The Relationship between Behavior Responses and Brain Acetylcholinesterase (AChE) Activity of Zebrafish (Danio rerio) in Cadmium Stress

Abstract

In this research, the toxic effects of Cadmium chloride (CdCl2 ), which can seriously pollute aquatic environment and threaten human health, are evaluated based on the behavior responses and the brain Acetylcholinesterase (AChE) activity of zebrafish (Danio rerio). The results showed that Behavior Strength (BS) of test groups (changed from 0.15 to 0.65), which was recorded using an online behavior monitoring system, was lower than the control groups (changed from 0.65 to 0.85). The behavior responses of zebrafish suggested that both dose and time effect relationships existed between Cd2+ stress and zebrafish BS. Meanwhile, the brain Acetylcholinesterase (AChE) activity of zebrafish were strongly inhibited by Cd2+: the AChE activities were lower than 60% after 0.5h Cd2+ exposure in both 1 TU (Toxic Unit) and 2 TU. The AChE activities in 0.1 TU Cd2+ treatment were about 60% in the first 2h and then increased to about 100% in 4h with a decrease tendency in the following exposure time (8h to 48h), which changed from 100% to 70%-80%. Totally, the brain AChE activities of zebrafish showed similar rules with BS after correlation analysis, which might provide an understanding of the ecotoxicological assessment of heavy metal Cd based on zebrafish.

Citation

Yang M, Ji L, Zhang X, Fan Y and Ren Z. The Relationship between Behavior Responses and Brain Acetylcholinesterase (AChE) Activity of Zebrafish (Danio rerio) in Cadmium Stress. Int J Fisheries Sci Res. 2017; 1(1): 1002.

Introduction

Heavy metals have received more and more attention due to their pollution and the persistence in the aquatic environment, which can affect species at all trophic levels and then endanger the balance of the water ecosystem [1-3]. In all heavy metals, Cadmium (Cd), which is one of the most toxic heavy metals with strong neurotoxicity and can cause great damage to the transduction signal [4], has seriously impacts aquatic environment and threaten human health [5]. As a by-product, Cd might be imported into aquatic environment after mining, forging and other industrial process [6]. Cd pollutions in environment can cause a variety of toxic effects to aquatic organisms, including behavior disturbances, physiological and biochemical changes, reproductive abnormalities, neurological deficits and immunological dysfunctions [7-10].

Acetylcholinesterase (AChE) activity has been widely used as an indicator of environmental stress. It is reported that AChE activity could be inhibited in mouse [11] and zebrafish [12] by heavy metal aluminum. As Cd can affect AChE activity and then induce the loss of nerve conduction ability [13], which can induce strong neurotoxicity on aquatic organisms and seriously damages nervous systems of animals [14], AChE activity analysis is a good approach to realize the understanding of the toxic effects of Cd on the neurotoxicity of aquatic organisms.

Behavior responses of organisms are frequently used as an endpoint to assess environmental stress due to the importance of altered behavior in toxicological studies [8,15,16]. Recent studies have suggested that the swimming behavior associated with low-level chemical toxicants can be observed earlier than changes in physiology [17]. Behavior responses have been widely used in contamination assessment and served as an indicator for environmental stress assessment [18,19].

In the toxic assessment of heavy metals, bioassays only depending on single analytical techniques [20] might be not enough to realize the purpose due to their strong neurotoxicity and bio-accumulation [21]. In fact, some researchers have applied some combined methods to realize the environmental stress assessment. Pereira et al. investigated the effects of endosulfan on the brain AChE activity and the swimming performance in adult zebrafish in 2012 [22], which suggested that that AChE activity inhibition is one of the endosulfan-induced toxicity pathway in fish brain and endosulfan can impairs behavior responses of zebrafish, which can potentially compromises their ecological and interspecific interaction. It is reported by Zhang et al., [23] that Aphanizomenon flosaquae can affect fish locomotor capacity by damaging the cholinergic system, which suggest that aquatic animals’ behavior responses and AChE activity can be used as indicators for investigating environment pollution in nature.

As model species, zebrafish (Danio rerio), which are very sensitive to the changes of the environmental stress [24], have been used to realize the water quality assessment due to their large-scale cultivation in water, short growth cycle, simple genomic background, and ability to adapt to a wide range of water conditions, and other characteristics [25]. It is reported that the brain AChE activities of zebrafish could be inhibited by heavy metals [26] and other chemicals [27]. Meanwhile, as the target site of neurotoxicity is brain, the brain AChE activity inhibition could accurately reflect real environmental stress [23]. On the other hand, the behavior responses of zebafish in different environmental stress have been reported [9,22,28], which suggest that biological behavior is the sensitive reactions in response to environmental changes [29]. Therefore, the relationship between behavior responses and brain Acetylcholinesterase (AChE) activity of zebrafish (Danio rerio) in Cd²⁺ stress is investigated to: 1) illustrate the difference of zebrafish behavior responses and the brain AChE activity inhibition in different Cd²⁺ treatments; 2) analyze the correlation between zebrafish behavior responses and the brain AChE activity after exposed to Cd²⁺ in different exposure time; 3) discuss the possibility of the ecotoxicological assessment of heavy metal Cd based on the behavior responses and the brain AChE activity of zebrafish. In this study, an online behavior monitoring system was used to automatically sample zebrafish swimming behavior, and then Bradford Protein Assay was used to determine the brain AChE activities in zebrafish in Cd treatments. The relationship between zebrafish swimming behavior and brain AChE activities was analyzed using Detrended Cross-Correlation Analysis (DCCA).

Materials and Methods

Test species and chemicals

The test zebrafish (Danio rerio) were obtained from the Institute of Environment and Ecology, Shandong Normal University, China. The stock populations were cultured in our laboratory in circulation system, under temperature of 26 ± 2°C with a photoperiod of 16h light (approximately 4000 lx) and 8h dark conditions. Culture medium was prepared according to the components of the Standard Reference Water. Zebrafish were fed twice a day with flake food (Trea®, Germany) with 8h interval (8:30 am. and 4:30 pm.). Healthy and uninjured adult zebrafish (3 ± 0.2 cm in length, 0.30 ± 0.05 g in weight) were selected randomly from the stock population. Feeding was stopped 24h before the experiment.

As a chemical compound of heavy metal Cd, Cadmium chloride (CdCl₂ ) presents distinctive physicochemical properties, and it can exert the same toxic effects as Cd on aquatic organisms [30]. CdCl₂ was purchased from Chinese standard sample center. Acetylthiocholine iodide (ATCh) and 5, 5-Dithio-2, 2-Nitrobenzoic Acid (DTNB) were purchased from Sigma - Aldrich. Coomassie brilliant blue G-250 and Bovine Serum Albumin (BSA) were purchased from SBH - Bio Corporation. All compounds were technical grade (>95% purity).

Experiment design

In this research, the laboratory conditions were consistent with the culturing room. All determinations were repeated three times. Forty eight hours static exposure experiments were performed using CdCl₂ to assess the environmental stress on zebrafish. According to previous research, LC50 - 48h of CdCl₂ on zebrafish, which is 42.6 mg/L with 95% confidence interval (41.096 - 43.712 mg/L) [9], were taken as one toxic unit (1.0 TU), and four concentrations (Control, 0.1 TU, 1.0 TU, and 2.0 TU) gradients were set up. Control treatments were used to analyze the normal behavior status of zebrafish during 48h. As LC50 - 48h of CdCl₂ on zebrafish, 1.0 TU concentration was always used to assess the acute toxic effects of different chemicals on organisms [18]. As sublethal concentration [31], 0.1 TU Cd²⁺ treatments were used to illustrate behavior responses and the brain AChE acitivity in lower concentration exposure. 2.0 TU Cd²⁺ treatments were used to illustrate behavior responses and the brain AChE acitivity in higher concentration exposure than 1.0 TU.

Forty eight hours exposure test was carried out to assess the effects of Cd on swimming behavior of zebrafish detected by the online behavior monitoring system. Test organisms were divided into four groups. Each group had three fish. The test organisms were placed in a flow-through test chamber (3 cm long, 2cm in diameter), which was closed off on both sides with nylon nets (250 mm) [8]. No food was fed during the tests. Behavior Strength (BS) of swimming behavior is sampled automatically during the exposure every second, and the average BS every 6 min is used to analyze behavioral changes via control database (Figure 1) [32].

Figure 1: Online behavior monitoring system and test chambers.

(a) The signal acquisition of OMS, (b) The normal signal analysis (BS),

(c) The signal analysis after Fast Fourier Transform and (d) test chambers.

The brain AChE activities in the different treatments were investigated. Fifty test organisms without feeding 24h before the experiment were exposed to Control, 0.1 TU, 1.0 TU, and 2.0 TU, respectively. Sampling time was set up as 0h, 0.5h, 1h, 2h, 4h, 8h, 16h, 32h, and 48h from the beginning of the exposure. The AChE activities in the homogenates were detected as followed: 50 μL enzyme and 50 μL ATCh (5 mm final concentration) were incubated at 30°C for 15 min in a final volume of 0.1 mL, and then the reaction was stopped by 0.125 mm DTNB-phosphate-ethanol reagent inside 0.9 ml (12.4 mg of DTNB dissolved in 125 mL 95% ethanol, 75 mL distilled water, and 50 mL 0.1 M phosphate buffer, pH 7.5) as the thiol indicator. OD value was measured by UV spectrophotometer at the wavelength of 412nm UV spectrophotometer OD values [33,34]. In this study, the brain AChE activity of zebrafish was detected according to the Bradford Protein Assay in the unit of nmol/min•mg [35,36].

Data analysis

All data are analyzed statistically in MATLAB environment (MATLAB 2010, © 1984-2009 The MathWorks, Inc.). The behavior data were analyzed by a three dimensional surface plotSurf (X, Y, Z). Surf (X, Y, and Z) creates a shaded surface, in which surface height Z stands for zebrafish BS, X stands for exposure time, and Y stands for Cd²⁺ concentration. In the brain AChE activity analysis, the relative AChE activity (% of each treatment at the beginning of the exposure) was used to analyze the toxic effects of Cd²⁺ on the AChE activity. As Detrended Cross-Correlation Analysis (DCCA) could be applied to quantify the correlation level of time series data [37], the correlation analysis between the brain AChE activity and BS of zebrafish in different Cd²⁺ treatments was analyzed using DCCA in MATLAB environments.

Results and Discussion

Behavior response of zebrafish

The behavior responses of zebrafish under the Cd²⁺ stress were shown in Figure 2.

Figure 2: Behavior responses of zebrafish exposed to Cd²⁺.

No obvious changes of BS were observed in the control group, which changed from 0.65 to 0.85. In different Cd²⁺ treatments, zebrafish BS changed from 0.15 to 0.70, which suggested an obviously lower result than in the control group. In 1.0 TU and 2.0 TU treatments, the swimming behavior showed evident up and-down variation tendency. But it showed obviously fluctuations in early period, and then followed by continuous decrease with no adaption ability because of the higher Cd²⁺ concentration in 5.0 TU, which showed similar behavior responses of Daphnia magna in other environmental stress [38].

In 0.1 TU Cd²⁺ treatments, which were regarded as sublethal concentration [31], the behavior responses of zebrafish showed some special characteristics: In the beginning of the exposure, BS was about 0.6 with an increase to about 0.7, a decrease to about 0.55 and then an increase to about 0.65 until the end of the exposure. The swimming behavior of zebrafish in 0.1 TU Cd²⁺ treatment showed evident stepwise behavior responses as described by Zhang et al. in 2013 [23], which included behavior stimulation, acclimation, (re-) adjustment and toxic effects.

Totally, some abnormal phenomena of zebrafish behavior responses in Cd²⁺ treatments could be recorded by the online behavior monitoring system, such as larger magnitude of BS (from 0.15 to 0.70) and the up and down behavior responses, especially the gradual BS increase from the beginning of the exposure, which could be illustrated by the avoidance behavior when aquatic organisms were under threaten of environmental stress [39]. Meanwhile, the behavior responses of zebrafish in Cd²⁺ treatments showed an evident dose- and time- effect relationship in lethal environmental stress, and stepwise behavior responses could be observed in sublethal environmental stress. These results suggested that behavior responses of zebrafish are sensitive to the changes of water quality, and BS could provide a reliable and real-time method for online assessment of aquatic heavy metal pollution [16].

AChE activity of zebrafish

Based on the relative values of each treatment at the beginning of the exposure (0h), the brain AChE activities of zebrafishin 48h Cd²⁺ exposure were shown in Figure 3.

Figure 3: Relative brain AChE activity of zebrafish exposed to Cd²⁺. Showed as M ± S.D., *p<0.5, ** p<0.01.

The overall brain AChE activity showed a general downward trend in 48h exposure. At the beginning, AChE activity decreased due to Cd²⁺ stress, and AChE activity was inhibited evidently in different concentrations after a period of exposure, which suggested that the trend was similar to the behavior responses as shown in Figure 2.

Based on the results shown in Figure 3, the brain AChE activities of zebrafish were strongly inhibited by Cd²⁺: the AChE activities were lower than 60% after 0.5h Cd²⁺ exposure in both 1 TU and 2 TU. The AChE activities in 0.1 TU Cd²⁺ treatment were about 60% in the first 2h and then increased to about 100% in 4h with a decrease tendency in the following exposure time (8h to 48h), which changed from 100% to 70% - 80%. Therefore, it was advised that the relative brain AChE activities changed with both exposure time and Cd²⁺ concentration, which suggested that the brain AChE activity can be used to evaluate Cd²⁺ stress as previous report [40].

Correlation analysis between BS and brain AChE activity

Previous studies have proved that AChE activity inhibition is correlated with the disorder of swimming behavior [41]. Cd²⁺ could affect AChE activity and Acetylcholine (ACh) synthesis rate; finally induce the collapse of nervous system [31]. In view of this, AChE inhibition could illustrate the intrinsic response mechanism of zebrafish.

In order to analyze correlation between BS and brain AChE activity of zebrafish in Cd²⁺ stress assessment, the DCCA were used based on the correlation coefficient (r) and significance (p). According to Ren et al., [41], r<0.3 means poor correlation, 0.30.5 means high correlation. So the correlation coefficient r was first checked to analyze how much they correlated. Then, p values were checked to see the significance between two variables, in which p<0.05 means significant correlation and p<0.01 means extreme significance. When r is absolutely higher than 0.5 with p<0.05, it means a significant correlation.

Figure 4: Cross correlation analysis of BS values and the brain AChE activity in different Cd²⁺ treatments.

In Figure 4, the cross correlation results using DCCA showed the relationship between BS values and the relative brain AChE activity of zebrafish in different Cd²⁺ treatments. The correlation could be illustrated by the regression line equation (1) with p = 2.916*e-07 and r = 0.737:

y = 0.004x + 0.241      (1)

This linear regression with the correlation coefficient (r>0.5) and significance (p<0.01) indicated that BS and the brain AChE activity of zebrafish exposed to Cd²⁺ exposure were highly correlated with extremely significance, which suggested that behavior disorder of zebrafish might be affected mainly by the inhibition of the nerve conduction, in which the AChE is the main neurotransmitter as reported [13]. These results could also be supported by previous researchers [4,14], which illustrated that Cd²⁺ could cause great damage to the transduction signal and then neurotoxicity due to the AChE activity inhibition.

AChE plays a major regulatory role in multiple physiological processes [42]. The activity of the cholinergic system plays an important role in normal behavior and muscle function [43]. In this study, our experimental results show a significant correlation between behavioral changes and brain AChE activity. The present study is consistent with other findings, whicn indicate that Cd²⁺ can affect the AChE activity of zebrafish and lead to change the behavior, which is consistent with previous findings on zebrafish gills [6]. Similar observations were found in the study by Zhang et al. [31], which show that there is a significant dose-effect and time effect at 1.0 TU and 2.0 TU exposure concentrations. It is also found that AChE activity is inhibited almost simultaneously in the brain, gill, muscle and liver under these two higher environmental stresses, whereas it shows a time lag in sublethal treatment (0.1 TU). The inhibition of AChE can also cause behavioral abnormalities. In addition, there are some researchers who have studied organophosphorus pesticides. The results showed that the inhibition of AChE activity may also lead to the loss of nerve conduction ability, eventually causing different types of behavioral changes [8]. Other experiments have also been conducted on rat pups with malathion. The experiment has shown that the AChE activity in the brain of the rat pups is inhibited and the locomotor activity of rat pups exposed to malathion decreased. It demonstrated that the AChE activity in the brain of rat pups exposed to malathion is involved in the change of the behavior [44].

Cd is a neurotoxin metal that has some enrichment in the organism and is not biodegradable. The organisms exposed to Cd have been observed to be barriers to behavioral and biochemical functions [13]. After sub-lethal doses of Cd treatment, AChE in the zebrafish brain and muscle was significantly inhibited and the extent of inhibition varied with the change of time and concentration [45].

Conclusion

As it is reported in Almeida et al., [46] Cd²⁺ is a high toxic poison to zebrafish and has a good dose-effect relationship. In this research, the toxic effects of Cd²⁺ were decided by concentrations and exposure time, which suggested that both dose and time effect relationships existed between Cd²⁺ stress and zebrafish BS and brain AChE activity (1.0 TU and 2.0 TU), while a process of adaptation, adjustment and readjustment of zebrafish BS and brain AChE activity was observed under sub-lethal conditions stress (0.1 TU). The correlation analysis using DCCA showed that BS and the brain AChE activity of zebrafish were highly correlated with extremely significance in Cd²⁺ stress, which suggested that behavior disorder of zebrafish might be affected mainly by the inhibition of the nerve conduction.

Cd²⁺ can enter the aquatic environment possibly through sludge and agricultural runoff, or through other industrial processes [45]. The limitation of Cd²⁺, which is a by-product of mining, forging and other industrial process, is 0.01mg/L in discharged wastewater according to the Environmental Quality Standards for Surface Water in China [47]. However, Cd²⁺ concentration in some discharged wastewater could reach 26 mg/L [48], which induce some heavy Cd²⁺ pollution in some place in China (359.8g/kg) [49]. Therefore, it is necessary to realize the ecotoxicological assessment of heavy metal Cd²⁺ pollution. As the brain AChE activities of zebrafish showed similar rules with BS after correlation analysis, the results in this study might provide an understanding of the ecotoxicological assessment of heavy metal Cd²⁺ based on zebrafish.

Acknowledgements

This study was financially supported by the Natural Science Foundation of China (21107135), the High-level Talent Project of Shandong Normal University, the Taishan Leader Talent Project of Shandong (tscy20150707) and the Oversea High-level Talent Project of Ji’nan (2013041).

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