This is unlikely as hemicatenane resolution can equally occur towards the inside of the DNA ring, generating a genuine catenane. The same molecular forms were also modified or lost by Top2 knockdown and inhibition by ciprofloxacin Figure 2A and Supplementary Figure S3A. Ciprofloxacin caused a dramatic effect on mtDNA topology, blocking replication initiation, reducing copy number and inhibiting mitochondrial transcription Figures 2B , 3A — E and 4A.
Its toxicity to mitochondria has been reported in various studies, suggesting a broad range of mechanisms including topoisomerase inhibition, oxidative stress, altered calcium handling and photosensitization 38— In our study, we observed ciprofloxacin to clearly reduce Top2 topoisomerase activity both in vitro and in vivo , but did not find any indication of increased mtDNA double-strand breaks Figure 3A — C.
As our detection method long-range PCR does not distinguish between strand-breaks, abasic sites or base alterations inhibiting Taq polymerase, the observed effect might be caused by oxidative damage, which fluoroquinolones have been reported to induce in a variety of cell types 41 , Interestingly, while doxorubicin, another known type II topoisomerase inhibitor, was inhibiting Top2 function stronger in vitro Supplementary Figure S2b-c , its effects on mtDNA replication in vivo were weaker Figure 3A , suggesting that ciprofloxacin specifically accumulates in mitochondria or its activity might be enhanced by metabolic alterations in vivo.
As ethidium bromide did not interfere with mtDNA topology at all in vivo Figure 3A , it can be concluded that DNA intercalation alone is not sufficient to inhibit Top2. As of note, each drug, ethidium bromide, ciprofloxacin and doxorubicin improved the resolution of the different mtDNA forms on gel electrophoresis, probably due the inhibition of mitochondrial transcription, resulting in the clearance of heterogeneous RNA forms from the preparations. These supercoils migrate slower and are less uniform in appearance than the supercoils present in untreated or ethidium bromide treated cells.
Ethidium bromide is known to induce negative supercoiling of DNA due to its intercalation between the two strands of the double helix, causing twisting of the molecule In bacteria supercoiling is an important genomic regulator, where negative supercoils are relaxed by the action of Topoisomerase I, while positive supercoils are relaxed by the type II topoisomerase DNA gyrase Although eukaryotic Top1 and Top1mt as well as Top2 can relax both negative and positive supercoils, mitochondria appear to possess a similar functional specialization as bacteria, as the loss of Top1mt specifically results in accumulation of negatively supercoiled mtDNA 18 , while we here show an accumulation of positively supercoiled mtDNA upon inhibition of Top2 Figure 3B.
In bacterial systems negative supercoiling is required for replication origin recognition 45 , 46 , with the binding of IHF or HU nucleoid proteins activating transcription and consequently replication origin activation. If relaxation is required for mtDNA replication initiation, while negative supercoiling favours transcription 51 , this could explain the loss of replication intermediates and 7S DNA in ciprofloxacin treated cells having positive supercoiled mtDNA Figure 4 and Supplementary Figure S5.
Both ciprofloxacin and doxorubicin also impaired mitochondrial transcription Figure 3D , although less efficiently than ethidium bromide. Ciprofloxacin did not only inhibit mtDNA synthesis, but also affected cellular growth and differentiation. The retardation of cell division was immediate and only present in cells with functional mitochondria, implying a retrograde signal from mitochondria to nucleus either caused by oxidative stress or impaired mtDNA replication.
Similarly, the differentiation of myoblasts to multinucleated muscle fibres was heavily impaired upon inhibition of mitochondrial, but not nuclear Top2 Figure 4E — F. This differentiation process does not require cellular proliferation, but is dependent on mitochondrial function 52 , further supporting a signalling connection between mitochondria and nucleus.
The severe side effects of ciprofloxacin and other fluoroquinolones include tendinopathies such as tendon rupture, joint inflammation, muscle weakness, central and peripheral neuropathies, epilepsy and psychological symptoms such as depression.
These symptoms have been proposed to be connected to enhanced oxidative stress 42 , 54 , 55 , but the molecular mechanism remained unclear. The reduction of mtDNA copy number and mitochondrial transcription caused by the altered topology of mtDNA might result in severe dysregulation of the electron transport chain complexes, as known to occur under ciprofloxacin treatment 56 , lead to respiratory chain dysfunction and cause the observed enhanced oxidative stress.
Ciprofloxacin has also been reported to interfere with physiologically significant cell differentiation processes, such as spermatogenesis 57 , brain development 41 , bone mineralization 58 , as well as to induce renal toxicity and heart arrhythmia While the molecular mechanisms of these adverse effects are yet unclear, mitochondria play a central role in all of these physiological processes, making mitochondrial impairment a likely culprit for the disturbed cellular physiology.
As a conclusion, the maintenance of topological homeostasis in mitochondria is required for gene expression and replication of mtDNA as it is for other genomes. Similar to Top1mt, Top2 regulates mtDNA supercoiling, and their division of labor could be linked to differential association of these proteins between transcriptionally active versus replicating nucleoids.
Although central in bacterial genome maintenance, the whole phenomena of DNA supercoiling and its functional implications are virtually unstudied in mitochondria and calls for future research. Our results identified mtDNA as a key target for ciprofloxacin-induced adverse effects through inhibition of Top2. As fluoroquinolone antibiotics are widely used and effective drugs against a number of important bacterial pathogens, their dosage, systemic enrichment and side-effects should be reviewed in the mitochondrial context, and their clinical use should be considered with great care.
We thank Dr Stefan Sobek and Prof. Author Contributions : All experiments were conducted by A. Academy of Finland [, to S. Pohjoismaki J. Of circles, forks and humanity: Topological organisation and replication of mammalian mitochondrial DNA. Google Scholar. Human heart mitochondrial DNA is organized in complex catenated networks containing abundant four-way junctions and replication forks. Alterations to the expression level of mitochondrial transcription factor A, TFAM, modify the mode of mitochondrial DNA replication in cultured human cells.
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Gonzalez R. Effects of conditional depletion of topoisomerase II on cell cycle progression in mammalian cells. Cell Cycle. Linka R. C-terminal regions of topoisomerase IIalpha and IIbeta determine isoform-specific functioning of the enzymes in vivo. Thakurela S. Gene regulation and priming by topoisomerase IIalpha in embryonic stem cells.
Haffner M. Androgen-induced TOP2B-mediated double-strand breaks and prostate cancer gene rearrangements. Tiwari V. Target genes of Topoisomerase IIbeta regulate neuronal survival and are defined by their chromatin state. Dorman C. DNA supercoiling is a fundamental regulatory principle in the control of bacterial gene expression. Pommier Y. Roles of eukaryotic topoisomerases in transcription, replication and genomic stability. Cell Biol. Dalla Rosa I. Adaptation of topoisomerase I paralogs to nuclear and mitochondrial DNA.
Zhang H. Negative regulation of mitochondrial transcription by mitochondrial topoisomerase I. Wang Y. Tsai H. Drosophila mitochondrial topoisomerase III alpha affects the aging process via maintenance of mitochondrial function and genome integrity.
Nicholls T. Topoisomerase 3alpha is required for decatenation and segregation of human mtDNA. Morham S. Targeted disruption of the mouse topoisomerase I gene by camptothecin selection.
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A variety of amino acid modifiers were initially tested. A Inhibition of helicase activity. The discontinuities in these gel images, denoted by a vertical line between treatments 8 and 9, indicates the location where an irrelevant treatment in the assay was electronically removed.
C The small molecules have variable effects on ssDNA binding. Filter binding assays were conducted as described in the Experimental Procedures section using nM of the indicated helicase. D Small molecule inhibition of helicase ATPase activity. ATPase activity was assayed as described in the Experimental Procedures section using nM final helicase hexamer concentration.
The treatment numbering in C and D are identical to those in A. The inhibitor effects of either ofloxacin A or ciprofloxacin B were tested on the DNA unwinding activity; the indicated helicases were all used at a final concentration of nM of complexes following preincubation with inhibitor. The effects of several previously identified helicase inhibitors were also examined.
The pyrimidinone—peptoid hybrid molecule MALb and the fluoroquinolones ofloxacin and ciprofloxacin have been previously reported to inhibit various TAg-mediated activities [ 10 , 11 ].
MALb inhibited all three helicases to a similar extent at 1 mM Figure 1 A, treatment 8 , but little or no inhibition of TAg helicase activity was observed with 1 mM ciprofloxacin or ofloxacin Figure 1 A, treatments 9 and 10; however, inhibition was observed at higher concentrations, see below. In contrast, 1 mM ciprofloxacin inhibited the helicase activity of both Mcm and Mcm Figure 1 A, treatment Because TAg subunits oligomerize only in the presence of ATP [ 23 ], and ATP preincubation probably causes a conformational change in Mcm [ 16 , 19 ], we also tested the effects of the potential inhibitors after the proteins were preincubated with ATP Figure 1 B.
Although this treatment had essentially no effect on either Mcm complex, it completely or partially protected TAg from all modifiers except Nbf Figure 1 B, treatment 6 and MALb Figure 1 B, treatment 8 , suggesting that at least one effect of the other inhibitors may be to block TAg oligomerization. Because helicase activity depends on ATP hydrolysis and ssDNA binding, the effects of the chemical modifiers and small molecules on both activities were examined.
Using previously established steady-state ATP hydrolysis [ 17 ] and ssDNA filter-binding [ 16 ] assays, the effect of the same panel of small molecules on each of the three helicases was examined.
With the exception of DCCD and ofloxacin, which failed to inhibit helicase activity, most of the remaining treatments severely inhibited the ATPase activities of all three helicases Figure 1 C. These data suggest that the inhibition of DNA unwinding is mediated by compromised function of one or several ATPase active sites.
Ciprofloxacin stands in sharp contrast: even though it completely inhibited Mcm helicase activity, it had only modest effects on ATP hydrolysis and ssDNA binding of the three helicases Figures 1 C and 1 D, treatment Together, these results suggest that ciprofloxacin inhibits a step or steps specifically required for DNA unwinding, possibly through selective inhibition of the Mcm regulatory subunits. This possibility is explored further below. We found that very high concentrations of ofloxacin inhibited both Mcm and Mcm with similar IC 50 s Figure 2 A : 4.
In contrast naladixic acid, the parent quinolone compound for both ciprofloxacin and ofloxacin, had essentially no effect on the activities of the three helicases at any concentration tested results not shown. This selectivity of ciprofloxacin for Mcm relative to TAg supports the proposal that Mcm-specific inhibitors may be found.
In addition, the selectivity of ciprofloxacin for Mcm relative to Mcm supports the proposal that active site-specific inhibitors of the Mcm complex can be identified. We reasoned that other fluoro quinolone derivatives might show enhanced Mcm specificity at potentially lower inhibitor concentrations.
As the fluoroquinolones are used as antibiotics reviewed in [ 24 ] , prior drug discovery efforts have resulted in the synthesis of chemically diverse libraries modeled on key elements found in the basic fluoroquinolone scaffold. Therefore we investigated a compound chemical library that contained either fluoro quinolone derivatives or molecules with various substructures found in ciprofloxacin and other marketed quinolones. Both fluoro quinolone and triaminotriazine-like inhibitors were identified.
Although a wide range of results were obtained, two general conclusions emerged from the data Supplementary Table S1 : 1 Few molecules exhibited robust inhibition of TAg, and those that did e.
Interestingly, although some of the inhibitors appeared to inhibit both Mcm and Mcm, the relative strength of this inhibition varied. One agent appeared to act like ciprofloxacin and preferentially inhibited Mcm , whereas others appear to preferentially inhibit Mcm e. Based on their differential inhibition of the three helicases, the inhibitors were classified into one of two groups:. Inhibitors that had approximately equal effects on all three helicases include MALb Figure 1 A and compounds , , and Table 1.
Interestingly, unlike any of the fluoro quinolones characterized, the triazole and the structurally related compound were more effective at inhibiting TAg than either Mcm complex Table 1. Two inhibitors and fall into this category. Although the limited solubility of prevented us from testing higher concentrations, we can conclude that the IC 50 against TAg is at least an order of magnitude greater than that of the Mcm complexes.
In contrast, preferentially inhibited Mcm relative to Mcm but had little effect on TAg. As noted above, DNA unwinding is the culmination of a variety of simpler biochemical activities. Thus, the seven representative inhibitors and ciprofloxacin may function by physically interacting with the helicase, the DNA substrate, or the ATP. To understand how all eight inhibitors block helicase activity, their effects on steady-state ATP hydrolysis were measured Figure 3 A.
Treatment order for each panel: 0, solvent control; 1, compound ; 2, ; 3, MALb; 4, ; 5, ; 6, ; 7, ; and 8, ciprofloxacin. This experiment was identical to that shown in Figure 1 C with the indicated helicase complexes used at nM concentration, but 1 mM of the indicated inhibitor was added prior to ATP addition. ATP and the indicated inhibitor were added together to Mcm without preincubation. C Ability of inhibitors to intercalate into DNA. After 1 h of Topo I treatment, 1mM of the indicated inhibitor was added and samples were incubated for an additional 1 h D Topo I activity inhibition assay.
This experiment was identical to C , except that Topo I and the indicated inhibitor were added at the same time. Topo I inhibition is indicated if addition of both inhibitor and topoisomerase together generates supercoiled DNA, while experiments shown in C generate relaxed plasmid.
E An intercalation assay performed with the indicated inhibitors at lower concentrations. These assays were similar to C Topo I added first, and the inhibitor added second , except the indicated concentration of inhibitor was used.
These results suggest one of three possible scenarios: First, the inhibitors with the possible exception of MALb might not target the ATPase active sites. Secondly, the inhibitors may deregulate or uncouple the activity of the enzyme rather than block ATP hydrolysis.
Thirdly, at least in the case of the Mcm complex, the inhibitors could preferentially target the ATPase active sites but are selective for the low-turnover regulatory sites.
Although the second and third possibilities are difficult to distinguish, the first explanation can be tested. Although we cannot rigorously test for competitive inhibition using our helicase endpoint assay, we can test if increased ATP concentration overcomes the inhibitory effects of these compounds Figure 3 B. Although doubling the ATP concentration in the absence of inhibitor caused a slight increase in helicase activity 1.
These results suggest that the inhibitors disrupt ATPase active sites in the Mcm complex in some manner. In contrast the inhibitory effects of , MALb, and could not be rescued by an increase in ATP concentration Figure 3 B, treatments , suggesting that these inhibitors operate independently of the ATPase active sites. Because these compounds are also planar double ring molecules, they could conceivably inhibit helicase activity via DNA intercalation.
To examine this model, we tested our inhibitors in a standard topoisomerase assay [ 21 ]. The rationale of this assay is that intercalating compounds will introduce supercoils into a fully relaxed plasmid.
Topo I will remove these introduced supercoils, but after quenching and gel electrophoresis the intercalator will diffuse away and produce a detectable compensatory supercoiling increase. Following plasmid relaxation, each inhibitor was added to 1 mM final concentration in the topoisomerase assay Figure 3 C, treatments 1—8.
The general inhibitors treatment 1 , treatment 2 , treatment 4 and treatment 5 cause extensive DNA intercalation, while in contrast, MALb treatment 3 and the more Mcm-selective inhibitors , and ciprofloxacin, treatments demonstrated little or no intercalation Figure 3 C. However, lack of apparent intercalation could also be caused by Topo I inhibition.
To test this possibility, the assay was repeated under conditions in which Topo I and each inhibitor were added to the reaction at the same time. Under these conditions, Topo I inhibition will only yield supercoiled plasmids Figure 3 D. The reasons for these side effects are yet unclear. A study carried out at the University of Eastern Finland and published in Nucleic Acids Research investigated the effect of ciprofloxacin on mitochondria, the important cell organelles in our body that produce the energy for cellular function.
Mitochondria possess their own small circular genome, which requires topoisomerase enzymes for its maintenance. Topoisomerases regulate the topology of DNA and untangle for instance knots and overwound stretches of a genome by cutting and reconnecting the DNA sequence. While fluoroquinolones are designed to inhibit the bacterial topoisomerase gyrase, which leads to the death of the bacterium, they also inhibit the topoisomerase 2 of our own cells. Ciprofloxacin stopped this normal maintenance and transcription of mitochondrial DNA by changing mtDNA topology, causing impaired mitochondrial energy production and blocking cellular growth and differentiation.
This dramatic impact on mitochondrial DNA is the likely cause for most negative side effects experienced by patients, and also a reason to use fluoroquinolone antibiotics with great caution. These are most commonly seen as pneumonia, bronchitis, and influenza and are characterized by shortness of breath, weakness, coughing, high fever, and fatigue. LRTI's can affect people of any age, but are most commonly seen in the pediatric and geriatric patient populations due to their propensity for less-than-optimal functioning immune systems.
Bacterial pneumonia should be treated with antibiotics; the selection of which is guided by the characteristics and type of bacteria causing the disease. A 68 year-old male presented to the urgent care unit with symptoms of shortness of breath, wheezing, cough, discolored sputum, and a fever. After microbiologic tests were performed to determine the pathogen as streptococcus pneumoniae, the physician wrote a prescription for oral ciprofloxacin, mg every 12 hours for 14 days.
One week later the patient returned to the urgent care unit with symptoms of severe drowsiness, fatigue, and low blood pressure. During a profile review, the pharmacist noted the concomitant use of tizanidine, as needed for muscle spasms. The pharmacist informed the physician that the patient's tizanidine may be interacting with the ciprofloxacin and was likely the causation of the patient's symptoms.
Pharmacologically, the concomitant use of ciprofloxacin with tizanidine may increase the serum concentration of tizanidine. Ciprofloxacin is an inhibitor of CYP1A2, and decreases its ability to perform the necessary metabolic functions for breakdown of other compounds in the body.
When CYP1A2 cannot exert its optimal activity, tizanidine cannot be metabolized as quickly, leading to a higher serum concentration of the drug. Consequently, this may lead to adverse drug reactions in the patient such as those seen in this case. Topoisomerase II is an enzyme that reduces the amount of supercoiling of the DNA double-stranded helix during the replication process, while the unlinking of the two daughter strands of DNA is a result of topoisomerase IV.
Therefore, Ciprofloxacin has bactericidal and bacteriostatic properties against both Gram-negative and Gram-positive bacterial pathogens.
Common Gram-negative pathogens that quinolones work against include Haemophilus, Salmonella, Pseudomonas and Enterobacter; Gram-positive infections include Staphylococci and Streptococci.
The action of ciprofloxacin is concentration-dependent; at low concentrations it reduces only the action of topoisomerase II. However, if it is given at higher doses, it is also able to inhibit topoisomerase IV. If the infection is caused by Gram-negative bacteria, then topoisomerase II becomes the primary target for the drug; if the pathogen is a Gram-positive bacterium, the primary target becomes topoisomerase IV.
In the case of Gram-negative organisms, ciprofloxacin exerts a long post-antibiotic effect PAE , meaning the drug remains active even after the patient stops taking it. Cipro 1-cyclopropylfluoro-1,4-dihydrooxopiperzino-quinolinecarboxylic acid began clinical trials in the early s and received patent approval in The original patent awarded to the drug company, Bayer AG, expired in Ciprofloxacin is classified as a quinolone antibiotic; a class of broad-spectrum anti-bacterial drugs.
Generally, these antibiotics selectively target topoisomerases, enzymes responsible for reducing the supercoiling of the DNA helix prior to replication. Quinolone interaction with DNA was primarily thought to be through Van der Waals forces and p-p stacking.
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