Oxidative stress, neurotoxicity, and intestinal microbial regulation after a chronic zinc exposure: an experimental study on adult zebrafish (Danio rerio)

The textile industry consumes a huge amount of water and is responsible for 17–20% of the total industrial pollution (Jegatheesan et al. 2016). Textile wastewater treatment plant effluent consists of a variety of complex chemicals such as acids, bases, salts, heavy metals, surfactants, oils, and fats. Especially, high levels of iron, copper, and zinc are included (El-Kassas & Mohamed 2014; Kishor et al. 2021; Nidheesh et al. 2022). Zinc is one of the heavy metals with a high ecological risk entropy in an aquatic environment (Supplementary Material, Figure S1). Elevated concentrations of zinc are also found in virtually all lakes and rivers in dense human-populated areas (Zheng et al. 2022). The high zinc concentrations in excess of 5 mg/L have been recorded in zinc-contaminated water environments, severely affecting the growth and development of aquatic life in rivers (Gozzard et al. 2011). However, most studies have focused on the toxic effects of zinc on fish embryos and larvae at high concentrations, while the mechanisms of chronic toxicity of zinc in adult zebrafish are still lacking.

Metalloenzymes and transcription factors contain zinc, which is essential for cellular growth, gene expression, protein synthesis, and cell division (Puar et al. 2021). However, zinc intake above nutritional levels poses a risk of toxicity to fish. It has been reported that zinc exposure at 1.5 and 4.9 mg/L resulted in delayed hatching in zebrafish and with the increase of zinc concentration, zebrafish mortality increased (Horie et al. 2020). Disruption of Ca, Mn, and Co homeostasis in zebrafish larvae by zinc exposure has also been observed (Puar et al. 2021). In addition, an investigation demonstrated that Etroplus suratensis developed significant necrotic lesions in the gills under 15.32 mg/L zinc exposure (Xavier et al. 2019).

The intestines, gills, and muscles are all critical organs for aquatic vertebrates. In addition to nutrient absorption and metabolism, the intestine also regulates the intrinsic immune system and maintains metal homeostasis (Zeng et al. 2019). Fish intestine can be used to evaluate toxicological effects when heavy metal contamination occurs (Zeng et al. 2019). Heavy metals mainly cause fish intestinal histopathological lesions (Dane & Sman 2020), imbalance of intestinal microbial community, damage of antioxidant enzymes and neural toxicity (Wang et al. 2020). Gills are involved in gaseous and ionic exchanges between fish and the water environment, and are easily affected by aqueous pollutants of their large surface area and small diffusion distance (Santos et al. 2022). Heavy metal exposure causes dilation of blood vessels in fish gills, affecting the blood supply to other organs (Luzio et al. 2021). Muscle is the largest tissue of fish and is the main effector of fish swimming behavior (Shahjahan et al. 2022). Damage to muscle cells can cause behavioral changes and abnormal feeding ability in zebrafish (Avallone et al. 2015). Therefore, abnormal changes in zebrafish intestines, muscles, and gills can be used as evidence to demonstrate the toxic effects of excessive zinc exposure in aquatic vertebrates.

To determine the mechanism of zinc accumulation and biotoxic effects in the intestines, muscles, and gills of fish following chronic zinc exposure, oxidative damage and neurotoxicity effects of chronic zine exposure in zebrafish organs were analyzed. Meanwhile, high-throughput sequencing was used to evaluate the effect of zinc on the zebrafish intestinal microbiome. By comparing the effects of different concentrations of zinc on zebrafish, it provides a new idea for dialectically regard with the harm of excessive zinc.

Adult wild-type zebrafish (AB, 3–4 months old) were purchased from the China Zebrafish Resource Center (Wuhan, China). Zebrafish were 3.0 ± 0.5 cm in body length and 0.4 ± 0.05 g in weight. The male-to-female ratio was 1:1. Before the formal experiment, zebrafish were uniformly acclimated in a large fish aquarium (65 L) for 7 d to adapt to the laboratory environment and ensure a natural mortality rate below 5%. Dechlorinated tap water, well aerated for 7 d, was used for the zebrafish culture. During the experiment, the water temperature was 25 ± 2 °C, pH was 7.7 ± 0.1, hardness was 200–230 mg/L CaCO₃, and a 12 h/12 h light–dark cycle was maintained. The zebrafish were fed hatched brine shrimp twice daily at 9:00 and 18:00. Dead fish, excrement, and food residues were removed timely. The culture solution was changed every 2 d to ensure a constant concentration of zinc in the fish tank. All procedures were approved by the Committee on the Ethics of Animal Experiments of Beijing Technology and Business University and conducted in accordance with the guidelines for the protection of animal welfare.

ZnCl₂ (CAS No. 7646-85-7, purity: 99.95%) was purchased from Beijing Mreda Technology Co., Ltd (Beijing, China) and used to regulate zinc concentration in culture water. Based on previous pre-experimental results (Supplementary Material, Figure S2) and environmental investigation data (Gozzard et al. 2011), the zinc concentration in the formal experiment was set to CG (control group), LG (low concentration group), and HG (high concentration group) with 0, 5, and 10 mg/L of zinc concentrations, respectively. Three replicate experiments were conducted for each concentration. After 7 d of acclimation, 270 adult zebrafish were equally divided into nine tanks (5 L) for a 25-d chronic exposure experiment. Two zebrafish were randomly collected from each tank and anesthetized with tricaine (MS-222; Aladdin Biochemical Technology Co., Ltd, Shanghai, China). After dissection, the intestines, muscles, and gills of fish were sampled for analyzing zinc levels and antioxidant enzyme assays. In addition, two fish samples were randomly collected from each tank and anesthetized with MS-222, and then only intestinal samples were taken for 16S rRNA sequencing. Samples were immediately frozen at −80 °C for subsequent analysis.

The zinc content in different zebrafish tissues was measured at 5 and 25 d. The tissue was accurately weighed and the volume of deionized water was added nine times to make a 10% tissue homogenate. It was centrifuged at 2,500 rpm for 10 min, and 25 μL of the supernatant was taken to measure the zinc ion concentration. Antioxidant index levels and acetylcholinesterase (AChE) activity were measured at exposure times of 5, 10, 15, 20, and 25 d, respectively. Zebrafish intestine, muscle, and gill tissue samples were weighed accurately, and pre-cooled saline was added at a ratio of weight (g):volume (mL) = 1:10. The samples were processed using a high-speed grinder, and 10% of the tissue homogenates were prepared. Samples were then centrifuged at 2,500 rpm for 10 min. After dilution, the supernatant was collected. Antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH)), malondialdehyde (MDA), and metallothionein (MT) were tested using assay kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) for indicating oxidative stress. In this study, AChE was analyzed using an assay kit (Nanjing Jiancheng Bioengineering Institute) as a recognized marker of neurotoxicity.

For intestinal microbiota composition analysis, the total DNA of the intestinal samples was extracted using the DNeasy Blood & Tissue Kit (Qiagen GmbH, Hilden, Germany; reference number 69506). The V3–V4 region of the bacterial 16S rRNA gene was amplified using primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGG GTWTCTAAT-3′) as previously described (Castrillo et al. 2017). After purification, all amplicons were pooled together with an equal molar amount from each sample and sequenced using an Illumina platform. The high-throughput sequencing of intestinal microbiome samples was all completed by Shanghai Personalbio Technology Co., Ltd.

Vsearch (v2.13.4_linux_x86_64) and cutadapt (v2.3) were used for further analysis of high-throughput sequencing data. After de-priming, splicing, quality filtering, and de-chimera, high-quality sequences were clustered at a sequence similarity threshold of 97%, and the representative sequence and OTU tables were output. Representative sequences of OTUs were annotated by species using the classify-sklearn algorithm of QIIME2 (Bokulich et al. 2018). Finally, differences in species diversity and community structure between samples were revealed through α- and β-diversity analyses (Ding et al. 2019). Principal coordinate analyses (PCoAs) were performed at the OTU level using the R 3.3.1 (The R Foundation for Statistical Computing, Vienna, Austria). A PCoA plot was obtained based on the Bray–Curtis distances between the samples. Rarefaction curves of sample abundance were plotted, and α-diversity was evaluated using the Chao1 richness and the Simpson diversity index.

Mean and standard error of the mean (SEM) are used to present all data for each group. To determine the significant differences between oxidative stress marker levels, zinc content, and AChE activity between groups, SPSS software was used to conduct a one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test (SPSS 25.0, IBM, Armonk, New York, NY, USA). Graphs were constructed using Origin 2019 (Microcal, Redmond, Washington, USA). The significance of differences in microbial communities was verified by the Kruskal–Wallis post hoc test (XL-Stat software, Addinsoft). Differences were considered statistically significant at p < 0.05.

Intestinal SOD activity of adult zebrafish exposed to 5 and 10 mg/L zinc was significantly higher than that of the control group at 20–25 d (p < 0.05; Figure 2(d)). However, no significant differences in intestinal SOD activity were observed between the control and exposure groups after 10–15 d. SOD activity in the zebrafish muscle was maintained at a similar level (Figure 2(e)). Although there was no significant difference in SOD activity in zebrafish gills among the three groups at 5 d, from day 10, a significant difference was observed between the experimental and control groups (p < 0.05; Figure 2(f)).

Exposure to different concentrations of zinc had significant effects on zebrafish intestinal CAT activity (Figure 2). Specifically, from days 5 to 15, intestinal CAT activities in the experimental groups were significantly higher than those in the control group (p < 0.05). However, opposite results were found at 20 and 25 d (Figure 2). Furthermore, CAT activity in the 10 mg/L experimental group showed a trend of first increasing and then decreasing over time (Figure 2). Short-term (5 d) zinc exposure did not significantly impact CAT activities in zebrafish muscles and gills (Figure 2 and 2(i)). At 25 d, a significant increase in CAT activity in muscles and gills was found between the experimental and control groups (Figure 2 and 2(i)).

The GSH contents in the intestine of zebrafish in the experimental groups were significantly lower than those in the control group (p < 0.05; Figure 2(j)). The effects of zinc exposure on intestinal GSH content in zebrafish were similar between the 5 and 10 mg/L groups during the initial 10 d, but significant differences were observed from day 15 onwards (p < 0.05). The GSH levels in the muscles of the experimental groups were significantly higher than those of the control group at all sampling times except the 10 mg/L group on 10 d (Figure 2k). GSH content in the gills of zebrafish was not statistically significant in the experimental groups compared with that in the control group (p > 0.05; Figure 2l).

MDA content in the zebrafish intestine decreased significantly in the 5 mg/L treatment group compared to that in the control, whereas a significant increase in MDA was found in the 10 mg/L group (p < 0.05; Figure 2m). The trends in MDA content in zebrafish muscle were similar to those in the intestine (Figure 2(n)). In contrast, no significant difference in MDA content was observed between the 5 and 10 mg/L groups at 10 d (p > 0.05; Figure 2(n)). The MDA content was significantly increased in zebrafish gills after exposure to 10 mg/L zinc (Figure 2(o)). Moreover, MDA content decreased significantly in the 5 mg/L treatment group at days 5–15 of exposure compared to that in the control (p < 0.05), and no significant change was observed at days 20 and 25 (p > 0.05; Figure 2(o)).

The levels of SOD, GSH, and MT in the intestinal tract of the experimental groups were higher than those in the muscles and gills (Figure 2), which is consistent with the accumulation of zinc (Figure 1). Correlation analysis (Table 2) demonstrated a highly significant correlation between zinc accumulation in organs and changes in SOD, GSH, MT, and AChE levels (p < 0.01). MDA and CAT levels in intestines, muscles and gills were not significantly correlated with zinc levels (Table 1). The differences in the distribution of MDA in the intestines, muscles, and gills were not significant. In addition, the activity of AChE in tissues was in the order of muscle > gill > intestine (Figure 3), but AChE was most significantly affected by zinc in the intestine (p < 0.05; Figure 3(a)).

In this study, to determine the microbiota α-diversity of zebrafish, the Chao1 and Simpson indices were used. The Chao1 index significantly increased in the 5 mg/L (LG) treatment group (p < 0.05; Figure 4(c)). Significantly lower Simpson indexes were found in both LG and HG than in the control group (p < 0.05; Figure 4(d)). In addition, there was no significant difference in the Simpson index between the LG and HG groups (p > 0.05; Figure 4(d)). The results of the PCoA based on the Bray–Curtis distance were consistent with those of the Simpson index. According to the PCoA, the 5 and 10 mg/L zinc exposure groups clearly deviated from the control group (Figure 4(b)). The zebrafish intestinal microbiota has been shown to change after chronic exposure to zinc. The LG and HG groups did not completely overlap, demonstrating that different concentrations of zinc produced different effects on intestinal microbiota.

At the genus level, Cetobacterium and Rhodobacter were the most abundant in the control group. Exposure to zinc in the experimental groups significantly decreased the relative abundance of Cetobacterium and Rhodobacter, which may be related to the occurrence of diseases (Figure 5(b)). In contrast, zinc exposure promoted an increase in the relative abundance of Rhizobium and Enterococcus. These changes were confirmed by linear discriminant analysis (LDA) and effect size (LEfSe) analysis (LDA ≥ 2, Figure 5(c)). Through LEfSe analysis, 35 distinct taxa were detected in the intestinal microbiota of the treatment groups compared with the control group. Additionally, the relative abundances of several other species of bacteria involved in vital physiological and biochemical processes changed significantly. For example, Aeromonadales, Gemmatimonadetes, and Rhizobiales had very high LDA scores (more than four orders of magnitude) in these samples (Figure 5(a) and 5(b)). It is noteworthy that the higher the LDA score, the greater the effect of this taxon on the differences between the groups. Heatmap based on dominant genus compositions of intestinal microbiota showed that the experimental groups were significantly different from the control group, and chronic exposure to zinc significantly altered the microbial community structure of the adult zebrafish intestine (Supplementary Material, Figure S7).

The process of transmission and accumulation of heavy metals in the gills, intestines, and muscles of fish is described. Fish gills are used for gas exchange in aquatic environments. Large gill surface areas with abundant active ion transport pumps are capable of efficiently absorbing heavy metals (Zhang et al. 2022). The absorbed zinc is exchanged with blood under the action of the gills, which promotes the distribution of zinc to the intestines and muscles (Juncos et al. 2019). This process prevents the excessive accumulation of zinc in the gills (Tsai & Liao 2006). The present study showed that after 25 d of zinc exposure, there was a significant accumulation of zinc in the organs of zebrafish, and the distribution of accumulation was in the order of intestine > gill > muscle. It has been reported that the accumulation level of cadmium in fish organs is in the order of intestine > kidney > liver > gill > muscle (Wang et al. 2020). This report can be cross-checked with the results of our study. In addition, the accumulation of toxic substances in the zebrafish intestine was found to be higher than that in the gills and muscles (Zhang et al. 2022).

The cumulative mortality of adult zebrafish approaching 30% during chronic experiments is the result of multiple mechanisms acting together in an environment of high zinc stress. The main causes of mortality are likely to be (1) excessive oxidative damage triggered by high zinc concentrations leading to the collapse of the antioxidant and immune systems, triggering death by immune disease (Capriello et al. 2021). (2) Dysbiosis of the intestinal microflora leads to disruption of the metabolic system and abnormal signaling of the nervous system, which seriously affects the normal life activities of zebrafish (McRae et al. 2016). (3) Excessive accumulation of Zn disturbs metal homeostasis, such as disrupting the calcium absorption mechanism, and eventually the fish suffer from hypocalcemia and die (Xu et al. 2019). (4) Excessive Zn transport through the gills causes branchial mucus secretion, which has a damaging effect on the gills, thus limiting the transport and absorption of oxygen by the gills (Skidmore 1970).

Zinc is a redox-inert divalent metal ion involved in the regulation of redox processes through interactions with cysteine sulfur in cellular proteins (Maret 2019). The cysteine-rich protein in cells is MT, which binds up to seven zinc ions in the fully reduced state (Hubner & Haase 2021). MT is used for the detoxification of heavy metals, such as zinc, and for antioxidant regulation in vivo. The main regulatory process involves zinc binding to reduced thiols and their release under oxidative conditions. The redox ligand formed by zinc as the central ion converts the redox signal to a zinc ion concentration (zinc signal) while participating in the redox process. Zinc signals can trigger an antioxidant response (Maret 2006). In addition, MT can directly scavenge harmful hydroxyl radicals and singlet oxygen to cooperate with oxidative detoxification (Gimenez et al. 2021).

The MT of the 10 mg/L group in the zebrafish intestine was maintained at a high level. This reflects the increasing damage to the intestinal tract when zinc levels are high. A large amount of MT is generated to scavenge the reactive oxygen species (ROS). At the early stage of zinc exposure (5 d) in the 5 mg/L group, MT in the gills increased most significantly, because the gills are the channel for ingesting zinc. MT is increased to bind more zinc ions for detoxification while scavenging hydroxyl and oxygen radicals more effectively. With prolonged exposure, the gills transfer the accumulated zinc ions to organs, such as the intestine, resulting in a decrease in MT in the gills and a significant increase in MT in the intestine. Consistent with the conclusions of the present study, zinc exposure has been reported to cause an increase in MT levels in the gills of zebrafish, followed by a decrease (Arini et al. 2015).

Excess zinc activates lipoamide dehydrogenase in zebrafish cells, which, in turn, catalyzes the massive production of hydrogen peroxide and superoxide radicals (Lee 2018). At this time, the balance between the production and elimination of ROS is disrupted, resulting in oxidative stress. Biological defenses against oxidative stress have evolved in fish (Sun et al. 2020). Antioxidative enzymes play crucial roles in cellular antioxidant processes. SOD converts highly reactive superoxide radicals into water and hydrogen peroxide, which are further decomposed into non-toxic oxygen and water by CAT (Shahjahan et al. 2022). In addition, the cysteine group contained in the reduction of GSH by non-enzymatic compounds has redox activity and participates in the eradication of hydrogen peroxide, together with antioxidant enzymes (Wu et al. 2019). However, the regulatory capacity of the antioxidant systems is limited. When the regulatory capacity of the antioxidant enzyme system is exhausted, damage owing to cellular lipid peroxidation cannot be avoided (Sun et al. 2020). One of the best indicators of oxidative damage is MDA, which is the final product of lipid peroxidation (Sun et al. 2019).

The present study showed that short-term (5 d) zinc exposure significantly increased SOD activity in the intestine, which is a marker for the activation of the antioxidant system. Zebrafish are stressed by zinc, and excessive ROS production in cells stimulates SOD activity to effectively regulate the balance of ROS (Sharma et al. 2022). In the mid-exposure period (10–15 d), under the action of SOD, the level of ROS gradually returned to a steady state, and SOD returned to its original state. However, SOD was reactivated with the excessive accumulation of zinc with prolonged exposure time. Muscles and gills also showed a decreased level of SOD activity. Enzyme activity is impaired or their generation is declining due to tissue damage, as evidenced by the decline in enzyme activity (Sharma & Jindal 2020). Significant changes in SOD activity after exposure to environmental pollutants have been reported (Jiang et al. 2022).

CAT activity in the intestines, muscles, and gills of zebrafish showed an overall trend of enhancement during zinc exposure, which was similar to that of SOD activity. This suggests a synergistic effect of SOD and CAT on ROS scavenging (Si et al. 2019). Increased CAT activity indicates activation of the cellular antioxidant defense system to repair oxidative stress damage by scavenging and breaking down excess H₂O₂ (Sharma et al. 2022). Specifically, intestinal CAT activity decreased significantly after 20–25 d. This may be because of the large amount of CAT used to eradicate H₂O₂ after a sustained H₂O₂ surge after continuous zinc exposure. When CAT production was slower than consumption, the antioxidant system was close to collapse. Thus, the conclusion that CAT activity is activated at low toxicity and inhibited at high toxicity was validated (Jiang et al. 2022).

A plausible explanation for the significant reduction of intestinal GSH of zebrafish in the experimental groups is that GSH peroxidase catalyzes the reduction of H₂O₂ at the expense of GSH (Capriello et al. 2021). Moreover, glutathione S-transferase induces the binding of electrophilic ions to GSH to form conjugates to maintain cellular redox homeostasis (Wu et al. 2019). GSH levels in the experimental groups were lower than those in the control group; this phenomenon is common in zebrafish studies. Oxidative stress responses induced by metals such as cadmium (Hu et al. 2022) and aluminum (Capriello et al. 2021) have also been observed. The present data showed that GSH levels were increased in zebrafish muscle. We speculate that this may be owing to the fact that muscle is less affected by oxidative stress, which is an initial response to ROS following the accumulation of trace toxicity of zinc.

The decreased MDA levels in the low concentration group implied that the defense effect of the antioxidant system was effectively exerted in the 5 mg/L zinc exposure group. The cells were not damaged by lipid peroxidation. Conversely, high levels of zinc induced significant ROS production. Invasion by ROS initiates antioxidant defense activities in the intestines, muscles, and gills of zebrafish. However, the defense system is insufficient for complete removal, leading to lipid peroxidation (Sharma et al. 2022). This was confirmed by the significant increase in MDA levels in the 10 mg/L experimental group. In a previous report on the effects of Cd on the liver of zebrafish, no significant difference in MDA levels was found at low concentrations, and high concentrations of Cd led to a significant increase in MDA (Hu et al. 2022).

AChE plays an important role in nerve impulse conduction and is a recognized biomarker of neurotoxicity in toxicology studies (Saiki et al. 2021). The enzyme immediately terminates the continuous excitatory effect of neurotransmitters by hydrolyzing acetylcholine, ensuring the normal transmission of nerve signals in the body (Tao et al. 2022). Zinc exposure significantly inhibits AChE activity in the intestines, muscles, and gills of zebrafish as was observed in the present study. This may be because of oxidative stress (Muthulakshmi et al. 2018). Furthermore, oxidative stress may alter AChE activity, and this finding is supported by other studies (Parlak 2018; Pullaguri et al. 2020). A significant enhancement in AChE activity in the muscle was observed after 25 d of exposure. A reasonable hypothesis is that lipid peroxidation damage leads to the rupture of presynaptic vesicle membranes and a large amount of acetylcholine is released, inducing a significant enhancement of AChE activity. Similar phenomena have been reported in another study (Zhang et al. 2021).

In the analysis of oxidative stress, the levels of SOD, CAT, GSH, and MT in the intestine of the experimental groups were higher than those in the muscles and gills. Our results validate that one of the major sites of zinc-induced oxidative stress injury and repair in zebrafish is the intestine. Muscles and gills are inferior in comparison. However, the distribution of MDA levels in the intestine, muscles, and gills was not significantly different, demonstrating a strong repair capacity in the intestine. The intestine protects zebrafish from heavy metals through immunogenic and non-immunogenic mechanisms (Marinsek et al. 2022). High AChE activity in gills may be a reflection of more muscle activity.

Several heavy metals are known to target the microbiota in the digestive system (Bist & Choudhary 2022). The metabolic capacity and immune functions of the intestine can be reduced by heavy metal invasion, which is caused by disturbances in the intestinal microbial community and may further increase the probability of intestinal damage and disease (Duan et al. 2020). A significant increase in the Chao1 index of the zinc-exposed group indicated an increase in microbial community richness. A decrease in the Simpson index reflects the diversity of the intestinal microbiota. Consistent results have been reported (Wu et al. 2021). In the PCoA, clearly separated clusters were observed in the exposure group, demonstrating that chronic exposure to zinc altered the structure of the zebrafish intestinal microbiota.

In studies of the effect of zinc on the structure of the zebrafish intestinal microbiota, significant changes in the relative abundance of microorganisms at the phylum and genus levels were the most direct evidence of an effect of zinc. High-throughput sequencing analysis of 16S rRNA showed that the dominant intestinal microbiota at the fish phylum level was Fusobacteria, Tenericutes, Proteobacteria, Firmicutes, and Bacteroidetes. Similar findings can be retrieved from other studies (Dulski et al. 2020; Zhang et al. 2020). At the phylum level, zinc exposure increased Proteobacteria in the intestine, potentially contributing to a disruption in zebrafish intestinal microbiota (Tan et al. 2020). In addition, an increased relative abundance of Proteobacteria has been associated with the development of inflammatory bowel disease (Lobionda et al. 2019). Firmicutes and Bacteroides play vital roles in host lipid metabolism. Significant increases in Firmicutes and Bacteroides may lead to abnormal lipid metabolism in zebrafish (Wang et al. 2021). Bacteroides is an opportunistic pathogen that may lead to endogenous infections (Dong et al. 2020). For example, butyric acid produced by Fusobacterium can improve the inflammatory status of the intestinal mucosa and inhibit colon carcinoma (Zhang et al. 2020). Decreases in Fusobacterium and Tenericutes may herald increased odds of intestinal diseases in zebrafish.

Changes in the dominant microbiota at the genus level are another manifestation of the effects of zinc exposure on intestinal microbiota. The dominant bacteria in the control group were Cetobacterium and Rhodobacter spp. The dominant bacteria in the experimental groups were Rhizobium and Enterococcus spp. Cetobacterium is an anaerobic bacterium involved in vitamin B12 synthesis (Bai et al. 2019). Cetobacterium and Rhodobacter are more abundant in the intestine of healthy fish than that in diseased individuals (Li et al. 2017; Xue et al. 2017). A significant reduction in the relative abundance of bacteria in these two genera could potentially increase the chance of pathogenicity in fish. Enterococcus can improve antioxidant enzyme activity and disease resistance in fish while enhancing immunity (Kakade et al. 2020). The significant increase in the abundance of Enterococcus may be related to intestinal oxidative stress. As a probiotic in the intestine, the reason for the significant increase in Rhizobium is presumed that chronic exposure to zinc accelerates the production of its product coenzyme Q10, which plays a role in enhancing host immunity (Xia et al. 2018).

There are links and interactions between oxidative stress, neurotoxicity, and altered gut microbiota in zebrafish resistance to zinc stress. In this paper, a Pearson correlation analysis was performed between neurotoxicity biomarkers and oxidative stress parameters. AChE activity was significantly negatively correlated with SOD, GSH, and MT levels after chronic exposure to zinc in zebrafish (p < 0.01), suggesting that the neurotoxicity of zinc in zebrafish may be mediated by oxidative stress (Guo et al. 2022). In addition, there are two main perspectives on the interaction between the nervous system and the gut microbial community. First, neurotoxic substances can be metabolically detoxified by the gut microbiota directly after production through reduction and hydrolysis/defixation reactions (Claus et al. 2016). Second, certain gut microbes can produce short-chain fatty acids and acetylcholine, which can act on receptors in neurons and regulate the normal transmission of neural signals (Dempsey et al. 2019). The significant increase in the relative abundance of enterococci associated with oxidative stress at the genus level is also potential evidence that the oxidative system and gut microbes collaborate with each other in detoxification.

In summary, this study demonstrates the toxic effects of chronic zinc exposure on oxidative stress, neurotransmitters, and intestinal microbiota in adult zebrafish. The changes of MT, SOD, CAT, GSH, and MDA levels in intestines, muscles, and gills were analyzed. Differences in the oxidative stress response of different organs to zinc exposure were found. The intestine is a more important site of antioxidant defense than gills and muscles. Inhibition of AChE activity by zinc exposure may be related to oxidative stress, which adversely affects the nervous system of zebrafish. Furthermore, the accumulation of zinc in zebrafish induces ecological changes in the intestinal microbiota that are twofold. This is reflected in the increased abundance and reduced diversity of the intestinal microbiota. Zinc exposure not only resulted in a significant reduction of potentially beneficial bacteria and an increase in opportunistic pathogens but also induced an increase in microorganisms associated with oxidative stress and enhanced intestinal immunity.

This study was funded by the National Key R&D Program of China (No. 2022YFE0104900), the Natural Science Foundation of Hebei Province (No. D2021402035), the Opening Project of Key Laboratory of Road Traffic Environmental Protection Technology, Ministry of Transport, PRC, the Opening Project of Key Laboratory of Environment Controlled Aquaculture (Dalian Ocean University) Ministry of Education (No. 202218), and the 2022 Graduate Research Capacity Enhancement Program of Beijing Technology and Business University.

Z.Y. administered the project, conducted investigation and funding acquisition, wrote the original draft, reviewed and edited the article. R.L. investigated the article, developed the methodology, and conducted formal analysis. S.L. investigated the article and performed visualization and funding acquisition. D.Q. performed visualization and supervised the work. G.L. performed visualization. C.W. administered the project, reviewed and edited the article, and performed funding acquisition. J.N. reviewed and edited the article. Y.S. administered the project and performed funding acquisition. H.H. reviewed and edited the article.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.