Cadmium-induced 8-hydroxydeoxyguanosine formation, DNA strand breaks and antioxidant enzyme activities in lymphoblastoid cells

CANCER LETTERS ELSEVIER

Cancer Letters 115 (1997) 141-148

Cadmium-induced %hydroxydeoxyguanosine formation, DNA strand breaks and antioxidant enzyme activities in lymphoblastoid cells Marina V. Mikhailova, Neil A. Littlefield, Bruce S. Hass, Lionel A. Poirier, Ming W. Chou* National

Center-for

Toxicological

Research.

FDA, Jefferson,

AR 72079,

USA

Received 14 January 1997; revision received 20 January 1997;accepted20 January 1997

Abstract The effect of cadmium ion (Cd) and ascorbic acid (Asc) on the induction of oxidative DNA damage and on the activities of antioxidant enzymes were investigated in human lymphoblastoid cells (AHH-I TK+/-). Cd at low concentrations of 5-35 ,uM induced the formation of 8-hydroxy-2’-deoxyguanosine (8-OHdG) and caused nuclear DNA strand breaks. The formation both of 8-OHdG and of DNA strand breaks was dose-dependent at the low Cd concentration; both parameters were linearly correlated with each other (R = 0.932 and P = 0.0209). 8-OHdG formation by Cd plateaued at a Cd concentration of 50 PM. Asc also induced 8-OHdG formation, but it had no synergistic effect with Cd on the formation of 8-OHdG or DNA strand breaks. Cd at the concentration of 50 yM induced the nuclear activity of the antioxidant enzymes, catalase and superoxide dismutase (SOD). Furthermore, Cd caused a decrease in the concentration of reduced glutathione (GSH) and an increase in concentration of the oxidized form (GSSG). While Asc had no observable effect on SOD activity, it did increase nuclear catalase activity in cells. This effect on catalase was synergistic with that of Cd. The linear correlation between 8-OHdG and DNA strand breaks induced by Cd at the lower Cd concentrations (550 PM), suggested that the extent of formation of DNA strand breaks induced by Cd may be offset by their induction of the formation of R-OHdG and antioxidant enzyme activities. 0 1997 Elsevier Science Ireland Ltd. Keywords: Oxidative enzymes

DNA damage; DNA strand breaks; 8-Hydroxy-2’-deoxyguanosine;

1. Introduction It is generally recognized that oxidants play an important role in carcinogenesis [1,2]. Although the mechanisms of oxidatively induced carcinogenesis are not known, the formation of oxidized DNA bases, such as 8-hydroxy-2’-deoxyguanosine (8OHdG), 5-hydroxy-2’-deoxyuridine, 5,6-dihydroxy5,6-dihydro-2’-deoxyuridine and 5-hydroxy-2’-deox* Correspondingauthor

ycytidine during carcinogenesis has been shown [3]. Oxidative DNA damage has been hypothesized to be initiated by the formation of reactive oxygen species, such as the superoxide anion (0;;) and hydrogen peroxide (H202) formed as by-products of ionizing radiation [4-61 and of cellular metabolism [ 1,l 1. The 0; and H202 then may generate DNA-damaging hydroxy radicals (.OH) through the Harber-Weiss reaction or the Fenton reaction [7,8] in the presence of a transitional metal ion such as iron or copper. 8-OHdG is an important biomarker for oxidative DNA damage. A

0304-3835/97/$17.00 0 1997 Elsevier Science Ireland Ltd. All rights reserved PII

SO304-3835(97)04720-4

Cadmium ions; Antioxidant

142

M.V. Mikhailova

et al. /Cancer Letters 115 (1997) 141-148

number of mutagens and carcinogens, including the metal ions, cadmium and nickel, may generate 8OHdG in DNA. Some classes of non-genotoxic carcinogens, such as peroxisome proliferators, also induce the formation of 8-OHdG in the liver DNA of rats [9]. Heavy metals constitute an important class of carcinogens, although the mechanisms underlying their activity are unclear [lo]. Among the transition metals, cadmium Ion (Cd) is a potent carcinogen in animals and is suspected to be a human carcinogen of increasing environmental and occupational concern [ 11,121. We have used the alkaline DNA unwinding technique [ 131to study the effect of Cd on DNA damage [ 141.In the present study we attempt to correlate the formation of 8-OHdG with the occurrence of DNA strand breaks by Cd, ascorbic acid (Asc), and mixtures of Cd and Asc (Cd/Asc). Employing a more sensitive method, the random oligonucleotide primer synthesis (ROPS) technique, we can determine the DNA strand breaks from the cells treated with CD at low concentrations. Since reactive oxygen species are directly related to the activities of antioxidant enzymes in cells, we also examined the effects of Cd, Asc, and Cd/Asc on catalase and superoxide dismutase (SOD). In addition, the levels of both reduced and oxidized forms of nuclear glutathione were also measured.

2. Materials

and methods

2. I. Chemicals Standard 8-OHdG was kindly provided by Dr. David H. Swenson of Louisiana State University, Baton Rouge, LA. Cadmium chloride, Asc, nitroblue tetrazolium (NTB), glutathione (both reduced and oxidized forms), nuclease Pl, alkaline phosphatase, methylglyoxal, 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) and other chemicals and enzymes required for DNA isolation were purchased from Sigma Chemical Co., St. Louis, MO. H202 was obtained from Fisher Scientific. Klenow fragment polymerase, dATP, dGTP, dCTP, and dTTP were from New England Biolabs, Beverly, MA. [32P]dCTP (spec. act. 3000 Ci/mmol) was purchased from Amersham Corp., Arlington Heights, IL. Bradford reagent was obtained from Bio-Rad Laboratories, Hercules, CA. Puregene DNA Isolation Kit was

obtained from Gentra Systems, Inc. All solvents used were of HPLC grade. 2.2. Cells and treatments The lymphocyte cell line (AHH-1 TK+/-) was obtained from Gentest Corp., Woburn, MA and grown in 1640 medium (Sigma Chemical Company, St. Louis, MO) containing 9% horse serum, 1% Lglutamine (200 mM), and 1% penicillin/streptomycin (v/v). For the study of the effect of low concentration of Cd on oxidative DNA damage, the cells were treated with 5, 15,25,35 and 50 PM CdClz for 4 h. For the high dose experiments, the cells were treated with 25, 50,75, and 100 PM CdCl;? in the presence and absence of 500 PM Asc for 4 h, respectively. After incubation. cells were harvested by low speed centrifugation and homogenized with 9 ~01s.of homogenizing buffer (50 mM T&0.25 M sucrose, pH 7.4). The nuclear and mitochondrial fractions were prepared by differential centrifugation. 2.3. Isolation qf DNA Genomic DNA was isolated by the method of Ausubel et al. [ 151. Approximately 10’ nuclei were incubated for 15 h at 50°C with 0.3 ml digestion buffer, containing 100 mM NaCl, 10 mM Tris-HCl (pH 7.9), 25 mM EDTA, 0.5% sodium dodecyl sulfate, and 0.3 mg/ml proteinase K. After incubation, the DNA was isolated through phenol/chloroform/isoamy1 alcohol (25:24:1) extraction. The resulting aqueous phase was then washed with diethyl ether to remove residual phenol. For each ml of DNA solution 5 pg DNase-free RNase was added and incubated at 37°C for 1 h to remove RNA. The DNA was dialyzed against the excess of Tris-EDTA buffer (10 mM Tris-HCl, pH 7.9, 1 mM EDTA) overnight. DNA concentration was determined by a spectrophotometer at 260 nm with purified calf-thymus DNA as the standard. The DNA used for 8-OHdG analysis was purified with the phenol-free Puregene DNA Isolation Kit in order to protect DNA from additional oxidative damage during and after isolation with phenol. 2.4. 8-hydroxydeoxyguanosine

analysis

Purified DNA was digested to nucleosides by

M.V. Mikhailova

nuclease Pl and alkaline phosphatase according to the method of Gehrke et al. [16]. 8-OHdG was separated from the rest of the deoxynucleoside mixture by HPLC (Model 600, Waters, Milford, MA, equipped with a T-Bondapak ODS column, 3.9 x 300 mm), equipped with both an electrochemical detector (Colouchem II, ESA), and a UV detector (PDA Model 996, Waters). The column was eluted with aqueous buffer containing 6.25 mM citric acid, 12.5 mM sodium acetate, 15.0 mM sodium hydroxide, 5.0 mM acetic acid (pH 5.3), and 10% methanol at the flow rate of 1 ml/min. The level of 8-OHdG in DNA was expressed as the ratio of 8-OHdG per lo5 dG. 2.5. DNA strand-break

143

et al. /Cancer Letters 115 (1997) 141-14X

assay

DNA strand breaks were measured by the random oligonucleotide primer synthesis (ROPS) assay [ 171. Briefly, 0.25 pg purified genomic DNA was incubated at 100°C for 5 min and then quickly cooled in an ice bath to denature DNA. The cold DNA solution was incubated with 10 mM Tris-HCl buffer (pH 7.5), containing 50 nCi [j2P]dCTP, 0.05 mM of dATP, dTTP and dGTP, 5 mM MgC12, 7.5 mM dithiothreitol, and 0.5 unit Klenow polymerase, in a total volume of 25 ~1, for 30 min at 16°C. Reaction was stopped by addition of an equal volume of 10 mM Tris-HCl (pH 7.5), containing 12.5 mM EDTA and 100 mM NaCl. Aliquots of 5 ~1 were applied onto DE8 1 filter paper. The filter paper was rinsed five times with 0.5 M Na-phosphate buffer (pH 6.8) to remove non-incorporated precursors. Labeled DNA absorbed on the paper was counted in a scintillation counter. Data on 32Pincorporation in the ROPS assay are presented in pmol [“P]dCTP incorporated per Fg DNA. 2.6. Enzyme assays

Catalase activity was measured as described by Cohen et al. 1181.The enzyme-catalyzed decomposition of H202 was recorded by a spectrophotometer at 480 nm. Enzyme activity was calculated as units/lo6 cells per min. SOD activity was measured by the inhibition of NTB reduction as previously described by Oberley and Spitz [ 191. One unit of activity is that amount of protein that gives half-maximal inhibition. The activity of SOD was expressed as units/lo6 cells per min.

Cellular reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were assayed according the method of Akerboom and Sies [20] by reaction with methyl glyoxal and DTNB, respectively. The content of GSH or GSSG was expressed as nmoV106 cells. Protein concentrations were determined by the method of Bradford [21] with BSA as the standard. 2.7. Statistical

analysis

Data were expressed as mean + SEM. Significant differences were compared between control groups (-Cd/ and -Asc) and the treated groups (-Cd/+Asc, tCd/ and -Asc, +Cdl+Asc) by one-way ANOVA. Linear regression was analyzed by using PC software, Microcal Origin.

3. Results The viabilities of the cells treated at each dose level of Cd and incubated for 4 h at 37°C were consistently greater than 90%. 3.1. HPLC separation

of 8-OHdG

The profiles of the separation of 8-OHdG from the other deoxyribonucleosides in the enzyme-digested DNA samples detected by a UV and electrochemical detector are shown in Fig. 1. The 8-OHdG peak (V, Fig. 1) was separated from dC, dG, dT and dA peaks (II, III, VI, and VII, respectively) (Fig. 1A). Peak I is a solvent peak. Peaks III and IV are unknown. 8-Hydroxydeoxyguanosine was the only nucleoside that was detected by the electrochemical (EC) detector (Fig. 1B). The EC detection of 8-OHdG from nuclear DNA hydrolysate showed a single peak at retention time of 15.1 min which could be co-eluted with standard 8-OHdG. The sensitivity of the detection of 8OHdG using the EC detector was in the range around 50 ng of 8-OHdG per injection. 3.2. ESfect of Cd on the formation DNA strand breaks

of 8-OHdG

and

The level of 8-OHdG in the cells not treated with Cd and Asc (control group) was low (0.9-1.5 per lo5 dG). Addition of Cd at low concentrations of 5, 15,25,

144

M. V. Mikhailova III

et al. / Cancer Letters 115 (I 997) 141- 148

A

dG

Retention

Time, min

Fig. 1. HPLC profile of the separation of 8-hydroxydeoxyguanosine (8-OHdG) monitored by a UV (A) and an electrochemical (B) detector.

35 and 50 PM significantly increased the 8-OHdG formation (Fig. 2A). Low doses of Cd (25 and 50

PM) also significantly caused the formation of single strand DNA breaks in the nuclear DNA of the cells (Fig. 2B). Both the increase of 8-OHdG and DNA strand breaks were proportional to the increase of Cd concentration. The oxidative DNA damages reached a maximum when cells were treated with Cd at the concentration of 35 PM. The formation of 8-OHdG and DNA strand breaks at low concentration Cd treatment correlated each other with R = 0.932 and P = 0.021 (Fig. 3). When the cells were treated with high concentration of Cd (>50 PM) the 8-OHdG formation (Fig. 4A) and DNA strand breaks (Fig. 4B) were not proportional to the dose and did not correlate with each other. Both 8-OHdG formation and DNA strand breaks induced by high concentration of Cd (>50 PM) reached a plateau at the Cd concentration of 50 PM. High concentration of Cd caused DNA fragmentation compared to the control groups detected by gel electrophoresis (data not shown), and resulted in greater variation in the assay of DNA strand breaks by ROPS. 3.3. ESfect of Asc and As&d mixture on the 8-OHdG formation and the DNA strand breaks Asc (0.5 n&l) alone induced an increase of the 8OHdG formation by 266% (Fig. 5A) compared with the control group (0 concentration of Cd in Fig. 3A).

I-

I-

0!

1

0

.

1

to

,

I

20

.

1

30

c410-%

.

I

40

i

cm

I+-

0

10

20

J)

40

90

c4 10S M

Fig. 2. Effect of cadmium (Cd) at low concentrations on the formation of 8-hydroxydeoxyguanosine (8-OHdG) (A), and nuclear DNA strand breaks (B) in cultured human lymphoblastoid cells. Each data point represents mean + SEM, n = 4. *Significantly different from the control group, P < 0.05.

M.V. Mikhailova

145

et al. / Cancer Letters 115 (1997) 141-148

a,

B

Fig. 3. Correlation of cadmium (Cd)-induced 8-hydroxydeoxyguanosine represent the concentrations of Cd in each treatment.

(8-OHdG) formation and DNA strand breaks. The numbers O-35

In the presence of 25, 50, and 75 PM of Cd, the Asc did not protect the Cd-induced oxidative DNA damages nor synergetically increased the 8-OHdG formation (Fig. 5A). AX alone also significantly induced DNA strand breaks (Fig. 5B) compared with the control group (0 concentration of Cd in Fig. 3B). Similar to the effect of Asc on the Cd-induced 8OHdG formation, Cd/Asc mixtures did not decrease nor synergistically increase the DNA strand breaks (Fig. 5B).

sured. The catalase activity associated with nuclei was significantly increased by Cd and Asc (Table 1). Cd and Asc separately increased nuclear catalase activity by 190% and 124%, respectively. Asc had a synergistic effect on the induction of nuclear catalase. The nuclear levels of both reduced (GSH) and oxidized (GSSG) glutathione were significantly altered by Cd treatment, with GSH levels being depressed

3.4. Effects of Cd and Ax on the activities of SOD and catalase and nuclear glutathione content The activities of cellular SOD and catalase are found mostly in the cytosolic fraction (>90%) of the cultured lymphoblastoid cells. The activities of SOD and catalase in cytosolic fraction were 18-28and 22--89-fold greater than those associated with the nuclei, respectively, in the cells with or without Cd/ Asc treatments (Table 1). No significant effect on the cytosolic SOD activity was found after cells were incubated with 50 PM of Cd and 500 PM of Asc for 4 h (Table 1). However, Cd significantly increased the nuclear SOD activity by 41% in the absence or presence of 0.5 mM of Asc. Asc alone ( -Cd/+Asc) did not affect the nuclear SOD activity. Similar trends were obtained when catalase activities were mea-

OB-

. 35

25

-02.,., 1.4

1.45

.

I. 1.50

I. I.55

I.

I.

I.

1.Q

l.a6

1.70

Log DNA Strand

.

6

Breaks

Fig. 4. Effect of cadmium (Cd) at higher concentrations on the formation of 8-hydroxydeoxyguanosine (8-OHdG) (A), and nuclear DNA strand breaks (B) in cultured human lymphoblastoid cells. Each data point represents mean f SEM, n = 4. *Significantly different from the control group, P < 0.05.

146

M. V. Mikhailova

with

Arc

et al. / Cancer Letters I15 (1997) 141-148

with

(MmM)

Asc

(0.5mM)

I-

,-

If

0

P

a

60

w

0

P

a

Cd 10” M

cd,

60

60

10"M

Fig. 5. Effect of ascorbic acid (0.5 mM) on cadmium (Cd)-induced 8-hydroxydeoxyguanosine (8-OHdG) formation (A), and nuclear DNA strand breaks (B) in cultured human lymphoblastoid cells. Each data point represents mean zk SEM, n = 4. *Significantly different from the control group (0 concentration of Cd in Fig. 4A,B), P < 0.05.

by Cd and GSSG being increased by Cd. Asc did not affect the nuclear GSH level and apparently did not restore the decrease of GSH by Cd (Table 1).

4. Discussion Oxidative modification of DNA, such as the formation of 8-OHdG and the generation of DNA strand breaks, are two types of DNA damage resulting from oxidative stress. Metal ions, such as Cd and Ni, induce such DNA damage [14,22], which has been proposed to contribute to the carcinogenicity of these metal ions. The aim of this study was to corre-

late the two types of DNA damage induced by Cd in cultured lymphoblastoid cells. The ROPS assay which we use is very sensitive to strand breaks caused by DNA oxidation [17]. It has been previously shown that 8-OHdG can be separated and quantitated by using HPLC monitored with an electrochemical detector [23]. In the control groups (-Cd/--Asc) the 8-OHdG level detected was low (0.9-1.5 B-OHdG/ 10’ dG) and comparable to that seen in a previous study [24] indicating that using phenol-free solvent minimized the exogenous S-OHdG formation during the DNA purification and analytical processes. Our results showed that in the presence of the lower Cd concentrations the dose-dependent formation of

Table 1 Effect of cadmium (Cd) and ascorbate (Asc) on the activities of superoxide dismutase (SOD) and catalase and levels of nuclear glutathione in AHH-1 TH+/- lymphoblastoid cellpb Treatment

-C&Asc +Cd/-Asc -Cdl+Asc +Cd/+Asc

SOD (units x 10-‘/106 cells)

Catalase (units x 10-3/106 cells)

Nuclear glutathione (nmol/105 cells)

Nuclei

Cytosol

Nuclei

Cytosol

GSH

GSSG

28.6 40.3 28.8 40.3

722 763 768 725

0.70 * 0.01 2.03 f 0.04* 1.57 k 0.12* 2.40 L!Z0.30*

62.0 62.1 53.0 53.7

15.5 f 0.6 9.8 k 0.4* 14.7 lb 0.1 11.4 * 1.0*

7.6 k 9.1 + 9.1 + 12.6 +

f 0.9 f 1.1* 3~0.7 + 1.3*

+ f + k

19 12* 19* 15

It i It k

2.9 2.5 6.9 1.2

aConcentrations of Cd and Asc used were 50 PM and 500 PM, respectively; treatment was for 4 h. bValues are mean + SEM, n = 7. *Significantly different from the control group (-Cd/-Asc).

0.5 0.5% 1.0 0.7*

M.V. Mikhuilovn et 01. /Cancer

both 8-OHdG and of DNA strand breaks in the cells increased significantly, indicating that the sites of 8OHdG formation may correlate the sites generating the 3’-OH end DNA strand breaks. However, at Cd concentrations greater than 50 PM, the two parameters did not correlate quantitatively, suggesting that high concentration of Cd may saturate the sites of 8-OHdG formation, and destabilize DNA causing DNA strand breaks and other DNA damage. Other factors such as the induction of DNA repair, effect on other mineral nutrients, zinc, iron or magnesium, or of antioxidant enzyme activities stimulated by the higher concentration of metal ions, may not be excluded. Indeed, cellular reducing agents, such as GSH and Asc, and antioxidant enzymes were affected by Cd treatment at the dose of 50 PM. The increase in SOD and catalase activities and the decrease in GSH was accompanied by a significant change in 8-OHdG formation but not in DNA strand breaks, further suggesting that different mechanisms may be involved in the formation of these changes. Most of the antioxidant enzyme activity was in the cytosolic fraction of the cultured lymphoblastoid cells. The activity of nuclear enzyme was l-5% of the total antioxidant enzyme activity. The increase in the nuclear SOD and catalase activity by Cd at the concentration of 50 PM was comparable to Cdinduced oxidative DNA damage. Asc did not affect either SOD activity nor the nuclear GSH content. However, signiticant enhancement in 8-OHdG and DNA strand breaks by Asc were found. Asc has been shown to protect against endogenous oxidative DNA damage in human sperm [25], and to protect against potassium bromate-induced oxidative DNA damage in rat kidney [26]. Cai et al. [27] found a dose-dependent ability of metals, e.g. Cu(II), Fe(I1) and Fe(E) to induce double-strand breaks in DNA in vitro in the presence of ascorbic acid. However, even 1 mM ascorbic acid did not prevent DNA strand breaks by 100 PM H202 [28]. Ascorbic acid did not prevent quercetin-induced DNA damage in the presence of equimolar concentrations of iron or copper, and stimulated the oxidative damage to nuclear macromolecules [ 291. Our results show that 500 PM Asc did not protect against the Cd-induced formation of 8-OHdG or DNA strand breaks in cultured lymphoblastoid cells. The Cd/Asc-induced DNA damage appears to be caused by induction of a Fenton-like

Letters 115 (19Y7) 141-148

157

reaction (Fe” + HZ02 -+ Fe”+ + HO- + HO.) which generates hydroxy radicals. Thus, the role of Asc in the formation of 8-OHdG may be to act as a reducing agent producing ferrous ions for the Fenton reaction. Since catalase is an iron-prophyrin enzyme, the increase in nuclear catalase activity by Asc thus can be interpreted. In conclusion, our results demonstrate that Cd induced 8-OHdG formation, increased antioxidant enzyme activity associated with nuclei and caused DNA strand breaks. The correlation of 8-OHdG formation with DNA strand breaks indicates that the formation of 8-OHdG may be the major factor leading to DNA strand breaks when cells were treated with low concentration of Cd. Other factors altered by Cd, such as DNA synthesis, DNA repair, and the fidelity of DNA replication, may also contribute to its damage to DNA.

References 111Ames, B.N. ( 1983) Dietary carcinogens and anticarcinogens. Oxygen radicals and degenerative diseases. Science. 22 I, l256- 1264. 121Cerutti, P.A. (1985) Prooxidant states and tumor promotion, Science, 227, 375-381. [31 Wagner, J.R., Hu, C-C. and Ames, B.N. ( 1992) Endogenous oxidative damage of deoxycytidine in DNA, Proc. Natl. Acad. Sci. USA, 80, 3384-3390. 141 Teoule, R. (1987) Radiation-induced DNA damage and its repair, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med., 5 I. 573-589. [51 Weitaman. S.A., Weitberg, A.B., Clark, E.P. and Stossel, T.P. (I 985) Phagocytes as carcinogens: malignant transformation produced by human neutrophils, Science, 227, 1231-1233. L61 Breimer, L.H. (1988) Ionizing radiation-induced mutagenesis, Br. J. Cancer, 57, 6- 18. t71 Halliwel, B. and Gutteridge, J.C.M. (1986) Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts, Arch. Biochem. Biophys.. 246. SOI532. Halliwell, B. and Aruoma, 0.1. ( 1991) DNA damage by oxygen-derived species. Its mechanism and measurement in mammalian systems, FEBS Lett., 281. 9.. 19. Kasai. H.. Okada, Y., Nishimura, S.. Rao, MS. and Keddy, J.K. (1989) Formation of 8-hydroxydeoxypuanosine in liver DNA of rats following long-term exposure to a pcroxisome proliferator, Cancer Res., 49, 2603-2605. Kasprzdk, KS. (1991) The role of oxidative damage in metal carcinogenicity, Chem. Res. Toxicol., 4, 605-615. Sharma, G., Nath, R. and Gill. K.D. t 199 I ) Effect of ethanol on cadmium-induced lipid peroxidation on antioxidant

148

M.V. Mikhailova

et al. /Cancer Letters 115 (1997) 141-148

enzyme in rat liver, B&hem. Pharmacol., 42 (Suppl.), S9Sl6. [ 121 Waalkes, M.P., Coogan, T.P. and Barter, R.A. (1992) Toxicological principles of metal carcinogenesis with special emphasis on cadmium, Crit. Rev. Toxicol., 22, 175-201. [ 13) Bimboim, H.C. and Javcak, J.J. (1981) A Ruorometric method for rapid detection of DNA strand breaks in human white cells produced by tow doses of radiation, Cancer Res., 41, 1889-1892. [14] Littleheld, N.A. and Hass, B.S. (1995) Damage to DNA by cadmium or nickel in the presence of ascorbate, Ann. Clin. Lab. Sci., 25. 485-492. ] 151 Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D, Seidman, J.G., Smith, J.A. and Struhl, K. (I 989) Current Protocols in Molecular Biology, 1, 3.5.9- 10, Wiley-Interscience, New York.

[16] Cehrke, C.W., McCune, R.A., Gama-Sosa, M.A., Ehrlich, M. and Kuo, KC. (1984) Quantitative reversed-phase high-performance liquid chromatography of major and modified nucleosides in DNA, J. Chromatogr., 301, 199-219. 1171 Basnahian, A.G. and James, S.J. (1996) Quantification of 3’. OH DNA breaks by random oligonucleotide-primed synthesis (ROPS) assay, DNA Cell Biol., 15, 255-262. ] 181 Cohen, G., Dembiec. D. and Marcus, J. (1970) Measurement of catatase activity in tissue extracts, Anal. Biochem.. 34, 3038. ] 191 Oberley, L.W. and Spitz, D.R. (1984) Nitroblue tetrazolium. In: Handbook of Methods for Oxy Radical Research, pp. 217-220. Editor: R. Greewald. CRC Press, Boca Raton, FL. [20] Akerboom, T.P.M. and Sies, H. (1981) Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples, Methods Enzymol., 77, 372-382. [21] Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72, 248254.

[22] Kawanishi, S., Inoue, S. and Yamamoto. K. (1994) Active oxygen species in DNA damage induced by cancerogenic metal compounds, Env. Health Perspect., 102, 17-20. [23] Barciszewski, J., Rattan, S.I.S., Siboska, GE., Otzen, D.E. and Clark, B.F.C. (1993) Reduction in the amount of 8hydroxy-2’-deoxyguanosine in the DNA of SV40-transformed human fibroblasts as compared with normal cells in culture, FEBS Lett., 318, 186-188. [24] Takeuchi, T., Nakajima, M., Ohta, Y., Mure, K., Takeshita. T. and Morimoto, K. (1994) Evaluation of S-hydroxydeoxyguanosine, a typical oxidative DNA damage, in human leukocytes, Carcinogenesis, 15, 1519-1523. [25] Fraga, C.G., Motchnik, P.A., Shigenaga, M.K., Helbock, H.J., Jacob, R.A. and Ames, B.N. (1991) Ascorbic acid protects against endogenous oxidative DNA damage in human sperm, Proc. Nat]. Acad. Sci. USA, 88, 11003-I 1006. [26] Sai, K., Umemura, T., Takagi, A., Hasegawa, R. and Kurokawa, Y. (1992) The protective role of glutathione, cysteine and vitamin C against oxidative DNA damage induced in rat kidney by potassium bromate. Jpn. J. Cancer Res., 83. 45-5 1. [27] Cai, L., Koropatnick, J. and Cherian. M.G. (1995) Metallothionin protects DNA from copper-induced but not ironinduced cleavage in vitro, Chem.-Biol. Interact., 96. 1333 145. [28] Baader, S.L., Bruchelt, G., Carmine, T.C., Lode, H.N., Reith, A.G. and Neithammer, D. (1994) Ascorbic-acid-mediated iron release from cellular ferritin and its relation to formation of DNA strand breaks in neuroblastoma ceils. J. Cancer Res. Clin. Oncol., 120, 415-421. [29] Sahu, S.C. and Washington, M.C. (1992) Effect of ascorbic acid and curcumin on quercetin-induced nuclear DNA damage, lipid peroxidation and protein degradation, Cancer Lett., 63, 237-241.