INDUCTION OF ESOPHAGEAL TUMORS IN ZINC-DEFICIENT RATS BY SINGLE LOW-DOSES OF N-NITROSOMETHYLBENZYLAMINE (NMBA) - ANALYSIS OF CELL-PROLIFERATION, AND MUTATIONS IN H-RAS AND P53 GENES

Dietary zinc deficiency in rats induces hyperplasia in the esophagus a nd increases N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumo r incidence. Previous work showed a direct relationship between epithe lial cell proliferation and esophageal tumor incidence in rats given m ultiple doses of NMBA. We investigated the effects of single low doses of NMBA in zinc-deficient rats since a single dose of 5.0 mg/kg was r eported to be non-carcinogenic in rats. Zinc-sufficient and deficient rats received a single i.g. dose of NMBA at 0.5 or 2.0 mg/kg. At week 14, tumor incidence was 50% with 0.8 +/- 1.0 tumors/rat, and 80% with 2.2 +/- 1.9 tumors/rat, in deficient groups, D-0.5 and D-2.0, that rec eived the lower and higher dose, respectively. In addition, two small papillomas were found in one out of eight untreated zinc-deficient rat s. None of the NMBA-treated or untreated zinc-sufficient rats had any tumors. Esophageal cell proliferation, as determined by proliferating cell nuclear antigen (PCNA) immunohistochemistry, showed that, irrespe ctive of NMBA treatment, deficient esophagi had significant increases in the number of labeled cells, the total number of cells, and the lab eling index, as compared with zinc-sufficient ones. Mutations in Ha-l as and p53 genes in esophageal tumors were detected by single strand c onformation polymorphism (SSCP) analysis. DNA sequencing of variant co nformers revealed a point mutation (GGA-->GAA, codon 12) in Ha-ras in 4/5 (80%) and 5/8 (63%) tumors, from D-0.5 and D-2.0 rats, respectivel y. Three out of eight tumors from D-2.0 rats exhibited SSCP mobility s hifts within p53 exons 5 and 7: two tumors (2/8, 25%) had missense mut ations and the third, a silent mutation. Of the two tumors with p53 mu tations, one had a double mutation (transition at codon 164, TCA-->TTA ; transversion at codon 241, AGT-->TGT), and the other tumor, a transi tion at codon 172 (AGA-->GGA), with amino acid changes in all cases. I n parallel with PCNA expression, elevated p53 expression was associate d with hyperplastic and dysplastic regions, as well as with tumors, in deficient esophagi. In short, these data indicate that dietary zinc d eficiency,with its associated sustained increased cell proliferation i n the esophagus, can drive an otherwise non-tumorigenic dose of NMBA i nto a highly tumorigenic one.