Fumonisins alone or mixed with other fusariotoxins increase the C22–24:C16 sphingolipid ratios in chickens and ducks, while deoxynivalenol and zearalenone have no effect

doi:10.21203/rs.3.rs-3725230/v1

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Fumonisins alone or mixed with other fusariotoxins increase the C22–24:C16 sphingolipid ratios in chickens and ducks, while deoxynivalenol and zearalenone have no effect

https://doi.org/10.21203/rs.3.rs-3725230/v1

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Background

The inhibitory effect of fumonisins on ceramide synthases results in several effects on the sphingolipidome, and recent results in chickens suggest that the C22–24:C16 sphingolipid ratios may be complementary biomarkers to Sa:So. Feeding diets containing fumonisins, deoxynivalenol, or zearalenone alone or in combination up to the maximum levels recommended by the European Commission for 35 days in chickens and 12 days in ducks resulted in no effects on performance or toxicity. The aim of this study was to investigate their effects on the liver sphingolipidome.

Results

Sphingolipids were characterised by UHPLC-MSMS. Numerous significant effects of fusariotoxins on sphingolipids were observed at the class and analyte levels. Fusariotoxins alone decreased sphingolipids in chickens but had weak effects in ducks. Feeding fumonisins in combination with deoxynivalenol and zearalenone increased sphingolipid levels in chickens but not in ducks. These increases were mainly due to an increase in C22–24 sphingolipids, whereas C16 sphingolipids decreased. The Sa:So ratio and the C22–24:C16 ratio for ceramides, sphingomyelins, monohexosylceramides, dihydroceramides, and dihydrosphingomyelins were unaffected in chickens and ducks fed the diets containing deoxynivalenol or zearalenone but increased in animals fed the fumonisin diets. Interestingly, while the effects of feeding fumonisins alone or in combination on the total amount of sphingolipids differed in chickens, the Sa:So and C22-24:C16 ratios were similar. Partial least squares-discriminant analysis of sphingolipids did not allow discrimination of the animals exposed to deoxynivalenol or zearalenone from the unexposed animals. By contrast, good discrimination of the animals fed the diets containing fumonisins was achieved. Sphinganine, d20 sphinganine, a large number of C16 sphingolipids, and dihydrosphingomyelins for which the fatty acid contains more than 18 carbon atoms, were important variables in the models obtained in chickens and ducks.

Conclusions

The C22–24:C16 ratios of sphingolipids are increased in chickens and ducks by intake of fumonisins but not deoxynivalenol or zearalenone. The increases were similar when fumonisins were fed alone or in combination with deoxynivalenol or zearalenone. The increase in the C22–24:C16 ratio may be an important factor in explaining the interspecies differences in sensitivity to fumonisin toxicity.

Chicken

Duck

Fusariotoxins

Sphingolipid

Biomarker

Fumonisins are mycotoxins found in food and feed in most countries of the world [1–4]. These compounds are carcinogenic to rodents and are classified as possibly carcinogenic to humans [5]. Most animal species are sensitive to fumonisin toxicity, but this toxicity varies greatly [1, 3, 6]. Although the origin of these differences remains unknown, the inhibition of dihydroceramide (DHCer) synthesis by fumonisin B1 (FB1) by blocking dihydroceramide synthases (DHCerS), due to a structural analogy between FB1, sphinganine (Sa) and fatty acids, is a common effect reported in all studies where it has been investigated [7]. Inhibition of the DHCerS results in the accumulation of sphinganine and inhibition of the de novo synthesis of all sphingolipids: ceramides (Cer), monohexosylceramides (HexCer), lactosylceramides (LacCer), and sphingomyelins (SM). This inhibition can lead to hydrolysis of membrane sphingolipids, the goal of which for the cell is to maintain its Cer pool. This mechanism is considered to be the main mechanism explaining the increase in sphingosine (So) frequently reported following fumonisin exposure [7]. The increase in So levels is even more pronounced because FB1 also blocks CerS. The variability in the effect of fumonisins on Sa and So concentrations makes the Sa:So ratio preferable to Sa as a biomarker of exposure in humans and animals [8].

The toxic effects of fumonisins are diverse and have been the subject of recent bibliographic reviews [1, 3, 4]. It has been suggested that all of the mechanisms of action considered may result from direct or indirect effects of fumonisins on sphingolipids, particularly ceramides [7]. Detailed characterisation of the effects of fumonisins on ceramides is all the more important as it has been shown that deletion of CerS2 in mice leads to a decrease in very-long-chain Cer and is accompanied by a "compensatory" increase in long-chain Cer, all of which reproduce the signs of fumonisins hepatotoxicity [9, 10]. Furthermore, analysis of the sphingolipidome in pigs exposed to fumonisins revealed a reduction of very-long-chain Cer in the lung, and this organ is the main target of fumonisins in this species [11]. On the other hand, no significant effect of fumonisins on very-long-chain Cer levels was detected in the liver, and this organ is hence less sensitive to fumonisin toxicity in this species. Interestingly, studies conducted on a porcine jejunum epithelial cell line also revealed that FB1 preferentially inhibited the expression of CerS2, leading to overexpression of endoplasmic reticulum stress markers, while CerS2 overexpression protected from FB1-induced damages [12]. More generally, alteration of the ratio between long-chain sphingolipids and very-long-chain sphingolipids has been reported in various human diseases, independent of the intake of fumonisins, and these ratios are considered key elements in terms of health [13–19].

The sensitivity of different species to fumonisin toxicity varies, but the origin of this variability remains unknown. Therefore, poultry, which are less sensitive to the toxic effects of fumonisins than pigs and horses, are a study model likely to reveal the mechanisms underlying the differences in sensitivity between species. Toxicokinetic studies have shown that the bioavailability of fumonisins is similar in most species and that the relative resistance of poultry to fumonisin toxicity cannot be explained by differences in toxicokinetics alone [20]. Several studies in chickens and turkeys have shown that administration of fumonisins leads to a decrease in long-chain ceramides and an increase in very-long-chain ceramides in the liver [21–24]. Very-long-chain Cer (incorporating a fatty acid of 22 to 26 carbon atoms, C22-26) are mainly synthesised by CerS type 2, whereas long-chain Cer (incorporating a fatty acid of 14 to 16 carbon atoms, C14-16) are synthesised in the liver by CerS type 5 [25–28]. The C22–24:C16 sphingolipid ratios were hence proposed as a complementary biomarker to the Sa:So ratio in the characterisation of the effects of fumonisins on the sphingolipidome [27].

The aim of this study was to determine the effect of the distribution of fumonisin-contaminated diets on sphingolipids in the liver under different experimental conditions that have not been investigated to date. These studies were performed I) in chickens exposed to fumonisins from birth to 35 days of age to reveal the effects of prolonged exposure and II) in ducks exposed for 12 days during force-feeding to reveal the effects of fumonisins on sphingolipids in this species. In order to determine the specificity of the effects observed, sphingolipids were also dosed in chickens and ducks fed diets containing deoxynivalenol or zearalenone. Finally, with the aim of revealing possible interactions between fusariotoxins that may be present together in diets [29–31], the sphingolipids were measured in chickens and ducks exposed to fumonisins in combination with deoxynivalenol and zearalenone. These studies were carried out at levels of fusariotoxin exposure close to the maximum levels recommended by the European Commission for poultry feed [32].

Analytes and reagents

The analytes and reagents used in this study were purchased from Sigma (Sigma-Aldrich Chimie SARL, Saint Quentin Fallavier, France) and Sharlab (Sharlab S.L., Sentmenat, Spain). The reagents and solvents used for separation and assay on sphingolipids were of UHPLC-MSMS quality, and the reagents and solvents used for extraction of analytes were of HPLC quality. The sphingolipid internal standards used correspond to "Ceramide/Sphingoid Internal Standard Mixture I" from Avanti Polar Lipids, consisting of C17-sphingosine, C17-sphinganine, C17-sphingosine-1P, C17-sphingosine-1P, C12:0-lactosyl(ß) ceramide, C12:0-sphingomyelin, C12:0-glucosyl(ß) ceramide, C12:0-ceramide, C12:0-ceramide-1P, and C25:0-ceramide, each at a concentration of 25µM in ethanol. This mixture was completed with m17:1/12:0, m17:0/12:0, m18:1/12:0, and C12:0-ceramide sulphate solubilised in ethanol, each at a concentration of 25 µM.

Biological samples

The chicken and duck liver samples used in this study were obtained from animals fed diets containing fumonisins, deoxynivalenol, or zearalenone, alone and as mixtures, according to the protocols described in [33] and [34], respectively. The experimental procedures were in accordance with the French national guidelines for the care and use of animals in research. The chicken experimental protocol was approved by the Ministry of Higher Education and Research and registered under number 02032.01. The duck protocol was approved by the INRAE UEPFG institutional review board under number PP-5-12.

The study in chickens was carried out on 75 male animals of the Ross PM3 strain, reared in individual cages at the ARVALIS experimental station (Institut du végétal, Villerable, France), according to the protocol described in [33]. The animals were divided into five groups that received, from birth to 35 days of age, the control diet (CON) considered free of mycotoxins, the diet containing fumonisins (FB) at a concentration of 23.1 mg FB1 + FB2/kg, the diet containing deoxynivalenol (DON) at a concentration of 4.72 mg/kg, the diet containing zearalenone (ZEN) at a concentration of 0.48 mg/kg, and the diet containing a mixture of fumonisins, deoxynivalenol, and zearalenone (FDZ) at concentrations of 19.23, 4.17, and 0.43 mg/kg, respectively. Experimental corn-soybean diets were prepared by the incorporation of powders containing the mycotoxins, as described in [33]. At 35 days of age, the animals were anaesthetised by electronarcosis and sacrificed by exsanguination. An autopsy was performed on all the animals, and liver samples were collected and stored at -80°C until analysis. Sphingolipid analysis was performed on 50 liver samples, with 10 animals per diet/group. No differences were observed between the groups with respect to performance, relative organ weights, biochemistry, histopathology, gut morphometrics, oxidative damage variables, and markers of testicular toxicity. The feed composition, including mycotoxins analysis, and the various parameters measured in this study are available in [33].

The study in ducks was carried out on 75 male Cairina moschata x Anas platyrhynchos of the commercial lines MMG PKL (Couvoir Ducournau, Bonnegarde, France) reared in individual cages at the Palmipole experimental station (Domaine d'Artiguères, Benquet, France) according to the protocol described in [34]. The ducks were fed mycotoxin-free diets from birth until 84 days of age. On day 84, the ducks were force-fed for 12 days according to the standard protocol [35]. Capsules without mycotoxins (CON) and capsules containing fusariotoxins, alone or as a mixture, were administered in the middle of each meal. The different doses of fusariotoxins administered were calculated to correspond to an exposure equivalent to the consumption of diets containing fumonisins (FB) at a concentration of 20 mg FB1 + FB2/kg, deoxynivalenol (DON) at a concentration of 5 mg/kg; zearalenone (ZEN) at a concentration of 0.5 mg/kg; and fumonisins, deoxynivalenol, and zearalenone as a mixture (FDZ) at concentrations of 20, 5, and 0.5 mg/kg, respectively. At 96 days of age, the animals were anaesthetised by electronarcosis and sacrificed by exsanguination. An autopsy was performed on all the animals, and liver samples were collected and stored at -80°C until analysis. Sphingolipid analysis was performed on 50 liver samples, with 10 animals per diet group. A slight decrease in body weight and feed conversion ratio, associated with a decrease in duodenal weight and jejunal length, was observed in ducks fed the FDZ diet. However, no effect was observed on the plasma biochemistry, histopathology, markers of oxidative damage at the liver and plasma levels, and markers of testicular function, irrespective of the diet given. The feed composition, including mycotoxins analysis, and the various parameters measured in this study are available in [34].

Analysis of sphingolipids

Extraction of sphingolipids in the liver was performed as previously described but with slight modification [24]. Briefly, 1 g of tissue was homogenised with a Potter–Elvehjem homogeniser in 3 mL of phosphate buffer (0.1 M, pH 7.4) and then centrifuged at 3000 × g for 15 min. Forty microliters of the supernatant was diluted with 120 µL of 0.9% NaCl, spiked with 10 µL of a solution containing the internal standards, each at a final concentration equivalent to 6250 pmol/g liver. Six hundred microliters were added, and the mixture was incubated overnight at 48°C. After returning to room temperature, 100 µL of 1 M KOH in methanolic solution was added and the samples were incubated for 2 hours at 37°C. After returning to room temperature, 10 µL of 50% acetic acid was added and the mixture was centrifuged at 4500 x g for 15 min. The supernatant was collected and the residue was extracted a second time with 600 µL methanol/chloroform (2/1, v/v). The supernatant was collected and added to the first supernatant, and the combined supernatants were evaporated to dryness. The dry residue was solubilised in 200 µL MeOH and filtered before injection into the chromatographic system.

The analytes were separated on a Poroshell 120 column (3.0 × 50 mm, 2.7 µm) using an Agilent 1260 binary pump (Agilent, Santa Clara, CA, USA), as previously described [24]. Sphingolipid detection was performed by dynamic multiple reaction monitoring (MRM) after positive ionisation at 300°C using an Agilent 6410 triple quadrupole spectrometer. The MRM transitions, fragmentation voltages, collision energies, and retention times of all analytes measured in this study are provided in Table S1.

Chromatograms were analysed using Agilent MassHunter quantitative analysis software with quadratic calibration using a 1/x² weighting factor. The linearity of the method was good over a wide range of concentrations (Table S2), consistent with previous results [27, 36]. The accuracy was considered acceptable, with a relative standard deviation (RSD) of 20%. The concentrations of sphingolipids not available as standards were calculated from the calibration curves of analytes of the same neighbouring mass class. The intra-day repeatability measured on the different internal standards is provided in Table S3. An RSD of 20% of the recovery was considered acceptable. The sphingolipid concentrations in the liver were corrected by the recovery measured for the different internal standards corresponding to the different sphingolipid classes.

Statistical analysis

Statistical analyses were performed using XLSTAT Biomed software (Addinsoft, 33000 Bordeaux, France). All of the statistical analysis were carried out separately in chickens and ducks. An effect was considered significant when the P value was < 0.05.

The effects of fusariotoxins on sphingolipids were evaluated globally using partial least squares-discriminant analysis (PLS-DA). Different analysis strategies were used. In an initial analysis, the effect of fusariotoxins was evaluated by comparing the animals fed the five different diets corresponding to the control diet (CON, n = 10), the fumonisin diet (FB, n = 10), the deoxynivalenol diet (DON, n = 10), the zearalenone diet (ZEN, n = 10), and the fumonisin, deoxynivalenol, and zearalenone mixture diet (FDZ, n = 10). In a second analysis, the animals were separated into three groups corresponding to animals fed the FB diet (n = 10), animals fed the FDZ diet (n = 10), and animals fed the diets without fumonisins (0, n = 30), with the latter corresponding to the grouping of animals fed the CON, DON, or ZEN diets. In a third analysis, the effect of fumonisins on sphingolipids was evaluated by comparing the animals divided into two groups consisting of animals exposed to fumonisins (1, n = 20), corresponding to the grouping of animals fed the FB or FDZ diets, and animals not exposed to fumonisins (0, n = 30), corresponding to the grouping of animals fed the CON, DON, or ZEN diets. This analysis strategy was also used to evaluate the effect of deoxynivalenol on sphingolipids. For this purpose, the animals were divided into two groups consisting of animals exposed to deoxynivalenol (1, n = 20), corresponding to animals fed the DON or FDZ diets, and animals not exposed to deoxynivalenol (0, n = 30), corresponding to the animals fed the CON, FB, or ZEN diets. Similarly, the effect of zearalenone on sphingolipids was evaluated by comparing animals divided into two groups consisting of animals exposed to zearalenone (1, n = 20), corresponding to the animals fed the ZEN or FDZ diets, and animals not exposed to zearalenone (0, n = 30), corresponding to the animals fed the CON, FB, or DON diets.

The effects of fusariotoxins on sphingolipids were also analysed in terms of total sphingolipids, corresponding to the sum of the analytes measured within the same class, in terms of sphingolipids for which the fatty acid contained 16 carbon atoms (C16), and on sphingolipids for which the fatty acid contained 22 to 24 carbon atoms (C22–24). The C22–24:C16 ratios were also calculated for the different classes of sphingolipids. In all of these analyses, the results were expressed as fold changes to facilitate comparison. The effects of the various diets on all of the analytes dosed are reported in Supplementary Tables S4 and S5. For all of these analyses, the results are presented as means ± the standard deviation. Differences between groups were compared using one-way ANOVA after testing for homogeneity of variance (Hartley test). When a significant difference was observed, individual means were compared (Duncan). Different letters indicate statistically different means.

In chickens, the administration of the diets containing fumonisins alone (FB) increased the level of hepatic sphinganine (Sa) and significantly decreased the levels of ceramides (Cer), sphingomyelins (SM), monohexosylceramides (HexCer), lactosylceramides (LacCer), dihydroceramides (DHCer), and dihydrosphingomyelins (DHSM) (Fig. 1A). Administration of the diets containing deoxynivalenol alone (DON) or zearalenone alone (ZEN) also reduced the levels of most sphingolipids. In chickens, the administration of diets containing fumonisins, deoxynivalenol, and zearalenone (FDZ) in combination greatly increased the sphinganine levels, had no effect on the Cer, HexCer, and LacCer levels, and increased the SM, DHCer, and DHSM levels (Fig. 1A). In ducks, administration of the FB diet increased the sphinganine levels and had no effect on the class level of sphingolipids (Fig. 1B). Feeding the DON or ZEN diets had no effect on the sphinganine concentration and only the level of SM was slightly increased. The FDZ diet had the same effects on the sphingolipid levels in the liver as the FB diets in ducks.

The effects of the various diets on the main sphingolipids assayed in this study are presented in Tables 1 and 2 for chickens and ducks respectively, while the effects on all of the analytes assayed are shown in Tables S4 and S5, respectively. The effects of fusariotoxins on a given class of sphingolipids do not reflect the effects observed on the other analytes. For example, in chickens fed FDZ, the d18:1/16:0 was significantly reduced, whereas d18:1/22:0 was significantly increased (Table 1). The sphingolipid concentrations in the liver were also highly variable depending on the animal species, the class of sphingolipid, and the chain length of the incorporated fatty acid. For example, C16 LacCer were not detectable in ducks, whereas C22–24 LacCer were not detectable in chickens in this study. Of note, as the effects of fumonisins on different sphingolipids have been reported to vary depending on the chain length of the incorporated fatty acid, a specific analysis of C16 sphingolipids and of the sum of the C22–24 sphingolipids was performed (Fig. 2). These effects are expressed as fold changes to facilitate comparison.

In chickens, administration of the FB diet greatly reduced the levels of Cer, SM, HexCer, LacCer, DHCer, and DHSM with C16, while sphingolipids levels with C22–24 were less affected (Figs. 2A and B). In ducks, the FB diet reduced the levels of Cer, SM, and HexCer with C16, but had no effect on the levels of DHCer and DHSM with C16 (Fig. 2C). Similarly, in ducks, the FB diet had no effect on the levels of Cer, SM, HexCer, DHCer, and DHSM with C22–24 (Fig. 2D). The effects of diets containing DON and ZEN were similar for C16 and C22–24 sphingolipid, but different in chickens and ducks (Fig. 2). A decrease in Cer, SM, HexCer, DHCer, and DHSM levels was observed in chickens (Figs. 2A and B), whereas these compounds tended to increase in ducks (Figs. 2C and D).

Table 1

Concentrations of the main sphingolipids measured in the livers of chickens fed a mycotoxin-free diet (CON) or diets containing deoxynivalenol (DON), zearalenone (ZEN), fumonisins (FB), or a mixture of these toxins (FDZ) at the maximum levels recommended by the European Commission¹.
Analytes/Feed	CON	DON	ZEN	FB	FDZ
Sphingoid bases
d18:0	1.21 ± 0.44^c	1.06 ± 0.44^c	0.91 ± 0.34^c	1.75 ± 0.46^b	4.06 ± 0.86^a
d18:1	19.4 ± 10.2^a	14.0 ± 4.7^abc	13 ± 2.9^bc	10.6 ± 2.2^c	18.3 ± 1.9^ab
d20:0 ²	95.8 ± 30.5^c	101 ± 14.3^c	91.3 ± 28.1^c	135 ± 37^b	174 ± 23.3^a
d20:1 ²	41.6 ± 10.7	39.8 ± 4.6	38.7 ± 5.6	37.8 ± 5.3	35.9 ± 2.6
t18:0	2.29 ± 0.65^a	2.09 ± 0.79^a	1.64 ± 0.59^ab	1.21 ± 0.3^b	2.31 ± 0.35^a
Dihydroceramides
18:0/16:0	42.1 ± 28.2^ab	25.4 ± 11.6^bc	27.1 ± 23.6^bc	17.3 ± 7.2^c	56.9 ± 20.3^a
18:0/22:0	2.25 ± 1.4^b	2.12 ± 0.9^b	1.89 ± 1.99^b	1.97 ± 0.65^b	5.76 ± 2.12^a
18:0/24:0	2.1 ± 0.82^b	1.68 ± 0.52^b	1.67 ± 1.58^b	1.7 ± 0.5^b	3.84 ± 0.87^a
Ceramides
18:1/14:0	1.23 ± 0.41^a	0.93 ± 0.25^b	0.9 ± 0.33^b	0.5 ± 0.09^c	0.93 ± 0.14^b
18:1/16:0	504 ± 128^a	415 ± 80^b	401 ± 117^b	192 ± 49^c	347 ± 50^b
18:1/18:0	127 ± 42^a	88 ± 19^bc	92.7 ± 35.0^bc	64.2 ± 18.8^c	103 ± 11^ab
18:1/20:0	30.0 ± 9.3^a	22.4 ± 5.1^b	22.4 ± 9.6^b	18.7 ± 4.8^b	33.1 ± 6.3^a
18:1/22:0	143 ± 34^b	118 ± 25^c	108 ± 32^c	109 ± 23^c	170 ± 24^a
18:1/24:0	75.7 ± 25.5^a	55.1 ± 11.8^b	48.6 ± 14.6^b	51.13 ± 11.47^b	88.7 ± 16.12^a
18:1/24:1	154 ± 42^a	114 ± 25^b	100 ± 31^b	103 ± 22^b	175 ± 11^a
18:1/24:2	102 ± 33^a	60.0 ± 15.7^b	50.4 ± 18.9^b	54.7 ± 18.6^b	107 ± 14.^a
Hexosylceramidesr
Hex18:1/16:0	11.5 ± 4^a	7.62 ± 3.41^b	7.91 ± 2.57^b	2.28 ± 0.88^c	7.3 ± 2.69^b
Hex18:1/18:0	6.69 ± 3.11^ab	5.63 ± 3.51^ab	3.92 ± 2.01^bc	2.24 ± 1.02^c	7.55 ± 2.54^a
Hex18:1/20:0	3.71 ± 1.13^b	2.58 ± 0.98^c	2.32 ± 1.05^c	1.87 ± 0.42^c	5.27 ± 1.15^a
Hex18:1/22:0	7.19 ± 2.37^b	4.44 ± 2.2^c	3.42 ± 1.79^c	2.73 ± 0.82^c	10.6 ± 2.5^a
Hex18:1/24:0	15.1 ± 3.3^b	11.4 ± 3.5^c	10.6 ± 3.4^c	9.43 ± 2.27^c	22.2 ± 3.3^a
Hex18:1/24:1	10.5 ± 5.4^b	6.01 ± 2.9^c	4.37 ± 1.9^c	3.75 ± 1.48^c	14.4 ± 5.4^a
Lactosylceramides
Lac18:1/16:0	12.2 ± 3.1^a	8.09 ± 2.12^b	7.72 ± 2.2^b	3.95 ± 1.21^c	8.36 ± 2.54^b
Lac18:1/18:0	4.03 ± 1.83^a	2.79 ± 0.76^ab	2.92 ± 1.13^ab	2.08 ± 1.18^b	3.69 ± 1.06^a
Lac18:1/20:0	36.5 ± 9.2^a	25.1 ± 7.6^b	26.8 ± 11.3^b	17.5 ± 5.7^c	31.5 ± 5.9^ab
Dihydrosphingomyelins
SM18:0/16:0	55.2 ± 33.3^b	44.3 ± 29.6^b	28.9 ± 20.8^b	25.1 ± 7.4^b	110 ± 37^a
SM18:0/18:0	1.76 ± 0.95^b	1.35 ± 0.78^b	1.15 ± 1.11^b	1.14 ± 0.38^b	4.37 ± 1.51^a
SM18:0/20:0	0.65 ± 0.26^b	0.56 ± 0.3^b	0.45 ± 0.33^b	0.49 ± 0.11^b	1.74 ± 0.62^a
SM18:0/22:0	10.2 ± 3.9^b	8.65 ± 4.44^b	6.38 ± 3.84^b	8.81 ± 1.92^b	33.7 ± 12^a
SM18:0/23:0	0.48 ± 0.25^b	0.39 ± 0.24^b	0.26 ± 0.16^b	0.39 ± 0.11^b	2.03 ± 0.89^a
SM18:0/24:0	1.78 ± 0.6^b	1.49 ± 0.72^b	1.05 ± 0.52^b	1.65 ± 0.37^b	5.05 ± 1.57^a
SM18:0/24:1	0.67 ± 0.41^b	0.5 ± 0.36^b	0.33 ± 0.22^b	0.46 ± 0.13^b	2.3 ± 1.1^a
Sphingomyelins
SM18:1/14:0	0.11 ± 0.1^ab	0.12 ± 0.15^ab	0.06 ± 0.03^b	0.06 ± 0.03^b	0.18 ± 0.08^a
SM18:1/16:0	100 ± 70^ab	81.7 ± 73.5^abc	43.8 ± 24^bc	33.8 ± 16.8^c	123 ± 50^a
SM18:1/18:0	98.4 ± 64.0^b	76.4 ± 56.8^b	47.1 ± 22.2^b	50.4 ± 20.8^b	161 ± 54^a
SM18:1/20:0	19.2 ± 11.3^b	15.3 ± 10.9^b	9.3 ± 4.63^b	12.3 ± 4.6^b	40.6 ± 12.4^a
SM18:1/22:0	228 ± 116^b	178 ± 99^bc	106 ± 47^c	159 ± 36^bc	449 ± 110^a
SM18:1/24:0	43.8 ± 22.4^b	32.9 ± 18.2^bc	19 ± 8.8^c	31.2 ± 8^bc	95 ± 27.2^a
SM18:1/24:1	55.7 ± 44.3^b	40.7 ± 35.9^bc	19 ± 11^c	32.9 ± 13.7^bc	119 ± 33^a
SM18:1/24:2	10.8 ± 10.1^b	7.28 ± 8.39^b	2.87 ± 1.85^b	6.24 ± 5.35^b	26.2 ± 9.4^a

¹The diets were administered from birth to 35 days of age, the concentrations of mycotoxins in the various diets are reported in the Materials and Methods. The results are expressed as nmol/kg or as ²pmol/kg of liver as means ± SD, n = 10 per dietary group. Differences between groups were assessed by one-way ANOVA (p < 0.05). Different letters in the same row indicate statistically different means (Duncan, p < 0.05). The results for all analytes measured are presented in Table S4.

Table 2

Concentrations of the main sphingolipids measured in the livers of ducks fed mycotoxin-free diets (CON) or diets containing deoxynivalenol (DON), zearalenone (ZEN), fumonisins (FB), or a mixture of these toxins (FDZ) at the maximum levels recommended by the European Commission¹.
Analytes/Feed	CON	DON	ZEN	FB	FDZ
Sphingoid bases
d18:0	0.51 ± 0.11^b	0.75 ± 0.2^b	0.61 ± 0.29^b	2.59 ± 1.69^a	2.95 ± 2.18^a
d18:1	2.6 ± 0.65	3.89 ± 1.44	2.93 ± 1.8	2.31 ± 0.77	3.99 ± 2.61
d20:0 ²	210 ± 30^b	212 ± 26^b	206 ± 26^b	372 ± 148^a	338 ± 106^a
d20:1 ²	153 ± 20	142 ± 27	152 ± 26	158 ± 30	139 ± 23
Dihydroceramides
18:0/16:0	4.45 ± 0.79	4.89 ± 1.46	4.45 ± 2	4.25 ± 1.03	4.83 ± 2.47
18:0/18:0	4.08 ± 1.08	5.92 ± 1.79	5.61 ± 3.87	6.21 ± 2.77	7.77 ± 4.22
18:0/20:0	1.81 ± 0.44	2.13 ± 0.35	2.16 ± 1.07	2.65 ± 0.88	2.91 ± 1.35
18:0/22:0	0.47 ± 0.08	0.61 ± 0.19	0.58 ± 0.31	0.73 ± 0.23	0.7 ± 0.39
18:0/24:0	1.69 ± 0.51	2.25 ± 0.92	1.9 ± 1.17	2.85 ± 1.45	2.68 ± 1.67
Ceramides
18:1/14:0	0.1 ± 0.02^ab	0.15 ± 0.05^a	0.11 ± 0.06^ab	0.08 ± 0.02^b	0.1 ± 0.06^ab
18:1/16:0	17.2 ± 3.7^ab	22.9 ± 8.1^a	17.9 ± 10.4^ab	10.4 ± 3.2^b	14.5 ± 9.8^ab
18:1/18:0	9.59 ± 2.04	15.1 ± 5.2	13.6 ± 8.6	9.9 ± 3.7	13.9 ± 9.1
18:1/20:0	4.67 ± 1.4	7.51 ± 2.63	6.53 ± 4.31	4.4 ± 1.58	6.04 ± 4.13
18:1/22:0	20.1 ± 5.3	30.1 ± 10	25.3 ± 15.7	18.3 ± 6.8	23.6 ± 14.2
18:1/24:0	14.6 ± 4.0	19.9 ± 6.5	15.7 ± 9.2	13.2 ± 4.6	16.8 ± 10.4
18:1/24:1	11.1 ± 3	15.5 ± 5.7	11.8 ± 6.8	10.2 ± 2.8	15.3 ± 11.2
Hexosylceramides
Hex18:1/16:0	1.05 ± 0.24^b	2.06 ± 0.99^a	1.35 ± 0.91^b	0.72 ± 0.25^b	1.02 ± 0.58^b
Hex18:1/20:0	1.14 ± 0.37	1.94 ± 0.95	1.84 ± 1.88	1.14 ± 0.66	1.21 ± 0.85
Hex18:1/22:0	4.22 ± 1.02	5.81 ± 1.78	4.52 ± 2.42	3.75 ± 1.78	4 ± 2.47
Hex18:1/24:0	2.46 ± 1.03	3.99 ± 2.05	2.87 ± 2.7	2 ± 1.2	2.6 ± 1.74
Lactosylceramides
Lac18:1/20:0	4.44 ± 1.03	7.49 ± 3.01	6.38 ± 4.26	4.53 ± 1.91	6.39 ± 4.69
Lac18:1/22:0	0.74 ± 0.41	1.23 ± 1.05	1.17 ± 1.13	1.01 ± 0.54	0.89 ± 0.64
Dihydrosphingomyelins
SM18:0/16:0	1.97 ± 0.31^b	3.68 ± 1.56^a	2.61 ± 1.7^ab	2.13 ± 0.8^b	2.26 ± 1.44^b
SM18:0/18:0	1.14 ± 0.23	2.45 ± 1	1.99 ± 1.68	2.12 ± 1.08	2.41 ± 1.84
SM18:0/20:0	0.3 ± 0.07	0.62 ± 0.26	0.46 ± 0.33	0.57 ± 0.28	0.65 ± 0.51
SM18:0/22:0	1.4 ± 0.42	2.8 ± 1.25	2.16 ± 1.67	3.38 ± 2.11	3.24 ± 2.47
SM18:0/24:0	0.3 ± 0.14	0.56 ± 0.29	0.43 ± 0.33	0.82 ± 0.51	0.76 ± 0.63
SM18:0/24:1	0.16 ± 0.04	0.34 ± 0.18	0.22 ± 0.17	0.41 ± 0.21	0.45 ± 0.43
Sphingomyelins
SM18:1/14:0	0.05 ± 0.01^b	0.08 ± 0.03^a	0.05 ± 0.03^b	0.04 ± 0.01^b	0.04 ± 0.01^b
SM18:1/16:0	12.5 ± 2.5^b	24.2 ± 11.5^a	16.0 ± 12^b	8.7 ± 3.31^b	8.53 ± 5.54^b
SM18:1/18:0	20.4 ± 5.5^b	49.7 ± 28.2^a	35.8 ± 31.4^ab	24.1 ± 13.1^ab	27 ± 24.5^ab
SM18:1/20:0	4.37 ± 1.29	10.5 ± 6.2	7.96 ± 6.69	5.43 ± 2.77	6.05 ± 5.8
SM18:1/22:0	33.4 ± 8.8	71 ± 38.5	49.7 ± 38.4	38.9 ± 20.5	41.2 ± 36.4
SM18:1/24:0	7.33 ± 2.11	13.6 ± 6.7	9.02 ± 6.4	8.94 ± 4.41	9.06 ± 7.1
SM18:1/24:1	8.03 ± 1.42	15.2 ± 7.0	10.4 ± 7.4	9.35 ± 3.14	9.33 ± 7.14

¹The diets were administered from 84 to 96 days of age, the concentrations of the mycotoxins in the various diets are reported in the Materials and Methods. The results are expressed as nmol/kg or as ²pmol/kg of liver as means ± SD, n = 10 per dietary group. Differences between groups were assessed by one-way ANOVA (p < 0.05). Different letters in the same row indicate statistically different means (Duncan, p < 0.05). The results for all analytes measured are presented in Table S5.

The effect of the FDZ diet on C16 and C22-24 sphingolipid levels differed between species. In chickens, feeding the FDZ diet led to a less pronounced reduction in the levels of Cer, HexCer, and LacCer with C16 than feeding of the FB diet (Fig. 2A). The FDZ diet had no significant effect on the levels of SM, DHCer, and DHSM with C16, nor on Cer and HexCer with C22–24, but it increased the levels of SM, DHCer, and DHSM with C22–24 (Fig. 2B). In ducks, the FDZ diet had the same effects on the levels of C16 and C22–24 sphingolipids as the FB diet (Figs. 2C and D). On the other hand, the effects of the DON and ZEN diets on the levels of the C16 and C22–24 sphingolipids were similar.

The Sa:So ratio and the C22–24:C16 ratios measured for the various sphingolipids were calculated and expressed as fold changes to facilitate comparison (Fig. 3) Administration of the FB and FDZ diets increased the Sa:So ratio, whereas administration of the DON and ZEN diets had no effect. In chickens, the effect of the FDZ diet was more pronounced than that of the FB diet, but the difference between the two diets was moderate compared to that observed for sphinganine. Administration of the FB and FDZ diets significantly increased the C22–24:C16 ratio of Cer, SM, DHCer, and DHSM in both species. The C22–24:C16 ratio measured for HexCer remained constant in ducks. The C22–24:C16 ratios could not be calculated for LacCer due to the low abundance of certain analytes mentioned above. In both species, administration of the DON and ZEN diets had no effect on the C22–24:C16 ratios of Cer, SM, HexCer, DHCer, and DHSM.

Since previous studies in chickens have reported the effects of fumonisins on the sphingolipidome at 4, 9, 14, and 21 days of exposure, these data were compared with those obtained in this study (Fig. 4). The levels of FB1 and FB2 in the diets and in the liver were also reported. As shown in Fig. 4, the effect of fumonisins on the Sa:So ratio varied greatly over time, whereas the C22–24:C16 ratios were increased by a factor close to 1.5 to 2 irrespective of the duration of the exposure to fumonisins. Thus, while all diets containing fusariotoxins can have significant effects at the sphingolipid classes or analytes levels (Fig. 1, Tables 1 and 2), only the FB and FDZ diets led to an increase in the Sa:So and the C22–24:C16 ratios. Partial least square-discriminant analyses (PLS-DA) were performed to determine whether the effects of fusariotoxins on the sphingolipidome observed at the analytes level were significant enough to discriminate animals on the basis of the administered diets.

A PLS-DA performed on chickens receiving the five diets did not allow correct discrimination of the animals according to the administered diets (Fig. S1), and it was not possible to distinguish chickens fed the DON or ZEN diets from those fed the CON diet. By contrast, this analysis suggested that it is possible to discriminate chickens fed the FB diet from those fed the FDZ diet. This observation was confirmed by a PLS-DA performed on three groups of chickens comprising chickens fed the FB diet (FB, n = 10), chickens fed the FDZ diet (FDZ, n = 10), and chickens not exposed to fumonisins fed the CON, DON, or ZEN diets (0, n = 30) (Fig. 5A). This analysis allowed good separation of the animals, as shown by the respective values of R²X, R²Y, and Q² of 0.853, 0.601, and 0.59, respectively, and the confusion matrix (Fig. 5B). Another PLS-DA was also performed by differentiating between chickens exposed to fumonisins fed the FB or FDZ diets (1, n = 20) and chickens not exposed to fumonisins fed the CON, DON, or ZEN diets (0, n = 30) (Fig. 5C). The Q² value of 0.71 and the confusion matrix (Fig. 5D) revealed that this model was more robust and more specific than the one based on the three groups (Fig. 5C). The most important variables in the projection for the first two components are shown in Figs. 5E and F, respectively. They corresponded to sphinganine, d20-sphinganine (d20-Sa), most Cer and GlyCer as C16: 18:1/16:0, m18:1/16:0, Hex18:1/16:0, and Lac18:1/16:0, and all DHSM for which the fatty acid contains 18 or more carbon atoms. A PLS-DA was also performed to determine if it was possible to discriminate chickens exposed to deoxynivalenol fed the DON or FDZ diets (1, n = 20) from chickens not exposed to deoxynivalenol fed the CON, ZEN, or FB diets (0, n = 30). The obtained Q² of 0.226 showed that this discrimination was not possible (Fig. S2). The same applies to the discrimination between chickens exposed to zearalenone and fed the ZEN or FDZ diets (1, n = 20) and chickens not exposed to zearalenone fed the CON, DON, or FB diets (0, n = 30) (Fig. S3).

Figure 5 Partial least squares-discriminant analysis (PLS-DA) of sphingolipids measured in the liver of (A, B) chickens fed the fumonisin-free diets CON, DON, or ZEN (0, n = 30), chickens fed the FB diet (FB, n = 10, and chickens fed the FDZ diet (FDZ, n = 10), (C, D) chickens fed the CON, DON, and ZEN diets, which are free of fumonisins (0, n = 30), and chickens fed the FB and FDZ diets, which contain fumonisins (1, n = 20). (A, C) Discrimination on the factor axes extracted from the original explanatory variables. (B, D) Model quality and confusion matrix for the training sample (variable groups). Values of the variables of importance in the projection (VIP) for the first (E) and second (F) components.

The analytical strategy used in chickens was also applied to ducks. A PLS-DA performed on ducks fed the five diets did not allow good separation of the animals receiving the CON diet from those fed the DON or ZEN diets (Fig. S4). Furthermore, the animals fed the FB diet did not appear to be separated from those fed the FDZ diet. This latter observation was confirmed by a PLS-DA performed on three groups formed by ducks exposed to fumonisins fed the FB diet (n = 10) or the FDZ diet (n = 10), and ducks unexposed to fumonisins fed the CON, DON, or ZEN diets (0, n = 30) (Figs. 6A and B). The obtained Q² of 0.3 revealed that it was not possible to discriminate ducks fed the FB diet from those fed the FDZ diet. By contrast, a PLS-DA done to compare ducks exposed to fumonisins (1, n = 20) and ducks that were not exposed (0, n = 30) showed good discrimination of the animals (Fig. 6C). The values of R²X, R²Y, and Q² of 0.8, 0.611, and 0.588, respectively, and the confusion matrix showed that this model was robust, specific, and sensitive (Fig. 6D). The important variables in the projection for the first two components are shown in Figs. 6E and F, respectively. They correspond to sphinganine, d20-Sa, a large number of sphingolipids with C16: 18:1/16:0, m18:1/16:0, Hex18:1/16:0, SM18:1/16:0, and SM18:1/16:1, and most DHCer and DHSM for which the fatty acid contains more than 18 carbon atoms. The results of the PLS-DA performed to discriminate ducks exposed to deoxynivalenol (1, n = 20) from those not exposed (0, n = 30) are shown in Fig. S5. The results of the PLS-DA performed to discriminate ducks exposed to zearalenone (1, n = 20) from those not exposed (0, n = 30) are presented in Fig. S6. None of these analyses allowed satisfactory discrimination of the animals.

Thus, although the effect of feeding the FDZ diet on the total sphingolipid content in chickens appeared to be different from that observed with the FB diet, PLS-DA showed only a partial separation of the two groups of animals. This observation is consistent with the fact that the Sa:So and C22–24:C16 ratios of Cer, SM, HexCer, DHCer, and DHSM are similar in chickens fed FB and FDZ diets. In ducks, PLS-DA showed that it was not possible to discriminate animals fed the FB diet from those fed the FDZ diet, whereas very good discrimination of animals exposed to fumonisins from not-exposed was observed. In both species, sphinganine and d20-sphinganine were the main two discriminators. On the other hand, sphingosine, although the most abundant sphingoid base in the liver, did not appear to be discriminant. Similarly, phytosphingosine (t18:0), which is the second most abundant sphingoid base in chicken liver, was not discriminatory. The analysis of VIPs also showed that C16 sphingolipids were highly discriminant variables in both species. A decrease in C16 sphingolipids was the main factor explaining the increase in the C22–24:C16 ratios (Figs. 2 and 3). PLS-DA performed on sphingolipids dosed in the livers of chickens and ducks exposed to deoxynivalenol showed that it was not possible to distinguish them from unexposed animals. The same applies to zearalenone.

Effect of fusariotoxins on the Sa:So ratio

In chickens, feeding the FB diet from birth to 35 days of age resulted in a 250% increase in the Sa:So ratio. In ducks, feeding the FB diet during 12 days of force-feeding resulted in a 510% increase in the Sa:So ratio. The increase in the Sa:So ratio observed in animals exposed to fumonisins is consistent with numerous studies in various species and its use as a biomarker [7, 24, 37–39]. The pronounced increase in the Sa:So ratio observed in ducks during force-feeding confirms the high sensitivity of this species to the effect of fumonisins [40, 40, 41]. Comparison of the effect of fumonisins on the Sa:So ratio measured in several studies in chickens (Fig. 4) showed that the effect of fumonisins varies depending on the duration of exposure. This finding is consistent with previous work in ducks [39].

Feeding the FDZ diets to chickens and ducks led to an increase in the Sa:So ratio similar to that observed with FB diets. By contrast, feeding the DON diets had no effect on the Sa:So ratio in chickens and ducks. The same applied to the feeding of the ZEN diets. These results are consistent with data obtained in turkeys [21]. They confirm the specificity of the Sa:So ratio in detecting exposure to fumonisins and show the absence of interaction between fumonisins, deoxynivalenol, and zearalenone on this biomarker.

Effect of fusariotoxins on C22–24:C16 ratios

Feeding the FB diet in chickens led to an increase in C22–24:C16 ratios of ceramides, sphingomyelins, hexosylceramides, dihydroceramides, and dihydrosphingomyelins. The same is generally observed in ducks. The effect of the FB diet on the C22–24:C16 ratios was mainly due to a reduction in C16 sphingolipids. These results are in agreement with previous findings in chicken liver and kidney [23, 24, 27]. The C22–24:C16 ratios, recalculated from data reported in turkey liver [21], were also increased after feeding a diet containing fumonisins. Taken together, these results suggest that the effect of fumonisins on the C22–24:C16 ratios is similar in chickens, ducks, and turkeys.

Feeding the FDZ diets to chickens and ducks led to an increase in the C22–24:C16 ratios similar to that observed with the FB diets, while feeding the DON and ZEN diets had no effect. These results are in line with previous work with turkeys [21]. They suggest that, with regard to the Sa:So ratio, the effect of fumonisins on the C22–24:C16 ratio is specific and that no interaction between fumonisins, deoxynivalenol, and zearalenone occurred in its determination. Interestingly, the effects of fumonisins on the C22–24:C16 ratios were similar irrespective of the total sphingolipid contents measured at the classes level. This observation is consistent with previous work [27]. It is also consistent with the fact that the Sa:So ratio is preferable to sphinganine to detect fumonisins exposure [7, 39].

An increase in C22–24:C16 ratios is of relevance because of the role of fatty acids in ceramide toxicity. Indeed, whereas genetic deregulation of sphingolipid metabolism has long been known to be responsible for pathologies, the role of ceramide fatty acid chain length in diseases has become increasingly evident. Studies conducted on non-alcoholic hepatic fatty liver disease, obesity, type 2 diabetes, and myocardial infarction have shown that the ratio of very-long-chain to long-chain ceramides is a biomarker of disease severity and prognosis [13–17, 19]. Epidemiological studies have also indicated that the very-long-chain to long-chain ceramides ratio is directly influenced by diet and that its reduction is associated with presumed pathologies and, more generally, reduced quality of life and increased mortality [18]. A recent bibliographic review has even suggested sequestration of C16-Cer as a strategy to combat these pathologies [26]. Moreover, a recent study of a porcine jejunum epithelial cell line revealed that FB1 preferentially inhibited the expression of CerS2, which is responsible for the synthesis of very-long-chain ceramides [12]. This inhibition led to overexpression of endoplasmic reticulum stress, while CerS2 overexpression protected from FB1-induced damage. Taken together, these results suggest that the increase in the C22–24:C16 ratio observed in chickens and ducks in this study, and previously reported in turkey livers [21], may be an important factor in explaining the interspecies differences in sensitivity to fumonisin toxicity.

Effect of fusariotoxins on sphingoid bases

A 50% and 230% increase in sphinganine content was observed in the livers of chickens fed FB and FDZ diets, respectively. This result is consistent with numerous studies in different species as well as with the inhibitory effect of fumonisins on CerS [7, 8]. However, the difference between chickens fed FB and FDZ diets cannot be explained by differences in fumonisin concentrations in the diet or even in the liver. In ducks, increases of 460% and 490% in the sphinganine concentration were observed in the livers of animals fed FB and FDZ diets, respectively. Thus, the effects of fumonisins on the sphinganine concentration in ducks fed the FB and FDZ diets were similar, as were the fumonisins concentrations in the diets. The similarity of the effects of the FB and FDZ diets on sphinganine concentration in duck liver is consistent with data in turkey liver [21].

Sphingosine was reduced by 45% in chickens fed the FB diet, whereas this analyte was only reduced by 5% in chickens fed the FDZ diet. As with sphinganine, the different effects of the two diets on sphingosine cannot be explained by the level of fumonisins in the diets or the livers. No significant effect of FB and FDZ diets on sphingosine levels in duck livers was observed. This latter result is in agreement with data obtained with turkeys [21].

The administration of diets containing deoxynivalenol or zearalenone led to a weak reduction in the levels of sphingoid bases in chicken liver, which was similar for all the sphingoid bases dosed. This effect was not observed in ducks. The difference between species could be related to the duration of the exposure, which was shorter in ducks than in chickens. This hypothesis is in agreement with previous work in turkeys that revealed that 14 days of exposure to diets containing deoxynivalenol or zearalenone had no effect on the concentrations of sphingoid bases in the liver [21].

Taken together, these results suggest that two concomitant but distinct mechanisms may be responsible for the effects on sphingoid bases observed in chickens fed the FB and the FDZ diets. One mechanism, resulting from the inhibition of DHCerS by fumonisins (Fig. 7), would be responsible for the increase in sphinganine concentration and the decrease in sphingosine concentration; and a second mechanism, observed in chickens fed FDZ, which would be responsible for an increase in hepatic synthesis of the sphingoid bases. This second mechanism would, therefore, have an additive effect to that of fumonisins on sphinganine concentrations and an antagonistic effect on sphingosine concentrations.

Effect of fusariotoxins on dihydroceramides and dihydrosphingomyelins

Feeding the FB diet led to significant decreases in the levels of dihydroceramides and dihydrosphingomyelins in chickens. These decreases are consistent with the inhibitory effect of fumonisins on DHCerS (Fig. 7). It is interesting to note that this effect was not observed in ducks, although a pronounced increase in sphinganine occurred. Administration of the FDZ diet led to a pronounced increase in the levels of dihydroceramides and dihydrosphingomyelins in chickens, whereas only a non-significant numerical increase was observed in ducks. These results, therefore, show that the effects of the FDZ diet on the dihydroceramides and dihydrosphingomyelins concentrations in the liver of chickens were different from those due to the FB diet. This observation is thus consistent with the hypothesis that two simultaneous mechanisms could be observed: the first, linked to the blocking of DHCerS by fumonisins, leading to a decrease in dihydroceramides and dihydrosphingomyelins, and the second corresponding to an increase in the de novo synthesis of sphingolipids.

The administration of DON and ZEN diets reduced the levels of dihydroceramides and dihydrosphingomyelins in chickens but had no effect in ducks. These results are, therefore, consistent with those observed for sphinganine and sphingosine, and the hypothesis that the difference between species could be due to the duration of exposure to mycotoxins is in agreement with the results obtained in turkeys [21].

Effect of fusariotoxins on ceramides, glycosylceramides, and sphingomyelins

Feeding the FB diet led to a reduction in the levels of ceramides, glycosylceramides, and sphingomyelins in chickens. This effect is consistent with the expected mechanism of action of fumonisins on sphingolipid synthesis. It is also consistent with the 0.062 µmol/kg FB1 measured in chicken liver and the IC₅₀ of 0.1 µM FB1 on ceramide synthesis in hepatocyte cultures [42]. In ducks, only a small non-significant numerical decrease in ceramide and glycosylceramide was observed. The lack of a significant effect of fumonisins on the ceramide levels in ducks could be due to the low FB1 content measured in the liver, which was only 0.018 µmol/kg. This observation is in agreement with previous data in chickens, where a slight decrease in the Cer level was observed in the liver at an FB1 concentration of 0.029 µmol/kg, whereas a concentration of 0.011 µmol/kg had no effect on Cer levels in the kidney [27].

The concentrations of ceramides and glycosylceramides measured in chickens fed the FDZ diets were not different from those observed in controls not exposed to fusariotoxins, whereas the concentrations of sphingomyelins were increased. These results are consistent with previous work with turkeys [21]. Moreover, comparison of the sphingolipid concentrations measured in chickens fed the FB and FDZ diets showed that the concentrations of ceramides, glycosylceramides, and sphingomyelins were higher in chickens fed the FDZ diet than in chickens fed the FB diet. In ducks, the effects of the FDZ diet exhibited the same trends as those in chickens, with only small, non-significant, numerical differences. Thus, with regard to sphingoid bases, dihydroceramides, and dihydrosphingomyelins, two concomitant mechanisms could underlie the changes in ceramide, glycosylceramide, and sphingomyelin levels in animals receiving the FDZ diet: One, leading to a decrease in their concentrations as a consequence of inhibition of the synthesis of sphingolipids by fumonisins on DHCerS, and the other, for which could be due to activation of the de novo synthesis of sphingolipids, thereby leading to an increase in their concentrations (Fig. 7).

The hypothesis of an increase in the de novo synthesis of sphingolipids in chickens fed the FDZ diet is supported by several findings. The effects of the FDZ diet measured on the concentrations of sphinganine, dihydroceramides, and dihydrosphingomyelins were greater than those observed on sphingosine, ceramides, and sphingomyelins. Greater increases in dihydrosphingolipids than sphingolipids have already been described in mice during non-infectious inflammatory peritonitis induced by zymosan administration, and by endoplasmic reticulum stress secondary to the administration of the calcium signalling modulator thapsigargin [43, 44]. Inflammatory effects of fusariotoxins have been reported in recent studies of fumonisins [45–49], deoxynivalenol [50–55], and zearalenone [56–63]. An additive or synergistic effect between deoxynivalenol and fumonisins was observed on intestinal inflammation in mice [64]. Similarly, a mixture of deoxynivalenol and zearalenone has synergistic effects on the levels of pro-inflammatory cytokines in piglets [65]. Endoplasmic reticulum stress has also been observed in numerous studies conducted with fumonisins [12, 46, 66–68], deoxynivalenol [69–73], and zearalenone [74–79]. This mechanism could play a key role in mitochondrial-induced apoptosis when deoxynivalenol and zearalenone were used in combination [80]. Finally, inflammation and endoplasmic reticulum stress together could be responsible for the effects of the FDZ diet on sphingolipids in chickens, and both mechanisms may be highly interconnected, as recently reviewed for other liver toxicants [81]. Taken together, all these results support the hypothesis that an increase in de novo synthesis of sphingolipids occurs in chickens exposed to an FDZ diet, with the difference between chickens and ducks likely being likely by the duration of the exposure, and this latter point is consistent with results obtained in turkeys [21]. Although the mechanism responsible for the increases in sphingolipids in chickens fed an FDZ diet remains to be established, it is important to highlight that the Sa:So and C22–C24: C16 ratios in the liver of chickens fed the FDZ diet were similar to those measured in the liver of chickens fed the FB diet.

In conclusion, this study revealed that the feeding of fumonisins to chickens and ducks increased the C22–24:C16 ratios measured for almost all classes of sphingolipids. This increase is consistent with previous findings in chickens and turkeys and was accompanied by an increase in the Sa:So ratio. It was not only observed in animals fed fumonisins alone, but also occurred in chickens fed fumonisins, deoxynivalenol, and zearalenone in combination. Interestingly, the effects of fumonisins on the C22–24:C16 and Sa:So ratios were similar when fumonisins were fed alone or in combination with deoxynivalenol and zearalenone, whereas the effects on the total amounts of sphingolipids within a class varied. The results at the analyte level revealed that the increase in the C22–24:C16 ratios were due to more pronounced inhibition of CerS producing C16 ceramides by fumonisins than that observed for CerS producing C22–24 ceramides. These effects are specific to fumonisins and were not observed when the diets contained deoxynivalenol or zearalenone alone. The results obtained in this study with chickens and ducks are consistent with previous results obtained with chickens exposed to fumonisins for a shorter time and the results obtained with turkeys exposed to fumonisins alone or in combination with deoxynivalenol and zearalenone. Because the long-chain to very-long-chain ceramide toxicity was reported to vary, the increase in the C22–24:C16 ratios may be an important factor in explaining the interspecies differences in sensitivity to fumonisin toxicity. Further studies are needed. However, to determine which mechanism is responsible for the increase in sphingolipids observed in chickens fed fumonisins, deoxynivalenol, and zearalenone in combination.

Sa: sphinganine; So: sphingosine, DHCerS: dihydroceramide synthase; CerS: ceramide synthase; HexCer: monohexosylceramide; LacCer: lactosylceramide; SM : sphingomyelin ; C16: long chain sphingolipids; C22-24: very long chain sphingolipids; FB1: fumonisins B1; FB2: fumonisins B2; CON: diet considered as free of mycotoxin; FB: diet containing fumonisins alone; DON: diet containing deoxynivalenol alone; ZEN: diet containing zearalenone alone; FDZ: diet containing fumonisins, deoxynivalenol and zearalenone; RSD: relative standard deviation; MRM: multiple reaction monitoring; PLS-DA: partial least squares-discriminant analysis

Ethics approval and consent to participate

The chicken experimental protocol was approved by the Ministry of Higher Education and Research and registered under number 02032.01. The duck protocol was approved by the INRAE UEPFG institutional review board under number PP-5-12.

Consent for publication

Not applicable.

Availability of data and material

All the datasets used and analyzed during the current study are included in the manuscript or supplementary material.

Funding

The experimental phase with animals was supported by CASDAR grants (project 11AIP077 MYCOVOL).

Competing interest

The authors declare that they have no competing interests.

Acknowledgments

The authors thank Sophie Domingues, 26 calle Gladiols, 43850 Cambrils, Spain, for language editing.

Authors’ contributions

PG: conceptualization, funding acquisition, methodology, validation, formal analysis, writing - original draft, writing - review & editing; EL: formal analysis, writing original draft; UBD: formal analysis, writing original draft; AT: conceptualization, funding acquisition.

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Fumonisins alone or mixed with other fusariotoxins increase the C22–24:C16 sphingolipid ratios in chickens and ducks, while deoxynivalenol and zearalenone have no effect

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Abstract

Background

Results

Conclusions

Figures

Background

Materials and Methods

Analytes and reagents

Biological samples

Analysis of sphingolipids

Statistical analysis

Results

Discussion

Effect of fusariotoxins on the Sa:So ratio

Effect of fusariotoxins on C22–24:C16 ratios

Effect of fusariotoxins on sphingoid bases

Effect of fusariotoxins on dihydroceramides and dihydrosphingomyelins

Effect of fusariotoxins on ceramides, glycosylceramides, and sphingomyelins

Conclusion

Abbreviations

Declarations

References

Supplementary Files

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