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Chem. Res. Toxicol. 2005, 18, 1242-1252
Functional, Biochemical, and Pathological Effects of Repeated Oral Administration of Ochratoxin A to Rats Angela Mally,† Wolfgang Vo¨lkel,† Alexander Amberg,‡ Michael Kurz,§ Paul Wanek,† Erwin Eder,† Gordon Hard,| and Wolfgang Dekant*,† Department of Toxicology, University of Wu¨ rzburg, Germany, Aventis Pharma, Drug Safety Evaluation, Hattersheim, Germany, Aventis Pharma, Chemistry, Frankfurt, Germany, and NTP Archives, Research Triangle Park, North Carolina Received December 16, 2004
Ochratoxin A (OTA), a mycotoxin produced by several fungi of Aspergillus and Penicillium species, may contaminate agricultural products, resulting in chronic human exposure. In rats, OTA is a potent nephrotoxin, and repeated administration of OTA for 2 years to rats in doses up to 0.21 mg/kg of body wt resulted in high incidences of renal tumors arising from the proximal tubular epithelial cells. The mechanism of tumor formation by OTA in the kidney is not well-defined, and controversial results regarding mode of action have been published. The aim of this study was to characterize dose-dependent changes induced by OTA by application of clinical chemistry, biochemical markers, and toxicokinetics for a better conclusion on modes of action. Administration of OTA (0, 0.25, 0.5, 1, and 2 mg/kg of body wt) to male F344 rats (n ) 3 per group) by oral gavage for 2 weeks resulted in a dose-dependent increase in OTA plasma concentrations and concentrations of OTA in both liver and kidney. Although oxidative stress has been implicated in OTA carcinogenicity, treatment with OTA did not induce overt lipid peroxidation or an increase in 8-oxo-7,8-dihydro-2′deoxyguanosine (8-OH-dG) in kidney. In the kidney, OTA-induced pathology was present at all dose levels administered, with a clear increase in severity related to dose. Pathology was restricted to the outer stripe of the outer medulla and consisted of disorganization of the tubule arrangement, frequent apoptotic cells, and abnormally enlarged nuclei scattered through the S3 tubules. Consistent with the histopathology, a dose-dependent increase in the expression of proliferating cell nuclear antigen (PCNA), indicative of cell proliferation, was observed in kidneys, but not in livers of treated animals. The most prominent change in the composition of urine induced by OTA analyzed by 1H NMR and principal component analysis consisted of a major increase in the excretion of trimethylamine N-oxide. However, typical changes observed with other proximal tubular toxins such as increased excretion of glucose were not observed at any of the doses administered. Similarly, treatment with OTA had no clear effects on clinical chemical parameters indicative of nephrotoxicity, although urinary volume was increased at the higher-dose groups. Taken together, the uncommon changes induced by OTA suggest that a unique mechanism may be involved in OTA nephrotoxicity and carcinogenicity.
Introduction 1
Ochratoxin A (OTA), (N-{[(3R)-5-chloro-8-hydroxy-3methyl-1-oxo-7-isochromanyl]-carbonyl}-3-phenyl-L-alanine), a mycotoxin produced by several fungi of Aspergillus and Penicillium species (1-3) may contaminate agricultural products. Because of the high stability and lipophilicity of OTA, consumption of contaminated cereals and grains results in chronic human exposure. * Author for correspondence: Prof. Dr. Wolfgang Dekant, Department of Toxicology, University of Wu¨rzburg, Versbacher Str. 9, 97078 Wu¨rzburg, Germany. Tel: +49-931-20148449. Fax: +49-931-20148865. E-mail:
[email protected]. † University of Wu ¨ rzburg. ‡ Aventis Pharma, Drug Safety Evaluation. § Aventis Pharma, Chemistry. | NTP Archives. 1 Abbreviations: OTA, ochratoxin A; OTalpha, ochratoxin alpha; OSOM, outer stripe of the outer medulla; 8-OH-dG, 8-oxo-7,8-dihydro2′deoxyguanosine; 4-HNE, trans-4-hydroxy-2-nonenal; FeNTA, iron(III)nitrilotriacetic acid; PCNA, proliferating cell nuclear antigen; GGT, γ-glutamyltranspeptidase; ASAT, aspartate aminotransferase; ALAT, alanine aminotransferase; ALP, alkaline phosphatase.
In rats, OTA is a potent nephrotoxin, and repeated administration of OTA for 2 years to rats in low doses results in high incidences of renal tumors arising from the proximal tubular epithelial cells (4, 5). The mechanism of tumor formation by OTA in the kidney is not welldefined, and controversial results regarding the mode of action have been published. Some authors proposed that genotoxicity plays a major role in OTA-induced tumorigenesis and have postulated the formation of DNA adducts as indicated by spots observed by 32P-postlabeling (6, 7). However, a number of studies indicate that covalent binding of OTA to DNA and biotransformationdependent mutagenicity of OTA are not involved in renal tumor formation by OTA (8-14). The kidney is an organ where several nongenotoxic agents have been shown to induce tumors, and the physiological response of the kidney to toxic insults, regenerative cell proliferation, chronic inflammation, and oxidative stress has been implicated as a mode of action (15-17). Therefore, pathological and functional changes in the kidney in-
10.1021/tx049651p CCC: $30.25 © 2005 American Chemical Society Published on Web 07/08/2005
Effects of Oral Administration of OTA
duced by an agent may have important consequences for tumor induction. OTA is also nephrotoxic after short-term administration to rodents, and some functional and pathological consequences of OTA administration have been characterized (5, 18-22). However, many of these studies used either very high, single doses of OTA or unusual routes of application. In this study, we attempted to characterize dose-dependent changes induced by gavage of OTA by application of clinical chemistry, urine analysis by 1H NMR and pattern recognition, and biochemical markers for a better conclusion on possible modes of action.
Material and Methods Animals and Treatment. Male Fischer 344 rats (170-190 g) were purchased from Charles-River Laboratories, Sulzfeld, Germany. The animals had free access to water and standard diet (Altromin) and were kept under standard conditions (12 h day/night cycle, temperature 21-23 °C, humidity 45-55%). All animal experimentation was performed under permit from the appropriate authorities in the approved animal care facility of the department. Rats were transferred to metabolic cages for collection of urine 3 days prior to dosing. Animals were administered OTA (0.25-2 mg/kg of body weight) dissolved in corn oil by oral gavage for 2 weeks, 5 days per week. Urine was collected every 12 h on day 1 of treatment and every 24 h thereafter on ice and stored at -20 °C until further analysis. Animals were sacrificed by CO2 asphyxiation and cervical dislocation 72 h after the final dose. This time-point was chosen as OTA is slowly eliminated and maximum DNA adduct concentrations have previously been reported in DNA extracted from kidneys 48-72 h after OTA administration (23, 24). Blood samples were obtained by cardiac puncture; plasma was separated by centrifugation and stored at -80 °C. Livers and kidneys were removed, flash-frozen in liquid nitrogen, and stored at -80 °C until further analysis. Aliquots of tissues were fixed in neutral-buffered formalin, embedded in paraffin, cut into 5 µm sections, and stained with haematoxylin and eosin for histopathological evaluation. Iron(III)nitrilotriacetic acid (FeNTA) was used as a positive control for an agent which causes nephrotoxicity and renal tumors presumably by oxidative stess (25, 26). Rats (n ) 3) were treated with a single dose of FeNTA (15 mg of Fe/kg of body wt) by ip injection. Dosing solutions were prepared as described (27). Control animals (n ) 3) received an equal volume of saline. Animals were sacrificed 5 h after administration by CO2 asphyxiation and cervical dislocation; blood was removed by cardiac puncture, and livers and kidneys were excised, flashfrozen in liquid nitrogen, and stored at -80 °C until analysis. Chemicals. Corn oil, methanol, and acetonitrile were purchased from Fluka, Sigma-Aldrich-Fluka, Taufkirchen, Germany. Purified water for LC/MS/MS was purchased from Roth, Karlsruhe, Germany. Chloroform, trifluoracetic acid, hydrochloric acid, and ethanol were purchased from Merck, Darmstadt, Germany. OTA was purchased from Sigma-Aldrich, Taufkirchen, Germany (Lot-No. 38H4120) and from Prof. Peter Mantle, Imperial College of Sciences, London, U.K. Purity of OTA was >99.9%, as assessed by HPLC with UV and fluorescence detection. Other chemicals were obtained from Sigma-Aldrich, Taufkirchen, Germany. Western-Blot Analysis of PCNA. Aliquots of frozen tissue were homogenized in 20 mM Tris, pH 7.4, containing 137 mM NaCl, 10% glycerol, and protease inhibitor cocktail (10 µL/mL, Sigma). Protein concentrations were determined using the BioRad DC Protein assay (Bio-Rad). Aliquots of lysate containing equal amounts of protein (50 µg) were separated by 12.5% SDSPAGE and transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat dry milk in TBST buffer (50 mM Tris, 150 mM NaCl, and 0.05% Tween-20, pH 7.5) overnight at 4 °C and subsequently incubated with monoclonal mouse anti-
Chem. Res. Toxicol., Vol. 18, No. 8, 2005 1243 PCNA antibody (PC-10, Santa Cruz, CA) [1:1000 in 0.5% nonfat dry milk in TBST, 2 h at room temperature] and goat antimouse IgG-HRP (Amersham Biosciences) [1:2000, in 0.5% nonfat dry milk in TBST for 1 h at room temperature]. Membranes were developed using the ECL Western-blotting detection system (Amersham Biosciences) and exposed to Hyperfilm (Amersham Biosciences). Clinical Chemistry. Excretion of γ-glutamyltranspeptidase (GGT) and creatinine was determined in urine samples of treated animals. Urine analysis and determination of aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), and alkaline phosphatase (ALP) in plasma was carried out at the Laboratory for Clinical Chemistry, University of Wu¨rzburg on a Vitros 700XR (Ortho-Clinical Diagnostics, Neckarsgemuend) using standard protocols for the determination of these parameters according to the manufacturers instructions. 1H NMR. Aliquots of urine (400 µL) were mixed with 200 µL of 0.2 M phosphate buffer (pH 7.4) and centrifuged for 10 min at 14 000 rpm. From the supernatant, 500 µL was mixed with 50 µL of a 10 mM solution of 3-(trimethylsilyl)proprionate2,2,3,3-d4 in D2O. 1H NMR spectra were recorded using a Bruker DPX 600 NMR operating at 600.13 MHz. The chemical shifts were normalized to 3(trimethylsilyl)proprionate-2,2,3,3,-d4 set at δ ) 0 ppm. The peak intensities were normalized to the creatinine resonance. The NMR spectra were corrected for phase and baseline distortions using XWINNMR software (Bruker, Germany) and reduced to 240 segments of equal width (0.04 ppm wide from δ 0.4-10.0 ppm) using AMIX software (Bruker, Germany). The regions of the spectra between δ 4.4 and 6.2 ppm were removed prior to statistical analysis to avoid variability due to water resonance. The data were tabulated into a single data table, and multivariate data analysis was performed using SimcaP 8.0 software (Umetrics, Sweden). DNA Isolation. DNA was isolated from livers and kidneys by the Nucleobond method (Macherey-Nagel, Dueren, Germany) according to the manufacturer’s instructions with minor modifications. Briefly, 300-400 mg of tissue was homogenized by an ultra-turrax, treated with proteinase K and RNase, and loaded onto a Nucleobond AX G 500 ion-exchange cartridge. After washing, the DNA was eluted from the cartridge using a modified elution buffer (1.5 M NaCl, 0.05 M Tris, and 15% ethanol, pH 7.0). The DNA was precipitated by the addition of 0.7 vol of 2-propanol. After washing with 70% ethanol, the DNA pellets were dissolved in H2O. Determination of 8-Oxo-7,8-dihydro-2′deoxyguanosine (8-OH-dG) by LC/MS/MS. Solutions containing 100 µg of DNA in 100 µL were incubated with 11 units nuclease P1 (Calbiochem) and 2.5 µL 1 M sodium acetate/45 mM zinc chloride buffer, pH 4.8, for 60 min at 37 °C. After addition of 10 µL 100 mM Tris (pH 8.0) and 7.5 units of alkaline phosphatase from calf intestine (Sigma, Deisenhofen, Germany), mixtures were incubated for an additional 30 min at 37 °C. Proteins were precipitated by the addition of an equal volume of chloroform and centrifugation at 1000g for 5 min. From the resulting aqueous layer, 50 µL wasinjected into the LC/MS/MS system. Separations were carried out on a YMC AQ QT HPLC column (2.1 mm × 150 mm; 4 µm, 100 A; YMC) using an Agilent 1100 autosampler and an Agilent 1100 HPLC-pump (Agilent, Waldbronn, Germany). The samples were separated by gradient elution with 10 mM ammonium acetate, pH 4.3 (solvent A), and methanol (solvent B) using the following conditions: 98% A for 5 min, followed by a linear increase to 85% A within 10 min, followed by a constant 85% A for 10 min at a flow-rate of 300 µL/min. The HPLC system was directly coupled to a triple stage quadrupole mass spectrometer (API 3000, Applied Biosystems, Darmstadt, Germany) equipped with a TurboIonSpray source. 8-OH-dG was detected in the positive-ion mode at a vaporizer temperature of 400 °C and a TurboIonSpray voltage of +4.0 kV. Spectral data were recorded with N2 (CAD ) 4) as collision gas, a declustering potential of 26 V, and a collision energy of 19 V. Data acquisition was performed in MRM mode monitoring the transition of m/z 284-168 and m/z 284-140 for 8-OH-dG. All
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Mally et al.
Table 1. Tissue and Plasma Levels of OTA after Administration for 2 Weeks (5 Days/Week)a OTA (mg/kg of body wt) plasma (nmol/mL) kidney (pmol/g tissue) liver (pmol/g tissue) a
0
0.25
0.5
1
2
0.008 ( 0.003 97% of the radioactivity present was recovered as OTA. Determination of OTA by LC/MS/MS. Parameters for the analysis of OTA and its known or presumed metabolites, sample workup, and separation conditions have been reported in detail previously (10, 29). In addition to the potential ochratoxin hydroquinone-derived glutathione conjugate, formation of the respective N-acetyl-cysteine conjugate was analyzed in plasma, urine, and tissue homogenates of animals treated with the highest dose using theoretical mass transitions (m/z 545-416) and constant neutral loss of 129 amu in the negative-ion mode based on the characteristic fragmentation pattern of mercapturic acids (30).
Results Repeated oral administration of OTA to rats for 5 days/ week for 2 weeks resulted in a dose-dependent increase in OTA plasma concentrations and also in a dose-related increase in OTA concentrations in both liver and kidney. Under the application scheme selected, concentrations of OTA in liver and kidney were very similar at terminal sacrifice (Table 1). In urine samples collected from exposed animals, the presence of both OTA, the metabolite ochratoxin alpha (OTalpha), and the previously described pentose and hexose conjugates of OTA were confirmed by LC/MS/MS. Over the time of the study, a dose- and time-dependent increase in the excretion of both OTA and OTalpha with urine was observed; however, excretion of both metabolites accounted for only a very small part of the total applied dose (Figure 1). No other metabolites were detected by LC/MS/MS using specific mass transitions based on synthetic standards or theoretical mass transitions for potential metabolites such as the ochratoxin hydroquinone-derived glutathione, mercapturic acid conjugates, and glucuronides (10, 29). Under the treatment conditions, a significant reduction in body weight gain was only observed at the highest dose of 2 mg/kg of body wt; however, relative liver, kidney, and spleen weights were not different to controls in the exposed animals at all doses (Table 2). Consistently, significant changes in clinical chemical parameters indicative of nephrotoxicity were only observed at the end of the study and after administration of the two highest doses (Table 3). However, it should be noted that urinary concentrations of GGT were still within the normal range and that the observed changes were not very marked compared to published values or previous experience in this laboratory with other renal proximal tubular toxins (31, 32). Consistent with the absence of liver damage as assessed by histopathology, no increase in plasma ALAT, ASAT, and ALP acitivities indicative of hepatic injury was observed at any of the doses applied, even with very high OTA concentrations present in the liver (Table 3). In the kidney, OTA-induced pathology was present at all dose levels administered with a clear increase in
Effects of Oral Administration of OTA
Chem. Res. Toxicol., Vol. 18, No. 8, 2005 1245
Table 2. Body and Relative Organ Weight (% of body wt) after Administration of OTA (0, 0.25, 0.5, 1, and 2 mg/kg of body wt) for 2 Weeksa OTA (mg/kg of body wt) body weight (g)
initial final
kidney liver spleen
0
0.25
0.5
1
2
177.3 ( 4.0 231.2 ( 3.7 0.76 ( 0.03 4.40 ( 0.41 0.23 ( 0.02
182.2 ( 0.5 235.7 ( 10.0 0.73 ( 0.01 4.55 ( 0.30 0.23 ( 0.01
180.9 ( 0.6 227.5 ( 7.2 0.73 ( 0.04 4.27 ( 0.09 0.23 ( 0.02
174.7 ( 0.4 216.4 ( 7.9 0.68 ( 0.06 4.15 ( 0.22 0.25 ( 0.02
179.3 ( 0.6 206.9 ( 11.8* 0.71 ( 0.06 4.47 ( 0.08 0.25 ( 0.01
a Statistical analysis was performed by ANOVA and Dunnett’s test. Significant changes compared to controls are indicated as *, p < 0.05 (n ) 3).
Table 3. Effects of Repeated OTA Administration on Clinical Chemical Parameters in Blood and Urine of Ratsa OTA (mg/kg of body wt) interval volume (mL/24 h) creatinine (mg/24 h) GGT (U/mg creatinine)
creatinine (mg/dL) urea (mg/dL) ASAT (U/L) ALAT (U/L) GGT (U/L) ALP (U/L)
day 1 day 7 day 13 day 1 day 7 day 13 day 1 day 7 day 13
0
0.25
0.5
1
2
7.5 ( 2.1 8.6 ( 3.0 10.3 ( 1.4 4.8 ( 0.5 5.4 ( 0.3 7.8 ( 0.5 0.16 ( 0.07 0.33 ( 0.13 0.09 ( 0.03
Urine 7.1 ( 0.3 9.8 ( 1.9 10.7 ( 1.9 nd nd nd nd nd nd
6.8 ( 1.0 9.5 ( 0.5 11.2 ( 3.5 nd nd nd nd nd nd
6.8 ( 0.8 10.3 ( 2.0 16.5 ( 0.9* 4.7 ( 0.2 5.7 ( 0.3 7.03 ( 0.4 0.03 ( 0.02* 0.23 ( 0.04 0.21 ( 0.4*
6.8 ( 3.6 11.8 ( 5.1 19.8 ( 4.2** 4.3 ( 1.0 5.3 ( 1.1 7.01 ( 0.4 0.02 ( 0.01* 0.30 ( 0.16 0.34 ( 0.06**
0.20 ( 0.00 50.2 ( 3.2 284.7 ( 110.8 100.5 ( 6.8
