Toxic effects of Ustilago maydis and fumonisin B1 in rats

The toxicity of Ustilago maydis and the possible synergism with fumonisin B1 (FB1) were studied in Fischer rats by evaluating pathological changes and biochemical parameters in blood serum (LDH, ALT, GGT, ChE) and tissue homogenate of brain and liver (AChE, ChE, GGT, ALP). One experimental group (US) consumed diet with 70% of U. maydis galls and the other group (US+FB1) was fed pellets containing 70% of U. maydis galls and 1 mg of FB1 per kg of diet for 17 days. Control group (C) consumed standard pellets. During the trial, experimental animals were more excited, showing hyperactivity. Body mass gains slightly increased in both groups compared to the control. Gross pathological changes in liver, lungs, uterus and ovaries were more pronounced in the US+FB1 than in the US group. Specific catalytic activities of AChE decreased by 61% and by 63% in the liver and brain homogenate of the US group (p<0.05) compared to the control, indicating neurotoxic activity of U. maydis. Also, specific catalytic concentration of AChE and ALP was significantly decreased in the liver of the US+FB(1) group (p<0.05). Activity of LDH in the blood serum was increased up to 166% and 165% in the US+FB1 group (p<0.05) compared to the control and US group values, respectively, which indicates that FB1 was responsible for the disruption of cell membrane integrity. These findings suggest that Ustilago maydis and FB1 showed neurotoxicity in Fischer rats, which could be related to the alkaloids of U. maydis and disruption of sphingolipid metabolism by FB1 activity.     

Efficacy of a commercial mycotoxin binder in alleviating effects of ochratoxin A, fumonisin B1, moniliformin and zearalenone in adult mink

The addition of nutritionally inert adsorbents to mycotoxin-contaminated animal feed has been a popular approach to decreasing toxicity in animals and carryover of mycotoxins from contaminated feed to animal by-products. Some studies suggest that esterified glucomannan derived from the cell wall of Saccharomyces cerevisiae is effective in reducing the bioavailability of at least some of the mycotoxins occurring in contaminated feed. Because cereal grains are important components of ranch mink diets, mycotoxicoses in mink is a potential problem faced by mink ranchers. We conducted a series of studies to determine if inclusion of a commercially available esterified glucomannan in ranch mink feed was effective in alleviating clinical signs indicative of exposure to ochratoxin A, fumonisin B1, moniliformin or zearalenone in adult mink. In 4 separate trials, mink were fed diets that contained 2.5, 5 or 10 mg ochratoxin A/kg feed, 200 mg fumonisin B1/kg feed, 20 mg moniliformin/kg feed, or 30 mg zearalenone/kg feed with or without 2 g esterified glucomannan/kg feed. Male mink fed diets containing ochratoxin A had significantly decreased feed intake as well as renal lesions characteristic of exposure to that mycotoxin. Inclusion of the esterified glucomannan did not ameliorate these effects. Male mink exposed to fumonisin B1 had increased urinary sphinganine concentration, which was not significantly reduced by the mycotoxin adsorbent. Male mink that consumed monilformin-contaminated diets had characteristic ultrastructural changes in the heart that were not reduced in severity by the esterified glucomannan. Female mink exposed to zearalenone had increased uterine weight, which was not reversed by inclusion of commercial mycotoxin binder in the contaminated feed. The results of this study suggest that a commercial esterified glucomannan was generally ineffective in alleviating effects indicative of exposure to ochratoxin A, fumonisin B1, monilformin and zearalenone in mink.     

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.     

Effect of fumonisin B1 on structure and function of macrophage plasma membrane

Fumonisin B1 (FB1), a mycotoxin produced by Fusarium moniliforme and related fungi, is nephrotoxic, neurotoxic, hepatotoxic, carcinogenic and immunosuppressive in animals and man. In this study we evaluate the modifications of fluidity, endocytosis and peroxidative damage of plasma membrane induced by FB1 in macrophage cell line J774A.1. In these immune cells FB1 (1-10 microM) enhances membrane fluidity and increases, time-dependently, the horseradish peroxidase (HRP) endocytosis. This effect is concentration-dependent, significant at 10 microM, and reverted by IFN-gamma (100 U/ml). Moreover, FB1 (1-10 microM) induces a membrane peroxidative damage as evident by the increase of malondialdehyde (MDA) production. All these mycotoxin effects provide additional insight into potential mechanism by which FB1, in macrophages, might enhance membrane damage and oxidative stress contributing to the pathogenesis of mycotoxin induced diseases.     

The stimulatory effects of caffeine, theophylline, lysine-theophylline and 3-isobutyl-1-methylxanthine on human sperm motility.

The potencies of caffeine, theophylline, lysine-theophylline and 3-isobutyl-1-methylxanthine (IBMX) in stimulating sperm motility have been compared, and we have found IBMX to be significantly more potent than the other three compounds, which did not exhibit significant differences in potency from each other.     

Prevention by vitamin E of DNA fragmentation and apoptosis induced by fumonisin B1 in C6 glioma cells

Fumonisin B₁ (FB₁), produced by the fungus Fusarium moniliforme, belongs to a class of sphingosine analogue mycotoxins that occur widely in the food chain. Epidemiological studies have associated consumption of Fusarium moniliforme-contaminated food with human oesophageal cancer in China and South Africa. FB₁ also causes equine leucoencephalomalacia. Evidence for induction of apoptosis by FB₁ was first obtained when C6 glioma cells were incubated with fumonisin B₁ (3–27 μM) causing DNA fragmentation profiles showing DNA laddering in gel electrophoresis and apoptotic bodies revealed by chromatin staining with acridine orange and ethidium bromide. Further confirmation experiments and comet assays have been performed under similar conditions. The results of the comet test show that FB₁ at 9 and 18 μM induces respectively 50 ± 2% and 40 ± 1% of cells with a comet with an increased tail length of 93 ± 9 μm and 102 ± 17 μm respectively. Under these concentrations, FB₁ induced DNA fragmentation and laddering and many apoptotic bodies. Pre-incubation of the cells with vitamin E (25 μM) for 24 h before FB₁ (18 μM) significantly reduced DNA fragmentation and apoptotic bodies induced by FB₁. Received: 30 August 1999 / Accepted: 18 January 2000     

Toxicity and apoptosis induced by the mycotoxins nivalenol, deoxynivalenol and fumonisin B1 in a human erythroleukemia cell line.

The toxicity of the mycotoxins nivalenol (NIV), deoxynivalenol (DON) and fumonisin B1 (FB1) were studied in the K562 human erythroleukemia cell line using the Trypan Blue, MTT and BrdU uptake analyses of cytotoxicity, cell metabolism and cell proliferation, respectively. Nuclear staining with propidium iodide and DNA analysis by flow cytometry were used to identify apoptosis and cell cycle distribution. By the MTT and BrdU tests, both NIV and DON were significantly more toxic than FB1 by at least one order of magnitude, with ID50s ranging from 0.5 microM for NIV to 70 microM for FB1. The MTT test indicated that NIV was significantly (approximately four times) more toxic than DON. In contrast, the Trypan Blue test did not reveal any effects of mycotoxin exposure suggesting that, at the concentrations tested, NIV, DON and FB1 did not induce cytotoxicity through plasma membrane damage. Cell cycle analysis suggested apoptotic cytotoxicity, revealing 100% cellular debris at the highest concentrations of NIV and DON and approximately 2.9 times more debris than control at the highest FB1 concentration. Morphological evidence of apoptosis was related to the toxicity of the substances, such that the more toxic NIV and DON resulted in more late stage apoptotic events than FB1. This study suggests that human blood cells are sensitive to mycotoxin exposure, that NIV is more toxic than DON which is more toxic than FB1, and that DNA damage and apoptosis rather than plasma membrane damage and necrosis may be responsible for the observed cytotoxicity.     

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.     

Effects of the mycotoxin fumonisin B(1) on cell death in human kidney cells and human lung fibroblasts in primary culture

Fumonisins are mycotoxins produced by Fusarium verticillioides. The toxic effects of fumonisin B(1) (FB(1)) at the cellular level consist of a mixture of both necrosis and apoptosis. We studied the effect of FB(1) in human lung fibroblasts (NHLF) and human kidney epithelial cells (RPTEC) in primary culture. Apoptotic and necrotic cell death, collagen and fibronectin secretion were determined mainly after 14 days' exposure. The protein content of NHLF and RPTEC cells was slightly increased after 14 days' exposure to low FB(1) concentrations (0.1 or 1 microm). Caspase-3 activity tended to increase in NHLF and to decrease in RPTEC cells with higher FB(1) concentrations after 14 days' exposure. LDH release was slightly decreased in both cell types after 14 days. Collagen I and III secretion was enhanced in NHLF cells. Collagen III was decreased in RPTEC. Collagen IV was not changed in both cell types. Fibronectin secretion was uninfluenced in RPTEC and interim increased in NHLF. Furthermore LC-MS/MS studies did not give any hints for a metabolism of FB(1). Therefore, the main risk of prolonged FB(1) exposure seems to be altered collagen secretion pattern.     

Comparative study of cytotoxicity and oxidative stress induced by deoxynivalenol, zearalenone or fumonisin B1 in human intestinal cell line Caco-2

Fusarium species infestations of cereals crops occur worldwide. Fusarium toxins such as, deoxynivalenol (DON), zearalenone (ZEN) and fumonisin B1 (FB1) have been shown to cause diverse toxic effects in animals and also suspected of disease causation in humans. From the literature and mechanistic point of view, DON binds to the ribosomal peptidyl-transferase and inhibits protein synthesis specifically and DNA synthesis consequently. ZEN known to be genotoxic, binds to 17-beta-estradiol receptors, induces lipid peroxidation, cell death and inhibits protein and DNA synthesis. FB1 disrupts sphingolipid metabolism, induces lipid peroxidation altering the cell membrane and causing cell death. We intended to compare DON, ZEN and FB1 (1-150 microM) cytotoxic effect and the pathways leading to cell death and related to oxidative stress and macromolecules syntheses in a human intestinal cell line in order to tentatively classify them according to their respective potential toxicity. The comparison reveals that all three mycotoxins bear, at variable degree, the capability of inducing lipid peroxidation (MDA production) and could be classified above 10 microM in decreasing potency order FB1>DON>ZEN. This effect seems to be related to their common target that is the mitochondria as revealed by MTT test and seemingly not related to sphingoids accumulation concerning FB1. DON and ZEN also adversely affect lysosomes in contrast to FB1. The three mycotoxins inhibit protein synthesis with respective IC50 of 5, 8.8 and 19 microM for DON, FB1 and ZEN confirming that protein synthesis is a specific target of DON. DNA synthesis is inhibited by DON, ZEN and FB1 with respective IC50 of 1.7, 10 and 20 microM. However at higher concentrations DNA synthesis seems to be restored for FB1 and DON suggesting a promoter activity. Altogether the potency of the three mycotoxins in macromolecules inhibition is DON>ZEN>FB1 in Caco-2 cells. It appears then that FB1 acts rather through lipid peroxidation while DON affects rather DNA and protein synthesis.     

Alteration of chromatin structure induced by the binding of adriamycin,daunorubicin and ethidium bromide

The results reported in this paper show the changes in chromatin structure caused by the binding of adriamycin (ADR), daunorubicin (DR) and ethidium bromide (EtdBr) to DNA in chromatin, either isolated or in nuclei or whole cells. Micrococcal nuclease was used as the structural probe of chromatin. The binding of the drugs to chromatin DNA induced two structural changes. First, it produced an unfolding of the overall chromatin structure as evidenced by the increased production of acid-soluble oligonucleotides for the drug-treated samples above the level of the control sample. Second, it caused a disruption of the core particle structure with increased production of DNA of subnucleosomal size and smearing of the nucleosome pattern. The effects were greatest for daunorubicin, followed by adriamycin and ethidium bromide.     

Differential effects of fumonisin B1 on cell death in cultured cells: the significance of the elevated sphinganine

Fumonisins are specific inhibitors of ceramide synthase in sphingolipid metabolism. An alteration in sphingolipid metabolism as a result of fumonisin exposure is related to cell death (Yoo et al., 1992). The objective of this study was to investigate whether elevated free sphinganine levels are related to the sensitivity of cultured cells to fumonisin exposure. Fumonisin B1 elevated the intracellular free sphinganine concentraions in both LLC-PK1 and Chinese hamster ovary (CHO) cells. However, CHO cells are resistant to fumonisin cytotoxicity at 50 microM, while LLC-PK1 cells are sensitive at concentrations greater than 35 microM. The intracellular concentration of free sphinganine in LLC-PK, cells treated at 50 microM fumonisin B1 for 72 h was approximately 1450 pmol/mg protein relative to the 37 pmol observed in the control culture. Under the same conditions, the population of apoptotic cells in the 50 M fumonisin B1-treated culture was approximately 37% of the total compared to 12% in the control. The caspase III-like activity after 72 h in the 50 microM fumonisin B1-exposed culture increased to approximately 50 pmol/mg protein/hr compared to 6 pmol/mg protein/hr in the control. L-cycloserine, a serine palmitoyltransferase inhibitor, reduced the fumonisin B1-stimulated caspase III-like activity down to the control level. Under the same culture conditions, the intracellular concentration of free sphinganine after L-cycloserine plus fumonisin B1 treatment was 140 pmol/mg protein compared to 1450 pmol/mg protein in fumonisin B1 alone. The intracellular concentration of free sphinganine in CHO cells treated with 50 microM fumonisin B1 for 72 h was approximately 460 pmol/mg protein, indicating that the mass amount of elevated free sphinganine in the CHO cells was about 32% of that in LLC-PK1 cells. Adding exogenous sphinganine to the CHO cells along with 50 microM fumonisin B1 treatment for 72 h caused both necrosis and apoptosis. In conclusion, the elevated endogenous sphinganine acts as a contributing factor to the fumonisin-induced cell death.     

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.     

Modulating effects of fumonisin B1 and ochratoxin A on leukocytes and messenger cytokines of the human immune system

Fumonisin B1 and ochratoxin A are mycotoxins of importance to public health and agro-economics. Although much is known about their cellular toxicity and carcinogenesis in animals, there are no reports of adverse effects on immune cells (leukocytes) or on the immune modulation of the molecular messengers (cytokines) in humans. This study was designed, therefore, to determine and compare the morphological effects of fumonisin B1 and ochratoxin A on lymphocytes and neutrophils harvested from the circulation of healthy volunteer subjects and patients with oesophageal and breast carcinomas. Both fumonisin B1 and ochratoxin A reduced the number of viable lymphocytes and neutrophils harvested from the circulation of volunteer subjects carcinoma patients in a dose-dependent manner. Leukocyte secretion of cytokines on exposure to the mycotoxins was evaluated by immunocytochemical methods. Expression of granulocyte-colony stimulating factor (G-CSF), tumour necrosis factor (TNF-alpha) and chemokine (CX3CR1) receptors were determined on the circulating leukocytes and the immunolabelling visualized by brightfield-and electron-microscopy. Cytokine levels were determined in the circulation of healthy volunteer subjects and in patients with oesophageal and breast carcinomas since they reflect the status of the immune system in humans. The findings of this study on immunocytes (leukocytes) and the immune molecular messengers (cytokines) suggest that fumonisin B1 and ochratoxin A have an immuno-suppressive effect in humans, in particular patients with cancer by impairing immune surveillance.     

In vivo and in vitro effects of hydralazine on cellular growth, differentiation, and chromatin structure

The effects of hydralazine (1-hydrazinophthalazine), an antihypertensive drug, on mammalian cell growth, viability, and differentiation were assessed using Friend leukemia cells, Chinese hamster ovary cells, human lymphocytes, and rat lymphocytes, testicular germ cells, and epididymal sperm. Cultured cells in exponential phase growth were more susceptible to hydralazine cytotoxicity than stationary phase (G0) cells. Growth inhibition was associated with a dose-related slowdown of cell progression through S phase and was observed prior to a decrease of cell viability. At high drug concentrations, progression in all phases of the cell cycle was partially or totally inhibited. Hydralazine did not have an effect on the proliferation and differentiation of testicular germ cells in spontaneously hypertensive rats receiving 0-90 mg/kg/day (up to 20 times the dose used in humans) of hydralazine for a 12-week period. Hydralazine-exposed, histone-containing somatic cells and protamine-containing sperm cells failed to show any alterations in stainability with a DNA-intercalating dye nor in the susceptibility of nuclear DNA to undergo acid-induced denaturation in situ. The data suggest that hydralazine causes a dose-related suppression of mammalian cell growth with S phase appearing to be the most susceptible to hydralazine cytotoxicity. Furthermore, the interaction of hydralazine with chromatin at concentrations leading to antigenicity did not inhibit DNA staining with the intercalating dye acridine orange, suggesting that the drug does not competitively intercalate at a detectable level. Association of hydralazine with chromatin did not cause a detectable level of stabilization or destabilization of the DNA to denaturation in situ.     

Implications of apoptosis for toxicity, carcinogenicity, and risk assessment: fumonisin B(1) as an example.

The rates of cell proliferation and cell loss in conjunction with the differentiation status of a tissue are among the many factors contributing to carcinogenesis. Nongenotoxic (non-DNA reactive) chemicals may affect this balance by increasing proliferation through direct mitogenesis or through a regenerative response following loss of cells through cytotoxic (oncotic) or apoptotic necrosis. In a recent NTP study in Fischer rats and B6C3F(1) mice, the mycotoxin fumonisin B(1) caused renal carcinomas in male rats and liver cancer in female mice. In an earlier study in male BD-IX rats, fumonisin B(1) caused hepatic toxicity and hepatocellular carcinomas. An early effect of fumonisin B(1) exposure in these target organs is apoptosis. However, there is also some evidence of oncotic necrosis following fumonisin B(1) administration, especially in the liver. Induction of apoptosis may be a consequence of ceramide synthase inhibition and disruption of sphingolipid metabolism by fumonisin B(1). Fumonisin B(1) is not genotoxic in bacterial mutagenesis screens or in the rat liver unscheduled DNA-synthesis assay. Fumonisin B(1) may be the first example of an apparently nongenotoxic (non-DNA reactive) agent producing tumors through a mode of action involving apoptotic necrosis, atrophy, and consequent regeneration.