DNA single-strand breaks and cytotoxicity induced by sodium chromate(VI) in hydrogen peroxide-resistant cell lines

Mutation Research, 299 (1993) 95-102

95

© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1218/93/$06.00

MUTGEN 01869

DNA single-strand breaks and cytotoxicity induced by sodium chromate(VI) in hydrogen peroxide-resistant cell lines Masayasu Sugiyama, Katsuyuki Tsuzuki and Nobuya Haramaki Department of Medical Biochemistry, Kurume University School of Medicine, Kurume, Japan (Received 24 July 1992) (Revision received 26 October 1992) (Accepted 30 October 1992)

Keywords: Chromate; Cadmium; Mercury; Active oxygen; Hydrogen peroxide-resistant cells

Summary Hydrogen peroxide-resistant Chinese hamster ovary (CHO R) cells were developed by exposing parental (CHO P) cells to sequential increases in H 2 0 2 concentration. Cytotoxicity as well as DNA single-strand breaks induced by Na2CrO 4 were then compared in CHO R and CHO P cell lines. Using the colony-forming assay, it was found that the cytotoxicity caused by Na2CrO 4 did not differ in the parent and resistant cells. However, alkaline elution studies showed that the production of DNA single-strand breaks in CHO R cells treated with Na2CrO 4 was reduced by about 50% as compared with that in CHO P cells. Similarly, electron spin resonance (ESR) studies revealed that the level of chromium(V) in CHO R cells during treatment with Na2CrO 4 was about 50% that in CHO P cells. CHO R cells were also found to be cross-resistant to the cytotoxicity and DNA breaks caused by other toxic metals such as CdCI 2 and HgCI 2. Catalase activity in resistant cells was 2-fold and the cellular content of glutathione was 3-fold that in parental cells. However, no obvious differences were seen in superoxide dismutase and glutathione reductase activity, although the contents of ascorbic acid or a-tocopherol were slightly decreased in CHO R cells, suggesting that the resistance in CHO R cells may be associated with the increase in both catalase activity and glutathione contents in cells. These results indicate that chromateinduced DNA breaks appear to be mediated by a different mechanism than that for the cytotoxicity of this metal, and also suggest that the formation of active oxygen species a n d / o r chromium(V) during reduction of chromium(VI) inside cells might be associated with the induction of the DNA strand breaks caused by the metal.

Carcinogenic and toxic chromium(VI) compounds have been shown to produce a variety of DNA lesions such as DNA single-strand breaks, Correspondence: Masayasu Sugiyama, Department of Medical Biochemistry, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830, Japan. Tel. 0942-35-3311 ext. 225; Fax 0942-31-4377.

alkali-labile sites and DNA-protein crosslinks in vivo and in cultured cells (reviewed in De Flora et al., 1990). Because chromium(VI) easily passes through the cell membrane and is consequently reduced through intermediates to chromium(III) by cellular reductants (De Flora and Wetterhahn, 1989; Sugiyama, 1992), the formation of the intermediate oxidation states, such as chromium(V)

96

and (IV), may play a role in the adverse biological effects of chromium(VI). Earlier studies have shown that this reduction process also causes the generation of active oxygen species such as hydroxyl radicals, with concomitant formation of chromium(V). For instance, chromium(VI) has been reported to react with H20 2 to form chromium(V), leading to the generation of hydroxyl radicals, which in vitro caused breaks and 8-hydroxydeoxyguanosine in DNA (Aiyar et al., 1989, 1990; Kawanishi et al., 1986). In addition, biologically generated chromium(V) complex has been reported to react with HzOz, in a Fentontype manner, to produce more hydroxyl radical than a similar reaction with chromium(VI), resulting in the induction of DNA damages in vitro (Aiyar et al., 1989, 1990; Shi and Dalal, 1990a,b). Moreover, enzymatic reduction of chromium(VI) by NADPH-dependent flavoenzyme was found to produce hydroxyl radicals, possibly through the reaction of chromium(V) and H 2 0 z formed during the reduction (Shi and Dalai, 1990c). Thus, the formation of active oxygen species, in particular H202, is suspected to be closely associated with the induction of biological effects caused by chromium(VI). However, there has been only one study that examined the involvement of active oxygen species in chromate-induced DNA damage and cytotoxicity in intact ceils (Snyder, 1988). In the present study, to help in understanding the mechanism of the toxic effect of chromium(VI), we established H2Oz-resistant Chinese hamster ovary (CHO R) cells and examined whether or not these CHO R cells had become resistant to the DNA single-strand breaks and cytotoxicity induced by chromium(VI). In addition, cellular levels of chromium(V) in CHO R and the parental cells (CHO P) were investigated directly by electron spin resonance (ESR) spectroscopy. Moreover, the induction of DNA breaks and cytotoxicity caused by other toxic metals such as cadmium and mercury was also examined in CHO R and CHO P cells, because these metal-induced damages in cultured cells have been shown to be mediated by formation of active oxygen species (Cantoni et al., 1984; Ochi et al., 1983; Snyder, 1988).

Materials and methods

Chemicals Na2CrO4"4H20, CdCI z and HgC12 were obtained from Nakarai Chemical Ltd. (Kyoto, Japan). The radioisotopes NazS1CrO4, t°9CdCl2 and 2°4Hg(NO3)2 were from Du Pont-New England Nuclear. Cell culture and H202-resistant cells CHO cells were maintained in a-minimal essential medium supplemented with 10% fetal bovine serum and a 1% solution of penicillin/ streptomycin (Gibco). CHO R cells were derived from the CHO e cells by adding progressively higher concentrations of H202 to the culture medium. Briefly, 24 h after cells were plated, H 2 0 2 w a s added to the culture medium. After 3-5 days, the cells were harvested and then plated. After 24 h H 2 0 2 w a s again added. These procedures were continuously repeated until the generation time of the resistant cells approached that of the parental cells. Selection for H20 2 resistance was initiated by the exposure of parental cells to 6.25/zM H202, and the concentration of H20 2 was increased up to 100 p.M in a stepwise fashion. To maintain the resistance, 100 /zM H20 2 was continuously added to the culture medium 24 h after plating. Treatment Twenty-four hours after plating, logarithmically growing cells were rinsed two times with salts-glucose medium (SGM; 50 mM Hepes (pH 7.2) containing 10 mM NaC1, 5 mM KCI, 2 mM CaCl2, and 5 mM glucose), and the cells were then treated with H20 2 for 1 h or with Na2CrO4, CdC12 and HgC12 for 2 h at 37°C in this maintenance medium. Cytotoxicity H20 2- and the metals-induced cytotoxicity were estimated by colony-forming assays as previously described (Sugiyama et al., 1989a,b). Alkaline elution The alkaline elution assay for DNA singlestrand breaks was carried out as previously de-

97

phate buffer (pH 7.0) and sonicated with a Sonitier Model 200 (Branson Sonic Power Co., Danbury, CT). They were pulsed seven times at setting 7, and centrifuged for 20 rain at 9000 × g. The supernatant was mixed with 10 mM H 2 0 2 and the decomposition of H 2 0 2 was followed directly by a decrease in absorbance at 240 nm. The enzyme activity was expressed as nmole of H 2 0 2 d e c r e a s e d / m i n / m g protein. Proteins were determined by the Bio-rad (Bio-rad Laboratories, Richmond, CA) protein assay. Superoxide dismutase (SOD) in the sonicated samples was measured using ESR spectrometry as previously described (Ogura et al., 1991). From the calibration curve prepared using SOD enzyme (Boehringer), the SOD activity was expressed as units of SOD activity/mg protein.

scribed (Sugiyama et al., 1988). D N A breaks were expressed in terms of the strand scission factor as described earlier (Sugiyama et al., 1987).

Cellular uptake Cellular uptake of the metals was measured by radioisotope SlCr, l ° 9 C d and 2°3Hg analysis. Briefly, cells were incubated with the radioisotope metals for 2 h in SGM, and harvested by trypsinization. The cell number was determined, and the cellular uptake of these metals was estimated by the radioactivity detected in a gamma counter.

Enzyme actiuity Catalase activity was measured as described by Aebi (1984) with minor modifications. Briefly, cells were suspended in 50 mM potassium phos-

(%)

(%)

100~ 100

lO

10

E 1' t3.

B

A

J

i

i

i

25

50

75

100

.1

i

i

20

40

Na2CrO 4 (gM)

H202 (gM) (%)

(%)

lOO

lOO

>

=

10 O9 n

CdCI

i

i

5

10 2 (~I.M)

.1 0.0

0.5

1.0

HgCI 2 (gM)

Fig. 1. Cytotoxicity of H 2 0 2 , Na2CrO4, CdCI 2 and HgCI 2. C H O P (e) and C H O R (©) cells were treated with H 2 0 2 for 1 h (A), or with Na2CrO 4 (B), CdC12 (C) and HgCI 2 (D) for 2 h in SGM. Following treatment, cytotoxicity was estimated with the colony-forming assay. Each value is the m e a n +_SD (n _> 3).

98

Glutathione reductase was measured as detailed in Sugiyama et al. (1989a,b). The enzyme activity was expressed as nmole of N A D P H oxidized/ min/mg protein.

and collected by centrifugation at 800 x g for 10 min at 4°C. After being washed twice in SGM, 1.5 x 107 cells were mixed with 400/zM Na2CrO 4 in this medium. The samples were rapidly placed in ESR fiat tubes and ESR measurements were carried out during the incubation. All ESR measurements were made at room temperature using a JES-FE3X spectrometer with a 100-kHz field modulation and 8 mW of microwave power.

Glutathione, a-tocopherol and ascorbic acid content The cellular content of total glutathione (oxidized and reduced), ascorbic acid and atocopherol was determined as previously described (Sugiyama et al., 1987, 1991a, 1992).

Results and discussion

ESR spectrometry To detect chromium(V) in ceils, cells were removed from the monolayer by trypsinization

Fig. 1A,B shows the cytotoxicity of H 2 0 2 and Na2CrO 4 in CHO a and C H O v ceils. The cytotoxicity of H 2 0 2 up to 100 /zM was completely 1.0

0.8-

0.8 "~

0.6 0.6

tO

69 69

0.4

0 69

0.4

t'-

0.2 0.2

0.0 ¸

0

,

,

,

,

,

5

10

15

20

25

0.0

i

i

i

L

i

200

400

600

800

1000

N a 2 Or0 4 (#M)

H 2 0 2 (p.M)

0.6

0.6

0.4

0.4'

0.2-

0.2"

0 0

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"5 69

-o c

0.0" 0

•

'

100

'

200 CdCI 2 (~M)

O.C

300

i

i

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2

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Fig. 2. D N A single-strand breaks of H 2 0 2 , Na2CrO 4, CdCI 2 and HgCI 2. C H O v (e) and C H O R ( 0 ) were treated with H 2 0 2 for 1 h (A), or with Na2CrO 4 (B), CdC12 (C) and HgCI 2 (D) for 2 h in SGM. Following treatment, cellular D N A was analyzed by alkaline elution. Each value is the m e a n + SD (n > 3).

99 TABLE 1 C E L L U L A R U P T A K E O F 51Cr, t°9Cd A N D 2°3Hg Treatment

CrO42 Cd 2+ Hg 2+

(/xM)

25 400 5 0.5

Uptake ( n m o l e / 1 0 6 cells) CHO P

CHO R

3.16_+0.09 9.53 _+0.70 0.58_+0.03 2.16_+0.40

3.10_+0.18 8.86 _+ 1.42 0.55_+0.03 2.28_+0.28

Cells were treated for 2 h with each of the metals in SGM, and cellular uptake was determined from the radioactivity present in 106 cells. Each value is the mean_+ SD (n >__3).

eliminated in C H O R cells as compared with CHO P ceils. Similar results were observed in C H O R cells after 30 times subculturing in the absence of H 2 0 2 in the culture medium (data not shown), indicating that the elevated resistance was stable. On the other hand, there was no significant difference in the cytotoxicity caused by Na2CrO 4 (5-50 ~ M ) between both cell lines. Fig. 1C,D shows that CHO R cells were cross-resistant to the cytotoxicity of both CdC12 and HgC12. These results suggest that the mechanism of chromate-induced cytotoxicity is completely different from that involved in the cytotoxicity of H 2 0 2 , cadmium(II) and mercury(II). Fig. 2A,B compares the induction of D N A single-strand breaks caused by H 2 0 2 or NazCrO 4 between C H O R and C H O P cells. As expected, the levels of DNA single-strand breaks induced by H 2 0 2 (6.25-25 /~M) were almost completely suppressed in the resistant cells. However, in contrast to cytotoxicity, the level of D N A singlestrand breaks in C H O R cells treated with NazCrO 4 (400 and 1000 /.LM) was reduced by about 50% as compared with that in C H O P cells.

Although relatively high concentrations of NazCrO 4 were necessary to demonstrate a difference in D N A breaks between C H O R and CHO P cell lines, these results indicate that chromium(VI)-induced D N A breaks may be mediated by a different mechanism than that for the cytotoxicity of this metal. CHO R cells were also cross-resistant to DNA single-strand breaks produced by CdC12 and HgCI 2 (Fig. 2C,D). It was hypothesized that the metal resistance may result from the inhibition of cellular uptake of metals. However, as shown in Table 1, there was no significant difference in the uptake of the metals between these cell lines. In the case of chromate, no difference in uptake was observed following 400 /.~M treatment at which D N A single-strand breaks were suppressed in the resistant cells. Thus, in the present study, the obtained metal resistance against D N A breaks a n d / o r cytotoxicity was not due to alterations of cellular uptake. The involvement of active oxygen species in HgC12- and CdC12-induced cellular damage has been reported in cultured mammalian cells (Cantoni et al., 1984; Ochi et al., 1983; Snyder, 1988), and the present results using hydrogen peroxide-resistant cells also indicate that these metal-induced D N A breaks and cytotoxicity may be mediated by active oxygen species inside cells. Concerning chromium(VI), active oxygen species may be partially involved in the induction of D N A breaks, but not in cytotoxicity, because C H O R cells were cross-resistant only to the D N A breakage caused by this metal. These results were similar to those of previous reports showing that extracellular treatment with catalase or SOD, which r e m o v e s H 2 0 2 or superoxide radicals, de-

TABLE 2 CELLULAR LEVEL OF ANTIOXIDANTS AND ANTIOXIDANT ENZYME ACTIVITY

CHO P CHO R

Catalase (nmole/min/ mg protein)

SOD (units/ mg protein)

Glutathione reductase (nmole/min/ mg protein)

Glutathione (/-~g/mg protein)

Ascorbic acid (p.g/mg protein)

a-Tocopherol (ng/mg protein)

10.7_+2.2 22.1_+1.0 *

2.94_+0.68 3.31_+0.89

27.8_+0.8 26.3_+1.4

1.87_+0.29 5.07_+0.44 *

0.77_+0.02 0.70_+0.02 **

6.13_+0.82 4.87_+0.36 * * *

Each value is the mean_+ SD (n >__3). * P < 0.01; ** P < 0.02; * * * P < 0.05 compared to C H O P cells.

100

creased D N A breakage without affecting the cytotoxicity induced by chromium(VI) (Snyder, 1988). Since catalase and SOD do not easily penetrate the cellular membrane, these results along with those obtained in the present study indicated that active oxygen species may be generated both outside and inside the cells during treatment with chromium(VI). It was thought that the difference obtained between these cell lines may arise from changes in the elimination of active oxygen species in the resistant ceils. Thus, we examined the antioxidant capacity in C H O l~ and CHO p cells, as summarized in Table 2. There was no difference in the content of proteins per cell between the two cell lines (data not shown). Catalase activity in the resistant cells was 2-fold and the cellular content of glutathione was about 3-fold that in parental ceils, while the activity of SOD and glutathione reductase did not differ between the two cell lines. These results were similar to those of previous studies in hydrogen peroxide-resistant cell lines (Sawada et al., 1988; Spitz et al., 1990). On the other hand, the content of antioxidant vitamins such as ascorbic acid and a-tocopherol was lowered by about 10 and 20%, respectively, in the A untreated

resistant cells. Thus, it would appear that catalase and glutathione play a role in protecting the resistant ceils against oxidative damage. With respect to chromium(VI), chromium(V) formed during reduction of chromium(VI) has been shown to be a reactive intermediate and to induce DNA breaks in vitro, through active oxygen species (Aiyar et al., 1989, 1990; Jones et al., 1991; Shi and Dalai, 1990a,b). We have previously shown using ESR spectrometry that a paramagnetic chromium(V) was formed following treatment with chromate in Chinese hamster V79 ceils, and that the levels of this intermediate in the cells were strongly related to the levels of DNA single-strand breaks produced by chromium(VI) (Sugiyama et al., 1989a,b, 1991a,b). Therefore, the formation of chromium(V) in both cell lines was examined. As shown in Fig. 3, the treatment of ceils with 400/zM Na2CrO 4 at room temperature resulted in the appearance of the ESR signal of chromium(V) complex with a maximum peak at g = 1.978. The ESR signal of chromium(V) increased proportionally with the time of exposure to chromium(VI) in both CHO p and CHO R cells. However, as was the case with DNA breakage, the level of chromium(V) was

B -

untreated

2min

2rnin

4.5min 4.5min ~ 7.0min

7.0rain ~ 9.5rain 9.5rain 12.0rain

12.0min

14.5rain

14.Srnin

I

f

lOG

~, g:1.978

g:1.978

Fig. 3. ESR spectra of chromium(V) complex in cells. ESR spectra were recorded during the incubation (2-14.5 min) of CHO P (A) and CHO R (B) cells with 400 ~ M Na2CrO 4 at room temperature.

101

about 2-fold lower in the resistant cells, indicating that the cellular reductive metabolism of chromium(VI) might be changed in the resistant cells. Previous studies have shown that the increase in intracellular vitamins such as a-tocopherol and ascorbic acid resulted in decreased cellular levels of chromium(V) (Sugiyama et al., 1989a, 1991a). In vitro studies have also shown that glutathioneand NADPH-dependent flavoenzymes such as glutathione reductase are capable of reducing chromium(VI) to form chromium(V), which reacts with H 2 0 2 to generate hydroxyl radicals (Aiyar et al., 1989, 1990; Jones et al., 1991; Shi and Dalai, 1990a,b,c). However, in resistant cell lines, the present results showed that glutathione levels were increased and the vitamin contents or the activity of glutathione reductase were slightly decreased or unchanged. Therefore, it is difficult to explain the mechanism of the inhibition of chromium(V) generation in the resistant cells at the present time. Studies are currently in progress using ESR spectrometry to evaluate what kind of antioxidants and chromate reductants, including catalase and glutathione, play a role in the formation of chromium(V) in intact cells. In conclusion, the present results showed that chromate-induced DNA single-strand breaks as well as the formation of chromium(V) were decreased in hydrogen peroxide-resistant cell lines, while the cytotoxicity of this metal was not affected, indicating that active oxygen species a n d / o r chromium(V) generation inside the cells might be associated with DNA strand breaks, but not with the cytotoxicity of chromium(VI). A cross-resistance was observed with respect to DNA breaks and cytotoxicity produced by toxic metals such as mercury(II) and cadmium(II).

Acknowledgements This work was supported in part by the Fukuoka Cancer Society, Ishibashi Research Foundation, Uehara Memorial Foundation, and Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. The authors would like to thank Mie Kitajima for excellent secretarial assistance.

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102 ity and metabolism of 4-hydroxy-2-nonenal and 2-nonenal in H202-resistant cell lines, Biochem. J., 267, 453-459. Sugiyama, M. (1992) Role of physiological antioxidants in chromium(VI)-induced cellular injury, Free Radical Biol. Med., 12, 397-407. Sugiyama, M., A. Ando, H. Furuno, N.B. Furlong, T. Hidaka and R. Ogura (1987) Effects of vitamin E, vitamin B 2 and selenite on DNA single strand breaks induced by sodium chromate(VI), Cancer Lett., 38, 1-7. Sugiyama, M., M. Costa, T. Nakagawa, T. Hidaka and R. Ogura (1988) Stimulation of polyadenosine diphosphoribose synthesis by DNA lesions induced by sodium chromate in Chinese hamster V-79 cells, Cancer Res., 48, 1100-1104.

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