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Spatial transcription of CYP1A in fish liver.

Olsvik PA, Lie KK, Saele Ø, Sanden M - BMC Physiol. (2007)

Bottom Line: Transcription of CYP1A and GST was higher in the middle section of the liver compared to the distal and proximal parts of the organ.Overall, the qRT-PCR and ISH results reported here suggest that gene expression analysis should be performed on as pure cell populations as possible.If bulk tissue samples are to be used, one should always check how evenly the target genes are expressed in tissue sections and organs in every study.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute of Nutrition and Seafood Research, N-5817 Bergen, Norway. pal.olsvik@nifes.no

ABSTRACT

Background: The aim of this work was to study how evenly detoxifying genes are transcribed spatially in liver tissue of fish. Ten Atlantic salmon Salmo salar were intraperitoneally injected with 50 mg/kg of the strong CYP1A inducer beta-naphthoflavone and liver tissue harvested seven days later. The liver from 10 control and 10 exposed fish were split into eight sections, RNA extracted and three reference (beta-actin, elongation factor 1AB (EF1AB)) and two detoxifying genes (CYP1A and GST) quantified with real-time RT-PCR. The cellular localization of the EF1AB and CYP1A mRNA in the liver of control and beta-naphthoflavone treated fish was then determined by in situ hybridization (ISH) using EF1AB and CYP1A biotinylated oligonucleotide probes.

Results: The study shows that genes encoding phase I and phase II conjugating enzymes are unevenly transcribed in different parts of the liver of Atlantic salmon seven days after a single-dose of beta-naphthoflavone exposure. Transcription of CYP1A and GST was higher in the middle section of the liver compared to the distal and proximal parts of the organ. The ISH data suggest that CYP1A transcription happens mainly in hepatocyte cells in the liver, and that hepatocytes in the vicinity of blood vessels respond stronger to beta-naphthoflavone than cells further away from the blood supply.

Conclusion: Overall, the qRT-PCR and ISH results reported here suggest that gene expression analysis should be performed on as pure cell populations as possible. If bulk tissue samples are to be used, one should always check how evenly the target genes are expressed in tissue sections and organs in every study.

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Related in: MedlinePlus

Mean normalized expression (MNE) of CYP1A (A) and GST (C) in eight sections (1–8) of Atlantic salmon Salmo salar liver from fish exposed to β-naphthoflavone. n = 6. Average MNE of CYP1A is shown in (B) and of GST in (D). n = 48. ● = exposed, □ = control. Note different axis for CYP1A in control and exposed fish.
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Figure 2: Mean normalized expression (MNE) of CYP1A (A) and GST (C) in eight sections (1–8) of Atlantic salmon Salmo salar liver from fish exposed to β-naphthoflavone. n = 6. Average MNE of CYP1A is shown in (B) and of GST in (D). n = 48. ● = exposed, □ = control. Note different axis for CYP1A in control and exposed fish.

Mentions: Fig. 2 shows the induction of CYP1A and GST mRNA in liver of Atlantic salmon seven days after BNF-exposure. When data from eight liver sections from each of six control and six exposed fish are compared, the results show that CYP1A mRNA was 121 fold higher expressed in the exposed animals (Fig. 2A) (t-test, P < 0.0001). In comparison, GST mRNA was only 3.6 times higher expressed in exposed animals than in control fish (Fig. 2C) (t-test, P < 0.0001). In these analyses each of the eight sections from the same fish were treated as individual measurements. Pooling the eight measurements from each fish (n = 6 in each group), and recalculating the data with a non-parametric test yields approximately the same result. Spatial distribution of CYP1A in the eight liver sections is shown in Fig. 2B. In non-exposed animals, CYP1A mRNA expression was evenly expressed throughout the liver, and no significant differences were observed between the sections. The results show that baseline levels of CYP1A mRNA are relatively low in un-stressed Atlantic salmon liver. In contrast, expression patterns in exposed fish show that CYP1A mRNA levels are higher in the middle sections of the liver compared to the proximal and distal regions. The differences in CYP1A mRNA expression between the eight sections of liver were significant (Kruskal-Wallis, P = 0.0001). For GST mRNA, the same general expression pattern was found (Fig. 2D). In control fish, GST was evenly expressed in all eight sections, with very low standard deviations within the groups. In stressed fish, however, GST mRNA expression was highest in the middle part and towards the distal part of the liver, with highest levels in section five and six. The mRNA expression differed significantly between the sections (Kruskal-Walls ANOVA, P < 0.0001). Posthoc tests showed that the biggest difference was between section six and section eight (Dunn's multiple comparison test).


Spatial transcription of CYP1A in fish liver.

Olsvik PA, Lie KK, Saele Ø, Sanden M - BMC Physiol. (2007)

Mean normalized expression (MNE) of CYP1A (A) and GST (C) in eight sections (1–8) of Atlantic salmon Salmo salar liver from fish exposed to β-naphthoflavone. n = 6. Average MNE of CYP1A is shown in (B) and of GST in (D). n = 48. ● = exposed, □ = control. Note different axis for CYP1A in control and exposed fish.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2238752&req=5

Figure 2: Mean normalized expression (MNE) of CYP1A (A) and GST (C) in eight sections (1–8) of Atlantic salmon Salmo salar liver from fish exposed to β-naphthoflavone. n = 6. Average MNE of CYP1A is shown in (B) and of GST in (D). n = 48. ● = exposed, □ = control. Note different axis for CYP1A in control and exposed fish.
Mentions: Fig. 2 shows the induction of CYP1A and GST mRNA in liver of Atlantic salmon seven days after BNF-exposure. When data from eight liver sections from each of six control and six exposed fish are compared, the results show that CYP1A mRNA was 121 fold higher expressed in the exposed animals (Fig. 2A) (t-test, P < 0.0001). In comparison, GST mRNA was only 3.6 times higher expressed in exposed animals than in control fish (Fig. 2C) (t-test, P < 0.0001). In these analyses each of the eight sections from the same fish were treated as individual measurements. Pooling the eight measurements from each fish (n = 6 in each group), and recalculating the data with a non-parametric test yields approximately the same result. Spatial distribution of CYP1A in the eight liver sections is shown in Fig. 2B. In non-exposed animals, CYP1A mRNA expression was evenly expressed throughout the liver, and no significant differences were observed between the sections. The results show that baseline levels of CYP1A mRNA are relatively low in un-stressed Atlantic salmon liver. In contrast, expression patterns in exposed fish show that CYP1A mRNA levels are higher in the middle sections of the liver compared to the proximal and distal regions. The differences in CYP1A mRNA expression between the eight sections of liver were significant (Kruskal-Wallis, P = 0.0001). For GST mRNA, the same general expression pattern was found (Fig. 2D). In control fish, GST was evenly expressed in all eight sections, with very low standard deviations within the groups. In stressed fish, however, GST mRNA expression was highest in the middle part and towards the distal part of the liver, with highest levels in section five and six. The mRNA expression differed significantly between the sections (Kruskal-Walls ANOVA, P < 0.0001). Posthoc tests showed that the biggest difference was between section six and section eight (Dunn's multiple comparison test).

Bottom Line: Transcription of CYP1A and GST was higher in the middle section of the liver compared to the distal and proximal parts of the organ.Overall, the qRT-PCR and ISH results reported here suggest that gene expression analysis should be performed on as pure cell populations as possible.If bulk tissue samples are to be used, one should always check how evenly the target genes are expressed in tissue sections and organs in every study.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute of Nutrition and Seafood Research, N-5817 Bergen, Norway. pal.olsvik@nifes.no

ABSTRACT

Background: The aim of this work was to study how evenly detoxifying genes are transcribed spatially in liver tissue of fish. Ten Atlantic salmon Salmo salar were intraperitoneally injected with 50 mg/kg of the strong CYP1A inducer beta-naphthoflavone and liver tissue harvested seven days later. The liver from 10 control and 10 exposed fish were split into eight sections, RNA extracted and three reference (beta-actin, elongation factor 1AB (EF1AB)) and two detoxifying genes (CYP1A and GST) quantified with real-time RT-PCR. The cellular localization of the EF1AB and CYP1A mRNA in the liver of control and beta-naphthoflavone treated fish was then determined by in situ hybridization (ISH) using EF1AB and CYP1A biotinylated oligonucleotide probes.

Results: The study shows that genes encoding phase I and phase II conjugating enzymes are unevenly transcribed in different parts of the liver of Atlantic salmon seven days after a single-dose of beta-naphthoflavone exposure. Transcription of CYP1A and GST was higher in the middle section of the liver compared to the distal and proximal parts of the organ. The ISH data suggest that CYP1A transcription happens mainly in hepatocyte cells in the liver, and that hepatocytes in the vicinity of blood vessels respond stronger to beta-naphthoflavone than cells further away from the blood supply.

Conclusion: Overall, the qRT-PCR and ISH results reported here suggest that gene expression analysis should be performed on as pure cell populations as possible. If bulk tissue samples are to be used, one should always check how evenly the target genes are expressed in tissue sections and organs in every study.

Show MeSH
Related in: MedlinePlus