Intracellular metabolome elucidates the time-of-day-dependent response to hydrogen peroxide in salmonid gill epithelial cells

The internal timekeeping system regulates the daily cycle of physiological and behavioural changes in living organisms. This rhythmic phenomenon also influences cellular responses to reactive oxygen species, such as hydrogen peroxide (H₂O₂). However, the temporal interaction between H₂O₂ and fish mucosal cells is not well understood. This study examined the temporal variations of immunological and physiological responses to H₂O₂ in salmonid gill cells using the RTgill-W1 cell line. The results showed that gene expression levels varied during a 24-h cycle but did not exhibit rhythmicity. The presence of a 12-h light-dark cycle (12L:12D) signal increased gene expression levels compared to a 24-h dark cycle (0L:24D). To investigate whether the time of day affects the defences in gills, cells were exposed to H₂O₂ at two different times (Zeitgebertime 2, ZT2, or ZT14). Although significant expression changes were observed in genes related to stress and NF-κB signalling, only a limited time-dependent pattern of response to H₂O₂ was observed. The intracellular metabolome of gill cells was primarily composed of organic acid and derivatives, organoheterocyclic compounds, benzoids, organic oxygen and nitrogen compounds. Exposure to H₂O₂ at ZT2 led to significant changes in the metabolome compared to the control group, while no such changes were observed at ZT14. Within the control groups, the concentrations of 11 metabolites significantly varied between ZT2 and ZT14, with higher levels at ZT14. These metabolites were involved in arginine biosynthesis, amino acid metabolism, and nitrogen metabolism. In contrast, the level of 26 metabolites significantly varied between ZT2 and ZT14 in H₂O₂-exposed groups, with lower levels at ZT14. Comparing control and H₂O₂-exposed groups at ZT2, 38 metabolites were affected, primarily organic acid and derivatives and organic oxygen compounds. Functional annotation revealed that these altered metabolites were involved in 15 different pathways, with valine, leucine, and isoleucine biosynthesis being the most affected. This study reveals the presence of a time-dependent response to H₂O₂ in salmonid gill cells, which is reflected in the intracellular metabolome. The findings provide new insights into the temporal regulation of mucosal defences in fish.