Cellular and molecular mechanisms in environmental and occupational inhalation toxicology.

The central issue of this review are inflammatory changes that take place in the mucous membranes of the respiratory tract as a result of inhaled pollutants. Of particular relevance are dusts, SO(2), ozone, aldehydes und volatile organic compounds. Bioorganic pollutants, especially fragments of bacteria and fungi, occur predominantly in indoor dusts. They activate the toll-like/IL-1 receptor and lead to the activation of the transcription factor NF-κB for the release of numerous proinflammatory cytokines. Metals are predominant in ambient air dust particles. They induce the release of reactive oxygen species that cause damage to lipids, proteins and the DNA of the cell. As well as NF-κB, transcription factors that foster proliferation are activated via stress activated protein kinases. Organic compounds such as polycyclic aromatic hydrocarbons and nitroso-compounds of incomplete combustion processes activate additional via the cytosolic arylhydrocarbon receptor for detoxification enzymes. Sulphur dioxide leads to acid stress, and ozone to oxidative stress of the cell. This is accompanied by the release of proinflammatory cytokines via stress activated protein kinases. Aldehydes and volatile organic compounds activate the vanilloid receptor of trigeminal nerve fibres and induce a hyperreactivity of the mucous membrane via the release of nerve growth factors. The mechanisms described work synergistically and lead to a chronic inflammatory reaction of the mucous membranes of the upper respiratory tract that is regularly demonstrable in inhabitants of western industrial nations. It is unclear whether we are dealing here with a physiological inflammation or with an at least partially avoidable result of chronic pollutant exposure.

1. Introduction

Illnesses result from a complex interplay between favourable individual factors (susceptibility), harmful agents (noxae) effecting individuals, additional external factors as well as individual assimilation of these variables (Fig. 1). Usually one of these factors is at the fore as a cause of the illness, e.g. genetic disposition in the case of hereditary diseases, anatomical variants in some forms of chronic sinusitis, an acute infection due to a defined causative agent or also a psychological conflict situation leading to physical symptoms. A large number of factors are not noticed in the clinic or in practice. Nevertheless they exist.

Figure 1

Intrinsic and extrinsic factors in the development of disease

For example, for a doctor in a pollution free rural area, sinusitis has exactly the same appearance as it would for a doctor in an area with severe air pollution. However, sinusitis clearly occurs more frequently in areas of high pollution than in low pollution areas [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11]. In a region with air pollution, environmental pollutants are thus involved in the emergence of sinusitis. However, this is not immediately recognizable and in individual cases cannot be quantified, either. Analogous to this, employees in particular professions suffer more frequently from malignancies of the upper aerodigestive tract than the population average. This difference also remains significant if the effects of alcohol and tobacco consumption are compensated for [12]. Here, too, the job effect on the emergence of the illness is in individual cases not immediately apparent and usually difficult to quantify.

These examples show that environmental and occupational pollutants play a role in disorders of the head and neck. This has been demonstrated for acute and chronic inflammation of the nose and paranasal sinuses, the middle ear, the throat and the larynx, allergies of the respiratory tract, disorders of the sense of smell, of hearing and of balance, as well as head and neck tumours [13]. Only in individual cases are environmental and occupational pollutants the decisive causal factor. Mostly it is a case of cofactors that further the emergence of a disorder. For various poisons, toxicological mechanisms have been examined with classic pharmacological methods. This does not apply to the effect mechanisms of many inhalation pollutants occurring in the environment and workplace. Right up until only a few years ago it was above all the morphological and functional changes that were described in specialist literature, the actual damage mechanisms remained unknown. Here, over the last few years a great deal of new knowledge has been gained through the use of cell biological techniques that are the subject of the present review.

2. Examples of inhaled pollutants relevant to ENT

Those chemical substances that are relevant for diseases of the head and neck are predominantly absorbed via inhalation; we are dealing here with substances transmitted by air. On the one hand they appear as gasses. For the state of a substance at a particular temperature, its volatility is a decisive factor. Boiling point is a simple indicator for volatility. Substances with a boiling point of over 200° are of secondary importance as inhalation gasses but can be absorbed by particles and so gain entry to the respiratory tract. The details of the concentration of gasses result in mass/volume, mostly in µg/m3. Gasses with environmental medical significance are SO2, NOx, ozone and formaldehyde. From an occupational medical point of view, volatile organic compounds (VOC), volatile complex mixtures containing hydrocarbon and irritant gasses are of additional significance.

The second form of pollutants transmitted by air is aerosols. Aerosols are fine dispersions of liquids or solid substances in a gas, frequently in air. Fog is liquid dispersant aerosols, airborne dusts are particulate dispersal aerosols and fumes are aerosols from combustion processes. Aerosol concentrations are also given in mass/volume, typically in µg/m3. Air pollutants in the form of particles have the highest environmental and industrial medical relevance as inhaled noxae (Fig. 2). Environmental dusts are complex particulate air pollutants from the real environment that emerge, for example, due to combustion processes, as a result of swirling up of dust from the ground, of condensation of gaseous air components or due to the mechanical rubbing off of various materials. Urban dusts are environmental dusts that collect in urban agglomeration zones. Combustion residue is a component of environmental and industrial dusts with particularly toxicological characteristics. They contain, amongst other things, polycyclic aromatic hydrocarbons (PAH), nitroso compounds and aromatic amines. Coal fly ash (CFA) is a product of combustion from hard coal heating with a high level of quartz and aluminium hydroxide, residual oil fly ash (ROFA) derives from from oil heating and has a high metal content. CFA and ROFA can be collected from the exhaust particles of coal- or oil-fired power stations for experimental investigations with appropriate filters. Diesel exhaust particles (DEP) come predominantly from motor vehicle traffic and can be produced for experimental purposes from the exhaust fumes of diesel engines. Bioorganic dusts come from organic materials that owe their origin biological processes. Toxicologically, fragments of bacteria and fungi are of prime importance. The extremely inhomogeneous group of substances includes, for example, livestock dusts, laboratory animal dusts, feedstuff and grain dusts, food dusts, wood dusts as well as dusts containing mites and fungi, for example from biologic waste. Environmental dusts from ambient air, and in particular indoor dusts, also contain bioorganic pollutants (bacteria, fungi pollen, spores, mite excrement) that are related to health disorders. Quartz and asbestos dusts belong to the common problem aerosols in industrial medicine. These are followed by, for example, combustion residue such as welding fumes, bioorganic dusts and cooling and lubrication agents.

Figure 2

Relevant environmental and occupational inhalative pollutants

3.Toxicokinetics of the upper respiratory tract

Toxicology is concerned with the harmful effects of chemical substances. Effects of radioactive or other radiation, as well as mechanical or thermal effects do not belong to toxicology in the narrow sense [14]. The harmful effect of a substance is dependent upon its concentration and duration of action at the structures affected. These two factors are characterised by absorption, distribution, metabolic conversion and excretion and are the subjects of toxicokinetics. In pharmacological and toxicological texts books, toxicokinetics, main emphases are enteral absorption quota, first pass effect in the liver, distribution volumes as well as renal and biliary elimination. In the case of inhalative absorption, one has to differentiate between two cases. On the one hand it can lead to systemic effects following inhalative absorption, for example with inhalative anaesthesia. In this case, except for enteral absorption, the mechanisms described above apply. Local effects on the mucus membranes of the respiratory tract, however, are frequently in the foreground. These are the subjects of inhalation toxicology. Toxicokinetic factors of particular relevance in inhalation toxicology are listed in table 1 (Tab. 1).

Table 1

Toxicokinetic factors in respiratory toxicology

3.1. Inhalability

Gasses can generally be inhaled. Under the influence of gravity gasses with a higher specific weight sink. For this reason the body size (children) or the position of the room (cellar) can influence the capacity of a gas to be inhaled. In the case of dusts, the mechanisms of inhalability are more complex. Dust in general is a disperse distribution of solid substances with particle sizes of below 200 µm [15]. Airborne dusts have a particle size of below 100 µm; larger particles sediment on the ground within a few seconds. Aerosols with a speck or droplet size of over 100 µm (USA: >35 µm) are for this reason not included in the fraction of those that can be inhaled.

3.2. Absorption and deposition of inhaled pollutants

The respiratory toxic effect of inhaled pollutants depends essentially upon the region of the respiratory tract they are either adsorbed or absorbed into. Here what is understood by adsorption is the attachment of a substance to a two-dimensional surface (deposition); what is understood by absorption is the intake into a three-dimensional matrix such as, for example, secretion or tissue. By resorption, absorption into the blood or lymph system is indicated. In the case of gasses, water solubility and reactivity are the main physical characteristics for absorption into the respiratory tract [16]. Gasses easily soluble in water are dissolved into the secretions of the upper respiratory tract and spread their effects here. In so far as they are not spread as aerosol droplets, only a small part of gasses easily soluble in water reach the lower respiratory tract. An overview of the absorption of a number of substances in the form of gas in the upper respiratory tract is provided in table 2 (Tab. 2).

Table 2

Absorption of gaseous pollutants in upper airways

In the case of aerosols, the deposition of particles is dependent upon the diameter, the form of the particles and their surface characteristics. Should one wish to describe the deposition by the particle size alone, then the influence of form and surface characteristics must be compensated for through calculation. To this purpose the particle sizes are converted into equivalent spherical diameters. This so-called aerodynamic diameter corresponds to the diameter of a sphere with the standard mass of 1g/cm3 and of the same sedimentation speed as the dust particle under observation. In which compartment of the respiratory tract the particles are predominantly deposited is dependent upon the aerodynamic diameter. The mass median of the aerodynamic diameter (MMAD) is the aerodynamic diameter that is either exceeded or fallen short of by 50% of the mass (not the amount) of the particle under observation. It is used for a simple characterisation of a dust mixture. Those dust fractions are termed total suspended particles (TSP) whose aerodynamic diameter is either smaller than or equal to 35 µm. This corresponds more or less to the inhalative fraction according to DIN ISO 7708. Fractions of up to 10 µm (particulate matter 10 [pm10]) are regarded as inhalative dust, which corresponds more or less to the thoracic fraction according to DIN ISO 7708. As a rule of thumb, the equation PM10 [µg/m3] = 0,55 x TSP [µg/m3] [17] is valid.

Dust particles characterised as respirable are up to 2·5 µm [pm2.5]. This corresponds with the above norm more or less to the alveolar fraction. Particles < 0·1 µm are characterised as ultrafine particles. The relation between aerodynamic diameter and deposition compartment in the respiratory tract according to DIN ISO 7708 in non-logarithmic presentation follows from illustration 3 (Fig. 3) [18].

Figure 3

Particle deposition according to ISO 7708

The inhaled fraction and the percentage deposited in various airway compartments is depicted in percent dependant on mean mass aerodynamnic diameter.

A significant factor for the harmful effect of an inhaled agent is first and foremost the concentration of the harmful substance in the air breathed in and the duration of exposure. In addition, the effect of inhaled pollutants is influenced by variable breathing parameters. Thus there are fundamental differences in the harmful effects of inhaled pollutants depending upon whether one is dealing with breathing through the nose or the mouth. The volume of breath and breathing frequency also play a considerable role. The surface of the mucous membrane with which the harmful substances inhaled come into contact is also relevant [19]. Due to this, the harmful effects of inhaled pollutants differ between children and adults and between rest or physical strain.

3.3. Interaction with the respiratory secretion

Respiratory secretion contains water (95%), electrolytes (1%), proteins (1%), phospholipids (including surfactant) and carbohydrates (approx. 1%) [20]. It forms a 10 - 16 µm thick layer above the tissues of the respiratory tract. As first described by Lucas and Douglas in 1934 [21], respiratory secretion consists of two layers. The lower low-viscous periciliar liquid layer (sol phase) is formed predominantly by transepithelial transport of ions and water from ciliated epithelia and brush cells [22]. What are dealt with here are similar and partly identical processes of water and ion transport as are found in the kidney. The highly viscous surface gel phase (mucous layer) shows a high share of integrated glycoproteins (mucins). They are formed predominantly in the submucous glands and the goblet cells. The 200-500 kD large glycoproteins (mucins) have an amino acid framework on which various short chain sugars (fucose, galactose) and amino sugars (N-acetylneuraminic acids, N-acetylglucosamine, N-acetylgalactosamine) are either o-gylcosidic or n-glycosidic attached. Apomucins are peptides that form the central framework of the mucins [23], [24]. The apomucins Muc1, Muc3, Muc4, Muc5A, Muc5B, Muc5AC, Muc7, Muc8 and Muc13 are expressed in the respiratory tract. The genes and their mRNAs are identified in epithelial cells of the submucous glands and in the goblet cells of the respiratory tract [25], [26]. The regulation of the gene expression of different mucins is subject to species differences [27]. As well as this, the mucins Muc1 and Muc4 are involved in cellular signal transduction [28]. Numerous inhaled pollutants stimulate the mucin expression in the respiratory tract and thus contribute to hypersecretion [29], [30]. In the case of the exposure to noxious substances, the constant activation of mucin genes leads to goblet cell hyperplasia, a characteristic feature of chronic inflammation of the respiratory tract [31]. Consecutively, this leads to the dysregulation of mucus composition, with hyperviscous secretion. This impairs the mucocilliary transport and encourages colonization of pathogenic organisms.

In order to gain access to the mucous membrane of the respiratory tract, a noxious substance must overcome the secretion barrier. This process differs with noxious substances in the form of gas or particles. For lipophilic gasses, the watery respiratory secretion is to a large degree impenetrable. An insignificant solution of lipophilic substances in respiratory secretion can be conveyed through phospholipids with amphiphilic properties. Water-soluble gasses go into solution in the respiratory secretion. At the same time, acids frequently form that can damage the epithelia of the respiratory tract. This is the case with SO2 or NO2, for example. Respiratory secretion has a buffer capacity in the range of 6 µmol H+-Ions per pH-level. This buffer capacity is provided to a great extent by mucins [32], [33]. Through the buffer effect of the secretion, the respiratory mucous membrane is protected from acidification caused, for example, by inflammatory processes [34], [35] or by the exposure to noxious substances and an optimal secretion transport in the neutral pH range is provided [36], [37].

Particulate air pollution is firstly adsorbed onto the mucous blanket of the upper respiratory tract and the proximal tracheobronchial tree. The mucous blanket functions from a technical point of view as an adhesion filter [38]. Adsorbed particles release water-soluble components such as salts, metals or proteins that then reach the epithelia of the respiratory tract. In this way, for example, water-soluble allergen proteins also pass into the mucous membrane of the respiratory tract. Through gaps in the mucous layer, however, smaller particles also reach the respiratory epithelia. Here they are phagocytosed mainly by local macrophages and neutrophils and to a lesser extent by epithelial cells [39].

Alongside its function as a mechanical barrier and acid/ base buffer, respiratory secretion can also neutralise a various exogenous and endogenous agents. This protective function is based partly on the activity of mucins. After the addition of mucins to the cell culture medium, the pro-inflammatory effect of environmental dusts on BEAS-2B cells was significantly reduced [40]. The effect of irritants is limited by peptidases resident in secretion. They degrade proinflammatory peptides, such as substance P released from nerve fibres, and thus exert an anti-inflammatory effect. Typical representative of these enzymes resident in secretion are neutral endopeptidases (NEP), angiotensin converting enzyme (ACE) or dipeptidylpeptidase IV (DPPIV) [41], [42]. Reactive oxygen species are neutralised by a variety of antioxidants resident in secretion. These include reduced glutathione, ascorbic acid, uric acid, a tocopherol, transferrin and ceruloplasmin [43], [44], [45]. On the one hand they intercept inhaled oxygen radicals such as ozone, but can also absorb released reactive oxygen species in the bounds of inflammation processes from respiratory cells. Lactoferrin stems from the specific granula of neutrophil granulocytes and is secreted from the seromucous glands of the respiratory tract. As a chelating agent, it has a high iron-binding capacity, but also binds other bivalent transitional metals and can thus reduce the toxicity of inhaled metal compounds. The lactoferrin metal complex, which is less poisonous in comparison to pure metal ion, is absorbed into the respiratory cell via a lactoferrin receptor and added to the cellular metal metabolism [46], [47]. Defensins are microbicide peptides formed above all in neutrophil granulocytes, but also in macrophages and respiratory epithelia. Human respiratory epithelia release β-Defensin 2 within 1-2 hours after contact with mucous forms of pseudomonas aeruginosa. The receptor for this is the toll-like receptor 2 [48], [49], [50].

3.4. Metabolism of foreign substances in mucous membrane of the respiratory tract

If the inhaled pollutants have overcome the secretion barrier, they are metabolized by the cellular detoxification system of the mucous membranes of the respiratory tract. The mucous membrane of the nose and tracheobronchial tree is, after the liver, the most active metabolic organ [51], [52], [53], [54], [55], [56]. If, for example, radioactively marked nitrosamines are applied either intraperitoneal or intravenously to the laboratory animal, they accumulate just as intensively in the mucous membrane of the nose as in the liver (Fig. 4) [57].

Figure 4

Radiography of a whole body section of the rat after intravenous injection of radioactive 1,2-dibromo-ethane

High metabolic activity is found in the liver and in the mucosa of the upper and lower airways.

Typical phase I and phase II biotransformation processes take place here [58]. The aim of these processes is the increase of the polarity and the water-solubility of xenobiotics so that they can be excreted via the kidneys or bile. Phase I and II enzymes also play a decisive role in the detoxification of toxins associated with tobacco smoke. A dysfunction of these detoxification enzymes, for example through gene polymorphisms, possibly furthers the development of head and neck tumours [59], [60], [61]. Detoxification enzymes in humans are mainly to be found in surface cells of the respiratory epithelia and in the sustentacular cells and Bowman glands of the olfactory epithelium [62].

The phase I enzymes belong predominantly to the group of cytochrome P-450 oxygenases (CYP P-450). The name comes from "pigment 450" as the reduced CYP P-450 shows an absorption band at 450 nm. In humans it is a case of at least 50 related enzymes with broadly overlapping substrate specificity. This enzyme group transmits oxygen atoms and can convert approx. 1 million different substrates. The resulting intermediate product, usually an epoxide, is very reactive and often more harmful than the initial substance (Fig. 5). It can cause damage to lipids and proteins of the cell and more particularly DNA damage. The intermediate product of phase I biotransformation is conjugated in a second metabolism stage through phase II enzymes with polar molecules such as, for example, glucuronic acids or glutathione to a molecule easily soluble in water and excreted.

Figure 5

Metabolism of benzene

A phase reaction results in the formation of a highly toxic itermediate product benzene epoxide, which is conjugated to to glucurnic acid to form a less toxic product, which can be renaly excreted.

Phase I enzymes that were identified in the mucous membrane of the nose are listed in table 3 (Tab. 3). Amongst others phase II enzymes, UDP glucuronyl transferases and glutathione S transferases were detected in the mucous membrane of the nose. UDP glucuronyl transferases transmit activated glucuronic acids to OH groups in, for example, phenols and alcohols, to COOH groups of organic acids or to NH2 groups of amines or primary amides. Glutathione S transferases transmit glutathione to electrophile centres, for example in epoxides, alkylhalides or chinones [63].

Table 3

Phase I enzymes detected in the upper airway mucosa

3.5. Elimination of inhaled pollutants

In the the upper respiratory tract and the tracheobronchial tree, the most important elimination mechanism is the mucociliary transport system. Noxious substances entrapped in respiratory secretion are transported at a speed of approx. 5 mm/min towards the throat and then swallowed. The driving force of the mucociliary transport system is the staggered beat activity of the cilia of respiratory epithelia. The cilia bend slightly to the side on the recovery beat, which allows them to carry out this movement entirely within the sol phase. On the active beat they stretch, reach into the surface gel phase with their ends and push it towards the throat (Fig. 6). The ciliary beat frequency of human nasal mucous membrane is around 10 Hz.

Figure 6

Mucociliary transport system of the upper airways

Cilia xxxxx in the watery periciliary fluid and grip into and propel the superficial mucus blanket during the effective stroke. The mucus blanket is thus moved to the nasopharynx, from where it is swallowed together with adherent pollutants.

Numerous inhalative pollutants interfere with the mucociliary transport. This results in the inhaled substances being able to remain for a longer time in a respiratory compartment and to accumulate there. In this way, their toxic function can be strengthened [64]. Although not quantifiable, one can assume that disturbances of the mucociliary transport also encourage the development of respiratory infections. The raised tendency for infection in patients with cystic fibrosis and ciliary dyskinesia bears this out.

In contrast to the upper respiratory tracts and the proximal tracheobronchial tree, the terminal sections of the bronchial tree and the alveolar space have no mucociliary transport. Particles that get this far are predominantly absorbed by macrophages and carried away via lymphatic drainage. This process is partly accompanied by a distinctive inflammatory reaction. The various elimination mechanisms result in the upper and lower respiratory tracts reacting differently to particle exposure. Whilst phagocytosis is very well characterised as an elimination mechanism for the lower respiratory tract, in the upper respiratory tract there is only few data for phagocytosis. As recent studies have shown, epithelial cells are also capable of phagocytosis [39], [65], [66], [67]. The significance of this observation for the elimination of inhaled pollutants is, however, still unclear. Further important elimination mechanisms of the upper respiratory tract are coughing, sneezing and profuse hypersecretion with which noxious substances can be expelled or flushed out from a respiratory compartment. It is true that these symptoms are an important expression of irritation syndromes, but they play a secondary role as elimination mechanisms in environmental and industrial medicine.

Resorbed via inhalation, that is to say systemically absorbed, inhaled pollutants are excreted predominantly via the exhalation. This is the basis of occupational threshold values in the air expelled, for example after benzene exposure. In addition, systemically absorbed inhaled pollutants are renally and biliarly eliminated.

3.6. Systemic resorption

Inhaled pollutants can have a harmful local effect on the respiratory tracts (airway toxicity) or on other organs after resorption. Typical examples for the effects of an inhaled agent far from the point of resorption are carbon monoxide poisoning or the narcotic effect of numerous volatile organic compounds such as inhalation anaesthetics. Included among the systemic effects on inhaled pollutants are neurotoxic and especially vestibulotoxic and ototoxic effects from organic solvents and complex mixtures containing hydrocarbons. Worthy of note in this context is the association of cardiovascular mortality and pollution caused by environmental dusts [68]. In a cohort study, the risk of dying within 16 years of diseases of the heart or circulation rose by up to eight percent per 10 µg/m3 pm2.5 [69]. The cause of this is probably systemic effects as a result of the release of inflammation mediators in the lower respiratory tract and alveolar space [68], [70].

4. Mechanisms of inhaled pollutants

The toxicity of a substance is a characteristic specific to it which determines in which way it interacts with living structures. Which harmful effects a substance causes in the organism and via which mechanisms it does it are the subjects of toxicodynamics.

4.1. Changes in respiratory mucous membranes

On the level of tissue, toxic effects of inhaled pollutants are described in detail (Fig. 7). Numerous environmental pollutants can be observed to lead to an accumulation of neutrophil granulocytes and macrophages in the respiratory mucous membranes, in other words triggering an unspecific inflammatory reaction [71], [72], [73], [74], [75], [76], [77], [78], [79], [80], [81]. Cytokines and chemokines released by cells of the respiratory mucous membrane after contact with the noxious agent play a central role in these inflammatory reactions [82], [83], [84], [85], [86], [87], [88], [89], [90], [91], [92], [93], [94], [95], [96], [97], [98], [99], [100], [101], [102], [103], [104], [105], [106], [107]. In respiratory toxicology, relevant cytokines derive from cells that can be found in the respiratory mucous membrane. Epithelial cells are most common. Their main task is the maintenance of a mechanic barrier between the outside world and the organism. As well as this they form a functional unit of the mucociliary apparatus. Over the past years it has been recognised that human respiratory epithelia can in addition induce an unspecific inflammatory reaction. They are capable of releasing various cytokines and chemokines that further inflammation as a reaction to external stimulus. These include IL-1, IL-6, IL-8, TNFα, G-CSF, RANTES, ENA-78 and GRO [39], [84], [87], [88], [108], [109], [110]. Amongst the numerous effects of these proteins are that endothelial cells express adhesion molecules in their cell membrane in neighbouring capillary vessels. These cell receptors mediate the migration of further inflammatory cells, predominantly of macrophages and neutrophil granulocytes into the tissue. These release cytotoxic substances such as reactive oxygen species (ROS), myeloperoxidase (MPO), prostaglandins (PG) and leukotrienes (LT). Cellular dysfunction with impaired mucociliary transport, disturbances of the cell-cell contact with a break up of tight junctions and the detachment of the cells from the basal membrane (epithelial shedding) all result from the inhales pollutants themselves, as well as the cytokines and the inflammatory mediators secondary released by infiltrating inflammatory cells. The barrier function of the epithelium is significantly disrupted. The epithelial damage introduce repair processes through fibroblasts and proliferating epithelia that result in structural changes in the respiratory mucous membrane with extensive deposition of extracellular matrix proteins such as collagen (remodelling, thickening of the basal membrane). Should the inflammatory stimulus persist, the proliferating cells no longer differentiate to ciliated epithelia but rather to goblet cells (goblet cell hyperplasia) and squamous epithelium (metaplasia). Damage of the protective epithelial layer, released inflammatory mediators and various inhaled agents themselves activate trigeminal nociceptors within the mucous membrane and induce airway hyperreactivity.

Figure 7

In the cells of the upper airway mucosa, inhalative pollutanta induce the release of proinflammatory cytokines.

They cause endothelial cells in neighboring vessels to express adhesion molecules in their cell membrane. Infalmmatory cells from the blood stich to those adhesion molecules and are transferred to the interstitial tissues and airway epithelium. Here, these cells release further inflammatory mediators including ROS, myeloperoxidase (MPO), prostaglandins (PG) and leukotrienes (LT). These mediators induce cellular dysfunction, disturbes cell-cell contacts and shedding of cells from the basal membrane. Sensible nerve endings are uncovered promoting airway hyperreactivity. Tissue repair promoted by fibroblasts may lead to structural changes with exaggerated production of collagen fibers rsulting in basal membrane thickening (remodelling). If inflammation persists, regenerated cells do not differentiate into ciliated, but into squamous epithelium (metaplasia) and goblet cells (goblet cell hyperplasia). Current research focuses on the mechanisms which induce the release of proinflammatory cyokines from airway cells (red circle). Pollutant binding to cellular receptors and and the concomitant release of ROS are supposed key mechansisms. ROS and additional intermediates damage the cells membrane and activate intracellular signal transduction cascades (stress activated protein kinases - SAPK) finally resulting in the release of cytokines. Not depicted are specific immune responses and changes due to DNA damage.

4.1.1. Basics of the cell response

These processes are described for various inhaled pollutants in detail. The subject of current research is the question of how inhaled pollutants initially cause the cells of the respiratory mucous membrane to release proinflammatory cytokines, for example. This can first be clarified on a cell-biological level. The airway cell must always be made to raise the synthesis of proteins, in this case cytokines.

To this end, the stimulus must first be recognised by the cell and the signal transmitted from the cell periphery into the cell nucleus (Fig. 8). This follows from cell receptors and cascade-like activation of intracellular signal proteins. The genetic information for the proteins of the cell is stored in the DNA in the cell nucleus. The conversion of the genetic information of a DNA segment into a target product, mostly a protein, is summarised under the term gene expression. According to the central dogma of cell biology, the path from DNA leads via the RNA to protein. In the cell nucleus, therefore, genes must be activated and transcribed into messenger RNA (mRNA). The influence of the transcription rate of genes (gene regulation) is a fundamental toxicity mechanism on inhaled pollutants. The gene regulation follows from regulatory transcription factors; a group of intracellular proteins that can either promote of silence the transcription of certain genes.

Figure 8

Altered gene expression following contact with an inhalative pollutant

By various mechanisms, the cell detects the inhaled pollutant (perception). This generates an intracellular signal, which is transferred by different cellular transduction systems and converge to transcription factors. Transcription factors are then transferred to the cell nucleus, where they bind to specific DNA-regions modifying the transcription into pre-mRNA. Following posttranscriptional modification, the resulting mature RNA leaves the nucleus into the cytoplasm, where the translation into a protein at the reibosoes takes place. The generated proteins can be excretes from the cell, integrate into the cell membrane to modify cell reactivity or serve as intracellular proteins various cell functions.

The mRNA must be passed on from the nucleus to the ribosomal protein synthesis apparatus in the cytosol of the cell. Here the translation into a protein takes place using the blueprint of the mRNA. Numerous proteins created in this way are channelled out of the cell. They may serve, for example, as components of the intermediary tissue substance (extracellular matrix ECM), or influence, as messengers, the behaviour of other cells (cytokines) or generate, as transmitters, a nerve action potential in other cells. Other proteins are incorporated into the cell membrane and transmit cell-cell or cell-matrix contacts or serve as cell receptors for the receiving of signals from other cells. The majority of the proteins formed remain in the cell. They carry out structural tasks as components of the cytoskeleton, for example, are involved in cell movement or provide for cellular metabolism, cell division or apoptosis. Also included in the proteins remaining in the cell are the transcription factors and cellular signal transduction molecules that as regulatory proteins together make up approx. 20% of the cell proteins.

Numerous intracellular proteins are involved in the intracellular signal transduction, for example, from a cell membrane receptor to the cell nucleus. Like blood clotting factors, these intracellular signal proteins are functionally connected one behind the other and are activated in the form of a cascade. The reversible protein activation and intracellular signal transduction are basic cell biological mechanisms that are given a central role in the conveyance of the harmful effects of inhaled pollutants.

4.1.2. Protein activation, protein kinases and intracellular signal transduction

In order to fulfil their complex task, cellular proteins must be able to reversibly alter their form and activity (allostery). These reversible changes result from the linking of small molecules to proteins [111]. The coupling of phosphate groups (phosphorylation), methyl groups or acetyl groups [112], nitric oxides (nitrosylation) [113], [114] or oxygen compounds [115] is typical [112]. As a result of the coupling, the protein changes its three-dimensional structure. The reversible change of intracellular proteins results particularly often as protein phosphorylation that is conveyed through enzymatic active proteins, the protein kinases. The phosphate groups are predominantly bound to amino acids that bear free hydroxyl (OH) groups. These include serine, threonine and tyrosine. Serine/threonine kinases are proteins that transmit a phosphate group to serine and/or threonine, tyrosine kinases transmit the phosphate group to tyrosine residue. Protein phosphorylation frequently results in a protein becoming enzymatically active itself and for its part catalyses the coupling of a phosphate group with another protein. This results in intracellular cascades of protein activation. The dephosphorylation and thus frequently the inactivation of proteins results from phosphatases. In the hypothetical resting state of a cell, activation maintains its balance through protein kinases and inactivation through phosphatases. An activation of the cell can thus result from the activation of protein kinases or the inactivation of phosphatases.

Phosphorylation can alter the activity of a protein in many ways. As well as protein kinase activity or phosphatase activity, binding sites for other proteins can be formed and proteins coupled with one and other, the linking to DNA molecules elicited, the permeability for example of ions altered, the enzyme activity in the context of metabolism processes influenced or movement processes performed. As well as the reversible protein activation through the linking of small groups of molecules, there is also irreversible protein activation resulting from the splitting off of protein parts, for example with blood clotting factors, the complementary system or caspases in the context of apoptosis.

Protein kinases and phosphatases are the main functional elements of intracellular signal transduction. Acting in concert, they provide intracellular signal pathways. One example is the mitogen activated protein kinases pathway (MAPK path) that leads from activated growth factor receptors to the activation of the extracellular signal regulated protein kinase 1 and 2 (ERK1/2). If mitogens (e.g. growth factors) bind to a cell membrane receptor, then a tyrosine kinase becomes active on that part of the receptor that projects into the interior of the cell (cytoplasmic domain). Via intermediate steps, a small GTP binding protein (Ras: Ras and Raf1 are the products of genes that in the case of a mutation can encourage tumour formation (protooncogenes). Should Ras or Raf1 proteins be formed through mutation that are constantly active, the cell is placed under constant growth and division pressure.) is activated that then activates the actual MAPK pathway. This consists of three proteins: RAF1 (a MAP-kinase-kinase-kinase or MAP 3 kinase), the MAP-kinase-kinase 1 (Syn MEK1, a MAP-2-kinase) and finally the MAP-kinase ERK1/2 (an MAP-1-kinase, as it were), that activates several transcription factors (e.g. Elk1, c-Myc, c-Fos). These transcription factors enhance the transcription of genes that promote cell growth and division. As well as the pure signal transmission, the MAPK pathway fulfils further functions. An activation threshold is built in, as MEK1 must be phosphorylated in several serine and threonine residues in order for its part to develop kinase activity. In addition the signal is amplified as Raf1 activates numerous MEK1 proteins. The MAP kinases of different signal transduction paths also influence each other, which allows a finely coordinated regulation of the cell response. The development of specific inhibitors of cellular signal transduction molecules is the subject of current pharmacological research.

A further relevant signal transduction pathway in inhalation toxicology is the stress-activated protein kinase (SAPK) path. Two closely related signal transduction pathways are subsumed under this term, i.e. the c Jun N-terminal kinase (JNK path) and the p38 pathway (Fig. 9). Cellular stressors such as heat, acidity or radiation lead to the activation of substances closely related to Ras, the small GTP binding proteins Rac1 and Cdc42. These activate analogously to the MAPK path SAP-3 kinases and SAP-2 kinases and finally the protein kinases JNK or p38. These two protein kinases activate several transcription factors (e.g. c-Jun, STAT1, STAT3, c-Myc, CREB) that stimulate cell growth and division and that are involved in the expression of pro-inflammatory cytokines. The intracellular signal transduction of toll-like/IL-1 (TIL) receptors proceeds more easily. This receptor has no intrinsic tyrosine kinase activity, but does activate the receptor-associated tyrosine kinases interleukin 1 receptor-associated kinase (IRAK) and TNF receptor-associated factor 6 (TRAF6). These activate the NF-κB inducing kinase (NIK) and the inhibitor of NF-κB kinase (IKK) that activates NF-κB via intermediate steps. With cytokine receptors, the coupling of the ligands leads to the activation of the receptor-associated protein kinases described as Janus kinases (JAK). These directly activate a group of transcription factors described as signal transducer and activator of transcription (STAT). Like NF-κB, they mediate the transcription of genes that promote inflammation. In the most simple of cases, the receptor itself serves as the transcription factor. This is the case with the cytosolic arylhydrocarbon receptor. It is activated via lipophilic organic compounds such as PAH and then passes into the cell nucleus where amongst other things it activates genes for detoxification enzymes.

Figure 9

The MAP-Kinase pathway, SAP-Kinasae, the activation of NF-κB following activation of the Toll-like/IL-1 Receptor, and activation of STAT via cytokine-receptors as paradigms of intracellular signal trandusction mechanisms (s. text).

Lipophilic noxae can pass the cell membrane and activate the cytosolic arylhydrocarbon-receptor (AhR) directly, which then serves as a transcription factor.

4.2. Toxicity mechanisms of bioorganic pollutants

The mechanisms of the initial cell activation, that is the perception of the harmful stimulus and the intracellular signal transduction, differ considerably between various inhaled pollutants. Respiratory, toxicologically relevant substances with exemplary character explain this initial cell activation in the following, where first of all particulate bioorganic pollutants are examined.

Dusts contain numerous fragments that are derived from living organisms. It concerns above all cell wall fragments of bacteria and fungi that have adhered to the surface of dust particles. They play a key role for the respiratory toxicity of dusts and are predominantly represented in the insoluble fraction PM2.5-10 [116]. The indicator substance for bioorganic pollutants in dusts are endotoxins. These are lipopolysaccharides (LPS) anchored to the cell wall of gram- bacteria that are composed of the immunologically active lipid A, a nuclear polysaccharide and a specific O-chain. Inhalation of endotoxins leads to the release of TNFα, IL-1β, IL-6 und IL-8 and consecutively to a neutrophil inflammatory reaction of the respiratory mucous membrane of the lower [117], [118], [119], and in higher concentrations also in the upper respiratory tracts [120], [121]. For people suffering from asthma or nasal allergies, these reactions are intensified [122].

Ambient air dusts have lower LPS activities than dusts found indoor. Utah valley dust, an ambient air dust from an industrial agglomeration zone rich in metals, has an endotoxin activity of 0.6 EU/mg [40]. Representative indoor dust from north German households has, according to our measurements, an endotoxin activity of 24 EU/mg dust, whilst dust from south German households with no livestock showed an activity of 30 EU/mg dust [123]. If we assume roughly an average indoor dust concentration of 50 - 250 µg/m3, then our results lie below 50 EU/m3 air. This corresponds to the Dutch limit for indoor endotoxin, i.e.approximately 5 ng/m3 [124]. In Dutch office blocks with sick-building syndrome, on average 250 ng/m3 endotoxin were found [125]. Bioorganic pollutants also play an outstanding role in occupational respiratory toxicology [126]. High LPS concentrations can be found in dust from working animals (stable dusts, on average 100 ng/m3) [127], laboratory animals, feedstuff and grain dusts, food dusts, wood dusts as well as in dusts containing mites and fungi, e.g. from the waste industry (75-575 EU/m3) [86]. In addition, high concentrations of endotoxins of up to 25000 ng/ml [128] occur in cooling and lubricating substances in the metal working industry.

Endotoxins can be neutralised by polymixin B or lipopolysaccharide binding protein. Through neutralisation studies, the endotoxin caused part of the inflammatory reaction can be estimated. The proinflammatory and cytotoxic effects of urban dusts from Zurich and Lugano were almost completely inhibited by polymixin B, and thus can above all be put down to bioorganic pollutants [95]. An at least partial neutralisation of the proinflammatory effects of environmental dusts by LPS neutralising substances has been established in several further studies [94], [116], [129], [130], [131], [132], [133], [134], [135], [136], [137], [138], [139], [140].

In respiratory epithelia, glucans from the cell wall of moulds trigger similar effects to LPS. This could be shown with the help of transcription profiles by DNA microarrays. The microarray technology makes possible the simultaneous recording of changes in transcription of several thousand genes. We have carried out a comparative examination of the effects of the two most widely spread bioorganic pollutants, LPS from gram- bacteria and 1,3-β-glucan from the cell wall of moulds on the respiratory epithelial cell line BEAS-2B. Various cytokines or chemokines released by the cells were also recorded. The reactions of epithelial cells to LPS and 1,3-β-glucan were very similar, both on the mRNA level as well as on the protein level. As well as LPS, further bacterial cell wall components are determined such as lipoteichoic acid, peptidoglycans, FMLP and so-called CpG motives (see 4.2.1. table 5). Along with glycans, mannans and mannanoproteins are also included in the pollution caused by fungi.

4.2.1. Cellular receptors for bioorganic pollution

An crucial initial step in respiratory inflammation through dusts is the phagocytosis of particles that are covered with bioorganic pollutants. By phagocytosis we understand the recognition, absorption and digestion of particle-shaped substances. Among the professional phagocytes are dendritic cells (DC), macrophages, monocytes and neutrophil granulocytes. To a lesser extent other cells, such as respiratory epithelia, are capable of phagocytosis [39]. The first step of phagocytosis is the recognition of the particle to be phagocytosed and its attachment to the cell membrane. This takes place on receptors on the surface of the cell membrane.

Here two cases must be distinguished between [141]. In the first case the bacteria and fungi particles on the particle surface are already known to the immune system and are opsonised by immunoglobulins. In this case the particle binding results via the FC-receptor (FcR) in the cell membrane of the phygocytes (Tab. 4). FcR for IgG signifies FcγR, for IgA FcαR and for IgE FcεR. The FcγR group is the most significant for phagocytosis, but a various number of subtypes have to be differentiated (Tab. 4). For IgM there are no Fc receptors. If the particle binds complement or IgM, then particle recognition can result via a complement receptor. The complement receptors CR1-CR5 (Tab. 4) exhibit different cell distribution patterns and functions [142]. If particles are phagocytosed via an Fc or complement receptor, a strong proinflammatory reaction is set off in the phagocyte.

Table 4

Characteristics of Fc- and complement-receptors

In the second case the particle is unknown to the immune system and not opsonised with immunoglobulin or complement. Parts of the bacteria or fungi on the particles are then recognised by special cell receptors. The cell wall of bacteria and fungi consists of regularly ordered modules of protein, fats and sugars that give their surface its typical pattern. These patterns differ from the surface of animal cells with no cell wall and were named 'pathogen associated molecular patterns' (PAMP) by Janeway and Medzhitov. The PAMPs are recognised by cells of the innate immune system with pattern recognition receptors (PRRs) (Tab. 5). This includes the toll-like receptors, the mannose receptor and the glucan receptor. As well as surface patterns, structure patterns of proteins or DNA modules of a number of different pathogens are also well known to the phagocytes.

Table 5

Pathogen-associated molecular patterns (PAMP) on particle-adsorbed bioorganic pollutants, related pathogens and pattern recognition receptors (PPR)

The coupling of a ligand to a toll-like receptor leads to numerous proinflammatory changes in the cell through activation of the transcription factor NF-κB.

4.2.2. Gene regulation, transcription factors and NF-κB

Gene regulation, that is the alteration of the transcription rate of genes through transcription factors, plays a central role in respiratory toxicology. An enzyme complex, the RNA-polymerase II complex (The basic possibility to transcribe a gene into mRNA depends upon the organisational state of the chromatin. Low grade condensed euchromatin can be transcribed whilst high grade condensed heterochromatin is not accessible to the transcription apparatus. This organisational state of the chromatin is transmitted on cell division to the daughter cell. This explains why a basal cell of the respiratory epithelium does not suddenly produce for instance a nerve cell [143].), manages the transcription of DNA into mRNA. Some genes are continuously (constitutively) active. These are household genes that encode, for example, proteins of basic metabolism or the cytoskeleton of the cell. For transcription they require proteins that are referred to as "basal transcription factors". They bind to the promoter region in the DNA strand and bring about the linking of the RNA polymerase II. Basal transcription factors are as a rule present in sufficient numbers and thus do not act in a regulatory manner. Amongst the basal transcription factors are, for example, the TATA binding protein, SP1, and NF1.

In contrast to the constitutively active genes, with inducible genes the transcription can be either enhanced or inhibited; in an extreme case they can be either turned on or off. With these genes, regulatory sequences called responsive elements are built into the DNA strand along with the promoters. The responsive elements that strengthen transcription are called enhancers, those that reduce the transcription rate silencers. They contain nucleotide sequences that arrange the binding of "regulatory transcription factors" to the DNA. Some frequently described regulatory transcription factors and the nucleotide sequences of the responsive element within the DNA strand are presented in table 6 (Tab. 6).

Table 6

Some nucleotide sequences of responsive elements recognized by transcription factors [357-360]

If a regulatory transcription factor links to an enhancing responsive element, it facilitates the binding of the RNA polymerase II complex to the promoter region. In this way the transcription rate of this gene is raised and more mRNA is formed, which frequently, but not always, leads to an increase in the release of the encoded protein.

The several hundred regulatory transcription factors at present known constitute approximately 10% of the amount of protein in a cell. They can be divided up into five groups according to characteristic structural and functional parts - so-called motifs - of the DNA binding domain. These are the helix-turn-helix motif, the zinc finger motif, the leucine zipper motif, the copper fist motif and the basal helix-turn-helix motif. These structural features fit to bays in the DNA molecule (Fig. 10) and link onto them when particular nucleotide sequences can be found there.

Figure 10

Strucutral motifs of the DNA-binding domain of regulatory transcription factors fit into grooves of the DNA molecule.

Depicted are the Helix-Turn-Helix-Motif and the Zink-Finger-Motif.

Respiratory inflammation caused by inhaled pollutants such as, for example, bioorganic pollutants, are substantially mediated by the transcription factor NF-κB [40], [90], [91], [97], [99], [102], [144], [145], [146], [147], [148], [149], [150], [151], [152], [153], [154]. NF-κB is composed of two proteins of a group of DNA binding proteins of which the combination of proteins p50 und RelA is the most common (Fig. 11). In resting cells NF-κB is bound to an inhibitor (IκB) present in the cytosol. The binding to the inhibitor prevents the translocation of NF-κB in the cell nucleus. Activation of toll-like receptors that, with regard to the intracellular signal transduction, are very closely related to the IL-1 receptor, leads via phosphorlation of NF-κB inducing kinase (NIK) to the activation of the IκB kinase. The activation of the IκB kinase (IKK) results in phosphorylation of IκB. Following this the NF-κB/IκB complex binds to ubiquitin that supplies it to a proteasome, i.e. a cystosolic protein degradation apparatus. The inhibitor is cleaved from the complex in the Proteasome, exposing an amino acid sequence (nuclear localisation peptide sequence [NLS]) that arranges the transport of NF-κB into the cell nucleus. In the cell nucleus, NF-κB binds to responsive elements with the sequence 5'-GGGGACTTTCC-3', whereby a certain variability is tolerated with the middle nucleotides [155], [156].

Figure 11

Activation of NF-κB

Ligand binding to different cell membrane receptors (Toll-like/IL-1, TNF) activates intracellular signal transduction molecules such as the receptor associated protein kinases Interleukin 1 Receptor-Associated Kinase (IRAK) and TNF receptor-associated factor (TRAF) and then the cytosolic proteinkinase NF-κB-inducing Kinase (NIK). This kinase phophorlyses IkB Kinase (IKK), which in turn activates IκB. Phosphorylsed IκB binds to Ubiquitin, which transfers the NF-κB/IκB-complex to cellular proteasomes for degradation. Following Abspaltung of IκB within the proteasome, NF-κB can be transferred into the nucleus and promote the transcription of genes with appropriate responsive elements. Reaktive oxygen species activate stress-activated-protein-kinases, which in turn activate auxiliary proteins such as CBP and p300. This enhances the NF-κB binding to its responsive elements.

NF-κB activates genes for several proinflammatory cytokines (Tab. 7) [157], [158]. As well as this it reinforces the expression of adhesion molecules, immune receptors and acute phase proteins [155]. NF-κB activation also leads to the upregulation of iNOS, cyclooxygenase 2 and the PAF receptor [159] and regulates the transcription of various genes for apoptosis and cell division.

Table 7

Gene transcription regulated by NF-κB

NF-κB is not only involved in the transcription of numerous proinflammatory signal molecules, several intracellular signal transduction pathways from several receptors also converge on NF-κB [157]. As well as the described pathway via the toll-like/IL-1 receptor, NF-κB can also be activated via the TNF receptor. In addition, NF-κB is activated through C5a via a receptor linked to a G protein [146], via bradykinin receptors [147], via arylhydrocarbon receptors [148], via ROS [102], [151], [160] and also via Fc receptors through the activation of the protein kinase C. For its part, NF-κB leads to the release of TNFα and IL-1 resulting in a self -reinforcing control loop. As well as the cytokines, the expression of the inhibitor is also increased, which checks the NF-κB activation as a negative regulatory loop.

4.2.3. Phagocytosis and oxidative burst

After the binding of the bacterial or fungal fragments to Fc, complement, or pattern recognition receptors, the particle is internalised. With this the second phase of the inflammatory cell activation through bioorganic pollutants begins. Pseudopodia push themselves around the particle and engulf it into a phagosome [161], [162]. Through the occupation of the receptor, small GTP binding Rho proteins Rac and CdC42 are activated. Via complex mechanisms, they act as intermediary in the accretion of actin monomers (G actin) to the receptor. Through further lengthening of the actin filaments, pseudopodia are pushed around the particle. In addition, activated tropmyosin-1 is possibly involved in these processes [163], [164], [165]. If the particle is completely internalised, then an oxidative burst occurs. This means the sudden, dramatic increase in reactive oxygen species in the cell within the scope of the phagocytosis processes. In this way, several proteins store together in the wall of the phagosomes to form the NADPH oxidase complex [166] that transmits electrons to molecular oxygen (Fig. 12). The oxygen species that abruptly results here decompose the internalised particle. The NADPH oxidase complex generates an H+-ion during the electron transmission that is channelled into the phagosome via a proton channel. The acidification in the phagosome speeds up the breakdown of the internalised particle.

Figure 12

During phagocytosis, intracellular reactive oxygen species rapidly increase, known as oxidative burst.

Following receptor binding od , e.g. LPS, and particle internalization, the protein p47phox is activated and integrates with the additional components p67phox and p40phox into the phagosome membrane, where it combines with flavocytochorme b to the active NADPH-oxidase complex. This enzyme complex catalyzes the generation of ROS and protons, which shift through proton-channels into the interior of the phagosome, where they destroy the internalized particle.

At the same time, the enzyme iNOS (inducible nitric oxide synthase) is activated and formed into high concentrations of nitrogen monoxide (●NO). This is a very reactive radical that introduces similar harmful effects such as ROS [167], [168], [169], [170]. Professional phagocytes have absorbed and to a large extent decomposed a particle in approximately 30-60 minutes. Here, considerable amounts of ROS can be released that along with the affected cell can even damage neighbouring cells and can induce a local inflammatory reaction. The formation of ROS also leads to the activation of NF-κB, as further described below.

4.2.4. Reactive oxygen species and nitrogen monoxide

Due to the particular significance of reactive oxygen species and nitrogen monoxide in respiratory toxicology, these substances should be looked at in more detail. The physiological generation of energy of the cell through molecular oxygen results primarily in the mitochondrial membrane as the oxidative phosphorlation of ADP to energy-rich ATP. The mediating enzyme is the NADH dehydrogenase complex of the respiratory chain. Reduced, highly reactive oxygen metabolites result as a by-product. These include superoxide or hyperoxide anion (●O2-), hydrogen peroxide (H2O2) and the particularly reactive hydroxide ion ●OH that forms in the presence of transition metals and that when dissolved appears as H3●O2- (The point next to the symbol for the element represents the unpaired electron of the radical.). These reduced O2 derivatives are more reactive than molecular oxygen and for this reason are referred to as reactive oxygen species (ROS) [171]. The term "oxygen radical" is inexact as molecular oxygen itself is a biradical. Aproximately 1 2% of these toxic metabolic by-products can be released during oxidative phosphorylation. Antioxidative enzymes in the cell rapidly break them down. In this way, superoxiddismutase (SOD) reduces the superoxide O2- to H2O2 and catalases and peroxidases, for example the glutathione peroxidase, H2O2 to H2O. Normally ROS and enzymes which break down ROS are in balance in the cell, however, through an increase in the amount of ROS or through a reduced activity of the ROS degrading enzymes, for example through glutathione depletion after SO2 exposure, the intracellular ROS concentration can increase. This is called oxidative stress.

As well as mitochondria and the phagosome within the scope of the oxidative burst, the smooth endoplasmic reticulum belongs to the intracellular ROS sources: ROS originate here, for example within the scope of the xenobiotics detoxification through cytochrome P-450 oxidases. Among the exogenic ROS sources are transition metals, γ-rays, UV rays, nutritional components and a variety of drugs [171]. ROS leads to damage of various cell components. Among the substances particularly sensitive to ROS are proteins, lipid membranes and DNA. With proteins, ROS leads to protein peroxidation. Aldehydes, ketones, and carbonyls are formed. Proteins modified in this way change their tertiary structure, lose their function and are quickly broken down inside cytosolic proteasomes. In this way a large number of cell functions such as energy generation, maintenance of membrane potential, movement processes, DNA repair and deactivation of signal proteins through phosphatase are impaired [171]. The weak point of the ROS in lipid membranes of the cell is their double bounds in unsaturated fatty acids. During peroxidation, reactive aldehydes, lipid hydroperoxides or cyclic endoperoxides occur that peroxidise further unsaturated fatty acids. A chain reaction occurs that can lead to a peroxidation of all the unsaturated fatty acids of a lipid membrane [171]. The third target structure of ROS is DNA molecules, whereby hydroxide ions have a particularly destructive effect. They cause single and double strand breaks, changes in the DNA bases, loss of purine bases, damage to the sugar backbone and DNA-protein adduct formation. Frequent adducts through ROS are 8-hydroxydeoxyguanosin (8-OhdG), 8-hydroxyadenin, thyminglycol or 5-hydroxymethyluracil [171]. 8-OhdG is a indicator for ROS-induced DNA damage commonly employed in environmental medicine.

In low concentrations, ROS have a stimulating effect of a large number of cell processes and are thus essential for the proper functioning of the cell. Amongst other things they act as intracellular signal molecules that reinforce the transcription of enzymes that break down ROS [115]. As well as this they influence the signal transduction ubiquitously by checking the activity of tyrosin phosphatases, thus reinforcing the tyrosinkinase effect [172]. They are involved in cell growth and cell division, as well as in the modulation of numerous biochemical processes, including the formation of prostaglandins. These positive ROS effects are probably the cause of the so-called "antioxidant paradox". Dietetic supplementation with antioxidants such as vitamin C, tocopheroles, polyphenoles and carotenoids in large studies with several thousand participants has produced no protection against illness, on the contrary, its has resulted in a raised incidence of illness [173], [174]. Concentrations of antioxidants that appear too high may also impair the redox balance and place the cell under reductive stress [171].

Nitrogen monoxide (●NO) is a gaseous, very reactive free radical. It is formed through NO synthases (NOS) from the amino acid arginin. Here NADPH and O2 are used. ●NO is broken down within a few seconds. It functions on the one hand as a cellular signal molecule in, amongst others, nerve cells, endothelial and epithelial cells. For this purpose, ●NO is continually formed ("constitutively expressed") in low concentrations from the neuronal nNOS or the endothelial eNOS. In neuronal systems it serves as a neurotransmitter, in blood vessels is has a vasodilatory effect (endothelium-derived relaxing factor) and in the bronchial, system it is bronchodilatory.

The function of ●NO catalysed through iNOS (inducible NOS) in the context of phagocytosis is very different. It is secreted in very high concentrations in the phagolysosome and has a similar effect to ROS [167], [168], [169], [170]. It also leaves the cell and can cause damage in neighbouring cells. In addition, it merges with exhaled air where it serves as a sensitive marker for inflammations of the mucous membrane. NO is also measurable in the nose. It stems almost exclusively from the nasal sinuses and is an exceptional marker for inflammatory alterations of the nasal sinuses [175], [176], [177], [178], [179]. The reaction of ●NO with superoxides leads to the formation of peroxynitrit, an extremely cytotoxic compound. The release of peroxynitrit into cytosol influences the function of numerous proteins through nitrosylation [113]. Amongst other things it reacts with the amino acid tyrosine to form nitrotyrosine (NO2Tyr). This compound can be called on to confirm the presence of reactive nitrogen species in tissue and secretions [180].

In the foreground of the ●NO effect, however, is less its short-term increase within the scope of phagocytosis processes, than the long-term increase through the increased supply of the enzyme iNOS. The transcription of iNOS is upregulated through endotoxins and various cytokines such as TNFα, IL-1β and IFN-γ. One important signal transduction pathway takes place via the activation of NF-κB and STAT1 [181]. Continuous ●NO increase influences the regulation of numerous cell processes and functions as a ubiquitous signal molecule independent of signal transduction pathways by nitrosylsing important cellular molecules [172].

4.3. Toxicity mechanisms of metals

In industrial agglomeration zones in particular, metals play a prominent role in the harmful effects of ambient air dusts [40], [153], [182], [183], [184]. The toxic effect of such dusts on the respiratory system can be almost completely inhibited by chelating agents [40], [84], [145]. The transition metal zinc, copper, iron, nickel and vanadium are at the forefront. Metals are mainly detectable on the soluble fraction of pm2.5 [116]. In small amounts, transition metals are essential for the proper functioning of the cell. The intake of transition metal into the cell results from active transport processes. Because of their toxicity, metals are bound intracellularly to storage proteins (ferritin, metallothionein) [185], [186], [187]. If the cellular metal storage capacity is exceeded, free metal ions occur in the cell. Within the cell, free transition metals transmit an electron to the hydrogen peroxide that is present in small amounts in accordance with the Fenton reaction (Fe2+ + H2O2 → Fe3+ + OH- + OH●). In this way a highly reactive hydroxide ion occurs and the cell is placed under oxidative stress [188], [189]. The released ROS lead to damage to proteins, lipids and DNA of the cell. The molecules altered by ROS trigger a stress response in the cell. They activate intracellular signal transduction cascades, whereby the stress activated kinase (SAPK) pathway is of particular significance [190], [191]. The induction of the stress activated kinase pathway through reactive oxygen species is a key mechanism, like metals, but also bioorganic compounds, lead to the release of proinflammatory cytokines. Here, the effects of metals and bioorganic compounds on the formation of ROS are synergistic [192]. Below, the stress response of the cell shall be observed in detail considering the metal-induced oxidative stress as an example.

4.3.1. Stress response of the cell

Changes in the surrounding condition of the cell interior require adaptation processes in the cell that are combined under the term cellular stress response. As well as oxidative stress, the stress response can also be produced by countless stimuli, for example by the withdrawal of nutrients and oxygen, or the heavy strain caused by typical cell functions such as muscle cell contractions. Other typical cell stressors are temporary heat (heat shock, e.g. 5 mins. at 42°C), ischemia/reperfusion, UV rays, DNA damage, hyperosmolarity, changes in pH values, proinflammatory cytokines, vasoactive peptides and also growth and differentiation signals. The varying stress responses are almost as numerous as the stressors themselves. Among these are apoptosis, cell cycle arrest, metabolic activation, release of proinflammatory cytokines and also growth, cell division or differentiation.

The stress activation of cells results from the stress activated protein kinase pathway (SAPK pathway). The activation of the SAPK pathway (Fig. 13) results via different mechanisms [193]. Cytokines and growth factors trigger the SAPK pathway in parallel to the typical signal transduction cascades activated by their specific receptors. Cell stressors, such as UV rays, osmotic stress, acid stress or ROS affecting the cell externally, react initially with parts of the lipid membrane. One typical chemical reaction is the hydrolysis of membrane sphingomyelins to ceramides. These act as second messenger and activate the SAPK pathway. Intracellular-active agents such as ROS or ●NO, as well as heat shock or toxic metabolites, for example in the breakdown of ethanol, lead to protein denaturations. Through protein denaturation, an indirect activation of the SPAK pathway probably results through suspension of the deactivating phosphatases [194]. Lipid peroxides also possibly bring about the activation of the SAPK pathway [145] in the cell membrane of cell organelles such as 4-hydroxy-2-nonenal (HNE) or malondialdehyde [195]. Ultimately DNA damage also leads via the tyrosine kinase c-Abl to the activation of the SAPK pathway [196], [197].

Figure 13

The stress activated protein-kinase path

SAP kinases are a group of cytosolic protein kinases activated in a cascade like fashion following cellular stress exposure. Their activation occurs basically via 4 mechanisms. Proimflammatory cytokines and growth factors activate SAP-kinases via interconnections with their main intracellular pathways mediated by small GTP binding proteins such as Rac1 or Cdc42. Extracellular stressors cause the generations of cermides in the within the cellular lipid membrane, which activate SAP-kinases as second messengers. DNA damage activates the tyrosinkinase c-Abl, which activates SAP-kinases via intermediary steps. Intracellular ROS denature phosphatases and thus interfere with the inactivation of SAP-kinases. In addition, ROS activate the transcriptionfactor NF-κB by mechanisms not yet understood. Activation of SAP kinases in turn activates several transcription factors, which then translocate into the nucleus. Here they activates genes coding for proteins involved in cell division, apoptosis, cytoskeleton, cell activity and inflammation. In addition, SAP-kinases activate phsopholipase A2 thus altering the release of prostaglandins and leukotrienes. Heat shock proteins belong to the chaperones, which repair denatured proteins (resoration of their three-dimensional structure). Heat skock proteins serve as an indicator for cellular stress.

The SAPK pathway leads to the activation of various transcription factors, whereby activator protein 1 (AP 1) takes on a key role. No direct activation of NF-κB takes place via the SAPK pathway. In several types of cells, ROS can activate NF-κB directly and without the involvement of the SAPK pathway and thus contribute to the release of different cytokines [155], [198]. The mechanisms of this activation are not exactly known [199], [200], [201], [202], [203]. One plausible explanation for this is activation of CBP/p300 via the SAPK pathway, a co-activator of NF-κB which occurs constitutively in small amounts in the nucleus. This phenomenon was observed among others with English environmental dusts and appears to be typical for the release of ROS in respiratory epithelia induced by metals [144], [145], [153].

The biological effects of the activation of the SAPK pathway differ between lymphoid and non-lymphoid cells. In the case of the majority of non-lymphoid cells, cell growth and division are limited and proinflammatory cytokines are released [197]. Thus via the formation of ROS, metals lead to the release of proinflammatory cytokines and chemokines (IL-1, IL-6, IL-8, TNFα) from respiratory epithelia [204]. In T lymphocytes, the SAPK pathway appears to be essential for the differentiation to effector cells and to effector-specific cytokine production [205]. Excessive cellular stress triggers off an apoptosis programme in the cell [206], [207]. Stress activation is, amongst other things, to be observed in with the exposure to asbestos. Asbestos induces oxidative stress in macrophages, possibly through the release of Fe2+. This results in a reduction of anti-oxidative intracellular glutathione and an upregulation of AP-1 [208]. An activation of NF-κB occurs in respiratory epithelia that can be reduced through the prior application of antioxidants [209]. Quartz dusts also lead via the release of reactive oxygen species to the activation of NF-κB and the release of IL-8 from human respiratory epithelia [150].

4.4. Organic compounds and combustion residues

As well bioorganic pollutants and metal, organic compounds, e.g. from combustion processes, also play a considerable proinflammatory role. Organic compounds in urban dusts account for about 20% of the mass at pm10, and about 45% at pm2.5 [206]. From the point of view of industrial medicine, workers in coking plants, the metal working industry, blacktop workers and welders are exposed to combustion residue and smoke to a considerable extent. Present in combustion residue are different groups of substances harmful to health such as metal, polycyclic aromatic hydrocarbons, halogenated hydrocarbons (e.g. dioxin) and nitrosamines. Particulate emissions from diesel engines (diesel exhaust particles or DEP) are very common and of great relevance for environmental medicine. This involves saturated and unsaturated aliphatic hydrocarbons and their substitution products such as aldehydes and ketones, anhydrides and carbon acids. A further considerable component is polycyclic aromatic hydrocarbons [PAH], as well as nitro-, amino- and hydroxo-combinations.

Diesel soot is probably responsible for the association between heavy goods traffic and allergic respiratory disorders that have been shown in numerous studies [210], [211], [212], [213], [214], [215], [216], [217], [218], [219]. As well as allergic respiratory disorders of the upper respiratory tract in residents of areas heavily affected by traffic, non-allergic disorders also occur in large numbers [220]. After many years exposure to diesel soot, chronic inflammatory and degeneratively altered nasal cytologies were found in Swiss customs officers [221]. The PAHs typical for DEP are, however, also found in normal environmental dusts in concentrations that can trigger off inflammatory reaction in the respiratory tract [206]. DEP induce changes in respiratory cells similar to bioorganic pollutants and metals, and thus lead to a release of ROS and in this way induce cellular stress [88], [222]. As a consequence, this results in an activation of the SAPK pathway [222] and the release of proinflammatory cytokines [223], [224].

4.4.1. The arylhydrocarbon receptor

The PAHs contained in the combustion residue, halogenated aromatics and N-substituted polycyclic hydrocarbons function via an additional specific toxicity mechanism. These substances are mostly lipophilic and can pass the cell membrane. Within the cell they bind to a cytosolic receptor, the arylhydrocarbon receptor (AhR) [225], [226]. Activation of the Ah receptor (Fig. 14) leads to its translocation in the cell nucleus the activation of genes that are regulated via the xenobiotic responsive element. Genes for detoxification enzymes such as CYP1A1, CYP1A2 and CYP1B1 and NAD(P)H quinon oxidoreductase and UDP-glucuronosyltransferase are more intensely transcribed and are also traceable as protein [227]. In addition haemoxygenase-1 is expressed that stabilises the cellular redox potential [58], [206], [222]. The AhR brings about proinflammatory impulses through its interaction with NF-κB [228], [229], [230], [231]. Activation of the AhR influences the cell cycle, amongst other things by activation of cyclin-dependent kinase 2 and inhibition of TGF-β [232]. The carcinogenic effect of typical AhR ligands such as benzo(a)pyrene is also transmitted via the AhR. It is no longer detectable in AhR deficient mice [233]. Pei and colleagues describe an upregulation of p53, probably via an analogous NF-κB dependent promoter region in the p53 gene [148]. The AhR also interacts via the nucleus translocation factor ARNT with hypoxia response elements that, for example, regulate the expression of erythropoietin [234]. The AhR is an essential mediator of cytotoxicity transmitted via cigarette smoke [235].

Figure 14

The arylhydrocarbon receptor (AhR) belongs to the Ligand activated basic helix-loop-helix transcription factors.

In the cytosol, it occurs as an tetrameric complex with heat shock protein (HSP) 90 and X-associated protein 2 (syn: ARA9). Ligands such as dioxin (TCCD) or polycyclic aromatic hydrocarbons dissolve the 2 HSP molecules from the complex. The remaining complex binds to the Arylhydrocarbon Receptor Nucleus Translocator (ARNT), is translocated in to the nucleus and enhance the transcription of xenobiotic response element dependent genes. The gene expression of several proteins including various detoxification enzymes is increased. In addition, cyclin kinase 2 is activated and TGF-β effects are inhibited, resulting in increased cell division.

4.5. Toxicity mechanisms of low toxicity particles

Dockery et al. were amongst the first to recognise the special respiratory toxicity of particulate air pollutants [236]. The question has to be answered as to whether the particles themselves are responsible for their respiratory toxicity as a result of physical and mechanical actions, or toxic dust constituents. In order to answer this question, numerous studies were conducted with dusts whose components are generally considered non-toxic. To be correct, these experimental dusts rarely found in the natural environment are termed "low toxic". Most animal experimental studies with low toxicity particles were carried out on the lower respiratory tract. In the lower respiratory tract definite, if comparatively weak inflammatory changes to the mucous membrane are triggered by low toxicity particles of, for example, melamine, polystyrene, titan dioxide or barium sulphate. This inflammatory reaction in the lower respiratory tract is predominantly determined by size and surface characteristics of the particle [237], [238], [239].

The key biological mechanism for the inflammatory reaction through low toxic particles in the lower respiratory tract is its phagocytosis through alveolar macrophages. This cell population comprises 40-50% of the cells in a bronchoalveolar lavage. For a long time it was unclear how macrophages manage to phagocyte particles without special immunological distinguishing features. The major role of scavenger receptors in these processes is now clear to a large extent [240], [241], [242], [243]. Scavenger receptors (SR) are the most primitive form of pattern recognition receptors [244]. They serve the phagocytosis of non-opsonized particles even if they do not have particular structural features. Due to the special collagenous makeup of their extramembranous domains, SR have a "sticky" effect and accordingly demonstrate a distinctive ligand promiscuity. They are to be found predominantly on macrophages but can also found on respiratory epithelia [245]. As well as the phagocytosis of gram+ und gram- bacteria, they convey the pathological effects of pollutants [246]. After their amino acid sequence they are assigned to types A, B and C. To type A receptors belong the groups SR-AI, SR-AII and MARCO. MARCO is to be found above all on lymph node and spleen macrophages. Their occurrence on alveolar macrophages is the subject of debate [242], [243], [247]. Type A SR only induce a weak inflammatory activation. With low toxic particles the phagocytosis is thus only accompanied by a weak inflammatory reaction [248]. This makes the scavenger receptor suitable for the neutral phagocytosis of apoptotic cells seen from the inflammatory aspect [142]. As well as low toxic particles, toxic particles from the environment also undergo phagocytosis via the SR [248]. Research in this area is facilitated by modern detection techniques [249], [250]. The proinflammatory effects with phagocytosis via SR are probably conveyed by a comparatively weak oxidative burst.

Whilst low toxicity dusts can induce an inflammatory reaction in the lower respiratory tracts, the upper respiratory tracts react to low toxic dusts via local reflex mechanisms. In the case of 32 healthy test persons at concentrations of 5000 µg/m3, low toxicity dusts (calcium carbonate with an average particle size of 15 µm) led, depending upon the dosage, to a reactive acceleration of mucociliary transport, an increase of the nasal respiratory resistance and feelings of nasal dryness [251]. These changes came about, however, without a nasal inflammatory reaction being detected. Two explanation present themselves for the lack of nasal inflammatory reaction on exposure in the upper respiratory tracts with respect to low toxicity particles: macrophages as professional phagocytes are considerably more scarce in the upper respiratory tracts than in the lower and the particles deposited in the nose are larger so that they cannot so easily undergo phagocytosis.

4.6. Sulphur dioxide and nitrogen dioxide

Sulphur dioxide is a pungent, colourless gas. It is highly water-soluble. In the upper respiratory tracts inhaled SO2 is almost completely dissolved in the respiratory secretion and reacts with H2O to form sulphurous acid. Less than 5% of the inhaled SO2 reaches the lower respiratory tracts. For this reason, it is mainly disorders of the upper respiratory tracts that occur with high ambient air-SO2 concentrations [8], [252], [253]. Central to this is the increased susceptibility for infections of the upper respiratory tracts. Changes in the lower respiratory tracts can be correlated either only in small measure or not at all with ambient air-SO2 concentrations [254], [255]. Changes in the lower respiratory tracts with comparatively high ambient air concentrations are, however, described [256].

After SO2 exposure, glycoproteins in the secretion first buffer the released protons. In this way the viscosity of the secretion is increased and the mucociliary transport decelerated [257], [258]. If the buffer capacity of the secretion is exhausted, the pH value drops and acid molecules come into contact with the cell and ciliary membrane of the respiratory epithelia. The exposure to protons leads to the damage to the epithelium. The sulphites do not damage the epithelium [259]. Through the slowing down of the ciliary beat the mucociliary transport is further impaired. Ultra-structurally, the development of fine stress fibres can first be observed and then the formation of small blebs in the cell and ciliary membrane (membrane blebs). At higher concentrations an increasing vacuolization can be recognised and then finally a break-up of the tight junctions [260], (Fig. 15).

Figure 15

Vacuolization and interruption of tight junctions (arrow) in a guinea pig trachea following exposure to 7,5 mg/m3 SO2

Analogous ultra-structural changes can be frequently observed when cells are placed under varying degrees of stress, as here in the case of acid stress. It involves active cellular processes as part of this stress response. Due to their load, acid molecules cannot penetrate the lipid membrane, but they denature proteins in the cell membrane [193]. This releases a large number of cellular reactions that lead to the stress response of the cell described above. Thus acid stress activates NF-κB in human tracheal epithelial cells that leads, amongst other things, to an increased expression of IL-1 and the PAF receptor. The raising of the PAF receptor to the surface of the cell increases the adherence of streptococcus pneumoniae to the epithelial cells and thus aids the development of respiratory infections [261].

Another mechanism of the SO2 effect is not based upon the acid activity, but rather on the reaction with cellular glutathione. Glutathione is a considerable factor towards the maintenance of the cellular redox balance. SO2 reacts with glutathione to form S-sulfo-glutathione and depletes cellular glutathione. What follows is a reduction of the activity of glutathione-dependent detoxification enzymes such as glutathione peroxidase or glutathione reductase. This reduces the cellular resistance to oxidative stress [262]. The slightly increased incidence of lung cancer mortality on SO2 exposure is probably to be seen in this context [263], [264], [265].

SO2 works as an irritant on the respiratory tracts. The cause of this is the permissive effect of protons on the Vanilloid receptor on trigeminal fibres of the respiratory mucous membrane (Fig. 16). These do not activate the receptor themselves, but result in a considerable increase in its sensitivity to numerous stimuli. In this way the receptors, under the influence of protons, are already activated at a temperature of 37°C [266]. Basic mechanisms of irritation of the mucous membrane are more closely described with the volatile organic compounds.

Figure 16

Proton activation of vanilloid-receptors

Vanilloid such as Capsaicin, various environmental irritants and heat (> 43°C) may open the Vanilloid receptor for Na+ and Ca2+ ions, whereas H+-Ions are not able to activate the receptor. However, protons bind to the receptor and increase its responsiveness to protons and irritants (permissive activity).

Nitric oxides are predominantly present in the environment in the form of NO2. Nitrogen monoxide reacts in the presence of oxygen in the air to form NO2. It is moderately water-soluble and reaches the lower respiratory tracts at about 50%. Nitric acid is formed in the water of the respiratory secretion following the reaction 3NO2 + H2O ↔ 2HNO3 + NO. In a carefully planned study, it was discovered that the incidence of atopic disorders in childhood does correlate with the NO2 content of ambient air, but not with individual NO2 exposure [212]. NO2 thus serves solely as an indicator substance for traffic congestion. With people it is only high (2000 µg/m3) NO2 concentrations that lead to inflammatory changes in the mucous membrane of the nose [267], whilst with persons suffering from an allergy, significantly lower NO2 concentrations can considerably enhance the allergic response [268], [269].

4.7. Ozone

Ozone is a strong oxidation agent. In urban agglomeration areas it occurs in toxic concentrations with UV light. Ozone is extremely reactive and is retained by reducing substances of respiratory secretion. Surfactant in respiratory secretion contains numerous unsaturated fatty acids that react with ozone to create more stable aldehydes, hydroxyhydroperoxides and endoperoxides. These lipid ozonation products, as well as small amounts of the inhaled ozone itself, penetrate through the respiratory secretion reaching the cell membrane of the cells in the respiratory tract [270], [271]. In the membrane of these cells, ozone and the still reactive lipid ozonation products that developed in the secretion lead in turn to the peroxidation of unsaturated fatty acids, whereby a chain reaction of lipid oxidations can occur. The reactive ozonation products of the membrane lipids (Fig. 17) place the cell under oxidative stress [272]. Numerous further reactive compounds occur including nonanal, hexanal [273], [274] and F2 isoprostane. This substances resembles prostaglandin with similar proinflammatory effects. It also is a good marker of the in vivo lipid peroxidation [275], [276]. As well as this, membrane proteins are denatured. Activation of the SAPK pathway follows with activation of phospholipase A2, C and D, of PI-3 kinase, protein kinase B, NF-κB, STAT-1, upregulation of the inducible NO synthase, to the formation of ●NO and the release of IL-8 and TNFα from macrophages, neutrophils and respiratory epithelia [272], [277], [278], [279], [280].

Figure 17

Peroxidation of membrane lipids (here: 1-palmitoyl-2-oleyl-sn-glycerol-3-phosphocholin (POPC)) and generation of lipid ozonation products by inhaled ozone attacks the double-bound in the right fatty acid (red circle) and epoxides it to an ozonide (yellow circle) or hydrolyzes it to an aldehyde and a hydroxyhydroperoxide (right, green circle).

After a few hours, the release of the chemokine IL-8 results in airway neutrophilia, the prominent feature in nasal cytologies after ozone exposure. The neutrophil granulocytes increase the epithelial damage induced by ozone [281]. In addition, ozone leads in vitro to the release of neuropeptides from nasal mucous membrane [282]. Mucous membrane irritation is probably not attributable to ozone itself, but rather to ozone oxidation products such as formaldehydes, peroxyacetyl nitrate, peroxybenzol nitrate, acrolein or, for example, ozonation products from terpenes [283].

Long-term or repeated ozone exposure leads to epithelial desquamation, probably as a result of an increased production of the interstitial substance tenascin C [284]. A mucoid degeneration of the mucous membrane with disturbances of mucociliary transport occurs, as well as a reactive proliferation of epithelia [285], [286], [287], [288], [289], [290]. In addition to a proliferation of respiratory epithelia, DNA damage was detected that is typical for ROS [291].

4.8. Aldehydes and volatile organic compounds

Aldehydes and volatile organic compounds are predominantly present indoor. Up to 50 different substances are frequently detectable in a concentration range of 1 µg/m3 each. Indoor formaldehyde frequently reaches concentrations above 50 µg/m3 [292], [293]. Toxicologically, their irritant effect on eyes and the respiratory tract are in the foreground. The Sensory Irritation Test can quantify the irritative effect of a substance [294]. A detailed list of the irritative properties of a variety of substances can be found in Bos et.al. [295]. One of the strongest known irritant agents of relevance for industrial medicine is toluol-2·4-diisocyanate (2·4-TDI). It was recently suggested that predominantly trigeminally transmitted nasal sensory irritants be separated from predominantly vagally transmitted pulmonary irritants. Nasal irritants typically result in an extended breathing rest with delayed expiration, whilst pulmonary irritants result in a breathing rest with delayed inspiration [296]. Whether we are really dealing here with characteristic substance features and receptor interactions, or whether, for example via differing solubility, different respiratory compartments are affected by the same mechanism, is at present not clear.

There are several new findings on the mechanisms of the irritant effect of chemical agents in the upper respiratory tracts and how they lead to neurogenic inflammation and hyperreactivity [297], [298], [299]. Via the respiratory secretion, water-soluble irritant agents gain access to the nerve endings of non-myelinated sensitive nerve fibres whose cell bodies lie in the trigeminal ganglion [300], [301]. Here, numerous irritant agents bind to a non-selective receptor of the axon membrane, the Vanilloid receptor of subtype 1 (VR 1) [302]. Dusts and ozone also activate VR 1 receptors [282], [303], [304]. This can take place anywhere in the nerve fibres pathway, not only at its end point. With the attachment of the ligands to the Vanilloid receptor, a receptor-operated ion channel opens and an influx of Ca2+ und Na+ takes place (Fig. 18). This leads to membrane depolarisation and to the discharge of a nerve action potential through stress-dependent Na+ channels in the membrane of the nerve fibres. In the trigeminal ganglion, SP and glutamate are released that transmit the irritant conduction towards the centre. In the mesencephalon, protective reflexes such as sneezing or coughing are triggered. However, nerve action potentials does not only run in an orthodromic direction towards the centre, but also in an antidromic line into the periphery where tachykinins such as substance P (SP), neurokinin A or B are released at nerve endings. These tachykinins have proinflammatory effects and project a neurogenic inflammatory reaction of the tissue. They lead to vascular dilation, oedema formation, hypersecretion and to an inflammatory cell influx [301], [305], [306].

Figure 18

Mucosal irritation and neurogenic inflammation

An irritant binds to a vanilloid type I receptor (VR1) on a trigeminal, non-myelinated group C fiber within the airway mucosa (nociceptor). Local influx of sodium and calcium ions results in circumscribed membrane depolarization, which in turn opens voltage gated sodium channels (VGSC) and generates an action potential along the nerve fiber. This action potential runs orthodromically (in the correct direction) toward the Gasserian ganglion, where transmitter substances including glutamate, Substance P (SP) und Calcitonin Gene Related Peptide (CGRP) transfers the signal to central neurons responsible for the perception of irritation and pain. The action potential also runs antidromically toward the periphery, where tachykinins such as SP, CGRP and Neurokinin A (NKA) are released. They activate the Neurokinin 1 (NK1) receptor on glandular cells, respiratory epithelium and endothelium of neighbouring vessels. Thus they mediate hypersecretion, vasodilation and inflammatory cell infiltration.

SP prefers the NK1 receptor, neurokinin A the NK2 receptor and neurokinin B the NK3 receptor. In the mucous membrane of the upper respiratory tracts, changes induced by tachykinins are projected for the most part via the NK1 receptor [307]. Predominantly, the NK2 receptor causes a bronchial constriction, the NK3 receptor has not yet been positively detected in human respiratory tracts [308]. The NK1 receptor belongs to the G protein-coupled receptors. Ligand binding leads to the activation of phospholipase C and adenylatcyclase [309] that leads to the contraction of smooth muscle cells of the vessel wall. The vasodilative effect thus results not through the effect on the smooth muscle in the vessel wall itself, but rather via the endothelium. In contrast to the smooth vascular muscle cells, the endothelial cells express the NK1 receptor. If these are activated, the endothelial cells then release prostacycline and NO, which secondarily leads to the relaxation of vessel smooth muscle cells (see background information nitrogen monoxide) [310], [311]. At the same time, the activation of the endothelial NK1 receptor leads to contraction of the endothelial cells. Gaps emerge in the vessel wall through which plasma proteins and water can pass over into the tissue [312]. This results in an inflammatory oedema. Glandular hypersecretion is conveyed predominantly by the NK1 receptors onto glandular cells [308].

The neurokinin system and the immune system have a brisk, two-way relationship. Via the NK1 receptor, SP has a predominantly chemotactic effect on neutrophils [313], [314], [315], [316], [317], [318], [319], it enhances their ROS production [320] and inhibits their apoptosis [321]. SP also has a chemotactic effect on T lymphocytes [322], monocytes [322] and dendritic cells [323]. Eosinophils are directly attracted by SP [314] or after previous peptide activation [324]. Secretoneurin, which is released into the respiratory tracts by nociceptive fibres, also has distinctive eosinophil-chemotactic features [325]; it was found in considerable amounts in nasal irrigation and in nasal mucous membrane biopsies of people suffering from allergies [326]. The NK1 receptor is only expressed on mast cells after preliminary treatment with IL-4 or SCF [327]. After this type of priming, however, SP can also bring about a release of histamine [328].

Longer-term exposition to irritants and inflammatory irritants, but also uniquely extreme exposition, can lead to a persistent hyperreactivity of the respiratory tracts [329], [330], [331], [332], [333]. After inhalation of sulphurous acids, guinea pigs develop a respiratory hyperreactivity lasting several weeks. It can be inhibited by a NK1 receptor antagonist [334]. Changes in the nociceptive system of the respiratory tracts form the basis of this persistent hyperreactivity, whereby the density of trigeminal group C fibres in the mucous membrane of respiratory tract apparently increases. Exposition with TDI [60 ppb] over 2 h results within 24 hours in an increased density of trigeminal group C fibres in the nasal mucous membrane, as well as an increase in SP and preprotachykinin in these fibres [300], [335]. Very similar changes take place after exposure to asphalt fumes [336].

The increased density of nociceptive fibres in the mucous membrane after exposure to irritants can probably be traced back to the release of nerve growth factors. NGF leads to the sprouting of nociceptive fibres [337], [338] and consecutively to hyperreactivity of the respiratory tract [339], [340]. As well as a short-term rise in SP after nasal irritation [341], an increased level of the nerve growth factor was measurable in the nasal lavage [335], [342]. As well as nerve fibres themselves, macrophages and epithelial cells contribute to the release of NGF [343], [344]. The signal transduction probably runs via protein kinase C and ERK1/2 [345], [346]. Transgenic mice that release NGF from mucous-producing bronchial epithelia (Clara cells) develop a marked bronchial hyperreactivity [347]. Transgenic mice without the NGF-p75 gene exhibit disturbances in the nerve fibres containing tachykinins and react less to sensory irritants [348]. With these animal models, the significance of nerve fibres containing tachykinins for the proinflammatory effect of ozone was examined. It was shown that the ozone-induced inflammatory reactions with animals deficient in tachykinins was considerable less common than was the case with normal animals, and proceeded considerably more strongly with animals with an overproduction of tachykinins. This would indicate that the irritant effect of ozone or its reaction products contributes significant to its inflammatory effect [349].

An important protective and regulatory function of the respiratory secretion is to be found in the rapid breakdown of biologically active tachykinins through varying amino peptides such as neutral endopeptidase (NEP), angiotensin converting enzyme (ACE) or dipeptidylpeptidase IV (DPPIV) [41]. A result of a lack of these enzymes that break down tachykinins in secretion can contribute greatly to the emergence of respiratory hyperreactivity [42], [350]. The value of oral neurokinin receptor inhibitors must first be proved [351], [352]. As well as their irritative effect, volatile organic compounds also have cytotoxic and genotixic features [81], [353] that in environmentally relevant concentrations, however, are not crucial.

4.9. Synopsis of the mechanisms of inhaled pollutants

Air pollutants lead to inflammatory changes in the mucous membrane of the respiratory tract. Mixtures of numerous toxic substances that can be assigned to several archetypes are inhaled. In the real exposure situation, these agents act in a synchronous or sequential manner and exhibit synergistic effects. Thus one can assume that in the real exposure situation all described cellular toxicity mechanisms are simultaneously activated to a greater or lesser degree.

Ozone and acid gasses such as SO2 denature molecules from the cell membrane. The denatured lipids and proteins activate an intracellular signal transaction cascade, the SAPK pathway. This leads to release of prostaglandins and leukotrienes that promote inflammation, to the activation of transcription factors such as AP-1 that activate genes encoding proteins for cell proliferation and extracellular matrix. As well as this, SAPK activate the transcription factor NF-κB that in turn activates genes for a large number of proteins with proinflammatory effects.

Metals are dissolved from inhaled dust particles and channelled into the cell by metal transporters. Here they bring about the formation of reactive oxygen species and nitrogen monoxide, that is to say highly reactive radicals. For their part, these then directly activate the SAPK pathway and also NF-κB via partly still unexplained mechanisms.

The indicator substance for bioorganic pollutants, mainly fragments of bacteria and fungi, is endotoxin. They bind to toll-like/IL-1 (TIL) receptors. This leads to the activation of NF-κB via intracellular signal molecules. As well as this, TIL receptors convey the phagocytosis of the particles with adherent endotoxins. This leads via the oxidative burst to the release of ROS and NO. Endotoxins also have positive features. They shape the TH1 -preferred immune response and thus work against the emergence of allergies of the respiratory tracts [136].

Organic compounds from combustion processes, for example polycyclic aromatic hydrocarbons (PAH), are lipophilic. They can penetrate the cell membrane and bind to the cytosolic arylhydrocarbon receptor. This moves into the cell nucleus and activates genes for detoxifications enzymes, but also encourages a proliferation and proinflammatory effect via interaction with NF-κB.

Irritant agents often belong chemically to aldehydes and volatile organic compounds. They release substance P (SP) from the nociceptors of the respiratory mucous membrane. SP has a high affinity interaction with the NK1 receptor. Subsequently, the respiratory cell releases nerve growth factor (NGF) that increases the nociceptor density in the respiratory mucous membrane. This contributes to the development of mucous membrane hyperreactivity. The intracellular signal transduction from the NK1 receptor to the activation of the gene for NGF is not conclusively explained, but there is a great deal of evidence that protein kinase C is involved. In addition, the transcription factor C-fos is apparently activated via the ERK1/2 pathway . As well as this, protein kinase C activates the transcription factor NF-κB after the coupling of SP to the NK1 receptor.

(Fig. 19)

Figure 19

Synopsis of frequent toxicity mechanisms of inhaled pollutants

Ozone and SO2 denature membrane components, which activate stress activated protein kinases (SAPK). This results in the release of prostaglandins (PG) and leukotrienes (LT) and in the activation of transcriptionfactors such as C-jun, which activates among others genes for cell proliferation. SAPK also activate NF-κB, a key transcription factor inducing inflammatory cell reactions. Metals are internalized via cellular metal transporter proteins and induce the release of reactive oxygen species (ROS) and nitrogen monoxide (NO), which in turn activate SAPK and NF-κB. Bioorganic pollutants bind to the Toll-like/IL-1 (TIL-) receptor and activate NF-κB via cytosolic signal transduction pathways. In addition, particles with adsorbed bioorganic pollutants are phagocytosed and induce the release of ROS and NO via the respiratory burst. Lipophilic pollutants such as polycyclic aromatic hydrocarbons (PAH) pass the cell membrane and bind to the cytosolic arylhydrocarbon receptor (ArH), which in addition to NF-κB activates genes for detoxification enzymes. Irritants release substance P from nociceptors, which bind to the neurokinin receptor 1 (NK1) and activate genes for nerve growth factors (neurotrophins), probably via activation of Proteinkinase C (PKC) and the transcription factor C-fos. This may result in airway hyperreactivity. In addition, NF-κB is activated via the NK1-receptor.

These mechanisms induce a chronic neutrophil-skewed inflammatory reaction in the mucous membrane of the upper respiratory tracts. These inflammatory reactions are detectable in practically every inhabitant of western industrial nations and were termed "physiological inflammation" by Niels Mygind, one of the pioneers of modern nasal mucous membrane research [354]. This implies that the steady exposition to toxic substances is providential and must be accepted as physiological. It is at present unclear to what extent ubiquitous nasal exposition to pollutants contributes to the fact that ca. 15% of the population of western industrial nations suffer from chronic disorders of the nose and sinuses [355].