Alternative developmental toxicity models for assessing the in vivo embryotoxicity of azoles

The implementation of regulations for protecting both humans and the environment from potential chemical health hazards, as well as the increase of global pressure for reducing, refining and replacing animal experiments promote the development and application of alternatives to in vivo developmental toxicity studies. Due to the complexity of the reproductive cycle, combined in vitro approaches, focusing on morphological, molecular and toxicokinetic parameters, could better define the developmental toxicity of chemicals. In this thesis, azoles, which are a group of chemicals with antifungal activity, are under investigation. These compounds show marked differences in developmental toxicity potency and similarities with retinoic acid (RA)- related teratogenicity. 

Chapter 1 of this thesis introduced information regarding the background of reproductive and developmental toxicology, including scientific concerns and the impact of past teratogenic outcomes on the society. For screening developmental teratogens, in vitro approaches have been proposed and successfully applied. Their combination may better mimic the in vivo embryo and, therefore, increase the accuracy in predicting possible developmental toxicants. Additional co-implementation of molecular approaches may give an insight in the mode of action underlying the observed effects. Azoles were selected in the present thesis due to evidence for possibly increasing developmental toxicity through dysregulating the balance of the RA pathways in the mammalian system. The chapter also described the objectives and outline of the research.        

In chapter 2, we examined the time- dependent developmental effects in rat embryos exposed in vitro to flusilazole (FLU), and their link to RA mediated pathways. To this end, we assessed the effects of 4-hour exposure of whole embryo culture (WEC) embryos to 300μM FLU during four developmental time windows (0-4, 4-8, 24-28 and 44-48 h), evaluated morphological parameters, as well as expression and localization of five genes directly or indirectly linked with the RA pathway. A stage- specific gene expression response of cultured rat embryos exposed to FLU was detected, which preceded the development of morphologically observable malformations. During all the tested time windows, the most pronounced effect was observed in the regulation of RA-related genes. Therefore, it was concluded that such biomarkers can be employed as useful tools for early detection of possible teratogenic properties of compounds that belong to the triazole- group or of compounds with a similar teratogenic mode of action.

Chapter 3 provides mechanistic insight into the embryotoxicity of six azoles tested in the rat WEC. Here, we evaluated dose-dependent embryotoxicity of azoles in the rat WEC, calculating the concentration at which the total morphological score (TMS) is 10% decreased (ID10). For the azoles tested we compared the in vitro ID10 for embryotoxicity to the in vivo effective doses, while we also performed a comparative analysis for understanding the toxicological and pharmacological mode of action of azoles in the rat WEC at the level of the transcriptome. Functional analysis of differential gene expression after 4 hours exposure at the ID10 revealed regulation of the sterol biosynthesis pathway and embryonic development genes, dominated by genes in the RA pathway, albeit in a differential way. FLU, ketoconazole and triadimefon were the most potent compounds affecting the RA pathway, while in terms of regulation of sterol function, difenoconazole and ketoconazole showed the most pronounced effects. A similar analysis at the 24-hour time point indicated an additional time-dependent difference in the aforementioned pathways regulated by FLU. Strong in vivo embryotoxic azoles showed also an increased regulation of the RA pathway when tested in vitro. On the other hand, weak or non- embryotoxic azoles showed a non-significant effect on genes that belong in the RA pathway. These observations led us to the conclusion that the toxicological mode of action of azoles was mediated through the RA pathway. In summary, the rat WEC assay in combination with transcriptomics could add mechanistic insight into the embryotoxic potency ranking and functional efficacy of the tested compounds, showing Cyp26a1 and Cyp51 as leader biomarkers of the off- and on- target effects, respectively.

Similarly to the previous chapter, in chapter 4, the potency ranking of the majority of the twelve tested azoles obtained based on the TMS in the WEC assay was in agreement with the in vivo potency ranking. Additionally, our expanded transcriptomics data, including gene specific responses of twelve azoles tested at their ID10 in the rat WEC for 4 hours, confirmed the observations of chapter 3 with another set of azoles. Potent embryotoxicants in both in vivo and in vitro assays caused more pronounced effects on the dysregulation of RA- mediated genes. Furthermore, azoles with more pronounced effects on the sterol biosynthesis mediated pathway were tested at a higher concentration, but with the same level of effect (ID10). Due to the increased concentration needed for reaching the same level of morphological effects and the absence of RA-mediated pathway regulation, these azoles were considered as more favourable candidates for clinical and agricultural use. Focusing on monitoring the fungicidal activity of azoles, we also detected an increased sensitivity of the expression of Msmo1, which is an enzyme participating in converting lanosterol for synthesizing cholesterol in the mammalian sterol biosynthesis, together with Cyp51and Nsdhl. This observation led us to the conclusion that Msmo1 could be a better biomarker of effect on the sterol biosynthesis pathway compared to the classical biomarker of this pathway, Cyp51, and this may be of use for further improvement of the assessment of fungicidal activity of azoles or chemicals with similar mode of action.

Chapter 5 shows the value of combining toxico-dynamic and -kinetic in vitro approaches for embryotoxicity testing of azoles. We also report on the alterations in gene expression induced by azoles. Both the WEC assay and the embryonic stem cells test (EST) predicted the in vivo potency ranking of the twelve tested azoles with moderate accuracy. Combining these results with relative placental transfer rates (Papp values) as determined in the BeWo cell culture model, increased the predictability of both WEC and EST, with R2 values increasing from 0.51 to 0.87 and from 0.35 to 0.60, respectively. The comparison of these in vitro systems correlated well (R2 = 0.67), correctly identifying the strong and weak embryotoxicants. Evaluating specific gene responses related with the toxicological and fungicidal mode of action of the tested azoles in WEC and EST, we observed that the differential regulation of Dhrs3 and Msmo1 reached higher magnitudes in both systems compared to Cyp26a1 and Cyp51. Establishing sensitive biomarkers across all the in vitro systems for studying the underlying mechanism of action of chemicals, such as azoles, is valuable for comparing alternative in vitro models and for improving insight in the mechanism of developmental toxicity of chemicals.

Chapter 6 of this thesis presented the general discussion and future perspectives on different topics raised based on the results obtained in the previously described experimental chapters. The results suggested that the combination of in vitro assays for screening the developmental toxicity of azoles may lead to predictions that are more accurate and in agreement with the in vivo observations. The addition of toxico-kinetics, which the BeWo placental transfer model offered, notably improved the correlations of in vivo and in vitro data. Furthermore, the co- implementation of transcriptomics and the identification of gene biomarkers revealed that despite the tested azoles were classified in the same chemical group, they might have a different mode of toxicological action. In conclusion, future combination of in vitro and in silico alternative approaches appear to be of advantage for screening and prioritizing chemical testing, in the process of assessing the consequences of chemical exposure for human health and the environment.