Nono‐titanium dioxide exposure during the adolescent period induces neurotoxicities in rats: Ameliorative potential of bergamot essential oil

Abstract Introduction In adolescence, the brain is still maturing, and disorders in maturation may affect the normal development of the brain. Exposure to titanium dioxide nanoparticles (TiO2 NPs) has various potential negative effects on the central nervous system. Bergamot essential oil (BEO) has been found to be effective for neuroprotection. Methods The rats were injected intraperitoneally with TiO2 NPs (20 mg/kg) and/or BEO (200 mg/kg). The endogenous antioxidant state and inflammatory parameters were estimated using ELISA kits, and then the memory ability and anxiety‐like behavior in rats were assessed. Results TiO2 NPs exposure during the adolescent period induced anxiety‐like behavior, cognitive impairment, neuroinflammation and oxidative damage in hippocampus, and BEO treatment could significantly ameliorate the neurotoxicities induced by TiO2 NPs exposure. Conclusion Our results suggest that the negative effects of TiO2 NPs exposure during the adolescent period on anxiety‐like behavior and cognitive function may be related to oxidative stress and neuroinflammation induced by TiO2 NPs exposure. In addition, BEO may ameliorate the neurotoxicities induced by TiO2 NPs exposure in adolescent rats through the antioxidant and anti‐inflammatory activity of BEO.


| INTRODUC TI ON
Nanoparticles are usually defined as particles with a size of less than 100 nanometers. Compared with normal-sized particles, nanoparticles have higher permeability (Takeuchi et al., 2017).
The strong permeability of nanoparticles can be effectively utilized. However, it also poses a potential threat to human health (Orr et al., 2019;Yang et al., 2019). Titanium dioxide nanoparticles (TiO 2 NPs) are widely used for their whiteness in cosmetics and food, such as sugar-coated chewing gum, whiten skim milk confectionery, sauces, cakes, pastries, and sunscreens Chen & Mao, 2007;Grande & Tucci, 2016;Shi et al., 2013;Warheit & Donner, 2015;Zhang et al., 2015). With the widespread use of this compound, more and more attention has been paid to the potential adverse effects of TiO 2 NPs on human health. A large number of animal studies have shown that TiO 2 NPs could accumulate in brain and induce negative impact on brain development (Czajka et al., 2015). Cui et al. (2014) exposed pregnant rats to TiO 2 NPs and examined the effects of TiO 2 NPs exposure on brain development in offspring. Results of the study showed that prenatal TiO 2 NPs exposure impaired the antioxidant status, caused significant oxidative damage to nucleic acids and lipids in the hippocampus of newborn pups, and enhanced the depressive-like behaviors in adulthood. Another study by Disdier et al. (2017) showed that TiO 2 NPs exposure in aged rats could induce oxidative stress, brain inflammation, blood-brain barrier dysfunction and neuronal synaptophysin decrease. In addition, Ze et al. (2014) show that TiO 2 NPs exposure impaired spatial memory in mice and activated the expression of inflammation cytokines, that is, TNFα, IKK1, IKK2, NF-κB, NF-κBP52, NF-κBP65, NIK, and IL-1β in hippocampus. In accordance with these animal studies, many in vitro studies also showed that TiO 2 NPs has neurotoxicity and can induce neuroinflammation response and apoptosis. Sheng et al. (2015) showed TiO 2 NPs treatment resulted in oxidative stress, destabilization of mitochondrial membrane potential (MMP), intracellular Ca 2+ elevation, and apoptosis in primary cultured hippocampal neurons.
In addition, Wu et al. (2010) showed that TiO 2 NPs treatment could induce dose-dependent generation of reactive oxygen species (ROS) and neuronal damage in PC12 cells.
Bergamot essential oil (BEO) has been widely used in perfumery and confections for its intense fragrance and freshness (Navarra et al., 2015). In addition, BEO is also used in aromatherapy to improve mood and mild symptoms of stress-induced disorders (Halcon, 2002;Navarra et al., 2015). With the widespread use of BEO, some biological effects of the BEO have been deciphered by some investigators. BEO has been found to be effective for neuroprotection, anti-inflammation, and immunomodulation (Corasaniti et al., 2007;Karaca et al., 2007;Navarra et al., 2015). It has been reported that intraperitoneal injection of BEO in rats can reduce the excitatory amino acid efflux and the infarct size of the striatum and motor cortex with middle cerebral artery occlusion (Amantea et al., 2009). Karaca et al. (2007 showed that BEO ameliorated the inflammation activity induced by carrageenan in rats. Meanwhile, it has been proved from a vitro study by Corasaniti et al. (2007) that BEO can prevent the accumulation of intracellular ROS and reduce cell death of human neuroblastoma (SH-SY5Y) induced by N-Methyl-D-aspartic acid (NMDA).
TiO2 NPs exert potential negative effects on the central nervous system while BEO is effective in neuroprotection and antiinflammation. Both BEO and TiO2 NPs are used in modern lifestyle products that adolescents like. In addition, adolescence is a critical period of brain maturation. Hippocampus is an important part of the brain related to emotion control and cognitive function, and it is still in the mature stage in adolescence (Hueston et al., 2017;Romeo et al., 2016;Tottenham & Galvan, 2016). Therefore, it would be interesting to assess the potential effects of TiO 2 NPs and BEO on the development of hippocampus in adolescents. In the present study, adolescent rats were exposed to TiO 2 NPs; the endogenous antioxidant state and inflammatory parameters in hippocampus, as well as the memory ability and anxiety-like behavior of adolescent rats, were evaluated; and the potential ameliorate effects of BEO on TiO 2 NPs exposure were assessed.

| Chemicals
TiO 2 NPs were obtained from Zhejiang Hangzhou Wanjing new material Co, Ltd. (Hangzhou, China). The details of the characterization of TiO 2 NPs were previously described by Ma et al. (2009). TiO 2 NPs were suspended in 0.9% sodium chloride at a concentration of 10 mg/ml and treated with ultrasound for more than 60 min before the start of experiments. BEO extracted from bergamot (Citrus medica cv. sarcodactylis), was obtained from Zhejiang Jinshoubao Biotechnology Ltd. The BEO samples were analyzed using GC and confirmed using GC-MS to identify the major compounds (60.91% limonene, 27.08% γ-terpinene, 1.71% α-pinene, 1.70% β-pinene, 1.58% β-myrcene, 1.33% cyclohexene). Emulsions of BEO were freshly prepared with soybean oil at a concentration of 100 mg/ml just before the start of experiments. University (Approval Number: 2,170,357). The rats were kept in a climate-controlled colony room at 24℃ on a 12/12 h reverse light/ dark cycle with free access to standard food and water. Rats were randomly divided into three groups (n = 12 for each group): TiO 2 NPs group (TiO 2 NPs + BEO vehicle); BEO group (TiO 2 NPs + BEO); control group (TiO 2 NPs vehicle + BEO vehicle). Rats were treated with TiO 2 NPs (20 mg/kg) (Hu et al., 2010;Younes et al., 2015) and BEO (200 mg/kg) (Gao & Tian, 2012) from PND 22 to PND 52. The TiO 2 NPs group was injected intraperitoneally once every two days with TiO 2 NPs (20 mg/kg) and given by gavage once a day with a dose of BEO vehicle. The BEO group was injected intraperitoneally once every two days with TiO 2 NPs (20 mg/kg) and given by gavage once a day with BEO (200mg/kg). The control group was injected intraperitoneally once every two days with a dose of TiO 2 NPs vehicle and given by gavage once a day with a dose of BEO vehicle. The concentration of TiO 2 NPs was 10 mg/ml and the amount of intraperitoneal injection was 0.16-0.6 ml. The concentration of BEO was 100 mg/ml and the gavage volume was 0.16-0.6 ml. After 31 days of TiO 2 NPs and BEO treatment, half of the rats (18 rats, n = 6 for each group) were used to perform the novel object recognition test on PND 53 and PND 54 and then sacrificed on PND 55 for immunohistochemistry test; the other half (18 rats, n = 6 for each group) were used to perform the open field test on PND 53 and then sacrificed on PND 54 for biochemical analysis. The timeline of the experiment is shown in Figure 1a.

| Novel object recognition test
The novel object recognition test is commonly used to assess memory. After the rats were exposed to TiO 2 NPs and BEO from PND 22 to PND 52, the memory of rats was evaluated in the novel object recognition test. Eighteen rats (n = 6 for each group) were used for the novel object recognition test in the morning of PND 53. The test room was uniformly illuminated by a red light. A square box (100 × 100 × 50 cm) and two kinds of objects (approximately 13 cm high) were used for the novel object recognition test. The shape, color and texture of the two kinds of objects were different.
The objects had enough weight and would not be moved by rats.
According to the published novel object recognition test protocols (Che et al., 2015;Leger et al. 2013;Lindsay, 2017), the experimental procedure of novel object recognition test included three periods: habituation, training, and testing. Briefly, the habituation trial was performed on PND 53. In the habituation trial, each rat was placed in the empty square box for 10 min. Training and testing were performed on PND 54. In the training trial, each rat was placed in the square box with two identical objects for 5 min. The discrimination index of left and right position preference is measured by the following formula: Tr stands for the right object and Tl for the left object.
After 1 hr, the testing trial was conducted, and each rat was placed back into the test box for another 5 min with one of the two identical objects replaced by a novel object. The discrimination index of familiar objects and new objects was measured by the following formula: Tn represents the exploration time devoted to the novel object and Tf represents the exploration time devoted to the familiar object.
In order to avoid the influence of natural preference and positions on the results as much as possible, as shown in Figure 1b

F I G U R E 1
The process of experimental operation. (a) Timeline of the experiment. A total of 36 male Sprague Dawley (SD) rats aged PND 21 were used in the experiment. Rats were treated with drug from PND 22 to PND 52. After 31 days of treatment, half of the rats (18 rats) were used to performed the novel object recognition test on PND 53 and PND 54 and then were sacrificed on PND 55 for immunohistochemistry test; the other half of the rats (18 rats) were used to performed the open field test on PND 53 and then were sacrificed on PND 54 for biochemical analysis. PND, postnatal day; OFT, open field test; NOT, novel object recognition test. (b) The experimental procedure of the novel object recognition test. In the training trial, each rat was placed in the square box with two identical objects for 5 min. After 1 hr, the testing trial was conducted, and each rat was placed back into the test box for another 5 min with one of the two identical objects replaced by a novel object. Choice of the objects to be explored and their positions were decided according to a random number table video camera. Total time spent to explore the familiar object and the novel object was measured with the video tracking system software (Noldus Information Technology Inc.). After each trial, the behavior box and objects were cleaned thoroughly with 70% ethanol.

| Open field test
The open field test is commonly used to assess anxiety-like behavior in rodents. After the rats were exposed to TiO 2 NPs and BEO from PND 22 to PND 52, the anxiety-like behavior of rats was evaluated in the open field test. Eighteen rats (n = 6 for each group) were used to perform the open field test in the morning of the PND 53. According to a published open field test protocol (Che et al., 2015). Briefly, the test room was uniformly illuminated by a red light. A square box (100 × 100 × 50 cm) was

| Biochemical analysis
After open field tests, the 18 rats (n = 6 for each group) were used for biochemical analysis according to a published protocol (Cui et al., 2014).
Briefly, rats were deeply anesthetized with ketamine (100 mg/kg), the hippocampus of each rat was rapidly isolated on an ice plate, homogenized in ice-cold phosphate buffered saline, and then centrifuged at 10,000 × g for 10 min at 4•C. The supernatant was used for biochemical analysis. The analysis of malondialdehyde (MDA), catalase activity (CAT), glutathione peroxidase activity (GSH-PX), and total antioxidant capacity (T-AOC) were performed using spectrophotometric methods with kits (Nanjing Jiancheng Bioengineering Institute). The IL-6, IL-1β, and TNFα productions were estimated using ELISA kits (Nanjing Jiancheng Bioengineering Institute).

| Immunohistochemistry
After novel object recognition tests, the 18 rats (n = 6 for each group) were used to detect the 8-hydroxy-deoxyguanosine (8-OHdG, an for 2 hr at room temperature, and mounted using antifade mounting medium. The fluorescence intensity of the hippocampus was counted. This intensity was calculated as follows:

| Data analysis
Statistical analysis was conducted using SPSS 16 (SPSS Inc). All results were presented as mean ± standard error of the mean.
Significant differences among groups were analyzed using one-way ANOVA followed by Newman-Keuls post hoc comparisons test.
Statistical significance was set as p < .05.

| Effect of BEO treatment on anxiety-like behavior of rats induced by TiO 2 NPs in open field test
As shown in Figure 2 field. In order to make the judgment of anxiety behavior reliable, the ratio of center distance to total distance was quantified (center area distance/total distance) to determine whether there is a disproportionate reduction in center area inquiry. Compared with the control group, the ratio of center distance to the total distance of the TiO 2 NPs exposure group decreased significantly (p < .05). Compared to TiO 2 NPs exposure group, BEO treatment group showed a significant increase in the frequency of center entries, distance traveled within the center area, total distance traveled, and the ratio of center distance to total distance (p < .05) but not in the number of rearings.

| Effect of beo treatment on memory decline of rats induced by TiO2 NPS in the novel object recognition test
During training, the time spent to explore the two objects (right and left) were recorded. As shown in Figure 3a, the discrimination index is near zero, indicating that the rats have no left or right position preference. During testing, it is illustrated in Figure 3b that the control group spent more time exploring the novel object than Fluorescence Intensity = Intensity of Hippocampus − Intensity of the Background.

F I G U R E 3
Effect of BEO treatment on memory decline of rats induced by TiO2 NPs in the novel object recognition test. (a) During training, the discrimination index is near zero. Rats have no obvious natural preference for the left or right position. (b) During testing, the control group spent more time exploring the novel object than the familiar one (compared to 0%), but not the TiO2 NPs exposure group and BEO group. Results were the mean ± SEM (N = 6). * p < .05; BEO, bergamot essential oil; TiO2 NPs, titanium dioxide nanoparticles; CTL, control the familiar one (compared to 0%), but not the TiO 2 NPs exposure group and BEO group. TiO 2 NPs exposure had significant effects on recognition memory in the novel object recognition test, while BEO treatment did not significantly improve the impairment of recognition memory.  (Table 1). TiO 2 NPs exposure was associated with a decline in the level of CAT, GSH-PX, and T-AOC (Table 1). BEO significantly increased the level of CAT (p < .05), but not the level of GSH-PX, and T-AOC (Table 1). The level of MDA (lipid peroxidation product) was significantly increased in TiO 2 NPs exposure group compared with the control group (p < .05), and BEO significantly decreased the level of MDA (p < .05) ( Table 1).

| Effect of BEO treatment on oxidative damage in hippocampus of rats induced by TiO 2 NPS
The DNA oxidative damage was evaluated by immunohistochemical staining with an antibody that recognizes 8-OHdG (DNA peroxidation product). There were significant differences of the DNA oxidative damage [F(2, 15) = 4.28, p = .039] (Figure 4) among control, TiO 2 NPs and BEO treatment groups. As shown in

| Effect OF BEO treatment on the increased amounts of cytokines in hippocampus of rats induced by TiO 2 NPS
As shown in Table 2 Note: Results were the mean ± SEM (N = 6).

| D ISCUSS I ON
The open field test is widely used to test animal emotional activity. Exploratory behaviors such as the frequency of access to central area, the distance traveled in the central area and the ratio of center distance to the total distance (center area distance/total distance) are used to demonstrate anxiety-like behavior (Abelaira et al., 2013;Pollak et al., 2010;Willner et al. 1992 anxiety-like behavior. Moreover, the novel object recognition test based on the natural proclivity of rodents to explore novelty has been used to assess cognitive function (Lindsay, 2017 (Lehrner et al., 2000;Ni et al., 2013;Wilkinson et al., 2007). These finding confirmed the anxiolytic-like properties of Adolescence is one of the "critical periods" of brain maturation (Ismail et al., 2017). The plasticity of the nervous system is particularly sensitive to multiple environmental factors in this "critical period" (Dalle & Mabandla, 2018;Schiavone et al., 2015). The age span of adolescence in rats is from PND 21 to PND 59 (Majcher-Maslanka et al., 2019;McCormick & Mathews, 2010). In the present study, TiO 2 NPs exposure from PND 22 to PND 52 leads to anxietylike behavior and memory impairment. The present results are consistent with the previous studies suggesting that chronic exposure to stressful environmental factors during the adolescent period may lead to emotional disorders and cognitive dysfunction (Romeo et al., 2016;Tottenham & Galvan, 2016).
Hippocampus, which is an important brain area related to emotional control and cognitive functions, is still maturing during the adolescent period. For example, the number of granule cells and the volume of hippocampus are increasing in this period (Hueston et al., 2017;Sousa et al., 1998). Hippocampal neurogenesis during the adolescent period could be altered by acute and long-term stress (Hueston et al., 2017). TiO 2 NPs can cross the blood-brain barrier, increase the generation of ROS (Fujishima et al. 2008), and induce oxidative stress (Gao et al., 2011;Hong et al., 2017). In the present study, we exposed rats to Note: Results were the mean ± SEM (N = 6).

| CON CLUS ION
The present results suggest that there may be negative effects of TiO 2 NPs exposure during adolescence on emotional control and cognitive functions, and these negative effects are associated with oxidative stress and neuroinflammation in hippocampus induced by TiO 2 NPs exposure. The negative effect of TiO 2 NPs exposure during the adolescent period should arouse our attention. On the other hand, BEO is effective in neuroprotection and could ameliorate the negative effects induced by TiO 2 NPs. However, in the present study, we only studied the effect of BEO treatment and TiO 2 NPs exposure on male animals, and we could not confirm whether it will have the same effect on female animals. We believe that the biological response effects of TiO 2 NPs exposure in the adolescent period and the neuroprotective activity of BEO may be complex and need further study.

CO N FLI C T O F I NTE R E S T
Authors declare no conflict of interest.

Yonghua Cui and Yi Che contributed to the experiments. Yonghua
Cui and Hongxin Wang contributed to the analyses of the data and the write up of the manuscript.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/brb3.2099.
[Correction added on March 20, 2021, after first online publication: Peer review history statement has been added.]

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.