Environmental Toxicology, University of Konstanz, Jacob-Burckhardtstr. 25, PO Box 5560 X918, D-78457, Konstanz, Germany
Received April 25, 2001; accepted June 20, 2001
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ABSTRACT |
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Key Words: ochratoxin A; nephropathy; renal cancer; uptake; cytostasis; toxicity.
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INTRODUCTION |
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Currently available data show large species- and sex differences in susceptibility towards OTA-induced nephrotoxicity and carcinogenicity. Chronic studies (210 µg/kg bw/day for 2 years) in rats have demonstrated a clear causal relationship between OTA exposure and the induction of renal cell tumors (RCT), with 60% of male rats developing carcinomas of cortical origin. This RCT was coupled with a distinct pathology of the pars recta of the proximal tubule. Female rats were found to be much less susceptible to OTA-mediated toxicity, displaying milder P3 pathology and only a 6% tumor incidence (Boorman, 1989; Boorman et al., 1992
; Castegnaro et al., 1998
; Rásonyi et al., 1999
). More pronounced sex differences were observed in the carcinogenic response of mice with 28% of male mice treated with 4800 µg/kg bw for 2 years presenting with RCT, while females were completely refractive (Bendele et al., 1985
; Grosse et al., 1997
). The mechanism(s) underlying these differences however, remain unknown. OTA toxicity at µM concentrations has been suggested to be due to DNA strand breaks, sister chromatid exchanges, DNA adduct formation, or reactive oxygen species (Galtier, 1991
; Rahimtula and Chong, 1991
). Other in vitro studies have been carried out either in the presence of heat inactivated serum (Creppy et al., 1995
), in the absence of serum (Bondy and Armstrong 1998
; Föllmann et al., 1995
; Gekle et al., 1995
) or more recently, at nM concentrations, under serum-free conditions in previously transformed cells (Gekle et al., 1998b
, 2000
). If the conditions of the in vitro system are to closely mimic the in vivo situation, investigation and characterization of the mechanisms involved in the acute toxicity of OTA in vitro should also include serum. However, the effects of nM concentrations of OTA (dietary relevant blood levels) in primary cell cultures and in the presence of serum have until now, not been investigated.
Control over cell growth and division is mediated through a carefully orchestrated balance of signals and checkpoints. Loss of this cell cycle control appears inherent to the etiology of various cancers. Furthermore, disruption in normal cell replication control could also be important in the etiology of the nephropathies associated with OTA exposure, for example either via a cytotoxic and/or cytostatic action on epithelial cells, thus allowing enhanced compensatory fibroblast proliferation and hence renal fibrosis. Such a mechanism has been hypothesized earlier in the case of the increased incidences of chronic interstitial nephropathy in Tunisia, which are associated with high blood levels of OTA (Maaroufi et al., 1995b; Simon et al., 1996
). The involvement of such an OTA-specific mechanism in the etiology of chronic interstitial nephropathy could be elucidated by the use of a range of relevant cell models of epithelial and fibroblast origin.
Thus, the aims of this study were to characterize the toxic effects (sum of antiproliferative, apoptotic, and necrotic effects) of relatively low concentrations of OTA in cell lines and in primary cells from both laboratory species and humans, in the presence of serum with a view to investigating their suitability for future studies into the mechanism(s) of OTA-mediated toxicity. Primarily, cells of epithelial origin were tested, however, the effects in the NRK-49F cell line (Normal Rat Kidney Fibroblast) was also examined to investigate the theory of epithelial cell replacement leading to the observed nephropathies as outlined above. For direct comparison with OTA, the toxic effects of ochratoxin B (OTB), a significantly less-toxic structural analogue of OTA, differing only by the lack of a chloride group on the isocumarin moiety (Fig. 1) was also investigated.
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MATERIALS AND METHODS |
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Cell culture and isolation of primary cells.
LLC-PK1 (ECACC No. 86121112), NRK-52E (DSMZ No. ACC 199), and NRK-42F (DSMZ No. ACC 172) cells were maintained under standard conditions in Dulbecco's modified Eagle's medium (DMEM) with 10% FCS and 2000 U/l penicillin and 2 mg/l streptomycin in a humidified atmosphere at 37°C, 5% CO2. The LLC-PK1 epithelial cell line was derived from renal cortex tissue from a juvenile Hampshire pig (Hull et al., 1976) and the NRK-52E and NRK-49F cell lines were cloned from a mixed culture of normal Osborne-Mendel rat renal cortex cells (DeLarco and Todaro, 1978).
Primary porcine cortical renal epithelial cells (PKC) from German hybrid pigs (local slaughterhouse) and primary cortical human renal epithelial cells (HKC) from human biopsy material (K. Hochberg, Klinikum Konstanz, Konstanz, Germany) were prepared essentially according to Pollock (Pollock and Field, 1993). Briefly, cells were isolated by 30 min digestion in collagenase (1 mg/ml) at 37°C and dissociated by passing through nylon meshes. Following collection by centrifugation and resuspension in MEM/Dval/10% FCS/ to suppress fibroblast growth, cells were assessed for viability by standard trypan blue exclusion and seeded at a density of 2 x 104/ml in Primaria cell culture material with MEM/Dval/10% FCS/2000 U/l penicillin and 2 mg/l streptomycin sulfate and incubated in a humidified atmosphere at 37°C, 5% CO2. Following 7 days in culture, the medium was replaced by DMEM/F12/10% FCS and then changed every 48 h until confluency was reached. Epithelial character was determined as described previously by us (Dietrich et al., 2001
). These cells were designated passage 0. Routine passaging was carried out using 0.025% trypsin/EDTA in PBS. All experiments involving primary cells were carried out using individual cell preparations generated from at least 3 different donors to ensure reproducibility and relevance of results. Primary cells of passage 12 and cell lines of passage < 20 only were used for experiments. Unless otherwise stated, cells were seeded at a density of 2 x 104 cells/ml and allowed a 24-h recovery before exposure.
Determination of toxic effects.
Toxic effects (sum of antiproliferative, apoptotic, and necrotic effects) were assessed by use of a standard nuclei-counting procedure. Briefly, freshly trypsinized cells were seeded into multiwell plates. Sterile stock solutions of OTA and OTB were prepared in 7% sodium bicarbonate and serially diluted to yield final exposure concentrations of 1 nM10 µM (OTA) and 10 nM100 µM (OTB). Vehicle concentration never exceeded 1%. Following 48-h exposure to OTA or OTB nuclei counting was performed in duplicate by replacing the medium with 500 µl of crystal violet (0.2%, w/v) in citric acid (0.1 M). The cells were incubated with this nuclei-releasing and -staining solution for 90 min at 37°C. Stained nuclei were then counted using a haemocytometer. Results were expressed as percentage control ± SD (n 3) and significant differences (*p < 0.05; **p < 0.01) calculated using ANOVA and Dunnett's post test. This method allows determination of cell numbers in a given population but does not however enable conclusions to be drawn on how the differences between treated or control cell numbers arise. Reductions in cell numbers following treatment could arise due to maintained proliferation coupled with increased apoptosis and/or necrosis, or via a cytostatic action. Therefore, although hereafter, the effects of OTA and OTB are referred to as "toxic," it should be noted that the actions of OTA and OTB could be of a cytotoxic or cytostatic nature.
Reversal of toxic effects.
Cells were seeded in 25 cm2 tissue culture flasks and exposed to 10 µM OTA or 80 µM OTB for 48 h (the respective concentrations above which, no further decrease in cell number could be observed in concentration/response assays). Medium was then removed and the cells washed 3 times in phosphate-buffered saline. One control and 1 treated flask were trypsinized and cell number determined using a haemocytometer. In the remaining flasks, full fresh medium was added and cell number determined 24 and 48 h subsequent to OTA/OTB removal. Results were expressed as the percentage of the respective 48-h control value ± SD (n 3). Significant differences (*p < 0.05; **p < 0.01) were determined using the Tukey-Kramer multiple comparisons test.
Measurement of [3H]-OTA uptake.
OTA uptake was measured by addition of 1 µCi [3H]-OTA per well to the culture medium. This concentration was selected as it had been demonstrated to have no effect in concentration-response experiments and also to be representative of normal serum OTA concentrations. At the indicated time points (10, 20, 30, 40, and 60 min), medium was removed and the cells were washed 3 times with ice-cold PBS to remove free [3H]-OTA and terminate uptake. Cells were then solubilized using 2% SDS, acidified and mixed with Beckman Ready Safe scintillation cocktail. [3H]-OTA uptake was determined by liquid scintillation counting (Beckman LS 6500 liquid scintillation counter). Results are expressed as dpm/cell ± SD (n 3). A previous study, using the same source and specific activity of 3H-OTA has previously determined the degradation rate of 3H-OTA generated by tritium exchange, to be less than 2% over 21 days at 37°C (Studer-Rohr et al., 2000
). Thus, the random labelling of OTA is stable under the conditions of the uptake studies carried out here.
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RESULTS |
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DISCUSSION |
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The results obtained in the current study strongly suggest that low nM OTA (but not OTB) concentrations in serum could elicit a "toxic" response in porcine and human primary renal epithelial but not mesenchymal (fibroblastic) cells, whereas rat primary cells and continuous pig epithelial cell lines are less susceptible to OTA-induced effects (Heussner et al., 2000). These observations suggest a succinct nephrotoxic activity of OTA primarily in humans and also in pigs. The effects observed in primary cells in this study were consistent in cells from several donors, suggesting them to represent a generalized phenomenon rather than idiosyncratic actions specific for 1 individual. The latter interpretation is further corroborated by the observation that tissue homogenates from the human renal cortex display a higher binding affinity and capacity for [3H]-OTA than the respective tissue homogenates from rat, mouse, or pig (Dietrich et al., 2000
). This specific binding appears unrelated to previously proposed organic transport systems for OTA (Groves et al., 1998
, 1999
).
Concentration-response curves for OTB versus cell number were in agreement with previously published results, which indicate OTB to be a factor 10100 less toxic than OTA (Heussner et al., 1998, 2000
; Rahimtula et al., 1988
; Schwöbel et al., 1999
). This supports the suggestion made by Xiao et al. (1996) that the toxic effects of OTA are mediated via a strict structure-activity relationship.
The methods employed in this study are simple and do not allow differentiation between inhibition of cell proliferation (cytostasis), apoptosis, and necrosis. Fundamental differences between cell types in their response to OTA exposure can however be delineated. In our laboratory, necrosis has been observed in cultured primary and transformed cells at medium to high OTA concentrations only (Dietrich et al., 2001; Heussner et al., 2000
). Apoptosis (Gekle et al., 2000
; Seegers, 1994
) appears to prevail in the low to medium concentration range while cytostasis and/or cellular dedifferentiation appears to predominate at very low concentrations (Heussner et al., 2000
). This interpretation is supported by the fact that pancreatic epithelial cells exposed to 2,3-dimethoxy-1,4-naphtoquinone demonstrated similar dose-dependent influence on cell growth, apoptosis, and necrosis (Dypbukt et al., 1994
). Indeed, nanomolar OTA concentrations appear to have a cytostatic effect in NRK-52E, LLC-PK1, and PKC(m) cell types. This cytostatic effect of OTA appears to be located to the G2 phase of the cell cycle (Dreger et al., 2000
). A G2/M specific cell cycle arrest is also suggested by the marked appearance of giant nuclei in P3 epithelial cells of rats (Maaroufi et al., 1999
; Rásonyi et al., 1999
) and pigs (Krogh et al., 1976
) treated with moderate OTA concentrations in vivo and in BEN patients with urothelial tumors (Godin et al., 1997
; Maaroufi et al., 1995a
,b
). Such a G2/M specific block would most likely occur subsequent to the inhibition or over-expression of a particular protein or gene ("molecular switch"), which needs to be either resynthesized or degraded, before the cells can begin to proliferate. This hypothesis is supported by the observation that with the exception of primary human cells from both sexes, the effects of OTA exposure were reversible in all cells, albeit coupled with a distinct lag-phase of 48 h (a significant increase in cell numbers was observed 48 h following OTA removal). The nature of the molecular "switch" mentioned above remains to be elucidated and deserves further attention. Possible candidates could be various members of the CDK family and their associated cyclins, particularly A and B, which are known to regulate G2/M transition, possibly in association with components of the PKC family that have also been shown to be closely associated with G2/M transition in vascular epithelial cells (Kosaka et al., 1996
).
As the differences observed in the OTA-mediated inhibition of proliferation could be due to differences in the amount of OTA incorporated into the cells, this was investigated using [3H]-OTA. Uptake remained relatively constant throughout the assay period, indicating the attainment of a plateau level within 10 min. Similar findings have been reported by Schwerdt and coworkers (Schwerdt et al., 1998) and by Tsuda et al. (1999). No statistically significant differences between the cell types were observed for [3H]-OTA uptake. Differences, however, were apparent between the uptake into continuous cell lines and primary cells from both pigs and humans. More OTA- sensitive cell types (primary human and porcine) consistently accumulated more radiolabeled OTA. The only exception to this was the NRK-49F cell line. These fibroblasts accumulated similar concentrations of OTA as their epithelial counterparts the NRK-52E cell line, but were not as susceptible to OTA- mediated effects as the epithelial cells. Thus, the decisive factor in cellular response to OTA exposure does not appear to be solely the amount of OTA gaining access to the cells, but how individual cell types process their respective OTA load. Conceivably NRK-49F cells and possibly fibroblasts in general, may possess a protein capable of neutralizing the effects of OTA or conversely, do not express the "molecular switch" apparently involved in OTA-mediated toxicity in epithelial cells. The latter observations lend support to the theory that BEN could be caused by a cytostatic/cytotoxic effect of OTA predominantly in epithelial cells, while fibroblasts are more refractive. In consequence this would lead to increased/maintained fibroblast proliferation coupled with reduced epithelial cell proliferation and/or epithelial cell death and hence the gradual replacement of healthy with fibrotic tissue and the characteristic onset of nephropathy. The observation that primary human cells from both sexes did not resume cell growth following OTA exposure strengthens the likelihood of the epithelial replacement by fibroblastic tissue and stresses further a causal involvement of OTA in the etiology of BEN and karyomegalic interstitial nephropathy.
In contrast, none of the cell types resumed proliferation following 48 h exposure to OTB suggesting that OTA and OTB, although almost identical in structure (see Figure 1), have some fundamental difference in their mechanism of action (Xiao et al., 1996
).
The fact that higher (high nM to low µM) concentrations or longer exposure times (96 h) to OTA does not further reduce cell numbers (Dreger et al., 2000) suggests the presence of a subpopulation of cells resistant to OTA. Selection of such a subpopulation has previously been described by Achanzar and coworkers in prostate epithelial cells exposed to cadmium (Achanzar et al., 2000
). These authors suggested that cadmium selected for apoptotic-defective prostate epithelial cells via direct or indirect effects on the expression of c-myc, p53, PCNA, and c-jun. Selection of an apoptotic-defective subpopulation was considered one of the primary steps involved in cadmium-induced tumor formation. If indeed low nM OTA concentrations, as used in the experiments discussed here and present in the serum of the populations in the areas endemic for BEN and UT, are capable of selecting apoptotic-defective epithelial cells, this subpopulation could be the long-sought link between OTA exposure and observed tumor formation. The latter hypothesis is currently under investigation in our laboratory using molecular methods.
In conclusion, the results presented are as yet, preliminary in nature, however, they provide support for the selection of primary renal epithelial cells rather than continuous/transformed cell lines mechanistic investigations on OTA-induced carcinogenesis and nephropathies. Furthermore, the data indicate ochratoxins to be toxic in vitro at dietary relevant concentrations and in the presence of serum
Future directions should include an analysis of the cell cycle events leading to cytostasis and cell differentiation/dedifferentiation, together with the expression levels of such factors as c-myc, p53, c-jun and PCNA, the CDK family and their associated cyclins and components of the PKC family following exposure to both OTA and OTB.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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