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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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Protein expression of cell death regulators was analysed after apical exposure of differentiated cocultures with aSNPs (NexSil20: 600 μg/ml) to mimic the situation after accidental inhalation of aSNPs. After 4 h incubation with aSNPs filter membranes were excised, lysed for 30 min and an apoptosis protein array was performed. Data are depicted as means ± S.D. from 2 independent experiments with n = 4 samples for each treatment. Quantitative analysis of the array revealed increased levels of a number of apoptosis markers, especially of the intrinsic pathway.
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Figure 7: Protein expression of cell death regulators was analysed after apical exposure of differentiated cocultures with aSNPs (NexSil20: 600 μg/ml) to mimic the situation after accidental inhalation of aSNPs. After 4 h incubation with aSNPs filter membranes were excised, lysed for 30 min and an apoptosis protein array was performed. Data are depicted as means ± S.D. from 2 independent experiments with n = 4 samples for each treatment. Quantitative analysis of the array revealed increased levels of a number of apoptosis markers, especially of the intrinsic pathway.

Mentions: The protein array for apoptosis markers demonstrated for the coculture that the expression of a number of intrinsic apoptosis markers was elevated in a time-dependent way after exposure to 600 μg/ml NexSil20 (data for 10 min and 1 h exposure are not shown). Figure 7 shows the percentage changes of apoptosis marker protein content related to the non-treated control after 4 h exposure. The assay revealed a phosphorylation of the p53-protein at Ser15, Ser46 and Ser392 (Ser15: 151 ± 7%, Ser46: 143 ± 7%, Ser392: 161 ± 9%). An increase of pro-apoptotic markers such as Bad (126 ± 1.4%) and Bax (144 ± 20.5%) and its ligands was also detected. In addition, the assay detected cytochrome c release (110 ± 0.9%) into the cytosol. Further major apoptotic markers, such as Fas (113 ± 0.6%), DR4 (174 ± 0.4%) and DR5 (164 ± 2.4%) were also upregulated. Concerning hypoxia, HIF-1α (140 ± 23%), an hypoxia inducible factor, showed a 1.4 fold increased level compared to the untreated control. Additionally, an on average 1.8 fold higher amount of cell cycle and proliferation regulator proteins p21 (174 ± 10%) as well as p27 (181 ± 13.8%) was observed compared to the untreated control. However, elevated levels of apoptosis inhibitory factors such as XIAP (X-linked Inhibitor of apoptosis Protein: 158 ± 10.8%) and survivin (153 ± 3.6%) were indicated by this array following NexSil20 exposure. The increase of paraoxonase-2 (Pon2: 145 ± 0.4%), which has a reductive function to reactive oxygen species, is an indication that the cells of the coculture are in a stressed condition after 4 h apical exposure to 600 μg/ml 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)

Protein expression of cell death regulators was analysed after apical exposure of differentiated cocultures with aSNPs (NexSil20: 600 μg/ml) to mimic the situation after accidental inhalation of aSNPs. After 4 h incubation with aSNPs filter membranes were excised, lysed for 30 min and an apoptosis protein array was performed. Data are depicted as means ± S.D. from 2 independent experiments with n = 4 samples for each treatment. Quantitative analysis of the array revealed increased levels of a number of apoptosis markers, especially of the intrinsic pathway.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Protein expression of cell death regulators was analysed after apical exposure of differentiated cocultures with aSNPs (NexSil20: 600 μg/ml) to mimic the situation after accidental inhalation of aSNPs. After 4 h incubation with aSNPs filter membranes were excised, lysed for 30 min and an apoptosis protein array was performed. Data are depicted as means ± S.D. from 2 independent experiments with n = 4 samples for each treatment. Quantitative analysis of the array revealed increased levels of a number of apoptosis markers, especially of the intrinsic pathway.
Mentions: The protein array for apoptosis markers demonstrated for the coculture that the expression of a number of intrinsic apoptosis markers was elevated in a time-dependent way after exposure to 600 μg/ml NexSil20 (data for 10 min and 1 h exposure are not shown). Figure 7 shows the percentage changes of apoptosis marker protein content related to the non-treated control after 4 h exposure. The assay revealed a phosphorylation of the p53-protein at Ser15, Ser46 and Ser392 (Ser15: 151 ± 7%, Ser46: 143 ± 7%, Ser392: 161 ± 9%). An increase of pro-apoptotic markers such as Bad (126 ± 1.4%) and Bax (144 ± 20.5%) and its ligands was also detected. In addition, the assay detected cytochrome c release (110 ± 0.9%) into the cytosol. Further major apoptotic markers, such as Fas (113 ± 0.6%), DR4 (174 ± 0.4%) and DR5 (164 ± 2.4%) were also upregulated. Concerning hypoxia, HIF-1α (140 ± 23%), an hypoxia inducible factor, showed a 1.4 fold increased level compared to the untreated control. Additionally, an on average 1.8 fold higher amount of cell cycle and proliferation regulator proteins p21 (174 ± 10%) as well as p27 (181 ± 13.8%) was observed compared to the untreated control. However, elevated levels of apoptosis inhibitory factors such as XIAP (X-linked Inhibitor of apoptosis Protein: 158 ± 10.8%) and survivin (153 ± 3.6%) were indicated by this array following NexSil20 exposure. The increase of paraoxonase-2 (Pon2: 145 ± 0.4%), which has a reductive function to reactive oxygen species, is an indication that the cells of the coculture are in a stressed condition after 4 h apical exposure to 600 μg/ml 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