The problem of maternal toxicity in developmental toxicity studies

Guidelines for developmental toxicity studies require that the highest dose(s) should induce some signs of maternal toxicity. However, the interpretation of the results is often difficult when developmentally toxic effects are recorded only at maternotoxic doses, as it is impossible to ascertain whether the developmental effects are maternally mediated or not. In order to avoid this source of misinterpretation we suggest to use in developmental toxicity tests for environmental chemicals the maximum dose unable to produce maternal toxic effects extrapolated by previous short term toxicity studies

Relative potency ranking of azoles altering craniofacial morphogenesis in rats : an in vitro data modelling approach

Facial malformations represent one of the most frequent abnormality in humans. The adverse outcome pathway involved in facial defects seems to be related to retinoic acid (RA) pathway imbalance. Environmental agents inducing craniofacial malformations in experimental models include pesticides (especially azole fungicides). By using the in vitro alternative method postimplantation rat whole embryo culture (WEC), we evaluated the intrinsic embryotoxic activity of some azole antifungals (cyproconazole, CYPRO; triadimefon, FON; flusilazole, FLUSI; and prochloraz, PCZ), in comparison to RA. All the tested molecules induced in a dose-related manner specific defects of the craniofacial structures (fused branchial arches), similar to those induced by RA. Collected data were modelled using PROAST 65.5 software to characterise the relative potency factors (RPFs) versus RA. In comparison to RA, all the evaluated azoles were less potent, showing among them a similar potency. Our data suggest a possible azole-related RA signalling perturbation to be further investigated. Moreover, the present results indicate the approach used in this work to be an interesting tool applicable to the hazard evaluation of novel compounds or the assessment of combined exposure to azoles or other dismorphogens

Teratogenic potential of nanoencapsulated vitamin A evaluated on an alternative model organism, the tunicate Ciona intestinalis

Nano-encapsulation is a technology used to pack substances in order to enhance their stability and bioavailability, but this packing may interact with living systems, causing unexpected toxicity. Vitamin A (vit A) is a substance that has received attention, because in developed countries, the increasing availability of supplements is leading to its excessive intake. This study aims to compare teratogenic effects caused by exposure to the traditional formulation of vit A versus nano-encapsulated vit A. We used ascidian embryos as an alternative model. Ascidians are marine organisms closely related to vertebrates that share with them a body plan and developmental programme, including the morphogenetic role of retinoic acid (RA). Our data showed that the adverse effects of exposure to the same concentration of the two formulations were different, suggesting that the nano-encapsulation increased the bioavailability of the molecule, which could be better absorbed and metabolised to RA, the effective teratogenic substance

Development of an adverse outcome pathway for cranio-facial malformations: A contribution from in silico simulations and in vitro data

Mixtures of substances sharing the same molecular initiating event (MIE) are supposed to induce additive effects. The proposed MIE for azole fungicides is CYP26 inhibition with retinoic acid (RA) local increase, triggering key events leading to craniofacial defects. Valproic acid (VPA) is supposed to imbalance RA-regulated gene expression trough histone deacetylases (HDACs) inhibition. The aim was to evaluate effects of molecules sharing the same MIE (azoles) and of such having (hypothetically) different MIEs but which are eventually involved in the same adverse outcome pathway (AOP). An in silico approach (molecular docking) investigated the suggested MIEs. Teratogenicity was evaluated in vitro (WEC). Abnormalities were modelled by PROAST software. The common target was the branchial apparatus. In silico results confirmed azole-related CYP26 inhibition and a weak general VPA inhibition on the tested HDACs. Unexpectedly, VPA showed also a weak, but not marginal, capability to enter the CYP 26A1 and CYP 26C1 catalytic sites, suggesting a possible role of VPA in decreasing RA catabolism, acting as an additional MIE. Our findings suggest a new more complex picture. Consequently two different AOPs, leading to the same AO, can be described. VPA MIEs (HDAC and CYP26 inhibition) impinge on the two converging AOPs

Antenatal Microbial Colonization of Mammalian Gut

The widely accepted dogma of intrauterine sterility and initial colonization of the newborn during birth has been blurred by recent observations of microbial presence in meconium, placenta, and amniotic fluid. Given the importance of a maternal-derived in utero infant seeding, it is crucial to exclude potential environmental or procedural contaminations and to assess fetal colonization before parturition. To this end, we analyzed sterilely collected intestinal tissues, placenta, and amniotic fluid from rodent fetuses and tissues from autoptic human fetuses. Total bacterial DNA was extracted from collected samples and analyzed by Next Generation Sequencing (NGS) techniques using hypervariable 16S ribosomal RNA (rRNA) regions (V3-V4). Colonizing microbes were visualized in situ, using labeled probes targeting 16S ribosomal DNA by fluorescent in situ hybridization. The NGS analysis showed the presence of pioneer microbes in both rat and human intestines as well as in rodent placentas and amniotic fluids. Microbial communities showed fetus- and dam-dependent clustering, confirming the high interindividual variability of commensal microbiota even in the antenatal period. Fluorescent in situ hybridization analysis confirmed the microbes' presence in the lumen of the developing gut. These findings suggest a possible antenatal colonization of the developing mammalian gut

Studio degli effetti miscela sullo sviluppo embrionale in vitro di due composti con diverso meccanismo d’azione (fluconazolo ed etanolo) utilizzando due diversi modelli sperimentali: coltura di embrioni di ratto e esposizione di embrioni di Ascidia

Scopo di questo lavoro \ue8 stato quello di valutare in vitro gli effetti miscela sul potenziale teratogeno della co-esposizione a Fluconazolo (FLUCO, fungicida azolico ampiamente utilizzato in ambito clinico) ed a concentrazioni sub-teratogene di Etanolo (Eth). Nella prima parte di questo studio, embrioni post-impianto di ratto sono stati esposti in vitro a concentrazioni crescenti di FLUCO (62.5-125-250-500\ub5M), alla concentrazione non effetto di Eth (1\ub5L/mL) o sono stati co-esposti a FLUCO 62.5-125-250-500\ub5M ed Eth (1\ub5L/mL). Al termine del periodo di coltura (48 ore) gli embrioni sono stati esaminati morfologicamente e processati per valutare l\u2019espressione dei geni specificamente coinvolti nel metabolismo dell\u2019Acido Retinoico (adh7, cyp26a1, cyp26b1, cyp26c1) o la distribuzione delle loro proteine nell\u2019embrione. Eth da solo non ha indotto effetti avversi sullo sviluppo embrionale. I gruppi esposti a FLUCO 125-250-500\ub5M mostravano specifiche anomalie a livello dell\u2019apparato branchiale, comparabili a quelle precedentemente documentate e correlate ad un probabile aumento di RA. Gli effetti erano dose-dipendenti sia per la frequenza degli embrioni malformati che per la gravit\ue0 delle malformazioni osservate. FLUCO 62.5 \ue8 stata individuata come la NOAEL (No Observed Adverse Effect Level). Gruppi co-esposti a FLUCO ed Eth mostravano un aumento significativo dell\u2019effetto teratogeno rispetto ai gruppi esposti al solo FLUCO. In seguito alla co-esposizione con Eth, FLUCO 62.5 \ub5M mostrava pi\uf9 del 35% degli embrioni anomali. Tale concentrazione non era pi\uf9 identificabile come NOAEL. L\u2019analisi dell\u2019espressione genica ha rivelato che la sola esposizione a Eth o a FLUCO 62.5 \ub5M alterava l\u2019espressione di geni coinvolti nel catabolismo di RA (cyp26a1, cyp26c1) e che tale alterazione peggiorava in seguito alla co-esposizione. La immunocolorazione per CYP26a1 e CYP26c1 non ha evidenziato differenze nelle aree embrionali coinvolte nella loro espressione, che risultavano sovrapponibili nell\u2019area cefalica ai territori di espressione del marker delle cellule delle creste neurali (CRABP1). I dati ottenuti mostrano che la co-esposizione con Eth alla concentrazione sub-teratogena pu\uf2 aumentare il potenziale teratogeno del FLUCO. Questi risultati sono di particolare rilevanza considerando che FLUCO ed Eth non condividono lo stesso meccanismo d\u2019azione: FLUCO interferisce con il catabolismo di RA, mentre Eth potrebbe interferire con la sintesi endogena di RA. L\u2019analisi dell\u2019espressione dei geni correlati al metabolismo di RA suggerisce che l\u2019effetto miscela indotto da FLUCO ed Eth potrebbe essere spiegato da un accumulo di RA dovuto all\u2019azione delle due sostanze. Nella seconda parte di questo studio, embrioni dell\u2019ascidia Ciona intestinalis (Chordata, Tunicata) sono stati esposti in vitro dallo stadio di neurula allo stadio di larva natante a concentrazioni crescenti di FLUCO (31.5-62.5-125-250-500\ub5M), alla concentrazione non effetto di Eth (1\ub5L/mL) o sono state co-esposte a FLUCO 62.5-125-250-500\ub5M ed Eth (1\ub5L/mL). Al termine del periodo di coltura (15 ore) le larve sono state esaminate morfologicamente. Eth da solo non ha indotto effetti avversi sullo sviluppo embrionale, mentre i gruppi esposti a FLUCO mostravano specifiche anomalie a livello delle strutture anteriori, comparabili a quelle gi\ue0 documentate e correlate all\u2019esposizione a RA. \uc8 stato osservato un significativo aumento di larve con malformazioni gravi nei gruppi co-esposti alle miscele di FLUCO ed Eth. I dati preliminari ottenuti sembrano indicare l\u2019ascidia come un adeguato modello alternativo per lo screening teratogeno delle miscele ottenute da fungicidi azolici: viene confermata l\u2019ipotesi di un ruolo chiave dell\u2019Eth nell\u2019aumentare il potenziale teratogeno del FLUCO, puntando l\u2019attenzione su un possibile impatto ambientale dei fungicidi azolici

Teratogenic Potential of Traditionally Formulated and Nano-Encapsulated Vitamin A in Two Vertebrate Models, Rattus norvegicus and Xenopus Laevis

Nano-encapsulation is applied for the preparation of functional food to preserve micronutrients degradation and to ameliorate their absorption. Being nano-encapsulation already related to increased vitamin A embryotoxicity, we aimed to evaluate the effect of traditionally formulated (BULK-A) and nano-encapsulated vitamin A (NANO-A) in two different vertebrate models: rat post implantation Whole Embryo Culture (WEC) and Frog Embryo Teratogenesis Assay-Xenopus (FETAX). After benchmark-dose modelling, WEC results showed that NANO-A was 7 times more effective than BULK-A, while FETAX results indicated that X. laevis development was affected only by NANO-A. The relative potency of WEC was 14 times the potency of FETAX, suggesting a minor role of preformed vitamin A in X. laevis development in respect to mammal embryogenesis. Results from this work prompt the necessity to monitor the use of food supplemented with NANO A, since even low doses can elicit teratogenic effects on vertebrate embryos due to its increased bioavailability

Comparison of teratogenic potency of azoles using in silico and in vitro methods

Craniofacial morphogenesis is affected in rodents by a number of clinical or agrochemical azole fungicides. The hypothesized mode of action of azoles includes abnormal neural crest cell specification and migration from hindbrain to the embryonic branchial region due to retinoic acid (RA) excess. Moreover, the additive effects after the multiple exposure to triazole fungicides account for a common mode of action. In analogy to their antifungal mode of action and hepatic side effects (inhibition of CYP isozymes), the proposed molecular initiating event for azole teratogenicity is the inhibition of embryonic CYP26 isozymes which are key proteins in RA catabolism. Experiments performed on postimplantation rat whole embryo cultures show that all tested azoles are teratogenic at micromolar concentrations, but characterized by different potencies (flusilazole=imazalil=ketoconazole>triadimefon=triadimenol>cyproconazole>tebuconazole>fluconazole). Molecular docking of eight azoles has been performed on CYP26a1, CYP26b1 and CYP26c1 isozymes, which play different roles in their teratogenic outcomes. Different affinities, consistent with the different azole teratogenic profiles and potencies, have been computed, confirming this hypothesis

Deciphering and modelling remyelinating mechanisms induced by clinically-used azole antifungals with exploitable repurposing properties

Recent evidence demonstrated the ability of some antifungals to induce (re)myelination in speci c experimental models. These compounds, already marketed for di erent pathologies, are interesting candidates for a repurposing strategy in neurodegenerative diseases, like multiple sclerosis (MS), characterized by strong demyelination. Our preliminary results show that the inhibition of enzymes involved in retinoic acid (RA) catabolism (CYP26), by miconazole and other azole antifungals, induces an increase of cellular RA concentration and that this increase may be behind the observed oligodendrocyte precursor cell (OPC) maturation and di erentiation. On this basis, our proposal is aimed at studying the molecular mechanisms of azole antifungals used in human pharmacology, both in silico and in vitro, and at identifying the most promising ones according to their ability to increase Myelin Basic Protein (MBP) expression on OPCs and to promote their di erentiation and myelination. In our investigation, the ability of 7 azoles to inhibit CYP26 isozymes was evaluated through molecular docking: in silico results were the basis for the selection of the drugs for the in vitro experiments. The selected azoles were tested in three di erent cell models, characterized by increasing complexity: i) OPC cultures, a maturation assay for myelin-producing cells; ii) OPC-dorsal root ganglion (DRG) neuron co-cultures, a myelination assay; iii) cerebral micromass cultures, a cell di erentiation assay for CNS. In parallel, the e ects of exogenous RA and of an inhibitor of the synthesis of RA (citral), were evaluated as well. In the next phase of the project, the outcomes from the di erent in vitro models will be statistically correlated with the pharmacological treatments. Our results will be useful: 1) for identifying the most promising azole antifungals with respect to their pro-myelinating activity, 2) for clarifying the molecular mechanisms underlying their reparative e ect and 3) for formalizing mathematical models to identify the most active concentrations for the most potent azoles

Comparison of four different colorimetric and fluorometric cytotoxicity assays in a zebrafish liver cell line

Background: A broad spectrum of cytotoxicity assays is currently used in the fields of (eco)toxicology and pharmacology. To choose an appropriate assay, different parameters like test compounds, detection mechanism, specificity, and sensitivity have to be considered. Furthermore, tissue or cell line can influence test performance. For zebrafish (Danio rerio), as emerging model organism, cell lines are now increasingly used, but few studies examined cytotoxicity in these cell systems. Therefore, we compared four cytotoxicity assays in the zebrafish liver cell line, ZFL, to test four differently acting model compounds. The tests comprised two colorimetric assays (MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, and the LDH assay detecting lactate dehydrogenase activity) and two fluorometric assays (alamarBlue® using resazurin, and CFDA-AM based on 5-carboxyfluorescein diacetate acetoxymethyl ester). Model compounds were the pharmaceutical Tamoxifen, its metabolite 4-Hydroxy-Tamoxifen, the fungicide Flusilazole and the polycyclic aromatic hydrocarbon Benzo[a]pyrene. Results: All four assays performed well in the ZFL cells and led to reproducible dose-response curves for all test compounds. Effective concentrations causing 10% or 50% loss of cell viability (EC10 and EC50 values) varied by a maximum factor of 7.0 for the EC10 values and a maximum factor of 1.8 for the EC50 values. The EC values were not statistically different between the four assays, which is due to the assessed unspecific effects of the compounds. However, most often, the MTT assay and LDH assay showed the highest and lowest EC values, respectively. Nevertheless, the LDH assay showed the highest intra- and inter-assay variabilities and the lowest signal-to-noise ratios. In contrast to MTT, the other three assays have the advantage of being non-destructive, easy to handle, and less time consuming. Furthermore, AB and CFDA-AM can be combined on the same set of cells without damaging the cells, allowing later on their use for the investigation of other endpoints. Conclusions: We recommend the alamarBlue and CFDA-AM assays for cytotoxicity assessment in ZFL cells, which can be applied either singly or combined.JRC.H.5-Rural, water and ecosystem resource