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Inhibition of 8-Hydroxyguanine Repair in Testes after Administration of Cadmium Chloride to GSH-Depleted Rats
TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.
147, 9–14 (1997)
TO978260
Inhibition of 8-Hydroxyguanine Repair in Testes after Administration of Cadmium Chloride to GSH-Depleted Rats Takeshi Hirano, Yoko Yamaguchi, and Hiroshi Kasai Department of Environmental Oncology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu, Fukuoka 807, Japan Received August 5, 1996; accepted July 15, 1997
has been shown to stimulate human polymorphonuclear leukocytes, which generate ROS that are mutagenic and carcinogenic (Zhong et al., 1990). According to these reports, a relationship seems to exist between Cd-induced cytotoxicity or carcinogenicity and ROS formation. However, the direct formation of a ROS from Cd has not been proven. Therefore, the mechanisms of its cytotoxicity or carcinogenicity based on ROS are still unclear. One notable effect of ROS is to cause DNA damage. The types of DNA damage caused by ROS include strand breaks (Minisini et al., 1994) and base modifications, such as thymine glycol (Leadon and Hanawalt, 1983), 5-hydroxymethyluracil (Teebor et al., 1982), 8-hydroxyadenine (Dizdaroglu and Bergtold, 1986), and 8-hydroxyguanine (8-OH-Gua). 8OH-Gua is one of the most abundant types of oxidative DNA damage, and it was first reported in 1984 (Kasai and Nishimura, 1984). Because 8-OH-Gua is known to cause GC to TA transversions in DNA (Basu et al., 1989; Cheng et al., 1992), it is important to evaluate the levels of 8-OHGua in nuclear DNA when exploring the mechanisms of Cd carcinogenesis. From these points of view, it is predicted that Cd-induced carcinogenicity might be mainly due to oxidative DNA damage. In fact, it was reported that CdCl2 induces DNA singlestrand scission, due to ROS (Mu¨ller et al., 1991). However, to our knowledge, there have been few studies of the mechanisms of Cd-induced carcinogenicity by oxidative DNA damage, although other metals, such as Ni(II), Fe(III)NTA, and Cr(VI), were reported to produce 8-OH-Gua in DNA (Kasprzak and Hernandez, 1989; Umemura et al., 1990; Aiyar et al., 1990). In this study, we measured 8-OH-Gua in nuclear DNA and its repair activity in CdCl2-treated rat testis and lung to study the mechanism of Cd-induced carcinogenicity. At the same time, we assayed superoxide dismutase (SOD) activities and LPO levels in the organs to understand the role of oxidative stresses in Cd-induced carcinogenicity. Furthermore, to examine the role of GSH in Cd-induced carcinogenicity, we analyzed rat organs in which GSH biosynthesis was inhibited by L-buthionine-[S, R]-sulfoximine (BSO).
Inhibition of 8-Hydroxyguanine Repair in Testes after Administration of Cadmium Chloride to GSH-Depleted Rats. Hirano, T., Yamaguchi, Y., and Kasai, H. (1997). Toxicol. Appl. Pharmacol. 147, 9–14. The main goal of this study is to investigate the mechanism of cadmium (Cd)-induced carcinogenesis by reactive oxygen species. Rats were divided into four groups and were treated with (i) saline (control), (ii) cadmium chloride (CdCl2), (iii) L-buthionine-[S, R]sulfoximine (BSO, an inhibitor of GSH biosynthesis), and (iv) CdCl2 and BSO, respectively. They were euthanized at 0, 24, 48, and 72 hr after these treatments, and the lungs and testes were analyzed. After treatment with both CdCl2 and BSO, the testicular 8-OH-Gua level increased (48 hr), its repair activity decreased (48 and 72 hr), the GSH content was markedly suppressed (48 and 72 hr), the superoxide dismutase activities slightly (48 and 72 hr) decreased, and the lipid peroxidation level increased (24 and 72 hr) in the testes as compared to the control levels. These results suggest that under GSH-depleted conditions, CdCl2 inhibits 8-OH-Gua repair activity in the rat testis and 8-OH-Gua accumulates in the DNA, which may pertain to testicular carcinogenesis. q 1997 Academic Press
Cadmium (Cd) is a highly toxic element that is naturally present in all parts of the environment, including food, water, and soil (Sherlock, 1984). It affects a wide variety of metabolic processes, such as energy metabolism (Mu¨ller, 1986), membrane transport (Verbost, 1989), and protein synthesis (Dudley et al., 1984). Several Cd compounds have been shown to have carcinogenic potential in humans and animals (Kazantzis, 1963), and the main target organs of Cd-induced carcinogenicity are lung, prostate, and testis (Waalkes and Poirier, 1985). However, the mechanisms of Cd-induced carcinogenicity are still not understood completely. Recently, it has been suggested that reactive oxygen species (ROS) play a major role in the mechanism of Cd-induced toxicity. It was reported that low doses of CdCl2 stimulated lipid peroxidation (LPO) without any evidence of acute damage in rat organs (Manca et al., 1991). Dietary ascorbic acid supplements were reported to reduce Cd toxicity in young Japanese quail (Fox et al., 1970). Cadmium sulfate (CdS) 9
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METHODS
The contents of GSH and GSSG in the supernatants were determined by the previous method (Hissin et al., 1976).
Animal preparation. Eight-week-old male Sprague–Dawley (SD) rats, weighing 271.4 { 13.4 g, were purchased from Seiwa Experimental Animals (Fukuoka, Japan), and were fed a CE pellet basal diet (Japan Clea Co., Tokyo, Japan) and tap water ad libitum. They were maintained in a temperature- and photoperiod-controlled room (247C, 12 hr/day). Chemicals. BSO, which is an inhibitor of glutathione biosynthesis, and cadmium chloride (CdCl2) were purchased from Sigma Chemical Co. (St. Louis, MO). BSO and CdCl2 were dissolved in sterile saline to 0.5 M and 20 mM, respectively. Spectrophotometric assay kits of superoxide dismutase activity (SOD-525) and lipid peroxidation (LPO-586) were purchased from Bioxytech S.A. (Bonneuil/Marne, France). [g-32P]ATP (Ç6000 Ci/mmol) were purchased from Amersham International Ltd. (Buckinghamshire, UK). Other chemicals were of the highest purity commercially available. Experimental protocol. Forty eight SD male rats were divided into four groups. The first group was a control group, in which rats were injected sc with 0.75 ml/kg body weight and ip with 10 ml/kg of sterile saline. Rats in the second group were injected sc with 15 mmol/kg of CdCl2 and ip with 10 ml/kg of sterile saline. Rats in the third group were injected sc with 0.75 ml/kg of sterile saline and ip with 5 mmol/kg of BSO. Rats in the last group were injected sc with 15 mmol/kg of CdCl2 and ip with 5 mmol/kg of BSO. Four rats from each group were euthanized under ether anesthesia at 24, 48, and 72 hr after treatment. Four more rats (no treatment) were euthanized as a time 0 control. Testes and lungs were removed immediately after sacrifice and were used for the experiments. Measurement of 8-OH-Gua. 8-OH-Gua levels in rat organs were determined as previously reported (Nakae et al., 1995), with slight modification (Yamaguchu et al., 1996). Briefly, the samples were homogenized in lysis buffer with a potter type homogenizer, and the nuclear DNA in the homogenate was extracted using the DNA Extractor WB kit. The extracted nuclear DNA was digested with nuclease P1 and acid phosphatase in a 10 mM sodium acetate solution (377C for 30 min). After the incubation, the mixture was treated with the iron exchange resin, Muromac (Muromachi kagaku, Tokyo, Japan) and was centrifuged at 15,000g for 5 min. The supernatant was transferred to a filter tube (Millipore, Samprep C, 0.2 mm), centrifuged at 5000g for 5 min, and injected onto a high-performance liquid chromatography column (Beckman, Ultrasphere-ODS, 5 mm, 4.6 1 250 nm) equipped with an electrochemical detector (ESA Coulochem II). As standard samples, 20 ml each of deoxyguanosine (0.5 mg/ml) and 8-hydroxydeoxyguanosine (5 ng/ml) solutions were injected. The value of 8-OH-Gua was calculated as the number per 105 guanine residues. Endonuclease nicking assay. Excision repair activities in rat organs were assayed by the previously described method (Hirano et al., 1995). Briefly, tissues removed from sacrificed rats were homogenized in 50 mM Tris–HCl buffer (pH 7.5) containing protease inhibitors (5 mg/ml each of pepstatin, leupeptin, antipain, and chymostatin) with a Dounce-type homogenizer. Homogenates were centrifuged at 10,000g to obtain the crude extracts. The total protein concentration of the crude extracts was adjusted to 5 mg/ml. A 22-mer double-stranded synthetic oligonucleotide containing 8-OH-Gua (upper strand, 5*-GGTGGCCTGACG*CATTCCCCAA-3*; G*, 8-OH-Gua) was used as the substrate for this assay. Crude extract (50 mg protein) was incubated with 0.05 pmol of the 32P end-labeled doublestranded DNA substrate at 257C for 1 hr. The cleaved fragment, generated as the consequence of base excision repair activity (glycosylase activity and AP endonuclease activity) was analyzed by 20% denaturing polyacrylamide gel electrophoresis. The repair activity is expressed as the ratio of the excised fragment activity to the total activity (substrate activity plus fragment activity). Measurement of GSH and GSSG content. Tissues were homogenized by a Dounce-type homogenizer, either alone or in combination with a Politron, in 0.1 M sodium phosphate buffer with 5 mM EDTA (pH 8.0), mixed with meta phospholic acid, and centrifuged at 12,000g for 30 min.
Measurement of SOD activity. Tissues were homogenized by a Dounce-type homogenizer, either alone or in combination with a Politron, in 10 mM Tris–HCl (pH 7.4) containing 0.25 M sucrose and 1 mM EDTA. Homogenates were centrifuged at 6000g for 30 min. SOD activities in the supernatants were measured using a spectrophotometric assay kit according to the manufacturer’s instructions. The SOD activities are expressed as units per milligram of protein.
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Assessment of lipid peroxidation by measurement of MDA. Tissues were homogenized by a Dounce-type homogenizer, either alone or in combination with a Politron, in 20 mM Tris–HCl (pH 7.4). Homogenates were centrifuged at 6000g for 30 min. The LPO levels in the supernatants were determined using a spectrophotometric assay kit according to the manufacturer’s instructions. The LPO values are expressed as nanomoles of malon dialdehyde (MDA) production per milligram of protein. Statistical analysis. Values in the text and tables represent the means { SD. The statistical significance was calculated using the Student t test. Probability values less than 0.05 were considered to indicate significant differences.
RESULTS
8-OH-Gua Levels in Testis and Lung DNA Using a rapid DNA extraction method with a commercial kit, the background level of 8-OH-Gua was reduced to 0.2– 0.3/105 Gua in this study. As seen in from Fig. 1, the 8OH-Gua levels in the nuclear DNA of CdCl2- and BSOtreated rat testis (48 hr) increased significantly as compared with those of other rat groups. On the other hand, the 8OH-Gua levels in rat lung nuclear DNA showed no significant change among the groups in this study (data not shown).
FIG. 1. Time course of 8-OH-Gua level (% of control) in the nuclear DNA of the rat testis. Mean values { SD, n Å 4. The absolute values (per 105 guanines) for control rats are 0.317 { 0.070 at time 0, 0.285 { 0.068 at 24 hr, 0.243 { 0.033 at 48 hr, and 0.268 { 0.020 at 72 hr. *Significantly different from the Cd treatment group (48 hr) at p õ 0.005 and the BSO treatment group (48 hr) at p õ 0.01.
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FIG. 2. Autoradiogram of an endonuclease nicking assay of rat testes preparations. (a) Substrate 22-mer oligonucleotides containing 8-OH-Gua. (b) Excised fragments. The 8-OH-Gua repair activities are expressed as the ratio of the radioactivity of the lower band to the total radioactivity.
8-OH-Gua Repair Activity in Testis and Lung At 24 hr after the treatments, no significant change was observed in the base excision repair activities of the testes of all rats. At 48 and 72 hr after the treatment, the 8-OHGua repair activity was quite depressed in the testes of the CdCl2- and BSO-treated rats (Figs. 2 and 3). The 8-OH-Gua repair activity showed no significant change in the testes of the rats in other groups. The repair activity in the rat lungs showed no significant change among the groups in this study (data not shown). Cellular GSH and GSSG Concentrations Since cellular GSH is supposed to work as a first defense against Cd-induced toxicity (Singhal et al., 1987), GSH bio-
FIG. 4. Time course of the cellular GSH concentrations in rat testes. The values are expressed as nmol/mg protein. Mean values { SD, n Å 4. Significantly different from the control group of the same observation time: *p õ 0.001, **p õ 0.01, ***p õ 0.05.
synthesis was selectively inhibited by BSO administration to investigate the relationship between GSH content and 8OH-Gua repair activity. Figure 4 shows that the cellular GSH content in CdCl2-treated rat testes decreased at 48 and 72 hr after administration. Furthermore, the GSH content in CdCl2- and BSO-treated rat testes decreased markedly, particularly at 48 and 72 hr after treatment, and it seems that CdCl2 and BSO work additively to decrease the GSH content. The GSSG content also was reduced in the testes of CdCl2- and BSO-treated rats (data not shown). As for the lungs, the GSH and GSSG levels showed tendencies of decreasing at 48 and 72 hr only in the BSO-treated and the CdCl2- and BSO-treated rats. No significant changes were seen in the lung GSH and GSSG levels of the rats treated with CdCl2 alone. It seems that Cd does not affect the GSH and GSSG contents in the rat lung (data not shown). SOD Activity in Testis
FIG. 3. Time course of the 8-OH-Gua repair activity value (% of control) in the rat testis. Mean values { SD, n Å 4. The absolute values for control rats are 0.331 { 0.091 at time 0, 0.403 { 0.016 at 24 hr, 0.396 { 0.032 at 48 hr, and 0.255 { 0.023 at 72 hr. *Significantly different from the Cd treatment group (48 hr) at p õ 0.05 and the BSO treatment group (48 hr) at p õ 0.005. **Significantly different from the Cd treatment group (72 hr) at p õ 0.01 and the BSO treatment group (72 hr) at p õ 0.005.
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To assess the balance of ROS production in the testis, the SOD activity was measured. SOD activities in the CdCl2and BSO-treated rat testes decreased at 48 and 72 hr after injection, and the minimum level was approximately 75% of the control at 72 hr after treatment. The activity in the BSO-treated rat testis increased at 48 and 72 hr after treatment, but no significant change was observed in the testes of other rat groups (Table 1). For all groups, there were no significant changes in the lung SOD activities (data not shown). LPO Production in Testis The cellular damage induced by ROS was estimated by monitoring the LPO levels, which are a well known indicator
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TABLE 1 SOD Activity in Rat Testes SOD activity (units/mg protein) Treatments
0 hr
Control Cd BSO Cd / BSO
0.259 { 0.010 — — —
24 hr 0.225 0.200 0.249 0.228
{ { { {
48 hr
0.037 0.025 0.015 0.042
0.230 0.215 0.278 0.189
{ { { {
0.028 0.019 0.027* 0.017*
72 hr 0.223 0.203 0.259 0.167
{ { { {
0.007 0.026 0.017* 0.014**
Note. Values are means { SD of four rats. * Significantly different from control group at p õ 0.05. ** Significantly different from control group at p õ 0.001.
of cellular damage by oxidative stress. In the testis, the MDA concentrations showed significant increases at 24 and 72 hr after both CdCl2 and BSO administration. There were also increases at 48 and 72 hr after CdCl2 administration and at 48 hr after both CdCl2 and BSO administration (Table 2). For all groups, there were no significant changes in the lung LPO levels (data not shown). DISCUSSION
We were interested in whether Cd-induced carcinogenicity is due to oxidative DNA damage. Cd compounds are known to induce various form of oxidative damage, such as an increase of LPO (Manca et al., 1991), a decrease of GSHPx (Sidhu et al., 1993), and DNA strand breaks (Mu¨ller et al., 1991). However, Cd is not thought to induce ROS directly, because Cd2/ is not capable of accepting or donating electrons under physiological conditions (Ochi et al., 1987). On the other hand, many reports (Piscator, 1981; Hallenbeck, 1984) indicated that some Cd compounds have carcinogenic potential in specific organs, such as lung, prostate, and testis. However, the direct role of Cd in carcinogenic mechanisms is not clear (Degraeve, 1981). In this study, we assayed the level of oxidative DNA damage, by monitoring 8-OH-Gua
formation and its repair activity, in CdCl2- and/or BSOtreated rats to investigate the relationship between oxidative DNA damage and Cd-induced carcinogenicity. As far as we know, no conclusive data have been published regarding Cdinduced cancer in rat testes. Therefore, we chose the CdCl2 dose based on a previous report, without regard to the carcinogenicity. Singhal et al. reported that BSO-treated mice also treated with 20 mmol Cd2//kg experienced 100% mortality, whereas BSO-treated mice also treated with 10 mmol Cd2//kg survived. Based on these data, we chose a 15 mmol/ kg CdCl2 dose. This dose might be extremely high in terms of carcinogenicity. However, because we designed a short time range experiment, we selected this dose. 8-OH-Gua is known to be induced by ionizing radiation or ROS-forming agents (Kasai and Nishimura, 1984; Dizdaroglu and Bergtold, 1985; Umemura et al., 1990; Aiyar et al., 1990). Since this form of DNA damage is reported to cause GC to TA transversions in DNA, it is believed to be responsible for point mutations in genomic DNA (Wood et al., 1990; Cheng et al., 1992). Hence, analysis of the 8-OHGua level is useful for the study of carcinogenic mechanisms. To obtain evidence that ROS, which cause an increase in 8OH-Gua, were actually produced, we assayed LPO production in rat testes. Since the dose of CdCl2 previously reported
TABLE 2 LPO Levels in Rat Testes MDA (nmol/mg protein) Treatments
0 hr
Control Cd BSO Cd / BSO
34.67 { 13.35 — — —
24 hr 45.38 31.26 50.58 130.82
{ { { {
48 hr
11.23 15.65 21.24 46.29*
35.02 312.88 48.81 349.31
{ 11.41 { 470.26 { 3.77 { 740.41
Note. Values are means { SD of four rats. * Significantly different from control group at p õ 0.05. ** Significantly different from control group at p õ 0.001.
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72 hr 56.22 754.96 19.14 1730.42
{ 48.60 { 789.93 { 6.35 { 214.94**
OXIDATIVE DNA DAMAGE AND CADMIUM CARCINOGENICITY
to stimulate LPO in rat was 500 mg CdCl2/kg (Manca et al., 1991) (Å2.7 mmol CdCl2/kg), we expected LPO production to be increased in the Cd-treated rat organs in our study. As shown in Table 2, LPO production increased in the testes of CdCl2- and BSO-treated rats. Although LPO production does not always indicate ROS production, the evidence might support the increase of oxidative DNA damage in the testes of CdCl2- and BSO-treated rats. In the testes of the same experimental groups, a decrease in SOD activity was also observed. The reduction of SOD activity is known to be due to the replacement of the Zn and/or Mn of the SOD molecule by Cd (Jacobson and Turner, 1980). In our analyses of the testicular SOD activities, we observed that CdCl2 alone had no effect, but the CdCl2- and BSO-treatment decreased them. We think that the lower SOD activity in the testes of CdCl2- and BSO-treated rats is due to cell damage. These results suggest that, in the testes of CdCl2- and BSOtreated rats, an imbalance of O20 and H2O2 might occur. Therefore, the decrease in the SOD activity might indirectly lead to an increase in oxidative DNA damage. Another possible mechanism for the increase of 8-OHGua would be that the 8-OH-Gua repair system is less efficient in rat testes after CdCl2- and BSO-treatment. To examine this hypothesis, we determined the levels of 8-OH-Gua repair activity in various organs. The assay method involved detection of the excised DNA fragment produced during the 8-OH-Gua repair process. The result of this endonuclease nicking assay showed no significant changes in CdCl2treated or BSO-treated rat testes and the rat lungs from all groups (Figs. 2 and 3). However, the activities were markedly depressed in CdCl2- and BSO-treated rat testes (48 and 72 hr) as compared to the control rat testes. These data suggest that CdCl2 might increase 8-OH-Gua level by inhibiting the 8-OH-Gua repair activity when GSH biosynthesis is inhibited. Since the repair activity was not suppressed by CdCl2 treatment alone, it seems that cellular GSH is required for the 8-OH-Gua repair activity. In fact, the GSH amounts in CdCl2- and BSO-treated rat testes were extremely reduced at 48 and 72 hr in comparison to those found in the testes of other rat groups, including the BSO-treated rats (Fig. 4). This pattern was almost the same as that of the 8-OH-Gua repair activity. However, although the GSH content in the testes of rats treated with CdCl2 alone was reduced, the repair activities did not show any change (Fig. 3). These results suggest that only a severe reduction in GSH might cause an inhibition of repair activity. Since the CdCl2 treatment did not increase the 8-OH-Gua level, we could not conclude that a direct relationship between CdCl2-induced carcinogenicity and oxidative DNA damage exists. However, our data suggest that if GSH depletion occurred, then the risk of CdCl2induced carcinogenicity would increase. Therefore, we suggest that GSH might, at least in part, regulate 8-OH-Gua
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repair activity. To our knowledge, GSH depletion in humans would occur in certain situations, such as excessive alcohol intake (Hirano et al., 1992). However, the exact role of GSH in 8-OH-Gua repair activity remains unclear. In this experiment, no notable changes of 8-OH-Gua levels and its repair activities were observed in the lung, another target organ of Cd carcinogenesis. We could not conclude that the mechanism of Cd-induced carcinogenicity in the lung is different from that in the testis, because target organ of Cd toxicity differ depending on the type of compound. To investigate the mechanism of carcinogenesis in the lung, we should use a CdCl2 aerosol (Takenaka et al., 1983) or other types of Cd compounds, such as CdS with various methods of administration, including inhalation. In conclusion, CdCl2 treatment increases 8-OH-Gua formation, possibly by suppression of the 8-OH-Gua repair activity, when GSH biosynthesis is inhibited. This implies that if these types of acute damage occur repeatedly, by Cd administration over a long period of time, they may result in the accumulation of replication errors that could lead to carcinogenesis, which might be one of the possible mechanisms of Cd-induced carcinogenicity in the testis. Since we did not measure the repair activity between 24 and 48 hr, we cannot discuss this temporal relationship. However, we suggest that an imbalance in the levels of ROS might increase the 8-OH-Gua level. Taken together, in the CdCl2and BSO-treated rat testis, both the imbalance of ROS and the decrease of 8-OH-Gua repair activity might increase 8OH-Gua accumulation. Since the CdCl2 dose we used in this study was higher than that used for carcinogenicity studies in rats, tissue damage, including cell death, might occur prior to the changes in 8-OH-Gua and its repair activity. However, the risk of replication errors leading to point mutations might be increased when the damaged tissue recovered. From this point of view, the severe depletion of 8-OH-Gua repair activity could be an important step in testicular carcinogenicity. However, the long-term effects of low dose Cd exposure on oxidative DNA damage merit further investigation. In this study, we suggest that CdCl2-induced testicular carcinogenicity is associated with oxidative DNA damage. However, since we did not measure ROS production directly, the strict relationship between Cd-induced carcinogenicity and ROS is still unclear. This relationship should also be investigated. ACKNOWLEDGMENTS This work was supported by a Grant from the University of Occupational and Environmental Health, Japan, and by a Grant-in Aid for Scientific Research on Priority Areas from The Ministry of Education, Science and Culture of Japan (05270102).
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stress protein synthesis induced by oxidative stress proceed independently in the human premonocytic line U937. Mutat. Res. 315, 169–179. Mu¨ller, L. (1986). Consequences of cadmium toxicity in rat hepatocytes: Mitochondrial dysfunction and lipid peroxidation. Toxicology 40, 285– 295. Mu¨ller, T., Schuckelt, R., and Jaenicke, L. (1991). Cadmium/zinc-metallothionein induces DNA strand breaks in vitro. Arch. Toxicol. 65, 20–26. Nakae, D., Mizumoto, Y., Kobayashi, E., Noguchi, O., and Konishi, Y. (1995). Improved genomic/nuclear DNA extraction for 8-hydroxy-deoxyguanosine analysis of small amounts of rat liver tissue. Cancer Lett. 97, 233–239. Ochi, T., Takahashi, K., and Ohsawa, M. (1987). Indirect evidence for the induction of a prooxidant state by cadmium chloride in cultured mammalian cells and a possible mechanism for the induction. Mutat. Res. 180, 257–266. Piscator, M. (1981). Role of cadmium in carcinogenesis with special reference to cancer of the prostate. Environ. Health Perspect. 40, 107–120. Sherlock, J. C. (1984). Cadmium in foods and the diet. Experientia 40, 152–156. Sidhu, M., Sharma, M., Bhatia, M., Awasthi, Y. C., and Nath, R. (1993). Effect of chronic cadmium exposure on glutathione S-transferase and glutathione peroxidase activities in Rhesus monkey: The role of selenium. Toxicology 83, 203–213. Singhal, R. K., Anderson, M. E., and Meister, A. (1987). Glutathione, a first line of defense against cadmium toxicity. FASEB J. 1, 220–223. Takenaka, S., Oldiges, H., Ko¨nig, H., Hochrainer, D., and Gu¨nter, O. (1983). Carcinogenicity of cadmium chloride aerosols in W rats. J. Natl. Cancer Inst. 70, 367–373. Teebor, G. W., Frenkel, K., and Goldstein, M. S. (1982). Identification of radiation-induced thymine derivatives in DNA. Adv. Enzyme Regul. 20, 39–54. Umemura, T., Sai, K., Takagi, A., Hasegawa, R., and Kurokawa, Y. (1990). Formation of 8-hydroxydeoxyguanosine (8-OH-dG) in rat kidney DNA after intraperitoneal administration of ferric nitrilotriacetate (Fe-NTA). Carcinogenesis 11, 345–347. Verbost, P. M. (1989). Cadmium inhibition of the erythrocyte Ca2/ pump. J. Biol. Chem. 264, 5613–5615. Waalkes, M. P., and Poirier, L. A. (1985). Interaction of cadmium with interstitial tissue of the rat testes. Biochem. Pharmacol. 34, 2513–2518. Wood, M. L., Dizdaroglu, M., Gajewski, E., and Essigmann, J. M. (1990). Mechanistic studies of ionizing radiation and oxidative mutagenesis: Genetic effects of a single 8-hydroxyguanine (7-hydro-8-oxoguanine) residue inserted at a unique site in a viral genome. Biochemistry 29, 7024– 7032. Yamaguchi, R., Hirano, T., Asami, S., Sugita, A., and Kasai, H. (1996). Increase in the 8-hydroxyguanine repair activity in the rat kidney after the administration of a renal carcinogen, Ferric Nitrilotriacetate. Environ. Health Persp. 104, 651–653. Zhong, Z., Troll, W., Koenig, K. L., and Frenkel, K. (1990). Carcinogenic sulfide salts of nickel and cadmium induce H2O2 formation by human polymorphonuclear leukocytes. Cancer Res. 50, 7564–7570.
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Inhibition of 8-Hydroxyguanine Repair in Testes after Administration of Cadmium Chloride to GSH-Depleted Rats Takeshi Hirano, Yoko Yamaguchi, and Hiroshi Kasai Department of Environmental Oncology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu, Fukuoka 807, Japan Received August 5, 1996; accepted July 15, 1997
has been shown to stimulate human polymorphonuclear leukocytes, which generate ROS that are mutagenic and carcinogenic (Zhong et al., 1990). According to these reports, a relationship seems to exist between Cd-induced cytotoxicity or carcinogenicity and ROS formation. However, the direct formation of a ROS from Cd has not been proven. Therefore, the mechanisms of its cytotoxicity or carcinogenicity based on ROS are still unclear. One notable effect of ROS is to cause DNA damage. The types of DNA damage caused by ROS include strand breaks (Minisini et al., 1994) and base modifications, such as thymine glycol (Leadon and Hanawalt, 1983), 5-hydroxymethyluracil (Teebor et al., 1982), 8-hydroxyadenine (Dizdaroglu and Bergtold, 1986), and 8-hydroxyguanine (8-OH-Gua). 8OH-Gua is one of the most abundant types of oxidative DNA damage, and it was first reported in 1984 (Kasai and Nishimura, 1984). Because 8-OH-Gua is known to cause GC to TA transversions in DNA (Basu et al., 1989; Cheng et al., 1992), it is important to evaluate the levels of 8-OHGua in nuclear DNA when exploring the mechanisms of Cd carcinogenesis. From these points of view, it is predicted that Cd-induced carcinogenicity might be mainly due to oxidative DNA damage. In fact, it was reported that CdCl2 induces DNA singlestrand scission, due to ROS (Mu¨ller et al., 1991). However, to our knowledge, there have been few studies of the mechanisms of Cd-induced carcinogenicity by oxidative DNA damage, although other metals, such as Ni(II), Fe(III)NTA, and Cr(VI), were reported to produce 8-OH-Gua in DNA (Kasprzak and Hernandez, 1989; Umemura et al., 1990; Aiyar et al., 1990). In this study, we measured 8-OH-Gua in nuclear DNA and its repair activity in CdCl2-treated rat testis and lung to study the mechanism of Cd-induced carcinogenicity. At the same time, we assayed superoxide dismutase (SOD) activities and LPO levels in the organs to understand the role of oxidative stresses in Cd-induced carcinogenicity. Furthermore, to examine the role of GSH in Cd-induced carcinogenicity, we analyzed rat organs in which GSH biosynthesis was inhibited by L-buthionine-[S, R]-sulfoximine (BSO).
Inhibition of 8-Hydroxyguanine Repair in Testes after Administration of Cadmium Chloride to GSH-Depleted Rats. Hirano, T., Yamaguchi, Y., and Kasai, H. (1997). Toxicol. Appl. Pharmacol. 147, 9–14. The main goal of this study is to investigate the mechanism of cadmium (Cd)-induced carcinogenesis by reactive oxygen species. Rats were divided into four groups and were treated with (i) saline (control), (ii) cadmium chloride (CdCl2), (iii) L-buthionine-[S, R]sulfoximine (BSO, an inhibitor of GSH biosynthesis), and (iv) CdCl2 and BSO, respectively. They were euthanized at 0, 24, 48, and 72 hr after these treatments, and the lungs and testes were analyzed. After treatment with both CdCl2 and BSO, the testicular 8-OH-Gua level increased (48 hr), its repair activity decreased (48 and 72 hr), the GSH content was markedly suppressed (48 and 72 hr), the superoxide dismutase activities slightly (48 and 72 hr) decreased, and the lipid peroxidation level increased (24 and 72 hr) in the testes as compared to the control levels. These results suggest that under GSH-depleted conditions, CdCl2 inhibits 8-OH-Gua repair activity in the rat testis and 8-OH-Gua accumulates in the DNA, which may pertain to testicular carcinogenesis. q 1997 Academic Press
Cadmium (Cd) is a highly toxic element that is naturally present in all parts of the environment, including food, water, and soil (Sherlock, 1984). It affects a wide variety of metabolic processes, such as energy metabolism (Mu¨ller, 1986), membrane transport (Verbost, 1989), and protein synthesis (Dudley et al., 1984). Several Cd compounds have been shown to have carcinogenic potential in humans and animals (Kazantzis, 1963), and the main target organs of Cd-induced carcinogenicity are lung, prostate, and testis (Waalkes and Poirier, 1985). However, the mechanisms of Cd-induced carcinogenicity are still not understood completely. Recently, it has been suggested that reactive oxygen species (ROS) play a major role in the mechanism of Cd-induced toxicity. It was reported that low doses of CdCl2 stimulated lipid peroxidation (LPO) without any evidence of acute damage in rat organs (Manca et al., 1991). Dietary ascorbic acid supplements were reported to reduce Cd toxicity in young Japanese quail (Fox et al., 1970). Cadmium sulfate (CdS) 9
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0041-008X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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METHODS
The contents of GSH and GSSG in the supernatants were determined by the previous method (Hissin et al., 1976).
Animal preparation. Eight-week-old male Sprague–Dawley (SD) rats, weighing 271.4 { 13.4 g, were purchased from Seiwa Experimental Animals (Fukuoka, Japan), and were fed a CE pellet basal diet (Japan Clea Co., Tokyo, Japan) and tap water ad libitum. They were maintained in a temperature- and photoperiod-controlled room (247C, 12 hr/day). Chemicals. BSO, which is an inhibitor of glutathione biosynthesis, and cadmium chloride (CdCl2) were purchased from Sigma Chemical Co. (St. Louis, MO). BSO and CdCl2 were dissolved in sterile saline to 0.5 M and 20 mM, respectively. Spectrophotometric assay kits of superoxide dismutase activity (SOD-525) and lipid peroxidation (LPO-586) were purchased from Bioxytech S.A. (Bonneuil/Marne, France). [g-32P]ATP (Ç6000 Ci/mmol) were purchased from Amersham International Ltd. (Buckinghamshire, UK). Other chemicals were of the highest purity commercially available. Experimental protocol. Forty eight SD male rats were divided into four groups. The first group was a control group, in which rats were injected sc with 0.75 ml/kg body weight and ip with 10 ml/kg of sterile saline. Rats in the second group were injected sc with 15 mmol/kg of CdCl2 and ip with 10 ml/kg of sterile saline. Rats in the third group were injected sc with 0.75 ml/kg of sterile saline and ip with 5 mmol/kg of BSO. Rats in the last group were injected sc with 15 mmol/kg of CdCl2 and ip with 5 mmol/kg of BSO. Four rats from each group were euthanized under ether anesthesia at 24, 48, and 72 hr after treatment. Four more rats (no treatment) were euthanized as a time 0 control. Testes and lungs were removed immediately after sacrifice and were used for the experiments. Measurement of 8-OH-Gua. 8-OH-Gua levels in rat organs were determined as previously reported (Nakae et al., 1995), with slight modification (Yamaguchu et al., 1996). Briefly, the samples were homogenized in lysis buffer with a potter type homogenizer, and the nuclear DNA in the homogenate was extracted using the DNA Extractor WB kit. The extracted nuclear DNA was digested with nuclease P1 and acid phosphatase in a 10 mM sodium acetate solution (377C for 30 min). After the incubation, the mixture was treated with the iron exchange resin, Muromac (Muromachi kagaku, Tokyo, Japan) and was centrifuged at 15,000g for 5 min. The supernatant was transferred to a filter tube (Millipore, Samprep C, 0.2 mm), centrifuged at 5000g for 5 min, and injected onto a high-performance liquid chromatography column (Beckman, Ultrasphere-ODS, 5 mm, 4.6 1 250 nm) equipped with an electrochemical detector (ESA Coulochem II). As standard samples, 20 ml each of deoxyguanosine (0.5 mg/ml) and 8-hydroxydeoxyguanosine (5 ng/ml) solutions were injected. The value of 8-OH-Gua was calculated as the number per 105 guanine residues. Endonuclease nicking assay. Excision repair activities in rat organs were assayed by the previously described method (Hirano et al., 1995). Briefly, tissues removed from sacrificed rats were homogenized in 50 mM Tris–HCl buffer (pH 7.5) containing protease inhibitors (5 mg/ml each of pepstatin, leupeptin, antipain, and chymostatin) with a Dounce-type homogenizer. Homogenates were centrifuged at 10,000g to obtain the crude extracts. The total protein concentration of the crude extracts was adjusted to 5 mg/ml. A 22-mer double-stranded synthetic oligonucleotide containing 8-OH-Gua (upper strand, 5*-GGTGGCCTGACG*CATTCCCCAA-3*; G*, 8-OH-Gua) was used as the substrate for this assay. Crude extract (50 mg protein) was incubated with 0.05 pmol of the 32P end-labeled doublestranded DNA substrate at 257C for 1 hr. The cleaved fragment, generated as the consequence of base excision repair activity (glycosylase activity and AP endonuclease activity) was analyzed by 20% denaturing polyacrylamide gel electrophoresis. The repair activity is expressed as the ratio of the excised fragment activity to the total activity (substrate activity plus fragment activity). Measurement of GSH and GSSG content. Tissues were homogenized by a Dounce-type homogenizer, either alone or in combination with a Politron, in 0.1 M sodium phosphate buffer with 5 mM EDTA (pH 8.0), mixed with meta phospholic acid, and centrifuged at 12,000g for 30 min.
Measurement of SOD activity. Tissues were homogenized by a Dounce-type homogenizer, either alone or in combination with a Politron, in 10 mM Tris–HCl (pH 7.4) containing 0.25 M sucrose and 1 mM EDTA. Homogenates were centrifuged at 6000g for 30 min. SOD activities in the supernatants were measured using a spectrophotometric assay kit according to the manufacturer’s instructions. The SOD activities are expressed as units per milligram of protein.
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Assessment of lipid peroxidation by measurement of MDA. Tissues were homogenized by a Dounce-type homogenizer, either alone or in combination with a Politron, in 20 mM Tris–HCl (pH 7.4). Homogenates were centrifuged at 6000g for 30 min. The LPO levels in the supernatants were determined using a spectrophotometric assay kit according to the manufacturer’s instructions. The LPO values are expressed as nanomoles of malon dialdehyde (MDA) production per milligram of protein. Statistical analysis. Values in the text and tables represent the means { SD. The statistical significance was calculated using the Student t test. Probability values less than 0.05 were considered to indicate significant differences.
RESULTS
8-OH-Gua Levels in Testis and Lung DNA Using a rapid DNA extraction method with a commercial kit, the background level of 8-OH-Gua was reduced to 0.2– 0.3/105 Gua in this study. As seen in from Fig. 1, the 8OH-Gua levels in the nuclear DNA of CdCl2- and BSOtreated rat testis (48 hr) increased significantly as compared with those of other rat groups. On the other hand, the 8OH-Gua levels in rat lung nuclear DNA showed no significant change among the groups in this study (data not shown).
FIG. 1. Time course of 8-OH-Gua level (% of control) in the nuclear DNA of the rat testis. Mean values { SD, n Å 4. The absolute values (per 105 guanines) for control rats are 0.317 { 0.070 at time 0, 0.285 { 0.068 at 24 hr, 0.243 { 0.033 at 48 hr, and 0.268 { 0.020 at 72 hr. *Significantly different from the Cd treatment group (48 hr) at p õ 0.005 and the BSO treatment group (48 hr) at p õ 0.01.
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FIG. 2. Autoradiogram of an endonuclease nicking assay of rat testes preparations. (a) Substrate 22-mer oligonucleotides containing 8-OH-Gua. (b) Excised fragments. The 8-OH-Gua repair activities are expressed as the ratio of the radioactivity of the lower band to the total radioactivity.
8-OH-Gua Repair Activity in Testis and Lung At 24 hr after the treatments, no significant change was observed in the base excision repair activities of the testes of all rats. At 48 and 72 hr after the treatment, the 8-OHGua repair activity was quite depressed in the testes of the CdCl2- and BSO-treated rats (Figs. 2 and 3). The 8-OH-Gua repair activity showed no significant change in the testes of the rats in other groups. The repair activity in the rat lungs showed no significant change among the groups in this study (data not shown). Cellular GSH and GSSG Concentrations Since cellular GSH is supposed to work as a first defense against Cd-induced toxicity (Singhal et al., 1987), GSH bio-
FIG. 4. Time course of the cellular GSH concentrations in rat testes. The values are expressed as nmol/mg protein. Mean values { SD, n Å 4. Significantly different from the control group of the same observation time: *p õ 0.001, **p õ 0.01, ***p õ 0.05.
synthesis was selectively inhibited by BSO administration to investigate the relationship between GSH content and 8OH-Gua repair activity. Figure 4 shows that the cellular GSH content in CdCl2-treated rat testes decreased at 48 and 72 hr after administration. Furthermore, the GSH content in CdCl2- and BSO-treated rat testes decreased markedly, particularly at 48 and 72 hr after treatment, and it seems that CdCl2 and BSO work additively to decrease the GSH content. The GSSG content also was reduced in the testes of CdCl2- and BSO-treated rats (data not shown). As for the lungs, the GSH and GSSG levels showed tendencies of decreasing at 48 and 72 hr only in the BSO-treated and the CdCl2- and BSO-treated rats. No significant changes were seen in the lung GSH and GSSG levels of the rats treated with CdCl2 alone. It seems that Cd does not affect the GSH and GSSG contents in the rat lung (data not shown). SOD Activity in Testis
FIG. 3. Time course of the 8-OH-Gua repair activity value (% of control) in the rat testis. Mean values { SD, n Å 4. The absolute values for control rats are 0.331 { 0.091 at time 0, 0.403 { 0.016 at 24 hr, 0.396 { 0.032 at 48 hr, and 0.255 { 0.023 at 72 hr. *Significantly different from the Cd treatment group (48 hr) at p õ 0.05 and the BSO treatment group (48 hr) at p õ 0.005. **Significantly different from the Cd treatment group (72 hr) at p õ 0.01 and the BSO treatment group (72 hr) at p õ 0.005.
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To assess the balance of ROS production in the testis, the SOD activity was measured. SOD activities in the CdCl2and BSO-treated rat testes decreased at 48 and 72 hr after injection, and the minimum level was approximately 75% of the control at 72 hr after treatment. The activity in the BSO-treated rat testis increased at 48 and 72 hr after treatment, but no significant change was observed in the testes of other rat groups (Table 1). For all groups, there were no significant changes in the lung SOD activities (data not shown). LPO Production in Testis The cellular damage induced by ROS was estimated by monitoring the LPO levels, which are a well known indicator
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TABLE 1 SOD Activity in Rat Testes SOD activity (units/mg protein) Treatments
0 hr
Control Cd BSO Cd / BSO
0.259 { 0.010 — — —
24 hr 0.225 0.200 0.249 0.228
{ { { {
48 hr
0.037 0.025 0.015 0.042
0.230 0.215 0.278 0.189
{ { { {
0.028 0.019 0.027* 0.017*
72 hr 0.223 0.203 0.259 0.167
{ { { {
0.007 0.026 0.017* 0.014**
Note. Values are means { SD of four rats. * Significantly different from control group at p õ 0.05. ** Significantly different from control group at p õ 0.001.
of cellular damage by oxidative stress. In the testis, the MDA concentrations showed significant increases at 24 and 72 hr after both CdCl2 and BSO administration. There were also increases at 48 and 72 hr after CdCl2 administration and at 48 hr after both CdCl2 and BSO administration (Table 2). For all groups, there were no significant changes in the lung LPO levels (data not shown). DISCUSSION
We were interested in whether Cd-induced carcinogenicity is due to oxidative DNA damage. Cd compounds are known to induce various form of oxidative damage, such as an increase of LPO (Manca et al., 1991), a decrease of GSHPx (Sidhu et al., 1993), and DNA strand breaks (Mu¨ller et al., 1991). However, Cd is not thought to induce ROS directly, because Cd2/ is not capable of accepting or donating electrons under physiological conditions (Ochi et al., 1987). On the other hand, many reports (Piscator, 1981; Hallenbeck, 1984) indicated that some Cd compounds have carcinogenic potential in specific organs, such as lung, prostate, and testis. However, the direct role of Cd in carcinogenic mechanisms is not clear (Degraeve, 1981). In this study, we assayed the level of oxidative DNA damage, by monitoring 8-OH-Gua
formation and its repair activity, in CdCl2- and/or BSOtreated rats to investigate the relationship between oxidative DNA damage and Cd-induced carcinogenicity. As far as we know, no conclusive data have been published regarding Cdinduced cancer in rat testes. Therefore, we chose the CdCl2 dose based on a previous report, without regard to the carcinogenicity. Singhal et al. reported that BSO-treated mice also treated with 20 mmol Cd2//kg experienced 100% mortality, whereas BSO-treated mice also treated with 10 mmol Cd2//kg survived. Based on these data, we chose a 15 mmol/ kg CdCl2 dose. This dose might be extremely high in terms of carcinogenicity. However, because we designed a short time range experiment, we selected this dose. 8-OH-Gua is known to be induced by ionizing radiation or ROS-forming agents (Kasai and Nishimura, 1984; Dizdaroglu and Bergtold, 1985; Umemura et al., 1990; Aiyar et al., 1990). Since this form of DNA damage is reported to cause GC to TA transversions in DNA, it is believed to be responsible for point mutations in genomic DNA (Wood et al., 1990; Cheng et al., 1992). Hence, analysis of the 8-OHGua level is useful for the study of carcinogenic mechanisms. To obtain evidence that ROS, which cause an increase in 8OH-Gua, were actually produced, we assayed LPO production in rat testes. Since the dose of CdCl2 previously reported
TABLE 2 LPO Levels in Rat Testes MDA (nmol/mg protein) Treatments
0 hr
Control Cd BSO Cd / BSO
34.67 { 13.35 — — —
24 hr 45.38 31.26 50.58 130.82
{ { { {
48 hr
11.23 15.65 21.24 46.29*
35.02 312.88 48.81 349.31
{ 11.41 { 470.26 { 3.77 { 740.41
Note. Values are means { SD of four rats. * Significantly different from control group at p õ 0.05. ** Significantly different from control group at p õ 0.001.
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{ 48.60 { 789.93 { 6.35 { 214.94**
OXIDATIVE DNA DAMAGE AND CADMIUM CARCINOGENICITY
to stimulate LPO in rat was 500 mg CdCl2/kg (Manca et al., 1991) (Å2.7 mmol CdCl2/kg), we expected LPO production to be increased in the Cd-treated rat organs in our study. As shown in Table 2, LPO production increased in the testes of CdCl2- and BSO-treated rats. Although LPO production does not always indicate ROS production, the evidence might support the increase of oxidative DNA damage in the testes of CdCl2- and BSO-treated rats. In the testes of the same experimental groups, a decrease in SOD activity was also observed. The reduction of SOD activity is known to be due to the replacement of the Zn and/or Mn of the SOD molecule by Cd (Jacobson and Turner, 1980). In our analyses of the testicular SOD activities, we observed that CdCl2 alone had no effect, but the CdCl2- and BSO-treatment decreased them. We think that the lower SOD activity in the testes of CdCl2- and BSO-treated rats is due to cell damage. These results suggest that, in the testes of CdCl2- and BSOtreated rats, an imbalance of O20 and H2O2 might occur. Therefore, the decrease in the SOD activity might indirectly lead to an increase in oxidative DNA damage. Another possible mechanism for the increase of 8-OHGua would be that the 8-OH-Gua repair system is less efficient in rat testes after CdCl2- and BSO-treatment. To examine this hypothesis, we determined the levels of 8-OH-Gua repair activity in various organs. The assay method involved detection of the excised DNA fragment produced during the 8-OH-Gua repair process. The result of this endonuclease nicking assay showed no significant changes in CdCl2treated or BSO-treated rat testes and the rat lungs from all groups (Figs. 2 and 3). However, the activities were markedly depressed in CdCl2- and BSO-treated rat testes (48 and 72 hr) as compared to the control rat testes. These data suggest that CdCl2 might increase 8-OH-Gua level by inhibiting the 8-OH-Gua repair activity when GSH biosynthesis is inhibited. Since the repair activity was not suppressed by CdCl2 treatment alone, it seems that cellular GSH is required for the 8-OH-Gua repair activity. In fact, the GSH amounts in CdCl2- and BSO-treated rat testes were extremely reduced at 48 and 72 hr in comparison to those found in the testes of other rat groups, including the BSO-treated rats (Fig. 4). This pattern was almost the same as that of the 8-OH-Gua repair activity. However, although the GSH content in the testes of rats treated with CdCl2 alone was reduced, the repair activities did not show any change (Fig. 3). These results suggest that only a severe reduction in GSH might cause an inhibition of repair activity. Since the CdCl2 treatment did not increase the 8-OH-Gua level, we could not conclude that a direct relationship between CdCl2-induced carcinogenicity and oxidative DNA damage exists. However, our data suggest that if GSH depletion occurred, then the risk of CdCl2induced carcinogenicity would increase. Therefore, we suggest that GSH might, at least in part, regulate 8-OH-Gua
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repair activity. To our knowledge, GSH depletion in humans would occur in certain situations, such as excessive alcohol intake (Hirano et al., 1992). However, the exact role of GSH in 8-OH-Gua repair activity remains unclear. In this experiment, no notable changes of 8-OH-Gua levels and its repair activities were observed in the lung, another target organ of Cd carcinogenesis. We could not conclude that the mechanism of Cd-induced carcinogenicity in the lung is different from that in the testis, because target organ of Cd toxicity differ depending on the type of compound. To investigate the mechanism of carcinogenesis in the lung, we should use a CdCl2 aerosol (Takenaka et al., 1983) or other types of Cd compounds, such as CdS with various methods of administration, including inhalation. In conclusion, CdCl2 treatment increases 8-OH-Gua formation, possibly by suppression of the 8-OH-Gua repair activity, when GSH biosynthesis is inhibited. This implies that if these types of acute damage occur repeatedly, by Cd administration over a long period of time, they may result in the accumulation of replication errors that could lead to carcinogenesis, which might be one of the possible mechanisms of Cd-induced carcinogenicity in the testis. Since we did not measure the repair activity between 24 and 48 hr, we cannot discuss this temporal relationship. However, we suggest that an imbalance in the levels of ROS might increase the 8-OH-Gua level. Taken together, in the CdCl2and BSO-treated rat testis, both the imbalance of ROS and the decrease of 8-OH-Gua repair activity might increase 8OH-Gua accumulation. Since the CdCl2 dose we used in this study was higher than that used for carcinogenicity studies in rats, tissue damage, including cell death, might occur prior to the changes in 8-OH-Gua and its repair activity. However, the risk of replication errors leading to point mutations might be increased when the damaged tissue recovered. From this point of view, the severe depletion of 8-OH-Gua repair activity could be an important step in testicular carcinogenicity. However, the long-term effects of low dose Cd exposure on oxidative DNA damage merit further investigation. In this study, we suggest that CdCl2-induced testicular carcinogenicity is associated with oxidative DNA damage. However, since we did not measure ROS production directly, the strict relationship between Cd-induced carcinogenicity and ROS is still unclear. This relationship should also be investigated. ACKNOWLEDGMENTS This work was supported by a Grant from the University of Occupational and Environmental Health, Japan, and by a Grant-in Aid for Scientific Research on Priority Areas from The Ministry of Education, Science and Culture of Japan (05270102).
REFERENCES Aiyar, J., Berkovits, H. J., Floyd, R. A., and Wetterhahn, K. E. (1990). Reaction of chromium (VI) with hydrogen peroxide in the presence of
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