Evaluating the Teratogenicity of Ritodrine and Nifedipine using a Frog Embryo Teratogenesis assay (FETAX)

The Frog Embryo Teratogenesis Assay—Xenopus (FETAX) was used to assess the teratogenic potential of two tocolytics. Embryos of the South African clawed frog, Xenopus laevis, were exposed to ritodrine or nifedipine. Exposure media were changed and monitored at 24-hour intervals. The 96-hour LC₅₀ (Lethal concentration), the 96-hour EC₅₀ (Malformation), and the No Observable Adverse Effect Concentrations (NOAEC) and the Lowest Observable Adverse Effect Concentration (LOAEC) for mortality, malformation and length were determined for each drug. Nifedipine was determined to be the more toxic and teratogenic than ritodrine, with a LC₅₀ of 0.606?µg/L, an EC₅₀ of 0.006?µg/L, and a teratogenicity Index (TI) value (LC₅₀/EC₅₀) of 101. On the other hand, the LC₅₀ of ritodrine was 28.571?mg/L. In addition; the LC₅₀, EC₅₀ and TI values for nifedipine in the 5?mg/L ritodrine?+?nifedipine combination group were determined as 1.050?µg/L, 0.868?µg/L and 1.5 respectively. For ritodrine, the NOAEC and LOAEC values were determined as 2?mg/L and 4?mg/L, respectively. For the nifedipine and the ritodrine?+?nifedipine groups; while the LOAEC values of these groups were 0.0001?µg/L and 0.1?µg/L, respectively. NOAEC value couldn’t be determined. Our results demonstrated that nifedipine administration was associated with higher levels of teratogenic and toxic effects. However, the ritodrine?+?nifedipine combination form reduced the toxic and teratogenic effects of nifedipine on Xenopus embryos. Further studies should be conducted in order to investigate the optimal combination concentrations of these substances for the treatment of preterm labor.     

Evaluation of the Developmental Toxicities of Ethanol,Acetaldehyde, and Thioacetamide Using FETAX

Abstract

Potential mechanisms of the developmental toxicities of ethanol, acetaldehyde, and thioacetamide were evaluated using frog embryo teratogenesis assay-Xenopus (FETAX). Early X. laevis embryos were exposed to ethanol and thioacetamide in two separate definitive concentration-response tests with and without differentially induced exogenous metabolic activation systems (MAS) or selectively inhibited MAS. Two concentration-response tests were also performed with ethanol metabolites, acetaldehyde and acetic acid. The MAS was treated with 3,4-amino-1,2,4-triazole to modulate CYP2E1 activity, and heat to inactivate flavin containing monooxygenases (FMO) activity. Results from these studies suggested that thioacetamide may be bioactivated by both CYP2E1 and the FMO systems. Ethanol also appeared to be bioactivated by CYP2E1. Acetaldehyde was markedly more potent as a developmental toxicant than ethanol or acetic acid. Binary joint mixture studies conducted with ethanol and acetaldehyde indicated that the parent compound and metabolite acetaldehyde acted in a response additive manner. These results warrant the continued use of FETAX as a means of evaluating mechanisms of developmental toxicity in vitro.     

Enhancing the predictive validity of Frog Embryo Teratogenesis Assay--Xenopus (FETAX)

Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a 4-day, alternative developmental toxicity assay designed to pre-screen chemicals and environmental mixtures. An approach used in the scoring of FETAX results, which focuses on the determination of characteristic abnormalities induced by a given test material, was used to evaluate results from previous validation efforts. Characteristic abnormalities are induced specifically by exposure to a given test material and are determined by the relationship between concentration and the response induced and the change in severity of response. Use of this approach eliminates non-specific effects that alter numerical endpoints, reduces intralaboratory variability, reduces the number of equivocal test results (gray area), helps to determine specific syndromes associated with teratogen exposure and, in some instances, increases the predictability of FETAX. In an effort to evaluate this approach, a 12-compound validation study that produced a relatively high degree of equivocal FETAX results was re-evaluated using the characteristic malformation criteria. Data were evaluated for repeatability, predictability and variability. Results from this study indicated that this scoring approach increased repeatability, test endpoint precision and predictability.     

Comparative sensitivity of Xenopus tropicalis and Xenopus laevis as test species for the FETAX model

The use of Xenopus tropicalis as an alternative test species for the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) model was evaluated. Five test substances with varying developmental toxicity potential were evaluated using the traditional FETAX (X. laevis) and a modified assay to accommodate the use of X. tropicalis. Two separate definitive concentration-response tests were performed with ethanol, semicarbazide, copper, 6-aminonicotinamide (6-AN) and atrazine. In order to evaluate the impact of culture temperature on species sensitivity, tests with X. tropicalis were performed concurrently at 27 degrees C (optimum temperature) and 23 degrees C (traditional FETAX temperature). Tests with X. laevis were performed only at 23 degrees C (optimal for X. laevis). Regardless of culture temperature, tests with X. laevis and X. tropicalis indicated that each of the compounds possessed teratogenic potential: semicarbazide>6-AN>atrazine approximately copper>ethanol. Results from these studies indicated that these two species responded similarly to the test compounds. Xenopus tropicalis was somewhat less sensitive to 6-AN, semicarbizide and atrazine when tested at 27 degrees C than at 23 degrees C. Ethanol, copper and atrazine were reasonably equipotent in X. tropicalis and X. laevis in terms of teratogenic response (EC50 for malformation), whereas 6-AN and semicarbizide were less potent in X. tropicalis than in X. laevis. No substantial differences (order of magnitude) in potency were observed between X. laevis and X. tropicalis with any of the test materials evaluated. Malformation syndromes induced in both species were similar in X. tropicalis and X. laevis. These results suggested that X. tropicalis could be used effectively as a test organism for the FETAX model.     

An assay to determine the sensitive window of embryos to chemical exposure using Xenopus tropicalis

The frog embryo teratogenesis assay‐Xenopus (FETAX) is an established method to evaluate the developmental toxicity of chemicals. In FETAX, a 48 h continuous exposure is usually conducted when the X. tropicalis embryo is used as the test model. In the present study, we exposed X. tropicalis embryos to nine known teratogens for four separate 12‐h periods. The embryos showed great variations in response to nine tested compounds during different exposure periods. Based on the value of the score of malformations, the most sensitive 12 h exposure periods of embryos were significantly distinguished for all the compounds with the exception of NiCl₂. The embryos were the most sensitive to retinols (e.g. all‐trans‐retinoic acid and 9‐cis‐retinoic acid) during 0–12 h and to metal compounds (e.g. triphenlytin and CdCl₂) during a 24 to 36 h exposure period. In the further 3 h exposure experiment, the most sensitive period could only be determined for one of three tested compounds. Based on the present results, we proposed an assay to determine a 12 h sensitive window of embryos to chemical exposure using Xenopus tropicalis. Copyright © 2015 John Wiley & Sons, Ltd.     

Toxicity of complex cyanobacterial samples and their fractions in Xenopus laevis embryos and the role of microcystins

This work evaluated the effects of various cyanobacterial fractions in Frog Embryo Teratogenesis Assay Xenopus (FETAX) with African clawed frog embryos. Fractions were prepared from five biomasses with different dominant genera (Microcystis, Aphanizomenon, Anabaena, Planktothrix) and different microcystin content. Effects of following fractions were investigated: (I) homogenate of complex cyanobacterial biomass, (II) cell debris (pellet) after centrifugation of complex biomass, (III) supernatant after centrifugation of complex biomass (=crude aqueous extract), (IV) permeate after passing of crude extract through C-18 column (fraction devoid of microcystins), and (V) eluate from C-18 column (containing microcystins, if present). Besides classical parameters evaluated in 96 h FETAX (mortality, growth inhibition, malformations), we have also assessed the effects on biochemical markers of oxidative stress and detoxification (glutathione pool, GSH; activity of glutathione peroxidase, GPx; glutathione reductase, GR; activity of glutathione-S-transferase, GST). Complex biomass (I) and aqueous extract (III) were generally the most toxic fractions in terms of mortality and growth inhibition, whereas eluates containing microcystins (V) were generally less toxic. On the other hand, the same fraction (eluates) induced significant malformations in low concentrations but the effects were not related to the content of microcystins. Biomarkers were affected in variable manner but no significant effect or clear relation to microcystin content was observed. Our data support the hypothesis that microcystins are not the only or major toxic compounds in the complex cyanobacterial samples (at least for some species) and that more attention should be paid to other components of complex cyanobacterial biomass including non-specific parameters such as oxygen content or toxic ammonia released during bacterial decay of organic material.     

Determination of DSP toxins: comparative study of HPLC and bioassay to reduce the observation time of the mouse bioassay

 The progressive increase of DSP toxic episodes in shellfish in the last few years has generated a series of criticisms centered on the suitability of the mouse bioassay as a reference method to regulate the harvest of mussels from the growth area. The observation time in injected mice is currently fixed in 12 h by the actual Spanish rules. The revision of this time period and the lack of a established DSP toxin threshold which permits the commercialization of mussels contaminated under certain levels, are some of the actual demands from the industry. In this study, the results obtained in a comparative study of DSP toxic mussels are shown using the HPLC method and the mouse bioassay. Based on these results, we consider feasible the reduction to 5 h of the observation times of the mouse bioassay currently established in the actual legislation, as well as the establishment of a DSP-toxin threshold of 2 μg okadaic acid/g hepatopancreas, which regulates the possibility of harvesting and commercialization of contaminated mussels. Received: 10 October 1995/Accepted: 7 December 1995     

Effects of specific dosages of magnesium and zinc on the teratogenicity of cadmium, nickel, and cobalt in Xenopus embryos, as assessed by the FETAX test

The objective of this study was to determine if exposure to divalent cations, Cd(2+), Ni(2+), and Co(2+) would lead to malformations in Xenopus laevis embryos, and whether addition of Mg(2+) and Zn(2+); separately and in combination, would reduce their toxicity and teratogenicity on the embryos of Xenopus laevis as assessed by 96-h FETAX tests. Results indicate that exposure to Cd(2+), Ni(2+) or Co(2+) lead to an increase in toxicity and teratogenicity in embryos, whereas Mg(2+), Zn(2+), or a combination of them reduced the toxic and teratogenic effects of these divalent cations. Modulation of Cd(2+), Ni(2+) or Co(2+) toxicity and teratogenicity by Mg(2+) and Zn(2+), varied with the metal. Zn(2+) was observed to be a better suppressor of Co(2+) toxicity and teratogenicity than Mg(2+). In contrast, Ni(2+), and Cd(2+) teratogenicity was reduced more prominently by Mg(2+). On the other hand, combination of Mg(2+) and Zn(2+) showed potentialization effect on all divalent cation toxicity and teratogenicity. We concluded that Mg(2+) and Zn(2+) reduced the toxicity and teratogenicity of Cd(2+), Ni(2+), Co(2+).     

用生物检定法研究力达霉素在小鼠和犬的药代动力学

目的用生物检定法测定力达霉素活性浓度和研究其药代动力学。方法体外细胞毒检测法测定血清力达霉素浓度。结果方法学确证基本符合药代动力学研究要求。小鼠iv力达霉素100,50和10 μg·kg^-1后浓度曲线符合二房室模型，α和β相t_1/2分别是0.77~1.8 min和5.6~7.2 min。AUC分别是2 851.3，887.8和166.4 μg·min·L^-1，随剂量非线性增加。AUC增加和抑瘤疗效增长趋势相似。犬iv力达霉素12 μg·kg^-1后浓度低并迅速下降，AUC仅16 μg·min·L^-1，比小鼠浓度低和消除快。首次给药后15 d进行第2次给药，第2次给药浓度低于首次。结论用生物检定法成功测定血清力达霉素浓度和研究其药代动力学。力达霉素药代动力学存在种属、单次及多次给药差别。     

The use of Lepidium sativum in a plant bioassay system for the detection of microcystin-LR.

Toxin-producing cyanobacteria pose a worldwide health threat to humans and animals due to their increasing presence in both drinking and recreational waters. Detection of microcystins in water generally relies on specialised equipment and a delay of several days for transport and analysis. Little work has, however, been done on establishing a simple, cost-effective and sensitive plant bioassay for the detection of microcystin-LR (MCLR) in water at the WHO Tolerable Daily Intake guideline level of 1 microg/l. We investigated the effect of a MCLR extract at 1 and 10 microg/l on the growth of Lepidium sativum over 6 days. Exposure to 10 microg/l MCLR resulted in a significant decrease in root and leaf lengths and fresh weights of seedlings when compared to the controls. These results were consistent with seedlings exposed to pure MCLR at 10 microg/l. Seedlings exposed to 1 microg/l MCLR showed a significant decrease in root development from day 2 to day 6. Glutathione S-transferase and glutathione peroxidase activities were also significantly raised in plants from days 5 and 4, respectively, at both toxin levels investigated.     

Development and validation of a quantitative cell-based bioassay for comparing the pharmacokinetic profiles of two recombinant erythropoietic proteins in serum

An in vitro cell-based bioassay was developed and validated to assess the pharmacokinetic profiles of two novel therapeutic recombinant proteins (EP1 and EP2) with erythropoiesis stimulating properties in Sprague-Dawley rats. While immunoassays are the standard choice for evaluating the pharmacokinetic parameters of drugs, no immunoassay was available for EP2, necessitating the need for a quantitative bioassay capable of measuring both EP1 and EP2 separately so that appropriate comparisons could be made. The bioassay described here utilizes a sub clone of the murine 32D cell line transfected with the gene encoding for the human erythopoietin (HuEPO) receptor. Erythropoietin (EPO), EP1 and EP2 exert their proliferative effect on the cell line by signaling through the HuEPO receptor. The proliferation induced by the erythropoietic proteins was measured by [methyl-(3)H]thymidine incorporation into the cellular DNA. The assay was conducted in 96-well microtiter plates and had relatively high throughput. The Guidelines of the International Conference on Harmonization (ICH) were followed for the validation of the different assay parameters including robustness, linearity, accuracy, precision, limit of quantitation (LOQ) and specificity. The robustness of the bioassay is demonstrated by the lack of an effect of age of the 32D cell culture on the performance of the EP2 bioassay. The bioassay demonstrated good linearity, yielding a coefficient of determination of 0.99 or higher for both EP1 and EP2. The assay showed reproducible dose-response curves for EP1 in the range of 0.039-2.5 ng/mL and for EP2 in the range of 0.125-8 ng/mL. The accuracy estimates ranged between 98% and 108% for EP1 and between 90% and 110% for EP2 in the reproducible range mentioned above. Intermediate precision (within-plate R.S.D.) in the same range was within 26% and 17% for the EP1 and EP2 bioassays, respectively. The validated bioassays for EP1 and EP2 were utilized to quantitatively analyze serum samples from a pharmacokinetic study conducted to compare the profiles of the two compounds in Sprague-Dawley rats.     

Repeatability and reproducibility of the luminescent bacteria bioassay

This paper presents the statistical analysis of the results of an inter-laboratory study for the luminescent bacteria toxicity bioassay. It also contains a discussion on the statistical methods for the presentation and refinement of the evaluation of the precision of an assay method (including intra- and inter-laboratory variability), with special emphasis on the rejection of outliers, and the use of standardized parameters, like the repeatability and reproducibility values.     

Prediction and assessment of mixture toxicity of compounds in antifouling paints using the sea-urchin embryo-larval bioassay

The ecotoxicological assessment of alternative "booster" biocides is urgently needed in order to develop environmentally acceptable antifouling paints. However, research has focused mainly on single compounds, and there is still a lack of data on their mixture toxicity. The present study investigated the single and mixture toxicity of three of the most widely used antifouling biocides: zinc pyrithione, chlorothalonil and Sea-Nine, using the sea-urchin (Paracentrotus lividus) embryo-larval bioassay. Also, the predictive ability of the concentration addition (CA) and independent action (IA) concepts for antifouling mixtures was evaluated. Both concepts failed to accurately predict the toxicity of the antifouling mixtures, with the exception of the zinc pyrithione and Sea-Nine mixture, which was accurately predicted by the IA concept, suggesting a dissimilar mode of action of those substances. In general, CA predicted consistently higher toxicity than IA; however, CA overestimated the toxicity of the studied mixtures by a factor of only 1.6, representing a reasonable worst-case approach to be used in the predictive hazard assessment of antifouling mixtures. Finally, the present study demonstrates that the risk of antifouling mixtures for the early developmental stages of sea urchin is higher than the risk of each single substance, and therefore, the inclusion of mixture considerations in the development of water quality criteria for antifouling compounds is strongly recommended.     

Assessment of environmental risks to groundwater ecosystems related to use of veterinary medicinal products

The current EU guidelines for the environmental risk assessment of veterinary pharmaceutical products (VMPs) in groundwater (GW) suggest an approach based on the comparison between the calculated concentration in GW (PEC_gw) and a threshold concentration of 0.1 μg/L. The latter is the upper limit of the concentration for pesticides in groundwater in the EU. If the calculated PEC_gw does not exceed the threshold, then the risk is considered acceptable. It is assumed that the concentration of 0.1 μg/L is by default safe for both humans and exposed GW organisms. On this basis, it is not clear whether the GW is recognized as an ecosystem or as a source of drinking water. Largely unrecognized biodiversity in GW is worthy of protection through the adoption of a more scientifically sound risk analysis, which should be based on the consideration of ecological criteria.Based on the evidence of their vulnerability, we propose that risk assessments of GW ecosystems should be a compulsory part of the overall risk assessment of VMPs (as well as pesticides, biocides and feed additives). Furthermore, we suggest the use of a risk quotient approach based on the PEC/PNEC ratio in which the PNEC is calculated including an additional safety factor of 10 to the calculated PNEC for surface water.     

Testing the toxicity of influents to activated sludge plants with the Vibrio fischeri bioassay utilising a sludge matrix

To protect the bioceonosis within activated sludge, a method of predicting the toxic effect of influents to the biological treatment stage of waste water treatment plants, based on DIN method 38412 L 34, has been developed. A population of the luminescent marine bacterium Vibrio fischeri was incorporated into a sludge testing matrix derived from a model laboratory and real activated sludge plants. The sludge was challenged with different concentrations of pure toxicants and complex aqueous samples, and light output by V. fischeri monitored. The results were compared to toxicant testing in the absence of sludge (standard test). The modified method was found to be less sensitive for some toxicants tested than the standard DIN and other bioluminescent tests, but considered more realistic as it provides buffering and takes into account sorption which can affect the sensitivity of the test towards some compounds. The method is comparable in terms of ease of use, speed, reproducibility and cost effectiveness to standard V. fischeri luminescence methods.     

Development and modification of a recombinant cell bioassay to directly detect halogenated and polycyclic aromatic hydrocarbons in serum.

Polycyclic and halogenated aromatic hydrocarbons (PAHs/HAHs) are a diverse group of widespread and persistent environmental contaminants that can cause a variety of detrimental effects in vertebrates. As most available methods to detect these contaminants are expensive, labor and time intensive, and require large amounts of tissue for extraction and analysis, several rapid mechanistically based bioassay systems have been developed to detect these chemicals. Here we describe application and optimization of a recently developed recombinant mouse cell bioassay system that responds to both PAHs and HAHs with the rapid induction of firefly luciferase for the detection of these chemicals in whole serum samples. This chemically activated luciferase expression (CALUX) bioassay has been modified to allow rapid (4-h) and direct analysis of small volumes (25-50 microl) of whole serum in a 96-well microtiter plate format without the need for solvent extraction. This bioassay can detect as little as 10 parts per trillion of the most potent HAH, 2,3,7,8-TCDD, and is also sensitive to other HAHs and PAHs. The use of simple procedures corrects for interplate and intraplate variability and the Ah receptor dependence of the induction response is accounted for by use of the antagonist 4-amino-3-methoxyflavone.     

Toxicity of some bis Mannich bases and corresponding piperidinols in the brine shrimp (Artemia salina) bioassay

Some acetophenone-derived bis Mannich bases were synthesized: bis[beta-benzoylethyl]ethylamine hydrochloride (IIa), bis[beta-(p-methylbenzoyl)ethyl]ethylamine hydrochloride (IIb), bis[beta-(p-chlorobenzoyl)ethyl]ethy- lamine hydrochloride (IId), bis[(2-thienylcarbonyl)ethyl]ethylamine hydrochloride (IIe); some corresponding piperidinol derivatives: 3-benzoyl-1-ethyl-4-phenyl-4-piperidinol hydrochloride (IIIa), 1-ethyl-3-(p-methyl- benzoyl)-4-(p-methylphenyl)-4-piperidinol hydrochloride (IIIb), 1-ethyl-3-(p-methoxybenzoyl)-4-(p-methoxy- phenyl)-4-piperidinol hydrochloride (IIIc), 1-ethyl-3-(p-chlorobenzoyl)-4-(p-chlorophenyl)-4-piperidinol hydrochloride (IIId), 1-ethyl-4-(2-thienyl)-3-(2-thienylcarbonyl)-4-piperidinol hydrochloride (IIIe); and some representative quaternary piperidinols: 3-benzoyl-1-ethyl-4-hydroxy-1-methyl-4-phenylpiperidinium iodide (IIIf), 1-ethyl-4-hydroxy-1-methyl-3-(p-methylbenzoyl)-4-(p-methylphenyl)piperidinium iodide (IIIg). Toxicity was tested by the brine shrimp bioassay as an intermediate test before further in vivo animal experiments. Piperidine derivatives were found to be more potent than bis Mannich bases. Quaternary piperidine derivatives IIIf and IIIg and also non-quaternary piperidine derivatives IIIb, IIIe, IIIc and IIId were more toxic than 5-fluorouracil in brine shrimp bioassay. Except for IIe, bis Mannich bases were not effective. Quaternization and conversion of bis Mannich bases to corresponding piperidines improved the toxicity. The lipid solubility of the compounds may not affect the toxicity. From these findings the quaternary piperidine derivatives IIIf and IIIg could be used in further drug development and also for in vivo experiments.     

A behavioural bioassay for impaired sea-water quality using the plantigrades of the common mussel Mytilus edulis L.: the response to copper

A bioassay has been developed, using the crawling and attachment behaviour of plantigrades of the common mussel, Mytilus edulis, for copper-impaired sea water. Potentiometric stripping analysis has been used to quantify the total and electrochemically labile species of copper. The threshold of sensitivity for the bioresponse lies between 13 ppb total, 8 ppb labile and 25 ppb total, 15 ppb labile copper, for a bioassay of 1 h duration. There is an example of hormesis for the bioresponse of the number of plantigrades that crawl for levels of copper at and below 13 ppb total, 8 ppb labile metal. The effects of light and gravity as directional cues and the effect of animal size and density in the bioassay have also been considered.