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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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In situ hybridization staining of CYP1A (brown) in liver of Atlantic salmon Salmo salar exposed to β-naphthoflavone (A) and control (B). Nucleus staining with methyl green. 1: hepatocytes, 2: blood vessel. Scale bar = 50 μm.
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Figure 6: In situ hybridization staining of CYP1A (brown) in liver of Atlantic salmon Salmo salar exposed to β-naphthoflavone (A) and control (B). Nucleus staining with methyl green. 1: hepatocytes, 2: blood vessel. Scale bar = 50 μm.

Mentions: Expression patterns of CYP1A were the same in the different liver sections (data not shown). CYP1A was only expressed in hepatocytes, in both groups (Fig. 5). In Fig. 5 it can be clearly seen that CYP1A is not expressed in connective tissue or bile duct lining cells. In the exposed group, hepatocytes in proximity to blood vessels displayed a stronger staining for CYP1A than hepatocytes more distant to blood circulation (Fig. 6A). This pattern was not observed in the same degree in the control group (Fig. 6B). No major differences were found between the different cell types in the expression of elongation factor (Fig. 7).


Spatial transcription of CYP1A in fish liver.

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

In situ hybridization staining of CYP1A (brown) in liver of Atlantic salmon Salmo salar exposed to β-naphthoflavone (A) and control (B). Nucleus staining with methyl green. 1: hepatocytes, 2: blood vessel. Scale bar = 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: In situ hybridization staining of CYP1A (brown) in liver of Atlantic salmon Salmo salar exposed to β-naphthoflavone (A) and control (B). Nucleus staining with methyl green. 1: hepatocytes, 2: blood vessel. Scale bar = 50 μm.
Mentions: Expression patterns of CYP1A were the same in the different liver sections (data not shown). CYP1A was only expressed in hepatocytes, in both groups (Fig. 5). In Fig. 5 it can be clearly seen that CYP1A is not expressed in connective tissue or bile duct lining cells. In the exposed group, hepatocytes in proximity to blood vessels displayed a stronger staining for CYP1A than hepatocytes more distant to blood circulation (Fig. 6A). This pattern was not observed in the same degree in the control group (Fig. 6B). No major differences were found between the different cell types in the expression of elongation factor (Fig. 7).

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