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Intestinal cellular localization of PCNA protein and CYP1A mRNA in Atlantic salmon Salmo salar L. exposed to a model toxicant.

Sanden M, Olsvik PA - BMC Physiol. (2009)

Bottom Line: PCNA protein and CYP1A mRNA were successfully localized in the intestinal cells (MI) of both experimental groups.At the cellular level, BNF significantly lowered intestinal cell proliferation and increased the CYP1A mRNA levels compared to the control group.The other examined genes did not show any differential regulation in the experimental fish group.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute of Nutrition and Seafood Research (NIFES), N-5817 Bergen, Norway. monica.sanden@nifes.no

ABSTRACT

Background: The aim of the study was to examine the intestinal cellular localization of proliferating cell nuclear antigen (PCNA) and cytochrome P450 A1 (CYP1A) expression in Atlantic salmon Salmo salar L. exposed to a model toxicant. The stress response was induced by intraperitoneal injection of four salmon with a single dose (50 mg/kg) of the CYP1A inducer beta-naphthoflavone (BNF) and intestinal tissue (mid and distal intestine; MI and DI) was sampled seven days later. Samples for histology and gene transcription analysis were collected from four exposed fish and four control fish. PCNA was assessed by immunohistochemistry, CYP1A mRNA was studied by in situ hybridization (ISH) and finally the transcription of five genes was quantified by real-time quantitative RT-PCR (real-time RT-PCR); two detoxifying genes (CYP1A and glutathione S-transferase; GST), a stress marker gene (heat shock protein 70; HSP70), PCNA and a gene marker of apoptosis (caspase 6A).

Results: PCNA protein and CYP1A mRNA were successfully localized in the intestinal cells (MI) of both experimental groups. At the cellular level, BNF significantly lowered intestinal cell proliferation and increased the CYP1A mRNA levels compared to the control group. The real-time RT-PCR data, which showed an increased mRNA expression both in the MI and DI of 139- and 62-fold, respectively, confirmed the increased cellular CYP1A mRNA levels detected using ISH. HSP70 expression was also up-regulated in the exposed fish. The other examined genes did not show any differential regulation in the experimental fish group.

Conclusion: This study showed that CYP1A mRNA had a specific intestinal cellular transcription pattern in Atlantic salmon exposed to BNF. At the cellular level CYP1A mRNA expression was always observed at or around the cell nucleus close to the basolateral cell membrane and at the tissue level CYP1A mRNA expression was most frequently observed in the basal and apex area of the intestinal folds. Taken together, a link between the intestinal detoxification system (CYP1A) and cell renewal system (PCNA) is indicated with these two processes being inversely correlated in BNF exposed fish.

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Light photomicrographs of CYP1A mRNA labelled mid intestine (MI) of Atlantic salmon (A) Negative control (sense). No CYP1A staining and only nuclear staining can be seen (B) Control group (antisense). CYP1A staining with faint and low mRNA levels especially in the basal area of the intestinal folds (C) Exposed group (antisense). CYP1A staining with strong and high mRNA levels especially in the basal area and the apex area of the intestinal fold. Inside the cells CYP1A are located at and around the nucleus. In the photographs CYP1A mRNA levels can be seen with a dark colour and all cell nuclei with a green staining. All pictures are stained with the nuclear stain; methyl green.
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Figure 3: Light photomicrographs of CYP1A mRNA labelled mid intestine (MI) of Atlantic salmon (A) Negative control (sense). No CYP1A staining and only nuclear staining can be seen (B) Control group (antisense). CYP1A staining with faint and low mRNA levels especially in the basal area of the intestinal folds (C) Exposed group (antisense). CYP1A staining with strong and high mRNA levels especially in the basal area and the apex area of the intestinal fold. Inside the cells CYP1A are located at and around the nucleus. In the photographs CYP1A mRNA levels can be seen with a dark colour and all cell nuclei with a green staining. All pictures are stained with the nuclear stain; methyl green.

Mentions: Results of CYP1A mRNA transcription analysis by ISH in the MI can be seen in Fig. 3. In the exposed groups (Fig 3C) CYP1A mRNA was mainly localized in the basal area and apex area of the intestinal folds. Only weak staining could be seen in the cell differentiation zone of the MI fold. This expression pattern was the same in all fish exposed to BNF. CYP1A mRNA was mainly seen in one intestinal cell type, the enterocytes. Another interesting observation was the intracellular localization of the CYP1A transcript around the nucleus close to the basolateral membrane. CYP1A transcripts were only seen as weak and random staining in the cytoplasm close to the apical membrane. This feature was prominent both in the basal area and apex area of the MI fold. Results of CYP1A mRNA transcription in the control fish can be seen in Fig. 3B. In this group only weak signals could be seen and with no particular cellular or tissue expression pattern. Fig. 3A shows a representative example of a sense probe control.


Intestinal cellular localization of PCNA protein and CYP1A mRNA in Atlantic salmon Salmo salar L. exposed to a model toxicant.

Sanden M, Olsvik PA - BMC Physiol. (2009)

Light photomicrographs of CYP1A mRNA labelled mid intestine (MI) of Atlantic salmon (A) Negative control (sense). No CYP1A staining and only nuclear staining can be seen (B) Control group (antisense). CYP1A staining with faint and low mRNA levels especially in the basal area of the intestinal folds (C) Exposed group (antisense). CYP1A staining with strong and high mRNA levels especially in the basal area and the apex area of the intestinal fold. Inside the cells CYP1A are located at and around the nucleus. In the photographs CYP1A mRNA levels can be seen with a dark colour and all cell nuclei with a green staining. All pictures are stained with the nuclear stain; methyl green.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667469&req=5

Figure 3: Light photomicrographs of CYP1A mRNA labelled mid intestine (MI) of Atlantic salmon (A) Negative control (sense). No CYP1A staining and only nuclear staining can be seen (B) Control group (antisense). CYP1A staining with faint and low mRNA levels especially in the basal area of the intestinal folds (C) Exposed group (antisense). CYP1A staining with strong and high mRNA levels especially in the basal area and the apex area of the intestinal fold. Inside the cells CYP1A are located at and around the nucleus. In the photographs CYP1A mRNA levels can be seen with a dark colour and all cell nuclei with a green staining. All pictures are stained with the nuclear stain; methyl green.
Mentions: Results of CYP1A mRNA transcription analysis by ISH in the MI can be seen in Fig. 3. In the exposed groups (Fig 3C) CYP1A mRNA was mainly localized in the basal area and apex area of the intestinal folds. Only weak staining could be seen in the cell differentiation zone of the MI fold. This expression pattern was the same in all fish exposed to BNF. CYP1A mRNA was mainly seen in one intestinal cell type, the enterocytes. Another interesting observation was the intracellular localization of the CYP1A transcript around the nucleus close to the basolateral membrane. CYP1A transcripts were only seen as weak and random staining in the cytoplasm close to the apical membrane. This feature was prominent both in the basal area and apex area of the MI fold. Results of CYP1A mRNA transcription in the control fish can be seen in Fig. 3B. In this group only weak signals could be seen and with no particular cellular or tissue expression pattern. Fig. 3A shows a representative example of a sense probe control.

Bottom Line: PCNA protein and CYP1A mRNA were successfully localized in the intestinal cells (MI) of both experimental groups.At the cellular level, BNF significantly lowered intestinal cell proliferation and increased the CYP1A mRNA levels compared to the control group.The other examined genes did not show any differential regulation in the experimental fish group.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute of Nutrition and Seafood Research (NIFES), N-5817 Bergen, Norway. monica.sanden@nifes.no

ABSTRACT

Background: The aim of the study was to examine the intestinal cellular localization of proliferating cell nuclear antigen (PCNA) and cytochrome P450 A1 (CYP1A) expression in Atlantic salmon Salmo salar L. exposed to a model toxicant. The stress response was induced by intraperitoneal injection of four salmon with a single dose (50 mg/kg) of the CYP1A inducer beta-naphthoflavone (BNF) and intestinal tissue (mid and distal intestine; MI and DI) was sampled seven days later. Samples for histology and gene transcription analysis were collected from four exposed fish and four control fish. PCNA was assessed by immunohistochemistry, CYP1A mRNA was studied by in situ hybridization (ISH) and finally the transcription of five genes was quantified by real-time quantitative RT-PCR (real-time RT-PCR); two detoxifying genes (CYP1A and glutathione S-transferase; GST), a stress marker gene (heat shock protein 70; HSP70), PCNA and a gene marker of apoptosis (caspase 6A).

Results: PCNA protein and CYP1A mRNA were successfully localized in the intestinal cells (MI) of both experimental groups. At the cellular level, BNF significantly lowered intestinal cell proliferation and increased the CYP1A mRNA levels compared to the control group. The real-time RT-PCR data, which showed an increased mRNA expression both in the MI and DI of 139- and 62-fold, respectively, confirmed the increased cellular CYP1A mRNA levels detected using ISH. HSP70 expression was also up-regulated in the exposed fish. The other examined genes did not show any differential regulation in the experimental fish group.

Conclusion: This study showed that CYP1A mRNA had a specific intestinal cellular transcription pattern in Atlantic salmon exposed to BNF. At the cellular level CYP1A mRNA expression was always observed at or around the cell nucleus close to the basolateral cell membrane and at the tissue level CYP1A mRNA expression was most frequently observed in the basal and apex area of the intestinal folds. Taken together, a link between the intestinal detoxification system (CYP1A) and cell renewal system (PCNA) is indicated with these two processes being inversely correlated in BNF exposed fish.

Show MeSH
Related in: MedlinePlus