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Assessmentofthegenotoxicityofquinoloneandfluoroquinolonescontaminatedsoilwiththe
Vicia faba micronucleustest

Article in Ecotoxicology and Environmental Safety · January 2012

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 Ecotoxicology and Environmental Safety 76 (2012) 187–192

Contents lists available at SciVerse ScienceDirect

Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Assessment of the genotoxicity of quinolone and fluoroquinolones

contaminated soil with the Vicia faba micronucleus test
A. Khadra a, E. Pinelli b,c,n, M.Z. Lacroix d,e, A. Bousquet-Melou d,e, H. Hamdi a,
G. Merlina b,c, M. Guiresse b,c, M. Hafidi a
a
Laboratoire d’Ecologie et Environnement (Unité associée au CNRST, URAC 32, Unité associée au CNERS), Département de Biologie,
Faculté des Sciences Semlalia, Université Cadi Ayyad, BP 2390, Marrakech, Maroc
b
Université de Toulouse, INPT, UPS, Laboratoire Ecologie Fonctionnelle et Environnement (EcoLab), ENSAT, Avenue de l’Agrobiopole,
31326 Castanet Tolosan Cedex, France
c
CNRS, EcoLab, 31326 Castanet Tolosan Cedex, France
d
INRA, UMR1331, Toxalim, F-31027 Toulouse, France
e
Université de Toulouse, INPT, ENVT, EIP, UPS, F-31076 Toulouse, France

a r t i c l e i n f o abstract

Article history: The genotoxicity of quinolone and fluroquinolones was assessed using the micronucleus (MN) test on
Received 5 May 2011 Vicia faba roots by direct contact exposure to a solid matrix. Plants were exposed to quinolones
Received in revised form (nalidixic acid) and fluoroquinolones (ciprofloxacin and enrofloxacin) alone or mixed with artificially
7 October 2011
contaminated soils. Four different concentrations of each of these antibiotics were tested (0.01, 0.1,
Accepted 8 October 2011
1 and 10 mg/Kg) for nalidixic acid and (0.005, 0.05, 0.5 and 5 mg/Kg) for ciprofloxacin and enrofloxacin.
Available online 1 November 2011
These antibiotics were also used in mixture. Exposure of Vicia faba plants to each antibiotic at the
Keywords: highest two concentrations showed significant MN induction. The lowest two concentrations had no
Antibiotics significant genotoxic effect. The mixture of the three compounds induced a significant MN induction
Nalidixic acid
whatever the mixture tested, from 0.02 to 20 mg/Kg. The results indicated that a similar genotoxic
Ciprofloxacin
effect was obtained with the mixture at 0.2 mg/Kg in comparison with each molecule alone at 5–10 mg/Kg.
Enrofloxacin
Genotoxicity Data revealed a clear synergism of these molecules on Vicia faba genotoxicity.
Soil contamination & 2011 Elsevier Inc. All rights reserved.

1. Introduction removed during the treatment processes. They also suggested

that sewage sludge was the main source and reservoir of FQs
In recent years, public and scientific concern about the residues (Golet et al., 2003). Sewage sludge is dispersed on the
relevance of trace amounts of pharmaceuticals that occur in the fields and the antibiotics may contaminate the soil and eventually
environment has been continuously increasing (Pico and Andreu, the ground water (Hamscher et al., 2005).
2007). Numerous pharmaceuticals have been detected in waste Many drugs used in hospitals (antibiotics, cytostatic drugs) are
and natural water resources, sediments, soil and aquatic biota. designed to exhibit DNA damage toward bacteria or eukaryotic
While the pharmaceuticals are found at relatively low concentra- cells, raising concern about the human and ecological hazard of
tions (Terns, 1998; Korpin et al., 2002), elevated concentrations of hospital wastewater (Giuliani et al., 1996). Ciprofloxacin was
antibiotics (several mg/Kg levels) have been reported in manure found in concentration ranging from 0.7 to 124.5 mg/L in hospital
(Hamscher et al., 2002), sewage sludge and sewage-treated soil effluents and was assumed to be the main source of genotoxic
(Golet et al., 2003). Indeed after administration to humans, fifty to effects measured with the UmuC test in these effluents
ninety percent of these pharmaceuticals or their primary meta- (Hartmann et al., 1999). Fluoroquinolones have been reported to
bolites are rapidly excreted into wastewater. In previous works induce unscheduled DNA synthesis, DNA strand breakage, chro-
(Golet et al., 2002, 2003), Fluoroquinolones (FQs) in raw sludge, mosome damage and micronuclei formation (Mc Queen et al.,
wastewater effluents and river water samples were analyzed in 1991; Holden et al., 1989; Bredberg et al., 1991; Ciaravinco et al.,
Switzerland. They found out that a large proportion of FQs 1993; Curry et al., 1996; Gibson et al., 1998). In addition, FQs
entering wastewater plants (89–92% of the FQs mass flow) was exert other toxic and genotoxic effects on animal and human
cells. For example, exposure of Chinese hamster ovary cells or
n
Syrian hamster embryo cells to nalidixic acid (NA) and ciproflox-
Corresponding author at: Université de Toulouse, INPT, UPS, Laboratoire
Ecologie Fonctionnelle et Environnement (EcoLab), ENSAT, Avenue de l’Agrobiopole,
acin (CIP) induced cytotoxic effects and micronucleus induction
31326 Castanet Tolosan Cedex, France. Fax: þ33 5 34 32 39 01. (Gibson et al., 1998). In vitro studies with human lymphocytes
E-mail address: [email protected] (E. Pinelli). exposed to enrofloxacin (ENR) and ciprofloxacin revealed an

0147-6513/$ - see front matter & 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2011.10.012
188 A. Khadra et al. / Ecotoxicology and Environmental Safety 76 (2012) 187–192

O O
O O
O O
OH F
OH F
OH
H 3C N N N N N N N N
N H 3C N
H3C H
Nalidixic acid (NA) Ciprofloxacin (CIP) Enrofloxacin (ENR)
(MW: 232) (MW: 331) (MW: 359)

Fig. 1. Chemical structures and molecular weights of the antibiotics studied.

increase in the chromosomal aberrations, detected as chromatid Table 1

and chromosome breaks and gaps (Gorla et al., 1999). FQs have Key properties of the LUFA standard soil.
been reported to cause neonatal alterations in articulation carti-
Organic carbon (%) 2.33
lages, bone growth and tendons, both in humans and animals pH (soil:water, 1:2.5) 5.7
(Patterson, 1991; Gough et al., 1992; Hildebrand et al., 1993; Cation exchange capacity (mval/100 g) 11
Forster et al., 1996; Simonin et al., 1999; Stahlmann, 2003; Lemus Particle size (mm) distribution according to USDA (%)
et al., 2009). o 0.002 7.9
The Vicia faba micronucleus test has been shown to be 0.002–0.05 14.2
sensitive in evaluating chromosomal aberrations and assessing 0.05–2.0 77.9
Soil type Loamy sand
genotoxicity from both organic and inorganic soil contaminants
(Cotelle et al., 1999), sediment (Chen and White, 2004), organic
material such as sewage sludge or composts (De Simone et al.,
2000) and water (Miao et al., 1999; Monarca et al., 2004). No
study has been conducted to test the genotoxic potential of Table 2A
quinolones and fluoroquinolones on Vicia faba plant roots. Chromatographic, UV detection and MRM parameters used for quinolone quanti-
Because these molecules are significantly present in septic tank fication in preparation solution and in mixture.
effluents pumped (STEP) wastewater and sludge, and because
UV detection MS detection
some countries use these products in plant cultivation, these
antibiotics and more particularly quinolones and fluoroquino- tr 7SD labs tr 7 SD MRM Ecoll (%)
lones became a real environmental concern. In this work, the (n¼ 6) (min) (nm) (n¼ 6) (min) transitions
genotoxicity of quinolones (nalidixic acid) and fluoroquinolones
Nalidixic acid 9.63 7 0.008 254 9.87 7 0.014 233 o 233 38
(ciprofloxacin and enrofloxacin) (Fig. 1), alone or in mixture, were
233 o 215
evaluated using the Vicia faba micronucleus test by direct contact Enrofloxacin 4.89 7 0.030 295 5.12 7 0.034 360 o 342 36
with artificially contaminated soils. 360 o 316
Ciprofloxacin 3.37 7 0.031 295 3.607 0.029 332 o 314 38
332 o 288
2. Materials and methods

2.1. Chemicals and reagents [Mþ H-44] þ corresponding to the loss of H2O and CO2, respectively, in accordance
with the fragmentations reported in the literature (Yang et al., 2008). Antibiotic
Ciprofloxacin (CIP), enrofloxacin (ENR), nalidixic acid (NA) and maleic hydra- preparation solution and mixture were directly quantified before soil contamina-
zide (MH) were purchased from Sigma-Aldrich. The purity of the chemicals used tion with either UV detection for the higher nominal concentrations of 1 or 2 and
in this study was 4 95% for all of the antibiotics. All the other chemicals used were 10 or 20 mg/L (calibration range: 0.5–50 mg/L) or mass spectrometry for the lower
of analytical grade. Stock solutions of ciprofloxacin, enrofloxacin, nalidixic acid concentrations of 0.1 or 0.2 and 1 or 2 mg/L (calibration range: 0.1–10 mg/L).
and mixture were prepared in deionised water.

2.2. Antibiotic analysis 2.3. Vicia faba micronucleus test

Antibiotic concentrations were checked by LC/UV/MS/MS. Chromatographic LUFA standard soil was used in all direct contact experiments (Song et al.,
analyses were performed on a Thermofinnigan Surveyors HPLC system with diode 2007). Some key characteristics of the LUFA standard soil are presented in Table 1.
s
array detector (DAD) and a LCQ Deca XP Max ion trap mass spectrometer The Vicia faba seeds were prepared according to Ma et al. (1995), El Hajjouji et al.
(Thermo Electron Corporation, Waltham, Mass, USA). Separation was carried out (2007) and Marcato-Romain et al. (2009). Dry Vicia faba seeds were soaked for
with a Luna C18 column (100  2.0 mm; 3 mm, Phenomenex, Torrance, CA, USA) at 24 h in deionised water, the seed coats were removed and the seeds left to
40 1C. A gradient elution was used at a flow rate of 200 mL/min with a mobile germinate between two layers of moist cotton. After 5 day, the primary roots,
phase of acetonitrile 0.1% formic acid (A) and water 0.1% formic acid (B) in about 2–3 cm in length, were selected for the MN assay and their tips were cut off
the following conditions: 0–1 min, 90% A; 1–8 min, 40% A; 8–10 min, 40% A; to promote the growth of the secondary roots. For each experiment, five plants
10–11 min, 90% A and 11–15 min, 90% A. The DAD wavelengths were set at were used as five independent replicates per treatment.
254 nm and 295 nm and molecules were ionized with an electrospray ionization After the germination period, the direct contact method developed by
source in positive mode (ESIþ ). The spray needle was set at a potential of 5 kV. Marcato-Romain et al. (2009) was used by placing germinated roots in the LUFA
Capillary voltage and temperature were 10 V and 350 1C, respectively. Sheath gas standard soil for the assessment of the effect of the three antibiotics. Four different
and auxiliary gas flow rate of nitrogen were set at 45 and 10 (arbitrary units), concentration levels of each of these molecules were tested (0.01, 0.1, 1 and
respectively. Helium was used in the trap as damping and collision gas. Collision 10 mg/Kg) for NA and (0.005, 0.05, 0.5 and 5 mg/Kg) for CIP and ENR. A two-fold
energies (Ecoll) were optimized for each antibiotic (Table 2A). CIP, ENR and NA higher concentration range was used for NA because its potency against suscep-
were assayed by HPLC with UV detection and mass spectrometry (MS) using the tible bacteria is at least two-fold lower than that of CIP and ENR. For each
selection reaction monitoring mode (SRM). The detection parameters are reported concentration, five replicates were processed. These antibiotics were also applied
in Table 2A. in mixture. In these cases, the mixture 0.02 mg/Kg corresponds to the mixture of
In mass spectrometry, the parent ion of CIP, ENR and NA was the protonated NA at 0.01 mg/Kg, CIP and ENR at 0.005 mg/Kg. The same mixtures were realized
molecular ions [Mþ H] þ and their fragment ions were [M þH-18] þ and/or with final concentrations of 0.2, 2 and 20 mg/Kg of each of these compounds.
 A. Khadra et al. / Ecotoxicology and Environmental Safety 76 (2012) 187–192 189

The maximal concentration corresponded to the fluoroquinolones analyzed in Table 3

the sewage sludge and sewage-treated soil (Golet et al., 2003). For each condition Evaluation of genotoxic effect of quinolones using the micronucleus test. Vicia faba
tested, moisture at 2/3 of water holding was maintained by introducing solutions roots were exposed by direct contact to soil contaminated by antibiotics at four
prepared of antibiotics at the bottom of the pots to avoid possible anoxia. Positive different concentrations.
controls were made of LUFA standard soil whose moisture was maintained using a
solution of maleic hydrazide (10  5 M). LUFA standard soil wetted with distilled % Mitotic cells % MN % Effect
water was used as negative control. The total duration of the experimentation was
3 days. Negative control 7.4 70.4 1.6 7 1.21 0
In all experiments, at least five root tips were collected per plant that is to say Positive control (MH) 5.9 70.9 24.6 7 1.42nn 100
at least 25 tips per treatment. Root tips were rinsed with distilled water, fixed in
aceto-ethanol (1:3, v/v) at 4 1C overnight, rinsed again with deionized water for Nalidixic acid
10 min and transferred to ethanol before storage. Then, root tips were hydrolyzed 0.01 mg/Kg 7.1 70.6 2.3 7 0.5 3.04
with 1 N HCl for 6 min at 60 1C and squash preparations were stained with 1% 0.1 mg/Kg 7.6 70.8 3.3 7 1.5 7.39
aceto-orcein for 3 min at 60 1C. Five slides were prepared for each of the five seeds 1 mg/Kg 7.2 71.1 6.5 7 0.5nn 21.3
(one slide per root tip) and at least 1500 cells were counted per seedling, i.e. MN 10 mg/Kg 7.5 71.2 12.8 7 2.8nn 48.7
frequency was obtained from at least six thousand cells per treatment. The Ciprofloxacin
interphase cells as defined by Ma et al. (1995) were scored for MN frequencies 0.005 mg/Kg 7.0 70.3 1.6 7 1.1 0.01
at 400  magnification. Mitotic index (MI) was expressed in % while MN 0.05 mg/Kg 6.5 70.6 2.1 7 0.8 2.17
frequencies were expressed in per 1000 cells. In order to avoid underestimation 0.5 mg/Kg 6.8 70.4 6.2 7 0.6nn 20.0
of MN frequency due to impaired cell proliferation rate, the MN test was 5 mg/Kg 6.7 70.8 11.9 7 1.2nn 44.8
performed only in the roots tips with a mitotic index greater than 2% (Ma et al.,
1995). Statistical analysis was performed on the data: the Mann–Whitney U-test Enrofloxacin
was used to determine the significance level against the negative control in each 0.005 mg/Kg 6.7 70.2 1.2 7 0.9 0.01
experimental MN test series (Béraud et al., 2007). 0.05 mg/Kg 7.1 70.4 2.3 7 0.5 3.04
0.5 mg/Kg 6.0 70.9 5.2 7 0.4nn 15.7
5 mg/Kg 6.6 70.6 8.5 7 1.3nn 30.0

3. Results Each value represents mean 7 SD of 5 independent experiments. n and nn represent

statistically significant value at p o0.05 and p o 0.01, respectively.
3.1. Quinolone and fluoroquinolones analysis

Table 4
NA, ENR and CIP measured concentrations of preparation Mitotic index and MN frequency values in Vicia faba root exposed by direct contact
solutions and mixtures are reported in Table 2B. Results of the to the mixture of the three antibiotics at different concentrations.
intermediate concentrations (1 or 2 mg/L) assayed by both LC/UV
and LC/MS showed a very high correspondence between the two Treatments % Mitotic cells % MN % Effect
methods (not shown). These results also showed good correspon-
Negative control 6.97 0.4 2.0 71.25 0
dence between nominal and measured concentrations. Positive control (MH) 6.47 0.5 22 71.75nn 100

Mixtures
3.2. Genotoxicity evaluation of antibiotic Mixture 0.02 mg/Kg 7.15 7 0.3 6.7 70.76nn 23.5
Mixture 0.2 mg/Kg 6.67 0.3 11 70.48nn 45.0
Mixture 2 mg/Kg 6.07 0.5 16 71.62nn 70.0
The genotoxic activities of quinolones (NA) and fluoroquino-
Mixture 20 mg/Kg 6.87 0.28 20.4 71.85nn 92.0
lones (CIP and ENR) evaluated by the Vicia faba Micronucleus test
are shown in Table 3. For all the antibiotics tested, the mitotic Each value represents mean 7 SD of 5 independent experiments. n and nn represent
index was greater than 2%, and not significantly different from the statistically significant value at p o0.05 and p o 0.01, respectively.
two controls. At the two lowest concentrations, the three anti-
biotics had no significant effect on MN frequency compared to the conditions, micronucleus induced by enrofloxacin increased by
negative control. At the two highest concentrations MN frequen- only 60% (5.2 to 8.5).
cies increased dose dependency. The most marked effect was
observed in Vicia faba roots exposed to the highest concentrations 3.3. Genotoxicity evaluation of antibiotic mixtures
(5 or 10 mg/Kg), reaching 30–49% of the effect obtained in the
positive control (Table 3). The 10-fold increase of concentrations The results of the genotoxicity assessment of quinolone
(from 1 to 10 mg/Kg for NA and from 1 to 5 mg/Kg for CIP) was mixtures are presented in Table 4. For all the experiments, the %
associated with a 2 fold increase of the number of micronucleus: of mitosis was always higher than 2% and not significantly
from 6.5 to 12.8% and 6.2 to 11.9%, respectively. In the same different from the negative control. Results revealed a significant
and concentration dependent MN induction whatever the mix-
Table 2B ture tested, from 0.02 to 20 mg/Kg. In these experimental condi-
Nominal concentrations of 10 and 20 mg/L: LC/UV assay. Nominal concentration tions, MN induction increased dose dependency. The maximal
of 1 and 2 mg/L: mean values of both LC/UV and LC/MS assays. Nominal
concentration of 0.1 and 0.2 mg/L: LC/MS assay.
genotoxic effect obtained with the mixture at 20 mg/Kg repre-
sented 92% of the effect obtained with the positive control.
Mean concentration (mg/L) and SD (n¼3) Interestingly, the range of maximal effects obtained with each
molecule alone at 5–10 mg/Kg (30–49%) was obtained with the
Nalidixic acid Enrofloxacin Ciprofloxacin
mixture at 0.2 mg/Kg (45%).
Nominal Measured Nominal Measured Nominal Measured
mean (SD) mean (SD) mean (SD)
4. Discussion
0.2 0.208 7 0.001 0.1 0.121 7 0.006 0.1 0.106 7 0.007
2 2.09 7 0.078 1 0.957 7 0.031 1 0.945 7 0.107
The compounds studied in this work were described to be
20 22.8 7 0.208 10 10.4 7 0.184 10 11.4 7 0.311
‘‘readily biodegradable’’, with a low biodegradation rate
Mixture (Kümmerer et al., 2000). These physico-chemical properties
0.2 0.275 7 0.055 0.1 0.118 7 0.001 0.1 0.101 7 0.002
2 2.12 7 0.214 1 1.1 7 0.124 1 0.924 7 0.144
coupled to these antibiotic effects explain their high concentra-
tions in sewage sludge and sewage-treated soil (Golet et al.,
190 A. Khadra et al. / Ecotoxicology and Environmental Safety 76 (2012) 187–192

2003). In view of the genotoxic character of quinolones and 0.2–0.4 mg/L for CIP. It is important to emphasize that all these
fluoroquinolones, it was important to evaluate their effect in soil, compounds are designed to inhibit the development of micro-
by direct contact with whole plant roots. For this purpose, the organisms and the high sensitivity observed remains normal.
Vicia faba micronucleus (MN) test has been used with a method For CIP, the concentrations were 1000 to 10,000 fold lower than
developed by Marcato-Romain et al. (2009). The Vicia faba those inducing chromosomic aberrations in human lymphocytes
micronucleus test is a very sensitive and useful method that (5–25 mg/L) or in V79 Chinese hamster lung cells (200 mg/L). The
allows detection of both clastogenic and aneugenic effects (El significant induction of MN obtained in our experimental condi-
Hajjouji et al., 2007). Micronuclei are the result of chromosome tions with concentration of quinolones ranging between 0.5 and
breaks or mitotic anomalies that require a passage through 1 mg/Kg corresponding to 1 and 2 mg/L, respectively, revealed the
mitosis to be recognizable. high sensitivity of this test, which is 10 to 100 fold more sensitive
The substances investigated represent a clinically important than the mammalian cell assay. The higher sensitivity observed
type of antibiotic drugs that are structurally related to nalidixic with the Vicia faba MN test compared to the mammalian cell
acid. These compounds target the A subunit of DNA gyrase and assay reveals firstly the rapid cross-reactivity of quinolones and
are effective inhibitors of this enzyme (Curry et al., 1996). The more particularly fluoroquinolones for the enzymes of the plants
most active compounds with the broad antibacterial spectrum are replication system and secondly the role of these two antibiotics
the C-6 fluorinated quinolones and 1-8-naphthyridines that have in the MN induction. These results also suggested (i) the mobility
a cyclic amino group at position 7 (Radl, 1990) as ENR and its of these compounds from soil to roots to promote MN induction,
principal metabolite CIP (Gorla et al., 1999). However, quinolones even if CIP and ENR have been described to be absorbed into the
have exhibited varying degrees of cross-reactivity with mamma- solid phase (Pico and Andreu, 2007), and (ii) the exposure
lian topoisomerase II, which is a gyrase-like enzyme, and certainly conditions with contaminated soil do not represent a limiting
with other replication enzymes (Bredberg et al., 1991). These factor for the genotoxicity assessment.
compounds stabilize DNA–gyrase complex and stimulate topoi- The results in Table 4 highlight a strong synergism in MN
somerase II cleavage of DNA (Robinson et al., 1991). Fluoroqui- induction when the three molecules were added altogether. In
nolones also showed cross reactivity with other enzymes involved this condition, MN induction was significant at the lowest con-
in the process of DNA replication (Bredberg et al., 1991). In centration tested. The number of MN induction observed at the
addition, these compounds induced a variety of genotoxic effect nominal concentration of 0.02 mg/Kg was more than 3 fold higher
due to their ability to inhibit topoisomerase II activities, including than the negative control, whereas each compound tested sepa-
induction of transient DNA strand breaks during replication, rately presented negative effects at concentrations of 0.1 mg/Kg
chromosome condensation and disjunction during meiosis (NA) and 0.05 mg/Kg (ENR, CIP). It is noteworthy that the level of
(Ferguson and Baguley, 1994; Heisig, 2009). In plants, topoisome- genotoxicity reached by the 0.02 mg/Kg mixture (23.5% of the
rase II exists, and exerts the same role in DNA replication and cell positive control) was achieved with each molecule alone at 0.5 or
proliferation (Fukata et al., 1986; Reddy et al., 1999). 1 mg/Kg (Tables 3 and 4). Such a result demonstrates a clear
The results presented in Table 3 revealed the genotoxicity of synergism of the mixture for genotoxic effects.
these compounds on Vicia faba root tips. MN induction appears In these experimentations, no sign of toxicity appears on the
significant for concentrations of 1 mg/Kg of NA, and 0.5 mg/Kg of root tips. In the same way, the number of mitoses remained
CIP or ENR. For a higher concentration of antibiotics, the number constant whatever the concentration of quinolones (NA) and
of MN increased about 5.5 to 7 fold with ciprofloxacin. Results fluoroquinolones (ENR and CIP) tested. In the last decades, several
obtained for NA are not in agreement with the negative in vitro studies suggested the concept of thresholds in genotoxicity
MN results reported by Albertini et al. (1995) and Gibson et al. (Lynch et al., 2003; Elhajouji et al., 2010). The MN test contributed
(1998) in hamster CHO-K cells exposed to this compound. In a to a large extent in understanding the dose–response relationship
review by Forte (1992), NA was reported to be negative in an for aneugens and clastogens. For topoisomerase inhibitor-induced
in vitro chromosome aberration assay. In contrast, NA was clastogenicity, this concept was also developed using MN induc-
reported to stimulate unscheduled DNA synthesis in rat splenic tion in L5178Y mouse lymphoma cells as a genetic end-point
cells and thymic cells in culture, but not in primary rat hepato- (Lynch et al., 2003). On the basis of this test, Lynch et al. (2003)
cyte. ENR and CIP have been described to induce chromosomal estimated the threshold for clastogenicity at 50 mg/mL for cipro-
aberrations in cultures of human lymphocytes at concentrations floxacin. This threshold depends on the time duration of exposure
varying between 5 and 50 mg/L for ENR and 5 and 25 mg/L for CIP and on the cell type. In Vicia faba root tips, the significant
(Gorla et al., 1999). A reduction in the mitotic index and fuzzy induction of MN appeared at the lower concentration when the
metaphases were observed at 50 mg/L of ciprofloxacin, indicating compounds were used simultaneously. These results suggest that
a cytotoxic effect of this compound. In previous studies, Albertini if a threshold exists for each molecule, the presence of the other
et al. (1995) and Curry et al. (1996) reported significant increases ones, which are known to inhibit the topoisomerase II, could act
in both chromosome aberration and in MN induction in the same synergistically to promote MN induction: 0.5/1 mg/Kg for the
range of ciprofloxacin concentration (0.3–1.8 mM) than those molecules alone vs. 0.005/0.01 mg/Kg for the mixture (Tables 3
observed by Gibson et al. (1998) in CHO-K1 cells. These results and 4). Two hypotheses could be proposed: (i) the mixture of the
indicated the higher genotoxicity of ENR and CIP in comparison three molecules reduced the threshold values of the other ones on
with NA. This is in agreement with our experimental conditions the topoisomerase II inhibition; (ii) the combination of the three
where CIP appears to be the most genotoxic compound. molecules induced other phenomena, not directly related to
The results of the literature also showed the low sensitivity of topoisomerase II inhibition. It is today difficult to clearly explain
the mammalian MN test in comparison to the SOS chromotest or this synergism, but many of these compounds exert various
the UmuC test, which revealed the positive effect of ciprofloxacin effects as mentioned above, that can interact at different levels
at 0.7 mg/L (Hartmann et al., 1999). Topoisomerases-II inhibitors in the replication system. Fluoroquinolones have been described
like CIP and ofloxacin are very strong genotoxicants in Escherichia to induce oxidative stress in bacteria (Becerra and Albesa, 2002)
coli PQ 37 (SOS chromotest) and highly mutagenic in Salmonella and in eukaryote cell lines (Pouzaud et al., 2004). The oxidative
typhimurium TA 102 (Ames test) in absence of exogenous meta- stress was described to play an important role in DNA-damage
bolizing system (Mersch-Sundermann et al., 1994). DNA dama- induction (Halliwel, 1990). So, the combination of different
ging effect could be observed in concentration of approximately adverse effects such as inhibition of topoisomerase II activities
 A. Khadra et al. / Ecotoxicology and Environmental Safety 76 (2012) 187–192 191

and oxidative stress, which also leads to DNA break-down, could Forster, C., Kociok, K., Shakibaei, M., Merker, H.J., Stahlmann, R., 1996. Quinolone
promote synergistic effect on MN induction. induced cartilage lesions are not reversible in rats. Arch. Toxicol. 70, 474–481.
Forte, F.L., 1992. Mutagenicity of quinolone antibacterials. Drug Saf. 7, 214–222.
So plant genotoxicity assays as the MN test on Vicia Faba roots Fukata, H., Ohgami, K., Fukasawa, H., 1986. Isolation and characterization
are sensitive tests and provide reliable and quantitative muta- of DNA topoisomerase II from cauliflower inflorescences. Plant Mol. Biol. 6,
genic data, that allow to detect new mutagens or combination of 137–144.
Gibson, D.P., Mo, X., Switzer, A.G., Murphy, V.A., Ardema, M.J., 1998. Comparative
mutagens (Kristen, 1997). They can be used to develop new
genotoxicity of quinolone and quinolonyl-lactam antibacterials in the in vitro
techniques for advancing mutagenic knowledge and alternative micronucleus assay in Chinese hamster ovary cells. Environ. Mol. Mutagenesis
first-tear assays in the evaluation of possible genetic damage of a 31, 345–351.
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They can also contribute to an in situ monitoring, which can be Golet, E.M., Alder, A.C., Giger, W., 2002. Environmental exposure and risk
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sensitivity of the Vicia faba MN test to evaluate genetic alterations
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due to these chemicals. This study also highlights the genotoxicity tetracycline residues in soil fertilized with liquid manure by high performance
of the mixture of the three molecules tested at very low and liquid chromatography with electrospray ionization tandem mass spectro-
realistic concentrations, which can be found in soil treated with metry. Anal. Chem. 74, 1509–1518.
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the present study demonstrates the necessity to investigate the liquid manure. Environ. Toxicol. Chem. 24, 861–868.
ecotoxicological effects of contaminants at low concentrations Hartmann, A., Golet, E.M., Gartiser, S., Alder, A.C., Koller, T., Widmer, R., 1999.
Primary DNA damage but not mutagenicity correlates with ciprofloxacin
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concentrations in German hospital waste waters. Arch. Environ. Contam.
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Hildebrand, H., Kempka, G., Schluter, G., Schmidt, M., 1993. Chondrotoxicity of
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The authors would like to thank the CNRS (France) and the Holden, H.E., Barett, J.F., Hutington, C.M., Muehebauer, P.A., Wahrenburg, M.G.,
1989. Genetic profile of nalidixic acid and analog: a model for the mechanism
CNRST (Marocco) for their financial support (Grant PRAD number
of sister chromatid exchange induction. Environ. Mol. Mutagenesis 13,
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