Abstract
The phenomenon of xenobiotic-activated induction of certain members of the CYP450 superfamily of haemoproteins, is the basis of this project concerning the response of mussels to pollution. This induction by chemicals such as organic environmental pollutants, elevates transcription of the CYP450 genes and subsequently yields a greater total amount of CYP450 enzyme activity within the cell. This mechanism of modulation can be monitored in a number of ways, including assay of the final enzyme activity, immunodetection of the enzyme protein and detection of the mRNA coding for the specific enzyme isoform. In this project I have used the detection of mRNA by Northern blot analysis in an attempt to quantify the response of mussel CYP1A levels to pollutants in the marine environment. However, invertebrate cDNA probes for CYP1A mRNA, suitable for this hybridisation technique, were not available. A CYP1A cDNA probe obtained from the rainbow trout was used to hybridise both mussel genomic DNA and mRNA (Heilmann et al., 1988). Cross-species hybridisation had been previously used by Spry (1991), in detecting CYP4A1 mRNA in M. edulis using a rat CYP4AI cDNA probe. I have attempted to follow up on this work and examine the expression of other members of the CYP450 superfamily in Mytilus sp. A number of CYP450 gene families were investigated, including CYP1A, CYP3A, CYP4A and CYP11A mRNA species with nucleotide sequence similarity to the vertebrate CYP1A, CYP3A, CYP4A and CYP 11A cDNA probes were shown to be expressed in this organism, although clear evidence for hybridising sequences related to CYP450 genes in genomic DNA was only found for CYP3A and CYP11A. A study was undertaken to investigate the seasonal changes in the levels of expression of CYP1A, CYP3A and CYP4A mRNAs. These studies were successful in showing varying mRNA expression over the period of a year, with the CYP450 cDNA probes revealing a distinct patterns of expression. The seasonal variation observed in M. edulis for putative CYP1A expression was correlated with microsomal B[a]P metabolism, indicating that the Mytilus sp. CYP1A orthologue may have a role in this monooxygenase activity. To determine if induction of CYP1A mRNA in mussels could be observed using classical mammalian CYP1A inducers, Mytilus sp. were treated with the PCB congener CB-138 and benzo[a]pyrene in separate studies. Clear evidence of modulation of CYP1A mRNA with these inducers was not found. Field studies in the Venice lagoon did however reveal modulation of the levels of mussel putative CYP1A mRNA at varying sites around the lagoon, which correlated with the levels of aliphatic hydrocarbons found in mussel tissues from these sites. An investigation of the CYP450 system in the alligator was attempted to see if the cross-species hybridisation technique could be extended to other aquatic species. Alligators were exposed to 3-MC, PB and clofibrate and the mRNA hybridised with the vertebrate cDNA probes for CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP4A. No clear elevation of mRNA levels could be determined from this study, mainly due to the low number of animals used for the control time points. In an attempt to optimise the CYP1A and CYP4A hybridisation techniques in the mussel, the mussel genomic library produced by Dr. A. Spry (1991) was screened in an attempt to find the mussel DNA sequences equivalent to these two CYP450s. PCR on genomic DNA and cDNA was also employed to perform the same function. Although there was strong evidence that sequences related to vertebrate CYP1A had been isolated using the PCR approach at the time, nucleotide sequencing has not substantiated this finding. In summary I have demonstrated that the use of the O. mykiss CYP1A probe could possibly be used to examine xenobiotic-induced modulation of CYP450 levels in the mussel, although as yet only one initial experiment in the Venice lagoon has shown promise. The reason for the discrepancies that occur in the results of all the experiments is probably due to non-specific hybridisation of the probe to other members of the CYP450 superfamily that may be present within the mussel.