Search

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.

Chronic obstructive pulmonary disease (COPD) is characterized by progressive destruction of the lung parenchyma resulting in emphysema and chronic inflammation of the airways. COPD is the fourth leading cause of death and more than 200 million people are affected worldwide. The major risk factor to develop COPD is cigarette smoke. Nowadays, lipopolysaccharide (LPS) is a widely used model compound which induces inflammation but cannot entirely reflect cigarette smoke induced toxicity and cytokine release in the lung. To understand the underlying mechanisms of cigarette smoke induced lung inflammation and destruction the need emerged to develop human relevant models. Aim of the study is to establish features of COPD in precision-cut lung slices (PCLS)of different species, including human, by exposure with cigarette smoke and cigarette smoke condensate (Csc). Murine, rat and human PCLS were prepared and exposed to Csc and LPS submersely or to cigarette smok eat air liquid interface (ALI). Induced toxicity was assessed by detection of lactate dehydrogenase, LIVE/DEAD® vitality staining and measurement of metabolic activity by usingWST-1 assay. Induction of pro-inflammatory immune responses was determined by ELISA. Therapeutical intervention was assessed by using dexamethasone. Concentration dependent toxicity was assessed after exposure to Csc with EC50 values of 85 mg/ml in murine PCLS, 228 mg/ml in rat PCLS and 121 mg/ml in human PCLS by measurement of metabolic activity. Csc exposure induced an increased release of pro-inflammatory cytokines tumor necrosis factor-a (TNF-a) and interleukin-1a/1v (IL-1a, IL-1v) in murine and human PCLS. For mouse, TNF-a secretion was three fold increased compared to medium control. Csc and cigarette smoke induced tissue damage in murine, rat and human PCLS or in rat PCLS, respectively. Therefore, PCLS from different species represent a promising model to provide translational data about toxic and pro-inflammatory aspects of cigarette smoke. Furthermore, pharmacological intervention against inflammation can be studied in PCLS.

Question: Human risk assessment of chemicals is currently based on the evaluation of in vivo animal studies. The use of high animal numbers and the suffering of animals in experiments such as repeated dose toxicity studies (RDT) demand for alternative test approaches. One alternative approach currently used in human risk assessment is read-across (RAX). In a RAX approach toxicity data of one or several tested source compound(s) are used to predict the toxicity of ""similar"" non-tested target compound(s). Similar compounds shall share structural- and physico-chemical proper-ties as well as a similar mode of action. This study was designed to evaluate the use of data from ex vivo experiments such as rator human precision-cut lung slices (PCLS). Three RAX-categories were tested, namely vicinal halogenides, naphthalene derivatives and vinyl esters. Each RAX category was selected based on shared structural properties and similar toxicological effects in RDT studies extracted from the FhG RepDose database (www.fraunhofer-repdose.de). Methods: Exposure of rat and human PCLS was performed on three days for three hours daily. The cytotoxicity of chemicals was assessed by LDH and WST-1 assay. Ex vivo IC50 values were calculated by sigmoidal curve fitting. These values were correlated to the LD50 values. Results: At least one substance in each category showed cyto-toxic effects in dose dependent manner. Linear regression analysis of public available in vivo LD50 values to the obtained ex vivo IC50values showed good linear correlation. Vicinal halogenides and naphthalene derivatives were less toxic in human lung sections compared to rat sections, where as the vinyl esters showed the comparable cytotoxicity in both species. Thus only one substance group showed similar cytotoxicity in both tested species, whereas two other groups reveal interspecies and metabolism diversity based on cytotoxicity endpoint. Conclusion: This first evaluation of rat and human and PCLs show that ex vivo chemical testing in human lung sections result in a promising approach for toxicity screening as no human in vivo reference data exist.