Dual mode cytotoxicities of reactive oxygens on L5178Y radiosensitive mutant M10

Free Radical Biology & Medicine, Vol. 13, pp. 299-304, 1992 0891-5849/92 $5.00 + .00 Printed in the USA. All rights reserved. Copyright © 1992 Pergamon Press Ltd.

Original Contribution

D U A L M O D E C Y T O T O X I C I T I E S O F R E A C T I V E O X Y G E N S

O N L 5 1 7 8 Y R A D I O S E N S I T I V E M U T A N T M 1 0

ATSUSHI ITO and KUNIO SHINOHARA

Department of Radiation Research, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113, Japan

(Received 22 August 1991 ; Revised 2 March 1992; Accepted 21 April 1992)

Abstract--Cytotoxic effects of O2 and H202 on mammalian cells were investigated in comparison with the relative sensitivity of mouse L5178Y cells and its radiosensitive mutant M 10. Both O~ and H202 exhibited two different modes of cytotoxic actions depending on their exposure rates: At a high exposure rate (4.3 nmol of O2/mL/min), M10 was more sensitive to O7 than L5178Y normal type cell, in agreement with the case of X-rays; while at a low exposure rate (five times less than the high exposure rate), M 10 became more resistant than L5178Y. Similar results were obtained with H202. Reactive species responsible for these two different cytotoxic actions were examined with special reference to the metal-catalyzed Haber-Weiss reaction, by using a metal chelator, 1,10-phenanthroline, and an "OH scavenger, dimethylsulfoxide (DMSO). Significant protection by l, 10-phenanthroline was observed, which indicates the presence of a metal-dependent process in both cytotoxic actions. DMSO showed a marked protective effect, except for the case of M l0 exposed at the low exposure rate, in which DMSO showed no protection. The resistance of M l0 to 02 and H202 observed at the low exposure rate suggests the possibility that reactive species other than "OH are involved in the cytotoxicity.

Keywords--Superoxide, Hydrogen peroxide, Hydroxyl radical, Radiosensitive mammalian mutant cell, Radical scavenger, Metal chelator, Metal-catalyzed Haber-Weiss reaction, Cytotoxicity, Free radicals

INTRODUCTION

In the elucidation of biological effects of H202, comparison with the effects of X-rays has been an issue of wide interest, ranging from molecular alterations to cellular effects, because both agents are considered to produce a common initial reactive species, "OH. The significant protection by "OH scavengers against H202-induced DNA single-strand breaks 1-4 and lethality 2,5 is the major evidence for this notion in mammalian cells.

Product analysis of damaged DNA has revealed the different effects of H202 and X-rays at molecular level: H202 produces greater numbers of single-strand breaks ''3 and double-strand breaks 6 per lethal event than X-rays and produces no DNA-protein cross- links, ~,4 in contrast to X-rays. 7

For cytotoxic effects on mammalian cells, cellular sensitivity to H202 has been compared between normal cells and X-ray-sensitive mutant cells. Ataxia-tel-

Address correspondence to: A. Ito, Department of Radiation Re- search, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan.

angiectasia (AT) cells were found to be not sensitive to H202 in four different cell lines, 8 while Shiloh et al. found that in the low survival region AT cells became more sensitive than normal ce l l s . 9 However, the re- duced inhibition of DNA synthesis compared to normal cells after X-irradiation was not observed at least in the survival close range of H202 treatment in both cases. Recent studies on mouse L5178Y mutant L5178Y-S and L5178Y-R having different radiosensitivity indicated that a radioresistant mutant L5178Y-R was more sensitive to H202 than a radiosensitive mutant L5178Y-SJ °'11 This result was consistent with the twofold amount of catalase in L5178Y-S compared with L5178Y-R. These studies suggest that H202 has a different cytotoxic mechanism in mammalian cells from those of X-rays.

We took a similar strategy in examining the cytotoxic effects of O~- or H202 by using X-ray-sensitive mutant M 10 of mouse L5178Y. Although the reason for hypersensitivity of M 10 to X-rays has not been clarified yet, M 10 was found to have a low glutathione content (about 60% of that in the normal cells) and to lack inhibitory factors against lipid peroxidation in cytosols.'2 In this respect, it is of particular interest to

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300 A. l'ro and K. SHINOHARA

examine the response of M I0 to oxidative stress in comparison with X-rays. In the previous report, we found that M 10 was more resistant to O2 and H202 than L5178Y when exposed at a low exposure rate, while at a high exposure rate M l0 was more sensitive than L5178Y in accordance with X-rays. 5 The exposure-rate-dependent change in the relative sensitivity of L5178Y and M 10 suggested that two distinct mechanisms are operating in the killing of O5 or H202: One is similar to that of X-rays and the other is unique to 05 or H202. In the present study, we further analysed the mechanisms underlying two different cytotoxic actions of 02 or H202 by using a radical scavenger and a metal chelator.

MATERIALS AND METHODS

Cell culture

Mouse leukemic cells, L5178Y, and radiosensitive mutant M 10 were grown in Fischer's medium supple- mented with 10% horse serum. Exponentially grow- ing cells (2-3 × 105 cells/mL) were used in all experiments. M I0 was kindly supplied by Dr. K. Sato, who originally isolated this mutant. 13

02, H202, and X-ray treatment

Cells were exposed to O£ generated from the hypoxanthine-xanthine oxidase system. Hypoxanthine is from Wako Chemical, Tokyo, and xanthine oxidase (grade IID is from Sigma Chemical Co. (St. Louis, MO). Three concentrations of xanthine oxidase were used: 0.01 units/mL for a low exposure rate, 0.05 units/mL for a high exposure rate, and 0.017 units/ mL for an intermediate exposure rate. The production rate ofO2 measured by the cytochrome c method was 0.86 nmol/mL/min for 0.01 units/mL. The amount of 05 produced was calculated following the formula presented by Johnston, Jr. ~4 05 treatment was started by adding xanthine oxidase to cell suspension containing 2 mM hypoxanthine. Cells were treated with H202 of concentrations ranging from 40 uM to 75 uM. X-rays, effective energy of 48 keV, were obtained from X-ray irradiation apparatus (MBR- 1505R, Hitachi, Tokyo, Japan) with a dose rate of about 80 R/min. All the experiments were carded out in Fischer's medium with 10% horse serum at am- bient temperature.

The surviving fraction of the cells was determined by colony-forming assay.15

Chelator and scavengers

Dimethylsulfoxide (DMSO, Wako Chemical, To- kyo) as an "OH scavenger and 1,10-phenanthroline

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Fig. I. Dose-survival curves for L5178Y normal cells and M 10 cells irradiated with X-rays. O: L5178Y; A: MI0.

(Wako Chemical, Tokyo) as a metal chelator were used because of their nature of entering cells. 2,16 Cata- lase and SOD are from Sigma. We added these agents into cell suspension 5 min before the 02 or H202 treatment.

RESULTS

Effects of X-rays on L5178 Y normal and M10

Figure 1 shows dose-survival curves for L5178Y and MI0 exposed to X-rays. M10 was 3.7 times more sensitive than L5178Y when the sensitivity was compared on the basis of the dose to give 10% survival (Di0).

Effects of 02 and H202 on L5178 Y normal and M10

Survival curves of L5178Y and MI0 treated with O5 of the three different exposure rates are shown in Figure 2. At the lowest exposure rate (0.01 units/mL), M 10 was found to be more resistant than L5178Y in contrast to X-rays. As the exposure rate increased, the relative sensitivity of the two cell lines became close. At the highest exposure rate (0.05 units/mL), opposite to the results at the lower exposure rate, M 10 was

Toxicity of reactive oxygen 301

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enhancement of the inactivation by 02 . At the high exposure rate, the lack of SOD enhancement is probably attributed to the occurrence of the rapid dispro- port ionation of 0 7 to H202 due to the high density of it.

Effects of 1, lO-phenanthroline on the inactivation of L5178Y normal and M10

l, 10-Phenanthroline exerted marked protective effects on all four cases tested (i.e., low and high exposure rates in LS178Y and M10; Fig. 5). 1,10-Phen- anthroline did not affect the production of 02 measured by the cytochrome c method. Therefore, these results demonstrate an important role of metal ions in the cell killing.

T r e a t m e n t t ime (min)

Fig. 2. Treatment time-survival curves for L5178Y and M 10 cells exposed to 02 with different exposure rates. Exposure rate is changed by using different concentration of xanthine oxidase in the unit ofunits/mL. ©, A: 0.01 units/mL; ~, A: 0.017 units/mL; O, A: 0.05 units/mE Circles denote the results of L5178Y, and triangles denote those of M 10.

more sensitive than L5178Y, in accordance with the case of X-rays.

Figure 2 was replotted into Figure 3 to show survival against accumulative O2 dose in the unit of production rate × exposure time (nmol/mL). In contrast with the independence of L5178Y in an exposure rate, it is clear that M l 0 became more readily inacti- vated as the exposure rate increased. Table 1 shows the relative sensitivity o f M l 0 and L5178Y exposed to O2 in the two different exposure rates. The ratio of Dso (the dose giving 50% survival) of L5178Y and M l0 was described in four separate experiments. These results confirmed the change in the relative sensitivity of L5178Y and M l0 between the high and low exposure rates.

H202 treatment gave the similar results to 0 2. The survivals at 5-rain treatment were shown in Table 2. The qualitatively similar concentration dependence of the relative sensitivity of the two strains was observed: At the highest concentration, 75 uM, M 10 was more sensitive than L5178Y, while at the lowest concentration, 30 #M, LS178Y had a higher sensitivity than M 10.

The similarity in the effects of O2 and H202 can be explained by the disproportionation of O2 into H202 . As shown in Figure 4, complete inhibition of OE-induced inactivation on L5178Y by catalase indicated that the effects of exogenously added O2 are attribut- able to those of H202 at both exposure rates. Further evidence for this explanation was obtained from SOD

Effects of DMSO on the inactivation ofL5178 Y normal and M10 by Or or H202

Table 3 compiles the effects of DMSO on the inactivation of L5178Y and M I 0 by O2 and H202. The ratio of Ds0 in the presence and absence of DMSO was used as an indication of the degree of protection. Ex- cept for the case of MI0 at the low exposure rate, DMSO protected the inactivation by O7 efficiently, indicating involvement of "OH probably generated via the metal-catalyzed Haber-Weiss reaction. Com- parison of the degree of protection at the high expo-

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Fig. 3. Exposure rate effects of O7 treatment on L5178Y and M 10. Survival was plotted against accumulative Or dose (exposure rate × treatment time). ©,/x: 0.01 units/mL; O, A: 0.017 units/mL; O, A: 0.05 units/mE Broken lines with circles represent the curve for LS178Y, and solid lines with triangles represent curves for M 10.

302 A. ITO and K. SHINOHARA

Table 1. Effects of O] with Low and High Production Rates

Dso(L5178Y)/Dso(M 10)

XO Concentration O7 Concentration Exp. 1 2 3 4 Mean

0.05 units/mL 4.3 nmol/mL 1.25 1.0 1.22 1.20 I. 17 __+ 0.10 0.01 0.86 0.54 0.51 0.74 0.59 0.60 _+ 0.10

sure rate between L5178Y and M10 indicated that M10 was more difficult to be protected by DMSO than L5178Y. This cellular response to DMSO was more markedly observed at the low exposure rate: The protection in M 10 was hardly detected.

In H202 experiments, M 10 was also hardly protected against inactivation at the low concentration. In addition, DMSO treatment alone did not cause any cellular lethality under the longest treatment time in the present experiments.

DISCUSSION

We found two modes in lethal effects of Or or H202 on M 10. Either effect could be dominantly exhibited depending on the exposure rate. At the high exposure rate, M 10 was more sensitive to O2 o r H 2 0 2

than L5178Y in accordance with the case of X-rays. On the other hand, at the low exposure rate, L5178Y became more sensitive than M10 in contrast to the case of the high exposure rate. The results of Shiloh et al. 9 seem consistent with our results, although experi- mental procedure was different: They exposed AT cells to H 2 0 2 with the varying treatment concentrations under the constant treatment time (l h). The concentration above which AT cells became more sensitive than normal cells was approximately 0.5 #g/ mL (i.e., 15 #M in their experiments), while in the present results it was 30 #M, which is quantitatively close to the value by Shiloh et al. 9 Bou~yk et al. found that at higher concentration of H202 the difference of sensitivity between L5178Y-R and L5178Y-S became small.~ ' However, this phenomenon may not be

Table 2. Concentration Effects of H202 on L5178Y and M10

H:O 2 Concentration Survival at 5 Min (uM) Cell Treatment (%)

30 L5178Y 68 MI0 82

50 L5178Y 15 MI0 21

60 L5178Y 7 M10 6

75 L5178Y 2.6 M10 0.95

comparable, because their results were obtained by the use of 2 mM H202, 10 to 50 times higher than in the present experiments, and also because their detec- tion method of cell death was the dye exclusion test.

The sensitivity of M 10 decreased with decreasing exposure rate, whereas L5178Y showed nearly the same sensitivity in the wide range of exposure rate tested (Fig. 3). In particular, the resistance of MI0 observed at the low exposure rate seems to be explained neither by the lack of inhibitory factors against lipid peroxidation found by Nakazawa et al. 12 nor by the low glutathione content in M 10. This evidence may be discussed with respect to the reactive species responsible for cell killing.

Reactive species

In the case of L5178Y and M10 at the high exposure rate, the protection by 1,10-phenanthroline and

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Fig. 4. Effects of catalase and SOD on O~- induced inactivation of L5178Y. O, A, D: 0.01 units/mL; e , A, B: 0.05 units/mL. O, e : control;/% A: catalase (200 #g/mL); I-7, B: SOD (200/zg/mL).

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Toxicity of reactive oxygen 303

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Fig. 5. Effects of 1,10-phenanthroline on O] induced inactivation ofL5178Y (a) and MI0 (b). ©, A, tO: 0.01 units/mL; O, A, B: 0.05 uni ts /mE Open symbols indicate data for control, triangles for 5 uM, and squares for 20 uM.

DMSO suggests that "OH produced via the metal-catalyzed Haber-Weiss reaction is the major initial species causing the lethal effects.

A model of the different spatial distribution of "OH produced by X-rays and H202 was proposed by Ward et al. to explain the different number of DNA single-strand breaks produced per lethal event by both agents. 3 According to their model, X-rays produce the small volumes (spurs, etc.) of dense "OH radicals. Consequently, the area would be filled with DNA damage, such as strand breaks and base damages,

and was named Locally Multiply Damaged Site (LMDS). 17 On the other hand, H202 was thought to produce "OH singly at the sites of metals at 4°C. As speculated by Ward et al., it is possible that at room temperature even H202 produces "OH in the form of LMDS like X-rays. Therefore, the observation of the relative sensitivity of L5178Y and M l0 similar to the case of X-rays at the high exposure rate at room temperature suggests the existence of a common pathway to cell death between H202 and X-rays initiated by the same initial species, including its spatial distribution.

At the low exposure rate, the participating species in M l 0 killing must be also produced via a metal-dependent process. The lack of DMSO protection may lead to the following two interpretations on the reactive species: One is that the production of "OH be- comes "site specific" where the distribution of "OH is localized in the vicinity of metal-bound target mole- cules. Such a situation would cause the limited protection by "OH scavengers. 18'~9 Consistent with this interpretation, in L5178Y less DMSO protection was also observed at the low exposure rate than at the high exposure rate (Table 3). However, it seems unlikely that site specificity explains the inefficient cell killing observed for M 10 at the low exposure rate.

The second explanation is that the contribution of reactive species other than "OH increases in the region of low exposure rate. Reactive species other than "OH have been suggested in some cases. 2°-22 In particular, since ferryl radical is less oxidizing than "OH 23 and is preventable by a metal chelator, it would be one of the most probable species in the present case. In either explanation, availability of active form of metal ions such as Fe E+ in L5178Y and M10 would be one of the critical factors to produce these reactive species.

In conclusion, a unique initial process leading to cell death by H20 2 was revealed through a radiosensitive mutant M l0 in comparison with that of X-rays.

Table 3. Effects of DMSO on 02 and H202 Induced Inactivation

Dso(+DMSO)/Dso(-DMSO)

XO DMSO Concentration (M) Cell Concentration 0 0.5 1.0 1.4

L5178Y 0.01 units/mL 1 1.24 1.48 1.50 0.05 1 1.45 1.66 1.97

M10 0.01 1 0.82 0.78 0.78 0.05 1 1.36 1.40 1.5 [

H202 Concentration

L5178Y 40 tzM 1 1.0 1.7 2.0 75 l 1.32 1.89 - -

M l 0 40 1 1.0 1.0 1.0 75 l 1.25 1.40 1.75

304 A. |TO and K. SHINOHARA

Particularly, the cytotoxic effects of H202 at the low exposure rate were found largely different f rom X- rays. Recently, the i n v o l v e m e n t of some physiological

changes in the lethali ty caused by H202 has been re- ported (i.e., the increase of in t racel lu lar Ca 2+ level

shown by the suppress ion of cell kil l ing by ca lc ium chelator, Qu in -2 [Ref. 24], and the decrease of cellular

A T P level a l though the concen t r a t i on of H202 for t r ea tmen t was rather high). 25-27 The two modes of cell

kil l ing presented here should be fur ther s tudied in this respect.

Acknowledgement--We thank Dr. K. Sato for supplying M10.

REFERENCES

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3. Ward, J. F.; Blakely, W. F.; Joner, E. I. Mammalian cells are not killed by DNA single-strand breaks caused by hydroxyl radicals from hydrogen peroxide. Radiat. Res. 103: 383-392; 1985.

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ABBREVIATIONS

AT--a tax ia - t e l ang iec tas i a D M S O - - d i m e t h y l s u l f o x i d e Hz 02 - - h y d r o g e n peroxide L M D S - - l o c a l l y mul t ip ly damaged site O 2 - - s u p e r o x i d e a n i o n • O H - - h y d r o x y l radical S O D - - s u p e r o x i d e d i smutase

Dual mode cytotoxicities of reactive oxygens on L5178Y radiosensitive mutant M10

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