. 2018 Jul 3;15:29. doi: 10.1186/s12989-018-0265-1

Table 4.

Toxicological endpoints used and/or recommended in in vitro and in vivo iENM toxicity investigations, and studies of the gut microbiome

Endpoint	Used and/or recommended assays or procedures	References
In vitro
Nanoparticle ion release and accumulation location	Fluorescent labeling of ions [156], ICP-MS	[63, 129]
Cell proliferation	Cell count using hemocytometer	[128]
Cellular energetics	WST-1, WST-8, live/dead kit, CellTiter-Glo, XTT, MTS, MTT, NRU, Prestoblue assay	[46, 69, 127]
ROS generation	Electron paramagnetic resonance, total glutathione content, DCFH-DA assay	[63, 64, 68, 127]
Cell membrane damage	LDH and trypan blue assay	[59]
Apoptosis initiation	Annexin V-FITC, monodansylcadaverine staining	[59, 64]
Necrosis	Sytox red and propidium iodide (PI) staining	[59, 63]
Pro-inflammatory and inflammatory cytokine release	ELISA, Wester blotting	[129]
DNA damage	Fpg-modified comet assay	[62, 68]
Brush border morphology	Immunocytochemistry, electron microscopy (TEM and SEM)	[66]
Barrier integrity	Trans-epithelial electrical resistance measurement	[46, 128]
Barrier permeability	Dextran-FITC and Lucifer yellow transport	[46, 128]
Gene expression	qRT-PCR	[46, 64, 66, 68]
In vivo
Coefficients of organs	Ratio of tissue (wet weight) to body weight	[21, 85]
Changes in tissues	Histopathological evaluation	[48, 123, 149]
Testicular toxicity	Sperm count, motility and % abnormal sperms	[65]
Tissue accumulation	ICMP-MS or ICP-AES	[100]
Reductive stress	GSH/GSSG ratio in plasma	[96]
Tissue function	Blood biochemical and hematological analysis	[123, 149]
Inflammatory cells quantification in blood and the GI segment of interest	Flow cytometry, imaging flow cytometry^a	[48, 50]
Apoptosis in the GI segment of interest	TUNEL assay	[81, 125]
Cytokine release in blood and the GI segment of interest	ELISA (IL-1β, IL-2, IL-6, TNF-α, IFN-γ, IL-8, IL-10, IL-17 and GM-CSF), Western blotting	[48, 49]
Tumor progression biomarkers in colon tissue	Immunohistochemistry (COX2, β-catenin and Ki67), ELISA^a, Western blotting^a	[49]
Intestinal permeability	⁵¹Cr-EDTA radioactivity	[50]
Aberrant crypts formation in the GI tract	Bird’s procedure [122]	[50]
Local tissue concentration	Micro X-ray fluorescence, NanoSIMS imaging	[46, 50]
Gut microbiome composition	16S rRNA pyrosequencing, Shotgun metagenomic sequencing^a, Microbial transcriptomics^a	[124]
Gut microbiome models
Gas production	Gas chromatography	[45]
Fatty acid production	Fatty acid methyl ester analysis	[45]
Microbiome diversity	16S rRNA 454 pyrosequencing, Shotgun metagenomic sequencing^a, Microbial transcriptomics^a	[45, 124]

^aRecommended assays or procedures – not used so far in the iENM toxicity literature