Differential expression of two CYP1A genes in rainbow trout (Oncorhynchys mykiss).

Differential expression of two rainbow trout CYP1A genes was measured in vivo and in vitro in response to treatment with the model CYP1A inducers beta-naphthoflavone (BNF), 3-methylcholanthrene (3-MC), isosafrole (ISF), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, only in vitro). Originally described by Berndtson and Chen (Arch. Biochem. Biophys. 310, 187-195, 1994) as CYP1A1 and CYP1A2, these genes were renamed CYP1A3 and CYP1A1, respectively, by the P450 nomenclature committee. A significant, differential, inducer-dependent induction of the two CYP1A mRNAs, as measured by RNase protection assay, was observed in vivo. CYP1A3 and CYP1A1 mRNA levels in liver were significantly induced 50- and 18-fold, respectively, following ip injection with BNF. Conversely, CYP1A3 and CYP1A1 mRNA levels were significantly induced 5- and 66-fold, respectively, following ip injection with 3-MC. Isosafrole had no significant effect on in vivo induction of CYP1A mRNA levels. In primary cultures of hepatocytes, BNF, 3-MC, ISF, as well as TCDD all significantly induced CYP1A3 and CYP1A1 mRNA levels compared to controls. The differential induction of the two CYP1A genes was not as evident in vitro as in vivo. In addition, reanalysis and sequence comparison of the these two trout CYP1A genes with the first trout CYP1A cDNA described by Heilmann et al. (DNA 7, 379-387, 1988) indicate that the Heilmann cDNA is a hybrid of the two trout genes. The 5' portion of the cDNA sequence (212 bp) was determined by sequencing of a genomic clone and is 100% identical to the trout CYP1A3 gene. The majority of the cDNA sequence (2377 bp), however, was sequenced from a partial cDNA clone and is 99.2% identical to trout CYP1A1. Although the nomenclature of these two trout CYP1A genes is undergoing revision, these results demonstrate a differential, inducer-dependent response to model mammalian CYP1A inducers.

Interaction of 2,2',4,4',5,5'-hexachlorobiphenyl with hepatic cytochrome P-4501A in rainbow trout (Oncorhynchus mykiss).

Di-ortho polychlorinated biphenyls (PCBs) are prominent environmental contaminants and their biological activity in fish may be more significant than previously thought. Four weeks after intraperitoneal (i.p.) injection with 50 or 250 microg 2,2',4,4',5,5'-hexachlorobiphenyl (2HxCB)/g fish, rainbow trout livers were removed and frozen at -80 degrees C or microsomes were prepared. Microsomal ethoxyresorufin O-deethylase (EROD) activity was approximately one and two orders of magnitude greater than controls in fish treated with 50 and 250 microg 2HxCB/g fish, respectively. Cytochrome P4501A (CYP1A) Western immunoblot relative optical density increased with 2HxCB dose. Hepatic CYP1A1 mRNA levels were approximately threefold greater in fish treated with 250 microg 2HxCB/g fish than in controls, while hepatic CYP1A1 mRNA levels in fish treated with 50 microg 2HxCB/g fish were not significantly induced. There was no increase of CYP1A3 mRNA in 2HxCB-treated fish. The study showed 2HxCB induced hepatic EROD activity, CYP1A protein, and CYP1A1 mRNA content in rainbow trout.

Transgenic fish and its application in basic and applied research.

Since 1985, transgenic fish have been successfully produced by microinjecting or electroporating desired foreign DNA into unfertilized or newly fertilized eggs using many different fish species. More recently, transgenic fish have also been produced by infecting newly fertilized eggs with pantropic, defective retroviral vectors carrying desired foreign DNA. These transgenic fish can serve as excellent experimental models for basic scientific investigations as well as in biotechnological applications. In this paper, we will review the current status of the transgenic fish research and its potential application in basic and applied research.