A PEGylated alternating copolymeric prodrug of sulfur dioxide with glutathione responsiveness for Irinotecan delivery.

In this study, an enhanced anticancer strategy combining the chemotherapy from antineoplastics with the oxidative damage from a sulfur dioxide (SO2) prodrug is presented. Based on the characteristics of a high glutathione (GSH) level in the tumor microenvironment, a novel GSH-responsive SO2 polymeric prodrug mPEG-b-P(PA-alt-GDNs) was designed and synthesized via a ring-opening alternating copolymerization and "click" reaction. The GSH-sensitive mechanism of the polymer was investigated in detail. Furthermore, Irinotecan was loaded into the polymeric prodrug nanoparticles by a self-assembly method with a drug loading content of 12.3 wt% and a loading efficiency of 42.2%. The drug-loaded nanoparticles showed a sensitive response to high concentrations of GSH in the tumor cells and rapidly released both Irinotecan and SO2. The depletion of GSH and the release of SO2 were supposed to increase the level of reactive oxygen species in the tumor cell, which, in combination with the released Irinotecan, exerted an enhanced anti-proliferative effect against HepG2 cells. Finally, Irinotecan-loaded nanoparticles exhibited a stronger antitumor effect than free antineoplastics in HepG2 cells. Thus, these results indicated that our polymeric prodrug SO2 is a promising candidate for chemotherapeutic drug delivery and would be a new weapon in anticancer treatment.

Reduction-responsive sulfur dioxide polymer prodrug nanoparticles loaded with irinotecan for combination osteosarcoma therapy.

Combination therapy can boost the therapeutic effectiveness of monotherapies by achieving synergy between therapeutic agents. Herein, a reduction-responsive sulfur dioxide (SO2) polymer prodrug was synthesized as a nanocarrier to load irinotecan (IRN) to be used in combination osteosarcoma therapy. The SO2 prodrug (denoted as mPEG-PLG (DNs)) was synthesized by coupling a small-molecule SO2 donor, N-(3-azidopropyl)-2,4-dinitrobenzenesulfonamide (AP-DNs), to the side chains of methoxy poly (ethylene glycol)-block-poly (γ-propargyl-L-glutamate) block copolymer. The mPEG-PLG (DNs) had the ability to self-assemble into micelles while simultaneously encapsulating IRN in aqueous media. The formed micelles led to enhanced SO2 and IRN release in reductive conditions. Using nile red as a model drug, the loaded micelles were efficiently internalized by cancer cells, demonstrated by confocal laser scanning microscopy and flow cytometry. The release of SO2 within nanoparticles (NPs) in tumor cells led to enhanced intracellular reactive oxygen species amounts together with induced oxidative destruction to cancer cells. Furthermore, the IRN-loaded SO2 polymer prodrug NPs mediated synergistic therapeutic effects against osteosarcoma cells, leading to improved biodistribution and enhanced tumor growth inhibition over control groups in a murine osteosarcoma model. Taken together, this work highlights the potential of SO2 polymer prodrugs as reduction-responsive nanocarriers to load chemotherapeutics for effective combination osteosarcoma therapy.

Cough and expiration reflexes elicited by inhaled irritant gases are intensified in ovalbumin-sensitized mice.

This study was designed to determine the effect of active sensitization with ovalbumin (Ova) on cough responses to inhaled irritant gases in mice. Conscious mice moved freely in a recording chamber, while the pressure change in the chamber and audio and video signals of the mouse movements were recorded simultaneously to measure the frequencies of cough reflex (CR) and expiration reflex (ER). To further verify the accuracy of cough analysis, the intrapleural pressure was also recorded by a telemetry sensor surgically implanted in the intrapleural space in a subgroup of mice. During the irritant gas inhalation challenge, sulfur dioxide (SO2; 200 and 400 ppm) or ammonia (NH3; 0.1% and 0.2%) was drawn into the chamber at a constant flow rate for 8 min. Ova sensitization and sham sensitization with vehicle (Veh) were performed over a 25-day period in separate groups of mice. Our results showed that 1) both SO2 and NH3 inhalation challenges increased CR and ER frequencies in a concentration-dependent manner before Ova sensitization; 2) the baseline CR frequency was significantly elevated after Ova sensitization, accompanied by pronounced airway inflammation; and 3) Ova sensitization also markedly augmented the responses of CR and ER to both SO2 and NH3 inhalation challenges; in sharp contrast, the cough responses did not change after sham sensitization in the Veh group. In conclusion, Ova sensitization caused distinct and lingering increases in baseline cough frequency, and also intensified both CR and ER responses to inhaled irritant gases, which probably resulted from an allergic inflammation-induced hypersensitivity of airway sensory nerves.

Inhaled sulfur dioxide causes pulmonary and systemic inflammation leading to fibrotic respiratory disease in a rat model of chemical-induced lung injury.

Inhalation of high concentrations of sulfur dioxide (SO2) affects the lungs and can be immediately dangerous to life. We examined the development of acute and long-term effects after exposure of SO2 in Sprague-Dawley rats, in particular inflammatory responses, airway hyperresponsiveness (AHR) and lung fibrosis. Animals were subjected to a single exposure of 2200ppm SO2 during 10min and treated with a single dose of the anti-inflammatory corticosteroid dexamethasone 1h following exposure. Exposed rats showed labored breathing, decreased body-weight and an acute inflammation with neutrophil and macrophage airway infiltrates 5h post exposure. The acute effects were characterized by bronchial damage restricted to the larger bronchi with widespread injured mucosal epithelial lining. Rats displayed hyperreactive airways 24h after exposure as indicated by increased methacholine-induced respiratory resistance. The inflammatory infiltrates remained in lung tissue for at least 14 days but at the late time-point the dominating granulocyte types had changed from neutrophils to eosinophils. Analysis of immunoregulatory and pro-inflammatory cytokines in serum and airways implicated mixed macrophage phenotypes (M1/M2) and T helper cell activation of both TH1 and TH2 subtypes. Increased expression of the pro-fibrotic cytokine TGFß1 was detected in airways 24h post exposure and remained increased at the late time-points (14 and 28 days). The histopathology analysis confirmed a significant collagen deposition 14 days post exposure. Treatment with dexamethasone significantly counteracted the acute inflammatory response but was insufficient for complete protection against SO2-induced adverse effects, i.e. treatment only provided partial protection against AHR and the long-term fibrosis.

Synergistic effects of particulate matter (PM10) and SO2 on human non-small cell lung cancer A549 via ROS-mediated NF-κB activation.

Since a real atmospheric scenario usually represents a system involving multiple pollutants, air pollution studies typically focused on describing adverse effects associated with exposure to individual pollutants cannot reflect actual health risk. Particulate matter (PM10) and sulfur dioxide (SO2) are two major pollutants derived from coal combustion processes and co-existing in coal-smoke air pollution, but their potentially synergistic toxicity remains elusive thus far. In this study, we investigated the cytotoxic responses of PM10 and SO2, singly and in binary mixtures, using human non-small cell lung cancer A549 cells, followed by clarifying the possible mechanisms for their interaction. The results indicated that the concomitant treatment of PM10 and SO2 at low concentrations led to synergistic injury in terms of cell survival and apoptosis occurrence, while PM10 and SO2 alone at the same concentrations did not cause damage to the cells. Also, radical oxygen species (ROS) production followed by nuclear factor kappa B (NF-κB) activation was involved in the above synergistic cytotoxicity, which was confirmed by the repression of the actions by an ROS inhibitor (NAC). This implies that assessment of health risk should consider the interactions between ambient PM and gaseous copollutants.

SO(2) inhalation modulates the expression of pro-inflammatory and pro-apoptotic genes in rat heart and lung.

SO(2) is a common air pollutant, and human exposure to SO(2) has become increasingly widespread due to the combustion of fossil fuels. The epidemiological studies have linked SO(2) exposure not only with many respiratory responses, but also with cardiovascular diseases. Also, its possible toxicity has been implicated by determining oxidative stress, DNA damage and membrane channel alteration in rat heart and lung. However, its detailed mechanisms remain unclear. In the present study, rats were treated with 7, 14 and 28 mg/m(3) SO(2) for 6h/day for 7 days, and the mRNA levels of TNF-α, IL-1ß, iNOS, ICAM-1, Bax and Bcl-2 and subsequent insults were determined in the heart and lung. The results indicate that SO(2) inhalation markedly elevated TNF-α and IL-1ß mRNA levels and secretions, enhanced iNOS and ICAM-1 mRNA levels and the ratio of Bax/Bcl-2 in a concentration-dependent manner, and induced occurrence of apoptosis. This suggests that SO(2) inhalation induced an inflammatory response and subsequent insults via modulating pro-inflammatory and pro-apoptotic genes in the heart and lung, which contributed to the increased risk of respiratory and cardiovascular diseases.

SO2 inhalation modulates the expression of apoptosis-related genes in rat hippocampus via its derivatives in vivo.

The possible neurotoxicity of SO(2) has been implicated by determining morphological change, oxidative stress, DNA damage and membrane channel alteration in previous studies, however, its detailed mechanisms remain unclear. In the present study, we investigated SO(2) inhalation-induced effects on the transcription and translation of several apoptosis-related genes (p53, bax, bcl-2, c-fos, and c-jun) in rat hippocampus, using real-time RT-PCR analysis and western blotting technique, respectively. The results demonstrate that SO(2) statistically increased p53 expression and the ratio of bax to bcl-2 in a concentration-dependent manner. Also, mRNA and protein levels of c-fos and c-jun significantly elevated in proportion to exposure concentration. Then, we treated primary cultured hippocampal neurons with SO(2) derivatives (bisulfite and sulfite, 3:1 M/M), and examined mRNA levels of above genes. The results show that P53, c-fos, c-jun mRNA expression and the ratio of bax to bcl-2 augmented as functions of SO(2) derivative concentration and exposure time, and confirm that SO(2) affected the transcription and translation process of apoptosis-related genes in central nervous system via its derivatives in vivo. The present data provide further evidence for SO(2)-caused neurological insults, and imply that two major pathways associated with p53 and AP-1 might play important roles in the pathogenesis.

Expression of oncogenes and tumor suppressor genes in lungs of rats exposed to sulfur dioxide and benzo(a)pyrene.

Concurrent exposure to SO(2) and benzo(a)pyrene (B(a)P) resulted in an increased incidence of lung tumors in rodents compared to exposure to B(a)P alone. A synergistic effect on the expression of c-fos and c-jun between SO(2) and B(a)P was observed in lungs after SO(2) and B(a)P exposure. However, tumorigenesis occurs by multiple events that may involve the activation of more than one oncogene, as well as the functional loss of the tumor suppressor gene. In order to further investigate the interactions between SO(2) and B(a)P, male Wistar rats were exposed via intratracheal instillation of B(a)P (3 mg) or SO(2) (56 mg/m(3)) inhalation, alone or together. The mRNA and protein levels of oncogenes (c-myc and H-ras) and tumor suppressor genes (p53, p16, and Rb) were analyzed in lungs by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot, respectively. The results showed that all treatments increased mRNA and protein expression levels of c-myc, H-ras, and p53, and reduced expression levels of p16 and Rb. In general, the combination of SO(2) and B(a)P was more effective in influencing these expression levels than either agent alone, except for H-ras expression. These findings indicate that multiple cell cycle regulatory proteins play key roles in the toxicity of SO(2) and B(a)P. It might involve the activation of c-fos, c-jun, c-myc, and p53. And the p16-Rb pathway might also participate in the progress. Elucidating the expression patterns of those factors after SO(2) and B(a)P exposure may be critical to understanding the mechanisms of SO(2) cocarcinogenesis and helpful for therapeutic intervention.

Vasodilatation of sulfur dioxide derivatives and signal transduction.

The vasodilatation of sulfur dioxide (SO(2)) derivatives on the rat thoracic aortic rings and its cell signal transduction pathway were studied. The levels of cAMP, cGMP, PGI(2), TXA(2) and activities of PKA and adenylyl cyclase (AC) in the rings exposed to SO(2) derivatives were determined. The results indicated that SO(2) derivatives could dose-dependently relax the isolated rat aorta rings with or without endothelium precontracted by NE, no difference was found between the rings with and without endothelium; Levels of cAMP, PGI(2), AC activity and PKA activity in the aortic rings were significantly increased by the derivatives in a dose-related way; No change of cGMP and TXA(2) levels in rings was observed; cAMP/cGMP and PGI(2)/TXA(2) ratios were increased significantly by the SO(2) derivatives. These results led to the conclusions that SO(2) derivatives might cause the endothelium-independent vasorelaxation effect, and the vasorelaxation was mediated in partly by the signal transduction pathway of PGI(2)-AC-cAMP-PKA.

The spontaneously hypertensive rat: an experimental model of sulfur dioxide-induced airways disease.

Chronic obstructive pulmonary disease (COPD) is characterized by airway obstruction, inflammation, and mucus hypersecretion, features that are common in bronchitis, emphysema, and often asthma. However, current rodent models do not reflect this human disease. Because genetically predisposed spontaneously hypertensive (SH) rats display phenotypes such as systemic inflammation, hypercoagulation, oxidative stress, and suppressed immune function that are also apparent in COPD patients, we hypothesized that SH rat may offer a better model of experimental bronchitis. We, therefore, exposed SH and commonly used Sprague Dawley (SD) rats (male, 13- to 15-weeks old) to 0, 250, or 350 ppm sulfur dioxide (SO(2)), 5 h/day for 4 consecutive days to induce airway injury. SO(2) caused dose-dependent changes in breathing parameters in both strains with SH rats being slightly more affected than SD rats. Increases in bronchoalveolar lavage fluid (BALF) total cells and neutrophilic inflammation were dose dependent and significantly greater in SH than in SD rats. The recovery was incomplete at 4 days following SO(2) exposure in SH rats. Pulmonary protein leakage was modest in either strain, but lactate dehydrogenase and N-acetyl glucosaminidase activity were increased in BALF of SH rats. Airway pathology and morphometric evaluation of mucin demonstrated significantly greater impact of SO(2) in SH than in SD rats. Baseline differences in lung gene expression pattern suggested marked immune dysregulation, oxidative stress, impairment of cell signaling, and fatty acid metabolism in SH rats. SO(2) effects on these genes were more pronounced in SH than in SD rats. Thus, SO(2) exposure in SH rats may yield a relevant experimental model of bronchitis.

DNA damage in mice treated with sulfur dioxide by inhalation.

Sulfur dioxide (SO2) is a ubiquitous air pollutant produced by the burning of fossil fuels. In this study, single-cell gel electrophoresis (the Comet assay) was used to evaluate the DNA damage produced by inhalation exposure of mice to SO2. Male and female mice were housed in exposure chambers and treated with 14.00 +/- 1.25, 28.00 +/- 1.98, 56.00 +/- 3.11, and 112.00 +/- 3.69 mg/m3 SO2 for 6 hr/day for 7 days, while control groups were exposed to filtered air. Comet assays were performed on blood lymphocytes and cells from the brain, lung, liver, spleen, kidney, intestine, and testicles of the animals. SO2 caused significant, dose-dependent increases in DNA damage, as measured by Olive tail moment, in all the cell types analyzed from both sexes of mice. The results indicate that inhalation exposure to SO2 damages the DNA of multiple organs in addition to the lung, and suggests that this damage could result in mutation, cancer, and other diseases related to DNA damage. Further work will be required to understand the ultimate toxicological significance of this damage. These data also suggest that detecting DNA damage in blood lymphocytes, using the Comet assay, may serve as a useful tool for evaluating the impact of pulmonary SO2 exposure in human biomonitoring studies.

Effect of sulfur dioxide inhalation on cytokine levels in lungs and serum of mice.

In order to elucidate the immunotoxic mechanism exerted by sulfur dioxide (SO(2)), we investigated the effect of SO(2), a major air pollutant, on the cytokine levels in lungs and serum of male mice. Levels of interlukin-6(IL-6), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta1 (TGF-beta1) in lungs and serum from male mice exposed to SO(2) at various concentrations were measured by the enzyme-linked immunosorbent assay. Sixty Kunming albino male mice were divided randomly into six equal groups: three groups exposed to SO(2) (14.00 +/- 1.25, 28.00 +/- 1.98, and 56.00 +/- 3.11 mg/m(3), which are 5 +/- 0.45, 10 +/- 0.71, and 20 +/- 1.11 ppm) and their respective control groups. The results were as follows: (1) For lung tissues of male mice, exposure to SO(2) at 14 mg/m(3) (5 ppm) caused statistically significant increase of levels of IL-6 and TNF-alpha (p < .05) compared with the control group; exposure at 28 mg/m(3) (10 ppm) caused a statistically highly significant increase of level of IL-6 (p < .01) and a significant increase of TNF-alpha (p < .05); and exposure at 56 mg/m(3) (20 ppm) caused no any significant increase of levels of IL-6 and TNF-alpha. SO(2) at all concentrations tested could not cause significant change of level of TGF-beta1 in lungs. (2) For serum from male mice, after exposure to SO(2) at 14 mg/m(3) (5 ppm), the level of TNF-a was significantly increased (p < .05) compared with the control group, but the changes of levels of IL-6 and TGF-beta1 were not significant. After exposure to SO(2) at 28 mg/m(3) (10 ppm) and 56 mg/m(3) (20 ppm), levels of IL-6 and TNF-alpha were increased nonsignificantly, but the level of TGF-beta1 was decreased nonsignificantly. These results imply that inflammation reaction could be induced in lung tissue by SO(2) inhalation and the inflammation reaction might relate to these cytokines. And determination of cytokines in lung may be more valuable than in serum when lung injury caused by SO(2).

Effect of sulfur dioxide inhalation on the glutathione redox system in mice and protective role of sea buckthorn seed oil.

This study investigated the effects of sulfur dioxide (SO2) inhalation and protection by sea buckthorn seed oil from oxidative damage caused by SO2 in male Kunming-strain mice. One approach was set up to study the effects of SO2 inhalation on changes of the mice antioxidant defense system. SO2 at different concentrations (22 +/- 2, 64 +/- 3, and 148 +/- 23 mg/m3) was administered to animals in treatment groups for 7 days, 6 h per day, while control groups were exposed to filtered air under the same condition. The activities of glutathione-S-transferase (GST) and glucose-6-phosphate dehydrogenase (G6PD) and the contents of reduced glutathione (GSH) in brain, lung, heart, liver, and kidney of mice were measured. In the case of inhalation of a SO2 concentration of 148 +/- 23 mg/m3, the activities of GST and G6PD and contents of GSH in the brain, lung, heart, liver, and kidney were significantly decreased. Dose-dependent relations were found between various SO2-exposed concentrations and the activities of GST and G6PD and the content of GSH. Meanwhile another approach was taken to determine whether sea buckthorn seed oil could maintain the glutathione redox system and prevent the oxidative damage of lung induced by SO2. In groups given a high dosage (6 or 8 ml/kg) intraperitoneally, the level of TBARS (thiobarbituric acid-reactive substances) was decreased significantly (p < 0.05) by the injection of sea buckthorn seed oil, and the activity of GST was increased significantly (p < 0.05). Overall GST activity and TBARS level exhibited a significant negative correlation (r = 0.891, p < 0.05). The observations showed that SO2 inhalation resulted in a significant change in the glutathione redox system and indicated that sea buckthorn seed oil could contribute to the antioxidant effects in the case of SO2 exposure.

[Induction effects of sulfur dioxide inhalation on chromosomal aberrations in mouse bone marrow cells].

OBJECTIVE: To investigate the induction effects of sulfur dioxide (SO(2)) inhalation on chromosomal aberrations (CA) in mouse bone marrow cells. METHODS: The mice were treated with SO(2) for 4 h/day x 7 days at various concentrations of SO(2), then mitotic indices and CA in the bone marrow cells were analyzed. RESULTS: SO(2) increase the frequencies of CA and aberrant cells in mouse bone marrow cells in dose-dependent manner. The frequencies (%) of the aberrant cells in mouse bone marrow cells induced by SO(2) at concentrations of 0, 14, 28, 56 and 84 mg/m(3) were 1.81, 3.00, 3.58, 4.26 and 4.86, respectively. SO(2) at low concentrations induced only chromatid-type CA, but at high concentrations it induced both chromatid-type and chromosome-type CA. SO(2) inhalation decreased the mitotic indices of the bone marrow cells. CONCLUSIONS: SO(2) inhalation may inhibit mitoses and increase CA frequencies of the bone marrow cells; therefore, it is a clastogenetic and genotoxic agent. It implies that long time exposure of SO(2) pollutant at low concentration in air may be a potential risk to induce damage of cytogenetic material in humans.

[Micronuclei induced by chronic inhalation of SO2 in mouse bone marrow cells].

In the chronic inhalation experiment of sulfur dioxide(SO2), micronuclei(MN) frequencies in the polychromatophilic erythroblasts(PCE) of mouse bone marrow and the frequencies of cells with MN were significantly increased in dose-dependent manner. There was a significant difference between the male and the female animals. The results also showed that SO2 inhibited urethone-induced MN formation. These results furtherly confirm that SO2 inhalation was a clastogenic and genotoxic agent to mammalian cells, and the combined effects of SO2 and other mutagens are complex.

Micronuclei induced by sulfur dioxide inhalation in mouse bone-marrow cells in vivo.

Induction effects of sulfur dioxide (SO2) inhalation on micronuclei (MN) formation in the polychromatic erythrocytes (PCE) of mouse bone marrow were studied in vivo. It was shown that SO2 inhalation caused an increase of MN frequencies in the PCE cells in a dose-dependent manner. These results from male and female mice were very similar; no sex difference was observed. These results imply that SO2 is a clastogenic and genotoxic agent.

Effect of sulfur dioxide inhalation on erythrocyte antioxidant status and lipid peroxidation in experimental diabetes.

The effect of sulfur dioxide (SO(2) ) on red cell antioxidant status and lipid peroxidation was examined in this research. Forty healthy male albino rats, aged three months, were divided into four equal groups: Control (C), SO(2) +C (CSO(2) ), diabetic (D) and SO(2) +D (DSO(2) ). Experimental diabetes mellitus was induced by i.v injection of alloxan with a dose of 50 mg/kg body weight. Ten ppm SO(2) was administered to the animals of SO(2) exposed groups in an exposure chamber for one hr/day x 7 days/wk x 6wks while other groups were exposed to filtered air in the same condition. SO(2) exposure, while markedly decreasing Cu, Zn-Superoxide dismutase (Cu, Zn-SOD) activity, significantly increased glutathione peroxidase (GSH-Px), catalase (CAT), glutathione (GSH) and glutathione-s-transferase (GST) activities and TBARS values in CSO(2) and DSO(2) groups compared with their respective control groups. From these results, it could be concluded that adaptative changes occurred in antioxidant systems that may counteract the free radical effect of SO(2) in the experimental groups.

Pulmonary endocrine cell hyperplasia and papilloma in rats induced by intratracheal injections of extract from particulate air pollutants.

We investigated the effect of intratracheal injections of an extract of suspended particulate matter (SPM) obtained from the urban ambient air of Tokyo, upon the development of proliferative lesions of pulmonary endocrine cells (PECs) in the rat. We also examined the modification effects of nitrogen dioxide, sulfur dioxide, or both of them on the PEC lesions. Male F344 rats were divided into six experimental groups of 5 animals each. Twenty animals were treated with intratracheal instillations of SPM admixed with carbon once a week for 4 weeks with or without additional gaseous exposure (6 ppm nitrogen dioxide or 4 ppm sulfur dioxide) 16 hrs a day for 11 months. Five animals were given intratracheal injections of carbon suspended in saline and the other five were untreated. The subcardiac lobes of the right lung were fixed with 4% paraformaldehyde, and embedded in paraffin. PEC hyperplasias and papillomas were counted in 200 serial sections, 4 microns thick. The average incidences of PEC hyperplasia in the untreated animals and in those treated with carbon were 194 and 200/cm3, respectively. The average incidences of PEC hyperplasia in the animals exposed to SPM tar only, SPM tar plus nitrogen dioxide and sulfur dioxide, SPM tar with nitrogen dioxide and SPM tar with sulfur dioxide were 376, 378, 372 and 349/cm3, respectively. These were significantly higher than the levels of the control animals, and additional gaseous stimuli had no effect on the incidence of PEC hyperplasia. Besides PEC hyperplasia, a few PEC papillomas were found in the animals treated with SPM tar, regardless of gaseous exposure, but in the control animals no papilloma was evident. Thus, compounds in airborne particulates are considered to be responsible for the development of PEC hyperplasias and papillomas.

Cell response in bronchoalveolar lavage fluid after sulfur dioxide exposure.

Environmental chamber exposure and bronchoalveolar lavage (BAL) were used to study the dose-response relationship between short-term exposure to sulfur dioxide (SO2) and inflammatory reactions in the human lung as reflected in BAL fluid. Healthy subjects were exposed to 10, 13, 20, or 30 mg/m3 for 20 min. BAL was performed several weeks preexposure and 24 h postexposure. Mast cells, lymphocytes, lysozyme positive macrophages, and the total number of macrophages were significantly increased after SO2 exposure. A dose-dependent increase in the cell response in BAL fluid was observed after exposure to 10-20 mg/m3, but no further increase was detected after 30 mg/m3. Inflammatory cell response was found in BAL fluid at SO2 levels that occur in industrial indoor environments worldwide, and cell response to SO2 was also seen below the short-term exposure limit of Sweden and many other countries (13 mg/m3).