Paul H. Beswick

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.

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.

Differential release of superoxide anions by macrophages treated with long and short fibre amosite asbestos is a consequence of differential affinity for opsonin.

OBJECTIVE--To investigate the ability of short and long fibre samples of amosite asbestos to stimulate superoxide production in isolated rat alveolar macrophages, and to determine how opsonisation with rat immunoglobulin might modify this response. METHODS--Macrophages were isolated from rat lung by bronchoalveolar lavage and challenged with both opsonised and non-opsonised long and short fibres of amosite asbestos. Release of superoxide anions was measured by the spectrophotometric reduction of cytochrome c, in the presence and absence of superoxide dismutase. RESULTS--Both long and short fibre samples of amosite asbestos without opsonisation were ineffective in stimulating isolated rat alveolar macrophages to release superoxide anions in vitro. After opsonisation with immunoglobulin, however, a dramatic enhancement of release of superoxide anion was seen with long fibres, but not short, which confirms the importance of fibre length in mediating biological effects. The increased biological activity of the long fibre sample is explained by increased binding of the opsonin to the fibre surface as, at equal mass, the long fibres bound threefold more immunoglobulin than the short fibres. CONCLUSION--Opsonisation is an important factor in modulation of the biological activity of fibres at the cellular level. Differences in binding of opsonin to samples of fibre previously considered to be identical apart from length, suggest that surface reactivity needs to be taken into account when fibres are compared. Binding of biological molecules, in vivo, may thus be an important modifying factor in the pathological processes initiated by fibres.

Induction of nuclear translocation of NF-κB in epithelial cells by respirable mineral fibres

A panel of mineral fibres has been studied for their ability to cause translocation of the transcription factor NF‐κB to the nucleus in A549 lung epithelial cells. On the basis of inhalation studies, three fibres were designated as being carcinogenic—amosite asbestos, silicon carbide and refractory ceramic fibre 1 (RCF1)—or non‐carcinogenic—man‐made vitreous fibre (MMVF10), Code 100/475 glass fibre, and RCF4. The experiments were carried out at equal fibre number. It was hypothesized that carcinogenic fibres have greater free radical activity than non‐carcinogenic fibres and that an oxidative stress produced in the lung after inhalation of fibres could cause translocation of the transcription factor NF‐κB to the nucleus, where transcription of pro‐inflammatory genes such as cytokines could occur. It was demonstrated that a simple oxidant, hydrogen peroxide, caused translocation in a time‐ and dose‐dependent manner. The three carcinogenic fibres produced a significant dose‐dependent translocation of NF‐κB to the nucleus, whereas the non‐carcinogenic fibres did not. Silicon carbide fibres were the most potent of the pathogenic fibres. MMVF10 was the most potent of the non‐pathogenic fibres, causing significant nuclear translocation of NF‐κB at high fibre number. Using three antioxidants, curcumin, pyrrolidine dithiocarbamate, and Nacystelin, translocation caused by carcinogenic fibres could be significantly reduced. The present study shows that a short‐term in vitro assay can discriminate between pathogenic and non‐pathogenic fibres in terms of a key pro‐inflammatory event in epithelial cells. The mechanism of the activation of NF‐κB by pathogenic fibres and its general applicability to other fibre types remain to be determined. Copyright

Enhancement of the Macrophage Oxidative Burst by Immunoglobulin Coating of Respirable Fibers: Fiber-Specific Differences Between Asbestos and Man-Made Fibers

The ability of long amosite asbestos fibers (LFA), vitreous fibers (MMVF 21 and CODE 100/475), and ceramic fibers (silicon carbide and RCF 1) to stimulate superoxide production in isolated rat alveolar macrophages is examined. The cells were exposed to both naked fibers (uncoated) and fibers coated with rat immunoglobulin (IgG), a normal component of lung lining fluid. The affinity for IgG of the various fibers was assessed by quantifying the binding of 125I-labeled IgG onto the fiber surface. Naked fibers stimulated a modest release of superoxide anion from alveolar macrophages, which was not obviously dose dependent. When IgG was adsorbed onto fibers of MMVF 21 and RCF 1, there was a dramatic increase in superoxide release, which correlated well with their high affinity for IgG.IgG-adsorbed code 100/475 and silicon carbide whiskers (SiCW) stimulated only modest superoxide release, and the fibers showed a correspondingly poor affinity for the opsonin. Conversely, the adsorbed fibers of LFA, generated a dramatic increase in superoxide release from the macrophages, despite a relatively poor adsorption of IgG. This study demonstrates the potential for components of the lung lining fluid to modify the response of alveolar macrophages to respirable natural and man-made fibers. It also draws attention to fiber-specific differences in adsorptive capacity and subsequent biological activity between these fiber types in vitro and, by implication, in vivo.

Chemical differences between long and short amosite asbestos: differences in oxidation state and coordination sites of iron, detected by infrared spectroscopy.

OBJECTIVES: Short fibres of amosite asbestos (SFA), obtained by ball milling of long fibres (LFA), have been shown to be less pathogenic than long fibres. Accumulating evidence suggests an important role for differences in surface chemistry between fibres. Iron has been implicated in the pathogenesis of asbestos fibres. In this study infrared (IR) spectroscopy was used to compare LFA and SFA in terms of the coordination and oxidation state of iron at the three cation sites (M1, M3, M1). METHODS: Infrared was used to examine LFA ad SFA, when dry and when hydrated in the presence and absence of the chelators desferroxamine and ferrozine. With appropriate software the proportions of iron and its oxidation states in the overlapping peaks were resolved and assigned, and the three coordination sites were identified. Data were obtained from 10 samples of both lengths of fibre for each of the four treatments. Iron release was also monitored. RESULTS: Iron was significantly more oxidised in LFA than SFA. Further oxidation of the dry fibres with water, ferrozine, or desferroxamine tended to abolish these differences. There were also significant differences between the proportions of iron held in the different coordination sites of the fibres. For LFA, a higher proportion of its iron was held in the cation sites coordinating less with iron and more with Mg. Interestingly, the sites coordinating single irons were significantly more oxidised than multiple sites. The single iron sites were more oxidised in LFA than SFA and were more readily oxidised by the treatments. CONCLUSIONS: Important chemical differences between LFA and SFA were found. There seemed to be some mobility of iron near the surface. Based on these data it is speculated that the 1 iron surface site may be important in pathogenesis.

Respirable Fibres: Surfactant Coated Fibres Release More Fe3+ than Native Fibres at Both pH 4.5 and 7.2

Exposure to asbestos is associated with several lung diseases. The carcinogenic action of asbestos is not fully understood but oxidative stress is considered to play a role. Iron on the surface can lead to Fenton chemistry and the Haber Weiss reaction producing free radicals such as the hydroxyl radical, which is likely to be important. Little is known of the pathogenic action of man-made fibres. This study involved the use of a panel of man-made fibres, some of which were shown to be pathogenic and others shown to be non-pathogenic in recent animal studies. A short term assay measuring Fe3+ release from the fibres over a 20 hour time period, and also a longer study of 12 week, found that pathogenic and non-pathogenic fibres could not be differentiated according to Fe3+ release only. Iron release from native fibres was compared with that from surfactant-coated fibres, and in all cases surfactant coated fibres released more Fe3+ inferring that in vivo lung lining fluid coats native fibres and therefore affects the fibre surface chemistry and hence reactivity.

Superoxide anion release by alveolar macrophages exposed to respirable industrial fibres: modifying effect of fibre opsonisation

Phagocyte-derived free radicals are considered to play a role in fibre-related pathology and the components of the lung lining fluid could modify the surface of fibres. Therefore we examined the ability of long amosite asbestos and a range of man-made fibres to stimulate release of superoxide anion from rat alveolar macrophages when they were in their native form (unopsonised) and opsonised by incubation in rat Immunoglobulin G. We also assessed the specific amount of opsonin adsorbed to each fibre type. In the uncoated form all of the fibres produced modest amounts of superoxide release from macrophages. When they were opsonised however there was an effect on stimulation of release of superoxide that was fibre-specific. Both MMVF21 and RCF 1 were dramatically enhanced in their ability to stimulate release and this was related to a high affinity of their surface for IgG. Code 100/475 and SiC were not substantially affected by opsonisation and this was reflected in their low affinity for IgG. Long amosite had low affinity for IgG but showed dramatic enhancement of capacity to stimulate superoxide release. These fibre-specific differences in the effect of a coating of material that is found in the lung lining points out the problems of interpretation of in vitro data and more work on this important area is warranted.

Release of TNFα in response to SiC fibres: differential effects in rodent and human primary macrophages, and in macrophage-like cell lines

Asbestos has been implicated in the pathogenesis of several lung diseases, but its mechanism of action is not fully understood. However, asbestos-induced oxidative stress and production of inflammatory cytokines may play a significant role. TNFalpha is an inflammatory cytokine which has a central role in inflammation and fibrosis due to its ability to stimulate fibroblasts and collagen deposition. In this study, a panel of fibres designated either pathogenic or non-pathogenic in recent animal studies, were utilized. The amount of TNFalpha released after a 16-hour exposure to the panel of fibres was compared in four different cell types; two primary macrophage cell types and two cell lines. TNFalpha release by cells exposed to the panel did not equate to pathogenicity, although the most pathogenic fibre caused three out of the four cell types tested, to produce the greatest amount of TNFalpha. Primary rat cells and primary human cells behaved in a similar manner as regards to TNFalpha production; the cell lines behaved quite differently to their primary counterparts with regards to TNFalpha production in this study.

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.