Quinolone antibacterials: a new class of photochemical carcinogens.

Ciprofloxacin-induced DNA damage in primary culture of rat astrocytes

A list of genes that are affected by quinolones, genes that interact with quinolones, which organisms that this quinlone-gene interaction been studied and a list of which cellular functions (GO terms) that are adversely affected by the quinolones.

Ciprofloxacin Induces an Immunomodulatory Stress Response in Human T Lymphocytes
Kristian Riesbeck,* Arne Forsgren, Agnethe Henriksson, and Anders Bredberg
Department of Medical Microbiology, Lund University, Malmö University Hospital, S-205 02 Malmö, Sweden
"Ciprofloxacin and other quinolones at >20 µg/ml inhibit peripheral blood lymphocyte (PBL) cell growth by 30 to 35%, causing impaired cell cycle progression through the S phase (8). Cell cycle analysis thus indicates DNA synthesis to be inhibited by fluoroquinolones at these concentrations."  

Mechanistic Study on Flumequine Hepatocarcinogenicity Focusing on DNA Damage in Mice
Yoko Kashida*, Yu F. Sasaki, Koh-ichi Ohsawa, Natsue Yokohama, Akiko Takahashi*, Takao Watanabe* and Kunitoshi Mitsumori
"... the results of the present study provide evidence that FL was not only a hepatic tumor promoter but also a hepatic tumor initiator."

Trovafloxacin, a quinolone antibiotic, is an idiosyncratic hepatotoxin. Further analysis revealed unique regulation of 142 genes, including some involved in RNA transcription.

Several gene programs are induced in ciprofloxacin-treated human lymphocytes as revealed by microarray analysis
Emily Eriksson, Arne Forsgren and Kristian Riesbeck
Department of Medical Microbiology, Lund University, Malmö University Hospital, Sweden "We conclude that the fluoroquinolone ciprofloxacin at high concentrations interferes with several gene programs, which is in accordance with a mammalian stress response."

Study of potential in vitro and in vivo genotoxicity in hepatocytes of quinolone antibiotics. McQueen CA, Way BM, Queener SM, Schluter G, Williams GM. American Health Foundation, Valhalla, New York 10595. 

4-Quinolone drugs affect cell cycle progression and function of human lymphocytes in vitro.

4-Quinolone antibiotics: positive genotoxic screening tests despite an apparent lack of mutation induction.

Studies on the interaction of 4-quinolones with DNA by DNA unwinding experiments.

Damage to mitochondrial DNA induced by the quinolone (Bay y 3118)  in embryonic turkey liver.

Journal of Antimicrobial Chemotherapy, Vol 17, 811-814, Copyright © 1986 by The British Society for Antimicrobial Chemotherapy
Influence of ofloxacin, norfloxacin, nalidixic acid, pyromidic acid and pipemidic acid on human gamma-interferon production and blastogenesis
C De Simone, L Baldinelli, M Ferrazzi, S De Santis, L Pugnaloni and F Sorice
"Several new quinolone derivatives were investigated for their influence on human lymphocyte blastogenesis and gamma-interferon production following concanavalin A stimulation. All the antimicrobials induced inhibition of lymphocyte DNA synthesis. The gamma-interferon measurements showed that nalidixic acid and norfloxacin have a negative influence on lymphokine production and release. "
 

"Nevertheless, some quinolones cause injury to the chromosome of eukaryotic cells.21,22 These findings prompted us to optimize the substituent at C-3, by..."
Gootz, T. D.; Barrett, J. F.; Sutcliffe, J. A.; Antimicrob. Agents
Chemother. 1990, 34, 8.

Rusquet, R., M. Bonhommet, and J. C. David. 1984. Quinolone
antibiotics inhibit eucaryotic DNA polymerase alpha and beta,
terminal deoxynucleotidyl transferase but not DNA ligase.
Biochem. Biophys. Res. Commun. 121:762-769.

Castora, F. J., and M. V. Simpson. 1979. Search for a DNA
gyrase in mammalian mitochondria. J. Biol. Chem. 254:11193-
11195.

Miller, K. G., L. F. Liu, and P. T. Englund. 1981. A homogeneous
type II DNA topoisomerase from HeLa cell nuclei. J.
Biol. Chem. 256:9334-9339.

Hussy, P., G. Maass, B. Tummler, F. Grosse, and U. Schomburg.
1986. Effect of 4-quinolones and novobiocin on calf thymus
DNA polymerase a primase complex, topoisomerases I and II,
and growth of mammalian lymphoblasts. Antimicrob. Agents
Chemother. 29:1073-1078.
 

Follow this link to view the original PDF file:

Inhibitory Effects of Quinolone Antibacterial Agents on Eucaryotic
Topoisomerases and Related Test Systems

The following is the text translation of the above PDF file:

Vol. 34, No. 1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1990, p. 8-12
0066-4804/90/010008-05$02.00/0
Copyright © 1990, American Society for Microbiology
Inhibitory Effects of Quinolone Antibacterial Agents on Eucaryotic
Topoisomerases and Related Test Systems
THOMAS D. GOOTZ,* JOHN F. BARRETT,t AND JOYCE A. SUTCLIFFE
Department of Immunology and Infectious Diseases, Pfizer Central Research,
Eastern Point Road, Groton, Connecticut 06340
 

INTRODUCTION
Two earlier reviews discussed the biochemical characteristics
and physiological importance of topoisomerases (3,
46). Details of various laboratory assays measuring the
effects of quinolones on both procaryotic and eucaryotic
topoisomerases were also reviewed. Biochemical and genetic
studies have linked the antibacterial effect of quinolones
to inhibition of the A subunit of DNA gyrase (46).
Much of the knowledge that has been gained about bacterial
gyrase as a target for quinolones has been used in attempts to
discover novel agents with improved antibacterial activity
(10, 15, 19, 28, 29, 43). Assays measuring the DNA supercoiling
and cleavage activity of bacterial gyrase in the
presence of quinolones have been used to find quinolones
with greater inhibitory activity against this enzyme (10).
Although it is generally accepted that quinolone antibacterial
agents are selective for DNA gyrase, there are relatively few
reports investigating the potential effects of these agents
against enzymes involved with eucaryotic DNA replication
and those maintaining DNA topology in cells. In addition,
there are reports of in vitro cytogenetic abnormalities associated
with some quinolones as well as inhibitory effects on
DNA replication in lymphocytes (21, 24). The purpose of
this review is to analyze some of the reported effects of
quinolones in these test systems and to examine the evidence
for potential quinolone involvement with eucaryotic
topoisomerases.
 

REPORTED EFFECTS OF QUINOLONES ON
EUCARYOTIC TOPOISOMERASES AND DNA
POLYMERASE * Corresponding author.
Present address: The R. W. Johnson Pharmaceutical Research
Institute (Ortho Division), Raritan, NJ 08869-0602.

Although the current quinolones are not considered to be
potent inhibitors of eucaryotic topoisomerases, some effects
on these and other enzymes involved with DNA replication
have been observed (7, 37, 42). The laboratory assay methods
used to measure the activity of topoisomerases have
been reviewed elsewhere (3). Miller et al. found that nalidixic
and oxolinic acids had 50% inhibitory concentrations
(IC50s) of 500 and 100 ,ug/ml, respectively, for topoisomerase
II isolated from Hela cell nuclei in a decatenation activity
assay (37). A similar degree of inhibition was found against
topoisomerase II isolated from calf thymus nuclei by Hussy
et al. (27). These investigators determined IC50s in the
catenation reaction for ciprofloxacin, norfloxacin, ofloxacin,
and nalidixic acid of 150, 300, 1,300, and 1,000 ,ug/ml,
respectively (27). Such studies have also indicated that there
is no correlation between the potency of quinolone inhibition
of bacterial DNA gyrase and their relative inhibition of
eucaryotic topoisomerase II (27). Using an assay for mea-
suring the relaxation activity of topoisomerase II isolated
from Drosophila melanogaster nuclei, Osheroff et al. found
K,s for nalidixic and oxolinic acids of 625 and 340 ,ug/ml,
respectively (41). These relatively high drug levels required
for inhibition may explain the failure of previous studies to
demonstrate inhibition of D. melanogaster and rat liver
topoisomerase II with low levels of nalidixic acid analogs
(16, 26, 27, 41). In contrast, coumermycin Al and novobiocin,
two DNA gyrase B-subunit inhibitors that act at the
ATP-binding site of DNA gyrase, were found to be 10- to
100-fold more potent inhibitors of eucaryotic topoisomerase
II relaxation activity in vitro than were quinolones (41). This
result is consistent with the observation that the sequence
homology at the ATP-binding sites between eucaryotic and
procaryotic type II topoisomerases is greater than that
observed in the corresponding consensus cleavage sites of
the enzymes, where quinolones exert their effects against
DNA gyrase (46, 49).
A limited number of studies that evaluate the interactions
of quinolones with eucaryotic topoisomerase I are available.
Quinolones have been shown to be less inhibitory for Escherichia
coli topoisomerase I than for DNA gyrase. Tabary et
al. (47) found that the IC50s of pefloxacin, ciprofloxacin,
norfloxacin, and ofloxacin against procaryotic topoisomerase
I ranged between 35 and 50 ,g/ml in the relaxation assay.
This level of relaxation inhibition was approximately 10-fold
lower than what was observed against DNA gyrase supercoiling
activity (47). It is not surprising, therefore, that little
inhibition of eucaryotic topoisomerase I has been observed
with quinolones (47). In one study (27), nalidixic acid and
ofloxacin were shown to have no inhibitory activity against
calf thymus nuclear topoisomerase I relaxation activity at
concentrations of up to 1,000 ,ug/ml. Norfloxacin and ciprofloxacin
exhibited limited inhibition in these tests, with IC50s
between 300 and 400 ,g/ml (27).
Other enzymes involved in DNA replication are somewhat
inhibited by nalidixic acid analogs. Nalidixic acid and 4-
quinolones have been shown to alter the chain length distribution
of replication products synthesized by eucaryotic
DNA polymerase a (13, 42). Yeast leucyl- and glycyltransfer
RNA synthetases are also inhibited by high concentrations
of nalidixic and oxolinic acids (51). All of these
inhibitory activities were only detected at drug levels 100- to
1,000-fold higher than that required to inhibit bacterial DNA
gyrase (13, 16, 37, 41, 51). Hussy et al. (27) examined the
effects of the newer 4-quinolones on enzymes involved in
eucaryotic DNA replication. When activated DNA was used
as a template primer, DNA synthesis by calf thymus 9S
DNA polymerase a primase complex was reversibly inhibited
at concentrations above 100 jig/ml (27). At a concentration
of 1,000 ,ug/ml, the activity of DNA polymerase a was
reduced 20% by ofloxacin, 60% by nalidixic acid, and greater
than 80% by ciprofloxacin (27). In contrast, the 4-quinolones
tested at these concentrations did not increase the error rate
of the DNA polymerase a primase complex (27).
The available in vitro data indicate that of the quinolones
tested so far, none appear to be potent inhibitors of eucaryotic
topoisomerases or DNA polymerase a. The selectivity
of quinolones for bacterial DNA gyrase is substantiated by
the fact that the drug levels required for inhibition of
eucaryotic topoisomerases are 100 to 10,000 times higher
than those required for inhibition of bacterial DNA gyrase
(41). It should be noted, however, that the majority of in
vitro results with the eucaryotic enzyme report drug concentrations
required to inhibit 50% of topoisomerase activity in
a given laboratory assay. It is not known what level of
topoisomerase inhibition may be relevant in vivo. In this
sense, levels of quinolones significantly below the calculated
IC50 have been shown to produce some inhibition of eucaryotic
topoisomerases (27, 47).

ABNORMAL CELLULAR RESPONSES ASSOCIATED
WITH QUINOLONES IN VITRO
Despite the reported in vitro selectivity of quinolones for
bacterial DNA gyrase, a number of abnormal eucaryotic
cellular responses have been observed with these compounds.
Oomori et al. (40) found a good correlation between
the cytotoxic effects of quinolones in vitro against HeLa
cells and their ability to inhibit the relaxation activity of
topoisomerase II purified from these cells. Other effects of
quinolones have been manifested as decreased growth rates
observed in mitogen-stimulated lymphocytes (21, 27). For
example, ofloxacin at 10 jig/ml delayed the onset of growth
of cultured lymphoblasts by 1 to 2 days, while ciprofloxacin
at 100 ,Lg/ml was found to inhibit cell growth completely (27).
Forsgren et al. (21) described a marked stimulation of
[3H]thymidine incorporation into T lymphocytes incubated
with phytohemagglutinin and a quinolone. Stimulation of
[3H]thymidine uptake was significantly above the response
obtained with phytohemagglutinin treatment alone and occurred
with ciprofloxacin, norfloxacin, ofloxacin, amifloxacin,
enoxacin, and pefloxacin at physiological concentrations
between 1.56 and 6.25 jig/ml (21). Nalidixic acid and
cinoxacin exhibited no effect in these studies at levels of up
to 25 ,ug/ml (21). Ciprofloxacin at 20 jig/ml was shown to
inhibit the progression of mitogen-stimulated lymphocytes
through the S and G2/M stages of the cell cycle and to
decrease the secretion of immunoglobulins G and M in
pokeweed mitogen-stimulated B cells (21). The stimulation
of [3H]thymidine uptake in human lymphocytes by quinolones
suggests an increase in DNA synthesis or inhibition of
de novo nucleotide biosynthesis. However, the lack of
progression of cells incubated with ciprofloxacin through the
S phase in cell cycle analysis indicated that DNA synthesis
was being inhibited at the drug concentrations tested (21).
Subsequent studies indicated that ciprofloxacin and other
quinolones do not directly inhibit pyrimidine nucleotide
biosynthesis in peripheral blood lymphocytes or deplete
pyrimidine nucleotide pools in cells (6). The mechanisms
responsible for the stimulation of [3H]thymidine uptake in
lymphocytes incubated with quinolones and the cell cycle
inhibition effects are not understood at this time (6, 20, 21).
Several other in vitro and in vivo tests have been used to
identify potential genotoxic (DNA-damaging) effects ofquinolones
in preclinical studies. Since quinolones inhibit bacterial
DNA gyrase and a related topoisomerase is found in
eucaryotes, negative genotoxicity test results provide a level
of confidence that the experimental quinolone will not inhibit
TABLE 1. Inhibition by quinolones in eucaryotic test systems

DNA metabolism in vivo. Some genotoxicity tests are designed
to identify mutagenic activity in specific genes or
more general chromosomal changes. Others are designed to
detect drug-associated DNA strand breakage and inhibition
of DNA replication. The most frequently cited mutagenicity
tests include the in vitro Ames test, Chinese hamster V79
cell test, mouse lymphoma cell test, plasmid shuttle vector
assays, and the in vivo micronucleus and dominant lethal
tests in mice (1, 11, 12, 18, 32, 34, 50). Additional tests
monitoring DNA strand breakage include the alkaline elution
procedure (30) and the unscheduled DNA synthesis (UDS)
test, the latter of which is run with quinolones incubated in
rat hepatocyte cell cultures and also with rat hepatocytes
removed from animals dosed with the test quinolone and
analyzed directly (38). A number of quinolones are inhibitory
in some of these in vitro genotoxicity tests, although
positive in vivo tests are rare (Table 1). It was reported that
ciprofloxacin, norfloxacin, ofloxacin, and pefloxacin were
mutagenic in the mouse lymphoma assay and also induced
DNA damage in the in vitro rat hepatocyte UDS assay (44,
45). These quinolones, however, did not show abnormal
effects in either the Ames or the V79 test for gene mutagenicity
or in the micronucleus and dominant lethal tests in
mice (44, 45).

The activity of ciprofloxacin in the UDS assay
VOL. 34, 1990 ANTIMICROB. AGENTS CHEMOTHER.
was confirmed in a separate study in which the compound,
when tested at 5 ,ug/ml, induced a positive UDS response in
human lymphocytes (6). A similar evaluation of norfloxacin
and fleroxacin (AM-833) showed that they were not mutagenic,
did not induce DNA strand breakage, and did not
elicit a UDS response in either human or mouse skin
fibroblasts (25).
As mentioned above, the alkaline elution test measures
DNA strand breakage (30) and has been used to test quinolones
for this activity, as described by Bredberg et al. (6).
The cellular DNA is prelabeled with [3H]thymidine and,
after incubation with a quinolone for 24 h, the cells are
washed and lysed on a 2-,um-pore-size filter. The doublestranded
DNA is unwound in alkaline buffer, and the single
strands are eluted through the filter. When the test drug
induces DNA strand breakage, the smaller DNA species
generated elute more freely through the filter, as detected by
increased counts of radioactivity in the eluate. With this
method, significant DNA breakage was obtained in lymphoblastoid
cells incubated with ciprofloxacin, ofloxacin, and
norfloxacin at test levels of 10, 80, and 160 ,ug/ml, respectively.
The DNA breakage effect observed with ciprofloxacin
was dose dependent, and at 80 pug of drug per ml, the
breakage approximated that obtained from exposure to 1,000
rads of X rays (6). Bredberg et al. described some potential
technical problems encountered in the alkaline elution procedure
that can drastically affect the degree of DNA breakage
observed (6). As an example, cells on a filter washed
with room temperature buffer exhibited less DNA breakage
than did cells kept on ice during the procedure. A significant
finding in this study (6) was that treated cells have the ability
to rapidly reverse DNA breakage when incubated at 37°C for
only 15 min in quinolone-free medium. This result suggests
that some eucaryotic cells have the ability to rapidly reverse
the DNA damage associated with exposure to quinolones,
and this ability may increase the chance of obtaining a
false-negative result with this in vitro assay. This result also
suggests that more quinolones may have the ability to induce
DNA breakage in cell cultures (6). It has also been noted that
in vitro genotoxicity test results can be significantly influenced
by variations in the ionic strength of the test medium
(45).

CORRELATIONS OF IN VIVO EFFECTS WITH IN
VITRO GENOTOXICITY TEST RESULTS
A major issue surrounding quinolone research involves
the relevance of positive in vitro genotoxicity tests for
measuring the safety of these compounds. There is currently
a good deal of debate in the literature concerning the
interpretation of positive in vitro genotoxicity results observed
in a single test system, because of the finding that
quinolones do not induce the same responses in in vivo tests
(9, 36, 39). McQueen and Williams (36) found that norfloxacin,
ofloxacin, pefloxacin, and ciprofloxacin induced DNA
breakage and repair in the in vitro rat hepatocyte assay at
levels between 300 and 500 p,g/ml. No in vivo response was
obtained in lymphocytes taken from rats given single subcutaneous
doses of ciprofloxacin at 30 or 190 mg/kg (36). These
authors contend that the negative in vivo results rendered
the positive in vitro test results physiologically irrelevant,
given the relatively high concentrations of test substance
required to elicit the in vitro response (36). Furthermore, in
a published clinical study, no cytogenetic abnormalities were
observed in peripheral blood lymphocytes isolated from
adults receiving 500 to 2,000 mg of ciprofloxacin daily for 1
to 10 weeks or with 200 mg of ofloxacin daily for 1 week (39).
An interesting relationship between in vitro and in vivo
genotoxicity test results has been described by Holden et al.
(24). These investigators described a new 6,8-difluoro-7-
pyridyl 4-quinolone, CP-67,015, that was active in genotoxicity
tests performed both in cultured cells and in animals
(24). Although CP-67,015 was a directly acting mutagen at
2100 ,ug/ml in the mouse lymphoma cell assay, the Chinese
hamster ovary cell-hypoxanthine-guanine phosphoribosyl
transferase gene (HGPRT) assay, and the V79 cell-HGPRT
forward mutation assay (24, 32), it was not mutagenic in the
Ames test (24). The compound also induced chromosome
aberrations in cultured human lymphocytes at levels of .50
,ug/ml and produced genetically abnormal bone marrow cells
in mice given five daily parenteral doses of 500 mg/kg per day
(24). Furthermore, CP-67,015 was found to be at least 10-fold
more potent than nalidixic acid, norfloxacin, oxolinic acid,
or ciprofloxacin at enhancing eucaryotic topoisomerase IImediated
DNA cleavage in vitro (J. J. Barrett, T. D. Gootz,
C. A. Farrell, S. A. Sokolowski, and M. Frescura, Program
Abstr. 27th Intersci. Conf. Antimicrob. Agents Chemother.,
abstr. no. 249, 1987). CP-67,015 is a 4-quinolone that is
inhibitory to both procaryotic and eucaryotic type II topoisomerases
and clearly induces genotoxic changes in standard
in vitro and in vivo tests at drug levels that approach
physiological relevance (Table 1). This is particularly true
since it has been observed that peak levels of quinolones in
the urinary tract range from 100 to 650 ,ug/ml (2) and can be
concentrated 4- to 20-fold above levels in serum in lymphocytes
and to variable degrees in other tissues such as kidney,
liver, and intestine (4).
 

CONCLUSIONS
In summary, the available data indicate that most quinolones
examined are not highly inhibitory for eucaryotic
topoisomerases or other enzymes involved in DNA replication.
Although a number of positive in vitro genotoxicity test
results have been documented with several different 4-
quinolones, the effective concentrations would be clinically
achieved predominantly in the urinary tract (4, 5). It has
been suggested that in vitro genotoxicity test results should
not be of concern, since corroborating positive in vivo test
results have not been observed with these antimicrobial
agents (36, 44, 45). There is also disagreement in the literature
concerning which genetic toxicology tests are relevant
for testing quinolones and more general disagreement over
the ability of a single positive test to predict the performance
of compounds in long-term carcinogenicity studies in rodents
(9, 12, 14, 18, 22, 23, 31, 33, 35, 36, 48, 50, 52). Given
the multiple physiological activities of topoisomerases (46)
and the diverse methods that have been developed to measure
their activity (3), it will be interesting to see whether all
of the many new and structurally diverse 4-quinolones retain
their selectivity for bacterial DNA gyrase and remain nontoxic
for eucaryotic cells. In this regard, it is important to
note that not all eucaryotic topoisomerases have been routinely
tested with 4-quinolones in published studies. The
topoisomerase II found in mitochondria, for example, has
been shown to be biochemically distinct from the commonly
studied enzyme found in the nuclei of eucaryotic cells (7, 8,
17). It appears that future research in the area of topoisomerases
will be helpful in understanding these issues.
ACKNOWLEDGMENTS
We thank Henry Holden and John Williams for a critical review of
the manuscript.
 

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