Frog Embryo Teratogenesis Assay Research Articles

Teratogenic assessment of four solvents using the Frog Embryo Teratogenesis Assay--Xenopus (FETAX).

The Frog Embryo Teratogenesis Assay--Xenopus (FETAX) was used to assess the teratogenic potential of four solvents. Embryos of the South African clawed frog, Xenopus laevis, were exposed for 96 h to ethanol, dimethyl sulfoxide (DMSO), formamide or glycerol formal. Exposure groups were maintained using a static renewal system in which the exposure media were changed at 24-h intervals. Survival was monitored at 24-h intervals. Length, as an indicator of growth effects, and developmental malformations were determined at the end of the assay (96 h). Using this information, the 96-h LC50, the 96-h EC50 (Malformation), and the no observable effect levels (NOELs) for mortality, malformation and length were determined for each solvent. The teratogenic index [TI = 96-h LC50/96-h EC50 (Malformation)] also was calculated for each of the solvents. DMSO appeared to be the least toxic or teratogenic solvent examined, with a pooled LC50 of 1.92%, a pooled EC50 (Malformation) of 1.57% and TI values of 1.20 and 1.24 in replicate trials. Formamide appeared to be the most toxic solvent, with a pooled LC50 of 1.04%. Data trends suggested that ethanol was the most teratogenic solvent tested, with a pooled EC50 (Malformation) of 1.04% and TI values of 1.42 and 1.50. The results obtained in the present work for ethanol and DMSO were compared to previously published FETAX results for these two solvents. The present results are in close agreement with these results from other laboratories, thus providing further evidence supporting the interlaboratory reproducibility of FETAX results.

Additive incidence of developmental malformation for Xenopus embryos exposed to a mixture of ten aliphatic carboxylic acids

A modified FETAX (Frog Embryo Teratogenesis Assay: Xenopus) protocol was used to assess the joint action of ten aliphatic carboxylic acids on Xenopus embryo development. Stock solutions of each acid alone, made up at twice the EC50 of the individual acids, were prepared for testing alone and in a mixture with an equal volume of each acid stock solution. For each treatment, five concentrations and a control dish, each with 25 embryos, were tested for 96 h, with solution renewal every 24 h. The embryos were then fixed and evaluated for gross malformations. For each dish, the number and types of malformations were recorded. An EC50 was calculated for each acid alone and this value was defined as 1.0 toxic unit (TU) for malformation induced by the acid. An EC50 was also calculated for the mixture. The concentration of each acid at the mixture EC50 and the TU values corresponding to these concentrations were then determined. A TU value of 0.990 (0.923-1.060) was obtained for the mixture by adding the TU values for each acid in the mixture. This represents a concentration additive rate of malformation. Microcephaly, TU = 1.09 (1.01-1.18), was the primary malformation, but did not completely account for the response. The concentration additive rate of malformation indicates that all ten acids are likely to induce malformation in Xenopus embryos in a similar manner. Quantitative structure-activity relationship (QSAR) analysis revealed developmental malformation induced by the acids was highly correlated (r2 = 0.979) with hydrophobicity and molar refractivity (r2 = 0.949). The approach has potential application in determining compounds that induce developmental malformations in a similar manner, when metabolism and pharmacokinetic factors are considered.

Altered developmental toxicity caused by three carrier solvents

Many aquatic bioassays rely on chemical solvents to solubilize water-insoluble test materials. Interactions between solvents and test materials can lead to false positive or negative results. For this reason, tests for interactions between solvents and test materials were performed. The Frog Embryo Teratogenesis Assay--Xenopus (FETAX) was chosen because of its capacity to assess three different endpoints; mortality, malformation and embryo growth. Three solvents--dimethylsulfoxide (DMSO), acetone and triethylene glycol (TG)--were tested with two teratogens--methylmercury chloride (MMC) and trichloroethylene (TCE). DMSO potentiated the lethal effect of both teratogens but did not alter significantly the rate of malformation. Acetone increased the mortality for both teratogens, but only increased the MMC malformation greater than the additive effects. TG only increased the mortality and malformation with TCE. There were additive effects for growth for all solvents with the teratogens. The carrier solvents caused interactions even at their no-observable-effect concentration (NOEC). Therefore, the choice of carrier solvent should be made with caution. This study shows that different results can occur depending on the solvent used, and that a difference in one endpoint does not necessarily change the other endpoints.

Evaluation of the frog embryo teratogenesis assay: Xenopus (FETAX) as a model system for mixture toxicity hazard assessment

AbstractThe joint toxic action for several binary mixtures was determined with FETAX (frog embryo teratogenesis assay Xenopus) to evaluate the malformation and lethality end points for potential value in mixture toxicity studies. Nine combinations of developmental toxicants were selected for testing at malformation‐inducing concentrations, based on presently accepted modes of teratogenesis of each chemical and on the primary types of malformations each had elicited in earlier tests. Two combinations were selected for embryolethal end point testing. Three mixture ratios (3 1, 1 1, 1 3) were tested for each combination, with each combination being tested on three or four separate occasions Individual tests of each chemical were simultaneously conducted for each combination to serve as positive controls (i.e., 1 0 and 0 1 solutions). Toxic unit analysis was used to determine the type of joint action for each combination. Concentration addition was observed for three combinations in which both chemicals were thought to have the same mode of teratogenesis. The other six combinations, with the chemicals having different modes of action, showed a response additive joint action Response addition was also seen for both combinations tested at the embryolethal end point, with an exception for one mixture ratio that was antagonistic. The results indicate FETAX should be useful for mixture toxicity hazard assessment, especially when the malformation end point is considered.

Teratogenicity of Ni2+ inXenopus laevis, assayed by the FETAX procedure

The teratogenicity of Ni2+ was tested by the FETAX (Frog Embryo Teratogenesis Assay: Xenopus) procedure in the South African frog, Xenopus laevis. In seven assays, beginning at 5 h postfertilization, groups of Xenopus embryos were incubated for 96 h in media that contained Ni2+ (added as NiCl2) at concentrations ranging from 1 x 10(-7) to 3 x 10(-3) mol/L; control groups were incubated in the same medium without added NiCl2. At 101 h postfertilization, surviving embryos were counted, fixed in formalin, and examined by microscopy to determine their developmental stages, malformations, and head-to-tail lengths. In control embryos, survival was greater than or equal to 95% and malformations were less than or equal to 7%. Malformations were found in greater than 95% of embryos exposed to Ni2+ concentrations greater than or equal to 5.6 mumol/L. The most frequent malformations in Ni(2+)-exposed embryos were ocular, skeletal, and intestinal deformities; less common malformations included facial, cardiac, and integumentary deformities. Other abnormalities, not categorized as malformations, included stunted growth, dermal hypopigmentation, and coelomic effusions or hemorrhages. The median embryolethal concentration (LC50) of Ni2+ was 365 (SE +/- 9) mumol/L; the median teratogenic concentration (EC50) was 2.5 (SE +/- 0.1) mumol/L; the Teratogenic Index (TI = LC50/EC50) was 147 (SE +/- 5), indicating that Ni2+ is a potent teratogen for Xenopus laevis. Experiments in which Ni(2+)-exposures were limited to specific 24 h periods showed that Xenopus embryos were most susceptible to Ni(2+)-induced malformations on the second and third days of life, during the most active period of organogenesis.

Structure-activity relationships for osteolathyrism: IV.Para-substituted benzoic acid hydrazides and alkyl carbazates

Nine benzoic acid hydrazides and carbazates were assayed for toxicity and teratogenicity by using early embryos of the frog Xenopus laevis. The results of the 96-h static tests were used for quantitative structure-activity relationship (QSAR) analyses. Each compound induced the connective tissue defect osteolathyrism. Regression analyses indicated toxicity (LC50) and teratogenicity (EC50) were best correlated with the STERIMOL width parameter B1, but due to redundancy in B1 values for the test chemicals and the relatively low r2 for the models, those equations should be used with caution. The mortality/malformation index (MMI) was negatively correlated with molar refractivity (MR). The relationships indicate that steric interactions may be important in explaining the variation in biological activity due to changes in chemical structure. Frog embryo teratogenesis assay: Xenopus (FETAX) may be useful in aquatic toxicology hazard assessment, evaluating developmental malformation.

Initial evaluation of developmental malformation as an end point in mixture toxicity hazard assessment for aquatic vertebrates

The joint toxic action of three binary mixtures was determined for the embryo malformation endpoint of the aquatic FETAX (frog embryo teratogenesis assay: Xenopus) test system. Osteolathyrogenic compounds and short-chain carboxylic acids, representing separate, distinct modes of action for induction of malformation, were selected for testing in 96-hr, static-renewal tests. Three mixtures were tested for each combination, with each combination being tested on three separate occasions. Using toxic unit analysis, the combination of osteolathyrogens and the combination of carboxylic acids produced strictly additive (concentration addition) rates of malformation, while the combination of an osteolathyrogen and a carboxylic acid was less-than-additive (response addition) for induction of malformation. Therefore, developmental malformation may have value as an endpoint in mixture toxicity hazard assessment.

Assessment of the Developmental Toxicity of Ascorbic Acid, Sodium Selenate, Coumarin, Serotonin, and 13–CIS Retlnolc Acid Using Fetax

The developmental toxicity of five compounds was evaluated with the Frog Embryo Teratogenesis Assay: Xenopus (FETAX) and the results were compared to mammalian literature. Small cell Xenopus laevis blastulae were exposed to ascorbic acid, sodium selenate, coumarin, serotonin and 13-cis retinoic acid for 96 hr. Three separate static-renewal assays were conducted for each compound. Teratogenic potential of the test materials was determined based on Teratogenic Index values [TI = LC50/EC50 (malformation)], types and severity of induced malformations and embryo growth. Ascorbic acid had little or no teratogenic potential. Sodium selenate and coumarin tested as having moderately positive teratogenic potential. Serotonin scored as having moderately strong teratogenic potential and 13-cis retinoic acid scored as having strong teratogenic potential. Results were consistent with mammalian data and support the use of FETAX for the screening of developmental toxicants.

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.

Teratogenicity of cobalt chloride in Xenopus laevis, assayed by the FETAX procedure

The teratogenicity of cobalt chloride (CoCl2) was tested by the FETAX (Frog Embryo Teratogenesis Assay: Xenopus) procedure in the South African frog, Xenopus laevis. In five assays, beginning at 5 h post-fertilization, groups of Xenopus embryos were incubated for 96 h in media that contained CoCl2 at concentrations ranging from 1.8 x 10(-6) to 1.8 x 10(-2) mol/L; control groups were incubated in the same medium without added CoCl2. At 101 h post-fertilization, surviving embryos were counted, fixed in formalin, and examined by microscopy to score malformations and measure head-to-tail lengths. In control embryos, survival was greater than or equal to 95% and malformations were less than or equal to 5%. Malformations were found in greater than 99% of embryos exposed to Co2+ levels greater than or equal to 56 mumol/L. Co2+)-exposed embryos showed a concentration-related pattern of malformations, comprising gut malrotation, ocular anomalies, kinked tail, craniofacial dysplasia, cardiac deformities, and dermal blisters. Other concentration-dependent abnormalities, not categorized as malformations, included stunted growth, edema, ventral distention, and hypopigmentation. The median embryolethal concentration (LC50) of CoCl2 was 10.4 (SE +/- 0.4) mmol/L; the median teratogenic concentration (EC50) was 25 (SE +/- 2) mumol/L; the teratogenic index (TI = LC50/EC50) was 416 (SE +/- 13), indicating that CoCl2 is a potent teratogen for Xenopus laevis.

Developmental toxicology of potato alkaloids in the frog embryo teratogenesis assay—Xenopus (FETAX)

Potatoes frequently contain growth inhibitors and toxic compounds including digestive enzyme inhibitors, lectins and glycoalkaloids. The literature suggests that Solanum alkaloids have the ability to induce neurological damage such as spina bifida and other malformations. As part of a programme of improvement in the safety of potatoes using molecular plant genetics and parallel food safety evaluation, we evaluated the effect of several potato glycoalkaloids and aglycones in the frog embryo teratogenesis assay—Xenopus (FETAX) with and without metabolic activation by Aroclor 1254-induced rat liver microsomes. The data suggest that the glycoalkaloid α-chaconine is teratogenic and more embryotoxic than α-solanine, in terms of the median lethal concentration (LC 50) after 96 hr of exposure, the concentration inducing gross terata in 50% of the surviving frog embryos (96-hr EC 50, malformation), and the minimum concentration needed to inhibit the growth of the embryos. Since these two compounds differ only in the nature of the carbohydrate side chain attached to the 3-OH group of solanidine, the side chain appears to be an important factor in governing teratogenicity. The aglycones demissidine, soladine and solasodine were less toxic than the glycosides α-chaconine and α-solanine. The in vitro teratogenesis assay should be useful for: (a) predicting the teratogenic potential of Solanaceae alkaloids, glycoalkaloids and related natural products, and (b) facilitating experimental approaches to suppress plant genes and enzymes that control the biosynthesis of the most toxic compounds.

Use of Frog Embryo Teratogenesis Assay- Xenopus and an exogenous metabolic activation system to evaluate the developmental toxicity of diphenylhydantoin

The teratogenic potential of diphenylhydantoin (DPH) and hydroxylated metabolites (HPPH) was evaluated with the Frog Embryo Teratogenesis Assay— Xenopus (FETAX). Embryos of the South African clawed frog, Xenopus laevis were exposed to DPH and HPPH in two separate static-renewal experiments with and without the presence of an exogenous metabolic activation system (MAS) for 96 hr. Two separate dose-response tests were also conducted with DPH and HPPH with a MAS modulated by various mixed functional oxidase inhibitors [carbon monoxide (CO) (broad spectrum cytochrome P450), cimetidine (mainly cytochrome P450), and ellipticine (cytochrome P448)] and an epoxide hydrolase inhibitor (cyclohexene oxide). Assessment of the potential teratogenic hazard was based on teratogenic indices [TI = 96-hr LC50/96-hr EC50 (malformation)], types and severity of malformations, and embryo growth endpoints. Addition of the intact MAS to DPH increased the 96-hr LC50 and EC50 (malformation) from approximately 74.5 and 32.4 mg/liter to 126.4 and 62.9 mg/liter, respectively. The TI was reduced 1.2-fold. Both p-HPPH and m-HPPH were much less developmentally toxic than DPH. CO and cimetidine inhibition of cytochrome P450 maintained much of the developmental toxicity of DPH, whereas ellipticine inhibition of cytochrome P448 was much less effective in maintaining the developmental toxicity of DPH. Cyclohexene oxide inhibition of epoxide hydrolase markedly increased DPH-induced embryotoxicity decreasing the 96-hr LC50 from approximately 74.5 to 38.6 mg/liter. These results suggest that unmetabolized DPH and an embryotoxic epoxide intermediate may serve as the teratogenic species in FETAX.

Analysis of the mechanism of isoniazid‐induced developmental toxicity with frog embryo teratogenesis assay: Xenopus (FETAX)

The developmental toxicity of isoniazid (INH) and the metabolites acetylhydrazide (AH) and isonicotinic acid (INA) were examined with the frog embryo teratogenesis assay-Xenopus (FETAX). Late Xenopus laevis blastulae were exposed to INH, AH, and INA for 96 h in two separate static-renewal tests with and without the presence of three differently induced metabolic activation systems (MAS). The MAS consisted of uninduced, Aroclor 1254-induced, and INH-induced rat liver microsomes. Addition of the INH-induced MAS decreased the 96 h LC50 of INH and AH approximately 1.6-fold and 7.9-fold, respectively. The 96 h EC50 (malformation) of INH was virtually unaffected; however, the INH-MAS decreased the teratogenic index (TI) [96 h LC50/96 h EC50 (malformation)] nearly 1.8-fold. The 96 h EC50 (malformation) of AH increased approximately 2.0-fold, decreasing the teratogenic index value 15.8-fold. INA yielded a teratogenic index value of 2.5. Neither the uninduced MAS nor the Aroclor 1254-induced MAS had an effect on any of the compounds tested and none of the MAS affected the developmental toxicity of INA. Results from this study suggest that mixed functional oxidase metabolism may alter the developmental toxicity of INH in vitro by producing a more embryolethal, but less teratogenic metabolite(s) than INH or AH themselves. Results are indicative of the utility and versatility of FETAX in evaluating toxicological mechanisms of teratogenesis in vitro.

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.

Use of Frog Embryo Teratogenesis Assay-Xenopus and an Exogenous Metabolic Activation System to Evaluate the Developmental Toxicity of Diphenylhydantoin

Use of Frog Embryo Teratogenesis Assay-Xenoptis and an Exogenous Metabolic Activation System to Evaluate the Developmental Toxicity of Diphenylhydantoin. FORT, D. J. AND BANTLE, J. A. (1990). Fundam. Appl. Toxicol. 14,720–733. The teratogenic potential of diphenylhydantoin (DPH) and hydroxylated metabolites (HPPH) was evaluated with the Frog Embryo Teratogenesis Assay—Xenopus (FETAX). Embryos of the South African clawed frog, Xenopus laevis were exposed to DPH and HPPH in two separate static-renewal experiments with and without the presence of an exogenous metabolic activation system (MAS) for 96 hr. Two separate dose-response tests were also conducted with DPH and HPPH with a MAS modulated by various mixed functional oxidase inhibitors [carbon monoxide (CO) (broad spectrum cyto-chrome P450), cimetidine (mainly cytochrome P450), and ellipticine (cytochrome P448)] and an epoxide hydrolase inhibitor (cyclohexene oxide). Assessment of the potential teratogenic hazard was based on teratogenic indices [TI = 96-hr LC50/96-hr EC50 (malformation)], types and severity of malformations, and embryo growth endpoints. Addition of the intact MAS to DPH increased the 96-hr LC50 and EC50 (malformation) from approximately 74.5 and 32.4 mg/liter to 126.4 and 62.9 mg/liter, respectively. The TI was reduced 1.2-fold. Both p-HPPH and m-H PPH were much less developmentally toxic than DPH. CO and cimetidine inhibition of cytochrome P450 maintained much of the developmental toxicity of DPH, whereas ellipticine inhibition of cytochrome P448 was much less effective in maintaining the developmental toxicity of DPH. Cyclohexene oxide inhibition of epoxide hydrolase markedly increased DPH-in-duced embryotoxicity decreasing the 96-hr LC50 from approximately 74.5 to 38.6 mg/liter. These results suggest that unmetabolized DPH and an embryotoxic epoxide intermediate may serve as the teratogenic species in FETAX.

Evaluation of the developmental toxicity of five compounds with the frog embryo teratogenesis assay: Xenopus (FETAX) and a metabolic activation system.

The potential teratogenic hazard of five compounds was evaluated using the Frog Embryo Teratogenesis Assay--Xenopus (FETAX) and a metabolic activation system. Embryos of the South African clawed frog, Xenopus laevis, were exposed to (i) three compounds suspected to be proteratogenic in mammalian test systems--[2-acetylaminofluorene (2-AAF), rifampicin (RA) and benzo[a]pyrene (BP)] for 96 h; (ii) one compound unaffected by mixed-functional oxidase (MFO) metabolism--ZnSO4; (iii) one compound thought to be inactivated by cytochrome P-450--cytochalasin D (CD). Two separate static renewal tests were conducted with and without the presence of an exogenous metabolic activation system (MAS). The metabolic activation system consisted of Aroclor 1254-induced rat liver microsomes. The teratogenic potential of each compound and the effects of metabolic activation were based on teratogenic indices [TI = 96 h LC50/96 h EC50 (malformation)], types and severity of malformation, and effects on embryo growth. Metabolic activation increased the potential teratogenic hazard of 2-AAF, RA and BP by TI factors of 1.3, 2.8 and 6.8, respectively. The teratogenic potential of ZnSO4 was virtually unaffected by the MAS. The MAS significantly reduced the teratogenic potential of CD by a TI factor of 2.7. These results demonstrate the utility and importance of a MAS for in vitro developmental toxicity screens such as FETAX. Consistent use of a MAS with FETAX should reduce the number of potential false-positive and false-negative test results.

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.

Structure-Activity Relationships for Osteolathyrism. I. Effects of Altering the Semicarbazide Structure

The toxic and osteolathyrogenic effects of 15 chemicals reflecting modifications of semicarbazide [H2N-N(H)-C(=O)-NH2] were evaluated using the Frog Embryo Teratogenesis Assay: Xenopus (FETAX). Osteolathyrism, the failure of connective tissue fibers to cross-link correctly, is expressed as gross alterations in the long axis of the embryo and a sinusoidal configuration of the notochord. Alteration (addition or subtraction) at the hydrazino (H2NNH-) end of the molecule sharply reduces osteolathyrogenic activity. Alteration at the amino (H2N-) end of the molecule results in a graded response. The replacement of oxygen with sulfur causes no loss of osteolathyrogenic activity. Among the myriad events constituting vertebrate organogenesis is the formation of intercellular connective tissue elements such as collagen and elastin. Failure of this developing collagen and elastin to polymerize properly is termed osteolathyrism (Selye, 1957). An osteolathyrogenic agent, therefore, is any chemical that produces increased connective tissue fragility and collagen extractability (Levene, 1973). This is thought to result because of inhibition of the copper-requiring enzyme lysyl oxidase (Harris et al., 1974) necessary for extracellular cross-linking in both collagen (Bailey, 1968) and elastin (Barrow et al., 1974) maturation. Studies of experimentally-induced osteolathyrism have utilized a number of 1These studies were supported by National Institute of Environmental Health Sciences Grant R01 ES04209 to T.W.S. 2 Supported by the U.S. Environmental Protection Agency under Cooperative Agreement CR813158 with T.W.S. 3 Supported in part by U.S. Environmental Protection Agency under Cooperative Agreement CR-813158 with T.W.S. and The University of Tennessee, College of Veterinary Medicine, Institute of Agriculture, Center of Excellence in Livestock Diseases and Human Health. TRANS. AM. MICROSC. SOC., 107(2): 113-126. 1988. ? Copyright, 1988, by the American Microscopical Society, Inc. This content downloaded from 207.46.13.110 on Fri, 09 Dec 2016 05:44:01 UTC All use subject to http://about.jstor.org/terms TRANS. AM. MICROSC. SOC. natural and synthetic chemicals (Levene, 1961). One such compound is semicarbazide. Neuman et al. (1956), in an effort to produce liver damage in chick embryos similar to that observed following administration of aminoguanidine, injected semicarbazide into incubating eggs. Although semicarbazide had no effect on the developing liver, it did induce skeletal abnormalities similar to those described by Chang et al. (1954) following exposure to beta-aminopropionitrile. This initial documentation of the osteolathyrogenic potential of semicarbazide was followed by the studies of Dasler (1958) using Sprague-Dawley rats and by Levy (1959) using salamander and toad embryos. These latter investigations described axial malformations in treated individuals and suggested that semicarbazide is a strong direct-acting teratogen. Recent work in our laboratory has documented in-depth the embryotoxic and teratogenic effects of semicarbazide (Schultz et al., 1985) and benzoyl hydrazine (Riggin & Schultz, 1986) using a frog embryo assay (FETAX). An in vitro bioassay using early embryos of Xenopus laevis and referred to as FETAX (Frog Embryo Teratogenesis Assay: Xenopus) was developed as a screening system (Dumont et al., 1983). FETAX has several advantages as a teratogenic screening system, including cost and time effectiveness. In addition, the frog embryo as a classic experimental embryological system is developmentally relevant to mammals undergoing events (i.e., cleavage, gastrulation, organogenesis) comparable to those of other vertebrates. In this investigation, we used FETAX to explore structure-activity relationships by examining the relative toxicity and osteolathyrogenicity of a series of 15 chemicals that are structural modifications of semicarbazide. MATERIALS AND METHODS Embryos were obtained from adult pairs of Xenopus laevis injected with human chorionic gonadotropin to induce ovulation and amplexus. Healthyappearing early to mid-blastula embryos were selected individually using a dissecting microscope. Groups of 25 embryos were each placed in 60 x 15mm Pyrex dishes in 10-ml solutions of graduated concentrations of the test chemical and FETAX solution (625 mg NaCl, 96 mg NaHCO3, 30 mg KCl, 15 mg CaC12, 60 mg CaSO4 2H20, and 75 mg MgSO4 per liter of distilled water) (Dawson & Bantle, 1987). Control embryos were placed in 100% FETAX solution. The tested chemicals were structural modifications of semicarbazide (Table I). Each was purchased from a commercial source (Aldrich Chemical Company, Milwaukee, Wisconsin, U.S.A.) and not repurified prior to use. Stock solutions of each test chemical at a concentration equal to the highest concentration tested were made fresh for each assay using FETAX solution as the diluent. The stock solutions were adjusted to pH 7.2 and final test solutions were made by appropriate dilutions of stock solutions. For concentration-effect experiments, exposure was for 96 h at 23?C ? 0.5 in an incubator with a 12L/12D photoperiod. A minimum of three replicates was tested for each concentration series. A 96 h, surviving embryos were fixed in 10% neutral buffered formalin. The mortality, number and types of mal114 This content downloaded from 207.46.13.110 on Fri, 09 Dec 2016 05:44:01 UTC All use subject to http://about.jstor.org/terms VOL. 107, NO. 2, APRIL 1988

Evaluation of the developmental toxicity of nicotine and cotinine with frog embryo teratogenesis assay: Xenopus.

The teratogenic potential of nicotine and a primary metabolite, cotinine, was examined with FETAX (Frog Embryo Teratogenesis Assay: Xenopus). Early embryos of Xenopus laevis were exposed for 96 hr to nicotine or cotinine in two separate static renewal tests of each compound without addition of the metabolic activation system (MAS). Two static renewal tests of nicotine with the MAS were also conducted. Addition of the MAS to nicotine reduced the LC50 from an average of 136 to 20 mg/L. However, the EC50 (malformation) was increased from 0.4 to 5.8 mg/L upon activation. The LC50 and EC50 values for cotinine averaged 4,340 and 720 mg/L, respectively. Based on mortality/malformation index values, growth end points, and the types and severity of the induced malformations, nicotine and cotinine scored as potential teratogens. Metabolism of nicotine to more polar metabolites increased the nicotine concentration required to induce terata. The results are indicative of the versatility of FETAX in developmental toxicity testing.

Development of a metabolic activation system for the frog embryo teratogenesis assay: Xenopus (FETAX).

FETAX (frog embryo teratogenesis assay: Xenopus) is a 96-hr teratogenesis screening assay using embryos of the South African clawed frog, Xenopus laevis. Since Xenopus embryos have limited xenobiotic metabolism through 96 hr of development, we have developed an in vitro metabolic activation system employing Aroclor 1254-induced rat liver microsomes. By adding an exogenous source of mixed functional oxidase (MFO) activity, we may more accurately assess the teratogenic risk of proteratogenic compounds. Xenopus embryos were cocultured with varying concentrations of cyclophosphamide (CP), Aroclor 1254-induced microsomal protein, an NADPH-generating system, and antibiotics in a static renewal fashion for 96 hr. Residual Aroclor 1254 remaining in the microsomes was successfully reduced during purification to levels that had no significant effect on embryo survival and development. The results of three definitive dose-response tests performed with CP revealed that activation reduced the 96 hr LC50 from 8.0 to 1.4 mg/ml (5.7-fold). The 96-hr EC50 (malformation) was reduced from 6.2 to 0.4 mg/ml (15.5-fold). Activation also increased the types and severity of malformation and reduced embryonic growth. Aroclor 1254-induced rat liver microsomes may be used as an acceptable in vitro metabolic activation system for FETAX.