Evaluation of temperature- and ethanol-related developmental degree variations by a new scoring system (FETAX-score) applicable to Frog Embryo Teratogenicity Assay: Xenopus.

The aim of the present work is to propose a new quantitative assessment method (FETAX-score) for determining the degree of Xenopus laevis embryo development intended for use in embryotoxicity studies. Inspired by a similar scoring system used to evaluate developmental delays (young-for-age phenotypes) in rat embryos cultured in vitro, the FETAX-score was established by considering seven morphological features (head, naris, mouth, lower jaw, tentacles, intestine, anus) that are easily evaluable in tadpoles during the late stages of development at the conclusion of the test. Given that X. laevis development is temperature-dependent and that temperatures below 14°C and above 26°C are teratogenic, the FETAX-score was tested in embryos maintained at 17, 20, 23 and 26°C. No abnormalities were observed in any group, while the total score was temperature-related, suggesting that the FETAX-score is sensitive to moderate distress that does not influence general morphology. Intestine and anus were the least sensitive structures to temperature variations. To assess the applicability of the FETAX-score in developmental toxicological studies, we evaluated FETAX-score in tadpoles exposed during the morphogenetic period to Ethanol (Eth) at concentrations of 0, 0.25, 0.5, 1, 1.5, and 2 % v/v. Gross malformations were observed only in tadpoles from the Eth 2 % group. By contrast, data analysis of the other Eth groups showed dose-related reductions in the FETAX-score. Tentacles were the most sensitive structures to Eth-related delays. These results support the use of the FETAX-score to quantitatively assess developmental deviations in FETAX embryotoxicity studies.

Effects of combined exposure to two bisphenol plasticizers (BPA and BPB) on Xenopus laevis development.

Due to its endocrine disruptive activity, the plastic additive Bisphenol A (BPA) is classified as substance of very high concern (EU ECHA 2017). A correlation between environmental exposure to BPA and congenital defects has been described in humans and in experimental species including the amphibian Xenopus laevis, where severe branchial defects were associated to lethality. The exposure of X. laevis embryos to the BPA analogue bisphenol B (BPB) was recently linked to similar teratogenic effects, with BPB having relative potency about 3 times higher than BPA. The combined BPA-BPB exposure is realistic as both BPA and BPB are detected in human samples and environment. Limited experimental data are available on the combined developmental toxicity of BPA and BPB. The aim of the present work is to evaluate the effects of BPA and BPB mixture in the X. laevis development model, using R-FETAX procedure. The exposure was limited to the first day of development (corresponding to the phylotypic developmental period, common to all vertebrates). Samples were monitored for lethal effects during the full six-day test period and the external morphology was evaluated at the end of the test. Mixture effects were described by modelling, using the PROAST software package. Overall data modelling showed that dose-addiction could not be rejected, suggesting a health concern for co-exposure.

Teratogenic and neuro-behavioural toxic effects of bisphenol A (BPA) and B (BPB) on Xenopus laevis development.

Bisphenol A (BPA) is a plastic additive with endocrine disruptive activity, classified in 2017 by EU ECHA as substance of very high concern. A correlation between environmental exposure to BPA and congenital defects has been described in humans and in experimental species, including the amphibian Xenopus laevis. Among BPA analogues, bisphenol B (BPB) is used as alternative in different not-EU countries, including US, but seems to share with BPA its endocrine disruptor properties. Aim of the present work is the evaluation of the effects of BPB versus BPA exposure in a X. laevis developmental model. A windowed exposure (R-FETAX method) was applied covering the developmental phylotypic period (teratogenicity window), or the late tailbud stages (neuro-behavioural toxicity window, corresponding to the spontaneous swimming acquisition period). Samples were monitored for lethal effects during the full test period. External morphology evaluation and deglutition functional test were applied in any group. Abnormal tadpoles were also processed for cartilage staining. In groups exposed during neuro-behavioural toxicity window the swimming test was also applied. Lethality and malformations were obtained only in samples exposed during the teratogenicity window; these data were modelled using PROAST software and BPB relative potency resulted about 3 times higher than BPA. The day-by-day evaluation revealed that lethality was correlated to embryonic abnormal development of gills and apoptosis in gill primordia. Teratogenicity was never detected in groups exposed during the neuro-behavioural toxicity window, where some significant neuro-behavioural deficits were detected in tadpoles exposed to the highest tested concentrations of BPA and BPB.

Sperm protein 17 is an oncofetal antigen: a lesson from a murine model.

Sperm protein 17 (Sp17) was originally identified in the flagellum of spermatozoa and subsequently included in the subfamily of tumor-associated antigens known as cancer-testes antigens (CTA). Sp17 has been associated with the motility and migratory capacity in tumor cells, representing a link between gene expression patterns in germinal and tumor cells of different histological origins. Here we review the relevance of Sp17 expression in the mouse embryo and cancerous tissues, and present additional data demonstrating Sp17 complex expression pattern in this murine model. The expression of Sp17 in embryonic as well as adult neoplastic cells, but not normal tissues, suggests this protein should be considered an "oncofetal antigen." Further investigations are necessary to elucidate the mechanisms and functional significance of Sp17 aberrant expression in human adult cells and its implication in the pathobiology of cancer.

Are azole fungicides a teratogenic risk for human conceptus?

Azole fungicides are widely used in agriculture and in human mycosis. Their antifungal activity is based on their ability to inhibit CYP51, a key enzyme in the formation of fungal wall. Several azole fungicides tested in laboratory animals have been found to possess a common teratogenic potential to induce facial, axial skeleton, and limb defects. The mechanism of the teratogenic effect has been hypothesized to be related to the capability of these substances to alter embryonic retinoic acid catabolism. Although a number of human epidemiological studies were unable to demonstrate a definite relationship between azole exposure during pregnancy and birth defects, some case reports indicate a possible teratogenic effect of high doses of azoles in humans. Because of their common mechanism of action, azole fungicides should be regarded with caution for use in pregnant women.

Molecular mechanism of teratogenic effects induced by the fungicide triadimefon: study of the expression of TGF-beta mRNA and TGF-beta and CRABPI proteins during rat in vitro development.

Azole derivatives are teratogenic in rats and mice in vitro and in vivo. The postulated mechanism for the dysmorphogenetic effects is the inhibition of retinoic acid (RA)-degrading enzyme CYP26. Azole-related abnormalities are confined to structures controlled by RA, especially the neural crest cells, hindbrain, cranial nerves, and craniofacial structures, through a complex signal cascade. The aim of this work is to study the expression of signal molecules activated by RA (TGF-betas) or involved in the modulation of cellular RA concentrations (CRABPI). E9.5 (9.5 day post coitum old embryos) rat embryos, exposed in vitro to triadimefon (FON) for 24 h, were examined or cultured in normal serum for extra 4, 16, and 24 h. RT-PCR was performed to quantify TGF-beta1, TGF-beta2, TGF-beta3, TGF-betaRI, TGF-betaRII, and TGF-betaRIII mRNA in the hindbrain after 24 h of culture. TGF-beta1, TGF-beta2, and TGF-betaRI were found significantly decreased by FON exposure, and consequently their protein expression was analyzed by Western blot and immunohistochemistry. In both controls and FON-exposed embryos, TGF-beta1 and TGF-betaRI were detected at 24 and 24+4 h; TGF-beta2 was present only at 24 h. Only TGF-beta1 was expressed at the level of hindbrain and branchial tissues. After quantization, TGF-beta1 was reduced in the FON group. The expression of CRABPI was observed at all developmental stages. However, in FON-exposed embryos, it was increased at 24 and 24+4 h. The hindbrain distribution of CRABPI-positive cells was abnormal in FON-exposed embryos. The results show that the two RA-related molecules (TGF-beta1 and CRABPI) are altered by FON exposure in vitro.

Study on the common teratogenic pathway elicited by the fungicides triazole-derivatives.

Triazole-derivatives alter the pharyngeal apparatus morphogenesis of rodent embryos cultured in vitro. The hindbrain segmentation and the rhombencephalic neural crest cell (NCCs) migration are altered by Fluconazole exposure in vitro. The aim of the present work is to identify if a common pathogenic pathway is detectable also for other molecules of this class of compounds. 9.5 days post coitum (d.p.c.) old rat embryos were exposed in vitro to the teratogenic concentrations of Flusilazole, Triadimefon and Triadimenol and cultured for 24, 48 or 60 h. The expression and localisation of Hox-b1 and Krox-20 proteins (used as markers for hindbrain segmentation) were evaluated after 24 h of culture. The localisation and distribution of NCC was evaluated after 24, 30 and 48 h of culture. The morphology of the embryos was analysed after 48 h, while the branchial nerve structures were evaluated after 60 h of culture. Hindbrain segmentation and NCC migration alteration as well as pharyngeal arch and cranial nerve abnormalities were detected after exposure of the tested molecules. A common severe teratogenic intrinsic property for the tested molecules of this chemical class has been found, acting through alteration of the normal hindbrain developmental pattern.

Histological study of timing and embryology of notochordal abnormalities in rat exposed in utero to Doxorubicin.

Experimental Doxorubicin-exposure in utero is correlated with foetal oesophageal atresia, tracheo-oesophageal fistula, axial alterations. While gastrointestinal and respiratory defects have been largely investigated, only sporadic data have been published to date on notochordal and vertebral defects. The aim of this work was the study of the genesis of chordal and vertebral abnormalities in rat embryos and foetuses exposed to Doxorubicin and the study of their correlation with oesophageal and tracheal defects. For this purpose, pregnant rats were i.p. injected with saline (control) or with 4 mg/Kg b.w. Doxorubicin on days 9.5 and 10.5 of gestation. Embryos and foetuses were morphologically analysed on days 10.5-15 and 16, 18, 20 of gestation respectively, fixed in formaldehyde and histologically processed. Slides were routinely stained with haematoxylin-eosin (11-15 days post coitum embryos and all foetuses) or specifically stained with aniline blue for the staining of basal laminae (10.5 days post coitum embryos). Moreover, some foetuses at term (20 days post coitum) were processed for bone and cartilage staining. The data obtained in the present work confirm the specificity of Doxorubicin in inducing gastro-intestinal and tracheal defects, describe the genesis of these defects step by step, describe the type and the genesis of notochordal abnormalities and their fate and exclude the role of Doxorubicin in inducing axial skeletal malformations.

Atlas of rat fetal skeleton double stained for bone and cartilage.

BACKGROUND: The double staining of fetal skeleton for bone and cartilage is a very useful method to evidence skeletal abnormalities in laboratory animals. However, this method has been rarely used in routine developmental toxicity tests. One reason could be the difficulty of comparing the single skeletal pieces and of having reference points. In this paper the fetal rat skeleton double stained with Alizarin red S and Alcyan Blue is described in detail to produce an atlas for developmental toxicity laboratories.

Antifungal triazoles induce malformations in vitro.

Triazole-derivatives are antimycotics used in agriculture as well as in clinical and veterinary therapy. The aim of the present work is the in vitro comparative study of the teratogenic activity of triazole (the parental compound), flusilazole (an agricultural triazole mono-derivative fungicide), and fluconazole (a clinically used bis-triazole derivative). Rat embryos, 9.5 days old (1 to 3 somites) were exposed in vitro to triazole 500 to 5000 microM, flusilazole 3.125 to 250 microM, or fluconazole 62.5 to 500 microM. After 48 h in culture, the embryos were morphologically examined and processed for histologic and biochemical analysis. Flusilazole and fluconazole showed similar teratogenic effects (abnormalities at the branchial apparatus level and cell death at the level of the branchial mesenchyme) at concentration levels of 6.25 microM and higher for flusilazole and of 125 microM and higher for fluconazole. By contrast, only slight developmental retardation and blood discoloration were observed at the highest concentrations of triazole, suggesting no teratogenic activity for the triazole group.

Teratogenic effects of Doxorubicin in rats at midgestation and at term.

Doxorubicin (DOXO) is an anticancer drug widely used against leukaemias and solid tumours. Doxorubicin is teratogenic in laboratory species, as demonstrated by a number of in vivo and in vitro experiments. In our in vitro study, the endoderm of the primitive gut and the caudal mesenchymes have been the main target tissues, while oxidative stress has been excluded as a mechanism in Doxorubicin-related abnormalities. The aims of the present work are the confirmation of our previous in vitro results (obtained on rat embryos cultured from 9.5 to 11.5 d.p.c.) in an in vivo model (midgestation) and the comparison of the abnormalities observed in the embryos at midgestation with malformations observable at term of pregnancy. For this purpose, pregnant rats were i.p. treated on 9.5 and 10.5 d.p.c. with DOXO. Embryos were analysed on 11 d.p.c., while foetuses were externally and viscerally analysed on 20 d.p.c. (term of gestation). The data collected at midgestation confirm the specific embryotoxic effects (particularly to the primitive gut and the caudal mesenchyme) previously observed in vitro. The data collected at term show the endoderm derivatives (gastrointestinal apparatus and respiratory tract) as the primary target organs, supporting the idea that the teratogenic activity of Doxorubicin is mediated by specific toxic effects directed to the primitive gut.

Acetaldehyde in vitro exposure and apoptosis: a possible mechanism of teratogenesis.

Alcohol abuse by pregnant women can result in fetal alcohol effects (FAE) and fetal alcohol syndrome (FAS). Both ethanol itself and its main metabolite, acetaldehyde (Ach), are able to produce specific FAS-related malformations. In previous in vitro studies, we documented that 10-day-old rat embryos exposed to Ach show a characteristic embryonic Ach syndrome, histologically characterized by marked cellular death. As both necrosis and pathological apoptosis are teratological mechanisms, the aim of this work was to evaluate if cellular death, observed in Ach-exposed embryos, can be related to necrotic or apoptotic events. Ten-day-old rat embryos were cultured in the presence of Ach 30-60 microg/ml and stained with the vital dye acridine orange to visualize apoptotic areas. After fixation, the TUNEL [3' terminal deoxynucleotide transferase (TdT)-mediated dUTP-biotin nick end labeling] method was used to histologically identify apoptosis. Both acridine orange and TUNEL staining showed signs of physiological apoptosis in controls and abnormal apoptotic regions in Ach-exposed embryos. Our results show a clear correlation between malformed organs and apoptotic embryonic districts, suggesting the role of apoptosis in Ach-induced abnormalities.

Expression of Fhit protein during mouse development.

Fhit protein has a putative tumor suppressor function in several types of human and experimental cancers. To assess whether Fhit is involved in fetal development we have examined the distribution of Fhit protein in the 12- through 16-day postcoitum mouse fetus and in postnatal day 0 mouse pups by immunocytochemistry. High levels of Fhit protein were observed in the endodermal derivatives, namely, bronchi, trachea, esophagus, stomach, and intestine, in the 12- to 16-day postcoitum mouse fetus and in the postnatal day 0 pup. Other tissues showed a more restricted pattern of Fhit protein expression. These results suggest that Fhit may play a role in the development of specific tissues during mouse development.

In vitro teratogenic potential of two antifungal triazoles: triadimefon and triadimenol.

The teratogenic potential of two antifungal triazoles (Triadimefon and Triadimenol) has been investigated in vitro by the rat postimplantation whole embryo culture method. Rat embryos 9.5 d old were cultured for 48 h in rat serum with Triadimefon (12.5-250 microM) or Triadimenol (6.25-125 microM) and then examined. Some embryos exposed to Triadimenol (6.25-125 microM) were cultured for 12 extra hours in control serum to improve their developmental degree and then immunostain cranial nerves and ganglia. The exposure to the highest doses of triazoles only moderately reduced some morphometrical developmental parameters. By contrast, 25-250 microM Triadimefon and 25-125 microM Triadimenol induced specific concentration-related teratogenic effects at the level of first and second branchial arches. After immunostaining, embryos exposed to 12.5-125 microM Triadimenol showed specific cranial nerve and ganglia abnormalities. The possible implication of neural crest cell alterations on triazole-related abnormalities is discussed.

Morphological alterations induced by sodium valproate on somites and spinal nerves in rat embryos.

The antiepileptic drug valproic acid is a well-known teratogenic agent; its main target organ is the neural tube, though skeletal malformations have also been described. In our recent work, respecifications of vertebrae were described in rat fetuses after treatment with 400 mg/kg of sodium valproate at specific somitogenic stages. The observed malformations were stage-dependent. Morphological segmental respecification was observed at the level of segments in formation at the moment of exposure and at the level of more posterior segments. Recently, specific alterations in the development of cranial nerves and ganglia were described in mouse embryos after in vitro exposure to VPA. The aim of the present work was to analyze dysmorphogenetic effects of VPA on embryonic metameric structures: somites, spinal and cranial nerves, and ganglia. Sodium valproate (400 mg/kg) was subcutaneously injected at specific gestational times corresponding to embryonic stages: presomitic or at about 2, 6, 10, 14, 18, or 22 somites. Females were sacrificed on the day 12 post coitum, and embryos were examined. Morphological examination of somites was performed by staining with acridine orange. Morphological examination of nerves and ganglia was performed by immunostaining, using monoclonal antibodies to the 160-kD neurofilament protein. No abnormalities were observed in the cranial nerves and ganglia. Specific and stage-dependent alterations were observed both at the level of the somites and at the level of the spinal nerves. The following characteristic malformations were observed: fusions, duplications, and reductions of somites and corresponding spinal nerves and ganglia. Our morphological data suggest a morphogenetic action of VPA at the level of the axial segments, with a possible respecification of the identity of the interested segments and their derivatives.

Development of rat embryos cultured in serum from diabetic rats.

Experimental studies carried out in vitro suggest a role of oxidative stress in diabetes-induced embryopathies. Glutathione is the main defense against free radicals in embryonic as it is in adult tissues. In this experiment, using postimplantation whole-embryo culture, we analyze: (1) the effects of serum from streptozotocin-induced diabetic rats on embryonic development and on glutathione distribution between the yolk sac and embryonic tissues and (2) the role of glutathione in preventing embryopathies (using the inhibitor of glutathione synthesis buthionine sulfoximine). Our data show that in rat embryos cultured in diabetic serum, the only observed effects are at the yolk sac level. No effects on the glutathione content were observed. The addition of buthionine sulfoximine reduced the glutathione content and produced signs of developmental delay in embryos cultured in diabetic serum, suggesting a role of the oxidative stress in producing diabetes-related embryotoxicity.

In vitro embryotoxicity study of n,n-dimethylacetamide and its main metabolite N-monomethylacetamide.

N,N-Dimethylacetamide (DMAC) is a widely used industrial solvent. Previous teratological studies in vivo reported discording results. Using the postimplantation rat whole embryo culture (WEC) method, the direct embryotoxic effects of DMAC and its main metabolite (N-monomethylacetamide, MMAC) have been investigated in the present work. Both chemicals showed specific embryotoxic and teratogenic effects at similar concentration levels. The no-observed-effect level (NOEL) was 0.85mm.Macroscopically, the main target organs were somites, brain and branchial bars. Histological examination revealed an increase in cell death at the effective concentrations on the neuroepithelium and branchial bars mesenchyme. The results of this work, together with those obtained in in vivo studies, suggest that the exposure limits in workplaces could be inappropriate for the safety of fertile women.

Postcoital antifertility activity of aminoalcohols.

Previously, postcoital antifertility effects of a number of aminoalcohols, including 2-(isopropylamino)-ethanol, have been demonstrated in rodents. In this experiment, we compared the antifertility activity of 2-(isopropylamino)-ethanol to the following analogs: hydroxyethylpiperidine, hydroxyethylpiridine, hydroxyethylpirrolidine, and hydroxyethylpirrolidone. Female rats were gavaged on Days 0 through 5 of gestation with 0.7 mmol/kg/d of these substances. Only 2-(isopropylamino)-ethanol and hydroxyethylpirrolidine showed a strong antifertility activity: females treated with 2-(isopropylamino)-ethanol had no signs of implantation, whereas those treated with hydroxyethylpirrolidine had 100% early resorptions. Treatments with these two substances during the periimplantation period (Days 4 and 5) produced 100% early resorptions. Histologic examination of the implantation sites showed signs of embryonic degeneration starting from Day 6.5 of gestation. The flushing of the uteri of females treated with 2-(isopropylamino)-ethanol on Days 0 through 3 post coitum showed 78% of the embryos at the stage of 1 to 3 blastomeres, whereas the embryos of females treated during the same period with hydroxyethylpirrolidine were normal blastocysts. Therefore, 2-(isopropylamino)-ethanol and hydroxyethylpirrolidine are able to kill embryos during the early implantation stages, whereas 2-(isopropylamino)-ethanol is also able to stop the development of preimplantation embryos.

Stage-dependent skeletal malformations induced by valproic acid in rat.

In this work we study the skeletal teratogenic response in rats exposed to NaVP at different embryonic stages. Crl:CD female rats were treated subcutaneously with 400 mg/Kg b.w. NaVP at presomitic stage (group II) or nearly at 2, 6, 10, 14, 18 or 22 somites (groups III-VIII). The females on group I were treated with saline and served as controls. No treatment-related effects were observed at the level of resorptions, live fetuses and fetal or placental weight. The skeletal examination showed characteristic patterns of malformations strictly related to the period of treatment. In particular, groups II and III showed a significant increase of alterations of cervical vertebrae (mainly 1st to 3rd segment) and a decrease of the frequency of extra lumbar ribs in comparison to control. Group IV showed severe abnormalities localized at the 4th to 7th cervical segment and at the level of the 1st and 2nd thoracic segments, including duplications of thoracic segments 1, 2 or 3. The fetuses of group V were characterized by several alterations of the thoracic segments distributed without a clear specificity. In group VI, the thoracic region was also affected with some specificity at the level of the segments 4th to 9th; in group VII, last thoracic and lumbar segments were affected (mainly duplications) and in group VIII only lumbo-sacral abnormalities were recorded. These results confirm the specific effect of NaVP at the level of the axial skeleton and suggest a possible interaction with the expression of genes identifying the vertebral segments.

Comparative embryotoxicity of four anthracyclines: In Vitro study on their effects on glutathione status.

The embryotoxicity of four different anthracyclines [doxorubicin (DOXO), epirubicin (EPI), daunorubicin (DAUNO) and idarubicin (IDA)] was compared using the postimplantation whole embryo culture method. Moreover, to investigate the role of free oxygen radical production in anthracycline-induced embryotoxicity, the reduced glutathione (GSH) status in embryos and extraembryonic membranes was measured. Rat embryos (1-3 somite stage) were cultured for 48 hr in heat-inactivated rat serum containing DOXO, EPI or DAUNO (0.025, 0.05, 0.075 or 0.1 muM) or IDA (0.0125, 0.025 or 0.05 muM). At the end of the culture period, the embryos were examined morphologically and processed for histological or biochemical examination. DOXO, EPI and DAUNO were found to have similar embryotoxic potential [the no-observed-effect level (NOEL) was 0.025 muM], whereas IDA had a greater effect (NOEL = 0.0125 muM). The primary target organ was identified macroscopically and microscopically for all compounds in the mesenchyme of the caudal region. No relationship was identified between embryonic or extraembryonic GSH content reduction and anthracycline-induced embryotoxicity, suggesting only a marginal role of free oxygen radical formation in anthracycline-related embryotoxic mechanisms.