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.

Comprehensive assessment of a chlorinated drinking water concentrate in a rat multigenerational reproductive toxicity study.

Some epidemiological studies report associations between drinking water disinfection byproducts (DBPs) and adverse reproductive/developmental effects, e.g., low birth weight, spontaneous abortion, stillbirth, and birth defects. Using a multigenerational rat bioassay, we evaluated an environmentally relevant "whole" mixture of DBPs representative of chlorinated drinking water, including unidentified DBPs as well as realistic proportions of known DBPs at low-toxicity concentrations. Source water from a water utility was concentrated 136-fold, chlorinated, and provided as drinking water to Sprague-Dawley rats. Timed-pregnant females (P0 generation) were exposed during gestation and lactation. Weanlings (F1 generation) continued exposures and were bred to produce an F2 generation. Large sample sizes enhanced statistical power, particularly for pup weight and prenatal loss. No adverse effects were observed for pup weight, prenatal loss, pregnancy rate, gestation length, puberty onset in males, growth, estrous cycles, hormone levels, immunological end points, and most neurobehavioral end points. Significant, albeit slight, effects included delayed puberty for F1 females, reduced caput epidydimal sperm counts in F1 adult males, and increased incidences of thyroid follicular cell hypertrophy in adult females. These results highlight areas for future research, while the largely negative findings, particularly for pup weight and prenatal loss, are notable.

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.

Immunotoxicity of perfluorooctanoic acid and perfluorooctane sulfonate and the role of peroxisome proliferator-activated receptor alpha.

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are environmentally widespread and persistent chemicals with multiple toxicities reported in experimental animals and humans. These compounds can trigger biological activity by activating the alpha isotype of peroxisome proliferator-activated receptors (PPARs), ligand-activated transcription factors that regulate gene expression; however, some biological effects may occur independently of the receptor. Activation of the peroxisome proliferator-activated receptor alpha (PPARalpha) modulates lipid and glucose homeostasis, cell proliferation and differentiation, and inflammation. Reported immunomodulation in experimental animals exposed to PFOA and PFOS has included altered inflammatory responses, production of cytokines and other proteins, reduced lymphoid organ weights, and altered antibody synthesis. Mounting experimental animal evidence suggests PPARalpha independence of some immune effects. This evidence originates primarily from studies with PPARalpha knockout models exposed to PFOA that demonstrate hepatic peroxisome proliferation, reduced lymphoid organ weights, and altered antibody synthesis. As human PPARalpha expression is significantly less than that of rodents, potential PPARalpha independence indicates that future research must explore mechanisms of action of these compounds, including PPARalpha-dependent and -independent pathways. This multiauthored review contains brief descriptions of current and recently published work exploring immunomodulation by PFOA and PFOS, as well as a short overview of other PPARalpha ligands of therapeutic and environmental interest.

Immunotoxicological profile of chloroform in female B6C3F1 mice when administered in drinking water.

Chloroform can be formed as a disinfection by-product during water chlorination, one of the primary modalities for purifying municipal water supplies for human consumption. The aim of this study was to characterize the immunotoxic effects of chloroform in female B6C3F1 mice when exposure occurred via the drinking water. Consistent with human exposure, female B6C3F1 mice were exposed to chloroform-containing drinking water at 2.5, 10, 25, 100, and 250 ppm for 28 days. The examined endpoints included the effects of chloroform on body and organ weights, water consumption, hematology, innate immunity, humoral immunity, and cell-mediated immunity. The functions of natural killer, B-, and T-cells were not altered by chloroform in drinking water at the concentrations tested, except that an increase in splenocyte basal proliferation was observed at chloroform levels of 100 and 250 ppm. Following chloroform administration, there was a decreased number of circulating neutrophils in the blood in all treatment groups, but neutrophil function in lung homogenates, as evaluated using an assay for myeloperoxidase activity following lipopolysaccharide and N-Formyl-Met-Leu-Phe stimulation, was not compromised. Further, the results of host resistance to Listeria monocytogenes infection also suggested that neutrophil function was normal. At the highest treatment level of chloroform (250 ppm), erythrocyte number and hemoglobin levels were significantly decreased. Some significant changes were also observed for body weights, water consumption, and organ weights; however, most of these effects were only observed at the highest treatment level of chloroform (250 ppm). Taken together, the results demonstrate that while chloroform administered via the drinking water affects body weight and selected hematological parameters at high dose levels, overall immune responses, as measured in several tests for immune function, are not compromised.

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.

Meeting report: moving upstream-evaluating adverse upstream end points for improved risk assessment and decision-making.

BACKGROUND: Assessing adverse effects from environmental chemical exposure is integral to public health policies. Toxicology assays identifying early biological changes from chemical exposure are increasing our ability to evaluate links between early biological disturbances and subsequent overt downstream effects. A workshop was held to consider how the resulting data inform consideration of an "adverse effect" in the context of hazard identification and risk assessment. OBJECTIVES: Our objective here is to review what is known about the relationships between chemical exposure, early biological effects (upstream events), and later overt effects (downstream events) through three case studies (thyroid hormone disruption, antiandrogen effects, immune system disruption) and to consider how to evaluate hazard and risk when early biological effect data are available. DISCUSSION: Each case study presents data on the toxicity pathways linking early biological perturbations with downstream overt effects. Case studies also emphasize several factors that can influence risk of overt disease as a result from early biological perturbations, including background chemical exposures, underlying individual biological processes, and disease susceptibility. Certain effects resulting from exposure during periods of sensitivity may be irreversible. A chemical can act through multiple modes of action, resulting in similar or different overt effects. CONCLUSIONS: For certain classes of early perturbations, sufficient information on the disease process is known, so hazard and quantitative risk assessment can proceed using information on upstream biological perturbations. Upstream data will support improved approaches for considering developmental stage, background exposures, disease status, and other factors important to assessing hazard and risk for the whole population.

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.

Host Resistance Assays.

The goal of immunotoxicity testing is to obtain data useful for immunotoxicity safety assessment. Guidance in the performance of immunotoxicity safety evaluations is provided in documents from the US EPA for chemicals and the ICH S8 document for pharmaceuticals. The ICH S8 document outlines a tiered approach that includes (1) standard toxicity studies with associated hematology, immune system organ weights, and histopathology data; (2) functional assays, such as cytotoxic T lymphocyte (CTL) assays, natural killer (NK) cell assays, respiratory burst, phagocytosis, and T-cell-dependent antibody response (TDAR) assays; and (3) host resistance assays. Host resistance assays are considered the gold standard in immunotoxicity testing and provide a critical overview of the extent to which innate, adaptive, and homeostatic regulatory immune functions are integrated to protect the host. Both comprehensive and targeted host resistance assays are available, each with distinct advantages. This chapter serves to provide a general overview of the various assays that may be used, as well as a summary of procedures.

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.

Immunotoxicogenomics: the potential of genomics technology in the immunotoxicity risk assessment process.

Evaluation of xenobiotic-induced changes in gene expression as a method to identify and classify potential toxicants is being pursued by industry and regulatory agencies worldwide. A workshop was held at the Research Triangle Park campus of the Environmental Protection Agency to discuss the current state-of-the-science of "immunotoxicogenomics" and to explore the potential role of genomics techniques for immunotoxicity testing. The genesis of the workshop was the current lack of widely accepted triggering criteria for Tier 1 immunotoxicity testing in the context of routine toxicity testing data, the realization that traditional screening methods would require an inordinate number of animals and are inadequate to handle the number of chemicals that may need to be screened (e.g., high production volume compounds) and the absence of an organized effort to address the state-of-the-science of toxicogenomics in the identification of immunotoxic compounds. The major focus of the meeting was on the theoretical and practical utility of genomics techniques to (1) replace or supplement current immunotoxicity screening procedures, (2) provide insight into potential modes or mechanisms of action, and (3) provide data suitable for immunotoxicity hazard identification or risk assessment. The latter goal is of considerable interest to a variety of stakeholders as a means to reduce animal use and to decrease the cost of conducting and interpreting standard toxicity tests. A number of data gaps were identified that included a lack of dose response and kinetic data for known immunotoxic compounds and a general lack of data correlating genomic alterations to functional changes observed in vivo. Participants concluded that a genomics approach to screen chemicals for immunotoxic potential or to generate data useful to risk assessors holds promise but that routine use of these methods is years in the future. However, recent progress in molecular immunology has made mode and mechanism of action studies much more practical. Furthermore, a variety of published immunotoxicity studies suggest that microarray analysis is already a practical means to explore pathway-level changes that lead to altered immune function. To help move the science of immunotoxicogenomics forward, a partnership of industry, academia, and government was suggested to address data gaps, validation, quality assurance, and protocol development.

The comparative immunotoxicity of five selected compounds following developmental or adult exposure.

It is well established that human diseases associated with abnormal immune function, including some common infectious diseases and asthma, are considerably more prevalent at younger ages. Although not established absolutely, it is generally believed that development constitutes a period of increased immune system susceptibility to xenobiotics, since adverse effects may occur at lower doses and/or immunomodulation may be more persistent, thus increasing the relative risk of xenobiotic exposure to the immunologically immature organism. To address this issue, a brief overview of immune maturation in humans is provided to demonstrate that functional immaturity alone predisposes the young to infection. Age-dependent differences in the immunotoxic effects of five diverse compounds, diethylstilbestrol (DES), diazepam (DZP), lead (Pb), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and tributyltin oxide (TBTO), which have undergone adult and developmental immunotoxicity testing in rodents, are then reviewed, as are human data when available. For all five chemicals, the developing immune system was found to be at greater risk than that of the adult, either because lower doses produced immunotoxicity, adverse effects were more persistent, or both.

"Omics" Technologies and the Immune System (a) , (b).

Recent advances in genomics-based identification of gene families and gene polymorphisms associated with immune system dysfunction have answered basic questions in immunology and have begun to move forward our understanding of immune-related disease processes. In toxicology, "omic" technologies have the potential to replace or supplement current immunotoxicological screening procedures, to provide insight into potential mode or mechanisms of action, and to provide data suitable for risk assessment. The application of omic technologies to the study of the immune system also has great potential to appreciably impact the diagnosis and treatment of immune-related diseases. This review focuses on the use of omic technologies in immunopharmacology and immunotoxicology, specifically considering the potential for these technologies to impact chemical hazard identification, risk characterization and risk assessment, and the development and application of novel therapeutics. The state of the science of omics technologies and the immune system is addressed in terms of a continuum of understanding of how omics technologies can and cannot yet be applied in the various aspects of immunopharmacology and immunotoxicology. Additionally, information gaps are identified that, once addressed, will move each area further down the continuum of understanding.

Associating Changes in the Immune System with Clinical Diseases for Interpretation in Risk Assessment.

This overview is an update of the unit originally published in 2004. While the basic tenets of immunotoxicity have not changed in the past 10 years, several publications have explored the application of immunotoxicological data to the risk assessment process. Therefore, the goal of this unit is still to highlight relationships between xenobiotic-induced immunosuppression and risk of clinical diseases progression. In immunotoxicology, this may require development of models to equate moderate changes in markers of immune functions to potential changes in incidence or severity of infectious diseases. For most xenobiotics, exposure levels and disease incidence data are rarely available, and safe exposure levels must be estimated based on observations from experimental models or human biomarker studies. Thus, it is important to establish a scientifically sound framework that allows accurate and quantitative interpretation of experimental or biomarker data in the risk assessment process.

Suppression of antigen-specific antibody responses in mice exposed to perfluorooctanoic acid: Role of PPARα and T- and B-cell targeting.

T-cell-dependent antibody responses (TDAR) are suppressed in female C57BL/6N mice exposed to ≥3.75 mg/kg of perfluorooctanoic acid (PFOA) for 15 days. To determine if suppression of humoral immunity by PFOA is peroxisome proliferator activated receptor alpha (PPARα)-dependent and if suppression is associated with specific targeting of T- or B-cells, three separate experiments were conducted: (1) female PPARα constitutive knockout (PPARα KO; B6.129S4-Ppar(tm1Gonz)N12) and wild-type controls (WT; C57BL/6-Tac) exposed to 0, 7.5, or 30 mg PFOA/kg for 15 days were immunized on Day 11 with a T-cell-dependent antigen and sera then collected for measures of antigen-specific IgM titers (TDAR) 5 days later; (2) female C57BL/6N WT mice exposed to 0, 0.94, 1.88, 3.75, or 7.5 mg PFOA/kg for 15 days were immunized with a T-cell-independent antigen on Day 11 and sera were then collected for analyses of antigen-specific IgM titers (TIAR) 7 days later; and (3) splenic lymphocyte phenotypes were assessed in unimmunized female C57BL/6N WT mice exposed to 0, 3.75, or 7.5 mg PFOA/kg for 10 days to investigate effects of PFOA in the absence of specific immunization. Separate groups of mice were immunized with a T-cell-dependent antigen after 11 days of exposure and splenic lymphocyte sub-populations were assessed after 13 or 15 days of exposure to assess numbers of stimulated cells. The results indicated that exposure to ≥1.88 mg PFOA/kg suppressed the TIAR; exposure to 30 mg PFOA/kg suppressed the TDAR in both PPARα KO and WT mice. The percentage of splenic B-cells was unchanged. Results obtained in the PPARα KO mice indicated that PPARα suppression of TDAR was independent of PPARα involvement. Suppression of the TIAR and the TDAR with minimal lymphocyte sub-population effects suggested that effects on humoral immunity are likely mediated by disruption of B-cell/plasma cell function.

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.

Developmental atrazine exposure suppresses immune function in male, but not female Sprague-Dawley rats.

Each year, 75 million pounds of the broadleaf herbicide atrazine (ATR) are applied to crops in the United States. Despite limited solubility, ATR is common in ground and surface water, making it of regulatory concern. ATR suppresses the immunomodulatory hormones prolactin (PRL) and the thyroid hormones (THs), with developmental exposure to ATR permanently disrupting PRL regulation. We hypothesized that ATR may cause developmental immunotoxicity through its disruption of PRL or THs. To test this hypothesis, pregnant Sprague-Dawley (SD) rats were exposed to 35-mg ATR/kg/d from gestational day (GD) 10 through postnatal day (PND) 23. Separate groups were exposed to bromocryptine (BCR) at 0.2 mg/kg/2x/day to induce hypoprolactinemia or to propylthiouracil (PTU) at 2 mg/kg/day to induce hypothyroidism. After the offspring reached immunologic maturity (at least 7 weeks old), the following immune functions were evaluated: natural killer (NK) cell function; delayed-type hypersensitivity (DTH) responses; phagocytic activity of peritoneal macrophages; and antibody response to sheep erythrocytes (SRBC). ATR decreased the primary antibody and DTH responses in male offspring only. Neither PTU nor BCR caused immunosuppression in any measured variable, although PTU increased phagocytosis by peritoneal macrophages. These results demonstrate that developmental exposure to ATR produced gender-specific changes in immune function in adult rats and suggest that immune changes associated with ATR are not mediated through the suppression of PRL or THs.

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.

Perfluorinated compounds: emerging POPs with potential immunotoxicity.

Perfluorinated compounds (PFCs) have been recognized as an important class of environmental contaminants commonly detected in blood samples of both wildlife and humans. These compounds have been in use for more than 60 years as surface treatment chemicals, polymerization aids, and surfactants. They possess a strong carbon-fluorine bond, which leads to their environmental persistence. There is evidence from both epidemiology and laboratory studies that PFCs may be immunotoxic, affecting both cell-mediated and humoral immunity. Reported effects of PFCs include decreased spleen and thymus weights and cellularity, reduced specific antibody production, reduced survival after influenza infection, and altered cytokine production. Immunosuppression is a critical effect associated with exposure to PFCs, as it has been reported to reduce antibody responses to vaccination in children. Mounting evidence suggests that immunotoxicity in experimental animals can occur at serum concentrations below, within, or just above the reported range for highly exposed humans and wildlife. Considering bioaccumulation and exposure to multiple PFCs, the risk of immunotoxicity for humans and wildlife cannot be discounted. This review will discuss current and recently published work exploring the immunomodulatory effects of PFCs in experimental animals and humans.

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.