Experimentally induced chromosome aberrations in plants. I. The production of chromosome aberrations by cyanide and other heavy metal complexing agents

The finding of Lilly and Thoday that potassium cyanide produces structural chromosome changes in root tips of Vicia faba was confirmed. Like mustards, diepoxides, and maleic hydrazide, potassium cyanide seems to act on cells at early interphase. A tendency of cyanide breaks to be concentrated in heterochromatic segments of the chromosomes was evident. The production of chromosome aberrations by cyanide proved to be practically unaffected by the temperature during treatment. In agreement with Lilly and Thoday, the effect of potassium cyanide was found to be dependent on oxygen tension during treatment. The effect of potassium cyanide increases with increasing oxygen concentration up to 100 per cent oxygen. In the absence of oxygen, potassium cyanide was not completely inactive, but produced a low, though significant frequency of aberrations. Pretreatments with 2.4-dinitrophenol did not influence the effect of potassium cyanide. When bean roots were treated with potassium cyanide before a treatment with 8-ethoxycaffeine, or at the same time as they were treated with 8-ethoxycaffeine, the effect of 8-ethoxycaffeine was almost completely suppressed. The effects of a number of other heavy metal complexing agents were also tested. Sodium fluoride, potassium thiocyanate, carbon monoxide, o-phenanthroline, 2.2-bipyridine, and sodium azide were without radiomimetic effect under the conditions employed, and so was a mixture of sodium azide and sodium fluoride. A low, but quite significant, radiomimetic effect was obtained after treatments with sodium diethyldithiocarbamate, cupferron, and 8-hydroxyquinoline. Under anaerobic conditions, the effects of cyanide and cupferron were both quantitatively and qualitatively indistinguishable. Unlike the effect of cyanide, the effect of cupferron was not enhanced by the presence of oxygen. The effects of the same heavy metal complexing agents were tested on the activities of the enzymes catalase and peroxidase. The activities of both of these enzymes were found to be totally inhibited only by potassium cyanide. In the other cases, little correlation was found between ability to inhibit the activities of these enzymes and ability to produce chromosome aberrations. In a number of experiments, hydrogen peroxide was found to be without radiomimetic effect, whether alone or in combination with potassium cyanide. t-Butyl hydroperoxide proved to be active. The effect of t-butyl hydroperoxide was substantially increased by pretreatments with 2.4.-dinitrophenol. The results are discussed, and it is concluded that the observations made do not support the hypothesis that hydrogen peroxide is involved in the production of chromosome aberrations by potassium cyanide. The possibility that organic peroxides are involved cannot be excluded on the bases of the experimental results. As an alternative hypothesis, it is suggested that iron or other heavy metals are present in the chromosomes and that cyanide and other heavy metal complexing agents produce chromosome aberrations by reacting with these metals.     

EXPERIMENTALLY INDUCED CHROMOSOME ABERRATIONS IN PLANTS : II. THE EFFECT OF CYANIDE AND OTHER HEAVY METAL COMPLEXING AGENTS ON THE PRODUCTION OF CHROMOSOME ABERRATIONS BY X-RAYS

The discovery of Lilly and Thoday, that the presence of potassium cyanide (KCN) increases the production of chromosome aberrations by x-rays in anoxia, but has no effect on the production of chromosome aberrations by x-rays in air, was confirmed. In the presence of cyanide, the effect of a given dose of x-rays in nitrogen was found to be even greater than the effect of the same dose of x-rays in air. The cyanide effect on x-ray breakage in nitrogen was obtained at cyanide concentrations as low as 2 x 10^–5 M. The breakage obtained after the combined x-ray-cyanide treatments was of the x-ray type, as evidenced by the distribution of breaks within and between the chromosomes. A number of other heavy metal complexing agents as well as some other compounds were tested for their ability to increase x-ray breakage in nitrogen and air. Of these compounds only cupferron proved to be effective. The results are discussed and it is concluded that the increased x-ray breakage in the presence of cyanide or cupferron cannot be due to an accumulation of peroxides. Instead it is suggested that the cyanide effect may be due to a complex formation between the active agents and heavy metals, presumably iron, within the chromosomes. The consequences of this hypothesis on the concept of the "oxygen effect," are discussed.     

Experimentally induced chromosome abberrations in plants. II. The effect of cyanide and other heavy metal complexing agents on the production of chromosome aberrations by x-rays

The discovery of Lilly and Thoday, that the presence of potassium cyanide (KCN) increases the production of chromosome aberrations by x-rays in anoxia, but has no effect on the production of chromosome aberrations by x-rays in air, was confirmed. In the presence of cyanide, the effect of a given dose of x-rays in nitrogen was found to be even greater than the effect of the same dose of x-rays in air. The cyanide effect on x-ray breakage in nitrogen was obtained at cyanide concentrations as low as 2 x 10(-5)M. The breakage obtained after the combined x-ray-cyanide treatments was of the x-ray type, as evidenced by the distribution of breaks within and between the chromosomes. A number of other heavy metal complexing agents as well as some other compounds were tested for their ability to increase x-ray breakage in nitrogen and air. Of these compounds only cupferron proved to be effective. The results are discussed and it is concluded that the increased x-ray breakage in the presence of cyanide or cupferron cannot be due to an accumulation of peroxides. Instead it is suggested that the cyanide effect may be due to a complex formation between the active agents and heavy metals, presumably iron, within the chromosomes. The consequences of this hypothesis on the concept of the "oxygen effect," are discussed.     

FACTORS AFFECTING THE PRODUCTION OF CHROMOSOME ABERRATIONS BY CHEMICALS

In the present paper, the results of a study on the influence of temperature, hydrogen ion concentration, and oxygen tension on the production of chromosome aberrations in Vicia root tips by maleic hydrazide (MH), di-(2, 3-epoxypropyl)ether (DEPE), and 8-ethoxycaffeine (EOC), are described. Variations in the hydrogen ion concentration of the treatment solutions did not significantly influence the effect of EOC and DEPE. In contrast, the MH effect was considerably diminished by raising the pH from 4.7 to 7.3. A marked increase in the frequencies of aberrations produced by DEPE and MH was obtained by raising the temperature from 3° to 25°C. The effect of EOC increased with rising temperature up to 12°C. With a further rise in temperature the effect of EOC decreased, so that at 25°C. it was of about the same magnitude as at 3°C. The effect of EOC was completely inhibited, and that of MH partly so, when during the treatment (1) oxygen was excluded from the solution, (2) respiration was inhibited by azide or cyanide, or (3) phosphorylation was uncoupled from respiration by 2, 4-dinitrophenol (DNP). Pretreatments with DNP had a similar effect, but posttreatments did not influence the frequencies of aberrations. The effect of DEPE was unchanged by anoxia. Pre- or posttreatments with DNP did not change the total number of aberrations produced by DEPE, but the appearance of the effect was considerably delayed. The results are discussed.     

The Effect of Respiratory Inhibitors and Chelating Agents on the Frequencies of Chromosomal Aberrations Produced by X-Rays in Vicia

Nitrosophenylhydroxylamine-ammonium (cupferron), potassium cyanide, sodium azide, ethylenediaminetetraacetate (EDTA), α,α'-dipyridyl, and o-phenanthroline were tested (1) for their ability to enhance the frequencies of chromosomal aberrations produced by x-rays in the root tip cells of the broad bean, Vicia faba, and (2) for their ability to inhibit oxygen consumption of excised roots of the same plant. In all cases a close correlation was found between the inhibitory effect on respiration and the enhancement of the sensitivity to x-rays at low oxygen pressures. EDTA, dipyridyl, and o-phenanthroline did not affect respiration to any greater extent, and they were without influence on the radiosensitivity. Cyanide, azide, and cupferron, which strongly inhibited respiration, also increased the frequencies of chromosome aberrations produced by x-rays at low oxygen pressures. The relation between oxygen concentration and radiosensitivity was determined both in the presence and the absence of the respiratory inhibitor cupferron. When cupferron was present, the radiosensitivity was influenced by oxygen concentrations 30 times lower than those effective in the absence of the inhibitor. In an atmosphere of pure oxygen, an increase of radiosensitivity of about 20 per cent was obtained with cupferron, EDTA, and potassium cyanide.     

Heterochromatin and chromosome aberrations

The chromosome breaking effect of mitomycin C, methyl methanesulfonate, maleic hydrazide, 8-ethoxycaffeine and gamma rays on the primary root meristematic cells of Nigella damascena was studied. All the agents tested except 8-ethoxycaffeine, produced relatively fewer aberrations, when compared to Vicia faba cells, though both the species have nearly similar total chromosomal length. Test for the presence of heterochromatin in Nigella gave negative results and it is interpreted that the observed differences between Vicia and Nigella are due to the presence and absence of heterochromatin in their chromosome complements respectively. The role of heterochromatin in the production of chromosome aberrations and its significance in evolution are briefly discussed.     

Genetic analysis of mitomycin C-induced interchange in Drosophila melanogaster females

A genetic analysis of an array of mitomycin-induced rearrangements in immature Drosophila oocytes is reported. Induced aberrations were recovered representing detachments of the compound-X chromosome, Y chromosome fragments, X chromosome loss and mosaicism. The spectrum of rearrangements induced by mitomycin C was very similar to that induced by X-ray treatment of immature oocytes. This work suggests that mitomycin C has two modes of action. The drug is radiomimetic for it induces the types of aberrations recovered after X-irradiation. Mitomycin C also seems to have a delayed effect which is reflected in the relatively high recovery of mosaics.     

METABOLIC INHIBITORS AND CHROMOSOME REJOINING

Beatty , Alvin V., and Jeanne W. Beatty . (Emory U., Atlanta, Ga.) Metabolic inhibitors and chromosome rejoining. Amer. Jour. Bot. 46(5): 317–323. 1959.—X ray-induced chromosomal aberrations of the dicentric and ring types in the first microspore division in Tradescantia paludosa were used to study the effects of temperature and chemical substances acting as metabolic modifiers on rejoining under both aerobic and anaerobic conditions. A total dose of 400 r of x rays was administered at 50 r/min. at temperatures ranging from 0.3°C. to 45°C. In the aerobic temperature experiments, 5% oxygen was used in either helium or carbon monoxide and in the anaerobic ones helium was used. In 5% oxygen in helium, the highest number of aberrations was produced at the lowest temperature. When the amount of oxygen in solution was made constant at all temperatures by adjusting the positive pressure of gas in experiments carried out above zero degrees, there was a temperature effect at 0.3°C. and 10°C., but no effect from 20°C. to 45°C. Temperature was believed not to influence chromosome breakage but only rejoining, through its influence on oxidative metabolism, thereby removing the source of energy. In the anaerobic temperature experiments, a low aberration yield of 0.14 occurred at 0.3°C., while a high of 0.66 was recorded at 40°C. Cytochrome oxidase was found not to be involved in the energy supply for rejoining in anaerobic experiments, since exposures in alkaline pyrogallate-washed carbon monoxide gas gave the same results as helium, indicating that both gases only provide anoxic conditions. Under anoxic conditions, pretreatment in M/1000 sodium iodoacetate or M/10,000 p-chloromercuribenzoate solutions had no effect on non-irradiated plants thus treated, but increased the average number of chromosome aberrations per cell produced by x rays at least 2-fold over the irradiated controls. The effect of these 2 salts, as used in these experiments, is believed to have taken place through their inactivation of triosephosphate dehydrogenase, thus inhibiting glycolysis at an early stage before any energy was released.     

Fluid secretion by isolated Malpighian tubules of the housefly Musca domestica

The Malpighian tubules of Musca domestica secrete a fluid with a high concentration of potassium and low concentrations of sodium, calcium, magnesium and chloride compared with the isolating medium.Low secretion rates are produced by low medium potassium concentrations (< 7 mM), with low sodium concentrations (up to 5 mM) increasing secretion; higher potassium concentrations produce higher secretion rates whilst higher sodium concentrations have no further effect. Calcium and magnesium are essential for secretion.The rate of tubule secretion is inversely proportional to the osmotic pressure of the isolating medium and the osmotic pressure of the secreted fluid is slightly hyper-osmotic to the medium over a range of medium osmotic pressures.The metabolic inhibitors cyanide, iodoacetate and 2,4-dinitrophenol inhibit secretion: Cu²⁺ ions, arsenate and ouabain have no effect whereas ethacrynic acid abolishes secretion. 5-hydroxytryptamine, cycle AMP and theophylline have no effect on secretion. Sodium thiocyanate stimulates fluid secretion and increases the osmotic pressure and the concentration of sodium and chloride, but not potassium, in the secreted fluid.     

THE RELATION BETWEEN DNA SYNTHESIS AND CHROMOSOME STRUCTURE AS RESOLVED BY X-RAY DAMAGE

Vicia faba root tip cells were treated for short periods with tritiated thymidine, either immediately before or after exposure of roots to x-rays, and autoradiograph preparations were analysed in an attempt to test the hypothesis that chromatid type (B') aberrations are induced only in those chromosome regions that have synthesized DNA prior to x-irradiation, whereas chromosome type (B') aberrations are induced only in unduplicated chromosome regions. Studying the relation between presence or absence of label at loci involved in aberrations, in cells irradiated at different development stages, and the pattern of labelling in cells carrying both types of aberration leads to the conclusion that B' aberrations are induced only in unreplicated chromosome regions. Following replication, only B' aberrations are induced, but these aberrations are also induced in chromosome regions preparing to incorporate DNA. It is suggested that the doubled response of the chromosome to x-rays prior to DNA incorporation might reflect a physical separation of replicating units prior to replication. The aberration yields in damaged cells which were irradiated in G₁ S, and early G₂ were in the ratio of 1.0:2.0:3.2. The data indicate that the increased yield of B' in early G₂ relative to S cells may be a consequence of changes in the spatial distribution of the chromosomes within the nucleus.     

Studies on the sodium and potassium transport in rabbit polymorphonuclear leukocytes

Rabbit polymorphonuclear leukocytes obtained from peritoneal exudates, incubated at 37°C. following exposure to 4°C., actively reaccumulate potassium while little or no net extrusion of sodium takes place. Preventing the utilization of oxidative metabolism with potassium cyanide, 2,4-dinitrophenol, or a nitrogen atmosphere does not inhibit the recovery process. Inhibitors blocking anaerobic glycolysis (sodium iodoacetate and sodium fluoride in low concentrations) completely abolish the capacity to reaccumulate potassium and cause a further dissipation of the sodium and potassium gradients. Water movements have been shown to be secondary to cation shifts. It is postulated that separate transport mechanisms exist for sodium and potassium and that the process of potassium reaccumulation relies on anaerobic glycolysis as a source of energy.     

Investigation of respiratory and ion transport properties of aging bean stem slices

Ion and oxygen uptake were studied on aging bean stem slices. Oxygen uptake was high immediately after slicing, decreased to a minimum at 100 minutes, and then increased again. Ion uptake per unit of O₂ uptake data suggested that metabolic energy was utilized almost exclusively for sodium transport in fresh tissue but was diverted to potassium transport as the slices aged. Oxygen and ion uptake in fresh slices was less sensitive to 2,4-dinitrophenol as compared to the aged slices, indicating major metabolic and physiological changes occurred during aging. This was further substantiated by the tissue response to cyanide and antimycin A. Oxygen uptake was decreased by cyanide (22% by 1 mm) and antimycin A (14% by 1 microgram per milliliter) in fresh slices but not in aged slices. Potassium uptake that developed during aging was sensitive to cyanide and antimycin A. The results are pertinent to understanding the role of the stem in regulating ion transport in plants.     

Chromosomenaberrationen bei Vicia paba und Ascitestumoren der Maus nach Einwirkung von N-nitroso-N-Methylharnstoff

Summary N-nitroso-N-methylurea was tested for its ability to induce chromosomal aberrations in root-tip meristems of Vicia faba and in two ascites tumour strains (Ehrlich mouse ascites carcinoma and S₂-sarcoma) of the mouse. In all cases the agent was found to be active (Table 1,8). The effectivity of the agent in aberration induction was strikingly different in the two tumour strains and possible reasons for that are discussed.In Vicia the radiomimetic activity of nitrosomethylurea was studied in more detail. The compound had a delayed effect (Table 2) and the aberrations induced were exclusively of the chromatid type. They were preferentially localized in the heterochromatic segments of the chromosomes and unevenly (not length proportional) distributed between the chromosomes. The distribution-ratio of aberrations between the short (5 pairs) and long chromosomes (one pair) was in favour of the short ones and dependent on concentration and duration of treatment (Table 1,2). The distribution of isolocus breaks and chromatid translocations between cells was in accordance with expectation on the basis of a Poisson-distribution (Table 3) and the effectivity of nitrosomethylurea was dependent on the temperature of the treatment solution (Table 4). The agent was almost inactive in the absence of oxygen and its activity was greatly reduced when the meristems were pretreated with the enzymeinhibitor sodium azide. Pretreatments with 2,4-dinitrophenol, KCN, hydroxylamine and chloromycetin did not affect significantly the rate of induced aberrations (Table 5). Pretreatment with the chelator EDTA sensitized to the radiomimetic effects of nitrosomethylurea (Table 6). Breaks induced by the compound stayed open for approximately 4–8 hours (Table 7).The experimental results are discussed and the tentative conclusion as to the mode of action of nitrosomethylurea is, that the aberrations become induced by a breakdown product of the agent the formation of which is dependent on the presence of oxygen and an enzyme containing heavy metal. Probably this decomposition product is not an alkylating agent.

---

---

Herrn Prof. Dr. Hans Bauer zum 60. Geburtstag.     

Mutants of Bacillus megaterium resistant to uncouplers of oxidative phosphorylation.

Mutants of Bacillus megaterium displaying malate-stimulated ATP synthesis resistant to uncouplers of oxidative phosphorylation were isolated and partially characterized. ATP synthesis in such mutants was resistant to carbonyl cyanide m-chlorophenyl hydrazone as well as to other uncouplers including 2,4-dinitrophenol, pentachlorophenol, and sodium azide. ATP synthesis in the wild type and in resistant mutants was sensitive to N,N'-dicyclohexylcarbodiimide, tributyltin, valinomycin plus potassium, and potassium cyanide. Active transport of glycine and glutamine which are sensitive to uncouplers in the wild type was also uncoupler-sensitive in the mutants.     

Influence of exogenous DNA on Ypenyl-treated chromosomes ofVicia faba L.

This paper is a study of the effect of exogenous DNA of different genetic origins on the repair of meristematic cells of primary roots ofVicia faba, damaged by 24 hour treatment with 0·01mm solution of Ypenyl. Both kinds of DNA,i.e. isologous and heterologous, stimulated cell proliferation which was decreased by the action of the radiomimetic and influenced both dynamics of production of chromosome aberrations and the interchromosomal distribution of induced damage. While heterologous DNA increased the frequency of aberrations after all recovery periods studied, isologous DNA significantly decreased the number of chromosomal aberrations. Heterologous DNA increased at the same time the relative number of breaks in the group of small chromosomes, while by the action of isologous DNA the number of aberrations related to this group of chromosomes was relatively decreased.     

Effect of Respiratory Inhibitors on the Motility of Pseudomonas fluorescens

The translational motility of Pseudomonas fluorescens was weakly inhibited by oligomycin, Dicumarol, 2,4-dinitrophenol, 2n-heptyl-4-hydroxyquinoline N-oxide, and potassium cyanide. Atabrine and antimycin A together with potassium cyanide immediately immobilized this bacterium, but antimycin A alone was without effect. Gramicidin D also immobilized P. fluorescens, but its action was inhibited by K(+) and NH(4) (+) ions. In like manner, the effect of p-chloromercuribenzoate could be counteracted with cysteine, thereby suggesting the involvement of -SH groups in flagellar motility processes. It appears that the energy required for motility of P. fluorescens is generated by oxidative phosphorylation mediated by the cytochrome system.     

Effects of Some Inhibitors on the Temperature-Dependent Component of Resting Potential in Lobster Axon

The resting membrane potential of the lobster axon becomes 5–8 mv more negative when the temperature of the perfusion solution is increased 10°C. This potential change is about twice that predicted if the axon membrane potential followed that expected for a potassium ion electrode potential. When the inhibitors, 2, 4-dinitrophenol, sodium cyanide, and sodium azide, were added separately to the perfusion medium the potential change was reduced to about 1.4 times that predicted for a potassium ion electrode potential. Assays of axons exposed to these inhibitors showed that ATP levels were reduced to about one-fourth that obtained for control axons. Ouabain added to the perfusion medium reduced the potential change to that expected for a potassium ion electrode potential. These results suggest that the resting potential changes with temperature as a result of the activity of an electrogenic ion pump.     

FISH analysis of telomeric repeat sequences and their involvement in chromosomal aberrations induced by radiomimetic compounds in hamster cells

The behaviour of telomeric repeat sequences in Chinese hamster CHO and CHE cell lines treated with the radiomimetic drugs bleomycin (BLM) and streptonigrin (STN) and the effect of these drugs on telomerase activity was investigated. Fluorescence in situ hybridisation revealed that 18% of the scored aberrations induced by BLM and 14% of those induced by STN in CHO cells exhibited telomeric repeat signals. In CHE cells, 29% of the total aberrations induced by BLM and 45% of those induced by STN involved telomeric repeat sequences. Acentric fragments labelled along their entire length and translocations of telomeric repeat sequences were also found in both cell lines. These results suggest that telomeric repeat sequences are preferentially involved in chromosome breakage, fragility and recombination induced by radiomimetic agents. In addition, some of the damaged CHE cells exhibited one or more chromosomes with additional zones of hybridisation, indicating the possible amplification of (TTAGGG)(n) repeats by telomerase. However, the fact that none of the radiomimetic compounds tested produced any effect on telomerase activity suggests that this enzyme is not related to the assumed amplification events induced by BLM and STN in CHE cells.     

Characterization of a lysine-specific active transport system in Rickettsia prowazeki.

Rickettsia prowazeki possesses an active transport system for lysine with a Kt of influx of 1 muM. Extraction and chromatographic analysis of the accumulated labeled material show the material to be lysine rather than a derivative. This intracellular lysine pool can be exchanged with external unlabeled substrates for at least 10 min; The lysine analogues L-aminoethyl cysteine, N-methyl lysine, hydroxylysine, and D-lysine competitively inhibit uptake of L-lysine, but cadaverine, diaminopimelate, arginine, ornithine, and epsilon-aminocaproate do not. Accumulation of lysine can be inhibited by the energy poisons potassium cyanide, triphenylmethyl phosphonium bromide, and 2,4-dinitrophenol. The effect of potassium cyanide, but not 2,4-dinitrophenol or triphenylmethyl phosphonium bromide, can be overcome by adenosine 5'-triphosphate. Both energy-dependent influx and energy-independent efflux are inhibited by the sulfhydryl reagents N-ethyl maleimide and p-chloromercuriphenyl sulfonic acid.     

ULTRASTRUCTURAL STUDIES OF RADIATION-INDUCED CHROMOSOME DAMAGE

The fine structure of radiation-induced chromosomal aberrations in Potorous tridactylis (rat kangaroo) cells was examined in situ by electron microscopy. The observations on the structure of terminal deletions (acentric fragments), anaphase bridges and "gaps," sidearm bridges, and specialized regions, such as the nucleolus organizer, are discussed in detail. Conclusions based on these observations are the following: (a) damage is physically expressed only at anaphase; (b) a gap region is composed of two subunits, each of which is about 800–1000 A in diameter and may correspond to a half-chromatid structure; (c) the ends of acentric fragments are structurally similar to normal chromosome ends, except where the break occurs in a specific region such as the secondary constriction; (d) at metaphase the fragment and the main portion of the chromosome move as a single unit to the equator, and the two units are disconnected only at the onset of anaphase; (e) sidearm bridges appear to be exchanges, involving a subchromatid unit. The interpretation of this evidence is consistent with the hypothesis that the chromosome is a multistranded structure.