Genotoxicity of streptonigrin: a review

Mutation Research 488 (2001) 25–37

Genotoxicity of streptonigrin: a review Alejandro D. Bolzán∗ , Martha S. Bianchi Laboratory of Cytogenetics and Mutagenesis, Instituto Multidisciplinario de Biolog´ıa Celular (IMBICE), C.C. 403, 1900 La Plata, Argentina Received 31 August 2000; accepted 21 November 2000

Abstract Streptonigrin (SN, CAS no. 3930-19-6) is an aminoquinone antitumor antibiotic isolated from cultures of Streptomyces flocculus. This compound is a member of a group of antitumor agents which possess the aminoquinone moiety and that includes also mitomycin C, porfiromycin, actinomycin, rifamycin and geldanamycin. Because of the potential use of SN in clinical chemotherapy, the study of its genotoxicity has considerable practical significance. SN inhibits the synthesis of DNA and RNA, causes DNA strand breaks after reduction with NADH, induces unscheduled DNA synthesis and DNA adducts and inhibits topoisomerase II. At the chromosome level, this antibiotic causes chromosome damage and increases the frequency of sister-chromatid exchanges. SN cleaves DNA in cell-free systems by a mechanism that involves complexing with metal ions and autoxidation of the quinone moiety to semiquinone in the presence of NADH with production of oxygen-derived reactive species. Recent evidence strongly suggests that the clastogenic action of this compound is partially mediated by free radicals. The present review aims at summarizing past and current knowledge concerning the genotoxic effects of SN. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Streptonigrin; Genotoxicity; Bruneomycin

1. Introduction Streptonigrin (SN, CAS no. 3930-19-6) is an aminoquinone antitumor antibiotic isolated from cultures of Streptomyces flocculus [1]. Its molecular structure, shown in Fig. 1, was identified by chemical analysis and mass spectrometry by Rao et al. in 1963 [2] and confirmed by X-ray diffraction by Chiu and Lipscomb in 1975 [3]. It was shown that the A, B, and C rings are very nearly co-planar, and D ring is virtually perpendicular to the other rings [3]. This finding was recently confirmed by Harding et al. [4] who ∗ Corresponding author. Tel.: +54-221-4210112; fax: +54-221-4253320. E-mail addresses: [email protected], [email protected] (A.D. Bolz´an).

observed that the major conformation of SN in solution coincides with the structure observed in the solid state. In 1968, Russian workers found that a substance called bruneomycin, isolated from Actinomyces albus var. bruneomycini, was identical to SN [5]. Recently, a mutant strain of Streptomyces helvaticus was also found to produce bruneomycin [6]. SN shows antitumor activity against a broad range of tumors, including breast, lung, head and neck cancer, lymphoma and melanoma [2,7–12]. Its use in cancer therapy is limited because it induces severe and prolonged bone marrow depression [13–18]. However, at least five studies report positive results with use of the antibiotic in the treatment of leukemias, lymphomas and melanomas [19–23]. Recently, it was found that SN can be efficiently bioactivated by DT-diaphorase, an enzyme showing increased

1383-5742/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 4 2 ( 0 0 ) 0 0 0 6 2 - 4

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The present review will attempt to summarize current knowledge concerning the genotoxic effects of this antibiotic, with emphasis on recent findings and developments.

2. DNA damage by streptonigrin

Fig. 1. Structure of streptonigrin (SN) as determined by Rao et al. [2].

activity in certain tumors such as human non-small cell lung cancer [24,25]. Hence, it was suggested that this compound could be used as a potential antineoplasic agent against tumors with increased DT-diaphorase function. SN is a member of a group of antitumor agents with an aminoquinone moiety. Other compounds in this group are: mitomycin C, porfiromycin, actinomycin, rifamycin and geldanamycin [26]. The antitumor activity of SN has been attributed to several different cellular mechanisms which include interference with cell respiration, impairment of DNA synthesis and direct damage to DNA [26–30]. Since the antitumor actions of SN are lost when the aminoquinone moiety is blocked (as in azastreptonigrin) it appears that the cell toxicity is mediated by the aminoquinone domain [31]. Due to its capacity to cause DNA damage in cell-free and in cellular systems via a targeted free radical mechanism [26,32–34], SN is considered a radiomimetic compound [32,35,36]. This antibiotic has been shown to inhibit synthesis of DNA and RNA [37–39], to cause DNA strand breaks both in vitro and in vivo [26,28,40–49], to induce unscheduled DNA synthesis and DNA adducts [50–52] and to inhibit topoisomerase II [53,54]. In addition, several studies carried out during the past two decades have shown that SN induces chromosomal aberrations and sister-chromatid exchanges (SCEs) in mammalian and other cell types [34,49,55–62]. The most recent reviews on SN or quinone antibiotics were written almost 20 years ago [63,64].

SN exhibits multiple metal complexation sites [65] and binds irreversibly to DNA in the presence of certain metal ions, such as zinc, copper, iron, manganese, cadmium and gold, via the formation of SN–metal–DNA complexes [47,66–71]. The absence of metal ions prevents the SN–DNA association [70]. SN titration with DNA at varying zinc molar equivalents revealed that one antibiotic molecule requires 5–7 mol of zinc and 20–25 mol of DNA-phosphorus for complexation [67]. Sinha [68] reported that incubation of chemically reduced SN with DNA in vitro resulted in irreversible binding of the antibiotic to DNA through complexes containing one molecule of SN per 250 nucleotides. The presence of zinc(II) considerably potentiates the binding, giving rise to complexes with a ratio of one molecule of the antibiotic per 80 nucleotides [68]. Sugiura et al. [47] analyzed the role of several SN–metal complexes in the binding and cleavage of plasmid pBR322 DNA and found that this antibiotic in the presence of NADPH and Cu(II) cleaves DNA. The Cu(II)–SN–NADPH system significantly stimulates double strand breaks of DNA to form linear DNA. In SN–DNA complexes the pyridine ring of the drug interact with purines producing a preferential cleavage at C-bases located 30 of the purine. The tricyclic phenanthidium ring system including the copper chelate ring was shown to contribute to the DNA interaction and cleavage by copper–SN [47]. DNA cleavage by other metal complexes of the antibiotic, which included Mn(II), Fe(II), Co(II), Ni(II), Zn(II), Fe(III), and Pt(II), were also tested. However, the effect of these complexes were remarkably small, comparable to DNA plus SN alone. The only exception was Pt(II), which formed a complex with the antibiotic that stimulated only weak DNA cleavage activity compared to the Cu(II) complex. These findings do not correlate with previous reports indicating that the presence of Zn(II), Cu(II), and Mn(II) enhances the cleavage of supercoiled DNA by SN

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into nicked and linear forms of DNA [70], and that Zn(II) potentiates the binding of SN to DNA [67,68]. Although causes for the above disagreements are not clear, they may result to the different experimental approaches employed. Complexed with Au, SN forms a 1:1 Au(III)–SN complex that is very stable as long as gold is in the trivalent state and exhibits antitumor activity against P-388 leukemia cells [69]. In conclusion, even though metals such as cadmium, platinum, gold and manganese are known to form complexes with SN, present evidence strongly suggest that zinc and copper play the most important role in the binding of this drug to DNA [47,66–68]. Besides complexing with metal ions, SN requires autoxidation of the quinone moiety to semiquinone in the presence of NADH with subsequent production of free radical species to produce its DNA-damaging effects [26,29,32,47]. Activation of the antibiotic can be accomplished via a one- or two-electron reductase to produce a semiquinone radical or hydroquinone, respectively. Either form can react with molecular oxygen to produce reactive oxygen species and regeneration of the parent quinone. The presence of metal ions catalyzes this reaction and produces the hydroxyl radicals through a Fenton-type reaction, which are believed to be the ultimate source of SN-induced DNA damage [26,34,46,48,72]. A secondary mode of action of SN involves the inhibition of topoisomerase II by stabilizing the transesterification intermediate of the enzyme (called cleavable complex) [53]. It was found that this antibiotic preferentially stabilizes topoisomerase II-mediated cleavages in DNA sequences with a thymine at position +2 and an adenine at position +3, respectively. SN is the first drug examined so far that exhibits base preferences that do not flank the cleavage site. In addition, this compound covalently binds to the minor groove of the DNA double helix, producing a tightening of the DNA helical twist [54,73]. These findings are consistent with direct interactions of the antibiotic with DNA as previously reported for other topoisomerase II poisons [74,75]. In addition to the aforementioned effects, SN has been found to be a potent inducer of DNA adducts, as shown by the presence of unscheduled DNA synthesis [50–52]. The proportion of covalent adduct formation compared to the total damage induced by SN was shown to increase with the dose of drug [52]. To our

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knowledge, none of the DNA adducts induced by SN have yet been identified. In summary, there are three main modes of action by which SN exerts its genotoxic effects at the DNA level. A primary mode of action involves the production of single and double strand breaks. A secondary mode of action, which enhances the primary action, is the inhibition of topoisomerase II by stabilizing cleavable complexes. Besides these effects, SN induces covalent DNA adducts. 3. Chromosomal effects 3.1. Induction of chromosomal aberrations and SCEs by SN The first study demonstrating the induction of chromosomal aberrations by SN was carried out by Cohen and coworkers [76] using human lymphocyte cultures. They found that even a concentration as low as 10−9 M of the antibiotic significantly increased the incidence of chromosomal aberrations. In 1964, Kihlman [77] analyzed the types of chromosomal rearrangements produced by the drug using root-tips of Vicia faba and found that sub-chromatid exchanges were produced in late G2 and prophase, chromatid type aberrations in S and G2, and chromosome type aberrations in cells that were assumed to be in Gl. This is in fact the type of effect produced by S-independent clastogens, like ionizing radiations and bleomycin. In good agreement with this observation, several authors [46,78,79] proposed that bleomycin and SN produce their biological effects by a similar mechanism. During the past two decades, numerous studies were conducted to investigate the effect of this antibiotic on chromosomes and the mechanism underlying its clastogenic action. SN was found to produce chromosome and chromatid aberrations in several test systems, including human lymphocytes, rabbit oocytes, Chinese hamster ovary (CHO) cells and other cell types [49,55–57,59–62,80]. Both chromosome- and chromatid-type aberrations (i.e. dicentrics, rings, deletions, exchanges, breaks and gaps) were observed but with a clear predominance of the latter. Besides confirming previous observations, these studies showed that SN also produces chromatid-type aberrations in the G1-phase [55]. In addition to this clastogenic

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effect, SN has been found to be a good inducer of SCEs, which seems to indicate an S-dependent mode of action [49,55,58,62,81,82]. However, unlike S-dependent agents, which induce SCEs at concentrations lower than those required for the induction of chromosomal aberrations [83], the increase in the frequency of SCEs induced by SN is obtained only at concentrations that also increase the frequency of chromosomal aberrations. No studies on its clastogenic effects using premature chromosome condensation (PCC) or micronucleus assays have yet been reported. The chromosomal effects of SN were further investigated using fluorescence in situ hybridization (FISH) [84]. These authors analyzed the induction of chromosomal aberrations in human lymphocytes using conventional staining techniques and chromosome painting. Using conventional Giemsa staining, they confirmed that SN induces mainly chromatid-type aberrations. Use of chromosome painting probes showed that SN induces reciprocal translocations, with a frequency significantly higher than that of dicentrics. Similar findings have been previously reported for ionizing radiations [85,86] bleomycin [87] and daunomycin [88]. However, the number of both types of aberrations was very limited. The frequency of cells carrying chromatid exchanges was low (maximum 17%) and of these, about 37% of chromatid exchanges seen after treatment of human lymphocytes with SN were symmetrical and complete [84]. Marshall and Obe [84] also found that at 72 h sampling time, the number of translocations and dicentrics induced by the antibiotic in human lymphocytes increased as a result of an additional contribution from aberrations derived from chromatid exchanges. Following FISH analysis, a significantly increased frequency of translocations in all time points studied (24, 72 and 120 h) was observed, but this frequency remained fairly constant over the period of sampling. Although the frequencies of aberrations detected with Giemsa stained preparations decreased over the sampling period, the numbers observed remained statistically significant even at 120 h after exposure [84]. This finding confirmed previous results of Linscombe et al. [60] and Testoni et al. [49,61] in the sense that SN has a delayed and persistent clastogenic effect. In summary, it is possible to conclude that like other agents with S-independent action, SN produces

sub-chromatid aberrations in prophase, chromatid aberrations in the G2-phase and in replicated S-phase chromatin, and chromosome-type aberrations in the G1-phase and in unreplicated S-phase chromatin. However, like S-dependent agents, SN also produces chromatid-type aberrations in the G1-phase, is an efficient inducer of SCEs, and has a delayed clastogenic effect. This suggests that this antibiotic is capable of producing chromosome damage both by S-independent and by S-dependent mechanisms. 3.2. Kinetics of chromosome and DNA damage by SN Several years ago, Testoni and coworkers studied the clastogenic effect of SN on CHO cells and found that the chromosomal damage in these cells increased with the lengthening of the interval between the end of the treatment and the harvesting time [61]. This delayed clastogenic action was further evidenced by the finding, in a more recent report, of chromatid-type aberrations in second and third mitosis cells [49]. This finding allowed these authors to conclude that SN has a delayed clastogenic action that lasts for at least three cell cycles. A similar finding was previously reported by Linscombe et al. [60], who studied the induction of chromosome damage in human lymphocytes that were pulse-treated with increasing doses of SN. They found that aberration yields at different time points (from 48 to 96 h) were statistically significant but almost the same magnitude, i.e. the frequency of SN-induced aberrations remained the same whether the cell populations examined were composed of as low as 46% or as high as 100% first division metaphases. Testoni et al. [49] also investigated the kinetics of DNA damage by SN in eukaryotic cells. They used alkaline unwinding to assess the kinetics of DNA damage in CHO cell populations and the single cell gel electrophoresis technique (comet assay) to evaluate the same process in individual CHO cells. These authors observed a dose-dependent decay of double-stranded DNA in the alkaline unwinding analysis and a dose-dependent increase in nuclear damage as detected by the comet assay. These data were interpreted as evidence of the capacity of SN to induce direct single strand breaks into the DNA molecule. Particularly interesting was the observation that a pulse treatment with SN elicited a triphasic response

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characterized by repair–damage–repair. As suggested by Testoni et al. [49], this response may be related to a continuous production of free radicals by the antibiotic. Closing of the single strand DNA breaks or gaps by the repair machinery would produce the first phase of repair. Saturation of repair enzymes and the persistent production of free radicals would initiate a second round of DNA degradation. A further recovery of the repair machinery would give rise to the second phase of DNA mending. Three decades ago, Mizuno and Gilboe [44] showed that SN covalently binds to DNA. Several years later, Andersson [55] hypothesized that if the semiquinone of the antibiotic is covalently bound to the DNA molecule, chromosomal aberrations would mostly be produced by lesions induced by reactive metabolites of oxygen and SCEs mostly by the DNA-bound drug molecule. As previously mentioned, Capranico et al. [73] showed that the drug actually binds to the minor groove of the DNA molecule. Based on this observation, Testoni et al. [49] suggested that the formation of a stable complex of SN–DNA may induce chromatid aberrations and an increase of SCEs by a DNA replication impairment during the S period and that such a complex, through a persistent cyclic redox process, may keep generating active oxygen species that could be an additional source of chromatid aberrations due to the induction of single strand breaks after chromosome replication. 3.3. Prevention of SN-induced chromosome and DNA damage 3.3.1. Antioxidant compounds The antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were found to totally inhibit DNA cleavage and deoxyribose degradation induced by SN in non-cellular systems [26,33]. In a more recent study, we examined the effect of SOD and CAT and the hydroxyl radical scavenger mannitol on the DNA and chromosome damage induced by the antibiotic in CHO cells [34]. Although the addition of these antioxidant compounds directly to the culture medium did not protect CHO [34] or V79 [48] cells from SN-induced clastogenesis, a marked protective effect was detected when the intracellular incorporation of the antioxidants was mediated by the use of liposome encapsulation [34].

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Very likely, SOD exerts its protective effect on chromosomes and DNA by eliminating the superoxide radical generated during SN reduction or semiquinone autoxidation, thereby preventing the reduction of metal ions and the Fenton reaction. Likewise, CAT, by removing hydrogen peroxide avoids the Fenton reaction and hydroxyl radical production which are responsible for site-specific damage to DNA. In addition, mannitol may prevent DNA degradation in a direct way by scavenging the hydroxyl radical released in the cytoplasm as well as those originated by SN–DNA redox cycling complexes in the nucleus. 3.3.2. Metal chelators Metal chelators were also found to prevent DNA and chromosome damage by SN. It was found that the chelating compounds desferrioxamine (DF) and 2,2-dipyridyl protect isolated DNA and bacterial cells against SN-induced toxic effects [89] and that 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TP, a nitroxide free radical with antioxidant properties) and DF protect Chinese hamster V79 cells against DNA double strand breaks and cytotoxicity induced by the antibiotic [48]. This protective effect of DF was also demonstrated in human colon carcinoma cells cultured in vitro [30]. These findings agree with previous reports indicating that intracellular transition metals are involved in SN cytotoxicity [33,90–93]. The effect of another metal chelating agent, 1,10phenanthroline (PNT), on the frequencies of SNinduced chromosomal aberrations and SCEs in CHO cells was recently investigated in our laboratory to test whether the aforementioned conclusions could be extrapolated to the chromosome level [72]. PNT is a compound that enters the cell and, by forming a complex with iron, prevents the Fenton reaction from occurring, thus, blocking the production of the hydroxyl radical [94]. It was found that pre-treatment of CHO cells with PNT significantly inhibited the induction of chromosomal aberrations and decreased the frequency of SCEs induced by SN, supporting the role of transition metals in its clastogenic action [72]. The protective effect of PNT indicates that very likely the Fenton reaction is involved in the induction of chromosomal aberrations and SCEs by SN. The fact that PNT did not completely inhibit the induction of SCEs suggests that this phenomenon might be due to a dual mechanism involving the production

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of free radicals and the formation and persistence of SN–DNA complexes [49,72]. These findings strongly support the hypothesis that SN induces chromosome damage through a redoxcycling process where O2 − radicals are generated from the interaction of oxygen and the SN hydroquinone. H2 O2 is formed from the dismutation of the superoxide radical, and the hydroxyl radical is produced via a Fenton reaction in the presence of reduced iron (Fe2+ ). • According to this view, the OH radical is the agent which ultimately causes the DNA and chromosome damage induced by the antibiotic. Thus, the mechanism underlying the chromosome damage by SN appears to be the same as the one previously indicated for DNA damage induced by the antibiotic in cellular and non-cellular systems [26,33,34,49]. 3.4. Resistance of insect chromosomes to SN An additional interesting point regarding the clastogenic action of SN is that in contrast to mammalian cells, insect cells have been shown to be very resistant to the induction of chromosomal aberrations and SCEs by this antibiotic [62]. It was found that SN doses inducing high levels of chromosome damage and a dose-dependent increase in SCEs in CHO cells fail to produce a significant increase of chromosomal aberrations and SCEs in mosquito cells [62]. Insect cells have been previously found to be highly resistant to the lethal effects of ionizing and non-ionizing radiations and the radiomimetic compound bleomycin [95–99]. This resistance has been ascribed to the differential chromosomal architecture between mammalian and insect cells as reported by Holmquist [100] and Bianchi et al. [101] and to the presence of a better and more efficient antioxidant system in insect cells. Insects cells have very large free amino acid pools in their hemolymph [102] and contain much higher levels of reduced glutathione (a well-known intracellular radical scavenger) than mammalian cells [103]. Very likely, in CHO but not in mosquito cells, the binding of the antibiotic to DNA may induce conformational changes in the chromatin fibril, enhancing the sensitivity of DNA to the free radicals generated by the drug [62] and giving rise to a partial or total replicon blockage which ultimately increases the SCEs observed in these cells [104].

3.5. Enhancement of SN-induced clastogenic effects by different compounds Like that of most DNA damaging agents, the effect of SN on the induction of chromosomal aberrations is potentiated by a number of compounds. Hydroxyurea, caffeine and 5-fluorodeoxyuridine were shown to increase the number of aberrations induced by the antibiotic in Vicia faba and human lymphocytes [36,105–108]. DNA substitution with halogenated deoxyuridine (BrdUrd and IdUrd) was also found to potentiate the chromosome damage induced by SN [109]. By exposing CHO cells to the antibiotic alone or in combination with halogenated compounds, Testoni et al. [109] were able to show that the combination of BrdUrd or IdUrd substitution plus SN treatments produces a very great increase in the frequency of breaks over the frequencies observed with the halogenated compounds or with SN only. The combined action of this radiomimetic drug and the halogenated compounds was found to be synergistic. Since SN has been shown to bind in the minor groove inducing a tightening of the DNA helical twist [75], Testoni et al. [109] postulated that the binding of this antibiotic to a substituted DNA increases the conformational changes due to the substitution, thereby enhancing the sensitivity of DNA to the active oxygen generated by SN. Several of the findings made by these authors [109] lend support to this hypothesis. Firstly, the synergistic clastogenesis was found to be more evident in bi- than in mono-substituted chromatids. Secondly, chromatid breaks showed a dose response in regard to the magnitude of DNA substitution. Thirdly, I, a halogen bulkier than Br, was more effective than Br in enhancing the sensitivity of DNA to SN. Continued studies on the type and frequency of chromosomal aberrations produced by SN coupled with molecular analysis of the mechanisms underlying the induction of DNA and chromosome damage by this compound will certainly enhance our understanding of the genotoxicity of this antibiotic.

4. Chromosome breakage and cytotoxicity It is likely that SN-induced cell killing is due to DNA double strand breakage, and the resulting loss of chromosome fragments, as previously suggested

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for ionizing radiations and bleomycin (see [110] for review). Evidence for DNA double strand breaks as the critical lesions in the cytotoxic effect of this antibiotic comes from studies with ataxia-telangiectasia (AT) cells. These cells are known to be sensitive to DNA-damaging agents which produce direct DNA double strand breaks, such as ionizing radiation and bleomycin. Cells from patients with AT were found to be about twice as sensitive as were normal cells in terms of survival and DNA double strand breakage [111]. The increased sensitivity of AT cells to SN was found to be fairly comparable to the sensitivity of these cells to ionizing radiation, bleomycin and neocarzinostatin [112,113]. The hypersensitivity of AT cells to SN was also observed by Shiloh et al. [32], and Taylor et al. [52] showed that despite the great sensitivity of AT cells to this compound, these cells can perform excision repair, as demonstrated by unscheduled DNA synthesis, at the same level as normal cells following exposure to the antibiotic. These results indicate that it is the DNA strand-breaking capacity of SN which is leading to cytotoxicity. In 1993, Jongmans et al. [114] studied the effects of radiomimetic agents on the radiosensitive Chinese hamster V79 cell mutants (V-C4, V-E5 and V-G8) which resemble human AT cells. The data on cell survival showed that all of these cell lines are hypersensitive to the agents tested, including SN (three-fold for V-G8 and V-C4, and 12-fold for V-E5). More recently, a Chinese hamster cell mutant (V-C8) sensitive to X-rays, alkylating agents, UV light and mitomycin C and which exhibits hampered DNA double strand break repair, was found to be also sensitive to SN (11-fold) and other radiomimetic compounds (bleomycin, hydrogen peroxide and etoposide) [115]. In summary, some indirect evidence suggests that DNA damage, and specifically double strand breakage, is involved in SN-induced cytotoxicity, although more studies are needed to confirm this assumption.

5. Mutagenesis 5.1. Bacterial assays To our knowledge, there are only two reports regarding the mutagenic properties of SN as determined by the Ames test [116,117].

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The genotoxicity of SN was tested in the Salmonella strains TA102 and TA104, strains where reversion to histidine prototrophy may occur by point mutation at an ochre codon. These strains are particularly useful for detecting mutagens that act preferentially at A:T basepairs, although mutations at G:C basepairs in suppressor tRNA genes are also recoverable. It was shown that SN induces basepair substitution mutation and that the mutational spectra characterized in Salmonella TA104 was predominated by AT → TA transversions [116,117]. It is worth noting that other oxidative mutagens such as bleomycin, X-rays and hydrogen peroxide also tested positive in TA102 strain [116,118]. SN was also found to be mutagenic in Pseudomonas aeruginosa mutants defective in ORFA, ORFB or ORFC genes, components of an operon implicated in the secretion of the siderophore pyoverdine [119]. 5.2. Mammalian and other eukaryotic cells The mutagenic effect of SN was also evaluated in mammalian and non-mammalian systems using other mutagenicity tests, although a survey of the literature reflects that little work has been done in mammalian systems. Hsie et al. [120,121] studied the mutagenicity and toxicity of physical and chemical agents in the CHO cell line K1-BH4 and its transformant, AS52, which lack the normal X-linked mammalian hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene but instead contain a single autosomally integrated copy of the bacterial equivalent, the xanthine-guanine phosphoribosyltransferase (gpt) gene. SN was found to induce a much higher frequency of 6-thioguanine-resistant mutants in the AS52 cell line than in CHO-K1-BH4 cells [120,121]. These authors postulated that the apparent hypermutability of AS52 cells probably results from a higher recovery of multi-locus deletion mutants in AS52 cells than in K1-BH4 cells, rather than a higher yield of induced mutants. In 1997, Sandhu and Birnboim [122] studied the induction of mutations by 137Cs radiation and radiomimetic drugs at the hprt locus of a murine tumor model (MN-11 cell line). At D0 (37% survival level), SN induced about 50 mutants per 105 viable cells, compared to 170 and 95 for radiation and bleomycin, respectively. Very recently, it has been reported that this antibiotic is positive in the Drosophila

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melanogaster “somatic mutation and recombination test” (SMART), using the Drosophila oregon K strain, which is known to be sensitive to chemical compounds that generate reactive oxygen species [123]. However, it was not determined whether the activity of SN in this assay is due to induced recombination or point mutation. Another recent study performed in V79-derived etoposide sensitive Chinese hamster cell mutants indicates that cellular responses to topoisomerase II inhibitors are complex and suggest that hypersensitivity to these kind of compounds may result from mutations in many different genes [124].

6. Conclusions Although SN is considered a radiomimetic compound, the results reported in the last 20 years regarding the genotoxic effects of this antibiotic have shown that there are important differences between this drug and ionizing radiation, despite the fact that both agents produce DNA and chromosome damage by liberation of free radicals. Even though SN acts on chromosomes mainly in an S-independent fashion, unlike ionizing radiations and classical radiomimetic compounds (such as bleomycin or neocarzinostatin) this antibiotic also acts in an S-dependent manner, producing mainly chromatid-type aberrations, even in the G1-phase and inducing SCEs [49,55–62,81,82]. In addition, SN has a persistent and delayed clastogenic effect [49,60,61]; the causes for this effect remain to be determined. The most plausible hypothesis is that it may be due to the formation and persistence of a SN–DNA complex which, through a continuous cyclic redox process, may keep generating free radicals that ultimately induce DNA damage [49]. Current and past studies have provided a large body of evidence indicating that SN directly interacts with DNA, binding covalently but not intercalating into the double helix and that this antibiotic produces its genotoxic effects through a mechanism that involves the generation of free radicals in the presence of metal ions such as iron, copper and zinc. Nevertheless, the scarcity of data regarding the mutagenic effects of this compound on mammalian and non-mammalian systems indicates that a more intensive work on this subject is needed. Although it has been reported that SN induces base substitutions in the Salmonella strains

TA102 and TA104, and multi-locus deletions in some mammalian cells [116,120,121], more detailed work on the mutagenic properties of this antibiotic remains to be done in order to determine the full spectra of SN-induced mutations and the mechanisms by which they are produced. While the induction of chromosomal aberrations by SN seems to be highly dependent on free radicals and metal ions [34,72], the mechanism of induction of SCEs by this drug remains to be fully elucidated. Although free radicals seem to play some role, present evidence indicates that the formation of SN–DNA complexes may be also involved [49,72]. From a cytogenetical point of view, it is clear that more studies are needed regarding the clastogenic effects of SN on mammalian cells. In particular, studies employing the FISH technique and combining the use of different types of DNA probes will provide more detailed information regarding the type, frequency and persistence of chromosomal aberrations induced by this radiomimetic compound as well as their mechanisms of induction, as previously reported by Natarajan et al. [125] and Deng and Lucas [126] for ionizing radiations. The resistance of insect cells to SN (and also to other mutagenic agents like ionizing radiations and bleomycin) is another point which merits further investigation. Although previous studies suggested that chromatin conformation and antioxidant defences present in the hemolymph of insects may be responsible for the high resistance of insect cells to SN as well as other mutagenic agents [62,99,102], the precise causes and mechanisms underlying this resistance remains to be determined. As mentioned in the introduction, despite SN’s antitumor activity against a broad range of tumors [2,7–12], its clinical use in cancer therapy is limited because it induces severe and prolonged bone marrow depression [13–18]. Although positive results have been reported for SN both as a single agent [19,20] and in combination therapy [21–23], the results reviewed show that in order to effectively use this antibiotic for clinical purposes more studies need to be done regarding its genotoxic effects and the factors that regulate them, especially in human cells. A critical unanswered question related to this subject is whether SN is carcinogenic. To our knowledge no data indicating SN-induced carcinogenesis have yet

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been reported. In summary, despite great advances in understanding the effects of this antibiotic on DNA and chromosomes, some important questions regarding its genotoxic effects remain to be answered.

Acknowledgements This work was supported by grants from CONICET and CIC of Argentina. The authors wish to thank Dr. Néstor Bianchi for critical reading of the manuscript and his helpful comments.

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