James R. Rayburn

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 alpha-chaconine is teratogenic and more embryotoxic than alpha-solanine, in terms of the median lethal concentration (LC50) after 96 hr of exposure, the concentration inducing gross terata in 50% of the surviving frog embryos (96-hr EC50, 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, solanidine and solasodine were less toxic than the glycosides alpha-chaconine and alpha-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.

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.

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.

Evaluation of Acetaminophen-Induced Developmental Toxicity Using Fetax

Potential mechanisms of acetaminophen-induced developmental toxicity were evaluated using FETAX (Frog Embryo Teratogenesis Assay-Xenopus). Early Xenopus laevis embryos were exposed to acetaminophen for 96-h in two definitive concentrations-response assays with and without an exogenous metabolic activation system (MAS). Two static renewal tests of acetaminophen and the MAS treated with carbon monoxide, cimetidine, ellipticine, diethyl maleate, and supplemented with glutathione were also performed. Addition of the MAS decreased the 96-h LC50 and EC50 (malformation) values of unactivated acetaminophen 3.9-fold and 7.1-fold, respectively. Addition of the carbon monoxide- and ellipticine-inhibited MAS, as well as the glutathione-supplemented MAS decreased the developmental toxicity of activated acetaminophen to levels near that of the unactivated parent compound. Cimetidine-inhibited MAS also reduced the developmental toxicity of acetaminophen, but not to the extent observed with the carbon monoxide- and ellipticine-inhibited, or glutathione-supplemented MAS. Addition of the diethyl maleate-treated MAS substantially increased the developmental toxicity of acetaminophen. Results indicate that a highly reactive intermediate formed as the result of MFO-mediated metabolism (possibly P-448) significantly increased the developmental toxicity of acetaminophen. Glutathione was also found to play a major role in intermediate detoxification in vitro.

Phase III Interlaboratory Study of Fetax, Part 2: Interlaboratory Validation of an Exogenous Metabolic Activation System for Frog Embryo Teratogenesis Assay-Xenopus (Fetax)

Interlaboratory validation of an exogenous metabolic activation system (MAS) developed for the alternative, short-term developmental toxicity bioassay, Frog Embryo Teratogenesis Assay-Xenopus (FETAX) was performed with cyclophosphamide and caffeine. Seven study groups within six separate laboratories participated in the study in which three definitive concentration-response experiments were performed with and without the MAS in a side-by-side format for each chemical. Since both chemicals had been previously tested in FETAX, the test concentrations were provided to each laboratory prior to testing. Interlaboratory coefficient of variation (CV) values for unactivated cyclophosphamide (no MAS) were 15%, 15%, 29%, and 25% for the 96-hr LC50, 96-hr EC50 (malformation), Minimum Concentration to Inhibit Growth (MCIG), and Teratogenic Index (TI) values, respectively. Addition of the MAS increased the CV values of each endpoint at least 3.9-fold. Interlaboratory CV values for unactivated caffeine were 31%, 18%, 31%, and 46% for the 96-hr LC50, 96-hr EC50 (malformation), MCIG, and TI values, respectively. Addition of the MAS decreased the CV values of each respective endpoint by at least 1.6-fold. Results indicated that bioactivated toxicants may be prone to greater variability in response amongst laboratories than compounds, which are detoxified. Even though more variability was noted with activated cyclophosphamide, results were within interlaboratory variation expected for other aquatic-based bioassays. Thus, results from these studies warrant the continued use and further refinement of FETAX for alternative developmental toxicity assessment.

FETAX Interlaboratory Validation Study: Phase III–Part 1 Testing

The Frog Embryo Teratogenesis Assay—Xenopus(FETAX) is a 96‐h whole embryo developmental toxicity screening assay that can be used in ecotoxicology and in detecting mammalian developmental toxicants when an in vitrometabolic activation system is employed. A standardized American Society for Testing and Materials (ASTM) guide for the conduct of FETAX has been published, along with a companion atlas that aids in embryo staging and identifying malformations. As part of the ASTM process, a three‐phase interlaboratory validation study was undertaken to evaluate the repeatability and reliability of FETAX. Seven different participants collaborated in the study. In Phase I, FETAX proved to be more repeatable and reliable than many bioassays. However, some excessive variation was observed in a few laboratories. An initial lack of assay experience by some technicians caused variation. Phase II showed far less intra‐ and interlaboratory variability than Phase I. Non‐teratogens showed the most consistent results, while more variability was observed for the two teratogens tested. Interlaboratory coefficient of variation values for all endpoints ranged from 7.3 to 54.7. Phase III‐Part 1, using coded samples and test concentration ranges selected by each laboratory, showed results similar to Phase I. Analysis of the causes of variation suggested that some technicians judged some embryos to be malformed while others consistently judged similar embryos as normal. Concentration ranges tested by some of the laboratories varied greatly and a new protocol for selecting concentrations for initial testing was written to reduce variation from this source. Testing to date suggests that FETAX is as repeatable and reliable as other standard bioassays.

L-cysteine, N-acetyl-L-cysteine, and glutathione protect Xenopus laevis embryos against acrylamide-induced malformations and mortality in the frog embryo teratogenesis assay.

Dietary acrylamide is largely derived from heat-induced reactions between the amino group of the free amino acid asparagine and carbonyl groups of glucose and fructose during heat processing (baking, frying) of plant-derived foods such as potato fries and cereals. After consumption, acrylamide is absorbed into the circulation and is then distributed to various organs, where it can react with DNA, neurons, hemoglobin, and essential enzymes. In the present study, we explored the potential of L-cysteine (CySH), N-acetyl-L-cysteine (NAC), reduced glutathione (GSH), and the amino acid glycine (Gly) to protect frog embryos against acrylamide-induced developmental toxicity in the frog embryo teratogenesis assay - Xenopus (FETAX). To test the antiteratogenic potential, based on concentration-response study ranging from 0.07 to 4.22 mM acrylamide in FETAX solution (pH 8.1), we selected concentrations of acrylamide that induced 100% malformations and mortality. At the end of 96 h, we counted survivors and malformed embryos and measured embryo length. The data show that CySH, NAC, and GSH protected the embryos against acrylamide induced malformations and mortality to different degrees. CySH and GSH protected the embryos against both malformations and mortality, whereas NAC protected only against mortality. Gly had no protective effect. Possible mechanisms of the protective effects and the dietary significance of the results of this and related studies for food safety and human health are discussed.

Synergism and Antagonism Induced by Three Carrier Solvents with t-Retinoic Acid and 6-Aminonicotinamide Using FETAX

The large number of water-insoluble chemicals requiring toxicity testing necessitates the development, validation and use of chemical cosolvents. Carrier solvents (cosolvents), such as dimethylsulfoxide (DMSO), acetone, and triethylene glycol (TG), are commonly used to solubilize hydrophobic compounds (Yalkowsky 1981). However, the use of solvents with in vitro bioassays may alter the developmental toxicity of test materials. Solvents interact with other compounds to change rates of reactions, membrane potentials, mutagenic activity, and many other cell processes (Coetzee and Ritchie 1969; Nemethy 1986; Demey et al. 1983; Gichner and Veleminsky 1987). For this reason solvent-compound interaction studies were performed to determine if the developmental toxicity of test materials was altered.

Potential protective effect of L-cysteine against the toxicity of acrylamide and furan in exposed Xenopus laevis embryos: an interaction study.

The embryo toxicities of two food-processing-induced toxic compounds, acrylamide and furan, with and without added L-cysteine were examined individually and in mixtures using the frog embryo teratogenesis assay-Xenopus (FETAX). The following measures of developmental toxicity were used: (a) 96 h LC50, the median concentration causing 50% embryo lethality; (b) 96 h EC50, the median concentration causing 50% malformations of the surviving embryos; and (c) teratogenic index (96 h LC50/96 h EC50), an estimate of teratogenic risk. Calculations of toxic units (TU) were used to assess possible antagonism, synergism, or response addition of several mixtures. The evaluated compounds demonstrated counterintuitive effects. Furan had lower than expected toxicity in Xenopus embryos and, unlike acrylamide, does not seem to be teratogenic. However, the short duration of the tests may not show the full effects of furan if it is truly primarily genotoxic and carcinogenic. L-Cysteine showed unexpected properties in the delay of hatching of the embryos. The results from the interaction studies between combination of two or three components (acrylamide plus L-cysteine; furan plus L-cysteine; acrylamide plus furan; acrylamide plus furan and L-cysteine) show that furan and acrylamide seem to have less than response addition at 1:1 toxic unit ratio in lethality. Acrylamide and L-cysteine show severe antagonism even at low 19 acrylamide/1 L-cysteine TU ratios. Data from the mixture of acrylamide, furan, and L-cysteine show a slight antagonism, less than would have been expected from binary mixture exposures. Bioalkylation mechanisms and their prevention are discussed. There is a need to study the toxicological properties of mixtures of acrylamide and furan concurrently formed in heat-processed food.