Table 7.
In vitro studies on OTA genotoxicity
| Test system | Cells | Concentration/Treatment time | Results | Comments | Reference |
|---|---|---|---|---|---|
| SSBs by comet assays | NIH/3T3 cells expressing human CYP450s, 1A1, 1A2, 2C9, 3A4 and human oxidoreductase (hOR) | 10, 25, 50, 100, 150, 200 μM (8 h) | Positive: Only in CYP2C9‐hOR. No toxicity (MTT and neutral red, no data presented) | Increased ROS. Poor presentation of the results | SimarroDoorten et al. (2006) |
| SSBs by comet assays +/− Fpg/EndoIII | Human renal proximal tubular epithelial cells (HK‐2) | 50, 100, 200, 400, 600 μM +/− rat liver S9 (3 and 6 h) | Positive: Slight increase at 6 h (400 and 600 μM, 50–60% survival); Survival and SSB unaffected by S9; SSB increased by Fpg and EndoIII (unconvincing data) | Increased ROS levels; slight protection by NAC | Arbillaga et al. (2007) |
| SSBs by comet assays, polyploidy by flow cytometry | CHO cells | 0.2, 0.8, 1 mM (3 h) |
Positive: Comet assay. Extremely toxic doses (cytotoxic at > 10 μM) Positive: polyploidy |
Dose‐dependent inhibition of TopoII (in vitro assay) | Cosimi et al. (2009) |
|
SSBs in comet assays; DSBs in γH2AX assay 8‐OHdG with HPLC‐ECD |
Human peripheral blood mononuclear cells | 5, 10, 20 μM (24 h) | Positive: SSB, DSBs, 8‐OHdG (dose‐dependent increases). No information on the toxicity of the treatment | Increased ROS at all doses. SSB partially inhibited by NAC‐pre‐treatment. Accumulation in G1 phase | Liu et al. (2012) |
| SSB by comet assays, DSBs with γH2AX (IF detection and western blotting), 8‐OHdG detection with HPLC‐ECD | Human gastric cell line (GES‐1) | 5, 10, 20 μM (24 h) | Positive: SSB, DSBs, 8‐OHdG (dose‐dependent increases). No information on the toxicity of the treatment | Increased ROS; G2 phase arrest; Phosphorylation of ATM, CHK2, p53 | Cui et al. (2013) |
| SSBs in comet assays +/− pg; MN | CHO and human TK6 cells | MN: 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25 μM (24‐27 h). Comet assays: 5, 10, 20, 30, 40, 50 μM (4 h) |
Positive: SSB +/− Fpg in TK6 Positive: SSB only + Fpg in CHO cells Positive: MN in TK6 and possibly in CHO cells (borderline levels of toxicity) |
Hypodiploid cells in both cell lines (scored in the MN, assays). Evidence of both direct and indirect DNA damage | Ali et al. (2011) |
| SSBs by comet assays; MN | Green monkey kidney Vero cell line | 10 μM (24 h) |
Positive: SSB Positive: MN. Experiments at 80% survival |
Limitation: Single dose observations | Ramyaa and Padma (2013) |
| SSBs in comet assays; MN | Human lymphocytes | Comet assays: 0.075, 0.15, 1.5, 5, 15 μM; MN: 0.075, 0.15, 1.5, 5 μM (3 h) |
Positive: Small increases in SSB at single points, no dose response Positive: Small increases in micronuclei (1.5 and 5 μM). Experiments at > 80% survival |
González‐Arias et al. (2014) | |
| SSBs in comet assays; DSBs by γH2AX | Human embryonic kidney cells (HEK 293) | 12.5, 25, 50 μM (24 h) | Positive: Dose‐dependent increase in SSB and DSBs. Limited toxicity (MTT assay) | Increased ROS; cell cycle arrest at S phase; downregulation of S‐phase proteins (Cyclin A2 and E1 and CDK2); decreased mitochondrial membrane potential | Yang et al. (2014) |
| SSBs in comet assays; CA | Human oesophageal epithelium immortalised cells (Het‐1A) | 2.5, 5, 10, 20 μM (24 h) |
Positive: dose response increase in SSB (IC50: 65.9 μM in MTT assay) Positive: dose response increase in CA |
G2 phase arrest (10–20 μM). Decreased expression of Cyclin B1, Cdc2 and Phospho‐Cdc2 | Liu et al. (2015) |
| SSBs in comet assays | Porcine epithelial kidney PK15 cells; human leucocytes | 1 and 5 μM (1 and 24 h) | Positive: dose‐ and time‐dependent increases (both cell lines). Non‐toxic doses by MTT assay | Combined effects with BEA | Klarić et al. (2010) |
| SSBs in comet assays | Primary rat hepatocytes | 2, 4, 6 nM (12 h) | Positive: at 4 and 6 nM (viability with MTT: 78% and 62%, respectively) | Limited study | Goyary et al. (2014) |
| SSBs in comet assays | Human peripheral blood lymphocytes | 1, 2, 4 μM (1 h) | Positive: SSB only at 4 μm/L (no toxicity with ethidium bromide/acridine orange) | Prevention by sodium copper chlorophyllin. Limited study | Domijan et al. (2015) |
|
SSBs in comet assays Plasmid DNA damage assay |
Mouse neuroblastoma Neuro‐2a cell line | 100, 250, 500 nM (24 h) | Positive: All doses (toxicity > 50%). Plasmid DNA damage assay (non‐informative) | Increased ROS; Small protection by NAC | Bhat et al. (2016) |
| SSBs by comet assays +/− Fpg; MN | Green monkey kidney Vero cell line | 7.5, 15, 25, 50 μM (24 h) |
Positive: SSB (only at 15μM in the presence of Fpg) Positive: MN (only at 25 μM) |
No dose response in MN and comet assays. Limited information | Costa et al. (2016) |
| DSBs by γH2AX (western blotting) | Human gastric cells (GES‐1) | 5, 10, 20 μM (24 h) | Positive: DSBs (small increase at 20 μM). No information on toxicity | Decreased Rad51 levels. Overexpression of Rad51 decreases OTA‐induced DSBs. Limited study | Lian et al. (2014) |
| MN | V79 cells and primary porcine urothelial bladder epithelial cells (PUBEC) | 0.01, 0.03, 0.1, 0.3, 1, 3, 10 μM (18 and 24 h for V79, 24 h for PUBEC) | Positive: V79 (0.3–10 μM but no dose response); PUBEC (3–10 μM) | Unclear levels of toxicity | Föllmann et al. (2007) |
| MN, SCE, CA endoreduplication, polyploidy | Human lymphocytes; V79 cells | 23.6‐1149 μM (+/− rat kidney and liver S9) |
Negative: SCEs, MN, CA Positive: Endoreduplications and polyploidy (V79 + kidney S9); (human lymphocytes +/− liver S9) |
Metaphases with abnormally separated chromatids | Mosesso et al. (2008) |
| Aberrant mitoses | Immortalised human kidney epithelial cells (IHKE) | 1, 10, 50 μM (12 and 24 h) | Positive: Dose‐response increase in giant cells with enlarged/multiple nuclei. Asymmetric/multipolar mitotic spindles | Inhibition of microtubule assembly in a cell‐free in vitro assay (200 & 400 μM) | Rached et al. (2006) |
| Chromosome condensation and loss of sister chromatid separation | Immortalised human kidney epithelial cells (IHKE) | 0, 1, 5, 10, 25, 50 μM (up to 15 h) | Positive: Chromosome over‐condensation and aberrant separation of sister chromatids | Decreased phosphorylation of histone H3Th3 and decreased acetylation of histones H3 and H4 | Czakai et al. (2011) |
| MN | Porcine epithelial kidney cells (PK15) | 0.05, 0.5, 5 μg/mL (corresponding to 0.12, 1.25, 12.5 μM (24 and 48 h) | Positive: All concentrations: No information on toxicity | Combined effects with BEA and FB1 | Klarić et al. (2008) |
| SCEs | Human hepatocellular carcinoma cells (Hep3B) | 10−12−2 × 10−4 M (72 h) | Positive: range 10−12–×10−6 M. No dose response; no information on toxicity (by MTT only at 24 and 48 h) | Combined effects with sterigmatocystin and citrinin | Anninou et al. (2014) |
| Mutations at Hprt and Tk genes | Chinese hamster V79 cells and mouse lymphoma cells (L5178Y) |
V79 (7, 11, 20, 35, 46, 108, 251 μM; 3h and 24 h); +/− rat kidney and liver S9 L5178Y (3, 81, 188, 438μM, 3 h) +/− rat kidney S9 |
Positive: Increased mutation frequency in both cell lines. No effects of S9. Mutational spectrum similar to the endogenous one (V79); point mutations and large deletions (L5178Y) | G2‐phase/M‐phase cell cycle arrest (V79 cells) | Palma et al. (2007) |
| Mutations in the SupF gene in plasmid Ps189 | Human embryonic kidney cells (Ad293) | 0.5, 1, 5 μM +/− rat liver microsomes or Fe(III)/Cu(II) metals | Positive: All doses, only + S9 or metal activation. (OTHQ: positive without metabolic activation) | No dose response. No proper controls for metal activation | Akman et al. (2012) |
| Mutations Tk+/− SSB by comet assays +/− Fpg | Mouse lymphoma cells (L5178Y) | 0, 5, 10, 25, 50, 100 μM +/− S9 rat liver (4 h) |
Positive: Dose‐dependent increase in mutations (from 25 μM) Positive: Dose‐dependent increase in SSB (increased by Fpg). No effect of S9 for both endpoints |
Large colonies (gene mutations). All assays at survival levels > 70% | Ali et al. (2014) |
| 8‐OHdG by oxyblot | Canine umbilical cord matrix‐mesenchymal stem cells (UCM‐MSC) | 0.025, 2.5 nM (4 and 7 days) | Positive: Similar increases in DNA 8‐OHdG levels at 0.025 and 2.5 nM. Less than 50% cell growth and cells showing fragmented chromatin | Rutigliano et al. (2015) | |
| 8‐OHdG by oxyblot | Bovine mammary epithelial (BME‐UV1) cells; Madin‐Darby canine kidney cells (MDCK) | 0.3, 0.6, 1.25 μg/mL (corresponding to 0.75, 1.5, 3.1 μM) |
Positive: BME‐UV1 (only 1.25 μg/mL, survival 40%) Negative: MDCK (range survival 100%–20%) |
No changes in the global content of 5‐meC | Giromini et al. (2016) |
| AP sites | Rat kidney cells (NRK) and rat hepatocytes | 3, 6 μM (24 h) | Positive: similar increases at 3 and 6 μM (unclear toxicity) | Non‐standard test | Cavin et al. (2007, 2009) |
| UDS | Swine lymphocytes, swine kidney cells (SK6) | 0.01–50 μM (72 h) |
Positive: swine lymphocytes (1 and 2 μM, cytotoxic concentration > 5 μM) ositive: SK6 (0.5–4 μM) |
Stec et al. (2006) |
8‐OHdG: 8‐hydroxydeoxyguanosine; AP: Apurinic/apyrimidinic; ATM; ataxia telangiectasia mutated gene; BEA: Beauvericin; CA: chromosome aberrations; cdc2; cell division control protein; CDK2: cyclin‐dependent kinase 2; CHK2; checkpoint kinase 2; CHO; Chinese hamster ovary; CTN: citrinin; CYP450: cytochrome P‐450); DSBs: Double strand breaks; EndoIII: endonuclease III; FB1: Fumonisin B1; fpg; formamido‐pyrimidine‐DNA glycosylase; γH2AX: Histone variant H2AX in which the Ser‐139 residue variant is phosphorylated (an early cellular response to induction of DSBs); G2: gap phase 2; HPLC‐ECD: High‐Performance Liquid Chromatography with Electrochemical Detection; IF: immunofluorescence; MN: micronuclei; MTT; 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide; NAC: N‐acetyl‐ L‐cysteine; OTHQ: ochratoxin hydroquinone metabolite; p53; tumour protein p53; ROS: reactive oxygen species; SSBs: single strand breaks; SCE: sister chromatid exchange; SupF: E. coli tyrosine amber suppressor tRNA gene; Tk: Thymidin kinase; TopoII; topoisomerase II; UDS: unscheduled DNA synthesis; V79 cells: Chinese hamster lung cells clone V79.