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Maternal exposure to titanium dioxide nanoparticles during pregnancy; impaired memory and decreased hippocampal cell proliferation in rat offspring
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Maternal exposure to titanium dioxide nanoparticles during pregnancy; impaired memory and decreased hippocampal cell proliferation in rat offspring Abbas Mohammadipour a , Alireza Fazel a , Hossein Haghir a,b , Fatemeh Motejaded a , Houshang Rafatpanah c , Hoda Zabihi d , Mahmoud Hosseini e , Alireza Ebrahimzadeh Bideskan a,∗ a
Department of Anatomy and Cell Biology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran b Medical Genetic Research Center (MGRC), School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran c Immunology Research Center, Buali Institute, Mashhad University of Medical Sciences, Mashhad, Iran d Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad, Iran e Neurocognitive Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
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Article history:
Titanium dioxide nanoparticles (TiO2 -NPs) are massively produced in the environment, and
Received 19 July 2013
because of their wide usage, they are a potential risk of damage to human health. TiO2 -NPs
Received in revised form
are often used as additives for paints, papers, and foods. The central nervous system (CNS),
16 January 2014
including hippocampal regions, is potentially susceptible targets for TiO2 -NPs. This study
Accepted 20 January 2014
aimed to determine the effects of exposure to TiO2 -NPs during pregnancy on hippocampal
Available online 30 January 2014
cell proliferation and the learning and memory of offspring. Pregnant Wistar rats received intragastric TiO2 -NPs (100 mg/kg body weight) daily from gestational day (GD) 2 to (GD) 21.
Keywords:
Animals in the control group received the same volume of distilled water via gavage. After
Titanium dioxide nanoparticles
delivery, the one-day-old neonates were deeply anesthetized and weighed. They were then
Maternal exposure
killed and the brains of each group were collected. Sections of the brains from the rat off-
Learning and memory
spring were stained using Ki-67 immunolabeling and the immunohistochemistry technique.
Hippocampal cell proliferation
Some of the male offspring (n = 12 for each group) were weaned at postnatal day (PND21), and
Neurotoxicity
housed until adulthood (PND60). Then the learning and memory in animals of each group were evaluated using passive avoidance and Morris water maze tests. The immunolabeling of Ki-67 protein as a proliferating cell marker showed that TiO2 -NPs significantly reduced cell proliferation in the hippocampus of the offspring (P < 0.05). Moreover, both the Morris water maze test and the passive avoidance test showed that exposure to TiO2 -NPs significantly impaired learning and memory in offspring (P < 0.05). These results may provide basic experimental evidence for a better understanding of the neurotoxic effects of TiO2 -NPs on neonatal and adult brains. © 2014 Elsevier B.V. All rights reserved.
∗ Corresponding author at: Department of Anatomy and Cell Biology, School of Medicine, Mashhad University of Medical Sciences, Azadi Sq., Vakilabad Blvd., P.O. Box 91779-48564 Mashhad, Iran. Tel.: +98 511 8002486; fax: +98 511 8002487. E-mail address: [email protected] (A.E. Bideskan). 1382-6689/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2014.01.014
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1.
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 617–625
Introduction
Titanium dioxide nanoparticles (TiO2 -NPs) are often used as additives in papers, paints, ceramics, plastics, and foods (Lomer et al., 2004; Oberdorster et al., 2005). The properties of absorption and reflection with ultra violet (UV) light also prompt the wide use of TiO2 -NPs in a variety of cosmetics. Moreover, they are used in the environmental decontamination of air, soil, and water (Wang et al., 2011). As ultrafine-sized materials, TiO2 -NPs can enter the human body through various routes such as inhalation, ingestion, and skin (Oberdorster et al., 2005; Jin et al., 2008). In recent years, studies have shown that after entering the body, TiO2 -NPs accumulate in the liver, kidneys, spleen, lungs, heart, and brain (Wang et al., 2007; Liu et al., 2009; Ma et al., 2010). Many studies have also shown that exposure to TiO2 -NPs may damage the central nervous system (CNS) (Wang et al., 2007; Hu et al., 2011). Still other studies have found that TiO2 -NPs promote the production of reactive oxygen species (ROS) (Long et al., 2006, 2007). Wang et al. found that TiO2 -NPs that migrated into the hippocampus led to oxidative stress and inflammation (Wang et al., 2008). The hippocampus is highly vulnerable to oxidative stress due to its high metabolic rate (Cui et al., 2004). The hippocampus is one of the important regions of the brain that has been implicated in learning and memory (Kim et al., 2009). Recent studies also confirmed that TiO2 -NPs can be transferred from mother to offspring via breastmilk and by passing through the placenta (Gao et al., 2011; Shimizu et al., 2009). Shimizu et al. also reported that maternal exposure to nano-particulate titanium dioxide during the prenatal period affects the expression of genes related to brain development (Shimizu et al., 2009). Moreover, Gao et al. reported that 100 mg/kg TiO2 exposure during development decreased hippocampal synaptogenesis in offspring (Gao et al., 2011). Thus, it seems that exposure to TiO2 -NPs during pregnancy may possibly affect the offspring hippocampus. For the study of hippocampal proliferation, the Ki-67 protein (as a proliferating cell marker) immunolabeling method is well-established (Seolhwa et al., 2011; Kim et al., 2009; Scholzen and Gerdes, 2000). This protein is expressed during mitosis. The formation of new neurons in the hippocampus occurs mainly during the gestational period (Scholzen and Gerdes, 2000). In the present study, the effect of maternal exposure to TiO2 -NPs during pregnancy on offspring hippocampal proliferation was investigated by Ki-67 immunolabeling. The Morris water maze and passive avoidance tests were used to study memory changes in pups.
2.
Materials and methods
2.1.
Chemicals and preparation
The TiO2 nanoparticles used in this study were a kind of nanopowder, anatase, with a particle size of 10 nm, surface area >150 m2 /g, purity +99%, and density 3.9 g/m3 , and were purchased from Nano Lima, Co. (Iran).
TiO2 -NPs were suspended in distilled water. Quantitative suspensions (100 mg/kg) were prepared fresh every day and fed to the rats with gavage.
2.2. Nanoparticle characterization by transmission electron microscopy The sizes of TiO2 -NPs were determined using transmission electron microscopy (TEM), which showed that particles diameters were in the nano-size range. An image of the sample is shown in Fig. 1.
2.3.
Animals and treatment
Male and female Wistar rats aged 3–4 months and weighing 250–300 g were purchased from the Animal Center of Mashhad University of Medical Sciences. The rats were housed under controlled conditions of temperature (20 ± 2 ◦ C) and lighting (12 h light:12 h dark photoperiod) and were permitted free access to food and water. The female rats were caged with male rats at a ratio of 3:1. The onset of pregnancy was confirmed by the presence of sperm in vaginal smears, and the pregnant dams were immediately housed in individual cages with free access to water and food until delivery. Experimental procedures were carried out in accordance with Mashhad University of Medical Sciences, Ethical Committee Acts. The pregnant rats were randomly divided into two groups of control and TiO2 . Animals in the TiO2 group (n = 6) received 100 mg/kg TiO2 orally (gavage) from prenatal day 2 to day 21. The animals in control group (n = 6) were administered distilled water (Gao et al., 2011). After delivery, two male pups from each litter were randomly sampled, weighed, and assigned to an experimental group. One pup was used for the determination of hippocampal titanium content, and another was used for an histological examination. Some of the male offspring (n = 12 for each group) were weaned at postnatal day (PND) 21, and housed until adulthood (PND 60). Then the learning and memory in animals of each group were evaluated using passive avoidance and Morris water maze tests.
2.4.
Hippocampus titanium contents determination
The whole hippocampus of each day-1 neonate was weighed, digested, and analyzed for titanium content. Briefly, prior to elemental analysis, the tissues of interest were digested in nitric acid overnight. After adding 0.5 ml H2 O2 , the mixed solutions were heated at about 160 ◦ C using a high pressure reaction container in an oven chamber until the samples were completely digested. Then, the solutions were heated to 120 ◦ C to remove the remaining nitric acid until the solutions were colorless and clear. Next, 3 ml of 2% nitric acid was added to the remaining solutions. Inductively coupled plasma-mass spectrometry (ICP-MS, HP4500, USA and Japan) was used to analyze the titanium concentration in the samples. Detected data were expressed as nanograms per gram of hippocampal tissue (Gao et al., 2011; Zhang et al., 2010).
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2.5.
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Coefficients of brain
After weighing the body and brains, the coefficient of brain to body weight was calculated as the ratio of tissues (wet weight, mg) to body weight (g).
2.6.
Behavioral tests
2.6.1.
Morris water maze
A circular black pool (136 cm diameter, 60 cm high, and 30 cm deep) was filled with water (23–25 ◦ C). A circular platform (10 cm diameter, 28 cm high) was placed within the pool and was submerged approximately 2 cm below the water surface in the center of the northeast quadrant. Outside the maze, fixed visual cues were present at various locations around the room (i.e., computer, hardware, and posters). The animals performed four trials on each of five consecutive days. Each trial began with the rat being placed in the pool and released facing the side wall at one of four positions (the boundaries of the four quadrants, labeled North (N), East (E), South (S), and West (W)). The released positions were randomly predetermined. For each trial, the rat was allowed to swim until it found and remained on the platform for 20 s. If 60 s passed and the animal had not found the platform, it was guided to the platform by the experimenter and allowed to stay there for 20 s. The rat was then removed from the pool, dried, and placed in its holding bin for 20 s. The latency to reach the platform and the length of the swimming path were recorded by a video tracking system. On the sixth day, the platform was removed, and the animals were allowed to swim for 60 s. The time spent in the target quadrants was compared between groups (Hosseini et al., 2011; Azizi-Malekabadi et al., 2012; Saffarzadeh et al., 2010).
2.6.2.
Passive avoidance test
The passive avoidance apparatus consisted of two light and dark (black) compartments of the same size (20 × 20 × 30 cm3 ) separated by a door. The floor of the dark compartment was made of stainless-steel bars (0.5 cm diameter) separated by a distance of 1 cm. All training and testing were carried out between 08:30 AM and 11:00 AM. Each animal was placed in the light
Fig. 1 – TEM image of the TiO2 particles.
Fig. 2 – The content of titanium in hippocampus of rat offspring after intragasteric treatment with TiO2 -NPs during pregnancy period; titanium in hippocampus of the control group was not detected. Bar marked with asterisks is statistically significant different from the control (P < 0.001). Values are represented as mean ± SEM.
compartment for 20 s, after which the door was raised and the time the animal waited before crossing to the dark (shock) compartment was recorded as the latency. The total time spent in light and dark compartments were also recorded. The animal was removed from the experiment when it waited for more than 180 s to cross to the other side. Once the animal completely crossed to the next compartment, the door was closed and an intermittent electric shock (50 Hz, 3 s, 1 mA intensity) was delivered to the floor of the dark compartment by an isolated stimulator. At 1, 24, and 48 h after training, retention tests were performed to examine memory. Each animal was placed in the light compartment for 20 s, the door was opened, and latency for entering the shock compartment as well as amount of time spent in the light and dark compartments were measured. During these sessions, no electric shock was applied (Pourmotabbed et al., 2011; Naghibi et al., 2012).
2.7.
Histopathological examination
2.7.1.
Tissue sampling
After delivery, one-day-old neonates were deeply anesthetized and weighed. Once the rats were killed, the brains were
Fig. 3 – The brain coefficients of rat offspring exposed to TiO2 -NPs during development. Bars marked with double asterisks are significantly different from the control (unexposed rats) at the 1% confidence level. Values are represented as mean ± SEM.
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Fig. 4 – Photomicrographs of the hippocampi of rat offspring. Maternal exposure to TiO2 -NPs during pregnancy reduced hippocampal cell proliferation in rat offspring. (A) CA1 control group, (B) CA1 TiO2 group, (C) CA3 control group, (D) CA3 TiO2 group, (E) DG control group, (F) DG TiO2 group, arrowheads point to representative Ki-67 positive cells. Cells were counterstained with Hematoxylin. Scale bars, 10 m.
collected from each group and fixed in 10% neutral buffered formalin for 3 days. The brain samples were processed for embedding in paraffin using routine histological protocols.
2.7.2.
Immunohistochemistry
For immunostaining, sections were deparaffinized with xylene, rehydrated through descending concentrations of ethanol, rinsed in retrieval antigen solution (citrate buffer 0.01 M, pH 6.0; S2031 Dako, England), and heated in a microwave for 15 min. The sections were then immersed in a methanol/H2 O2 solution (1:100) for 20 min in the dark to block
endogenous peroxidase. Then they were rinsed in 0.05 M PBS plus 0.025% Trition X-100 (5 min, 3 times) at room temperature. To decrease background staining, all sections were treated with 10% normal goat serum (ab 7481, abcam, USA) in PBS (goat as the host of the secondary antibody) for 1 h. The sections were then covered with proliferating cell marker, anti Ki-67 (ab16667, Abcam, USA), and with (diluted 10 in 1000 with 1% BSA) as the primary antibody, were kept in a humidified chamber overnight at 4 ◦ C. After incubation, the slides were washed extensively with PBS containing 0.025% Trition X-100. The sections were then applied with HRP-conjugated secondary
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antibody (goat anti-rabbit IgG, ab6721, Abcam, USA), diluted to 10:700 concentrations in PBS with 1% BSA for 60 min at room temperature. All sections were washed extensively with PBS for 3 min and treated with DAB (DAB kit, Sigma Aldrich, USA) solution (0.03 g DAB in 100 ml PBS and 200 l H2 O2 /100 ml PBS) for 15 min at room temperature in the dark. After being washed under running water, all sections were counterstained with a solution of Harris Hematoxylin for 2 min. Finally, the sections were dehydrated in increasing graded ethanol, cleared in xylene, and mounted on glass slides.
2.8.
Quantification of Ki-67+ cells
The areas of the hippocampi were measured using Image software (Olympus BX51, Japan). The number of Ki-67 immunoreactive cells was counted under 40× objective and by using rectangular grids placed randomly on the investigated areas. Morphometrical methods were used to count Ki-67+ cells per unit area in the CA1, CA2, CA3, and dentate gyrus of the hippocampus. The mean number of Ki-67+ cells per unit area (NA) in different regions of the hippocampus was calculated using the following formula (Sadeghi et al., 2013; Rajabzadeh et al., 2011): NA =
Q
a/f ·
P
In this formula “ Q” is the sum of counted particles in the sections, “a/f” is the area associated with each frame, and “ P” is the sum of frame-associated points hitting the defined space.
Statistics
Statistical analysis was performed using the SPSS 16 software for Windows. The data were analyzed statistically using repeated measures ANOVA for behavioral data and independent t test for histological data. P-values of <0.05 were considered to be statistically significant.
3.
Results
3.1.
Titanium content
The content of hippocampal titanium is shown in Fig. 2. The results indicated that the titanium accumulated in the hippocampus of the TiO2 -NPs group (P < 0.001) while in the unexposed rats, titanium was not detected.
3.2.
The increased coefficients indicate that injury was induced in the offspring after exposure to TiO2 -NPs (Fig. 3).
3.3.
2.9.
Fig. 5 – The influence of prenatal exposure to TiO2 on cell proliferation in the hippocampus in one-day-old pups, Bars marked with an asterisk or double asterisks are significantly different from the control at the 5% or 1% confidence level, respectively. The data are presented as the mean ± SEM.
Coefficient of brain
After delivery, one-day neonates were anesthetized and the body and brain weights were measured. Then the body weight coefficients of the brain which were expressed in mg (wet weight of tissue)/g (body weight) were analyzed. In the group treated with TiO2 -NPs, the coefficients of the brain were significantly higher (P < 0.01) than those of the control. Moreover, in the control group, the average body weight was 7.64 ± 0.17 g while it was 5.97 ± 0.18 g in the TiO2 treated group (P < 0.001).
Effects of TiO2-NPs on Ki-67+ cells
To elucidate the effect of TiO2 -NPs on hippocampal cell proliferation, the Ki-67 (a proliferating cell marker) was examined in the hippocampi of the rat offspring. Ki-67-positive cells were observed in the hippocampi of all offspring (Fig. 4). After the administration of TiO2 -NPs, the number of Ki-67-positive cells in the CA areas and DG of the hippocampus declined sharply (Figs. 4 and 5).
3.4.
Evaluation of spatial recognition memory
The length of the swimming path during 5 days and latency were significantly higher in the progeny of the TiO2 group compared to the control group (P < 0.01) (Fig. 6A and B). In the probe trial, time spent in the target quadrant (Q1) was also significantly lower in the progeny of the TiO2 group compared to that of the control group (P < 0.05) (Fig. 6C). Trial-by-trial analysis of day-1 data of the time showed that there was no difference between the two groups in the first trial, but other trials in the control group had significantly lower results than the TiO2 group (Fig. 6D). There was no significant difference in speed between the two groups (Fig. 6E).
3.5.
Evaluation of passive avoidance test
As shown in Fig. 7A, before receiving the shock, there was no significant difference between groups in latency to enter the dark compartment. One, 24, and 48 h after receiving the shock, latency to enter the dark compartment was lower in the TiO2 group than in the control group (P < 0.01). Total time spent in the dark compartment by the animals in the TiO2 group was significantly more than that of the control group at all three times after receiving the shock (Fig. 7B, P < 0.01); however, there was no significant difference before the shock.
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Fig. 6 – The effect of exposure to TiO2 -NPs during pregnancy on spatial recognition memory in offspring. (A and B) The length of the swimming path during 5 days and latency were significantly higher in the progeny of the TiO2 group compared with the control group (P < 0.01). (C) The time spent in the target quadrant (Q1) was significantly lower in the progeny of the TiO2 group than that of the control group (P < 0.05). (D) The trial-by-trial analysis of day-1 data of the time. The data showed that there was no difference between the two groups in the first trial, but other trials in the control group were significantly lower than the TiO2 group. (E) shows that there was no significant difference in speed between the two groups.
4.
Discussion
The aim of the present study was to assess the effects of exposure to TiO2-NPs > during pregnancy on hippocampal cell proliferation, learning, and memory performance of rat offspring. Our data indicate that exposure of pregnant mothers to TiO2 -NPs can impair hippocampal cell proliferation in newborn offspring. Moreover, we found that these nanoparticles impair the memory and learning of pups in adulthood. Today, TiO2 -NPs are widely used in daily life. Several studies reported that a daily intake of TiO2-NPs > may lead to its accumulation in various organs (Wang et al., 2007; Liu et al., 2009; Zhang et al., 2010). Nanoparticles can enter the body through
inhalation, dermal contact, and ingestion. The oral route may be important in many consumer products such as toothpaste, food additives, kitchen utensils, and water decontamination (Lomer et al., 2004; Wang et al., 2011). Because of this, oral administration was used in the present study. It has been shown that during the developmental period, the development of individuals is influenced by environmental factors (Hayashi et al., 1998; Iqbal et al., 2004). Recent studies have confirmed that TiO2-NPs > can be transferred from mother to offspring (Gao et al., 2011; Shimizu et al., 2009). Previous studies also proved that TiO2-NPs > can cross through the blood–brain barrier, enter the brain, and partially deposit in the hippocampus (Wang et al., 2008; Zhang et al., 2010). The current study showed that nanotitanium can transfer from
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Fig. 7 – The effect of prenatal exposure to TiO2 -NPs on passive avoidance learning, after receiving the shock, latency to enter the dark compartment was lower in the TiO2 group than in the control group (A). Moreover, time spent in the dark compartment was longer in the TiO2 group than in the control group (B) (P < 0.01). Data are expressed as ±SEM.
mother to the offspring’s hippocampus, confirming previous studies (Gao et al., 2011; Shimizu et al., 2009). The present study also confirmed that maternal exposure to titanium leads to titanium accumulation in the offspring’s hippocampus. Hu et al. reported that exposing animals to TiO2 -NPs leads to titanium accumulation in the brain (Hu et al., 2011). Gao et al. reported that maternal exposure to TiO2 -NPs during pregnancy leads to titanium accumulation in offspring hippocampus (Gao et al., 2011). After exposure to TiO2 -NPs, K+ content and Na/K-ATPase activity significantly decreased, and Na+ content increased in brain. Therefore, Na/K-ATPase cannot export intracellular Na+ and/or import extracellular K+ normally, which would result in the accumulation of intracellular Na+ , loss of intracellular K+ , and consequently, a reduction in the Na+ and K+ electrochemical gradient. This may disturb ionic homeostasis and the physiological functions of neurons (Renping et al., 2010). Moreover, exposure to TiO2 -NPs decreased the zinc content in rat brains. Zinc plays an important role in neurotransmission by modulating the activity of glutamate and gamma aminobutyric acid receptors (Renping et al., 2010). It is also believed to play an important role in cognition and memory via its function as a neuronal messenger, modulator of synaptic transmission, and cortical plasticity (Nakashima and Dyck, 2009). It is well-known that a reduction in brain zinc content can impair spatial memory in adult rats (Tahmasebi Boroujeni et al., 2009). These nanoparticles can also stimulate ROS generation in the brain (Long et al., 2007). In addition, a recent study reported that after animals were exposed to TiO2 -NPs (100 mg/kg), the activities of anti-oxidative enzymes were reduced in their brains (Ma et al., 2010). The hippocampus is a main target of TiO2 -NPs, and hippocampal cells are highly sensitive to ROS following exposure to nanoparticles (Yang et al., 2010). Oxidative stress can cause damage to cellular components such as membranes, DNA, and proteins (Beal, 1996). Moreover, the formation of ROS affects cell proliferation or the survival of new neurons. As oxidative stress is implicated in numerous neuropsychiatric disorders, the pathologies of such disorders may potentially be mediated through the impairment of proliferation by the excessive accumulation of ROS in the adult brain (Taupin, 2010). So, oxidative stress may undermine cell proliferation in the offspring brain. It has also been
reported that TiO2 -NPs may have developmental neurotoxicity on the physiological functions of the hippocampus. Studies have also indicated that exposure to TiO2 -NPs (100-NPs (100 mg/kg) from prenatal day 2 to day 21 may disrupt synaptogenesis in the developing hippocampus (Gao et al., 2011). However, no study has been published on the effects of embryonic exposure to TiO2 -NPs on cell proliferation and memory. This study found that cell proliferation, memory, and learning are affected by developmental exposure to these nanoparticles. It has been well-documented that neurogenesis occurs during developmental periods, including prenatal and immediately after birth, which affects behavior during adulthood (Gil-Mohapel et al., 2010). According to the results of the present study, it seems that the memory impairment seen in adult rats was, at least in part, due to neurogenesis impairment during the developmental period. In the present study, the length of swimming path during 5 days and latency were significantly higher in the TiO2 group than in the control group. Trial-by-trial analysis of day-1 data of the time showed that there was no difference between the two groups in the first trial, but results of other trials in the control group were significantly lower than the TiO2 group. This confirms that there were no pre-existing effects and animals had no mobility problems, but learning tasks decreased in the TiO2 group. In this study, the effects of TiO2 -NPs on a limited number of animals were investigated. Only one dose of TiO2 -NPs was given to the rats in the present study; however, further, broader studies need to be done in this regard. It is recommended that other methods such as Western Blot and PCR be used in further studies. In the past, 5-bromo-2-deoxyuridin (BrdU) was used as the principal mitotic marker to determine the rate of cell proliferation (Kempermann and Gage, 2002; Schauwecker, 2006). However, BrdU can be incorporated into dividing neurons (Gould et al., 1998), and numerous other side effects have been reported, such as stress during injection and mutagenesis following incorporation (Kee et al., 2002). Ki-67, an endogenous marker for proliferating cells, is a reliable marker, because it is expressed during mitosis and has a short half-life (Scholzen and Gerdes, 2000). In addition, Ki-67 can be easily detected by immunohistochemistry. Therefore, in this study, the effect of
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maternal exposure to TiO2 -NPs on hippocampal proliferation was investigated by Ki-67 immunoreactivity. The 1969 report of the World Health Organization (WHO) was consulted about dose selection in the current study. According to the report, the LD50 of TiO2 for rats is larger than 12,000 mg/kg BW after oral administration (Renping et al., 2010). In this study, rats were exposed to 100 mg/kg BW TiO2 NPs every day. That is equal to about 6–7 g TiO2 -NPs per 60–70 kg body weight for humans, which is a relatively safe dose. Although this dose was less than the dose reported by the WHO, it still had side effects on the hippocampus. Therefore, long-term applications of products containing TiO2 -NPs on human fetuses should be carried out cautiously. In conclusion, the results of the present study showed that exposure to TiO2 -NPs during pregnancy reduced hippocampal cell proliferation in rat offspring. Furthermore, it was observed that spatial and inhibitory memory and learning decreased in offspring after the maternal administration of TiO2 -NPs. Therefore, the data draw attention to the effects of TiO2 -NP exposure on brain development as an area of concern needing further study.
Conflict of interest The authors declare that there are no conflicts of interest.
Transparency document The Transparency document associated with this article can be found in the online version.
Acknowledgement The results described in this paper were extracted from a Ph.D. student thesis and research protocol (900843) which was supported financially by the Vice Chancellor for Research, Mashhad University of Medical Sciences, Mashhad, Iran.
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Maternal exposure to titanium dioxide nanoparticles during pregnancy; impaired memory and decreased hippocampal cell proliferation in rat offspring Abbas Mohammadipour a , Alireza Fazel a , Hossein Haghir a,b , Fatemeh Motejaded a , Houshang Rafatpanah c , Hoda Zabihi d , Mahmoud Hosseini e , Alireza Ebrahimzadeh Bideskan a,∗ a
Department of Anatomy and Cell Biology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran b Medical Genetic Research Center (MGRC), School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran c Immunology Research Center, Buali Institute, Mashhad University of Medical Sciences, Mashhad, Iran d Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad, Iran e Neurocognitive Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
a r t i c l e
i n f o
a b s t r a c t
Article history:
Titanium dioxide nanoparticles (TiO2 -NPs) are massively produced in the environment, and
Received 19 July 2013
because of their wide usage, they are a potential risk of damage to human health. TiO2 -NPs
Received in revised form
are often used as additives for paints, papers, and foods. The central nervous system (CNS),
16 January 2014
including hippocampal regions, is potentially susceptible targets for TiO2 -NPs. This study
Accepted 20 January 2014
aimed to determine the effects of exposure to TiO2 -NPs during pregnancy on hippocampal
Available online 30 January 2014
cell proliferation and the learning and memory of offspring. Pregnant Wistar rats received intragastric TiO2 -NPs (100 mg/kg body weight) daily from gestational day (GD) 2 to (GD) 21.
Keywords:
Animals in the control group received the same volume of distilled water via gavage. After
Titanium dioxide nanoparticles
delivery, the one-day-old neonates were deeply anesthetized and weighed. They were then
Maternal exposure
killed and the brains of each group were collected. Sections of the brains from the rat off-
Learning and memory
spring were stained using Ki-67 immunolabeling and the immunohistochemistry technique.
Hippocampal cell proliferation
Some of the male offspring (n = 12 for each group) were weaned at postnatal day (PND21), and
Neurotoxicity
housed until adulthood (PND60). Then the learning and memory in animals of each group were evaluated using passive avoidance and Morris water maze tests. The immunolabeling of Ki-67 protein as a proliferating cell marker showed that TiO2 -NPs significantly reduced cell proliferation in the hippocampus of the offspring (P < 0.05). Moreover, both the Morris water maze test and the passive avoidance test showed that exposure to TiO2 -NPs significantly impaired learning and memory in offspring (P < 0.05). These results may provide basic experimental evidence for a better understanding of the neurotoxic effects of TiO2 -NPs on neonatal and adult brains. © 2014 Elsevier B.V. All rights reserved.
∗ Corresponding author at: Department of Anatomy and Cell Biology, School of Medicine, Mashhad University of Medical Sciences, Azadi Sq., Vakilabad Blvd., P.O. Box 91779-48564 Mashhad, Iran. Tel.: +98 511 8002486; fax: +98 511 8002487. E-mail address: [email protected] (A.E. Bideskan). 1382-6689/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2014.01.014
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1.
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Introduction
Titanium dioxide nanoparticles (TiO2 -NPs) are often used as additives in papers, paints, ceramics, plastics, and foods (Lomer et al., 2004; Oberdorster et al., 2005). The properties of absorption and reflection with ultra violet (UV) light also prompt the wide use of TiO2 -NPs in a variety of cosmetics. Moreover, they are used in the environmental decontamination of air, soil, and water (Wang et al., 2011). As ultrafine-sized materials, TiO2 -NPs can enter the human body through various routes such as inhalation, ingestion, and skin (Oberdorster et al., 2005; Jin et al., 2008). In recent years, studies have shown that after entering the body, TiO2 -NPs accumulate in the liver, kidneys, spleen, lungs, heart, and brain (Wang et al., 2007; Liu et al., 2009; Ma et al., 2010). Many studies have also shown that exposure to TiO2 -NPs may damage the central nervous system (CNS) (Wang et al., 2007; Hu et al., 2011). Still other studies have found that TiO2 -NPs promote the production of reactive oxygen species (ROS) (Long et al., 2006, 2007). Wang et al. found that TiO2 -NPs that migrated into the hippocampus led to oxidative stress and inflammation (Wang et al., 2008). The hippocampus is highly vulnerable to oxidative stress due to its high metabolic rate (Cui et al., 2004). The hippocampus is one of the important regions of the brain that has been implicated in learning and memory (Kim et al., 2009). Recent studies also confirmed that TiO2 -NPs can be transferred from mother to offspring via breastmilk and by passing through the placenta (Gao et al., 2011; Shimizu et al., 2009). Shimizu et al. also reported that maternal exposure to nano-particulate titanium dioxide during the prenatal period affects the expression of genes related to brain development (Shimizu et al., 2009). Moreover, Gao et al. reported that 100 mg/kg TiO2 exposure during development decreased hippocampal synaptogenesis in offspring (Gao et al., 2011). Thus, it seems that exposure to TiO2 -NPs during pregnancy may possibly affect the offspring hippocampus. For the study of hippocampal proliferation, the Ki-67 protein (as a proliferating cell marker) immunolabeling method is well-established (Seolhwa et al., 2011; Kim et al., 2009; Scholzen and Gerdes, 2000). This protein is expressed during mitosis. The formation of new neurons in the hippocampus occurs mainly during the gestational period (Scholzen and Gerdes, 2000). In the present study, the effect of maternal exposure to TiO2 -NPs during pregnancy on offspring hippocampal proliferation was investigated by Ki-67 immunolabeling. The Morris water maze and passive avoidance tests were used to study memory changes in pups.
2.
Materials and methods
2.1.
Chemicals and preparation
The TiO2 nanoparticles used in this study were a kind of nanopowder, anatase, with a particle size of 10 nm, surface area >150 m2 /g, purity +99%, and density 3.9 g/m3 , and were purchased from Nano Lima, Co. (Iran).
TiO2 -NPs were suspended in distilled water. Quantitative suspensions (100 mg/kg) were prepared fresh every day and fed to the rats with gavage.
2.2. Nanoparticle characterization by transmission electron microscopy The sizes of TiO2 -NPs were determined using transmission electron microscopy (TEM), which showed that particles diameters were in the nano-size range. An image of the sample is shown in Fig. 1.
2.3.
Animals and treatment
Male and female Wistar rats aged 3–4 months and weighing 250–300 g were purchased from the Animal Center of Mashhad University of Medical Sciences. The rats were housed under controlled conditions of temperature (20 ± 2 ◦ C) and lighting (12 h light:12 h dark photoperiod) and were permitted free access to food and water. The female rats were caged with male rats at a ratio of 3:1. The onset of pregnancy was confirmed by the presence of sperm in vaginal smears, and the pregnant dams were immediately housed in individual cages with free access to water and food until delivery. Experimental procedures were carried out in accordance with Mashhad University of Medical Sciences, Ethical Committee Acts. The pregnant rats were randomly divided into two groups of control and TiO2 . Animals in the TiO2 group (n = 6) received 100 mg/kg TiO2 orally (gavage) from prenatal day 2 to day 21. The animals in control group (n = 6) were administered distilled water (Gao et al., 2011). After delivery, two male pups from each litter were randomly sampled, weighed, and assigned to an experimental group. One pup was used for the determination of hippocampal titanium content, and another was used for an histological examination. Some of the male offspring (n = 12 for each group) were weaned at postnatal day (PND) 21, and housed until adulthood (PND 60). Then the learning and memory in animals of each group were evaluated using passive avoidance and Morris water maze tests.
2.4.
Hippocampus titanium contents determination
The whole hippocampus of each day-1 neonate was weighed, digested, and analyzed for titanium content. Briefly, prior to elemental analysis, the tissues of interest were digested in nitric acid overnight. After adding 0.5 ml H2 O2 , the mixed solutions were heated at about 160 ◦ C using a high pressure reaction container in an oven chamber until the samples were completely digested. Then, the solutions were heated to 120 ◦ C to remove the remaining nitric acid until the solutions were colorless and clear. Next, 3 ml of 2% nitric acid was added to the remaining solutions. Inductively coupled plasma-mass spectrometry (ICP-MS, HP4500, USA and Japan) was used to analyze the titanium concentration in the samples. Detected data were expressed as nanograms per gram of hippocampal tissue (Gao et al., 2011; Zhang et al., 2010).
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2.5.
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Coefficients of brain
After weighing the body and brains, the coefficient of brain to body weight was calculated as the ratio of tissues (wet weight, mg) to body weight (g).
2.6.
Behavioral tests
2.6.1.
Morris water maze
A circular black pool (136 cm diameter, 60 cm high, and 30 cm deep) was filled with water (23–25 ◦ C). A circular platform (10 cm diameter, 28 cm high) was placed within the pool and was submerged approximately 2 cm below the water surface in the center of the northeast quadrant. Outside the maze, fixed visual cues were present at various locations around the room (i.e., computer, hardware, and posters). The animals performed four trials on each of five consecutive days. Each trial began with the rat being placed in the pool and released facing the side wall at one of four positions (the boundaries of the four quadrants, labeled North (N), East (E), South (S), and West (W)). The released positions were randomly predetermined. For each trial, the rat was allowed to swim until it found and remained on the platform for 20 s. If 60 s passed and the animal had not found the platform, it was guided to the platform by the experimenter and allowed to stay there for 20 s. The rat was then removed from the pool, dried, and placed in its holding bin for 20 s. The latency to reach the platform and the length of the swimming path were recorded by a video tracking system. On the sixth day, the platform was removed, and the animals were allowed to swim for 60 s. The time spent in the target quadrants was compared between groups (Hosseini et al., 2011; Azizi-Malekabadi et al., 2012; Saffarzadeh et al., 2010).
2.6.2.
Passive avoidance test
The passive avoidance apparatus consisted of two light and dark (black) compartments of the same size (20 × 20 × 30 cm3 ) separated by a door. The floor of the dark compartment was made of stainless-steel bars (0.5 cm diameter) separated by a distance of 1 cm. All training and testing were carried out between 08:30 AM and 11:00 AM. Each animal was placed in the light
Fig. 1 – TEM image of the TiO2 particles.
Fig. 2 – The content of titanium in hippocampus of rat offspring after intragasteric treatment with TiO2 -NPs during pregnancy period; titanium in hippocampus of the control group was not detected. Bar marked with asterisks is statistically significant different from the control (P < 0.001). Values are represented as mean ± SEM.
compartment for 20 s, after which the door was raised and the time the animal waited before crossing to the dark (shock) compartment was recorded as the latency. The total time spent in light and dark compartments were also recorded. The animal was removed from the experiment when it waited for more than 180 s to cross to the other side. Once the animal completely crossed to the next compartment, the door was closed and an intermittent electric shock (50 Hz, 3 s, 1 mA intensity) was delivered to the floor of the dark compartment by an isolated stimulator. At 1, 24, and 48 h after training, retention tests were performed to examine memory. Each animal was placed in the light compartment for 20 s, the door was opened, and latency for entering the shock compartment as well as amount of time spent in the light and dark compartments were measured. During these sessions, no electric shock was applied (Pourmotabbed et al., 2011; Naghibi et al., 2012).
2.7.
Histopathological examination
2.7.1.
Tissue sampling
After delivery, one-day-old neonates were deeply anesthetized and weighed. Once the rats were killed, the brains were
Fig. 3 – The brain coefficients of rat offspring exposed to TiO2 -NPs during development. Bars marked with double asterisks are significantly different from the control (unexposed rats) at the 1% confidence level. Values are represented as mean ± SEM.
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Fig. 4 – Photomicrographs of the hippocampi of rat offspring. Maternal exposure to TiO2 -NPs during pregnancy reduced hippocampal cell proliferation in rat offspring. (A) CA1 control group, (B) CA1 TiO2 group, (C) CA3 control group, (D) CA3 TiO2 group, (E) DG control group, (F) DG TiO2 group, arrowheads point to representative Ki-67 positive cells. Cells were counterstained with Hematoxylin. Scale bars, 10 m.
collected from each group and fixed in 10% neutral buffered formalin for 3 days. The brain samples were processed for embedding in paraffin using routine histological protocols.
2.7.2.
Immunohistochemistry
For immunostaining, sections were deparaffinized with xylene, rehydrated through descending concentrations of ethanol, rinsed in retrieval antigen solution (citrate buffer 0.01 M, pH 6.0; S2031 Dako, England), and heated in a microwave for 15 min. The sections were then immersed in a methanol/H2 O2 solution (1:100) for 20 min in the dark to block
endogenous peroxidase. Then they were rinsed in 0.05 M PBS plus 0.025% Trition X-100 (5 min, 3 times) at room temperature. To decrease background staining, all sections were treated with 10% normal goat serum (ab 7481, abcam, USA) in PBS (goat as the host of the secondary antibody) for 1 h. The sections were then covered with proliferating cell marker, anti Ki-67 (ab16667, Abcam, USA), and with (diluted 10 in 1000 with 1% BSA) as the primary antibody, were kept in a humidified chamber overnight at 4 ◦ C. After incubation, the slides were washed extensively with PBS containing 0.025% Trition X-100. The sections were then applied with HRP-conjugated secondary
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antibody (goat anti-rabbit IgG, ab6721, Abcam, USA), diluted to 10:700 concentrations in PBS with 1% BSA for 60 min at room temperature. All sections were washed extensively with PBS for 3 min and treated with DAB (DAB kit, Sigma Aldrich, USA) solution (0.03 g DAB in 100 ml PBS and 200 l H2 O2 /100 ml PBS) for 15 min at room temperature in the dark. After being washed under running water, all sections were counterstained with a solution of Harris Hematoxylin for 2 min. Finally, the sections were dehydrated in increasing graded ethanol, cleared in xylene, and mounted on glass slides.
2.8.
Quantification of Ki-67+ cells
The areas of the hippocampi were measured using Image software (Olympus BX51, Japan). The number of Ki-67 immunoreactive cells was counted under 40× objective and by using rectangular grids placed randomly on the investigated areas. Morphometrical methods were used to count Ki-67+ cells per unit area in the CA1, CA2, CA3, and dentate gyrus of the hippocampus. The mean number of Ki-67+ cells per unit area (NA) in different regions of the hippocampus was calculated using the following formula (Sadeghi et al., 2013; Rajabzadeh et al., 2011): NA =
Q
a/f ·
P
In this formula “ Q” is the sum of counted particles in the sections, “a/f” is the area associated with each frame, and “ P” is the sum of frame-associated points hitting the defined space.
Statistics
Statistical analysis was performed using the SPSS 16 software for Windows. The data were analyzed statistically using repeated measures ANOVA for behavioral data and independent t test for histological data. P-values of <0.05 were considered to be statistically significant.
3.
Results
3.1.
Titanium content
The content of hippocampal titanium is shown in Fig. 2. The results indicated that the titanium accumulated in the hippocampus of the TiO2 -NPs group (P < 0.001) while in the unexposed rats, titanium was not detected.
3.2.
The increased coefficients indicate that injury was induced in the offspring after exposure to TiO2 -NPs (Fig. 3).
3.3.
2.9.
Fig. 5 – The influence of prenatal exposure to TiO2 on cell proliferation in the hippocampus in one-day-old pups, Bars marked with an asterisk or double asterisks are significantly different from the control at the 5% or 1% confidence level, respectively. The data are presented as the mean ± SEM.
Coefficient of brain
After delivery, one-day neonates were anesthetized and the body and brain weights were measured. Then the body weight coefficients of the brain which were expressed in mg (wet weight of tissue)/g (body weight) were analyzed. In the group treated with TiO2 -NPs, the coefficients of the brain were significantly higher (P < 0.01) than those of the control. Moreover, in the control group, the average body weight was 7.64 ± 0.17 g while it was 5.97 ± 0.18 g in the TiO2 treated group (P < 0.001).
Effects of TiO2-NPs on Ki-67+ cells
To elucidate the effect of TiO2 -NPs on hippocampal cell proliferation, the Ki-67 (a proliferating cell marker) was examined in the hippocampi of the rat offspring. Ki-67-positive cells were observed in the hippocampi of all offspring (Fig. 4). After the administration of TiO2 -NPs, the number of Ki-67-positive cells in the CA areas and DG of the hippocampus declined sharply (Figs. 4 and 5).
3.4.
Evaluation of spatial recognition memory
The length of the swimming path during 5 days and latency were significantly higher in the progeny of the TiO2 group compared to the control group (P < 0.01) (Fig. 6A and B). In the probe trial, time spent in the target quadrant (Q1) was also significantly lower in the progeny of the TiO2 group compared to that of the control group (P < 0.05) (Fig. 6C). Trial-by-trial analysis of day-1 data of the time showed that there was no difference between the two groups in the first trial, but other trials in the control group had significantly lower results than the TiO2 group (Fig. 6D). There was no significant difference in speed between the two groups (Fig. 6E).
3.5.
Evaluation of passive avoidance test
As shown in Fig. 7A, before receiving the shock, there was no significant difference between groups in latency to enter the dark compartment. One, 24, and 48 h after receiving the shock, latency to enter the dark compartment was lower in the TiO2 group than in the control group (P < 0.01). Total time spent in the dark compartment by the animals in the TiO2 group was significantly more than that of the control group at all three times after receiving the shock (Fig. 7B, P < 0.01); however, there was no significant difference before the shock.
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Fig. 6 – The effect of exposure to TiO2 -NPs during pregnancy on spatial recognition memory in offspring. (A and B) The length of the swimming path during 5 days and latency were significantly higher in the progeny of the TiO2 group compared with the control group (P < 0.01). (C) The time spent in the target quadrant (Q1) was significantly lower in the progeny of the TiO2 group than that of the control group (P < 0.05). (D) The trial-by-trial analysis of day-1 data of the time. The data showed that there was no difference between the two groups in the first trial, but other trials in the control group were significantly lower than the TiO2 group. (E) shows that there was no significant difference in speed between the two groups.
4.
Discussion
The aim of the present study was to assess the effects of exposure to TiO2-NPs > during pregnancy on hippocampal cell proliferation, learning, and memory performance of rat offspring. Our data indicate that exposure of pregnant mothers to TiO2 -NPs can impair hippocampal cell proliferation in newborn offspring. Moreover, we found that these nanoparticles impair the memory and learning of pups in adulthood. Today, TiO2 -NPs are widely used in daily life. Several studies reported that a daily intake of TiO2-NPs > may lead to its accumulation in various organs (Wang et al., 2007; Liu et al., 2009; Zhang et al., 2010). Nanoparticles can enter the body through
inhalation, dermal contact, and ingestion. The oral route may be important in many consumer products such as toothpaste, food additives, kitchen utensils, and water decontamination (Lomer et al., 2004; Wang et al., 2011). Because of this, oral administration was used in the present study. It has been shown that during the developmental period, the development of individuals is influenced by environmental factors (Hayashi et al., 1998; Iqbal et al., 2004). Recent studies have confirmed that TiO2-NPs > can be transferred from mother to offspring (Gao et al., 2011; Shimizu et al., 2009). Previous studies also proved that TiO2-NPs > can cross through the blood–brain barrier, enter the brain, and partially deposit in the hippocampus (Wang et al., 2008; Zhang et al., 2010). The current study showed that nanotitanium can transfer from
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Fig. 7 – The effect of prenatal exposure to TiO2 -NPs on passive avoidance learning, after receiving the shock, latency to enter the dark compartment was lower in the TiO2 group than in the control group (A). Moreover, time spent in the dark compartment was longer in the TiO2 group than in the control group (B) (P < 0.01). Data are expressed as ±SEM.
mother to the offspring’s hippocampus, confirming previous studies (Gao et al., 2011; Shimizu et al., 2009). The present study also confirmed that maternal exposure to titanium leads to titanium accumulation in the offspring’s hippocampus. Hu et al. reported that exposing animals to TiO2 -NPs leads to titanium accumulation in the brain (Hu et al., 2011). Gao et al. reported that maternal exposure to TiO2 -NPs during pregnancy leads to titanium accumulation in offspring hippocampus (Gao et al., 2011). After exposure to TiO2 -NPs, K+ content and Na/K-ATPase activity significantly decreased, and Na+ content increased in brain. Therefore, Na/K-ATPase cannot export intracellular Na+ and/or import extracellular K+ normally, which would result in the accumulation of intracellular Na+ , loss of intracellular K+ , and consequently, a reduction in the Na+ and K+ electrochemical gradient. This may disturb ionic homeostasis and the physiological functions of neurons (Renping et al., 2010). Moreover, exposure to TiO2 -NPs decreased the zinc content in rat brains. Zinc plays an important role in neurotransmission by modulating the activity of glutamate and gamma aminobutyric acid receptors (Renping et al., 2010). It is also believed to play an important role in cognition and memory via its function as a neuronal messenger, modulator of synaptic transmission, and cortical plasticity (Nakashima and Dyck, 2009). It is well-known that a reduction in brain zinc content can impair spatial memory in adult rats (Tahmasebi Boroujeni et al., 2009). These nanoparticles can also stimulate ROS generation in the brain (Long et al., 2007). In addition, a recent study reported that after animals were exposed to TiO2 -NPs (100 mg/kg), the activities of anti-oxidative enzymes were reduced in their brains (Ma et al., 2010). The hippocampus is a main target of TiO2 -NPs, and hippocampal cells are highly sensitive to ROS following exposure to nanoparticles (Yang et al., 2010). Oxidative stress can cause damage to cellular components such as membranes, DNA, and proteins (Beal, 1996). Moreover, the formation of ROS affects cell proliferation or the survival of new neurons. As oxidative stress is implicated in numerous neuropsychiatric disorders, the pathologies of such disorders may potentially be mediated through the impairment of proliferation by the excessive accumulation of ROS in the adult brain (Taupin, 2010). So, oxidative stress may undermine cell proliferation in the offspring brain. It has also been
reported that TiO2 -NPs may have developmental neurotoxicity on the physiological functions of the hippocampus. Studies have also indicated that exposure to TiO2 -NPs (100-NPs (100 mg/kg) from prenatal day 2 to day 21 may disrupt synaptogenesis in the developing hippocampus (Gao et al., 2011). However, no study has been published on the effects of embryonic exposure to TiO2 -NPs on cell proliferation and memory. This study found that cell proliferation, memory, and learning are affected by developmental exposure to these nanoparticles. It has been well-documented that neurogenesis occurs during developmental periods, including prenatal and immediately after birth, which affects behavior during adulthood (Gil-Mohapel et al., 2010). According to the results of the present study, it seems that the memory impairment seen in adult rats was, at least in part, due to neurogenesis impairment during the developmental period. In the present study, the length of swimming path during 5 days and latency were significantly higher in the TiO2 group than in the control group. Trial-by-trial analysis of day-1 data of the time showed that there was no difference between the two groups in the first trial, but results of other trials in the control group were significantly lower than the TiO2 group. This confirms that there were no pre-existing effects and animals had no mobility problems, but learning tasks decreased in the TiO2 group. In this study, the effects of TiO2 -NPs on a limited number of animals were investigated. Only one dose of TiO2 -NPs was given to the rats in the present study; however, further, broader studies need to be done in this regard. It is recommended that other methods such as Western Blot and PCR be used in further studies. In the past, 5-bromo-2-deoxyuridin (BrdU) was used as the principal mitotic marker to determine the rate of cell proliferation (Kempermann and Gage, 2002; Schauwecker, 2006). However, BrdU can be incorporated into dividing neurons (Gould et al., 1998), and numerous other side effects have been reported, such as stress during injection and mutagenesis following incorporation (Kee et al., 2002). Ki-67, an endogenous marker for proliferating cells, is a reliable marker, because it is expressed during mitosis and has a short half-life (Scholzen and Gerdes, 2000). In addition, Ki-67 can be easily detected by immunohistochemistry. Therefore, in this study, the effect of
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maternal exposure to TiO2 -NPs on hippocampal proliferation was investigated by Ki-67 immunoreactivity. The 1969 report of the World Health Organization (WHO) was consulted about dose selection in the current study. According to the report, the LD50 of TiO2 for rats is larger than 12,000 mg/kg BW after oral administration (Renping et al., 2010). In this study, rats were exposed to 100 mg/kg BW TiO2 NPs every day. That is equal to about 6–7 g TiO2 -NPs per 60–70 kg body weight for humans, which is a relatively safe dose. Although this dose was less than the dose reported by the WHO, it still had side effects on the hippocampus. Therefore, long-term applications of products containing TiO2 -NPs on human fetuses should be carried out cautiously. In conclusion, the results of the present study showed that exposure to TiO2 -NPs during pregnancy reduced hippocampal cell proliferation in rat offspring. Furthermore, it was observed that spatial and inhibitory memory and learning decreased in offspring after the maternal administration of TiO2 -NPs. Therefore, the data draw attention to the effects of TiO2 -NP exposure on brain development as an area of concern needing further study.
Conflict of interest The authors declare that there are no conflicts of interest.
Transparency document The Transparency document associated with this article can be found in the online version.
Acknowledgement The results described in this paper were extracted from a Ph.D. student thesis and research protocol (900843) which was supported financially by the Vice Chancellor for Research, Mashhad University of Medical Sciences, Mashhad, Iran.
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