Cumulative risk: toxicity and interactions of physical and chemical stressors.

Recent efforts to update cumulative risk assessment procedures to incorporate nonchemical stressors ranging from physical to psychosocial reflect increased interest in consideration of the totality of variables affecting human health and the growing desire to develop community-based risk assessment methods. A key roadblock is the uncertainty as to how nonchemical stressors behave in relationship to chemical stressors. Physical stressors offer a reasonable starting place for measuring the effects of nonchemical stressors and their modulation of chemical effects (and vice versa), as they clearly differ from chemical stressors; and "doses" of many physical stressors are more easily quantifiable than those of psychosocial stressors. There is a commonly held belief that virtually nothing is known about the impact of nonchemical stressors on chemically mediated toxicity or the joint impact of coexposure to chemical and nonchemical stressors. Although this is generally true, there are several instances where a substantial body of evidence exists. A workshop titled "Cumulative Risk: Toxicity and Interactions of Physical and Chemical Stressors" held at the 2013 Society of Toxicology Annual Meeting provided a forum for discussion of research addressing the toxicity of physical stressors and what is known about their interactions with chemical stressors, both in terms of exposure and effects. Physical stressors including sunlight, heat, radiation, infectious disease, and noise were discussed in reference to identifying pathways of interaction with chemical stressors, data gaps, and suggestions for future incorporation into cumulative risk assessments.

Potential risk of asthma associated with in utero exposure to xenobiotics.

The incidence of asthma, a complex disease and significant public health problem, has been increasing over the last 30 years for unknown reasons. Changes in environmental exposures or lifestyle may be involved. In some cases asthma may originate in utero or in early life. Associations have been found between in utero exposures to several xenobiotics and increased risk of asthma. There is convincing evidence that maternal smoking and/or in utero and perinatal exposure to environmental tobacco smoke are associated with increased risk of asthma. Similar effects have been demonstrated in animal models of allergic asthma. Evidence also suggests that in utero and/or early-life exposures to various ambient air pollutants may increase the risk of asthma although supporting animal data are very limited. A few studies have suggested that in utero exposure to acetaminophen is associated with increased risk of asthma; however, animal data are lacking. Various vitamin deficiencies and supplements during pregnancy have been studied. In general, it appears that vitamins A, C, and E have protective effects and vitamins D and B may, in some instances, increase the risk, but the data are not conclusive. Some studies related to in utero exposures to polychlorinated biphenyls and bisphenol A and asthma risk are also reported. The underlying mechanisms for an association between xenobiotic exposures and asthma remain a matter of speculation. Genetic predisposition and epigenetic changes have been explored. The developing immune, respiratory, and nervous systems are potential targets. Oxidative stress and modulation of inflammation are thought to be involved.

Immunotoxicology and its application in risk assessment.

Immunotoxicology is the study of undesired modulation of the immune system by extrinsic factors. Toxicological assessments have demonstrated that the immune system is a target following exposure to a diverse group of xenobiotics including ultraviolet radiation, chemical pollutants, therapeutics, and recreational drugs. There is a well-established cause and effect relationship between suppression of the immune response and reduced resistance to infections and certain types of neoplasia. In humans, mild-to-moderate suppression of the immune response is linked to reduced resistance to common community-acquired infections, whereas opportunistic infections, which are very rare in the general population, are common in individuals with severe suppression. Xenobiotic exposure may also result in unintended stimulation of immune function. Although a cause and effect relationship between unintended stimulation of the immune response and adverse consequences has yet to be established, evidence does suggest that hypersensitivity, autoimmunity, and pathological inflammation may be exacerbated in susceptible populations exposed to certain xenobiotics. Xenobiotics can act as allergens and elicit hypersensitivity responses, or they can modulate hypersensitivity responses to other allergens such as pollen or dust mite by acting as adjuvants, enhancing the development or expression of hypersensitivity. Allergic contact dermatitis, allergic rhinitis, and asthma are the most commonly encountered types of hypersensitivity reactions resulting from chemical exposure. The immunologic effectors and mechanisms involved in autoimmune reactions are the same as those associated with responses to foreign antigens; however, the reactions are directed against the host's own cells. Thus, chemicals that induce immune suppression, nonspecific immunostimulation, or hypersensitivity may also impact autoimmunity. Risk assessment for immunotoxicity should be performed using the same approaches and principles for other noncancer effects. However, since xenobiotics may have effects on more than one aspect of immune function, immunotoxicity data should be evaluated separately for evidence of suppression, stimulation, hypersensitivity, and autoimmunity.

Decision trees for evaluating skin and respiratory sensitizing potential of chemicals in accordance with European regulations.

Guidance for determining the sensitizing potential of chemicals is available in EC Regulation No. 1272/2008 Classification, Labeling, and Packaging of Substances; REACH guidance from the European Chemicals Agency; and the United Nations Globally Harmonized System (GHS). We created decision trees for evaluating potential skin and respiratory sensitizers. Our approach (1) brings all the regulatory information into one brief document, providing a step-by-step method to evaluate evidence that individual chemicals or mixtures have sensitizing potential; (2) provides an efficient, uniform approach that promotes consistency when evaluations are done by different reviewers; (3) provides a standard way to convey the rationale and information used to classify chemicals. We applied this approach to more than 50 chemicals distributed among 11 evaluators with varying expertise. Evaluators found the decision trees easy to use and recipients (product stewards) of the analyses found that the resulting documentation was consistent across users and met their regulatory needs. Our approach allows for transparency, process management (e.g., documentation, change management, version control), as well as consistency in chemical hazard assessment for REACH, EC Regulation No. 1272/2008 Classification, Labeling, and Packaging of Substances and the GHS.

Suppression of pulmonary host defenses and enhanced susceptibility to respiratory bacterial infection in mice following inhalation exposure to trichloroethylene and chloroform.

Numerous epidemiological studies have associated episodes of increased air pollution with increased incidence of respiratory disease, including pneumonia, croup, and bronchitis. Trichloroethylene (TCE) and chloroform are among 33 hazardous air pollutants identified by the U.S. Environmental Protection Agency as presenting the greatest threat to public health in the largest number of urban areas. Also, both are common indoor air pollutants. Here, we assessed the potential effects of TCE and chloroform on resistance to pulmonary bacterial infection and related alveolar macrophage (AM) function. CD-1 mice were exposed by inhalation to filtered air (control) or concentrations of TCE ranging from 5 to 200 ppm, or concentrations of chloroform ranging from 100 to 2000 ppm. Immediately following exposure, mice were challenged with an aerosol of Streptococcus zooepidemicus and monitored for clearance of bacteria from the lung and mortality. In separate experiments, exposed mice were injected intratracheally with viable bacteria and phagocytic function was evaluated in macrophages obtained from lung washes 30 min later. The NOEL for enhanced mortality to infection was 25 ppm for TCE and 500 ppm for chloroform. Relative to the air controls, differences in clearance of bacteria from the lung were noted in mice exposed to TCE (NOEL = 50 ppm) and to chloroform (NOEL 100 ppm), and differences in AM phagocytic index were noted for TCE (NOEL = 100 ppm) and for chloroform (NOEL < 100 ppm). The data support the utility of the S. zooepidemicus infectivity model in assessing potential increased risk of respiratory infection and suggest that delayed clearance of bacteria from the lung or decreased phagocytosis are viable alternatives to mortality as an endpoint. Collectively, these endpoints are among the most sensitive health effects reported for TCE.

Human serum IgE reacts with a Metarhizium anisopliae fungal catalase.

BACKGROUND: Previous studies have demonstrated that Metarhizium anisopliae extract can induce responses characteristic of human allergic asthma in a mouse model. The study objectives were (1) to identify and characterize the M. anisopliae mycelia extract (MYC) proteins that are recognized by mouse serum IgE, (2) to determine if human serum IgE reacts with these proteins, and (3) to determine if these IgE-reactive proteins are found in other fungi. METHODS: Asthmatic human serum IgE, M. anisopliae crude antigen (MACA) immunized mouse serum IgE, and anti-catalase antibodies were used to probe one- and two-dimensional gel electrophoresis blots of MYC. RESULTS: Mass spectrometry analysis identified catalase as a mouse IgE-reactive protein. This identification was confirmed by assaying catalase activity in the extract and extract immunoblots probed with anti-catalase antibody. Six adult asthmatic sera contained IgE, but not IgG, that was reactive with mycelia extract proteins. A similar protein profile was seen when blots were probed with either mouse anti-MACA IgE or anti-bovine liver catalase antibodies. Furthermore, these mouse anti-MACA and anti-catalase antibodies were cross-reactive with other mold extracts (skin prick testing mix) and Aspergillus niger catalase. CONCLUSIONS: Some human asthmatics have developed IgE that reacts with an M. anisopliae catalase, most likely due to cross-reactivity (minimal IgG development). The cross-reactivity among fungal catalases suggests that IgE-reactive catalase might be useful for exposure assessment. Additionally, the similarity of protein profiles visualized with both human and mouse serum IgE suggests that allergy hazard identification can be facilitated using a mouse model.

Safety assessment of biotechnology products for potential risk of food allergy: implications of new research.

Food allergy is a potential risk associated with use of transgenic proteins in crops. Currently, safety assessment involves consideration of the source of the introduced protein, in silico amino acid sequence homology comparisons to known allergens, physicochemical properties, protein abundance in the crop, and, when appropriate, specific immunoglobulin E binding studies. Recently conducted research presented at an International Life Sciences Institute/Health and Environmental Sciences Institute-hosted workshop adds to the scientific foundation for safety assessment of transgenic proteins in five areas: structure/activity, serum screening, animal models, quantitative proteomics, and basic mechanisms. A web-based tool is now available that integrates a database of allergenic proteins with a variety of computational tools which could be used to improve our ability to predict allergenicity based on structural analysis. A comprehensive strategy and model protocols have been developed for conducting meaningful serum screening, an extremely challenging process. Several animal models using oral sensitization with adjuvant and one dermal sensitization model have been developed and appear to distinguish allergenic from non-allergenic food extracts. Data presented using a mouse model suggest that pepsin resistance is indicative of allergenicity. Certain questions remain to be addressed before considering animal model validation. Gel-free mass spectrometry is a viable alternative to more labor-intensive approaches to quantitative proteomics. Proteomic data presented on four nontransgenic varieties of soy suggested that if known allergen expression in genetically modified crops falls within the range of natural variability among commercial varieties, there appears to be no need to test further. Finally, basic research continues to elucidate the etiology of food allergy.

Utility of rodent models for evaluating protein allergenicity.

Animal models are needed to assess novel proteins produced through biotechnology for potential dietary allergenicity. The exact characteristics that give certain foods allergenic potential are unclear, but must include both the potential to sensitize (induce IgE) as well as the capacity to avoid induction of oral tolerance (specific inhibition of IgE production). EPA has developed two complementary mouse models; one which distinguishes allergenic from non-allergenic food extracts using oral sensitization with adjuvant (cholera toxin) and another which further distinguishes highly potent allergens following oral administration without adjuvant based on the development (or not) of tolerance. For the foods tested thus far (roasted or raw peanut, Brazil nut, egg white, turkey, and spinach), the ability to sensitize and/or tolerize in these models are consistent with observed allergenicity as well as persistence and severity among allergens. Additionally, in vitro pepsin-resistance analysis of these food extracts suggests an association between sensitization capacity and proteins which are stable to gastric digestion. A subcutaneous exposure model did not distinguish allergenic from non-allergenic foods and does not appear useful for assessing potential allergenicity.

Identifying food proteins with allergenic potential: evolution of approaches to safety assessment and research to provide additional tools.

A safety assessment process exists for genetically engineered crops that includes the evaluation of the expressed protein for allergenic potential. The objectives of this evaluation are twofold: (1) to protect allergic consumers from exposure to known allergenic or cross-reactive proteins, and (2) protect the general population from risks associated with the introduction of genes encoding proteins that are likely to become food allergens. The first systematic approach to address these concerns was formulated by Metcalfe et al. [Metcalfe, D.D., Astwood, J.D., Townsend, R., Sampson, H.A., Taylor, S.L., and Fuchs, R.L. 1996. Assessment of the allergenic potential of foods from genetically engineered crop plants. Crit. Rev. Food Sci. Nutr. 36(5), 165-186.] and subsequently Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO) [FAO/WHO, 2001. Evaluation of allergenicity of genetically modified foods. Report of a Joint FAO/WHO Expert Consultation on Allergenicity of Foods Derived from Biotechnology. January 22-25, 2001. Rome, Italy]. More recently, Codex [Codex Alimentarius Commission, 2003. Alinorm 03/34: Joint FAO/WHO Food Standard Programme, Codex Alimentarius Commission, Twenty-Fifth Session, Rome, Italy, 30 June-5 July, 2003. Appendix III, Guideline for the conduct of food safety assessment of foods derived from recombinant-DNA plants, and Appendix IV, Annex on the assessment of possible allergenicity. pp. 47-60], noting that no single factor is recognized as an identifier for protein allergenicity, suggested a weight of evidence approach be conducted that takes into account a variety of factors and approaches for an overall assessment of allergenic potential. These various recommendations are based on what is known about allergens, including the history of exposure and safety of the gene(s) source; amino acid sequence identity to human allergens; stability to pepsin digestion in vitro; protein abundance in the crop and processing effects; and when appropriate, specific IgE binding studies or skin-prick testing. Similarities and differences between these various suggested recommendations, as well as data gaps, are discussed. The US Environmental Protection Agency (EPA)'s Office of Research and Development (ORD) has initiated a targeted research effort to address data gaps and improve the various recommended methods/endpoints for assessing the allergenic risks associated with plant incorporated pesticides (PIPs) through both intramural and extramural (grant supported) research. The areas of primary focus for EPA include: (1) development and evaluation of animal models; (2) targeted or specific serological assays; and (3) structure-activity relationships. Details on the current as well as proposed EPA funded research are discussed. More recently US EPA has partnered with the National Institute of Allergy and Infectious Disease (NIAID), National Institutes of Health to support research in areas of mutual interest with respect to food allergy.

Failure to induce oral tolerance in mice is predictive of dietary allergenic potency among foods with sensitizing capacity.

Animal models are needed to assess novel proteins produced through biotechnology for potential dietary allergenicity. Currently proposed rodent models evaluate sensitizing potential of food extracts or proteins following parenteral administration or oral administration with adjuvant. However, food allergy requires not only the potential to induce immunoglobulin (Ig) E but also the capacity to avoid induction of oral tolerance (specific inhibition of IgE production). Here we describe a mouse model that assesses the potential of food extracts to induce oral tolerance. Adult C3H/HeJ mice were exposed orally to food extracts (without adjuvant) and subsequently challenged with the extract ip. Reduction of antigen-specific serum IgE relative to appropriate controls was used to indicate tolerance. Foods associated with persistent, severe allergy (peanut, Brazil nut), and nonallergens (turkey, spinach) were less tolerizing than foods associated with frequently resolving allergy (egg white). Digestibility was assessed in vitro, and pH alterations or encapsulation were used to modify solubility or digestibility. Egg white, peanut, and Brazil nut proteins were resistant to gastric enzyme (pepsin) degradation; turkey and spinach were not. Among pepsin-resistant proteins, peanut and Brazil nut appeared more sensitive to intestinal enzyme than egg white. For the extracts tested, full gastric digestion appeared to prevent induction of tolerance. Once through the stomach, only proteins resistant to intestinal enzymes induced tolerance. Limiting gastric digestion with sodium bicarbonate enhanced tolerance to peanut and Brazil nut. This model represents a complementary method of assessing potential allergenicity. Also, the conditions under which the test protein is encountered may impact experimental outcome.

Skin sensitization in chemical risk assessment: report of a WHO/IPCS international workshop focusing on dose-response assessment.

An international workshop was held in 2006 to evaluate experimental techniques for hazard identification and hazard characterization of sensitizing agents in terms of their ability to produce data, including dose-response information, to inform risk assessment. Human testing to identify skin sensitizers is discouraged for ethical reasons. Animal-free alternatives, such as quantitative structure-activity relationships and in vitro testing approaches, have not been sufficiently developed for such application. Guinea pig tests do not generally include dose-response assessment and are therefore not designed for the assessment of potency, defined as the relative ability of a chemical to induce sensitization in a previously naive individual. In contrast, the mouse local lymph node assay does include dose-response assessment and is appropriate for this purpose. Epidemiological evidence can be used only under certain circumstances for the evaluation of the sensitizing potency of chemicals, as it reflects degree of exposure as well as intrinsic potency. Nevertheless, human diagnostic patch test data and quantitative elicitation data have provided very important information in reducing allergic contact dermatitis risk and sensitization in the general population. It is therefore recommended that clinical data, particularly dose-response data derived from sensitized patients, be included in risk assessment.

Assessing the health effects and risks associated with children's inhalation exposures--asthma and allergy.

Adults and children may have different reactions to inhalation exposures due to differences in target tissue doses following similar exposures, and/or different stages in lung growth and development. In the case of asthma and allergy both the developing immune system and initial encounters with common allergens contribute to this differential susceptibility. Asthma, the most common chronic childhood disease, has significant public health impacts and is characterized by chronic lung inflammation, reversible airflow obstruction, and immune sensitization to allergens. Animal studies described here suggest that air pollutants exacerbate asthma symptoms and may also play a role in disease induction. Changes characteristic of asthma were observed in rhesus monkeys sensitized to house dust mite antigen (HDMA) as infants and exposed repeatedly thereafter to ozone (O3) and HDMA. O3 exposure compromised airway growth and development and exacerbated the allergen response to favor intermittent airway obstruction and wheeze. In Brown Norway rats a variety of air pollutants enhanced sensitization to HDMA such that symptoms elicited in response to subsequent allergen challenge were more severe. Although useful for assessing air pollutants effects on initial sensitization, the rodent immune system is immature at birth relative to humans, making this model less useful for studying differential effects between adults and children. Because computational models available to address children's inhalation exposures are limited, default adjustments and their associated uncertainty will continue to be used in children's inhalation risk assessment. Because asthma is a complex (multiple genes, phenotypes, organ systems) disease, this area is ripe for systems biology approaches.

Differences in allergenic potential of food extracts following oral exposure in mice reflect differences in digestibility: potential approaches to safety assessment.

An animal model for food allergy is needed to assess genetically modified food crops for potential allergenicity. The ideal model must produce allergic antibody (IgE) to proteins differentially according to known allergenicity before being used to accurately identify potential allergens among novel proteins. The oral route is the most relevant for exposure to food antigens, and a protein's stability to digestion is a current risk assessment tool based on this natural route. However, normal laboratory animals do not mount allergic responses to proteins administered orally due to oral tolerance, an immunologic mechanism which specifically suppresses IgE. To circumvent oral tolerance and evoke differential IgE responses to a panel of allergenic and nonallergenic food extracts, female C3H/HeJ mice were exposed subcutaneously or orally with cholera toxin as an adjuvant. All foods elicited IgE by the subcutaneous route. Oral exposure, however, resulted in IgE to allergens (peanut, Brazil nut, and egg white) but not to nonallergens (spinach and turkey), provided that the dose and exposures were limited. Additionally, in vitro digestibility assays demonstrated the presence of digestion-stable proteins in the allergenic food extracts but not in the nonallergenic foods. Our results suggest that the subcutaneous route is inadequate to distinguish allergens from nonallergens, but oral exposure under the appropriate experimental conditions will result in differential allergic responses in accordance with known allergenicity. Moreover, those foods containing digestion-resistant proteins provoke allergic responses in this model, supporting the current use of pepsin resistance in the decision tree for potential allergenicity assessment.

Immunotoxicity: the risk is real.

Several papers published over the last year represent significant progress in closing the gap between rodent immunotoxicity data and human risk and indicate that, at least for the developing immune system, the concern raised by rodent data is justified. The studies reviewed here show that suppression of immune responses in rodents is predictive of suppression of immune responses in humans and that there is a relationship between immune suppression following developmental exposure to the toxicants and enhanced risk of infectious or neoplastic disease in humans. The three cases highlighted here are remarkable in that they all deal with real-world environmental exposures that represent different media -- air (cigarette smoke), water (arsenic), and food (polychlorinated biphenyls [PCBs]) -- and constitute very real risks. Moreover, the arsenic and PCB studies actually demonstrate a quantitative relationship between human exposure and immune suppression. There is evidence that in utero exposure to cigarette smoke and arsenic but not PCBs is associated with increased risk of allergic disease as well. There is clearly potential for designing studies that could address both issues.

Animal models for protein respiratory sensitizers.

Protein induced respiratory hypersensitivity, particularly atopic disease in general, and allergic asthma in particular, has increased dramatically over the last several decades in the US and other industrialized nations as a result of ill-defined changes in living conditions in modern western society. In addition, work-related asthma has become the most frequently diagnosed occupational respiratory illness. Animal models have demonstrated great utility in developing an understanding of the etiology and mechanisms of many diseases. A few models been developed as predictive models to identify a protein as an allergen or to characterize its potency. Here we describe animal models that have been used to investigate and identify protein respiratory sensitizers. In addition to prototypical experimental design, methods for exposure route, sample collection, and endpoint assessment are described. Some of the most relevant endpoints in assessing the potential for a given protein to induce atopic or allergic asthma respiratory hypersensitivity are the development of cytotropic antibodies (IgE, IgG1), eosinophil influx into the lung, and airway hyperresponsiveness to the sensitizing protein and/or to non-antigenic stimuli (Mch). The utility of technologies such as PCR and multiplexing assay systems is also described. These models and methods have been used to elucidate the potential for protein sources to induce allergy, identify environmental conditions (pollutants) to impact allergy responsiveness, and establish safe exposure limits. As an example, data are presented from an experiment designed to compare the allergenicity of a fungal biopesticide Metarhizium anisopliae (MACA) crude extract with the one of its components, conidia (CON) extract.

Induction of asthma and the environment: what we know and need to know.

The prevalence of asthma has increased dramatically over the last 25 years in the United States and in other nations as a result of ill-defined changes in living conditions in modern society. On 18 and 19 October 2004 the U.S. Environmental Protection Agency and the National Institute of Environmental Health Sciences sponsored the workshop "Environmental Influences on the Induction and Incidence of Asthma" to review current scientific evidence with respect to factors that may contribute to the induction of asthma. Participants addressed two broad questions: a) What does the science suggest that regulatory and public health agencies could do now to reduce the incidence of asthma? and b) What research is needed to improve our understanding of the factors that contribute to the induction of asthma and our ability to manage this problem? In this article (one of four articles resulting from the workshop), we briefly characterize asthma and its public health and economic impacts, and intervention strategies that have been successfully used to prevent induction of asthma in the workplace. We conclude with the findings of seven working groups that focus on ambient air, indoor pollutants (biologics), occupational exposures, early life stages, older adults, intrinsic susceptibility, and lifestyle. These groups found strong scientific support for public health efforts to limit in utero and postnatal exposure to cigarette smoke. However, with respect to other potential types of interventions, participants noted many scientific questions, which are summarized in this article. Research to address these questions could have a significant public health and economic impact that would be well worth the investment.

Assessing the potential to induce respiratory hypersensitivity.

Acute and repeat dose inhalation studies have been an important part of the safety assessment of drugs, chemicals, and other products throughout the world for many years. It is known that damage to the respiratory tract can be triggered either by nonspecific irritation or by specific immune-mediated pathogenesis, and it is acknowledged that traditional inhalation studies are not designed to address fully the impact of the latter. It is also recognized that different types of immune-mediated responses can be triggered by different classes of compounds and that some immune reactions in the lung are life threatening. As such, it is important to understand as fully as possible the basis for the immune-mediated damage to the lung in order to characterize adequately the risks of individual chemicals or proteins. It is against this background that a review of the methods used to assess the potential for immune-mediated respiratory hypersensitivity was conducted. The primary objectives of this review are to discuss appropriate methods for identifying and characterizing respiratory hypersensitivity hazards and risks; and to identify key data gaps and related research needs with respect to respiratory hypersensitivity testing. The following working definition of respiratory hypersensitivity was formulated: a hypersensitivity response in the respiratory tract precipitated by a specific immune response, mediated by multiple mechanisms, including IgE antibody. Because of the importance played by various classes of compounds, the subsequent sections of this review will consider protein-specific, chemical-specific, and drug-specific aspects of respiratory hypersensitivity.

Dose-dependent allergic responses to an extract of Penicillium chrysogenum in BALB/c mice.

Indoor mold has been associated with the development of allergic asthma. Penicillium chrysogenum, a common indoor mold, is known to have several allergens and can induce allergic responses in a mouse model of allergic penicilliosis. Our hypothesis is that soluble components of P. chrysogenum (PCE) can dose-dependently induce responses typical of allergic asthma in BALB/c mice. Mice were exposed to 10, 20, 50, or 70 microg of PCE by involuntary aspiration four times over a 4-week period. Serum and bronchoalveolar lavage fluid (BALF) were collected before (day 0), and at days 1 and 3 following the final exposure. PCE-exposed mice demonstrated dose-dependent increases in: BALF total cell numbers including eosinophil, serum and BALF total IgE levels, BALF IL-5 levels, and increased severity of histopathologic lesions. A single exposure to the highest dose of PCE resulted in edema and cellular damage but not immune responses. Four exposures to Metarhizium anisopliae crude antigen (10 microg, positive control) resulted in equivalent or greater allergic asthma-like responses than those demonstrated by multiple exposures to 50 or 70 microg of PCE. Multiple exposures to 70 microg of PCE showed increased allergen-triggered immediate respiratory responses as well as non-specific airway hyperresponsiveness to methacholine as assessed by barometric whole-body plethysmography. Taken together, repeated pulmonary challenge with P. chrysogenum extract induced dose-dependent allergic asthma-like responses in mice.