Table 1.
Representative in silico and in vitro assays to measure the key characteristics of carcinogens
| Endpoint | In silico or non-human in vitro assay | Human in vitro assay |
|---|---|---|
| KC 1: Is Electrophilic | ||
| Electrophilic reactivity | In silico prediction (111) | |
| Protein adducts | Fluorescence-based (MSTI) assay (18) Glutathione depletion assay (112) *Chemoproteomics (113) |
|
| DNA adducts | DNA adduct measurement by HPLC (114) | |
| KC 2: Is Genotoxic | ||
| Mutation/single nucleotide variants | In silico prediction (115) Bacterial reverse mutation (Ames) (OECD 471) |
|
| Mammalian mutation assay (mouse lymphoma assay, human HPRT mutation assay) (OECD 476) | ||
| *Error-corrected Next-Generation Sequencing (116) | ||
| Structural chromosome alterations/DNA strand breaks (clastogenicity); aneugenicity | Chromosome aberration assay (OECD 473/475) Micronucleus assay (OECD 487) Comet assay (OECD 489) |
|
| *TGX-DDI biomarker in Human TK6 cells* (117) | ||
| KC 3: Alters DNA Repair or Causes Genomic Instability | ||
| Copy Number Variations (Duplications, Deletions, Amplifications, Insertions) | Comparative Genome Hybridization (CGH, Array based) (118) Next Generation High-throughput Sequencing for somatic mutation detection (43) |
|
| Inter-/intra-chromosomal translocations | Spectral Karyotyping (SKY), Karyotyping (50) Next Generation High-throughput Sequencing for somatic mutation detection (43) |
|
| Microsatellite instability | Fluorescent Multiplex PCR-based method using DNA (119) | |
| DNA repair capacity | Unscheduled DNA synthesis (120) Host cell reactivation for evaluation of nucleotide excision repair, mismatch repair, base excision repair, nonhomologous end joining, homologous recombination, and methylguanine methyltransferase (121,122) Topoisomerase I and II enzymatic activity analysis using gel electrophoresis (123) |
|
| KC 4: Induces Epigenetic Alterations | ||
| Global and locus-specific DNA methylation | High-performance liquid chromatography and ELISA-based methods (124) Enzyme activity assays for “writers, erasers, editors and readers” (125) Bisulfite sequencing (BS-seq) (126) |
|
| Histone modifications | ChIP-Seq (61) Stable isotope labeling by amino acid in culture (SILAC) (127) High throughput histone mapping (HiHiMap) (128) |
|
| Chromatin remodeling | Transposase-accessible chromatin using sequencing (ATAC-seq) (129) | |
| Changes in non-coding RNAs | RNA-Seq (130) in situ / FISH detection of small RNAs (131) and long non-coding RNAS (132,133) |
|
| KC 5: Induces Oxidative Stress | ||
| Oxidative DNA damage | 8-OHdG adducts via HPLC-electrochemical detection (134,135) Comet assay modified with lesion-specific repair endonucleases (e.g., 8-oxoguanine DNA glycosylase [OGG1], formamidopyrimidine (fapy)-DNA glycosylase [FPG], Endonuclease III [Nth]) (136) |
|
| Reactive oxygen species (ROS) formation | Electron paramagnetic resonance (137) | |
| Glutathione oxidation | Measurement of GSH/GSSG ratio (138) | |
| Nrf2-ARE-dependent gene expression response | Antioxidant enzyme activity (superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase) (139)) Nrf2/ARE-dependent gene expression (140) |
|
| Lipid peroxidation | Thiobarbituric acid reactive substances assay for detection of malondialdehyde, 4-Hydroxynonenal hydroperoxides and isoprostanes (141) | |
| KC 6: Induces Chronic Inflammation | ||
| Inflammatory signaling | *Colonic organoid-based cell transformation assay (142) | |
| KC 7: Is Immunosuppressive | ||
| T cell activation and proliferation | Mitogen and/or antibody mediated proliferation (143) | |
| Cytotoxic T-lymphocyte (CTL) activity | *BiTE®-mediated CTL assay (144) | |
| Natural killer cell activity | Missing-self cytotoxicity assay (145) | |
| KC 8: Modulates Receptor-mediated Effects | ||
| Interacts with receptors | Androgen and estrogen receptor binding assay ((146), OECD TG 493) | |
| Receptor activation | Estrogen receptor Transactivation (147) Androgen receptor Transactivation (148) Aryl hydrocarbon receptor transactivation (149) |
|
| Alters ligand synthesis | Aromatase enzyme activity (US EPA 890.1200) H295R steroidogenesis assay (OECD TG 456) |
|
| KC 9: Causes Immortalization | ||
| Alters in vitro transformation activity | Cell transformation assays (150) | |
| Alters cellular senescence markers | Changes in B-galactosidase, p16, p21, and p53 protein levels (151) | |
| Telomere length and telomerase activity | Telomerase activity assay (152) | |
| Alterations in stem cell genes | Expression of MYC, Oct3/4, Klf4, Sox-2 (153,154) | |
| KC 10: Alters Cell Proliferation, Cell Death or Nutrient Supply | ||
| Cell proliferation | DNA labeling (e.g., EdU, 3H-thymidine) (155) Cell cycle markers (e.g., Ki-67, propidium iodide) (155) Metabolic activity (e.g., MTT) (155) Cell number/microscopy (e.g., Hemocytometer) (155) Colony formation (156) |
|
| Evasion or reduction of apoptosis | Evasion of apoptosis (157) by TUNEL, Annexin-V, PARP cleavage or others *Changes in expression of pro and anti-apoptotic factors (158) |
|
| Angiogenesis | Endothelial cell proliferation, migration and differentiation (159) Transwell cell invasion (Boyden) assay (160) Aortic ring assay (161) |
|
| Glycolytic (Warburg) shift | Cellular respiration and acidification (Seahorse) assay (162) | |
*
Emerging assay or biomarker. Abbreviations: 8-OHdG, 8-hydroxydeoxyguanosine; BITE, Bispecific T-cell Engager; ChIP-Seq, chromatin immunoprecipitation-sequencing; ELISA, enzyme-linked immunosorbent assay; GSH, glutathione; GSSG, glutathione-S-S-glutathione; HPLC, high-performance liquid-chromatography; Klf4, Krüppel-like factor 4; LC, liquid chromatography; MSTI, (E)-2-(4-mercaptostyryl)-1,3,3-trimethyl-3H-indol1-ium; nrf2/ARE, nuclear erythroid 2-related factor 2/antioxidant response element; Oct3/4, octamer-binding transcription factor 3 or 4; PARP, poly (ADP-ribose) polymerase; Sox-2, SRY-Box 2; TGx-DDI, toxicogenomics-DNA damage-inducing; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling.