DNA damage induced in vivo evaluated with a non-radioactive alkaline elution technique

Cancer Letters, 6 (1979) 221--226

221

© Elsevier/North-Holland Scientific Publishers Ltd.

DNA DAMAGE INDUCED IN VIVO EVALUATED WITH A NON-RADIOACTIVE ALKALINE ELUTION TECHNIQUE

M. SCI"IWARZ,J. HUMMEL, K.E. APPEL, R. RICKART and W. KUNZ Institute of ~iochemistry, German Cancer Research Centre, Heidelberg (F.R.G.) (Received 6 November 1978)

~Aecepted 7 December 1978)

SUMMARY A modification of the alkaline filter elution test was used to study damage to liver DNA of NMRI mice following in vivo treatment with various hepatocarcinogens and drugs causing liver enlargement. Liver cell nuclei were prepared and lysed on top of polyvinyl filters. D N A w ~ eluted, and the amounts both remaining on the filter znd in the eluate were measured colorimetrically. Dimethyinitrosamine (DMN)~ diethylnitrosamine (DEN),N-nitrosomorpholine and methyl methanesulfonate (MMS) caused significant enhancement of DNA passage through the filters, whereas N-acetylaminofluorene (AAF), 4-dimethylaminoazobenzene (DAB), phenobarbital, halothane and CC14 did not. The applicability of this short term test is discussed.

INTRODUCTION Chemical carcinogenesis is commonly believed to result from the interaction if a carcinogen or of an active metabolite therefrom with target cell DNA, leadin~ to cellular trm~sl~ormation. Measurement o f alterations to DNA followhag exposure to f o r e i ~ compounds therefore appears to be a suitable tool l~or detecting potential carcinogens. Damage to DNA following '~rea~ment of cells cultured in vitro with a great variety of miscellaneous compounds, including es~,ablished carcinogens, has been demonstrated by Swenberg et al. [9] by meamL of the alkaline filter elution method o r / ~ ! l y developed by Kohn ~ad Grimek-Ewig [4]. More Address all correspondence to: Michsel Schwarz, Institute of Biochemistry, German Cancer

Research Centre, Ira Neuenheimer Feld 280, 6900 Heidelberg, F.R.G. Abbreviations: AAF, N-ace~ylaminofluorene; DAB, 4-dimethylaminoazobenzene ; DABA, 3,5-diamlnobenzoic acid. DEN, diethylnitrosamine, DMN, dimethylnitrcsamine; DMSO, dimethyl sulfo.~ide;MMS, methyl methanesulfonate; PCA, pereblorie acid; TEAH, tetraethylammonium hydroxide.

222 recently, this technique has been successfully used to demonstrate D N A d~mage after exposure of ra1~sto carcinogens in vivo [7]. A major disadvantage of the testsdescribed is that the D N A must be radioactively labelled prior to alkalineelution. A non-radioactive method to detect carcinogen-induced strand breaks in D N A by means of alkaline sucrose gradient centrifugation has been used by Zubroff and Sarrna [12]. In this commumcation a modification of the alkaline filterelution method is presented whereby D N A is measured colorimetfically,thereby avoiding the need for D N A pzelabelling. MATERIALS A N D M E T H O D S Chemicals

DMN, DEN, MMS, DAB, 3,5-diaminobenzoic a.'id (DABA) and tetraethylammonium hydroxide (TEAH) were obtained from Merck-Schuchardt, Miinchen, A A F was purchased from Deutsche Hc.ffmann ha Roche, Grenzach; halothane was procured from ICI, Plankstadt; phenobarbital-Na was purchased from Pharma Zentrat GmbH, Herdecl~e; i~-nitrosomorpholine was the generous gift o f Prof. Bannasch, this centre; polyvinyl filters were purchased from the Millipore Corporation, Bedford, Massachusetts, USA. Treatment o f an imals

Male and female mice of the NMRI strain were maintained on a standar~ diet (Altromin pellets), Water was continually available. DMN, DEN, d!~-nitrosomorphotine and MMS were diluted in 0.15 M saline to a volume o f about 100 ~l/i.p. injection. MMS was used immediately after diluti~')n. A A F was dissolved in dimethyl sulfoxide (DMSO), DAB in DMSO or in olive oil. The compounds were administered either by means of a stomach tube ~)r b y i.p. injection. Controls received vehicles orfly. F o r induction of liver enzymes mice received phenobarbital-Na (0.1% in the drinking water) for 5 days, or were exposed to b alothane vapour (1% in air) on 6 consecutive days for i h a day. Animals were sacrificed by decapitation; liver~ were rapidly removed and gently triturated in a mortar with a porcelain pestle. The resulting suspension was centri£uged at about 6 X g for 1 rain; aliquots of the supezmatants were used for alkaline elution. Microscopic examination showed that this preparative procedure yielded well presereed nuclei with little c o n t a m i n a t i o n b y adherent cytoplasm. Fi,Iter el~Ition Alkalh~e e!lutionof D N A from polyvinylfilterswas carried out e~sentially according to the method of K o h n and Grimek-Ewig [4] with the modifications described by Swenberg et al. [9]. Briefly, nuclei (3 X I0~--7 X 10 '6)suspended in i ml of "~heN a C I / E D T A buffer were deposited on the filterat a flow rate of 2 ml/min, and washed with 2 ml of the sm'ne buffer. The flow rate was then

223 changed to 0.3 ml/min, and the nuclei were lysed with 6 ml of 0.2% Triton-X100 plus 0.02 M ~3DTA in 2 M NaC1, pH 8.2. The lysis medium w:s washed out with 5 ml 1 mM EDTA, pH 7.8, and the filters were eluted with 13 ml of the TEAH-based buffer, pH 12.2, containing 0.02 M EDTA. TEAH in 10% solution was added to give the required pH of the buffer. The last 18 ml of eluate were collected in centrifuge tubes in the cold.

DATA determination In routine measurements amounts of DNA both remaining on the filter and in the eluate were determined with the diphenylamine method of Burton [2]. DNA in the eluate was precipitated by the addition of 4 ml of 4 N PCA. After standing for 15--30 min in the cold, DNA was centrifuged down at 500 × g for 15 rain, and the supernatant was carefully drawn off. From this point filters and eluates were treated identically; 0.5 ml of 0.6 N perchloric acid (PCA) were added to each tube, and the DNA hydrolysed by heating at 95°C for 15 rain. After coolin|~,, 1 rnl of Button's reagent was added, and the extivLctions measured at 580 nm after standing for 17--20 h at 30°C. The filters alone gave only blank values. Standard curves over the range of 10--100 #g/DNA revealed linear relationsh ips. DNA amounts were sometimes estimated by a more sensitive fluorometric technique [3]. After elution at pH values described later, the filters were further eluted with 2 ml of 0.6 N TCA, 2 ral of cold 0.1 M potassium acetate in ethanol, 2 ml ethanol at 60°C, 2 ml cold ethanol and 1 ml 0.6 N PCA. The DNA in the eluate was precipitated as described above, and further purified using the same procedure as for the filters, except that all washing steps were performed in a centrifuge tube. DABA dihydrochloride solution (0.5 ml) was added to the precipitated DNA of both filter and eluate, and allowed to react for 30 rain at 60°C. Fluorescence was determined in a Perkin Elmer MPF 44 fluorescence spectrophotome~er after diluting with 3 ml 0.6 N PCA. The excitation wavelength was 405 nm; the: fluorescence was read at 520 nm. The fluorimetric method was very sensitive, b u t had the disadvantage that the filters alone gave a high background fluorescence with DABA dihydrochloride. RESULTS

The effect of pH on the course of DNA elution from the filters is shown in Fig. 1. No DNA from DMN-treated mice (10 mg/kg} passed through the filters at pH 10.5, whereas at pH values above 13 the dameged DNA could be eluted extensively. Unfortunately, considerable amounts of control DNA could be eluted as well. Optimal elution was obtained at pH 12.2; at this pH control DNA remained almost completely on the filters, whereas about 50% of DNA from DMN-treated animal passed through. Under these conditions a dose-dependent increase in alkaline elution of DNA was found in mice pretreated with various amounts of DMN (Fig. 2a). This

224 100, \ .~50'

T

alkaline elution

N

e|uted Fig, 1. ~ffeet of pH on the elution of DNA, Mice were exposed to 10 mg/kg DMN for 4 h. Homogenisation of the liver and DNA elution were as described in the text. Fractions of the eluates (3 mI) were collected, an~l the amounts of DNA both remaining on the filters and in the eluate were measured after ccnversion t o a fluorescent derivative by reaction with diaminoben~oie acid dihydrochloride; *, pH 10.5 ; *,~pH 11.5; e, pH 12.2; o. control 12.2, The arrows indicate the start and duration of elution with the appropriate alkaline buffer.

damage was partly repaired in the course o f the next 24 h (Fig. 2b). Both these results correlate well with data obtained by e]ution of radioactively prelabe]led rat liver DNA under comparable condition~ [7]. Administration of all oth~.r alkylating carcinogens investigated resulted in significant increases in DNA elution (Table 1). MMS was found to be most effective, at the high dose of 150 mg/kg. The hepatocarcinogenic aromatic amines DAB and AAF, however, failed to alter the elu:ion characteristics of liver DNA. The same was true for the hepatocarcinogen CCI4, even when given in doses sufficiently high to cause massive liver cell necrosis. Administration of phcn.~barbital and halothane under conditions which effectively induce liver growth associaCed with DNA synthesis produced no changes in DNA elution. 100

100"

B

A

.~.

N

6 ~

g--

1~

DMN{rngtkgl

Z

¢2

Time after treatment (houPsl

2~-

Fig. 2. (A) Effect o f different doses of DMN on the alkaline elution charactezisties o f liver DNA. DMN was injected i.p. and allowed to remain for 4 h. (B) Time-dependent changes in the elation rates of |iver D N A following a single dose o f 10 mg/kg DMN. The dashed tines represent the lower limit of control D N A eluted under identical conditions. Livers were homogenized and DNA elution carried o u t as described in the text. DNA both remaining ass the filters and in the eluat~s w ~ determined using the diphenylamine method o f Burton.

225

TABLE 1 EFFECT OF VARIOUS FOREIGN C O M P O U N D S ADMINISTERED IN VIVO O N THE ALKALINE ELUTION CHARACTERISTICS OF M O U S E LIVER D N A Compound

Dose

Mode of admini-

Duration of

stratiovL

treatment

Co~troI

Percent remaining

na

on the filter~ S.D.

10 mg/kg 100 mg/kg 100mg/kg

i.p. i.p, i.p.

I h 1h 4 h

Saline or oil or DMSO Saline Saline Saline

100 mg/kg

Lp.

4 h

Saline

61 ± 3.8

5

i.p. i.p.

12 h 4h

Saline Saline

67 ± 3.5 18 -+ 2.3

5 5

DAB

100 mg/kg 150mg/kg 20--I00 mg/kg 100 mg/kg

i.p. or p.o. i.p. or p.o.

4--24 h 4h

98 ± 2.1 92 ± 2.5

10 5

CCI4

2.5 ml/kg

p.o.

4h

95 -+ 3.8

5

Phenobarbital

0.1%

Drinkng

5 days

DMSO Oi1 or DMSO Undiluted or oil Water

96 ± 1,4

5

99 ~ I

5

D~N Dt:N DEN Nitrosomorpholine Nitrosomorpholine MMS AAF

up to 24 h

Vehicle

96 ± 2.8

15

38 ± 7.7 56 ± 7.9 74 ± 5.G

10 5 5

water Halothane

1%

Inhalation

6 day/ 1 h daily

Air

Controls receivedvehiclesonly. D N A was measured by the diphenylamine method. n, number of animals tested.

DISCUSS ION

The major advantage of the technique described is that damage to D N A can be studied without using either radioactive carcinogens or radioactive D N A . Thus, those mutagens or carch~ogens suspected of being able to react with D N A to produce alkali-labilesites can be tested cheaply and simply in non-proliferating tissues of various animal species. In principle D N A strand breaks can be generated either enzymatically or chemically 16,11]. Alkali-labile reaction products of D N A with covalently binding carcinogens have been described[ [1,5,8,10]. Indeed, all alkylating compounds tested were found to enhance D N A elution under the alkaline conditions used (Table !). However, administration of other hepatocarcinogens investigated gave no hint that alkali~sensitive reaction products are formed in mouse liver (Table 1). Regardless of whether such alkali-labile reaction products might not be formed in vivo or

226 f o r m e d t o such a small e x t e n t as t o b e u n d e t e c t a b l e b y t h i s t e c h n i q u e , t h e f i n d i n g s i m p o s e a l i m i t o n t h e value o f t h e p r e s e n t m e t h o d f o r large-scale s c r e e n i n g o f c h e m i c a l carcinogens. N o n e t h e l e s s , t h e t e c h n i q u e appears useful to study the influences of va~ous substances on the metabolic activation and i n a c t i v a t i o n o f a l k y l a t i n g c a r c i n o g e n s in v a r i o u s organs, a n d also enables a b a l a n c e t o b e s t r u c k b e t w e e n D N A d a m a g e a n d r e p a i r in c o n s i d e r a b l y shortened periods of time. ACKNOWLEDGEMENTS T h e a u t h o r s gratefully a c k n o w l e d g e Dr. R. ,/ones a n d Dr. H.W. T h i e l m a n n f o r h e l p f u l discussions. REFERENCES 1 Bannon, P. and Verly, W. (1972) Alkylation of phosphate and stability of phosphate triesters in DNA. Eur. J. Biochem., 31,103--111. 2 Burton, K.A. (1956) A study of the conditions and mechanis~r~s of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J., 65, 315--323. 3 Kissane, J.M. andRobins, E.(1958)Thefluorimetricmeasuret.':~n~ofdeoxyribonucleic acid in animal tissues with ~special reference to the central nervous system. J. BioI. Chem., 233, 184--188. 4 Kohn, K.W. and Grimek-Ewig, R.A. (1973) Alkaline elution analysis, a new approach to the study of DNA single-strand interruptions in cells. Cancer Res., 33, 1849--1863. 5 Lawley, P.D. and Brookes, P. (1963) Further studies on the alkylation of nuclei acids and their constituent nucleotides. Biochem. J., 89, 127--138. 6 Peterson, A.R, Bertram, J.S. z~nd Heidelhe:ger, C. (1974) DNA damage ard its repair in transformable mouse fibrobla~ts treated with N-methyl-N'-nitro-N-~osoguanidlne. Cancer Res., 34, 1592--1599. 7 Petzold, G.L. and Swenberg, J.A. (1978) Detection of DNA damage induced in viuo following exposure of rats to c:vxcinogens. C ~ c e r Res., 38, 1589--1594. 8 Shooter, K.V. (1976) The kin:etics of the alkaline hydrolysis of phosphotrieaters in DNA. Chem.-Biol. Interact., 1~, 151--163. 9 Swenberg, J.A., Petzold, G.L..~nd Harbach, P.R. (1976) In vitro DNA damage/alkaline elution assay for predicting caJ~cinogenic potential. Biophys. Biochem. Res. Commun., 72, 732--738. 10 Tamm, C., Shapiro, H.S., Lips]~:itz, R. and Chargaff, E. (1953) Distribution of nucleotides within a deoxyribonucleic acid chain. J. Biol. Chem., 203, 673--688. 11 Thielmann, H.W. (1977) Dete~:tion of strand breaks in 0X174 RFI and PM2 DNA reacted with ultimate and pro~:~mate carcinogens. Z. Krebsforsch., 90, 37--69. 12 Zubroff, J. and Sarma, D.S.R. (1~,76) A non-radioactive method for mea6uring DNA damage and its repair in non-proliferating tissues. Anal. Biochem., 70, 387--396.