Horses are the most sensitive species to FB1 toxicity, and FB1 concentrations of 0.02 and 0.12 μg/g in feed are already able to cause outbreaks in this species [53
], even if the concentration range found in outbreaks is wide, from 0.1 to 126 μg/g [54
]. The neurotoxic (leukoencephalomalacia—Equine Leuko Encephalo Malacia (ELEM)) and the hepatotoxic forms are the two syndromes, which can occur simultaneously or independently, described in horses with FB1-mycotoxicoses. Central nervous system, liver and heart are involved as target organs. ELEM typical signs are: sweating, inability to swallow, muscle fasciculation and weakness, in coordination and ataxia, hypermetria, circling, head pressing, tonic-clonic seizures, paresis, hyperexcitability or depression, blindness, dilated pupils, and absence of a pupillary light reflex [53
]. Early moderate neurological signs such as tongue paralysis and ataxia may precede by hours or days the onset of more severe signs. The ELEM is characterized by a rapid clinical course (hours or days) and a high mortality. Sometimes, death can occur without sign of disease. The histopathological findings are mainly a liquefactive necrosis with an influx of macrophages of the white matter of the cerebral hemispheres with edema and hemorrhage [18
]. Equines are the only species that suffer from this neurological syndrome related to FB1 intoxication. The hepatotoxic syndrome, which is less frequent than the neurotoxic form, is characterized by hyporexia, depression, icterus and edema of the head. The main histopathological sign is a centrilobular necrosis with periportal fibrosis [18
Regarding the most recently recognized cardiotoxicity, Smith and colleagues documented for the first time in 2002 that horses with fumonisin B1
-induced neurologic syndrome have cardiovascular dysfunction. In a study performed by these authors, in which FB1 was administered intravenously (0.20 mg/kg), signs included decreased cardiac output, negative chronotropic and inotropic effects and a reduction in the arterial pulse pressure. Moreover, all the animals showed an increased sphingosine and sphinganine concentration in the myocardium [56
]. Although the mechanism of action of fumonisins is not completely understood, it has been demonstrated that high concentrations of sphingosine inhibit L-type calcium channels in several mammalian species myocardial cells [57
], leading to a decrease in Ca ion release with a reduction of cardiac activity. It can also be hypothesized that the development of leukoencephalomalacia may be related to the decreased cardiovascular function with cerebral vessel damage.
A study was performed in vitro on primary isolated epidermal and dermal hoof cells to evaluate the influence of FB1 on the lamellar tissue and sphingolipid metabolism [61
]. A significant increase in sphinganine concentrations was found in the supernatant of the explants with a simultaneous decrease in the lamellar integrity, confirming in vitro the in vivo findings.
Regarding the reproductive toxicity, effects were observed only at doses that induce clear clinical signs in the mares [62
]. In vitro toxicity of fumonisin B1
on fresh and frozen-thawed semen has also been assessed. Sperm viability and motility, chromatin stability and reactive oxygen species (ROS) production was assessed in a study by Minervini et al. [63
]. No effects on viability were found in fresh sperm after exposure to FB1 up to 25 µM while reduction of total and progressive motility was recorded at a concentration similar to those reported as neurotoxic (10 µg/g). Just one frozen-thawed sample showed chromatin damage after FB1 exposure. Moreover, the study found that the action of FB1 on sperm functional parameters was subject-dependent.
Ruminants appear to be considerably less sensitive to FB1 than horses and pigs and, in cases of highly contaminated feed, some animals may develop mild biochemical alterations and microscopic liver (diffuse mild hydropic degeneration in a periacinar pattern) [64
] and renal lesions [65
]. In a study by Osweiler and colleagues [64
], serum liver enzymes showed a mild elevation after consumption for 10 days of a contaminated feed (148 microg/g): changes were too small to be suggestive of severe liver disease, but they suggested an effect of the mycotoxin on hepatocyte and biliary excretion. Moreover, after a chronic feeding period, lymphocyte blastogenesis was impaired.
Studies suggested that the low sensitivity of ruminants to FB1 may be due to its low bioavailability after oral administration [66
] and, contrarily to other mycotoxins, FB1 does not undergo rumen microbial degradation and passes the rumen virtually unchanged [67
]. Moreover, FB1 does not affect the production of short-chain fatty acids in rumen, so this mycotoxin had no toxic effects on the ruminal microflora [68
Reproductive effects of FB1, alone and in combination with other mycotoxins, have been investigated using bovine granulosa cell models. When tested alone, FB1 did not show effects on granulosa cell proliferation and had no significant effect on progesterone production at any dose, whereas at a concentration between 1 and 3 µM weakly inhibited estradiol production [69
], thus demonstrating that FB1 may impair reproductive function in cattle. The decrease in estradiol production was not linked to a significant modification on CYP19A1 expression in bovine granulosa cells, while it was observed in porcine granulosa cells [70
], indicating that another mechanism of action should be implied (i.e., estradiol metabolism and/or FSH/IGF1-receptors). It has been reported that FB1 can modulate the endocrine system by antagonism of nuclear receptor transcriptional activity [71
], but whether this is how FB1 is acting in granulosa cells will require further elucidation. Moreover, many in vitro studies concerning the reproductive effects in cattle have shown that the co-exposure plays a fundamental role, and that doses tested have a strong influence on the entity and type of exerted effects [72
Pigs are, together with horses, the most sensitive species to fumonisins. A typical and species-specific presentation of FB1 acute mycotoxicosis in pigs is pulmonary edema, a sign that has been reported only in this species. It was described for the first time in 1981 after the experimental administration of feed contaminated by F. verticilloides
]. After that, in 1989, an outbreak due to consumption of corn contaminated by fumonisins resulted in the deaths of several pigs. The main sign was a huge pulmonary edema, thus the syndrome was called “porcine pulmonary edema” (PPE), whereas some animals also showed reproductive anomalies (e.g., abortion).
Further studies were able to induce the same pulmonary edema by administering orally and intravenously purified FB1 [74
]. The pathogenesis of the toxicosis-induced pulmonary edema is linked primarily to an increase in pulmonary capillary hydrostatic pressure due to an acute left-sided heart failure and to an increase in the vascular permeability caused by damages to the alveolar capillary endothelium and alveolar epithelium. Other signs of fumonisin toxicosis are hepatic, cardiovascular and immune system damages together with the alteration of the sphingolipid metabolism. Development of lethal pulmonary edema is reported in pigs fed with concentrations of 16 mg/kg of FB1 for 4–7 days or intravenously injected [31
]. Clinical signs are: inactivity, respiratory distress shown by increased respiratory rate, abdominal respiratory effort and open mouth breathing, and a decreased heart rate, which occur during the 12-h period that precedes pulmonary edema and death [76
]. In some cases, gastrointestinal signs have also been reported.
The cardiovascular signs include a negative chronotropic and inotropic effect, and the relaxation of the vascular smooth muscle that causes a reduction of the cardiovascular reserve, effects that are probably mediated via the increase in sphingosine concentration that inhibits the L-type calcium channels. The hepatic damage (characterized by cholestasis, hepatic necrosis, cell proliferation) is dose- and time-dependent and is characterized by an increase in the activity of the hepatic enzymes in serum, icterus, anorexia and hepatic encephalopathy.
In swine, as in the other species, the FB1 causes an inhibition of the ceramide synthase with a resultant increase in the sphingoid bases, sphinganine and sphingosine, in serum and tissues within 24 h [77
], which is the earliest and most specific time- and dose-dependent sign of FB1 toxicosis [76
]. Hypercholesterolemia also is a sensitive and specific parameter to detect FB1 exposure. Necropsy of pigs that died of PPE showed a pulmonary interstitial edema with widened interlobular septa in the lungs and the presence of modified transudate in the airways and in the thoracic cavity [77
]. The major histopathological signs were presence of fluid in the connective tissue of bronchi, vessels, subpleural space and interlobular septa. No signs of inflammation were present. Some endothelial cells of lung vascular system appeared damaged and apoptotic and a dense accumulation of membranous material was found within the lung intravascular macrophages, which was probably the result of the phagocytosis of dead endothelial cells. This lesion is species-specific: comparable endothelial lesions were observed only in pigs [78
In vitro studies were performed on porcine lung endothelial cell cultures to clarify the action of FB1. An association between the increase in albumin permeability detected within 12 h of treatment with FB1 and cell death was recorded, therefore the authors hypothesized that cell death played a role in the permeability increase [79
]. Nevertheless, the in vitro increase in permeability was not considered physiologically relevant in vivo [79
] because studies in pigs with PPE showed that only a few cells were affected by degeneration, and the number of involved cells was not sufficient to justify the huge increase in permeability. On the contrary, the significant time- and dose-dependent increase in sphingoid bases observed in lung and endothelial cells, in vivo and in vitro studies [77
] was considered more relevant. It has been hypothesized that the described accumulation of membranous material in lung capillary is directly caused by the alteration of the sphingolipid metabolism induced by FB1 that acts on Golgi and endoplasmic reticulum causing an impairment of these cell organelles.
While PPE occurs only in case of high-level exposure, a chronic exposure to a lower concentration administered orally was found to be associated to a right ventricular hypertrophy and medial pulmonary arterial hypertrophy indicative of pulmonary hypertension [80
]. Other lesions described with FB1 chronic intoxication include hyperplasia of the basal cell layer of the esophageal mucosa, sometimes with hyperplastic regions and gastric ulceration. These findings were particularly interesting because of the described association between FB1 ingestion and the occurrence esophageal cancer in humans [81
Moreover, FB1 has been implicated in poor reproductive performance in pigs. In vitro studies were performed to analyze the effect of FB1 on the reproductive functions, using cultures of porcine granulosa cells. FB1 decreased the granulosa cell number with a dose 10–14 µM but had no effect at lower doses [70
]. Comparable results were obtained with many porcine cell lines, such as renal (LLC-PK1) [82
] and intestinal (IPEC-1) [83
] cell lines and also in primary cells (i.e., porcine lymphocytes) [84
]. Regarding the effects on granulosa cell steroidogenesis, FB1 stimulated progesterone production (not mediated by an increased activity of the mitochondrial enzyme cytochrome P450scc) while it had no significant effect on estrogen production [70