Int. J. Rad. Biol., Vol. 10, No. 4, 329-342

The effect of sulphoguanidine and p-nitrobenzoic acid onthe chromosome breakage-reunion process by y-, X-

and -irradiation in Vicia faba

ANDREI LAZANYI

The Academy of the Socialist Republic Romania,Centre of Biological Researches, Cluj

(Received 9 August, 1965; revision received 8 October 1965)

Primary roots of Vicia faba seedlings were irradiated with gamma-,x- and beta-rays in a sulphoguanidine solution. Analysis of anaphaseaberrations showed that the breakage-reunion index (which is the numberof anaphase fragments per bridges) in the middle of the first mitosis afterirradiation, is constant, and at low doses is independent of the intensity andquality of ionizing radiation.

The breakage-reunion index indicates that after irradiation in the presenceof sulphoguanidine, twice as many breaks enter into reunion as in distilledwater. This cytological effect of the sulphoguanidine was suspended byp-aminobenzoic acid, folic acid, arginine and guanine, as well as by 2,4-dinitrophenol and chloramphenicol. Therefore, the sulphoguanidineeffect seems to be connected with the chromosomal histone and DNA-synthesis. p-nitrobenzoic acid has the same effect as the sulphoguanidine.The cytological effect of these compounds was linked with a radiosensitizingeffect, manifested in root-growth inhibition.

1. INTRODUCTION

According to Sax's (1939) and Lea's (1946) hypotheses, a transverse ' break'of the chromosome is produced by the track of an ionizing particle. The 'brokenends' may:

(a) restitute in the initial state (in this case no microscopically visibleaberration-products are formed),

(b) remain permanently unjoined, i.e. they may produce real fragments, or(c) rejoin other broken ends (in this case the reunion products are visible as

intra-, or inter-change figures).Wolff and Luippold (1955, 1956) have shown that the reunion of the

chromosome breaks produced by x-rays is a process closely linked with cellmetabolism. According to Wolff's hypothesis, the x-ray-induced breaks wouldstay open and become available for reunion for a longer time, if ATP-production,or protein-synthesis is inhibited between two dose fractions by 2,4-DNP andchloramphenicol respectively (Wolff 1960).

Accepting the hypothesis of Wolff and Luippold, according to which thebreakage-reunion process may be influenced by cellular metabolism, we haveinvestigated those biochemical mechanisms of the breakage-reunion processin which some anti-metabolites may interfere. In previous papers we havereported on the biological effects of sulphanilamides (Laz/nyi et al. 1961, 1962).

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Our presumption was that some sulphanilamides do not act as antagonists ofp-amino-benzoic-acid only, but that through the lateral chain they may alsointerfere as purine-antagonists with DNA-synthesis. Cytological studiesconcerning the effect of seven different sulphanilamide-compounds on Viciafaba have revealed the following:

The sulphanilamide treatment exerts an inhibitory effect on cell division inthe first half of the period of DNA-synthesis of interphase and also induces alagging of the chromatids, as a consequence of centromere inactivation. Two tofour hours later, these lagging chromatids are transformed into micronuclei.Sulphanilamides do not produce chromosomal aberrations and fragmentations,as do ionizing radiations or radiomimetic chemicals. As to the effects describedabove, there is no essential difference between these sulphanilamides (LazAnyi1965 a). Nevertheless an interference of the sulphanilamides in the chromosomalbreakage-reunion process after irradiation might be possible.

A previous short communication (Laz/nyi 1965b) reported that sulpho-guanidine has a radiosensitizing effect on Vicia faba. Six other sulphanilamidecompounds were inactive; thus this effect is probably connected with the lateralguanidine-radical of the sulphoguanidine molecule.

We assumed that evidence about the effect of sulphoguanidine on thechromosomal breakage-reunion process could be obtained by investigating thenumerical relation between the breaks and reunions of the chromatids. Havingthis in view, the scoring of the chromatid fragments and bridges at anaphaseseemed to be more useful, since at anaphase, the number of fragments showsthe number of breakages which became permanent, while the number of bridgesindicates the reunions of the centric fragments. It is true that the scoring ofaberrations at anaphase is biased with other errors. Thus some small fragmentsmay be rings which are themselves the products of a rejoining process. Conger(1965) has shown that a considerable proportion of bridges break at anaphase,and that the proportion increases with increasing dose. It must be mentioned,too, that the reunion of acentric fragments, as well as those between the acentricand centric fragments, cannot be scored at anaphse. But all these errors weightsimilarly all the variants of our experiment, and therefore may be neglected.In these experiments a solution has been sought to the following questions:

(1) Does a constant relation exist between the total number of breakages andreunions for irradiations with different low doses (120 r to 240 r), and for ionizingradiations of different qualities, such as cobalt gamma-rays, x-rays and beta-raysfrom strontium-90.

(2) If this relation exists, can it be changed by a sulphoguanidine treatment ?

(3) What is the biochemical mechanism of these processes ?

2. MATERIAL AND METHODS

Main roots of Viciafaba seedlings were treated with a solution of 7 per centsaccharose followed by distilled water, for 15 min each; the whole procedurewas then repeated. This was necessary to increase the permeability of thenuclear membrane through repeated plasmolysis and deplasmolysis (Haberlandt1919), thus facilitating the penetration of the substances with which treatmentof the roots was continued.

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The roots were then immersed in the solutions mentioned below, and 15 minlater were irradiated in the same solution, after which they were kept for a further30 min in the same solution. The following solutions were used:

1. Sulphoguanidine, 4 mmol (in the table= SG).2. p-aminobenzoic acid, 4 mmol (= PABA).3. Folic acid, saturated solution (= Fol).4. Arginine, 4 mmol (= Arg).5. Histidine, 4 mmol( = Hist).6. Guanine, sat. sol. (= Guan).7. 2,4-dinitrophenol, 001 mmol (= DNP).8. Chloramphenicol, 005 mmol (= Clph).9. p-nitrobenzoic acid, 4 mmol (= PNBA).

Each of these substances was dissolved in phosphate-buffer, at pH = 6.4 to 70.In each variant, 12 roots were irradiated as follows:(a) 150r/15min x-rays (180kv, 05mm Cu filter), focus-object distance

53 cm; irradiation parallel to the length of roots.(b) 240r/180min gamma-rays emitted from a 60°Co source with 5 Curie

activity; irradiation perpendicular to the length of roots.(c) 120 to 240r/3 min beta-rays, emitted from a 9 0Sr- 90Y beta-ray medical

applicator, with 4cm2 radiating surface; irradiation parallel to the length ofroots which were held in two lots of 6 between two parallel stretched thin gum-sheets 02 mm thick; the variation of dose was partly due to technicallyinevitable variations of distance of the centre of the active-meristem from theradiation surface (1 to 2mm) and partly to variation of the dose throughout theroot due to absorption of the beta-rays (see Friedell, Thomas and Krohmer 1954).

After this treatment the seedlings were kept for recovery in distilled water at22°c. Twenty-four hours later, fixation in alcohol : acetic acid (3: 1), followed.The scoring of anaphase aberrations was performed on Feulgen-squash-Euparalpreparations.

Figure 1 (c), (d), (e) shows the most suitable anaphase states for the aberrationscoring. Those anaphases in which the chromatid ends are not distant enoughone from another (fig. 1 (a), (b)), or in which the despiralization has alreadystarted (figure 1 (f)), should not be scored. Likewise, the so-called ' spider-foot '-anaphases (figure 1 (a)) are unsuitable for aberration scoring.

Parallel with these, 12 seedlings of each variant of treatment were kept inHoagland's nutritive solution for 8 days, after which the length of the roots wasmeasured. These measurements indicated whether the treatment had or hadnot a radiosensitizing effect, manifested in the growth inhibition of roots.

3. RESULTS AND DISCUSSION

Table 1 shows the variation of the proportion of reunions 24 hrs. afterirradiation with y-rays either in distilled water, or in the presence of sulpho-guanidine. This proportion was expressed as the 'breakage-reunion index'(BR-index), which represents the number of fragments per bridges.

BR-index= fragmentsbridges

In the case of irradiation in distilled water, the BR-index is about 10, and insulphoguanidine solution about 5. This means that in the presence of

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sulphoguanidine, the breaks entering into reunion are twice as many as in distilledwater. In these two variants of treatment, the total number of fragments plusbridges was approximately equal; the primary radiation-damage was nottherefore affected by the sulphoguanidine.

Figure 2 and table 2 show the variation of the BR-index during the firstmitosis after irradiation with 240 r cobalt gamma-rays and 150 r x-rays in distilledwater, or in the presence of sulphoguanidine. No mitoses occur before 12 hours.of recovery time. At the beginning, the two curves show higher BR-indexvalues, and between 21 and 27 hours of recovery-time, both BR-indexes becomeconstant. In the end, when the most damaged and delayed anaphases arrived,the two curves converge.

Thoday found that the different types of aberrations, produced by x- andalpha-rays-forexample, the isolocus breaks and the isolocus-isolocus exchanges-appear at different intervals after irradiation (see Read 1959, figure 33 (a), (c)).Thus the shape of the curves in figure 2 may be explained by the first arrivalafter treatment of those aberrations produced at interphase, when relativelyfewer reunions occur, and therefore the BR-index presents higher values.

These data demonstrate that the sulphoguanidine effect consists in a realenhancement of the reunions, and not in a shift in the cell-population.

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Figure 2. The variation of the breakage-reunion index during the first mitosis afterirradiation. Open triangles: 240 r gamma-rays per 3 hours in distilled water.Filled triangles: 150 r x-rays per 15 min in distilled water. Open circles: 240 rgamma-rays per 3 hours in sulphoguanidine 4 mmol. Filled circles: 150 r x-raysper 15 min in sulphoguanidine 4 mmol.Note: In the x-ray irradiation the smaller' minute' fragments were not scored.

Later, the BR-index was investigated using fl-rays.Table 3 shows that at the variable dose and higher intensity of -irradiation

(120r to 240 r per 3 min), the BR-index presents the same values as in the caseof protracted y-irradiation (240r per 3 hours). Thus, it seems that between120r and 240r, the BR-index is independent of the intensity and quality ofionizing radiation.

It was interesting to test the biochemical mechanism of the sulphoguanidine-effect. Table 4 shows that the effect of sulphoguanidine is suspended not only

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by p-aminobenzoic acid and folic acid (the general sulphanilamide antagonists)but also by arginine and guanine; histidine had a feeble and uncertain anta-gonistic effect against the sulphoguanidine.

The effect of sulphoguanidine was also suspended by 2,4-dinitrophenol andchloramphenicol, which suspend ATP-production and protein-synthesis, res-pectively.

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From our data it follows that, probably at the reunion of break ends, thesulphoguanidine temporarily blocks the incorporation of arginine and guanine,thus maintaining the broken surface as an active end, capable of combiningitself with another active end. To elucidate this mechanism, other treatmentswill be necessary, using radioisotope-labelled compounds.

The interpretation of the BR-index values is a more difficult problem. Wecannot simply consider as a pure random event that the BR-index is 10 forirradiation in distilled water, decreasing to 5 in the presence of sulphoguanidine,and increasing again to 10, when the sulphoguanidine-effect is suspended by thenatural metabolites (p-aminobenzoic acid, arginine, folic-acid, or guanine). It isprobable that the 10 and 5 values of BR-index indicate that at length of the DNA

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Sulphoguanidine effect in irradiation of Vicia faba

and histone molecules, i.e. in the nucleotide and amino-acid sequence of thesemolecules, there is a certain regularity (periodicity) in the distribution of sites,which are available for reunion after breakage. This indicates also that even ifthe breaks are distributed at random, the reunions occur exclusively between twobroken surfaces, which have a certain chemical structure.

It also remains to clarify what is the cause at the molecular level, why thebroken ends of fragments become inactive, i.e. no longer capable for reunion.However, from our data, it appears that the reunion of broken ends is a resultof a DNA-and chromosomal-histone-synthesis.

From the results shown in table 4 one can also conclude that the inactivationof the broken ends, and the occurence of reunions in a ratio of 10:1 is a quickprocess, lasting less than 5-10 min, and that it is independent of a simultaneousATP-production and protein-synthesis, the BR-index being 10 in the presenceof both 2,4-dinitrophenol and chloramphenicol.

Compared to this, the enhancement of the reunions caused by sulpho-guanidine is a cellular metabolic process, dependent on ATP-production andprotein-synthesis. The sulphoguanidine cannot interfere after the inactivationof the broken ends (see variant No. 13 of table 4, which is irradiation in distilledwater for 3 min followed immediately by treatment with sulphoguanidine).

Since sulphoguanidine is a p-amino-benzoic-acid-antagonist, another anta-gonist, namely p-nitrobenzoic acid was tested. This compound has the sameeffect on the BR-index as sulphoguanidine, although the guanidine radical islacking from the p-nitrobenzoic acid molecule.

Table 5 shows that the cytological effect of p-nitrobenzoic acid is alsosuspended by the natural metabolites, p-aminobenzoic acid, arginine and guanine.At present we cannot explain the mechanism of the action of p-nitrobenzoic acid.

Table 6 shows that the effect of sulphoguanidine and p-nitrobenzoic acid ismanifest also in a more marked inhibition of root-growth. This radiosensitizingeffect is additive and is suspended by p-aminobenzoic acid, which re-establishesat 10 the BR-index. However, the other metabolites which suspend thecytological effect of sulphoguanidine and of p-nitrobenzoic acid, cannot efficientlycounteract their growth-inhibitory effect.

The comparison of the radiosensitizing effect of sulphoguanidine andp-nitrobenzoic acid, manifested in root-growth inhibition, with their cytologicaleffect indicates that the increase of reunion yield explains insufficiently thisgrowth inhibition. Thus it may be presumed that the two radiosensitizingcompounds may intensify other forms of radiation damage to the cells, whichare undetectable with the Feulgen technique. Primarily, damage induced inthe histone- and RNA-synthesis is suggested.

Finally, we must mention that in an experiment executed on mouse Ehrlichascites tumour (Schoneich and Lazanyi, unpublished paper), we have noted thatsulphoguanidine has a radiosensitizing effect upon the tumour cells, too.

ACKNOWLEDGMENTS

The author wishes to thank Professor Dr. B. Gyirffy and ProfessorDr. A. Balogh for discussions and suggestions concerning the biochemicalproblems of this paper.

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Sulphoguanidine effect in irradiation of Vicia faba

Des racines primaires de Vicia faba ont te irradies aux des rayons gamma, x et betadans une solution de sulphoguanidine. L'analyse des aberrations anaphases montrent quel'indice rupture-reunion (qui est le nombre de fragments anaphases par ponts) au milieude la premiere mitose apres irradiation, est constant, et petites doses est ind6pendant del'intensit6 et de la quality des radiations ionisantes.

L'indice rupture-r6union montre qu'apr6s irradiation en presence de sulphoguanidine,deux fois plus de ruptures se mettent en runion compare l'eau distillee. L'effet cytolo-gique de la sulphoguanidine on t6 arr&t6 par l'acide aminobenzoique-p, 'acide folique,l'arginine et la guanine aussi bien que par le dinitrophenol 2,4 et le chloramphenicol. Parconsequent, 'effet de la sulphoguanidine semble &tre relief avec 'histone chromosomiqueet la synthese ADN. L'acide nitrobenzoique-p a le meme effet que la sulphoguanidine.L'effet cytologique de ces composes tait relief a l'effet radio-sensibilisant, manifesto dansla paralysie de la croissance de racines.

Die prinimaren Wurzeln von Vicia faba-Samlingen wurden mit Gamma-R6ntgen-und Beta-Strahlen in einer Sulfoguanidin-Losung bestrahlt. Die Analyse der Anaphasenabweichungen zeigte, dab der Index von Wiedervereinigungen nach Bruch (d.h. dieAnzahl von Anaphasen Fragmenten pro Briucken) in der Mitte der ersten Mitose nachBestrahlung konstant war und bei niedrigen Dosierungen von der Intensiftait und Qualiittder ionisierenden Besstrahlung nicht beeinflult wurde.

Der Index von Wiedervereinigungen nach Bruch zeigt an, da13 nach Bestrahlung inGegenwart von Sulfoguanidin, doppelt so viele Briuche als im destillierten Wasser sichwieder vereinigen. Diese zytologische Wirkung des Sulfoguanidins wurde mittelsPara-Aminobenzoesaure, Folicsiure, Arginin and Guanin sowie mittels 2,4-Di-Nitrophenol und Chloramphenicol unterbrochen. Die Sulfoguanidin-Wirkung scheintdaher mit dem Chromosom-Histon und der DNA-Synthese verbunden zu sein. Para-Nitrobenzoesaure hat dieselbe Wirkung wie das Sulfoguanidin. Die zytologische Wirkungdieser Verbindungen ist mit der Radio-Sensibilisierungswirkung verbunden, die sich durchdie Hemmung des Wurzelwachstums offenbart.

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