Peter G Mantle
email: p.mantle@imperial.ac.uk.

Virginie Faucet-Marquis

Richard A Manderville

Bianca Squillaci

Annie Pfohl-Leszkowicz

Structures of Covalent Adducts between DNA and Ochratoxin A: A New Factor in Debate about Genotoxicity and Human Risk Assessment

The potent renal carcinogenicity of ochratoxin A (OTA) in rats, principally in the male, raises questions about mechanism. Chromatographic evidence of DNA adducts after 32 P-postlabeling analysis contrasts with experimental attempts to demonstrate the absence of OTA in such adducts. Proffered schemes for alternative epigenetic mechanisms in OTA carcinogenicity remain unsatisfying, while structural data substantiating DNA-OTA adducts has also been lacking. We report refined 32 P-postlabeling methodology revealing one principal adduct isolated in small amounts from the kidneys of all five Fischer and five Dark Agouti rats to which OTA had been given on four consecutive days. We also describe structural data for the principal adduct from OTA/DNA interaction in vitro and its subsequent preparative isolation by the postlabeling methodology (as C-C8 OTA 3′dGMP), essentially creating an ochratoxin B-guanine adduct. Reasoning for the unsuitability of experimental protocols in published evidence claiming nongenotoxicity of OTA is given. In vivo exposure of renal DNA to cycles of adduction with OTA, necessarily protracted for carcinogenesis to occur, can reasonably explain an occasional focal neoplasm from which metastasizing carcinoma could develop.

Introduction

Whether the nephrotoxic mycotoxin ochratoxin A (OTA) exerts its potent nephrocarcinogenicity in male rats by direct interaction with DNA has long been a matter of debate. First positive claims came from the application of 32 P-postlabeling methodology to DNA isolated from mice given OTA [START_REF] Pfohl-Leszkowicz | DNA-Adduct Formation, in Mice Treated with Ochratoxin A, in Mycotoxins, Endemic Nephropathy and Urinary Tract Tumours[END_REF]. Autoradiographic evidence of radiolabeled compounds separated after contact transfer on two-dimensional chromatograms was proposed as showing that OTA had made covalent bonds with nucleotides in vivo. Subsequently, postlabeling methodology has consistently detected adduct spots on chromatograms which were attributed to exposure of rats, mice, and pigs to OTA in laboratories in Strasbourg, Bordeaux, Lyon (IARC) and Toulouse. Little or no evidence for this conclusion was experienced in other laboratories (e.g., see ref 2), thus fueling the current reluctance to accept positive postlabeling evidence as an indicator of genotoxicity of OTA [START_REF] Efsa | Opinion of the scientific panel on contaminants in the food chain on a request from the Commission related to ochratoxin A in food[END_REF]. Academic debate about whether or not OTA conforms to the current definition of a genotoxin might have rested as a minor unresolved esoteric controversy if it were not for concerns that experimental carcinogenicity in male rats and mice pointed to the possibility that OTA could be a human carcinogen [START_REF] Boorman | Toxicology and Carcinogenesis Studies of Ochratoxin A[END_REF][START_REF] Bendele | Ochratoxin A carcinogenesis in the (C57BL/6J x C3H)F1 mouse[END_REF][START_REF] Castegnaro | Sex-and strainspecific induction of renal tumours by ochratoxin A in rats correlates with DNA adduction[END_REF]. Clear demonstration that a chemical carcinogen is a genotoxin influences legislative attitude, whereby a 10-fold reduced human risk differential may be applied when assessing the extent of exposure to a nongenotoxic carcinogen rather than to a carcinogen concerning which adducts of defined structure are known. Food and drinks industries with commercial interests in the significance of natural and largely unavoidable traces of OTA in some agricultural commodities, and a need to comply with statutory limits, will naturally prefer that OTA is shown not to be a genotoxin.

In recent years, several publications consistently claiming the nonexistence of direct OTA/DNA adducts have been noted (summarized in ref 7); concurrently, several other publications have claimed the opposite (summarized in refs 8 and 9). However, there has been little critical appraisal of the experimental material used in publications claiming that DNA/OTA covalent adducts do not occur. Meanwhile, we have addressed the difficult challenge of obtaining structural data relating to rat kidney DNA adducts. Demonstration of the structure of a principal DNA/OTA adduct, prepared in vitro from calf thymus DNA and isolated preparatively via postlabeling methodology, here extends proof of covalent binding of OTA on guanine beyond the quite compelling cochromatographic implication described in ref 10.

Materials and Methods

Rats and OTA. Ten 8-week old males of the Fischer and Dark Agouti strains (B & K Universal Ltd., Grimston, Hull, UK) were ∼220 and 180 g, respectively, and were maintained on standard (14.4% protein) diet (SDS Services, Witham, Essex, UK).

Design of OTA dosing strategy was based on previous experience [START_REF] Miljkovic | Comparative responses to mode of oral administration and dose of ochratoxin A or nephrotoxic extract of Penicillium polonicum in rats[END_REF] of short-term administration with only mild clinical effects and best incidence of renal DNA adducts achieved through daily oral gavage. OTA (77 mg) was dissolved in ethanol (1.2 mL) and the solution made up to 20 mL with 3% w/v NaHCO 3 in water. Then 0.4 mL (1.5 mg OTA) was given from this solution to each of seven rats, at the same time of day by oral gavage on three consecutive days. The daily dose was therefore ∼8.3 and 6.8 mg/ kg b.w. for Dark Agouti and Fischer rats, respectively. Controls were given 0.4 mL of vehicle (5% ethanol in 3% aqueous NaHCO 3 ). Twenty-four hours after the last dose, four treated rats from each strain and all controls were euthanized, and blood was withdrawn from the heart. The kidneys were excised, separated into quarters, flash frozen in liquid N 2 , transferred into separate Eppendorf tubes, and stored at -20 °C. Blood was centrifuged immediately and plasma separated for quantitative OTA analysis at the Central Science Laboratory, York, UK. The mean weights of the remaining three OTA-treated Fischer and Dark Agouti rats were 310.7 ( 12.1 g and 261.7 ( 10 g, respectively, 5 weeks after the first OTA dose. After 3 months (370 ( 17.3 g and 278.3 ( 4.7 g, respectively), the rats were euthanized and the blood collected as before. A narrow central transverse section was removed from each kidney for H & E-stained histology and the remaining tissues flash frozen in liquid N 2 and stored at -20 °C pending 32 P-postlabeling analysis.

All kidney samples were coded for blind analysis for adducts, to avoid suggestion of any analytical bias, and transferred personally by air in dry ice between London and Toulouse.

DNA Extraction and Postlabeling Separation of DNA Adducts. Materials. The following enzymes [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 purchased from Sigma (Saint Quentin Fallavier, France); spleen phosphodiesterase (centrifuged before use) was from Calbiochem (VWR, France), and nuclease P1 (NP1) and T4 polynucleotide kinase were from Roche diagnostics (Meylan, France). [γ 32 P-ATP] (444 Tbq/mmol, 6000 Ci/mmol) was from Amersham (Les Ullis, France). Rotiphenol (phenol saturated with TRIS-HCl at pH 8) was from Rothsichel (Lauterbourg, France); cellulose MN 301 was from Macherey Nagel (Du ¨ren, Germany); polyethyleneimine (PEI) was from Corcat (Virginia Chemicals, Portsmouth, VA); Whatman No 1 paper (ref 6130932) was from VWR (France), and PEI/cellulose TLC plates used for 32 Ppostlabeling analyses were prepared in the laboratory at Toulouse, France. All reagents (potassium chloride, sodium hydrogen carbonate, sulfuric acid, phosphoric acid, hydrochloric acid, acetic acid, and sodium dihydrogen phosphate) were of normal grade.

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 (10000g), 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 (10000g). 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) was 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 10000g 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.

Postlabeling Method. The equivalent of 7 µg of DNA was dried in vacuo, 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 ZnCl 2 (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 the addition of 3 µL of Tris base 500 mM. The DNA adducts were labeled as follows: to the nuclease P1 digest, 5 µL of the reaction mixture containing 2 µL of bicine buffer [bicine (800 µM), dithiothreitol (400 mM), MgCl 2 (400 mM), and spermidine (400 mM) adjusted to pH 9.8 with NaOH], 10 U of polynucleotide kinase T4, and 100 µCi of [γ-32 P]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 3 M NaH 2 PO 4 buffer, pH 5.7, overnight. The origin (2 cm) areas containing labeled 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. A further (D3) migration was performed after turning the plate 90°anticlockwise in 0.6 M NaH 2 PO 4 and 5.95 M urea, pH 6.4, for 3 h. Finally, the chromatogram was washed in the same direction in 1.7 M NaH 2 PO 4 , pH 6, for 2 h (D4). Adduct profiles were analyzed qualitatively and semiquantitatively 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). In parallel to the adduct analysis, a series of controls were performed to verify the labeling efficiency of adducts and the efficiency of dephosphorylation of the nuclease P1. Refinement of adduct separation by use of a 3 M phosphate buffer, instead of the former 2.3 M buffer, in the D1 migration, and subsequent transfer of only 2 cm of the origin, is shown in Figure 1. Steps in the refined procedure to resolve two of our synthetic model adducts are illustrated in Figure 2.

Synthesis of OTA-dG and OTA-3′-dGMP. A sample of the OTA-dG nucleoside adduct was prepared from the photoreaction of OTA in the presence of excess dG, as outlined previously [START_REF] Dai | Ochratoxin A forms a carbon-bonded C8-deoxyguanosine nucleoside adduct: Implications for C8 reactivity by a phenolic radical[END_REF][START_REF] Frenette | Structure-activity relationships for the fluorescence of ochratoxin A: Insight for detection of ochratoxin A metabolites[END_REF]. A reaction mixture (1 mL total volume) of 500 µM OTA and 40 mol equiv of dG in a mixture of 25 vol. % DMSO and 75 vol. % 100 mM aqueous phosphate buffer (pH 7.4) was irradiated at 350 nm for 15 min using a Rayonet Chamber Reactor, Model RPR-200. The OTA-dG nucleoside adduct was isolated using an Agilent 1200 series HPLC equipped with an autosampler, autocollector, and diode array detector, and a mobile phase consisting of 40 mM ammonium formate (pH 6.1)/acetonitrile and a semipreparative Phenomenex Kromasil C8 column (10 mm × 250 mm) at a flow rate of 5 mL/min. The isolated sample of OTA-dG had MS and UV-vis characteristics identical to those previously reported [START_REF] Dai | Ochratoxin A forms a carbon-bonded C8-deoxyguanosine nucleoside adduct: Implications for C8 reactivity by a phenolic radical[END_REF][START_REF] Frenette | Structure-activity relationships for the fluorescence of ochratoxin A: Insight for detection of ochratoxin A metabolites[END_REF].

The protocol for the photochemical synthesis of OTA-3′-dGMP was the same as that outlined previously [START_REF] Faucet | Evidence for covalent DNA adduction by ochratoxin A following chronic exposure to rat and subacute exposure to pig[END_REF]. A reaction mixture (1 mL total volume) of 100 mM OTA and 50 mol equiv of 3′-dGMP in 100 mM phosphate buffer (pH 7.4) was irradiated for 2 min at 350 nm. The isolated OTA-3′-dGMP adduct had MS and UV-vis characteristics identical to those reported previously [START_REF] Faucet | Evidence for covalent DNA adduction by ochratoxin A following chronic exposure to rat and subacute exposure to pig[END_REF] and was used as a postlabeling standard.

Photoreaction of OTA with DNA. A reaction mixture (1 mL total volume) of 500 µM OTA and 100 µg of calf thymus DNA (repurified) in a mixture of 10 vol. % DMSO and 90 vol. % 100 mM aqueous phosphate buffer (pH 7.0) was irradiated at 350 nm for 13 min using a Rayonet Chamber Reactor, Model RPR-200. The sample was then divided into four 250 µL fractions, and 1 mL of 100% ethyl alcohol was added to each fraction in microcentrifuge tubes. After incubation at -20 °C for 10 h, samples were centrifuged for 15 min at 12000 rpm and 6 °C in an AccuSpin centrifuge. The DNA pellet was washed with 70% ethanol, then dried under vacuum and sent to the Pfohl-Leszkowicz laboratory for 32 P-postlabeling analysis and preparative isolation of two prominent adducts.

Preparative Isolation of Adducts from the Photoreaction of OTA and DNA. The protocol for postlabeling was exactly the same as that for rat DNA, except that unlabeled ATP was used instead of that radiolabeled with 32 P. For postlabeling of 10 µg of DNA, 20 pmol of ATP was used. After postlabeling, a preparative chromatogram was prepared in which typically (Figure 3) were applied radioactive spots of our standard C-C8 adduct, products of the calf thymus DNA-OTA interaction postlabeled with 32 P ATP, our standard O-C8, and a mixture of C-C8 and O-C8 adducts. In the central region of the plate, a stripe of products of the calf thymus DNA-OTA interaction postlabeled only with unlabeled ATP was applied. Chromatography in the D1 direction was run with NaH 2 PO 4 (2.3 M, pH 5.7) for 16 h. The plate was washed twice by soaking in ultrapure water, dried, and autoradiographed. Cellulose in the central area corresponding to spots of interest was scraped off, the cellulose suspended in methanol, shaken, centrifuged, the solution, and the extraction cycle repeated. Residual cellulose was then similarly extracted with triethanolamine. Combined extracts were evaporated to dryness in vacuo. The preparative eluates from spots 1 and 2 (Figure 3) were sent, blind as samples 1 and 2, respectively, for LC-MS analysis.

Chemicals for HPLC/MS Analysis of Adducts. OTA from Aspergillus ochraceus was purchased from Sigma-Aldrich Co. (Dorset, UK), formic acid was purchased from Perbio Science (Northumberland, UK), HPLC grade acetonitrile was purchased from Fisher Scientific (Loughborough, UK), and HPLC grade water was produced by an Elga Maxima Ultra Pure Water system (Buckinghamshire, UK). All solvents and chemical standards were of the highest grade commercially available. Authentic standards of C-C8 OTA 3′-dGMP (Figure 4) and O-C8 OTA 3′-dGMP (Figure 5), and OTA-DNA samples were synthesized in Guelph.

Buffers, Reagents, and Standards. A 10 µg/mL MS standard of OTA in 50:50 acetonitrile/water was prepared by diluting a 10 µL of a 1 mg/mL solution of OTA in water with 990 µL of 50:50 acetonitrile/water. A 0.1% formic acid solution was prepared by diluting 1 µL of concentrated formic acid with 999 µL of water. For all analytes, the instrument was tuned to give the best possible MS performance. HPLC/MS n analysis was performed on an Agilent 1100 HPLC system (Santa Clara, USA) consisting of a binary pump and column thermostat, coupled to a Waters QTof F Premier mass spectrometer (Manchester, UK) equipped with an electrospray ionization (ESI) source with separate Lockmass interface and operated in negative ion mode. Samples were introduced to the HPLC via a CTC-Analytics PAL autosampler (Zwingen, Switzerland) consisting of a 100 µL loop. The autosampler, HPLC system, and mass spectrometer were controlled using Waters software MassLynx version 4.1 (Manchester, UK). Calibration over the range m/z 50-1000 was carried out using a NaI/CsI calibration standard purchased from Waters. MS tuning was performed using direct infusion of a 10 µg/mL standard solution of OTA, at a flow rate of 10 µL/min, using the integrated instrument syringe pump. The following mass spectrometer conditions were common to all LC/ MS experiments performed: capillary voltage, 1.5 kV; sampling cone, 35 V; source temperature, 140 °C; desolvation temperature, 350 °C; cone gas flow, 50 L/h; desolvation gas flow, 400 L/h; and LM and HM resolution, 15 arbitrary units. A 1 µg/mL solution of leucine enkephalin (m/z 554.2615) in 50:50 acetonitrile/water was pumped into the lockmass interface at a rate of 20 µL/min using a dedication HPLC pump (Shimadzu UK Limited, Manchester, UK). Lockmass data was collected with a scan time of 0.5 at 10 s intervals and averaged over 10 scans. Mass spectral data was collected in MS centroid mode with a scan time of 1 s and interscan time of 0.02 s, over a mass range of m/z 100-1000.

Method Development of MS Analysis of Model Synthetic Adducts in Preparation for the Analysis of Products of in

All samples were pretreated with concentrated formic acid (50 µL), vortex mixed, and heated on a heating block at 80 °C for 60 min. The samples were then reduced to dryness under N 2 gas, resuspended in 100 µL of 0.1% formic acid (aqueous), and vortex mixed. Ten microliters of each sample was injected onto a C18 column (Thermo Hypersil Gold 5 µm, 4.6 mm i.d. × 250 mm, Thermo Fisher Scientific, Waltham, USA); 0.1% formic acid (solvent A) and acetonitrile (solvent B) were used as mobile phases. The samples were separated using a gradient elution between 5% and 95% B in 15 min. The column was kept at 95% B for 5 min and then reconditioned at 5% B for 10 min.

Results

Pharmacokinetics in Rats Treated with OTA. Plasma OTA concentration in the four Fischer rats treated with OTA was 45.7 ( 2.6 µg/mL and in the Dark Agoutis was 36.8 ( 6.4 µg/mL. At first sight, the lower value in the smaller rats given the same amount of OTA appears incongruous. Purely on a body weight basis, the plasma concentration in the Dark Agoutis might have been ∼56 µg/mL. Subsequent discovery that Dark Agoutis have much shorter plasma OTA half-life than that of Fischer rats [START_REF] Mantle | Interpretation of the pharmacokinetics of ochratoxin A in blood plasma of rats, during and after acute or chronic ingestion[END_REF] can account for the apparent disparity in accumulation in plasma after the three consecutive doses. However, substantial potential for delivery of OTA via vascular circulation to kidneys in both rat strains was demonstrated. Histology of kidneys from animals euthanized 3 months after giving OTA showed sparse karyomegaly diffused across the outer stripe of the outer medulla, consistent in our experience with a typical response in renal tubule epithelia to circulating OTA.

Results of Postlabeling Analysis. Autoradiographs relating to the kidney of each animal were copied electronically from Toulouse to London for matching to the blind code. No spots were present in samples from control (untreated) rats or from treated rats euthanized 3 months after the administration of OTA. This applied to all replicates of both strains of rats. In contrast, for the kidneys of all OTA-treated rats analyzed after the three consecutive doses, one or two radioactive spots were present in otherwise very clean autoradiographs of chromatograms, indicating the usual 32 P-postlabeling evidence of adducted DNA arising from consistent delivery of OTA to nephron epithelia (Figure 6). Notably, there was close replication of the strainspecific pattern of DNA adducts. In Fischer rats, there was predominantly one adduct, while in Dark Agoutis the same adduct was evident together with a minor component with slightly greater chromatographic mobility. The abundance of the principal adduct across the 10 animals was in the range of 20-70 adducts per 10 9 nucleotides. MS Analysis. Preparative isolation of two adducts from in vitro interaction between OTA and DNA, occurring in approximately equal amounts, provided material for LC-MS analysis coded only as samples 1 and 2. An approximate limit of detection of 1 ng of material on column had been determined during method development.

No peaks were identified in sample 1 at mass m/z 713, the expected mass of the C-C8 OTA 3′dGMP adduct. However, a prominent peak was identified in the m/z 517 extracted ion chromatogram at 11.7 min. The mass spectrum under the peak is shown in Figure 7. The base peak ion at m/z 517 corresponded to the loss of the phosphate conjugated deoxyribose sugar moiety from the C-C8 OTA 3′dGMP, which is consistent with hydrolysis due to the addition of formic acid in the sample pretreatment step.

Extracted ion chromatograms of m/z 517 for samples 1 and 2, together with the analysis of the authentic standard of C-C8 OTA 3′dGMP cochromatographed at 11.7 min, confirming the deconjugated aglycone moiety, were present in all three analyses (Figure 8).

The mass spectrum under the peak at 11.7 min in OTA-DNA sample 2 is shown in Figure 9.

The peak at m/z 517 corresponds to the loss of the phosphate conjugated deoxyribose sugar moiety from the C-C8 OTA 3′dGMP as seen for OTA-DNA sample 1. However, additional adduct related ions are also observed in the spectrum. The ion m/z 655 corresponds to the sodium adduct of the C-C8 OTA 3′dGMP which has lost the phosphate conjugate but still has the deoxyribose sugar moiety intact (Figure 10a). The ion at m/z 815 represents the sodium adduct of the 3′,5′-bisphosphate OTA-dGMP, showing additional phosphate conjugation on the 5′ hydroxyl group of the deoxyribose sugar (Figure 10b).

The structure of the deconjugated aglycone observed in OTA-DNA sample 2 was confirmed by MS/MS analysis of the precursor ion m/z 517 (Figure 11). MS/MS data was generated on the Waters QT of Premier in continuum mode over the mass range m/z 100-600 at a collision energy of 25 eV, a scan time of 1 s, and an interscan time of 0.02 s.

The ions m/z 473 and m/z 429 represent the loss of the carboxylic acid group as CO 2 , with a further loss of CO 2 from the lactone moiety, thus confirming the presence of the OTA adduct in OTA-DNA sample 2. Matching of analytical findings to the blind sample code showed sample 1 to be C-C8 OTA 3′dGMP and sample 2 to be its triphosphorylated homologue.

Discussion

The present findings have taken the search for definitive evidence on the structures of adducts, revealed consistently in the Pfohl-Leszkowicz laboratory by 32 P-postlabeling in renal tissue after exposure to OTA, as far as possible with limited research resources. Limited resource of renal tissue precluded preparative isolation for LC-MS of sufficient amounts of either of the two key adducts revealed only by the 2D chromatography of 32 P-postlabeling after contact transfer. At least 1 ng would be needed on the column and would require ∼100 rats. The present finding confirms previous recognition of C-C8-dG-OTA as an in vivo DNA-OTA adduct by cochromatography in a 2D system after contact transfer with an authentic standard [START_REF] Faucet | Evidence for covalent DNA adduction by ochratoxin A following chronic exposure to rat and subacute exposure to pig[END_REF]. We also show (Figure 12) similarly consistent chromatographic behavior through five sequential steps in two dimensions for C-C8-dG-OTA as a product of DNA-OTA interaction both in vitro and in vivo. However, research effort may better be focused on understanding the genetic changes in rat renal tumors caused by OTA and the mechanism for marked gender difference in sensitivity and pharmacokinetics [START_REF] Mantle | Interpretation of the pharmacokinetics of ochratoxin A in blood plasma of rats, during and after acute or chronic ingestion[END_REF][START_REF] Vettorazzi | A different kinetic profile of ochratoxin A in mature male rats[END_REF]. Approximately 10 18 molecules of OTA necessarily pass via a male rat's kidneys during several months to cause any renal cancer [START_REF] Mantle | Minimum tolerable exposure period and maximum threshold dietary intake of ochratoxin A for causing renal cancer in Dark Agouti rats[END_REF].

The experimental design described in ref 17 is presumably based on previous OTA toxicokinetics [START_REF] Zepnik | Toxicokinetics of the mycotoxin ochratoxin A in F344 rats after oral administration[END_REF] in which a single dose (500 µg/kg body weight) was given by oral gavage. For male rats in that study, assuming that the quoted rat weight (∼250 g) is more reliable than the stated 8 week age (cf. Charles River Web site for Fischer rats), approximately 130 µg of OTA had been given per os in 500 µL of corn oil. Maximum concentration of OTA in blood, measured in plasma 48 h later, had reached ∼1.8 µg/mL, corresponding to ∼0.1 µg/g measured in kidney from which blood had been flushed by syringing with physiological saline after excision. Thus, in a rat given 130 µg of OTA, less than 0.2% was actually found resident in its kidneys. If blood containing 1.8 µg OTA/mL of the plasma component circulates through the kidney in which blood constitutes 20-25% of tissue [START_REF] Garcia-Sanz | Three-dimensional microcomputed tomography of renal vasculature in rats[END_REF], OTA concentration in the organ would be ∼0.4 µg/g. Since removal of blood from the kidney can only be achieved efficiently, particularly from the peritubular capillaries, by perfusion in situ under normal blood pressure [START_REF] Garcia-Sanz | Three-dimensional microcomputed tomography of renal vasculature in rats[END_REF], the concentration of OTA in nonvascular tissue within the kidney would have been considerably less than the 0.2% of the given dose recorded in ref 18. The similar dosing of three adult male rats in ref 17, with assumed weight of ∼250 g and each rat given ∼130 µg 14 C-OTA (0.25 mCi/mmol), thus delivered ∼0.08 µCi 14 C to each animal. This translates to ∼170,000 dpm of 14 C, of which only about 50 dpm might have been intracellular in the 300-400 mg of kidney used for the extraction of DNA for ACMS analysis. Unfortunately, there was no measurement of 14 C content of kidney of the dosed animals. Even with very efficient extraction and isolation (which seems not to have been the case), the total content of radioactivity of the DNA above background, about one 14 C atom to disintegrate per second, could hardly have significantly changed its natural abundance even if all potentially disintegrating carbon atoms had been involved in DNA/OTA adduct(s). This simple calculation should have been possible from the authors' own data. It is also unfortunate that no preliminary studies to optimize the delivery of 14 C-OTA to kidney had been performed [START_REF] Mally | Ochratoxin A: Lack of formation of covalent DNA adducts[END_REF], considering the costly resource investment made elsewhere to produce the 14 C-OTA. Optimum concentration of OTA circulating in blood may only be achieved after several weeks of continuous daily intake of the amount of 14 C-OTA given in ref 17, as indicated by in vivo rat data [START_REF] Mantle | Interpretation of the pharmacokinetics of ochratoxin A in blood plasma of rats, during and after acute or chronic ingestion[END_REF][START_REF] Mantle | Minimum tolerable exposure period and maximum threshold dietary intake of ochratoxin A for causing renal cancer in Dark Agouti rats[END_REF][START_REF] Castegnaro | Balkan endemic nephropathy: role of ochratoxin A through biomarkers[END_REF] and corroborated by predictive pharmacokinetic modeling [START_REF] Vettorazzi | A different kinetic profile of ochratoxin A in mature male rats[END_REF]. All these factors compound the unsatisfactory design of the radiolabeled study, which, together with failure to have the planned positive control evidence of successful 32 P-postlabeling of parallel material in the Pfohl-Leszkowicz laboratory, fails to justify the claim of nongenotoxicity. The claim was also premature because substitution, of commercial 14 C phenylalanine for the corresponding unlabeled moiety in the 14 C-OTA prepared biosynthetically [START_REF] Nestler | Experimental Renal Ochratoxicosis: A Molecular and Pathological Study[END_REF], by well-established methodology [START_REF] Hult | Ochratoxin A labelled with 14 C or 3 H in the phenylalanine moiety[END_REF][START_REF] Breitholz-Emanuelsson | Distribution of 14C-ochratoxin A and 14C-ochratoxin B in Rats: A Comparison Based on Whole Body Autoradiography, in Mycotoxins, Endemic Nephropathy and Urinary Tract Tumours[END_REF][START_REF] Breitholz-Emanuelsson | Synthesis of 14C-ochratoxin A and 14C-ochratoxin B and a comparative study of their distribution in rats using whole body radiography[END_REF] was not done.

Experiments described in ref 25 showed integrated findings on concentration of OTA in blood and ochratoxin R (OTalpha) in urine over a 2-week period of 5-day-a-week gavage dosing in vegetable oil. This information provides insight into pharmacokinetics applicable to the NTP study and the renal tumors produced. Clearly, with increased dose, blood OTA concentration increased progressively irrespective of the two nondosing days in the middle and end of the 2-week experimental period. This is consistent with strong binding of OTA to serum albumin and a plasma half-life of about 10 days in the Fischer rat [START_REF] Mantle | Interpretation of the pharmacokinetics of ochratoxin A in blood plasma of rats, during and after acute or chronic ingestion[END_REF]. However, the pattern of OTalpha excretion is striking and informative, although no discussion of this finding was made in ref 25. OTalpha in urine indicates metabolism of OTA in nephron epithelia and, at higher OTA doses, this activity is prominent. However, OTalpha excretion falls quickly nearly to zero during the two-day nondosing period, but exactly the same pattern is repeated during the second week. Thus, repeated pulses of OTA dosed by oral gavage are rather quickly subject to metabolism in nephron epithelia. Therefore, waiting for 3 days after a single OTA dose in ref 17 can be seen as counterproductive in an experiment designed to find evidence of DNA/OTA adducts in whole kidney.

The general lack of DNA adduct detection in several publications [START_REF] Gautier | Metabolism of ochratoxin A: Absence of formation of genotoxic derivatives by human and rat enzymes[END_REF][START_REF] Mally | Ochratoxin A: Lack of formation of covalent DNA adducts[END_REF][START_REF] Mally | Ochratoxin A causes DNA-damage and cytogenetic effects, but no DNA-adducts in rats[END_REF][START_REF] Schlatter | Carcinogenicity and kinetic aspects of ochratoxin A[END_REF] could be explained by several drawbacks in the methodologies used. Indeed, the experimental conditions and the methodologies used in these papers are not optimal and did not reproduce those described by the others. Notably, there is no quality verification of the DNA used for ACMS analysis or 32 P-postlabeling in the key publication [START_REF] Mally | Ochratoxin A: Lack of formation of covalent DNA adducts[END_REF] used to underpin much of the current claims that OTA is not a genotoxin. For some in vivo experiments, a low dose (0.2-1 mg/kg b.w.) was used for a short exposure time, 24 h; and for ACMS detection, the conditions were 0.5 mg/kg b.w. for 72 h. It has long been stated clearly that most of the adducts at a low (0.6 mg/kg b.w.) dose or a medium (1.2 mg/kg b.w.) dose become undetectable by 72 h [START_REF] Pfohl-Leszkowicz | DNA-Adduct Formation, in Mice Treated with Ochratoxin A, in Mycotoxins, Endemic Nephropathy and Urinary Tract Tumours[END_REF][START_REF] Pfohl-Leszkowicz | Differential DNA adduct formation and disappearance in three mice tissues after treatment by the mycotoxin, ochratoxin A[END_REF]. Thus, the conditions were not optimal for adduct recognition. Moreover, the ACMS measurement had been performed without piloting authentic samples for comparison, and they used a Turbo Ionspray source and heated the samples to 400 °C. Most of the DNA adducts and OTA-GSH will probably decompose at such high temperatures. For generation of OTA-DNA adduct standards, the photoreaction was carried out with OTA at 500 mM and dG at 20 mM, conditions that are at an exceedingly high concentration and do not match the conditions reported in ref 12 for successful isolation and characterization of the adduct standards. Also, it appears that no purification of the photoreaction was carried out and, at best, the solution would have been a mixture of C-C8 and O-C8 adducts.

Conditions for optimizing DNA extraction are not correct in ref 17. Mainly for the purification, they used a Qiagen column, a technique shown in three interlaboratory studies on postlabeling to induce a loss of DNA adduct (EU project 5 V0448 Comparison and validation of 32 P-postlabeling method; EU project BEQALM; EU project OTA Risk assessment QKL1-2001-0614). The purity of the DNA was also not good enough, probably being contaminated by RNA and protein, which would have decreased postlabeling efficiency [START_REF] Pfohl-Leszkowicz | Further arguments in favour of direct covalent binding of Ochratoxin A (OTA) after metabolic biotransformation[END_REF]. Figure 13 shows comparison of the UV spectrum of DNA prepared in the Wurzburg laboratory contemporaneously with the studies of ref 17 and markedly contaminated with RNA with that from which the adduct data in Figure 6 was obtained. Other spectra showed contamination with protein. The chromatographic conditions of separation were also not adequate for OTA-DNA adduction. The chromatographic pH conditions for 32 P-postlabeling analysis, with D1 at pH 6.8 and D3 at pH 3.5, do not conform to standard published procedures, where D1 is run at a pH value below 6.0, and D3 is run at pH ∼6.4 [START_REF] Faucet | Evidence for covalent DNA adduction by ochratoxin A following chronic exposure to rat and subacute exposure to pig[END_REF]. Indeed, in ref 17, the separation conditions described were those for polycyclic aromatic hydrocarbons (PAH). PAH conditions were too strong for OTA and, even for the aristolochic acid positive control, the pattern was not optimal. The authors speculate that the DNA adduct observed is due to the cytotoxicity of oxygen reactive species leading for example to 8-oxoguanine. This modification can, however, not be detected with the postlabeling method using nuclease P1 enrichment, as it detects only bulky lipophilic adducts and not hydroxylated hydrophilic nucleotides. In the same way, etheno bases cannot be detected by this technique [START_REF] Bartsch | Ultrasensitive and specific detection methods for exocyclic DNA adducts: markers for lipid peroxidation and oxidative stress[END_REF]. Recently, we explained how an oxidative pathway leads to the covalent OTA-DNA adduct via biotransformation of OTA into the quinone derivative OTHQ [START_REF] Faucet | Evidence for covalent DNA adduction by ochratoxin A following chronic exposure to rat and subacute exposure to pig[END_REF][START_REF] Manderville | Bioactivation and DNA adduction as a rationale for ochratoxin A carcinogenesis[END_REF][START_REF] Tozlovanu | Genotoxicity of the hydroquinone metabolite of ochratoxin A: Structure-activity relationships for covalent DNA adduction[END_REF].

If histopathological change is a general indicator of renal cells that are significant active or passive sites of OTA uptake and may be sites of persistent genetic change necessary for tumorigenesis, the outer stripe of the outer medulla is ideally the preferred tissue for finding adducts because prominent aneuploid karyomegaly in response to some mycotoxins is generally located in and around this region [START_REF] Boorman | Toxicology and Carcinogenesis Studies of Ochratoxin A[END_REF][START_REF] Mantle | Persistent karyomegaly caused by Penicillium nephrotoxins in the rat[END_REF][START_REF] Turner | The Effect of Penicillium Moulds on the Kidney and Their Relationship with Balkan Nephropathy[END_REF][START_REF] Brown | DNA ploidy distribution in renal tumours induced in male rats by dietary ochratoxin A[END_REF]. Even this region is not absolutely defined; packing of nephron P3 segments does not rigidly conform to a sharp line of demarcation in renal tissue, particularly concerning those from peripheral glomeruli. Recognition of precise anatomical transition in segment structure and function from P2 to P3 in a single nephron cannot easily be made in histology sections. There is no doubt that OTA can cause genetic damage both in vitro and in vivo. Demonstration of this [START_REF] Mally | Ochratoxin A causes DNA-damage and cytogenetic effects, but no DNA-adducts in rats[END_REF] in the kidney of rats dosed for a short period has been complemented by data from cells in tissue culture [START_REF] Mosesso | In vitro cytogenetic results supporting a DNA nonreactive mechanism for ochratoxin A, potentially relevant for its carcinogenicity[END_REF]; similar demonstration was also made (Mosesso, unpublished data) in kidneys at the 12-month stage of the lifetime experiment described in ref 36. More recent analysis by isotopic dilution to determine whether OTA forms DNA adducts in vivo has also concluded that it does not [START_REF] Delatour | Absence of 2′-deoxyguanosine-carbon 8-bound ochratoxin A adduct in rat kidney DNA monitored by isotope dilution LC-MS/ MS[END_REF]. However, as with the findings in ref 17, data from sophisticated spectrometric methodology can only be as reliable as is the quality of the material analyzed. The use of kidney from other protocols [START_REF] Mally | Ochratoxin A causes DNA-damage and cytogenetic effects, but no DNA-adducts in rats[END_REF][START_REF] Delatour | Absence of 2′-deoxyguanosine-carbon 8-bound ochratoxin A adduct in rat kidney DNA monitored by isotope dilution LC-MS/ MS[END_REF], not specifically designed to optimize the recognition of very small amounts of adducted nucleotide, is disappointing. Kidneys from rats given the higher OTA dose [START_REF] Mally | Ochratoxin A causes DNA-damage and cytogenetic effects, but no DNA-adducts in rats[END_REF] were obtained 3 days after the last OTA dose and from animals with marked polyuria, which indicated some renal dysfunction. It is unreasonable to assume efficient delivery of OTA to proximal tubule epithelia if there is overt evidence of renal dysfunction. We calculate that circulating OTA concentration in blood plasma can account for all measured OTA in kidneys so that no renal parenchyma accumulation of OTA could be perceived.

Material sourced from a 90-day study [START_REF] Rached | Ochratoxin A: 13-week oral toxicity and cell proliferation in male F344/N rats[END_REF] for the study of ref 37 had plasma OTA concentration for the highest dose group, designed to mimic that of the NTP study ( 4), only at a mean value of 3 µg/mL (with rather large standard deviation). In any case, such animals would probably not have had a sufficient period of OTA exposure for renal tumorigenesis to have been put in place [START_REF] Mantle | Minimum tolerable exposure period and maximum threshold dietary intake of ochratoxin A for causing renal cancer in Dark Agouti rats[END_REF], and there was no evidence that plasma OTA concentration had reached a steady state. This is an important consideration since covalent binding of OTA to DNA could relate only rather specifically to renal tumorigenicity. Adducts can occur without implication that they constitute an actual carcinogenic risk if the toxin insult is below threshold [START_REF] Waddell | Concordance of thresholds for carcinogenicity of N-nitrosodiethylamine[END_REF]. DNA adducts were recognized by 32 P-postlabeling in our studies on kidneys of rats with plasma OTA concentration stabilized around 8 µg/mL [START_REF] Garcia-Sanz | Three-dimensional microcomputed tomography of renal vasculature in rats[END_REF], which was typical of that associated with longterm renal tumorigenesis in male Fischer rats [START_REF] Mantle | Renal tumourigenesis in male rats in response to chronic dietary ochratoxin A[END_REF]. The rats were at the 12 month stage from a large cohort continuing for life on dietary OTA and causing renal cancer [START_REF] Mantle | Renal tumourigenesis in male rats in response to chronic dietary ochratoxin A[END_REF], but at that time, it was not realized that further exposure to OTA might be unnecessary for tumorigenesis because no one had yet explored latency for this toxin. This emphasizes that sensitive 32 Ppostlabeling analysis should have been applied to the kidney tissues selected for the study of ref 37 to give assurance that spectrometric methodologies could be used with confidence to assess whether adducts were present. Otherwise, acquisition of negative evidence from protocols that have not been challenged concerning their fitness for purpose is an extremely weak basis for constructing a putative mechanism in carcinogenesis.

It has recently been pointed out that the work presented in ref 37 was flawed because the limit of detection (LOD) (10 fmol dG-OTA, corresponding to 3.5 adducts per 10 9 nucleotides) was obtained from DNA hydrolysates (500 µg DNA equivalent) and not from the actual rat kidney sample for which the negative results were presented [START_REF] Pfohl-Leszkowicz | Formation of 2′-deoxyguanosine-carbon 8-bound ochratoxin A adduct in kidney DNA[END_REF]. This argument stemmed from the fact that a high amount of sample spiking (1250 fmol) with an isotopically labeled dG-OTA adduct of 96-98% isotopic purity was used, and the nonisotopically labeled dG-OTA adduct impurity was observed in the DNA hydrolysates but not in the rat kidney sample. Clearly, the LOD for the DNA hydrolysates versus the rat kidney sample were not the same. DNA purification from the rat tissue sample is more complicated, requiring extra steps, and interfering species could dramatically change the LOD. It was also pointed out that in the rat kidney sample a peak for the dG-OTA adduct following 90 days of incubation was actually observed in the ref 37 study using the m/z 633 f 429 fragmentation transition in MS/MS detection. This apparent peak was not attributed to the dG-OTA adduct because an adduct peak was not observed using the transition m/z 633 f 517. In response to the criticism, the authors argued (41) that their method of quantification was sound, but they avoided discussing whether they were justified in their assumption that the LOD from the DNA hydrolysates could serve as an accurate LOD for the rat kidney sample. They also point out that the absence of a signal at transition m/z 633 f 517 is a strong argument against the presence of dG-OTA in the sample, even though an adduct peak was observed using the m/z 633 f 429 transition. However, the m/z 633 f 429 transition is more intense than the m/z 633 f 517 (20% greater), and here a clear adduct peak above background was observed that comigrates with the adduct standard. Given that the m/z 633 f 429 transition provides a more intense adduct signal and showed positive evidence for dG-OTA in rat kidneys (for OTAtreated rat at a dose of 210 µg/kg b.w. for 90 days), we can argue that the findings from the ref 37 study are in agreement with our positive evidence for dG-OTA presented here.

However, a genotoxicity mechanism operating in animals in which renal tumors will form and in which genetic damage has been demonstrated will evoke a relatively simple concept. The concept turns on a reasonable probability of occasional persistent genetic change related to tumorigenesis residing in perhaps just one nucleus in the ∼30,000 highly differentiated nephrons [START_REF] Solomon | Developmental changes in nephron number, proximal tubular length and superficial nephron glomerular filtration rate of rats[END_REF] in just one of a rat's kidneys. Such could certainly be expected after a lifetime of continual bombardment of rat tubular epithelial cells with OTA molecules, as in the principal lifetime carcinogenicity studies to date [START_REF] Boorman | Toxicology and Carcinogenesis Studies of Ochratoxin A[END_REF][START_REF] Castegnaro | Sex-and strainspecific induction of renal tumours by ochratoxin A in rats correlates with DNA adduction[END_REF][START_REF] Mantle | Renal tumourigenesis in male rats in response to chronic dietary ochratoxin A[END_REF]. It is not unreasonable that other recent studies have shown that lifetime exposure is not mandatory [START_REF] Mantle | Minimum tolerable exposure period and maximum threshold dietary intake of ochratoxin A for causing renal cancer in Dark Agouti rats[END_REF] but that less than half a lifetime or even as little as 6 months of continuous toxic insult during the half of an ∼2-year rat lifespan would also be sufficient to determine that renal tumorigenesis could arise in some individuals. Experimental OTA tumorigenesis seems to be highly focal in rat kidneys, according to observations on about 50 tumorous kidneys throughout recent studies in London. Bilateral OTA tumorigenesis seems, in our experience in the rat, to be doserelated, and this would be consistent with the statistical probability of causing one focus of the necessary genetic damage in each kidney of an animal.

All experimental dose-response data for OTA's renal carcinogenesis, combined and plotted in the format of ref 43, makes a compelling case for OTA being a thresholded carcinogen in the male rat [START_REF] Mantle | Minimum tolerable exposure period and maximum threshold dietary intake of ochratoxin A for causing renal cancer in Dark Agouti rats[END_REF]. There should be no philosophical conflict that the mycotoxin can also make covalent adducts with DNA and thereby, after many months of exposure to toxin molecules passing through renal epithelium, occasionally cause the particular combination of persistent DNA change that programs an occasional renal cell toward tumorigenesis, discovered late in life. Recently, it has been demonstrated (44) that genotoxic thresholds can exist for some genotoxins. Thus, determining the lowest dose that does not increase background mutation rate is essential and represents an important new aspect of genotoxicology. We recently showed that continuous treatment of Dark Agouti rats with feed contaminated with 50 ng OTA/kg for 4 weeks led to the formation of DNA adduct [START_REF] Castegnaro | Balkan endemic nephropathy: role of ochratoxin A through biomarkers[END_REF][START_REF] Pfohl-Leszkowicz | Further arguments in favour of direct covalent binding of Ochratoxin A (OTA) after metabolic biotransformation[END_REF]; this dosage is 8-fold less than that having no adverse clinical effect during lifetime exposure [START_REF] Mantle | Minimum tolerable exposure period and maximum threshold dietary intake of ochratoxin A for causing renal cancer in Dark Agouti rats[END_REF].

The current findings relate solely to defining the fundamental genotoxicity of OTA as revealed by 32 P-postlabeling and the structure determination of the principal adduct to demonstrate direct covalent binding of OTA to DNA. Further understanding of the precise mutations involved in rat renal tumorigenesis in response to OTA will assist evidence-based application of animal model data to human risk assessment.

Figure 1 .

 1 Figure 1. Comparison of the kidney DNA profile of an OTA-treated rat. (a) D1 2.3 M phosphate buffer, + transfer of 4 cm. (b) D1 3 M phosphate buffer, + transfer of 2 cm.

  Vitro OTA-DNA Interaction. Authentic synthetic standards of model OTA-guanine compounds, C-C8 OTA 3′-dGMP, and O-C8 OTA 3′-dGMP were used to develop a suitable HPLC/MS n method.

Figure 2 .

 2 Figure 2. Autoradiographic illustration of steps in chromatographic resolution of two of our model synthetic adducts after 32 P-postlabeling, showing discrete separation resolved from stray radioactivity after a second D4 step. Transition from D1-D2 was by contact transfer of the radioactive region after the D1 step.

Figure 3 .

 3 Figure 3. Annotated autoradiograph of a model chromatogram (D1 separation) illustrating the preparative isolation of samples 1 and 2 used for structural analysis. Lane 1 is synthetic C-C8dG-OTA processed via standard 32 P-postlabeling. Lanes 2 and 3 are products of calf thymus DNA/OTA photoirradiation processed via standard 32 P-postlabeling to guide preparative excision of unlabeled compounds from the central region. Lane 4 is synthetic O-C8dG-OTA also processed via 32 P-postlabeling. It was necessary to avoid 32 P contamination of samples for LC-MS.

Figure 4 .

 4 Figure 4. Structure of C-C8 OTA 3′-dGMP.

Figure 5 .

 5 Figure 5. Structure of O-C8 OTA 3′-dGMP.

Figure 6 .

 6 Figure 6. Representative autoradiographic displays from Dark Agouti and Fischer rats illustrating similar adduct patterns for each strain but consistent dominance of an adduct attributed to C-C8 OTA 3′-dGMP.

Figure 7 .

 7 Figure 7. Background subtracted mass spectrum for OTA-DNA sample 1.

Figure 8 .

 8 Figure 8. Extracted ion chromatograms of m/z 517 for (A) OTA-DNA sample 1, (B) OTA-DNA sample 2, and (C) the authentic standard of C-C8 OTA 3′dGMP after pretreatment with formic acid. The data for the authentic standard is weak but clearly shows a peak at the correct retention time.

Figure 9 .

 9 Figure 9. Background-subtracted mass spectrum for OTA-DNA of sample 2.

Figure 10 .

 10 Figure 10. (a) Structure of desphosphate C-C8 OTA 3′dGMP (m/z 655) and (b) structure of 3′,5′-bisphosphate OTA-dGMP (m/z 815).

Figure 11 .

 11 Figure 11. MS/MS spectrum (collision energy 25 eV) of m/z 517, showing CID fragmentation of the deconjugated aglycone of C-C8 OTA 3′dGMP. The spectrum shows the two principal fragment ions m/z 473 and m/z 429.

Figure 12 .

 12 Figure 12. Autoradiographic comparison of chromatographic mobility after 32 P-postlabeling of synthetic C-C8dG-OTA, the product of photoradiation of calf thymus DNA with OTA, and kidney DNA of a Dark Agouti rat given the oral OTA regimen. (A) D1 separation, showing sources of contact transfer of spot 1 to new plates for subsequent D2, D3, and D4 steps and (B) outcome of the completed protocol showing the mobility of the standard adduct similar to that of a principal radioactive component after both in vitro and in vivo interaction with OTA.

Figure 13 .

 13 Figure 13. Comparison of UV-spectra of rat kidney DNA isolated by EU partners in Wurzburg (above) and Toulouse (below) as part of a 2003 interlaboratory study, as illustrated in attachments 11 and 7, respectively, submitted to the EU project coordinator for the official report but not included. Spectra are matched here for x-axis scale.Considering the DNA quality criterion that UVmax should not be <257 nm and that the UV absorbance ratio (260:280 nm) should be 1.8-2.0, all Wurzburg samples were contaminated by RNA; the spectrum illustrated here represents nearly pure RNA (UVmax 252; 260:280 nm absorbance ratio 2.2).

Acknowledgment. Part of this work was funded by the 5th RTD Framework Programme of the European Union, Project QLK1-2001-01614. R.A.M. thanks the Natural Sciences and Engineering Research Council of Canada for funding. A.P.-L. thanks Region Midi-Pyre ´ne ´es 'food safety program', Ligue Nationale Franc ¸aise Contre le Cancer (2007) and Association pour la Recherche Contre le Cancer (2005; 2008) for funding. We thank Mariana Tozlovanu and Maud Juzio for skilled technical assistance in Toulouse and Guelph, respectively, and the Drug Metabolism and Pharmacokinetics Department, Glaxo-SmithKline for the use of their analytical facilities.


From competition to cooperation: Visual neglect across the hemispheres

Keywords: Post-stroke cognitive deficits, brain connectivity, visual attention, recovery, rehabilitation

Visuospatial neglect is a frequent and disabling consequence of injuries to the right hemisphere. Patients with neglect show signs of impaired attention for left-sided events, which depends on dysfunction of fronto-parietal networks. After unilateral injury, such as stroke, these networks and their contralateral homologs can reorganize following multiple potential trajectories, which can be either adaptive or maladaptive. This article presents possible factors influencing the profile of evolution of neglect towards recovery or chronicity, and highlights potential mechanisms that may constrain these processes in time and space.

The integrity of white matter pathways within and between the hemisphere appears to pose crucial connectivity constraints for compensatory brain plasticity from remote brain regions. Specifically, the availability of a sufficient degree of inter-hemispheric connectivity might be critical to shift the role of the undamaged left hemisphere in spatial neglect, from exerting maladaptive effects, to promoting compensatory activity.

INTRODUCTION

Lesion-induced cognitive deficits place a huge burden on the daily life of patients and their families, and have a substantial impact on public health. Brain damage, even when it is focal in nature, such as in stroke, has effects which are not only local. It typically disrupts activity in highly distributed, large-scale neural networks, connected by long-range white matter tracts [START_REF] Bartolomeo | The quest for the 'critical lesion site' in cognitive deficits: problems and perspectives[END_REF][START_REF] Corbetta | Common Behavioral Clusters and Subcortical Anatomy in Stroke[END_REF]. After damage, these networks can reorganize following multiple potential trajectories [START_REF] Bartolomeo | Let thy left brain know what thy right brain doeth: Inter-hemispheric compensation of functional deficits after brain damage[END_REF], which can be either adaptive or maladaptive [START_REF] Bartolomeo | Spatially biased decisions: Toward a dynamic interactive model of visual neglect[END_REF].

Dysfunction of fronto-parietal networks in the right, non-language dominant hemisphere often provokes signs of visual neglect, a disabling condition whereby patients do not pay attention to left-sided objects, and may behave as if the left part of the world did not exist [START_REF] Heilman | Neglect and related disorders[END_REF][START_REF] Mesulam | A cortical network for directed attention and unilateral neglect[END_REF][START_REF] Bartolomeo | Visual and motor neglect: Clinical and neurocognitive aspects[END_REF][START_REF] Bartolomeo | Visual neglect[END_REF][START_REF] Bartolomeo | Attention disorders after right brain damage: Living in halved worlds[END_REF][START_REF] Husain | [END_REF]. Neglect predicts a poor functional outcome [START_REF] Denes | Unilateral spatial neglect and recovery from hemiplegia: A follow-up study[END_REF][START_REF] Nijboer | Predicting functional outcome after stroke: the influence of neglect on basic activities in daily living[END_REF]; clinical management and social reinsertion of these patients represent substantial societal problems. Subcortical damage to long-range white matter tracts has emerged as a major cause of visual neglect and other post-stroke cognitive deficits [START_REF] Bartolomeo | The quest for the 'critical lesion site' in cognitive deficits: problems and perspectives[END_REF][START_REF] Corbetta | Common Behavioral Clusters and Subcortical Anatomy in Stroke[END_REF][START_REF] Bartolomeo | Left unilateral neglect as a disconnection syndrome[END_REF][START_REF] Ffytche | Beyond localization: from hodology to function[END_REF]. Damage to the tightly packed white matter fascicles is likely to be more disruptive for network activity than equivalent damage to cortical areas [START_REF] Bartolomeo | Left unilateral neglect as a disconnection syndrome[END_REF]. Today, diffusion MRI and white matter tractography permit the detailed anatomical study of white matter tracts in vivo. Such techniques are becoming increasingly important in studies of stroke-induced network dysfunctions [START_REF] Urbanski | DTI-MR tractography of white matter damage in stroke patients with neglect[END_REF][START_REF] De Schotten | Direct evidence for a parietal-frontal pathway subserving spatial awareness in humans[END_REF][START_REF] Urbanski | Brain networks of spatial awareness: Evidence from diffusion tensor imaging tractography[END_REF].

Visusospatial attention relies on large-scale fronto-parietal networks in the human brain [START_REF] Corbetta | The reorienting system of the human brain: from environment to theory of mind[END_REF], linked by three branches of the superior longitudinal fasciculus (SLF I-III) [START_REF] Makris | Segmentation of subcomponents within the superior longitudinal fascicle in humans: a quantitative, in vivo, DT-MRI study[END_REF][START_REF] De Schotten | A lateralized brain network for visuospatial attention[END_REF]. These branches display anatomical signs of hemispheric asymmetries favoring the right hemisphere [START_REF] Bartolomeo | Hemispheric lateralization of attention processes in the human brain[END_REF], with a ventro-dorsal gradient of asymmetry, progressively decreasing from SLF II to SLF I [START_REF] De Schotten | A lateralized brain network for visuospatial attention[END_REF]. A further, more ventral network is connected by the inferior frontooccipital fasciculus (IFOF) [START_REF] Catani | Virtual in vivo interactive dissection of white matter fasciculi in the human brain[END_REF] (Fig. 1). Partially overlapping fronto-parietal networks also control nonspatial aspects of attention, such as alertness [START_REF] Sturm | On the functional neuroanatomy of intrinsic and phasic alertness[END_REF], which is typically impaired in spatial neglect [START_REF] Robertson | Phasic alerting of neglect patients overcomes their spatial deficit in visual awareness[END_REF]. Increasing alertness alleviates spatial bias in neglect patients [START_REF] Robertson | Phasic alerting of neglect patients overcomes their spatial deficit in visual awareness[END_REF], and also improves perceptual awareness of near-threshold targets in normal participants [START_REF] Kusnir | Phasic auditory alerting improves visual conscious perception[END_REF].

A vast amount of evidence from neurological patients indicates that network dysfunction in the SLF II-III and IFOF networks, rather than focal brain damage, is the critical precursor of many signs of visual neglect [START_REF] Bartolomeo | Visual and motor neglect: Clinical and neurocognitive aspects[END_REF][START_REF] Bartolomeo | Left unilateral neglect as a disconnection syndrome[END_REF][START_REF] De Schotten | Direct evidence for a parietal-frontal pathway subserving spatial awareness in humans[END_REF][START_REF] Doricchi | The anatomy of neglect without hemianopia: a key role for parietal-frontal disconnection?[END_REF][START_REF] Doricchi | White matter (dis)connections and gray matter (dys)functions in visual neglect: Gaining insights into the brain networks of spatial awareness[END_REF][START_REF] Bartolomeo | Brain networks of visuospatial attention and their disruption in visual neglect[END_REF][START_REF] Corbetta | Spatial neglect and attention networks[END_REF][START_REF] Lunven | Attention and spatial cognition: Neural and anatomical substrates of visual neglect[END_REF]. The most frequent cause of such patterns of network dysfunction is stroke [START_REF] Urbanski | DTI-MR tractography of white matter damage in stroke patients with neglect[END_REF][START_REF] Urbanski | Brain networks of spatial awareness: Evidence from diffusion tensor imaging tractography[END_REF][START_REF] Verdon | Neuroanatomy of hemispatial neglect and its functional components: a study using voxel-based lesionsymptom mapping[END_REF][START_REF] De Schotten | Damage to white matter pathways in sub-acute and chronic spatial neglect: a group study and two single-case studies with complete virtual "in-vivo" tractography dissection[END_REF], but signs of neglect can also be observed in other neurological conditions, such as brain tumors [START_REF] Charras | Functional reorganization of the attentional networks in low-grade glioma patients: A longitudinal study[END_REF][START_REF] Bartolomeo | Spatial cognition[END_REF][START_REF] Jackson | Case of large cerebral tumour without optic neuritis and with left hemiplegia and imperception In: Taylor J, editor Selected Writings of John Hughlings Jackson[END_REF], brain surgery [START_REF] De Schotten | Direct evidence for a parietal-frontal pathway subserving spatial awareness in humans[END_REF][START_REF] Shinoura | Damage to the right superior longitudinal fasciculus in the inferior parietal lobe plays a role in spatial neglect[END_REF][START_REF] Vallar | Cerebral correlates of visuospatial neglect: a direct cerebral stimulation study[END_REF], or neurodegenerative diseases [START_REF] Andrade | Visual neglect in posterior cortical atrophy[END_REF][START_REF] Andrade | Visuospatial deficits in posterior cortical atrophy: structural and functional correlates[END_REF][START_REF] Andrade | Visuospatial deficits and hemispheric perfusion asymmetries in posterior cortical atrophy[END_REF][START_REF] Migliaccio | Brain networks in posterior cortical atrophy: A single case tractography study and literature review[END_REF][START_REF] Silveri | Unilateral spatial neglect in degenerative brain pathology[END_REF]. Specific lesional sites, such as the occipito-temporal cortex [START_REF] Khurshid | Reperfusion of specific cortical areas is associated with improvement in distinct forms of hemispatial neglect[END_REF] or the cingulate gyrus (in addition to fronto-parietal damage [START_REF] Lecce | Cingulate neglect in humans: Disruption of contralesional reward learning in right brain damage[END_REF]), may give rise to specific forms of neglect (respectively, stimulus-centered and motivational neglect). White matter damage may also produce specific patterns of neglect, depending on the affected circuits [START_REF] Verdon | Neuroanatomy of hemispatial neglect and its functional components: a study using voxel-based lesionsymptom mapping[END_REF][START_REF] Toba | Common brain networks for distinct deficits in visual neglect. A combined structural and tractography MRI approach[END_REF]. Other component deficits of neglect, however, such as 'magnetic' attraction of attention towards the ipsilesional stimuli [START_REF] Mark | Hemispatial neglect affected by non-neglected stimuli[END_REF][START_REF] D'erme | Early rightwards orienting of attention on simple reaction time performance in patients with left-sided neglect[END_REF][START_REF] Gainotti | Early orientation of attention toward the half space ipsilateral to the lesion in patients with unilateral brain damage[END_REF], and impaired spatial working memory [START_REF] Wansard | Visual neglect: is there a relationship between impaired spatial working memory and recancellation?[END_REF][START_REF] Wansard | Support for distinct subcomponents of spatial working memory: a double dissociation between spatial-simultaneous and spatial-sequential performance in unilateral neglect[END_REF][START_REF] Malhotra | Spatial working memory capacity in unilateral neglect[END_REF] have proven more difficult to link to specific lesional patterns [START_REF] Toba | Component deficits of visual neglect: "Magnetic" attraction of attention vs. impaired spatial working memory[END_REF].The term motor neglect designates yet another condition, whereby patients underuse their contralesional limbs, even in the absence of elementary motor deficits [START_REF] Laplane | Motor neglect[END_REF][START_REF] Bartolomeo | Motor neglect[END_REF], sometimes to a degree mimicking hemiplegia. The lesional correlates of motor neglect, and its relationship to visual neglect, remain underdetermined [START_REF] Migliaccio | Damage to the medial motor system in stroke patients with motor neglect[END_REF][START_REF] Toba | Quantitative Assessment of Motor Neglect[END_REF].

The network-based nature of visual neglect offers potential perspectives for its compensation, whether spontaneous or based on rehabilitation procedures [START_REF] Halligan | Visual Neglect[END_REF]. After the injury, brain networks can follow multiple trajectories during post-stroke reorganization in different patients [START_REF] Bartolomeo | Let thy left brain know what thy right brain doeth: Inter-hemispheric compensation of functional deficits after brain damage[END_REF]. These trajectories can be either adaptive or maladaptive [START_REF] Bartolomeo | Spatially biased decisions: Toward a dynamic interactive model of visual neglect[END_REF]. Disrupted activity of lesioned network nodes can potentially be compensated for by activity in different nodes, provided that the compensatory nodes can communicate with the rest of the system [START_REF] Bartolomeo | Visual neglect: getting the hemispheres to talk to each other[END_REF]. Network activity within and across the hemispheres can be restored or rebalanced, by acting on the intact nodes of the damaged networks, or on their homologs in the healthy hemisphere [START_REF] Halligan | Visual Neglect[END_REF][START_REF] Vico Fallani | Network neuroscience for optimizing brain-computer interfaces[END_REF]]. An intriguing possibility emerging from these considerations is that the connectivity profiles of individual patients may constitute crucial determinants of their individual response to injury and treatment.

Importantly, the identification of biomarkers of the likely trajectory of post-stroke cognitive deficits can improve the clinicians' ability to stratify patients and to reduce variability in trial outcomes. Biomarkers can assist clinical decisions for individual patients, by predicting the potential for recovery and by enabling clinicians to choose an appropriate rehabilitation strategy [START_REF] Boyd | Biomarkers of Stroke Recovery: Consensus-Based Core Recommendations from the Stroke Recovery and Rehabilitation Roundtable[END_REF]. The following sections will examine potential factors influencing post-stroke recovery in time and space, as well as possible biomarkers targeting these factors.

WHEN: NEGLECT COMPENSATION IN TIME

The time since lesion occurred has a deep influence on compensation processes [START_REF] Bartolomeo | Let thy left brain know what thy right brain doeth: Inter-hemispheric compensation of functional deficits after brain damage[END_REF][START_REF] Cramer | Repairing the human brain after stroke: I. Mechanisms of spontaneous recovery[END_REF].

During the acute/subacute phase of a stroke, remote effects of the lesion are described as diaschisis phenomena. The concept of diaschisis was introduced by von Monakov [START_REF] Monakow Cv | Die Lokalisation im Grosshirn und der Abbau der Funktion durch kortikale Herde[END_REF] to describe the effects of focal brain damage in anatomically intact areas that are connected to the lesion site [START_REF] Carrera | Diaschisis: past, present, future[END_REF][START_REF] Feeney | Diaschisis[END_REF]. Diaschisis effects implicate not only remote loss of excitability, but also disinhibition leading to increased excitability, both in the lesioned hemisphere (ipsilateral diaschisis) and in the unaffected hemisphere (transcallosal or transhemispheric diaschisis) [START_REF] Buchkremer-Ratzmann | Electrophysiological Transcortical Diaschisis After Cortical Photothrombosis in Rat Brain[END_REF]. Murine models suggest that ipsilateral diaschisis is associated to the downregulation of gamma-aminobutyric acid (GABA)A receptors alpha1, alpha2, alpha5 and gamma2 within the lesioned hemisphere, whereas up-regulation of receptors alpha3 in the contralateral hemisphere may lead to transhemispheric diaschisis. Thus, a sequential order of receptor modulation seems to occur in either hemisphere in the acute phase of a stroke [START_REF] Paik | Role of GABA plasticity in stroke recovery[END_REF].

Cortical hyperexcitability tends to increase in the early weeks in both hemispheres after a stroke, and to decrease thereafter. These processes may contribute to spontaneous functional recovery. However, when they persist after the first weeks post-stroke they can assume a maladaptive role and hinder recovery [START_REF] Cramer | Repairing the human brain after stroke: I. Mechanisms of spontaneous recovery[END_REF]. Thus, the significance of post-stroke hyperactivity for functional recovery varies according to time. Four partially overlapping temporal epochs can be identified [START_REF] Bartolomeo | Let thy left brain know what thy right brain doeth: Inter-hemispheric compensation of functional deficits after brain damage[END_REF]: (1) an hyperacute phase within hours of the stroke, with local inflammation, oedema and distal diaschisis; (2) a subacute phase during the first weeks after the stroke, when most of the spontaneous recovery occurs and then reaches a plateau; (3) a chronic phase, beginning weeks to months after the stroke, associated with a 'normalization' of the activity and a stabilization of the deficits [START_REF] Cramer | Repairing the human brain after stroke: I. Mechanisms of spontaneous recovery[END_REF]; (4) finally, several months after the injury, rewiring of white matter fibers may occur, with axonal sprouting near the site of ischemic injury [START_REF] Dancause | Extensive cortical rewiring after brain injury[END_REF]. These mechanisms, which develop over different time frames, may be associated with different forms of functional recovery after stroke [START_REF] Bartolomeo | Let thy left brain know what thy right brain doeth: Inter-hemispheric compensation of functional deficits after brain damage[END_REF]. For instance, whereas recovery of motor deficits usually peaks within 30 days, language and other high cognitive functions may improve well beyond this phase, up to a year after stroke [START_REF] Cramer | Repairing the human brain after stroke: I. Mechanisms of spontaneous recovery[END_REF]. This difference in recovery rate suggests that partly distinct mechanisms may be at work in motor recovery or in cognitive recovery [START_REF] Grefkes | Cortical Reorganization After Stroke: How Much and How Functional?[END_REF]. For example, the late occurrence of cortical rewiring might especially contribute to the recovery of cognitive functions [START_REF] Bartolomeo | Let thy left brain know what thy right brain doeth: Inter-hemispheric compensation of functional deficits after brain damage[END_REF].

Animal models of post-stroke recovery of function suggested the idea of time-limited windows of neuroplasticity following stroke in the adult brain. These time periods share molecular mechanisms with critical time windows in development [START_REF] Murphy | Plasticity during stroke recovery: from synapse to behaviour[END_REF]. For example, rehabilitation of motor deficits in the first few weeks after experimental stroke was more likely to be successful, in parallel with an increased number of branches and complexity of layer V neurons occurring at this early post-lesion period [START_REF] Biernaskie | Efficacy of rehabilitative experience declines with time after focal ischemic brain injury[END_REF].

Although at the much longer time scale of several months, the finding that shorter lesion-rehabilitation intervals tended to predict better response to prism adaptation therapy in patients with chronic neglect [START_REF] Lunven | Anatomical predictors of successful prism adaptation in chronic visual neglect[END_REF] seems broadly consistent with these notions; the earlier the intervention, the greater the potential for prism adaptation to induce compensation of neglect. This result suggests that anterior inter-hemispheric communication may favorably influence patients' response to prism adaptation especially during early temporal windows of opportunity.

Despite these considerations, however, the windows of neuroplasticity never completely close, and (suboptimal) recovery can still occur after the critical post-stroke period. Furthermore, the possibility exists of reopening developmental time windows in the adult brain [START_REF] Heimler | Are critical periods reversible in the adult brain? Insights on cortical specializations based on sensory deprivation studies[END_REF]; thus, critical periods of plasticity might be reactivated even in the lesioned adult brain, e.g. by using noninvasive brain stimulation [START_REF] Pino | Modulation of brain plasticity in stroke: a novel model for neurorehabilitation[END_REF] or behavioral techniques such as prism adaptation therapy [START_REF] Lunven | Anatomical predictors of successful prism adaptation in chronic visual neglect[END_REF].

WHERE: NEGLECT COMPENSATION IN THE BRAIN SPACE

In general, the localization of the lesions in the brain is the best predictor of the occurrence of post-stroke cognitive impairment [START_REF] Weaver | Strategic infarct locations for post-stroke cognitive impairment: a pooled analysis of individual patient data from 12 acute ischaemic stroke cohorts[END_REF]. However, the importance of localization should not be construed as supporting a localistic view of regional specialization in the brain. Rather, strategically placed lesions, for example affecting the tightly packed fibers of long-range white matter tracts [START_REF] Bartolomeo | Left unilateral neglect as a disconnection syndrome[END_REF], are more likely than cortical lesions to disrupt the large-scale brain networks whose functioning is at the basis of cognitive processes [START_REF] Bartolomeo | The quest for the 'critical lesion site' in cognitive deficits: problems and perspectives[END_REF]. Thus, the brain space should be conceived as a complex network of structural connections, or 'connectome' [START_REF] Sporns | The human connectome: A structural description of the human brain[END_REF],

The functional repertoires resulting from lesions to the connectome, or "disconnectome", are not only defined by white matter connections, but also by the highly stereotyped spatial distribution of strokes [START_REF] De Schotten | Brain disconnections link structural connectivity with function and behaviour[END_REF], depending on the vascular architecture of the brain.

A first spatial factor which may influence recovery is lesion size; large lesions are more likely than small lesions to impact the functioning of several brain systems, and hinder potential compensation from adjacent circuits. For example, the resolution of the ischemic penumbra may unmask compensatory activity from perilesional regions [START_REF] Murphy | Plasticity during stroke recovery: from synapse to behaviour[END_REF]. Follow-up of motor recovery after experimental lesions of the internal capsule in two macaques suggested that the size of the lesion can induce distinct compensatory mechanisms for flaccid paralysis.

Functional recovery required neural plasticity in the ipsilesional hemisphere for the smaller infarction, and in the contralesional hemisphere for the larger lesion [START_REF] Kato | Functional near-infraredspectroscopy-based measurement of changes in cortical activity in macaques during post-infarct recovery of manual dexterity[END_REF]. Thus, lesion volume might influence the mechanisms of recovery, with predominantly intrahemispheric compensation for small lesions, and recruitment of circuits in the opposite, healthy hemisphere for larger lesions. More relevant to neglect, a mouse model of ipsilesional spatial bias after focal damage to the medial agranular cortex demonstrated that the extent of the postlesional bias, but not that of recovery, correlated with the lesion size [START_REF] Ishii | Ipsilesional spatial bias after a focal cerebral infarction in the medial agranular cortex: A mouse model of unilateral spatial neglect[END_REF]. This pattern of results suggests that recovery was based on neural plasticity not within the peri-infarct area, but in distal regions in the same or contralateral hemisphere. Broadly consistent with these notions, early evidence on human neglect patients showed that the volume of lesion influenced the presence and degree of post-stroke neglect [START_REF] Levine | Left spatial neglect: Effects of lesion size and premorbid brain atrophy on severity and recovery following right cerebral infarction[END_REF].

A recent longitudinal study [START_REF] Lunven | White matter lesional predictors of chronic visual neglect: a longitudinal study[END_REF], which assessed neglect in 45 right-brain damaged patients in the subacute (<6 months) and chronic (> 1 year) phases of stroke, further specified the role of lesion volume in the evolution of neglect. Patients with chronic, persistent neglect tended to have larger lesions as compared with non-neglect patients; they had, however, similar lesion volumes as patients who eventually recovered from neglect, suggesting that lesion size per se is not a major determinant of the evolution of neglect in time. The results remain, however, consistent with an indirect role of lesion size; as stated above, large lesions are more likely than small lesion to impact several brain circuits, thus decreasing compensatory capacities.

Inter-and intra-hemispheric loci of lesions are more directly linked with the number and identity of lesioned circuits; as such, lesion location is an important factor determining the persistence of neglect signs in time. The laterality of the hemispheric lesion is perhaps the most obvious aspect. It has long been known that signs of contralesional neglect are more frequent, severe and persistent after right hemisphere damage than after lesions in the left hemisphere [START_REF] Stone | The assessment of visuo-spatial neglect after acute stroke[END_REF][START_REF] Beis | Right spatial neglect after left hemisphere stroke: Qualitative and quantitative study[END_REF]. Right-sided neglect after left hemisphere damage may recover promptly because of the compensatory capacities of the right hemisphere attention networks, which may be able to take account information coming from both sides of space, whereas the competence of left hemisphere networks is more restricted to the right space [START_REF] Bartolomeo | Hemispheric lateralization of attention processes in the human brain[END_REF][START_REF] Mesulam | Attention, confusional states and neglect[END_REF][START_REF] Heilman | Right hemisphere dominance for attention: the mechanism underlying hemispheric asymmetries of inattention (neglect)[END_REF][START_REF] Bartolomeo | Left unilateral neglect or right hyperattention?[END_REF][START_REF] Bartolomeo | Anatomy and disorders of the spatial attention system[END_REF].

Consistent with this model, persistent right-sided neglect has been described after bilateral hemispheric damage, whether due to vascular strokes [START_REF] Weintraub | Right sided hemispatial neglect and bilateral cerebral lesions[END_REF][START_REF] Brink | Differences between left-and right-sided neglect revisited: A large cohort study across multiple domains[END_REF], or to neurodegenerative conditions [START_REF] Andrade | Visual neglect in posterior cortical atrophy[END_REF][START_REF] Andrade | Visuospatial deficits in posterior cortical atrophy: structural and functional correlates[END_REF][START_REF] Andrade | Visuospatial deficits and hemispheric perfusion asymmetries in posterior cortical atrophy[END_REF][START_REF] Bartolomeo | Right-side neglect in Alzheimer's disease[END_REF]. Thus, some degree of right hemisphere dysfunction might be necessary even for signs of right-sided neglect to occur. The rightlateralized SLF III network is a possible candidate site for such right hemisphere dysfunction in patients with right-sided neglect [START_REF] Bartolomeo | Hemispheric lateralization of attention processes in the human brain[END_REF].

Relatively less is known about the intra-hemispheric determinants of neglect evolution. Although, as mentioned above, neglect is a network-based condition, and can in principle occur from damage anywhere in the trajectory of dorso-rostral SLF II-III and IFOF, most patients with persistent neglect have retrorolandic lesions. A possible reason for this occurrence is that SLF II and III have spatially close origins in the inferior parietal lobule, but diverge when traveling rostrally; thus, damage at or near the inferior parietal lobule [START_REF] Mort | The anatomy of visual neglect[END_REF] is likely to concurrently disrupt both the SLF II and the SLF III networks [START_REF] Bartolomeo | Brain networks of visuospatial attention and their disruption in visual neglect[END_REF]. In a study on 58 patients with strokes in the territory of the right middle cerebral artery [START_REF] De Schotten | Damage to white matter pathways in sub-acute and chronic spatial neglect: a group study and two single-case studies with complete virtual "in-vivo" tractography dissection[END_REF], SLF II disconnection was the most likely lesion to predict chronic spatial neglect. However, 7 of the 38 patients showing chronic spatial neglect did not show neuroimaging signs of frontoparietal disconnection; their lesions instead overlapped within the latero-dorsal portion of the thalamus.

Concerning more ventral lesions, Lunven et al. made a detailed description of a patient with chronic severe neglect on cancellation tasks at more than 8 years from a stroke in the occipito-temporal regions of the right hemisphere [START_REF] Lunven | Effector-dependent neglect and splenial disconnection: a spherical deconvolution tractography study[END_REF]. Neglect was, however, only present when the patient performed cancellation tasks with her right hand (controlled by the left hemisphere); performance reverted to normal when the patient used her left hand. White matter tractography demonstrated damage to the splenium of the corpus callosum, as well as a relative preservation of the right fronto-parietal network. Chronic, effector-dependent neglect may have emerged because the splenial disconnection deprived the right frontoparietal networks from visual information processed by the left hemisphere.

An important variable for neglect compensation is the status of inter-hemispheric communication. Although surgical section of the corpus callosum does not typically determine signs of neglect [START_REF] Berlucchi | Rightward attentional bias and left hemisphere dominance in a cue-target light detection task in a callosotomy patient[END_REF] (but see Ref. [START_REF] Corballis | Now you see it, now you don't: Variable hemineglect in a commissurotomized man[END_REF]), splenial disconnection may contribute to neglect in patients with right hemisphere damage, whether it occurred before [START_REF] Heilman | Callosal neglect[END_REF], or concurrently [START_REF] Lunven | Effector-dependent neglect and splenial disconnection: a spherical deconvolution tractography study[END_REF][START_REF] Tomaiuolo | Selective visual neglect in right brain damaged patients with splenial interhemispheric disconnection[END_REF]. In this case, callosal disconnection might prevent the left hemisphere from compensating for the deficits induced by right hemisphere damage, by taking charge of left-sided events [START_REF] Bartolomeo | Left unilateral neglect as a disconnection syndrome[END_REF]. In agreement with these notions, microstructural damage of the posterior corpus callosum has been shown to correlate with the clinical severity of neglect [START_REF] Bozzali | Microstructural damage of the posterior corpus callosum contributes to the clinical severity of neglect[END_REF]. In the above-mentioned longitudinal study of 45 patients with unilateral strokes in the right hemisphere [START_REF] Lunven | White matter lesional predictors of chronic visual neglect: a longitudinal study[END_REF], the presence of signs of posterior callosal disconnection was able to predict the chronic persistence of neglect. The authors concluded that splenial disconnection may prevent fronto-parietal networks in the left hemisphere from taking into account visual information coming from the left hemispace. In a subsequent study [START_REF] Lunven | Anatomical predictors of successful prism adaptation in chronic visual neglect[END_REF] on patients with splenial disconnection and chronic neglect, the integrity of more anterior callosal connections appeared to be crucial for patients to respond to prism-based rehabilitation [START_REF] Rossetti | Prism adaptation to a rightward optical deviation rehabilitates left hemispatial neglect[END_REF]. These results (summarized in Fig. 2) support the hypothesis that the healthy hemisphere has an important role in the compensation for stroke-induced, chronic neuropsychological deficits [START_REF] Bartolomeo | Let thy left brain know what thy right brain doeth: Inter-hemispheric compensation of functional deficits after brain damage[END_REF], and suggest that prism adaptation can foster this role by exploiting sensorimotor/prefrontal circuits for neglect compensation. Thus, the status of different sectors of the corpus callosum may represent possible connectional biomarkers to predict neglect recovery, and to choose the appropriate rehabilitation procedures.

Further, confirmatory evidence on the role of the healthy hemisphere in neglect compensation came from a study on 20 patients with brain tumors affecting the right hemisphere, who were followed up before and after awake brain surgery [START_REF] Charras | Functional reorganization of the attentional networks in low-grade glioma patients: A longitudinal study[END_REF]. Patients showed evidence for transient neglect signs in the acute post-operative phase; however, all of them had fully recovered when retested 3 months after surgery. It is likely that both the preservation of long-range white matter pathways within the right hemisphere, obtained thanks to intraoperative mapping [START_REF] De Schotten | Direct evidence for a parietal-frontal pathway subserving spatial awareness in humans[END_REF][START_REF] Bartolomeo | Mapping of visuo-spatial functions during brain surgery: a new tool to prevent unilateral spatial neglect[END_REF], and of the callosal connections with the left hemisphere, contributed to prevent the occurrence of chronic neglect in this population.

CONCLUSIVE REMARKS

Despite these encouraging results on the identification of connectional biomarkers for recovery of neglect, a word of caution is necessary. It is well known that patients who attain normal performance on paper-and-pencil tests may nevertheless keep showing clinical signs of neglect in everyday life [START_REF] Sala | Persistent neglect in everyday life[END_REF]. This possibility calls for more ecological tests of neglect, which for example require patients to interact with real 3D objects [START_REF] Tham | The baking tray task: A test of spatial neglect[END_REF]. Technologyenhanced versions of these tests are now being developed [START_REF] Cerrato | a technology-enhanced platform with tangible objects for the assessment of visual neglect: A multiple single-case study[END_REF], which provide clinicians with convenient, fast, and relatively automatized procedures, that patients can even perform at home to follow-up the effects of rehabilitation.

Apparently recovered patients may also show subtle signs of spatial bias on paperand-pencil tests. For example, they may keep starting to perform visual search tasks from the rightmost targets, even if they eventually find out all the targets [START_REF] Gainotti | Early orientation of attention toward the half space ipsilateral to the lesion in patients with unilateral brain damage[END_REF][START_REF] Azouvi | Sensitivity of clinical and behavioural tests of spatial neglect after right hemisphere stroke[END_REF]. Normal participants tend instead to start their search from the left side [START_REF] Bartolomeo | The relationship between visuospatial and representational neglect[END_REF], possibly as a consequence of physiological pseudoneglect [START_REF] Gigliotta | Pseudoneglect in Visual Search: Behavioral Evidence and Connectional Constraints in Simulated Neural Circuitry[END_REF]. Moreover, these patients often keep showing deficits on more stringent tests of spatial or nonspatial attention, such as speeded response time tests, or on dual task paradigms [START_REF] Bartolomeo | The novelty effect in recovered hemineglect[END_REF][START_REF] Bartolomeo | Inhibitory processes and compensation for spatial bias after right hemisphere damage[END_REF][START_REF] Bonato | Neglect and extinction depend greatly on task demands: a review[END_REF].

Thus, functional recovery from neglect may be incomplete, as it often happens when behavioral recovery is sustained by compensatory processes. It is important to be aware of the possibility of these "subclinical" deficits, which do have clinical implications, for example in taking decisions about the patient's ability to drive. Patients with normal performance of paper-and-pencil tests, but left-right asymmetries of response times, might well be at risk of road accidents and should not be permitted to resume driving or to use dangerous mechanical devices.

In addition, neglect compensation can be domain-selective. For example, some neglect patients show signs of neglect for both real and imagined scenes [START_REF] Bisiach | Unilateral neglect of representational space[END_REF][START_REF] Bartolomeo | Time to imagine space: a chronometric exploration of representational neglect[END_REF], perhaps as a result of disconnections between right-hemisphere attention networks and lefthemisphere systems important for the generation of visual mental images [START_REF] Rode | Representation and disconnection in imaginal neglect[END_REF]. A few of these patients have been shown to selectively recover from visual neglect, while keeping to demonstrate neglect for their visual mental images [START_REF] Bartolomeo | The relationship between visuospatial and representational neglect[END_REF][START_REF] Coslett | Neglect in vision and visual imagery: A double dissociation[END_REF]. Such patients might have learned with time (and possibly the help of people around them) to compensate for their neglect in the visuospatial domain, but not in the less ecological imaginal domain. As a matter of fact, neglect patients are often reminded by relatives and hospital staff to explore the visual scene thoroughly, and could learn to appreciate the consequences of their omissions (e.g., while eating or reading a newspaper), but this is less likely to happen for imagined scenes [START_REF] Bartolomeo | Levels of impairment in unilateral neglect[END_REF]. Such 'dynamic' dissociations between preserved and impaired performance might also rely on changes in functional long-range brain connectivity. Further, detailed reports on individual patients using advanced behavioral and neuroimaging techniques are required to assess these hypotheses.

Fig. 1 .

 1 Fig. 1. Schematic representation of long-range white matter pathways in the right hemisphere

Fig. 2 .

 2 Fig. 2. Schematic representation of the callosal regions whose anatomical integrity is important