Effects of β- and γ-carboline derivatives on DNA topoisomerase activities

Fundamental and Molecular Mechanisms of Mutagenesis

ELSEVIER

Mutation

Research 349

( 1996) I X3- 19 I

Effects of p- and y-carboline derivatives on DNA topoisomerase activities Yasunori Funayama a, Kazuto Nishio ‘, Keiji Wakabayashi b, Minako Nagao ‘, Kayoko Shimoi d, Tatsuo Ohira a, Shizuo Hasegawa e, Nagahiro Saijo a.* ” Pharmacology

Dir,i.sion. National

h Riochenzist~

Cancer Center Research Institute, Cancer Center Research Institute.

’ Curcino,genesis

Dif,ision.

National

of Food

Hygiene,

School of Food cmd Nutritional

’ Laboratory ’ Dit,ision

Dir~ision. National

of’Ptr/monaq

Medicine.

Cancer Center Research Institute.

Insirute of Clinical

Medicine.

I-l I-l l-l

Tsukiji 5-chome, Chuo-ku. Tsukiji &home, Tsukiji 5.chome,

Sciences. tinirersity Unil,ersi~

Chuo-ku,

of TwI&a.

Chuo-X-u. Tokyo 104. Japan

of Shizuoka. 52-l I- I Amakuho

Tokyo 104. Japan Tokyo 104, Japan

Yada. Shiwoka

2-chotne,

422. Japan

T.sukuha-shi, lharaki

305.

Jrtpan

Received 26 June 1995: revised 79 August 1995: accepted 9 September 1995

Abstract P-Carbolines, harman (I-methyl-9H-pyrido[3,4-hlindole) and norharman (9H-pyrido[3,4-blindole) and y-carbolines, 3-amino-l,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-l) and 3-amino-4-methyl-SH-pyrido[4,3-b]indole (Trp-P-2). are present in cooked foods and cigarette smoke. We studied the effects of these heterocyclic amines on the activity of DNA topoisomerases. Trp-P-l and Trp-P-2 inhibited topoisomerase I (topo I) activity with ED,, values of 1.48 and 1.55 pg/ml, respectively, in a relaxation assay. Harman and norharman inhibited topo I activity but with much higher ED,, values, 23.8 and 34.4 pg/ml, respectively. Trp-P-l and Trp-P-2 also inhibited topoisomerase II (topo II> activity at about 50 pg/ml, in a decatenation assay. Harman and norharman showed a much lower inhibitory effect on topo II activity. None of these compounds stabilized the cleavable complex mediated by topo II. Trp-P-l and Trp-P-2 intercalated into DNA at concentrations inhibitory to topoisomerases. We considered that the intercalation with DNA and the inhibition of DNA topoisomerases by heterocyclic amines might be partly related to their inhibition of DNA excision repair and their enhancing effect on UV- or chemically induced mutagenic activity.

1. Introduction

Abbreviations: DNA, deoxyribonucleic acid: UV, ultraviolet; Topo I. topoisomerase I: Topo II. topoisomerase II: SDS, sodium dodecyl sulfate: BPB. bromophenol blue: EDTA. ethylenediaminetetraacetic acid; ATP. adenosine 5’Qriphosphate Corresponding author. Tel.: 81-3-3542-25 I I : Fax: 81-3-3542.

I886 0027.5107/96/$15.00

0 1996 Elsevier Science B.V. All rights reserved

SSDI 0027-5107(95)00176-X

P-Carbolines, harman (l-methyl-9Wpyrido[3,4b]indole) and norharman (9H-pyido[3,4-blindole) and y-carbolines. 3-amino-1.4-dimethvl-SH-, pyrido[4,3-blindole (Trp-P-l), and 3-amino- lmethyl-5H-pyrido[4,3&]indole (Trp-P-2) are compounds present in cooked foods and cigarette smoke (Fig. 1). Trp-P-l and Trp-P-2 show high mutagenic

\ c&Q /N

CY

Harman

H

Norharman

CY

/ cx$L N

NH?

Trp-P-l Fig.

Trp-P-2

I Structures of Trp-P- 1, Trp-P-2, harman and norharman

activity to Snlrnorrella ~~~z~~~~j~~~ in the presence of metabolizing enzymes (Sugimura et al.. 1989). They also produce DNA damage in mammalian cells. and have been proven to be carcinogenic in rodents (Matsukura et al.. 198 I). TrpP-1 has been reported to inhibit DNA excision repair following UV-induced DNA damages in E. coli (Mori et al., 1993). This inhibitory effect of these y-carboline derivatives has been considered to be a mechanism of enhancing effects of these compounds on UV- or chemically induced mutagenesis (Shimoi et al., 19921. It has also been reported that in cultured mammalian cells, the number of chromosome aberrations induced by UV is increased after treatment with Trp-P-l or Trp-P-2 (Sasaki et al.. 1992). Harman and norharman are not mutagenic by themselves, but they showed co-mutagenicity to S. tyhimurium TA98 with S9 mix, enhancing the mutagenicity of dimethylaminoazobenzene (Nagao et al., 1977). Norharman also shows mutagenicity in the presence of nonmutagenic compounds such as aniline (Nagao et al.. 1977) and diphenylamine remarkably (Wakabayashi et al., 1982). However, the mechanism of the comutagenicity of P-carbolines is still unclear. DNA topoisomerases are enzymes that control and modify the topological states of DNA. Topoisomerase I (topo 11 catalyzes the passage of the DNA single strand through a transient single-strand break. while topoisomerase II (topo II) catalyzes passages of DNA double strands through a transient double1985). These enzymes are strand break (Wang, known to be involved in many important physiological functions of DNA, including replication, recom-

bination, transcription, and chromosome segregation at mitosis (Liu. 1989). Recently, it has been reported that DNA topoisomerases affect DNA excision repair (Jones et al., 19911; for example, the 180-kDa form of topo II is involved in the steps preceding repair-specific DNA incision (Popanda and Thielmann. 19921, and thus, it is possible that the inhibition of DNA repair by heterocyclic amines is related to DNA topology. In the present study, we examined the effects of TrpP- I and TrpP-2 on the activity of DNA topoisomerases in relevance to the inhibition of DNA excision repair. In addition, the effects of P-carbolines, norharman and harman, were also examined under the same conditions and compared their effects with those of y-carbolines.

2. Materials and methods 2. I. Chemids Trp-P- 1 CH ,COOH and Trp-P-2 CH ,COOH were purchased from Nard Institute, Osaka. Norharman and harman were obtained from Katsura Chemii cals (Tokyo) and Sigma (St. Louis, MO, USA), respectively. Etoposide, adriamycin and m-AMSA were obtained from Nihon Kayaku (Tokyo, Japan), Kyowa-Hakko Kogyo (Tokyo, Japan) and Sigma (St. Louis, MO, USA), respectively. Drugs were dissolved in dimethyl sulfoxide (Wako Pure Chemical Industries. Tokyo. Japan) at 50 mg/ml (harman, norharman). 25 mg/ml (Trp-P-l . CH,COOH, Trp P-2 . CH,COOHl. 20 mg/ml (etoposide), and 50 mM (~r7-AMSA), or in distilled water at 10 mM (adriamycin). They were stocked at - 20°C until use. Calcium- and magnesium-free Dulbecco’s phosphate-buffered saline, RPM1 1640 and fetal bovine serum (FBS) were purchased from Daiichi Pure Chemicals (Tokyo. Japan), Nissui Pharmaceuticals (Tokyo, Japan) and Biobeck Laboratories (Toronto. Canada), respectively. Proteinase K, [ a-” P]dCTP. pBR322 plasmid DNA, Klenow fragment of DNA polymerase I and T4 DNA ligase, were provided by Merck AG (Darmstadt, Germany), Amersham Japan (Tokyo, Japan). Toyo Boseki (Tokyo, Japan), Takara Shuzo and Boehringer-Mannheim (Germany). rem spectively. Trypanosomatoid kinetoplast DNA

(kDNA1 and sodium dodecyl sulfate (SDS) were purchased from Wako Pure Chemical Industries (Tokyo, Japan). Hind111 and EcoRI were purchased from Takara Shuzo. Human type I and II topoisomerases were obtained from TopoGen (Columbus. OH, USA). Other materials were purchased from Sigma (St. Louis. MO, USA). 2.2. Cell lines A human small cell lung cancer cell line, H69 and a human breast cancer cell line, MCF-7 were grown in RPM1 1640 medium supplemented with 10% FBS. penicillin (100 U/ml) and streptomycin (100 pg/ml) in a humidified atmosphere of 5% CO,/95% air at 37°C. 2.3. Prepnratioil

of crude nucleur extructs

Crude nuclear extracts were prepared according to the method described by Defie et al. (1989). Briefly, about 1 X lo* cells in early log-phase culture were collected by centrifugation (H69) or detached by treatment with trypsin-EDTA (MCF-7). and washed twice by centrifugation with cold NB solution containing 2 mM K,PO,, 5 mM MgCl,, 150 mM NaCl, 1 mM EGTA and 0.1 mM dithiothreitol, and the pH was adjusted to 6.5. Cell pellets were then lysed in 1 ml of cold NB solution and 9 ml of NB solution containing 0.35% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride (PMSF) on ice for 10 min. The suspension was centrifuged at 1000 X g for 10 min, and washed once with Triton-free NB solution. The nuclear pellets were resuspended in 0.3 ml of cold NB solution, then an equal volume of NB containing 0.7 M NaCl (final concentration of NaCl, 0.35 Ml was added and kept on ice for 60 min. The mixture was then centrifuged at 17 000 X g for 15 min. The protein concentration in the extracts was determined by the method of Bradford (Bradford, 1976). The extract was stored in 50% glycerol at -80°C until use. 2.4. DNA relaxation with topoisomeruses

und DNA clear,age

reactions

DNA relaxation and cleavage activities of topoisomerases were examined according to the method

described by Yamashita et al. (19911 with some modifications. For topo I reactions, the reaction buffer consisted of 10 mM Tris . HCl (pH 7.5), 100 mM KCl, 1 mM PMSF, 10 mM mercaptoethanol, 50 pg/ml bovine serum albumin, pBR322 plasmid DNA (0.15 pg in 1 ~1 of Tris-EDTA buffer) and topo I. and various concentrations of drugs were incubated at 37°C for 15 min. For the relaxation assay. 0.3 U of topo I was used and the reactions were terminated by addition of 5 X stop solution (ScTr SDS, 0.25% BPB, 50% sucrose), then the samples were electrophoresed through 1% agarose in Tris-acetate-EDTA at 60 V for 3 h. The gels were stained with ethidium bromide and photographed under UV light. For DNA cleavage assay, 6 U of topo I was used and the reactions were terminated with 1% SDS and 0.5 mg/ml proteinase K, followed by incubation for an additional 30 min at 50°C. After addition of 6 X loading buffer (0.25% BPB, 0.25% xylene cyanol, 50% glycerol), the samples were analyzed by agarose gel electrophoresis as described above. Topo II reactions were performed in the reaction buffer (50 mM Tris . HCl (pH 8.0). 120 mM KCl, 10 mM MgCl?. 0.5 mM ATP, 0.5 mM dithiothreitol, 30 mM bovine serum albumin), 0.15 pg of pBR322 DNA, 0.6 U of topo II, and various concentrations of drugs. The reaction mixtures were incubated at 30°C for 10 min for the relaxation and cleavage assays. Other procedures were the same as for the topo I assays described above. After photography under UV light. the amount of DNA was quantified by scanning with an Image Master (Pharmacia Biotech). 2.5. 1~~~~~~~~~ ,for DNA decatenation

actici8

of top II

The catalytic activities of topo II were assayed by decatenation of kDNA to minicircular DNA (Marini et al., 1980). The reaction buffer consisted of 50 mM Tris (pH 8.01, 120 mM KCl, 10 mM MgCl?, 0.5 mM ATP, 0.5 mM dithiothreitol and 30 mM bovine serum albumin. The 15 ~1 of reaction mixture containing the buffer, 0.1 pg kDNA, 0.4 U of topo II and various concentrations of drugs was incubated at 30°C for 15 min. The reaction was stopped by addition of 4 ~1 of the 5 X stop solution. Then the samples were electrophoresed through 1% agarose in Tris-acetate-EDTA at 60 V for 3 h. The gels were

Y. Fwznwma

186

et ul./Mutntior~

stained with ethidium bromide and photographed under UV light. For the combination of harman and Trp - P - 1, the reaction mixture containing the reaction buffer, 0.2 pg kDNA. 50 pg/ml nuclear extract, 50 or 200 pg/ml harman, and various concentrations of Trp - P - I was incubated at 30°C for 30 min. 2.6. Cleavable

complex ,formation

assay

The assay of cleavable complex formation was performed by the method of Liu et al. (1983). Briefly, 0.3 pg of pBR322 plasmid was linearized by digestion with HindIII. The linear plasmid was then labeled at the 3’ ends with [ CY-~’P]dCTP by the Klenow fragment of DNA polymerase I. and the 3”-labeled DNA was digested with EcoRI again.

(511

1 2

Research 349 (19961 183-191

Unincorporated nucleotides were removed by three sequential precipitations with ethanol/ammonium acetate. Then 3’-labeled DNA was incubated at 37°C for 15 min with an appropriate amount of topo II in the presence or absence of various concentrations of drugs in 30 ~1 of the reaction mixture containing 10 mM Tris (pH 7.0). 1 mM MgCl?, 0.5 mM EDTA and 10 pg/ml bovine serum albumin. The reaction was terminated by addition of 60 ~1 of a stop solution containing 2% SDS. 2 mM EDTA and 0.5 mg/ml salmon testis DNA, and heating at 65°C for 10 min. The cleavable complex was then precipitated by addition of 30 ~1 of 0.25 M KCI and incubation on ice for IO min. The precipitates were collected by centrifugation. and the pellet was washed with 200 ~1 of a solution containing 10 mM Tris (pH 8.0). 100 mM KCI. 1 mM EDTA and 100 pg/ml salmon

3 4 5 6 7 8 9 101112131415

(b)

161718192021

222324252627

‘O”] 60

4

x 0

60

8 Z? t

40 -

5 8

20 -

“3.3

1

o-1 drug

100 concentration

(pgiml)

Fig. 2. Inhibitory effects of Trp-P- 1. Trp-P-2, harman and norharman on topo I-mediated DNA relaxation. (a) Lane I, supercoiled pBR322 DNA; lane 2, linear DNA: lane 3. no drug; lanes 4-9. Trp-P- I : lanes IO- 15, Trp-P-2: lanes 16-2 I, harman; lanes 22-27, norharman. Drug concentrations were as follows: lanes 4, 10, 16 and 22.0.08 pg/ml: lanes 5. 1 I. 17 and 23. 0.4 pg/ml; lanes 6. 12. 18 and 24. 2.0 pg/ml: (b) The proportion of lanes 9. 15, 21 and 27. 250.0 pg/ml. lanes 7, 13, 19 and 25, 10.0 Kg/ml: lanes 8, 14, 20 and 26, 50.0 pg/ml: relaxed DNA to total DNA measured by scanning with an Image Master (Pharmacia Biotech). Horizontal axis represents the concentration of Trp-P-l, Trp-P-2, harman and norharman. and the vertical axis represents the proportion of relaxed DNA. (0) Trp-P-l, (0) Trp-P-2. ( A ) harman. ( A ) norharman.

Y. Funayama et al./Mututim

Then, the reaction mixtures were incubated with 2 U of T4 DNA ligase at 15°C for 60 min, and the reactions were stopped by addition of EDTA at 20 mM final concentration. The drugs bound to DNA were removed by extraction with phenol and precipitation with ethanol. Then the samples were solubilized in 20 ~1 of distilled water, and electrophoresed in 1.0%) agarose gel after addition of 4 ~1 of 6 X loading buffer.

testis DNA at 65°C for 10 min. After recentrifugation, the pellet was solubilized in 200 ~1 of distilled water by heating at 65°C for 10 min and analyzed for radioactivity in a liquid scintillation spectrometer. Etoposide was used instead of other drugs as the positive control. 2.7. DNA unbinding

away

DNA unwinding effects of the compounds were assayed according to the method of Camillone et al. (19861. Briefly, pBR322 plasmid DNA was linearized by Hind111 digestion, extraction with phenol and ethanol precipitation. The pBR322 DNA (0.6 pg in 20 ~1 of distilled water) were equilibrated with various concentrations of drugs in the reaction buffer (66 mM Tris. HCl. pH 7.5, 6 mM MgCl?, 10 mM dithiothreitol, 0.7 mM ATP) at 15°C for 10 min.

(a >

1 2

187

Reseurch 349 119961 1X3-191

3. Results 3.1. Effects of /3- and y-carboline catalytic actkity of top0 I

deriratir,es on the

Effects of Trp-P-l, Trp-P-2, norharman and harman on topo I was determined by measuring relax-

3 4 5 6 7 8 9 101112131415

161719192021222324252627

‘*A* =c supercoiled e

loo-

o-

. . ..

..-I

.

10-l

drug

.

. . . . ..I

100

. . . . ..I

,......I

IO'

concentration

1

.

o2

..-T

i03

(yglml)

Fig. 3. Inhibitory effects of Trp-P-l. Trp-P-2, harman and norharman on topo II-mediated DNA relaxation. (a) Lane I. supercoiled pBR322 DNA; lane 2. linear DNA; lane 3, no drug; lanes 4-9. Trp-P-l; lanes 10-1.5, Trp-P-2: lanes 16-21, harman; lanes 22-27, norharman. Drug concentrations were as follows: lanes 4, 10, 16 and 22. 0.08 pg/ml: lanes 5. 11. 17 and 23, 0.4 wg/ml; lanes 6, 12, 18 and 24, 2.0 /*.g/ml: lanes 9. 15, 21 and 27, 250.0 pg/ml. (b) The proportion of lanes 7, 13, 19 and 25. 10.0 pg/ml; lanes 8. 14, 20 and 26, 50.0 pg/ml: relaxed DNA to total DNA measured by scanning with an Image Master (Phannacia Biotech). Horizontal axis represents the concentration of Trp-P-l, Trp-P-2. harman and norharman. and the vertical axis represents the proportion of relaxed DNA, (0) Trp-P-l. (0) Trp-P-2. ( A ) harman. ( A ) norharman.

188

catenated II)

decatenated

I+)

Fig. 4. Inhibition of decatenation of kDNA by TrpP-I, Trp-P-2, harman and norharman. Capalytic activity of topo II was assayed by decatenation of trypanosomatid kinetoplast DNA (1 pg) as described in Materials and methods. Lane I. untreated kDNA control: lane 2, no drug; lanes 3-8, Trp-P- 1; lanes 9-14, Trp-P-2: lanea 15-20. harman: lanes 21-26, norharman. Drug concentrations were as follows: lanes 3. 9. 15, and 21, 0.08 kg/ml: lanes 4, 10. 16, and 22, 0.4 Kg/ml; lanes 5, Il. 17. and 23. 2.0 Fg/ml: lanes 6, 12, 18. and 24, 10 ,ug/ml; lanes 7, 13, 19. and 25, 50.0 pg/ml; lanes 8. 14. 20, and 26. 250.0 kg/ml. Positions of catenated and decatenated DNA are shown by arrows

ation of pBR322 DNA. These four compounds decreased formation of the relaxed form of DNA in dose-dependent manners (Fig. 2). Levels of the relaxed form were decreased to 50% by Trp-P-l and Tip-P-2 at concentrations of 1.48 and 1.55 pg/ml, respectively. On the other hand, harman and norharman decreased formation of relaxed DNAs to 50% at concentrations of 23.8 and 34.4 pg/ml, respectively (Fig. 2b). However, no cleavage of DNA was detected with these four compounds (data not shown).

50% at concentrations of 1.3 and 7.9 pg/ml, respectively. On the other hand, harman and norharman, decreased the relaxed DNA levels to 50% at concentrations of 137.4 and 223.0 pg/ml, respectively (Fig. 3b). By decatenation assay, topo II catalytic activity was inhibited by Tip-P-1 and Trp-P-2 at about 50 pg/ml. However, harman and norharman did not inhibit the decatenation of kDNA even at 250 pg/ml (Fig. 4). These compounds did not affect the topo II-mediated DNA cleavage activity (Fig. 5).

3.2. Effects of /I- and y-carboline catalytic activity of top0 II

3.3. Effects of Trp-P-l complex formation

derivatives

on the

Catalytic activity of topo II was measured by cleavage of pBR322 and decatenation of kDNA to minicircular DNA (Fig. 3 and 4). Trp-P- I and Trp-P2 inhibited the topo II activity in a dose dependent manner, and relaxed DNA level was decreased to

and Trp-P-2

on cleatuble

Most topo II inhibitors such as etoposide and adriamycin are known to stabilize covalent DNA and topo II binding (cleavable complex). Therefore, we examined whether Trp-P-l and Trp-P-2 formed a cleavable complex. Etoposide which was used as a

Fig. 5. The effects of Trp-P-l, Trp-P-2, harmdn and norharman on topo II-mediated DNA cleavage. Lane 1. CCC-DNA control; lane 2, linear DNA control; lane 3, no drug; lanes 4. 5, etoposide as a positive control; lanes 6. 7, Trp-P-l: lanes 8, 9. Trp-P-2: lanes 10, 11, 3 20 pg/ml; lanes 5, 7, 9, 1 I and 13. 400 harman: lanes 12, 13. norharman. Drug concentrations were as follows: lanes 4, 6, 8, 10 and I_. pg/ml.

“,L 10.2

1

100

o-1

drug

10'

concentration

102

103

(pg/ml)

Fig. 6. Effects of Trp-P- I. Trp-P-2. harman and norharman on DNA-top0 II cleavable complex formation. Human topo II and 3‘-end labeled [“P]pBR322 were incubated with various concentrations of drug>. and the complex was precipitated with SDS-KC1 as described in Materials and methods. Horizontal axis represents the concentrations of drugs, and vertical axis represents the relative radioactivity. Trp-P-l (0). Trp-P-2 (0). harman (A) and norharman (A ) did not form the cleavable complex. Etoposide (0 ) was the positive control.

positive control, increased dose dependently the amount of cleavable complex formed (Liu et al., 1983). On the other hand, Trp-P-l or Trp-P-2 did not stabilize the cleavable complex in a concentration range of 0.l- 100 pug/ml (Fig. 6). 3.4. Intercalation deriratiL,es

uctihty

of

/3- artd y-carboline

To investigate whether these heterocyclic amines intercalate into DNA, an unwinding assay was performed using linearized pBR322 DNA and T4 DNA ligase. In this assay, m-AMSA, a weak intercalator, and adriamycin, a strong intercalator, were included as positive controls. Adriamycin produced positively

supercoiled DNA at concentrations greater than 2.0 ,ug/ml (Fig. 7. lane 12). and m-AMSA did so at a concentration of 50 pg/mI (Fig. 7, lane 8). In the presence of adriamycin at concentrations greater than 10.0 pg/ml, the linear DNA substrate remained unchanged (Fig. 7. lanes 13-1.51, indicating that strong intercalation activity of adriamycin caused inhibition of T4 DNA ligase enzyme activity (Montecucco et al., 1988). Trp-P-l and Trp-P-2 produced DNA band shifts in a concentration-dependent manner, indicating that they changed the DNA linking number (Fig. 7. lanes 16-27). Trp-P-I was more effective than Trp-P-2. and these compounds were stronger intercalators than m-AMSA but milder intercalators than adriamycin. On the other hand. harman and norharman were less effective than mAMSA, and were able to change the linking number of DNA only at concentrations exceeding 250 /*g/ml (Fig. 7, lane 33 and lane 39).

4. Discussion Inhibitors of DNA topo I and II are classified into at least four different groups (Popanda and Thielmann, 1992; Ross et al., 1994; Ferguson and Baguley, 1994): (1) inhibition of the ATPase subunit of topo II (novobiocin, coumermycin A,); (2) DNA intercalation or binding to the DNA minor groove (quinacrine, ethidium bromide, distamycin); (3) intercalation and binding to the cleavable DNA-topoisomerase complex (m-AMSA, doxorubicin); and (4) trapping the cleavable DNA-top0 II complex (etoposide, teniposide). In the present study, y-carbolines (Trp-P-l, TrpP-2) and P-carbolines (harman. norharman) were

1 2 3 4 5 6 7 8 9 1011 12131415161718192021222324252627182930313233343536373839

Fig. 7. Unwinding of DNA by jn-AMSA, adriamycin, Trp-P-l, Trp-P-2, harman and norharman. Unwinding measurements were done as described in Materials and methods. Lane 1. supercoiled pBR322 DNA control: lane 2, linear DNA control; lane 3, no drug; lanes 4-9. m-AMSA; lanes 10-15. adriamycin; lanes 16-21. Trp-P-l; lanes 22-27, Trp-P-2: lanes 38-33, harman; lanes 34-39, norharman. Drug concentrations were as follows: lanes 4, 10, 16, 22, 28 and 34, 0.08 pg/ml: lanes 5, 11, 17. 23. 29 and 35, 0.4 pg/ml: lanes 6, 12. 18, 24. 30 and 36. 2.0 @g/ml: lanes 7, 13. 19. 25, 31 and 37, 10.0 pg/ml: lanes 8. 14. 20. 26. 32 and 38, 50.0 pg/ml: lanes 9. 15. 2 1. 27. 33 and 39. 250.0 pg/ml.

190

Y. Funayama

et al./Murarion

demonstrated to inhibit the DNA relaxation activity of topo I and topo II at l-10 and 20-250 pg/ml, respectively. Trp-P-l and Trp-P-2 also inhibited decatenation of kDNA. However, these compounds did not inhibit DNA cleavage activities of topo I and topo II and did not stabilize the cleavable complex by topo 1 and topo II. Harman and norharman were weaker inhibitors than Trp-P-l or Trp-P-2 in all of the activities in which TrpP- 1 and Trp-P-2 exhibited inhibitory effects. Harman and norharman were previously demonstrated to intercalate into DNA (Hayashi et al., 1977). Trp-P-l and Trp-P-2 were also demonstrated to intercalate into DNA by CD spectral analysis (Inohara et al., 1993). In this study we confirmed that these compounds were DNA intercalators in DNA unwinding assay using linearized pBR322 DNA and T4 DNA ligase with an order of Try-P-l > Try-P-2 > norharman > harman. It was reported that the intercalators, ethidium bromide, quinacrine and doxorubicin are topo II inhibitors (Tewey et al., 1984; Leman, 1963). Therefore, the inhibition of topoisomerases by Trp-P- 1 and Trp-P-2 may be due to their intercalating activity. Shimoi et al. (1992) described that Trp-P-l enhanced UV or chemically induced mutagenesis by inhibiting DNA excision repair in E. coli. Further, the inhibition of the removal of UV photolesions from the DNA by Trp-P-l was demonstrated by ELISA (Mori et al., 1993). These results suggested that Trp-P- 1 inhibited UvrABC enzymes which work in the nucleotide excision repair in a coordinated manner in E. coli. On the other hand, it has been demonstrated that topo II inhibitors affect UV-induced DNA repair; novobiocin and coumermycin A, which block the ATPase activity of topo II, diminished removal of pyrimidine dimers (Snyder, 1987). Quinacrine also reduced the number of repair-specific strand breaks considerably due to the structural perturbations of DNA that render it a poor substrate for UV endonuclease (Thielmann et al., 1991). Thielmann et al. reported that majority of topo II inhibitors except nalidixic acid, oxolinic acid and etoposide were able to suppress DNA-repair synthesis in UV-irradiated human fibroblasts and assumed that the 180-kDa form of topo II was the main target enzyme involved in steps preceding repair-specific DNA incision (Popanda and Thielmann, 1992). Therefore, there is a possibility that Trp-P-l and

Rrsrarch

34

CIYY6) 183-IYI

Trp-P-2 which intercalate into DNA and cause DNA unwinding suppress DNA repair due to the inhibition of topoisomerases. In our environment, there are many compounds interacting with DNA, with or without mutagenicity by themselves. Examination of combined effects of many mutagens/carcinogens, for example, UV and Trp-P-l are needed for prevention of mutations in somatic cells and germ cells.

References Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72,248-254 Camillone, G.. F.D. Seta, R. Negri, A.G. Ficca and E.D. Mauro i 1986) Structure of RNA polymerase II promoters. Conformational alterations and template properties of circularized Sot,charongws cererisiar GAL1 -GAL IO divergent promoters, EMBO J.. 5. 763-771. Defie. A.M., J.K. Batra and G.J. Goldenberg (1989) Direct correlation between DNA topoisomerase II activity and cytotoxicity in Adriamycin-sensitive and -resistant P388 leukemia cell lines, Cancer Res., 49, 58-62. Ferguson, L.R. and B.C. Baguley (1994) Topoisomerase II enzymes and mutagenicity, Environ. Mol. Mutagen., 24, 245261. Hayashi. K.. Nagao. M. and T. Suglmura (1977) lnteracrions of norharman and harman with DNA. Nucleic Acids Res.. 4, 3679-3685. Inohara, T.. M. Tarui. M. Doi, M. lnoue and T. Ishida (1993) Interaction of mutagenic tryptophan pyrolysate with DNA CD spectral study on the binding specificity, FEBS Lett.. 324. 301-304. Jones, J.C., T. Stevnsner. M.R. Mattern and V.A. Bohe (1991) Effct of specific enzyme inhibitors on replication. total genome DNA repair and on gene-specific DNA repair after UV irrddiation in CHO cells, Mutation Res.. 255. 155-162. Leman. L.S. (1963) The structure of the DNA-a&dine complex. Proc. Natl. Acad. SC]. USA. 49, 94-102. Liu. L.F. (1989) DNA topoisomeraae poisons as antitumor drugs. Annu. Rev. Biochem., 58, 351-375. Liu. L.F., T.C. Rowe, L. Yang, K.M. Tewey and G.L. Chen (1983) Cleavage of DNA by mammalian DNA topoisomerase II, J. Biol. Chem., 258. 15365-15370. Marini, J.C.. K.G. Miller and P.T. Englund (1980) Decatenation of kinetoplast DNA by topoisomerases, J. Biol. Chem.. 255. 4976-4979. Matsukura, N.. T. Kawachl, K. Marina, H. Ohgaki. T. Sugimura and S. Takayama (1981) Carcinogenicity in mice of mutatagenie compounds from a rryptophan pyrolysate, Science, 2 13. 346-347. Montecucco. A., G.P. Noy. S. Spadari, E. Zanolin and G. Ciarrocchi (1988) DNA unwinding and inhibition of T4 DNA ligase by anthracyclines, Nucleic Acids Res.. 16. 3907-3918.

Mori. T., K. Shrmoi. Y.F. Sasaki, K. Wakabayashi, M. Nagao and N. Kinae ( 19931 3-Amino-l,4-dimethyl-5H-pyrido[4.3-h]indole (Trp-P-l) inhibits the removal of both cyclobutane dimers and (6-41 photoproducts from the DNA of ultraviolet-erradiated E. co/i. Carcinogenesis, 13. 1475-147X. Nagao. M., T. Yahagi. M. Honda. Y. Seino, T. Matsushima and T. Sugimura (19771 Demonstration of mutagenicity of anilme and +toluidine by norharmen. Proc. Jpn. Acad., 53B. 35-37. Popanda, 0. and H.W. Thielmann (19921 The function of DNA tpoisomerases in UV-induced DNA excision repair: studies with specific inhibitiors in permeabilized human fibroblasts. Carcinogenesis. 13. 232 I-2318. Ross. J.A.. J.D. Potter. L.L. Robison (1994) Infztt leukemia. topoisomerase II inhibitors, and the MLL gene. J. Natl. Cancer Inst., 86. 167% 1680. Sasaki, Y.F.. H. Yamada, K. Shimoi. N. Kinae. 1. Tomita, H. Matsumura. T. Ohm and Y. Shirasu (1992) Enhancing effects of heterocyclic amines and fl-carbolines on the induction of chromosome aberrations in cultured mammalian cells. Mutation Res.. 269. 79-95. Shimoi. K.. H. Kawabata and I. Tomita (19931 Enhancing effect of heterocyclic amines and P-carbolines on UV or chemically induced mutagenesis in E. toli. Mutation Res.. 268. 287-295. Snyder, R.D. (1987) Is DNA topoisomerase involved in the UV excision repair process’? New evidence from studies with DNA

intercalating

and

non-intercalating

antitumor

agents,

Pho-

tochem. Photobiol.. 45. 105- I 11. Sugimura. T., K. Wakabayashi, M. Nagao and H. Ohgaki (19891 Heterocyclic amines in cooked food, in: S.L. Talor and R.A. Scanlan fEds.1. Food toxicology. a perspective on the relative risks, Dekker. New York. pp. 31-55. Tewey. K.M.. T.C. Rowe. L. Yang. B.D. Halligan and L.F. Liu i 1984)Adriamycin-induced DNA damage mediated by mammallan DNA topoisomerase II, Science. 226. 466-468. Thielmann. H.W.. 0. Popanda and L. Edler ( 1991) The effects of inhibitors of topoisomeraae II and quinacrine on ultravioletlight-induced DNA incision in normal and xeroderma pigmento\um fibroblasts. J. Cancer Res. Clin. Oncol.. I 17. 19-26. Wakabayashi. K., M. Nagao, T. Kawachi and T. Sugimura (19821 Mechanism of appearance of mutagenicity of N-nitrosodiphenylamine with norharman. in: H. Bartsch (Ed.). N-Nitroso Compounds: Occurrence and Biological Effects, IARC Sci. Publ. 41. IARC. Lyon. pp. 695-707. Wang, J.C. (19851 DNA topoisomerases. Annu. Rei. Biochem.. 5-l. 665-697. Yamashita. Y.. S. Kawada. N. Fujii and H. Nakano (19911 Induction of Mammalian DNA topoisomerase I and II mediated DNA cleavage by saintopin, a new antitumor agent from fungus. Biochemistry, 30. 5838-5815.