carcinogens

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Mutation Research, 63 ( 1 9 7 9 ) 3 9 3 - - 4 0 0 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

STUDIES OF DNA-STRAND BREAKS INDUCED IN HUMAN FIBROBLASTS BY CHEMICAL MUTAGENS/CARCINOGENS

M. N O R D E N S K J { ~ L D 1 , , S. S O D E R H A L L 1 a n d P. MOLDI~US 2

Department o f Clinical Genetics 1 and Department o f Forensic Medwine 2, Karolinska Instituter, S-104 01 Stockholm (Sweden) ( R e c e i v e d 10 M a r c h 1 9 7 9 ) (Revision received 26 J u n e 1 9 7 9 ) (Accepted 5 July 1979)

Summary A m e t h o d for the study of DNA-strand breaks using alkaline denaturation followed b y hydroxylapatite chromatography has been modified and used for the detection of chemically induced DNA-strand breaks. A new procedure for the incubation of human fibroblasts with a metabolizing system and the detection of DNA-strand breaks is presented. With this method the induction and repair of DNA-strand breaks have been studied in human fibroblasts exposed to methyl methanesulphonate, melphalan, benzo [a ] pyrene and cyclophosphamide. These agents all give rise to DNA-strand breaks. In cells exposed to methyl methanesulphonate, melphalan or b e n z o [ a ] p y r e n e these breaks disappeared within 21 h after removal of the drug. In cells exposed to the bifunctional alkylating agent cyclophosphamide, studies of DNA-strand breaks suggest the presence of inter~strand cross-links. Measurements o f strand breaks in DNA is a well-established m e t h o d for the study of DNA damage [12,15]. However, the gradient methods for such determinations are time-consuming and they present technical problems in the handling of DNA of very high molecular weight. During the last years two new methods [2,10,18] have been described for the determination of the number of chain breaks in DNA. These are based on a changed behaviour of DNA in an alkaline solution if strand breaks are present. Both methods are very sensitive and suitable for studies of changes in the a m o u n t of chain breaks in DNA * To whom reprint requests should

be sent.

Abbreviations: BP, benzo[a]pyrene; 3-OH-BP, 3-hydroxybenzo[a]pyrene; BSS, Hanks's balanced salt solutaon; DMSO, dimethyl sulphoxlde; Eagle's MEM, Eagle's mlmmal essential medium, h e p e s , N-2-hydroxyethylplperazme-N'-2-ethanesulphomc acld~ MC, 3-methylcholanthrene; MMS, methyl methanesulphonate; 3H-TdR, [Me-3H]thymldine.

394 [1,10]. The method of AhnstrSm and Erixon [2] and Rydberg [18] is especially well suited for multiple determinations owing to its simplicity. It has been used for studies of strand breaks induced by ultraviolet and X-irradiation [1,2,5,18]. In the present study we have applied the method of Ahnstr5m and Erixon for studies of chemically induced strand breaks in human cells in the presence and absence of a metabolizing system. DNA-strand breaks were induced in normal human fibroblast DNA by two directly acting alkylating agents (methyl methanesulphonate and melphalan) and two procarcinogens (benzo[a]pyrene and cyclophosphamide). Materials and methods

Chemicals Benzo[a]pyrene (BP), isocitrate, NADP and dimethyl sulphoxide (DMSO) were obtained from Sigma Chemical Company (St. Louis, MO, U.S.A.), cyclophosphamide from Pharmacia AB (Uppsala, Sweden), melphalan from Burroughs Wellcome and Co. (London, Great Britian), methyl methanesulphonate (MMS) from Merck (Darmstadt, German Federal Republic), isocitrate dehydrogenase from Boering Mannheim GmbH (Mannheim, German Federal Republic), hydroxylapatite (Bio-gel HTP) from Bio-Rad laboratories (Richmond, CA, U.S.A.), methyl-tritiated thymidine (3H-TdR, 5 Ci/mmol) from the Radiochemical Centre (Amersham, Great Britain), Eagle's minimal essential medium (Eagle's MEM), Hanks's balanced salt solution (BSS)and foetal calf serum from Flow laboratories (Irvine, Scotland), and Instagel scintillation cocktail from Packard Instr. Company (Downers Grove, IL, U.S.A.). All other chemicals were obtained from commercial sources and were of analytical grade. Rat-liver microsomes Male Sprague-Dawley rats (200--250 g) were allowed food and drink ad libitum. Rats treated with 3-methylcholanthrene were given 20 mg MC/g body weight (0.5% solution in corn oil) intraperitoneally once daily for 3 days. MCtreated rats were killed 36 h after the last injection. Phenobarbital-treated rats were given phenobarbital, 20 mg/g, in water, intraperitoneally once daily for 3 days before they were killed. Liver microsomes (105 000 × g pellet) were prepared according to Ernster et al. [6]. Cell culture and incubations Human fibroblasts from healthy donors were grown on 1.2-cm 2 glass plates and labelled to confluency with 3H-TdR (0.2 pCi/ml). The plates with fibroblasts were incubated in glass tubes containing Eagle's MEM supplemented with N-2hydroxyethyl-piperazine-N'-2~ethanesulphonic acid (hepes, 20 mM), streptomycin (150 pg/ml) and benzylpenicillin (85 pg/ml). BP was added in 10 pl DMSO, cyclophosphamide and MMS in Hanks's balanced salt solution. Melphalan was dissolved in 15 M ethanol containing 1.55 M HC1 (1 ttg melphalan per 10 pl) and diluted in BSS. All incubations were performed at 37°C and pH 7.4. The microsomal metabolism system contained microsomal protein (0.7 mg/ ml), NADP (1 mM), isocitrate (8 mM) and isocitrate hydrogenase (100 pg/ml)

395 in Eagle's MEM. Incubations with BP were performed under yellow light. After exposure to the drug, fibroblasts were washed twice in BSS and fresh Eagle's MEM supplemented with hepes, streptomycin, penicillin and foetal calf serum (10%). The incubations were terminated by washing the cells twice in ice-cold Dulbecco's phosphate-buffered saline.

Detectzon of DNA-strand breaks --alkali-labile sites DNA-strand breaks were determined by the method of AhnstrSm and Erixon [2] modified as follows. Cells on glass plates were incubated in 1.5 ml 0.03 M NaOH in 0.97 M NaC1 in the dark on ice for 30 min followed by adjustment of the pH to 6.8 by forceful addition of a b o u t 0.6 ml 0.067 M HC1 in 0.02 NaH:PO4. After sonication with a Branson sonifier (microtip, 30 W, 15 sec) and addition of sodium dodecyl sulphate (0.25 g/100 ml final concentration), DNA was chromatographed on 0.5-ml columns of hydroxylapatite that had been boiled in phosphate buffer [14]. Single-stranded DNA was eluted with 3.5 ml 0.1 M potassium phosphate buffer (pH 6.8) and double-stranded DNA with 3.0 ml 0.25 M potassium phosphate buffer (pH 6.8). Of the applied radioactivity, 85--90% was recovered in these fractions. The radioactivity in each fraction was determined in a Packard scintillation counter with 4 ml Instagel scintillation cocktail. Alterations in the number of DNA-strand breaks are expressed as percentage of total radioactivity eluted in the single-strand fraction [18]. The experimental background of around 25% single-stranded DNA is related to the denaturation conditions used [ 18 ].

Metabolism Benzpyrene metabolism was measured by determination of 3-hydroxyb e n z o [ a ] p y r e n e according to Atlas et al. [3] in an Aminco Bowman spectrofluorometer. Results

(A ) Directly acting alkylating agents MMS and melphalan Directly acting alkylating agents such as MMS are known to induce breaks and alkali-labile sites in DNA strands [11,21]. Bifunctional or polyfunctional agents are also known to induce a certain a m o u n t of inter-strand cross-links. To study the relation of induction of strand breaks with time, drug concentration and incubation conditions a monofunctional (MMS) and a bifunctional alkylating agent (melphalan) were chosen for primary tests of the method. The timerelated change in DNA-strand breaks in human fibroblasts after exposure to MMS is shown in Fig. 1. There was a dose-related rapid increase in strand breaks with, time. After omission of the drug at 30 min the a m o u n t of strand breaks decreased during a 21-h period to background levels, indicating DNA repair. The corresponding effects of melphalan treatment are shown in Fig. 2. Here, too, there was an increase in DNA-strand breaks although it was smaller

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than that found for MMS. After omission of melphalan a recovery to background levels occurred within 21 h.

(B) Metabolic activation Benzo[a]pyrene To study the effect of chemical agents that are metabolized by the micro-

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Fig. 2. lnductaon o f DNA-strand break~ in human fabroblasts by melphaJan. Cells were incubated at 37°C m complete medium w i t h o u t (o) or w i t h melphalan at 1 X 10 -5 M (v), 1 × 10 -4 M (~) and 1 X 10 -3 M (ram) c o n c e n t r a t l o n s . O p e n s y m b o l s i n d i c a t e i n c u b a t i o n s f r o m w h i c h t h e d r u g w a s o m i t t e d a f t e r 30 m m o f i n c u b a t i o n (arrow). S o h d s y m b o l s mdlcate i n c u b a t i o n s m which the drug was present t h r o u g h o u t the mcubatlon.

397 somal mixed-function oxidases, rat-liver microsomes were included in the incubation. The production of 3-hydroxy-benzo[a]pyrene (3-OH-BP) in incubations with liver microsomes from rats treated with methylcholanthrene is shown in Fig. 3a. The production of 3-OH-BP decreased after 10 min in incubations with 8 and 25 pM BP, indicating that most of the BP had been metabolized b y this time. Human fibroblasts were included in the same type of incubation, and the a m o u n t of DNA strand breaks was analysed. Fig. 3b shows that there was an induction of strand breaks in the fibroblasts exposed to 8, 25 and 80 pM BP in the presence of rat-liver microsomes. A lag between the time when all BP was metabolized (Fig. 3a) and the maximal a m o u n t of induced strand breaks was noted in the 8 pM incubations (Fig. 3b). When NADP was omitted from the incubation, aeither BP metabolism (not shown) nor any induction of strand

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Fzg. 3a. B e n z o [ a ] p y r e n e m e t a b o l i s m . F o r m a t i o n o f 3 - h y d r o x y - b e n z o [ a ] p y r e n e f r o m b e n z o [ a ] p y r e n e b y liver m m r o s o m e s (0,7 m g p r o t e i n / m l ) f r o m M C - t r e a t e d rats. B e n z o [ a ] p y r e n e c o n c e n t r a t i o n s : 8 # M (v), 2 5 # M (A), 80 #M (m). Fig. 3b. I n d u c t z o n of D N A - s t r a n d b r e a k s m h u m a n f i b r o b l a s t s b y b e n z o [ a ] p y r e n e . Ceils w e r e i n c u b a t e d w i t h h v e r m i c r o s o m e s (0.7 m g p r o t e i n / m l ) f r o m M C - t r e a t e d rats, a n N A D P H - g e n e r a t m g s y s t e m a n d b e n z o [ a ] p y r e n e . N o DMSO (o); 8 # M ( v ) ; 25 juM (4): 80 #M ( a ) . O p e n s y m b o l s i n d i c a t e i n c u b a t i o n s f r o m w h i c h t h e d r u g was o m z t t e d a f t e r 3 0 rain o f i n c u b a t i o n . S o h d s y m b o l s i n d i c a t e i n c u b a t i o n m w h i c h the d r u g was p r e s e n t t h r o u g h o u t t h e i n c u b a t i o n . Fig. 4. I n d u c t i o n o f D N A - s t r a n d b r e a k s in h u m a n f i b r o b l a s t s b y c y c l o p h o s p h a m z d e . Cells w e r e i n c u b a t e d w i t h liver m i c r o s o m e s (0.7 m g p r o t e m / m l ) f r o m p h e n o b a r b i t a l - t r e a t e d rats, an N A D P H - g e n e r a t m g s y s t e m a n d c y c l o p h o s p h a m z d e . 0 (o), 5 × 1 0 -5 M (v), 5 × 10 .-4 M (4), 5 × 10 -3 M (o). O p e n s y m b o l s i n d i c a t e i n c u b a t i o n s f r o m w h i c h t h e d r u g was o m i t t e d a f t e r 30 m m o f i n c u b a t z o n ( a r r o w ) . Solid s y m b o l s i n d i c a t e i n c u b a t i o n in w h i c h t h e d r u g w a s p r e s e n t t h r o u g h o u t t h e i n c u b a t z o n .

398 breaks (not shown) was recorded. A DNA repair process was completed after removal of BP by changing the medium as indicated by the decrease in strand breaks (Fig. 3b).

Cyclophosphamide To study the effect of an indirectly acting bifunctional alkylating agent in the system, human fibroblasts were incubated with cyclophosphamide in the presence of rat-liver microsomes. The dose and time relation for cyclopnosphamide-induced DNA-strand breaks is shown in Fig. 4. The fibroblasts were incubated with microsomes from rats treated with phenobarbital. Cyclophosphamide caused an early and slight increase in strand breaks at a concentration of 5 × 10 -s M. This is in contrast with the decrease in single-stranded DNA eluted in incubations with 5 X 10 -3 M cyclophosphamide at 10 min. At longer incubation times a high level of strand breaks was detected in the 5 × 10 -3 M incubation. No effect of cyclophosphamide was seen m the absence of NADP (not shown). In contrast with the other drugs studied, the level of strand breaks did n o t decrease within 21 h after removal of cyclophosphamide at 60 min.

Discussion DNA repair can be used as an indirect way of detecting DNA damage [4,13, 20]. The known DNA-repair mechanisms involve, at some stage of the process, one or more strand breaks per repaired site. Thus, measurements of DNAstrand breaks at various times after production of DNA damage may give information about the various steps involved in DNA-repair processes. In this study a significant increase in the a m o u n t of DNA-strand breaks was recorded after exposure to MMS and melphalan. Similar results were obtained for BP and cyclophosphamide when a rat-liver metabolizing system was present. The monofunctional alkylating agents (MMS and BP products) gave rise to a rapid increase in strand breaks. The difference noted in the time required for elimination of the strand breaks are most likely due to differences in the spect r u m of DNA damage induced by the various drugs. BP is known to give rise to at least two different DNA-binding products [9], and these show distinct differences in repair kinetics [17]. Exposing the cells to melphalan and cyclophosphamide resulted m a slower increase and a lower maximal value of apparent strand breaks. This may to a certain extent be due to inter-strand cross-linking caused by these bifunctional alkylating agents, resulting in enhanced renaturation after alkaline treatment of DNA. After a certain a m o u n t of chain breaks has occurred, the presence of cross-links will result in an increase in the DNA fraction eluted from hydroxylapatite as double-stranded, and thus an apparently lower a m o u n t of strand breaks will be recorded. As Fig. 4 illustrates, 5 × 10 -3 M cyclophosphamlde gave rise to an initial decrease in single-stranded DNA eluted, suggesting the presence of inter-strand cross-links. Using alkaline elution, Swenberg et al. [22] found induction of DNA-strand breaks in Chinese hamster fibroblasts (V79) after treatment with MMS and BP. However, in contrast with our findings, no breaks were observed after cyclo-

399 phosphamide treatment in the presence of rat-liver microsomes ($9 fraction) in that system [22]. This discrepancy might be explained by differences in (i) the metabolizing systems, (ii) the type of cell studied, (iii) the interference of crosslinks in the system used, and by the better resolution in the present study with multiple timepoints. In contrast with the results from other chemicals studied we did not observe a total elimination of the chain breaks induced after treatment with cyclophosphamide within 21 h. The reason for this difference is not clear, and from the present data a concentration effect cannot be excluded. However, a similar persistence of strand breaks has been found in rat gliosarcoma cells after exposure in vivo to 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU) [8]. These findings indicate that further detailed studies of the repair of DNA damage induced by cyclophosphamide and CCNU may be of interest. The method described can be used for studies of DNA-repair mechanisms [17] and for qualitative studies of the genotoxic activity of chemical agents. Our experimental set-up makes it possible to use different metabolizing systems. Thus, it is possible to incubate isolated hepatocytes with human fibroblasts in order to study the cell-to-cell transfer of reactive metabolites [16]. The role of metabolic processes other than those present in isolated microsomes, e.g. conjugation, can then be studied. As illustrated in Fig. 3, the incubation procedure also enables studies to be made of the metabolism in the system. Thus, it may be possible to relate the formation of a specific reactive metabolite to the formation of DNA damage in the same incubation. The importance of different metabolic mechanisms for the generation of DNA damage can then be analysed. The system described may be further developed for a study of the mechanisms of DNA repair after chemically induced damage by using repair-deficient cells and/or drugs that interfere with certain steps in the DNA repair process, e.g. hydroxyurea [5], 2-chloroethylisocyanate [7], and dideoxythymidine [19].

Acknowledgements This work was supported by the Swedish Medical Research Council (No. 3681), the Swedish Cancer Society (l180-k78-01X) and NIH (Contract 1 CP 33363). We thank Kerstin Willander for technical assistance.

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