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Enhanced hepatocarcinogenesis in acatalasemic mice treated with diethylnitrosamine
Hepatology Research 12 (1998) 217 – 224
Enhanced hepatocarcinogenesis in acatalasemic mice treated with diethylnitrosamine Da-Hong Wang *, Yuka Funamori, Satoru Ikeda, Masaki Sato, Shohei Kira, Kazuhisa Taketa Department of Public Health, Okayama Uni6ersity Medical School, 2 -5 -1 Shikata-cho, Okayama 700 -8558, Japan Received 1 May 1998; received in revised form 29 June 1998; accepted 14 July 1998
Abstract Diethylnitrosamine (DEN)-induced hepatocellular carcinogenesis was compared between C3H/AnLCbs Cbs (acatalasemic) and C3H/AnLCas Cas (normal) mice. A total of 31 normal and 38 acatalasemic male mice, average age 9 weeks, received a single intraperitoneal injection of DEN (75 mg/kg body wt) weekly for 6 weeks. All animals that survived until the end of week 31 (25th week after the last injection of DEN) were sacrificed and the development of liver tumors was found. They grossly varied from one to multiple in number and from 1 to 15 mm in diameter in both groups. The numbers and size of liver tumors per liver were significantly greater (PB0.05) in acatalasemic mice (mean 9 S.D.: 3.8 92.0 in number per liver; 9.6 9 7.4 in mm diameter per liver) than in normal mice (1.8 9 1.1 in number per liver; 2.89 1.9 in mm diameter per liver). Histological examination of the tumor tissues revealed the development of well-differentiated hepatocellular carcinomas. We suggest that H2O2 or OH formed from H2O2 is likely to be involved in DEN-induced hepatocarcinogenesis and catalase plays a critical role in the prevention of hepatocarcinogenesis. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acatalasemic mice; Diethylnitrosamine (DEN); Hepatocarcinogenesis; Catalase; Hydrogen peroxide; Hydroxyl radical
* Corresponding author. Tel.: +81 86 2357184; fax: + 81 86 2260715. 1386-6346/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S1386-6346(98)00065-5
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D.-H. Wang et al. / Hepatology Research 12 (1998) 217–224
1. Introduction Catalase is an important enzyme in the regulation of intracellular H2O2 level in biological systems [1,2]. C3H/AnLCbs Cbs (acatalasemic) mouse has been reported to possess only one-tenth catalase levels in blood and one-third in liver of those in C3H/AnLCas Cas (normal) mice [3–5]. In our previous studies we found an enhanced CCl4-induced liver injury in acatalasemic mice and an inhibition of the injury after iron deprivation in comparison with normal mice, indicating that H2O2 or OH radicals formed from H2O2 was probably produced by the Fenton reaction or the iron-catalysed Haber–Weiss reaction in the presence of ferric or ferrous ions and catalase played an important role in preventing CCl4-induced hepatotoxicity [5–7]. Reddy et al. [8] reported that the incidence of nafenopin-induced hepatocellular carcinoma in acatalasemic mice was higher than that of the normal mice, suggesting the low catalase level in acatalasemic mice was probably responsible for the higher incidence of hepatocellular carcinoma. Many studies have shown lowered antioxidant enzyme levels in tumor cells, which indicates the possible involvement of reactive oxygen species (ROS) in the processes of carcinogenesis and the protective influence of antioxidant enzymes against the carcinogenesis [9–11]. The present study attempted to see if there was any difference in incidence of diethylnitrosamine (DEN)-induced liver tumors between acatalasemic and normal mice due to the difference of the liver catalase levels.
2. Materials and methods
2.1. Animals and treatments A total of 31 normal and 38 acatalasemic male mice, average age 9 weeks, were randomly housed 3–5 per cage of each strain, in polycarbonate cages with hardwood chips for bedding and allowed access to a standard mouse chow (Oriental MF, Oriental Yeast, Tokyo) and water ad libitum. All animals were weighed before treatments and received a single intraperitoneal injection of 75 mg/kg body wt of DEN (Sigma, St Louis, MO) weekly for 6 weeks (a modified procedure of Matsuzaki et al. [12]). The animals were maintained in an air-conditioned room at a temperature of 23 9 2°C. Animals were observed daily for abnormalities and body weight was recorded once a week during the first 10 weeks of the experiment and once every 2–4 weeks thereafter. All survivors were sacrificed at the end of week 31 of the experiment (25th week after the last injection of DEN). Animals that survived until the end of experiment were included in the effective numbers. The livers of all animals were excised, weighed and fixed in 10% neutral formalin. All tumors \ 1 mm diameter in the liver lobes were measured and counted.
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2.2. Histology Random samples of the liver tumors in each group of animals were stained with hematoxylin and eosin and examined under light microscope.
2.3. Statistical analysis Results were expressed in means9 S.D. Statistical comparisons in hepatocarcinogenesis between groups were performed using the Mann–Whitney U-test. Kaplan– Meier survival curve was employed to calculate the mortality of all experimental animals and the difference in survival time between two groups was compared by the log-rank test. 3. Results All animals that survived at the end of the experiment in both groups developed liver tumors. Liver tumors grossly varied from one to multiple in number and from 1 to 15 mm in diameter. There were significantly more tumors per liver in acatalasemic mice than in normal mice. The average numbers of liver tumors were 3.892.0 (mean9S.D.) per liver in acatalasemic mice and 1.89 1.1 pre liver in normal mice. The average tumor size was also significantly larger in acatalasemic mice (9.697.4 in mm diameter per liver) than in normal mice (2.89 1.9 in mm diameter per liver) (Table 1). Histological examination of liver tissues revealed the development of well-differentiated hepatocellular carcinomas (HCC) in both animal groups and the liver tumors appeared HCC with clear cell change, including vacuolated foci (Fig. 1). Grossly extrahepatic metastases were not detected. Severe changes of body weight were observed in both groups of animals during the initial 6-week DEN treatments period, particularly in normal mice (Fig. 2). Liver weight were determined in animal surviving to the end of the experiment. Average liver weight of acatalasemic mice were heavier than those of the normal mice (Table 1), constituting 6.6 and 5.4% of the body weight in acatalasemic and normal mice, respectively (P B 0.05). A total of 83.9% normal and 26.3% acatalasemic mice died during the experiment, especially during the initial 6-week period for multiple initiation. There was a significant difference in cumulative survival between the two groups of animals (Fig. 3). 4. Discussion So far, little information is available on whether there is any difference in susceptibility to liver carcinogens between the normal and acatalasemic mice. The present results demonstrated an enhanced tumorigenesis in livers of acatalasemic mice treated by DEN, in terms of size and number, compared with normal mice, which showed the role of catalase in the prevention of hepatocarcinogenesis.
29.09 3.0 (31) 27.1 9 2.2 (38)*
Normal Acatalasemic * PB0.05 (Mann–Whitney U-test).
Body wt before DEN treatment (n)
Mice
26.491.5 (5) 28.592.2 (28)*
Body (n)
Final weight (g)
5.4 9 0.6 6.6 91.2*
Liver (% of body wt.)
Table 1 Effect of Diethylnitrosamine on hepatocarcinogenesis of acatalasemic and normal mice
1.891.1 3.8 92.0*
2.89 1.9 9.69 7.4*
Tumor no./liver Tumor size (diameter, mm)/liver
220 D.-H. Wang et al. / Hepatology Research 12 (1998) 217–224
D.-H. Wang et al. / Hepatology Research 12 (1998) 217–224
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Catalase and glutathione peroxidase are the most important enzymes in the regulation of intracellular H2O2 level in biological systems. The former is regarded as playing a major role in the breakdown of overproduced H2O2 in the liver [1,2]. The liver catalase activities in acatalasemic mice were observed as one-third that of normal mice [5] and there was no significant difference in glutathione peroxidase activity in blood between acatalasemic and normal mice [4]. Many studies have suggested that reactive oxygen species are probably involved in the processes of carcinogenesis by DNA damage and that antioxidant enzymes are capable of preventing tumor development [9–11,13]. Results of the present study showed the importance of catalase in protecting normal mice from developing HCC, indicating that H2O2 was probably produced in the processes of DEN-induced hepatocarcinogenesis. The low catalase activities in acatalasemic mice would have caused increased tissue or cellular levels of H2O2 by DEN treatments, resulting in the overproduction of OH radicals by the Fenton reaction or the iron-catalysed Haber – Weiss reaction in the presence of transition metals. This may give an explanation of the observed enhancement of hepatocarcinogenesis in acatalasemic mice.
Fig. 1. Liver histopathology (hematoxylin – eosin staining). Liver from diethylnitrosamine-treated (a single i.p. injection of 75 mg/kg body wt of DEN weekly for 6 weeks) acatalasemic mice sacrificed at the end of the experiment (the 31st week). (a) Well-differentiated hepatocellular carcinoma occupies most of the lower portion of the photograph (63). (b) The liver tumors appeared HCC with clear cell change and vacuolated foci (125).
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Fig. 2. Body weight alterations of acatalasemic ( ) and normal ( ) mice before and after DEN treatment. Each value represents mean 9 S.D. of all mice per treatment group. *P B0.05.
DEN has been commonly used as a liver carcinogen in animal hepatocarcinogenicity studies. In the present study, the fact that multiple initiation by DEN led to a high incidence (100%) of HCC in both acatalasemic and normal mouse groups after 31 experimental weeks, suggested that multiple treatments with DEN were an efficient way for rapid development of hepatic tumor models in mice. A higher mortality in normal mice than in acatalasemic mice after multiple DEN initiations were observed in the present experiment, which was compatible with the result of Reddy et al. [8], in which the mortality of normal mice was higher compared with acatalasemic mice during the experiment of nefenopin-induced HCC. The cause of early death of animals in the present experiment is probably due to the acute toxicity of DEN. At the necropsy, gross atrophy of organs with congestion were observed but no specific pathological findings were present in the early death of mice. According to the present results, we conclude that acatalasemic mice are more susceptible to DEN, resulting in enhanced hepatocarcinogenesis compared with normal mice and catalase plays a critical role in protecting the liver from the reactive oxygen species in DEN-induced hepatocarcinogenesis.
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Fig. 3. Cumulative survival curve of acatalasemic and normal mice after DEN treatment. — , acatalasemic mice (n= 38 at the beginning and n = 28 at the end of the experiment); ---, normal mice (n =31 at the beginning and n=5 at the end of the experiment). **PB0.01 (log-rank test).
Acknowledgements We are indebted to S. Ariyoshi, A. Yokomizo and S. Kunisada for animal maintenance. This study was supported partially by the Ryoubi Teien Memorial Foundation, 1996, Japan.
References [1] Cohen G, Hochstein P. Glutathione peroxide: The primary agent for the elimination of hydrogen peroxide in erythrocytes. Biochemistry 1963;2:1420. [2] Tibayrenc M. Population genetics of parasitic protozoa and other microorganisms. In: Barker JR, Muller R, Rollinson D, editors. Advances in Parasitology, vol. 36, 1995:48 – 115. [3] Feinstein RN, Braun JT, Howard JB. Acatalasemic and hypocatalasemic mouse mutants. Arch Biochem Biophys 1967;120:165–9. [4] Ogata M, Kobayashi H, Ioku N, Ishii K. Methemoglobin concentration in the blood of acatalasemic mice. Proc Jpn Acad 1986;62:360 – 71. [5] Wang DH, Ishii K, Zhen LX, Taketa K. Enhanced liver injury in acatalasemic mice following exposure to carbon tetrachloride. Arch Toxicol 1996;70:189 – 94. [6] Ishii K, Wang DH, Zhen LX, Satho M, Taketa K. An enhanced liver injury induced by carbon tetrachloride in acatalasemic mice. In: Rana SVS, Taketa K, editors. Liver and Environmental Xenobiotics. New Delhi: Narosa Publishing House, 1997:40 – 52. [7] Wang DH, Ishii K, Taketa K. Inhibition of carbon tetrachloride-induced liver injury in acatalasemic mice by hepatic iron deprivation. Hepatol Res 1998;10:237 – 47. [8] Reddy JK, Rao MS, Moody DE. Hepatocellular carcinomas in acatalasemic mice treated with nafenopin, a hypolipidemic peroxisome proliferator. Cancer Res 1976;36:1211 – 7.
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[9] Senturker S, Karahalil B, Inal M, Yilmaz H, Muslumanoglu H, Gedikoglu G, Dizdaroglu M. Oxidative DNA base damage and antioxidant enzyme levels in childhood acute lymphoblastic leukemia. FEBS Lett 1997;416:286–90. [10] Jaruga P, Zastawny TH, Skokowski J, Dizdaroglu M, Olinski R. Oxidative DNA base damage and antioxidant enzyme activities in human lung cancer. FEBS Lett 1994;341:59 – 64. [11] Toyokuni S, Okamoto K, Yodoi J, Hiai H. Persistent oxidative stress in cancer. FEBS Lett 1995;358:1–3. [12] Matsuzaki T, Murase N, Yagahashi A, Shinozuka H, Shimizu Y, Furuya T, Burrell N, Iwatsuki S, Starzl TE. Liver transplantation for diethylnitrosamine-induced hepatocellular carcinoma in rats. Transplant Proc 1992;24:748–51. [13] Ishii K, Zhen LX, Wang DH, Funamori Y, Ogawa K, Taketa K. Prevention of mammary tumorigenesis in acatalasemic mice by vitamin E supplementation. Jpn J Cancer Res 1996;87:680 – 4.
.
Enhanced hepatocarcinogenesis in acatalasemic mice treated with diethylnitrosamine Da-Hong Wang *, Yuka Funamori, Satoru Ikeda, Masaki Sato, Shohei Kira, Kazuhisa Taketa Department of Public Health, Okayama Uni6ersity Medical School, 2 -5 -1 Shikata-cho, Okayama 700 -8558, Japan Received 1 May 1998; received in revised form 29 June 1998; accepted 14 July 1998
Abstract Diethylnitrosamine (DEN)-induced hepatocellular carcinogenesis was compared between C3H/AnLCbs Cbs (acatalasemic) and C3H/AnLCas Cas (normal) mice. A total of 31 normal and 38 acatalasemic male mice, average age 9 weeks, received a single intraperitoneal injection of DEN (75 mg/kg body wt) weekly for 6 weeks. All animals that survived until the end of week 31 (25th week after the last injection of DEN) were sacrificed and the development of liver tumors was found. They grossly varied from one to multiple in number and from 1 to 15 mm in diameter in both groups. The numbers and size of liver tumors per liver were significantly greater (PB0.05) in acatalasemic mice (mean 9 S.D.: 3.8 92.0 in number per liver; 9.6 9 7.4 in mm diameter per liver) than in normal mice (1.8 9 1.1 in number per liver; 2.89 1.9 in mm diameter per liver). Histological examination of the tumor tissues revealed the development of well-differentiated hepatocellular carcinomas. We suggest that H2O2 or OH formed from H2O2 is likely to be involved in DEN-induced hepatocarcinogenesis and catalase plays a critical role in the prevention of hepatocarcinogenesis. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acatalasemic mice; Diethylnitrosamine (DEN); Hepatocarcinogenesis; Catalase; Hydrogen peroxide; Hydroxyl radical
* Corresponding author. Tel.: +81 86 2357184; fax: + 81 86 2260715. 1386-6346/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S1386-6346(98)00065-5
218
D.-H. Wang et al. / Hepatology Research 12 (1998) 217–224
1. Introduction Catalase is an important enzyme in the regulation of intracellular H2O2 level in biological systems [1,2]. C3H/AnLCbs Cbs (acatalasemic) mouse has been reported to possess only one-tenth catalase levels in blood and one-third in liver of those in C3H/AnLCas Cas (normal) mice [3–5]. In our previous studies we found an enhanced CCl4-induced liver injury in acatalasemic mice and an inhibition of the injury after iron deprivation in comparison with normal mice, indicating that H2O2 or OH radicals formed from H2O2 was probably produced by the Fenton reaction or the iron-catalysed Haber–Weiss reaction in the presence of ferric or ferrous ions and catalase played an important role in preventing CCl4-induced hepatotoxicity [5–7]. Reddy et al. [8] reported that the incidence of nafenopin-induced hepatocellular carcinoma in acatalasemic mice was higher than that of the normal mice, suggesting the low catalase level in acatalasemic mice was probably responsible for the higher incidence of hepatocellular carcinoma. Many studies have shown lowered antioxidant enzyme levels in tumor cells, which indicates the possible involvement of reactive oxygen species (ROS) in the processes of carcinogenesis and the protective influence of antioxidant enzymes against the carcinogenesis [9–11]. The present study attempted to see if there was any difference in incidence of diethylnitrosamine (DEN)-induced liver tumors between acatalasemic and normal mice due to the difference of the liver catalase levels.
2. Materials and methods
2.1. Animals and treatments A total of 31 normal and 38 acatalasemic male mice, average age 9 weeks, were randomly housed 3–5 per cage of each strain, in polycarbonate cages with hardwood chips for bedding and allowed access to a standard mouse chow (Oriental MF, Oriental Yeast, Tokyo) and water ad libitum. All animals were weighed before treatments and received a single intraperitoneal injection of 75 mg/kg body wt of DEN (Sigma, St Louis, MO) weekly for 6 weeks (a modified procedure of Matsuzaki et al. [12]). The animals were maintained in an air-conditioned room at a temperature of 23 9 2°C. Animals were observed daily for abnormalities and body weight was recorded once a week during the first 10 weeks of the experiment and once every 2–4 weeks thereafter. All survivors were sacrificed at the end of week 31 of the experiment (25th week after the last injection of DEN). Animals that survived until the end of experiment were included in the effective numbers. The livers of all animals were excised, weighed and fixed in 10% neutral formalin. All tumors \ 1 mm diameter in the liver lobes were measured and counted.
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2.2. Histology Random samples of the liver tumors in each group of animals were stained with hematoxylin and eosin and examined under light microscope.
2.3. Statistical analysis Results were expressed in means9 S.D. Statistical comparisons in hepatocarcinogenesis between groups were performed using the Mann–Whitney U-test. Kaplan– Meier survival curve was employed to calculate the mortality of all experimental animals and the difference in survival time between two groups was compared by the log-rank test. 3. Results All animals that survived at the end of the experiment in both groups developed liver tumors. Liver tumors grossly varied from one to multiple in number and from 1 to 15 mm in diameter. There were significantly more tumors per liver in acatalasemic mice than in normal mice. The average numbers of liver tumors were 3.892.0 (mean9S.D.) per liver in acatalasemic mice and 1.89 1.1 pre liver in normal mice. The average tumor size was also significantly larger in acatalasemic mice (9.697.4 in mm diameter per liver) than in normal mice (2.89 1.9 in mm diameter per liver) (Table 1). Histological examination of liver tissues revealed the development of well-differentiated hepatocellular carcinomas (HCC) in both animal groups and the liver tumors appeared HCC with clear cell change, including vacuolated foci (Fig. 1). Grossly extrahepatic metastases were not detected. Severe changes of body weight were observed in both groups of animals during the initial 6-week DEN treatments period, particularly in normal mice (Fig. 2). Liver weight were determined in animal surviving to the end of the experiment. Average liver weight of acatalasemic mice were heavier than those of the normal mice (Table 1), constituting 6.6 and 5.4% of the body weight in acatalasemic and normal mice, respectively (P B 0.05). A total of 83.9% normal and 26.3% acatalasemic mice died during the experiment, especially during the initial 6-week period for multiple initiation. There was a significant difference in cumulative survival between the two groups of animals (Fig. 3). 4. Discussion So far, little information is available on whether there is any difference in susceptibility to liver carcinogens between the normal and acatalasemic mice. The present results demonstrated an enhanced tumorigenesis in livers of acatalasemic mice treated by DEN, in terms of size and number, compared with normal mice, which showed the role of catalase in the prevention of hepatocarcinogenesis.
29.09 3.0 (31) 27.1 9 2.2 (38)*
Normal Acatalasemic * PB0.05 (Mann–Whitney U-test).
Body wt before DEN treatment (n)
Mice
26.491.5 (5) 28.592.2 (28)*
Body (n)
Final weight (g)
5.4 9 0.6 6.6 91.2*
Liver (% of body wt.)
Table 1 Effect of Diethylnitrosamine on hepatocarcinogenesis of acatalasemic and normal mice
1.891.1 3.8 92.0*
2.89 1.9 9.69 7.4*
Tumor no./liver Tumor size (diameter, mm)/liver
220 D.-H. Wang et al. / Hepatology Research 12 (1998) 217–224
D.-H. Wang et al. / Hepatology Research 12 (1998) 217–224
221
Catalase and glutathione peroxidase are the most important enzymes in the regulation of intracellular H2O2 level in biological systems. The former is regarded as playing a major role in the breakdown of overproduced H2O2 in the liver [1,2]. The liver catalase activities in acatalasemic mice were observed as one-third that of normal mice [5] and there was no significant difference in glutathione peroxidase activity in blood between acatalasemic and normal mice [4]. Many studies have suggested that reactive oxygen species are probably involved in the processes of carcinogenesis by DNA damage and that antioxidant enzymes are capable of preventing tumor development [9–11,13]. Results of the present study showed the importance of catalase in protecting normal mice from developing HCC, indicating that H2O2 was probably produced in the processes of DEN-induced hepatocarcinogenesis. The low catalase activities in acatalasemic mice would have caused increased tissue or cellular levels of H2O2 by DEN treatments, resulting in the overproduction of OH radicals by the Fenton reaction or the iron-catalysed Haber – Weiss reaction in the presence of transition metals. This may give an explanation of the observed enhancement of hepatocarcinogenesis in acatalasemic mice.
Fig. 1. Liver histopathology (hematoxylin – eosin staining). Liver from diethylnitrosamine-treated (a single i.p. injection of 75 mg/kg body wt of DEN weekly for 6 weeks) acatalasemic mice sacrificed at the end of the experiment (the 31st week). (a) Well-differentiated hepatocellular carcinoma occupies most of the lower portion of the photograph (63). (b) The liver tumors appeared HCC with clear cell change and vacuolated foci (125).
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Fig. 2. Body weight alterations of acatalasemic ( ) and normal ( ) mice before and after DEN treatment. Each value represents mean 9 S.D. of all mice per treatment group. *P B0.05.
DEN has been commonly used as a liver carcinogen in animal hepatocarcinogenicity studies. In the present study, the fact that multiple initiation by DEN led to a high incidence (100%) of HCC in both acatalasemic and normal mouse groups after 31 experimental weeks, suggested that multiple treatments with DEN were an efficient way for rapid development of hepatic tumor models in mice. A higher mortality in normal mice than in acatalasemic mice after multiple DEN initiations were observed in the present experiment, which was compatible with the result of Reddy et al. [8], in which the mortality of normal mice was higher compared with acatalasemic mice during the experiment of nefenopin-induced HCC. The cause of early death of animals in the present experiment is probably due to the acute toxicity of DEN. At the necropsy, gross atrophy of organs with congestion were observed but no specific pathological findings were present in the early death of mice. According to the present results, we conclude that acatalasemic mice are more susceptible to DEN, resulting in enhanced hepatocarcinogenesis compared with normal mice and catalase plays a critical role in protecting the liver from the reactive oxygen species in DEN-induced hepatocarcinogenesis.
D.-H. Wang et al. / Hepatology Research 12 (1998) 217–224
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Fig. 3. Cumulative survival curve of acatalasemic and normal mice after DEN treatment. — , acatalasemic mice (n= 38 at the beginning and n = 28 at the end of the experiment); ---, normal mice (n =31 at the beginning and n=5 at the end of the experiment). **PB0.01 (log-rank test).
Acknowledgements We are indebted to S. Ariyoshi, A. Yokomizo and S. Kunisada for animal maintenance. This study was supported partially by the Ryoubi Teien Memorial Foundation, 1996, Japan.
References [1] Cohen G, Hochstein P. Glutathione peroxide: The primary agent for the elimination of hydrogen peroxide in erythrocytes. Biochemistry 1963;2:1420. [2] Tibayrenc M. Population genetics of parasitic protozoa and other microorganisms. In: Barker JR, Muller R, Rollinson D, editors. Advances in Parasitology, vol. 36, 1995:48 – 115. [3] Feinstein RN, Braun JT, Howard JB. Acatalasemic and hypocatalasemic mouse mutants. Arch Biochem Biophys 1967;120:165–9. [4] Ogata M, Kobayashi H, Ioku N, Ishii K. Methemoglobin concentration in the blood of acatalasemic mice. Proc Jpn Acad 1986;62:360 – 71. [5] Wang DH, Ishii K, Zhen LX, Taketa K. Enhanced liver injury in acatalasemic mice following exposure to carbon tetrachloride. Arch Toxicol 1996;70:189 – 94. [6] Ishii K, Wang DH, Zhen LX, Satho M, Taketa K. An enhanced liver injury induced by carbon tetrachloride in acatalasemic mice. In: Rana SVS, Taketa K, editors. Liver and Environmental Xenobiotics. New Delhi: Narosa Publishing House, 1997:40 – 52. [7] Wang DH, Ishii K, Taketa K. Inhibition of carbon tetrachloride-induced liver injury in acatalasemic mice by hepatic iron deprivation. Hepatol Res 1998;10:237 – 47. [8] Reddy JK, Rao MS, Moody DE. Hepatocellular carcinomas in acatalasemic mice treated with nafenopin, a hypolipidemic peroxisome proliferator. Cancer Res 1976;36:1211 – 7.
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[9] Senturker S, Karahalil B, Inal M, Yilmaz H, Muslumanoglu H, Gedikoglu G, Dizdaroglu M. Oxidative DNA base damage and antioxidant enzyme levels in childhood acute lymphoblastic leukemia. FEBS Lett 1997;416:286–90. [10] Jaruga P, Zastawny TH, Skokowski J, Dizdaroglu M, Olinski R. Oxidative DNA base damage and antioxidant enzyme activities in human lung cancer. FEBS Lett 1994;341:59 – 64. [11] Toyokuni S, Okamoto K, Yodoi J, Hiai H. Persistent oxidative stress in cancer. FEBS Lett 1995;358:1–3. [12] Matsuzaki T, Murase N, Yagahashi A, Shinozuka H, Shimizu Y, Furuya T, Burrell N, Iwatsuki S, Starzl TE. Liver transplantation for diethylnitrosamine-induced hepatocellular carcinoma in rats. Transplant Proc 1992;24:748–51. [13] Ishii K, Zhen LX, Wang DH, Funamori Y, Ogawa K, Taketa K. Prevention of mammary tumorigenesis in acatalasemic mice by vitamin E supplementation. Jpn J Cancer Res 1996;87:680 – 4.
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