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Amiodarone is a clinically effective antiarrhythmic drug shown to cause lung damage in humans and animals. While the mechanism of this pulmonary toxicity is unknown, it may be associated with the accumulation of amiodarone and its principal metabolite, desethylamiodarone, by alveolar macrophages. In the present study, characteristics of the uptake of these drugs by rat alveolar macrophages in vitro were examined. The alveolar macrophages were collected by pulmonary lavage from male Fischer 344 rats. Amiodarone and desethylamiodarone were incubated separately (2.5 microM) with the cells in culture for 1, 2, 4 and 18 hr. High performance liquid chromatography was used to measure drug uptake. At 1 and 2 hr, the uptake of desethylamiodarone by alveolar macrophages was significantly greater (P less than 0.05) than that of amiodarone, but over time, the accumulation of amiodarone began to approach that of desethylamiodarone and was not significantly different by 4 hr. To simulate a more physiological situation, plasma levels achieved in the adult male rat after 1 week of amiodarone treatment (150 mg/kg) were used. Amiodarone (1.95 micrograms/mL) and desethylamiodarone (0.80 microgram/mL) were added together into the cell culture. At 1 and 18 hr, the ratio of desethylamiodarone/amiodarone uptake was significantly greater (P less than 0.05) than in incubation medium containing no cells, indicating an enhanced uptake of desethylamiodarone. Metabolic inhibitors (KCN, 2,4-dinitrophenol, and ouabain) and other cationic, amphiphilic drugs (chlorcyclizine, chlorphentermine, and imipramine) were added individually to the cell cultures containing amiodarone or desethylamiodarone. During 1 hr of incubation, these agents had no effect in blocking the accumulation of amiodarone and desethylamiodarone in the cells. The efflux of amiodarone or desethylamiodarone was measured from cells following incubation for 4 hr with each drug. After this time, the medium was replaced with drug-free medium, and the cells were incubated for another 24 hr. Sixty-three percent of amiodarone was lost as compared to only 31% of desethylamiodarone over the 24-hr period (P less than 0.05). The results of this study are suggestive of a preferential uptake and retention of desethylamiodarone as compared to amiodarone. The accumulation of the drugs appears not to be due to active transport or associated with any carrier protein involved in the transport of other structurally-related compounds.

Pulmonary infections have been reported to be increased in welders. Previous animal studies have indicated that manual metal arc, stainless steel welding fume (MMA-SS) increased susceptibility to lung infections. MMA-SS is composed of a complex of metals (e.g., iron, chromium, nickel). The objective was to determine which metal component of MMA-SS welding fume alters lung defense responses. At Day 0, rats were intratracheally instilled one time with saline or MMA-SS at a concentration of 2 mg/rat. Additional rats were treated with the metal constituents, Fe(2)O(3), NiO, or Cr(2)Na(2)O(7) alone or in combination, at concentrations that are present in the dose used for MMA-SS treatment. At Day 3, rats were intratracheally inoculated with 5 x 10(3) Listeria monocytogenes. At Days 6, 8 and 10, homogenized left lungs were cultured, and colony-forming units were counted after an overnight incubation to assess pulmonary bacterial clearance. At Day 3 (prior to infection) and at Days 6, 8 and 10, right lungs were lavaged to recover cells and fluid from the airspaces to measure lung injury, inflammation, and cytokine secretion. The production of reactive oxygen species by phagocytes recovered from the lungs was measured. Exposure to MMA-SS, soluble Cr, or the mixture of all three metals before infection significantly increased bacterial lung burden and tissue damage when compared to control. Animals treated with NiO or Fe(2)O(3) did not differ from control. Animals pre-treated with soluble Cr had alterations in inflammation and in the production of different cytokines (TNFalpha, IL-6, IL-2, and IL-12) involved in lung immune responses. This study indicates that soluble Cr present in MMA-SS is likely the primary component responsible for the suppression of lung defense responses associated with stainless steel welding fumes.

Many workers worldwide are continually exposed to complex aerosols generated from welding processes. The objective was to assess the effect of inhalation exposure to mild steel (MS) welding fume on lung injury, inflammation, and defense responses. Male Sprague-Dawley rats were exposed to MS fume at a concentration of 40 mg/m(3) x 3 h/day x 3 or 10 days using a robotic welding fume generator. Controls were exposed to filtered air. To assess lung defense responses, a group of animals were intratracheally inoculated with 5 x 10(4) Listeria monocytogenes 1 day after the last daily exposure. Welding particles were collected during exposure, and chemical composition and particle size were determined. After exposure, lung injury, inflammation, and host defense (bacterial clearance) were measured. The particles were composed of iron (80.6 %) and manganese (14.7 %) with a mass median aerodynamic diameter of 0.31 microm. No significant difference was observed in lung injury or inflammation after MS fume inhalation at 1, 4, and 11 days after the last exposure. However, there were significantly more bacteria at 3 days after infection in the lungs of the animals exposed to MS fume compared to air controls. Acute exposure of rats to MS fume had no effect on injury and inflammation, but suppressed lung defense responses after infection. More chronic inhalation studies are needed to further examine the immune effects and to elucidate the possible mechanisms of the suppressed lung defense response to infection associated with the inhalation of MS welding fume.

There is a growing demand for a cost-effective, efficient, and high-throughput method for measuring cytokines. Currently, many studies are using flow cytometric bead-based multiplex assays in the measurement of cytokines. However, limited data are available regarding the performance of these cytometric bead assays versus enzyme-linked immunosorbent assay (ELISA) or correlation with mRNA expression using real time reverse transcriptase-polymerase chain reaction (RT-PCR). In one of our studies, cytometric bead array (CBA) was used to measure inflammatory cytokine protein levels in bronchoalveolar lavage (BAL) samples from mice exposed to welding fume, an inflammatory particulate. The results were then compared to whole lung mRNA levels of the same cytokines measured by real time RT-PCR in the same mouse model. It was found that the trends in cytokine profiles measured via CBA agreed with the whole lung mRNA results. In a separate experiment, we used a rat zymosan infectivity model to induce a pulmonary immunomodulatory response and determined cytokine concentrations in recovered BAL fluid by ELISA and two different types of cytometric bead-based assays, CBA and FlowCytomix (FC). The sample-to-sample correlation was good between ELISA and CBA with correlation coefficient R values of 0.76, 0.66, and 0.92 for rat IFN-gamma, TNF-alpha, and IL-6, respectively. ELISA only correlated significantly with the FC assay for TNF-alpha with R=0.43. Patterns of cytokine response in our rat model also differed among the assays but overall, the ELISA and CBA yielded similar results. For a method-to-method comparison, we assayed supplied cytokine standards from ELISA kits using both ELISA and CBA to determine the R values and found it to be greater than 0.90 for all the cytokines tested. It was found that the ELISA was more sensitive in the low range of the standard curve while the bead assays were capable of detecting higher protein concentrations, which would allow for direct measurement of concentrated samples. There was a lack of agreement between the absolute protein values for the ELISA and flow cytometric bead-based assays; in most cases, the latter method tended to give higher protein concentrations than ELISA. In conclusion, direct comparisons between absolute protein values did not agree among the assays tested in this study, but patterns of cytokine response generally agreed between ELISA and CBA. In the case of the mouse CBA, a companion measurement is recommended if samples with low concentrations of an analyte are reported and extrapolated below sensitivity or zero.

Exposure to diesel exhaust particles (DEP) was shown to increase the susceptibility of the lung to bacterial infection in rats. In this study, the effects of DEP on alveolar macrophage (AM) phagocytic and bactericidal functions and cytokine secretion by AM and lymphocytes in response to Listeria monocytogenes infection were investigated in vitro and the roles of different DEP components in these processes were compared. Exposure to DEP or the organic extracts of DEP (eDEP) significantly decreased the phagocytosis and killing of L. monocytogenes by AM obtained from normal rats. Washed DEP (wDEP) also decreased AM phagocytosis and bacterial killing to a lesser extent, whereas carbon black (CB) reduced AM phagocytosis but had no significant effect on AM bactericidal activity. DEP or eDEP concentration-dependently suppressed L. monocytogenes-induced secretion of tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-12 by AM and of IL-2 and interferon-gamma by lymphocytes obtained from L. monocytogenes-infected rats, but augmented the AM secretion of IL-10. wDEP or CB, however, exerted little or no effect on these L. monocytogenes-induced cytokines. These results provide direct evidence that DEP, through the actions of organic components, suppresses AM phagocytic and bactericidal functions in vitro. Inhibition of AM phagocytic function and alterations of AM and lymphocyte cytokine secretion by DEP and DEP organic compounds may be implicated in the diminished AM bactericidal activity and the lymphatic arm of the host immune system, thus resulting in an suppressed pulmonary clearance of L. monocytogenes and an increased susceptibility of the lung to bacterial infection.

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and a leading cause of death worldwide. COPD combines two lung diseases, emphysema and chronic bronchitis. Both conditions frequently exist together and cause obstruction of airflow that interferes with normal breathing. Long-term cigarette smoking is the most important risk factor for COPD. Spirometry is required to make a diagnosis of COPD. Even though there is no cure for COPD, pharmacologic and non-pharmacologic treatments have improved adverse respiratory symptoms, quality of life, and exercise capacity, as well as modifying the disease progression.

It has been suggested that welding fume (WF) exposure increases lung cancer risk in welders. Epidemiology studies have failed to conclude that WF alone causes lung cancer and animal studies are lacking. We examined the course of inflammation, damage, and repair in the lungs of A/J mice, a lung tumor susceptible strain, caused by stainless steel WF. Mice were exposed by pharyngeal aspiration to 40 mg/kg of WF, silica, or saline. Bronchoalveolar lavage (BAL) was performed 24 hours, 1 and 16 weeks to assess lung injury and inflammation and histopathology was done 1, 8, 16, 24, and 48 weeks postexposure. Both exposures increased inflammatory cells, lactate dehydrogenase and albumin at 24 hr and 1 week. At 16 weeks, these parameters remained elevated in silica-exposed but not WF-exposed mice. Histopathologic evaluation at 1 week indicated that WF induced bronchiolar epithelial hyperplasia with associated cellular atypia, alveolar bronchiolo-alveolar hyperplasia (BAH) in peribronchiolar alveoli, and peribronchiolar lymphogranulomatous inflammation. Persistent changes included foci of histiocytic inflammation, fibrosis, atypical bronchiolar epithelial cells, and bronchiolar BAH. The principle changes in silica-exposed mice were histiocytic and suppurative inflammation, fibrosis, and alveolar BAH. Our findings that WF causes persistent bronchiolar and peribronchiolar epithelial changes, suggest a need for studies of bronchiolar changes after WF exposure.

Respiratory effects observed in welders have included lung function changes, metal fume fever, bronchitis, and a possible increase in the incidence of lung cancer. Many questions remain unanswered regarding the causality and possible underlying mechanisms associated with the potential toxic effects of welding fume inhalation. The objective of the present study was to construct a completely automated, computer-controlled welding fume generation and inhalation exposure system to simulate real workplace exposures. The system comprised a programmable six-axis robotic welding arm, a water-cooled arc welding torch, and a wire feeder that supplied the wire to the torch at a programmed rate. For the initial studies, gas metal arc welding was performed using a stainless steel electrode. A flexible trunk was attached to the robotic arm of the welder and was used to collect and transport fume from the vicinity of the arc to the animal exposure chamber. Undiluted fume concentrations consistently ranged from 90-150 mg/m(3) in the animal chamber during welding. Temperature and humidity remained constant in the chamber during the welding operation. The welding particles were composed of (from highest to lowest concentration) iron, chromium, manganese, and nickel as measured by inductively coupled plasma atomic emission spectroscopy. Size distribution analysis indicated the mass median aerodynamic diameter of the generated particles to be approximately 0.24 microm with a geometric standard deviation (sigma(g)) of 1.39. As determined by transmission and scanning electron microscopy, the generated aerosols were mostly arranged as chain-like agglomerates of primary particles. Characterization of the laboratory-generated welding aerosol has indicated that particle morphology, size, and chemical composition are comparable to stainless steel welding fume generated in other studies. With the development of this novel system, it will be possible to establish an animal model using controlled welding exposures from automated gas metal arc and flux-cored arc welding processes to investigate how welding fumes affect health.

Serious questions have been raised regarding a possible causal association between neurological effects in welders and the presence of manganese in welding fume. An experimental model is needed that could examine the potential neurotoxic effect of manganese after pulmonary exposure to welding fume. The National Institute for Occupational Safety and Health (NIOSH) has recently finished construction of a completely automated, computer controlled welding fume generation and inhalation exposure system for laboratory animals. The system is comprised of a programmable six-axis robotic welding arm and a water-cooled arc welding torch. A flexible trunk has been attached to the robotic arm of the welder and is used to collect and transport fume from the vicinity of the arc to the animal exposure chamber. Preliminary fume characterization studies have indicated that particle morphology, size, and chemical composition were comparable to welding fume generated in the workplace. Animal inhalation studies are currently underway. With the development of this novel system, an animal model has been established using controlled welding exposures to investigate the possible mechanisms by which welding fume may affect the central nervous system.

Uncontrolled apoptosis has been associated with several pulmonary disorders; however, the molecular mechanism underlying this process and the fate of apoptotic cells in vivo are unclear. Here we show that direct administration of apoptotic cells to the lungs of rats caused pulmonary inflammation and fibrosis, as indicated by emigration of inflammatory cells to the air spaces, TNF-α immunoreactivity, and connective tissue accumulation, indicating a direct relationship between apoptotic cells and the observed lung pathologies. To determine how the lungs process the accumulated apoptotic cells, normal or apoptotic cells from autologous donor rats were labeled with fluorescent nanobeads and intratracheally instilled into the lungs of rats. Probe distribution and lung cell apoptosis were determined at various times over a 28-day period by confocal fluorescence microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, respectively. Labeled apoptotic cells were cleared by lung macrophages within 1 wk after the treatment. However, the total number of apoptotic cells in the lung remained high at 28 days posttreatment. The results indicate a continuous induction of secondary apoptosis by apoptotic cell instillation, which may contribute to the observed lung pathology. Analysis of lung cell apoptosis by caspase assays showed an elevation of caspase-8 but not caspase-9 in the treatment group at 28 days posttreatment, indicating involvement of the death receptor-mediated pathway in the apoptotic process. Together, our results demonstrate a direct effect of apoptotic cell accumulation on inflammatory and fibrotic pulmonary responses and the continuous induction of lung cell apoptosis by apoptotic cell instillation.

Questions exist concerning the potential carcinogenic effects after welding fume exposure. Welding processes that use stainless steel (SS) materials can produce fumes that may contain metals (e.g., Cr, Ni) known to be carcinogenic to humans. The objective was to determine the effect of in vitro and in vivo welding fume treatment on free radical generation, DNA damage, cytotoxicity and apoptosis induction, all factors possibly involved with the pathogenesis of lung cancer. SS welding fume was collected during manual metal arc welding (MMA). Elemental analysis indicated that the MMA-SS sample was highly soluble in water, and a majority (87%) of the soluble metal was Cr. Using electron spin resonance (ESR), the SS welding fume had the ability to produce the biologically reactive hydroxyl radical (•OH), likely as a result of the reduction of Cr(VI) to Cr(V). In vitro treatment with the MMA-SS sample caused a concentration-dependent increase in DNA damage and lung macrophage death. In addition, a time-dependent increase in the number of apoptotic cells in lung tissue was observed after in vivo treatment with the welding fume. In summary, a soluble MMA-SS welding fume was found to generate reactive oxygen species and cause DNA damage, lung macrophage cytotoxicity and in vivo lung cell apoptosis. These responses have been shown to be involved in various toxicological and carcinogenic processes. The effects observed appear to be related to the soluble component of the MMA-SS sample that is predominately Cr. A more comprehensive in vivo animal study is ongoing in the laboratory that is continuing these experiments to try to elucidate the potential mechanisms that may be involved with welding fume-induced lung disease.

Welding fumes are a complex mixture composed of different metals. Most welding fumes contain a small percentage of manganese. There is an emerging concern among occupational health officials about the potential neurological effects associated with the exposure to manganese in welding fumes. Little is known about the fate of manganese that is complexed with other metals in the welding particles after inhalation. Depending on the welding process and the composition of the welding electrode, manganese may be present in different oxidation states and have different solubility properties. These differences may affect the biological responses to manganese after the inhalation of welding fumes. Manganese intoxication and the associated neurological symptoms have been reported in individual cases of welders who have been exposed to high concentrations of manganese-containing welding fumes due to work in poorly ventilated areas. However, the question remains as to whether welders who are exposed to low levels of welding fumes over long periods of time are at risk for the development of neurological diseases. For the most part, questions remain unanswered. There is still paucity of adequate scientific reports on welders who suffered significant neurotoxicity, hence there is a need for well-designed epidemiology studies that combine complete information on the occupational exposure of welders with both behavioral and biochemical endpoints of neurotoxicity.

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 shortterm 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/m 3 ) 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/ m 3 ), 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.

Matrix metalloproteinases (MMPs) are the principle enzymes that initiate degradation of collagen. We examined the role of MMPs during alveolar wall fibrosis and fibrotic nodule formation from silica exposure. Rats were exposed to filtered air or 15 mg/m3silica by inhalation for 5 days/wk, 6 h/day. Lungs were preserved by intratracheal instillation of fixative at 20, 40, 60, 79, and 116 days of exposure. Additional groups were fixed after 20, 40, and 60 days of exposure followed by 36 days of recovery. The number of nodules, defined by a collagenous core and a bounding cell layer detached from the alveolar wall, was determined by morphometry. Lungs showed increased alveolar wall collagen and fibrotic nodules at 79 and 116 days of exposure with increased collagenase and gelatinase activity. The number of nodules per lung in exposed groups increased from 619 ± 447 at 40 days to 13,221 ± 1,096 at 116 days (means ± SE, n = 5). No nodules were seen in control lungs. Silica-exposed rats with a 36-day recovery in filtered air showed enhanced MMP activity over exposure to silica for the same duration with no recovery. MMP-2 and MMP-9 were significantly elevated in alveolar macrophages after 40-day exposure. Stromelysin expression was demonstrated in alveolar macrophages and cells within fibrotic nodules. TIMP-1 expression was not significantly altered. In summary, MMP activity was upregulated at 40 days of silica exposure and progressively increased during ensuing fibrotic responses. Early expression of stromelysin was found in fibrosing alveolar walls and fibrotic nodules.

Diesel exhaust particles (DEP) have been shown to suppress alveolar macrophage (AM)-mediated pulmonary immune responses to Listeria monocytogenes in vivo. In this study, effects of DEP-derived reactive oxygen species (ROS) and heme oxygenase (HO)-1 on AM-mediated immune responses to L. monocytogenes were investigated. Brown Norway rats were intratracheally inoculated with 100,000 L. monocytogenes, and AM were isolated at 7 days post-infection. Exposure to DEP or their organic extract (eDEP), but not the washed DEP (wDEP) or carbon black, increased intracellular ROS and HO-1 expression in AM. Induction of ROS and HO-1 by eDEP was partially reversed by alpha-naphthoflavone, a cytochrome P450 1A1 inhibitor, and totally blocked by N-acetylcysteine. In addition, exposure to eDEP, but not wDEP, inhibited lipopolysacchride-stimulated secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin-12 (IL-12), but augmented production of IL-10 by AM. Kinetic studies showed that modulation of cytokines by eDEP was preceded by ROS and HO-1 induction. Furthermore, pretreatment of AM with superoxide dismutase (SOD) or zinc protoporphrin IX (Znpp), which attenuated eDEP-induced HO-1 expression/activity, substantially inhibited eDEP effect on IL-10. Finally, direct stimulation with pyrogallol (PYR), a superoxide donor, upregulated HO-1 and IL-10 but decreased secretion of IL-12 in L. monocytogenes-infected AM. These results show that DEP, through eDEP-mediated ROS, induce HO-1 expression and IL-10 production and at the same time inhibit AM production of TNF-alpha and IL-12 to dampen the host immune responses. The results also suggest that HO-1 may play an important role in regulating production of IL-10 by DEP-exposed and L. monocytogenes-infected AM.

Epidemiology suggests that inhalation of welding fumes increases the susceptibility to lung infection. The effects of chemically distinct welding fumes on lung defense responses after bacterial infection were compared. Fume was collected during gas metal arc (GMA) or flux-covered manual metal arc (MMA) welding using two consumable electrodes: stainless steel (SS) or mild steel (MS). The fumes were separated into water-soluble and -insoluble fractions. The GMA-SS and GMA-MS fumes were found to be relatively insoluble, whereas the MMA-SS was highly water soluble, with the soluble fraction comprised of 87% Cr and 11% Mn. On day 0, male Sprague-Dawley rats were intratracheally instilled with saline (vehicle control) or the different welding fumes (0.1 or 2 mg/rat). At day 3, the rats were intratracheally inoculated with 5 x 10(3) Listeria monocytogenes. On days 6, 8, and 10, left lungs were removed, homogenized, cultured overnight, and colony-forming units were counted to assess pulmonary bacterial clearance. Bronchoalveolar lavage (BAL) was performed on right lungs to recover phagocytes and BAL fluid to measure the production of nitric oxide (NO) and immunomodulatory cytokines, including tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-2, IL-6, and IL-10. In contrast to the GMA-SS, GMA-MS, and saline groups, pretreatment with the highly water soluble MMA-SS fume caused significant body weight loss, extensive lung damage, and a dramatic reduction in pulmonary clearance of L. monocytogenes after infection. NO concentrations in BAL fluid and lung immunostaining of inducible NO synthase were dramatically increased in rats pretreated with MMA-SS before and after infection. MMA-SS treatment caused a significant decrease in IL-2 and significant increases in TNF-alpha, IL-6, and IL-10 after infection. In conclusion, pretreatment with MMA-SS increased production of NO and proinflammatory cytokines (TNF-alpha and IL-6) after infection, which are likely responsible for the elevation in lung inflammation and injury. In addition, MMA-SS treatment reduced IL-2 (involved in T cell proliferation) and enhanced IL-10 (involved in inhibiting macrophage function) after bacterial infection, which might result in a possible suppression in immune response and an increase in susceptibility to infection.

Exposure to certain particulate hexavalent chromium [Cr(VI)] compounds, such as lead chromate (PbCrO4), has been associated with lung cancer and respiratory tract toxicity. Previous studies indicate that the solubility of Cr(VI)-compounds is an important factor in Cr(VI)-induced carcinogenesis. The present study investigates reactive oxygen species (ROS) generation by PbCrO4 particles and cellular responses using RAW 264.7 cells. A mixture containing PbCrO4 and RAW 264.7 cells generated hydroxyl radical ((.)OH), using cellularly generated H2O2 as a precursor, as measured by electron spin resonance (ESR) spin trapping in combination with H2O2 and (.)OH scavengers, catalase and sodium formate. The effect of ascorbic acid on (.)OH radicals was also measured using ESR. Confocal microscopy showed that particles could become either bound to the cell surface or engulfed over a 120 min time period. H2O2 generation and O2 consumption were also increased after treatment of the cells with PbCrO4. Both NF-kappaB and AP-1 were activated after exposure to PbCrO4 particles as measured by the NF-kappaB or AP-1 luciferase reporter plasmid assay. Our investigation thus demonstrated that the RAW 264.7 cells phagocytized the PbCrO4 particles leading to accumulation of the particles within vacuoles in the cytoplasm. These particles could induce chronic production of ROS and activation of NF-kappaB and AP-1. Such induction of transcription pathways may be involved in the inflammatory and carcinogenic responses induced by Cr(VI)-containing particles.

It is estimated that more than 1 million workers worldwide perform some type of welding as part of their work duties. Epidemiology studies have shown that a large number of welders experience some type of respiratory illness. Respiratory effects seen in full-time welders have included bronchitis, siderosis, asthma, and a possible increase in the incidence of lung cancer. Pulmonary infections are increased in terms of severity, duration, and frequency among welders. Inhalation exposure to welding fumes may vary due to differences in the materials used and methods employed. The chemical properties of welding fumes can be quite complex. Most welding materials are alloy mixtures of metals characterized by different steels that may contain iron, manganese, chromium, and nickel. Animal studies have indicated that the presence and combination of different metal constituents is an important determinant in the potential pneumotoxic responses associated with welding fumes. Animal models have demonstrated that stainless steel (SS) welding fumes, which contain significant levels of nickel and chromium, induce more lung injury and inflammation, and are retained in the lungs longer than mild steel (MS) welding fumes, which contain mostly iron. In addition, SS fumes generated from welding processes using fluxes to protect the resulting weld contain elevated levels of soluble metals, which may affect respiratory health. Recent animal studies have indicated that the lung injury and inflammation induced by SS welding fumes that contain water-soluble metals are dependent on both the soluble and insoluble fractions of the fume. This article reviews the role that metals play in the pulmonary effects associated with welding fume exposure in workers and laboratory animals.

Inhalation of residual oil fly ash (ROFA) has been shown to impair lung defense mechanisms in laboratory animals and susceptible populations. Bioavailability of soluble transition metals has been shown to play a key role in lung injury caused by ROFA exposure. The goal of this study was to evaluate the effect of soluble metals on lung defense and injury in animals preexposed to ROFA followed by pulmonary challenge with a bacterial pathogen. ROFA was suspended in saline (ROFA-TOTAL), incubated overnight at 37 degrees C, and separated by centrifugation into soluble (ROFA-SOL) and insoluble (ROFA-INSOL) fractions. A portion of the soluble sample was treated with the metal-binding resin Chelex for 24 h at 37 degrees C. Sprague-Dawley rats were intratracheally dosed at d 0 with ROFA-TOTAL (1.0 mg/100 g body weight), ROFA-INSOL, ROFA-SOL, saline, saline + Chelex, or ROFA-SOL + Chelex. At d 3, 5 x 10(5) Listeria monocytogenes were intratracheally instilled into rats from each treatment group. At d 6, 8, and 10, left lungs were removed, homogenized, and cultured to assess bacterial clearance. Histopathological analysis was performed on the right lungs. Pulmonary exposure of ROFA-TOTAL or ROFA-SOL before infection led to a marked increase in lung injury and inflammation at all three time points after inoculation, and an increase in morbidity in comparison to saline control rats. Treatment with ROFA-INSOL, saline + Chelex, or ROFA-SOL + Chelex caused no significant increases in lung damage and morbidity when compared to control. By d 10, the ROFA-SOL and ROFA-TOTAL groups had approximately 200-fold more bacteria in the lung than saline control, indicating the inability of these groups to effectively respond to the infection. None of the other treatment groups had significant impairments in bacterial clearance when compared to saline. In conclusion, exposure to ROFA-TOTAL and ROFA-SOL significantly suppressed the lung response to infection. These results suggest that soluble metals present in ROFA may play a key role in increased susceptibility to pulmonary infection in exposed populations.

The goals of this study were to examine acute lung damage and inflammation, as well as free radical production, caused by welding fumes of different chemical compositions and solubilities. The fumes were from a gas metal arc welding using a mild-steel (GMA-MS) or stainless-steel electrode (GMA-SS) and a manual metal arc welding using a stainless-steel electrode (MMA-SS), which was further separated into soluble and insoluble fractions. The MMA-SS was the only fume to contain soluble chromium. Free radical production was observed only in suspensions of MMA-SS fume under various conditions. Male Sprague-Dawley rats were intratracheally instilled with either a welding fume suspension at 2 mg/rat or a saline vehicle, and various parameters of inflammation and damage were measured at 3 h and days 1, 3, and 6. Only the MMA-SS treatment caused a continued increase in lung weight until day 6 and elevated lipid peroxidation at day 3. All of the fumes caused increases in macrophages and neutrophils obtained by lavage, but the increased cellularity was extended through day 6 following the MMA-SS treatment only. Only the MMA-SS treatment led to an increased recovery of eosinophils and damage to the alveolar-capillary barrier. While all of the fumes produced increases in cytotoxicity, the MMA-SS treatment caused the maximal response at day 3. These findings indicate that different welding fumes caused varied responses in the lungs of rats, correlated to their metal composition and ability to produce free radicals. Additionally, both the soluble and insoluble fractions of the MMA-SS fume were required to produce most effects, indicating that the responses are not dependent exclusively on the soluble metals.

Amiodarone (AD) is gaining support as a first-line antiarrhythmic drug despite its potentially fatal pulmonary toxicity involving inflammation and fibrosis. We previously reported a model for this amiodarone-induced pulmonary toxicity (AIPT) in which F344 rats were intratracheally (i.t.) instilled with AD (6.25 mg/kg) in sterile water on days 0 and 2, which led to transient pulmonary inflammation and lung damage and subsequent fibrosis. The goals of this study were to determine the direct effect of the drug in the lung damage occurring after i.t. AD administration, to identify its location, and to examine its potential mechanisms. Using bronchoalveolar lavage and laser-scanning confocal microscopy, it was discovered that AD instillation produces rapid and massive damage to the alveolar-capillary barrier and damage or death to lung airway and parenchymal cells. While AD in solution was found to be capable of generating hydroxyl radicals, protection from AD-induced damage could not be obtained by incorporating water-soluble antioxidants in the drug solution. However, damage induced by free-radicals could still occur after AD partitions into lipid membranes. AD could also be directly disrupting cellular membranes via its amphiphilic structure. It is not known if the mechanism(s) of damage following i.t. AD treatment are similar to the mechanisms that underlie human AIPT. Therefore these data suggest that investigators should use caution in extrapolating results from animal studies that utilize i.t. administration of AD to human AIPT.

Inhalation of residual oil fly ash (ROFA) increases pulmonary morbidity in exposed workers. We examined the role of reactive oxygen species (ROS) in ROFA-induced lung injury. ROFA was collected from a precipitator at Boston Edison Co., Everett, MA, USA. ROFA (ROFA-total) was suspended in saline, incubated for 24 h at 37 degrees C, centrifuged, and separated into its soluble (ROFA-sol.) and insoluble (ROFA-insol.) fractions. Sprague-Dawley rats were intratracheally instilled with saline or ROFA-total or ROFA-sol. or ROFA-insol. (1 mg/100 g body wt.). Lung tissue and bronchoalveolar lavage cells were harvested at 4, 24, and 72 h after instillation. Chemiluminescence (CL) of recovered cells was measured as an index of ROS production, and tissue-lipid-peroxidation was assessed to determine oxidative injury. Significant amounts of Al, Fe, and Ni were present in ROFA-sol., whereas ROFA-insol. contained Fe, V, and Al. Using electron spin resonance (ESR), significantly more hydroxyl radicals were measured in ROFA-sol. as compared to ROFA-insol. None of the ROFA samples had an effect on CL or lipid peroxidation at 4 h. Treatment with ROFA-total and ROFA-insol. caused significant increases in both CL (at 24 h) and lipid peroxidation (at 24 and 72 h) when compared to saline control value. ROFA-sol. significantly reduced CL production at 72 h after treatment and had no effect on lipid peroxidation at any time point. In summary, ROFA, particularly its soluble fraction, generated a metal-dependent hydroxyl radical as measured by a cell-free ESR assay. However, cellular oxidant production and tissue injury were observed mostly with the ROFA-total and ROFA-insol. particulate forms. ROS generated by ROFA-sol. as measured by ESR appear not to play a major role in the lung injury caused after ROFA exposure.

Many of the epidemiology studies performed are difficult to compare because of differences in worker populations, industrial settings, welding techniques, duration of exposure, and other occupational exposures besides welding fumes. Some studies were conducted in carefully controlled work environments, others during actual workplace conditions, and some in laboratories. Epidemiology studies have shown that a large number of welders experience some type of respiratory illness. Respiratory effects seen in full-time welders have included bronchitis, airway irritation, lung function changes, and a possible increase in the incidence of lung cancer. Pulmonary infections are increased in terms of severity, duration, and frequency among welders. Although epidemiological studies have demonstrated an increase in pulmonary illness after exposure to welding fumes, little information of the causality, dose-response, and possible underlying mechanisms regarding the inhalation of welding fumes exists. Even less information is available about the neurological, reproductive, and dermal effects after welding fume exposure. Moreover, carcinogenicity and short-term and long-term toxicology studies of welding fumes in animals are lacing or incomplete. Therefore, an understanding of possible adverse health effects of exposure to welding fumes is essential to risk assessment and the development of prevention strategies and will impact a large population of workers.

Induction of apoptosis has been associated with a variety of exposures which result in inflammatory and fibrotic lung disorders. Macrophages are key regulatory cells in the lung; however, the role of apoptotic macrophages in those pulmonary disorders is not well characterized. In the present investigation, apoptotic macrophages were instilled into the lungs of rats to study directly the pulmonary responses to apoptotic cells. The effects of apoptotic macrophages on lung inflammation and fibrosis, as well as associated protein expression of TNF-alpha, TGF-beta, and matrix metalloproteinases (MMPs) were examined. Induction of macrophage apoptosis was carried out in vitro using a variety of known apoptosis inducers. Intratracheal administration of apoptotic macrophages (5 x 10(6) cells/rat) into the lung of rats caused an increase in pulmonary infiltration of macrophages and lung cell apoptosis 4 weeks after the treatment as indicated by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. In contrast, pulmonary instillation of saline or normal control macrophages had no effect. Histological analysis of lung sections showed collagen deposition and fibrotic lesions after apoptotic cell treatment but not in control groups. Immunohistochemical studies revealed increased expression of TNF-alpha, TGF-beta, MMP2, and MMP9 in the treatment group 4 weeks after the treatment. These results suggest a role for macrophage apoptosis in the initiation of these lung disorders. This study provides direct evidence that apoptotic macrophages can induce lung inflammation and fibrosis and that this induction may be associated with increased expression of TNF-alpha, TGF-beta, MMP2, and MMP9. Published 2002 Wiley-Liss, Inc.

Pulmonary fibrosis is a serious lung disorder that in certain cases may be difficult to quantify. It was our objective to evaluate the use of laser scanning confocal microscopy (LSCM) in quantifying fibrosis after exposure to amiodarone (AD) and bleomycin (BLM), two commonly used therapeutic drugs known to cause debilitating lung fibrosis in humans. Male F344 rats were intratracheally dosed with AD (6.25 mg/kg on days 0 and 2), BLM (0.25 and 1.0 mg/kg on day 0), or their respective vehicle controls. The right lung was assayed for hydroxyproline, a biochemical measure of collagen, at day 21 for the BLM groups and day 28 for the AD groups. The left lung was fixed, sectioned into blocks, dehydrated, stained with Lucifer yellow (LY, 0.1 mg/ml), and embedded in Spurr resin. The area of lung tissue stained by LY was quantified by LSCM. A fibrotic response in the AD and BLM groups was confirmed by histopathological assessment and a significant increase (p < 0.05) in total right lung hydroxyproline above control values. The area of connective tissue stained by LY of the two drug-treated groups appeared as bright linear bands in the alveolar septae and was significantly increased (p < 0.05) as measured by image analysis when compared with their respective controls. LSCM, with its advanced image analysis system, is an alternate method to quantify fibrotic lung disease. LSCM could be particularly useful when tissue quantity is limited, such as when tissue has been archived from previous studies, or when analyzing human lung biopsy samples for disease diagnosis, where biochemical analysis is difficult.

Welding processes that generate fumes containing toxic metals, such as hexavalent chromium (Cr(VI)), manganese (Mn), and nickel (Ni), have been implicated in lung injury, inflammation, and lung tumor promotion in animal models. While federal regulations have reduced permissible worker exposure limits to Cr(VI), this is not always practical considering that welders may work in confined spaces and exhaust ventilation may be ineffective. Thus, there has been a recent initiative to minimize the potentially hazardous components in welding materials by developing new consumables containing much less Cr(VI) and Mn. A new nickel (Ni) and copper (Cu)-based material (Ni-Cu WF) is being suggested as a safer alternative to stainless steel consumables; however, its adverse cellular effects have not been studied. This study compared the cytotoxic effects of the newly developed Ni-Cu WF with two well-characterized welding fumes, collected from gas metal arc welding using mild steel (GMA-MS) or stainless steel (GMA-SS) electrodes. RAW 264.7 mouse macrophages were exposed to the three welding fumes at two doses (50 µg/ml and 250 µg/ml) for up to 24 hours. Cell viability, reactive oxygen species (ROS) production, phagocytic function, and cytokine production were examined. The GMA-MS and GMA-SS samples were found to be more reactive in terms of ROS production compared to the Ni-Cu WF. However, the fumes from this new material were more cytotoxic, inducing cell death and mitochondrial dysfunction at a lower dose. Additionally, pre-treatment with Ni-Cu WF particles impaired the ability of cells to phagocytize E. coli, suggesting macrophage dysfunction. Thus, the toxic cellular responses to welding fumes are largely due to the metal composition. The results also suggest that reducing Cr(VI) and Mn in the generated fume by increasing the concentration of other metals (e.g., Ni, Cu) may not necessarily improve welder safety.

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.

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.

Background Dosimetry for toxicology studies involving carbon nanotubes (CNT) is challenging because of a lack of detailed occupational exposure assessments. Therefore, exposure assessment findings, measuring the mass concentration of elemental carbon from personal breathing zone (PBZ) samples, from 8 U.S.-based multi-walled CNT (MWCNT) manufacturers and users were extrapolated to results of an inhalation study in mice. Results Upon analysis, an inhalable elemental carbon mass concentration arithmetic mean of 10.6 μg/m3 (geometric mean 4.21 μg/m3) was found among workers exposed to MWCNT. The concentration equates to a deposited dose of approximately 4.07 μg/d in a human, equivalent to 2 ng/d in the mouse. For MWCNT inhalation, mice were exposed for 19 d with daily depositions of 1970 ng (equivalent to 1000 d of a human exposure; cumulative 76 yr), 197 ng (100 d; 7.6 yr), and 19.7 ng (10 d; 0.76 yr) and harvested at 0, 3, 28, and 84 d post-exposure to assess pulmonary toxicity. The high dose showed cytotoxicity and inflammation that persisted through 84 d after exposure. The middle dose had no polymorphonuclear cell influx with transient cytotoxicity. The low dose was associated with a low grade inflammatory response measured by changes in mRNA expression. Increased inflammatory proteins were present in the lavage fluid at the high and middle dose through 28 d post-exposure. Pathology, including epithelial hyperplasia and peribronchiolar inflammation, was only noted at the high dose. Conclusion These findings showed a limited pulmonary inflammatory potential of MWCNT at levels corresponding to the average inhalable elemental carbon concentrations observed in U.S.-based CNT facilities and estimates suggest considerable years of exposure are necessary for significant pathology to occur at that level.

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.

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.

Advances in molecular and cellular biology have identified the cellular mediators that regulate many disease processes and have facilitated the development of new therapeutic agents that control these events. However, the size, complexity, and cellular inaccessibility of these therapeutic agents make their cellular delivery difficult. Here, we describe an efficient cellular delivery system that exploits the membrane-translocating ability of signal peptides to import functional peptides into cells. Molecular conjugates consisting of the signal import peptide (IP) and nuclear localization sequence (NLS) of the transcription factor NF-kappaB were synthesized. Electrophoretic mobility shift and enzyme-linked immunosorbent assays were used to assess the inhibitory effects of these synthetic peptides on agonist-induced NF-kappaB nuclear translocation and transcriptional activation. Our results indicated that the peptides were effective in inhibiting both the nuclear translocation and transcriptional activation of NF-kappaB. However, their effects required the presence of the IP moiety for efficient cellular entry of the NLS. Structural analysis of IP showed that the hydrophobic domain, and to a lesser extent the N-terminal domain, was responsible for the membrane translocating activity of IP.

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.

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.

Background The potential use of quantum dots (QD) in biomedical applications, as well as in other systems that take advantage of their unique physiochemical properties, has led to concern regarding their toxicity, potential systemic distribution, and biopersistence. In addition, little is known about workplace exposure to QD in research, manufacturing, or medical settings. The goal of the present study was to assess pulmonary toxicity, clearance, and biodistribution of QD with different functional groups in rats after pulmonary exposure. Methods QD were composed of a cadmium-selenide (CdSe) core (~5nm) with a zinc sulfide (ZnS) shell functionalized with carboxyl (QD-COOH) or amine (QD-NH2) terminal groups. Male Sprague–Dawley rats were intratracheally-instilled (IT) with saline, QD-COOH, or QD-NH2 (12.5, 5.0, or 1.25 μg/rat). On days 0, 1, 3, 5, 7, 14, and 28 post-IT, the left lung, lung-associated lymph nodes (LALN), heart, kidneys, spleen, liver, brain, and blood were collected for metal analysis of Cd content by neutron activation to evaluate clearance and biodistribution. One right lobe was ligated and fixed for microscopy and histopathological analysis. The remaining right lobes from rats in each group were subjected to bronchoalveolar lavage (BAL) to retrieve BAL fluid and cells for analysis of injury and inflammation. Results Lung injury and inflammation was found to be dose-dependent and peaked at days 7 and 14 post-exposure for both forms of QD, with slight variations in degree of toxicity at early and later time points. Both QD appeared to lose their fluorescent properties and destabilize after 1 week in the lung. Cd persisted up to 28 days for both forms of QD; however, clearance rate was slightly greater for QD-COOH over time. No Cd was detected in the liver, spleen, heart, brain, or blood at any time point. Cd appeared in the LALN and kidneys beginning at 1–2 weeks post-exposure. Conclusions QD-COOH and QD-NH2 differed in clearance rate and differed slightly in degree of toxicity at different time points; however, the overall pattern of toxicity and biodistribution was similar between the two particles. Toxicity may be dependent on the dissolution rate and bioavailability of free Cd.

Chemical alteration of the glucocorticoid, methylprednisolone, has led to the introduction of a new class of compounds called the 21-aminosteroids (21-ASs). The purpose of this study was to investigate the effect of the 21-AS, U74389G, on silica-induced acute lung injury. Male Fischer 344 rats were treated intraperitoneally with saline or U74389G in a total dose of 15 mg/kg divided into three injections of 5 mg/kg separated by 4 h. Following the first treatment, animals from the two groups were intratracheally instilled with silica (10 mg/100 g body wt in 0.5 ml of saline) or saline vehicle (0.5 ml). Twenty-four hours after the instillations, bronchoalveolar lavage (BAL) was performed. In the animals not receiving U74389G, marked increases in total protein, beta-glucuronidase, and lactate dehydrogenase (LDH) activities and number of neutrophils (PMNs) were demonstrated in the BAL fluid of the silica-treated animals compared to their controls. Silica also caused dramatic increases in the luminol-dependent chemiluminescence (CL) of lung tissue and BAL cells. The CL reaction was decreased by superoxide dismutase (SOD) and N-nitro-L-arginine methyl ester hydrochloride (L-NAME), a nitric oxide (NO) synthase inhibitor. In animals treated with U74389G, there was attenuation of the silica-induced increases in biochemical, cellular, and chemiluminescent indices of damage. This study demonstrates that U74389G significantly reduces acute lung injury caused by the intratracheal instillation of silica, and this drug may be of potential value for treatment of lung diseases in which damage caused by reactive oxygen species has been implicated.

The inhalation of silica has been shown to produce a dramatic inflammatory and toxic response within the lungs of humans and laboratory animals. A variety of cellular and biochemical parameters are used to assess the silica-induced lung injury. The purpose of this paper is to introduce the use of luminol-dependent chemiluminescence as a new method to study inflammation in both phagocytic cells and lung tissue recovered from silica-exposed animals. Chemiluminescence, or the emission of light, accompanies the release of reactive forms of oxygen when phagocytic cells are challenged. In this study, male Fischer 344 rats were intratracheally instilled with either silica (10 mg/100 g bw) or saline vehicle. One day after the instillations, a marked increase in the chemiluminescence was observed in the lung tissue and bronchoalveolar lavage cells recovered from the silica-treated animals when compared with the saline controls. The light reaction was markedly decreased by either superoxide dismutase of N-nitro-arginine methyl ester hydrochloride. Superoxide dismutase is involved in the enzymatic breakdown of superoxide anion, while N-nitro-L-arginine methyl ester hydrocholoride, a nitric oxide synthase inhibitor, prevents the formation of nitric oxide. When superoxide anion and nitric oxide react, they form the highly oxidizing substance peroxynitrite. This study then implicates peroxynitrite as an agent that may be responsible for some of the oxidant lung injury that is associated with silica exposure. The use of luminol-dependent chemiluminescence may prove valuable as a method to measure the earliest events in the inflammatory process, and may be an adjunct in studying the mechanisms that produce inflammation.

Alveolar macrophages (AM) exposed to cytokines or bacterial lipopolysaccharide (LPS) produce the free radical nitric oxide (NO.) by an inducible nitric oxide synthase (iNOS). They also release reactive oxygen free radicals following exposure to silica dust. The purpose of the present study was to determine whether NO. is produced by rat AM and/or recruited leukocytes following the intratracheal (IT) instillation of silica. Male Sprague-Dawley rats (175 to 225 g) were IT instilled with either silica dust (10 mg/100 g body wt) or LPS (0.25 mg/100 g body wt). After 24 h, bronchoalveolar lavage cells (BALC) and lavaged lung tissue were assayed for iNOS mRNA. Cell counts of BALC and iNOS-dependent (N omega-nitro-L-arginine methyl ester [L-NAME]-inhibitable) chemiluminescence generated by AM were also determined. Northern blot analysis demonstrated that the steady-state levels of BALC iNOS mRNA were significantly increased by 3-fold following IT silica and by 7-fold following IT LPS. Partially enriched fractions of either AM or leukocytes from silica-treated rats both exhibited significantly elevated iNOS mRNA in Northern analysis. iNOS-dependent chemiluminescence was significantly increased in AM by 36-fold following IT silica and by 89-fold following IT LPS. Differential counts of BALC showed that AM numbers did not change in any of the treatments; however, red blood cells increased by 30-fold following IT silica and by 23-fold following IT LPS. Total leukocytes (polymorphonuclear leukocytes plus lymphocytes) increased by 58-fold following IT silica and by 274-fold following IT LPS.(ABSTRACT TRUNCATED AT 250 WORDS)

The objective of our study was to investigate whether coating the surface of silica with Survanta, a commercially available, bovine pulmonary surfactant, would reduce the in vitro cytotoxicity to alveolar macrophages (AMs), as well as attenuate lung damage in vivo following intratracheal instillation of silica. In the in vitro studies, alveolar macrophages from male Fischer 344 rats were incubated for 1 and 24 h with native or Survanta-treated silica (0.5 mg/ml). At both time points, the native, uncoated silica caused a dramatic loss of AM viability. The AMs were protected, however, when the silica was treated with the Survanta surfactant. This protective effect was significantly greater after 1 h when compared with 24 h. In the in vivo studies, a high dose of silica (10 mg/100 g body weight) was suspended in Survanta and intratracheally instilled into the lungs of male Fischer 344 rats. A number of biochemical and cellular parameters were measured within the bronchoalveolar lavage fluid (BALF) 1 and 14 d after the instillation exposures to assess lung damage. One day after the instillations, the suspension of silica in Survanta resulted in significant reductions in the silica-induced increases in total protein, beta-glucuronidase activity, and influx of neutrophils (PMNs) into the airspaces of the lungs. Fourteen days after the instillation exposures, this protective effect was lost. When Survanta was instilled into the lungs 15 min after the intratracheal instillation of silica, a significant reduction also was demonstrated in the silica-induced elevations in BALF total protein, beta-glucuronidase activity, and influx of PMNs 1 d after the instillation exposures. In an attempt to protect silica-exposed lungs over a longer period of time, Survanta was instilled into the lung 15 min after the silica instillation, and then every other day over a 7-d treatment period. Twenty-four hours after the last Survanta instillation, slight but significant decreases in the silica-induced elevations in BALF total protein and beta-glucuronidase activity were observed. The Survanta treatment, however, had no effect in preventing the infiltration of PMNs into the airspaces of the lungs. The results of this study indicate that artificially coating the silica with surfactant phospholipid offers short-term protection against its toxicity under both in vitro and in vivo conditions.

Background Welding fumes consist of a wide range of complex metal oxide particles which can be deposited in all regions of the respiratory tract. The welding aerosol is not homogeneous and is generated mostly from the electrode/wire. Over 390,000 welders were reported in the U.S. in 2008 while over 1 million full-time welders were working worldwide. Many health effects are presently under investigation from exposure to welding fumes. Welding fume pulmonary effects have been associated with bronchitis, metal fume fever, cancer and functional changes in the lung. Our investigation focused on the generation of free radicals and reactive oxygen species from stainless and mild steel welding fumes generated by a gas metal arc robotic welder. An inhalation exposure chamber located at NIOSH was used to collect the welding fume particles. Results Our results show that hydroxyl radicals (.OH) were generated from reactions with H2O2 and after exposure to cells. Catalase reduced the generation of .OH from exposed cells indicating the involvement of H2O2. The welding fume suspension also showed the ability to cause lipid peroxidation, effect O2 consumption, induce H2O2 generation in cells, and cause DNA damage. Conclusion Increase in oxidative damage observed in the cellular exposures correlated well with .OH generation in size and type of welding fumes, indicating the influence of metal type and transition state on radical production as well as associated damage. Our results demonstrate that both types of welding fumes are able to generate ROS and ROS-related damage over a range of particle sizes; however, the stainless steel fumes consistently showed a significantly higher reactivity and radical generation capacity. The chemical composition of the steel had a significant impact on the ROS generation capacity with the stainless steel containing Cr and Ni causing more damage than the mild steel. Our results suggest that welding fumes may cause acute lung injury. Since type of fume generated, particle size, and elapsed time after generation of the welding exposure are significant factors in radical generation and particle deposition these factors should be considered when developing protective strategies.

Hard metal is an alloy of tungsten carbide and cobalt along with other components such as chromium carbide, molybdenum carbide, tantalum carbide, and nickel. Chronic exposure to hard metal particles by inhalation causes alveolitis leading to interstitial fibrosis, the pathogenesis of which is still undefined. The initial inflammatory response includes a change in epithelial cell permeability barrier function (1) which has been shown to be regulated by the state of assembly and organization of the actin cytoskeletal network (2, 3). Therefore, the objective of this study was to evaluate the effect hard metal particles have on F-actin organization of rat lung epithelial cells in an in vitro culture system.Rat lung epithelial cells (L2: ATCC, CCL-149) were grown to confluence on glass coverslips and exposed to various concentrations of hard metal particles for 24 hours. The effect on F-actin organization was visualized by confocal microscopy following Bodipy-Phallacidin staining, while changes in cell morphology were assessed by phase contrast microscopy. Hard metal particles of cobalt, tungsten carbide, and tungsten carbide/cobalt (6 % cobalt) were tested at concentrations of 1, 3, and 5 μg/ml. There was a dose-dependent change in the F-actin organization in the cells. The actin microfilaments lost their uniform distribution and aggregated into homogeneous masses of F-actin staining. Significant change in F-actin state was observed even at a 1 μg/ml concentration of tungsten carbide/cobalt particles. This is consistent with previous observations that pathological effects of tungsten carbide/cobalt particles are more pronounced compared to either metal alone. Phase contrast microscopy revealed no significant change in the cell morphology at this short incubation time.

Background Welding fume has been categorized as "possibly carcinogenic" to humans. Our objectives were to characterize the lung response to carcinogenic and non-carcinogenic metal-containing welding fumes and to determine if these fumes caused increased lung tumorigenicity in A/J mice, a lung tumor susceptible strain. We exposed male A/J and C57BL/6J, a lung tumor resistant strain, by pharyngeal aspiration four times (once every 3 days) to 85 μg of gas metal arc-mild steel (GMA-MS), GMA-stainless steel (SS), or manual metal arc-SS (MMA-SS) fume, or to 25.5 μg soluble hexavalent chromium (S-Cr). Shams were exposed to saline vehicle. Bronchoalveolar lavage (BAL) was done at 2, 7, and 28 days post-exposure. For the lung tumor study, gross tumor counts and histopathological changes were assessed in A/J mice at 48 and 78 weeks post-exposure. Results BAL revealed notable strain-dependent differences with regards to the degree and resolution of the inflammatory response after exposure to the fumes. At 48 weeks, carcinogenic metal-containing GMA-SS fume caused the greatest increase in tumor multiplicity and incidence, but this was not different from sham. By 78 weeks, tumor incidence in the GMA-SS group versus sham approached significance (p = 0.057). A significant increase in perivascular/peribronchial lymphoid infiltrates for the GMA-SS group versus sham and an increased persistence of this fume in lung cells compared to the other welding fumes was found. Conclusion The increased persistence of GMA-SS fume in combination with its metal composition may trigger a chronic, but mild, inflammatory state in the lung possibly enhancing tumorigenesis in this susceptible mouse strain.

Background Debate exists as to whether welding fume is carcinogenic, but epidemiological evidence suggests that welders are an at risk population for the development of lung cancer. Recently, we found that exposure to welding fume caused an acutely greater and prolonged lung inflammatory response in lung tumor susceptible A/J versus resistant C57BL/6J (B6) mice and a trend for increased tumor incidence after stainless steel (SS) fume exposure. Here, our objective was to examine potential strain-dependent differences in the regulation and resolution of the lung inflammatory response induced by carcinogenic (Cr and Ni abundant) or non-carcinogenic (iron abundant) metal-containing welding fumes at the transcriptome level. Methods Mice were exposed four times by pharyngeal aspiration to 5 mg/kg iron abundant gas metal arc-mild steel (GMA-MS), Cr and Ni abundant GMA-SS fume or vehicle and were euthanized 4 and 16 weeks after the last exposure. Whole lung microarray using Illumina Mouse Ref-8 expression beadchips was done. Results Overall, we found that tumor susceptibility was associated with a more marked transcriptional response to both GMA-MS and -SS welding fumes. Also, Ingenuity Pathway Analysis revealed that gene regulation and expression in the top molecular networks differed between the strains at both time points post-exposure. Interestingly, a common finding between the strains was that GMA-MS fume exposure altered behavioral gene networks. In contrast, GMA-SS fume exposure chronically upregulated chemotactic and immunomodulatory genes such as CCL3, CCL4, CXCL2, and MMP12 in the A/J strain. In the GMA-SS-exposed B6 mouse, genes that initially downregulated cellular movement, hematological system development/function and immune response were involved at both time points post-exposure. However, at 16 weeks, a transcriptional switch to an upregulation for neutrophil chemotactic genes was found and included genes such as S100A8, S100A9 and MMP9. Conclusions Collectively, our results demonstrate that lung tumor susceptibility may predispose the A/J strain to a prolonged dysregulation of immunomodulatory genes, thereby delaying the recovery from welding fume-induced lung inflammation. Additionally, our results provide unique insight into strain- and welding fume-dependent genetic factors involved in the lung response to welding fume.

Welding fume is an exposure that consists of a mixture of metal-rich particulate matter with gases (ozone, carbon monoxide) and/or vapors (VOCs). Data suggests that welders are immune compromised. Given the inability of pulmonary leukocytes to properly respond to a secondary infection in animal models, the question arose whether the dysfunction persisted systemically. Our aim was to evaluate the circulating leukocyte population in terms of cellular activation, presence of oxidative stress, and functionality after a secondary challenge, following welding fume exposure. Rats were intratracheally instilled (ITI) with PBS or 2 mg of welding fume collected from a stainless steel weld. Rats were sacrificed 4 and 24 h post-exposure and whole blood was collected. Whole blood was used for cellular differential counts, RNA isolation with subsequent microarray and Ingenuity Pathway Analysis, and secondary stimulation with LPS utilizing TruCulture technology. In addition, mononuclear cells were isolated 24 h post-exposure to measure oxidative stress by flow cytometry and confocal microscopy. Welding fume exposure had rapid effects on the circulating leukocyte population as identified by relative mRNA expression changes. Instillation of welding fume reduced inflammatory protein production of circulating leukocytes when challenged with the secondary stimulus LPS. The effects were not related to transcription, but were observed in conjunction with oxidative stress. These findings support previous studies of an inadequate pulmonary immune response following a metal-rich exposure and extend those findings showing leukocyte dysfunction occurs systemically.