Iron-catalyzed Reactive Oxygen Species Research Articles

Rhodamine-based Fluorescent Probe With Quick Response and High Selectivity for Imaging Labile Ferrous Iron in Living Cells and Zebrafish.

The transition between its various oxidation states of Iron plays a crucial part in various chemical transformation of cells. Misregulation of iron can give rise to the iron-catalyzed reactive oxygen species disorder which have been linked to a variety of diseases, so it is crucial to monitor the labile iron pool in vivo for clinical diagnosis. According to iron autoxidation and hydrogen abstraction reaction, we reported a novel "off-on" fluorescent probe to response to ferrous (Fe2+) both in solutions and biological systems. The probe responds to Fe2+ with good selectivity toward competing metal ions. What's more, the probe presents significant fluorescent enhancement to Fe2+ in less than 1min, making real-time sensing in biological system possible. The applications of the probe in bioimaging revealed the changes in labile iron pool by iron autoxidation or diverse stimuli.

Iron Chelation in Murine Models of Systemic Inflammation Induced by Gram-Positive and Gram-Negative Toxins

Iron is an essential element for various physiological processes, but its levels must remain tightly regulated to avoid cellular damage. Similarly, iron plays a dual role in systemic inflammation, such as with sepsis. Leukocytes utilize iron to produce reactive oxygen species (ROS) to kill bacteria, but pathologically increased iron-catalyzed ROS production in sepsis can lead to damage of host cells, multi-organ failure and death. Temporary reduction in bioavailable iron represents a potential therapeutic target in sepsis. This study investigates the effect of the novel iron chelator, DIBI, in murine models of systemic (hyper-)inflammation: C57BL/6 mice were challenged with toxins from Gram-positive (Staphylococcus aureus: lipoteichoic acid, peptidoglycan) and Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae: lipopolysaccharide). Intravital microscopy (IVM) was performed to assess immune cell activation and its impact on microvascular blood flow in vivo in the microcirculation of the gut. Plasma inflammatory mediators were measured via multiplex assay, and morphologic change in intestinal tissue was evaluated. DIBI treatment decreased leukocyte (hyper-)activation induced by Gram-positive and Gram-negative toxins. In some cases, it preserved capillary perfusion, reduced plasma inflammatory markers and attenuated tissue damage. These findings support the utility of DIBI as a novel treatment for systemic inflammation, e.g., sepsis.

TGFβ2-Hepcidin Feed-Forward Loop in the Trabecular Meshwork Implicates Iron in Glaucomatous Pathology

PurposeElevated levels of transforming-growth-factor (TGF)-β2 in the trabecular meshwork (TM) and aqueous humor are associated with primary open-angle glaucoma (POAG). The underlying mechanism includes alteration of extracellular matrix homeostasis through Smad-dependent and independent signaling. Smad4, an essential co-Smad, upregulates hepcidin, the master regulator of iron homeostasis. Here, we explored whether TGF-β2 upregulates hepcidin, implicating iron in the pathogenesis of POAG.MethodsPrimary human TM cells and human and bovine ex vivo anterior segment organ cultures were exposed to bioactive TGF-β2, hepcidin, heparin (a hepcidin antagonist), or N-acetyl carnosine (an antioxidant), and the change in the expression of hepcidin, ferroportin, ferritin, and TGF-β2 was evaluated by semiquantitative RT-PCR, Western blotting, and immunohistochemistry. Increase in reactive oxygen species (ROS) was quantified with dihydroethidium, an ROS-sensitive dye.ResultsPrimary human TM cells and bovine TM tissue synthesize hepcidin locally, which is upregulated by bioactive TGF-β2. Hepcidin downregulates ferroportin, its downstream target, increasing ferritin and iron-catalyzed ROS. This causes reciprocal upregulation of TGF-β2 at the transcriptional and translational levels. Heparin downregulates hepcidin, and reduces TGF-β2-mediated increase in ferritin and ROS. Notably, both heparin and N-acetyl carnosine reduce TGF-β2-mediated reciprocal upregulation of TGF-β2.ConclusionsThe above observations suggest that TGF-β2 and hepcidin form a self-sustained feed-forward loop through iron-catalyzed ROS. This loop is partially disrupted by a hepcidin antagonist and an anti-oxidant, implicating iron and ROS in TGF-β2-mediated POAG. We propose that modification of currently available hepcidin antagonists for ocular use may prove beneficial for the therapeutic management of TGF-β2-associated POAG.

Anticoagulation, ferrotoxicity and the future of translational lung cancer research.

Numerous studies have shown that elements of coagulation reactions mediate tumor cell proliferation, motility (invasiveness), tissue remodeling and metastasis. Coagulation activation is virtually a universal feature of human malignancy that differs from the clotting response to injury in that it is self-perpetuating rather than self-attenuating. Coagulation activation participates in tumor matrix deposition and local inflammation, and predicts subsequent cancer risk and adverse cancer outcomes. Several clinical trials of anticoagulants have shown improved outcomes in small cell carcinoma of the lung (SCCL) that have been correlated with assembly on the tumor cells of an intact coagulation pathway. However, variable efficacy of anticoagulant therapy has raised doubts about the coagulation hypothesis. Recently, initiators of coagulation and fibrinolytic pathways have been identified that mediate tumor inception and progression. Notable among these is oxidative stress driven by iron-catalyzed reactive oxygen species that may be the basis for local coagulation activation, tumor matrix deposition, inflammation and aberrant properties characteristic of the malignant phenotype. Recognition of important biological characteristics of individual tumor types, disease stage, choice of standard therapy including chemotherapy and the iron status of the host may clarify mechanisms. All of these are subject to modification based on controlled clinical trial design. Further tests of the coagulation hypothesis may lead to novel, low cost and relatively non-toxic approaches to treatment of malignancy including lung cancer that contrast with certain current cancer treatment paradigms.

220. AAV-Mediated miR122 Overexpression Rescues Hepatic Inflammatory Response Induced by Acute Iron-Overload

Frequent blood transfusions among patients with thalassaemia and sickle cell disease can lead to acute iron overload (IO), also known as transfusional haemosiderosis. Excessive iron is initially distributed to the liver, the primary organ for iron recycling and storage, and then gradually leads to damage of other major organs such as the heart and endocrine organs. IO frequently increases hepatic inflammatory infiltration and expression of pro-inflammatory cytokines. Recently, hepcidin, a well-known iron-regulatory hormone, and iron-catalyzed reactive oxygen species were proven to be independent of the IO-mediated inflammation. These findings triggered us to further pursue the potential underlying molecular mechanisms.In our preliminary studies, we observed that IO significantly reduced the expression of liver-specific microRNA, miR122, through the inhibition of a nuclear receptor, hepatocyte nuclear factor 4 alpha (HNF4α), but not other regulators of miR122. Furthermore, we demonstrated that the chemokine (C-C motif) ligand 2 (CCL2), which recruits monocytes, memory T cells, and dendritic cells to the sites of inflammation, is negatively regulated by miR122. Thus, we hypothesized that overexpression of hepatic miR-122 may relieve the inflammatory responses to the acute IO. To this end, a recombinant adeno-associated virus serotype 8 (rAAV8) vector was generated, in which a miR-122 expression cassette was embedded in the intron region between a Guassia Luciferase (GLuc) transgene and a chicken β-actin promoter. Another rAAV8 vector, which has the exact GLuc transgene cassette but without miR-122, was used as a negative control. C57BL/6 mice were intra-peritoneal challenged with iron-dextran at 200mg/kg mouse weight or PBS as a control, at Day 0 and Day 8. The IO group was sub-divided into three sub-groups, in which PBS, rAAV8-GLuc, or rAAV8-miR122-GLuc vectors were tail-vein injected at 10^11 viral genome/mouse, respectively, at Day 6. All mice were sacrificed at Week 4. It was evident that following systemic administration, rAAV8-miR122-GLuc vectors led to a 12-fold increase in the expression of miR122. On the other hand, only a modest increase (<2-fold) in the HNF4α expression was observed. Interestingly, although in vivo overexpression of miR-122 had no effect on serum iron indices or hepatic expression of iron-related molecules, it significantly reduced the expression of several inflammatory genes, such as IL-6 and IL-1β, in the IO mice. Western blot analysis also revealed that overexpression of miR-122 resulted in a significant reduction of CCL2 and NF-kB proteins in the IO mice. Consequently, both the hepatic inflammation scores and serum activities of AST and ALT in the IO mice were significantly reduced upon miR-122 overexpression.In sum, our model (Figure 1Figure 1) and studies should be important and informative for elucidating the mechanism of IO-induced hepatic inflammation and for developing clinical strategies to prevent them in the patients with major haemoglobinopathy.View Large Image | Download PowerPoint Slide

Lysosomal iron liberation is responsible for the vulnerability of brain microglial cells to iron oxide nanoparticles: comparison with neurons and astrocytes

Iron oxide nanoparticles (IONPs) are used for various biomedical and neurobiological applications. Thus, detailed knowledge on the accumulation and toxic potential of IONPs for the different types of brain cells is highly warranted. Literature data suggest that microglial cells are more vulnerable towards IONP exposure than other types of brain cells. To investigate the mechanisms involved in IONP-induced microglial toxicity, we applied fluorescent dimercaptosuccinate-coated IONPs to primary cultures of microglial cells. Exposure to IONPs for 6 h caused a strong concentration-dependent increase in the microglial iron content which was accompanied by a substantial generation of reactive oxygen species (ROS) and by cell toxicity. In contrast, hardly any ROS staining and no loss in cell viability were observed for cultured primary astrocytes and neurons although these cultures accumulated similar specific amounts of IONPs than microglia. Co-localization studies with lysotracker revealed that after 6 h of incubation in microglial cells, but not in astrocytes and neurons, most IONP fluorescence was localized in lysosomes. ROS formation and toxicity in IONP-treated microglial cultures were prevented by neutralizing lysosomal pH by the application of NH4Cl or Bafilomycin A1 and by the presence of the iron chelator 2,2′-bipyridyl. These data demonstrate that rapid iron liberation from IONPs at acidic pH and iron-catalyzed ROS generation are involved in the IONP-induced toxicity of microglia and suggest that the relative resistance of astrocytes and neurons against acute IONP toxicity is a consequence of a slow mobilization of iron from IONPs in the lysosomal degradation pathway.

Uptake, metabolism and toxicity of iron oxide nanoparticles in cultured microglia, astrocytes and neurons

Iron oxide nanoparticles (IONPs) are frequently used for biomedical applications including magnetic resonance imaging, drug delivery or tumor treatment by hyperthermia. Since IONPs can reach the brain from periphery or are directly applied into the brain, the investigation of potential adverse effects of IONPs on properties and functions of the different types of brain cells is an important task. In order to directly compare different types of brain cells concerning the uptake and toxicity of IONPs, we synthesized fluorescent IONPs and characterized these BODIPY-labeled particles regarding their physicochemical properties. In the medium used for the cell studies, these particles had a hydrodynamic diameter of around 158 ± 28 nm and a negative surface charge with a zeta-potential of -10 ± 2 mV. Exposure of primary cultures of rat astrocytes, neurons and microglial cells revealed that among these cell types, microglial cells accumulated IONPs most rapidly. However, microglial cells were also most vulnerable towards acute IONP-induced stress while astrocytes and neurons were not acutely damaged by IONPs. In microglial cells, but not in astrocytes or neurons, IONPs were found to be localized in lysosomes and large amounts of reactive oxygen species (ROS) were observed after IONP-exposure. The IONP-induced toxicity in microglial cells was prevented by neutralizing lysosomes or by chelation of intracellular ferrous iron ions, suggesting that the toxic potential of IONPs in microglia involves rapid particle uptake, liberation of ferrous iron from the internalized IONPs in the acidic lysosomal compartment and iron-catalyzed ROS formation. These data suggest that also in brain IONPs may harm microglial cells and compromise microglial functions.

Hypoxia induced changes in expression of proteins involved in iron uptake and storage in cultured lens epithelial cells

Hypoxia inducible factor (HIF) regulates expression of over 60 genes by binding to hypoxia response elements (HRE) located upstream of the transcriptional start sites. Many genes encoding proteins involved in iron transport and homeostasis are regulated by HIF. Expression of iron handling proteins can also be translationally regulated by binding of iron regulatory protein (IRP) to iron responsive elements (IREs) on the mRNA of ferritin chains and transferrin receptor (TfR). Lens epithelial cells (LEC) function in a low oxygen environment. This increases the risk of iron catalyzed formation of reactive oxygen species (ROS) and oxidative cell damage. We examined changes in expression of ferritin (iron storage protein) and Tf/TfR1 (iron uptake proteins) in LEC cultured under hypoxic conditions. Ferritin consists of 24 subunits of two types, heavy (H-chain) and light (L-chain) assembled in a cell specific ratio. Real-time PCR showed that 24 h exposure to hypoxia lowered transcription of both ferritin chains by over 50% when compared with normoxic LEC. However it increased the level of ferritin chain proteins (20% average). We previously found that 6 h exposure of LEC to hypoxia increased the concentration of cytosolic iron which would stimulate translation of ferritin chains. This elevated ferritin concentration increased the iron storage capacity of LEC. Hypoxic LEC labeled with 59FeTf incorporated 70% more iron into ferritin after 6 h as compared to normoxic LEC. Exposure of LEC to hypoxia for 24 h reduced the concentration of TfR1 in cell lysates. As a result, hypoxic LEC internalized less Tf at this later time point. Incorporation of 59Fe into ferritin of hypoxic LEC after 24 h did not differ from that of normoxic LEC due to lower 59FeTf uptake. This study showed that hypoxia acutely increased iron storage capacity and lowered iron uptake due to changes in expression of iron handling proteins. These changes may better protect LEC against oxidative stress by limiting iron-catalyzed ROS formation in the low oxygen environment in which the lens resides.

The role of iron and reactive oxygen species in cell death

The transition metal iron is essential for life, yet potentially toxic iron-catalyzed reactive oxygen species (ROS) are unavoidable in an oxygen-rich environment. Iron and ROS are increasingly recognized as important initiators and mediators of cell death in a variety of organisms and pathological situations. Here, we review recent discoveries regarding the mechanism by which iron and ROS participate in cell death. We describe the different roles of iron in triggering cell death, targets of iron-dependent ROS that mediate cell death and a new form of iron-dependent cell death termed ferroptosis. Recent advances in understanding the role of iron and ROS in cell death offer unexpected surprises and suggest new therapeutic avenues to treat cancer, organ damage and degenerative disease.

Mechanisms of Anthracycline Cardiac Injury: Can We Identify Strategies for Cardioprotection?

Anthracycline antibiotics have saved the lives of many cancer victims in the 50 plus years since their discovery. A major limitation of their use is the dose-limiting cardiotoxicity. Efforts focusing on understanding the biochemical basis for anthracycline cardiac effects have provided several strategies currently in clinical use: limit dose exposure, encapsulate anthracyclines in liposomes to reduce myocardial uptake, administer concurrently with the iron chelator dexrazoxane to reduce free iron-catalyzed reactive oxygen species formation; and modify anthracycline structure in an effort to reduce myocardial toxicity. Despite these efforts, anthracycline-induced heart failure continues to occur with consequences for both morbidity and mortality. Our inability to predict and prevent anthracycline cardiotoxicity is, in part, due to the fact that the molecular and cellular mechanisms remain controversial and incompletely understood. Studies examining the effects of anthracyclines in cardiac myocytes in vitro and small animals in vivo have demonstrated several forms of cardiac injury, and it remains unclear how these translate to the clinical setting. Given the clinical evidence that myocyte death occurs after anthracycline exposure in the form of elevations in serum troponin, myocyte cell death seems to be a probable mechanism for anthracycline-induced cardiac injury. Other mechanisms of myocyte injury include the development of cellular “sarcopenia” characterized by disruption of normal sarcomere structure. Anthracyclines suppress expression of several cardiac transcription factors, and this may play a role in the development of myocyte death as well as sarcopenia. Degradation of the giant myofilament protein titin may represent an important proximal step that leads to accelerated myofilament degradation. An interesting interaction has been noted clinically between anthracyclines and newer cancer therapies that target the erbB2 receptor tyrosine kinase. There is now evidence that erbB2 signaling in response to the ligand neuregulin regulates anthracycline uptake into cells via the multidrug-resistance protein. Therefore, up-regulation of cardiac neuregulin signaling may be one strategy to limit myocardial anthracycline injury. Moreover, assessing an individual's risk for anthracycline injury may be improved by having some measure of endogenous activity of this and other myocardial protective signals.

Sequence analysis of dolphin ferritin H and L subunits and possible iron-dependent translational control of dolphin ferritin gene

BackgroundIron-storage protein, ferritin plays a central role in iron metabolism. Ferritin has dual function to store iron and segregate iron for protection of iron-catalyzed reactive oxygen species. Tissue ferritin is composed of two kinds of subunits (H: heavy chain or heart-type subunit; L: light chain or liver-type subunit). Ferritin gene expression is controlled at translational level in iron-dependent manner or at transcriptional level in iron-independent manner. However, sequencing analysis of marine mammalian ferritin subunits has not yet been performed fully. The purpose of this study is to reveal cDNA-derived amino acid sequences of cetacean ferritin H and L subunits, and demonstrate the possibility of expression of these subunits, especially H subunit, by iron.MethodsSequence analyses of cetacean ferritin H and L subunits were performed by direct sequencing of polymerase chain reaction (PCR) fragments from cDNAs generated via reverse transcription-PCR of leukocyte total RNA prepared from blood samples of six different dolphin species (Pseudorca crassidens, Lagenorhynchus obliquidens, Grampus griseus, Globicephala macrorhynchus, Tursiops truncatus, and Delphinapterus leucas). The putative iron-responsive element sequence in the 5'-untranslated region of the six different dolphin species was revealed by direct sequencing of PCR fragments obtained using leukocyte genomic DNA.ResultsDolphin H and L subunits consist of 182 and 174 amino acids, respectively, and amino acid sequence identities of ferritin subunits among these dolphins are highly conserved (H: 99–100%, (99→98) ; L: 98–100%). The conserved 28 bp IRE sequence was located -144 bp upstream from the initiation codon in the six different dolphin species.ConclusionThese results indicate that six different dolphin species have conserved ferritin sequences, and suggest that these genes are iron-dependently expressed.

Deferiprone Does Not Protect against Chronic Anthracycline Cardiotoxicity in Vivo

Anthracycline cardiotoxicity ranks among the most severe complications of cancer chemotherapy. Although its pathogenesis is only incompletely understood, "reactive oxygen species (ROS) and iron" hypothesis has gained the widest acceptance. Besides dexrazoxane, novel oral iron chelator deferiprone has been recently reported to afford significant cardioprotection in both in vitro and ex vivo conditions. Therefore, the aim of this study was to assess whether deferiprone 1) has any effect on the anticancer action of daunorubicin and 2) whether it can overcome or significantly reduce the chronic anthracycline cardiotoxicity in the in vivo rabbit model (daunorubicin, 3 mg/kg i.v., weekly for 10 weeks). First, using the leukemic cell line, deferiprone (1-300 microM) was shown not to blunt the antiproliferative effect of daunorubicin. Instead, in clinically relevant concentrations (>10 microM), deferiprone augmented the antiproliferative action of daunorubicin. However, deferiprone (10 or 50 mg/kg administered p.o. before each daunorubicin dose) failed to afford significant protection against daunorubicin-induced mortality, left ventricular lipoperoxidation, cardiac dysfunction, and morphological cardiac deteriorations, as well as an increase in plasma cardiac troponin T. Hence, this first in vivo study changes the current view on deferiprone as a potential cardioprotectant against anthracycline cardiotoxicity. In addition, these results, together with our previous findings, further suggest that the role of iron and its chelation in anthracycline cardiotoxicity is not as trivial as originally believed and/or other mechanisms unrelated to iron-catalyzed ROS production are involved.

Protective Effects of an Antioxidant Derived from Serine and Vitamin B6 on Skin Photoaging in Hairless Mice

The generation of reactive oxygen species (ROS) by ultraviolet radiation (UVR) accelerates skin aging, which is known as photoaging. Because cutaneous iron catalyzes ROS generation, sequestering iron by chelating agents is thought to be an effective approach toward preventing photoaging. Previously, N-(4-pyridoxylmethylene)-l-serine (PYSer) was designed as an antioxidant to suppress iron-catalyzed ROS generation by its iron-sequestering activity. In this study, PYSer showed protective effects against skin damage in hairless mice irradiated with ultraviolet B (UV-B). Topical application of PYSer to the skin significantly delayed and/or decreased the visible wrinkle formation induced by chronic UV-B irradiation. A histological study indicated that UV-B-induced epidermal hypertrophy and lymphocytic infiltration were suppressed by PYSer. Moreover, PYSer showed suppressive activity against the UV-B-induced increase in glycosaminoglycans (GAG). These results indicate that PYSer is a promising antioxidant for the prevention of chronic skin photoaging by its iron-sequestering activity.

Protective Effects of an Antioxidant Derived from Serine and Vitamin B6 on Skin Photoaging in Hairless Mice¶

The generation of reactive oxygen species (ROS) by ultraviolet radiation (UVR) accelerates skin aging, which is known as photoaging. Because cutaneous iron catalyzes ROS generation, sequestering iron by chelating agents is thought to be an effective approach toward preventing photoaging. Previously, N-(4-pyridoxylmethylene)-l-serine (PYSer) was designed as an antioxidant to suppress iron-catalyzed ROS generation by its iron-sequestering activity. In this study, PYSer showed protective effects against skin damage in hairless mice irradiated with ultraviolet B (UV-B). Topical application of PYSer to the skin significantly delayed and/or decreased the visible wrinkle formation induced by chronic UV-B irradiation. A histological study indicated that UV-B–induced epidermal hypertrophy and lymphocytic infiltration were suppressed by PYSer. Moreover, PYSer showed suppressive activity against the UV-B–induced increase in glycosaminoglycans (GAG). These results indicate that PYSer is a promising antioxidant for the prevention of chronic skin photoaging by its iron-sequestering activity.

Antioxidant protection against iron in children with meningococcal sepsis

To assess antioxidant protection against iron-catalyzed reactive oxygen species in meningococcal sepsis and to establish whether severity of illness is related to deficiencies in these antioxidant systems. Prospective, controlled study. Pediatric intensive care unit of a postgraduate teaching hospital. Twenty children aged 6 months to 15 yrs (median, 5 yrs) with meningococcal septic shock were studied. Paired convalescent samples taken 8-10 wks after discharge were available in nine children. Routine management for meningococcal sepsis. Patients were classified for disease severity using the Glasgow Meningococcal Septicaemia Prognostic Score. Paired acute and convalescent samples were compared. Transferrin level (1.77 +/- 0.08 g/L) and total iron-binding capacity (46.2 +/- 2.0 microM) were significantly decreased in acute patients compared with paired convalescent samples (2.85 +/- 0.10 g/L and 74.4 +/- 2.5 microM, respectively; p <.0001). The iron saturation of transferrin was significantly increased in acute disease (36.9% +/- 2.5%) compared with convalescence (18.8% +/- 1.5%; p =.0003). Iron-binding antioxidant protection was not significantly different in acute (81.4% +/- 1.7%) and paired convalescent samples (85.6% +/- 2.5%; p =.54). However, patients with more severe meningococcal septicemia (GMSPS, >10; n = 12) had significantly diminished protection (77.5% +/- 2.4%) compared with less severe disease (87.1% +/- 1.6%; p =.0028), and there was a significant correlation between disease severity and iron-binding antioxidant protection (R =.48; p =.00067) in acute disease. Paired ceruloplasmin levels were available in six patients and were decreased in acute disease (0.29 +/- 0.02 g/L) compared with convalescence (0.40 +/- 0.04 g/L), although not statistically significant (p =.076). However, there was a significant correlation between plasma ceruloplasmin and disease severity (Pearson product moment correlation, p =.038) in the acute patients. Iron-oxidizing antioxidant assays were performed in four paired samples and were diminished in acute patients (53.3 +/- 4.4%) compared with convalescence (67.8 +/- 3.2%; p =.015). Acute samples demonstrated a significant relationship between iron-oxidizing antioxidant protection and both disease severity (r =.30; p =.012) and plasma ceruloplasmin levels (r =.48; p =.00067). Children with meningococcal septicemia exhibit abnormal plasma iron chemistry and decreased protection against iron-catalyzed oxidative damage. Such deficiencies correlate with disease severity.

Asbestos-induced alveolar epithelial cell apoptosis: role of mitochondrial dysfunction caused by iron-derived free radicals.

Asbestos causes asbestosis and malignancies by mechanisms that are not fully understood. Alveolar epithelial cell (AEC) injury by iron-derived reactive oxygen species (ROS) is one important mechanism implicated. We previously showed that iron-catalyzed ROS in part mediate asbestos-inducedAEC DNA damage and apoptosis. Mitochondria have a critical role in regulating apoptosis after exposure to agents causing DNA damage but their role in regulating asbestos-induced apoptosis is unknown. To determine whether asbestos causes AEC mitochondrial dysfunction, we exposed A549 cells to amosite asbestos and assessed mitochondrial membrane potential changes (delta(psi)m) using a fluorometric technique involving tetremethylrhodamine ethyl ester (TMRE) and mitotracker green. We show that amosite asbestos, but not an inert particulate, titanium dioxide, reduces delta(psi)m after a 4 h exposure period. Further, the delta(psi)m after 4 h was inversely proportional to the levels of apoptosis noted at 24 h as assessed by nuclear morphology as well as by DNA nucleosome formation. A role for iron-derived ROS was suggested by the finding that phytic acid, an iron chelator, blocked asbestos-induced reductions in A549 cell delta(psi)m and attenuated apoptosis. Finally, overexpression of Bcl-xl, an anti-apoptotic protein that localizes to the mitochondria, prevented asbestos-induced decreases in A549 cell delta(psi)m after 4 h and diminished apoptosis. We conclude that asbestos alters AEC mitochondrial function in part by generating iron-derived ROS, which in turn can result in apoptosis. This suggests that the mitochondrial death pathway is important in regulating pulmonary toxicity from asbestos.

Asbestos causes apoptosis in alveolar epithelial cells: Role of iron-induced free radicals

Asbestos causes asbestosis and malignancies by mechanisms that are not fully understood. Alveolar epithelial cell (AEC) injury by iron-induced reactive oxygen species (ROS) is one important mechanism. To determine whether asbestos causes apoptosis in AECs, we exposed WI-26 (human type I–like cells), A549 (human type II–like cells), and rat alveolar type II cells to amosite asbestos and assessed apoptosis by terminal deoxynucleotidyl transferase–mediated deoxyuridine-5′-triphosphate-biotin nick end labeling (TUNEL) staining, nuclear morphology, annexin V staining, DNA nucleosome formation, and caspase 3 activation. In contrast to control medium and TiO2, amosite asbestos and H2O2 each caused AEC apoptosis. A role for iron-catalyzed ROS was suggested by the finding that asbestos-induced AEC apoptosis and caspase 3 activation were each attenuated by either an iron chelator (phytic acid and deferoxamine) or a ·OH scavenger (dimethyl-thiourea, salicylate, and sodium benzoate) but not by iron-loaded phytic acid. To determine whether asbestos causes apoptosis in vivo, rats received a single intratracheal instillation of amosite (5 mg) or normal saline solution, and apoptosis in epithelial cells in the bronchoalveolar duct regions was assessed by TUNEL staining. One week after exposure, amosite asbestos caused a 3-fold increase in the percentage of apoptotic cells in the bronchoalveolar duct regions as compared with control (control, 2.1% ± 0.35%; asbestos, 7.61% ± 0.15%; n = 3). However, by 4 weeks the number of apoptotic cells was similar to control. We conclude that asbestos-induced pulmonary toxicity may partly be caused by apoptosis in the lung epithelium that is mediated by iron-catalyzed ROS and caspase 3 activation. (J Lab Clin Med 2001;137:330-9)

Asbestosis: clinical spectrum and pathogenic mechanisms.

Asbestosis is a diffuse pulmonary fibrotic process caused by the inhalation of asbestos fibers. Despite extensive investigations, the precise mechanisms regulating asbestos-induced lung damage are not fully understood. This review summarizes the important clinical manifestations and pathogenic mechanisms of asbestosis. We focus on the relatively new information that has emerged over the last several years. The diagnosis of asbestosis is often easily established by well-characterized criteria. Pulmonary physiologic testing and high-resolution computed tomography can detect clinically occult disease. The finding of asbestos bodies in the bronchoalveolar lavage fluid confirms that an individual has been exposed to asbestos but is of unclear significance in diagnosing asbestosis. Evidence reviewed herein suggests that asbestos pulmonary toxicity is due in part to the physical properties of the fibers, iron-catalyzed reactive oxygen species (ROS), and macrophage-derived cytokines and growth factors. Special emphasis is given to the hypothesis that iron-catalyzed hydroxyl radicals (HO.-) have a pivotal role in causing asbestosis. Definitive proof of this hypothesis is difficult to obtain since HO.- are highly reactive and their deleterious effects to cells may have occurred years prior to disease presentation. Despite these limitations, considerable data firmly support the notion that ROS have an important role in causing asbestos toxicity. Further, the iron content of asbestos or the redoxactive iron associated with or mobilized from the surface of the fibers is important in generating HO.- as well as in activating inflammatory cells. There also appears to be a close association between asbestos-induced ROS production and cellular toxicity and DNA damage. The full expression of asbestos-induced diseases likely involves the contribution of cytokines, growth factors, proteases, and other inflammatory cell products. Many of the mechanisms by which asbestos- and inflammation-induced ROS activate specific genes in pulmonary cells remain to be elucidated.

Iron and reactive oxygen species in the asbestos-induced tumor necrosis factor-alpha response from alveolar macrophages.

Free radicals and other reactive oxygen species (ROS) are important mediators in asbestos-induced lung toxicity. Asbestos fibers are thought to stimulate cells to generate ROS via iron that is present on fibrous silicates. The pathophysiologic responses in the lung after asbestos exposure are characterized by the accumulation of macrophages at the site of fiber deposition and the release of growth factors and proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha). We have examined the role of iron-catalyzed ROS in asbestos induction of TNF-alpha from rat alveolar macrophages. Treatment of alveolar macrophage cultures with asbestos stimulated dose-dependently TNF-alpha secretion, which was inhibited by the addition of deferoxamine, an iron chelator. Asbestos fibers, pretreated with deferoxamine to remove iron from the fibers before addition to alveolar macrophages, also significantly reduced the TNF-alpha response. Consistent with the role of iron on asbestos fibers in catalyzing hydroxyl radical generation, membrane-permeable hydroxyl radical scavengers (tetramethylthiourea, dimethyl sulfoxide) inhibited the asbestos-induced TNF-alpha response. The asbestos-induced increase in TNF-alpha, as well as in interleukin-1 alpha, and their inhibition by tetramethylthiourea occurred at the transcriptional level. The role of ROS in signaling TNF-alpha stimulation was confirmed by use of free radical-generating systems (hypoxanthine-xanthine oxidase, hydrogen peroxide, glucose-glucose oxidase, or ferrous plus hydrogen peroxide). These results suggest that intracellularly generated ROS can stimulate TNF-alpha in alveolar macrophages and that asbestos-induced TNF-alpha gene expression and secretion are mediated by iron-catalyzed product of ROS.