Regulation of GeCu/Zn-SOD, GeMn-SOD, GeHsp90, and GeMT in Gymnocypris eckloni in Response to Copper and Lead Ion Challenges

Zhao, Jing; Li, Zixuan; Li, Changzhong; Wang, Rong; Zhao, Zifeng; Gou, Huayu; Li, Lanying; Xie, Anbin; Ren, Haotian; Qiu, Bo; Li, Xiaodie; Chen, Yanxia; Wang, Zhenji; Wang, Guojie; Jin, Wenjie

doi:https://doi.org/10.1155/2024/5716920

Journal of Applied Ichthyology

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Abstract Introduction Materials and Methods Results Discussion Conclusion Data Availability Ethical Approval Conflicts of Interest Authors’ Contributions Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2024 | Article ID 5716920 | https://doi.org/10.1155/2024/5716920

Regulation of GeCu/Zn-SOD, GeMn-SOD, GeHsp90, and GeMT in Gymnocypris eckloni in Response to Copper and Lead Ion Challenges

Jing Zhao,¹Zixuan Li,¹Changzhong Li,¹Rong Wang,¹Zifeng Zhao,¹Huayu Gou,¹Lanying Li,¹Anbin Xie,¹Haotian Ren,¹Bo Qiu,¹Xiaodie Li,¹and Yanxia Chen¹ et al.

Academic Editor: Mohamed Abdelsalam

Received06 Oct 2023

Revised13 Feb 2024

Accepted21 Feb 2024

Published07 Mar 2024

Abstract

The contamination of aquatic environments by heavy metals can have detrimental effects on fish, impacting their growth and overall health, including the regulation of antioxidant genes. An investigation was carried out to assess the distribution and habitat of Gymnocypris eckloni in the Yellow River basin. Simultaneously, heavy metal concentrations in its habitat and in selected locations within the upper Yellow River were measured. In an effort to explore the potential roles of specific genes in antioxidant responses, G. eckloni was exposed to low concentrations of copper (Cu²⁺) and lead (Pb²⁺) for varying durations (12, 24, and 48 hours). The mRNA levels of GeCu/Zn-SOD, GeMn-SOD, GeHsp90, and GeMT were quantified in the gills, kidneys, and liver through qRT-PCR. The findings suggest that the habitat of G. eckloni is generally safe; however, occasional exceedances of safety standards could pose a potential threat to its growth. Importantly, the expression of GeCu/Zn-SOD, GeMn-SOD, GeHsp90, and GeMT exhibited responses to the low concentrations of copper-induced and lead-induced stress. Notably, GeCu/Zn-SOD, GeMn-SOD, and GeMT demonstrated heightened sensitivity to lead compared to copper. Furthermore, the expression of these genes displayed tissue-specific responses under identical metal stress conditions. These results indicate that GeCu/Zn-SOD, GeMn-SOD, GeHsp90, and GeMT genes have the potential to serve as early, sensitive biomarkers for the detection of metal toxicity induced by Cu²⁺ and Pb²⁺. This study also provides valuable insights into the functioning of antioxidant genes under oxidative stress in fish.

1. Introduction

Gymnocypris eckloni Herzenstein, a freshwater fish, is a member of the Gymnocypris genus within the Schizothoracinae subfamily. These fish, native to the Qinghai-Tibet Plateau, are primarily found in the upper reaches of the Yellow River, including locations such as Zhaling Lake, Erling Lake, and the Naqi River basin in the Qaidam basin [1, 2]. Unfortunately, due to human activities and man-made hydroelectric projects, the habitat of G. eckloni has suffered significant damage, resulting in a reduction in its natural distribution [3]. In an effort to mitigate the decline in the wild population of G. eckloni, successful artificial reproduction of this species has been carried out in Qinghai province, China, since 2008.

Heavy metals, characterized by their high density, exceeding five times that of water [4], encompass elements such as lead (Pb), cadmium (Cd), zinc (Zn), copper (Cu), mercury (Hg), silver (Ag), chromium (Cr), ferrum (Fe), and more. Prolonged or sudden exposure to heavy metal pollution in water can significantly impact fish physiology, growth, reproduction, and even population dynamics [5, 6]. Fish can accumulate heavy metals within their bodies through predator-prey relationships, resulting in the degeneration of gill fibers and stem cells. X-ray examinations have revealed that heavy metal stress induces slight curvature of the fish’s spine and reduces calcium and phosphorus levels, leading to bone deformities [7]. Through the food chain, heavy metals can accumulate in fish, birds, and mammals and even pose risks to human health [8].

Biomarkers are more sensitive than general biological detection indexes. Thus, the key genes or gene clusters of algae, plankton, and fish can be tested to detect the health status of aquatic organisms and infer the true state of water quality. Metallothionein (MT), as a biomarker of heavy metal pollution in aquatic organisms, is a heavy metal binding protein [9, 10]. In general, the detoxification of MT depends to a large extent on its affinity with heavy metals in the following order: silver > copper > cadmium > lead > zinc > cobalt > iron [11, 12]. Superoxide dismutases (SODs) are a class of antioxidant enzymes, which can catalyze the disproportionation of superoxide anion free radicals to balance the oxygen free radicals in the body [13]. Cu/Zn-SOD and Mn-SOD are the important components in the antioxidant system of aquatic animals [14]. SOD activity was enhanced under heavy metal stress, which is a self-protecting mechanism of the body and can minimize oxidative stress injury [15]. The concentration of Cu ions in the feed affected not only the growth of fish but also its antioxidant capacity, which demonstrated that higher Cu ions in the feed of Larimichthys croceus reduced Cu/Zn-SOD activity and total antioxidant capacity in the livers [16]. When the content of copper in the feed decreased, the Cu/Zn-SOD activity in the Ictalurus punctatus liver decreased significantly [17]. Heat shock proteins (HSPs) are a family of protein molecules produced by cells under heat induction and other external stimuli [18, 19]. According to amino acid sequences, molecular weights, and functions, HSPs can be divided into several major families: HSP110, HSP90, HSP70, HSP60, and several small HSPs [20]. HSPs can not only respond to high-temperature stress but also be induced to express by environmental factors that may cause protein damage [21].

The heavy metals in the Yellow River basin are mainly accumulated in the sediments of lakes and rivers entering the lakes. Copper and lead are the most common heavy metals in the water, bottom mud, soil, and animals and plants in the basin. In this study, we conducted an assessment of key water quality parameters, dissolved oxygen, pH, and Cu²⁺ and Pb²⁺ at 11 monitoring sites situated in the upper reaches of the Yellow River within Qinghai province. Moreover, we selected concentrations of 0.01 mg/L of Cu²⁺ and 0.05 mg/L of Pb²⁺ in accordance with the China Water Quality Standard for Fisheries (GB 11607-1989) to expose Gymnocypris eckloni (G. eckloni) to these heavy metals. Subsequently, we examined the expression levels of GeCu/Zn-SOD, GeMn-SOD, GeHsp90, and GeMT in the gills, liver, and kidneys of these fish. The findings of this study indicate that the responses of fish to heavy metal stress, involving various regulatory mechanisms at different concentration levels, offer a more accurate representation of how aquatic organisms react to low-dose heavy metal pollutants. This approach holds a practical significance for early warning systems aimed at monitoring heavy metal pollution in water quality and the environment.

2. Materials and Methods

2.1. Detection of Heavy Metal Ions in Distribution Areas of G. eckloni

To understand the water quality status of the upper reaches of the Yellow River and its tributaries in Qinghai province, water environment monitoring was conducted in 11 monitoring sites in July every year for the following 4 years (Figure 1).

At the beginning of July, water quality parameters (dissolved oxygen and pH) were periodically measured using a CTD probe (Ocean Seven 310 CTD; Idronaut, Brugherio, Italy). At each sampling point, three samples were taken and placed in high-density polyethylene bottles, which were then taken back to the laboratory and stored in a refrigerator at 4°C. Determination of copper and lead ion concentration was performed by an atomic absorption spectrometer (ZEEnit 700P, Germany) within 24 h according to the China Water Quality Standard for Fisheries (GB 11607-1989). The Cu²⁺ standard solution (GNM-SCU-002-2013, 100 μg/ml with 5% HNO₃) (National Center for Analysis and Testing of Nonferrous Metals and Electronic Materials, China) was prepared with 2 mg/L mother solution and diluted in a concentration gradient of 0, 0.001, 0.002, 0.005, 0.010, and 0.020 mg/L, respectively. The Pb²⁺ standard solution (GNM-SPB-002-2013, 100 μg/ml with 5% HNO₃) (National Center of Analysis and Testing for Nonferrous Metals and Electronic Materials, China) was prepared with 2 mg/L mother solution and diluted in a concentration gradient of 0, 0.005, 0.010, 0.020, 0.050, and 0.100 mg/L, respectively.

2.2. Animals

In September, 120 three-year-old Gymnocypris eckloni, weighing 100 ± 2.5 g and 19 ± 3.4 cm in length, were obtained from the Qinghai Provincial Fishery Environmental Monitoring Center, Xining, China. The fish were kept in the tank (L: 48.0 cm, W: 25.0 cm, and H: 45.5 cm), and 40 L of 15 ± 0.5°C aerated dechlorinated tap water was put in, the water was changed every 12 hours, and the fish were fed once a day, and the light and dark cycle was maintained for 14 h/10 h.

2.3. Heavy Metal Stress

CuSO₄·5H₂O was purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA). Pb(NO₃)₂ was supplied by Sinopharm Chemical Reagent Co. (Shanghai, China). 1000 mg/L Cu²⁺ or Pb²⁺ solution was prepared using double-distilled water, and the working liquid of 0.01 mg/L Cu²⁺ and 0.05 mg/L Pb²⁺ was prepared using 40 L aerated and dechlorinated tap water, respectively.

After seven days of acclimatization to the laboratory environment, 90 healthy fish were randomly assigned to nine groups. Three groups were selected as controls, while the other six groups suffered from heavy metal stress, 0.01 mg/L Cu²⁺ or 0.05 mg/L Pb²⁺. Each pressure group had three repeats of 10 fish at a time. After heavy metals were treated for 12, 24, and 48 h, the gills, liver, and kidneys were dissected immediately following ether anesthesia (euthanasia) and stored at −80°C until use.

2.4. Heavy Metal Analysis

0.2 g (accurate to 0.001 g) of frozen tissue samples was put into microwave digestion tubes, and 6 ml of HNO₃ and 2 ml of H₂O₂ were added to each tube. Put them into a microwave digestion system (MASTERr-40, China) for 10 min at 150°C and then for 25 min at 180°C and allow to cool to room temperature. After cooling, remove the digestion tubes and drive the acid to 1 ml at 150°C on the acid drive meter. The digestion liquid was transferred to a 25 ml volumetric bottle in which they were diluted using ultrapure water 2-3 times. Following acid digestion, all the samples were analyzed with AAS (AA-6880, China). The excitation wavelength of the lead ion was 283.3 nm, and the excitation wavelength of the copper ion was 324.8 nm.

2.5. Expression of GeCu/Zn-SOD, GeMn-SOD, GeHsp90, and GeMT

The GenBank accession numbers and reference of primers are given in Table 1. qPCR was conducted using the LightCycler® 96 (Roche, Basel, Switzerland) in a final volume of 25 μl containing 1 μl (10 μM) each of forward and reverse primer, 12.5 μl of SYBR Premix Ex Taq II (2×), 2 μl of cDNA, and 8.5 μl of ddH₂O. The relative abundance of genes was normalized by the expression of β-actin [22] and calculated using the 2^−ΔΔCt method.

2.6. Statistical Analyses

Expression levels of GeCu/Zn-SOD, GeMn-SOD, GeHsp90, and GeMT were assessed using one-way ANOVA followed by an LSD test with SPSS statistics 17.0 software. All data were represented as mean ± SD as shown in the figures. A probability value of 0.05 or below was considered significant.

3. Results

3.1. Detection of Copper and Lead Ions in the Distribution Area of G. eckloni

The detection data of multiple heavy metal ions from 11 monitoring stations in the distribution area of G. eckloni in Qinghai province (Table 2) showed that the measured values of Cu²⁺ and Pb²⁺ had little changes and were all within the maximum range of the China Water Quality Standard for Fisheries (GB 11607–1989, the permitted maximal value is Cu²⁺ ≤ 0.01 mg/L and Pb²⁺ ≤ 0.05 mg/L). In general, the dissolved oxygen and pH values of the 11 test sites meet the requirements of the national fishery and aquaculture standards, but the concentrations of copper and lead ions in a few test sections occasionally approach or exceed the requirements of the national fishery and aquaculture standards. For example, the maximum concentration of Pb²⁺ in Zhaling Lake is 0.051 mg/L, which is slightly higher than the GB 11607-1989 of no more than 0.05 mg/L. The Pb²⁺ is 0.044 mg/L in the Jishixia Reservoir tailwater, which is close to the GB 11607-1989. In addition, the highest concentration of Cu²⁺ in the Laxiwa Reservoir tailwater reached 0.01 mg/L of the GB 11607-1989, while the concentration of Cu²⁺ in Zhaling Lake, Erling Lake, Maduro Yellow River Bridge, and Gongboxia Reservoir area was at risk of approaching the GB 11607-198.

3.2. Detection of Copper and Lead Ion Content in Experimental Water and Fish

In order to better understand the transcriptional response to Cu²⁺ stress and Pb²⁺ stress by Gymnocypris eckloni, we introduced Cu²⁺ and Pb²⁺ to the aerated and dechlorinated water at a theoretical Cu²⁺ concentration of 0.01 mg/L and Pb²⁺ concentration of 0.05 mg/L. Given that Cu²⁺ and Pb²⁺ may be present in water, resulting in an actual value that differs from the theoretical value, we employed an atomic absorption spectrometer to measure Cu²⁺ and Pb²⁺ concentrations in water of both the experimental and control groups. The results showed that the concentrations of Cu²⁺ and Pb²⁺ were 0.0085 ± 0.0001 mg/L and 0.0019 ± 0.0002 mg/L, respectively, in the blank group, whereas 0.01 mg/L of Cu²⁺ solution and 0.05 mg/L of Pb²⁺ solution contained an actual concentration of 0.0119 ± 0.0006 mg/L and 0.0501 ± 0.0001 mg/L (Figure 2).

(a)

(b)

(c)

(d)

Similarly, in order to further determine the content of copper and lead ions in G. eckloni, AAS results showed that the content of Cu and Pb ions in the gills, kidneys, and liver at 12 h, 24 h, and 48 h were close to 0.01 mg/kg and 0.05 mg/kg (Table 3).

3.3. Expression of GeCu/Zn-SOD after Cu²⁺ stress and Pb²⁺ Stress

In the gills, as shown in Figure 3, GeCu/Zn-SOD’s initial response was less intense after Cu²⁺ stress but increased significantly at 24 h (n = 3, ). However, it increased rapidly and substantially under Pb²⁺ stress, especially at 12 h when the expression was 24.18-fold higher than that in the control group. In the kidney, the expression of GeCu/Zn-SOD under Cu²⁺ stress for 48 h and Pb²⁺ stress for 24 h was 70.65 and 11.74 times lower than that of the control group, respectively, and the expression of GeCu/Zn-SOD was significantly upregulated and reached the maximum under Pb²⁺ stress for 48 h (n = 3, ). In the liver, the expression of GeCu/Zn-SOD decreased to varying degrees after Cu²⁺ stress, while under Pb²⁺ stress, GeCu/Zn-SOD expression increased first, then decreased rapidly, and then significantly upregulated to the maximum (n = 3, ).

In general, the response rate of GeCu/Zn-SOD was the fastest in the liver and kidney, and the response intensity of GeCu/Zn-SOD to Pb²⁺ was stronger than that of Cu²⁺ stress in the gills, kidneys, and liver. Interestingly, compared with the control group, the expression of GeCu/Zn-SOD was sharply downregulated after Cu²⁺ stress for 48 h, but it was sharply downregulated earlier to 24 h under Pb²⁺ stress.

3.4. Expression of GeMn-SOD after Cu²⁺ stress and Pb²⁺ Stress

In the gills, as shown in Figure 4, the expression of GeMn-SOD was slow after Cu²⁺ stress, while it was upregulated rapidly and strongly after Pb²⁺ stress and reached a peak at 48 h (n = 3, ). In the kidney, the expression of GeMn-SOD initially increased and then decreased after Pb²⁺ stress and Cu²⁺ stress, but it was an abnormally high expression and reached the highest value after 48 h of Pb²⁺ stress (n = 3, ). In the liver, the response of GeMn-SOD was slow and significantly downregulated and reached the lowest value after 48 h of Cu²⁺ stress (n = 3, ), while the response was sharply downregulated after 24 h of Pb²⁺ stress and recovered to normal level after 48 h. In general, the response strength and speed of GeMn-SOD to Pb²⁺ stress were superior to that of Cu²⁺ stress.

3.5. Expression of GeHsp90 after Cu²⁺ stress and Pb²⁺ Stress

In the gills, as shown in Figure 5, the expression of GeHsp90 was first significantly upregulated and significantly downregulated after Cu²⁺ stress (n = 3, ) and then recovered to the normal level, while the expression of GeHsp90 after Pb²⁺ stress was opposite to that after Cu²⁺ stress. In the kidney, the expression of GeHsp90 increased first, then decreased, and finally upregulated after Pb²⁺ stress and Cu²⁺ stress. In the liver, the expression of GeHsp90 was increased gradually and reached a peak at 48 h after Cu²⁺ stress, while the expression was first significantly downregulated and significantly upregulated after Pb²⁺ stress, and it was significantly lower than that in the blank control group (n = 3, ). In general, GeHsp90 has a strong response to both Cu²⁺ stress and Pb²⁺ stress. In addition, the expression line of GeHsp90 in the gills and kidneys was upregulated and downregulated after Cu²⁺ stress, while it was continuously upregulated in the liver.

3.6. Expression of GeMT after Cu²⁺ stress and Pb²⁺ Stress

In the gills, as shown in Figure 6, the expression of GeMT was first significantly upregulated to reach the peak and significantly downregulated and then significantly upregulated to a state higher than that of the blank control group after Cu²⁺ stress (n = 3, ). After Pb²⁺ stress, the expression of GeMT was mild, upregulated at 12 h, reached the peak at 24 h, and recovered to normal after 48 h. In the kidney, the expression of GeMT was upregulated significantly after Cu²⁺ stress and Pb²⁺ stress, respectively. In the liver, GeMT expression was significantly downregulated after Cu²⁺ stress but significantly upregulated after Pb²⁺ stress (n = 3, ). In general, the response strength of GeMT in gills to Cu²⁺ stress was greater than that to Pb²⁺ stress. However, the response intensity of GeMT to Cu²⁺ stress and Pb²⁺ stress in both the kidney and the liver was very high, but the response mode was different. For example, the expression of GeMT in the kidney was significantly upregulated after Cu²⁺ stress and Pb²⁺ stress, while the expression of GeMT in the liver was significantly downregulated after Cu²⁺ stress. In addition, the expression of GeMT after Pb²⁺ stress was opposite to that after Cu²⁺ stress.

3.7. Temporal and Spatial Expression Patterns of SODs, HSP90, and MT after Cu²⁺ stress and Pb²⁺ Stress

The expression of GeCu/Zn-SOD in the gills of the blank control group was taken as a reference value, and the expressions of GeCu/Zn-SOD, GeMn-SOD, GeHsp90, and GeMT in different tissues were analyzed (Figure 7). It was found that the response speed of GeCu/Zn-SOD in the liver was the fastest. The response speed and intensity of GeCu/Zn-SOD to Pb²⁺ in the gills, kidneys, and liver were superior to Cu²⁺ stress. The response intensity and response speed of GeMn-SOD to Pb²⁺ stress were superior to Cu²⁺ stress, except that the response speed of GeMn-SOD to Cu²⁺ was faster than that of Pb²⁺ in the kidney. The response intensity of GeHsp90 to Cu²⁺ and Pb²⁺ was very strong. In addition, the expression of GeHsp90 was upregulated and downregulated in the gills and kidneys after Cu²⁺ stress, while the expression of GeHsp90 was continuously upregulated in the liver. The response of GeMT to Cu²⁺ stress was stronger than that to Pb²⁺ stress. In the kidney and liver, the response intensity of GeMT to Cu²⁺ stress and Pb²⁺ stress was very high, but the response mode was different.

4. Discussion

In this study, the 11 sampling sites in the Qinghai region of the upper reaches of the Yellow River involve the source segment with an average altitude of 4000 m and the gorge segment with an average altitude of 2000–4000 m. Monitoring sites no. 1–3 are located in the source section of the river, where there are few human activities, no industrial production activities, and the pollution of agricultural production to the water body is weak; thus, Cu²⁺ and Pb²⁺ mainly come from natural sources such as soil parent material. Starting from monitoring site no. 3, the surrounding terrain of the downstream monitoring site is dominated by valleys and cliffs, coupled with urbanization, reservoir construction, and weathering rock becoming a new source of pollution, and other factors that lead to dynamic changes of heavy metal ions in the river channel [25]. The dissolved oxygen level increases from upstream to downstream, and there may be no. 1–3 sample sites located in the source region of the Yellow River, with an average elevation of 4500 m, low temperature, slow water exchange, thin air, and relatively low dissolved oxygen level [26]. From no. 4 to no. 11 monitoring points, the altitude decreases, the oxygen content in the air increases, the water column drop is large, the water velocity is fast, and the dissolved oxygen level increases correspondingly. Overall, the detection data indicated that the measured values were all within the China Water Quality Standard for Fisheries (GB 11607-1989), which had little effect on aquatic organisms. However, some monitoring sites occasionally approach or exceed the standard. For example, the maximum concentration of Pb²⁺ in Zhaling Lake was 0.051 mg/L, and the content of Pb²⁺ in the tailwater of Jishixia Reservoir was 0.044 mg/L. In addition, the concentration of Cu²⁺ in the tailwater of Laxiwa Reservoir reaches up to 0.01 mg/L, and the concentration of Cu²⁺ in Zhaling Lake, Erling Lake, Maduro Yellow River Bridge, and Gongboxia Reservoir area was in danger of approaching GB 11607-198. This may be caused by pollutants entering rivers through identifiable point sources such as urban and industrial wastewater and diffusion sources closely related to meteorological factors such as surface runoff, erosion, and atmospheric deposition [27, 28]. These results indicate that heavy metal pollution exists in the upper reaches of the Yellow River, and heavy metal concentrations must be measured regularly to reduce the risk of chronic poisoning in the future. In addition, as an indigenous fish living in water, the behavior and physiological changes of G. eckloni can also directly reflect whether heavy metals in the effluent environment exceed the standard. However, the adaptive regulation mechanism of G. eckloni to abnormal changes of heavy metal ions in the water environment and the screening of biomarker molecules that can be used for early warning still need further systematic studies.

Traditional toxicology often uses semilethal and lethal concentrations to determine heavy metal stress concentrations and explore the impact of heavy metals on fish. However, before the concentration of heavy metals reaches the semilethal concentration, genes such as the molecular partner, metal chelation, and immune response are already activated in the fish. When the lethal concentration is reached, the safety of the aquatic ecosystem and the growth of aquatic organisms are seriously affected, indicating that the signal molecules screened by traditional detection methods could not provide accurate warning signals for the protection of the aquatic ecosystem. Here, 0.01 mg/L of Cu²⁺ and 0.05 mg/L of Pb²⁺ concentrations were selected according to the GB 11607-1989 to treat G. eckloni, and the expression of genes related to antioxidant stress (GeCu/Zn-SOD, GeMn-SOD, and GeHsp90) and metal chelation (MT) was detected.

The activity of Cu/Zn-SOD in an organism tends to decrease under severe stress but increases under mild stress [29, 30]. The expression of Cu/Zn-SOD in the brain of fish was decreased after 0.60 mg/L of Cu²⁺ stress for 4 d [31]. On the contrary, the expression of Cu/Zn-SOD was increased in Carassius auratus gibelio after 0.005 mg/L or 0.050 mg/L of Cu²⁺ stress [32]. A similar phenomenon was observed in Daphnia magna, where Cu/Zn-SOD expression was increased when exposed to the mixture of copper, unionized ammonia, and low dissolved oxygen for 48 h [33]. Qiang et al. [31] found that Cu/Zn-SOD expression was increased rapidly and expressed intensively in the gills and liver and under Cu²⁺ stress. Similarly, we found that the expression of GeCu/Zn-SOD was the fastest and more intense in the liver. This is related to the liver as the main detoxification organ, carrying out heavy metal detoxification. Under 0.01 mg/L of Cu²⁺ and 0.05 mg/L of Pb²⁺ stress, the expression of GeCu/Zn-SOD was significantly increased, and the expression of GeCu/Zn-SOD under Pb²⁺ stress was significantly stronger than that under Cu²⁺ stress. Copper may serve as an important element in living organisms and is beneficial to living organisms at the right concentration [34]. However, lead is the nonessential and most toxic metal in the aquatic environment, and even very low concentrations of lead cause immune impairment in Anguilla [35]. Interestingly, after Cu²⁺ stress for 48 h and Pb²⁺ stress for 24 h, GeCu/Zn-SOD expression was significantly downregulated compared with the control group. Similar results were also observed in Esomus detritus [36]. These results may be related to Cu²⁺ promoting ROS overproduction and destroying SOD enzyme through the Fenton reaction [37]. Overall, Cu²⁺ stress and Pb²⁺ stress elicited oxidative stress, and the expression of Cu/Zn-SOD can protect organisms against future severe stress situations and it was more strongly under Pb²⁺ than Cu²⁺. Also owing to the accumulation effect of heavy metals in fish being different, the expression of Cu/Zn-SOD shows certain tissue specificity.

Mn-SOD is one of the members of the first line of defense to remove ROS. Its expression and regulation play a crucial role in the maintenance of equilibrium redox homeostasis [38]. Studies have shown that the mRNA level of Mn-SOD generally increases in fish and mammals during metal ion or heat stress [39–41]. Under Cu²⁺, Cd²⁺, or Pb²⁺ stress, the expression of Mn-SOD was significantly upregulated in the gills, liver, and kidneys and showed a certain time dependence [14, 41, 42]. Similar results were found in this study, where both GeCu/Zn-SOD and GeMn-SOD were susceptible to Cu²⁺ and Pb²⁺, and the response rate of GeCu/Zn-SOD and GeMn-SOD in the liver was faster than that in the gills and kidneys, which indicated that SODs could be useful as possible biomarkers for monitoring heavy metal contamination [43].

Heat shock proteins (HSPs) have been widely found to play an important role in protecting organisms from heat stress through combining heat shock factor 1 (HSF1) with heat shock elements (HSEs) to upregulate HSP90 [44, 45]. Meanwhile, the expression of HSP90 is regulated by salinity, reduced oxygen level, food scarcity, and heavy metals [44–46]. Interestingly, according to earlier research, HSP90 expression showed a time and concentration dependence under stress conditions. For example, after Cu²⁺ stress and Cd²⁺ stress for 30 days, Hsp90 mRNA approximately increased and reached maximum levels at 10 days and 15 days, respectively [47]. Similarly, previous studies indicate that the expression of HSP90 under salt stress was upregulated from 0 to 40 ppt and reached a peak at 40 ppt in the muscle of Huso dauricus [44]. Here, GeHsp90 expression was upregulated in the liver after 0.01 mg/L of Cu²⁺ stress, which consists of the previous study that significantly upregulated in the liver of fish after exposure to Cr⁶⁺ [48]. While, GeHsp90 expression in the kidney was upregulated and downregulated after reaching the peak after 0.05 mg/L of Pb²⁺ stress. In addition, compared with Cu²⁺ stress, the expression of GeHsp90 mRNA was more obvious in the gills, liver, and kidneys after Pb²⁺ stress. HSP90 mRNA expression after heavy metal ion stress has certain tissue specificity and concentration dependence.

Metallothioneins (MTs) are small proteins rich in cysteine amino acids, which are responsible for the regulation and transportation of metal ions such as cadmium, copper, and zinc in living organisms [49]. Research has found that MT plays a crucial role in regulating essential metal levels within cells, preventing metal toxicity, and carrying out necessary physiological functions such as antioxidant activity and immune modulation [50]. In addition, MT expression has been associated with different diseases, such as cancer, cardiovascular ailments, and neurodegenerative disorders [51]. Cadmium (Cd) is known to be one of the most potent inducers of metallothioneins (MTs) in various organisms including fish and has long been used as a model metal for studying MT induction [52]. Studies conducted in Kryptolebias marmoratus, a species of hermaphroditic fish, have shown that exposure to trace levels of cadmium can cause a strong induction of MT expression in a dose-dependent manner [53]. Here, the response strength of GeMT in the gills to Cu²⁺ stress was greater than that to Pb²⁺ stress. However, the response intensity of GeMT to Cu²⁺ stress and Pb²⁺ stress in both the kidney and the liver was very high, but the response mode was different. In general, the response of fish to heavy metal exposure varies depending on many different factors, including the species of fish, the duration and degree of exposure, as well as other environmental factors such as water temperature, pH, and dissolved oxygen levels [54]. Different species of fish may have different sensitivities to heavy metals and may respond differently to exposure, and the same species of fish may respond differently depending on the duration and degree of the exposure.

5. Conclusion

The objective of this research was to comprehensively evaluate water quality and heavy metal concentrations in the upper reaches of the Yellow River and its tributaries in Qinghai province. In addition, we sought to investigate the expression patterns of GeCu/Zn-SOD, GeMn-SOD, GeHsp90, and GeMT in response to exposure to 0.01 mg/L of Cu²⁺ and 0.05 mg/L of Pb²⁺ within the tissues of Gymnocypris eckloni (G. eckloni). Our findings reveal that, despite occasional instances of Cu²⁺ and Pb²⁺ levels exceeding recommended limits, the overall water quality of the Yellow River meets the Water Quality Standard. In response to Cu²⁺ and Pb²⁺ exposure, specific genes in G. eckloni were activated to combat oxidative stress. Notably, GeCu/Zn-SOD, GeMn-SOD, GeHsp90, and GeMT displayed heightened sensitivity to heavy metal stress in distinct tissues. Under Cu²⁺ stress, GeCu/Zn-SOD, GeMn-SOD, and GeHsp90 exhibited more pronounced responses in the liver, while GeMT responded predominantly in the kidney. Conversely, under Pb²⁺ stress, GeCu/Zn-SOD and GeMn-SOD showed stronger reactions in the kidney, while GeHsp90 and GeMT displayed greater sensitivity in the liver. Intriguingly, the response of GeCu/Zn-SOD, GeMn-SOD, and GeMT to Pb²⁺ stress exceeded their response to Cu²⁺ stress. These findings indicate that GeCu/Zn-SOD, GeMn-SOD, GeHsp90, and GeMT demonstrate tissue and heavy metal specificity, making them valuable biomarkers for monitoring pollution levels in the Yellow River.

Data Availability

The data used to support the findings of this study are included within the article.

Ethical Approval

All experiments involving Gymnocypris eckloni were conducted in compliance with the relevant regulations of the Science and Technology Ethics Committee of Qinghai University (Approval no. SL-2021029).

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Wenjie Jin, Jing Zhao, Zixuan Li, and Changzhong Li carried out the experiment design, verification, and manuscript writing. Rong Wang, Zifeng Zhao, Huayu Gou, Lanying Li, and Anbin Xie were responsible for water sample monitoring and experimental fish farming. Haotian Ren, Bo Qiu, and Xiaodie Li carried out sample collection and experiment. Yanxia Chen, Zhenji Wang, and Guojie Wang provided experimental animals and provided technical support for the smooth development of the experiment. Jing Zhao, Zixuan Li, and Changzhong Li contributed equally to this paper and are the co-first authors. The authors agreed to data publication and informed about the publisher rules and terms.

Acknowledgments

The authors would like to thank Mr. Zhengang Wang and Mr. Baojun Lu from the Fisheries Environmental Monitoring Station (Xining, China) for their assistance in specimen collection and transportation. The teaching staff and graduate students from the College of Eco-Environmental Engineering, Qinghai University, who participated in this NSFC project, are also greatly appreciated. The research project received financial support from the National Natural Science Foundation of China (NSFC, 31460700) and the Youth Project of the Natural Science Foundation of Qinghai (2024-ZJ-994).

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Copyright © 2024 Jing Zhao et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Journal of Applied Ichthyology

Regulation of GeCu/Zn-SOD, GeMn-SOD, GeHsp90, and GeMT in Gymnocypris eckloni in Response to Copper and Lead Ion Challenges

Abstract

1. Introduction

2. Materials and Methods

2.1. Detection of Heavy Metal Ions in Distribution Areas of G. eckloni

2.2. Animals

2.3. Heavy Metal Stress

2.4. Heavy Metal Analysis

2.5. Expression of GeCu/Zn-SOD, GeMn-SOD, GeHsp90, and GeMT

2.6. Statistical Analyses

3. Results

3.1. Detection of Copper and Lead Ions in the Distribution Area of G. eckloni

3.2. Detection of Copper and Lead Ion Content in Experimental Water and Fish

3.3. Expression of GeCu/Zn-SOD after Cu2+ stress and Pb2+ Stress

3.4. Expression of GeMn-SOD after Cu2+ stress and Pb2+ Stress

3.5. Expression of GeHsp90 after Cu2+ stress and Pb2+ Stress

3.6. Expression of GeMT after Cu2+ stress and Pb2+ Stress

3.7. Temporal and Spatial Expression Patterns of SODs, HSP90, and MT after Cu2+ stress and Pb2+ Stress

4. Discussion

5. Conclusion

Data Availability

Ethical Approval

Conflicts of Interest

Authors’ Contributions

Acknowledgments

References

Copyright

3.3. Expression of GeCu/Zn-SOD after Cu²⁺ stress and Pb²⁺ Stress

3.4. Expression of GeMn-SOD after Cu²⁺ stress and Pb²⁺ Stress

3.5. Expression of GeHsp90 after Cu²⁺ stress and Pb²⁺ Stress

3.6. Expression of GeMT after Cu²⁺ stress and Pb²⁺ Stress

3.7. Temporal and Spatial Expression Patterns of SODs, HSP90, and MT after Cu²⁺ stress and Pb²⁺ Stress