53

Mutation Research, 35 ( 1 9 7 6 ) 5 3 - - 6 4 © Elsevier S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

INHIBITION OF TRANSCRIPTION BY ISONICOTINIC ACID HYDRAZIDE *

O L A F L. K L A M E R T H

lnstitut fiir Humangenetik der Universitiit Heidelberg, D-69 Heidelberg (Germany) ( R e c e i v e d J u l y 21st, 1975) ( A c c e p t e d N o v e m b e r 2rid, 1975)

Summary Isonicotinic acid hydrazide (INH) reacts with the CMP moiety of a polynucleotide at slightly acid and alkaline pH. The reaction product, when used as a template in the cell-free transcription step, greatly diminishes the incorporation of GMP. In this system we were not able to show that INH -- in contrast with hydrazine -- is potentially capable of producing point mutations, since noncomplementary incorporation could not be observed with a poly(C)/INH adduct as template. Hyclrazine could not be detected when INH was incubated with liver-cell fractions.

Introduction

The well-known tuberculostaticum isoniacid (isonicotinic acid hydrazide) (INH), which has even been used occasionally as an antidepressivum [10,23], is suspected Of having cancerogenic and mutagenic properties [26]. Several authors have postulated the formation of hydrazine, known to be mutagenic, as the effective agent of INH in animals, although INH has not proved to be mutagenic in the dominant lethal test with mice [26]. In human lymphocytes, as well as in bone marrow cells of mice, chromosomal aberrations were noted only after extremely high doses [8,22]. In 1974 Marfey and Li reported an enhanced binding ratio of INH-treated lymphocyte chromosomes, particularly of the A group, to polylysine even after small doses of INH, suggesting an otherwise not noticeable change in chromosomal DNA behavior under the influence of INH. * This research was supported by the Deutsche Forschunpgemeinschaft (SFB SS). SSC 0.15 M NaCI--O.015 M Na citrate, pH 7; TKM buffer, 0.05 M Tris--HCl Coil 7.5), 0.025 M KCI, 0.005 M MgCI2.

Abbreviations:

54 In the host-mediated assay, however, INH showed slightly elevated back mutation frequencies over the control, suggesting a tendency to induce point or suppressor mutations [26]. Results recently reported by Buselmaier and Herbold [6] with the microsomal system of Ames et al. [1] seem to confirm these tendencies. But in these experiments, too, the mutagenic frequency after application of INH increased in only one out of five test strains and only to maximally three to four times that of the controls at the highest dose applied (50 pg/ml), an increase not sufficiently high to ensure the certainty of point mutations in the broader sense of a change in biochemical function. Because of all these more or less inconclusive reports, it seemed worth while to investigate the influence of INH in a simulated transcription system as a screening test in vitro for the investigation of potentially mutagenic substances. Despite the known arguments against the use of such a system, there are a number of factors in favor of it. It is undisputed that the biological function of DNA is reflected in its template activity in the replication and transcription steps as well as in its ability to guide the repair process. All these processes are affected by the influence of mutagens. To mention only some of the pertinent literature, Ono et al. [24] have investigated the influence of UV irradiation, and Phillips et ai. [25] that of hydroxylamine, on the template properties of polynucleotides, later extensively studied by Budowsky et al. [4]. Troll et al. [30] described the influence of mutagenic alkylating agents on these properties, and Hagen et al. [14] reported on the effects of X-rays on the template in the transcription step. The influence of the mutagen Trenimon on the transcription reaction and the resultant modification in RNA synthesis were investigated by Klamerth and Kopun [17] following a similar study dealing with malondialdehyde by Klamerth and Lewinsky [16]. In addition, in my opinion, the cell-free system can also be used to test the potential ability of a mutagen to induce point mutations in the restricted sense (erroneous base pairing), in that it measures the extent of complementary and non-complementary transcription after reaction of the template with the compound to be tested [18,19]. Ludlum [20,21] has shown that the introduction of a methyl group into a CMP moiety containing polynucleotide and into the corresponding deoxy derivative alters pairing characteristics in such a way that in the transcription and]or polymerization process not only guanine, but also uracil and adenine are incorporated into the resulting polymer. Similar observations regarding the misreading of chemically altered poly(C) by mutagens have been made in the extensive studies of Fraenkel-Conrat and Singer [9]. The tendency of Cytoxan to induce point mutations, suggested by the hostmediated assay, could be confirmed in the cell-free system on the basis of the abnormal base pairings observed [18]. Recently, Sirover and Loeb [28] have shown that the potent mutagen and carcinogen ~-propiolactone effectively changes the template function of a synthetic deoxyribopolynucleotide to favor the incorporation of non-complementary bases. Thus a chemical mutagen and carcinogen may directly induce mutations during DNA replication in normal cells. While the above-named mutagens react as alkylating agents with the guanine and adenine moieties in DNA, it has been found that INH reacts with cytosine in the same way as hydrazine, i.e. by binding to the amino group at C4 of the

55 base with release of ammonia [15]. Originally, we had planned to synthesize mixed polymers by reaction of INH-treated cytosine diphosphate in the presence of CDP with ribonucleoside phosphorylase, and to investigate their template activity for GMP and AMP. This method proved impracticable, however, because of the very low yield of the reaction product, and therefore the influence of INH on the template activity of DNA and poly(C) was studied. Materials and methods All reagents used were A grade. Poly(C), bovine serum albumin, and unlabeled ribonucleoside phosphates were obtained from Serva Feinbiochemica (Heidelberg) and from Boehringer (Mannheim). Radioactive nucleoside triphosphates came from New England Nuclear Corp. (Boston), ~4C-labeled INH was purchased from Amersham-Buchler (Braunschweig). RNA polymerase (EC 2.7.7.6) was initially obtained from Boehringer. Since this product was found to contain traces of poly(A) -- source bacteria harvested in log phase -- the enzyme from Searle (High Wycombe) was used for measurement of UMP incorporation and subsequent experiments. Determination of INH was carried out as described by Watt and Chrisp [31] and Boenicke and Reif [3] with slight modifications. Optical measurements were made at 485 nm. Phosphorus was determined according to Chen et al. [7]. All readings were taken on a Zeiss PMQ II Spectrophotometer. Ultracentrifugation analyses were performed with the analytical ultracentrifuge Spinco, Model E. Radioactivity was measured with the Tricarb scintillation spectrometer (Packard) under a toluene-PPO-POPOP cocktail.

Reaction of INH with ribonucleotides 30/~mol CMP were incubated at 37°C with 300 pmol INH in 0.5 M acetate buffer (pH 4.25). The reaction mixture was kept at 37°C for 72 h, concentrated, and an aliquot taken for thin-layer chromatography. The rest was applied to a Dowex (1 × 8, HCOa form) column (20 cm X 1.2 cm) and eluted with increasing concentrations of ammonium hydrocarbonate (pH 8.7). Fractions (2 ml) were collected and combined according to the extinction quotients at 270 and 345 nm (at pH 9), where the adduct shows a second maximum. About 11 A270 (1.5 /2mol) INH/CMP were obtained. In a similar manner, 14/~mol CDP (lithium salt) were incubated with 3 ml 0.7 M INH in 0.25 M ammonium formate (pH 4.3), and the compounds formed were separated after 72 h at 37°C by DEAE cellulose column chromatography (20 cm × 1.5 cm). The yield of the adduct eluted with 1 M ammonium hydrocarbonate (pH 7.8), however, amounted to less than 5% of the theoretical, and its purity was obscured by hydrolytically formed CMP.

Reaction of poly(C) with INH (determination of binding capacity) About 15 A270 poly(C) were dissolved in 1 ml 0.02 Tris--HC1 at pH 7--8 and incubated at 37°C with 6.5 ml 0.25 M acetate buffer (pH 4.3) containing 7.5% w/v INH together with 15 pCi [14C] INH (final specific activity 10840 cpm/ pmol INH). The same experiment was also performed at pH 5.3. After the

56 CMP/INH

CMP/tNH • 180

22 20

-160

19 - 140 18

120

16

14

100 12

8d

10 8

+

6 4O

4 2O

2

hrs

Fig. 1. Time and pH d e p e n d e n c e of binding of INH to poly(C) after i n c u b a t i o n at pH 4 . 2 5 (+) (CMP/INH ratio at left) and at pH 5.3 (o) (at right).

number of hours indicated in Fig. 1, a 2.5 ml aliquot was taken, concentrated to about 0.5 ml in the cold (Amicon Mini-Dialysator), and applied to a 1.2 cm × 26 cm Sephadex (G 25 fine) column and eluted with water. The first fractions contained more than 95% o f the high-molecular polynucleotide moiety and, after concentration to a small volume, were precipitated with 2 vol pre-cooled ethanol 1% w/v potassium acetate at low temperature. After the mixture had been kept overnight in ice, the sediment was separated by centrifugation, washed with 66% alcohol until no radioactivity was found in the supernatant, and dissolved in 1 ml 0.02 M Tris--HCl (pH 7.5). In the clarified solution the content of phosphorus was determined, and a 100/~l aliquot applied to 3 cm X 18 cm strips of Whatman paper and eluted for 18 h with a mixture of 1 M ammonium acetate (pH 5.1) and ethanol (1 : 1) to remove any remaining traces of INH (while the high-molecular c o m p o u n d remained at the starting point). The paper surrounding the applied spots was washed with alcohol and ether, the spots were cut o u t and counted. The nucleotide/INH ratio was calculated on the basis of the previously determined phosphorus content of the solution and the a m o u n t of radioactivity on the filter (Fig. 1). A corresponding experiment was conducted with poly(U) at pH 5 and 8.25, but under these conditions no binding of INH to poly(U) could be detected (binding ratio > 1 : 2,000).

Identification of reaction product To identify the reaction product, 4 mg poly(C) previously dissolved in 1 ml water were incubated at pH 4.38 and 37 ° with 5 ml INH (10% v/w) for 72 h and freed from INH as described. Potassium hydroxide was added to the con-

57 centrated and neutralized solution (final concentration 0.3 N), and the mixture incubated over-night at 37 ° C. After addition of Dowex 50 (pyridine form), the mixture was shaken for one h and the neutral solution separated from the ion exchanger by centrifugation. The resin was eluted repeatedly with diluted ammonium hydroxide. After concentration of the combined supernatants in vacuo, about 5 A2?0 (~5 #l) were taken for thin-layer chromatography (20 cm × 20 cm, DC-Kieselgel, Merck) together with 3'-CMP and the reaction product from 3'-CMP and INH. The chromatogram was developed in 1 M ammonium acetate (pH 5.1)/ethanol (3 : 7 v/v) and the spots were located at 254 nm.

Poly(C)/INH adducts Poly(C)/INH adducts were obtained after various incubation times and pH, as described. Each incubation mixture was freed from INH by 8ephadex (G 25 fine) chromatography and dialysis of the first fractions with subsequent reduction in volume. Purification by precipitation with alcohol was not necessary to obtain a product virtually free of unbound INH. An analogous experiment using hydrazine instead of INH was conducted at pH 5.8. Incubation of DNA with INH About 17 A~o DNA from thymus (e~p~ ~ 6700) dissolved in 0.1 × SSC were incubated at 37°C with 5 ml 10% (w/v) INH in 0.25 M ammonium acetate (pH 8.25) for 100 h. The reaction mixture was processed as described above, the precipitate dissolved in 0.1 × SSC and dialyzed against the same fluid. The reacam ~ 7760 and showed diminished incorporation tion product had a value of e(p) of GMP in the transcription reaction with all four nucleotides (Table I). Transcription experiments The reaction mixture contained in 0.3 ml: 0.025 M Tris--HC1 (pH 8.0), 130 mM ammonium sulfate, 1.0 mM dithiothreitol, 1.25 mM MnC12, 60/~g serum albumin, 1.0 mM each of the nucleotide triphosphate pairs (in the experiment with DNA as template, all four triphosphates were used, with the addition of 8 TABLE I T R A N S C R I P T I O N OF DNA A F T E R INCUBATION WITH INH I n c u b a t i o n time (rain)

GMP i n c o r p o r a t e d cpm (background d e d u c t e d ) Control Treated

5 10 20

623 906 1440

175 360 618

T h e r e a c t i o n m i x t u r e c o n t a i n e d in 0.5 m l p o l y m e r i z a t i o n m e d i u m supplemente/t by 8 mM MgCI2 : 100 /~mol t h y m u s DNA (prevloualy i n c u b a t e d w i t h INH (9% w/v) for 100 h a t S7°C, pH 8.25), 0.875 mM ATP, CTP and UTP each as well as 0.03 p m o l [3H] GTP (specific activity 2.5 x 103 c p m / p m o l ) . The react i o n was started b y the a d d i t i o n of 2 . 6 6 u n i t s ( a b o u t 13 pg p r o t e i n ) R N A polymerase. I n c u b a t i o n was a t 37°C. At the appropldate time, 100 ~I aliquots w e r e t a k e n and, after a d d i t i o n of 20 ~I s a t ura t e d s o d i u m , p y r o p h o s p h a t e and 1 00 #g albumin, p r e c / p i t a t e d in the cold w i t h 3 m l 5% (w/v) TCA. The insoluble matetdal was processed as deserlbed (filter m e t h o d ) . I n c u b a t i o n of DNA w i t h INH a t pI-I 4~}Sfor I 0 0 h resulte d in a l m o s t c o m p l e t e loss of activity of the t e m p l a t e for b o t h c o n t r o l and sample ( e ~ ~ 8450).

58 mM MgC12), 1.2 pCi of the appropriate radioactive triphosphate (specific activity indicated in the figures), and 100 ~zmol (as nucleotide P) treated and nontreated poly(C). The reaction was started at 37°C by the addition of 10 /~l RNA polymerase (EC 2.7.7.6), equivalent to 3.3 units. Difficulties encountered in the determination of AMP incorporation because of a very low but constant content in the labelled ATP of oligo or poly(A) (0.31% of the total radioactivity) were overcome by pre-purification of [3H] ATP on a DEAE cellulose column (1 cm × 5 cm), where high-molecular compounds were retained. Where AMP or UMP incorporation was to be measured, the following procedure was used. At appropriate intervals, 100 pl samples were taken from the reaction mixture, quickly cooled, and mixed with 20 pl pre-cooled saturated sodium pyrophosphate (pH 7.0) to stop the reaction. From the ice-cold mixture, 50 /~l aliquots were applied to strips of Whatman 3 MM paper (3 cm × 42 cm) and processed as described above. Some samples were applied to Whatman DEAE paper strips and eluted with 0.3 M ammonium formate at pH 5.0. Although the results did not differ greatly, the latter method gave somewhat lower background counts (9--12 cpm). The starting regions of the strips were washed in ethanol--ether (1 : 2 v/v) and dried; the UV-located spots were cut out and counted. This procedure diminishes the background count to less than 15 cpm and makes it possible to measure the very low incorporation rates which could theoretically be expected for AMP and UMP. For control, the region below the applied spot was also treated as above. The counting rates exceeded those for a blank spot by less than 4--5 cpm. The filter-paper technique for the measurement of GMP incorporation alone described elsewhere [18] was usually adopted.

Treatment of INH with liver homogenate fractions After perfusion in situ with cold saline, two rat livers (14.5 g) were homogenized and the homogenate was fractionated according to Blobel and Potter [2]. The washed nuclear sediment was incubated at 37°C with TKM buffer (pH 7.5) containing 10 mM INH and dialyzed with frequent changes of the outer fluid against the same buffer with decreasing concentrations of INH. The microsomal fraction was treated in the same manner after the addition of 4 mM dithiothreitol. The combined outer fluids from both experiments were acidified with acetic acid to pH 3.8, concentrated under reduced pressure, and freed of any turbidity by centrifugation. The content of the dialysis tube was concentrated in the cold, TCA (final concentration 5% w/v)added at 0°C, and the solution clarified by centrifugation. A preliminary experiment had shown that under these conditions INH does not hydrolyze to hydrazine. The supernatant of each of the fractions was then brought to pH 8--9 by the addition of solid sodium hydrocarbonate and an aliquot taken for quantitative estimation of INH (including hydrazine, if formed). Another 100/zl aliquot was combined with 200/~l absolute pyridine and 10 ~umol dansyl chloride in 200/~1 dimethylformamide or acetone were added. The mixture was treated for 1 h at 45°C in a closed vial. The reaction product was applied in 10--20 #l aliquots to a 20 cm X 20 cm Kieselgel thin-layer plate (Merck) and developed with a mixture of benzene and acetone (1 : 1). Control samples of hydrazine and INH after dansylation were run simultaneously.

59 Results and discussion

A preliminary experiment had shown that, with DNA, INH diminishes the transcription of GTP (Table I). For the investigation of non-complementary transcription, however, we used poly(C). The reaction product of poly(C) and INH, under various pH and time conditions, was resistant to dialysis and relatively acid-stable. At short wave-lengths and alkaline pH, the compound showed a different spectrum from that of poly(C) {Fig. 2), whereas no differences were observed at neutral or acid pH. The hydrolysis product in 0.3 M KOH contained a compound identical with the reaction product of 3'-CMP and INH (Fig. 3). This compound had an R~ value of 0.59 as compared with 0.44 for 3'CMP and showed a fluorescent cap in the UV (Fig. 3). Interaction of poly(C) with radioactive INH revealed a binding ratio which varied according to the pH and incubation times selected between 12 and 160 nucleotides per INH and gave an asymptotic curve (Fig. 1). Ultracentrifugation analyses to determine the sedimentation constants suggested a gradual, time-dependent chain break in the macromolecule (Table II). In the trancription experiment, a strong inhibition of the template function for complementary transcription was observed both at slightly acid a n d alkaline pH, which increased with incubation time. The template properties of the various poly(C)/INH adducts are shown in Fig. 4. Poly(U) did not react with INH under our experimental conditions at pH 5. At pH 8, the INH/UMP ratio amounted to no more than 1 : 2000. Since INH is a derivative of hydrazine and reacts with cytosine in analogous fashion, it seems reasonable to expect similar behavior in the transcription reaction. The ability of hydrazine to produce point mutations is attributed to the

0800

0.700

,/~

0600 050C

."

z

0,40C

0.30(: 0.20C

\ (\

0.10(:

%

. ~ x - . . . . . x--"x---x~ . . . . . x

~..~ %,==7_'~-x . . . I_ . - . x

250

260

270

280

290

300

310

. . . . . . . x. . . . . . . . . . . . . . . . . . .

320

330

340

350

rnF

x

360

Fig. 2. E x t i n c t i o n c u r v e o f t h e p r o d u c t o b t a i n e d a f t e r 4 8 h o f i n c u b a t i o n o f poly(C) and INH a t p H 4 . 2 5 ° m e u u r e d i n b u f f e r e d i o l u t i o n s a t : p H 4 . 8 (X X ); p H 7 . 0 ( X - - • --X ); p H 9 . 0 ( X - - --X ); p o l y ( C ) c o n t r o l a t p H 4 . 8 (X . . . . . . X); p o l y ( C ) c o n t r o l a t p H 9 . 0 (X . . . . . . X ).

60

G

@

0

0

6

@

I

I

I

CMP/INH

Poly(C) / INH Hydrolys0te

CMP

Fig. 3. S e p a r a t i o n b y thin-layer c h r o m a t o g r a p h y o n DC-Kiese]gel o f t h e r e a c t i o n p r o d u c t s o f I N H w i t h CMP and INH w i t h p o l y ( C ) after h y d r o l y s i s (0.3 M KOH). A b o u t 6 A270 o f t h e respective r e a c t i o n mixt~Lre w e r e applied and d e v e l o p e d for 5 h w i t h 1M a m m o n i u m acetate (pH 5.1) in e t h a n o l (1 : 1).

fact that its reaction product with cytosine leads to tautomerization. The derivative formed (N4-aminocytosine) can be regarded as a tautomer of the hydrazide of uracil, and is thus potentially capable of pairing with adenine. Neither for adenine nor for uracil, however, could we detect incorporation -- although more than fifty experiments were carried out under various conditions. That the in vitro system as such is competent is proved by the experiment with hydrazine, in which it not only diminished the template function of the treated poly(C) for GMP incorporation, but also enhanced the incorporation of AMP (Fig. 5). An explanation of the lack of non~omplementary binding in the experiment with INH may be that the isonicotinic acid group prevents the formation o f hydrogen bonds with adenine, whereas hydrazine does not change the three~limensional structure or distort the planarity of the pyrimidine ring, both of which are essential to function [4]. We were unable to confirm a hydrolysis of INH to hydrazine with rat liver-

TABLE II SEDIMENTATION ANALYSIS OF POLY(C) A F T E R INCUBATION WITH INH AT pH 4.4 and 8.15 (h)

S~vX 10 -13

Approximate m o l . w t . × 103

0 24 a 100 130

6.04 5.50 5.70 4.79

100 89 92 70

S e d i m e n t a t / o n a n a l y s e s w e r e p e r f o r m e d a c c o r d i n g t o S c h a c h m a n [ 2 7 ] . T h e values are c o r r e c t e d for 2 0 ° C . a A t p H 8.15.

61

cpm x l O 3 jx

x111

4

x

H---~ H---~ Ip--¢

H~--k 4O Fig. 4. Time and pH d e p e n d e n c e of GMP incorporation into the p o l y m e r f o r m e d by RNA p o l y m e r a s e with poly(C) as template after i n c u b a t i o n with INH. The reaction m i x t u r e c o n t a i n e d in 0.S ml 1.2/~Ci [ 3 H ] G T P (specific activity about 4 #Ci/#mol). Control (X); 24 h, pH 4.3 (ix); 72 h, pH 4.3 (o); 100 h, pH 4.3 (o); 100 h, pH 8.2 (u).

cell fractions, despite our using the highly sensitive thin-layer chromatographic method after dansylation of the dialyzable compounds of the incubation mixture. The dansylated INH spot in the dialyzate from the nuclear fraction was easily discernible, whereas that of hydrazine, whose Rf values (0.80 and 0.91 respectively) differ markedly from that of dansylated INH (0.59), could not be detected. In the experiment with the microsomal fraction, the INH content (spot after dansylation) was faintly visible, while none could be observed for hydrazine. It cannot be entirely excluded, however, that the hypothetically formed hydrazine may react in statu nascendi with liver~cell components which, under our experimental condition, would not be hydrolyzed to hydrazine. According to Goedde et al. [12], the mammalian organism excretes applied INH as 2'-acetyl-INH. The same observation, acetylation but no formation of hydrazine, was made by Grebenik [13] (see also Weber and Cohen [32] ). The differing velocity of this reaction is a well-known example of polymorphism in humans [11]. It should be mentioned, however, that Toida [29] has described the isolation, from an INH-resistant strain of Mycobacterium t.b. avium, of an enzyme fraction which proved to be a very active hydrazido-hydrolase for a number of heterocyclic hydrazides, such as nicotinic acid hydrazide and INH. Our negative results as far as the induction of errors in the replication or transcription steps is concerned, and--on the other hand--the demonstrated reaction with the polynucleotide, suggest that INH may play a role in the repair process. In a manner similar to that of caffeine, INH may influence the postreplication process by inhibiting the gap-filling step through binding to the dam-

62

105I n~JmolincorporatldX I0-3

3 2

10 3

1 O;

3 2

i ii

i 6

v 12

~-

i 2~

i 45

i 72

ht|.

Fig. 5. C o m p a r i s o n o f c o m p l e m e n t a r y a n d n o n - c o m p l e m e n t a r y t r a l ~ c r i p t i o n (expremled in #~nol X 10 -3 i n c o r p o r a t e d ) w i t h p o l y ( C ) as t e m p l a t e a f t e r i n c u b a t i o n w i t h I N H o r h y d r a z i n e f o r t h e ~times i n d i c a t e d . T h e specific a c t i v i t i e s v a r i e d b e t w e e n 4 4 6 a n d 4 8 7 0 e p m / # m o l n u c l e o t i d e t r l p h o s p h a t e . R e a c t i o n t i m e w a s 6 0 rain. R e s u l t s a r e m e a n v a l u e s o f s e v e r a l i n d e p e n d e n t e x p e r i m e n t & A M P ( h y d r a z i n e ) (~); A M P ( I N H ) ( s ) ; U M P ( I N H ) ( × ) ; G M P ( h y d r a z i n e ) ( e ) ; G M P ( I N H ) (o).

aged sites in D N A (free ends). Investigation of this hypothesis is n o w under way. Even though results in vitro are n o t necessarily transferable to living cells, on the basis of our findings in the transcription experiment, the harmful effects of INH on the chromosomal substance can be assumed with a high degree of probability.

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