Aflatoxin M1 effects on Xenopus laevis development

BACKGROUND: The principal Aflatoxin B(1) (AFB(1)) hydroxylated metabolite excreted in milk is Aflatoxin M(1) (AFM(1)) classified in group 2B by the International Agency for Research on Cancer (IARC). Human exposure to AFM(1) is due to the consumption of contaminated dairy products and partly to endogenous production through AFB(1) liver metabolism. METHODS: Since no data are available on AFM(1) embryotoxicity, its lethal and teratogenic potential was investigated using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). Stage-8 blastulae were exposed to AFM(1) at 1, 4, 16, 64, and 256 microg/L concentrations until stage 47, free-swimming larva. RESULTS: A slight increase of mortality and malformed larva percents was found in AFM(1)-exposed groups but these differences were not statistically significant in comparison with the controls. CONCLUSIONS: Therefore, AFM(1) is a non-embryotoxic compound when evaluated with a FETAX model at concentrations under the conditions tested. However, AFM(1) merits further studies using mammals as experimental models to identify a possible risk during human pregnancy.     

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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."     

Identification of developmental toxicants using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX)

Because growth and development are processes sensitive to the action of many chemicals, bioassays that screen for developmental toxicants may be more indicative of chronic effects than acute toxicity assays. FETAX is a 96 h whole embryo static renewal test employing the embryos of the frog Xenopus laevis. Endpoints are mortality, malformation and growth. Because of the frog's fecundity, its extensive use in basic research and the ability to obtain embryos year-round, it is an ideal organism to use in screening for developmental toxicants. By validating using known mammalian teratogens and the use of rat liver microsomes to stimulate mammalian metabolism, we have extended the use of the system for the prescreening of human developmental toxicants. In past validation work, we have correctly identified the teratogenicity of 15 to 17 compounds used in validation for a predictive accuracy of approximately 88%. In the present study, the ability of FETAX to detect developmental toxicants in groundwater samples taken from an industrial waste dump was evaluated. FETAX showed that it was sensitive enough to detect developmental toxicants in samples without prior concentration. In some samples, less than half the LC50 concentration was required to cause significant malformation. In some cases, a dose-response curve was not obtainable but the test results nonetheless indicated some developmental toxicity. The results of this study indicate that it is necessary to routinely screen for developmental toxicants when establishing water quality criteria for the preservation of species and for human health.     

Adverse reproductive and developmental effects in Xenopus from insufficient boron

Frog embryo teratogenesis assay—Xenopus (FETAX) was utilized as a model system to evaluate the effects on embryo-larval development at various low boron (B) exposure levels in the culture media. Concentrations tested ranged from <1 to 5000 μg B/L. A statistically significant (P < 0.05) increase in malformations was observed at ≤ 3 μg B/L, but not at the greater concentrations. Abnormal development of the gut, craniofacial region and eye, visceral edema, and kinking of the tail musculature (abnormal myotome development) and notochord were observed. In subsequent studies, adult frogs were maintained for 28 d on two diets: (1) low B (LB, 62 μg B/kg) or (2) boric acid supplemented (BA, 1851 μg B/kg); the frogs were subsequently mated, and their offspring were cultured in media containing various levels of B. Results of the 28-d depletion studies indicated that frogs maintained under LB conditions produced a greater proportion of (1) necrotic eggs and (2) fertilized embryos, which abnormally gastrulated at a greater rate and were substantially less viable than embryos from frogs fed the BA diet. Malformations similar to those seen in the initial study were observed in embryos from the B-depleted adults maintained in an LB environment; 28 d on the LB diet enhanced the incidence of malformations associated with the LB culture media. These abnormalities were not observed in embryos cultured in ≥4 μg B/L from adults cultured on the BA diet. These studies showed that insufficient B reproducibly interfered with normalXenopus laevis development during organogenesis, substantially impaired normal reproductive function in adult frogs, and thus represent the first studies demonstrating the nutritional essentiality of B in an amphibian species.     

Zn2+-induction of metallothionein in myotomal cell nuclei during somitogenesis of Xenopus laevis

The localization of metallothionein in control and Zn-exposed embryos of Xenopus laevis was studied by whole-mount immunohistochemical staining. The embryos were grown according to the FETAX (Frog Embryo Teratogenesis Assay: Xenopus) protocol from N/F stage 8 to stage 47, with or without addition of ZnCl₂ (300 μM) to the medium. At stages 27, 38, 42, 45, and 47, control and Zn-exposed embryos were fixed in buffered formalin, and whole mounts were stained by an immunoperoxidase technique, using monoclonal murine antibody to equine metallothionein. Staining of metallothionein was evident in myotomal cell nuclei of developing somites by stage 27, stomatodeum, oropharynx, and gills by stage 38, developing kidneys (mesonephros) by stage 45, and liver by stage 47. The staining of metallothionein at these sites was more intense in Zn-exposed embryos than controls. The central nervous system (especially the spinal cord) and the yolk mass were faintly stained for metallothionein in controls and Zn-exposed embryos. Staining of metallothionein in myotomal cell nuclei was most prominent at stage 38, diminished at stages 42 and 45, and practically disappeared by stage 47. This is the first report that metallothionein is expressed in myotomal cell nuclei of Xenopus embryos during normal somitogenesis and becomes increased when the embryos are exposed to teratogenic levels of Zn². © 1996 Wiley-Liss, Inc.     

Investigation of the teratogenic potential of ochratoxin A and B using the FETAX system

BACKGROUND: Ochratoxin A (OTA) is a mycotoxin produced by certain Aspergillus and Penicillium species. It has been observed to be teratogenic in a number of animal models including rat, mouse, hamster, and chick, with reduced birth weight and craniofacial abnormalities being the most commonly observed malformations. Neither the potential of OTA to cause malformations in humans nor its teratogenic mode of action is known. The FETAX system is an embryotoxicity assay system, with a high correlation to animal models and epidemiological data. Analysis of OTA-mediated teratogenesis using this system could provide a useful tool for the generation of high numbers of samples for mechanistic studies. METHODS: Using the standard ASTM 96-hr exposure protocol, the effect of OTA and its structural analogue OTB on the development of Xenopus laevis embryos in vitro was assessed. The accumulation of both substances in Xenopus embryos was also examined using tritiated OTA and OTB. RESULTS: Both OTA and OTB caused craniofacial malformations, while OTA also caused reduced embryo growth. As expected, OTA was far more potent in inducing these effects than OTB. This could at least in part be due to greater levels of OTA being accumulated within the embryos. CONCLUSIONS: The ability of FETAX to differentiate between close structural analogues indicates the assay has great potential for the elucidation of the embryotoxic and teratogenic mechanisms of action. Hence, the model could provide a suitable system for the investigation of other known teratogens or for the pre-screening of new agents for teratogenic potential.     

Evaluation of aflatoxin B1 embryotoxicity using the frog embryo teratogenesis assay-Xenopus and bio-activation with microsome activation systems

BACKGROUND: Aflatoxins are a group of mycotoxins produced by Aspergillus, A. flavus, and A. parasiticus. Aflatoxin B1 (AFB1) should be a strong teratogen in hamsters, but its effect in rats is equivocal and extremely limited in mice. Therefore, the AFB1 embryotoxic potential in mammals remains unclear. METHODS: Little is known about the AFB1 effects on amphibians, therefore its embryotoxic potential was evaluated using the frog embryo teratogenesis assay-Xenopus (FETAX). X. laevis blastulae were exposed to: 1) positive controls for bio-activation (4 g/L cyclophosphamide monohydrate, Cy, and 4 g/L Cy+30 mg/L MAS-rat; 4 g/L Cy+30 mg/L MAS-human); 2) positive controls for MAS (30 mg/L MAS-rat and 30 mg/L MAS-human); 3) exposed groups to AFB1 (1 mg/L AFB1); and 4) AFB1 bio-activation (1 mg/L AFB1+30 mg/L MAS-rat and 1 mg/L AFB1 +30 mg/L MAS-human). RESULTS: In MAS-rat and human, Cy did not induce a statistically significant increase of mortality and malformed larvae percentage, but when bio-activated Cy increased the percentage of mortality. Instead, MAS-rat and human alone did not show any increase of mortality and malformed larvae percentages. When bio-activated by MAS-rat and human, AFB1 increased significantly both the mortality and malformed larvae percentages. The malformed larvae were mainly plurimalformed, i.e., affected by generalized edema, abnormal gut coiling, and microphthalmia. CONCLUSIONS: This research shows that AFB1 alone is not embryotoxic but, when bio-activated with MAS-rat or MAS-human the percentage of mortality and malformed larvae increased significantly. These results also show that AFB1 must be bio-activated to exert its embryotoxic effects.     

Adverse developmental and reproductive effects of copper deficiency in Xenopus laevis

The effect of copper (Cu) deficiency on the reproduction and development in Xenopus laevis was evaluated, culminating in the development of a defined concentration-response relationship. Separate groups of four adult frog pairs were fed one of three diets for 28 d: (1) low-copper (−Cu); (2) copper supplemented (+Cu); and (3) ASTM standard beef liver and lung (BLL). Embryos collected from frogs administered the −Cu diet had markedly decreased egg masses and viability rates and an increased rate of necrosis when compared to the other dietary treatments. Malformations in −Cu larvae included maldevelopment of the heart, eye, craniofacial region, brain, and notochord. Larvae from adults administered the −Cu diet showed delayed abnormal hindlimb development, characterized as selective reductive deficiencies distal to the femur, with poor cartilaginous development. A U-shaped dose-response curve characteristic of nutritional essentiality was developed for Cu. Overall, these studies indicated that embryos produced from frogs administered a −Cu diet are substantially less viable than embryos from frogs administered a +Cu or copper-adequate (BLL) diet.     

Determination of myoseverin embryotoxic potential by using FETAX

BACKGROUND: Since MYS is a microtubular poison with a reversible activity, Xenopus blastulae were exposed to MYS to verify the eventual drug-related developmental suspension and the reversibility of this effect. METHODS: Lethal and teratogenic effects of myoseverin (MYS) were evaluated using the FETAX. Embryos were exposed to different MYS concentrations from stage 8 to stage 47. RESULTS: Probit analysis gave 12.14 microM LC50 and 7.67 microM TC50 from which 1.58 T.I. is derived. Several malformations were observed such as facial abnormalities, abnormal tail flexure, heart ventricle chamber enlargement and external appendix. MYS led to an arrest of living embryo development. Before MYS removing, exposed blastulae showed the lack of mitotic spindles along with different nuclei alterations. Living embryos, moved in control solution, mainly died around the hatching showing severe malformations likely ascribable to the altered planes of newly occurring mitosis. CONCLUSION: In spite of the low T.I, MYS has to be considered a highly teratogenic compound.