Effects of mixtures of azole fungicides in postimplantation rat whole-embryo cultures.

The effect of mixtures of azole fungicides on development of postimplantation rat whole-embryos cultured in vitro has been tested. On the basis of bench mark dose (BMD) modeling of the in vitro effect in rat embryo, the potency of 7 azoles was determined and compared. Then, relative potency factors have been derived based on either the NOAEL or on the BMD curve. Alternatively, each compound was used as index compound (IC), and IC-equivalent concentrations have been calculated for each mixture. Expected effects of such IC-equivalent concentrations of the mixture were derived from the appropriate BMD curve. Test mixture includes the agrochemicals triadimefon and imazalil (MIX2) or triadimefon, imazalil, and the clinically used fluconazole (MIX3) at their previously determined no-effect concentration, corresponding to approximately a benchmark response of 5-10 %. Subsequently, we tested the effect of a mixture of the agrochemicals triadimefon, imazalil, triadimenol, cyproconazole, tebuconazole, and flusilazole (MIX6) at concentration levels derived from their established human acceptable daily intake. MIX6 was also added with fluconazole at concentration levels indicated as the minimum therapeutically effective plasmatic concentration (MIX7A) or ten times this level (MIX7B). Generally, the experimental response was higher than the estimated one, by a factor of 2-6. Our data suggest that it is in principle correct to assume that azoles act as teratogens via a common mode of action and therefore should be grouped together for risk assessment. The synergistic effect needs to be confirmed with more combinations of concentrations/compounds in vitro and with specific in vivo experiments.

Methionine pretreatment enhances the effects of valproate on axial development in a CD1 mouse model.

BACKGROUND: The present study was performed to evaluate the effect of methionine (Met) pretreatment on valproate (VPA) axial defects, induced in CD1 mice by a single intraperitoneal (i.p.) injection of 400 mg/kg VPA on E8 dams. This VPA dosage regimen has been in the past related to a specific pathogenic pathway cascade: (1) VPA in utero exposure, (2) H4 histone hyperacetylation (hAC) at the level of somites, (3) expression of pro-apoptotic factors in somite tissues, (4) apoptosis of somite cells, and (5) axial defects in embryos (abnormal or fused somites) and fetuses (fusions, duplications, respecifications of vertebrae, and/or ribs). METHODS: On the basis of literature suggestion, E8 CD1 mice were i.p. injected with 70 mg/kg Met 30 min before the i.p. injection with 400 mg/kg VPA. Some females were sacrificed, 1 or 3 hr after the VPA injection, embryos explanted, and used to evaluate the H4 histone hAC. The remaining females were sacrificed at term (E18) and fetuses processed for external and skeletal examination. RESULTS: The pretreatment with Met worsened the axial skeletal malformative picture in fetuses (we observed a larger number of affected segment per fetus in respect to the groups treated with VPA alone). In embryos, Met pretreatment increased the H4 hAC index and shifted the timing of the H4 hAC peak. CONCLUSIONS: Our data suggest that Met pretreatment enhances the effects of VPA in deregulating the epigenetic control of gene expression in somites, and by consequence, induces more extended dysmorphogenic effects along the axial axis.

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.

Early genetic control of craniofacial development is affected by the in vitro exposure of rat embryos to the fungicide triadimefon.

BACKGROUND: Previous published experiments reported that in vitro exposure of postimplantation rat embryos to the triazole fungicide triadimefon (FON) resulted in specific abnormalities at the branchial apparatus and that the sensitive period is restricted to the first 24 hr of culture and is associated with the abnormal expression of TGF family genes (some of a large panel of genes regulated by retinoic acid (RA) and involved in branchial arch morphogenesis). The aim of this study is the determination of the sensitive window to FON-induced abnormalities during in vitro development and the evaluation of the expression of some genes controlled by RA and involved in early branchial arch morphogenesis (Gsc, Msx1, Msx2, Dlx1, Dlx2, Shh, Patched (the main Shh receptor)). METHODS: Rat embryos were exposed in vitro to the FON under condition known to be able to induce 100% of abnormal embryos (250 µM) at different stages and examined after 48 hr of culture. The sensitive window for FON-induced abnormalities was during the hours E9 h8.00 PM-E10 h8.00 AM. To evaluate the expression of selected genes, embryos exposed during the sensitive stages were processed to perform quantitative PCR after 18 and 24 hr of culture. RESULTS: FON was able to affect the expression of some genes in a stage-specific manner: earlier embryos were characterized by the downregulation of Msx2 and Gsc, later embryos showed the downregulation of Gsc, Shh, and Patched. The obtained data suggest that FON-induced abnormalities are mediated, at least in part, through the imbalance of the expression of RA-related signals.

Expression analysis of some genes regulated by retinoic acid in controls and triadimefon-exposed embryos: is the amphibian Xenopus laevis a suitable model for gene-based comparative teratology?

BACKGROUND: The use of nonmammal models in teratological studies is a matter of debate and seems to be justified if the embryotoxic mechanism involves conserved processes. Published data on mammals and Xenopus laevis suggest that azoles are teratogenic by altering the endogenous concentration of retinoic acid (RA). The expression of some genes (Shh, Ptch-1, Gsc, and Msx2) controlled by retinoic acid is downregulated in rat embryos exposed at the phylotypic stage to the triazole triadimefon (FON). In order to propose X. laevis as a model for gene-based comparative teratology, this work evaluates the expression of Shh, Ptch-1, Gsc, and Msx2 in FON-exposed X. laevis embryos. METHODS: Embryos, exposed to a high concentration level (500 µM) of FON from stage 13 till 17, were examined at stages 17, 27, and 47. Stage 17 and 27 embryos were processed to perform quantitative RT-PCR. RESULTS: The developmental rate was never affected by FON at any considered stage. FON-exposed stage 47 larvae showed the typical craniofacial malformations. A significant downregulation of Gsc was observed in FON-exposed stage 17 embryos. Shh, Ptch-1, Msx2 showed a high fluctuation of expression both in control and in FON-exposed samples both at stages 17 and 27. CONCLUSION: The downregulation of Gsc mimics the effects of FON on rat embryos, showing for this gene a common effect of FON in the two vertebrate classes. The high fluctuation observed in the gene expression of the other genes, however, suggests that X. laevis at this stage has limited utility for gene-based comparative teratology.

Microtubule dysfunction precedes transport impairment and mitochondria damage in MPP+ -induced neurodegeneration.

Dysfunction of the microtubule (MT) system is an emerging theme in the pathogenesis of Parkinson's disease. This study was designed to investigate the putative role of MT dysfunction in dopaminergic neuron death induced by the neurotoxin 1-methyl-4-phenylpiridinium (MPP(+)). In nerve growth factor-differentiated PC12 cells, we have analyzed post-translational modifications of tubulin known to be associated with differently dynamic MTs and show that MPP(+) causes a selective loss of dynamic MTs and a concomitant enrichment of stable MTs. Through a direct live cell imaging approach, we show a significant reduction of MT dynamics following exposure to MPP(+) and a reorientation of MTs. Furthermore, these alterations precede the impairment of intracellular transport as revealed by changes in mitochondria movements along neurites and their accumulation into varicosities. We have also analyzed activation of caspase 3 and mitochondrial injury, well-known alterations induced by MPP(+), and found that they are noticeable only when MT dysfunction is already established. These data provide the first evidence that axonal transport impairment and mitochondrial damage might be a consequence of MT dysfunction in MPP(+) -induced neurodegeneration, lending support to the concept that alterations of MT organization and dynamics could play a pivotal role in neuronal death in Parkinson's disease.

New sulfurated derivatives of valproic acid with enhanced histone deacetylase inhibitory activity.

One dithiolthione and two new methanethiosulfonate derivatives of valproic acid (VPA) were synthesized and tested in vitro as histone deacetylase (HDAC) inhibitors. The new molecules, as well as their sulfurated moieties, exhibited a much stronger inhibition of HDAC enzymatic and antiproliferative activities and histone hyperacetylation than VPA. ACS 2 is the most interesting compound among the new VPA derivatives and its sulfurated moiety, 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione, also known to be a metabolite of anethole trithione, seems to contribute significantly to its activity. This is the first time that HDAC inhibitory activity is described for dithiolethiones and thiosulfonates.

Relationship between embryonic histonic hyperacetylation and axial skeletal defects in mouse exposed to the three HDAC inhibitors apicidin, MS-275, and sodium butyrate.

Some histone deacetylase inhibitors (HDACi) have recently been related to teratogenic effects in rodents. Skeletal defects have been directly associated with embryonic hyperacetylation of somitic nuclei after valproic acid or trichostatin A exposure in vivo. Albeit the antitumoral activity of HDACi has been classically related to chromatin condensation due to histonic lysine hyperacetylation, nonhistonic proteins have also been suggested as an HDACi target. The aim of this work was the study of the effects of three HDACi (apicidin, API; MS-275; sodium butyrate, BUT) on mouse development and their activity on embryonic histonic and nonhistonic proteins. Pregnant mice were ip treated with 10 mg/kg body weight API, 25 mg/kg MS-275, 2000 mg/kg BUT or with the vehicle alone on day 8 post coitum. Embryos were extracted 1, 2, or 3 h after treatment and Western blotting (using antibodies antihyperacetylated histone H4, antiacetylated lysine, or antitubulin) and immunohistochemistry (using the antibody antihyperacetylated histone H4) were performed. Fetuses, explanted at term of gestation, were double stained for bone and cartilage to detect skeletal abnormalities. The studied HDACi were teratogenic. The specific axial skeletal malformations were fusions or homeotic respecifications. These molecules induced hyperacetylation restricted to somitic histones. The hyperacetylation index of histone H4 as well as immunohistochemical and skeletal analyses indicated BUT as the less active molecule. These new data on effects of API, MS-275, and BUT on development suggest histonic hyperacetylation as the mechanism for the induction of the observed skeletal abnormalities.

Krox20 is down-regulated following triazole in vitro embryonic exposure: a polycompetitor-based assay.

This study was conducted in order to analyse gene-expression alterations in rat embryos following exposure to triazoles, using an easy-handling approach. Triazole derivatives have been shown to alter the morphology of cranio-facial structures and to induce abnormalities in hindbrain patterning and neural crest cell migration. Specification of hindbrain segments is regulated by retinoic acid and the hox code. Krox20 was chosen as molecular marker for its specific distribution in the anterior neural tube. In fact, this zinc-finger protein is expressed in rhombomere 3 and 5. Mis-regulation of Krox20 levels have shown to induce severe alterations in the correct patterning of the rhomboencephalon and the derived structures. In order to analyse Krox20 mRNA levels in rat embryos exposed in vitro to the triazole derivative triadimefon, a semi-quantitative approach utilising the competitive RT-PCR was chosen. A lambda phage-based plasmid construct that could compete with target and internal standard gene at the same time during enzymatic reaction was generated. Results were confirmed by real-time RT-PCR analysis on the same samples. Our data show a down-regulation of Krox20 transcript levels after exposure to the triazole derivative, implying a key role of this molecule in the pathogenic pathway induced by triazole exposure.

Citral, an inhibitor of retinoic acid synthesis, attenuates the frequency and severity of branchial arch abnormalities induced by triazole-derivative fluconazole in rat embryos cultured in vitro.

The clinically used antimycotic fluconazole (fluco) is teratogenic in rodents. Exposure in vitro to fluco, other investigated azoles (triadimefon, triadimenol, flusilazole, ketoconazole and imazalil) or retinoic acid (RA), is correlated to branchial arch abnormalities. Inhibition of RA degradation has been suggested as the azole-related mechanism. Citral is a RA synthesis inhibitor. E9.5 rat embryos were cultured for 48 h in normal serum or exposed in vitro to fluco 125 microM, citral 200 microM or co-exposed to the two molecules to test the hypothesis that citral attenuates fluco-related teratogenic effects. Some embryos were cultured for 12 extra hours, and cranial nerves immunodetected. Fluco induced typical abnormalities, including branchial arch and cranial nerve defects. The co-exposure to fluco+citral was significantly effective in reducing branchial arch and cranial nerve defects, supporting the hypothesis that citral balances the fluco-induced RA concentration increase. However, other fluco-related effects were unalterated by citral.

Dysmorphogenic effects of some fungicides derived from the imidazole on rat embryos cultured in vitro.

Like triazole-derivatives, imidazole-derivatives exert their antifungal and toxicological properties by inhibiting P450 enzymes (Cyps). At the embryonic level, Cyp enzymes are involved also in the catabolism of the retinoic acid. Specific effects of triazole-derivatives have been reported on developing rodent embryos, and were correlated to an imbalance of the retinoid homeostasis. The aim of this work was to investigate if imidazole-derivatives are able to induce specific malformations similar to those observed after triazole-derivative exposure. Post implantation rat embryos were exposed in vitro to 1,000 microM Imidazole and to 5-100 microM of the imidazole-derivatives Ketoconazole and Enilconazole. After 48 h in culture, the embryos exposed to the imidazole-derivatives showed specific malformations, quite similar to those observed after triazole-derivative exposure. The common dysmorphogenic effects of the azole-derivatives of the two classes could be due to the inhibition of retinoid catabolism. From this point of view, the contemporaneous exposure to these substances or their therapeutic use could be considered as potentially dangerous for human conceptuses.

Postulated pathogenic pathway in triazole fungicide induced dysmorphogenic effects.

Triazole fungicides are used in medicine as well as in agricultural treatment of mycoses. The pharmacological mechanism is related to the inhibition of CYP enzymes involved in the formation of the fungal walls. A similar inhibition of human CYP enzymes has been suggested as the cause of triazole side effects in humans. An important role of some CYP isoforms (CYP26 isoforms) expressed during mammalian development is the catabolism of retinoic acid, a known morphogen in vertebrates and invertebrates. The adverse effects on morphogenesis, observed after exposure of mammalian, amphibian and ascidiacea, are compared to the reported effects of triazole in humans. The possible pathogenic pathway in triazole-related teratogenesis is discussed on the basis of different experimental approaches.

Teratogenic effects of two antifungal triazoles, triadimefon and triadimenol, on Xenopus laevis development: craniofacial defects.

Triadimefon and triadimenol, fungicides used in agriculture, are suspected of producing craniofacial malformations. As the results of FETAX analysis showed that Triadimefon was highly teratogenic, we studied the action of these triazoles on the development of the branchial apparatus in Xenopus, using early molecular markers and analysis of the cartilaginous-muscular elements of tadpoles. Teratogenic effects were observed, after exposure at the neurula stage, at the level of cartilages and muscles of the 1st and 2nd branchial arches. By in situ hybridization, we observed that in exposed specimens the territories of the branchial arches are perturbed. From these results, we considered the craniofacial malformations related to the influence of triazoles on the differentiation of branchial arches. Comparing the anomalies caused by triazoles and by retinoic acid (RA) led us to the conclusion that triazoles can affect endogenous RA content, as has been shown for mammals.

The use of in vitro testing to refine cumulative assessment groups of pesticides: The example of teratogenic conazoles.

The most relevant issues in cumulative risk assessment (CRA) are the identification of cumulative assessment groups and the hypothesis of dose-additivity, at relevant human exposures. In vitro methods can provide meaningful data to help solving those issues. Integration of in vitro studies, selected in vivo studies, and PBPK modeling for teratogenic conazoles confirmed that in vitro studies may give results in a cheaper and faster fashion. In particular, in vitro studies with explanted rat embryos provided support for dose-additivity for conazoles causing cranio-facial malformations. Although this could not be immediately quantitatively transferred to the in vivo situation, they provided indication on how to conduct targeted in vivo studies. In addition, by means of PBPK modeling, it was possible to estimate the dose in humans associated with a defined teratogenic risk and also to conclude that for cumulative risk assessment only exposures occurring within a short period of time (a day or less) need to be cumulated. Although PBPK modeling cannot be widely applied, at least in the short term, it should be considered if available. It is recommended to incorporate in vitro testing and PBPK modeling, whenever available and feasible in the process of risk assessment, particularly of CRA.

Comparative study of sodium valproate-induced skeletal malformations using single or double staining methods.

The teratogenic activity of xenobiotics is usually investigated by examining visceral and skeletal abnormalities of term fetuses. Although the rodent fetal skeleton is only partially ossified, the single stain for bone is the most commonly used method in routine teratology testing, while the double stain for evaluation of both bone and cartilage is often used only in basic research. The present work compares data obtained from rat fetuses using the two methods after exposure to the teratogenic agent sodium valproate at specific embryonic stages of development. Pregnant rats were treated with 400mg/kg sodium valproate and sacrificed at term of pregnancy. Even if both methods were able to identify sodium valproate as a teratogenic molecule, correct and complete interpretation of data was possible only by using the double stain. Our results show the inability of the single stain to correctly discriminate between major and minor abnormalities.

Relationship between hindbrain segmentation, neural crest cell migration and branchial arch abnormalities in rat embryos exposed to fluconazole and retinoic acid in vitro.

Fluconazole (FLUCO) and retinoic acid (RA) can perturb morphogenesis of the branchial apparatus in rodent embryos exposed in vitro. The aim of the present study was to compare the effects induced by in vitro exposure to FLUCO or to RA on rhombomere organisation, neural crest cell (NCC) migration and cranial nerve differentiation using specific antibodies. For this purpose 9.5 d.p.c. rat embryos were exposed to teratogenic concentrations of FLUCO or RA; another group was exposed to no-effect concentrations of both agents. Expression of Hox-b1 and Krox20 (markers of specific rhombomeres) was altered after FLUCO and RA exposure. Furthermore, FLUCO and RA showed a synergistic effect. These results suggest that the observed branchial abnormalities are due to anomalous NCC migration related to incorrect organisation of specific rhombomeres.

Effects of excess and deprivation of serotonin on in vitro neuronal differentiation.

The neurotransmitter serotonin (5HT) possesses developmental functions in vertebrates and invertebrates. Rodent embryos express 5HT receptors even before neural development, but the role of this neurochemical seems to be particularly important during axonal morphogenesis and differentiation and in neural crest cell migration. Moreover, 5HT inhibitors are teratogenic in mammals, inducing brain and heart abnormalities. The aim of this study was to investigate the effects of nonphysiological concentrations of 5HT (5HT excess as well as deprivation) on developing rat neural cells using the micromass method. This simple and rapid micromass method allows the culture of mesencephalic cells capable of achieving and maintaining a significant degree of differentiation. Mesencephalic cells from 13 d post coitum (pc) rat were cultured and exposed to exogenous 5HT (1, 10, 50, or 100 microM) or to the specific 5HT2 receptor inhibitor mianserin (0.5, 5, 25, or 50 microM) during the whole culture period (5 d). The micromass morphology, the cytoskeletal organization, the pathological apoptosis, and the differentiative capability of cultured mesencephalic cells have been analyzed. The results show that 10-100 microM 5HT and 0.5-50 microM mianserin are able to disrupt the normal micromass morphology; 5HT and mianserin are unable to interfere with the cytoskeletal structures; mianserin (but not 5HT) induces pathological apoptosis on micromass cells at concentration levels of 0.5-50 microM; 5HT (but not mianserin) alters the neural differentiation at concentration levels of 10-100 microM. In conclusion, our results demonstrate that an excess of 5HT inhibits the capability of mesencephalic neurons to differentiate as shown by the alterations of the expression of the neuronal differentiative proteins glial-derived neurotrophic factor and Neu-N; on the other hand, the blocking of 5HT2 receptors induces apoptosis in differentiating neurons.

Teratogenic activity of HDAC inhibitors.

Modification of the terminal tails of histones is considered one of the documented mechanisms for epigenetic control of gene expression. Histone deacetylase inhibitors (HDACi) lead to a state of hyperacetylation of histone, a condition that can affect normal gene transcription. Furthermore, HDACi have many other protein targets involved in regulation of gene expression, cell proliferation and cell death. For these properties some HDACi are nowadays used as anticancer drugs with promising results. Several molecules with HDACi properties (valproic acid, trichostatin A, apicidin, MS-275, sodium butyrate, boric acid, salicylic acid) have been found to induce congenital malformations associated with hyperacetylation of histones in the target organs. Cell death is the major event in the target organs a few hours after embryonic exposure to HDACi. Gene deregulation, oxidative stress, DNA demethylation, and/or retinoic acid imbalance are the modes of action postulated for HDACi-induced teratogenesis.

Microtubule alterations occur early in experimental parkinsonism and the microtubule stabilizer epothilone D is neuroprotective.

The role of microtubule (MT) dysfunction in Parkinson's disease is emerging. It is still unknown whether it is a cause or a consequence of neurodegeneration. Our objective was to assess whether alterations of MT stability precede or follow axonal transport impairment and neurite degeneration in experimental parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in C57Bl mice. MPTP induced a time- and dose-dependent increase in fibres with altered mitochondria distribution, and early changes in cytoskeletal proteins and MT stability. Indeed, we observed significant increases in neuron-specific ßIII tubulin and enrichment of deTyr tubulin in dopaminergic neurons. Finally, we showed that repeated daily administrations of the MT stabilizer Epothilone D rescued MT defects and attenuated nigrostriatal degeneration induced by MPTP. These data suggest that alteration of ΜΤs is an early event specifically associated with dopaminergic neuron degeneration. Pharmacological stabilization of MTs may be a viable strategy for the management of parkinsonism.

Biomarkers of teratogenesis: suggestions from animal studies.

Biomarkers of effect are measurable biochemical, physiological or other alterations within an organism that can be recognized as causing an established or potential impairment of embryo-fetal development. They may be identified studying the mechanisms of action of teratogens. Hyperacetylation of histones, oxidative stress, cholesterol and retinoic acid unbalance are some of the identified mechanisms of action of some known teratogens. Nevertheless, their use is not currently applicable in human pregnancy because of the difficulty of the choice of biological material, the time when the material must be obtained, and the invasivity of methods. Furthermore, before using them in human pregnancy studies, biomarkers should be validated in experimental animals and in epidemiologic studies. On the contrary, some biomarkers could be useful in the screening of developmental toxicity of chemicals and drugs, comparing molecules of the same chemical class or with the similar pharmacologic activity, and using adequate in vitro tests, in order to reduce the use of experimental animals.