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Mutation Research, 55 ( 1 9 7 8 ) 1 2 1 - - 1 4 5 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

GENETIC TOXICOLOGY OF BLEOMYCIN

B A L D E V K. V I G * ' * * a n d R O G E R L E W I S * * *

* Nevada Mental Health Institute, P.O. Box 2460, Reno, N V 89505, ** Department o f Biology, and *** Department o f Biochemistry, University o f Nevada, Reno, N V 89557 (U.S.A.) ( R e c e i v e d 31 M a y 1 9 7 8 ) ( R e v i s e d r e c e i v e d 19 S e p t e m b e r 1 9 7 8 ) ( A c c e p t e d 20 S e p t e m b e r 1 9 7 8 )

Co~e~s Summary ................................................... Introduction ................................................. Action on DNA ............................................... Effects on DNA metabolism ....................................... Repair of bleomycin-induced damage to DNA ........................... Inactivation of bleomycin ........................................ G e n e r a l e f f e c t s o n cell m o r p h o l o g y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distribution .................................................. E f f e c t s o n cell-cycle p r o g r e s s i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects on chromatin ............................................ Effects on chromosomes ......................................... In vitro studies . . . ........................................... Numerical aberrations ......................................... I n vivo e f f e c t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potentiation of effect on induced chromosomal aberrations ................ Induction of micronuclei ....................................... Induction of sister-chromatid exchanges ............................. Mutagenic effects on lower eukaryotes ................................ Conclusions .................................................. Acknowledgements ............................................. References ..................................................

121 122 124 126 126 127 127 128 128 131 132 132 135 136 137 138 138 138 139 140 140

Summary Bleomycin (BLM), an antibiotic obtained from Streptomyces verticillus, is of significance as an antineoplastic agent. The c o m p o u n d is actually the mixture * Address reprint requests to Dr. B.K. Vig, N e v a d a Mental Health Institute, P.O. B o x 2 4 6 0 , R e n o , NV 89505 (U.S.A.). A b b r e v i a t i o n s : BLM, b l e o m y c i n ; PCC, p r e m a t u r e c h r o m o s o m e c o n d e n s a t i o n ; SCE, sister-chromatid exchange.

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of some 200 related forms which differ from each other in the amine moiety. The drug, at low concentrations, can cause elimination of bases, particularly thymine. This causes strand breakage of DNA and inhibition of cell growth. The influence of BLM on cell growth may be unrelated to the effects on DNA. In general, mitotically dividing cells show more DNA damage than ~$on-dividing cells. G2 seems to be the most sensitive phase indicating that cell death may not be related to a direct effect of BLM on DNA replication. The antibiotic shows specific effects on chromatin and causes chromosomal damage in all sub-phases of interphase. It can affect early prophase chromosomes also. Suggestion has been made that BLM-induced breakage and cell death are similar to those induced by densely ionizing radiations. Whereas the antibiotic affects the frequency of somatic crossing over and produces micronuclei, the data on mutation induction and production of sister-chromatid exchanges do not permit classifying BLM as a potent inducer of these phenomena. The genetic effects of BLM can be modified quantitatively by thiol compounds, caffeine, hyperthermia and H202. It is concluded that the available data do not permit assessment of genetic damage in the offsprings of BLMtreated patients. Such studies are urgently needed, as are the studies to find out the effects of BLM on meiotic phenomena.

Introduction

The bleomycins are a group of basic, water soluble antibiotics, similar in physicochemical properties and chemical structure [114,155]. This class of compounds was first identified by Umezawa et al. [118] in culture filtrates of Streptomyces verticillus. Chemical analysis of the antibiotics revealed that they all contain 1-gulose, 3-O-carbomoyl-D-mannose, 5 amino acids and an amine. The amine moiety is characteristic o f each BLM in the series, of which there are approximately 200. Fig. 1 shows the general structure of BLM [113]. BLM-A2, for example, possesses 3-aminopropyl dimethylsulfonium ion as its amine, whereas, in BLM-B2 the amine is agmatine. The significance of BLM resides in its antineoplastic and antibacterial activity in vivo and in vitro. The drug has been reported to be effective against squamous cell carcinomas and transplantable mouse tumors. Specifically, it has been shown to inhibit the growth of small cell t y p e lung cancer, squamous cell type lung cancer, testicular cancer, cervical cancer, hepatoma AH-66, HeLa cells in culture and Sarcoma 180 cells in mice (see refs. [16] and [96]). The mechanism of action in causing cell death is n o t fully understood, as is generally the case with chemotherapeutic agents, but has been related to DNA strand breaks, chromosomal aberrations, and inhibition of synthesis of specific proteins, among others. The present review seeks to discuss the effects of this antibiotic almost exclusively on those parameters of cellular function and structure which are of consequence from the point of view of genetic toxicology, and no effort is made to enter detailed discussions of the chemistry of the molecule or its antineoplastic activities.

O

NH2

N/ N

O

123

CH~ ,

O

H

°

o~HN

OH

t~~Ot!

a

O N, - - ~ R

H

OH

OH

O~ ~NH:

H2N'-CHa'-CH3 H2N--CH2--CH 2--CH2--NHz H2N'CH2--~H--'CH3 NH2 (BI ° = R--NH--CH2-~H--CH~) NH2 H2N--(CH2)~--NH--CH3 (BI" = R--NH--(CH2h--NH--CH~) CH~ / H2N--(CH2h--N\ CH3 H2N--(CH2)3--N(CHjI3 HzN--(CH2)3--NH--(CH2)3--N(CH3)z HaN--(CHz)3--N--(CH2)3--NH2 H2N--(CH2)~--NH--CH(CH~)--(CH2)2--NH2 (BI" = R--NH--(CH3h--NH--CH(CH3)--(CH2)2--NH2) H2N--(CH2)~--NC H2N--(CH2)3--~ H2N--(CH2)3--N

O

H2N--(CH2h--N~ 11: N--(CH2h--NH--(CH2)3--OH H2N--(CH2h--NH--(CH2)3--OCH3

HzN--(CH2).~--NH--CH2~ H2N--(CH1)3--NH--~H~ H2N--CH2-~-~

H2N--(CH2)j--NH~ b

Pig. 1. (a) Structure of the bleomyein molecule, with R (the terminal moiety) in bleomyeinie acid=OH. (b) Examples of amines incorporated into the terminal amine moiety of the bleomyein molecule. (Prom

124 Action on DNA The ability of BLM to alter DNA metabolism appears to be due to events which take place on DNA. That [3H]BLM binds to DNA has been d o c u m e n t e d in studies with native E. coli DNA [102]. When mixtures of the antibiotic and DNA were subjected to Sephadex column chromatography it was clearly demonstrated that a stable hybrid of the t w o molecules was formed. Furthermore, (i) the binding of BLM was specific for DNA, with no interaction with R N A being detected, (ii) the interaction was enhanced by the addition of 2-mercaptoethanol, and (iii) the association was decreased by the addition of Cu 2+ or Zn 2÷. Equilibrium dialysis [102] and difference spectroscopy measurements [49,67] confirm that an interaction between BLM and DNA takes place. It is not known if such a mechanism is also responsible for the liberation of DNA from BLM-treated DNA-membrane complexes [ 87 ]. It has been suggested [73] that the interaction of BLM and DNA involves interaction o f a thiazole ring in the BLM structure between the stacked bases of DNA. A particular interaction of the thiazole base with thymine is proposed to be stabilized by an intermolecular electron transfer between the N-3 atom of thymine and the sulfur atom of the thiazole moiety. Additionally, this interaction is probably stabilized b y ionic interactions between the negatively charged phosphates of DNA and positively charged amine moiety of the BLM molecule [75]. The antagonism of BLM to DNA metabolism is not due solely to its binding to DNA, but seems to reside in events which occur after the formation of the BLM--DNA complex. However, the mechanism by which BLM alters the chemical structure of DNA is complicated; at least two measurable phenomena, strand scission of the DNA and elimination of pyrimidine and purine bases from the DNA, are known to take place. Among the first reports that BLM acted u p o n DNA were those of Suzuki et al. [102,104], who showed that BLM induced strand breakage. By alkaline sucrose gradient centrifugation of DNA from BLM-treated E. coli and HeLa cells, it was determined that BLM caused single-stranded scissions in the cellular DNA. This fragmentation process has been studied in vitro and is shown to require the presence of no added factors [98,117]; however, it is enhanced by 2-mercaptoethanol [76], dithiothreitol [67] or hydrogen peroxide [79]. On the other hand, strand cleavage is inhibited by Zn 2+ and Cu 2÷ [78,117] which can effectively chelate with the antibiotic thus rendering it unable to complex with DNA. General confirmation and the study of various conditions under which BLM causes fragmentation of DNA has been since reported using a wide variety of cell types [18,27,32,62,67,87,95,106]. At low concentrations of the drug single-strand breaks occur, b u t at high concentrations, double-strand breaks are observed [62]. Double-strand breaks are probably independent events which take place near each other on the duplex DNA. Single-strand s~issions have also been observed with ~ X 174 DNA [79], and with the synthetic polymers, p o l y [ d G , dC] and p o l y [ d A , dT] [32, 76,79]. A report [64], that BLM-induced DNA strand scission is optimal at pH 9.0, suggests that an unprotonated amine is essential for activity. Investigations employing L1210 cells have shown that BLM causes extensive

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fragmentation in some parts of the cellular DNA while other segments of the macromolecule are affected to a lesser degree [39]. This might indicate that the cellular DNA possesses some sensitive and resistant segments in its chromosomal structure. Alternately, perhaps only a part of the cell population is susceptible to the drug's action while other cells are resistant. As an alternate m e t h o d to study the BLM-induced fragmentation of DNA, measurements of the melting temperatures of treated versus untreated DNA have been employed, lTsing such techniques Nagai and co-workers [76--78] have shown that the Tm of DNA treated with BLM in the presence of either 2-mercaptoethanol or hydrogen peroxide produced single-strand scissions in the DNA with a concomitant decrease in the observed Tm. The other major p h e n o m e n o n observed in BLM-treated DNA is that of base elimination. Treatment of DNA either in vivo or in vitro causes the liberation of pyrimidine or pyrimidine and purine bases from that DNA. At lower concentrations of the drug (below 100 pg/ml in the presence of dithiothreitol) only thymine is released [32,40,67,105]. The release of thymine is known to be dependent upon the composition of DNA, i.e. the greater the mole fraction of dA : dT in the DNA, the more thymine is liberated [67]. However, at higher levels of treatment (12 mg/ml in the presence of 2-mercaptoethanol) all four bases are released [33]. The elimination of bases from lac operator DNA reveal two interesting results. Firsty, 70% of the bases removed were thymine, 22% were cytosine and 8% were adenine; guanine remained intact. Secondly, of the thymines which were eliminated, 92% were adjacent to a guanine [72]. In spite of the fact that strand scission was the first and most c o m m o n l y observed result of BLM action on DNA, it is quite possible that strand breakage is n o t the direct consequence of BLM action. More likely it is an event which occurs non-specifically with the BLM-produced apyrimidinic and/or apurinic DNA. The concept that BLM causes fragmentation of DNA was primarily derived from studies in which the drug-treated DNA was isolated by alkaline sucrose gradients. The basic problem with the use of this technique is the alkaline treatment of the DNA. It is well d o c u m e n t e d that chain cleavage readily occurs in apurinic D N A which is exposed to hydroxide ion [7,14]. The idea that hydroxide ion initiates the cleavage of BLM-treated DNA is supported by the fact that when strand scissions are measured by the filter assay technique, in the absence o f hydroxide ion and in the presence of 5 pg/ml and 2 mM dithiothreitol, no significant strand scission is observed. However, upon addition of KOH, the BLM-treated DNA appears to be hydrolyzed [71]. Furthermore, when neutral sucrose gradients are employed to detect breaks in BLMtreated superhelical DNA, no breaks are measured. Likewise, the addition of dithiothreitol produced strand scissions when measured by this technique [94]. This result is not unexpected in light of the suggestion by Kent et al. [41] that thiol c o m p o u n d s react with purine deoxyribosyl linkages in DNA giving a mecaptalated DNA, the phosphodiester bond of which is easily hydrolyzed. Finally, the fact that BLM-fragmented DNA contains 5'-phosphoryl and 3'-hydroxyl termini, is consistent with the concept of alkali mediated cleavage of apurinic or apyrimidinic DNA [53].

126 Effects on DNA metabolism The biological action of BLM is accomplished by the ability of this antibiotic to alter normal DNA structure and metabolism. That DNA metabolism is influenced by BLM was shown by Umezawa and co-workers [52,103], who demonstrated that the drug inhibits incorporation of radioactive thymidine into the DNA of HeLa, Ehrlich carcinoma, and E. coli cells. In contrast, the incorporation o f tritiated uridine into RNA and [14C]leucine into proteins was unaffected b y BLM treatment. More recently, studies with mouse L cells [123] and L 1 5 1 7 8 Y l y m p h o m a cells [66] confirmed these observations. Muller et al. [68,69] also reported that BLM was inhibitory to DNA and RNA synthesis in isolated enzyme systems, b u t not to either RNA-dependent DNA polymerase or to cell free protein synthesis. In E. coli and Ehrlich carcinoma cells, an increase in DNA polymerase activity following treatment with 40 pg/ml BLM and 1 pM 2-mercaptoethanol has been reported [61,126]. The stimulation lasted for only a short period and was presumably due to the creation of additional 3'-hydroxyl ends (primers) on the template DNA. After a short stimulatory burst, BLM treatment became markedly inhibitory to the polymerization reaction. Investigations by Muller et al. [69] have shown that when DNA synthesis is directed by DNA rich in dA and dT, BLM causes an overall inhibition of polymerization; however, when the reaction employs a template high in dG and dC content, DNA synthesis is stimulated. That BLM stimulates the incorporation of [ 3H] TTP into the DNA of isolated liver and h e p a t o m a nuclei has been shown by Spangler et al. [101]. A 13-fold increase in incorporation was measured for host liver and hepatoma 16 nuclei, 8-fold for hepatoma 7800 nuclei, and 3-fold for hepatoma 777 nuclei. A similar stimulation was measured for nuclei denuded of membranes by triton X-100, as well as when polymerase activity was measured employing chromatin or calf t h y m u s DNA as template and primer. It was not shown whether this stimulation was due to BLM's action directly on the DNA or whether direct action on one or more of the proteins important to DNA synthesis was involved. However, since BLM-induced stimulations of the [3H]TTP incorporation were similar in both nuclei and in preparations devoid of membranes, it is clear that in this study the nuclear membrane did not play an important role in the action o f BLM. Thus, these data are more probably the result of the repair of thymine bases which were liberated from the template DNA b y BLM treatment [31,39, 66,103] and, therefore, do not involve a direct action of BLM on the proteins required for [3H]TTP incorporation. Two enzymes important in DNA metabolism are altered by BLM treatment. At concentrations t o o low to cause strand scission in DNA (0.01--0.1 #g/ml) BLM-A2 inhibits DNA ligase [61]. Pancreatic DNAase exhibits a slight stimulation of activity caused by drug treatment [70,126]. This latter effect is reportedly lost in the presence of dithiothreitol. Repair o f bleomycin-induced damage to DNA The ability of mammalian cells to repair DNA damage induced b y BLM was first demonstrated b y Terasima et al. [109] and has since been confirmed [93,

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95]. These investigators measured the rejoining of single-strand breaks in DNA up to 3 h after the removal of the drug when incubation of cells was continued in a drug-free medium. After 24 h of incubation, double-strand breaks were repaired. Fujiwara and Kando [27] showed a small unscheduled incorporation of [3H]thymidine into non-S phase HeLa cells after 30 min exposure to BLM. In rate liver, in situ studies by Cox et al. [18] showed that the drug-induced damage to the liver DNA was repaired after the removal of the drug. It is known t h a t BLM has no effect on replication but induces extensive repair synthesis of DNA [93]. Attempts made to relate the repair of BLM-induced DNA damage to the damage which follows radiation treatment are contradictory. That BLM and X-ray treatments cause additive influences on HeLa cell repair has been reported [10]. On the other hand, studies with mouse m a m m a r y carcinoma cells showed a synergistic response between the BLM and X-ray treatment [59]. When Bleehen et al. [11] tested a variety of cell types, including bacterial, mouse m a m m a r y and HeLa cells, t h e y observed that upon t r e a t m e n t with BLM followed by X-ray exposure, all cell types showed an additive response for the two types of repair of DNA damage. However, when X-radiation was followed by BLM treatment, some cells exhibited an additive response, whereas others were synergistic. It was, therefore, suggested that the synergistic response is possibly due to an inhibition of the radiation repair system by BLM. o n the contrary, Byfleld et al. [15] found, that in HeLa cells, as well as in rat t u m o r cells, BLM did not inhibit the repair of X-ray induced DNA single strand breaks and that X-ray and BLM damage is quite similar, though independent, in induction and repair. Using radiation sensitive mutants of E. coli, Yamagami et al. [125] found that UV-sensitive and UV- and X-ray-sensitive strains are equally sensitive to BLM as the wild strain. This suggests that the BLM-induced damage to DNA is different from t h a t of X-ray. Furthermore, a UV-sensitive m u t a n t obtained from a BLM-sensitive strain of E. coli was far more suspectible to BLM damage than the strain which was sensitive to BLM only. Recently, Zollner et al. [130] have shown t h a t an alkaline DNAase isolated from cultured l y m p h o m a cells is activated by BLM t r e a t m e n t of the cells. Since this alkaline DNAase shows a preference for denatured DNA as a substrate, it is believed to be an enzyme which might participate in the repair process. Inactivation o f bleomycin Normal resistance to BLM action is observed in various cells and appears to vary in magnitude as cell types are compared. This resistance is at least partially due to the presence of an enzyme which has the ability to degrade the antibiotic. BLM hydroxylase acts by catalyzing the hydrolysis of the free amine m o i e t y of the drug thus rendering the antibiotic inactive. A correlation between the cell suspectibility to the drug and enzyme content has been established specifically for squamous cell carcinoma and sarcoma induced by 3-methylcholanthrene [119] and for AH66 and AH66F h e p a t o m a cells [62]. General effects on cell morphology BLM induced alterations of the cell range from fine structural changes, like lack of electron dense chromatin, to production of hypertrophied organelles

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like mitochondria and Golgi bodies and an increase in cell volume with multiple nuclei [50]. The latter results from the continuation of DNA synthesis in a system which does n o t undergo cytokinesis [50,80,81]. Nucleolar components may shrink [50], presumably due to the fact that the chemical causes loss of ribosomal DNA sequences [20]. It is interesting in this regard that, in spite of similar molecular weights, the nucleolar DNA is far more (as much as 20--30 times) sensitive, than nucleoplasmic DNA, to degradation by BLM-A2 [19,20]. Distribution The distribution of, and toxicity to, BLM in various tissues of the b o d y as well as various organelles of the cell is not uniform. Considering tissue specificity, the antibiotic is far more toxic to skin and lungs. This is in conformity with the observations that distribution of [3H]BLM in various organs of mice indicated a low inactivity of the drug in these tissues. Similarly, b y comparison, the mouse squamous cell carcinoma shows a higher concentration of the label than does sarcoma. The inactivity, suggested to be due to enzymatic reduction of the BLM molecule to a non-toxic substance, is age-dependent, at least in mice [116], and raises the interesting possibility o f differential induction of genetic damage by this antibiotic in individuals of different ages. Ascites t u m o r cells, removed at appropriate times from tumor-bearing mice that had received radioactively labelled BLM (sp. act. 27 #Ci/mM), were studied for the intracellular distribution of 14C located at the 3-aminopropyl dimethylsulfonium site [25]. Autoradiographs showed that 2 h after injection the BLM molecule was adsorbed to the surface of cell membrane and 4 h later the molecule was in the cell, mainly close to the nuclear membrane. After longer exposures which permitted geographic stabilization of the molecule, no BLM was detectable in the nucleoplasm. Even in the cells which had been severely effected to the extent of being disorganized and necrotic because of a high level of intracellular localization of BLM, no drug was observed in the nucleus [25]. In another study, however, the label was detected on the chromosomes [26]. The concentration of BLM on the nuclear membrane appears to be closely associated with the degree of cell injury, including chromosomal aberrations, and even though prophase chromosomes can be damaged by exposure to BLM [45], some studies have shown a total lack of incorporation of isotope into metaphase chromosomes [25]. The cell death, chromosomal aberrations, cell injury and DNA degradation, can then be linked to the observations that the related molecule, phleomycin, can bring a b o u t the release of membrane-associated DNA and cause DNA degradation in Bacillus subtillus where it acts on the cell wall [92]. The BLM-induced strand scission o f DNA b y activation of DNA ligase can explain cell injury and support the hypothesis that chromosomal aberrations induced by chemicals m a y originate at the nuclear envelop which aUedgedly has heterochromatic zones attached to it [ 120]. Effects on cell-cycle progression Both copper-free and copper-chelated BLM's show similar activity in inhibiting bacterial growth and growth of various cultured mammalian cells [116].

129 The concentrations of BLM which apparently have no effect on R N A or DNA synthesis, as shown b y lack of inhibition of [3H]uridine or [3H]TdR incorporation, or on protein synthesis, as shown by leucine-labeling experiments, do inhibit cell division of HeLa ceils [52,110]. The blockage of HeLa $3 cells in G2 is prominent ([80], see also ref. [110]) even though some cells pass on to the next S without cytokinesis and thus appear with 4X DNA content. The studies by Barranco and Bolton [1] and Barranco and Humphrey [4] have shown that the most sensitive stage to BLM damage is mitosis, since DNA strand breakage is the greatest in this stage, and that the order of sensitivity of dividing cells in other phases is G2 > early S > l a t e S > G , . The concentrations of BLM which may not be effective in G1, S or G2 phases can inhibit cells in mitosis. However, the low concentrations which may n o t effect G1, S or G2 cells immediately, reduce the replication activity in the following S [ 17 ]. It is difficult to pin down the cause of differential survival of BLM-treated ceils in various phases. It could be the differential damage or differential ability to repair damage [17]. However, the potentially lethal damage apparently is not related to DNA strand breakage since cells in mitosis which show greater DNA strand breakage than non-dividing cells, and can progress to ensuing GI [4], are less sensitive than the latter. Also non-dividing cells, unlike mitotic cells, do n o t recover from potentially lethal damage. Such cells may show twice as much lethality as dividing cells [6]. It appears that a fixed amount of recovery can occur regardless of the amount of original damage [1]. Nonetheless, it had also been suggested that ceils treated in mitosis cannot recover from B LM-induced potentially lethal damage ([ 3], also see ref. [ 30]). The treatment of synchronous populations of mouse L ceils has shown a lack of effect of BLM on early S phase as determined Joy autoradiographic detection of incorporated [3H]TdR [124]. However, the completion of DNA synthesis phase is blocked by the drug and the cells cannot move on to G2. Earlier studies b y Barranco et al. [5] are in confirmity with the later findings [1,6] that human t u m o r cells treated in vivo show accumulation at the S--G2 boundary while other phases are not so severely effected. Such accumulation, upon removal of BLM block, may lead the cells through mitosis and ensuing S witho u t cytokinesis [79]. It is not conclusively proven if S--G2 blockage is in very late S or soon after the completion of DNA synthesis in very early G2. Though it might be a question o f semantics, one must recognize the possibility of involvement of DNA or some phase-specific proteins. According to T o b e y [111], the BLM inhibits cell progression in G2 without having any effect on genome replication, thereby suggesting the involvement of proteins only. BLM, like other agents, arrests the cells at a number of distinct stages in G2 due to its effect on division-specific proteins [112]. Also the cells in mitosis show sensitivity differentials within various parts of mitosis which may last only an hour or so [4]. Whereas, as discussed above, studies with cell-cycle progression do show that mitosis is the most sensitive phase of cell cycle, the cells do not always accumulate at S--G2 boundary nor is there general agreement about the relative degree of sensitivity of various phases. In one study Linden and colleagues [56] have shown that cells accumulate at G2-mitosis phase and Terasima et al. [109] reported sensitivity of HeLa $3 cells in order of M > e a r l y S > l a t e S >G2, G1.

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Similarly, in contrast to some studies discussed above [4], Bhuyan and Fraser [9] determined such order to be M >G~ > e a r l y S > m i d S >G2. The data by Barranco and Humphrey [4] show a lack of any effect on S cells which had entered G~ 1.5 h before BLM (100 pg/ml for 30 min) was added to the growth medium. These ideas are not in total conformity with some which suggest that BLM blocks cells in G1 and G2 phases and cell death occurs directly in these parts of cell cycle. In these flow microfluorometric analyses of cells the G~ block has been suggested to be at the point of initiation of DNA synthesis. However, Tobey [111] has shown t h a t BLM treatment of Chinese hamster cells has no effect on either the initiation or termination of DNA synthesis; the cells treated within the 56 min of prophase (late G2) continue to divide. But when inhibitory, the G2 effect of the chemical was not readily reversible even when leucine, uridine and thymidine incorporation was at 90, 85 and 80% of the control, respectively, thus defying a correlation between inhibition of cell-cycle progression and macromolecule synthesis. The effect of BLM on cell kinetics in regard to the type of damage appears to be similar to that of X-rays [22]. A similar general conclusion was drawn by Byfield and associates [15] who postulated that the enzymatic mechanism for repair may also be quite similar in the two cases. The observations that the kinetics of induced chromosomal aberrations by the two agents is also similar [21], supports the idea. However, comparison of survival curves obtained by some Japanese workers (see ref. [85]) shows that in HeLa cells and V79 Chinese hamster cell lines the recovery patterns in the two treatments are not similar; the BLM effect producing what has been called the "reverse Elkind recove r y " pattern. Additionally the response of human l y m p h o m a cells [22] as well as human melanoma cells [5] is very different to BLM when compared to their response to 1,3,bis(2-chloroethyl)-l-nitrosourea, in spite of the observation that the latter chemical produces effects on survival curves similar to those produced by X-rays [22]. The broad shoulder curve produced by nitrosourea indicates an accumulation of damage before the killing effect is expressed; BLM produces a biphasic response with no shoulders [4] indicating differential recovery of the components of the treated population. The colony-forming ability of BLM-treated L1210 cells has been shown to be effected in parallel to the induction of single-strand breaks in DNA [47]. Such damage is partially repaired upon incubation [39] and apparently is not effected by changes in pH between 6.6 and 7.6 [47]. The breaks in DNA are repaired within 3 h when incubated in medium free of antibiotics [95]. This might explain why fractionation of BLM doses lowers the survival of the treated cells compared to what can be expected on theoretical, additive basis. The major depression in recovery, however, is observed when the doses (say of 2.5 to 50--100 pg/ml for 30 min each are separated by about 1 h. Of course, the BLM-induced potentially lethal damage is also influenced by the ability of the cell to repair such damage. This repair results in higher survival, if the time between drug exposure and replating (i.e. stimulation for cell proliferation) is increased [30]. Whereas enhancement of BLM effect has been reported for agents such as H202 [77], and thiol compounds like dithiothreitol [62,78], the most dramatic effect on cell killing has been shown to be related to increased temperature dur°

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ing BLM treatment of Chinese hamster cells. Hahn and colleagues [30] determined the effect of BLM on cell survival at 37 vs. 43°C. The HA1 cells treated at 43°C showed far lower survival than those treated at 37 or 41°C. In one experiment the survival after 1 h exposure to 30 #g/ml BLM was 4 X 10 -4 at 43 ° C, which is lower by a factor of 500 when compared to the predicted BLM and hyperthermia treatments, separately. Under these conditions, the 37°C treatment with BLM gave a survival value of 40%. The data for both densityinhibited cultures as well as for exponentially growing cells appear similar. The sensitization and cell death due to BLM-induced potentially lethal damage is apparently related, partially, to inhibition of repair mechanisms, and is n o t related to highest uptake of the molecule at 43°C [13], as has been demonstrated for adriamycin. The idea is t h a t 43°C fixes potential lethal damage which is not repaired, and is rather enhanced by hyperthermia. Such damage may be due to protein denaturation and also involves strand scission of DNA [30,31]. Adriamycin, which also shows a similar hyperthermic potentiation effect, however, does not seem to utilize such a repair mechanism(s) [30]. In spite of different mechanisms proposed for adriamycin- and BLM-induced hyperthermic cell death, the lack of effect on repair responsible for rejoining of adriamycin-induced chromosome exchanges at 43°C [122] is similar to that recently found by the senior author for BLM (unpublished). In studies with BLM applied at 43 ° C one would like to find o u t the phase of cell cycle which is most sensitive to combination treatment of hyperthermia and BLM and to know if non-cycling cells in the tumors would also be vulnerable to such effects. This can make a great contribution to cancer chemotherapy. The repair of damage apparently is mediated by several factors. In one study the inhibitors of DNA synthesis, e.g., cytosine arabinoside, or those of protein synthesis, e.g., cyclohexamide, did not effect recovery from BLM-induced potentially lethal damage, whereas inhibitors of RNA synthesis e.g.. actinomycin D, did. Such inhibition of recovery was immediate and complete [1], but may not be differentiable in origin from actinomycin D binding to DNA even at doses which inhibit only RNA synthesis. Effects on chromatin There is a general lack of studies dealing with effect of BLM on chromatin. In one such a t t e m p t Muller and Zahn [72,73] studied the influence of BLM on isolated quail oviduct chromatin. They reported that treatments with 200 pg/ ml BLM and 5 mM dithiothreitol caused the release of t h y m i n e from the nucleoprotein complex. When chromatin from proliferating and non-proliferating tissues were compared, the chromatin isolated from the growing cells was found to be more sensitive to BLM treatment. The accessibility of BLM to the nucleoprotein is increased by a KC1 pretreatment of the chromatin. Thus certain proteins which are extracted by the salt t r e a t m e n t appear to be important in the blocking of the drug from its site o f interaction. In comparison to the activity of BLM to salt-extracted chromatin, it is suggested t h a t histones, which bind in the minor groove, block the activity of BLM [72,73]. Recent studies by Kuo and Hsu [54] indicate a similarity of action of BLM and nucleases in that BLM attacks internucleosome regions (approx. 40--60

132 base pair long strand between nu-bodies) but does not digest the nu-bodies per se. In contrast to nucleases, however, the antibiotic does not produce a series of bands with multiplicity of 10 nucleotides. This leads to the interesting conclusion that BLM might be " m o r e specific in recognition of sequences" than nucleases. In isolated chromatin the observed effects of BLM are similar to those of antineoplastic protein, neocarninostatin. Effects on chromosomes Most chemicals which inhibit DNA synthesis and cause DNA strand breakage also exhibit their effect on chromosomes, almost exclusively during S phase. BLM, however, in spite of its above-mentioned effects on DNA, does not appear to have a phase-specific type of effect during cell cycle. These effects, as discussed below, range from the action of the chemical on Go, G~, S to G2 and even on prophase cells. The chemical consequently has a " n o n - d e l a y e d " type of effect on chromosomes and thus resembles X-rays or UV in this regard.

In vitro effects The first report of BLM-induced chromosome damage appeared in 1970 by Ohama and Kadotani [83] who treated short-term cultures of h u m a n leukocytes with 0.5, 2, 5, 10, 20, and 30 pg/ml of the drug. In these studies the doses of 5 pg/ml or more for 70 h were found to almost totally suppress mitotic activity; but at lower doses a high frequency of aberrations were recovered. These were, as indicated by other studies on similar systems [51,91,107], primarily chromatid-type breaks (showing that the chemical had its major affect on post-G~ chromatin) besides some chromosome breaks and exchanges as well as pulverized chromosomes [36]. Even though no data are available in Ohama and Kadotani's paper [83] on the frequency of chromatid exchanges, the 70 h treated material showed a few dicentrics indicating that rejoining can occur and that either some cells could move onto the second cell cycle or that G1 chromatin was effected. Similar results have been reported for other animal systems, e.g. Chinese hamster [45,46], mouse fibroblasts [88], human HeLa and CERF-CEM cells [88] as well as plant root tips, e.g., Vicia faba [45,100]. In these studies, a respectable frequency of chromatid exchanges was also found. Besides chromatid gaps, chromatid breaks, deletions and exchanges, dicentrics [107] and ring chromosomes [88] have also been recovered with rather high frequency in lymphocytes and L-929 mouse fibroblasts recovered between 20 and 22 h after treatment initiation [88]. The data, as presented, do not permit a critical evaluation of whether these chromosome-type aberrations were found in the cell going through first mitosis or the cells were already in the second cell cycle so t h a t these chromosome-type exchanges were the result of transformation of chromatid-type exchanges. Also, high concentrations of BLM have been reported to cause extensive fragmentation and pulverization of chromosomes of mouse, man and Chinese hamster [36,38,88]. Differential sensitivity of cell lines to BLM-induced aberration has been known. Thus cells of human origin (HeLa and CCRF-CEM) are far more resistant to BLM than mouse (L929) cells, both for the fraction of cell population

133 showing aberrations as well as number of aberrations per cell [88]. Furthermore, between cells of human origin, HeLa cells turned o u t to be more sensitive than CCRF-CEM cells. Such differential sensitivity has been observed for other antibiotic antineoplastic agents also (see for example [121] ), besides differential effect of BLM on strains of E. coli [86]. It is n o t known if these differences are truly intrinsic or are linked to differential permeability of the cell membrane as demonstrated for BLM sensitivity in E. coli [23]. However, it is of interest in this regard that sensitive strains of ascites hepatoma cells show 817 times higher BLM binding to DNA, in spite of the fact that these cells take up only 1.2 times more BLM than resistant ones [62]. O f all the studies which have come to the attention of the authors only one [21] deals with the effect of BLM on Go cells. In view of the fact that unstimulated lymphocytes treated with BLM for 3 h may produce more than 20 times as many dicentrics as chromatid translocations (and twice as many dicentrics as all chromatid aberrations pooled together) this is a strong indication that unreplicated Go chromosome can be effected by BLM in such a way as to effect the entire chromonema laterally (double-stranded breaks in DNA), besides producing partial, lateral breakage (single-strand break in DNA). These data also show distinction from G2-effects in which the primary aberrations are of chromatid type. If chromosomal aberrations induced by BLM use similar mechanisms as utilized for BLM-induced cell death, then one expects a parallelism between BLMand X-ray induced aberrations. This is expected in view of the fact that cell killing b y two agents has a similar pattern of response [108] (see Effects on cellcycle progression). The studies by Schmid and colleagues [21] demonstrate such parallelism showing a linear quadratic dose relation for dicentrics and acentric fragments induced by X-rays [98] and a linear dose relation of BLMinduced aberration in Go [21]. Similar observations have been made for micronuclei, premature chromosome condensation of micronuclear contents and mitosis with aberrations [51]. "Consequently it is likely that the cytogenetic effect of BLM can be better compared with that of densely ionizing radiation" [21]. However, a dose--response curve could not be established by HayezDelette and Freemans [ 36] for BLM-induced aberrations. The production of chromosome-type aberration within the first cell cycle, as observed by treatment with S-independent, non-delayed, effects produced by BLM (as also for closely related phleomycin [44,160] ) has been attributed to the action of the drug on G~ chromatin [45]. A recovery of 18 h after BLM treatment of Vicia faba r o o t tips produces chromosome-type exchanges and isochromatid breaks [45] which are best accounted for as having originated in G~. Their frequency is dramatically enhanced b y post-treatment with caffeine (as those of chromatid and sub-chromatid aberrations induced b y BLM in G2 or early prophase). Studies by Tamura and associates [107] have also shown the vulnerability of human G, chromatin in producing n o t only chromosome-type exchanges but also acentric fragments and chromatid exchanges. As shown by Dresp et al. [21] for the Go effect of BLM, the induced chromatid breaks and chromosome exchanges show linear increases with concentrations of BLM to which G, cells are exposed. The production of chromatid aberration in G~ cells exposed to BLM may be explained b y the single-strand breaks in DNA which

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would result into chromatid discontinuities, or residual effect of the drug which can stay in the cell even after washing. In view of the observations that DNA polymerase reaction is not inhibited by BLM [126], that on S-phase cells BLM is much less effective than on G2phase cells [1,17] and that the BLM effect is S-independent [45], it is not surprising t h a t little attention has been focused on the study of S-phase sensitivity of BLM-induced chromosomal aberrations. However, a few studies available do indicate that S-phases cells, e.g. those from mouse fibroblasts and HeLa [88], are far more sensitive than those in G2. Sensitivity of S-phase cells to induced chromosomal aberrations and caffeine-induced enhancement in Vicia faba have also been demonstrated [45,46]. That caffeine can induce enhancement of BLM-induced aberrations can be explained, partly, by a synergistic effect of the chemicals at this stage. The data from Promchainant [91] and Ohama and Kadotani [83] also indicate the action of BLM on S-chromatin, even though no experiments were carried out to particularly study the specificity of the chemical on this phase. It appears, in general, that in contrast to alkylating agents or other S-specific chemicals [43,44] this phase contributes only modestly to the overall chromosome-breaking action of BLM. Studies on cell kinetics indicate that BLM can effect a cell in G2 [1,110-112]. That G2 cells are more sensitive to this antibiotic than S or G1 cells [9], and can be irreversibly inhibited, [111] may indicate susceptibility of chromatin at this stage. The recovery of such aberrations in BLM-treated root tips of Vicia faba [45, 46], HeLa cells [88] and h u m a n lymphocytes [21] recovered 3--5 h after treatm e n t initiation has been taken as evidence of the effect of the drug on G2 chromosomes. This interpretation was based, additionally, on the lack of recovery of labeled HeLa cells when BLM was administered simultaneously with [3H]TdR. The aberrations in these cells included chromatid exchanges, which according to some [24] should not be possible unless the cell synthesizes at least a small a m o u n t of DNA required for reunions. The premature chromosome condensation (PCC) technique as used by Hittleman and Rao [37] has demonstrated the ability of BLM to effect G2 chromosomes of Chinese hamster. Immediate visualization of treated chromosomes by the PCC technique, and the comparison of aberration frequency with the population allowed to pass on to first metaphase after treatment, showed that about one-third of all G2 lesions which give rise to aberrations are repaired by the time the cell reaches metaphase. This repair time has been estimated to be about 60 min. However, the incidence of all types of aberration (gaps, breaks and exchanges reported) was about 5--9 times higher in the BLM-treated PCC material compared to the routine preparation. A possible reason for these differences could be that extensively damaged G2 cells may not progress into metaphase, indicating that a cell may carry only a certain m a x i m u m chromosomal damage. The types of aberration in cells treated in G2, as expected, are primarily of chromatid type [21,108]. However, the G2 cells appear to be at least twice as sensitive as Go cells [21]. These observations agree well with those of Barranco and Humphrey [4] who found BLM to be most effective in G2 phase of the Chinese hamster ovary cells in interphase. Another point of interest is the pul-

135 verization of G2 chromosomes b u t not those of Go. BLM effect along the length of the genome is n o t uniform. Even though the region specificity is not shown, it has been clear from studies b y Ohama and Kadotani [83] and Ohno [84] that n o t all human chromosomes are affected uniformly (see also ref. [12]). Chromosome 3 is most resistant to the effect of the chemical. To compare, chemicals like adriamycin, daunomycin and streptonigrin [121] also appear to be less effective on chromosome 3 than other chromosomes in the human genome. Such differences might reflect either structural and/or functional differences between chromosomes and should be of help in understanding the differential intragenomic effect of chemicals. However, Promchainant's data [91] showed that long arms of chromosomes were effected more frequently than the short arms and larger chromosomes belonging to groups A and D were generally more frequently effected than chromosomes of smaller size, with almost total lack of effect on chromosomes 19, 20, 21, 22 and Y, and the centromeres. The information obtained by Schubert and Rieger [100] using Vicia faba, however, contrasts to some extent from that available for animal cells. Whereas BLM effect on Vicia genome is definitely non-random, there is no specific region which can be labelled a "hotspot", as seen for example, in case of effect of maleic hydrazide on segment 4 or ethyl alcohol effect on segment 19. The hot spots, if any induced by BLM, are more evenly distributed along the length of the genome of Vicia; of course, with aberration clustering in the heterochromatic regions. This effect of BLM, which has non-delayed t y p e of action regarding production of chromosome breakage, differs from that of chemicals expressing delayed effect e.g. mitomycin C, cytostasan, triethylmelamine, and maleic hydrazide. Therefore, S-phase specific "mutagens showing a delayed dffect suggest that S phase specific chromosome structural features and]or processes may have differential, and chromosome region specific, influence on aberration clustering: more so than is the case with non-delayed, non-S-specific mutagens, e.g. BLM or X-rays" [100].

Numerical aberrations Most of the effort of cytogeneticists dealing with induced chromosomal aberrations have centered on looking for structural damage. Information of importance can be obtained by analyzing treated cells during the 2nd ensuing metaphase to find o u t if numerical errors have occurred as result of interference by the drug with activity of spindle fibers or that of the centromeric regions. It is therefore of significance to note that BLM can cause an unequal distribution of chromosomes at least in cells treated for 70 h. In one instance [83], whereas 94% of the untreated cultured leukocytes showed a regular complement of 46 chromosomes, the cultures treated with BLM (2/~g/ml for 70 h) showed only 84% population with normal complement. In the absence of any correlative data between structural damage and numerical aberrations, it would be of interest to find any relationship between the t w o end-points as these might again relate to structural specificity of the chemical. In case of the studies mentioned above as well as one b y Tamura et ai. [107] the frequency of cells with numerical inequalities was only a third of cells with structural aberrations. In one study, however, using 1 pg/ml BLM

136 treatment for 22 h Tamura et al. [107] showed the frequency of cells with breaks and exchanges to be equal to the frequency of cells expressing non-disjunction. It is of concern, however, that even the control showed 7.3% cells with numerical aberrations. Apparently, BLM effects cytokinesis so that polyploid cells accumulate in the treated population [51,91]. This indicates that at concentrations which effect proteins (or whatever) associated with the division of the cytoplasm, the nuclear DNA continues synthesis resulting into 4C, 8C or even higher DNA content of the nucleus [80,81]. In some cells the nuclear division followed by failure of cytokinesis has been known to lead to multinucleate cells [88]. In vivo effects BLM, when administered intravenously at therapeutic doses, is capable of causing chromosomal damage in leukocytes. In one study [12] 4 individuals given 0.3 mg BLM/kg b.w./12 h for 4 days showed chromatid and isochromatid gaps, hypochromatic lesions, and breaks even when cultures were prepared from blood drawn as late as 1 month after therapy. However, no exchanges of any t y p e were reported. This is in contrast with the recovery of dicentrics in preparations of chromosomes by Schinzel and Schmid [97] made from blood of BLM-treated patients with 2 weeks recovery. Besides, as shown by these studies, in 72-h cultures most cells from these BLM-treated patients were in first mitosis. When compared to other antitmmor antibiotics, the effect of BLM appears to last longer in human peripheral lymphocytes. Using patients exposed to a host of antineoplastics, Schinzel and Schmid [97] found that whereas there were no, or very few, aberrations observable in cultured leukocytes obtained from patients using one or more chemotherapeutic agents other than BLM, those on BLM therapy exhibited rather extensive damage even 2 weeks after the treatment was stopped. One can only guess at the possible damage to the gonadal cells of the individuals and the transmissibility of the damage to future offsprings. The cytogenetic evaluation of BLM-induced damage in bone-marrow cells of 2 patients permitted Dresp et al. [21] to tentatively conclude that di- and tricentric chromosomes observed from l y m p h o c y t e preparations resulted from the action of the drug on Go chromosomes. However, in spite of their demonstration of a Go effect in in vitro experiments, the rationalization for an in vivo effect is hard to make. In conformity with these observations, though, are the data showing that dicentrics and isochromatid breaks recovered from BLMtreated Chinese hamster cells may be due to the effect o f chromosomes in pre-S phase. An in vivo effect of BLM is the production of PCC, observed in bone-marrow cells of Chinese hamsters given injections of the drug at doses of 7.5, 15, and 30 mg/animal (30 g b o d y weight/animal). The major source of such PCC has been interpreted to be the BLM-induced micronuclei which express asynchronous replication pattern compared to the main macronucleus. This "imbalance in the cell cycle of different chromatins included in the same cytoplasm" results in chromatin condensing in G~ or S phase [51]. Similarly, Obe et al. [82] have shown that PCC could be induced in vitro in human leukocytes

137 treated with BLM {also with X-ray or A139). In this case the micronuclei had been interpreted to be in G2 or S, but n o t G1 phase. Treatments of these cells with 2/~g/ml BLM for 24 or 48 h showed as many as 3.4% of these cells with.PCC. In both in vivo and in vitro experiments the G1, S and G2 chromatin expressing PCC can be so identified by morphological characteristics of the condensed interphase chromatin [ 51,82 ].

Potentiation o f effect on induced chromosomal aberrrations Information obtained on co-mutagenic action of two or more agents is available for some anticancer, antibiotics (see ref. [122]) and the limited data now appearing in the literature strongly indicate that BLM is no exception. Using Vicia faba root tips for study of BLM-induced chromosomal aberrations, Kihlman et al. [45,46] showed asynergistic effect of BLM and caffeine. In one study, for example (see Table IV in ref. [45], 1-h treatment of roots with 1.75 pg/ml of BLM produced an average of 0.27 aberrations/cell when a 5-h recovery was given. Similar treatment, with additional exposure of roots to 2.5 × 10-3M caffeine during this period of recovery, increased the aberration frequency more than 5-fold, to 1.42 aberrations/cell. The data not only show that G2 (or prophase) chromosomes are vulnerable to the effect of BLM, and that the BLM effect is ~reatly potentiated by caffeine, but also t h a t caffeine, which is t h o u g h t to be primarily an S-phase-specific agent (p. 58 in ref. [43]), could interfere with BLM-induced damage in post-S cells. The interactive result, however, is similar to that of X-rays and caffeine and may not be the result of caffeine treatment on post-replication repair. The potentiation, however, might not be unique to induction of chromosomal aberrations. Griggs and colleagues [28,29] have shown that caffeine treatment can increase cell death and DNA degradation of Escherichia coli, treated with the closely related chemical phleomycin. Perhaps the explanation given for phleomycin that "if caffeine is present it binds to DNA where normal hydrogen bonding has been weakened by the distortion and thereby causes local cooperative d e n a t u r a t i o n " [28] also applies to potentiation of BLMinduced chromosomal aberrations by caffeine. The effect of BLM-induced chromosomal damage at 37°C vs. 43°C has been a subject of recent study in our laboratory. Using concentrations which would produce chromosomal aberrations in about ~ to ~ of the population of Chinese hamster cells treated with BLM at 37 ° C, the frequency of cells with aberrations as well as the number of aberrations per cell has been dramatically increased when treatment was carried out at 43 ° C. In one instance, t r e a t m e n t at 37°C with 2 ttg/ml BLM produced 20% cells with average aberration frequency of 0.39, in contrast to 95% cells with 4.79 aberrations/cell when t r e a t m e n t was carried out at 43 ° C. The frequency of exchanges in material treated at 43°C was found not to be adversely effected as could be expected from Hahn et al.'s [ 30] suggestion that BLM-induced cell death at 43°C is due to lack of repair. Not only can the effect of BLM be potentiated, but BLM can also potentiate the effect of ~/-radiation in induction of chromosomal damage produced in Nigella damascena [65]. At a concentration of 0.5 mg/1, the chemical causes no, or very little, potentiation when doses of radiation are limited to ! krad for dry seed or 400 rad for soaked seed. However, at these doses of radiation treat-

138 ment with 1 mg/1 BLM potentiates the effect, as does 0.5 mg/1 at higher doses of radiation, e.g., 6--10 krad to dry seed or 1000 tad to soaked seed. The moisture content of the seed appears to have considerable influence on BLMinduced potentiation in this case. The main increase, however, is in the frequency of breaks and "micro-fragments". Considering Nagai et al.'s [78] suggestion BLM might "considerably reduce protective effectiveness normally exerted by sulfhydryl groups" and this may lead to potentiation of chromosomal damage.

Induction of micronuclei The induction of micronuclei results from lagging chromosomes, or fragments, and can be induced by a variety of chemicals. However, using BLM at concentrations b e t w e e n 0.1 and 40 mg/kg b o d y weight on ICR strain of mice, Schmid's laboratory [57] showed that a total of 6% of the monochromatic as well as polychromatic erythrocytes expressed micronuclei. This value does not differ much from the control. On the other hand, BLM-induced micronuclei have been reported for in vivo studies using Chinese hamster bone-marrow cells [51,57] and in vitro experiments with CH-C12 cells of Chinese hamster origin [38], as well as human leukocytes [36]. Therefore, the reason for failure to induce micronuclei in mice even at doses far higher than those effective in Chinese hamster bone marrow [51] or those needed to induce traditional aberrations in Chinese hamster cells in culture [38] should be looked into in further detail. The micronuclei so induced could undergo PCC resulting from aynchronous division with the main nucleus. Such micronuclei have been shown [36, 38] to incorporate [3H]TdR when macronucleus may or may not show simultaneous labeling.

Induction of sister-chromatid exchanges (SCEs) Whereas S-phase-specific chemicals are very p o t e n t in inducing SCEs, even at doses at which no tradiational chromosomal aberrations can be observed, BLM, at best, is a poor inducer of the phenomenon. In the first study of SCE induction utilizing m o d e m techniques of sister-chromatid differentiation [48,89,90], Perry and Evans [89] had to use concentrations as high as 10 "s M to substantially increase their frequency over the control range. Thus, at concentrations of 3 × 10 -6 M only 451 SCEs in a sample of 20 Chinese hamster cells were observed against 244 such events in the comparable control, in spite of the fact that the drug was present throughout two cell cycles along with BrdUr. A better idea can be had when one considers 2555 SCEs obtained with similar treatment with 10 -2 M mitomycin C. Kihlman et al. [46], using Vicia faba, also showed that BLM would induce SCEs only at concentrations which also produce a rather high frequency of chromosomal aberrations, again in contrast to alkylating agents which produced SCEs at doses producing no chromosomal aberrations. In a recent study [27a] up to 4 pg/ml of BLM failed to induce this p h e n o m e n o n in human lymphocytes. Mutagenic effects on lower euk~ryotes

The studies of mutation induction in Salmonella have proven to be negative [8] regarding the effect of BLM on this genetic parameter. In similar studies

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using the yeast, Saccharomyces cerevisiae, Hannan and Nasim [34] showed general lack of mutagenic effect on the ade ÷ to ade- systems as well as lack o f reversion from a u x o t r o p h y to p r o t o t r o p h y for t r y p t o p h a n and lysine reversions in strain XV185-14C, and for arginine in a highly UV-revertible strain C1 17813A [55]. A few histidine prototrophs, however, were recovered in XV 18514C strain, b u t at a survival level of 31.2--13.9%. More recently, however, Moore [63] has presented evidence that BLM treatment of the strain CM-1194 at a b o u t 25% survival results in increased reverse mutations, from isoleucine requirements to p r o t o t r o p h y , over 300-fold. All these revertants may not, though, be the result of reverse mutations, and allele non-specific suppressor mutations [129] may contribute to the results observed. Additionally, increased reversion frequencies of two ochre alleles (lys 2-1 and cyc 1-9), and t w o missense alleles (his 1 and trp 2), were also induced [63]. In these studies, however, 50% survival level was accompanied by only 2- to 3-fold increase. To this should be added the case of increased histidine p r o t o t r o p h y at 27--68% survival, but a decreased frequency of spontaneous mutations for lysine p r o t o t r o p h y [35]. Thus, this chemical, like actinomycin [121], is mutagenic as well as antimutagenic. Making allowance for the genetic background of the test organism, one might conclude that BLM effects are highly locus specific and, in line with many other p o t e n t antitumor agents, BLM appears to be only weakly mutagenic. BLM favorably affects the induction of gene conversion and somatic recombination in yeast [45,63]. In strain D7, t r y p t o p h a n non-requiring colonies were produced on selective medium [94] and in CM-1194, Trp 5 and Cyc 1 gene convertants were induced [63], besides both types of recombinations in this d o u b l y heteroaUelic strain. The ade ÷, diploid D7 strain, when treated with BLM, produces several different types of aberrant colonies [45]. Following Zimmermann's suggestion [128], the red/pink sectors so obtained were considered to result from mitotic crossing over between ade 2-119 and ade 2-40 allels. In contrast to lack of such colonies in the control, treatment with 100 pg/ml BLM produced 2 2 / 1 7 0 4 such colonies at 30.2% survival and 6/768 at 7.7% survival. These studies have been confirmed [63] using the same markers in strain CM-1194. Strain D81 showed considerably higher sensitivity in the production of red/white sectors in comparison to red/pink sectors in CM-1194, at equivalent surviving fractions. Considering that BLM is capable of inducing DNA strand breaks (see Action on DNA), the induction o f mitotic recombination can be expected to result from the repair that follows [39]. Conclusions

The discussion presented above does not permit unequivocal statements to be made about the t o x o c i t y of BLM to the genetic material in man. The study of in vitro as well as in vivo effects on chromosomes of somatic cells observed at therapeutic doses of this antibiotic is n o t enough to suggest that the drug might also produce transmissible genetic changes. It has been k n o w n that the production of genetic damage, specifically chromosomal aberrations, in somatic

140 cells, e.g. bone marrow, does n o t guarantee the production of similar, quantitatively equivalent, damage to gametes (see for discussion ref. [121]), and there are no data available to show that in spite of high incidence of aberrations induced in l y m p h o c y t e s of patients treated with BLM [97] there is any transmissible, heritable, change induced in the sex cells of these patients. However, if mutagenicity and carcinogenicity are closely related, there may be a danger of malignant transformation by BLM therapy as has been well documented by Marquardt and Marquardt [58], in spite of the fact that BLM is incapable of inducing mutations in tradiational test systems using Salmonella [8]. Thus, an agent like BLM, which is strongly effective in producing genetic aberrations in eukaryotes could be easily missed as mutagen (and carcinogen) in preliminary tests with akaryotes, or even by sister-chromatid exchange test when low doses are used. This review is aimed at providing information about the work done with BLM, but also brings h o m e the point that a lot remains to be accomplished to understand the potentials of the drug as a mutagenic agent. It would be rewarding to study the details of mechanisms b y which cell death is caused and to see if the production of chromosomal aberrations contributes to this end, and if the two end-results have some similarities of mechanisms involved. In this respect it might be profitable to study the effect of hyperthermia on the production of aberrations requiring rejoining since, according to Hahn et al. [30], the increased incidence of cell death by BLM treatment at 43°C can be attributed to lack of inhibition of repair. Also, as mentioned above, it would be importance to know if BLM-induced genetic damage in vivo is age related as shown for cell death. One might also suggest the study of meiotic processes, e.g., chromosome pairing, chiasma formation, induction of non-disjunction and related processes as might be influenced by BLM. The paucity of information about the effects of BLM on genetic material of lower eukaryotes is understandable in view of the early data showing lack of effect on akaryotic genome. The efforts now initiated in this direction [35] need to be continued and extended to the study of effects on non-nuclear parameters, e.g., mitochondrial DNA. It is only when data from such above-mentioned studies become available, along with the information on co-mutagenicity with other agents, that geneticists would be able to make a critical evaluation of risks associated with BLM therapy to the progeny of those in reproductive age. Acknowledgements We are thankful to Mr. John Wassom and Ms. Beth Owens for p r o m p t help in searching the literature through EMIC files, and to Ms. Beverly Nation and Sherry Stevens for typing the manuscript. References 1 B a r r a n c o , S.C., a n d W . E . B o l ~ o n , Cell c y c l e p h a s e r e c o v e r y f r o m b l e o m y c i n - i n d u c e d p o t e n t i a l l y lethal damage, Cancer Res., 37 (1977) 2589--2591. 2 B a r r a n c o , S.C., B. D r e w i n k o a n d R . M . H u m p h r e y , D i f f e r e n t i a l r e s p o n s e b y h u m a n m e l a n o m a cells t o 1,3-bis(2-chloroethyl)-l-nitrosourea and bleomycin, Mutation Res., 19 (1973) 277--280. 3 B a r ~ a n c o , S.C., G . R . H a e n e l t a n d W.E. B o l t o n , I n h i b i t i o n o f r e c o v e r y f r o m b l e o m y c i n - i n d u c e d p o t e n t i a l l y l e t h a l d a m a g e , J. N a t l . C a n c e r I n s t . , 5 9 ( 1 9 7 7 ) 1 6 8 5 - - 1 6 9 1 .

141 4 B a r r a n c o , S.C., a n d R . M . H u m p h r e y , T h e e f f e c t s o f b l e o m y c i n o n survival a n d cell p r o g r e s s i o n in C h i n e s e h a m s t e r cells in v i t r o , C a n c e r R e s . , 31 ( 1 9 7 1 ) 1 2 1 8 - - 1 2 2 3 . 5 B a r r a n c o , S.C.. J . K . L u c e , M.M. R o m s d a h l a n d R . M . H u m p h r e y , B l e o m y c i n as a p o s s i b l e s y n c h r o n i z i n g a g e n t f o r h u m a n t u m o r cells in v i v o , C a n c e r R e s . , 3 3 ( 1 9 7 3 ) 8 8 2 - - 8 8 7 . 6 B a r r a n c o , S.C., J . K . N o v a k a n d R . M . H u m p h r e y , S t u d i e s o n r e c o v e r y f r o m c h e m i c a l l y i n d u c e d d a m a g e in m a m m a l i a n cells, C a n c e r R e s . , 3 5 ( 1 9 7 5 ) 1 1 9 4 - - 1 2 0 4 . 7 B a y l e y , C . R . , K.W. B r a m m e r a n d A.S. J o n e s , T h e n u c l e o t i d e s e q u e n c e in d e o x y r i b o n u c l e i c a c i d s , V. T h e a l k a l i n e d e g r a d a t i o n o f a p u r i n i e a c i d s , J. C h e m . S o c . , ( 1 9 6 1 ) 1 9 0 3 - - 1 9 0 7 . 8 B e n e d i c t , W., M.S. B a k e r , L. H a r o u n , E. C h o i a n d B . N . A m e s , M u t a g e n i e i t y o f c a n c e r c h e m o t h e r a peutic agents in the SalmoneUa/microsome test, Cancer Res., 37 (1977) 2209--2213. 9 B h u y a n , B . K . , a n d T . J . F r a s e r , C y t o t o x i c i t y o f a n t i t u m o r a g e n t s in a s y n c h r o n o u s m a m m a l i a n cell s y s t e m , C a n c e r C h e m o t h e r . R e p . ( P a r t I), 5 8 ( 1 9 7 4 ) 1 4 9 - - 1 5 5 . 1 0 B i e n k o w s k a , Z.M., K . B . D a w s o n a n d J . H . P e a c o c k , A c t i o n o f a c t i n o m y c i n s , b l e o m y c i n a n d X - r a y s o n H e L a cells, Br. J. R a d i o l . , 4 6 ( 1 9 7 3 ) 6 1 9 - - 6 2 2 . 11 B l e e h e n , N . M . , N . E . Gilleis a n d P . R . T w e n t y m a n , T h e e f f e c t s o f b l e o m y c i n a n d r a d i a t i o n in c o m b i n a t i o n o n b a c t e r i a l a n d m a m m a l i a n cells in c u l t u r e , Br. J. R a d i o l . , 4 7 ( 1 9 7 4 ) 3 4 6 - - 3 5 1 . 1 2 B o r n s t e i n , R . S . , D . A . H u n g e r f o r d , G. H a i l e r , P . F . E n g s t r o m a n d J . W . Y a r b o r o , C y t o g e n e t i c e f f e c t s o f bleomycin therapy in man, Cancer Res., 31 (1971) 2004--2007. 1 3 B r a u n , J , S . , a n d G . M . H a h n , E n h a n c e d cell k i l l i n g b y b l e o m y e i n a n d 4 3 ° h y p e r t h e r m i a a n d t h e i n h i bition of recovery from potentially lethal damage, Cancer Res., 35 (1975) 2921--2927. 1 4 B r o w n , D.M., a n d A . R . T o d d , E v i d e n c e o n t h e n a t u r e o f t h e c h e m i c a l b o n d s in n u c l e i c a c i d s , in: E. C h a r g a f f a n d J . N . D a v i d s o n ( E d s . ) , T h e N u c l e i c A c i d s , Vol. 7, A c a d e m i c Press, N e w Y o r k , 1 9 5 3 , pp. 4O9--455. 1 5 B y f i e l d , J . E . , Y . C . Lee, L. T u a n d F. K u l h a n i a n , M o l e c u l a r i n t e r a c t i o n o f t h e c o m b i n e d e f f e c t s o f b l e o m y c i n a n d X - r a y s o n m a m m a l i a n cell survival, C a n c e r R e s . , 3 6 ( 1 9 7 6 ) 1 1 3 8 - - 1 1 4 3 . 1 6 C a r t e r , S . K . , C a n c e r t r e a t m e n t t o d a y a n d its i m p a c t o n d r u g d e v e l o p m e n t w i t h s p e c i a l e m p h a s i s o n t h e p h a s e II clinical trials, J . N a t l . C a n c e r I n s t . , 57 ( 1 9 7 6 ) 2 3 5 - - 2 4 4 . 17 C l a r k s o n , J . M . , a n d R . M . H u m p h r e y , T h e s i g n i f i c a n c e o f D N A d a m a g e in t h e cell c y c l e s e n s i t i v i t y o f C h i n e s e h a m s t e r o v a r y cells t o b l e o m y c i n , C a n c e r R e s . , 3 6 ( 1 9 7 6 ) 2 3 4 5 - - 2 3 4 9 . 1 8 C o x , R . A . , A. D a o u d a n d C.C. Irving, D a m a g e o f r a t liver d e o x y r i b o n u c l e i c a c i d b y b l e o m y c i n , Biochem. Pharmacol., 23 (1974) 3147--3151. 1 9 C r o o k e , S . T . , I. D a s k a l , R . K l o m p a n d H. B u s c h , N u c l e o l a r a l t e r a t i o n s i n d u c e d i n N o v i k o f f h e p a t o m a ascites cells b y b l e o m y c i n , F e d . P r o c . , 3 3 ( 1 9 7 4 ) 5 8 1 . 2 0 C r o o k e , S.T., T . O . Sitz, M. B a n n o n a n d H. B u s c h , B i o c h e m i c a l e f f e c t s o f b l e o m y c i n A 2 in N o v i k o f f h e p a t o m a a s c i t e s cells, P h y s . C h e m . P h y s . , 7 ( 1 9 7 5 ) 1 7 7 - - 1 9 0 . 21 D r e s p , J., E. S c h m i d a n d M. B a u c h i n g e r , T h e e y t o g e n e t i c e f f e c t o f b l e o m y c i n o n h u m a n p e r i p h e r a l l y m p h o c y t e s in v i t r o a n d i n v i v o , M u t a t i o n R e s , , 5 6 ( 1 9 7 8 ) 3 4 1 - - 3 5 3 . 2 2 D r e w i n k o , B., J . K . N o v a k a n d S.C. B a r r a n c o , T h e r e s p o n s e of h u m a n l y m p h o m a cells in v i t r o t o bleomycin and 1,3-bis(2-chloroethyl)-l-nitrosourea, Cancer Res., 32 (1972) 1206--1208. 2 3 E n d o , H . , H . Y a m a g a m i , M. I s h i z u k a a n d K . H o r i b a t a , B l e o m y c i n s e n s i t i v i t y a n d cell m e m b r a n e , in: A d v a n c e s in A n t i m i c r o b i a l a n d A n t i n e o p l a s t i c C h e m o t h e r a p y V o l . II, P r o c . V I I I n t e r n . C o n g r . Chemotherapy, Prague, 1971, pp. 293--298. 2 4 E v a n s , H . J . , M o l e c u l a r m e c h a n i s m s in t h e i n d u c t i o n o f c h r o m o s o m e a b e r r a t i o n s , i n : D. S c o t t , B . A . Bridges and F.H. Sobels (Eds.), Progress in Genetic Toxicology, Elsevier/North-Holland, Amsterdam, 1977, pp. 57--74. 2 5 F u j i m o t o . J . , R a d i o a u t o g ~ a p h i c s t u d i e s o n t h e i n t r a - e e l l u l a r d i s t r i b u t i o n o f b l e o m y c i n - 1 4 C in m o u s e t u m o r cells, C a n c e r R e s . , 3 4 ( 1 9 7 4 ) 2 9 6 9 - - 2 9 7 4 . 2 6 F u j i m o t o , J . , H. H i g a s h i a n d G. K a s a k i , I n t r a e e l l u l a r d i s t r i b u t i o n o f [ 1 4 C ] b l e o m y c i n a n d t h e c y t o k i n e t i c e f f e c t s o f b l e o m y c i n in t h e m o u s e t u m o r , C a n c e r R e s . , 3 6 ( 1 9 7 6 ) 2 2 4 8 - - 2 2 5 3 . 2 7 F u j i w a r a , Y. a n d T, K a n d o , S t r a n d s c i s s i o n o f H e L a cell d e o x y r i b o n u c l e i c a c i d b y b l e o m y c i n in v i t r o a n d in vivo, B i o c h e m . P h a r m a c o l . , 2 2 ( 1 9 7 3 ) 3 2 3 - - 3 3 3 . 2 7 a G e b h a r t , E., a n d H. K a p p a u f , B l e o m y c i n a n d s i s t e r - c h r o m a t i d e x c h a n g e i n h u m a n l y m p h o c y t e c h r o mosomes, Mutation Res., 58 (1978) 121--124. 2 8 G r i g g , G . N . , A m p l i f i c a t i o n o f p h l e o m y c i n i n f l u e n c e d d e a t h a n d D N A b r e a k d o w n b y c a f f e i n e in Escherichia coli, Mol. G e n . G e n e t . , 1 0 7 ( 1 9 7 0 ) 1 6 2 - - 1 7 2 . 2 9 G r i g g , G . W . , M.J. E d w a r d a n d D . J . B r o w n . E f f e c t s o f c o u m a r i n , t h i o p u r i n e s , a n d p y r o n i n e o n a m p l i f i c a t i o n o f p h l e o m y c i n - i n d u e e d d e a t h a n d d e o x y r i b o n u c l e i c a c i d b r e a k d o w n in E s c h e r i c h i a culi, J. Bacteriol., 107 (1971) 599-609. 3 0 H a h n , G., J. B r a u n a n d I. H a r - K e d a r , T h a r m o e h e m o t h e r a p y : s y n e r g i s m b e t w e e n h y p e r t h e r m i a ( 4 2 - 4 3 ° ) a n d a d r i a m y c i n ( o r b l e o m y c i n ) in m a m m a l i a n cell i n a c t i v a t i o n , P r o c . N a t l . A e a d . Sci. ( U . S . A . ) , 72 (1975) 937--940. 31 H a h n , G . M . , G . R . R a y , L . F . G o r d o n a n d R . F . K o l m a n , R e s p o n s e o f s o l i d t u m o r cells e x p o s e d t o c h e m o t h e r a p e u t i c a g e n t s i n vivo: cell survival a f t e r 2- a n d 2 4 - h o t t r e x p o s u r e , J. N a t l . C a n c e r I n s t . , 5 0 (1973) 529--533.

142 3 2 H a i d i e , C.W., F r a g m e n t a t i o n o f d e o x y r i b o n u c l e i c a c i d b y b l e o m y c i n , Mol. P h a r m a c o l . , 7 ( 1 9 7 1 ) 645--652. 3 3 H a i d l e , C.W., K.K. Weiss a n d M.T. K u o , R e l e a s e o f free b a s e s f r o m d e o x y r i b o n u c l e i c a c i d a f t e r r e a c t i o n w i t h b l e o m y c i n , Mol. P h a r m a c o l . , 8 ( 1 9 7 2 ) 5 3 1 - - 5 3 7 . 3 4 H a n n a h ° M . A . , a n d A. N a s i m , G e n e t i c a c t i v i t y o f b l e o m y c i n : d i f f e r e n t i a l e f f e c t s o n m i t o t i c r e c o m b i n a t i o n a n d m u t a t i o n s in y e a s t , M u t a t i o n R e s . , 5 3 ( 1 9 7 8 ) 3 0 9 - - 3 1 6 . 3 5 H a n n a h , M . A . , A. N a a i m a n d T. B r y c h e y , M u t a g e n i c a n d a n t i m u t a g e n i c e f f e c t s ' o f b l e o m y c i n in Saccharornyces cerevisiae, M u t a t i o n R e s . , 5 8 ( 1 9 7 8 ) 1 0 7 - - 1 1 0 . 3 6 H a y e z - D e l e t t e , F . , a n d W. F r e e m a n s , S t u d y o f c h r o m o s o m a l a b e r r a t i o n s i n d u c e d in v i t r o b y b l e o m y c i n o n h u m a n l y m p h o c y t e s , Bull. C a n c e r R e s . , 6 2 ( 1 9 7 5 ) 2 9 - - 3 6 . 37 H i t t i e m a n , W., a n d P . N . R a o , B l e o m y c i n - i n d u c e d d a m a g e in p r e m a t u r e l y c o n d e n s e d c h r o m o s o m e s a n d i t s r e l a t i o n s h i p t o cell c y c l e p r o g r e s s i o n in C H O cells, C a n c e r R e s . , 3 4 ( 1 9 7 4 ) 3 4 3 3 - - 3 4 3 9 . 3 8 I k e u c h i , T., C h r o m o s o m e p u l v e r i z a t i o n in C h i n e s e h a m s t e r m u l t i n u c l e a t e cells i n d u c e d b y b l e o m y c i n a n d c y t o c h a l a s t i n B, J a P . J. G e n e t . , 4 8 ( 1 9 7 3 ) 3 9 1 - - 4 0 5 . 3 9 I q b a l , Z.M., K.W. K a h n , R . A . G . E w i g a n d A . J . J o m a c e , J r . , Single s t r a n d s c i s s i o n a n d r e p a i r o f D N A in m a m m a l i a n cells b y b l e o m y c i n , C a n c e r R e s . , 3 6 ( 1 9 7 6 ) 3 8 3 4 - - 3 8 3 8 . 4 0 I s h i d a , R . , a n d T. T a k a h a s h i , I n c r e a s e d D N A c h a i n b r e a k a g e b y c o m b i n e d a c t i o n o f b l e o m y c i n a n d superoxide radical, Biochem. Biophys. Res. Commun., 66 (1975) 1432--1438. 41 K e n t , P.W., J . A . L u e y a n d P . F . U . W a r d , D i s t r i b u t i o n o f p y r i m i d i n e b a s e s in M e r i n g - R o e d e o x y r i b o n u c l e i c a c i d , B i o c h e m . J . , 61 ( 1 9 5 5 ) 5 2 9 - - 5 3 4 . 42 Kihlman, B.A., Effects of caffeine on the genetic material, Mutation Res., 26 (1974) 53--71. 4 3 K i h l m a n , B . A . , R o o t t i p s o f Vicia faba f o r t h e s t u d y o f t h e i n d u c t i o n o f c h r o m o s o m a l a b e r r a t i o n , M u t a t i o n Res,, 3 1 ( 1 9 7 5 ) 4 0 1 - - 4 0 2 . 4 4 K i h l m a n , B . A . , G. O d m a r k a n d B. H a r t l y , S t u d i e s o n t h e e f f e c t s o f p h l e o m y c i n o n c h r o m o s o m e s t r u c t u r e a n d n u c l e i a c i d s y n t h e s i s i n Vicia faba, M u t a t i o n R a s . , 4 ( 1 9 6 7 ) 7 8 3 - - 7 9 0 . 4 5 K i h l m a n , B . A . , S. S t u r e l i d , B. I-Iartly-Asp a n d K . N i l s o n , T h e e n h a n c e m e n t b y c a f f e i n e o f t h e freq u e n c i e s o f c h r o m o s o m a l a b e r r a t i o n s i n d u c e d i n p l a n t a n d a n i m a l cells b y c h e m i c a l a n d p h y s i c a l agents, Mutation Res., 26 (1974) 105--122. 4 6 K i h l m a n , B . A . , S. S t u r e l i d , F. P a l l t t i a n d A. B e c c h e t t i , E f f e c t s o f c a f f e i n e , a n i n h i b i t o r o f p o s t - r e p l i c a t i o n r e p a i r i n m a m m a l i a n cells, o n t h e f r e q u e n c i e s o f c h r o m o s o m a l a b e r r a t i o n s a n d sister c h r o matid exchanges induced by mutagenic agents, Mutation Res, 46 (1977) 130. 47 K o h n , K . W . , a n d R . A . G . E w i g , E f f e c t o f p H o n t h e b l e o m y c i n i n d u c e d D N A single s t r a n d scission in L 1 2 1 0 cells a n d t h e r e l a t i o n t o cell survival, C a n c e r R e s . , 3 6 ( 1 9 7 6 ) 3 8 3 9 - - 3 8 4 1 . 4 8 K o r e n b e r g , J., a n d E . F . F r e e l e n d e r , G i e s m a t e c h n i q u e f o r t h e d e t e c t i o n o f sister c h r o m a t i d exchanges, Chromosoma, 48 (1974) 355--360. 4 9 K r a u g e r , W.C., L . M . P s c h i g o d a a n d F. R e u s s e r , I n t e r a c t i o n s o f D N A w i t h z o b r a m y c i n , p h l e o m y c i n a n d b l e o m y c i n , U l t r a v i o l e t a b s o r p t i o n a n d c i r c u l a r d i c h r o i s m m e a s u r e m e n t s , J. A n t i b i o t . , 2 6 ( 1 9 7 3 ) 424--428. 5 0 K i r s h a n , A., B l e o m y c i n - i n d u c e d fine s t r u c t u r a l a l t e r a t i o n s in c u l t u r e d m o u s e f i b r o b l a s t s a n d h u m a n lymphocytes of neoplastic origin, Cancer Res., 33 (1973) 777--785. 51 K u e r s t i n , S., a n d G . O b e , P r e m a t u r e c h r o m o s o m e c o n d e n s a t i o n in t h e b o n e m a r r o w o f C h i n e s e h a m s t e r s a f t e r a p p l i c a t i o n o f b l e o m y c i n in vivo, M u t a t i o n R e s . , 27 ( 1 9 7 5 ) 2 8 5 - - 2 9 4 . 5 2 K u n i m o t o , T., M. H o r i a n d H . U m e z a w a , M o d e o f a c t i o n o f p h l e o m y c i n , b l e o m y c i n a n d f o r m y c i n o n H e L a $3 cells in s y n c h r o n i z e d c u l t u r e s , J. A n t i b i o t . , 2 0 A ( 1 9 6 7 ) 2 7 7 . 5 3 K u o , M . T . , a n d C.W. H a i d l e , C h a r a c t e r i z a t i o n o f c h a i n b r e a k a g e in D N A i n d u c e d b y b l e o m y c i n , Biochim. Biophys. Acta, 335 (1973) 109--114. 5 4 K u o , M . T . , a n d T . C . H s u , B l e o m y c i n c a u s e s release o f n u c l e o s o m e s f r o m c h r o m a t i n a n d c h r o m o somes, Nature (London), 271 (1978) 84. 5 5 L e m o n t t , J . F . , M u t a n t s o f y e a s t d e f e c t i v e in m u t a t i o n i n d u c e d b y u l t r a v i o l e t l i g h t , G e n e t i c s , 6 8

(1971) 21--23. 5 6 L i n d e n , W . A . , S. B. R e d d y a n d F. Z y w i e t z , E f f e c t s o f d a u n o m y c i n , b l e o m y c i n , c y t o s i n e a r a b i n o s i d e , a c r o m y c i n e a n d i o n i z i n g r a d i a t i o n o f t h e cell c y c l e in v i t r o a n d in v i v o , Br. J . C a n c e r , 3 2 ( 1 9 7 5 ) 7 5 2 . 57 Maier, P., a n d W. S c h m i d , T e n m o d e l m u t a g e n s e v a l u a t e d b y t h e m i c r o n u c l e u s t e s t , M u t a t i o n R e s . , 40 (1976) 325--338. 5 8 M a r q u a r d t , H . , a n d H . M a r q u a r d t , I n d u c t i o n o f m a l i g n a n t t r a n s f o r m a t i o n a n d m u t a g e n e s i s i n cell cultures by cancer chemotherapeutic agents, Cancer, 40 (1977) 1930--1934. 5 9 M a t s u z a w a , T., M. O n o z a w a , K . M o r i t a a n d M. K a k e h i , R a d i o s e n s i t i z a t i o n o f b l e o m y c i n o n l e t h a l e f f e c t s o f m o u s e c a n c e r cell in v i t r o , S t r a h l e n t h e r a p i e , 1 4 4 ( 1 9 7 2 ) 6 1 4 - - 6 1 7 . 6 0 M a t t i n g l y , E., I n d u c t i o n o f c h r o m o s o m e - a n d c h r o m a t i d - t y p e a b e r r a t i o n s b y p h l e o m y c i n , M u t a t i o n Res., 4 (1967) 51--57. 6 1 M i y a k i , M., T. O n o a n d H. U m e z a w a , I n h i b i t i o n o f llgase r e a c t i o n b y b l e o m y c i n , J. A n t i b i o t . , 2 4 (1971) 587--592. 6 2 M i y a k i , M., T. O n o , S. H o r i a n d H. U m e z a w a , B i n d i n g o f h l e o m y c i n t o D N A in b l e o m y c i n - s e n s i t i v e a n d r e s i s t a n t r a t a s c i t e s h e p a t o m a cells, C a n c e r R e s . , 3 5 ( 1 9 7 5 ) 2 0 1 5 - - 2 0 1 9 .

143 6 3 M o o r e , C.W., B l e o m y c i n i n d u c e d m u t a t i o n a n d r e c o m b i n a t i o n i n Saccharomyces cerevisiae, M u t a tion Res., 58 (1978) 41--49. 6 4 M o r i s h i m a , H . , T. S a w a , T. T a k i t a , T. A o y a g i , T. T a k e u c h i a n d H . U m e z a w a , B i o s y n t h e t i c s t u d i e s o n pepstatin, Biosynthesis of (3s,4s)-4-amino-3-hydroxy-6-methyl zeptamore acid moiety, J. Antibiot., 27 ( 1 9 7 4 ) 2 6 7 - - 2 7 2 . 6 5 M o u t s c h e n , J., G i l o t - D e l h a l l e a n d R . G . T h a k a r e , M o d i f i c a t i o n b y m e a n s o f a n a n t i b i o t i c o f t h e e f f e c t o f Co 60 g a m m a r a d i a t i o n a t t h e c h r o m o s o m a l level, K e r n t e c h n . I s o t o p t e c h n . C h e m i c . , 1 5 ( 1 9 7 3 ) 72--74. 6 6 M u l l e r , W . E . G . , A. T o t s u k a , I. N u s s e r a n d R . Z a h n , B l e o m y c i n i n h i b i t i o n o f D N A s y n t h e s i s in isol a t e d e n z y m e s y s t e m s a n d i n i n t a c t cell s y s t e m s , B i o c h e m . P b a r m a c o l . , 2 4 ( 1 9 7 5 ) 9 1 1 - - 9 1 5 . 6 7 Muller, W . E . G . , Z. Y a m a z a k i , H . J . B r e t e r a n d R . K . Z a h n , A c t i o n o f b l e o m y c i n o n D N A a n d R N A , E u r . J. B i o e h e m . , 31 ( 1 9 7 2 ) 5 1 8 - - 5 2 5 . 6 8 Muller, W . E . G . , Z. Y a m a z a k i a n d R . C . Zahn, B l e o m y c i n , a selective i n h i b i t o r o f D N A - d e p e n d e n t D N A p o l y m e r a s e f r o m o n c o g e n i c R N A viruses, B i o c h e m . B i o p h y s . R e s . C o m m u n . , 4 6 ( 1 9 7 2 ) 1 6 6 7 - 1673. 6 9 Muller, W . E . G . , Z. Y a m a z a k i a n d R . K . Z a h n , A c t i o n o f b l e o m y e i n o n p r o g r a m m e d s y n t h e s i s . Influence on enzymatic DNA, RNA and protein synthesis, Biochim. Biophys. Acta, 308 (1973) 412--421. 7 0 Muller, W . E . G . , Z. Y a m a z a k i , J . E . Z o e l i n e r a n d R . K . Z a h n , A c t i o n o f b l e o m y c i n o n p r o g r a m m e d synthesis, Influence on DNA and RNA nucleases, FEBS Lett., 31 (1973) 217--221. 71 Muller, W . E . G . , a n d R . K . Z a h n , B l e o m y e i n : m o d e o f a c t i o n o n D N A , G a n n , 1 9 ( 1 9 7 6 ) 5 1 - - 6 2 . 7 2 Muller, W . E . G . , a n d R . K . Z a h n , E f f e c t o f b l e o m y c i n o n D N A , R N A , p r o t e i n , c h r o m a t i n a n d ceil t r a n s f o r m a t i o n b y o n c o g e n i c R N A viruses, P r o g r . B i o c h e m . P h a r m a c o l . , 1 1 ( 1 9 7 6 ) 2 8 - - 4 7 . 7 3 Muller, W . E . G . , a n d R . K . Z a h n , B l e o m y c i n , a n a n t i b i o t i c t h a t r e m o v e s t h y m i n e f r o m d o u b l e s t r a n d e d D N A , P r o g r , N u c l . A c i d . R e s . Mol. Biol., 2 2 ( 1 9 7 7 ) 2 1 - - 5 7 . 7 4 M u r a k a m i , H . , H . M o r i a n d S. T a i r a , A c t i v e c e n t r e m o d e l o n t h e s t r u c t u r e a n d f u n c t i o n o f t h e b l e o m y c i n m o l e c u l e as t h e p r o t o t y p e o f e s t e r a t i c e n z y m e s , J . T h e o r . Biol., 4 2 ( 1 9 7 3 ) 4 4 3 - - 4 6 0 . 7 5 M u r a k a m i , H . , H. M o r i a n d S. T a i r a , M o l e c u l a r e m b r a c e o f b l e o m y c i n w i t h h e l i c a l D N A , J. T h e o r . Biol., 5 9 ( 1 9 7 6 ) 1 - - 2 3 . 7 6 N a g a i , K., H . S u z u k i , N. T a n a k a a n d H . U m e z a w a , D e c r e a s e o f m e l t i n g t e m p e r a t u r e a n d s i n g l e s t r a n d scission o f D N A b y b l e o m y c i n in t h e p r e s e n c e o f 2 - m e r c a p t o e t h a n o l , J . A n t i b i o t . , 2 2 ( 1 9 6 9 ) 5 9 6 - 573. 77 N a g a i , K., H . S u z u k i , N. T a n a k a a n d H. U m e z a w a , D e c r e a s e o f m e l t i n g t e m p e r a t u r e a n d single s t r a n d s c i s s i o n o f D N A b y b l e o m y c i n in t h e p r e s e n c e o f h y d r o g e n p e r o x i d e , J . A n t i b i o t . , 2 2 ( 1 9 6 9 ) 6 2 4 - 628. 78 Nagai, K., H. Yamaki, H. Suzuki, N. Tanaka and H. Umezawa, The combined effects of bleomycin and sulfhydryl compounds on the thermal denaturation of DNA, Biochim. Biophys. Acta, 179 (1969) 165--171. 7 9 N a g a t s u , M., T. O k a g a k i , R . R i c h a r t a n d A . L a m b e r t , E f f e c t s o f b l e o m y c i n o n n u c l e a r D N A i n t r a n s p l a n t a b l e V X - 2 c a r c i n o m a o f r a b b i t , C a n c e r R e s . , 31 ( 1 9 7 1 ) 9 9 2 - - 9 9 6 . 8 0 N a g a t s u , M., R . M . R i c b a r t a n d A. L a m b e r t , E f f e c t o f b l e o m y c i n o n t h e cell c y c l e o f E h r l i c h a s c i t e s carcinoma, Cancer Res., 32 (1972) 1966--1970. 81 O g a w a , K . , a n d T. O n o e , N u c l e a r c h a n g e s p r o d u c e d b y b l e o m y c i n in t h e 3 - m e t b y l c h o l a n t h r e n e i n d u c e d m o u s e e p i d e r m a l c a r c i n o m a cell, G a n n , 6 0 ( 9 1 6 9 ) 5 0 3 - - 5 0 7 . 8 2 O b e , G., G. B e e k a n d V. G o p a l V a i d y a , T h e h u m a n l e u k o c y t e t e s t s y s t e m , 3. P r e m a t u r e c h r o m o s o m e condensation from chemically and X-ray induced micronuclei, Mutation Res., 27 (1975) 89--101. 83 Ohama, K., and Kadotani, T., Cytologic effects of bleomycin on cultured human leukocytes, Jap. J. Human Genet., 14 (1970) 293--297. 8 4 O h n o , T., C h r o m o s o m a l a b e r r a t i o n s o f H e L a cells t r e a t e d w i t h b l e o m y c i n , A c t a O b s t e t . G y n e c o l . Japan, 19 (1972) 73. 8 5 O k a d a , S., R e p a i r s t u d i e s at t h e m o l e c u l a r , c h r o m o s o m a l a n d c e l l u l a r levels: a r e v i e w o f c u r r e n t w o r k in Japan, Radiation Res., Proc., 5th (1974) Int. Congr. (1975) 694--702. 8 6 O n i s h i , T . , K . S h i m a d a a n d Y. T a k a g l , E f f e c t s o f b l e o m y c i n o n Escherichia coli s t r a i n s w i t h v a r i o u s sensitivities t o r a d i a t i o n s , B i o c h i m . B i o p h y s . A c t a , 3 1 2 ( 1 9 7 3 ) 2 4 8 - - 2 5 8 . 87 O n o , T., M. M i y a k i a n d J . K u r o d a , S t u d i e s o n b l e o m y c i n - - D N A . R e p o r t a t t h e A n n u a l M e e t i n g o f Japan Cancer Society at Nagaya, 1972. 8 8 P a i k a , K . , a n d A. K r i s h a n , B l e o m y c i n i n d u c e d c h r o m o s o m a l a b e r r a t i o n s i n c u l t u r e d m a m m a l i a n cells, Cancer Res., 33 (1973) 961---965. 8 9 P e r r y , P.. a n d H . J . E v a n s , C y t o l o g i c a l d e t e c t i o n o f m u t a g e n c a r c i n o g e n e x p o s u r e b y sister c h r o m a t i d exchange, Nature (London) 258 (1975) 121--125. 9 0 P e r r y , P,, a n d S. W o l f f , N e w G i e m s a m e t h o d f o r t h e d i f f e r e n t i a l s t a i n i n g o f sister c h r o m a t i d s , N a t u r e (London), 258 (1974) 156--158. 91 P r o m c h a i n a n t , C., C y t o g e n e t i c e f f e c t o f b l e o m y c i n o n h u m a n l e u k o c y t e s in v i t r o , M u t a t i o n Res.0 2 8 (1975) 107--112.

144 9 2 R e i t e r , H., M. M i l e w s k i y a n d P. K e l l e y , M o d e o f a c t i o n o f p h l e o m y c i n o n Bacillus subtilis, J. B a c t e riol., 1 1 1 ( 1 9 7 2 ) 5 8 6 - - 5 9 2 . 9 3 R o s s , S . L . , a n d R . E . Moses, E f f e c t o f b l e o m y c i n o n d e o x y r i b o n u c l e i c a c i d s y n t h e s i s in t o l u e n e t r e a t e d E s c h e r i c h i a coli cells, A n t i m i c r o b . A g e n t s C h e m o t h e r . , 9 ( 1 9 7 6 ) 2 3 9 - - 2 4 6 . 9 4 R o s s , S . L . , a n d R . E . Moses, T w o a c t i o n s o f b l e o m y c i n o n s u p e r h e l i c a l D N A , B i o c h e m i s t r y , 17 (1978) 581--586. 9 5 S a i t o , M., a n d T. A n d o h , B r e a k a g e o f a D N A p r o t e i n c o m p l e x i n d u c e d b y b l e o m y c i n a n d t h e i r r e p a i r in c u l t u r e d m o u s e f i b r o b l a s t s , C a n c e r R e s . , 3 3 ( 1 9 7 3 ) 1 9 6 9 - - 1 7 0 0 . 9 6 S a r t i a n o , G . L . , M . L . C o e t z e e , K . K l e i n a n d P. Ove, E f f e c t o f c a m p t o t h e c i n a n d b l e o m y c i n - i n d u c e d t r i t i a t e d t h y m i d i n e t r l p h o s p h a t e i n c o r p o T a t i o n in a r a t n u c l e a r s y s t e m , J. N a t l . C a n c e r Inst., 5 8 (1977) 1357--1364. 9 7 S c h i n z e l , A . , a n d W. S c h m i d , L y m p h o c y t e s c h r o m o s o m e s t u d i e s in h u m a n s e x p o s e d t o c h e m i c a l m u t a g e n s , T h e v a l i d i t y o f t h e m e t h o d in 67 p a t i e n t s u n d e r c y t o s t a t i c t h e r a p y , M u t a t i o n R e s . , 4 0 (1976) 139--166. 9 8 S h i r a k a w a , I., M. A z e g a m i , S. Ishii a n d H. U m e z a w a , R e a c t i o n o f b l e o m y c i n w i t h D N A , S t r a n d s c i s s i o n o f D N A in t h e a b s e n c e o f s u l f h y d r y l o r p e r o x i d e c o m p o u n d s , J. A n t i b i o t . , 2 4 ( 1 9 7 1 ) 7 6 1 - 766. 9 9 S c h m i d , E., M. B a u c h i n g e r a n d W. M e r g e n t h a l e r , A n a l y s i s o f t h e t i m e r e l a t i o n s h i p o f t h e i n t e r a c t i o n o f X - r a y i n d u c e d p r i m a r y b r e a k s in t h e f o r m a t i o n o f d i c e n t r i c c h r o m o s o m e s , I n t . J. Biol., 3 0 ( 1 9 7 6 ) 339--346. 1 0 0 S c h u b e r t , I., a n d R . R i e g e r , O n t h e e x p r e s s i v i t y o f a b e r r a t i o n h o t s p o t s a f t e r t r e a t m e n t w i t h m u t a gens showing delayed or non-delayed effects, Mutation Res., 44 (1977) 337--344. 1 0 1 S p a n g l e r , M., G . P . S a r t i a n o , M.T. C o e t z e e a n d P. O r e , E f f e c t o f b l e o m y c i n o n [ 3 H ] t h y m i d i n e - 5 - t r i p h o s p h a t e i n c o r p o r a t i o n i n t o h o s t liver a n d h e p a t o m a n u c l e i , C a n c e r R e s . , 3 6 ( 1 9 7 6 ) 1 3 3 9 - - 1 3 4 6 . 1 0 2 S u z u k i , H., K. N a g a i , E. A k u t s u , H. Y a m a k i , N. T a n a k i a n d H. U m e z a w a , O n t h e m e c h a n i s m o f a c t i o n o f b l e o m y c i n s t r a n d s c i s s i o n o f D N A c a u s e d b y b l e o m y c i n a n d its b i n d i n g t o D N A in v i t r o , J. A n t i b i o t . , 2 3 ( 1 9 7 0 ) 4 7 3 - - 4 8 0 . 1 0 3 S u z u k i , H., K. N a g a i , H. Y a m a k i , N. T a n a k a a n d H. U m e z a w a , M e c h a n i s m o f a c t i o n o f b l e o m y c i n , S t u d i e s w i t h t h e g r o w i n g c u l t u r e o f b a c t e r i a l a n d t u m o r cells, J. A n t i b i o t . , 21 ( 1 9 6 8 ) 3 7 9 - - 3 8 6 . 1 0 4 S u z u k i , H . , K. N a g a i , H . Y a m a k i , N. T a n a k a a n d H. U m e z a w a , O n t h e m e c h a n i s m o f a c t i o n o f b l e o m y c i n s c i s s i o n o f D N A s t r a n d s in v i t r o a n d in vivo, J . A n t i b i o t . , 2 2 ( 1 9 6 9 ) 4 4 6 - - 4 4 8 . 1 0 5 T a k e s h i t a , M., A . P . G r o l l m a n a n d S.B. H o r w i t z , E f f e c t o f A T P a n d o t h e r n u e l e o t i d e s o n t h e b l e o mycin-induced degradation of vaccinia virus DNA, Virology, 69 (1976) 453--563. 1 0 6 T a k e s h i t a , M., S.B. H o r w i t z a n d A,P. G r o l l m a n , B l e o m y c i n , a n i n h i b i t o r o f v a c c i n i a v i r u s r e p l i c a t i o n , Virology, 60 (1974) 455--465. 1 0 7 T a m u r a , H . , Y. S u g i y a m a a n d T. S u g e h a r a , E f f e c t s o f b l e o m y c i n o n t h e c h r o m o s o m e s o f h u m a n l y m p h o e y t e s at v a r i o u s cell p h a s e s , G a n n , 6 5 ( 1 9 7 4 ) 1 0 3 - - 1 0 7 . 1 0 8 T e r a s i m a , T . , a n d H. U m e z a w a , L e t h a l e f f e c t o f b l e o m y c i n o n c u l t u r e d m a m m a l i a n cells, J. A n t i blot., 23 (1970) 300. 1 0 9 T e r a s i m a , T., M. Y a s u k a w a a n d H. U m e z a w a , B r e a k s a n d r e j o i n i n g o f D N A in c u l t u r e d m a m m a l i a n cells t r e a t e d w i t h b l e o m y c i n , G a n n , 6 1 ( 1 9 7 0 ) 5 1 3 - - 5 1 6 . 110 Tobey, R.A., A simple, rapid technique for detection of the effects of chemotherapeutic agents on m a m m a l i a n cell c y c l e t r a v e r s e , C a n c e r R e s . , 3 2 ( 1 9 7 2 ) 3 0 9 - - 3 1 6 . 1 1 1 T o b e y , R . A . , A r r e s t o f C h i n e s e h a m s t e r cells i n G 2 f o l l o w i n g t r e a t m e n t w i t h t h e a n t i t u m o r d r u g b l e o m y c i n , J . Cell P h y s i o l . , 7 9 ( 1 9 7 2 ) 2 5 9 - - 2 6 5 . 1 1 2 T o b e y , R . A . , D i f f e r e n t d r u g s a r r e s t cells a t a n u m b e r o f d i s t i n c t s t a g e s in G 2, N a t u r e ( L o n d o n ) , 2 5 4 (1975) 245--247. 1 1 3 U m e z a w a , H., N a t u r a l a n d a r t i f i c i a l b l e o m y c i n s : c h e m i s t r y a n d a n t i - t u m o r a c t i v i t y , P u r e A p p l . Chem., 28 (1971) 665--680. 114 Umezawa, H., Studies on bleomycin: Chemistry and the biological action, Biomedicine, 18 (1973) 459--475. 115 Umezawa, H., Chemistry and mechanism of action of bleomycin, Fed. Proc., 38 (1974) 2296--2302. 1 1 6 U m e z a w a , H . , B l e o m y c i n , in: J . W . C o r e o r a n a n d F . H a h n ( E d s . ) , A n t i b i o t i c s , III. M e c h a n i s m o f Action of Antimicrobial and Antitumor Agents, Springer, Berlin, 1975, pp. 21--33. 1 1 7 U m e z a w a , H., H . A s k u r a , K . O d a , S. H o r i a n d M. H o r i , T h e e f f e c t s o f h l e o m y c i n o n 8 V 4 0 D N A : C h a r a c t e r i s t i c s o f b l e o m y c i n a c t i o n w h i c h p r o d u c e a single s t r a n d seission in a s u p e r h e l i c a l f o r m o f S V 4 0 D N A , J. A n t i b i o t . , 2 6 ( 1 9 7 3 ) 5 2 1 ~ 5 2 7 . 1 1 8 U m e z a w a , H., K . M a e d a , T. T a k e u c h i a n d Y. O k a m i , N e w a n t i b i o t i c s , b l e o m y e i n A a n d B, J. A n t i b i o t . (See. A), 1 9 ( 1 9 6 6 ) 2 0 0 - - 2 0 9 . 1 1 9 U m e z a w a , H . , T. T a k e u c h i , S. H o r i , T. S a w a , M. I s h i z u k a , T. I c h i k a w a a n d T. K o m i , S t u d i e s o n t h e m e c h a n i s m o f a n t i t u m o r e f f e c t s o f b l e o m y c i n o n s q u a m o u s cell c a r c i n o m a , J. A n t i b i o t . , 2 5 ( 1 9 7 2 ) 409--420. 1 2 0 V i g , B . K . , C h r o m a t i n - n u c l e a r m e m b r a n e a t t a c h m e n t in r e l a t i o n t o D N A r e p l i c a t i o n a n d c h r o m o s o m e a b e r r a t i o n s : A n e w h y p o t h e s i s , J. T h e o r . Biol., 5 4 ( 1 9 7 5 ) 1 9 1 - - 1 9 9 .

145

1 2 1 Vig, B . K . , G e n e t i c t o x i o l o g y o f m i t o m y c i n C, a c t i n o m y c i n s , d a u n o m y e i n a n d a d r i a m y c i n , M u t a t i o n Res., 49 (1977) 189--238. 1 2 2 Vig, B . K . , E f f e c t o f h y p e r t h e r n i a a n d h y p e r t h e r m i a o n t h e i n d u c t i o n o f c h r o m o s o m e a b e r r a t i o n s b y a d r i a m y c i n in h u m a n l e u k o c y t e s , C a n c e r R e s . , 3 8 ( 1 9 7 8 ) 5 5 0 - - 5 5 5 . 1 2 3 W a t a n a b e , M., Y. T a k a b e a n d T. K a t s u m a t a , R e s p o n s e in m a c r o m o l e c u l a r s y n t h e s i s o f m o u s e L cells to bleomycin with special reference to cell-antibiotic interaction, J. Antibiot., 26 (1973) 417--423. 1 2 4 W a t a n a b e , M., Y. T a k a b e , T. K a t s u m a t a a n d T. T e r a s i m a , E f f e c t s o f b l e o m y c i n o n p r o g r e s s i o n t h r o u g h cell c y c l e o f m o u s e L-ceils, C a n c e r R e s . , 3 4 ( 1 9 7 4 ) 8 7 8 ~ 8 8 1 . 1 2 5 Y a m a g a m i , H., M. I s h i z a w a a n d H., E n d o , P h e n o t y p i c a n d g e n e t i c c h a r a c t e r i s t i c s o f b l e o m y c i n - s e n s i tive s t r a i n o f Escherichia coli, G a n n , 6 5 ( 1 9 7 4 ) 6 1 - - 6 7 . 1 2 6 Y a m a k i , H., S u z u k i , H. N a g a i , N T a n a k a a n d H. U m e z a w a , E f f e c t o f b l e o m y c i n A 2 o n d e o x y r i b o n u clease, D N A p o l y m e r a s e a n d ligase r e a c t i o n , J. A n t i b i o t . , 2 4 ( 1 9 7 1 ) 1 7 8 - - 1 8 4 . 1 2 7 Y a t a g a m a s , X., a n d B.S. C l a r k s o n , F l o w m i c r o f l u o r o m e t r i c a n a l y s i s o f cell killing w i t h c y t o t o x i c drugs, J. Histochem. Cytochem., 22 (1974) 661--659. 1 2 8 Z i m m e r m a n n , F . K . , A y e a s t s t r a i n f o r a visual s c r e e n i n g f o r t h e t w o r e c i p r o c a l p r o d u c t s o f m i t o t i c c r o s s i n g o v e r , M u t a t i o n R e s . , 21 ( 1 9 7 3 ) 2 6 3 - - 2 6 9 . 1 2 9 Z i m m e x T n a n n , F.]~:., R . K e r n a n d H. R o s e n b e r g , A y e a s t s t r a i n f o r s i m u l t a n e o u s d e t e c t i o n o f i n d u c e d m i t o t i c c r o s s i n g o v e r , g e n e c o n v e r s i o n a n d reverse m u t a t i o n , M u t a t i o n R e s . , 2 8 ( 1 9 7 5 ) 3 8 1 - - 3 8 8 . 1 3 0 Z o n n e r , E . G . , D. W e i n b l u m , J. O v e r m e i e r a n d R . K . Z a h n , I n f l u e n c e o f U V i r r a d i a t i o n , 4 - n i t r o q u i noline-l-oxide, methylmethane sulfonate and bleomycin on the activity of an alkaline deoxyribonuclease f r o m h u m a n l y m p h o c y t e s , E x p . Cell Res., 9 9 ( 1 9 6 6 ) 1 8 5 - - 1 8 9 .