Characterization of the presence and seasonal variation of a CYP1A-like enzyme in digestive gland of the common mussel, Mytilus edulis

Abstract

The presence and putative catalytic properties of a CYP1A-like enzyme in the digestive gland of Mytilus edulis L. were investigated by molecular biological and seasonal studies. Reverse-transcriptase PCR using oligonucleotide primers to amplify a sequence around the conserved haem binding cysteine region of hepatic CYP1A1 of trout ( Oncorhynchus mykiss) produced several cDNA bands resolved by electrophoresis, including major bands of about 220 and 280 bp compared to the predicted size for O. mykiss of 208 bp. Following Southern blotting and probing with a cDNA probe to O. mykiss CYP1A1 (pfP 1450-3′ probe modified by removal of 3′-non-coding region by digestion by Cla1), a single band (280 bp) only was detected using moderate stringency conditions of sequence recognition (i.e. hybridization at 42 °C followed by washing at 55 °C in 1 × SSC containing 0.1% SDS), providing evidence for the presence of a CYP1A orthologous gene sequence. The seasonal variation in levels of putative CYP1A mRNA (Northern analysis using the modified cDNA probe for O. mykiss CYP1A1) over 1 year showed some similarity to seasonal patterns of change in microsomal metabolism of 3H-benzo[a]pyrene (BaP) to polar metabolites (dials, diones and phenols resolved by HPLC). Maxima for putative CYP1A mRNA and BaP metabolism levels were in late spring-early summer. However, differences were also apparent, possibly indicative of other P450s contributing to BaP metabolism. Overall the results indicate the existence of a CYP1A-like enzyme which is, at least, partly responsible for the mono-oxygenase activity of BaP metabolism.