Zonula Occludens-1 alterations and enhanced intestinal permeability in methotrexate-treated rats.
OBJECTIVE
The molecular mechanisms that underlie the methotrexate (MTX)-mediated disruption of intestinal barrier function have not been fully characterized. Epithelial barrier function is determined in large part by a multiprotein complex located at the most apical part of the lateral membrane, which is referred to as a tight junction (TJ). In the present study, we examined the alteration of zonula occludens-1 (ZO-1), which is a scaffolding protein that plays a pivotal role in the formation of TJs, to identify an additional molecular mechanism for epithelial barrier dysfunction.


METHODS
Male Wistar rats were administered MTX (15 mg kg(-1)) orally once daily for 3-5 days. Intestinal mucosal permeability was determined using the in vitro everted intestinal sac technique. Mucosal inflammation was assessed by myeloperoxidase activity and production of reactive oxygen species. Altered expression, tyrosine phosphorylation, and localization of ZO-1 were evaluated by RT-PCR, Western blotting, immunoprecipitation, and immunohistochemistry.


RESULTS
A barrier function study revealed increased intestinal permeability in rats treated with MTX for 4 days, as indicated by enhanced fluorescein isothiocyanate-dextran flux. In addition, mucosal inflammation was linked to enhanced intestinal permeability. Quantitative analysis of ZO-1 expression showed the absence of significant differences in MTX-treated rats, whereas tyrosine dephosphorylation of ZO-1 was observed. Moreover, we also detected an obvious reduction of ZO-1 immunostaining along the apical membrane of intestinal villi.


CONCLUSIONS
These results indicate that, in MTX-treated rats, ZO-1 alterations may contribute to disturbance of the TJ barrier, which leads to enhanced intestinal permeability.
Hamada
Kazuma
K
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan.
Shitara
Yoshihisa
Y
Sekine
Shuichi
S
Horie
Toshiharu
T
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000009:adverse effects*; D015153:Blotting, Western; D003911:Dextrans / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000378:metabolism; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D007249:Inflammation / Q000378:metabolism; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000378:metabolism* / Q000473:pathology; D007421:Intestine, Small / Q000187:drug effects* / Q000378:metabolism; D049449:Luminescence; D008297:Male; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism*; D008727:Methotrexate / Q000009:adverse effects*; D010539:Permeability / Q000187:drug effects; D010750:Phosphoproteins / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019108:Tight Junctions / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
D000964:Antimetabolites, Antineoplastic; D003911:Dextrans; D008565:Membrane Proteins; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; C015219:fluorescein isothiocyanate dextran; D014443:Tyrosine; D016650:Fluorescein-5-isothiocyanate; D008727:Methotrexate