Frog Embryo Teratogenesis Assay-Xenopus Protocol Research Articles

Mortality, teratogenicity and growth inhibition of three glyphosate formulations using Frog Embryo Teratogenesis Assay-Xenopus.

Ample evidence around the world exists suggesting a link between exposure to glyphosate, toxicity and perturbed physiological functions in non-target organisms. Although glyphosate formulations are widely used for weed and alien plant management, their ecotoxicological information remain scanty. Using the 96-hour Frog Embryo Teratogenesis Assay-Xenopus protocol, embryotoxicity and teratogenicity of three glyphosate-based formulations were assessed. Embryos of Xenopus laevis were exposed to Roundup, Kilo Max and Enviro Glyphosate at concentration of 0.3-1.3, 130-280 and 320-560mg acid equivalent (a.e.)/L respectively. The results showed Roundup to be more toxic than the other formulations with a 96-hour LC50 of 1.05mg a.e/L. compared with 207mg a.e./L, and 466mg a.e./L for Kilo Max and Enviro Glyphosate respectively. Although, both Roundup and Kilo Max formulations show inhibition on growth of the embryo-larva (P˂.05), the minimum concentration inhibiting growth ratios of the three formulations was >0.30 baseline, indicating no significant growth inhibiting effect in the formulations. For teratogenicity, Roundup and Enviro Glyphosate formulations exhibited increasing teratogenic traces, with the teratogenic index at 1.7 and 1.6 respectively. Kilo Max formulation shows low teratogenicity with the teratogenic index at 1.4. Characteristic malformation induced by these formulations included generalized edema, cardiac and abdominal edema, improper gut formation and axial malformations. This study confirms that these formulations could be a potential physiological and ecological health disruptor, particularly concerning teratogenicity and growth disruption. Further studies to characterize the contributions of their surfactants will be invaluable.

Determination of metals and pharmaceutical compounds released in hospital wastewater from Toluca, Mexico, and evaluation of their toxic impact

Due to the activities inherent to medical care units, the hospital effluent released contains diverse contaminants such as tensoactives, disinfectants, metals, pharmaceutical products and chemical reagents, which are potentially toxic to the environment since they receive no treatment or are not effectively removed by such treatment before entering the drain. They are incorporated into municipal wastewater, eventually entering water bodies where they can have harmful effects on organisms and can result in ecological damage. To determine the toxicological risk induced by this type of eflluents, eight metals and 11 pharmaceuticals were quantified, in effluent from a hospital. Developmental effects, teratogenesis and oxidative stress induction were evaluated in two bioindicator species: Xenopus laevis and Lithobates catesbeianus. FETAX (frog embryo teratogenesis assay–Xenopus) was used to obtain the median lethal concentration (LC50), effective concentration inducing 50% malformation (EC50), teratogenic index (TI), minimum concentration to inhibit growth (MCIG), and the types of malformation induced. Twenty oocytes in midblastula transition were exposed to six concentrations of effluent (0.1, 0.3, 0.5, 0.7, 0.9, 1%) and negative and positive (6-aminonicotinamide) controls. After 96 h of exposure, diverse biomarkers of oxidative damage were evaluated: hydroperoxide content, lipid peroxidation, protein carbonyl content, and the antioxidant enzymes superoxide dismutase and catalase. TI was 3.8 in X. laevis and 4.0 in L. catesbeianus, both exceed the value in the FETAX protocol (1.2), indicating that this effluent is teratogenic to both species. Growth inhibition was induced as well as diverse malformation including microcephaly, cardiac and facial edema, eye malformations, and notochord, tail, fin and gut damage. Significant differences relative to the control group were observed in both species with all biomarkers. This hospital effluent contains contaminants which represents a toxic risk, since these substances are teratogenic to the bioindicators used. The mechanism of damage induction may be associated with oxidative stress.

Evaluation of the effects of the marine toxin okadaic acid by using FETAX assay

The Frog Embryo Teratogenesis Assay Xenopus (FETAX), is a screening assay using embryos at gastrula stage of the anuran Xenopus laevis to identify substances that may pose a developmental hazard in humans. The FETAX assay evaluates three parameters, i.e. mortality, delayed growth and embryo malformation. In the present investigation, the FETAX protocol was applied to the marine toxin okadaic acid (OA) and the experiments show that OA affects the above parameters in a dose-correlated manner. The morpho-functional modifications induced in embryo organs by OA were also studied. The nervous system, tail skeletal musculature, intestine and kidney appeared particularly damaged, with the former being the most sensitive. On the whole, various advantages emerge from using the FETAX assay: different parameters can be tested simultaneously, the indication of the presence of a potentially dangerous substance is rapid and the assay is a valid alternative to mammalian systems.

Application of frog embryo teratogenesis assay‐Xenopus to ecological risk assessment

An expert workshop recently was convened to consider the frog embryo teratogenesis assay-Xenopus (FETAX) as a screening method for identifying the potential developmental toxicity of single chemicals and chemical mixtures. One recommendation from the workshop was that, in order to determine the utility of FETAX for ecological risk assessments, additional consideration of how the assay is conducted is necessary. In addition, a comparative evaluation would be useful of FETAX endpoints (i.e., survival, malformations, growth) versus each other, endpoints from aquatic toxicity tests using more commonly tested species of cladocerans and fish, and tests with other amphibian species. This review provides an evaluation and critique of the current FETAX protocol from two perspectives: Practical considerations relative to conducting the test and sensitivity of the assay (and associated endpoints) compared to tests with other species. Several aspects of the current standard protocol, including test temperature, diet, loading rates, and chemical exposure options, need to be modified to ensure that the assay is robust technically. Evaluation of FETAX data from the open literature indicates that growth is the most sensitive endpoint in the assay, followed by malformations and then survival; unfortunately, the growth endpoint often is not considered or reported in the assay. Comparison of FETAX data with acute toxicity data from tests with other amphibians or traditional aquatic test species indicates FETAX is relatively insensitive. This suggests that environmental risk assessments using acute hazard data from tests with traditional aquatic test species usually would be more protective of native amphibian species than risk assessments that use hazard data from FETAX.

FETAX, a versatile tool in toxicology, can be conveniently integrated with molecular biology techniques

The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a powerful and flexible bioassay for developmental toxicants that makes use of the embryos of the anuran Xenopus laevis. FETAX, thanks to its three endpoints (i.e., mortality, malformation, and growth inhibition), can also detect the xenobiotics that affect embryonic development, a weak link in the life cycle of an organism. The FETAX protocol, however, is amenable to modification in several ways and to being integrated with molecular biology techniques that considerably increase the capability of the test. As exposure to xenobiotics may alter gene expression and therefore mRNA and protein patterns, transcriptome and proteome modifications can be studied with the aim to obtain new insights on the mechanisms of embryotoxicity and teratogenesis or simply to obtain molecular markers of exposure useful in the early diagnosis of environmental stress.

Application of the FETAX protocol to assess the developmental toxicity of nonylphenol ethoxylate to Xenopus laevis and two Australian frogs

The Frog Embryo Teratogenesis Assay– Xenopus (FETAX) protocol has recently been adopted as a valuable tool for evaluating the embryotoxicity of environmental contaminants in amphibians. The bioassay utilises Xenopus laevis as a test species, but there are few comparative studies to evaluate whether data collected in this species is applicable to other amphibians. In this study, the embryotoxicity of the nonionic surfactant, nonylphenol ethoxylate, was determined in X. laevis and the Australian frogs, Litoria adelaidensis and Crinia insignifera using the FETAX protocol. The 96-h LC 50, EC 50 and minimum concentration to inhibit growth (MCIG) values for X. laevis were 3.9–5.4, 2.8–4.6 and 1.0–3.0 mg/l, respectively. The 140-h LC 50, EC 50 and MCIG values for L. adelaidensis were 9.2, 8.8 and 5.1–6.0 mg/l, respectively. The 134-h LC 50, EC 50 and MCIG values for C. insignifera were 6.4, 4.5 and 4.0 mg/l, respectively. Teratogenicity indices for the three species ranged between 1.0 and 1.6, indicating either no or low teratogenicity. Growth inhibition as assessed by embryo length was the most sensitive indicator of effect in all three species. X. laevis was the more sensitive of the three species and the only species that displayed indisputable terata.

Effects of water quality on development of Xenopus laevis: A frog embryo teratogenesis assay—Xenopus assessment of surface water associated with malformations in native anurans

AbstractThe purpose of this work was to determine if surface water from a site in Minnesota, USA, with malformed anurans was able to elicit adverse developmental effects in the frog embryo teratogenesis assay: Xenopus (FETAX) and to isolate and identify the factors responsible for the effects. In the standard FETAX test, 25 organisms are placed in a 10‐ml volume of solution that is renewed daily. The organisms are assessed for abnormal development and the test is terminated at 96 h. Under these conditions, the site water adversely affected craniofacial development, gastrointestinal development, and growth. However, these effects were eliminated by each of several different treatments, including diluting the surface‐water sample with the standard testing solution, adding dry salts directly to the surface water, testing the organisms in larger volumes of site water, and evaporating site water to increase the concentration of total dissolved solids. These results are consistent with the hypothesis that the low ion concentrations found in this particular site water were responsible for the developmental effects observed in FETAX. This hypothesis was strengthened further when a reconstituted water, made with deionized water and reagent‐grade salts to mimic the content of the site water, affected survival, growth, and development similarly to the surface water. In addition, substantial ammonia accumulation in the FETAX tests was documented. These data suggest that the inherent variability in water quality of field‐collected samples is important and could result in artifactual developmental effects when using FETAX. As such, the developmental toxicity observed in these FETAX studies is probably not relevant to malformations observed in native anuran species. To obviate this type of problem, the volumes used in the FETAX protocol should be increased for better organism performance and to reduce the possibility of ammonia toxicity.