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Induction of Genotoxic Effects and Modulation of the Intracellular Calcium Level in Syrian Hamster Embryo (SHE) Fibroblasts caused by Ochratoxin A
Food and Chemical Toxicology 37 (1999) 713±721
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Research Section
Induction of Genotoxic Eects and Modulation of the Intracellular Calcium Level in Syrian Hamster Embryo (SHE) Fibroblasts Caused by Ochratoxin A E. DOPP1*, J. MUÈLLER1, C. HAHNEL2 and D. SCHIFFMANN1 1
Department of Biology, Animal Physiology, Division of Cellular Pathophysiology, University of Rostock and 2Department of Ophthalmology, University of Rostock, Germany (Accepted 13 January 1999)
AbstractÐThe mycotoxin ochratoxin A (OTA) is a naturally occuring contaminant of food. The genotoxic status of OTA is still controversial because contradictory results were obtained in various microbial and mammalian gene mutation assays. In this study, OTA was investigated to examine its potency to induce micronuclei (MN) in SHE cells. The SHE-micronucleus assay revealed that OTA induces MN in a dose- and time-dependent manner. The results of kinetochore analysis revealed that mainly clastogenic events are involved in OTA genotoxicity. Induction of mitotic disturbances can be closely related to changes of the intracellular calcium concentration ([Ca2+]i). The investigated time course of OTA-induced [Ca2+]i changes revealed that the obtained signal is a short spike signal resembling physiological responses. In the absence of extracellular calcium, a long-lasting signal indicates possible damage to intracellular calcium stores or channels. Our data show that the OTA-induced [Ca2+]i rise is caused by Ca2+-release from intracellular stores as well as Ca2+ in¯ux from extracellular area. Finally, the in¯uence of the changed intracellular calcium level on the actin cytoskeleton was investigated. Visualization of the actin ®laments revealed time- and concentration-dependent eects. Cell shrinkage and depolymerized ®laments were observed. We conclude that OTA disrupts actin ®laments by a direct irreversible binding to actin. # 1999 Elsevier Science Ltd. All rights reserved Keywords: micronuclei; ochratoxin A; kinetochores; intracelluar calcium; actin ®laments. Abbreviations: [Ca2+]i = intracellular calcium concentration; CHO cells = Chinese hamster ovary cells; CREST = calcinosis, Raynaud's phenomenon; oesophageal motility abnormalities; sclerodactyly and telangiectasia, DMEM = Dulbecco's modi®ed Eagle's medium; DMSO = dimethylsulfoxide; FITC = ¯uorescein isothiocyanate; MN = micronuclei; OSV = ovine seminal vesicle cell cultures; OTA = ochratoxin A; PBS/CMF = phosphate buered saline/calcium and magnesium free; SCE = sister chromatid exchange; SHE = Syrian hamster embryo ®broblasts; TBS = Tris buered saline.
INTRODUCTION
Ochratoxin A (OTA) is a naturally occurring mycotoxin produced by several species of the fungal genera Aspergillus and Penicillium. Its occurrence in plant and animal products has been well documented (Krogh, 1987). It consists of a chlorinated dihydroisocoumarin moiety linked through its 7-carboxy group by an amide bound to L-b-phenylalanine (Fig. 1). OTA has been shown to have a number of *Corresponding author at: University of Rostock, Department of Biology, Institute of Animal Physiology, Division of Cellular Pathophysiology, UniversitaÈtsplatz 2, 18055 Rostock, Germany.
toxic eects, the most prominent is nephrotoxic (Krogh, 1992). In addition to the kidney, the urinary tract is a target organ of OTA (PetkovaBocherova and Castegnaro, 1991). OTA is also immunosuppressive, teratogenic and carcinogenic (Creppy et al., 1985, Dirheimer and Creppy, 1991). Based on the current literature, the toxicity of OTA may be the result of three major eects: (1) inhibition of ATP synthesis; (2) inhibition of protein synthesis; and (3) enhanced lipid peroxidation (Marquardt and Frohlich, 1992). The genotoxic status of OTA was controversial since almost all microbial and mammalian test assays were negative (Ueno and Kubota, 1976; Wehner et al., 1978).
0278-6915/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(99)00057-5
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Fig. 1. Chemical structure of ochratoxin A.
Contradictory results were found for sister-chromatid exchange in CHO cells (for review, see KuiperGoodmann and Scott, 1989). Nevertheless, newer studies by FoÈllmann et al. (1995) have clearly shown that OTA induces SCEs in cultured porcine urinary bladder epithelial cells. Manolova et al. (1990) demonstrated that chromosomal aberrations occur, particularly on X chromosomes, produced in human lymphocytes in culture by OTA at a concentration of 15 nM. DNA adduct formation was found in vitro (e.g. Grosse et al., 1995) as well as in vivo (e.g. Petkova-Bocharova et al., 1998). Degen et al. (1997) studied the eects of OTA on micronucleus induction in vitro using ovine seminal vesicle cell cultures. These authors have shown a dosedependent induction of micronuclei in this test system. Hoehler et al. (1996) suggested that OTA induces free radical production. This eect may enhance the permeability of the cellular membrane to Ca2+. Chong and Rahimtula (1992) reported that administration of a single high dose or multiple lower doses of OTA to rats resulted in an increase of the renal cortex endoplasmatic reticulum ATP-dependent calcium pump activity. In connection with intracellular Ca2+ changes by bradykinin, disturbances of the actin stress ®bres have been described by WoÈll et al. (1993). Activation of calcium sensitive K+ channels is an element of cell volume regu-
lation and has been shown to induce altered mophology of cells in a variety of tissues. Syrian hamster embryo (SHE) ®broblasts are known as a useful model system for the analysis of chemically-induced primary chromosomal changes (Fritzenschaf et al., 1993). The results of these and a number of other investigations have shown that the SHE-micronucleus assay is a short-term test of a high predictive value (Schimann und DeBoni, 1991; Schmuck et al., 1988). In combination with immuno¯uorescent staining of kinetochores in micronuclei, clastogenic events, as well as the loss of whole chromosomes, are detectable (Degrassi and Tanzarella, 1988). In this study, we investigated the genotoxic potency of OTA to induce micronuclei in SHE cells and to characterize the induced micronuclei by kinetochore staining (CREST analysis). For further characterization of the cytotoxic eects of OTA, measurements of [Ca2+]i and observations of the actin cytoskeleton were performed.
MATERIALS AND METHODS
Cell cultures All experiments were performed with cultures derived from 13-day-old SHEs. Cell cultures were grown in a humidi®ed atmosphere with 12% CO2 in air at 378C. The culture medium used was IBR [modi®ed Dulbecco's modi®ed Eagle's medium (DMEM)] medium (GIBCO, Karlsruhe, Germany) supplemented with 15% foetal bovine serum (GIBCO, Karlsruhe, Germany), 3.7 g NaHCO3/litre (7.5%), 1% glucose, 10,000 IU penicillin/litre, 10 mg streptomycin/litre and 0.01% tylosin (Sigma, Germany).
Fig. 2. Occurence of micronuclei in SHE cells after treatment with dierent concentrations of ochratoxin A. Each data point represents the mean of three to ®ve counts of 2000 nuclei of dierent experiments.
OTA induces genotoxic and [Ca2+]i changing eects
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Table 1. Formation of micronuclei in SHE cells after treatment with ochratoxin A (10 mM and 15 mM) in dependence on exposure time No. of micronuclei/2000 cells Exposure time
10.0 mM OTA
Untreated control
3 hr 6 hr 18 hr 36 hr 54 hr 72 hr
21.02 0.7 32.02 0.7 48.72 2.6 50.52 1.9 47.72 5.8 46.02 2.7
15.0 mM OTA
32.0 21.4* 43.0 23.5** 76.0 28.6*** 91.6 22.0*** 78.6 215.8*** 68.7 215.2***
40.0 21.4** 55.0 23.5*** 80.6 27.3*** 109.3 26.8*** 91.3 28.0*** 82.3 219.6***
Signi®cance tested by the t-test: *P E0.05; **PE 0.01; ***PE0.001.
Micronucleus assay and kinetochore analysis SHE cells were grown on coverslips, and after treatment with ochratoxin A (Sigma, Germany; stock solution 1 mM in DMSO; ®nal concentrations in the culture medium 5 to 20 mM), cells were ®xed and stored in cold methanol (ÿ208C) for at least 30 min. After washing of cells with phosphate buffered saline (PBS), nuclei were stained with bisbenzimide (Hoechst 33258, concentration: 1 mg/ml, 4 min). The slides were then mounted for microscopy and examined for the presence of micronuclei. Each data point represents the mean of at least four treated cultures from dierent experiments with 2000 nuclei evaluated in each case. For further analysis of the induced micronuclei, kinetochores were stained by incubating the ®xed cell preparations with CREST serum (Chemicon, Temecula, CA, USA) for 1 hr in a humidi®ed chamber at 378C. After rinsing with phosphate buffered saline, the cells were incubated with ¯uorescein isothiocyanate (FITC) conjugated goat antihuman antibodies (Sigma, Germany) before applying bisbenzimide. At least 100 micronuclei were examined for the presence of kinetochores in each case.
magnesium free) and DMEM. An equivalent volume of DMSO was added to the control cultures. For each OTA concentration, 100 images in 53 sec were acquired at 488 nm excitation wave length and 515 nm emission wave length. The [Ca2+]i of SHE cells was measured by determining the overall ¯uorescence intensity (50 cells/frame) with the Intervision Software (Noran) on a Silicon Graphics Workstation. Rhodamin/phalloidin staining OTA-treated (10, 20, 30 mM; 18 and 36 hr) and untreated SHE cells were ®xed in paraformaldehyde (3%) for 15 min and treated with TBS + 0.1% Triton X-100 for permeablization of the cell membrane. The cells were incubated with a blocking solution [1% bovine serum albumin and 2% normal goat serum in tris buered saline (TBS)] for 5 min at room temperature and subsequently incubated with rhodamine/phalloidin (Sigma, Germany) for 15 min. After washing with TBS again, the cells were mounted with antifade and the actin skeleton was observed directly using a ¯uorescence microscope.
Measurement of intracellular calcium concentration SHE cells were plated and grown on Petriperm dishes. For [Ca2+]i measurements, the culture medium was removed and cells were incubated with PBS containing the calcium indicator ¯uo-3 AM (®nal concentration: 16 mM) for 30 min at 378C. After washing with PBS and treatment with OTA (dissolved in DMSO, stock solution: 1 mM), the ¯uorescence intensity of cells was measured using a confocal laser scanning microscope (Noran Odyssey). For various ®nal concentrations of OTA (15, 30, 60 mM) the time course was observed in PBS/CMF (phosphate buered saline/calcium and
RESULTS
The results of the micronucleus assay showed that OTA induces micronuclei in SHE cells in a dose-dependent manner (Fig. 2). A comparison of the dierences between MN formation in untreated and OTA-treated SHE cells revealed that these dierences are signi®cant at OTA concentrations of 5 mM to 20 mM and exposure times of 18 hr to 72 hr (Fig. 2). The time course of MN formation revealed that the number of induced MN is statistically signi®cant even after 3 hr of exposure and an OTA concentration of 10 mM (Table 1).
Table 2. Results of kinetochore staining of SHE cells after treatment with ochratoxin A
Ochratoxin A Control a
Concentration (mM)
Exposure time (hr)
No. of cells scored
No. of micronucleia
% CREST+b (mean 2SD)
5.0±15.0 ±
36 36
13868 6966
631 209
33.6 2 2.5 28.7 2 0.4
Number of micronuclei scored for presence of kinetochores. Percentage of detected micronuclei that reacted positively to antikinetochore serum.
b
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Fig. 3. Fluorescence intensity of OTA-treated and untreated SHE cells after incubating of cells with the calcium indicator ¯uo-3 AM for intracellular calcium measurements in calcium/magnesium free medium (PBS/CMF). The intracellular calcium concentration of untreated cells was about 100 nM. The point (*) indicates OTA administration.
OTA induces genotoxic and [Ca2+]i changing eects
The maximum induction of micronuclei is reached at 36 hr exposure and 15 mM OTA (Table 1). At higher concentrations and longer exposure times, the frequency of micronuclei decreased again as a result of increased cytotoxicity (Fig. 2). The results of the kinetochore analysis showed that OTA induces both kinetochore-positive
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(CREST+) and kinetochore-negative (CRESTÿ) micronuclei in SHE cells (Table 2). 33.6% of induced micronuclei contain whole chromosomes or chromatids indicating aneuploidy. The higher percentage of kinetochore-negative micronuclei (CRESTÿ: 66.4%) indicates that these mainly originate from clastogenic events. The dierence between kinetochore containing micronuclei of trea-
Plate 1. Staining of actin ®laments of OTA-treated (i) 20 mM OTA, 18 hr exposure) and untreated (ii) SHE cells with rhodamin/phalloidin.
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Fig. 4. Fluorescence intensity of OTA-treated and untreated SHE cells after incubating of cells with the calcium indicator ¯uo-3 AM for intracellular calcium measurements in calcium containing medium (DMEM). The point (*) indicates OTA administration.
OTA induces genotoxic and [Ca2+]i changing eects
ted and untreated cells is statistically not signi®cant (Table 2). Our investigations have also shown that the treatment of SHE cells with OTA results in disturbances of intracellular Ca2+ homeostasis. Measurements of SHE cells in PBS/CMF (medium without calcium) and DMEM (medium containing calcium) showed an increase of [Ca2+]i within the ®rst seconds after treatment with OTA. The obtained ¯uorescence intensities of untreated and OTA-treated SHE cells are shown in Figs 3 and 4. In PBS/CMF, the [Ca2+]i is signi®cantly increased at OTA concentrations of 30 mM (Fig. 3b). At higher concentrations (60 mM) the [Ca2+]i is not further increased compared with the untreated control (Fig. 3c). In PBS/CMF, the [Ca2+]i level is maintained at a constantly elevated level, whereas in DMEM (extracellular calcium concentration: 1.8 mM) the [Ca2+]i elevation appears as a short spike signal within seconds after OTA administration, and it decreases again below the normal level (Fig. 4f). The intracellular calcium rise is about 45% higher in SHE cells treated with 60 mM OTA in calcium containing medium in comparison to SHE cells treated with 30 mM OTA in calciumfree medium. Visualization of the actin ®laments showed strong eects on actin ®bres of OTA treated SHE cells (Plate 1). Actin staining was performed at 10, 20, 30 mM OTA concentrations and 18 hr and 36 hr exposure time (the cells were incubated with OTA in DMEM). The observed eects were time and concentration dependent (no more data shown). Cell shrinkage and depolymerized ®laments increased with higher concentrations and longer exposure times. These eects were not reversible up to 36 hr recovery time. Longer recovery times (>36 hr) were not tested. DISCUSSION
Previous studies showed that OTA may inhibit synthesis of protein and DNA (Creppy et al., 1986; Gekle et al., 1995) and induces alterations in calcium homeostasis (Rahimtula and Chong, 1991). Neither the time course of intracellular calcium changes nor changes of the actin cytoskeleton after OTA treatment were investigated so far. Degen et al. (1997) reported about induction of micronuclei with OTA in ovine seminal vesicle cell cultures (OSV). OTA was found to induce dose-dependently micronuclei in cytokinesis-blocked binucleated OSV. A maximum of MN formation was achieved at 10 mM OTA and 6 hr of exposure. However, Degen et al. did not test exposure times other than 6 hr. When induced MN were characterized in our investigations by indirect immuno¯uorescence microscopy using antikinetochore (CREST) antibodies, the OTA-treated fraction of kinetochorepositive MN was similar to that observed in solvent
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controls. These data indicate that OTA induces MN apparently by a mixed, although predominantly clastogenic, mode of action. It is possible that OTA induces DNA damage by disturbances of chromosomes/chromatin in a direct or an indirect way (production of free radicals by OTA, Hoehler et al., 1996). OTA also induced an intracellular calcium rise. The time course of this eect in calcium-containing medium is rather typical for a physiological response and not for a ``damaging signal''. The role of Ca2+ as an intracellular regulator of many physiological processes is well established. Ca2+ can also play a determinant role in pathological processes (Nicotera et al., 1992). It has been proposed that intracellular Ca2+ accumulation may be a common step in the development of cytotoxicity because disturbances of Ca2+-regulated mechanisms are often early events in the development of cell injury (Hoehler et al., 1996). Normally, the Ca2+ concentration in the cytosol of unstimulated cells is maintained between 0.05 and 0.2 mM. Extracellular Ca2+ levels are approximately 1.3 mM. This produces a large electrochemical gradient that is mainly balanced by active Ca2+ extrusion through the plasma membrane and by co-ordinated activity of Ca2+-sequestering systems located in the mitochondrial, endoplasmatic reticular and nuclear membranes (Nicotera et al., 1992). Disturbances of these processes can result in enhanced Ca2+ in¯ux, release from intracellular stores and inhibition of Ca2+ extrusion at the plasma membrane. Calcium sensitive channels can also be in¯uenced. Hoehler et al. (1996) suggested that OTA induces free radical production by enhancing the permeability of the cellular membrane to Ca2+. Our experiments have shown that the enhanced [Ca2+]i caused by OTA treatment is a result of the Ca2+ release from intracellular stores as well as of damaged cell membranes with an increased in¯ux of extracellular Ca2+. After a short spike, the [Ca2+]i decreased again below the normal level in calcium-containing medium. This can be caused by an increase of the calcium pump activity. Rahimtula and Chong (1991) also observed an increase in renal endoplasmatic reticulum calcium pump activity after administration of OTA to rats in vivo. In contrast to the increased calcium pump activity of OTA treated SHE cells in calcium containing medium, the calcium pump activity seems to be inhibited in calcium-free medium, or intracellular calcium stores are damaged. The [Ca2+]i level remains elevated in PBS/CMF. Rahimtula and Chong (1991) published evidence that OTA administration to rats in vivo resulted in an decreased renal mitochondrial state-3 respiration and calcium uptake. These authors hypothesized that a decrease in microsomal calcium pump activity could lead to higher cytosolic calcium levels.
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An increase of intracellular calcium concentration can also contribute to cell shrinkage. WoÈll et al. (1993) descibed that an inhibition of K+ and Clÿ channels by barium (5 mM) and OTA (5 mM) prevents an intracellular alkalinization. Calcium triggers calcium-sensitive K+ channels, and presumably Clÿ channels, the subsequent loss of cellular KCl leads to cell shrinkage which, in turn, activates Na+/H+ exchange. Yokoshiki et al. (1997) concluded from their investigations with cytochalasin D that the disruption of the actin cytoskeleton attenuates the ability of the sulfonylurea receptor to inhibit the opening of ATP-sensitive K+ channels. OTA seems to have similar eects on actin ®laments like cytochalasin D. Van Deurs et al. (1996) described an actin ®lament-depolarization after treatment with cytochalasin D (10 mM). In contrast to OTA, the ®lament formation is already reversible after 2 hr of cytochalasin D incubation in about 20% of cases. We suggest that the disruption of actin ®laments after OTA treatment do not correlate with the increased [Ca2+]i. It is more likely that OTA binds directly and irreversibly to actin. In summary, OTA was found to induce dosedependently micronuclei in SHE ®broblasts. The characterization of the induced MN revealed that mainly kinetochore-negative MN were induced. We conclude that OTA has a predominantly clastogenic mode of action. Measurements of the intracellular calcium concentration have shown an increase of [Ca2+]i within the ®rst seconds after OTA treatment. This time course is typical for a physiological response. Ca2+-release from intracellular stores as well as Ca2+ in¯ux from extracellular media are involved in OTA cytotoxicity. Observations of the actin cytoskeleton of OTA-treated SHE cells revealed time- and concentration-dependent eects, such as cell shrinkage and depolymerized ®laments. These studies reveal that OTA is an eective inducer of DNA damage, of disturbances of the intracellular calcium homeostasis, and of disruption of the actin stress ®bres. AcknowledgementÐThe authors thank Mrs Jutta Saedler for excellent technical assistance. REFERENCES
Chong X. and Rahimtula A. D. (1992) Alterations in ATP-dependent calcium uptake by rat renal cortex microsomes following ochratoxin A administration in vivo or addition in vitro. Biochemical Pharmacology 44, 1401±1409. Creppy E. E., Kane A., Dirheimer G., Lafarge-Frayssinet C., Moussett S. and Frayssinet C. (1985) Genotoxicity of ochratoxin A in mice: DNA single strand break evalution in spleen, liver and kidney. Toxicology Letters 28, 29±35. Creppy E. E., Kane A., Giessen-Crouse E., Roth A., Roschenthaler R. and Dirheimer G. (1986) Eect of ochratoxin A on enzyme activities and macromolecules synthesis in MDCK cells. Archives of Toxicology Suppl. 9, 310±314.
Degen G. H., Gerber M. M., Obrecht-P¯umio S. and Dirheimer G. (1997) Induction of micronuclei with ochratoxin A in ovine seminal vesicle cell cultures. Archives of Toxicology 71, 365±371. Degrassi F. and Tanzarella C. (1988) Immuno¯uorescent staining of kinetochores in micronuclei: a new assay for the detection of aneuploidy. Mutation Research 203, 339±345. Dirheimer G. and Creppy E. E. (1991) Mechanism of the Action of Ochratoxin A. IARC Scienti®c Publications no. 115, pp. 171±186. International Agency for Research on Cancer, Lyon. FoÈllmann W., Hillebrand I. E., Creppy E. E. and Bolt H. M. (1995) Sister chromatid exchange frequency in cultured isolated porcine urinary bladder epithelial cells (PUBEC) treated with ochratoxin A and alpha. Archives of Toxicology 69, 280±286. Fritzenschaf H., Kohlpoth M., Rusche B. and Schimann D. (1993) Testing of known carcinogens in the Syrian Hamster embryo (SHE) micronucleus test in vitro; correlations with in vivo micronucleus formation and cell transformation. Mutation Research 319, 47±53. Gekle M., Pollock C. A. and Silbernagl S. (1995) Timeand concentration-dependent biphasic eect of ochratoxin A on growth of proximal tubular cells in primary culture. Journal of Pharmacology and Experimental Therapeutics 275, 397±404. Grosse Y., Baudrmont I., Castegnaro M., Betbeder A. M., Creppy E. E., Dirheimer G. and Pfohl-Leszkowicz A. (1995) Formation of ochratoxin A metabolites and DNA-adducts in monkey kidney cells. Chemico± Biological Interactions 95, 175±187. 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). Journal of Biological Chemistry 271, 27388±27394. Krogh P. (1987). Ochratoxins in food. In Mycotoxins in Food, ed. P. Krogh, pp. 97±121. Academic Press, London. Krogh P. (1992) Role of ochratoxin in disease causation. Food and Chemical Toxicology 30, 213±224. Kuiper-Goodman T. and Scott P. M. (1989) Risk assessment of the mycotoxin A. Biomedical and Environmental Science 2, 179±248. Manolova Y., Manolov G., Parvanova L., PetchkovaBocharova T., Castegnaro M. and Chernozemsky I. (1990) Induction of characteristic chromosomal aberrations, particulary X-trisomy, in cultured human lymphocytes treated by ochratoxin A, a mycotoxin implicated in Balkan endemic nephropathy. Mutation Research 231, 143±149. Marquardt R. R. and Frohlich A. A. (1992) A review of recent advances in understanding ochratoxicosis. Journal of Animal Science 70, 3968±3988. Nicotera P., Bellomo G. and Orrenius S. (1992) Calciummediated mechanisms in chemically induced cell death. Annual Review of Pharmacology and Toxicology 32, 449±470. Petkova-Bocharova T. and Castegnaro M. (1991) Ochratoxin A in Human Blood in Relation to Balkan Endemic Nephropathy and Urinary Tract Tumours in Bulgaria. IARC Scienti®c Publications no. 115, pp. 135±137. International Agency for Research on Cancer, Lyon. Petkova-Bocharova T., Stoichev I. I., Chernozemsky I. N., Castegnaro M. and Pfohl-Leszkowicz A. (1998) Formation of DNA adducts in tissues of mouse progeny through transplacental contamination and/or lactation after administration of a single dose of ochratoxin A to the pregnant mother. Environmental and Molecular Mutagenesis 32, 155±162?. Rahimtula A. D. and Chong X. (1991) Alterations in Calcium Homeostasis as a Possible Cause of Ochratoxin
OTA induces genotoxic and [Ca2+]i changing eects A Nephrotoxicity. International Agency for Research on Cancer, Lyon , 207±214. Schimann D. and DeBoni U. (1991) Dislocation of chromatin elements in prophase induced by diethylstilbestrol: a novel mechanism by which micronuclei can arise. Mutation Research 246, 113±122. Schmuck G., Lieb G., Wild D., Schimann D. and Henschler D. (1988) Characterization of an in vitro micronucleus assay with Syrian hamster embryo ®broblasts. Mutation Research 203, 397±404. Ueno Y. and Kubota K. (1976) DNA-atacking ability of carcinogenic mycotoxins in recombination-de®cient mutant cells of Bacillus subtilis. Cancer Research 36, 445±451.
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Van Deurs B., von Bulow F., Vilhardt F., Holm P. K. and Sandvig K. (1996) Destabilization of plasma membrane structure by prevention of actin polymerization. Microtubule-dependent tubulation of the plasma membrane. Journal of Cell Science 109, 1655±1665. Wehner F. C., Thiel P. G., Van Rensburg S. J. and Demasius I. P. P. C. (1978) Mutagenicity to Salmonella typhimurium of some Aspergillus and Penicilium toxins. Mutation Research 58, 193±203. WoÈll E., Ritter M., Scholz W., Haussinger D. and Lang F. (1993) The role of calcium in cell shrinkage and intracellular alkalinization by bradykinin in Ha-ras oncogene expressing cells. FEBS Letters 322, 261±265.
www.elsevier.com/locate/foodchemtox
Research Section
Induction of Genotoxic Eects and Modulation of the Intracellular Calcium Level in Syrian Hamster Embryo (SHE) Fibroblasts Caused by Ochratoxin A E. DOPP1*, J. MUÈLLER1, C. HAHNEL2 and D. SCHIFFMANN1 1
Department of Biology, Animal Physiology, Division of Cellular Pathophysiology, University of Rostock and 2Department of Ophthalmology, University of Rostock, Germany (Accepted 13 January 1999)
AbstractÐThe mycotoxin ochratoxin A (OTA) is a naturally occuring contaminant of food. The genotoxic status of OTA is still controversial because contradictory results were obtained in various microbial and mammalian gene mutation assays. In this study, OTA was investigated to examine its potency to induce micronuclei (MN) in SHE cells. The SHE-micronucleus assay revealed that OTA induces MN in a dose- and time-dependent manner. The results of kinetochore analysis revealed that mainly clastogenic events are involved in OTA genotoxicity. Induction of mitotic disturbances can be closely related to changes of the intracellular calcium concentration ([Ca2+]i). The investigated time course of OTA-induced [Ca2+]i changes revealed that the obtained signal is a short spike signal resembling physiological responses. In the absence of extracellular calcium, a long-lasting signal indicates possible damage to intracellular calcium stores or channels. Our data show that the OTA-induced [Ca2+]i rise is caused by Ca2+-release from intracellular stores as well as Ca2+ in¯ux from extracellular area. Finally, the in¯uence of the changed intracellular calcium level on the actin cytoskeleton was investigated. Visualization of the actin ®laments revealed time- and concentration-dependent eects. Cell shrinkage and depolymerized ®laments were observed. We conclude that OTA disrupts actin ®laments by a direct irreversible binding to actin. # 1999 Elsevier Science Ltd. All rights reserved Keywords: micronuclei; ochratoxin A; kinetochores; intracelluar calcium; actin ®laments. Abbreviations: [Ca2+]i = intracellular calcium concentration; CHO cells = Chinese hamster ovary cells; CREST = calcinosis, Raynaud's phenomenon; oesophageal motility abnormalities; sclerodactyly and telangiectasia, DMEM = Dulbecco's modi®ed Eagle's medium; DMSO = dimethylsulfoxide; FITC = ¯uorescein isothiocyanate; MN = micronuclei; OSV = ovine seminal vesicle cell cultures; OTA = ochratoxin A; PBS/CMF = phosphate buered saline/calcium and magnesium free; SCE = sister chromatid exchange; SHE = Syrian hamster embryo ®broblasts; TBS = Tris buered saline.
INTRODUCTION
Ochratoxin A (OTA) is a naturally occurring mycotoxin produced by several species of the fungal genera Aspergillus and Penicillium. Its occurrence in plant and animal products has been well documented (Krogh, 1987). It consists of a chlorinated dihydroisocoumarin moiety linked through its 7-carboxy group by an amide bound to L-b-phenylalanine (Fig. 1). OTA has been shown to have a number of *Corresponding author at: University of Rostock, Department of Biology, Institute of Animal Physiology, Division of Cellular Pathophysiology, UniversitaÈtsplatz 2, 18055 Rostock, Germany.
toxic eects, the most prominent is nephrotoxic (Krogh, 1992). In addition to the kidney, the urinary tract is a target organ of OTA (PetkovaBocherova and Castegnaro, 1991). OTA is also immunosuppressive, teratogenic and carcinogenic (Creppy et al., 1985, Dirheimer and Creppy, 1991). Based on the current literature, the toxicity of OTA may be the result of three major eects: (1) inhibition of ATP synthesis; (2) inhibition of protein synthesis; and (3) enhanced lipid peroxidation (Marquardt and Frohlich, 1992). The genotoxic status of OTA was controversial since almost all microbial and mammalian test assays were negative (Ueno and Kubota, 1976; Wehner et al., 1978).
0278-6915/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(99)00057-5
714
E. Dopp et al.
Fig. 1. Chemical structure of ochratoxin A.
Contradictory results were found for sister-chromatid exchange in CHO cells (for review, see KuiperGoodmann and Scott, 1989). Nevertheless, newer studies by FoÈllmann et al. (1995) have clearly shown that OTA induces SCEs in cultured porcine urinary bladder epithelial cells. Manolova et al. (1990) demonstrated that chromosomal aberrations occur, particularly on X chromosomes, produced in human lymphocytes in culture by OTA at a concentration of 15 nM. DNA adduct formation was found in vitro (e.g. Grosse et al., 1995) as well as in vivo (e.g. Petkova-Bocharova et al., 1998). Degen et al. (1997) studied the eects of OTA on micronucleus induction in vitro using ovine seminal vesicle cell cultures. These authors have shown a dosedependent induction of micronuclei in this test system. Hoehler et al. (1996) suggested that OTA induces free radical production. This eect may enhance the permeability of the cellular membrane to Ca2+. Chong and Rahimtula (1992) reported that administration of a single high dose or multiple lower doses of OTA to rats resulted in an increase of the renal cortex endoplasmatic reticulum ATP-dependent calcium pump activity. In connection with intracellular Ca2+ changes by bradykinin, disturbances of the actin stress ®bres have been described by WoÈll et al. (1993). Activation of calcium sensitive K+ channels is an element of cell volume regu-
lation and has been shown to induce altered mophology of cells in a variety of tissues. Syrian hamster embryo (SHE) ®broblasts are known as a useful model system for the analysis of chemically-induced primary chromosomal changes (Fritzenschaf et al., 1993). The results of these and a number of other investigations have shown that the SHE-micronucleus assay is a short-term test of a high predictive value (Schimann und DeBoni, 1991; Schmuck et al., 1988). In combination with immuno¯uorescent staining of kinetochores in micronuclei, clastogenic events, as well as the loss of whole chromosomes, are detectable (Degrassi and Tanzarella, 1988). In this study, we investigated the genotoxic potency of OTA to induce micronuclei in SHE cells and to characterize the induced micronuclei by kinetochore staining (CREST analysis). For further characterization of the cytotoxic eects of OTA, measurements of [Ca2+]i and observations of the actin cytoskeleton were performed.
MATERIALS AND METHODS
Cell cultures All experiments were performed with cultures derived from 13-day-old SHEs. Cell cultures were grown in a humidi®ed atmosphere with 12% CO2 in air at 378C. The culture medium used was IBR [modi®ed Dulbecco's modi®ed Eagle's medium (DMEM)] medium (GIBCO, Karlsruhe, Germany) supplemented with 15% foetal bovine serum (GIBCO, Karlsruhe, Germany), 3.7 g NaHCO3/litre (7.5%), 1% glucose, 10,000 IU penicillin/litre, 10 mg streptomycin/litre and 0.01% tylosin (Sigma, Germany).
Fig. 2. Occurence of micronuclei in SHE cells after treatment with dierent concentrations of ochratoxin A. Each data point represents the mean of three to ®ve counts of 2000 nuclei of dierent experiments.
OTA induces genotoxic and [Ca2+]i changing eects
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Table 1. Formation of micronuclei in SHE cells after treatment with ochratoxin A (10 mM and 15 mM) in dependence on exposure time No. of micronuclei/2000 cells Exposure time
10.0 mM OTA
Untreated control
3 hr 6 hr 18 hr 36 hr 54 hr 72 hr
21.02 0.7 32.02 0.7 48.72 2.6 50.52 1.9 47.72 5.8 46.02 2.7
15.0 mM OTA
32.0 21.4* 43.0 23.5** 76.0 28.6*** 91.6 22.0*** 78.6 215.8*** 68.7 215.2***
40.0 21.4** 55.0 23.5*** 80.6 27.3*** 109.3 26.8*** 91.3 28.0*** 82.3 219.6***
Signi®cance tested by the t-test: *P E0.05; **PE 0.01; ***PE0.001.
Micronucleus assay and kinetochore analysis SHE cells were grown on coverslips, and after treatment with ochratoxin A (Sigma, Germany; stock solution 1 mM in DMSO; ®nal concentrations in the culture medium 5 to 20 mM), cells were ®xed and stored in cold methanol (ÿ208C) for at least 30 min. After washing of cells with phosphate buffered saline (PBS), nuclei were stained with bisbenzimide (Hoechst 33258, concentration: 1 mg/ml, 4 min). The slides were then mounted for microscopy and examined for the presence of micronuclei. Each data point represents the mean of at least four treated cultures from dierent experiments with 2000 nuclei evaluated in each case. For further analysis of the induced micronuclei, kinetochores were stained by incubating the ®xed cell preparations with CREST serum (Chemicon, Temecula, CA, USA) for 1 hr in a humidi®ed chamber at 378C. After rinsing with phosphate buffered saline, the cells were incubated with ¯uorescein isothiocyanate (FITC) conjugated goat antihuman antibodies (Sigma, Germany) before applying bisbenzimide. At least 100 micronuclei were examined for the presence of kinetochores in each case.
magnesium free) and DMEM. An equivalent volume of DMSO was added to the control cultures. For each OTA concentration, 100 images in 53 sec were acquired at 488 nm excitation wave length and 515 nm emission wave length. The [Ca2+]i of SHE cells was measured by determining the overall ¯uorescence intensity (50 cells/frame) with the Intervision Software (Noran) on a Silicon Graphics Workstation. Rhodamin/phalloidin staining OTA-treated (10, 20, 30 mM; 18 and 36 hr) and untreated SHE cells were ®xed in paraformaldehyde (3%) for 15 min and treated with TBS + 0.1% Triton X-100 for permeablization of the cell membrane. The cells were incubated with a blocking solution [1% bovine serum albumin and 2% normal goat serum in tris buered saline (TBS)] for 5 min at room temperature and subsequently incubated with rhodamine/phalloidin (Sigma, Germany) for 15 min. After washing with TBS again, the cells were mounted with antifade and the actin skeleton was observed directly using a ¯uorescence microscope.
Measurement of intracellular calcium concentration SHE cells were plated and grown on Petriperm dishes. For [Ca2+]i measurements, the culture medium was removed and cells were incubated with PBS containing the calcium indicator ¯uo-3 AM (®nal concentration: 16 mM) for 30 min at 378C. After washing with PBS and treatment with OTA (dissolved in DMSO, stock solution: 1 mM), the ¯uorescence intensity of cells was measured using a confocal laser scanning microscope (Noran Odyssey). For various ®nal concentrations of OTA (15, 30, 60 mM) the time course was observed in PBS/CMF (phosphate buered saline/calcium and
RESULTS
The results of the micronucleus assay showed that OTA induces micronuclei in SHE cells in a dose-dependent manner (Fig. 2). A comparison of the dierences between MN formation in untreated and OTA-treated SHE cells revealed that these dierences are signi®cant at OTA concentrations of 5 mM to 20 mM and exposure times of 18 hr to 72 hr (Fig. 2). The time course of MN formation revealed that the number of induced MN is statistically signi®cant even after 3 hr of exposure and an OTA concentration of 10 mM (Table 1).
Table 2. Results of kinetochore staining of SHE cells after treatment with ochratoxin A
Ochratoxin A Control a
Concentration (mM)
Exposure time (hr)
No. of cells scored
No. of micronucleia
% CREST+b (mean 2SD)
5.0±15.0 ±
36 36
13868 6966
631 209
33.6 2 2.5 28.7 2 0.4
Number of micronuclei scored for presence of kinetochores. Percentage of detected micronuclei that reacted positively to antikinetochore serum.
b
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Fig. 3. Fluorescence intensity of OTA-treated and untreated SHE cells after incubating of cells with the calcium indicator ¯uo-3 AM for intracellular calcium measurements in calcium/magnesium free medium (PBS/CMF). The intracellular calcium concentration of untreated cells was about 100 nM. The point (*) indicates OTA administration.
OTA induces genotoxic and [Ca2+]i changing eects
The maximum induction of micronuclei is reached at 36 hr exposure and 15 mM OTA (Table 1). At higher concentrations and longer exposure times, the frequency of micronuclei decreased again as a result of increased cytotoxicity (Fig. 2). The results of the kinetochore analysis showed that OTA induces both kinetochore-positive
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(CREST+) and kinetochore-negative (CRESTÿ) micronuclei in SHE cells (Table 2). 33.6% of induced micronuclei contain whole chromosomes or chromatids indicating aneuploidy. The higher percentage of kinetochore-negative micronuclei (CRESTÿ: 66.4%) indicates that these mainly originate from clastogenic events. The dierence between kinetochore containing micronuclei of trea-
Plate 1. Staining of actin ®laments of OTA-treated (i) 20 mM OTA, 18 hr exposure) and untreated (ii) SHE cells with rhodamin/phalloidin.
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Fig. 4. Fluorescence intensity of OTA-treated and untreated SHE cells after incubating of cells with the calcium indicator ¯uo-3 AM for intracellular calcium measurements in calcium containing medium (DMEM). The point (*) indicates OTA administration.
OTA induces genotoxic and [Ca2+]i changing eects
ted and untreated cells is statistically not signi®cant (Table 2). Our investigations have also shown that the treatment of SHE cells with OTA results in disturbances of intracellular Ca2+ homeostasis. Measurements of SHE cells in PBS/CMF (medium without calcium) and DMEM (medium containing calcium) showed an increase of [Ca2+]i within the ®rst seconds after treatment with OTA. The obtained ¯uorescence intensities of untreated and OTA-treated SHE cells are shown in Figs 3 and 4. In PBS/CMF, the [Ca2+]i is signi®cantly increased at OTA concentrations of 30 mM (Fig. 3b). At higher concentrations (60 mM) the [Ca2+]i is not further increased compared with the untreated control (Fig. 3c). In PBS/CMF, the [Ca2+]i level is maintained at a constantly elevated level, whereas in DMEM (extracellular calcium concentration: 1.8 mM) the [Ca2+]i elevation appears as a short spike signal within seconds after OTA administration, and it decreases again below the normal level (Fig. 4f). The intracellular calcium rise is about 45% higher in SHE cells treated with 60 mM OTA in calcium containing medium in comparison to SHE cells treated with 30 mM OTA in calciumfree medium. Visualization of the actin ®laments showed strong eects on actin ®bres of OTA treated SHE cells (Plate 1). Actin staining was performed at 10, 20, 30 mM OTA concentrations and 18 hr and 36 hr exposure time (the cells were incubated with OTA in DMEM). The observed eects were time and concentration dependent (no more data shown). Cell shrinkage and depolymerized ®laments increased with higher concentrations and longer exposure times. These eects were not reversible up to 36 hr recovery time. Longer recovery times (>36 hr) were not tested. DISCUSSION
Previous studies showed that OTA may inhibit synthesis of protein and DNA (Creppy et al., 1986; Gekle et al., 1995) and induces alterations in calcium homeostasis (Rahimtula and Chong, 1991). Neither the time course of intracellular calcium changes nor changes of the actin cytoskeleton after OTA treatment were investigated so far. Degen et al. (1997) reported about induction of micronuclei with OTA in ovine seminal vesicle cell cultures (OSV). OTA was found to induce dose-dependently micronuclei in cytokinesis-blocked binucleated OSV. A maximum of MN formation was achieved at 10 mM OTA and 6 hr of exposure. However, Degen et al. did not test exposure times other than 6 hr. When induced MN were characterized in our investigations by indirect immuno¯uorescence microscopy using antikinetochore (CREST) antibodies, the OTA-treated fraction of kinetochorepositive MN was similar to that observed in solvent
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controls. These data indicate that OTA induces MN apparently by a mixed, although predominantly clastogenic, mode of action. It is possible that OTA induces DNA damage by disturbances of chromosomes/chromatin in a direct or an indirect way (production of free radicals by OTA, Hoehler et al., 1996). OTA also induced an intracellular calcium rise. The time course of this eect in calcium-containing medium is rather typical for a physiological response and not for a ``damaging signal''. The role of Ca2+ as an intracellular regulator of many physiological processes is well established. Ca2+ can also play a determinant role in pathological processes (Nicotera et al., 1992). It has been proposed that intracellular Ca2+ accumulation may be a common step in the development of cytotoxicity because disturbances of Ca2+-regulated mechanisms are often early events in the development of cell injury (Hoehler et al., 1996). Normally, the Ca2+ concentration in the cytosol of unstimulated cells is maintained between 0.05 and 0.2 mM. Extracellular Ca2+ levels are approximately 1.3 mM. This produces a large electrochemical gradient that is mainly balanced by active Ca2+ extrusion through the plasma membrane and by co-ordinated activity of Ca2+-sequestering systems located in the mitochondrial, endoplasmatic reticular and nuclear membranes (Nicotera et al., 1992). Disturbances of these processes can result in enhanced Ca2+ in¯ux, release from intracellular stores and inhibition of Ca2+ extrusion at the plasma membrane. Calcium sensitive channels can also be in¯uenced. Hoehler et al. (1996) suggested that OTA induces free radical production by enhancing the permeability of the cellular membrane to Ca2+. Our experiments have shown that the enhanced [Ca2+]i caused by OTA treatment is a result of the Ca2+ release from intracellular stores as well as of damaged cell membranes with an increased in¯ux of extracellular Ca2+. After a short spike, the [Ca2+]i decreased again below the normal level in calcium-containing medium. This can be caused by an increase of the calcium pump activity. Rahimtula and Chong (1991) also observed an increase in renal endoplasmatic reticulum calcium pump activity after administration of OTA to rats in vivo. In contrast to the increased calcium pump activity of OTA treated SHE cells in calcium containing medium, the calcium pump activity seems to be inhibited in calcium-free medium, or intracellular calcium stores are damaged. The [Ca2+]i level remains elevated in PBS/CMF. Rahimtula and Chong (1991) published evidence that OTA administration to rats in vivo resulted in an decreased renal mitochondrial state-3 respiration and calcium uptake. These authors hypothesized that a decrease in microsomal calcium pump activity could lead to higher cytosolic calcium levels.
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An increase of intracellular calcium concentration can also contribute to cell shrinkage. WoÈll et al. (1993) descibed that an inhibition of K+ and Clÿ channels by barium (5 mM) and OTA (5 mM) prevents an intracellular alkalinization. Calcium triggers calcium-sensitive K+ channels, and presumably Clÿ channels, the subsequent loss of cellular KCl leads to cell shrinkage which, in turn, activates Na+/H+ exchange. Yokoshiki et al. (1997) concluded from their investigations with cytochalasin D that the disruption of the actin cytoskeleton attenuates the ability of the sulfonylurea receptor to inhibit the opening of ATP-sensitive K+ channels. OTA seems to have similar eects on actin ®laments like cytochalasin D. Van Deurs et al. (1996) described an actin ®lament-depolarization after treatment with cytochalasin D (10 mM). In contrast to OTA, the ®lament formation is already reversible after 2 hr of cytochalasin D incubation in about 20% of cases. We suggest that the disruption of actin ®laments after OTA treatment do not correlate with the increased [Ca2+]i. It is more likely that OTA binds directly and irreversibly to actin. In summary, OTA was found to induce dosedependently micronuclei in SHE ®broblasts. The characterization of the induced MN revealed that mainly kinetochore-negative MN were induced. We conclude that OTA has a predominantly clastogenic mode of action. Measurements of the intracellular calcium concentration have shown an increase of [Ca2+]i within the ®rst seconds after OTA treatment. This time course is typical for a physiological response. Ca2+-release from intracellular stores as well as Ca2+ in¯ux from extracellular media are involved in OTA cytotoxicity. Observations of the actin cytoskeleton of OTA-treated SHE cells revealed time- and concentration-dependent eects, such as cell shrinkage and depolymerized ®laments. These studies reveal that OTA is an eective inducer of DNA damage, of disturbances of the intracellular calcium homeostasis, and of disruption of the actin stress ®bres. AcknowledgementÐThe authors thank Mrs Jutta Saedler for excellent technical assistance. REFERENCES
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