Mutation Research, 257 (1991) 127-143 © 1991 Elsevier Science Publishers B.V. 0165-1110/91/$03.50 ADONIS 016511109100057E
127
MUTREV 07295
Genotoxicity of bleomycin Lawrence F. Povirk and M.J. Finley Austin Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298 (U.S.A.)
(Received 6 July 1990) (Accepted 18 October 1990)
Keywords: Bleomycin, genotoxicity; Blenoxane
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemistry of DNA damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uptake and metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromosomal effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromosome breakage and cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bacterial assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mammalian cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction Bleomycin is a radiomimetic antitumor agent with unique genotoxic properties. The drug is a glycopeptide (Fig. 1) which contains several highly unusual amino acids and sugars, and a terminal amine which varies a m o n g bleomycin analogues. Few significant differences in action have been f o u n d between the various analogues. T h e clinical formulation Blenoxane is a mixture of c o m p o nents, primarily bleomycins A 2 and B 2.
Correspondence: Dr. Lawrence F. Povirk, Medical College of Virginia, P.O. Box 230, MCV Station, Richmond, VA 23298 (U.S.A.).
127 128 130 131 133 135 135 135 136 137 137 139 139
Because o f the fairly extensive use of this drug in clinical chemotherapy, its genetic toxicity has considerable practical significance. In addition, the ability of bleomycin to inflict oxidative d a m a g e at a specific position in D N A , i.e., the C - 4 ' position of deoxyribose, offers the potential to assess the role of such sugar d a m a g e in mediating the effects of other oxidative mutagens. As can be seen f r o m the genetic activity profile of bleomycin (Fig. 2), the drug tests positive in the great majority of genetic toxicity assays. N o t a b l e exceptions, discussed in m o r e detail below, are assays with most A m e s tester strains, and the sister c h r o m a t i d exchange (SCE) assay. This review will a t t e m p t to summarize current knowledge concerning the genotoxic effects of bleomycin, with emphasis on new developments in
128
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Bleomycins AZ: R = ~ - N / ~ cH'~ H "CHs B2: R= ~'N~'~/HN"~ N" H CH NH I~pleomycin i ~ R= " N ~ N / ' " . ~ / ~ Fig. 1. Structure of bleomycin, showing (1) double bond which is replaced by a single bond in phleomycin, (2) site of attachment of an additional talose sugar derivative in the analogue tallysomycin, and (3) amide which is deaminated to give a carboxylic acid in desamidoblcomycin, an inactive metabolite. The area enclosed by the dashed fine is considered to be the DNA-binding domain of the molecule. The lelt-hand portion of the molecule is involved in metal chelation.
the past decade, and especially the past few years. Earlier studies have been reviewed by Vig and Lewis (1978).
Chemistry of DNA damage The chemistry of bleomycin activation and of the intermediates in DNA damage has recently been the subject of some extensive and detailed reviews (Hecht, 1986; Stubbe and Kozarich, 1987), and will be described only briefly here. Addition of oxygen to Fe(II)-bleomycin or addition of H202 to Fe(III) • bleomycin results in formation of "activated bleomycin" (probably bleomycin ferric peroxide), a chelate complex which is identified by its distinct EPR spectrum (Burger et al., 1981; Kuramochi et al., 1981). It is the last bleomycin
species detectable prior to DNA damage. This species, or an extremely short-lived product thereof, is believed to specifically abstract hydrogen from C-4' of deoxyribose. The resulting free radical on C-4' partitions into one of two pathways (Fig. 3). Hydroxylation of C-4' (left pathway) results in sugar ring opening and base release, leaving a chemically modified apurinic/ apyrimidinic (AP) site with a ketone at C-4' and an aldehyde at C-I' (Sugiyama et al., 1985; Rabow et al., 1986); the source of the hydroxyl is solvent water (Rabow et al., 1990). The sugar of the AP site presumably exists in equilibrium between ring-closed and ring-opened forms. Alternatively, (right pathway) addition of 02 to C-4' forms a peroxyl radical species, which decomposes to give a strand break with 5'-phosphate and 3'-phosphoglycolate termini, with release of a base-propenal (Giloni et al., 1981). Despite uncertainty in the molecular details of the formation of these two lesions, it is generally agreed that both are products of an initial C-4' free radical (Kozarich and Stubbe, 1987). Under physiological conditions the two products are formed in a ratio of approximately 1:1 (Povirk et al., 1977). Both lesions are formed predominantly at pyrimidines in G-C and G-T sequences (D'Andrea and Haseltine, 1978; Takeshita et al., 1978; Steighner and Povirk, 1990a). However, the relative susceptibilities of individual G-Py sites, which vary widely, are not readily predicted on the basis of sequence and presumably depend on subtle, sequence-dependent variations in DNA conformation. Cytosine methylation can greatly inhibit bleomycin-induced cleavage in the immediate vicinity of the methylated bases (Hecht, 1986). In addition to these single lesions, bleomycin specifically induces double strand breaks, as well as AP sites with closely opposed strand breaks, at a frequency far greater than expected from coincidence of the single lesions (Povirk et al., 1977, Lloyd et al., 1978, Povirk and Houlgrave, 1988b). The chemistry of these bivalent lesions, however, appears to be identical to that of the single lesions (Povirk et al., 1989; Steighner and Povirk, 1990a). Studies from our laboratory have recently shown that the great majority of double strand breaks have either blunt ends or noncomplementary single-base 5' extensions. In general, only one of
129
G-Py site in the complementary strand, at which single strand cleavage is rare (Povirk et al., 1989).
the breaks comprising each double strand break follows the normal G-Py specificity of bleomycin; we refer to this as the " p r i m a r y " cleavage site. The "secondary" break typically occurs at a non-
Remarkably, double strand breaks involving the two C residues in self-complementary G-C se-
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Fig. 2. The genetic activity profile of bleomycin. Assays which gave positive results are indicated in three-letter codes at the top of the figure, and those which gave negative results at the bottom. The vertical scale gives a measure of genotoxic potency in each assay, with a value of 8 indicating a minimum effective dose of 0.001 # g / m l and a value of 1 indicating 10,000 #g/ml. Negative values indicate the highest concentrations used in assays which gave negative results, with - 1 representing 10 # g / m l and - 5 representing 100,000 #g/ml. Vertical and horizontal bars show average values, and values reported in individual studies, respectively (see Waters et al., 1988 for further details). The three letter codes for positive assays are: PRB, prophage induction, SOS repair, DNA strand breaks or crosslinks; ECB, E. coil strand breaks, cross-links or repair; ERD and BRD, differential toxicity to E. cob recA strains and to other repair-deficient bacteria; SA2, Ames test, strain AT102; ECF, E. coli forward mutation; ECK, E. cob K12, forward or reverse mutation; SCG, SCH and SCR, gene conversion, homozygosis and reverse mutation in yeast; ANG and ANF, gene conversion and forward mutation in Aspergillus; VCF, chromosome aberrations in Vicia faba; DMG, DMM, DMX, DMC and DMN, genetic crossing-over, somatic mutation, sex-linked recessive lethal mutation, chromosome aberrations and aneuploidy in Drosophila; DIA, strand breaks in cultured cells; URP and UIA, unscheduled DNA synthesis in rat hepatocytes and in other cultured cells; GCO, gene mutation in CHO cells; SIC, SCE in hamster cells; CIC, CIM and CIT, chromosome aberrations in cultured Chinese hamster, mouse and other transformed cells; AIA and TCM, aneuploidy and cell transformation in culture; DIH, DNA strand breaks in human cells; UHL, unscheduled DNA synthesis in human lymphocytes; SHT and SIH, SCE in transformed and in other human cultured cells; CHL, CHT and CIH, chromosome aberrations in human lymphocytes, transformed cells, and other human cells; SVA, SCE in animal cells in vivo; MVM, micronucleus test, mice in vivo; CBA and CBC, chromosomal aberrations in animal bone marrow and in spermatogonia; MHT, mouse heritable transiocation test; CLH, chromosomal aberrations in human lymphocytes in vivo. Assays which gave negative assays were: SAO, SA9 and SAS, Ames test with strains AT100, AT98 and other strains; SCF, forward mutation in yeast; VFS, SCE in Vicia faba; DMH, heritable translocation in Drosophila; G9H, mutation at the HGPRT locus in V79 cells; UHT, unscheduled DNA synthesis in transformed human cells; SHL, SCE in human lymphocytes. See International Agency for Research on Cancer (1986) for individual references.
130 DNA
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reactivated during the primary cleavage (Povirk et al., 1989). As discussed by Steighner and Povirk (1990a), this model is supported by the finding that, in the case of AP sites with closely opposed breaks, the AP site invariably occurs at the secondary site, and the break at the primary site. The ratio of double strand to single strand breaks is much lower for phleomycin than for bleomycin (Huang et al., 1981), but the reason for this difference is not known. It has recently been reported that bleomycin also produces 8-hydroxyguanine in DNA, with a yield about one-one hundredth that of strand cleavage (Kohda et al., 1989). To our knowledge, no other bleomycin-induced base damage has been detected.
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quences rarely if ever occur, even where both C residues are prominent sites of single strand cleavage. To explain the finding that bleomycin-induced double strand breaks are formed with single-hit kinetics, we recently proposed a model of double strand cleavage in which bleomycin is
Most pharmacokinetic studies have employed the radiolabeled chelate complex 57Co(III)bleomycin. This species preferentially accumulates in tumors, and has even been used for tumor imaging, but the basis for this preferential accumulation has yet to be determined (Alberts et al., 1979). Studies with metal-free bleomycin have been hampered by the general unavailability of radiolabeled drug, but early investigations (Umezawa et al., 1972) did not show any strong preferential accumulation in tumors. Thus, it is possible that metal-free bleomycin and Co(Ill). bleomycin differ substantially in pharmacokinetics (Alberts et al., 1979). Roy and Horwitz (1984) prepared [3H]bleomycin by chemical postlabeling and found that, as might be expected for a large hydrophilic compound, bleomycin was not efficiently taken up by cultured ceils. At low drug concentrations, only 0.1% of the [3H]bleomycin in the medium became associated with HeLa cells grown in monolayer, during a 30 min incubation. Association was temperature-dependent, but was unaffected by metabolic poisons, suggesting that energy was not required. Similarly, Lyman et al. (1986) showed that, even after several hours of treatment, the concentration of [3H]bleomycin in Ehrlich cells remained lower than the concentration in the medium. They also found that, in agreement with earlier animal studies, the inactive Co(Ill).
131 bleomycin complex was much more efficiently taken up than metal-free bleomycin or the Fe(III), Cu(II) or Zn(II) complexes. Sidik and Smerdon (1990) showed that transient permeabilization of cells with lysophosphatidylcholine dramatically increased bleomycin cytotoxicity. Thus, a number of studies (see also Ozawa et al., 1987) suggest that the drug is very poorly taken up by intact cells. The process by which it enters cells is still largely unknown. Since metal-free bleomycin is effective in killing a variety of cultured cells, it is assumed that the drug chelates iron and becomes activated intracellularly. Little is known about the process leading to activation, although it is likely to involve Fe(II) • bleomycin as an intermediate. Under physiological conditions, unchelated iron would be expected to be in the Fe(III) form, although some could be transiently converted to Fe(II) by reaction with glutathione or other reducing agents. Direct reduction of Fe(III) • bleomycin to Fe(II) • bleomycin by glutathione is very inefficient, especially in the presence of DNA (Povirk, 1979), and in recent years the possibility of enzymatic reduction has received considerable attention. Mahmutoglu and Kappus (1988) showed that purified cytochrome b5 reductase effected reduction of Fe(III). bleomycin in an NADH-dependent reaction, and there have been at least two reports of NADPHdependent Fe(III)-bleomycin activation by cytochrome P-450 reductase (Scheulen et al., 1981; Kilkuskie et al., 1984). In contrast, Ciriolo et al. (1987) reported that rat liver microsomes did not reduce Fe(III). bleomycin, although they did enhance the conversion of Fe(II). bleomycin to activated bleomycin. This apparent discrepancy may have been due to the very high DNA concentration used in the studies of Ciriolo et al. Besides metal chelation, the most important feature of bleomycin metabolism is its conversion to the inactive derivative desamidobleomycin (see Fig. 1) by the enzyme bleomycin hydrolase. Several studies have shown a correlation between the resistance of certain tumors or cultured cells to bleomycin and their levels of bleomycin hydrolase (Umezawa et al., 1972; Akiyama et al., 1981). High levels of this enzyme in bone (Umezawa et al., 1972) probably explain the lack of myelosuppression. The bleomycin hydrolase gene has re-
cently been cloned (Sebti et al., 1989); the deduced amino acid sequence indicates that the enzyme belongs to the cysteine proteinase family, but its normal function is not known. Chromosomal effects
In 1970, Ohama and Kadotani reported the induction of chromosomal aberrations in cultures of human leukocytes treated with various concentrations of bleomycin. During the ensuing decade numerous studies were conducted, in vitro and in vivo, on the effects of bleomycin on chromosomes (reviewed by Vig and Lewis, 1978, and by Povirk, 1983). Bleomycin was found to produce both chromosome- and chromatid-type aberrations (deletions, dicentrics, rings, exchanges, breaks and gaps) in all of the systems studied. These systems included the use of different cell types of human origin as well as other animal and plant sources, and the standard technique of scoring stained metaphase figures. The effects of bleomycin were also studied with premature chromosome condensation (PCC), and with micronucleus and sister chromatid exchange (SCE) assays. Though bleomycin induces chromosomal aberrations in standard and PCC preparations, and also induces formation of micronuclei, it appears to have little effect on the level of SCEs. Based on the results reviewed it was concluded that bleomycin acted on chromosomes in an S-independent fashion since bleomycin was observed to produce aberrations when administered in any period of interphase. Furthermore, the types of aberrations produced by treatment in various phases of the cell cycle were those expected to result from production of both double and single strand breaks by bleomycin. Many parallels have been drawn between the effects of bleomycin and those of ionizing radiation; both agents act in an S-independent manner and both produce chromosomal aberrations presumably as a result of induced DNA single and double strand breaks. The induction of chromosomal aberrations and SCEs in two X-ray-sensitive mutants by bleomycin and other DNA damaging agents has been recently reported (Darroudi and Natarajan, 1989). The two mutant lines, xrs 5 and xrs 6, were both found to be more
132
sensitive than wild-type cells to the induction of chromosomal aberrations by bleomycin. An increase in SCE production was observed for one of the lines, xrs 6, while no increase was seen in xrs 5 or the wild-type fine, CHO-K1. In general bleomycin has been found to be a poor inducer of SCEs (reviewed by Vig and Lewis, 1978) and more current work (the above exception noted) continues to corroborate this finding. In in vitro studies with a variety of cell types bleomycin induced either no increase in SCEs or only a small increase at very high doses (Sono and Sakaguchi, 1978; Banerjee and Benedict, 1979; Clare et al., 1982; Morgan and Crossen, 1982). The same trends are seen in studies of patients undergoing cytostatic therapy with bleomycin alone or in combination with other agents (Lambert et al., 1978; DiCker, 1981; Clare et al., 1982; Abe et al., 1984). A possible model to explain the lack of SCE induction by agents which induce strand breaks has been proposed by Painter (1980). Early use of the PCC technique revealed that approximately 1/3 of bleomycin-induced G 2 aberrations are repaired before cells reach mitosis (reviewed by Vig and Lewis, 1978). Employing both PCC and alkaline elution Sognier et al. (1982) observed a 2-component profile, with a more sensitive response at low doses, for the formation of bleomycin-induced aberrations. The 2-component profile was most easily seen with alkaline elution but it was also clearly evident with PCC; however it was not as apparent in data obtained from scoring mitotic figures. Also, the number of aberrations seen in mitotic preparations was greatly reduced as compared to the Gz-PCC. The reduction of visible aberrations in mitosis is most likely due to repair of strand breaks before cells reach mitosis. These authors postulated that the 2-component profile was indicative of a nonrandom interaction of bleomycin with DNA and that the underestimation of aberrations observed with mitotic preparations prevented the detection of the nonlinear kinetics. Bleomycin-induced chromosome damage and repair was studied in quiescent mononuclear human blood cells and fibroblasts with the PCC technique (Sen and Hittelman, 1984). The G 1 cells were treated with bleomycin for 30 rain and washed free of drug. Following various periods of
repair, cells were fused to induce PCC and aberrations were scored. The repair profile displayed fast- and slow-repair components, with a significant reduction in chromosome damage within 30 minutes. After two hours the repair of aberrations slowed. Though the frequency of damage differed in the two cell types the kinetic profiles were similar. At the molecular level bleomycin is now known to induce single strand breaks. This is reflected at the chromosomal level in the appearance of chromatid-type aberrations in cells treated with bleomycin in G 1. Further cytogenetic evidence for the production of single strand breaks has come from exposing cells first treated with X-irradiation or bleomycin during G 2 to Neurospora endonuclease, an enzyme specific for single stranded regions, and observing an increase in all types of chromosomal aberrations in the following mitosis (Natarajan and Obe, 1978; Nowak et al., 1984; Nowak and Obe, 1984). Presumably, the Neurospora endonuclease converted single strand breaks induced by either agent to double strand breaks. Like that of most, if not all DNA damaging agents, the effect of bleomycin on the induction of chromosomal aberrations is potentiated by a number of agents a n d / o r culture conditions. In the 1970's both caffeine and hyperthermia were shown to increase the number of aberrations induced by bleomycin in cells in culture (Kihlman and Sturelid, 1978; Vig and Lewis, 1978; Vig, 1979). The hyperthermic potentiation of bleomycin-induced aberrations was found to be synergistic in cultured human lymphocytes and Chinese hamster cells (Vig et al., 1982). More recently, it has been reported that the concomitant treatment of cells with hyperthermia and trifluoperazine, a calmodulin inhibitor, induces chromatin changes resulting in an increased accumulation of bleomycininduced DNA damage due to a decrease in repair functions (Smith et al., 1986b). The frequency of bleomycin-induced aberrations in vitro has also been demonstrated to be enhanced by hydroxyurea (Hansson and HartleyAsp, 1981), isoptin (Scheid et al., 1984), 3-aminobenzamide (Zwanenburg et al., 1985), and ethanol and acetaldehyde (Lin et al., 1989). In contrast, the anticlastogens fl-aminoethyl-isothiouronium and homocysteinethiolactone greatly reduced the
133 numbers of aberrations induced in human lymphocytes treated simultaneously with these agents and bleomycin as compared to cultures treated with bleomycin alone (Gebhart, 1978). More recently, Pohl and Reidy (1989) found a reduction in bleomycin-induced aberrations in the cultured lymphocytes of individuals after receiving dietary supplementation with 1 g of vitamin C per day for two and four weeks. Pretreatment of human lymphocyte cultures with a low dose (0.01-0.1 /~g/ml) of bleomycin has been reported to invoke resistance to the clastogenic effects of X-rays or of a high concentration (1.5 /~g/ml) of bleomycin (Vijayalaxmi and Burkart, 1989). Bleomycin not only induces structural aberrations in mitotic cells but it has also been found to cause aneuploidy (reviewed by Vig and Lewis, 1978). Because these effects have been observed in mitotic cells concern has been generated about possible correlates occurring in germ cells, as well as potential teratogenic consequences, of individuals treated with bleomycin. In a study of the teratogenic potential of bleomycin a single injection of 80 mg/kg into pregnant females was found to induce chromosomal aberrations in cells derived from post-implantation mouse embryos only 3 hours after the transplacental treatment (Adler, 1983). Though the frequency decreased a significant number of aberrations were still present 18 hours post-treatment. The induction of aneuploidy by bleomycin in Drosophila melanogaster oocytes has been observed with the aneuploidy pattern method (Traut, 1984). Bleomycin has also been found to induce both reciprocal translocations and aneuploidy in the spermatogonia of mice (van Buul and Goudzwaard, 1980; De Luca et al., 1988; Sen and Liang, 1989). Even before the application of banding techniques the nonrandom nature of bleomycin-induced chromosomal damage was evident (Vig and Lewis, 1978). DNA damage by bleomycin has also been shown to be concentrated in nucleosomal linker regions in isolated nuclei (Kuo and Hsu, 1978). Kuo (1981) reported that bleomycin acted preferentially on active chromatin. Using restriction enzyme digestion of DNA from bleomycintreated oviduct cell and erythrocyte nuclei along with Southern blotting he found that the active
ovalbumin gene was more sensitive to damage by bleomycin than the inactive globin gene. More recently, Yunis et al. (1987) found that bleomycin along with caffeine as an enhancer induced the expression of common chromosomal fragile sites, constant chromosomal locations where aberrations are induced nonrandomly by specific conditions, in cultured human lymphocytes. A high concordance was noted between the bleomycin-induced sites and fragile sites induced by aphidicolin as well as a variety of DNA damaging agents and other synthesis inhibitors. Several of the agents studied by this group have been reported to preferentially attack active DNA regions and Austin et al. (1989) recently found an association between X chromosomal aphidicolin-inducible fragile site locations and gene activity on the human X chromosome. Thus, presence of actively transcribed genes appears to be one essential characteristic of chromosomal fragile sites, though other factors must be involved as well. Continued studies of chromosomal aberrations produced by bleomycin coupled with molecular analyses will certainly enhance the understanding of mechanisms involved in DNA damage and repair as they relate specifically to bleomycin as well as in general. An understanding of the nonrandom nature of damage induced by bleomycin and other agents may provide key insights into the processes underlying mutagenesis and carcinogenesis.
Chromosome breakage and cytotoxicity It is likely that bleomycin-induced cell killing is due to DNA double strand cleavage, and the resulting loss of chromosome fragments. The primary impetus for this view comes from analogy to extensive studies with ionizing radiation, which show that at least in some systems, there is a one-to-one correspondence between chromosome aberrations and cell death (Dewey et al., 1970; Joshi et al., 1982; Cornforth and Bedford, 1987). However, while there are extensive qualitative similarities between bleomycin and ionizing radiation in their clastogenic and cytotoxic effects, the two agents often show quite different behavior quantitatively. In a particularly intriguing study
134
with V79 cells, Scott and Zampetti-Bosseler (1985) found that, at equivalent levels of cytotoxicity, bleomycin induced 5- to 10-fold fewer chromosome aberrations than ionizing radiation, and also induced much less mitotic delay. In fact, at a dose of bleomycin which killed 80% of the cells, less than 5% of the cells had visible aberrations, over a wide range of sampling times following treatment. These authors came to the rather disquieting conclusion that chromosome breakage plays little or no role in bleomycin cytotoxicity. On the other hand, with L5178Y mouse leukemia cells, Doerr et al. (1989) found a fairly close correspondence between the fraction of surviving cells and the fraction of cells with no visible aberrations, over a range of bleomycin concentrations. Interpretation of such quantitative studies, and particularly the comparisons with ionizing radiation, are further complicated by several unexplained features of bleomycin-induced D N A damage in cells, including the nonrandom nature of the damage, as mentioned above. Alkaline elution studies with L1210 cells (Iqbal et al., 1976) have indicated that a fraction of cellular D N A typically suffers severe damage, while the remainder is much less affected; the fraction of heavily degraded D N A increases with bleomycin dose. Although other studies (Kuo, 1981; Beckmann et al., 1987) have indicated that actively transcribed genes are particularly susceptible to bleomycin damage, these domains would constitute far less than the fraction of heavily degraded cellular D N A observed by Iqbal et al. Even greater heterogeneity of damage was found in an alkaline elution study with Ehrlich ceils (Byrnes et al., 1990), in which it was suggested that cytotoxicity was correlated with the amount of very heavily degraded D N A which eluted immediately in the lysis fraction. Even more disturbing are experiments involving measurements of D N A damage by nucleoid electrophoresis, which indicate extreme cell to cell differences in the extent of D N A damage (0stling and Johanson, 1987). All of these heterogeneities should clearly be taken into account in attempting to assess the role of D N A damage in cell death, but it is difficult to do so since the basis of heterogeneity is largely unknown.
Other evidence for double strand breaks as the critical lesions in the cytotoxic effect of bleomycin comes from the hypersensitivity of ataxia telangiectasia (AT) cells. These cells are sensitive to DNA-damaging agents which produce direct DNA double strand breaks, such as ionizing radiation, neocarzinostatin and bleomycin, suggesting that the cells have a defect in double strand break repair (see McKinnon, 1987 for a review). This conclusion is supported by the finding that these agents induce more chromosome breaks and other aberrations in AT cells than in normal cells. However, attempts to demonstrate a repair defect with biochemical methods (primarily neutral and alkaline filter elution) have been less successful. With few exceptions (e.g., Coquerelle et al., 1987), the great majority of these studies have shown no deficiency in strand break repair in AT cells (McKinnon, 1987). On the other hand, AT cells do show a defect in repair of radiation-induced breaks in PCC assays, with a much greater fraction of the initial breakage remaining unrepaired several hours after irradiation than in normal cells (Cornforth and Bedford, 1985). These results suggest that only a small fraction of the double strand breaks measured biochemically have the potential to produce chromosome breaks, and that AT cells do have a defect in repair of this subpopulation of breaks. In a similar experiment involving measurement of bleomycin-induced breaks by PCC, deficiencies in rate or extent of repair were seen in AT cells, but the differences were less pronounced than those seen with X-rays, and wide variations were seen among individual AT lines (Hittelman and Sen, 1988). Other cell lines with hypersensitivity to X-rays, some of which show biochemically demonstrable defects in strand break repair, are also hypersensitive to bleomycin. These include the C H O mutants xrs 5, xrs 6, XR-1 and EM9 (Stamato et al., 1983; Darroudi and Natarajan, 1989), and the variant L5178Y strains M10 and LX830 (Sato et al., 1986). The BLM-2 variant of CHO-K1 cells, which was specifically selected for sensitivity to bleomycin, shows a deficiency in repair of bleomycin-induced single and double strand breaks (Robson et al., 1989). In addition, inhibitors of poly(ADP)-ribose polymerase (Huet and Laval, 1985) and of calmodulin (Smith et al., 1986a; Scheid and Traut,
135
1988), both of which have been implicated in DNA repair, have been shown to potentiate bleomycin cytotoxicity (for a review see Kennedy et al., 1986). In summary, a large body of indirect evidence suggests that DNA damage, and specifically double strand breakage, is involved in bleomycin-induced cytotoxicity. Yet, there remain many inconsistencies and unexplained findings, and the disparate quantitative data comparing chromosome breaks and cytotoxicity have been particularly confusing. Despite intensive effort, little progress has been made toward resolving these inconsistencies, which continue to cast doubt on the significance of DNA breakage in cell killing by bleomycin.
Mutagenesis Bacterial assays A number of studies have shown that bleomycin is negative in most of the Ames tester strains (Benedict et al., 1977; Seino et al., 1978; Pak et al., 1979; Umezawa et al., 1987). A notable exception is the AT102 strain, which contains the hisG428 allele on a multicopy plasmid. Most of the bleomycin-induced revertants in this strain are 3or 6-base deletions of the amber codon; the remainder were uncharacterized (Levin et al., 1984). Intriguingly, no bleomycin-induced revertants were found in the TA2638 strain, which contains the same hisG428 allele in the bacterial chromosome (Leon et al., 1982). A second exception is the trpE8 allele, which is efficiently reverted by bleomycin, although the revertants have not been characterized in detail (Podger and Grigg, 1983). As discussed previously, the failure of many Ames strains to be reverted by bleomycin may be explained by the fact that none of the nucleotide positions that are targets for reversion are G-C or G-T sequences (Povirk and Goldberg, 1987). Intact bacteriophage are completely refractory to bleomycin (presumably due to inability to penetrate the phage coat), but lambda phage repackaged from DNA treated with bleomycin in vitro show a substantial increase in the frequency of cI mutants. The mutagenesis is largely SOS-dependent (Povirk and Houlgrave, 1988a). A collection of sequenced cI mutants (Povirk, 1987) con-
stitutes the only available spectrum of bleomycininduced mutations. These mutations were predominantly base substitutions, with a minor component of - 1 frameshifts. Most of the mutations occurred at pyrimidines in G-C (particularly PyG-C) and G-T sequences, reflecting the specificity of bleomycin-induced DNA lesions (strand breaks and AP sites). C-G-C-C sequences were hotspots for bleomycin-induced mutagenesis. In vitro studies have indicated that each of the three C-G-C-C hotspots, as well as a single C-G-T-T hotspot, were frequent targets for formation of both lone AP sites, and AP sites accompanied by directly opposed strand breaks (Steighner and Povirk, 1990a). Other experiments (Steighner and Povirk, 1990b) with repackaged lambda phage suggest a major role for AP sites with closely opposed strand breaks, rather than lone AP sites, in bleomycin-induced mutagenesis. Specifically, posttreatment of bleomycin-damaged lambda DNA with putrescine, which cleaves all AP sites, resulted in a 60% decrease in mutation frequency, while posttreatment with endonuclease IV, which cleaves only lone AP sites, had no effect. Furthermore, a substantial fraction of the bleomycin-induced cI mutations occurred at non-G-Py nucleotide positions which could only be "secondary" sites of bleomycin attack; at such sites, damage only occurs in the context of a closely opposed strand break at a primary site in the complementary strand (Povirk, 1987; Povirk et al., 1989; Steighner and Povirk, 1990a). Mammalian cells Bleomycin-induced mutagenesis in cultured mammalian cells varies greatly according to the cell line, the genetic locus and the treatment protocol employed. Studies reporting mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus are shown in Table 1. Although the reasons for the disparate results obtained by different investigators are largely unknown, there is some suggestion that the treatment time may be an important variable, with very short treatments producing higher mutation frequencies than the 16-h treatments normally used in the standard C H O / H G P R T assay. Mutagenesis was reported to be inhibited by superoxide dismutase (Cunningham et al., 1984) and by the
136
radioprotective agent 2-[(aminopropyl)amino]ethanethiol (WR2721) (Nagy and Grdina, 1986). Since diffusible free radicals appear to have no role in bleomycin-induced DNA damage, these results are difficult to interpret. One possibility is that the observed mutagenesis may have resulted from free radicals generated by extracellular bleomycin, rather than from direct interaction of bleomycin with cellular DNA. It is also possible that the scavengers may have had an effect on bleomycin uptake or activation. Surprisingly, with the exception of a single 28kb deletion detected in MH3-XPD cells (Wood and Moses, 1989), no analysis of bleomycin-induced HGPRT mutants by Southern blot, to determine whether they are predominantly point mutations or large deletions, has yet been reported. However, like other anticancer drugs and oxidative DNA-damaging agents, bleomycin produces greater mutagenesis at the XPRT locus in CHO-AS52 cells (a line stably transfected with the E. coli gpt gene) than at the HGPRT locus in normal CHO cells (Hsie et al., 1986), and it induces predominantly small-colony T K - mutants in the mouse TK ÷/- assay (Doerr et al., 1989). Both these patterns are considered to be characteristic of agents which preferentially induce large deletions (DeMarini et al., 1989). The AK (adenosine kinase) locus is also much more muta-
ble by bleomycin (and other cancer drugs) than is the HGPRT locus (Singh and Gupta, 1983a). Our laboratory has tried, without success, to induce mutations at the APRT locus in the hemizygous CHO strain D422. However, the drug is apparently effective in inducing mutants in the APRT heterozygous strain D423 (Veigl et al., 1989). This difference could likewise be attributed to a preferential production of large deletions, although allele loss by induced mitotic recombination could produce the same results. Bleomycin is only marginally effective in inducing ouabain-resistant mutants, suggesting that it is a weak base substitution mutagen (Singh and Gupta, 1983b). Bleomycin did not produce detectable mutagenesis in the SV40-based shuttle vector pZ189 grown in human 293 host ceils; however, in a similar shuttle vector grown in COS-1 cells, bleomycin induced small deletions, as well as a minor component of base substitutions, most of which were targeted at G-C or G-T sites (R.A.O. Bennett and L.F. Povirk, unpublished).
Other eukaryotes One of the first reports of bleomycin-induced mutagenesis was that of Moore (1978), showing reversion of nonsense and missense mutations in yeast, but the revertants were not characterized at the molecular level and little subsequent work has
TABLE 1 BLEOMYCIN-INDUCED MUTAGENESIS AT THE HGPRT LOCUS a Cell line (Ref.)
V79 (1) V79 (2) CHO-K1 (3) CHO-W-14 (4) CHO-W-14 (5)
Bleomycin concentration
Treatment time
Mutation frequency b ( M u t a n t s / 1 0 6 survivors)
(#g/ml)
(h)
Untreated
Treated
1000 c 200 50 40 15
0.5 3 1 16 16
20 NR d 20 5 3
140 20 e 880 13 12
Survival (%)
NR d 3 9
127
MUTREV 07295
Genotoxicity of bleomycin Lawrence F. Povirk and M.J. Finley Austin Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298 (U.S.A.)
(Received 6 July 1990) (Accepted 18 October 1990)
Keywords: Bleomycin, genotoxicity; Blenoxane
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemistry of DNA damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uptake and metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromosomal effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromosome breakage and cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bacterial assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mammalian cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction Bleomycin is a radiomimetic antitumor agent with unique genotoxic properties. The drug is a glycopeptide (Fig. 1) which contains several highly unusual amino acids and sugars, and a terminal amine which varies a m o n g bleomycin analogues. Few significant differences in action have been f o u n d between the various analogues. T h e clinical formulation Blenoxane is a mixture of c o m p o nents, primarily bleomycins A 2 and B 2.
Correspondence: Dr. Lawrence F. Povirk, Medical College of Virginia, P.O. Box 230, MCV Station, Richmond, VA 23298 (U.S.A.).
127 128 130 131 133 135 135 135 136 137 137 139 139
Because o f the fairly extensive use of this drug in clinical chemotherapy, its genetic toxicity has considerable practical significance. In addition, the ability of bleomycin to inflict oxidative d a m a g e at a specific position in D N A , i.e., the C - 4 ' position of deoxyribose, offers the potential to assess the role of such sugar d a m a g e in mediating the effects of other oxidative mutagens. As can be seen f r o m the genetic activity profile of bleomycin (Fig. 2), the drug tests positive in the great majority of genetic toxicity assays. N o t a b l e exceptions, discussed in m o r e detail below, are assays with most A m e s tester strains, and the sister c h r o m a t i d exchange (SCE) assay. This review will a t t e m p t to summarize current knowledge concerning the genotoxic effects of bleomycin, with emphasis on new developments in
128
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Bleomycins AZ: R = ~ - N / ~ cH'~ H "CHs B2: R= ~'N~'~/HN"~ N" H CH NH I~pleomycin i ~ R= " N ~ N / ' " . ~ / ~ Fig. 1. Structure of bleomycin, showing (1) double bond which is replaced by a single bond in phleomycin, (2) site of attachment of an additional talose sugar derivative in the analogue tallysomycin, and (3) amide which is deaminated to give a carboxylic acid in desamidoblcomycin, an inactive metabolite. The area enclosed by the dashed fine is considered to be the DNA-binding domain of the molecule. The lelt-hand portion of the molecule is involved in metal chelation.
the past decade, and especially the past few years. Earlier studies have been reviewed by Vig and Lewis (1978).
Chemistry of DNA damage The chemistry of bleomycin activation and of the intermediates in DNA damage has recently been the subject of some extensive and detailed reviews (Hecht, 1986; Stubbe and Kozarich, 1987), and will be described only briefly here. Addition of oxygen to Fe(II)-bleomycin or addition of H202 to Fe(III) • bleomycin results in formation of "activated bleomycin" (probably bleomycin ferric peroxide), a chelate complex which is identified by its distinct EPR spectrum (Burger et al., 1981; Kuramochi et al., 1981). It is the last bleomycin
species detectable prior to DNA damage. This species, or an extremely short-lived product thereof, is believed to specifically abstract hydrogen from C-4' of deoxyribose. The resulting free radical on C-4' partitions into one of two pathways (Fig. 3). Hydroxylation of C-4' (left pathway) results in sugar ring opening and base release, leaving a chemically modified apurinic/ apyrimidinic (AP) site with a ketone at C-4' and an aldehyde at C-I' (Sugiyama et al., 1985; Rabow et al., 1986); the source of the hydroxyl is solvent water (Rabow et al., 1990). The sugar of the AP site presumably exists in equilibrium between ring-closed and ring-opened forms. Alternatively, (right pathway) addition of 02 to C-4' forms a peroxyl radical species, which decomposes to give a strand break with 5'-phosphate and 3'-phosphoglycolate termini, with release of a base-propenal (Giloni et al., 1981). Despite uncertainty in the molecular details of the formation of these two lesions, it is generally agreed that both are products of an initial C-4' free radical (Kozarich and Stubbe, 1987). Under physiological conditions the two products are formed in a ratio of approximately 1:1 (Povirk et al., 1977). Both lesions are formed predominantly at pyrimidines in G-C and G-T sequences (D'Andrea and Haseltine, 1978; Takeshita et al., 1978; Steighner and Povirk, 1990a). However, the relative susceptibilities of individual G-Py sites, which vary widely, are not readily predicted on the basis of sequence and presumably depend on subtle, sequence-dependent variations in DNA conformation. Cytosine methylation can greatly inhibit bleomycin-induced cleavage in the immediate vicinity of the methylated bases (Hecht, 1986). In addition to these single lesions, bleomycin specifically induces double strand breaks, as well as AP sites with closely opposed strand breaks, at a frequency far greater than expected from coincidence of the single lesions (Povirk et al., 1977, Lloyd et al., 1978, Povirk and Houlgrave, 1988b). The chemistry of these bivalent lesions, however, appears to be identical to that of the single lesions (Povirk et al., 1989; Steighner and Povirk, 1990a). Studies from our laboratory have recently shown that the great majority of double strand breaks have either blunt ends or noncomplementary single-base 5' extensions. In general, only one of
129
G-Py site in the complementary strand, at which single strand cleavage is rare (Povirk et al., 1989).
the breaks comprising each double strand break follows the normal G-Py specificity of bleomycin; we refer to this as the " p r i m a r y " cleavage site. The "secondary" break typically occurs at a non-
Remarkably, double strand breaks involving the two C residues in self-complementary G-C se-
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Fig. 2. The genetic activity profile of bleomycin. Assays which gave positive results are indicated in three-letter codes at the top of the figure, and those which gave negative results at the bottom. The vertical scale gives a measure of genotoxic potency in each assay, with a value of 8 indicating a minimum effective dose of 0.001 # g / m l and a value of 1 indicating 10,000 #g/ml. Negative values indicate the highest concentrations used in assays which gave negative results, with - 1 representing 10 # g / m l and - 5 representing 100,000 #g/ml. Vertical and horizontal bars show average values, and values reported in individual studies, respectively (see Waters et al., 1988 for further details). The three letter codes for positive assays are: PRB, prophage induction, SOS repair, DNA strand breaks or crosslinks; ECB, E. coil strand breaks, cross-links or repair; ERD and BRD, differential toxicity to E. cob recA strains and to other repair-deficient bacteria; SA2, Ames test, strain AT102; ECF, E. coli forward mutation; ECK, E. cob K12, forward or reverse mutation; SCG, SCH and SCR, gene conversion, homozygosis and reverse mutation in yeast; ANG and ANF, gene conversion and forward mutation in Aspergillus; VCF, chromosome aberrations in Vicia faba; DMG, DMM, DMX, DMC and DMN, genetic crossing-over, somatic mutation, sex-linked recessive lethal mutation, chromosome aberrations and aneuploidy in Drosophila; DIA, strand breaks in cultured cells; URP and UIA, unscheduled DNA synthesis in rat hepatocytes and in other cultured cells; GCO, gene mutation in CHO cells; SIC, SCE in hamster cells; CIC, CIM and CIT, chromosome aberrations in cultured Chinese hamster, mouse and other transformed cells; AIA and TCM, aneuploidy and cell transformation in culture; DIH, DNA strand breaks in human cells; UHL, unscheduled DNA synthesis in human lymphocytes; SHT and SIH, SCE in transformed and in other human cultured cells; CHL, CHT and CIH, chromosome aberrations in human lymphocytes, transformed cells, and other human cells; SVA, SCE in animal cells in vivo; MVM, micronucleus test, mice in vivo; CBA and CBC, chromosomal aberrations in animal bone marrow and in spermatogonia; MHT, mouse heritable transiocation test; CLH, chromosomal aberrations in human lymphocytes in vivo. Assays which gave negative assays were: SAO, SA9 and SAS, Ames test with strains AT100, AT98 and other strains; SCF, forward mutation in yeast; VFS, SCE in Vicia faba; DMH, heritable translocation in Drosophila; G9H, mutation at the HGPRT locus in V79 cells; UHT, unscheduled DNA synthesis in transformed human cells; SHL, SCE in human lymphocytes. See International Agency for Research on Cancer (1986) for individual references.
130 DNA
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reactivated during the primary cleavage (Povirk et al., 1989). As discussed by Steighner and Povirk (1990a), this model is supported by the finding that, in the case of AP sites with closely opposed breaks, the AP site invariably occurs at the secondary site, and the break at the primary site. The ratio of double strand to single strand breaks is much lower for phleomycin than for bleomycin (Huang et al., 1981), but the reason for this difference is not known. It has recently been reported that bleomycin also produces 8-hydroxyguanine in DNA, with a yield about one-one hundredth that of strand cleavage (Kohda et al., 1989). To our knowledge, no other bleomycin-induced base damage has been detected.
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0 01~" 0
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quences rarely if ever occur, even where both C residues are prominent sites of single strand cleavage. To explain the finding that bleomycin-induced double strand breaks are formed with single-hit kinetics, we recently proposed a model of double strand cleavage in which bleomycin is
Most pharmacokinetic studies have employed the radiolabeled chelate complex 57Co(III)bleomycin. This species preferentially accumulates in tumors, and has even been used for tumor imaging, but the basis for this preferential accumulation has yet to be determined (Alberts et al., 1979). Studies with metal-free bleomycin have been hampered by the general unavailability of radiolabeled drug, but early investigations (Umezawa et al., 1972) did not show any strong preferential accumulation in tumors. Thus, it is possible that metal-free bleomycin and Co(Ill). bleomycin differ substantially in pharmacokinetics (Alberts et al., 1979). Roy and Horwitz (1984) prepared [3H]bleomycin by chemical postlabeling and found that, as might be expected for a large hydrophilic compound, bleomycin was not efficiently taken up by cultured ceils. At low drug concentrations, only 0.1% of the [3H]bleomycin in the medium became associated with HeLa cells grown in monolayer, during a 30 min incubation. Association was temperature-dependent, but was unaffected by metabolic poisons, suggesting that energy was not required. Similarly, Lyman et al. (1986) showed that, even after several hours of treatment, the concentration of [3H]bleomycin in Ehrlich cells remained lower than the concentration in the medium. They also found that, in agreement with earlier animal studies, the inactive Co(Ill).
131 bleomycin complex was much more efficiently taken up than metal-free bleomycin or the Fe(III), Cu(II) or Zn(II) complexes. Sidik and Smerdon (1990) showed that transient permeabilization of cells with lysophosphatidylcholine dramatically increased bleomycin cytotoxicity. Thus, a number of studies (see also Ozawa et al., 1987) suggest that the drug is very poorly taken up by intact cells. The process by which it enters cells is still largely unknown. Since metal-free bleomycin is effective in killing a variety of cultured cells, it is assumed that the drug chelates iron and becomes activated intracellularly. Little is known about the process leading to activation, although it is likely to involve Fe(II) • bleomycin as an intermediate. Under physiological conditions, unchelated iron would be expected to be in the Fe(III) form, although some could be transiently converted to Fe(II) by reaction with glutathione or other reducing agents. Direct reduction of Fe(III) • bleomycin to Fe(II) • bleomycin by glutathione is very inefficient, especially in the presence of DNA (Povirk, 1979), and in recent years the possibility of enzymatic reduction has received considerable attention. Mahmutoglu and Kappus (1988) showed that purified cytochrome b5 reductase effected reduction of Fe(III). bleomycin in an NADH-dependent reaction, and there have been at least two reports of NADPHdependent Fe(III)-bleomycin activation by cytochrome P-450 reductase (Scheulen et al., 1981; Kilkuskie et al., 1984). In contrast, Ciriolo et al. (1987) reported that rat liver microsomes did not reduce Fe(III). bleomycin, although they did enhance the conversion of Fe(II). bleomycin to activated bleomycin. This apparent discrepancy may have been due to the very high DNA concentration used in the studies of Ciriolo et al. Besides metal chelation, the most important feature of bleomycin metabolism is its conversion to the inactive derivative desamidobleomycin (see Fig. 1) by the enzyme bleomycin hydrolase. Several studies have shown a correlation between the resistance of certain tumors or cultured cells to bleomycin and their levels of bleomycin hydrolase (Umezawa et al., 1972; Akiyama et al., 1981). High levels of this enzyme in bone (Umezawa et al., 1972) probably explain the lack of myelosuppression. The bleomycin hydrolase gene has re-
cently been cloned (Sebti et al., 1989); the deduced amino acid sequence indicates that the enzyme belongs to the cysteine proteinase family, but its normal function is not known. Chromosomal effects
In 1970, Ohama and Kadotani reported the induction of chromosomal aberrations in cultures of human leukocytes treated with various concentrations of bleomycin. During the ensuing decade numerous studies were conducted, in vitro and in vivo, on the effects of bleomycin on chromosomes (reviewed by Vig and Lewis, 1978, and by Povirk, 1983). Bleomycin was found to produce both chromosome- and chromatid-type aberrations (deletions, dicentrics, rings, exchanges, breaks and gaps) in all of the systems studied. These systems included the use of different cell types of human origin as well as other animal and plant sources, and the standard technique of scoring stained metaphase figures. The effects of bleomycin were also studied with premature chromosome condensation (PCC), and with micronucleus and sister chromatid exchange (SCE) assays. Though bleomycin induces chromosomal aberrations in standard and PCC preparations, and also induces formation of micronuclei, it appears to have little effect on the level of SCEs. Based on the results reviewed it was concluded that bleomycin acted on chromosomes in an S-independent fashion since bleomycin was observed to produce aberrations when administered in any period of interphase. Furthermore, the types of aberrations produced by treatment in various phases of the cell cycle were those expected to result from production of both double and single strand breaks by bleomycin. Many parallels have been drawn between the effects of bleomycin and those of ionizing radiation; both agents act in an S-independent manner and both produce chromosomal aberrations presumably as a result of induced DNA single and double strand breaks. The induction of chromosomal aberrations and SCEs in two X-ray-sensitive mutants by bleomycin and other DNA damaging agents has been recently reported (Darroudi and Natarajan, 1989). The two mutant lines, xrs 5 and xrs 6, were both found to be more
132
sensitive than wild-type cells to the induction of chromosomal aberrations by bleomycin. An increase in SCE production was observed for one of the lines, xrs 6, while no increase was seen in xrs 5 or the wild-type fine, CHO-K1. In general bleomycin has been found to be a poor inducer of SCEs (reviewed by Vig and Lewis, 1978) and more current work (the above exception noted) continues to corroborate this finding. In in vitro studies with a variety of cell types bleomycin induced either no increase in SCEs or only a small increase at very high doses (Sono and Sakaguchi, 1978; Banerjee and Benedict, 1979; Clare et al., 1982; Morgan and Crossen, 1982). The same trends are seen in studies of patients undergoing cytostatic therapy with bleomycin alone or in combination with other agents (Lambert et al., 1978; DiCker, 1981; Clare et al., 1982; Abe et al., 1984). A possible model to explain the lack of SCE induction by agents which induce strand breaks has been proposed by Painter (1980). Early use of the PCC technique revealed that approximately 1/3 of bleomycin-induced G 2 aberrations are repaired before cells reach mitosis (reviewed by Vig and Lewis, 1978). Employing both PCC and alkaline elution Sognier et al. (1982) observed a 2-component profile, with a more sensitive response at low doses, for the formation of bleomycin-induced aberrations. The 2-component profile was most easily seen with alkaline elution but it was also clearly evident with PCC; however it was not as apparent in data obtained from scoring mitotic figures. Also, the number of aberrations seen in mitotic preparations was greatly reduced as compared to the Gz-PCC. The reduction of visible aberrations in mitosis is most likely due to repair of strand breaks before cells reach mitosis. These authors postulated that the 2-component profile was indicative of a nonrandom interaction of bleomycin with DNA and that the underestimation of aberrations observed with mitotic preparations prevented the detection of the nonlinear kinetics. Bleomycin-induced chromosome damage and repair was studied in quiescent mononuclear human blood cells and fibroblasts with the PCC technique (Sen and Hittelman, 1984). The G 1 cells were treated with bleomycin for 30 rain and washed free of drug. Following various periods of
repair, cells were fused to induce PCC and aberrations were scored. The repair profile displayed fast- and slow-repair components, with a significant reduction in chromosome damage within 30 minutes. After two hours the repair of aberrations slowed. Though the frequency of damage differed in the two cell types the kinetic profiles were similar. At the molecular level bleomycin is now known to induce single strand breaks. This is reflected at the chromosomal level in the appearance of chromatid-type aberrations in cells treated with bleomycin in G 1. Further cytogenetic evidence for the production of single strand breaks has come from exposing cells first treated with X-irradiation or bleomycin during G 2 to Neurospora endonuclease, an enzyme specific for single stranded regions, and observing an increase in all types of chromosomal aberrations in the following mitosis (Natarajan and Obe, 1978; Nowak et al., 1984; Nowak and Obe, 1984). Presumably, the Neurospora endonuclease converted single strand breaks induced by either agent to double strand breaks. Like that of most, if not all DNA damaging agents, the effect of bleomycin on the induction of chromosomal aberrations is potentiated by a number of agents a n d / o r culture conditions. In the 1970's both caffeine and hyperthermia were shown to increase the number of aberrations induced by bleomycin in cells in culture (Kihlman and Sturelid, 1978; Vig and Lewis, 1978; Vig, 1979). The hyperthermic potentiation of bleomycin-induced aberrations was found to be synergistic in cultured human lymphocytes and Chinese hamster cells (Vig et al., 1982). More recently, it has been reported that the concomitant treatment of cells with hyperthermia and trifluoperazine, a calmodulin inhibitor, induces chromatin changes resulting in an increased accumulation of bleomycininduced DNA damage due to a decrease in repair functions (Smith et al., 1986b). The frequency of bleomycin-induced aberrations in vitro has also been demonstrated to be enhanced by hydroxyurea (Hansson and HartleyAsp, 1981), isoptin (Scheid et al., 1984), 3-aminobenzamide (Zwanenburg et al., 1985), and ethanol and acetaldehyde (Lin et al., 1989). In contrast, the anticlastogens fl-aminoethyl-isothiouronium and homocysteinethiolactone greatly reduced the
133 numbers of aberrations induced in human lymphocytes treated simultaneously with these agents and bleomycin as compared to cultures treated with bleomycin alone (Gebhart, 1978). More recently, Pohl and Reidy (1989) found a reduction in bleomycin-induced aberrations in the cultured lymphocytes of individuals after receiving dietary supplementation with 1 g of vitamin C per day for two and four weeks. Pretreatment of human lymphocyte cultures with a low dose (0.01-0.1 /~g/ml) of bleomycin has been reported to invoke resistance to the clastogenic effects of X-rays or of a high concentration (1.5 /~g/ml) of bleomycin (Vijayalaxmi and Burkart, 1989). Bleomycin not only induces structural aberrations in mitotic cells but it has also been found to cause aneuploidy (reviewed by Vig and Lewis, 1978). Because these effects have been observed in mitotic cells concern has been generated about possible correlates occurring in germ cells, as well as potential teratogenic consequences, of individuals treated with bleomycin. In a study of the teratogenic potential of bleomycin a single injection of 80 mg/kg into pregnant females was found to induce chromosomal aberrations in cells derived from post-implantation mouse embryos only 3 hours after the transplacental treatment (Adler, 1983). Though the frequency decreased a significant number of aberrations were still present 18 hours post-treatment. The induction of aneuploidy by bleomycin in Drosophila melanogaster oocytes has been observed with the aneuploidy pattern method (Traut, 1984). Bleomycin has also been found to induce both reciprocal translocations and aneuploidy in the spermatogonia of mice (van Buul and Goudzwaard, 1980; De Luca et al., 1988; Sen and Liang, 1989). Even before the application of banding techniques the nonrandom nature of bleomycin-induced chromosomal damage was evident (Vig and Lewis, 1978). DNA damage by bleomycin has also been shown to be concentrated in nucleosomal linker regions in isolated nuclei (Kuo and Hsu, 1978). Kuo (1981) reported that bleomycin acted preferentially on active chromatin. Using restriction enzyme digestion of DNA from bleomycintreated oviduct cell and erythrocyte nuclei along with Southern blotting he found that the active
ovalbumin gene was more sensitive to damage by bleomycin than the inactive globin gene. More recently, Yunis et al. (1987) found that bleomycin along with caffeine as an enhancer induced the expression of common chromosomal fragile sites, constant chromosomal locations where aberrations are induced nonrandomly by specific conditions, in cultured human lymphocytes. A high concordance was noted between the bleomycin-induced sites and fragile sites induced by aphidicolin as well as a variety of DNA damaging agents and other synthesis inhibitors. Several of the agents studied by this group have been reported to preferentially attack active DNA regions and Austin et al. (1989) recently found an association between X chromosomal aphidicolin-inducible fragile site locations and gene activity on the human X chromosome. Thus, presence of actively transcribed genes appears to be one essential characteristic of chromosomal fragile sites, though other factors must be involved as well. Continued studies of chromosomal aberrations produced by bleomycin coupled with molecular analyses will certainly enhance the understanding of mechanisms involved in DNA damage and repair as they relate specifically to bleomycin as well as in general. An understanding of the nonrandom nature of damage induced by bleomycin and other agents may provide key insights into the processes underlying mutagenesis and carcinogenesis.
Chromosome breakage and cytotoxicity It is likely that bleomycin-induced cell killing is due to DNA double strand cleavage, and the resulting loss of chromosome fragments. The primary impetus for this view comes from analogy to extensive studies with ionizing radiation, which show that at least in some systems, there is a one-to-one correspondence between chromosome aberrations and cell death (Dewey et al., 1970; Joshi et al., 1982; Cornforth and Bedford, 1987). However, while there are extensive qualitative similarities between bleomycin and ionizing radiation in their clastogenic and cytotoxic effects, the two agents often show quite different behavior quantitatively. In a particularly intriguing study
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with V79 cells, Scott and Zampetti-Bosseler (1985) found that, at equivalent levels of cytotoxicity, bleomycin induced 5- to 10-fold fewer chromosome aberrations than ionizing radiation, and also induced much less mitotic delay. In fact, at a dose of bleomycin which killed 80% of the cells, less than 5% of the cells had visible aberrations, over a wide range of sampling times following treatment. These authors came to the rather disquieting conclusion that chromosome breakage plays little or no role in bleomycin cytotoxicity. On the other hand, with L5178Y mouse leukemia cells, Doerr et al. (1989) found a fairly close correspondence between the fraction of surviving cells and the fraction of cells with no visible aberrations, over a range of bleomycin concentrations. Interpretation of such quantitative studies, and particularly the comparisons with ionizing radiation, are further complicated by several unexplained features of bleomycin-induced D N A damage in cells, including the nonrandom nature of the damage, as mentioned above. Alkaline elution studies with L1210 cells (Iqbal et al., 1976) have indicated that a fraction of cellular D N A typically suffers severe damage, while the remainder is much less affected; the fraction of heavily degraded D N A increases with bleomycin dose. Although other studies (Kuo, 1981; Beckmann et al., 1987) have indicated that actively transcribed genes are particularly susceptible to bleomycin damage, these domains would constitute far less than the fraction of heavily degraded cellular D N A observed by Iqbal et al. Even greater heterogeneity of damage was found in an alkaline elution study with Ehrlich ceils (Byrnes et al., 1990), in which it was suggested that cytotoxicity was correlated with the amount of very heavily degraded D N A which eluted immediately in the lysis fraction. Even more disturbing are experiments involving measurements of D N A damage by nucleoid electrophoresis, which indicate extreme cell to cell differences in the extent of D N A damage (0stling and Johanson, 1987). All of these heterogeneities should clearly be taken into account in attempting to assess the role of D N A damage in cell death, but it is difficult to do so since the basis of heterogeneity is largely unknown.
Other evidence for double strand breaks as the critical lesions in the cytotoxic effect of bleomycin comes from the hypersensitivity of ataxia telangiectasia (AT) cells. These cells are sensitive to DNA-damaging agents which produce direct DNA double strand breaks, such as ionizing radiation, neocarzinostatin and bleomycin, suggesting that the cells have a defect in double strand break repair (see McKinnon, 1987 for a review). This conclusion is supported by the finding that these agents induce more chromosome breaks and other aberrations in AT cells than in normal cells. However, attempts to demonstrate a repair defect with biochemical methods (primarily neutral and alkaline filter elution) have been less successful. With few exceptions (e.g., Coquerelle et al., 1987), the great majority of these studies have shown no deficiency in strand break repair in AT cells (McKinnon, 1987). On the other hand, AT cells do show a defect in repair of radiation-induced breaks in PCC assays, with a much greater fraction of the initial breakage remaining unrepaired several hours after irradiation than in normal cells (Cornforth and Bedford, 1985). These results suggest that only a small fraction of the double strand breaks measured biochemically have the potential to produce chromosome breaks, and that AT cells do have a defect in repair of this subpopulation of breaks. In a similar experiment involving measurement of bleomycin-induced breaks by PCC, deficiencies in rate or extent of repair were seen in AT cells, but the differences were less pronounced than those seen with X-rays, and wide variations were seen among individual AT lines (Hittelman and Sen, 1988). Other cell lines with hypersensitivity to X-rays, some of which show biochemically demonstrable defects in strand break repair, are also hypersensitive to bleomycin. These include the C H O mutants xrs 5, xrs 6, XR-1 and EM9 (Stamato et al., 1983; Darroudi and Natarajan, 1989), and the variant L5178Y strains M10 and LX830 (Sato et al., 1986). The BLM-2 variant of CHO-K1 cells, which was specifically selected for sensitivity to bleomycin, shows a deficiency in repair of bleomycin-induced single and double strand breaks (Robson et al., 1989). In addition, inhibitors of poly(ADP)-ribose polymerase (Huet and Laval, 1985) and of calmodulin (Smith et al., 1986a; Scheid and Traut,
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1988), both of which have been implicated in DNA repair, have been shown to potentiate bleomycin cytotoxicity (for a review see Kennedy et al., 1986). In summary, a large body of indirect evidence suggests that DNA damage, and specifically double strand breakage, is involved in bleomycin-induced cytotoxicity. Yet, there remain many inconsistencies and unexplained findings, and the disparate quantitative data comparing chromosome breaks and cytotoxicity have been particularly confusing. Despite intensive effort, little progress has been made toward resolving these inconsistencies, which continue to cast doubt on the significance of DNA breakage in cell killing by bleomycin.
Mutagenesis Bacterial assays A number of studies have shown that bleomycin is negative in most of the Ames tester strains (Benedict et al., 1977; Seino et al., 1978; Pak et al., 1979; Umezawa et al., 1987). A notable exception is the AT102 strain, which contains the hisG428 allele on a multicopy plasmid. Most of the bleomycin-induced revertants in this strain are 3or 6-base deletions of the amber codon; the remainder were uncharacterized (Levin et al., 1984). Intriguingly, no bleomycin-induced revertants were found in the TA2638 strain, which contains the same hisG428 allele in the bacterial chromosome (Leon et al., 1982). A second exception is the trpE8 allele, which is efficiently reverted by bleomycin, although the revertants have not been characterized in detail (Podger and Grigg, 1983). As discussed previously, the failure of many Ames strains to be reverted by bleomycin may be explained by the fact that none of the nucleotide positions that are targets for reversion are G-C or G-T sequences (Povirk and Goldberg, 1987). Intact bacteriophage are completely refractory to bleomycin (presumably due to inability to penetrate the phage coat), but lambda phage repackaged from DNA treated with bleomycin in vitro show a substantial increase in the frequency of cI mutants. The mutagenesis is largely SOS-dependent (Povirk and Houlgrave, 1988a). A collection of sequenced cI mutants (Povirk, 1987) con-
stitutes the only available spectrum of bleomycininduced mutations. These mutations were predominantly base substitutions, with a minor component of - 1 frameshifts. Most of the mutations occurred at pyrimidines in G-C (particularly PyG-C) and G-T sequences, reflecting the specificity of bleomycin-induced DNA lesions (strand breaks and AP sites). C-G-C-C sequences were hotspots for bleomycin-induced mutagenesis. In vitro studies have indicated that each of the three C-G-C-C hotspots, as well as a single C-G-T-T hotspot, were frequent targets for formation of both lone AP sites, and AP sites accompanied by directly opposed strand breaks (Steighner and Povirk, 1990a). Other experiments (Steighner and Povirk, 1990b) with repackaged lambda phage suggest a major role for AP sites with closely opposed strand breaks, rather than lone AP sites, in bleomycin-induced mutagenesis. Specifically, posttreatment of bleomycin-damaged lambda DNA with putrescine, which cleaves all AP sites, resulted in a 60% decrease in mutation frequency, while posttreatment with endonuclease IV, which cleaves only lone AP sites, had no effect. Furthermore, a substantial fraction of the bleomycin-induced cI mutations occurred at non-G-Py nucleotide positions which could only be "secondary" sites of bleomycin attack; at such sites, damage only occurs in the context of a closely opposed strand break at a primary site in the complementary strand (Povirk, 1987; Povirk et al., 1989; Steighner and Povirk, 1990a). Mammalian cells Bleomycin-induced mutagenesis in cultured mammalian cells varies greatly according to the cell line, the genetic locus and the treatment protocol employed. Studies reporting mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus are shown in Table 1. Although the reasons for the disparate results obtained by different investigators are largely unknown, there is some suggestion that the treatment time may be an important variable, with very short treatments producing higher mutation frequencies than the 16-h treatments normally used in the standard C H O / H G P R T assay. Mutagenesis was reported to be inhibited by superoxide dismutase (Cunningham et al., 1984) and by the
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radioprotective agent 2-[(aminopropyl)amino]ethanethiol (WR2721) (Nagy and Grdina, 1986). Since diffusible free radicals appear to have no role in bleomycin-induced DNA damage, these results are difficult to interpret. One possibility is that the observed mutagenesis may have resulted from free radicals generated by extracellular bleomycin, rather than from direct interaction of bleomycin with cellular DNA. It is also possible that the scavengers may have had an effect on bleomycin uptake or activation. Surprisingly, with the exception of a single 28kb deletion detected in MH3-XPD cells (Wood and Moses, 1989), no analysis of bleomycin-induced HGPRT mutants by Southern blot, to determine whether they are predominantly point mutations or large deletions, has yet been reported. However, like other anticancer drugs and oxidative DNA-damaging agents, bleomycin produces greater mutagenesis at the XPRT locus in CHO-AS52 cells (a line stably transfected with the E. coli gpt gene) than at the HGPRT locus in normal CHO cells (Hsie et al., 1986), and it induces predominantly small-colony T K - mutants in the mouse TK ÷/- assay (Doerr et al., 1989). Both these patterns are considered to be characteristic of agents which preferentially induce large deletions (DeMarini et al., 1989). The AK (adenosine kinase) locus is also much more muta-
ble by bleomycin (and other cancer drugs) than is the HGPRT locus (Singh and Gupta, 1983a). Our laboratory has tried, without success, to induce mutations at the APRT locus in the hemizygous CHO strain D422. However, the drug is apparently effective in inducing mutants in the APRT heterozygous strain D423 (Veigl et al., 1989). This difference could likewise be attributed to a preferential production of large deletions, although allele loss by induced mitotic recombination could produce the same results. Bleomycin is only marginally effective in inducing ouabain-resistant mutants, suggesting that it is a weak base substitution mutagen (Singh and Gupta, 1983b). Bleomycin did not produce detectable mutagenesis in the SV40-based shuttle vector pZ189 grown in human 293 host ceils; however, in a similar shuttle vector grown in COS-1 cells, bleomycin induced small deletions, as well as a minor component of base substitutions, most of which were targeted at G-C or G-T sites (R.A.O. Bennett and L.F. Povirk, unpublished).
Other eukaryotes One of the first reports of bleomycin-induced mutagenesis was that of Moore (1978), showing reversion of nonsense and missense mutations in yeast, but the revertants were not characterized at the molecular level and little subsequent work has
TABLE 1 BLEOMYCIN-INDUCED MUTAGENESIS AT THE HGPRT LOCUS a Cell line (Ref.)
V79 (1) V79 (2) CHO-K1 (3) CHO-W-14 (4) CHO-W-14 (5)
Bleomycin concentration
Treatment time
Mutation frequency b ( M u t a n t s / 1 0 6 survivors)
(#g/ml)
(h)
Untreated
Treated
1000 c 200 50 40 15
0.5 3 1 16 16
20 NR d 20 5 3
140 20 e 880 13 12
Survival (%)
NR d 3 9
