Partial pronase digestion of rat gastric mucosa isolates cells undergoing replicative DNA synthesis.
A pronase digestion procedure for the isolation of gastric mucosal cells was evaluated for its usefulness in measuring unscheduled DNA synthesis (UDS). The method has been claimed to be suited for assessing the genotoxicity potential of compounds. Compounds were given orally to rats. After 13 h [3H]thymidine was injected and after another hour the animals were killed. The dissected stomachs were treated with pronase for 45 min and the incorporation of radioactivity into DNA was determined. Autoradiography was also performed both on the mucosa and on the isolated cell suspension. The cell suspensions were found to include cells undergoing normal replicative (S phase) DNA synthesis. Of all cells isolated, 4.8 +/- 0.6% consisted of S phase cells. The total amount of DNA recovered (corresponding to the number of cells isolated) was variable and ranged from 20 to 671 micrograms DNA, i.e. approximately 30-fold variation. Neither omeprazole (10, 30 and 80 mg/kg) nor ranitidine (215 mg/kg) had any effect on [3H]thymidine incorporation. The known carcinogen 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) increased incorporation. Refeeding of fasted animals increased incorporation of [3H]thymidine almost 4-fold, showing that animal feeding status influences incorporation. Hydroxyurea, the selective inhibitor of S phase DNA synthesis, inhibited [3H]thymidine incorporation in control and omeprazole-treated animals as well as that induced by MNNG by 92-98%. The results clearly show that the pronase digestion procedure used releases cells undergoing normal, replicative DNA synthesis and can therefore neither be used for measurements of UDS nor for the assessment of the genotoxic potential of drugs.
Larsson
H
H
Department of Biology, AB Hässle, Mölndal, Sweden.
Fryklund
J
J
Helander
H F
HF
Wallmark
B
B
D000284:Administration, Oral; D000818:Animals; D001345:Autoradiography; D002469:Cell Separation / Q000379:methods*; D004261:DNA Replication / Q000187:drug effects; D005215:Fasting; D005753:Gastric Mucosa / Q000166:cytology*; D006918:Hydroxyurea / Q000494:pharmacology; D008297:Male; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D009152:Mutagenicity Tests / Q000379:methods*; D009853:Omeprazole / Q000494:pharmacology; D011402:Pronase / Q000378:metabolism*; D011899:Ranitidine / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D016196:S Phase
D008769:Methylnitronitrosoguanidine; D011899:Ranitidine; D011402:Pronase; D009853:Omeprazole; D006918:Hydroxyurea