Are fish and standardized FETAX assays protective enough for amphibians? A case study on Xenopus laevis larvae assay with biologically active substances present in livestock wastes.

Biologically active substances could reach the aquatic compartment when livestock wastes are considered for recycling. Recently, the standardized FETAX assay has been questioned, and some researchers have considered that the risk assessment performed on fish could not be protective enough to cover amphibians. In the present study a Xenopus laevis acute assay was developed in order to compare the sensitivity of larvae relative to fish or FETAX assays; veterinary medicines (ivermectin, oxytetracycline, tetracycline, sulfamethoxazole, and trimethoprim) and essential metals (zinc, copper, manganese, and selenium) that may be found in livestock wastes were used for the larvae exposure. Lethal (LC(50)) and sublethal effects were estimated. Available data in both, fish and FETAX studies, were in general more protective than values found out in the current study, but not in all cases. Moreover, the presence of nonlethal effects, caused by ivermectin, zinc, and copper, suggested that several physiological mechanisms could be affected. Thus, this kind of effects should be deeply investigated. The results obtained in the present study could expand the information about micropollutants from livestock wastes on amphibians.

Gene expression responses for detecting sublethal effects of xenobiotics and whole effluents on a Xenopus laevis embryo assay.

In the present study, the authors investigated the effects of bisphenol A, chlorpyrifos, methylparaben, and 2 effluent samples from wastewater treatment plants located in the province of Madrid, Spain, on the messenger RNA expression of specific genes involved in early development (ESR1, pax6, bmp4, and myf5) and a gene involved in the general stress response (hsp70) during Xenopus laevis embryo development. Gene expression was analyzed after 4 h, 24 h, and 96 h of exposure by semiquantitative reverse-transcriptase-polymerase chain reaction. Concentration ranges of the compounds and dilutions for the samples were selected to cause morphological alterations in embryos after 96 h of exposure. Transcript levels of ESR1, pax6, and hsp70 were differentially altered at early developmental stages with patterns specific to the contaminant and the exposure time. However, further studies are needed to establish transcript levels of specific genes as biomarkers of sublethal effects in an environmental risk-assessment framework. Besides, studies including more generic responses, such as genes encoding antioxidant enzymes, together with genes related to embryonic development have to be developed to look for a battery of mechanistic endpoints for the evaluation of chemical exposure at the molecular level in a first-tier assessment.

Gene expression of heat shock protein 70, interleukin-1ß and tumor necrosis factor α as tools to identify immunotoxic effects on Xenopus laevis: a dose-response study with benzo[a]pyrene and its degradation products.

The exposure to benzo[a]pyrene (B[a]P) results in an alteration of immune function in mammals and fish, and the analysis of cytokine mRNA levels has been suggested for predicting the immunomodulatory potential of chemicals. To obtain evidence of the innate immune responses to B[a]P in Xenopus laevis, the present study monitored the mRNA expression of interleukin 1-ß (IL-1ß), tumor necrosis factor α (TNF-α) and heat shock protein 70 (HSP70) in a laboratorial exposure. Tadpoles exposed to 8.36, 14.64, 89.06 and 309.47 µg/L of B[a]P,were used for detecting hsp70, IL-1ß and TNF-α mRNA induction. A dose-response increase in the expression of hsp70 and IL-1ß mRNA was found. The results of this study confirmed the use of hsp70 and IL-1ß, but not TNF-α, as sensitive indicators of immunotoxic effect of B[a]P in X. laevis. Further research would be required for the validation of these endpoints.

Assessment of potential immunotoxic effects caused by cypermethrin, fluoxetine, and thiabendazole using heat shock protein 70 and interleukin-1ß mRNA expression in the anuran Xenopus laevis.

The current study describes the effect of cypermethrin, fluoxetine, and thiabendazole, at environmentally relevant concentrations, on the expression of heat shock protein 70 (HSP70) and interleukin 1ß (IL-1ß), using Xenopus laevis larvae as animal model. Cytokines and interleukins are considered good predictors of the immunotoxic potential of xenobiotics. Tadpoles at stage 47 (normal tables of X. laevis) were exposed under static conditions to: 0.3 and 30 µg/L fluoxetine, 0.7 µg/L thiabendazole, and 0.24 µg/L cypermethrin. The effects were evaluated at 7, 24, and 72 h, and 6 and 9 d. Randomly chosen tadpoles were used as genetic material for detection of hsp70 and IL-1ß mRNA induction through reverse transcription PCR. Tadpoles exposed to 30 µg/L fluoxetine showed mRNA expression of both genes at all exposure times, whereas at 0.3 µg/L a peak response for hsp70 was observed after 24 h, and the increase in IL-1ß mRNA was statistically significant with respect to the control 72 h after exposure. Thiabendazole induced a high expression of mRNA for both hsp70 and IL-1ß at all exposure times. Cypermethrin increased the hsp70 mRNA levels, with a peak at 24 h, and provoked high expression of IL-1ß mRNA at all exposure times. Considering the relationship between HSP70 and IL-1ß and their involvement (mainly of IL-1ß) in immune responses, certain changes observed in their expression could be considered warning indicators of potential immunotoxic effects of these substances on Xenopus.