Target cell types with stem/progenitor function to isolate for in vitro reconstruction of human bronchiolar epithelia.

Recent advancement in research on stem/progenitor cells of respiratory organs is breathtaking, benefiting from the rapid development of technology to create transgenic mice. There is now a great deal of knowledge capable of direct translation from mice to humans. Nevertheless, one has to be careful, since there may be unexpected pitfalls. First of all, there are differences anatomically, histologically and ultrastructurally in the airway epithelia of the two species. In parallel with these structural differences, regionally specific cell types behave and function, particularly in regenerative instances, differently between the two species, at least to some extent. From the viewpoint of important human respiratory diseases, one of the most susceptible regions of the respiratory tract is the bronchiole. In our approach to develop in vitro systems utilizing human bronchiolar epithelial cells, we are currently leaning on the data obtained from mouse studies in spite of the above-mentioned species differences. With the help of such in vitro systems we should be able to investigate the damaging effects and mechanisms of environmental pollutants in the human respiratory epithelium and consequently achieve results useful for quantitative analyses of the impact on human respiratory health. While pursuing this goal, the mouse data have suggested that it should be worthwhile to pay close attention to the stem/progenitor cells contained in the human bronchiolar epithelia and eventually make use of them. The mouse data have further shown that these stem/progenitor cells possess a very close association with the immature and variant club cells and the neuroendocrine cells, and our own unpublished preliminary data with human cells are, apparently, at least partly consistent with what the mouse data are telling us.

Pulmonary tumor types induced in Wistar rats of the so-called "19-dust study".

The incidences of primary lung tumor types histologically diagnosed in 28 groups of Wistar rats of the so-called "19-dust study" are described, the total study having been already presented by Pott and Roller (Carcinogenicity study with nineteen granular dusts in rats. Eur J Oncol, 2005; 10: 249-81). Each exposed group was repeatedly instilled intratracheally with a suspension of one type and dose of 13 non-mining dusts differing in at least one of the following properties: chemical composition, density, specific surface area, and mean particle size. Eleven of the 13 dusts were classified as respirable granular bio-durable particles without known significant specific toxicity (abbreviation of the nine-word definition: GBP). In 579 (58%) lungs of 1002 rats which survived more than 26 weeks after the first instillation of GBP, at least one primary lung tumor type was observed, and in 306 (31%) at least two types. Three benign tumor types were diagnosed in the 579 tumor-bearing rats: bronchiolo-alveolar adenoma in 46%, cystic keratinizing epithelioma in 53%, and non-keratinizing epithelioma in 2.6% of the rats. Two of three malignant tumor types (bronchiolo-alveolar carcinoma and squamous cell carcinoma) occurred in 46% and 31% of the tumor-bearing rats, respectively, and adenosquamous carcinoma was diagnosed in 0.9%. Numerous lungs with a malignant tumor also showed one or more benign tumor types. In addition, single or multiple metastases from primary tumors of other sites (mainly carcinoma of the uterus) were diagnosed in 14% of the 1002 lungs. The proportionate incidences of the four predominantly diagnosed tumor types were compared with three summarized experimental groups which were exposed either to carbon black (two size classes), to titanium dioxide (two size classes), or to the total of the other nine GBP. A significant difference was not detected. The combination of dust volume with particle size correlated best with the carcinogenic effect, in contrast to dust mass and surface area.

Mosquito coil smoke inhalation toxicity. Part II: subchronic nose-only inhalation study in rats.

This paper addresses the results of a subchronic inhalation study in rats exposed to the smoke of burning mosquito coils manufactured in Indonesia. The objective of the study was a comparative assessment of different mosquito coils, including a blank coil, utilizing the OECD No. 413 testing paradigm, however, with the focus on hazard identification at a single maximum tolerated exposure concentration rather than concentration-response. Groups of rats were nose-only exposed 6 h a day, 5 days a week for 13 weeks to an average particulate concentration of 30 mg m(-3) from either blank coils or coils that contain the insecticidal ingredient transfluthrin. Nose-only air-exposed rats served as a control. A range of markers of exposure have been characterized to define the most critical exposure metrics with regard to total suspended particulate matter (TSP) and potentially noxious volatile products of combustion. During the course of the exposure period the smoke-exposed rats showed clinical signs suggestive of acute upper respiratory tract sensory irritation. Body weights were mildly affected in the male rats, but food and water consumption were indistinguishable amongst the groups. Carboxyhemoglobin concentrations were approximately 11% throughout the exposure period in smoke exposed rats. Hematology, clinical pathology and urinalysis as well as the analysis of organ weights and histopathology of extrapulmonary organs and the lung did not reveal any evidence of adverse systemic or local effects, whereas in the anterior region of the nasal passages, and to some extent also in the larynx, irritant-related changes typical for water-soluble upper respiratory irritants were found. Markers of pulmonary inflammation or increased phagocytosis and lysosomal activity in bronchoalveolar lavage were indistinguishable amongst the groups. gamma-Glutamyltranspeptidase was significantly increased in the smoke exposure groups, which is taken as indirect evidence of an adaptive upregulation of the pulmonary antioxidant glutathione. In rats exposed to mosquito coil smoke containing transfluthrin, a somewhat increased frequency of alveolar macrophages with foamy appearance was identified through cytodifferentiation but not histopathology compared with the blank coil. From the specific staining of intracellular phospholipids, the notion is supported that this equivocal finding is probably related to an increased uptake of modified pulmonary surfactant rather than increased engulfment of insoluble particulate matter since pigmentation or clustering or intra-alveolar cells did not occur. The results of this subchronic inhalation study support the conclusion that smoke from burning mosquito coils in concentrations high enough to elicit acute upper respiratory tract irritation due to the presence of common wood-combustion products (such as aliphatic aldehydes) did not cause any adverse effect in the lower respiratory tract or any other extrapulmonary organ. The most critical mode of action is related to acute and readily perceivable sensory irritation. The concentration tested was estimated to be well above that occurring under more realistic exposure conditions. Therefore, overnight exposure to the smoke from burning mosquito coils (manufactured in Indonesia) is unlikely to be associated with any unreasonable health risk.

Maternal effects and cancer risk in the progeny of mice exposed to X-rays before conception.

To investigate in an animal model whether preconceptual X-ray exposure leads to an altered tumor rate and spectrum in the offspring, a transgeneration carcinogenesis study was carried out. Female mice received X-ray irradiation (2 x 2 Gray) 2 weeks prior to mating with untreated males. After weaning, half of the descendants were exposed for 6 months to the immunomodulating and tumor-promoting compound cyclosporine A (CsA) by diet, the others remained untreated. The animals were maintained for their entire lifespan, terminal sacrifices were carried out after 28 months. Complete autopsy was performed, and three protocol organs (lung, liver and spleen) were examined histologically, together with any suspicious lesions in other organs. Fertility and the lifetime of the maternal mice were reduced by the X-ray irradiation, and their incidence of lung and liver tumors was increased as compared to non-irradiated mice. The descendants of all groups revealed comparable body weights and mortality rates. The incidence of hematopoietic/lymphoreticular tissue tumors increased in the female hybrids by 6 months of CsA-treatment. A higher incidence of lung and liver tumors in the sham-treated male progeny of irradiated mothers was detected, pointing to a possible germ cell-transmitted alteration initiated by the preconceptual maternal X-ray exposure.

Experimental approaches to evaluate respiratory allergy in animal models.

Asthma is defined as a chronic disease of the entire lung and asthma attacks may either be immediate, delayed or dual in onset. Allergic asthma is a complex chronic inflammatory disease of the airways and its etiology is multifactorial. It involves the recruitment and activation of many inflammatory and structural cells, all of which release mediators that result in typical pathological changes of asthma. A wealth of clinical and experimental data suggests that allergic asthma is due to an aberrant lung immune response mediated through T-helper type 2 (Th2) cells and associated cytokine-signaling pathways. The pathology of asthma is associated with reversible narrowing of airways, associated with prominent features that involve structural changes in the airway walls and extracellular matrix remodeling including abnormalities of bronchial smooth muscle, eosinophilic inflammation of the bronchial wall, hyperplasia and hypertrophy of mucous glands. The primary objective of respiratory allergy tests is to determine whether a low-molecular-weight chemical (hapten) or high-molecular-weight compound (antigen) exhibits sensitizing properties to the respiratory tract. This may range from reactions occurring in the nose (allergic rhinitis), in the bronchial airways (i.e., allergic bronchitis, asthma) or alveoli (e.g., hypersensitivity pneumonitis). Current assays utilize several phases, viz. an induction phase, which includes multiple exposures to the test compound (sensitization) via the respiratory tract (e.g., by intranasal or intratracheal instillations), by inhalation exposures or by dermal contact, and a single or multiple challenge or elicitation phase. The challenge can either be with the chemical (hapten), the homologous protein conjugate of the hapten or the antigen. The choice depends both on the irritant potency and the physical form (vapor, aerosol) of the hapten. The appropriate selection of concentrations (dosages) both for the induction and elicitation of respiratory allergy appears to be paramount for the outcome of test. Endpoints to characterize positive response range from the induction of immunoglobulins, cytokine or lymphokine patterns in serum (or the lung) to (patho-)physiological reactions typifying asthma. None of the currently applied animal models duplicate all features of human asthma. Accordingly, the specific pros and cons of the selected animal model, including protocol variables, animal species and strain selected, must be interpreted cautiously in order to arrive at a meaningful extrapolation for humans.

A modified CULTEX system for the direct exposure of bacteria to inhalable substances.

Increasing attention is being paid to the impact on human health of inhaled gaseous compounds and complex mixtures such as cigarette smoke. The evaluation of the genotoxicity of such materials is mostly based on experiments with model substances or mixtures and condensates in the standard Ames assay. Due to the methodological difficulties of testing air contaminants in their natural gaseous or aerosolised state, there are no generally accepted concepts and techniques for effective exposure of bacteria under such conditions. Therefore, we established a novel experimental approach using an exposure device based on the cell exposure system CULTEX. This allows us to investigate chemically and physically unchanged atmospheres like mainstream cigarette smoke by exposing bacteria of Salmonella typhimurium strains directly on the surface of culture media. The CULTEX exposure device can be connected to gas or aerosol generating systems. The introduction of this exposure device in the field of inhalation genotoxicology offers new test strategies for the in vitro evaluation of a wide range of inhalable substances in both laboratory and ambient situations. A patent was applied for this technical solution.

The north american control animal database: a resource based on standardized nomenclature and diagnostic criteria.

Historical control data have been shown to be valuable in the interpretation and evaluation of results from rodent carcinogenicity studies. Standardization of terminology and histopathology procedures is a prerequisite for meaningful comparison of control data across studies and analysis of potential carcinogenic effects. Standardization is particularly critical for the construction of a database that includes incidence data from different studies evaluated by pathologists in different laboratories. Standardized nomenclature and diagnostic criteria have been established for neoplasms and proliferative lesions. Efforts of the National Toxicology Program, the Society of Toxicologic Pathology (STP), and the Registry of Industrial Toxicology Animal-data (RITA) have led to a harmonized pathology nomenclature for the rat and the mouse. This nomenclature with detailed descriptions of lesions is available in publications by the STP and International Agency for Research on Cancer (IARC). A listing of these terms is available on the World Wide Web. Utilizing the model established by RITA and working with the International Life Sciences Institute (ILSI), companies with laboratories in North America formed a working group in 1994 to establish and maintain a database of neoplastic and proliferative lesions from control animals in carcinogenicity studies. The rationale for development of the North American Control Animal Database (NACAD), the factors that influence tumor incidence, operation of the database, and the benefits to be realized by using a standardized approach are discussed.

The value of historical control data-scientific advantages for pathologists, industry and agencies.

Historical control tumor data are useful in the interpretation of long-term rodent carcinogenicity bioassays, especially to assess the occurrence of rare tumors and marginally increased tumor incidences. The major prerequisites to compare historical control data with studies under evaluation are the validity and consistency of the respective databases. The RITA (Registry of Industrial Toxicology Animal-data) database for historical data of tumors and pre-neoplastic lesions collects data according to highly standardized procedures including tissue sampling and trimming, histopathology according to internationally harmonized nomenclature and diagnostic criteria, and peer review. All lesions that are entered are unanimously diagnosed according to IARC (Intermational Agency for Research on Cancer)/WHO criteria. The validity of data is additionally confirmed by a complete peer review performed by a database pathologist. Equivocal diagnoses and selected cases are additionally submitted to a panel of RITA pathologists. In the RITA database, there are currently 10,896 rats from 106 studies with more than 17,604 primary tumors and 16,551 pre-neoplastic lesions. The RITA database for historical control data for Wistar and Sprague Dawley rats as well as for different mouse strains is briefly described. Based upon RITA background data, the survival rate of Wistar rats has been consistent over a period of 10 years. The occurrence of tumor-bearing animals also shows a stable percentage over a decade. Additionally, examples of how historical control data may support carcinogenic risk assessment in cases of rare tumors or marginally increased incidences of tumors and pre-neoplastic lesions are given.

Respiratory hypersensitivity in guinea pigs sensitized to 1,6-hexamethylene diisocyanate (HDI): comparison of results obtained with the monomer and homopolymers of HDI.

This study used guinea pigs that were sensitized to the biuret or isocyanurate type homopolymers of 1,6 hexamethylene diisocyanate (HDI). Induction was either by intradermal injection or repeated inhalation exposures. For comparison, groups of guinea pigs were sensitized to monomeric HDI. Naive animals served as negative controls. Two and three weeks following induction, animals were challenged by inhalation with the hapten and homologous protein conjugate of the hapten, respectively. Assessments were based on changes in respiratory rate, serum IgG(1)-antibody titer, and influx of eosinophilic granulocytes in airways. Guinea pigs induced and challenged with the HDI-monomer did not display appreciable changes in respiratory rate, whilst the re-challenge with the HDI-protein conjugate caused unequivocal changes in respiratory patterns, including a marked bronchial influx of eosinophilic granulocytes. In contrast, animals induced and challenged with either the free or conjugated aerosols of HDI-homopolymers failed to elicit specific physiological or morphological pulmonary responses. IgG(1) antibodies were observed in all groups receiving monomeric HDI or HDI-homopolymers. Based on the comparative assessment of antibody titers following intradermal injections, it appeared that monomeric HDI was more potent to induce specific IgG(1) antibodies than the homopolymers of HDI. In summary, with respect to induction of allergy and asthma, the data presented here suggest that the homopolymeric forms of HDI appear to be less potent asthmagens, if any, than monomeric HDI.