DNA strand breakage, cytotoxicity and mutagenicity of hydrogen peroxide treatment at 4°C and 37°C in L5178Y sublines

Fundamental and Molecular Mechanisms of Mutagenesis

ELSEVIER

Mutation Research 308 (1994) 233-241

DNA strand breakage, cytotoxicity and mutagenicity of hydrogen peroxide treatment at 4°C and 37°C in L5178Y sublines Marcin Kruszewski a,,, Michael H.L. Green b, Jillian E. Lowe b, Irena Szumiel a "Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-195 Warsaw, Poland b MRC Cell Mutation Unit, Universityof Sussex, Brighton BN1 9RR, UK

Received 23 July 1993; revision received 18 January 1994; accepted 26 January 1994

Abstract

Cells from the L5178Y routine lymphoma subline LY-R are twofold more resistant to killing by ionizing radiation than the subline LY-S. In contrast, LY-R cells are more sensitive to killing by hydrogen peroxide. Cells of the two sublines in logarithmic growth phase were treated with hydrogen peroxide in phosphate-buffered saline for 1 h at 4°C or 37°C. From the comparison of D Ovalues it followed that at 37°C LY-R were 3.6 times more sensitive to the killing effect of H 2 0 2 than LY-S cells; at 4°C they were 11 times more sensitive. Treatment with hydrogen peroxide at 4°C gave a considerable sparing effect, which was substantially greater for the LY-S subline; for LY-S cells D Owas 5.7 times lower at 37°C than at 4°C, for LY-R cells only 1.9 times. The mutation frequency (HGPRT) in LY-R cells was increased in proportion to H 2 0 2 concentration and was the same at both treatment temperatures. In contrast, mutation frequencies initially increased, then decreased with increasing H 2 0 2 concentration in LY-S cells treated at 4 or 37°C. The concentration at which the decline was initiated was higher at 4 than at 37°C. DNA damage after H 2 0 2 treatment (both temperatures, 5 min) was estimated from the 'comet' assay (single-cell gel electrophoresis). The initial damage, but not the residual damage, differed significantly in LY sublines. A period of slower repair (between 3 and 10 rain) was found in LY-R cells. Key words: Hydrogen peroxide; Single-cell gel electrophoresis; L5178Y murine lymphoma sublines

1. Introduction Pairs of cell lines, usually parental line and mutant, that differ in sensitivity to D N A - d a m a g ing agents are a convenient tool for examination of molecular and cellular mechanisms of sensitivity (e.g. Sato et al., 1986; Jaworska et al., 1987; Spitz et al., 1989; Bou£,yk et al., 1991; Vaughan

* Corresponding author.

and Gordon, 1992). In this respect, the pair of L5178Y (LY) sublines is exceptional, because of the unique inverse cross-sensitivity to X-rays and hydrogen peroxide. The high sensitivity of LY-S cells to X-rays (D = 0.5 Gy) is reasonably explained by the impairment of double-strand break (DSB) rejoining (Wlodek and Hittelman, 1987). Another cell line with a similar repair defect, xrs-5, derived from Chinese hamster K1 cells, is susceptible to X-rays and hydrogen peroxide, and the latter feature is explained by the same deft-

0027-5107/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0027-5107(94)00036 -5

234

M. Kruszewski et al. / Mutation Research 308 (1994) 233-241

ciency in DNA repair (Jeggo and Kemp, 1983; Prise et al., 1989; Vaughan and Gordon, 1992). Also, a mutant derived from L5178Y lymphoma, M10, and two mouse mammary carcinoma mutants, SX9 and SX10, described by Sato et al. (1986) are cross-sensitive to hydrogen peroxide and X-rays. The considerable difference in the lethal effect of hydrogen peroxide treatment in LY sublines (BouZ'yk et al., 1991) and the inverse cross-sensitivity to X-rays are not compatible with this uniform picture and point to other cellular features as equally important in the response to oxidative damage. The purpose of this work has been to analyze the nature of such features and to connect the initial DNA damage and its repair with the damage manifested at the cellular level. Hydrogen peroxide produces different effects at different doses with respect to cell death type (Bou2yk et al., 1991); longer treatment intervals are advisable in order to mimic 'natural' exposure to oxidants, whereas DNA repair experiments require pulse treatment. With the different endpoints examined (survival, DNA damage and repair, mutation frequency), using methods with varying degrees of sensitivity, it is unavoidable to deal with various doses of hydrogen peroxide in order to obtain the optimal damage range for each method. Consequences of this awkward requirement are somewhat mitigated by the fact that we compare relative responses in two cell sublines rather than the absolute relations between molecular, subcellular and cellular levels of damage.

2. Materials and methods

Cell cultures Murine leukemic lymphoblasts LY-R and LY-S were maintained in suspension cultures in Fischer's medium supplemented with 8% bovine serum, as described by Szumiel (1979). Asynchronous populations in exponential phase of growth were used in all experiments. Treatment with H2O 2 In survival and mutation frequency experiments cells were centrifuged, resuspended in

phosphate-buffered saline (PBS; 10 ml; 2 x 105 cells/ml) and hydrogen peroxide was added for 1 h in the amount appropriate to produce the desired concentration. After incubation at 4°C or 37°C 1 ml of calf serum was added, cells were centrifuged and resuspended in Fischer's medium. For cell survival estimations cells were plated out immediately after treatment, whereas for mutation frequency 7 days incubation was allowed for expression before plating out (see below). To minimize repair processes, in the 'comet' assay the cells (10 ml, 2 x 105 in PBS) were treated with hydrogen peroxide for only 5 min at 4°C or 37°C and 1 ml of calf serum was added. Following the treatment, the cells were centrifuged and resuspended in RPMI 1640 without phenol red and immediately processed, as described in section 2.4, or resuspended in Fischer's medium, left at 37°C for repair intervals indicated in Fig. 5 and then processed, as described below. Estimation of survival and mutation frequency Survival was estimated from cloning in soft agar (Szumiel, 1979). Survival curves were fitted to a linear model. The mutation frequency of the HGPRT locus was determined as described by Knaap and Simons (1975). Treated cells were grown in Fischer's medium for 7 days to achieve mutation expression (Knaap and Simons, 1975). The total cell number per sample was always greater than 2 x 10 7. Subsequently, cells were plated in soft agar with 5 ~zg/ml 6-thioguanine as the selecting factor. According to the expected mutation frequencies of LY-R and LY-S cells, 2 X 10 6 and 3 X 106 cells per Petri dish (10 cm diameter) were plated; in parallel, the same cells were plated in non-selecting medium on 5 cm diameter Petri dishes to determine the plating efficiency. 'Comet' assay The 'comet' assay was performed as described by Singh et al. (1988) with slight modifications by Green et al. (1992). Image analysis of data was by the Fencomet from Fenestra Vision v. 1.4 software package (Confocal Technologies, Liverpool, UK). This package determines a variety of parameters, including tail moment, computed as

M. Kn~zewski et a l . / Mutation Research 308 (1994) 233-241

described by Olive et al. (1990). Fifty cells were scored per experimental point. Slides with treated cells were placed in ice-cold lysing solution (2.5 M NaC1, 100 mM EDTA, 10 mM Tris, 1% sodium lauryl sarcosine, 1% Triton X-100, 10% DMSO, pH 10) for 1 h at 4°C. Then slides were placed in a horizontal gel box in electrophoresis buffer (300 mM NaOH, 1 mM EDTA) and left for 40 min for unwinding at 10°C. Electrophoresis (24 min, 20 V) was carried out at 10°C. All steps of the 'comet' assay preceding electrophoresis were per-

235

formed on ice to prevent repair and effects of metabolic processes.

Statistical analysis Significance of differences between data sets was determined by multiply pairwise comparisons of means using two-tailed Student's t-test. To determine the extent of intracellular heterogeneity within the pooled data, the ratio of the range to standard deviation (SD) was calculated: values which are below 2 or above 6 indicate that data

Table 1 Summary of numerical data characterizing the response of LY sublines to hydrogen peroxide treatment

Feature

L5178Y-R 37oc

Survival D o [/zM]

Mutation data Control ( n u m b e r of experiments) Effective survival N u m b e r of m u t a n t s per plate

1.8

L5178Y-S 4oc 3.4

37°C

4oc

6.5

37.4

M F per 10 s survivors

4 0.22 + 0.02 29.61 + 2.13 2.56 + 0.31

4 0.26 + 0.02 27.94 + 1,70 2.31 -1- 0,30

4 0.20 + 0.02 43.12 + 1.24 3.01 5- 0.21 ##

4 0.19 + 0.02 24.86 + 1.20 1.95 + 0.56 ##

H 2 0 2 0.1 IzM ( n u m b e r of experiments) Effective survival N u m b e r of m u t a n t s per plate M F per 105 survivors

3 0.16 + 0.01 27.85 5- 3.35 2.14 5- 0.36

3 0.23 + 0.01 27.55 5- 2.45 2.05 5- 0.24

3 0.20 + 0.01 48.24 + 4.87 3.79 5- 0.51

3 0.21 + 0.02 29.83 5- 1.89 2.64 _+ 0.34

H 2 0 2 1 / z M ( n u m b e r of experiments) N u m b e r of m u t a n t s per plate M F per 105 survwors

3 0.16 + 0.02 27.24 5- 3.42 2.40 + 0.35

3 0.06 + 1 - 3 23.63 5- 4.21 2.11 + 0.29

3 0.05 5- 1 - 3 25.63 5- 2.58 1.78 + 0.10 ##

3 0.12 + 1 - 3 45.80 + 4.55 3.04 + 0.32 ##

H 2 0 2 5 / ~ M ( n u m b e r of experiments) Effective survival N u m b e r of m u t a n t s per plate M F per 105 survivors

3 1 - 3 5- 1 - 4 44.63 5- 4.68 4.02 + 0.52

3 0.01 5- 3 - 4 43.41 5- 3.27 3.68 + 0.21

3 6 -4 5- 2 -5 ND ND

ND ND ND

H 2 0 2 1 0 / z M ( n u m b e r of experiments) Effective survival N u m b e r of m u t a n t s per plate M F per 105 survwors

3 1 - 4 5- 2 -5 60.90 5- 7.23 4.91 5- 0.32 ##

3 3 5 -3 + 1-4 4 - 4 5- 2"5 103.09 + 11.49 ND 8.63 5- 0.74 ##** N D

3 0.05 -t- 2 -3 53.66 + 10.15 4.63 + 1.07 **

H 202 50/~ M ( n u m b e r of experiments) Effective survival N u m b e r of m u t a n t s per plate M F per 10 s survwors

ND ND ND

ND ND ND

3 0.02 + 1 - 3 31.23 + 4.67 2.83 + 0.56

Effective survival

ND ND ND

All data are mean + SE. ND, not determined. ** Significant difference LY-R vs. LY-S, 0.01 < p < 0.05; ## significant difference 37"C vs. 4°C, 0.01 < p < 0.05.

M. Kruszewski et al. / Mutation Research 308 (1994) 233-241

236

L~

1.01

4--'

"~

ol

o Un c-

LY- R

1.0E-2

•

~'

m

4o0

•

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zx--zx

7:

•--a

37°C

~'

4oc

]

/

370C

LY-S

(23 1.0E-4

T

8-

4Oc

A

:_~ I.OE-3

1.0E-5

10"

o 91

O

"5 li

37oc '

'

' .....

" i

3~

:

:

:

::::~1

,

10

,

:

:::::;

0~

:

: ::::::'.m #:

: ::::::'.

"

; ::::::1

: :::::::

:

: ::::::~

100 Control

H202 [/zM] Fig. 1. Survival of LY-R and LY-S cells treated with H 2 0 2 at 4°C or 37°C. M e a n results from 3 - 4 experiments; standard error indicated where larger than the point plotted.

O. 1

1

10

H202 E/zM~ Fig. 2. Mutation frequency at the H G P R T locus in LY-R cells treated with H 2 0 2 at 4°C or 37°C. Mean results from 3 - 4 experiments; standard error indicated where larger than the point plotted.

are extremely homo- or heterogeneous, respectively.

3. Results

Fig. 1 presents dose-survival curves of LY cells treated with H 2 0 2 for 1 h at 4°C or 37°C. As seen in the figure, LY-S cells were consistently more resistant to H 2 0 2 treatment than LY-R cells, and the lethal effect of the treatment was higher at 37°C than at 4°C. First order regression for all data points could be fitted only in a double-log plot. Nevertheless, values of D O from a log-linear plot (only for the data points in the concentration range 1-50 /zM hydrogen peroxide, as those for lower concentrations did not fit) are given in Table 1, with other numerical data, characterizing the response of LY sublines to hydrogen peroxide treatment. While the fit for LY-S cells treated at 37°C was very good in the double-log plot (r = 0.99), that in the log-linear plot was poor (r = 0.83). From the comparison of D Ovalues (Table 1) it followed th~at at 37°C LY-R were 3.6 times more sensitive to the killing effect of H 2 0 z than LY-S cells; at 4°C they were 11 times more sensitive. Treatment at 4°C gave a considerable sparing effect as compared with that at 37°C: for LY-S cells D Owas 6.7 times lower at 37°C than at 4°C, and for LY-R cells the D O ratio was 1.9. Mutation frequencies for the above treatment schedules were estimated at the H G P R T locus by

selection in 6-thioguanine-containing Fischer's medium. Fig. 2 shows the dose-dependent increase in mutants of LY-R cells treated with H 2 0 2 at both temperatures. With the exception of 10/xM H 2 0 2 , mutant frequencies were identical at 4°C and 37°C. The pattern of mutation frequencies in LY-S cells is strikingly different (Fig. 3). A slight increase with increasing H 2 0 2 concentrations at both temperatures of treatment was followed by a decrease at higher concentrations; at 4°C the decrease took place above 10 /zM, whereas at 37°C it occurred above 0.1 /zM. Table 1 presents the respective numerical data and their statistical evaluation.

10-

o 9. "~ tt~ Cb

8.

o--0

37°C

e--e

4oc

6, 1

13t:: O

o

•

o

2+ 0

,

1,----,m

Control

0.1

1

10

H202 [/zM] Fig. 3. Mutation frequency at the H G P R T locus in LY-S cells treated with H 2 0 2 at 4°C or 37°C. M e a n results from 3 - 4 experiments; standard error indicated where larger than the point plotted.

M. Kruszewski et al. / Mutation Research 308 (1994) 233-241

DNA damage was estimated in H2Oz-treated LY cells with the use of the 'comet' (single-cell gel electrophoresis) assay. Tail moment (amount

237

of DNA in the 'comet' tail multiplied by the tail's length, Olive et al., 1991) or relative tail moment, calculated by dividing by the tail moment of the

Table 2 Summary of numerical data characterizing the response of LY sublines to hydrogen peroxide treatment Feature

LY-R

LY-S

37oc

4oc

37°C

4°C

6.16 ± 0.56 * 90.21 ± 9.00

5.80 ± 0.51 * 90.85 ± 9.12

8.13 ± 0.79 * 88.81 ± 8.90

8.10 ± 0.81 * 89.35 ± 8.91

17.03 ± 1.43 #** 82.83 ± 8.56

14.43 ± 1.36 #* 84.61 ± 8.56

13.29 ± 1.70 ~** 83.88 ± 12.07

8.90 ± 0.87 #* 88.03 ± 12.37

20.90 ± 1.98 #* 76.56 ± 7.81

16.99 ± 1.43 #* 80.47 + 8.39

16.18 ± 1.98 #* 79.63 ± 11.66

8.95 ± 0.90 #* 86.03 ± 11.08

29.51 ± 2.67 # 63.32 + 6.56

20.26 ± 1.74 # 74.52 ± 7.70

30.20 + 63.04 ±

20.68 _+ 1.95 # 73.76 ± 7.35

ND ND

71.39 ± 10.17 * 30.45 + 1.68

ND ND

61.98 ± 6.20 * 37.36 ± 3.72

9.63 + 0.45 88.95 ± 0.47

10.78 ± 0.53 * 86.21 ± 0.55

8.70 ± 0.37 88.21 + 0.50

7.77 ± 0.45 * 90.45 ± 0.52

27.77 + 1.20 # 63.91 ± 1.11

36.96 + 59.98 ±

1.52 #* 1.27

28.64 ± 64.33 ±

1.22 1.17

30.02 ± 63.61 ±

1.23 * 1.15

29.24 ± 2.04 #* 64.69 ± 2.07

37.09 ± 59.85 ±

1.58 #* 1.30

22.12 ± 73.99 +

1.34 * 1.58

23.79 + 68.68 ±

1.51 * 1.52

26.31 ± 1.58 ##* 68.20 + 1.44

30.95 ± 65.27 ±

1.29 ##* 1.05

17.00 ± 0.92 ~* 79.78 + 0.94

21.91 _+ 1.05 #* 71.83 ± 1.15

29.63 + 2.16 * 66.77 + 1.88

28.59 + 0.96 * 66.95 ± 0.92

14.86 ± 0.83 #* 82.17 ± 0.89

20.90 ± 75.68 ±

1.29 #* 1.27

24.24 + 2.40 * 73.21 + 2.19

24.03 + 1.00 * 71.45 + 0.96

13.99 ± 0.67 * 84.18 + 0.78

16.01 + 80.85 +

1.22 * 1.44

12.42 + 0.57 ## 85.58 + 0.49

10.88 + 0.45 ## 86.98 + 0.50

12.97 + 0.51 ## 85.70 + 0.60

11.52 + 0.48 ## 85.74 + 0.56

11.36 + 0.62 87.71 + 0.49

10.29 + 0.70 89.00 + 0.63

11.23 + 0.57 87.60 + 0.64

11.41 _+ 0.67 85.90 + 0.78

Initial damage Control Tail moment Head D N A 1/~M H 2 0 2 Tail moment Head D N A 5/~M H 2 0 2 Tail moment Head D N A 10/zM H 2 0 2 Tail moment Head D N A 50/~M H 2 0 2 Tail moment Head D N A

2.75 # 6.49

DNA damage repair Control Tail moment Head D N A 0 min Tail moment Head D N A 1 mln Tail moment Head D N A 3 mm Tail moment Head D N A 5 mm Tail moment Head D N A 10 mm Tail moment Head D N A 30 mm Tail moment Head D N A 60 mm Tail moment Head D N A

All data are mean + SEM. ND, not determined. * Significant difference LY-R vs. LY-S, p < 0.01; ** significant difference LY-R vs. LY-S, 0.01 < p < 0.05; # significant difference 37°C vs. 4°C, p < 0.01; '~'~ significant difference 37°C vs. 4°C, 0.01 < p < 0.05.

M. Kruszewski et al. / Mutation Research 308 (1994) 233-241

238

control, was taken as the measure of damage. Numerical data and their statistical evaluation are given in Table 2. Fig. 4 presents the initial D N A damage in L Y - R and LY-S cells exposed to hydrogen peroxide at both temperatures. In LY-S cells there was less damage at 4°C than at 37°C at all H 2 0 z concentrations; the same applied to LY-R cells with the exception of 1 ~ M H 2 0 2, however, the difference between the effect of H 2 0 2 at the two temperatures was smaller than that in LY-S cells. Owing to its sensitivity, the ' c o m e t ' method could be applied only to a limited range of H 2 0 2 concentrations: 50 /zM H 2 0 2 at 37°C was beyond this range, although damage inflicted at this concentration at 4°C was still measurable. Therefore, we could not confirm the bell-shaped relation of D N A damage vs. H 2 0 2 concentration described by Iliakis et al. (1992). Differences in initial D N A damage between LY sublines were smaller than could be expected from the differential susceptibility to H 2 0 2 ; nevertheless, at H 2 0 2 concentrations up to 10 p.M the differences were statistically significant (Table 2). The rate of D N A repair, measured by the same 'comet' assay, was very close in LY-S cells treated at 4°C and 37°C (Table 2 and Fig. 5); in LY-R cells the restoration of D N A structure was delayed with the most marked difference between 5 and 10 min of the repair interval, especially after treatment at 37°C. However, after 30 min the amount of residual damage was the same in

10 9 O

[~] 1

LY-S, 37°C LY-S, 4°C LY-R, 37°C LY-R, 4°C

0 7 =E 6 5 4

__

3 2 1

nIHH Control

1

n,i,l,jn 5

10

50

H202 [/~M] Fig. 4. Initial DNA damage to LY cells produced by H202 treatment at 4°C or 37°C. Mean results from two experiments _+range.

4.0-

(9

E

3.5-

A--zx

3.0-

A--&

LY-R

0--0

LY-S 3 7 ° C

0

2.5~

•~

20~

(9 .> -4-, 0

1,0

~

~AT

0

10

L Y - R 37°C

4°C

1.5

0.5 o.o

20

30

]~ime

[min]

40

50

60

Fig. 5. Repair of DNA damage in LY cells treated with H202 at 4°C or 37°C. Mean results from two experiments + range.

both sublines regardless of the t e m p e r a t u r e of treatment.

4. Discussion Cellular sensitivity to hydrogen peroxide may depend on several factors: (1) inherent 'protective enzyme' (catalase and glutathione peroxidase) activities, (2) extent of O H radical generation to which the initial D N A damage, critical for survival, is related, (3) D N A repair rate and fidelity. The contributions of these factors to the response of LY sublines to hydrogen peroxide treatment are discussed below. Protective enzymes

The potential importance of catalase for cellular sensitivity to hydrogen peroxide has been noticed in a series of stable hydrogen peroxide-resistant variants of Chinese hamster ovary ( H a - l ) cells, differing in ploidy and expression of catalase (Spitz et al., 1989). In this respect, the pair of LY sublines fit the picture, as LY-S cells, more resistant to hydrogen peroxide, have about twice higher catalase activity than L Y - R cells (Jaworska et al., 1987). On the other hand, LY sublines do not differ in glutathione peroxidase activity (Jaworska et al., 1987). Catalase may protect the cytoplasmic compartments, especially mitochondria, on whose A T P synthesis the cell's integrity is dependent when subject to oxidative stress (Halleck et al., 1992).

M. Kruszewski et al. / Mutation Research 308 (1994) 233-241

Also, the protective effect of catalase, if any, should be reflected in the initial DNA damage, determined after hydrogen peroxide treatment. As discussed below (also Table 2, Fig. 4), the overall damage (expressed as relative tail moment in the 'comet' assay) differs in LY sublines significantly enough to be explained by some protection by catalase.

Initial DNA damage In vitro, H202 induces DNA breaks (Ward, 1988; Ward et al., 1985, 1987; Prise et al., 1989; Mello-Filho and Meneghini, 1984; Sandstr6m, 1991). In the Haber-Weiss (or Fenton) reaction involving transition metal ions hydrogen peroxide gives rise to the highly reactive OH radical (Mello-Filho and Meneghini, 1984). Its reaction with DNA generates single-strand (SSB) (MelloFilho and Meneghini, 1984, 1985, 1991; Meneghini, 1988; Ward et al., 1985; Baker and He, 1991) and double-strand (DSB) (Prise et al., 1989) breaks and thymine glycol residues (Blakely et al., 1990). On the other hand, Ward et al. (1985) suggested that the cyto- and genotoxic effect of H 2 0 2 is mainly due to locally multiply damaged sites (LMDS). The 'comet' assay applied here detects DNA strand breaks (Singh eta!., 1988; McKelvey-Martin et al., 1993); it would also detect LMDS and alkali-labile sites, but base damage was beyond the scope of this study. A difference in the mean and relative tail moments (Table 2, Fig. 4) in hydrogen peroxidetreated LY cell populations does not exclude the existence of further, qualitative differences in DNA damage between LY-R and LY-S cells. Induction of base damage may differ and account in part for survival and mutational patterns of response. This aspect of H202-induced damage is under investigation. The extent of the Fenton reaction may depend on available transition metal ions and reducing equivalents. OH radicals are generated at sites containing reduced metal ions (Cu ÷, Fe2+). At temperatures that inhibit cellular metabolism, these ions do not undergo repeated reduction ('recycling effect'), because the reducing equivalents are not produced. Hence, some information on the pre-requirements for the Fenton reaction

239

can be obtained by examination of the temperature effect in hydrogen peroxide-treated cells (Ward et al., 1985; Jonas et al., 1989). The relatively high survival (Fig. 1) and low DNA damage (Fig. 4) found for LY-S cells treated with hydrogen peroxide at 4°C as compared to those treated at 37°C indicate that their metabolic activity constitutes a sensitizing factor by providing reducing equivalents. In contrast, the difference in response of LY-R cells treated at 37°C and 4°C (Figs. 1, 4) is small, indicating that the damage is extensive enough without contribution of reducing equivalents. This result allows us to conclude that transition metal ions are available for entering the Fenton reaction to a greater extent in LY-R than in LY-S cells.

DNA repair rate and fidelity DNA repair proceeded in both sublines to the same level of residual damage, 30 min after placing the cells at 37°C. The only feature that would be compatible with the higher susceptibility of LY-R as compared to LY-S cells was the slower repair between 5 and 10 min of incubation (Fig. 5). Such delay may enable damage interaction, as often assumed in the case of DNA repair in X-irradiated ceils (e.g. Dikomey and Jung, 1993). Measurements of the repair rates did not provide any information on repair fidelity. Some conclusions on misrepair may be drawn from mutation frequencies. The killing and mutagenic effects of H 2 0 2 in LY cells treated at 4°C and 37°C form a consistent pattern that indicates different proportions between error-prone and error-free repair and the unrepairable damage in LY sublines. A higher yield of hydrogen peroxide-induced chromatid aberrations in LY-R than in LY-S cells (Bou£,yk et al., 1992) supports this assumption. According to Ward et al. (1985), at 4°C LMDS are formed less efficiently than at 37°C, hence the smaller killing effect in both LY sublines. At 37°C LMDS and DSB are formed in higher numbers. In LY-R ceils this does not affect the mutation frequency per 105 survivors (Fig. 3), except at the highest H 2 0 2 concentration, but the relation between mutational and lethal events differs at the two temperatures; the number of mutations increases with the increase

240

M. Kn~zewski et aL /Mutation Research 308 (1994) 233-241 10 0

._>

T

9.

?. 8. ~ 7.

u'3 0 ~

A--A

LY-R 37°C

~'~ A--A

LY-R

4°C

6. 5-

'

3.

~

0

,

I

'

0

I

'

I

'

I

'

I

J

I

'

I

'

I

L

I

4 5 6 7 8 Lethal events per cell

1

2

3

~

I

'

9

Fig. 6. Mutation frequency at the HGPRT locus in LY-R cells treated with H 2 0 2 at 4°C or 37°C plotted vs. number of lethal events per cell. Number of lethal events per cell = - I n (surviving fraction).

in the number of lethal events per cell ( - I n (surviving fraction)), indicating a very high mutagenicity of lesions induced at 4°C (Fig. 6). LY-S cell populations sustain more lethal damage and mutation frequency declines with increase in H 2 0 2 concentration (Fig. 3) or number of lethal events per cell (Fig. 7). This may be due to numerous lethal mutations, possibly multilocus deletions, as a consequence of the DSB repair defect in these cells (Wlodek and Hittelman, 1987) in analogy to mutations induced in these ceils by X-rays, UV radiation and alkylating agents (Clive et al., 1980; Moore et al., 1982; Evans et al., 1986). In LY-R cells mutations are usually more frequent after treatment with DNA-damaging agents 10 0

.>

9

~ 8 ~

~ 0 ~

O--O

LY-S 37°C

• --e

LY-S

4°C

7 6

l

5

~ 4 133 C

2

0

1

2

3

4

5

6

7

8

g

L e t h a l e v e n t s p e r cell

Fig 7. Mutation frequency at the HGPRT locus in LY-S cells treated with H 2 0 2 at 4°C or 37°C plotted vs. number of lethal events per cell. Number of lethal events per cell = - I n (surviving fraction).

(Beer et al., 1984; Evans et al., 1986) than in LY-S ceils; in the case of hydrogen peroxide, the difference between sublines is only in the decline in mutation frequency (LY-S cells) or its lack (LY-R cells) at higher hydrogen peroxide concentrations (Figs. 3 and 4). In summary, such factors as activity of 'protecting enzymes' and the amount of initial DNA damage contribute to the differential sensitivity pattern of LY sublines to hydrogen peroxide. This does not exclude the character (strand breaks vs. base damage) a n d / o r location of DNA lesions as other reasons for the difference in sensitivity. Consistent with this assumption would be a difference in abundance and availability of transition metal ions between LY sublines. Preliminary results (to be reported elsewhere, Kruszewski et al., in preparation) point to a considerably higher sensitivity of LY-R than LY-S cells to the iron ion chelator desferrioxamine.

Acknowledgements The authors thank Dr. Andrew Collins for helpful discussions on the comet assay. This work was supported by KBN Grant 4.0451.91.01. M.K. was a recipient of the European Science Foundation Fellowship in Toxicology.

References Baker, M.A, and S. He (1991) Elaboration of cellular DNA breaks by hydroperoxides, Free Radical Biol. Med., 11, 563-572. Beer, J.Z., E.D. Jacobson, H.H. Evans and I. Szumiel (1983) X-ray and UV mutagenesis in two L5178Y cell strains differing in tumorigenicity, radiosensitivity and DNA repair, Br. J. Cancer, 49, 107-111. Blakely, W.F., A.F. Fuciarelli, B.J. Wegher and M. Dizdaroglu (1990) Hydrogen peroxide-induced base damage in deoxyribonucleic acid, Radiat. Res., 12, 338-343. Bou~yk, E., I. Buraczewska, O. Rosiek, B. Sochanowicz and I. Szumiel (1991) Hydrogen peroxide induced reproductive and interphase death in two strains of L5178Y murine lymphoma differing in radiation sensitivity, Radiat. Environ. Biophys., 30, 105-116. Clive, D., K.O. Johnson, J.F.S. Spector, A.G. Batson and M.M.M. Brown (1980) Validation and characterization of

M. Kruszewski et al. / Mutation Research 308 (1994) 233-241

the L5178Y/TK +/- mouse lymphoma mutagen assay system, Mutation Res., 59, 61-108. Dikomey, E., and H. Jung (1993) Correlation between thermal radiosensitization and heat-induced loss of DNA polymerase /3 activity in CHO cells, Int. J. Radiat. Biol., 63, 215-221. Evans, H.H., J. Mencl, M.-F. Horng, M. Ricanati, C. Sanchez and J. Hozier (1986) Locus specificity in the mutability of L5178Y mouse lymphoma cells: The role of multilocus lesions, Proc. Nat. Acad. Sci. USA, 83, 4379-4383. Green, M.H.L., J.E. Lowe, S.A. Harcourt, P. Akinluyi, T. Rowe, J. Cole, A.V. Anstey and C.F. Arlett (1992) UV-C sensitivity of unstimulated and stimulated human lymphocytes from normal and xeroderma pigmentosum donors in the comet assay: a potential diagnostic technique, Mutation Res., 273, 137-144. Halleck, M.M., J.H. Richburg and F.C. Kauffman (1992) Reversible and irreversible oxidant injury to PC12 cells by hydrogen peroxide, Free Radical Biol. Med., 12, 137-144. Iliakis, G.E., G.E. Pantelias, R. Okayasu and W.F. Blakely (1992) Induction by H 2 0 z of DNA and interphase chromosome damage in plateau-phase Chinese hamster ovary cells, Radiat. Res., 131, 192-203. Jaworska, A., O. Rosiek and K. Witkowska (1987) Activities of superoxide dismutase and catalase in two L5178Y murine lymphoma cell strains with different radiosensitivities, Nukleonika, 32, 193-201. Jeggo, P.A., and L.M. Kemp (1983) X-ray-sensitive mutants of Chinese hamster ovary cell line: Isolation and cross-sensitivity to other DNA-damaging agents, Mutation Res., 112, 313-327. Jonas, S.K., P.A. Riley and R.L. Willson (1989) Hydrogen peroxide cytotoxicity: Low-temperature enhancement by ascorbate or reduced lipoate, Biochem. J., 264, 551-655. Knaap, A.G.A.C., and J.W.I.M. Simons (1980) A mutational assay system for L5178Y mouse lymphoma cells, using hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT) deficiency as marker. The occurrence of a long expression time for mutations induced by X-rays and EMS, Mutation Res., 30, 97-110. McKelvey-Martin, V.J., M.H.L. Green, P. Schmezer, B.L. Pool-Zobel, M.P. De Meo and A.R. Collins (1993) The single-cell gel electrophoresis (SCGE) assay (comet assay): a European review, Mutation Res., 288, 47-64. Mello-Filho, A.C., and R. Meneghini (1984) In vivo formation of single strand breaks in DNA by hydrogen peroxide is mediated by the Haber-Weiss reaction, Biochim. Biophys. Acta, 781, 56-63. Mello-Filho, A.C., and R. Meneghini (1985) Protection of mammalian cells by o-phenanthroline from lethal and DNA-damaging effects produced by active oxygen species, Biochim. Biophys. Acta, 847, 82-89.

241

Mello-Filho, A.C., and R. Meneghini (1991) Iron is the intracellular metal involved in the production of DNA damage by oxygen radicals, Mutation Res., 251, 109-113. Meneghini, R. (1988) Genotoxicity of active oxygen species in mammalian cells, Mutation Res., 195, 215-230. Moore, M.M., and D. Clive (1982) The quantitation of TK - / and HGPRT mutants of L5178Y/TK +/- mouse lymphoma cells at varying times post-treatment, Environ. Mutagen., 4, 499-519. Olive, P.L., D. Wlodek and J.P. Banath (1991) DNA doublestrand breaks measured in individual cells subjected to gel electrophoresis, Cancer Res., 51, 4671-4676. Prise, K.M., S. Davies and B.D. Michael (1989) Cell killing and DNA damage in Chinese hamster V79 ceils treated with hydrogen peroxide, Int. J. Radiat. Biol., 55, 583-592. Sandstrom, B.E. (1991) Induction and rejoining of DNA single-strand breaks in relation to cellular growth in human cells exposed to three hydroperoxides at 0°C and 37°C, Free Radical Res. Commun., 15, 79. Sato, K., A. Ito, T. Shiomi and H. Hama-Inaba (1986) X-raysensitive mutants of mouse mammary carcinoma cells are hypersensitive to bleomycin and hydrogen peroxide, Jpn. J. Cancer Res., 77, 456-461. Singh, N.P., M.T. McCoy, R.R. Tice and E.L. Schneider (1988) A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res., 175, 184-191. Spitz, D.R., G.C. Li, M.L. McCormick, Y. Sun and L.W. Oberley (1988) Stable hydrogen peroxide-resistant variants of CHO fibroblasts demonstrate increases in catalase activity, Radiat. Res., 114, 114-124. Szumiel, I. (1979) Response of two strains of L5178Y cells to c/s-dichlorobis-(cyclopentylamine) platinum II, Chem.-Biol. Interact., 24, 51-72. Vaughan, A.T.M., and D.J. Gordon (1992) Hydrogen peroxide lethality is associated with a decreased ability to maintain positive DNA supercoiling, Exp. Cell Res., 202, 376380. Ward, J.F. (1988) DNA damage produced by ionizing radiation in mammalian cells: Identities, mechanisms of formation and repairability, Nucleic Acids Res., 35, 95-125. Ward, J.F., W.F. Blakely and E.I. Joner (1985) Mammalian cells are not killed by DNA single-strand breaks caused by hydroxyl radicals from hydrogen peroxide, Radiat. Res., 103, 383-392. Ward, J.F., J.W. Evans, C.L. Limoli and P.M. Calabro-Jones (1987) Radiation and hydrogen peroxide induced free radical damage to DNA, Br. J. Cancer, 55, 105-112. Wlodek, D., and W.N. Hittelman (1987) The repair of double-strand breaks correlates with radiosensitivity of L5178Y-S and L5178Y-R cells, Radiat. Res., 112, 146-155.