Apoptotic Changes, Oxidative Stress and Immunomodulatory Effects in the Liver of Japanese Seabass (Lateolabrax japonicus) Induced by Ammonia-Nitrogen Stress during Keep-Live Transport

Simple Summary Excreta produced by fish during preservation transport can increase ammonia-nitrogen (NH3-N) levels in transport water and reduce survival rates. However, studies on the effects of NH3-N stress on Japanese seabass during transport are limited. The objective of this study was to investigate the effects of NH3-N stress on tissue damage, oxidative stress, apoptosis and immunity in Japanese seabass during transport, and to determine tolerance and response to NH3-N toxicity. The results of this study provide insight into the impact of environmental factors on Japanese seabass during maintenance of live transport, which can be considered during transport in terms of improving the quality of transport water, among other things, to develop optimal procedures for live fish transport and improve survival rates during transport. Abstract This study investigated the effects of NH3-N on antioxidant responses, histoarchitecture, and immunity of Japanese seabass (Lateolabrax japonicus) during keep-live transport. The findings suggest that NH3-N stress transport alters the transcription of P53, Caspase 9, Bcl2, Caspase 3 and Bax genes, demonstrating that NH3-N stress can trigger the apoptotic pathway of P53-Bax-Bcl2 and Caspase and induce apoptosis. NH3-N stress transport also evoked transcriptional upregulation of inflammatory cytokines (tumor necrosis factor α (TNF-α), Toll-like receptor 3 (TLR-3), nuclear factor kappa β (NF-κB), interleukin 6 (IL-6) and interleukin 1β (IL-1β)) and increased complement C3, C4, lysozyme (LZM) and immunoglobulin (IgM) levels, activating the innate immunological system during keep-live transport. In addition, NH3-N stress transport altered changes in the levels of superoxide dismutase (SOD), catalase (CAT), glutathione-related enzymes, and heat shock proteins 70 and 90 in the liver, indicating that the antioxidant system and Hsp protected the cells from NH3-N-induced oxidative stress. When excess ROS were not removed, they caused the body to respond with immunological and inflammatory responses, as well as apoptosis and tissue damage. This helps towards understanding the effect of NH3-N levels on sea bass during keep-live transport.


Introduction
In China, Japanese seabass (Lateolabrax japonicus) is an economically important cultured fish. Nevertheless, high-density seabass keep-live transport causes ammonia-nitrogen

Experimental Fish
Japanese seabass (mean weight 400 ± 3.5 g, mean length 35 ± 1.0 cm) were obtained from the seafood market in Shanghai. The seabass were temporarily incubated in a 300 L food-grade tank in the laboratory for 36 h before transport experiments. Temporary incubation conditions were as follows: the water temperature was 20 • C, salinity was 16‰, pH was 7.5 and dissolved oxygen (DO) concentration was above 7.0 mg/L. To maintain high water quality, the water was replaced every day. After the transient period, the water was cooled from 20 to 12 • C at a rate of 3 • C/h.

Experiment to Determine LC 50 and Experimental Procedure:
A NH 4 Cl master mix was used during the experiment to adjust the NH 3 -N concentration to reach the required value for each experimental group. The range of NH 3 -N concentration was obtained from the lowest concentration where no fish died in 48 h (2 mg/L) to the highest concentration where all fish died (40 mg/L) by the pre-experiment and divided logarithmically. Six concentration gradients were established-2 mg/L, 3.6411 mg/L, 6.6289 mg/L, 12.0684 mg/L, 21.9712 mg/L, and 40 mg/L. Nine fish were used in each experimental group and this was repeated three times. The number of dead Japanese seabass was recorded on the 12th, 24th, 36th and 48th h. Dead fish were removed promptly (considered dead if they were stationary and did not respond to the surrounding stimuli) and water quality management was the same as during the transient period. The LC 50 = 21.32 mg/L at 48 h was calculated based on the survival rate.
LC 50 = 21.32 mg/L was obtained for sea bass by pre-experiment. Four groups were set up in the formal experiment: a control group (CK), a low NH 3 -N group (LA, 6.4 mg/L), a medium NH 3 -N group (MA, 12.8 mg/L), and a high NH 3 -N group (HA, 19.2 mg/L), which were equivalent to 30%, 60% and 90% of the 48 h LC50, respectively (Table 1). NH 3 -N concentration was measured using an NH 3 -N detector (DWS-296, Shanghai Insa Scientific Instruments Co., Ltd., Shanghai, China) to achieve the specified NH 3 -N levels. The fish were reared in sealed tanks with a fish-to-water ratio of 1:2. The dissolved oxygen (DO) concentration was guaranteed to exceed 7.0 mg/L, the water temperature was 20 • C, salinity was 16‰ and pH was 7.5 during transport. Live fish were simulated transport in a vibration conveyor in terms of 1 h on a class B road, followed by 5 h on a class A road. The cycles were repeated 8 times [12]. The numbers of dead fish were recorded at 0, 12th, 24th, 36th and 48th h, respectively. After 48 h transport, the surviving fish recovered for 24 h in seawater under transient conditions.

Sample Collection and Processing
Samples were taken once at the 0, 12th, 24th, 36th, and 48th h of NH 3 -N exposure and at the 12th and 24th h of recovery, respectively. Eighteen fish were sampled per experimental group at a time, for a total of three replicate groups, using nine fish per replicate. The livers of sea bass were immediately removed, washed with saline and divided into two parts. One part of the liver tissue samples were immersed for 24 h and fixed for histological and TUNEL assays, the remaining samples were stored at −80 • C qPCR index determination and enzyme index measurement.
Malondialdehyde (MDA) in the liver was measured using the TBA method according to the kit (A003-1-2, Jiancheng Bioengineering Institute, Nanjing, China) instructions and in nmol/mg protein. MDA is a peroxidized lipid degradation product that condenses with thiobarbituric acid (TBA) to form a red product with a maximum absorption peak of 532 nm.
The level of ROS in the liver was detected with fluorescent probe 2 ,7 -dichlorofluorescein diacetate (DCFH-DA) (E004-1-1, Jiancheng Bioengineering Institute, Nanjing, China). DCFH-DA can be oxidized by esterase into strong green fluorescent substance DCF (2 ,7 -dichlorofluorescein) in the presence of reactive oxygen species. The liver tissue was homogenized and made into a single cell suspension. 2 ,7 -dichlorofluorescein yellow diacetate was added to the suspension and then incubated for 1 h at 37 • C, centrifuged for 10 min, and single cells were collected and washed in PBS solution. The excitation wavelength of 500 nm and the fluorescence wavelength of 525 nm were used to determine the amount of ROS. These values were expressed as the following equation: ROS content (%) = f luorescence intensity o f treated cells f luorescence intensity o f control × 100

Apoptosis Detection
Samples were processed according to the method of He et al. [13]. Paraffin sections were made from liver tissue preserved in 4% formaldehyde solution. After dewaxing, hydration and cell permeation, the sections were assayed using a kit (Roche, Basel, Switzerland). TUNEL-positive cells were observed using a fluorescence microscope (Nikon, Tokyo, Japan). Positive apoptotic nuclei are green.

Histological Examination
The liver was dried, translucent, and embedded after being fixed for 24 h, and it was then sliced into 6 m slices using a slicer (Leica, Wetzlar Germany). The liver was examined under a light microscope (Leica, Wetzlar, Germany) after being stained with hematoxylin and eosin (HE). Then, the severity score is assigned to the degree and range of change: (−) no histopathology; (−) no histopathology; (+) <20% histopathology in the visual field; (++) 20-60% histopathology in the visual field; (+++) >60% histopathology in visual field [14].

Real-Time PCR
Total RNA was extracted from the liver sample using an RNA extraction solution (Xavier Biotechnology Co., Ltd., Wuhan, China). Ultramicro spectrophotometer (Nan-oDrop2000, Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the quantity and quality of RNA. Using 10 µL of total RNA (200 ng/µL), the single-stranded cDNA was created by the directions of Servicebio ® RT cDNA Synthesis Kit (G3330) (Xavier Biotechnology Co., Wuhan, China). The mRNA expression level of inflammatory factors and apoptotic genes was determined using RT-PCR. The total volume of the amplification reaction, which contained 2.0 µL of retrotranscription product (cDNA), 1.5 µL of 2.5 µM gene primer, 4.0 µL of water nuclease-free, and 7.5 µL of 2× qPCR Mix, was 15 µL. The reaction parameters were shown in Table 2. Primer sequences were designed according to the previously reported sequences (Table 3), and the 2 −∆∆ct technique was used to assess the degree of gene expression [15]. All samples were set in triplicate.

Oxidative Stress Response
The CAT, SOD, and ROS contents of each sample increased continuously with the transportation time, and the MN and HN groups had higher CAT, SOD, and ROS contents after 48 h of transportation ( Figure 1). The CAT, SOD, and ROS contents of all groups decreased to the initial levels. The Hsp 70 and Hsp 90 contents of each group increased with the transport time and reached the peak after 36 h of transport, and the Hsp 70 and Hsp 90 contents of the MN and HN groups were significantly higher than those of the CK group (p < 0.05).

Glutathione-Related Reactions
During the recovery process, GR and GSSG contents in ammonia stress-treated samples showed a decreasing trend, while GSH contents showed an increasing trend. The contents of GSH-Px and GST in the liver of the NH 3 -N stress group increased with the transport time and reached the peak at 48th h. The contents of GSH-Px and GST decreased with the recovery time ( Figure 2).

Tissue Damage and Liver Pathology
The results in Figure 3 show that the ALT, AST, and MDA contents of all groups increased continuously with the transportation time, and the ALT, AST, and MDA contents of the HN group were the highest after 48 h of transportation (p < 0.05), and the ALT, AST, and MDA contents of all groups showed a decreasing trend in the recovery period.
The liver color of the LN, MN, and LN groups was significantly darker than that of the CK after 48 h of NH3-N stress transport, especially the LN group. After 24 h of recovery, the livers of the LN, MN, and LN groups were significantly lighter compared to the 48 h of transport, but still darker compared to the CK (Figure 3).
In addition, histopathological damage of the seabass liver occurred to different degrees after 48 h of NH3-N stress transport ( Table 4). The CK liver showed normal hepatocytes with the neat arrangement, normal morphology, clear and intact boundaries, and nuclei located in the center of the cells (Figure 4). Hepatic tissues in the LN and MN groups showed hepatocyte vacuolization and hepatic sinusoidal dilatation compared with the CK (Table 4). Liver tissues in the HN group were more severely damaged, showing more hepatocyte vacuolization and hepatic sinusoidal dilatation, and the nuclei shifted to the periphery and the cell boundaries blurred.

Tissue Damage and Liver Pathology
The results in Figure 3 show that the ALT, AST, and MDA contents of all groups increased continuously with the transportation time, and the ALT, AST, and MDA contents of the HN group were the highest after 48 h of transportation (p < 0.05), and the ALT, AST, and MDA contents of all groups showed a decreasing trend in the recovery period.
The liver color of the LN, MN, and LN groups was significantly darker than that of the CK after 48 h of NH 3 -N stress transport, especially the LN group. After 24 h of recovery, the livers of the LN, MN, and LN groups were significantly lighter compared to the 48 h of transport, but still darker compared to the CK (Figure 3).
In addition, histopathological damage of the seabass liver occurred to different degrees after 48 h of NH 3 -N stress transport ( Table 4). The CK liver showed normal hepatocytes with the neat arrangement, normal morphology, clear and intact boundaries, and nuclei located in the center of the cells (Figure 4). Hepatic tissues in the LN and MN groups showed hepatocyte vacuolization and hepatic sinusoidal dilatation compared with the CK (Table 4). Liver tissues in the HN group were more severely damaged, showing more hepatocyte vacuolization and hepatic sinusoidal dilatation, and the nuclei shifted to the periphery and the cell boundaries blurred.

Immune Response and Inflammatory Factors
The results in Figure 5 show that the contents of C3, C4, IgM, and LZM decreased continuously with the transport time. The contents of C3, C4, IgM, and LZM increased during the recovery process. After 24 h of recovery, these parameters were still lower in the HN group than in the CK.
The expressions of TLR-3, NFκB, TNF-α, IL-6 and 1L-1β were first increased and then decreased in all groups. With the increase in NH 3 -N transport stress time, the expression of TLR-3, NFκB, TNF-α, IL-6 and 1L-1β in the CK and LN groups peaked at 24th h, while the gene expression of each pro-inflammatory factor in the MN and HN groups peaked at 36th h ( Figure 5).

Apoptosis
As shown in Figure 6, positive TUNEL signals were detected in all groups after 48 h of NH3-N stress transport. The number of apoptotic cells was HN < MN < LN < CK. After 24 h of recovery, the number of positive cells declined in each group.
The peak expression levels of P53, Caspase 3, Caspase 9 and Bax genes in the CK and

Apoptosis
As shown in Figure 6, positive TUNEL signals were detected in all groups after 48 h of NH 3 -N stress transport. The number of apoptotic cells was HN < MN < LN < CK. After 24 h of recovery, the number of positive cells declined in each group.  The peak expression levels of P53, Caspase 3, Caspase 9 and Bax genes in the CK and LN groups appeared at 24th h, while the peak expression levels of P53, Caspase 3, Caspase 9 and Bax genes in the CK and LN groups appeared at 36th h (Figure 7).

The impact of NH3-N Stress Transport on the Antioxidant Defense System of the Liver of Japanese Seabass
ROS are increased in concentration by ammonia, which causes oxidative stress in living things [1]. Organisms reduce ROS by increasing the levels of antioxidant enzymes (e.g., CAT, GST and SOD) to reduce ROS-induced oxidative damage. The initial line of defense in preventing the harm that oxidative stress causes is thought to be SOD-CAT and SOD-GPX [22]. Superoxide is converted to H2O2 by SOD, which is then reduced to water by CAT or GPX [23]. Because it maintains metabolic homeostasis and removes toxins, the liver is the ideal organ for assessing oxidative stress [24]. ROS produced by Japanese seabass under NH3-N stress transport could activate its antioxidant defense system, which prevented the damage caused by NH3-N and transport stress to the organism. A similar finding was made by Cheng et al. [1], where excessive ROS production caused by NH3-N production resulted in increased antioxidant enzyme activity in red puffer fish. Liu et al. [14] discovered that during the start of NH3-N stress, the juvenile golden pompano (Trachinotus ovatus) had an increase in antioxidant enzyme activity, and the antioxidant enzyme activity decreased when it returned to the normal environment. ROS induced by higher NH3-N concentrations were not eliminated by the organisms after 48 h. Chen et al. [25] found that snapping turtle (Chelydra serpentina) exposed to high

The impact of NH 3 -N Stress Transport on the Antioxidant Defense System of the Liver of Japanese Seabass
ROS are increased in concentration by ammonia, which causes oxidative stress in living things [1]. Organisms reduce ROS by increasing the levels of antioxidant enzymes (e.g., CAT, GST and SOD) to reduce ROS-induced oxidative damage. The initial line of defense in preventing the harm that oxidative stress causes is thought to be SOD-CAT and SOD-GPX [22]. Superoxide is converted to H 2 O 2 by SOD, which is then reduced to water by CAT or GPX [23]. Because it maintains metabolic homeostasis and removes toxins, the liver is the ideal organ for assessing oxidative stress [24]. ROS produced by Japanese seabass under NH 3 -N stress transport could activate its antioxidant defense system, which prevented the damage caused by NH 3 -N and transport stress to the organism. A similar finding was made by Cheng et al. [1], where excessive ROS production caused by NH 3 -N production resulted in increased antioxidant enzyme activity in red puffer fish. Liu et al. [14] discovered that during the start of NH 3 -N stress, the juvenile golden pompano (Trachinotus ovatus) had an increase in antioxidant enzyme activity, and the antioxidant enzyme activity decreased when it returned to the normal environment. ROS induced by higher NH 3 -N concentrations were not eliminated by the organisms after 48 h. Chen et al. [25] found that snapping turtle (Chelydra serpentina) exposed to high ambient NH 3 -N had a high level of basic antioxidant defense, that most likely relied mostly on the GSH system to eliminate extra ROS. GSH/GSSG is also considered one of the essential antioxidant systems in cells, and its stable state is crucial for maintaining the normal physiological functions of cells. NH 3 -N stress transport resulted in a decline in GSH content and a gradual rise in GSSG content in the liver of seabass in the current study, which is due to GSH being converted to GSSG by GSH-Px and GST in order to scavenge intracellular free radicals and peroxides, thus maintaining normal cell and tissue function and metabolism [23]. After 48 h of NH 3 -N stress transport, GR activity increased significantly and subsequently decreased. However, the increase in GSSG content was still higher than the decrease in GSH content, indicating that the increase in GR did not offset the GSH produced by GST and GSH-Px. After a period of recovery, GSH and GSSG did not reach their initial states and the glutathione system had not recovered for short-time equilibrium.
Molecular chaperones, such as Hsp, play an essential role in preventing cellular damage from oxidative stress by ensuring the correct folding of proteins to regulate cellular homeostasis [26]. High NH 3 -N promotes the production of Hsp90 and Hsp70, according to studies [27], which suggests that the buildup of too much ROS and denatured proteins causes the elevation of Hsp levels during NH 3 -N stress. According to the study's findings, Hsp70 and Hsp 90 levels were significantly elevated during NH 3 -N stress transport compared to CK. These findings are in line with the patterns of Hsp70 and Hsp90 gene expression after NH 3 -N exposure in kuruma shrimp (Marsupenaeus japonicus) and European seabass (Dicentrarchus labrax) [27,28]. Hsp has a key function in the prevention of protein synthesis, repair and degradation as well as apoptosis in Japanese seabass exposed to NH 3 -N stress. Hsp upregulation may be an effective measure to protect cells from oxidative stress [29].

The Impact of NH 3 -N Stress Transport on Liver Tissue Damage in Japanese Seabass
Excess ROS produced by NH 3 -N stress are not scavenged in time and are responsible for lipid peroxidation, inflammation and cellular damage [30]. The accumulation of ROS generates lipid peroxides (e.g., MDA) that cause cellular damage [31]. MDA levels increase after NH 3 -N stress transport and reflect the NH 3 -N generated in the liver of Japanese seabass oxidative stress, which can lead to liver damage. Peacock fish (Poecilia reticulate) were shown to exhibit elevated levels of ROS and MDA as well as altered antioxidant enzyme activity during NH 3 -N stress in a study by Zhang et al. [32]. However, the MDA content started to decrease after 24 h of recovery, showing that the harm caused by NH 3 -N stress transport was being gradually counteracted. After a period of recovery, the transport-induced tissue damage caused by NH 3 -N stress can revert to its original state.
The toxicity of a substance can be determined by measuring elevated AST and ALT activity, which are important markers of tissue damage [22,33]. Ali Taheri Mirghaed et al. [34] found the AST and ALT levels increased in common carp (Cyprinus carpio) after 24 h of NH 3 -N stress. Zhao et al. [35] found that NH 3 -N stress exposure resulted in elevated ALT and AST levels, indicating NH 3 -N stress caused tissue damage in juvenile yellow catfish (Pelteobagrus fulvidraco). In comparison to the CK group, the HN group's liver was much darker and more congested. NH 3 -N poisoning can cause changes in the color and appearance of the liver during NH 3 -N stress [36,37]. NH 3 -N stress transport resulted in hepatotoxicity, which was characterized by histopathological lesions, such as vacuolation of hepatocytes, dilated hepatic sinusoids, indistinguishable cell contours, deviated nuclei, and nuclei disintegration. The damage to liver tissue was significantly reduced through photograms and histopathological optical microscopy, but it was not enough to recover to the initial level within 48 h. This indicates that the damage caused by NH 3 -N toxicity is reversible. Furthermore, Yan et al. [23] found that tissue sections of bighead carp (Aristichthys nobilis) after nitrite stress also revealed that liver damage caused by nitrite toxicity could be alleviated to some extent after a recovery period.

The Impact of NH 3 -N Stress Transport on Immunological and Inflammatory Responses in Japanese Seabass
The immunological response is a self-protective mechanism for fish against various infections. To combat infectious challenges, fish have evolved different immune systems, including lysozyme and complement. LZM and IgM maintain a healthy state in fish by eliminating invading bacteria and other pathogens while activating the complement system [38]. Studies have shown that a variety of environmental pollutants can inhibit complement C3, complement C4, IgM levels and LZM activities, thus affecting the immunological parameters of scleractinian fish [14,39]. Esam et al. [11] found the mRNA levels of complement C3 and LZM decreased, resulting in immunotoxicity when the combined ammonia-nitrogen and heat stress was applied to Nile tilapia (Oreochromis niloticus). In our study, complement C3, complement C4, IgM levels and LZM activities of seabass decreased after 48 h NH 3 -N stress transport, indicating a continued decline in immunological function. Gao et al. [40] also found that Takifugu rubripes (T. rubripes) showed a considerable decline in complement C3, complement C4, IgM levels and LZM activities after 12 h of NH 3 -N stress at different concentrations. After 24 h of recovery, the immunological parameters increased due to the weakening of oxidative stress and the gradual recovery of the immunological system from the toxic environment.
Fish that are under oxidative stress develop an inflammatory reaction. To control inflammation, TLR activation triggers the expression and release of cytokines. The results of this research concur with those of Gao et al. [41], who discovered that NH 3 -N exposure raised the gene level of TLR-3 and activated the TLR-3 signaling pathway. TNF-α is regarded as a crucial component of the TNF family, which causes inflammation and stimulates the immune system. The activated TNF-α could activate NF-κB channels. NF-κB, as a signaling factor, can activate several pro-inflammatory factors, including IL-6 and IL-1β. In addition, The NF-κB pathway can also be activated by an excessive buildup of ROS, which can worsen the inflammatory response. Our experimental results suggest that both NH 3 -N stress and transport stress activated the NF-κB inflammatory pathway to exert anti-inflammatory effects. This was in line with previous studies on gibel carp (Carassius gibelio) and guppies (Poecilia reticulate), where it was discovered that NH 3 -N stress induced the expression of genes linked to inflammation, resulting in an inflammatory response [32,42]. Furthermore, we found that the different timing of peak gene expression of anti-inflammatory factors may vary depending on the NH 3 -N concentration.

The Impact of NH 3 -N Stress Transport on Apoptosis in Japanese Seabass
In addition to inducing inflammation, oxidative stress also leads to apoptosis. The oncogene P53 is a crucial regulator of apoptosis, and ROS production brought on by NH 3 -N stress can modulate the activity of P53. In response to stress, P53 alters the expression of Bcl2 and Bax to induce apoptosis. The increase in the ratio of Bax to Bcl2 can be used to determine the apoptosis of the organism [1]. In the current research, the Bax/Bcl2 ratio increased in all experimental groups during NH 3 -N stress transport, suggesting that NH 3 -N stress transport could cause cell apoptosis via the P53-Bax-Bcl2 pathway. To identify stress-induced apoptosis, Caspase expression is a crucial marker. According to Li et al. [43], that NH 3 -N exposure upregulated genes Caspase 3 and Caspase 9 and induced cell apoptosis. The current work showed that the expression of Caspase 9 and Caspase 3 was raised by both NH 3 -N exposure and transport stress, which also suggested that Caspase-dependent apoptotic pathways might be implicated in the cell death generated by transport stress of NH 3 -N.
The results of TUNEL staining showed green fluorescent spots, which verified the hypothesis that NH 3 -N stress transport leads to apoptosis. The P53-Bax/Bcl2 apoptotic route and the Caspase-dependent apoptotic pathway may both be affected by NH 3 -N stress in Yellow catfish (Pelteobagrus fulvidraco) [36]. This is in line with what the current experimental study's findings show. These findings also imply that oxidative stress and inflammation may be intimately related to cell apoptosis brought on by simultaneous exposure to these two stressors.

Conclusions
This research was the first to investigate the influence of NH 3 -N on oxidative stress, apoptosis, tissue damage and immunological response in Japanese seabass during keep-live transport. The histopathological and TUNEL results confirmed that ammonia and nitrogen stress during transport could produce ROS, resulting in oxidative stress, and that eliminated ROS through the glutathione system, but this system failed to provide adequate protection when exposed to highly oxidizing conditions. Excess ROS was involved in NH 3 -N stressinduced apoptosis through the P53-Bax-Bcl2 pathway and the cysteine-dependent apoptosis pathway and induced an inflammatory response via the NF-κB pathway. These defenses declined during the recovery period, but did not return to the normal levels compared to CK. The findings of this research help towards a thorough understanding of how environmental factors effect seabass during keep-live transport, which helps towards developing optimal procedures for transport and improving the survival rate during transport.