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Strain variation in renal carcinogenesis by N-ethyl-N-hydroxyethylnitrosamine in F1 (Wistar–Fischer) rats
Cancer Letters 170 (2001) 125±130
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Strain variation in renal carcinogenesis by N-ethyl-Nhydroxyethylnitrosamine in F1 (Wistar±Fischer) rats Kenji Takashima a, Seiichiro Ozono a,*, Isamu Nakanou a, Masahiko Ota a, Masahiro Tanaka a, Mitsuru Tani a, Kazuya Hirao a, Yoshihiko Hirao a, Shinichi Kuwashima b, Yoshio Hiasa c a
Department of Urology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara, Japan Department of Pathology, Nara Prefectunal Nara Hospital, 1-30-1 Hiramatu-cho Nara, Nara, Japan c The Second Department of Pathology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara, Japan b
Received 13 April 2001; received in revised form 17 May 2001; accepted 21 May 2001
Abstract The present study was conducted to compare the incidences of renal tumors in Wistar (W), Fischer (F) and F1 rats (WF: female Wistar rats £ male Fischer rats; FW: female Fischer rats £ male Wistar rats) induced by N-ethyl-N-hydroxyethylnitrosamine (EHEN). Levels of 8-OHdG in renal DNA were also investigated in Wistar and Fischer rats. After 2000 ppm of EHEN was administered orally for 2 weeks, the animals were fed basal diet until week 32. Wistar males and females demonstrated signi®cantly higher sensitivity regarding induction of renal lesions, while both WF and FW rats had similar incidences, generally intermediate between those for the two parent strains. The formation of 8-OHdG was maximal 60±180 min after an intraperitoneal dose of 750 mg/kg to Wistar and Fischer rats, which correlates with the increase tending to the incidence of renal tumors in male and female Wistar and Fischer rats. The results suggest that EHEN induction of renal tumors is related to oxygen radical damage and that the genes in the Wistar strain responsible for the sensitivity are not inherited in a sex-dependent fashion, despite the male being more susceptible. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Renal carcinogenesis; N-Ethyl-N-hydroxyethylnitrosamine; F1; 8-OHdG; Oxidative DNA damage
1. Introduction N-Ethyl-N-hydroxyethylnitrosamine (EHEN) is known to induce renal epithelial tumors in rats [1,2] with species-, strain- and sex-related differences present in the incidences of lesions. The Wistar rat strain is more sensitive to EHEN induction of renal carcinogenesis than Fischer rats, and males are more prone to the development of lesion than females of the * Corresponding author. Tel.: 181-744-22-3051 ext. 2338; fax: 181-744-22-9280. E-mail address: [email protected] (S. Ozono).
same strain [3]. Administration of EHEN has recently been reported to increase the formation of 8-OHdG, a marker of oxidative damage to DNA serving as an index of carcinogenesis in the renal cortex [4]. The present study was conducted to examine the incidences of renal tumors in Wistar, Fischer and F1 rats (WF: female Wistar rats £ male Fischer rats; FW: female Fischer rats £ male Wistar rats) using an experimental model of EHEN-induced renal carcinogenesis [1,3]. We examined levels of 8-OHdG in renal DNA in Wistar and Fischer rats.
0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00608-5
126
K. Takashima et al. / Cancer Letters 170 (2001) 125±130
2. Materials and methods 2.1. Chemicals EHEN was purchased from Nacalai Tesque (Kyoto, Japan), Nuclease Pl, Proteinase K and alkaline phosphatase were purchased from Sigma (St. Louis, MO) and an 8-OHdG Check kit was purchased from Nikken Food (Shizuoka, Japan). 2.2. Experiment I 2.2.1. Animals and experimental design Ten-week-old Wistar (10 male and 20 female) and Fischer (10 male and 20 female) rats were purchased from Charles River Co. Japan Inc. (Atsugi, Japan). After 1 week of acclimatization, one male Wistar or Fischer rat and two female Fischer rats or two female Wistar rats were housed in the same cage and allowed to produce F1 rats. A total of 222 animals at 6 weeks of age (26 male and 26 female rats each of Wistar and Fischer strains, 27 male and 40 female WF rats, and 25 male and 26 female FW rats) were housed in plastic cages at 24 ^ 18C and 55 ^ 5% relative humidity. All rats were housed two to a cage and fed on powdered basal diet (Oriental M Powder; Oriental
Yeast, Osaka, Japan). They received 2000 ppm of EHEN in tap water ad libitum for the ®rst 2 weeks from week 6, and then tap water (chemical-free) ad libitum for the 30 week duration of the experiment. The experiments were performed according to the Principles of Laboratory Animal Care of the NIH. 2.2.2. Histopathological studies All rats were sacri®ced under ether anesthesia at the end of week 32. Whole body and kidney weights were recorded. Kidneys were preserved in 10% phosphatebuffered formalin solution. After overnight ®xation, slices were processed for embedding in paraf®n, sectioned at 5 mm, and stained with hematoxylin± eosin (HE). The criteria for histopathological classi®cation of the kidney lesions were as described previously for simple hyperplasia, adenomatous hyperplasia and renal cell neoplasms [4,5]. 2.3. Experiment II 2.3.1. Animals and experimental design Groups of 18 5-week-old male and female Wistar and Fischer rats were fed on basal diet and tap water ad libitum for 1 week. All rats were housed in plastic cages at 24 ^ 18C and 55 ^ 5% relative humidity.
Fig. 1. Experimental design of Experiment II.
K. Takashima et al. / Cancer Letters 170 (2001) 125±130
127
Table 1 Weight of body and kidney and EHEN uptake for rats treated with EHEN Group
1 WF 2 WC 3 FF 4 FC 5 WFF 6 WFC 7 FWF 8 FWC
No. of rats
26 26 26 26 27 40 25 25
Body weight (g)
Kidney weight
Initial
Final
Absolute (g)
Relative (%)
Uptake
Uptake per 100 g of initial body weight
240.4 174.9 154.4 118.0 175.5 145.7 180.4 133.7
625.8 352.9 388.4 199.2 550.4 292.3 550.4 303.5
2.0 1.0 1.1 0.6 1.5 0.8 1.6 0.8
0.31 0.28 0.28 0.30 0.27 0.27 0.29 0.26
0.482 0.255 0.129 0.115 0.147 0.125 0.184 0.126
0.20 0.15 0.084 0.097 0.084 0.086 0.10 0.094
After 1 week, the animals treated with EHEN at 750 mg/kg body weight and control groups were given water. The experimental design for Experiment II is shown in Fig. 1. Rats were sacri®ced at 0.5, 1, 2, 3 or 4 h after intraperitoneal injection of EHEN. Control rats were sacri®ced at 2 h after injection of water. Three rats in each group were killed at each time-point and their livers and kidneys were excised and frozen in liquid nitrogen until use. 2.3.2. Determination of 8-OHdG formation The renal cortex was separated and milled under liquid nitrogen and then subjected to proteolytic treatment with Proteinase K before extraction of DNA by a conventional method with a mixture of phenol, chloroform and isoamyl alcohol, and subsequent puri®cation by ethanol precipitation. The DNA concentration was determined from absorbance at 260/280 nm.
Total EHEN (g)
For the 8-OHdG assay, DNA samples were dissolved in distilled water at a concentration of 50 mg/50 ml, heat-denatured at 958C for 5 min and cooled on ice. The samples (50 ml) were mixed with 1.5 ml of 1 M sodium acetate buffer (pH 4.8) and 2.5 units of nuclease Pl and digested by incubation at 378C for 1 h in the dark. To the solution were added 9 ml of 1 M Tris± HCl buffer (pH 7.5) and 1.5 units of alkaline phosphatase and the products were digested by incubation at 378C for 1 h. Aliquots (50 mg DNA/59.5 ml) of this mixture were assayed by ELISA using an 8-OHdG check kit. 2.4. Statistical analysis Quantitative data were analyzed for statistically signi®cant differences using Student's t-test for multiplicity and the x 2-test for incidence.
Table 2 Incidences of renal lesions in male rats treated with EHEN Group
1 WF 3 FF 5 WFF 7 FWF a b c
No. of rats
26 26 27 25
Simple hyperplasia
Adenomatous hyperplasia
Renal cell neoplasm
No. of rats (%)
Total no. (/rat)
No. of rats (%)
Total no. (/rat)
No. of rats (%)
26 (100.0) 23 (88.5) 25 (92.6) 24 (96.0)
438 (16.8) a,b,c 57 (2.2) 158 (5.9) a 146 (5.8) a
13 (50.0) a 2 (7.7) 7 (25.9) 7 (28.0)
20 (0.77) a 2 (0.08) 9 (0.33) 9 (0.36)
10 0 4 5
Signi®cantly different from group 3 (P , 0:05). Signi®cantly different from group 5 (P , 0:05). Signi®cantly different from group 7 (P , 0:05).
(38.4) a (0.0) (14.8) (20.0)
Total no. (/rat) 21 0 8 7
(0.81) a (0.0) (0.30) a (0.28) a
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K. Takashima et al. / Cancer Letters 170 (2001) 125±130
Table 3 Incidences of renal lesions in female rats treated with EHEN Group
2 WC 4 FC 6 WFC 8 FWC a b
No. of rats
26 26 40 26
Simple hyperplasia
Adenomatous hyperplasia
Renal cell neoplasm
Total no. (/rat)
No. of rats (%)
Total no. (/rat)
7 (0.27) a,b 0 (0.00) 1 (0.03) 5 (0.19)
5 (19.2) a 1 (3.8) 3 (7.5) 3 (11.5)
8 1 3 4
No. of rats (%)
Total no. (/rat)
No. of rats (%)
21 (80.8) a 2 (7.7) 34 (85.0) a 19 (73.1) a
75 (2.9) a 2 (0.8) 90 (2.3) a 60 (2.3) a
6 0 1 4
(23.1) a,b (0.0) (2.5) (15.4)
(0.31) a (0.038) (0.075) (0.15)
Signi®cantly different from group 4 (P , 0:05). Signi®cantly different from group 6 (P , 0:05).
3. Results 3.1. Experiment I The mean body weights, organ weights and EHEN uptake for each group at 32 experimental weeks are summarized in Table 1. In both males and females, the greatest EHEN uptake was observed in Wistar rats. In terms of EHEN uptake per 100 g of initial body weight, however, no differences were observed between F1 rats and Fischer rats in both males and females (Table 1). The incidences of kidney hyperplasias and tumors are shown in Tables 2 and 3. Among the male rat groups (Table 2, Fig. 2), male Wistar, WF and FW rats showed relatively high frequencies of renal lesions with EHEN administration when compared to Fischer rats. Only in Fischer rats was renal cell tumor development not induced. Incidences of renal lesions in male WF and FW rats did not differ with incidences at levels intermediate between those for Wistar and Fischer rats. In the female rat groups
(Table 3, Fig. 2), Wistar rats again showed relatively high frequencies for renal lesions while they were almost absent in the Fischer strain. Female F1 rats again showed intermediate values. Like the parent strains, F1 males showed high frequencies of renal cell neoplasms compared with F1 females (Fig. 2). 3.2. Experiment II Changes in 8-OHdG levels in the DNA of the renal cortex at various time-points (0.5±4 h) after treatment are shown in Table 4. The formation of 8-OHdG in kidney was time-dependently increased reaching a maximum 2 h after an injection of EHEN. Thereafter, the levels declined gradually. Peaks for 8-OHdG formation were 28.1, 83.1, 34.1, and 8.2 ng/50 mg DNA for female and male Wistar rats and female and male Fischer rats, respectively (Table 4). Maximum increases in 8-OHdG, which were calculated by difference between 8-OHdG after EHEN injection and after water injection, were 16.3, 42.6, 2.55, and 8.39 ng/50 mg DNA for female and male Wistar rats and female and male Fischer rats, respectively. The greatest increase was obtained in male Wistar rats, showing the same tendency as the incidence of renal tumors. In terms of the increase in 8-OHdG after 2 h, a signi®cant difference (P , 0:05) was observed between male Wistar rats and all other groups, and between female Fischer rats and all other groups. 4. Discussion
Fig. 2. Sex differences in the number of renal lesions (total number) in rats treated with EHEN. aSigni®cantly different from females of the same strain (P , 0:05).
Druckrey et al. [2] ®rst reported that EHEN induces tumors in kidneys, liver and ovaries of rats. Hiasa et al. [1] showed that renal tubular cell tumors selec-
K. Takashima et al. / Cancer Letters 170 (2001) 125±130
129
Table 4 8-OHdG levels in kidney DNA from rats treated with EHEN and water Group
1 WF 2 WC 3 FF 4 FC
Body weight (g)
214.8 158.4 133.6 104.7
8-OHdG (ng/50 mg DNA) after EHEN injection
8-OHdG after water injection
0.5 h
1h
2h
3h
4h
2h
40.2 12.9 25.5 5.5
75.3 12.9 27.5 8.2
83.1 27.9 34.1 6.8
67.1 28.1 29.5 6.3
58.6 16.0 26.1 6.5
40.6 11.8 25.7 5.7
tively develop at high incidence, without other kinds of renal tumors, in Wistar rats with 0.1% EHEN added to the diet for 2 weeks. Differences in the susceptibility to carcinogenesis of certain organs in various animal strains and species have been widely examined. Konishi et al. [3] showed higher sensitivity of the rat kidney to EHEN than mouse and hamster renal tissue. It further appears that rats of the Wistar strain are more susceptible with regard to EHEN renal carcinogenesis than are Fischer rats, and that males are more sensitive than females of the same strain. Various modi®ers have been detected with mediumterm organ bioassays in rats treated with EHEN [4±8]. Regarding the mechanism of action, an importance for DNA alkylation in nitrosamine carcinogenesis is widely accepted but there is ample evidence that this alone is not suf®cient to account for its targetorgan speci®city [9]. Several studies have shown that deoxyguanosine residues in DNA are hydroxylated at the C-8 position of 2 0 -deoxyguanosine to produce 8-OHdG by various carcinogens that produce active oxygen species [10±18]. The hydroxyl radical is well known to cause DNA damage [19] and to induce the formation of 8-OHdG, which is now considered an excellent indicator of oxidative DNA damage [14,20]. Its accumulation due to EHEN treatment could be an important factor in enhancing the mutation rate leading to renal epithelial tumors [4,11,12,14,15,17,21±25]. The present study (Experiment I) con®rmed that Wistar rats are more prone to EHEN renal carcinogenesis than are Fischer rats, and that males are more sensitive than females of the same strain. In this connection, Deguchi et al. [26] also reported that the sex difference observed in the incidence of renal tumors induced by ferric nitrilotriacetate might depend upon the sex hormones. Therefore, it is
expected that the WF and FW rats had the same incidence of renal tumors. In addition, incidences of renal lesions in WF and FW rats did not differ, so that equal effects of the father and mother were apparent, induction being intermediate between those for Wistar and Fischer rats. Comparison of EHEN uptake by F1 rats with that by other groups (per 100 g of initial body weight) revealed no differences. These ®ndings suggest involvement of genetic variation which is not inherited in a parental sex-dependent fashion. A time- and dose-dependent increase in 8-OHdG in male Wistar rat kidney DNA after EHEN administration has been previously documented [4]. The present study (Experiment II) showed similar results for 8OHdG in kidney DNA of female Wistar and Fischer rats, with levels tending to correlate with the incidence of renal tumors. As for 8-OHdG assay in WF and FW rats, although we did not examine this in the present study, no signi®cant difference of the formation of 8-OHdG would be anticipated between these two groups, showing the same tendency as the incidence of renal tumors. There are distinct sex- and race-related differences in the incidence of renal cell carcinoma in humans. In Japan there were 5.09 cases (7.1 males and 3.1 females) per 100 000 people as of 1997 [27]. Incidences are reported to be higher in North European countries. The results of the present study provide interesting pointers to future clinical epidemiological studies of renal cell carcinomas. Acknowledgements We thank Dr Yoshiteru Kitahori for expert technical assistance. We also wish to thank Dr Malcolm A. Moore for his critical review of the manuscript.
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K. Takashima et al. / Cancer Letters 170 (2001) 125±130
References [1] Y. Hiasa, M. Ohshima, C. Iwata, T. Tanikake, Histopathological studies on renal tubular cell tumors in rats treated with N-ethyl-N-hydroxyethylnitrosamine, Gann 70 (1979) 817± 820. [2] H. Druckrey, D. Steinhoff, R. Preussmann, S. Ivankovic, Erzeugung von Krebs durch eine einmalige Dosis von Methylnitroso-Harnstoff und verschiedenen Dialkylnitrosaminen an Ratten, Z. Krebsforsch. 66 (1964) 1±10. [3] N. Konishi, I. Hayashi, S. Nakaoka, Y. Kitahori, T. Tsuzuki, H. Matsuda, M. Kitamura, Y. Hiasa, Rodent species and strain difference in kidney and lung pathology induced by N-ethylN-hydroxyethylnitrosamine, J. Toxicol. Pathol. 7 (1994) 67± 71. [4] M. Sato, Y. Kitahori, Y. Nakagawa, N. Konishi, M. Cho, Y. Hiasa, Formation of 8-hydroxydeoxyguanosine in rat kidney DNA after administration of N-ethyl-N-hydroxyethylnitrosamine, Cancer Lett. 124 (1998) 111±118. [5] Y. Hiasa, N. Konishi, S. Nakaoka, T. Nakamura, K. Nishii, M. Ohshima, Promoting effects of potassium dibasic phosphate on early-stage renal carcinogenesis in unilaterally nephrectomized rats treated with N-ethyl-N-hydroxyethyl-nitrosamine, Jpn. J. Cancer Res. 83 (1992) 688±694. [6] N. Konishi, K. Nishii, I. Hayashi, S. Nakaoka, K. Matsumoto, T. Yabuno, Y. Kitahori, Y. Hiasa, Inhibitory effect of potassium citrate on rat renal tumors induced by N-ethyl-N-hydroxyethylnitrosamine followed by potassium dibasic phosphate, Jpn. J. Cancer Res. 84 (1993) 128±134. [7] Y. Hiasa, N. Konishi, S. Nakaoka, M. Nakamura, S. Nishii, Y. Kitahori, M. Ohshima, Possible application to medium-term organ bioassays for renal carcinogenesis modi®ers in rats treated with N-ethyl-N-hydroxyethylnitrosamine and unilateral nephrectomy, Jpn. Cancer Res. 82 (1991) 1385±1390. [8] T. Munehisa, T. Sakata, Y. Nakano, Y. Takagi, Y. Yamada, H. Hibi, K. Miyake, T. Shirai, Initiation-stage enhancement by uracil of N-ethyl-N-hydroxyethylnitrosamine-induction of kidney carcinogenesis in rats, Cancer Lett. 87 (1994) 151± 157. [9] E. Von Hofe, I. Schmerold, W. Lijinsky, W. Jeltsch, P. Kleihues, DNA methylation in rat tissues by a series of homologous aliphatic nitrosamines ranging from Nnitrosodimethylamine to N-nitrosomethyldodecylamine, Carcinogenesis 8 (1987) 1337±1341. [10] F.L. Chung, Y. Xu, Increased 8-oxodeoxyguanosine levels in lung DNA of A/J mice and F344 rats treated with the tobaccospeci®c nitrosamine 4-(methyl-nitrosamine)-1-(3-pyridyl)-1butanone, Carcinogenesis 13 (1992) 1269±1277. [11] B. Halliwell, O.I. Aruoma, DNA damage by oxygen-derived species. Its mechanism and measurement in mammalian systems, FEBS Lett. 281 (1991) 9±19. [12] T. Ichinose, Y. Yajima, M. Nagashima, S. Takenoshita, Y. Nagamachi, M. Sagai, Lung carcinogenesis and formation of 8-hydroxydeoxyguanosine in mice by diesel exhaust particles, Carcinogenesis 18 (1997) 185±192. [13] M. Inagake, T. Yamane, Y. Kitao, K. Oya, H. Matsumoto, N. Kikuoka, H. Nakatani, T. Takahashi, H. Nishimura, A.
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Strain variation in renal carcinogenesis by N-ethyl-Nhydroxyethylnitrosamine in F1 (Wistar±Fischer) rats Kenji Takashima a, Seiichiro Ozono a,*, Isamu Nakanou a, Masahiko Ota a, Masahiro Tanaka a, Mitsuru Tani a, Kazuya Hirao a, Yoshihiko Hirao a, Shinichi Kuwashima b, Yoshio Hiasa c a
Department of Urology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara, Japan Department of Pathology, Nara Prefectunal Nara Hospital, 1-30-1 Hiramatu-cho Nara, Nara, Japan c The Second Department of Pathology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara, Japan b
Received 13 April 2001; received in revised form 17 May 2001; accepted 21 May 2001
Abstract The present study was conducted to compare the incidences of renal tumors in Wistar (W), Fischer (F) and F1 rats (WF: female Wistar rats £ male Fischer rats; FW: female Fischer rats £ male Wistar rats) induced by N-ethyl-N-hydroxyethylnitrosamine (EHEN). Levels of 8-OHdG in renal DNA were also investigated in Wistar and Fischer rats. After 2000 ppm of EHEN was administered orally for 2 weeks, the animals were fed basal diet until week 32. Wistar males and females demonstrated signi®cantly higher sensitivity regarding induction of renal lesions, while both WF and FW rats had similar incidences, generally intermediate between those for the two parent strains. The formation of 8-OHdG was maximal 60±180 min after an intraperitoneal dose of 750 mg/kg to Wistar and Fischer rats, which correlates with the increase tending to the incidence of renal tumors in male and female Wistar and Fischer rats. The results suggest that EHEN induction of renal tumors is related to oxygen radical damage and that the genes in the Wistar strain responsible for the sensitivity are not inherited in a sex-dependent fashion, despite the male being more susceptible. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Renal carcinogenesis; N-Ethyl-N-hydroxyethylnitrosamine; F1; 8-OHdG; Oxidative DNA damage
1. Introduction N-Ethyl-N-hydroxyethylnitrosamine (EHEN) is known to induce renal epithelial tumors in rats [1,2] with species-, strain- and sex-related differences present in the incidences of lesions. The Wistar rat strain is more sensitive to EHEN induction of renal carcinogenesis than Fischer rats, and males are more prone to the development of lesion than females of the * Corresponding author. Tel.: 181-744-22-3051 ext. 2338; fax: 181-744-22-9280. E-mail address: [email protected] (S. Ozono).
same strain [3]. Administration of EHEN has recently been reported to increase the formation of 8-OHdG, a marker of oxidative damage to DNA serving as an index of carcinogenesis in the renal cortex [4]. The present study was conducted to examine the incidences of renal tumors in Wistar, Fischer and F1 rats (WF: female Wistar rats £ male Fischer rats; FW: female Fischer rats £ male Wistar rats) using an experimental model of EHEN-induced renal carcinogenesis [1,3]. We examined levels of 8-OHdG in renal DNA in Wistar and Fischer rats.
0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00608-5
126
K. Takashima et al. / Cancer Letters 170 (2001) 125±130
2. Materials and methods 2.1. Chemicals EHEN was purchased from Nacalai Tesque (Kyoto, Japan), Nuclease Pl, Proteinase K and alkaline phosphatase were purchased from Sigma (St. Louis, MO) and an 8-OHdG Check kit was purchased from Nikken Food (Shizuoka, Japan). 2.2. Experiment I 2.2.1. Animals and experimental design Ten-week-old Wistar (10 male and 20 female) and Fischer (10 male and 20 female) rats were purchased from Charles River Co. Japan Inc. (Atsugi, Japan). After 1 week of acclimatization, one male Wistar or Fischer rat and two female Fischer rats or two female Wistar rats were housed in the same cage and allowed to produce F1 rats. A total of 222 animals at 6 weeks of age (26 male and 26 female rats each of Wistar and Fischer strains, 27 male and 40 female WF rats, and 25 male and 26 female FW rats) were housed in plastic cages at 24 ^ 18C and 55 ^ 5% relative humidity. All rats were housed two to a cage and fed on powdered basal diet (Oriental M Powder; Oriental
Yeast, Osaka, Japan). They received 2000 ppm of EHEN in tap water ad libitum for the ®rst 2 weeks from week 6, and then tap water (chemical-free) ad libitum for the 30 week duration of the experiment. The experiments were performed according to the Principles of Laboratory Animal Care of the NIH. 2.2.2. Histopathological studies All rats were sacri®ced under ether anesthesia at the end of week 32. Whole body and kidney weights were recorded. Kidneys were preserved in 10% phosphatebuffered formalin solution. After overnight ®xation, slices were processed for embedding in paraf®n, sectioned at 5 mm, and stained with hematoxylin± eosin (HE). The criteria for histopathological classi®cation of the kidney lesions were as described previously for simple hyperplasia, adenomatous hyperplasia and renal cell neoplasms [4,5]. 2.3. Experiment II 2.3.1. Animals and experimental design Groups of 18 5-week-old male and female Wistar and Fischer rats were fed on basal diet and tap water ad libitum for 1 week. All rats were housed in plastic cages at 24 ^ 18C and 55 ^ 5% relative humidity.
Fig. 1. Experimental design of Experiment II.
K. Takashima et al. / Cancer Letters 170 (2001) 125±130
127
Table 1 Weight of body and kidney and EHEN uptake for rats treated with EHEN Group
1 WF 2 WC 3 FF 4 FC 5 WFF 6 WFC 7 FWF 8 FWC
No. of rats
26 26 26 26 27 40 25 25
Body weight (g)
Kidney weight
Initial
Final
Absolute (g)
Relative (%)
Uptake
Uptake per 100 g of initial body weight
240.4 174.9 154.4 118.0 175.5 145.7 180.4 133.7
625.8 352.9 388.4 199.2 550.4 292.3 550.4 303.5
2.0 1.0 1.1 0.6 1.5 0.8 1.6 0.8
0.31 0.28 0.28 0.30 0.27 0.27 0.29 0.26
0.482 0.255 0.129 0.115 0.147 0.125 0.184 0.126
0.20 0.15 0.084 0.097 0.084 0.086 0.10 0.094
After 1 week, the animals treated with EHEN at 750 mg/kg body weight and control groups were given water. The experimental design for Experiment II is shown in Fig. 1. Rats were sacri®ced at 0.5, 1, 2, 3 or 4 h after intraperitoneal injection of EHEN. Control rats were sacri®ced at 2 h after injection of water. Three rats in each group were killed at each time-point and their livers and kidneys were excised and frozen in liquid nitrogen until use. 2.3.2. Determination of 8-OHdG formation The renal cortex was separated and milled under liquid nitrogen and then subjected to proteolytic treatment with Proteinase K before extraction of DNA by a conventional method with a mixture of phenol, chloroform and isoamyl alcohol, and subsequent puri®cation by ethanol precipitation. The DNA concentration was determined from absorbance at 260/280 nm.
Total EHEN (g)
For the 8-OHdG assay, DNA samples were dissolved in distilled water at a concentration of 50 mg/50 ml, heat-denatured at 958C for 5 min and cooled on ice. The samples (50 ml) were mixed with 1.5 ml of 1 M sodium acetate buffer (pH 4.8) and 2.5 units of nuclease Pl and digested by incubation at 378C for 1 h in the dark. To the solution were added 9 ml of 1 M Tris± HCl buffer (pH 7.5) and 1.5 units of alkaline phosphatase and the products were digested by incubation at 378C for 1 h. Aliquots (50 mg DNA/59.5 ml) of this mixture were assayed by ELISA using an 8-OHdG check kit. 2.4. Statistical analysis Quantitative data were analyzed for statistically signi®cant differences using Student's t-test for multiplicity and the x 2-test for incidence.
Table 2 Incidences of renal lesions in male rats treated with EHEN Group
1 WF 3 FF 5 WFF 7 FWF a b c
No. of rats
26 26 27 25
Simple hyperplasia
Adenomatous hyperplasia
Renal cell neoplasm
No. of rats (%)
Total no. (/rat)
No. of rats (%)
Total no. (/rat)
No. of rats (%)
26 (100.0) 23 (88.5) 25 (92.6) 24 (96.0)
438 (16.8) a,b,c 57 (2.2) 158 (5.9) a 146 (5.8) a
13 (50.0) a 2 (7.7) 7 (25.9) 7 (28.0)
20 (0.77) a 2 (0.08) 9 (0.33) 9 (0.36)
10 0 4 5
Signi®cantly different from group 3 (P , 0:05). Signi®cantly different from group 5 (P , 0:05). Signi®cantly different from group 7 (P , 0:05).
(38.4) a (0.0) (14.8) (20.0)
Total no. (/rat) 21 0 8 7
(0.81) a (0.0) (0.30) a (0.28) a
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K. Takashima et al. / Cancer Letters 170 (2001) 125±130
Table 3 Incidences of renal lesions in female rats treated with EHEN Group
2 WC 4 FC 6 WFC 8 FWC a b
No. of rats
26 26 40 26
Simple hyperplasia
Adenomatous hyperplasia
Renal cell neoplasm
Total no. (/rat)
No. of rats (%)
Total no. (/rat)
7 (0.27) a,b 0 (0.00) 1 (0.03) 5 (0.19)
5 (19.2) a 1 (3.8) 3 (7.5) 3 (11.5)
8 1 3 4
No. of rats (%)
Total no. (/rat)
No. of rats (%)
21 (80.8) a 2 (7.7) 34 (85.0) a 19 (73.1) a
75 (2.9) a 2 (0.8) 90 (2.3) a 60 (2.3) a
6 0 1 4
(23.1) a,b (0.0) (2.5) (15.4)
(0.31) a (0.038) (0.075) (0.15)
Signi®cantly different from group 4 (P , 0:05). Signi®cantly different from group 6 (P , 0:05).
3. Results 3.1. Experiment I The mean body weights, organ weights and EHEN uptake for each group at 32 experimental weeks are summarized in Table 1. In both males and females, the greatest EHEN uptake was observed in Wistar rats. In terms of EHEN uptake per 100 g of initial body weight, however, no differences were observed between F1 rats and Fischer rats in both males and females (Table 1). The incidences of kidney hyperplasias and tumors are shown in Tables 2 and 3. Among the male rat groups (Table 2, Fig. 2), male Wistar, WF and FW rats showed relatively high frequencies of renal lesions with EHEN administration when compared to Fischer rats. Only in Fischer rats was renal cell tumor development not induced. Incidences of renal lesions in male WF and FW rats did not differ with incidences at levels intermediate between those for Wistar and Fischer rats. In the female rat groups
(Table 3, Fig. 2), Wistar rats again showed relatively high frequencies for renal lesions while they were almost absent in the Fischer strain. Female F1 rats again showed intermediate values. Like the parent strains, F1 males showed high frequencies of renal cell neoplasms compared with F1 females (Fig. 2). 3.2. Experiment II Changes in 8-OHdG levels in the DNA of the renal cortex at various time-points (0.5±4 h) after treatment are shown in Table 4. The formation of 8-OHdG in kidney was time-dependently increased reaching a maximum 2 h after an injection of EHEN. Thereafter, the levels declined gradually. Peaks for 8-OHdG formation were 28.1, 83.1, 34.1, and 8.2 ng/50 mg DNA for female and male Wistar rats and female and male Fischer rats, respectively (Table 4). Maximum increases in 8-OHdG, which were calculated by difference between 8-OHdG after EHEN injection and after water injection, were 16.3, 42.6, 2.55, and 8.39 ng/50 mg DNA for female and male Wistar rats and female and male Fischer rats, respectively. The greatest increase was obtained in male Wistar rats, showing the same tendency as the incidence of renal tumors. In terms of the increase in 8-OHdG after 2 h, a signi®cant difference (P , 0:05) was observed between male Wistar rats and all other groups, and between female Fischer rats and all other groups. 4. Discussion
Fig. 2. Sex differences in the number of renal lesions (total number) in rats treated with EHEN. aSigni®cantly different from females of the same strain (P , 0:05).
Druckrey et al. [2] ®rst reported that EHEN induces tumors in kidneys, liver and ovaries of rats. Hiasa et al. [1] showed that renal tubular cell tumors selec-
K. Takashima et al. / Cancer Letters 170 (2001) 125±130
129
Table 4 8-OHdG levels in kidney DNA from rats treated with EHEN and water Group
1 WF 2 WC 3 FF 4 FC
Body weight (g)
214.8 158.4 133.6 104.7
8-OHdG (ng/50 mg DNA) after EHEN injection
8-OHdG after water injection
0.5 h
1h
2h
3h
4h
2h
40.2 12.9 25.5 5.5
75.3 12.9 27.5 8.2
83.1 27.9 34.1 6.8
67.1 28.1 29.5 6.3
58.6 16.0 26.1 6.5
40.6 11.8 25.7 5.7
tively develop at high incidence, without other kinds of renal tumors, in Wistar rats with 0.1% EHEN added to the diet for 2 weeks. Differences in the susceptibility to carcinogenesis of certain organs in various animal strains and species have been widely examined. Konishi et al. [3] showed higher sensitivity of the rat kidney to EHEN than mouse and hamster renal tissue. It further appears that rats of the Wistar strain are more susceptible with regard to EHEN renal carcinogenesis than are Fischer rats, and that males are more sensitive than females of the same strain. Various modi®ers have been detected with mediumterm organ bioassays in rats treated with EHEN [4±8]. Regarding the mechanism of action, an importance for DNA alkylation in nitrosamine carcinogenesis is widely accepted but there is ample evidence that this alone is not suf®cient to account for its targetorgan speci®city [9]. Several studies have shown that deoxyguanosine residues in DNA are hydroxylated at the C-8 position of 2 0 -deoxyguanosine to produce 8-OHdG by various carcinogens that produce active oxygen species [10±18]. The hydroxyl radical is well known to cause DNA damage [19] and to induce the formation of 8-OHdG, which is now considered an excellent indicator of oxidative DNA damage [14,20]. Its accumulation due to EHEN treatment could be an important factor in enhancing the mutation rate leading to renal epithelial tumors [4,11,12,14,15,17,21±25]. The present study (Experiment I) con®rmed that Wistar rats are more prone to EHEN renal carcinogenesis than are Fischer rats, and that males are more sensitive than females of the same strain. In this connection, Deguchi et al. [26] also reported that the sex difference observed in the incidence of renal tumors induced by ferric nitrilotriacetate might depend upon the sex hormones. Therefore, it is
expected that the WF and FW rats had the same incidence of renal tumors. In addition, incidences of renal lesions in WF and FW rats did not differ, so that equal effects of the father and mother were apparent, induction being intermediate between those for Wistar and Fischer rats. Comparison of EHEN uptake by F1 rats with that by other groups (per 100 g of initial body weight) revealed no differences. These ®ndings suggest involvement of genetic variation which is not inherited in a parental sex-dependent fashion. A time- and dose-dependent increase in 8-OHdG in male Wistar rat kidney DNA after EHEN administration has been previously documented [4]. The present study (Experiment II) showed similar results for 8OHdG in kidney DNA of female Wistar and Fischer rats, with levels tending to correlate with the incidence of renal tumors. As for 8-OHdG assay in WF and FW rats, although we did not examine this in the present study, no signi®cant difference of the formation of 8-OHdG would be anticipated between these two groups, showing the same tendency as the incidence of renal tumors. There are distinct sex- and race-related differences in the incidence of renal cell carcinoma in humans. In Japan there were 5.09 cases (7.1 males and 3.1 females) per 100 000 people as of 1997 [27]. Incidences are reported to be higher in North European countries. The results of the present study provide interesting pointers to future clinical epidemiological studies of renal cell carcinomas. Acknowledgements We thank Dr Yoshiteru Kitahori for expert technical assistance. We also wish to thank Dr Malcolm A. Moore for his critical review of the manuscript.
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