Jan Knebel

Development of an in vitro system for studying effects of native and photochemically transformed gaseous compounds using an air/liquid culture technique

An experimental in vitro model was established to study the effects of environmentally relevant gaseous compounds on lung cells. The technical unit consists of a gas reaction chamber (2400 l) with a sun-simulator to produce and photochemically transform gaseous mixtures and compounds at the upper limit of environmentally relevant concentrations. Rat lung cells were exposed on transwells in a perspex chamber inside an incubator, into which the gaseous mixtures were conducted. Analysis of the gas phase was performed inside the reaction chamber and at the outlet of the exposure box to assess the effective exposure concentrations. The growth of the cells on PET-membranes allowed direct cell exposure with a minimal barrier for contact between gas and cells. To assess the cytotoxicity, the following biochemical markers for the cellular status after exposure were determined: amount of dsDNA, WST, BrdU-incorporation after exposure, LDH release into the culture medium, activity of glutathione S-transferases and esterases. Using this system, dose-dependent cytotoxicity was found for NO2 in the concentration range from 80 to 360 ppb and strong cytotoxic effects for ozone in the concentration range from 225 to 500 ppb. Exposure to purified air did not show significant effects. In addition, some irradiated gas mixtures (photo smog) showed cytotoxicity whereas non-irradiated mixtures did not.

Requirement of Monooxygenase Induction in Cultured Lung Epithelial Cells for Metabolism of PAH

Abstract In vitro mammalian lung cell systems should mimic the cells of native organs as closely as possible. Particularly so, when the metabolic activity for PAH and other precarcinogens is studied. Although fetal (embryonic) cells have more proliferative capacity than adult ones in vitro, the fetal cells usually lack the induced enzyme activity for PAH metabolism. This may be one of the reasons why the fetal cells in culture need longer exposure periods for PAH activation when compared with freshly isolated adult cells. It appears that such potent inducers as benzo[a]pyrene and benz[a]anthracene enhance the level of CYP450 synthesis in the initial phase of exposure to an extent that this increased CYP450 subsequently can convert benzo[a]pyrene itself effectively. When phenanthrene, an incompetent inducer, is studied for its in vitro conversion, however, a pre-exposure of cells with potent monooxygenase inducers such as benzo[a]pyrene or benz[a]anthracene is required to attain sufficient levels of CYP450...

Metabolism of polycyclic aromatic hydrocarbons in fetal human, rat and hamster epithelial lung cells

The main objectives which have been envisaged by the investigations on the metabolism of various PAH in cultivated fetal human, rat and hamster epithelial cells were (i) to characterize the metabolite profile in order to compare it to that recorded in the urinary excretion ; (ii) to simulate the in vivo situation by an in vitro system, or in other terms, to check to what extent the metabolism taking place in the intact organ is reflected by a cell system obtained from this tissue ; (iii) to study the species-specificity in order to validate animal experiments.

Investigations of the Biological Effects of Airborne and Inhalable Substances by Cell-Based In Vitro Methods: Fundamental Improvements to the ALI Concept

The state of the art for cell-based in vitro investigations of airborne and inhalable material is “air-liquid interface” (ALI) technology. Cell lines, primary cells, complex 3D models, or precision-cut lung slices (PCLS) are used to represent the lung or skin by way of an in vitro barrier model. These models have been applied in toxicity or pharmacological testing. However, contrasting with a clear demand for alternative methods, there is still no widely accepted procedure for cell-based in vitro testing of inhalable substances. In the light of this, an analysis was undertaken of common drawbacks of current approaches. Hence, the pivotal improvements aimed at were the cellular exposure environment, overall performance and applicability, operability of online investigations during exposure and routine setup. It resulted in an improved device (P.R.I.T. ExpoCube) based on an “all-in-one-plate” concept including all phases of the experiment (cell culture, exposure, and read-out) and all experimental groups (two test gas groups, controls) in one single commercial multiwell plate. Verification of the concept was demonstrated in a first experimental series using reference substances (formaldehyde, ozone, and clean air). The resulting ALI procedure enables the application of inhalable substances and mixtures under highly effective exposure conditions in routine utilization.

PAH Metabolism in Cultured Mammalian Lung Epithelial Cells

Abstract In vitro cultures of mammalian cells have an advantage over animal experiments in that human cells can be directly compared with various other mammalian cells. In this study we compared the metabolism of several polycyclic aromatic hydrocarbons (PAH) (benzo[a]pyrene, chrysene, pyrene, phenanthrene, benz[a]anthracene) in epithelial cells from hamster, rat and human lung. The cells investigated in our system yielded more or less qualitatively similar metabolic profiles for all PAH mentioned above except benz[a]anthracene in the three species. Additionally, species-specific differences were prominent between human and rodent cells with regard to the ratio of phase I and phase II metabolites. Indications have been obtained that monooxygenase induction is required in fetal cells prior to metabolic conversion of those PAH which lack an inductive potential for CYP450.