First-line defense of antioxidant system
SOD and CAT activity were compared according to the response level in the organs as shown in Fig. 2. SOD activity in muscle and gill were observed to be significantly lower (p < 0.05) compared to the liver in Setiu Wetland. The same pattern was noticed for the response of SOD activity in Semerak with muscle and gill were significantly lower (p < 0.05) compared to the liver. CAT showed the same pattern of response with liver remained as the organ with the highest response. Significant results were observed for the response of CAT between organs for all sites (p < 0.05). As shown in Fig. 2 the response of CAT in liver was significantly higher compared to both muscle and gill in Setiu Wetland. Meanwhile, in Semerak and Tumpat liver showed a significant higher response compared to both gill and muscle with significant higher response of gill (p < 0.05) compared to muscle.
Further, the response level of SOD and CAT were compared as shown in Fig. 3. From the results, it showed that despite the superior SOD response compared to CAT for gill and liver in all studied sites. Muscle appeared to be in the opposite, with significant higher of CAT response (p < 0.05) for both Setiu and Semerak followed by a significant strong correlation (Setiu Wetland: 0.91, Semerak: 0.79) (p < 0.01) as shown in Table 1.
Table 1
Pearson correlation of GST and GR in muscle, gill and liver of L.calcarifer
Setiu Wetland |
Muscle | 0.91** |
Gill | -0.08 |
Liver | 0.07 |
Semerak |
Muscle | 0.02 |
Gill | 0.26 |
Liver | 0.65 |
Tumpat |
Muscle | 0.78** |
Gill | 0.37 |
Liver | 0.004 |
** indicates significant correlation (p < 0.01), * indicates correlation (p < 0.05) |
This study emphasized on the response of oxidative stress of different tissues in L.calcarifer inhabiting different aquaculture farms. The pattern of SOD and CAT in L.calcarifer showed a consistent and convincing outcome that these two biomarkers will respond the most in the liver, followed by gill and muscle. This agrees with studies reported by (Ameur et al., 2012; Beg et al., 2014; Ballesteros et al., 2017) in Mugil cephalus, Dicentrachus labrax, Acanthopagrus latus, Cynoglossus arel and Jenynsia multidentata. From these results it further explains, regardless differences a certain habitat have the response of SOD and CAT in L.calcarifer will respond following the capability of the organ to take up the pollutants as well as the route of xenobitics to get into the body system (Ahmad et al, 2000).
Muscle is often neglected because it is expected to show the least response and it is rarely seen to be used as a target organ by researchers. The present study showed the least SOD and CAT response in muscle as expected which agrees to previous reported studies (dos Santos Carvalho et al., 2012; Maulvault et al., 2018) performed on the muscle of Oreochromis niloticus and Dicenthrachus labrax (European seabass). However, it is important to take into account the importance of this organ since it reflects the route of pollutants into the fish by providing the level of response for each body compartment for a better understanding of how these enzymes respond in a certain organ (Oliveira et al., 2008). Pointing out from this, the response of SOD and CAT in muscle was evidenced to be different from sensitive organs, gill and liver.
The response in gill and liver agreed to a study reported by (Abhijith et al., 2016) in gill and liver of carp, Catla catla. From this, it explains that both enzymes at the front line of antioxidant system work together but respond differently in different body compartment as evidenced by the present study. This is further supported by the strong correlation shown in muscle. Therefore, it is very important to have these enzymes together as targeted biomarkers because their responses are correlated to each other.
Glutathione-dependent Enzymes System
The response level of both GST and GR in muscle, gill and liver was compared for each study site as shown in Fig. 4. Overall, the most response was shown in the liver for both enzymes followed by muscle being the least responded organ for all sites. A significant higher response of GST (p < 0.05) was observed in the liver for both sites Setiu Wetland and Semerak compared to the muscle. The response of GST in gill for Semerak also observed to be significantly lower (p < 0.05) compared to liver. Regarding GR, the responses in liver for all sites were significantly higher (p < 0.05) compared to both muscle and gill. The response of both GST and GR were tested using Pearson correlation analysis as shown in Table 2. There was a strong significant correlation observed for the response of GST and GR in all targeted tissues from Tumpat, muscle: 0.89, gill: 0.95 and liver: 0.54 (p < 0.01, p < 0.05). Strong significant correlations also observed in both sites Setiu Wetland (0.72) and Semerak (0.79) for muscle (p < 0.01).
Table 2
Pearson correlation of GST and GR in muscle, gill and liver of L.calcarifer
Setiu Wetland |
Muscle | 0.72** |
Gill | 0.24 |
Liver | -0.08 |
Semerak |
Muscle | 0.79** |
Gill | 0.25 |
Liver | 0.38 |
Tumpat |
Muscle | 0.89** |
Gill | 0.95** |
Liver | 0.54* |
** indicates significant correlation (p < 0.01), * indicates correlation (p < 0.05) |
It has been proven in human, rats and reptiles that GST could indicate the organ damages through their defensive responses since GST is considered as non-specific oxidant enzymes that help to detoxify various oxidants (Hermes-Lima & Storey, 1993; Kilty et al., 1998; El-Demerdash et al., 2009). The results showed liver experienced the most damages compared to gill and muscle. This is also proved by previous studies performed on fish as done by (Simonato et al., 2016; Samantha et al., 2018) since liver acts as a depository organ for various pollutants. The level of GST response was higher compared to GR. This agrees with the previous reported studies (Song et al., 2012; Jaafar et al., 2015; Gianng et al., 2018).
GST activity was expressed in umol/min/mg protein whilst GR activity was expressed in nmol/min/mg protein. This showed that the difference of their responses was apparent. GST is amendable towards various numbers of electrophilic compounds (Habig, 1983; Armstrong, 1997; Strange et al., 2001). This allows it to catalyse non-specific oxidants and result in a higher activity response. The presence of pollutants leads to reduction in the level of GSH and GSH-GSSG ratio and causing reduction in GR activity (Monteiro et al., 2010). Besides, GR is responsible to cater GSH to ensure a proper function of other GSH utilizing enzymes which include GST, Glutathione peroxidase (GPx), Glutaredoxins, glyoxalases 1 and 2 (Hayes et al., 2005; Deponte, 2013; Csiszár et al., 2016). Therefore, the higher responses showed by GST followed their specific functionality in antioxidant system.
Following the specific functionality of both GST and GR, their responses will correlate to each other. This is proven by the strong correlation index obtained. Many studies focussed on different pollutants exposure onto testing organisms with different exposure time that exhibited various induction and reduction of GST and GR responses in different body compartment as reported by (Oliva et al., 2012; Song et al., 2012). However, study on how these two enzymes responses correlated to each other is scarce. Gondhowiardjo (1993) reported a correlation of GST and GR activity in a normal and separate group of corneal disorders in mammal. A similar response could be expected in fish but further studies should be implemented.
Thiols Protein And Carbonyl Protein (protein Damages)
In general, a consistent pattern was observed for the total thiols containing protein for all three sites (Fig. 5). The response of oxidized thiols in the tissues of L.calcarifer was consistent with muscle remained as the least oxidized tissue followed by gill and liver for all sites. A higher significant result (p < 0.05) was observed in the liver as compared to gill and muscle in Setiu Wetland. However, no significant results were observed for Semerak and Tumpat.
In contrary to thiols, carbonyl containing protein showed inconsistent results in the targeted tissues as shown in Fig. 5. Insignificant consistent result was observed in Setiu Wetland with liver remained as the most oxidized organ followed by gill and muscle. The same pattern of results was observed for Semerak and Tumpat with muscle as the most oxidized followed by liver and gill. Muscle was significantly (p < 0.05) more oxidized than gill in Semerak and gill was significantly (p < 0.05) less oxidized than muscle and liver in Tumpat.
The response of thiols showed that sensitive organs are more susceptible to damages as evidenced by the consistent oxidized thiols in liver for all study sites. The trend of responses for thiols is different from other targeted biomarkers in the present study considering its mechanism of action. Thiols molecules are present on the side chain of protein and they are very sensitive to the presence of ROS that tend to oxidize them (Dalle-Donne et al., 2005; Rossi et al., 2006). Therefore, the decrease in their numbers indicates damages that happened to the targeted species (Rainville, 2015). The response level of thiols in Mytilus edulis reported by (Todesco et al., 2012) is more pronounced compared to this study and it was evident as a suitable biomarker for biomonitoring. However, the present study only managed to evaluate the pattern and level of oxidative damages that occurred in different body compartments. Nevertheless, the consistent responses in muscle, gill and liver from all aquaculture farms suggest that the antioxidant defenses are correspondingly efficient in each organ of L. calcarifer to prevent from reactive oxygen species (ROS) attack. The other reason that supported the responses consistency also related to the possible pathway of pollutants to get into the body compartment of the fish. The response of thiols is comparable with the study done by (Parvez, & Raisuddin, 2006; Jaafar et al., 2015; Menezeset al., 2016) on different marine species. However, there was no publication reported on the responses of thiols in L.calcarifer. Thus, this could be a baseline data for the response of thiols in this commercially important species.
The more apparent damages indicated by high carbonyl level occurred in muscles for Semerak and Tumpat. The increase of protein damages followed by the decrease of antioxidant enzymes activity showed inefficiency of antioxidant defense (Almroth et al., 2019). Therefore, the results suggesting that the antioxidant defense in muscle is less efficient compared to gill and liver. However, enzymatic antioxidant responses were recorded in the present study and all of them were consistent respectively. Tabassum et al. (2016) has reported an increase in the response of carbonyl content with decreasing response of non-enzymatic antioxidant in Channa punctata Bloch. Therefore, inefficiency might happen in the response of non-enzymatic antioxidant such as (glutathione, ascorbic acid and uric acid) and affected the response of carbonyl. The robustness of antioxidants defense system limited the extent of carbonyl and Lipid peroxidification (LPO) (Barros et al., 2017). Thus, this explains the high response of carbonyl content in muscle for both Semerak and Tumpat were due to the low response of non-enzymatic antioxidant in the present study.