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.

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.

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.

Reproductive toxicity of exemestane, an antitumoral aromatase inactivator, in rats and rabbits.

Exemestane is an orally active, irreversible inactivator of aromatase, structurally related to the natural substrate androstenedione, in clinical use at 25 mg daily for the treatment of advanced breast cancer in postmenopausal women. The reproductive and developmental toxicity of exemestane was assessed in rats and rabbits with oral administration. Pivotal experiments included a fertility study (Segment I), in which female rats received exemestane doses of 4, 20, or 100 mg/kg/day from two weeks premating until GD 20 (cesarean-sectioned dams), or until GD 15 and then from D 1 to D 21 postpartum (dams allowed to deliver), and developmental toxicity studies (Segment II), in which rats and rabbits were treated from GD 6 through GD 17 (rats) or GD 18 (rabbits) at doses of 10, 50, 250, or 810 mg/kg/day and 30, 90, or 270 mg/kg/day, respectively. All rabbits and two-thirds of the rats were cesarean sectioned toward the end of pregnancy to determine litter parameters and examine structural abnormalities in the fetuses; the remaining one-third of the rats was allowed to litter and rear pups to weaning. No pivotal male fertility or peri- and postnatal studies were performed, taking into consideration the therapeutic use. Postnatal effects on the first generation offspring were assessed in both studies in rats, in the portion of dams allowed to deliver. Their F1 offspring were raised to adulthood, when they were evaluated for reproductive performance, and the F1 females were terminated on GD 20. The dosing schedule for the Segment I study in rats, which included a postnatal component, was established to exclude exposure before and during parturition (by withdrawing treatment from GD 16 until the end of parturition). This withdrawal of treatment was put in place because in a preliminary study with treatment including the peripartum period, doses from 5 to 200 mg/kg/day prolonged gestation and interfered with parturition.Overall, studies in rats showed that female fertility was not affected up to 100 mg/kg/day, but doses higher than 4 mg/kg/day, which is approximately the pharmacologically active dose (ED50 = 3.7 mg/kg), prolonged gestation and impaired parturition, leading to maternal deaths in labor and perinatal deaths of offspring. Rats killed on GD 20 showed nondose-related increases in resorptions at doses higher than 10 mg/kg/day, a reduction in fetal body weights at 20 and 100 mg/kg/day (fertility study) and 810 mg/kg/day (developmental toxicity study), and an increase in placental weights at all doses. Female fetuses exposed in utero until GD 20 at 100 mg/kg/day showed an increase in the anogenital distance, very likely related to an increase of the potent androgen DHT as a consequence of aromatase inhibition. Morphologic examinations in fetuses and born pups that were exposed in utero up to the end of the organogenesis period, as well as postnatal investigations on offspring up to adulthood, showed no treatment-related effects. In a developmental toxicity study in rabbits, treatment at 270 mg/kg/day affected maternal food intake and body weight gain, caused abortion or total resorption in about 30% of pregnant females, and reduced body weight and numbers of live fetuses, but did not affect fetal morphology. It was concluded that exemestane did not affect parturition in rats at 4 mg/kg/day or pregnancy in rabbits at 90 mg/kg/day (about 1.5 and 70 times the human dose, respectively, on a mg/m2 basis) and was not teratogenic in rats and rabbits. Exemestane is marketed for use only in postmenopausal women. Its labeling includes a contraindication to use in pregnant or lactating women.

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.

Teratogenic effects of sodium valproate in mice and rats at midgestation and at term.

This experiment was carried out with the aims of comparing the embryotoxic potential of valproic acid (VPA) in rats and mice, better defining the malformation pattern in these species, and comparing the embryotoxic effects detectable in mid-pregnancy to those observed in fetuses at term. Pregnant CD:Crl rats were treated subcutaneously (s.c.) at 08:00, 16:00, and 00:00 on day 9 of gestation with 0, 150, or 300 mg/ kg VPA; pregnant NMRI mice were treated s.c. at 00:00 on day 7 of gestation, and at 08:00 and 16:00 on day 8 of gestation with 0,75, 150, or 300 mg/kg VPA. Groups of females were killed on day 9 (mice) or day 11 (rats) of pregnancy and their embryos were carefully examined under a dissecting microscope. The remaining females were killed 1 day before parturition and their fetuses were examined for external, visceral, and skeletal malformations. A very high frequency (84%) of malformed embryos was recorded in the group of mice treated with 300 mg/kg, including open brain folds (73%), somite defects (36%), and heart malformations (20%). The rat embryos were less sensitive: only 43% of them were malformed after treatment with 300 mg/ kg, however, the pattern of malformations was quite similar to that observed in mice. The treatment with 150 mg/kg produced about 32% malformed embryos in mice and only 8.5% in rats. More than 84% of mouse fetuses from mothers treated with the highest dose showed a severe form of exencephaly. The axial skeleton was also severely affected. The postimplantation loss reached 52%. Exencephaly and skeletal malformations were also recorded in mouse fetuses from mothers exposed to 150 mg/kg. The dose of 75 mg/kg was without effects. Exencephaly was not observed in rat fetuses at term. In this species the axial skeleton was the most severely affected region at 300 mg/kg, while the lowest dose produced only sporadic malformations. These results confirm that the mouse is the more sensitive species for the teratogenic effects of VPA. Furthermore, it has been shown that, in both species, the axial skeleton is a system which is very sensitive to the teratogenic effects of VPA. The observed alterations show a possible link between axial specification and VPA and suggest further studies of embryos exposed to VPA for the expression of genes controlling the identity of vertebral segments.

Glutathione status in diabetes-induced embryopathies.

The mechanism involved in diabetes-induced embryotoxicity is still unclear. Mitochondrial alterations probably produced by oxidative stress have been described in embryos developing in a diabetic environment. Furthermore, oxygen radicals-scavenging enzymes can reduce the embryotoxic effects induced by diabetic conditions. In this work we tried to test if glutathione (GSH), a tripeptide implicated in cellular protection against reactive oxygen species, is involved in diabetes-related embryotoxicity. Rat embryos were explanted on day 11 on gestation from normal and from streptozotocin-diabetic mothers. The embryos were examined morphologically, then protein, DNA and GSH were determined both in embryos and in their visceral yolk sacs. The embryos explanted from diabetic mothers showed signs of developmental retardation and 16% were morphologically abnormal. GSH content was reduced in these embryos in comparison to control, but the GSH/protein in the visceral yolk sacs of conceptuses explanted from diabetic mothers was higher than in control visceral yolk sacs. Our hypothesis is that the reduction of embryonic GSH is a consequence of the alteration in GSH transport across the yolk sac endodermal cells damaged by diabetic conditions. The observed reduction in embryonic GSH could reduce the protection against the oxidative stress condition described in diabetic pathology.

In vitro development of rat embryos obtained from diabetic mothers.

Rat embryos of 9.5 or 10 days of gestation were removed from control or streptozotocin-diabetic mothers and cultured in normal rat serum (180 mg% glucose) or in diabetic serum (600 mg% glucose). The development of control embryos in normal serum was adequate. Embryos from normal mothers cultured in diabetic serum showed signs of developmental retardation. The development of embryos obtained from diabetic mothers was severely impaired, regardless of the gestational age or the culture medium. These results suggest that a diabetic maternal milieu produces irreversible effects in the embryo very early in gestation.

Glutathione and N-acetylcysteine protection against acetaldehyde embryotoxicity in rat embryos developing in vitro.

Previous in vitro studies demonstrated that acetaldehyde (ACHO) is able to produce specific morphological alterations related to the foetal alcohol syndrome. Intracellular reduced glutathione (GSH) has been shown to modulate the embryotoxicity elicited by various chemicals in vivo and in vitro. The present study evaluates the role played by endogenous and exogenous GSH and its precursor N-acetylcysteine (NAC) on the embryotoxicity induced by ACHO using the rat whole embryo culture system. In the first experiment embryos at gestation day (GD) 9.5 were cultured in rat serum medium for 18 hr in the presence of 1 mm l-buthionine-S,R-solfoximine (BSO), a specific inhibitor of GSH synthesis. Following pretreatment, conceptuses were cultured for a further 30 hr in the presence of 30 mug ACHO/ml. Pretreatment with BSO significantly enhanced the embryotoxic effects of ACHO and markedly reduced the GSH level only in the yolk sac. In the second experiment GSH or NAC (8 mum) were added to the medium by two different procedures in an attempt to reduce ACHO-induced embryotoxicity. In one case the embryos were exposed to ACHO for 8 hr and then transferred to media containing NAC or GSH for the remaining time of culture (22 hr); in another, the embryos were maintained for the entire culture period (30 hr) in a medium containing ACHO plus NAC or GSH. Only in the first case did exposure to NAC significantly reduce the frequency of abnormal embryos; in the second case the concurrent exposure to ACHO and thiols only marginally reduced ACHO-induced effects. Significant variations in the GSH content were recorded only at the level of the yolk sac. This result suggests that the yolk sac GSH can play a major role in the protection of the embryo against the toxic effects produced by xenobiotics.