AbstractsBiology & Animal Science

Epithelial cell lines, RTgill-W1 and RTL-W1 from respectively the gill and liver of rainbow trout, Onchorhynchus mykiss (Walbum), were used to investigate the role of p53 in the cellular survival pathways of fish and to evaluate the potential impact on fish of the emerging contaminants, benzotriazoles (BTRs) and benzothiazoles (BTHs). For studying p53, RTgill-W1 was used with two p53 inhibitors, which are termed pifithrins- (PFT-) or 2-phenylethynesulfonamide (PES) and pifithrin-α (PFT-α). Both agents were developed for cancer chemotherapy but also have been used widely to explore p53 functions in mammals but not in fish. PFT- or 2-phenylethynesulfonamide (PES) was identified as an inhibitor of p53 translocation to the mitochondria but subsequently shown to be a HSP70 inhibitor as well. PFT-α was recognized as an inhibitor of p53-mediated transcription. Cellular toxicity was evaluated for seven BTRs: 1H-Benzotriazole (BTR), 4-methyl-1H-benzotriazole (4MBTR), 5-methyl-1H-benzotriazole (5MBTR), tolytriazole (TT), 5,6-Dimethyl-1H-benzotriazole monohydrate (DM), 5-Chlorobenzotriazole (5CBTR) and Hydroxybenzotriazole (OHBTR). The BTHs were Benzothiazole (BTH), 3,3’-diethylthia dicarbocyanine iodide (DTDC), C.I. Sulphur orange 1 (SO), 2-Mercaptobenzothiazole (2MBTH), Zinc 2-Mercaptobenzothiazole (ZincMBTH), Sodium 2-Mercaptobenzothiazole (NaMBTH), 2-Hydroxy-benzothiazole (OHBTH), 2-Aminobenzothiazole (2ABTH), C.I. Vat yellow 2 (VY), N,N-Dicyclohexyl-2-benzothiazolsulfene amide (NNA), 2,2'-Dithiobis (benzothiazole) (DBTH) and 2-(p-aminophenyl)-6-methylbenzothiazole-7-sulfonic acid (MBTHS). PES had complex actions on RTgill-W1. As judged by three viability assays, cells were killed by 24 h exposures to PES, but cell death was blocked by the anti-oxidant N-acetylcysteine (NAC). Cell death had several hallmarks of apoptosis: DNA laddering, nuclear fragmentation, Annexin V staining, mitochondrial membrane potential decline, and caspase activation. Reactive oxygen species (ROS) production peaked in several hours after the addition of PES and before cell death. HSP70 and BiP levels were higher in cultures treated with PES for 24 h, but this was blocked by NAC. As well, PES treatment caused HSP70, BiP and p53 to aggregate and become detergent-insoluble, and this too was prevented by NAC. Of several possible scenarios to explain the results, the following one is the simplest: PES enhances the generation of ROS, possibly by inhibiting the anti-oxidant actions of p53 and HSP70. ER (Endoplasmic reticulum) stress arises from the ROS and from PES inhibiting the chaperone activities of HSP70. The ER stress in turn initiates the unfolded protein response (UPR), but this fails to restore ER homeostasis so proteins aggregate and cells die. Despite these multiple actions, PES should be useful for studying fish cellular survival pathways. PFT-α had unexpected and expected actions on RTgill-W1. When dosed indirectly into RTgill-W1 cultures, PFT-α did not reduce cell viability but caused a transient rise in the mitotic index and a…