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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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Membrane integrity was determined by the LDH assay for H441 in coculture. The H441 of the coculture were incubated with aSNP (Ludox TM-40: light grey, NexSil20: dark grey) for 4 h in serum-free medium. aSNPs were then removed and cells were cultivated for further 20 h. The assays were conducted after both time points (4 h exposure and 4 h exposure with 20 h recovery). Data are depicted as percentage of the total LDH amount of the cells (lysis control). Results are shown as means ± S.D. (n = 6-9) of 2-3 independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to the untreated control
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Figure 2: Membrane integrity was determined by the LDH assay for H441 in coculture. The H441 of the coculture were incubated with aSNP (Ludox TM-40: light grey, NexSil20: dark grey) for 4 h in serum-free medium. aSNPs were then removed and cells were cultivated for further 20 h. The assays were conducted after both time points (4 h exposure and 4 h exposure with 20 h recovery). Data are depicted as percentage of the total LDH amount of the cells (lysis control). Results are shown as means ± S.D. (n = 6-9) of 2-3 independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to the untreated control

Mentions: An LDH leakage was also measured after 4 h apical (upper well) exposure and also 20 h recovery of the coculture (Figure 2). After 4 h a concentration of 600 μg/ml aSNPs caused a non-significant release of LDH (Ludox TM-40: 19 ± 10% and NexSil20: 15 ± 5%), whereas after 20 h recovery a significant, but much lower release compared to the monoculture, was detected for 600 μg/ml aSNPs, apically exposed (Ludox TM-40: 53 ± 11% and NexSil20: 35 ± 5%). Incubation with 6000 μg/ml, however, showed a considerable LDH release at 4 h (Ludox TM-40: 72 ± 0.85% and NexSil20: 90 ± 11%) as well as after 20 h recovery (Ludox TM-40: 97 ± 3.5% and NexSil20: 95 ± 0.24%). The coculture revealed a lower responsiveness towards aSNP exposure concerning LDH release. Compared to the conventional monocultures the response towards aSNP treatment was shifted by about a factor of 10 of aSNP concentration (from 600 μg/ml to 6000 μg/ml).


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)

Membrane integrity was determined by the LDH assay for H441 in coculture. The H441 of the coculture were incubated with aSNP (Ludox TM-40: light grey, NexSil20: dark grey) for 4 h in serum-free medium. aSNPs were then removed and cells were cultivated for further 20 h. The assays were conducted after both time points (4 h exposure and 4 h exposure with 20 h recovery). Data are depicted as percentage of the total LDH amount of the cells (lysis control). Results are shown as means ± S.D. (n = 6-9) of 2-3 independent experiments. *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 2: Membrane integrity was determined by the LDH assay for H441 in coculture. The H441 of the coculture were incubated with aSNP (Ludox TM-40: light grey, NexSil20: dark grey) for 4 h in serum-free medium. aSNPs were then removed and cells were cultivated for further 20 h. The assays were conducted after both time points (4 h exposure and 4 h exposure with 20 h recovery). Data are depicted as percentage of the total LDH amount of the cells (lysis control). Results are shown as means ± S.D. (n = 6-9) of 2-3 independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to the untreated control
Mentions: An LDH leakage was also measured after 4 h apical (upper well) exposure and also 20 h recovery of the coculture (Figure 2). After 4 h a concentration of 600 μg/ml aSNPs caused a non-significant release of LDH (Ludox TM-40: 19 ± 10% and NexSil20: 15 ± 5%), whereas after 20 h recovery a significant, but much lower release compared to the monoculture, was detected for 600 μg/ml aSNPs, apically exposed (Ludox TM-40: 53 ± 11% and NexSil20: 35 ± 5%). Incubation with 6000 μg/ml, however, showed a considerable LDH release at 4 h (Ludox TM-40: 72 ± 0.85% and NexSil20: 90 ± 11%) as well as after 20 h recovery (Ludox TM-40: 97 ± 3.5% and NexSil20: 95 ± 0.24%). The coculture revealed a lower responsiveness towards aSNP exposure concerning LDH release. Compared to the conventional monocultures the response towards aSNP treatment was shifted by about a factor of 10 of aSNP concentration (from 600 μg/ml to 6000 μg/ml).

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