Pulmonary toxicity following acute coexposures to diesel particulate matter and α-quartz crystalline silica in the Sprague-Dawley rat.

The effects of acute pulmonary coexposures to silica and diesel particulate matter (DPM), which may occur in various mining operations, were investigated in vivo. Rats were exposed by intratracheal instillation (IT) to silica (50 or 233 µg), DPM (7.89 or 50 µg) or silica and DPM combined in phosphate-buffered saline (PBS) or to PBS alone (control). At one day, one week, one month, two months and three months postexposure bronchoalveolar lavage and histopathology were performed to assess lung injury, inflammation and immune response. While higher doses of silica caused inflammation and injury at all time points, DPM exposure alone did not. DPM (50 µg) combined with silica (233 µg) increased inflammation at one week and one-month postexposure and caused an increase in the incidence of fibrosis at one month compared with exposure to silica alone. To assess susceptibility to lung infection following coexposure, rats were exposed by IT to 233 µg silica, 50 µg DPM, a combination of the two or PBS control one week before intratracheal inoculation with 5 × 105 Listeria monocytogenes. At 1, 3, 5, 7 and 14 days following infection, pulmonary immune response and bacterial clearance from the lung were evaluated. Coexposure to DPM and silica did not alter bacterial clearance from the lung compared to control. Although DPM and silica coexposure did not alter pulmonary susceptibility to infection in this model, the study showed that noninflammatory doses of DPM had the capacity to increase silica-induced lung injury, inflammation and onset/incidence of fibrosis.

Pulmonary toxicity of indium-tin oxide production facility particles in rats.

Indium-tin oxide (ITO) is used to make transparent conductive coatings for touch-screen and liquid crystal display electronics. Occupational exposures to potentially toxic particles generated during ITO production have increased in recent years as the demand for consumer electronics continues to rise. Previous studies have demonstrated cytotoxicity in vitro and animal models have shown pulmonary inflammation and injury in response to various indium-containing particles. In humans, pulmonary alveolar proteinosis (PAP) and fibrotic interstitial lung disease have been observed in ITO facility workers. However, which indium materials or specific processes in the workplace may be the most toxic to workers is unknown. Here we examined the pulmonary toxicity of three different particle samples that represent real-life worker exposures, as they were collected at various production stages throughout an ITO facility. Indium oxide (In2O3), sintered ITO (SITO) and ventilation dust (VD) particles each caused pulmonary inflammation and damage in rats over a time course (1, 7 and 90 days post-intratracheal instillation), but SITO and VD appeared to induce greater toxicity in rat lungs than In2O3 at a dose of 1 mg per rat. Downstream pathological changes such as PAP and fibrosis were observed in response to all three particles 90 days after treatment, with a trend towards greatest severity in animals exposed to VD when comparing animals that received the same dose. These findings may inform workplace exposure reduction efforts and provide a better understanding of the pathogenesis of an emerging occupational health issue.

Reactive oxygen species- and nitric oxide-mediated lung inflammation and mitochondrial dysfunction in wild-type and iNOS-deficient mice exposed to diesel exhaust particles.

Pulmonary responses to diesel exhaust particles (DEP) exposure are mediated through enhanced production of reactive oxygen species (ROS) and nitric oxide (NO) by alveolar macrophages (AM). The current study examined the differential roles of ROS and NO in DEP-induced lung injury using C57B/6J wild-type (WT) and inducible NO synthase knockout (iNOS KO) mice. Mice exposed by pharyngeal aspiration to DEP or carbon black particles (CB) (35 mg/kg) showed an inflammatory profile that included neutrophil infiltration, increased lactate dehydrogenase (LDH) activity, and elevated albumin content in bronchoalveolar lavage fluid (BALF) at 1, 3, and 7 d postexposure. The organic extract of DEP (DEPE) did not induce an inflammatory response. Comparing WT to iNOS KO mice, the results show that NO enhanced DEP-induced neutrophils infiltration and plasma albumin content in BALF and upregulated the production of the pro-inflammatory cytokine interleukin 12 (IL-12) by AM. DEP-exposed AM from iNOS KO mice displayed diminished production of IL-12 and, in response to ex vivo lipopolysaccharide (LPS) challenge, decreased production of IL-12 but increased production of IL-10 when compared to cells from WT mice. DEP, CB, but not DEPE, induced DNA damage and mitochondria dysfunction in AM, however, that is independent of cellular production of NO. These results demonstrate that DEP-induced immune/inflammatory responses in mice are regulated by both ROS- and NO-mediated pathways. NO did not affect ROS-mediated mitochondrial dysfunction and DNA damage but upregulated IL-12 and provided a counterbalance to the ROS-mediated adaptive stress response that downregulates IL-12 and upregulates IL-10.

Cooperation of the inducible nitric oxide synthase and cytochrome P450 1A1 in mediating lung inflammation and mutagenicity induced by diesel exhaust particles.

Diesel exhaust particles (DEPs) have been shown to activate oxidant generation by alveolar macrophages (AMs), alter xenobiotic metabolic pathways, and modify the balance of pro-antiinflammatory cytokines. In this study we investigated the role of nitric oxide (NO) in DEP-mediated and DEP organic extract (DEPE) -mediated inflammatory responses and evaluated the interaction of inducible NO synthase (iNOS) and cytochrome P450 1A1 (CYP1A1). Male Sprague-Dawley rats were intratracheally (IT) instilled with saline, DEPs (35 mg/kg), or DEPEs (equivalent to 35 mg DEP/kg), with or without further treatment with an iNOS inhibitor, aminoguanidine (AG; 100 mg/kg), by intraperitoneal injection 30 min before and 3, 6, and 9 hr after IT exposure. At 1 day postexposure, both DEPs and DEPEs induced iNOS expression and NO production by AMs. AG significantly lowered DEP- and DEPE-induced iNOS activity but not the protein level while attenuating DEPE- but not DEP-mediated pulmonary inflammation, airway damage, and oxidant generation by AMs. DEP or DEPE exposure resulted in elevated secretion of both interleukin (IL) -12 and IL-10 by AMs. AG significantly reduced DEP- and DEPE-activated AMs in IL-12 production. In comparison, AG inhibited IL-10 production by DEPE-exposed AMs but markedly increased its production by DEP-exposed AMs, suggesting that NO differentially regulates the pro- and antiinflammatory cytokine balance in the lung. Both DEPs and DEPEs induced CYP1A1 expression. AG strongly inhibited CYP1A1 activity and lung S9 activity-dependent 2-aminoanthracene mutagenicity. These studies show that NO plays a major role in DEPE-induced lung inflammation and CYP-dependent mutagen activation but a lesser role in particulate-induced inflammatory damage.

Exposure of brown Norway rats to diesel exhaust particles prior to ovalbumin (OVA) sensitization elicits IgE adjuvant activity but attenuates OVA-induced airway inflammation.

Exposure to diesel exhaust particles (DEP) during the sensitization process has been shown to increase antigen-specific IgE production and aggravate allergic airway inflammation in human and animal models. In this study, we evaluated the effect of short-term DEP exposure on ovalbumin (OVA)-mediated responses using a post-sensitization model. Brown Norway rats were first exposed to filtered air or DEP (20.6 +/- 2.7 mg/m3) for 4 h/day for five consecutive days. One day after the final air or DEP exposure (day 1), rats were sensitized with aerosolized OVA (40.5 +/- 6.3 mg/m3), and then again on days 8 and 15, challenged with OVA on day 29, and sacrificed on days 9 or 30, 24 h after the second OVA exposure or the final OVA challenge, respectively. Control animals received aerosolized saline instead of OVA. DEP were shown to elicit an adjuvant effect on the production of antigen-specific IgE and IgG on day 30. At both time points, no significant airway inflammatory responses and lung injury were found for DEP exposure alone. However, the OVA-induced inflammatory cell infiltration, acellular lactate dehydrogenase activity and albumin content in bronchoalveolar lavage (BAL) fluid, and numbers of T cells and their CD4+ and CD8+ subsets in lung-draining lymph nodes were markedly reduced by DEP on day 30 compared with the air-plus-OVA exposure group. The OVA-induced nitric oxide (NO) in the BAL fluid and production of NO, interleukin (IL)-10, and IL-12 by alveolar macrophages (AM) were also significantly lowered by DEP on day 30 as well as day 9. DEP or OVA alone decreased intracellular glutathione (GSH) in AM and lymphocytes on days 9 and 30. The combined DEP and OVA exposure resulted in further depletion of GSH in both cell types. These results show that short-term DEP exposure prior to sensitization had a delayed effect on enhancement of the sensitization in terms of allergen-specific IgE and IgG production, but caused an attenuation of the allergen-induced airway inflammatory responses.

Effect of diesel exhaust particles on allergic reactions and airway responsiveness in ovalbumin-sensitized brown Norway rats.

We have previously demonstrated that exposure to diesel exhaust particles (DEP) prior to ovalbumin (OVA) sensitization in rats reduced OVA-induced airway inflammation. In the present study, Brown Norway rats were first sensitized to OVA (42.3 +/- 5.7 mg/m3) for 30 min on days 1, 8, and 15, then exposed to filtered air or DEP (22.7 +/- 2.5 mg/m3) for 4 h/day on days 24-28, and challenged with OVA on day 29. Airway responsiveness was examined on day 30, and animals were sacrificed on day 31. Ovalbumin sensitization and challenge resulted in a significant infiltration of neutrophils, lymphocytes, and eosinophils into the lung, elevated presence of CD4+ and CD8+ T lymphocytes in lung draining lymph nodes, and increased production of serum OVA-specific immunoglobulin (Ig)E and IgG. Diesel exhaust particles pre-exposure augmented OVA-induced production of allergen-specific IgE and IgG and pulmonary inflammation characterized by marked increases in T lymphocytes and infiltration of eosinophils after OVA challenge, whereas DEP alone did not have these effects. Although OVA-sensitized rats showed modest response to methacholine challenge, it was the combined DEP and OVA exposure that produced significant airway hyperresponsiveness in this animal model. The effect of DEP pre-exposure on OVA-induced immune responses correlated with an interactive effect of DEP with OVA on increased production of reactive oxygen species (ROS) and nitric oxide (NO) by alveolar macrophages (AM) and alveolar type II (ATII) cells, NO levels in bronchoalveolar lavage fluid, the induction of inducible NO synthase expression in AM and ATII cells, and a depletion of total intracellular glutathione (GSH) in AM and lymphocytes. These results show that DEP pre-exposure exacerbates the allergic responses to the subsequent challenge with OVA in OVA-sensitized rats. This DEP effect may be, at least partially, attributed to the elevated generation of ROS in AM and ATII cells, a depletion of GSH in AM and lymphocytes, and an increase in AM and ATII cell production of NO.

Sustained effect of inhaled diesel exhaust particles on T-lymphocyte-mediated immune responses against Listeria monocytogenes.

Studies have shown that exposure to diesel exhaust particles (DEP) suppresses pulmonary host defense against bacterial infection. The present study was carried out to characterize whether DEP exposure exerts a sustained effect in which inhaled DEP increase the susceptibility of the lung to bacterial infection occurring at a later time. Brown Norway rats were exposed to filtered air or DEP by inhalation at a dose of 21.2 +/- 2.3 mg/m3, 4 h/day for 5 days, and intratracheally instilled with saline or 100,000 Listeria monocytogenes (Listeria) 7 days after the final DEP exposure. Bacterial growth and cellular responses to DEP and Listeria exposures were examined at 3 and 7 days post-infection. The results showed that inhaled DEP prolonged the growth of bacteria, administered 7 days post DEP exposure, in the lung as compared to the air-exposed controls. Pulmonary responses to Listeria infection were characterized by increased production of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-12, and IL-10 by alveolar macrophages (AM) and increased presence of T lymphocytes and their CD4+ and CD8+ subsets in lung draining lymph nodes that secreted elevated levels of IL-2, IL-6, IL-10, and interferon (IFN)-gamma. Diesel exhaust particles were found to inhibit Listeria-induced production of IL-1beta and TNF-alpha, which are responsible for the innate immunity, and IL-12, which initiates the development of T helper (Th)1 responses, but enhance Listeria-induced AM production of IL-10, which prolongs Listeria survival in these phagocytes. The dual action of DEP on AM production of IL-12 and IL-10 correlated with an inhibition of the development of bacteria-specific T lymphocytes by DEP. Cytokine production by lymphocytes from DEP- and Listeria-exposed rats showed a marked decrease in the production of IL-2, IL-10, and IFN-gamma compared to Listeria infection alone, suggesting either that DEP inhibit the production of cytokines by lymphocytes or that these lymphocytes contained T-cell subsets that are different from those of Listeria infection alone and less effective in mediating Th1 immune responses. This study demonstrates that inhaled DEP, after a 7-day resting period, increase the susceptibility of the lung to bacterial infection occurring at a later time by inhibiting macrophage immune function and suppressing the development of T-cell-mediated immune responses. The results support the epidemiological observations that exposure to DEP may be responsible for the pulmonary health effects on humans.

Time course of gene expression of inflammatory mediators in rat lung after diesel exhaust particle exposure.

Diesel exhaust particles (DEPs) at three concentrations (5, 35, and 50 mg/kg body weight) were instilled into rats intratracheally. We studied gene expression at 1, 7, and 30 days postexposure in cells obtained by bronchoalveolar lavage (BAL) and in lung tissue. Using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), we measured the mRNA levels of eight genes [interleukin (IL)-1beta, IL-6, IL-10, iNOS (inducible nitric oxide synthase), MCP-1 (monocyte chemoattractant protein-1), MIP-2 (macrophage inflammatory protein-2), TGF-beta1 (transforming growth factor-beta1), and TNF-alpha (tumor necrosis factor-alpha )] in BAL cells and four genes [IL-6, ICAM-1 (intercellular adhesion molecule-1), GM-CSF (granulocyte/macrophage-colony stimulating factor), and RANTES (regulated upon activation normal T cell expressed and secreted)] in lung tissue. In BAL cells on day 1, high-dose exposure induced a significant up-regulation of IL-1beta, iNOS, MCP-1, and MIP-2 but no change in IL-6, IL-10, TGF-beta1, and TNF-alpha mRNA levels. There was no change in the mRNA levels of IL-6, RANTES, ICAM-1, and GM-CSF in lung tissue. Nitric oxide production and levels of MCP-1 and MIP-2 were increased in the 24-hr culture media of alveolar macrophages (AMs) obtained on day 1. IL-6, MCP-1, and MIP-2 levels were also elevated in the BAL fluid. BAL fluid also showed increases in albumin and lactate dehydrogenase. The cellular content in BAL fluid increased at all doses and at all time periods, mainly due to an increase in polymorphonuclear leukocytes. In vitro studies in AMs and cultured lung fibroblasts showed that lung fibroblasts are a significant source of IL-6 and MCP-1 in the lung.

Respirable coal dust particles modify cytochrome P4501A1 (CYP1A1) expression in rat alveolar cells.

Cytochrome P4501A1 (CYP1A1) metabolizes polycyclic aromatic hydrocarbons in cigarette smoke to DNA-binding reactive intermediates associated with carcinogenesis. Epidemiologic studies indicate that the majority of coal miners are smokers but have a lower risk of lung cancer than other smokers. We hypothesized that coal dust (CD) exposure modifies pulmonary carcinogenesis by altering CYP1A1 induction. Therefore, male Sprague Dawley rats were intratracheally instilled with 2.5, 10, 20, or 40 mg CD/rat or vehicle (saline); and 11 d later, pulmonary CYP1A1 was induced by intraperitoneal injection of beta-naphthoflavone (BNF; 50 mg/kg). Fourteen days after CD exposure, CYP1A1 protein and activity were measured by Western blot and 7-ethoxyresorufin-O-deethylase activity, respectively. CYP1A1 and the alveolar type II markers, cytokeratins 8/18, were localized and quantified in lung sections by dual immunofluorescence with morphometry. The area of CYP1A1 expression in alveolar septa and alveolar type II cells in response to BNF was reduced by exposure to 20 or 40 mg CD compared with BNF alone. CD exposure significantly inhibited BNF-induced 7-ethoxyresorufin-O-deethylase activity in a dose-responsive manner. By Western blot, induction of CYP1A1 protein by BNF was significantly reduced by 40 mg CD compared with BNF alone. These findings indicate that CD decreases BNF-induced CYP1A1 protein expression and activity in the lung.

Inhalation exposure of rats to asphalt fumes generated at paving temperatures alters pulmonary xenobiotic metabolism pathways without lung injury.

Asphalt fumes are complex mixtures of various organic compounds, including polycyclic aromatic hydrocarbons (PAHs). PAHs require bioactivation by the cytochrome P-450 monooxygenase system to exert toxic/carcinogenic effects. The present study was carried out to characterize the acute pulmonary inflammatory responses and the alterations of pulmonary xenobiotic pathways in rats exposed to asphalt fumes by inhalation. Rats were exposed at various doses and time periods to air or to asphalt fumes generated at paving temperatures. To assess the acute damage and inflammatory responses, differential cell counts, acellular lactate dehydrogenase (LDH) activity, and protein content of bronchoalveolar lavage fluid were determined. Alveolar macrophage (AM) function was assessed by monitoring generation of chemiluminescence and production of tumor necrosis factor-alpha and interleukin-1. Alteration of pulmonary xenobiotic pathways was determined by monitoring the protein levels and activities of P-450 isozymes (CYP1A1 and CYP2B1), glutathioneS-transferase (GST), and NADPH:quinone oxidoreductase (QR). The results show that acute asphalt fume exposure did not cause neutrophil infiltration, alter LDH activity or protein content, or affect AM function, suggesting that short-term asphalt fume exposure did not induce acute lung damage or inflammation. However, acute asphalt fume exposure significantly increased the activity and protein level of CYP1A1 whereas it markedly reduced the activity and protein level of CYP2B1 in the lung. The induction of CYP1A1 was localized in nonciliated bronchiolar epithelial (Clara) cells, alveolar septa, and endothelial cells by immunofluorescence microscopy. Cytosolic QR activity was significantly elevated after asphalt fume exposure, whereas GST activity was not affected by the exposure. This induction of CYP1A1 and QR with the concomitant down-regulation of CYP2B1 after asphalt fume exposure could alter PAH metabolism and may lead to potential toxic effects in the lung.

Alteration of pulmonary immunity to Listeria monocytogenes by diesel exhaust particles (DEPs). I. Effects of DEPs on early pulmonary responses.

It has been hypothesized that diesel exhaust particles (DEPs) aggravate pulmonary bacterial infection by both innate and cell-mediated immune mechanisms. To test this hypothesis, we investigated the effects of DEP exposure on the functions of alveolar macrophages (AMs) and lymphocytes from lung-draining lymph nodes using a rat Listeria monocytogenes infection model. In the present study, we focused on the effects of DEP exposure on AM functions, including phagocytic activity and secretion of proinflammatory cytokines. The Listeria infection model was characterized by an increase in neutrophil count, albumin content, and acellular lactate dehydrogenase activity in the bronchoalveolar lavage (BAL) fluid at 3 and 7 days postinfection. Short-term DEP inhalation (50 and 100 mg/m(3), 4 hr) resulted in a dose-dependent suppression of lung clearance of Listeria, with the highest bacteria count occurring at day 3. This aggravated bacterial infection was consistent with the inhibitory effect of DEPs on macrophage functions. DEPs suppressed phagocytosis and Listeria-induced basal secretion of interleukin-1ss (IL-1ss) and IL-12 by AMs in a dose-dependent manner. The amount of IL-1ss and IL-12 in the BAL fluid was also reduced by DEP exposure. In addition, DEPs decreased Listeria-induced lipopolysaccharide-stimulated secretion of tumor necrosis factor-alpha (TNF-alpha), IL-1ss, and IL-12 from AMs. These results suggest that DEPs retard bacterial clearance by inhibiting AM phagocytosis and weaken the innate immunity by inhibiting AM secretion of IL-1ss and TNF-alpha. DEPs may also suppress cell-mediated immunity by inhibiting AM secretion of IL-12, a key cytokine for the initiation of T helper type 1 cell development in Listeria infection.

Transport and utilization of arginine and arginine-containing peptides by rat alveolar macrophages.

PURPOSE: To demonstrate that rat alveolar macrophages (AM) exhibited the PepT1-like transporter for the uptake of arginine (Arg)-containing small peptides and utilized these peptides as direct substrates for nitric oxide (NO) production. NO is an important mediator that, on one hand, protects the lung from bacteria infection and, on the other hand, augments inflammatory lung injury. METHOD: The uptake of small peptides by rat AM was evaluated using fluorescein isothiocyanate (FITC)-labeled (*) peptides (Arg-Lys*, Gly-Sar-Lys*, and beta-Ala-Lys*), high-performance liquid chromatography (HPLC) analysis of potential peptide degradation, and known inhibitors of Arg and PepT1 transport. NO production by AM through Arg and Arg-containing peptides was studied with and without inhibition by transport inhibitors. The presence of PepT1-like transporter on AM was evaluated using anti-PepT1 antisera and Western blot analysis. The substrate specificity of Arg-Gly and Arg-Gly-Asp was determined using purified inducible NO synthase (iNOS). The availability of Arg-containing peptides in the lung was determined by HPLC analysis of bronchoalveolar lavage (BAL) fluid. RESULTS: The FITC-labeled peptides were internalized by AM without degradation. The uptake of Arg-Lys*, beta-Ala-Lys*, and Gly-Sar-Lys* was blocked (approximately 50%) by cephradine (an inhibitor of PepT1 for peptide transport) but not by Lys (an inhibitor on cationic amino acid transporter 2B for Arg transport). The NO production by AM through Arg-containing peptides was significantly blocked only by PepT1 inhibitors and by an anti-PepT1 antibody in a dose-dependent manner. These inhibitors had no effect on the AM production of NO using Arg as a substrate. Arg-Gly and Arg-Gly-Asp were found to be direct substrates for iNOS with similar Km and Vmax values to those of Arg. But, the production of NO by AM using these peptides as substrates was 2-fold higher than using Arg as a substrate. Both Arg-Gly and Arg-Gly-Asp were found in the BAL fluid. The presence of a PepT1-like transporter on AM was confirmed by Western blotting. CONCLUSION: This study shows that AM exhibit PepT1-like transporter for small peptide uptake. Arginine-containing peptides, through the PepT1 transporter system, can serve as direct substrates of iNOS for the production of NO by AM.

The effect of diesel exhaust particles (DEP) and carbon black (CB) on thiol changes in pulmonary ovalbumin allergic sensitized Brown Norway rats.

Brown Norway rats were exposed by intratracheal instillation of saline, carbon black (CB), or diesel exhaust particles (DEP) (5 mg/kg) on day 1, followed by exposure to ovalbumin (OVA, 90 mg/m(3)) or saline for 30 minutes on days 1, 8, 15, and 29. Animals were sacrificed on day 30. The DEP, CB, or OVA exposure alone did not result in abnormal levels of inflammatory cells, lactate dehydrogenase (LDH), or total protein in the lavage fluid. In combined OVA-DEP or OVA-CB exposure, however, these markers were significantly increased. The adjuvant effect of CB and DEP on OVA sensitization was evidenced by the marked increases in serum OVA-specific IgG (5.6-fold) and IgE (3.5-4 fold) levels, and the increase in interleukin-4 (IL-4) mRNA levels in lung tissue. The OVA exposure markedly reduced glutathione (GSH) levels in both cell types. In combined DEP-OVA exposure, the level of GSH in lymphocytes was further decreased, indicating a possible interactive effect between DEP and OVA exposures. These results show that both DEP and CB augmented OVA-induced allergic sensitization, and that particle composition of DEP may not be a critical factor for the adjuvant effect. OVA exposure causes significant depletion of intracellular GSH in lymphocytes, which may play a key role in OVA-mediated immune responses.

Alteration of intracellular cysteine and glutathione levels in alveolar macrophages and lymphocytes by diesel exhaust particle exposure.

The purpose of this study was to characterize the effects of diesel exhaust particles (DEP) on thiol regulation in alveolar macrophages (AM) and lymphocytes. We obtained AM and lymph node (thymic and tracheal) cells (LNC) (at different time points) from rats exposed intratracheally to DEP (5 mg/kg) or saline, and measured inflammatory markers, thiol levels, and glutathione reductase (GSH-R) activity. DEP exposure produced significant increases in neutrophils, lactate dehydrogenase, total protein, and albumin content in the lavage fluid. AM from DEP-exposed rats showed a time-dependent increase in intracellular cysteine (CYSH) and GSH. In LNC the intracellular GSH reached peak level by 24 hr, declining toward control levels by 72 hr after exposure. LNC-CYSH and AM-CYSH and GSH were increased at both 24 and 72 hr. Both Sprague-Dawley and Brown Norway rats showed similar trends of responses to DEP exposure as per measurement of the inflammatory markers and thiol changes. AM and, to a lesser degree, LNC were both active in cystine uptake. The DEP exposure stimulated GSH-R activity and increased the conversion of cystine to CYSH in both cell types. The intracellular level of GSH in DEP-exposed AM was moderately increased compared with the saline control, and was further augmented when cells were incubated with cystine. In contrast, the intracellular level of GSH in DEP-exposed LNC was significantly reduced despite the increased CYSH level and GSH-R activity when these cells were cultured for 16 hr. DEP absorbed 23-31% of CYSH, cystine, and GSH, and only 8% of glutathione disulfide when incubated in cell free media. These results indicate that DEP exposure caused lung inflammation and affected thiol levels in both AM and LNC.

Dose-dependent thiol and immune responses to ovalbumin challenge in Brown Norway rats.

Dose-dependent specific antibody production, antigen-dependent pulmonary inflammation, and thiol changes in the lung and associated lymph nodes were examined in a Brown Norway rat model of pulmonary sensitization. Cysteine (CYSH), glutathione (GSH), and markers of inflammation in bronchoalveolar lavage fluid (BALF) were measured following ovalbumin (OVA) inhalation challenge. Alveolar macrophages (AM) and pulmonary-associated lymph node cells (LNC) were isolated and intracellular CYSH and GSH assessed. OVA-specific IgE and IgG antibodies were quantified from sera. A dose-dependent biphasic response was noted with respect to OVA-specific IgE. OVA-specific IgG concentrations were maximal at 68 mg (OVA)/m3. OVA challenge to sensitized rats induced increases in BALF albumin, total protein, lactate dehydrogenase, CYSH and GSH that were independent of serum antibody concentrations. AM thiols were modestly elevated at low OVA challenge doses, but sharply reduced at the higher OVA challenge doses. In contrast, both thiols were dose dependently elevated in BALE CYSH, but not GSH, was elevated in LNC of OVA challenged rats. In summary, antigen exposure caused a dose-dependent alteration of inflammatory, thiol and immune parameters in OVA sensitized and challenged rats. Changes in thiol levels did not correlate with antibody responses. While the results of the present study do not support a functional role for thiols in the immune response, it is important to note the dose-dependent dramatic alteration seen in thiols following sensitization and challenge.