Teratogenic Activity Research Articles

Hydroxamic acid and fluorinated derivatives of valproic acid: Anticonvulsant activity, neurotoxicity and teratogenicity

Purpose Fluorinated and non-fluorinated valproic acid (VPA) analogues with hydroxamic acid moieties were tested for their teratogenic, anticonvulsant and neurotoxic potencies in mice. Methods Compounds were synthesized from their corresponding acids. The induction of neural tube defects (exencephaly) of the resulting hydroxamates (applied on day 8.25 of gestation) was tested in the offspring of pregnant animals (Han:NMRI mice). The anticonvulsant activity was evaluated in the subcutaneous pentylenetetrazole (PTZ) seizure threshold test and neurotoxicity in the rotorod neurotoxicity test. Results All tested hydroxamates showed no or greatly reduced teratogenic potency in mice compared to the free acids. Furthermore all compounds exhibited anticonvulsant activity with ED 50 doses ranging from 0.16 mmol/kg to 0.59 mmol/kg (VPA 0.57 mmol/kg). Neurotoxicity of the hydroxamates was increased compared to VPA. TD 50 doses range from 0.70 mmol/kg to 1.42 mmol/kg (VPA 1.83 mmol/kg). Conclusion Hydroxamic acid derivatives of VPA with improved protective index and little or undetectable teratogenic potency compared to the free acids are described. α-fluorination of VPA also resulted in loss of teratogenic activity. Such fluorination of the hydroxamic acids also led to compounds with an improved anticonvulsant profile compared to non-fluorinated hydroxamates. The non-chiral 2-Fluoro-VPA-hydroxamic acid was the most promising compound with a protective index (ratio of TD 50 to ED 50) of 4.4 compared to 3.2 for VPA. This compound combines an improved ratio of anticonvulsant potency/neurotoxicity with the advantage of not being teratogenic in the mouse neural tube defect model used.

Fetal ethanol exposure activates protein kinase a and impairsShhexpression in prechordal mesendoderm cells in the pathogenesis of holoprosencephaly

In humans, fetal ethanol exposure can cause holoprosencephaly (HPE), one of the most common birth defects that is characterized by brain, facial, and oral abnormalities. However, the pathogenesis of HPE is not clear. In the present study, we investigated the teratogenic mechanism of ethanol-induced brain and facial malformations in mice. Pregnant C57BL/6J mice were administered ethanol on E7 and facial and brain malformations were characterized on E10.5. We examined the effect of fetal ethanol exposure on Shh expression and activation of protein kinase A (PKA) because mutations in the human Shh gene are the most frequent cause of autosomal-dominant inherited HPE and PKA is a potent endogenous antagonist of Shh signaling. Fetal ethanol exposure on E7 induced severe midline defects characteristic of HPE. Ethanol exposure impaired Shh expression and induced excessive apoptosis only along the anterior edge of the prechordal mesendoderm (PME). In addition, ethanol activated PKA in anterior PME cells. Pretreatment of embryos with antioxidants, such as vitamins C or E, prevented the development of ethanol-induced HPE. Shh expression in PME cells is involved in the pathogenesis of ethanol-induced HPE. Ethanol may impair Shh expression indirectly by activating PKA. The inhibition of excessive apoptosis in PME cells by antioxidants implies that oxidative stress may underlie the teratogenic actions of ethanol. Thus, antioxidant treatment may be a simple preventative measure that could reduce the incidence of HPE following fetal ethanol exposure.

New Proposals for Testing Drugs with IKr-Blocking Activity to Determine Their Teratogenic Potential

Drugs blocking the potassium current IKr, either as an intended pharmacologic effect (eg antiarrhythmics dofetilide and almokalant) or as an unwanted side-effect (eg antihistamine astemizole, propulsive drug cisapride, antidepressive drugs and macrolide antibiotics) are potential human teratogens. It is the contention of this paper that the existing repeat dose regimen used in teratology studies to fulfil regulatory requirements, does not properly identify the teratogenic risk of these drugs. Results from conventional studies for dofetilide and almokalant showed high rates of postimplantation embryonic death with few malformed fetuses. For astemizole and cisapride only embryonic death was seen. These latter results were not considered important because they occurred either in the presence of maternal toxicity and/or at high doses. Subsequent studies have shown that IKr-blockers are highly teratogenic when administered on single gestational days (GD) during a sensitive period of rat pregnancy (GD 10-14) when they induce a high incidence of stage-specific malformations. This teratogenic activity of astemizole and cisapride was missed in the original teratology studies. Mechanistically IKr-blockers cause bradycardia and arrhythmia of the embryonic heart and while an embryo may be able to survive a single day exposure to a teratogenic dose, repeat dosing often leads to death of the embryo. With this review we suggest that new drugs identified at the preclinical stage of development as having IKr-blocking properties, should undergo more comprehensive teratology testing including single GD dosing and studies using embryo culture. This would further help identify and characterise their teratogenic potential.

Valproic acid-induced neural tube defects.

Antiepileptic drug therapy with valproic acid (VPA) during early pregnancy can result in a 1-2% incidence of spina bifida aperta, a closure defect of the posterior neural tube in the human. The predominant defect produced by VPA in the mouse is exencephaly, a closure defect of the anterior neural tube. An appropriate dosing regimen (consecutive doses of VPA on Day 9 of gestation) can also result in a low incidence of spina bifida aperta and a high incidence of spina bifida occulta in the mouse. It is likely that the parent drug and not a metabolite is the proximate teratogen. Structure-activity relationships show a strict structural requirement for high teratogenic potency: the molecule must contain an alpha-hydrogen atom, a carboxyl function and branching on C-2 with two chains containing three carbon atoms each for maximum activity. If these two carbon chains are different, then enantiomers are present. Pairs of enantiomers were synthesized and shown to be significantly different in regard to teratogenic potency. Both enantiomers of each compound reach the embryo to the same degree, therefore, the intrinsic teratogenic activity of the enantiomers differs. This suggests that stereoselective interaction occurs between the drugs and a chiral structure within the embryo. The molecular mechanism of the teratogenicity of VPA is not known; one hypothesis is that VPA interacts with embryonic folate metabolism.

Molecular biomonitoring of a population of nurses handling antineoplastic drugs

Many antineoplastic drugs have been found to have carcinogenic, mutagenic and teratogenic activity and so hospital personnel handling these substances are potentially exposed to health risk. Understanding this risk derived from protracted occupational exposure has great relevance even if the workers normally adopt individual and environmental protective measures. To address this question we have studied the presence of DNA and chromosome damage in a population of nurses employed in Italian oncology units and in matched controls. We used the comet assay to evidence the presence of DNA strand breaks, due to both acute and chronic exposure, and the micronucleus (MN) test, which is a measure of clastogenic and aneugenic events. Furthermore, since the individual response to the exogenous insults may be genetically determined, we studied the possible influence of single nucleotide polymorphism in XRCC1 and XRCC3 DNA repair genes on induced genetic damage. We also considered the effects of confounding factors like smoking, age and gender. The results indicated that the exposed subjects had significantly high levels of genetic damage. Age and gender were associated with increased values in MN, both in control and in exposed groups; the smoking habit affects MN frequency in controls, but not in workers. Furthermore we found that exposed subjects bearing at least one XRCC1 variant allele (399Gln) show higher values of MN. The present data provide the evidence to show that occupational exposure to antineoplastic drugs, even if in safety controlled conditions, represents a serious health risk. Furthermore we have shown that the presence of XRCC1 genetic polymorphism could contribute to increase the genetic damage in susceptible individuals who are occupationally exposed to dangerous substances.

A developmental toxicity study of 20 kHz magnetic field exposure on chick embryos—A 7-day, 1.1 mT magnetic field exposure did not modify teratogenic action of retinoic acid

A developmental toxicity study of 20 kHz magnetic field exposure on chick embryos—A 7-day, 1.1 mT magnetic field exposure did not modify teratogenic action of retinoic acid

Studies of the reproductive toxicity of Rumalaya

The reproductive toxicity of the complex plant preparation Rumalaya was studied in experiments on white mongrel rats. Rumalaya at doses of 400 and 2000 mg/kg given via the intragastric route with simultaneous application of cream to the skin was found to have no embryotoxic or teratogenic actions and had no significant effect on the postnatal development of offspring or the generative functions of female or male rats. Thus, these data identified no contraindications to the clinical use of this agent in pregnant women.

Increased Susceptibility to Phenytoin Teratogenicity: Excessive Generation of Reactive Oxygen Species or Impaired Antioxidant Defense?

Phenytoin is a human and animal teratogen. Accumulating evidence suggests that the teratogenicity is associated with a potential of phenytoin to cause embryonic cardiac arrhythmia and resultant generation of toxic reactive oxygen species via hypoxia-reoxygenation mechanisms. The A/J mouse is more susceptible to phenytoin teratogenicity than other mouse strains. The aim of this study was to investigate whether A/J mice have other antioxidant enzyme activities than C57BL/6J and CD-1 mice. Also, strain differences in phenytoin effects on embryonic heart rate and rhythm were determined. Another objective was to determine whether a spin trapping agent with capacity to capture reactive oxygen species alter the developmental toxicity of phenytoin. Treatment with this agent resulted in a marked decrease in phenytoin teratogenicity, which supports the idea that reactive oxygen species are important mediators for the teratogenic action of phenytoin. The A/J mice embryos were most susceptible to the adverse cardiac effects of phenytoin and had the highest activity of superoxide dismutase and glutathione peroxidase, while the activity of catalase was the same in embryos of the three different strains. The high activities of antioxidant enzymes in the A/J stain indicate that the sensitivity to develop malformations is caused by excessive arrhythmia-related generation of reactive oxygen species rather than impaired antioxidant defense.

Induction of the homeotic gene Hoxa1 through valproic acid's teratogenic mechanism of action

Background Valproic acid (VPA) exposure in utero has been associated with an increased risk of both neural tube defects and autism spectrum disorders (ASDs). The terata induced by VPA suggest interference with pattern formation. Retinoic acid produces similar terata and is known to act in part by increasing the expression of Hoxa1. We tested the hypotheses that exposure to VPA would alter the expression of Hoxa1 in rat embryos during times of normal Hoxa1 expression (d10.5–13.5) and that exposure at earlier and later stages would induce inappropriate expression. Method Hoxa1 expression levels were determined by real-time PCR in individual embryos 1 h after exposure on gestational d10, 12, 13, 14, or 15. Additionally, teratogenic (4- yn-VPA) and nonteratogenic analogs of VPA (IE-VPA), retinoic acid (RA), and saline were compared for effects on Hoxa1 expression on d12. Embryos were allowed to develop for 1, 2, 4, 6, or 24 h, to follow the time course of effects. Results In utero exposure to VPA on gestational d10 and on d12–14 significantly increased the level of Hoxa1 expression compared to saline-exposed embryos at developmental ages prior to, during and after the normal expression period for this gene. On gestational d12, exposures to VPA and 4- yn-VPA significantly increased Hoxa1 expression at all sacrifice times, compared to saline-exposed embryos. RA significantly elevated Hoxa1 expression at all time points except 24-h post-treatment. The nonteratogenic VPA analog, IE-VPA, did not affect Hoxa1 expression. Conclusions VPA and 4- yn-VPA exposures elevated Hoxa1 mRNA during its normal expression period and induced expression outside of the normal period. This may explain, in part, how VPA disrupts development.

Atp7a determines a hierarchy of copper metabolism essential for notochord development

The critical developmental and genetic requirements of copper metabolism during embryogenesis are unknown. Utilizing a chemical genetic screen in zebrafish, we identified small molecules that perturb copper homeostasis. Our findings reveal a role for copper in notochord formation and demonstrate a hierarchy of copper metabolism within the embryo. To elucidate these observations, we interrogated a genetic screen for embryos phenocopied by copper deficiency, identifying calamity, a mutant defective in the zebrafish ortholog of the Menkes disease gene (atp7a). Copper metabolism in calamity is restored by human ATP7A, and transplantation experiments reveal that atp7a functions cell autonomously, findings with important therapeutic implications. The gene dosage of atp7a determines the sensitivity to copper deprivation, revealing that the observed developmental hierarchy of copper metabolism is informed by specific genetic factors. Our data provide insight into the developmental pathophysiology of copper metabolism and suggest that suboptimal copper metabolism may contribute to birth defects.

Gene expression in Xenopus laevis embryos after Triadimefon exposure

The triazole derivative Triadimefon (FON) is a systemic fungicide used to control powdery mildews, rusts, and other fungal pests. Some data have already been published about the teratogenic activity of this compound: craniofacial malformations were found in mouse, rat, and Xenopus laevis embryos exposed to FON. These alterations were correlated to defective branchial arch development possibly caused by abnormal neural crest cell (NCC) migration into the branchial mesenchyme. As the migration of NCCs is controlled by the HOX code and by an anteroposterior retinoic acid (RA) gradient, we analyzed the expression of CYP26, a key enzyme in RA metabolism, following FON exposure. The increased expression of this gene and the ability of citral (a RA inhibitor) to reduce the teratogenic effects of the fungicide support the notion that endogenous RA is involved in the mechanism of action of FON. Moreover, by in situ hybridization, we studied the effects of FON exposure at gastrula stage on the expression of some genes involved in craniofacial development, hindbrain patterning, and NCC migration. We observed abnormal localization of xCRABP, Hoxa2 and Xbap signal expression at the level of migrating NCC domains, whereas in the hindbrain, we did not find any alteration in Krox20 and Hoxa2 expression.

H2O2 induces abnormal tail flexure in Xenopus embryos: similarities with Paraquat teratogenic effects

As previously shown, Paraquat (PQ) treatments of Xenopus developing embryos mainly induce a characteristic developmental alteration we named "abnormal tail flexure." PQ oxidative activity has been indicated as the cause of this malformation. Since PQ evokes reactive oxygen species (ROS), among which hydroxyl radicals (OH(*)), and H(2)O(2) can be converted to (OH(*)) via Fenton reaction, we compared here the lethal and teratogenic potentials of both oxidants by using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX), in order to grasp eventual similarities in their teratogenic activity. Xenopus embryos were exposed, from stage 8 to stage 47, at 368, 491, 612, and 735 microM H(2)O(2) and 0.388 microM PQ. The probit analysis of H(2)O(2) mortality and malformed larva percents gave a 598.82 microM Lethal Concentration 50% (LC(50)) and 536.04 microM Teratogenic Concentration 50% (TC(50)) from which a 1.11 Teratogenic Index (T.I.) has been calculated. This T.I. value should allow the classification of H(2)O(2) as a non-teratogenic compound. A comparison of H(2)O(2) mortality and malformed larva percents with those obtained from PQ exposure showed the higher embryotoxicity of PQ, but, markedly, both compounds mainly induced the "abnormal tail flexure." Histological analysis of both H(2)O(2) and PQ malformed embryo tails showed a similar distorted morphology of both somites and myocytes. Some of muscle cells were necrotic and affected by an apical enlargement as well as a detachment from the connective tissue of intersomitic boundaries. In our opinion, both of the tested chemicals likely weaken the mechanical bridge connecting the myocyte contractile apparatus to the extracellular matrix, therefore causing the detachment of some of tail myocytes from their connectival septum as well as their apical enlargement. This could lead to the unbalance of tail tensional forces and, in turn, to the appearance of the "abnormal tail flexure."

Triadimefon Causes Branchial Arch Malformations in Xenopus laevis Embryos (5 pp)

Triazole-derivatives are potent antifungal agents used as systemic agricultural fungicides and against fungal diseases in humans and domestic animals. They act by inhibiting the cytochrome P-450 conversion of lanosterol to ergosterol, thus resulting in faulty fungal cell wall synthesis. Some data have been published about the teratogenic activity of triazoles on rodent embryos: Hypoplasias, abnormal shape, agenesis of the branchial arches, for example, were reported as typical induced malformations. Unfortunately, no data are available on the embryotoxicity of these compounds in amphibians, despite the increasing concern among the scientific community about the phenomenon of global amphibian population declines. The aim of the present work is to evaluate the embryo-lethal and teratogenic potentials of Triadimefon (FON), a triazole-derivative widely used as an antimycotic in agriculture, by the test FETAX (Frog Embryos Teratogenic Assay, Xenopus) with particular attention being paid to the analysis of branchial arch malformations. Xenopus laevis embryos were exposed continuously from stage 9 to increasing concentrations of FON and analyzed at stage 47 for mortality and teratogenicity (group I) to determine the median lethal (LC50) and teratogenic (TC50) concentrations. Another two pools of larvae were exposed to FON for a 2 hour period at early gastrula (Group II) or neurula (Group III) stages to verify which period of development is the most sensitive to FON. The malformations observed were further investigated by histological section and cartilage staining with Alcian blue. The assay has estimated LC50 and TC50 values of 63.8 microM and 2.73 microM, respectively; the resulting TI (Teratogenic Index = LC50/TC50) value of 23.4 has underlined the very high teratogenic risk associated with this compound. Neurulation was more sensitive to FON exposure than gastrulation, since the TC50 estimated values for group III (neurula exposed) specimens was 7.6 times lower than those of group II (gastrula exposed). Interestingly, for each group analyzed, 100% of malformed embryos showed alterations at branchial arch derived cartilages: Anterior cartilages were reduced, missing, fused or incorrectly positioned while gill cartilages were altered only in the most severely affected specimens. In some cases these malformations were associated with hyperpigmentation. Our results support the hypothesis that FON can interfere with Neural Crest Cell (NCC) migration, since craniofacial components and melanophores are derived from neural crest material. In conclusion, our data show Triadimefon to be a potent teratogen able to induce specific craniofacial malformation in Xenopus laevis embryos, probably interfering with the NCC migration into the branchial mesenchyme. These results are also interesting for ecotoxicological reasons as FON, as well as other pesticides, are likely to be present in water systems near agricultural or urban areas which may serve as habitats for developing amphibians and fishes. Our results are in agreement with the data obtained on in vitro cultured rat embryos suggesting that the FON mechanism of action involves strongly conserved molecules. The choice of Xenopus laevis as the model organism allows us to extend the toxicological and teratological observations to a molecular level, in order to search for novel genes regulated by FON exposure.

MONITORING PERSPECTIVES OF CHILDREN WITH CONGENITALS DEVELOPMENT ABNORMALITIES IN GOMEL REGION

The growth of congenital abnormalities in Belarus is an urgent issue, especially on the background of a worse ecological situation and decreased birth rate. Monitoring systems are involved into investigation of reasons of growth and both temporal and geographic trends of a frequency of inborn abnormalities for timely prevention measures. An increased sensitivity of the monitoring system of the Republic of Belarus due to expansion of the group of prior analyzed abnormalities will reduce the time to evaluate the severity of the mutation process and the action of possible teratogens.

Effect of ethanol on the development of visceral yolk sac

Prenatal ethanol exposure can cause development retardation and malformations in human offspring. Before the formation of chorioallantoic placenta, yolk sac plays an important role in transporting nutrients from the mother to the embryo. Functional suppression of yolk sac is found to be relevant to the malformations in mammalian embryos. Female 8.5-day C57BL/6J mouse embryos were cultured in vitro and exposed to different doses of ethanol. The development of visceral yolk sac (VYS) was examined with light and electron microscopes. The expression profiles of some vasculogenesis-related genes were detected with reverse transcription-PCR. A dose-dependent toxicity to the VYS was found, including reduced diameter, decreased protein and DNA contents, and suppressed development of vitelline vessels. The hypogenesis of VYS agreed with the retarded development and/or malformations found in the embryos. Histological and functional alterations were found in the ethanol-exposed VYS endodermal cells. The expressions of vasculogenesis-related genes, fetal liver kinase 1 (Flk1) and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 (Tie2), were repressed by ethanol. Impaired structural and functional development of VYS may contribute to the teratogenic action of ethanol in mice, which may also provide a clue to the study of fetal alcohol syndrome in humans.

Ercc6l, a gene of SNF2 family, may play a role in the teratogenic action of alcohol

The expression profile of a newly identified mouse nucleotide excision repair (NER) gene, Ercc6l, was investigated in a mouse model of fetal alcohol syndrome (FAS). In test 1, whole-mount in situ hybridization showed Ercc6l expressed mainly in the neural tube and heart of 10.5-day embryo. However, the expressions in both of the two organs were significantly down regulated after in uterus alcohol exposure from embryonic day (ED) 6–10, which was in accordance with the result of semi-quantitative RT-PCR. In test 2, the dams were given alcohol intragastrically from ED 6–15, and Northern blot of Ercc6l mRNA was carried out with five major embryo organs on ED 15.5, which were heart, brain, kidney, liver and lung. Ercc6l expression in 15.5-day embryonic brain and heart, which are the most commonly affected organs of FAS, were both decreased by alcohol exposure. The expressions in the other three organs were unaffected. From the results, we considered that Ercc6l might play a role in the teratogenic action of alcohol.

A COMPARATIVE STUDY OF THE ESSENTIAL OILS OF FOUR GONIOTHALAMUS SPECIES

A COMPARATIVE STUDY OF THE ESSENTIAL OILS OF FOUR GONIOTHALAMUS SPECIES

Identification of early-responsive genes correlated to valproic acid-induced neural tube defects in mice

Valproic acid (VPA) causes the failure of neural tube closure in newborn mice. However, the molecular mechanism of its teratogenesis is unknown. This study was conducted to investigate the genomewide effects of VPA disruption of normal neural tube development in mice. Microarray analysis was performed on the head part of NMRI mouse embryos treated for 1 hr with VPA on gestational day (GD) 8. Subsequently, we attempted to isolate genes that changed in correlation with the teratogenic action of VPA by employing reduced teratogenic VPA analogs, valpromide (VPD) and valnoctamide (VCD), in a real-time PCR study. Microarray results demonstrated that during neurulation, many genes, some of whose functions are known and some unknown, were either increased or decreased after VPA injection. Some genes were affected by VPD or VCD in the same way as VPA, but others were not changed by the analogs. In this way, our system identified 11 increased and 20 decreased genes. Annotation analysis revealed that the increased genes included gadd45b, ier5, per1, phfl3, pou3f1, and sox4, and the decreased genes included ccne2, ccnl, gas5, egr2, sirt1, and zfp105. These findings demonstrate that expression changes in genes having roles in the cell cycle and apoptosis pathways of neural tube cells were strongly expected to relate to the teratogenic, but not antiepileptic, activity of VPA. Our approach has allowed the expansion of the catalog of molecules immediately affected by VPA in the developing neural tube.

B56 Regulatory Subunit of Protein Phosphatase 2A Mediates Valproic Acid-Induced p300 Degradation

Transcriptional coactivator p300 is required for embryonic development and cell proliferation. Valproic acid, a histone deacetylase inhibitor, is widely used in the therapy of epilepsy and bipolar disorder. However, it has intrinsic teratogenic activity through unidentified mechanisms. We report that valproic acid stimulates proteasome-dependent p300 degradation through augmentation of gene expression of the B56gamma regulatory subunits of protein phosphatase 2A. The B56gamma3 regulatory and catalytic subunits of protein phosphatase 2A interact with p300. Overexpression of the B56gamma3 subunit leads to proteasome-mediated p300 degradation and represses p300-dependent transcriptional activation, which requires the B56gamma3 interaction domain of p300. Conversely, silencing of the B56gamma subunit expression by RNA interference increases the stability and transcriptional activity of the coactivator. Our study establishes the functional interaction between protein phosphatase 2A and p300 activity and provides direct evidence for signal-dependent control of p300 function.

Effect in rats of simultaneous prenatal exposure to ochratoxin A and aflatoxin B1. I. Maternal toxicity and fetal malformations

Ochratoxin A (OA) and Aflatoxin B1 (AFB1), the food borne mycotoxins are produced by several fungal species of the genera Aspergillus and Penicillium. To determine the teratogenic effects, these mycotoxins were administered orally either individually or in combination to the pregnant Wistar rats on days 6-15 of gestation. OA and AFB1 were dissolved in corn oil and different doses of OA (0.125, 0.25, 0.50, and 0.75 mg/kg), AFB1 (0.125, 0.25, 0.50, and 1.00 mg/kg), and a combination of OA+AFB1 (0.125+0.125; 0.25+0.50; 0.50+0.25 mg/kg) were given by gastric intubation to rats. During dosing period, the body weight and body weight gains significantly decreased at a higher dosage, in both individual and combined treatments. In all the combination treatments, the percent implants resorbed, fetal body weights, and crown-rump lengths were comparable to those of controls and with the individual mycotoxin treatment. The number of dead fetuses was significantly increased in the high OA combination (OA+AFB1 0.50+0.25) group as compared with the other two combinations. OA and AFB1 alone and in combination caused various gross, skeletal, and visceral anomalies. The occurrence was considerably less pronounced in fetuses of AFB1 and combination groups as compared with those of OA group fetuses. The exencephaly, incomplete closure of skull, wavy and fused ribs, agenesis of the ischium bone, and enlarged renal pelvis, recorded in OA treatment and ear abnormality and incomplete ossification of skull bones observed in AFB1 when given individually, were not seen in combination groups. However, new manifestations, such as gastroschisis and syndactyly were observed and the incidence of cardiac defects was increased in fetuses due to the combined treatment. The results of the present study indicated that there is some interaction between these mycotoxins that resulted in reduced teratogenic activity of OA in the presence of AFB1. Apparently, new manifestations observed in combination treatment points to the potential threat of teratogenicity in terms of public health hazards.