The new vertebrate CYP1C family: cloning of new subfamily members and phylogenetic analysis.

Two novel CYP1 genes from teleost fish constituting a new subfamily have been cloned. These paralogous sequences are designated CYP1C1 and CYP1C2. Both genes were initially obtained from untreated scup Stenotomus chrysops tissues by RT-PCR and RACE. Scup CYP1C1 and CYP1C2 code for 524 and 525 amino acids, respectively, and share 80-81% identity at the nucleotide and amino acid levels. Orthologues of CYP1C1 and CYP1C2 were identified in genome databases for other fish species, and both CYP1B1 and CYP1C1 were cloned from zebrafish (Danio rerio). Phylogenetic analysis shows that CYP1Cs and CYP1Bs constitute a sister clade to the CYP1As. Analysis of sequence domains likely to have functional significance suggests that the two CYP1Cs in scup may have catalytic functions and/or substrate specificity that differ from each other and from those of mammalian CYP1Bs or CYP1As. RT-PCR results indicate that CYP1C1 and CYP1C2 are variously expressed in several scup organs.

Induction of cetacean cytochrome P4501A1 by beta-naphthoflavone exposure of skin biopsy slices.

Marine mammals can accumulate environmental contaminants in their blubber at concentrations harmful to laboratory animals. Induction of the cytochrome P450 1A1 (CYP1A1) enzyme is widely used as a biomarker of exposure and molecular effects in animal species, yet the validity of this biomarker has not been established in marine mammals. In vivo studies are generally precluded in these protected species, but skin biopsies (epidermis and dermis) can be collected in a minimally invasive way. We developed an in vitro assay using skin biopsy slices to examine CYP1A1 protein induction in marine mammals in response to chemical exposure. Skin biopsies from sperm whale (Physeter macrocephalus) were exposed for 24 h to beta-naphthoflavone (BNF), a prototypical CYP1A1 inducer, and CYP1A1 induction was detected by immunohistochemical staining in endothelial cells, smooth muscle cells, and fibroblasts. Biopsy slices were exposed to a range of BNF concentrations (0.6-600 microM), and a significant concentration-effect relationship was observed in both endothelial and smooth muscle cells (p = 0.05). This is the first study using skin biopsy slices to examine exposure of cetacean tissue to a CYP1A1 inducer. It demonstrates a causal relationship between chemical exposure and CYP1A1 induction and therefore validates the use of CYP1A1 expression in skin biopsies as a biomarker in cetaceans. Our protocol can be adapted to the investigation of chemicals, mixtures, concentrations, incubation times, or biological endpoints of choice. This should prove particularly relevant for these and other protected species that cannot be studied in the laboratory.