Induction patterns of new CYP1 genes in environmentally exposed rainbow trout

Abstract

The cytochrome P4501 (CYP1) gene family comprises four subfamilies in fish: CYP1A, CYP1B, CYP1C, and CYP1D. Only two CYP1 genes, CYP1A1 and CYP1A3, are so far known in rainbow trout (Oncorhynchus mykiss). The present study aimed to identify other CYP1 subfamily genes in rainbow trout, to establish methods for quantitative mRNA expression analysis of these genes, and to determine their basal and induced mRNA expression in gills and liver. Another goal was to examine their mRNA expression in environmentally exposed fish. We cloned four new transcripts, denoted rbCYP1B1, rbCYP1C1, rbCYP1C2, and rbCYP1C3. Levels of these and the previously known rbCYP1A transcripts were determined by real-time PCR in unexposed fish, fish exposed to the potent aryl hydrocarbon receptor (AhR) agonist 3,3',4,4',5-pentachlorobiphenyl (PCB126), and fish caged in various waters in the Uppsala region (Sweden). The mRNA expression patterns observed in unexposed rainbow trout (basal levels) were markedly similar to those reported for orthologous genes in other species. All six transcripts were induced by PCB126 in gills and liver, suggesting all genes to be AhR regulated. The caged fish showed clear rbCYP1 induction in gills at all monitoring sites (up to 70-fold the basal level), whereas the liver responses were weak; induction (up to 5-fold) was recorded only at the Uppsala municipal sewage treatment plant outlet. Gill filament EROD activity was induced at all caging sites. Most interestingly, the rbCYP1 gene response patterns in gills differed among caging sites and among subfamilies. The EROD induction seemed to only reflect induction of rbCYP1A transcription. Response patterns of multiple CYP1 genes in gills and liver could provide an improved monitoring strategy. Such patterns could be used to characterize complex mixtures of AhR agonists and antagonists in aquatic environments.

Figure 1

Map of the Uppsala region showing the locations for the four caging sites used in the environmental exposure experiment. Roads are shown with black and rivers and lakes with blue (grey).

Figure 2

Alignment of the deduced amino acid sequences of the cloned CYP1B1, CYP1C1, CYP1C2, and CYP1C3 transcripts in rainbow trout (Oncorhynchus mykiss) with orthologous sequences in zebrafish (Danio rerio) and human (Homo sapiens) made using ClustalW (Hall, 1999). The location for the heme binding site (blue) and the substrate recognition sites (SRS1–6) (red) (Lewis et al., 2003) of the proposed enzymes are indicated by shading. Accession numbers are available in the supplemental information.

Figure 3

Molecular phylogeny of rbCYP1A1, rbCYP1A3, rbCYP1B1, rbCYP1C1, rbCYP1C2, and rbCYP1C3 deduced amino acid sequences with selected other CYP1 sequences including assembled or renamed salmon sequences derived from GenBank (Salmo_1B1_part, Salmo_1C2_EST, Salmo_1C3). Both Bayesian and maximum likelihood techniques recover the topology presented here. Uncertainties in the topology are due to the alterative positioning of the zebrafish CYP1C1 sequence. Clade support values presented at each node represent the Bayesian posterior probability calculated after 3e6 generations and the maximum likelihood bootstrap support calculated from 100 replicates. Accession numbers are available in the supplemental information.

Figure 4

Relative levels of rbCYP1A1 rbCYP1A3 rbCYP1B1 rbCYP1C1 rbCYP1C2, and rbCYP1C3 mRNA expression in gills (A), in liver (B), and in gills relative to liver (C) in unexposed rainbow trout (basal expression). Basal mRNA expression was calculated as a percentage of rbCYP1A1 mRNA expression in gills or liver (% of mean rbCYP1A1), or for each of the rbCYP1s in liver (% of mean rbCYP1 in liver). EF1α was used as reference gene. The dotted lines indicate 100%. Statistically significant differences between levels of rbCYP1 transcripts within a tissue were examined by one-way ANOVA followed by Tukey’s post hoc test and are shown by different letters. Statistically significant differences between transcript levels in gills and liver were determined by t-test with the Welch correction and are shown by stars (*** = p<0.001 and * = p<0.05), and n=5–6.

Figure 5

Relative transcriptional induction of rbCYP1A1 rbCYP1A3 rbCYP1B1, rbCYP1C1 rbCYP1C2, and rbCYP1C3 in gills (A) and liver (B) of PCB126-exposed rainbow trout (n=5–6). Calculations were made using EF1α as the reference gene and the mean values of the different rbCYP1s in unexposed controls as calibrators. Statistically significant differences compared with the unexposed control were examined by one-way ANOVA followed by Bonferroni's post hoc test for selected pairs and are shown by stars (*** = p<0.001 and ** = p<0.01).

Figure 6

Relative mRNA expression patterns of rbCYP1A1 rbCYP1A3 rbCYP1B1 rbCYP1C1 rbCYP1C2, and rbCYP1C3 in gills (A) and liver (B) of environmentally exposed rainbow trout. Groups of 12 fish were exposed by two days of caging at four freshwater sites in the Uppsala region. Calculations were made using EF1α as the reference gene and the mean values of the different rbCYP1s in unexposed controls as calibrators. Statistically significant differences among the rbCYP1 genes at a caging site were examined by one-way ANOVA followed by Tukey’s post hoc test and are shown by different letters (p<0.05). Statistically significant differences compared with the unexposed control were examined by one-way ANOVA followed by Bonferroni's post hoc test for selected pairs and are shown by stars (*** = p<0.001, ** = p<0.01, and * = p<0.05) and n=5–6.

Figure 7

mRNA expression patterns for rbCYP1A (A), rbCYP1B (B), rbCYP1C (C) subfamily genes and EROD activity (D) in gills of environmentally exposed rainbow trout (n=5–6). Fish were exposed by two days of caging at four freshwater sites in the Uppsala region. Calculations were made using EF1α as the reference gene and the mean values of the different rbCYP1s at the reference site (Hålsjön) as calibrators. Statistically significant differences in rbCYP1 mRNA expression among trout caged at different sites and unexposed controls were examined by one-way ANOVA followed by Tukey’s post hoc test and are shown by different letters (p<0.05).

Figure 8

Summary of all results on CYP1 mRNA expression in gills of rainbow trout caged in the Uppsala region (n=5–6). The bars represent mean values of relative expression data (fold-unexposed control).