CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY,1071-412X/02/$04.00�0 DOI: 10.1128/CDLI.9.1.149–155.2002

Jan. 2002, p. 149–155 Vol. 9, No. 1

Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Immunobiological Effects of Fumonisin B1 in ExperimentalSubchronic Mycotoxicoses in Rats

M. G. Theumer,1 A. G. Lopez,2 D. T. Masih,1 S. N. Chulze,3 and H. R. Rubinstein1*Micologıa, Departamento de Bioquımica Clınica, Facultad de Ciencias Quımicas, Universidad Nacional de Cordoba,

Ciudad Universitaria, Cordoba,1 Instituto de Ciencia y Tecnologıa de los Alimentos, ICTA, Facultad de CienciasExactas Fısicas y Naturales, Universidad Nacional de Cordoba, Cordoba,2 and Departamento de

Microbiologıa e Inmunologıa, Facultad de Ciencias Exactas Fısico-Quımicas y Naturales,Universidad Nacional de Rıo Cuarto, Ria Cuarto,3 Argentina

Received 20 March 2001/Returned for modification 8 June 2001/Accepted 13 September 2001

Fumonisin B1 (FB1), the principal secondary metabolite produced by the fungus Fusarium verticillioides(Gibberella fujikuroi mating population A), is a potent toxin that can be found in fungus-contaminated corn andcorn-based food products. We have investigated the immunobiological effects of subchronic dietary exposure toFB1 in male Wistar rats. Animals were fed with diets containing 0 (control) or 100 ppm of FB1 for 12 weeks.The total FB1 intake on day 90 was 810 mg/kg of body weight. Food consumption, body weight, and body weightgain on day 90 were reduced in animals exposed to FB1. Histopathologic changes consisted of histiocyticperivascular infiltrate and an increased number of Kupffer cells in the liver, necrosis and apoptosis of tubularepithelial cells in the kidney, and increased mitotic figures and lymphocytic infiltrate in the small intestine.Serum enzyme alkaline phosphatase was significantly elevated in rats fed FB1, while triglyceride levelsdecreased compared to controls. Treatment with FB1 in vivo or in vitro did not have a significant effect onmitogen-induced proliferation of spleen mononuclear cells. However, increased levels of interleukin-4 (IL-4)and decreased levels of IL-10 were released by these cells in culture compared to controls. FB1 in vivo or invitro decreased the hydrogen peroxide (H2O2) released by peritoneal macrophages, while no changes in levelsof superoxide anion produced by total peritoneal cells were detected. The results from the present workdemonstrate that subchronic FB1 intake could affect the small intestine and alter the interleukin profile andsome main functions of macrophages in antitumor activity.

Fumonisins are produced by toxicogenic strains of the genusFusarium and are synthesized mainly in media where there arenitrogen-limited conditions (37). These mycotoxins have achemical structure similar to that of ceramides, and it has beenshown that 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) strainMRC 826, an interest in these toxins has arisen (3).

Diseases induced by mycotoxins cause acute, chronic, andsubchronic toxicities, which depend on different factors such asthe animal species, age, sex, strain, dosage, and administrationroute (18, 41). Fumonisins have been associated with differentkinds of mycotoxicoses in domestic animals, such as leukoen-cephalomalacia in equines (34), pulmonary edema in pigs (10),and hepatocellular carcinoma in rats (15). Animals, as well ashumans, are exposed to mycotoxins through consumption ofcontaminated food in the diet, which can be considered thegateway to cases of natural intoxication by these compounds(17, 19). Contamination with mycotoxins has been detected indifferent countries in most agricultural products, such as cere-als and corn-based food products (16, 25). Of the fumonisins

known, only FB1, FB2, and FB3 produce high levels of con-tamination in naturally contaminated products (16).

During the last few years, different researchers have re-ported infection levels produced by toxicogenic stocks ofFusarium, Aspergillus, and Penicillium in cereals and in foodbased on grains produced in Argentina. In these studies Fusar-ium was found in a high percentage of the analyzed samples.The fumonisin producers F. nygamai (G. fujikuroi mating pop-ulation G) and F. verticillioides were the main species found (9,14), with FB1 being the toxin present in the highest concen-tration (16).

Among the toxins produced by Fusarium, fumonisins, syn-thesized mainly by F. verticillioides and F. proliferatum (G.fujikuroi mating population D), are the most important be-cause of epidemiological evidence that links them to a highincrease of esophageal cancer in humans (33). Marasas et al.have demonstrated a high prevalence of cereals infected by F.verticillioides in African areas where there is a higher incidenceof esophageal cancer compared to those with a low incidenceof 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 fu-monisins are able to modulate the in vivo immune function inbroiler chicks. A decrease of viability of lymphocytes in chick-ens fed an FB1- and FB2-contaminated diet has been reported(12). On the other hand, FB1 and FB2 in vitro are able to

* Corresponding author. Mailing address: Micologıa, Departamentode Bioquımica Clınica, Facultad de Ciencias Quımicas, UniversidadNacional de Cordoba, Ciudad Universitaria (5000), Cordoba, Argen-tina. Phone: 054-351-4334164. Fax: 054-351-4334187. E-mail: hectorru@bioclin.fcq.unc.edu.ar.

149

on February 28, 2020 by guest

http://cvi.asm.org/

Dow

nloaded from

induce NO2 production by rat splenic macrophages and tostimulate T-cell proliferation (11).

Other mycotoxins produced by Fusarium, such as vomitoxin(deoxynavaleriol), are able to overinduce interleukin secretionin CD4� cell cultures, at the same time and in addition to thecell proliferation inhibition (1). Vomitoxin in an experimentalmacrophage model in vitro also appears to interfere with theassociated functions of activated macrophages regulatingH2O2 production, depending on the dosage used (20).

Furthermore, a series of microscopic alterations in targetorgans, such as liver and kidney, has been described. It hasbeen observed that F344 female and male rats that consumedbetween 0 and 484 ppm of FB1 for 28 days showed apoptosisin liver hyperplasia of the bile ducts and apoptosis of tubularepithelial cells of the kidney (38). According to Bondy et al.(4), the kidney was one of the organs most affected by fumo-nisin toxicity in male Sprague-Dawley rats, in which they wereable to observe necrosis of tubular epithelial cells of the innercortex, cytoplasmatic basophilia, and atrophy of tubular epi-thelial cells.

Diets in animals and humans can be contaminated with lowlevels of fumonisins, producing chronic mycotoxicoses that canalter immunologic mechanisms. The experimental models usedto date to study mycotoxin effects on laboratory animals arebased mainly on the production of acute mycotoxicoses; how-ever, the alterations at the immunologic level have been stud-ied in only a few cases.

The main objective of this study was to evaluate the immu-nologic effects caused in rats by an FB1 administration similarto that occurring in nature.

MATERIALS AND METHODS

Animals. Male Wistar inbred rats, 6 to 8 weeks old, were housed in age-matched pairs in stainless-steel cages. The cages were kept in environmentallycontrolled rooms with a 12-h light-dark cycle. Animals were housed and cared forin the animal resource facilities of the Department of Clinical Biochemistry,Faculty of Chemical Sciences, National University of Cordoba, in accordancewith institutional guidelines.

Preparation of fumonisin extracts. FB1 was produced using maize as a sub-stratum layer. Wheat (300 g) was placed in 1,000-ml Erlenmeyer flasks at 35%humidity and sterilized for two consecutive days in an autoclave at 121°C for 15min. A culture of F. verticillioides M 7075 obtained from agar-carnation leaves bymonosporic isolation was used as an inoculum. Incubation was for 28 days in thedark at 25°C, with manual stirring during the first 5 days. Separation and puri-fication of the toxin were performed with the fermented wheat, according to amodification of the methodology of Voss et al. (42).

FB1 quantification. Samples (100 �l) obtained from the extracts were dilutedwith acetonitrile (100 �l). Before the quantification assays, the samples werediluted 1/50 with acetonitrile-water (1:1). The quantification of the diluted ex-tracts was performed by the methodology proposed by Shephard et al. (36).Briefly, an aliquot (50 �l) of this solution was derivatized with 200 �l of o-phthaldialdehyde. This solution was obtained by adding 5 ml of 0.1 M sodiumtetraborate and 50 �l of 2-mercaptoethanol to 1 ml of methanol containing 40mg of o-phthaldialdehyde. The derivatized samples were analyzed with a high-pressure liquid chromatograph (Hewlett-Packard) equipped with a fluorescencedetector. The wavelengths used for excitation and emission were 335 and 440 nm,respectively. An analytical reverse-phase C18 column (150 by 4.6 mm [internaldiameter]; 5-�m particle size) connected to a C18 precolumn (20 by 4.6 mm;5-�m particle size) was used. The mobile phase was methanol–0.1 M NaH2PO4

(75:25), the pH was set at 3.35 � 0.2 with ortho-phosphoric acid, and a flow rateof 1.5 ml/min was used. The quantification of FB1 was carried out by comparingthe peak areas obtained for rats fed FB1 with those corresponding to standardsof 10.5, 5.25, and 2.625 �g of FB1 per ml (Programme on Mycotoxins andExperimental Carcinogenesis, Tygerberg, Republic of South Africa).

Diets. (i) Control diet. The control diet was prepared by adding 435 ml ofaqueous extract of maize without inoculation of F. verticillioides to a solution of

agar (Difco) (15 g) in 435 ml of distilled water. This mixture was warmed untilthe agar dilution was completed and then cooled to 50°C. Next, 1,000 g ofbalanced rat-mouse food (Cargill S.A. C.I., Saladillo, Buenos Aires, Argentina),finely ground and free of mycotoxins, was continuously shaken until a homoge-neous mixture was obtained. Pieces of approximately 20 g each were molded, andafter solidification they were stored at �18°C until they were used. The finalconcentration of FB1 in the food was �0.3 ppm.

(ii) Diet with FB1. The diet with FB1 was prepared as the control diet was,using fumonisin extract as described above. The final FB1 concentration in thefood was 100 ppm.

Experimental model. Two groups of rats were used. One (control) (n � 6) wasfed a control diet, and the other (n � 6) was fed a diet with FB1. Animals werehoused in pairs in different cages and fed for 90 days. The food ration wasreplaced daily, and the weights of food portions given and left uneaten after 24 hwere determined. Animals were weighed on the 30th, 60th, and 90th days ofbeing fed. After this period, blood samples were obtained by intracardiac punc-ture and the animals were killed by cervical dislocation.

Determination of food consumption, body weight, body weight gain, and fu-monisin consumption. The food consumption per day was calculated from thedifference between the weights of the portions given and uneaten. The bodyweight was determined on a scale (Ohaus, Florham Park, N.J.) with a precisionof 0.05 g. The body weight gain of each animal was determined on the 30th, 60th,and 90th days of feeding as the weight difference in comparison to the weight inthe previous month. The total fumonisin consumption on days 30, 60, and 90 forthe group given FB1 was calculated by taking into account the food consumptionand the toxin concentration in the food. The results are expressed in relation tobody weight.

Examination of tissues. Specimens of lungs, spleen, liver, kidney, and smallintestine were obtained on the 90th day of feeding. For examination by lightmicroscopy, tissues were fixed in 10% neutral buffered formalin (pH 7.2). Par-affin sections (4 �m) of tissues were stained with hematoxylin and eosin. Pho-tomicrographs were taken with a Zeiss Axiophot instrument using Kodak Plus-Xpan (PX 135 to 24) film. In the small intestine 10 crypts were examined asrepresentatives of each sample, and the numbers of mitotic cells found in thecrypt bases of control animals and those fed with FB1 were obtained.

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

SMCs. Spleen mononuclear cell (SMC) suspensions were prepared by themethod described by Kisaki et al. (22). Briefly, spleens were removed asepticallyfrom animals, minced, and passed through stainless-steel mesh to obtain single-cell suspensions. The cells were washed with RPMI 1640 medium (Sigma) andresuspended in sterile RPMI 1640 medium supplemented with 10% heat-inac-tivated fetal calf serum (Gibco), gentamicin (50 �g/ml), and �-mercaptoethanol(5 � 10�5 M). Suspensions of spleen cells were prepared aseptically and adjustedto 6 � 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. Resident cells were collected from rats fed with control dietand from rats fed the FB1 diet.

Mitogenic responses of SMCs. Cell suspensions (50 �l; 6 � 106 cells/ml; 3 �105 cells) were dispensed into each well of 96-well culture plates containing 100�l of culture medium (RPMI 1640). Concanavalin A (ConA) (type IV; Sigma)and lipopolysaccharide (LPS) (055:B5; Sigma) were added at optimal final con-centrations of 10 and 40 �g/ml, respectively. The viability of cells was assessed bythe trypan blue (0.1%) exclusion test. For in vitro assays, FB1 was added at anoptimal final concentration of 10 �M. The cultures were incubated with themitogens at 37°C in an atmosphere containing 5% CO2 and were labeled duringthe last 18 h of 96-h cultures with 1 �Ci of [3H]thymidine (Comision Nacional deEnergıa Atomica). These cells were harvested 18 h thereafter on a glass fiberfilter using an automated cell harvester (Skatron; Molecular Devices, Sunnyvale,Calif.). Incorporation of tritiated thymidine into cell DNA was measured intriplicate using a beta liquid scintillation counter.

Cytokine measurement. SMC suspensions were cultured in RPMI 1640 me-dium supplemented with 10% heat-inactivated fetal calf serum (Gibco), genta-micin (50 �g/ml), �-mercaptoethanol (5 � 10�5 M), and ConA (type IV; Sigma),at an optimal final concentration of 10 �g/ml. Supernatants from cultures werecollected after 24 h for determination of interleukin-2 (IL-2) and after 72 h fordetermination of IL-4 and IL-10 and were frozen at �70°C until analyzed.

150 THEUMER ET AL. CLIN. DIAGN. LAB. IMMUNOL.

on February 28, 2020 by guest

http://cvi.asm.org/

Dow

nloaded from

Interleukins were measured using an sandwich enzyme-linked immunosorbentassay protocol (35). Briefly, a purified fraction of anti-IL-2, anti-IL-4, and anti-IL-10 antiserum (PharMingen) was used as capture antibody in conjunction withthe biotinylated anti-rat IL-2, IL-4, and IL-10 monoclonal antibody. Dilutions ofrecombinant rat IL-2, IL-4, and IL-10 were used as standards. After beingwashed four times with phosphate-buffered saline–Tween 20, the plates werereacted with horseradish peroxidase-streptavidin (Sigma) and o-phenylenedi-amine was added. After 5 to 20 min, the reaction was stopped with 25 �l ofsulfuric 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 mi-croassay was used. Briefly, cells (8 � 106/ml) were placed in 96-well plates andleft to stand for 2 h at 37°C in 5% CO2. The medium was then replaced with 250�l of PRS buffer (NaCl [140 mM], dextrose [5.5 mM], phenol red [280 �M], andperoxidase [Sigma] [EC 1.11.1.7] [8.5 U/ml] in phosphate-buffered saline, pH7.0). For the in vitro assays, FB1 was added at an optimal final concentration of10 �M (7.21 �g/ml). Wells were treated with phorbol 12-myristate 13-acetate(PMA) (100 ng/ml) and incubated for 45 min at 37°C in 5% CO2. The reactionwas stopped with 10 �l of 1 N NaOH, and the reactive wells were read in amicroplate reader (Bio-Rad) with a 595-nm filter. Results are expressed asnanomoles of H2O2 released by 106 cells in 30 min.

Detection of O2� released by resident peritoneal cells. Superoxide anion was

quantitatively determined by nitroblue tetrazolium reduction. Peritoneal cells(4 � 106) were incubated in the dark for 30 min at 37°C with 5% CO2 in thepresence of nitroblue tetrazolium (0.1%) with or without PMA at an optimalfinal concentration of 100 ng/ml. The reaction was stopped with 0.4 ml of 0.1 NHCl. Cells were centrifuged, and insoluble formazan was extracted twice with 1ml of 1,4-dioxane. Optical densities in supernatants were determined at 560 nm,and results are expressed as the percentage of optical densities developed incontrol tubes.

Statistical evaluation. Data from these studies were analyzed by one-wayanalysis of variance. Results giving P values of �0.05 were considered signifi-cantly different.

RESULTS

Food consumption, body weight, body weight gain, and fu-monisin consumption. Daily observations for 90 days did notindicate detectable alterations in the general state of any of theanimals. The average food consumptions until the 30th, 60th,and 90th days were 36.8, 40.2, and 41.5 g, respectively, forcontrol group animals, while the average consumptions for ratsfed FB1 were 35.9, 39.8, and 34.3 g, respectively. Significantdifferences in food consumption, body weight, and body weightgain at days 30 and 60 were not observed. On day 90, decreasesin food consumption (P � 0.05), in weight (P � 0.001), and inweight gain (P � 0.001) were detected in the group fed FB1with respect to the control group. The total average FB1 con-sumptions on days 30, 60, and 90 were 319, 544, and 810 mg ofFB1/kg of body weight, respectively.

Examination of tissues. In the histopathologic examinationthe tissues of organs showed little modification in the cellstructures of lungs and esophagus in FB1-fed animals com-pared with controls. On the other hand, in liver samples ofFB1-fed rats, a perivascular histiocytic infiltrate (Fig. 1a) andan increased number of Kupffer cells and changes in the nor-mal structure (Fig. 1c) in comparison with control animals(Fig. 1b and d, respectively) were detected. In the kidneys ofrats fed FB1, apoptotic bodies and necrotic alterations in tu-bular epithelial cells (Fig. 1e), an increase in the capsular space(Fig. 1f), and the presence of proteinogenous material in thetubular lumen (lights) were observed. These kidney alterationswere not detected in control rats. In addition, the microscopicexamination showed an increase in the average number ofmitotic cells in the base of the crypt (Fig. 2a) and a majorlymphocytic infiltrate (Fig. 2b) in the small intestine in rats fedFB1.

Serum biochemical measurements. The data obtained fromthe biochemical profile are shown in Table 1. In the sera ofanimals fed FB1, an increase of ALP activity (P � 0.001) anda decrease of TG levels (P � 0.05) in comparison with controlrats were observed No significant changes in Chol or Ca levelsor in ALT, AST, and GGT activities were observed.

Mitogenic responses of SMCs. To examine the effects pro-duced in the immunologic system by the subchronic FB1 in-toxication, SMCs from control rats and from those fed FB1 inthe basal state and in the presence of ConA and LPS werecultured. No significant differences were observed in [3H]thy-midine uptake by SMCs in the basal state or in the presence ofConA or LPS between 72 and 96 h of culture. In in vitro assays,there were no important changes in the [3H]thymidine uptakewhen SMCs of normal rats were cultured with FB1 at 10 �M(7.21 �g/ml) in comparison to the basal proliferation or whenthey were cultured with FB1 at 10 �M (7.21 �g/ml) plus ConAat 10 �g/ml, in contrast to SMCs cultured with ConA at 10�g/ml (data not shown).

Cytokine measurement. After 72 h of culture, supernatantsof SMCs from animals fed FB1 had significantly higher con-centrations of IL-4 (P � 0.01) and lower concentrations ofIL-10 (P � 0.01) than controls (Fig. 3). There were no alter-ations in IL-2 levels produced by cells from FB1-fed rats incomparison with controls (data not shown). In in vitro assays,there were no changes in the levels of IL-2, IL-4, and IL-10produced by SMCs of normal rats in the presence of FB1 (10�M) with respect to controls.

H2O2 and O2� released by resident peritoneal cells. The

levels of H2O2 produced by adherent peritoneal cells and thelevels of anion superoxide produced by total peritoneal cells inthe 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 levels ofH2O2 (P � 0.01) than controls stimulated with PMA, whilethere were no differences in H2O2 levels produced in the basalstate (Fig. 4A). In in vitro assays, adherent peritoneal cellsfrom normal animals produced significantly lower concentra-tions of H2O2 (P � 0.01) in the presence of FB1 (10 �M) andwhen stimulated by PMA than controls (Fig. 4B). There wereno changes in anion superoxide production in the basal state orin the presence of PMA (data not shown).

DISCUSSION

In this study we have detected immunobiological alterationsproduced by ingestion of FB1 in a model of experimentalsubchronic mycotoxicosis in rats. In this model, the total in-gestion was 303 mg of FB1 during 90 days, producing signifi-cant decreases in food consumption (17.4%), body weight(17.9%), and body weight gain (125.7%). Under similar exper-imental conditions in a murine model, our group observed thatwith a total ingestion of 7.37 mg of FB1 there were also lossesin weight and in weight gain in the animals; however, in con-trast to what happened in rats, the food consumption washigher (5). On the other hand, Voss et al. have reported adecrease in food consumption, body weight, and body weightgain in a model of rats that consumed 228 ppm of FB1 plus 58ppm of FB2 plus 17 ppm of FB3 for a 3-week period (43).These observations suggest that FB1 can modify these param-

VOL. 9, 2002 EFFECTS OF FUMONISINS IN RATS 151

on February 28, 2020 by guest

http://cvi.asm.org/

Dow

nloaded from

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) for90 days. In the livers of animals fed FB1, a perivascular histiocytic infiltrate (a) with respect to control rats (b) and an increased number of Kupffercells (c) compared to those of control rats (d) were the main alterations found. Histological findings in the kidney included apoptosis (arrows) andnecrosis (arrowheads) of tubular epithelial cells (e), and increased capsular space (f) was also found. Magnifications, �200 (a and b), �100 (c, d,and f), and �400 (e).

152 THEUMER ET AL. CLIN. DIAGN. LAB. IMMUNOL.

on February 28, 2020 by guest

http://cvi.asm.org/

Dow

nloaded from

eters in different ways, depending on the animal model and theexperimental scheme 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 increased av-erage number of mitotic cells found by the crypt base in theintestine were present in all samples of animals fed FB1. De-spite the fact that the small intestine is not one of the organsmost affected by this mycotoxin, it is exposed to the same FB1concentrations via oral administration. FB1 is able to havetoxic activity on the intestinal cell by interference in the lipidmetabolism (13), causing alteration of the cellular cycle (29)and increasing the cell number in different phases of mitosis(Fig. 2a). These findings would be related to a major suscep-tibility to infections by pathogens that enter via the oral route(39).

The level of serum TGs is influenced by fats introduced inthe diet and endogenous synthesis in the liver and intestine (2).In this work, a decrease in the TG concentration in animals fedFB1 was observed (Table 1). Bondy et al. (4) have reported asimilar finding in a study of acute toxicity in rats in which thefood consumption and therefore the fats in the diet were di-minished for only a short time. On the other hand, Enongene

et al. (13) have reported alterations in lipid metabolism in theepithelial cells of the small intestine and hepatocytes. Theseresults indicate that FB1 could be a cause of the decreasedlevels of TGs in serum in our model, interfering in the biosyn-thesis of 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 intes-tinal metabolism is stimulated, the intestinal isoenzyme couldrepresent an important factor (21). A similar effect is obtaineddue to cellular alterations in the proximal convoluted tubulesof the kidney, which may contribute to the total serum ALPactivity (21). These results are related to the histopathologicfindings in the kidney (Fig. 1e).

The failure to observe changes in SMC proliferation in an-imals fed FB1 (in the basal state or stimulated), as well as inthe proliferation of normal SMCs exposed in vitro to FB1, isdue to the mycotoxin concentration used. These results arerelated to the observations of Tryphonas et al. (39) that thedaily ingestion of 25 mg of FB1/kg of body weight/day for 14days did not produce changes in the proliferative response ofrat lymphocytes. Charoenpornsook et al. (6), using bovine pe-ripheral blood mononuclear cells, have observed a 50% de-crease of proliferation in the presence of ConA when the cells

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

RatsMean (SEM) level

Chol (mg/dl) TGs (mg/dl) Ca (mg/dl) AST (kata/liter) ALT (kat/liter) GGT (kat/liter) ALP (kat/liter)

Controlb 73.50 (3.22) 157.50 (15.82) 11.37 (0.13) 2.10 (0.19) 0.65 (0.02) 0.09 (0.02) 2.64 (0.13)FB1c 71.67 (6.44) 103.00d (15.62) 11.37 (0.08) 2.33 (0.19) 0.92 (0.11) 0.09 (0.01) 4.53e (0.53)

a One katal catalyzes 1 mol of product/s under defined conditions.b Rats fed control diet for 90 days.c Rats fed diet with FB1 for 90 days.d P � 0.05.e P � 0.001.

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, �400 (a) and �100 (b).

VOL. 9, 2002 EFFECTS OF FUMONISINS IN RATS 153

on February 28, 2020 by guest

http://cvi.asm.org/

Dow

nloaded from

were exposed to 35 �g of FB1 per ml. Taking into account thepharmacokinetic data reported by Martinez-Larranaga et al.(27), in our experimental model the major FB1 concentrationsthat could arise in blood would be 5 to 10 �g/ml. Even if thereare differences among species, higher FB1 concentrations inrats than the one used in this work (7.21 �g/ml) would beneeded to produce alterations in the normal blostomytogenicresponse of lymphocytes.

Little is known about the function of interleukins in a my-cotoxicosis produced by fumonisins. In our work, higher con-centrations of IL-4 and lower concentrations of IL-10 in su-

pernatants of SMCs in rats fed with FB1 were found withrespect to controls (Fig. 3). This increase of IL-4 could bestimulated by the presence of FB1 and/or the accumulation ofsphingoid bases (sphingosine) in the intracellular space bymeans of an unknown mechanism (28). Therefore, the inges-tion of FB1 during a subchronic period could produce a breakin the balance of Th1 and Th2 subsets. In models of chronicFB1 intoxication, the main expression of some interleukinscould be related to the evasion of tumor cells from immuno-logic surveillance (24). Furthermore, it was determined thatamong the functions of IL-10, this cytokine could act as acostimulator for the growth of mature thymocytes. It also func-tions as a cytotoxic-T-cell differentiation factor, promoting ahigher number of IL-2-activated cytotoxic-T-lymphocyte pre-cursors to proliferate and differentiate into cytotoxic effectorcells (7). It has also been suggested that IL-10 is an essentialimmunoregulator of the intestinal tract and that the general-ized bowel inflammation in IL-10-deficient animals is due touncontrolled immune responses stimulated by enteric antigens(23). The decrease of IL-10 found in animals fed FB1 couldcontribute to the alterations observed in the small intestine.On the other hand, the absence of modifications in the IL-2levels in these animals in comparison with controls would berelated to the results obtained on the proliferation of SMCs inthe presence of ConA.

The presence of some Th2 profile cytokines could have alsobeen modulating the macrophage function (32). The hydrogenperoxide and anion superoxide produced by these cells have animportant role in the host defense against tumors and micro-organisms. In our experimental model, peritoneal macro-phages exposed in vivo and in vitro to FB1 produced lesshydrogen peroxide (Fig. 4); however, alterations in the pro-duction of anion superoxide were not found. These resultssuggest that FB1 can have immunosuppressive effects on some

FIG. 3. Interleukins released by SMCs obtained from rats fed acontrol diet or a diet with FB1 (Problem). Cytokine levels in superna-tants of cells cultured for 72 h after recovery from spleens were de-termined by enzyme-linked immunosorbent assay. Error bars indicatestandard errors. �, P � 0.01.

FIG. 4. H2O2 released by adherent peritoneal cells. Macrophages were incubated with or without PMA as a stimulant. Results are expressedas 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 controldiet 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 fournormal rats was used. Cells were incubated with (Problem) or without (Control) 10 �M FB1. �, P � 0.05; ��, P � 0.01.

154 THEUMER ET AL. CLIN. DIAGN. LAB. IMMUNOL.

on February 28, 2020 by guest

http://cvi.asm.org/

Dow

nloaded from

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 tox-icity in rats. Further, in this model the small intestine is clearlyaffected. With the doses used, FB1 is able to produce a mod-ification of the excretion of interleukins, acting on macrophagefunction. A more extensive study on the accumulation of sphin-goid bases in the immune cell system and its functionalitywould be able to clarify the mechanisms acting in the patho-genesis of this intoxication.

ACKNOWLEDGMENT

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

REFERENCES

1. Azcona-Olivera, J. I., Y-L. Ouyang, R. L. Warner, J. E. Linz, and J. J. Pestka.1995. Effects of vomitoxin (deoxynivalenol) and cycloheximide on IL-2, 4, 5and 6 secretion and RNA levels in murine CD4� cells. Food Chem. Toxicol.33:433–441.

2. Bachorick, P. S., R. I. Levy, and B. M. Rifkind. 1993. Lıpidos y dislipopro-teinemias, p. 195–221. In J. B. Henry (ed.), Diagnostico y tratamiento clıni-cos por el laboratorio, 19th ed. W. B. Saunders Co., Philadelphia, Pa.

3. Blackwell, B. A., O. E. Edwards, A. Fruchier, J. W. ApSimon, and J. D.Miller. 1996. NMR structural studies of fumonisin B1 and related com-pounds from Fusarium moniliforme. Adv. Exp. Med. Biol. 392:75–91.

4. Bondy, G., M. Barker, R. Mueller, S. Fernie, J. D. Miller, C. Armstrong, S. L.Hierlihy, P. Rowsell, and C. Suzuki. 1996. Fumonisin B1 toxicity in maleSprague-Dawley rats. Adv. Exp. Med. Biol. 392:251–264.

5. Casado, J. M., M. Theumer, D. T. Masih, S. Chulze, and H. R. Rubinstein.2000. Experimental subchronic mycotoxicoses in mice. Individual and com-bined effects of dietary exposure to fumonisins and aflatoxin B1. Food Chem.Toxicol. 39:579–586.

6. Charoenpornsook, K., J. L. Fitzpatrick, and J. E. Smith. 1998. The effects offour mycotoxins on the mitogen stimulated proliferation of bovine periph-eral blood mononuclear cells in vitro. Mycopathologia 143:105–111.

7. Chen, W. F., and A. Zlotnik. 1991. IL-10: a novel cytotoxic T cell differen-tiation factor. J. Immunol. 147:528–534.

8. Choi, C. Y., H. Nakajima-Adachi, S. Kaminogawa, and Y. Sugita-Konishi.2000. Nivalenol inhibits total and antigen-specific IgE production in mice.Toxicol. Appl. Pharmacol. 165:94–98.

9. Chulze, S. N., M. L. Ramirez, A. Torres, and J. F. Leslie. 2000. Geneticvariation in Fusarium section Liseola from no-till maize in Argentina. Appl.Environ. Microbiol. 66:5312–5315.

10. Colvin, B. M., and L. R. Harrison. 1992. Fumonisin-induced pulmonaryedema and hydrothorax in swine. Mycopathologia 117:79–82.

11. Dombrink-Kurtzman, M. A., R. Gomez-Flores, and R. J. Weber. 2000. Ac-tivation of rat splenic macrophage and lymphocyte functions by fumonisinB(1). Immunopharmacology 49:401–409.

12. Dombrink-Kurtzman, M. A., T. Javed, G. A. Bennet, J. L. Richard, L. M.Cote, and W. B. Buck. 1993. Lymphocyte cytotoxicity and erythrocytic ab-normalities induced in broiler chicks by fumonisin B1 and B2 and monili-formin from Fusarium proliferatum. Mycopathologia. 124:47–54.

13. Enongene, E. N., R. P. Sharma, N. Bhandari, K. A. Voss, and R. T. Riley.2000. Disruption of sphingolipid metabolism in small intestines, liver andkidney of mice dosed subcutaneously with fumonisin B(1). Food Chem.Toxicol. 38:793–799.

14. Etcheverry, M., A. Nesci, G. Barros, and S. Chulze. 1999. Occurrence ofAspergillus section flavi and aflatoxin B1 in corn genotypes and corn meal inArgentina. Mycopathologia 147:37–41.

15. Gelderblom, W. C., M. E. Cawood, S. D. Snyman, and W. F. Marasas. 1994.Fumonisin B1 dosimetry in relation to cancer initiation in rat liver. Carci-nogenesis 15:209–214.

16. Gonzales, H. H., E. J. Martinez, A. M. Pacin, S. L. Pacin, S. L. Resnik, andE. W. Sydenham. 1999. Natural co-occurrence of fumonisins, deoxynivalenol,zearalenone and aflatoxins in field trial corn in Argentina. Food Addit.Contam. 16:565–569.

17. Gutema, T., C. Munimbazi, and L. B. Bullerman. 2000. Occurence of fumo-nisins and moniliformin in corn and corn-based food products of U.S. origin.J. Food Prot. 63:1732–1737.

18. Hengstler, J. G., B. Van de Burg, P. Steinberg, and F. Oesch. 1999. Inter-species differences in cancer susceptibility and toxicity. Drug Metab. Rev.31:917–970.

19. Hennigen, M. R., S. Sanchez, N. M. Di Benedetto, A. Longhi, J. E. Torroba,

and L. M. Valente Soares. 2000. Fumonisin levels in commercial corn prod-ucts in Buenos Aires, Argentina. Food Addit. Contamin. 17:55–58.

20. Ji, G. E., S. Y. Park, S. S. Wong, and J. J. Pestka. 1998. Modulation of nitricoxide, hydrogen peroxide and cytokine production in a clonal macrophagemodel by the tricothecene vomitoxin (deoxynivalenol). Toxicology 125:203–214.

21. Kaplan, M. M. 1993. Laboratory test, p. 108–144. In L. Schiff and E. R. Schiff(ed.), Diseases of the liver, 7th ed. Lippincott, Philadelphia, Pa.

22. Kisaki, T., S. Kobayashi, K. Ogasawra, and N. K. Day. 1991. Immunesuppression induced by protoscoleces of E. multilocularis in mice. Evidencefor the presence of CD8dull suppressor cells in spleens of mice intraperito-neally infected with E. multilocularis. J. Immunol. 147:1659–1663.

23. Kuhn, R., J. Lohler, D. Rennick, K. Rajewsky, and W. Muller. 1993. Inter-leukin-10-deficient mice develop chronic enterocolitis. Cell 75:263–274.

24. Liu, J., Z. Tian, and R. Sun. 1998. The predominant expression of Th2 typecytokines in human tumor cells. Chung. Hua. Chung. Liu. Tsa. Chih. 20:105–107.

25. Machinski, M., Jr., and L. M. Soares. 2000. Fumonisins B1 and B2 inBrazilian corn-based food products. Food Addit. Contam. 17:875–879.

26. Marasas, W. F., K. Jaskiewicz, F. S. Venter, and D. J. Schalkwyk. 1988.Fusarium moniliforme contamination of maize in oesophageal cancer areasin Transkei. S. Afr. Med. J. 74:110–114.

27. Martinez-Larranaga, M. R., A. Anadon, M. J. Diaz, M. L. Fernandez Cruz,M. A. Martinez, M. T. Frejo, M. Martinez, R. Fernandez, R. M. Anton, M. E.Morales, and M. Tafur. 1999. Toxicokinetics and oral bioavailability offumonisin B1. Vet. Hum. Toxicol. 41:357–362.

28. Martinova, E. A. 1997. Influence of sphingolipids on T lymphocyte activa-tion. Biochemistry (Moscow). 63:102–110.

29. Merrill, A. H., E. M. Schmelz, D. L. Dillehay, S. Spiegel, J. A. Shayman, J. J.Schroeder, R. T. Riley, K. A. Voss, and E. Wang. 1997. Sphingolipids—theenigmatic lipid class: biochemistry, physiology, and pathophysiology. Toxicol.Appl. Pharmacol. 142:208–225.

30. Merril, A. H., E. Wang, T. R. Vales, E. R. Smith, J. J. Schroeder, D. S.Menaldino, C. Alexander, H. M. Crane, J. Xia, D. C. Liotta, F. I. Meredith,and R. T. Riley. 1996. Fumonisin toxicity and sphingolipid biosynthesis. Adv.Exp. Med. Biol. 392:297–306.

31. Moon, E. Y., D. K. Rhee, and S. Pyo. 1999. Inhibition of various functions inmurine peritoneal macrophages by aflatoxin B1 exposure in vivo. Int. J. Im-munopharmacol. 21:47–58.

32. Nemoto, Y. T. Otsuka, H. Niiro, K. Izuhara, K. Yamahoka, H. Nakashima,and Y. Niho. 1999. Differential effects of interleukin-4 and interleukin-10 onnitric oxide production by murine macrophages. Inflamm. Res. 48:643–650.

33. Pitt, J. I. 2000. Toxigenic fungi and mycotoxins. Br. Med. Bull. 56:184–192.34. Ross, P. F., A. E. Ledet, D. L. Owens, L. G. Rice, H. A. Nelson, G. D.

Osweiler, and T. M. Wilson. 1993. Experimental equine leukoencephaloma-lacia, toxic hepatosis, and encephalopathy caused by corn naturally contam-inated with fumonisins. J. Vet. Diagn. Investig. 5:69–74.

35. Sabder, B., I. Hoiden, U. Andersson, E. Moller, and J. S. Abrams. 1993.Similar frequencies and kinetics of cytokine producing cells in murine pe-ripheral blood and spleen. J. Immunol. Methods 166:201–214.

36. Shephard, G. S., E. W. Sydenham, P. G. Thiel, and W. C. A. Gelderblom.1990. Quantitative determination of fumonisins B1 and B2 by high-perfor-mance liquid chromatography with fluorescence detection. J. Liquid Chro-matogr. 13:2077–2087.

37. Shim, W. B., and C. P. Woloshuk. 1999. Nitrogen repression of fumonisin B1biosynthesis in Gibberella fujikuroi. FEMS Microbiol. Lett. 177:109–116.

38. Tolleson, W. H., K. L. Dooley, W. G. Sheldon, J. D. Thurman, T. J. Bucci,and P. C. Howard. 1996. The mycotoxin fumonisin induces apoptosis incultured human cells and in livers and kidneys of rats. Adv. Exp. Med. Biol.392:237–250.

39. Tryphonas, H., G. Bondy, J. D. Miller, F, Lacroix, M. Hodgen, P. McGuire,S. Fernie, D. Miller, and S. Hayward. 1997. Effects of fumonisin B1 on theimmune system of Sprague-Dawley rats following a 14-day oral (gavage)exposure. Fundam. Appl. Toxicol. 39:53–59.

40. van der Westhuizen, L., G. S. Shephard, S. D. Snyman, S. Abel, S. Swan-evelder, and W. C. Gelderblom. 1998. Inhibition of sphingolipid biosynthesisin rat primary hepatocyte cultures by fumonisin B1 and other structurallyrelated compounds. Food Chem. Toxicol. 36:497–503.

41. Voss, K. A., R. T. Riley, C. W. Bacon, W. J. Chamberlain, and W. P. Norred.1996. Subchronic toxic effects of Fusarium moniliforme and fumonisin B1 inrats and mice. Nat. Toxins 4:16–23.

42. Voss, K. A., R. D. Plattner, C. W. Bacon, and W. P. Norred. 1990. Compar-ative studies of hepatotoxicity and fumonisin B1 and B2 content of water andchloroform/methanol extracts of Fusarium moniliforme strain MRC 826culture material. Mycopathologia 112:81–92.

43. Voss, K. A., R. D. Plattner, R. T. Riley, F. I. Meredith, and W. P. Norred.1998. In vivo effects of fumonisin B1 nonproducing Fusarium moniliformeisolates are similar: fumonisins B2 and B3 cause hepato- and nephrotoxicityin rats. Mycopathologia 141:45–58.

44. Yamada, A., T. Kataoka, and K. Nagai. 2000. The fungal metabolite glio-toxin: immunosuppressive activity on CTL-mediated cytotoxicity. Immunol.Lett. 71:27–32.

VOL. 9, 2002 EFFECTS OF FUMONISINS IN RATS 155

on February 28, 2020 by guest

http://cvi.asm.org/

Dow

nloaded from