Targeted Sphingolipid Analysis in Heart, Gizzard, and Breast Muscle in Chickens Reveals Possible New Target Organs of Fumonisins

Alteration of sphingolipid synthesis is a key event in fumonisins toxicity, but only limited data have been reported regarding the effects of fumonisins on the sphingolipidome. Recent studies in chickens found that the changes in sphingolipids in liver, kidney, lung, and brain differed greatly. This study aimed to determine the effects of fumonisins on sphingolipids in heart, gizzard, and breast muscle in chickens fed 20.8 mg FB1 + FB2/kg for 9 days. A significant increase in the sphinganine:sphingosine ratio due to an increase in sphinganine was observed in heart and gizzard. Dihydroceramides and ceramides increased in the hearts of chickens fed fumonisins, but decreased in the gizzard. The dihydrosphingomyelin, sphingomyelin, and glycosylceramide concentrations paralleled those of ceramides, although the effects were less pronounced. In the heart, sphingolipids with fatty acid chain lengths of 20 to 26 carbons were more affected than those with 14–16 carbons; this difference was not observed in the gizzard. Partial least squares-discriminant analysis on sphingolipids in the heart allowed chickens to be divided into two distinct groups according to their diet. The same was the case for the gizzard. Pearson coefficients of correlation among all the sphingolipids assayed revealed strong positive correlations in the hearts of chickens fed fumonisins compared to chickens fed a control diet, as well as compared to gizzard, irrespective of the diet fed. By contrast, no effect of fumonisins was observed on sphingolipids in breast muscle.


Effect of Fumonisins According to the Class of Sphingolipids
The concentrations of sphingolipids measured in heart, gizzard, and breast muscle of chickens fed the control diet and chickens fed a diet containing 20.8 mg FB1 + FB2/kg over a period of 9 days are presented in Table 1. The effects of fumonisins according to the class of sphingolipids are presented in Figure 2.
Partial least square-discriminant analysis was conducted to determine whether changes in the heart sphingolipidome were sufficient to separate chickens into two groups according to their exposure to fumonisins. As shown in Figure 3C, a good separation of chickens was observed in this analysis. The values of the R 2 Y and R 2 X indices were 0.608 and 0.632, respectively, indicating that the selected sphingolipids could predict the group that the chickens belonged to. The value of Q 2 with the two first components was 0.403, indicating that the model was of medium quality ( Figure 3D). Not surprisingly, sphin- , and glycosylceramides (GlyCer) in the heart, gizzard, and breast muscle of chickens fed the control diet free of mycotoxins (C) and chickens fed 9 days with a diet containing 20.8 mg FB1 + FB2/kg (FB). * 0.05< p <0.01 and *** p < 0.001.
Partial least square-discriminant analysis was conducted to determine whether changes in the heart sphingolipidome were sufficient to separate chickens into two groups according to their exposure to fumonisins. As shown in Figure 3C, a good separation of chickens was observed in this analysis. The values of the R 2 Y and R 2 X indices were 0.608 and 0.632, respectively, indicating that the selected sphingolipids could predict the group that the chickens belonged to. The value of Q 2 with the two first components was 0.403, indicating that the model was of medium quality ( Figure 3D). Not surprisingly, sphingolipids that were important in the projection corresponded to variables that differed significantly in chickens fed fumonisins versus the controls. It is also interesting to note that SM18:1/14:0 and SM18:1/16:0 were important in explaining the repartition of chickens into the two groups ( Figure 3A,B), whereas only a very weak difference was noted in the mean concentration of these analytes in heart compared to the controls (Table 1). Other variables important in the projection corresponded to sphingolipids that were increased by fumonisins for which the changes measured were considered not significant by ANOVA. golipids that were important in the projection corresponded to variables that differed significantly in chickens fed fumonisins versus the controls. It is also interesting to note that SM18:1/14:0 and SM18:1/16:0 were important in explaining the repartition of chickens into the two groups ( Figure 3A,B), whereas only a very weak difference was noted in the mean concentration of these analytes in heart compared to the controls (Table 1). Other variables important in the projection corresponded to sphingolipids that were increased by fumonisins for which the changes measured were considered not significant by ANOVA.   Table S1.   Table S1.    Table S1.
Only weak correlations were observed between Sa and So, among the sphingoid bases and the other sphingolipids in control chickens (i.e., unexposed to fumonisins) ( Figure 4A). Moreover, the correlations among the different classes of sphingolipids were weak, except for ceramides and glycosylceramides, which exhibited significant positive correlations, and ceramides and sphingomyelins, which exhibited significant negative correlations. By contrast, significant positive correlations were observed within the same class among dihydroceramides, ceramides, sphingomyelins, hexosylceramides, and lactosylceramides, which have similar fatty acid chain lengths. The feeding of fumonisins strongly increased the positive correlations measured among sphingolipids in the heart ( Figure 4B). Notably, significant positive correlations among the different classes of sphingolipids were observed in chickens fed fumonisins, which was not the case in the controls. Moreover, the coefficients of correlation found among the same class of sphingolipids with similar fatty acid chain lengths were generally increased (Table S1B). The correlations among unsaturated sphingolipids and other analytes were close to those observed for the corresponding saturated forms. The correlations among C25 and C26 sphingolipids and other analytes were close to those observed for C24 sphingolipids ( Table S1).
All of these results suggest a significant effect of fumonisins on sphingolipids in the heart. This effect was dominated by an increase in dihydroceramides and ceramides and, to a lesser extent, an increase in glycosylceramides. This was accompanied by strong positive correlations between the different classes and subclasses of sphingolipids. The effects of fumonisins also varied according to the fatty acid chain length. C20-C26 ceramides were significantly increased by the addition of the toxins to the feed (p = 0.029), whereas C14-C16 ceramides were not (p = 0.112). Additionally, Hex20-Hex26 hexosylceramides, Lac20-Lac26 lactosylceramides, and SM20-SM26 sphingomyelins tended to increase, whereas Hex16, Lac16, and SM14SM16 were unaffected.

Effects of Fumonisins on Sphingolipids in Gizzard
As shown in Table 1, the sphinganine concentration in the gizzards of chickens fed a diet containing 20.8 mg FB1 + FB2/kg over a period of 9 days was significantly increased. A significant increase in N-acetylsphinganine (d18:0/2:0) was also observed. By contrast, the levels of most of the dihydroceramides and most of the ceramides assayed were decreased by fumonisins, and this effect was significant for 18:1/14:0, 18:1/16.0, 18:0/16:0, 18:0/18:0, 18:1/22:2, 18:0/22:0, 18:1/23:0, 18:1/24:2, 18:1/26:2, 18:1/26:1, and 18:1/26:2. No significant difference between groups was observed in terms of the dihydrosphingomyelin and sphingomyelin levels in gizzard. Hex18:1/24:1 and Lac18:1/16:0 were significantly decreased by fumonisins, and most of the other glycosylceramides tended to decrease, but the effect was not significant (Table 1). Figure 5 shows the results of the partial least square-discriminant analysis conducted on the sphingolipids in gizzard. A good separation of the chickens according to the feed was observed ( Figure 5C). The values of the R 2 Y, R 2 X, and Q 2 indices, which were 0.755, 0.615, and 0.671, respectively, indicated the good predicted appurtenance of the chickens to the different groups and the good quality of the model. The confusion matrix confirmed the model was highly sensitive and specific ( Figure 5D). The sphingolipids that were important in the projection mainly corresponded to the dihydroceramides, ceramides, and glycosylceramides that were significantly decreased in chickens fed fumonisins. SM18:1/16:0 and SM18:1/18:0 also had VIP scores above 1.1 in this analysis ( Figure 5A,B), although their concentrations in gizzards did not differ from the controls (Table 1) The correlations measured among the assayed sphingolipids in gizzards in this study are reported in Figure 6 and Table S2. Strong significant positive correlations were observed among So and dihydroceramides and among So and ceramides, whereas weak negative correlations were observed among Sa and dihydroceramides in chickens unexposed to fumonisins ( Figure 6A). Significant positive correlations were observed among sphingolipids of the same class with similar fatty acid chain lengths. Additionally, a significant positive correlation was observed between dihydroceramides and ceramides and between sphingomyelins and hexosylceramides. Most of the correlations between ceramides and sphingomyelins, ceramides and hexosylceramides, and between hexosylceramides and lactosylceramides were weak and not significant. Feeding fumonisins only resulted in weak effects on the correlations observed among sphingolipids ( Figure  6B). The correlations among unsaturated sphingolipids and other analytes were close to those observed for the corresponding saturated forms. The correlations among C25 and C26 sphingolipids and other analytes were close to those observed for C24 sphingolipids (Table S2).
These results suggest that there was a significant effect of fumonisins on sphingolipids in gizzards in this study. The effects observed were dominated by a decrease in dihydroceramides and ceramides. Sphingomyelins and glycosylceramides also tended to decrease in gizzards of chickens fed fumonisins, although the effects were not significant The correlations measured among the assayed sphingolipids in gizzards in this study are reported in Figure 6 and Table S2. Strong significant positive correlations were observed among So and dihydroceramides and among So and ceramides, whereas weak negative correlations were observed among Sa and dihydroceramides in chickens unexposed to fumonisins ( Figure 6A). Significant positive correlations were observed among sphingolipids of the same class with similar fatty acid chain lengths. Additionally, a significant positive correlation was observed between dihydroceramides and ceramides and between sphingomyelins and hexosylceramides. Most of the correlations between ceramides and sphingomyelins, ceramides and hexosylceramides, and between hexosylceramides and lactosylceramides were weak and not significant. Feeding fumonisins only resulted in weak effects on the correlations observed among sphingolipids ( Figure 6B). The correlations among unsaturated sphingolipids and other analytes were close to those observed for the corresponding saturated forms. The correlations among C25 and C26 sphingolipids and other analytes were close to those observed for C24 sphingolipids (Table S2).
at the class level and varied with the analyte assayed. The feeding of fumonisins did not profoundly change the correlations among the sphingolipids in the gizzard.   Table S2.
These results suggest that there was a significant effect of fumonisins on sphingolipids in gizzards in this study. The effects observed were dominated by a decrease in dihydroceramides and ceramides. Sphingomyelins and glycosylceramides also tended to decrease in gizzards of chickens fed fumonisins, although the effects were not significant at the class level and varied with the analyte assayed. The feeding of fumonisins did not profoundly change the correlations among the sphingolipids in the gizzard.

Effects of Fumonisins on Sphingolipids in Breast Muscle
The concentrations of sphingolipids measured in breast muscle of chickens fed a control diet free of mycotoxins and chickens fed 9 days with a diet containing 20.8 mg FB1 + FB2/kg are reported in Table 1. Sphingosine was the only sphingolipid assayed for which the concentration in gizzard was significantly affected by fumonisins, and its concentration increased. There were only weak non-significant changes in the concentrations of dihydroceramides, ceramides, sphingomyelins, hexosylceramides, and lactosylceramides.
Partial least square-discriminant analysis was conducted to determine whether this analysis would be able to discriminate chickens according to the diet that they had been fed ( Figure S1). As shown in Figure S1C, there was a good separation of chickens. The values of the R 2 Y and the R 2 X indices were satisfactory, and the confusion matrix revealed the model was highly sensitive and specific ( Figure S1D). However, the value of Q 2 obtained with the two first components was only 0.185, indicating that the model can vary greatly depending on the sphingolipids selected for the modeling. Additionally, the standard deviations observed for the score of the variables important in the projection (VIP) were high in breast muscle ( Figure S1A,B), confirming high variability in the modeling.
Finally, all together, the total amount of sphingolipids (Figure 2), the concentration of the various analytes assayed (Table 1), and the comparison of the repartition of the chickens into groups according to the diet that they had been fed using PLS-DA ( Figure S1) revealed that the presence of fumonisins in the feed only had a minor effect on the sphingolipids in breast muscle in this study.

Discussion
Feeding 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 palmitoyltransferase 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.

Conclusions
In conclusion, this study revealed for the first time that feeding fumonisins at a concentration that did not alter performances in chickens nonetheless resulted in several changes in the sphingolipid composition of muscles that varied according to the type of muscle. Whereas no effects of fumonisins were noted in breast muscle, an increase in sphingoid bases, dihydroceramides, ceramides, and glycosylceramides, and to a lesser extent also sphingomyelins, was observed in the myocardium. These changes were more pronounced for sphingolipids with fatty acid chains of 20 to 26 carbons than for those with 14-16 carbon chain lengths. By contrast, a decrease in dihydroceramides and ceramides, and to a lesser extent also sphingomyelins, was observed in the gizzard. The decreases in sphingolipid levels in the gizzard appeared to be the same irrespective of the fatty acid chain length. Taken together, these results confirmed that the effects of fumonisins on sphingolipids at the scale of the organism are complex and probably involve cell-or metabolite-mediated effects in association with the inhibition of CerS activity. Further studies are necessary to understand these mechanisms and their consequences on health.

Tissue Samples
Heart, 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.

Sphingolipids in Tissues
The 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.

Analytes and Reagents
One-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 < p < 0.01, (**) 0.01 < p < 0.001, and (***) p < 0.001. Partial least squares-discriminant analysis (PLS-DA) was carried out to reveal the appurtenance of the chickens to the control and the fumonisins-fed group and to identify the sphingolipids that were important in the model. A score above 1.1 was retained to select the variables important in the projection (VIP) for heart and gizzard, whereas a score above 0.8 was retained for breast muscle. Pearson coefficients among the sphingolipids assayed in this study were measured and reported in bold font when significant in Figures S1 and S2. All the statistical analyses were carried out with XLSTAT Biomed software (Addinsoft, Bordeaux, France).

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/toxins14120828/s1, Table S1: Pearson coefficients of correlation among sphingolipids dosed in heart; Table S2: Pearson coefficients of correlation among sphingolipids dosed in gizzard; Figure S1: Partial least squares discriminant analysis (PLS-DA) of sphingolipids measured in the breast muscle of chickens fed 9 days with a control diet free of mycotoxins or a diet containing 20.  Table S3: Linearity of the method measured for the sphingolipids available as standards; Table S4: Recovery of the internal standards of sphingolipids measured in the heart, gizzard, and breast muscle of chickens.