Immunobiological Effects of Fumonisin B1 in Experimental Subchronic Mycotoxicoses in Rats

Fumonisin B1 (FB1), the principal secondary metabolite producedby the fungus Fusarium verticillioides (Gibberella fujikuroimating population A), is a potent toxin that can be found infungus-contaminated corn and corn-based food products. We haveinvestigated the immunobiological effects of subchronic dietaryexposure to FB1 in male Wistar rats. Animals were fed with dietscontaining 0 (control) or 100 ppm of FB1 for 12 weeks. The totalFB1 intake on day 90 was 810 mg/kg of body weight. Food consumption,body weight, and body weight gain on day 90 were reduced inanimals exposed to FB1. Histopathologic changes consisted ofhistiocytic perivascular infiltrate and an increased numberof Kupffer cells in the liver, necrosis and apoptosis of tubularepithelial cells in the kidney, and increased mitotic figuresand lymphocytic infiltrate in the small intestine. Serum enzymealkaline phosphatase was significantly elevated in rats fedFB1, while triglyceride levels decreased compared to controls.Treatment with FB1 in vivo or in vitro did not have a significanteffect on mitogen-induced proliferation of spleen mononuclearcells. However, increased levels of interleukin-4 (IL-4) anddecreased levels of IL-10 were released by these cells in culturecompared to controls. FB1 in vivo or in vitro decreased thehydrogen peroxide (H₂O₂) released by peritoneal macrophages,while no changes in levels of superoxide anion produced by totalperitoneal cells were detected. The results from the presentwork demonstrate that subchronic FB1 intake could affect thesmall intestine and alter the interleukin profile and some mainfunctions of macrophages in antitumor activity.

INTRODUCTION

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Introduction

Fumonisins are produced by toxicogenic strains of the genusFusarium and are synthesized mainly in media where there arenitrogen-limited conditions (37). These mycotoxins have a chemicalstructure similar to that of ceramides, and it has been shownthat they interfere in the lipid metabolism of the cell (30,40). After isolation and characterization of fumonisin B1 (FB1)and FB2 from cultures of Fusarium verticillioides (F. monoliforme;Gibberella fujikuroi mating population A) strain MRC 826, aninterest in these toxins has arisen (3).

Diseases induced by mycotoxins cause acute, chronic, and subchronictoxicities, which depend on different factors such as the animalspecies, age, sex, strain, dosage, and administration route(18, 41). Fumonisins have been associated with different kindsof mycotoxicoses in domestic animals, such as leukoencephalomalaciain equines (34), pulmonary edema in pigs (10), and hepatocellularcarcinoma in rats (15). Animals, as well as humans, are exposedto mycotoxins through consumption of contaminated food in thediet, which can be considered the gateway to cases of naturalintoxication by these compounds (17, 19). Contamination withmycotoxins has been detected in different countries in mostagricultural products, such as cereals and corn-based food products(16, 25). Of the fumonisins known, only FB1, FB2, and FB3 producehigh levels of contamination in naturally contaminated products(16).

During the last few years, different researchers have reportedinfection levels produced by toxicogenic stocks of Fusarium,Aspergillus, and Penicillium in cereals and in food based ongrains produced in Argentina. In these studies Fusarium wasfound in a high percentage of the analyzed samples. The fumonisinproducers F. nygamai (G. fujikuroi mating population G) andF. verticillioides were the main species found (9, 14), withFB1 being the toxin present in the highest concentration (16).

Among the toxins produced by Fusarium, fumonisins, synthesizedmainly by F. verticillioides and F. proliferatum (G. fujikuroimating population D), are the most important because of epidemiologicalevidence that links them to a high increase of esophageal cancerin humans (33). Marasas et al. have demonstrated a high prevalenceof cereals infected by F. verticillioides in African areas wherethere is a higher incidence of esophageal cancer compared tothose with a low incidence of the disease (26).

Dietary exposure to various mycotoxins results in decreasesof antibody production, T-lymphocyte proliferative response,cytotoxic action of T lymphocytes, and production of oxygenderivatives by peritoneal cells (8, 31, 44). There is some recentevidence suggesting that FB1 or other structurally related fumonisinsare able to modulate the in vivo immune function in broilerchicks. A decrease of viability of lymphocytes in chickens fedan FB1- and FB2-contaminated diet has been reported (12). Onthe other hand, FB1 and FB2 in vitro are able to induce NO₂production by rat splenic macrophages and to stimulate T-cellproliferation (11).

Other mycotoxins produced by Fusarium, such as vomitoxin (deoxynavaleriol),are able to overinduce interleukin secretion in CD4⁺ cell cultures,at the same time and in addition to the cell proliferation inhibition(1). Vomitoxin in an experimental macrophage model in vitroalso appears to interfere with the associated functions of activatedmacrophages regulating H₂O₂ production, depending on the dosageused (20).

Furthermore, a series of microscopic alterations in target organs,such as liver and kidney, has been described. It has been observedthat F344 female and male rats that consumed between 0 and 484ppm of FB1 for 28 days showed apoptosis in liver hyperplasiaof the bile ducts and apoptosis of tubular epithelial cellsof the kidney (38). According to Bondy et al. (4), the kidneywas one of the organs most affected by fumonisin toxicity inmale Sprague-Dawley rats, in which they were able to observenecrosis of tubular epithelial cells of the inner cortex, cytoplasmaticbasophilia, and atrophy of tubular epithelial cells.

Diets in animals and humans can be contaminated with low levelsof fumonisins, producing chronic mycotoxicoses that can alterimmunologic mechanisms. The experimental models used to dateto study mycotoxin effects on laboratory animals are based mainlyon the production of acute mycotoxicoses; however, the alterationsat the immunologic level have been studied in only a few cases.

The main objective of this study was to evaluate the immunologiceffects caused in rats by an FB1 administration similar to thatoccurring in nature.

MATERIALS AND METHODS

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Materials and Methods

Animals. Male Wistar inbred rats, 6 to 8 weeks old, were housed in age-matchedpairs in stainless-steel cages. The cages were kept in environmentallycontrolled rooms with a 12-h light-dark cycle. Animals werehoused and cared for in the animal resource facilities of theDepartment of Clinical Biochemistry, Faculty of Chemical Sciences,National University of Córdoba, in accordance with institutionalguidelines.

Preparation of fumonisin extracts. FB1 was produced using maize as a substratum layer. Wheat (300g) was placed in 1,000-ml Erlenmeyer flasks at 35% humidityand sterilized for two consecutive days in an autoclave at 121°Cfor 15 min. A culture of F. verticillioides M 7075 obtainedfrom agar-carnation leaves by monosporic isolation was usedas an inoculum. Incubation was for 28 days in the dark at 25°C,with manual stirring during the first 5 days. Separation andpurification of the toxin were performed with the fermentedwheat, according to a modification of the methodology of Vosset al. (42).

FB1 quantification. Samples (100 µl) obtained from the extracts were dilutedwith acetonitrile (100 µl). Before the quantificationassays, the samples were diluted 1/50 with acetonitrile-water(1:1). The quantification of the diluted extracts was performedby the methodology proposed by Shephard et al. (36). Briefly,an aliquot (50 µl) of this solution was derivatized with200 µl of o-phthaldialdehyde. This solution was obtainedby adding 5 ml of 0.1 M sodium tetraborate and 50 µl of2-mercaptoethanol to 1 ml of methanol containing 40 mg of o-phthaldialdehyde.The derivatized samples were analyzed with a high-pressure liquidchromatograph (Hewlett-Packard) equipped with a fluorescencedetector. The wavelengths used for excitation and emission were335 and 440 nm, respectively. An analytical reverse-phase C₁₈column (150 by 4.6 mm [internal diameter]; 5-µm particlesize) connected to a C₁₈ precolumn (20 by 4.6 mm; 5-µmparticle size) was used. The mobile phase was methanol-0.1 MNaH₂PO₄ (75:25), the pH was set at 3.35 ± 0.2 with ortho-phosphoricacid, and a flow rate of 1.5 ml/min was used. The quantificationof FB1 was carried out by comparing the peak areas obtainedfor rats fed FB1 with those corresponding to standards of 10.5,5.25, and 2.625 µg of FB1 per ml (Programme on Mycotoxinsand Experimental Carcinogenesis, Tygerberg, Republic of SouthAfrica).

Diets. (i) Control diet. The control diet was prepared by adding 435 ml of aqueous extractof maize without inoculation of F. verticillioides to a solutionof agar (Difco) (15 g) in 435 ml of distilled water. This mixturewas warmed until the agar dilution was completed and then cooledto 50°C. Next, 1,000 g of balanced rat-mouse food (CargillS.A. C.I., Saladillo, Buenos Aires, Argentina), finely groundand free of mycotoxins, was continuously shaken until a homogeneousmixture was obtained. Pieces of approximately 20 g each weremolded, and after solidification they were stored at -18°Cuntil they were used. The final concentration of FB1 in thefood was <0.3 ppm.

(ii) Diet with FB1. The diet with FB1 was prepared as the control diet was, usingfumonisin extract as described above. The final FB1 concentrationin the food was 100 ppm.

Experimental model. Two groups of rats were used. One (control) (n = 6) was feda control diet, and the other (n = 6) was fed a diet with FB1.Animals were housed in pairs in different cages and fed for90 days. The food ration was replaced daily, and the weightsof food portions given and left uneaten after 24 h were determined.Animals were weighed on the 30th, 60th, and 90th days of beingfed. After this period, blood samples were obtained by intracardiacpuncture and the animals were killed by cervical dislocation.

Determination of food consumption, body weight, body weight gain, and fumonisin consumption. The food consumption per day was calculated from the differencebetween the weights of the portions given and uneaten. The bodyweight was determined on a scale (Ohaus, Florham Park, N.J.)with a precision of 0.05 g. The body weight gain of each animalwas determined on the 30th, 60th, and 90th days of feeding asthe weight difference in comparison to the weight in the previousmonth. The total fumonisin consumption on days 30, 60, and 90for the group given FB1 was calculated by taking into accountthe food consumption and the toxin concentration in the food.The results are expressed in relation to body weight.

Examination of tissues. Specimens of lungs, spleen, liver, kidney, and small intestinewere obtained on the 90th day of feeding. For examination bylight microscopy, tissues were fixed in 10% neutral bufferedformalin (pH 7.2). Paraffin sections (4 µm) of tissueswere stained with hematoxylin and eosin. Photomicrographs weretaken with a Zeiss Axiophot instrument using Kodak Plus-X pan(PX 135 to 24) film. In the small intestine 10 crypts were examinedas representatives of each sample, and the numbers of mitoticcells found in the crypt bases of control animals and thosefed with FB1 were obtained.

Serum biochemical measurements. The levels of total cholesterol (Chol), triglycerides (TGs),and calcium (Ca) and the enzymatic activities of aspartate aminotransferase(AST), alanine aminotransferase (ALT), gamma glutamyltransferase(GGT), and alkaline phosphatase (ALP) in serum obtained fromintracardiac puncture were determined by using a Technicon RA-1000autoanalyzer.

SMCs. Spleen mononuclear cell (SMC) suspensions were prepared by themethod described by Kisaki et al. (22). Briefly, spleens wereremoved aseptically from animals, minced, and passed throughstainless-steel mesh to obtain single-cell suspensions. Thecells were washed with RPMI 1640 medium (Sigma) and resuspendedin sterile RPMI 1640 medium supplemented with 10% heat-inactivatedfetal calf serum (Gibco), gentamicin (50 µg/ml), and ß-mercaptoethanol(5 x 10^-5 M). Suspensions of spleen cells were prepared asepticallyand adjusted to 6 x 10⁶ cells/ml.

Peritoneal cells. Peritoneal cells were obtained by sterile washing with KrebsRinger phosphate dextrose buffer (pH 7.0) containing gentamicin(50 mg/liter) and heparin (20 U/ml). Cells were washed twice,resuspended in culture medium, counted, and diluted. Residentcells were collected from rats fed with control diet and fromrats fed the FB1 diet.

Mitogenic responses of SMCs. Cell suspensions (50 µl; 6 x 10⁶ cells/ml; 3 x 10⁵ cells)were dispensed into each well of 96-well culture plates containing100 µl of culture medium (RPMI 1640). Concanavalin A (ConA)(type IV; Sigma) and lipopolysaccharide (LPS) (055:B5; Sigma)were added at optimal final concentrations of 10 and 40 µg/ml,respectively. The viability of cells was assessed by the trypanblue (0.1%) exclusion test. For in vitro assays, FB1 was addedat an optimal final concentration of 10 µM. The cultureswere incubated with the mitogens at 37°C in an atmospherecontaining 5% CO₂ and were labeled during the last 18 h of 96-hcultures with 1 µCi of [³H]thymidine (ComisiónNacional de Energía Atómica). These cells wereharvested 18 h thereafter on a glass fiber filter using an automatedcell harvester (Skatron; Molecular Devices, Sunnyvale, Calif.).Incorporation of tritiated thymidine into cell DNA was measuredin triplicate using a beta liquid scintillation counter.

Cytokine measurement. SMC suspensions were cultured in RPMI 1640 medium supplementedwith 10% heat-inactivated fetal calf serum (Gibco), gentamicin(50 µg/ml), ß-mercaptoethanol (5 x 10^-5 M),and ConA (type IV; Sigma), at an optimal final concentrationof 10 µg/ml. Supernatants from cultures were collectedafter 24 h for determination of interleukin-2 (IL-2) and after72 h for determination of IL-4 and IL-10 and were frozen at-70°C until analyzed. Interleukins were measured using ansandwich enzyme-linked immunosorbent assay protocol (35). Briefly,a purified fraction of anti-IL-2, anti-IL-4, and anti-IL-10antiserum (PharMingen) was used as capture antibody in conjunctionwith the biotinylated anti-rat IL-2, IL-4, and IL-10 monoclonalantibody. Dilutions of recombinant rat IL-2, IL-4, and IL-10were used as standards. After being washed four times with phosphate-bufferedsaline-Tween 20, the plates were reacted with horseradish peroxidase-streptavidin(Sigma) and o-phenylenediamine was added. After 5 to 20 min,the reaction was stopped with 25 µl of sulfuric acid (1:9,vol/vol). The reactions were read in a microplate reader (Bio-Rad),and results are expressed as nanograms per milliliter.

Detection of H₂O₂ released by adherent cells. The phenol red oxidation microassay was used. Briefly, cells(8 x 10⁶/ml) were placed in 96-well plates and left to standfor 2 h at 37°C in 5% CO₂. The medium was then replacedwith 250 µl of PRS buffer (NaCl [140 mM], dextrose [5.5mM], phenol red [280 µM], and peroxidase [Sigma] [EC 1.11.1.7][8.5 U/ml] in phosphate-buffered saline, pH 7.0). For the invitro assays, FB1 was added at an optimal final concentrationof 10 µM (7.21 µg/ml). Wells were treated with phorbol12-myristate 13-acetate (PMA) (100 ng/ml) and incubated for45 min at 37°C in 5% CO₂. The reaction was stopped with10 µl of 1 N NaOH, and the reactive wells were read ina microplate reader (Bio-Rad) with a 595-nm filter. Resultsare expressed as nanomoles of H₂O₂ released by 10⁶ cells in30 min.

Detection of O₂^- released by resident peritoneal cells. Superoxide anion was quantitatively determined by nitrobluetetrazolium reduction. Peritoneal cells (4 x 10⁶) were incubatedin the dark for 30 min at 37°C with 5% CO₂ in the presenceof nitroblue tetrazolium (0.1%) with or without PMA at an optimalfinal concentration of 100 ng/ml. The reaction was stopped with0.4 ml of 0.1 N HCl. Cells were centrifuged, and insoluble formazanwas extracted twice with 1 ml of 1,4-dioxane. Optical densitiesin supernatants were determined at 560 nm, and results are expressedas the percentage of optical densities developed in controltubes.

Statistical evaluation. Data from these studies were analyzed by one-way analysis ofvariance. Results giving P values of $<=$ 0.05 were considered significantlydifferent.

RESULTS

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Results

Food consumption, body weight, body weight gain, and fumonisin consumption. Daily observations for 90 days did not indicate detectable alterationsin the general state of any of the animals. The average foodconsumptions until the 30th, 60th, and 90th days were 36.8,40.2, and 41.5 g, respectively, for control group animals, whilethe average consumptions for rats fed FB1 were 35.9, 39.8, and34.3 g, respectively. Significant differences in food consumption,body weight, and body weight gain at days 30 and 60 were notobserved. On day 90, decreases in food consumption (P $<=$ 0.05),in weight (P $<=$ 0.001), and in weight gain (P $<=$ 0.001) were detectedin the group fed FB1 with respect to the control group. Thetotal average FB1 consumptions on days 30, 60, and 90 were 319,544, and 810 mg of FB1/kg of body weight, respectively.

Examination of tissues. In the histopathologic examination the tissues of organs showedlittle modification in the cell structures of lungs and esophagusin FB1-fed animals compared with controls. On the other hand,in liver samples of FB1-fed rats, a perivascular histiocyticinfiltrate (Fig. 1a) and an increased number of Kupffer cellsand changes in the normal structure (Fig. 1c) in comparisonwith control animals (Fig. 1b and d, respectively) were detected.In the kidneys of rats fed FB1, apoptotic bodies and necroticalterations in tubular epithelial cells (Fig. 1e), an increasein the capsular space (Fig. 1f), and the presence of proteinogenousmaterial in the tubular lumen (lights) were observed. Thesekidney alterations were not detected in control rats. In addition,the microscopic examination showed an increase in the averagenumber of mitotic cells in the base of the crypt (Fig. 2a) anda major lymphocytic infiltrate (Fig. 2b) in the small intestinein rats fed FB1.

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FIG. 1. Hematoxylin-and-eosin-stained sections of rat organs exposed to a diet containing 100 ppm of FB1 or a diet without FB1 (control) for 90 days. In the livers of animals fed FB1, a perivascular histiocytic infiltrate (a) with respect to control rats (b) and an increased number of Kupffer cells (c) compared to those of control rats (d) were the main alterations found. Histological findings in the kidney included apoptosis (arrows) and necrosis (arrowheads) of tubular epithelial cells (e), and increased capsular space (f) was also found. Magnifications, x200 (a and b), x100 (c, d, and f), and x400 (e).

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FIG. 2. Hematoxylin-and-eosin-stained sections of rat organs exposed to a diet containing 100 ppm of FB1 for 90 days. In the small intestine, an increased number of mitotic cells (arrows) (a) and lymphocytic infiltrate (b) were present. Magnification, x400 (a) and x100 (b).

Serum biochemical measurements. The data obtained from the biochemical profile are shown inTable 1. In the sera of animals fed FB1, an increase of ALPactivity (P $<=$ 0.001) and a decrease of TG levels (P $<=$ 0.05) incomparison with control rats were observed No significant changesin Chol or Ca levels or in ALT, AST, and GGT activities wereobserved.

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TABLE 1. Serum parameters for rats (n = 6) on day 90

Mitogenic responses of SMCs. To examine the effects produced in the immunologic system bythe subchronic FB1 intoxication, SMCs from control rats andfrom those fed FB1 in the basal state and in the presence ofConA and LPS were cultured. No significant differences wereobserved in [³H]thymidine uptake by SMCs in the basal stateor in the presence of ConA or LPS between 72 and 96 h of culture.In in vitro assays, there were no important changes in the [³H]thymidineuptake when SMCs of normal rats were cultured with FB1 at 10µM (7.21 µg/ml) in comparison to the basal proliferationor when they were cultured with FB1 at 10 µM (7.21 µg/ml)plus ConA at 10 µg/ml, in contrast to SMCs cultured withConA at 10 µg/ml (data not shown).

Cytokine measurement. After 72 h of culture, supernatants of SMCs from animals fedFB1 had significantly higher concentrations of IL-4 (P $<=$ 0.01)and lower concentrations of IL-10 (P $<=$ 0.01) than controls (Fig.3). There were no alterations in IL-2 levels produced by cellsfrom FB1-fed rats in comparison with controls (data not shown).In in vitro assays, there were no changes in the levels of IL-2,IL-4, and IL-10 produced by SMCs of normal rats in the presenceof FB1 (10 µM) with respect to controls.

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FIG. 3. Interleukins released by SMCs obtained from rats fed a control diet or a diet with FB1 (Problem). Cytokine levels in supernatants of cells cultured for 72 h after recovery from spleens were determined by enzyme-linked immunosorbent assay. Error bars indicate standard errors. *, P $<=$ 0.01.

H₂O₂ and O₂^- released by resident peritoneal cells. The levels of H₂O₂ produced by adherent peritoneal cells andthe levels of anion superoxide produced by total peritonealcells in the basal state and in the presence of PMA were quantified.The levels of H₂O₂ found are shown in Fig. 4. The peritonealcells of animals fed FB1 produced significantly lower levelsof H₂O₂ (P $<=$ 0.01) than controls stimulated with PMA, while therewere no differences in H₂O₂ levels produced in the basal state(Fig. 4A). In in vitro assays, adherent peritoneal cells fromnormal animals produced significantly lower concentrations ofH₂O₂ (P $<=$ 0.01) in the presence of FB1 (10 µM) and whenstimulated by PMA than controls (Fig. 4B). There were no changesin anion superoxide production in the basal state or in thepresence of PMA (data not shown).

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FIG. 4. H₂O₂ released by adherent peritoneal cells. Macrophages were incubated with or without PMA as a stimulant. Results are expressed as mean (standard error) nanomoles of H₂O₂ released by 10⁶ cells in 30 min. (A) In vivo exposure to FB1 (n = 6 rats). Control, rats fed control diet for 90 days; Problem, rats fed diet with FB1 for 90 days. (B) In vitro exposure to FB1. For in vitro assays a pool of peritoneal cells from four normal rats was used. Cells were incubated with (Problem) or without (Control) 10 µM FB1. *, P $<=$ 0.05; **, P $<=$ 0.01.

DISCUSSION

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Discussion

In this study we have detected immunobiological alterationsproduced by ingestion of FB1 in a model of experimental subchronicmycotoxicosis in rats. In this model, the total ingestion was303 mg of FB1 during 90 days, producing significant decreasesin food consumption (17.4%), body weight (17.9%), and body weightgain (125.7%). Under similar experimental conditions in a murinemodel, our group observed that with a total ingestion of 7.37mg of FB1 there were also losses in weight and in weight gainin the animals; however, in contrast to what happened in rats,the food consumption was higher (5). On the other hand, Vosset al. have reported a decrease in food consumption, body weight,and body weight gain in a model of rats that consumed 228 ppmof FB1 plus 58 ppm of FB2 plus 17 ppm of FB3 for a 3-week period(43). These observations suggest that FB1 can modify these parametersin different ways, depending on the animal model and the experimentalscheme used.

In histopathologic examination alterations similar to thosedescribed by other authors were found, indicating that the liverand kidney are the principal target organs for FB1 action inrats. Furthermore, the lymphocytic infiltrate and increasedaverage number of mitotic cells found by the crypt base in theintestine were present in all samples of animals fed FB1. Despitethe fact that the small intestine is not one of the organs mostaffected by this mycotoxin, it is exposed to the same FB1 concentrationsvia oral administration. FB1 is able to have toxic activityon the intestinal cell by interference in the lipid metabolism(13), causing alteration of the cellular cycle (29) and increasingthe cell number in different phases of mitosis (Fig. 2a). Thesefindings would be related to a major susceptibility to infectionsby pathogens that enter via the oral route (39).

The level of serum TGs is influenced by fats introduced in thediet and endogenous synthesis in the liver and intestine (2).In this work, a decrease in the TG concentration in animalsfed FB1 was observed (Table 1). Bondy et al. (4) have reporteda similar finding in a study of acute toxicity in rats in whichthe food consumption and therefore the fats in the diet werediminished for only a short time. On the other hand, Enongeneet al. (13) have reported alterations in lipid metabolism inthe epithelial cells of the small intestine and hepatocytes.These results indicate that FB1 could be a cause of the decreasedlevels of TGs in serum in our model, interfering in the biosynthesisof endogenous TGs.

Among the parameters studied in the biochemical profile, anincrease of ALP activity was found. Although the liver is themajor source of this enzyme, in some cases in which the intestinalmetabolism is stimulated, the intestinal isoenzyme could representan important factor (21). A similar effect is obtained due tocellular alterations in the proximal convoluted tubules of thekidney, which may contribute to the total serum ALP activity(21). These results are related to the histopathologic findingsin the kidney (Fig. 1e).

The failure to observe changes in SMC proliferation in animalsfed FB1 (in the basal state or stimulated), as well as in theproliferation of normal SMCs exposed in vitro to FB1, is dueto the mycotoxin concentration used. These results are relatedto the observations of Tryphonas et al. (39) that the dailyingestion of 25 mg of FB1/kg of body weight/day for 14 daysdid not produce changes in the proliferative response of ratlymphocytes. Charoenpornsook et al. (6), using bovine peripheralblood mononuclear cells, have observed a 50% decrease of proliferationin the presence of ConA when the cells were exposed to 35 µgof FB1 per ml. Taking into account the pharmacokinetic datareported by Martinez-Larranaga et al. (27), in our experimentalmodel the major FB1 concentrations that could arise in bloodwould be 5 to 10 µg/ml. Even if there are differencesamong species, higher FB1 concentrations in rats than the oneused in this work (7.21 µg/ml) would be needed to producealterations in the normal blostomytogenic response of lymphocytes.

Little is known about the function of interleukins in a mycotoxicosisproduced by fumonisins. In our work, higher concentrations ofIL-4 and lower concentrations of IL-10 in supernatants of SMCsin rats fed with FB1 were found with respect to controls (Fig.3). This increase of IL-4 could be stimulated by the presenceof FB1 and/or the accumulation of sphingoid bases (sphingosine)in the intracellular space by means of an unknown mechanism(28). Therefore, the ingestion of FB1 during a subchronic periodcould produce a break in the balance of Th1 and Th2 subsets.In models of chronic FB1 intoxication, the main expression ofsome interleukins could be related to the evasion of tumor cellsfrom immunologic surveillance (24). Furthermore, it was determinedthat among the functions of IL-10, this cytokine could act asa costimulator for the growth of mature thymocytes. It alsofunctions as a cytotoxic-T-cell differentiation factor, promotinga higher number of IL-2-activated cytotoxic-T-lymphocyte precursorsto proliferate and differentiate into cytotoxic effector cells(7). It has also been suggested that IL-10 is an essential immunoregulatorof the intestinal tract and that the generalized bowel inflammationin IL-10-deficient animals is due to uncontrolled immune responsesstimulated by enteric antigens (23). The decrease of IL-10 foundin animals fed FB1 could contribute to the alterations observedin the small intestine. On the other hand, the absence of modificationsin the IL-2 levels in these animals in comparison with controlswould be related to the results obtained on the proliferationof SMCs in the presence of ConA.

The presence of some Th2 profile cytokines could have also beenmodulating the macrophage function (32). The hydrogen peroxideand anion superoxide produced by these cells have an importantrole in the host defense against tumors and microorganisms.In our experimental model, peritoneal macrophages exposed invivo and in vitro to FB1 produced less hydrogen peroxide (Fig.4); however, alterations in the production of anion superoxidewere not found. These results suggest that FB1 can have immunosuppressiveeffects on some of the macrophage immunologic mechanisms, diminishingtheir cytotoxic capacity, which would be related to a lowerantitumor activity.

The results obtained in this work indicated that FB1 has theliver and kidney as principal target organs for subchronic toxicityin rats. Further, in this model the small intestine is clearlyaffected. With the doses used, FB1 is able to produce a modificationof the excretion of interleukins, acting on macrophage function.A more extensive study on the accumulation of sphingoid basesin the immune cell system and its functionality would be ableto clarify the mechanisms acting in the pathogenesis of thisintoxication.

ACKNOWLEDGMENTS

This work was supported by Agencia Nacional de Ciencia y Tecnologíagrant FONCYT-PICT 09-03688.

FOOTNOTES

* Corresponding author. Mailing address: Micología, Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria (5000), Córdoba, Argentina. Phone: 054-351-4334164. Fax: 054-351-4334187. E-mail: hectorru{at}bioclin.fcq.unc.edu.ar.

REFERENCES

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