http://informahealthcare.com/nan ISSN: 1743-5390 (print), 1743-5404 (electronic) Nanotoxicology, 2014; 8(S1): 85–91 ! 2014 Informa UK Ltd. DOI: 10.3109/17435390.2013.857734

ORIGINAL ARTICLE

Effects of silver nanoparticles on pregnant dams and embryo-fetal development in rats Wook-Joon Yu1*, Jung-Mo Son2*, Jinsoo Lee1, Sung-Hwan Kim2, In-Chul Lee2, Hyung-Seon Baek2, In-Sik Shin2,3, Changjong Moon2, Sung-Ho Kim2, and Jong-Choon Kim2 Korea Institute of Toxicology, KRICT, Daejeon, Republic of Korea, 2College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea, and 3Korea Research Institute of Bioscience and Biotechnology, Chungbuk, Republic of Korea

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Abstract

Keywords

Although the potential risk of silver nanoparticles (AgNPs) to humans has recently increased due to widespread application, the potential effects of AgNPs on embryo-fetal development have not yet been determined. This study investigated the potential effects of AgNPs on pregnant dams and embryo-fetal development after maternal exposure on gestational days (GD) 6–19 in rats. AgNPs were administered to pregnant rats by gavage at concentrations of 0, 100, 300, and 1000 mg/kg/day. All dams were subjected to Cesarean section on GD 20 and the fetuses were examined for signs of embryotoxic and teratogenic effects. Examinations of hepatic oxidant/antioxidant balance and serum biochemistry were also added to the routine developmental toxicity study. Treatment with AgNPs caused a decrease in catalase and glutathione reductase activities at 100 mg/kg/day and a reduction in glutathione content at 1000 mg/kg/day in maternal liver tissues. However, no treatment-related deaths or clinical signs were observed in any of the animals treated with AgNPs. No treatment-related differences in maternal body weight, food consumption, gross findings, serum biochemistry, organ weight, gestation index, fetal deaths, fetal and placental weights, sex ratio, or morphological alterations were observed between the groups. The results show that repeated oral doses of AgNPs during pregnancy caused oxidative stress in hepatic tissues at 100 mg/kg/day, but did not cause developmental toxicity at doses of up to 1000 mg/kg/day. The no-observed-adverse-effect level of AgNPs is considered to be 5100 mg/kg/day for dams and 1000 mg/kg/day for embryo-fetal development.

Developmental toxicity, maternal toxicity, nanomaterials, oxidative stress, teratogenicity

Introduction The physicochemical properties of nanomaterials are attributable to their small size, chemical composition, surface structure, solubility, shape, and aggregation. The novel properties of nanomaterials raise several questions with regard to their safety and environmental impact. The adverse effects of nanomaterials have been intensively investigated with increasing interest in their potential toxicity (Sharifi et al., 2012). Many studies have demonstrated that nanomaterials readily travel throughout the body, deposit in target organs, penetrate cell membranes, lodge in mitochondria, and may induce adverse effects in reproductive organs and embryos (Ahamed et al., 2010; Ema et al., 2010; Johnston et al., 2010; Nel et al., 2006). Accordingly, their ultimate usage may be limited by potential adverse effects on human health and the environment (Ahamed et al., 2010; Stensberg et al., 2011). Silver nanoparticles (AgNPs) have increasingly been used for a wide range of practical applications as antibacterial/antifungal agents in biotechnology and bioengineering, textile engineering,

*These authors contributed equally to this work as co-first authors. Correspondence: Jong-Choon Kim, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea. Tel: +82 62 530 2827. Fax: +82 62 530 2809. E-mail: [email protected]

History Received 9 July 2013 Revised 13 October 2013 Accepted 17 October 2013 Published online 22 November 2013

water treatment, and silver-based consumer products due to their unique size-dependent optical, electrical and magnetic properties (Sintubin et al., 2012). Therefore, they have attracted huge academic and industrial interest, evidenced by the thousands of studies on AgNPs published every year. However, despite of such appealing features, their usage may be limited by the possible risks to the environment and human health (Nel et al., 2006; Johnston et al., 2010). Consequently, considerable effort has been dedicated to understanding the health effects of these nanomaterials before they are widely used in consumer products where the potential for general public exposure would be increased. It is evident that AgNPs represent a potential hazard to human health and have an environmental impact. However, the potential effects of AgNPs on human health are still unclear and have been relatively unexplored. Recent studies have reported that AgNPs cause inflammatory, oxidative, genotoxic, and cytotoxic effects (Ahamed et al., 2010; Johnston et al., 2010; Kim et al., 2010a). A number of investigators have reported that cytotoxicity, DNA damage, and apoptosis induced by AgNPs are mediated through membrane lipid peroxidation, reactive oxygen species (ROS) generation, and oxidative stress (Choi et al., 2010; Kim et al., 2009; Lima et al., 2012). Previous studies have demonstrated that AgNPs lead to reproductive failure, embryonic death, and morphological abnormalities in zebrafish (Asharani et al., 2008; Powers et al., 2011), Japanese medaka (Wu et al., 2010), fathead minnow (Laban et al., 2009), and oysters

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(Ringwood et al., 2010). Common causes of AgNP-induced reproductive toxicity include oxidative stress, DNA damage, and apoptosis (Ahamed et al., 2010; Choi et al., 2010). It has been well described that oxidative stress and DNA damage can induce embryotoxicity and/or teratogenicity in experimental animals (Hansen, 2006; Kim et al., 2012; Kovacic & Somanathan, 2006). Thus, we hypothesized that exposure to AgNPs during pregnancy might generate adverse effects on pregnant dams and embryo-fetal development through oxidative stress and DNA damage. According to a combined repeated-dose toxicity study with reproductive/developmental toxicity of AgNPs, oral administration of AgNPs before mating, during the mating and gestation period, and during 4 days of lactation at 250 mg/kg/day does not produce any obvious effects on reproduction or development in rats (Hong et al., 2013). However, the potential effects of AgNPs on pregnant rats and embryo-fetal development have not been examined rigorously. The possible risks of AgNPs to human health and the environment have recently increased due to enhanced production and widespread use, which may result in severe health impacts due to environmental contamination. Therefore, it is important to investigate the potential effects of AgNPs on pregnant dams and embryo-fetal development. The aim of this study was to determine the potential effects of AgNPs on pregnant dams and embryo-fetal development in SpragueDawley rats administered AgNPs from days 6–19 of gestation.

Materials and methods Animal husbandry and maintenance Male and nulliparous female Sprague-Dawley rats aged 10 weeks were obtained from a specific pathogen-free colony at Orient Bio Inc. (Seoul, Korea) and used after one week of quarantine and acclimatization. The animals were housed in a room maintained at a temperature of 23  3 C and a relative humidity of 50  10% with artificial lighting from 08:00 to 20:00 and 13 to 18 air changes per hour. For mating, two females were placed in a cage with a male rat overnight. Successful mating was confirmed by the presence of sperm in the vaginal smear, and the following 24 h was designated as day 0 of gestation (GD 0). The mated females were subsequently housed individually in clear polycarbonate cages with stainless steel wire lids and were provided UV-irradiation sterilized tap water and fed commercial rodent chow (PMI Nutritional International Inc., Brentwood, MO, USA) ad libitum. This experiment was conducted in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care International and all procedures were approved by our Institutional Animal Care and Use Committee. Test article The test article examined in this study was commercially available AgNPs (NANOMIXTM) purchased from NanoPoly Co. Ltd (Seoul, Korea). Information provided by manufacturer states that the particle size measured by transmission electron microscopy (TEM) is 7.5  2.5 nm. The test article was suspended at concentrations of 100, 300, or 1000 mg/10 ml in 0.5% carboxymethylcellulose (Sigma Aldrich Co., St. Louis, MO, USA) aqueous solution and was prepared immediately before treatment. The stock solution of 1000 mg/10 ml was prepared in a Pyrex bottle using Millipore water (ion free) and sonicated in an ultrasonicator (VCX-130, Sonics and Materials, Newtown, CT) for 3 min (130 watt, 20 kHz, pulse 59/1). To obtain the size and morphology of the AgNPs, TEM characterization was performed using a JEM-1210 (JEOL, Tokyo, Japan) at an accelerating voltage of 200 kV. The samples were deposited on carbon-coated nickel grids and were air dried overnight before TEM analysis.

Nanotoxicology, 2014; 8(S1): 85–91

Experimental groups Forty-four healthy female rats were randomly assigned to four experimental groups: three AgNP treatment groups receiving 100, 300, or 1000 mg/kg/day and a control group (n ¼ 11 inseminated females per group). Treatment The test mixture was administered daily by gavage to pregnant rats from GD 6–19 with a dose volume of 10 ml/kg body weight of the rats. Control rats received an equivalent volume of vehicle alone. The daily application volume was calculated in advance based on the most recently recorded body weight of the individual animal. Although human exposure to environmental materials can occur through a variety of mechanisms, the oral route was selected in this study. Because of their widespread use in foods, packaging and cosmetics, oral ingestion of AgNPs is of special concern (Philbrook et al., 2011; Sintubin et al., 2012). Once in the environment due to waste from consumer products, they could be ingested by a host of organisms. Individuals who work in the AgNP industry or nanoparticle research laboratories may also be at increased risk. Observation of dams All pregnant females were examined daily throughout the gestation period for mortality, morbidity, general appearance, and behavior. Maternal body weights were measured on GD 0, 6, 9, 12, 15, 17, and 20, and individual food consumption was determined on GD 0, 6, 9, 12, 15, 17, and 20. Post mortem examination Cesarean sections were performed on GD 20. The ovaries and uterus of each female were removed and examined for the number of corpora lutea and the status of all implantation sites, i.e. live and dead fetuses, early and late resorptions, and total implantations. Uteri that showed no evidence of implantation were further evaluated by staining with 2% sodium hydroxide solution to identify the presence of early implantation losses (Yamada et al., 1988). If no stained implantation sites were present, the rat was considered not pregnant. Resorption was classified as ‘‘early’’ when only a resorption site resembling a dark brown blood clot and with no embryonic tissue was visible, and ‘‘late’’ when both the placental and embryonic tissues were visible at the postmortem examination. All live fetuses were weighed individually, sexed, and examined for any external morphological abnormalities, including a cleft palate. Alternate fetuses were selected for either a skeletal or visceral examination. Half of the live fetuses from each litter were fixed in 5% formalin solution, eviscerated, and then processed for skeletal staining with Alizarin Red S using the modified Dawson’s method (1926) for the subsequent skeletal examination. The other half were preserved in Bouin’s solution and examined for internal soft tissue changes using a freehand razor sectioning technique (Wilson, 1965) and Nishimura’s method (1974). The fetal morphological alterations observed in this study were classified as developmental malformations or variations. A malformation was defined as a permanent structural change likely to adversely affect survival or health (Chahoud et al., 1999). The term ‘‘variation’’ was defined as a change within the normal population under investigation unlikely to adversely affect survival or health. In fetuses with supernumerary ribs (SNR), the lengths of ossified portions of the supernumerary ribs were measured with an ocular micrometer. We used an actual length of 0.6 mm to separate short (rudimentary) from long (extra) SNR according to the method of Rogers et al. (2004). Terminology suggested in an internationally developed glossary

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of terms was used to classify structural developmental abnormalities (Makris et al., 2009).

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Pathology At the time of Caesarean section, all pregnant females were euthanized by carbon dioxide inhalation for blood sample collection. Blood samples were drawn from the posterior vena cava using a syringe with a 24-gauge needle. The samples were centrifuged at 1256 g for 10 min within 1 h after collection. The sera were stored in a 80  C freezer before they were analyzed. The following parameters were measured by an autoanalyzer (Shimadzu CL-7200, Shimadzu Co., Kyoto, Japan): aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine phosphokinase (CPK), glucose, total protein (TP), albumin (ALB), blood urea nitrogen (BUN), creatinine (CRTN), triglyceride (TG), phospholipid (PL), total cholesterol (T-CHO), total bilirubin (T-BIL), calcium (Ca), and inorganic phosphorus (IP). A complete gross postmortem examination was performed. The absolute and relative (organ-to-body weight ratio) weights of the brain, pituitary gland, adrenal glands, liver, spleen, kidneys, heart, and ovaries were measured. Weighed frozen liver tissue was homogenized in a glass-Teflon homogenizer with 50 mM phosphate buffer (pH 7.4) to obtain 1:9 (w/v) whole homogenates. The homogenates were then centrifuged at 11 000  g for 15 min at 4 C to discard any cell debris. The supernatant was used to measure malondialdehyde (MDA), reduced glutathione (GSH), glutathione reductase (GR), catalase (CAT), glutathione peroxidase (GPx), and glutathione-S-transferase (GST). The concentration of MDA was assayed by monitoring thiobarbituric acid reactive substance formation by the method of Berton et al. (1998). Glutathione was measured by the method of Moron et al. (1979). The activities of antioxidant enzymes including GR (Carlberg & Mannervik, 1986), CAT (Aebi, 1984), GPx (Uchiyama & Mihara, 1978), and GST (Habig et al., 1984) were also determined. Total protein contents were determined by the method of Lowry et al. (1951), using bovine serum albumin as the standard. Statistical analyses The unit for statistical measurement was the pregnant female or the litter. Quantitative continuous data such as maternal body weight, food consumption, average fetal body weight per litter, and placental weight were subjected to one-way analysis of variance (ANOVA). A Scheffe’s multiple comparison test was carried out when the differences were significant. The number of corpora lutea, total implantations, live and dead fetuses, and gender ratio were evaluated statistically using the Kruskal–Wallis nonparametric ANOVA, followed by the Mann–Whitney U-test, where appropriate. The proportions of litters with malformations and developmental variations were compared using the chi-square test and Fisher’s exact probability test. The statistical analysis was performed by comparing the treatment groups with the control group using the Path/Tox System ver. 4.2.2 (Xybion Medical Systems Co., Cedar Knolls, NJ) and SAS software ver. 9.1 (SAS Institute, Cary, NC). Significant probability values are represented as p50.05 (*) and p50.01 (**).

Results

Figure 1. A representative transmission electron micrograph image of AgNPs. The size distribution of AgNPs in suspension (1000 ppm) was measured using a submicron particle size analyzer. The average particle size was 6.45  2.55 nm when measured immediately after preparation.

Dose-range finding study Doses of 10, 100, and 1000 mg/kg/day were given to six pregnant rats per group. There were no treatment-related effects related to maternal and developmental parameters at any of the doses tested. Therefore, 1000 mg/kg/day, the limit dose recommended by the Organization for Economic Co-operation and Development Test Guideline 414, was used as the high dose, and doses of 300 and 100 mg/kg/day were selected as medium and low doses, respectively. Effects of AgNPs on dams Although hair loss was observed in one case in the control group, two cases in the 100 mg/kg group and one case in the 1000 mg/kg group, this clinical sign was transiently observed and was not dose-dependent in either incidence or severity. No significant differences in body weight (Table 1) or food consumption (Supplementary Table I) were observed between the groups. At the scheduled autopsy, no treatment-related gross findings were observed in dams of any group. Absolute brain weights in all treatment groups increased significantly compared with those in the control group (Table 2). No significant difference was observed between the treatment groups and controls regarding any of the serum biochemical parameters examined in pregnant dams (Supplementary Table II). As shown in Table 3, liver CAT and GR activities in all treatment groups and glutathione content in the 1000 mg/kg group decreased significantly when compared with the control group.

TEM analysis of AgNPs Analysis of the vehicle-based solution of AgNPs by TEM indicated a size distribution 510 nm, with a mean and standard deviation of 6.45  2.55 nm (Figure 1).

Effects of AgNPs on embryo-fetal development Table 4 summarizes the reproductive findings for the pregnant rats treated with AgNPs on days 6 through 19 of pregnancy.

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Nanotoxicology, 2014; 8(S1): 85–91

Table 1. Body weight changes of pregnant rats treated with AgNPs during gestational days 6–19. AgNPs (mg/kg/day) Parameters

0

No. of rats mated No. of pregnant rats Gestational day 0 Gestational day 6 Gestational day 9 Gestational day 12 Gestational day 15 Gestational day 17 Gestational day 20 (Covariate-adjusted mean) Weight gain during pregnancy

100

11 8 251.5  11.49a 290.6  16.80 304.4  16.33 322.1  15.03 341.2  13.75 367.1  13.75 418.1  16.75 (411.1) 166.6  12.70

300

11 10 247.7  13.56 295.8  25.67 308.2  21.95 327.3  22.43 345.1  20.27 366.3  22.52 412.5  26.50 (413.7) 164.9  22.80

1000

11 11 246.7  13.27 297.1  26.79 310.2  24.01 331.1  22.03 348.3  25.58 373.2  26.54 421.3  27.58 (415.4) 174.6  18.97

11 11 247.6  12.78 289.7  19.84 302.6  18.89 320.7  17.26 335.5  21.41 356.2  26.95 393.4  39.00 (409.5) 145.8  38.52

a

Values are presented as means  SD (g).

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Table 2. Absolute and relative organ weights of pregnant rats treated with AgNPs during gestational days 6–19. AgNPs (mg/kg/day) Parameters No. of dams Body weight at term Brain (g) Per body weight (%) Pituitary gland Per body weight (%) Adrenal glands (g) Per body weight (%) Liver (g) Per body weight (%) Spleen (g) Per body weight (%) Kidneys (g) Per body weight (%) Heart (g) Per body weight (%) Ovaries (g) Per body weight (%)

0

100

300

1000

8 410.8  16.51a 1.83  0.072 0.45  0.022 0.015  0.0019 0.004  0.0004 0.064  0.0067 0.016  0.0018 15.42  1.038 3.76  0.223 0.68  0.104 0.17  0.027 2.03  0.190 0.49  0.036 1.07  0.054 0.26  0.014 0.12  0.015 0.03  0.004

10 404.9  27.66 1.96  0.098** 0.49  0.038 0.013  0.0035 0.003  0.0010 0.066  0.0073 0.016  0.0024 14.89  0.979 3.69  0.301 0.68  0.099 0.17  0.023 2.09  0.183 0.52  0.059 1.13  0.095 0.28  0.031 0.12  0.013 0.03  0.005

11 415.1  25.84 1.92  0.037* 0.46  0.031 0.013  0.0025 0.003  0.0006 0.071  0.0109 0.017  0.0035 15.31  1.174 3.69  0.162 0.73  0.063 0.18  0.017 2.14  0.175 0.52  0.023 1.10  0.075 0.27  0.017 0.12  0.011 0.03  0.004

11 385.2  39.42 1.92  0.058* 0.49  0.043 0.013  0.0027 0.003  0.0007 0.064  0.0098 0.016  0.0022 14.15  1.592 3.62  0.265 0.67  0.066 0.17  0.023 2.09  0.211 0.54  0.063 1.06  0.076 0.27  0.017 0.12  0.014 0.03  0.005

a

Values are presented as means  SD. *Significant difference at p50.05 level when compared with the control group. **Significant difference at p50.01 level when compared with the control group.

Table 3. Antioxidant enzymes, glutathione and lipid peroxidation levels in the livers of pregnant rats treated with AgNPs during gestational days 6–19. AgNPs (mg/kg/day) Parameters No. of dams Catalase (unit/mg protein) Glutathione reductase (unit/mg protein) Glutathione-S-transferase (unit/mg protein) Glutathione peroxidase (unit/mg protein) Glutathione (nmol/mg protein) Malondialdehyde (mmol/mg protein)

0

100

300

1000

8 1228.8  257.82a 1.16  0.166 40.3  4.79 4.36  0.481 775.9  97.89 113.8  11.06

10 397.9  80.26** 0.73  0.105** 36.4  5.50 4.73  0.352 662.1  70.84 118.5  16.62

11 327.5  82.06** 0.55  0.115** 41.8  6.04 4.87  1.010 676.5  48.93 123.7  12.55

11 354.1  82.36** 0.46  0.078** 44.1  10.59 4.62  0.222 647.5  73.68* 127.4  18.87

a

Values are presented as means  SD. *Significant difference at p50.05 level when compared with the control group. **Significant difference at p50.01 level when compared with the control group.

Overall pregnancy rates were comparable across all dosage groups, ranging from 72.7 to 100%. No totally resorbed litters were found in any group. The number of corpora lutea, implantations, post-implantation loss rates, fetal deaths, litter size, gender ratio of live fetuses, fetal body weight, and placental

weight were similar in the treatment groups and the control group, whereas pre-implantation loss rate in the 1000 mg/kg group was significantly higher than that in the controls. Supplementary Table III summarizes the types and incidence of external and visceral malformations and variations observed

Developmental toxicity of silver nanoparticles

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Table 4. Cesarean section data in pregnant rats treated with AgNPs during gestational days 6–19. AgNPs (mg/kg/day) Parameters No. of dams No. of corpora lutea No. of implantation sites Pre-implantation loss (%)b Fetal deaths Resorption: early late Dead fetuses Post-implantation loss (%)c Litter size Gender ratio at birth (% male) Fetal weight (g) Male (Covariate-adjusted mean) Female (Covariate-adjusted mean)

0 8 15.4  1.51a 15.0  1.41 2.4  3.30 0.5  0.76 0.4  0.52 0 0.1  0.33 3.3  4.73 14.5  1.41 46 4.1  0.20 (4.2) 3.9  0.14 (4.0)

100 10 16.0  1.49 13.7  4.24 14.5  24.58 0.8  1.32 0.5  0.85 0.3  0.67 0 5.7  8.63 12.9  4.25 51 4.1  0.31 (4.1) 3.9  0.28 (3.9)

300 11 15.6  1.29 15.0  1.10 3.8  6.07 0.7  1.01 0.7  1.01 0 0 5.0  7.07 14.3  1.74 50 4.0  0.16 (4.1) 3.8  0.22 (3.8)

1000 11 15.7  2.69 11.5  4.34 25.5  28.29* 0.4  0.50 0.3  0.47 0 0.1  0.30 4.1  7.60 11.2  4.33 54 4.2  0.22 (4.1) 3.9  0.18 (3.9)

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a

Values are presented as means  SD. Pre-implantation loss (%) ¼ [(No. of corpora lutea  No. of implantation sites)/No. of corpora lutea]  100. c Post-implantation loss (%) ¼ [(No. of implantation sites  No. of live embryos)/No. of implantation sites]  100. *Significant difference at p50.05 level when compared with the control group. b

in F1 fetuses. Although some external and visceral abnormalities were found in the treatment groups, the incidence of these findings was comparable between the groups. Supplementary Table IV summarizes the types and incidences of fetal skeletal malformations and variations observed in the F1 fetuses. Only one case of skeletal malformation (i.e. short 13th rib) was observed in the 1000 mg/kg group. Although some types of skeletal variations, including incomplete ossification of the parietal, incomplete ossification of the interparietal, wavy ribs, short supernumerary ribs, full supernumerary ribs, misshapen sternebrae, misaligned sternebrae, bipartite ossification of the sternebrae, dumbbell ossification of the sternebra, unossified thoracic centrum, bipartite ossification of the thoracic centrum, and dumbbell ossification of the thoracic centrum were found in the treatment groups, no significant differences in the number of fetuses with developmental variations or in the number of litters with affected fetuses were observed between the groups.

Discussion and conclusions This study was conducted to investigate the maternal and developmental toxic potential of AgNPs administered orally to Sprague–Dawley rats at doses of 0, 100, 300, and 1000 mg/kg/day from GD 6–19. Examinations of hepatic oxidant/antioxidant balance and serum biochemistry were added to the routine developmental toxicity study, as AgNP exposure can induce cytotoxicity and hepatic dysfunction through oxidative damage (Choi et al., 2010; Kim et al., 2009; Lima et al., 2012; Tiwari et al., 2011), which may play a major role in maternal toxicity and teratogenesis (Hansen, 2006; Kim et al., 2012; Kovacic & Somanathan, 2006). Our results show that 14-day oral repeated dosing of AgNPs during pregnancy caused oxidative stress in maternal hepatic tissues, as evidenced by a decrease in liver CAT and GR activities at a dose of 100 mg/kg/day, but did not cause any developmental toxicity at doses up to 1000 mg/kg/day in rats. Toxicity of AgNPs has been extensively studied over the last decade using in vitro and in vivo test systems. The primary site of AgNP accumulation has been consistently demonstrated to be the liver; therefore, it is relevant that a number of in vitro and in vivo investigations have focused on this target organ. According to a report by Hussain et al. (2005), AgNP exposure results in membrane damage, reduced GSH levels, and an increase in ROS production in BRL 3 A rat liver cells. It was demonstrated that

AgNP treatment resulted in a decrease in DNA content in the human Huh-7 hepatoma cell line (Cha et al., 2008). Kim et al. (2009) reported that AgNPs are toxic to human hepatoma cells and that the cytotoxicity is primarily the result of oxidative stress. Kawata et al. (2009) found that non-cytotoxic doses of AgNPs induce the expression of genes associated with cell cycle progression and apoptosis in HepG2 human hepatoma cells. Arora et al. (2009) also found that AgNPs enter fibroblasts and liver cells and cause DNA damage and apoptosis. Most recently, Piao et al. (2011) reported that AgNPs induced the generation of ROS and deplete intracellular GSH in human Chang liver cells, leading to damaged cellular components. The results of these studies clearly show that AgNP exposure in vitro causes cytotoxicity through oxidative stress in liver cells. The present study also showed that AgNPs treatment of pregnant rats caused some oxidative stress in hepatic tissues, as evidenced by the decrease in CAT and GR activities at doses 100 mg/kg/day and a decrease in glutathione content at a dose of 1000 mg/kg/day. Unlike in vitro studies, in vivo experimental studies have provided inconsistent evidence concerning the type and severity of hepatotoxicity by AgNPs. According to a 28-day oral toxicity study of AgNPs in rats (Kim et al., 2008), minimal hepatotoxicity, including increased serum values of ALP and cholesterol and an increased incidence of histopathological changes such as bile duct hyperplasia, inflammatory cell infiltration, and dilatation of central veins was observed at doses 300 mg/kg/day. Slight hepatotoxic effects of AgNPs were also found at 125 mg/kg/day in a rat 90-day oral toxicity study (Kim et al., 2010b), but the ALP and cholesterol values observed in the treatment groups were within the limits of normal biological variation (Lee et al., 2012; Petterino & Argentino-Storino, 2006; Wolford et al., 1986) and were not associated with any other indicators of hepatic dysfunction. Hepatic histopathological changes and serum biochemical alterations have also been observed in dermal and intravenous toxicity studies of AgNPs (Korani et al., 2011; Tiwari et al., 2011). In contrast, no changes in liver function parameters or histopathology were reported in a combined repeated-dose toxicity study of AgNPs with reproductive/developmental toxicity (Hong et al., 2013). Hadrup et al. (2012) also reported that 28-day repeated oral dose of 9 mg AgNPs/kg/day did not induce any hepatotoxic effects in rats. In the present study, although 14-day repeated oral doses of AgNPs to pregnant rats caused an increase in hepatic oxidative stress at 100 mg/kg/day, no treatment

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related effects, including clinical signs, body weight changes, food intake, gross findings, or serum biochemistry were observed at any dose tested. Increased absolute brain weights observed in the treatment groups were also considered incidental and not treatment-related, as the changes did not show a dose-response relationship and were within the limits of normal biological variations (Lee et al., 2012). Our results agree with those of previous studies (Hadrup et al., 2012; Hong et al., 2013). The apparent discrepancies among studies may be related to many factors including exposure route, level, and duration, organ doses, and physicochemical properties of the AgNPs such as size, shape, chemical composition, solubility, surface area and surface charge. Nevertheless, the results of this study and the existing literature suggest that toxic potency of AgNPs for oral exposure in vivo is much lower than that in in vitro experimental systems and that AgNPs are relatively safe for human consumption. We have recently demonstrated that repeated oral doses of multi-wall carbon nanotubes during pregnancy is minimally maternotoxic, but not embryo–fetotoxic even at 1000 mg/kg/day in rats (Lim et al., 2011). Philbrook et al. (2011) have reported that oral administration of AgNPs on GD 9 decreased viability of fetal mice at lowest dose (10 mg/kg), but not at higher doses (100 or 1000 mg/kg). A number of previous studies have demonstrated that AgNPs lead to reproductive failure, embryonic death, and developmental malformations in a number of non-mammalian animal models (Ahamed et al., 2010; Johnston et al., 2010; Stensberg et al., 2011). Philbrook et al. (2011) reported that AgNPs increased mortality of fetal mice when given orally to pregnant mice at 10 mg/kg on GD 9. However, the fetotoxic effects were of questionable significance because the increased number of non-viable fetuses was slight and no obvious signs of maternal and developmental toxicity were observed at the higher dose levels of 100 and 1000 mg/kg. In the present study, no treatment-related effects were found for any reproductive or developmental parameter examined. Although some fetal morphological alterations were observed in the treatment groups, they occurred at a low incidence and were similar to those commonly observed in normal control rat fetuses (Kim et al., 2001; Morita et al., 1987). The increased pre-implantation loss rate observed in the high dose group was not considered related to AgNP treatment because the test article was administered after the completion of embryo implantation (Quinn et al., 2008) and the finding did not exhibit a dose-response relationship. This interpretation was also supported by the results of a combined repeated-dose toxicity study with reproductive/ developmental toxicity (Hong et al., 2013). In that study, the 52day repeated oral doses of AgNPs from 2 weeks before mating, during the mating and gestation period, and during 4 days of lactation at dose levels of 62.5, 125 and 250 mg/kg/day did not cause any treatment-related effects on reproduction or development in rats. On the other hand, it has been well described that the remnants of embryos that die soon after implantation are not apparent at gross examination of the uterus at terminal necropsy (Salewski, 1964; Yamada et al., 1988). Early postimplantation losses may not be detected near term even by staining the uterus with sodium hydroxide or ammonium sulfide. It has been reported that in vitro AgNP treatment of mouse blastocysts causes a decrease in the implantation success rate and is associated with increased resorption of post-implantation embryos and decreases in fetal weight (Li et al., 2010). Therefore, it is possible that the increased pre-implantation loss observed in the high dose group could result from the AgNP treatment. However, the exact cause of the increased preimplantation loss is not known at present. Further more focused studies are necessary to elucidate the cause-effect relationship

Nanotoxicology, 2014; 8(S1): 85–91

between the AgNP exposure and implantation failure observed in this study. It has been reported recently that free silver ions are released during preparation of dosing suspensions and that the biological activity of freshly prepared and aged AgNPs is strongly different due to the different amounts of silver ions released (Kittler et al., 2010; Loeschner et al., 2011). In the present study, AgNPs were stored in the dry state to prevent dissolution and they were freshly prepared daily before treatment. Although the concentration of ionic silver in the AgNP suspensions was not analyzed, it is considered that a minimum amount of free silver ions was included in the dosing suspensions. Overall, it can be concluded that repeated oral doses of AgNPs during pregnancy caused oxidative stress in hepatic tissues at doses 100 mg/kg/day, but did not cause developmental toxicity at doses up to 1000 mg/kg/day. Under these experimental conditions, the no-observed-adverse-effect level of AgNPs is 5100 mg/kg/day for dams and 1000 mg/kg/day for embryo-fetal development. This study was performed according to the current test guidelines, except 8–11 litters examined per group. Although the number of litters per group was relatively lower than that recommended in current guidelines for developmental toxicity studies, the results of our study will provide valuable information on the developmental toxic effects and target organ toxicity of AgNPs via repeated oral exposure, which can aid in the process of risk assessment.

Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (NRF-2013R1A1A2010835). The animal experiment in this study was supported by the Animal Medical Institute of Chonnam National University.

Declaration of interest The authors report no conflicts of interest. The authors are solely responsible for the content and writing of the paper.

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Supplementary material available online Supplementary Tables I–IV