Effects of diphenhydramine on crayfish cytochrome P450 activity and antioxidant defence mechanisms: First evidence of CYP2C- and CYP3A-like activity in marbled crayfish

Growing evidence has reported that diphenhydramine (DPH), an ionisable antihistamine, is widely present in surface waters across the world. Relative to vertebrates studied, its impact on invertebrates, particularly concerning cytochrome P450 (CYP) metabolism and oxidative stress, remains poorly understood. In this study, we aimed to investigate the effects of 2, 20, and 200 µg/L DPH on marbled crayfish (Procambarus virginalis) after 96-h exposure. Specifically, we assessed CYP activity, antioxidant enzyme responses, and acetylcholinesterase (AChE) activity in gills, muscle, and hepatopancreas. The crayfish CYP metabolised fluorogenic CYP-metabolic substrates of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) and dibenzylfluorescein (DBF), which evidenced the activity of CYP2C and CYP3A isoforms, well known in mammalian detoxification metabolism. Both BFC and DBF dealkylations showed a positive correlation with each other but were negatively correlated to water and haemolymph DPH concentrations. Exposure to 200 µg/L DPH elicited an apparent inhibition trend, albeit not significant, in BFC- and DBF-transformation activities in crayfish. Other tested 7-benzyloxyresorufin and 7-pentoxyresorufin substrates were poorly metabolised, suggesting their relatively low activity or the lack of mammalian-like CYP1A and CYP2B isoforms in marbled crayfish. The significant modulation of antioxidant enzymes was demonstrated in gills and hepatopancreas. The exposure to DPH did not alter the activity of AChE. Integrated biomarker response version 2 showed the highest cumulative effect of DPH exposure on gills, implying that gill tissue is the most reliable matrix for evaluating DPH toxicity. Activities of glutathione peroxidase and glutathione-S-transferase were the most deviated determinants among the investigated biomarkers, providing insights into the DPH toxicity in crayfish. This study brought the first insight into utilising the fluorogenically active substrates BFC and DBF to demonstrate the CYP involvement in the detoxification metabolism in marbled crayfish. Further, our results provided information on valuable antioxidant defence mechanisms and biomarker responses for a future DPH toxicity assessment in aquatic organisms.