The
Ochratoxin A (OTA) is a mycotoxin produced by several food-borne species of
Human epidemiology has been inconclusive: a number of studies have suggested a correlation between exposure to OTA and Balkan Endemic Nephropathy (BEN) and mortality from urinary tract tumors [
In absence of adequate human data, risk assessments have relied on animal data. Kidney has been considered as the key target organ of OTA toxicity. In all animal species studied, OTA was found to produce renal toxicity, while in rodents renal carcinogenicity was clearly established. Recently, OTA renal and hepatic carcinogenicity was also observed in chicks [
In 2008, JECFA applied a benchmark dose (BMD) modeling approach using carcinogenicity data [
A recent health risk assessment, performed by Health Canada [
In the risk assessment of carcinogenic substances, consideration of the mode of action (MOA) is essential, determining the method to be applied in order to define levels of exposure below which a low safety concern is expected. The key events analysis framework of the MOA has not yet been formally applied to OTA. However, the approach used by most expert groups (EFSA, JECFA, ILSI) to establish the safe level of exposure of OTA (based on uncertainty factors) implies the consideration of key events compatible with a threshold effect. For these groups, amongst the mechanisms of action highlighted as possible, oxidative stress has been presented as one of the most probable [
Over the last decades, studies aimed at elucidating the modes of action implicated in OTA toxicity and carcinogenicity have been published [
Although genotoxicity is likely to play a role in OTA carcinogenicity [
The present paper is not intended to provide a thorough review of the complex and controversial scientific literature on DNA-adduct formation. However, it is important to keep in mind that DNA adducts are increasingly considered as markers of exposures and not only of effects [
The focus of the present short paper was to collect and highlight the evidence associated with a role for oxidative stress as a plausible mechanism to consider for OTA. A list of the studies used to illustrate the main messages of the present paper is provided in Table
OTA oxidative stress-related studies.
Model | Gender | Via | Time treatment | Dose | Aim | Results/Conclusion | Ref. |
---|---|---|---|---|---|---|---|
BALB/c macrophage J774a cell | 24, 48, 72 h | 30 nM–100 | OTA immunotoxicity and modulation inflammatory process | Induction of iNOs, COX-2 and NF- | [ | ||
Porcine kidney tubuli cells LL-PK1 | 6–24 h | 1–100 | Characterization effect of OTA on Nrf2 response | Nrf2 potential signal transduction pathway by which OTA impairs its own detoxification | [ | ||
Porcine kidney tubuli cells LL-PK1 | 24 h | 1–100 | Impact of OTA on Nrf2, AP-1 activity, antioxidant enzymes and GST | Enhanced production of ROS, GST impairment. Nrf2 and AP-1 disruption by OTA. Impairment of the detoxification machinery | [ | ||
Rat Sprague-Dawley | male | diet | 15 days | 3.0 mg/kg bw | Oxidative stress protection study | OTA-induced oxidative stress chemoprotection by | [ |
Rat F344 | male | gavage | 7 and 21 days | 0.5 mg/kg bw | Mechanism of action study-microarrays | Oxidative stress, calcium homeostasis, cytoskeleton structure | [ |
Human hepatocytes HepG2; Monkey kidney Vero cells | 0–100 | Decrease GSH | No induction of heat shock protein (HSP) | [ | |||
Rat Wistar | male | diet | 15 days | 5 ng/g bw 50 ng/kg bw | Oxidative damage study (proteins and lipids) | Malondialdehyde (MDA) and protein carbonylation (PC) increase in kidney > liver | [ |
Chinese Hamster lung V79 cells; Lymphoma mouse LY5178 cells | 0–438 | OTA mutagenicity | OTA is mutagenic at cytotoxic doses in mammalian cells via oxidative DNA damage induction. | [ | |||
Rats Sprague-Dawley | male | diet | 4 weeks | 200 ppb | Oxidative stress protection study | OTA-induced oxidative stress and DNA damage chemoprotection by | [ |
Pig kidney cell line LLC-PK1 | 24 h | 0, 10, 15, 20 | Oxidative stress protection study | OTA-induced ROS. Scavenging by cathechins (epigallocathechin gallate (EGCG) and epicatechin gallate (ECG)) | [ | ||
Human epithelial colorectal adenocarcinoma Caco-2 cells | 100 | Effect of OTA on barrier function impairment | Loss of microdomains associated with tight junctions maybe due to oxidative events | [ | |||
Neural stem/progenitor cells (NSCs) | 0.01–100 | Vulnerability of brains mouse cells to OTA | Robust increased in total and mitochondrial SOD activity. OTA impaired hippocampus neurogenesis | [ | |||
Rats | Male/liver and kidney | Diet (drinking water) | 4 weeks | 289 | Oxidative stress protection study | Melatonin protection against OTA-induced oxidative damage in liver and kidney. CoQ protective in liver. | [ |
Human renal cell line HK-2 | 6 and 24 h | 50 | Mechanism of action study-microarrays | Significant increase in ROS level and oxidative DNA damage. | [ | ||
Human renal proximal tubular epithelial cell line HK-2 | 50–800 | Evaluate single-strand DNA breaks and oxidative damage induction by OTA | Oxidative stress precedes cytotoxicity and genotoxicity | [ | |||
Male Fischer 344; Primary hepatocytes; adherent proximal tubules epithelial NRK cells; rat liver RL-34 | Rats 2 years; | 300 | Demonstration of cellular defense reduction by OTA | OTA induces depletion of antioxidant defense by inhibition of Nrf2 responsible of oxidative stress response | [ | ||
Eker and wild type rats | male | gavage | 1–14 days | 210 | Early carcinogen-specific gene expression study | Oxidative DNA damage response genes, general stress response, and cell proliferation | [ |
Wistar rats | gavage | 90 days | 289 | Early effects of chronic OTA administration in liver | Reduction in the ability to counteract oxidative stress in liver | [ | |
Swiss ICR | male | i. p | 6, 24, 72 hours | 0–6 mg/kg bw | Oxidative stress and OTA neurotoxicity | Acute depletion of striate DA on a background of globally increased oxidative stress and transient inhibition of oxidative DNA repair | [ |
Swiss mice | male | I.p; infusion | 2 weeks | Acute 3.5 mg/kg; chronic 4, 8, 16 mg/kg | Effect of chronic low dose OTA exposure on regional brain oxidative stress and stratial DA metabolism | Low doses exposure caused global oxidative stress | [ |
F344 rats | male | diet | 7 and 21 d; 4, 7 and 12 months | 300 mg/kg bw | OTA mechanism of action-microarrays study in liver and kidney | Oxidative stress and metabolic response modulated involving mainly Nrf2 and HNF4 | [ |
Swiss mice | male | oral | 24 hours | 10 mg/kg | Immune cells response after acute OTA exposure | OTA-induced oxidative stress response responsible of its own toxicity. | [ |
Wistar rats | female | Intraperitoneally | 7, 14 and 21 days | 0.5 mg/kg bw/day | Genotoxic potential of OTA measuring DNA strand breaks (comet assay) in the kidney | OTA-induced DNA strand breaks were detected, OTA concentration in the kidney and duration of the treatment correlated with severity of the DNA damage | [ |
Wistar rats | male | Oral | 15 days | 5 ng; 0.05 mg; 0.5 mg/bw | Effect of OTA on DNA damage | Oxidative stress responsible for OTA-DNA damage as shown by Fpg-modified comet assay | [ |
Pig kidney microsomes, human bronchial epithelial cells, human kidney cells | Cells: 2, 7, 24 hours | 0.5, 1.0, 2.5 | Genotoxicity of the hydroquinone (OTHQ) metabolite of OTA | OTQ-mediated adduct spots form in a dose-and-time-dependent manner | [ | ||
Wistar rats | female | oral | 7, 14 and 21 days | 0.5 mg/kg bw | Effect of OTA on protein oxidation in kidney and liver | Increased protein carbonyls in the kidney and liver | [ |
F344 rats | male | gavage | 0.03, 0.10, 0.30 mg/kg bw | Evaluate relevance of OTA-induced oxidative damage on nephrotoxicity and carcinogenicity | Tumours in rat kidney may be attributable to oxidative DNA damage in combination with cell-specific cytotoxic and proliferation-stimulating effects as cell-signaling response | [ | |
V79 (Chinese hamster lung fibroblasts) cells, CV-1 (African green monkey, kidney) cells, primary rat kidney cells | 1–24 hours | 2.5, 100 | Relevance of OTA-induced oxidative damage in nephrotoxicity and carcinogenicity | Cytotoxicity and oxidative DNA damage already at low doses could be a relevant factor for the nephrocarcinogenicity | [ | ||
Rat lymphoid cells | 1 hour | 0.5, 2, 20 | OTA immune function modification | Protein synthesis inhibition, oxidative metabolism of OTA, prostaglandin synthesis implicated in NK cells toxicity | [ | ||
Human hepatoma—derived cell lines (HepG2), human colonic adenocarcinoma cell line (Caco-2) | 24, 48, 72 hours | 0–100 | Oxidative stress protection study | OTA-induced oxidative stress damage. Protective effect by Cyanidin-3- | [ | ||
F344 Fischer rats | male | gavage | 2 weeks | 0–2000 | Genotoxicity of OTA | DNA strand breaks in target and nontarget tissues probably involving oxidative stress mechanism | [ |
Human hepatoma—derived cell line (HepG2) | 48–72 hours | 35–10 mM | Oxidative damage protection study | No cytotoxicity protection observed with Vitamine E, polyphenols | [ | ||
Sprague-Dawley | male | diet | 15 days | 3 mg/kg | Oxidative stress protection | Preventive effect against OTA-induced oxidative stress and lipid peroxidation by melatonin | [ |
Human fibroblast cells | 48–72 hours | 0–50 | Oxidative stress protection study | Reduction of free radical species production and DNA damage prevention by cyanin 3- | [ | ||
Fetal rat telencephalon aggregating cells | 24–48 hours, 9 days | 0–20 nM | Adverse effect of OTA in brain | Brain inflammatory response induction of HO-1, iNOs, PPAR | [ | ||
Human hepatoma-derived cell line (HepG2) | 24 hours | 0–40 | Genotoxicity of OTA | Dose-dependent induction of DNA single strand breaks (comet assay) and micronuclei (MN) | [ | ||
Primary proximal tubules renal (PT) cells, proximal tubular cell line (LLC-PK1) | 0–24 hours | 0–100 | OTA mediated oxidative stress response in proximal tubular cells, oxidative stress protection | Oxidative stress contributes to tubular toxicity. Antioxidants ( | [ | ||
Wistar rats | male | gavage | 10, 30, 60 days | 120 | Kidney low dose OTA response: sequence of events leading to cell death | Low dose induces oxidative stress, apoptosis in proximal, and distal tubule kidney cells | [ |
Human hepatoma—derived cell line (HepG2) | 1 hour, 24 hours | 0–50 | Genotoxicity of OTA | No inductions of mutations in the Ames assay, a dose-dependent induction of micronuclei in the MN assay, and DNA migration (comet assay) were detected | [ | ||
Proximal tubular cells (PTC), Wistar rats | male | gavage | 24–72 hours ( | 5.0 | [ | ||
Dark Agouti (DA), Lewis rats | male | Intragastric intubation | 0.4 mg/kg bw | Life-time | Life-time study to evaluate if MESNA leads to a more effective reduction of OTA-induced tumour development or urinary tract damage | Lack of effect of mesna on OTA-induced urinary tract damage or renal tumor development | [ |
Dark Agouti (DA), Lewis rats | male | Intragastric intubation | 0.4 mg/kg bw | 2 years | Life-time study to evaluate the potential protective effect of 2 mercaptoethane sulfonate (MESNA) and N-acetyl cysteine (NAC ) | MESNA decreased karyomegalies in kidney, but had no beneficial effect on renal tumour incidence | [ |
Fischer rats | male | gavage | 4, 8, 24, 48 hours | 0–2.0 mg/kg bw | Chemical and biological markers induced by OTA exposure associated with oxidative stress | Oxidative stress may contribute to mechanism of OTA renal toxicity and carcinogenicity in rats over long term exposure | [ |
Bronchial epithelial cells incubated with microsomes of seminal vesicles of pig | 4 hours | 10 | Roles of cyclooxygenase and lipoxygenase in ochratoxin A genotoxicity in human epithelial lung cells | OTA is biotransformed into genotoxic metabolite via a lipoxygenase, whereas prostaglandin—H-synthetase (PGHS) decreases OTA genotoxicity | [ | ||
Sprague-Dawley liver microsomes, liver mitochondria and hepatocytes cells | female | 2.5 mM | Free radical generation by OTA in hepatocytes, mitochondria, and microsomes using electron paramagnetic resonance (EPR) | Oxidative damage may be one of the manifestations of cellular damage in the toxicity of OTA | [ | ||
10 min | 1 mg/mL | Study free radical generation in bacteria as model system | OTA induces free radical production, enhancing permeability of the cellular membrane to Ca2+ | [ | |||
Swiss mice | Male | Gastric intubation | 48 hours | 2 mg/kg bw | Effects of vitamins on genotoxicity of OTA | Vitamins E, A, and C also reduced OTA-DNA adduct formation in mice kidney | [ |
Wistar rat | male | Gastric intubation | Every 48 hours/3 weeks | 289 | Protective effect of superoxide dismutase (SOD) and catalase | SOD + catalase prevents the nephrotoxicity induced by OTA in rats | [ |
Wistar rat liver microsomes, kidney microsomes | male | 6 mg/kg bw | Lipid peroxidation induction by OTA | lipid peroxidation may play a role in the observed toxicity of ochratoxin A | [ |
Production of reactive oxygen species (ROS) leading to oxidative stress and macromolecular damage is known to contribute to the pathogenesis of age-related as well as chronic diseases including cancer [
Several oxido-reduction mechanisms elicited by OTA have been proposed. In a reconstituted system consisting of phospholipid vesicles, the flavoprotein NADPH-cytochrome P450 reductase and Fe3+, OTA was found to chelate ferric ions (Fe3+), facilitating their reduction to ferrous ions (Fe2+), which in the presence of oxygen, provided the active species initiating lipid peroxidation [
The generation of an OTA hydroquinone/quinone couple from the oxidation of OTA (phenol oxidation) by electrochemical, photochemical, and chemical processes was reported [
OTA was found to reduce the expression of several genes regulated by nuclear factor-erythroid 2 p45-relatetd factor (Nrf2) [
ROS, such as hydroxyl radicals and nitric oxide, are capable of forming oxidized DNA bases that directly produce diverse types of DNA damage [
Carbonylation of proteins occurs through a variety of oxidative pathways [
Lipid peroxidation is among the most extensively investigated processes induced by free radicals. Of these, the by-products, 4-hydroxy-2-nonenal (HNE), the tautomer malondialdehyde (MDA), acrolein and crotonaldehyde have been widely studied. The ability of these reactive electrophiles to modify DNA bases, yielding promutagenic lesions, is considered to contribute to the mutagenic and carcinogenic effects associated with oxidative stress-induced lipid peroxidation. HNE and MDA have increasingly been implicated in carcinogenesis [
It is widely acknowledged that reactive oxygen and nitrosative species can trigger biological responses such as stimulation or inhibition of signal transduction and gene expression. Such biological responses are considered to contribute to the expression of the carcinogenic potential of the reactive chemicals. A number of
ROS induces release of calcium from intracellular stores, resulting in the activation of kinases, such as protein kinase C (PKC). ROS species play also a critical role in the selective mobilization of other cell signaling responses. Cell signaling phosphoproteins of mitogen-activated protein (MAP) kinases including ERK, c-Jun N-terminal kinases (JNK), and p38 kinases are involved in proliferation, differentiation, and apoptosis. Activation of these molecules has been observed in response to changes in the cellular redox balance and are considered as vectors of ROS biological effects [
Numerous reports have characterized interactions of ROS and RNS with activity of transcription factors [
A strong correlation between the ability of a compound to block cell-to-cell communication in cultured cells and its ability to induce rodent tumors through nongenotoxic mechanisms has been documented [
As mentioned earlier, OTA was found to reduce the expression of antioxidant enzymes through inhibition of Nrf2 activity [
Several studies have been performed to try to counteract the adverse effects of oxygen radicals generated under OTA-treatment. A number of molecules with various antioxidant properties were tested, including
Indomethacin and aspirin were found to prevent OTA genotoxicity in the urinary bladder and kidney of mice [
The carcinogenic mycotoxin OTA has been reviewed by a number of expert groups [
It has been clearly shown that OTA generates oxidative stress predominantly in kidney through potentially direct (redox cycling) and indirect (reduction of cellular antioxidant defenses) mechanisms. Interestingly, these two mechanisms may interact with each other. The reduction of defense may amplify the impact of the direct production of radicals. Consequences resulting from the production of oxidative stress were observed at different levels. High kidney susceptibility to oxidative stress conditions may explain the target-specific toxicity of OTA. Oxidative stress has been incriminated in a number of kidney pathological pathways [
As depicted in Figure
Scheme to illustrate the oxidative stress-mediated mode of action proposed for OTA. Increased production of ROS, RNS, and RCS is likely to originate either from direct redox reactions involving OTA or through the inhibition of cellular defenses such as through the inhibition of transcription factors as Nrf2 which regulates enzymes with antioxidant properties. The generation of radicals will induce macromolecular oxidative damage such as oxidized DNA bases which may be converted into mutation resulting into generation of transformed cells. In addition, radicals will trigger biological responses which may impair intercellular communication and induce cell proliferation as well as reduction in cellular defense in oxidative stress. This last effect is likely to amplify the oxidative stress-mediated effects of OTA. Altogether, these molecular mechanisms will result in cancer development.
In conclusion, the evidence for the induction of an oxidative stress response resulting from OTA exposure can be considered strong. Because the contribution of the oxidative stress response in the development of cancers is well established, a role in OTA carcinogenicity is plausible. Altogether, the data reviewed above support the application of a threshold-based approach to establish safe level of dietary human exposure to OTA.