* Department of Pathology, New York Medical College, Valhalla, New York 10595; and
Department of Biology, Mount Holyoke College, South Hadley, Massachusetts 01075
Received January 9, 2002; accepted April 26, 2002
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ABSTRACT |
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Key Words: human topoisomerase II; fluoroquinolones; photogenotoxicity.
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INTRODUCTION |
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At high concentrations, some FQs have been reported to exhibit genotoxic effects in eukaryotic systems as a result of topoisomerase inhibition (Kohlbrenner et al., 1992; Robinson et al., 1991
). The FQs CP-67,804 and CP-115,953 were shown to induce topoisomerase II-mediated DNA cleavage by enhancing pre- and post-strand DNA breaks (Robinson et al., 1991
). Ciprofloxacin and CP-67,015 were also found to inhibit the catalytic DNA strand passage activity (Barrett et al., 1989
). Since FQs can associate with mammalian topoisomerases, it is possible that this interaction could be enhanced by UV irradiation. In support of this, the phototoxic effects of several FQs, including lomefloxacin, were almost completely inhibited in Chinese hamster V79 cells pretreated with sodium azide, which is reported to inactivate the catalytic activity of topoisomerase II (Ju et al., 2001
; Snyder and Cooper, 1999
). Based on this observation, it was proposed that UV-dependent toxicities of FQs involve a common mechanism of topoisomerase II-induced DNA double strand breaks (Snyder and Cooper, 1999
). However, sodium azide is not a specific inhibitor of topoisomerase II, since it has been shown to interfere with the mitochondrial electron transport chain depleting cells of ATP (Muneyuki et al., 1993
). To examine directly whether the photochemical genotoxicity of FQs involves topoisomerase II inhibition, the UV-dependent inhibition of human topoisomerase II
activity was measured in vitro for a spectrum of FQs with different UV-dependent genotoxicities (Jeffrey et al., 2000
; Spratt et al., 1999
). While one potent photogenotoxic FQ induced UV-mediated inhibition of topoisomerase II
, another did not, indicating that topoisomerase inhibition does not appear to be the only mechanism of FQ photogenotoxicity.
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MATERIALS AND METHODS |
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To confirm whether plasmid cleavage involved the inhibition of the topoisomerase II or a UVA-mediated reaction independent from the enzyme, inhibition assays were conducted in the absence of enzyme, ATP, or magnesium. Enzyme assays were also performed in the presence of 100 µM TEMPO or under anaerobic conditions using a nitrogen atmosphere to examine whether the generation of reactive oxygen species participated in the UVA-FQinduced topoisomerase inhibition.
Religation of cleaved pBR322.
DNA religation was followed using the heat-induced religation protocol described by Robinson et al.(1991). Briefly, DNA cleavage/religation equilibrium was established as described above in the presence of 100 µM y3118 dosed with 360 mJ/cm2 UVA. Following UVA exposure, the DNA:topoisomerase complexes were trapped with 0.8 µl of 250 mM EDTA. Following the addition of 0.6 µl of 5 M NaCl, the samples were shifted from 37° to 55°C, and the reaction was stopped at various time points by the addition of 2 µl of 10% SDS. The samples were extracted once with phenol:chloroform (1:1), ethanol-precipitated, and electrophoresed in a 1% agarose gel. The gels were stained with ethidium bromide, and densitometry of the reaction products was analyzed using the AlphaImager system.
Binding of radiolabelled FQs to pBR322 and topoisomerase II.
To examine the degree of association of photoactivated fluoroquinolones to DNA or DNA:topoisomerase complexes, [14C] y3118 (3.52 MBq/mg) or [14C] Mox (2.94 MBq/mg) were incubated in cleavage buffer containing pBR322 alone or pBR322 plus human topoisomerase II, and irradiated with 360 mJ/cm2 of UVA. Following UVA exposure, the reaction mixture was extracted with phenol:chloroform (1:1), and the DNA was precipitated in 2.5 volumes of ethanol. The DNA pellet was rinsed twice in 70% ethanol, resuspended in 0.1% SDS, and radioactivity finally measured using a Wallac scintillation counter (Perkin Elmer, Gaithersburg, MD).
[14C]-Labeled y3118 and Mox were also incubated in cleavage buffer at 37°C for 30 min with 20 picograms of human topoisomerase II, alone in the dark or under 360 mJ/cm2 UVA. Following the incubation period, the topoisomerase enzyme was immunoprecipitated using a polyclonal topoisomerase II
antibody. Briefly, the reaction mixture was incubated for 1 h at 4°C with 20 picograms of antibody. Following the incubation with primary antibody, 10% protein A-agarose beads were added to the samples and the mixture was incubated at 4°C with rocking for 1 h. The antigen-antibody complexes, bound to the protein A-agarose beads, were centrifuged at 10,000 x g for 5 min at 4°C. The supernatant was discarded and the pellets were resuspended 3 times in RIPA buffer (150 nM NaCl, 1% NP40, 0.1% SDS, and 50 mM TrisHCl pH 8.0) and centrifuged at 10,000 x g. Following the final wash, the pellets were resuspended in 0.1% SDS and boiled for 10 min, then radioactivity was measured using a Wallac scintillation counter.
Statistics.
Samples from unirradiated and irradiated groups were compared by analysis of variance (ANOVA). Unless specified, the data represent the mean ± SEM of samples obtained from three experiments.
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RESULTS |
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The pBR322 plasmid preparation utilized in these studies contained 2 plasmid states, supercoiled (closed circular) and relaxed nicked (open circular) double stranded DNA. The closed circular DNA migrates faster in agarose gel than the open circular DNA, as shown in Fig. 2. Topoisomerases catalyze the relaxation of supercoiled DNA through the transient cleavage of DNA, mediated by covalent binding between the enzyme and DNA followed by DNA religation and dissociation of the topoisomerase (Berger, 1998
; Burden et al., 1998; Ferguson et al., 1994; Jacob et al., 2001
; Nitiss, 1998
). In the presence of topoisomerase II
, an equilibrium between the open circular and closed circular plasmids was established (Fig. 2
). When treated with the topoisomerase II
inhibitor etoposide, the half-life of the cleaved DNA is lengthened by preventing religation (Ferguson and Baguley, 1994
). The cleaved DNA can then be identified in gels as a band that migrates between the open circular and closed circular DNA, which consists of linearized plasmid (Fig. 2
).
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DISCUSSION |
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To examine the mechanism by which photoactivated FQs inhibit the human topoisomerase II enzyme, studies were conducted using the photoactive y3118, which strongly inhibited topoisomerase II
in the presence of UVA. Irradiation of y3118, DNA or topoisomerase alone or combinations of y3118:DNA or y3118:topoisomerase yielded no DNA double strand breaks. DNA double strand breaks were only observed when y3118, DNA, and topoisomerase II
were simultaneously present in the enzyme assay. This suggests that photoactivated y3118 could be exerting its inhibitory effects by forming a ternary FQ-enzyme-DNA complex requiring ATP, as has been described for etoposide and m-AMSA in the absence of UV irradiation (Anderson et al., 1994; Nelson et al., 1984
; Robinson et al., 1990). Quinolone-induced inactivation of topoisomerase II
at high concentrations was proposed to involve the direct binding of quinolones to DNA and not to the enzyme (Shen et al., 1985, 1989; Tornaletti and Pedrini, 1988
). Such binding of FQs to DNA was proposed to inhibit topoisomerase II
activity resulting in DNA damage (Shen and Pernet, 1985
; Tornaletti and Pedrini, 1988
). In contrast, our studies using radiolabeled Mox and y3118 revealed that both FQs are capable of associating with both the enzyme and the DNA. A nonsignificant association of Mox or y3118 with DNA or the human topoisomerase II
incubated individually in the presence of these FQs was observed in the absence of UVA. However, in the presence of UVA, the association of the photostable FQ Mox and the highly photoreactive FQ y3118 to the DNA:topoisomerase complexes was increased 2-fold and 100-fold, respectively. This finding suggests that the degree of association of photoactive FQs to DNA:topoisomerase complexes could determine the degree of DNA damage, as proposed previously for other topoisomerase II poisons (Snyder and Cooper, 1999
).
The DNA cleavage induced by photoactivated y3118 was irreversible, in contrast to studies performed by Robinson et al. using Drosophila melanogaster topoisomerase II and high concentrations of the FQs CP-115,953 and CP-67,804 without irradiation (Robinson et al., 1991). It is likely that the inability of topoisomerase II
to religate the double strand breaks may result from the photomediated increase in binding of y3118 to both the topoisomerase II
and the DNA. Extensive binding of y3118 not only could prevent topoisomerase II
from recognizing and ligating DNA cleaved sites but could also induce a conformational change of the enzyme leading to its inactivation. Most likely, the binding of FQs to the DNA:topoisomerase complexes could result in the stabilization of the "cleavable complex," as described for a number of topoisomerase II poisons (Burden et al., 1999; Boos and Stopper, 2000
; Corbett and Osheroff, 1993
; Nelson et al., 1984
; Robinson and Osheroff, 1990
)
UVA irradiation of FQs results in the formation of reactive oxygen species, and this effect has been proposed as a possible mechanism involved in FQ photochemical toxicity (Martínez et al., 1998; Robertson et al., 1991
; Umezawa et al., 1997
) and genotoxicity (Rosen et al., 1996
; Spratt et al., 1999
). Our experiments, using the antioxidant TEMPO or conducted under a nitrogen atmosphere, excluded the involvement of this mechanism in topoisomerase inhibition and DNA cleavage by y3118. Moreover, in previous experiments using high fluences of UVA in which oxidative damage is produced, Lmx was more efficient than y3118 in producing DNA strand breaks in pBR322 (Spratt et al., 1999
), in contrast to the greater inhibitory activity of y3118 in the present experiments. Nevertheless, the overall photochemical genotoxicity of a particular FQ may involve DNA oxidation as well as inhibition of topoisomerase II.
In summary, the photochemical genotoxic effects of some, but not all FQs could be attributed to their capacity to inhibit the topoisomerase II enzyme with UVA irradiation. Thus, in the elucidation of the overall mechanism of photochemical genotoxicity, investigation of DNA binding, oxidative DNA damage, and inhibition of topoisomerases and possibly other enzymes, such as DNA repair enzymes, will be required.
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ACKNOWLEDGMENTS |
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NOTES |
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