The procoagulant potential of environmental particles (PM10).

BACKGROUND AND AIMS: Epidemiology studies have shown that cardiovascular (CV) disease is primarily responsible for the mortality associated with increased pulmonary environmental particle (PM10) exposure. The mechanisms involved in PM10 mediated CV effects are unknown although changes in plasma viscosity and in the homoeostasis of blood coagulation have been implicated. It was hypothesised that PM10 exposure would result in an inflammatory response and enhance the activation of the extrinsic coagulation mechanisms in pulmonary and vascular cells in culture. METHODS: Primary human monocyte derived macrophages and human umbilical cord vein endothelial, human alveolar type II epithelial (A549), and human bronchial epithelial (16HBE) cells were tested for their inflammatory and procoagulant response to PM10 exposure. IL-8, tissue factor (TF), and tissue plasminogen activator (tPA) gene expression and protein release, and coagulation enhancing ability of culture media were determined 6 and 24 hours following exposure. RESULTS: The culture media from macrophages and 16HBE bronchial epithelial cells, but not A549 cells, exposed to PM10 had an enhanced ability to cause clotting. Furthermore, H2O2 also increased the clotting activity. Apoptosis was significantly increased in macrophages exposed to PM10 and LPS as shown by annexin V binding. TF gene expression was enhanced in macrophages exposed to PM10, and HUVEC tissue factor and tPA gene and protein expression were inhibited. CONCLUSIONS: These data indicate that PM10 has the ability to alter macrophage, epithelial, and endothelial cell function to favour blood coagulation via activation of the extrinsic pathway and inhibition of fibrinolysis pathways.

Calcium and ROS-mediated activation of transcription factors and TNF-alpha cytokine gene expression in macrophages exposed to ultrafine particles.

Ultrafine (Uf) particles are a component of particulate air pollution suggested to be responsible for the health effects associated with elevations of this pollutant. We have previously suggested that Uf particles, through the induction of oxidative stress, may induce inflammation in the lung, thus exacerbating preexisting illness in susceptible individuals. Alveolar macrophages are considered to play a key role in particlemediated inflammation and lung disease. The effect of Uf particles on rat alveolar macrophages and human blood monocytes was investigated with reference to the roles of calcium and reactive oxygen species (ROS). TNF-alpha protein release, intracellular calcium concentration, TNF-alpha mRNA expression, and transcription factor activation were studied as end points after treatment of rat alveolar macrophages or peripheral blood monocytes. The calcium channel blocker verapamil, the intracellular calcium chelator BAPTA-AM, the calmodulin inhibitor W-7, and the antioxidants Trolox and Nacystelin (NAL) were included in combination with Uf particles. Verapamil reduced intracellular calcium concentration in rat alveolar macrophages on stimulation with Uf particles. This effect was also apparent with transcription factor AP-1 activation. All antagonists and antioxidants reduced Uf-stimulated nuclear localization of the p50 and p65 subunits of NF-kappaB in human monocytes. Verapamil, BAPTA-AM, and NAL reduced Uf-stimulated TNF-alpha protein release, whereas only verapamil reduced Uf-stimulated mRNA expression in rat alveolar macrophages. In human monocytes, verapamil, Trolox, BAPTA-AM, and W-7 reduced Uf-stimulated TNF-alpha protein release. These findings suggest that Uf particles may exert proinflammatory effects by modulating intracellular calcium concentrations, activation of transcription factors, and cytokine production through a ROS-mediated mechanism.

PM(10)-exposed macrophages stimulate a proinflammatory response in lung epithelial cells via TNF-alpha.

There is now considerable evidence for an association between the levels of particulate air pollution [particulate matter <10 microm in aerodynamic diameter (PM(10))] and various adverse health endpoints. The release of proinflammatory mediators from PM(10)-exposed macrophages may be important in stimulating cytokine release from lung epithelial cells, thus amplifying the inflammatory response. A549 cells were treated with conditioned media from monocyte-derived macrophages stimulated with PM(10), titanium dioxide (TiO(2)), or ultrafine TiO(2). We demonstrate that only conditioned media from PM(10)-stimulated macrophages significantly increased nuclear factor-kappaB and activator protein-1 DNA binding, enhanced interleukin-8 (IL-8) mRNA levels as assessed by RT-PCR, and augmented IL-8 protein levels, over untreated controls. Furthermore, PM(10)-conditioned media also caused transactivation of IL-8 as determined by an IL-8-chloramphenicol acetyl transferase reporter. Analysis of these conditioned media revealed marked increases in tumor necrosis factor-alpha (TNF-alpha) and protein levels and enhanced chemotactic activity for neutrophils. Preincubation of conditioned media with TNF-alpha-neutralizing antibodies significantly reduced IL-8 production. These data suggest that PM(10)-activated macrophages may amplify the inflammatory response by enhancing IL-8 release from lung epithelial cells, in part, via elaboration of TNF-alpha.

Aluminium lactate treatment of DQ12 quartz inhibits its ability to cause inflammation, chemokine expression, and nuclear factor-kappaB activation.

In 1997, an IARC Working Group classified quartz (crystalline silica) as a Group 1 lung carcinogen, but only in some industries, i.e., the quartz hazard is a variable entity. The reactivity of the quartz surface may underlie its ability to cause inflammation, and treatments that ameliorate this reactivity will reduce the quartz hazard. In this study we treated quartz (Q) with aluminium lactate (AL), a procedure that is reported to decrease the quartz hazard, and explored the effect this had on the highly reactive quartz surface and on proinflammatory events in rat lungs. Aluminium lactate-treated quartz showed a reduced surface reactivity as measured by electron spin resonance and the hemolysis assay. Eighteen hours after instillation of Q into the rat lung, there was massive inflammation as indicated by the number of neutrophils in the bronchoalveolar lavage (BAL). In addition, Q induced an increase in BAL macrophage inflammatory protein-2 (MIP-2) while ALQ had no significant effect compared to control. Epithelial damage, as indicated by BAL protein and gamma glutamyl transpeptidase, also increased with Q but not with ALQ. Furthermore, Q induced an increase in MIP-2 mRNA by BAL cells while ALQ had no effect compared to controls. There was an increase in nuclear binding of the transcription nuclear factor kappaB (NF-kappaB) in the Q-exposed BAL cells and again no effect on nuclear NF-kappaB binding in BAL cells from ALQ-exposed rats. In conclusion, treatment of the quartz surface with aluminium lactate reduced the reactivity of the particles both in terms of hydroxyl radical generation and in terms of the induction of molecular signaling events leading to inflammation.

Oxidant-mediated lung epithelial cell tolerance: the role of intracellular glutathione and nuclear factor-kappaB.

The airway epithelium is injured by oxidants inhaled as atmospheric pollutants or produced during inflammatory responses. We studied the effect of modulating the antioxidant intracellular glutathione, both using thiol compounds and by the adaptive effect of hyperoxia, on oxidant-induced injury and activation of the nuclear factor-kappaB (NF-kappaB) in two cell lines: the human bronchial (16HBE) and type II alveolar epithelial cells (A549). The thiol antioxidants glutathione (GSH) and glutathione monoethyl ester (GSH-MEE) [2 mM] increased GSH levels (nmol/mg protein) in A549 cells (GSH 383 +/- 26 and GSH-MEE 336 +/- 23 vs control 171 +/- 13, P < 0.001) and in 16HBE cells (GSH 405 +/- 33, GSH-MEE 362 +/- 37 vs control 198 +/- 12, P < 0.001, N = 3). Treatment of hyperoxia (95% oxygen) also increased GSH levels between 4 and 24 hr exposure compared with control (P < 0.01). Hydrogen peroxide (H(2)O(2)) (0.01 mM) induced NF-kappaB activation, whereas hyperoxia exposure did not affect NF-kappaB activation in either cell line. Pretreatment with dl-buthionine (SR)-sulfoximine, which decreased intracellular glutathione, increased NF-kappaB binding induced by H(2)O(2) and increased lactate dehydrogenase (LDH) release (P < 0.001). Pretreatment with the thiol compounds and hyperoxia totally inhibited H(2)O(2)-induced NF-kappaB binding and cell injury as measured by LDH release. These data indicate the importance of intracellular glutathione and inhibition of NF-kappaB in both protection/tolerance against oxidant-induced epithelial cell injury, and NF-kappaB activation in response to oxidative stress which may be important in lung inflammation. Thus, increasing intracellular glutathione may be of therapeutic relevance if able to modulate NF-kappaB activation and hence attenuate inflammation.

Adenoviral E1A primes alveolar epithelial cells to PM(10)-induced transcription of interleukin-8.

The presence of the adenoviral early region 1A (E1A) protein in human lungs has been associated with an increased risk of chronic obstructive pulmonary disease (COPD), possibly by a mechanism involving amplification of proinflammatory responses. We hypothesize that enhanced inflammation results from increased transcription factor activation in E1A-carrying cells, which may afford susceptibility to environmental particulate matter < 10 microm (PM(10))-mediated oxidative stress. We measured interleukin (IL)-8 mRNA expression and protein release in human alveolar epithelial cells (A549) transfected with the E1A gene (E1A+ve). Both E1A+ve and -ve cells released IL-8 after incubation with TNF-alpha, but only E1A+ve cells were sensitive to LPS stimulation in IL-8 mRNA expression and protein release. E1A+ve cells showed an enhanced IL-8 mRNA and protein response after treatment with H(2)O(2) and PM(10). E1A-enhanced induction of IL-8 was accompanied by increases in activator protein-1 and nuclear factor-kappa B nuclear binding in E1A+ve cells, which also showed higher basal nuclear binding of these transcription factors. These data suggest that the presence of E1A primes the cell transcriptional machinery for oxidative stress signaling and therefore facilitates amplification of proinflammatory responses. By this mechanism, susceptibility to exacerbation of COPD in response to particulate air pollution may occur in individuals harboring E1A.

Systemic Effect of Particulate Air Pollution.

The association in epidemiological studies between particulate air pollution (PM10) and increased mortality and morbidity from cardiovascular disease is well established. However, the mechanism(s) by which PM10 produces these effects is unknown. We have developed a hypothesis that ultrafine components of PM10 cause lung inflammation by creating local lung oxidative stress, which activates transcription factors such as NF-kB and hence the transcription of genes for inflammatory mediators. In a series of studies in vivo in the rat and using cultured airspace epithelial cells in vitro, we have tested this hypothesis using environmental PM10 and fine (CB 260 nm in diameter) and ultrafine (UfCB, 14 nm in diameter) carbon particles, since carbon is a major component of PM10 We have shown that compared with CB, which produces trivial effects, UFCB and PM10 produce local lung inflammation, increased epithelial permeability, and evidence of oxidative stress. We have also shown that PM10 also activates NF-kB in airspace epithelial cells. We also hypothesized that the local lung inflammation produced by PM10, may result in systemic effects, in particular systemic oxidative stress and enhanced blood coagulation, which may have a role in the adverse cardiovascular effects induced by PM10. To test this hypothesis we measured oxidative stress and changes in coagulation factors in plasma following the inhalation of UFCB, CB, or instillation ofPM,10 in the rat. Immediately after 7 h inhalation of UFCB (1000 (g/m(3)) there was a significant decrease in the antioxidant capacity in rat plasma, which fell further 16 and 48 h after cessation of the inhalation. A similar fall in plasma antioxidant capacity was shown 6 h after instillation of PM10 (125 tig). In contrast, there were no significant changes in antioxidant capacity in rats after 7 h of inhalation of CB at similar concentrations. The levels of factor VII in plasma, which is a key factor in the intrinsic pathway of coagulation cascade, increased at time points from 6 h to 7 days after inhalation of UFCB, but did not change after CB exposure. There were no changes in plasma fibrinogen or other coagulation factors after inhalation of particles. Thus, inhalation of ultrafine carbon black particles and instillation of PMio in rats decreases plasma antioxidant capacity as an indication of systemic oxidative stress. Ultrafine carbon black particles also cause increased factor VII levels in the plasma, a known risk factor for adverse cardiovascular events. These studies help to explain the relationship between the levels of particulate air pollutants and cardiovascular morbidity/mortality.

Isolation of a second recombinant human respiratory syncytial virus monoclonal antibody fragment (Fab RSVF2-5) that exhibits therapeutic efficacy in vivo.

A second human respiratory syncytial virus (RSV)-neutralizing monoclonal antibody was isolated and its binding site was identified. Fab F2-5 is a broadly reactive fusion (F) protein-specific recombinant Fab generated by antigen selection from a random combinatorial library displayed on the surface of filamentous phage. In an in vitro plaque-reduction test, the Fab RSVF2-5 neutralized the infectivity of a variety of field isolates representing viruses of both RSV subgroups A and B. The Fab recognized an antigenic determinant that differed from the only other human anti-F monoclonal antibody (RSV Fab 19) described thus far. A single dose of 4.0 mg of Fab RSVF2-5/kg of body weight administered by inhalation was sufficient to achieve a 2000-fold reduction in pulmonary virus titer in RSV-infected mice. The antigen-binding domain of Fab RSVF2-5 offers promise as part of a prophylactic regimen for RSV infection in humans.

In vivo and in vitro proinflammatory effects of particulate air pollution (PM10).

Epidemiologic studies have reported associations between fine particulate air pollution, especially particles less than 10 mm in diameter (PM10), and the development of exacerbations of asthma and chronic obstructive pulmonary disease. However, the mechanism is unknown. We tested our hypothesis that PM10 induces oxidant stress, causing inflammation and injury to airway epithelium. We assessed the effects of intratracheal instillation of PM10 in rat lungs. The influx of inflammatory cells was measured in bronchoalveolar lavage (BAL). Airspace epithelial permeability was assessed as total protein in bronchoalveolar lavage fluid (BALF) in vivo. The oxidant properties of PM10 were determined by their ability to cause changes in reduced glutathione (GSH) and oxidized glutathione (GSSG). We also compared the effects of PM10 with those of fine (CB) and ultrafine (ufCB) carbon black particles. Six hours after intratracheal instillation of PM10, we noted an influx of neutrophils (up to 15% of total BAL cells) in the alveolar space, increased epithelial permeability, an increase in total protein in BALF from 0.39 +/- 0.01 to 0.62 +/- 0.01 mg/ml (mean +/- SEM) and increased lactate dehydrogenase concentrations in BALF. An even greater inflammatory response was observed after intratracheal instillation of ufCB, but not after CB instillation. PM10 had oxidant activity in vivo, as shown by decreased GSH in BALF (from 0.36 +/- 0.05 to 0.25 +/- 0.01 nmol/ml) after instillation. BAL leukocytes from rats treated with PM10 produced greater amounts of nitric oxide, measured as nitrite (control 3.07 +/- 0.33, treated 4.45 +/- 0.23 mM/1 x 10(6) cells) and tumor necrosis factor alpha (control 21.0 +/- 3.1, treated 179.2 +/- 29.4 unit/1 x 10(6) cells) in culture than BAL leukocytes obtained from control animals. These studies provide evidence that PM10 has free radical activity and causes lung inflammation and epithelial injury. These data support our hypothesis concerning the mechanism for the adverse effects of particulate air pollution on patients with airway diseases.

Free radical activity of PM10: iron-mediated generation of hydroxyl radicals.

The purpose of this study was to test the hypothesis that particulate matter < or = 10 microns in aerodynamic diameter (PM10) particles have the ability to generate free radical activity at their surface. We collected PM10 filters from the Edinburgh, United Kingdom, Enhanced Urban Network sampling site, removed particles from the filter, and tested their ability to cause free radical damage to supercoiled plasmid DNA. We found that the PM10 particles did cause damage to the DNA that was mediated by hydroxyl radicals, as shown by inhibition of the injury with mannitol. The PM10-associated hydroxyl radical activity was confirmed using a high-performance liquid chromatography-based assay to measure the hydroxyl radical adduct of salicylic acid. Desferrioxamine abolished the hydroxyl radical-mediated injury, which suggests that iron was involved. Analysis of PM10 filters confirmed the presence of large amounts of iron and leaching studies confirmed that the PM10 samples could release substantial amounts of Fe(III) and lesser amounts of Fe(II). To investigate the size of the particles involved in the hydroxyl radical injury, we centrifuged the suspension of PM10 to clarity, tested the clear supernatant, and found that it had all of the suspension activity. We conclude, therefore, that the free radical activity is derived either from a fraction that is not centrifugeable on a bench centrifuge, or that the radical generating system is released into solution.

Free radical activity of industrial fibers: role of iron in oxidative stress and activation of transcription factors.

We studied asbestos, vitreous fiber (MMVF10), and refractory ceramic fiber (RCF1) from the Thermal Insulation Manufacturers' Association fiber repository regarding the following: free radical damage to plasmid DNA, iron release, ability to deplete glutathione (GSH), and activate redox-sensitive transcription factors in macrophages. Asbestos had much more free radical activity than any of the man-made vitreous fibers. More Fe3+ was released than Fe2+ and more of both was released at pH 4.5 than at pH 7.2. Release of iron from the different fibers was generally not a good correlate of ability to cause free radical injury to the plasmid DNA. All fiber types caused some degree of oxidative stress, as revealed by depletion of intracellular GSH. Amosite asbestos upregulated nuclear binding of activator protein 1 transcription factor to a greater level than MMVF10 and RCF1; long-fiber amosite was the only fiber to enhance activation of the transcription factor nuclear factor kappa B (NF kappa B). The use of cysteine methyl ester and buthionine sulfoximine to modulate GSH suggested that GSH homeostasis was important in leading to activation of transcription factors. We conclude that the intrinsic free radical activity is the major determinant of transcription factor activation and therefore gene expression in alveolar macrophages. Although this was not related to iron release or ability to deplete macrophage GSH at 4 hr, GSH does play a role in activation of NF kappa B.

Adverse health effects of PM10 particles: involvement of iron in generation of hydroxyl radical.

OBJECTIVES: Environmental particles < 10 microns average aerodynamic diameter (PM10) are associated with mortality, exacerbation of airways diseases, and decrement in lung function. It is hypothesised that PM10 particles, along with other pathogenic particles, generate free radicals at their surface in reactions involving iron, and that this is a factor in the pathogenicity of PM10 particles. Identification of free radical activity in PM10 and examination of the content and role of iron in this process was undertaken. METHODS: Free radical activity was detected with a supercoiled plasmid, phi X174 RF1 DNA, and measured as scission of the supercoiled DNA (mediated by free radicals) by scanning laser densitometry. The role of the hydroxyl radical was confirmed by the use of the specific scavenger mannitol, and the role of iron investigated with the iron chelator desferrioxamine-B (DSF-B). Iron released from PM10 particles at pH 7.2 and pH 4.6 (to mimic conditions on the lung surface and in macrophage phagolysosomes, respectively) was assessed spectrophotometrically with the Fe++ chelator ferrozine and the Fe+ + + chelator DSF-B. RESULTS: PM10 particles showed significant free radical activity by their ability to degrade supercoiled DNA. A substantial part of this activity was due to the generation of hydroxyl radicals, as shown by partial protection with mannitol. Similarly, DSF-B also conferred protection against the damage caused to plasmid DNA indicating the role of iron in generation of hydroxyl radicals. Negligible Fe++ was released at either pH 7.2 or pH 4.6 by contrast with Fe+ + +, which was released in substantial quantities at both pHs, although twice as much was released at pH 4.6. CONCLUSIONS: PM10 particles generate the hydroxyl radical, a highly deleterious free radical, in aqueous solution. This occurs by an iron dependent process and hydroxyl radicals could play a part in the pathogenicity of PM10 particles. Iron release was greatest at the pH of the lysosome (pH 4.6) indicating that iron may be mobilised inside macrophages after phagocytosis, leading to oxidative stress in the macrophages.

Free radical activity and pro-inflammatory effects of particulate air pollution (PM10) in vivo and in vitro.

BACKGROUND: Epidemiological evidence has implicated fine particulate air pollution, particularly particles less than 10 microns in diameter (PM10), in the development of exacerbations of asthma and chronic obstructive pulmonary disease (COPD) although the mechanism is unknown. The hypothesis that PM10 particles induce oxidant stress, causing inflammation and injury to airway epithelium, was tested. METHODS: The effects of intratracheal instillation of PM10 was assessed in rat lungs (three per group). Inflammatory cell influx was measured by bronchoalveolar lavage (BAL) and air space epithelial permeability was assessed as the total protein in BAL fluid in vivo. The oxidant properties of PM10 particles were determined by their ability to cause damage to plasmid DNA and by changes in reduced (GSH) and oxidised (GSSG) glutathione. The effects of PM10 particles were compared in some experiments with those of fine (CB) and ultrafine (ufCB) carbon black particles. RESULTS: Six hours after intratracheal instillation of PM10 there was an influx of neutrophils (up to 15% of total cells in BAL fluid) into the alveolar space, increased epithelial permeability, the mean (SE) total protein in the BAL fluid increasing from 0.39 (0.01) to 0.62 (0.01) mg/ml, and increased lactate dehydrogenase (LDH) concentrations in the BAL fluid. An even greater inflammatory response was seen following intratracheal instillation of ufCB but not following CB instillation. PM10 particles had free radical activity in vivo, as shown by a decrease in GSH levels in the BAL fluid from 0.36 (0.05) to 0.25 (0.01) nmol/ml following instillation. The free radical activity of PM10 was confirmed in vitro by its ability to deplete supercoiled plasmid DNA, an effect which could be reversed by mannitol, a specific hydroxyl radical scavenger. BAL fluid leucocytes from rats treated with PM10 produced greater amounts of nitric oxide (NO), measured as nitrite (control 3.07 (0.33), treated 4.45 (0.23) microM/1 x 10(6) cells), and tumour necrosis factor alpha (control 21.0 (3.1), treated 179.2 (29.4) units/l x 10(6) cells) in culture than those obtained from control animals. Since the PM10 preparation was contaminated with small amounts of filter fibres due to the extraction process, the effects of instillation of filter fibres alone was assessed. These studies showed that filter fibres did not account for the proinflammatory and injurious effects of the PM10 suspension. CONCLUSIONS: These findings provide evidence that PM10 has free radical activity and causes lung inflammation and epithelial injury. These data support the proposed hypothesis for the mechanism by which particulate air pollution causes adverse effects in patients with airways diseases.

Free radical activity associated with the surface of particles: a unifying factor in determining biological activity?

Using a sensitive phi X174 RF plasmid DNA assay, free radical activity was detected at the surface of normal and ultrafine titanium dioxide (TiO2), environmental particles (PM-10), asbestos and a range of man-made fibres. There were differences in the amount of free radical activity that was detected, with ultrafine TiO2 being much more active than normal-sized TiO2; PM-10 also had substantial free radical activity. Amphibole asbestos samples were highly active, whilst man-made fibres were much less active than asbestos. For all of the particles, the DNA damage could be ameliorated by mannitol, showing that hydroxyl radicals were involved. The ability of particles to generate free radicals at or near their surface, and thereby impose oxidant stress in key target cells, could be central to determining their pathogenicity.

Detection of surface free radical activity of respirable industrial fibres using supercoiled phi X174 RF1 plasmid DNA.

The ability of a number of respirable industrial fibres, amosite and crocidolite asbestos, refractory ceramic fibres (RCFs) and man-made vitreous fibres (MMVFs) to cause free radical injury to plasmid phi X174 RFI DNA was assessed. The oxidative DNA damage was observed as depletion of supercoiled DNA after fibre treatment was quantified by scanning laser densitometry. The mechanism of fibre-mediated damage was determined by the use of the specific hydroxyl radical scavenger mannitol and the iron chelator desferrioxamine-B. The amosite and crocidolite asbestos caused substantial damage to DNA that was dose-related. The free radicals responsible for the asbestos-mediated DNA damage were hydroxyl radicals, as determined by inhibition with mannitol. Asbestos fibre-mediated damage to DNA was completely ameliorated by the chelation of fibre-associated iron with desferrioxamine-B. The amount of Fe(II) and Fe(III) released by equal numbers of the different fibre types at equal fibre number was determined. The fibres released very small amounts of Fe(II) and there were no significant differences between the fibre types. The fibres released substantial amounts of Fe(III); MMVF 21 released significantly more Fe(III) than any of the other fibres and short fibre amosite also released more Fe(III) than three of the MMVFs and two of the RCFs. When ability to release Fe(II) and Fe(III) was compared with ability to cause DNA damage there was not a good correlation, because only the long amosite and crocidolite caused substantial free radical injury to DNA; this contrasts with MMVF 21 and short amosite being the two fibres that released the greatest amounts of iron. The loss of ability to damage DNA in DSF-B-treated asbestos fibres shows that iron at the surface of asbestos fibres definitely has a role in generating hydroxyl radicals. However, it is clear that some fibres, such as short amosite and MMVF 21, release large quantities of iron without causing free radical damage, whilst neither long amosite nor crocidolite released more iron than the other fibres. The exact role of iron in fibre reactivity therefore remains unresolved, but fibre-bound iron not released from the surface of asbestos could be important. Further research is under way to investigate this possibility.

Supercoiled plasmid DNA as a model target for assessing the generation of free radicals at the surface of fibres.

The ability of respirable amosite and crocidolite asbestos, refractory ceramic fibres (RCFs) and man made vitreous fibres (MMVFs) to cause free radical injury to plasmid, phiX174 RFI DNA was assessed. The amosite and crocidolite asbestos caused substantial damage to the DNA and, in the main, the free radicals responsible for the asbestos-mediated DNA damage were hydroxyl radicals as determined by inhibition with mannitol. Asbestos fibre-mediated damage to the DNA was completely ameliorated by the chelation of fibre-associated iron by pre-treatment of fibres with desferrioxamine-B, confirming the importance of iron in the production of free radicals. MMVFs and RCFs produced modest free radical damage to the DNA, which was prevented by mannitol but not by iron chelation.