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Interlaboratory evaluation of in vitro cytotoxicity and inflammatory responses to engineered nanomaterials: the NIEHS Nano GO Consortium.

Xia T, Hamilton RF, Bonner JC, Crandall ED, Elder A, Fazlollahi F, Girtsman TA, Kim K, Mitra S, Ntim SA, Orr G, Tagmount M, Taylor AJ, Telesca D, Tolic A, Vulpe CD, Walker AJ, Wang X, Witzmann FA, Wu N, Xie Y, Zink JI, Nel A, Holian A - Environ. Health Perspect. (2013)

Bottom Line: MWCNTs did not produce cytotoxicity, but stimulated lower levels of IL-1β production in THP-1 cells, with the original MWCNT producing the most IL-1β.The results provide justification for the inclusion of mechanism-linked bioactivity assays along with traditional cytotoxicity assays for in vitro screening.In addition, the results suggest that conducting studies with multiple relevant cell types to avoid false-negative outcomes is critical for accurate evaluation of ENM bioactivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of NanoMedicine, Center for Environmental Implications of Nanotechnology, California Nanosystems Institute, University of California at Los Angeles, Los Angeles, California, USA.

ABSTRACT

Background: Differences in interlaboratory research protocols contribute to the conflicting data in the literature regarding engineered nanomaterial (ENM) bioactivity.

Objectives: Grantees of a National Institute of Health Sciences (NIEHS)-funded consortium program performed two phases of in vitro testing with selected ENMs in an effort to identify and minimize sources of variability.

Methods: Consortium program participants (CPPs) conducted ENM bioactivity evaluations on zinc oxide (ZnO), three forms of titanium dioxide (TiO2), and three forms of multiwalled carbon nanotubes (MWCNTs). In addition, CPPs performed bioassays using three mammalian cell lines (BEAS-2B, RLE-6TN, and THP-1) selected in order to cover two different species (rat and human), two different lung epithelial cells (alveolar type II and bronchial epithelial cells), and two different cell types (epithelial cells and macrophages). CPPs also measured cytotoxicity in all cell types while measuring inflammasome activation [interleukin-1β (IL-1β) release] using only THP-1 cells.

Results: The overall in vitro toxicity profiles of ENM were as follows: ZnO was cytotoxic to all cell types at ≥ 50 μg/mL, but did not induce IL-1β. TiO2 was not cytotoxic except for the nanobelt form, which was cytotoxic and induced significant IL-1β production in THP-1 cells. MWCNTs did not produce cytotoxicity, but stimulated lower levels of IL-1β production in THP-1 cells, with the original MWCNT producing the most IL-1β.

Conclusions: The results provide justification for the inclusion of mechanism-linked bioactivity assays along with traditional cytotoxicity assays for in vitro screening. In addition, the results suggest that conducting studies with multiple relevant cell types to avoid false-negative outcomes is critical for accurate evaluation of ENM bioactivity.

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Related in: MedlinePlus

Phase I/II comparisons for RLE-6TN and THP-1 cells using MTS assay data. (A) Percent viable RLE-6TN cells relative to no-particle control for each individual laboratory in phase I. (B) The ENM distorted OD readings in the MTS assay: with the ENM in the culture well (left); with the media supernatant removed and replaced in wells without particle interference (right); OD, optical density. (C) Percent viable RLE-6TN cells relative to no-particle control for each individual laboratory in phase II. (D,E) Percent viable cells relative to no-particle control for THP-1 phase I conditions (D) and for THP-1 phase II conditions (E). (F) Changes in error of the mean (left) and measure in error (right) from phase I to phase II trials for MTS assay data. Data are expressed as mean ± SE.*p < 0.05 compared with other particles at the same concentration and/or the “no-particle” control.
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f2: Phase I/II comparisons for RLE-6TN and THP-1 cells using MTS assay data. (A) Percent viable RLE-6TN cells relative to no-particle control for each individual laboratory in phase I. (B) The ENM distorted OD readings in the MTS assay: with the ENM in the culture well (left); with the media supernatant removed and replaced in wells without particle interference (right); OD, optical density. (C) Percent viable RLE-6TN cells relative to no-particle control for each individual laboratory in phase II. (D,E) Percent viable cells relative to no-particle control for THP-1 phase I conditions (D) and for THP-1 phase II conditions (E). (F) Changes in error of the mean (left) and measure in error (right) from phase I to phase II trials for MTS assay data. Data are expressed as mean ± SE.*p < 0.05 compared with other particles at the same concentration and/or the “no-particle” control.

Mentions: MTS assays. The CPPs discovered substantial variations among the replicates within individual laboratories in the phase I MTS assays using BEAS-2B and RLE-6TN cells. Figure 2A shows the low consistency for ZnO MTS data using RLE-6TN cells in phase I experiments. The CPPs determined that ENMs interfered directly with the optical density readings (Figure 2B, left). Therefore, the CPPs eliminated the optical interference by adding a centrifugation procedure to isolate the suspended ENM at the bottom of the cell culture dish, then transferring the supernatants to a fresh plate to conduct the absorbance readings (Figure 2B, right). Figure 2C shows the phase II results of MTS assay in RLE-6TN cells using the updated protocols, and the results demonstrated high consistency with low variability among different laboratories.


Interlaboratory evaluation of in vitro cytotoxicity and inflammatory responses to engineered nanomaterials: the NIEHS Nano GO Consortium.

Xia T, Hamilton RF, Bonner JC, Crandall ED, Elder A, Fazlollahi F, Girtsman TA, Kim K, Mitra S, Ntim SA, Orr G, Tagmount M, Taylor AJ, Telesca D, Tolic A, Vulpe CD, Walker AJ, Wang X, Witzmann FA, Wu N, Xie Y, Zink JI, Nel A, Holian A - Environ. Health Perspect. (2013)

Phase I/II comparisons for RLE-6TN and THP-1 cells using MTS assay data. (A) Percent viable RLE-6TN cells relative to no-particle control for each individual laboratory in phase I. (B) The ENM distorted OD readings in the MTS assay: with the ENM in the culture well (left); with the media supernatant removed and replaced in wells without particle interference (right); OD, optical density. (C) Percent viable RLE-6TN cells relative to no-particle control for each individual laboratory in phase II. (D,E) Percent viable cells relative to no-particle control for THP-1 phase I conditions (D) and for THP-1 phase II conditions (E). (F) Changes in error of the mean (left) and measure in error (right) from phase I to phase II trials for MTS assay data. Data are expressed as mean ± SE.*p < 0.05 compared with other particles at the same concentration and/or the “no-particle” control.
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672931&req=5

f2: Phase I/II comparisons for RLE-6TN and THP-1 cells using MTS assay data. (A) Percent viable RLE-6TN cells relative to no-particle control for each individual laboratory in phase I. (B) The ENM distorted OD readings in the MTS assay: with the ENM in the culture well (left); with the media supernatant removed and replaced in wells without particle interference (right); OD, optical density. (C) Percent viable RLE-6TN cells relative to no-particle control for each individual laboratory in phase II. (D,E) Percent viable cells relative to no-particle control for THP-1 phase I conditions (D) and for THP-1 phase II conditions (E). (F) Changes in error of the mean (left) and measure in error (right) from phase I to phase II trials for MTS assay data. Data are expressed as mean ± SE.*p < 0.05 compared with other particles at the same concentration and/or the “no-particle” control.
Mentions: MTS assays. The CPPs discovered substantial variations among the replicates within individual laboratories in the phase I MTS assays using BEAS-2B and RLE-6TN cells. Figure 2A shows the low consistency for ZnO MTS data using RLE-6TN cells in phase I experiments. The CPPs determined that ENMs interfered directly with the optical density readings (Figure 2B, left). Therefore, the CPPs eliminated the optical interference by adding a centrifugation procedure to isolate the suspended ENM at the bottom of the cell culture dish, then transferring the supernatants to a fresh plate to conduct the absorbance readings (Figure 2B, right). Figure 2C shows the phase II results of MTS assay in RLE-6TN cells using the updated protocols, and the results demonstrated high consistency with low variability among different laboratories.

Bottom Line: MWCNTs did not produce cytotoxicity, but stimulated lower levels of IL-1β production in THP-1 cells, with the original MWCNT producing the most IL-1β.The results provide justification for the inclusion of mechanism-linked bioactivity assays along with traditional cytotoxicity assays for in vitro screening.In addition, the results suggest that conducting studies with multiple relevant cell types to avoid false-negative outcomes is critical for accurate evaluation of ENM bioactivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of NanoMedicine, Center for Environmental Implications of Nanotechnology, California Nanosystems Institute, University of California at Los Angeles, Los Angeles, California, USA.

ABSTRACT

Background: Differences in interlaboratory research protocols contribute to the conflicting data in the literature regarding engineered nanomaterial (ENM) bioactivity.

Objectives: Grantees of a National Institute of Health Sciences (NIEHS)-funded consortium program performed two phases of in vitro testing with selected ENMs in an effort to identify and minimize sources of variability.

Methods: Consortium program participants (CPPs) conducted ENM bioactivity evaluations on zinc oxide (ZnO), three forms of titanium dioxide (TiO2), and three forms of multiwalled carbon nanotubes (MWCNTs). In addition, CPPs performed bioassays using three mammalian cell lines (BEAS-2B, RLE-6TN, and THP-1) selected in order to cover two different species (rat and human), two different lung epithelial cells (alveolar type II and bronchial epithelial cells), and two different cell types (epithelial cells and macrophages). CPPs also measured cytotoxicity in all cell types while measuring inflammasome activation [interleukin-1β (IL-1β) release] using only THP-1 cells.

Results: The overall in vitro toxicity profiles of ENM were as follows: ZnO was cytotoxic to all cell types at ≥ 50 μg/mL, but did not induce IL-1β. TiO2 was not cytotoxic except for the nanobelt form, which was cytotoxic and induced significant IL-1β production in THP-1 cells. MWCNTs did not produce cytotoxicity, but stimulated lower levels of IL-1β production in THP-1 cells, with the original MWCNT producing the most IL-1β.

Conclusions: The results provide justification for the inclusion of mechanism-linked bioactivity assays along with traditional cytotoxicity assays for in vitro screening. In addition, the results suggest that conducting studies with multiple relevant cell types to avoid false-negative outcomes is critical for accurate evaluation of ENM bioactivity.

Show MeSH
Related in: MedlinePlus