Effects of Simulated Smog Atmospheres in Rodent Models of Metabolic and Immunologic Dysfunction.

Air pollution is a diverse and dynamic mixture of gaseous and particulate matter, limiting our understanding of associated adverse health outcomes. The biological effects of two simulated smog atmospheres (SA) with different compositions but similar air quality health indexes were compared in a nonobese diabetic rat model (Goto-Kakizaki, GK) and three mouse immune models (house dust mite (HDM) allergy, antibody response to heat-killed pneumococcus, and resistance to influenza A infection). In GK rats, both SA-PM (high particulate matter) and SA-O3 (high ozone) decreased cholesterol levels immediately after a 4-h exposure, whereas only SA-O3 increased airflow limitation. Airway responsiveness to methacholine was increased in HDM-allergic mice compared with nonallergic mice, but exposure to SA-PM or SA-O3 did not significantly alter responsiveness. Exposure to SA-PM did not affect the IgM response to pneumococcus, and SA-O3 did not affect virus titers, although inflammatory cytokine levels were decreased in mice infected at the end of a 7-day exposure. Collectively, acute SA exposures produced limited health effects in animal models of metabolic and immune diseases. Effects of SA-O3 tended to be greater than those of SA-PM, suggesting that gas-phase components in photochemically derived multipollutant mixtures may be of greater concern than secondary organic aerosol PM.

Effects of Libby amphibole asbestos exposure on two models of arthritis in the Lewis rat.

Epidemiological data suggest that occupational exposure to the amphibole-containing vermiculite in Libby, MT, was associated with increased risk for developing autoimmune diseases and had an odds ratio of 3.23 for developing rheumatoid arthritis (RA). The collagen-induced arthritis (CIA) and the peptidoglycan-polysaccharide (PG-PS) models of RA were employed to determine whether exposure to Libby amphibole (LA) induced a more rapid onset, increased expression, or prolonged course of RA. Female Lewis rats were intratracheally instilled with total doses of 0.15, 0.5, 1.5, or 5 mg LA or 0.5 or 1.5 mg amosite asbestos, and arthritis was induced with either the PG-PS or CIA model. Neither LA nor amosite exposure affected the disease course in the CIA model, or the production of rheumatoid factor (RF) or anti-cyclic citrullinated peptide (CCP) antibodies. LA exposure reduced swelling in the PG-PS model and decreased anti-PG-PS and total immunoglobulin M (IgM) antibody titers. Both amosite and LA exposure increased the number of rats with circulating anti-nuclear antibodies (ANA), the majority of which presented a speckled staining pattern. However, this ANA enhancement was not dose responsive. These results failed to show a positive correlation between LA exposure and RA disease in two animal models, although upregulated ANA suggest an altered immunological profile consistent with other systemic autoimmune diseases.

Perfluorooctanoic acid-induced immunomodulation in adult C57BL/6J or C57BL/6N female mice.

BACKGROUND: Perfluorooctanoic acid (PFOA), an environmentally persistent compound of regulatory concern, has been reported to reduce antibody responses in mice at a single dose. OBJECTIVE: The aim of this study was to evaluate PFOA effects on humoral and cellular immunity using standard assays for assessing immune function, and to derive dose-response data. METHODS: C57BL/6J mice received 0 or 30 mg PFOA/kg/day for 10 days; half of the exposed groups were switched to vehicle and half continued on PFOA for five days. C57BL/6N mice received 0-30 mg/kg/day of PFOA in drinking water for 15 days. Mice were immunized with sheep red blood cells or sensitized to bovine serum albumin in Freund's complete adjuvant on day 10 of exposure; immune responses were determined 1 day post-exposure. RESULTS: We found that 30 mg PFOA/kg/day given for 10 or 15 days reduced IgM synthesis; serum collected 1 day postexposure contained 8.4 x 10(4) or 2.7 x 10(5) ng PFOA/mL, respectively. IgM synthesis was suppressed at exposures > or = 3.75 mg PFOA/kg/day in a dose-dependent manner, and IgG titers were elevated at 3.75 and 7.5 mg PFOA/kg/day. Serum PFOA at 3.75 mg/kg/day was 7.4 x 10(4) ng/mL 1 day postexposure, or 150-fold greater than the levels reported in individuals living near a PFOA production site. Using a second-degree polynomial model, we calculated a benchmark dose of 3 mg/kg/day, with a lower bound (95% confidence limit) of 1.75 mg/kg/day. Cell-mediated function was not affected. CONCLUSIONS: IgM antibodies were suppressed after PFOA exposure. The margin of exposure for reduced IgM antibody synthesis was approximately 150 for highly exposed human populations.

An organotin mixture found in polyvinyl chloride (PVC) pipe is not immunotoxic to adult Sprague-Dawley rats.

Organotin compounds used in polyvinyl chloride (PVC) pipe production are of concern to the U.S. Environmental Protection Agency (EPA) because they leach from supply pipes into drinking water and are reported multisystem toxicants. Immune function was assessed in male Sprague-Dawley rats exposed to the mixture of organotins used in PVC pipe production. Although several of these organotins are reported immunotoxicants, their immunotoxicity as a mixture when given by drinking water has not been evaluated. Adult male rats were given drinking water for 28 d containing a mixture of dibutyltin dichloride (DBTC), dimethyltin dichloride (DMTC), monobutyltin trichloride (MBT), and monomethyltin trichloride (MMT) in a 2:2:1:1 ratio, respectively, at 3 different concentrations (5:5:2.5:2.5, 10:10:5:5, or 20:20:10:10 mg organotin/L), MMT alone (20 or 40 mg MMT/L), or plain water as a control. Delayed-type hypersensitivity, antibody synthesis, and natural killer cell cytotoxicity were evaluated in separate endpoint groups (n = 8/dose; 24/endpoint) immediately after exposure ended. The evaluated immune functions were not affected by the mixture or by MMT alone. Our data suggest that immunotoxicity is unlikely to result from the concentration of organotins present in drinking water delivered via PVC pipes, as the concentrations used were several orders of magnitude higher than those expected to leach from PVC pipes.

Immune function is not impaired in Sprague-Dawley rats exposed to dimethyltin dichloride (DMTC) during development or adulthood.

Organotins are used commercially as pesticides, antifouling agents and stabilizers for polyvinyl chloride (PVC) pipe. Mono- and di-substituted butyltins, used in PVC pipe production, are of concern to the US EPA because they leach from supply pipes into drinking water and are reported multisystem toxicants. We assessed several immune functions in Sprague-Dawley rats after adult or developmental dimethyltin dichloride (DMTC) exposure because various organotins have been reported to be immunotoxic. Adult male and female rats were given drinking water containing 0, 20 or 40 mg DMTC/L (0, 1.7, or 3.4 mg DMTC/kg body weight [BW]) for 28 days. Pregnant females were given the same DMTC drinking water concentrations for a total of 37 days, from gestational day (GD) six through weaning of pups (0, 2.4, or 4.6 mg DMTC/kg BW during gestational exposure; 0, 3.6, or 6.9 mg DMTC/kg BW during postnatal exposure). On postnatal day (PND) two, litters were sexed, weighed, and culled to four males and four females per dam. Delayed-type hypersensitivity (DTH), antibody synthesis, and natural killer (NK) cell activity were evaluated in adults (N=8/sex/group) and in immunologically mature offspring (N=6/sex/group). Although water consumption was decreased in all of the DMTC dose groups, the immune functions evaluated were not affected. Our data suggest that these immune functions are not sensitive to the levels of DMTC anticipated to occur in drinking water delivered via PVC pipe as the concentrations we used were several orders of magnitude higher than those expected to leach from PVC pipes.

Toxicological assessments of rats exposed prenatally to inhaled vapors of gasoline and gasoline-ethanol blends.

The primary alternative to petroleum-based fuels is ethanol, which may be blended with gasoline in the United States at concentrations up to 15% for most automobiles. Efforts to increase the amount of ethanol in gasoline have prompted concerns about the potential toxicity of inhaled ethanol vapors from these fuels. The well-known sensitivity of the developing nervous and immune systems to ingested ethanol and the lack of information about the neurodevelopmental toxicity of ethanol-blended fuels prompted the present work. Pregnant Long-Evans rats were exposed for 6.5h/day on days 9-20 of gestation to clean air or vapors of gasoline containing no ethanol (E0) or gasoline blended with 15% ethanol (E15) or 85% ethanol (E85) at nominal concentrations of 3000, 6000, or 9000 ppm. Estimated maternal peak blood ethanol concentrations were less than 5mg/dL for all exposures. No overt toxicity in the dams was observed, although pregnant dams exposed to 9000 ppm of E0 or E85 gained more weight per gram of food consumed during the 12 days of exposure than did controls. Fuel vapors did not affect litter size or weight, or postnatal weight gain in the offspring. Tests of motor activity and a functional observational battery (FOB) administered to the offspring between post-natal day (PND) 27-29 and PND 56-63 revealed an increase in vertical activity counts in the 3000- and 9000-ppm groups in the E85 experiment on PND 63 and a few small changes in sensorimotor responses in the FOB that were not monotonically related to exposure concentration in any experiment. Neither cell-mediated nor humoral immunity were affected in a concentration-related manner by exposure to any of the vapors in 6-week-old male or female offspring. Systematic concentration-related differences in systolic blood pressure were not observed in rats tested at 3 and 6 months of age in any experiment. No systematic differences were observed in serum glucose or glycated hemoglobin A1c (a marker of long-term glucose homeostasis). These observations suggest a LOEL of 3000 ppm of E85 for vertical activity, LOELs of 9000 ppm of E0 and E85 for maternal food consumption, and NOELs of 9000 ppm for the other endpoints reported here. The ethanol content of the vapors did not consistently alter the pattern of behavioral, immunological, or physiological responses to the fuel vapors. The concentrations of the vapors used here exceed by 4-6 orders of magnitude typical exposure levels encountered by the public.

Mortality in dioxin-exposed mice infected with influenza: mitochondrial toxicity (reye's-like syndrome) versus enhanced inflammation as the mode of action.

Increased mortality following influenza A infection was reported in B6C3F1 mice exposed to a low (0.01 micro g/kg) dose of dioxin. However, mortality was not associated with increased viral load and antibody titers to the virus were not decreased at doses of TCDD < or = 10 micro g/kg, suggesting that viral overgrowth, secondary to immunosuppression, was not the proximate cause of death. We tested the hypothesis that mitochondrial toxicity and dysfunction, similar to Reye's syndrome (RS) in humans, is responsible for increased mortality in dioxin-exposed, infected B6C3F1 female mice, based on similarities in the biochemical and immunological events that occur in RS and in TCDD-exposed animals. Endpoints were also included to test the hypothesis that increased pulmonary inflammation following dioxin exposure, in the absence of mitochondrial toxicity, was associated with increased mortality. Dose-related effects of TCDD alone, infection with influenza A alone, and combined TCDD exposure/influenza infection were evaluated. Mice were given a single ip injection of 0, 0.001, 0.01, 0.1, or 1.0 micro g TCDD/kg, 7 days before infection by intranasal instillation of an estimated LD(10-20) of influenza A Hong Kong/8/68 (H3N2) and were terminated 1, 7, and 10 days after infection. Serum, bronchoalveolar lavage fluid (BALF), and lung tissue were collected for various measurements, including clinical chemistries, cell counts, cytokine analysis, and viral titers. Exposure to < or = 1.0 micro g TCDD/kg did not increase mortality; virus titers were similar at all doses of TCDD and there was no dioxin-related effect on serum NH(3) or glucose concentrations, two prominent indicators of the altered mitochondrial oxidative metabolism typically observed in RS. A study was therefore conducted over a wider range of TCDD doses. A single injection of 0, 0.025, 0.5, or 10 micro g TCDD/kg preceded infection by 7 days; subgroups of noninfected control and highest dose group (10 micro g TCDD/kg) mice were also evaluated for biochemical and immunological endpoints on the equivalent of infection day 4 to provide baseline data. Five days after infection the same endpoints described above were evaluated. The 10 micro g TCDD/kg dose increased mortality, but once again did not increase virus titer; as in previous experiments, serum biochemistry endpoints did not support mitochondrial dysfunction. These results suggest that RS is an unlikely explanation for increased influenza mortality in TCDD-exposed mice. Rather, constituents in BALF implicate increased pulmonary inflammation as the mode of TCDD action.

Toxicological outcomes in rats exposed to inhaled ethanol during gestation.

Recent legislation has encouraged replacing petroleum-based fuels with renewable alternatives including ethanol, which is typically blended with gasoline in the United States at concentrations up to 10%, with allowances for concentrations up to 85% for some vehicles. Efforts to increase the amount of ethanol in gasoline have prompted concerns about the potential toxicity of inhaled ethanol vapors from these fuels. The well-known sensitivity of the developing nervous and immune systems to ingested ethanol, and the lack of information about its toxicity by inhalation prompted the present work on its potential developmental effects in a rat model. Pregnant Long-Evans rats were exposed for 6.5h/day on days 9-20 of gestation to clean air or ethanol vapor at concentrations of 5000, 10,000, or 21,000 ppm, which resulted in estimated peak blood ethanol concentrations (BECs) of 2.3, 6.7, and 192 mg/dL, respectively. No overt toxicity in the dams was observed. Ethanol did not affect litter size or weight, or postnatal weight gain in the pups. Motor activity was normal in offspring through postnatal day (PND) 29. On PND 62, the 5000 and 21,000 ppm groups were more active than controls. On PND 29 and 62, offspring were tested with a functional observational battery, which revealed small changes in the neuromuscular and sensorimotor domains that were not systematically related to dose. Cell-mediated and humoral immunity were not affected by ethanol exposure in 6-week-old offspring. Systolic blood pressure was increased by 10,000 ppm ethanol in males at PND 90 but not at PND 180. No differences in lipoprotein profile, liver function, or kidney function were observed. In summary, prenatal exposure to inhaled ethanol caused some mild changes in physiological and behavioral development in offspring that were not clearly related to inhaled concentration or BEC, and did not produce detectable changes in immune function. This low toxicity of inhaled ethanol may result from the slow rise in BEC by the inhalation route.

Evaluation of anti-nuclear antibodies and kidney pathology in Lewis rats following exposure to Libby amphibole asbestos.

The prevalence of anti-nuclear antibodies (ANA) and self-reported systemic autoimmune diseases were increased in residents of Libby, MT, as was the incidence of ANA in Lewis rats exposed to Libby amphibole (LA) asbestos. However, rats induced to develop rheumatoid arthritis (RA) did not develop autoantibodies associated with RA, nor was RA exacerbated by LA exposure, suggesting that increased ANA expression might be related to some other autoimmune process. Libby residents self-reported increased numbers of physician-diagnosed cases of systemic lupus erythematosus (SLE). Thus, the goal of this study was to determine if the increased incidence of ANA in Lewis rats exposed to LA is related to the development of SLE-like disease. Female Lewis rats were intratracheally instilled bi-weekly for 13 weeks with total doses of 0.15, 0.5, 1.5, or 5.0 mg of LA or 0.5 or 1.5 mg of a positive control fiber, amosite. ANA incidence was significantly increased in all asbestos dose groups, although no dose response was observed. The occurrence of proteinuria was increased in LA 0.5, LA 5.0, and amosite 0.5 dose groups; however, the microscopic appearance of the kidneys was normal, no binding of autoimmune complexes to glomerular surfaces was observed, and antibodies to double-stranded DNA were not elevated. Therefore, an increased prevalence of ANA in rats exposed to asbestos does not appear to correlate with disease markers typically observed in SLE. Analysis of ANA specificity for extractable nuclear antigens (ENA) determined that 98% of ENA(+) samples were specific for the Jo-1 antigen. Autoantibodies to Jo-1 have been reported in patients with interstitial lung disease, suggesting that autoantibodies to Jo-1 may be a biomarker for asbestos-related pulmonary disease.

Suppression of humoral immunity by perfluorooctanoic acid is independent of elevated serum corticosterone concentration in mice.

The T-cell-dependent antibody response is suppressed in mice exposed to 3.75, 7.5, 15, and 30 mg PFOA (perfluorooctanoic acid)/kg body weight (bw). Reduced bw accompanied immunosuppression at 15 and 30 mg/kg. We investigated the hypothesis that the observed immunosuppression is secondary to elevated serum corticosterone levels by assessing immune function in adrenalectomized (adx) or sham-operated C57BL/6N female mice exposed to 0, 7.5, or 15 mg PFOA/kg bw in drinking water for 10 days. Bw, primary antibody responses to a T-dependent antigen, clinical serum chemistries related to liver health, and serum corticosterone levels were evaluated. Exposure to 15 mg/kg decreased bw by approximately 10% after 8 days of dosing and until 2 days postdosing in both adx and sham animals; bw of adx animals were still reduced 5 days postdosing. IgM antibody titers were statistically reduced by 15% in sham animals and 18% in adx animals exposed to 15 mg/kg and by 11.8% in adx animals exposed to 7.5 mg/kg. Corticosterone concentrations were elevated by 157% in dosed sham animals relative to control animals and were reduced by 27% in dosed adx animals relative to control animals (neither changes were statistically significant). Clinical serum chemistries related to liver health were not statistically altered by either dose or adrenalectomy. The failure of adrenalectomy to protect mice from the immunosuppressive effects of PFOA indicates that suppression of antibody synthesis is not the result of liver toxicity or stress-related corticosterone production.

Developmental Exposure to 1.0 or 2.5 mg/kg of Dibutyltin Dichloride Does Not Impair Immune Function in Sprague-Dawley Rats.

Organotins are used commercially as pesticides, antifouling agents, and stabilizers for polyvinyl chloride (PVC) pipe. Mono-and di-substituted butyltins, used in PVC pipe production, are of concern to the United States EPA, they leach from supply pipes into drinking water and are reported multisystem toxicants. We assessed immune function in Sprague-Dawley rats after developmental dibutyltin dichloride (DBTC) exposure. Pregnant rats were given drinking water containing 0, 10, or 25 mg/L of DBTC (final concentration) in 0.5% Alkamuls from gestational Day (GD) 6 through weaning of pups (37 days total). Approximate doses to dams: 1 and 2.5 mg DBTC/kg body weight (BW) during gestation, or 2.0 and 4.4 mg DBTC/kg BW while nursing. Litters were sexed, weighed, and culled to 4 males and 4 females per dam on postnatal Day (PND) 2. Beginning on PND3, litters of half of the dams per dose were gavaged with 0, 1.0, or 2.5 mg DBTC/kg BW 3X/week for 10 doses (maternal + direct treatment); remaining litters were exposed indirectly via lactation (maternal treatment). BW of litters exposed to 2.5 mg DBTC/kg BW was 10-20% lower (p < or = 0.05) relative to other groups from PND14 (males) or PND17 (females) through PND37. Delayed-type hypersensitivity (DTH), antibody synthesis, and natural killer (NK) cell activity were evaluated in immunologically mature offspring (N = 6/sex/group). DTH responses and antibody synthesis did not differ by dose, sex, or exposure. NK cell activity in the 10 mg DBTC/L maternal only group was greater in male offspring than in female. In female offspring from the maternal + direct group, cytotoxicity increased by dose at the 25:1 effector:target cell ratio. Our data suggest that developmental immunotoxicity from DBTC-tainted drinking water is unlikely as the concentrations we used were several orders of magnitude higher than concentrations expected to leach from PVC pipes.

Immune responses in sprague-dawley rats exposed to dibutyltin dichloride in drinking water as adults.

Organotins are used commercially as agricultural pesticides, antifouling agents, and stabilizers for polyvinyl chloride (PVC) pipe. Mono- and di-substituted methyl and butyltins, used in PVC pipe production, are of concern as they leach from supply pipes into drinking water and have been reported to cause multisystem toxicity, including immunotoxicity. As part of an ongoing study to evaluate immunotoxic effects of organotins, we assessed immune function in adult Sprague-Dawley (CD) rats after exposure to dibutyltin dichloride (DBTC). Individually-housed adult male and female CD rats were given drinking water containing 0, 10, or 25 mg DBTC/L (final concentration) in 0.5% Alkamuls for 28 days. Water bottles were changed and water consumption was monitored twice weekly and body weights (BW) were recorded weekly. Delayed-type hypersensitivity (DTH), primary and secondary antibody responses to sheep red blood cells, and natural killer (NK) cell activity were evaluated in separate groups of treated and control animals on day 29 of exposure. Water consumption was significantly decreased in both sexes at 25 mg DBTC/L. BW, immune organ weights, the DTH response, and NK cell activity did not vary by dose. Different results for antibody responses in male rats were obtained in two experimental replicates. In the first replicate, IgG was elevated at the highest dose whereas in the second replicate, IgM was suppressed. However, as these effects occurred at the high dose of 25 mg DBTC/L, which is a concentration a million times higher than levels of DBTC reported in drinking water, our data suggest that DBTC is unlikely to cause immunotoxicity at concentrations found in drinking water supplies.