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

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.

Figure 1

SEM images of ENMs: (A) TiO₂-P25, (B) TiO₂-NB, (C) TiO₂-A, (D) ZnO, (E) O-MWCNT, (F) P-MWCNT, and (G) F-MWCNT.

Figure 2

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.

Figure 3

Phase I/II comparisons for RLE-6TN and THP-1 cells using LDH assay data. (A,B) Percent LDH release in RLE-6TN cells relative to total cell lysis (100% cell death) for each individual laboratory in phase I (A) and in phase II (B). (C,D) Percent LDH release relative to total lysis for THP-1 phase I conditions (C) and for THP-1 phase II conditions (D). (E) Changes in error of the mean (left) and measure in error (right) from phase I to phase II trials for LDH 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.

Figure 4

Phase I/II comparisons for THP-1 cells using IL-1β assay data. (A) Percent IL-1β release from THP-1 cells for each individual laboratory in phase I. (B) THP-1 cell differentiation technique altered cell morphology: THP-1 cells pretreated with 1.62 µM PMA for 24 hr formed clumps (left), whereas cells pretreated with vitamin D3 overnight and then treated with 5 nM PMA were evenly dispersed (right). (C) IL-1β release from THP-1 cells for each individual laboratory in phase II. (D,E) Summary IL-1β release for phase I conditions (D) and for 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 IL-1β 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. ^#p < 0.001 for dose response (each laboratory’s data analyzed independently).