Toxicological effects in rats chronically fed low dietary levels of fumonisin B(1)

The toxicity of low dietary levels of fumonisin B(1) (FB(1)), i.e. 1, 10 and 25 mg FB(1)/kg diet, were monitored in rats over a period of 24 months. No effects on the body weight gain and feed intake profiles were noticed, while the relative liver weight was significantly (P<0.05) reduced in the FB(1)-treated rats. Mild toxic effects, including single cell necrosis (apoptosis), proliferation of bile duct epithelial cells (DEC), and early signs of fibrosis, bile duct hyperplasia and in one case, adenofibrosis, were noticed in the liver of the rats fed the highest (25 mg/FB(1)/kg diet) dietary level. A significant (P<0.05) increase in the level of oxidative damage was also noticed in the liver of the rats of high dosage dietary group. The toxic effects were less severe in the 10 mg FB(1)/kg dietary group, whilst only a few ground glass foci were observed in the 1 mg FB(1)/kg dietary group. Hepatocyte nodules, staining positively for glutathione-S-transferase (placental form, PGST), were observed macroscopically in the 25 mg FB(1)/kg treated group and to a lesser extent in the 10 mg FB(1)/kg treated rats. The most prominent toxic lesions by FB(1) (10 and 25 mg FB(1)/kg dietary groups) in the kidneys were restricted to the tubular epithelium manifesting as granular cast, necrosis, apoptosis, calcification and the presence of regenerative foci in the proximal convoluted tubules. The existence of a cytotoxic/proliferative threshold with respect to cancer induction by FB(1) in rat liver became apparent, with a dietary level of <10-mg FB(1)/kg diet as a no effect threshold for the induction of hepatocyte nodules.     

Initial studies on the toxicokinetics of fumonisin B1 in rats.

Fumonisin B1 (FB1), the major compound in the fumonisin group of secondary metabolites of Fusarium moniliforme Sheldon, is associated with some human and animal diseases. After intraperitoneal dosing to rats (7.5 mg/kg), FB1 was rapidly absorbed and reached a maximum concentration in plasma within 20 min after injection. Thereafter, it underwent rapid removal from plasma, displaying a mono-exponential elimination phase that fitted a one-compartment model with a half-life of 18 min. Collection of 24- and 48-hr urine samples indicated that only 16% of the applied dose was eliminated unmetabolized in urine, all within the first 24-hr period following dosing. In contrast to this, a similar dose of FB1 given by gavage resulted in the recovery of only 0.4% of the FB1 in urine.     

The effects of mycotoxins,fumonisin B1 and aflatoxin B1, on primary swine alveolar macrophages

Mycotoxins were fungal metabolites that were widely present in feed and food; some of them were known to associate with human and animal disease. In the present study, the effects of fumonisin B1 (FmB1) and aflatoxin B1 (AFB1) on swine alveolar macrophages (AM) were examined by exposing primary cultures of swine AM to various concentrations of mycotoxins. Incubation of AM with 5 microg/ml of FmB1 for 72 h led to a reduction in the number of viable cells to 65% of the control levels. In the presence of 1.5 microg/ml of AFB1, the viability of AM falls to less than 41% of controls after 24 h exposure. FmB1, but not AFB1, induced the apoptosis of swine AM with evidence of DNA laddering and nuclear fragmentation. However, both FmB1 and AFB1 exposure induced the expression of apoptosis-related heat shock protein 72 (HSP 72) in AM. Swine AM treated with 50 ng/ml of FmB1 and 100 ng/ml of AFB1 for 24 h led to a reduction in phagocytic ability to approximately 55 and 36% of the control levels, respectively. Incubation of AM with FmB1 (2 and 10 microg/ml) for 24 h dramatically decreased the mRNA levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). However, AFB1 treatment did not affect the expression of IL-1beta and TNF-alpha mRNA. The results suggest that both FmB1 and AFB1 are immunotoxic to swine AM but that they exert their toxic effects via different biochemical mechanisms.     

Toxicokinetics and oral bioavailability of fumonisin B1

The kinetics of fumonisin B1 (FB1) after single doses of 10 mg FB1/kg (po) or 2 mg FB1/kg (i.v.) were studied in male Wistar rats. Serial blood samples were obtained after p.o and i.v. administration. Liver and kidney tissue samples were also obtained after p.o administration. Plasma, liver and kidney concentrations of FB1 were determined by a reversed-phase high-performance liquid chromatographic assay using precolumn 0-phthaldialdehyde derivatisation with fluorescence detection. The FB1 plasma profile could be adequately described by a 2-compartment open model. For FB1, the elimination half-life from plasma was 1.03 h after i.v. and 3.15 h after p.o administration. The apparent volume of distribution and volume of distribution at steady state for FB1 were 0.11 and 0.072 L, respectively, after i.v. administration. The total plasma clearance of FB1 was the same for both the p.o and i.v. routes, 0.072 L/h. After the single p.o dose, FB1 was rapidly absorbed with a Tmax of 1.02 h. The maximum plasma concentration of FB1 was 0.18 microgram/mL. The p.o bioavailability of FB1 was 3.5%. The tissue concentration time data for FB1 fit a 1-compartment open model. Considerable concentrations of FB1 were found in the liver and kidney tissues. The elimination half-lives for FB1 were longer for liver (4.07 h) and kidney (7.07 h) than for plasma (3.15 h). Tissue accumulation of FB1 was evidenced by the tissue/plasma area under the concentration-time curve (AUC) ratios; the AUCtissue/AUCplasma for FB1 was 2.03 in liver and 29.89 in kidney.     

Toxic effects induced by mycotoxin fumonisin B1 on equine spermatozoa: Assessment of viability,sperm chromatin structure stability,ROS production and motility

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium species that exerts its toxic effect through interference with the sphingolipid pathway by inhibiting ceramide synthase. A FB1-dependent sperm toxicity was reported in boars. No information on FB1-related reproductive toxicity in stallions, the most sensitive animal species, has been reported. The aim of the present study was to assess the in vitro toxicity of FB1 on fresh and frozen-thawed equine spermatozoa by analyzing sperm viability, chromatin stability (SCSA) and reactive oxygen species (ROS) production by flow cytometry and sperm motility by CASA system. Fumonisin B1 did not affect viability of fresh spermatozoa after 2 h exposure up to 25 μM. Damage on sperm chromatin structure was observed only in one frozen sample after exposure up to 2.5 × 10⁻⁵ μM FB1 without associated increase of ROS. Increase of ROS, at FB1 levels up to 2.5 × 10⁻⁴ μM, was found on another frozen-thawed sperm sample, may be as a consequence of seminal plasma removal. At 7.5 and 15 μM, FB1 induced reduction of total and progressive motility.     

Ustilipids,acylated beta-D-mannopyranosyl D-erythritols from Ustilago maydis and Geotrichum candidum

Ustilago species produce an extracellular oil that shows activity in various pharmaceutical assays. We isolated several complexes of this heterogeneous glycolipid from cultures of Ustilago maydis DSM 11494 and Geotrichum candidum ST 002515, and determined the chemical structures of these new compounds, termed ustilipids, on the basis of NMR experiments, mass spectra, and fatty acid analyses. They all possess a 4-O-beta-D-mannopyranosyl D-erythritol basic framework, the configuration of which was confirmed, after initial solvolysis, by a single-crystal X-ray structure analysis. All the investigated ustilipids and related compounds are similarly constructed: the three hydroxy groups of the erythritol side chain are free in all cases, whereas the hydroxy groups of the mannose residue are for the most part acylated. Medium-chain fatty acids have for the first time been detected as components of glycolipids produced by Ustilaginales. While the 2-hydroxy group of the mannose residue is esterified with a C2-C8 carboxylate side chain, the 3-hydroxy group is in all cases esterified by a longer, C12-C20 fatty acid residue. The oxygen functionalities at the 4 and 6 positions are either acetylated or present as free hydroxy groups. Ustilipids antagonize dopamine D3 receptors in micromolar quantities; other members of this class of compounds have been found to possess an inhibitory action on the neurotensin receptor. The hemolytic activity of ustilipids is low.     

Biochemical and morphological effects of fumonisin B(1) on primary cultures of rat cerebrum

Chronic dietary consumption of the mycotoxin fumonisin B(1) (FB(1)) is associated with leukoencephalomalacia and neuronal degeneration, but identification of the cellular mechanisms underlying this neurotoxicity is difficult due to concurrent adverse systemic changes. For this reason, the present investigation used an in vitro approach to assess the short-term consequences of direct FB(1) (0. 5-75 microM) exposure on astrocytes and oligodendrocytes in primary cultures of rat cerebrum. Beginning at 5 days in vitro, the cultures were exposed to FB(1) at five concentrations (0.5-75 microM), and the cultures were evaluated at 10 and 15 days in vitro. The levels of the sphingolipid-associated constituents sphingosine and sphinganine were determined with a high-performance liquid chromatography. Relative to untreated cultures, exposure to FB(1) diminished the levels of sphingosine at 15 days in vitro, whereas FB(1)-exposed cultures showed significantly increased sphinganine levels and sphinganine/sphingosine ratios. In addition to these changes in sphingolipid constituents, FB(1)-exposed (0.5-75 microM) cultures exhibited a two-fold increase in the number of process-bearing cells by 15 days in vitro. Also, the activity of 2', 3'-cyclic nucleotide 3'-phosphohydrolase, an enzyme associated with myelin and oligodendrocytes, was increased in FB(1)-treated cultures. This study suggests that short-term exposure to FB(1) may modify the proliferation or differentiation of glial cells.     

Sequential dietary exposure to aflatoxin B1 and fumonisin B1 in F344 rats increases liver preneoplastic changes indicative of a synergistic interaction

Dietary co-exposure to aflatoxin B1 (AFB1) and fumonisin B1 (FB1) and their interaction on hepatocellular carcinogenesis is of particular concern in toxicology and public health. In this study we evaluated the liver preneoplastic effects of single and sequential dietary exposure to AFB1 and FB1 in the F344 rat carcinogenesis model. Serum biochemical alterations, liver histopathological changes, and the formation of liver glutathione S transferase positive (GST-P+) foci were the major outcome parameters examined. Compared to the AFB1-only treatment, the FB1-only treatment induced less dysplasia, and more apoptosis and mitoses. Sequential AFB1 and FB1 treatment lead to increased numbers of dysplasia, apoptosis and foci of altered hepatocytes, as compared to either mycotoxin treatment alone. More importantly, sequential exposure to AFB1 and FB1 synergistically increased the numbers of liver GTP-P+ foci by approximately 7.3-and 12.9-fold and increased the mean sizes of GST-P+ foci by 6- and 7.5-fold, respectively, as compared to AFB1- or FB1-only treatment groups. In addition, liver ALT and AST levels were significantly increased after sequential treatment as compared to single treatment groups. The results demonstrate the interactive effect of dietary AFB1 and FB1 in inducing liver GST-P+ foci formation and provide information to model future intervention studies.     

Standard and Fpg-modified comet assay in kidney cells of ochratoxin A- and fumonisin B(1)-treated rats

The effect of ochratoxin A (OTA), fumonisin B(1) (FB(1)), and their combinations on DNA damage was studied using the standard alkaline comet assay and the Fpg-modified comet assay. Rats were orally receiving OTA (5 ng/kg b.w., 0.05 mg/kg b.w., and 0.5mg/kg b.w., respectively) for 15 days, FB(1) (200 ng/kg b.w., 0.05 mg/kg b.w., and 0.5mg/kg b.w., respectively) for 5 days, and the combinations of two lower OTA and FB(1) doses. The tail length, tail intensity, and Olive tail moment (OTM) obtained with the standard comet assay and Fpg-modified comet assay were significantly higher in treated animals than in controls, even at the lowest dose of OTA or FB(1) (p<0.01). The Fpg-modified comet assay showed significantly greater tail length, tail intensity, and OTM in all treated animal than did the standard comet assay (p<0.05), which suggests that oxidative stress is likely to be responsible for DNA damage. DNA damage detected by the standard comet assay at all OTA or FB(1) doses indicates that some other mechanism is also involved. Combined OTA+FB(1) treatment measured either by the standard comet or the Fpg-modified comet assay showed a synergistic increase in the tail intensity and OTM in kidney cells, even at doses that correspond to the daily human exposure in Europe.     

Combined cytotoxic and genotoxic effects of ochratoxin A and fumonisin B1 in human kidney and liver cell models

Food products can be contaminated by several fungi species and each species may produce different mycotoxins, leading to human combined exposure. Although predictions about the joint toxic effects of mycotoxins can be made from their individual toxicities, experimental data is still limited to allow a reliable hazard assessment. Thus, this study aimed to characterize the combined cytotoxic and genotoxic effects of ochratoxin A (OTA) and fumonisin B1 (FB₁) in cell lines representative of their target organs, kidney and liver. Interactions were ascertained using mathematical extensions to the reference models of concentration addition and independent action. Cytotoxicity (MTT assay) data modeling revealed a synergistic pattern for low doses of both FB₁ and OTA shifting to antagonism at higher concentration levels, irrespectively of the reference model applied. Concerning genotoxicity assessment, neither OTA nor FB₁, individually or in combination, induced a prominent increase in DNA damage (comet assay) or oxidative DNA damage (FPG-comet assay). In conclusion, this study revealed a synergistic cytotoxic effect for OTA and FB₁ at low concentration levels. Given that human co-exposure to these two mycotoxins is probable to occur at low doses, these results raise concerns regarding their potential health outcomes that seem to differ from those predicted by an additive model.     

In vitro ruminal disappearance of fumonisin B1 and its effects on in vitro dry matter disappearance.

Fumonisins were produced by inoculating corn with Fusarium moniliforme M-1325 and incubating for 5 w. Fumonisin B1 (FB1) concentration determined by high performance liquid chromatography was 5,087 ppm. Ruminal fluid inoculum obtained from 2 ruminally cannulated steers and tubes containing 0, 50 or 100 ppm of FB1 were incubated in vitro with ruminal fluid and artificial saliva for 72 h in a 39 C oscillating incubator. Supernatant was analyzed for FB1 concentration, and in vitro dry matter disappearance (IVDMD) was calculated in the remaining precipitates. There was minimal degradation of FB1 by ruminal microbes (about 10%) irrespective of concentration of FB1 used when incubated for the 72 h. No differences in IVDMD rates, were found between treatments, and the rates were normal, indicating that microbial efficiency was unaffected by the presence of FB1 at diet concentrations up to 100 ppm.     

Fate of a single dose of the 14C-labelled mycotoxin, fumonisin B1, in rats.

The fate of the mycotoxin, fumonisin B1, (FB1) dosed to rats by i.p. injection and by gavage was traced using 14C-labelled FB1. Twenty-four hours after i.p. injection, 66% of the radioactivity was recovered in faeces, 32% in urine, 1% in liver and trace amounts (less than 1%) in kidney and red blood cells. When dosed by gavage, all (101%) radioactivity was recovered in faeces and trace amounts were found in urine, liver, kidney and red blood cells. The bulk of the radioactivity recovered was unmetabolized FB1.     

Gender-dependent immunosuppression following subacute exposure to fumonisin B1

The effects of fumonisin B1 (FB1), a mycotoxin produced by Fusarium lertcillioides, on the immune system are controversial; FB1 exposure causing immunosuppression in poultry, swine, bovine and rodents species and immunostimulation in rodent species. The current study was conducted to examine the effects of FB1 on the immune system of BALB/c mice and to determine if there is sex specificity. Female and male mice (five per group) received five daily subcutaneous (s.c.) injections of 2.25 mg/kg/day of FB1, on the following day tissues were collected for immunological examinations. FB1 treatment dramatically reduced relative spleen and thymus weights in females, whereas there was no effect on organ weights in males. Exposure to FB1 reduced splenic cellularity and the basal rate of lymphocyte proliferation in females only. In addition, phytohemagglutinin (PHA-P)-induced T-lymphocyte and LPS-induced B-lymphocyte proliferation were reduced in female mice. Splenocytes from female mice exposed to FB1 showed a reduced expression of interleukin-2 mRNA. These changes occurred in the absence of alterations in tumor necrosis factor alpha or interleukin-1beta mRNA expression. Phenotypic analysis indicated that FB treatment caused a relative increase in the T-lymphocyte population in the spleen of female mice only. In contrast, FB1 dramatically reduced the immature CD4+/CD8+ double positive cell population in the thymus of females. No changes were evident in the thymocyte populations of male mice treated with FB1. The results of this study indicate that FB1 is immunosuppressive in mice; the magnitude of FB1-induced immunosuppression is highly dependent on sex, females being more susceptible than males.     

Comparative acute and combinative toxicity of aflatoxin B1 and fumonisin B1 in animals and human cells.

Aflatoxin B(1) (AFB(1)) and fumonisin B(1) (FB(1)) are important food-borne mycotoxins. The co-contamination of food stuffs with these two mycotoxins is well known and has been possibly implicated in the development of human hepatocellular carcinoma in high risk regions around the world. In this study the acute and combinative toxicity of AFB(1) and FB(1) were tested in F-344 rats, mosquitofish (Gambusia affinis), immortalized human hepatoma cells (HepG2) and human bronchial epithelial cells (BEAS-2B). Preliminary experiments were conducted in order to assess the acute toxicity and obtain LD(50), LC(50) and IC(50) values for individual toxins in each model, respectively. This was followed by testing combinations of AFB(1) and FB(1) to obtain LD(50), LC(50) and IC(50) values for the combination in each model. All models demonstrated a significant dose response in relation to toxin treatment. The potency of the mixture was gauged through the determination of the interaction index metric. Results of this study demonstrate that these two toxins interacted to produce alterations in the toxic responses with a strong additive interaction noted in the cases of F344 rats and mosquitofish. It can be gathered that this combination may pose a significant threat to public health and further research needs to be completed addressing alterations in metabolism and detoxification that may influence the toxic manifestations in combination. These results will provide foundational knowledge for future studies on long-term combinative toxic and health effects of these mycotoxins.