Examination of the exposome in an animal model: The impact of high fat diet and rat strain on local and systemic immune markers following occupational welding fume exposure.

The goal of this study was to investigate the impact of multiple exposomal factors (genetics, lifestyle factors, environmental/occupational exposures) on pulmonary inflammation and corresponding alterations in local/systemic immune parameters. Accordingly, male Sprague-Dawley (SD) and Brown Norway (BN) rats were maintained on either regular (Reg) or high fat (HF) diets for 24wk. Welding fume (WF) exposure (inhalation) occurred between 7 and 12wk. Rats were euthanized at 7, 12, and 24wk to evaluate local and systemic immune markers corresponding to the baseline, exposure, and recovery phases of the study, respectively. At 7wk, HF-fed animals exhibited several immune alterations (blood leukocyte/neutrophil number, lymph node B-cell proportionality)-effects which were more pronounced in SD rats. Indices of lung injury/inflammation were elevated in all WF-exposed animals at 12wk; however, diet appeared to preferentially impact SD rats at this time point, as several inflammatory markers (lymph node cellularity, lung neutrophils) were further elevated in HF over Reg animals. Overall, SD rats exhibited the greatest capacity for recovery by 24wk. In BN rats, resolution of immune alterations was further compromised by HF diet, as many exposure-induced alterations in local/systemic immune markers were still evident in HF/WF animals at 24wk. Collectively, HF diet appeared to have a greater impact on global immune status and exposure-induced lung injury in SD rats, but a more pronounced effect on inflammation resolution in BN rats. These results illustrate the combined impact of genetic, lifestyle, and environmental factors in modulating immunological responsivity and emphasize the importance of the exposome in shaping biological responses.

Inhalation of iron-abundant gas metal arc welding-mild steel fume promotes lung tumors in mice.

Welding fumes were reclassified as a Group 1 carcinogen by the International Agency for Research on Cancer in 2017. Gas metal arc welding (GMAW) is a process widely used in industry. Fume generated from GMAW-mild steel (MS) is abundant in iron with some manganese, while GMAW-stainless steel (SS) fume also contains significant amounts of chromium and nickel, known carcinogenic metals. It has been shown that exposure to GMAW-SS fume in A/J mice promotes lung tumors. The objective was to determine if GMAW-MS fume, which lacks known carcinogenic metals, also promotes lung tumors in mice. Male A/J mice received a single intraperitoneal injection of corn oil or the initiator 3-methylcholanthrene (MCA; 10 µg/g) and, one week later, were exposed by whole-body inhalation to GMAW-MS aerosols for 4 hours/day x 4 days/week x 8 weeks at a mean concentration of 34.5 mg/m3. Lung nodules were enumerated by gross examination at 30 weeks post-initiation. GMAW-MS fume significantly increased lung tumor multiplicity in mice initiated with MCA (21.86 ± 1.50) compared to MCA/air-exposed mice (8.34 ± 0.59). Histopathological analysis confirmed these findings and also revealed an absence of inflammation. Bronchoalveolar lavage analysis also indicated a lack of lung inflammation and toxicity after short-term inhalation exposure to GMAW-MS fume. In conclusion, this study demonstrates that inhalation of GMAW-MS fume promotes lung tumors in vivo and aligns with epidemiologic evidence that shows MS welders, despite less exposure to carcinogenic metals, are at an increased risk for lung cancer.

Effects of pulmonary exposure to chemically-distinct welding fumes on neuroendocrine markers of toxicity.

Exposure to welding fumes may result in disorders of the pulmonary, cardiovascular, and reproductive systems. Welders are also at a greater risk of developing symptoms similar to those seen in individuals with idiopathic Parkinson's disease. In welders, there are studies that suggest that alterations in circulating prolactin concentrations may be indicative of injury to the dopamine (DA) neurons in the substantia nigra. The goal of these studies was to use an established model of welding particulate exposure to mimic the effects of welding fume inhalation on reproductive functions. Since previous investigators suggested that changes in circulating prolactin may be an early marker of DA neuron injury, movement disorders, and reproductive dysfunction, prolactin, hypothalamic tyrosine hydroxylase (TH) levels (a marker of DA synthesis), and other measures of hypothalamic-pituitary-gonadal (HPG) function were measured after repetitive instillation of welding fume particulates generated by flux core arc-hard surfacing (FCA-HS), manual metal arc-hard surfacing (MMA-HS) or gas metal arc-mild steel (GMA-MS) welding, or manganese chloride (MnCl2). Exposure to welding fume particulate resulted in the accumulation of various metals in the pituitary and testes of rats, along with changes in hypothalamic TH and serum prolactin levels. Exposure to particulates with high concentrations of soluble manganese (Mn) appeared to exert the greatest influence on TH activity levels and serum prolactin concentrations. Thus, circulating prolactin levels may serve as a biomarker for welding fume/Mn-induced neurotoxicity. Other reproductive measures were collected, and these data were consistent with epidemiological findings that prolactin and testosterone may serve as biomarkers of welding particulate induced DA neuron and reproductive dysfunction.

Effect of exposure to diesel exhaust particles on the susceptibility of the lung to infection.

There are at least three mechanisms by which alveolar macrophages play a critical role in protecting the lung from bacterial or viral infections: production of inflammatory cytokines that recruit and activate lung phagocytes, production of antimicrobial reactive oxidant species, and production of interferon (an antiviral agent). In this article we summarize data concerning the effect of exposure to diesel exhaust particles on these alveolar macrophage functions and the role of adsorbed organic chemicals compared to the carbonaceous core in the toxicity of diesel particles. In vitro exposure of rat alveolar macrophages to diesel exhaust particles decreased the ability of lipopolysaccharide (LPS), a bacterial product] to stimulate the production of inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha). Methanol extract exhibited this potential but methanol-washed diesel particles did not. Exposure of rats to diesel exhaust particles by intratracheal instillation also decreased LPS-induced TNF-alpha and IL-1 production from alveolar macrophages. In contrast, carbon black did not exhibit this inhibitory effect. Exposure of rats to diesel exhaust particles by inhalation decreased the ability of alveolar macrophages to produce antimicrobial reactive oxidant species in response to zymosan (a fungal component). In contrast, exposure to coal dust increased zymosan-stimulated oxidant production. In vivo exposure to diesel exhaust particles but not to carbon black decreased the ability of the lungs to clear bacteria. Inhalation exposure of mice to diesel exhaust particles but not to coal dust depressed the ability of the lung to produce the antiviral agent interferon and increased viral multiplication in the lung. These results support the hypothesis that exposure to diesel exhaust particles increases the susceptibility of the lung to infection by depressing the antimicrobial potential of alveolar macrophages. This inhibitory effect appears to be due to adsorbed organic chemicals rather than the carbonaceous core of the diesel particles.

Effect of age on respiratory defense mechanisms: pulmonary bacterial clearance in Fischer 344 rats after intratracheal instillation of Listeria monocytogenes.

STUDY OBJECTIVES: To examine the lung defense mechanisms of both young and aged rats before and after pulmonary challenge with a bacterial pathogen. DESIGN: Male Fischer 344 rats, either 2.5 months or 20 months of age, were intratracheally inoculated with 5 x 10(3), 5 x 10(4), or 5 x 10(5) Listeria monocytogenes, and the effects on mortality, lung inflammation, pulmonary bacterial clearance, alveolar macrophage (AM) function, and T-lymphocyte characterization were determined. MEASUREMENTS AND RESULTS: In noninfected control animals, the older rats had lower numbers of AMs on lavage and a lower percentage of total T, CD4+, and CD8+ cells. No difference was observed between noninfected young and old rats in AM function, assessing both chemiluminescence and nitric oxide (NO) production. After bacterial challenge, aged rats exhibited an increase in mortality, pulmonary infection, and edema, and lung lesions, which were more extensive than those observed in the younger rats. Interestingly, AM chemiluminescence was enhanced, while AM NO, a highly important antibacterial defense product, was abrogated in the aged rats as compared to the young rats. CONCLUSIONS: This study demonstrated that advanced age is associated with alterations in lung defense mechanisms and increased susceptibility to pulmonary bacterial infection marked by elevated mortality, slowed pulmonary bacterial clearance, and altered AM function, specifically a decrease in NO production. These observations are indicative of reduced pulmonary defense function in an older population of rats.

Diesel exhaust particles suppress macrophage function and slow the pulmonary clearance of Listeria monocytogenes in rats.

In this study, we tested the hypothesis that exposure to diesel exhaust particles (DEP) may increase susceptibility of the host to pulmonary infection. Male Sprague-Dawley rats received a single dose of DEP (5 mg/kg), carbon black (CB, 5 mg/kg), or saline intratracheally. Three days later, the rats were inoculated intratracheally with approximately 5,000 Listeria monocytogenes and sacrificed at 3, 5, and 7 days postinfection, and we determined the number of viable Listeria in the left lobe of lungs. The remaining lungs underwent bronchoalveolar lavage (BAL) and the retrieved BAL cells were identified and counted. Luminol-dependent chemiluminescence, a measure of reactive oxygen species (ROS) formation, generated by BAL cells was monitored and the levels of nitric oxide and tumor necrosis factor (TNF)-[alpha] produced by macrophages in culture were determined. At 7 days postinfection, we excised the lung-draining lymph nodes and phenotyped the lymphocyte subpopulations. Exposure of rats to DEP, but not to CB, decreased the clearance of Listeria from the lungs. Listeria-induced generation of luminol-dependent chemiluminescence by pulmonary phagocytes decreased by exposure to DEP but not CB. Similarly, Listeria-induced production of NO by alveolar macrophages was negated at 3, 5, and 7 days after inoculation in DEP-exposed rats. In contrast, CB exposure had no effect on Listeria-induced NO production at 3 days after infection and had a substantially smaller effect than DEP at later days. Exposure to DEP or CB resulted in enlarged lung-draining lymph nodes and increased the number and percentage of CD4(+) and CD8(+) T cells. These results showed that exposure to DEP decreased the ability of macrophages to produce antimicrobial oxidants in response to Listeria, which may play a role in the increased susceptibility of rats to pulmonary infection. This DEP-induced suppression is caused partially by chemicals adsorbed onto the carbon core of DEP, because impaired macrophage function and decreased Listeria clearance were not observed following exposure to CB.

Cadmium-induced cell transformation and tumorigenesis are associated with transcriptional activation of c-fos, c-jun, and c-myc proto-oncogenes: role of cellular calcium and reactive oxygen species.

The molecular mechanisms of carcinogenesis by cadmium were studied using BALB/c-3T3 cell transformation and nude mouse tumorigenesis models. BALB/c-3T3 cells transformed with cadmium chloride were subcutaneously injected into nude mice to develop tumors and the cell lines derived from these tumors were used in the present study. The proto-oncogenes c-fos and c-jun were overexpressed in 100% (10 out of 10) of the cell lines, while a statistically significant overexpression of c-myc was observed in 40% (4 out of 10) of the cell lines. Analysis of tumor cells stained with fluorescent dyes specific for reactive oxygen species revealed that these cells possessed markedly higher levels of superoxide anion and hydrogen peroxide compared with the nontransformed cells. Similarly, the intracellular calcium level was higher in the tumor cells compared with the nontransformed cells. Overexpression of the proto-oncogenes in these cells was blocked by treating the cells with superoxide dismutase, catalase, and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra acetoxy methyl ester (BAPTA/AM), which are scavengers of superoxide anion, hydrogen peroxide, and calcium, respectively. This confirmed that the overexpression of the proto-oncogenes in the tumor cells required elevated intracellular levels of reactive oxygen species and calcium. In addition to the scavengers of reactive oxygen species and calcium, inhibitors specific for transcription (actinomycin D), protein kinase C (RO-31-8220), and MAP kinase (PD 98059) also blocked the cadmium-induced overexpression of the proto-oncogenes in the tumor cells. Exposure of the nontransformed BALB/c-3T3 cells to 20 microM cadmium chloride for 1 h caused elevated intracellular levels of superoxide anion, hydrogen peroxide, and calcium, with corresponding increases in the expression levels of c-fos, c-jun, and c-myc. As in the case of the tumor cells, treating the nontransformed cells with the various modulators prior to their exposure to cadmium chloride resulted in inhibition in the expression of the proto-oncogenes. Based on these data, we conclude that the cadmium-induced overexpression of cellular proto-oncogenes is mediated by the elevation of intracellular levels of superoxide anion, hydrogen peroxide, and calcium. Further, the cadmium-induced overexpression of the proto-oncogenes is dependent on transcriptional activation as well as on pathways involving protein kinase C and MAP kinase.

Pulmonary responses to single versus multiple intratracheal instillations of silica in rats.

The pulmonary toxicity of particles is often studied using a single intratracheal instillation of the material. It was hypothesized that smaller multiple intratracheal administrations of silica would result in differences in pulmonary responses as compared to a single large intratracheal administration. In the first of a series of experiments, the pulmonary responses in male F344 rats to a single intratracheal instillation of crystalline silica (5 mg/100 g body weight) given on d 0 were compared with those resulting from 5 consecutive daily intratracheal administrations of the dust (1 mg/100 g body weight/d) with the initial dose given on d 0. Controls received saline intratracheally. In the second experiment, the dose was reduced to 1 mg/100 g body weight for the single-dose protocol and 0.2 mg/100 g body weight/d for 5 consecutive days for the multiple-dose protocol. In both experiments, responses were assessed on d 14. In the third experiment, the doses were the same as the first experiment, but the responses were assessed on d 28. The indices of toxicity were cellular differentials recovered by bronchoalveolar lavage, which is an index of inflammation, and the level of albumin in the bronchoalveolar lavage fluid, a measure of damage to the capillary-epithelial barrier. At the higher dose of silica, similar levels of inflammation and lung damage were evident in both dosing protocols. Less severe responses occurred at the lower dose. The comparative pattern between the single and multiple dosing protocols was similar in all three experiments. Since only minor differences were noted in the pulmonary responses when the responses to the single- and multiple-dose protocols were compared, data indicate that the multiple-dose protocol does not offer any advantages over the single-dose protocol.

Strain-related differences of nonspecific respiratory defense mechanisms in rats using a pulmonary infectivity model.

A number of animal studies have assessed pulmonary host defense mechanisms by inoculating the lungs with the bacterial agent, Listeria monocytogenes. Most studies use only a single strain of the animal to be tested; however, strain-related differences in responsiveness to pulmonary toxicants have been well documented. It was the goal of this current investigation to measure the pulmonary defense responses of two different strains of rats in a lung infectivity model. Fischer 344 (F344) and Sprague-Dawley (SD) rats were instilled intratracheally with 5 x 10(3) or 5 x 10(5) L. monocytogenes, and the effect on mortality, lung injury and inflammation, pulmonary bacterial clearance, and alveolar macrophage (AM) function was determined at 3, 5, and 7 days after bacteria treatment. Pulmonary inoculation with the higher (5 x 10(5) L. monocytogenes) dose proved to be highly pneumotoxic to the F344 rats as evidenced by an increase in mortality and more severe lung injury and inflammation when compared with the SD rats. After intratracheal instillation with the lower (5 x 10(3) L. monocytogenes) dose, pulmonary bacterial clearance was slowed and an increase in pulmonary responsiveness was observed for the F344 rats as compared to the SD rats. Specifically, the total number of neutrophils recovered from the lungs and tumor necrosis factor-alpha secreted by AMs were elevated for the F344 group throughout the 7 days, while cellular chemiluminescence, an index of reactive oxygen species production, and lung albumin and lactate dehydrogenase, indicators of injury, were increased at 3 and 5 days after bacterial instillation. This study demonstrated that respiratory defense function was compromised in F344 rats as evidenced by elevated mortality, slowed pulmonary bacterial clearance, and altered AM function. F344 rats may then represent a sensitive model for the examination of respiratory defense mechanisms after bacterial challenge.

Subchronic silica exposure enhances respiratory defense mechanisms and the pulmonary clearance of Listeria monocytogenes in rats.

Both Listeria monocytogenes infection and silica exposure have been shown to significantly alter immune responses. In this study, we evaluated the effect of preexposure to silica on lung defense mechanisms using a rat pulmonary L. monocytogenes infection model. Male Sprague-Dawley rats were instilled intratracheally with saline (vehicle control) or silica using either an acute treatment regimen (5 mg/kg; 3 days) or a subchronic treatment protocol (80 mg/kg; 35 days). At 3 or 35 days after silica instillation, the rats were inoculated intratracheally with either approximately 5000 or 500,000 L. monocytogenes. At 3, 5, and 7 days postinfection, the left lung was removed, homogenized, and cultured on brain heart infusion agar at 37 degrees C. The numbers of viable L. monocytogenes were counted after an overnight incubation. Bronchoalveolar lavage (BAL) was performed on the right lungs, and BAL cell differentials, acellular lactate dehydrogenase (LDH) activity and albumin content were determined. Alveolar macrophage (AM) chemiluminescence (CL) and phagocytosis were assessed as a measure of macrophage function. Lung-associated lymph nodes were removed, and lymphocytes were recovered and differentiated. Preexposure to silica significantly increased the pulmonary clearance of L. monocytogenes as compared to saline controls. Exposure to silica caused significant increases in BAL neutrophils, LDH and albumin, and lymph-nodal T cells and natural killer (NK) cells in infected and noninfected rats. CL and phagocytosis were also elevated in silica-treated rats. In summary, the results demonstrated that exposure of rats to silica enhanced pulmonary immune responses, as evidenced by increases in neutrophils, NK cells, T lymphocytes, and macrophage activation. These elevations in pulmonary immune response are likely responsible for the increase in pulmonary clearance of L. monocytogenes observed with preexposure to silica.

Changes in F-actin organization induced by hard metal particle exposure in rat pulmonary epithelial cells using laser scanning confocal microscopy.

Chronic inhalation of hard metal (WC-Co) particles causes alveolitis and the eventual development of pulmonary fibrosis. The initial inflammatory response includes a change in the alveolar epithelial cell-capillary barrier, which has been shown to be regulated by the state of assembly and organization of the actin cytoskeletal network. The objective of this study was to evaluate the effect WC-Co particles have on F-actin organization of lung epithelial cells in an in vitro culture system. Rat lung epithelial (L2) cells were exposed to 5, 25, and 100 microg/mL of WC-Co particles, as well as the individual components (Co and WC) of the hard metal mixture particles for 24 h. The effect on F-actin organization was visualized by laser scanning confocal microscopy (LSCM) following Bodipy-Phallacidin staining. Minimal changes in the F-actin microfilaments of L2 cells were observed by LSCM after exposure to WC and WC-Co at 5 and 25 microg/mL, while at 100 microg/mL, there was a noticeable disruption in the uniform distribution of L2 cell F-actin microfilaments. After exposure to Co, a dose-dependent change in the F-actin organization of the L2 cells was observed. Little change in F-actin assembly was observed after treatment with 5 microg/mL of Co (the concentration equivalent to the 5% amount of Co commonly present in 100 microg/mL of the WC-Co sample mixture). However, at 100 microg/mL of Co, the microfilaments aggregated into homogeneous masses within the cells, and a significant loss in the organization of L2 F-actin was observed. These dramatic alterations in F-actin organization seen after exposure to the higher doses of Co were attributed to an increase in L2 cell injury as measured by lactate dehydrogenase and trypan blue exclusion. We conclude the pulmonary response evoked in the lung by inhalation of high levels of WC-Co particles is unlikely due to alterations in the F-actin microfilaments of lung-epithelial cells.

Quantitative image analysis of lung connective tissue in murine silicosis.

Pulmonary fibrosis is a disabling consequence of many lung diseases but is difficult to quantify. Lucifer yellow CH fluorescent dye (LY) appears to stain connective tissue matrix macromolecules selectively. Laser scanning confocal microscopy can quantify the intensity of fluorescence and determine the area of fluorescent material. We hypothesized that the abundance of lucifer yellow-stained matrix macromolecules in lung tissue sections could be measured by laser scanning confocal microscopy, would reflect differences between varying degrees of pulmonary fibrosis, and could be compared directly to biochemical measurements of lung collagen. We exposed C57B1/6 and 129 strains of mice by aerosol to cristobalite silica (70 mg/m3, 12 days, 5 hours/day) or sham-air and examined them 2 and 16 weeks after exposure. The area of LY-stained matrix in tissue sections was quantitated by laser scanning confocal microscopy, and total lung collagen was measured biochemically as hydroxyproline (OH-proline). The LY-stained connective tissue matrix appeared as bright linear bands in the alveolar septae, and was increased significantly by image analysis in C57B1/6 and 129 mice with silicosis 16 weeks after exposure. Total lung OH-proline was significantly increased in silica-exposed mice from both stains at both time points. Comparing all 8 groups, there was a significant linear correlation between the average area of connective tissue measured by LY stain and the total OH-proline per lung measured by chemical analysis (r = .72, P = .042). LY staining and confocal microscopy with image analysis offers a rapid technique for quantitative measurements of the extent of pulmonary fibrosis.

Superinduction of CYP1A1 gene expression. Regulation of 2,3,7, 8-tetrachlorodibenzo-p-dioxin-induced degradation of Ah receptor by cycloheximide.

Cycloheximide superinduces the transcription of CYP1A1 in the presence of an agonist for the Ah receptor (AhR). To investigate the molecular target for "superinduction," we analyzed the agonist-induced degradation of AhR. Whereas 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), a potent agonist of AhR, induces a rapid reduction of the AhR protein, cycloheximide blocks the down-regulation of steady state AhR. Analyses of the turnover of AhR reveal that cycloheximide blocks the shortening of the half-life of AhR by TCDD. Blocking of the TCDD-induced AhR degradation requires inhibition of protein synthesis, because (a) cycloheximide inhibits protein synthesis at the concentration at which it causes superinduction and inhibition of AhR degradation; and (b) puromycin, an inhibitor of protein synthesis by mimicking aminoacyl-tRNA, also blocks the TCDD-induced AhR degradation. The blocking of the TCDD-induced AhR degradation correlates with the superinduction of CYP1A1 gene expression in a time- and dose-dependent manner. Furthermore, cycloheximide is shown to increase the accumulation of the TCDD-activated AhR and the functional AhR x Arnt complex in nucleus. Collectively, our results reveal a mechanism of superinduction by cycloheximide by enhancing the stability of agonist-activated AhR. The finding that inhibition of protein synthesis blocks the TCDD-induced AhR turnover implicates a cycloheximide-sensitive, labile factor (designated as AhR degradation promoting factor, or ADPF) in controlling the removal of agonist-activated AhR in nucleus.

Inhaled particle-bound sulfate: effects on pulmonary inflammatory responses and alveolar macrophage function.

Acid sulfate-coated solid particles are a significant environmental hazard produced primarily by the combustion of fossil fuels. We have previously described a system for the nascent generation of carbonaceous particles surface coated with approximately 140 microg/m(3) acid sulfate [cpSO(4)(2-); 10 mg/m(3) carbon black (CB) and 10 ppm sulfur dioxide (SO(2)) at 85% relative humidity (RH)]. The effects of inhaled cpSO(4)(2-) on pulmonary host defenses are assessed in the present work. Mice were acutely exposed (4 h) to either 10 mg/m(3) CB, 10 ppm SO(2), or their combination at 10% or 85% RH in a nose-only inhalation chamber. No evidence of an inflammatory response was found following any of the exposures as assessed by total cell counts and differential cell counts from bronchoalveolar lavage fluid. However, alveolar macrophage Fc receptor-mediated phagocytosis decreased only following exposure to 140 microg cpSO(4)(2-), significant suppression occurred after 24 h, maximal suppression occurred at 3 days postexposure, and recovery to preexposure levels required 7-14 days. Intrapulmonary bactericidal activity (IBA) was also suppressed only after exposure to 140 microg cpSO(4)(2-); suppression was maximal at 1 day postexposure and recovered by day 7. To assess the effects of lower cpSO(4)(2-) concentrations, mice were repeatedly exposed to 1 mg/m(3) CB and 1 ppm SO(2) at 85% RH ( approximately 20 microg/m(3) cpSO(4)(2-) for 4 h/day) for up to 6 days. A significant decrement in IBA was observed following 5 and 6 days of exposure. These studies indicated that acute or repeated exposure to cpSO(4)(2-) could alter pulmonary host defense mechanisms.

Effect of silica inhalation on the pulmonary clearance of a bacterial pathogen in Fischer 344 rats.

Silica inhalation predisposes workers to bacterial infection and impairments in pulmonary defense function. In this study, we evaluated the effect of pre-exposure to silica on lung defense mechanisms by use of a rat pulmonary Listeria monocytogenes infection model. Male Fischer 344 rats were exposed by inhalation to filtered air or silica (15 mg/m3 x 6 h/day x 5 days/wk). After 21 or 59 days of silica exposure, the rats were inoculated intratracheally with 5 x 10(3) L. monocytogenes. At 0 (noninfected controls), 3, and 7 days after infection, the left lungs were removed, homogenized, and the number of viable L. monocytogenes was counted after an overnight culture at 37 degrees C. Bronchoalveolar lavage (BAL) was performed on the right lungs. Alveolar macrophages (AM) were collected, and the AM production of chemiluminescence (CL), an index of reactive oxygen species generation, was measured. The number of lavagable neutrophils (PMNs) and acellular BAL lactate dehydrogenase (LDH) activity were determined as indices of inflammation and injury, respectively. Pre-exposure to silica for 59 days caused substantial increases in PMN number and LDH activity compared with the air controls, whereas silica inhalation for both 21 and 59 days significantly enhanced the pulmonary clearance of L. monocytogenes compared with air controls. Dramatic elevations were also observed in zymosan- and phorbol myristate acetate (PMA)-stimulated CL production by lung phagocytes recovered from rats pre-exposed to silica for 59 days. These results demonstrate that short-term exposure to inhaled silica particles activates lung phagocytes, as evidenced by increases in reactive oxygen species. This up-regulation in the production of antimicrobial oxidants is likely responsible for the enhancement in pulmonary clearance of L. monocytogenes observed with short-term silica inhalation.

Effect of welding fume solubility on lung macrophage viability and function in vitro.

It was shown previously that fumes generated from stainless steel (SS) welding induced more pneumotoxicity and were cleared from the lungs at a slower rate than fumes collected from mild steel (MS) welding. These differences in response may be attributed to the metal composition of SS and MS welding fumes. In this study, fumes with vastly different metal profiles were collected during gas metal arc (GMA) or flux-covered manual metal arc (MMA) welding using two different consumable electrodes, SS or MS. The collected samples were suspended in saline, incubated for 24 h at 37 degrees C, and centrifuged. The supernatant (soluble components) and pellets (insoluble particulates) were separated, and their effects on lung macrophage viability and the release of reactive oxygen species (ROS) by macrophages were examined in vitro. The soluble MMA-SS sample was shown to be the most cytotoxic to macrophages and to have the greatest effect on their function as compared to the GMA-SS and GMA-MS fumes. Neither the soluble nor insoluble forms of the GMA-MS sample had any marked effect on macrophage viability. The flux-covered MMA-SS fume was found to be much more water soluble as compared to either the GMA-SS or the GMA-MS fumes. The soluble fraction of the MMA-SS samples was comprised almost entirely of Cr. The small fraction of the GMA-MS sample that was soluble contained Mn with little Fe, while a more complex mixture was observed in the soluble portion of the GMA-SS sample, which contained Mn, Ni, Fe, Cr, and Cu. Data show that differences in the solubility of welding fumes influence the viability and ROS production of macrophages. The presence of soluble metals, such as Fe, Cr, Ni, Cu, and Mn, and the complexes formed by these different metals are likely important in the pulmonary responses observed after welding fume exposure.

Application of laser scanning confocal microscopy in the analysis of particle-induced pulmonary fibrosis.

Laser scanning confocal microscopy (LSCM) allows us to simultaneously quantitate the degree of lung fibrosis and distinguish various pathological lesions of intact lung tissue. Lucifer Yellow has been shown an ideal fluorescent stain to examine the connective tissue matrix components of embedded lung tissue with LSCM. We evaluated the use of LSCM in quantitating lung fibrosis and compared this procedure with the more traditional method of assessing fibrosis by measuring hydroxyproline, a biochemical assay of collagen. CD/VAF rats were intratracheally dosed with silica (highly fibrogenic), Fe2O3 (non-fibrogenic), and saline (vehicle control) at a high dose of 10-mg/100 g body weight. At 60 days post-instillation, the left lung was dissolved in 6 M HCl and assayed for hydroxyproline. Silica induced increases of 58% and 94% in hydroxyproline content over the Fe2O3 and control groups, respectively. The right lung lobes were fixed, sectioned into blocks, dehydrated, stained with Lucifer Yellow (0.1 mg/ml), and embedded in Spurr plastic. Using LSCM and ImageSpace software, the tissue areas of ten random scans from ten blocks of tissue for each of the three groups were measured, and three-dimensional reconstructions of random areas of lung were generated. The silica group showed increases of 57% and 60% in the lung areas stained by Lucifer Yellow over the Fe2O3 and control groups, respectively. Regression analysis of hydroxyproline vs. lung tissue area demonstrated a significant positive correlation (p < 0.05) with a correlation coefficient of 0.91. Histological analysis of right lung tissue revealed a marked degree of granulomatous interstitial pneumonitis for the silica group, which was absent in the Fe2O3 and control groups. No significant differences (p < 0.05) in hydroxyproline content and measured tissue area were observed between the Fe2O3 and control groups. LSCM, and its associated advanced image analysis and three-dimensional capabilities, is an alternative method to both quickly quantitate and examine fibrotic lung disease without physical disruption of the tissue specimen.

In situ microscopic analysis of asbestos and synthetic vitreous fibers retained in hamster lungs following inhalation.

Hamsters breathed, nose-only, for 13 weeks, 5 days/week, 6 hr/day, either man-made vitreous fiber (MMVF)10a, MMVF33, or long amosite asbestos at approximately 300 World Health Organization (WHO) fibers/cc or long amosite at 25 WHO fibers/cc. [World Health Organization fibers are longer than 5 microm and thicker than 3 microm, with aspect ratio >3.] After sacrifice, fiber burden was estimated (left lungs) by ashing and scanning electron microscopy (ashing/SEM) or (right middle lobes) by confocal laser scanning microscopy (CLSM) in situ. In situ CLSM also provided three-dimensional views of fibers retained, undisturbed, in lung tissue. Fibers of each type were lodged in alveoli and small airways, especially at airway bifurcations, and were seen fully or partly engulfed by alveolar macrophages. Amosite fibers penetrated into and through alveolar septa. Length densities of fibers in parenchyma (total length of fiber per unit volume of lung) were estimated stereologically from fiber transsections counted on two-dimensional optical sections and were 30.5, 25.3, 20.0, and 81.6 mm/mm3 for MMVF10a, MMVF33, and low- and high-dose amosite, respectively. Lengths of individual fibers were measured in three dimensions by tracking individual fibers through series of optical sections. Length distributions of amosite fibers aerosolized, but before inhalation versus after retention in the lung were similar, whether determined by ashing/SEM or in situ CLSM. In contrast, the fraction of short MMVF10a and MMVF33 fibers increased and the geometric mean fiber lengths of both MMVFs decreased by approximately 60% during retention. Most likely due to fiber deposition pattern and differences in sampling, fiber burdens [MMVF10a, MMVF33, and amosite (high dose; 269 WHO fibers/cc)] determined by ashing/SEM were 1.4, 1. 5, and 3.5 times greater, respectively, than those calculated from in situ CLSM data. In situ CLSM is able to provide detailed information about the anatomic sites of fiber retention and also fiber lengths and burdens in good agreement with ashing/SEM results.

Freshly generated stainless steel welding fume induces greater lung inflammation in rats as compared to aged fume.

It has been previously reported that both short- and long-lived reactive oxygen species (ROS) are present on the surface of freshly generated fumes. The objective of this study was to determine if freshly formed welding fume induces greater lung inflammation and injury in rats due to the presence of reactive oxygen species than aged welding fume. Fume was collected during gas metal arc welding using a stainless steel consumable electrode and found to be of respirable size with a mean diameter of 0.77 microm +/- 0.48. Male CD/VAF rats were dosed intratracheally with the welding fume 30 min (fresh) and 1 and 7 days (aged) after fume collection at a dose of 1.0 mg/100 g b wt. Bronchoalveolar lavage (BAL) was performed 24 h post-instillation. Lung injury and inflammation were assessed by measuring the concentration of neutrophils, albumin, lactate dehydrogenase (LDH), and glucosaminidase (GLU) in the recovered BAL fluid. More neutrophils and enhanced GLU activity were observed for the 'fresh' group as compared to both 'aged' groups (P < 0.05). Slight, but not significant, elevations were seen in albumin content and LDH activity for the 'fresh' group as compared to the 'aged' groups. No significant differences were observed for any of the parameters when fume aged for 1 and 7 days were compared. When the 'fresh' and 'aged' fumes (12.5, 25, and 50 microg/ml) were suspended in dichlorofluorescin (15 microM), a probe which becomes fluorescent when oxidized, the concentration-dependent increases in fluorescence were greater for the 'fresh' fume versus the 'aged' fumes. We have demonstrated that freshly generated stainless steel welding fume induces greater lung inflammation than 'aged' fume. This is likely due to a higher concentration of ROS on fresh fume surfaces.

Responses to welding fumes: lung injury, inflammation, and the release of tumor necrosis factor-alpha and interleukin-1 beta.

Possible mechanisms were examined whereby welding fumes may elicit injury and inflammation in the lungs. The effects of different welding fumes on lung macrophages and on the in vivo production of two inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta), were assessed. Fume was collected during flux-covered manual metal are welding using a stainless steel consumable electrode (MMA-SS) and gas metal are welding using a mild steel electrode (GMA-MS). For the in vitro study, bronchoalveolar lavage was performed on untreated rats to recover lung macrophages, and the effects of the welding fumes on macrophage viability and respiratory burst were examined. In vivo, additional rats were intratracheally instilled with the welding fumes at a dose of 1 mg/100 g body weight. These rats were lavaged 1, 14, and 35 days postinstillation, and indicators of lung damage (cellular differential, albumin. TNF-alpha and IL-1 beta release, and lactate dehydrogenase and beta-n-acetyl glucosaminidase activities) were measured. In vitro, the MMA-SS fume was more cytotoxic to the macrophages and induced a greater release of reactive oxygen species as measured by the respiratory burst compared to the GMA-MS fume. In vivo, evidence of lung damage was observed for both fumes 1 day postinstillation. By 14 days, lung responses to the GMA-MS fume had subsided and were not different from the saline vehicle control group. Significant lung damage was still observed for the MMA-SS group at 14 days, but by 35 days, the responses had returned to control values. One day after the instillations, both welding fumes had detectable levels of TNF-alpha and IL 1 beta within the lavage fluid. However, the MMA-SS particles caused a significantly greater release of both cytokines in the lavage fluid than did the GMA-MS group. The results demonstrate that MMA-SS fume caused more pneumoloxicity than GMA-MS. This increased response may reflect enhanced macrophage activation, the increased production of reactive oxygen species, as well as secretion of TNF-alpha and IL-1 beta.