DRUG SAFETY CONCEPTS
Drug Safety 7 (3): 214-222, 1992 0114-5916/ 92/0005-0214/$04.50/0 © Adis International Limited. All rights reserved. DRS1
Mutagenicity of Quinolone Antibacterials Farrel L. Fort Department of Toxicology, Abbott Laboratories, Abbott Park, Illinois, USA
Contents 214
215 215 215 216 216 216 216 217 217 217 217 218 218
Summary
Summary I. DNA Damage Assays 1.1 Bacterial Cells 1.2 Unscheduled DNA Synthesis in Mammalian Cells L3 DNA Strand Breakage in Mammalian Cells 2. Sister Chromatid Exchanges 3. Chromosomal Aberrations 3.1 In Vitro 3,2 In Vivo
4. Mutagenicity 4.1 Bacterial 4.2 Mammalian In Vitro 4.3 In Vivo
5. Discussion
The literature is summarised on the activity of quinolone antibacterial compounds in assays which are commonly used for risk assessment of new pharmaceuticals. These include assays for DNA damage, sister chromatid exchanges, chromosome aberrations and mutation induction. The general pattern of activity exhibited by these compounds is induction of DNA damage in both prokaryotic and eukaryotic cells, and induction of mutations in DNA repair-proficient bacteria and at the thymidine kinase locus in mammalian cells. They do not appear as a class to induce mutations at the hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) or Na+,K+-ATPase loci or to cause chromosome aberrations. It is suggested that these actions may be the result of interference with eukaryotic topoisomerase and that this interference differs in some respects from the topoisomerase interference caused by certain anti tumour compounds. The postulated mechanism of action has important implications for assessment of risk from consumption of quinolone antibacterials. The risk of adverse genotoxic events should vary directly with the concentration of drug reaching the intracellular enzyme target and the affinity of the drug for the target. Results of carcinogenicity studies conducted to date with the quinolone antibacterials suggest minimal risk from long term consumption of the newer, second-generation compounds.
Mutagenicity of Quinolones
The quinolone antibacterials have proved useful in a variety of urinary, respiratory and gastrointestinal tract, and skin, bone and joint infections. They are primarily effective against Gram-negative and positive aerobic bacteria and are particularly useful against infections resistant to other classes of antibiotics. They are the first orally active agents against Pseudomonas and other resistant Gramnegative bacteria. These compounds act by inhibition of bacterial DNA gyrase, an essential type II DNA topoisomerase, thus interfering with bacterial replication (Norris & Mandell 1988). Although considered to be specific for the bacterial enzyme, the possibility exists that some of the quinolone antibacterials might also affect mammalian DNA metabolism as mammalian cells also contain an essential type II DNA topoisomerase. In addition to cytotoxicity, this could theoretically lead to undesirable mutagenic sequelae. The purpose of this review is to summarise and discuss the available literature on the mutagenicity of quinolone antibacterials in order to determine any patterns of activity which may be apparent, and to assess the potential risk to humans from systemic exposure to these compounds. As a class the quinolone antibacterials tend to give positive results in various tests for genetic toxicity; however, the results are not uniform among quinolones or across the mutagenicity tests reported.
1. DNA Damage Assays 1.1 Bacterial Cells DNA damage assays in bacteria referred to here are the so-called DNA repair assays. In these, the compound is tested for killing of DNA repairdeficient strains relative to repair-proficient parent strains. Increased killing of the DNA repair-deficient strain relative to the repair-proficient strain is evidence of induction of DNA damage which could not be repaired by the repair-deficient strain prior to cell division, thus leading to cell death (Kada et at. 1984). Norfloxacin, fleroxacin (AM 833) and ofloxacin
215
were positive in a DNA repair assay in Bacillus subtilis (Corrado et at. 1987; Hosomi et at. 1988; Irikura & Hosomi 1981; Mayer & Bruch 1986; Physicians' Desk Reference 1991 b; Shimada et at. 1984). Nalidixic acid and oxolinic acid were positive in Escherichia coli, Salmonella typhimurium and B. subtilis DNA repair tests (Akema et at. 1978; McCoy et at. 1980; McDaniel et at. 1978; Rosenkranz & Leifer 1980), while ciprofloxacin was negative in E. coli (Physicians' Desk Reference 199Ia). 1.2 Unscheduled DNA Synthesis in Mammalian Cells Unscheduled DNA synthesis (UDS) assays measure the uptake of radiolabelled thymidine or thymidine analogues into DNA of nonreplicating cells (Hanawalt 1977). When using cells which would normally undergo S phase DNA synthesis during the assay it is common to apply an inhibitor of replicative DNA synthesis, such as hydroxyurea. However, when using cells such as rat liver hepatocytes, such an S phase inhibitor is usually not used since these cells do not normally undergo cell division. This model is popular because of the metabolic capabilities inherent in the cell and because both in vitro and in vivo/in vitro (drug treatment in vivo and UDS measurement in vitro) assays are easily performed. A positive UDS result is interpreted as a DNA repair response (nonreplicative DNA synthesis), which occurs during excision DNA repair as a result of DNA damage inflicted by the test agent. Ciprofloxacin, pefloxacin, norfloxacin and ofloxacin were all positive in the in vitro primary hepatocyte UDS assay (Mayer 1987; McQueen & Williams 1987; Physicians' Desk Reference 199Ia). However, norfloxacin has also been reported to be negative for UDS in human and mouse skin fibroblasts as well as rat hepatocytes (Hosomi et at. 1988). Similarly, ofloxacin was negative when tested for UDS in human diploid fibroblasts in culture (Mayer 1987; Shimada et at. 1984) and ciprofloxacin was negative in an in vivo/in vitro UDS assay (Schluter 1986). Ciprofloxacin was positive for UDS when tested in human lymphocytes in culture
216
(Bredberg et al. 1989). Nalidixic acid was shown to stimulate UDS in rat splenic and thymic cells in culture (Tempel & Spath 1987), but was negative for UDS in primary rat hepatocyte cultures (McQueen & Williams 1987). Aeroxacin was negative for UDS in human and mouse skin fibroblasts as well as rat hepatocytes (Hosomi et al. 1988). However, DNA gyrase-like activity has been observed in rat liver mitochondria (Castora et al. 1983). It has therefore been suggested that inhibition of mitochondrial DNA synthesis by DNA gyrase inhibitors may lead to false positive results in UDS assays by reducing the cytoplasmic background grain counts that are normally subtracted from nuclear counts to determine net nuclear counts, thus falsely elevating the net nuclear grain counts (Christ et al. 1988). Nalidixic acid and oxolinic acid inhibit rat liver mitochondrial DNA gyrase (Castora, et al. 1983). Ciprofloxacin did not inhibit DNA synthesis in rat liver mitochondria (Forsgren et al. 1989). Therefore, the above argument may hold for some quinolones but not for others. This could only be determined by reporting cytoplasmic background counts with UDS results, which is virtually never done. 1.3 DNA Strand Breakage in Mammalian Cells DNA strand breakage may be induced directly by a chemical or physical (e.g. ionising radiation) agent or may occur secondary to enzymatic action occurring subsequent to DNA damage (e.g. during DNA excision repair). In the case of DNA gyrase inhibitors this may occur secondary to interference with topoisomerase II (see discussion). Ciprofloxacin, ofloxacin and norfloxacin caused DNA strand breakage in a human Iymphoblastoid cell line in culture (Bredberg et al. 1989). Ciprofloxacin caused DNA strand breakage in human lymphocytes in vitro (Forsgren et al. 1987). Nalidixic acid has been shown to cause DNA crosslinking in human amnion cells in culture (Rosenkranz & Lambek 1965) but apparently did not cause DNA strand breakage in rat splenic and thymic cells
Drug SafelY 7 (3) 1992
even though UDS was stimulated in these cells (Tempel & Spath 1987). Oxolinic and pipemidic acid caused DNA strand breakage in granuloma pouch cells when given orally to rats (Maura & Pino 1988). Norfloxacin caused DNA strand breakage in fetal tissues of rats in vivo when given at 30 times the clinical dose (Pi no et al. 1991). However, cinoxacin did not cause strand breakage in the liver or kidney in vivo in rats when given orally (pino et al. 1989), and fleroxacin and norfloxacin did not cause DNA strand breakage in mouse L-121O or human IMR-90 cells in culture (Hosomi et al. 1988).
2. Sister Chromatid Exchanges Sister chromatid exchanges (SCE) result from the exchange of equal regions of chromatin between homologous chromatids of a chromosome during late S phase of the cell cycle. Although the significance or mechanisms of the phenomenon are not known, SCE are known to commonly occur in response to DNA-damaging agents (Perry & Thomson 1984). Nalidixic acid was positive for the induction of sister chromatid exchanges in human lymphocytes from drug-treated patients (Kowalczk 1980). Norfloxacin and ofloxacin were negative in this assay in both human lymphocytes and Chinese hamster lung cells cultured in vitro (Corrado et al. 1987; Irikura & Hosomi 1981 ; Kullich et al. 1988; Mayer 1987; Shimada et al. 1984). CP-67015 was weakly positive in vitro in human lymphocytes but negative in mouse bone marrow in vivo (Holden et al. 1989).
3. Chromosomal Aberrations 3.1 In Vitro Norfloxacin, ofloxacin, fleroxacin, cinoxacin, ciprofloxacin and nalidixic acid were negative for induction of chromosome aberrations in vitro in cultured cells (Corrado et al. 1987; Forsgren et al. 1987; Hosomi et al. 1988; Irikura & Hosomi 1981 ; Shimada et al. 1984; Shiratori & Takase 1980; Stenchever et al. 1970). However, CP-67015 was
Mutagenicity of Quinolones
positive in human lymphocytes and CHO cells (Holden et al. 1989). 3.2 In Vivo Norfloxacin was negative for chromosome aberrations in vivo in rat and hamster bone marrow (lrikura et al. 1981; Physicians' Desk Reference 1991 b). Cinoxacin was negative for chromosomal aberrations in mouse bone marrow (Shiratori et al. 1980). Ciprofloxacin and ofloxacin did not cause chromosomal aberrations in lymphocytes from patients treated with the drugs (Mayer 1987; Mitel man et al. 1988). Ciprofloxacin, cinoxacin, CP-670 15 and ofloxacin were negative in the mouse micronucleus test (Carlin 1975; Holden et al. 1989; Mayer 1987; Physicians' Desk Reference 1991 a; Shimada et al. 1984). Micronuclei are chromatin fragments that become separated from the nuclear material during mitosis and end up in the cytoplasm of a daughter cell. These are most commonly measured in polychromatic erythrocytes (PCE) in the bone marrow of rodents. Normal rodent PCE have no nucleus since this is the stage immediately after nuclear expulsion in erythrocyte development. Nuclear material detected by staining in these cells represents micronuclei which are the end products of either chromosome fragmentation or interference with the mitotic spindle apparatus (Heddle et al. 1984). Ciprofloxacin, norfloxacin, ofloxacin and nalidixic acid were negative in the mouse dominant lethal test (Corrado et al. 1987; Irikura et al. 1981; Mayer 1987; Physicians' Desk Reference 199Ia,b; Shimada et al. 1980, 1985). The dominant lethal test is conducted by treating males with test compound for I week and then mating them to untreated females over a period of about 8 weeks using a different group of females each week. The end-point is the number of early postimplantation deaths in pregnant females. An increase in such deaths is evidence for germ line chromosomal damage in the male (Bateman 1984).
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4. Mutagenicity 4.1 Bacterial Nalidixic acid was positive in a multigene sporulation assay in B. subtilis (a test for mutation induction in bacteria) [Sacks & MacGregor 1982]. Ciprofloxacin, fleroxacin, enoxacin, norfloxacin, ofloxacin, oxolinic acid, nalidixic acid and CP67015 were negative for mutation induction in S. typhimurium (Ames test) using DNA repair-deficient strains (Corrado et al. 1987; Gocke 1991; Holden et al. 1989; lrikura & Hosomi 1981; Mayer 1987; McCoy et al. 1980; Physicians' Desk Reference 1991 a, b; Sacks & MacGregor 1982; Shimada et al. 1984; Ysern et al. 1990). However, nalidixic acid, norfloxacin, oxolinic acid, ciprofloxacin, enrofloxacin, enoxacin, fleroxacin and ofloxacin were positive in strains which were DNA repair-proficient (Gocke 1991; Levin et al. 1984; Phillips et al. 1987; Ysern et al. 1990). Ciprofloxacin, norfloxacin, fleroxacin, nalidixic acid and ofloxacin were negative for reverse mutation in DNA repairdeficient E. coli (Hosomi et al. 1988; lrikura & Hosomi 1981; McCoy et al. 1980; Physicians' Desk Reference 1991 a; Shimada et al. 1984; Witkin & Wermundsen 1979). However, nalidixic acid was positive for mutation induction in proliferating cultures of DNA repair-proficient E. coli (Cook et al. 1966; Witkin & Wermundsen, 1979). These results are interpreted to mean that DNA repair in bacteria is required in order for the actions of the DNA gyrase inhibitors to be converted to mutations. A sequence of events can be envisioned where: (a) the gyrase inhibitors inflict DNA damage as demonstrated by the DNA repair test mentioned above; (b) the DNA damage tends to cause cell killing unless repaired, and (c) errors are made during the repair process which lead to the fixation of mutations in the DNA. 4.2 Mammalian In Vitro Ciprofloxacin, ofloxacin, fleroxacin, nalidixic acid and norfloxacin were negative in an hypoxanthine-guanine-phosphoribosyltransferase (H GPR T) forward mutation assay in Chinese hamster V79
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cells (Corrado et al. 1987; Hosomi et al. 1988; Mayer 1987; Physicians' Desk Reference 1991a,b; Schluter 1986). Cinoxacin, oxolinic acid and pipemidic acid were negative for HGPRT forward mutation in rat granuloma pouch cells when injected directly into the granuloma pouch or given orally (Maura & Pino 1988; Pino et al. 1989). However, CP-67015 was positive for HGPRT mutation in CHO and V79 cells (Holden et al. 1989). Both norfloxacin and fleroxacin were negative for induction of ouabain-resistant mutants in Chinese hamster V79 cells (Hosomi et al. 1988). Ciprofloxacin, norfloxacin, ofloxacin, pefloxacin and nalidixic acid were positive in the mouse lymphoma thymidine kinase forward mutation assay (Physicians' Desk Reference 1991 a; Schluter 1986). 4.3 In Vivo
Nalidixic acid was mutagenic in vivo in the sexlinked recessive lethal test in Drosophila (Filippova & Efremova 1974). This was the only test reported for mutation induction in vivo in any species with quinolones. The above results are summarised in table I by listing a drug as positive for a test category if reported positive in any of the reports cited above. A drug may have been reported negative for a particular test category in 1 or more papers, but if it was reported positive in at least 1 paper for the test category, it is listed as positive in the table.
5. Discussion The inconsistent assay results obtained from the literature were surely influenced by the assay conditions used, in particular by the concentrations of test compound, the test system and the protocol for a given test. Study designs by different investigators varied widely in these aspects. The variability in results among quinolones studied and among test systems could at least partially be due to the affinity of the various quinolones for mammalian topoisomerase (they were designed and selected on the basis of activity against bacterial rather
Drug Safety 7 (3) 1992
than mammalian topoisomerase) and the ability of the drugs to enter mammalian cells in order to affect the enzymes. Nevertheless, a general pattern seems to emerge that, with exceptions, the quinolone antibacterials as a class tend to be positive in assay systems for DNA damage (such as UDS and strand breakage), mutagenicity in DNA repair-proficient bacteria and at the thymidine kinase locus in mouse lymphoma cells, but negative in assays for chromosomal aberrations or mutagenicity at the HGPRT or Na+,K+-ATPase loci in mammalian cells. It is not surprising that compounds of this class might be positive in tests for DNA damage since they inhibit topoisomerase II (DNA gyrase) in bacterial cells [Shen et al. 1989] and may have activity against similar enzymes in eukaryotic cells. For example, nalidixic acid, oxolinic acid and pefloxacin inhibit several eukaryotic enzymes involved in DNA replication (Rusquet et al. 1984). Ciprofloxacin, norfloxacin, nalidixic acid and ofloxacin have been shown to inhibit calf thymus topoisomerase I and II (Hussy et al. 1986; Sato et al. 1989). Reroxacin has also been reported to inhibit calf thymus topoisomerase II (Sato et al. 1989). However, nalidixic acid and oxolinic acid did not inhibit topoisomerases I and II from rat liver (Duguet et al. 1983). Eukaryotic topoisomerase II allows unwinding and relaxation of DNA supercoils during DNA replication and transcription (Vosberg 1985). As part of its action the enzyme creates double strand breaks in the DNA held together by the enzyme (Cozzarelli 1980). Interference with this process could result in the appearance of single or double strand breaks in the DNA. Additionally, it is known that protein-DNA crosslinks can be recovered from mammalian cells when topoisomerase enzymes are blocked (Liu et al. 1983; Nelson et al. 1984). When the enzyme creates double strand breaks and holds the DNA together at the site of these breaks, it does so by means of DNA-protein crosslinks between the DNA and the enzyme (Cozzarelli 1980). It is not unreasonable to expect that DNA double strand breaks and DNA-protein crosslinks created by interference with topoisomerase enzymes would be
Mutagenicity of Quinolones
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Table I. Quinolone mutagenicity summary NOR
DNA damage Bacterial + Mammalian in vitro UDS + in vivo/in vitro UDS DNA breakage + SCE Chromosome aberrations In vitro In vivo Mutagenicity Bacterial repair deficient repair proficient Mammalian Na+,K+-ATPase HGPRT thymidine kinase + Drosophila
=
CIP
OFL
CIN
PEF
PIP
OXO
+ +
+
+
+
NAL
FLE
+
+
ENR
CPS
ENO
+
+
+
+
+ +
+ +
+
+
+
+
+
+
+
+
+ +
+ +
+
=
+
=
Abbreviations: UDS unscheduled DNA synthesis; SCE sister chromatid exchange; HGPRT hypoxanthine-guaninephosphoribosyltransferase; NOR norfloxacin; CIP ciprofloxacin; OFL ofloxacin; PEF pefloxacin; CIN cinoxacin; PIP pipemidic acid; OXO oxolinic acid; NAL nalidixic acid; FLE fleroxacin; ENR enrofloxacin; CP6 CP-67015;
=
= = ENO = enoxacin; + = positive; - = negative.
=
=
detected as DNA damage by assays which detect damage directly (e.g. DNA strand breakage) and indirectly (e.g. DNA repair or unscheduled DNA synthesis). The mouse lymphoma thymidine kinase assay is known to be more sensitive to certain mutagens because of the shorter expression time required with the cell line and because the thymidine kinase locus assay also detects chromosomal mutagens, whereas the HGPRT and Na+,K+ATPase loci do not (Applegate & Hozier 1987). The pattern of positive results at the thymidine kinase locus and mostly negative results at other loci (HGPRT and Na+,K+-ATPase) in mammalian cells can most likely be interpreted to suggest that multilocus effects are being induced by these agents (Applegate & Hozier 1987; Evans et al. 1986). Interference with topoisomerase II is believed to be involved in the antitumour and cytotoxic action of amsacrine (m-AMSA) and o-AMSA (DeMarini et al. 1987a; Nelson et al. 1984), teni-
=
=
=
=
= =
poside and etoposide (DeMarini et al. 1987b; Yang et al. 1985) and doxorubicin (adriamycin) [Tewey et al. 1984]. However, the pattern of mutagenic activity with these compounds appears to be different from that with the quinolone antibacterials with the exception of CP-67015. The antitumour compounds are clastogenic (i.e. cause chromosome aberrations) [DeMarini et al. 1987a,b; Wilson et al. 1984], whereas the quinolones (except for CP67015) apparently lack this capability. Teniposide and m-AMSA induce SCE in cell cultures (Lim et al. 1986), whereas among the 3 quinolones tested for SeE induction only nalidixic acid and CP-67015 possess this property. Although both the quinolone antibacterials and the antitumour agents listed above are likely to exert their genotoxic effects in mammalian cells by interference with topoisomerase action, the discrepancy between their patterns of activity in the various assays for genotoxic activity suggest that the 2 classes of compounds may
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cause their genotoxic effects via different (albeit unknown) mechanisms. CP-67015 clearly presents an exceptional case and may represent a third category with activities of both the anti tumour agents mentioned above as well as of the majority of quinolone antibacterials. Interestingly, in an assay for calf thymus to poi somerase II-induced DNA cleavage, ciprofloxacin, norfloxacin, oxolinic acid and nalidixic acid exhibited concentrations required for 50% cleavage (CC50) approximately WOO-fold greater than the anti tumour compounds ellipticine and etoposide, whereas the CC50 for CP-76015 was only about 10fold higher (Barrett et al. 1989). This is in contrast to the potency of CP-670 15 to inhibit other actions of to poi some rase II. For example, the concentrations required to cause 50% inhibition (IC50) for inhibition of P4 DNA unknotting and concatenation of pBR322 DNA were similar for DP-67015, ciprofloxacin and norfloxacin (Barrett et al. 1989). The mechanism of action postulated for positive results in the mutagenicity assays cited above, i.e. interference with enzyme action rather than a direct DNA damaging effect, has significance in terms of the assessment of risk to people exposed to quinolone antibacterials. That is, any effects leading indirectly to DNA or chromosome damage should depend on the concentration of compound reaching the enzyme-DNA target within the nucleus of the cell as wel1 as the affinity of the compound for the enzyme. This means that although some of the quinolones were positive in some of the assays for genetic toxicity, the significance of these findings to humans can be estimated on the basis of the concentrations of drug required to elicit the positive results compared to the levels of drug expected. For example, ciprofloxacin, norfloxacin, nalidixic acid and ofloxacin inhibit calf thymus topoisomerase I and topoisomerase II at concentrations at least 100 times those required to inhibit bacterial DNA gyrase (Hussy et al. 1986). Ofloxacin, ciprofloxacin, fleroxacin and nalidixic acid have also been reported to inhibit calf thymus topoisomerase II (IC50) at concentrations over 1000 times those required to inhibit E. coli DNA gyrase (minimum
Drug Safety 7 (3) 1992
inhibitory concentration) [Sato et al. 1989]. In separate studies, the ratio of the IC50S for inhibition of calf thymus topoisomerase II versus E. coli DNA gyrase were 1000 to 2000 for ofloxacin, fleroxacin and ciprofloxacin, 300 to 600 for tosufloxacin and lomefloxacin, about 50 for enoxacin and about 20 for CI-934 and nalidixic acid (Hoshino et al. 1989). These data indicate that therapeutic concentrations should be much lower than those that would pose a risk for interference with mammalian to poi some rase II activity. As mentioned earlier, only nalidixic acid has been reported to induce some evidence for mutagenic activity (SCE) in humans (Kowalczk 1980). Further estimates of the significance of positive mutagenicity results to people who ingest these compounds comes from carcinogenicity studies completed with several quinolones. Carcinogenicity studies have been completed in rats and mice with ciprofloxacin, norfloxacin and nalidixic acid and with piromidic acid in mice (Jida et al. 1977; Kurokawa et al. 1986; Morrissey et al. 1991; Physicians' Desk Reference 1991 b; Reed 1988). Ciprofloxacin and norfloxacin were negative in both rats and mice. Piromidic acid was negative in mice. Nalidixic acid was negative in CFD, mice but was equivocal1y positive in B6C3F, mice, inducing an increase in subcutaneous fibromas or fibrosarcomas in male mice, and was positive in rats, inducing preputial gland tumours in male rats and clitoral gland tumours in female rats. Since the newer quinolones (i.e. all except nalidixic acid) are more closely similar on the basis of chemical structure to norfloxacin and ciprofloxacin, it seems reasonable for prediction of risk with the newer quinolones to discount the results of carcinogenicity studies with nalidixic acid, which is the prototype first generation quinolone antibacterial. In summary, on the basis of the available data, the most likely mechanism for positive genetic toxicity results with quinolones is interference with enzyme (topoisomerase) action rather than a direct effect on DNA. Since interference with enzyme action should be a concentration-dependent phenomenon, any risk to humans of genetic toxicity from these compounds depends on the con centra-
Mutagenicity of Quinolones
tions achieved in vivo. The negative carcinogenicity data for the newer quinolones that have been tested suggest little or no risk to humans from consumption of these compounds at therapeutic concentrations.
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effects as a potential source of clinical toxicity. Reviews of Infectious Diseases II (Suppl. 2): SI382-S1389, 1989 Gocke E. Mechanism of quinolone mutagenicity in bacteria. Mutation Research 248: 135-143, 1991 Hanawalt Pc. DNA repair processes: an overview. In Nichols & Murphy (Eds) DNA repair processes, pp. 1-19, Symposia Specialists Inc., Miami, 1977 Heddle JH, Stuart E, Salamone MF. The bone marrow micronucleus test. In Kilbey et al. (Eds) Handbook of mutagenicity test procedures, 2nd ed., pp. 441-457, Elsevier Science Publishers, Amsterdam, 1984 Holden HE, Barrett JF, Huntington CM, Muehlbauer PA, Wahrenburg MG. Genetic profile of a nalidixic acid analog: a model for the mechanism of sister chromatid exchange induction. Environmental anq-Molecular Mutagenesis 13: 238-252, 1989 Hoshino K, Sato K, Une T, Osada Y. Inhibitory effects of quinolones on DNA gyrase of Escherichia coli and topoisomerase II of fetal calf thymus. Antimicrobial Agents and Chemotherapy 33: 1816-1818, 1989 Hosomi J, Maeda A; Oomori Y, Irikura T, Yokota T. Mutagenicity of norfloxacin and AM-833 in bacteria and mammalian cells. Reviews of Infectious Diseases 10 (Suppl. I): SI48-S149, 1988 Hussy P, Maass G, Tummler BL, Grosse FL, Schomburg U. Effect of 4-quinolones and novobiocin on calf thymus DNA polymerase" primase complex, topoisomerases I and II, and growth of mammalian Iymphoblasts. Antimicrobial Agents and Chemotherapy 29: 1073-1078, 1986 !ida M, Nakajima F, Abe M, Ohnishi K, Tatsumi H. Carcinogenicity study of piromidic acid in mice. Iyakuhin Kenkyu 8: 287-295, 1977 lrikura T, Hosomi J. The mutagenicity of AM-715 in vitro. Chemotherapy (Tokyo) 29 (Suppl. 4): 938-945, 1981 lrikura T, Suzuki H, Sugimoto T. Mutagenicity studies of AM715 in animals. Chemotherapy (Tokyo) 29 (Suppl. 4): 932-937, 1981 Kada T, Sadaie Y, Sakamoto Y. Bacillus subtilis repair test. In Kilbey et al. (Eds) Handbook of mutagenicity test procedures, 2nd ed., pp. 13-313, Elsevier Science Publishers, Amsterdam, 1984 Kowalczk J. Sister-chromatid exchanges in children treated with nalidixic acid. Mutation Research 77: 371-375, 1980 Kullich W, Brugger P, Klein G. Zur Beurteilung des genotoxischen Risikos durch den gyrasehemmer Ofloxacin mittels der Bestimmung von Schwesterchromatidaustauschraten. Wiener Medizinische Wochenschrift 138: 107-109, 1988 Kurokawa Y, Matsushima Y, Imazawa T, Takamura N, Maekaw A, et al. Long-term' in vivo carcinogenicity study of nalidixic acid in CDFI mice. Food and Chemical Toxicology 24: 319323, 1986 Levin DE, Marnett LJ, Ames BN. Spontaneous and mutageninduced deletions: mechanistic studies in Salmonella tester strain T A 102. Proceedings of the National Academy of Sciences of the United States of America 81: 4457-4461,1984 Lim M, Liu LF, Jacobson-Kram D, Williams JR. Induction of sister chromatid exchanges by inhibitors of topoisomereases. Cell Biology and Toxicology 2: 485-494, 1986 Liu LF, Rowe TC, Yang L, Tewey K, Chen GL. Cleavage of DNA by mammalian DNA topoisomerase II. Journal of Biological Chemistry 258: 15365-15370, 1983 Maura A, Pi no A. Evaluation of the DNA-damaging and mutagenic activity of oxolinic and pipemidic acids by the granuloma pouch assay. Mutagenesis 3: 397-401, 1988 Mayer D, Bruch K. Kein Hinweis fur Mutagenitat von Ofloxacin. Infection 14 (Suppl. I): S 108-S 109, 1986 Mayer DG. Overview of toxicological studies. Drugs 34 (Suppl. I): 150-153, 1987 McCoy EC, Petrulb LA, Rosenkranz HS. Non-mutagenic geno-
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toxicants: novobiocin and nalidixic acid, two inhibitors of DNA gyrase. Mutation Research 79: 33-43, 1980 McDaniel LS, Rogers LH, Hill WE. Survival of recombinationdeficient mutants of Escherichia coli during incubation with nalidixic acid. Journal of Bacteriology 134: 1195-1198, 1978 McQueen CA, Williams GM . Effects of quinolone antibiotics in tests for genotoxicity. American Journal of Medicine 82 (Suppl. 4A): 94-96, 1987 Mitelman F, Kolnig AM, Strombeck B, Norrby R, Kromann-Andersen B, et al. No cytogenetic effects of quinolone treatment in humans. Antimicrobial Agents and Chemotherapy 32: 936937, 1988 Morrissey RE, Eustis S, Haseman JK, Huff J, Bucher JR. Toxicity and carcinogenicity studies of nalidixic acid in rodents. Drug and Chemical Toxicology 14: 45-66, 1991 Nelson EM, Tewey KM, Liu LF. Mechanism of antitumor drug action: poisoning of mammalian DNA topoisomerase II on DNA by 4'-(9-acridinylamino)-methanesulfon-m-anisidide. Proceedings of the National Academy of Sciences of the United States of America 81: 1361-1365, 1984 Norris S, Mandell GL. The quinolones: history and overview. In Andriole (Ed.) The quinolones, pp. 1-22, Academic Press, New York, 1988 Perry PE, Thomson EF. The methodology of sister chromatid exchanges. In Kilbey et al. (Eds) Handbook of mutagenicity test procedures, 2nd ed. , pp. 495-529, Elsevier Science Publishers, Amsterdam, 1984 Pino A, Maura A, Grillo P. Absence of cinoxacin-induced DNA fragmentation and mutations in the rat granuloma pouch. Environmental and Molecular Mutagenesis 13: 112-115, 1989 Pino A, Maura A, Villa F, Masciangelo L. Evaluation of DNA damage induced by norfloxacin in liver and kidney of adult rats and in fetal tissues after transplacental exposure. Mutation Research 264: 81-85, 1991 Phillips I, Culebras E, Moreno F, Baquero F. Induction of SOS response by new 4-quiolones. Journal of Antimicrobial Chemotherapy 20: 631-638, 1987 Physicians' Desk Reference, 44th ed. , pp. 1542-1544, 1991a Physicians' Desk Reference, 44th ed., pp. 1474-1476, 1991b Reed TG. Revised labeling dated May 20, 1988: ciprofloxacin. Obtained from FDA via Freedom of Information Act, 1988 Rosenkranz H, Lambek e. In vivo effect of nalidixic acid (NegGram) on the DNA of human diploid cells in tissue culture. Proceedings of the Society for Experimental Biology and Medicine 120: 549-552, 1965 Rosenkranz HS, Leifer A. Determining the DNA-modifying activity of chemicals using DNA-polymerase-deficient Escherichia coli. In deSerres (Ed.) Chemical mutagens, pp. 109-147, Plenum Press, New York, 1980 Rusquet R, Bohommet M, David Je. Quinolone antibiotics inhibit eucaryotic DNA polymerase a and /3) terminal deoxynucleotidyl transferase but not DNA ligase. Biochemical and Biophysical Research Communications 121: 762-769, 1984 Sacks LE, MacGregor JT. The B. subtilis multigene sporulation test for mutagens: detection of mutagens inactive in the Salmonella his reversion test. Mutation Research 95: 191-202, 1982
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Sato K, Hoshino K, Une T, Osaka, Y. Inhibitory effects of ofloxacin on DNA gyrase of Escherichia coli and topoisomerase II of bovine calf thymus. Reviews of Infectious Diseases 2 (Suppl. 5): S915-S916, 1989 Schluter G. Toxicology of ciprofloxacin. In New & Weuta (Eds) Proceedings of the First International Ciprofloxacin Workshop, Amsterdam, pp. 61-70, Excerpta Medica, 1986 Shen LL, Lester AM, Sharma PN, O'Donnell TJ, Chu DWT, et al. Mechanism of inhibition of DNA gyrase by quinolone antibacterials: a cooperative drug-DNA binding model. Biochemistry 28: 3886-3894, 1989 Shimada H, Morita H, Akimoto T. Lack of induction of dominant lethal mutations in male mice by nalidixic acid. Toxicology Letters 7: 165-170, 1980 Shimada H, Yutaka E, Kurasawa Y, Arauchi T. Mutagenicity studies of DL-8280, a new antibacterial drug. Chemotherapy (Tokyo) 32 (Suppl. I): 1162-1170, 1984 Shimada H, Ebine Y, Sato T, Kurosawa Y, Arauchi T. Dominant lethal study in male mice treated with ofloxacin, a new antimicrobial drug. Mutation Research 144: 51-55, 1985 Shiratori 0, Takase S. Mutagenic activity tests on cinoxacin in vitro and in vivo cytogenetic tests in mammalian cells. Chemotherapy (Tokyo) 28 (Suppl. 4): 523-529, 1980 Stenchever M, Powell W, Jarvis J. Effect of nalidixic acid on human chromosome integrity. Journal of Obstetrics and Gynecology' 107: 329-330, 1970 Tempel K, Spath A. Stimulation of DNA repair synthesis of rat thymocytes by novobiocin and nalidixic acid in vitro without detectable DNA damage. Archives of Toxicology 60: 287-292, 1987 Tewey KM, Rowe TC, Yang L, Halligan BD, Liu LF. Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II. Science 226: 466-468, 1984 Vosberg HP. DNA topoisomerases: enzymes that control DNA conformation. Current Topics in Microbiology and Immunology 114: 19-102, 1985 Wilson WR, Harris NM, Ferguson LR. Comparison of mutagenic and clastogenic activity of amsacrine and other DNA-intercalating drugs in cultured V79 Chinese hamster cells. Cancer Research 44: 4420-4431 , 1984 Witkin EM, Wermundsen, IE. Targeted and untarteted mutagenesis by various inducers ofSOS functions in Escherichia coli. Cold Spring Harbor Symposia 43: 88 I-886, 1979 Yang L, Rowe TC, Liu LF. Identification of DNA topoisomerease" as an intracellular target of antitumor epipodophyllotoxins in simian virus 40-infected monkey cells. Cancer Research 45: 5872-5876, 1985 Ysern P, Clerch B, Castano M, Isidre G, Barbe J, et al. Induction of SOS genes in Escherichia coli and mutagenesis in Salmonella typhimurium by fluoroquinolones. Mutagenesis 5: 63-66, 1990
Correspondence and reprints: Dr Farrel L. ForI, Department of Toxicology, D468, Abbott Laboratories, Abbott Park. IL 60064, USA.
Drug Safety 7 (3): 214-222, 1992 0114-5916/ 92/0005-0214/$04.50/0 © Adis International Limited. All rights reserved. DRS1
Mutagenicity of Quinolone Antibacterials Farrel L. Fort Department of Toxicology, Abbott Laboratories, Abbott Park, Illinois, USA
Contents 214
215 215 215 216 216 216 216 217 217 217 217 218 218
Summary
Summary I. DNA Damage Assays 1.1 Bacterial Cells 1.2 Unscheduled DNA Synthesis in Mammalian Cells L3 DNA Strand Breakage in Mammalian Cells 2. Sister Chromatid Exchanges 3. Chromosomal Aberrations 3.1 In Vitro 3,2 In Vivo
4. Mutagenicity 4.1 Bacterial 4.2 Mammalian In Vitro 4.3 In Vivo
5. Discussion
The literature is summarised on the activity of quinolone antibacterial compounds in assays which are commonly used for risk assessment of new pharmaceuticals. These include assays for DNA damage, sister chromatid exchanges, chromosome aberrations and mutation induction. The general pattern of activity exhibited by these compounds is induction of DNA damage in both prokaryotic and eukaryotic cells, and induction of mutations in DNA repair-proficient bacteria and at the thymidine kinase locus in mammalian cells. They do not appear as a class to induce mutations at the hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) or Na+,K+-ATPase loci or to cause chromosome aberrations. It is suggested that these actions may be the result of interference with eukaryotic topoisomerase and that this interference differs in some respects from the topoisomerase interference caused by certain anti tumour compounds. The postulated mechanism of action has important implications for assessment of risk from consumption of quinolone antibacterials. The risk of adverse genotoxic events should vary directly with the concentration of drug reaching the intracellular enzyme target and the affinity of the drug for the target. Results of carcinogenicity studies conducted to date with the quinolone antibacterials suggest minimal risk from long term consumption of the newer, second-generation compounds.
Mutagenicity of Quinolones
The quinolone antibacterials have proved useful in a variety of urinary, respiratory and gastrointestinal tract, and skin, bone and joint infections. They are primarily effective against Gram-negative and positive aerobic bacteria and are particularly useful against infections resistant to other classes of antibiotics. They are the first orally active agents against Pseudomonas and other resistant Gramnegative bacteria. These compounds act by inhibition of bacterial DNA gyrase, an essential type II DNA topoisomerase, thus interfering with bacterial replication (Norris & Mandell 1988). Although considered to be specific for the bacterial enzyme, the possibility exists that some of the quinolone antibacterials might also affect mammalian DNA metabolism as mammalian cells also contain an essential type II DNA topoisomerase. In addition to cytotoxicity, this could theoretically lead to undesirable mutagenic sequelae. The purpose of this review is to summarise and discuss the available literature on the mutagenicity of quinolone antibacterials in order to determine any patterns of activity which may be apparent, and to assess the potential risk to humans from systemic exposure to these compounds. As a class the quinolone antibacterials tend to give positive results in various tests for genetic toxicity; however, the results are not uniform among quinolones or across the mutagenicity tests reported.
1. DNA Damage Assays 1.1 Bacterial Cells DNA damage assays in bacteria referred to here are the so-called DNA repair assays. In these, the compound is tested for killing of DNA repairdeficient strains relative to repair-proficient parent strains. Increased killing of the DNA repair-deficient strain relative to the repair-proficient strain is evidence of induction of DNA damage which could not be repaired by the repair-deficient strain prior to cell division, thus leading to cell death (Kada et at. 1984). Norfloxacin, fleroxacin (AM 833) and ofloxacin
215
were positive in a DNA repair assay in Bacillus subtilis (Corrado et at. 1987; Hosomi et at. 1988; Irikura & Hosomi 1981; Mayer & Bruch 1986; Physicians' Desk Reference 1991 b; Shimada et at. 1984). Nalidixic acid and oxolinic acid were positive in Escherichia coli, Salmonella typhimurium and B. subtilis DNA repair tests (Akema et at. 1978; McCoy et at. 1980; McDaniel et at. 1978; Rosenkranz & Leifer 1980), while ciprofloxacin was negative in E. coli (Physicians' Desk Reference 199Ia). 1.2 Unscheduled DNA Synthesis in Mammalian Cells Unscheduled DNA synthesis (UDS) assays measure the uptake of radiolabelled thymidine or thymidine analogues into DNA of nonreplicating cells (Hanawalt 1977). When using cells which would normally undergo S phase DNA synthesis during the assay it is common to apply an inhibitor of replicative DNA synthesis, such as hydroxyurea. However, when using cells such as rat liver hepatocytes, such an S phase inhibitor is usually not used since these cells do not normally undergo cell division. This model is popular because of the metabolic capabilities inherent in the cell and because both in vitro and in vivo/in vitro (drug treatment in vivo and UDS measurement in vitro) assays are easily performed. A positive UDS result is interpreted as a DNA repair response (nonreplicative DNA synthesis), which occurs during excision DNA repair as a result of DNA damage inflicted by the test agent. Ciprofloxacin, pefloxacin, norfloxacin and ofloxacin were all positive in the in vitro primary hepatocyte UDS assay (Mayer 1987; McQueen & Williams 1987; Physicians' Desk Reference 199Ia). However, norfloxacin has also been reported to be negative for UDS in human and mouse skin fibroblasts as well as rat hepatocytes (Hosomi et at. 1988). Similarly, ofloxacin was negative when tested for UDS in human diploid fibroblasts in culture (Mayer 1987; Shimada et at. 1984) and ciprofloxacin was negative in an in vivo/in vitro UDS assay (Schluter 1986). Ciprofloxacin was positive for UDS when tested in human lymphocytes in culture
216
(Bredberg et al. 1989). Nalidixic acid was shown to stimulate UDS in rat splenic and thymic cells in culture (Tempel & Spath 1987), but was negative for UDS in primary rat hepatocyte cultures (McQueen & Williams 1987). Aeroxacin was negative for UDS in human and mouse skin fibroblasts as well as rat hepatocytes (Hosomi et al. 1988). However, DNA gyrase-like activity has been observed in rat liver mitochondria (Castora et al. 1983). It has therefore been suggested that inhibition of mitochondrial DNA synthesis by DNA gyrase inhibitors may lead to false positive results in UDS assays by reducing the cytoplasmic background grain counts that are normally subtracted from nuclear counts to determine net nuclear counts, thus falsely elevating the net nuclear grain counts (Christ et al. 1988). Nalidixic acid and oxolinic acid inhibit rat liver mitochondrial DNA gyrase (Castora, et al. 1983). Ciprofloxacin did not inhibit DNA synthesis in rat liver mitochondria (Forsgren et al. 1989). Therefore, the above argument may hold for some quinolones but not for others. This could only be determined by reporting cytoplasmic background counts with UDS results, which is virtually never done. 1.3 DNA Strand Breakage in Mammalian Cells DNA strand breakage may be induced directly by a chemical or physical (e.g. ionising radiation) agent or may occur secondary to enzymatic action occurring subsequent to DNA damage (e.g. during DNA excision repair). In the case of DNA gyrase inhibitors this may occur secondary to interference with topoisomerase II (see discussion). Ciprofloxacin, ofloxacin and norfloxacin caused DNA strand breakage in a human Iymphoblastoid cell line in culture (Bredberg et al. 1989). Ciprofloxacin caused DNA strand breakage in human lymphocytes in vitro (Forsgren et al. 1987). Nalidixic acid has been shown to cause DNA crosslinking in human amnion cells in culture (Rosenkranz & Lambek 1965) but apparently did not cause DNA strand breakage in rat splenic and thymic cells
Drug SafelY 7 (3) 1992
even though UDS was stimulated in these cells (Tempel & Spath 1987). Oxolinic and pipemidic acid caused DNA strand breakage in granuloma pouch cells when given orally to rats (Maura & Pino 1988). Norfloxacin caused DNA strand breakage in fetal tissues of rats in vivo when given at 30 times the clinical dose (Pi no et al. 1991). However, cinoxacin did not cause strand breakage in the liver or kidney in vivo in rats when given orally (pino et al. 1989), and fleroxacin and norfloxacin did not cause DNA strand breakage in mouse L-121O or human IMR-90 cells in culture (Hosomi et al. 1988).
2. Sister Chromatid Exchanges Sister chromatid exchanges (SCE) result from the exchange of equal regions of chromatin between homologous chromatids of a chromosome during late S phase of the cell cycle. Although the significance or mechanisms of the phenomenon are not known, SCE are known to commonly occur in response to DNA-damaging agents (Perry & Thomson 1984). Nalidixic acid was positive for the induction of sister chromatid exchanges in human lymphocytes from drug-treated patients (Kowalczk 1980). Norfloxacin and ofloxacin were negative in this assay in both human lymphocytes and Chinese hamster lung cells cultured in vitro (Corrado et al. 1987; Irikura & Hosomi 1981 ; Kullich et al. 1988; Mayer 1987; Shimada et al. 1984). CP-67015 was weakly positive in vitro in human lymphocytes but negative in mouse bone marrow in vivo (Holden et al. 1989).
3. Chromosomal Aberrations 3.1 In Vitro Norfloxacin, ofloxacin, fleroxacin, cinoxacin, ciprofloxacin and nalidixic acid were negative for induction of chromosome aberrations in vitro in cultured cells (Corrado et al. 1987; Forsgren et al. 1987; Hosomi et al. 1988; Irikura & Hosomi 1981 ; Shimada et al. 1984; Shiratori & Takase 1980; Stenchever et al. 1970). However, CP-67015 was
Mutagenicity of Quinolones
positive in human lymphocytes and CHO cells (Holden et al. 1989). 3.2 In Vivo Norfloxacin was negative for chromosome aberrations in vivo in rat and hamster bone marrow (lrikura et al. 1981; Physicians' Desk Reference 1991 b). Cinoxacin was negative for chromosomal aberrations in mouse bone marrow (Shiratori et al. 1980). Ciprofloxacin and ofloxacin did not cause chromosomal aberrations in lymphocytes from patients treated with the drugs (Mayer 1987; Mitel man et al. 1988). Ciprofloxacin, cinoxacin, CP-670 15 and ofloxacin were negative in the mouse micronucleus test (Carlin 1975; Holden et al. 1989; Mayer 1987; Physicians' Desk Reference 1991 a; Shimada et al. 1984). Micronuclei are chromatin fragments that become separated from the nuclear material during mitosis and end up in the cytoplasm of a daughter cell. These are most commonly measured in polychromatic erythrocytes (PCE) in the bone marrow of rodents. Normal rodent PCE have no nucleus since this is the stage immediately after nuclear expulsion in erythrocyte development. Nuclear material detected by staining in these cells represents micronuclei which are the end products of either chromosome fragmentation or interference with the mitotic spindle apparatus (Heddle et al. 1984). Ciprofloxacin, norfloxacin, ofloxacin and nalidixic acid were negative in the mouse dominant lethal test (Corrado et al. 1987; Irikura et al. 1981; Mayer 1987; Physicians' Desk Reference 199Ia,b; Shimada et al. 1980, 1985). The dominant lethal test is conducted by treating males with test compound for I week and then mating them to untreated females over a period of about 8 weeks using a different group of females each week. The end-point is the number of early postimplantation deaths in pregnant females. An increase in such deaths is evidence for germ line chromosomal damage in the male (Bateman 1984).
217
4. Mutagenicity 4.1 Bacterial Nalidixic acid was positive in a multigene sporulation assay in B. subtilis (a test for mutation induction in bacteria) [Sacks & MacGregor 1982]. Ciprofloxacin, fleroxacin, enoxacin, norfloxacin, ofloxacin, oxolinic acid, nalidixic acid and CP67015 were negative for mutation induction in S. typhimurium (Ames test) using DNA repair-deficient strains (Corrado et al. 1987; Gocke 1991; Holden et al. 1989; lrikura & Hosomi 1981; Mayer 1987; McCoy et al. 1980; Physicians' Desk Reference 1991 a, b; Sacks & MacGregor 1982; Shimada et al. 1984; Ysern et al. 1990). However, nalidixic acid, norfloxacin, oxolinic acid, ciprofloxacin, enrofloxacin, enoxacin, fleroxacin and ofloxacin were positive in strains which were DNA repair-proficient (Gocke 1991; Levin et al. 1984; Phillips et al. 1987; Ysern et al. 1990). Ciprofloxacin, norfloxacin, fleroxacin, nalidixic acid and ofloxacin were negative for reverse mutation in DNA repairdeficient E. coli (Hosomi et al. 1988; lrikura & Hosomi 1981; McCoy et al. 1980; Physicians' Desk Reference 1991 a; Shimada et al. 1984; Witkin & Wermundsen 1979). However, nalidixic acid was positive for mutation induction in proliferating cultures of DNA repair-proficient E. coli (Cook et al. 1966; Witkin & Wermundsen, 1979). These results are interpreted to mean that DNA repair in bacteria is required in order for the actions of the DNA gyrase inhibitors to be converted to mutations. A sequence of events can be envisioned where: (a) the gyrase inhibitors inflict DNA damage as demonstrated by the DNA repair test mentioned above; (b) the DNA damage tends to cause cell killing unless repaired, and (c) errors are made during the repair process which lead to the fixation of mutations in the DNA. 4.2 Mammalian In Vitro Ciprofloxacin, ofloxacin, fleroxacin, nalidixic acid and norfloxacin were negative in an hypoxanthine-guanine-phosphoribosyltransferase (H GPR T) forward mutation assay in Chinese hamster V79
218
cells (Corrado et al. 1987; Hosomi et al. 1988; Mayer 1987; Physicians' Desk Reference 1991a,b; Schluter 1986). Cinoxacin, oxolinic acid and pipemidic acid were negative for HGPRT forward mutation in rat granuloma pouch cells when injected directly into the granuloma pouch or given orally (Maura & Pino 1988; Pino et al. 1989). However, CP-67015 was positive for HGPRT mutation in CHO and V79 cells (Holden et al. 1989). Both norfloxacin and fleroxacin were negative for induction of ouabain-resistant mutants in Chinese hamster V79 cells (Hosomi et al. 1988). Ciprofloxacin, norfloxacin, ofloxacin, pefloxacin and nalidixic acid were positive in the mouse lymphoma thymidine kinase forward mutation assay (Physicians' Desk Reference 1991 a; Schluter 1986). 4.3 In Vivo
Nalidixic acid was mutagenic in vivo in the sexlinked recessive lethal test in Drosophila (Filippova & Efremova 1974). This was the only test reported for mutation induction in vivo in any species with quinolones. The above results are summarised in table I by listing a drug as positive for a test category if reported positive in any of the reports cited above. A drug may have been reported negative for a particular test category in 1 or more papers, but if it was reported positive in at least 1 paper for the test category, it is listed as positive in the table.
5. Discussion The inconsistent assay results obtained from the literature were surely influenced by the assay conditions used, in particular by the concentrations of test compound, the test system and the protocol for a given test. Study designs by different investigators varied widely in these aspects. The variability in results among quinolones studied and among test systems could at least partially be due to the affinity of the various quinolones for mammalian topoisomerase (they were designed and selected on the basis of activity against bacterial rather
Drug Safety 7 (3) 1992
than mammalian topoisomerase) and the ability of the drugs to enter mammalian cells in order to affect the enzymes. Nevertheless, a general pattern seems to emerge that, with exceptions, the quinolone antibacterials as a class tend to be positive in assay systems for DNA damage (such as UDS and strand breakage), mutagenicity in DNA repair-proficient bacteria and at the thymidine kinase locus in mouse lymphoma cells, but negative in assays for chromosomal aberrations or mutagenicity at the HGPRT or Na+,K+-ATPase loci in mammalian cells. It is not surprising that compounds of this class might be positive in tests for DNA damage since they inhibit topoisomerase II (DNA gyrase) in bacterial cells [Shen et al. 1989] and may have activity against similar enzymes in eukaryotic cells. For example, nalidixic acid, oxolinic acid and pefloxacin inhibit several eukaryotic enzymes involved in DNA replication (Rusquet et al. 1984). Ciprofloxacin, norfloxacin, nalidixic acid and ofloxacin have been shown to inhibit calf thymus topoisomerase I and II (Hussy et al. 1986; Sato et al. 1989). Reroxacin has also been reported to inhibit calf thymus topoisomerase II (Sato et al. 1989). However, nalidixic acid and oxolinic acid did not inhibit topoisomerases I and II from rat liver (Duguet et al. 1983). Eukaryotic topoisomerase II allows unwinding and relaxation of DNA supercoils during DNA replication and transcription (Vosberg 1985). As part of its action the enzyme creates double strand breaks in the DNA held together by the enzyme (Cozzarelli 1980). Interference with this process could result in the appearance of single or double strand breaks in the DNA. Additionally, it is known that protein-DNA crosslinks can be recovered from mammalian cells when topoisomerase enzymes are blocked (Liu et al. 1983; Nelson et al. 1984). When the enzyme creates double strand breaks and holds the DNA together at the site of these breaks, it does so by means of DNA-protein crosslinks between the DNA and the enzyme (Cozzarelli 1980). It is not unreasonable to expect that DNA double strand breaks and DNA-protein crosslinks created by interference with topoisomerase enzymes would be
Mutagenicity of Quinolones
219
Table I. Quinolone mutagenicity summary NOR
DNA damage Bacterial + Mammalian in vitro UDS + in vivo/in vitro UDS DNA breakage + SCE Chromosome aberrations In vitro In vivo Mutagenicity Bacterial repair deficient repair proficient Mammalian Na+,K+-ATPase HGPRT thymidine kinase + Drosophila
=
CIP
OFL
CIN
PEF
PIP
OXO
+ +
+
+
+
NAL
FLE
+
+
ENR
CPS
ENO
+
+
+
+
+ +
+ +
+
+
+
+
+
+
+
+
+ +
+ +
+
=
+
=
Abbreviations: UDS unscheduled DNA synthesis; SCE sister chromatid exchange; HGPRT hypoxanthine-guaninephosphoribosyltransferase; NOR norfloxacin; CIP ciprofloxacin; OFL ofloxacin; PEF pefloxacin; CIN cinoxacin; PIP pipemidic acid; OXO oxolinic acid; NAL nalidixic acid; FLE fleroxacin; ENR enrofloxacin; CP6 CP-67015;
=
= = ENO = enoxacin; + = positive; - = negative.
=
=
detected as DNA damage by assays which detect damage directly (e.g. DNA strand breakage) and indirectly (e.g. DNA repair or unscheduled DNA synthesis). The mouse lymphoma thymidine kinase assay is known to be more sensitive to certain mutagens because of the shorter expression time required with the cell line and because the thymidine kinase locus assay also detects chromosomal mutagens, whereas the HGPRT and Na+,K+ATPase loci do not (Applegate & Hozier 1987). The pattern of positive results at the thymidine kinase locus and mostly negative results at other loci (HGPRT and Na+,K+-ATPase) in mammalian cells can most likely be interpreted to suggest that multilocus effects are being induced by these agents (Applegate & Hozier 1987; Evans et al. 1986). Interference with topoisomerase II is believed to be involved in the antitumour and cytotoxic action of amsacrine (m-AMSA) and o-AMSA (DeMarini et al. 1987a; Nelson et al. 1984), teni-
=
=
=
=
= =
poside and etoposide (DeMarini et al. 1987b; Yang et al. 1985) and doxorubicin (adriamycin) [Tewey et al. 1984]. However, the pattern of mutagenic activity with these compounds appears to be different from that with the quinolone antibacterials with the exception of CP-67015. The antitumour compounds are clastogenic (i.e. cause chromosome aberrations) [DeMarini et al. 1987a,b; Wilson et al. 1984], whereas the quinolones (except for CP67015) apparently lack this capability. Teniposide and m-AMSA induce SCE in cell cultures (Lim et al. 1986), whereas among the 3 quinolones tested for SeE induction only nalidixic acid and CP-67015 possess this property. Although both the quinolone antibacterials and the antitumour agents listed above are likely to exert their genotoxic effects in mammalian cells by interference with topoisomerase action, the discrepancy between their patterns of activity in the various assays for genotoxic activity suggest that the 2 classes of compounds may
220
cause their genotoxic effects via different (albeit unknown) mechanisms. CP-67015 clearly presents an exceptional case and may represent a third category with activities of both the anti tumour agents mentioned above as well as of the majority of quinolone antibacterials. Interestingly, in an assay for calf thymus to poi somerase II-induced DNA cleavage, ciprofloxacin, norfloxacin, oxolinic acid and nalidixic acid exhibited concentrations required for 50% cleavage (CC50) approximately WOO-fold greater than the anti tumour compounds ellipticine and etoposide, whereas the CC50 for CP-76015 was only about 10fold higher (Barrett et al. 1989). This is in contrast to the potency of CP-670 15 to inhibit other actions of to poi some rase II. For example, the concentrations required to cause 50% inhibition (IC50) for inhibition of P4 DNA unknotting and concatenation of pBR322 DNA were similar for DP-67015, ciprofloxacin and norfloxacin (Barrett et al. 1989). The mechanism of action postulated for positive results in the mutagenicity assays cited above, i.e. interference with enzyme action rather than a direct DNA damaging effect, has significance in terms of the assessment of risk to people exposed to quinolone antibacterials. That is, any effects leading indirectly to DNA or chromosome damage should depend on the concentration of compound reaching the enzyme-DNA target within the nucleus of the cell as wel1 as the affinity of the compound for the enzyme. This means that although some of the quinolones were positive in some of the assays for genetic toxicity, the significance of these findings to humans can be estimated on the basis of the concentrations of drug required to elicit the positive results compared to the levels of drug expected. For example, ciprofloxacin, norfloxacin, nalidixic acid and ofloxacin inhibit calf thymus topoisomerase I and topoisomerase II at concentrations at least 100 times those required to inhibit bacterial DNA gyrase (Hussy et al. 1986). Ofloxacin, ciprofloxacin, fleroxacin and nalidixic acid have also been reported to inhibit calf thymus topoisomerase II (IC50) at concentrations over 1000 times those required to inhibit E. coli DNA gyrase (minimum
Drug Safety 7 (3) 1992
inhibitory concentration) [Sato et al. 1989]. In separate studies, the ratio of the IC50S for inhibition of calf thymus topoisomerase II versus E. coli DNA gyrase were 1000 to 2000 for ofloxacin, fleroxacin and ciprofloxacin, 300 to 600 for tosufloxacin and lomefloxacin, about 50 for enoxacin and about 20 for CI-934 and nalidixic acid (Hoshino et al. 1989). These data indicate that therapeutic concentrations should be much lower than those that would pose a risk for interference with mammalian to poi some rase II activity. As mentioned earlier, only nalidixic acid has been reported to induce some evidence for mutagenic activity (SCE) in humans (Kowalczk 1980). Further estimates of the significance of positive mutagenicity results to people who ingest these compounds comes from carcinogenicity studies completed with several quinolones. Carcinogenicity studies have been completed in rats and mice with ciprofloxacin, norfloxacin and nalidixic acid and with piromidic acid in mice (Jida et al. 1977; Kurokawa et al. 1986; Morrissey et al. 1991; Physicians' Desk Reference 1991 b; Reed 1988). Ciprofloxacin and norfloxacin were negative in both rats and mice. Piromidic acid was negative in mice. Nalidixic acid was negative in CFD, mice but was equivocal1y positive in B6C3F, mice, inducing an increase in subcutaneous fibromas or fibrosarcomas in male mice, and was positive in rats, inducing preputial gland tumours in male rats and clitoral gland tumours in female rats. Since the newer quinolones (i.e. all except nalidixic acid) are more closely similar on the basis of chemical structure to norfloxacin and ciprofloxacin, it seems reasonable for prediction of risk with the newer quinolones to discount the results of carcinogenicity studies with nalidixic acid, which is the prototype first generation quinolone antibacterial. In summary, on the basis of the available data, the most likely mechanism for positive genetic toxicity results with quinolones is interference with enzyme (topoisomerase) action rather than a direct effect on DNA. Since interference with enzyme action should be a concentration-dependent phenomenon, any risk to humans of genetic toxicity from these compounds depends on the con centra-
Mutagenicity of Quinolones
tions achieved in vivo. The negative carcinogenicity data for the newer quinolones that have been tested suggest little or no risk to humans from consumption of these compounds at therapeutic concentrations.
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Correspondence and reprints: Dr Farrel L. ForI, Department of Toxicology, D468, Abbott Laboratories, Abbott Park. IL 60064, USA.
