The main objective of this work is to study the effect of chronic administration of cadmium (Cd) on the level of depression-like, anxiety-like, memory state and oxidative stress in male and female Wistar rats. For this purpose, this study was conducted with 24 rats for each gender. Four groups were constituted: (Group 1: Control): received saline solution NaCl (0.9%), (Group 2: Cd-0.25; Group 3: Cd-0.5; Group 4: Cd-1): received daily 0.25 mg/kg, 0.5 mg/kg and 1 mg/kg of Cd respectively during 8 weeks. After treatment period, animals were tested in the open-field, elevated plus maze tests for anxiety-like behavior, and forced swimming test for depression-like behavior. The Y maze was used to evaluate the working memory and the Morris Water Maze, to evaluate space learning and spatial memory. The results revealed that in males, all doses of Cd provoke depression-like, while in females only the group treated with 1 mg/kg Cd shows elevated depression-like behavior. In regard to anxiety-like behavior, Cd induces an anxiogenic effect in both genders tests. In the Y-Maze test, both males and females expressed a low percentage of alternations, suggesting that working memory was affected by Cd at 1 mg/kg. In the Morris Water Maze test, the space learning and spatial memory were significantly impaired in the group Cd-1. Neurochemical analysis showed that levels of nitric oxide and lipid peroxidation in the hippocampus were significantly increased after Cd treatments. Overall analysis of our data revealed that Cd caused significant alterations in the examined parameters that were sex-dependent and dose-dependent.
Cd is one of the most toxic elements that bio-accumulates in the environment [
At peripheral level, prolonged exposure to Cd will cause toxic effect due to its accumulation over time in a variety of tissues, including kidneys, liver, central nervous system (CNS), and peripheral neuronal systems [
In this target, the present study was designed to determine the effects of chronic administration of Cd on animal behavior, especially on anxio-depressive disorders and memory deficits, and on levels of oxidative stress in male and female rats.
This study was performed on adult male and female Wistar rats aged 8 to 9 weeks and having an average weight of 120 ± 20 g from breeding of Faculty of life sciences, University Ibn Tofaïl. Animals of the same experimental group were housed together by six rats in each cage (36 cm long, 20 cm wide and 15 cm high) and this was consistent for all rats. The space reserved for the breeding and the survival of the rats respects the well-being of the animals. All rats were maintained under LD 12/12 (12 h Light/12 h Darkness) and at a standard temperature (21 ± 1)˚C. Water and food were provided ad libitum. All experimental procedures were approved by the University Ethics Committee for Animal Experiments.
The rats are divided into 4 groups of 6 animals each (6 males and 6 females). For each sex, the different groups of rats are distributed as follows:
・ 1st group: rats control receiving daily an intraperitoneal injection of Nacl 0.9%.
・ 2nd group: rats receiving daily a dose of 0.25 mg/kg of Cd.
・ 3rd group: rats receiving daily a dose of 0.5 mg/kg of Cd.
・ 4th group: rats receiving daily 1 mg/kg of Cd.
Saline solution or CdCl2 (obtained from SIGMA-ALDRICH) used in the present work were injected intraperitoneally and chronically at the rate of one injection per day and this during 8 weeks according. All injections are carried out between 16:00 and 16:30. The rats were blindly treated and tested throughout the experiment. The route of Cd was selected according with previous works from literature [
Twenty four hours after end of the 8 weeks corresponding to the treatment period, the animals were submitted to behavioral tests at the rate of one test per day, in the following order: the Open field test (OFT) followed by the Elevated plus maze (EPM), Forced swimming test (FST), Y maze and Morris water maze test (MWM), respectively [
・ Open Field Test:
The OFT is used to measure the anxiety-like behavior in rodents [
・ Test of the Elevated Plus Maze (EPM):
The EPM is an ethological model of anxiety in rodents provoked by the novelty and repulsion as a result of elevation and illumination of the maze [
The FST is an excellent maze used to assess the depressive-like behavior [
・ Y-maze test:
Spatial working memory was assessed by using the Y-maze spontaneous alternation test, as previously described [
・ Morris Water Maze Test:
The water maze [
Rats were tested in the Morris water maze in four phases: acquisition, reversal, probe trial and visible platform trial [
One day after end of behavioral tests, all animals were firstly anesthetized and then sacrificed by decapitation. Brains were quickly removed and maintained at low temperature on ice cold. The hippocampus was rapidly and gently removed and separated from surrounding tissues and homogenized in phosphate buffer at PH: 7.4 (W/V), centrifuged at 1500 rpm for 10 min and the resulting supernatant was used in the biochemical assays [
・ Nitrite/nitrate assay:
In biological systems conversion of oxide nitric (NO) in aqueous solution to nitrite and nitrate is thought to favour nitrite production. The concentrations of nitrite in tissue homogenates were measured by using the diazotization method based on the Griess reaction, which is an indirect assay for NO production [
・ Lipid peroxidation assay:
The formation of lipid peroxides during lipid peroxidation process were analysed by measuring the thiobarbituric-acid-reacting substances (TBARS) in cells, as previously described by Draper and Hadley [
Behavioral data and biochemical parameters were analyzed by two-way ANOVA using SPSS version 22. Post hoc comparisons were made using the Tukey’s test. ANOVA repeat measures were used for the Morris water maze test. All data are expressed as the means ± standard error of the means (S.E.M.). In order to estimate the gender effect, we expressed the results for each sex as a percentage of the basal level (% BL) represented by the respective control and considered as being 100%. Differences were considered significant when p < 0.05, very significant when p < 0.01 and highly significant when p < 0.001.
・ Time spent in the central area (TCA) (
The results summarized in
In males, Cd affects TCA in dose-dependent manner between 0.25 and 1 mg/kg in comparison with the control group (Cont/Cd-0.25: p < 0.05, Cont/Cd-0.5: p < 0.001 and Cont/Cd-1: p < 0.001 respectively). Cd induced mean average decrease of 29%, 57% and 55% at doses of 0.25, 0.5 and 1 mg/kg
respectively. In addition, there is a statistically significant difference between the groups Cd-0.25/Cd-0.5 and Cd-0.25/Cd-1 (p < 0.05). In contrast, no difference was noted between Cd-0.5/Cd-1 groups (p > 0.05).
In females, Cd affects TCA at doses of 0.25, 0.5 and 1 mg/kg in comparison with the control group (Cont/Cd-0.25: p < 0.01, Cont/Cd-0.5: p < 0.01 and Cont/Cd-1: p < 0.001 respectively). Cd induced mean average decrease of 32%, 38% and 47% at doses of 0.25, 0.5 and 1 mg/kg respectively. No statistically significant difference was observed with comparing different treated Cd groups (p > 0.05).
The sex effect was more visible when considering the relative comparison (TCA% BL) between respective treated Cd groups in males and females. Indeed, the males of the groups Cd-0.5 showed a TCA significantly lower compared to females of similar groups (p < 0.01).
・ Number of Returns to the Center (NRC) (
The treatment factor (F(3.32) = 7.93, p < 0.001) significantly affected the NRC. The effect of Cd is observed in males with referring to NRC parameter. At doses of 0.25, 0.5 and 1 mg/kg, Cd significantly reduced the NRC compared with the control group (Cont/Cd-0.25: p < 0.05, Cont/Cd-0.5: p < 0.01 and Cont/Cd-1: p < 0.01 respectively). Cd induced mean average decrease of 29%, 57% and 55% at doses of 0.25, 0.5 and 1 mg/kg respectively. While in females Cd did not induce any significant change in this parameter (p > 0.05). It induced mean average decrease of 32%, 38% and 47% at doses of 0.25, 0.5 and 1 mg/kg respectively. In both sexes, no statistically significant difference was observed with comparing different treated Cd groups (p > 0.05).
The sex effect was clear when considering the relative comparison (NRC % BL) between respective treated Cd groups in males and females. Indeed, the males of the group Cd-0.5 showed a NRC significantly lower compared to females of similar group (p < 0.05).
・ Number of total squares (NTS) (
Locomotors activity was unaffected by any treatment (F(3.32) = 1.04, p > 0.05), and no effect of sex (F(1.32) = 1.49, p > 0.05) was noted. The values of all groups were comparable. Even though is not significant, we observed a slight increase in the total activity of females compared to males.
・ Time Spent in Open Arms (TOA) (
Statistical analysis showed that TOA was significantly affected by the Cd treatment (F(3.32) = 48.30, p < 0.001).
In males, Cd decrease significantly TOA in all treated group in comparison with the control group (p < 0.001). Cd induced mean average decrease of 48%, 46% and 57% at doses of 0.25, 0.5 and 1 mg/kg respectively. No statistically significant difference was observed with comparing different treated Cd groups (p > 0.05).
In females, Cd affects TOA in dose-dependent manner, since at doses of 0.5 and 1 mg/kg it decreases the TOA compared with the control group (p < 0.05, p < 0.001 respectively), whereas at 0.25 mg/kg Cd was not effective (p > 0.05). The metal induced mean average decrease of 9%, 21% and 38% at doses of 0.25, 0.5 and 1 mg/kg respectively. In addition, there is a difference statistically significant between Cd-0.25/Cd-1 groups (p < 0.01). In contrast, no difference was noted between Cd-0.25/Cd-0.5 and Cd-0.5/Cd-1 groups (p > 0.05).
The sex effect was more visible when considering the relative comparison (TOA% BL) between respective treated Cd groups in males and females. Indeed, the males showed a TOA significantly lower compared to females of similar groups (p < 0.01).
・ Entry to Open Arms (EOA) (
This parameter was affected by Cd treatment (F(3.32) = 8.97, p <0.001): In males, at doses of 0.5 and 1 mg/kg, Cd decreases significantly the EOA in comparison with control group (p < 0.05), while at dose of 0.25 mg/kg this metal did not induce any significant change in this parameter (p > 0.05). In addition, there is a difference statistically significant between Cd-0.25/Cd-1 groups (p < 0.05). In contrast, no difference was noted between Cd-0.25/Cd-0.5 and Cd-0.5/Cd-1 groups (p > 0.05).
In females, at dose of 1 mg/kg, Cd decreases significantly the EOA in comparison with control group (p < 0.05), while at doses of 0.25 and 0.5 mg/kg this metal did not induce any significant change in this parameter (p > 0.05). No statistically significant difference was observed with comparing different treated Cd groups (p > 0.05).
Similar results were observed when considering the relative comparison (EOA % BL) between treated Cd and control groups. Thus, Cd induced mean average decrease of 5%, 55% and 60% in males; of 40%, 40% and 58% in females respectively, at doses of 0.25, 0.5 and 1 mg/kg respectively. Also, the sex effect was visible when considering the relative comparison (EOA% BL) between respective treated Cd groups in males and females. Indeed, the males of the group Cd-0.25 showed a NRC significantly lower compared to females of similar group (p < 0.05).
・ Total entries in arms (TEA) (
In contrast to TOA and EOA parameters, Cd was no significant effect on locomotors activity (TEA) represented whatever the dose considered (p > 0.05).
・ Immobility Time (TIM) (
Statistical analysis showed that TIM was significantly affected by sex factor (F(1.32) = 8.32, p < 0.01) and the Cd treatment (F(3.32) = 23.5, p < 0.001).
In males, Cd significantly affects TIM in dose-dependent manner, since at doses of 0.25, 0.5 and 1 mg/kg it increases the TIM compared with the control group (p < 0.05, p < 0.05 and p < 0.001 respectively). Cd induced mean average increase of 42%, 49% and 123% at doses of 0.25, 0.5 and 1 mg/kg respectively. In addition, there is a difference statistically significant between Cd-0.25/Cd-1, Cd-0.5/Cd-1 groups (p < 0.01). In contrast, no difference was noted between Cd-0.25/Cd-0.5 groups (p > 0.05).
In females, at dose of 1 mg/kg, Cd increases significantly TIM in comparison with control group (p < 0.05), while at doses of 0.25 and 0.5 mg/kg this metal did not induce any significant change in this parameter (p > 0.05). It induced mean
average increase of 0%, 5% and 39% at doses of 0.25, 0.5 and 1 mg/kg respectively. No statistically significant difference was observed with comparing different treated Cd groups (p < 0.05).
The sex effect was clear when considering the relative comparison (TIM% BL) between respective treated Cd groups in males and females. Indeed, the males showed a TIM significantly higher compared to females of similar groups (p < 0.001).
・ Struggling Time (TST) (
This parameter was affected by Cd treatment (F(3.32) = 13.67, p < 0.001) : The dose-dependent effect of Cd is observed in males with referring to TST parameter. At doses of 0.25, 0.5 and 1 mg/kg, Cd decreases the TST compared with the control group (p < 0.05, p < 0.001 and p < 0.001 respectively). No statistically significant difference was observed with comparing different treated Cd groups (p > 0.05).
In females, at dose of 1 mg/kg, Cd decreases the TST in comparison with control group (p < 0.05), while at doses of 0.25 and 0.5 mg/kg this metal did not induce any significant change in this parameter (p > 0.05). In addition, there is a difference statistically significant between Cd-0.25/Cd-1 groups (p < 0.05). No difference was noted between Cd-0.25/Cd-0.5 and Cd-0.5/Cd-1 groups (p > 0.05).
Similar results were observed when considering the relative comparison (TST % BL) between treated Cd and control groups. Thus, Cd induced mean average decrease of 11%, 20% and 18% in males; of 7%, 24% and 18% in females respectively at doses of 0.25, 0.5 and 1 mg/kg respectively. The sex effect was observed when considering the relative comparison (TST% BL) between respective treated Cd groups in males and females. Indeed, the males of the group Cd-0.5 showed a TST significantly lower compared to females of similar group (p < 0.05).
Spontaneous alternation percentage was affected by Cd treatment (F(3.32) = 6.12, p < 0.01), but not by sex factor (F(1.32) = 0.75, p > 0.05).
In males and females, chronic treatment with Cd at dose of 1 mg/kg was associated with an decrease of spontaneous alternation percentage statistically significant compared to the control group (p < 0.05), while at doses of 0.25 and 0.5 mg/kg this metal did not induce any significant change in this parameter (p > 0.05). Indeed, no statistically significant difference was observed with comparing different treated Cd groups (p > 0.05).
The relative comparison (Spontaneous alternation percentage % BL) between treated Cd and control groups shows that Cd induced mean average decrease of 6%, 11% and 33% in males; of 15%, 19% and 29% in females respectively at doses of 0.25, 0.5 and 1 mg/kg respectively.
・ Acquisition and reversal training (
No significant differences in latency to reach the hidden platform in both phases acquisition trials (days 1 - 3) and reversal trials (days 4 - 6) between the treated and control groups (p > 0.05). Despite this, the group Cd-1 showed a non-significant increase in latency to reach the platform over days in acquisition and reversal learning.
・ Percentage time spent in the correct quadrant during the probe trial (
The statistical analysis showd that the percentage of time spent in the correct quadrant is significantly affected the treatment with Cd (F(3,32) = 7.79; p < 0.01).
In males and females, Cd at dose of 1 mg/kg was associated with a significant decrease of the percentage of time spent in the correct quadrant statistically significant compared to the control group (p < 0.05), while at doses of 0.25 and 0.5 mg/kg this metal did not induce any significant change in this parameter (p > 0.05). Indeed, no statistically significant difference was observed with comparing different treated Cd groups (p > 0.05).
The relative comparison (percentage of time spent in the correct quadrant % BL) between treated Cd and control groups shows that Cd induced mean average decrease of 12 %, 8% and 29% in males; of 6%, 12% and 21% in females respectively at doses of 0.25, 0.5 and 1 mg/kg respectively.
・ Visible platform test (
Latency to find the visible platform was unaffected by any treatment (F(3.32) = 0.84, p > 0.05), and no effect of sex (F(1.32) = 0.008, p > 0.05). The values of all groups were comparable.
・ LPO in hippocampus (
Statistical analysis showed that LPO reflected by TBARS levels was significantly affected by sex factor (F(1.32) = 6.81, p = 0.0153 < 0.05)), and the Cd treatment (F(3.32) = 42.15, p < 0.001).
The results summarized in
In females, at dose of 1 mg/kg, Cd increases significantly the LPO levels in comparison with control group, while at doses of 0.25 and 0.5 mg/kg this metal did not induce any significant change in this parameter (p < 0.001, p > 0.05 and p > 0.05 respectively). There is a difference statistically significant between Cd-0.25/Cd-1 and Cd-0.5/Cd-1 groups (p < 0.01). No difference was noted between Cd-0.25/Cd-0.5 groups (p > 0.05).
Similar results were observed when considering the relative comparison (TBARS % BL) between treated Cd and control groups. Thus, Cd induced mean average increase of 34%, 146% and 153% in males; of 28%, 31% and 91% in females respectively at doses of 0.25, 0.5 and 1 mg/kg respectively. In addition, the sex effect was observed when considering the relative comparison (TBARS% BL) between respective treated Cd groups in males and females. Indeed, the males of the groups Cd-0.5 and Cd-1 showed a TBARS levels significantly higher compared to females of similar groups (p < 0.001 and p < 0.05 respectively).
・ NO concentrations in hippocampus (
This parameter was affected by Cd treatment (F(3.32) = 59.79, p < 0.001), but not by sex factor (F(1.32) = 0.22, p > 0.05). No interaction was found between treatment and sex F(3.32) = 0.72, p > 0.05).
In males and also females, Cd affects the NO levels in dose-dependent manner, since at doses of 0.25 and 1 mg/kg it increases the nitrite/nitrate levels (nitric oxide; NO) in comparison with the control group (p < 0.01, p < 0.001 and p < 0.001 respectively). The metal induced mean average increase of 83%, 130% and 147% in males, of 80%, 107% and 113% in females, at doses of 0.25, 0.5 and 1 mg/kg respectively. In addition, in males there is a statistically significant difference between the groups Cd-0.25/Cd-1 (p< 0.01) and no difference was noted between Cd-0.25/Cd-0.5 and Cd-0.5/Cd-1 groups (p > 0.05). In contrast, in females no statistically significant difference was observed with comparing different treated Cd groups (p > 0.05).
The sex effect was observed when considering the relative comparison (NO% BL) between respective treated Cd groups in males and females. Indeed, the males of the groups Cd-1 showed a NO levels significantly higher compared to females of similar groups (p < 0.05).
The main objective of this study was to determine the effects of chronic exposure to Cd on animal behavior, in particular on affective, cognitive disorders and on levels of oxidative stress. The assessment of anxiety-like and depression-like behaviors is based on the use of validated OFT, EPM and FST behavioral tests. While spatial working memory and spatial learning have been evaluated using respectively the Y-maze test and the Morris water maze test. Oxidative stress has been determined by measurement of NO and TBARS directly associated with LPO.
The evaluation of the anxiety levels obtained in the OFT is still confirmed by the use of EPM, one of the most used anxiety models [
The present study also showed that chronic administration of Cd caused an increase in TIM in a dose-dependent manner between 0.25 and 1 mg/kg. Since, the locomotor activity measured in the OFT was not affected by this treatment, it was suggested that the behavioral differences in the Porsolt test would induced by changes in the depressive-like state. Our study was corroborated by the observation of Haider (2014) which reported that in FST, the TIM was significantly increased by acute administration of Cd at a dose of 3 mg/kg as well as other two dose, indicating dose-dependent effects (1 - 2 mg/kg) [
The question is how the Cd acts to induce its effects on anxio-depressive behavior. Cd, after chronic exposure, could affect the integrity or permeability of the BBB consequently penetrating in the brain of developing and adult rats, leading to brain accumulation and cellular dysfunction [
In this study, we also reported that learning and working memory were affected by Cd. Generally, animals that have undergone hippocampal lesions have poor performance in Y maze and Morris water maze test [
Our study supported by the work mentioned above confirmed that learning and memory was impaired by chronic Cd administration. These impairments of spatial memory, which are generally caused by hippocampal lesions [
Another important aspect to be discussed here is the effect of Cd on the cholinergic system, which plays a crucial role in the process of learning and memory [
Another explication of Cd action on affective and cognitive disorders was found in the results of examination of same parameters in hippocampus reported in this study. We demonstrate that Cd exposure increased LPO levels, accompanied by a significant increase in NO levels in the hippocampus in male and female rats. This results are in agreement with several recent studies showing an increase in LPO and NO levels in the hippocampus which suggest an elevation of oxidative stress, following Cd administration in rat [
Our hypothesis is that behavioral dysfunction observed in the present study (depression-like, anxiety-like and memory deficit) after chronic administration of Cd, might be linked to an increase in LPO and NO levels in the hippocampus and/or other brain areas. With regard to affective disorder, this could cause death of 5 HT or other neurotransmitter neurons and consequently a decrease in 5 HT brain levels. In this sense, a decrease in levels of 5-HT, dopamine in all regions of the brain including the anterior brain (hippocampus and striatum) has been shown 24 h after exposure to Cd [
In addition, in our study, dose and sex dependent effect was well established; the effects of Cd on depression-like and anxiety-like being more pronounced in males than in females. This sexual dimorphism suggests the involvement of sexual hormones in the modulation of affective disorders by regulating neurotransmitter synthesis release or actions. In this sense, gonadal hormones contribute to the anatomical or functional characteristics of several neurotransmitter systems such as serotoninergic (5-HT) and gabaergic (GABA) systems that regulate anxiety and depression [
On the other hand, numerous studies showed that uptake and accumulation of Cd were more in the males when compared to female rats [
This study suggests that people living in heavy metal pollution environments and continuous exposure to Cd may eventually lead to behavioral pathologies such as affective and cognitive disorders.
This work was supported by CNRST and faculty of Sciences University Ibn Tofaïl, kenitra. Thanks to Mr, A. REZQUAOUI, O. ZGHARI from Unit of Nervous and Endocrine Physiology, Laboratory of Genetics, Neuroendocrinology and Biotechnology, University Ibn Tofail, Kenitra, Morocco, for their help and assistance.
Lamtai, M., Chaibat, J., Ouakki, S., Berkiks, I., Rifi, El-H., El Hessni, A., Mesfioui, A., Tadlaoui Hbibi, A., Ahyayauch, H., Essamri, A. and Ouichou, A. (2018) Effect of Chronic Administration of Cadmium on Anxiety-Like, Depression-Like and Memory Deficits in Male and Female Rats: Possible Involvement of Oxidative Stress Mechanism. Journal of Behavioral and Brain Science, 8, 240-268. https://doi.org/10.4236/jbbs.2018.85016