Prevention of lipid peroxidation induced by ochratoxin A in Vero cells in culture by several agents

ELSEVlER

Chemico-Biological Interactions 104 (1997) 29-40

Prevention of lipid peroxidation induced by ochratoxin A in Vero cells in culture by several agents I. Baudrimont,

R. Ahouandjivo,

E.E. Creppy *

Laboratoire de Toxicologic et d’Hygi&ze appliqke, UFR des Sciences Pharmaceutiques, UniLersitP Victor Segalen Bordeaux 2- 146 rue LCo Saignat, 33076 Bordeaux cedex, France

Received 28 August 1996; received in revised form 24 January 1997; accepted 28 January 1997

Abstract Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus ochraceus as well as other moulds. This mycotoxin contaminates animal feed and food and is nephrotoxic for all animal species studied so far. OTA is immunosuppressive, genotoxic, teratogenic and carcinogenic. It is a structural analogue of phenylalanine and contains a chlorinated dihydroisocoumarinic moiety. Ochratoxin A inhibits protein synthesis by competition with phenylalanine in the phenylalanine-tRNA aminoacylation reaction. Recently lipid peroxidation induced by OTA has been reported, indicating that the lesions induced by this toxin could also be related to oxidative damage. An attempt to prevent its toxic effect, mainly the lipid peroxidation, has been made using aspartame (L-aspartyl-L-phenylalanine methyl ester) a structural analogue of both OTA and phenylalanine, piroxicam, a non steroidal anti-inflammatory drug and superoxide dismutase + catalase (endogenous oxygen radical scavengers). Lipid peroxidation was assayed in monkey kidney cells (Vero cells) treated by increasing concentrations of OTA (S-50 ,uM). After 24 h incubation OTA induced lipid peroxidat;:on in Vero cells in a concentration dependent manner, as measured by malonaldehyde (MDA) production. The MDA production, in Vero cells, was significantly increased by 50.5% from 694.1 4 21.0 to 1041.5 f 23.5 pmol/mg of protein. In the presence of superoxide dismutase (SOD) + catalase (25 pg/ml each) the MDA production induced by OTA was

* Corres,>onding author. Tel.: + 33 56 986606; fax: + 33 56 986685. 0009-2797/‘37/$17.00

0 1997 Elsevier Science Ireland Ltd. All rights reserved

PII SOOOS-2797(97)03764-2

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significantly decreased. At 50 ,uM of OTA concentration (optimal production of MDA) the MDA production decreased from 1041.5 k 23.5 to 827.5 + 21.3 pmol/mg of protein. SOD and catalase, when applied prior to the toxin, seemed to prevent lipid peroxidation more efficiently than piroxicam (at a ten-fold higher concentration than OTA) and aspartame (at equimolar concentration). These molecules also partially prevented the OTA-induced leakage of MDA in the culture medium. 0 1997 Elsevier Science Ireland Ltd. Keywords: Ochratoxin A; Lipid peroxidation; Malonaldehyde; tame, piroxicam and superoxide dismutase + catalase

Preventive

effects

of aspar-

1. Introduction Ochratoxin A (OTA) is a mycotoxin produced by several species of fungi, especially from the Aspergillus and Penicillium genera [l]. It is a natural contaminant of mouldy feed and food (mainly cereals, dried fruits, beans, coffee and cocoa). OTA has also been identified in the blood of animals and humans after consumption of contaminated food, in the Balkans, Scandinavia, Germany, France, Canada, Japan and Northern Africa [2-61. OTA has a number of toxic effects, the most prominent being nephrotoxicity. In animals it induces a tubulo-interstitial nephropathy [7,8]. It is also presumed to be involved in a fatal human chronic kidney disease called balkan endemic nephropathy (BEN), a chronic tubulo-interstitial nephropathy [2,9]. Many people dying from the disease have also developed tumours in the the urinary tract [2,10]. In addition to this nephrotoxicity, ochratoxin A disturbs blood coagulation [ll], glucose metabolism [12] and is immunosuppressive [13,14], teratogenic [15,16], genotoxic [17,18] and carcinogenic [19,20]. Ochratoxin A is a structural analogue of phenylalanine and consists of a chlorinated dihydroisocoumarin moiety linked, through its 7-carboxyl group, by an amide bound, to L-p-phenylalanine (Fig. 1). OTA inhibits protein synthesis by competition with phenylalanine in the phenylalanyl-tRNA synthetase catalysed reactions [2 1~ 231. It also induces oxidative damages by enhancing lipid peroxidation [24,25]. OTA enhances lipid peroxidation when added to rat liver or kidney microsomes, in vitro, or when administered, in vivo, to rats [25]. Transition metals and iron ions are involved in lipid peroxidation [26]. In a reconstituted microsomal system, containing NADPH-CYP 450 reductase, EDTA, iron ions and NADPH, OTA stimulates lipid peroxidation by chelating Fe’+ ions, resulting in an 0TApFe3+ complex which is readily reducible by an NADPH-CYP 450 reductase to an 0TApFe2+ complex. The rate of reduction of Fe-’ + to Fe2 + largely increases in the presence of OTA [24,25]. In the presence of oxygen the OTA-Fe2+ complex provides the active species which initiates lipid peroxidation. Once this process is initiated, it can be easily propagated in the cellular environment where polyunsaturated fatty acids and oxygen are present. The oxidation of lipids by oxygen continues in a chain of

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31

radical reactions. As a consequence of this biochemical process, a wide range of degradat:ion compounds such as malonaldehyde and other oxidation products are formed [:27-291. Most of these products are chemically very reactive and produce structural tissue injuries [27,30]. Considering the implication of OTA in public health and the difficulty to completely prevent the proliferation of toxinogenic moisture it seems necessary to search for a way of preventing OTA-induced toxic effects. Since lipid peroxidation is one of the main mechanisms of OTA-induced cytotoxicity, and the kidney being the specific target, we tested the capability of (1) piroxicam, a non-steroidal anti-inflammatory drug (Fig. 1); (2) of aspartame, a structural analogue of both OTA and phenylalanine (Fig. 1); and of (3) superoxide dismutase (SOD) + catalase, endogenous oxygen radicals scavengers; to prevent ochratoxin A-induced lipid peroxidation in monkey kidney cells (Vero cells). In addition, the action of these potentiel protective agents on OTA-induced leakage of MDA in the culture medium was also determined.

Ochratoxin A

I

CO-0CH3 CH2-CH-NH-CO-CH-NH2 I CH, - COOH

Aspartame

Piroxicam

Fig. 1. Chemical

structures

of ochratoxin

A, aspartame

and piroxicam

32

I. Baudrimont

2. Materials

and methods

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There are a variety of methods available for detection and quantification of lipid peroxidation in biological samples. They are all based on the detection of intermediates or end-products of lipid peroxidation (decrease in polyunsaturated fatty acids, oxygen consumption, determination of aldehyde, malonaldehyde and ethane exhalation) [31,32]. However, all of these methods are not appropriated to in vitro models. The determination of malonaldehyde, an end-product formed during lipid peroxidation is an excellent index of tissue damage induced by oxygen free radicals [31,33]. Several methods for MDA determination have been reported recently for the measurement of lipid peroxidation in oxidized lipids or biological samples. Due to its simplicity and its sensitivity the most widely used assay for in vitro detection of MDA level is based on its reaction with thiobarbituric acid (TBA). This method consists of inducing a fluorescent complex between TBA and MDA [34]. The MDA-TBA chromophore absorbs at 515 nm and can be detected by fluorometry. But this test is not very specific because other compounds may give a positive reaction with TBA. Many aldehydes can react with TBA to produce a fluorescent complex. To improve sensitivity and specificity our method consists of a butanolic extraction, a purification and a separation of the MDA-TBA complex, by HPLC. In these conditions no complexes of TBA and other reactive substances are detected. 2.1. Cell culture conditions Vero cell lines (Biovalori, France), originating from monkey kidney [35], were cultured, in monolayer, in RPM1 1640 medium (Sigma, St Louis MO), supplemented with 4% L-glutamine (Sigma, St Louis, MO), gentamycine (80 @g/ml) (Shering-Plough, France) and 5% of foetal calf serum (FCS) (Biovalori, France). Cells were routinely incubated, for 24 h, in 6-well culture discs (Greiner, Labortechnik, Germany), in an humidified 5% CO,/95% air mixture, at 37°C. Three wells were used for each concentration, in 3 ml culture medium per well containing lo5 cells/ml. In a first set of cells, OTA (CSIR, South Africa) dissolved in NaHCO, 0.1 M pH 7.4 ranging from 5 to 50 ,uM final concentration was added to the culture medium for an incubation period of 24 h. A second set of cells was pretreated respectively with aspartame (500 PM) (Searle, France), piroxicam (50 PM) (KRKA, Novo Mesto, Slovenia) and SOD (25 pug/ml) + catalase (25 pg/ml) (Sigma, St Louis, MO) for 24 h, 24 h and 4 h respectively, before adding OTA. The control wells only received NaHCO, 0.1 M pH 7.4, for 24 h, with or without these potential protective agents. After 24 h of incubation with or without the toxin the culture medium was removed (and kept for the assay of MDA in the culture medium) and the cells were collected, by scraping, using a cell scraper (Costar), into fresh RPM1 medium (lacking serum). They were centrifuged, for 10 min, at 600 x g. The pellets were resuspended in 250 ~1 of 50 mM Tris-HCl buffer solution, pH 8, containing 0.1 M

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NaCl anId 20 mM EDTA. Afterwards, 25 ,~l of sodium dodecyl sulfate (SDS) 7% (w/v), 300 ,ul of HCl 0.1 N, 40 ,~l of phosphotungstic acid 1% (w/v) and 300 ,~l of thiobarbnuric acid 0.67% (w/v) were added to each sample. The mixture was shaken and after 1 h incubation in the dark, at 80°C the cell lysates were cooled in an ice water bath (O’C) for 10 min. 2.2. Extraction and puriJication of MDA-TBA

adduct by HPLC

300 ~1 of n-butanol was added to each tube and the mixture was vigorously shaken. After centrifugation, 10 min at 3000 x g, the n-butanol layer was taken off for HPLC purification and the aqueous phase was kept for the protein assay. A standard curve (125-500 pmol/ml) was prepared using a 20 mmol/l MDA solution in the salme medium, and treated similarly to OTA and control wells for extraction and quantification of MDA. Analysis of the MDA-TBA adducts was performed, at room temperature, by high performance liquid chromatography (HPLC) and microfluorometric detection, at emission/excitation wavelengths respectively of 515/553 nm, using methanol-water 40:60 (v/v) pH 8.3 as the mobile phase, at a flow rate of 0.5 ml/min. The HPLC system consisted of a Bischoff Model A 2200 pump, an Alcott Model 738 Autosampler injector, a Spherisorb ODS column Cl8 (250 x 8 mm), 6 ,uM and a Jasco 821-FP fluorescence HPLC monitor. Analytical data were collected, stored and processed using the software Pit 3 developed by ICS (Instrumentation Consommable Service, France). 2.3. Determination of the protein concentration Protein concentration was quantified in the culture homogenates using the Bradford method [36].

medium

and in the cellular

2.4. Statistical analysis All the results were expressed as the mean f S.E. on the mean (S.E.M.) in pmol of MDA/mg of proteins. Statistical analysis was carried out using the Wilcoxon Rank Sum Test.

3. Results and discussion Vero cell lines established from monkey kidney were used in this study because they had been found to be sensitive to OTA and showed good metabolic activities for this toxin [37]. Lipid peroxidation is one of the cellular pathways involved in oxidative damage induced by OTA [32-341. The reactive oxygen species and free radicals also induced a wide range of le(0,. -> OH., ROO . ), initiated by lipid peroxidation, sions on target tissues such as membranes, proteins, nucleic acid. These oxygen free

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radicals are able to interact with DNA to cause oxidative DNA damage [38]. The first consequences of this harmful mechanism are bases modification, denaturation of the DNA and formation of DNA-adducts, which may induce genotoxic, mutagenic and perhaps carcinogenic processes. Previously, it has been reported that the oxidative pathway was probably responsible for the formation of the majority of DNA-adducts induced by OTA [38]. Since lipid peroxidation is one of the main mechanisms of OTA-induced toxicity several molecules, which were found to largely prevent in vivo the nephrotoxicity and the genotoxicity of OTA [39-411, were tested for their possible beneficial effects on OTA-induced lipid peroxidation. The compounds studied, in this purpose, were chosen among many candidates. These were radical scavengers such as SOD + catalase, structural analogues such as piroxicam and aspartame (Fig. 1) which also prevented the inhibition of protein synthesis induced by OTA. Aspartame, the L-aspartyl-L-phenylalanine methyl ester, being used as sweetner for at least two decades, has been chosen because of its structural analogy to OTA (Fig. 1). Superoxide dismutase removes reactive oxygen by converting it into hydrogen peroxide. This enzyme works in conjunction with catalase which removes hydrogen peroxide by conversion into water. It has also been shown that: (i) these two enzymes given together to rats inhibit the nephrotoxicity induced by cyclosporine A [42]; (ii) they are now used in humans to reduce the adriamycine-induced adverse effects and (iii) some myocardic and renal ischemia in which the action of free radicals is suspected, are also being treated by SOD + catalase [43]. Piroxicam, a structural analogue of OTA (Fig. l), is a powerful inhibitor of prostaglandin 2 (PG2) synthesis as other NSAIDs [44]. This property of piroxicam could be relevant in the prevention of OTA induced toxic effects mediated by the oxidative pathway. Thus piroxicam, which in addition has also some structural analogy to OTA, could be expected to prevent OTA-induced genotoxicity and carcinogenicity since it has already been shown to prevent the carcinogenicity of a nitrosamine derivative and that of the 2-acetyl aminofluorene [45,46]. In previous investigations the addition of aspartame 24 h prior to ochratoxin A proved to be helpful in the preventive effect of this compound [47]. Piroxicam was also added prior to OTA in some experiments for the same purpose. Concerning the enzymes (SOD + catalase) they were applied subcutaneously by Wolf et al [42], 1 h before injection of cyclosporine in rat and this procedure was beneficial for their preventive action. These data prompted us to follow similar procedure since maximal protective effects were expected. Under our experimental conditions, the lowest amount of MDA detected by HPLC system was about 50 pmol/ml of extract or of cell homogenates. The lowest amount detected, in Vero cell controls (untreated cells), was about 650 pmol/mg of protein (100000 cells/ml). Our method is sufficiently specific and sensitive for the detection of lipid peroxidation in Vero cells. After 24 h incubation, Vero cells in culture spontaneously produced 676.2 _+ 17.5 pmol/mg of protein of MDA (Fig. 2). This value could be considered as the basic value. After 24 h incubation with increasing concentration of OTA (O-50 PM) the

I. Baudrimont et al. / Chemico-Biological Interactions 104 (1997) 29-40

Untreated

Controls (NaHC03)

5

10

OTA concentrations Fig. 2. Lipid peroxidation measured by quantification

35

50

25

(m

induced by increasing concentrations of ochratoxin of malonaldehyde, after 24 h incubation.

A, in Vero

ceils, as

MDA production in Vero cells was significantly increased (P = 0.01) from 694.2 f 21.0 to 1041.5 f 23.5 pmol/mg of protein (Fig. 2). The MDA production increased 14.3-50.5% by 10 and 50 ,uM respectively of OTA as compared to the controls (NaHCO,). It should be pointed out that under the same experimental conditions, after 24 h of incubation with the toxin, the protein synthesis was inhibited by about 45% and 85% with 10 and 50 PM respectively of OTA concentration [47] while the production and release of MDA were respectively increased by only 14 and 50%. After a rapid set up of protein synthesis inhibition (30 min to 1 h), the inhibition gradually increased to reach a plateau which remained unchanged after 24 h [23]. So lipid peroxidation seemed to appear as a consequence of the inhibition of protein synthesis in Vero cells since inhibition of protein synthesis could reduce enzymatrc systems involved in detoxication of oxygen radicals. When Vero cells were pretreated with SOD + catalase (25 pg/ml each) 4 h before OTA (5550 ,uM) intoxication, the MDA production decreased significantly as compared to the OTA alone-treated cells (Table 1). In the presence of SOD + catalase the MDA production was significantly decreased from 1041.5 f 23.5 to 827.5 + 21.3 pmol/mg of protein at 50 ,uM of OTA concentration (optimal production of MDA). When added to the culture medium 4 h before OTA intoxication, these two enzymes seemed to be efficient in preventing OTA induced lipid peroxidation in Vero cells but they didn’t completely prevent the MDA production. Results in

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Table 1 Effect of superoxide cells

dimutase

OTA concentration

(pM)

combined

to catalase

Untreated controls Controls (NaHCO,) SOD + catalase OTA 5 PM OTA 10 ELM OTA 25 ELM OTA 50 /.LM OTA (5 pM)+ SOD+catalase OTA (10 p M) + SOD + catalase OTA (25 p M) + SOD + catalase OTA (50 p M) + SOD + catalase

on the lipid peroxidation

MDA

concentration

616.2 694.1 677.8 743.5 793.1 907.3 1041.5 690.0 741.2 783.3 827.5

k + + f k + k k + + k

(pmoljmg

induced

by OTA in Vero

of protein)

17.5 21.0 19.4* 27.3* 29.2*+ 28.4** 23.5** 14.3*** 1.5*** 16.5*** 21.3***

Superoxide dismutase (25 fig/ml), catalase (25 pg/ml) and OTA (5-50 PM) was used. Results after 24 h incubation. * Not significantly different from controls (NaHCO,), P = 0.01 (Wilcoxon Rank Sum Test). ** Significantly different from controls (NaHCO,), P = 0.01. *** Significantly different from OTA alone-treated cells, P = 0.01.

recorded

Table 1 strongly suggest that at least two classes of radicals are involved: active oxygen radicals and other free radicals. One of these is easily scavenged by SOD + catalase whereas the other is not. This is the reason why there is always a certain rate of lipid peroxidation with OTA high concentration, even when the scavengers are present. When Vero cells were pretreated for 24 h before OTA intoxication (50 PM) with aspartame (500 ,uM) or piroxicam (50 PM), the MDA production was significantly decreased as compared to the OTA alone-treated cells (Table 2). In the presence of Table 2 Effect of piroxicam

or aspartame

on the lipid peroxidation

Treatment Untreated Controls Piroxicam Aspartame OTA (50 OTA (50 OTA (50

MDA controls (NaHCO,) (50 PM) (500 PM) PM) p M) + piroxicam yM)+aspartame

(50 p M) (500 PM)

646.0 635.5 656.0 641.4 963.4 882.7 833.1

concentration k f + k k + +

induced

by OTA in Vero cells

(pmol/mg

of protein)

22.1 17.5 11.2* 23.5* 14.0** 9.05*** 8.3***

Piroxicam (50 PM), aspartame (500 PM) and OTA (50 PM) were used. incubation. * Not significantly different from controls (NaHCO,), P = 0.01 (Wilxocon ** Significantly different from controls (NaHCO,), P = 0.01. *** Significantly different from OTA alone-treated cells, P = 0.01.

“/o increase

3.2 1.8 51.6 38.9 31.1 Results

recorded

Rank

Sum Test).

after

24 h

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piroxicam (50 PM) the MDA production was significantly decreased from 963.4 + 14.0 to 882.7 f 9.5 pmol/mg of protein at 50 ,LLMof OTA concentration (Table 2). In the presence of aspartame (500 PM), the MDA production was significantly decreased from 963.4 + 14.0 to 833.1 + 8.3 pmol/mg of protein. If lipid peroxidation is considered as a consequence of protein synthesis inhibition, the protective effects of aspartame and of piroxicam could be explained by their efficiency in preventing the OTA-induced inhibition of protein synthesis in Vero cells as shown previously [47]. Assuming that OTA is activated by prostaglandin synthetase pathway [48] and since piroxicam is a specific inhibitor of prostaglandin synthesis [44], this molecule could avoid activation in Vero cells and consequently prevent the damage induced by the activated OTA-compounds. Chelation of iron by the ochratoxin molecule could be a mechanism leading to lipid peroxidation. It is not known whether aspartame and piroxicam also chelate iron but we cannot exclude it. However both affect the metabolism of OTA by increasing its detoxication into OTa mainly [49]. When applied prior to the toxin, SOD + catalase seemed to prevent the lipid peroxidation more efficiently than piroxicam and aspartame. The combination of several of these protective agents could be considered. The MDA production was also assayed in the culture medium after 24 h of incubation of Vero cells with increasing concentrations of OTA (5-50 PM). After 24 h of incubation, the toxin (25, 50 PM) induced the leakage of MDA in the culture medium as compared to the controls. This leakage of MDA in the culture medium, which is concentration dependent, could be explained by the attack of the membrane by reactive oxygen radicals leading to the alteration of the membrane integrity and the increase of the cellular permeability. This was confirmed by a neutral red dye test which showed 70% of cytotoxicity with 50 ,uM of OTA in the culture medium [SO]. When the cells were pretreated for 4 h, 24 h and 24 h respectively with SOD + catalase (25 pg/ml), aspartame (500 PM) and piroxicam (50 PM) the amounts of MDA released in the culture medium were significantly decreased indicating that the membranes were less damaged. All these results showed that after 24 h incubation, OTA induced the production of free reactive oxygen species in Vero cells. These oxygen species were responsible for the -peroxidation of membrane lipids. SOD + catalase, piroxicam and aspartame, when added to the culture medium prior to OTA addition to the medium, seemed to be efficient in preventing lipid peroxidation, one of the main mechanisms of OTA-,induced cytotoxicity in Vero cells.

Acknowledgements The authors are grateful to the Minis&&-e de I’Education Nationale et de la Recherche Scientifique (contrat DRED no. 5696) and to the University of Bordeaux 2. Thanks to Mrs Mary C. Ward from Ireland, for linguistic advice.

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References [I]

K.J. Van der Merwe, P.S. Steyn, L. Fourrie, D.B. Scott and J.J. Theron, Ochratoxin A, a toxic metabohte produced by Aspergillus ochraceus Wilh, Nature, 205 (1965) 1112-1113. I.N. Chernozemsky and’ M. Castegnaro, Ochratoxin A in human blood in PI T. Petkova-Bocharova, relation to Balkan Endemic Nephropathy and urinary system tumours in Bulgaria, Food Addit. Contam., 5 (1988) 299-301. and P.M. Scott, Risk assessment of mycotoxin ochratoxin A, Biomed. [31 T. Kuiper-Goodman Environ. Sci., 2 (1989) 1799248. 141A. Breitholtz, M. Olsen, A. Dahlback and K. Huh, Plasma ochratoxin A levels in three Swedish populations surveyed using an ion-pair HPLC technique, Food Addit. Contam., 8 (1991) 1833192. H. Bartsch, P. Monchar151 E.E. Creppy, A.M. Betbeder, A. Gharbi, J. Counord, M. Castegnaro, mom, B. Fouillet, P. Chambon and G. Dirheimer, Human ochratoxicosis in France, In: M. Castegnaro, R. Plestina, G. Dirheimer, I.N. Chernozemsky and H. Bartsch (Eds.), Mycotoxin, Endemic Nephropathy and Urinary Tract Tumors. IARC Sci. pub. Lyon Int. Agency Cancer Res., 115 1991, pp. 145151. et ochratoxiPI H. Bacha, K. Maaroufi, A. Achour, M. Hammami and E.E. Creppy, Ochratoxines cases humaines en Tunisie, In: E.E. Creppy, M. Castegnaro, G. Dirheimer (Eds.), Human ochratoxicosis and its pathologies, Colloque INSERM/John Libbey Eurotext Ltd, 231, 1993, pp. III-121 B. Hald, J. Hyldgaard-Jensen, A.E. Larsen, [71 P. Krogh, N.H. Axelsen, F. Elling, N. Gyard-Hansen, A. Madsen, H.P. Mortensen, T. Moller, O.K. Petersen, U. Ravnskov, M. Rostgaard and 0. Aalund, Experimental porcine nephropathy changes of renal function and structure induced by ochratoxin A-contaminated feed, Acta Pdthol. Microbial. Stand. Section A, Suppl., 246 (1974) I-21. and G. Dirheimer, Changes in urinary and renal tubular 181 A. Kane, E.E. Creppy, R. Roschenthaler, enzymes caused by subchronic administration of ochratoxin A in rats, Toxicology, 42 (1986) 2333243. and food born [91 P. Krogh, B. Hald, R. Plestina and S. Ceovic, Balkan Endemic Nephropathy ochratoxin A: Preliminary results of survey of foodstuffs, Acta. Pathol. Microbial. Stand. Section B, 85 (1977) 2388240. H. Bartsch and I.N. Chernozemsky, Endemic nephropathy and urinary tract UOI M. Castegnaro, tumors in the Balkans, Cancer Res., 47 (1987) 360883609. G. Bodin, M. Alvinerie and J. More, Physiopathology of [I 11 P. Galtier, B. Boneu, J. Charpenteau, haemorrhagic syndrom related to ochratoxin A intoxication in rats, Food Cosmet. Toxicol., 17 (1979) 49953. [I21 M.J. Pitout, The effect of ochratoxin A on glycogen storage in rat liver, Toxicol. Appl. Pharmacol. 13 (1968) 299-306. lmmunosuppression by ochratoxin 1133 H.D. Haubeck, G. Lorkowski, E. Kolsch and R. Roschenthaler, A and its prevention by phenylalanine, Appl. Environ. Microbial., 41 (1981) 1040~1042. and G. Dirheimer, Effects of two metabolites of [I41 E.E. Creppy, F.C. Starmer, R. Roschenthaler ochratoxin A, [4R]-4-hydroxy ochratoxin A and ochratoxin A, on the immune response in mice, Infect. Immun., 39 (1983b) 101551018. u51 R.G. Arora, and H. Friilen, Interference of mycotoxins with prenatal development of the mouse. II. Ochratoxin A induced teratogenic effects in relation to the dose and stage of gestation, Acta. Vet. Stand., 22 (1981) 5355552. [161 K. Mayura, R. Parker, W.O. Berndt and T.D. Phillips, Dchrdtoxin A induced teratogenesis in rats: partial protection by phenylalanine, Appl. Environ. Microbial., 48 (1984) I 186-I 188. and S. Mousset, Genotoxicity of [I71 E.E. Creppy, A. Kane, G. Dirheimer, C. Lafarge-Frayssinet ochratoxin A in mice: DNA single-stand breaks. Evaluation in spleen, liver and kidney, Toxicol. Lett. 28 (1985) 29935. K. Chakor, E.E. Creppy and G. Dirheimer, DNA adduct formation in mice [I81 A. Pfohl-Leszkowicz, treated with ochratoxin A. In: M. Castegnaro, R. Plestina, G. Dirheimer, I.N. Chernozemsky, and

I. Buudrimont

[I91 [20]

[21] [22]

[23]

[24] [25] [26] [27] [28] [29] [30]

[31] [32]

[33] [34]

[35] [36] [37]

[38]

[39]

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104 (1997) 29-40

39

H. Bartsch (Eds.), Mycotoxin, endemic nephropathy and urinary tract tumors. IARC Sci. pub. Lyon Int. Agency Cancer Res., 115, 1991, pp. 245-253. AM Bendele, W.W. Carlton, P. Krogh and E.B. Lillehoj, Ochratoxin A carcinogenesis in the (C 57 BL/6JxC3H)Fl mouse, J.N.C.I., 75 (1985) 733-739. NTP, Technical Report on the Toxicology and Carcinogenesis Studies of Ochratoxin A. (CAS No. 303-4.7-g) in F 344/N rats (gavage studies), In: G. Boorman, (Ed.), (NIH Publication No. 89-2813) Research Triangle Park, NC, US Department of Health and Human Services, 1989. I. Bunge, G. Dirheimer and R. Riischenthaler, In vivo and in vitro inhibition of protein synthesis in B~!cillus stearothermophilus by ochratoxin A, Biochem. Biophys. Res. Comm. 83 (1978) 3988405. E.E. Creppy, A.A.J. Lugnier, F. Fasiolo, K. Heller, R. Roschenthaler and G. Dirheimer, In vitro inhibition of yeast phenylalanine-tRNA synthetase by ochratoxin A, Chem. Biol. Interact., 24 (1979) 257-262. E.E. Creppy, D. Kern, P.S. Steyn, R. Vleggaar, R. Riischenthaler and G. Dirheimer, Comparative study of the effect of ochratoxin analogues on yeast aminoacyl-tRNA synthetases and on the growth and protein synthesis of hepatoma cells, Toxicol. Lett., 19 (1983b) 217-224. R.F. Omar, B.B. Hasinoff, F. Mejilla and A.D. Rahimtula, Mechanism of ochratoxin A stimulated lipid peroxidation, Biochem. Pharmacol., 40, 6 (1990) 118331191. R.F. Omar, A.D. Rahimtula and H. Bartsch, Role of cytochrome P450 in ochratoxin A-stimulated lipid peroxidation, J. Biochem. Toxicol., 6, 3 (1991) 2033209. B. Halliwell, Reactive oxygen species in living systems: source, biochemistry and role in human disease, Am. J. Med., 91 (suppl. 3C) (1991) 14-22. B. Halliwell and J.M.C. Gutteridge, Free radicals and antioxydant protection: Mechanisms and signijiciance in toxicology and disease, Hum. Toxicol., 7 (1988) 7713. H. Esterbauer, Cytotoxicity and genotoxicity of lipid-oxidation products, J. Clin. Nutr., 57 (1993) 779SS786S. G. \‘an Ginkel and A. Sevanian, Lipid peroxidation-induced membrane structural alterations, Methods Enzymol., 233 (1994) 273-288. F. El Ghissassi, A. Barbin, J. Nair and H. Bartsch, Formation of I-N6-Ethenoadenine and 3-N4-Etheno cytosine by lipid peroxidation products and nucleic acid bases, Chem. Res. Toxicol., 5 (15’95) 278-283. R.P. Bird and H.H. Draper, Comparative studies on different methods of malonaldehyde determinaticn, In: L. Parker (Ed.), Methods Enzymol., Academic press, Orlando, 105, 1984, pp. 2999305. K.H. Cheeseman, Methods of measuring lipid peroxidation in biological systems: an overview, In: A. Crastes de Paulet, L. Douste-Blazy and R. Paoletti (Eds.), Free radicals, lipoproteins, and membrane lipids, Plenum Press, New York, 1990, pp. 1433152. M. Tomita and T. Okuyama, Determination of malonaldehyde in oxidized biological materials by high performance liquid chromatography, J. Chromatogr., 526 (1990) 174-179. F. Masugi and T. Na kamura, Measurement of thiobarbituric acid value in liver homogenate solubilized with sodium dodecyl sulfate and variation of the values affected by vitamin E and drugs, Vitamins, 51 (1977) 21-29. T. Terasima and M. Yasukawa, Biological properties of vero cells derived from the present stock, School of Medicine, Chiba University, (1988) 32-35. M.M. Bradford, A rapid sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye, Anal. Biochem., 72 (1976) 2488254. Y. Grosse, I. Baudrimont, M. Castegnaro, A.M. Betbeder and E.E. Creppy, Formation of ochratoxin A metabolites and DNA-adducts in monkey kidney cells, Chem. Bio. Interact., 95 (1995) 157m 187. A. Pfohl-Leszkowicz, Y. Grosse, A. Kane, A. Gharbi, I. Baudrimont, S. Obrecht, E.E. Creppy and G. Dirheimer, Is the oxidative pathway implicated in the genotoxicity of ochratoxin A? In: E.E. Creppy, M. Castegnaro, G. Dirheimer, (Eds.), Human Ochratoxicosis and its Pathologies. John Libbey Eurotext INSERM, 231 1993, pp. 1777187. 1. Baudrimont, A.M. Betbeder, A. Gharbi, A. Pfohl-Leszkowicz, G. Dirheimer and E.E. Creppy, Effect of superoxide dismutase and catalase on the nephrotoxicity induced by subchronical administration of ochratoxin A in rats, Toxicology, 89 (1994) 101~ II I.

40

I. Baudrimont

et al. / Chemico-Biological

Interactions

104 (1997) 29-40

[40] I. Baudrimont, M. Murn, A.M. Betbeder, J. Guilcher and E.E. Creppy, Effect of piroxicam on the nephrotoxicity induced by ochratoxin A in rats, Toxicology, 95 (1995) 1477154. [41] E.E. Creppy, I. Baudrimont and A.M. Betbeder, Prevention of nephrotoxicity of ochratoxin A, a food contaminant, Toxicol. Lett., 82/83 (1995) 8699877. [42] A. Wolf, M. Tschopp and B. Ryffel, Inhibition of sandimhum (cyclosporin A)-induced adverse effects in rat kidneys by superoxide dismutase and catalase-evidence for the involvement of free reactive oxygen species in the pathomechanism of the drug, Toxicol. Lett., 97 suppl., (1992). [43] C. Deby and J. Pincemail, Toxicite de I’oxygene, radicaux libres et moyens de defense, La presse medicale, 15, 31 (1985) 1468-1474. [44] T.J. Carty, J.S. Stevens, J.G. Lombardino, M.J. Parry and M.J. Randall, Piroxicam a structurally novel anti-inflammatory compound. Mode of prostaglandin synthesis inhibition, Prostaglandins, 19 (1980) 671-682. [45] T. Tanaka, T. Kojima, A. Okumara, S. Sugie and H. Mori, Inhibitory effect of a non-steroidal anti inflammatory drugs, indomethacin and piroxicam on 2-acetylaminofluorene-induced hepatocarcinogenesis in male ACI/N rats, Cancer Lett., 68 (1993) 111-I 18. [46] R.C. Moon, G.J. Kelloff, C.J. Detrisac, V.E. Steele, C.F. Thomas and CC. S&man, Chemoprevention of OH-BBN-induced bladder in mice by piroxicam, Carcinogenesis, 14, 7 (1993) 1487-1489. [47] I. Baudrimont, A.M. Betbeder and E.E. Creppy, Reduction of the ochratoxin A-induced cytotoxicity in Vero cells by Aspartame, Arch. Toxicol., (1996) in press. [48] S. Obrecht, Y. Grosse, A. Pfohl-Leszkowicz and G. Dirheimer, Protection by indomethacin and aspirin against genotoxicity of ochratoxin A, particulary in the urinary bladder and kidney, Arch Toxicol., (1996) 70, 244-248. [49] I. Baudrimont, Variation de la nephrotoxicite de l’ochratoxine A sous l’effet de facteurs metaboliques et non metaboliques, PhD thesis, no. 2, Bordeaux (1995). [50] I. Baudrimont and E.E. Creppy, Effect of aspartame on OTA-induced cytotoxicity in Vero cells, Cell Biol. Toxicol. (1996) 12, 4-6, 392.