Emerging mycotoxins induce hepatotoxicity in pigs' precision-cut liver slices and HepG2 cells.

Emerging mycotoxins are currently gaining more attention due to their high frequency of contamination in foods and grains. However, most data available in the literature are in vitro, with few in vivo results that prevent establishing their regulation. Beauvericin (BEA), enniatins (ENNs), emodin (EMO), apicidin (API) and aurofusarin (AFN) are emerging mycotoxins frequently found contaminating food and there is growing interest in studying their impact on the liver, a key organ in the metabolization of these components. We used an ex vivo model of precision-cut liver slices (PCLS) to verify morphological and transcriptional changes after acute exposure (4 h) to these mycotoxins. The human liver cell line HepG2 was used for comparison purposes. Most of the emerging mycotoxins were cytotoxic to the cells, except for AFN. In cells, BEA and ENNs were able to increase the expression of genes related to transcription factors, inflammation, and hepatic metabolism. In the explants, only ENN B1 led to significant changes in the morphology and expression of a few genes. Overall, our results demonstrate that BEA, ENNs, and API have the potential to be hepatotoxic.

Deoxynivalenol induces apoptosis and inflammation in the liver: Analysis using precision-cut liver slices.

Deoxynivalenol (DON) is one of the most common mycotoxins in cereals and their by-products. Its adverse effects on animal and human health have been extensively studied in the intestine, but little attention has been paid to another target organ for mycotoxins, the liver that is potentially exposed after intestinal absorption and enterohepatic circulation. To assess DON's toxicity in an ex vivo model structurally and physiologically closer to the whole liver, we developed a pig precision-cut liver slices (PCLS) model. PCLS contain all cell types and maintain intercellular and cell-matrix interactions, among other architectural features of the liver. The human HepG2 cell line was used for comparison. We observed that after a short exposure, DON reduced the cell viability of HepG2 cells and induced the expression of genes involved in apoptosis, inflammation and oxidative stress. When PCLS were exposed to DON, damage to the tissues was observed, with no changes in markers of liver function or injury. Exposure to the toxin also triggered liver inflammation and apoptosis, effects already observed in pigs fed DON-contaminated diets. Overall, these data demonstrate that DON had toxic effects on a liver cell line and on whole liver tissue, consistent with the effect observed during in vivo exposure. They also indicate that pig PCLS is a relevant and sensitive model to investigate the liver toxicity of food contaminants.

Intestinal toxicity of the new type A trichothecenes, NX and 3ANX.

NX and its acetylated form 3ANX are two new type A trichothecenes produced by Fusarium graminearum whose toxicity is poorly documented. The aim of this study was to obtain a general view of the intestinal toxicity of these toxins. Deoxynivalenol (DON), which differs from NX by the keto group at C8, served as a benchmark. The viability of human intestinal Caco-2 cells decreased after 24 h of exposure to 3 µM NX (-21.4%), 3 µM DON (-20.2%) or 10 µM 3ANX (-17.4%). Histological observations of porcine jejunal explants exposed for 4 h to 10 µM of the different toxins showed interstitial edema and cellular debris. Explants exposed to NX also displayed cell vacuolization, a broken epithelial barrier and high loss of villi. Whole transcriptome profiling revealed that NX, DON and 3ANX modulated 369, 146 and 55 genes, respectively. Functional analyses indicated that the three toxins regulate the same gene networks and signaling pathways mainly; cell proliferation, differentiation, apoptosis and growth, and particularly immune and pro-inflammatory responses. Greater transcriptional impacts were observed with NX than with DON. In conclusion, our data revealed that the three toxins have similar impacts on the intestine but of different magnitude: NX > DON ≫ 3ANX. NX and 3ANX should consequently be included in overall risk analysis linked to the presence of trichothecenes in our diet.

Versicolorin A enhances the genotoxicity of aflatoxin B1 in human liver cells by inducing the transactivation of the Ah-receptor.

Aflatoxins are a group of mycotoxins that have major adverse effects on human health. Aflatoxin B1 (AFB1) is the most important aflatoxin and a potent carcinogen once converted into a DNA-reactive form by cytochrome P450 enzymes (CYP450). AFB1 biosynthesis involves the formation of Versicolorin A (VerA) which shares structural similarities with AFB1 and can be found in contaminated commodities, often co-occurring with AFB1. This study investigated and compared the toxicity of VerA and AFB1, alone or in combination, in HepG2 human liver cells. Our results show that both toxins have similar cytotoxic effects and are genotoxic although, unlike AFB1, the main genotoxic mechanism of VerA does not involve the formation of DNA double-strand breaks. Additionally, we show that VerA activates the aryl hydrocarbon receptor (AhR) and significantly induce the expression of the CYP450-1A1 (CYP1A1) while AFB1 did not induce AhR-dependent CYP1A1 activation. Combination of VerA with AFB1 resulted in enhanced genotoxic effects, suggesting that AhR-activation by VerA influences AFB1 genotoxicity by promoting its bioactivation by CYP450s to a highly DNA-reactive metabolite. Our results emphasize the need for expanding the toxicological knowledge regarding mycotoxin biosynthetic precursors to identify those who may pose, directly or indirectly, a threat to human health.

Comparative sensitivity of proliferative and differentiated intestinal epithelial cells to the food contaminant, deoxynivalenol.

The intestinal epithelium is a functional and physical barrier formed by a cell monolayer that constantly differentiates from a stem cell in the crypt. This is the first target for food contaminants, especially mycotoxins. Deoxynivalenol (DON) is one of the most prevalent mycotoxins. This study compared the effects of DON (0-100 µM) on proliferative and differentiated intestinal epithelial cells. Three cell viability assays (LDH release, ATP content and neutral red uptake) indicated that proliferative Caco-2 cells are more sensitive to DON than differentiated ones. The establishment of transepithelial electrical resistance (TEER), as a read out of the differentiation process, was delayed in proliferative cells after exposure to 1 µM DON. Transcriptome analysis of proliferative and differentiated exposure to 0-3 µM DON for 24 h revealed 4862 differentially expressed genes (DEG) and indicated an effect of both the differentiation status and the DON treatment. KEGG enrichment analysis indicated involvement of metabolism, ECM receptors and tight junctions in the differentiation process, while ribosome biogenesis, mRNA surveillance, and the MAPK pathway were involved in the response to DON. The number of differentially expressed genes and the amplitude of the effect were higher in proliferative cells exposed to DON than that in differentiated cells. In conclusion, our study shows that proliferative cells are more susceptible than differentiated ones to DON and that the mycotoxin delays the differentiation process.

Acute Exposure to Zearalenone Disturbs Intestinal Homeostasis by Modulating the Wnt/ß-Catenin Signaling Pathway.

The mycotoxin zearalenone (ZEN), which frequently contaminates cereal-based human food and animal feed, is known to have an estrogenic effect. The biological response associated with exposure to ZEN has rarely been reported in organs other than the reproductive system. In the intestine, several studies suggested that ZEN might stimulate molecular changes related to the activation of early carcinogenesis, but the molecular mechanisms behind these events are not yet known. In this study, we investigated gene expression and changes in protein abundance induced by acute exposure to ZEN in the jejunum of castrated male pigs using an explant model. Our results indicate that ZEN induces the accumulation of ER but not ER, modulates Wnt/ß-catenin and TGF- signaling pathways, and induces molecular changes linked with energy sensing and the antimicrobial activity without inducing inflammation. Our results confirm that the intestine is a target for ZEN, inducing changes that promote cellular proliferation and could contribute to the onset of intestinal pathologies.

Versicolorin A, a precursor in aflatoxins biosynthesis, is a food contaminant toxic for human intestinal cells.

Aflatoxin B1 (AFB1) is the most potent carcinogen among mycotoxins. Its biosynthesis involves the formation of versicolorin A (VerA), whose chemical structure shares many features with AFB1. Our data revealed significant levels of VerA in foodstuff from Central Asia and Africa. Given this emerging food risk, it was of prime interest to compare the toxic effects of the two mycotoxins against cells originating from the intestinal tract. We used human colon cell lines (Caco-2, HCT116) to investigate the cytotoxic process induced by the two mycotoxins. Contrary to AFB1, a low dose of VerA (1 µM) disturbed the expression level of thousands of genes (18 002 genes). We show that the cytotoxic effects of low doses of VerA (1-20 µM) were stronger than the same low doses of AFB1 in both Caco-2 and HCT116 cell lines. In Caco-2 cells, VerA induced DNA strand breaks that led to apoptosis and reduced DNA replication of dividing cells, consequently inhibiting cell proliferation. Although VerA was able to induce the p53 signaling pathway in p53 wild-type HCT116 cells, its toxicity process did not mainly rely on p53 expression since similar cytotoxic effects were also observed in HCT116 cells that do not express p53. In conclusion, this study provides evidence of the risk of food contamination by VerA and shed light on its toxicological effect on human colon cells.

Prediction of human prenatal exposure to bisphenol A and bisphenol A glucuronide from an ovine semi-physiological toxicokinetic model.

Bisphenol A (BPA) risk assessment is hampered by the difficulty of determining the extent of internal exposure in the human fetus and uncertainties regarding BPA toxicokinetics (TK) in the maternal-fetal unit. A feto-maternal TK model describing BPA and BPA glucuronide (BPAG) disposition in sheep was humanized, using human TK data obtained after d6-BPA administration on a cookie, to predict BPA and BPAG kinetics in the human mother-fetus unit. Validation of the model predictions included the assessed dose proportionality of BPA and BPAG disposition and the similarity between the simulated and measured time courses of BPA and BPAG in fetal rhesus monkeys after BPA maternal dosing. The model predicted fluctuations in fetal BPA concentrations associated with typical maternal exposure to BPA through the diet, with similar trough (0.011 ng/L vs 0.014 ng/L) and lower peak BPA concentrations (0.023 ng/L vs 0.14 ng/L) in fetal than in maternal plasma. BPAG concentrations in fetal plasma were predicted to increase over time to reach a steady value (29 ng/L) reflecting the cumulative BPA dose received by the fetus. Model-predicted BPAG concentrations in fetal plasma are consistent with reported levels in human cord blood that may be considered as relevant markers of the BPA dose entering blood throughout fetal life.

Development of an on-line solid phase extraction ultra-high-performance liquid chromatography technique coupled to tandem mass spectrometry for quantification of bisphenol S and bisphenol S glucuronide: Applicability to toxicokinetic investigations.

Regulatory measures and public concerns regarding bisphenol A (BPA) have led to its replacement by structural analogues, such as Bisphenol S (BPS), in consumer products. At present, no toxicokinetic investigations have been conducted to assess the factors determining human internal exposure to BPS for subsequent risk assessment. Toxicokinetic studies require reliable analytical methods to measure the plasma concentrations of BPS and its main conjugated metabolite, BPS-glucuronide (BPS-G). An efficient on-line SPE-UPLC-MS/MS method for the simultaneous quantification of BPS and BPS-G in ovine plasma was therefore developed and validated in accordance with the European Medicines Agency guidelines for bioanalytical method validation. This method has a limit of quantification of 3ngmL-1 for BPS and 10ngmL-1 for BPS-G, an analytical capacity of 200 samples per day, and is particularly well suited to toxicokinetic studies. Use of this method in toxicokinetic studies in sheep showed that BPS, like BPA, is efficiently metabolized into its glucuronide form. However, the clearances and distributions of BPS and BPS-G were lower than those of the corresponding unconjugated and glucuroconjugated forms of BPA.

Evidence for bisphenol A-induced disruption of maternal thyroid homeostasis in the pregnant ewe at low level representative of human exposure.

Many uncertainties remain regarding the potential of bisphenol A (BPA) as a thyroid disruptor in mammals and the relevance of experimental data to humans. The relevance of the exposure schemes used in experimental in vivo studies is also a major source of uncertainty when analysing the risk of BPA exposure for human health. In this context, the goals of our study, conducted in an ovine model relevant to human gestation and thyroid physiologies, were to: 1) determine the equivalence of subcutaneous and dietary exposures and 2) determine if environmentally relevant doses of BPA can alter gestational and newborn thyroid functions. The difference between the two routes of exposure was mainly related to the overall BPA exposure and much less to the peak serum concentrations. Interestingly, BPA-GLUC (the main metabolite of BPA) internal exposure via both routes was almost identical. The decrease in thyroid hormones concentration overtime was more accentuated in ewes treated with BPA, particularly with the medium dose (50 µg/(kg.d); SC) for which the maximum BPA concentrations were predicted to be within the 1-10 ng/mL range i.e. very similar to the highest blood concentrations reported in humans. The balance between TT4 and rT3 varied differently between the vehicle and the medium dose group. The mechanisms underlying those modifications of maternal thyroid homeostasis remain to be determined. Our study did not evidence significant modification of TSH secretion or binding to serum proteins but might suggest an effect at the level of deiodinases.

Characterization of the contribution of buccal absorption to internal exposure to bisphenol A through the diet.

The gavage route is often used for the toxicological evaluation of food contaminants. This route does not take into account absorption of the toxicants through the buccal mucosa, as evidenced in dogs for bisphenol A (BPA). Our goal was to determine the functional significance of buccal BPA absorption during dietary exposure. Four ewes received BPA by nasogastric gavage (100 mg/kg) and through food pellets (10 mg/kg), 13 days apart. The time course of serum concentrations of BPA and its main metabolite BPA-G was submitted to non-compartmental analysis. The dietary route led to 3-fold higher bioavailability as compared to gavage. The ratio of BPA-G to BPA concentrations varied greatly over time after the food administration, but not after gavage, suggesting a delayed metabolism of BPA after dietary exposure. The maximum entrance rate of BPA in the systemic circulation, determined by deconvolution analysis, was much higher after dietary administration than after gavage and a biphasic pattern of BPA entry was observed in 3 of the 4 ewes. Our results evidenced a dual mechanism of BPA absorption (buccal and digestive) after dietary exposure and highlight the necessity to take buccal absorption into account when evaluating food contaminants.

Bisphenol A glucuronide deconjugation is a determining factor of fetal exposure to bisphenol A.

Previous studies in experimental animals have shown that maternal exposure to bisphenol A (BPA) during late pregnancy leads to high plasma concentrations of BPA glucuronide (BPAG) in fetus compared to mother due to the inability of BPAG to cross the placental barrier. A recent in vitro study has reported that BPAG can exert adipogenic effect underlining the need for characterization of the fetal disposition of BPAG. Experiments were conducted in chronically catheterized fetal sheep to determine the contribution of BPAG hydrolysis to BPA to the elimination of BPAG from the fetal compartment and its resulting effect on the overall fetal exposure to free BPA. Serial sampling of fetal arterial blood, amniotic fluid, maternal venous blood and urine was performed following separate single doses of BPA and BPAG administered intravenously to eight fetal/maternal pairs after cesarean section, and repeated BPAG doses given to two fetal sheep. On average 67% of the BPA entering the fetal circulation was rapidly eliminated through fetal to maternal clearance, with a very short half-life (20 min), while the remaining fraction (24%) was glucuronoconjugated. BPA conjugation-deconjugation cycling was responsible for a 43% increase of the overall fetal exposure to free BPA. A very specific pattern of fetal exposure to free BPA was observed due to its highly increased persistence with a hydrolysis-dependent plasma terminal free BPA half-life of several tens of hours. These findings suggest that although the high fetal to maternal clearance of free BPA protects the fetus from transient increases in free BPA plasma concentrations associated with maternal BPA intake, low but sustained basal free BPA concentrations are maintained in the fetus through BPA conjugation-deconjugation cycling. The potential health implications of these low but sustained basal concentrations of free BPA in fetal plasma should be addressed especially when considering time-dependent effects.

Influence of intraspecific variability and abiotic factors on mycotoxin production in Penicillium roqueforti.

Penicillium roqueforti has the ability to produce secondary metabolites, including roquefortine C (ROQC) and mycophenolic acid (MPA). In a previous study, the presence of these mycotoxins, alone or in co-occurrence, has been reported in blue-veined cheese. A high variability of mycotoxin content has also been observed, although the majority of samples exhibited relatively low concentrations. The observed variability raises the question of the factors impacting ROQC and MPA production. In this context, the mycotoxigenic potential of 96 P. roqueforti strains (biotic factor) and the effect of some abiotic factors (pH, temperature, NaCl and O2 contents, and C/N ratio) on mycotoxin production were evaluated. A high intraspecific diversity, established via genotypic (RAPD) and phenotypic (FTIR) approaches, was observed. It was associated with mycotoxigenic potential variability and may thus explain part of the observed variability in mycotoxin content of blue-veined cheese. Moreover, a significant decrease of ROQC and MPA production was observed for conditions (temperature, C/N ratio, O2 and NaCl concentrations) encountered during cheese-making compared with optimal growth conditions. The results also highlighted that there was no significant effect of addition of ROQC amino-acid precursor on the production of both mycotoxins whereas a pH increase from 4.5 to 6.5 slightly reduced MPA but not ROQC production.

Allometric scaling for predicting human clearance of bisphenol A.

The investigation of interspecies differences in bisphenol A (BPA) pharmacokinetics (PK) may be useful for translating findings from animal studies to humans, identifying major processes involved in BPA clearance mechanisms, and predicting BPA PK parameters in man. For the first time, a large range of species in terms of body weight, from 0.02 kg (mice) to 495 kg (horses) was used to predict BPA clearance in man by an allometric approach. BPA PK was evaluated after intravenous administration of BPA in horses, sheep, pigs, dogs, rats and mice. A non-compartmental analysis was used to estimate plasma clearance and steady state volume of distribution and predict BPA PK parameters in humans from allometric scaling. In all the species investigated, BPA plasma clearance was high and of the same order of magnitude as their respective hepatic blood flow. By an allometric scaling, the human clearance was estimated to be 1.79 L/min (equivalent to 25.6 mL/kg.min) with a 95% prediction interval of 0.36 to 8.83 L/min. Our results support the hypothesis that there are highly efficient and hepatic mechanisms of BPA clearance in man.

Conjugation and deconjugation reactions within the fetoplacental compartment in a sheep model: a key factor determining bisphenol A fetal exposure.

The widespread human exposure to bisphenol A (BPA), an endocrine disruptor targeting developmental processes, underlines the need to better understand the mechanisms of fetal exposure. Animal studies have shown that at a late stage of pregnancy BPA is efficiently conjugated by the fetoplacental unit, mainly into BPA-glucuronide (BPA-G), which remains trapped within the fetoplacental unit. Fetal exposure to BPA-G might in turn contribute to in situ exposure to bioactive BPA, following its deconjugation into parent BPA at the level of fetal sensitive tissues. The objectives of our study were 1) to characterize the BPA glucurono- and sulfoconjugation capabilities of the ovine fetal liver at different developmental stages, 2) to compare hepatic conjugation activities in human and sheep, and 3) to evaluate the extent of BPA conjugation and deconjugation processes in placenta and fetal gonads. At an early stage of pregnancy, and despite functional sulfoconjugation activity, ovine fetuses expressed low hepatic BPA conjugation capabilities, suggesting that this stage of development represents a critical window in terms of BPA exposure. Conversely, the late ovine fetus expressed an efficient detoxification system that metabolized BPA into BPA-G. Hepatic glucuronidation activities were quantitatively similar in adult sheep and humans. In placenta, BPA conjugation and BPA-G deconjugation activities were relatively balanced, whereas BPA-G hydrolysis was systematically higher than BPA conjugation in gonads. The possible reactivation of BPA-G into BPA could contribute to an increased exposure of fetal sensitive tissues to bioactive BPA in situ.

Bisphenol A disposition in the sheep maternal-placental-fetal unit: mechanisms determining fetal internal exposure.

The widespread human exposure to bisphenol A (BPA), a xenoestrogen interfering with developmental processes, raises the question of the mechanisms determining fetal exposure to BPA. A physiological model was developed in ewes to determine whether the pregnancy-associated physiological changes and the metabolic specificities of the fetal-placental unit can influence BPA toxicokinetics (TK) and fetal exposure to BPA. In a first longitudinal study, BPA was infused (2 mg/[kg·day] i.v. for 1 day) into ewes before breeding, at early and late stages of gestation, and after lambing. In a second study, BPA and BPA-glucuronide (BPA-G) were infused intravenously into pregnant ewes or into fetuses at 4 mo of gestation. BPA and its metabolites were assayed in maternal and fetal plasma and amniotic fluid sampled at steady state and after the end of the infusion. The pregnancy status did not modify the TK parameters of BPA and of BPA-G. Five percent of the BPA dose infused into the pregnant ewe was transferred across the placenta to the fetus. The fetal-placental unit was very efficient in metabolizing BPA into conjugated compounds; those metabolites remained trapped in the fetal-placental compartment, leading to a high fetal exposure to BPA conjugates. Taking into account a body weight adjustment, the ovine fetus in late pregnancy is exposed to a BPA dose similar to that of its mother. In contrast to its mother, the fetus exhibits much higher and sustained exposure to BPA metabolites without evidence of their hydrolysis.

Maternal and fetal exposure to bisphenol a is associated with alterations of thyroid function in pregnant ewes and their newborn lambs.

The putative thyroid-disrupting properties of bisphenol A (BPA) highlight the need for an evaluation of fetal exposure and its consequence on the mother/newborn thyroid functions in models relevant to human. The goals of this study were to characterize in sheep a relevant model for human pregnancy and thyroid physiology, the internal exposures of the fetuses and their mothers to BPA and its main metabolite BPA-glucuronide (Gluc), and to determine to what extent it might be associated with thyroid disruption. Ewes were treated with BPA [5 mg/(kg · d) sc] or vehicle from d 28 until the end of pregnancy. Unconjugated BPA did not appear to accumulate in pregnant ewes, and its concentration was similar in the newborns and their mothers (0.13 ± 0.02 and 0.18 ± 0.03 nmol/ml in cord and maternal blood, respectively). In amniotic fluid and cord blood, BPA-Gluc concentrations were about 1300-fold higher than those of BPA. Total T(4) concentrations were decreased in BPA-treated pregnant ewes and in the cord and the jugular blood of their newborns (30% decrease). A similar difference was observed for free T(4) plasma concentrations in the jugular blood of the newborns. Our results show in a long-gestation species with a similar regulatory scheme of thyroid function as humans that BPA in utero exposure can be associated with hypothyroidism in the newborns. If such an effect were to be confirmed for a more relevant exposure scheme to BPA, this would constitute a major issue for BPA risk assessment.

CYP450-dependent biotransformation of the insecticide fipronil into fipronil sulfone can mediate fipronil-induced thyroid disruption in rats.

In rats, the widely used insecticide fipronil increases the clearance of thyroxine (T(4)). This effect is associated with a high plasma concentration of fipronil sulfone, the fipronil main metabolite in several species including rats and humans. In sheep, following fipronil treatment, fipronil sulfone plasma concentration and thyroid disruption are much lower than in rats. We postulated that fipronil biotransformation into fipronil sulfone by hepatic cytochromes P450 (CYP) could act as a potential thyroid disruptor. The aim of this study was to determine if fipronil sulfone treatment could reproduce the fipronil treatment effects on T(4) clearance and CYP induction in rats. Fipronil and fipronil sulfone treatments (3.4 µmol/kg/day per os, 14 days) increased total and free T(4) clearances to the same extent in THX + T(3), euthyroid-like rats. Both treatments induced a 2.5-fold increase in Ugt1a1 and Sult1b1 messenger RNA (mRNA) expressions and a twofold increase in UGT1A activity suggesting that T(4) elimination was mediated, at least in part, by hepatic uridine 5'-diphospho-glucuronosyltransferases (UGT) and/or sulfotransferases (SULT) induction. Both treatments induced a 10-fold increase in Cyp3a1 and Cyp2b2 mRNA expressions concomitant with a threefold increase in CYP3A immunoreactivity and a 1.7-fold increase in antipyrine clearance, a biomarker of CYP3A activity. All these results showed that fipronil sulfone treatment could reproduce the fipronil treatment effects on T(4) clearance and hepatic enzyme induction in rats. The potential of fipronil sulfone to act as a thyroid disruptor is all the more critical because it persists much longer in the organism than fipronil itself.