Respiratory effect of acute and subacute exposure to endotoxin-contaminated metal working fluid (MWF) aerosols on Sprague-Dawley rats

Male Sprague-Dawley rats were exposed to a water-soluble metal working fluid (MWF) (5% v/v) contaminated with endotoxins (10,000 eu/ml or 100,000 eu/ml) at 10 mg/m³ for six hours per day for three days (acute exposure) or two weeks (subacute exposure). The geometric mean diameter of the MWF aerosols was 1.56 m, and the airborne endotoxin concentrations ranged from 1,231 to 2,173 eu/m³ (10,000 eu/ml in the bulk MWF) for the low dose and 19,263–27,386 eu/m³ (100,000 eu/ml in the bulk MWF) for the high dose. Minimal effects were observed after exposure to 10 mg/m³ of the MWF without endotoxins for three days or two weeks. However, an increase in the number of polymorphonuclear cells (PMNs) and the level of protein was noted in the bronchoalveolar lavage (BAL) fluid from the rats acutely exposed to the MWF with endotoxins. The acute exposure produced a greater increase in the number of PMNs and total cell number in the BAL fluid than the subacute exposure. The number of white blood cells in the peripheral blood and the weight of the lungs both increased after the subacute exposure to the MWF aerosol with endotoxins, indicating increased vascular permeability in response to the endotoxin exposure. The levels of cyotokines such as IL-4, INF-, and IL-1 in the BAL fluid from the rats exposed to the MWF with or without endotoxins remained unchanged. Although the level of nitric oxide (NO_x) in the BAL supernatant did not show any change, the induction of NO_x from the alveolar macrophages increased in the rats acutely or subacutely exposed to the MWF contaminated with endotoxins. The ConA-induced proliferation response showed no change, yet the LPS-induced proliferation response was significantly increased in the splenocytes from the rats subacutely exposed to the MWF with and without endotoxins. The level of TNF- in the spleen cell culture obtained from the rats exposed to the MWF with or without endotoxins increased without changing the levels of IL-1, IL-4, and INF-. The level of endotoxin-specific IgE in the serum obtained from the rats exposed to the MWF with endotoxins increased dose-dependently, while the levels of total immunoglobulins (IgG₁, IgG_2a and IgE) and endotoxin-specific IgG₁ and IgG_2a remained unchanged. Accordingly, the current results indicate that lung inflammation can be immediately induced by acute or subacute exposure to an MWF contaminated with endotoxins, and macrophages would appear to play a role in the induction of inflammation along with B-cell functions rather than T-cell functions, after subacute exposure to an MWF with endotoxins. In addition, endotoxin-specific IgE is an early marker for endotoxin exposure in the workplace.     

DERMAL AND SYSTEMIC TOXICITY AFTER APPLICATION OF SEMISYNTHETIC METAL-WORKING FLUIDS IN B6C3F1 MICE

About 10 million industrial workers of both sexes are exposed to metal-working fluids (MWFs) via inhalation, skin or both. Our preliminary results, following dermal application of 200 µl of 50% unused (neat) semisynthetic MWF (pH 7 or pH 9.7) to the unshaved backs of 6-wk-old B6C3F1 mice, twice a week for 6 wk, produced significant increase in weights of the liver of both sexes. The purpose of the present study was to determine if this weight change was related to oxidative stress subsequent to MWF exposure and also to determine whether ethanol intake influences this effect. Therefore, 6-mo-old mice of both sexes were exposed to MWFs following the protocol just described, except that the topical application was with 5% MWFs (pH 7 and 9.7, 5 d/wk) with or without adding 5% ethanol to their drinking water (7 d/wk) for 13 wk. The skin histamine levels and mast-cell numbers were significantly increased in the female group treated with 5% MWF (pH 7). The ascorbic acid levels in the liver (both sexes) (all groups except 5% MWF pH 9.7 males) and testes were reduced significantly. Malondialdehyde levels in the male liver were significantly increased with topical MWF exposure. Glutathione levels were reduced significantly in both male and female liver after 5% MWF (pH 7). Alcohol dehydrogenase activity of the male liver increased significantly after MWF (pH 7). These results suggest that MWFs are absorbed through the skin and produce toxicity in the liver of both sexes and in the male gonads. This may represent an important health risk to MWF-exposed industrial workers, and ethanol may exacerbate this risk.     

Enhanced oxidative stress in the skin of vitamin E deficient mice exposed to semisynthetic metal working fluids

Metal working fluids (MWFs) are widely used in industry for metal cutting, drilling, shaping, lubricating, and milling. Many occupational health concerns have arisen for workers exposed to MWFs. It has been reported earlier that occupational exposure to MWFs causes allergic and irritant contact dermatitis. Previously, we have shown that dermal exposure of female and male B6C3F1 mice to 5% MWFs for 3 months resulted in accumulation of mast cells and elevation of histamine in the skin. Topical exposure to MWFs also resulted in elevated oxidative stress in the liver of both sexes and the testes in males. The goal of this study was to evaluate whether preexisting oxidative stress in the skin exacerbated mast cell influx after MWFs treatment. Oxidative stress in the skin of B6C3F1 mice was generated by dietary vitamin E deprivation. Mice were given vitamin E deficient (5-10 i.v./kg of vitamin E) or basal (50 i.v./kg of vitamin E) diets for 34 weeks. Topical treatment with MWFs (100 microl, 30%) started after 18 weeks of alimentary vitamin E deprivation. Histology of the skin after 16 weeks of exposure to MWFs revealed a 53% increase in mast cell accumulation in vitamin E deficient diets compared to mice given a vitamin E sufficient diet. Total antioxidant reserve in skin of vitamin E deprived mice treated with MWFs was decreased by 66% as compared to those mice given a vitamin E sufficient diet. GSH and protein thiols in the dermis of vitamin E deprived mice exposed to MWFs were also decreased 39 and 42%, respectively, as compared to mice given basal diet. This study clearly delineates the role of oxidative stress in enhancing mast cell accumulation caused by topical exposure to MWFs.     

Dermal and systemic toxicity after application of semisynthetic metal-working fluids in B6C3F1 mice

About 10 million industrial workers of both sexes are exposed to metal-working fluids (MWFs) via inhalation, skin or both. Our preliminary results, following dermal application of 200 microl of 50% unused (neat) semisynthetic MWF (pH 7 or pH 9.7) to the unshaved backs of 6-wk-old B6C3F1 mice, twice a week for 6 wk, produced significant increase in weights of the liver of both sexes. The purpose of the present study was to determine if this weight change was related to oxidative stress subsequent to MWF exposure and also to determine whether ethanol intake influences this effect. Therefore, 6-mo-old mice of both sexes were exposed to MWFs following the protocol just described, except that the topical application was with 5% MWFs (pH 7 and 9.7, 5 d/wk) with or without adding 5% ethanol to their drinking water (7 d/wk) for 13 wk. The skin histamine levels and mast-cell numbers were significantly increased in the female group treated with 55% MWF (pH 7). The ascorbic acid levels in the liver (both sexes) (all groups except 5%, MWF pH 9.7 males) and testes were reduced significantly. Malondialdehyde levels in the male liver were significantly increased with topical MWF exposure. Glutathione levels were reduced significantly in both male and female liver after 5% MWF (pH 7). Alcohol dehydrogenase activity of the male liver increased significantly after MWF (pH 7). These results suggest that MWFs are absorbed through the skin and produce toxicity in the liver of both sexes and in the male gonads. This may represent an important health risk to MWF-exposed industrial workers, and ethanol may exacerbate this risk.     

Metal working fluids: sub-chronic effects on pulmonary functions in B6C3F1 mice given vitamin E deficient and sufficient diets

Metal working fluids (MWFs) have been widely known to cause asthma and neoplasia of the larynx, pancreas, rectum, skin and urinary bladder (Textbook of Clinical Occupational and Environmental Medicine (1994) 814; Am. J. Ind. Med. 32 (1997) 240; Am. J. Ind. Med. 33 (1997) 282; Am. J. Ind. Med. 22 (1994) 185). Other non-neoplastic respiratory effects in industrial workers attributed to MWFs include increased rates of cough, phlegm production, wheeze, chronic bronchitis and chest tightness (Eur. J. Resir. Dis. 63(118) (1982), 79; J. Occup. Med. 24 (1982) 473; Am. J. Ind. Med. 32 (1997) 450). The epidemic and endemic nature of immune mediated lung morbidity commonly known as hypersensitivity pneumonitis in workers from several different industries using MWFs has been well documented (J. Allergy clin. Immunol. 91 (1993) 311; Chest 108 (1995) 636; MMWR45 (1996) 606; Am. J. Ind. Med. 32 (1997) 423). We studied morphological/functional and antioxidant outcomes in lungs after inhalation exposure of vitamin E deficient mice to MWF (27 mg m(-3) 17 weeks, 5 days a week, 6 h a day). Mice were given vitamin E deficient (<10 IU kg(-1) vitamin E) or basal diets (50 IU kg(-1) vitamin E) for 35 weeks. Inhalation exposure to MWF started after 18 weeks on diet. Microscopic observation of lungs from mice given vitamin E deficient or sufficient diets revealed no inflammation or morphological alteration after exposure to MWF. Mice given vitamin E deficient diet exhibited a significant decrease (P<0.05) in breathing rate, peak inspiratory/expiratory flow, minute ventilation, and tidal volume compared with sufficient controls. However, no differences were found after exposure to MWF in pulmonary function, with the exception of tidal volume which also significantly decreased (P<0.05). Exposure to MWF reduced vitamin E, protein thiol and ascorbate level in lungs. Exposure to MWF in combination with a vitamin E deficient diet resulted in significantly enhanced accumulation of peroxidative products compared with vitamin E deficient controls. This is the first report that describes the increase of oxidative stress in the lungs after MWF exposure.     

Effects of gasoline engine emissions on preexisting allergic airway responses in mice

Gasoline-powered vehicle emissions contribute significantly to ambient air pollution. We hypothesized that exposure to gasoline engine emissions (GEE) may exacerbate preexisting allergic airway responses. Male BALB/c mice were sensitized by injection with ovalbumin (OVA) and then received a 10-min aerosolized OVA challenge. Parallel groups were sham-sensitized with saline. Mice were exposed 6 h/day to air (control, C) or GEE containing particulate matter (PM) at low (L), medium (M), or high (H) concentrations, or to the H level with PM removed by filtration (high-filtered, HF). Immediately after GEE exposure mice received another 10-min aerosol OVA challenge (pre-OVA protocol). In a second (post-OVA) protocol, mice were similarly sensitized but only challenged to OVA before air or GEE exposure. Measurements of airway hyperresponsiveness (AHR), bronchoalveolar lavage (BAL), and blood collection were performed approximately 24 h after the last exposure. In both protocols, M, H, and HF GEE exposure significantly decreased BAL neutrophils from nonsensitized mice but had no significant effect on BAL cells from OVA-sensitized mice. In the pre-OVA protocol, GEE exposure increased OVA-specific IgG(1) but had no effect on BAL interleukin (IL)-2, IL-4, IL-13, or interferon (IFN)-gamma in OVA-sensitized mice. Nonsensitized GEE-exposed mice had increased OVA-specific IgG(2a), IgE, and IL-2, but decreased total IgE. In the post-OVA protocol, GEE exposure reduced BAL IL-4, IL-5, and IFN-gamma in nonsensitized mice but had no effect on sensitized mice. These results suggest acute exposure to the gas-vapor phase of GEE suppressed inflammatory cells and cytokines from nonsensitized mice but did not substantially exacerbate allergic responses.     

Skin absorption of six performance amines used in metalworking fluids

Every year, 10 million workers are exposed to metalworking fluids (MWFs) that may be toxic. There are four types of MWFs: neat oils and three water‐based MWFs (soluble oil, semisynthetic and synthetic), which are diluted with water and whose composition varies according to the mineral oils ratio. MWFs also contain various additives. To determine the absorption of six amines used as corrosion inhibitors and biocides in MWFs, porcine skin flow‐through diffusion cell experiments were conducted with hydrophilic ethanolamines (mono‐, di‐ and triethanolamine, MEA, DEA and TEA respectively) and a mixture of lipophilic amines (dibutylethanolamine, dicyclohexylamine and diphenylamine). The six amines were dosed in four vehicles (water and three generic water‐based MWF formulations) and analyzed using a scintillation counter or gas chromatography/mass spectrometry. These 24 h studies showed that dermal absorption significantly (P < 0.05) increased from water for the six amines (e.g. 1.15 ± 0.29% dose; DEA in water) compared to other formulations (e.g. 0.13 ± 0.01% dose; DEA in semisynthetic MWF) and absorption was greatest for dibutylethanolamine in all the formulations. The soluble oil formulation tended to increase the dermal absorption of the hydrophilic amines. The permeability coefficient was significantly higher (P < 0.05) with TEA relative to the other hydrophilic amines (e.g. 4.22 × 10^–4 ± 0.53 × 10^–4 cm h^–1 [TEA in synthetic MWF] vs. 1.23 × 10^–4 ± 0.10 × 10^–4 cm h^–1 [MEA in synthetic MWF]), except for MEA in soluble oil formulation. Future research will confirm these findings in an in vivo pig model along with dermatotoxicity studies. These results should help MWF industries choose safer additives for their formulations to protect the health of metalworkers. Copyright © 2014 John Wiley & Sons, Ltd.     

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

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

Enhancement of the pulmonary allergic granulocyte recruitment in rats exposed to DMTI-II, a Kunitz-type inhibitor isolated from Dimorphandra mollis seeds

DMTI-II (23-kDa trypsin inhibitor purified from Dimorphandra mollis seeds) promotes acute inflammation accompanied by an early infiltration of eosinophils, a critical cell type involved in allergic diseases. We have evaluated here the capacity of DMTI-II to enhance the allergic pulmonary inflammation, looking over time to the leukocyte trafficking from bone marrow to peripheral blood, and their recruitment into the allergic airways. Male Wistar rats were sensitized and challenged with ovalbumin (OVA). At 2 to 16h prior to OVA challenge, animals were exposed to DMTI-II (10μg). Bronchoalveolar lavage fluid (BAL), circulating blood and bone marrow were examined at 24h post-OVA challenge. Challenge with OVA significantly increased the influx of total inflammatory cells, neutrophils and eosinophils in BAL and lung tissue. Pre-exposure to DMTI-II potentiated total inflammatory cell and neutrophil recruitment (p<0.05). Neutropoiesis and neutrophilia accompanied pulmonary cell influx. Pre-exposure to DMTI-II also significantly increased eosinophil recruitment to BAL, an effect starting at 4h, remaining markedly elevated at 16h (p<0.05). Eosinopoiesis and eosinophilia (seen within 2 to 4h) were also observed. Exposure to DMTI-II alone increased the IL-4 levels, and further increased the IL-4 levels in OVA-challenged rats. The levels of IgE, LTB(4) and eotaxin in OVA-challenged rats were greater compared with non-sensitized rats, but DMTI-II exposure failed to further enhance such levels. In summary, our study shows that DMTI-II itself presents granulocytopoietic activity, and enhances allergen-induced neutrophil and eosinophil mobilization from bone marrow to lung tissues that is accompanied by enhanced IL-4 production.     

Airway inflammation and adjuvant effect after repeated airborne exposures to di-(2-ethylhexyl)phthalate and ovalbumin in BALB/c mice

BACKGROUND: Epidemiological studies have suggested an association between exposure to phthalate plasticizers, including di-(2-ethylhexyl)phthalate (DEHP), and increased prevalence of asthma, rhinitis or wheezing. Furthermore, studies in mice have demonstrated an adjuvant effect from DEHP after parenteral administration with the model allergen ovalbumin (OVA). OBJECTIVE: Exposures to DEHP were investigated for adjuvant effects and airway inflammation in a mouse inhalation model. METHODS: BALB/cJ mice were exposed to aerosols of 0.022-13 mg/m(3) DEHP and 0.14 mg/m(3) OVA 5 days/week for 2 weeks and thereafter weekly for 12 weeks. Mice exposed to OVA alone or OVA+Al(OH)(3) served as control groups. Finally, all groups were exposed to a nebulized 1% OVA solution on three consecutive days. Serum, bronchoalveolar lavage (BAL) fluid, and draining lymph nodes were collected 24h later. RESULTS: In the OVA+Al(OH)(3) group, significantly increased levels of OVA-specific IgE and IgG1 in serum as well as of eosinophils in BAL fluid were observed. DEHP affected OVA-specific IgG1 production in a concentration-dependent manner, whereas little effect was seen on IgE and IgG2a. Dose-dependent increases in inflammatory cells were observed in BAL fluids, leading to significantly higher lymphocyte, neutrophil and eosinophil numbers in the OVA+13 mg/m(3) DEHP group. Ex vivo cytokine secretion by cultures of draining lymph nodes suggested that DEHP has a mixed Th1/Th2 cytokine profile. CONCLUSION: Airborne DEHP is able to increase serum IgG1 and lung inflammatory cell levels, but only at very high concentrations. Realistic DEHP levels do not have an adjuvant effect or induce allergic lung inflammation in the present mouse model.     

Adjuvant effects of inhaled mono-2-ethylhexyl phthalate in BALB/cJ mice

Phthalates, including di(2-ethylhexyl) phthalate (DEHP), are widely used and have been linked with the development of wheezing and asthma. The main metabolite of DEHP, mono-2-ethylhexyl phthalate (MEHP), was investigated for adjuvant effects in a mouse inhalation model. BALB/cJ mice were exposed to aerosols of 0.03 or 0.4 mg/m(3) MEHP 5 days/week for 2 weeks and thereafter weekly for 12 weeks together with a low dose of ovalbumin (OVA) as a model allergen. Mice exposed to OVA alone or OVA+Al(OH)(3) served as negative and positive controls, respectively. Finally, all groups were exposed to a nebulized 1% OVA solution on 3 consecutive days to investigate the development of an inflammatory response. Serum, bronchoalveolar lavage (BAL) fluid, and draining lymph nodes were collected 24h later. In the OVA+Al(OH)(3) group, significantly increased levels of OVA-specific IgE and IgG1 in serum as well as of eosinophils in BAL fluid were observed. OVA-specific IgG1 production in both MEHP groups was significantly increased. OVA-specific IgE and IgG2a were not increased significantly. A dose-dependent increase in inflammatory cells was observed in BAL fluid, leading to significantly higher lymphocyte and eosinophil numbers in the OVA+0.4 mg/m(3) MEHP group. Ex vivo cytokine secretion by cultures of draining lymph nodes suggested a T(H)2 profile of MEHP. In conclusion, MEHP acted as a T(H)2 adjuvant after inhalation. However, it is suggested that the inflammation in the MEHP groups was primarily mediated by an IgG1-dependent mechanism. To address implications for humans, a margin-of-exposure was estimated based on the lack of significant effects on IgE production and inflammation after exposures to 0.03 mg/m(3) MEHP observed in the present study and estimated human exposure levels.     

Effects of sub-chronic exposure to palladium (as potassium hexachloro-palladate) on cytokines in male Wistar rats

Palladium (Pd) is a heavy metal belonging to the platinum group elements. It has been shown that Pd and its compounds can cause sensitization, asthma and dermatologic disorders. In this study, Wistar rats were exposed for 3 months to 0, 1, 10, 100, and 250 ng/ml of Pd (as potassium hexachloro-palladate) in drinking water. At the end of exposure, possible changes in two type-1 cytokines [interleukin (IL)-2, interferon (INF)-gamma] and one type-2 cytokine (IL-4) in the serum were measured. After the sub-chronic exposure, analysis of variance of IL-2 and INF-gamma response shows that the difference between mean values in the groups of animals exposed reaches statistical significance for IL-2 (P = 0.001), showing a J-shaped dose-response relationship. At the higher dose of 250 ng/ml Pd, it was observed a significant increase in IL-2 production when compared with the controls. Furthermore at 1 and 10 ng/ml of Pd we observed an increase of INF-gamma production that was significant at 100 ng/ml of Pd, while at the higher dose of 250 ng/ml of Pd the response was indistinguishable from the control. At the doses investigated, Pd has been shown not to determine a modification of IL-4 response (P = 0.10). These preliminary findings indicate the need to carry out further investigations regarding the effect of other Pd compounds and the measurement of other types of cytokines both in the animal model used in our study and other models.     

An approach for evaluating the respiratory irritation of mixtures: application to metalworking fluids

 Recently, the sensory and pulmonary irritating properties of ten metalworking fluids (MWF) were assessed using a mouse bioassay. Relative potency of the MWFs was estimated, but it was not possible to identify the component(s) responsible for the the respiratory irritation induced by each MWF. One of the ten fluids, MWF “E”, produced sensory and pulmonary irritation in mice, and it was of moderate potency in comparison to the other nine MWFs. MWF “E” had three major components: tall oil fatty acids (TOFA), sodium sulfonate (SA), and paraffinic oil (PO). In the present study, the sensory and pulmonary irritating properties of these individual components of MWF “E” were evaluated. Mixtures of the three components were also prepared and similarly evaluated. This analysis revealed that the sensory irritation from MWF “E” was largely due to TOFA, whereas SA produced the pulmonary irritation observed with MWF “E”. Both TOFA and SA were more potent irritants than was MWF “E”, and the potency of TOFA and/or SA was diminished through combination with PO. There was no evidence of synergism of the components when combined to form MWF “E”. This approach for identifying the biologically “active” component(s) in a mixture should be useful for other MWFs. Furthermore, the approach should be easily adapted for other applications involving concerns with mixtures. Received: 2 November 1994/Accepted: 16 March 1995     

Effects of Gasoline Engine Emissions on Preexisting Allergic Airway Responses in Mice

Gasoline-powered vehicle emissions contribute significantly to ambient air pollution. We hypothesized that exposure to gasoline engine emissions (GEE) may exacerbate preexisting allergic airway responses. Male BALB/c mice were sensitized by injection with ovalbumin (OVA) and then received a 10-min aerosolized OVA challenge. Parallel groups were sham-sensitized with saline. Mice were exposed 6 h/day to air (control, C) or GEE containing particulate matter (PM) at low (L), medium (M), or high (H) concentrations, or to the H level with PM removed by filtration (high-filtered, HF). Immediately after GEE exposure mice received another 10-min aerosol OVA challenge (pre-OVA protocol). In a second (post-OVA) protocol, mice were similarly sensitized but only challenged to OVA before air or GEE exposure. Measurements of airway hyperresponsiveness (AHR), bronchoalveolar lavage (BAL), and blood collection were performed ～24 h after the last exposure. In both protocols, M, H, and HF GEE exposure significantly decreased BAL neutrophils from nonsensitized mice but had no significant effect on BAL cells from OVA-sensitized mice. In the pre-OVA protocol, GEE exposure increased OVA-specific IgG₁ but had no effect on BAL interleukin (IL)-2, IL-4, IL-13, or interferon (IFN)-γ in OVA-sensitized mice. Nonsensitized GEE-exposed mice had increased OVA-specific IgG_2a, IgE, and IL-2, but decreased total IgE. In the post-OVA protocol, GEE exposure reduced BAL IL-4, IL-5, and IFN-γ in nonsensitized mice but had no effect on sensitized mice. These results suggest acute exposure to the gas–vapor phase of GEE suppressed inflammatory cells and cytokines from nonsensitized mice but did not substantially exacerbate allergic responses.     

Developmental immunotoxicity of dexamethasone: comparison of fetal versus adult exposures

Dexamethasone-21 phosphate was administered (s.c.) to pregnant CD rats at days 6-21 of gestation (0, 0.0625, 0.125, 0.25, and 0.5 mg/kg/day) with identical exposure of non-pregnant adult females. Some reproductive (anogenital distance) and growth (body weight) measures of pups were altered. In the juvenile (5 weeks), the delayed type hypersensitivity response to KLH was significantly reduced at all doses examined and this pattern continued into adulthood (13 weeks). In contrast, the DTH response of adults exposed to DEX was unaltered even at the highest dose. Few DEX-induced changes were seen in offspring or adult blood parameters or in splenocytes analyzed for cell surface makers (by flow cytometry). The thymus of both exposed pups (both ages) and adults showed a marked reduction in the medulla/lobe area beginning with the 0.125 mg/kg/day DEX exposure level. Macrophage production of TNF and NO was only marginally affected as was splenocyte production of IL-4 and IFN-gamma. In contrast, pups assessed as juveniles were significantly depressed in splenic IL-2 and IL-10 production. DEX exposure altered serum antibody levels across age groups with an increase of KLH-specific IgG (beginning with the 0.0125 mg/kg/day dose) while total IgE was reduced. These results suggest that while DEX exposure produces some common alterations following in utero versus adult exposure, fetal exposure (even at the lowest doses tested) produces marked and persistent functional loss (DTH) not evident in exposed adults. Furthermore, there was no apparent advantage in delaying immune assessment until the offspring reached adulthood.     

Development of pulmonary tolerance in mice exposed to zinc oxide fumes.

As a result of repeated exposures to inhaled toxicants such as zinc oxide (ZnO), numerous individuals acquire tolerance to the exposures and display reduced symptoms. To ascertain whether tolerance is developed in an animal model, NIH-Swiss mice were exposed to 1.0 mg/m(3) ZnO for 1, 3, or 5 days (1X, 3X, or 5X), and polymorphonuclear leukocyte (PMN) and protein levels in bronchoalveolar lavage (BAL) were measured. Mice acquired tolerance to neutrophil infiltration into the lungs, as total PMNs returned near baseline in 5X-exposed animals as compared to that of the 1X exposure group (1X = 2.7 +/- 0.4 x 10(4), 5X = 0.2 +/- 0.1 x 10(4), mean +/- SE, p < 0.05). Development of tolerance to changes in lavageable protein, however, was not observed (1X = 313 +/- 29 microg/ml, 5X = 684 +/- 71 microg/ml, p < 0.05). Tolerance to PMN influx did not persist following re-exposure to ZnO after 5 days of rest. In contrast to ZnO exposure, following single and repeated exposure to aerosolized endotoxin there was development of tolerance to protein in BAL (1X = 174 +/- 71 microg/ml, 5X = 166 +/- 14 microg/ml, p > 0.05), but not to PMN influx (1X = 5.5 +/- 1.7 x 10(4), 13.9 +/- 1.7 x 10(4), p < 0.05). Induction of lung metallothionein (MT) was also observed in mice exposed once or repeatedly exposed to ZnO, suggesting that MT may play a role in its molecular mechanism.     

Time course of pulmonary response of rats to inhalation of crystalline silica: NF-kappa B activation,inflammation, cytokine production,and damage

In vitro studies suggest that silica-induced lung disease may be linked to processes regulated by nuclear factor-kappa B (NF-kappa B) activation, but this has not been examined in vivo. Rats were exposed to a silica aerosol of 15 mg/m(3) (6 h/day, 5 days/wk) for 116 days, and bronchoalveolar lavage (BAL) was conducted at various times during the exposure. Silica-induced pulmonary inflammation and damage were determined by measuring BAL cell differentials and first BAL fluid lactate dehydrogenase (LDH) activity and serum albumin concentrations, respectively. NF-kappa B activation and production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) by BAL cells were also measured. The results demonstrate that NF-kappa B activation occurred after 5 days exposure, and continued to increase thereafter. BAL cell production of IL-1 and TNF-alpha had increased incrementally by 10 and 30 days of exposure, respectively. This elevation continued through 79 days of exposure before further increasing at 116 days of exposure. Pulmonary inflammation and damage in silica-exposed rats were also significantly elevated at 5 days of exposure, further increased at a slow rate through 41 days of exposure, and dramatically increased thereafter. Taken together, the results indicate that the initial molecular response of NF-kappa B activation in BAL cells occurs in response to low levels of silica deposition in the lung and increases more rapidly versus exposure duration than silica-induced pulmonary inflammation, cellular damage, and cytokine production by BAL cells. This suggests that NF-kappa B activation in BAL cells may play an important role in the initiation and progression of silica-induced pulmonary inflammation, cellular damage, and fibrosis.     

Differences in pulmonary biochemical and inflammatory responses of rats and guinea pigs resulting from daytime or nighttime, single and repeated exposure to ozone.

Rats and guinea pigs were exposed to 0.8 mg ozone (O3)/m3 (approximately 0.4 ppm) for 12 hr during the daytime, 12 hr during the nighttime, or continuously to investigate circadian variation in O3-induced pulmonary toxicity during single and repeated O3 exposures. Biomarkers in bronchoalveolar lavage (BAL) fluid and lung tissues were measured as indicators of biochemical and inflammatory responses. Nighttime O3 exposure of rats resulted in larger increases of protein, albumin, and inflammatory cells in BAL fluid compared to those after daytime O3 exposure and this daytime-nighttime difference was statistically significant (p < 0.05). Single daytime or nighttime O3 exposure of guinea pigs resulted in comparable increases of BAL fluid proteins and inflammatory cells without a daytime-nighttime difference. Nighttime and continuous O3 exposure of rats for 3 days resulted in comparable increases in lung antioxidant enzyme activities, both of which differed statistically from effects from daytime O3 exposures (p < 0.05). Continuous O3 exposure of guinea pigs for 3 days caused, in general, statistically larger increases in lung tissue parameters compared to nighttime O3 exposures (p < 0.05). These results suggest that the extent of O3-induced acute pulmonary biochemical and inflammatory responses is directly related to the level of physical and respiratory activity. For rats, effects from continuous O3 exposure appear to be controlled by the nighttime, physically active period. In guinea pigs, the comparable responses following daytime or nighttime O3 exposure seem in accordance with their random behavioral daily activity pattern. This study supports the view that physical activity-related increases in inhaled dose significantly enhance the pulmonary O3 responses.     

Higher faecal excretion and lower tissue accumulation of mercury in Wistar rats from contaminated fish than from methylmercury chloride added to fish.

A short-term low level exposure experiment was conducted on rats in order to determine urinary and faecal excretion, accumulation, and biological responses to methylmercury from fish products. Male Wistar rats were fed fish-meal diets containing methylmercury contaminated fish (1.45 or 2.61 mgHg/kg as methylmercury), uncontaminated fish supplemented with methylmercury chloride (CH3HgCl) at similar levels (1.24 and 2.49 mgHg/kg, respectively) or uncontaminated fish as a control (0.052 mgHg/kg) for 4 weeks (n=6 rats per treatment). After 2 and 4 weeks of exposure, rats were placed in metabolic chambers for 48 h to assess overall faecal and urinary excretion of mercury. The overall faecal excretion in rats fed fish supplemented with CH3HgCl (12%) was significantly lower (P <0.05) than rats fed methylmercury in fish muscle (19%) or rats fed control diet (76%). Urinary excretion did not differ among the experimental groups. Rats fed the highest level of CH3HgCl had a significantly higher (P <0.05) blood, liver, kidney and brain mercury contamination compared to rats fed methylmercury contaminated fish or rats fed control diet. Metallothionein levels in kidney were significantly higher in CH3HgCl-fed rats compared to rats fed contaminated fish. The results indicate a higher faecal excretion and lower tissue accumulation, and metallothionein induction in rats following exposure to methylmercury naturally incorporated in fish compared to methylmercury chloride added to the same matrix.