Mutation Research, 204 (1988) 645-648 645 Elsevier

MTR 01268

Chromosomal damage induced by maleic hydrazide in m a m m a l i a n cells in vitro and in vivo

R. Meschini 1, M.T. Quaranta 1, M. Fiore 1, C. Polcaro 2, E. Possagno 2 and F. Palitti 1,3 Centro di Genetica Evoluzionistica - C.N.R., c /o Dipartimento di Genetica e Biologia Molecolare,

Universita "La Sapienza" Piazzale A. Moro, 00185 Roma (Italy), 2 lstituto di Cromatografia and Istituto di Chimica Nucleare - C.N.R., Area della Ricerca di Roma, C.P. 10, 00016 Monterotondo Stazione, Roma (Italy), and 3 Dipartimento di Biologia Animale,

Universita di Catania, Via Androne 81, 95124 Catania (Italy)

(Received 13 May 1987) (Revision received 1 October 1987)

(Accepted 9 October 1987)

Keywords: Maleic hydrazide; Chinese hamster cells; Sister-chromatid exchanges; Chromosomal aberrations.

Summary

The induction of sister-chromatid exchanges (SCE) and chromosomal aberrations (Ch.Ab.) by the herbicide maleic hydrazide (MH) has been investigated in Chinese hamster ovary (CHO) cells grown in vitro and in bone marrow cells of mice treated in vivo. MH induces SCE and Ch.Ab. in CHO cells without metabolic activation; however, no induction of SCE was found in the in vivo experiments.

The herbicide maleic hydrazide (1,2-dihydro- 3,6-pyridazinedione) (MH) is a well-known clasto- genic agent in plant material (Swietlinska and Zuk, 1978). Studies performed using root-tip cells of Vicia f a b a indicated that MH induces chro- mosomal aberrations (Ch.Ab.) by an S-dependent mechanism (Evans and Scott, 1964). In the same plant system, the increase of sister-chromatid ex- changes (SCE) and chromosomal aberrations by MH has been found to be pH-dependent (Kihl- man, 1975). Furthermore, MH requires metabolic activation by plant mixed-function oxidase to damaging metabolites in order to interact with the DNA (Plewa and Gentile, 1982). Among MH's

Correspondence: Prof. Fabrizio Palitti, Centro di Genetica Evoluzionistica CNR, Dipartimento di Genetica e Biologia Molecolare, Universita 'La Sapienza', 00185 Rorna (Italia).

metabolites, hydrazine is a well-known mutagen (Kimbal, 1977). The results of MH mutagenicity studies in animal cells are conflicting (Perry and Evans, 1975; Swietlinska and Zuk, 1978; Yang et al., 1981) and its carcinogenic activity in rodent bioassays is not well characterised (IARC, 1974). The ability of MH to induce SCE and Ch.Ab. has been studied in Chinese hamster ovary (CHO) cells grown in vitro and in bone marrow cells of in vivo treated mice. The possible contamination of MH with hydrazine and other compounds has been investigated using samples of MH of differ- ent origins and purity.

Materials and methods

Test chemicals

MH from 3 different sources was used: 98% pure from Merck (DI365), crystalline from Sigma (M0500) and Merck purified in ethanol/water.

0165-1218/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

646

MH was dissolved in Ham's F-10 medium (Flow) without serum at 40 °C for in vitro experi- ments. For in vivo experiments, MH was dissolved in hot water (50 ° C) and administered orally to the animals in 0.6-ml quantities.

In vitro assays Chinese hamster ovary (CHO) cells were grown

in screw-capped 25-cm flasks (Sterilin) in 5 ml medium (Ham's F-10, Flow) supplemented with 15% newborn calf serum (Flow) and antibiotics.

SCE induction. An appropriate number of cultures was treated for 24 h with medium con- taining 5 . 1 0 - 6 M 5-bromodeoxyuridine (BrdUrd) and MH (Merck 98% pure) at various concentra- tions (750, 500, 250, 166 and 83/ tg /ml) . Another group of cultures was treated for 24 h with MH of different samples (Merck, Sigma and Merck puri- fied) at various doses (950, 750 and 500/~g/ml). One set of cultures was treated with MH (Sigma) for 12 h during the first cell cycle or for 12 h during the second cell cycle.

For each experimental point, the frequency of SCE was analysed in 40 diffc.:zntially stained cells prepared according to the fluorochrome-plus- Giemsa technique (Perry and Wolff, 1974).

Chromosomal aberration induction. Some cul- tures were treated with MH (950/~g/ml) for 1.5 h at either pH 7 or pH 5.5, after which the cells were washed once with Hanks' balanced salt solution and returned to 37°C for 9 h. In the last 3 h before fixation the ceils were treated with col- chicine (5- 10 - 7 M ) and routine air-dried prepara- tions were made. In order to lower the pH, HC1 was added to the culture medium. As MH by itself lowers the pH of the medium, this drop in pH was neutralized by adding NaOH.

For each experimental point, the frequency of Ch.Ab. was analysed in 100 cells.

In vivo assays 3-month-old albino Swiss male mice (CD-1)

(Charles Rivers) weighing 30-35 g were used. Mice were housed under controlled environmental con- ditions (20 o C, 60% relative humidity and an 8-h photoperiod). All mice were bedded on sawdust; they were fed with standard food pellets (Italiana

Mangimi) and water was available ad libitum. To obtain differentially stained chromatids in bone marrow cells, agar-coated tablets containing 25 mg BrdUrd were implanted subcutaneously (King et al., 1981). 8 h after tablet implantation, MH (Merck 98% pure) was administered orally to the mice (2 mice for each dose level). After 16 h the mice received an intraperitoneal injection of col- chicine (3 m g /k g b.w.) and 3 h later they were killed by cervical dislocation.

Air-dried preparations were made according to the technique of Ford and Hamerton (1956).

The staining was done according to the method of Perry and Wolff (1974). For each dose, 40 well-spread metaphases were analysed for SCEs (20 for each mouse).

Statistical analysis Mean SCEs per cell for each treatment were

compared with the concurrent negative control using the 2-sided Student's t test, accepting p < 0.05 as significant.

Results

Table 1 shows that 24-h treatment with MH at various doses induced a dose-related increase in SCE in CHO cells.

No difference was found in the induction of SCE after 24-h treatment with MH obtained from different sources (Table 2). Table 3 shows that 12-h treatment with MH produces the same in- crease in SCE frequency whether administered in the first cell cycle or in the second cell cycle after BrdUrd treatment.

TABLE 1

EFFECT OF A 24-h TREATMENT WITH D I F F E R E N T

DOSES OF MH ON THE FREQUENCIES OF SCE IN CHINESE HAMSTER CELLS G R O W N IN VITRO

Treatment (# g / m l ) SCE/ce l l + S.D.

Control 9.40 + 4.98 83 8.55 + 3.01

166 9.80 + 2.77

250 11.40 + 3.57 500 16.20 + 4.18 * * * 750 28.60 + 7.74 * * *

*** p < 0.001.

TABLE 2

EFFECT OF A 24-h TREATMENT WITH MH OF DIF- FERENT SOURCES ON THE FREQUENCIES OF SCE IN CHINESE HAMSTER CELLS GROWN IN VITRO

Treatment (/~g/ml) SCE/cell 5: S.D.

Control 7.30 + 2.51

MH (Crystalline) 500 14.10 + 4.37 * * * MH (Merck) 500 17.65 + 5.53 * * * MH (Sigma) 500 16.05 +4.45 * * *

MH (Crystalline) 750 21.05 + 8.99 * * * MH (Merck) 750 30.20 + 9.53 * * * MH (Sigma) 750 30.65 + 6.36 * * *

MH (Crystalline) 950 29.00 + 6.74 * * * MH (Merck) 950 33.80 + 7.52 * * * MH (Sigma) 950 26.00 + 7.72 * * *

*** p < 0.001.

T h e 2 4 - h t r e a t m e n t , as s h o w n i n T a b l e 1, i n -

d u c e s a h i g h e r i n c r e a s e i n t he f r e q u e n c y o f S C E

c o m p a r e d to t h e 1 2 - h t r e a t m e n t .

T a b l e 4 s h o w s t h a t s h o r t t r e a t m e n t w i t h M H

TABLE 3

EFFECT OF A TREATMENT WITH MH (750 /~g/ml) ON THE FREQUENCIES OF SCE DURING THE FIRST OR THE SECOND CELL CYCLE OR DURING 2 CONSECU- TIVE CELL CYCLES

Treatment SCE/cell + S.D.

First cycle Second cycle

12 h BrdUrd + 12 h BrdUrd 5.95 + 2.74 12 h BrdUrd+MH + 12 h BrdUrd+MH 21.05+6.19 *** 12 h BrdUrd + MH + 12 h BrdUrd 17.85 + 6.89 * * * 12 h BrdUrd + 12 h BrdUrd + MH 16.55 + 7.02 * * *

*** p < 0.001.

647

TABLE 5

EFFECT OF AN ORAL TREATMENT WITH DIFFERENT DOSES OF MH ON THE FREQUENCIES OF SCE IN BONE MARROW CELLS OF SWISS MICE a

Treatment (mg/kg b.w.) SCE/cell + S.D.

Control 3.60 + 1.90 333 3.95 + 1.69 500 3.42 + 2.04 666 3.42 + 1.79

1000 3.52 + 2.14

a Two mice were treated for each dose and a total of 40 cells (20 for each animal) was analysed.

(1.5 h ) h a s a n e f f ec t o n t h e i n d u c t i o n o f C h . A b .

w h i c h is p H - d e p e n d e n t .

T h e i n v i v o e x p e r i m e n t s s h o w t h a t M H d o e s

n o t i n d u c e S C E i n b o n e m a r r o w cel ls o f m i c e

t r e a t e d o r a l l y i n t h e r a n g e o f t h e t e s t e d d o s e s

( 3 3 3 - 1 0 0 0 m g / k g b .w . ) ( T a b l e 5).

Discussion

O u r d a t a s h o w t h a t t h e h e r b i c i d e M H i n d u c e s

g e n o t o x i c d a m a g e i n a d o s e - d e p e n d e n t m a n n e r i n

C H O cel l s i n t h e a b s e n c e o f a n e x o g e n o u s m e t a -

b o l i c a c t i v a t i o n s y s t e m ( T a b l e 1).

T h e p o s s i b i l i t y t h a t t h e m u t a g e n i c a c t i v i t y o f

M H was d u e t o t h e p r e s e n c e o f i m p u r i t i e s s u c h as

h y d r a z i n e o r o t h e r c o m p o u n d s w a s c o n s i d e r e d .

T h e r e f o r e , C H O cel l s w e r e t r e a t e d w i t h 3 d i f f e r e n t

s a m p l e s o f M H of v a r y i n g g r a d e s o f p u r i t y . T h e

m a g n i t u d e o f S C E i n d u c t i o n w a s s i m i l a r f o r t h e 3

d i f f e r e n t s a m p l e s , s u g g e s t i n g t h a t t h e e f f ec t was

n o t d u e to t h e p r e s e n c e o f i m p u r i t i e s ( T a b l e 2). A s

i t h a s b e e n s h o w n b y N a t a r a j a n e t al. ( 1981) t h a t

f a l s e - p o s i t i v e r e s u l t s i n t h e S C E a s s a y m a y b e

TABLE4

INDUCTION OF CHROMOSOMAL A B E R R A ~ O N S I N CHO CELLSTREATED WITH M H ( 9 5 0 # g / m l ) FOR1 .5h A T p H 7 OR5.5 a

Treatment Abnormal Gaps Chromatid Isochromatid Chromatid Total aberra- metaphases breaks breaks exchanges tions + gaps

p H 7 9 5 3 0 0 3+ 5 pH 7 + M H 10 5 2 3 1 6+ 5 pH 5.5 8 6 2 0 0 2+ 6 pH 5.5 + MH 49 37 23 7 20 50 + 37

a Hundred cells were scored for each point.

648

caused by inhib i tors of po ly - (ADP- r ibose ) -po ly - merase, the effect of M H t rea tment in the first or in the second cell cycle was evalua ted (Table 3). Since there was no signif icant difference in the induct ion of SCE be tween the 2 t r ea tment p ro to - cols, M H is not an inh ib i to r of p o l y - ( A D P - r ibose)-polymerase , and its mechan i sm of ac t ion is p r o b a b l y s imilar to a ' c ross- l ink agent ' (Na t a r a j an et al., 1983).

There was a p H - d e p e n d e n t effect on the induc- t ion of Ch.Ab.

These da t a are in good agreement with those ob ta ined in Vicia faba b y K ih lman et al. (1975). The low p H used in our p ro toco l d id no t seem to induce ch romosomal damage by itself.

Un t i l now, M H has been cons idered negat ive in animal cells for the induc t ion of SCE. This lack of effect was p r o b a b l y due to the use of shor t t rea t - men t t imes (Perry and Evans, 1975; Yang et al., 1981); we also have ob ta ined a negat ive result with M H for SCE induc t ion using a t r ea tment t ime of only 1.5 h (da ta not shown).

Recent ly , Gonza les -Gi l l and Nava r r e t e (1986) have shown that in roo t -mer i s t em cells of Allium cepa, M H induces SCE more eff ic ient ly in cells t rea ted dur ing the S phase. Therefore, long t reat- men t with M H will cer ta in ly cover the S pe r iod while pulse t rea tment with M H will p r e s u m a b l y damage few cells which are in the S phase.

As it has been shown (Brusik, 1986) that hyper - osmot ic med ium can induce ch romosomal damage, the osmot ic pressure of the M H solu t ion used in our exper iments was measured. The result shows tha t the osmot ic pressure of the m e d i u m is no t changed in the presence of MH, and canno t be the cause of the induc t ion of ch romosoma l damage.

Maleic hydraz ide does not seem to induce SCE in bone mar row of mice t rea ted in vivo. These da t a ob ta ined in vivo b y us canno t be cons idered conclusive, as it is well known that cells o ther than bone mar row may be the sensit ive cell popu la t ion .

These p re l iminary exper iments show that M H is genotoxic in m a m m a l i a n cells in vi t ro bu t ap- pa ren t ly no t in vivo.

Acknowledgements

This invest igat ion was f inancia l ly suppo r t ed by Proget to F ina l izza to C N R Medic ina Prevent iva e Riab i l i t a t iva No. 82.0225 6.56, and Proget to F i n a -

l izzato del C N R Oncologia . W e thank Dr. P. Mosseso for his suppor t .

References

Brusick, D. (1986) Genotoxic effects in cultured mammalian cells produced by low pH treatment conditions and in- creased ion concentrations, Environ. Mutagen., 8, 879-886.

Evans, H.J., and D. Scott (1964) Influence of DNA synthesis on the production of chromatid aberrations by X-ray and maleic hydrazide in Vicia faba, Genetics, 49, 17-38.

Ford, C.E., and J.L. Hamerton (1956) A colchicine, hypotonic citrate, squash sequence for mammalian chromosomes, Stain Technol., 31,247-251.

Gonzalez-Gil, G., and M.H. Navarrete (1986) Induction of SCE by maleic hydrazide throughout the cell cycle, Muta- tion Res., 163, 57-61.

IARC (1974) Monographs on the evaluation of carcinogenic risk to man, Some aromatic amines, hydrazine and related substances, N-nitroso compounds and miscellaneous al- kylating agents, Vol. 4, International Agency for Research on Cancer, Lyon, pp. 173-179.

Kihlman, B.A. (1975) Root tips of Vicia faba for the study of the induction of chromosomal aberrations, Mutation Res., 31, 401-412.

Kimball, R.F. (1977) The mutagenicity of hydrazine and some of its derivatives, Mutation Res., 39, 111-126.

King, M.T., D. Wild, E. Gocke and F. Eckhardt (1981) 5- Bromodeoxyuridine tablets with improved depot effect for analysis in vivo of SCE in bone marrow and spermato- gonial cells, Mutation Res., 97, 117-119.

Natarajan, A.T., I. Csukas and A.A. van Zeeland (1981) Con- tribution of incorporated 5-bromodeoxyuridine in DNA to the frequencies of sister-chromatid exchanges induced by inhibitors of poly-(ADP-ribose)-polymerase, Mutation Res., 84, 125-132.

Natarajan, A.T., A.D. Tates, M. Meijers, I. Neuteboom and N. de Vogel (1983) Induction of sister-chromatid exchanges (SCEs) and chromosomal aberrations by mitomycin C and methyl methanesulfonate in Chinese hamster ovary cells. An evaluation of methodology for detection of SCEs and persistent DNA lesions towards the frequencies of observed SCEs, Mutation Res., 121, 211-223.

Perry, P., and S. Wolff (1974) New Giemsa method for dif- ferential staining of sister chromatid exchanges, Nature (London), 251, 156-158.

Perry, P., and H.J. Evans (1985) Cytological detection of mutagen-carcinogen exposure by sister chromatid ex- changes, Nature (London), 258, 121-125.

Plewa, M.J., and J.M. Gentile (1982) The activation of chem- icals into mutagens by green plants, in: F.J. de Serres and A. Hollaender (Eds.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 7, Plenum, New York, pp. 401-420.

Swietlinska, Z., and J. Zuk (1978) Cytotoxic effects of maleic hydrazide, Mutation Res., 55, 15-30.

Yang, D.P. , F. Graupensperger, L.C. Minecci and B.A. Rubin (1981) Induction of sister chromatid exchanges in human diploid fibroblasts by mutagens with and without rat liver microsomal activation, Environ. Mutagen., 3, 45-52.