Cadmium(II), unlike nickel(II), inhibits 8-oxo-dGTPase activity and increases 8-oxo-dG level in DNA of the rat testis, a target organ for cadmium(II) carcinogenesis

Karol Bialkowski1,3, Aneta Bialkowska2,4 and Kazimierz S. Kasprzak1,5

1 Laboratory of Comparative Carcinogenesis, National Cancer Institute and
2 Intramural Research Support Program, SAIC Frederick, FCRDC, Frederick, MD 21702, USA
3 On leave from Department of Clinical Biochemistry and
4 Department of Pathophysiology, University School of Medical Sciences, 85092 Bydgoszcz, Poland


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
8-Oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) is an enzyme which prevents incorporation into DNA of promutagenic 8-oxo-2'-deoxyguanosine (8-oxo-dG) from a deoxynucleotide pool damaged by endogenous oxidants. Its inhibition may thus be carcinogenic. We previously found that Cd(II) inhibited 8-oxo-dGTPase in both cell free systems and cultured cells. To verify this finding in a relevant animal model, we investigated the effects of Cd(II) on cellular 8-oxo-dGTPase activity and nuclear DNA 8-oxo-dG levels in the rat testis, a target organ for Cd(II) carcinogenesis. Ni(II), which does not induce testicular tumors in rats and is a weaker in vitro inhibitor of 8-oxo-dGTPase than Cd(II), was investigated as a comparison. Male F344/NCr rats were given a single s.c. dose of 20 µmol Cd(II) acetate, 90 µmol Ni(II) acetate or 180 µmol sodium acetate (controls) per kg body wt and killed 2, 8, 24 or 48 h later (three rats/time point). Cd(II) caused a gradual decrease in testicular 8-oxo-dGTPase activity with time. It became significant only after 8 h post-injection (P < 0.05) and resulted in a final 50% loss of the enzyme activity at 48 h (P < 0.01). Although the results for Ni(II) at 8 h and later were apparently lower than the controls, the decrease did not reach statistical significance. Treatment of rats with Cd(II) led to an early and progressive increase (from 130% at 2 h to 200% at 48 h versus the controls) of the 8-oxo-dG level in testicular DNA (P < 0.05 or better). Ni(II) acetate also tended to raise the testicular 8-oxo-dG level, but the increase was transient, with an apparent maximum at 8 h, and did not approach statistical significance (P < 0.2). Thus, Cd(II), unlike Ni(II), is able to inhibit 8-oxo-dGTPase activity and to raise 8-oxo-dG levels in rat testicular DNA. However, the time course of both effects indicates that 8-oxo-dGTPase inhibition is most likely not the sole cause of the increase in 8-oxo-dG.

Abbreviations: CHO, Chinese hamster ovary; 8-oxo-dG, 8-oxo-2'-deoxyguanosine (8-hydroxy-2'-deoxyguanosine); 8-oxo-dGMP, 8-oxo-2'-deoxyguanosine 5'-monophosphate; 8-oxo-dGDP, 8-oxo-2'-deoxyguanosine 5'-diphosphate; 8-oxo-dGTP, 2'-deoxyguanosine 5'-triphosphate; 8-oxo-dGTPase, 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cadmium is toxic and carcinogenic to humans and animals (1,2). One possible pathogenic pathway of its action includes mediation of promutagenic DNA damage, as demonstrated by various assays, including DNA strand break and 8-oxo-2'-deoxyguanosine (8-oxo-dG) determination (37). Since Cd(II) is not redox active under physiological conditions, this type of damage can be inflicted by Cd(II) only indirectly, e.g. through inflammatory cells and/or inhibition of cellular antioxidant and DNA repair systems (5,6,810).

Besides being generated directly in the DNA, an 8-oxo-dG lesion may be incorporated into DNA from 2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) produced in the deoxynucleotide pool through oxidation of dGTP by endogenous metabolic oxidants (11,12). Such a potentially mutagenic incorporation is countered in cells by a specific 8-oxo-dGTP pyrophosphohydrolase (8-oxo-dGTPase) (11,12). Hence, inhibition of this protective enzyme should enhance mutagenesis and cancer. Our previous in vitro experiments revealed that 8-oxo-dGTPase was sensitive to inhibition by several carcinogenic metals, including Cd(II) (13). Cd(II) was also inhibitory toward 8-oxo-dGTPase activity in cultured Chinese hamster ovary (CHO) cells (14). The goal of the present study was to test this Cd(II) effect in vivo, in an appropriate animal model. To achieve this goal, 8-oxo-dGTPase activity and 8-oxo-dG levels were determined in rat testes, which is the main target organ for Cd(II)-induced carcinogenesis (1,2,15), at time intervals up to 48 h following a single s.c. injection of Cd(II) acetate. Similar effects of nickel(II), which was shown to slightly inhibit 8-oxo-dGTPase activity in vitro (13) and exert limited toxic effects (carcinogenicity was not sought) in the rat testis (16), were investigated for comparison.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
Ni(II) acetate tetrahydrate, Cd(II) acetate dihydrate and anhydrous sodium acetate were purchased from J.T. Baker Chemical Co. (Phillipsburg, NJ). 8-Oxo-dGTP and 8-oxo-2'-deoxyguanosine 5'-monophosphate (8-oxo-dGMP) were synthesized as described in our previous paper (14). 8-Oxo-dG was synthesized by Dr Victor Nelson of SAIC Frederick (Frederick, MD). All other chemicals and reagents were purchased from Sigma Chemical Co. (St Louis, MO).

Animals and treatment
Male F344/NCr rats, 6–7 weeks old, weighing 120–200 g, were randomly divided into three groups of 12 animals. The animals were housed on hardwood bedding in a room with a 12 h fluorescent light/12 h dark cycle, at 24 ± 2°C and 50 ± 5% relative humidity. They were fed NIH-31 Open Formula 6% Modified Diet (Zeigler Brothers, Gardners, PA) and had free access to drinking water. The rats were given a single dose of one of the following salts: Cd(II) acetate, 20 µmol/kg body wt; Ni(II) acetate, 90 µmol/kg body wt; sodium acetate, 180 µmol/kg body wt (control group). These doses of metal acetates, except that of sodium acetate, are known to initiate carcinogenesis in rats (cadmium in testes, nickel in kidneys) (reviewed in refs 1,2,10). The injections were s.c. at the nape of the neck, in 2 ml of water/kg body wt. Three rats of each group were killed with CO2 2, 8, 24 and 48 h post-injection. Their testes were collected immediately and frozen in liquid nitrogen.

Determination of 8-oxo-dGTPase activity
8-Oxo-dGTPase activity was determined in one testis of each rat according to the following protocol based on the general method developed previously by us for cultured cells and mouse tissues (14,17). The whole testis was homogenized for 10 s in 3.5 ml of ice-cold 20 mM Tris–HCl, pH 7.4, with a Brinkmann Polytron homogenizer PT 10/35 equipped with a 1 cm (diameter) foam reducing generator with saw teeth. The homogenate was centrifuged for 15 min at 2800 g (4°C). The resulting supernatant was ultracentrifuged for 1 h at 100 000 g (4°C). Three 150 µl portions of each supernatant, termed below `extract', were ultrafiltered through 30 kDa cut-off, low protein-binding regenerated cellulose membrane (Ultrafree-MC Filtration Units; Millipore, Bedford, MA). This step separates the assayed 8-oxo-dGTP pyrophosphatase activity from the interfering 8-oxo-dGTP phosphatase and 8-oxo-dGMP phosphatase activities (Figure 1Go). The extracts and ultrafiltrates were stored at –70°C for subsequent determinations of protein concentration and 8-oxo-dGTPase activity. The former was done by the biuret method (18), while the latter was accomplished as follows. A 60 µl volume of the reaction solution containing 40 µM 8-oxo-dGTP, 5 mM MgCl2, 100 mM Tris–HCl (pH 8.5) and ultrafiltrate was incubated at 37°C for 30 min. The reaction was initiated by addition of the ultrafiltrate (5 µl) and terminated by addition of 20 µl 50 mM Na2EDTA. Blank samples were prepared with Na2EDTA introduced before the ultrafiltrate, followed by incubation at 37°C.



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Fig. 1. HPLC separation of the substrate (1) and products (24) of 8-oxo-dGTP hydrolysis catalyzed by (A) a total protein extract of normal rat testis and (B) a <=30 kDa ultrafiltrate of that extract. Mixtures (60 µl) containing 40 µM 8-oxo-dGTP, 5 mM MgCl2, 100 mM Tris–HCl (pH 8.5) and 5 µl of testicular protein extract or its ultrafiltrate were incubated at 37°C for 60 min. The hydrolysis was stopped by addition of 20 µl 50 mM Na2EDTA. Aliquots of 20 µl of the mixtures were chromatographed as described in Materials and methods. 1, 8-oxo-dGTP; 2, 8-oxo-dGDP; 3, 8-oxo-dGMP; 4, 8-oxo-dG.

 
The reaction solutions were analyzed by HPLC for the amount of 8-oxo-dGMP formed. Our Waters HPLC system consisted of two pumps (model 510), an autosampler (model 717 plus), a UV-VIS photodiode array detector (model 996), a Supelcosil LC-18-T column (250x4.6 mm, 5 µm grain; Supelco, Bellefonte, PA) and was controlled by a Millennium32 Chromatography Manager. Aliquots (20 µl) of the reaction mixtures were chromatographed isocratically with 100 mM NaH2PO4–NaOH buffer, pH 5.5, methanol (95:5), at a flow rate of 1 ml/min. Solutions of known concentrations of 8-oxo-dGMP, ranging from 1.25 to 15 µM, were used for calibration. For quantification of the reaction product, chromatograms acquired at 295 nm were integrated.

The enzymatic activity unit (U) was defined as the amount of enzyme converting 1 pmol 8-oxo-dGTP to 8-oxo-dGMP per min under the above reaction conditions. The mean 8-oxo-dGTPase activity in the tissue extract was calculated from determinations in three separate ultrafiltrates of the same extract and finally expressed in relation to total protein concentration in the extract.

Determination of 8-oxo-dG level in nuclear DNA
The level of 8-oxo-dG in nuclear DNA was determined in the second testis of each rat, using the enzymatic hydrolysis procedure according to Adachi et al. (19). Separate dilutions of genuine 8-oxo-dG and deoxyguanosine were used as standards. DNA hydrolysates were analyzed by a HPLC system consisting of a Hewlett Packard 1050 pump, a Waters Intelligent Sample Processor model 710B, a Waters 490E Programmable Multiwavelength Detector, an ESA Coulochem II 5200A electrochemical detector (guard cell, 700 mV, standard analytical cell model 5010; working electrode E1 at 300 mV) and a Supelcosil LC-18-S (250x4.6 mm, 5 µm grain) column equipped with a 2 cm guard column. Aliquots (20 µl) of the DNA hydrolysates were chromatographed at 1 ml/min flow rate, with 100 mM sodium acetate–orthophosphoric acid, pH 5.2, methanol (92:8) as eluent. Chromatograms were acquired and integrated by an ESA 500 Chromatography Data System.

Statistical analysis
The significance of differences between means was tested using Student's t-test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
8-Oxo-dGTPase activity
As illustrated in Figure 1Go, 8-oxo-dGTPase activity in the rat testis could be measured using our assay developed originally for cultured cells (14). Unlike in the whole testis extract (Figure 1AGo), no degradation of 8-oxo-dGTP to products other than 8-oxo-dGMP was observed in the ultrafiltrate (Figure 1BGo). Thus, like the mouse liver and lungs (17), the rat testis does not contain proteins with phosphatase activity which could pass the 30 kDa cut-off ultrafilters. It is also important to notice that the metal treatments did not change the specificity of the enzymatic activity in the ultrafiltrates, as signified by the presence of 8-oxo-dGMP and absence of other possible products, e.g. 8-oxo-2'-deoxyguanosine 5'-diphosphate (8-oxo-dGDP) and/or 8-oxo-dG, of 8-oxo-dGTP hydrolysis (Figure 2Go).



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Fig. 2. The treatment of rats with metal salts does not change the specificity of 8-oxo-dGTP decomposition by ultrafiltrates of testicular protein extracts. Exemplary HPLC chromatograms demonstrating the presence of pure 8-oxo-dGTP pyrophosphatase activity in the ultrafiltrates of rats 24 h after a single s.c injection of 180 µmol/kg body wt Na(I) acetate (control), 90 µmol/kg body wt Ni(II) acetate or 20 µmol/kg body wt Cd(II) acetate. Numbers indicate the same compounds as in Figure 1Go.

 
Treatment of rats with Cd(II) acetate resulted in a significant gradual decrease in testicular 8-oxo-dGTPase activity with time after injection (Figure 3Go). This decrease first became apparent as early as 8 h post-injection (P < 0.05) and finally resulted in a 50% loss of enzyme activity at 48 h (P < 0.01). Although the results for Ni(II) at 8 h and later after the injection were lower than the controls, the differences did not reach statistical significance. Likewise, the slight variations in 8-oxo-dGTPase activity observed with time in the control testes were not significant (Figure 3Go).



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Fig. 3. 8-Oxo-dGTPase activity in testes of rats 2, 8, 24 and 48 h after a single s.c. injection of 180 µmol/kg body wt Na(I) acetate (control), 90 µmol/kg body wt Ni(II) acetate or 20 µmol/kg body wt Cd(II) acetate. Error bars denote SE. *P < 0.05 or better versus the corresponding control value.

 
8-oxo-dG levels in nuclear DNA
As shown in Figure 4Go, treatment of rats with Cd(II) acetate led to a marked increase in the 8-oxo-dG level in testicular DNA that remained statistically significant over the entire period of the experiment, with P < 0.05 or better. It reached nearly 200% of the control value at 48 h. Ni(II) acetate also tended to increase the testicular 8-oxo-dG level, but that increase was transient with an apparent maximum at 8 h and its difference from the control did not approach statistical significance (P < 0.2 versus the corresponding control for 8 h).



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Fig. 4. The levels of 8-oxo-dG in DNA of the rat testes 2, 8, 24 and 48 h after a single s.c. injection of 180 µmol/kg body wt Na(I) acetate (control), 90 µmol/kg body wt Ni(II) acetate or 20 µmol/kg body wt Cd(II) acetate. Error bars denote SE. *P < 0.05 or better versus the corresponding control value.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
8-Oxo-dGTPase is an enzyme hydrolyzing 8-oxo-dGTP, one of the mutagenic products of active oxygen attack on the nucleotide pool (11,12), to 8-oxo-dGMP and pyrophosphate. Unlike 8-oxo-dGDP, which may be derived from 8-oxo-dGTP by phosphatase hydrolysis, 8-oxo-dGMP cannot be rephosphorylated back to the triphosphate (12). Therefore, 8-oxo-dGTPase is regarded as an antimutagenic enzyme `sanitizing' the cellular nucleotide pool (20). Its inhibition is expected to increase incorporational mutagenicity of 8-oxo-dGTP, signified by AT->CG transversions (11,12,21).

The major problem in determination of 8-oxo-dGTPase activity in tissue extracts has been interference from other cellular phosphatases which degrade the substrate triphosphate through intermediate phosphates, 8-oxo-dGDP and 8-oxo-dGMP, to 8-oxo-dG (Figure 1AGo). Thus, the substrate and product of the 8-oxo-dGTPase-catalyzed reaction are hydrolyzed by other enzymes. Furthermore, 8-oxo-dGDP is a very strong inhibitor of 8-oxo-dGTPase (14). Fortunately, the 18 kDa rat 8-oxo-dGTPase (22) can be easily separated from the larger interfering enzymes by simple ultrafiltration through 30 kDa cut-off, low protein-binding, regenerated cellulose membranes (Figure 1BGo). In our previous experiment, this procedure proved successful for intact animal tissues (17). The present study confirms the effectiveness of ultrafiltration and also the entire novel assay in analysis of a tissue damaged by toxic metals (Figure 2Go).

The 8-oxo-dGTP signature AT->CG transversion, mentioned above, is frequent among point mutations identified in the hypoxanthine (guanine) phosphoribosyl-transferase gene of cadmium-exposed CHO cells (7). Cd(II) was also found to inhibit bacterial and human 8-oxo-dGTPases in vitro (13) and the hamster enzyme in cultured CHO cells (14), as well as to increase 8-oxo-dG levels in DNA of cultured cells (4) and in testes, but not lung, of glutathione-depleted rats (3). In the latter paper, oxidative DNA damage has been related to the inhibition by Cd(II) of DNA repair when glutathione biosynthesis is also inhibited. Since Cd(II), being non-redox active under physiological conditions, cannot oxidize DNA bases directly, these observations, as well as our present results, are consistent with, or at least do not contradict, the general notion that the observed oxidative DNA damage (by metabolic oxidants) is assisted by this metal through inhibition of cellular antioxidant and DNA protection/repair systems, of which 8-oxo-dGTPase is a member (23). It is, however, very likely that Cd(II) can also enhance oxidative DNA damage by boosting endogenous oxidation, e.g. by triggering inflammation (10,24,25). Our results, which differ from those reported by Hirano et al. (3) in the sense that we observed elevation of testicular 8-oxo-dG by Cd(II) in rats even without prior depletion of glutathione, may support that notion. The difference might be due to a different strain of rat and/or higher Cd(II) dose used in the present experiment. Species-, strain- and dose-related variations in response to Cd(II), including the severity of the inflammatory/necrotizing reactions in testes, have been reported (26 and references therein).

The decrease in 8-oxo-dGTPase activity with time after injection was concurrent in our experiment with increasing level of 8-oxo-dG. It would be tempting, therefore, to speculate about a causative relationship between these two effects. However, the dynamics of these effects appear to be dissimilar enough to cast doubt upon the existence of such a relationship: 2 h after Cd(II) injection, the 8-oxo-dGTPase activity remained unchanged while the 8-oxo-dG level was already significantly elevated above the control. Thus, at least at this time point, the increase in oxidative DNA damage could not be a result of inhibition of the enzyme. Nonetheless, a contribution of the latter to the elevation of 8-oxo-dG level at later times, especially past 24 h, cannot be excluded.

Ni(II) tended to suppress 8-oxo-dGTPase activity versus that of the control at >=8 h after treatment. The very limited extent of this effect seems to be consistent with the much lower inhibitory potential of Ni(II) toward 8-oxo-dGTPase observed in vitro (13), as well as a generally weaker toxicity of Ni(II) in testes (16,27) as compared with that for Cd(II) (1,2,16,26). The testicular 8-oxo-dG levels in Ni(II)-treated rats, consistently higher than in the controls [with an apparent maximum 8 h after Ni(II), with P < 0.2 versus the control for this time point but P < 0.05 versus the mean of all control values], might indicate some oxidative damage to the DNA. However, under the present experimental conditions, this effect was too weak to be significant. It should be verified on larger groups of rats. A rationale for a closer look at possible generation of oxidative DNA damage by Ni(II) in the testis comes from the strong oxidation-mediating properties of the Ni(II) complex with protamine P2, a DNA-binding protein abundantly present in the testes and sperm heads (28,29).

In conclusion, Cd(II) acetate treatment results in marked inhibition of cellular 8-oxo-dGTPase activity and increases in the nuclear 8-oxo-dG level in the rat testis, the target organ of Cd(II)-induced carcinogenesis. The increase in oxidative DNA damage precedes the decrease in 8-oxo-dGTPase activity, indicating that the first appearance of 8-oxo-dG in DNA is not due to its incorporation from the nucleotide pool. However, the results do not exclude the possibility that a further decrease in 8-oxo-dGTPase activity with time after Cd(II) treatment may contribute to such incorporation. The very limited effects of Ni(II) observed in the present study, although suggestive of some damaging trends toward testicular 8-oxo-dGTPase and DNA, did not reach statistical significance and await confirmation on larger animal groups.


    Acknowledgments
 
The authors wish to thank Dr Gregory S.Buzard for helpful critical comments on this manuscript and Ms Kathy Breeze for editorial assistance. This project was funded in part with Federal funds from the National Cancer Institute under contract no. NO1-CO-56000. Animal care was provided in accordance with the procedure outlined in the Guide for the Care and Use of Laboratory Animals (NIH publication no. 86-23, 1985). The content of this publication does not necessarily reflect the views and policies of the Department of Health and Human Services nor does mention of trade names, commercial products or organizations imply endorsement by the US Government. The publisher or recipient acknowledges the right of the US Government to retain a non-exclusive, royalty-free licence in and to any copyright covering this article.


    Notes
 
5 To whom correspondence should be addressed Email: kasprkaz{at}mail.ncifcrf.gov Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 9, 1999; revised May 5, 1999; accepted May 6, 1999.