CANCER GENOMICS & PROTEOMICS 5: 319-332 (2008) Ochratoxin A Lowers mRNA Levels of Genes Encoding for Key Proteins of Liver Cell Metabolism CHRISTOPH HUNDHAUSEN 1, CHRISTINE BOESCH-SAADATMANDI 1, NICOLE MATZNER 2, FLORIAN LANG 2, RALF BLANK 3, SIEGFRIED WOLFFRAM 3, WOLFGANG BLASCHEK 4 and GERALD RIMBACH 1 Institutes of 1Human Nutrition and Food Science and 3Animal Nutrition and Physiology, and 4Department of Pharmaceutical Biology, Institute of Pharmacy, Christian Albrechts University, Kiel; 2Department of Physiology, Eberhard Karls University, Tuebingen, Germany Abstract. Ochratoxin A (OTA) is a nephro- and hepatotoxic Ochratoxin A (OTA) is produced by several Aspergillus and mycotoxin that frequently contaminates food and feedstuffs. Penicillium species and was first isolated from Aspergillus Although recent studies have indicated that OTA modulates ochraceus (1 ). Chemically, OTA consists of a dihydroiso- renal gene expression, little is known regarding its impact on coumarin moiety linked to phenylalanine (Figure 1). Over differential gene expression in the liver. Therefore a microarray recent years, OTA, which is frequently present in cereals (2), study of the HepG2 liver cell transcriptome in response to OTA has received increasing attention because it has also been exposure (0, 0.25, 2.5 μmol/l for 24 h) was performed using detected in relatively large amounts in non-cereal plant Affymetrix GeneChip technology. Selected microarray results products including coffee, nuts, spices, olives, grapes and beans were verified by real-time PCR and Western blotting as (3, 4). Moreover, OTA has also been found in meat (5, 6) and independent methods. Out of 14,500 genes present on the in human blood, urine and milk (7-10). Numerous animal microarray, 13 and 250 genes were down-regulated by 0.25 and studies have demonstrated predominantly nephrotoxic, but also 2.5 μmol/l OTA, respectively. Reduced mRNA levels of hepatotoxic, teratogenic, immunosuppressive and recently also calcineurin A beta (PPP3CB), which regulates inflammatory genotoxic effects of OTA (11-14). The International Agency signalling pathways in immune cells, and of the uncoupling for Research on Cancer (IARC) has classified OTA as a protein 2 (UCP2), which has been suggested to control the putative human carcinogen (15). production of reactive oxygen species (ROS), were observed in The toxicity mechanisms of OTA are incompletely response to 0.25 μmol/l OTA. A particularly strong down- understood. Although it has been shown that OTA damages regulation due to 2.5 μmol/l OTA was evident for the mRNA the mitochondria (16), inhibits protein biosynthesis (17) and levels of insulin-like growth factor binding protein 1 (IGFBP1) causes oxidative stress (18, 19), little is known about the and tubulin beta 1 (TUBB1) which have been demonstrated to mode of action of OTA at the molecular level. In particular, function as a pro-survival factor in hepatocytes and as an the impact of OTA on hepatic gene expression has remained important cytoskeletal component, respectively. In addition, elusive. many genes involved in energy and xenobiotic metabolism, A recent microarray study in primary rat kidney cells and including phosphoglycerate kinase 1 (PGK1), stearoyl- a continuous kidney cell line has revealed an influence of Coenzyme A desaturase 1 (SCD), and glutathione S-transferase OTA on the transcription of genes involved in DNA repair, omega 1 (GSTO1), were down-regulated by OTA. Furthermore, antioxidative defence and inflammation (20). Additionally, OTA significantly inhibited the capacitative calcium entry into the study demonstrated that OTA modulates the same set of the HepG2 cells, indicating an alteration of calcium genes in vitro and in vivo . Moreover, recent data have homeostasis. Overall, OTA dose-dependently affects multiple suggested an impairment of the nuclear factor (erythroid- genes encoding for key proteins of liver cell metabolism. derived 2)-like 2 (Nrf2)-dependent gene expression (21, 22). Only a few hepatic genes have been reported to be affected by OTA, including genes involved in the detoxification of reactive oxygen species (ROS) ( e.g. Correspondence to: Gerald Rimbach, Institute of Human Nutrition superoxide dismutase) and in tumour development ( e.g. and Food Science, Christian Albrechts University, Hermann- Rodewald-Strasse 6, D-24118 Kiel, Germany. Tel: +49 431880 connexin) (23). Since the molecular targets of OTA are 2583, Fax: +49 4318802628, e-mail: [email protected] largely unknown, a large-scale gene expression profile study was performed to study the impact of OTA on the liver Key Words: Ochratoxin A, gene expression, liver, HepG2. transcriptome using gene chip technology. HepG2 cells were 1109 -6535 /2008 $2.00+.40 319 CANCER GENOMICS & PROTEOMICS 5: 319-332 (2008) for glutathione- S-transferase omega 1 (GSTO1), phosphoglycerate kinase 1 (PGK1), stearoyl- coenzyme A desaturase 1 (SCD), tubulin beta 1 (TUBB1), insulin-like growth factor-binding protein 1 (IGFBP1) and the uncoupling protein 2 (UCP2) with standard software tools such as Spidey, Primer3, NCBI/BLAST (National Center for Biotechnology Information/Basic Local Alignment Search Tool) and synthesized by MWG Biotech AG (Ebersberg, Germany). Primer sequence information is given in Table I. Real-time PCR was performed as a one-step procedure with a QuantiTect SybrGreen Kit (Qiagen) on a Rotorgene RG-3000 thermo cycler (Corbett Life Science, Sidney, Australia). Gene Figure 1. Chemical structure of ochratoxin A. expression was normalized to the housekeeping gene 18SRNA (small ribosomal subunit, 18 Svedberg units) (Qiagen) and the results were calculated as a percent age of the control cells. Microarray data analysis. The raw data files (Cel files) were used since they are of human origin, well characterized, processed and normalized by dChip software (29). The gene widely used for toxicity studies (24), and sensitive towards expression values (arbitrary units, a.u.) were calculated with the OTA treatment (25, 26). Furthermore, HepG2 cells are an perfect match (PM) / mismatch (MM) difference model. In the excellent model for investigating mitochondrial toxicity following “compare samples” analysis the two control arrays were firstly compared with the two “low-dose” and secondly with the “high- because of their high content of organelles and mitochondrial dose” arrays. For each gene the ratio of the mean expression values of DNA (27). However, a limitation of the HepG2 model is that the control and the experiment were calculated, resulting in the so- these cells do not express some phase II enzymes to the same called fold change (FC) value. From the list of genes obtained, genes extent as primary hepatocytes (28). with detectable (“present”) expression values were discriminated from undetectable (“absent”) ones . Genes were selected on the basis of the Materials and Methods following criteria: “present” signal on all arrays; “present” signal on both control arrays and “absent” signal on both experiment al arrays; Cell culture, RNA isolation and microarray production. HepG2 cells “absent” signal on both control arrays and “present” signal on both (DSMZ GmbH, Braunschweig, Germany) were seeded at a density experiment al arrays. Furthermore, those genes that showed a fold of 2.5×10 5 cells/ml in six-well plates (3 ml/well) and cultivated change ≥+2.0, or ≤ –2.0 with p<0.05 were selected. In order to exclude under standard conditions (RPMI-1640 medium with 10% fetal calf noise signals, the genes with a mean expression level lower than 27 a.u., representing 5% of the mean control expression level, were not serum, 37˚C, 5% CO 2). At confluence, two wells were treated identically with 0, 0.25 or 2.5 μmol/l OTA (Sigma, Deisenhofen, considered. Finally, genes were selected on the basis of reproducibility Germany) for 24 h. After washing twice with phosphate-buffered by considering only those genes that revealed similar expression values saline (PBS), the cells of the identically treated wells were pooled within the two control or the two experiment al arrays (coefficient of and the total RNA was extracted by use of a RNAeasy mini kit variation of expression value ≤25%). Classification analysis of (Qiagen, Hilden, Germany). The treatment was repeated once in an selected genes was performed with DAVID (Database for Annotation, independent experiment, resulting in a total of six RNA samples, Visualization and Integrated Discovery) software. two for each experimental condition (0, 0.25 and 2.5 μmol/l OTA) (n=2). Agarose gel electrophoresis was performed to check the Western blotting. HepG2 cells were incubated with 0, 0.25 or 2.5 μmol/l RNA quality. The purity and concentration of RNA were determined OTA for 48 h, harvested in ice-cold PBS and centrifuged. The remaining spectrophotometrically at 260 nm (Beckman Coulter, Fullerton, cell pellets were resuspended in RIPA (radio immunoprecipitation Kalifornien, USA). The six RNA samples were further processed assay) buffer (25 mmol/l Tris/HCl pH 7.6, 150 mmol/l NaCl, 1% NP- for microarray hybridization as described in the “Affymetrix 40, 1% sodium deoxycholate, 0.1% SDS) with protease inhibitor GeneChip Expression Analysis Technical Manual” by a regional (Protease Inhibitor Cocktail; Sigma) and incubated on ice for 30 min. German Affymetrix Service Provider, KFB - Center of Excellence After centrifugation, the supernatants were removed and stored at –80˚C for Fluorescent Bioanalytics (Regensburg, Germany), resulting in until further analysis. The