The Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.38, No.4, 525-530, 2013
525 Letter
Exposure to zinc oxide nanoparticles affects reproductive development and biodistribution in offspring rats Eunhye Jo, Gyounbaek Seo, Jung-Taek Kwon, Mimi Lee, Byoung cheun Lee, Igchun Eom, Philje Kim and Kyunghee Choi Environmental Health Research Department, National Institute of Environmental Research, Incheon 404-708, Korea (Received March 15, 2013; Accepted May 4, 2013)
ABSTRACT — Understanding reproductive development effects and transferable properties to next generation of zinc oxide nanoparticles is necessary for prevention of its potential risks. To accomplish this, rats were exposed to zinc oxide nanomaterials (500 mg/kg bw) of less than 100 nm beginning 2 weeks before mating to postnatal day 4. In addition, body distribution of zinc concentration was evaluated in dams and offspring. Rat treated with nano-zinc oxide showed reduced number of born/live pups, decreased body weights of pups and increased fetal resorption. Zinc oxide nanomaterials were also distributed to organs such as mammary tissue of dams and liver and kidney of pups. These results indicate that zinc oxide nanoparticles-exposure before and during pregnancy and lactation could pose health risks to pregnant women and their fetus. Key words: Zinc oxide, Nanoparticles, Reproductive and developmental toxicity, Distribution
INTRODUCTION The recent increasing use of nanosized particles has been purported to found in several products, such as drug/gene deliveries, pesticides, fertilizers, foods and cosmetics. Several articles about nanomaterials investigated the potential side effect of nanosized particles. These studies may appear to be more dangerous to the fetus, with the size below 1/1000 th and the width of a human hair. For example, silica and titanium nanoparticles with diameters less than 100 nm can cause pregnancy complications when injected intravenously into pregnant mice, and also accumulate in the placenta, fetal liver and fetal brain (Yamashita et al., 2011). Prenatal titanium dioxide exposure also results in alteration to the cerebral cortex, olfactory bulb and some regions intimately related to dopamine systems of offspring mice (Umezawa et al., 2012). Fullerene distributed to the placenta and fetuses of exposed pregnant dams and to the milk and pups of exposed lactating dams (Sumner et al., 2010) Yosida et al. (2008) noted that carbon black nanoparticles adversely affect mouse spermatogenesis caused the consequent fluctuation of sperm testosterone levels. Moreover, researchers argue that nanomaterials found in many products affected poor
fetal growth and low birth weight in unborn and newborn infants (Yamashita et al., 2011; Wang et al., 2010). Even these results showed that placenta or breast milkmediated exposure to several nanomaterials may induce adverse effects in the next generation and, no studies have examined animal exposure to zinc oxide nanoparticles. Zinc oxide nanoparticles have been commonly used in ceramic manufacture, photocatalysis, UV filters and food industry (Yon et al., 2011). Zinc oxides are used in consumer products such as sunscreen, additives and packing agents due to their antimicrobial properties, fungicides, anticancer drug, and imaging in medical purpose, can lead to ingestion exposure (He et al., 2010; Rasmussen et al., 2010). The oral exposure of zinc oxide nanoparticles (300 mg/kg) during 14 days resulted that the accumulation of nanoparticles in the liver and induced oxidative stress which led to DNA damage and apoptosis in liver cells (Sharma et al., 2012). Mouse embryos exposed with zinc oxide nanoparticles (< 100 nm) during embryonic organogenesis exhibited severe growth and development retardation such as diminishment of yolk sac diameter, crown-rump length and head length (Yon et al., 2011). In this study, we investigated the reproductive and development toxicity and tissue distribution of offspring following oral exposure in rats. To assess this, histopatho-
Correspondence: Eunhye Jo (E-mail:
[email protected])
Vol. 38 No. 4
526 E. Jo et al.
logical changes were examined by oral exposure in reproductive organs. MATERIALS AND METHODS Zinc oxide nanoparticle Zinc oxide nano dispersion (particle size < 100 nm; size (DLS, dynamic light scattering), 35 nm average particle size 50 wt % water; Sigma-Aldrich Korea Ltd., Yongin, Korea) was directly administered to animals. Animals and treatment Specific-pathogen free male and female (7-weeks-old) SD rats purchased from OrientBio (Sungnam, Korea) were maintained in our laboratory animal facility (23 ± 2°C, 50 ± 20% relative humidity and 12-hr light/dark cycle). The animals were acclimatized for 7 days prior to beginning the study. The OECD guideline 421 for testing chemicals (Reproductive and development toxicity screening test) is useful to obtain initial information on possible effects on reproduction and development (Ema et al., 2010). This study was performed using relatively small numbers of animals in limited group referring to the OECD TG 421. The rats were randomly divided into two groups (control and treatment) and each group consisted of 12 male and 12 female rats. The test materials was administered by gavage at dose levels of 0 or 500 mg/kg bw/day to male rats for 6 weeks including 2 weeks before mating, and to females from 2 weeks before mating to day 4 of lactation including the gestation period. The dose was selected based on the results of the repeated dose 13-week oral toxicity study of zinc oxide nanoparticle (KFDA, 2010). All methods used in this study were approved by the Animal Care and Use Committee of National Institute of Environmental Research (NIER-12-1). Observations Throughout the test period, clinical observations conducted at once a day. Males and females were weighed on the first day of dosing, at least weekly thereafter, and at termination. The number of stillbirths, live births, runts and the presence of gross abnormalities of each litter were examined. On day 3 of lactation, live pups were counted and weighed. At the time of sacrifice, adult animals were examined macroscopically for any abnormalities or pathological changes. The testes, epididymides, ovaries and uterus of adult animals were weighed. Biodistribution of nanoparticles A small piece of liver, kidney, uterus and mammary Vol. 38 No. 4
tissue in dams and brain, liver, kidney, stomach including weaned milk and blood were collected under anesthesia and froze at -20¶C overnight. Each sample was weighted and then the zinc burdens in tissues were quantified with and inductively coupled plasma mass spectrometry (ICP-MS, Varian 820-MS, Australia) after microwave-assisted digestion of samples with HNO3 using a microwave digestion system. Histopatholosical observation A small piece of testis and epididymis of male rats and ovary and uterus of female rats was fixed by 10% formalin, the embedded into paraffin, sectioned at 5 um thickness and mounted on the glass microscope slides. The slides were stained with hematoxylin-eosin and examined by microscope. Statistical analysis The Student’s t-test (Graphpad Software, San Diego, CA, USA) and two-way ANOVA (IBM SPSS statics 19, Chicago, IL, USA) analysis was used to compare results obtained from the exposed groups with results obtained from the control group. RESULTS Reproductive and development toxicity The rats gastrointestinally administered with 500 mg/kg bw nanoscaled zinc oxide showed symptoms of diarrhea and alopecia from the beginning day after treatment. Two weeks later, those symptoms in both control and treatment groups were disappeared, but three male rats in zinc oxide nanoparticles-group were dead at 4, 13 and 25 days after administration. All mortalities were preceded by body weight loss and anorexia. No death occurred in the treated female rats. The body weight gain of the treated rats by zinc oxide nanoparticles was about up to 16% lower than the control rats. There was no significant difference in the body weights (data not shown). Similarly, no obvious difference was observed in the testes, epididymides and ovaries between two groups (Tables 1 and 2). However, a significant (p < 0.05) increase in the uterus weight was found in female rats treated 500 mg/kgGzinc oxide nanomaterials as compared to the control (Table 2). Zinc oxide nanomaterials at a dose of 500 mg/kg had no effect on male and female fertility and mating/gestation period. The number of implants was similar between groups, indicating that pregnancy rate, mating performance and postimplantation were unaffected (Table 3). However, the number of newborn and live pups after
527 Development effects and biodistribution of ZnO NP in offspring rats
Table 1. Absolute organ weight changes in Male SD rat after oral administration of ZnO NPs (unit : g) Number of animals examined
Testis (L)
Testis (R)
Epididymis (L)
Epididymis (R)
Control
12
1.661 ± 0.065
1.647 ± 0.067
0.631 ± 0.046
0.627 ± 0.042
ZnO NPs 500 mg/kg
9
1.599 ± 0.097
1.619 ± 0.099
0.623 ± 0.070
0.630 ± 0.039
Table 2. Absolute organ weight changes in Female SD rat after oral administration of ZnO NPs (unit : g)
Control
Number of animals examined
Uterus
Ovary (L)
12
0.708 ± 0.111
0.080 ± 0.018
0.073 ±
0.008
0.112 ± 0.137
0.078 ±
0.014
ZnO NPs 12 1.790 ± 1.467* 500 mg/kg *, significant differences (p < 0.05) from the control group
(A)
Ovary (R)
(B) ໌/g
໌/g
*
*
Fig. 1.
Expression of the zinc concentration in tissues of (A) dam rats and (B) pups (* indicating p < 0.05).
4 days of birth was reduced in the treated group. The number of pups surviving from birth to weaning was reduced gradually during lactation and pups gained less weight in the exposed group (Table 4). At necropsy, offspring born to dams in treatment group had no abnormal incidence. Zinc concentration in tissues To analyze the transferability of properties to fetus and
sucking pups via milk, zinc concentration in biological samples was checked accurately. A significant (p < 0.05) increase in the zinc content was found in the mammary tissue of adult rats in the zinc oxide nanomaterials-treated group compared with control (Fig. 1A). The liver, kidney and uterus of female adult rats also showed increases in the zinc content but not statistically significant. The offspring exposed to zinc oxide nanomaterials at a dose of 500 mg/kg showed significantly (p < 0.05) higher levels Vol. 38 No. 4
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12
12
No. of females mated 10
100
No. of females Mating index (%) pregnant 83.3
Male fertility index (%)
13.8 ± 1.6
5.1
94.9
Implantation Live birth index loss rate (%) (%) 13.1 ± 1.6
No. of Pups Born (day 0) 13.1 ± 1.6
100.0
27.2
100.0
11
10
Normal delivery
2.9 ± 1.1
2.3 ± 1.2
Mating period (days)
No. of live pups Viability index Gestation index (day 4) day 4 (%) (%)
91.7
91.7
Female fertility index (%)
ZnO NPs 12.3 ± 2.7 52.8 47.2 5.8 ± 5.8 1.3 ± 2.7 22.4 500 mg/kg Implantation loss rate (%) : ((No. of implantations No. live neonates)/No. of implantations) × 100 Live birth index (%) : (No. of live pups on postnatal day 0/No. of implantations) × 100 Viability index day 4 (%) : (No. of pups surviving on postnatal day 4/No. of pups born alive in postnatal day 0) × 100 Gestation index (%) : (No. of females with live pups/No. of pregnant females) × 100
Control
No. of Implantations
Table 4. Delivery and pups observations in rats exposed ZnO NPs
ZnO NPs 12 12 11 100 83.3 500 mg/kg Mating index (%) : (No. of females mated/No. of females placed with males) × 100 Male fertility index (%) : (No. of males impregnating a female/No. of males placed with females) × 100 Female fertility index (%) : (No. of females pregnant/No. of females placed with males) × 100
Control
No. of females placed with males
Table 3. Mating and gestation results in rats exposed ZnO NPs
7.80 ± 2.87
12.02 ± 0.97
Body weight of F1 (day4)
22.0 ± 0.4
21.9 ± 0.3
Gestation period (days)
528 E. Jo et al.
529 Development effects and biodistribution of ZnO NP in offspring rats
n.
n.
A Fig. 2.
B Histopathology of the uterus of the control and Zinc nanoparticles administered rats. Animal No.=A, F1 (Con); B, F3 (ZnO). H&E, Magnification = X100 for A and B.
of zinc contents in liver and kidney compared to control group (Fig. 1B). The zinc content of stomach including milk and blood in pups exposed to zinc oxide nanomaterials increased but it was not significantly different. In contrast, there was no difference in zinc content in the brain of treated and control pups. Histopathological changes No abnormal findings were observed in the testes, epididymides and ovaries. However, the uterus of adult female rats orally exposed to zinc oxide nanomaterials (500 mg/kg) showed pathological lesions. In uterus, multifocal granulation tissues were noted in both groups. The lesions were characterized by localized necrosis (n.), histiocyte infiltration (arrows in Fig. 2B) and thrombosis (arrows in Fig. 2A) as well as inflammation. These regions were considered to be the implantation site of fetus, and they were on the process of repairing after fetal delivery. DISCUSSION Zinc has been reported to have functional properties such as antioxidant, anti-inflammatory and antiapoptotic effects with its immunoregulator effects on animals and humans (Costa et al., 2012), Especially, the prenatal zinc supplementation confers a beneficial effect on the infants’ neurobehavioural development and immune functions, or the rate of head growth in infants (Surkan et al., 2012). However there is concern that nano-sized materials exhibit unknown biological or environmental effects, even if their bulk counterparts are known to be safe (Yoshida et
al., 2009). The developmental toxicity is one of the major emerging issues on the hazard of nanomaterials (Fujitani et al., 2012) This study specifically provides insights into the consequences of administered zinc oxide nanomaterials on reproductive and development in rats and biotransferred effects on this nanoparticles-related fetal accumulation. In the mating, fertility of male/female, mean mating duration and gestation period were not affected by treatment of zinc oxide nanoparticles in adult rats. The decreased body weight gain in male/female adult rats was observed, but was insignificant changes comparing with control. However, the death of three adult males and adverse clinical effects such as diarrhea and alopecia were found in treated group. These symptoms were presented in the mice exposed with nano-zinc powders and zinc salts (Wang et al., 2006). But more consideration of the deaths by nanozinc oxide is required, since the NOAEL value of nanozinc oxide was 268.4 mg/kg bw in previous studies using SD rats (KFDA, 2010). Another study represented that nano-zinc oral exposure (5,000 mg/kg bw) induced severe intestinal obstruction because nanoscaled materials easing aggregate in animal body (Wang et al., 2006). This nanomaterial specific effect induced anorexia and three deaths of male adult rats. Additionally, slight damages of reproductive organs were detected in histopathological examinations that localized necrosis was seen in uterus of treated group. These results were observed in 20% of animals and then not considered to be related to nano-zinc oxide (data not shown). In development study, the number of implantations Vol. 38 No. 4
530 E. Jo et al.
was unaffected, but increased post-implantation loss rate, and decreased live birth index and the number of pups born were observed at a dose of 500 mg/kg zinc oxide nanoparticles. After delivery, viability of pups and mean weight gain of litters were highly decreased in F1 generation treated. In the previous studies, the oral exposure to zinc oxide nanoparticles in mice mainly leads to accumulation in liver and kidneys (Sharma et al., 2012; Lee et al., 2012). As shown in Figure, increased zinc levels were detected in the dam liver, kidney, uterus and mammary tissue, but only mammary tissue reached a level of statistical significance. When we investigated the absorption and distribution to fetus or offspring, zinc levels significantly increased in the liver and kidney of pups, indicating similar toxicokinetic behavior to that of adult rats. The zinc contents in pups’ brain, on the other hand, were not changed, indicating that nano-zinc could not pass through the blood brain barrier. Summarizing these findings, the liver and kidney were found to be the possible target organs for accumulation of zinc oxide nanoparticles. Also, it seems likely that transplacental and breastfeeding transport of nano-zinc oxide occurred actively between pregnant mother and pups. The similar possible mechanism by nano-zinc oxide induced adverse effects on the offspring of several species (Nations et al., 2011; Kulvietis et al., 2011; Ma et al., 2013). In conclusion, the evidence of reproductive developmental toxicity of zinc oxide nanoparticles was found, is consistent with theirs transference and accumulation over the lactation and placenta barrier. The fertility and mating parameters were unaffected by zinc oxide nanoparticles, but exposed dams to nano-zinc oxide during before pregnant, gestation and lactation led to clear indications of developmental toxicity in the offspring. Further detailed data on exposure, teratogenicity and toxicological mechanism of nano-zinc, such as teratogenicity study and kinetics on pups, are necessary to determine whether it has human risk or not. REFERENCES Costa, C., Brazao, V., Kuehn, C., Oliveira, L., Junior, J., Sala, M. and Abrahao, A. (2012): Zinc and pregnancy: Marked changes on the immune response following zinc therapy for pregnant females challenged with Trypanosoma cruzi. Clin. Nutr., in press. Ema, M., Kobayashi, N., Naya, M., Hanai, S. and Nakanishi, J. (2010): Reproductive and developmental toxicity studies of manufactured nanomaterials. Reprod. Toxicol., 30, 343-352. Fujitani, T., Ohyama, K., Hirose, A., Nishimura, T., Nakae, D. and Ogata, A. (2012): Teratogenicity of multi-wall carbon nanotube (MWCNT) in ICR mice. J. Toxicol. Sci., 37, 81-89. He, L., Liu, Y., Mustapha, A. and Lin, M. (2010): Antifungal activity of zinc oxide nanoparticles against Botrytis cinerea and Penicillium expansum. Microbiol. Res., 166, 209-215. Vol. 38 No. 4
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