Further arguments in favour of direct covalent binding of Ochratoxin A (OTA) after metabolic biotransformation

Abstract

Ochratoxin A (OTA) is nephrotoxic to all animal species, carcinogenic for rats and mice and probably implicated in human Balkan endemic nephropathy and the associated urothelial tract tumour. Controversial results concerning genotoxicity and biotransformation of OTA have been generated. By ³²P post-labelling technique, a dose- and time-dependent DNA adduct formation is observed in vivo and in vitro. Use of several inducers or inhibitors of biotransforming enzymes (including cytochrome P 450, cyclooxygenase, lipoxygenase, glutathione-S-transferase), demonstrated that OTA is biotransformed into genotoxic derivatives damaging for DNA. Authentic C8dG-OTA standards have been synthesized by photo-oxidation. Both of them (C-C8 & O-C8) co-migrate on TLC with two adducts formed by in vitro incubation of OTA in the presence of kidney microsomes, and in vivo in kidney of pig or rodent fed OTA as well as in kidney and bladder tumour of humans exposed to OTA. Several OTA metabolites have been isolated from tissues or cells treated by OTA. The open ring lactone (OP-OTA) and quinone OTA (OTQ) are genotoxic.

Keywords:

• Ochratoxin A
• genotoxicity
• DNA adduct
• OTA metabolites

Introduction

Ochratoxin A (OTA) is a potent mycotoxin produced by Penicillium in cool/temperate climate and Aspergillus in tropical latitudes (for review see Pfohl-Leszkowicz et al. 2002a; Molinié et al. 2005). It is a common contaminant of foodstuffs (JECFA 2002; Monaci & Palmisano 2004). OTA is very toxic to several animal species, the kidney being the main target organ (for review, see Pfohl-Leszkowicz et al. 2002a). OTA is associated with Balkan endemic nephropathy (BEN), a chronic kidney disease characterized by renal failure and a high incidence of urinary tract tumours, and is restricted to areas of the Balkans where high levels of OTA are found in food (for a review see Pfohl-Leszkowicz et al. 2002a). OTA causes kidney and liver tumours in mice and rats (review in IARC 1993a; Castegnaro et al. 1998; Pfohl-Leszkowicz et al. 2002b). In humans, OTA is classified by the International Agency for Research on Cancer (IARC) as a possible carcinogen (group 2B), based on sufficient evidence for carcinogenicity in animal studies and inadequate evidence in humans (IARC 1993a) because the group considered that the data were not convincing since BEN and UTT patients had not been separated. However, this is difficult, because the two diseases are very closely linked. On the basis of nephrotoxic effects in pigs as the endpoint, JECFA has set a provisional tolerable weekly intake of OTA at 100 ng/kg body weight (JECFA 2002).

DNA-xenobiotic binding is considered to be a critical step in the initiation of mutagenesis and carcinogenesis. The process of chemical carcinogenesis is initiated by the covalent binding of carcinogens or their reactive metabolites to DNA, thus forming DNA-adducts (Miller & Miller 1981). In all animals there is a good correlation between DNA-adducts formation and the frequency of mutations (for a review see Lutz & Gaylor 1996) and with the incidence of tumours (Poirier & Beland 1992). To interact with cellular macromolecules and thus initiate carcinogenesis, most chemical carcinogens require metabolic activation (Miller & Miller 1981). DNA adduct detection is a useful technique to demonstrate genotoxicity of compounds that sometimes are not positive with other classical tests accepted by regulatory authorities (Brambilla & Martelli 2004; Farmer 2004). Several publications indicated that ochratoxin A induces the formation of DNA adducts in vitro and in vivo, but the nature of the bound “entity” is controversial. This raises the question “Does OTA induce adduct formation by direct binding or by an indirect process (oxidative stress)”? The overall goal of our work was to determine the nature of DNA adducts formed in vitro in presence of microsomes from various tissues after exposure to ochratoxin A and the relationship of these adducts to the metabolic fate of ochratoxin A in vivo and thus ascertain the existence of the “true” adducts.

Material and methods

Chemicals

OTA (benzene free), dithiothreitol, spermidine, bicine, N-acetylcysteine (NAC), acivicin, buthionine sulfoximine (BSO), arachidonic acid (AA), vitamin A, vitamin E, 2-mercaptoethane sulfonate (MESNA), salmon testes DNA were purchased from Sigma-Aldrich, (Saint-Quentin Fallavier, France) and re-purified. The enzymes were treated as follows: proteinase K (used as received), RNase A, RNase T1 (boiled 10 min at 100°C to destroy DNases), and microccocal nuclease (dialyzed against deionized water) were from Sigma (Saint-Quentin Fallavier, France); spleen phosphodiesterase from Calbiochem (VWR, France) was centrifuged before use; nuclease P1 and T4 polynucleotide kinase were from Roche diagnostics (Meylan, France) and used as received. NADPH₂ was from Boehringer (Mannheim, Germany); rotiphenol (phenol saturated with Tris-HCl, pH 8) was from Rothsichel (Lauterbourg, France); cellulose MN 301 was from Macherey Nagel (Düren, Germany); polyethyleneimine (PEI) was from Corcat (Virginia Chemicals, Portsmouth, VA); Whatman no. 1 paper was from VWR (France); [^γ32P-ATP] (444 Tbq/mmol, 6000 Ci/mmol) from Amersham and PEI/cellulose TLC plates were prepared in the laboratory of Toulouse. All reagents (potassium chloride, sodium hydrogen carbonate, sulphuric acid, phosphoric acid, hydrochloric acid, acetic acid, sodium dihydrogen phosphate) were of normapur grade. All solvents (methanol, chloroform, acetonitrile, propan-2-ol, n-hexane) were of HPLC grade from ICS (Lapeyrouse-Fossat, France). Deionized water prepared freshly from a Milli-Q system (Millipore, France) was used for HPLC analysis, dialysis of the enzymes and preparation of all aqueous solutions.

In vitro incubation to determine DNA adduct formation or metabolites

The incubation was done in presence 0–100 μM OTA as described in Pinelli et al. (1999). Two controls were added: (1) incubation of DNA without microsomes, and (2) incubation of microsomes alone. All incubations were performed in duplicate.

DNA adduct detection by ³²P-postlabelling method

DNA extraction

Tissues (500 mg) were homogenized in 0.8 ml of a solution containing NaCl (0.1 M), EDTA (20 mM), and Tris-HCl, pH 8 (50 mM) (SET) in an ice bath. To the homogenate, 200 μl of a 10% solution of sodium dodecylsulfate was added, and following incubation for 10 min at 65°C, 800 μl of potassium acetate (6 M, pH 5) was added. The reaction mixture was kept at 0°C for 30 min. After centrifugation for 25 min at 0°C (10,000  g ), the supernatant, which contained nucleic acids, was collected and nucleic acids were precipitated overnight at −20°C by adding 2 volumes of cold ethanol. The DNA pellets were collected and washed once with 1 ml of 90% ethanol and dissolved in 500 μl of SET (15 min at 37°C). The total extract was mixed with 10 μl of a mixture of RNase A (20 mg/ml) and RNase T1 (10,000 U/ml) and incubated for 1 h at 37°C; this treatment was repeated twice. Samples were then treated with 25 μl of proteinase K solution (20 mg/mL SET) for 1 h at 37°C. After digestion, 500 μl of rotiphenol® was added and the mixture was moderately shaken for 20 min at room temperature and centrifuged for 15 min at 15°C (10,000  g ). The aqueous phase was collected after two extractions. After a final extraction with one volume of chloroform/isoamyl alcohol (24 : 1), the aqueous phase was collected and 50 μl of sodium acetate (3 M, pH 6) were added. The DNA was precipitated by the addition of two volumes of cold ethanol overnight at −20°C, and the precipitate was collected by centrifugation at 10,000  g for 30 min. The DNA pellet was washed four times with 90% ethanol. DNA was dissolved in deionized water and tested for purity by recording UV spectra between 220 and 320 nm. Example spectra concerning the quality of DNA purification are presented in Figure 1(a).

Figure 1. Tests of purification and postlabeling (A) UV spectrum to test purity of DNA; (B) post-labeling efficiency; (C) nuclease P1 efficiency; (D) exemples of good and bad DNA adduct patterns.

³²P-Postlabelling analysis of OTA-mediated DNA adduction

The equivalent of 7 µg DNA was dried in vacuum, dissolved in 10 μl of the mix containing 1 μl of micrococcal nuclease (2 mg/ml corresponding to 500 U), spleen phosphodiesterase (15 mU/µg DNA), 1 μl of sodium succinate (200 mM), and 1 μl of calcium chloride (100 mM, pH 6) and digested at 37°C for 4 h. The digested DNA was then treated with 5 μl of the mix containing 1.5 μl of nuclease P1 (4 mg/ml), 1.6 μl of ZnCl₂ (1 mM), and 1.6 μl of sodium acetate (0.5 M, pH 5) at 37°C for 45 min. The reaction was stopped by addition of 3 μl of Tris base 500 mM. The DNA adducts were labelled as follows: to the NP1 digest, 5 μl of the reaction mixture containing 2 μl of bicine buffer [Bicine (800 μM), dithiothreitol (400 mM), MgCl₂ (400 mM), and spermidine (40 mM) adjusted to pH 9.8 with NaOH], 10 U of polynucleotide kinase T4, and 100 μCi of [γ-³²P]ATP (specific activity 6000 Ci/mmol) was added and the mixture incubated at 37°C for 45 min. Normal nucleotides, pyrophosphate, and excess ATP were removed by chromatography on PEI/cellulose TLC plates (D1) in 2.3 M NaH₂PO₄ buffer, pH 5.7, overnight (See Figure 2A). The origin (4 cm height) areas containing labelled adducted nucleotides were cut out and transferred to another PEI/cellulose TLC plate, which was run (D2) in 4.8 M lithium formate and 7.7 M urea pH 3.5 for 3 h (Figure 2B). A further (D3) migration was performed after turning the plate 90° anticlockwise in 0.6 M NaH₂PO₄ and 5.95 M urea, pH 6.4 for 3 h (Figure 2B). Finally, the chromatogram was washed in the same direction in 1.7 M NaH₂PO₄, pH 6, for 2 h (D4). Adduct profiles were analysed qualitatively and semi-quantitatively by autoradiography of the plates, carried out at −80°C for 48 h in the presence of an intensifying screen, using a radio-analytical system of image analysis (AMBIS, Lablogic).

Figure 2. Influence of the molarity and pH on migration of DNA adduct. (A) Influence of the molarity on D1 migration of the C8 dG-OTA adduct; (B) separation of AA (aristolochic acid) and OTA (ochratoxin A) as function of molarity and pH of D2 & D3 solvents of migration.

In parallel to the adduct analysis a series of controls were performed to verify the labelling efficiency of adducts (Figure 1B) and the efficiency of dephosporylation of the NP1 (Figure 1C). In addition, as depicted in Figures 1D, the final autoradiograms allows the quality of the purification (not always discovered beforehand because all the tests and analysis are run in parallel) and the efficiency of the labelling by T4-PNK to be assessed. In all cases the resulting “faulty samples” were not considered.

Several modifications which required adjustment of the analytical parameters were incorporated since the first publication in 1991:

1. The quality of cellulose was changed, which required modification of the preparation of the plates;

2. Spleen phosphodiesterase (SPD) from Sigma was withdrawn from the market, it was dialysed before use and used at 2.6 mU/µg DNA; now the SPD provided by Calbiochem is centrifuged before use and used at 15 mU/µg);

3. Now the migration of TLC plates is performed in a thermostatted area while previously it was performed at room temperature;

4. The specific activity of the ATP was 3000 Ci/mmole, now it is 6000 Ci/mmole;

5. Spots were first located on the cellulose plate from the autotradiograms; the cellulose corresponding to spots was scraped off and their radioactivity counted by the Cerenkov procedure, now the radioactivity of the spot is evaluated by a bioimager using the core software AMBIS. In both cases, appropriate blank count rates were substracted from sample count rates. The frequency of radioactive nucleotides was expressed as relative adduct level (RAL), numerically as number of adducts/ 10⁹ nucleotides.

OTA and metabolites extraction from tissues, biologic fluids and cell supernatant

For all tissues, blood, serum, urine, cell media incubated with OTA and corresponding controls, the tissues/media were extracted as described by Petkova-Bocharova et al. (2003a, 2003b).

HPLC analyses

Initially an isocratic method for separation of metabolites was used (Petkova-Bocharova et al. 2003b). In order to obtain a separation to base line with a solvent compatible with use of mass spectrometry and in a short time (less than 60 min) new HPLC conditions were developed. The metabolites were separated on prontosil 250 × 4 mm, 3 μ using the following gradient: solvent A: MeOH/acetonitrile/6.5 mM ammonium formate (200 : 200 :600) adjusted to pH 3 with formic acid; solvent B: MeOH/acetonitrile/6.5 mM ammonium formate (350 : 350 : 300) adjusted to pH 3 with formic acid. Program: T0 100% A; T10 100% A; T25 30% A; T30 30% A; T45 0% A; T55 0% A; T58 100% A.

Results and discussion

Method of DNA adduct detection by ³²P post-labelling method

Genotoxicity of chemicals could be evaluated by the detection of DNA-adduct using ³²P post-labelling method originally developed by Reddy and Randerath (1986). The condition of hydrolysis and adducts separations should be adapted to the compound studied (IARC 1993b). Interpretation of results could be done only if the quality of the data is reached. Figure 1 describes the controls needed. Indeed, if DNA is not pure (Figure 1A), notably if column such as Qiagen tips or nucleobond are used for purification, several problems could arise: (1) poor efficiency of post labelling (see Figures 1B and 1C) (2) incomplete hydrolysis (3) high background (Figure 1D). DNA of high purity is critical for reliable results on DNA adduct (Phillips & Castegnaro 1999; Turesky & Vouros 2004; Esaka et al. 2003). After the outbreak of mad cow disease, enzymes such as the calf spleen phosphodiesterase were withdrawn from the market. The replacement by the spleen phosphodiesterase (SPD) from Calbiochem required greater amounts of this enzyme to be used as the activity is ten times lower than the previous SPD from Sigma. In the same way as separations are done on polyethylene-imines, cellulose thin layer chromatography (PEI-TLC) which is based an anion exchange; solvent composition is of the greatest importance. As shown in Figure 2(A), increase of the molarity of NaH₂PO₄ allows a better retention of some DNA adducts, especially those from OTA with known structure. In the same way, use of solvents (D2 and D3) developed for polycyclic aromatic compounds are not applicable for OTA-DNA adducts (Figure 2B).

Thus it is now clear that the method of DNA isolation, enzymatic conditions for DNA hydrolysis, variations in TLC plates, or pH changes in chromatography solvents can affect the recoveries and detection of OTA-DNA adducts. We have found that other ³²P-postlabelling studies that have failed to detect OTA-mediated DNA adduction have suffered a number of methodological problems that may have precluded detection of OTA-DNA adducts. (Gautier et al. 2001; Mally et al. 2004).

Dose and time dependent DNA adducts formation

DNA adducts detected by ³²P-post-labelling are formed in the kidney of several animal species treated with OTA (for a recent review see Manderville & Pfohl-Leszkowicz 2005). Figure 3 shows typical DNA adduct profiles. Some DNA adducts are common to all animals treated by OTA either by gavage or in feed, and are also found in the kidney or bladder of humans having developed kidney or bladder tumours, and nephropathy.

Figure 3. Comparison of OTA-DNA adduct formed in various animal species and in human tumours. MK, mouse kidney; RK. rat kidney; PK, pig kidney; CK, chicken kidney; HK, Human kidney accidental death; HBT, human bladder tumour; HKT, human kidney tumour.

Some others are specific to the animals or the organ. Our attention will focus on adducts numbered 1–4. Single doses of OTA administered to mice and rat by gavage induced DNA adducts in kidney in a dose- and time-dependent manner (Pfohl-Leszkowicz et al. 1991, 1993a). DNA adduct No. 1 is the main adduct. Its formation is dose dependent (see Figure 4A). This adduct is detected in mouse with the lowest dose tested (3.5 µg/kg b.w.) but is below the limit of quantification (0.1 adduct/10⁹ nucleotides). In rat kidney, this adduct is not detectable for the two lowest doses tested. The highest levels have been found in kidney DNA where some adducts persist after high dosing (2 mg/kg bw) for more than 16 days, while adducts are repaired in liver and spleen after 5 days (Pfohl-Leszkowicz et al. 1993a, 1993b).

Figure 4. Dose-dependent OTA-DNA adduct formation: (A) OTA-DNA adduct (No. 1) in kidney of mouse and rat treated by gavages, one dose, Panel A grey = mouse, white = rat; (B) OTA-DNA adduct (No. 1) in monkey kidney cells; (C) Comparison of Total DNA adduct formation in kidney of rat after gavage or feed contamination, Panel grey = 0.2 mg/kg bw white = 1 mg/kg bw; (D) Total DNA adduct in kidney of Dark Agouty rat fed 28 days.

The formation of adduct No. 1 is also dose dependent in monkey kidney cells (Figure 4B) and parallelled metabolite formation (Grosse et al. 1995). The amount of DNA adducts is lower when animals are treated via contaminated food than oral gavage (Figure 4C), nevertheless the adduct profile is similar and a dose relationship could be observed (Miljkovic et al. 2002). Interestingly, feeding of rat for one month with low doses (0.5–100 µg/kg bw/day) induced increasing amount of kidney DNA adduct (Figure 4D). The same amounts of kidney DNA adduct (11 adduct/10⁹ nucleotides) were observed in rats fed five days with 1 mg/kg bw/day or rat fed one month with 40 μg/kg bw/day. Administration of OTA to pregnant mother on the day 17 of gestation, induced DNA adducts in mother, foetus and pups (Petkova-Bocharova et al. 1998). In order to elucidate the metabolic pathway leading to DNA adduct formation, a series of in vitro experiments have been undertaken. Only kidney microsomes were capable of inducing DNA adduct in a time and dose dependent manner (Pfohl-Leszkowicz et al. 1993b). Incubation of rat kidney microsomes with increasing amount of OTA shows that DNA adduct are formed with dose higher than 5 μM. DNA adducts No. 1 & 4 appeared and increased with doses. For high dose (100 μM), the DNA adduct pattern is complex, with individual adducts in addition to a diagonal zone revealing cross links (Pfohl-Leszkowicz et al. 1993). No adducts can be formed by in vitro incubation using hepatic microsomes. These data are in agreement with Gross-Steinmeyer et al. (2002) who did not find adducts in hepatocytes and Gautier et al. (2001), who could not form DNA adducts using liver microsomes. The formation of metabolites by in vitro incubation in presence of pig kidney microsomes is time dependent. At least ten different metabolites are formed. Some of them have been identified by co-migration and by mass spectrometry such as open ring OTA (OP-OA), OTB (dechlorinated OTA), 4-OH-OTA, 10-OH-OTA. The formation was linear the first 10 min and reached a plateau at about 25 min.

The nature of bases modified was studied using polydeoxynucleotides. Poly dA, poly dC, poly dT, poly dG-dC and DNA were incubated with OTA in the presence of pig kidney microsomes. In the absence of microsomes no adducts were formed on polynucleotides or DNA (Figure 5).

Figure 5. Typical DNA modification pattern observed after in vitro incubation of polynucleotides or DNA in presence pig kidney microsomes.

No adducts were formed in poly dC and in poly dT, whereas several individual adducts were formed in poly dA, notably the adduct numbered No. 4, and in poly dC-dG. As OTA does not form adducts on poly dC, the observed adducts (e.g. adduct No. 1) in this latter polynucleotide is due to adduct formation on guanine. In this polymer, in addition to several individual adducts, a diagonal radioactive zone is observed indicating that cross links are induced by OTA. With DNA, the DNA-adducts observed are similar to those of guanine (adduct No. 1, 2, 3) and adenine (adduct No. 4). The prevalence of guanine adduct formation was previously described by Obrecht-Pflumio and Dirheimer (2000, 2001). The main adduct formed in the presence of microsomes from human kidney tumours also corresponds to the guanine adduct (Figure 6).

Figure 6. Correlation between DNA adduct and biotransformation enzyme. (A) DNA + microsome without OTA; (B) DNA + OTA without microsome; HHK, human healthy kidney microsome; HPTK, human peri-tumoral kidney microsome; HTK, human tumoral kidney microsme; MK, kidney from mouse pre-treated with vitamin A.

Correlation between DNA adducts and biotransformation

Because kidney is rich in peroxidases and OTA catalyzes formation of reactive oxygen species (ROS) and lipid peroxidation in kidney of rats (Rahimtula et al. 1988; Omar et al. 1990), the implication of an oxidative metabolic pathway was investigated. Prevention of DNA adduction in kidney of mice was obtained by pre-treatment of animals by superoxide dismutase and catalase prior to OTA administration (Pfohl-Leszkowicz et al. 1993c) and by antioxidant vitamins (Grosse et al. 1997a). In kidney, the enzymes of biotransformation are mainly represented by cyclooxygenase (COX), lipoxygenase (LOX) and epoxygenase (notably CYP 2C) capable of co-oxidation of xenobiotics (Rendic & Di Carlo 1997; Eling et al. 1990). Inhibition of LOX and COX₂ pathways decreases OTA genotoxicity in vivo (Obrecht-Pflumio et al. 1996) and in vitro (Pinelli et al. 1999). Induction of LOX and COX₂ by vitamin A increases the formation of the main OTA-DNA adduct (adduct No. 1) in kidney of mice treated by OTA (Grosse et al. 1997a, and Figure 6). The implication of LOX is reinforced by the absence of OTA-DNA adducts when cells were pre-treated with nor dihydroguaretic acid (NDGA) and high concentrations of indomethacin (10 μM) which inhibits all arachidonic acid (AA) pathways (Pinelli et al. 1999). In vitro incubation of human kidney microsomes arising from different parts of tumorous kidney (Figure 6) show that the DNA adduct pattern is dependent on expression of biotransformation enzymes. With healthy parts expressing CYP 2C9 and COX1, four adducts are observed, including adduct No. 1. With peritumoral tissue where expression of CYP2C9 and COX₁ was induced, we observed amplification of adduct No. 1 and adduct No. 4 related to adenine adduct. With tumour tissue, where LOX and COX₂ are expressed whereas CYP2C9 is inhibited, the main adduct was considerably enhanced. Use of microsomes from kidney of Balb C/6 mice (C57) or knock out Ah mice pre-treated or not by betanaphthoflavone (BNF) confirmed the involvement of these enzymes in the genotoxicity of OTA. The enzymes of the Ah gene battery [CYP 1 family, NAD(P)H quinone (menadione) reductase, aldehyde dehydrogenase, UDP-glucuronosyltransferase and glutathione-S-transferase] are induced by BNF via the Ah receptor. Recently, it has been demonstrated that cyclooxygenase (COX) is induced only if the Ah receptor is functional, whereas BNF induces the other enzymes involved in the AA cascade (i.e. CYP 450 epoxygenation, lipoxygenases) independently of the Ah receptor. Thus CYP 1B1, 2C9, LOX, COX₂ and NAD(P)H quinone (menadione) reductase are induced in C57 pretreated with BNF whereas only CYP 2C and LOX are induced in knock out mice. The absence of the Ah receptor modifies neither the number of DNA adducts nor their total levels. Incubation with microsomes from BNF-pre-treated mice, induced a significant increase of the total DNA-adduct levels, mainly adducts No. 1 and 4, independently of the co-substrate (1.5-fold in presence of C57 microsomes and 2.7-fold in presence of AhR microsomes).

This experiment leads to the following conclusions:

1. OTA is preferentially biotransformed into genotoxic derivatives by pathways linked to AA cascade;

2. The increase of DNA adduct formation by βNF is due to enhancement of enzymes not regulated by the Ah receptor;

3. The higher increase in presence of Ah knock-out microsomes indicates that some enzymes regulated by this receptor display a protective effect against OTA genotoxicity.

In a carcinogenicity study, we demonstrated that the susceptibility of male dark agouti (DA) rats is due to CYP 2C11 corresponding to human CYP 2C9, which is able to metabolize debrisoquine (Pfohl-Leszkowicz et al. 1998). Some of these samples have been analysed in order to differentiate the OTA DNA adduct pattern and the aristolochic acid (AA) pattern (Arlt et al. 2001, 2002). Implication of CYP 2C has been shown in other experiments. For example, induction of CYP 2C by Phenobarbital (PB) in rabbit and in bronchial epithelial cells induced an increase of DNA adduct formation, especially adduct No. 4, which is also formed when BEAS-2B cells expressing human CYP 2C9 were incubated with OTA (El Adlouni et al. 2000). It has been demonstrated recently that in cells expressing CYP2C9, OTA exerts an increased cytotoxicity as measured by neutral red (Simarro Doorten et al. 2004), and mutation frequency is increased (De Groene et al. 1996). It has also been suggested that CYP3A4 might be involved in the biotransformation of OTA ( Grosse et al. 1997b; Zepnik et al. 2001), which is particularly relevant for xenobiotic metabolism because of its broad substrate specificity and abundant expression in the human liver, intestine and kidney. The CYP 3A5*1 allele was more prevalent in BEN patients with a frequency of 9.38% compared to 5.36% in controls and was associated with a higher risk for BEN (OR 2.41) (Atanasova et al. 2005). These authors demonstrated that no correlation of metabolism was found with CYP 2D6 even though a correlation exists with debrisoquine metabolism as suggested by previous phenotyping analysis in BEN (Nikolov et al. 1991). These latter data are of particular interest because they support our results concerning the high susceptibility of rat in relation to debrisoquine hydroxylation capacity (mainly due to CYP 2C11 and not to CYP 2D6) (Pfohl-Leszkowicz et al. 1998), and the fact that CYP 3A was only induced in the most susceptible animals (Pfohl-Leszkowicz et al. 1998). A close relationship exists between these two enzymes. Indeed, CYP 2C is induced by sex hormones (Gustafsson et al. 1983; Kamataki et al. 1983, 1985) which are under the control of CYP 3A.

One important enzyme in the genotoxicity of OTA is leucotriene C4 synthase. This enzyme is a member of the group of non-haem Fe-containing enzymes capable of oxidizing GSH to GSSG, simultaneously generating reactive oxygen species responsible for co-oxidation of xenobiotics and export of metabolites from liver to kidney. In cell culture, we demonstrated that etacrynic acid, a specific inhibitor of this enzyme, inhibited the formation of some adducts, whereas some others such as adduct No. 4, persisted. Often glutathione transferases are involved in dehalogenation. The first step is the formation of an epoxide, and in the second step the epoxide is converted into phenol, which in the case of OTA can lead to OTHQ and/or OTB. Treatment of mice with PB, an inducer of GST in addition to CYP, decreased nephrotoxicity (Moroi et al. 1985) but increased hepatic tumour formation (Suzuki et al. 1986). This suggests that biotransformation of OTA by enzymes modulated by PB generates OTA metabolites less nephrotoxic than the parent OTA, but which are genotoxic. Formation of OTB is increased by PB treatment. Since MESNA protect rats against nephrotoxicity and carcinogenicity induced by oxidative stress by increasing free thiol groups in kidney, the potential protective effect of MESNA on renal toxicity and carcinogenicity induced by OTA was examined in a long-term rat study. We have shown that the kidney karyomegalies were prevented by MESNA but an increase of the incidence of kidney tumours was observed (Pfohl-Lezkowicz et al. 2002b). Using other antioxidants and modulators of the glutathione pathway [N-acetylcysteine (NAC), buthionine sulfoximine (BSO), alpha-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid (acivicin)] we demonstrated that two different biotransformation pathways are implicated. Some OTA-DNA adducts disappeared whereas some others correspond to adducts No. 1, 2, 3, which are always found in kidneys of animals having developed a kidney tumour and are similar to those formed in human kidney or human urinary bladder tumour from patient suffering nephropathy after ingestion of diet contaminated by OTA (Pfohl-Leszkowicz et al. 1993d). After acivicin pre-treatment (an inhibitor of gamma glutamyl transpeptidase which blocks the cytotoxicity of hydroquinone-S-conjugates) (Lash & Anders 1986) before OTA administration, only one adduct persists and has the same chromatographic properties as adduct No. 1. Modulation of the glutathione pathway by these substances modified the OTA-metabolite profiles in Opossum kidney cell, (see Figure 7A and 7B).

Figure 7. Differences in OTA biotransformation in OK cells treated by several modulators [A] Example of separation of OTA derivatives —— OTA alone —— after treatment with acivicin; [B] Comparison of metabolites formed after pretreatment with

OTA alone

+ MESNA

+ NAC

+ BSO

+ Acivicin.

Interestingly, pre-treatment with acivicin induced the formation of a large amount of OTB and another dechlorinated OTA derivatives (see Figure 8). Other OTA metabolites, notably OTHQ, also have been observed. Oxidative activation of OTA generates the phenoxyl radical and the hydroquinone/quinone redox couple, resulting in dechlorination of OTA (Gillman et al. 1998; Calcutt et al. 2001; Dai et al. 2002). These species can form adducts with sulfhydryl groups and deoxyguanine (Dai et al. 2003). Recent studies confirm the presence of the hydroquinone (OTHQ) in the urine of rat following administration of OTA by gavage (Mally et al. 2004). Recently we have isolated several OTA metabolites, including 4-OH-OTA from urine of rat fed OTA in the diet for 28 days.

Figure 8. Mass spectrum of a dechlorinated derivative of OTA.

To confirm the nature of the DNA adducts observed in our study, two guanine OTA-DNA adducts (O-C8-dG-OTA & C-C8-dG-OTA) have been synthesized. In postlabelling analysis, both adducts comigrate with OTA-DNA adducts formed in kidney of rats developing tumours (Faucet et al. 2004). C-C8-dG-OTA is also formed in the kidney of pig fed OTA (Dai et al. 2004; Faucet et al. 2004).

Conclusion

Altogether, these data on the mechanism of action of OTA indicate clearly that OTA is genotoxic after metabolic activation. OTA induces the formation of DNA adducts in the kidney of mice, rats, pigs and chickens in a dose- and time-dependent manner. Four adducts are always detected in all species; these seem to be directly involved in the genotoxicity and carcinogenicity of OTA, because they are detected in tumours of rodents and humans, and can be generated by in vitro incubation with several kidney microsomes, but not liver microsomes. Adducts are mainly formed on guanine. One adduct is also formed on adenine. Repetitive ingestion by DA rat of small amounts (40 nanog/kg bw) of OTA induces the formation of the main DNA adduct indicating that accumulation of OTA is dangerous.

The biotransformation of OTA, which is a chlorinated compound, is complex and involves several biotransforming enzymes such as cytochrome P450s, but also glutathione transferases and lipoxygenase, enzymes present in large amount in kidney. The metabolites conjugated to GSH and/or UDP are excreted in bile and in kidney. At least 10 different metabolites of OTA including OTB, OTC, OH-OTA, OP-OTA, OTα, OTβ and metabolites of unknown structure were detected.

Modulations of DNA adduct patterns and amounts by OTA metabolism demonstrates that the genotoxicity of OTA and the formation of DNA adducts are the result of biotransformation of OTA. DNA-binding of OTA is related to the formation of some metabolites, some of them being already identified by mass spectrometry. The main adduct comigrates with the authentic standard C-C8 dG OTA and is probably formed in the body via the quinone pathway (for a review see Manderville & Pfohl-Leszkowicz 2005).

Acknowledgments

The authors thank their collaborators Faucet-Marquis Virginie; Molinié Anne; Petkova-Bocharova Theodora; El Adlouni Chakib; Manderville Richard; Dai Jian; Stormer Frederik; Pont Frédéric; Mantle Peter; Nestler Sandra; Schmeiser Heinz; Arlt Volker and several groups in hospital in Toulouse and Bulgaria who provided us with human material. European Union for grant “Ochratoxin A-risk assessment” QLK1-2001-01614, “Région Midi-Pyrénées” and French Ministry of research.