Article
Effects of prenatal exposure to single-wall carbon nanotubes on reproductive performance and neurodevelopment in mice
Toxicology and Industrial Health 1–9 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233714555388 tih.sagepub.com
Saeed Ivani1, Isaac Karimi2, Seyed Reza Fatemi Tabatabaei1 and Leila Syedmoradi3 Abstract Carbon nanotubes with extraordinary properties may become a novel drug and gene delivery tool in nanomedicine; however, insufficient information is available regarding their biosafety. Therefore, this work was performed to study the effect of prenatal exposure of single-walled carbon nanotubes (SWCNTs) on reproductive and neurobehavioral endpoints in mice. Thirty pregnant female mice were assigned to three groups (n ¼ 10 for each group). The two treated groups were injected intraperitoneally (i.p.) with 1 or 10 mg/kg body weight (b.w.) of SWCNTs suspended in 1 ml of phosphate buffer saline (PBS) on gestational days 0 and 3. The control group was injected i.p. with an equal volume of PBS. The neurobehavioral ontogeny of pups was evaluated using a modified Fox battery. A decrease in litter size on postnatal day 2 was observed in the group treated with 10 mg/kg b.w. of SWCNTs whereas no significant differences between groups were observed in any other parameters. The behavioral development of pups did not show significant differences during growth except for the surface righting reflex, which showed significant delay compared to control in the group treated with 1 mg/kg b.w. SWCNTs. Moreover, exposed offspring (10 mg/kg b.w. SWCNTs) displayed enhanced anxiety in the elevated plus maze; however, other ethological analysis (Morris water maze and open field test) did not show behavioral changes in the experimental groups. In conclusion, the present results demonstrated small changes in offspring sensory and motor development following exposure to SWCNTs and support the idea that SWCNT risk assessment merits further investigation. Keywords Carbon nanotubes, prenatal exposure, reproductive endpoints, neurobehavioral development, fox battery, mice
Introduction Single-wall carbon nanotubes (SWCNTs) or nanometer-scale seamless cylinders of a single graphene sheet are new forms of crystalline carbon with extraordinary chemical and electromechanical properties that have attracted intense research efforts (Anantram and Leonard, 2006; Iijima, 2002; Koziol et al., 2007; Ma et al., 2009). They usually have diameters ranging from 0.4 to 3.0 nm and their lengths are typically greater than 1 m. Expanding production and widespread applications of these engineered nanoparticles (ENPs) may lead to significant exposure of humans, animals, and environment to these substances (Lam et al., 2006; Mahmood et al., 2012).
1
Division of Physiology, Department of Basic Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran 2 Laboratory of Molecular and Cellular Biology 1214, Department of Basic Veterinary Sciences, School of Veterinary Medicine, Razi University, Kermanshah, Iran 3 Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran Corresponding author: Leila Syedmoradi, Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran 1417755469, Iran. Email: [email protected]
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Figure 1. TEM image of SWCNTs (a). SEM image of SWCNTs (b). TEM: transmission electron microscopy; SWCNT: single-walled carbon nanotube; SEM: scanning electron microscopy.
Recently, special attention is being paid to the utilization of carbon nanotubes (CNTs) for the development of efficient drug and gene delivery tools (McDevitt et al., 2007). This breakthrough seems to be possible because CNTs have great potential to host therapeutic genes or peptides used in molecular therapy of diseases (Cui and Gao, 2003; Cui et al., 2004; Gao et al., 2003; Mahmood et al., 2012). Although CNTs are good candidates as drug delivery devices, their developmental and neurobehavioral toxicities are poorly understood in the in vivo condition. More systematic studies are needed on the basic safety issues surrounding the usage of SWCNTs. Much experience shows that in utero exposure to toxicants can have greater consequences than similar exposures at other life stages (Jackson et al., 2012; Tripathi et al., 2008). In this regard, Tripathi et al. (2008) using alkaline comet assay demonstrated multiorgan genotoxicity of known transplacental genotoxins like cyclophosphamide, mitomycin-C, and zidovudine in newborn mice born to received these compounds in the late gestational period (16–20th days of pregnancy). More specifically, Jackson et al. (2012) with the aid of an integrated battery of biochemical assay concluded that inflammation in maternal lung after pulmonary exposure to carbon black Printex 90 led to some kinds of metabolic disorders in the offspring. Currently, one of the great concerns about nanotechnology products is that ENPs may produce latent functional and overt toxic effects in the human nervous system owing to their ability to pass through biological membranes (Yang et al., 2010). In this context, a
significant increase in lipid peroxidation was reported in the brain of juvenile largemouth bass following exposure to fullerenes (Oberdorster, 2004). In another study, SWCNTs significantly decreased the overall DNA content in mixed neuroglial cultures (Belyanskaya et al., 2009). To our knowledge, no systematic research exists addressing the question of whether SWCNTs alter the neurobehavioral endpoints of in utero exposed individuals. Therefore, this work was performed to study the effects of SWCNT exposure during gestation on reproductive and behavioral endpoints in the mouse.
Materials and methods Particle characteristics SWCNTs (1–2 nm diameter, approximately 10 m length, and purity >98%) were purchased from Research Institute of Petroleum Industry, Tehran, Iran. The nanotubes were produced using chemical vapor deposition (Vajtai et al., 2004). Characterization of SWCNTs was done using both transmission and scanning electron microscopy (SEM; Figure 1). The heat-treated particles were suspended in sterile PBS, and manually vortexed just before administration as described previously (Muller et al., 2009).
Animals This study was reviewed and approved by the Committee of the School of Veterinary Medicine, Razi University, Kermanshah, Iran. Male, aged 5–7 weeks,
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(n ¼ 12) and virgin NMRI female mice (n ¼ 30), were procured from the Central Laboratory Animal Facility of the School of Veterinary Medicine, Razi University, Kermanshah, Iran. The animals were housed in standard plastic cages (five mice/cage), and maintained on a standard 12-h light/12-h dark cycle, at temperature of 22 + 2 C and humidity of 55 + 5% with free access to tap water and food (Dan-e-pars Co., Kermanshah, Iran). After 1 week of acclimatization, five female mice were housed with two males overnight and were examined the next morning for vaginal plug (this morning was considered gestational day (GD) 0). Pregnant females were divided to three groups (n ¼ 10/group): control group which was injected intraperitoneally (i.p.) with 1 ml PBS pH 7.4 and two SWCNT-treated groups that were i.p. exposed to 1 and 10 mg/kg of SWCNTs on GD 0 and 3. The doses of SWCNTs used in this study were adopted from previous study (Muller et al., 2009).
Reproductive performance of dams Body weights of the pregnant females were recorded on GD 1, 4, 7, 14, and delivery day (GD 21 or 22). Reproductive indices were evaluated using a modified version of the methods designed by Ratnasooriya et al. (2003) as described by Yousofi et al. (2011): gestation index ¼ (number of pregnant females with alive pups on birthday/number of total females); sex ratio ¼ (number of male pups/number of female pups); pup survival rate ¼ (number of pups alive on postnatal day (PND) 4/ number of pups alive on PND 1); lactation rate ¼ (weight of pups alive on day 21/weight of pups on PND 4) and milk production was determined by the method of Sampson and Jansen (1984) by the following equation: milk production ¼ [0.0322 þ 0.0667 (pup weight, g) þ 0.877 (pup weight gain/ day)]. Here, weight gain on PND 2 and PND 3 were considered for this equation (Ratnasooriya et al., 2003; Yousofi et al., 2011).
Developmental milestones and reflex development Body weights of the pups were taken on delivery day and PND 1, 4, 7, 14, and 21 (weaning). Two pups of the opposite sex from each litter were observed daily for the expression of developmental indications like pinna unfolding, lower incisor eruption, eye opening, fur development, and body weight changes. The absence and presence of different developmental
indications were assessed by two investigators blinded to the exposure status of the offspring from PND 1 to PND 15. Measurements of reflex ontogeny were assessed according to the Fox battery of tests (Fox, 1965) as described by Yousofi et al. (2011) as following: (a) surface righting reflex test—a pup was gently placed in the supine position and was timed until it had righted itself and all four limbs were in contact with the surface; (b) cliff aversion reflex— pup withdraws from the edge of a flat surface when its snout and forepaws are placed over the cliff within 30 s; (c) palm grasping reflex—pup grips a wooden pencil with its forepaws, and this test was positive, if pups stayed suspended for 30 s; (d) vibrissae placing reflex—pup places its forepaw on a cotton swab stroked across its vibrissae; (e) level or vertical stick reflex—pup grasps onto a wire mesh when gently pulled across the mesh by the tail; if it displayed resistance this test was considered positive; (f) auditory startle reflex—pup shows a whole-body startle response when a loud clap of the hands occurs less than 15 cm away; (g) air righting reflex—pup turns in mid-air to land ventrally after being dropped from an inverted position, 35 cm above a cotton wool pad; and (h) homing test—wood shavings from the litter home cage were evenly spread over one side of a new plastic cage, and the pup was placed close to the wall on the opposite side. If the pup took less than 60 s to get to the goal area (home bedding), this test was considered positive.
Behavioral tests Open field test (OFT). Locomotors activity and anxiety were evaluated by OFT on PND 22. Mice were tested for 5 min in an open field apparatus which consists of a bright square with a diameter measuring 100 cm. The time of movement including walking or running (ambulation) and the time spent in the inner area of the arena (>5 cm away from any wall) were monitored as locomotor activity and anxiety indices, respectively (Kihara, 1991; Wolansky and Harrill, 2008). All parameters used to assess behaviors were recorded using a camera located at the right angle above the open field apparatus. Morris water maze (MWM). Spatial memory was tested in MWM on PND 22 to 27. The water maze consisted of a circular pool of water, 150 cm in diameter, 60 cm in height, which was filled with tap water to a depth of 45 cm from the brim. The pool was made opaque by
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the addition of non-toxic black paint. A stable platform was submerged 1 cm below the water surface so that mouse could easily climb onto to escape from water. Each session was comprised of four trials with an inter-trial interval of 30 s for a learning period of 5 days. Pup was placed at the designated starting position and the animal was allowed to swim until it either came upon the hidden platform or until 60 s had elapsed. Then at the 6th training day, platform positions were changed for assessment of new learning. During the probe trial the animal was released into the quadrant opposite to the one that had previously contained the platform and allowed to swim in the maze for 1 min. The latency for finding the platform and time spent in each quadrant were videotaped. Elevated plus maze (EPM). The anxiety was tested by EPM on PND 22. The wooden, plus-shaped apparatus was elevated to a height of 50 cm, and consisted of two 50 10 cm2 open arms and two 50 10 50 cm3 enclosed arms, each with an open roof. Then with the aid of a camera that was suspended above the maze, mouse’s behavior was recorded and films were evaluated for following parameters: (1) time spent in the open arms, (2) time spent in the closed arms, (3) number of entries into the open arms, and (4) number of entries into the closed arms during the 5 min test period. Open-arm activity was quantified as the amount of time that the pup spent in the open arms relative to the total amount of time spent in any arm (open/total 100), and the number of entries into the open arms was quantified relative to the total number of entries into any arm (open/total 100). The numbers of entries in both arms were also interpreted as an index of locomotors activity to ensure that there are no differences in overall activity levels in the experimental groups (Van Meer and Raber, 2005).
Statistical analysis Statistical analyses were performed using the statistical package for the social sciences (SPSS) program (SPSS Inc., Chicago, IL, USA; Version 16). Data are presented as mean + SEM. The normality of variables was tested using the Shapiro–Wilk test at the 5% level of significance. Non-normal data were analyzed using the non-parametric Kruskal–Wallis followed by a pair wise Mann–Whitney U test where the overall Kruskal–Wallis test revealed a significant difference between groups. For normally distributed data analysis of variance (ANOVA) was used to
Figure 2. Effect of exposure to SWCNT on dams’ body weights. Values are mean + SEM. SWCNT: single-walled carbon nanotube.
compare control and treated groups. Postnatal body weights were evaluated using analysis of covariance (covariant was the litter size). The escape latency in different trials of MWM was analyzed using a repeated-measure ANOVA. p values less than 0.05 were considered significant.
Results Reproductive performance of dams There were no significant changes in the body weight of dams exposed to 1, or 10 mg/kg SWCNTs compared to the control group (Figure 2). The reproductive parameters are presented in Table 1. Females exposed to 10 mg/kg SWCNTs delivered significantly less pups than control dams (12.2 + 0.67 vs. 14.8 + 0.34, p < 0.05). Other parameters such as gestational index, gestational period, lactation rate, pup survival rate, sex ratio and the milk production were not significantly different between the control and SWCNTs groups.
Neurobehavioral and developmental milestones As shown in Figure 3, no significant differences in the weight of litters were found between groups. Results of the developmental milestones and neurobehavior of pups are shown in Table 2. The surface righting reflex showed a significant delay in the 1 mg/kg
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Table 1. Reproductive parameters of dam mice exposed to SWCNTS. Parameters
Maternal doses of SWCNTs (mg/kg b.w.)
Number of time-mated females Gestational index Gestational period (day) Lactation rate Pup survival rate Sex ratio (males/females) Litter size (PND 2) Milk production (g/day)
0 (control) 10 0.8 21.42 + 0.42 3.87 + 0.14 0.99 + 0.01 1.61 + 0.32 14.8 + 0.34 0.39 + 0.009
1 10 0.8 21.87 + 1.93 4.01 + 0.21 0.98 + 0.01 1.72 + 0.27 13.1 + 1.04 0.37 + 0.014
10 10 0.8 22.00 + 0.39 3.94 + 0.11 0.99 + 0.02 1.25 + 0.17 12.2 + 0.67b 0.38 + 0.002
SWCNT: single-walled carbon nanotube; PND: postnatal day; b.w.: body weight. a Values are mean + SEM. b Significantly different from the control group (p < 0.05).
differences between the exposed and control offspring for the locomotor activity and time spent in the center of arena (Figures 6 and 7). Moreover, in MWM, spatial performances in three groups were comparable (Figure 8).
Discussion
Figure 3. Effect of prenatal exposure to SWCNT on the cumulative litter body weights. Values are mean + SEM. SWCNT: single-walled carbon nanotube.
SWCNT group compared to control group (3.7 + 0.31 vs. 1.3 + 0.18, p < 0.05) while no significant differences were observed in other parameters between groups. Analysis of EPM showed that exposed offspring (10 mg/kg SWCNT) spent significantly more time (216.9 + 11.5 vs. 169.0 + 11.1 s, p < 0.05) in the closed arms and less time (47.6 + 6.8 vs. 95.6 + 10.4 s, p < 0.05) in the open arms than control group (Figure 4). Prenatal exposure to SWCNTs did not alter the locomotor activity as measured by the number of entries into the open and closed arms in the EPM (Figure 5). In the OFT, there were no significant
To the best of our knowledge, this is the first report concerning the neurobehavioral and reproductive performance effects of maternal exposure to SWCNTs in mouse offspring. In this study, prenatally exposed offspring displayed changes in the surface righting reflex test whereas no changes were observed in the other battery of neurodevelopmental endpoints. Also we found that maternal exposure to SWCNTs at the high dose level decreased the number of pups; however, other reproductive parameters were not altered significantly. Consistent with our findings, previous studies showed that maternal exposure to CNTs did not adversely affect embryo-fetal development. For example, Lim and coworkers reported no toxicological effects of oral administration of multi-walled CNTs (MWCNTs; 40, 200, and 1000 mg/kg b.w./day) to pregnant rats (Lim et al., 2011b). Another study showed that acute and repeated oral doses of SWCNTs and MWCNTs did not cause harmful effects in rats (Matsumoto et al., 2012). Moreover, it has been reported that maternal pulmonary exposure to carbon nanoparticles (Printex 90) had no effect on gestation, lactation, and offspring development (Jackson et al., 2011). Following the behavioral testing, offspring prenatally exposed to 1 mg/kg b.w. SWCNTs showed significant delays in the surface righting reflex test
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Table 2. Behavioral tests in pups exposed prenatally to single wall carbon nanotubes.a Behavioral testes Growth and reflex Litters (n) Pinna unfolding Incisor eruption Eye opening Development of fur Air righting reflex Palm grasping reflex Surface righting reflex Cliff aversion test Level stick reflex Vibrissae placing reflex Auditory startle reflex Homing test
Maternal doses of SWCNTs (mg/kg bw) 0 (control)
1
10
7 4.7 + 0.18
8 5.8 + 0.76
10 4.4 + 0.33
9.0 + 0.81
9.7 + 1.16 10.1 + 0.5
15.4 + 0.29 6.2 + 0.28
14.5 + 1.37 15.2 + 0.24 7.5 + 1.10 6.7 + 0.26
11.3 + 0.7
12.2 + 0.7
13.5 + 0.1
5.1 + 0.4
6.5 + 0.2
5.6 + 0.2
1.3 + 0.18
3.7 + 0.31b
2.5 + 0.26
1.7 + 0.26
1.3 + 0.26
1.9 + 0.27
3.4 + 0.36
4.0 + 0.43
3.4 + 0.3
4.1 + 0.68
5.5 + 0.90
5.1 + 0.34
11.7 + 1.16
13.4 + 0.26 12.5 + 0.34
12.7 + 0.56
11.8 + 0.51 13.2 + 0.44
Figure 4. Effect of prenatal exposure to SWCNT on anxiety level in the elevated plus maze. Data are shown as mean + SEM (n ¼ 7). *p:
Effects of prenatal exposure to single-wall carbon nanotubes on reproductive performance and neurodevelopment in mice
Toxicology and Industrial Health 1–9 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233714555388 tih.sagepub.com
Saeed Ivani1, Isaac Karimi2, Seyed Reza Fatemi Tabatabaei1 and Leila Syedmoradi3 Abstract Carbon nanotubes with extraordinary properties may become a novel drug and gene delivery tool in nanomedicine; however, insufficient information is available regarding their biosafety. Therefore, this work was performed to study the effect of prenatal exposure of single-walled carbon nanotubes (SWCNTs) on reproductive and neurobehavioral endpoints in mice. Thirty pregnant female mice were assigned to three groups (n ¼ 10 for each group). The two treated groups were injected intraperitoneally (i.p.) with 1 or 10 mg/kg body weight (b.w.) of SWCNTs suspended in 1 ml of phosphate buffer saline (PBS) on gestational days 0 and 3. The control group was injected i.p. with an equal volume of PBS. The neurobehavioral ontogeny of pups was evaluated using a modified Fox battery. A decrease in litter size on postnatal day 2 was observed in the group treated with 10 mg/kg b.w. of SWCNTs whereas no significant differences between groups were observed in any other parameters. The behavioral development of pups did not show significant differences during growth except for the surface righting reflex, which showed significant delay compared to control in the group treated with 1 mg/kg b.w. SWCNTs. Moreover, exposed offspring (10 mg/kg b.w. SWCNTs) displayed enhanced anxiety in the elevated plus maze; however, other ethological analysis (Morris water maze and open field test) did not show behavioral changes in the experimental groups. In conclusion, the present results demonstrated small changes in offspring sensory and motor development following exposure to SWCNTs and support the idea that SWCNT risk assessment merits further investigation. Keywords Carbon nanotubes, prenatal exposure, reproductive endpoints, neurobehavioral development, fox battery, mice
Introduction Single-wall carbon nanotubes (SWCNTs) or nanometer-scale seamless cylinders of a single graphene sheet are new forms of crystalline carbon with extraordinary chemical and electromechanical properties that have attracted intense research efforts (Anantram and Leonard, 2006; Iijima, 2002; Koziol et al., 2007; Ma et al., 2009). They usually have diameters ranging from 0.4 to 3.0 nm and their lengths are typically greater than 1 m. Expanding production and widespread applications of these engineered nanoparticles (ENPs) may lead to significant exposure of humans, animals, and environment to these substances (Lam et al., 2006; Mahmood et al., 2012).
1
Division of Physiology, Department of Basic Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran 2 Laboratory of Molecular and Cellular Biology 1214, Department of Basic Veterinary Sciences, School of Veterinary Medicine, Razi University, Kermanshah, Iran 3 Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran Corresponding author: Leila Syedmoradi, Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran 1417755469, Iran. Email: [email protected]
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Figure 1. TEM image of SWCNTs (a). SEM image of SWCNTs (b). TEM: transmission electron microscopy; SWCNT: single-walled carbon nanotube; SEM: scanning electron microscopy.
Recently, special attention is being paid to the utilization of carbon nanotubes (CNTs) for the development of efficient drug and gene delivery tools (McDevitt et al., 2007). This breakthrough seems to be possible because CNTs have great potential to host therapeutic genes or peptides used in molecular therapy of diseases (Cui and Gao, 2003; Cui et al., 2004; Gao et al., 2003; Mahmood et al., 2012). Although CNTs are good candidates as drug delivery devices, their developmental and neurobehavioral toxicities are poorly understood in the in vivo condition. More systematic studies are needed on the basic safety issues surrounding the usage of SWCNTs. Much experience shows that in utero exposure to toxicants can have greater consequences than similar exposures at other life stages (Jackson et al., 2012; Tripathi et al., 2008). In this regard, Tripathi et al. (2008) using alkaline comet assay demonstrated multiorgan genotoxicity of known transplacental genotoxins like cyclophosphamide, mitomycin-C, and zidovudine in newborn mice born to received these compounds in the late gestational period (16–20th days of pregnancy). More specifically, Jackson et al. (2012) with the aid of an integrated battery of biochemical assay concluded that inflammation in maternal lung after pulmonary exposure to carbon black Printex 90 led to some kinds of metabolic disorders in the offspring. Currently, one of the great concerns about nanotechnology products is that ENPs may produce latent functional and overt toxic effects in the human nervous system owing to their ability to pass through biological membranes (Yang et al., 2010). In this context, a
significant increase in lipid peroxidation was reported in the brain of juvenile largemouth bass following exposure to fullerenes (Oberdorster, 2004). In another study, SWCNTs significantly decreased the overall DNA content in mixed neuroglial cultures (Belyanskaya et al., 2009). To our knowledge, no systematic research exists addressing the question of whether SWCNTs alter the neurobehavioral endpoints of in utero exposed individuals. Therefore, this work was performed to study the effects of SWCNT exposure during gestation on reproductive and behavioral endpoints in the mouse.
Materials and methods Particle characteristics SWCNTs (1–2 nm diameter, approximately 10 m length, and purity >98%) were purchased from Research Institute of Petroleum Industry, Tehran, Iran. The nanotubes were produced using chemical vapor deposition (Vajtai et al., 2004). Characterization of SWCNTs was done using both transmission and scanning electron microscopy (SEM; Figure 1). The heat-treated particles were suspended in sterile PBS, and manually vortexed just before administration as described previously (Muller et al., 2009).
Animals This study was reviewed and approved by the Committee of the School of Veterinary Medicine, Razi University, Kermanshah, Iran. Male, aged 5–7 weeks,
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Ivani et al.
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(n ¼ 12) and virgin NMRI female mice (n ¼ 30), were procured from the Central Laboratory Animal Facility of the School of Veterinary Medicine, Razi University, Kermanshah, Iran. The animals were housed in standard plastic cages (five mice/cage), and maintained on a standard 12-h light/12-h dark cycle, at temperature of 22 + 2 C and humidity of 55 + 5% with free access to tap water and food (Dan-e-pars Co., Kermanshah, Iran). After 1 week of acclimatization, five female mice were housed with two males overnight and were examined the next morning for vaginal plug (this morning was considered gestational day (GD) 0). Pregnant females were divided to three groups (n ¼ 10/group): control group which was injected intraperitoneally (i.p.) with 1 ml PBS pH 7.4 and two SWCNT-treated groups that were i.p. exposed to 1 and 10 mg/kg of SWCNTs on GD 0 and 3. The doses of SWCNTs used in this study were adopted from previous study (Muller et al., 2009).
Reproductive performance of dams Body weights of the pregnant females were recorded on GD 1, 4, 7, 14, and delivery day (GD 21 or 22). Reproductive indices were evaluated using a modified version of the methods designed by Ratnasooriya et al. (2003) as described by Yousofi et al. (2011): gestation index ¼ (number of pregnant females with alive pups on birthday/number of total females); sex ratio ¼ (number of male pups/number of female pups); pup survival rate ¼ (number of pups alive on postnatal day (PND) 4/ number of pups alive on PND 1); lactation rate ¼ (weight of pups alive on day 21/weight of pups on PND 4) and milk production was determined by the method of Sampson and Jansen (1984) by the following equation: milk production ¼ [0.0322 þ 0.0667 (pup weight, g) þ 0.877 (pup weight gain/ day)]. Here, weight gain on PND 2 and PND 3 were considered for this equation (Ratnasooriya et al., 2003; Yousofi et al., 2011).
Developmental milestones and reflex development Body weights of the pups were taken on delivery day and PND 1, 4, 7, 14, and 21 (weaning). Two pups of the opposite sex from each litter were observed daily for the expression of developmental indications like pinna unfolding, lower incisor eruption, eye opening, fur development, and body weight changes. The absence and presence of different developmental
indications were assessed by two investigators blinded to the exposure status of the offspring from PND 1 to PND 15. Measurements of reflex ontogeny were assessed according to the Fox battery of tests (Fox, 1965) as described by Yousofi et al. (2011) as following: (a) surface righting reflex test—a pup was gently placed in the supine position and was timed until it had righted itself and all four limbs were in contact with the surface; (b) cliff aversion reflex— pup withdraws from the edge of a flat surface when its snout and forepaws are placed over the cliff within 30 s; (c) palm grasping reflex—pup grips a wooden pencil with its forepaws, and this test was positive, if pups stayed suspended for 30 s; (d) vibrissae placing reflex—pup places its forepaw on a cotton swab stroked across its vibrissae; (e) level or vertical stick reflex—pup grasps onto a wire mesh when gently pulled across the mesh by the tail; if it displayed resistance this test was considered positive; (f) auditory startle reflex—pup shows a whole-body startle response when a loud clap of the hands occurs less than 15 cm away; (g) air righting reflex—pup turns in mid-air to land ventrally after being dropped from an inverted position, 35 cm above a cotton wool pad; and (h) homing test—wood shavings from the litter home cage were evenly spread over one side of a new plastic cage, and the pup was placed close to the wall on the opposite side. If the pup took less than 60 s to get to the goal area (home bedding), this test was considered positive.
Behavioral tests Open field test (OFT). Locomotors activity and anxiety were evaluated by OFT on PND 22. Mice were tested for 5 min in an open field apparatus which consists of a bright square with a diameter measuring 100 cm. The time of movement including walking or running (ambulation) and the time spent in the inner area of the arena (>5 cm away from any wall) were monitored as locomotor activity and anxiety indices, respectively (Kihara, 1991; Wolansky and Harrill, 2008). All parameters used to assess behaviors were recorded using a camera located at the right angle above the open field apparatus. Morris water maze (MWM). Spatial memory was tested in MWM on PND 22 to 27. The water maze consisted of a circular pool of water, 150 cm in diameter, 60 cm in height, which was filled with tap water to a depth of 45 cm from the brim. The pool was made opaque by
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the addition of non-toxic black paint. A stable platform was submerged 1 cm below the water surface so that mouse could easily climb onto to escape from water. Each session was comprised of four trials with an inter-trial interval of 30 s for a learning period of 5 days. Pup was placed at the designated starting position and the animal was allowed to swim until it either came upon the hidden platform or until 60 s had elapsed. Then at the 6th training day, platform positions were changed for assessment of new learning. During the probe trial the animal was released into the quadrant opposite to the one that had previously contained the platform and allowed to swim in the maze for 1 min. The latency for finding the platform and time spent in each quadrant were videotaped. Elevated plus maze (EPM). The anxiety was tested by EPM on PND 22. The wooden, plus-shaped apparatus was elevated to a height of 50 cm, and consisted of two 50 10 cm2 open arms and two 50 10 50 cm3 enclosed arms, each with an open roof. Then with the aid of a camera that was suspended above the maze, mouse’s behavior was recorded and films were evaluated for following parameters: (1) time spent in the open arms, (2) time spent in the closed arms, (3) number of entries into the open arms, and (4) number of entries into the closed arms during the 5 min test period. Open-arm activity was quantified as the amount of time that the pup spent in the open arms relative to the total amount of time spent in any arm (open/total 100), and the number of entries into the open arms was quantified relative to the total number of entries into any arm (open/total 100). The numbers of entries in both arms were also interpreted as an index of locomotors activity to ensure that there are no differences in overall activity levels in the experimental groups (Van Meer and Raber, 2005).
Statistical analysis Statistical analyses were performed using the statistical package for the social sciences (SPSS) program (SPSS Inc., Chicago, IL, USA; Version 16). Data are presented as mean + SEM. The normality of variables was tested using the Shapiro–Wilk test at the 5% level of significance. Non-normal data were analyzed using the non-parametric Kruskal–Wallis followed by a pair wise Mann–Whitney U test where the overall Kruskal–Wallis test revealed a significant difference between groups. For normally distributed data analysis of variance (ANOVA) was used to
Figure 2. Effect of exposure to SWCNT on dams’ body weights. Values are mean + SEM. SWCNT: single-walled carbon nanotube.
compare control and treated groups. Postnatal body weights were evaluated using analysis of covariance (covariant was the litter size). The escape latency in different trials of MWM was analyzed using a repeated-measure ANOVA. p values less than 0.05 were considered significant.
Results Reproductive performance of dams There were no significant changes in the body weight of dams exposed to 1, or 10 mg/kg SWCNTs compared to the control group (Figure 2). The reproductive parameters are presented in Table 1. Females exposed to 10 mg/kg SWCNTs delivered significantly less pups than control dams (12.2 + 0.67 vs. 14.8 + 0.34, p < 0.05). Other parameters such as gestational index, gestational period, lactation rate, pup survival rate, sex ratio and the milk production were not significantly different between the control and SWCNTs groups.
Neurobehavioral and developmental milestones As shown in Figure 3, no significant differences in the weight of litters were found between groups. Results of the developmental milestones and neurobehavior of pups are shown in Table 2. The surface righting reflex showed a significant delay in the 1 mg/kg
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Ivani et al.
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Table 1. Reproductive parameters of dam mice exposed to SWCNTS. Parameters
Maternal doses of SWCNTs (mg/kg b.w.)
Number of time-mated females Gestational index Gestational period (day) Lactation rate Pup survival rate Sex ratio (males/females) Litter size (PND 2) Milk production (g/day)
0 (control) 10 0.8 21.42 + 0.42 3.87 + 0.14 0.99 + 0.01 1.61 + 0.32 14.8 + 0.34 0.39 + 0.009
1 10 0.8 21.87 + 1.93 4.01 + 0.21 0.98 + 0.01 1.72 + 0.27 13.1 + 1.04 0.37 + 0.014
10 10 0.8 22.00 + 0.39 3.94 + 0.11 0.99 + 0.02 1.25 + 0.17 12.2 + 0.67b 0.38 + 0.002
SWCNT: single-walled carbon nanotube; PND: postnatal day; b.w.: body weight. a Values are mean + SEM. b Significantly different from the control group (p < 0.05).
differences between the exposed and control offspring for the locomotor activity and time spent in the center of arena (Figures 6 and 7). Moreover, in MWM, spatial performances in three groups were comparable (Figure 8).
Discussion
Figure 3. Effect of prenatal exposure to SWCNT on the cumulative litter body weights. Values are mean + SEM. SWCNT: single-walled carbon nanotube.
SWCNT group compared to control group (3.7 + 0.31 vs. 1.3 + 0.18, p < 0.05) while no significant differences were observed in other parameters between groups. Analysis of EPM showed that exposed offspring (10 mg/kg SWCNT) spent significantly more time (216.9 + 11.5 vs. 169.0 + 11.1 s, p < 0.05) in the closed arms and less time (47.6 + 6.8 vs. 95.6 + 10.4 s, p < 0.05) in the open arms than control group (Figure 4). Prenatal exposure to SWCNTs did not alter the locomotor activity as measured by the number of entries into the open and closed arms in the EPM (Figure 5). In the OFT, there were no significant
To the best of our knowledge, this is the first report concerning the neurobehavioral and reproductive performance effects of maternal exposure to SWCNTs in mouse offspring. In this study, prenatally exposed offspring displayed changes in the surface righting reflex test whereas no changes were observed in the other battery of neurodevelopmental endpoints. Also we found that maternal exposure to SWCNTs at the high dose level decreased the number of pups; however, other reproductive parameters were not altered significantly. Consistent with our findings, previous studies showed that maternal exposure to CNTs did not adversely affect embryo-fetal development. For example, Lim and coworkers reported no toxicological effects of oral administration of multi-walled CNTs (MWCNTs; 40, 200, and 1000 mg/kg b.w./day) to pregnant rats (Lim et al., 2011b). Another study showed that acute and repeated oral doses of SWCNTs and MWCNTs did not cause harmful effects in rats (Matsumoto et al., 2012). Moreover, it has been reported that maternal pulmonary exposure to carbon nanoparticles (Printex 90) had no effect on gestation, lactation, and offspring development (Jackson et al., 2011). Following the behavioral testing, offspring prenatally exposed to 1 mg/kg b.w. SWCNTs showed significant delays in the surface righting reflex test
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Toxicology and Industrial Health
Table 2. Behavioral tests in pups exposed prenatally to single wall carbon nanotubes.a Behavioral testes Growth and reflex Litters (n) Pinna unfolding Incisor eruption Eye opening Development of fur Air righting reflex Palm grasping reflex Surface righting reflex Cliff aversion test Level stick reflex Vibrissae placing reflex Auditory startle reflex Homing test
Maternal doses of SWCNTs (mg/kg bw) 0 (control)
1
10
7 4.7 + 0.18
8 5.8 + 0.76
10 4.4 + 0.33
9.0 + 0.81
9.7 + 1.16 10.1 + 0.5
15.4 + 0.29 6.2 + 0.28
14.5 + 1.37 15.2 + 0.24 7.5 + 1.10 6.7 + 0.26
11.3 + 0.7
12.2 + 0.7
13.5 + 0.1
5.1 + 0.4
6.5 + 0.2
5.6 + 0.2
1.3 + 0.18
3.7 + 0.31b
2.5 + 0.26
1.7 + 0.26
1.3 + 0.26
1.9 + 0.27
3.4 + 0.36
4.0 + 0.43
3.4 + 0.3
4.1 + 0.68
5.5 + 0.90
5.1 + 0.34
11.7 + 1.16
13.4 + 0.26 12.5 + 0.34
12.7 + 0.56
11.8 + 0.51 13.2 + 0.44
Figure 4. Effect of prenatal exposure to SWCNT on anxiety level in the elevated plus maze. Data are shown as mean + SEM (n ¼ 7). *p:
