Food and Chemical Toxicology 48 (2010) 2891–2897

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Food and Chemical Toxicology

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Isolation, purification, LC–MS/MS characterization and reactive oxygen speciesinduced by fumonisin B1 in VERO cells

Giuseppe Meca a,*, Maria José Ruiz a, Monica Fernandez-Franzón a, Alberto Ritieni b, Jordi Manes a

a Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of València, Avenue Vicent Andrés Estellés s/n, 46100 Burjassot, Spainb Department of Food Science, University of Naples ‘‘Federico II”, Via Universitá 100, 80055 Portici, Napoli, Italy

a r t i c l e i n f o

Article history:Received 4 May 2010Accepted 16 July 2010

Keywords:Fumonisin B1

LC–MSFermentationReactive oxygen speciesVERO cells

0278-6915/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.fct.2010.07.023

* Corresponding author. Tel.: +34 963544959; fax:E-mail address: giuseppe.meca@uv.es (G. Meca).

a b s t r a c t

Fumonisins are mycotoxins produced by Fusarium verticillioides that commonly contaminate maize andmaize products. The present work shows the results of a comparative study of three different fermenta-tion’s techniques (solid and liquid medium of corn and a solid agarized medium) for the production offumonisins B1, B2 and B3 with strains of F. verticillioides. The solid medium of corn was the most effectivein the production of fumonisins, being Fumonisin B1 the one produced with higher concentration, so theextract obtained by solid fermentation process was used for FB1 purification. Fumonisins characterizationand quantification were performed with reversed-phase high-performance liquid chromatography withelectrospray ionization triple quadrupole tandem mass spectrometry. The role of production of reactiveoxygen species (ROS) in Fumonisin B1 mediated toxicology has not been fully addresses in studies explor-ing FB1 toxicity. It is evaluated the level of ROS production in kidney cell line (VERO) exposed to 1, 5 and10 lM of FB1 for 0.5–100 min. The ROS level was detected using a fluorescence probe, 20 ,70-dichloroflu-orescein diacetate (DCFH-DA), which could be converted to highly fluorescent dichlorofluorescein(DCF) with the presence of intracellular ROS. Significant increase of ROS products was observed in VEROcells at 10 lM dose. These results indicate that ROS production by FB1 on renal cells is a mechanism offumonisin mediated toxicity.

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1. Introduction

Fumonisins are a family of mycotoxins originally characterizedfrom culture material of Fusarium verticillioides (Bezuidenhoutet al., 1988; Gelderblom et al., 1988). The natural occurrence offumonisins in maize and market maize-based products, such ascorn grits, corn flakes, snacks, corn flour, is a widespread problem(D’Arco et al., 2009; Molinié et al., 2005; Visconti et al., 2001).These toxins have also been found in wheat, barley, sorghum, rice,etc. (Mateo and Jiménez, 2000).

The structural similarity between free sphinganine and fumon-isins lead to the discovery that fumonisins are potent and specificinhibitors of the mammalian acyl CoA-dependent ceramide syn-thase, a key enzyme in the de novo sphingolipid biosynthesis path-way. Such inhibition of ceramide synthase also occurs in plants(Riley et al., 2001; Williams et al., 2006).

Many studies have demonstrated the relation between fumon-isins exposition and human esophageal cancer (Gelderblom et al.,1988) as well as several animal feeding studies have shown a caus-ative role of these mycotoxins in leukoencephalomalacia in horses

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+34 96354954.

(Domijan et al., 2008), pulmonary edema in swine, hepatic cancerin rats (Bouhet et al., 2004) and nephrotoxic effects in rats (Vosset al., 2001). The International Agency for Research on Cancer(IARC, 2002) has included FB1 in group 2B (possibly carcinogenicto humans).

The cellular mechanisms behind FB1-induced toxicity includedthe induction of oxidative stress, apoptosis and cytotoxicity. How-ever, the role of production of ROS in FB1-mediated toxicity has notbeen fully studied (Stockmann-Juvala and Savolainen, 2008). Inparticular, Stockmann-Juvala et al. (2004a) studied the productionof reactive oxygen species (ROS) by dichlorodihydrofluoresceindiacetate (H2-DCFDA) assay and the lipid peroxidation processwas evidenced by the malondialdehyde (MDA) assay on U-118MG glioblastoma cells exposed to the FB1 at 0.1, 1, 10 and100 lM after 4, 24, 48, 72 and 144 h of treatment.

The oxidative damage caused by fusarotoxins was also evi-denced in other cell lines, in particular FB1 on human fibroblast cellline (Galvano et al., 2002a); mouse GT1-7 hypothalamic cells andrat C6 glioblastoma cells (Stockmann-Juvala et al., 2004b); deoxy-nivalenol (DON) on human hepatocellular liver carcinoma cell line(HepG2) (Zhang et al., 2009), Enniatins (ENs) and Beauvericin(BEA) on human lung cancer cell line (A549 and GLC-4), epidermalcarcinoma-derived cell line (KB3) and promyelocytic leukemia cell

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line (HL-60) (Dornetshuber et al., 2009); and BEA, patulin(PAT) and zearalenone (ZEA) on Chinese hamster ovary cell line(CHO-K1) (Ferrer et al., 2009).

As regards to the production and purification of fumonisinsfrom Fusarium spp., Cawood et al. (1991), described a method forthe quantitative purification of FB1 and FB2 from solid culture ofcorn fermented by Fusarium moniliforme strain MRC 826 applyinga purification procedure composed by preliminary separation onAmberlite XAD 2 column followed by two purification steps on sil-ica gel column, and a final purification of FB1 and FB2 on reversephase C18 column.

Dantzer et al. (1996) studied a procedure for the production andpurification from liquid culture of F. proliferatum strain M5991 uti-lizing a chromatography approach that starts with a preliminaryportioning of the toxins with the low pressure liquid chromatogra-phy using a amberlite XAD-16 column, followed by a liquid chro-matography separation with a C8 column. The separation processwas completed with other two steps; the first one operated witha liquid chromatography utilizing a DEAE-Sepharose Ion Exchangecolumn and the second one consisted in the final purification of theextract with a liquid chromatography operated with a C18 column.

The isolation and purification of fumonisins was also studied byPoling and Plattner (1999) that obtained the complete separationof the singles fumonisins and also of the hydrolyzed fumonisin uti-lizing the NH2-cartridge, using a normal phase and then in the re-versed phase modes.

The aims of this study were to use a strain of F. verticillioides forthe production of the fumonisins B1, B2 and B3 with three differentfermentation’s techniques, and using the best procedure for purify-ing the FB1 produced by the microorganism with a low pressure li-quid chromatography (LPLC) on reverse and normal phase columnsfollowed by a LC–MS/MS characterization of the compound.

We have recently shown that high levels of FB1 decrease cellviability and increase the lipid peroxidation as measured by MDAproduction in renal cell line. To complete these data, we studiedROS production by VERO cells using a procedure based on an anti-oxidant sensitive fluorescenic probe dichlorodihydrofluoresceindiacetate (H2-DCFDA), to detect intracellular ROS as early stagemarker for toxin induced oxidative stress in mammalian cells. Thismethod is exceptionally sensitive and it detects intracellular oxida-tive in mycotoxins injured cells with a low cost and time.

2. Materials and methods

2.1. Chemicals

A stocks standard solutions of FB1, FB2 provided by Sigma–Aldrich (Madrid,Spain) and FB3 was supplied by PROMEC (Programme on Mycotoxins and Experi-mental carcinogenesis, Tygerbei, Republic of South Africa) that were up to 98% pur-ity were prepared by dissolving 1 mg of each fumonisin in 1 mL of pureacetonitrile–water (50:50 v/v), obtaining 1 mg/mL (1000 lg/mL) of each solution.These stock solutions were then diluted with pure acetonitrile–water (50:50 v/v)in order to obtain the appropriated work solutions. All solutions were stored indarkness at 4 �C until the chromatographic analysis. Acetonitrile, methanol, ethylacetate (all of HPLC grade) and acetic acid were purchased from Merck (Darmstadt,Germany), amberlite XAD-7, silica gel, sodium sulphate and hydrocloridric acid wasprovided by Sigma–Aldrich (Madrid, Spain).

2.2. Fermentation of F. verticillioides on solid and liquid medium

A solid medium of corn was utilized in this study. The medium was preparedweighting 100 g of solid corn in a 2 L Erlenmeyer flasks, and adding 2 L of water.The obtained suspensions were boiled for 15 min, and filtered with paper filter Phe-nomenex no. 4 (Madrid, Spain) to separate the liquid fraction from the solid (De laLuz et al., 2007). The solid and liquid fractions were autoclaved for 20 min at 121 �C,and a suspension of conidia in concentration of 106 conidia m L�1 of F. verticillioidesCECT 2982, was used for the inoculation of the mediums. Conidial concentrationwas measured by optical density at 600 nm (Kelly et al., 2006).

The strain of F. verticillioides CECT 2982 is deposited at the Spanish Type CultureCollection (CECT Valencia, Spain), in steriled 18% glycerol.

Fermentations were performed at 25 �C on an orbital shaker (IKA Ks 260 basic,Stanfen, Germany) in batch culture for 30 days. At the end of the fermentations thesolid and the liquid cultures were extracted for the analysis of fumonisins.

2.3. Fermentation of F. verticillioides on PDA plates

A suspension of 106 conidia m L�1 of F. verticillioides CECT 2982, was used forthe inoculation in potato dextrose agar (PDA), obtained from Oxoid (Basingstoke,UK). Fermentations were conducted in triplicate at 30 �C for 11 days. At the endof this procedure the agar plates were used for the extraction and determinationby LC–MS/MS of fumonisins.

2.4. Fumonisin extraction from solid medium

A modified method of Chelkowski et al. (2007) was carried out for toxin extrac-tions. Fifty grams of dried samples were homogenized by shaking with 500 mL of amixture methanol–water (70:30 v/v) with Ika T18 basic Ultraturrax (Staufen, Ger-many) for 5 min. Samples were then filtered through Phenomenex no. 4 filter paper,and the solvent was removed under reduced pressure. The extract was dissolved in50 mL of methanol–water (70:30 v/v) and filtered through a paper filter Phenome-nex before toxin identification and quantification by liquid chromatography (LC) asdescribed by D’Arco et al. (2009).

2.5. Fumonisin extraction from liquid medium

After the incubation of 21 days of the mediums, the extractions of fumonisinswere operated with the method described by Nilanonta et al. (2002). The cultureswere filtered, and the obtained residues (mycelia cake) were extracted with meth-anol 200 mL. After partial concentration of the extracts to 100 mL with a rotaryevaporator (Buchi, Postfach, Switzerland), 10 mL of water milli-Q was added, theextracts were washed with hexane 60 mL, and the aqueous methanol layers wereconcentrated under reduced pressure with rotary evaporator (Buchi, Postfach, Swit-zerland). The residual oils were dissolved in 20 mL of methanol, filtered with0.22 lm filter and 20 lL of the extract was injected in the LC–MS/MS.

2.6. Fumonisin extraction from PDA plate

A semiquantitative method based on agar plug (Smedsgaard, 1997) was devel-oped for the detection of the selected toxins of this study. Five agar plugs (diame-ter = 6 mm) were cut out of the colony from the center and in a radius toward theedge of the colony. The five agar plugs were placed in a 2 mL glass vial, 750 lL of amixture acetonitrile in water (80:20, v/v) was added, and the plugs were extractedfor fumonisins for during 50 min using ultrasonication. The extracts were filteredusing a PFTE 0.45 lm filter and analyzed by LC–MS/MS.

2.7. Purification of FB1

The extraction residue produced in the fermentation on solid medium of cornwas applied to an Amberlite XAD-7 column (7 � 65 cm; 500 g) previously equili-brated with methanol/water (80:20 v/v). The column was washed successively with1 L of methanol water (70:30 v/v) and 1 L of methanol/water (50:50 v/v), fumoni-sins were eluted with 1 L methanol.

The residue obtained from the methanol fraction (12 g) was dried under re-duced pressure with a rotary evaporator at 40 �C and fractionated on a silica gel col-umn (5.5 � 50 cm; 600 g) using chloroform/methanol/acetic acid (60:30:10 v/v/v)as the mobile phase. The sample was dissolved in the (70 mL) eluant and appliedto the column which contained anhydrous Na2SO4 (ca. 50 g) on top of the silicagel. After elution of 1 L of mobile phase at a flow rate of 3.5 mL/min, fractions(8 mL) were collected and analyzed by LC–MS/MS. Fractions containing FB1 andFB2 (5–19), FB2 and FB3 (30–50), and FB1, FB3 (51–90) were combined separatelyand the solvents evaporated at 50 �C.

The fraction intervals 5–19 and 51–90 were used for the final purification of FB1

that was achieved on a RP C18 column (1.5 � 50 cm; 200 g) using methanol/water aseluant. The eluant flow was maintained at 1.5 mL/min with a peristaltic pump Gil-son (Middelton, USA). The pH of the sample, dissolved in 20 mL methanol/water(50:50 v/v), was adjusted to 3.5 with 1 N HCl prior to the application to the column.Fractionation was obtained by running a 800-mL linear gradient from methanol/water (50:50 v/v) to methanol/water (90:10 v/v) at a flow rate of 1.5 mL/min. Dif-ferent fractions of 10 mL were collected after 400 mL of the gradient had elutedfrom the column. The purity of FB1 (1.2 g), which eluted as a single peak from thecolumn (fractions 8–15), was determined by LC–MS/MS.

2.8. LC–MS/MS analysis of FB1 instruments

LC separation was carried out on a Luna C18 analytical column(150 mm � 4.6 mm I.D., 5 lm) preceded by a C18 security guard cartridge(4 mm � 2 mm I.D., 5 lm), both from Phenomenex (Madrid, Spain). The analyticalseparation was performed using gradient elution with water as mobile phase Aand methanol as mobile phase B, both containing 0.5% formic acid. After an isocratic

Table 1Production of the FB1, B2 and B3 with the three different fermentation techniques.

Medium FB1 (mg) FB2 (mg) FB3 (mg)

Corn 73.32 31.43 11.21Liquid medium of corn 31.43 17.80 7.88PDA 11.21 5.55 1.50

G. Meca et al. / Food and Chemical Toxicology 48 (2010) 2891–2897 2893

step of 65% B for 3 min, it was gradually increased to 95% B in 4 min and held con-stantly for 3 min. Afterwards, the initial conditions were maintained for 10 min.Flow rate was maintained at 0.3 mL min�1. A TQ mass spectrometer Quattro LCfrom Micromass (Manchester, UK), equipped with an LC Alliance 2695 system(Waters, Milford, MA), consisted of an autosampler, a quaternary pump, a pneumat-ically assisted electrospray probe, a Z-spray interface, and a Mass Lynx NT software4.1 was used for data acquisition and processing. Parameters were optimized bycontinuous infusion of a standard solution (10 lg mL�1) via a syringe pump at aflow rate of 10 lL min�1. The analysis was performed in positive ion mode. TheESI source values were as follows: capillary voltage, 3.20 kV; cone, 50 V; extractor,3 V; RF lens, 0.2 V; source temperature, 125 �C; desolvation temperature, 300 �C;desolvation gas (nitrogen, 99.99% purity) flow, 500 L/h; cone (gas flow) 50 L/h.The analyzer settings were: resolution, 12.0 (unit resolution) for the first and thirdquadrupoles; ion energy, 0.5; entrance and exit energies, �3 and 1; multiplier,650 V; collision gas (argon, 99.995%) pressure 3.74 � 10�3 mbar; interchannel de-lay, 0.02 s; total scan time, 1.0 s. The mass spectrometer was operated in scan, prod-uct ion scan, and multiple reaction monitoring (MRM) modes. All themeasurements were carried out by triplicate.

2.9. Experimental solutions and cell culture

The reagent grade chemicals and cell culture components used, Dulbecco’smodified Eagle’s medium (DMEM), antibiotics, trypsin/EDTA solution, HEPES, 2,7-dichlorodihydrofluoroescein diacetate (H2-DCFDA) were Sigma products (SigmaCo. St. Louis Mo. USA). Fetal calf serum (FCS) was from Cambrex Company (Bel-gium). Deionised water (resistivity < 18 MX cm) was obtained using a Milli-Q waterpurification system (Millipore, Bedford, MA, USA).

VERO cells (ATCC CCL-81) kidney epithelial cell line derived from African greenmonkey were cultured in monolayer in 9 cm2 polystyrene tissue culture dishes withDMEM supplemented with 25 mM HEPES buffer (pH 7.4), 10% heat inactivated FCS,100 U/mL penicillin and 100 mg mL�1 streptomycin. Incubation conditions were37 �C, 5% CO2 and 95% relative humidity atmosphere.

2.10. Determination of intracellular reactive oxygen species by H2-DCFDA

Mycotoxin concentrations to determine intracellular ROS production by H2-DCFDA assay was chosen on the basis of concentrations that reduced cell viabilityas determined by neutral red assays. The median lethal concentration (LD50) val-ues obtained for FB1 was 55.5 lM (Meca et al., 2010). The FB1 was assayed at theconcentrations of 1, 5, and 10 lM. Intracellular ROS accumulation was monitoredin VERO cells by adding the H2-DCFDA according to Ruiz-Leal and George (2004).In brief, 2 � 104 cells/well were seeded in a 96-well black culture microplate. Oncecells exhibited 65% confluence, the culture medium was replaced and cells wereloaded with 20 lL H2-DCFDA for 20 min and then, the medium with H2-DCFDAwas removed and washed with PBS before the addition of medium/1% methanol(control) or medium with mycotoxins concentration (1, 5 or 10 lM). H2-DCFDAis non-fluorescent until it is hydrolyzed by intracellular esterases and readily oxi-dized to the highly fluorescent DCF in the presence of ROS. Increases in fluores-cence were measured at excitation and emission wavelengths of 485 and535 nm, respectively during 100 min with a 1420 Multilabel Counter (Perkin El-mer, Madrid, Spain).

2.11. Statistical analysis

Statistical analysis of data was carried out using SPSS version 13 (SPSS, Chicago,IL, USA), statistical software package. Data were expressed as mean ± SD of fourindependent experiments. The statistical analysis of the results was performed byStudent’s t-test for paired samples. Differences between groups were analyzed sta-tistically with one-way ANOVA followed by the Tukey HDS post hoc test for multi-ple comparisons. Different levels of signification (P 6 0.05 to P6 0.0001) in the MDAassay were considered statistically significant.

3. Results and discussion

3.1. Evaluation of the fumonisins produced on solid, liquid and agarmedium

In this study the strain of F. verticillioides CECT 2982 was utilizedto compare the FBs production utilizing three different fermenta-tions techniques and in particular the fermentation on solid and li-quid medium of corn and the bioproduction on an agarizedmedium (PDA).

As it is shown in Table 1, the higher amount of FBs was pro-duced on the solid medium of corn, whereas the liquid mediumof corn has resulted the second for the quantity of the production

of these metabolites. Only a few quantities of FBs were producedwith the technique of the agar plug.

In details the fermentation operated on the solid medium ofcorn, F. verticillioides CECT 2982, produced 73.32 mg of FB1,31.43 mg of FB2, and only 11.21 mg of FB3. The major metaboliteobtained with this fermentation technique was FB1 produced inthe double of quantity respect at the FB2

In the fermentation operated with a liquid medium of corn thestrain of F. verticillioides CECT 2982 produced 31.43, 17.80, and7.88 mg, respectively of FB1, FB2, and FB3.

In the fermentation executed on the agarized medium (PDA),the strain has produced a very low quantity of these fumonisins,obtaining at the end of the fermentation 11.21 mg for the FB1,5.50 mg for the FB2, and 1.50 mg for the FB3.

Considering the high value of the FB1 produced by the strain ob-ject of this study on the solid medium of corn, the extracts corre-sponding at this fermentation were utilized for the purification ofthe FB1.

The first part of this study was focalized on the influence of themedium for the production of the FBs.

Marín et al. (1999) studied the production of the FB1 by differ-ent strains of Fusarium spp. on a solid medium of corn during 30fermentation days The maximum concentrations produced of thisfusarotoxin were variable from 17.62 to 28.61 mg/kg.

Hinojo et al. (2006) worked on the production of the FB1 and FB2

utilizing a solid medium of rice fermented by several strains of F.verticillioides with a fermentations time of 35 days. The concentra-tions of FB1 and FB2 found in rice cultures inoculated with theseisolates were variable from 5 and 3840 mg/kg for the FB1, and from1 to 210 mg/kg for the FB2 using also different incubation temper-atures and water activity values.

The other fermentation technique employed in this study wasthe method of the agar plug. Frisvad et al. (2007), monitored theproduction of the FB1, FB2, and FB3 produced by Aspergillus nigerand F. verticillioides on 13 different agarized mediums. The limita-tion of this technique is that there is not a real quantification of thefumonisins produced. The hypothetical quantity produced is ex-pressed with the intensity of the ions corresponding at the threefumonisins detected by the LC/MS–MS.

F. verticillioides MRC 826 produced large amounts of FB1 onplant-based media such as malt extract media as MEA (malt ex-tract agar), MEAB (malt extract agar with Merck malt extract),MEAG (malt extract agar with Biokar malt extract), RC (rice andcorn steep agar), OAT (oatmeal agar), and PDA (potato dextroseagar). Lower amounts of FB1 were produced on PCA (potato carrotagar). FB2 was produced in highest amounts on malt extract media,PDA, and OAT. Lower amounts FB2 were produced on RC agar andPCA. For F. verticillioides MRC 826, FB1 was produced in the higherquantity, and the amounts of FB2 were higher than the amounts ofFB3 on most of the media used (Cawood et al., 1991).

As concern the production of the fumonisins in submerged cul-ture, Jackson and Bennett (1990) were the first to use a chemicallydefined liquid medium for the production of fumonisins. Liquidmedia has the advantage over solid media that impurities derivedfrom solid cultures such as maize and rice, co-elute with FB1 (Mill-er et al., 1994). However, most Fusarium mycotoxins are producedat lower levels in liquid than on solid media (Blackwell et al., 1994;Marín et al., 1999).

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Inert materials such as vermiculite and resins have been usedsuccessfully to support fungal growth and mycotoxin productionin liquid media (Xu et al., 2009).

Vismer et al. (2004) studied the production of the fumonisins byF. verticillioides in solid and in submerged culture. Levels of the to-tal fumonisins produced on solid culture ranged from 1500 to3900 mg/kg in rice and 2900–12,500 mg/kg in maize cultureswhereas the production in submerged culture had permitted to ob-tain a concentration of total fumonisins variable from 40 to590 mg/L.

3.2. Purification and LC/MS–MS characterization of the FB1

In the present study the fundamental extraction and purifica-tion steps reported previously (Cawood et al., 1991) were used,with minor modifications, for the isolation of FB1.

Table 2Quantification of the FB1 during the purification steps.

Step Sample (g) FB1 (mg) Recovery (%)

Solvent extraction 1.25 73.32 96.5Amberlite XAD-7 0.12 68.80 94.4Silica gel 0.70 67.45 92.6C18 column 0.01 66.70 91.2

Fig. 1. Graphical representation of the exp

The extraction of the FB1 with the mixture methanol–water(70:30 v/v) has permitted to obtain a recovery of 96% of FB1 fromthe corn fermented by the strain of F. verticillioides CECT 2982(Table 2).

This study evidence that the aqueous methanol solution is agood extraction solvent mixture for FB1 extraction, as also con-firmed by the study of Cawood et al. (1991).

Chromatographic separation on the Amberlite XAD-7 columnwas performed by methanol as eluting solvent after the equilibra-tion of the column with methanol–water (80:20; 70:30; 50:50 v/v).

The bulk (>94%) of the fumonisins was recovered in the finalmethanol eluate while a little degree of purification was achieved.

The chromatographic procedure on the Amberlite column isvery important in the purification process because removes inter-ferences, although no separation between individual fumonisins isobtained. In addition, polar components that would interfere withthe subsequent purifications on silica gel were effectively removedfrom the extract.

The most effective separation between the different fumonisinswas obtained on silica gel by using chloroform–methanol–aceticacid (60:30:10) as mobile phase. When this purification step wasomitted, some of the pigments were present in the final FB1

extract.The subsequent RP C18 column step was used mainly for

removing the inorganic material while the gradient elution of FB1

also removed some yellow pigments that coeluted with FB1 fromthe silica gel column. A high recovery (91%) of FBI was achieved

erimental plan realized in this study.

G. Meca et al. / Food and Chemical Toxicology 48 (2010) 2891–2897 2895

in this chromatographic purification step. The resume of the FB1

quantity recovered in each purification step is represented inTable 1. The purity of the fractions containing the FB1 obtainedwith the final purification step on the C18 column was determinateby LC–MS/MS (see Fig. 1).

At the moment our attemption was focalized on the study of thepurification of the FB1 but other isolates of F. verticillioides are un-der investigation in an attempt to select those isolates which willallow optimal purification of each individual fumonisin. Other sol-vent systems for the purification of fumonisins on silica gel are alsounder investigation to prevent the formation of the structurally re-lated compounds, thereby increasing the final yield of the purefumonisins.

The LC–MS/MS analysis was performed using a C18 column anda water–MeOH mobile phase acidified with 0.1% HCOOH, using agradient elution. The fumonisin B1, purified was analyzed by LC/MS–MS on the triple quadrupole instrument.

The full scan MS spectrum (Fig. 2) shows a protonated moleculeat m/z 722 for fumonisin B1. The spectrum is similar to that re-ported previously (D’Arco et al., 2009) but showed significantlyless ion formation due to sodium and potassium adduction.

Fig. 2. Electrospray ionization (ESI/M

Fig. 3. Time dependence of ROS-induced fluorescence in VERO cells exposed to FB1. VERwell) and then exposed to test agent or vehicle (control). Fluorescence of oxidized DCFreplicates. Significantly different from the control, P 6 0.05 (*), P 6 0.001 (**), P 6 0.000

Three successive dehydrations involving the hydroxyl groupson the fumonisin backbone lead to the ions at m/z 704 [M-H2O]+,m/z 686 [M-2H2O]+ and m/z 668 [M-3H2O]+ respectively. The ionat m/z 564 [M-TCA]+ resulted from loss of one of the tricarboxylicacid (TCA) side chains from [M + H]+, four losses of water then leadto the ions at m/z 546 [M-TCA-H2O]+, m/z 528 [M-TCA-2H2O]+, m/z510 [M-TCA-3H2O]+ and m/z 492 [M-TCA-4H2O]+, respectively. Theion at m/z 370 [M-2TCA]+ results from elimination of both TCA sidechains with the associated double loss of water from the backbone.Three further losses of water then lead to the ions at m/z 352[M-2TCA-H2O]+, m/z 334 [M-2TCA-2H2O]+ and m/z 316 [M-2TCA-3H2O]+, respectively. The ions evidenced in the Fig. 2 confirm thepresence of the pure FB1 in the final extract and the efficiency ofthe purification method utilized.

3.3. Intracellular ROS production

To determine the changes in the redox status in response to FB1,VERO cells were exposed to different concentrations of this myco-toxin from 0 to 100 min (Fig. 3). The increase of ROS productionwas time and concentration dependent, when compared to the

S) product ion spectrum of FB1.

O cells were loaded with H2-DCFDA for 20 min in 96-well plates (20,000 cells perwas followed by emission at 535 nm and the excitation of 485 nm. Mean ± SD, 24(***). Control: 1 lM; 5 lM; 10 lM.

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basal rate. The highest relative intensity of fluorescence in VEROcells was evidenced by the FB1 at 10 lM that shows a significativedifference (P 6 0.000) respect to the control, from 0 to 80 min, andevidencing values of ROS production variables from 23.08 to125.21 mg of MDA/mg protein.

Another important data evidenced with this assay are that: (a)FB1 at 10 lM, shows a significative difference (P 6 0.000) of 1.5and 1.2 times highest respect to the FB1 at 1 lM in the times as-sayed of 5 and 80 min and (b) the FB1 at the concentration of10 lM shows a significative difference (P 6 0.000) compared tothe FB1 at 5 lM in the assay time range between 20 and100 min. The values of the ROS production evidenced by the FB1

at 10 lM resulted variables from 1.1 to 1.4 times higher respectto the value evidenced by the FB1 at 5 lM.

Oxidative stress may be defined as the alteration of the steady-state balance between oxidant and antioxidant agents in cells. Theincrease of the ROS generation induced lipid peroxidation. ROShave been implicated as a subcellular messenger in multiple sig-naling pathways and in particular, it has been demonstrated thatROS could be a cause or consequence of the mitochondrial altera-tions and, probably the cause of the apoptotic cell death. Moreover,ROS generation plays a role in the molecular events leading to celldamage particularly by the induction of lipid peroxidation (Bouazizet al., 2008; Wu et al., 2008).

In this study, it is provided the evidence that VERO cells rap-idly generate ROS after FB1 treatment (Fig. 3). Although nephro-toxic effects have been reported in laboratory animals, this isthe first report about the ROS formation generated by this fungalmetabolite on renal cell line. Cytotoxicity of FB1 and of the othersfusarotoxins has been tested in other cell lines. The generation ofprimary peroxidation products in cells treated with the FB1, wasdocumented by Stockmann-Juvala et al. (2004a), that studied theformation of ROS determinate with the dichlorodihydrofluoresce-in diacetate (H2-DCFDA) assay on U-118MG glioblastoma cellsexposed to the FB1 in the concentration of 0.1, 1, 10 and100 lM, showing a significant effects after culture times from48 to 144 h at dose levels of 10 or 100 lM. Moreover, they dem-onstrated that ROS production was probably the cause of theapoptotic death cell. No ROS production were observed afterexposure of 10–100 lM FB1 on human non immortalized fibro-blast cell lines during 72 h (Galvano et al., 2002a) or in primaryculture of rat astrocytes exposed to that concentration for 6 days(Galvano et al., 2002b). High dose of FB1 clearly increased ROSproduction on C6 cells and especially in GT1-7 at 48 h and72 h, whereas in SH-SY5Y cells, production of ROS was not signif-icantly altered by the FB1 exposure (Stockmann-Juvala et al.,2004b). These data corroborate that ROS production by FB1 variesbetween different cell lines and cell lines from different species(Stockmann-Juvala and Savolainen, 2008).

The formation of ROS and the mechanisms that participate atthe formation of cell lipid peroxidation was evaluated by Zhanget al. (2009). The authors studied the production of ROS by HepG2cells exposed to the mycotoxin deoxinivalenol (DON) utilized inthe concentration of 15, 30 and 60 lM during 1 h applying theH2-DCFDA assay. Significant increase in the level of ROS was ob-served in HepG2 cells at a higher concentration (60 lM). The DCFfluorescence intensity in HepG2 cells at the highest dose of DON(60 lM) was found to be 1.4-fold higher than the solvent control(ethanol). Dornetshuber et al. (2009) elucidate the oxidative celldamage induced by a mixture of enniatins (ENs) A, A1, B, B1, andby BEA on the human lung cancer cell lines A549 and GLC-4, onthe epidermal carcinoma-derived cell line KB-3-1and on the pro-myelocytic leukemia cell line HL-60, exposed at the concentrationof 10 lM during 15 min. In general, no detectable increase in theamount of ROS with respect to control after ENN or BEA exposurein each cell line tested was observable.

The induction of ROS in CHO-K1 cells exposed to beauvericin(BEA), patulin (PAT) and zearalenone (ZEA), was studied by Ferreret al. (2009). To determine the intracellular production of ROS, theintensity of fluorescence emitted from the probe H2-DCFDA wasmeasured. The relative intensity of fluorescence from cells incu-bated with BEA, PAT and ZEA was approximately 4-, 7- and 4-foldhigher in comparison with control cells at 0 min, respectively. Gen-erally the increase of ROS generation induced the cell death pro-cess by GSH depletion and direct damage to DNA, proteins andlipids (Ferrer et al., 2009).

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by the Science and Education SpanishMinistry Projects (CTQ2007-291 63186 and AGL2007-61493), andby the pre PhD program of University of Valencia ‘‘Cinc segles”.

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