Objective: To assess whether fine and ultrafine particles (nanoparticles) have the capacity to activate factors in serum that would induce macrophage migration. This is a model previously reported to investigate complement activation by other respirable particles and fibres. Method: Foetal bovine serum was exposed to varying doses of fine and nanoparticle carbon black as well as the oxidant tertbutyl hydroperoxide (tBHP). The subsequent potential of the serum to induce macrophage migration was measured using a macrophage chemotaxis assay. Results: Treatment of serum with 10 mg/ml of nanoparticle carbon black generated substances that induced a 1.8-fold increase in macrophage migration (P < 0.001) compared with untreated serum. This effect was partially inhibited by antioxidant intervention. Serum treated with an equivalent mass of fine carbon black did not display any chemotactic potential. tBHP treatment of the serum did not result in the generation of macrophage chemotactic factors. Conclusions: High doses of nanoparticle carbon black have the capacity to cause chemotactic factor generation in serum, by a mechanism involving ROS generation, although ROS alone, in the form of tBHP are not adequate to generate chemotactic factors in serum. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Background: Ultrafine particles have been hypothesised to be an important contributing factor in the toxicity and adverse health effects of particulate air pollution (PM10) and nanoparticles are used increasingly in industrial processes. Aims: To compare the ability of ultrafine and fine particles of titanium dioxide and carbon black to induce inflammation, cause epithelial injury, and affect the alveolar macrophage clearance functions of phagocytosis and chemotaxis in vivo. Methods: Rats were instilled with fine and ultrafine carbon black and titanium dioxide. Inflammation was quantified by bronchoalveolar lavage; the ability of the macrophages to phagoytose indictor fluorescent beads and to migrate towards aC5a were determined. Results: Ultrafine particles induced more PMN recruitment, epithelial damage, and cytotoxicity than their fine counterparts, exposed at equal mass. Both ultrafine and fine particles significantly impaired the phagocytic ability of alveolar macrophages. Only ultrafine particle treatment significantly enhanced the sensitivity of alveolar macrophages to chemotact towards C5a. Conclusions: Ultrafine particles of two very different materials induced inflammation and epithelial damage to a greater extent than their fine counterparts. In general, the effect of ultrafine carbon black was greater than ultrafine titanium dioxide, suggesting that there are differences in the likely harmfulness of different types of ultrafine particle. Epithelial injury and toxicity were associated with the development of inflammation after exposure to ultrafines. Increased sensitivity to a C5a chemotactic gradient could make the ultrafine exposed macrophages more likely to be retained in the lungs, so allowing dose to accumulate.

A range of different particle types cause excessive lung inflammation that is thought to play a role in the various types of pathology they produce. Recently attention has been focused on ultrafine particles and the phenomenon of rat lung overload. The authors and their collaborators have shown previously that the surface area metric drives the overload response. Acute inflammatory response following instillation of particles has also been used to evaluate hazard but has been criticized because of the non-physiological delivery and the problems of local overload. We have instilled a number of low-toxicity dusts of various particle sizes and assessed neutrophil influx into the lung at 18–24 h. The extent of inflammation has been shown to be a function of the surface area instilled. Since ultrafine particles present a 'special' case of high surface area, they are relatively inflammogenic. There is no evidence that ultrafine particles of carbon black, titanium dioxide or latex have any special reactivity in addition to their large surface area. We tested whether we could use this approach to model the reactivity of highly toxic dusts. Rats were instilled with either DQ12 quartz or aluminium lactate-treated DQ12 and, as anticipated, the high specific surface toxicity of DQ12 meant that it was much more inflammogenic than was predicted using the relationship described for low-toxicity dusts. By contrast, aluminium lactate-treated DQ12 fell on to the line of 'low-toxicity' dusts. This approach presents the possibility of modelling potential toxicity for nuisance dusts based on the inflammatory response of a given instilled surface area dose.

Ultrafine particles are a component of air pollution, derived from primary combustion sources, and so we have undertaken a programme of study on the mechanisms of lung injury caused by ultrafine particles. Ultrafine particles made of low-solubility, low-toxicity materials are more inflammogenic in the rat lung than fine respirable, particles made from the same material. Ultrafine particles can cause inflammation via processes independent of the release of transition metals, as shown by the fact that soluble products from ultrafine carbon black have no ability to cause inflammation. The property that drives the greater inflammogenicity of ultrafines is unknown but very likely relates to particle surface area and involves oxidative stress. Increases in intracellular Ca(++) may underlie the cellular effects of ultrafines, although the mechanism whereby ultrafines have this effect is not understood. However, increased influx of Ca(++) into macrophages occurs via the membrane Ca(++) channels following contact with ultrafine particles, and involves oxidative stress. Increased Ca(++) in macrophages exposed to ultrafines can lead to the transcription of key pro-inflammatory genes such as TNFalpha. Ultrafine particles can also impair the ability of macrophages to phagocytose and clear other particles, and this may be pro-inflammogenic.

here is a long history of disease caused by inhaled particles that stretches from Agricola and Paracelsus in the 14th century up to the present. 1 In this century the twin scourges of asbestos and crystalline silica (quartz) have exerted a terrible toll of death and disease.The bad old days when these dust related lung diseases were unbridled are gone but as we enter the 21st century a new particle type-the ultrafine particle-has emerged as one with a potential role in causing disease.Ultrafine particles have been the subject of several recent reviews. 2 3 0 c ULTRAFINE PARTICLES Particles that are less than 100 nm in diameter are commonly defined as ultrafine.However, all particle populations that are found in the air are polydispersed-that is, they comprise particles with ranges of diameters that are usually summarised as mean or median diameter.There is no reason at the moment to think that there is much diVerence between a 90 nm and a 110 nm particle in ability to have an adverse eVect and there is little evidence for the cut oV point for the ultrafine eVect.Ultrafine particles are very small compared with the cellular structures and so this may be important in the apparent problems they present to the lung (fig 1).Particles of diesel exhaust may be taken as an example of an ultrafine particle, albeit complex.They are largely insoluble because of the particulate carbon core, commonly the size of singlet diesel particles, 10-20 nm.However, they tend to group into aggregates with sulphates, metals, and hydrocarbons. 4 Particles of diesel soot are ubiquitous in urban air and contribute to particulate air pollution of aerodynamic diameter 10 µm (PM 10 ).However, ultrafine particles in urban air could come from a range of sources other than diesel soot.For experimental purposes toxicologists have used several standard ultrafine particles such as titanium dioxide and carbon black for their studies.

We investigated whether slowed clearance after exposure to ultrafine particles was due to a failure in alveolar macrophage phagocytosis. This was achieved by measuring the ability of a macrophage cell line (J774.2 MPhi) to phagocytose 2-microg indicator latex beads following 8-h exposures to a number of test particles. Particles utilized were fine titanium dioxide (TiO2), ultrafine titanium dioxide (UTiO2), carbon black (CB), or ultrafine carbon black (UCB). Cytotoxicity of particles was measured by means of MTT activity. In a preliminary study, we assessed the effects of conditioned medium from particle-treated macrophages on the phagocytic ability of naive macrophages. Ultrafine and fine particles had no significant cytotoxic effects on J774.2 MPhi. A significant reduction in the ability of macrophages to phagocytose the indicator beads occurred after exposure to 0.39 microg/mm(2) (p < 0.001) of UCB and 0.78 microg/mm(2) (p < 0.001) of all particle types compared to the control. Furthermore, ultrafine particles were shown to significantly (p < 0.001) impair macrophage phagocytosis at a lower dose than their fine counterparts (0.39 and 0.78 microg/mm(2), respectively). At all doses, UCB resulted in a greater number (p < 0.001) of nonphagocytic macrophages compared to the other test particles. We tested whether a diffusable mediator being released from particle-exposed cells inhibited the phagocytic activity of adjacent macrophages. The conditioned medium from particle-exposed macrophages had no significant effect on the phagocytic ability of macrophages, suggesting that cell-cell contact is responsible for the pattern of failed phagocytosis (data not shown). We have demonstrated that ultrafine particles impair macrophage phagocytosis to a greater extent than fine particles compared on a mass basis. Therefore, we conclude that slowed clearance of particles, specifically the ultrafines, can in part be attributed to a particle-mediated impairment of macrophage phagocytosis.

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 κB (NF-κB) in the Q-exposed BAL cells and again no effect on nuclear NF-κB 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.

Modification of the quartz surface during the history of the particle is a powerful idea in understanding the variability of the quartz hazard. Interactions between quartz and other minerals are likely to occur in sediments, during industrial processing, or in matrix-bound quartz. We discuss new evidence regarding the basis of changes in the quartz surface that relate to changes in its ability to cause inflammation. Different samples of quartz were subjected to various biological assays. Endpoints included instillation of quartz into the tracheobronchial tree and measurement of PMN numbers in bronchoalveolar lavage (BAL) and in lung tissue, levels of the chemokine MIP-2 in BAL, and nuclear translocation of the transcription factor NF-kappaB in BAL cells. In vitro biological assays included cytotoxicity to epithelial cells, hemolytic activity, and radical activity of the particle surface as measured by electron spin resonance. Treatment of quartz with aluminium lactate impaired its ability to cause PMN recruitment, chemokine release, and NF-kappaB nuclear translocation in BAL. Workplace quartzes had no proinflammatory activity, which correlated with their ability to cause hemolysis but not their electron spin resonance (ESR) activity. Quartz in a matrix with coalmine dust or fly-ash showed different effects. In fly-ash, the toxicity was masked, but coalmine dusts were more toxic to epithelial cells than pure quartz in vitro; however, after instillation, the long-term inflammation was not related to the in vitro activity. Amelioration of quartz surface activity can occur in workplace samples of quartz and quartz samples whose surface is protected, to the extent that they have very little inflammogenic activity and display an inability to activate key subcellular pathways that lead to inflammation. Quartz from a workplace whose surface has been affected, or in a matrix such as coalmine dust or fly-ash, can have its toxicity modulated. These effects are due to minerals and organic compounds that can both decrease (e.g., aluminium salts) or enhance (e.g., coalmine dust matrix) biological activity and thus may contribute to toxicity in a complex way that is not easily predicted. Iron is a good example. There are reports that it can enhance quartz toxicity, or it may have little role to play in its toxicity, as shown here for almost pure quartz particles. A broad program of further research is needed before we have a sound understanding of the mechanisms of quartz toxicity.

In this paper we discuss evidence regarding the basis of changes in the quartz surface that relate to changes in the ability to cause key biological responses such as inflammation and genotoxicity. Different samples of quartz, coal and coal fly ashes were subjected to various acellular and biological assays. In vivo endpoints included instillation of samples and measurement of PMN numbers in bronchoalveolar lavage (BAL) and incidence of lung tumours in rats. In vitro assays included cytotoxicity to epithelial cells, release of pro-inflammatory mediators from various cells and radical (generating) activity of the particle surface as measured by electron spin resonance (EPR). We demonstrate that amelioration of the quartz surface activity can occur in workplace samples of quartz, such that they have very little inflammogenic activity, despite their ability to generate OH-radicals from their surface. These effects, also shown in coal mine dusts and coal fly-ashes, are due to minerals and organic compounds which can both decrease or enhance biological activity and thus may contribute to toxicity in a complex way that is not easily predicted. A broad programme of further research is needed before we have a sound understanding of the mechanisms of quartz toxicity and can achieve differential standard setting for mixed-dusts containing quartz.

In 1997, the IARC (International Agency for Research on Cancer) reevaluated its quartz classification from a class 2 carcinogen, to that of a class 1, stating sufficient evidence for carcinogenicity in both humans and experimental animals. However, tumor development did not occur across all occupational settings. It is probable that this is due to the considerable differences in toxicity between workplace quartz in comparison to quartz used in experimental studies. We therefore hypothesized that workplace quartz samples differ in toxicity from standard experimental quartz samples at equal mass. To test this hypothesis we compared 2 workplace quartz samples (RH1 and OM) with standard experimental quartz (DQ12) in several assays commonly used in particle toxicology. The sizes of the quartz samples were as closely matched as possible. The endpoints of this study were inflammation in the rat lung following intratracheal instillation (1000 microg or 250 microg for 3 or 14 days), release of soluble iron, cytotoxicity to cells in culture, and surface reactivity as assessed by hemolysis and ESR. The workplace samples did not cause inflammation at any dose or time point. DQ12 quartz caused marked inflammatory responses, as measured by an increased number of neutrophils in the lungs of instilled animals for both time points and doses. Protein in the bronchoalveolar lavage also increased in animals exposed to DQ12 but not the workplace samples. In vitro, DQ12 had the greatest hemolytic activity but only RH1 released substantial amounts of soluble iron. The increased inflammogenicity of DQ12 was not wholly explained by a greater surface area, by diameter, or by releasable iron. The hemolytic activity of DQ12, while not being informative in terms of understanding the mechanism of carcinogenicity, was the best in vitro predictor for in vivo activity. Therefore the surface reactivity of DQ12 appears to drive its inflammogenicity.

A new nose-only inhalation facility for rodents has been designed and built for operation within a high containment glove box facility. All operations using the equipment, whether concerned with aerosol generation or animal handling and exposure are conducted under high containment with total operator protection. The facility has been used to investigate known carcinogenic fibres such as the amphiboles. It has been designed to be resistant to most chemicals, under the conditions of an experiment, and can be used with radioactive material within the limitations which would be imposed for radiological protection. This paper describes the construction and validation of the equipment using titanium dioxide.

The aims of this study were to determine if freshly isolated bronchiolar Clara cells retained biotransformation potential and whether components of phase 1 and/or phase 2 metabolism were present in the extracellular lining fluid of the lung airways. Approximately 550 μl of lavage fluid was obtained from the mouse lung, which had been totally perfused of blood in order to facilitate the isolation of Clara cells from the same animal. Samples of acellular lavage fluid and aliquots of purified Clara cells were frozen at — 20°C prior to analysis. Minimum numbers of cells and lavage fluid volumes, pooled from 2 CD-1 mice, were assayed for ethoxycoumarin-O-deethylase, NADPH-dependent cytochrome c reductase, glutathione-S-transferase(s) and non-protein sulphydryls (mostly reduced glutathione). Isolated Clara cells retained a high activity/level of all these parameters confirming their functional status for subsequent studies of xenobiotic metabolism in vitro. Acellular lavage fluid, from all the healthy animals, also contained mono-oxygenase, reductase and transferase activities and high non-protein sulphydryl levels suggesting that phase 1 and 2 reactions with xenobiotics could take place in the extracellular environment of the lung. Clara cells are known to undergo apocrine secretion and this removal of the apical cap containing secretory granules and smooth endoplasmic reticulum could account for the airway biotransformation potential that was observed.

The inhalation of inorganic dusts and fibers is associated with chronic inflammatory processes in the lower respiratory tract dominated by alveolar macrophages, which release, inter alia, increased amounts of inflammatory products and cytokines. The effect of these various mediators on other cells may be important in asbestos-related epithelial cell injury. To defend against such injury the lung airways and parenchyma contain a number of antioxidant species for their protection, of which the dominant one is glutathione. This compound can be regarded as a paradigm for the antioxidant protection system of the lung, and a decrease in its level as a result of any toxic action would leave the lung vulnerable to damage. Intracellular glutathione tends to be located in Clara cells, type II pneumocytes, and alveolar macrophages and can be secreted under stress or lost by cell damage.-Glutamyl transpeptidase (-GT) is found in the Clara and type II pneumocytes of the rat lung, but almost none is found in macrophage...