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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 PCNA labelled nuclei in the mid intestine (MI) of Atlantic salmon (A) In the control group, proliferating cells (PCNA) can be seen (strong black nuclear staining) in several cells in the basal area of the intestinal folds (B) In the treated group, proliferating cells (PCNA) can be seen (weak faint nuclear staining) only in a few cells in the basal area of the intestinal fold.
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Figure 2: Light photomicrographs of PCNA labelled nuclei in the mid intestine (MI) of Atlantic salmon (A) In the control group, proliferating cells (PCNA) can be seen (strong black nuclear staining) in several cells in the basal area of the intestinal folds (B) In the treated group, proliferating cells (PCNA) can be seen (weak faint nuclear staining) only in a few cells in the basal area of the intestinal fold.

Mentions: A single dose of β-naphthoflavone (BNF) significantly decreased the intestinal cell proliferation in the mid intestine (MI). Fish exposed to BNF had a significantly lower density (%) of PCNA-positive cells compared to fish in the control group (t-test: % PCNA, p < 0.03). Exposed fish had a PCNA index of 19.8 ± 4.5 (mean ± SD) compared to the PCNA index of 27.3 ± 4.1 (mean ± SD) in the control group (only for the MI). The relative quantification of PCNA positive cells confirmed the visual differences between the two experimental groups (Fig. 2). For both groups proliferating cells were observed in the basal area of the MI folds with only a few PCNA stained cells located in the cell differentiation zone. For quantification, only strongly stained cells located in the basal area 15 cells from the middle basal area were considered. Fig. 2A shows a representative section of cell proliferation in fish from the control group. Several cells in the basal area are strongly stained and assumed to be in the S-phase of the cell cycle. Fig. 2B shows a representative section of cell proliferation in fish from the exposed group and only weak PCNA staining can be seen. Only a few cells were strongly stained in the examined sections and generally the proliferative compartment length (PCL) (not measured) was smaller compared to the PCL in the exposed group. A brief description of intestinal PCL measurements can be found in Bakke-McKellep et al. [8].


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 PCNA labelled nuclei in the mid intestine (MI) of Atlantic salmon (A) In the control group, proliferating cells (PCNA) can be seen (strong black nuclear staining) in several cells in the basal area of the intestinal folds (B) In the treated group, proliferating cells (PCNA) can be seen (weak faint nuclear staining) only in a few cells in the basal area of the intestinal fold.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Light photomicrographs of PCNA labelled nuclei in the mid intestine (MI) of Atlantic salmon (A) In the control group, proliferating cells (PCNA) can be seen (strong black nuclear staining) in several cells in the basal area of the intestinal folds (B) In the treated group, proliferating cells (PCNA) can be seen (weak faint nuclear staining) only in a few cells in the basal area of the intestinal fold.
Mentions: A single dose of β-naphthoflavone (BNF) significantly decreased the intestinal cell proliferation in the mid intestine (MI). Fish exposed to BNF had a significantly lower density (%) of PCNA-positive cells compared to fish in the control group (t-test: % PCNA, p < 0.03). Exposed fish had a PCNA index of 19.8 ± 4.5 (mean ± SD) compared to the PCNA index of 27.3 ± 4.1 (mean ± SD) in the control group (only for the MI). The relative quantification of PCNA positive cells confirmed the visual differences between the two experimental groups (Fig. 2). For both groups proliferating cells were observed in the basal area of the MI folds with only a few PCNA stained cells located in the cell differentiation zone. For quantification, only strongly stained cells located in the basal area 15 cells from the middle basal area were considered. Fig. 2A shows a representative section of cell proliferation in fish from the control group. Several cells in the basal area are strongly stained and assumed to be in the S-phase of the cell cycle. Fig. 2B shows a representative section of cell proliferation in fish from the exposed group and only weak PCNA staining can be seen. Only a few cells were strongly stained in the examined sections and generally the proliferative compartment length (PCL) (not measured) was smaller compared to the PCL in the exposed group. A brief description of intestinal PCL measurements can be found in Bakke-McKellep et al. [8].

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