Novel approaches for studying pulmonary toxicity in vitro.

The in vitro study of adverse cellular effects induced by inhaled pollutants poses a special problem due to the difficulties of exposing cultured cells of the respiratory tract directly to test atmospheres that can include complex gaseous and particulate mixtures. In general, there is no widely accepted in vitro exposure system. However, in vitro methods offer the unique possibility for use of human cells, developed and validated cell culture and exposure device (CULTEX(1)) using the principle of the air/liquid exposure technique. Cells of the respiratory tract are grown on porous membranes in transwell inserts. After removal of the medium, the cells can be treated on their superficial surfaces with the test atmosphere, and at the same time they are supplied with nutrients through the membrane below. In comparison with other experimental approaches, the goal of our studies is to analyze the biological effects of test atmospheres under environmental conditions, i.e. without humidifying the atmosphere or adding additional CO(2). The system used is small and flexible enough independent of a cultivation chamber and thus offers the opportunity for onsite study of indoor and outdoor atmospheres in the field. The efficacy of the exposure device has already been demonstrated in the analysis of dose-dependent cytotoxic and genotoxic effects of exposure of epithelial lung cells to complex mixtures such as native diesel exhaust and side-stream smoke.

Exposure of human lung cells to native diesel motor exhaust--development of an optimized in vitro test strategy.

To investigate the effects of native diesel motor exhaust on human lung cells in vitro, a new experimental concept was developed using an exposure device on the base of the cell cultivation system CULTEX (Patent No. DE19801763.PCT/EP99/00295) to handle the cells during a 1-h exposure period independent of an incubator and next to an engine test rig. The final experimental set-up allows the investigation of native (chemically and physically unmodified) diesel exhaust using short distances for the transportation of the gas to the target cells. The analysis of several atmospheric compounds as well as the particle concentration of the exhaust was performed by online monitoring in parallel. To validate the complete system we concentrated on the measurement of two distinct viability parameters after exposure to air and undiluted, diluted and filtered diesel motor exhaust generated under different engine operating conditions. Cell viability was not influenced by the exposure to clean air, whereas dose-dependent cytotoxicity was found contingent on the dosage of exhaust. Additionally, the quality of exhaust, represented by two engine operating conditions (idling, higher load), also showed well-distinguishable cytotoxicity. In summary, the experimental set-up allows research on biological effects of native engine emissions using short exposure times.

In vitro efficacy of chemotherapeutics as determined by 50% inhibitory concentrations in cell cultures of mammary gland tumors obtained from dogs.

OBJECTIVE: To determine the 50% inhibitory concentration (IC-50) of carboplatin, cisplatin, and doxorubicin in cell cultures of mammary gland tumors obtained from dogs and to assess whether in vitro efficacy was within the range of clinically relevant concentrations, SAMPLE POPULATION: 30 mammary gland tumors excised from dogs. PROCEDURE: Cell cultures were established from the 30 tumors. Cultures then were treated with carboplatin, cisplatin, or doxorubicin. Growth inhibition of cultures was assessed via DNA measurement 24, 48, and 72 hours after treatment. The IC-50 values were calculated by use of linear interpolation. RESULTS: Cultures varied in their pattern of susceptibility. Doxorubicin induced significantly lower IC-50 values than the platinum derivatives. Cisplatin and carboplatin had comparable effects. The IC-50 values for carboplatin and doxorubicin were in the range of clinically relevant concentrations, but only part of the cisplatin cultures had IC-50 values within clinically relevant concentrations. We did not detect differences in the in vitro susceptibility among subtypes of tumors (ie, adenocarcinoma, solid carcinoma, malignant mixed tumor). CONCLUSION AND CLINICAL RELEVANCE: The IC-50 values determined in this study allowed assessment of in vitro drug efficacy of chemotherapeutics in cultures of mammary gland tumors obtained from dogs. Variations in susceptibility were evident and emphasize the importance of assessing susceptibility and resistance patterns for each tumor. Prospective studies to assess direct correlations between in vitro and in vivo efficacy must be performed to determine the clinical predictive value of this in vitro chemosensitivity assay for treatment of dogs with mammary gland tumors.

A method for in vitro analysis of the biological activity of complex mixtures such as sidestream cigarette smoke.

Studies of the cytotoxicity of air contaminants such as gaseous or particulate compounds and complex mixtures have traditionally used in animal experiments because of the difficulties in exposing cell cultures directly to these substances. New cultivation and exposure techniques enhance the efficiency of in vitro methods, as demonstrated by a new system called CULTEX* which uses a transwell membrane technique for direct exposure of complex mixtures like sidestream cigarette smoke at the air/liquid interface. The factors influencing the susceptibility of human bronchial epithelial cells (e.g. gas flow rate or duration of exposure) were studied and the cells were finally exposed for one hour to clean air or different concentrations of sidestream smoke. The biological parameters estimated were number of cells, metabolic activity and glutathione concentration. After exposure of the cells to sidestream cigarette smoke, dose-dependent effects were measured. Thus, the introduction of these cultivation and exposure techniques offers new testing strategies for the toxicological evaluation of a broad range of airborne and inhalable compounds.

In vitro exposure of isolated cells to native gaseous compounds--development and validation of an optimized system for human lung cells.

An exposure system for adherent growing cells to native gaseous compounds was developed using air/liquid culture techniques on the basis of the Cultex system'. In contrast to other exposure systems the reproducible testing of native environmentally relevant gases without changing their physical or chemical properties including heating, CO2- content and humidity is possible. Specially designed systems for medium flow and gas support guarantee the nutrification and humidification as well as the direct gas contact of the exposed cells which are cultivated on microporous membranes (0.4 microm pore size). The system works independently of a cell culture incubator offering the possibility to analyze any relevant gas mixture directly under indoor or outdoor conditions. Several experimental approaches were carried out to characterize the properties of the system. In exploratory experiments without cells, the reproducibility and quality of the gas/membrane contact could be demonstrated. Exposures of human lung fibroblasts (Lk004 cells) and human lung epithelial cells (HFBE-21 cells) to synthetic air, ozone (202 ppb, 510 ppb) and nitrogen dioxide (75 ppb to 1,200 ppb) established that cells could be treated for 120 minutes without significant loss of cellular viability. At the same time, the experiments confirmed that such exposure times are long enough to detect biological effects of environmentally relevant gas mixtures. The analysis of viability (viable cell number, tetrazoliumsalt cleavage) and intracellular end-points (oxidized/reduced glutathione, ATP/ADP) showed that both gases induced relevant cellular changes. In summary, the efficiency and practicability of this newly developed exposure system for adherent human lung cells could be clearly demonstrated.

Use of cultivated osteoprogenitor cells to increase bone formation in segmental mandibular defects: an experimental pilot study in sheep.

The hypothesis of the present experimental pilot study was that autogeneous cultivated osteoprogenitor cells in porous calcium phosphate scaffolds can increase bone formation in segmental defects of the mandible. The autogenous osteoprogenitor cells of eight sheep were cultivated from bone biopsies from the iliac crest and seeded into cylindrical scaffolds of pyrolized bovine bone of an overall length of 35 mm and 13 mm in diameter. Segmental defects of 35 mm length were created unilaterally in the mandibles of the animals. Reconstruction was performed using cylinders with cultivated osteoprogenitor cells in four animals and empty scaffolds in the remaining four sheep, which served as controls. After 5 months, the mandibles were retrieved and the reconstructed areas were analyzed by qualitative and quantitative histology in serial undecalcified thick-section specimens. There was significantly more bone formation in the group that had received scaffolds with cultivated bone cells (P=0.028). Bone formation was present in 34.4% of the evaluated cross-sectional units in the seeded scaffolds, while it was found in 10.4% in the control group. Although the spatial distribution of bone formation was significantly different across the scaffold in both groups, osteoprogenitor cells appeared to have increased bone formation, particularly in the centre of the defect when compared to the control group. It is concluded that the repair of segmental defects of the mandible can be enhanced by the transplantation of autogenous osteoprogenitor cells in a porous calcium phosphate scaffold.

Development of a cell culture model system for routine testing of substances inducing oxidative stress.

The toxicological model of oxidative stress is a mechanism which is currently thought to be involved in the formation and development of many serious human diseases. Little is known about cellular responses to oxidative damage and the related specific toxicological properties of substances such as chemicals or components of the polluted urban atmosphere. On the basis of biological pathways involved in cellular antioxidative mechanisms, we developed a biological model for studying the oxidative properties of substances. This includes human lung cells and methods for biochemical analysis of cellular endpoints. Antioxidative and glycolysis-related enzyme activities (glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase, phosphofructokinase, enolase, glucose-6-phosphate dehydrogenase), ATP/ADP/AMP and glutathione (oxidized and reduced) are determined. Routine testing of substances with high reproducibility and fast screening is provided by adapting methods for biochemical analysis to determinations using cells grown on microtitre plates. First experiments with standard model substances inducing oxidative stress such as H(2)O(2) and tert-butylhydroperoxide show that enzymatic activities can be measured with good reproducibility and their changes can be followed kinetically. The results indicate the relevance of the determined parameters for such toxicological events and the usefulness of the biological indicator system for routine testing.

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.

Micronucleus induction in V79 cells after direct exposure to whole cigarette smoke.

Previous investigations on the effects of cigarette smoke on cultured cells have used mainly smoke condensate dissolved in culture medium. A system has been designed which allows direct exposure of cells to fresh cigarette smoke, without an intervening layer of growth medium between the cells and the smoke. Preliminary results have been obtained which demonstrate the viability of the system. V79 cells were cultured on porous membranes (Transwell; Costar). During smoke exposure only the lower surface of each Transwell is supplied with culture medium from the bottom of the culture chambers. In this way the cells had direct contact with the atmosphere at the upper surface and could be exposed directly to the test compound. The constructed exposure system consists of a smoke generator and an exposure unit containing six Transwells, the latter contained in an incubator. Cigarette smoke was generated using a standard 2 s, 35 ml puff once per min. The puff is diluted with conditioned air from the incubator and injected into the exposure unit. Following exposure of the cells to air only for 3 h there was no effect upon V79 cell viability. However, after exposure to smoke containing between 88 and 224 mg/m3 particulate matter, an inhibition of cell proliferation and induction of micronuclei was measured. When a Cambridge filter pad was placed between the cigarette and the cell exposure system to remove particulate matter cell proliferation was also reduced and an increased frequency of micronuclei above the control value was measured.

Experimental reconstruction of the mandible using polylactic acid tubes and basic fibroblast growth factor in alloplastic scaffolds.

PURPOSE: This study evaluated the use of a mitogenic growth factor in combination with barrier membranes and porous alloplastic scaffolds for the repair of segmental defects of the mandible. MATERIALS AND METHODS: Fifteen Göttingen minipigs were used for the study. In five animals, mandibular defects of 2 cm were created on both sides of the mandible and bridged by a system of polylactic acid (PLA) tubes and pyrolized bovine bone. On one side of the mandible, the alloplastic scaffolds were loaded with 115 microg recombinant human basic fibroblast growth factor (rhbFGF). In five other animals, defects 4 cm in length were created bilaterally, similarly bridged, and loaded with 230 microg of rhFGF. In five control animals, bilateral 2-cm defects were created that were left empty on one side and bridged with an empty PLA tube on the other. Mitogenic efficacy of the growth factor was assessed on fibroblast cultures by di-methyl-thiazol-2-tetrazolium-bromide assay before implantation. RESULTS: After 5 months, there was negligible bone regeneration in the control defects, regardless of whether they had been left completely empty or bridged by empty PLA tubes. The 2-cm defects showed bridging in 8 of 10 tubes, with complete consolidation by bone ingrowth in four defects. The 4-cm defects showed bony union in six cases, with complete bone fill in two tubes, and four defects incompletely filled. The bFGF had no appreciable effect with regard to velocity, quantity, and three-dimensional structure of bone formation, neither in the short nor in the long defects despite clear in vitro efficacy. CONCLUSION: Repair of segmental defects using bioresorbable membranes appears to be possible. However, a single-dose application of bFGF is apparently ineffective, possibly because of rapid dilution.

Sensitivity of a hamster lung cell line to direct and indirect acting carcinogens.

Cytotoxicity of benzo(a)pyrene (B(a)P), 7,12-dimethylbenz(a)anthracene (DMBA), aflatoxin B1 (AB1), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was estimated in vitro by using a hamster lung cell line. The studies were conducted to assess the usefulness of an organ-specific cell culture system for demonstrating differences in the cytotoxic potency of diverse chemical carcinogens. Cytotoxicity was determined by using the succinate dehydrogenase assay (MTT assay) after different incubation times and concentrations with the corresponding carcinogens. The effective concentration EC50 as well as the slope of the regression line were used as parameters for the biological effects. The results from these studies indicate a clear dose-dependent reaction after incubation of the cells with aflatoxin B1 (EC50: 2.3 microM) and MNNG (EC50: 4.0 microM). For the polycyclic hydrocarbons benzo(a)pyrene and DMBA, a dose-independent reaction was found. These results indicate that consideration of the EC50 values only might not be sufficient to characterize differences in the cytotoxic activity of different substances. Chemicals can lead to equal values in the EC50, but cells can differ significantly in their biological sensitivity, meaning that the extent of reduction in cell proliferation depends on the chemical used. By considering the two above-mentioned parameters, a ranking for the analyzed substances will be possible in the following way: AB1, MNNG, DMBA and B(a)P. Taken together, our experiments show that it is possible to reveal differences in the cytotoxic potency of chemicals by using in vitro methods.

Comparison of biological effects of different polycyclic aromatic hydrocarbons in lung cells of hamster and rat in vitro.

The cytotoxicity and frequencies of transformation induced by 5 environmental polycyclic aromatic hydrocarbons (PAH) in hamster (M3E3/C3) and rat (WRB K3) lung cells were compared. Both cell strains investigated here retain major metabolic characteristics of the target cells in vivo and are thus able to effectively metabolize, i.e. activate, PAH. Cytotoxic effects of the carcinogen were determined in colony-forming assays and the PAH tested induced dose-dependent cytotoxic responses in the M3E3/C3 and WRB cells. They could then be classified into strong and weak cytotoxicity. Compared to the hamster cell system, the WRB cells were generally shown to be more sensitive. The transforming capacity of the compounds was determined by a soft agar colony formation assay detecting cells with anchorage independency (AI). All PAH investigated induced transformation to AI growth in both cell systems. The transforming activity of the PAH, relative to benzo[a]pyrene (B[a]P) as a reference substance, was determined to facilitate their ranking. This order of transforming potency appears to be similar to that observed in animal studies.