ToxSci Advance Access originally published online on November 16, 2006
Toxicological Sciences 2007 96(1):30-39; doi:10.1093/toxsci/kfl169
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Reduction in Antioxidant Defenses may Contribute to Ochratoxin A Toxicity and Carcinogenicity
* Quality and Safety Department, Nestlé Research Center, CH-1000 Lausanne 26, Switzerland
Biomedical Research Centre, University of Dundee, Dundee DD1 9SY, United Kingdom
Nephro-Urology Unit, Institute of Child Health, University College London, London WC1E 1EH, United Kingdom
Department of Environmental Science and Technology, Imperial College London, London SW7 2AZ, United Kingdom
1 To whom correspondence should be addressed at Quality and Safety Department, Nestle Research Center, P.O. Box 44, Vers-chez-les Blanc, CH-1000 Lausanne 26, Switzerland. Fax: +41 21-785-85-53. E-mail: christophe.cavin{at}rdls.nestle.com.
Received July 26, 2006; accepted November 14, 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Ochratoxin A (OTA) is a renal carcinogen in rodents. Its human health significance is unclear. It likely depends upon the mechanism of carcinogenesis. In a previous microarray study a reduction in nuclear factor-erythroid 2 p45-related factor 2 (Nrf2)–dependent gene expression was observed in the kidney but not in the liver of rats fed OTA up to 12 months. Nrf2 regulates detoxification and antioxidant gene expression. The present report shows that OTA decreased the protein expression of several markers of the Nrf2-regulated gene battery in kidney in vivo indicating that the effects observed at mRNA level may be of biological significance. The OTA-mediated Nrf2 response could be reproduced in an NRK renal cell line and in primary hepatocyte cultures. In in vitro systems, an OTA-mediated inhibition of Nrf2 activity was demonstrated by electrophoretic mobility shift and Antioxidant Regulatory Element–driven luciferase reporter assays. The reduction of Nrf2-regulated gene expression resulted in oxidative DNA damage as evidenced by formation of abasic sites in vitro and confirmed in kidney in vivo. All OTA-mediated effects observed were prevented by pretreatment of cell cultures with inducers of Nrf2 activity. Our data suggest that reduction of cellular defense against oxidative stress by Nrf2 inhibition may be a plausible mechanism of OTA nephrotoxicity and carcinogenicity.
Key Words: mycotoxin; ochratoxin A; nuclear factor-erythroid 2 p45-related factor 2 (Nrf2); oxidative stress; nephrotoxicity; carcinogenicity.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Ochratoxin A (OTA) is a naturally occurring mycotoxin produced by several species of Aspergillus and Penicillium fungi. Its widespread occurrence in a variety of food commodities such as cereals, green coffee, cocoa, dried fruits, and meat products results in a continuous exposure of the human population (Benford et al., 2001
; EFSA [European Food Safety Authority], 2006; Fink-Gremmels, 2005). OTA causes various toxic effects, the most relevant being nephrotoxicity and nephrocarcinogenicity in rodents (Benford et al., 2001; see EFSA, 2006; O'Brien and Dietrich, 2005). Chronic studies have shown a clear causal relationship between OTA administration and the incidence of renal tumors in rats (O'Brien and Dietrich, 2005). Similar effects were observed in mice, although at much higher doses (Bendele et al., 1985; Grosse et al., 1997). It has been speculated that OTA may be associated with the human disease Balkan endemic nephropathy (BEN) and the onset of urinary tract tumors (Castegnaro et al., 2006; Petkova-Bocharova et al., 1988; Radic et al., 1997). However, in a recent workshop organized by the European branch of the International Life Sciences Institutes, it was concluded that there is no convincing evidence from human epidemiology to confirm the association between OTA exposure and the prevalence of BEN or Urinary Tract Tumors (Fink-Gremmels, 2005). The Scientific Panel on Contaminants in the Food Chain of the European Food Safety Authority considered that epidemiological data are incomplete and do not justify the classification of OTA as a human renal carcinogen (EFSA, 2006). Therefore, the actual significance of OTA exposure in human health remains unclear.
The mechanism of OTA carcinogenicity in animal models has not been fully characterized. Its elucidation would allow application of the most appropriate risk evaluation procedure to address the health significance of OTA contamination of foods. Several authors and expert groups have concluded that OTA is unlikely to act through a direct genotoxic mechanism (Benford et al., 2001; EFSA, 2006; O'Brien and Dietrich, 2005) since various laboratories have failed to detect any OTA-DNA adducts in treated animals, either through radiolabeled OTA-DNA binding or analytical chemistry using mass spectrometry (Mally et al., 2004; Turesky, 2005). These results were supported by other data showing that OTA is poorly metabolized and is unlikely to generate electrophilic intermediates (Gross-Steinmeyer et al., 2002; Turesky, 2005). However, there is no scientific consensus on OTA metabolism and DNA binding. Using a 32P-postlabeling method, some authors have found low levels of DNA lesions in various organs of OTA-treated animals (Castegnaro et al., 1998; Pfohl-Leskowicz et al., 1993) suggesting that OTA may be activated into reactive metabolites which may then react with DNA to form adducts (Faucet et al., 2004; Faucet-Marquis et al., 2006; Pfohl-Leskowicz and Castegnaro, 2005). EFSA (2006) did not consider this evidence as sufficient to demonstrate the actual formation of OTA-DNA adducts in rats in vivo. Interestingly, the observed OTA-mediated DNA damage (e.g., 32P-postlabeling lesions, clastogenicity) may not necessarily require direct OTA-DNA binding, but they may be related to other, indirect mechanisms, such as oxidative stress and cytotoxicity (Gautier et al., 2001; Petrik et al., 2003; Schaaf et al., 2002).
Gene expression profiles were studied in kidney and liver of male rats fed OTA for 21 days and 12 months as part of a 2-year carcinogenicity study with a final renal tumor incidence of 25% (Mantle et al., 2005). Surprisingly, in kidney samples, many genes expected to be induced by oxidative stress were significantly downregulated by OTA, whereas no effect was observed in liver samples (Marin-Kuan et al., 2006). Many of these genes were found to contain an Antioxidant Regulatory Element (ARE) in their promoter region. The ARE-motif is recognized by the nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), a member of the "Cap-n-Collar" family of basic-region leucine zipper transcription factors. Nrf2 is involved in both the basal expression and induction of many genes, including genes encoding for detoxification, cytoprotective, and antioxidant enzymes (Kensler et al., 2007; Lee and Johnson, 2004). These findings led to the hypothesis that OTA might inhibit the Nrf2-mediated gene expression pathway. Considerable biological consequence would be anticipated from such an effect. Inhibition of Nrf2 might be expected to impair the antioxidant defense of the cells making them more susceptible to oxidative damage. Low concentration of oxygen species has been demonstrated to play an important role in cell proliferation and carcinogenesis (Klaunig and Kamendulis, 2004). Therefore, inhibition of defense is a highly plausible new mechanism, which could contribute to OTA carcinogenicity.
In the present study, OTA-mediated inhibition of cellular defense mechanisms in rat has been further characterized. Cell culture systems were applied to define the role of Nrf2 in this effect. Moreover, biological significance of a reduction in cellular defense on DNA damage has been studied.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Chemicals
Cafestol and Kahweol (C + K) were prepared from coffee oil according to the procedure of Bertholet (1987)
. The mixture contained C + K in the proportion of 52.5:47.5 and its purity was greater than 98%. Other chemicals were of the highest grade and readily available commercially. OTA and coumarin (Cou) were purchased from Sigma-Aldrich and Co., St-Louis, Atlanta, L-4,5-3H Leucine (specific activity: 6 TBq/mmol) was from Amersham Bioscience Ltd (Buckinghamshire, UK) Standardized OTA production was performed by growing Aspergillus ochraceus (isolate D2306) in shaken solid substrate fermentations at 28°C for 2 weeks to yield a product containing 5-6 mg OTA/g as described in detail previously (Mantle et al., 2005). Fermentation product was homogenized into powdered standard commercial rat feed (Special Diets Services, UK) to a final concentration appropriate for the required OTA-intake per animal. Each animal was given 20 g of contaminated feed daily, which was always fully consumed.
OTA Rat Study and Cell Culture
Rat Study
Male Fischer 344 rats in groups of five were administered OTA in diet given daily over 2 years. The initial daily dietary intake was 300 µg OTA/kg body weight (bw) but was held at 100 µg/rat after animals reached 333 g (Mantle et al., 2005). Over the period of the study, animals were housed in cages on absorbent paper under tightly controlled conditions (21 ± 1°C, 55 ± 10% relative humidity, air-exchange, 12-h light-dark cycle). Animal growth and welfare were monitored by regular weighing and daily surveillance. For the present study, two time points were selected for analyzing early (21 days) and late responses (12 months). At both time points, control and treated animals were randomly chosen for tissue harvest. Kidneys and liver from these rats were immediately snap frozen in liquid nitrogen. All handling and procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986.
Cell Culture
Primary rat hepatocytes.
Primary isolated hepatocytes were obtained by perfusion of the liver in adult male Sprague-Dawley (230 ± 20 g, fed ad libitum with Nafag diet) with a collagenase solution as described previously (Sidhu et al., 1993). Cell viability, estimated by Trypan blue exclusion test, was found to range between 90 and 95%. Cells were seeded at a density of 106 cells/ml on 60-mm plastic tissue culture dishes in 3 ml of William's E medium supplemented with 2mM L-glutamine, 10mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), pH 7.4, 1% ITS+ (Insulin, Transferring, Selenious acid [ITS+], bovine serum albumin [BSA], linoleic acid) (BD Biosciences, Bedford), 100 U/ml penicillin/streptomycin, 100nM dexamethasone, and fetal bovine serum (FBS) 5% (Hyclone, Logan, Utah). Hepatocytes were allowed to attach for 2 h and then washed with Earle's balanced salt solution to remove debris and unattached cells. Fresh serum -free medium containing 250nM dexamethasone was added followed by application of an overlay of matrigel (233 µg/ml). Fresh matrigel was added to the cultures every other day following medium change. To study the effects of OTA on specific markers, the component was added to culture media containing ITS+ 0.2% 24 h after cells seeding over periods of 24 or 48 h.
NRK cells.
NRK cells were purchased from the ATCC's Cell Biology Collection (ATCC CRL-6509). NRK cells are adherent proximal tubule epithelial cells isolated from normal kidney of Osborne Mendel rats. NRK cells were cultivated in minimal essential medium (MEM) containing 2mM L-glutamine supplemented with MEM nonessential amino acids, 100 Units/ml penicillin/streptomycin, and 10% FBS for cell growth or 1% FBS for treatments with OTA.
RL-34 cells.
Rat liver RL-34 cells (Japanese Cancer Research Resources Bank) were grown in Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) heat inactivated FBS, 50 units/ml penicillin/streptomycin mix, and 2mM L-glutamine (Nioi et al., 2003).
Western Blot Analysis
Kidney tissues were homogenized and cell culture (NRK cells and hepatocytes) lysates were prepared by treating cells with lysis buffer (150mM NaCl, 10mM Tris-HCl, pH 8, 1% Tween-20, 1mM ethylenediaminetetraacetic acid [EDTA], pH 8, proteases inhibitors: 4,2-amino-ethyl-benzoisulfonyl-fluorid-hydrochloride (PEFA) [Merck, Darmstadt, Germany], N,N-diethyldithiocarbamic acid (DETC) [Aldrich, St Louis, Atlanta]). Lysates were sonicated for 5 s and centrifuged at 10,000 x g for 5 min. Protein concentrations were estimated using the Bradford assay, which was standardized using BSA (Bio Rad, Richmond, CA).
Protein extracts (10–25 µg) were boiled in Laemmli sample buffer and resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 10% polyacrylamide gels. Proteins were electroblotted onto nitrocellulose membranes. Nonspecific binding sites were blocked by incubating the membrane in 5% dried milk phosphate-buffered saline (PBS) solution containing 0.1% of Tween-20 overnight at 4°C. Blots were hybridized with antisera raised in rabbits against rat GSTM1, GSTP1 (Biotrin, Ireland), GSTA5, NQO1, and AKR7A1 and glutamate cysteine ligase (GCLC) (the anti-GCLC serum was a gift kindly supplied by Dr Lesley I. McLellan, University of Dundee, UK). Antisera were used at a dilution of between 1/1000 and 1/5000 in a solution of 5% (wt/vol) dried milk in PBS; reactions were allowed to proceed for 1 h. Subsequently, blots were washed before being probed with a secondary antibody conjugated to horseradish peroxidase. After washing, protein expression was detected using enhanced chemiluminescence (ECL) Western blotting reagents (Amersham, Buckinghamshire, UK).
Glutathione Assay
Total glutathione (GSH) content of NRK cell and hepatocyte cultures was measured with a kinetic assay which utilizes the continuous GSH reductase-catalyzed reduction of the sufhydryl reagent 5,5'-dithiobis-2 nitrobenzoic acid to the chromophoric product 2-nitro-5-thiobenzoic acid according to the method of Gallagher et al. (1994). Detection of the chromophore was monitored spectrophotometrically at 412 nm. Quantitation was achieved by comparison with a standard curve of known GSH concentration carried out in parallel.
Cytotoxicity Assay
Cytotoxicity of OTA in cell cultures (NRK cell line and primary hepatocytes) was determined by the amount of lactate dehydrogenase (LDH) leakage into the medium after 24 or 48 h of treatment. In protection experiments with the coffee diterpenes C + K, cells were initially treated with 2–4 µg/ml C + K for 24 h before OTA treatment (1.5–9µM) for 24 h. LDH activity was measured spectrophotometrically by analyzing the appearance of lactate at 340 nm using the Biomérieux kit which provides the substrate pyruvate and the cofactor nicotinamide adenine dinucleotide (reduced) (NADH). The procedure was automated using a Cobas Mira S plus.
ARE-Luciferase Assay
RL-34 cells were seeded into six-well plates at a density of 3 x 105 cells per well. After 24 h, when 70% confluency was achieved, cells were transfected using Lipofectamine 2000 (Invitrogen, Carlstadt, CA) with either empty pGL3-promoter (Promega, Madison, Wiscontin) luciferase reporter vector or –1016/nqo5'-luc (0.2 µg) or mut-1016/nqo5'-luc (0.2 µg), along with pcDNA3.1/V5mNrf2 (0.2 µg), and pRL-TK (Nioi et al., 2003). pRL-TK I reporter vector was used as a control for transfection efficiency. Cells were treated 24-h posttransfection with OTA (3 or 6µM) or vehicle (7.5% NaHCO3) (Invitrogen, Carlstadt, CA) for 24 h. Cells were lysed and Renilla and firefly luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega) and a Turner Designs TD-20/20 luminometer. Luciferase activities were normalized to Renilla internal control luminescence. In protection experiments, cells were pretreated with C + K, 15 µg/ml, for 1 h before 3µM OTA was added for 24 h.
Electrophoretic Mobility Shift Assay
Nuclear proteins were extracted from hepatocyte cultures by adding 300 µl of cold hypotonic buffer (10mM HEPES, pH8, 1.5mM MgCl2, 10mM KCl, 200mM Sucrose, 0.5% Nonidet P-40, 0.5mM phenylmethyl–sulfonyl fluoride [PMSF], 0.5mM dithiotreitol [DTT], 10 µg/ml E64, chymostatin, leupeptin, aprotinin, pepstatin). After incubation on ice for 30 min., cell pellets were centrifuged at 3000 x g for 30 min at 4°C. The supernatant formed the cytosolic fraction and the pellets were resuspended in 50 µl of cold hypertonic buffer (20mM HEPES pH 8, 1.5mM MgCl2, 420mM NaCl, 0.2mM EDTA, 20% glycerol, 0.5mM PMSF, 0.5mM DTT, 10 µg/ml E64, chymostatin, leupeptin, aprotinin, pepstatin). Cell debris were removed by centrifugation at 12,000 x g for 30 min at 4°C. The supernatant fraction containing the nuclear proteins was stored at – 80°C. Binding reactions were performed on ice for 40 min with 15 µg of nuclear protein in 20 µl of binding buffer (10mM Tris-HCl pH 7.5, 20mM NaCl, 1mM EDTA, 5% glycerol, 1mM DTT, 1 µg poly (dI-dC) (Roche Diagnostics GmbH, Germany), 30,000 cpm 32P-labeled oligonucleotides (Amersham Pharmacia Biotech) labeled with T4 polynucleotide kinase (Roche) and 
32P-adenosine triphosphate (5000 Ci/mmol). DNA-protein complexes were separated from the unbound DNA probe on a 5% polyacrylamide gel. The gels were vacuum dried and exposed to Konica film at – 80°C for 6–16 h. The ARE-like sequence in rat GSTP1, called GPEI 5'-AGT AGT CAG TCA CTA TGA TTC AGC AAC A-3') (Hayes et al., 2005) and the ARE in rat NQO-1 5'-AGC TCT AGA GTC ACA GTG ACT TGG CAA AA-3' sequence were synthesized by MWG-Biotech AG, Germany as double-stranded oligonucleotides.
Analysis of Abasic Sites by Aldehyde Reactive Probe-Slot Blot
Abasic sites in DNA were measured in cell cultures (NRK cells and hepatocytes) and in rat kidney. The analysis was adapted from Nakamura et al. (1998). Typically, DNA was extracted from rat hepatocytes or kidney cells with a kit by Dojindo Molecular Technologies, Kumamoto, Japan. DNA (15 µg) was incubated with the aldehyde reactive probe reagent (1mM, total volume 150 µl) at 37°C for 15 min in the TE buffer (10mM Tris-HCl, 1mM EDTA, pH 7.2). Then, DNA was precipitated with 45 µl of 1M sodium acetate and 1 ml of ethanol prior to centrifugation. DNA pellets were washed with 1.5 ml 70% ethanol, and dried in vacuo. Pellets were dissolved in 500 µl of TE buffer (10mM Tris, 1mM EDTA, pH 7.2), and DNA was quantified (for 1 ml, 1 AU at 260 nm corresponds to 50 µg DNA). Samples were diluted in TE buffer to a final concentration of 10 µg/ml, and incubated 10 min at 100°C. Samples were cooled on ice, before an equal volume of 2M ammonium acetate was added. The BAS-85 NC membrane was soaked in water (5 min) prior to further 5 min in 1M ammonium acetate. DNA (1 µg) was applied into slots which were subsequently run dry. Slots were washed with 250 µl of 1M ammonium acetate, and run dry for 10 min. The membrane was soaked with the saline sodium citrate (SSC) buffer (0.75M NaCl, 75mM trisodium citrate) at 37°C for 15 min prior to irradiation with the FLX-20C device (Vilber Lourmat, Marnes la Vallée, France) for 1 min at 0.364 J/cm2 maximum. The membrane was preincubated at room temperature for 1 h in 50 ml Tris-NaCl buffer (20mM Tris-HCl pH 7.5, 0.1M NaCl, 1mM EDTA, 0.1% Tween) containing 2% BSA. The membrane was washed with 30 ml of the Tris-NaCl buffer and incubated overnight in the Tris-NaCl buffer containing 0.6% BSA with streptavidin-conjugated horseradish peroxidase (dilution factor 1600). The membrane was soaked in wash buffer (20mM Tris-HCl pH 7.5, 0.26M NaCl, 1mM EDTA, 0.1% Tween) for 5 min before the enzymatic activity was visualized with the ECL reagents (Amersham).
Statistical Analyses
Statistical significance was determined by one-way ANOVA using the Newman-Keuls multiple comparison test with a 5% critical value.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
OTA Inhibition of Nrf2-Regulated Protein Expression
The effects of OTA on the expression of members of the ARE-gene battery were evaluated by Western blotting. In the kidney, OTA reduced the levels of all the proteins examined (Fig. 1), including GCLC, the rate-limiting protein in GSH synthesis and the GSH transferase subunits GSTP1, GSTA5, and GSTM1. A similar effect was found at both time points analyzed.
|
To further study the mechanism responsible for the OTA-mediated inhibition of Nrf2-regulated protein expression, two in vitro cell culture systems were applied, a rat proximal tubule epithelial cell line from normal kidney (NRK cells) and primary hepatocytes. In an initial step, the validity of the culture models for our purposes was confirmed by showing that effects of OTA observed in vivo could be reproduced in vitro. In both systems, OTA produced a dose-dependent cytotoxic response, as assayed by LDH release, and a significant inhibition of protein synthesis (data not shown). In addition, in both systems a decreased expression of various Nrf2-regulated protein markers NQO1, GSTP1, GSTM1 and GSTA5, GCLC and AKR7A1 was observed as a result of 48 h OTA treatment at noncytotoxic concentration (Fig. 2). Interestingly, the reduced expression in GCLC was associated with a significant decrease in the cellular content of reduced GSH after 48 h of OTA treatment (Fig. 3). The similar responses of the two in vitro models allowed application of both in further mechanistic investigations.
|
|
Inhibition of Nrf2 Activity by OTA and Effects on DNA Damage
The effect of OTA on the DNA binding activity of the transcription factor Nrf2 was investigated by electrophoretic mobility shift assay (EMSA) using a rat GSTP1 (GPEI) as a probe. Rat primary hepatocytes were treated with OTA for 6 and 48 h. A dose- and time-dependent inhibition of the Nrf2-DNA binding was found. Slight inhibition was observed at 6µM OTA after 6 h of treatment, whereas a clear dose-dependent inhibition was observed after 48 h of OTA treatment (Fig. 4A). Rat liver RL-34 cells transiently transfected with the –1016/nqo5'-luc reporter construct were employed to further determine whether Nrf2 activity might be reduced by OTA. In RL-34 cells transfected with the mouse NQO1 reporter, OTA was found to produce a dose-dependent repression of luciferase reporter activity after just 24 h treatment with the mycotoxin (Fig. 4B). As control experiment, no luciferase expression was found in both control and treated RL-34 cells transfected with a mutant NQO1 reporter.
|
Depletion of Nrf2-mediated gene expression is anticipated to reduce the cellular defense against oxidative stress resulting in increased oxidative damage. This hypothesis was tested by analyzing abasic sites as a marker of DNA damage. A dose-dependent increase in abasic site formation was observed in both models, as a result of OTA treatment for 48 h (Fig. 5A, B). The in vivo significance of this in vitro effect was then addressed through the analysis of abasic sites in rat kidney. As shown in Figure 6, an increase in abasic site formation was observed in kidney after solely 21 days. However, statistically significant increase was found after 12 months of OTA treatment.
|
|
Prevention of OTA Toxicity and DNA Damage by Nrf2 Activators
To confirm a direct link between the depletion of Nrf2-regulated gene expression and the increased formation of abasic sites, the effects of OTA were studied in presence of known activators of the Nrf2 pathway, such as the coffee diterpenes C + K and Cou. In EMSA using the ARE-like sequence in rat GSTP1, GPE1 probe, the OTA inhibition of Nrf2-binding activity was confirmed (Fig. 7A). In contrast, no inhibition but an increased Nrf2-binding activity was observed when cells were either cotreated with C + K (4 µg/ml) and OTA (3µM) for 24 h, or cotreated after an initial 24-h pretreatment with C + K (4 µg/ml). Similarly, in RL-34 cells stably transfected with the wild-type –1016/nqo5'-luc reporter construct and cotreated with OTA (3 and 6µM) and C + K (30 µg/ml) for 24 h, the luciferase expression was similar to or higher than in control cells (Fig. 7B), thus preventing the OTA-mediated repression of luciferase expression. The effect of a suppression of OTA-dependent inhibition of Nrf2 activity on the expression of Nrf2-regulated proteins was investigated. As shown in Figure 8, in cells pretreated with C + K prior to a cotreatment with OTA and C + K for 48 h, the expression of the Nrf2 markers (GSTA5, GSTP1, NQO1, GCLC, AKR7A1) was similar to or higher than in control cells, and at levels significantly higher than those observed in OTA-treated cells. Similar results were found with Cou (250µM) (data not shown).
|
|
The data described above indicate that the application of Nrf2 activators compensated OTA-mediated depletion of Nrf2-regulated gene expression. This may logically result in a restoration of cell defense capacity. This hypothesis was tested by measuring the effects of OTA on abasic site formation in presence of Nrf2 activators. The pretreatment of cell cultures with the Nrf2 activators C + K (4 µg/ml) or Cou (250µM) for 24 h followed by a treatment with OTA (3µM) and Nrf2 activators for 48 h prevented the increased formation of abasic sites induced by OTA (Fig. 9). Interestingly, pretreatment of the cells with the Nrf2 activator C + K was also associated with a protection against the cytotoxic effects of OTA as assayed by LDH release (Fig. 10). Similar results were obtained with Cou (data not shown).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In rodents, OTA has been clearly identified as a kidney carcinogen, while the health significance of low levels of human dietary exposure has still to be established (Benford et al., 2001
; EFSA, 2006; Fink-Gremmels, 2005). Evaluation of the safety of OTA exposure in humans would be greatly helped by elucidation of the mechanism(s) responsible for its toxicity and carcinogenicity.
In an in vivo long-term feeding study, applying gene profiling analysis (Marin-Kuan et al., 2006), OTA produced in the kidney, but not in liver, some effects that could result from a disruption of pathways regulated by the transcription factors Nrf2 and Hepatocyte Nuclear Factor 4 alpha (HNF4
). Genes affected by Nrf2 provide direct antioxidants, encode enzymes that directly inactivate oxidants and increase levels of cofactors involved in oxidant inactivation (Kensler et al., 2007). Since the production of oxidative stress has been increasingly proposed as playing a key role in OTA carcinogenicity (Benford et al., 2001; EFSA, 2006), the effect of OTA on Nrf2 was considered of key relevance to further understand the mechanism implicated. Therefore, focus was made on the biological mechanism involved in the OTA-mediated disruption of the Nrf2 pathway and on the oxidative stress consequence of such a disruption. However, a role of Nrf2-regulated genes implicated in other biological functions including chemical detoxification and removal of damaged proteins cannot be excluded and requires further investigations. This remark also applies to HNF4, another transcription factors identified as potentially modulated by OTA (Marin-Kuan et al., 2006) and to any other effects observed in kidney but not in liver.
OTA was reported to induce oxidative stress in vivo and in vitro. For example, an increased formation of malondialdehyde (MDA) was observed in the kidney of rats exposed to 120 µg/kg bw/day of OTA for 60 days (Petrik et al., 2003). Other studies confirmed that lipid peroxidation results from OTA exposure (Omar et al., 1990). In contrast, treatment of male rats with OTA (up to 2 mg/kg bw administered by gavage) did not increase the formation of biomarkers of oxidative damage such as the lipid peroxidation marker MDA in plasma, kidney, and liver, or the DNA damage marker 8-oxo-7,8-dihydro-2'deoxyguanosine in kidney DNA (Gautier et al., 2001). However, a significant increase in the expression of HSP32 protein was observed in the kidney but not in the liver suggesting that the mycotoxin can affect an oxidative stress response in target tissue. Surprisingly, the induction of such a response was not confirmed in a recent study using DNA microarray technology, in which many genes expected to be induced by oxidative stress were actually downregulated (Marin-Kuan et al., 2006). Several genes downregulated by OTA contain one or more AREs in their promoter regions. The ARE-motif is recognized by the Nrf2 transcription factor. Nrf2 has been reported to transactivate detoxifying enzymes and antioxidant proteins (Jaiswal, 2004; Kensler et al., 2007; Lee and Johnson, 2004). In the present study, the loss of expression of Nrf2-regulated genes was further confirmed at the protein level in rat kidney in vivo and in cell culture in vitro, indicating that the OTA-mediated effects observed previously at the mRNA level are likely to be of biological significance. In contrast to the in vivo situation, the renal target selectivity was not observed in cell culture in vitro. The same effects were obtained in both renal and hepatocyte cultures, although higher OTA concentrations were required to produce a significant Nrf2 response in hepatic cells. Other authors have observed oxidative damage in liver and other tissues of rats treated with OTA (Gagliano et al., 2006; Kamp et al., 2005; Mally et al., 2005). The direct comparison with our data is difficult since different endpoints were applied. In addition, in these studies OTA was administered by gavage and often at high doses. Gavage is known to increase OTA toxicity as compared to dietary administration (Mantle et al., 2005). Overall, the data available suggest that as already highlighted by others (Schwerdt et al., 1996; Zepnik et al., 2003), the high renal selectivity of OTA effects observed in vivo is compatible with an increased concentration of this mycotoxin in kidney cells as a result of active transport (Schwerdt et al., 1996; Zepnik et al., 2003). However, if intracellular concentration reaches a sufficiently high level, any other cell type would be expected to respond in a similar way.
Since the downregulation of the Nrf2 genes observed in vivo could be reproduced in vitro, both cell cultures systems were then employed to further address mechanistic questions. The data obtained indicated that OTA treatment was associated with an inhibition of cellular Nrf2 activity. This inhibition was demonstrated through the application of two different and independent approaches, a gel shift retardation assay performed with a GPEI probe and a NQO1 (ARE) reporter luciferase assay. From these studies, the exact mechanism involved cannot be determined but several hypotheses may be envisaged. Literature suggests that the protein kinase C enzymes could be involved in the activation of Nrf2 activity (Huang et al., 2002; Jaiswal, 2004). OTA may therefore interfere with this upstream regulatory pathway. A portion of the ARE sequence in several genes regulated by Nrf2 is closely related structurally to the response element for the activator protein 1 (AP-1). AP-1 factors have been shown also to bind to ARE but without activating ARE-dependent transcription. Presumably, AP-1 factors may prevent the binding of other signaling proteins such as Nrf2 to the same promoter site (Li and Jaiswal, 1992). Overexpression of AP-1 factors was shown to repress the expression of an ARE reporter gene in human HepG2 cells (Venugopal and Jaiswal, 1996). Since OTA was previously shown to activate AP-1 in rat embryonic midbrain cells (Hong and Lee, 2002) and in rat primary hepatocytes (data not shown), an inhibition of Nrf2 activity following activation of AP-1 appears a plausible mechanism for OTA action.
OTA-mediated downregulation of many Nrf2-regulated genes is likely to have important toxicological consequences. It is well documented that Nrf2 activators inhibit carcinogenesis in animal models, and potentially in humans (Kensler et al., 1998; Ramos-Gomez et al., 2004). Downregulation of these genes is expected to significantly reduce the cellular defense against various xenobiotics and oxidative stress, resulting potentially in an increased risk of cancer as reported in Nrf2 knockout mice (Kensler et al., 2007; Ramos-Gomez et al., 2001). In response to OTA treatment, GCLC, the rate-limiting enzyme in GSH synthesis, was inhibited and the cellular content of gluthatione was decreased, as already reported (Schaaf et al., 2002). Because of its major role as an antioxidant, a reduction in the intracellular GSH concentration is anticipated to compromise significantly the defense of the cells against oxidative injuries. Inhibition of the expression of other enzymes with protective properties may further increase the negative impact of the OTA-mediated reduction in GSH. For example, the expression of Nrf2-regulated GSH transferase subunit GSTP1 was decreased following OTA treatment. GSTP1 is thought to play a role in the detoxification of the lipid peroxidation product 4-hydoxynonenal (4-HNE) by conjugating efficiently with GSH (Hartley et al., 1995). 4-HNE is a reactive chemical known to bind to macromolecules including DNA (Bartsch and Nair, 2004). Taken together, our data support the hypothesis that OTA-induced depletion of the cellular antioxidant defense mechanism may result in increased oxidative damage and cytotoxicity.
The potential adverse consequences of Nrf2 depletion was supported by our data showing that, in vivo and in vitro, OTA induced a small but significant increase in the formation of DNA abasic sites. In vivo, significant DNA damage was found in renal tissues after 12 months of treatment. The direct link between the depletion of Nrf2-regulated gene expression and the increased formation of abasic sites was established in vitro. In the presence of the Nrf2 activator C + K, OTA-induced inhibition of Nrf2-regulated gene expression was abrogated and C + K prevented the formation of DNA abasic sites resulting from OTA treatment. Nrf2 depletion is also associated with the cytotoxicity mediated by OTA since pretreatment of cells with C + K was found to protect against the cytotoxic effects of OTA.
Many genes that are regulated by Nrf2 and are downregulated by OTA appear to be involved in detoxification and transport processes. They include several GSH S-transferases, uridine diphosphate–glucuronyltransferases, and NAD(P)H:quinone oxido-reductase and various transport proteins (Marin-Kuan et al., 2006). Their reduced expression may result in reduced detoxification and excretion of OTA or other xenobiotics. An increased concentration of a variety of xenobiotics in the cell may result in synergistic toxicity, as already suggested between OTA and other mycotoxins (Stoev et al., 2001). This indicates that the role of OTA as a modulator of the toxic effects of other chemicals deserves further investigations.
In summary, the production of oxidative stress has been thought to play an important role in OTA carcinogenicity but exact mechanisms involved have not yet been elucidated. In the evaluation of OTA, several potential relevant oxido-reduction mechanisms need to be considered. 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 (Omar et al., 1990). A role for cytochrome P450 in this reaction was also suggested (Omar et al., 1991). Other authors have reported that the OTA hydroquinone/quinone couple was generated from the oxidation of OTA (phenol oxidation) by electrochemical, photochemical, and chemical processes (Dai et al., 2002; Gillman et al., 1999) and also possibly by biological reactions in vitro (Faucet-Marquis et al., 2006). The quinone can undergo reductions to form semiquinone and hydroquinone. Such events are likely to result in redox cycling and in the generation of reactive oxygen species (Dai et al., 2002; Gillman et al., 1999). Our studies suggest that a depletion of antioxidant defense by inhibition of the Nrf2 regulatory pathway may be a novel and plausible mechanism, by which OTA may increase oxidative stress and induce the formation of tumors. Indeed, low levels of chronic oxidative stress have been associated with carcinogenesis (Klaunig and Kamendulis, 2004). The source of oxidants can be either endogenous (normal physiological processes) or exogenous (from xenobiotics or OTA itself). It is the first time that chemical induced oxidative stress and associated adverse effects can be attributed to such a mechanism.
In respect to human risk assessment, it has to be noted that our newly proposed mechanism of OTA toxicity does not involve direct binding of the toxin to DNA. It is dependent upon gene expression and is therefore thresholded. This conclusion is in agreement with the recent OTA evaluation conducted by EFSA (2006) which concluded that the various genetics effects seen in vitro and in vivo studies are compatible with the hypothesis of DNA damage induced by oxidative stress rather than indicating a direct interaction of OTA with cellular DNA. Therefore, the panel applied a threshold-based approach in its risk assessment. However, it has to be acknowledged that the issue regarding the genotoxicity of OTA remains controversial since results obtained with 32P-postlabeling-technique have suggested the existence of DNA adducts (Pfohl-Leskowicz and Castegnaro, 2005) whose structure still needs to be confirmed.
|
NOTES |
|---|
This work was supported by the EU-Grant#QLK1-CT-2001-011614.
|
ACKNOWLEDGMENTS |
|---|
We acknowledge staff of Imperial College Central Biomedical Services animal husbandry.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bartsch H and Nair J. (2004) Oxidative stress and lipid peroxidation-derived DANN-lesions in inflammation driven carcinogenesis. Cancer Detect. Prev. 28:385–391.[CrossRef][Web of Science][Medline]
Bendele AM, Carlton WW, Krogh P, Lillehoj EB. (1985) Ochratoxin A carcinogenesis in the (C57BL/6J x C3H)F1 mouse. J. Natl. Cancer Inst. 75:733–742.[Web of Science][Medline]
Benford D, Boyle C, Dekant W, Fuchs R, Gaylor DW, Hard G, Mc Gregor DB, Pitt JJ, Plestina R, Sheppard G, et al. (2001) Ochratoxin A, Safety Evaluation of Certain Mycotoxins in Food WHO Food Additives, Series 47(WHO, Genève) 74: pp. 281–415.
Bertholet R. (1987) Preparation of cafestol. United States Patent, No. 4692534.
Castegnaro M, Canadas D, Vrabcheva T, Petkova-Bocharova T, Chernozemsky IN, Pfohl-Leszkowicz A. (2006) Balkan endemic nephropathy: Role of ochratoxin A through biomarkers. Mol. Nutr. Food Res. 50:519–529.[CrossRef][Web of Science][Medline]
Castegnaro M, Mohr U, Pfohl-Leszkowicz A, Esteve J, Steinmann J, Tillmann T, Michelon J, Bartsch H. (1998) Sex- and strain-specific induction of renal tumors by ochratoxin A in rats correlates with DNA adduction. Int. J. Cancer 77:70–75.[CrossRef][Web of Science][Medline]
Dai J, Park G, Wright MW, Adams M, Akman SA, Manderville RA. (2002) Detection and characterization of a glutathione conjugate of ochratoxin A. Chem. Res. Toxicol. 15:1581–1588.[CrossRef][Web of Science][Medline]
EFSA (European Food Safety Authority) Scientific Panel on Contaminants in the Food Chain. (2006) Opinion of the on scientific panel on contaminants in the food chain on a request from the commission related to ochratoxin A in food. EFSA J. 365:1–56.
Faucet V, Pfohl-Leskowicz A, Dai J, Castegnaro M, Manderville A. (2004) Evidence for covalent DNA adduction by ochratoxin A following chronic exposure to rat and subacute exposure to pig. Chem. Res. Toxicol. 17:1289–1296.[CrossRef][Web of Science][Medline]
Faucet-Marquis V, Pont F, Stomer C, Rizk T, Castegnaro M, Pfohl-Leskowicz A. (2006) Evidence of a new dechlorinated ochratoxin A derivative formed in opossum kidney cell cultures after pretreatment by modulators of glutathione pathways: Correlation with DNA-adduct formation. Mol. Nutr. Food Res. 50:530–542.[CrossRef][Web of Science][Medline]
Fink-Gremmels J. (2005) Conclusion from the workshops on ochratoxin A in food: Recent developments and significance, organized by ILSI Europe in Baden (Austria), 29 June–1 July 2005. Food Addit. Contam. Suppl. 1, 1–5.
Gagliano N, Dalle Donne I, Torri C, Migliori M, Grizzi F, Milzani A, Filippi C, Annoni G, Colombo P, Costa F, et al. (2006) Early cytotoxic effects of Ochratoxin A in rat liver: A morphological biochemical and molecular study. Toxicology 225:214–224.[CrossRef][Web of Science][Medline]
Gallagher EP, Kavanagh TJ, Eaton DL. (1994) Glutathione, oxidized glutathione and mixed disulfides in biological samples. Methods in Toxicology(Academic Press, New York) 1B: pp. 349–366.
Gautier J-C, Holzhaeuser D, Markovic J, Gremaud E, Schilter B, Turesky RJ. (2001) Oxidative damage and stress response from ochratoxin A exposure to rats. Free Radic. Biol. Med. 30:1089–1098.[CrossRef][Web of Science][Medline]
Gillman IG, Clark TN, Manderville RA. (1999) Oxidation of ochratoxin A by an Fe-porphyrin system: Model for enzymatic activation and DNA cleavage. Chem. Res. Toxicol. 12:1066–1076.[CrossRef][Web of Science][Medline]
Grosse Y, Chekir-Ghedira L, Huc A, Obrecht-Pflumio S, Dirheimer G, Bacha H, Pfohl-Leszkowicz A. (1997) Retinol, ascorbic acid and alpha-tocopherol prevent DNA adduct formation in mice treated with the mycotoxins ochratoxin A and zearalenone. Cancer Lett. 114:225–229.[CrossRef][Web of Science][Medline]
Gross-Steinmeyer K, Weymann J, Hege HG, Metzler M. (2002) Metabolism and lack of DNA reactivity of the mycotoxin ochratoxin a in cultured rat and human primary hepatocytes. J. Agric. Food Chem. 50:938–945.[CrossRef][Web of Science][Medline]
Hartley DP, Ruth JA, Petersen DR. (1995) The hepatocellular metabolism of 4-hydroxynonenal by alcohol dehydrogenase, aldehyde dehydrogenase, and glutathione S-transferase. Arch. Biochem. Biophys. 316:197–205.[CrossRef][Web of Science][Medline]
Hayes JD, Flanagan JU, Jowsey IR. (2005) Glutathione transferases. Annu. Rev. Pharmacol. Toxicol. 45:51–88.
Hong JT and Lee MK. (2002) Inhibitory effect of peroxisome proliferator-activated receptor gamma agonist on Ochratoxin A-induced cytotoxicity and activation of transcription factors in cultured rat embryonic midbrain cells. J. Toxicol. Environ. Health 65:407–418.[CrossRef][Web of Science]
Huang HC, Nguyen T, Pickett CB. (2002) Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription. J. Biol. Chem. 277:42769–42774.
Jaiswal AK. (2004) Nrf2 signaling in coordinated activation of antioxidant gene expression. Free Radic. Biol. Med. 36:1199–1207.[CrossRef][Web of Science][Medline]
Jeong W-S, Jun M, Kong AH-NGT. (2006) Nrf2: A potential molecular target for cancer chemoprevention by natural compounds. Antioxid. Redox Signal. 8:99–106.[CrossRef][Web of Science][Medline]
Kamp HG, Eisenbrand G, Janzowski C, Kiossev J, Latendresse JR, Schlatter J, Turesky R. (2005) Ochratoxin A induces oxidative DNA damage in liver and kidney after oral dosing to rats. Mol. Nutr. Food. Res. 49:1160–1167.[CrossRef][Web of Science][Medline]
Kensler TW, He X, Otieno M. (1998) Oltipraz chemoprevention in Quidong, People's Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol. Biomarkers Prev. 7:127–134.[Abstract]
Kensler TW, Wakabayashi N, Biswal S. (2007) Cell survival response to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu. Rev. Pharmacol. Toxicol. 47:6.1–6.28.
Klaunig JE and Kamendulis LM. (2004) The role of oxidative stress in carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 44:239–267.
Lee JM and Johnson J. (2004) A. An important role of Nrf2-ARE pathway in the cellular defense mechanism. J. Biochem. Mol. Biol. 37:139–143.[Web of Science][Medline]
Li Y and Jaiswal AK. (1992) Regulation of human NAD(P)H:quinone oxido-reductase gene. Role of AP-1 binding site contained within human antioxidant response element. J. Biol. Chem. 267:15097–15104.
Mally A, Volkel W, Amberg A, Kurtz M, Wanek P, Eder E, Hard G, Dekant W. (2005) Functional, biochemical, and pathological effects of repeated oral administration of ochratoxin A to rats. Chem. Res. Toxicol. 18:81242–1252.[CrossRef][Web of Science][Medline]
Mally A, Zepnik H, Wanek P, Eder E, Dingley K, Ihmels H, Völkel W, Dekant W. (2004) Ochratoxin A: Lack of formation of covalent DNA adducts. Chem. Res. Toxicol. 17:234–242.[CrossRef][Web of Science][Medline]
Mantle PG, Kulinskaya E, Nestler S. (2005) Renal tumourigenesis in male rats in response to chronic dietary ochratoxin A. Food Addit. Contam. 22:58–64.
Marin-Kuan M, Nestler S, Verguet C, Bezençon C, Piguet D, Mansourian R, Holzwarth J, Grigorov M, Delatour T, Mantle P, et al. (2006) A toxicogenomics approach to identify new plausible epigenetic mechanisms of Och ratoxin A carcinogenicity in rat. Toxicol. Sci. 89:120–134.
Nakamura J, Walker VE, Upton PB, Chiang S-Y, Kow YW, Swenberg JA. (1998) Highly sensitive apurinic/apyrimidinic site assay can detect spontaneous and chemically induced depurination under physiological conditions. Cancer Res. 58:222–225.
Nioi P, McMahon M, Itoh K, Yamamoto M, Hayes JD. (2003) Identification of a novel Nrf2-regulated antioxidant response element (ARE) in the mouse NAD(P)H:quinone oxidoreductase 1 gene: Reassessment of the ARE consensus sequence. Biochem. J. 374:337–348.[CrossRef][Web of Science][Medline]
O'Brien E and Dietrich DR. (2005) Ochratoxin A: The continuing enigma. Crit. Rev. Toxicol. 35:33–60.[CrossRef][Web of Science][Medline]
Omar RF, Hasinoff BB, Mejilla F, Rahimtula AD. (1990) Mechanism of ochratoxin A stimulated lipid peroxidation. Biochem. Pharmacol. 40:1183–1191.[CrossRef][Web of Science][Medline]
Omar RF, Gelboin HV, Rahimtula AD. (1996) Effect of cytochrome P450 induction on the metabolism and toxicity of ochratoxin A. Biochem. Pharmacol. 51:3207–216.[CrossRef][Web of Science][Medline]
Petkova-Bocharova T, Chernozemsky IN, Castegnaro M. (1988) Ochratoxin A in human blood in relation to Balkan endemic nephropathy and urinary system tumours in Bulgaria. Food Addit. Contam. 5:299–301.[Web of Science][Medline]
Petrik J, Zanic-Grubisic T, Barisic K, Pepeljnjak S, Radic B, Ferencic Z, Cepelak I. (2003) Apoptosis and oxidative stress induced by ochratoxin A in rat kidney. Arch. Toxicol. 77:685–693.[CrossRef][Web of Science][Medline]
Pfohl-Leskowicz A and Castegnaro M. (2005) Further arguments in favour of direct covalent binding of ochratoxin A after metabolic biotransformation. Food Addit. Contam. 22:175–87.
Pfohl-Leskowicz A, Grosse Y, Kane A, Creppy EE, Dirheimer G. (1993) Differential DNA adduct formation and disappearance in three mouse tissues after treatment with the mycotoxin ochratoxin A. Mutat. Res. 289:265–273.[CrossRef][Web of Science][Medline]
Radic B, Fuchs R, Peraica M, Lucic A. (1997) Ochratoxin A in human sera in the area with endemic nephropathy in Croatia. Toxicol. Lett. 91:105–109.[CrossRef][Web of Science][Medline]
Ramos-Gomez M, Dolan PM, Itoh K, Yamamoto M, Kensler TW. (2004) Interactive effects of nrf2 genotype and oltipraz on benzo[a]pyrene-DNA adducts and tumor yield in mice. Carcinogenesis 24:461–467.
Ramos-Gomez M, Kwak M, Dolan PM, Itoh K, Yamamoto M, Talalay P. (2001) Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proc. Natl. Acad. Sci. U.S.A. 98:3410–3415.
Schaaf GJ, Nijmeijer SM, Maas RFM, Roestenberg P, de Groene EM, Fink-Gremmels J. (2002) The role of oxidative stress in the ochratoxin A-mediated toxicity in proximal tubular cells. Biochim. Biophys. Acta. 1588:149–158.[Medline]
Schwerdt G, Bauer K, Gekle M, Silbernagl S. (1996) Accumulation of ochratoxin A in rat kidney in vivo and in cultivated renal epithelial cells in vitro. Toxicology 114:177–185.[CrossRef][Web of Science][Medline]
Sidhu JS, Farin FM, Omiecinski CJ. (1993) Influence of extracellular matrix overlay on Phenobarbital-mediated induction of CYP 2B1, 2B2 and 3A1 genes in primary adult rat hepatocyte culture. Arch. Biochem. Biophys. 301:103–113.[CrossRef][Web of Science][Medline]
Stoev SD, Vitanov S, Anguelov G, Petkova-Bocharova T, Creppy EE. (2001) Experimental mycotoxic nephropathy in pigs provoked by a diet containing ochratoxin A and penicillic acid. Vet. Res. Commun. 25:205–223.[CrossRef][Web of Science][Medline]
Turesky RJ. (2005) Perspective: Ochratoxin A is not a genotoxic carcinogen. Chem. Res. Toxicol. 18:1082–1090.[CrossRef][Web of Science][Medline]
Venugopal R and Jaiswal AK. (1996) Nrf1 and Nrf2 positively and c-Fos and Fra-1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxido-reductase 1 gene. Proc. Natl. Acad. Sci. U.S.A. 93:14960–14965.
Zepnik H, Völkel W, Dekant W. (2003) Toxicokinetics of the mycotoxin ochratoxin A in F 344 rats after oral administration. Toxicol. Appl. Pharmacol. 192:36–44.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. Cavin, T. Delatour, M. Marin-Kuan, F. Fenaille, D. Holzhauser, G. Guignard, C. Bezencon, D. Piguet, V. Parisod, J. Richoz-Payot, et al. Ochratoxin A-Mediated DNA and Protein Damage: Roles of Nitrosative and Oxidative Stresses Toxicol. Sci., July 1, 2009; 110(1): 84 - 94. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. P. Grollman and B. Jelakovic Role of Environmental Toxins in Endemic (Balkan) Nephropathy J. Am. Soc. Nephrol., November 1, 2007; 18(11): 2817 - 2823. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Rached, G. C. Hard, K. Blumbach, K. Weber, R. Draheim, W. K. Lutz, S. Ozden, U. Steger, W. Dekant, and A. Mally Ochratoxin A: 13-Week Oral Toxicity and Cell Proliferation in Male F344/N Rats Toxicol. Sci., June 1, 2007; 97(2): 288 - 298. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Stemmer, H. Ellinger-Ziegelbauer, H.-J. Ahr, and D. R. Dietrich Carcinogen-Specific Gene Expression Profiles in Short-term Treated Eker and Wild-type Rats Indicative of Pathways Involved in Renal Tumorigenesis Cancer Res., May 1, 2007; 67(9): 4052 - 4068. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||