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


CONCLUSIONS
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.
Sanden
Monica
M
National Institute of Nutrition and Seafood Research (NIFES), N-5817 Bergen, Norway. monica.sanden@nifes.no
Olsvik
Pål A
PA
D000818:Animals; D053178:Caspase 6 / Q000032:analysis / Q000235:genetics; D002455:Cell Division / Q000187:drug effects; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics*; D042783:Endothelial Cells / Q000737:chemistry / Q000187:drug effects; D004847:Epithelial Cells / Q000737:chemistry / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D005982:Glutathione Transferase / Q000032:analysis / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis / Q000235:genetics; D017403:In Situ Hybridization; D007274:Injections, Intraperitoneal; D007413:Intestinal Mucosa / Q000737:chemistry* / Q000166:cytology; D016133:Polymerase Chain Reaction; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis*; D020317:Salmo salar / Q000378:metabolism*; D019324:beta-Naphthoflavone / Q000008:administration & dosage / Q000494:pharmacology / Q000633:toxicity*
D018840:HSP70 Heat-Shock Proteins; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D019324:beta-Naphthoflavone; D019363:Cytochrome P-450 CYP1A1; D005982:Glutathione Transferase; D053178:Caspase 6