Volume 6, Issue 3, Pages 149-156 (December 1999) Apoptosis and 8-hydroxydeoxyguanosine levels in renal tissue after an injection of ferric nitrilotriacetate, a renal carcinogen, in male Wistar rats maintained on vitamin E- deficient, -normal or -supplemented diets  Kaori Eguchi, Takashi Akiyama, Takahiro Kiriu, Yukiko Minamiyama, Shigeru Okada  Pathophysiology  Volume 6, Issue 3, Pages 149-156 (December 1999) DOI: 10.1016/S0928-4680(99)00006-1

Fig. 1 Vitamin E concentrations in the rat kidney, liver and serum at the time of sacrifice. In the deficient animals, the levels of vitamin E were nearly zero in serum, and decreased in the kidney and liver. The data are mean±S.D. (n=6). *P<0.05, **P<0.01 (Student’s t-test). Pathophysiology 1999 6, 149-156DOI: (10.1016/S0928-4680(99)00006-1)

Fig. 2 The survival rates of rats given different doses of Fe-NTA injection. The injected doses of Fe-NTA were 1, 3 and 8 mg Fe per kg body weight (each n=6 in each of the three vitamin E groups). The vitamin E-deficient animals given Fe-NTA 3 mg (–––) or 8 mg (—) Fe per kg body weight died within 3.5 h after the injection. All of the vitamin E-normal and -supplemented animals given 8 mg Fe per kg body weight and the deficient animals given 1 mg Fe per kg body weight Fe-NTA survived (---). Pathophysiology 1999 6, 149-156DOI: (10.1016/S0928-4680(99)00006-1)

Fig. 3 (A) The levels of hemoglobin in serum and (B) the hematocrit. The vitamin E-deficient group showed high levels of hemoglobin and low hematocrits. Hemolysis was observed in the vitamin E-deficient rats. Pathophysiology 1999 6, 149-156DOI: (10.1016/S0928-4680(99)00006-1)

Fig. 4 Hematoxylin–eosin staining of rat renal tissues 1 h after injection of intraperitoneal saline or Fe-NTA 8 mg Fe per kg body weight. (A–C) The saline injected animals showed no obvious histological differences among the three groups (A, vitamin E-deficient; B, vitamin E-normal; C, vitamin E-supplemented rats). (D–F) In Fe-NTA injected animals, the vitamin E-deficient (D) and -normal (E) rats showed pyknosis or karyorrhexis of the proximal tubular cells in the cortex, and the vitamin E-deficient rats also showed necrosis. (F) In the vitamin E-supplemented rats, the renal tissue injuries were very slight. Original magnification (A–F): ×400. Pathophysiology 1999 6, 149-156DOI: (10.1016/S0928-4680(99)00006-1)

Fig. 5 Demonstration of apoptosis by TUNEL 1 h after an 8 mg Fe per kg Fe-NTA injection. In agreement with the pyknotic or karyorrhexic nuclei shown by hematoxylin–eosin staining, numerous positively stained nuclei were seen in proximal tubular cells in the cortex (A, vitamin E-deficient; B, vitamin E-normal). (C) In the vitamin E-supplemented group, positively stained nuclei were few. Original magnification (A–C): ×400. Pathophysiology 1999 6, 149-156DOI: (10.1016/S0928-4680(99)00006-1)

Fig. 6 Immunohistochemical staining of 8-OHdG (N45.1) 1 h after an intraperitoneal injection of Fe-NTA 8 mg Fe per kg body weight. Positive nuclear staining of proximal (arrow) and distal (double arrows) tubular cells was seen in both the vitamin E-deficient (A) and vitamin E-normal (B) rats. The nuclei that had ungergone apoptosis or necrosis were negative for N45.1. (C) In the vitamin E-supplemented rats, only a few positively stained nuclei of the distal tubular cells were observed. Original magnification (A–C): X400. Pathophysiology 1999 6, 149-156DOI: (10.1016/S0928-4680(99)00006-1)

Fig. 7 Determination of 8-OHdG in rat renal tissue by HPLC-ECD. One hour after an Fe-NTA injection of 8 mg Fe per kg, the animals showed increased levels of 8-OHdG compared to the saline-treated groups. In the vitamin E-supplemented group, the generation of 8-OHdG was inhibited compared with the other groups. There was no significant differences in the level of 8-OHdG between the vitamin E-deficient and -normal groups. The columns show mean±S.D. (n=13). *P<0.05, **P<0.01, ***P<0.001 (Student’s t-test). Pathophysiology 1999 6, 149-156DOI: (10.1016/S0928-4680(99)00006-1)