Contrasting effects of a classic Nrf2 activator, tert-butylhydroquinone, on the glutathione-related antioxidant defenses in Pacific oysters, Crassostrea gigas.

Nrf2 is a well-known transcription factor controlling a number of antioxidant defense-related genes, which is understudied in bivalves. In this study, oysters Crassostrea gigas were exposed for 24, 48 and 96 h to 10 or 30 µM tert-butylhydroquinone (tBHQ), a classic Nrf2 activator. At 96 h, a clear induction of GSH-related antioxidant defenses was observed in gills of tBHQ-exposed animals, including GSH, glutathione S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR). Unexpectedly, the activities of GST, GPx and GR were significantly decreased 24 h after tBHQ treatment, suggesting a possible inhibition, which was supported by in vitro experiments. GR mRNA (24 h) and protein levels (24 and 96 h) were increased by tBHQ treatment, confirming its induction, possibly by the Nrf2 pathway. The conserved domains at C. gigas Keap1 and Nrf2 proteins and the clear induction of GSH-related antioxidant defenses by tBHQ, a classical Nrf2 inducer, support the idea of a functional Nrf2/Keap1 pathway in bivalves. tBHQ also proved to be a tool to explore redox regulatory mechanisms in bivalves.

Upregulating Nrf2-dependent antioxidant defenses in Pacific oysters Crassostrea gigas: Investigating the Nrf2/Keap1 pathway in bivalves.

Analysis of the Pacific oyster Crassostrea gigas annotated genome revealed genes with conserved sequences belonging to typical cap 'n' collar Nrf2 domain, a major player in antioxidant protection, and domains belonging to Nrf2 cytoplasmic repressor (Keap1), but little is known about Nrf2/Keap1 induction in bivalves. C. gigas were exposed to waterborne 10 and 30µM curcumin, a known inducer of the mammalian Nrf2. Curcumin disappeared from the seawater after 10h, and accumulated in the gills (10h) and digestive gland (10-96h). A clear induction of glutathione (GSH)-related antioxidant defenses was observed at 96h in the gills of curcumin exposed animals (10 and 30µM), including GSH levels, and the activity of glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST). This response was completely absent in the digestive gland, in line with the idea that bivalve gills act as a major site for antioxidant protection under acute exposure. The relative mRNA levels coding glutamate-cysteine ligase, GR, GPx2 and GSTpi were clearly induced by curcumin treatment (30µM, 24h). Curcumin pre-treatment for 96h increased oyster resistance to cumene hydroperoxide, but neither Nrf2 nor Keap1 genes were modulated by curcumin. However, the conserved sequences belonging to typical Nrf2 and Keap1 domains, and the notorious induction of antioxidant defense-related genes known to be controlled by Nrf2 in mammals, indicates a functional Nrf2/Keap1 pathway in bivalves, and curcumin seems to be a new tool to investigate the antioxidant response in bivalves.