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Experimental and Toxicologic Pathology 67 (2014) 21–29

Contents lists available at ScienceDirect

Experimental and Toxicologic Pathology

j ourna l h omepage: www.elsev ier .de /e tp

valuation of hepatotoxic and genotoxic potential of silveranoparticles in albino rats

agdy Mohamed El Mahdya, Taher Ahmed Salah Eldinb,c,∗, Halima Sayed Alyd,aten Fathy Mohammeda,∗, Mohamed Ibrahim Shaalana

Department of Pathology, Faculty of Veterinary Medicine, Cairo University, Giza, EgyptNanotechnology & Advanced Materials Central Lab, Agricultural Research Center, Giza, EgyptRegional Center for Food and Feed (RCFF), Agricultural Research Center, Giza, EgyptDepartment of Cell Biology, National Research Center, Giza, Egypt

r t i c l e i n f o

rticle history:eceived 31 May 2014ccepted 24 September 2014

eywords:lbino ratsilver nanoparticlesepatocellular histopathologyxidative stresshromosomal aberrations

a b s t r a c t

Silver nanoparticles (AgNPs) have wide medical applications regarding their antimicrobial effects. Theyare applied also in appliances such as refrigerators and washing machines. For assessment of toxicologicalpotential of silver nanoparticles 20 mature female albino rats were divided into four groups (five rats pereach). Animals were injected i/p by different doses of approximately 8.7 nm silver nanoparticles (1, 2 and4 mg/kg b.w) daily for 28 days in addition to control group which were injected by deionized water only.Indicators of oxidative stress in liver tissue, determination of silver nanoparticles tissue concentration,description of hepatic histopathological alterations and detection of possible chromosomal aberrations inbone marrow were carried out. Results revealed various hepatic histopathological lesions that were dosedependent. The effect of Ag-NPs on hepatic malondialdhyde (MDA) and glutathione (GSH) levels werevariable in different treated groups compared with the control. The tissue residues of silver nanoparticles

were found in hepatic tissue and related to original treated dose. Finally, silver nanoparticles inducedvariable chromosomal aberrations that were dose dependent. Conclusion: Silver nanoparticles had theability for inducing various hepatic histopathological alterations indicating hepatocytotoxicity presum-ably by oxidative stress, in addition to the induction of chromosomal aberrations in bone marrow cellsdenoting the genotoxicity of nanosilver particles.

© 2014 Elsevier GmbH. All rights reserved.

. Introduction

Nanomaterials are any materials that have at least one dimen-ion <100 nm and they can be divided into two large groups;ltrafine nano sized particles that are not intentionally produced

nd engineered and nanoparticles that are produced in a con-rolled and engineered way (Oberdörster et al., 2005). One of theidely used nanomaterials is nano silver, its particles have a size

Abbreviations: AgNPs, silver nanoparticles; ANOVA, analysis of variance; GSH,educed glutathione; kV, kilo volt; MDA, malondialdhyde; PAS, periodic acid Schiff;VP, polyvinyl pyrrolidone; ROS, reactive oxygen species; TBA, thiobarbituriccid; TCA, trichloroacetic acid; TEM, transmission electron microscopy; UV–vis,ltraviolet–visible light; XRD, x-ray diffraction.∗ Corresponding authors at: Department of Pathology, Faculty of Veterinaryedicine, Cairo University, El-Gamaa Street, 12211 Giza, Egypt.

el.: +20 1003542636/+20 1001030534.E-mail addresses: T1salah@hotmail.com (T.A.S. Eldin), fatenfathy21@yahoo.com,

atenfathy@staff.cu.edu.eg (F.F. Mohammed).

ttp://dx.doi.org/10.1016/j.etp.2014.09.005940-2993/© 2014 Elsevier GmbH. All rights reserved.

ranging from 1 to 100 nm. Silver nanoparticles represent a promi-nent nanoproduct with potential applications in medicine andhygiene because of the antibacterial effects (Lok et al., 2006; Kimet al., 2007; Ayala-Núnez et al., 2009) antiviral actions (Elechiguerraet al., 2005; Mehrbod et al., 2009) and antifungal activity (Kimet al., 2008a). They also promote wound healing by playing arole in cytokine modulation (Wong et al., 2009). The use of sil-ver nanoparticles is not only restricted on medical application butalso extended to various issues related to environment and con-sumer products. There are many applications like disinfection ofdrinking water (Li et al., 2008; Lv et al., 2009), swimming poolsanti-fouling (Yang et al., 2009a) and as a promising antibacterialadditive to water-based paints (Holtz et al., 2012). The problemis that nasal and throat sprays, or contraceptive foams which con-tain nanosilver might leave remarkable residue in the human body;

Also coatings of surfaces in contact with the human skin (textiles)or food will increase human exposure and uptake of nanosilver(Quadros and Marr, 2010; Hansen et al., 2008; Yang et al., 2009b).Several researchers have studied the hepatotoxic effect of silver

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anoparticles via different routes and doses; hepatic alterationsere induced by inhalation of silver nanoparticles in Sprague-awley rats for 28 days (Ji et al., 2007). On the other hand bile-ductyperplasia, with or without necrosis, fibrosis, and/or pigmenta-ion studied was observed after oral toxicity of silver nanoparticles56 nm) over a period of 90 days in F344 male and female ratshich were given different doses (30, 125 and 500 mg/kg) (Kim

t al., 2010). Nanoparticles were detected in Kupffer cells lining,alls of the venous sinusoids, venous endothelial cells and in small

oci of inflammatory reaction after intravenous injection of silveranoparticles (AgNPs) with different sizes (20 nm and 100 nm) was

or 28 days (De Jong et al., 2013). Although it is still question-ble, whether the silver nanoparticles (AgNPs) cause damage tohe genetic material of treated germinated onion root tips (Alliumepa) with different concentrations (10, 20, 40 and 50 ppm) (Babut al., 2008), in vitro study demonstrated DNA damage in mam-alian cells with exposure to silver nanoparticles (Cha et al., 2008;sharani et al., 2009).

Our article investigates the hepatotoxicity and genotoxicity ofilver nanoparticles in female albino rats via evaluating the hepaticistopathological alterations, determining the hepatic oxidativetress parameters and detecting the possible chromosomal aber-ations occurring in bone marrow cells.

. Materials and methods

.1. Preparation of silver nanoparticles

Silver nanoparticles were prepared using chemical reductionethod according to (Van Dong et al., 2012) with some modi-

cation. AgNPs were synthesized by using sodium borohydrideNaBH4) and polyvinyl pyrrolidone (PVP) as reducing and stabi-izing agents, respectively. First, 0.272 g of AgNO3 was dissolvedn 344 mL deionized water and then put on magnetic stirrer for5 min. A mixture of 2.912 g of trisodium citrate and 0.504 g ofolyvinyl pyrrolidone (PVP) was dissolved in 48 mL deionizedater, stirred for 15 min and then added to the prepared AgNO3

olution.NaBH4 solution is prepared by dissolving 1.89 g of sodium boro-

ydride in 50 mL deionized water and is then put in refrigeratornd 8 mL of freshly prepared cold aqueous solution of NaBH4 wasuickly added to the formed solution with continuous stirring for0 min, the reduction reaction occurs with change of color to darkellow and brown.

. Characterization of silver nanoparticles

.1. UV-Visible absorption spectroscopy

Absorption spectra were recorded using a double beam UV–Vispectrophotometer (Cary 5000, Varian, Australia). The absorptionpectra of diluted solutions of the prepared AgNPs in aque-us medium were recorded within the appropriate scan range350–800 nm). The spectra of pure solvent were taken as aalibrating reference. Measurements were performed at room tem-erature.

.2. Transmission electron Microscope (TEM)

The morphology of AgNPs and their particle sizes were exam-ned using TEM (Tecnai, FEI, The Netherlands), operating at an

ccelerating voltage of 200 kV. A drop from a dilute sample solutionas deposited on an amorphous carbon coated-copper grid and left

o evaporate at room temperature forming a monolayer. Analysis ofarticle size diameters of the prepared AgNPs were estimated using

xicologic Pathology 67 (2014) 21–29

the software program Image J over several shots of TEM images forthe target sample (Ross and Dykstra, 2003).

3.3. Particle sizer

The particle size, size distribution were measured by zeta sizer(Malvern zeta sizer Nano ZS) based on the dynamic light scatteringtechnique.

3.4. X-ray diffraction

Samples were air dried, powdered and used for XRD analysis. X-ray diffraction patterns were recorded in the scanning mode onan X‘pert PRO PAN analytical instrument operated at 40 kV anda current of 30 mA with Cu K� radiation (� = 1.54 A). The diffrac-tion intensities were recorded from 35◦ to 79.93◦, in 2� angles. Thediffraction intensities were compared with the standard JCPDS files.The software gave the information about the crystal structure of theparticle (Bragg, 1914).

3.5. Experimental animal design and conditions

A total of 20 mature female albino rats were purchased fromResearch Institute of Ophthalmology and were kept in the Depart-ment of Animal Care and Management-Faculty of VeterinaryMedicine, Cairo University. Rats were acclimated for 14 days beforestarting the experiment. During this period and the experimentperiod, rats were housed in plastic cages (5 rats per each cage) inroom temperature and humidity in well-ventilated room with nat-ural light. The rats were fed commercial pelleted feed and givenwater ad libitum. The rats were divided into four experimentalgroups 5 per each. The treated groups were injected i/p with dif-ferent doses of silver nanoparticles (1, 2 and 4 mg/kg b.w) daily for28 days. The control groups were injected with deionized waterto exclude the factor of injection stress. Rats were weighed at thebeginning of the study and weekly throughout the experimentalperiod and the final body weights were also recorded at the end ofthe study. All procedures of using laboratory animals in this studymet the regulations of Ethics of Research Committee at Faculty ofVeterinary Medicine, Cairo University and received the approvalnumber: Cu F Vet/F/PAT/2013/12.

3.6. Oxidative stress analysis

- Protein assay: Crude enzyme protein content was determinedaccording to method described by (Bradford, 1976).

- MDA and GSH assay: Liver samples were kept frozen at −20 ◦C.and tissue homogenate was made in glass homogenizer using 10%(w/v) cold 1.15% KCl for (MDA) according to (Ding et al., 2002) and5% 5-suphosalicylic acid for (GSH) according to (Anderson, 1985).

- Estimation of malondialdhyde (MDA) and GSH in liver was doneaccording to (Albro et al., 1986) and (Ellman, 1959), respectively,and results expressed as nmol/mg protein.

3.7. Histopathological examination

The rats were observed throughout the experimental period;then the rats were euthanized using chloroform as anestheticreagent and finally cervical dislocation was done after 28 days ofexposure, Liver was removed carefully, weighed, and fixed in 10%

hol, then cleared with xylene and embedded in paraffin, sectionedat 5 �m thickness and stained by H & E stain, Masson’s trichromestain, periodic acid Schiff’s (PAS) and Prussian blue stains and thenexamined microscopically (Bancroft and Gamble, 2008).

nd Toxicologic Pathology 67 (2014) 21–29 23

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M.M. El Mahdy et al. / Experimental a

.8. Chromosome aberrations assay (CA)

Bone marrow metaphases were prepared according to theethod of Yosida and Amano (1975). Both treated and control

nimals were injected with 0.5 mg/kg colchicine 2 h before euth-nization. The colchicine is injected intraperitoneally to arresthe cell division at the metaphase stage. Femoral bone marrowas flushed with physiological saline solution (0.9% sodium chlo-

ide). The cells were centrifuged at 1000 rpm for 10 min and theupernatant was discarded. The cells were suspended in hypotonicolution of 0.56% potassium chloride, incubated at 37 ◦C for 30 minnd then centrifuged for 10 min at 100 rpm and the supernatantas discarded and fixed at room temperature in methyl-acetic acidxative (3:1 methyl alcohol: glacial acetic acid) for 30 min thenentrifugation and washing were done twice in fixative. Finallyhe cells were suspended in small amount of fixative; few dropsf the cell suspension were dropped on a clean cold slide storedn 70% ethyl alcohol and then dried on flame; after complete dry-ng, slides were stained in 10% phosphate buffered Giemsa stain for0–45 min, washed twice (5 min each) in buffer, and then mountedith mounting media (Permount) and covered with clean and dry

over slips. Slides were examined microscopically at 1000× magni-cation. At least 250 good metaphases of each animal were studied,coring the different types of chromosomal aberrations such asaps, breaks, deletions, end to end associations, centromeric atten-ations and numerical aberrations, with selection being based onniform staining quality, lack of overlapping chromosomes andhromosome number (42 ± 3 chromosomes).

.9. Determination of silver residues

Pure perchloric acid (70%) and nitric acid (70%) were pur-hased from SDFCL Company, India for digestion of liver tissues toetermine silver residues. Tissue samples were prepared for flametomic absorption spectroscope according to the method described

y Kehoe et al. (1988) where known amount of liver tissue fromach Ag-NPs treated group was weighed and digested by a mixturef perchloric and nitric acids. Pooling of samples from each groupas conducted.

Fig. 2. HRTEM Image showing spherical shape of prepared silver n

Fig. 1. UV–vis spectrum for silver nanoparticles showing peak absorption (1.652)at wavelength 406.00 nm.

3.10. Statistical analysis

Oxidative stress indicators as well as chromosomal aberrationswere evaluated statistically according to statistical analysis:

Data were presented as mean ± SEM. Variables were statisticallyanalyzed by one-way ANOVA test, using software SPSS (version 16).When differences were significant, LSD (Least Significant Differ-ence) was performed to find the individual differences betweengroups. Statistical analyses were performed according to Snedecorand Cochran (1982).

4. Results

4.1. Characterization of silver nanoparticles

4.1.1. UV-Visible absorption spectroscopySpectrophotometer measurements showed sharp absorption

peak at 406.00 nm which corresponding to the plasmonic absorp-tion band of spherical shaped silver nanoparticles as shown in Fig. 1.

4.1.2. High resolution transmission electron microscope (HRTEM)Transmission electron microscope image showed that spherical

silver nanoparticles with average size 15 ± 5 nm as shown in Fig. 2.

anoparticles with average size 15 ± 5 nm, scale bar is 20 nm.

24 M.M. El Mahdy et al. / Experimental and Toxicologic Pathology 67 (2014) 21–29

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Table 1Effects of i/p injection silver nanoparticles on liver weight:total body weight ratio.

Dose (mg/kg b.w) Liver weight: total body weight ratio (M ± SEM)

Control 2.85 ± 0.02b

1 3.67 ± 0.21a

2 3.81 ± 0.16a

4 3.52 ± 0.19a

Values are expressed as Means ± SEM. Different letters in the same column aresignificantly different (p ≤ 0.05).

Table 2Effects of i/p injection of silver nanoparticles on MDA & GSH level (nmol/mg protein)in rat liver.

Dose (mg/kg b.w) MDA (M ± SEM) GSH (M ± SEM)

Control 36,542.63 ± 7034.23b 638.58 ± 31.47b

1 32,273.37 ± 15,781.71bc 1161.44 ± 211.63a

2 58,795.32 ± 1085.28a 899.59 ± 170.46ab

4 20,459.02 ± 3234.86c 616.07 ± 62.29b

Fig. 3. Particle size distribution of prepared silver.

.1.3. Particle sizerZeta particle sizer measurements were found in accordance with

he spectrophotometric measurements and transmission electronicroscopic imaging where the average of the particle size was

.7 nm diameter as illustrated in Fig. 3.

.1.4. X-ray diffractionStructural characterization was performed using XRD analysis

nd the typical XRD pattern for silver nanoparticles was obtaineds shown in Fig. 4. The four characteristic peaks correspondingf Ag are located at 2� equal 38.91◦, 44.39◦, 64.58◦ and 77.58◦,espectively. The result indicates the formation of crystalline silveranoparticles with cubic unit cell (Card No: 04-003-5625).

.1.5. Effects of i/p injection of silver nanoparticles on total bodyeight of rats

Concerning the total body weight, there was no significant dif-erence in the total body weight between different treated groupsompared with the control group

.1.6. Effects of i/p injection of silver nanoparticles on liver weightn relation to body weight

The results showed significant increase in all treated groups ashown in Table 1.

Fig. 4. XRD pattern of si

Values are expressed as Means ± SEM. Different letters in the same column aresignificantly different (p ≤ 0.05).

4.1.7. Effect of i/p injection of silver nanoparticles on oxidativestress indicators in tissues of rats

Results were summarized in Table 2, concerning MDA, there wasnon-significant difference in MDA level in the 1 mg/kg b.w Ag-NPstreated group compared with the control. However, in 2 mg/kg b.wtreated group showed significant increase of MDA level comparedwith dose treated and control groups, while there was a signif-icant decline in MDA level in the 4 mg/kg b.w treated group incomparison with control.

Concerning GSH, there was an increase in GSH level in 1 and2 mg/kg b.w dose treated groups, this increase was significant atdose of 1 mg/kg b.w and non significant at dose of (2 mg/kg b.w)compared with control group while, no significant difference inGSH level at dose of 4 mg/kg b.w compared with control.

4.1.8. Hepatic histopathological alterationsCholangiopathy was the most prevalent hepatic alteration in all

treated groups and was found to be dose dependent. The lesion

lver nanoparticle.

M.M. El Mahdy et al. / Experimental and Toxicologic Pathology 67 (2014) 21–29 25

Fig. 5. Rat liver: (a) Control group showing normal histological structure of the portal area (H&E x400). (b) 1 mg dose treated group showing minimal cholangiofibrosis (H&Ex 4 mg

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400). (c) 2 mg dose treated group showing slight cholangiofibrosis (H&E x400). (d)reated group showing bile duct hyperplasia with formation of bile ductules assocroup positive stained fibrous tissue with Masson‘s trichrome stain (×200).

as characterized microscopically by hyperplasia of the bile ductith the presence of newly formed bile ductules associated with

val cell hyperplasia, these findings appeared to be more pro-ounced at a dose of 4 mg/kg b.w. compared with control. The lesionas associated with mild to moderate cholangiofibrosis, fibrosis

tained positively with Masson’s trichrome stain (Fig. 5a–f),On thether hand hepatocellular alterations were variable including hep-tocellular necrosis (Fig. 6a), occasional apoptosis was observedn the 1 and 2 mg/kg treated groups (Fig. 6b). The portal bloodessels were congested together with areas of sinusoidal dilata-ion and leucocytosis was observed in all Ag-NP treated groups.ine brown pigments that were noticed in the kupffer cell lin-

ng the sinusoids negatively stained with Prussian blue or PAS

ere observed in all Ag-NP treated groups (Fig. 6c) and these pig-ents were also observed in the abdominal fat at the injected area

Fig. 6d).

dose treated group showing moderate cholangiofibrosis (H&E x400). (e) 4 mg doseith portal congestion and oval cell hyperplasia (H&E x400). (f) 4 mg dose treated

4.1.9. Detection of chromosomal aberrations induced by i/padministration of silver nanoparticles

The statistical analysis of the total number and types of chro-mosomal aberrations in different treated groups is shown inTable 3. There were various chromosomal aberrations that hadbeen observed in the bone marrow metaphase cells in all Ag-NPtreated groups that were dose dependent, there was a significantincrease in the total number of structural chromosomal aberrationsat 1 mg/kg b.w and 2 mg/kg b.w dose treated groups while a signifi-cant increase in chromosomal aberrations was detected in 4 mg/kgb.w dose treated group. A normal metaphase from the bone marrowof control rat is shown in (Fig. 7a). Centromeric attenuations were

the most frequent structural aberrations observed (Fig. 7b) thatshowed a significant increase in all treated groups compared withcontrol group and showed an increase in incidence with advancingdose, while chromatid deletions were also observed (Fig. 7c) but

26 M.M. El Mahdy et al. / Experimental and Toxicologic Pathology 67 (2014) 21–29

Fig. 6. Rat liver (a)1 mg dose treated group showing hepatocellular necrosis with sinusoidal leucocytosis and Kupffer cell activation (H&EX400). (b) 4 mg dose treated groups 400).a owini

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howing occasional apoptosis associated with mild sinusoidal leukocytosis (H&E ×ctivated Kupffer cell lining the hepatic sinusoids (H&E ×400) (d) Abdominal fat shntense lymphocytic infiltration and dilatation of blood capillaries (H&E ×400).

ith no significant difference between 1 mg/kg b.w treated groupsompared with the control group. However, 2 and 4 mg/kg b.whowed significant increase in comparison with the control group.hromatid gaps were noticed (Fig. 7d) with no significant differ-nce between the control and all treated groups. Sporadic cases ofumerical chromosomal aberrations hypoploidy and hyperploidyere also recorded.

.2. Determination of silver residues

The chart in Fig. 8 showed a dose dependent increase in residualilver concentration in liver tissue (�g/g).

. Discussion

The wide range of use of AgNPs in medical devices, clothing,

ousehold water filters, contraceptives, antibacterial sprays, cos-etics, detergents, cooking utensils, cell phones, computers and

hildren’s toys is likely to result in an increase in the concentrationf AgNPs discharge to our ecosystems (Marambio-Jones and Hoek,

able 3tatistical analysis of chromosomal aberration in bone marrow cells of rats treated i/p wi

Dose (mg/kgb.w)

No. ofexamined cells

Structural aberrations (M ± SEM)

Gap Deletion Centr

Control 250 0.2 ± 0.2 a 0.4 ± 0.24 c 0.4 ±1 250 0.2 ± 0.2 a 1.2 ± 0.2 bc 4.2 ±2 250 0.2 ± 0.2 a 2.8 ± 0.97 a 1.8 ±4 250 0.2 ± 0.2 a 1.8 ± 0.2 ab 6.2 ±alues are expressed as means ± SEM. Different letters in the same column are significan

(c) 2 mg dose treated group showing deposition of brown colored pigment in theg extensive deposition of brown stained pigment free or inside macrophages with

2010) in addition the silver nanoparticles had found their way tomany consumer products, starting from water-based paints (Holtzet al., 2012) to drinking water disinfectants (Li et al., 2008; Lv et al.,2009) and food containers (Benn et al., 2010). There is strong evi-dence that microorganisms and plants are capable of concentratingnanoparticulate materials, which increases the potential for AgNPsto accumulate in the food chain (Oberdörster et al., 2005). Severalstudies were conducted on the toxicity of different sizes and con-centrations of silver nanoparticles in vitro and to less extent in vivostudies. At the present study there was no significant differencein the total body weight between different treated groups withdifferent doses and between the control group as constant bodyweight gain was similar indicating that AgNPs did not affect bodyweight this result agreed with (Kim et al., 2009, 2010), while theliver weight related to body weight was significantly increased inall dose treated groups that were related to hepatic histopatholog-

ical alterations, this results partially agreed with (Kim et al., 2010)who recorded no significant organ-weight changes in either themale or female rats after 90 days. No significant difference wasobserved in MDA levels in liver of the Ag-Nps treated group when

th different doses of silver nanoparticles.

Numerical aberrations

omeric attenuation Total 2n 4n >2n <2n

0.24 e 1.0 ± 0.00 e 250 0 0 0 0.37 b 5.6 ± 0.51 b 248 0 1 1 0.37 d 4.8b ± 1.07 c 249 0 0 1 0.37 a 8.2 ± 0.37 a 250 0 0 0

tly different (p ≤ 0.05).

M.M. El Mahdy et al. / Experimental and Toxicologic Pathology 67 (2014) 21–29 27

F rmal mc treateS errati

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reports by (Sung et al., 2009; Kim et al., 2010).The genotoxic potential of silver nanoparticles was represented

by various types of chromosomal aberrations that were dose

ig. 7. metaphase spread from bone marrow of rat (Giemsa stain, ×1000) (a) Noentromeric attenuations type of chromosomal aberrations (c) silver nano particlesilver nano particles treated group showing chromatid gap type of chromosomal ab

ompared with control group. These findings are in agreement withhe results recorded by (Ebabe Elle et al., 2013). Concerning GSHevel, the results showed significant increase in GSH level in the

mg/kg b.w treated group and non significant increase in 2 mg/kg.w treated group compared with control group suggesting to be arotective response elicited by low level of oxidative stress inducedy the lower doses of Ag-NPs and that was supported by Arorat al. (2009) while the reduction in GSH level that was recordedn 4 mg/kg b.w was in accordance with the significant reduction in

DA level. Another finding which confirms the oxidative potentialf silver nanoparticles is MDA level in tissue. The increase of MDAevel in the 1, 2 mg/kg treated group revealed the lipid peroxidationrocess while the exact cause of reduction in MDA level that wasecorded in 4 mg/kg w dose treated group is unclear. Histopatholog-cal examination of liver revealed that various alterations denotinghe hepatotoxic effect of silver nanoparticles including hepato-ellular degeneration, necrosis and individual apoptosis were theost recognized hepatic changes that were dose dependent. Sev-

ral studies confirmed that liver is the target organ for the effectf silver nanoparticles (Ji et al., 2007; Gopinath et al., 2008; Kimt al., 2008b; Sung et al., 2009), also (Hussain et al., 2005) reportedhat silver nanoparticles were highly toxic in rat’s liver cells. Silveranoparticles reduced the activity of mitochondria which results

n reduction of available energy for cells. Moreover, (Sardari et al.,012) discussed the mechanism of silver nanoparticles inducedepatotoxicity by repeated oral administration and they found thatanoparticles are removed from the liver by macrophages due tohagocytosis process and the repetition of this process produced

igher oxygen radicals while (Loghman et al., 2012) concludedhat the cytotoxicity of silver nanoparticles to the mitochondrialctivity increased with the increase in the concentration of silveranoparticles inducing drastic reduction of mitochondrial function,

etaphase spread control group. (b) Silver nano particles treated group showingd group showing chromatid deletion type of chromosomal aberrations (arrow). (d)ons (arrow). Nanoparticles.

increased membrane leakage, necrosis and induction of apopto-sis. Kupffer call activation with brown pigment deposition wasobserved in all treated groups this pigment was negatively stainedby Prussian blue or PAS indicating that it was silver nanoparti-cles not hemosiderin or lipofuscin pigment similar findings weredescribed by (Danscher, 1981) who found that uncleared silver hasbeen shown to be deposited in Kupffer cells and sinusoid endothe-lium cells in the liver. Cholangiopathy was the most prominenthepatic alteration in this study ranging from bile duct hyperplasiawith presence of newly formed bile ductules to oval cell prolifera-tion. The results of the current study are consistent with previous

Fig. 8. The following chart showed increase in residual silver concentration in livertissue (�g/g) with increase the dose of injected silver nanoparticles (mg/kg).

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ependent with significant increase at higher doses which may bettributed to oxidative stress created by silver nanoparticles expo-ure as previously discussed, various in vitro and in vivo studiesere conducted to evaluate the genotoxicity of silver nanopar-

icles that confirms the findings obtained in the present studyr contrasting the results of this study, in vitro and in vivo tox-city studies confirmed the potential of silver nanoparticles toause chromosomal aberrations and DNA damage, entering theells resulting in cellular damage and were capable of induc-ng proliferation arrest in cell lines of zebra fish (Ji et al., 2007;sharani et al., 2009; Hussain et al., 2005) but the exact mecha-ism through which silver nanoparticles induce genotoxicity stillemains unclear although (Asharani et al., 2009) found that AgNPsan damage DNA (in this case from human lung fibroblasts anduman glioblastoma cells) indirectly by increasing ROS productionr by decreasing ATP production inducing mitochondrial damagehich in turn impairs energy-dependent DNA repair mechanisms.lso, (Cha et al., 2008; Yang et al., 2009b) assumed that direct DNAamage occurred by Ag+ released by AgNPs themselves. Asare et al.2012) confirmed results of the present study as they recorded aoncentration dependent increase in DNA-strand breaks in humanesticular embryonic carcinoma cells damage to DNA by increasen reactive oxygen species the data were obtained from the studyuggesting the AgNPs inducing genotoxicity by increasing reactivexygen metabolites level that was recorded in tissue homogenates.n contrast Kim et al. (2008b) found no statistically significantffects in bone marrow micronucleus test after the oral adminis-ration of 60 nm silver nanoparticles for 28 days at various dosesr after exposure of rats to 18 nm silver particles via inhalation atoncentrations of 0.7 × 106 particles/cm3, 1.4 × 106 particles/cm3,r 2.9 × 106 particles/cm3, 6 h/day for 90 days (Kim et al., 2011).

. Conclusion

Silver nanoparticles had the ability for inducing variousistopathological alterations in the liver indicating hepatocyto-oxicity presumably by oxidative stress. Also, the induction ofhromosomal aberrations in bone marrow cells denoting the geno-oxicity of nanosilver particles even with small doses ranging from

to 4 mg/kg which were injected i/p for 28 days. The severityf hepatic lesions and silver concentration in liver tissues wasncreased proportionally.

cknowledgement

Thanks are extended to all members of Nanotechnology &dvanced Materials Central Lab for their support and help.

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