Toxins, Vol. 14, Pages 828: Targeted Sphingolipid Analysis in Heart, Gizzard, and Breast Muscle in Chickens Reveals Possible New Target Organs of Fumonisins

1. IntroductionFumonisins are mycotoxins produced by molds of the genus Fusarium, mainly F. verticillioides. This class of contaminant is found worldwide in maize, maize byproducts, and animal feed [1,2]. Among the various types of fumonisins, fumonisins B are the most abundant, and among fumonisins B, fumonisin B1 (FB1), and to a lesser extent fumonisin B2 (FB2), are the most abundant, toxic, and regulated. Fumonisins B are considered carcinogenic in rodents and probable carcinogens for humans, with a provisional maximum tolerable daily intake (PMTDI) being set for humans and maximum levels of fumonisins being recommended in animal feed [2,3,4,5]. Fumonisins have multiple toxic effects in animals as they are neurotoxic in horses, pneumotoxic in pigs, hepatotoxic, and nephrotoxic in most animal species, including poultry. At the cellular level, oxidative stress, mitochondrial dysfunction, and induction of apoptosis or cell proliferation have been reported [6,7,8,9]. Although the effects of FB1 vary greatly, inhibition of ceramide synthase (CerS) due to structural analogy between FB1 and sphingoid bases is considered one of the main mechanisms underlying the toxicity [10,11]. This inhibition leads to reduction of the de novo synthesis of ceramides and an increase in sphinganine (Sa) concentrations, while sphingosine (So) concentrations tend to decrease (Figure 1). Ceramide levels in cells can be maintained by hydrolysis of sphingomyelins, which is known as the salvage pathway for ceramide synthesis.The pronounced effect of fumonisins on sphingolipids has led to widespread use of the Sa:So ratio as an exposure biomarker in humans and animals, including poultry [9,11,12]. There have, however, only been a limited number of studies to date aimed at characterization of the effects of fumonisins on the sphingolipidome. Sphingolipidomic studies in chickens and turkeys conducted at the maximum recommended concentration for fumonisins in feed in the European Union have revealed numerous alterations in sphingolipids in the liver [13,14]. These changes are consistent with the previously reported inhibition of CerS in vitro and in vivo [10,11]. Because the effects of fumonisins on sphingolipids in chicken and turkey livers were characterized by a decrease in C14–C16 ceramides and an increase in C20–C24 ceramides, it has been hypothesized that inhibition of CerS may be more pronounced for CerS5 than that for CerS2 (Figure 1), whereas the opposite has been suggested to occur in mammals [11,15]. This difference is important because a decrease in C20–C24 ceramides, and a compensatory increase in C16 ceramide, have been reported in CerS2 knockout mice, and these changes were responsible for hepatotoxicity that resembles fumonisins toxicity [16,17]. Sphingolipid analysis in chickens and turkeys also revealed that sphingomyelin levels did not decrease in liver [13,14]. This latter result is also important for explaining the lack of toxicity of fumonisins in these studies, with a decrease in sphingomyelins being reported in tissue and cell cultures at toxic concentrations of fumonisins, and this event was found to precede ceramide-induced apoptosis [11,15,18,19,20,21].Targeted sphingolipids analysis conducted in the kidneys, lungs, and brains of chickens revealed that the effects of fumonisins were not limited to changes in sphingolipids in the liver, and that the effects varied according to the organ studied [22]. The effects in kidneys were similar to those observed in livers (Figure 1). By contrast, sphinganine and ceramides were unchanged in the lungs and brain, suggesting no inhibition of CerS in these organs. However, major changes in sphingolipid concentrations in lung and brain were found in chickens fed with a diet that was contaminated with fumonisins compared to controls [22]. In the lungs, these changes corresponded to an increase in sphinganine-1-phosphate and a decrease in glycosylceramides. In the brain, these changes corresponded to an increase in deoxysphinganine, sphingosine, ceramides, and sphingomyelins (Figure 1). Interestingly, the most affected sphingolipids in lung and in brain in chickens fed fumonisins corresponded to compounds that have key functions in these organs in human diseases [23,24,25,26]. As only traces of FB1 were found in lungs and no FB1 was detected in brains, it has been hypothesized that the effects of fumonisins are due to indirect effects, metabolites, or cell-mediated (Figure 1) [22].Although fumonisins have been reported to exert cardiac toxicity in mammals and avian species [27,28,29,30], the effect of fumonisins on sphingolipids in the heart remain unknown. These effects seem interesting to study as sphingolipids have a key role in the onset of cardiovascular diseases in humans [31]. Additionally, diets containing fumonisins have been reported to damage the gizzard and to alter its weight [32,33,34,35,36], although there have been no studies to date regarding the effect of fumonisins on sphingolipids in the gizzard. Moreover, sphingolipids are known to play a key role in skeletal muscle [37,38,39,40], but the effects of fumonisins on this tissue remain unknown. The purpose of this study was to investigate the effects of 20.8 mg FB1 + FB2/kg fed to chicken for 9 days on sphingolipids in the heart, gizzard, and breast muscle. Targeted sphingolipid analysis was conducted by UHPLC-MSMS of samples obtained from animals by a method that has already been used to characterize the effects of fumonisins on sphingolipids in liver, kidney, lung, and brain [13,22]. Figure 1. Schematic outline of the sphingolipid synthesis pathways [11]. Ceramide synthases (CerS) specificity and tissue expression were obtained from [41]. Fumonisins B (FB) are inhibitors of CerS (red arrow). 1-Deoxysphinganine is generated from alanine [42]. The effects of fumonisins on sphingolipids in liver, kidney, lung, and brain are summarized from [13,22]. 1: Serine palmitoyl-transferase; 2: Reductase; 3: Sphinganine kinase; 4: Phosphatase; 5: (Dihydro)ceramide synthase; 6: Dihydroceramide desaturase; 7: Ceramide kinase; 8: Phosphatase; 9: Ceramidase; 10: Ceramide synthase; 11: Sphingomyelin synthase; 12: Sphingomyelinase; 13: Glucosylceramide synthase; 14: β-glucosidase; 15: Lactosylceramide synthase; 16: Ceramide galactosyltransferase; 17: Galactosylase; 18: Cerebroside sulfotransferase; and 19: Arylsulfatase. Figure 1. Schematic outline of the sphingolipid synthesis pathways [11]. Ceramide synthases (CerS) specificity and tissue expression were obtained from [41]. Fumonisins B (FB) are inhibitors of CerS (red arrow). 1-Deoxysphinganine is generated from alanine [42]. The effects of fumonisins on sphingolipids in liver, kidney, lung, and brain are summarized from [13,22]. 1: Serine palmitoyl-transferase; 2: Reductase; 3: Sphinganine kinase; 4: Phosphatase; 5: (Dihydro)ceramide synthase; 6: Dihydroceramide desaturase; 7: Ceramide kinase; 8: Phosphatase; 9: Ceramidase; 10: Ceramide synthase; 11: Sphingomyelin synthase; 12: Sphingomyelinase; 13: Glucosylceramide synthase; 14: β-glucosidase; 15: Lactosylceramide synthase; 16: Ceramide galactosyltransferase; 17: Galactosylase; 18: Cerebroside sulfotransferase; and 19: Arylsulfatase. Toxins 14 00828 g001 3. DiscussionFeeding 20.8 mg FB1 + FB2/kg over a period of 9 days did not lead to significant differences in the performances or the biochemistry in the chickens in this study [43]. This observation is consistent with the regulatory guidelines, which define a maximum tolerable level of fumonisins in feed of 20 mg FB1 + FB2/kg [1,4], whereas feeding 2.5 mg FB1 + FB2/kg of feed was reported to alter the length of intestinal crypts in chickens after 21 days of exposure, and feeding 5.3 mg FB1 + FB2/kg induced oxidative damage in the liver at 17 days, and histological damage to the liver and lungs at 21 days, effects on growth were only observed at 21 days [2,44].Targeted analysis of sphingolipids in heart, gizzard, and breast muscle revealed that the effects of fumonisins varied greatly with the tissue studied. A significant increase in the Sa:So ratio was observed in the heart, and this increase was due to an increase in Sa, with a small increase in So also being observed. The change in sphingoid bases was accompanied by an increase in ceramides and, to a lesser extent, an increase in glycosylceramides and sphingomyelins. Most of the effects observed for dihydroceramides and dihydrosphingomyelins in the heart paralleled those found for ceramides and sphingomyelins. The effect of fumonisins on Sa is consistent with its well-known property of inhibiting CerS (Figure 1), and de novo synthesis of ceramides [10]. An increase in So concomitant with an increase in Sa has previously been reported to be the consequence of sphingomyelin hydrolysis occurring to maintain ceramide concentrations in cells [11], with this mechanism being known as the salvage synthesis pathway of ceramides (Figure 1). In this study, an increase rather than a decrease in sphingomyelins was observed, suggesting that sphingomyelin hydrolysis cannot explain the observed increase in So and ceramides. Moreover, glycosylceramides are derived from ceramides, and because glycosylceramides were increased in this study, the hypothesis of an increase in ceramides in heart secondary to hydrolysis of sphingomyelins appears unlikely (Figure 1). Other mechanisms that could explain an increase in ceramide concentrations in heart include: (1) change in the availability of the substrates used for their de novo synthesis [45,46]; (2) activation of de novo synthesis by different mechanisms linked with an inflammatory response such as Toll-like receptor (TLR) or tumor necrosis factor alpha (TNFα) activation [47]; and (3) increased delivery of sphingolipids from plasma, which includes recycling of sphingolipids from the gut microbiota [46,48].Irrespective of the mechanism involved, sphingolipid concentrations were increased in the hearts of chickens in this study that had been fed fumonisins. PLS-DA revealed that the changes in sphingolipids were enough to distinguish chickens fed the control diet from chickens fed the fumonisin diet. Analysis of the correlations among sphingolipids also revealed that nearly all the sphingolipids positively correlated together in chickens fed fumonisins, which was not the case in the controls, all suggesting that pronounced changes in sphingolipid homeostasis occurred in the hearts of chickens fed fumonisins. Moreover, it is interesting to note that the increases in sphingolipids for which the fatty acids had 20–26 carbon chain lengths were more pronounced than for those with 14–16 carbon chain lengths. This observation is consistent with previous results in liver and kidney suggesting that fumonisins preferentially inhibit CerS5/6 activity in chickens [13,22]. Thus, it can be hypothesized that a general increase in sphingolipid synthesis occurred in the hearts of animals fed fumonisins, with this increase being less pronounced for sphingolipids for which the fatty acid chain length was 14 or 16 carbons due to partial inhibition in CerS5/6 activity.Changes in sphingolipid concentrations in the hearts of chickens fed fumonisins could have negative consequences on health. Indeed, recent studies have revealed a prominent role of sphingolipids in cardiovascular diseases and heart failure [31]. Increased concentrations of ceramides in the myocardium have been reported in a rat model of ischemic reperfusion injury [49], and measurement of the expression of serine palmitoyl-transferase in mice suggested an increase in the de novo synthesis of sphingolipids in the infarct area and in the area at risk [50]. By contrast, sphingosine 1-phosphate has been reported to have a protective function in the myocardium [31]. Interestingly, a pronounced increase in So1P was observed in the plasma of chickens fed fumonisins, and a rapid increase in So1P has been reported in transient ischemia and reperfusion injury in humans [13,51]. Unfortunately, So1P was only found at trace levels in the myocardium in this study, and it was hence not possible to quantify its concentration. Only a few studies to date have reported cardiac alterations in animals fed fumonisins. A reduced heart rate has been reported to occur in pigs before the development of pulmonary edema and death [27]. Hypertrophy of the heart has been reported in pigs at very high doses of fumonisins in feed without histopathologic alterations despite an increase in the Sa:So ratio [28]. In contrast, a recent study of low doses of fumonisins revealed no alteration in heart weight, whereas histopathologic lesions characterized by hemorrhage and lymphocyte inflammatory infiltrate were observed [30]. Myocardial infiltration by lymphocytes leading to inflammatory damage is consistent with the hypothesis of activation of de novo synthesis of sphingolipids by TLR or TNFα [30,47]. Cardiac alterations characterized by macroscopic thinning of the heart and thinning of cardiomyocytes have been observed in Japanese quails fed fumonisins [29]. Transmission electron microscopy also revealed that the number of mitochondria was increased and that the mitochondria appeared swollen and pleomorphic in fumonisins-fed quails, although the outer membrane remained intact [29]. Alteration of the heart mitochondria of Japanese quails fed fumonisins is of considerable relevance because an in vitro study has revealed that ceramides cause mitochondrial dysfunction, oxidative stress, and cell death in cardiomyocytes [52]. Interestingly, overexpression of CerS2 induced oxidative stress, mitophagy, and apoptosis, which were prevented by depletion of CerS2. By contrast overexpression of CerS5 did not affect these processes, suggesting a chain-length dependent impact of ceramides on mitochondrial function [52].A minor decrease in the Sa concentration was observed in the gizzards of chickens fed fumonisins, whereas fumonisins had no effect on the sphingolipid concentrations in breast muscle in this study. There are no data to compare the effects of fumonisins on sphingolipids in gizzard and breast muscle with our present results. Fumonisins have been reported to induce gizzard ulceration in chickens and have been reported to increase the relative weights of gizzards in chickens, turkeys, and ducks, but the mechanism underlying these alterations remains unknown [32,33,34,35,36]. In this study, the decrease in Sa observed in the gizzards of chickens receiving fumonisins was accompanied by a small decrease in So, so the Sa:So remained unchanged. Total ceramides in gizzards were decreased by fumonisins, and all of the ceramides appeared to decrease. The concentrations of dihydroceramides, sphingomyelins, dihydrosphingomyelins, hexosylceramides, and lactosylceramides paralleled those of ceramides. Interestingly, the effects of fumonisins on sphingolipids in gizzards appeared to be independent of the fatty acid carbon chain length. PLS-DA allowed the chickens to be separated into two groups according to the presence or absence of fumonisins in the feed, but the correlations measured among the sphingolipids in gizzards did not differ greatly in fumonisin-fed chickens and in controls. Finally, the effects of fumonisins on gizzards appeared to be consistent with the hypothesis of a decrease in the de novo synthesis of ceramides even though there was no accumulation of Sa. The decrease in sphingolipids in gizzards appeared to be independent of the fatty acid chain, which is different from what was observed in the liver and kidneys and to a lesser extent in the heart [22].Sphingolipids have been shown to have important roles in skeletal muscle in obesity and aging. Accumulation of C16 ceramide occurs during the development of insulin resistance and CerS6 silencing may be a potential target for the treatment of insulin resistance, obesity, and type 2 diabetes [37]. C18 ceramide, produced mainly by CerS1, also accumulated in mice fed with a high-fat diet that promotes systemic insulin resistance [38,40]. By contrast, a decrease in C16 and C18 ceramides appears to be important in the skeletal muscle and myocardium of aged mice and humans [39]. Histological analysis of muscle in CerS1- and CerS5-deficient mice revealed reduced caliber sizes in slow (type 1) and fast (type 2) fibers of quadriceps femoris [39]. All of these results suggest that the decrease in Cer concentrations observed in gizzards could affect its contractility, even though no clinical consequences were observed. 5. Materials and Methods 5.1. Analytes and Reagents

The analytes and reagents used in this study were obtained from Sharlab (Sharlab S.L., Sentmenat, Spain) or Sigma (Sigma Aldrich Chimie, Saint-Quentin-Fallavier, France). The separation of the analytes by UHPLC-MSMS was done with LC-MS grade solvents, whereas all other reagents were HPLC grade. The 42 sphingolipids used as standards were obtained from Sigma or Bertin (Bertin Technologies, Montigny-Le-Bretonneux, France) and corresponded to: deoxysphingosine (dSo = m18:1); deoxysphinganine (dSa = m18:0); sphingosine (So = d18:1); sphinganine (Sa = d18:0); sphingosine-1-P (d18:1P); sphinganine-1-P (d18:0P); glucosylsphingosine (GluSo); lysosphingomyelin (LysoSM); lactosylsphingosine (LacSo); N-acetylsphingosine (18:1/2:0); N-acetylsphinganine (18:0/2:0); ceramides: 18:1/14:0, 18:1/16:0, 18:1/18:0, 18:1/20:0, 18:1/22:0, 18:1/24:1, and 18:1/24:0; ceramide-1P: 18:1/16:0P; dihydroceramides: 18:0/16:0, and 18:0/24:0; deoxyceramides: m18:1/16:0, m18:1/22:0, m18:1/24:1, m18:1/24:0; deoxydihydroceramides: m18:0/22:0, m18:0/24:1, m18;0/24:0; glucosylceramides: Glu18:1/16:0 and Glu18:1/24:1; lactosylceramides: Lac18:1/16:0 and Lac18:1/24:1; ceramides sulfatides: ST18:1/24:1 and ST18:1/24:0; sphingomyelins: SM18:1/14:0, SM18:1/16:0, SM18:1/18:0, SM18:1/18:1, SM18:1/20:0, SM18:1/22:0, SM18:1/24:1, and SM18:1/24:0. The 12 sphingolipids used as IS corresponded to the 10 IS mixture “Ceramide/Sphingoid Internal Standard Mixture I” from Avanti Polar Lipids, which contains 25 µM C17 sphingosine (d17:1), C17 sphinganine (d17:0), C17 sphingosine-1-P (d17:1P), C17 sphinganine-1-P (d17:0P), lactosyl (ß) C12 ceramide (Lac18:1/12:0), C12 sphingomyelin (SM18:1/12:0), glucosyl (ß) C12 ceramide (Glu18:1/12:0), 12:0 ceramide (18:1/12:0), 12:0 ceramide-1-P (18:1/12:0P), and 25:0 ceramide (18:1/25:0) in ethanol solution. This mixture was completed by C12 deoxyceramide (m18:1/12:0) and C12 ceramide sulfatide (ST18:1/12:0) solubilized in ethanol at a concentration of 25 µM.

5.2. Tissue SamplesHeart, gizzard, and breast muscle were obtained from a Ross chicken study for which the animal maintenance conditions, feed formulation, and results of the effects of fumonisins on health and performance were detailed in [43]. This study was completed at Cebiphar (Cebiphar, ondettes, France) under number V9152 as a randomized, parallel, monocenter study under project number 2017062111426641 accepted by the French Ministry of Higher Education, Research, and Innovation (Paris, France) on 6 November 2017. Briefly, the experimental diets were formulated on a corn-soybean basis to best meet the nutritional needs of the chickens. Corn containing fumonisins was incorporated to a final concentration of FB1, FB2, and FB3 in the feed of 15.1, 5.6, and 0.9 mg/kg, respectively. Mycotoxin-free corn was used as the control diet. Mycotoxins in the raw materials and in the diets were measured by LC-MSMS according to AFNOR V03-110 [53]. Drinking water and feed were provided ad libitum. A diet containing fumonisins was provided to 10 chickens per group from the day of age 14 to the day of age 21. A control diet free of mycotoxins was provided to 10 other chickens until the day of age 21. On day 21, the feed was removed for eight hours before euthanasia and tissue collection. Heart, gizzard, and breast muscle were stored at −80 °C until analysis. 5.3. Sphingolipids in TissuesThe sphingolipids in heart, gizzard, and breast muscle were measured as previously described in [13] and completed in [22] for the determination of deoxyceramides, deoxydihydroceramides, and ceramides sulfatides. Briefly, 0.5 g of tissue was homogenized with a Potter grinder in 1.5 mL of phosphate buffer (0.1 M, pH 7.4) and centrifuged for 15 min at 3000× g. A 40 µL aliquot of the supernatant was collected and 120 µL of NaCl 0.9%, 600 µL of methanol/chloroform (2:1), and 10 µL of a solution containing the IS mixture were added to obtain a final concentration of each IS equivalent to 6250 pmol/g of tissue. The IS mixture was composed of the “Ceramide/Sphingoid Internal Standard Mixture I” that was completed with m18:1/12:0 and ST18:1/12:0. Samples were incubated overnight at 48 °C. After cooling to room temperature, 100 µL of KOH (1 M in methanol) was added and the samples were incubated for 2 h at 37 °C to hydrolyze glycerophospholipids, which could otherwise interfere with the determination of sphingolipids. A 10 µL aliquot of 50% acetic acid was added and the samples were centrifuged for 15 min at 4500× g. The supernatant was collected, and the residue was extracted again with 600 µL of methanol/chloroform (2:1). The second supernatant was added to the first and then evaporated to dryness. The dry residue was solubilized in 200 µL methanol, and a 5 µL aliquot was injected into the UHPLC-MSMS system comprising a 1260 binary pump, an autosampler, and an Agilent 6410 triple quadrupole spectrometer (Agilent, Santa Clara, CA, USA). The analytes were separated on a Poroshell 120 column (3.0 × 50 mm, 2.7 µm). The mobile phase was (A) methanol/acetonitrile/isopropanol (4/1/1) and (B) water, each containing 10 mM ammonium acetate and 0.2% formic acid. The mobile phase was delivered using a gradient of elution at a flow rate of 0.3 mL/min, as previously described [13]. Sphingolipids were detected after positive electrospray ionization under the following conditions: temperature 300 °C, flow rate of 10 L/min, pressure of 25 psi, capillary voltage 4000 V. Transitions, fragmentor voltages, and collision energies were reported in [22]. Agilent Mass-Hunter quantitative analysis software (B.05.00 SP03/Build 5.0.291.7) was used to analyze the chromatograms. The precision of the method expressed as a relative standard deviation (RSD) was considered acceptable for an RSD of 20%. As shown in Table S3, the linearity of the method of analysis was good over a relatively large range of concentrations, in agreement with previous results [13,22]. The recovery of the 12 IS in heart, gizzard, and breast muscle is presented in Table S4. The recovery varied according to the tissue and the analyte. The lowest recoveries of the IS were measured in breast muscle, whereas the recoveries in heart and gizzard were similar. A high recovery attributed to a positive matrix effect was observed for 18:1/12P and to a lesser extent for d17:1P and d17:0P, which is in agreement with previous results involving other tissues [13,22]. The lowest recovery was measured for 18:1:25:0, which is in agreement with previous results involving other tissues [13,22]. The repeatability of the method was considered good, with an RSD of 20%. Good repeatability was observed for all the IS in heart, gizzard, and breast muscle except for 18:1/25:0 in gizzard, for which an RSD of 25% was found. This value was considered acceptable, with a high RSD being already found for this analyte in other tissues [13,22].

The sphingolipid concentrations were determined from the calibration curves obtained with the standards. The final concentrations in tissues were corrected by the recovery measured for the corresponding IS. No correction was used for 18:1/2:0, 18:0/2:0, GluSo, LysoSM, and LacSo. The concentrations of sphingolipids not available as standards were calculated using the calibration curves obtained with standards of the same class with the closest mass and similar abundance. The final concentrations in tissues were corrected as carried out for the sphingolipids available as standards.

5.4. Analytes and ReagentsOne-way ANOVA was carried out to compare the sphingolipid concentrations in the controls and in the chickens fed fumonisins after determination of the homogeneity of variance (Hartley’s test). Significant differences were reported as follows: (*) 0.05 pppFigures S1 and S2. All the statistical analyses were carried out with XLSTAT Biomed software (Addinsoft, Bordeaux, France).

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