Amedeo ; Pietri

Terenzio Bertuzzi
email: terenzio.bertuzzi@unicatt.it

Barbara Agosti

G Donadini

Transfer of aflatoxin B1 and fumonisin B1 from naturally contaminated raw

Keywords: aflatoxin B 1, fumonisin B 1, brewing, beer, maize grit, malted barley. 25

1

Introduction 2

In Italy it is common practice to brew beer from a mixture of malted barley and maize grit; 3 according to the Italian Regulation (Gazzetta Ufficiale della Repubblica Italiana 1962) and its 4 subsequent modifications, adjuncts (usually maize-derived products), can replace malted barley up to a 5 maximum of 40%. The natural occurrence in maize of mycotoxins such as aflatoxins, fumonisins, 6 trichothecenes and zearalenone, has been reported in various studies. Nearly 91% of the Italian 7 production of maize is grown in northern Italy (Istat 2006). In this area, which ranges from Piedmont 8 to Friuli Venezia Giulia, the mycotoxins that deserve special attention are aflatoxins, for their extreme 9 acute and chronic toxicity, and fumonisins for their high and widespread contamination, that cause 10 several disease states in animals and could affect human health [START_REF] Pietri | Occurrence of mycotoxins and ergosterol in maize 3 harvested over 5 years in Northern Italy[END_REF]Battilani et al. 11 2005). 12 Aflatoxins B 1 , B 2 , G 1 and G 2 are mainly produced by Aspergillus flavus and A. parasiticus, 13 fungal species that grow in warm conditions in tropical and sub-tropical regions of the world. 14 Aflatoxin B 1 (AFB 1 ), the prevalent and most toxic aflatoxin, is a potent genotoxic carcinogen in 15 laboratory animals and there is strong evidence that it is a liver carcinogen in humans. The 16 International Agency for Research on Cancer (IARC) has classified AFB 1 as a Class 1 (human 17 carcinogen) (IARC 1993). Food and health bodies have not set a tolerable daily intake (TDI) as AFB 1 18 is a genotoxic carcinogen; therefore, contamination in food should be reduced to the lowest possible 19 level. 20 Fumonisins B 1 , B 2 and B 3 are a group of closely related compounds mainly produced by Fusarium 21 verticillioides and F. proliferatum, fungi that commonly contaminate maize in many growing regions 22 of the world. Fumonisins have been shown to be hepatocarcinogenic in male rats and female mice and 23 nephrocarcinogenic in male rats [START_REF] Voss | An overview of rodent toxicities: liver an kidney effects of fumonisins and 10 Fusarium moniliforme[END_REF]. Fumonisin B 1 (FB 1 ), the prevalent and most toxic 24 fumonisin, has been associated with the high incidence of human oesophageal cancer in some regions 25 of South Africa and China (Sydenham et al. 1990; [START_REF] Sun | Fumonisin B 1 21 contamination of home-grown corn in high risk areas for esophageal and liver cancer in China[END_REF]). Recent findings suggest that FB 1 26 might increase the risk of neural tube defects in populations consuming large amounts of fumonisin-1 contaminated maize [START_REF] Missmer | Exposure 15 to fumonisins and the occurrence of neural tube defects along the Texas-Mexico border[END_REF]. FB 1 has been categorised by the IARC as class 2B 2 (possible human carcinogen) (IARC 2002). 3 As a consequence of the use of maize adjuncts, normally maize grit, in the brewing process, 4 aflatoxins and fumonisins may potentially occur in beer; moreover, aflatoxins can be occasionally 5 found also in malted barley [START_REF] Fukal | Radiochemical aflatoxin determination in barley, malt and beer[END_REF]). Surveys on different types of beer reported that AFB 1 6 was detected in 5 of 8 samples imported from Mexico and in one from Brazil (0.0028-0.049 µg l -1 ) 7 (Scott and Lawrence 1997), in 2 of 22 (0.0005 and 0.0008 µg l -1 ) and in 11 of 94 (0.0005-0.0831 8 µg l -1 ) Japanese and imported beer samples [START_REF] Nakajima | A survey of ochratoxin A and aflatoxins in domestic and 18 imported beers in Japan by immunoaffinity and liquid chromatography[END_REF]. Successively, in a survey on 304 9 samples of Canadian and imported beer, the toxin was found in 4 of 5 samples from India (0.096-10 0.230 µg l -1 ), 2 of 6 from Spain, 1 of 5 from Portugal and in 5 of 49 from Mexico (0.0044-0.018 µg l -1 ) 11 [START_REF] Mably | 11 Survey of aflatoxins in beer sold in Canada[END_REF]. Regarding FB 1 , several surveys reported concentrations generally lower than 100 12 µg l -1 (Scott and Lawrence 1995; [START_REF] Kawashima | Fumonisin B 1 and ochratoxin A in beers made 9 in Brazil[END_REF][START_REF] Torres | Occurrence of fumonisins in Spanish beers analyzed by an 4 enzyme-linked immunosorbent assay method[END_REF]; Hlywka and Bullerman 13 1999; [START_REF] Dasko | Determination of fumonisins B 1 and B 2 in beer[END_REF]). Generally, with the exception of a survey on 75 bottled Kenyan lager beers 14 [START_REF] Mbugua | The contamination of Kenyan lager beers with Fusarium 13 mycotoxins[END_REF], much higher levels of AFB 1 and FB 1 were found in some types of 15 traditional African beer (aflatoxins: 200-400 µg l -1 ; FB 1 : 38-1066 µg l -1 ) [START_REF] Scott | Mycotoxins transmitted into beer from contaminated grains during brewing[END_REF]Odhav and 16 Naicker 2002;[START_REF] Shephard | 16 Fumonisin mycotoxins in traditional Xhosa maize beer in South Africa[END_REF]. 17

Since aflatoxins (AFs) and fumonisins (FBs) can be present in beer, the aim of this study was to 18 investigate, during full scale industrial brewing process, the carry-over of AFs and FBs from naturally 19 contaminated raw materials to the different intermediate and final products. 20

21

Materials and methods 22

Brewing process 23

The study was carried out in an industrial brewery, located in northern Italy, that produces bottom 24 fermented pilsner type beer from a mixture of malted barley and de-germed maize grit (Figure 1) with 25 a particle size > 500 µm. First, finely ground malted barley and maize grit were mashed in order to 1 convert starch into fermentable sugars and dextrins and to provide mashing liquors with nitrogenous 2 fractions which can be assimilated by yeast. The mashing process was performed according to a single 3 decoction process, which requires two different kettles to be completed. In the adjunct kettles, a 4 fraction of the finely ground malted barley and maize grit were boiled with water (cooked mash); in 5 the second tun, finely ground malted barley and water were mixed and heated at two different 6 saccharification rest temperatures according to a specific protocol (malt mash). In this process the 7 content of the cooked mash was added back to the main malt mash to increase the main malt mash 8 temperature up to the first saccharification rest (decoction process). The following are the essentials of 9 the mashing operations. Malted barley (≅800 kg), maize grit (≅5000 kg) and water (≅15000 l) were 10 mixed in the adjunct kettle and held at 65 °C for 17 min. Then, the temperature was increased to 99. 5 11 °C in 40 min and the mash was boiled for 20 min. In the main mash tun, finely ground malted barley 12 (≅7000 kg) and water (≅20000 l) were mixed at 37 °C for 15 min and the mixture was allowed to stand 13 at 37 °C for 15 min. Then, the cooked mash was added and the temperature was raised to 63 °C. A 14 first stand for 45 min was programmed. Successively, the temperature was increased to 69 °C and a 15 second shorter stand (20 min) programmed. Finally, heating up to 78 °C (mashing off) in 20 min 16 preceded the separation of the sweet wort. During the process, the pH of the malt mash was adjusted to 17 5.6±0.1 by means of phosphoric acid. No addition of exogenous enzymes occurred during mashing 18 nor acidification by means of lactic acid bacteria. [START_REF] Belajova | Retention of ochratoxin A and fumonisin FB 1 and FB 2 from 20 beer on solid surfaces: comparison of efficiency of adsorbents with different origin[END_REF] The mashing liquors were separated from spent grains by means of a Meura filter, which is a thin 20 bed membrane-assisted mash filter (lautering). Lautering occurred in two stages: 1) the running off of 21 the first wort, that is the aqueous solution of the extract recovered from mashing; 2) the sparging of the 22 spent grains at 78 °C to wash out the extract retained by the spent grains. Sparging was performed 23 until the last run off wort concentration was about 1.6 °P. At the end of the filtration process, spent 24 grains were discharged. Sweet wort and spargings were gathered in a buffer tank which served the 25 wort boiling kettle. Successively, sweet wort was boiled for 1 h and hopped (boiling and hopping) 1 using a combination of CO 2 hop extract (53.4% α-acids per 500 g of product) and type 90 pellets (15% 2 α-acids per 1 kg of product) in a fixed proportion, in order to obtain a cast wort of 35 European 3 Brewery Convention (EBC) bitter units. During boiling, a small amount of water evaporated 4 (maximum 4%). Then, the cast wort was treated in a whirlpool at 90 °C, for 20 min in order to 5 separate protein-polyphenol aggregates formed during wort boiling (hot trub). After removal of the hot 6 trub (about 4000 l), the wort was cooled at 15 °C, aerated to avoid adverse yeast behaviors during cell 7 division and pitched (pitching rate 0,007 kg l -1 ) with a lager strain of Saccharomyces cerevisiae, 8 originating from a former Hürlimann brewery collection (fermentation). 9

The fermentation was performed in 140000 l horizontal stainless steel vessels at 15 °C for an 10 average period of 10 days; normally a small amount of water was added (about 2500 l). Fermentation 11 time may vary according to the reduction of the diacetyl level in green beer below 50 µg kg -1 . Then, 12 the green beer was cooled at 7 °C for 2 days. At the end of fermentation, beer was centrifuged. Then, 13 beer was clarified and stabilized by using diatomaceous earth and polyvinyl polypyrrolidone (PVPP) 14 as the main filter aids, respectively. A sheet type filter was used for clarification, supplied with a fully 15 automatic kieselguhr dosing unit. Stabilization with PVPP was performed in a recycling process. The 16 stabilizing solution was made dissolving 350 kg of PVPP in 5000 l of water at 80 °C; PVPP dosing 17 into beer was set at 0.35 g l -1 (stabilization). Successively, dilution to the final beer alcohol content 18 was performed by means of de-aerated water. A trap filter, installed downstream, helped retain 19 kieselguhr particles that bleed through the sheet filter and PVPP fines. Finally, the finished beer was 20 recovered in 100000 l horizontal bright beer tanks; after bottling, the beer was tunnel pasteurized; the 21 bottled beer was kept at 64 °C for 20 min and then cooled to room temperature. connected the weighing machines to the silos. Using this flux deviator, 40 incremental samples were 10 collected in a time period as long as the time required for the off-take of a single brew; two aggregate 11 samples of about 8 kg of malted barley and maize grit were obtained. Samples of intermediate 12 products and finished beer were taken at the end of the corresponding process (numbered in figure 1). 13

For these products, two samples of 1.5 kg or liter were collected and put together. Solid samples 14 (malted barley and maize grit) were ground with a hammer mill (1 mm sieve) and homogenized for 10 15 min using a horizontal mixer. From the industrial plant were collected: malted barley (1), maize grit 16

(2), malt mash (3), sweet wort (4), boiled wort (5), wort after whirlpool process (6), hot trub [START_REF]Scientific Committee on Food[END_REF], green 17 beer (8), beer after kieselguhr filtration (9), beer after PVPP stabilization (10), beer after water dilution 18 and trap filtration [START_REF]Natural Toxins. Aflatoxins[END_REF], bottled beer (12). All the samples were stored at -20 °C until the time of 19 analysis. For each brewing, the amounts of malted barley and maize grit and the volumes of water, 20 wort and beer are shown in table 1. The volumes were measured in l with an approximation of ±200 l. 21 These data derived from measurements carried out by plant-technicians at the time of brewing. The bottled beer was a standard product, whose main parameters are shown in table 2. The 1 parameters were measured by means of an Alcolyzer Plus (Anton Paar GmbH, Graz, Austria) coupled 2 with a DMA 4500 density meter and with a SP-1m automatic sample changer. Bitterness and diacetyl 3 were measured in compliance with Analytica EBC methods 9.8 and 9.24.1, respectively (European 4

Brewery Convention, 1998). [START_REF] Visconti | Determination of fumonisins B 1 and B 2 in corn and 6 corn flakes by liquid chromatography with immunoaffinity column cleanup: collaborative 7 study[END_REF] 6

Analytical Standards 7

Aflatoxin and fumonisin standards were obtained from Sigma-Aldrich (St. Louis, MO, USA). For 8 each aflatoxin, a stock solution of 5-8 µg ml -1 was prepared in benzene:acetonitrile (98:2, v/v, 2 ml) 9 and stored at -20°C. The solution was calibrated spectrophotometrically at 350 nm (AOAC 10 International, 2005a). The working standard solution was prepared after evaporation under nitrogen of 11 an aliquot (50 µl) of the stock solution and re-dissolution in chloroform (10 ml) by ultrasonication. An 12 aliquot (50 or 100 µl) of this solution was evaporated under nitrogen and re-dissolved in the HPLC 13 mobile phase (1-25 ml), to obtain calibrant solutions at individual concentrations of AFB 1 , AFB 2 , 14 AFG 1 , AFG 2 between 0.1 and 6 µg l -1 . FB 1 and FB 2 (10 mg) were separately dissolved in 50 ml 15 acetonitrile:water (1:1, v/v). These solutions were diluted to obtain HPLC calibrant solutions in 16 acetonitrile:water (30:70, v/v) at individual concentrations of FB 1 and FB 2 between 2.5 and 50 µg l -1 . 17 For liquid samples (wort and beer), 20 ml was diluted with 20 ml of phosphate buffered saline 3 (PBS, pH 7.4) and the solution was shaken for about 1 min. The pH was controlled and adjusted, if 4 necessary, between 6.5 and 7.5 using NaOH 0.1 N; then the solution was purified through the 5 immunoaffinity column. 6

After washing of the column with 5 ml of distilled water, aflatoxins were eluted into a graduated 7 glass vial with methanol (2.5 ml). The eluate, evaporated under a gentle stream of nitrogen, was 8 redissolved in 1 ml of acetonitrile:water (25:75, v/v), and vortex-mixed for a few seconds; then, the 9 extract was filtered (Millipore Corporation, Bedford, Massachussetts, USA, HV 0.45 µm) and injected 10 (30 or 100 µl). 11

Analysis was performed using an HPLC instrument, consisting of two PU-1580 chromatographic 12 pumps, an AS 1555 sampling system, an FP 1520 fluorescence detector and a post-column 13 derivatization system (Jasco Corporation, Tokyo, Japan); the instrument was controlled by Borwin 1. 5 14 software (Jasco). A Superspher RP-18 column (4 µm particle size, 125x4 mm i.d., Merck) was used at 15 ambient temperature, with a mobile phase of water:methanol:acetonitrile (64:23:13, v/v/v), at 1.0 ml 16 min -1 . A solution of pyridinium bromide perbromide (25 mg in 500 ml of HPLC-grade water) was 17 used as a derivatizing agent. The flow of the post-column derivatizing solution was set at 0.1 ml min -1 18 and the reaction tubing was 500 µl. The detector was set at λ ex =365 nm and λ em =440 nm. [START_REF] Belajova | Retention of ochratoxin A and fumonisin FB 1 and FB 2 from 20 beer on solid surfaces: comparison of efficiency of adsorbents with different origin[END_REF] 20

Analysis for fumonisins 21

Regarding solid samples (malted barley, maize grit, spent grains), fumonisins were analysed 22 according to the method proposed by [START_REF] Visconti | Determination of fumonisins B 1 and B 2 in corn and 6 corn flakes by liquid chromatography with immunoaffinity column cleanup: collaborative 7 study[END_REF]. Fumonisins were extracted from 20 g of 23 sample in a plastic centrifuge bottle with 100 ml of acetonitrile:methanol:water (25:25:50, v/v/v). 24

After extraction for 45 min using a rotary-shaking stirrer and centrifugation at 4500 g for 6 min, the 25 supernatant was poured into a flask. Another 100 ml of the same solution was added to the residue in 1 the centrifuge bottle, and a second extraction performed for 30 min; then, the extracts were combined. 2

For liquid samples with solid materials (cooked and malt mash), 40 ml of acetonitrile:methanol (50:50 3 v/v) were added to a homogenized aliquot of 50 g; the extraction was performed using a rotary-4 shaking stirrer for 45 min. After filtration of the extracts through a folded filter-paper, an aliquot of the 5 filtrate (2 ml) was diluted with 20 ml of PBS and purified through an immunoaffinity column (R-6

Biopharm Rhône LTD, Glasgow, Scotland, UK). 7

For liquid samples, 20 ml was diluted with 20 ml of PBS and the solution was shaken for about 1 8 min. The pH was controlled and adjusted, if necessary, between 6.5 and 7.5 using NaOH 0.1 N; then 9 the solution was purified through the immunoaffinity column. 10

After washing the column with PBS (2 ml), fumonisins were slowly eluted (0.5 ml min -1 ) with 11 methanol (6 ml) into a graduated glass vial; subsequently, the eluate was concentrated to 2 ml under a 12 gentle stream of nitrogen. [START_REF]Natural Toxins. Aflatoxins. Official Methods of Analysis of the 14 International Association of Official Analytical Chemists[END_REF] Analysis was carried out using a LC-MS/MS system, consisting of a LC 1.4 Surveyor pump 14 (Thermo-Fisher Scientific, San Jose, CA, USA), a PAL 1.3.1 sampling system (CTC Analitycs AG, 15 Zwingen, Switzerland) and a Quantum Discovery Max triple-quadrupole mass spectrometer; the 16 system was controlled by Excalibur 1.4 software (Thermo-Fisher). After dilution of the extract (0.1 ml 17 brought to 1 ml) with acetonitrile:water (30:70 v/v), fumonisins were separated on a Betasil RP- 18 18 column (5 µm particle size, 150x2.1 mm, Thermo-Fisher) with mobile-phase gradient acetonitrile-19 water (both acidified with 0.4% acetic acid) from 25:75 to 55:45 in 9 min, then isocratic for 3 min; the 20 flow rate was 0.2 ml min -1 . Ionisation was carried out with an ESI interface (Thermo-Fisher) in 21 positive mode as follows: spray capillary voltage 4.0 kV, sheath and auxiliary gas 35 and 14 psi, 22 respectively, temperature of the heated capillary 270 °C. The mass spectrometric analysis was 23 operated in selected reaction monitoring (SRM). For fragmentation of [M+H] + ions (722 and 706 m/z 24 for FB 1 and FB 2 , respectively), the argon collision pressure was set to 1.5 mTorr and the collision 25 energy to 36 V. The selected fragment ions were 704, 352 and 334 m/z for FB 1 and 688, 336 and 318 1 m/z for FB 2 . Quantitative determination was performed using LC-Quan 2.0 software (Thermo-Fisher). 2

Both for aflatoxins and fumonisins, three replicates were analysed for each sample. 3 4

Other mycotoxins 5

Malted barley and maize grit were also analyzed for deoxynivalenol (DON), zearalenone (ZEA) and 6 ochratoxin A (OTA) by methods routinely used in our laboratory [START_REF] Rossi | 5 Preliminary survey on composition and quality of conventional and organic wheat[END_REF][START_REF] Pietri | Occurrence of mycotoxins and ergosterol in maize 3 harvested over 5 years in Northern Italy[END_REF]); 7 since very low levels were found in all samples (DON < 200 µg kg -1 , ZEA < 20 µg kg -1 and OTA < 8 0.2 µg kg -1 ), the carry-over of these mycotoxins was not investigated. 9 10

Results and discussion 11

Performance of the methods 12

Percentage recoveries were determined by spiking known blank maize grit samples at levels of 2 and 13 1000 µg kg -1 and blank beer samples at levels of 0.1 and 50 µg kg -1 for aflatoxins and fumonisins, 14 respectively. Average recovery values were: 97.8 ±1.6% and 98.3±1.5% for AFB 1 , 94.9±2.3% and 15 97.6±1.8% for AFB 2 , 95.5±1.9% and 97.6±1.7% for FB 1 , 93.7±3.0% and 95.5±1.9% for FB 2 , in maize 16 grit and beer, respectively. Recovery values higher than 90% were also obtained for malt mash and 17 boiled wort; for the other liquid intermediate products, being very similar to boiled wort and beer, 18 recovery experiments were not carried out. [START_REF] Belajova | Retention of ochratoxin A and fumonisin FB 1 and FB 2 from 20 beer on solid surfaces: comparison of efficiency of adsorbents with different origin[END_REF] Certified reference materials (ground corn, R-Biopharm Rhône LTD), with AFB 1 and fumonisin 20 contamination of 4.1±1.0 µg kg -1 and 2500±600 µg kg -1 (FB 1 =1600, FB 2 =700, FB 3 =200 µg kg -1 ), 21 respectively, were also analysed; our results were 4.2±0.2 µg kg -1 for AFB 1 , 1553±39 and 721±21 µg 22 kg -1 for FB 1 and FB 2 , respectively. The results of the analyses were not corrected for recovery; the 23 relative standard deviations were always <13% for AFB 1 and <11% for FB 1 . The limits of detection 24 (LOD, signal-to-noise ratio of 3:1) and of quantification (LOQ, signal-to-noise ratio of 10:1) were 25 respectively: 0.005 and 0.01 µg kg -1 for AFB 1 , 0.01 and 0.02 µg kg -1 for AFB 2 , 30 and 50 µg kg -1 for 1 FB 1 and FB 2 , in maize grit; 0.001 and 0.002 µg l -1 for AFB 1 , 0.002 and 0.004 µg l -1 for AFB 2 , 1 and 2 2.5 µg l -1 for FB 1 and FB 2 , in beer. 3 4 Aflatoxin B 1 5 AFB 1 ranged in maize grit from 0.31 to 14.85 µg kg 1 ; it was also found in malted barley, at levels 6 ranging from 0.20 to 4.07 µg kg -1 (table 3). Malted barley and maize grit used for brewing A and 7 maize grit for brewing B showed concentrations higher than 2 µg kg -1 , the maximum level fixed by EC 8 Regulation for all cereals and all products derived from cereals, including processed cereal products 9 (Commission of the European Communities 2006b). As regards the other aflatoxins, only AFB 2 was 10 detected in maize grits used for brew A and B production (1.2 and 0.7 µg kg -1 , respectively); however, 11 it resulted <LOD in the bottled beer and therefore the very low levels found in some intermediate 12 products are not reported. For each stage of the brewing, the AFB 1 percentage in the product, referring 13 to the total amount in the raw materials (TARM), was calculated ((conc product *kg(or l) product )/(conc malted 14 barley *kg malted barley +conc maize grit *kg maize grit )*100). A relatively small amount of the toxin was transferred 15 to the liquid phase during mashing; in fact, averaging the 4 brewing results, only 14.3±2.8% of the 16 AFB 1 TARM was found in the liquor after mashing: this is probably due to the low solubility of the 17 toxin in water. In the sweet wort the AFB 1 level was reduced both because of the dilution of the 18 product, and mainly because of the filtration step: in effect, lautering caused a consistent reduction (-19 45.4±14.3%) of the AFB 1 present in the liquor and only an average of 7.9±3.0% of the AFB 1 TARM 20 was found in the sweet wort. In the following steps, the reductions were more moderate (boiling 21 -28.5±10.5%, whirlpooling -2.6±14.9%, fermentation -14.3±9.5%), while a further strong decrease 22 (-59.6±17.3%) was observed passing from green to bottled beer. The percentages of AFB 1 TARM 23 found in the bottled beer varied from 0.6 to 2.2% (mean 1.5±0.8%) and the concentrations, because of 24 the high dilution, were in the low part-per-trillion level. These results are in agreement with a previous 25 study [START_REF] Steiner | Aflatoxins in preparation of malt and beer[END_REF], where about 5% of AFB 1 and AFB 2 were recovered in the final 1 beer brewed from fortified malt. Differently, [START_REF] Chu | Stability of Aflatoxin B1 and ochratoxin A in 23 brewing[END_REF], in a micro-brewing process, after 2 addition of AFB 1 (dissolved in chloroform) at 1 and 10 µg g -1 , at the beginning of mashing, found 18 3 and 27% of the toxin, respectively, in bottled beer; Okoye (1986) found a carry-over of 41% from red 4 guinea corn into a Nigerian native beer. These higher recoveries were likely due to the modality of the 5 trials and to the very high level of spiking. 6 7

Fumonisin B 1 8

The FB 1 levels in maize grit varied from 1146 to 3194 µg kg -1 (table 4); FB 2 occurred as well, at 9 levels from 347 to 908 µg kg -1 . Since FB 2 was not detected in the bottled beer, the very low levels 10 found in some intermediate products are not reported. Only in the case of maize grit used for brewing 11 B (FB 1 +FB 2 =1493 µg kg -1 ), was the concentration of fumonisins close to the level fixed by the EC 12 Regulation 1126/2007 (Commission of the European Communities 2007) for milling fractions of 13 maize with particle size > 500 µm not intended for direct human consumption (1400 µg kg -1 ); on the 14 contrary, the fumonisin level exceeded the limit in the case of the grit used in the other brewing 15 processes. Fumonisins were not detected in malted barley. For each stage of the brewing, the FB 1 16 percentage in the product, referring to the TARM, was calculated ((conc product *kg(or l) product )/(conc maize 17 grit *kg maize grit )*100). Unlike AFB 1 , most of the FB 1 entering the brewing was extracted into the 18 mashing liquors (average 84.3±10.3%). During the following steps, the reduction was quite low: 19 -7.3±2.5% after lautering, -8.3±3.6% at the end of the boiling process, -10.3±2.5% after whirlpooling. 20

Likewise, the fermentation process did not appreciably decrease the amount of FB 1 (-4.2±2.6%); this 21 was in agreement with the study of Scott et al. (1995), in which a loss of FB 1 in the range 3-28% was 22 estimated. Finally, a further low reduction (on average -16.4±10.0%) was observed from green beer to 23 bottled beer. The clarification and stabilization processes, which employed kieselguhr and PVPP, 24 respectively, did not appreciably reduce the amount of FB 1 . These materials showed a nearly full 25 retentive indifference towards FB 1 ; this held true also for celite used for the brewing process, as 1 reported by [START_REF] Belajova | Retention of ochratoxin A and fumonisin FB 1 and FB 2 from 20 beer on solid surfaces: comparison of efficiency of adsorbents with different origin[END_REF]. In bottled beer 50.7±4.7% of the FB 1 TARM was recovered; the 2 concentrations varied from 37 to 89 µg l -1 . These results indicate that FB 1 can occur in beer when 3 maize-based solid adjuncts are used. FB 1 occurrence in finished beer is mainly due to its high 4 solubility in water and to the relative stability to heat treatment. 5 6

Conclusions 7

The four industrial brewing processes gave quite similar results; as expected, AFB 1 and FB 1 8

showed different behaviors during brewing, mainly due to their different solubility in water; however, 9 also the process determined a different transfer rate of the two solubilised toxins into the bottled beer 10 (10.5 vs. 60.1% for AFB 1 and FB 1 , respectively). Consequently, the concentration of AFB 1 retrieved in 11 bottled beer was very low. In Italy the consumption of beer is about 30 l per person year -1 (Assobirra 12 2007), corresponding to 0.082 l per person day -1 ; if a consumer drinks beer with contamination like 13 that obtained from brewing A, the AFB 1 intake would be 1.8 ng or 0.03 ng kg -1 b.w., that is 3.6-0.5% 14 of the estimated range of daily exposure to total aflatoxins in Italy (0.838-1.934 ng kg -1 b.w.; EFSA 15 2007). When the EU limits for AFB 1 in raw materials are respected, like in brewing C+D, the 16 corresponding figures for a consumer are 0.12 ng of AFB 1 or 0.002 ng kg -1 b.w., that is 0.2-0.1% of the 17 estimated range of daily exposure in Italy. In the latter case, the contribution of beer to exposure is 18

negligible. 19

Analogous precautions should be taken for the fumonisin contamination in maize adjuncts. If the 20 fumonisin contamination clearly exceeds the EU limit, as in maize adjuncts used in brewing C+D, a 21 daily exposure of 0.12 µg kg -1 b.w. can be calculated for a consumer, that represents about 6% of the 22 TDI of 2 µg kg -1 b.w., fixed by the Scientific Committee on Food (2000). If the EU limit is respected, 23 as in maize adjuncts used in brewing B, the corresponding figure for a consumer is 0.05 µg kg -1 b.w. 24 per day, that represents only about 2% of the TDI. 25 However, it must be considered that in Italy about 2% of males and about 3% of young males (25-1 44 years) consume more than 0.5 l of beer; for this fraction of population, the calculated mean 2 exposure must be multiplied by six or more. 3 It is evident that when the raw materials used in the brewing process exceed the EU limits for 4 AFB 1 and FB 1 , beer can become a more significant source of exposure to the two toxins. (1) (conc product *kg(or l) product )/(conc maize grit *kg maize grit )*100.

22 23

 22 Samples 24In accordance with the brewing program, four brews (A, B, C and D) were brewed on a 65000 l 25 A and B were fermented in two different horizontal fermentors with a capacity of 140000 1 l. Wort C and D were jointly fermented using a horizontal fermentor of equal full capacity. The 2 utilization of fermentors of such size, usually used for the production of extremely limited volumes of 3 specialty beer in substitution of classical 450000 l cylindroconical units, helped us optimize the 4 sampling plan and avoided there being an overwhelming number of samples to be monitored for each 5 brew at the fermentation stage. Sampling of malted barley and maize grit was performed according to 6 the EC Regulation 401/2006 (Commission of the European Communities 2006a). Primary samples of 7 about 200 g of malted barley and maize grit were separately collected in continuum at a fixed and 8 constant flow rate by means of a sampling flux deviator applied to the raw material conveyors, which 9

18

  Analysis for aflatoxins 19Regarding solid samples, aflatoxins were extracted from 25 g of sample with 250 ml acetone:water 20 (85:15 v/v) according to the AOAC method 2003.02 (AOAC International 2005b). For liquid samples 21 with solid materials (cooked and malt mash), 120 ml of acetone was added to a homogenized aliquot 22 of 50 g. The extraction was performed using a rotary-shaking stirrer for 45 min. After filtration of the 23 extracts through a folded filter-paper, an aliquot of the filtrate (5 ml) was diluted with distilled water 24 ) and the solution was purified through an immunoaffinity column (R-Biopharm Rhône LTD, 1 Glasgow, Scotland, UK). 2

Figure 1 :

 1 Figure 1: layout of the industrial plant showing sampling points (numbered). Page 23 of 23

Table 1 .

 1 Amounts (kg) of malted barley and maize grit and volumes (l) of wort and beer. Table. 2. Main parameters of bottled beer.

	5
	6
	7	Aknowledgement
	8	The authors thank Roberto Pastori, President of the Italian Association for Food Protection and
	9	Environmental Hygiene, for funding this study.

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table 3 .

 3 Transfer of aflatoxin B 1 (mean of three replicates) from raw materials to the different intermediate and final products during the brewing processes. 1) (conc product *kg(or l) product )/(conc malted barley *kg malted barley +conc maize grit *kg maize grit )*100.

	Page 21 of 23			Food Additives and Contaminants
		brewing A	brewing B	brewing C	brewing D
		µg kg -1	Percentage	µg kg -1	Percentage	µg kg -1	Percentage	µg kg -1	Percentage
		or µg l -1	(%) (1)	or µg l -1	(%) (1)	or µg l -1	(%) (1)	or µg l -1	(%) (1)
	Malted barley Maize grit	4.07 14.85	100	1.36 10.93	100	0.38 0.92	100	0.20 0.31	100
	Malt mash	0.437	15.6	0.202	10.7	0.038	17.3	0.012	13.6
	Sweet wort	0.178	11.8	0.046	4.8	0.009	8.4	0.003	6.8
	Boiled wort	0.114	7.5	0.033	3.4	0.008	7.3	0.002	4.2
	Wort	0.089	5.5	0.029	2.8	0.005	4.2	0.002	4.2
	Hot trub	0.141	0.5	0.024	0.2	0.008	0.4	0.003	0.4
							brewing C+D
						µg l -1	Percentage (%)
	Green beer	0.082	5.3	0.021	2.1	0.003		3.6
	Filtered beer	0.060	3.8	0.019	1.9	0.003		3.6
	Stabilized beer	0.058	3.7	0.019	1.9	0.003		3.6
	Diluted beer	0.046	3.6	0.013	1.6	0.002		2.9
	Bottled beer	0.022	1.7	0.005	0.6	0.0015		2.2

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Table 4 .

 4 Transfer of fumonisin B 1 (mean of three replicates) from maize grit to the different intermediate and final products during the brewing processes.

		brewing A	brewing B	brewing C	brewing D
		µg kg -1	Percentage	µg kg -1	Percentage	µg kg -1 or	Percentage	µg kg -1	Percentage
		or µg l -1	(%) (1)	or µg l -1	(%) (1)	µg l -1	(%) (1)	or µg l -1	(%) (1)
	Malted barley Maize grit	< 30 1741	100	< 30 1146	100	< 30 2880	100	< 30 3194	100
	Malt mash	207	89.2	116	70.3	347	83.4	424	94.2
	Sweet wort	102	81.7	56	66.0	162	79.6	191	84.6
	Boiled wort	97	77.5	52	60.3	156	74.7	170	73.4
	Wort	95	71.7	50	54.5	145	65.4	159	64.7
	Hot trub	134	5.8	66	4.6	227	6.3	211	5.3
							brewing C+D
						µg l -1	Percentage (%)
	Green beer	88	68.4	47	53.7		142		60.8
	Filtered beer	83	64.4	46	52.4		137		57.7
	Stabilized beer	81	62.8	45	51.3		132		55.6
	Diluted beer	62	58.1	38	52.3		98		50.5
	Bottled beer	59	55.2	37	50.9		89		45.9

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Fabiano E Xavier

Javier Blanco-Rivero

Esther Sastre

Lina Badimón

Dr Gloria Balfagón
email: gloria.balfagon@uam.es

Simultaneous inhibition of TxA 2 and PGI 2 synthesis increases NO release in mesenteric resistance arteries from cirrhotic rats

Keywords: Cirrhosis, nitric oxide, prostaglandins, thromboxane A 2, prostaglandin I 2

Our study examines, in mesenteric resistance arteries, possible vasodilation alterations, and the role of NO and COX-derivates in cirrhosis. Vasodilator response to acetylcholine was analysed in segments from control and cirrhotic rats. The effects of the unspecific COX inhibitor indomethacin, of the specific COX-1 inhibitor SC-560 and of the specific COX-2 inhibitor NS-398 were analysed in segments from both groups of rats. NO release was measured and eNOS, phosphorylated eNOS (P-eNOS), iNOS, COX-1 and COX-2 expressions were also analysed. The effects of TP receptor antagonist SQ 29548, TxA 2 synthesis inhibitor furegrelate, of PGI 2 synthesis inhibitor TCP or of TCP-plus-furegrelate were only determined in segments from cirrhotic rats. Vasodilator response to acetylcholine was higher in segments from cirrhotic rats. Indomethacin, SC-560 and NS-398 did not modify vasodilator response in control rats However, indomethacin, NS-398 and TCP-plus-furegrelate increased, SC-560 did not modify, and SQ 29548, furegrelate or TCP decreased the vasodilator response to acetylcholine in cirrhotic rats. NO release was higher in cirrhotic rats. Furegrelate decreased, while TCP-plus-furegrelate increased the NO release in segments from cirrhotic rats. eNOS and COX-1 expression were not modified, while P-eNOS, iNOS and COX-2 expression were higher in cirrhotic rats. The increased iNOS expression and eNOS activity mediate increases in endothelial NO release. The COX-2 derivates TxA 2 and PGI 2 act simultaneously, producing a compensatory effect that reduces NO release and may limit hyperdynamic circulation.

Introduction

Cirrhosis is associated with a hyperdynamic circulatory syndrome characterised by high cardiac output and reduced systemic vascular resistance and arterial pressure. Vascular resistance in circulatory districts other than the splanchnic system can be normal or increased, depending on the degree of portal hypertension, while splanchnic arterial vasodilation produces the hyperdynamic circulatory syndrome. The effects of endothelial factors, mainly nitric oxide (NO) and cyclooxygenase (COX)-derivatives, such as prostacyclin (PGI 2 ) [START_REF] Gatta | Vasoactive factors and hemodynamic mechanisms in the pathophysiology of portal hypertension in cirrhosis[END_REF] are reported to be the mechanism(s) leading to the splanchnic vasodilation.

Various endothelial factors are involved in the regulation of vascular resistance in normal and altered haemodynamic conditions and we believe that these factors probably also influence resistance arteries in cirrhosis. NO is synthesised by a family of enzymes, which is divided into two classes: constitutive (cNOS) and inducible (iNOS) [START_REF] Moncada | Mechanisms of disease: The L-arginine-nitric oxide pathway[END_REF][START_REF] Lowenstein | Nitric oxide: A physiologic messenger[END_REF]. Many investigations have demonstrated that enhanced NO release contributes to the hyperdynamic process ocurring in cirrhosis [START_REF] Morales-Ruiz | Increased nitric oxide synthase expression in arterial vessels of cirrhotic rats with ascites[END_REF][START_REF] Iwakiri | The molecules: mechanisms of arterial vasodilatation observed in the splanchnic and systemic circulation in portal hypertension[END_REF], but exactly which NOS isoform(s) and to what extent is still controversial.

There are few studies analysing the role of COX derivates in cirrhosis. Some of these studies have found PGI 2 release to be altered, and this alteration seems to affect the hyperdynamic circulation observed in cirrhosis [START_REF] Iwakiri | The molecules: mechanisms of arterial vasodilatation observed in the splanchnic and systemic circulation in portal hypertension[END_REF]. Although enhanced production of vasoconstrictive COX derivates has been reported to contribute to the increased vascular tone of cirrhotic livers [START_REF] Gracia-Sancho | Enhanced vasoconstrictor prostanoid production by sinusoidal endothelial cells increases portal perfusion pressure in cirrhotic rat livers[END_REF], hardly any report has considered their possible role in hyperdynamic splanchnic circulation. NOS and COX are involved in vascular tone regulation and changes in one are accompanied by changes in the other [START_REF] Mollace | Modulation of prostaglandin biosynthesis by nitric oxide and nitric oxide donors[END_REF].

Thus, our study determines, in mesenteric resistance arteries, possible alterations in vasodilation and the role of NO and COX-derivates in cirrhosis. vessels would have if they were exposed to a passive tension that was equivalent to the tension produced by a transmural pressure of 100 mmHg [START_REF] Mulvany | Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats[END_REF]. .

Experimental Protocols

After a 30 min equilibration period, arteries were exposed twice to 120 mmol/L KCl to check their functional integrity. Afterwards, concentration-response curves to acetylcholine (1 nmol/L -1 µmol/L) were made in arterial segments previously contracted with noradrenaline at a concentration that produced close to 50% of the contraction induced by KCl (120 mmol/L). This curve was performed in absence and in presence of the NO synthase inhibitor L-NAME (100 Pmol/L).

To determine the role of COX derivates in the vasodilator response to acetylcholine, either the unspecific COX inhibitor indomethacin (10 Pmol/L), the constitutive COX-1 inhibitor SC-560 (50 nmol/), or the inducible COX-2 inhibitor, NS-398 (10 Njmol/L), were added to the organ chamber 30 min before the concentrationresponse curve to acetylcholine was performed. In another set of experiments, segments were incubated with the TP receptor antagonist SQ 29548 (1 Pmol/L), with the PGI 2 synthesis inhibitor tranylcypromine (TCP, 10 Njmol/L), with the thromboxane A 2 (TxA 2 ) synthase inhibitor, furegrelate (1 Njmol/L), or with a combination of both TCP and furegrelate, to analyze the possible involvement of these vasoactive factors in relaxation to acetylcholine.

To assess possible differences in NO vasodilator response and TxA 2 vasoconstrictor response, concentration-response curves to either the NO donor dyethylamine NONOate (DEA-NO, 0.1 nmol/L-0.1 mmol/L) or the TxA 2 mimetic U46619 (0.1 nmol/L-3 Pmol/L) were performed in segments from control and cirrhotic animals.

Nitric Oxide release

NO release was measured as previously described [START_REF] Xavier | Endothelium modulates vasoconstrictor response to prostaglandin I2 in rat mesenteric resistance arteries: interaction between EP1 and TP receptors[END_REF]. . In order to study the effect of acetylcholine on NO release the second, third and fourth branches of mesenteric artery were incubated for 60 minutes in 3 mL of HEPES buffer, containing in mmol/L: 119 NaCl, 20 HEPES, 4. Afterward, arteries were incubated with the fluorescent probe 4,5-diaminofluorescein (2 µmol/L) for 45 min and medium was collected to measure basal NO release. Once the organ bath was refilled, a dose-response curve to acetylcholine was performed, as described in the Experimental Protocol. The medium was collected only at the end of the concentration-response curve to acetylcholine. The fluorescence of the medium was measured at room temperature using a spectrofluorimeter (LS50 Perkin Elmer Instruments, FL WINLAB Software) with excitation wavelength set at 492 nm and emission wavelength at 515 nm. The stimulated NO release was calculated by subtracting the basal NO release from that evoked by acetylcholine. Also, blank measurement samples were collected from medium without resistance mesenteric segments in order to calculate and subtract the background emission. Some assays were performed in the presence of L-NAME (0.1 mmol/L), TCP (10 Pmol/L), furegrelate (1 Pmol/L) or TCP-plus-furegrelate to assess the effect of these drugs on NO release, but only in segments from cirrhotic rats. The amount of NO released was expressed as arbitrary units mg tissue -1 . rats were equilibrated for 30 min in HEPES buffer at 37ºC, transferred to test tubes that contained 1 mL HEPES buffer (pH 7.4) containing lucigenin (250 µmol/L) and then kept at 37 o C. The luminometer was set to report arbitrary units of emitted light; repeated measurements were collected during 5 min at 10 s intervals and averaged. 4,5-Dihydroxy-1,3-benzene-disulphonic acid ''Tiron'' (10 mmol/L), a cell permeant and non-enzymatic scavenger of the superoxide anion, was added to quench the superoxide anion-dependent chemiluminescence. Also, blank measurement samples without mesenteric segments were collected in the same way to subtract background emission. The amount of superoxide anion released was expressed as chemoluminiscence units min -1 mg tissue -1 .

Prostanoids and CRP (C-reactive protein) production

To measure the release of CRP, and the stable metabolites of TXA 2 and PGI 2 , TXB 2 and 6-keto PGF 1Į respectively, a rat CRP ELISA kit (BD Biosciences), TXB 2 and 6-keto-PGF 1Į EIA kits (Cayman Chemical) were used. The second, third and fourth branches of mesenteric resistance arteries from control and cirrhotic rats were preincubated for 45 minutes in 200 µL of KHS at 37°C and continuously gassed with a 95% O 2 -5% CO 2 mixture (stabilisation period). Afterward, three washout periods of seven minutes in a bath of 200 µL of KHS were run before incubation with acetylcholine (0.1 nmol/L -0.1 µmol/L). The medium was collected only at the end of concentration-response curve to acetylcholine. Some samples were collected in the presence of 1 Pmol/L furegrelate in order to determine possible variations produced by this inhibitor. The different assays were performed following the manufacturer's instructions. Results were expressed as ng CRP mL -1 mg tissue -1 , or pg.prostanoid mL -1 mg wet tissue -1 .

Western blot

For Western blot analysis, the second, third and fourth branches of mesenteric artery from both groups of rats were homogenised in a boiling buffer composed of 1 mmol/L sodium vanadate, 1% SDS, and 0.01 mol/L pH 7.4 Tris-HCl. Homogenates containing 30 Pg protein were electrophoretically separated on a 7.5% SDSpolyacrylamide gel, and then transferred to polyvinyl difluoride membranes (Bio Rad Immun-Blot) overnight at 4ºC, 230 mA, using a Bio-Rad Mini Protean III system (Bio-Rad Laboratories, Hercules, CA, USA) containing 25 mmol/L Tris, 190 mM glycine, 20% methanol, and 0.05% SDS. The membrane was blocked for 1 h at room temperature in a Tris-buffered-saline (TBS) solution (100 mmol/L Tris-HCl, pH 7.4, 0.9% w/v NaCl, 0.1% SDS) with 5% powdered fat-free milk before being incubated overnight at 4º C with mouse monoclonal antibody against iNOS (1:5000 dilution, Transduction Laboratories), mouse monoclonal antibody against eNOS (1:2500 dilution, Transduction Laboratories) rabbit polyclonal antibody against phosphorylated eNOS (P-eNOS, assessed by its phosphorylation at serine 1177, 1:500, Abcam Laboratories), mouse monoclonal antibody against COX-1 (1:500, Cayman Chemical), or rabbit polyclonal antibody against COX-2 (1:500, Cayman Chemical). After washing, the membrane was incubated with the appropriate secondary horseradish peroxidase-conjugated immunoglobulin G antibody (Amersham International Plc). The membrane was thoroughly washed and the immunocomplexes were the detected using an enhanced horseradish peroxidase/luminol chemiluminescence system (ECL Plus, Amersham International Plc, Little Chalfont, UK) and subjected to autoradiography (Hyperfilm ECL, Amersham International Plc). Signals on the immunoblot were quantified using a computer program (NIH Image V1.56, National Institute of Health, Bethesda, MD, USA). The same membrane was used to determine D-actin expression, and the content of the latter was used to correct protein expression in each sample by means of a monoclonal antibody anti D-actin (1:2000 dilution, Sigma).

Drugs and Solutions

The drugs used were L-NA hydrochloride, acetylcholine chloride, diethylamine NONOate diethilammonium salt, U46619, SQ 29548, L-NAME hydrochloride, indomethacin, TCP, NS-398, SC-560, SQ 29548, furegrelate, tiron and lucigenin. Stock solutions (10 mmol/L) of drugs were made in distilled water except for noradrenaline, which was dissolved in a NaCl (0.9%)-ascorbic acid (0.01% w/v) solution; indomethacin, U46619, SQ 29548 and SC-560, which were solubilized in ethanol and administered from a prepared stock in such a way that the maximal ethanol concentration of the medium was less than 0.001% (vol./vol.); and NS-398, which was solubilised in DMSO. All stock solutions were kept at í20°C, and appropriate dilutions were made in KHS on the day of the experiment.

Statis ical Analysis t

Results are expressed as mean ± SEM for the number of rats indicated. Statistical analysis compared the curve obtained in the presence of the different substances with the control curve by means of two-way analysis of variance (ANOVA). For the NO, prostanoids and CRP release experiments, the statistical analysis employed Student's t test for unpaired experiments. A P value of less than 0.05 was considered significant.

Results

Animal evolution

Liver cirrhosis did not modify systemic blood pressure, but body weight was lower in cirrhotic animals. All cirrhotic animals showed spleen hypertrophy and hepatomegaly (Table 1). The liver histology of cirrhotic animals showed a finely granulated surface and the histological features of cirrhosis (Data not shown).

In the liver cirrhosis group, rats presented portosystemic collateral circulation (pararectal, paraesophageal, splenorenal and portohepatic collateral vessels).

Both GPT and GOT levels were increased in our experimental model. Additionally, LDL levels increased, HDL and triglycerides decreased, and total cholesterol levels were not modified after the establishment of liver cirrhosis (Table 2).

Vascular reactivity experiments

To provide maximal activation for each preparation segments were contracted with 120 mol/L KCl; responses obtained were similar in vessels from control and cirrhotic rats (Control: 2.81±0.11 vs. Cirrhotic: 2.88±0.15 mN/mm, P>0.05). Acetylcholine caused a cumulative concentration-dependent relaxation in noradrenaline-precontracted rat mesenteric resistance arteries (MRA) that was greater in segments from cirrhotic rats (Figure 1); in vessels from both groups, preincubation with L-NAME significantly reduced this response (Figure 1). In presence of L-NAME, relaxation to acetylcholine was similar in vessels from both control and cirrhotic rats (Figure 1) Pretreatment with indomethacin, SC-560 or NS-398 did not modify vasodilator response in mesenteric segments from control rats (Figure 2A). However, both indomethacin and NS-398 increased vasodilator response to acetylcholine, while SC-560 did not modify the acetylcholine-induced vasodilation in segments from cirrhotic rats (Figure 2B).

Preincubation with SQ 29548, furegrelate or TCP decreased vasodilator response in segments from cirrhotic rats (Figure 3). Simultaneous preincubation with both TCP and furegrelate together increased concentration-response vasodilation to acetylcholine in segments from cirrhotic rats (Figure 3). In arteries from control rats, relaxation to acetylcholine remained unmodified in the presence of SQ 29548, furegrelate, TCP or TCP-plus-furegrelate (results not shown).

Vasodilator response to DEA-NO and vasoconstrictor response to U46619 were similar in both experimental groups (Data not shown).

Nitric oxide release

Both basal and acetylcholine-induced NO release were higher in segments from cirrhotic rats (Figure 4). Preincubation with TCP did not modify either basal or acetylcholine-stimulated NO release in segments from cirrhotic rats (Figure 4). Furegrelate decreased both basal and acetylcholine-induced NO release in segments from cirrhotic rats (Figure 4). A combination of TCP-plus-furegrelate increased both basal and acetylcholine-induced NO release in segments from cirrhotic rats (Figure 4).

Superoxide anion release

Liver cirrhosis increased superoxide anion release by mesenteric resistance arteries (Control: 10.50 + 1.85 U min -1 mg tissue -1 ; Cirrhotic: 29.25 + 3.12 U min -1 mg tissue -1 ; P< 0.05; n=5 in each group).

CRP release

Basal CRP release was increased by liver cirrhosis (Control: 4.083 + 0.78 ng ml í1 mg tissue -1 ; Cirrhotic: 23.20 + 6.65 ng ml í1 mg tissue -1 ; P<0.05; n=5 in each group). Acetylcholine increased CRP release in both groups, but the release was higher in segments from cirrhotic rats (Control: 14.93 + 3.48 ng ml í1 mg tissue -1 ; Cirrhotic: 47.67 + 12.27 ng ml í1 mg tissue -1 ; P<0.05; n=5 in each group)

Prostanoids release

Liver cirrhosis did not modify basal, while decreased acetylcholine-induced TxB 2 release in mesenteric resistance arteries (Figure 5A). Both basal and acetylcholine-induced 6-keto PGF 1D levels were decreased by liver cirrhosis (Figure 5B). Preincubation with furegrelate did not modify 6-keto PGF 1D levels in mesenteric segments from cirrhotic rats (Results not shown).

Western blot

Liver cirrhosis did not modify eNOS or COX-1 expression (Figure 6), while increased P-eNOS, iNOS and COX-2 expression (Figure 6).

Discussion

CCl 4 treatment was maintained for 9 weeks. All CCl 4 -treated rats used in these experiments macroscopically showed macro/micronodular cirrhosis of the liver, hepatomegaly and spleen hypertrophy. No rat showed ascites, which has only been described after 12-20 weeks CCl 4 administration in these animals [START_REF] Wiest | Splanchnic and systemic vasodilation: the experimental models[END_REF]. Transaminase levels (GOT and GPT) were increased after CCl 4 treatment. Additionally, in our experimental model, HDL and triglycerides levels were decreased, LDL increased but total cholesterol was not modified in serum samples. These data agree with previous studies performed in portal hypertensive rats [START_REF] Sánchez-Patán | Chronic prehepatic portal hypertension in the rat: is it a type of metabolic inflammatory syndrome?[END_REF]. . All these alterations confirm that our experimental model develops liver cirrhosis.

In this study, we have observed that, as reported in conductance mesenteric arteries [START_REF] Vaughan | Vasoconstrictor responses are normal but prostanoid-mediated vasodilatation is enhanced in human cirrhotic mesenteric arteries[END_REF], cirrhosis increases vasodilation induced by acetylcholine in mesenteric resistance arteries. This effect is associated with an increase in NO release while the vasodilator response to DEA-NO remained unmodified, confirming the major role played by NO release in the development of hyperdynamic circulation. This effect was accompanied by an increase in iNOS expression, as previously described in aorta and superior mesenteric arteries [START_REF] Malyshev | Discrepant effects of inducible nitric oxide synthase modulation on systemic and splanchnic endothelial nitric oxide synthase activity and expression in cirrhotic rats[END_REF]. Additionally, in spite of a non-modification in eNOS expression, we observed that eNOS phosphorylation at serine 1177 was increased in cirrhotic rats, indicating increased eNOS activation produced by liver cirrhosis. These observations do not agree with previous reports of both a decrease and a no modification in superior mesenteric artery eNOS expression [START_REF] Vaughan | Vasoconstrictor responses are normal but prostanoid-mediated vasodilatation is enhanced in human cirrhotic mesenteric arteries[END_REF][START_REF] Malyshev | Discrepant effects of inducible nitric oxide synthase modulation on systemic and splanchnic endothelial nitric oxide synthase activity and expression in cirrhotic rats[END_REF], and a decrease on eNOS activity in aorta and superior mesenteric artery [START_REF] Malyshev | Discrepant effects of inducible nitric oxide synthase modulation on systemic and splanchnic endothelial nitric oxide synthase activity and expression in cirrhotic rats[END_REF] has also been described. This discrepancy can be due to a difference on the vascular bed analysed.

Vascular alterations could be a consequence of oxidative stress [START_REF] Rodriguez-Villarrupla | Current concepts on the pathophysiology of portal hypertension[END_REF][START_REF] Bomzon | Oxidative stress and vascular smooth muscle cell function in liver disease[END_REF], so we analysed the release of superoxide anions in our experimental conditions. The results obtained showed, as has been observed in cirrhotic livers [START_REF] Gracia-Sancho | Increased oxidative stress in cirrhotic rat livers: A potential mechanism contributing to reduced nitric oxide bioavailability[END_REF], an increase on superoxide anion levels in mesenteric segments from cirrhotic rats.

One of the most commonly used inflammatory markers is CRP, which can predict a risk of coronary events [START_REF] Aller | Inflammation: a way to understanding the evolution of portal hypertension[END_REF][START_REF] Bucova | C-reactive protein, cytokines and inflammation in cardiovascular diseases[END_REF]. This marker has been reported to rise in short-time PH with and without cirrhosis [START_REF] Georgopoulos | Is portal hypertension associated with protein-losing enteropathy?[END_REF]. The increased CRP levels observed in our experimental conditions confirm an increase in inflammatory processes due to liver cirrhosis. Since a number of studies have already focused on the role of COX-derived PGI 2 and TxA 2 in the inflammatory process observed in cirrhosis [START_REF] Iwakiri | The molecules: mechanisms of arterial vasodilatation observed in the splanchnic and systemic circulation in portal hypertension[END_REF][START_REF] Birney | Eicosanoids in cirrhosis and portal hypertension[END_REF], the next objective was to analyse the possible participation of COX-derivates in the vasodilator response induced by acetylcholine in mesenteric resistance arteries from cirrhotic rats. Indomethacin, NS-398 or SC-560 do not have any effect on the vasodilator response in control segments, indicating the non-participation of COXderivates in this experimental condition, as previously reported [START_REF] Xavier | Aldosterone induces endothelial dysfunction in resistance arteries from normotensive and hypertensive rats by increasing thromboxane A2 and prostacyclin[END_REF], while vasodilation increases in cirrhotic segments preincubated with indomethacin or NS-398, but not with SC-560. These results therefore indicate that COX-2-derived arachidonic acid metabolites modulate the endothelium-dependent relaxation in mesenteric resistance arteries from cirrhotic rats, but not from healthy ones. Supporting these results is the fact that COX-2 is overexpressed in segments from cirrhotic rats while COX-1 expression is not modified. Taken all together these results support the existence of an inflammatory process that would underlie the development of liver cirrhosis [START_REF] Aller | Inflammation: a way to understanding the evolution of portal hypertension[END_REF].

Our next objective was to analyze which COX-2 derivate was implicated in this effect. In our experimental model we have seen that PGI 2 levels increased after the establishment of liver cirrhosis, as has been reported regarding circulating PGI 2 levels in cirrhotic patients and portal hypertensive rats [START_REF] Yin | Plasma prostacyclin (PGI2) levels in peripheral venous, arterial and portal venous blood in cirrhotic patients with portal hypertension and their clinical implication[END_REF][START_REF] Cao | Expression of cyclooxygenase in hyperdynamic portal hypertensive rats[END_REF]. Numerous studies have demonstrated that PGI 2 promotes vasodilation in various vascular beds by stimulating IP receptors and thereby increasing the intracellular cyclic-AMP concentration. In mesenteric arteries from cirrhotic rats the relaxation to acetylcholine was diminished by incubation with the PGI 2 synthesis inhibitor TCP, indicating that PGI 2 has a functional role on the vasodilator response to acetylcholine and would consequently participate in the increased vasodilation associated to cirrhosis. NO release was not modified by TCP treatment, thus ruling out possible NO participation in this response. This result contrasts with early reports in a similar experimental model that indicated a minor role for PGI 2 in the development of hyperdynamic circulation [START_REF] Xu | Role of nitric oxide synthase and cyclooxygenase in hyperdynamic splanchnic circulation of portal hypertension[END_REF].

Changes in TxA 2 release have also been suspected in cirrhosis. In the present work we have observed that liver cirrhosis decreases TxA 2 levels. Additionally, the TxA 2 analogue U46619 induces vasoconstriction through TP receptor activation. Incubation with TP receptor antagonist SQ 29548 increases the vasodilator response to acetylcholine in the cirrhotic arteries, suggesting that TxA 2 could participate as a vasoconstrictor in the response to this endothelium-dependent vasodilator agent. However, inhibition of TxA 2 synthesis by furegrelate induces a decrease in the vasodilator response to acetylcholine, suggesting that inhibition of TxA 2 synthesis induces some other mechanism that would affect the vasomotor response to acetylcholine. Interactions between COX derivatives are known to be complex; the synthesis of one prostanoid is usually accompanied by changes in the synthesis of the others. This imbalance can be shifted towards hypo-or hyper-compensatory responses [START_REF] Martorell | Orchidectomy increases the formation of prostanoids and modulates their role in the acetylcholineinduced relaxation in the rat aorta[END_REF]. Therefore we analysed the release of the PGI 2 metabolite 6-keto PGF 1Į after preincubation with furegrelate in segments from cirrhotic rats. Our results show that 6-keto PGF 1Į release was not modified by preincubation with furegrelate, ruling out the possibility that furegrelate had induced a new equilibrium in prostanoid synthesis in segments from cirrhotic animals. Increasing evidences indicate that there is considerable "cross talk" between the NO and COX-derivate biosynthetic pathways, resulting in an active modulation in several situations [START_REF] Mollace | Modulation of prostaglandin biosynthesis by nitric oxide and nitric oxide donors[END_REF][START_REF] Blanco-Rivero | Longterm portal hypertension increases the vasodilator response to acetylcholine in rat aorta. Role of prostaglandin I2[END_REF]. . These data lead us to hypothesize a possible modulation of NO synthesis by TxA 2 in arteries from cirrhotic rats. Our results indicate that NO release was diminished in the presence of the TxA 2 synthesis inhibitor furegrelate, thus explaining the decrease of vasodilator response to acetylcholine after T X A 2 synthesis inhibition.

These last results seem to show that both COX-2 derivates, PGI 2 directly, and TxA 2 indirectly by increasing NO formation, play a role in maintaining the hyperdynamic circulation observed in resistance mesenteric arteries from cirrhotic rats; the results also suggest that inhibition of both TxA 2 and PGI 2 would have an additive effect. Thus, our next objective was to analyse the effect of the simultaneous inhibition of both TxA 2 and PGI 2 synthesis on the response to acetylcholine. Surprisingly, we observed that the relaxation induced by acetylcholine was significantly increased in the presence of TCP-plus-furegrelate. In line with this possible COX-NO interaction we hypothesised that NO release would increase if TxA 2 and PGI 2 synthesis were simultaneously inhibited. Our hypothesis was confirmed: preincubation with furegrelate combined with TCP increased NO release, and this would explain the increase in the vasodilator response to acetylcholine.

Additionally, the fact that simultaneous TxA 2 and PGI 2 synthesis inhibition produces exactly the same effect on the vasodilator response to acetylcholine as COX-2 inhibition excludes the participation of the other COX-2 derivates in this effect. To our knowledge, this is the first time that the joint action of two COX-2 derivates, PGI 2 and TxA 2 , has been described to induce a different effect on NO release than either of them alone. Additionally, these results indicate that activation of both NOS and of COX-2 plays a relevant role in the modulation of vasodilator response to acetylcholine in cirrhosis, and that they lead to opposite effects: while NO seems to participate in hyperdynamic circulation development, the COX-2 derivates TxA 2 and PGI 2 act together, seeming to produce a compensatory mechanism inhibiting NO.

The use of COX inhibitors like indomethacin has been suggested to avoid liver fibrosis as well as to attenuate the increased portal hypertension produced in liver cirrhosis [START_REF] Tu | Antifibrotic activity of rofecoxib in vivo is associated with reduced portal hypertension in rats with carbon tetrachloride-induced liver injury[END_REF][START_REF] Kim | Effect of selective cyclooxygenase-2 inhibitor meloxicam on liver fibrosis in rats with ligated common bile ducts[END_REF]. However, our results show that, as well as these beneficial effects, the use of COX inhibitors could worsen the hyperdinamic circulation associated with liver cirrhosis.

In conclusion, the present study shows that in mesenteric resistance arteries from cirrhotic rats increased iNOS expression and eNOS activity mediate increases in endothelial NO release. The COX-2 derivates TxA 2 and PGI 2 act simultaneously, producing a compensatory effect that reduces NO release and could limit the hyperdynamic circulation. A c c e p t e d M a n u s c r i p t

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

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  Clinical Science Immediate Publication. Published on 11 May 2010 as manuscript CS20090536 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090536 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited

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  Clinical Science Immediate Publication. Published on 11 May 2010 as manuscript CS20090536 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090536 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited

9

  Clinical Science Immediate Publication. Published on 11 May 2010 as manuscript CS20090536 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090536 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited

10 Clinical

 10 Science Immediate Publication. Published on 11 May 2010 as manuscript CS20090536 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090536 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited

11 Clinical

 11 Science Immediate Publication. Published on 11 May 2010 as manuscript CS20090536 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090536 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited

Figure 1 .

 1 Figure 1. Vasodilator response to acetylcholine in mesenteric resistance arteries from control and cirrhotic rats in the absence and presence of 100 Pmol/L L-NAME. Results are expressed as mean ± S.E.M. Number of animals used is indicated in parentheses.

Figure 2 .

 2 Figure 2. Effect of preincubation with 10 Pmol/L indomethacin, 10 Pmol/L NS-398 or 50 nmol/L SC-560 on the vasodilator response to acetylcholine in mesenteric resistance arteries from control (A) and cirrhotic (B) rats. Results are expressed as mean ± S.E.M. Number of animals used is indicated in parentheses.

Figure 3 .

 3 Figure 3. Effect of preincubation with 1 Pmol/L furegrelate, 10 Pmol/L TCP, TCP-plusfuregrelate, or 1 Pmol/L SQ 29548 on the vasodilator response to acetylcholine in mesenteric resistance arteries from cirrhotic rats. Results are expressed as mean ± S.E.M. Number of animals used is indicated in parentheses.

Figure 4 .

 4 Figure 4. Basal and acetylcholine-induced NO release in segments from control and cirrhotic rats. Effect of TCP (10 Pmol/L), furegrelate (1 µmol/L), or TCP-plusfuregrelate on the basal-and acetylcholine-induced NO release in mesenteric artery segments from cirrhotic animals. Results (Means ± S.E.M.) were expressed as arbitrary units (A.U.) mg tissue -1 . n = 4-7 each experimental condition. * P < 0.05 vs. Basal; # P<0.05 vs. control rats; # P < 0.05; + P< 0.05 vs. cirrhotic rats.

Figure 5 .

 5 Figure 5. Effect of liver cirrhosis on basal and acetylcholine-induced TxB 2 (A) and 6keto PGF 1D (B) release. Results (Means ± S.E.M.) were expressed as pg mL -1 mg tissue -1 . n=4-6 animals each experimental condition. *P< 0.05 vs. Basal; # P< 0.05 vs. Control rats.

Figure 6 .

 6 Figure 6. Representative Western blot for COX-1, COX-2, eNOS, P-eNOS and iNOS expression in resistance mesenteric segments from control and cirrhotic rats. The figure is representative of four separate experiments. Lower panel shows densitometric analysis of the different blots. Results (Means ± S.E.M.) are expressed as the ratio between the signal for the protein expression and the signal for Į-actin. *P<0.05 control vs. cirrhotic.

  6 KCl, 1 MgSO 4 .7H 2 O, 0.15 Na 2 HPO 4 .12H 2 O, 0.4 KH 2 PO 4 , 5 NaHCO 3 , 1.2 CaCl 2 .2H 2 O, 5.2 glucose, at 37°C (stabilisation period).

Table 1

 1 Effect of liver cirrhosis on body weight, systemic blood pressure, spleen weight and liver weight.

			Body weight	Blood	Spleen weight/	Liver weight/
				(g)	pressure	Body weight	Body weight
					(mm Hg)	(%)	(%)
	Control	525.5 + 6.4	149.6 + 4.1	0.1 + 0.008	3 + 0.1
	Cirrhotic	487.9 + 6.9 * *	144.4 + 3.4	0.2 + 0.009 * *	4.1 + 0.1 * *
	Results are expressed as mean + S.E.M. * P< 0.05 vs. control. N= 10 animals each
	group.
	Table 2
	Effect of liver cirrhosis on GPT, GOT, LDL, HDL, triglycerides and cholesterol levels
		GPT	GOT	LDL	HDL	Triglycerides	Cholesterol
		(U/L)	(U/L)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)
	Control	53.20 +	92.50 +	5.25 +	31.73+	58.40 + 5.2 92.00 + 7.1
		4.9		6.6	0.4	4.2
	Cirrhotic 100.30 +	135.20 +	10.80 +	20.94 +	34.20 + 4.6* * 86.80 + 9.9
		11.8 * *	14.6* *	0.8* *	1.2 * *
	Results are expressed as mean + S.E.M. * P< 0.05 vs. control. N= 10 animals each
	group.

16 Clinical Science Immediate Publication. Published on 11 May 2010 as manuscript CS20090536 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090536

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2010 The Authors Journal compilation © 2010 Portland Press Limited

Acknowledgements

We thank Mr. Pablo Catalina for his technical assistance.

Funding

This work was supported by grants from Comisión Interministerial de Ciencia y Tecnología de España (SAF2009-10374 and DEP2006-56187-C04-04).