Prediction of acute fish toxicity (AFT) and fish embryo toxicity (FET) tests by cytotoxicity assays using liver and embryo zebrafish cell lines (ZFL and ZEM2S).

Fish cell-based assays represent potential alternative methods to vertebrates' use in ecotoxicology. In this study, we evaluated the cytotoxicity of thirteen chemicals, chosen from OECD guidelines 236 and 249, in two zebrafish cell lines (ZEM2S and ZFL). We aimed to investigate whether the IC50 values obtained by viability assays (alamar blue, MTT, CFDA-AM, and neutral red) can predict the LC50 values of Acute Fish Toxicity (AFT) test and Fish Embryo Toxicity (FET) test. There was no significant difference between the values obtained by the different viability assays. ZFL strongly correlated with AFT and FET tests (R2AFT = 0.73-0.90; R2FET48h = 0.79-0.90; R2FET96h = 0.76-0.87), while ZEM2S correlated better with the FET test (48h) (R2 = 0.70-0.86) and weakly with AFT and FET tests (96h) (R2AFT = 0.68-0.74 and R2FET96h = 0.62-0.64). The predicted LC50 values allowed the correct categorization of the chemicals in 76.9% (AFT test) - 90.9% (FET test) using ZFL and in 30.7% (AFT test) - 63.6% (FET test) using ZEM2S considering the US EPA criterion for classifying acute aquatic toxicity. ZFL is a promising cell line to be used in alternative methods to adult fish and fish embryos in ecotoxicity assessments, and the method performed in 96-well plates is advantageous in promoting high-throughput cytotoxicity assessment.

An integrated in silico-in vitro investigation to assess the skin sensitization potential of 4-Octylphenol.

One of the major challenges in chemical toxicity testing is the possibility to protect human health against adverse effects with non-animal methods. In this paper, 4-Octylphenol (OP) was tested for skin sensitization and immunomodulatory effects using an integrated in silico-in vitro test approach. In silico tools (QSAR TOOLBOX 4.5, ToxTree and VEGA) were used together with several in vitro tests including HaCaT cells (quantification of IL-6; IL-8; IL-1α and IL-18 by ELISA and expression of genes TNF, IL1A, IL6 and IL8 by RT- qPCR), RHE model (quantification of IL-6; IL-8; IL-1α and IL-18 by ELISA) and THP-1 activation assay (CD86/CD54 expression and IL-8 release). Additionally, the immunomodulatory effect of OP was investigated using lncRNAs MALAT1 and NEAT1 expression and LPS-induced THP-1 activation (CD86/CD54 expression and IL-8 release). The in silico tools predicted OP as a sensitizer. In vitro tests are also concordant with the in silico prediction. OP increased IL-6 expression (HaCaT cells); IL-18 and IL-8 expressions (RHE model). An irritant potential was also shown by a great expression of IL-1α (RHE model); and increased expression of CD54 marker and IL-8 in THP-1 cells. Immunomodulatory effects of OP were demonstrated by the downregulation of NEAT1, MALAT1 (epigenetic markers), IL6 and IL8; and an increase in LPS-induced CD54 and IL-8 expressions. Overall, results indicate that OP is a skin sensitizer, being positive in three key events of the AOP for skin sensitization, also showing immunomodulatory effects.

Single and mixture toxicity evaluation of three phenolic compounds to the terrestrial ecosystem.

Endocrine-disrupting chemicals (EDCs) are primarily studied regarding endocrine-mediated effects in mammals and fish. However, EDCs can cause toxicity by mechanisms outside the endocrine system, and, as they are released continuously into soils, they may pose risks to terrestrial organisms. In this work, the plant Allium cepa and the earthworm Eisenia foetida were used as test systems to evaluate the toxicity and cyto-/geno-toxicity of three environmental phenols known as EDCs (Bisphenol A - BPA, Octylphenol - OP, Nonylphenol - NP). The tested phenols were evaluated in environmentally relevant concentrations (µg/L) and in single forms and mixture. BPA, OP, and NP did not inhibit the seed germination and root development in A. cepa in their single forms and mixture. However, all single forms of the tested phenols caused cellular and DNA damages in A. cepa, and although these effects persist in the mixtures, the effects were verified at lower levels. These phenols caused acute toxicity to E. foetida after 48 h of exposure and at both conditions evaluated (single forms and mixture); however, unlike A. cepa, in earthworms, mixtures and single forms presented the same level of effects, indicating that interspecies physiological different might influence the mixture toxicity. In summary, our results suggest that BPA, OP, and NP are toxicants to earthworm and cyto-/geno-toxicants to monocotyledonous plants at low concentrations. However, interaction among these phenols reduces the magnitude of their individual effects (antagonistic effect) in the plant test system. Therefore, this study draws attention to the need to raise knowledge about the ecotoxicity of phenolic compounds to help predict their ecological risks and protect non-target terrestrial species.