1 INTRODUCTION

Reactive oxygen species (ROS) are commonly formed in all aerobic biological systems, but the overproduction of ROS in organisms can induce oxidative injury, lipid peroxidation, cell membrane destruction, apoptosis, and cell death (Nordberg and Arnér, 2001; An et al., 2008; Nwani et al., 2013; Kim et al., 2019). To antagonize the detrimental effects due to excessive ROS, aerobic organisms, through evolution, have developed complex antioxidant defense systems using antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and peroxidases (Afonso et al., 2007; Yu et al., 2017). SOD catalyzes the dismutation of superoxides into oxygen (O₂) and hydrogen peroxide (H₂O₂), which is then alleviated by CAT and GPx (Zhang et al., 2018).

CAT, a major antioxidative oxidoreductase that exists virtually in all aerobic organisms is involved in several cellular processes, such as apoptosis, mutagenesis, and inflammation (Bandyopadhyay et al., 1999; Klotz and Loewen, 2003). To date, numerous catalase genes isolated from different organisms have been cloned and studied (Gerhard et al., 2000; Yamamoto et al., 2005; Li et al., 2008). CAT isolated from several species of fish, including Danio rerio (Ken et al., 2000), Sebastes schlegelii (Elvitigala et al., 2015), Liza haematocheila (Qi et al., 2015), Oplegnathus fasciatus (Elvitigala et al., 2013), Megalobrama amblycephala Yih (Sun et al., 2014), and Larimichthys crocea (Yan et al., 2017) have been cloned and expressed. To study the roles and molecular evolution of fish CAT, characterization of CAT from a diverse range of species is required.

Turbot (Scophthalmus maximus), a large-sized demersal fish naturally distributed in the coasts of Europe at Northeast Atlantic is known for its rapid growing speed and low-temperature resistance (Qin et al., 2008). Turbot has been bred with an increasing scale since its introduction to China in 1992. However, vibriosis, especially the infection caused by Vibrio anguillarum has caused tremendous economic loss to the turbot breeding industry. A study has confirmed that the pathogenicity of V. anguillarum is related to various internal virulence factors, such as extracellular protease, lipopolysaccharide, flagella, and hemolysin (Ge et al., 2014). Among these factors, extracellular protease, which is a zinc-containing metalloprotease has been shown to be associated with strong pathogenicity (Denkin and Nelson, 2004; Rock and Nelson, 2006). Extracellular metalloprotease has been considered as a pivotal virulence factor involved in the pathogenic mechanism of V. anguillarum. Chen et al. (2009) have found that extracellular metalloprotease purified from V. amguillarum can induce tissue damage and death of the fish. Chi (2006) has confirmed that extracellular metalloprotease of V. anguillarum is obviously toxic to flounder gill cells by inducing apoptotic corpuscles and cell disaggregation. Extracellular metalloprotease isolated and purified from V. anguillarum has been found to be lethal to mice (Inamura et al., 1985). The above studies imply that extracellular metalloprotease of pathogens can generate toxicity and pathogenicity to organisms. The protease may attack the defense system of the host organism to induce tissue damage and create conditions that facilitate infection of the pathogen (Wei et al., 2002; Chen, 2003). To date, there is no data about the effects of metalloprotease on the antioxidative status and ROS index, as well as on apoptosis in the head kidney cells of turbot.

The aim of this study was to clone full-length cDNA encoding CAT from the liver of S. maximus and compare its sequence with other known CATs. Then we wished to: clarify the expression profile of S. maximus catalase (SmCAT) in various tissues from S. maximus, analyze the expression profiles of manganese superoxide dismutase (MnSOD), CAT, and GPx in head kidney cells of S. maximus after metalloprotease treatment, measure ROS and mitochondrial membrane potential from head kidney cells, and finally evaluate apoptosis in these cells by fluorescent microscopy. These results provide a systematic method to understand the mechanism of metalloprotease-induced toxicity, especially apoptosis, in the head kidney cells of S. maximus.

2 MATERIAL AND METHOD 2.1 Animals and isolation of head kidney cells

Turbots weighing about 45 g were obtained from a local farm in Changli, China. The turbots, ten per 160-L breeding tank, were raised in aerated clean seawater with 19 salinity and > 6.0-mg/L dissolved O₂ at 16±1 ℃.

Turbot primary head kidney cells were cultured as previously described (Wang et al., 2010; Bain and Schuller, 2012) but with slight modification. Briefly, the fish were anesthetized with clove oil and surfacedisinfected with 75% ethanol, following which the head kidney tissues were collected and transferred to a clean bench. The kidney tissues were rinsed several times with phosphate buffer solution (pH 7.4), and added with 400-U/mL penicillin and 400-μg/mL streptomycin before being cut with scissors. The tissues were then placed in 15-mL centrifugal tubes where 10 volumes of 0.25% parenzyme were added for digestion for 30 min at room temperature. Trypsinization was terminated using 2 mL of L-15 complete medium (200-U/mL penicillin, 200-μg/mL streptomycin, 20% fetal bovine serum, 100-ng/mL beta fibroblast growth factor). After filtration using a 50-mesh filter screen, the resulting cell suspension was transferred into 15-mL centrifuge tubes for centrifugation at 1 600 r/min for 5 min. After discarding the supernatant, the cells were adjusted to a concentration of 5×10⁵ cells/mL before being inoculated on cell culture plates and cultured in a CO₂ incubator at 25 ℃. The L-15 complete medium of the cell culture was semi-exchanged at an interval of 2–3 d.

2.2 Total RNA isolation and cDNA preparation

Total liver RNA was immediately isolated from freshly harvested cells using an RNAiso Plus device (TaKaRa, Japan). The quantity and quality of isolated RNA were evaluated using a ND-1000UV spectrophotometer (NanoDrop, USA) and 1.0% agarose electrophoresis, respectively. The OD260/ OD280 ratio of isolated RNA varied from 1.8 to 2.0. First-strand cDNA was synthesized using M-MLV reverse transcriptase (TaKaRa). The 5' and 3' rapid amplification of cDNA ends (RACE) cDNA templates were obtained using a SMART^TM cDNA kit (TaKaRa). All procedures were conducted as recommended by the manufacturers.

2.3 Cloning of S. maximus CAT

Full-length cDNA of SmCAT was obtained by reverse-transcription polymerase chain reaction (RTPCR) and RACE. Conserved regions in the central fragment of SmCAT cDNA sequence were detected with two degenerate primers CAT-F2 and CAT-R2 (Table 1) and by sequence alignment with the corresponding genes of O. fasciatus, Rachycentron canadum, S. schlegelii, Siniperca chuatsi, Sparus aurata, and Totoaba macdonaldi. The following thermal cycling condition was used: initial denaturation at 94 ℃ for 5 min, followed by 33 cycles of denaturation at 94 ℃ for 35 s, annealing at 57 ℃ for 35 s and elongation at 72 ℃ for 70 s, and final extension at 72 ℃ for 8 min before conservation at 4 ℃. Finally, CAT cDNA with a length of about 1 046 bp was amplified.

Based on the partial sequence of CAT, the 5' and 3' ends of the above fragment sequence of SmCAT were acquired using SMART^TM RACE cDNA kit (Clontech). The amplification reactions for both 5' and 3' ends involved the use of RACE cDNA templates from liver RNA, GSP-R1, and universal anchor primer (UPM, Table 1). The PCR was conducted as recommended by Clontech.

Amplified products were analyzed on 1% agarose gel, in which the DNA fragment of interest was excised and purified using gel isolation kit (TaKaRa). Purified DNA was then subcloned into pMD18-T cloning vector as per manufacturer's introduction. The resulting DNA construct was used for Escherichia coli DH5α transformation. Positive clones containing the DNA insert were screened by PCR before being verified by DNA sequencing (Sangon Biotech. Co. Ltd., Beijing, China).

2.4 Sequence analyses

Comparative investigation of nucleotide and estimated amino acid sequences of SmCAT cDNA were analyzed using BLAST (www.blast.ncbi.nlm.nih.gov/Blast). Translation and protein analysis were performed using ExPASy tools (//us.expasy.org/tools/). Prediction of signal peptide was carried out using Signal P-4.1 (www.cbs.dtu.dk/services/SignalP/). Multi-alignment of turbot CAT was executed using DNAMAN. Phylogenetic tree of CAT was conducted by neighbor-joining on MEGA 4.0. Statistical support for reliability was tested using 1 000 bootstrap replications (Tamura et al., 2007).

2.5 Differential expression profiles of SmCAT by qRT-PCR

To detect the tissue distributions of SmCAT expression in healthy turbots, total RNA from healthy S. maximus tissues (blood, gill, kidney, liver, stomach, muscle, heart and intestine) was reverse transcribed into cDNA using gDNA Eraser (TaKaRa, China) in accordance to the manufacturer's recommended protocol. The expression levels of CAT mRNA transcript in each tissue were evaluated by quantitative RT-PCR (qRT-PCR). The PCR primers of SmCAT (Q-CAT-F and Q-CAT-R) were chosen based on analysis by Primer Premier 5.0. The expression of β-actin was used as internal housekeeping control (GenBank accession number: AY008305). The primer sequences of β-actin-F and -R (Table 1) were based on a previously published study (Yun et al., 2012).

2.6 Expression of antioxidant genes after metalloprotease challenge

Extracellular metalloprotease of V. anguillarum was obtained as previously described (Yang et al., 2007). The protein concentration was quantified using Bradford method with bovine serum albumin as the reference.

Cells collected from cultivation flasks were subjected for qRT-PCR analysis using SYBR premix Ex Taq^TM (TaKaRa, China) with SYBR green I in a CFX96 device (Bio-Rad, US). S. maximus head kidney cells, after being cultured for 3, 6, 12, or 24 h, were sampled using 0-, 1.6-, 8.0-, or 40.0-μg/mL metalloprotease, following which the expression levels of MnSOD, CAT, and GPx were examined. The sequence-pecific primers for SmMnSOD (Q-MnSOD-F and Q-MnSOD-R), SmCAT, and SmGpx (Q-GPx-F and Q-GPx-R) used in qRT-PCR were based on analysis using Primer Premier 5.0 (Table 1).

Each qRT-PCR reaction system (10 μL), performed in triplicates, comprised SYBR Premix Ex Taq^TM Ⅱ (TaKaRa; 5 μL), RT solution (1 μL), each primer (10 μmol/L, 0.2 μL) and ultrapure water (3.6 μL). The thermal cycling conditions were performed as per recommended by the manufacturer. The threshold cycle value (C_t) of each PCR thermal profile estimated on CFX96^TM real-time PCR 1.0 was used to calculate ΔC_t of each sample. The relative expression of SmMnSOD, SmCAT, and SmGPx were determined by 2^-ΔΔCt (Livak and Schmittgen, 2001).

2.7 Detection of ROS levels

The levels of ROS were detected using 2', 7'-dichlorofluorescein-diacetate (DCFH-DA, Beyotime, China) using the manufacture's recommended protocol. Briefly, head kidney cells cultivated in 96-well plates (5×10⁵ cells/well) were treated with 40-μg/mL metalloprotease. At different time points after metalloprotease treatment, the cells were cultivated with 10-mmol/L DCFH-DA at 25 ℃ for 20 min in darkness, and then washed 2 times with PBS and filtrated through a 200-mesh nylon mesh. The fluorescent signal intensity of DCF was analyzed by a flow cytometer (Becton Dickinson, US).

2.8 Measurement of mitochondrial membrane potential (Δψ_m)

Variations in Δψ_m were assessed using JC-1 kit (Beyotime, China) in accordance to the protocol recommended by the manufacturer. Briefly, cells cultured in 96-well plates were treated with metalloprotease. After different time points, the Δψ_m of metalloprotease-treated cells was measured in triplicates. Prior to Δψ_m measurement, cells were rinsed with PBS, collected and incubated with the JC-1 staining solution (1×) for 20 min at 25 ℃. Cells were then rinsed with JC-1 buffer two times before being analyzed by flow cytometry. The Δψ_m of each test group was computed as the ratio of red (aggregates) to green (monomers) fluorescence signals as previously described (Cui et al., 2011).

2.9 Hoechst 33342 assay

To distinguish normal cells from apoptotic cells at 12 and 24 h, cells from the normal and metalloprotease-treated groups (8.0 and 40.0 μg/mL) were stained with 5.0-μg/mL Hoechst 33342 (Beyotime) for 20 min at 25 ℃ according to the protocol recommended by the manufacturer. Cells were then washed in PBS twice. The morphological changes in the cell nucleus were observed under a fluorescence microscope (Nikon, Japan) to distinguish between the condensed chromatin of apoptotic cells and the looser chromatin of living cells.

2.10 Statistical analyses

All experimental data are shown as mean ± standard deviation (n=3). The data of qRT-PCR were analyzed by one-way analysis of variance (ANOVA). Different treatments were compared using multiple-comparison (Tukey) on SPSS 20.0. P < 0.05 implies statistical significance.

3 RESULT 3.1 Characterization and bioinformatic analysis of SmCAT sequence

The full-length SmCAT cDNA obtained by RACE (Fig. 1) was submitted to GenBank database (No. MG253621). The 3 174-bp SmCAT cDNA with a poly(A) end contains a 1 584-bp open reading frame (ORF) that encodes a 527-amino-acid protein (Fig. 1) with a molecular mass of 59.44 kDa and a theoretical isoelectric point of 6.83. Analysis by PROSITE revealed that the SmCAT amino acid sequence contains a proximal active-site signature sequence (⁶⁴FDRERIPERVVHAKGAG⁸⁰), a proximal hemeligand signature sequence (³⁵⁴RLFSYPDTH³⁶²), and three conserved catalytic amino acid residues (His⁷⁵, Asn¹⁴⁸, and Tyr³⁵⁸).

3.2 Homology and phylogeny analyses of SmCAT

Multiple sequence alignment showed that the deduced sequence of SmCAT was highly similar with CAT of Paralichthys olivaceus (92.4%), Lates calcarifer (91.8%), O. fasciatus (91.3%), Rachycentron canadum (91.3%), Seriola dumerili (92.0%), Bos taurus (77.8%), Portunus trituberculatus (67.6%), and Litopenaeus vannamei (66.9%). The signature sequences (⁶⁴FDRERIPERVVHAKGAG⁸⁰ and ³⁵⁴RLFSYPDTH³⁶²) and catalytic residues (His⁷⁵, Asn¹⁴⁸, and Tyr³⁵⁸) were found to be largely conserved in diverse species (Fig. 2).

The identity and phylogeny of SmCAT were further validated using phylogenetic tree built on MEGA 4.0 with selected CAT sequences (Fig. 3). The CAT gene family can be broadly separated into invertebrates and vertebrates. SmCAT was found to be highly related to the CATs of some fish species, but less related to the CATs of Homo sapiens, Cavia porcellus, B. taurus, and Mus musculus.

3.3 Basal tissue expression of SmCAT mRNA

The mRNA expression of SmCAT in eight tissue types of healthy turbot, including blood, liver, kidney, intestine, stomach, heart, gill, and muscle was evaluated (Fig. 4). Our results show that CAT was ubiquitously expressed in all tested tissues. The dissociation curves of SmCAT and β-actin only showed one peak, indicating that the amplifications were specific. SmCAT was found to have the lowest expression level in muscle tissues and the highest expression level in blood. The expression of SmCAT was observed to be moderate in other tissue types.

3.4 Effects of metalloprotease on the expression of antioxidant genes

Temporal expression of antioxidant genes (SmMnSOD, SmCAT, and SmGPx) in head kidney cells at designated time points after metalloprotease treatment are displayed in Fig. 5. Whilst the expression of SmMnSOD in different concentration groups were unchanged at 3 h after treatment (P > 0.05); the expressions of SmCAT and SmGPx were dramatically higher (P < 0.05) than that of the control group. The gene expression of SmMnSOD, SmCAT, and SmGPx in cells treated with metalloprotease at all tested concentrations from 12 to 24 h were found to be dramatically downregulation. Hence, metalloprotease induces the expression of SmMnSOD, SmCAT, and SmGPx in head kidney cells at different levels within the 24-h exposure.

3.5 Effects of metalloprotease exposure on ROS level

ROS production in head kidney cells after treatment with metalloprotease is shown in Fig. 6. Flow cytometry indicated that the accumulation of ROS is higher in metalloprotease-treated cells relative compared to that of untreated cells (Figs. 6–7). ROS production in head kidney cells, compared to that of the control group, was found to be increased significantly in all treatment groups (P < 0.05), except for the 3-h treatment group.

3.6 Effects of metalloprotease exposure on mitochondrial membrane potential (Δψ_m)

To explore the participation of mitochondria-modulated pathway in metalloprotease-induced apoptosis, the Δψ_m in head kidney cells was measured through flow cytometry and JC-1 staining. Our results showed that metalloprotease dose-dependently caused the loss of Δψ_m, which dropped considerably from 6 to 24 h compared to that of the control group (Fig. 8)

3.7 Effects of metalloprotease treatment on cell morphology

Next, the morphology of metalloprotease-induced head kidney cells was visualized by fluorescent microscopy. As shown in Fig. 9a & d, untreated cells were stained uniformly with blue fluorescence, suggesting that the chromatin was evenly distributed in the nucleolus, implying a normal healthy shape. Conversely, as shown in Fig. 9b, c, e, & f, cells after incubation with 8.0- or 40.0-μg/mL metalloprotease for 12 and 24 h displayed condensed chromatin and shrunken nucleus to some extent. The number of Hoechst-positive cells was increased, depending on metalloprotease treatment time and dosage, compared to that of untreated cells.

4 DISCUSSION

As a major prevalent bacterial pathogen, V. anguillarum has been reported as the cause of severe infections and high mortality in many marine and freshwater fishes (Planas et al., 2005; Toranzo et al., 2005). Extracellular metalloprotease, a critical virulence index of V. anguillarum has been shown to be lethal to a number of fish species (Varina et al., 2008; Han et al., 2011). To our knowledge, there is currently no report about the effects of V. anguillarum extracellular metalloprotease on the expression of antioxidant genes and on cell apoptosis in turbots. To better understand the antioxidative resistance of turbots in response to the virulence factor, we cloned the cDNA of SmCAT, and then investigated the expression of antioxidant enzymes (SmMnSOD, SmCAT, and SmGPx), ROS level, Δψ_m, and apoptosis in the head kidney cells of S. maximus under metalloprotease stress.

In this study, we found that the SmCAT amino acid sequence contained a proximal heme ligand-binding site (³⁵⁴RLFSYPDTH³⁶²), an active-site signature sequence (⁶⁴FDRERIPERVVHAKGAG⁸⁰) and three conserved catalytic amino acid residues (His⁷⁵, Asn¹⁴⁸, and Tyr³⁵⁸). The catalytic and peroxidase activities of CATs may rely on a charge-relay network to stabilize the reactive intermediates in order to repair the resulting damage by enhancing the peroxide cleavage (Sellaththurai et al., 2019). Residues His⁷⁵, Asn¹⁴⁸, and Tyr³⁵⁸ that are pivotal in the charge-relay network have been reported as the basic residues required to complete the enzymatic activity (Heck et al., 2010). Our phylogenetic analysis further illustrates the existence of two extensive categories of CATs (vertebrates and invertebrates) and SmCAT that was found to be clustered into the vertebrate category was most closely related to the enzyme from O. fasciatus. The observed relationships within CAT homologues indicate that SmCAT may be evolutionally distant from CATs of invertebrates.

The evaluation of tissue expression distribution showed that SmCAT is expressed in all tissue types examined. The basal expression level of SmCAT was found the highest in blood followed by liver, mainly due to the different metabolic activities associated with ROS production among these tissues (Ekanayake et al., 2008). Blood cells and phagocytes are critical in removing pathogen invasion through oxygen consumption to form ROS (Lee and Söderhäll, 2002). The liver is also a site where various oxidative reactions occur (Gül et al., 2004; Avci et al., 2005), hence, liver cells are prone to oxidative stress due to the high metabolic activity (Kamata et al., 2005). It has been suggested that blood and liver tissues are important for modulating redox homeostasis in turbots, and that CAT may play an essential role in detoxifying oxygen-free radicals and avoiding their overproduction in organisms (Tavares-Sánchez et al., 2004). Additionally, differentially expressed SmCAT mRNA transcripts among various turbot tissues may be associated with the oxidative levels of those tissues (Sellaththurai et al., 2019).

Antioxidative resistance systems are critical in preventing oxidative damage due to excess ROS (Kim et al., 2012). As the major constituents of antioxidative defense mechanism, SOD, CAT, and GPx can orchestrate cell defense against oxidative stress-induced cell injury, prevent peroxidation and balance cellular redox status (Lortz et al., 2000). In this study, the expression of SmCAT and SmGPx in cells at 3 h after exposure to different concentrations of metalloprotease dramatically surpassed that of untreated cells, indicating that the expression of antioxidant enzymes can be induced by metalloprotease. The sharp increase in the mRNA transcript levels of SmCAT and SmGPx at 3 h may be an adaptive response known as hormesis by Stebbing (1982), that is induced to counteract ROS toxicity and production (Woo et al., 2009). Similar observations have been reported in L. vannamei (Qian et al., 2014) and Crassostrea gigas (Jo et al., 2008). With prolonged metalloprotease exposure time, the gene expression of SmMnSOD, SmCAT, and SmGPx in cells were found to be dramatically declined from 12 to 24 h. A possible interpretation is that the formation of excessive radicals may have surpassed the neutralizing ability of antioxidant enzymes, therefore causing oxidative destruction that damages normal cell functions (Jo et al., 2008; Ren et al., 2015). The induction or inhibition of antioxidant enzyme expression is also associated with the degree of metalloprotease-induced oxidative injuries in head kidney cells. However, to better understand the role of antioxidant enzymes in the prevention of free radical production, or reactive species production initiated by metalloprotease, future studies looking at the mRNA and protein levels of MnSOD, CAT, and GPx in order to offset metalloprotease activity are required.

In fish tissues, although the antioxidation defense mechanisms are capable of modulating the production and removal of cellular ROS, but the dynamic balance of ROS can be altered by a number of stress factors. In this study, the ROS levels in head kidney cells were found to be significantly increased in all metalloprotease treatment groups from 6 to 24 h, indicating that excessive ROS can be induced through metalloprotease exposure. This suggests a possible mechanism for metalloprotease-induced toxicity via mitochondria dysfunction that causes increased ROS levels. Additionally, the expression of antioxidant enzymes (MnSOD, CAT, and GPx) were shown to be completely inhibited at 12 to 24 h, probably due to the generation of excessive metalloproteinase-induced ROS.

Mitochondria have been suggested as the key organelle that modulate apoptosis (Desagher and Martinou, 2000). ROS, as a byproduct of cell metabolism, is mainly produced in the mitochondria, causing damages to mitochondrial components (Martindale and Holbrook, 2002). Mitochondria are highly sensitive to oxidative stress, and can become dysfunctional under excessive ROS in many cell types (Lee et al., 2009; Guo et al., 2013). Changes in Δψ_m is typically observed in apoptotic cells under many stimuli. In this study, a significant drop in Δψ_m was found in all metalloprotease-treated groups from 6 to 24 h, indicating that metalloprotease can decrease Δψ_m to initiate mitochondrial injuries in head kidney cells, probably caused by excessive ROS generation. Previous studies have indicated that arsenic trioxide and enrofloxacin can promote the alteration of Δψ_m and elevate the production of ROS in fish cells (Selvaraj et al., 2013; Cui, 2014).

Extracellular metalloprotease is a critical virulent factor of pathogens that can cause death in turbot and flounder when injected intraperitoneally (Mo et al., 2002; Varina et al., 2008; Han et al., 2011). A previous study has confirmed that the metalloproteinase of V. anguillarum is toxic and can cause apoptosis in gill cells (Chi, 2006). Staining using Hoechst 33342 has been extensively applied in genotoxicity test in vitro to evaluate nuclear fragmentation and condensation due to different stress factors (Speit and Rothfuss, 2012). In this study, Hoechst 33342 fluorescent staining of head kidney cells treated with 8.0- or 40.0-μg/mL metalloprotease for 12 or 24 h showed that the cells were apoptotic to different extents after different treatments. Moreover, our results from the evaluation of cell apoptosis rate and Caspase 3 gene expression demonstrated that cells undergo apoptosis at different levels under the tested metalloprotease concentrations and exposure time (data not shown). Taken together, these findings suggest that extracellular metalloproteases may inhibit expression of antioxidant genes in vivo, induce oxidative damage, and ultimately cell apoptosis.

5 CONCLUSION

The full-length cDNA of SmCAT that belongs to the conserved CAT family was cloned the first time. With the highest level of expression in blood, SmCAT was found to be extensively expressed in all tested tissue types. The expression of SmCAT, SmMnSOD, and SmGPx in head kidney cells were observed to be significantly inhibited in response to metalloprotease treatment from 12 to 24 h. Metalloprotease can cause mitochondrial damage in head kidney cells by lowering the Δψ_m and by increasing the production of ROS. Hoechst 33342 staining showed metalloprotease can cause apoptosis in head kidney cells. To our knowledge, this is the first study that demonstrates the toxic effects of metalloprotease in the inhibition of antioxidant enzyme expression and in the initiation of apoptosis and oxidative stress in living cells. Moreover, we show that the increase of metalloprotease-induced intracellular ROS levels is an essential component that triggers the apoptosis of head kidney cells by inhibiting the expression of antioxidant enzymes.

6 DATA AVAILABILITY STATEMENT

The datasets generated during and/or analyzed during this study are available from the corresponding author on reasonable request.