Detection of micronucleated cells and gene expression changes in glandular stomach of mice treated with stomach-targeted carcinogens.
To assess the genotoxicity of chemicals on the stomach, we developed in vivo assays that can detect micronucleus induction and gene expression changes in epithelial cells of the glandular stomach in mice. Male BALB/c mice were orally given a single dose (100 mg/kg) of N-nitroso-N-methylurea (MNU) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as stomach-targeted carcinogens. The glandular stomach was excised at 4h, 3 and 4 days after administration, and a single cell suspension of epithelial cells was prepared from the everted glandular stomach by EDTA treatment. For determination of micronucleus induction, gastric epithelial cells on days 3 and 4 after administration were fixed with 10% neutral-buffered formalin, stained with a combination of AO-DAPI, and analyzed under fluorescence microscopy. We also examined the induction of micronuclei in peripheral blood of these mice on days 2 and 3 after administration. Moreover, total RNA was extracted from gastric epithelial cells at 4h after administration, and p21 and plk2 expression was analyzed using a quantitative RT-PCR technique. 1) A significant increase of micronucleated cells was observed in the glandular stomach in mice treated with N-nitroso-N-methylurea (MNU) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) compared to mice treated with vehicle. 2) In peripheral blood, induction of micronuclei was observed in mice treated with MNU but not with MNNG. 3) p21 and plk2, which related to cell cycle arrest, were up-regulated in the glandular stomach in mice treated with MNU or MNNG compared to mice treated with vehicle. The present study showed that these assays using glandular stomach may help to evaluate the genotoxicity of chemicals after oral administration.
Okada
Emiko
E
Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi-shi, Tokyo 186-8650, Japan. emiko-okada@yakult.co.jp
Fujiishi
Yohei
Y
Yasutake
Nobuyoshi
N
Ohyama
Wakako
W
D000818:Animals; D002273:Carcinogens / Q000494:pharmacology*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D005753:Gastric Mucosa / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D008297:Male; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D008770:Methylnitrosourea / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D015162:Micronucleus Tests; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D013270:Stomach / Q000166:cytology / Q000187:drug effects*
D002273:Carcinogens; D050759:Cyclin-Dependent Kinase Inhibitor p21; D008769:Methylnitronitrosoguanidine; D008770:Methylnitrosourea; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C444640:serum-inducible kinase