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JOURNAL OF APPLIED TOXICOLOGYJ. Appl. Toxicol. 2007; 27: 380–390Published online 30 January 2007 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/jat.1217

A sub-acute study of metronidazole toxicity assessedin Egyptian Tilapia zillii

Wagdy K. B. Khalil,1,* Mahmoud A. Mahmoud,2 Malak M. Zahran1 and Karima F. Mahrous1

1 Cell Biology Department, National Research Center, 12622 Dokki, Giza, Egypt2 Pathology Department, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt

Received 10 August 2006; Revised 27 November 2006; Accepted 29 November 2006

ABSTRACT: Metronidazole (MTZ), an antiparasitic and antibacterial compound, is one of the world’s most widely used

drugs. Despite being considered as a rodent mutagen and a carcinogen, it is still widely used in humans for the treatment

of infections with anaerobic organisms. Therefore, the main objective of the current study was to evaluate the in vivo

toxicity of MTZ using the micronucleus (MN) assay and random amplified polymorphism DNA (RAPD-PCR) analysis as

well as histopathological examination in Tilapia zillii. Moreover, the protective effect of vitamin C (VitC) against toxi-

city of MTZ was investigated in the present study. Fish were treated with three doses of MTZ (5, 10 and 20 mg l−−−−−1) alone

or in combination with VitC (200 mg kg−−−−−1 food) at several time intervals (2 days, 7 days and 14 days). The results of the

present study showed a significant effect of MTZ on micronucleus formation and changes in polymorphic band patterns

as well as induction of different histopathological alterations in Tilapia zillii. The effects of the drug were reduced when

fish were exposed to a combination of MTZ and VitC. Copyright © 2007 John Wiley & Sons, Ltd.

KEY WORDS: fish; metronidazole; RAPD-PCR; micronucleus; histopathology; vitamin C

studied in humans and rodents using different end-

points, however, controversial results have been reported

(Bendesky et al., 2002; Fahrig and Engelke, 1997). It has

been shown that fish may act as a sentinel organism in

genetic toxicology studies, as an alternative to laboratory

mammals (Cavas and Ergene-Gözükara, 2005; Van der

Oost et al., 2003; Kleinjans and Van Schooten, 2002;

Al-Sabti and Metcalfe, 1995). MN test is an effective

method for detecting unstable chromosome aberrations

(Celik et al., 2003; Choudhury et al., 2000). One of its

advantages is that it can be applied to any proliferating

cell population regardless of its karyotype. The micro-

nucleus test in fish cells (piscine micronucleus test) has

been effectively used to monitor the genotoxic potentials

of different chemicals and their complex mixtures under

both field and controlled laboratory conditions (Cavas

and Ergene-Gözükara, 2003a,b; Kolpoth et al., 1999;

Hayashi et al., 1998). Moreover, the use of molecular

markers has provided important advances in the charac-

terization and genetic variation in many species, includ-

ing yeast and mammals, as well as fish (Assem and

El-Zaeem, 2005; Gallego et al., 2005; Horng et al.,

2004). PCR-based techniques, such as RAPDs, have

previously allowed the discrimination as well as estima-

tion of genetic variation attributed to genotoxic elements.

Over the course of evolution, many animals have lost

the ability to synthesize specific substances that neverthe-

less continue to play critical roles in their metabolism

(Premkumar and Bowlus, 2003; Halliwell, 2001). Vita-

mins are among the substances required in micronutrient

Introduction

Nitroimidazoles are a well-established group of anti-

protozoan and antibacterial agents. Metronidazole (MTZ,

1-[2-hydroxyethyl]-2-methyl-5-nitroimidazole) is an anti-

bacterial and antiprotozoal drug that has been in use for

over 35 years. Currently, it is among the top 100 most

prescribed drugs in the USA (Bendesky et al., 2002) and

one of the 10 most widely used drugs during pregnancy

(Thapa et al., 1998). It appears on the essential drug list

of the World Health Organization (WHO, 1999). MTZ

was introduced as a trichomonicide in Europe by Cosar

and Julou (1959). Since then, its clinical applications

have been growing and it is now the principal treatment

for Helicobacter pylori infections, amebiasis, giardiasis,

trichomoniasis and Crohn’s disease. It has also been used

extensively to treat bacterial vaginosis and several other

anaerobic bacterial infections and as a prophylactic anti-

biotic in surgical interventions (Freeman et al., 1997).

The genotoxic activity of MTZ has been studied in

different in vivo and in vitro assays. It is mutagenic for

bacteria and induces gene mutations and recombination in

fungi (De Meo et al., 1992). Mutagenicity is an undesir-

able side effect of clinically prescribed drugs and raises

the question of their potential carcinogenicity and

genotoxicity. The genotoxic effects of MTZ have been

* Correspondence to: Dr Wagdy Khalil, Cell Biology Department, National

Research Center, El Tahrir Street, 12622 Dokki Giza, Egypt.

E-mail: wagdykh@yahoo.com

METRONIDAZOLE TOXICITY IN TILAPIA ZILLII 381

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DOI: 10.1002/jat

amounts in the diet. Vitamin deficiencies in the human

diet are generally thought to lead to DNA damage

(Franke et al., 2005). Vitamin C found in fresh fruits and

vegetables is an important micronutrient, mainly required

as a co-factor for enzymes involved in oxi-reduction reac-

tions (Edenharder et al., 2003; Ames, 2001; Halliwell,

2001). It has been studied for its protective action against

different diseases (Edenharder et al., 2003, 2002;

Vijayalaxmi and Venu, 1999). The mechanisms by which

ascorbic acid acts include bio-antimutagenic (Guha and

Khuda-Bukhsh, 2002) and desmutagenic activities (Sram

et al., 1983) as well as regulation of DNA-repair

enzymes (Lunec et al., 2002). Many of the results

reported are based on in vitro studies. Vitamin C has

been insufficiently studied for its ability to interact, either

directly or indirectly, with mutagens, especially in view

of the controversial results of its consumption on genome

stabilization (Ames, 2001; Halliwell, 2001).

The main objective of this investigation was to verify

the possible genotoxic and cytotoxic effects of MTZ as

well as to study the protective action of vitamin C against

MTZ-induced DNA damage and histopathological altera-

tions in Tilapia zillii in vivo.

Materials and Methods

Chemicals

Metronidazole (MTZ) was commercially obtained as

Amrizole in tablet form, from Amriya Pharm (Egypt).

Each tablet contains 500 mg MTZ as its active ingredient.

All reagents and chemicals were of the highest purity.

Fish

Tilapia zillii fish weighing 75 ± 10 g were purchased

from the private El-Wafaa fish farm located in Giza

governorate, Egypt. Fish were transported in large plas-

tic water containers supplied with battery aerators as a

source of oxygen. Fish were maintained on ad libitum

standard fish food at the Animal House, Faculty of

Veterinary Medicine, Cairo University (Giza, Egypt).

After an acclimation period of 1 week, fish were divided

into six experimental groups (nine fishes per group) and

each group was individually placed into a fish aquarium

containing de-chlorinated tap water, the average water

temperature was 14.5 ± 3.7 °C and the pH was in the

range of 7.2–8.2.

Experimental Design

Table 1 is intended to demonstrate the experimental design.

A total number of 54 fish were classified into six groups

(nine fishes per group). Group 1, untreated control; group

2, treated with 5 mg l−1 of MTZ; group 3, treated with

10 mg l−1 of MTZ; group 4, treated with 20 mg l−1 of MTZ;

group 5, treated with 10 mg l−1 of MTZ combined with

VitC (200 mg kg−1 food); group 6, treated with 20 mg l−1 of

MTZ combined with VitC (200 mg kg−1 food). Three fish

samples were collected from each group on day 2, 7 and

14 for DNA extraction, MN test and histopathological

examination, respectively. Blood samples were collected

from the gills of fish at the end of each experimental

period for DNA isolation. Gills of fish were collected for

MN test while tissue samples were collected from differ-

ent organs for histopathological examination.

Micronucleus Test

A drop of blood from the gills was mixed with a drop of

fetal calf serum on a glass slide and air-dried. The slide

was fixed in methyl alcohol for 5 min, then stained with

5% Giemsa for 10 min. Two thousand erythrocytes were

examined for each fish to determine the incidence of

micronucleated polychromatic erythrocytes (MnPCEs)

(Adler, 1984).

Molecular Analysis

The genomic DNA was isolated using phenol/chloroform

extraction and ethanol precipitation method with minor

modifications (Sambrook et al., 1989).

RAPD-PCR profiles from tilapia DNA were generated

using 20 primers (10-mer random primers, Table 2)

from Operon Technologies (Alameda, CA, USA). DNA

Table 1. Experimental design for assessment of MTZ toxicity in Tilapia zillii

Group Treatment Concentration (mg l−1) Time of sampling after treatmenta

1 Control —

2 MTZ 5 2, 7 and 14 days

3 10

4 20

5 MTZ + VitC 10 + 200

6 20 + 200

MTZ, metronidazole; VitC, vitamin C (mg kg−1 food); a Three fish were collected at each time of sampling (2, 7 and 14 days), No. of fish per group = 9 fish.

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cal Analysis System (SAS Institute Inc., 1982) followed

by Scheffé-test to assess significant differences among

groups (Dammaschke et al., 2005). The values are

expressed as mean ± SEM. All statements of significance

were based on probability of P < 0.05.

Results

Micronucleus Assay

MnPCEs of T. zillii treated with MTZ alone or MTZ

combined with VitC at different time intervals are

summarized in Table 3. MTZ treatments increased the

number of MnPCEs with respect to the controls. This

increase was statistically significant for the two highest

doses (10 and 20 mg l−1, Table 3). Dose–duration interac-

tions were also found to be significant in 1- and 2-week

treatment groups (P < 0.05 and P < 0.01, respectively,

Table 3). VitC was able to reduce the number of

MnPCEs in the T. zillii resulting from MTZ administra-

tion in all dose- and duration-groups (Table 3).

RAPD Fingerprinting Pattern

To evaluate the genetic variability among the treated

fish genomes and their control, 20 primers (10-mer

random primers, Table 2) were used to determine DNA

fingerprinting in T. zillii. Only seven of these primers

gave positive and detectable bands (Figs 1 and 2). They

provided a total of 348 different bands with an average

of 16.6 ± 1.4 bands per primer. Nearly the same results

were obtained when the PCR assay was performed

for each sample within each group (three fish). Of the

scorable bands, one band (0.3%) was monomorphic

(primer C20 at 631 bp), because it was present in

all groups, while 43 bands (12.6%) were monomorphic

for control and MTZ (10 and 20 mg l−1) combined

with VitC (Figs 1 and 2). However, the DNA of the

samples treated with MTZ alone revealed 192 (55.2%)

Table 2. Sequence of primers employed

Primer Sequence Primer Sequence

A01 5′-CAGGCCCTTC-3′ A15 5′-TTCCGAACCC-3′A02 5′-TGCCGAGCTG-3′ A20 5′-GTTGCGATCC-3′A03 5′-AGTCAGCCAC-3′ C03 5′-GGGGGTCTTT-3′A04 5′-AATCGGGCTG-3′ C05 5′-GATGACCGCC-3′A05 5′-AGGGGTCTTG-3′ C06 5′-GAACGGACTC-3′A07 5′-GAAACGGGTG-3′ C07 5′-GTCCCGACGA-3′A08 5′-GTGACGTAGG-3′ C09 5′-CTCACCGTCC-3′A09 5′-GGGTAACGCC-3′ C12 5′-TGTCATCCCC-3′A10 5′-GTGATCGCAG-3′ C15 5′-GACGGATCAG-3′A12 5′-TCGGCGATAG-3′ C20 5′-ACTTCGCCAC-3′

Table 3. Micronucleated polychromatic erythrocytes (MnPCEs) in T. zillii exposed to different treatments (mean ±standard error)

2 days 7 days 14 days

Treatment Conc. (mg l−1) MnPCEs/2000 PCEs

Control — 4.0 ± 0.0 4.7 ± 0.9b 6.7 ± 1.2b

MTZ 5 7.3 ± 1.9 9.7 ± 1.5ab 12.3 ± 1.8ab

10 8.0 ± 2.0 11.0 ± 2.1ab 13.3 ± 0.9ab

20 9.7 ± 2.3 15.7 ± 2.6a 15.7 ± 1.2a

MTZ + VitC 10 + 200 7.0 ± 1.5 8.0 ± 3.2ab 11.3 ± 0.7ab

20 + 200 8.3 ± 1.8 9.7 ± 0.3ab 11.7 ± 1.8ab

MTZ: Metronidazole; VitC: Vitamin C (mg kg−1 food); Conc.: MTZ concentration; Each treatment contains 3 fishes; Total counted PCEs were 6000 cells

per a group; a,b values with different superscripts within columns represent significant statistical differences (P < 0.05, Scheffé-Test); ab values with different

superscripts within columns represent no significant statistical differences (P < 0.05, Scheffé-Test).

amplification reactions were performed under conditions

reported by Luceri et al. (2000). PCR amplification was

conducted in 50 µl reaction volume containing 100 ng

genomic DNA; 100 µM dNTPs; 40 nm primer; 2.5 units

of Taq DNA polymerase and 5 µl promega 10X Taq DNA

polymerase buffer. The reactions were carried out in a

thermocycler (Perkin-Elmer 9700) programmed first for

denaturation of 5 min at 94 °C, followed by 45 cycles of

0.5 min at 94 °C, 1 min at 36 °C and 2 min at 72 °C and

finally, one cycle at 72 °C for 5 min. The PCR product was

analysed by electrophoresing 15 µl of the amplified mixture

on agarose gel. The Gel-Pro Analyzer (Media Cybernetics)

was used to document ethidium bromide DNA gels.

Histopathological Examination

For histopathological study, specimens from the liver,

spleen, kidneys, intestine and testis were collected from

each fish and fixed in 10% neutral buffered formalin.

Tissue specimens were processed routinely for paraffin

sections of 4–5 µm thickness, stained with hematoxylin

and eosin (Bancroft et al., 1996).

Statistical Analysis

All data for the micronucleus test were analysed using

the General Linear Models (GLM) procedure of Statisti-

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Figure 1. Comparison of RAPD fingerprinting profiles of different T. zillii genomic DNA treated with MTZ. (a, b,c) represent PCR products with primer A09; (d, e) represent PCR products with primer A12; (f, g, h) represent PCRproducts with primer A20. Lane 1 represents DNA marker. Lane 2 represents negative control, lanes 3, 4 and5 represent T. zillii treated with 5, 10 and 20 mg l−1 of MTZ, respectively, lane 6 represents T. zillii treated with10 mg l−1 of MTZ plus vitamin C (200 mg kg−1 food), lane 7 represents T. zillii treated with 20 mg l−1 of MTZ plusvitamin C (200 mg kg−1 food), and lane 8 represents untreated T. zillii

384 W. K. B. KHALIL ET AL.

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Figure 2. Comparison of RAPD fingerprinting profiles of different T. zillii genomic DNA treated with MTZ. (a, b,c) represent PCR products with primer C06; (d, e, f) represent PCR products with primer C07; (g, h, i) represent PCRproducts with primer C15, (j, k) represent PCR products with primer C20. Lane 1 represents DNA marker. Lane 2represents negative control, lanes 3, 4 and 5 represent T. zillii treated with 5, 10 and 20 mg l−1 of MTZ, respec-tively, lane 6 represents T. zillii treated with 10 mg l−1 of MTZ plus vitamin C (200 mg kg−1 food), lane 7 representsT. zillii treated with 20 mg l−1 of MTZ plus vitamin C (200 mg kg−1 food), and lane 8 represents untreated T. zillii

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polymorphic bands, which did not appear in the DNA

samples of normal or VitC protected T. zillii (Figs 1 and

2). These new bands could be considered as ‘genus diag-

nostic’ markers which attributed to MTZ treatment.

Pathological Findings

Gross pathology and clinical signs

There was no mortality observed within the span of the

experiment. Gross examination of the fish within the time

period of the experiment revealed no obvious gross

lesions except dark discoloration of the fish after 2 and 7

days, respectively, which was observed in the group

treated with the highest dose of MTZ. Some behavioral

and clinical signs were noticed, such as erratic movement

and the presence of mucous shreds from the intestinal

tract during swimming in the same group.

Histopathological Results

The histopathological examination of all organs collected

from fish exposed to 5 mg l−1 of MTZ showed a normal

structure. However, pathological lesions of fish exposed

to 10 mg l−1 of MTZ were prominent in different organs

after 2 days. These lesions were noticed in the liver,

intestine, spleen and testis. The liver showed vacuolar

degeneration of the hepatocytes and melanophore aggreg-

ation in the area of hepatopancrease. The melanophores

were prominent around the pancreatic duct with promi-

nent pyknosis of some nuclei (Fig. 3a). In the intestine,

the epithelial lining showed intraepithelial aggregation of

melanophores (Fig. 3b) together with hemorrhage. In the

spleen, marked activation of the melanomacrophage

centers was observed where the centers appeared large

and contained a larger number of melanophores (Fig. 3c).

Testicular degeneration was a prominent picture in this

group where the seminiferous tubules were lined by

sertoli cells and a few spermatogonia. The lumen of the

tubules was devoid of mature sperms (Fig. 3d). In such

cases, few melanophores were noticed in the testi-

cular interstitial tissue (Fig. 3e). Similar lesions were

noticed on days 7 and 14 in this group.

In fish exposed to 20 mg l−1 of MTZ, the lesions were

also noticed after 2 days. The liver showed marked

vacuolation and necrosis in the area of hepatopancrease

together with melanophore aggregation (Fig. 3f). The

intestines of this group showed marked necrosis and

desquamation of the epithelial lining (Fig. 4a). Testicular

degeneration was also noticed while the melanomacr-

ophage centers of the spleen showed marked depletion

of both melanophores and lymphocytes (Fig. 4b). After

7 days, the fish in this group showed advanced pathologi-

cal lesions. In the liver, marked necrosis in both hepatic

tissue and hepatopancrease was a common picture. The

cells in the necrotic part appeared more eosinophilic with

fragmented nuclei (Fig. 4c).

Fish exposed to 10 mg l−1 of MTZ plus VitC showed

reduced lesions after 7 days, but with evidence of degen-

erative changes in renal tissue together with necrosis and

hemorrhage in the hepatic tissue (Fig. 4d), while the tes-

ticular tissue was apparently normal. These lesions were

less prominent than that of MTZ only group. The most

common lesions in fish exposed to 20 mg l−1 of MTZ

plus VitC were detected in the kidneys after 2 days. The

glomeruli showed marked thickening of the capillary

basement membrane of the glomerular capillary tufts and

interstitial hemorrhage among renal tubules (Fig. 4e). At

this time, the testicular tissue appeared normal (Fig. 4f).

All organs of the non treated-fish were apparently normal

(Fig. 5).

Discussion

Most antibiotics and their metabolites are excreted by

animals or humans after administration and therefore

reach the municipal sewage in the form of excretion.

Moreover, it was recognized that genotoxic and cytotoxic

effects of these substances may represent a health hazard

to humans but also may affect organisms in the environ-

ment. Therefore, the aim of this work was to study the

effect of one clinically important antibiotic drug, MTZ,

and the protective effect of VitC against possible MTZ-

induced micronuclei formation, DNA damage as well as

histopathological alterations in T. zillii.

The potential biological side effects of most antibiotics

are mainly related to organism exposure, the level of

which depends on the quantities used, mode of applica-

tion and technologies applied (Lalumera et al., 2004). In

the current study several doses of MTZ based on the work

of Cavas and Ergene-Gözükara (2005) were selected.

Generally, conflicting results have been obtained on

the genotoxicity of MTZ. Some studies show that MTZ

has no genotoxic effects, especially in human subjects

(Fahrig and Engelke, 1997; Mitelman et al., 1980). On

the other hand, clastogenic activity of MTZ has been

reported in several studies such as Mitelman et al.

(1976), who found an increase in the frequency of chro-

mosomal aberrations in cells of patients treated with

MTZ. Similarly, Elizondo et al. (1996) reported that

MTZ can induce chromatid and isochromatid breaks in

the lymphocytes of subjects receiving therapeutic doses.

Mudry et al. (1994) reported that MTZ has a clastogenic

effect and significantly increased the frequencies of

chromosomal aberrations, micronuclei and abnormal

metaphases. The clastogenic activity of this drug may be

due to the unpaired strand breaks expressed at the chro-

mosomal level as the cell cycle proceeds (Horvathova

et al., 1998).

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Previous studies on aquatic organisms have pointed

out that nitroimidazoles may cause environmental pro-

blems such as toxic effects (Macri et al., 1988). It was

shown that MTZ is non-biodegradable and quite water

soluble (Kümmerer et al., 2000). Therefore, it would

be expected that primarily aquatic ecosystems would be

exposed after emission to the environment. Very little

information is outlined in the literature concerning the

toxic effects of MTZ on fish species (Willford, 1996). An

acute test on Bradio rerio with MTZ showed no effect on

survival (Lanzky and Halling-Sorensen, 1997). In the

present study no mortality in the fish was observed

Figure 3. Photomicrographs of Tilapia zillii treated with MTZ for 2 days: (a) liver of Tilapia zillii exposed to10 mg l−1 of MTZ showing vacuolar degeneration of the hepatocytes (arrow), necrosis and pyknotic nuclei ofhepatopancrease. Note: melanophore aggregation around pancreatic duct (2 arrows, H & E stain × 400). (b) Intes-tine of Tilapia zillii exposed to 10 mg l−1 of MTZ showing intraepithelial aggregation of melanophores (arrows,H & E stain × 400). (c) Spleen of Tilapia zillii exposed to 10 mg l−1 of MTZ showing marked activation ofmelanomacrophage centers (arrows, H & E stain × 400). (d) Testis of Tilapia zillii exposed to 10 mg l−1.metronidazole showing testicular degeneration characterized by sertoli cells lining the seminiferous tubulesand lumen devoid of sperms (arrows, H & E stain × 400). (e) Testis of Tilapia zillii exposed to 10 mg l−1 of MTZshowing melanophores aggregation in the interstitial tissue (arrows, H & E stain × 1000). (f) Liver of Tilapia zilliiexposed to 20 mg l−1 of MTZ showing necrosis and pyknotic nuclei of hepatopancrease (arrows). Note:melanophore aggregation (H & E stain × 400). This figure is available in colour online at www.interscience.wiley.com/journal/jat

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throughout the experiment. However, Cavas and Ergene-

Gözükara (2005) showed an increase in the micronucleus

frequencies of immature peripheral erythrocytes of O.

niloticus treated with 10 and 15 mg l−1 of MTZ for 24, 48

and 72 h. In the present study, the results showed that

MTZ at the two highest doses (10 and 20 mg l−1) for 1-

and 2-week treatments was able to induce micronucleus

formation and the appearance of some polymorphic bands

as markers linked to MTZ treatment in fish. To our

knowledge, this is the first study relevant to genotoxic

effects of MTZ in T. zillii.

The action mechanism of MTZ-genotoxicity may be

attributed to cytochrome P450 activity. In this concern,

Cavas and Ergene-Gözükara (2005) suggested that MTZ

Figure 4. Photomicrographs of Tilapia zillii treated with MTZ: (a) intestine of Tilapia zillii exposed to 20 mg l−1 ofMTZ after 2 days showing necrosis and desquamation of epithelial lining (arrows, H & E stain × 200). (b) Spleen ofTilapia zillii exposed to 20 mg l−1 of MTZ after 7 days showing depletion of melanophores and lymphocytes in thearea of melanomacrophage centers (arrows, H & E stain × 400). (c) Liver of Tilapia zillii exposed to 20 mg l−1 ofMTZ after 7 days showing necrosis of the hepatocytes and pyknotic nuclei. Note: Eosinophilic appearance of thecells (arrows) in the necrotic part (H & E stain × 400). (d) Liver of Tilapia zillii exposed to 10 mg l−1. metronidazoleand vitamin C after 7 days necrosis (arrows) and hemorrhage (H & E stain × 400). (e) Kidney of Tilapia zillii ex-posed to 20 mg l−1 of MTZ plus vitamin C after 2 days showing thickening of glomerular capillary basement mem-brane and interstitial hemorrhage (arrows, H & E stain × 400). (f) Testis of Tilapia zillii exposed to 20 mg l−1 of MTZplus vitamin C after 2 days showing normal testicular structure (H & E stain × 200). This figure is available in colouronline at www.interscience.wiley.com/journal/jat

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DOI: 10.1002/jat

is biotransformed to a hydroxylated metabolite by the

cytochrome P450 enzyme system. The presence of an

OH group in this metabolite increases the reactivity with

macromolecules such as DNA, RNA and proteins. It is

known that this MTZ metabolite is much more mutagenic

and DNA damaging than MTZ itself (Dobias et al.,

1994). Similar metabolic pathways of MTZ have also

been shown in other fish species by Sorensen and Hansen

(2000), who reported that the residues of MTZ and its

metabolite hydroxy-MTZ have appeared in muscle and

skin tissues of rainbow trout in as little as 24 h after the

administration period.

The current study found that MTZ was able to induce

micronucleus formation and the appearance of some

changes in polymorphism band patterns after treatment

lasting longer than two days. These results were consist-

ent with those of Cavas and Ergene-Gözükara (2005),

who reported that the effect of MTZ was more pro-

nounced after 48 h and 72 h than after 24 h of treatment.

This was explained by the fact that the genotoxic

metabolites of MTZ produced by the P450 enzyme sys-

tem appeared in tissues after the first 24 h. The authors

added that they had observed a sharp increase in the

micronucleus formation at 48 h or 72 h, which could be

Figure 5. Photomicrographs of Tilapia zillii untreated control group: (a) Normal hepatocytes (arrow) andhepatopancreas (two arrows). (b) Normal intestine. Note: intestinal villi without necrosis or desquamation (arrows).(c) Normal spleen without change in melanomacrophage centers (arrow). (d) Normal testicular tissue with largenumber of sperms in the lumen of seminiferous tubules (arrows). (e) Normal kidney without change in glomeruli(arrow) or renal tubules (two arrows) (H & E stain × 200). This figure is available in colour online at www.interscience.wiley.com/journal/jat

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attributed to the effects of hydroxy metabolites produced

in the first 24 h.

From the pathological point of view, the study indi-

cated signs of toxicity manifested by degenerative and

necrobiotic changes in different parenchymatous organs.

These results are already pointed out by Mendez et al.

(2002), who detected a toxic effect of MTZ as a result

of hydroxyl metabolites of this antibacterial agent. On

the other hand, Sandra et al. (2004) studied the effect

of MTZ on human lymphocytes cell line and found in-

creases in the mitotic index accompanied by metabolic

activation of MTZ. However, different studies indicated

cellular hyperplastic proliferation in different organs and/

or tumor formation accompanying administration of MTZ

(Elizondo et al., 1996; Bendesky et al., 2002; Cavas and

Ergene-Gözükara, 2005). In contrast, in our study there

was no evidence of such lesions; the absence of tumors

or hyperplastic lesions may be time and/or species

dependent. Thus, long term future study is required to

detect the effect of vitamin C on neoplastic-induced

lesions due to MTZ treatment.

In our study, the damage to testicular tissue was pro-

minent. In this regard, Ralph et al. (1992) reported

ornidazole as one of 14 reproductive toxicants causing

testicular damage in rat. On the other hand, a reduction

of the pathological lesions was noticed after the use of

vitamin C.

Because the pathological findings indicated the occur-

rence of some vascular related lesions as renal hemorr-

hages and necrosis which may occur as a result of blood

circulation hindrance, vitamin C may play a role in the

lesion reduction as it improves endothelium dependent

vasodilation (Taddei et al., 1998) and thus improves

circulation in the damaged tissue. Another mechanism

which could explain the effect of vitamin C was men-

tioned by Terova et al. (1998), who demonstrated the

effects of vitamin C on collagen synthesis. As collagen

is the main component of blood vessels, the enhanced

collagen synthesis could be reflected positively on dam-

aged tissue.

The present study showed that vitamin C may suppress

MTZ toxicity, where MnPCEs were decreased in number

and alterations on DNA bands; histopathological lesions

were reduced as well. Kim and Lee (2006) found that

VitC was able to improve the gene expression and activ-

ity of hepatic microsomal cytochrome P450, which was

decreased by polymicrobial sepsis. The authors suggest

that VitC improves hepatic drug metabolizing dysfunction

caused by oxidant stress and lipid peroxidation.

Due to the fact that MTZ has been found to be toxic

to some biological systems (as mentioned by Cavas and

Ergene-Gözükara, 2005; Elizondo et al., 1996) including

our study, we recommend using alternative drugs with

the same biological response and similar structural char-

acteristics to MTZ but with a lower toxicity or using

MTZ mixed with vitamin C.

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