Mycotoxin ochratoxin A (OTA) is produced as a secondary metabolite by certain Penicillum and Aspergillum fungal species and is a common contaminant of human foodstuffs and animal feed. OTA occurs in a variety of plant products and can enter the human food chain directly through cereals, grains, beans, spices and by food products such as coffee, grape juice, wine, beer and breadReference Galvano, Ritieni, Piva, Pietri and Diaz1. The carcinogenicity of OTA in rats and mice is well established. OTA induces renal tumours in rats of both sexes and in male miceReference Bendele, Carlton, Krogh and Lillehoj2, Reference Boorman, McDonald, Imoto and Persing3. In man, exposure to high levels of OTA in the diet has been suggested as a causative factor in ‘Balkan endemic nephropathy’ and in the development of urinary tract tumoursReference Radovanovic, Jankovic and Jevremovic4–Reference Pfohl-Leszkowicz, Petkova-Bocharova, Chernozemskyn and Castegnaro7, although a direct causal relationship is still under debate and the mechanism of OTA carcinogenicity is not knownReference Galvano, Ritieni, Piva, Pietri and Diaz1.
In a recent review by O'Brien & DietrichReference O'Brien and Dietrich8 on its toxicological properties, OTA has justly been called ‘the continuing enigma’. According to the authors, it is not yet clear whether the predominant toxic mechanism of OTA is of a genotoxic or epigenetic nature, such as induced cytotoxicity, oxidative cell stress or increased cell proliferation due to an imbalance between proliferative and antiproliferative intracellular signal pathways.
Both carcinogenicity and cytotoxicity of OTA have been related to free radical-mediated oxidative cell damageReference Gautier, Holzhaeuser, Markovoc, Gremaud, Schilter and Turesky9–Reference Rahimtula, Bereziat, Bussacchini-Griot and Bartsch14. Schaaf et al. Reference Schaaf, Nijmeijer, Maas, Roestenberg, de Groene and Fink-Gremmel10 attributed proximal tubule cell damage caused by OTA to the formation of reactive oxygen species such as the superoxide anion (O2. − ), hydroxyl radical (.OH) and peroxide (ROO.) which induce a wide range of lesions to cell components. Other authors reported that cytochrome P450 is able to stimulate OTA-dependent lipid peroxidation and that this action is mediated by OTA–Fe3+/2+ complexesReference de Groene, Hassing, Blom, Seinen, Fink-Gremmels and Horback15–Reference Baudrimont, Betbeder and Creppy17.
In view of these reports, it is conceivable that nutritional factors may affect the susceptibility to OTA's effects, since most of the natural antioxidants occurring in the diet can act both as free radical scavengers and as Fe chelators.
The list of natural compounds acting as antioxidants includes anthocyanins, a widespread group of water-soluble plant constituents collectively known as flavonoids. The anthocyanins' health properties are due to their particular chemical structure; their electron deficiency makes them very reactive towards reactive oxygen species. The most common anthocyanins in plants are cyanidins, largely present in the human diet through beans, fruits, vegetables and red wine. Cyanidin antioxidant and Fe-chelating properties have been demonstrated in numerous studies using several methods, both in vivo and in vitro Reference Galvano, La Fauci, Lazzarino, Fogliano, Ritieni, Ciappellano, Battistini, Tavazzi and Galvano18, Reference Acquaviva, Russo, Galvano, Galvano, Barcellona, Li Volti and Vanella19, and a recent study carried out in our laboratory demonstrated that cyanidin 3-O-β-d-glucoside (C3G) is able to protect human fibroblasts against OTA-induced DNA damageReference Russo, La Fauci, Acquaviva, Campisi, Raciti, Scifo, Renis, Galvano, Vanella and Galvano20.
The ability of several natural antioxidants to induce haeme oxygenase (HO)-1 expression has recently been reportedReference Motterlini, Foresti, Bassi and Green21–Reference Balogun, Hoque, Gong, Kileen, Green, Foresti, Alam and Motterlini23. HO is the rate-limiting enzyme in haeme degradation to carbon monoxide, Fe and bilirubin. To date, three isoforms of HO have been identified: HO-1, or inducible enzyme, HO-2 or constitutive isoform and HO-3Reference Maines, Trakshel and Kutty24–Reference McCoubrey, Huang and Maines28.
HO-1, also known as stress protein HSP32, is present in normal conditions in the spleenReference Braggins, Trakshel, Kutty and Maines29. Its expression can, however, increase in many tissues following stressful stimuli including heavy metals, heat shock, glutathione depletion, UVA radiation, endotoxin, hypoxia, hyperoxia, ischaemia–reperfusion and a wide range of conditions characterised by alteration of the cellular redox stateReference Keyse and Tyrrell30–Reference Ryter, Tyrrell, Forman and Cadenas38. A substantial body of evidence demonstrates that HO-1 induction represents an essential step in cellular adaptation to stress subsequent to pathological eventsReference Raju and Maines34, Reference Nath, Balla, Vercellotti, Balla, Jacob, Levitt and Rosenberg39–Reference Foresti and Motterlini41. Although HO-1 hyper-expression can be considered a marker of cellular stress, this enzyme can be also regarded as a potential therapeutic target in a variety of oxidant- and inflammatory-mediated diseases. In fact, it has recently been reported that naturally occurring antioxidants potently induce HO-1 expression, leading to an increased resistance to oxidative stress-mediated damageReference Scapagnini, Foresti, Calabrese, Giuffrida Stella, Green and Motterlini22, Reference Balogun, Hoque, Gong, Kileen, Green, Foresti, Alam and Motterlini23, Reference Motterlini, Foresti, Bassi and Green42. As a consequence, it has been hypothesised that the beneficial actions attributed to several natural substances could be due to their intrinsic ability to activate the HO-1 pathwayReference Scapagnini, Foresti, Calabrese, Giuffrida Stella, Green and Motterlini22, Reference Balogun, Hoque, Gong, Kileen, Green, Foresti, Alam and Motterlini23, Reference Motterlini, Foresti, Bassi and Green42.
The present in vivo study was performed to verify whether the chronic oral administration of C3G might counteract damage induced by chronic exposure to OTA in rats and if its effect may be mediated by HO-1.
Material and methods
OTA from Aspergillus ochraceus was purchased from Sigma-Aldrich (Milan, Italy) and C3G was purchased from Polyphenols Laboratories (Sandnes, Norway). The Qiamp DNA mini kit was purchased from Qiagen (Löhne, Germany). Monoclonal HO-1 antibody was from Stressgen Biotechnologies (Victoria, BC, Canada), anti-actin antibody was from Sigma Aldrich Co. (St Louis, MO, USA) and secondary horseradish peroxidase-conjugated anti-mouse antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The enhanced chemiluminescence system for developing immunoblots and nitrocellulose membranes were purchased from Amersham Pharmacia Biotech (Milan, Italy). All other chemicals were purchased from Merck (Frankfurt, Germany).
Animals and treatment
The experiments reported in the present paper complied with current Italian law and met the guidelines of the Institutional Animal Care and Use Committee of Sacred Heart Catholic University of Piacenza (Italy). The experiments were performed in male Sprague–Dawley albino rats (150 g body weight and age 45 d at the beginning of experiments). They had free access to water and were kept at room temperature with a natural photo-period (12 h light–12 h dark cycle). Rats were subdivided into four groups (each group consisted of ten animals; each animal was placed into a separate cage) and received the test compounds orally, via their food pellets, for 4 weeks. A control group received a commercial balanced standard diet, group C3G received the standard control diet supplemented with C3G (1 g/kg feed), group OTA received the standard control diet supplemented with 200 parts per billion of OTA, group OTA+C3G received the standard diet of the OTA group supplemented with C3G (1 g/kg feed). Both OTA and C3G dosage were chosen accordingly to overall literature data relating to toxic chronic effect and antioxidant properties, respectively. After 4 weeks of daily treatment, animals underwent euthanasia by an overdose of anaesthetic (ethyl urethane, intraperitoneally) and the liver, kidneys and brain of each rat were rapidly removed in a cold room and immediately frozen ( − 80°C). Samples were processed within 1 week of collection.
Tissues were homogenised in 9 volumes of cold PBS. Samples of homogenate were used to evaluate non-proteic thiol groups (RSH) and lipid hydroperoxide (LOOH) levels, HO-1 expression and DNA fragmentation.
LOOH levels were measured in tissue homogenates following the oxidation of Fe+2 to Fe+3 in the presence of xylenol orange at λ = 560 nmReference Wolff and Packer43. The assay mixture contained, in a total volume of 1 ml: 100 μl homogenate, 100 μm-xylenol orange, 250 μm-ammonium ferrous sulfate, 90 % methanol, 4 mm-butylated hydroxytoluene and 25 mm-H2SO4. After 30 min incubation at room temperature, the absorbance at λ = 560 nm was measured using a U2000 Hitachi spectrophotometer (Hitachi, Ibaraki, Japan). Calibration was obtained using H2O2 (0·2–20 μm). Results are expressed as μmol/mg proteins.
Non-proteic thiol groups
Levels of RSH were measured, in 200 μl homogenate, using Hu's methodReference Hu and Packer44 partially modified. This spectrophotometric assay is based on the reaction of thiol groups with 2,2-dithio-bis-nitrobenzoic acid at λ = 412 nmReference Hu and Packer44 in absolute ethanol (εM = 13 600). Results are expressed as nmol/mg proteins.
Western blotting for haeme oxygenase-1 expression
Protein levels were visualised by immunoblotting with antibodies against HO-1. Briefly, samples of homogenates corresponding to 50 μg proteins were separated by SDS–PAGE and transferred to a nitrocellulose membrane. In order to block non-specific binding sites, the membranes were incubated overnight with 5 % non-fat dry milk in 10 mm-tri(hydroxymethyl)-aminomethane-HCl (pH 7·4), 150 mm-NaCl, 0·05 % Tween 20 buffer at 4°C. After washing with the buffer, the membranes were incubated with a 1:1000 dilution of anti-HO-1 antibody for 1 h at room temperature under constant shaking. The filters were then washed and subsequently probed with horseradish peroxidase-conjugated anti-mouse IgG for HO-1 at a dilution of 1:2000. Chemiluminescence detection was performed using an enhanced chemiluminescence detection kit according to the manufacturer's instructions. Results were quantified and normalised to β-actin by using image analysis software (Scion Image; Scion Corp., Frederick, MD, USA).
Protein content was evaluated according to the method of Lowry et al. Reference Lowry, Rosebrough, Farr and Randal45.
Genomic DNA was isolated from liver, kidney and brain homogenates using the Qiamp DNA micro kit (Qiagen) according to the manufacturer's instructions, electrophoresed on a 2 % agarose gel and stained by ethidium bromideReference Zhang, Shan, Otterbein, Alam, Flavell, Davis, Choi and Lee46. The gels were then photographed under UV luminescence. Under these conditions, damaged DNA appears as a smear consisting of DNA fragments, whereas intact DNA has high molecular weight and does not migrate very far into the gel.
Arithmetical means and standard deviations of all measured parameters were calculated. The results were analysed for statistical significance using ANOVA. A value of P < 0·001 was taken as significant.
During the experiment rats showed neither reduced vitality nor evident symptom of toxicity. No significant differences were observed as regards body weight and feed intake (data not shown) and no mortality was recorded. Fig. 1 reports the values of RSH content in kidney, liver and brain. In the OTA-treated group, a significant decrease in RSH content was observed in both kidney and liver, while values observed in brains of OTA-treated rats were similar to controls. According to the observed reduced levels of RSH, chronic administration of OTA led to a significant increase in LOOH levels (Fig. 2) in all the tissues under study.
In animals fed on the diet supplemented with C3G, kidney and brain but not hepatic RSH contents were slightly higher than controls (Fig. 1). However, the same group of animals showed significantly reduced LOOH levels in all three tissues (Fig. 2), suggesting that the antioxidant effect of C3G is not merely due to its ability to preserve endogenous stores of RSH.
Chronic supplementation with C3G was also able to counteract the toxic effect of OTA. In fact, in the OTA+C3G group both RSH content and LOOH levels were similar to those observed in control rats (Figs. 1 and 2).
Fig. 3 reports results regarding HO-1 expression by Western blot analysis. A significant induction of HO-1 is evident in kidney and liver of both OTA and C3G groups; however, the most relevant HO-1 expression was observed in kidney of C3G-supplemented rats. The simultaneous intake of both substances caused an increase in the expression of HO-1 in liver but not in kidney when compared with controls. None of the dietary treatments significantly affected brain HO-1 expression.
The results obtained from DNA fragmentation indicated that following chronic consumption of OTA, DNA damage occurred in all three tissues examined (Fig. 4). Fig. 4 clearly shows the protective effect of C3G.
It has been shown that oxidative damage contributes to the wide range of toxic effects of the mycotoxin ochratoxinReference Baudrimont, Betbeder and Creppy17, Reference Belmadani, Steyn, Tramu, Betbeder, Baudrimont and Creppi47–Reference Rahimtula, Bereziat, Busacchini-Griot and Bartsch49. The alterations in LOOH and RSH levels observed in the present study confirm the involvement of oxidative pathway in damage induced by OTA in kidney and liver. They also evidence the ability of the mycotoxin to affect brain tissue, as demonstrated by significantly increased LOOH levels and DNA damage found in brains of the OTA group. These results confirm that kidney is the target organ, but also demonstrate that OTA toxicity to other organs should not be underestimated.
Our working hypothesis was that natural antioxidants such as C3G might counteract OTA-induced damage. Data from these experiments demonstrated that chronic supplementation with C3G is able to efficiently counteract OTA-mediated oxidative changes. In fact, animals fed with a diet containing both OTA and C3G showed both RSH and LOOH levels similar to control values. In addition, the present results also suggest that the protective effects of C3G may be attributable not only to its antioxidant ability, but also to the HO-1-inducing property of the molecule. HO has both catabolic and anabolic roles in the cell. In the former capacity, it regulates cellular haeme and haemoprotein levels, including that of cytochrome P450Reference Maines50. In its anabolic role, HO produces bile pigments, carbon monoxide and Fe, all of which are biologically active: bile pigments function as antioxidantsReference Stocker, Yamamato, McDonagh, Glaser and Ames51, carbon monoxide has recently been suggested to have a function in the generation of cGMP and in regulation of vascular toneReference Maines52–Reference Morita, Perrella, Lee and Kourembanas56, and Fe regulates the expression of various genes, including that of HO-1 itselfReference Maines and Kappas57.
In the present study, an increased expression of HO-1 was observed in liver and kidneys of C3G-supplemented rats. However, from data regarding HO-1 expression it is also evident that both OTA and C3G are able to induce the heat-shock protein 32 in rat liver and kidney, even if the maximal increase was observed in the C3G group. The ability of both molecules to induce HO-1 expression may be the result of different molecular actions: C3G could increase HO-1 expression due to its molecular structure. It has been reported, in fact, that plant-derived polyphenolic compounds possess chemical features required to trigger the induction of antioxidant and defensive genesReference Motterlini, Foresti, Bassi and Green21, Reference Scapagnini, Foresti, Calabrese, Giuffrida Stella, Green and Motterlini22; then as reported for other polyphenols, C3G may activate the HO-1 gene via regulation of Nrf2 and/or the antioxidant-responsive elementReference Balogun, Hoque, Gong, Kileen, Green, Foresti, Alam and Motterlini23.
Although HO-1 hyper-expression may be considered a marker of cell stress, in view of the increasing evidence corroborating the importance of carbon monoxide and bilirubin (products of HO-1) in counteracting stress conditions, molecules with HO-1-inducing activity could also represent pharmacological strategies to increase cellular response and to increase cytoprotection. In fact, several reports showed the crucial participation of HO-1 gene expression in alleviating organ dysfunction and counteracting metabolic disorders in mammals, so this enzyme may be regarded as a potential target in a variety of oxidant-mediated diseasesReference Abraham, Drummond, Lutton and Kappas35, Reference Choi and Alam36, Reference Ryter, Tyrrell, Forman and Cadenas38–Reference Poss and Tonegawa40, Reference Maines50, Reference Stocker, Yamamato, McDonagh, Glaser and Ames51, Reference Motterlini, Foresti, Intaglietta and Winslow58–Reference Clark, Foresti, Sarathchandra, Kaur, Green and Motterlini62.
Conversely, HO-1-inducing activity of OTA may be attributable to its RSH-decreasing effect. A correlation between HO and glutathione has in fact been documented, where HO-1 levels are augmented in conditions of glutathione depletionReference Lautier, Luscher and Tyrrell31, Reference Rizzardini, Carelli, Cabello Porras and Cantoni33, Reference Ewing and Maines63 and among exogenous chemical agents inducing HO-1, molecules that directly complex intracellular glutathione are includedReference Ryter, Tyrrell, Forman and Cadenas38, Reference Oguro, Hayashi, Numazawa, Asakawa and Yoshida64–Reference Applegate, Luscher and Tyrrel66.
Data regarding the OTA+C3G group showed that hepatic and renal HO-1 expression is less pronounced compared with that observed in the OTA or C3G groups, but higher than in control. This result might be attributable to the capacity of chronic consumption of C3G to counteract RSH depletion induced by OTA. However, in view of the multiple response elements within HO-1 promoter and the multiplicity of interactions between components of the cell-signalling cascadeReference Maines and Gibbs67 we cannot rule out the involvement of other molecular mechanisms mediated by the complex intracellular signalling network implicated in the induction of stress proteins.
The present study aimed to verify the hypothesis that chronic consumption of C3G might counteract OTA-mediated injury. The results reported here showed that chronic consumption of C3G, a naturally occurring antocyanin, potently induced liver and kidney HO-1 expression and increased RSH levels, leading to enhanced resistance to OTA-derived oxidative stress-mediated damage.
Coherently with results regarding brain RSH levels, none of the diet regimens induced significant changes in brain HO-1 expression even if a relevant increase in LOOH was observed in the OTA group. This result further supports the hypothesis that OTA-induced hyper-expression of HO-1 may be a consequence of decreased RSH levels. The lack of modification in brain HO-1 expression in C3G-supplemented rats may be not surprising in consideration of data in the literature reporting the tissue selectivity of several HO-inducersReference Maines and Kappas57; moreover, it has to be considered that in the brain the prominent form of HO protein is the HO-2 isoformReference Chang, Wong, Vreman, Igarashi, Galo, Sharp, Stevenson and Noble-Haeusslein68, Reference Maines69, whose transcription levels are sensitive to cellular redox stateReference Hawkins, Zhuo and Arancio70, Reference McCoubrey, Eke and Maines71.
Analysis of DNA fragmentation evidenced that, following chronic consumption of OTA, DNA damage occurred in all three tissues under examination (liver, kidney and brain). These results may support the hypothesis that OTA toxicity is not limited to organs dedicated to its metabolism and excretion. At the present we cannot discriminate the type of mechanism involved (apoptotic or necrotic); however, based on results of experiments in progress in our laboratory, we are inclined to deem that DNA damage observed in the OTA group is apoptotic (F. Galvano, R. Acquaviva, L. La Fauci, C. Di Giacomo, unpublished results). In addition, our data indicated a clear DNA protection exerted by C3G, in agreement with previous reports by Acquaviva et al. Reference Acquaviva, Russo, Galvano, Galvano, Barcellona, Li Volti and Vanella19 demonstrating a protective effect of C3G on DNA cleavage. Overall, the results obtained demonstrate that C3G is able to counteract the deleterious effects of chronic consumption of OTA and provide evidence that its nutritional supplementation may contribute in preventing OTA-induced damage.
This work has been supported by funding from MIUR - Prin 2005 (Prof. Fabio Galvano) and, in part, by Provincia Regionale di Catania (Italy), research project on the antioxidant properties of Sicilian pigmented oranges (Prof. Giacomo Galvano).