Vorgabe neu Journal of Applied Botany and Food Quality 80, 179 - 186 (2006) (1)Institute of Human Nutrition and Food Science, Christian-Albrechts-University Kiel (2)Institute of Animal Nutrition and Physiology, Christian-Albrechts-University Kiel Ochratoxin A-induced cytotoxicity in liver (HepG2) cells: Impact of serum concentration, dietary antioxidants and glutathione-modulating compounds* Christine Bösch-Saadatmandi(1), Christoph Hundhausen(1), Laia Jofre-Monseny(1), Ralf Blank(2), Siegfried Wolffram(2), Gerald Rimbach(1) (Received August 15, 2006) * The paper was presented at the 41th meeting of the „Deutsche Gesellschaft für Qualitätsforschung (Pflanzliche Nahrungsmittel) DGQ e. V.“ Abbreviations BSO, buthionine sulfoximine; CAT, catechin; DMSO, dimethyl sulfoxide; DTNB, dithio-bis-nitrobenzoic acid; EGCG, epigallo- catechin gallate; FCS, foetal calf serum; GSH, glutathione; IARC, international agency for research on cancer; NAC, N-acetylcysteine; NO, nitric oxide; NR, neutral red; OATP, organic anion-transporting polypeptide; OTA, ochratoxin A; PBS, phosphate buffered saline; QUE, quercetin; ROS, reactive oxygen species; ROSAC, rosmarinic acid; RPMI, roswell park memorial institute; α-TOC, α-tocopherol; α-TOC-P, α-tocopherol phosphate Summary Ochratoxin A (OTA) is a nephro- and hepatotoxic mycotoxin pro- duced by various species of the genera Aspergillus and Penicillium. OTA is known to bind with high affinity to plasma proteins which may have a substantial impact on its bioavailability and, thus, on its toxicity. However, the underlying mechanisms of OTA-induced cellular toxicity have not yet been fully elucidated. It has been suggested that oxidative damage contributes to its cytotoxic effects. Dietary antioxidants such as vitamin E and polyphenols may there- fore counteract OTA-induced cell death. Furthermore, compounds influencing the intracellular level of glutathione (L-gamma-glutamyl- L-cysteinylglycine, GSH), the most abundant thiol antioxidant in mammalian cells, may have an impact on OTA-induced cytotoxicity. In this study we investigated the effects of serum concentrations as well as different dietary antioxidants on the viability of OTA-exposed liver (HepG2) cells. Additionally, we determined the intracellular GSH-levels after incubation with OTA and N-acetylcysteine (NAC, a precursor of GSH) or buthionine sulfoximine (BSO, an inhibitor of gamma-glutamylcysteinyl synthetase). Incubation of human hepatoma cells (HepG2) for 24 h with increasing concentrations of OTA (0.25 - 50 µmol/l) in the presence of 0, 2.5, 5, or 10% foetal calf serum (FCS) resulted in a dose-dependent decrease in cell viability. Decreasing the serum concentrations in the cell culture medium led to increased cell mortality. Pre-treatment for 24 h with RRR-α-tocopherol (α-TOC), RRR-α-tocopherolphosphat (α-TOC- P), epigallocatechin gallate (EGCG), quercetin (QUE), catechin (CAT), and rosmarinic acid (ROSAC) at concentrations of 25, 50 and 100 µmol/l did not prevent OTA-induced toxicity. α-TOC-P, EGCG, and QUE even amplified the cytototoxic effects of OTA in HepG2. Supplementation with NAC and BSO in the presence of OTA did not substantially change cell viability, although BSO treatment resulted in depletion of cellular GSH. OTA treatment increased GSH levels at concentrations of 50 and 100 nmol/l, but decreased cellular GSH at the higher concentrations (250, 500, and 1000 nmol/l). The decrease in cellular GSH concentrations was less pronounced than for BSO. Our results indicate that the cytotoxicity of OTA in HepG2 cells, which is strongly dependent on the protein concentration in the cell culture medium, can not be prevented by pre-incubation with dietary antioxidants. Although cellular GSH levels are influenced by OTA incubation, mechanisms other than oxidative stress are likely to be involved in OTA-induced cell death in HepG2 cells. Introduction The mycotoxin ochratoxin A (OTA), mainly produced by Aspergillus ochraceus and Penicillium verrucosum, is the most toxic of the three known members of the ochratoxin family (ochratoxin A, B, and C). OTA was discovered and its chemical structure described in 1965 (VAN DER MERWE et al., 1965). Chemically, OTA consists of a dihydroisocumarin moiety joined by a peptide bond to 1-phenyl- alanine (Fig. 1). OTA can be found in many foods and beverages, e.g. cereal grains, coffee, and wine (FAZEKAS et al., 2002; STUDER- ROHR et al., 1995; VAN EGMONT et al., 1994). Due to its ubiqui- tous occurrence in foods, it is virtually impossible to completely avoid ingestion of OTA. Both in animals and humans, a wide range of toxic effects of OTA has been reported, including renal and hepatic toxicity (BENDELE et al., 1985), genotoxicity (CREPPY et al., 1985; BOSE and SINHA, 1994), neurotoxicity (MIKI et al., 1994; BRUININK et al., 1998), and immunotoxicity (HARVEY et al., 1992; PETZINGER and ZIEGLER, 2000). Severe human renal diseases, such as Endemic Nephropathy (BEN), chronic interstitial nephritis and karyomegalic interstitial nephritis, may be a result of continued exposure to OTA (SIMON et al., 1996). Moreover, the International Agency for Research on Cancer has classified OTA as a class 2B carcinogen (possible human carcinogen) (IARC, 1991). Fig. 1: Chemical structure of ochratoxin A The toxicity of OTA is largely determined by its protein binding properties. In blood, 99% of OTA is bound to serum proteins (mainly albumin), which prolongs its systemic half-life, delays its elimination and may, therefore, amplify its toxicity (CHU, 1971; CHU, 1974). The exact mode of action of OTA toxicity is as yet unclear. The toxicity of OTA has been explained by three major mechanisms: 1. inhibition of protein synthesis via inhibition of phenylalanine-tRNA synthetases (CREPPY et al., 1979; CREPPY et al., 1998), 2. inhibition of mitochondrial function (MOORE and TRUELOVE, 1970; MEISNER, and CHAN, 1974; WEI et al., 1985), and 3. generation of reactive oxygen species (ROS) which may oxidise DNA, proteins, lipids, and other macromolecules (RAHIMTULA et al., 1988; OMAR et al., 1990; SCHAAF et al., 2002; DOMIJAN et al., 2005; KAMP et al., 2005). Therefore, antioxidants may counteract OTA-induced cytotoxicity. Recent studies have shown that supplementation with vitamin E or several polyphenols partly diminished the toxic effects of OTA (SCHAAF et al., 2002; BALDI et al., 2004; RENZULLI et al., 2004; GUERRA et al., 2005). Furthermore, an involvement of the glutathione (GSH) system in OTA cytotoxicity, has been suggested. GSH (γ- glutamylcysteinylglycine) functions as a free radical scavenger, and serves as a nucleophilic co-substrate for glutathione transferases in the detoxification of xenobiotics. It has been shown that pre- incubation with N-acetylcysteine (NAC), a precursor of GSH, com- pletely prevented OTA-induced cell death in kidney (LLC-PK1) cells (SCHAAF et al., 2002). The objective of the present study was to assess the impact of serum protein concentration in the cell culture medium and the effect of dietary antioxidants on OTA-induced cytotoxicity in HepG2 cells. Furthermore, we aimed at investigating whether modulation of cellular GSH levels affects OTA-induced cytotoxicity in human liver cells in culture. Materials and methods Cell culture and determination of OTA-cytotoxicity The human hepatoblastoma derived cell line HepG2 was cultured in RPMI medium supplemented with 10% FCS, 2 mmol/l glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin under standard conditions (37°C, 5% CO 2 ). Confluent cells were harvested by tryp- sination and seeded at 1 x 105 cells per well in 24-well plates. The medium was changed every 48 h. In dose-response experiments, HepG2 cells were exposed to in- creasing concentrations of OTA ranging from 0.25 - 50 µmol/l for 24 h. The FCS concentrations in the medium were 0, 2.5, 5 or 10%. In a separate experiment, HepG2 cells were incubated with OTA at concentrations of 10 - 1000 nmol/l to find out the highest non-toxic OTA concentration under serum-free conditions. The cytotoxicity of OTA was determined employing the neutral red (NR) assay (VALACCHI et al., 2001). This test is based on the ability of viable cells to incorporate NR in lysosomes which is photometrically measured at 540 nm. Cell viability was expressed as percentage of solvent-exposed control (0.1% methanol) survival. For each in- cubation period, two independent experiments were performed in triplicate (n = 6). Pre-incubation with test substances To study the potential protective effects of vitamin E and polyphenols on OTA-induced cytotoxicity, cells were pre-treated with RRR- α-tocopherol (α-TOC), RRR-α-tocopherolphosphate (α-TOC-P), epigallocatechin gallate (EGCG), quercetin (QUE), catechin (CAT), and rosmarinic acid (ROSAC) (Fig. 2) for 24 h at concentrations of 25, 50, and 100 µmol/l (non-toxic concentrations for HepG2 cells, data not shown). 100 mmol/l stock solutions were prepared in dimethyl sulfoxide (DMSO), ethanol or an acetic acid:ethanol mixture (1:3) for the polyphenols, α-TOC and α-TOC-P, respectively. Stock solutions were further diluted in culture medium containing 10% FCS with a maximum solvent concentration of 0.1%. Cells were washed twice with PBS and incubated for another 24 h with 1, 5 or 15 µmol/l OTA in serum-free RPMI. Additionally, HepG2 were pre-incubated for 24 h with 0, 0.2, 0.5, 1, 2, or 4 mmol/l NAC or 0, 5, 25, 50, 100, or 500 µmol/l BSO. BSO and NAC were pre- pared as 100 mmol/l stock solutions in PBS. The pH of the NAC stock solution was adjusted to pH 7 with NaOH. Subsequently, cells were washed twice with PBS and incubated for 24 h with 1, 5 or 15 µmol/l OTA in FCS-free medium. Two or three independent replicates were performed in triplicate. Determination of cellular glutathione levels Total cellular GSH content was determined by the method of Griffith with minor modifications (GRIFFITH, 1980). HepG2 cells were grown at a density of 9 x 105 cells per well in 6-well plates for 48 hours and incubated with OTA, BSO, or NAC at increasing concentrations. After 24 h, the incubation medium was aspirated; cells were washed with PBS and harvested by trypsination. Due to the limited cell material, cells of three wells were pooled together. Cell pellets were collected after centrifugation and stored immediately at -80°C. To measure glutathione concentrations, cell pellets were resuspended in lysis buffer (PBS, 0.05% Triton-X (w/v), 0.05 mmol/l EDTA) and homo- genized. The cell suspension was treated with an equal volume of 10% sulfosalicylic acid (w/v) and separated by centrifugation (12,000 x g, 5 min, 4°C). 270 µL of the supernatant was mixed with 20 µL of a 150 mmol/l phosphate buffer (including 1% bovine serum albumin, pH 7.5) and 30 µL triethanolamin (50%). Sample (75 µL) was added to the reaction mixture containing 350 µL of 0.28 mmol/l NADPH and 50 µL of 6 mmol/l DTNB (5-5'dithio-bis- 2-nitro-benzoic acid). The reaction was started by addition of 50 µl glutathione-reductase (10 U/ml) and the increase in absorption at 412 nm was followed for 180 min in 20 sec intervals. Each sample was measured in duplicate. Results were calculated by comparison of the difference in absorbance per minute with data from a standard curve generated with oxidised glutathione. The protein content of the cell suspension was determined with the commercially available BCA Protein Kit from Pierce. Three independent replicates were performed in duplicate. Statistical analysis Data are presented as mean with standard deviation. The statistical program SPSS (version 13.0) was used to assess the effects of various treatments by analysis of variance (ANOVA) followed by the post hoc test Dunnett. In the case of inhomogeneous variances multiple t-tests were applied. Differences were considered significant if p < 0.05. Results Cytotoxicity of OTA Incubation of HepG2 cells with concentrations of OTA ranging from 0.25 to 50 µmol/l for 24 h resulted in a dose-dependent decline in cell viability (Fig. 3). Decreasing FCS concentrations were paralleled by a marked increase in OTA-induced cytotoxicity, with the strongest effects at OTA concentrations between 0.25 and 15 µmol/l. In cells cultured in serum-free medium the strongest cytotoxicity was observed at OTA concentrations between 10 (100% viability) and 250 nmol/l (53% viability). However, in medium containing 5 or 10% serum, 24 h exposure to 1 µmol/l OTA did not lead to cell death at all, while 0 or 2.5% serum decreased cell viability to 52 and 87%, respectively. The minimum level of cell viability (approximately 40%) was reached upon incubation with 15 µmol/l OTA and did not 180 Christine Bösch-Saadatmandi, Christoph Hundhausen, Laia Jofre-Monseny, Ralf Blank, Siegfried Wolffram, Gerald Rimbach decrease further even upon incubation with as much as 50 µmol/l OTA in FCS-free medium (Fig. 3).The highest non-toxic OTA concentration was 10 nmol/l (Fig. 4). Due to the higher bioavailability of OTA under these conditions, serum-free medium was chosen for further experiments. Pre-incubation experiments with antioxidants and glutathione-modulators A 24 h pre-incubation of HepG2 cells with α-TOC, α-TOC-P, EGCG, QUE, CAT, and ROSAC at concentrations of 25, 50, or 100 µmol/l and a subsequent incubation with 1, 5, or 15 µmol/l OTA for 24 h did not prevent the cytotoxic effects of OTA (Fig. 5). Treatment with α- TOC-P, EGCG, and QUE even enhanced OTA cytotoxicity dose- dependently. No changes in cell viability were observed upon pre- Fig. 2: Chemical structures of the antioxidants α-Tocopherol Catechin α-Tocopherol phosphate (disodium salt) Epigallocatechin gallate Rosmarinic acidQuercetin Ochratoxin A (µmol/lOchratoxin A (µmol/lOchratoxin A (µmol/lOchratoxin A (µmol/l)))) Fig. 3: Influence of foetal calf serum (FCS) concentration on the viability of HepG2 cells after 24 h incubation with ochratoxin A (0 - 50 µmol/l). Data are mean ± SD from two experiments in triplicate (n = 6). vi a b ili ty vi a b ili ty vi a b ili ty vi a b ili ty (% ) (% ) (% ) (% ) Ochratoxin A (nmol/lOchratoxin A (nmol/lOchratoxin A (nmol/lOchratoxin A (nmol/l)))) Fig. 4: Viability of HepG2 cells after 24 h incubation with ochratoxin A (0 - 1000 nmol/l). Data are mean ± SD from two experiments in tri- plicate (n = 6). Effect of antioxidants on OTA-induced cytotoxicity in hepatocytes 181 incubation with α-TOC, CAT and ROSAC. Cellular α-TOC levels remained unchanged in response to OTA treatment (data not shown), cellular concentrations of EGCG, QUE, CAT, and ROSAC were not determined. To further investigate the involvement of cellular glutathione levels in OTA-induced cytotoxicity, additional pre-incubations with the glutathione modulators BSO and NAC were carried out. Incubation with NAC did not protect OTA-treated cells from cytotoxicity (Fig. 6A). Pre-incubation with 500 µmol/l BSO decreased cell viability by 10%, while lower concentrations of BSO were without effect (Fig. 6B). Effect on cellular glutathione levels As shown in Fig. 7A, incubation with BSO for 24 h resulted in a substantial and dose-dependent depletion of cellular glutathione levels in HepG2 cells. Concentrations as low as 5 µM decreased cellular GSH by about 40% compared to untreated controls. A complete GSH- depletion was observed at 100 µmol/l BSO. Supplementation with NAC did not increase cellular GSH (data not shown). Incubation with OTA decreased GSH levels at concentrations of > 0.25 µmol/l (Fig. 7B). 1 µmol/l OTA led to an approximately 30% decrease in cellular GSH as compared to controls. In contrast, lower concentrations (0.05 or 0.1 µmol/l) of OTA resulted in a moderate increase in GSH levels in HepG2 cells. Discussion In line with previous studies our data indicate that OTA induces cytotoxicity in HepG2 cells in a dose-dependent manner (BALDI et al., 2004; RENZULLI et al., 2004; HUNDHAUSEN et al., 2005). More- 0000 10101010 20202020 30303030 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 0000 20202020 10101010 30303030 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) 1111 5555 15151515 1111 5555 15151515 OchratoxinOchratoxinOchratoxinOchratoxin A (A (A (A (µµµµmol/l)mol/l)mol/l)mol/l) OchratoxinOchratoxinOchratoxinOchratoxin A (A (A (A (µµµµmol/l)mol/l)mol/l)mol/l) ----TOCTOCTOCTOC ----TOCTOCTOCTOC----PPPP 0000 10101010 20202020 30303030 50505050 40404040 60606060 70707070 80808080 100100100100 90909090 0000 10101010 20202020 40404040 30303030 50505050 60606060 70707070 80808080 100100100100 90909090 v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) 1111 5555 15151515 1111 5555 15151515 OchratoxinOchratoxinOchratoxinOchratoxin A (A (A (A (µµµµmol/l)mol/l)mol/l)mol/l) OchratoxinOchratoxinOchratoxinOchratoxin A (A (A (A (µµµµmol/l)mol/l)mol/l)mol/l) EGCGEGCGEGCGEGCG QUEQUEQUEQUE 0000 10101010 30303030 20202020 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 0000 10101010 20202020 30303030 50505050 40404040 60606060 70707070 80808080 100100100100 90909090 v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) 1111 5555 15151515 1111 5555 15151515 OchratoxinOchratoxinOchratoxinOchratoxin A (A (A (A (µµµµmol/l)mol/l)mol/l)mol/l) OchratoxinOchratoxinOchratoxinOchratoxin A (A (A (A (µµµµmol/l)mol/l)mol/l)mol/l) CATCATCATCAT R OSACROSACROSACROSAC 0000 10101010 20202020 30303030 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 0000 20202020 10101010 30303030 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) 1111 5555 15151515 1111 5555 15151515 OchratoxinOchratoxinOchratoxinOchratoxin A (A (A (A (µµµµmol/l)mol/l)mol/l)mol/l) OchratoxinOchratoxinOchratoxinOchratoxin A (A (A (A (µµµµmol/l)mol/l)mol/l)mol/l) ----TOCTOCTOCTOC ----TOCTOCTOCTOC----PPPP 0000 10101010 20202020 30303030 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 0000 10101010 20202020 30303030 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 10101010 20202020 30303030 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 0000 20202020 10101010 30303030 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) 1111 5555 151515151111 5555 15151515 1111 5555 151515151111 5555 15151515 OchratoxinOchratoxinOchratoxinOchratoxin A (A (A (A (µµµµmol/l)mol/l)mol/l)mol/l) OchratoxinOchratoxinOchratoxinOchratoxin A (A (A (A (µµµµmol/l)mol/l)mol/l)mol/l) ----TOCTOCTOCTOC ----TOCTOCTOCTOC----PPPP 0000 10101010 20202020 30303030 50505050 40404040 60606060 70707070 80808080 100100100100 90909090 0000 10101010 20202020 40404040 30303030 50505050 60606060 70707070 80808080 100100100100 90909090 v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) 1111 5555 15151515 1111 5555 15151515 OchratoxinOchratoxinOchratoxinOchratoxin A (A (A (A (µµµµmol/l)mol/l)mol/l)mol/l) OchratoxinOchratoxinOchratoxinOchratoxin A (A (A (A (µµµµmol/l)mol/l)mol/l)mol/l) EGCGEGCGEGCGEGCG QUEQUEQUEQUE 0000 10101010 20202020 30303030 50505050 40404040 60606060 70707070 80808080 100100100100 90909090 0000 10101010 20202020 40404040 30303030 50505050 60606060 70707070 80808080 100100100100 90909090 v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) 1111 5555 151515151111 5555 15151515 1111 5555 151515151111 5555 15151515 OchratoxinOchratoxinOchratoxinOchratoxin A (A (A (A (µµµµmol/l)mol/l)mol/l)mol/l) OchratoxinOchratoxinOchratoxinOchratoxin A (A (A (A (µµµµmol/l)mol/l)mol/l)mol/l) EGCGEGCGEGCGEGCG QUEQUEQUEQUE 0000 10101010 30303030 20202020 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 0000 10101010 20202020 30303030 50505050 40404040 60606060 70707070 80808080 100100100100 90909090 v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) 1111 5555 15151515 1111 5555 15151515 OchratoxinOchratoxinOchratoxinOchratoxin A (A (A (A (µµµµmol/l)mol/l)mol/l)mol/l) OchratoxinOchratoxinOchratoxinOchratoxin A (A (A (A (µµµµmol/l)mol/l)mol/l)mol/l) CATCATCATCAT R OSACROSACROSACROSAC 0000 10101010 30303030 20202020 40404040 50505050 60606060 70707070 80808080 100100100100 90909090 0000 10101010 20202020 30303030 50505050 40404040 60606060 70707070 80808080 100100100100 90909090 v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) v ia b il it y v ia b il it y v ia b il it y v ia b il it y (% ) (% ) (% ) (% ) 1111 5555 151515151111 5555 15151515 1111 5555 151515151111 5555 15151515 OchratoxinOchratoxinOchratoxinOchratoxin A (A (A (A (µµµµmol/l)mol/l)mol/l)mol/l) OchratoxinOchratoxinOchratoxinOchratoxin A (A (A (A (µµµµmol/l)mol/l)mol/l)mol/l) CATCATCATCAT R OSACROSACROSACROSAC * * vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) vi a b ili ty (% ) Ochratoxin A (µmol/lOchratoxin A (µmol/lOchratoxin A (µmol/lOchratoxin A (µmol/l)))) Ochratoxin A (µmol/lOchratoxin A (µmol/lOchratoxin A (µmol/lOchratoxin A (µmol/l)))) Ochratoxin A (µmol)/lOchratoxin A (µmol)/lOchratoxin A (µmol)/lOchratoxin A (µmol)/l)))) Ochratoxin A (µmol/lOchratoxin A (µmol/lOchratoxin A (µmol/lOchratoxin A (µmol/l)))) Ochratoxin A (µmol/lOchratoxin A (µmol/lOchratoxin A (µmol/lOchratoxin A (µmol/l)))) Ochratoxin A (µmol/lOchratoxin A (µmol/lOchratoxin A (µmol/lOchratoxin A (µmol/l)))) * ** * * * * * * * * * * * * * * α α Fig. 5: Cytotoxicity of ochratoxin A (24 h, 1 - 15 µmol/l) in HepG2 cells after pre-incubation with RRR-α-tocopherol (α-TOC), RRR-α-tocopherolphosphate, epigallocatechin gallate (EGCG), quercetin (QUE), catechin (CAT), and rosmarinic acid (ROSAC) (24 h, 0 , 25 , 50 , and 100 µmol/l). Data are mean ± SD from two experiments in triplicate (n = 6). *Indicates significant differences when compared to control (p < 0.05). 182 Christine Bösch-Saadatmandi, Christoph Hundhausen, Laia Jofre-Monseny, Ralf Blank, Siegfried Wolffram, Gerald Rimbach Fig. 7: Total glutathione (GSH, in % of control) after 24 h incubation with buthionine sulfoximine (BSO, A) or ochratoxin A (OTA, B) at the given concentrations. Data are mean ± SD from two experiments in duplicate (n = 4, A) or triplicate (n = 6, B). *Indicates significant differences when compared to control (p < 0.05). 0 10 20 30 40 50 60 70 80 90 100 0 5 10 25 50 100 BSO (µM) to ta l G S H ( % o f c o n tr o l) 0 10 20 30 40 50 60 70 80 90 100 0 5 10 25 50 100 BSO (µM) to ta l G S H ( % o f c o n tr o l) A 0 20 40 60 80 100 120 140 0 0.05 0.1 0.25 0.5 1 OTA (µM) to ta l G S H ( % o f c o n tr o l) 0 20 40 60 80 100 120 140 0 0.05 0.1 0.25 0.5 1 OTA (µM) to ta l G S H ( % o f c o n tr o l) B BSO (µmol/l) OTA (µmol/l) * * * over, we demonstrated a strong impact of serum protein concentration in the cell culture medium on the viability of OTA-exposed HepG2 cells. A decrease in serum concentration led to a dramatic increase in OTA-induced cell death in HepG2 cells. Importantly, under serum- free conditions even nanomolar OTA-concentrations resulted in a considerable loss of cell viability. Concerning the protein binding properties of OTA, a high affinity of OTA for plasma proteins, both in animals and in humans, has been reported (RINGOT et al., 2006). In a wide range of species, including quail, mouse, rat and monkey, the fraction of free OTA in plasma was found to be less than 0.2% (HAGELBERG et al., 1989). Further- more, it is well-known that OTA toxicity depends on its half-life in the body (O’BRIEN and DIETRICH, 2005), which in turn has been suggested to be influenced by binding of OTA to proteins (CHU, 1971; CHU, 1974). This is supported by findings that albumin- deficient rats had a 20- to 70-times faster OTA clearance from the systemic circulation than non-deficient rats (KUMAGAI, 1985). However, studies investigating the dose-response relationship between the protein concentration in the cell culture medium and OTA toxicity are lacking. For this reason, a major focus of the present study was to determine the impact of different FCS levels in the culture medium on OTA-induced cell death in hepatocytes. We observed that a decrease of serum concentration in the medium was paralleled by a dramatic increase in OTA toxicity, which may indicate strong protein binding of OTA, possibly linked with inhibited cellular uptake and, thus, lower toxicity in HepG2 cells. Consistently, a previous study revealed decreased neurotoxic effects of OTA by protein binding to bovine serum albumin in embryonic chick brain and neural retina cell cultures (BRUININK and SIDLER, 1997). On the other hand, protein binding alone does not always decrease OTA Fig. 6 Cytotoxicity of ochratoxin A (24 h, 1 - 15 µmol/l) on HepG2 cells after pre-incubation with N-acetylcysteine (NAC, A) (24 h, 0 - 4 mmol/l) or buthionine sulfoximine (BSO, B) (24 h, 0 – 500 µmol/l). Data are mean ± SD from two experiments in triplicate (n = 6).*Indicates significant differences when compared to control (Dunnett; p < 0.05). 0 10 20 30 40 50 60 70 80 90 100 Ochratoxin A (µmol/l) v ia b il it y % NAC 0 mmol/l NAC 0.2 mmol/l NAC 0.5 mmol/l NAC 1 mmol/l NAC 2 mmol/l NAC 4 mmol/l 5 1 15 0 10 20 30 40 50 60 70 80 90 100 Ochr atoxin A (µmol/l) v ia b il it y % BSO 0 µmol/l BSO 5 µmol/l BSO 25 µmol/l BSO 50 µmol/l BSO 100 µmol/l BSO 500 µmol/l 5 1 15 0 10 20 30 40 50 60 70 80 90 100 Ochratoxin A (µmol/l) v ia b il it y % NAC 0 mmol/l NAC 0.2 mmol/l NAC 0.5 mmol/l NAC 1 mmol/l NAC 2 mmol/l NAC 4 mmol/l 5 1 15 0 10 20 30 40 50 60 70 80 90 100 Ochr atoxin A (µmol/l) v ia b il it y % BSO 0 µmol/l BSO 5 µmol/l BSO 25 µmol/l BSO 50 µmol/l BSO 100 µmol/l BSO 500 µmol/l 5 1 15 * * * Fig. 6: Cytotoxicity of ochratoxin A (24 h, 1 - 15 µmol/l) on HepG2 cells after pre-incubation with N-acetylcysteine (NAC, A) (24 h, 0 - 4 mmol/l) or buthionine sulfoximine (BSO, B) (24 h, 0 - 500 µmol/l). Data are mean ± SD from two experiments in triplicate (n = 6). *Indicates significant differences when compared to control (Dunnett; p < 0.05). A B Effect of antioxidants on OTA-induced cytotoxicity in hepatocytes 183 toxicity (STOJKOVIC et al., 1984). It has been suggested that binding of OTA to two small serum proteins (molecular mass 20.000 Da), being able to pass through the glomerular membrane, might explain the OTA-mediated nephrotoxic effect in mammals (STOJKOVIC et al., 1984). Taken together, variations in the degree of protein binding as well as selective binding of OTA to distinct proteins may contribute to the often reported species-specific differences in OTA toxicity (DIETRICH et al., 2001; O’BRIEN et al., 2001; HEUSSNER et al., 2002). In the present study, we investigated the potential cytotoxic effects of OTA at nanomolar concentrations. Previous studies have revealed that nanomolar OTA concentrations induced damage to renal and brain cells, due to an induction of apoptosis (GEKLE et al., 2000) and a loss of neuronal enzyme activity (MONNET-TSCHUDI et al., 1997). Our study, however, is the first to demonstrate that OTA causes cell death in hepatocytes at nanomolar concentrations, which is probably due to the absence of foetal calf serum (FCS) in the culture medium. Under serum-free conditions, incubation with 50, 100, or 250 nmol/l OTA for 24 hours resulted in an approximately 10, 40, or 60% decrease in viability of HepG2 cells, respectively. We observed toxicity with concentrations as low as 50 nmol/l OTA, which is with- in the range of OTA detected in human blood and serum. Between 6 and 26% of human blood and serum samples from the Balkan area contained OTA in the range of 1 - 35 ng/ml (2.5 - 88 nmol/l) and 1 - 40 ng/ml (2.5 - 100 nmol/l) (PETKOVA-BOCHAROVA et al., 1988), respectively, paralleled by an increased occurrence of the Balkan Endemic Nephropathie (BEN) (PETKOVA-BOCHAROVA et al., 1988). Considering the elimination half-life of OTA in humans of approxi- mately 36 days (STUDER-ROHR et al., 2000), which is substantially longer than that reported for other species – mice 40 h; rats 55-120 h (GALTIER et al., 1979; HAGELBERG et al., 1989); pigs 72-120 h (GALTIER et al., 1981); monkeys 820 h (KUIPER-GOODMAN and SCOTT, 1989) – it is apparent that OTA constitutes a higher risk in humans. Our study also demonstrated that at least 45% of the cells were still viable after incubation with 50 µmol/l OTA for 24 h. In serum-free medium this maximum toxicity was reached at OTA concentrations as low as 0.25 µmol/l. Incubation with 1 - 50 µmol/l OTA did not further increase OTA-induced cell death in HepG2 cells. In ac- cordance with our results it has been shown in LLC-PK1 cells that higher OTA-concentrations (high nmol/l to low µmol/l) or longer times of exposure (96 h) did not further decrease cell viability (DREGER et al., 2000). One hypothesis, which might explain the survival of a considerable number of HepG2 cells, suggests the existence of OTA carrier systems being saturated at low OTA- concentrations and, thus, not being able to increase intracellular OTA-transport at higher OTA-concentrations. Such OTA carriers, belonging to the family of human organic anion transporters, are well-known in proximal tubule cells from mice (JUNG et al., 2001; BABU et al., 2002). However, studies investigating the transport of OTA in liver cells are rare. It has been proposed, however, that the organic anion-transporting polypeptide (OATP) is involved in OTA- uptake in hepatocytes (KONTAXI et al., 1996). Neither the toxicokinetics of OTA, nor its precise toxicity mecha- nisms have been established to date. Nevertheless, the involvement of reactive oxygen species (ROS) in OTA toxicity seems to be likely (RAHIMTULA et al., 1988; GAUTIER et al., 2001; SCHAAF et al., 2002). Therefore, one objective of our study was to counteract OTA toxicity by pre-incubation of HepG2 cells with various antioxidant test substances. We pre-incubated liver cells with 25, 50, or 100 µmol/l of the respective antioxidants. Concerning α-TOC these concen- trations are similar to physiological plasma concentrations, which have been reported in the range of 25 - 40 µmol/l (HENSLEY et al., 2004), referring to flavonoid concentrations used in the cell culture experiments however exceeded physiological plasma concentrations (0.1 - 10 µmol/l) (MANACH et al., 2004). To avoid interactions between OTA and the test compounds in the culture medium, cells were washed twice with PBS before incubation with OTA. None of the test compounds prevented HepG2 cells from OTA-induced cell death. This is in contrast to studies reporting protective effects of anti- oxidants towards OTA-induced cell damage. For example, 24 h of pre-treatment with 50 µmol/l cyanidin-3-O-beta-glucopyranoside or 50 µmol/l rosmarinic acid inhibited the cytotoxicity of 10 µmol/l OTA in HepG2 cells by 25 and 35%, respectively (RENZULLI et al., 2004; GUERRA et al., 2005). Another experiment, also using OTA in serum-free medium, showed that three hours of pre-incubation with 1 nM α-TOC decreased OTA-induced cytotoxicity in bovine mam- mary epithelium cells by 10% (BALDI et al., 2004). The reasons for these conflicting results remain unclear, although cell-specific dif- ferences might be one plausible explanation. We also found that EGCG, QUE, and α-TOC-P enhanced the toxic effects of OTA. α-TOC-P inhibits cell proliferation (OGRU et al., 2004) and modulates membrane fluidity (REZK et al., 2004). The latter might be the reason for a facilitated uptake of OTA into HepG2 cells, and thus, for the observed synergistic toxic effects of α-TOC-P and OTA. Moreover, it has been demonstrated that both EGCG and QUE are able to exert not only antioxidant, but also pro-oxidant activity, partly due to the formation of hydrogen peroxide (METODIEWA et al., 1999; ELBLING et al., 2005; FERRARESI et al., 2005), which might explain the increased cytotoxicity of OTA in HepG2 cells. We further investigated the importance of the intracellular anti- oxidant glutathione for the prevention of OTA toxicity. Several studies report a depletion of glutathione by buthionine sulfoximine (BSO), often paralleled by either loss of cell viability or increased suscepti- bility to compounds exerting oxidative stress (WRIGHT et al., 1998; ANDERSON et al., 1999; LONG et al., 2000; ANDERSON and REYNOLDS, 2002; HONDA et al., 2004). However, it has been shown that HepG2 cells are able to survive BSO treatment, which may be explained by an up-regulation of Bcl-2, a protein preventing apoptosis (D’ALESSIO, 2004). In the present study we confirmed the resistance of HepG2 cells to BSO treatment and showed, additionally, that there were no synergistic effects of OTA and BSO on cytotoxicity in HepG2 cells. Although GSH was dose-dependently depleted by both BSO and OTA (Fig. 7), pre-treatment with BSO did not enhance cell death (Fig. 6). Furthermore, NAC did not increase cellular GSH levels (data not shown), and was not able to prevent HepG2 cells against the toxic effects of OTA (Fig. 6). Consistent with these findings are the results of a study with cultured rat embryonic cells demonstrating a decrease of GSH induced by OTA, but no protection due to exogenous GSH supplementation (HONG et al., 2000). On the other hand, it has been shown that NAC completely protected LLC-PK1 from OTA- induced cell death (SCHAAF et al., 2002). Interestingly, we observed a biphasic effect of OTA on intracellular GSH levels in HepG2 cells. 50 and 100 nmol/l OTA increased cellular GSH to 116 and 121% compared to controls, respectively, while OTA at concentrations between 0.1 and 1 µmol/l dose-dependently de- creased cellular GSH levels to approximately 70%. This effect may indicate that GSH is involved in the detoxification of OTA, possibly by directly binding to OTA as proposed by DAI and co-workers (2002). On the other hand, it has been shown for brain cells that OTA-exposure leads to an increased generation of nitric oxide (NO) (ZURICH et al., 2005), which in turn is detoxified through conjugation with GSH. In conclusion, the presented data indicate a dose-dependent effect of foetal calf serum concentrations in the cell culture medium on OTA-induced cytotoxicity in HepG2 cells. Supplementation with dietary antioxidants did not counteract cytotoxic effects of OTA. Although cellular GSH levels are modulated by OTA, mechanisms other than oxidative stress are likely to be involved in OTA-induced cell death in HepG2 cells. Acknowledgements The authors gratefully thank Susan Pollard from the School of Food Biosciences, University of Reading, UK and Dr. Jan Frank from this department for their critical reading of the manuscript and helpful comments. C. Bösch-Saadatmandi and C. Hundhausen contributed equally to this manuscript. References ANDERSON, C.P., REYNOLDS, C.P., 2002: Synergistic cytotoxicity of buthionine sulfoximine (BSO) and intensive melphalan (L-PAM) for neuroblastoma cell lines established at relapse after myeloablative therapy. Bone Marrow Transplant. 30, 135-140. ANDERSON, C.P., TSAI, J.M., MEEK, W.E., LIU, R.M., TANG, Y., FORMAN, H.J., REYNOLDS, C.P., 1999: Depletion of glutathione by buthionine sulfoxine is cytotoxic for human neuroblastoma cell lines via apoptosis. Exp. Cell Res. 246, 183-192. BABU, E., TAKEDA, M., NARIKAWA, S., KOBAYASHI, Y., ENOMOTO, A., TOJO, A., CHA, S.H., SEKINE, T., SAKTHISEKARAN, D., ENDOU, H., 2002: Role of human organic anion transporter 4 in the transport of ochratoxin A. Biochim. Biophys. Acta. 1590, 64-75. BALDI, A., LOSIO, M.N., CHELI, F., REBUCCI, R., SANGALLI, L., FUSI, E., BERTASI, B., PAVONI, E., CARLI, S., POLITIS, I., 2004: Evaluation of the 184 Christine Bösch-Saadatmandi, Christoph Hundhausen, Laia Jofre-Monseny, Ralf Blank, Siegfried Wolffram, Gerald Rimbach protective effects of alpha-tocopherol and retinol against ochratoxin A cytotoxicity. Br. J. Nutr. 91, 507-512. BENDELE, A.M., CARLTON, W.W., KROGH, P., LILLEHOJ, E.B., 1985: Ochra- toxin A carcinogenesis in the (C57BL/6J X C3H)F1 mouse. J. Natl. Cancer Inst. 75, 733-742. BOSE, S., SINHA, S.P., 1994: Modulation of ochratoxin-produced genotoxicity in mice by vitamin C. Food Chem. Toxicol. 32, 533-537 BRUININK, A., RASONYI, T., SIDLER, C., 1998: Differences in neurotoxic effects of ochratoxin A, ochracin and ochratoxin-alpha in vitro. Nat. Toxins. 6, 173-177. BRUININK, A., SIDLER, C., 1997: The neurotoxic effects of ochratoxin-A are reduced by protein binding but are not affected by l-phenylalanine. Toxicol. Appl. Pharmacol. 146, 173-179. CHU, F.S., 1971: Interaction of ochratoxin A with bovine serum albumin. Arch. Biochem. Biophys. 147, 359-366. CHU, F.S., 1974: A comparative study of the interaction of ochratoxins with bovine serum albumin. Biochem. Pharmacol. 23, 1105-1113. CREPPY, E.E., BAUDRIMONT, I., ANNE, M., 1998: How aspartame prevents the toxicity of ochratoxin A. J. Toxicol. Sci. 23 (Suppl. 2), 165-172. CREPPY, E.E., KANE, A., DIRHEIMER, G., LAFARGE-FRAYSSINET, C., MOUSSET, S., FRAYSSINET, C., 1985: Genotoxicity of ochratoxin A in mice: DNA single-strand break evaluation in spleen, liver and kidney. Toxicol. Lett. 28, 29-35. CREPPY, E.E., LUGNIER, A.A., FASIOLO, F., HELLER, K., ROSCHENTHALER, R., DIRHEIMER, G., 1979: In vitro inhibition of yeast phenylalanyl-tRNA synthetase by ochratoxin A. Chem. Biol. Interact. 24, 257-261. D’ALESSIO, X.Y.??, 2004: Glutathione depletion up-regulates Bcl-2 in BSO- resistant cells. Faseb J. 18, 1609-1611. DOMIJAN, A.M., RUDES, K., PERAICA, M., 2005: The effect of ochratoxin A on the concentration of protein carbonyls in rats. Arh. Hig. Rada Toksi- kol. 56, 311-315. ELBLING, L., WEISS, R.M., TEUFELHOFER, O., UHL, M., KNASMUELLER, S., SCHULTE-HERMANN, R., BERGER, W., MICKSCHE, M., 2005: Green tea extract and (-)-epigallocatechin-3-gallate, the major tea catechin, exert oxidant but lack antioxidant activities. Faseb J. 19, 807-809. FERRARESI, R., TROIANO, L., ROAT, E., LUGLI, E., NEMES, E., NASI, M., PINTI, M., FERNANDEZ, M.I., COOPER, E.L., COSSARIZZA, A., 2005: Essential requirement of reduced glutathione (GSH) for the anti-oxidant effect of the flavonoid quercetin. Free Radic. Res. 39, 1249-1258. GALTIER, P., ALVINERIE, M., CHARPENTEAU, J.L., 1981: The pharmacokinetic profiles of ochratoxin A in pigs, rabbits and chickens. Food Cosmet. Toxicol. 19, 735-738. GALTIER, P., CHARPENTEAU, J.L., ALVINERIE, M., LABOUCHE, C., 1979: The pharmacokinetic profile of ochratoxin A in the rat after oral and intra- venous administration. Drug Metab. Dispos. 7, 429-434. GAUTIER, J.C., HOLZHAEUSER, D., MARKOVIC, J., GREMAUD, E., SCHILTER, B., TURESKY, R.J., 2001: Oxidative damage and stress response from ochratoxin a exposure in rats. Free Radic. Biol. Med. 30, 1089-1098. GEKLE, M., SCHWERDT, G., FREUDINGER, R., MILDENBERGER, S., WILFLINGS- EDER, D., POLLACK, V., DANDER, M., SCHRAMEK, H., 2000: Ochratoxin A induces JNK activation and apoptosis in MDCK-C7 cells at nanomolar concentrations. J. Pharmacol. Exp. Ther. 293, 837-844. GRIFFITH, O.W., 1980: Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal. Biochem. 106, 207-212. GUERRA, M.C., GALVANO, F., BONSI, L., SPERONI, E., COSTA, S., RENZULLI, C., CERVELLATI, R., 2005: Cyanidin-3-O-beta-glucopyranoside, a natural free-radical scavenger against aflatoxin B1- and ochratoxin A-induced cell damage in a human hepatoma cell line (Hep G2) and a human colonic adenocarcinoma cell line (CaCo-2). Br. J. Nutr. 94, 211-220. HAGELBERG, S., HULT, K., FUCHS, R., 1989: Toxicokinetics of ochratoxin A in several species and its plasma-binding properties. J. Appl. Toxicol. 9, 91-96. HARVEY, R.B., ELISSALDE, M.H., KUBENA, L.F., WEAVER, E.A., CORRIER, D.E., CLEMENT, B.A., 1992: Immunotoxicity of ochratoxin A to growing gilts. Am. J. Vet. Res. 53, 1966-1970. HENSLEY, K., BENAKSAS, E.J., BOLLI, R., COMP, P., GRAMMAS, P., HAMDHEY- DARI, L., MOU, S., PYE, Q.N., STODDARD, M.F., WALLIS, G., WILLIAMSON, K.S., WEST, M., WECHTER, W.J., FLOYD, R.A., 2004: New perspectives on vitamin E: gamma-tocopherol and carboxyelthylhydroxychroman metabolites in biology and medicine. Free Radic. Biol. Med. 36, 1-15. HONDA, T., COPPOLA, S., GHIBELLI, L., CHO, S.H., KAGAWA, S., SPURGERS, K.B., BRISBAY, S.M., ROTH, J.A., MEYN, R.E., FANG, B., MCDONNELL, T.J., 2004: GSH depletion enhances adenoviral bax-induced apoptosis in lung cancer cells. Cancer Gene Ther. 11, 249-255. HONG, J.T., PARK, K.L., HAN, S.Y., PARK, K.S., KIM, H.S., OH, S.D., LEE, R.D., JANG, S.J., 2000: Effects of ochratoxin A on cytotoxicity and cell differentiation in cultured rat embryonic cells. J. Toxicol. Environ. Health A. 61, 609-621. HUNDHAUSEN, C., BOSCH-SAADATMANDI, C., AUGUSTIN, K., BLANK, R., WOLFFRAM, S., RIMBACH, G., 2005: Effect of vitamin E and polyphenols on ochratoxin A-induced cytotoxicity in liver (HepG2) cells. J. Plant Physiol. 162, 818-822. IARC, 1991: Ochratoxin A. In: IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 489-521. IARC Scientific Publ. No. 56. Lyon, France. JUNG, K.Y., TAKEDA, M., KIM, D.K., TOJO, A., NARIKAWA, S., YOO, B.S., HOSOYAMADA, M., CHA, S.H., SEKINE, T., ENDOU, H., 2001: Charac- terization of ochratoxin A transport by human organic anion transporters. Life Sci. 69, 2123-2135. KAMP, H.G., EISENBRAND, G., JANZOWSKI, C., KIOSSEV, J., LATENDRESSE, J.R., SCHLATTER, J., TURESKY, R.J., 2005: Ochratoxin A induces oxidative DNA damage in liver and kidney after oral dosing to rats. Mol. Nutr. Food Res. 49, 1160-1167. KONTAXI, M., ECHKARDT, U., HAGENBUCH, B., STIEGER, B., MEIER, P.J., PETZINGER, E., 1996: Uptake of the mycotoxin ochratoxin A in liver cells occurs via the cloned organic anion transporting polypeptide. J. Pharmacol. Exp. Ther. 279, 1507-1513. KUIPER-GOODMAN, T., SCOTT, P.M., 1989: Risk assessment of the mycotoxin ochratoxin A. Biomed. Environ. Sci. 2, 179-248. LONG, L.H., CLEMENT, M.V., HALLIWELL, B., 2000: Artifacts in cell culture: rapid generation of hydrogen peroxide on addition of (-)-epigallocatechin, (-)-epigallocatechin gallate, (+)-catechin, and quercetin to commonly used cell culture media. Biochem. Biophys. Res. Commun. 273, 50-53. MANACH, C., SCALBERT, A., MORAND, C., REMESY, C., JIMENEZ, L., 2004: Polyphenols: food sources and bioavailability. Am. J. Clin. Nutr. 79, 727- 747. MEISNER, H., CHAN, S., 1974: Ochratoxin A, an inhibitor of mitochondrial transport systems. Biochemistry 13, 2795-2800. METODIEWA, D., JAISWAL, A.K., CENAS, N., DICKANCAITE, E., SEGURA- AGUILAR, J., 1999: Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal product. Free Radic. Biol. Med. 26, 107-116. MIKI, T., FUKUI, Y., UEMURA, N., TAKEUCHI, Y., 1994: Regional difference in the neurotoxicity of ochratoxin A on the developing cerebral cortex in mice. Brain Res. Dev. Brain Res. 82, 259-264. MONNET-TSCHUDI, F., SORG, O., HONEGGER, P., ZURICH, M.G., HUGGETT, A.C., SCHILTER, B., 1997: Effects of the naturally occurring food myco- toxin ochratoxin A on brain cells in culture. Neurotoxicology. 18, 831- 839. MOORE, J.H., TRUELOVE, B., 1970: Ochratoxin A: inhibition of mitochondrial respiration. Science 168, 1102-1103. O’BRIEN, E., DIETRICH, D.R., 2005: Ochratoxin A: the continuing enigma. Crit. Rev. Toxicol. 35, 33-60. OGRU, E., LIBINAKI, R., GIANELLO, R., WEST, S., MUNTEANU, A., ZINGG, J.M., AZZI, A., 2004: Modulation of cell proliferation and gene expression by alpha-tocopheryl phosphates: relevance to atherosclerosis and in- flammation. Ann. N. Y. Acad. Sci. 1031, 405-411. OMAR, R.F., HASINOFF, B.B., MEJILLA, F., RAHIMTULA, A.D., 1990: Mecha- nism of ochratoxin A stimulated lipid peroxidation. Biochem. Pharma- Effect of antioxidants on OTA-induced cytotoxicity in hepatocytes 185 col. 40, 1183-1191. PETKOVA-BOCHAROVA, T., CHERNOZEMSKY, I.N., 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. PETZINGER, E., ZIEGLER, K., 2000: Ochratoxin A from a toxicological per- spective. J. Vet. Pharmacol. Ther. 23, 91-98. RAHIMTULA, A.D., BEREZIAT, J.C., BUSSACCHINI-GRIOT, V., BARTSCH, H., 1988: Lipid peroxidation as a possible cause of ochratoxin A toxicity. Biochem. Pharmacol. 37, 4469-4477. RENZULLI, C., GALVANO, F., PIERDOMENICO, L., SPERONI, E., GUERRA, M.C., 2004: Effects of rosmarinic acid against aflatoxin B1 and ochratoxin-A- induced cell damage in a human hepatoma cell line (Hep G2). J. Appl. Toxicol. 24, 289-296. REZK, B.M., HAENEN, G.R., VAN DER VIJGH, W.J., BAST, A., 2004: The extraordinary antioxidant activity of vitamin E phosphate. Biochim. Biophys. Acta. 1683 (1-3), 16-21. RINGOT, D., CHANGO, A., SCHNEIDER, Y.J., LARONDELLE, Y., 2006: Toxi- cokinetics and toxicodynamics of ochratoxin A, an update. Chem. Biol. Interact. 159, 18-46. SCHAAF, G.J., NIJMEIJER, S.M., MAAS, R.F., ROESTENBERG, P., DE GROENE, E.M., FINK-GREMMELS, J., 2002: The role of oxidative stress in the ochratoxin A-mediated toxicity in proximal tubular cells. Biochim. Bio- phys. Acta. 1588, 149-158. SIMON, P., GODIN, M., FILLASTRE, J.P., 1996: Ochratoxin a: a new environ- mental factor which is toxic for the kidney? Nephrol. Dial. Trans- plant. 11, 2389-2391. STOJKOVIC, R., HULT, K., GAMULIN, S., PLESTINA, R., 1984: High affinity binding of ochratoxin A to plasma constituents. Biochem. Int. 9, 33-38. STUDER-ROHR, I., SCHLATTER, J., DIETRICH, D.R., 2000: Kinetic parameters and intraindividual fluctuations of ochratoxin A plasma levels in humans. Arch. Toxicol. 74, 499-510. VALACCHI, G., RIMBACH, G., SALIOU, C., WEBER, S.U., PACKER, L., 2001: Effect of benzoyl peroxide on antioxidant status, NF-kappaB activity and interleukin-1alpha gene expression in human keratinocytes. Toxi- cology 165, 225-234. VAN DER MERWE, K.J., STEYN, P.S., FOURIE, L., SCOTT, D.B., THERON, J.J., 1965: Ochratoxin A, a toxic metabolite produced by Aspergillus ochraceus Wilh. Nature 205, 1112-1113. WEI, Y.H., LU, C.Y., LIN, T.N., WEI, R.D., 1985: Effect of ochratoxin A on rat liver mitochondrial respiration and oxidative phosphorylation. Toxi- cology 36, 119-130. WRIGHT, S.C., WANG, H., WEI, Q.S., KINDER, D.H., LARRICK, J.W., 1998: Bcl-2-mediated resistance to apoptosis is associated with glutathione- induced inhibition of AP24 activation of nuclear DNA fragmentation. Cancer Res. 58, 5570-5576. ZURICH, M.G., LENGACHER, S., BRAISSANT, O., MONNET-TSCHUDI, F., PELLERIN, L., HONEGGER, P., 2005: Unusual astrocyte reactivity caused by the food mycotoxin ochratoxin A in aggregating rat brain cell cultures. Neuroscience 134, 771-782. Address of the authors: Christine Bösch-Saadatmandi, Christoph Hundhausen, Laia Jofre Monseny, Gerald Rimbach1, Institute of Human Nutrition and Food Science, Christian- Albrechts-University, Hermann-Rodewald-Strasse 6, D-24118 Kiel, Germany. Ralf Blank, Siegfried Wolffram, Institute of Animal Nutrition and Physiology, Christian-Albrechts-University, Hermann-Rodewald-Strasse 9, D-24118 Kiel, Germany. 1Corresponding author: rimbach@foodsci.uni-kiel.de 186 Christine Bösch-Saadatmandi, Christoph Hundhausen, Laia Jofre-Monseny, Ralf Blank, Siegfried Wolffram, Gerald Rimbach << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /All /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Warning /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJDFFile false /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails false /EmbedAllFonts true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveEPSInfo true /PreserveHalftoneInfo false /PreserveOPIComments false /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputCondition () /PDFXRegistryName (http://www.color.org) /PDFXTrapped /Unknown /Description << /FRA /ENU (Use these settings to create PDF documents with higher image resolution for improved printing quality. The PDF documents can be opened with Acrobat and Reader 5.0 and later.) /JPN /DEU /PTB /DAN /NLD /ESP /SUO /ITA /NOR /SVE >> >> setdistillerparams << /HWResolution [2400 2400] /PageSize [612.000 792.000] >> setpagedevice