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High Frequency and Heterogeneous Distribution of p53 Mutations in Aflatoxin B₁-induced Mouse Lung Tumors¹

Department of Pharmacology and Toxicology, Queen’s University, Kingston, Ontario, Canada K7L 3N6 [A. S. T., T. E. M.], and Laboratory of Experimental Pathology [J. F. F., R. R. M.] and Molecular Toxicology Group [T. R. D.], National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Inactivation of the p53 tumor suppressor gene is one of the most frequent genetic alterations observed in human lung cancers. However, p53 mutations are more rarely detected in chemically induced mouse lung tumors. In this study, 62 female AC3F1 (A/J x C3H/HeJ) mice were treated with aflatoxin B₁ (AFB₁; 150 mg/kg i.p. divided into 24 doses over 8 weeks). At 6–14 months after dosing, mice were killed, and tumors were collected. A total of 71 AFB₁-induced lung tumors were examined for overexpression of p53 protein by immunohistochemical staining. Positive nuclear p53 staining was observed in 79% of the AFB₁-induced tumors, but the pattern was highly heterogeneous. In approximately 73% of the positively stained tumors, fewer than 5% of cells demonstrated positive staining; in the other 27%, between 10% and 60% of the cells stained positively, with staining localized to the periphery of the tumors in many cases. Single-strand conformational polymorphism analysis of the evolutionarily conserved regions of the p53 gene (exons 5–8) from AFB₁-induced whole lung tumor DNA revealed banding patterns consistent with point mutations in 20 of 76 (26%) tumors, with 85% of the mutations in exon 7 and 15% of the mutations in exon 6. Identification of point mutations could not be confirmed by direct sequence analysis because bands representing putative mutations appeared only weakly on autoradiograms. This was presumably due to the heterogeneous nature of the DNA analyzed. Single-strand conformational polymorphism analysis of DNA from laser capture microdissected cells of paraffin-embedded AFB₁-induced tumor tissue sections stained for p53 produced banding patterns consistent with point mutations in 18 of 30 (60%) DNA samples. Direct sequencing of the microdissected samples revealed mutations at numerous different codons in exons 5, 6, and 7. Of 26 mutations found in microdissected regions from adenomas and carcinomas, 9 were G:CA:T transitions, 11 were A:TG:C transitions, and 5 were transversions (2 G:CT:A, 2 T:AA:T, and 1 A:TC:G), whereas 1 deletion mutation was identified. The concordance between immunostaining and molecular detection of p53 alterations was 72% when laser capture microdissection was used versus 17% based on whole tumor analysis. The high mutation frequency and heterogeneous staining pattern suggest that p53 mutations occur relatively late in AFB₁-induced mouse lung tumorigenesis and emphasize the value of analyzing different staining regions from paraffin-embedded mouse lung tumors.

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