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Inflammatory and cytotoxic responses of an alveolar-capillary coculture model to silica nanoparticles: comparison with conventional monocultures.

Kasper J, Hermanns MI, Bantz C, Maskos M, Stauber R, Pohl C, Unger RE, Kirkpatrick JC - Part Fibre Toxicol (2011)

Bottom Line: Attention should primarily be focussed on SNP effects on biological barriers.This may mimic the early inflammatory events that take place in the pulmonary alveoli after aSNP inhalation.Furthermore, a number of apoptosis markers belonging to the intrinsic pathway were upregulated in the coculture following aSNP treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: University Medical Centre, Institute of Pathology, Mainz, Germany.

ABSTRACT

Background: To date silica nanoparticles (SNPs) play an important role in modern technology and nanomedicine. SNPs are present in various materials (tyres, electrical and thermal insulation material, photovoltaic facilities). They are also used in products that are directly exposed to humans such as cosmetics or toothpaste. For that reason it is of great concern to evaluate the possible hazards of these engineered particles for human health. Attention should primarily be focussed on SNP effects on biological barriers. Accidentally released SNP could, for example, encounter the alveolar-capillary barrier by inhalation. In this study we examined the inflammatory and cytotoxic responses of monodisperse amorphous silica nanoparticles (aSNPs) of 30 nm in size on an in vitro coculture model mimicking the alveolar-capillary barrier and compared these to conventional monocultures.

Methods: Thus, the epithelial cell line, H441, and the endothelial cell line, ISO-HAS-1, were used in monoculture and in coculture on opposite sides of a filter membrane. Cytotoxicity was evaluated by the MTS assay, detection of membrane integrity (LDH release), and TER (Transepithelial Electrical Resistance) measurement. Additionally, parameters of inflammation (sICAM-1, IL-6 and IL-8 release) and apoptosis markers were investigated.

Results: Regarding toxic effects (viability, membrane integrity, TER) the coculture model was less sensitive to apical aSNP exposure than the conventional monocultures of the appropriate cells. On the other hand, the in vitro coculture model responded with the release of inflammatory markers in a much more sensitive fashion than the conventional monoculture. At concentrations that were 10-100fold less than the toxic concentrations the apically exposed coculture showed a release of IL-6 and IL-8 to the basolateral side. This may mimic the early inflammatory events that take place in the pulmonary alveoli after aSNP inhalation. Furthermore, a number of apoptosis markers belonging to the intrinsic pathway were upregulated in the coculture following aSNP treatment. Analysis of the individual markers indicated that the cells suffered from DNA damage, hypoxia and ER-stress.

Conclusion: We present evidence that our in vitro coculture model of the alveolar-capillary barrier is clearly advantageous compared to conventional monocultures in evaluating the extent of damage caused by hazardous material encountering the principle biological barrier in the lower respiratory tract.

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Transmembrane electrical resistance was measured for cocultures of H441 with ISO-HAS-1 (H441/ISO-HAS-1) as well as for cocultures of primary isolated cells (alveolar type II and HPMEC (ATII/HPMEC)). During 4 h incubation with aSNPs (NexSil20, Ludox TM-40 at concentrations of 6, 60, 600, and 6000, μg/ml) TER-values are depicted as % of time-point t0 (TER-value prior to aSNP treatment). Results are shown as means ± S.D. of 3 independent experiments with n = 2 samples for each treatment. For statistical analysis using Dunnett's Multiple Analyzing test, the 4 h value of the untreated samples was used as control. Treatment with 600 and 6000 μg/ml of both aSNP revealed a time-dependent decrease of TER after 4 h incubation. *P < 0.05, ** P < 0.01 and *** P < 0.001 compared to the untreated control
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Figure 5: Transmembrane electrical resistance was measured for cocultures of H441 with ISO-HAS-1 (H441/ISO-HAS-1) as well as for cocultures of primary isolated cells (alveolar type II and HPMEC (ATII/HPMEC)). During 4 h incubation with aSNPs (NexSil20, Ludox TM-40 at concentrations of 6, 60, 600, and 6000, μg/ml) TER-values are depicted as % of time-point t0 (TER-value prior to aSNP treatment). Results are shown as means ± S.D. of 3 independent experiments with n = 2 samples for each treatment. For statistical analysis using Dunnett's Multiple Analyzing test, the 4 h value of the untreated samples was used as control. Treatment with 600 and 6000 μg/ml of both aSNP revealed a time-dependent decrease of TER after 4 h incubation. *P < 0.05, ** P < 0.01 and *** P < 0.001 compared to the untreated control

Mentions: As shown in figure 5 the TER of untreated controls cultivated in parallel with the aSNP-stimulated groups over 4 hours decreased from t0 to t4h to approximately 70% (72 ± 7%) due to the change from serum-containing to serum-free medium. Due to the reduced osmolarity of the serum-free medium compared to the medium containing serum a slight decrease of the TER value can be expected. For both aSNPs (NexSil20 and Ludox TM-40) treatment with 6-60 μg/ml aSNPs gave similar results to the untreated control, while 600 μg/ml aSNP caused a reduction of TER to approximately 20% (Ludox TM-40: 17.4 ± 9% and NexSil20: 17.9 ± 13%) of the initial TER-value at t0. A concentration of 6000 μg/ml caused a complete disruption of the barrier after 2 h exposure for both aSNPs. Additionally, the effect of aSNPs on TER of primary isolated pulmonary microvascular endothelial cells in coculture with human alveolar type II/type I-like cells was studied as gold standard model of the alveolocapillary barrier. The behaviour of both cocultures, that is the cell lines (H441/ISO-HAS-1) and also the primary cells (ATII/HPMEC), upon aSNP exposure was comparable (600 μg/ml reduced TER to 12.5 ± 5% for Ludox TM-40 and to 6 ± 0.9% for NexSil20).


Inflammatory and cytotoxic responses of an alveolar-capillary coculture model to silica nanoparticles: comparison with conventional monocultures.

Kasper J, Hermanns MI, Bantz C, Maskos M, Stauber R, Pohl C, Unger RE, Kirkpatrick JC - Part Fibre Toxicol (2011)

Transmembrane electrical resistance was measured for cocultures of H441 with ISO-HAS-1 (H441/ISO-HAS-1) as well as for cocultures of primary isolated cells (alveolar type II and HPMEC (ATII/HPMEC)). During 4 h incubation with aSNPs (NexSil20, Ludox TM-40 at concentrations of 6, 60, 600, and 6000, μg/ml) TER-values are depicted as % of time-point t0 (TER-value prior to aSNP treatment). Results are shown as means ± S.D. of 3 independent experiments with n = 2 samples for each treatment. For statistical analysis using Dunnett's Multiple Analyzing test, the 4 h value of the untreated samples was used as control. Treatment with 600 and 6000 μg/ml of both aSNP revealed a time-dependent decrease of TER after 4 h incubation. *P < 0.05, ** P < 0.01 and *** P < 0.001 compared to the untreated control
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3040689&req=5

Figure 5: Transmembrane electrical resistance was measured for cocultures of H441 with ISO-HAS-1 (H441/ISO-HAS-1) as well as for cocultures of primary isolated cells (alveolar type II and HPMEC (ATII/HPMEC)). During 4 h incubation with aSNPs (NexSil20, Ludox TM-40 at concentrations of 6, 60, 600, and 6000, μg/ml) TER-values are depicted as % of time-point t0 (TER-value prior to aSNP treatment). Results are shown as means ± S.D. of 3 independent experiments with n = 2 samples for each treatment. For statistical analysis using Dunnett's Multiple Analyzing test, the 4 h value of the untreated samples was used as control. Treatment with 600 and 6000 μg/ml of both aSNP revealed a time-dependent decrease of TER after 4 h incubation. *P < 0.05, ** P < 0.01 and *** P < 0.001 compared to the untreated control
Mentions: As shown in figure 5 the TER of untreated controls cultivated in parallel with the aSNP-stimulated groups over 4 hours decreased from t0 to t4h to approximately 70% (72 ± 7%) due to the change from serum-containing to serum-free medium. Due to the reduced osmolarity of the serum-free medium compared to the medium containing serum a slight decrease of the TER value can be expected. For both aSNPs (NexSil20 and Ludox TM-40) treatment with 6-60 μg/ml aSNPs gave similar results to the untreated control, while 600 μg/ml aSNP caused a reduction of TER to approximately 20% (Ludox TM-40: 17.4 ± 9% and NexSil20: 17.9 ± 13%) of the initial TER-value at t0. A concentration of 6000 μg/ml caused a complete disruption of the barrier after 2 h exposure for both aSNPs. Additionally, the effect of aSNPs on TER of primary isolated pulmonary microvascular endothelial cells in coculture with human alveolar type II/type I-like cells was studied as gold standard model of the alveolocapillary barrier. The behaviour of both cocultures, that is the cell lines (H441/ISO-HAS-1) and also the primary cells (ATII/HPMEC), upon aSNP exposure was comparable (600 μg/ml reduced TER to 12.5 ± 5% for Ludox TM-40 and to 6 ± 0.9% for NexSil20).

Bottom Line: Attention should primarily be focussed on SNP effects on biological barriers.This may mimic the early inflammatory events that take place in the pulmonary alveoli after aSNP inhalation.Furthermore, a number of apoptosis markers belonging to the intrinsic pathway were upregulated in the coculture following aSNP treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: University Medical Centre, Institute of Pathology, Mainz, Germany.

ABSTRACT

Background: To date silica nanoparticles (SNPs) play an important role in modern technology and nanomedicine. SNPs are present in various materials (tyres, electrical and thermal insulation material, photovoltaic facilities). They are also used in products that are directly exposed to humans such as cosmetics or toothpaste. For that reason it is of great concern to evaluate the possible hazards of these engineered particles for human health. Attention should primarily be focussed on SNP effects on biological barriers. Accidentally released SNP could, for example, encounter the alveolar-capillary barrier by inhalation. In this study we examined the inflammatory and cytotoxic responses of monodisperse amorphous silica nanoparticles (aSNPs) of 30 nm in size on an in vitro coculture model mimicking the alveolar-capillary barrier and compared these to conventional monocultures.

Methods: Thus, the epithelial cell line, H441, and the endothelial cell line, ISO-HAS-1, were used in monoculture and in coculture on opposite sides of a filter membrane. Cytotoxicity was evaluated by the MTS assay, detection of membrane integrity (LDH release), and TER (Transepithelial Electrical Resistance) measurement. Additionally, parameters of inflammation (sICAM-1, IL-6 and IL-8 release) and apoptosis markers were investigated.

Results: Regarding toxic effects (viability, membrane integrity, TER) the coculture model was less sensitive to apical aSNP exposure than the conventional monocultures of the appropriate cells. On the other hand, the in vitro coculture model responded with the release of inflammatory markers in a much more sensitive fashion than the conventional monoculture. At concentrations that were 10-100fold less than the toxic concentrations the apically exposed coculture showed a release of IL-6 and IL-8 to the basolateral side. This may mimic the early inflammatory events that take place in the pulmonary alveoli after aSNP inhalation. Furthermore, a number of apoptosis markers belonging to the intrinsic pathway were upregulated in the coculture following aSNP treatment. Analysis of the individual markers indicated that the cells suffered from DNA damage, hypoxia and ER-stress.

Conclusion: We present evidence that our in vitro coculture model of the alveolar-capillary barrier is clearly advantageous compared to conventional monocultures in evaluating the extent of damage caused by hazardous material encountering the principle biological barrier in the lower respiratory tract.

Show MeSH
Related in: MedlinePlus