Assessing the impact of antiviral drugs commonly utilized during the COVID-19 pandemic on the embryonic development of Xenopus laevis.

The antiviral drugs favipiravir and oseltamivir are widely used to treat viral infections, including coronavirus 2019 (COVID-19), and their levels are expected to increase in the aquatic environment. In this study, the potential toxic and teratogenic effects of these drugs were evaluated using the frog embryo teratogenesis assay Xenopus (FETAX). In addition, glutathione S-transferase (GST), glutathione reductase (GR), catalase, carboxylesterase (CaE), and acetylcholinesterase (AChE) enzyme activities and malondialdehyde levels were measured as biochemical markers in embryos and tadpoles for comparative assessment of the sublethal effects of the test compounds. Prior to embryo exposure, drug concentrations in the exposure medium were measured with high-performance liquid chromatography. The 96-h median lethal concentration (LC50) was 137.9 and 32.3 mg/L for favipiravir and oseltamivir, respectively. The teratogenic index for favipiravir was 4.67. Both favipiravir and oseltamivir inhibited GR, CaE, and AChE activities in embryos, while favipiravir increased the GST and CaE activities in tadpoles. In conclusion, favipiravir, for which teratogenicity data are available in mammalian test organisms and human teratogenicity is controversial, inhibited Xenopus laevis embryo development and was teratogenic. In addition, sublethal concentrations of both drugs altered the biochemical responses in embryos and tadpoles, with differences between the developmental stages.

Toxicity of commercial and pure forms of three nonsteroidal anti-inflammatory drugs in Xenopus laevis embryos before and after ozonation.

In this study, the toxic and teratogenic effects of three commercial drugs and their active ingredients on Xenopus laevis embryos before and after ozonation were evaluated using the Frog Embryos Teratogenesis Assay-Xenopus (FETAX). First, the median lethal concentration (LC50) and, if data were available, the median effective concentration, teratogenic index and minimum growth inhibitory concentration were determined for each drug substance without ozonation. Then, the active substance amounts of three selected nominal concentrations (LC50/2, LC50, and LC50×2) of each test substance before ozonation were measured by HPLC analysis and the toxicity of these substances was evaluated after 2, 3, 4, and 5 h of ozonation. In addition, degradation products that may occur during ozonation were evaluated by LC-MS analysis. The 96-h LC50s of Dolphin-diflunisal, Dichloron-diclofenac sodium, and Apranax-naproxen drug-active substance pairs were determined to be 22.3 and 11.1, 25.7 and 18.7, and 47.8 mg active substance/L and 45.3 mg/L, respectively. According to the FETAX test results, the Dolphin-diflunisal drug-active ingredient pair did not cause growth retardation in exposed embryos. Dichloron-diclofenac sodium and Apranax-naproxen drug-active ingredient pairs were both teratogenic and growth inhibitory. In the second stage of the study, in which the effectiveness of ozonation in eliminating the toxic effects of drugs is evaluated, it is seen that ozonation is partially successful in eliminating the toxic effects of Dolphin-diflunisal and Dichloron-diclofenac sodium pairs, but insufficient for eliminating the effects of the Apranax-naproxen pair.

Biochemical Studies to Understand Teratogenicity and Lethality Outcomes in Modified-FETAX.

The frog embryo teratogenesis assay-Xenopus (FETAX) is a standardized test used to assess the toxic and teratogenic effects of xenobiotics. With this test, toxic and/or teratogenic concentrations of xenobiotic substances can be determined using morphological parameters such as lethality, length, and malformations in stage 8-11 Xenopus laevis embryos after 96 h exposure. These parameters enable the determination of the median lethal and effective concentrations (LC50 and EC50), minimum concentration to inhibit growth (MCIG), and teratogenic index of the tested chemical to reveal the short-term effects of relatively high concentrations. On the other hand, although FETAX provides quantitative and qualitative data on teratogenicity and toxicity, the biochemical and molecular mechanisms of these effects cannot be explained. Recent studies have tried to elucidate the mechanisms causing malformations and to explain the underlying causes of toxicity and teratogenicity by biochemical marker analysis. This chapter describes methods to analyze modified-FETAX and some detoxification and oxidative stress-related biomarkers during the early embryonic development of X. laevis.

Developmental, toxicological effects and recovery patterns in Xenopus laevis after exposure to penconazole-based fungicide during the metamorphosis process.

Fungicides are a group of chemicals causing pollution of freshwater ecosystems due to their widespread use in agriculture. However, their endocrine disrupting effects are less studied than herbicides and insecticides. The aim of this study was to evaluate the developmental and toxicological effects and recovery patterns of penconazole-based fungicide (PBF) during Xenopus laevis metamorphosis. For this purpose, firstly, the 96 h median lethal (LC50) and effective (EC50) concentrations and minimum concentration to inhibit growth (MCIG) values of PBF were estimated for X. laevis as 4.97, 3.55 and 2.31 mg/L respectively, using Frog Embryo Teratogenesis Assay-Xenopus (FETAX) on Nieuwkoop-Faber (NF) stage 8 embryos. FETAX results showed PBF formulation was slightly teratogenic with a 1.4 teratogenic index; most recorded malformations were gut, abdominal edema, and tail curvature. The Subacute Amphibian Metamorphosis Assay (AMA) was modified based on acute FETAX results, and used to evaluate toxic effects and recovery patterns of relatively low PBF concentrations on metamorphosis using morphological and biochemical markers. NF Stage 51 tadpoles were exposed to two separate groups of each concentration for seven days in the AMA. Secondly, tadpoles of one group of each concentration continued to be exposed to PBF for the next 7 and 14 days while the other group was kept in a pesticide-free environment (depuration/recovery). Various morphological and biochemical markers were measured homogenate samples of tadpoles from exposure and recovery groups. Continuous exposure to relatively low PBF concentrations caused oxidative stress, toxic, and endocrine disrupting effects in the AMA, leading us to conclude that it has negative effects on frog health and development during the recovery period when PBF exposure is terminated. The glutathione S-transferase, glutathione reductase, catalase, carboxylesterase, and acetylcholinesterase activities were higher than the control group transferred to pesticide-free media for 14 days after the 7 days exposure and indicate persistent PBF impact.

Developmental and lethal effects of glyphosate and a glyphosate-based product on Xenopus laevis embryos and tadpoles.

Effects of pure glyphosate and a glyphosate-based product were evaluated comparatively using two embryonic development stages of Xenopus laevis as model system. When pure glyphosate was applied in pH adjusted media, lethal or developmental effects were not observed at concentrations up to 500 mg L-1. The 96 h LC50 values for the commercial herbicide, in contrast, were 32.1 and 35.1 mg active ingredient L-1 for embryos and tadpoles, respectively. Since pure glyphosate has no effect on the selected biomarkers, it is thought that developmental toxic effects caused by glyphosate-based products are increased mainly due to formulation additives.

Photocatalytic degradation of azo dye using core@shell nano-TiO2 particles to reduce toxicity.

Clean and safe water is fundamental for human and environmental health. Traditional remediation of textile dye-polluted water with chemical, physical, and biological processes has many disadvantages. Due to this, nano-engineered materials are drawing more attention to this area. However, the widespread use of nano-particles for this purpose may lead to photocatalytic degradation of xenobiotics, while increasing the risk of nano-particle-induced ecotoxicity. Therefore, we comparatively evaluated the toxicity of novel synthesized core@shell TiO2 and SiO2 nano-particles to embryonic stages of Danio rerio and Xenopus laevis. The ability of photocatalytic destruction of the synthesized nano-particles was tested using toxic azo dye, disperse red 65, and the effects of reducing the toxicity were evaluated. The reflux process was used to synthesize catalysts in the study. The samples were characterized by scanning electron microscopy, X-ray fluorescence spectroscopy, X-ray diffractometry, BET surface area, and UV-vis-diffuse reflectance spectra. It was determined that the synthesized nano-particles had no significant toxic effect on D. rerio and X. laevis embryos. On the other hand, photocatalytic degradation of the dye significantly reduced lethal effects on embryonic stages of the organisms. Therefore, we suggest that specific nano-particles may be useful for water remediation to prevent human health and environmental impact. However, further risk assessment should be conducted for the ecotoxicological risks of nano-particles spilled in aquatic environments and the relationship of photocatalytic interaction with nano-particles and xenobiotics.