Carbamazepine transmits immune effect by activation of gut-liver axis and TLR signaling pathway from parental zebrafish to offspring

Abstract

Carbamazepine (CBZ) has been identified in the aquatic environment as an emerging contaminant. Its immune effect across generations at environmentally relevant concentrations is little known. We aim to elucidate the effects of CBZ on the immune system in zebrafish (Danio rerio), hypothesizing the effects caused by CBZ exposure in the parental generation can be passed on to its offspring, leading to impairment of innate immune function and defense against pathogen weakened. A suite of bioassays (including a test with added lipopolysaccharide) was used to measure the effects of environmentally relevant levels of CBZ (1, 10, and 100 μg/l) on zebrafish at multiple biological levels, and across 2 successive generations (21 days exposure for F₀; 5 and 21 days exposure or nonexposure for F₁). The results showed that CBZ affected homeostasis in the immune system, caused liver vacuolization, increased the inflammation-related microbiota proportion in gut, and decreased reproduction, by induction of oxidative stress and modulation of Toll-like receptors (TLR) signaling pathway on gut-liver axis. The effects of exposure to CBZ over 21 days in F₀ could be passed to the next generation. Intergenerational effects on TLR and antioxidant defense system were also observed in nonexposed F₁ at 5 days post-fertilization (5 dpf), but diminished at 21 dpf. The finding provided evidence to unravel immune response by gut-liver axis mediated and oxidative stress under 4 test conditions. The study has raised a potential concern about the multigenerational immune effects of environmental pollutants and calls for a focus on the risk of synergetic pathogen infection.

Carbamazepine (CBZ) is a widely used antidepressant drug. The drug has a high annual production (>1000 tons) and discharge volume, for instance, it has been estimated that since entry into the market, 55 metric tons of CBZ has been accumulated in the Baltic Sea (Björlenius et al., 2018; Fraz et al., 2019). Low removal efficiency of CBZ (<2%) was in conventional sewage treatment plant effluents (Pohl et al., 2020). It was highly valued and considered as a possible anthropogenic marker in urban wastewater (Clara et al., 2004). The detected concentrations and distribution of CBZ in treated municipal wastewater effluents (18–199.1 μg/l) and surface water (1.1–1.09 μg/l) were collected in Supplementary Table 1. Studies have shown that CBZ accumulated in phytoplankton, zooplankton, macroinvertebrates, fish, and birds (Baali and Cosio, 2022; Valdes et al., 2016). Liu et al. (2021b) measured concentrations of CBZ, diclofenac, fluoxetine, and sertraline in tissues of Nile tilapia (0.5–8 days) and found that the concentrations of CBZ were higher in the gut (10.7–29.1 ng/g) and liver (14.0–28.9 ng/g). Recently, studies have focused on the reproductive and developmental toxicity in fish (Qiang et al., 2016). A study has reported changes in the feeding behavior and survival rate in zebrafish embryo due to 63 days exposure of parental adults to this compound (Santos et al., 2018). Another study showed that after exposure (6 weeks) to 10 µg CBZ/l, adult zebrafish displayed a trend of reduced fecundity, decreased sperm quality as indicated by reduced motility and velocity (Galus et al., 2013). Furthermore, a study reported that CBZ may reduce excitability of neurons and subsequently lead to reduced neuronal stimulation to reproductive organs and gonadal steroid synthesis (Galus et al., 2013; Seda et al., 2007). These studies have reported the direct effects of CBZ on fish reproduction, however, the immune suppression will indirectly decrease survival and the number of population. For instance, polychlorinated biphenyls threaten the survival of >50% of killer whales due to impaired reproduction and immune function (Desforges et al., 2018). As a result, the lower environmentally relevant concentration of CBZ may affect immune functions, increase the susceptibility to disease and lead to adverse outcomes after a long-term exposure (Koller, 2001).

Gut-liver axis is an important bidirectional regulating shaft between the liver and intestine (Albillos et al., 2020; Lin et al., 2023; Wang et al., 2023; Zhu et al., 2022). Some studies focused on the gut-liver axis to elucidate effects on immune functions by xenobiotics (Adamovsky et al., 2020; Qiu et al., 2022). Due to gut microbiota dysbiosis, lipopolysaccharide (LPS) will be released and induce hepatic inflammation by active the Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling pathway (Tian et al., 2023). A recent study reviewed a number of studies of CBZ triggered by antioxidant enzymatic activity (Baali and Cosio, 2022), CBZ induces the production of reaction oxygen species (ROS) and modifies the activity of antioxidant enzymes in aquatic organisms. In common carp, CBZ has been reported to decrease superoxide dismutase (SOD) and catalase activity in liver after a short-term exposure (Gasca-Perez et al., 2019). However, due to the presence of pathogens in surface waters, the immune response such as defense against infection and inflammatory reaction is still uncertain originating from micro-CBZ exposure. When aquatic animals suffer from long-term exposure to CBZ, unexpected immune responses and health hazards might be life stage-specific due to the asynchronous developmental process of innate immune system and adaptive immune system. Pathogenic bacteria will impact aberrant immune defense caused by pollutant exposure.

Therefore, we hypothesize that oxidative stress and immune response mediated by gut-liver axis will be the underlying toxicity mechanism. Experiments were designed to evaluate the immune responses of inflammation and defense infection and oxidative stress in liver as well as the changes of gut microbiota composition on parental (F₀) zebrafish for 21 days, and the intergenerational effects that CBZ exposure exerts on the early life stages (5 and 21 dpf) of the unexposed F₁ generation and exposed F₁ generation, and after adding LPS (10 µg/ml, which induces mild immune responses that are not harmful to fish) to exposed F₁ generation. To provide additional insights into the immune response sensitive stages and to reveal the underlying toxicity pathway of CBZ at environmentally relevant concentration exposure across 2 generations.

Materials and methods

Experimental design

All use and handling procedures with zebrafish were approved by animal ethical and welfare committee in a previous ethical review process.

To investigate the 2-generational effects of environmentally relevant concentration of CBZ (CAS: 298-46-4; 99.9% purity; Nanjing MCE, China), F₀ zebrafish (3-month-old) were exposed to 3 CBZ nominal concentrations (in triplicate): 1, 10, 100 µg/l, and a control group (in triplicate) for 21 days. Totally, 144 F₀ zebrafish were housed using the 2 l glass beaker and each beaker contained 6 fish, females and males were allocated in a 1:1 ratio (the details of zebrafish maintenance were provided in Supplementary Text 1). Dosing solution of CBZ in 3 treatment groups was prepared in dimethyl sulfoxide (DMSO; CAS: 67-68-5; 99% purity; Shanghai Generay Biotech, China) and added water to achieve 0.01% DMSO. Water change was performed every 2 days with dosing renewal in a semi-static. To evaluate the potential immune effects caused by parental exposure of CBZ, the embryos resulting from F₀ exposure were raised using the 1 l glass beaker and divided into 4 groups (group I, II, III, and IV; Figure 1): group I is done in duplicate, placed in CBZ-free water and CBZ-solvent water until reaching 5 dpf, separately; group II is the same as group I, but its exposure time has been extended to 21 dpf; group III is also as group I, but added 10 μg/ml LPS at 4 dpf and then cultured until 5 dpf to assess the impact on mediators of host defense of CBZ; group IV is also as group II, but added 10 μg/ml LPS at 19 dpf and then cultured until 21 dpf.

In order to evaluate CBZ effects on 2 generations of zebrafish, the assays of reproduction and immune response were conducted. Firstly, a breeding assay was performed after 21 days exposure of F₀ zebrafish to evaluate CBZ effects in fecundity (n = 24, each pair as the ratio of female and male is 1:1 was randomly selected from CBZ treatment and the control group). Livers were preserved in RNALater at −80°C for later qRT-PCR (n = 24, n = 18 for 3 CBZ treatments and n = 6 for the control group; gene sequence shown in Supplementary Table 2, prepared from National Library of Medicine) and cytokine measurement (n = 24, n = 6 for each CBZ treatments and the control group). Livers of male fish (n = 12, 3 per replicate) were only preserved to observe histological examination and other samples of F₀ were not distinguished as 1 sample was from a female and a male. Gut samples of F₀ (n = 24) were preserved in −80°C for microbial community analysis.

To assess the intergenerational effects of CBZ for F₁, 3 different F₁ studies as shown in Figure 1 were conducted. In group I, survival and hatching rate at 5 dpf were recorded and then F₁ larvae were sampled for measuring respiratory burst (n = 48, 4 per replicate), cytokine (n = 48), and gene expression (n = 48) in EX. and NX, separately. In groups II, IV, and III, RNA from livers was individually isolated from F₁ 21 dpf (n = 48) and F₁ 5 dpf (n = 48) in EX and NX, separately. In groups I and III, behavioral test which tracks movement of zebrafish were conducted (n = 36, 3 per replicate; detailed in Supplementary Text 2).

Chemical analysis

Actual CBZ concentrations were determined in water samples by High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS; Agilent 1260 Series system; Agilent Technologies, Palo Alto, California). More information was detailed description in Supplementary Text 3. The measured concentrations of CBZ were within a ± 20% range of the nominal concentrations (Supplementary Table 3). Therefore, the nominal concentrations were used in the following sections.

Measurement of gene expression

To evaluate and quantify the effects of CBZ at the molecular level, qRT-PCR was used in liver samples from F₀ and F₁ preserved in RNALater (Qiagen Co. Ltd, Germany) at −80°C, according to the sampling protocol as described earlier. The qRT-PCR assay (the total RNA extraction, cDNA synthesis and quantitative real-time PCR) was conducted according to the manufacturer’s protocol (Vazyme Biotech Co., Ltd, Nanjing, China). Quantification and verification of RNA and procedure of qRT-PCR were performed as reported previously (Ma et al., 2016). The categories of genes measured were mainly oxidative stress-related genes, immune development-related genes, lysozyme (lyz) and complement C3a (c3), and innate immunity genes. The primer sequences of genes are listed in Supplementary Table 2. The amplification efficiency was examined by LinRegPCR (Version: 2013.1, Amsterdam, Netherlands). gapdh was selected as a housekeeping gene and the expression of mRNA for each target gene was standardized. The fold change of mRNA expression of related genes was calculated by the 2^−ΔΔCt method (Livak and Schmittgen, 2001).

Measurement of cytokine, LYS, and respiratory burst

To observe the changes of immune regulation, the cytokines (tumor necrosis factor-α, TNF-α; interleukin-1β, IL-1β; interleukin-8, IL-8) and lysozyme (LYS) of F₀ and F₁ in group I were measured. Each sample from F₀ and F₁ zebrafish was homogenized with 20 μl PBS and then centrifuged for 10 min at 3000 rpm to collect the supernatants for measurement. The content of TNF-α, IL-1β, IL-8, and LYS were quantified using commercial Enzyme-Linked Immunosorbent Assay (ELISA, Jiangsu Meimian Industrial Co., Ltd) kits according to the manufacturer’s instructions.

The method for measuring respiratory burst was modified from published methods (Xu et al., 2015). Briefly, the production of ROS was measured in whole live embryos, using 96-well microplates (blackboard) in which each well contained 1 F₁ larva from group I in 100 μl of embryo water. In each well containing embryos, 2′,7′-dihydrodichlorofluorescein diacetate (H2DCFDA) and phorbol-12-myristate-13-acetate (PMA) were added to each well containing embryos for a final concentration of 500 and 10 ng/ml, receptively. Fluorescence was measured in a microplate reader with excitation and emission filters set at 485 and 535 nm, respectively.

Liver histopathology

The livers of F₀ male zebrafish were examined in CBZ treatments and the control group. Briefly, the samples were fixed in the 10% paraformaldehyde solution. Then, the tissue was embedded in paraffin with different ethanol solutions. Finally, the tissue was sectioned and stained with hematoxylin and eosin. More information can be obtained from our previous study (Liu et al., 2021a).

Gut microbial community analysis

The zebrafish intestines were sent to Novogene, China for microbial diversity analysis. The primer for the 16S V4 region was 515F-806R, the 18S V4 region was 528F-706R, the 18S V9 region was 1380F-1510R, the ITS1 region was ITS5-1737F and ITS2-2043R, and the ITS2 region was ITS3-2024F and ITS4-2409R. The library was constructed using the Illumina TruSeq DNA PCR-Free Library Preparation Kit. The constructed libraries were quantified and tested by Qubit, and then sequenced using NovaSeq6000. The reads of each sample were spliced using FLASH (V1.2.7, http://ccb.jhu.edu/software/FLASH/) after truncating the Barcode and primer sequences (Magoc and Salzberg, 2011), and the obtained spliced sequences were the original Tags data (Raw Tags); the spliced Raw Tags need to be strictly filtered to obtain high quality Tags data (Bokulich et al., 2013). Clustering of Operational Taxonomic Units (OTUs) with 97% agreement (Identity). OTUs were analyzed for the relative abundance of microbiota at different biological classification levels.

Statistical analysis

GraphPad Prism 8 (GraphPad Software Inc., California) was utilized to analyze data. All data were shown as the mean ± standard error. All results were checked for normality using Shapiro-Wilk test and homogeneity of variance using Brown-Forsythe test. Statistically significant differences between each treatment and the control group were determined by the 1-way analysis of variance followed by Tukey’s multiple comparisons or Dunnett’s multiple comparisons test.

Results and discussion

Because the immune effects of CBZ on nontarget organisms and life-stage still remain under-investigated, the present study focuses on different immune developmental stages (innate immune stage and the adaptive immune development stage) to assess the effects of CBZ on parental exposed organism (F₀) and F₁ progenies (intergenerational effects) in zebrafish. A suite of endpoints, such as respiratory burst, cytokines, and immune-related gene expression, were included to provide insights into the potential hazards of CBZ.

Effects of CBZ exposure on F₀

Effects on zebrafish fecundity

After 21 days exposure, obvious decreases in the spawning of F₀ exposed to 1 and 10 μg/l are shown in Figure 2A, leading to adverse reproductive outcomes in adult zebrafish (F₀). This is consistent with the previous study where a concentration-dependent reduction in spawning was found in adult zebrafish after 6 weeks exposure to 0.5 and 10 μg CBZ/l (Galus et al., 2013). The data of CBZ concentration detected from wastewater and surface water (Supplementary Table 1) was generated to a frequency distribution curve, and then the relative fecundity of F₀ exposed to 3 concentrations in this study were also summarized in Supplementary Figure 2. It was found that the concentration of 1 μg/l had about 20% occurrence where the fecundity was inhibited to 80%, and the concentration of 10 μg/l had about 5% occurrence where the fecundity was inhibited to 60%, thus, the reproductive capacity of F₀ generation may be affected at lower concentration.

Effects on gut microbiome

The gut microbiome plays a key role in the immune systems and protects host from pathogen invasion, and the gut microbial changes may be responsible for inflammatory increase (Ruff et al., 2020; Schluter et al., 2020; Zheng et al., 2020). In this study, the gut microbial community structure and the number of species in F₀ were significantly affected after 100 μg CBZ/l exposure, potentially leading to dysbiosis in the gut microenvironment. Aberrant to gut flora can lead to immune system disorders, inflammatory bowel disease, and various metabolic diseases (Fan and Pedersen, 2021). Gut microbiota sequencing demonstrated that Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidota were the dominant microbial phyla in the gut of all F₀ treatments and control group (Figure 2B). The CBZ-exposed fish gut had a markedly higher proportion of Proteobacteria (1.45 times as many as the control group). Proteobacteria contain pathogen so the increasing relative abundance will disrupt gut balance and further affect immune system (Shin et al., 2015). In addition, a decrease in phyla Firmicutes and Bacteroidota was observed at 1 and 10 μg CBZ/L. Firmicutes and Bacteroidota are both associated with the production of butyric acid which can increase growth performance, intestinal barrier function, and anti-inflammatory capacity (van Kessel et al., 2011). The increase of species Aermonasveronii, Shewanella putrefaciens, Escherichia coli, and Plesiomonas shigelloides was observed (Figure 2C), and they may result in inflammation. One study reported that Aermonasveronii and Shewanella putrefaciens were prevalent in diseased (Red-Operculum Disease) fish (Li et al., 2017a). Additionally, mature intestinal mucosa differentiates between pathogens and commensal bacteria via pattern recognition receptor (PRR) such as TLR receptors, which then activate signaling cascades to modulate the immune response (Yang et al., 2023).

Effects on liver by TLR signaling pathway

Distinct histological changes were identified in the liver of F₀ fish. Compared with healthy fish hepatocytes, which normally have irregular polygons with large and round nucleus in the center of cells, a large amount of loose or vacuolated hepatic cytoplasm were observed in zebrafish exposed to the 3 treatments (Figs. 2D–G).

To gain more mechanistic insights into the effects of CBZ on immune responses, we analyzed LYS and cytokines (pro-inflammatory cytokines: TNF-α, IL-1β, IL-8), a group of proteins involved in immunological and inflammatory reactions. The levels of IL-1β, IL-8, and LYS significantly increased in F₀ at lower exposure concentrations of CBZ but decreased at 100 μg CBZ/L (Figure 2H). TNF-α was significantly suppressed by 1 μg CBZ/L (Figure 2H). Additionally, the expression of cytokines-related genes (il-1β, il-8, and tnf-α) and complements-related gene (lyz and c3, which have the ability to complement antibodies and clear pathogens) were analyzed (Qiu et al., 2016). Despite no significant change in tnf-α, the expression of other genes (il-1β, il-8, lyz, and c3) were upregulated at lower concentrations and inhibited at the highest concentration, which was roughly consistent with the responses of the inflammation cytokines (Figure 3 and Supplementary Figure 3). Thus, CBZ-mediated inflammatory responses in F₀ were further supported by the changes in cytokines and LYS at all 3 test concentrations. Inflammatory cytokine can be used to indicate the severity of organ inflammation due to exposure to toxicants. LYS is one of the key maternal transferable immune factors that can combat infection and is also known to play a role in the defense of bonefish juveniles against pathogens (Dong et al., 2018). Studies have shown that the activity of LYS changes with the presence of toxic substances. Exposure to bisphenol A (BPA), bisphenol S (BPS), and bisphenol F (BPF) increased LYS activity in the offspring zebrafish and interfered with the immunity development in offspring (Dong et al., 2018).

We hypothesized that exposure to CBZ may affect both innate and adaptive immune systems in adult zebrafish (F₀), especially innate immunity that is the first line to defense against external invasion. The innate immune system recognized pathogens-associated molecular patterns (PAMPs) relying on PRRs. TLRs is one of the best characterized groups of PRRs. TLRs recognize and bind the corresponding PAMPs, and it is principally responsible for the recognition and response to pathogen ligands such as LPS from Gram-negative bacteria (TLR4 ligand) (Li and Wu, 2021; Li et al., 2017b). Activation of signal transduction pathways by TLRs leads to the induction of various genes that function in host defense, including inflammatory cytokines. The genes [including Toll-like receptor 2 (tlr2), tlr3, tlr4, Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (trif), myeloid differentiation factor 88 (myd88), and Type I interferon (ifn)] which are related to TLRs signal pathway were evaluated by transcriptional responses. Additionally, the genes such as recombination activating 1 (rag-1), and ikaros were selected to evaluate the development of adaptive immune system (Lam et al., 2004). The pathway of recognition pathogen (tlr4, trif) and the development of adaptive immune system (ikaros and rag-1) were inhibition at low concentration (Supplementary Figure 3). On the contrary, TLR signaling pathway (tlr2, tlr4, trif, and myd88) and rag-1 were significantly upregulated by exposure to 100 μg CBZ/L triggering the release of downstream inflammatory factors (Supplementary Figure 3). In addition, il-1β was significantly upregulated in 10 μg/l treatment and il-8 was significantly upregulated in 1 μg/l treatment, indicating that CBZ stimulates inflammatory responses in the liver of zebrafish.

Effects on hepatic oxidative stress

Oxidative stress induced cytokine gene expression and activated cytokine production (Driscoll et al., 1997). ROS was released to eliminate the invading agents in inflammation response, which is not able to discriminate microbial and host targets and so oxidative stress unavoidably have collateral damage to host tissues (Biller-Takahashi et al., 2015; Bogdan et al., 2000; Medzhitov, 2008). The antioxidant system, such as CAT, SOD, and glutathione peroxidase (GPX) are the first line of defense against ROS and part of the innate immune defense (Zhang et al., 2017). In the present study, the transcriptional responses of genes related to antioxidant defense in F₀ zebrafish, such as myeloperoxidase (mpx) and glutathione peroxidase 1a (gpx1a) were significantly downregulated (fold change over 4 times in 10 μg/l treatment than the control group), whereas cat was significantly upregulated in 10 µg/l treatment (Figure 3 and Supplementary Figure 3). In early 1994, 1 study found that CBZ affected immune function through a chemically reactive metabolite of activated leukocytes and MPX enzyme system (Furst, 1994). Additionally, GPX activity was higher in liver in 96 h acute toxicity test of juvenile rainbow trout (weighing about 65 g) (Li et al., 2011). These collectively suggest that exposure to CBZ may weaken the antioxidant defense in fish. The downregulation of antioxidant defense system may also persist in the F₁ generation (Figure 3).

Effects on CBZ-exposed or nonexposed F₁

As whether direct exposed to CBZ and whether adding LPS, F₁ were grouped to 4 test conditions (group I, II, III, and IV; Figure 1), and then from these 4 groups we analysis the effect on F₁ induced by CBZ.

F₁ exposed or nonexposed to CBZ until 5 dpf (group I)

The 2-generational effects from exposed F₀ generation to the F₁ progenies were assessed by 2 assays on the early life stages (F₁ exposed or nonexposed to CBZ until 5 dpf). CBZ had an effect on the survival rate of exposed F₁ larvae (Supplementary Figure 4). Survival rate decreased significantly in 1 and 10 μg/l treatments (Galus et al., 2013) and increased significantly at the highest concentration while F₁ exposed to CBZ. Compared with adult zebrafish that have fully developed immune systems, the F₁ larvae rely on innate immune systems and partially developed immune defense mechanisms, from the early-stage embryos prior to the development of functional organ systems (Esteban and Cerezuela, 2015), thus they are more susceptible to toxicants. In line with the previous knowledge, the present results illustrated that exposure to CBZ led to delay pre-hatching of F₁ embryos at 48 h post-fertilization (Supplementary Figure 4) (Qiang et al., 2016).

Under the parental exposure of CBZ, the contents of IL-1β, IL-8, and LYS in F₁ zebrafish increased significantly 3 CBZ treatments, either in the exposed group or unexposed group (Figs. 3A and 4), in line with those data in exposed F₀. Therefore, inflammatory responses may be transmitted by CBZ exposure of F₀ generation. A previous study showed that LYS activity was elevated in the offspring of both generations of zebrafish exposed to BPA, BPS, and BPF, disrupting the immune systems in the offspring (Dong et al., 2018). In exposed F₁, 1 and 10 µg CBZ/l caused a significant decrease in the content of TNF-α, but 100 μg CBZ/l caused a significant increase to recruit the leukocytes to the site of infection (van der Vaart et al., 2012). In the nonexposed F₁, there was no significant difference in TNF-α (Figure 4 and Supplementary Figure 5). The TLR signal pathway was inhibited in F₁ generation, tlr3, tlr4, and myd88 were significantly downregulated at 10 and 100 μg/l and tlr2 was significantly downregulated at 10 μg/l in the unexposed F₁ generation. Genes associated with the development of the adaptive immune system such as ikaros was significantly decreased (from 100 μg/l treatment of F₀) and rag-1 was significantly downregulated (from 1 and 100 μg/l treatment of F₀). In the exposed F₁ generation, tlr2 and tlr3 were significantly downregulated at 10 μg/l and myd88 and ikaros were significantly downregulated at 10 and 100 μg/l, respectively (Supplementary Figure 5). However, the TLR signaling pathway downstream genes (il-1β and lyz) upregulated in unexposed F₁ generation at 5 dpf. These indicated that CBZ-induced inflammation in larvae zebrafish may not only be induced by the TLR signaling pathway. Some studies thought the inflammatory pathway was induced by other PRRs. Infection of cells by microorganisms activates the inflammatory response. The initial sensing of infection is mediated by innate PRRs, which include Toll-like receptors (TLR), RIG-I-like receptors, NOD-like receptors, and C-type lectin receptors. The intracellular signaling cascades triggered by these PRRs lead to transcriptional expression of inflammatory mediators that coordinate the elimination of pathogens and infected cells (Magnadottir, 2006; Takeuchi and Akira, 2010). Other supported that oxidative stress affected and was correlative to inflammatory response, so we analyzed the expression of antioxidant system-related genes and respiratory burst activity.

The expression of antioxidant system-related genes was basically downregulated and then excessive ROS production have led to oxidative stress. The expression of antioxidant genes especially mpx (10 and 100 μg/l) was downregulated in nonexposed F₁ and exposed F₁ (same with exposed F₀) which likely were from exposed F₀ generation, whereas gpx1a was upregulated in unexposed F₁ and downregulated in exposed F₁ (same with exposed F₀) so it was more likely affected by CBZ direct exposure (Figure 3A and Supplementary Figure 5). mpx, which acts as a ROS scavenger in neutrophils during the development of zebrafish embryos and acts upstream of or within inflammatory response, was significantly downregulated in groups without continued 10 μg BPA/L exposure in F₁ (Dong et al., 2018). In another study, the activities of SOD and GPX in Tinca embryos were significantly reduced after 35 days exposure to 60 μg CBZ/L (Stancova et al., 2017). However, the mRNA levels of mn-sod and cat showed significant downregulation in exposed F₁ (10 and 100 μg/l). The expression of cat in nonexposed F₁ which were from F₀ exposed to 10 and 100 μg CBZ/L were down-regulated (Supplementary Figure 5).

Oxidative stress and respiratory burst were promoted, resulting to inflammatory response increased. Respiratory burst activity had a significant difference between the nonexposed and exposed F₁ at 5 dpf (Figure 5). In the nonexposed group, respiratory burst activity significantly increased in a typical concentration-response relationship at 3 concentrations. In the exposed group, respiratory burst in exposed F₁ showed a bell-shaped concentration-response relationship (Figure 5). Lower concentrations of CBZ induced excessive ROS production and caused oxidative stress in F₀ zebrafish, whereas at higher concentrations, CBZ affected host immunity and reduced ROS levels. Respiratory burst activity was also found to be reduced in the unexposed offspring of zebrafish after parental exposure to BPA (Xu et al., 2013), but found to be increased when zebrafish embryos were directly exposed to BPA (Dong et al., 2018).

F₁ exposed or nonexposed to CBZ until 21 dpf (group II)

The mainly effect of CBZ on the TLR signaling pathway as transmitted from parental exposure is likely reversible. Along with the development of adaptive immune system, the inhibitory effect of TLR by CBZ in unexposed F₁ was recovered at 21 dpf than at 5 dpf (group I). When F₁ were transferred in CBZ-free water to recovery until 21 dpf, the inhibition of the TLR signal pathway (tlr2, tlr3, tlr4, and myd88), rag-1, and cat was reversed. However, exposed F₁ generation was inhibited in the TLR signal pathway and immune system development (Figure 3A). F₁ exposed to CBZ until 21 dpf, tlr4 was significantly down-regulated at 3 concentrations; tlr2, myd88, trif, ifn, il-1β, il-8, and tnf-α were significantly downregulated at 10 and 100 μg/l; tlr3, lyz, rag-1, and ikaros were significantly downregulated at 100 μg/l (Supplementary Figure 6). However, only the mRNA expression of ikaros was significantly downregulated at 100 μg/l in exposed and nonexposed F₁ at 21 dpf (Supplementary Figure 6). IKAROS are used to encode early lymphoid development (Lam et al., 2004; Langenau and Zon, 2005; Trede et al., 2004; Yoder et al., 2002) and its change may be due to the parental (F₀) zebrafish because the downregulation range of ikaros was same in the exposed and nonexposed F₁ at 5 and 21 dpf (Figure 3A). Thus, parental exposure of 100 µg CBZ/L inhibited the development of lymphocytes in offspring and affected the development of the immune system, which did not disappear and was difficult to recovery.

The antioxidant defense system was a key feature to understand the immune response of zebrafish. CBZ exposure promoted ROS by the antioxidant defense system (especially mpx and gpx1a) inhibition. At 21 dpf, downregulations of mpx in 3 treatments and mn-sod, cat, and gpx1a at 10 and 100 μg CBZ/l were also observed in exposed F₁ zebrafish (Figure 3A). Moreover, the effects on antioxidant defense system in larvae zebrafish can recovery in clean water as the time went by (from 5 to 21 dpf). The high expression of inflammatory cytokines together with the inhibition of antioxidant system genes was resulted in the decrease of spawning. Studies have shown that inflammation and oxidative stress are associated with pregnancy, reproduction, and development (Hedger et al., 2018). The mouse model was used to demonstrate that the damage of placenta caused by placental inflammation and compromised antioxidant defense was the main cause of placental dysfunction (Raunig et al., 2011).

Problem scenario: F₁ exposed to CBZ with LPS until 5 dpf (group III) and 21 dpf (group IV)

The immune response is a defensive reaction against harmful substances such as viruses or bacteria, which enter the body (Liu et al., 2017). Normally, when fish come into with bacteria such as LPS, the immune system is activated to eliminate them so as to maintain their health, but when other toxic substances affect the immune system, resistance decreases resulting in adverse consequences (Novoa et al., 2009). Hence, LPS was added to test that larvae zebrafish will face pathogens in their life when they were laid by adult zebrafish exposed to environmentally relevant concentration CBZ. LPS stimulated antioxidant defense system and TLR signaling pathway that were previously suppressed by CBZ to respond, and the impairment of recognition and response pathogen in larvae zebrafish can be restored in CBZ-free water within a certain period of time. There was no significant difference in the survival rate of zebrafish at 5 and 21 dpf when adding LPS, which indicated that the adverse effects of CBZ on zebrafish immune system were not enough to cause death at this level of bacteria. F₁ exposed to CBZ had no effect on the movement trajectory at 5 dpf, however, when there was LPS invasion, the movement trajectory at 5 dpf significantly changed in the dark (Figure 6). Compared with the control group, the 5 dpf zebrafish was significantly excited after dark stimulation. It is strongly suggested that exposure to CBZ may lead to changes in individual behavior by affecting the immune system and inhibiting its resistance.

At 5 dpf, the inhibition of antioxidant genes (mn-sod, gpx1a, and mpx) and TLR pathway (tlr2, tlr3, tlr4, and myd88) had a recovery and upregulation trend in the nonexposed F₁ (10 μg/l) generation added LPS than that without LPS (Figure 3B and Supplementary Figure 5). These effects basically recovered after delaying time placed in CBZ-free water added LPS until 21 dpf (Supplementary Figs. S5 and S6). It may also be due to the late addition of bacteria in 4 dpf (19 dpf) or the lower concentration of LPS.

The attack of LPS can cause oxidative stress in organisms, and the subsequent antioxidant response can be used to resist the infection of LPS (Liu et al., 2010). Under the attack of LPS, the antioxidant stress gene in exposed F₁ zebrafish are generally inhibited. In F₁ zebrafish (5 dpf) exposed to CBZ and infected with LPS, mn-sod was significantly down-regulated at 10 μg/l and cat was significantly down-regulated at 10 and 100 μg/l while gpx1a was significantly upregulated at 100 μg/l (Supplementary Figure 5). mpx due to bacterial stimulation was significantly down- and upregulated at all 3 concentrations for 5 and 21 dpf, respectively. In addition, gpx1a was downregulated at all 3 concentrations for 21 dpf (Supplementary Figure 6). Further, due to the exposure of CBZ, LPS is not eliminated by the immune system but aggravates the damage to the immune system.

LPS can be recognized by TLR receptors, activating inflammatory genes and cytokines to resist simulation (Dong et al., 2018; Sigh et al., 2004; Watzke et al., 2007). In this study, CBZ destroyed the response of TLR signaling pathway to bacteria in F₁ zebrafish (5 dpf) continuously exposed to CBZ at 3 concentrations, and its inhibitory effect was still observed at the highest concentration in the nonexposed group. In addition, the stimulation of LPS will lead to the overexpression of tlr4 in exposed F₁ and nonexposed F₁ zebrafish until 21 dpf, which in turn leads to a significant increase of downstream cytokine IL-1β and stimulates inflammatory response. With the growth of zebrafish, the effect of CBZ exposure to the recognition function of tlr4 for LPS have changed from inhibition to over-activation. Therefore, more attention should be paid to the immune responses to resist pathogen in organism.

Additionally, the immunotoxicity pathway on F₀ zebrafish and F₁ larvae was summarized according to the above mechanisms as shown on Figure 7. We further mapped our results to relevant adverse outcome pathway (AOP) in the AOP-Wiki (www.aopwiki.org) to obtain more evidence support for the current findings. AOP 362 “Immune mediated hepatitis” and AOP 492 “Glutathione conjugation leading to reproductive dysfunction via oxidative stress” further supported that TLR signaling pathway mediated-inflammatory response and oxidative stress induced the hepatitis and impairment of fertility in adult zebrafish and decreased the survival of offspring.

Immune response and antioxidant system was affected in adult zebrafish (F₀) by CBZ exposure at environmentally relevant concentrations, mediated by TLR signaling pathway on gut-liver axis. The gut microbiome was affected by CBZ and increased the proinflammatory environment of the liver and triggered a proinflammatory cascade that worsens hepatic inflammation by TLR signaling pathway. These effects were similar in unexposed or exposed F₁ generation, which means these effects in offspring likely attributed to F₀ transmission. It also revealed the innate immune system at 5 dpf was sensitive window to observe F₁ immune response and other apical endpoints than at 21 dpf. Such transmission of effects, however, warrants further investigation, as our current study was not able to distinguish the F₁ effects from indirect exposure as germ cells or through inheritable epigenetic mechanisms. Consisted with the previous hypothesis, the impairment of defense against pathogens in F₀ generation transmitted to its offspring, but this impairment recovered as the immune system develops (Desforges et al., 2018; Dong et al., 2018; Lin et al., 2020). However, larva zebrafish live in the aquatic environment with ubiquitous CBZ and pathogens, when F₁ larva continued to be exposed to CBZ, the TLR signaling pathway to recognize LPS and the capacity of defense against pathogens was inhibited and not be able to recovered, which led to significant hyperactivity and even death. That provided a distinctive sight for us to care about chemical detected frequently with a lower concentration and the complex effects between chemical and stressors (eg, temperature, pathogens) in the field.

Supplementary data

Supplementary data are available at Toxicological Sciences online.

Funding

National Natural Science Foundation of China (22176095, 21677073); National Key Research and Development Program of China (2018YFC1801505).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Author contributions

Xuan Liu: Conceptualization, Writing—original draft, Writing—review & editing, Validation, Formal analysis, Visualization. Fan Liu: Investigation, Writing—original draft, Validation, Formal analysis. Li Liu: Investigation, Formal analysis. You Song: Supervision, Writing—review & editing. Hongling Liu: Conceptualization, Funding acquisition, Supervision, Writing—review & editing.

References