Mycopathologia 151: 147–153, 2000. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

147

In vitro and in vivo studies to assess the effectiveness of cholestyramine as a binding agent for fumonisins ∗ Michele Solfrizzo1 , Angelo Visconti1, Giuseppina Avantaggiato1, Adriana Torres2 & Sofia Chulze2 1 Istituto

Tossine e Micotossine da Parassiti Vegetali, CNR, 70125 Bari, Italy; 2 National University of Rio Cuarto, Department of Microbiology and Immunology, Rio Cuarto, Argentina

Received 10 November 2000; accepted 1 June 2001

Abstract Several adsorbent materials were tested at 1 mg/ml for their in vitro capacity to adsorb fumonisin B1 (FB1 ) from aqueous solutions. Cholestyramine showed the best adsorption capacity (85% from a solution containing 200 µg/ml FB1 ) followed by activated carbon (62% FB1 ). Bentonite adsorbed only 12% of the toxin from a solution containing 13 µg/ml FB1 , while celite was not effective even at the lowest tested FB1 concentration (3.2 µg/ml). Cholestyramine was tested in vivo to evaluate its capacity to reduce the bioavailability of fumonisins (FBs) in rats fed diet contaminated with toxigenic Fusarium verticillioides culture material. Rats were exposed for one week to FBs-free diet, FBs-contaminated diet containing 6 or 20 µg/g FB1 + FB2 and the same FBs-contaminated diet added of 20 mg/g cholestyramine. The increase of sphinganine/sphingosine (SA/SO) ratio in urine and kidney of treated rats was used as specific and sensitive biomarker of fumonisin exposure. The addition of cholestyramine to the FBs-contaminated diets consistently reduced the effect of FBs by reducing significantly (P < 0.05) both urinary and renal SA/SO ratios. Key words: biomarker, cholestyramine, detoxification, fumonisins, sphinganine Abbreviations: FB1 = fumonisin B1 ; FB2 = fumonisin B2 ; FBs = fumonisins; SA = sphinganine; SO = sphingosine; CH = cholestyramine; HPLC = high performance liquid chromatography; SAX = strong anion exchange; OPA = o-phthaldialdehyde

Introduction Procedures for the decontamination of fumonisincontaminated corn and corn-based feed are necessary since these mycotoxins are widespread in most Countries of the world at levels that may be of concern for animal and human health [1–3]. Acute and chronic toxicity of fumonisins, including carcinogenic activity, have been clearly documented [4–7]. Although the prevention of fumonisin formation in the field is the best option, during certain environmental conditions of temperature and humidity the formation of these mycotoxins is unavoidable. Fumonisins are relatively stable under most food processing conditions ∗ Published in 2001.

and can be detected in most corn based foods [8]. Nixtamalization of maize dough (cooking with Ca(OH)2 ) reduces consistently the fumonisin levels of contaminated corn, but the product of fumonisin degradation (hydrolyzed fumonisin) seems to retain the original toxicity when fed to rats [9]. Similarly, no reduction in toxicity was found when ammoniated fumonisincontaining feed was fed to rats although the fumonisin B1 (FB1 ) contamination level was reduced by 30– 45% in the treated material [10]. Fumonisins are also resistant to autoclaving and bleach treatments [11]. Reaction of FB1 with fructose seems to prevent FB1 induced hepatotoxicity and promotion of hepatocarcinogenesis while stimulating liver-associated natural killer cell activity in rats [12]. Fumonisin B1 is rapidly degraded by ozone gas (O3 ), but degradation of FB1

148 do not correlate with detoxification since FB1 solutions treated with O3 were still positive in two bioassay systems [13]. Attempts to detoxify fumonisins through the use of commercial enzymes or cytochrome P-450 monooxygenase targeting amino groups failed since FB1 retained its reactivity with o-phthaldialdehyde reagent after incubation with enzyme or cytochrome P-450 monooxygenase [14, 15]. Black yeast and a bacterium strain isolated from corn stalk have been found to completely metabolize FB1 with release of CO2 , however, only the fumonisin esterase was expressed in the relevant transgenic maize plant [16, 17]. This means that maize transgenic plants can degrade FB1 to its hydrolysis product (aminopentol), a compound that retains most of the toxicity of FB1 . Gamma irradiation at 15 KGy reduced fumonisins (FB1 and FB2 ) levels in corn with 20% [18]. An appropriate approach to detoxification of mycotoxins involves the use of adsorbent materials with the capacity to tightly bind and immobilize mycotoxins in the gastro-intestinal tract of animals, then reducing toxin bioavailability. The problem that in vitro efficacy does not always predict the efficacy of adsorbent materials in vivo has been reported for different adsorbents used for several mycotoxins [19]. For instance, activated carbons have been found to adsorb FB1 from water solutions [20], but resulted ineffective when tested in vivo [21]. In this paper we report the in vitro evaluation of cholestyramine, activated carbon, celite and bentonite for their capacity to adsorb FB1 from water solutions and in vivo evaluation of the efficacy of the best adsorbent resulted from the in vitro study by using Wistar rats as model. Sphinganine to sphingosine (SA/SO) ratio has been used as biomarker of fumonisin bioavailability. Materials and methods In vitro measurement of adsorbent’s capacity Cholestyramine (CH), celite and bentonite were purchased by Sigma (St. Louis, MO, USA) and activated carbon by Carlo Erba (Milan, Italy). Solid FB1 and FB2 were purchased by PROMEC (Medical Research Council, South Africa). Experiments were performed by incubating at 25 ◦ C for 1 h each adsorbent material (1 mg) with water solutions (1 ml) containing different concentrations of FB1 (from 3.25 to 260 µg/ml). Duplicate experiments were performed for each fumonisin concentration; solutions were manually stirred

every 15 min and centrifuged at 2500 × g at the end of incubation. The initial FB1 concentration and the unadsorbed FB1 remaining in the solution were measured by HPLC analysis of its o-phthaldialdehydeFB1 derivative [22]. The amount of FB1 adsorbed by each testing material was calculated by difference between the starting and final concentration of FB1 in the testing solutions. In order to assess the capacity of cholestyramine to reduce the water extractability of fumonisins from contaminated corn flour, naturally contaminated corn samples with two different levels of fumonisins (Corn A and Corn B, with 3 µg/g and 162 µg/g of water extractable FB1 + FB2 , respectively) were divided in three parts. The first part was extracted with water, cleaned-up through strong anion exchange (SAX) column and analyzed by HPLC for FB1 and FB2 (22), whereas the second and third parts were mixed with 10 and 100 mg/g of CH, respectively, then extracted, cleaned-up and analysed by HPLC as reported above. Diets Blank control diet was obtained from a standard commercial diet (Rata-Ratón, Córdoba, Argentina). Six kg of each experimental diet were prepared by thoroughly mixing blank control diet with appropriate amounts of Fusarium verticillioides (= F. moniliforme Sheldon) strain M-7075 (Department of Microbiology and Immunology, National University of Rio Cuarto, Argentina) corn culture material in order to obtain diets containing 6 and 20 µg fumonisins (FB1 + FB2 )/g total diet. Corn culture of F. verticillioides was prepared and analyzed as reported elsewhere [23]. To check the homogeneous distribution of fumonisins in the diet, three samples of each experimental diet were collected randomly and analyzed by the method of Sydenham et al. [22] with a minor modification, i.e., using 10 g test portion size (instead of 50 g) and acetonitrile+water (50 + 50, v + v) as extraction solvent (instead of methanol + water 75 + 25, v + v). Fifty per cent of the homogeneous fumonisin contaminated diets were thoroughly mixed with 20 mg CH/g diet. Animals, husbandry and feeding protocol Male or female Wistar rats (3 month-old, supplied by National University of Rio Cuarto, Argentina) were housed in cages (4 rats/cage) with free access to control or experimental diets, and tap water. Cages were kept in environmentally controlled rooms (23 ◦ C and 50% humidity) with a 12 h light/dark cycle. Prior to

149 feeding experimental diets, rats were maintained on a blank control diet for 1 week. Three groups (4 rats/group) of female rats were fed either control diet or FBs-contaminated (20 µg/g of FB1 + FB2 ) diet or FBs-contaminated diets mixed with 20 mg CH/g diet for one week. Three groups (4 rats/group) of male rats were fed either control diet or FBs-contaminated (6 µg/g of FB1 + FB2 ) diet or FBs-contaminated diet mixed with 20 mg CH/g diet for one week. Rats were weighed at the start and end of the study period (one week). At the end of the feeding period, rats were individually transferred in metabolic cages in order to collect urine for a period of 24 h and soon after they were anaesthetized with ether, and sacrificed to collect liver and kidney. These organs were weighed and stored at −20 ◦ C together with urine samples until analysis of sphinganine (SA) and sphingosine (SO). The study with experimental animals was performed in accordance with the Argentinian ethics legislation governing these experiments (National Administration of Drug, Food and Medical Technology of Argentina, disposition No. 6344/96).

below 2 ml/min), than loaded with 2 ml CHCl3 extract and washed with 1 ml CHCl3 discarding the eluates. SA and SO were eluted with 4 ml of CHCl3 + MeOH + NH4 OH (50 + 50 + 2, v + v + v). After solvent evaporation at 60 ◦ C under a stream of nitrogen, samples were redissolved in 250 µl MeOH + water (9 + 1, v + v), derivatized with 50 µl OPA reagent and analyzed by reversed phase HPLC as described by Solfrizzo et al., 1997 [24]. Apparatus The HPLC apparatus consisted of a Waters 625 LC quaternary pump (Waters, Milford, MA, USA) equipped with a Rheodyne Model 9125 injection valve (Rheodyne , Cotati, CA, USA), a Perkin Elmer LC 240 fluorometer detector and a Turbochrom 4.0 data system (Perkin Elmer, Norwalk, CT, USA). The analytical column was a reversed phase Discovery C18 (15 cm × 4.6 mm, 5 µm particles) (Supelco, Bellefonte, PA, USA) preceded by a Rheodyne guard filter (0.5 µm). Fluorescence detector was set at 335 nm excitation and 440 nm emission.

Analysis of sphinganine and sphingosine Statistical analysis Free SA and SO concentrations in rat urine samples were analyzed according to Solfrizzo et al. (24). Liver and kidneys were analyzed by using the same procedure appropriately modified. Briefly, 90 mg of tissue were added to 3 ml phosphate buffer (8.0 g NaCl, 1.2 g Na2 HPO4 , 0.2 g KH2 PO4 and 0.2 g KCl in approximately 990 ml water, adjust pH to 7.0 with concentrated HCl and bring to 1 l with distilled water), homogenized with Potter-Elvehjam and centrifuged. The supernatant was separated, diluted with 2 ml methanol, alkalinized with 1.2 ml of 0.35 N NH4 OH, extracted with 4 ml CHCl3 and centrifuged. The upper phase was discarded and the CHCl3 phase transferred into a clean vial avoiding to carry the white film formed at the interphase. The CHCl3 phase was washed twice with 4 ml of alkaline solution (0.6 ml 0.35 N NH4 OH in 250 ml distilled water). After water removal, 2 ml CHCl3 extract was transferred into a clean vial. The CHCl3 extract was cleaned up through a minicolumn consisting of 5 g anhydrous Na2 SO4 crystals (Baker, Deventer, The Netherlands) packed on the top of 0.2 g silica gel 60 (15–40 µm) (Merck, Darmstadt, Germany) in a 12 mm diameter polypropylene column containing a polyethylene frit on the bottom (Alltech Italia srl, Milan, Italy). The minicolumn was preconditioned with 3 ml CHCl3 (maintaining flow rate

Comparison among groups (of four rats each) was made by one-way analysis of variance using Instat (software (Instat
, San Diego, CA). Tukey-Kramer multiple comparisons test was performed and differences were considered significant at P < 0.05. Values reported in the tables are means ± standard error.

Results and discussion In vitro experiments The results of in vitro experiments aimed to ascertain the capacity of CH, activated carbon, bentonite and celite to adsorb FB1 from water solution are reported in Table 1. Celite did not adsorb FB1 , even at the lowest FB1 concentration (3.2 µg/ml), while bentonite showed a relatively low affinity for FB1 , with ca. 12% adsorption from a solution containing 13 µg/ml FB1 ; therefore higher FB1 concentrations were not tested with these two adsorbents. Activated carbon and CH exhibited a good adsorption capacity even at high toxin concentrations. Although CH seemed to be less efficient than activated carbon at FB1 concentration of 3.25, 13.0 and 26.0 µg/ml, it showed

150 Table 1. In vitro capacity of different materials to adsorb fumonisin B1 (FB1 ) from aqueous solutions Adsorbent (1 mg/ml)

FB1 initial conc. (µg/ml)

Unadsorbed FB1 (µg/ml)a

Adsorbed FB1 (µg/mg)b

Adsorbed FB1 (%)

Cholestyramine

3.25 13.0 26.0 130.0 200.0 260.0

0.39 0.65 0.50 6.50 30.25 70.20

2.86 12.35 25.50 123.50 168.75 189.80

88 95 98 95 85 73

Activated carbon

3.25 13.0 26.0 200.0

0 0 0 76.0

3.25 13.0 26.0 124.0

100 100 100 62

Bentonite

3.25 13.0

0.03 11.37

3.22 1.63

99 12

Celite

3.25 13.0

3.25 13.0

0 0

0 0

a Mean concentration of FB residue in water solution (n = 2). 1 b Calculated by substracting the unadsorbed FB from the initial FB concentration. 1 1

better performance than activated carbon at higher FB1 concentrations (85% vs 62%, at 200 µg/ml). The addition of CH to fumonisin contaminated corn flour reduced the amount of fumonisins extractable with water. Water was used as extraction solvent in order to obtain results comparable with the above experiments. The results obtained with fumonisin contaminated corn flour materials (Corn A and Corn B) mixed with different amounts of CH (10 and 100 mg/g) are reported in Table 2. In particular, the amount of fumonisins extracted with water from contaminated corn flour was considerably reduced when CH was added to corn flour and the relevant percentages of fumonisins adsorbed by CH ranged from 16 to 95% for FB1 and from 56 to 100% for FB2 , respectively. These results demonstrate that CH, up to certain levels, can adsorb fumonisins from water solution even in presence of corn. In vivo experiments Following the above in vitro experiments, and the negative results obtained in vivo with activated carbon [21], CH remained the sole interesting material to be tested in vivo in order to confirm its efficacy. The evaluation of the in vivo effectiveness of CH in reducing gastrointestinal fumonisin absorption was performed by measuring SA/SO ratios in urine and kidney of treated rats. This biomarker is particularly important because it is indicative for both the exposure to fumonisins and the toxic effects derived from a biochemical

change (disruption of sphingolipid metabolism) generated directly by fumonisins on the exposed animal. The biomarker has been validated by proving that the increase of the SA/SO ratio in pigs and rats showed a consistent relationship with regard to either dose or time of exposure to fumonisins in the diet [25, 26]. Fumonisin concentrations tested in this study were selected on the basis of realistic values of natural contamination observed in feeds worldwide. The blank control diet was fumonisin free (<0.05 µg/g) and gave SA/SO <0.5 in urine and kidney of rats fed with this diet. Figure 1 shows the SA/SO increase in kidney and urine of male rats exposed to 6 µg/g FBs with and without the addition of CH, while Figure 2 shows similar results obtained with female rats used for the experiment at 20 µg/g FBs. The addition of 20 mg/g of CH to FBs-contaminated diets resulted effective in reducing the absorption of fumonisins in the gastrointestinal tract as demonstrated by the reduction of SA/SO ratios, both in urine and kidney, after the addition of CH. The protective effect of CH was evident in both male and female rats exposed to FBscontaminated diets. In particular, mean kidney and urinary SA/SO ratios decreased (P < 0.05) from 1.50 to 0.52 and from 1.03 to 0.63, respectively, when CH was added to a diet containing 6 µg/g FBs. Again, in rats fed with 20 µg/g FBs, mean SA/SO ratios in kidney and urine decreased (P < 0.05) from 1.83 to 0.78 and from 2.82 to 1.36, respectively, when 20 mg/g of CH was added to FBs-contaminated diet.

151 Table 2. Extractable fumonisins concentrations from contaminated corn flour, with and without the addition of cholestyramine (CH) Sample

FB1 1 (µg/g)

FB2 1 (µg/g)

Adsorbed FB1 (%)

Adsorbed FB2 (%)

Corn A

2.45

0.62

–

–

Corn A + CH (10 mg/g) Corn A + CH (100 mg/g)

0.83 0.12

0.07 0

66 95

88 100

Corn B

151.9

10.2

–

–

Corn B + CH (10 mg/g) Corn B + CH (100 mg/g)

127.6 66.9

4.5 0

16 56

56 100

1 Samples were extracted with water, purified on SAX column and analysed by HPLC.

Figure 1. SA/SO ratios in urine and kidney of male rats exposed for one week to FBs-free diet (Control), FBs-contaminated diet (6 µg/g FB1 + FB2 ), and FBs-contaminated diet mixed with cholestyramine (CH) (20 mg/g). Bars represent mean ± standard error relevant to four rats. ∗ Different from FBs-contaminated diet (P < 0.05).

In vivo experiments with female rats were also performed at 6 µg/g FBs, however a significant increase (P < 0.05) of the SA/SO ratio in urine and kidney was only observed with diets containing 20 µg/g FBs. Results relevant to liver did not show any SA/SO increase in male or female rats fed FBs-contaminated diets as compared to controls (data not shown). This result confirms previous data reporting that, in in vivo experiments, kidney is a more sensitive organ than liver in respect to SA/SO increase produced by the ingestion of fumonisins [27]. Data relevant to organ weight of rats fed with 6 µg/g and 20 µg/g FBs are reported in Tables 3 and

Figure 2. SA/SO ratios in urine and kidney of female rats exposed for one week to FBs-free diet (Control), FBs-contaminated diet (20 µg/g FB1 + FB2 ), and FBs-contaminated diet mixed with cholestyramine (CH) (20 mg/g). Bars represent mean ± standard error relevant to four rats. ∗ Different from FBs-contaminated diet (P < 0.05).

4, respectively. From the tables it is evident that one week exposure to these levels of fumonisin contamination did not affect liver and kidney weight, since no significant difference was found when the relative weights (organ weight to body weight ratio) were compared. The increase of SA/SO ratio is the only tool that allows to display the biological effect of fumonisins at the concentrations used in this study. On the other hand, it is known that the positive response to fumonisin exposure by this biomarker appears at fumonisin dietary levels lower than those necessary to elicit other serum chemical markers [28].

152 Table 3. Organ weight of male Wistar rats fed fumonisin-free and fumonisins (FB1 + FB2 ) contaminated diets for one week with and without the addition of cholestyramine (CH)

Absolute liver weight (g) Relative liver weight (mg/g)1 Absolute kidney weight (g) Relative kidney weight (mg/g)1

Control

FB1 + FB2 (6 µg/g)

FB1 + FB2 (6 µg/g) + CH (20 mg/g)

13.0 ± 0.20a∗ 34.1 ± 0.34a

10.5 ± 0.82b 36.3 ± 1.10a

11.2 ± 0.34ab 35.2 ± 0.50a

3.6 ± 0.06a 9.5 ± 0.29a

2.6 ± 0.30b 9.1 ± 0.58a

2.8 ± 0.12b 8.7 ± 0.48a

∗ Different letters in individual row indicate significant difference (P < 0.05). 1 Organ weight (mg) to body weight (g) ratio.

Table 4. Organ weight of female Wistar rats fed fumonisin-free and fumonisins (FB1 + FB2 ) contaminated diets for one week with and without the addition of cholestyramine (CH) Control Absolute liver weight (g) Relative liver weight (mg/g)1 Absolute kidney weight (g) Relative kidney weight (mg/g)1

FB1 + FB2 (20 µg/g)

FB1 + FB2 (20 µg/g) + CH (20 mg/g)

25.8 ± 1.12a

7.4 ± 0.75a 28.5 ± 2.52a

7.4 ± 0.59a 28.8 ± 2.02a

1.5 ± 0.06a 5.5 ± 0.22a

1.6 ± 0.06a 6.4 ± 0.19a

1.6 ± 0.19a 6.2 ± 0.66a

7.0 ± 0.42a∗

∗ Different letters in individual row indicate significant difference (P < 0.05). 1 Organ weight (mg) to body weight (g) ratio.

Conclusions The use of adsorbent materials binding mycotoxins could be considered the most effective procedure commercially applicable for detoxification of contaminated cereals or feeds because it is simple and relatively inexpensive. Many mycotoxin-binding agents have been investigated (for a review see [29]) and several have been made commercially available mainly based on their in vitro efficacy. However, the efficacy of most adsorbent materials which are effective in vitro in binding mycotoxins fails when confirmation of in vivo activity is tested [19]. Our data demonstrate that CH is an effective adsorbent material both in vitro and in vivo and can be proposed as a suitable tool for detoxification of fumonisin contaminated grains and feeds. The effectiveness of CH, a highly charged quaternary ammonium (strong anion exchange) resin, could be due to the sum of two factors: the ion exchange capacity of the adsorbent and its capacity to physically entrap the fumonisins in the polymeric matrix. Considering also previous reports on the effectiveness of CH in reducing the bioavailability of zearalenone [30, 31] and ochratoxin A [32], it can be concluded that this adsorbent material has the potential to be used as multi-mycotoxins binding agent. However further studies are necessary to optimize, in relation to the levels and kind of mycotoxin contamin-

ation, the concentrations that may render this material useful for commercial purposes.

Acknowledgment This work was supported in part by a bilateral project for scientific cooperation between CNR, Italy and CONICET, Argentina.

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