Prevalidation of the ex-vivo model PCLS for prediction of respiratory toxicity.

In acute inhalation toxicity studies, animals inhale substances at given concentrations. Without additional information, however, appropriate starting concentrations for in-vivo inhalation studies are difficult to estimate. The goal of this project was the prevalidation of precision-cut lung slices (PCLS) as an ex-vivo alternative to reduce the number of animals used in inhalation toxicity studies. According to internationally agreed principles for Prevalidation Studies, the project was conducted in three independent laboratories. The German BfR provided consultancy in validation principles and independent support with biostatistics. In all laboratories, rat PCLS were prepared and exposed to 5 concentrations of 20 industrial chemicals under submerged culture conditions for 1h. After 23 h post-incubation, toxicity was assessed by measurement of released lactate dehydrogenase and mitochondrial activity. In addition, protein content and pro-inflammatory cytokine IL-1α were measured. For all endpoints IC50 values were calculated if feasible. For each endpoint test acceptance criteria were established. This report provides the final results for all 20 chemicals. More than 900 concentration-response curves were analyzed. Log10[IC50 (µM)], obtained for all assay endpoints, showed best intra- and inter-laboratory consistency for the data obtained by WST-1 and BCA assays. While WST-1 and LDH indicated toxic effects for the majority of substances, only some of the substances induced an increase in extracellular IL-1α. Two prediction models (two-group classification model, prediction of LC50 by IC50) were developed and showed promising results.

Assessment of immunotoxicity induced by chemicals in human precision-cut lung slices (PCLS).

Occupational asthma can be induced by a number of chemicals at the workplace. Risk assessment of potential sensitizers is mostly performed in animal experiments. With increasing public demand for alternative methods, human precision-cut lung slices (PCLS) have been developed as an ex vivo model. Human PCLS were exposed to increasing concentrations of 20 industrial chemicals including 4 respiratory allergens, 11 contact allergens, and 5 non-sensitizing irritants. Local respiratory irritation was characterized and expressed as 75% (EC25) and 50% (EC50) cell viability with respect to controls. Dose-response curves of all chemicals except for phenol were generated. Local respiratory inflammation was quantified by measuring the production of cytokines and chemokines. TNF-α and IL-1α were increased significantly in human PCLS after exposure to the respiratory sensitizers trimellitic anhydride (TMA) and ammonium hexachloroplatinate (HClPt) at subtoxic concentrations, while contact sensitizers and non-sensitizing irritants failed to induce the release of these cytokines to the same extent. Interestingly, significant increases in T(H)1/T(H)2 cytokines could be detected only after exposure to HClPt at a subtoxic concentration. In conclusion, allergen-induced cytokines were observed but not considered as biomarkers for the differentiation between respiratory and contact sensitizers. Our preliminary results show an ex vivo model which might be used for prediction of chemical-induced toxicity, but is due to its complex three-dimensional structure not applicable for a simple screening of functional and behavior changes of certain cell populations such as dendritic cells and T-cells in response to allergens.

Determination of genotoxicity by the Comet assay applied to murine precision-cut lung slices.

Precision-cut lung slices (PCLSs) are an organotypic lung model that is widely used in pharmacological, physiological, and toxicological studies. Genotoxicity testing, as a pivotal part of early risk assessment, is currently established in vivo in various organs including lung, brain, or liver, and in vitro in cell lines or primary cells. The aim of the present study was to provide the three-dimensional organ culture PCLS as a new ex vivo model for determination of genotoxicity using the Comet assay. Murine PCLS were exposed to increasing concentrations of ethyl methane sulfonate 'EMS' (0.03-0.4%) and formalin (0.5-5mM). Tissue was subsequently dissociated, and DNA single-strand breaks were quantified using the Comet assay. Number of viable dissociated lung cells was between 4×10(5) and 6.7×10(5)cells/slice. Even treatment with EMS did not induce toxicity compared to untreated tissue control. As expected, DNA single-strand breaks were increased dose-dependently and significantly after exposure to EMS. Here, tail length rose from 24µm to 75µm. In contrast, formalin resulted in a significant induction of DNA cross-links. The effects induced by EMS and formalin demonstrate the usefulness of PCLS as a new ex vivo lung model for genotoxicity testing in the early risk assessment of airborne substances in the future.

Bronchoconstriction in nonhuman primates: a species comparison.

Bronchoconstriction is a characteristic symptom of various chronic obstructive respiratory diseases such as chronic obstructive pulmonary disease and asthma. Precision-cut lung slices (PCLS) are a suitable ex vivo model to study physiological mechanisms of bronchoconstriction in different species. In the present study, we established an ex vivo model of bronchoconstriction in nonhuman primates (NHPs). PCLS prepared from common marmosets, cynomolgus macaques, rhesus macaques, and anubis baboons were stimulated with increasing concentrations of representative bronchoconstrictors: methacholine, histamine, serotonin, leukotriene D4 (LTD4), U46619, and endothelin-1. Alterations in the airway caliber were measured and compared with previously published data from rodents, guinea pigs, and humans. Methacholine induced maximal airway constriction, varying between 74 and 88% in all NHP species, whereas serotonin was ineffective. Histamine induced maximal bronchoconstriction of 77 to 90% in rhesus macaques, cynomolgus macaques, and baboons and a lesser constriction of 53% in marmosets. LTD4 was ineffective in marmosets and rhesus macaques but induced a maximum constriction of 44 to 49% in cynomolgus macaques and baboons. U46619 and endothelin-1 caused airway constriction in all NHP species, with maximum constrictions of 65 to 91% and 70 to 81%, respectively. In conclusion, PCLS from NHPs represent a valuable ex vivo model for studying bronchoconstriction. All NHPs respond to mediators relevant to human airway disorders such as methacholine, histamine, U46619, and endothelin-1 and are insensitive to the rodent mast cell product serotonin. Only PCLS from cynomolgus macaques and baboons, however, responded also to leukotrienes, suggesting that among all compared species, these two NHPs resemble the human airway mechanisms best.

Natural innate cytokine response to immunomodulators and adjuvants in human precision-cut lung slices.

Prediction of lung innate immune responses is critical for developing new drugs. Well-established immune modulators like lipopolysaccharides (LPS) can elicit a wide range of immunological effects. They are involved in acute lung diseases such as infections or chronic airway diseases such as COPD. LPS has a strong adjuvant activity, but its pyrogenicity has precluded therapeutic use. The bacterial lipopeptide MALP-2 and its synthetic derivative BPPcysMPEG are better tolerated. We have compared the effects of LPS and BPPcysMPEG on the innate immune response in human precision-cut lung slices. Cytokine responses were quantified by ELISA, Luminex, and Meso Scale Discovery technology. The initial response to LPS and BPPcysMPEG was marked by coordinated and significant release of the mediators IL-1ß, MIP-1ß, and IL-10 in viable PCLS. Stimulation of lung tissue with BPPcysMPEG, however, induced a differential response. While LPS upregulated IFN-γ, BPPcysMPEG did not. This traces back to their signaling pathways via TLR4 and TLR2/6. The calculated exposure doses selected for LPS covered ranges occurring in clinical studies with human beings. Correlation of obtained data with data from human BAL fluid after segmental provocation with endotoxin showed highly comparable effects, resulting in a coefficient of correlation >0.9. Furthermore, we were interested in modulating the response to LPS. Using dexamethasone as an immunosuppressive drug for anti-inflammatory therapy, we found a significant reduction of GM-CSF, IL-1ß, and IFN-γ. The PCLS-model offers the unique opportunity to test the efficacy and toxicity of biological agents intended for use by inhalation in a complex setting in humans.

Effects of acute in vitro exposure of murine precision-cut lung slices to gaseous nitrogen dioxide and ozone in an air-liquid interface (ALI) culture.

The aim of this study was to establish an air-liquid interface (ALI) culture of precision-cut lung slices (PCLS) for direct exposure of lung cells to gaseous contaminants. Nitrogen dioxide (NO(2)) and ozone (O(3)) were selected as model gas compounds. Acute pro-inflammatory and toxic effects of NO(2) and O(3) on live lung tissue were investigated. Murine PCLS were exposed to different flow rates (3-30mL/min) of synthetic air, O(3) (3.5-8.5ppm), or NO(2) (1-80ppm). Tissue survived ex vivo in ALI culture and resisted exposure to NO(2) (1-10ppm) and O(3) (3.5-8.5ppm) for 1h. Longer exposure to NO(2) resulted in a clear loss of viability, whereas exposure to O(3) was less effective. Exposure to NO(2) dose-dependently induced release of the pro-inflammatory IL-1alpha (40%), whereas RANTES, IL-12, and eotaxin remained unchanged. Early secretion of IL-1alpha (80%), RANTES (>800%), MIP-1beta (44%), and MCP-1 (60%) was already detected after 1h of exposure to O(3). The obtained data showed that direct exposure to O(3) and NO(2) induced cytotoxicity and pro-inflammatory responses in PCLS with ALI culture. This provides a model that more closely resembles in vivo exposure of airborne contaminants, and thus should be appropriate for toxicity testing.

A toxicological evaluation of inhaled solid lipid nanoparticles used as a potential drug delivery system for the lung.

Inhalation is a non-invasive approach for both local and systemic drug delivery. This study aimed to define the therapeutic window for solid lipid nanoparticles (SLNs) as a drug delivery system by inhalation from a toxicological point of view. To estimate the toxic dose of SLNs in vitro, A549 cells and murine precision-cut lung slices (PCLS) were exposed to increasing concentrations of SLNs. The cytotoxic effect of SLNs on A549 cells was evaluated by MTT and NRU assays. Viability of lung tissue was determined with WST assay and by life/dead staining using calcein AM/EthD-1 for confocal microscopy (CLSM) followed by quantitative analysis with IMARIS. Inflammation was assessed by measuring chemokine KC and TNF-alpha levels. The in vivo effects were determined in a 16-day repeated-dose inhalation toxicity study using female BALB/c mice, which were daily exposed to different concentrations of SLN30 aerosols (1-200 microg deposit dose). Local inflammatory effects in the respiratory tract were evaluated by determination of total protein content, LDH, chemokine KC, IL-6, and differential cell counts, performed on days 4, 8, 12, and 16 in bronchoalveolar lavage fluid. Additionally, a histopathological evaluation of toxicologically relevant organs was accomplished. The in vitro and ex vivo dose finding experiments showed toxic effects beginning at concentrations of about 500 microg/ml. Therefore, we used 1-200 microg deposit doses/animal for the in vivo experiments. Even after 16 days of challenge with a 200-microg deposit dose, SLNs induced no significant signs of inflammation. We observed no consistent increase in LDH release, protein levels, or other signs of inflammation such as chemokine KC, IL-6, or neutrophilia. In contrast, the particle control (carbon black) caused inflammatory and cytotoxic effects at corresponding concentrations. These results confirm that repeated inhalation exposure to SLN30 at concentrations lower than a 200-microg deposit dose is safe in a murine inhalation model.

Ex vivo lung function measurements in precision-cut lung slices (PCLS) from chemical allergen-sensitized mice represent a suitable alternative to in vivo studies.

A wide range of industrial chemicals can induce respiratory allergic reactions. Hence, there is an urgent need for methods identifying and characterizing the biological action of chemicals in the lung. Here, we present an easy, reliable alternative method to measure lung function changes ex vivo after exposure to chemical allergens and compare this to invasive in vivo measurements after sensitization with the industrial chemicals trimellitic anhydride (TMA) and 2,4-dinitrochlorobenzene (DNCB). Female BALB/c mice were sensitized epicutaneously with the respiratory allergen TMA and the contact sensitizer DNCB. The early allergic response to TMA and DNCB was registered in vivo and ex vivo on day 21 after inhalational challenge with dry standardized aerosols or after exposure of precision-cut lung slices (PCLS) to dissolved allergen. Airway hyperresponsiveness (AHR) to increasing doses of methacholine (MCh) was measured on the next day in vivo and ex vivo. Bronchoalveolar lavage (BAL) was performed for immunological characterization of local inflammation. TMA-sensitized mice showed AHR to MCh in vivo (ED(50): 0.06 microg MCh vs. 0.21 microg MCh in controls) and in PCLS (EC(50): 0.24 microM MCh vs. 0.4 microM MCh). TMA-treated animals showed increased numbers of eosinophils (12.8 x 10(4) vs. 0.7 x 10(4)) and elevated eotaxin-2 concentrations (994 pg/ml vs. 167 pg/ml) in BAL fluid 24 h after allergen challenge. In contrast, none of these parameters differed after sensitization with DNCB. The present study suggests that the effects of low molecular weight allergens, like TMA and DNCB, on ex vivo lung functions tested in PCLS reflect the in vivo situation.

Ex vivo testing of immune responses in precision-cut lung slices.

The aim of this study was the establishment of precision-cut lung slices (PCLS) as a suitable ex vivo alternative approach to animal experiments for investigation of immunomodulatory effects. For this purpose we characterized the changes of cytokine production and the expression of cell surface markers after incubation of PCLS with immunoactive substances lipopolysaccharide (LPS), macrophage-activating lipopeptide-2 (MALP-2), interferon gamma (IFNgamma), and dexamethasone. Viability of PCLS from wild-type and CD11c-enhanced yellow fluorescent protein (CD11-EYFP)-transgenic mice was controlled by measurement of lactate dehydrogenase (LDH) enzyme activity and live/dead fluorescence staining using confocal microscopy. Cytokines and chemokines were detected with Luminex technology and ELISA. Antigen presenting cell (APC) markers were investigated in living mouse PCLS in situ using confocal microscopy. LPS triggered profound pro-inflammatory effects in PCLS. Dexamethasone prevented LPS-induced production of cytokines/chemokines such as interleukin (IL)-5, IL-1alpha, TNFalpha, IL-12(p40), and RANTES in PCLS. Surface expression of MHC class II, CD40, and CD11c, but not CD86 was present in APCs of naive PCLS. Incubation with LPS enhanced specifically the expression of MHC class II on diverse cells. MALP-2 only failed to alter cytokine or chemokine levels, but was highly effective in combination with IFNgamma resulting in increased levels of TNFalpha, IL-12(p40), RANTES, and IL-1alpha. PCLS showed characteristic responses to typical pro-inflammatory stimuli and may thus provide a suitable ex vivo technique to predict the immunomodulatory potency of inhaled substances.