Cytotoxicity Assays with Zebrafish Cell Lines.

Fish cell lines have become increasingly used in ecotoxicity studies, and cytotoxicity assays have been proposed as methods to predict fish acute toxicity. Thus, this protocol presents cytotoxicity assays modified to evaluate cell viability in zebrafish (Danio rerio) embryo (ZEM2S) and liver (ZFL) cell lines in 96-well plates. The cytotoxicity endpoints evaluated are mitochondrial integrity (Alamar Blue [AB] and MTT assays), membrane integrity via esterase activity (CFDA-AM assay), and lysosomal membrane integrity (Neutral Red [NR] assay). After the exposure of the test substances in a 96-well plate, the cytotoxicity assays are performed; here, AB and CFDA-AM are carried out simultaneously, followed by NR on the same plate, while the MTT assay is performed on a separate plate. The readouts for these assays are taken by fluorescence for AB and CFDA-AM, and absorbance for MTT and NR. The cytotoxicity assays performed with these fish cell lines can be used to study the acute toxicity of chemical substances on fish.

Genetic variants affecting chemical mediated skin immunotoxicity.

The skin is an immune-competent organ and this function may be impaired by exposure to chemicals, which may ultimately result in immune-mediated dermal disorders. Interindividual variability to chemical-induced skin immune reactions is associated with intrinsic individual characteristics and their genomes. In the last 30-40 years, several genes influencing susceptibility to skin immune reactions were identified. The aim of this review is to provide information regarding common genetic variations affecting skin immunotoxicity. The polymorphisms selected for this review are related to xenobiotic-metabolizing enzymes (CYPA1 and CYPB1 genes), antioxidant defense (GSTM1, GSTT1, and GSTP1 genes), aryl hydrocarbon receptor signaling pathway (AHR and ARNT genes), skin barrier function transepidermal water loss (FLG, CASP14, and SPINK5 genes), inflammation (TNF, IL10, IL6, IL18, IL31, and TSLP genes), major histocompatibility complex (MHC) and neuroendocrine system peptides (CALCA, TRPV1, ACE genes). These genes present variants associated with skin immune responses and diseases, as well as variants associated with protecting skin immune homeostasis following chemical exposure. The molecular and association studies focusing on these genetic variants may elucidate their functional consequences and contribution in the susceptibility to skin immunotoxicity. Providing information on how genetic variations affect the skin immune system may reduce uncertainties in estimating chemical hazards/risks for human health in the future.