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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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Gastrointestinal tract of Atlantic salmon (≈ 100 grams) showing oesophagus, stomach, pyloric caeca, mid intestine (MI) and distal intestine (DI). A) Arrow showing where the DI samples were taken for gene expression B) Arrow showing where the MI samples were taken for gene expression, immunohistochemistry and in situ hybridization.
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Figure 1: Gastrointestinal tract of Atlantic salmon (≈ 100 grams) showing oesophagus, stomach, pyloric caeca, mid intestine (MI) and distal intestine (DI). A) Arrow showing where the DI samples were taken for gene expression B) Arrow showing where the MI samples were taken for gene expression, immunohistochemistry and in situ hybridization.

Mentions: The aim of the study was to examine the cellular localization of CYP1A mRNA and PCNA protein in a stress-induced intestine. Tissues from the middle part of the intestine (MI) and the distal part of the intestine (DI) were sampled for analysis (Fig. 1). To confirm that the model toxicant successfully induced a stress reaction in the intestine, the expression of five genes encoding proteins involved in cellular response to stressors were examined; CYP1A, HSP70, GST, PCNA and caspase 6A by real-time RT-PCR. The histological parameters PCNA protein and CYP1A mRNA, were only examined in the MI since previous observations indicate a higher metabolic capacity in the proximal intestine of fish compared to the distal parts [10,11,18].


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)

Gastrointestinal tract of Atlantic salmon (≈ 100 grams) showing oesophagus, stomach, pyloric caeca, mid intestine (MI) and distal intestine (DI). A) Arrow showing where the DI samples were taken for gene expression B) Arrow showing where the MI samples were taken for gene expression, immunohistochemistry and in situ hybridization.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Gastrointestinal tract of Atlantic salmon (≈ 100 grams) showing oesophagus, stomach, pyloric caeca, mid intestine (MI) and distal intestine (DI). A) Arrow showing where the DI samples were taken for gene expression B) Arrow showing where the MI samples were taken for gene expression, immunohistochemistry and in situ hybridization.
Mentions: The aim of the study was to examine the cellular localization of CYP1A mRNA and PCNA protein in a stress-induced intestine. Tissues from the middle part of the intestine (MI) and the distal part of the intestine (DI) were sampled for analysis (Fig. 1). To confirm that the model toxicant successfully induced a stress reaction in the intestine, the expression of five genes encoding proteins involved in cellular response to stressors were examined; CYP1A, HSP70, GST, PCNA and caspase 6A by real-time RT-PCR. The histological parameters PCNA protein and CYP1A mRNA, were only examined in the MI since previous observations indicate a higher metabolic capacity in the proximal intestine of fish compared to the distal parts [10,11,18].

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