Modulation of toxicity effects of CuSO4 by sulfated polysaccharides extracted from brown algae (Sargassum tenerrimum) in Danio rerio as a model

Copper is widely used in agriculture and aquaculture due to its high disinfection properties and relatively low cost. However, the increase in copper concentration due to evaporation can lead to water reservoir pollution, which can harm the health of consumers. The present study aimed to determine the role of sulfated polysaccharides (SPs) extracted from Sargassum tenerimum algae in reducing lesions caused by the heavy metal copper. Zebrafish (Danio rerio) were used as a human model in five treatments. The negative and positive control groups were fed a diet containing zero percent of SPs, while the experimental groups were fed 0.5%, 1%, and 1.5% of SPs in three treatments for 56 days, finally CuSO4 was exposed only to the positive control group and the groups fed with SPs. Results showed a significant decrease in the activity level of ALT enzymes (39–16 U/mL), AST (67–46 U/mL), and ALP (485–237 U/mL), confirming the results obtained from histopathological studies in CuSO4 exposed groups. The addition of SPs to the diet resulted in a significant reduction (sig < 0.05) of mortalities due to the decrease of tissue damage. Additionally, due to the anti-inflammatory properties and the protective effect of SPs, a significant decrease (sig < 0.05) was observed in the relative expression of Il-1β and Tnf-α genes.


Fish and treatment
Zebra fish (Danio rerio) were purchased from the breeding center of ornamental fish.Initially, the fish were cultivated for a period of two weeks to facilitate acclimatization and ensure their health.During this interval, they were provided with a standard commercial diet.After that SPs supplement diet was used for 8 weeks in 5 treatments (each treatment with 3 replicates) including negative control (N.Con.), positive control (P.Con.) fed without SPs, the group fed with 0.5%, 1% and 1.5% of SPs, then they were exposed to CuSO 4 at the end of culture period for 1 week.After anaesthetization of the fish for gene expression studies and whole-body extract (WBE) assays, whole-body samples were dried with clean and sterilized cloths to remove excess water and then immediately immersed in liquid nitrogen.The WBE was extracted in phosphate buffer (pH 7.4) and supernatant was used for WBE assays and RNA was extracted using the Wizol extraction kit 12 .For histological studies, liver sampling was done.During the rearing period, water temperature, dissolved oxygen and total hardness were measured as 26 ± 1.5 °C, 6 ± 0.98 mg/L, and 470 ± 25 mg/L, respectively.

Food preparation
The sulphated polysaccharide used in this study was extracted from Sargassum tenerimum algae using the method of Yang et al. 13 .The required amounts of SPs were weighed, dissolved in distilled water, and added to the diet as a spray.Daily feeding consisted of 3% of the biomass for the first 3 weeks and 5% of the biomass for the last 5 weeks of the culture period for each treatment.

Exposure to copper sulfate
CuSO 4 was purchased in powder form with Sigma brand.According to the water conditions of the reservoirs before the experiment, to determine the LC 50 , a pre-test was conducted, in which 60 fish were exposed to different concentrations of CuSO 4 (0, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, and 0.5 mg/L) in triplicate.the LC 50 value was calculated as 0.2 mg/L of CuSO 4 in 96 h by examining the wide concentrations [14][15][16] At the end of culture period with experimental diets, 60 fish were randomly selected for each treatment and then exposed to CuSO 4 .Concentration of CuSO 4 in main study was used as 1 2 of the LC 50 for each tank (0.1 mg/L) for one-week exposure time.

Whole-body extract assays
Total protein, albumin, cholesterol, glucose, ALP, ALT, and AST enzymes were measured by a commercial biochemistry kit using the spectrophotometric method (Biochrom, libra S12) based on the instructions inside the kits approved by Zeist Chem diagnostics, Iran.The amount of total immunoglobulin was calculated by dividing the protein concentration in the initial sample and the protein concentration after precipitation with polyethylene glycol 17 .

Gene expression studies
After beating, the whole-body tissue samples were sonicated according to the instructions of the RNA extraction kit approved by Wizol kit.cDNA synthesis was performed using GENET BIO's cDNA synthesis Master-mix according to the kit's instructions.In order to measure the quality of extracted RNA, cDNA and confirm the quality of primers (the sequence and other specifications of the primers are shown in Table 1), normal PCR test

Statistical studies, data analysis and ethical issues
To analyze the data, one-way analysis of variance was performed with SPSS 22 software, and Duncan's test was used to compare the means.The difference between the means in different treatments was determined with a confidence level of 95%.The results of the relative gene expression were calculated using the 2 −ΔΔCt method in Excel software compared to the β-actin reference gene.All experimental procedures were approved by the GUASNR ethics committee (Gorgan University of Agricultural Sciences and Natural Resources).It confirmed that all experiments were performed in accordance with relevant guidelines and regulations as described by the ARRIVE guidelines (PLoS Bio 8(6), e1000412, 2010).

Ethical approval
All experimental procedures related to the fish were in accordance with ethical standards for ethical review of fish welfare in GUASNR (Gorgan University of Agricultural Sciences and Natural Resources). The

Survival
According to Table 2, the results of the study show the average percentage of mortality after exposure to copper sulfate in the negative control treatment was zero, in the positive control treatment it was 89.1%, in the treatments 0.5%, 1%, and 1.5% were 73.9%, 47.8%, and 39.1% respectively.

Glucose and cholesterol
The amount of glucose (Fig. 1) in the negative control treatment was significantly lower compared to other groups, and the highest level was in the group fed with a diet containing 1.5% SPs (sig < 0.05).Exposure to CuSO 4 caused a significant decrease (sig < 0.05) in the blood cholesterol in the positive control and the group fed with 0.5% SPs, but no significant difference (sig < 0.05) was observed among other treatments (Fig. 1).

Total protein, total immunoglobulin and albumin
According to Fig. 2, the highest and lowest significant levels (sig < 0.05) of total protein were measured in the group fed with 1% SPs and positive control group, respectively.No significant difference was observed between the negative control group and group fed with 1.5% SPs (sig < 0.05).The results of total immunoglobulin (Fig. 2) between the positive control group and the group fed with 0.5% SPs showed no significant difference (sig < 0.05), www.nature.com/scientificreports/but total immunoglobulin level of these two groups were significantly lower compared to the other groups (sig > 0.05).In the groups under CuSO 4 exposure (Fig. 2), the positive control group showed the lowest amount of albumin, and with the increase in the concentration of SPs, the level of albumin also significantly increased (sig < 0.05) so the highest albumin level was measured for the negative control group and the group fed with 1.5% SPs (sig < 0.05).

ALT, AST and ALP enzymes
Amount of ALT enzyme (Fig. 3) show that in groups under CuSO 4 exposure, it decreases significantly (sig < 0.05) with the increase in the concentration of SPs.The level of ALT enzyme in the negative control group and the 1.5% SPs group showed no significant difference (sig < 0.05).The comparison of AST enzyme level (Fig. 3) between the groups showed a significant difference in the positive control group and the negative control group (sig < 0.05).Also, a significant difference was seen in the negative and positive control groups with the 0.5% and 1% SPs groups (sig < 0.05).The level of ALP enzyme (Fig. 3) was the lowest in the negative control group (sig < 0.05) and the highest level was in the positive control group (sig < 0.05), which significantly decreased with the increase in the concentration of SPs (sig > 0.05).

Expression of lysozyme, Tnf-α and Il-1β genes
According to Fig. 4, the highest relative expression level of lysozyme gene was in positive control and 1.5% SPs groups (sig < 0.05).The relative expression level of Tnf-α gene (Fig. 4) showed that the highest significant level (sig < 0.05) corresponds to the positive control group and the lowest significant level (sig < 0.05) corresponds to the 1 and 1.5% groups.According to Fig. 4, the highest significant level (sig < 0.05) of the relative expression of Il-1β gene found for the positive control group and the lowest level was also for the group 1 and 1.5% SPs (sig < 0.05).

Liver histopathology
Exposure to CuSO 4 in zebrafish causes tissue damage in the liver.By increasing the concentration of SPs, the observed tissue damage was decreased (Table 3).The main damage observed in the liver tissue of treatments under CuSO 4 exposure were liver hepatocyte vacuolation and necrosis in the positive control group and the 0.5% SPs groups (Fig. 5).

Discussion and conclusion
Seaweeds are inexpensive sources of bioactive compounds that provide various properties such as growth stimulation, appetite stimulation, increased immunity and increased resistance to diseases in consumers 21 .In recent years, the use of bioactive compounds in various industries has gained special importance.However, the use of new compounds as nutritive-medicinal compounds requires a lot of research and research about the effect of these compounds on the physiological state and health of animals, so studying the various properties of bioactive compounds is very important.

Survival rate and resistance to CuSO 4 exposure
The resistance to CuSO 4 exposure depends on factors such as water hardness, temperature, pH, species, the amount of manipulation, size and age, different biological stages and nutritional conditions.The occurrence of Table 3. Determination of liver lesions in zebra fish exposed under CuSO 4 and fed with different levels of SPs.
This study shows the positive effect of SPs extracted from S. tenerimum algae on the tolerance of exposures conditions caused by the heavy metal copper in zebra fish.During this experiment, 89.1% of the positive control group died in 94 h under CuSO 4 exposure.By increasing the concentration of SPs, the mortality trend showed a significant decrease, so that by adding 0.5% of SPs, the mortality rate was 73.9%, and by increasing the concentration of SPs to 1.5%, the mortality rate was decreased to 39.1%.

Liver histopathology
In general, damage to the liver parenchyma causes an increase in serum transaminases 25 , and therefore an increase in AST, ALT and ALP enzymes is a sign of primary cell damage, which reflects the level of this enzyme in the blood serum.Heart, liver, kidney and spleen are among the primary organs that are affected by metabolic reactions caused by various substances.The liver, which is a key organ in the metabolism and detoxification of xenobiotic compounds 25 , may be vulnerable to various compounds.Liver is one of the main organs for plasma purification, homeostasis and removal of toxins and toxins from the body.Liver histological studies demonstrate the ability of SPs to increase resistance to the heavy metal copper [26][27][28] .The most liver damage was related to the positive control group that were fed only with the basic diet, and the amount of tissue damage decreased with the increase in the concentration of SPs.SPs has high antioxidant ability and strong chelation of heavy metals, which may have prevented it from increasing damage with its antioxidant properties when exposed to CuSO 4 26-28 .

Serological analysis
Given the involvement of body fluids in various metabolic processes, changes in their composition can serve as useful indicators of an animal's physiological status 29 .As such, serum evaluations are a critical diagnostic tool for assessing the health of living organisms 29 .

Glucose and cholesterol
Blood glucose is a highly variable parameter that is strongly influenced by manipulative and environmental exposure such as nutritional status, seasonal changes, and sexual maturity 30 .Glucose level is widely used as an indicator of exposure to heavy metal in fish 31 .An increase in glucose concentration is a secondary response to stressors, so the amount of glucose increase indicates the body's response to metals exposure 32 .Cholesterol is a waxy substance with a lipid nature, which is necessary as a precursor for the synthesis of some hormones and vitamin D, which their major part is made by the liver and the intestinal wall.
In stress conditions, the blood glucose level can increase, according to the results of the positive control group, due to severe liver damage, glucose and cholesterol metabolism showed a significant decrease.The amount of exposure increased and cholesterol production decreased significantly.In the treatments receiving SPs due to its protective properties of liver cells [26][27][28] , less tissue damage was observed and the amount of exposure decreased, therefore, with increasing concentration of SPs, glucose and cholesterol levels approached normal levels, but did not decrease.The increase in glucose level compared to the negative control group may be due to two reasons, the sugary nature of SPs and the presence of CuSO 4 exposure, as well as tissue damage and reduced liver metabolism 33,34 .

Total protein, total immunoglobulin and albumin
A decrease in total protein, albumin and total immunoglobulin is a prominent feature of many diseases and may occur due to liver and kidney failure, decreased or loss of protein absorption [35][36][37][38] .In this study, by CuSO 4 exposure and liver tissue damage, the amount of total protein, followed by the amount of albumin and total immunoglobulin in the positive control treatments and 0.5% group was significantly lower than other treatments.In groups under CuSO 4 exposure and receiving 1 and 1.5 percent of SPs, the levels of total protein, albumin, and total immunoglobulin increased significantly.This may be due to the protective and antioxidant properties of this compound, which prevented the occurrence of impaired protein synthesis by the liver [26][27][28] .

Activity of AST, ALT and ALP enzymes
ALT is a liver-specific enzyme that usually increases in response to liver tissue damage, whereas AST enzyme increases not only due to liver damage but also in cases of heart and muscle injuries.ALP is most active in an alkaline pH and is found in tissues such as the spleen, intestinal wall, thymus gland, and testicles, although its highest concentration is in the liver [35][36][37][38][39][40] .Normally, this enzyme's level rises in liver lesions and in cases of bile duct obstruction, cysts, and liver abscesses.In this study, adding SPs to the diet resulted in a significant decrease in the activity of AST, ALT, and ALP enzymes in all experimental treatments compared to the positive control group, indicating that SPs reduced the damage caused by CuSO 4 and prevented tissue damage from spreading.The metal ion chelation and antioxidant properties of SPs are likely responsible for this effect 41,42 .As a result, the levels of AST, ALT, and ALP enzymes significantly decreased.The increase in the level of AST, ALT, and ALP enzymes in the positive control group suggests that CuSO 4 induces stress conditions and impairs liver function, ultimately leading to mortality in this group.

Expression of lysozyme, Tnf-α and Il-1β genes
Lysozyme hydrolyzes glycosidic bonds and is known as a lytic enzyme, and its activity level is usually considered as one of the parameters of innate immunity 23 .Il-1β is known as one of the pro-inflammatory factors that defends www.nature.com/scientificreports/ the body during the conflict with microbial pathogens and tissue damage by stimulating the increase of phagocyte activity, increasing the number of macrophages and the production of lysozyme 43 .Tnf-α also helps the innate defense system by stimulating cells to chemotaxis and phagocytosis in eliminating various pathogens 44 .Although an increase or decrease in the level of gene expression does not necessarily mean an increase or decrease in the production of the final product 45 , it can be said that in exposure induction, the level of transcription of the gene is changed to translate into a specific protein in order to carry out a specific reaction and overcome exposures conditions [46][47][48] .In this study, the relative expression level of Lysozyme gene in the treatments does not depend on the treatments, but it is possible that due to the CuSO 4 exposure, the level of gene expression has increased in order to be able to cope with the invasion of pathogens.By dietary adding SPs, the living organism had a greater ability to tolerate exposures conditions and also has less liver damage.In this case, it may be due to the protective and antioxidant property of polysaccharide 41,42 , the expression level of Il-1β and Tnf-α genes had shown a significant decrease in doses of 1% and 1.5% of SPs to deal with inflammation and phagocytotic response.

Conclusion
Further detailed studies are necessary to draw definitive conclusions regarding the effectiveness of SPs as a supplement for commercial, industrial, and pharmaceutical uses.However, in the present study, the observation of reduced tissue damage and a significant decrease in the activity level of ALT, AST, and ALP enzymes, which is supported by the results of histological studies, suggests that SPs, with their chelating and antioxidant properties, have the potential to protect against copper exposure and may serve as an effective supplement.

Figure 1 .Figure 2 .
Figure 1.Glucose and Cholesterol in body extracts of zebra fish exposed under CuSO 4 and fed with different levels of SPs extracted from S. tenerimum algae.Non-similar lowercase English letters on each column indicate a significant difference at the 0.05 level.Data are mean ± standard deviation.

Figure 3 .
Figure 3. ALT, AST and ALP enzyme activity levels in body extracts of zebra fish exposed under CuSO 4 and fed with different levels of SPs extracted from S. tenerimum algae.Non-similar lowercase English letters on each column indicate a significant difference at the 0.05 level.Data are mean ± standard deviation.

Figure 4 .
Figure 4. Relative expression level of lysozyme gene, tumor necrosis factor (Tnf-α) gene and interleukin gene (Il-1β) in zebra fish exposed under CuSO 4 and fed with different levels of SPs extracted from S. tenerimum algae.Non-similar lowercase English letters on each column indicate a significant difference at the 0.05 level.Data are mean ± standard deviation. https://doi.org/10.1038/s41598-023-38549-0

Figure 5 .
Figure 5. Liver tissue damage caused by CuSO 4 exposure in zebra fish fed with different concentrations of SPs extracted from S. tenerimum algae.Figure (A) and (B) Liver tissue in negative control group (no exposure), normal bile duct (a), normal liver hepatocytes (b).Figure (C) and (D) Liver tissue in positive control group fish (CuSO 4 exposure), bile duct (a), nucleus (b), intense vacuolation of liver hepatocytes (c), degeneration and necrosis in liver hepatocytes (d).Figure (E) and (F) Liver tissue of fish exposed to CuSO 4 and fed with 0.5% SPs, normal bile duct (a), nucleus (b), vacuolation of liver hepatocytes (c), degeneration and the beginning of the process of necrosis in hepatocytes.Liver (d), central vein (e), blood cells (f).Figure (G) and (H) Liver tissue of fish exposed to CuSO 4 and fed with 1% SPs, sinusoidal space (a), vacuolation of liver hepatocytes (b), vein with blood cells (c).Figure (I) and (J) Liver tissue of fish exposed to CuSO 4 and fed with 1.5% SPs, normal bile duct (a), liver hepatocytes (b), sinusoidal space (c), beginning of vacuolation of liver hepatocytes (d).
Figure 5. Liver tissue damage caused by CuSO 4 exposure in zebra fish fed with different concentrations of SPs extracted from S. tenerimum algae.Figure (A) and (B) Liver tissue in negative control group (no exposure), normal bile duct (a), normal liver hepatocytes (b).Figure (C) and (D) Liver tissue in positive control group fish (CuSO 4 exposure), bile duct (a), nucleus (b), intense vacuolation of liver hepatocytes (c), degeneration and necrosis in liver hepatocytes (d).Figure (E) and (F) Liver tissue of fish exposed to CuSO 4 and fed with 0.5% SPs, normal bile duct (a), nucleus (b), vacuolation of liver hepatocytes (c), degeneration and the beginning of the process of necrosis in hepatocytes.Liver (d), central vein (e), blood cells (f).Figure (G) and (H) Liver tissue of fish exposed to CuSO 4 and fed with 1% SPs, sinusoidal space (a), vacuolation of liver hepatocytes (b), vein with blood cells (c).Figure (I) and (J) Liver tissue of fish exposed to CuSO 4 and fed with 1.5% SPs, normal bile duct (a), liver hepatocytes (b), sinusoidal space (c), beginning of vacuolation of liver hepatocytes (d).
Figure 5. Liver tissue damage caused by CuSO 4 exposure in zebra fish fed with different concentrations of SPs extracted from S. tenerimum algae.Figure (A) and (B) Liver tissue in negative control group (no exposure), normal bile duct (a), normal liver hepatocytes (b).Figure (C) and (D) Liver tissue in positive control group fish (CuSO 4 exposure), bile duct (a), nucleus (b), intense vacuolation of liver hepatocytes (c), degeneration and necrosis in liver hepatocytes (d).Figure (E) and (F) Liver tissue of fish exposed to CuSO 4 and fed with 0.5% SPs, normal bile duct (a), nucleus (b), vacuolation of liver hepatocytes (c), degeneration and the beginning of the process of necrosis in hepatocytes.Liver (d), central vein (e), blood cells (f).Figure (G) and (H) Liver tissue of fish exposed to CuSO 4 and fed with 1% SPs, sinusoidal space (a), vacuolation of liver hepatocytes (b), vein with blood cells (c).Figure (I) and (J) Liver tissue of fish exposed to CuSO 4 and fed with 1.5% SPs, normal bile duct (a), liver hepatocytes (b), sinusoidal space (c), beginning of vacuolation of liver hepatocytes (d).
Figure 5. Liver tissue damage caused by CuSO 4 exposure in zebra fish fed with different concentrations of SPs extracted from S. tenerimum algae.Figure (A) and (B) Liver tissue in negative control group (no exposure), normal bile duct (a), normal liver hepatocytes (b).Figure (C) and (D) Liver tissue in positive control group fish (CuSO 4 exposure), bile duct (a), nucleus (b), intense vacuolation of liver hepatocytes (c), degeneration and necrosis in liver hepatocytes (d).Figure (E) and (F) Liver tissue of fish exposed to CuSO 4 and fed with 0.5% SPs, normal bile duct (a), nucleus (b), vacuolation of liver hepatocytes (c), degeneration and the beginning of the process of necrosis in hepatocytes.Liver (d), central vein (e), blood cells (f).Figure (G) and (H) Liver tissue of fish exposed to CuSO 4 and fed with 1% SPs, sinusoidal space (a), vacuolation of liver hepatocytes (b), vein with blood cells (c).Figure (I) and (J) Liver tissue of fish exposed to CuSO 4 and fed with 1.5% SPs, normal bile duct (a), liver hepatocytes (b), sinusoidal space (c), beginning of vacuolation of liver hepatocytes (d). https://doi.org/10.1038/s41598-023-38549-0

Table 1 .
Characteristics of the primers used in investigating the relative expression of lysozyme, Tnf-α and Il-1β genes in zebrafish.