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Toxicity evaluation of manufactured CeO2 nanoparticles before and after alteration: combined physicochemical and whole-genome expression analysis in Caco-2 cells.

Fisichella M, Berenguer F, Steinmetz G, Auffan M, Rose J, Prat O - BMC Genomics (2014)

Bottom Line: Gene expression profiles obtained from cells exposed to NPs before and after their alteration were compared, to highlight differences in cellular functions.No change in the cerium redox state was observed for altered NPs.Conversely, Nanobyk 3810™ coated with ammonium citrate did not display any adverse effect at the same concentration.It can be assumed that the safe design of engineered nanoparticles could include robust protective layers conferring on them greater resistance to alteration during their life cycle.

View Article: PubMed Central - PubMed

Affiliation: CEA, IBEB, SBTN, Laboratoire d'Etude des Protéines Cibles, F-30207 Bagnols-sur-Cèze, France. odette.prat@cea.fr.

ABSTRACT

Background: Engineered nanomaterials may release nanosized residues, by degradation, throughout their life cycle. These residues may be a threat for living organisms. They may be ingested by humans through food and water. Although the toxicity of pristine CeO2 nanoparticles (NPs) has been documented, there is a lack of studies on manufactured nanoparticles, which are often surface modified. Here, we investigated the potential adverse effects of CeO2 Nanobyk 3810™ NPs, used in wood care, and their residues, altered by light or acid.

Results: Human intestinal Caco-2 cells were exposed to residues degraded by daylight or in a medium simulating gastric acidity. Size and zeta potential were determined by dynamic light scattering. The surface structure and redox state of cerium were analyzed by transmission electronic microscopy (TEM) and X-ray absorption spectroscopy, respectively. Viability tests were performed in Caco-2 cells exposed to NPs. Cell morphology was imaged with scanning electronic microscopy. Gene expression profiles obtained from cells exposed to NPs before and after their alteration were compared, to highlight differences in cellular functions.No change in the cerium redox state was observed for altered NPs. All CeO2 NPs suspended in the culture medium became microsized. Cytotoxicity tests showed no toxicity after Caco-2 cell exposure to these various NPs up to 170 μg/mL (24 h and 72 h). Nevertheless, a more-sensitive whole-gene-expression study, based on a pathway-driven analysis, highlighted a modification of metabolic activity, especially mitochondrial function, by altered Nanobyk 3810™. The down-regulation of key genes of this pathway was validated by qRT-PCR. Conversely, Nanobyk 3810™ coated with ammonium citrate did not display any adverse effect at the same concentration.

Conclusion: The degraded nanoparticles were more toxic than their coated counterparts. Desorption of the outside layer was the most likely cause of this discrepancy in toxicity. It can be assumed that the safe design of engineered nanoparticles could include robust protective layers conferring on them greater resistance to alteration during their life cycle.

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Caco-2 cell viability tests. Caco-2 cells were grown in 96-well plates and differentiated for 21 days. Cells were then exposed for 24 h or 72 h to concentrations of CeO2 NPs ranging from 21.25 to 170 μg/mL. Left side) ATP tests: cell viability was determined by reading the level of bioluminescence (CellTiter-Glo luminescent cell viability assay, Promega). Right side) XTT tests: cell viability was determined by mitochondrial enzyme activity via XTT reagent (In Vitro toxicology assay kit XTT based, Sigma-Aldrich). An experimental positive control was obtained by exposing cells to H2O2 in both tests. Cell viability was not altered for concentrations up to 170 mg/L.
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Fig2: Caco-2 cell viability tests. Caco-2 cells were grown in 96-well plates and differentiated for 21 days. Cells were then exposed for 24 h or 72 h to concentrations of CeO2 NPs ranging from 21.25 to 170 μg/mL. Left side) ATP tests: cell viability was determined by reading the level of bioluminescence (CellTiter-Glo luminescent cell viability assay, Promega). Right side) XTT tests: cell viability was determined by mitochondrial enzyme activity via XTT reagent (In Vitro toxicology assay kit XTT based, Sigma-Aldrich). An experimental positive control was obtained by exposing cells to H2O2 in both tests. Cell viability was not altered for concentrations up to 170 mg/L.

Mentions: The studies were conducted on the well-established Caco-2 cell line, differentiated for 21 days. The integrity of the cell layer was assessed by measurement of the transepithelial electrical resistance (TEER), stabilized at 500 ohms.cm2 after 21 days. We performed two cytotoxicity tests at two exposure times, 24 h and 72 h, to take into account the kinetic parameters. As shown in Figure 2, with the ATP assay (Left side), the presence of CeO2 NPs did not induce adverse effects on Caco-2 cells after 24 h or 72 h exposure, even at high concentration (170 μg/mL). The XTT assay (Right side) confirmed the lack of apparent toxicity of Nanobyk NPs and pristine cerium NPs in Caco-2 cells. For degraded Nanobyk NPs (NB-DL and NB-DA) only mild toxicity was observed using the XTT test, and only at the highest concentration (170 μg/mL) after 72 h exposure.Figure 2


Toxicity evaluation of manufactured CeO2 nanoparticles before and after alteration: combined physicochemical and whole-genome expression analysis in Caco-2 cells.

Fisichella M, Berenguer F, Steinmetz G, Auffan M, Rose J, Prat O - BMC Genomics (2014)

Caco-2 cell viability tests. Caco-2 cells were grown in 96-well plates and differentiated for 21 days. Cells were then exposed for 24 h or 72 h to concentrations of CeO2 NPs ranging from 21.25 to 170 μg/mL. Left side) ATP tests: cell viability was determined by reading the level of bioluminescence (CellTiter-Glo luminescent cell viability assay, Promega). Right side) XTT tests: cell viability was determined by mitochondrial enzyme activity via XTT reagent (In Vitro toxicology assay kit XTT based, Sigma-Aldrich). An experimental positive control was obtained by exposing cells to H2O2 in both tests. Cell viability was not altered for concentrations up to 170 mg/L.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4150968&req=5

Fig2: Caco-2 cell viability tests. Caco-2 cells were grown in 96-well plates and differentiated for 21 days. Cells were then exposed for 24 h or 72 h to concentrations of CeO2 NPs ranging from 21.25 to 170 μg/mL. Left side) ATP tests: cell viability was determined by reading the level of bioluminescence (CellTiter-Glo luminescent cell viability assay, Promega). Right side) XTT tests: cell viability was determined by mitochondrial enzyme activity via XTT reagent (In Vitro toxicology assay kit XTT based, Sigma-Aldrich). An experimental positive control was obtained by exposing cells to H2O2 in both tests. Cell viability was not altered for concentrations up to 170 mg/L.
Mentions: The studies were conducted on the well-established Caco-2 cell line, differentiated for 21 days. The integrity of the cell layer was assessed by measurement of the transepithelial electrical resistance (TEER), stabilized at 500 ohms.cm2 after 21 days. We performed two cytotoxicity tests at two exposure times, 24 h and 72 h, to take into account the kinetic parameters. As shown in Figure 2, with the ATP assay (Left side), the presence of CeO2 NPs did not induce adverse effects on Caco-2 cells after 24 h or 72 h exposure, even at high concentration (170 μg/mL). The XTT assay (Right side) confirmed the lack of apparent toxicity of Nanobyk NPs and pristine cerium NPs in Caco-2 cells. For degraded Nanobyk NPs (NB-DL and NB-DA) only mild toxicity was observed using the XTT test, and only at the highest concentration (170 μg/mL) after 72 h exposure.Figure 2

Bottom Line: Gene expression profiles obtained from cells exposed to NPs before and after their alteration were compared, to highlight differences in cellular functions.No change in the cerium redox state was observed for altered NPs.Conversely, Nanobyk 3810™ coated with ammonium citrate did not display any adverse effect at the same concentration.It can be assumed that the safe design of engineered nanoparticles could include robust protective layers conferring on them greater resistance to alteration during their life cycle.

View Article: PubMed Central - PubMed

Affiliation: CEA, IBEB, SBTN, Laboratoire d'Etude des Protéines Cibles, F-30207 Bagnols-sur-Cèze, France. odette.prat@cea.fr.

ABSTRACT

Background: Engineered nanomaterials may release nanosized residues, by degradation, throughout their life cycle. These residues may be a threat for living organisms. They may be ingested by humans through food and water. Although the toxicity of pristine CeO2 nanoparticles (NPs) has been documented, there is a lack of studies on manufactured nanoparticles, which are often surface modified. Here, we investigated the potential adverse effects of CeO2 Nanobyk 3810™ NPs, used in wood care, and their residues, altered by light or acid.

Results: Human intestinal Caco-2 cells were exposed to residues degraded by daylight or in a medium simulating gastric acidity. Size and zeta potential were determined by dynamic light scattering. The surface structure and redox state of cerium were analyzed by transmission electronic microscopy (TEM) and X-ray absorption spectroscopy, respectively. Viability tests were performed in Caco-2 cells exposed to NPs. Cell morphology was imaged with scanning electronic microscopy. Gene expression profiles obtained from cells exposed to NPs before and after their alteration were compared, to highlight differences in cellular functions.No change in the cerium redox state was observed for altered NPs. All CeO2 NPs suspended in the culture medium became microsized. Cytotoxicity tests showed no toxicity after Caco-2 cell exposure to these various NPs up to 170 μg/mL (24 h and 72 h). Nevertheless, a more-sensitive whole-gene-expression study, based on a pathway-driven analysis, highlighted a modification of metabolic activity, especially mitochondrial function, by altered Nanobyk 3810™. The down-regulation of key genes of this pathway was validated by qRT-PCR. Conversely, Nanobyk 3810™ coated with ammonium citrate did not display any adverse effect at the same concentration.

Conclusion: The degraded nanoparticles were more toxic than their coated counterparts. Desorption of the outside layer was the most likely cause of this discrepancy in toxicity. It can be assumed that the safe design of engineered nanoparticles could include robust protective layers conferring on them greater resistance to alteration during their life cycle.

Show MeSH
Related in: MedlinePlus