Strategies for clean air and health.

An international colloquium, "Strategies for Clean Air and Health," was organized by the Network of Environmental Risk Assessment and Management (NERAM) and the AIRNET European Network on Air Pollution and Health to identify directions for air quality policy development and research priorities to improve public health. A conference statement was prepared to provide guidance from the perspective of an international group of scientists, regulators, industries, and interest groups on a path forward to improve the interface between science and clean air policy strategies to protect public health. The statement represents the main findings of two breakout group discussion sessions, supported by perspectives of keynote speakers from North America and Europe on science-policy integration and views of the delegates expressed in plenary discussions. NERAM undertook a carefully considered process to try to ensure that the statement would accurately reflect the conference discussions, including documentation of supporting comments from the proceedings and inviting delegates' comments on two draft versions of the statement.

Ozone-induced airway hyperresponsiveness in patients with asthma: role of neutrophil-derived serine proteinases.

Proteinase inhibitors may be of potential therapeutic value in the treatment of respiratory diseases such as chronic obstructive pulmonary disease (COPD) or asthma. Our aim was to study the role of neutrophils, and neutrophil-derived serine proteinases in an acute model in patients with asthma. Exposure to ozone induces an acute neutrophilic inflammatory reaction accompanied by an increase in airway hyperresponsiveness. It is thought that these two effects of ozone are linked, and that neutrophil-derived serine proteinases (i.e. elastase) may play a role in the ozone-induced airway hyperresponsiveness. Therefore, we examined the effect of recombinant antileukoprotease (rALP), one of the major serine proteinase inhibitors in the lung, on ozone-induced changes in airway hyperresponsiveness in this model. We observed that 16 h after exposure to ozone, airway hyperresponsiveness to methacholine was increased both following placebo and rALP treatment. There was no significant difference between placebo and rALP treatment (change in area under the dose-response curve to methacholine: 117.3+/-59.0 vs 193.6+/-59.6 % fall x DD; p=.12). Moreover, the immediate decrease in FEV1 after ozone exposure was not significantly different between the two groups (placebo: -29.6+/-6.7%; rALP: -20.9+/-3.8%; p=.11). In addition, no significant differences were observed in plasma levels of fibrinogen degradation products generated by neutrophil serine proteinases before and after exposure to ozone. We conclude that neutrophil-derived serine proteinases are not important mediators for ozone-induced hyperresponsiveness.

Ozone-induced inflammation assessed in sputum and bronchial lavage fluid from asthmatics: a new noninvasive tool in epidemiologic studies on air pollution and asthma.

We investigated correlations between ozone-induced increases in inflammatory markers in induced sputum and in bronchial lavage fluid. Sixteen volunteers with intermittent asthma participated in a placebo-controlled parallel study with two exposures. Six days before and 16 h after the first exposure to ozone (0.4 ppm during 2 h) sputum was induced with hypertonic saline. This resulted in a significant increase in the sputum levels of eosinophil cationic protein (ECP; 1.8-fold; p = .03), neutrophil elastase (5.0-fold; p = .005) and the total cell number (1.6-fold; p = .02). After 4 weeks, a second exposure was randomized for air or ozone. Six days before and 16 h after the second exposure a bronchial lavage was performed. ECP values in sputum and in bronchial lavage fluid obtained after ozone correlated significantly (Rs = .79; p = .04), as did interleukin-8 (IL-8) values (Rs = .86; p = .01), and the percentage eosinophils (Rs = .89; p = .007). Moreover, the ozone-induced changes in percentage eosinophils observed in sputum and lavage fluid were highly correlated (Rs = .93; p = .003). In conclusion, changes in eosinophils, IL-8, and ECP markers induced by ozone and measured in sputum reflect the inflammatory responses in the lower airways of asthmatics, and may provide a noninvasive tool in epidemiologic studies on air pollution and asthma.

Homolateral cerebrocortical changes in neuropeptide and receptor expression after minimal cortical infarction.

A cortical infarct of 2 mm diameter was obtained in the parietal cortex after a craniotomy, disruption of the dura mater and topical application of 3 M KCl. It has been shown previously that the presence of a small cortical infarct induces an increase in immediate early gene messenger RNA expression followed by an increase in neuropeptide and glutamic acid decarboxylase messenger RNA expression. Glutamate, acting at N-methyl-D-aspartate receptors, is held responsible for these changes, since they are blocked by pretreatment with dizocilpine. In the present study, we have analysed the consequences of the dramatic changes in messenger RNA expression on the level of immediate early gene products c-fos and zif 268, and on that of neuropeptides by using immunohistochemistry. After just 1 h, an increase in c-fos- and zif 268-like immunoreactivity is observed in the entire cortical hemisphere homolateral to the infarct, and is no longer detected after 6 h. An increase in cholecystokinin octapeptide-, substance P-, neuropeptide Y- and somatostatin-like immunoreactivity is observed in the entire cortical hemisphere homolateral to the infarct after three days, and is no longer detected after 30 days. To investigate if these dramatic increases in neuropeptide immunoreactivities may have functional consequences, we studied the level of cholecystokinin receptors by autoradiographic binding using [125I]cholecystokinin-8S and in situ hybridization for the detection of cholecystokinin-b receptor messenger RNA. A decrease in cholecystokinin binding sites and cholecystokinin-b receptor messenger RNA is observed in the entire cortical hemisphere homolateral to the infarct after three days, and is no longer detected after nine days. This study shows that a topical stimulation has diffuse effects, reaching regions far from the site of the lesion, and some of them are still strongly present after nine days. The increase in neuropeptide messenger RNAs is followed by an increase in the protein products of these genes, which may modify the neurotransmission. As a corollary to this, a decrease in cholecystokinin binding sites occurs. This may have further consequences on signal transduction pathways. This decrease in cholecystokinin binding sites is associated with a decrease in the cholecystokinin-b receptor messenger RNA, and this is the first example of a decrease in messenger RNA levels in this experimental model.

Homolateral cerebrocortical increase of immediate early gene and neurotransmitter messenger RNAs after minimal cortical lesion: blockade by N-methyl-D-aspartate antagonist.

A small surgical lesion of the parietal cortex induces an increase in the expression of several messenger RNAs varying from 172 to 980% in the entire homolateral cerebral cortex, as detected by quantitative in situ hybridization histochemistry. The messenger RNAs encoding the immediate early genes of the leucine zipper family (c-fos, c-jun, jun-B), the Zinc finger family (zif268), the glucocorticoid receptor family (NGFI-B) and the interferon family (PC4) are increased within 2 h after the lesion and return to normal levels at 6 h. The messenger RNAs encoding cholecystokinin, neuropeptide Y, somatostatin and the synthetizing enzyme of the neurotransmitter GABA, glutamate decarboxylase, are elevated within one day and return to normal levels after six days. An intraperitoneal injection of the N-methyl-D-aspartate receptor antagonist dizocilpine maleate, 30 min before surgery, prevented either the induction of immediate early gene expression or the increase of neuropeptide and glutamate decarboxylase messenger RNA expression. This study demonstrates that a minimal cortical lesion induces extensive changes in gene expression and that the mechanism(s) leading to these changes involves the action of glutamate at the N-methyl-D-aspartate receptor. These modifications may be of importance in explaining diffuse changes not related to neuronal circuitry in several conditions.

NMDA receptor antagonist MK-801 down-regulates rat striatal proenkephalin and protachykinin mRNAs.

Using quantitative in situ hybridization, a significant decrease in expression of proenkephalin (27.1%) and of protachykinin mRNAs (20.0%) is observed in the rat caudate-putamen 14 days after daily intraperitoneal administration of N-methyl-D-aspartate receptor antagonist MK-801, 2 mg/kg.

Changes in striatal neuropeptides and GAD67 expression following a minimal cortical lesion.

We have analyzed the effects of a small cortical infarct which is known to induce dramatic changes in gene expression in the entire cerebral cortex, on the gene expression in the striatum, a target structure of cortical neurons. Striatal glutamic acid decarboxylase (GAD67) and enkephalin expressions were increased in the striatum ipsilateral to the lesion. Conversely, neuropeptide Y- and somatostatin-like immunoreactivity were decreased in the ipsilateral striatum and this decrease was only related to a decrease in the labeling of processes with no changes in the number of labeled neurons. A minimal cortical lesion may therefore induce changes in gene expression in a subcortical structure through hyperactivity of glutamatergic synaptic inputs. One should therefore remember these extensive and long-lasting effects when surgical manipulations are performed on rat brain for stereotaxic surgery and placement of electrodes or probes.

beta-Adrenoceptor studies. 6. Further investigations on the hybrid nature of the rat adipocyte beta-adrenoceptor.

The nature of the rat adipocyte beta-adrenoceptor was studied in further detail using a selected series of tolamol-type beta-adrenoceptor antagonists. Isoprenaline antagonism by these compounds was evaluated on adipocytes, right atrium, left atrium and left hemidiaphragm of the rat. Adipocyte pA2 values were corrected for binding to bovine serum albumin determined separately for each antagonist. A strong correlation between adipocyte and diaphragm pA2 values was found with those antagonists which had an identical N-substituent but a different substitution pattern in the phenoxypropanolamine ring. This relationship was absent with those compounds which had the same 2-methylphenoxypropanolamine moiety but a different N-substituent. With the latter antagonists, however, adipocyte pA2 values correlated significantly with the two cardiac pA2 values. These results support the idea that the interaction site of the rat adipocyte beta-adrenoceptor for the aromatic moiety of arylethanolamines and aryloxypropanolamines has beta 2-characteristics whereas the alkanolamine side-chain interaction site has a beta 1-nature. Finally, it was ascertained that the apparently dualistic (beta 1 and beta 2) character of the rat adipocyte adrenoceptor is not due to the presence of both a beta 1- and a beta 2-receptor population on the fat cell plasma membrane.

Capsaicin treatment induces muscarinic hyperreactivity in guinea pig trachea: a warning.

Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is a widely used tool for the depletion of neuropeptides from sensory C-fibres. Upon capsaicin treatment tachykinins are released, resulting in a variety of responses in the airways. We showed that after capsaicin (0.3 microM; 30 min) treatment of guinea pig tracheal smooth muscle preparations, the maximal contraction of the trachea after methacholine stimulation was strongly increased (capsaicin: 1.147 +/- 0.050 g vs. control: 0.717 +/- 0.047 g). This effect was completely nullified after pretreatment with capsazepine (2-[2-(4-chlorophenyl)ethyl-amino-thiocarbonyl]-7,8-dihydroxy-2,3, 4,5-tetrahydro-1H-2benzazepine; a vanilloid receptor antagonist) and YM38336 (a dual tachykinin NK1 and tachykinin NK2 receptor antagonist). Our results serve as a warning against using capsaicin as a putatively clean pharmacological tool to deplete the neuropeptides from pools on the C-fibres because we showed that capsaicin also strongly influences basal mechanisms in tracheal smooth muscle control.

Distribution of immediate early gene zif-268, c-fos, c-jun and jun-D mRNAs in the adult cat with special references to brain region related to vision.

The distribution of immediate early gene zif-268, c-fos, c-jun and jun-D mRNAs was investigated in the visual cortex, dorsal lateral geniculate nucleus and hippocampus of the adult cat brain with in situ hybridization. In area 17, zif-268, c-jun and jun-D were found predominantly in layers II-III and VI, while c-fos mRNA was abundant in layer VI. In area 18, the zif-268, c-fos and c-jun labelling pattern was identical to that of area 17, this was not true for jun-D. In area 19, only c-jun retained the lamination pattern of areas 17 and 18, while zif-268, c-fos and jun-D were homogeneously distributed. In the dorsal lateral geniculate nucleus, only c-fos and jun-D resulted in labelling. In the pyramidal layer of hippocampus, zif-268 was found in CA1-4, c-jun in CA1-3, and jun-D in CA2-4. In the dentate gyrus, c-jun was abundant, jun-D moderate and zif-268 faint. C-fos labelling was absent in the hippocampal formation.

The 3-methylcholanthrene-mimetic effect of 4,4'-dichlorobiphenyl-treatment on phenacetin-induced hepatic glutathione depletion and liver microsomal phenacetin O-deethylation in rats.

Both 4,4'-dichlorobiphenyl (4,4'-DCB) and 3-methylcholanthrene (3-MC) caused a substantial increase of phenacetin-induced hepatic glutathione (GSH) depletion, whereas phenobarbital (PB) had no effect, suggesting that 4,4'-DCB possesses cytochrome P-448 inducing activity. The O-deethylation of phenacetin by liver microsomes from control and PB- and 4,4'-DCB-treated rats showed biphasic Michaelis-Menten kinetics, in contrast to the monophasic course after pretreatment with 3-MC. Hepatic phenacetin levels indicated that in vivo interaction with only a high affinity site is involved in the O-deethylation of phenacetin. 4,4'-DCB and 3-MC caused marked increases in intrinsic clearance and extraction ratio of phenacetin, whereas control values were obtained after PB-treatment. Because of an absence of a spectral change at low phenacetin concentrations, it could not be demonstrated whether the observed differences in metabolism should be ascribed to a change in binding of phenacetin to cytochrome P-450. The results of this study indicate that after pretreatment with various enzyme inducers the phenacetin-induced hepatic GSH depletion strongly correlates with microsomal phenacetin O-deethylation. Further, these findings suggest a discrepancy between 4,4'-DCB and PB in cytochrome P-450 inducing activity, as 4,4'-DCB mimics 3-MC in the induction of phenacetin O-deethylase. The difference between 4,4'-DCB and PB is discussed in relation to the multiplicity and induction of cytochrome P-450 isoenzymes.

Effects of ozone on cellular ATP levels in rat and mouse alveolar macrophages.

Hydrogen peroxide (H2O2) is thought to be a major intermediate in the toxicity of ozone. In a previous study we demonstrated that ATP depletion may play an important role in the H2O2-induced inhibition of the phagocytic functions of alveolar macrophages. Ozone exposure can adversely affect the phagocytic capacities of alveolar macrophages. In the present study we investigated whether a decrease in the cellular ATP concentration may underly the effects of ozone on alveolar macrophages. Neither following single (6 and 12 h) exposure nor repeated (12 h/day for 3 and 7 days) exposures of mice or rats to 0.4 ppm ozone, were decreased levels of ATP found in the alveolar macrophages. In contrast, repeated exposures of mice for 7 days to ozone led to a significant increase (1.5-fold) in the ATP content of the alveolar macrophages. In vitro ozone exposure of rat and mouse alveolar macrophages also did not lead to a decrease in the cellular ATP concentration. These results showed that ATP depletion does not play a role in the toxicity mechanism of ozone for alveolar macrophages.

Changes in neuroreceptor function of tracheal smooth muscle following acute ozone exposure of guinea pigs.

We studied the effect of in vivo ozone inhalation (3 ppm, 2 h) on neuroreceptor function in guinea pig tracheal smooth muscle in vitro and the role of the epithelial layer in this process. Changes in smooth muscle tension after stimulation of the muscarinic- and beta-adrenergic receptor were recorded isometrically and stained tracheal tissue sections were histologically evaluated for changes in the epithelial and smooth muscle layer. Ozone exposure resulted in an increase in maximal contraction following stimulation of the muscarinic receptor, whereas pD2 values remained unchanged. After stimulation of the beta-adrenergic receptor no increase in maximal relaxation but only an increase in pD2 value was observed after correction for differences in precontraction level in control- and ozone-exposed situations. Mechanical removal of the epithelial layer resulted in a slight increase of the maximal contraction level after stimulation with methacholine in the control situation, whereas exposure to ozone resulted in a strong decrease of the maximal contraction level under these conditions. Histological stainings showed a slight and focal influx of neutrophilic granulocytes in the epithelial layer, submucosal layer and airway lumen after exposure to ozone. These data support the idea that ozone is able to increase the maximal degree of airway narrowing upon muscarinergic stimulation, i.e. a hyperreactivity response. The results also suggest that functionally altered epithelium plays an important role in the process of ozone-induced hyperreactivity, possibly linked with an early inflammatory response.

Glutathione pathway enzyme activities and the ozone sensitivity of lung cell populations derived from ozone exposed rats.

Rats were exposed to 1.5 +/- 0.1 mg ozone/m3 for 4 days and the activities of glucose-6-P dehydrogenase (G6PDH), glutathione reductase (GR) and glutathione peroxidase (GSHPx) were measured in the cytosolic fraction of lungs from exposed and control rats. Enzyme activities were also measured in isolated alveolar macrophages and type II cells. After ozone exposure enzyme activities, expressed per gram of protein, showed the following changes. G6PDH activity was increased (P less than 0.001) in the whole rat lung and showed the same tendency in isolated alveolar macrophages and type II cells. GR activity did not change significantly neither in whole lungs, nor in isolated cell populations. GSHPx activity was increased (P less than 0.001) in whole lung homogenates, and was also markedly increased in both alveolar macrophages (P less than 0.05) and type II cells (P less than 0.01) isolated from ozone-exposed rats. From these results it was concluded that biochemical changes measured in whole lung homogenates might reflect biochemical changes that occur within specific cell types. Furthermore, it was demonstrated, using an in vitro ozone exposure system, that lung cell populations isolated from ozone-exposed rats, in spite of their marked increase in GSHPx activity, did not show a decreased ozone sensitivity compared to cells from unexposed rats, as determined by trypan blue exclusion or phagocytosis. So an increase in GSHPx activity might not be related to an increased cellular resistance to ozone.

Protection against paracetamol-induced hepatotoxicity by acetylsalicylic acid in rats.

Acetylsalicylic acid (ASA) given simultaneously with paracetamol decreased paracetamol-induced hepatotoxicity (measured by plasma transaminase activities as well as histology) without any effect on glutathione depletion, indicating that ASA prevents a process (or processes) subsequent to the metabolic activation of paracetamol. Delayed treatment with ASA also reduced paracetamol-induced liver toxicity, suggesting that reduction of the absorption rate of paracetamol does not contribute essentially to the protection by ASA. Combinations of paracetamol and ASA may have potential use in the development of safer analgesic combinations containing paracetamol (or ASA).

Interaction of environmental chemicals with respiratory sensitization.

The acute effects of the inhalation of air polluting agents have been examined by many research groups in both animal models and human beings. For instance, it is evident that exposure to ozone has toxic effects and can lead to lung function disturbances. For this reason it is likely that individuals suffering from COPD or asthma are groups especially at risk with respect to the effects of ozone. The majority of studies dealing with effects of air pollutants on pulmonary allergy are restricted to IgE mediated allergy (type I allergy). Again for ozone, in animal models for type I allergy it has been demonstrated that exposure can affect the induction as well as the effector phase of this type of hyperimmune reaction (e.g. allergic asthma). Recently it has been demonstration in animal models that non-IgE mediated "asthma' T cells, and notably Th1 cells, may play a crucial role. In a murine model it was demonstrated that low molecular weight compounds can induce delayed type hypersensitivity-like reactions in the respiratory tract, and that these reactions are associated with the induction of airway hyperreactivity. Such compounds include toluene diisocyanate (TDI), to which immune responses can be readily mounted, and which can cause occupational asthma through its sensitizing capability, but to which IgE is only detected in a minority of patients suffering from TDI-associated asthma. Effects of air pollutants on Th1 responses in the respiratory tract have not been studied so far. We have demonstrated that ozone can inhibit resistance to a an intratracheal challenge with Listeria monocytogenes, indicating suppression of Th1 immune responses. In addition, we have shown that ozone exposure suppresses pulmonary delayed type hypersensitivity induced by small molecular weight compounds, as well as the tracheal hyperreactivity that is induced during the development of these immune responses, again supporting the hypothesis of suppression of Th1 responses by ozone exposure. These phenomena may be due to activation of Th2 cell dependent reactions that in turn lead to a downregulation of Th1 mediated immunity, or to a direct effect on Th1 cells or other cell types that are crucial for delayed type hypersensitivity and related airway hyperresponsiveness in this model. These data indicate that exposure to air pollutants may have differential consequences on different types of immune responses in the respiratory tract.

Acute exposure to ozone does not influence neuroreceptor density and sensitivity in guinea pig lung.

The effects of acute exposure of guinea pigs to 3 ppm of ozone for 2 h on the receptor density and sensitivity of the muscarinergic-, the histaminergic- and the beta-adrenergic receptor systems were studied, in order to provide more insight in the complex mechanisms underlying the well known ozone-induced changes in receptor functionality. The exposure to ozone did not change either the total amount of receptors present in lung tissue, nor the receptor sensitivity of the systems studied. Although no effects were observed, this does not yet fully exclude the receptor system for being a target of ozone exposure. The receptor function can be changed after exposure to ozone, e.g., the coupling with the G-protein can be influenced. Furthermore, the G-protein itself may have been altered or changes can occur at lower levels in the receptor signal transmission route leading to functional changes after stimulation of the receptor with an agonist.

Attenuation of acute lung injury by ozone inhalation--the effect of low level pre-exposure.

The attenuating influence of a pre-exposure of rats to a low concentration of ozone (O3) for 7 days on a subsequent O3 challenge was investigated. Effects of O3 were quantified by measuring indicators of lung permeability and inflammation in bronchoalveolar lavage fluid. The results suggest that pre-exposure to relatively low levels of O3 produces a diminished permeability response in lower airways of rats upon a following challenge with a higher level of O3. Extrapolated to human exposure situations, these data suggest that health effect evaluation of repeated exposure periods of enhanced O3 levels is rather complex and needs further investigation.

Dose-effect models for ozone exposure: tool for quantitative risk estimation.

Short-term ozone exposure causes lung function decrements, increased airway reactivity, airway inflammation, increased respiratory symptoms and hospital admissions. Exposure to long-term elevated ozone levels seems to be associated with reduced lung function (aging), increase of respiratory symptoms, exacerbation of asthma, and airway cell and tissue changes. Health risk caused by exposure to ozone has been evaluated mainly in a qualitative way by comparing ozone air quality data with health-based guidelines or standards. A preliminary approach to quantifying health risk from short-term exposure to oxidant air pollution has been taken by expert judgement, describing known or expected effects at specific levels of ozone. For quantitative assessment of the health impact of distinct ozone exposure conditions (acute, repeated daily, chronic) specific exposure-dose-response models are being developed which can be linked to human exposure data. Exposure-(dose-)response models using data from epidemiological, human-clinical and animal toxicity studies are presented.

Reduction by acetylsalicylic acid of paracetamol-induced hepatic glutathione depletion in rats treated with 4,4'-dichlorobiphenyl, phenobarbitone and pregnenolone-16-alpha-carbonitrile.

The role of enzyme induction in the reduction by acetylsalicylic acid (ASA) of paracetamol-induced hepatic glutathione (GSH) depletion has been studied in rats. Administration of an overdose of paracetamol to control rats resulted in an appreciable decrease of GSH concentration. Pretreatment with the enzyme inducers phenobarbitone, 3-methylcholanthrene (3-MC), pregnenolone-16-alpha-carbonitrile (PCN) and 4,4'-dichlorobiphenyl (4,4'-DCB) significantly potentiated the paracetamol-induced depletion of GSH. Simultaneous administration of an equimolar dose of ASA resulted in a reduction of the paracetamol-induced depletion of GSH in all instances except for those rats that were not pretreated and those given 3-MC. Benorylate, the ASA ester of paracetamol, depressed rat liver GSH to levels comparable to those produced by the combination of paracetamol and ASA. ASA itself caused only minor changes in liver GSH concentrations. The results demonstrate that ASA causes a diminution of paracetamol-induced GSH depletion in rats with phenobarbitone type of enzyme induction. Inhibition of the formation of the reactive metabolite of paracetamol or reduction of the absorption rate of paracetamol seem to be unlikely as mechanisms underlying the ASA-induced effect. An ASA-mediated effect via changes of the hepatic thiol status is proposed.