Detection of Apoptosis, p53, and Bax Protein Expression in Various Tissues of Neonatal and Adult Rats after Exposure to Gamma Irradiation or Aflatoxin B1

Abstract

Levels of apoptosis induction, nuclear p53 expression, and cytoplasmic Bax protein expression were evaluated in various tissues before and after exposure of 2 neonatal and 6 adult rats to gamma irradiation. These studies served to provide positive control material for an evaluation of the effects of aflatoxin B1 administered to adult rats at several doses followed by sacrifice at several timepoints. The irradiation work also was run to determine any differences between neonate and adult expression of apoptosis, p53 and Bax. For the aflatoxin B1 study, only dUTP-biotin nick end labeling (TUNEL) staining to demonstrate apoptotic bodies and irnmunohistochemical staining to localize p53 nuclear protein were performed. In neonates, untreated control liver showed slight apoptosis. Following irradiation, apoptosis increased markedly in liver; other neonate tissues showed no apoptosis before or after irradiation. Results in untreated adult rats revealed slight apoptosis in some tissues with high cell turnover rates (thymus, spleen, intestine, and lymph node). Little apoptosis was evident in liver or kidney. Following irradiation, apoptosis increased in all tissues examined, especially at the 6 hr timepoint. In aflatoxin B1-treated adults, apoptosis evaluated by TUNEL appeared to increase slightly 24 hr post-dose, although morphological assessment showed increases at 3, 6, and 24 hr postdose sacrifices. The degree of apoptosis was directly proportional to increasing aflatoxin B1 dose. In neonates, p53 showed a significant increase only in thymus following irradiation, especially at the 4 hr timepoint. In adults, irradiation produced significant increases of p53 expression in all tissues except kidney. Following aflatoxin administration, p53 was present only in rat liver at 24 hr post-dose; and 1, 3, and 10 mg/kg doses showed slight dose-related increases in the number of positive cells. Bax protein staining was ubiquitous in adults and was present in all tissues; its detection was not affected by irradiation. (The J Histotechnol 23:11, 2000)

Keywords:

• immunohistochemistry
• positive controls
• TUNEL