Intermoult duration affects the susceptibility of shore crabs Carcinus maenas (L.) to pyrene and their ability to metabolise it

Abstract

We have studied pyrene (PYR) toxicity and the ability to metabolise and eliminate PYR in two colour forms of shore crabs Carcinus maenas (Linnaeus, 1758). In addition, we analysed differences in the expression of expressed sequence tags (ESTs) encoding specific cytochrome P450s (CYPs) by quantitative realtime PCR. Green and red intermoult crabs are considered to represent different adaptational life stages, allocating energy into growth (green) and reproduction (red), respectively. PYR injection resulted in significantly higher mortality in red crabs than in green crabs during a 51 days period. PYR is an ideal model PAH compound as only 1 phase I metabolite, 1-hydroxypyrene (1-HP) is formed, which is further conjugated to form various phase II metabolites. In this study, 1-HP was detected only after deconjugation of total PYR derived metabolites indicating that PYR hydroxylation (putatively CYP catalysed) conceivably is the rate-limiting step in PYR metabolism. Investigation of the accumulation of PYR and 1-HP (after deconjugation), in different tissues revealed a significantly higher accumulation of PYR in muscle and epidermis of red crabs compared to green crabs. Consistent with this observation, green crabs had significantly higher levels of 1-HP in the hepatopancreas than red crabs. This indicates that a larger portion of the injected PYR was metabolised into 1-HP in green crabs compared to red crabs. 1-HP was mainly detected in the hepatopancreas confirming its major role in the biotransformation of lipophilic compounds. CYP enzymes typically mediate the phase I hydroxylations of lipohilic contaminants such as PYR. In agreement with the higher rate of conversion of PYR into 1-HP in green compared to red crabs, increased abundance of several CYP transcripts was observed in green crabs. Furthermore, in vitro pyrene hydroxylase assays revealed significantly higher NADPH-depedent pyrene hydroxylase activity in hepatopancreas microsomes of green crabs (18.4 ρmol min −1 mg −1 protein) compared to red crabs (8 ρmol min −1 mg −1 protein). The present study demonstrates that the susceptibility of shore crabs to PYR and their ability to metabolise it is life stage dependent, conceivably due to life stage related differences in the expression of certain CYP genes, suggesting a mechanistic explanation of the observed life stage differences in PYR toxicity.