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Classical conditioning of oxidative DNA damage in rats
Neuroscience Letters 288 (2000) 13±16
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Classical conditioning of oxidative DNA damage in rats Masahiro Irie a,*, Shinya Asami b, Shoji Nagata a, Masakazu Miyata c, Hiroshi Kasai b a
Department of Mental Health, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan b Department of Environmental Oncology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan c Department of Neurology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan Received 31 March 2000; received in revised form 13 May 2000; accepted 13 May 2000
Abstract This study investigated whether the formation of 8-hydroxydeoxyguanosine (8-OH-dG), a known oxidative DNA modi®cation relevant to carcinogenicity, can be classically conditioned to a novel taste in order to clarify the possible role of the central nervous system (CNS) or psychological stress on cancer initiation via a classical conditioning mechanism. Male Wistar rats underwent one or two conditioned taste aversion (CTA) experiments in which ferric nitrilotriacetate (Fe-NTA), which has renal toxicity and can induce renal cell carcinoma, served as a visceral unconditioned stimulus (US), and a saccharin solution (SAC) was used as a conditioned stimulus (CS). The 8-OH-dG levels in the group conditioned with the combination of SAC and Fe-NTA signi®cantly increased as compared to those of the uncombined groups by two repeats of the conditioning procedure (P 0:013). The rats that showed a painful response at the Fe-NTA administration had signi®cantly higher values of 8-OH-dG than those without pain (P 0:003). These results not only provide the ®rst evidence regarding classical conditioning of oxidative DNA damage using the CTA procedure, but also suggest the involvement of the CNS and psychological stress in the pathogenesis of cancer via oxidative DNA damage. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Conditioned taste aversion; 8-Hydroxydeoxyguanosine; Oxidative DNA damage; Stress; Pain; Cancer
It has been proposed that reactive oxygen species (ROSs) play a signi®cant role in the pathogenesis of cancer through several kinds of nuclear DNA injuries [3]. Among the more abundant types of base modi®cations, 8-hydroxydeoxyguanosine (8-OH-dG, 7,8-dihydro-8-oxodeoxyguanosine), produced by the oxidation of deoxyguanosine, is considered as the most sensitive and useful marker of oxidative DNA adducts [8]. It has been shown that 8-OH-dG is closely associated with various diseases, including cancer, and is produced by exposure to various carcinogens and hazardous materials [8]. Psychological stress has also been reported to increase the formation of 8-OH-dG in the rat liver [1]. However, the underlying pathway of stress-induced oxidative DNA damage is not yet clearly understood. Classical or Pavlovian conditioning phenomenon has been considered to be a part of stress responses and has often been used to identify noxious stimuli [2]. A condi* Corresponding author. Tel.: 181-93-691-7475; fax: 181-93692-5419. E-mail address: [email protected] (M. Irie).
tioned taste aversion (CTA) is a major form of classical conditioning, in which animals learn to avoid a novel taste (conditioned stimulus; CS) that has been previously followed by transient poisoning (unconditioned stimulus; US). With the use of this paradigm, it was demonstrated that cyclophosphamide-induced immune suppression can be conditioned [2]. Several kinds of CTAs were identi®ed thereafter, using a variety of agents, including carcinogens instead of cyclophosphamide [12]. Classical conditioning may contribute to the relationships between psychological stress and the formation of 8-OH-dG in rats; however, it has never been studied whether the CTA paradigm would affect oxidative DNA damage, such as 8-OH-dG. If conditioning of 8-OH-dG can be established, then it suggests that the central nervous system (CNS) can play a signi®cant role in oxidative DNA damage. Furthermore, it is assumed that the causes and the aggravating factors of cancer and other diseases involved in this oxidative DNA damage are partly mediating, conditioned reactions. Ferric nitrilotriacetate (Fe-NTA) is a well-known iron-
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 19 4- 0
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M. Irie et al. / Neuroscience Letters 288 (2000) 13±16
chelator that causes iron-catalyzed free radical reactions in its target organ, the kidney [4,11,13,15]. Many groups have reported that Fe-NTA induces renal proximal tubular damage followed by a high incidence of renal cell carcinoma (RCC) in rodents [4,11]. Moreover, Fe-NTA-induced RCCs in rodents have been found to resemble human RCCs in several points, such as frequent invasion and metastasis [11]. It has been reported that the 8-OH-dG levels in renal tissues immediately increased at 1 h and decreased to the basal levels within three days after a single intraperitoneal administration of Fe-NTA (15 mg Fe/kg body weight) [13,15]. Thus, it is reasonable to use Fe-NTA, which is a carcinogen with a known time sequence for 8-OH-dG formation, as an unconditioned stimulus in the experiment of oxidative DNA damage relevant to cancer initiation due to classical conditioning. In the present study, we examined whether classical conditioning can be found regarding the formation of 8OH-dG in rats. Fe-NTA was initially used as an US, and a saccharin solution (SAC) served as a CS in the CTA procedure. This study was conducted in two ways, with either one conditioning (Experiment 1) or two conditionings (Experiment 2). The subjects were six-week old male Wistar rats (Seiwa Experimental Animal Institute, Fukuoka, Japan). After a one-week acclimatization to the housing environment, the time of the daily water supply was restricted gradually over one week, and thereafter, the rats were allowed to drink water only for 1 h, from 09:00 to 10:00 in the morning. The animals were gently handled for ®ve minutes on at least ®ve occasions, which were randomly scheduled during this period. On the conditioning day, the rats were randomly divided into four groups, according to the conditioning protocol described in Table 1. One half of the rats received SAC (0.2%) in place of water from 09:00 to 10:00, followed immediately by an intraperitoneal (i.p.) injection of Fe-NTA (CS1 US1 group) at a dose of 15 mg Fe/kg body weight or its equivalent volume of saline (CS1 US2 group), respectively. The rest of the rats were given water during the same period, followed immediately by an i.p. injection of FeNTA (CS2 US1 group) or saline (CS2 US2 group), respectively. In all groups, the animals were fasted for 2 h after the injection of Fe-NTA or saline. As there were rats Table 1 Conditioning procedure Group a
Adaptation period
Conditioning day b
Test day
CS1US1 CS1US2 CS2US1 CS2US2
H2O H2O H2O H2O
SAC 1 Fe-NTA SAC 1 SAL H2O 1 Fe-NTA H2O 1 SAL
SAC 1 SAL SAC 1 SAL H2O 1 SAL H2O 1 SAL
a
CS, conditioned stimulus; US, unconditioned stimulus. Fe-NTA, ferric nitrilotriacetate (15 mg Fe/kg body weight, i.p.); SAC, saccharin (0.2%); SAL, saline. b
showing clearly painful behaviors, such as crying and aggressive actions at the time of the Fe-NTA administration, the presence of pain was recorded. Subsequently, the restriction of drinking water to 1 h a day and the 2-h fasting were continued thereafter. One week later, either SAC or water, whichever was used with the conditioning procedures, was administered to the rats, followed by an i.p. injection of saline. All of the animals were fasted for 2 h after the injection and were then sacri®ced. The animals were killed by suf®cient exsanguination. Both kidneys were removed and a portion of the organ was stored at 2808C. In Experiment 2, the procedures and methodology were the same as in Experiment 1 except as noted below. The same conditioning procedure as in Experiment 1 was repeated twice at oneweek intervals. The solution of Fe-NTA was prepared according to the method of Yamaguchi et al. [15]. Fe(NO3)3 and Na2NTA (Wako Biochemicals, Osaka, Japan) were each dissolved in Milli-Q water and then mixed at a molar ratio of 1:4. The pH was adjusted with NaHCO3 to 7.4. These solutions were prepared immediately before use. The methods for analyzing the formation of 8OH-dG were described elsewhere [15]. In brief, the nuclear DNA was extracted from 50 mg of each kidney with the DNA Extractor WB Kit (Wako Biochemicals, Osaka, Japan). The extracted nuclear DNA was digested with nuclease P1 and acid phosphatase (378C for 30 min), and injected onto a HPLC column (Beckman, Ultrasphere-ODS, 5 mm, 4:6 £ 250 nm) coupled to an electrochemical detector (ESA Coulochem II: detector 1, 0.15 V; detector 2, 0.30 V). A one-way analysis of variance (ANOVA) with the Fisher's PLSD test, Student's t-test, and Spearman's rank correlations were performed to estimate statistical differences with the use of SPSS 7.5 for Windows. In Experiment 1, no signi®cant differences in the 8-OHdG levels were found among the four groups (F(3,32) 0.69; P 0.566), although the group conditioned with the combination of SAC and Fe-NTA (CS1 US1) showed slightly higher 8-OH-dG levels than the other groups. However, when the US-treated animals were analyzed further, by division into painful and non-painful groups, the painful group (n 8) showed signi®cantly higher 8OH-dG levels than the non-painful group (n 10) (t 3:53; P 0:003; Fig. 1). The painful group consisted of 4 CS1 and 4 CS2 animals. In Experiment 2, there were signi®cant differences regarding the levels of 8-OH-dG among the four groups (F 3; 36 4:11; P 0:013; Fig. 2). Multiple comparisons showed that the levels of 8-OH-dG in the CS1 US1 group were signi®cantly higher than those of the CS1 US2 group (P 0:003), the CS2 US2 group (P 0:007), and the CS2 US1 group (P 0:042), respectively. There was no signi®cant correlation between the 8-OH-dG levels and the body weights after the conditioning procedures in both experiments. Also, no differences were found regarding body weights before and after the conditioning procedures among the four groups in both experiments. The effect of pain at the time of US on the
M. Irie et al. / Neuroscience Letters 288 (2000) 13±16
Fig. 1. Comparison of 8-OH-dG levels between painful and nonpainful groups at the time of Fe-NTA administration. Each bar represents mean ^ SEM.
8-OH-dG levels was not examined in Experiment 2, because most of the US-treated animals showed painful behavior at least once. Numerous studies have shown possible classical conditionings of various biological parameters, such as neurological, endocrinological, and immune functions [2,5,6]. Besides these biological studies on classical conditionings, research is now being extended to cover its relationship to genetics. As for cancer, some researchers have found that natural killer cell activity, a defense against cancer progression, can be conditioned [5]. Classical conditioning of tumor growth was also reported [6]. However, it has scarcely been investigated as to whether conditioning plays a signi®cant role in cancer initiation through genetic alterations relevant to carcinogenicity. We report here the ®rst evidence that the formation of representative oxidative DNA damage, 8-OH-dG, increased in response to the CS using the CTA procedure. Our data provide one mechanism whereby the CNS modulates the formation of 8-OH-dG. Since an association of such DNA damage with carcinogenicity is evident [3,8], it seems possible that the conditioning mechanism plays an etiological role in the initiation of cancer. Furthermore, the results support the involvement of stress in the pathogenesis of cancer through oxidative DNA damage. Although the underlying mechanism of how the formation of 8-OH-dG can be classically conditioned is unclear, some investigators found positive associations between stress and the formation of ROSs [9,10]. Classical conditioning of ROSs appears to have not been investigated. Further studies are required to con®rm this possibility. However, the degree of the involvement of the classical conditioning mechanism was not estimated to be very high, even if it affected the formation of 8OH-dG, because the 8-OH-dG level at 2 h after the injection of saline in the CS1 US1 group in Experiment 2 was about 1/3 or 1/4 of that after the administration of Fe-NTA [15].
15
It is necessary to consider the effects of Fe-NTA and saline themselves on the formation of 8-OH-dG in discussing our ®ndings. In previous studies [13,15], the formation of 8-OH-dG in renal tissues increased 1 h after an intraperitoneal injection of Fe-NTA (15 mg Fe/kg body weight) to rats, but it returned to the basal level within three days. In contrast, the repair enzyme activity maintained a higher level for ®ve days and returned to the basal level within two weeks. The injection of saline did not induce a signi®cant increase in the level of 8-OH-dG. Based on these fundamental studies, the 8-OH-dG levels in the kidneys were measured at a one-week interval after conditioning with the administration of Fe-NTA in the present study, so that there was no possibility of residual 8-OH-dG synthesized in the renal tissues affecting the measured values. This view was also veri®ed by the ®ndings that the absence or presence of Fe-NTA administration did not show a signi®cant difference in Experiment 1. Even if the 8-OH-dG repair enzyme was increased at one week after the administration of Fe-NTA, it should have acted to decrease the formation of 8-OH-dG and, therefore, is not considered to be a factor for the increase in the 8-OH-dG levels by conditioning. It is not clear whether the 8-OH-dG levels in renal tissues at one week after the Fe-NTA administration returned to the preadministration levels when Fe-NTA was given twice at a oneweek interval. However, the 8-OH-dG levels after two injections of Fe-NTA unpaired with SAC did not differ from those without the Fe-NTA administration, and were signi®cantly lower than those of the group that received two injections of Fe-NTA paired with SAC. Therefore, the possibility of accumulation of 8-OH-dG by two administrations of Fe-NTA was considered to be negligible. The positive relationship between painful behaviors and the 8-OH-dG levels seems to support the previous results that stress induced oxidative DNA damage [1]. While the
Fig. 2. Comparison of 8-OH-dG levels among the four groups according to the two different pairings of CS 2 US. Each bar represents mean ^ SEM; n 10 rats/group; *P , 0:05, **P , 0:01, as compared to the SAC 1 Fe-NTA group by Fisher's PLSD test. See Table 1 for more details.
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M. Irie et al. / Neuroscience Letters 288 (2000) 13±16
possibility that the use of SAC confounded the relationship was considered, the presence of pain was not associated with the use of SAC. Our results indicate that stress plays a signi®cant role in enhancing the probability of a conditioned oxidative DNA damage. There have been some other reports in which the effects of stress on the conditionability of biological parameters were evaluated. For example, Wood and Shors [14] reported that stress facilitates the effect of classical conditioning in male rats, but impairs it in female rats through the activational effects of ovarian hormones. This sex difference was interesting, because male rats were found to produce more 8-OH-dG than female rats when treated with carcinogens [7]. Thus, the use of male rats seem to contribute to the susceptibility to the classical conditioning and the formation of 8-OH-dG in the present study. Our ®ndings suggest that not only the CTA paradigm, but also pain, contributed to the formation of 8-OH-dG. While there is a report [1] stating that the 8-OH-dG levels in the rat hepatic and renal tissues were increased by psychological stress, the psychological stress due to anxiety induced by at least two sessions of 5 h of electric stimulation was needed in the case of the liver, and four sessions were used in the case of the kidney. Taking this report into consideration, although the experimental method differs, the effect of psychological stress on 8-OH-dG formation may be insuf®cient with one or two conditionings. However, in contrast to the repeated loading of psychological stress, it is dif®cult to repeat the administration of Fe-NTA to rats, since this agent increases kidney damage and could possibly affect the measurement values by the damage alone. Similar methodological limitations are expected when other carcinogens are employed. In view of the different effects of psychological stress on the formation of 8-OH-dG between the kidney and the liver, the kidney may be less sensitive to stress than the liver. Therefore, it will be necessary to carry out studies using other, less toxic carcinogens or other organs, such as the liver, in the future to con®rm our ®ndings. [1] Adachi, S., Kawamura, K. and Takemoto, K., Oxidative damage of nuclear DNA in liver of rats exposed to psychological stress, Cancer Res., 53 (1993) 4153±4155. [2] Ader, R. and Cohen, N., Psychoneuroimmunology: conditioning and stress, Annu. Rev. Psychol., 44 (1993) 53±85.
[3] Dreher, D. and Junod, A.F., Role of oxygen free radicals in cancer development, Eur. J. Cancer, 32A (1996) 30±38. [4] Ebina, Y., Okada, S., Hamazaki, S., Ogino, F., Li, J.L. and Midorikawa, O., Nephrotoxicity and renal cell carcinoma after use of iron-and aluminum-nitrilotriacetate complexes in rats, J. Natl. Cancer Inst., 76 (1986) 107±113. [5] Ghanta, V.K., Hiramoto, R.N., Solvason, H.B. and Spector, N.H., Neural and environmental in¯uences on neoplasia and conditioning of NK activity, J. Immunol., 135 (Suppl. 2) (1985) 848s±852s. [6] Gorczynski, R.M., Kennedy, M. and Ciampi, A., Cimetidine reverses tumor growth enhancement of plasmacytoma tumors in mice demonstrating conditioned immunosuppression, J. Immunol., 134 (1985) 4261±4266. [7] Guo, N., Conaway, C.C., Hussain, N.S. and Fiala, E.S., Sex and organ differences in oxidative DNA and RNA damage due to treatment of Sprague±Dawley rats with acetoxime or 2-nitropropane, Carcinogenesis, 11 (1990) 1659±1662. [8] Kasai, H., Analysis of a form of oxidative DNA damage, 8hydroxyguanosine, as a marker of cellular oxidative stress during carcinogenesis, Mutat. Res., 387 (1997) 147±163. [9] Kihara, H., Teshima, H., Sogawa, H. and Nakagawa, T., Stress and superoxide production by human neutrophils, Ann. N.Y. Acad. Sci., 15 (1992) 307±310. [10] Kovacs, P., Juranek, I., Stankovicova, T. and Svec, P., Lipid peroxidation during acute stress, Pharmazie, 51 (1996) 51± 53. [11] Nishiyama, Y., Suwa, H., Okamoto, K., Fukumoto, M., Hiai, H. and Toyokuni, S., Low incidence of point mutations in H-, K-and N-ras oncogenes and p53 tumor suppressor gene in renal cell carcinoma and peritoneal mesothelioma of Wistar rats induced by ferric nitrilotriacetate, Jpn. J. Cancer Res., 86 (1995) 1150±1158. [12] Riley, A.L. and Tuck, D.L., Conditioned taste aversions: a behavioral index of toxicity, Ann. N.Y. Acad. Sci., 443 (1985) 272±292. [13] Umemura, T., Sai, K., Takagi, A., Hasegawa, R. and Kurokawa, Y., Formation of 8-hydroxydeoxyguanosine (8-OHdG) in rat kidney DNA after intraperitoneal administration of ferric nitrilotriacetate (Fe-NTA), Carcinogenesis, 11 (1990) 345±347. [14] Wood, G.E. and Shors, T.J., Stress facilitates classical conditioning in males, but impairs classical conditioning in females through activational effects of ovarian hormones, Proc. Natl. Acad. Sci. USA., 95 (1998) 4066± 4071. [15] Yamaguchi, R., Hirano, T., Asami, S., Chung, M.H., Sugita, A. and Kasai, H., Increased 8-hydroxyguanine levels in DNA and its repair activity in rat kidney after administration of a renal carcinogen, ferric nitrilotriacetate, Carcinogenesis, 17 (1996) 2419±2422.
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Classical conditioning of oxidative DNA damage in rats Masahiro Irie a,*, Shinya Asami b, Shoji Nagata a, Masakazu Miyata c, Hiroshi Kasai b a
Department of Mental Health, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan b Department of Environmental Oncology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan c Department of Neurology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan Received 31 March 2000; received in revised form 13 May 2000; accepted 13 May 2000
Abstract This study investigated whether the formation of 8-hydroxydeoxyguanosine (8-OH-dG), a known oxidative DNA modi®cation relevant to carcinogenicity, can be classically conditioned to a novel taste in order to clarify the possible role of the central nervous system (CNS) or psychological stress on cancer initiation via a classical conditioning mechanism. Male Wistar rats underwent one or two conditioned taste aversion (CTA) experiments in which ferric nitrilotriacetate (Fe-NTA), which has renal toxicity and can induce renal cell carcinoma, served as a visceral unconditioned stimulus (US), and a saccharin solution (SAC) was used as a conditioned stimulus (CS). The 8-OH-dG levels in the group conditioned with the combination of SAC and Fe-NTA signi®cantly increased as compared to those of the uncombined groups by two repeats of the conditioning procedure (P 0:013). The rats that showed a painful response at the Fe-NTA administration had signi®cantly higher values of 8-OH-dG than those without pain (P 0:003). These results not only provide the ®rst evidence regarding classical conditioning of oxidative DNA damage using the CTA procedure, but also suggest the involvement of the CNS and psychological stress in the pathogenesis of cancer via oxidative DNA damage. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Conditioned taste aversion; 8-Hydroxydeoxyguanosine; Oxidative DNA damage; Stress; Pain; Cancer
It has been proposed that reactive oxygen species (ROSs) play a signi®cant role in the pathogenesis of cancer through several kinds of nuclear DNA injuries [3]. Among the more abundant types of base modi®cations, 8-hydroxydeoxyguanosine (8-OH-dG, 7,8-dihydro-8-oxodeoxyguanosine), produced by the oxidation of deoxyguanosine, is considered as the most sensitive and useful marker of oxidative DNA adducts [8]. It has been shown that 8-OH-dG is closely associated with various diseases, including cancer, and is produced by exposure to various carcinogens and hazardous materials [8]. Psychological stress has also been reported to increase the formation of 8-OH-dG in the rat liver [1]. However, the underlying pathway of stress-induced oxidative DNA damage is not yet clearly understood. Classical or Pavlovian conditioning phenomenon has been considered to be a part of stress responses and has often been used to identify noxious stimuli [2]. A condi* Corresponding author. Tel.: 181-93-691-7475; fax: 181-93692-5419. E-mail address: [email protected] (M. Irie).
tioned taste aversion (CTA) is a major form of classical conditioning, in which animals learn to avoid a novel taste (conditioned stimulus; CS) that has been previously followed by transient poisoning (unconditioned stimulus; US). With the use of this paradigm, it was demonstrated that cyclophosphamide-induced immune suppression can be conditioned [2]. Several kinds of CTAs were identi®ed thereafter, using a variety of agents, including carcinogens instead of cyclophosphamide [12]. Classical conditioning may contribute to the relationships between psychological stress and the formation of 8-OH-dG in rats; however, it has never been studied whether the CTA paradigm would affect oxidative DNA damage, such as 8-OH-dG. If conditioning of 8-OH-dG can be established, then it suggests that the central nervous system (CNS) can play a signi®cant role in oxidative DNA damage. Furthermore, it is assumed that the causes and the aggravating factors of cancer and other diseases involved in this oxidative DNA damage are partly mediating, conditioned reactions. Ferric nitrilotriacetate (Fe-NTA) is a well-known iron-
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 19 4- 0
14
M. Irie et al. / Neuroscience Letters 288 (2000) 13±16
chelator that causes iron-catalyzed free radical reactions in its target organ, the kidney [4,11,13,15]. Many groups have reported that Fe-NTA induces renal proximal tubular damage followed by a high incidence of renal cell carcinoma (RCC) in rodents [4,11]. Moreover, Fe-NTA-induced RCCs in rodents have been found to resemble human RCCs in several points, such as frequent invasion and metastasis [11]. It has been reported that the 8-OH-dG levels in renal tissues immediately increased at 1 h and decreased to the basal levels within three days after a single intraperitoneal administration of Fe-NTA (15 mg Fe/kg body weight) [13,15]. Thus, it is reasonable to use Fe-NTA, which is a carcinogen with a known time sequence for 8-OH-dG formation, as an unconditioned stimulus in the experiment of oxidative DNA damage relevant to cancer initiation due to classical conditioning. In the present study, we examined whether classical conditioning can be found regarding the formation of 8OH-dG in rats. Fe-NTA was initially used as an US, and a saccharin solution (SAC) served as a CS in the CTA procedure. This study was conducted in two ways, with either one conditioning (Experiment 1) or two conditionings (Experiment 2). The subjects were six-week old male Wistar rats (Seiwa Experimental Animal Institute, Fukuoka, Japan). After a one-week acclimatization to the housing environment, the time of the daily water supply was restricted gradually over one week, and thereafter, the rats were allowed to drink water only for 1 h, from 09:00 to 10:00 in the morning. The animals were gently handled for ®ve minutes on at least ®ve occasions, which were randomly scheduled during this period. On the conditioning day, the rats were randomly divided into four groups, according to the conditioning protocol described in Table 1. One half of the rats received SAC (0.2%) in place of water from 09:00 to 10:00, followed immediately by an intraperitoneal (i.p.) injection of Fe-NTA (CS1 US1 group) at a dose of 15 mg Fe/kg body weight or its equivalent volume of saline (CS1 US2 group), respectively. The rest of the rats were given water during the same period, followed immediately by an i.p. injection of FeNTA (CS2 US1 group) or saline (CS2 US2 group), respectively. In all groups, the animals were fasted for 2 h after the injection of Fe-NTA or saline. As there were rats Table 1 Conditioning procedure Group a
Adaptation period
Conditioning day b
Test day
CS1US1 CS1US2 CS2US1 CS2US2
H2O H2O H2O H2O
SAC 1 Fe-NTA SAC 1 SAL H2O 1 Fe-NTA H2O 1 SAL
SAC 1 SAL SAC 1 SAL H2O 1 SAL H2O 1 SAL
a
CS, conditioned stimulus; US, unconditioned stimulus. Fe-NTA, ferric nitrilotriacetate (15 mg Fe/kg body weight, i.p.); SAC, saccharin (0.2%); SAL, saline. b
showing clearly painful behaviors, such as crying and aggressive actions at the time of the Fe-NTA administration, the presence of pain was recorded. Subsequently, the restriction of drinking water to 1 h a day and the 2-h fasting were continued thereafter. One week later, either SAC or water, whichever was used with the conditioning procedures, was administered to the rats, followed by an i.p. injection of saline. All of the animals were fasted for 2 h after the injection and were then sacri®ced. The animals were killed by suf®cient exsanguination. Both kidneys were removed and a portion of the organ was stored at 2808C. In Experiment 2, the procedures and methodology were the same as in Experiment 1 except as noted below. The same conditioning procedure as in Experiment 1 was repeated twice at oneweek intervals. The solution of Fe-NTA was prepared according to the method of Yamaguchi et al. [15]. Fe(NO3)3 and Na2NTA (Wako Biochemicals, Osaka, Japan) were each dissolved in Milli-Q water and then mixed at a molar ratio of 1:4. The pH was adjusted with NaHCO3 to 7.4. These solutions were prepared immediately before use. The methods for analyzing the formation of 8OH-dG were described elsewhere [15]. In brief, the nuclear DNA was extracted from 50 mg of each kidney with the DNA Extractor WB Kit (Wako Biochemicals, Osaka, Japan). The extracted nuclear DNA was digested with nuclease P1 and acid phosphatase (378C for 30 min), and injected onto a HPLC column (Beckman, Ultrasphere-ODS, 5 mm, 4:6 £ 250 nm) coupled to an electrochemical detector (ESA Coulochem II: detector 1, 0.15 V; detector 2, 0.30 V). A one-way analysis of variance (ANOVA) with the Fisher's PLSD test, Student's t-test, and Spearman's rank correlations were performed to estimate statistical differences with the use of SPSS 7.5 for Windows. In Experiment 1, no signi®cant differences in the 8-OHdG levels were found among the four groups (F(3,32) 0.69; P 0.566), although the group conditioned with the combination of SAC and Fe-NTA (CS1 US1) showed slightly higher 8-OH-dG levels than the other groups. However, when the US-treated animals were analyzed further, by division into painful and non-painful groups, the painful group (n 8) showed signi®cantly higher 8OH-dG levels than the non-painful group (n 10) (t 3:53; P 0:003; Fig. 1). The painful group consisted of 4 CS1 and 4 CS2 animals. In Experiment 2, there were signi®cant differences regarding the levels of 8-OH-dG among the four groups (F 3; 36 4:11; P 0:013; Fig. 2). Multiple comparisons showed that the levels of 8-OH-dG in the CS1 US1 group were signi®cantly higher than those of the CS1 US2 group (P 0:003), the CS2 US2 group (P 0:007), and the CS2 US1 group (P 0:042), respectively. There was no signi®cant correlation between the 8-OH-dG levels and the body weights after the conditioning procedures in both experiments. Also, no differences were found regarding body weights before and after the conditioning procedures among the four groups in both experiments. The effect of pain at the time of US on the
M. Irie et al. / Neuroscience Letters 288 (2000) 13±16
Fig. 1. Comparison of 8-OH-dG levels between painful and nonpainful groups at the time of Fe-NTA administration. Each bar represents mean ^ SEM.
8-OH-dG levels was not examined in Experiment 2, because most of the US-treated animals showed painful behavior at least once. Numerous studies have shown possible classical conditionings of various biological parameters, such as neurological, endocrinological, and immune functions [2,5,6]. Besides these biological studies on classical conditionings, research is now being extended to cover its relationship to genetics. As for cancer, some researchers have found that natural killer cell activity, a defense against cancer progression, can be conditioned [5]. Classical conditioning of tumor growth was also reported [6]. However, it has scarcely been investigated as to whether conditioning plays a signi®cant role in cancer initiation through genetic alterations relevant to carcinogenicity. We report here the ®rst evidence that the formation of representative oxidative DNA damage, 8-OH-dG, increased in response to the CS using the CTA procedure. Our data provide one mechanism whereby the CNS modulates the formation of 8-OH-dG. Since an association of such DNA damage with carcinogenicity is evident [3,8], it seems possible that the conditioning mechanism plays an etiological role in the initiation of cancer. Furthermore, the results support the involvement of stress in the pathogenesis of cancer through oxidative DNA damage. Although the underlying mechanism of how the formation of 8-OH-dG can be classically conditioned is unclear, some investigators found positive associations between stress and the formation of ROSs [9,10]. Classical conditioning of ROSs appears to have not been investigated. Further studies are required to con®rm this possibility. However, the degree of the involvement of the classical conditioning mechanism was not estimated to be very high, even if it affected the formation of 8OH-dG, because the 8-OH-dG level at 2 h after the injection of saline in the CS1 US1 group in Experiment 2 was about 1/3 or 1/4 of that after the administration of Fe-NTA [15].
15
It is necessary to consider the effects of Fe-NTA and saline themselves on the formation of 8-OH-dG in discussing our ®ndings. In previous studies [13,15], the formation of 8-OH-dG in renal tissues increased 1 h after an intraperitoneal injection of Fe-NTA (15 mg Fe/kg body weight) to rats, but it returned to the basal level within three days. In contrast, the repair enzyme activity maintained a higher level for ®ve days and returned to the basal level within two weeks. The injection of saline did not induce a signi®cant increase in the level of 8-OH-dG. Based on these fundamental studies, the 8-OH-dG levels in the kidneys were measured at a one-week interval after conditioning with the administration of Fe-NTA in the present study, so that there was no possibility of residual 8-OH-dG synthesized in the renal tissues affecting the measured values. This view was also veri®ed by the ®ndings that the absence or presence of Fe-NTA administration did not show a signi®cant difference in Experiment 1. Even if the 8-OH-dG repair enzyme was increased at one week after the administration of Fe-NTA, it should have acted to decrease the formation of 8-OH-dG and, therefore, is not considered to be a factor for the increase in the 8-OH-dG levels by conditioning. It is not clear whether the 8-OH-dG levels in renal tissues at one week after the Fe-NTA administration returned to the preadministration levels when Fe-NTA was given twice at a oneweek interval. However, the 8-OH-dG levels after two injections of Fe-NTA unpaired with SAC did not differ from those without the Fe-NTA administration, and were signi®cantly lower than those of the group that received two injections of Fe-NTA paired with SAC. Therefore, the possibility of accumulation of 8-OH-dG by two administrations of Fe-NTA was considered to be negligible. The positive relationship between painful behaviors and the 8-OH-dG levels seems to support the previous results that stress induced oxidative DNA damage [1]. While the
Fig. 2. Comparison of 8-OH-dG levels among the four groups according to the two different pairings of CS 2 US. Each bar represents mean ^ SEM; n 10 rats/group; *P , 0:05, **P , 0:01, as compared to the SAC 1 Fe-NTA group by Fisher's PLSD test. See Table 1 for more details.
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M. Irie et al. / Neuroscience Letters 288 (2000) 13±16
possibility that the use of SAC confounded the relationship was considered, the presence of pain was not associated with the use of SAC. Our results indicate that stress plays a signi®cant role in enhancing the probability of a conditioned oxidative DNA damage. There have been some other reports in which the effects of stress on the conditionability of biological parameters were evaluated. For example, Wood and Shors [14] reported that stress facilitates the effect of classical conditioning in male rats, but impairs it in female rats through the activational effects of ovarian hormones. This sex difference was interesting, because male rats were found to produce more 8-OH-dG than female rats when treated with carcinogens [7]. Thus, the use of male rats seem to contribute to the susceptibility to the classical conditioning and the formation of 8-OH-dG in the present study. Our ®ndings suggest that not only the CTA paradigm, but also pain, contributed to the formation of 8-OH-dG. While there is a report [1] stating that the 8-OH-dG levels in the rat hepatic and renal tissues were increased by psychological stress, the psychological stress due to anxiety induced by at least two sessions of 5 h of electric stimulation was needed in the case of the liver, and four sessions were used in the case of the kidney. Taking this report into consideration, although the experimental method differs, the effect of psychological stress on 8-OH-dG formation may be insuf®cient with one or two conditionings. However, in contrast to the repeated loading of psychological stress, it is dif®cult to repeat the administration of Fe-NTA to rats, since this agent increases kidney damage and could possibly affect the measurement values by the damage alone. Similar methodological limitations are expected when other carcinogens are employed. In view of the different effects of psychological stress on the formation of 8-OH-dG between the kidney and the liver, the kidney may be less sensitive to stress than the liver. Therefore, it will be necessary to carry out studies using other, less toxic carcinogens or other organs, such as the liver, in the future to con®rm our ®ndings. [1] Adachi, S., Kawamura, K. and Takemoto, K., Oxidative damage of nuclear DNA in liver of rats exposed to psychological stress, Cancer Res., 53 (1993) 4153±4155. [2] Ader, R. and Cohen, N., Psychoneuroimmunology: conditioning and stress, Annu. Rev. Psychol., 44 (1993) 53±85.
[3] Dreher, D. and Junod, A.F., Role of oxygen free radicals in cancer development, Eur. J. Cancer, 32A (1996) 30±38. [4] Ebina, Y., Okada, S., Hamazaki, S., Ogino, F., Li, J.L. and Midorikawa, O., Nephrotoxicity and renal cell carcinoma after use of iron-and aluminum-nitrilotriacetate complexes in rats, J. Natl. Cancer Inst., 76 (1986) 107±113. [5] Ghanta, V.K., Hiramoto, R.N., Solvason, H.B. and Spector, N.H., Neural and environmental in¯uences on neoplasia and conditioning of NK activity, J. Immunol., 135 (Suppl. 2) (1985) 848s±852s. [6] Gorczynski, R.M., Kennedy, M. and Ciampi, A., Cimetidine reverses tumor growth enhancement of plasmacytoma tumors in mice demonstrating conditioned immunosuppression, J. Immunol., 134 (1985) 4261±4266. [7] Guo, N., Conaway, C.C., Hussain, N.S. and Fiala, E.S., Sex and organ differences in oxidative DNA and RNA damage due to treatment of Sprague±Dawley rats with acetoxime or 2-nitropropane, Carcinogenesis, 11 (1990) 1659±1662. [8] Kasai, H., Analysis of a form of oxidative DNA damage, 8hydroxyguanosine, as a marker of cellular oxidative stress during carcinogenesis, Mutat. Res., 387 (1997) 147±163. [9] Kihara, H., Teshima, H., Sogawa, H. and Nakagawa, T., Stress and superoxide production by human neutrophils, Ann. N.Y. Acad. Sci., 15 (1992) 307±310. [10] Kovacs, P., Juranek, I., Stankovicova, T. and Svec, P., Lipid peroxidation during acute stress, Pharmazie, 51 (1996) 51± 53. [11] Nishiyama, Y., Suwa, H., Okamoto, K., Fukumoto, M., Hiai, H. and Toyokuni, S., Low incidence of point mutations in H-, K-and N-ras oncogenes and p53 tumor suppressor gene in renal cell carcinoma and peritoneal mesothelioma of Wistar rats induced by ferric nitrilotriacetate, Jpn. J. Cancer Res., 86 (1995) 1150±1158. [12] Riley, A.L. and Tuck, D.L., Conditioned taste aversions: a behavioral index of toxicity, Ann. N.Y. Acad. Sci., 443 (1985) 272±292. [13] Umemura, T., Sai, K., Takagi, A., Hasegawa, R. and Kurokawa, Y., Formation of 8-hydroxydeoxyguanosine (8-OHdG) in rat kidney DNA after intraperitoneal administration of ferric nitrilotriacetate (Fe-NTA), Carcinogenesis, 11 (1990) 345±347. [14] Wood, G.E. and Shors, T.J., Stress facilitates classical conditioning in males, but impairs classical conditioning in females through activational effects of ovarian hormones, Proc. Natl. Acad. Sci. USA., 95 (1998) 4066± 4071. [15] Yamaguchi, R., Hirano, T., Asami, S., Chung, M.H., Sugita, A. and Kasai, H., Increased 8-hydroxyguanine levels in DNA and its repair activity in rat kidney after administration of a renal carcinogen, ferric nitrilotriacetate, Carcinogenesis, 17 (1996) 2419±2422.