Induction of micronuclei in cultured murine splenocytes exposed to elevated levels of ferrous ions, hydrogen peroxide and ultraviolet irradiation

Mutation Research, 244 (1990) 337-343

337

Elsevier MUTLET 0384

Induction of micronuclei in cultured murine splenocytes exposed to elevated levels of ferrous ions, hydrogen peroxide and ultraviolet irradiation Ivor E. Dreosti, Peter A. Baghurst, Eric J. Partick and Julie Turner Division of Human Nutrition, Commonwealth Scientific and Industrial Research Organisation, Adelaide, South Australia 5000 (Australia)

(Accepted 3 April 1990)

Keywords." Micronuclei; Free radicals; Pro-oxidants; Mouse splenocytes;Ferrous ions; Hydrogen peroxide

Summary The frequency of micronuclei in cultured mouse splenocytes increased positively and in a dose-related manner to exposure to ferrous ions and ultraviolet irradiation, but not to hydrogen peroxide. Combined treatments, especially when ferrous ions were present with hydrogen peroxide or with ultraviolet irradiation, led to a synergistic enhancement in micronucleus frequency. The results indicate that a significant level of c h r o m o s o m e damage is associated with exposure to ultraviolet light and to general cellular pro-oxidative stress, and indicate that under these conditions the micronucleus assay can provide an effective in vitro model for the study of genotoxicity in relation to oxygen-derived free radicals.

Chromosome damage is conventionally measured by visual inspection of chromosome spreads. However, the technique is laborious and less sensitive than an evaluation of the micronucleus frequency, especially when ascertained using the cytokinesis-block (CB) method (Fenech and Morley, 1985). Micronuclei are extranuclear DNAcontaining bodies resulting from chromosome or chromatid fragments, as well as aberrant chromosomes, which have been excluded from the daughter nuclei following cell division. They reflect in particular, D N A damage resulting in acentric c h r o m o s o m e fragments and are therefore good inCorrespondence: Dr. I.E. Dreosti, Division of Human Nutrition, CSIRO, Kintore Avenue, Adelaide, South Australia 5000 (Australia).

dicators of the extent of double-stranded D N A scission (Heddle et al., 1978), or whole chrom o s o m e loss, depending on the nature of the inducing agent. Micronuclei are frequently found in increased numbers in target tissues after a carcinogenic insult, and provide a useful assessment of genomic damage and of the potential cancer risk (Stich, 1987). The validity of equating micronuclei with genotoxicity has been demonstrated for X-rays and with mutagenic chemicals both in vivo and in vitro (Heddle et al., 1978; Fenech and Morley, 1986; R a m a l h o et al., 1988). Since much of the genomic damage associated with X-irradiation and a number of clastogenic chemicals has been attributed to an attack on D N A by active free radicals, notably the hydroxyl radical (Cochrane et

0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

338 al., 1988), the question arises whether other sources of active oxygen may also damage DNA in a manner reflected by an increase in the number of micronuclei. Accordingly, attention was focused in this study on the applicability of the micronucleus technique to assess genotoxicity in murine splenocytes exposed in vitro to a series of putative hydroxyl radicalgenerating systems. The systems used included ferrous ions (Fe 2+), hydrogen peroxide (HzO2) and ultraviolet light irradiation. The demonstration in most cases of a dose-related response points to an effect of these systems on micronucleus formation, and offers an effective in vitro model for the study of free radical-related genotoxicity, and of factors which might modify the extent of the chromosomal damage. Materials and methods

Animals. In vitro studies were performed on splenocytes obtained from young adult (10-week) male mice (C57BL/6J) fed ad libitum on a stock colony rodent diet (Pro-Rat, Milling Industries, Adelaide, Australia), and housed under conditions of constant temperature and equal (12 h) light/ dark cycles. Splenocyte preparation and experimental treatments. Spleen cells were dissociated and suspended in McCoy's 5A modified medium prior to exposure to one of 3 pro-oxidant treatments, (Fe 2÷ [Analar, FeSO4.7H20], H202 [Univar, 100 vol.], UV irradiation [Wotan Sunlamp, Germany: UVA, 6 . 3 x 103 W/cm2; UVB, 2 . 6 x 103 W/crn2]) either individually or in combination as indicated in Tables 1-3. At least 4 splenocyte preparations, each derived from a separate animal were used in each pro-oxidant treatment group. Splenocytes subjected to irradiation were suspended in Hanks' balanced salt solution during exposure to avoid absorption of UV by the phenol red indicator in the McCoy's medium. After treatment, all cells were pelleted by centrifugation and suspended ( 2 x 10 6 cells/ml) in McCoy's 5A modified medium containing concanavalin A (2.5 #g/ml; Sigma Chemi-

cals, U.S.A.) and supplemented with mitogenic conditioning factors as described by Fenech and Morley (1986).

Cytokinesis-block micronucleus assay (Fenech and Morley, 1986). Splenocyte cultures in the mitogenic medium were incubated for 28 h at 37°C in a humidified atmosphere containing 10070 COz, in order to initiate mitosis and to allow time for cells to progress into the cell cycle. At that stage cytochalasin B (4.5 /~g/ml; Sigma Chemicals, U.S.A.) was added to the medium to prevent microfilament assembly and to block cytokinesis, and cells were cultured for a further 18 h to increase the number of cells arrested at the binucleate stage. Cytological examination of splenocytes for micronuclei was made on slides prepared by cytocentrifugation and stained with Diff-Quick stain (Lab-Aids, Australia). Micronuclei were counted in 1000 binucleate cells at x 1000 magnification under oil-immersion light microscopy by two separate observers.

Statistical analysis. Counts of micronuclei (per 1000 binucleate cells examined) were assumed to be Poisson random variables and the data were analysed by fitting linear models on a log scale, using maximum likelihood methods (Alvey et al., 1983). The Poisson model may not be optimal, given that the residual deviance (a measure of how well a model fits) was usually high for the saturated model, which included main effect terms and their interactions - - and reported significance levels should be regarded as approximate only. Nevertheless, the effects were, in most instances, so strong that the conclusions are most unlikely to be affected by the overdispersion, which appears to be characteristic of micronuclei count data. The treatment combinations tested may be conveniently regarded for analysis purposes as 3 factorial experiments: (1) 3 levels of iron by 3 levels of UV exposure (Table 1), (2) 3 levels of iron by 2 levels of hydrogen peroxide (Table 2), (3) 3 levels of UV exposure by 3 levels of hydrogen peroxide (Table 3). Some treatment combinations involving the zero

339 TABLE 1 E F F E C T OF IRON SPLENOCYTES

AND

ULTRAVIOLET

Fe 2 + concentration (mM)

LIGHT

ON

MICRONUCLEUS

FORMATION

IN

CULTURED

MOUSE

Micronucleus frequency (count/1000 binucleate cells) Duration of UV exposure (sec) 0

5

10

Observer 1

0 0.5 1.0

7.2 (13) a 17.1 (8) 18.9 (8)

16.4 (8) 20.3 (4) 28.5 (4)

38.0 (7) 82.5 (4) 107.8 (4)

Observer 2

0 0.5 1.0

9.6 (11) 21.4 (8) 21.4 (8)

15.5 (8) 29.5 (4) 50.5 (8)

28.4 (7) 68.5 (4) 83.8 (4)

a Number of incubations indicated in parentheses.

Summary analysis of deviance Observer 1 model

df

residual deviance

df

change in deviance

constant constant + Fe

59 57

1494.6 1230.8

0 2

* 263.8

p < 0.001

constant + Fe + UV constant + Fe + UV + Fe + UV

55 51

281.8 263.7

2 4

949.0 18.2

p < 0.001 p = 0.001

model

df

residual deviance

df

change in deviance

constant constant + Fe constant + Fe + UV constant + Fe + UV + Fe + UV

57 55 53 49

895.1 599.0 140.8 127.0

0 2 2 4

* 296.1 458.2 13.7

Observer 2

level of an exposure factor are c o m m o n to two experiments, which are therefore not entirely independent of one another. Separate analyses were performed for each observer. Results The frequency of micronuclei was increased by most treatments and often showed a measure of potentiation when two pro-oxidant systems were imposed concurrently. Both Fe 2+ and UV irradiation individually elicited an increase in the number of micronuclei (Table 1), with cell preparations in each case re-

p < 0.001 p < 0.001 p = 0.008

flecting a deterioration in quality and reliability at higher exposures ( > 1 m M Fe 2* and 10 sec UV). At Fe 2 + levels of 1.0 mM the micronucleus incidence approximately doubled, without the loss of slide quality or increase in cell debris which occurred at 2.0 m M . Exposure to UV for 5 sec increased the number of micronuclei 2-fold, and after 10 sec by about 4-fold, with no evidence of accompanying cell damage. A very marked rise in the micronucleus frequency (approx. 10-fold) occurred when UV (10 sec) and Fe 2 * (0.5-1.0 mM) were imposed together, but the effect was accompanied by severe deterioration in slide quality. H202 (20/~M) alone had little effect on the micronucleus frequency and did not appear to be disruptive to the cell prepara-

340 TABLE 2 EFFECT OF IRON AND HYDROGEN PEROXIDE ON MICRONUCLEUS FORMATION IN CULTURED MOUSE SPLENOCYTES Fe2÷ concentration (mM)

Micronucleus frequency (count/1000 binucleate cellls) HzO2 concentration (/~M) 0

20

Observer 1

0 0.5 1.0

7.2 (13)a 17.1 (8) 18.9 (8)

9.0 (7) 40.8 (4) 32.8 (8)

Observer 2

0 0.5 1.0

9.6 (11) 21.4 (8) 21.4 (8)

13.6 (7) 39.3 (4) 40.8 (4)

a Number of incubations indicated in parentheses.

Summary analysis of deviance Observer 1 model

df

residual deviance

df

change in deviance

constant constant + Fe constant + Fe + peroxide constant + Fe + peroxide + Fe- peroxide

43 41 40 38

338.5 147.7 80.1 69.4

0 2 1 2

* 190.8 67.6 10.7

model

df

residual deviance

df

change in deviance

constant constant + Fe constant + Fe + peroxide + Fe. peroxide

41 39 38 36

241.0 96.2 30.0 26.9

0 2 1 2

* 144.8 66.2 3.2

p < 0.001 p < 0.001 p = 0.005

Observer 2

p < 0.001 p < 0.001 p = 0.21

t i o n (Tables 2 and 3). T o g e t h e r , Fe z+ ( 0 . 5 - 1 . 0

a n d cell debris. Th e ratio o f single:multiple m i c r o -

m M ) an d H202 (20 izM) increased the m i c r o n u c l e u s

nuclei r a n g e d f r o m a r o u n d 5-6:1 in c o n t r o l an d

c o u n t 3 - 4 - f o l d w i t h o u t loss o f cell viability, a n d

H 2 O z - t r e a t e d cultures, to b et w een 1 an d 3:1 in

with n o increase in cell debris w h e n p r e p a r e d for

p r e p a r a t i o n s exhibiting a significant overall eleva-

m i c r o s c o p i c e x a m i n a t i o n (Fig. 1). N o detectable enhancement of UV-related micronucleus forma-

t i o n in the t o t al m i c r o n u c l e u s c o u n t . T h e ratio o f m o n o n u c l e a t e : b i n u c l e a t e cells was fairly c o n s t a n t

t i o n was achieved by c o n c u r r e n t t r e a t m e n t with

in all t r e a t m e n t g r o u p s an d held at a r o u n d 7:3.

H202 (10 an d 20 #M) (Table 3). T h e f r e q u e n c y with which cells c o n t a i n e d multi-

Discussion

ple r a t h e r t h a n single m i c r o n u c l e i t e n d e d to rise c o n c u r r e n t l y with the t o t a l n u m b e r o f m i c r o n u c l e i

T h e i m p o r t a n c e o f o x y g e n - d e r i v e d free radicals,

present, which was o f t e n , b u t n o t always, a c c o m -

especially h y d r o x y l radicals, in D N A d a m a g e is

p a n i e d by a higher level o f cellular d i s i n t e g r a t i o n

widely r e c o g n i z e d (Brawn an d F r i d o v i c h ,

1981;

341 TABLE 3 EFFECT OF ULTRAVIOLET L I G H T A N D H Y D R O G E N P E R O X I D E ON MICRONUCLEUS F O R M A T I O N IN CULTURED MOUSE SPLENOCYTES

H202 concentration (ttM)

Micronucleus frequency (count/1000 binucleate cells) Duration of UV exposure (sec) 0

Observer 1

0 10 20

Observer 2

0 10 20

7.2 (13) a 8.4 (5) 9.0 (7) 9.6 (11) 10.0 (5) 13.6 (7)

5

10

16.4 (8) 18.3 (4) 17.5 (4)

38.0 (7) 32.5 (4) 26.5 (4)

15.5 (8) 17.3 (4) 20.3 (4)

28.4 (7) 29.0 (4) 27.8 (4)

a Number of incubations indicated in parentheses.

Summary analysis of deviance Observer 1 model

df

residual deviance

df

change in deviance

constant constant + peroxide constant + UV + peroxide constant + UV + peroxide + UV. peroxide

55 53 51 47

517.5 514.1 178.5 167.2

0 2 2 4

*

model

df

residual deviance

df

change in deviance

constant constant + peroxide constant + peroxide + UV + peroxide. UV

53 51 49 45

252.9 249.0 96.7 91.5

0 2 2 4

*

3.4 335.6 11.2

p = 0.18 p < 0.001 p = 0.02

Observer 2

Freeman and Crapo, 1982; Simic, 1986; Halliwell, 1987; Cochrane, 1988) and has been reviewed recently (Dreosti, 1989). In the present study, mouse splenocytes were exposed to Fe 2 +, H202 and UV-irradiation, each of which alone, and in various combinations, has been proposed to act as a cellular pro-oxidant, largely through the production of reactive hydroxyl radicals (Freeman and Crapo, 1982; Symons, 1985; Cerutti, 1985; Halliwell, 1987; Cochrane, 1988). Chromosome damage was assessed in relation to the micronucleus count, which was found to be raised by two of the putative hydroxyl radical generating systems (Fe 2÷, UV),

3.9 152.3 5.2

p = 0.14 p < 0.001 p = 0.27

and to be further elevated in some cases when prooxidative treatments were combined to provide an expected increase in hydroxyl radical flux. The data are of interest in that they extend existing biochemical evidence of free radical-releated clastogenicity to include the cytological demonstration o f accompanying chromosomal damage. The significant increase in micronucleus count in the presence of Fe 2 ÷ accords well with the postulated production o f hydroxyl radicals via an ironcatalysed Fenton-type reaction, possibly involving a D N A - F e 2÷ complex located directly on the chromatin structure (Floyd, 1981; Dreosti, 1969).

342

associated with c o n c u r r e n t exposure to Fe 2 ÷ a n d H202 a n d to UV a l o n e affords a versatile cellular m o d e l for the study o f factors affecting the genotoxicity of oxygen-centred free radicals, a n d o f c o n d i t i o n s which might m o d u l a t e their activity.

References

Fig. 1. Micronucleus in mouse splenocyte exposed to Fe2+ (1.0 mM) and H202 in culture prior to assay by the cytokinesis block method. Magnification × 2000.

UV irradiation substantially increased the m i c r o n u c l e u s c o u n t when applied as the only treatm e n t , which provides s u p p o r t for the view that D N A strand scission c o n t r i b u t e s significantly to the g e n o m i c d a m a g e associated with U V A irradiation (Cadet et al., 1986). W h e n c o m b i n e d with H202, UV i r r a d i a t i o n resulted in severe d i s r u p t i o n o f cells which was a p p a r e n t l y n o t a c c o m p a n i e d by a further increase in the m i c r o n u c l e u s c o u n t . Possibly the general cytotoxicity o f this c o m b i n a t i o n arises f r o m the widespread f o r m a t i o n o f hydroxyl radicals f r o m h y d r o g e n peroxide by rupture o f the O - O b o n d as suggested by S y m o n s (1985). T r e a t m e n t with H202, c o n t r a r y to theoretical predictions based o n its p o t e n t i a l as a source of hydroxyl radicals, did n o t m e a s u r a b l y affect the n u m b e r of micronuclei, b u t this m a y be due to the relatively low levels at which it was included in the present study. Use o f the C B - m i c r o n u c l e u s t e c h n i q u e in these studies has d e m o n s t r a t e d its effective a p p l i c a t i o n for the in vitro assessment o f genetic d a m a g e associated with a variety o f free radical generating systems. The sharp increase in m i c r o n u c l e u s c o u n t

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