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Differential expression of two CYP1A genes in rainbow trout (Oncorhynchys mykiss)
C M Råbergh1, N H Vrolijk, M M Lipsky
1Department of Pathology, Program in Toxicology, University of Maryland at Baltimore, 10 South Pine Street, Baltimore, Maryland 21201, USA. christina.rabergh@utu.fi
Abstract:
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.
Insights
Rainbow trout exhibit differential gene expression in CYP1A3 and CYP1A1 in response to chemical inducers like beta-naphthoflavone and 3-methylcholanthrene. This study clarifies their distinct responses in vivo and in vitro, aiding toxicological research.
Area of Science:
- Environmental Toxicology
- Molecular Biology
- Biochemistry
Background:
- Cytochrome P450 (CYP) enzymes play a crucial role in xenobiotic metabolism.
- Rainbow trout (Oncorhynchus mykiss) possess multiple CYP1A genes with potentially differential responses to environmental contaminants.
- Previous nomenclature for trout CYP1A genes has been revised, with CYP1A3 and CYP1A1 being the current designations.
Purpose of the Study:
- To investigate the differential expression of two rainbow trout CYP1A genes (CYP1A3 and CYP1A1) in response to known CYP1A inducers.
- To compare the in vivo and in vitro responses of these genes to various chemical treatments.
- To clarify the nature of a previously described trout CYP1A cDNA sequence.
Main Methods:
- Quantitative analysis of CYP1A3 and CYP1A1 mRNA levels using RNase protection assay.
- Treatment of rainbow trout with inducers beta-naphthoflavone (BNF) and 3-methylcholanthrene (3-MC) in vivo.
- Treatment of primary hepatocyte cultures with BNF, 3-MC, isosafrole (ISF), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vitro.
- Sequence analysis and comparison of trout CYP1A genes and cDNA clones.
Main Results:
- Significant, inducer-dependent induction of both CYP1A3 and CYP1A1 mRNA was observed in vivo.
- BNF and 3-MC showed differential induction patterns for CYP1A3 and CYP1A1 mRNA in vivo.
- In vitro, all tested inducers (BNF, 3-MC, ISF, TCDD) significantly induced both genes, but with less pronounced differential responses compared to in vivo.
- Reanalysis revealed the Heilmann et al. cDNA is a hybrid of trout CYP1A3 and CYP1A1 genes.
Conclusions:
- Rainbow trout CYP1A3 and CYP1A1 genes exhibit differential, inducer-dependent expression patterns.
- The in vivo response to CYP1A inducers is more distinct between the two genes than the in vitro response.
- The findings contribute to a better understanding of fish toxicology and gene regulation by xenobiotics.