Hepatic versus extrahepatic expression of CYP3A30 and CYP3A56 in adult killifish (Fundulus heteroclitus)

Members of the CYP3A subfamily represent the largest portion of CYP proteins in liver and intestine in vertebrates. The CYP3A enzymes are involved in metabolic clearance of numerous chemically diverse compounds including toxins, carcinogens, pesticides, therapeutic drugs, dietary products and hormones. Most studies of CYP3A have been performed in mammals, whereas relatively little is known of that in non-mammalian species. We have investigated CYP3A expression in the marine and estuarine killifish (Fundulus heteroclitus). We isolated a novel CYP3A cDNA sequence, denoted CYP3A56, from killifish intestine. The CYP3A56 sequence shared 98% nucleotide and amino acid sequence identity with CYP3A30, previously isolated from killifish liver. We hypothesize that a recent gene duplication event has occurred within the killifish CYP3A subfamily. The CYP3A30 and CYP3A56 genes were co-expressed in liver, intestine, gill, kidney, spleen, brain and ovary in several individuals. The hepatic versus extrahepatic CYP3A30/56 mRNA expression was analyzed in adult killifish of both sexes, using conventional as well as real-time semi-quantitative RT-PCR. Tissues expressing CYP3A30/56 mRNA were in a descending order of magnitude: liver > intestine much greater than gill > spleen > kidney > brain. Furthermore, inter-individual differences (up to 18%) in CYP3A30/56 mRNA expression were evident in killifish, in particular in extrahepatic organs. For comparison, CYP3A protein expression levels were determined using polyclonal antibodies (PAb) against rainbow trout (Oncorhynchus mykiss) CYP3A. Sexually dimorphic expression of hepatic and extrahepatic CYP3A30/56 mRNA and CYP3A proteins was observed in killifish. For example, males displayed up to 2.5-fold higher CYP3A protein expression compared with females. In agreement with CYP3A30/56 mRNA analysis, highest CYP3A protein levels were observed in liver and intestine. Low CYP3A protein levels were seen in gill, kidney and spleen. Cellular localization of CYP3A protein expression was investigated using immunohistochemistry analysis and PAb against rainbow trout CYP3A. Strong CYP3A protein staining was seen in intestinal enterocytes, in gill filaments and in renal tubular epithelial cells. Moderate CYP3A staining was seen in hepatocytes in the liver, whereas mild staining was observed in hematopoietic cells in the spleen and in follicles in the ovary. Thus, similar to mammals, CYP3A expression in fish is prominent in the digestive- and respiratory tracts and may be important for the first-pass metabolism of xenobiotics. Moreover, CYP3A expression also is evident in brain and ovary in killifish, which suggests a role for CYP3A enzymes in biotransformation of xenobiotics and fine-tuning levels of steroid hormones in situ in extrahepatic organs. (C) 2003 Elsevier Science B.V. All rights reserved.