molecular weight chelates, may be very important in eliciting some of the biological effects of asbestos in vivo.

Mobilization of iron from crocidolite asbestos by certain chelators results in enhanced crocidolite-dependent oxygen consumption.

The reactivity of iron on crocidolite asbestos with dioxygen was determined and compared with iron mobilized from crocidolite. Ferrozine, a strong Fe(II) chelator, was used to demonstrate that iron on crocidolite was redox active. More Fe(II) was mobilized from crocidolite (1 mg/ml) by ferrozine anaerobically (11.2 nmol/mg crocidolite/h) than aerobically (6.6 nmol/mg/h) in 50 mM NaCl, pH 7.5, suggesting that Fe(II) on crocidolite reacts with O2 upon aqueous suspension. However, suspension of crocidolite in 50 mM NaCl, pH 7.5, did not result in a measurable amount of O2 consumption. The addition of reducing agents (1 mM) increased the amount of Fe(II) on crocidolite, and addition of ascorbate resulted in 0.4 nmol O2 consumed/mg crocidolite/min. Therefore, iron on crocidolite had limited redox activity in the presence of ascorbate. However, mobilization of iron from crocidolite increased its redox activity. Citrate, nitrilotriacetate (NTA), or EDTA (1 mM) mobilized 79, 32, or 58 microM iron, respectively, in preincubations up to 76 h, and increased O2 consumption upon addition of ascorbate to 2.8, 7.6, or 22.0 nmol O2 consumed/mg/min, respectively. This activity depended only upon the presence of a component(s) mobilized from crocidolite by the chelators. Pretreatment of crocidolite with the iron chelator desferrioxamine B (10 mM) inhibited O2 consumption. The results of the present study suggest that iron on or in crocidolite is responsible for the redox activity of crocidolite, but that mobilization of iron by chelators such as citrate, NTA, or EDTA greatly enhances its redox activity. Thus, iron mobilization from crocidolite in vivo by low-molecular-weight chelators may lead to the increased production of reactive oxygen species which may damage biomolecules, such as DNA.

Iron mobilization from asbestos by chelators and ascorbic acid.

The ability of chelators and ascorbic acid to mobilize iron from crocidolite, amosite, medium- and short-fiber chrysotile, and tremolite was investigated. Ferrozine, a strong Fe(II) chelator, mobilized Fe(II) from crocidolite (6.6 nmol/mg asbestos/h) and amosite (0.4 nmol/mg/h) in 50 mM NaCl, pH 7.5. Inclusion of ascorbate increased these rates to 11.4 and 4.9 nmol/mg/h, respectively. Ferrozine mobilized Fe(II) from medium-fiber chrysotile (0.6 nmol/mg/h) only in the presence of ascorbate. Citrate and ADP mobilized iron (ferrous and/or ferric) from crocidolite at rates of 4.2 and 0.3 nmol/mg/h, respectively, which increased to 4.8 and 1.0 nmol/mg/h in the presence of ascorbate. Since ascorbate alone mobilized iron from crocidolite (0.5 nmol/mg/h), the increase appeared to result from additional chelation by ascorbate. Citrate also mobilized iron from amosite (1.4 nmol/mg/h) and medium-fiber chrysotile (1.6 nmol/mg/h). Mobilization of iron from asbestos appeared to be a function not only of the chelator, but also of the surface area, crystalline structure, and iron content of the asbestos. These results suggest that iron can be mobilized from asbestos in the cell by low-molecular-weight chelators. If this occurs, it may have deleterious effects since this could result in deregulation of normal iron metabolism by proteins within the cell resulting in iron-catalyzed oxidation of biomolecules.