Ochratoxin A: 13-Week Oral Toxicity and Cell ... - Oxford Journals

TOXICOLOGICAL SCIENCES 97(2), 288–298 (2007) doi:10.1093/toxsci/kfm042 Advance Access publication March 6, 2007

Ochratoxin A: 13-Week Oral Toxicity and Cell Proliferation in Male F344/N Rats ¨ zden,* Eva Rached,* Gordon C. Hard,† Kai Blumbach,‡ Klaus Weber,‡ Regina Draheim,‡ Werner K. Lutz,* Sibel O Ulrich Steger,§ Wolfgang Dekant,* and Angela Mally*,1 *Department of Toxicology, University of Wu¨rzburg, 97078 Wu¨rzburg, Germany; †Private Consultant, Tairua 2853, New Zealand; ‡RCC Ltd, CH-4414 Fu¨llinsdorf/CH-4452 Itingen, Switzerland; and §Department of Surgery, University of Wu¨rzburg, 97078 Wu¨rzburg, Germany Received February 5, 2007; accepted March 2, 2007

Ochratoxin A (OTA) is nephrotoxic and a potent renal carcinogen. Male rats are most susceptible to OTA toxicity, and chronic administration of OTA (70 and 210 mg/kg bw) for 2 years has been shown to induce high incidences of adenomas and carcinomas arising from the straight segment of the proximal tubule epithelium. In contrast, treatment with a lower dose of 21 mg/kg bw did not result in increased tumor rates, suggesting a nonlinear dose response for renal tumor formation by OTA. Since the mechanism of OTA carcinogenicity is still largely unknown, this study was conducted to investigate early functional and pathological effects of OTA and to determine if sustained stimulation of renal cell proliferation plays a role. Male F344/N rats were treated with OTA for up to 13 weeks under conditions of the National Toxicology Program (NTP) bioassay. Cell proliferation in the renal cortex and outer stripe of the outer medulla (OSOM) was determined using bromodeoxyuridine incorporation and immunohistochemistry. Histopathological examination showed renal alterations in mid- and high-dose–treated animals involving single-cell death and prominent nuclear enlargement within the straight proximal tubules. Treatment with OTA at doses of 70 and 210 mg/kg bw led to a marked dose- and time-dependent increase in renal cell proliferation, extending from the medullary rays into the OSOM. No effects were evident in kidneys of low-dose–treated animals or in the liver, which is not a target for OTA carcinogenicity. A no observed effect level in this study was established at 21 mg/kg bw, correlating with the dose in the NTP 2-year bioassay that did not produce renal tumors. The apparent correlation between enhanced cell turnover and tumor formation induced by OTA indicates that stimulation of cell proliferation may play an important role in OTA carcinogenicity and provides further evidence for an epigenetic, thresholded mechanism. Key Words: ochratoxin A; kidney; carcinogenicity; cell proliferation; karyomegaly.

Ochratoxin A (OTA) (N-{[(3R)-5-chloro-8-hydroxy3-methyl-1-oxo-7-isochromanyl]-carbonyl}-3-phenyl-L-alanine) is an ubiquitous mycotoxin and natural food contaminant, which 1 To whom correspondence should be addressed at Department of Toxicology, University of Wu¨rzburg, Versbacher Strasse 9, 97078 Wu¨rzburg, Germany. Fax: þ49-931-20148865. E-mail: [email protected].

occurs in a variety of food items including cereals, wine, beer, grapes, and coffee. Due to its presence in food, humans are chronically exposed to OTA with mean daily intakes estimated to range between 2 and 3 ng/kg bw/day for the average adult consumer and 6 and 8 ng/kg bw/week for high consumers within the European Union member states (EFSA, 2006; SCOOP, 2002). OTA is nephrotoxic in all animal species tested and has in the past been suspected of being involved in the pathogenesis of endemic nephropathy, a chronic tubulointerstitial kidney disease which occurs in geographically limited areas of the Balkan region and is associated with an increased risk for the development of urothelial cancers. In rodents, OTA is also a potent renal carcinogen, with male rats being most susceptible to renal tumor formation by OTA. In a 2-year carcinogenicity bioassay, relatively low doses of OTA (70 and 210 lg/kg bw) were shown to induce high incidences of renal adenomas and carcinomas in male F344 rats (NTP, 1989). In contrast, treatment with a low dose of 21 lg/kg bw did not result in increased tumor rates. Tumors in rats develop from the straight segment of the proximal tubule (P3) in the outer stripe of the outer medulla (OSOM), the target site of OTA nephrotoxicity, and are characterized by an unusual, aggressive phenotype and high potential to metastasize (Boorman et al., 1992; NTP, 1989). Although controversial results regarding mode of action have been published, increasing evidence suggests that OTA is not a mutagenic, DNA-reactive carcinogen. Considering the lack of evidence for the existence of specific OTA-DNA adducts, the nonlinear dose response for tumor formation in the rat and the conclusion that nephrotoxicity appears to be the most sensitive end point for OTA toxicity, the European Food Safety Authority (EFSA) Scientific Panel on Contaminants in the Food Chain applied a threshold approach for OTA risk assessment and established a tolerable weekly intake of 120 ng/kg bw, which corresponds to a safety factor of 1300 applied to the no observed effect level (NOEL) for OTA carcinogenicity in male rats (EFSA, 2006). However, the molecular events leading to nephrotoxicity and renal tumor formation by OTA are still poorly understood. Over the last 30 years, several epigenetic mechanisms potentially involved in OTA toxicity and carcinogenicity have been proposed, including inhibition of protein synthesis,

The Author 2007. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: [email protected]

OTA TOXICITY AND RENAL CELL PROLIFERATION

mitochondrial toxicity, modulation of cell signaling, and oxidative stress (Schilter et al., 2005). Recently, OTA was found to alter expression of a battery of genes regulated by the transcription factor NF-E2 p45–related factor 2 (Nrf2), known to be involved in detoxification and antioxidant defense (Marin-Kuan et al., 2006). Disruption of Nrf2 activity and subsequent reduction of Nrf2-dependent gene expression was evident in kidneys, but not livers, of treated animals, suggesting that reduction of antioxidant defense may be involved in OTA nephrocarcinogenicity (Cavin et al., 2007). DNA strand breaks suggestive of oxidative DNA damage were also detected in rat kidney in response to OTA treatment using the comet assay, while other markers of oxidative stress, such as protein carbonyls and lipid peroxidation end products, were not elevated (Kamp et al., 2005; Mally et al., 2005a,b). However, in contrast to disruption of the Nrf2 pathway, oxidative DNA damage was also consistently detected in liver, which is not a target for OTA carcinogenicity (Kamp et al., 2005; Mally et al., 2005a). Considering the apparent lack of target organ specificity, the contribution of oxidative stress to renal tumor formation by OTA remains unclear. For many carcinogens that act via an indirect, epigenetic mechanism, a close correlation between stimulation of cell division and tumorigenesis is well established (Alden, 2000; Klaunig et al., 2000; Lutz et al., 1997). In the kidney, cytotoxicity to proximal tubule cells may trigger regenerative cell proliferation, which may cause tumor induction. A major role for cytotoxicity and compensatory cell proliferation has been demonstrated for several renal carcinogens which bind to a2lglobulin (Dietrich and Swenberg, 1991b; Dill et al., 2003; Prescott-Mathews et al., 1997). However, Rasonyi et al. (1999) demonstrated that accumulation of the male rat–specific urinary protein a2l-globulin and associated stimulation of regenerative cell proliferation does not occur in response to OTA treatment (1 mg/kg bw for seven consecutive days) and thus does not appear to be responsible for the higher susceptibility of male rats to OTAinduced tumor formation. In contrast, treatment with 0.5–2 mg OTA/kg bw for 2 weeks resulted in a small but significant and dose-dependent increase in the expression of proliferating cell nuclear antigen (PCNA) in the kidney, but not in the liver, indicating that OTA may enhance renal cell turnover (Mally et al., 2005b). However, these short-term studies were conducted at relatively high dose levels, and a detailed analysis of early changes induced by OTA under conditions of the National Toxicology Program (NTP) carcinogenicity bioassay has not been performed. Therefore, this study was conducted to investigate the functional and pathological effects of OTA in male rats, which are most susceptible to OTA toxicity, and to determine if sustained stimulation of renal cell proliferation might play a role in the mechanism of OTA carcinogenicity. MATERIALS AND METHODS Chemicals. OTA (CAS No. 303-47-9; 99% purity) was purchased from Axxora, Gru¨nberg, Germany (batch no. L16528/a). Corn oil and 5-bromo-2#-

289

deoxyuridine (BrdU) were from Sigma, Taufkirchen, Germany. All other chemicals were from Roth, Karlsruhe, Germany. Animals. Male F344/N rats (6–7 weeks old) were purchased from HarlanWinkelmann, Borchen, Germany. Animals were housed in groups of five in Macrolon cages and allowed free access to pelleted standard rat maintenance diet (SSNIFF, Soest, Germany) and tap water. Room temperature was maintained at 22 ± 2C with a relative humidity of 55 ± 10% and a day/night cycle of 12 h. Food and water consumption were determined weekly. Body weight measurements were conducted twice weekly. All animals were observed twice daily for clinical signs of toxicity. Dose formulation. OTA dosing solutions were prepared weekly or every 2 weeks (see NTP, 1989). OTA was weighed and dissolved in an appropriate volume of corn oil by sonication. The homogeneity of OTA in the corn oil solution was checked with an ultraviolet lamp prior to dosing and dilution with more corn oil for the lower doses. Study design. Following a week of acclimatization, rats (five per group) were administered OTA dissolved in corn oil at doses of 0, 21, 70, or 210 lg/kg bw by gavage for 14, 28, or 90 days, 5 days/week. For cell proliferation studies, osmotic minipumps (ALZET, 2ML1, flow rate 9.8 ll/h; Charles River Laboratories, Sulzfeld, Germany) containing 2 ml of a sterile solution of BrdU (15 mg/ml in phosphate-buffered saline, pH 7.4) were sc implanted into animals under ketamine (90 mg/kg bw, ip)/xylazine (10 mg/kg bw ip) anesthesia 1 week prior to necropsy. The animals were housed separately after the insertion of the pumps. For urine collection, animals were transferred into metabolic cages 48 h prior to necropsy. Animals were fasted for 20 h but allowed free access to drinking water. An aliquot of urine was immediately used for clinical chemistry analyses. The remaining urine was aliquoted and stored at 20C until further analysis. After urine collection, blood samples were drawn from the retro-orbital plexus under light isoflurane anesthesia. Samples for hematology were collected into EDTA tubes, and clinical chemistry samples were collected into heparinized tubes. Both urine and blood samples were transferred on ice to RCC (Fu¨llinsdorf, Switzerland) for clinical biochemistry, urine analysis, and hematology. Animals were killed by CO2 asphyxiation, and blood was collected by cardiac puncture into heparinized tubes for determination of OTA concentrations. Blood samples were centrifuged, and plasma was stored at 20C. Organs (liver, kidney, spleen, and bladder) were removed and weighed. Kidneys were cut longitudinally. One half of the right kidney, an aliquot of the liver, and all other organs were fixed in 10% neutral buffered formalin for histological evaluation. The remaining parts of kidneys and livers were aliquoted, flash frozen in liquid nitrogen, and stored at 80C. Determination of OTA in tissues and plasma. Aliquots of frozen tissue (200–400 mg) were homogenized in four volumes of ice-cold 50mM sodium phosphate buffer, pH 6.5. Proteins were precipitated by addition of an equal volume of ice-cold ethanol and centrifugation at 15,800 3 g at 4C for 30 min. Plasma samples were mixed with an equal volume of ice-cold ethanol and centrifuged at 15,800 3 g at 4C for 30 min to precipitate proteins. The resulting supernatant was either diluted with H2O or injected directly into the liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) system. Determination of OTA by LC/MS/MS was performed as previously described (Mally et al., 2005b; Zepnik et al., 2003). Urine analysis and clinical chemistry. Standard parameters in plasma were determined at RCC on a Hitachi 917 analyzer (Roche Diagnostics, Rotkreuz, Switzerland) using standard protocols for the determination of these parameters according to the manufacturer’s instructions. Urine analysis was performed using a Miditron semiautomated urine chemistry analyzer and reagent test strips (Roche Diagnostics). Specific gravity and osmolality of urine samples were determined using a Clinical Refractometer SU-202 (Kernco Instruments, El Paso, TX) and an Osmometer Fiske Type 2400 (IG Instrumenten-Gesellschaft, Zurich, Switzerland). Hematology. Hematological analyses were performed at RCC using an ADVIA 120 Hematology System (Bayer Healthcare, Zurich, Switzerland) and

290

RACHED ET AL.

standard protocols for the determination of these parameters according to the manufacturer’s instructions. Histopathology. One half of the right kidney and a sample of the left liver lobe were fixed in 10% neutral buffered formalin and subsequently embedded in paraffin. Sections were stained with hematoxylin and eosin for histopathological assessment by two independent pathologists. Cell proliferation. Cell proliferation in livers and kidneys was assessed using the BrdU technique, which determines the rate of DNA synthesis. BrdUpositive cells in kidney and liver were visualized on formalin-fixed paraffinembedded sections by using the BrdU in situ detection kit (BD Biosciences, Heidelberg, Germany) according to the manufacturer’s instructions. Cell proliferation was determined by light microscopic counts of proximal tubule cells which acquired brown nuclear staining per unit area of renal cortex and OSOM. On each section, 10 randomly selected fields of view at 3200 magnification were counted. For renal cortex, fields were chosen to avoid medullary rays. In the liver, BrdU-positive cells were determined in 20 fields of view at 3200 magnification. Statistical analysis. Data are expressed as mean ± SD of five individual animals. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s test or Kruskal-Wallis followed by the Steel test. A p value < 0.05 was considered statistically significant.

RESULTS

Concentration of OTA in Plasma and Tissues Consistent with previous reports, low concentrations of OTA (0.036 ± 0.026 lM; 0.026 ± 0.013lM; 0.023 ± 0.011 lM after 14, 28, and 90 days, respectively) were detected in plasma but not in tissues of control animals. Treatment with OTA resulted in a time- and dose-dependent increase in OTA concentrations in plasma, kidney, and liver (Fig. 1). After 90 days, OTA plasma concentrations in the mid- and low-dose–treated group appeared to reach a steady-state level of ~0.64lM for the 21 lg/kg bw group and 2.34 lM for the 70 lg/kg bw group. In contrast, OTA plasma concentrations continued to increase in the high-dose–treated group, reaching 7.42 lM after 90 days. Consistent with previous findings (Mally et al., 2005b), similar OTA concentrations were detected in liver and kidney 24 h after the final dose of OTA at all time points. Tissue concentrations appeared to reach a steady state in low-dose–treated animals but continued to rise in the mid- and high-dose–treated groups until the end of the study. Body and Organ Weight No clinical signs of toxicity were observed throughout the study. OTA treatment had no effect on food and water consumption, and no differences in body weight gain and relative organ weight of liver and spleen were evident in OTAtreated animals compared to controls (Table 1). In contrast, kidney weight was significantly reduced in a time- and dosedependent manner (Table 1). Clinical Chemistry and Urine Analysis No signs of nephrotoxicity were evident by serum clinical chemistry and urine analysis (Table 2 and supplementary data)

FIG. 1. Plasma and tissue concentrations of OTA after repeated administration of OTA at doses of 0, 21, 70, and 210 lg/kg bw for 14, 28, and 90 days. Data are presented as mean ± SD (n ¼ 5 animals per dose group).

except for a small but statistically significant increase in lysosomal N-acetyl-b-D-glucosaminidase activity in urine and increased serum creatinine in high-dose–treated animals, indicating mild impairment of kidney function after continuous treatment with high doses of OTA. In contrast to most nephrotoxic agents which target the proximal tubule, urinary activity of the brush-border enzyme c-glutamyl transferase (GGT) was reduced after treatment with 70 or 210 lg/kg OTA. In addition, a dose-dependent increase in urinary erythrocyte and leucocyte number, possibly indicative of damage to the kidneys or urinary tract, was observed in mid- and high-dose– treated animals. Interestingly, treatment with 70 or 210 lg/kg OTA for 13 weeks resulted in a small but significant increase in plasma calcium, which was also associated with a decrease in urinary calcium concentrations. Consistent with previous studies (Mally et al., 2005b; NTP, 1989), administration of

291


TABLE 1 Body and Relative Organ Weight after Repeated Administration of 0, 21, 70, or 210 lg/kg bw OTA for 14, 28, or 90 days. Data Are Presented as Mean ± SD of Five Individual Animals per Dose Group. Statistical Analysis Was Performed by ANOVA and Dunnett’s Test. Statistically Significant Changes Are Indicated by *p < 0.05 and **p < 0.01 OTA (lg/kg bw) Interval (days) Body weight (g) Initial Final Change (g) Final weight (%) Relative body weight increase (%) Relative organ weight (% of body weight) Left kidney

0 90

0

217.4 331.4 114.0 100.0 152.4

± ± ± ± ±

21

10.6 25.7 19.1 7.8 7.9

219.6 347.0 127.4 104.7 158.2

± ± ± ± ±

70

9.8 11.4 9.2 3.4 5.6

219.0 342.4 123.4 103.3 156.3

± ± ± ± ±

10.1 24.2 16.2 7.3 6.1

210

215.0 336.8 121.8 101.6 156.7

± ± ± ± ±

8.9 12.8 6.1 3.9 2.8

14 28 90

0.35 ± 0.00 0.34 ± 0.00 0.30 ± 0.00

0.34 ± 0.02 0.33 ± 0.01 0.28 ± 0.01

0.34 ± 0.01 0.32 ± 0.01 0.26 ± 0.01**

0.35 ± 0.02 0.31 ± 0.01** 0.23 ± 0.01**

Right kidney

14 28 90

0.33 ± 0.00 0.32 ± 0.00 0.29 ± 0.00

0.31 ± 0.03 0.32 ± 0.01 0.27 ± 0.01

0.35 ± 0.01 0.31 ± 0.03 0.26 ± 0.02*

0.33 ± 0.02 0.29 ± 0.01 0.22 ± 0.01**

Liver

14 28 90

4.72 ± 0.20 4.06 ± 0.20 2.94 ± 0.20

4.12 ± 0.65 4.03 ± 0.23 3.05 ± 0.08

4.49 ± 0.16 3.86 ± 0.18 3.04 ± 0.15

4.50 ± 0.24 3.80 ± 0.19 2.74 ± 0.09

Spleen

14 28 90

0.23 ± 0.00 0.21 ± 0.00 0.19 ± 0.00

0.23 ± 0.02 0.21 ± 0.00 0.18 ± 0.01

0.22 ± 0.01 0.21 ± 0.01 0.19 ± 0.01

0.23 ± 0.01 0.21 ± 0.01 0.18 ± 0.01

OTA did not result in increased activities of serum toxicity marker enzymes indicative of liver damage. However, 28-day treatment with OTA led to a slight decrease in alkaline phosphatase activity which persisted until the end of the study. Inconsistent changes were observed with some of the other parameters measured, such as serum potassium or triglycerides, but these were not considered to be compound related due to the lack of dose response. (Detailed clinical chemistry data are provided as supplementary data.) Hematology Only minor differences in blood counts of OTA-treated animals versus controls were observed. Rats treated with 70 or 210 lg/kg OTA showed decreased numbers of reticulocytes after 4 weeks, but this effect was not detected after 90 days. In contrast, leucocytes were increased in a dose-related manner after 4 weeks. This effect was also evident after 13 weeks, albeit to a lesser extent. Differential leukocyte counts suggested a dose-dependent decrease in neutrophils and eosinophils along with a concomitant increase in lymphocytes and large unstained cells that could not be identified by standard hematological analyses. (Data are provided as supplementary data.) Histopathology OTA produced renal alterations involving single-cell death of lining cells, initially in the proximal part of the straight

proximal tubules (S3) in the OSOM and medullary rays, and prominent nuclear enlargement, presumably through inhibition of cytokinesis (Fig. 2). There were signs of regeneration of tubular epithelium in the form of tubular basophilia at locations where apoptosis/degeneration was recorded. Despite cell proliferation occurring to replace cell loss in the affected tubules, simple tubule hyperplasia did not appear to be a component of the response. The lesions progressed with increasing dose and with time. Kidneys of rats treated with 70 lg/kg bw for 2 weeks were generally indistinguishable from kidneys of controls. However, minimal to faint basophilia involving S3 tubules was evident in the cortical medullary rays and outer part of the OSOM after 28 and 90 days. Affected tubules showed minimal (28 days) to moderate (90 days) lining cell degeneration in the form of condensed cells with dense eosinophilic cytoplasm and contracted nuclei, appearing to be detached into the tubule lumen. There was also minimal (28 days) to mild (90 days) nuclear variability, implying a slight increase in nuclear size scattered through affected tubules and an increase in mitotic figures. The same histopathological changes were evident as early as 2 weeks after treatment with 210 lg OTA/kg bw. After 28 and 90 days, irregular areas of faint tubule basophilia in the outer OSOM increased in size and extended into the deeper OSOM and medullary rays. Enlarged nuclei and nuclei categorized as karyomegaly, possibly representing octaploid (or greater) cells, were scattered through the affected tubules.

292

RACHED ET AL.

TABLE 2 Urine Analysis and Serum Clinical Chemistry after Repeated Administration of 0, 21, 70, or 210 lg/kg bw OTA for 14, 28, or 90 Days. Data Are Presented as Mean ± SD of Five Individual Animals per Dose Group. Statistical Analyses Were Performed Using ANOVA and Dunnett’s Test or Kruskal-Wallis and Steel Test. Statistically Significant Changes Are Indicated by *p < 0.05 and **p < 0.01 for ANOVA þ Dunnett’s and þp < 0.05 for Kruskal-Wallis and Steel Test. Additional Clinical Chemistry Data Are Provided as Supplementary Data OTA (lg/kg bw) Interval (days) Urine analysis Volume (ml)

Relative density (rel. 1)

Osmolality (mosmol/kg)

pH

Creatinine (mg/20 h)

Protein (g/l)

b-NAG (mU/mg creatinine)

GGT (U/mg creatinine)

Ketones (mM)

Erythrocytes (per ll)

Leucocytes (per ll)

RBC score 0/3 WBC score 0/3 Sodium (mM)

Potassium (mM)

Calcium (mM)

Phosphorus (mM)

Serum clinical chemistry Urea (mM)

14 28 90 14 28 90 14 28 90 14 28 90 14 28 90 14 28 90 14 28 90 14 28 90 14 28 90 14 28 90 14 28 90 90 90 14 28 90 14 28 90 14 28 90 14 28 90 14 28 90

0

13.5 9.1 8.8 1.03 1.03 1.03 430 746 655 7.2 6.6 6.8 5.83 6.19 7.94 0.10 0.20 0.20 11.4 12.2 8.2 2.22

21

70

210

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

6.0 4.0 4.0 0.02 0.01 0.01 168 284 248 0.8 0.4 0.3 1.03 0.70 0.86 0.14 0.11 0.11 2.2 1.4 1.2 0.59

16.0 11.1 5.2 1.02 1.02 1.04 403 609 1014 7.7 6.8 6.6 6.00 5.78 6.83 0.05 0.25 0.35 10.2 10.9 6.5 1.64

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

10.8 5.3 3.5 0.01 0.01 0.01 356 276 370 0.7 0.3 0.4 1.37 1.67 1.90 0.11 0.00 0.22 3.0 1.5 1.0 0.28

15.4 6.2 8.2 1.02 1.03 1.03 377 951 744 7.3 6.5 6.9 6.08 5.63 8.27 0.15 0.25 0.25 11.6 11.5 8.1 2.15

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

6.4 1.4 4.4 0.01 0.00 0.01 109 97 157 0.7 0.4 0.2 0.79 1.13 1.48 0.14 0.00 0.00 3.3 0.7 0.7 0.86

13.6 5.8 9.9 1.02 1.03 1.02 479 906 564 7.4 7.2 6.7 5.84 3.93 8.57 0.20 0.25 0.25 15.3 20.8 10.6 2.56

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.98 ± 0.1 ± 0.6 ± 0.3 ± 4± 13 ± 11 ± 10 ± 20 ± 20 ± 0 0 17.3 ± 40.8 ± 28.5 ± 65.5 ± 75.7 ± 69.9 ± 1.12 ± 1.31 ± 1.23 ± 10.63 ± 15.15 ± 13.72 ±

0.36 0.2 0.6 0.3 6 7 9 14 11 11

1.76 0.1 0.5 0.5 2 8 21 5 10 25

1.16 0.1 1.0 0.3 2 21 40 5 38 85

0.48 0.1 0.9 0.0 25 50 45 20 85 70

19.8 35.6 47.1 56.5 77.1 123.4 1.61 0.81 2.64 6.71 11.01 22.00

± ± ± ± ± ± ± ± ± ± 0 1 ± ± ± ± ± ± ± ± ± ± ± ±

0.09** 0.2 0.6 0.3 5 8 14 11 43 34

4.8 13.7 14.1 33.2 19.4 30.8 1.06 0.53 0.51 11.12 10.55 2.89

± 0.13 ± 0.2 ± 0.6 ± 0.6 ±5 ±5 ± 18 ± 11 ± 14 ±0 1 0 ± 17.5 ± 13.8 ± 25.5 ± 68.5 ± 31.7 ± 47.0 ± 1.19 ± 0.74 ± 1.88 ± 14.13 ± 9.22 ± 21.79

4.3 22.9 13.7 13.4 38.8 20.6 2.28 0.23 0.38 520.77 4.71 6.27

18.8 70.3 25.7 59.5 78.7 57.9 2.56 0.85 0.63 7.72 16.32 19.69

± 0.04** ± 0.2 ± 0.6 ± 0.0 ± 0þ ± 0þ ± 11þ ± 11 ± 34 ± 41 0 1þ ± 3.4 ± 17.2 ± 7.11 ± 15.9 ± 46.5 ± 10.9 ± 1.87 ± 0.22 ± 0.17 ± 12.74 ± 9.88 ± 6.22

4.84 ± 0.29 7.32 ± 0.49 6.25 ± 1.10

4.68 ± 0.67 5.83 ± 0.90** 5.79 ± 0.97

17.2 58.7 35.1 53.0 84.3 83.6 2.62 0.86 1.43 238.68 28.27 13.49

5.11 ± 0.24 7.69 ± 0.88 6.06 ± 0.49

3.3 1.5 1.7 0.01 0.01 0.00 192 135 74 0.9 1.0 0.3 0.52 1.27 0.65 0.11 0.00 0.00 1.6 2.0** 1.3** 0.69

5.12 ± 0.24 6.67 ± 0.24 5.38 ± 1.01

293


TABLE 2—Continued OTA (lg/kg bw) Interval (days) Creatinine (mg/l)

Calcium (mM)

14 28 90 14 28 90

0 2.43 2.83 3.29 2.43 2.44 2.58

± ± ± ± ± ±

21 0.13 0.20 0.30 0.06 0.06 0.06

2.56 2.95 3.35 2.40 2.42 2.52

± ± ± ± ± ±

70 0.11 0.15 0.34 0.07 0.05 0.08

4.63 2.59 3.61 2.48 2.37 2.70

210 ± ± ± ± ± ±

5.03 0.14 0.09 0.08 0.02 0.05*

5.52 2.69 4.00 2.54 2.41 2.71

± ± ± ± ± ±

4.92 0.23 0.19** 0.03* 0.04 0.06*

Note. b-NAG, N-acetyl-b-D-glucosaminidase.

Degenerated cells remained present but were not as frequent after 90 days as at earlier time points. In contrast to the midand high-dose–treated group, kidneys of animals treated with 21 lg/kg bw were indistinguishable from kidneys of controls at all time points, establishing 21 lg/kg bw as a NOEL for nephrotoxicity in this study. There was no effect of OTA on rat livers, except for morphological variations reflecting changes in glycogen status. Proliferation Repeated administration of OTA at doses of 70 and 210 lg/kg bw resulted in a marked time- and dose-dependent, target organ–specific increase in cell proliferation in the kidney. In control and low-dose–treated animals, the pattern of dividing tubular epithelial cells in the proximal OSOM and cortex was random, and proliferation decreased over time as a consequence

of maturation. The pattern and frequency of staining in low-dose–treated animals (21 lg/kg bw) were not distinguishable from those of the kidneys of controls at the equivalent time points. In contrast, administration of 70 or 210 lg/kg OTA led to a definite change in the labeling pattern from controls, consisting of scattered individual or clusters of P3 tubules in the proximal OSOM and medullary rays with multiple nuclei labeled (Fig. 3). The size of these clusters increased over time. At the same time, a reduction of cortical staining (apart from the medullary rays) compared to kidneys of controls was observed. Quantification of BrdU-positive cells in the proximal OSOM demonstrated highly significant changes in cell proliferation after 4 and 13 weeks in response to 210 and 70 lg/kg bw OTA (Fig. 4). In contrast, a dose- and time-dependent decrease in cell proliferation was evident in the cortex (Fig. 4). No increase in cell proliferation was measured in the 21-lg/kg group or in the liver, which is not a target for OTA carcinogenicity (Fig. 5).

DISCUSSION

FIG. 2. Hemotoxylin and eosin–stained kidney sections of a control rat (a) and of rats treated with 21 (b), 70 (c), and 210 (d) lg/kg bw OTA for 90 days. At 70 or 210 lg/kg bw, OTA induced single-cell degeneration (thin arrow) and nuclear enlargement (thick arrow) in proximal tubular epithelial cells in the OSOM. No effects were seen in animals exposed to 21 lg/kg bw compared to controls. Magnification: 3200.

OTA is one of the most potent renal carcinogens studied to date (Lock and Hard, 2004) but the mechanism by which OTA induces tumors in rats still remains unsolved. To obtain a better understanding of the early structural and functional changes which occur in rat kidney under conditions of carcinogenesis, male F344 rats, which are most susceptible to renal tumor formation by OTA, were treated with OTA for up to 90 days using a dosing regimen identical to the NTP 2-year bioassay (NTP, 1989). In addition to histopathological analysis and routine measurement of markers of kidney injury, stimulation of cell proliferation as a potential epigenetic mechanism of tumorigenesis was determined in kidney and liver of OTAtreated animals. Early effects of OTA treatment observed in this study consisted of cell loss accompanied by increased cell proliferation and prominent nuclear enlargement within the straight segment of the proximal tubule epithelium (P3) in the OSOM, from which OTA-induced tumors arise (JECFA, 2001). In contrast, none of these effects were observed in the

294

RACHED ET AL.

FIG. 3. OTA-induced cell proliferation in kidneys of male rats treated with 0 (a, b), 21 (c, d), 70 (e, f), and 210 (g, h) lg/kg bw OTA for 90 days. BrdUpositive cells were visualized on formalin-fixed paraffin-embedded sections by using an anti-BrdU antibody. Clear changes in the frequency and pattern of BrdU labeling are evident in the OSOM of animals treated with 70 (e, f) or 210 (g, h) lg/kg bw OTA as compared to controls (a, b) and low-dose–treated animals (21 lg/kg bw) (c, d). Stimulation of cell proliferation was restricted to the OSOM and medullary rays. Magnification: 3100 (a, c, e, g); 3200 (b, d, f, h).

liver throughout the study. The marked increase in renal cell proliferation is consistent with previous results, which showed increased expression of PCNA in kidney but not liver of rats treated with up to 2 mg/kg bw OTA (Mally et al., 2005b). Although Rasonyi et al. (1999) did not observe changes in renal cell proliferation following treatment with OTA, it is possible that the shorter treatment period (7 days), albeit at higher doses as compared to the present study (1 mg/kg bw vs. 70 and 210 lg/kg bw), might have been insufficient to trigger a proliferative response.

FIG. 4. Quantification of BrdU-positive cells in renal OSOM and cortex of rats treated with 0, 21, 70, or 210 lg/kg bw OTA. Repeated administration of 70 or 210 lg/kg bw OTA resulted in a strong increase in BrdU-positive cells in the renal OSOM. In contrast, a dose-dependent decrease in cell proliferation was evident in the cortex after treatment for 90 days. Data are presented as mean ± SD (n ¼ 5 animals per group). Statistical analysis was performed by ANOVA þ Dunnett’s post hoc test. Statistically significant changes compared to controls are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

It is well established that cell proliferation is a critical step within the complex, multistage process of carcinogenesis, causing conversion of DNA damage into mutations and clonal expansion of initiated cells (Dietrich and Swenberg, 1991a; Lutz, 1990). Sustained induction of cell proliferation may also


FIG. 5. Representative images of BrdU immunostaining of control rat liver (a) and liver obtained from a rat exposed to 210 lg/kg bw OTA for 90 days (b). (c) Quantitative analysis of BrdU-positive cells in livers of rats treated with 0, 21, 70, or 210 lg/kg bw OTA for 90 days clearly shows that OTA does not affect cell proliferation in the liver. Data are presented as mean ± SD (n ¼ 5 animals per group). Statistical analysis was performed by ANOVA þ Dunnett’s post hoc test.

contribute to carcinogenesis through inhibition of DNA repair (Butterworth and Bogdanffy, 1999). Independent of their primary mechanism, stimulation of cell proliferation is a common feature of many nongenotoxic carcinogens (Alden, 2000; Klaunig et al., 2000). In principle, chemicals which induce tumor formation through modulation of cell proliferation can be divided into two subclasses, mitogens and cytotoxicants (Jones et al., 1996; Klaunig et al., 2000). In the kidney, many nongenotoxic carcinogens are thought to act via stimulation of compensatory cell proliferation to replace cell loss caused by (direct or a2l-globulin mediated) cytotoxicity (Lock and Hard, 2004). However, OTA does not cause a2l-globulin accumulation (Rasonyi et al., 1999), and although administration of OTA at doses of 70 and 210 lg/kg bw for up to 90 days resulted in a time- and dose-dependent decrease in relative kidney weight and single-cell degeneration within the straight segment of the proximal tubule, only minor changes in clinical chemistry parameters indicative of nephrotoxicity were observed. This was also evident by a decrease in urinary GGT activity in response to the 90-day OTA treatment, which is in apparent contrast to other nephrotoxins that target the proximal tubule and normally cause release of brush-border enzymes through damage to the tubule epithelium. Similar effects on GGT activity were previously reported in OTA-treated pigs (Krogh et al., 1988)

295

and may be due to loss of brush-border and reduced expression of GGT in response to OTA. Moreover, simple tubule hyperplasia, which frequently occurs in response to nephrotoxins that produce sustained cytotoxicity and compensatory regeneration, does not appear to be a feature of OTA-induced kidney pathology under conditions of carcinogenicity. In contrast, the frequency and early onset of karyomegaly appear to be unique to OTA even though sporadic nuclear enlargement is occasionally recorded in regions affected by tubule cell injury/regeneration following chronic treatment with nongenotoxic/cytotoxic renal carcinogens, such as chloroform and trichlorethylene (Lock and Reed, 2006; Mally et al., 2006b; Templin et al., 1996). Increased nuclear size is indicative of blocked nuclear division during mitosis and subsequent inhibition of cytokinesis. Disruption of cell division, leading to the production of polyploid nuclei instead of simple tubule hyperplasia, may provide a potential explanation for the apparent disparity between kidney pathology induced by OTA compared to other nephrotoxins, and one might even speculate that aberrant mitosis may represent the principle cause of cell degeneration and subsequent trigger for cell proliferation. Abnormal mitotic figures and formation of giant cells were detected both in vitro (Rached et al., 2006; Steyn et al., 1975) and in vivo (Boorman et al., 1992; Maaroufi et al., 1999; Mally et al., 2005b) after OTA treatment. Although the contribution of these aberrant cells to renal tumor formation by OTA remains to be determined, it is well established that polyploid cells are genetically unstable and constitute potential tumor precursors (Storchova and Pellman, 2004). Normally, these cells are held in a quiescent state and do not undergo mitosis (Storchova and Pellman, 2004). However, checkpoint override and uncontrolled cell division may occur in the presence of strong mitogenic stimuli, as might be the case following OTA treatment. This is supported by the observation that enlarged nuclei were labeled by BrdU, indicating multiple rounds of DNA synthesis in the absence of cytokinesis. Although the molecular events leading to disruption of mitosis in kidney tubule cells exposed to OTA have not been identified, the marked proliferative response to OTA and concomitant presence of mitotic abnormalities suggest that the mechanism of OTA carcinogenicity involves disruption of complex signaling pathways which control entry and progression of the cell cycle. There is some evidence to suggest that OTA may mediate some of its effects by altering intracellular Ca2þ homeostasis (Dopp et al., 1999; Hoehler et al., 1996; Khan et al., 1989; Mally et al., 2006a). In human kidney epithelial cells, OTA was shown to stimulate cell proliferation by interfering with intercellular Ca2þ (Benesic et al., 2000). Dopp et al. (1999) investigated alterations in Ca2þ homeostasis in response to OTA treatment and demonstrated that OTA induces short spikes in intracellular Ca2þ concentrations, resembling physiological responses. Although we are only beginning to

296

RACHED ET AL.

understand how spatiotemporal changes in intercellular Ca2þ signaling affect cell function, it is well established that intracellular Ca2þ signals play an important role in the regulation of cell growth and proliferation (Munaron et al., 2004; Santella et al., 2005). Thus, it is possible that disruption of calcium homeostasis may lead to disruption of cell cycle control and/or serve as a trigger for cell proliferation in rat kidney. Interestingly, treatment with 70 or 210 lg/kg bw OTA for 90 days resulted in a significant increase in plasma Ca2þ concentrations, together with a small, although not statistically significant decrease in urinary Ca2þ. Disruption of Ca2þ homeostasis in rat kidney by OTA is further supported by a recent microarray analysis, which demonstrates changes in the expression of genes involved in calcium signaling, including regucalcin (Luhe et al., 2003; Marin-Kuan et al., 2006). In these studies, OTA was also found to alter expression of genes associated with mitogenic growth factor signaling, such as insulin-like growth factor-1, insulin receptor, and GTPbinding protein. Thus, temporal changes in intracellular Ca2þ may occur as a result of growth factor receptor activation and transduction of extracellular signals, which involve Ca2þ as a second messenger, or direct interaction of OTA with Ca2þ and/or regulators of Ca2þ homeostasis. Irrespective of the primary mechanisms leading to altered tubule cell growth in response to OTA, the early effects on cell replication in the absence of significant changes in renal function indicate that stimulation of cell proliferation may be a critical event involved in renal tumor formation by OTA. Moreover, the fact that OTA triggers cell division specifically in the kidney, the target organ of OTA carcinogenicity, at the specific target site, the straight segment of the proximal tubules, suggests a close link between enhanced cell proliferation and OTA carcinogenicity. This is further supported by the very tight correlation between tumor incidence (NTP, 1989) and cell proliferation in the OSOM (Fig. 6). In this study, no significant differences between OTA concentrations in kidney (target) and liver (non-target) were evident at terminal sacrifice. Although we cannot exclude that higher peak concentrations may occur in kidney or that OTA may accumulate in specific cell types within the kidney from which tumors arise (i.e., the S3 segment), these findings are in line with previous results after repeated administration of OTA at doses up to 2 mg/kg bw (Mally et al., 2005b). Consistent with the similar degree of OTA accumulation in both organs, DNA breakage indicative of (oxidative) DNA damage has been detected in target (kidney) and non-target (liver) tissues of rats continuously exposed to OTA using the comet assay in the presence of formamidopyrimidine-DNA glycosylase, which is known to convert oxidative DNA base modifications, particularly 8-oxo-7,8-dihydro-2#-deoxyguanosine, into strand breaks (Mally et al., 2005b). The apparent lack of target organ specificity for oxidative DNA damage suggests that oxidative stress is not a major event in OTA carcinogenicity, although it is possible that renal tumor formation in rats may occur as a result

FIG. 6. Correlation between numbers of BrdU-positive cells in renal OSOM of male rats treated with OTA for 90 days and kidney tumor incidence after chronic administration of OTA for 2 years.

of (oxidative) DNA lesions combined with a sustained increase in cell proliferation, serving to convert DNA damage into permanent mutations. However, the significance of cell proliferation compared to oxidative stress is underscored by the fact that oxidative DNA damage, but not cell proliferation, was shown to occur in rats treated with extremely low doses of OTA, which do not cause tumor formation (Kamp et al., 2005). In conclusion, the close correlation between enhanced cell turnover and tumor formation induced by OTA provides further evidence for an epigenetic, thresholded mechanism of OTA carcinogenicity. The apparent threshold implies that there is a dose below which no adverse health effects are expected to occur. In this study, 21 lg/kg bw OTA was clearly established as a NOEL for both kidney pathology and renal cell proliferation. While OTA is known to accumulate in lipid-rich organs due to its lipophilicity and long plasma half-life, the lack of effects at this dose suggests that the critical tissue concentration for OTA toxicity is not reached under these conditions. Indeed, determination of OTA concentrations in kidney showed that repeated administration of 21 lg/kg bw OTA resulted in a steady state between uptake, distribution, and elimination, while OTA tissue concentrations continued to rise in the mid- and high-dose–treated groups. It is important to note that plasma concentrations in rats treated with 21 lg/kg bw for 90 days did not exceed 0.64lM, which is still a factor of ~200–1400 higher than mean OTA plasma concentrations in humans resulting from dietary exposure to OTA (0.18–1.19 ng/ml corresponding to 0.45–2.95nM) (SCOOP, 2002). Based on food consumption data and OTA serum concentrations, it appears that human exposure—even in areas with relatively high dietary exposure to OTA such as endemic villages—is several orders of magnitude below doses known to cause adverse effects in laboratory animals.


SUPPLEMENTARY DATA

Detailed clinical chemistry and hematology data of the 90-day oral toxicity study on ochratoxin A in male F344/N rats are available online at http://toxsci.oxfordjournals.org/.

297

Kamp, H. G., Eisenbrand, G., Janzowski, C., Kiossev, J., Latendresse, J. R., Schlatter, J., and 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. Khan, S., Martin, M., Bartsch, H., and Rahimtula, A. D. (1989). Perturbation of liver microsomal calcium homeostasis by ochratoxin A. Biochem. Pharmacol. 38, 67–72.

ACKNOWLEDGMENTS

Klaunig, J. E., Kamendulis, L. M., and Xu, Y. (2000). Epigenetic mechanisms of chemical carcinogenesis. Hum. Exp. Toxicol. 19, 543–555.

Parts of this work were supported by RCC Ltd, Itingen, Switzerland, and the Deutsche Forschungsgemeinschaft. The authors would also like to thank Susan Schnarwiler, Caroline Kro¨cher, Elisabeth Ru¨b-Spiegel, and Michaela Bekteshi for excellent technical assistance.

Krogh, P., Gyrd-Hansen, N., Hald, B., Larsen, S., Nielsen, J. P., Smith, M., Ivanoff, C., and Meisner, H. (1988). Renal enzyme activities in experimental ochratoxin A-induced porcine nephropathy: Diagnostic potential of phosphoenolpyruvate carboxykinase and gamma-glutamyl transpeptidase activity. J. Toxicol. Environ. Health 23, 1–14.

REFERENCES

Lock, E. A., and Hard, G. C. (2004). Chemically induced renal tubule tumors in the laboratory rat and mouse: Review of the NCI/NTP database and categorization of renal carcinogens based on mechanistic information. Crit. Rev. Toxicol. 34, 211–299.

Alden, C. L. (2000). Safety assessment for non-genotoxic rodent carcinogens: Curves, low-dose extrapolations, and mechanisms in carcinogenesis. Hum. Exp. Toxicol. 19, 557–560; discussion 571–552.

Lock, E. A., and Reed, C. J. (2006). Trichloroethylene: Mechanisms of renal toxicity and renal cancer and relevance to risk assessment. Toxicol. Sci. 91, 313–331.

Benesic, A., Mildenberger, S., and Gekle, M. (2000). Nephritogenic ochratoxin A interferes with hormonal signalling in immortalized human kidney epithelial cells. Pflugers Arch. 439, 278–287.

Luhe, A., Hildebrand, H., Bach, U., Dingermann, T., and Ahr, H. J. (2003). A new approach to studying ochratoxin A (OTA)-induced nephrotoxicity: Expression profiling in vivo and in vitro employing cDNA microarrays. Toxicol. Sci. 73, 315–328.

Boorman, G. A., McDonald, M. R., Imoto, S., and Persing, R. (1992). Renal lesions induced by ochratoxin A exposure in the F344 rat. Toxicol. Pathol. 20, 236–245. Butterworth, B. E., and Bogdanffy, M. S. (1999). A comprehensive approach for integration of toxicity and cancer risk assessments. Regul. Toxicol. Pharmacol. 29, 23–36.

Lutz, U., Lugli, S., Bitsch, A., Schlatter, J., and Lutz, W. K. (1997). Dose response for the stimulation of cell division by caffeic acid in forestomach and kidney of the male F344 rat. Fundam Appl Toxicol. 39, 131–137. Lutz, W. K. (1990). Dose-response relationship and low dose extrapolation in chemical carcinogenesis. Carcinogenesis 11, 1243–1247.

Cavin, C., Delatour, T., Marin-Kuan, M., Holzhauser, D., Higgins, L., Bezencon, C., Guignard, G., Junod, S., Piguet, D., Richoz-Payot, J., et al. (2007). Reduction in antioxidant defences may contribute to ochratoxin A toxicity and carcinogenicity. Toxicol. Sci. 96, 30–39.

Maaroufi, K., Zakhama, A., Baudrimont, I., Achour, A., Abid, S., Ellouz, F., Dhouib, S., Creppy, E. E., and Bacha, H. (1999). Karyomegaly of tubular cells as early stage marker of the nephrotoxicity induced by ochratoxin A in rats. Hum. Exp. Toxicol. 18, 410–415.

Dietrich, D. R., and Swenberg, J. A. (1991a). Preneoplastic lesions in rodent kidney induced spontaneously or by non-genotoxic agents: Predictive nature and comparison to lesions induced by genotoxic carcinogens. Mutat. Res. 248, 239–260.

Mally, A., Decker, M., Bekteshi, M., and Dekant, W. (2006a). Ochratoxin A alters cell adhesion and gap junction intercellular communication in MDCK cells. Toxicology 223, 15–25.

Dietrich, D. R., and Swenberg, J. A. (1991b). The presence of alpha 2u-globulin is necessary for d-limonene promotion of male rat kidney tumors. Cancer Res. 51, 3512–3521. Dill, J. A., Lee, K. M., Renne, R. A., Miller, R. A., Fuciarelli, A. F., Gideon, K. M., Chan, P. C., Burka, L. T., and Roycroft, J. H. (2003). Alpha 2u-globulin nephropathy and carcinogenicity following exposure to decalin (decahydronaphthalene) in F344/N rats. Toxicol. Sci. 72, 223–234. Dopp, E., Muller, J., Hahnel, C., and Schiffmann, D. (1999). Induction of genotoxic effects and modulation of the intracellular calcium level in syrian hamster embryo (SHE) fibroblasts caused by ochratoxin A. Food Chem. Toxicol. 37, 713–721. EFSA. (2006). Opinion of the Scientific Panel on Contaminants in the Food Chain on a request from the Commission related to ochratoxin A in food, EFSA-Q-2005-154. EFSA J. 365, 1–56. Hoehler, D., Marquardt, R. R., McIntosh, A. R., and Xiao, H. (1996). Free radical generation as induced by ochratoxin A and its analogs in bacteria (Bacillus brevis). J. Biol. Chem. 271, 27388–27394. JECFA. (2001). Safety Evaluation of Certain Mycotoxins in Food. Fifty-Sixth Meeting of the JOINT FAO/WHO Expert Committee on Food Additives (JECFA). WHO Food Additives Series No. 47, 281–415. WHO, Geneva. Jones, H. B., Eldridge, S. R., Butterworth, B. E., and Foster, J. R. (1996). Measures of cell replication in risk/safety assessment of xenobiotic-induced, nongenotoxic carcinogenesis. Regul. Toxicol. Pharmacol. 23, 117–127.

Mally, A., Pepe, G., Ravoori, S., Fiore, M., Gupta, R. C., Dekant, W., and Mosesso, P. (2005a). Ochratoxin A causes DNA damage and cytogenetic effects but no DNA adducts in rats. Chem. Res. Toxicol. 18, 1253–1261. Mally, A., Volkel, W., Amberg, A., Kurz, M., Wanek, P., Eder, E., Hard, G., and Dekant, W. (2005b). Functional, biochemical, and pathological effects of repeated oral administration of ochratoxin A to rats. Chem. Res. Toxicol. 18, 1242–1252. Mally, A., Walker, C. L., Everitt, J. I., Dekant, W., and Vamvakas, S. (2006b). Analysis of renal cell transformation following exposure to trichloroethene in vivo and its metabolite S-(dichlorovinyl)-l-cysteine in vitro. Toxicology. 224, 108–118. Marin-Kuan, M., Nestler, S., Verguet, C., Bezencon, 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 ochratoxin A carcinogenicity in rat. Toxicol. Sci. 89, 120–134. Munaron, L., Antoniotti, S., and Lovisolo, D. (2004). Intracellular calcium signals and control of cell proliferation: How many mechanisms? J. Cell. Mol. Med. 8, 161–168. NTP. (1989). Toxicology and carcinogenesis studies of ochratoxin A (CAS No. 303-47-9) in F344/N rats (gavage studies). Natl. Toxicol. Program Tech. Rep. Ser. 358, 1–142. Prescott-Mathews, J. S., Wolf, D. C., Wong, B. A., and Borghoff, S. J. (1997). Methyl tert-butyl ether causes alpha2u-globulin nephropathy and enhanced

298

RACHED ET AL.

renal cell proliferation in male Fischer-344 rats. Toxicol. Appl. Pharmacol. 143, 301–314. Rached, E., Pfeiffer, E., Dekant, W., and Mally, A. (2006). Ochratoxin A: Apoptosis and aberrant exit from mitosis due to perturbation of microtubule dynamics? Toxicol. Sci. 92, 78–86. Rasonyi, T., Schlatter, J., and Dietrich, D. R. (1999). The role of alpha2uglobulin in ochratoxin A induced renal toxicity and tumors in F344 rats. Toxicol. Lett. 104, 83–92. Santella, L., Ercolano, E., and Nusco, G. A. (2005). The cell cycle: A new entry in the field of Ca2þ signaling. Cell. Mol. Life Sci. 62, 2405–2413. Schilter, B., Marin-Kuan, M., Delatour, T., Nestler, S., Mantle, P., and Cavin, C. (2005). Ochratoxin A: Potential epigenetic mechanisms of toxicity and carcinogenicity. Food Addit. Contam. 22(Suppl. 1), 88–93. SCOOP. (2002). Reports on Tasks for Scientific Cooperation: Assessment of Dietary Intake of Ochratoxin A by the Population of EU Member

States. Directorate-General Health and Consumer Protection. Available at: http://ec.europa.eu/food/fs/scoop/3.2.7_en.pdf. Accessed March 16, 2007. Steyn, P. S., Vleggaar, R., Du Preez, N. P., Blyth, A. A., and Seegers, J. C. (1975). The in vitro toxicity of analogs of ochratoxin A in monkey kidney epithelial cells. Toxicol. Appl. Pharmacol. 32, 198–203. Storchova, Z., and Pellman, D. (2004). From polyploidy to aneuploidy, genome instability and cancer. Nat. Rev. Mol. Cell. Biol. 5, 45–54. Templin, M. V., Larson, J. L., Butterworth, B. E., Jamison, K. C., Leininger, J. R., Mery, S., Morgan, K. T., Wong, B. A., and Wolf, D. C. (1996). A 90-day chloroform inhalation study in F-344 rats: Profile of toxicity and relevance to cancer studies. Fundam. Appl. Toxicol. 32, 109–125. Zepnik, H., Volkel, W., and Dekant, W. (2003). Toxicokinetics of the mycotoxin ochratoxin A in F 344 rats after oral administration. Toxicol. Appl. Pharmacol. 192, 36–44.