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Table 4 Toxicological endpoints used and/or recommended in in vitro and in vivo iENM toxicity investigations, and studies of the gut microbiome

From: Ingested engineered nanomaterials: state of science in nanotoxicity testing and future research needs

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 cytometrya [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), ELISAa, Western blottinga [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 sequencinga, Microbial transcriptomicsa [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 sequencinga, Microbial transcriptomicsa [45, 124]
  1. aRecommended assays or procedures – not used so far in the iENM toxicity literature