C© 2015 Wiley Periodicals, Inc. Birth Defects Research (Part B) 0:1–10 (2015)

Research Article

Biomonitoring of the Genotoxic and HepatotoxicEffects and Oxidative Stress Potentials of Itraconazole

in Pregnant Rats

Abdel-Fattah El-Shershaby,1 Ahmed I. Dakrory,1,2 Mai H. El-Dakdoky,3 Jehane Ibrahim,1and Fatma Kassem1∗

1Zoology Department, Faculty of Science, Cairo University, Cairo, Egypt2Biology Department, Faculty of Science, Taif University, Taif, Kingdom of Saudi Arabia

3Zoology Department, Women’s College for Arts, Science & Education, Ain Shams University, Cairo, Egypt

BACKGROUND: Pregnant women are more susceptible to both vaginal colonization and infection by yeast. One hundredmillion fungal infected patients have been treated worldwide with itraconazole (Caputo, 2003. METHOD: Itraconazolewas administrated orally to pregnant rats at doses of 75, 100, or 150 mg/kg during gestational days (GD) 1 to 7 or GD8 to 14 or GD 14 to 20. The genotoxicity and hepatotoxicity of the antifungal drug itraconazole were assessed duringdifferent periods of pregnancy using different methods. RESULTS: It was found that itraconazole was a genotoxic drugfor both mothers and fetuses. This finding was observed via significant elevation in the estimated comet assay param-eters (percentage of fragmented DNA, tail moment, and olive moment), percentage of fragmented DNA measured bydiphenylamine assay and mixed smearing and laddering of DNA fragments of liver samples. In addition, itraconazolecaused significant elevation in the level of hepatic malondialdehyde and depletion in the catalase activity and glutathionelevel. Furthermore, itraconazole induced histopathological alterations in the hepatic tissues of both mothers and fetuses.CONCLUSION: These findings indicate that itraconazole administration at doses of 75, 100, or 150 mg/kg during preg-nancy induced maternal and fetal toxicity that could be induced by the genotoxicity and the oxidative damage. BirthDefects Res (Part B) 0:1–10, 2015. C© 2015 Wiley Periodicals, Inc.

Key words: gestational periods; genotoxicity; hepatotoxicity; itraconazole;oxidative stress; rat

INTRODUCTIONItraconazole is one of the triazole compounds with broad-spectrum antifungal activity. It evokes its role throughinhibition of fungal cytochrome P450 sterol C-14 al-pha demethylation. Itraconazole is an important drug inobstetrics and gynecology for treatment of vaginal can-didiasis. It has been recently estimated that more than100 million patients have been treated worldwide withitraconazole (Caputo, 2003). Clinical trials in drug devel-opment generally exclude pregnant women for ethicalreasons but leave questions about the safety of medica-tions for the developing fetus and the pregnant womanherself. Fletcher et al. (2000) found that antifungal drugtreatment during pregnancy is problematic because of therisk of possible teratogenic effects. It was reported in sev-eral publications that itraconazole had teratogenic effectin mice, rats, and rabbit (Van Cauteren et al., 1989; Tiboniet al., 2006). Moreover, our previous work showed that theeffect of itraconazole on fetuses varied according to the

time of exposure (El-Shershaby et al., 2014). On the otherhand, Cornely et al. (2007) reported that hepatotoxic reac-tions are among the most common and potentially seriousadverse effects associated with triazole therapy. It mayalso disrupt steroid hormone homeostasis (Amber andDavid, 2009). The mechanism of the detrimental influenceof itraconazole on pregnancy and on fetal developmenthas yet to be identified. It has been postulated that ox-idative damage is one of the main mechanisms involvedin most antifungal drugs (Liu et al., 2002; Rodriguez andBuckholz, 2003; Amin and Hamza, 2005; Amin, 2008). Ithas also been proposed that the genotoxic effect of the an-tifungal drugs could underlie such defects (Fucic et al.,

∗Correspondence to: Fatma Kassem, Zoology Department, Faculty of Science,Cairo University, Cairo, Egypt. 12613E-mail: fatmakassem_86@yahoo.com

Received 05 December 2014; Accepted 22 March 2015

Published online in Wiley Online Library (wileyonlinelibrary.com/journal/bdrb) DOI: 10.1002/bdrb.21138

2 EL-SHERSHABY ET AL.

2008), but there are very limited available data regard-ing the genotoxicity of the antifungal triazole derivatives(Yuzbasioglu et al., 2008). Based on Product Information(2004), itraconazole causes a dose-related increase in ma-ternal toxicity and embryotoxicity at dosage levels of 40 to160 mg/kg/day (5–20 times the maximal recommendedhuman dose). However, the underlying mechanisms ofitraconazole developmental toxicity and the most sensi-tive period of pregnancy to this drug remain unclear.

Therefore, the aim of the present study was to assess theunderlying mechanisms of itraconazole embryotoxicityand teratogenesis in rats at different gestational periodsthrough investigating the induction of DNA damage viasingle-cell gel electrophoresis assay, diphenylamine assay,and gel electrophoresis technique as well as through in-vestigating the oxidative stress and histopathological al-terations of the liver of pregnant rats and their fetuses, asliver is highly exposed to the toxicant and also responsiblefor its removal.

MATERIAL AND METHODS

Drug PreparationSporanox capsules (CAS Number: 84625-61-6) contain-

ing itraconazole (purity more than 99%) as an activeingredient produced by JANSSEN-CILAG pharmaceu-tica (Beerse, Belgium) were dissolved in 4% dimethylsulphoxide. Concentrations were prepared to deliver 75,100, or 150 mg/kg body weight in 0.5 ml/dose by oraladministration to pregnant rats. Controls received 4%dimethyl sulphoxide vehicle.

Experimental AnimalsAdult female albino rats Rattus norvegicus weighing 140

to 170 g were purchased from the National Institute forResearch, Giza, Egypt. A standard diet and water ad li-bitum were used. After 2 weeks of acclimatization, fe-male rats were housed overnight with adult males of thesame strain. The day of detection of sperm in the vagi-nal smear was assumed as the first day of gestation (ges-tational day (GD) 1). The protocols for animal use andthe procedures needed for the experiments described herewere approved by the Animal Ethics Committee of theNational Institute for Research, Egypt.

Experimental DesignPregnant rats were randomly divided into four groups

(15 rats per group), each treated orally with itraconazoleat doses 0 (vehicle), 75, 100, or 150 mg/kg b.wt. It hasbeen reported that exposure of 150 mg/kg of itraconazolewas found to be the lowest observed adverse effect levelfor inducing malformations in the mouse to avoid highmortality in the generation (Pharmaceutica, 1997; Tiboniet al., 2006; Marotta and Tiboni, 2010). Haria et al. (1996)reported that human dose of 400 to 600 mg per day wererequired for adequate vaginal candidiasis treatment.

Each group was further divided into three subgroupsand orally received the corresponding treatment oncedaily at different exposure periods as follows: subgroup(1) rats were received itraconazole during GD 1 to 7(preimplantation-implantation period); subgroup (2) ratswere given itraconazole during GD 8 to 14 (organogene-

sis period)), and subgroup (3) rats were treated with itra-conazole during GD 14 to 20 (fetal developmental period).

On the morning GD 21, rats were sacrificed by cervi-cal dislocation, cesarean sections were done. The liver ofpregnant rats and their fetuses were removed and usedfor genotoxic and oxidative stress assessments as well ashistopathological examination.

Genotoxic AssessmentsIt is necessary to use more than one test system to ob-

tain an adequate evaluation of the genotoxicity of a drugor its metabolites (Yuzbasioglu et al., 2008). For these rea-sons, we aimed to provide additional genotoxicity datafor the antifungal drug itraconazole, investigating the in-duction of DNA damage via single-cell gel electrophoresisassay, diphenylamine assay, and gel electrophoresis tech-nique using appropriate pieces of the liver of all dams andthat of four fetuses/litter of vehicle and treated groups asfollows.

Single-cell gel electrophoresis (comet assay).The liver of mothers and fetuses of control and treatedgroups were used for evaluation of the genotoxic effectof itraconazole using single-cell gel electrophoresis tech-nique (comet assay). The used method was performed un-der alkaline conditions as described by Singh et al. (1988).

Quantitative estimation of fragmented DNA us-ing diphenylamine assay. This method is based onquantification of the fragmented DNA in the liver accord-ing to the method described by Hickey et al. (2001). Theamount of fragmented DNA was expressed as the per-centage of total DNA appearing in the supernatant.

Qualitative estimation of fragmented DNA us-ing gel electrophoresis technique. Genomic DNAwas prepared from the hepatic tissue using a salting-outprocedure performed as described by Miller et al. (1988).Fragmented DNA was detected by running 5 �g of ge-nomic DNA on a 1.5% ethidium bromide-treated agarosegel (Sigma, UK) according to the standard protocol de-scribed by Sambrook et al. (1989). The gel was run at 80V (power supply Biorad, Model 200/2.0), and visualizedunder UV transillminator (Statagene, California).

Estimation of Oxidative StressLiver pieces of all mothers and at least three fe-

tuses/litter of control and itraconazole-treated dams wereweighed and homogenized in 10 mmol per liter phos-phate buffer saline as 10% (W/V) at pH 7.4. The ho-mogenates were centrifuged and the supernatants weretaken to estimate of levels of malondialdehyde (MDA)and reduced glutathione (GSH) and the activity of cata-lase. Lipid peroxides were estimated by thiobarbituricacid reaction as described by Ohkawa et al (1979) usingsaturated thiobarbituric acid (0.37%) and trichloroaceticacid (15%) at the ratio of 1:3. Reduced GSH was mea-sured by modification of the method described by Ell-man (1959). A total of 2500 �l of each sample was addedto a tube containing 1.8 ml of Ellman’s Reagent Solu-tion (prepared by dissolving 0.1 mM of 5-5’-dithiobis [2-nitrobenzoic acid] in 0.3 M phosphate buffer (pH 7.4))and the absorbance of each sample was measured with aspectrophotometer set to 412 nm. Catalase activity (CAT)was determined by the method of Aebi (1983). A total of

Birth Defects Research (Part B) 0:1–10, 2015

ITRACONAZOLE GENOTOXICITY AND HEPATOTOXICITY IN PREGNANT RATS 3

0.5 ml of phosphate buffer, 0.1 ml of sample and 0.4 mlof distilled water were mixed in test tubes, and then thetubes were incubated at 37°C for 2 min. The reaction forassaying CAT was initiated by adding 0.5 ml hydrogenperoxide. Therefore, Absorbances of the samples wereread against distilled water immediately at zero time andwere read again after 30 sec at 240 nm.

Histopathological ExaminationSpecimens from the liver of all mothers and two fe-

tuses/litter from control and itraconazole-treated groupswere fixed in neutral-buffered formalin (10%) for 24 hr.Then, the specimens were washed in tap water, dehy-drated in ascending series of ethyl alcohol (70, 80, 90, 95,and 100%), cleared in pure xylene, and then embedded inparaffin wax (58–60°C). Sections of 5 �m thickness wereprepared and stained with hematoxylin and eosin (Druryand Wallington, 1973).

Statistical AnalysesThe data were presented as mean ± SE. One-way anal-

ysis of variance was performed followed by post hoc leastsignificant difference test for multiple comparisons usingthe statistical package for social science (SPSS, Chicago,IL) version 17. Differences were considered statisticallysignificant when p < 0.05.

RESULTSItraconazole was found to reduce maternal body

weight gain, especially through GD 14 to 20 (data notshown). Also, vaginal bleeding and complete resorptionof all implanted embryos were observed in all dams thatreceived the high dose of itraconazole (150 mg/kg b.wt.)during the preimplantation-implantation and organogen-esis periods as well as those received the mid-dose(100 mg/kg b.wt.) during the preimplantation-implantation period.

Evaluation of the Genotoxicity of Itraconazolein Dams and Fetuses

Quantitative evaluation of DNA damage.

Single-cell gel electrophoresis. Statistically significantincreases were observed in the measured comet param-eters (percentage of tail DNA, tail moment, and olive mo-ment) in the hepatocytes of pregnant rats (Table 1) andtheir surviving fetuses (Table 2) of groups exposed toitraconazole during the three different gestation periodsrelative to the vehicle-treated groups and all measuresdemonstrated a dose response such that DNA damageincreased with increasing dose (Figs. 1 and 2). However,dams treated with the low dose of this drug (75 mg/kgb.wt.) showed no significant change in the mean of thepercentage of DNA in the tail in comparison with thevehicle-treated group (Table 1).

Diphenylamine assay. Table 3 revealed that the hepatictissues of dams treated with itraconazole and their fetusesshowed significant increases in the percentage of DNAfragmentation (p < 0.001). The degree of fragmentationincreased with increasing the dose.

Tabl

e1

DN

AD

amag

ein

the

Liv

erof

Preg

nant

Rat

sM

easu

red

Usi

ngth

eC

omet

Ass

ayaf

ter

Ora

lAd

min

istr

atio

nof

Thr

eeD

iffe

rent

Dos

esof

Itra

cona

zole

(0,7

5,10

0,an

d15

0m

g/kg

b.w

t.)d

urin

gD

iffe

rent

Peri

ods

ofPr

egna

ncy

Dos

e(m

g/kg

/d

ay)

Veh

icle

(0)

Low

dos

e(7

5m

g/kg

)M

ediu

md

ose

(100

mg/

kg)

Hig

hd

ose

(150

mg/

kg)

Ges

tati

onin

terv

alIm

plan

tati

onO

rgan

ogen

esis

Feta

lIm

plan

tati

onO

rgan

ogen

esis

Feta

lIm

plan

tati

onO

rgan

ogen

esis

Feta

lIm

plan

tati

onO

rgan

ogen

esis

Feta

l

DN

Ain

tail

(%)

16.2

3±

3.55

16.2

±2.

217

.3±

2.9

18.6

6±

2.80

16.0

5±

2.09

19.3

7±

0.70

31.2

6±

2.71

***

30.5

0±

0.70

***

34.0

9±

3.02

***

34.4

4±

0.54

***

31.4

0±

0.80

***

34.3

3±

1.29

***

Tail

mom

ent

0.36

±0.

100.

39±

0.09

0.41

±0.

11.

17±

0.13

*1.

38±

0.09

*1.

14±

0.10

*1.

27±

0.10

*1.

66±

0.13

**1.

25±

0.12

*2.

24±

0.74

***

2.44

±0.

06**

*2.

41±

0.20

***

Oliv

em

omen

t0.

64±

0.12

0.59

±0.

20.

62±

0.18

1.72

±0.

12**

*1.

31±

1.10

**1.

36±

0.14

**1.

34±

0.11

**1.

66±

0.08

***

1.63

±0.

08**

*1.

56±

0.07

***

1.38

±0.

16**

*1.

91±

0.18

***

Val

ues

are

pres

ente

das

mea

n±

SE.

Com

pare

dw

ith

the

cont

rolg

roup

:* p<

0.05

;**p

<0.

01;**

* p<

0.00

1.

Birth Defects Research (Part B) 0:1–10, 2015

4 EL-SHERSHABY ET AL.

Tabl

e2

DN

AD

amag

ein

the

Liv

erof

Fetu

ses

Mat

erna

llyTr

eate

dw

ith

Thr

eeD

iffe

rent

Dos

esof

Itra

cona

zole

(0,7

5,10

0,an

d15

0m

g/kg

b.w

t.)d

urin

gD

iffe

rent

Peri

ods

ofPr

egna

ncy

Dos

e(m

g/kg

/d

ay)

Veh

icle

(0)

Low

dos

e(7

5m

g/kg

)M

ediu

md

ose

(100

mg/

kg)

Hig

hd

ose

(150

mg/

kg)

Ges

tati

onin

terv

alIm

plan

tati

onO

rgan

ogen

esis

Feta

lIm

plan

tati

onO

rgan

ogen

esis

Feta

lIm

plan

tati

onO

rgan

ogen

esis

Feta

lIm

plan

tati

onO

rgan

ogen

esis

Feta

l

DN

Ain

tail

(%)

10.2

0±

0.98

8.69

±2.

39.

83±

1.55

28.3

5±

2.54

***

28.6

1±

10.3

7***

24.8

4±

1.20

**N

D33

.82

±1.

71**

*33

.85

±0.

43**

*N

DN

D73

.72

±5.

05**

*

Tail

mom

ent

(arb

itra

ryun

its)

0.54

±0.

070.

43±

0.1

0.52

±0.

091.

31±

0.7**

1.54

±0.

21**

*1.

27±

0.18

**N

D2.

39±

0.18

***

2.51

±0.

14**

*N

DN

D3.

80±

0.85

***

Oliv

em

omen

t0.

51±

0.12

0.43

±0.

098

0.57

±0.

11.

83

±0.

12*

1.88

±0.

23**

2.08

±0.

32*

ND

3.08

±0.

46**

*3.

61±

0.18

***

ND

ND

4.95

±0.

64**

*

Val

ues

are

pres

ente

das

mea

n±

SE.

Com

pare

dw

ith

the

cont

rolg

roup

:* p<

0.05

;**p

<0.

01;**

* p<

0.00

1.N

D:n

otd

one

(ear

lyre

sorb

edfe

tuse

s).

Fig. 1. Photomicrographs of maternal hepatic tissue showing typ-ical nuclei with various degrees of DNA damage observed ascomet. This damage was seen in all studied groups with dif-ferent frequencies (magnification 400×). (A) Undamaged controlmaternal hepatocyte. (B–D) Different degrees of damaged ma-ternal hepatocytes treated with the applied doses of itracona-zole (1: 75 mg/kg; 2: 100 mg/kg, and 3: 150 mg/kg) during: (B)preimplantation-implantation period, (C) organogenesis period,and (D) fetal developmental period.

Fig. 2. Photomicrographs of fetal hepatic tissue showing typicalnuclei with various degree of DNA damage observed as comet.This damage was seen in all studied groups with different fre-quencies (magnification 400×). (A) Undamaged control fetal hep-atocyte. (B–D) Different degrees of damaged fetal hepatocytesobtained from dams treated with the applied doses of itracona-zole (1: 75 mg/kg; 2: 100 mg/kg, and 3: 150 mg/kg) during: (B)preimplantation-implantation period, (C) organogenesis period,and (D) fetal developmental period.

Qualitative evaluation of DNA damage. Itwas found that the extracted genomic DNA from thesamples from dams treated with the low, medium, andhigh doses of itraconazole showed internucleosomal frag-mentation that appeared as a ladder pattern mixed witha smear-like pattern, generally regarded as a molecu-lar hallmark of apoptosis and necrosis (Fig. 3). How-ever, DNA extracted from fetuses of dams treated withthe three different doses of itraconazole (75, 100, or150 mg/kg) during the three different periods of gestation(preimplantation-implantation, organogenesis, and fetaldevelopment) showed a smear-like pattern in the agarosegel (Fig. 4).

Evaluation of Oxidative Stress MarkersThe levels of MDA, reduced GSH, and CAT were eval-

uated in the hepatic tissues of pregnant rats and their

Birth Defects Research (Part B) 0:1–10, 2015

ITRACONAZOLE GENOTOXICITY AND HEPATOTOXICITY IN PREGNANT RATS 5

Tabl

e3

Perc

enta

geof

Frag

men

ted

DN

A(%

)in

the

Hep

atoc

ytes

ofPr

egna

ntR

ats

and

The

irFe

tuse

sTr

eate

dw

ith

Thr

eeD

iffe

rent

Dos

esof

Itra

cona

zole

(0,7

5,10

0,an

d15

0m

g/kg

b.w

t.)th

roug

hth

eT

hree

Dif

fere

ntG

esta

tion

alPe

riod

sU

sing

Dip

heny

lam

ine

Ass

ay

Dos

e(m

g/kg

/d

ay)

Veh

icle

(0)

Low

dos

e(7

5m

g/kg

)M

ediu

md

ose

(100

mg/

kg)

Hig

hd

ose

(150

mg/

kg)

Ges

tati

onin

terv

alIm

plan

tati

onO

rgan

ogen

esis

Feta

lIm

plan

tati

onO

rgan

ogen

esis

Feta

lIm

plan

tati

onO

rgan

ogen

esis

Feta

lIm

plan

tati

onO

rgan

ogen

esis

Feta

l

Perc

enta

geO

fm

ater

nalD

NA

frag

men

tati

on

16.4

0±

1.20

16.4

0±

1.20

16.4

0±

1.20

24.6

6±

3.11

*26

.26

±4.

85*

29.8

9±

2.41

**34

.96

±5.

02**

*35

.07

±2.

61**

*34

.43

±3.

88**

*41

.06

±4.

30**

*43

.49

±3.

07**

*44

.78

±4.

02**

*

Perc

enta

geO

ffe

talD

NA

frag

men

tati

on

7.31

±0.

528.

44±

0.39

8.65

±0.

8011

.88

±2.

13*

18.2

2±

3.04

***

8.82

±0.

80N

D28

.03

±1.

88**

*14

.89

±1.

99**

ND

ND

26.8

9±

2.3**

*

Val

ues

are

pres

ente

das

mea

n±

SE.

Com

pare

dw

ith

the

cont

rolg

roup

:* p<

0.05

;**p

<0.

01;**

* p<

0.00

1.N

D:n

otd

one

(ear

lyre

sorb

edfe

tuse

s).

Fig. 3. Representative 1.5% agarose gel electrophoretograms ofextracted genomic DNA (showing fragmentation that appear asladder pattern mixed with smear-like pattern) from the liver ofcontrol and itraconazole-treated rats I (75): rats treated with 75mg/kg during preimplantation-implantation period. O (75): ratstreated with 75 mg/kg during organogenesis period. FD (75):rats treated with 75 mg/kg during fetal development period.I (100): rats treated with 100 mg/kg during preimplantation-implantation period. O (100): rats treated with 100 mg/kg dur-ing organogenesis period. FD (100): rats treated with 100 mg/kgduring fetal development period. I (150): rats treated with 150mg/kg during preimplantation-implantation period. O (150):rats treated with150 mg/kg during organogenesis period. FD(150): rats treated with 150 mg/kg during fetal development pe-riod

Fig. 4. Representative 1.5% agarose gel electrophoretograms ofextracted genomic DNA (showing fragmentation that appear assmear-like pattern) from the liver of control and itraconazole ma-ternally treated fetuses I (75): maternally treated fetuses with 75mg/kg during preimplantation-implantation period O (75): ma-ternally treated fetuses with 75 mg/kg during organogenesis pe-riod FD (75): maternally treated fetuses with 75 mg/kg during fe-tal development period O (100): maternally treated fetuses with100 mg/kg during organogenesis period FD (100): maternallytreated fetuses with 100 mg/kg during fetal development periodFD (150): maternally treated fetuses with 150 mg/kg during fetaldevelopment period

Birth Defects Research (Part B) 0:1–10, 2015

6 EL-SHERSHABY ET AL.

fetuses treated with different doses of itraconazole (75,100, or 150 mg/kg) during the three gestational periods(Tables 4 and 5).

Significant increases were observed in the hepatic MDAlevels of dams and fetuses whose mothers treated withthe three applied doses of itraconazole through the threegestational periods when compared with the vehicle-treated group. The degree of change was dose and perioddependent.

The activity of the antioxidant enzyme CAT was sig-nificantly decreased (p < 0.001) in the hepatic tissuesof pregnant female rats treated orally with 75, 100, or150 mg/kg of itraconazole through the three gestationalperiods comparison to the vehicle-treated group (Table 4).However, the hepatic CAT of fetuses maternally treatedwith itraconazole showed no significant changes from thevehicle group (Table 5).

Treatment with different doses of itraconazole throughthe three gestational periods resulted in significant reduc-tion in the level of GSH of the hepatic tissues of dams andtheir fetuses (Tables 4 and 5).

Histopathological Changes of Hepatic TissuesLiver sections of the vehicle-treated group showed nor-

mal hepatic lobular architecture (Fig. 5C). The microscop-ical examination of liver sections of dams treated with thelow dose of itraconazole (75 mg/kg) showed that most ar-eas of the liver tissue exhibited normal architecture (sim-ilar to the vehicle control group); however, a few areasshowed degeneration. These degenerated areas showeddissociated hepatocytes, dilated and congested sinusoids,and vacuolization of the cytoplasm of many hepatocytes(Fig. 5I, O, and D (75)). However, more severe histologi-cal alterations were observed in the liver sections of preg-nant rats administered with the medium dose of itracona-zole (100 mg/kg) as compared to low dose treated groups.Inflammatory foci consisting of necrotic cells and lympho-cytic infiltration (Fig. 5I (100)) and hypertrophied Kupffercells were observed. In addition, disorganized and dis-rupted strands of deteriorated hepatocytes (Fig. 5D and O(100)) were observed. A necrotic mass of hepatocytes to-gether with many lymphocytes and hemorrhagic patches,fatty vacoulation, dilated sinusoids with increased and ac-tivated Kupffer cells were observed in the liver sectionsof rats treated with the high dose of itraconazole (Fig. 5Oand D (150)). Fibrosis was recognized very near to somehepatic blood vessels and showing some fibroblast cellsin the liver tissues of dams receiving the high dose of itra-conazole (150 mg/kg; Fig. 5I (150)).

Histological examination of liver sections from GD 21fetuses obtained from dams treated with the low dose ofitraconazole (75 mg/kg) during the three studied peri-ods showed areas of more or less normal hepatic archi-tecture and areas with clear signs of histopathological al-terations (Fig. 6I(75), O(75), D(75)). Fetuses of mothers re-ceiving the medium dose of itraconazole 100 mg/kg b.wt.showed dilation and congestion of the central vein; its lu-men was filled with masses of collapsed erythrocytes ad-hered together, disruption of its wall at certain regions,and disorganization of the hepatic strands with appear-ances of necrotic masses (Fig. 6O(100), D(100)). High-doseitraconazole treatment revealed numerous inflammatory

Tabl

e4

Mal

ond

iald

ehyd

eC

once

ntra

tion

,Cat

alas

eA

ctiv

ity,

and

Red

uced

Glu

tath

ione

Lev

elof

the

Hep

atic

Tiss

ues

ofPr

egna

ntR

ats

Ora

llyA

dm

inis

tere

dw

ith

Dif

fere

ntD

oses

ofIt

raco

nazo

le(0

,75,

100,

or15

0m

g/kg

b.w

t.)

Dos

e(m

g/kg

/d

ay)

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icle

(0)

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dos

e(7

5m

g/kg

)M

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ose

(100

mg/

kg)

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(150

mg/

kg)

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esis

Feta

l

MD

Aco

ncen

trat

ion

(nm

ol/

gw

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ssue

)

31.4

±2.

734

.2±

0.6

30±

1.2

50.4

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1*58

.3±

2.6**

50.8

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0*60

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2.8**

63.3

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8**46

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116.

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***

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±4.

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GSH

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0.9

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234

.1±

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27.6

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0***

19.7

±1.

7***

17.6

±3.

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18.3

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20.8

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0***

18.6

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6***

16.2

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4***

Cat

alas

eac

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ty(n

mol

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ue)

0.05

7±

0.01

0.00

21±

0.01

0.06

8±

0.01

0.01

37±

0.00

4***

0.01

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0.01

***

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0.00

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:* p<

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* p<

0.00

1.

Birth Defects Research (Part B) 0:1–10, 2015

ITRACONAZOLE GENOTOXICITY AND HEPATOTOXICITY IN PREGNANT RATS 7

Tabl

e5

Mal

ond

iald

ehyd

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once

ntra

tion

,Cat

alas

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ctiv

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l

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trat

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(nm

ol/

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etti

ssue

)32

.03

±2.

0840

.23

±0.

138

.2±

0.1

56.5

7±

2.77

***

53.6

9±

1.89

***

57.3

7±

2.34

***

ND

108.

88±

1.35

***

86.4

1±

6.05

***

ND

ND

127.

77±

9.10

***

GSH

leve

l(�

g/g

tiss

ue)

476.

76±

89.9

345

0.53

±7.

1136

0.21

±13

.01

338.

86±

28.3

6*19

5.07

±15

.53**

*30

1.73

±74

**N

D15

7.95

±29

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*27

2.18

±42

.84**

ND

ND

161.

61±

22.7

8***

Cat

alas

eac

tivi

ty(n

mol

/se

c/g

tiss

ue)

0.01

6±

0.00

40.

021

±0.

003

0.01

8±

0.00

20.

01±

0.00

50.

013

±0.

003

0.01

5±

0.00

3N

D0.

006

±0.

001

0.02

±0.

014

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ND

0.00

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1

Val

ues

are

pres

ente

das

mea

n±

SE.

Com

pare

dw

ith

the

cont

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roup

:* p<

0.05

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1.N

D:n

otd

one

(ear

lyre

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edfe

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s).

Fig. 5. Photomicrographs of liver sections on the 21st day of ges-tation of C: Control rat showing normal hepatic architecture I:Itraconazole-treated rats during implantation period I (75): With75 mg/kg showing dilated sinusoids and central vein, enlargedKuppfer and endothelial cells, degenerated hepatocytes and vac-uolated cytoplasm I (100): With 100 mg/kg showing granuloma-like structure with cell debris and numerous lymhocytes I (150):With 150 mg/kg showing fibrosis and hemorrhage in the livertissue O: Itraconazole-treated rats during organogenesis periodO (75): With 75 mg/kg showing very thick walled portal veinbranch and its lumen is filled with blood thrombosis O (100):With 100 mg/kg showing necrotic mass with an enhanced degreeof deterioration and high degree of dissolution O (150): With 150mg/kg showing necrotic area displayed large centrally locatedempty spaces with deteriorated cells at its vicinity and lipolyticor hydropic degeneration. D: Itraconazole-treated rats during fe-tal development period D (75): With 75 mg/kg showing the pres-ence of a group of degenerated hepatocytes with pyknotic nucleiwithin the dilated and congested portal vein branch D (100): With100 mg/kg showing n area of highly vacuolated and degener-ated hepatocytes, dilation and congestion of the central vein withthickened wall and enlargement of its endothelial lining D (150):With 150 mg/kg showing masses of deteriorated cells and largeempty spaces and areas of liquefactive necrosis (N). B: binucle-ated cell; Bi. D: bile ductule; Cn: chromyolytic nuclei; C.V: cen-tral vein; DS: dilated blood sinusoids; De.H: degenerated hep-atocytes; Di.H: dissociated hepatocytes; E: endothelial lining; F:fibrosis; F.D: fatty degeneration; G: granuloma; H: hepatocytes;He: hemorrhage; K: enlarged Kuppfer cells; L.Inf: lymphocyticinfiltration; N: necrotic mass; Pn: pyknotic nuclei; PV: portal vein;S: sinusoids; VD: vacuolated degeneration; Vn: vacuolated nu-clei.

Birth Defects Research (Part B) 0:1–10, 2015

8 EL-SHERSHABY ET AL.

Fig. 6. Photomicrographs of liver sections (400×) on the 21st dayof gestation of C: Control rat fetus showing normal hepatic archi-tecture I (75): Maternally itraconazole-treated rat fetus during—implantation period with 75 mg/kg showing dilated central veinand blood sinusoids and degenerated endothelial lining O: Ma-ternally itraconazole-treated rat fetus during organogenesis pe-riod O (75): With 75 mg/kg showing dilated and congestedcentral vein and blood sinusoids, rupture of the central veinwall (arrow) and degenerated hepatocytes O (100): With 100mg/kg showing dilated central vein and its lumen is filled withblood thrombosis and degenerated hepatocytes D: Maternallyitraconazole-treated rat fetus during fetal development periodD (75): With 75 mg/kg showing degenerated endothelium, de-generated hepatocytes, vacuolated cytoplasm, and dilated sinu-soids D (100): With 100 mg/kg showing necrotic mass. D (150):With 150 mg/kg showing dilated central vein with perivesicu-lar lymphocytic infiltration. CV: central vein; D.E: degeneratedendothelial lining; DS: dilated blood sinusoids; De.H: degener-ated hepatocytes; Ec: erythrocytes; H: hepatocytes; He: hemor-rhage; L.Inf: lymphocytic infiltration; N: necrotic mass; Nor: nor-mocytes; Nor.B: normoblast; Pn: pyknotic nuclei; S: sinusoids; V:vacuolated cytoplasm.

lymphocytes around a highly dilated branch of the portalvein. The wall of this vein was somewhat thick and its lu-men showed numerous erythrocytes and cell debris (Fig.6D(150)).

DISCUSSIONIn the current study, a significant elevation was ob-

served in DNA damage in hepatocytes of pregnant ratsorally administrated different doses of itraconazole dur-ing different periods of gestation. This was indicatedby a significant increase in estimated comet assay pa-rameters (percentage of tail DNA, tail moment, andolive moment), in the incidence of fragmented DNA us-

ing diphenylamine assay and by mixed smearing andladdering of DNA fragments of liver samples. It is sug-gested that itraconazole may have the ability to generatereactive oxygen species (ROS) that induced DNA damagethrough direct genotoxic mechanisms, possibly oxidativestress (Sozen et al., 2014). In addition, Emerit (1994) re-ported that free radicals can induce chromosome break-age. Breen and Murphy (1995) added that ROS can reactwith DNA bases leading to alterations in their structure.In addition, it was reported by Packer (1999) that increas-ing production of lipid peroxides in different tissues in-duced the generation of clastogenic factors which couldlead to chromosome alterations and fetal malformations.

In this respect, Wang et al. (2006) reported that ROSmay play an essential role in DNA damage and apop-tosis. Some other authors reported the genotoxicity ofsome antifungal drugs (Hassan, 1997; Yuzbasioglu et al.,2008) and of itraconazole (El-Shershaby et al., 2014), how-ever, other authors reported the absence of genotoxicity ofother antifungal drugs (Perez-Rivera et al., 2009).

In addition, to the best of our knowledge, this is thefirst study to evaluate the genotoxic effect of itracona-zole on embryonic tissues (liver) of fetuses maternallyexposed to this drug throughout the three periods of ges-tation using comet assay and diphenylamine assay. Asignificant increase was shown in measured comet pa-rameters (percentage of tail DNA, tail moment, and olivemoment) and in the percentage of fragmented DNA intheir hepatocytes. Therefore, there may be an associationbetween the level of DNA damage and hepatotoxicity.However, precise mechanisms behind this operation arenot fully understood and need further investigation. It canbe suggested that itraconazole may evoke its genotoxicitythrough its placental transfer from mothers to embryonictissues which may have a direct or indirect effect involvedin the generation of ROS in fetal tissues that are associ-ated with oxidative tissue damage including lipid peroxi-dation and DNA damage.

Iannaccone (1986) suggested that preimplantationmouse embryos are more susceptible to oxygen toxicity(oxygen radicals). Furthermore, Vogel and Speilmann(1986) added that during the preimplantation period,defects in DNA repair enzymes and low antioxidantenzyme levels lead to high DNA fragmentation andpreimplantation embryos may have a lower capacity forDNA repair than adult tissues. These observations maybe supported by the study that was done by El-Shershabyet al. (2014) who found that fetuses whose mothers weretreated during the preimplantation-implantation periodwith 100 and 150 mg/kg of itraconazole and through theorganogenesis period with 150 mg/kg of itraconazolewere resorbed early. Furthermore, Tripathi et al. (2008)revealed that the fetus and newborn are more susceptibleto genetic damage in comparison to the adult, due toan underdeveloped metabolizing enzyme profile andalso due to the decrease in the DNA repair capacityin newborn as compared to adult. In addition, Fucicet al. (2008) reported that young and newborn animalstransplacentally exposed to a dose of 12 mg/kg of flu-conazole showed a significant increase in micronucleusfrequency in comparison with adult animals.

The current work revealed that treatment of preg-nant rat with different doses of itraconazole during the

Birth Defects Research (Part B) 0:1–10, 2015

ITRACONAZOLE GENOTOXICITY AND HEPATOTOXICITY IN PREGNANT RATS 9

three gestational periods induced oxidative stress damageobserved via significant elevation of the level of MDAand significant decrease in the activity of CAT and thelevel of GSH. This effect might be due to the generationof ROS which interact with biological macromoleculescausing enzyme inactivation, DNA damage and initiatinglipid peroxidation leading to the hepatotoxic effect (Sozenet al., 2014).

It was reported that triazoles inhibit CYP26, whichis one of the cytochrome P450, responsible for thecatabolism of retinoic acid (RA; Menegola et al., 2006).Any excess or deficiency of RA induces ROS production,oxidative stress, and embryonic malformation (Hansen,2006). In addition, retinol and RA exhibit cytotoxic effectof concentrations above 50 �M, while lower doses signif-icantly inhibited both cytotoxicity and mutation rate.

According to Francois et al. (2006), the generation ofROS is important for the antifungal activity of miconazolewhich inhibits the enzymes implicated in breakdown ofperoxide radicals and hydrogen peroxide, that is peroxi-dase and catalase. The oxidative stress damage caused byitraconazole is consistent with previous studies using dif-ferent antifungals (Liu et al., 2002; Rodriguez and Buck-holz, 2003; Amin and Hamza, 2005; Amin, 2008). Sozenet al. (2014) found that the use of itraconazole increasedoxidative stress through significant increases of myeloper-oxidase and nitric oxide levels and significant decreasesin the antioxidant activity of superoxide dismutase andGSH-peroxidase.

In the current experiment, administration of itracona-zole to pregnant dams caused significant elevation in theembryonic hepatic level of lipid peroxidation and signif-icant reduction in the reduced GSH concentration. How-ever, the activity of embryonic catalase was not affected.According to Wells et al. (2005), the embryonic level ofmost antioxidative enzymes is only around 5% of the ma-ternal activity (might be considered too low for antiox-idative protection). This may also reflect the observationsthat antioxidant enzyme activity starts in the embryo withlow level and increases as organogenesis proceeds. In theearly organogenesis period, the embryo may not be ableto respond effectively to oxidative imbalances, leading toteratogenicity (Choe et al., 2001; Hansen, 2006). In the cur-rent study, the level of fetal MDA was higher than its levelin their mothers. This finding was consistent with that ofBeuret et al. (2005) who found that higher lipid peroxida-tion in the fetus than in the mother is one of the reasonsfor greater biological effects of xenobiotics on the fetus,and reflects lower activity of antioxidant enzymes.

Concerning the histopathological alterations in the liverof pregnant rats and their fetuses, the present study hasshown that administration of the antifungal drug itra-conazole with the three applied doses plays a critical rolein the appearance of deleterious histopathological disor-ders in the liver of the treated pregnant rats and their fe-tuses. According to Talwalkar et al. (1999), itraconazolemetabolism occurs primarily in the liver; therefore, anyadverse effects of this drug are shown primarily in theliver. The observed histopathological alterations rangedfrom moderate to severe changes according to the doseused and to the length of the period of administration. Thefindings of many human and animal studies using severalantifungal drugs were consistent with the present results

(Adriaenssens et al., 2001; Liu et al., 2002; Chien et al.,2003; Kim et al., 2003). Histopathological changes such asinflammation and fibrosis were also observed in the liverof rats that received repeated doses of itraconazole (Nor-shahida, 2004). In addition, Somchit et al. (2004) revealedthat the liver of rats treated subchronically with itracona-zole demonstrated dose-dependent severe degenerationof centrolobular and midzonal regions, cloudy swellingand apoptosis. Treatment with the high dose (100 mg itra-conazole/kg per day) induced bile duct hyperplasia, bil-iary cirrhosis, focal necrosis, and severe infiltration of neu-trophils with detection of giant cells. Sozen et al. (2014)found that itraconazole led to significant hepatocyte de-generation, bile duct hyperplasia, formation of bile ductplugs, inflammation of the portal/periportal area, and thepresence of apoptotic cells.

The mechanism underlying the hepatotoxicity of itra-conazole remains unclear. According to Somchit et al.(2009), it was suggested that cytochrome P450 may playa key role in preventing the liver toxicity of certain azoles,especially itraconazole. The authors added that it canbe strongly suggested that the hepatotoxicity could bedue to metabolic idiosyncrasy (a reaction to a medicationthat is unusual and unpredictable, specific to a particularperson).

In conclusion, itraconazole induced genotoxicty andhepatotoxicty in both pregnant dams and their fetuses ina dose-dependent manner. Maternal exposure to doses ofitraconazole higher than the maximum recommended hu-man dose may represent a potential threat to the preg-nant woman and her fetus. Further studies should beconducted to demonstrate the genotoxic potential of itra-conazole in other mammalian test systems.

CONFLICT OF INTERESTNo conflict of interest.

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