Download The Frequency of Sister Chromatid Exchanges in Cultured Human

Drug and Chemical Toxicology, 1:85–94, 2006
Copyright © Taylor & Francis LLC
ISSN: 0148-0545 print
DOI: 10.1080/01480540500408663
The Frequency of Sister
Chromatid Exchanges in
Cultured Human Peripheral
Blood Lymphocyte Treated
with Metronidazole in Vitro
1525-6014
0148-0545
LDCT
Drug
and Chemical Toxicology,
Toxicology Vol. 29, No. 01, November 2005: pp. 0–0
Frequency
Çelik
and Ate[#x0015F]
of Sister Chromatid Exchanges
Ayla Çelik 1 and Nurcan Aras Ates2
1
Department of Biology, Faculty of Science and Letters, Mersin University, Mersin,
Turkey
2
Department of Medical Biology and Genetics, Faculty of Medicine, Mersin University
Mersin, Turkey
5-Nitroimidazoles are a group of antiprotozoal and antibacterial agents. Thanks to
their antimicrobial activity, these chemotherapeutic agents inhibit the growth of both
anaerobic bacteria and certain anaerobic protozoa. One of the useful drugs used in the
treatment of infections caused by Trichomonas vaginalis, Entamoeba histolytica, and
Giardia lamblia is metronidazole (MTZ). The mutagenicity of metronidazole [1(hydroxyethyl)-2-methyl-5-nitroimidazole] has been previously shown in different
prokaryotic systems but not in eukaryotic systems. The objective of this study is to
determine the mutagenic and cytotoxic effects of MTZ at plasma concentration and
higher in vitro. In this study, we evaluated the mutagenicity and cytotoxicity of MTZ in
cultured human peripheral blood lymphocytes. Doses were selected according to
plasma concentration of drug. End points analyzed included mitotic index (MI), replication index (RI), and sister chromatid exchange (SCE). An analysis of variance test
(ANOVA) was performed to evaluate the results. A significant decrease (p < 0.001) in
MI and RI as well as an increase in SCE frequency (p < 0.001) was observed compared
with control cultures. Our results indicate the genotoxic and cytotoxic effect of MTZ in
cultured human peripheral blood lymphocytes at plasma concentrations slightly higher
than encountered therapeutically
Keywords Human peripheral blood lymphocytes, Metronidazole, Mitotic index,
Replication index, Sister chromatid exchange
Address correspondence to Dr. Ayla Çelik, Mersin University, Faculty of Science and
Letters, Department of Biology, 33342 Mersin, Turkey; Fax: +90 324 361 00 47;
E-mail: [email protected]
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Çelik and Ates
INTRODUCTION
Nitroheterocyclic chemicals have a wide variety of applications, ranging
from food preservatives to antibiotics. 5-Nitroimidazoles are a well-established group of antiprotozoan and antibacterial agents. They have a heterocyclic structure consisting of an imidazole-based nucleus with a nitro group,
NO2, in position 5. These chemotherapeutic agents inhibit the growth of both
anaerobic bacteria and certain anaerobic protozoa, such as Trichomonas vaginalis, Entamoeba histolytica, and Giardia lamblia. 5-Nitroimidazoles are generally administered before various surgery operations (Castelli et al., 1997).
Since introduction of metronidazole [MTZ; 1-(2-hydroxyethyl)-2-methyl-5nitroimidazole); CAS 443-48-1] into clinical applications, the effectiveness of
nitroimidazoles in the treatment of various diseases was counterbalanced by
the increasing evidence of their mutagenicity, carcinogenicity, and embryolethality (Dobias et al., 1994; Edwards, 1993; Knight et al., 1978; Ludlum et al.,
1988; Mudry et al., 2001).
MTZ was originally introduced to treat Trichomonas vaginalis infections.
Moreover, MTZ has been proposed as a drug of choice against Helicobacter
pylori, which is considered the etiological agent for active chronic gastritis. It is
reported that this nitroimidazole derivative binds covalently to guanine and
cytosine and produces single- and double-strand DNA breaks in different organisms (Edwards, 1993). Furthermore, MTZ has been found to induce DNA repair
mechanism such as SOS in bacteria cells and unscheduled DNA synthesis
(UDS) in human hepatocytes (De Meo, 1992). It is reported that this drug and
its metabolites have shown mutagenic activity in bacteria (Martelli et al., 1990).
MTZ has been reported to interact, bind, and damage DNA directly; however,
there are controversial data on the capacity of MTZ to induce gene mutations in
mammalian cells. According to the International Agency for Research on
Cancer (IARC), MTZ causes cancer in experimental animals, but there is no
adequate evidence in relation to carcinogenesis in humans (IARC, 1987).
Among the nitroimidazoles, metronidazole is one of the best-investigated
compounds for mutagenicity. This drug is mutagenic in bacteria. The serum
levels in man after intake of this drug are sufficient to produce mutations in
bacteria (Voogd, 1981). Controversial results have been obtained in some
studies performed with MTZ. MTZ induces an increase in the frequency of sister chromatid exchange (SCE) in an intestinal Chinese hamster cell line (Neal
and Probst, 1984). On the contrary, neither MTZ nor its metabolites induced
point mutations, SCEs, or chromosomal aberrations (CA) in mammalian cells
in vitro (Dayan and Crajer, 1982; Hartley-Asp, 1979; Lambert and Lindblad,
1980; Lambert et al., 1979; Mahood and Wilson, 1981). Considered its carcinogenic potential, it is determined that MTZ increases definite cancer types in
mice and in rats (Elizondo et al., 1996), even though negative cancer results
have also been reported in some organisms (Cohen et al., 1973).
Frequency of Sister Chromatid Exchanges
Genotoxic agents have the potential to interact with DNA and may cause
DNA damage. SCE occurs spontaneously in proliferating cells and is regarded
as a manifestation of damage to the genome. It has been commonly used as a
test of mutagenicity in order to evaluate cytogenetic responses to chemical
exposure, and dose-response relationships for different chemicals have been
reported in both in vivo and in vitro studies (Bal et al., 1998). The objective of
the current study was to evaluate the cytotoxicity by measuring the mitotic
index and the effects on cell proliferation kinetics by measuring the replication index and mutagenicity of MTZ on cultured human peripheral blood lymphocytes using the SCE test system.
MATERIALS AND METHODS
Chemical
MTZ was commercially obtained as Flagyl (parenteral solution) from
Eczacibasi Drug Company (Istanbul, Turkey).
Microscopic Evaluation
Lymphocyte Cultures and Sister Chromatid Exchange Analysis in Peripheral
Blood Lymphocytes
The study was carried out by using blood samples from two healthy nonsmoking male donors, aged 26 and 24 years. In two donors, results of clinical
routine laboratory analyses were in normal range, and the absence of exposure to known genotoxicants was considered. The dose range was selected
according to plasma concentrations of MTZ under therapeutic conditions.
Peak plasma concentration of approximately 10 μg/mL is achieved 1 h after a
single dose of 500 mg of MTZ. Therefore, parenteral solution of drug was used.
Four different concentrations of MTZ (10 μg/mL, 30 μg/mL, 100 μg/mL, and
300 μg/mL of peripheral blood lymphocyte cultures) were prepared.
Lymphocyte cultures were prepared according to the technique of
Moorhead et al. (1960) with slight modifications. Heparinized whole-blood (0.8
mL) was added to 6 mL of culture medium F 10 (Gibco, USA), supplemented
with 20% of fetal calf serum (Gibco, USA), 0.1% mL phytohemagglutinin
(Gibco), and antibiotics (10,000 IU/mL penicillin and 10,000 IU/mL streptomycin). 5-Bromo-2-deoxyuridine (9 μg/mL, BrdU, Sigma) was added to cultures
at the beginning of the 72-h incubation period at 37°C for SCE analysis. Lymphocytes were cultured in the dark for 72 h, and metaphases were blocked
during the last 1.5 h with colcemid at final concentration of 0.2 μg/mL. Mitomycin C (2 μg/mL) was used as positive control. The cells were harvested
by replacing the culture medium with KCl (0.075 M) in which cells were
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incubated for 20 min at 37°C. The cells were fixed in Carnoy’s fixative (methanol:acetic acid, 3:1 v:v) five times, and slides were kept at room temperature
overnight. Air-dried slides were stained according to fluoresence-plus Giemsa
method by Pery and Wolf (1974) with slight modification. The number of SCEs
was counted in 50 second-metaphase cells from each of the duplicate cultures
on coded slides. Thus, 100 cells were scored in blind per dose for SCEs.
Cell Proliferation Kinetic (CPK) and Mitotic Index
The mitotic index (MI) was calculated as proportion of metaphases among
the total cell population by counting a total of 1000 cells. The cell proliferation
kinetics was defined as the proportion of the relative frequency of firstdivision metaphases (M1, identifiable by uniform staining of both the sister
chromatids), second-division metaphases (M2, identifiable by differential
staining of the sister chromatids), and third and subsequent division
metaphases (M3, identifiable by nonuniform pattern of staining). Replication
index or proliferation index (RI) was calculated according to Ivett and Tice
(1982). RI is the average number of replications completed by metaphase cells
and is calculated as follows:
RI = 1×(% first division metaphases) + 2× (% second division metaphases)
+ 3× (% subsequent division metaphases)/100
Statistical Analysis
Data were compared by one-way variance analysis. Statistical analysis
was performed using the SPSS for Windows 9.05 package program. Post hoc
analysis was performed by least significant difference (LSD) test.
RESULTS AND DISCUSSION
The results of the frequency of SCEs and the values RI and MI for each dose
and control groups are depicted in Table 1. All doses of MTZ caused the
increase in the frequency of SCE and a decrease in the frequency of MI and RI.
Various screening tests has been used as an indicator of potential mutagenicity in genetic damage. Including the sister chromatid exchange assay. The
SCE test involves exchange of DNA segments between two sister chromatids
in a chromosome during replication in the cell. The increase in frequency of
SCE indicates the mutagenic effect of chemical substances. We demonstrated
an increase in the SCE frequency for four different concentrations of MTZ
compared with controls.
Because of adverse effects of MTZ, other nitroimidazoles, such as tinidazole
and ornidazole, were developed (Lamp et al., 1999; López-Nigro et al., 2003).
The genotoxicity of MTZ has been extensively studied in prokaryotic systems.
MTZ has been reported to damage DNA (Espinosa-Aquirre, 1996; Martelli
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4.79
4.98
6.64
6.69
7.17
18.30
Control
MTZ 10 μg/mL
30 μg/mL
100 μg/mL
300 μg/mL
Positive control
MMC (2 μg/mL)
2.34
2.01
2.06
1.86
1.79
1.49
RI (%)
Donor A
6.25
5.61
4.86
4.19
3.86
3.54
MI (%)
4.80
5.68
6.66
6.70
7.18
18.40
SCE/M
2.32
2.00
2.04
1.88
1.81
1.51
RI (%)
Donor B
6.26
5.62
4.84
4.21
3.84
3.56
MI (%)
RI (%)
2.33 ± 0.01
2.00 ± 0.005**
2.05 ± 0.01**
1.87 ± 0.01**
1.80 ± 0.01**
1.50 ± 0.01**
SCE/M
4.79 ± 0.005
5.33 ± 0.35*
6.65 ± 0.01**
6.69 ± 0.005**
7.17 ± 0.005**
18.35 ± 0.05**
Mean ± SE
6.25 ± 0.01
5.61 ± 0.005**
4.85 ± 0.01**
4.20 ± 0.01**
3.85 ± 0.01**
MI (%)
MMC, mitomycin C (positive control); SCE, sister chromatid exchange; MI, mitotic index; RI, proliferation index; MTZ, metronidazole; M,
metaphase.
Values are represented as mean ± SE.
*p < 0.05, **p < 0.001 compared with control cultures.
SCE/M
Groups
in vitro.
Table 1: Results of frequencies of SCE and the values RI and MI of cultured human peripheral blood lymphocytes treated with MTZ
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Çelik and Ates
et al., 1990; Tocker and Edwards, 1994), to induce single- and double-strand
breaks preferentially in adenine thymine sites (Edwards, 1993), to form
adducts with cytosine and guanine, to induce repair synthesis, and to lead
GC-CG transversions and DNA strand breaks (Reitz et al., 1991; Trinh and
Reysset, 1998). In a study performed by Ferreiro et al. (2002), it was reported
that three nitroimidazoles were genotoxic. Although antimicrobial activity
and genotoxicity of nitroimidazoles is produced by reduction of nitro group in
anaerobic conditions, genetic damage in mammals is thought to be induced
under aerobic conditions by generation of reactive oxygen species in the cytochrome P-450 (microsomal NADPH-dependent enzymes) or in mitochondria
(Edwards, 1993). It is usually believed that nitro group reduction is also
responsible for the expression of mutagenicity (Walsh et al., 1987). Genotoxicity studies performed with nitroimidazole derivatives show that the reduction
of the nitro group is responsible for mutagenic activity (De Meo et al., 1992),
and the binding of a hydroxyl group to the parent molecule causes a increase
in its mutagenic activity (Connor et al., 1977).
A significant decrease (p < 0.001) in the frequency of mitosis was detected
for treatment with MTZ under our experimental conditions. A dose-dependent
decrease was observed in the mitotic index frequency. A significant difference
was found between the doses of MTZ (10, 30, 100, 300 μg/mL) and negative
control (p < 0.001).
MI and RI are used as indicators of sufficient cell proliferation (Anderson
et al., 1988; Scott et al., 1991). MTZ exhibits a cytotoxic effect in vitro because
of its inhibition of mitotic activity. The decrease in the mitotic activity is dosedependent. These findings generally support most of the data of study
reported by López-Nigro (2003).
In RI values, significant differences were observed between negative control and cultures treated with MTZ (p < 0.001). Responses at increasing concentrations were different with statistical difference between 10 μg/mL and 30
μg/mL dose of MTZ of p < 0.05 level. On the other hand, a significant decrease
in RI is an indicator of cytostatic effect. Our results have demonstrated that
there are modifications on CPK values expressed as RI in lymphocyte cultures
exposed to MTZ. Therefore, it may be said that MTZ has cytostatic effects on
human peripheral blood lymphocyte cultures in vitro because it causes delay
in the cell cycle. Among the end points describing genotoxic damage, a significant induction of SCE was observed in all cultures treated with MTZ compared with negative control cultures. No significant difference only was
observed between the lowest doses MTZ (30 μg/mL) and medium doses (100
μg/mL).
SCEs are the cytological indication of interchanges between DNA replication products. SCE analysis appears to be a very good screning test and excellent dosimeter for exposure in in vitro and in vivo short-term experiments
(Painter, 1980; Tucker and Preston, 1996). The interpretation of SCE results
Frequency of Sister Chromatid Exchanges
as indicator of mutagenic effect is based on either a significant difference in
the SCE frequency compared with controls or a statistically significant
increase at any dose. Cytogenetic assay systems based on the detection of SCE
in peripheral blood lymphocytes are widely advocated as a sensitive screening
method for assessing genotoxic potential of suspected human mutagens and
carcinogens. Its sensitivity and reliability have made this technique one of the
most popular methods in toxicology and human biomonitoring (Çelik and
Akba0, 2005).
Our results agree with other reports obtained from in vitro and in vivo
studies that evaluated effects of nitroimidazole drugs (Ferreiro et al., 2002;
Mudry et al., 1994; Neal and Probst, 1984). Neal and Probst (1984) found that
MTZ induced a dose-related increase in SCE formation in intestinal epithelium but not in bone marrow in vivo. Ferreiro et al. (2002) reported that
nitroimidazole derivatives, such as MTZ, are DNA-damaging agents and
induce single-strand breaks in DNA under in vitro test conditions. In a study
performed by Ré et al. (1997), MTZ induced genotoxicity in human peripheral
blood lymphocytes as measured by the comet assay in vitro. In several studies
performed on animals and humans, it was reported that MTZ did not increase
the frequency of SCE (Neal and Probst, 1983). In the study reported by LópezNigro et al. (2003), two nitroimidazoles, MTZ and ornidazole (ONZ), were
investigated for genotoxicity. They reported that MTZ and ONZ have potential genotoxic and cytotoxic effect in human peripheral blood lymphocyte cultures in vitro.
In our study, 100 μg/mL dose of MTZ and 300 μg/mL dose of MTZ has an
identical effect on RI. In this context, it is concluded that the highest dose that
causes the decrease in RI is 10-fold plasma concentration of MTZ (10 μg/mL).
In addition to this, there is no difference between 100 μg/mL dose and 30 μg/
mL dose of MTZ. The data obtained from the current study reflect that MTZ
has cytostatic and cytotoxic properties and produces genotoxic effects in
human peripheral blood lymphocyte cultures in vitro.
These results indicate that there is a strong interaction between metronidazole and DNA at concentrations exceeding recommended plasma concentrations of MTZ. As indicated by Martinez-Palamo and Espinosa-Castellano
(1998), these drugs are widely used and may be used repeatedly because of
reinfections (situations sometimes worsen due to automedication). Therefore,
the identification of potential mutagenic/genotoxic characteristics of drugs
contributes to the protection of human health. Further study should be considered in patients under long-term treatment with this drug.
REFERENCES
Anderson, D., Jenkinson, P. C., Dewdeney, R. S., Franis, A. J., Godbert, P., Butterworth, K. R. (1988). Chromosome aberrations, mitogen-induced blastogenesis and
91
92
Çelik and Ates
proliferative rate index in peripheral lymphocytes from 106 control individuals of
U.K. population. Mutat. Res. 204:407–420.
Bal, F., Sahin, F. I., Yirmibes, M., Balcý, A., Menevse, S. (1998). The in vitro effect of bcarotene and mitomycin C on SCE frequency in Down’s syndrome lymphocyte cultures. Tohoku J. Exp. Med. 184:295–300.
Castelli, M., Malagoli, M., Ruberto, A. I., Baggio, A., Casolari, C., Cermelli, C., Bossa,
M. R., Rossi, T., Paolucci, F., Roffia, S. (1997). In-vitro studies of two 5-nitroimidazole derivatives. J. Antimicrob. Chemother. 40:19–25.
Çelik, A., Akba0, E. (2005). Evaluation of sister chromatid exchange and chromosomal
aberration frequencies in peripheral blood lymphocytes of gasoline station attendants. Ecotoxicol. Environ. Safety. 60:106–112.
Cohen, S. M., Ertürk, E., Von Esch, A. M., Crovetti, A. J., Bryan, J. T. (1973). Carcinogenicity of 5-nitrofurans, 5-nitroimidazoles, 4-nitrobenzenes and related compounds. J. N. C. I. 51:403–417.
Connor, T. H., Stoeckel, M., Evrard, J., Legator, M. S. (1977). The contribution of metronidazole and two metabolities to the mutagenic activity detected in urine of
treated humans and mice. Cancer Res. 37:629–633.
Dayan, J., Crajer, M. C., Deguingand, S. (1982). Mutagenic activity of 4 active-principle forms of pharmaceutical drugs. Comparative study in the Salmonella typhimurium microsome test and the HGPRT and Na+ /K+ ATPase systems in cultured
mammalian cells. Mutat. Res. 102:1–12.
De Meo, M., Vanelle, P., Bernardi, E., Laget, M., Maldonado, J., Jentzer, O., Crozet, M.
P., Dumenil, G. (1992). Evaluation of mutagenic and genotoxic activities of 48
nitroimidazoles and related imidazole derivatives by the Ames test and the SOS
chromotest. Environ. Mol. Mutagen. 19:167–170.
Dobias, L., Cerná, M., Rösner, P., Srám, R. (1994). Genotoxicity and carcinogenicity of
metronidazole. Mutat. Res. 317:177–194.
Edwards, D. I. (1993). Nitroimidazole drug-resistance mechanisms. Mechanisms of
action. J. Antimicrob. Chemother. 31:9–20.
Elizondo, G., Gonsebatt, M. E., Salazar, A. M., Lares, I., Herrera, J., Hong, E., OsrowskyWeigman, P. (1996). Genotoxic effects of metronidazole. Mutat. Res. 370:75–80.
Espinosa-Aquirre, J. J., Torre, R. A., Lares, I., Rubio, J., Dorado, V., Wong, M.,
Hernández, J. J. (1996). Bacterial mutagens in the urine of patients under tinidazole treatnent. Mutat. Res. 359:133–140.
Ferreiro, G. R., Badías, L. C., Nigro-Lopez, M., Palermo, A., Mudry, M., Elio, G. P.,
Carballo, A. M. (2002). DNA single strand break in peripheral blood lymphocytes
induced by three nitroimidazole derivatives. Toxicol. Lett. 132:109–115.
Hartley-Asp, B. (1979). Metronidazole exhibits no cytogenetic effect in the micronucleus test in mice or on human lymphocytes. Mutat. Res. 67:193–196.
IARC (International Agency for Research on Cancer). (1987). IARC Monographs on the
Evaluation of Carcinogenic Risk to Humans. Suppl. 7. Overall Evaluations of Carcinogenicity: An Updating of IARC Monographs Volumes 1 to 42. Lyon: International Agency for Research on Cancer, pp. 250–252.
Ivett, J. L., Tice, R. R. (1982). Average generation time: a new method of analysis and
quantitation of cellular proliferation kinetics. Environ. Mutagen. 4:358.
Knight, R. C., Solimowsky, I. M., Edwards, D. I. (1978). The interaction of reduced metronidazole with DNA. Biochem. Pharmacol. 27:2089–2093.
Frequency of Sister Chromatid Exchanges
Lambert, B., Lindblad, A. (1980). The effects of metronidazole on the frequency of sister chromatid exchanges and chromosomal aberrations in human lymphocytes in
vitro and in vivo. Mutat. Res. 74:230.
Lambert, B., Lindblad, A., Ringborg, U. (1979). Absence of genotoxic effects of metronidazole
and its urinary metabolites on human lymphocytes in vitro. Mutat. Res. 67:281–287.
Lamp, K. C., Freeman, C. D., Klutman, N. E., Lacy, M. K. (1999). Pharmacokinetics
and pharmacodynamics of the nitroimidazole antimicrobials. Clin. Pharmacol.
36:353–375.
López-Nigro, M. M., Palermo, A. M., Mudry, M. D., Carballo, M. A. (2003). Cytogenetic
evaluation of two nitroimidazole derivatives. Toxicol. in Vitro. 17:35–40.
Ludlum, D. B., Colinas, R. J., Kirk, M. C., Metha, J. R. (1988). Reaction of reduced metronidazole with guanosine to form an unstable adduct. Carcinogenesis 9:593–596.
Mahood, J. S., Wilson, R. L. (1981). Failure to induce sister chromatid exchanges (SCE)
with metronidazole. Toxicol. Lett. 8:359–361.
Martelli, A., Allavena, A., Robbiano, L., Mattioli, F., Giovanni, B. (1990). Comparison
of sensitivity of human and rat hepatocytes to genotoxic effects of metronidazole.
Pharmacol. Toxicol. 66:329–334.
Martinez-Palamo, A., Espinoza-Castellano, M. (1998). Amoebiasis: new understanding
and new goals. Parasitol. Today. 14:1–3.
Moorhead, P. S., Novell, W. J., Wellman, D. M., Batlips, D., Hungerford, A. (1960).
Chromosome preparations of leucocytes cultured from peripheral blood. Exp. Cell.
Res. 20:613–616.
Mudry, M. D., Carballo, M., Labal De Vinuesa, M., Gonzáles Cid, M., Larripa, I. (1994).
Mutagenic bioassay of certain pharmacological drugs: III Metronidazole (MTZ).
Mutat. Res. 305:127–132.
Mudry, M., Martinez-Flores, I., Palermo, A., Carballo, M. A., Egozcue, J., García
Caldés, M. (2001). Embryolethality induced by metronidazole (MTZ) in Rattus
norvegicus. Teratogen. Carcinogen. Mutagen. 21:197–205.
Neal, S. B., Probst, G. S. (1983). Chemically-induced sister chromatid exchange in vivo
in bone marrow of Chinese hamsters: an evaluation of 24 compounds. Mutat. Res.
113:33–43.
Neal, S. B., Probst, G. S. (1984). Assessment of sister chromatid exchange in spermatogonia and intestinal epithelium in Chinese hamster. Basic Life Sci 29:613–628.
Painter, R. B. (1980). A replication model for sister-chromatid exchange. Mutat. Res.
70:37–341.
Perry, P., Wolff, S. (1974). New Giemsa method for the differential staining of sister
chromatids. Nature 251:156–158.
Ré, J. L., De Meo, M. P., Laget, M., Guiraud, H., Castegnaro, M., Vanelle, P., Duménil,
G. (1997). Evaluation of the genotoxic activity of metronidazole and dimetridazole
in human lymphocytes by the comet assay. Mutat. Res. 375:147–155.
Reitz, M., Rumpf, M., Knitza, R. (1991). DNA single strand-breaks in lymphocytes
after metronidazole. Arzneimittelforschung 41:155–156.
Scott, D., Galloway, S., Marshall, R., Ishidate, M., Brusick, D., Ashby, J., Myhr, B.
(1991). Genotoxicity under extreme culture conditions. Mutat. Res. 257:147–205.
Tocher, J. H., Edwards, D. I. (1994). Evidence for the direct interaction of reduced metronidazole derivatives with DNA bases. Biochem. Pharmacol. 48:1089–1094.
93
94
Çelik and Ates
Trinh, S., Reysset, G. (1998). Mutagenic action of nitroimidazoles: in vivo induction of
GC → CG transversion in two Bacteroides fragilis reporter genes. Mutat. Res.
398:55–65.
Tucker, J. D., Preston, R. J. (1996). Chromosome aberrations, micronuclei, aneuploidy,
sister chromatid exchange, and cancer risk assessment. Mutat. Res. 365:147–159.
Voogd, C. E. (1981). On the mutagenicity of nitroimidazole. Mutat. Res. 86:243–277.
Walsh, J. S., Wang, R., Bagan, E., Wang, C. C., Wislocki, P., Miwa, G. T. (1987). Structural alterations that differentially affect the mutagenic and antitrichomonal
activities of 5-nitroimidazoles. J. Med. Chem. 30:150–156.