Douglas J. Fort

Further Validation of Fetax: Evaluation of the Developmental Toxicity of Five Known Mammalian Teratogens and Non-Teratogens

The developmental toxicity of five compounds was evaluated with the Frog Embryo Teratogenesis Assay: Xenopus (FETAX). Late Xenopus laevis blastulae were exposed to 5-azacytidine, methotrexate, pseudoephedrine, aspartame, and amaranth for 96 h. Three separate static-renewal assays were conducted for each compound. Based on Teratogenic Index [LC50/EC50 (malformation)] values, types and severity of induced malformations, and embryo growth, 5-azacytidine and methotrexate tested as having strong teratogenic potential. Pseudoephedrine scored as having moderate teratogenic potential, but amaranth and aspartame had little or no teratogenic potential. Results support the use of FETAX for the screening of developmental toxicants.

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.

Developmental Toxicity Testing with Fetax:Evaluation of Five Compounds

The teratogenic potential of five compounds was evaluated with FETAX (Frog Embryo Teratogenesis Assay: Xenopus). Early embryos of Xenopus laevis were exposed for 96 h to 6-aminonicotinamide (6-AN), isoniazid (INH), urethane, nitrilotriacetate (NTA) or sodium cyclamate, in two separate static-renewal tests of each compound. Based on Teratogenic Index values, growth endpoints and the types and severity of the induced malformations, 6-AN, INH and urethane scored as having strong teratogenic potential. NTA and sodium cyclamate had little or no teratogenic potential. The results support continued evaluation of FETAX as a screening assay for chemical teratogens.

Effects of pond water, sediment, and sediment extracts from minnesota and vermont, USA, on early development and metamorphosis of xenopus

In recent studies, a high incidence of amphibian mortality and malformation has been reported in the field, suggesting that toxic and/or bioactive agents are present in the environment of the affected amphibians. This study provides evidence for this hypothesis, because it applies to several affected ponds in Minnesota and Vermont, USA. Three developmental bioassays were carried out on samples from three reference and three test sites in Minnesota and one reference and three test sites, in Vermont. The bioassays utilized Xenopus as a model system, measuring altered developmental patterns during the first 4 d of development (frog embryo teratogenesis assay-Xenopus [FETAX]), hind-limb development over a 30-d period, and tail length resorption over a 14-d period. Strong correlations were observed among the results for all three in vitro bioassays, as well as between adverse developmental effects in vitro and in the field.

Preliminary validation of a short-term morphological assay to evaluate adverse effects on amphibian metamorphosis and thyroid function using Xenopus laevis

Short‐term static‐renewal studies were performed on Xenopus laevis embryos with 16 selected test materials from day 50 (stage 60) to day 64 (stage 66) (14‐day test) to evaluate effects on tail resorption and thyroid function. Of the 16 test materials, nine were found to inhibit significantly the rate of tail resorption, four were found to stimulate metamorphosis and three had no appreciable effect on the rate of metamorphosis. In an effort to determine if the morphological effects observed were related to alteration in thyroid activity, measurement of triiodothyronine (T3) in the test organisms and co‐administration studies using thyroxine (agonist) or propylthiouracil (antagonist) were performed based on the morphological response noted during tail resorption. Of the nine compounds found to inhibit the rate of tail resorption, six were found to reduce the levels of T3. In each case, the inhibitory response could be at least partially alleviated by the co‐administration of thyroxine. Larvae exposed to the four stimulatory agents had somewhat elevated levels of T3 and were responsive to propylthiouracil antagonism. These results suggest that 12 of the 14 compounds tested in this study that altered the rate of tail resorption did so via the thyroid axis. Overall, the X. laevis model appeared to be a suitable system for evaluating the impact of environmental agents and chemical products on thyroid function. Copyright

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 frogs 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.

Triclosan and Anuran Metamorphosis: No Effect on Thyroid-Mediated Metamorphosis in Xenopus laevis

Nieuwkoop and Faber stage 51 Xenopus laevis larvae were exposed for 21 days to four different concentrations of triclosan (TCS): <0.2 (control), 0.6, 1.5, 7.2, or 32.3 microg TCS/l. Primary endpoints were survival, hind limb length, body length (whole; snout to vent), developmental stage, wet whole body weight, and thyroid histology. Thyroid hormone (TH) concentrations were determined in whole thyroid and plasma samples from stage-matched exposure day 21 specimens. TH receptor-beta (TRbeta) expression was measured in stage-matched tail fin tissue samples collected at exposure days 0 and 21. Reduced larval growth occurred at exposure day 21 with 1.5 microg/l treatment. Larval developmental stage at exposure day 21 was not significantly different from controls based on observed parameters. Thyroid histology was not affected by TCS, and thyroxine (T4) levels in thyroid glands or plasma were not different from controls. A concentration-dependent increase in TRbeta expression in exposure day 21 larvae was not detected. However, increased expression was found in stage-matched larvae exposed to 1.5 or 7.2 microg TCS/l. Our study indicates that environmentally relevant TCS concentrations do not alter the normal course of thyroid-mediated metamorphosis in this standard anuran model.

PROGRESS TOWARD IDENTIFYING CAUSES OF MALDEVELOPMENT INDUCED IN XENOPUS BY POND WATER AND SEDIMENT EXTRACTS FROM MINNESOTA, USA

In previously conducted laboratory studies with the South African clawed frog (Xenopus laevis), pond water and sediment samples collected from various sites in Minnesota, USA, were demonstrated to have the potential to induce a variety of developmental abnormalities, including early embryo-larval maldevelopment, abnormal limb development, and disruption of metamorphosis. The results of exposure of X. laevis to suspect pond water and sediment samples supported the hypothesis that these samples were capable of inducing these abnormalities as the result of either the presence of developmental toxicants or the absence of essential micronutrients. Physicochemical characterization of the causes of abnormal frog embryo-larval and limb development were performed using the frog embryo teratogenesis assay-Xenopus (FETAX). Specific compounds were subsequently identified within the complex mixture fractions and tested by dilution in a control solution and native reference water using both the 4- and 30-d treatment protocols. Results from these studies suggested that a complex mixture of both naturally occurring and man-made compounds was primarily responsible for the effects observed in X. laevis. The potency of several compounds was also enhanced by the site water, thus indicating that the water matrix deserves consideration as a contributing factor for both laboratory and field studies.

Assessing the efficacy of an Aroclor 1254-induced exogenous metabolic activation system for FETAX.

The developmental toxicity of N-nitrosodimethylamine (NDMA) and trichloroethylene (TCE) was assessed with Frog Embryo Teratogenesis Assay: Xenopus (FETAX). Late Xenopus laevis blastulae were exposed to NDMA and TCE for 96-h in two separate static-renewal tests with and without the presence of three differently induced exogenous metabolic activation systems (MAS). The MAS consisted of Aroclor 1254-induced (Aroclor 1254 MAS), isoniazid-induced (INH MAS), and a post-isolation mixture (mixed MAS) of Aroclor 1254- and isoniazid-induced rat liver microsomes. Addition of the INH MAS and the mixed MAS increased the Teratogenic Index [TI = LC50/EC50 (malformation)] of NDMA and TCE nearly 2.0- and 2.1-fold and 2.1- and 1.7-fold, respectively. Inclusion of the Aroclor 1254 MAS did not alter the developmental toxicity of either compound. Based on TI values, embryo growth, and types and severity of induced malformations, both NDMA and TCE were developmentally toxic. Use of post-microsome isolation mixtures from differentially induced rat livers increased the efficacy of the exogenous MAS routinely used by FETAX.

Assessing the predictive validity of frog embryo teratogenesis assay-Xenopus (FETAX).

The ability of frog embryo teratogenesis assay - Xenopus (FETAX) to identify the potential developmental toxicity of a group of diverse chemicals was evaluated by comparison with results from in vivo studies in rats. A total of 12 chemicals, three of which were shown to be teratogenic in vivo, four of which were embryolethal (but not teratogenic) in vivo, and five which did not produce any developmental toxicity in vivo in the rat were evaluated using FETAX. Results of the FETAX test with these 12 blind-coded compounds correctly predicted that three chemicals had strong teratogenic potential, four had low teratogenic hazard potential but were embryolethal, and five posed little if any developmental toxicity hazard. In addition, this study concluded that within a family of chemistry analogs could be ranked according to relative teratogenic hazard and that for the teratogenic compounds the types of malformations induced in Xenopus mimicked the abnormalities induced in vivo in rats. In summary, these results confirmed that the FETAX assay is predictive and can be useful in an integrated biological hazard assessment for the preliminary screening of chemicals. Teratogenesis Carcinog. Mutagen. 20:87-98, 2000.