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BY 4.0 license Open Access Published by De Gruyter July 15, 2021

Antioxidant potential of bulk and nanoparticles of naringenin against cadmium-induced oxidative stress in Nile tilapia, Oreochromis niloticus

  • Nouf Abdallah Mreat Al-Ghamdi , Promy Virk EMAIL logo , Awatif Hendi , Manal Awad and Mai Elobeid

Abstract

The study assessed the attenuating effect of citrus flavonone, naringenin, and its nanoformulation against cadmium (Cd) toxicity in Nile tilapia (Oreochromis niloticus). Nanoparticles of naringenin (NNn) were synthesized; size 165.1 nm (PDI 0.396) in variable shapes; cluster widespread, spherical accumulated, and tubular bacillary. Parallel, mature male Nile tilapia (n = 120 fish) were used for the exposure study. Group I was negative control. The groups II, III, and IV were exposed to 5 ppm of cadmium chloride monohydrate (CdCl2·H2O) for 21 days. Group III was treated with bulk naringenin (BNn) (3 ppm) and group IV was treated with NNn (3 ppm). Group V was exposed only to NNn (3 ppm). Cd-induced oxidative stress was evident from a significant increase in the hepatic malondialdehyde (MDA) levels and modulation of antioxidant enzymes in the liver and kidney. A significant increase in the hepatic metallothionein and HSP70 levels in the gills was observed. Treatment with both BNn and NNn significantly (p ≤ 0.05) reversed the Cd-induced alterations. However, the protective effect of nano naringenin was more profound on the bioaccumulation of Cd in liver and levels of HSP70 in the gills. These key findings could add to the commercial exploitation of naringenin and its nanosized form as a dietary therapeutic molecule.

Graphical abstract

Effect of naringenin and its nanoparticles on Cd-induced toxicity in Nile tilapia.

1 Introduction

Cadmium (Cd) is a nonessential, toxic and a carcinogenic element. Cadmium contamination of the aquatic environment, both marine and freshwater, has been in the forefront of research [1]. Aquatic ecosystem impacts of Cd have been widely reported. A major concern associated with heavy metal contamination in the aquatic environment is the ability of aquatic organisms, such as fish, to bioaccumulate the metal [2,3,4], which eventually enters the food chain and has adverse effects on human health [5]. There is an analogy between the adverse effects of cadmium on aquatic organisms and humans, which includes skeletal deformities and impaired renal function. Since in saltwater Cd combines with chlorides to form a molecule that makes it less available in solution, its toxicity is therefore more pronounced in fresh water [6]. Oxidative stress, and generation of reactive oxygen species (ROS), has been widely implicated in Cd toxicology [7]. It has also been reported to be instrumental in the pathogenicity of Cd-induced toxicity in several fish species. Previous studies on a few species such as Oreochromis mossambicus [8], freshwater catfish (Clarias batrachus) [9], gilt-head (sea) bream (Sparus aurata) [10], O. niloticus [11], and rare minnow (Gobiocypris rarus) [12] have reported the ameliorative role of antioxidants against Cd toxicity. Replete research evidence suggests that the antioxidants intervene in the generation of free radicals and subsequently modulate and decrease the oxidative stress. Flavonoids are a large group of polyphenolic compounds found extensively in plants and are recognized for their bioactive effects such as chelating and antioxidant properties [13]. The commonly used therapeutic strategies in metal intoxication are the use of chelates, antioxidants, and phytocompounds [14]. Towards the end of twentieth century, there has been a resurgence in the interest in food phenolics due to their antioxidant and free radical-scavenging abilities [15]. Naringenin is one such important naturally occurring flavonoid, predominantly found in some edible fruits, like citrus species and tomatoes, grapefruit, cherries, and figs belonging to smyrna-type Ficus carica [16]. Chemically known 2,3-dihydro-5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one (Figure 1) is a flavanone that is derived from naringin, its glycone precursor on hydrolysis [16].

Figure 1 
               Chemical structure of naringenin (C15H15O5).
Figure 1

Chemical structure of naringenin (C15H15O5).

Naringenin has been widely reported for its broad pharmacological effects on human and animal health which include a decrease in lipid peroxidation biomarkers, protein carbonylation, increase in antioxidant defenses, scavenging reactive oxygen species, modulating immune system activity, in addition to being anti-inflammatory in nature [16]. Previous studies have also reported its pronounced nephroprotective and hepatoprotective effect attributed to its potent antioxidant activity [17,18,19]. The only drawback that limits the use of this bioflavonoid is its poor absorption on oral ingestion, with only 15% of ingested naringenin absorbed in the human gastrointestinal tract [20], which has led to several studies on developing strategies to enhance its bioavailability. Combining herbal medicine with nanostructures or nanonization strategies has been widely used in the field of nanomedicine to enhance the targeted delivery and reduce the systemic side effects, which the conventional treatments fail to meet [21]. The concept of nanomedicine in fish culture has been primarily limited to the use of nanospheres or polymeric nanoparticles. Several modes of nanoparticle synthesis have been employed in the recent times, out of which “green synthesis” is an emerging field of bionanotechnology, as it offers a safer and more eco-friendly alternative while retaining the efficacy of the nanosized active compounds [22].

With this premise, the present study aimed at fabricating an eco-friendly nanoparticle of naringenin as nanonaringenin. The biosynthetic method used in the study has been granted a patent [23]. Further, the antioxidative potential of bulk naringenin relative to nanoparticles of naringenin was also assessed against Cd-induced toxicity in adult Nile tilapia (Oreochromis niloticus), a widely cultured freshwater fish in Saudi Arabia.

2 Materials and methods

2.1 Preparation of nanoparticles of naringenin

Naringenin powder (300 mg) was dissolved in 70 mL of methanol. Once the solution was prepared, it was added dropwise at a flow rate of 0.2 mL/min for 5 min to boiling water kept in a beaker (150 mL). The drop flow was significant for both the formation of nanoparticles and maintaining uniformity in their size. This was done under ultrasonic conditions in an ultrasonic cleaner (Roop Telsonic, India) with an ultrasonic power of 100 W and a frequency of 30–60 kHz for 50–60 min. Finally, the mixture in the beaker was further mixed with a magnetic stirrer for 50–60 min at 30–50°C. Thereafter, the solution was freeze dried [23].

2.2 Characterization of nanoparticles

The synthesized nanoparticles of naringenin (NNn) were characterized using Zetasizer, Nano series, HT Laser, ZEN3600 from Molvern Instruments, UK. Transmission electron microscopy (TEM) (JEM-1011, JEOL, Japan) was used to characterize the shape and morphology of formed nanoparticles. TEM was operated at accelerating voltage of 80 kV.

2.3 Experimental design

All experiments were performed in accordance with the requirements of the Institutional Research Ethics Committee, Deanship of Scientific Research, King Saud University, Riyadh. Adult Nile tilapia, Oreochromis niloticus (n = 120 fish), with an average range of 20–24 cm and weight of 150–200 g were procured from a local fish farm near Riyadh City. They were placed in 10 glass tanks (100 L) containing non-chlorinated water. The fish were acclimated to the laboratory conditions with a 12 h light/dark cycle, tank water with a pH range 6.95–7.60 and temperature ranging from 20°C to 24°C for 15 days. Commercial fish feed (crude protein-35%) was used to feed the fish once daily (2% body weight) and the residues were siphoned every 48 h. The fish were divided into five experimental groups in duplicates. Each group comprising 12 fishes was kept in separate glass tanks. The exposure period was 3 weeks. Group I was the control group, fish were kept in dechlorinated tap water. In group II, fish were exposed to a sublethal concentration of 5 ppm CdCl2 (equivalent to 2.14 ppm Cd) [9]. Group III – fish were exposed to a sublethal concentration of 5 ppm CdCl2·H2O and treated with nanonaringenin (NNn) (3 ppm). Group IV – fish were exposed to a sublethal concentration of 5 ppm CdCl2·H2O (equivalent to 2.14 ppm Cd) and treated with bulk naringenin extract (BNn) (3 ppm). Group V – fish were exposed to only nanonaringenin (NNn) (3 ppm). At the end of the exposure period, the fish were sacrificed from each group. The liver, kidneys, gills, and muscle were excised out. All tissues were preserved at −80°C till further analysis.

2.4 Determination of cadmium in tissue

The tissue samples were acid digested by a mixture of (nitric acid) HNO3 or with (perchloric acid) HClO4. The concentration of Cd in digested tissue samples was analyzed in an atomic absorption spectrophotometer (220 FS Varian, Australia) at wavelength of 228.8 nm (detection limit 0.005 μg/mL) with 4.0 mA current. A calibration curve with standard solutions was plotted. The average reading of blanks was subtracted from standard and test sample and then final concentration (ng/g) was calculated.

2.5 Biochemical analysis

2.5.1 Determination of lipid peroxides (malonaldehyde levels)

Malondialdehyde (MDA) levels in serum were determined using the Alliance Waters high performance liquid chromatography (HPLC) 2,695 system and a multi fluorescence detector (Model 2475, USA). This system was operated by a Dell Optiplex GX1 computer and Empower software. The reversed-phase analytical HPLC column was a ODS Hypersil from Thermo Scientific (4.6 mm × 25.0 cm × 5 μm). A guard column, Waters Symmetry TM C18 (4.6 mm × 2 cm, 5 μm particle size), with the same packing materials was placed in front of the analytical column for protection. The elution was carried out at a flow rate of 1.0 mL/min. The column effluent was quantified at an excitation and emission wavelengths of 515 and 553 nm, respectively. Run time per sample was 4.0 min. The intermediate working standards were prepared by diluting the MDA stock solution with water to concentrations of 0.5, 1.0, 2.5, 5.0, 7.5, and 15 nmol/mL. The MDA levels in the samples were expressed as nmol/mL of serum [24].

2.5.2 Determination of catalase activity

Tissue homogenates for liver and kidney from each experimental group were prepared in accordance with the protocol provided with the ELISA kits (Cayman Chemicals, USA).

The CAT activity in the liver and kidney was measured in terms of formaldehyde concentration following a modified method of Cohen et al. [25]. For this, 0.5 g of tissue (liver and kidney) was homogenized on ice in 2–5 mL of cold phosphate buffered saline (PBS). Here after, the contents were centrifuged at 10,000×g for 15 min at 4°C. The supernatant was stored on ice and used for the assay.

2.5.3 Determination of superoxide dismutase (SOD) activity

Cayman’s SOD ELISA kit was used to measure the SOD activity following the method of Minami and Yoshikawa [26] with certain modifications. For this, 0.5 g of tissue (kidney, liver) was homogenized in 3–6 mL of cold 20 mM HEPES buffer, pH 7.2 containing 1 mM EGTA, 210 mM mannitol, and 70 mM sucrose. The contents were then centrifuged at 1,500×g for 5 min at 4°C. The supernatant was stored on ice and used for the assay.

2.5.4 Determination of metallothionein levels in the liver

Measurement of fish metallothionein (MT-2) in the liver was performed using a pre-coated ELISA kit (Mybiosource, USA). The 96-well polystyrene microtiter plates were supplied pre-coated with antibody specific to fish MT-2.

2.5.5 Determination of heat shock protein (HSP70) in gills

Measurement of fish heat shock protein (HSP70) in gills was performed using a pre-coated ELISA kit (Mybiosource, USA). The 96-well polystyrene microtiter plates were supplied pre-coated with antibody specific to fish HSP70.

2.6 Statistical analysis

All presented data are expressed as mean values ± standard deviation (SD). One-way analysis of variance (ANOVA) was performed followed by Tukey’s post hoc test used to evaluate group differences. Numerical data were correlated with SPSS 22.0 statistical software (Chicago, IL, USA). The significance level was set to p ≤ 0.05.

3 Results

Exposure to waterborne 5 ppm cadmium chloride (CdCl2), bulk naringenin, and nanoparticles of naringenin used in the present study did not cause any mortality in fish throughout the experimental period.

The particle size distribution of the prepared naringenin nanoparticles (NNn) is shown in (Figure 2). The mean particle size as measured by dynamic light scattering on Zetasizer was 165.1 nm with a poly dispersity index (PDI) of 0.369, with two contiguous peaks. The TEM analysis showed the presence of distinct variable forms of the nanoparticles, cluster widespread, spherical accumulated, and tubular bacillary (Figure 3).

Figure 2 
               Size characterization and distribution by intensity of naringenin nanoparticles. Notice the contiguous peaks.
Figure 2

Size characterization and distribution by intensity of naringenin nanoparticles. Notice the contiguous peaks.

Figure 3 
               TEM images of the nanoparticles of naringenin with methanol. Nanoparticles had distinct variable forms (a) cluster widespread (b) spherical accumulated (c) the tubular bacillary.
Figure 3

TEM images of the nanoparticles of naringenin with methanol. Nanoparticles had distinct variable forms (a) cluster widespread (b) spherical accumulated (c) the tubular bacillary.

3.1 Cadmium concentration in the tissues

The concentration of cadmium in liver, kidney, muscle, and gills was significantly (p ≤ 0.05) higher in cadmium-exposed groups in comparison to the control group. There was no significant difference observed between the group exposed to nanoparticles of naringenin only and the control. A significant increase was observed in the Cd levels in the liver from the group exposed to Cd only (1113.33 ± 48.39 ng/g) in comparison to the control (205.48 ± 62.92 ng/g). Treatment with nanonaringenin (Cd + NNn) was efficacious as it significantly (p ≤ 0.05) decreased the Cd levels in the liver. In contrast, Cd levels in the liver were not statistically different on treatment with bulk naringenin (Cd + BNn). A significant increase in the Cd concentration in the kidneys (5375.41 ± 84.46 ng/g) was observed on Cd exposure when compared to the control (414.72 ± 35.32 ng/g) (Table 1). Treatment with both bulk (Cd + BNn) and nanonaringenin (Cd + NNn) significantly (p ≤ 0.05) reduced the Cd concentration in comparison to the group exposed to Cd only. A significant (p ≤ 0.05) increase in the Cd concentration was observed in the gills (1122.17 ± 147.22 ng/g) in the Cd-exposed group in comparison to the control (55.01 ± 5.60 ng/g). However, treatment with nanonaringenin (Cd + NNn) and bulk naringenin (Cd + BNn) had no significant change in the cadmium concentration in the gills (Table 1).

Table 1

Mean (±SD) cadmium concentration (ng/g) in target tissues of fish exposed to 5 ppm CdCl2‧H2O and treated with nanonaringenin (Cd + NNn) and bulk naringenin (Cd + BNN)

Experimental groups Liver Kidneys Gills Muscle
Control 205.48 ± 62.92a 414.72 ± 35.32a 55.01 ± 5.60a 29.54 ± 5.55a
Cd 1113.33 ± 48.39b 5375.41 ± 84.46b 1122.1667 ± 147.22b 239.52 ± 23.42b
Cd + NNn 795.16 ± 121.32c 4918.76 ± 69.29c 839.5667 ± 165.38b 238.74 ± 55.67b
Cd + BNn 1044.19 ± 94.61b 1692.10 ± 282.89d 849.0267 ± 230.70b 189.60 ± 19.41b
NNn 233.55 ± 36.25a 120.73 ± 18.82a 61.9400 ± 5.88a 28.54 ± 5.85a

Different letters within a column indicate significant differences between the experimental groups (p ≤ 0.05).

After 21 days of exposure, a significant (p ≤ 0.05) increase in the Cd concentration was observed in the muscle from the group exposed to Cd only (239.52 ± 23.42 ng/g) in comparison to the control group (29.54 ± 5.55 ng/g). However, both treatments, nanonaringenin (Cd + NNn) and bulk naringenin (Cd + BNn), showed no significant effect (Table 1).

3.2 Biochemical analysis

3.2.1 MDA levels in liver

The MDA levels in liver were significantly (p ≤ 0.05) higher in the group exposed to Cd only (158.36 ± 15.57 nmol/mL), in comparison to the control (31.97 ± 4.04 nmol/mL). On treatment with both bulk naringenin (Cd + BNn) and nano naringenin (Cd + NNn), a significant (p ≤ 0.05) decrease was observed in the MDA concentration in comparison to the group exposed to Cd only. However, within the treatment groups (Cd + NNn and Cd + BNn), there was no significant difference observed on the MDA concentration in the liver (Figure 4).

Figure 4 
                     Mean (±SD) MDA concentration (nmol/mL) in the liver of fish exposed to 5 ppm CdCl2‧H2O treated with nanonaringenin (Cd + NNn) and bulk naringenin (Cd + BNn). Different letters indicate significant differences between Cd and the treated groups (p ≤ 0.05).
Figure 4

Mean (±SD) MDA concentration (nmol/mL) in the liver of fish exposed to 5 ppm CdCl2‧H2O treated with nanonaringenin (Cd + NNn) and bulk naringenin (Cd + BNn). Different letters indicate significant differences between Cd and the treated groups (p ≤ 0.05).

3.2.2 Catalase activity

After an exposure period of 21 days, the group exposed to Cd only showed a significant (p ≤ 0.05) increase in the catalase activity in the kidneys (1437.38 ± 40.32 U/g) in comparison to the control group (1233.12 ± 11.65 U/g). Treatment with nanonaringenin (Cd + NNn) and bulk naringenin (Cd + BNn) significantly (p ≤ 0.05) reduced the catalase activity in comparison to the group exposed to Cd only. However, the effect of treatment with both nanonaringenin (Cd + NNn) and bulk naringenin (Cd + BNn) was comparable (Figure 5). There was a significant (p ≤ 0.05) decrease observed in the catalase activity in the liver from the group exposed to the Cd only (1077.63 ± 140.52 U/g) in comparison to the control (1258.88 ± 51.33 U/g). The treatment with nanonaringenin (Cd + NNn) and bulk naringenin (Cd + BNn) significantly (p ≤ 0.05) enhanced the catalase activity in comparison to the non-treated Cd-exposed group. However, the efficacy of both treatments was comparable (Figure 5).

Figure 5 
                     Mean (±SD) CAT activity (U/g of wet tissue) in kidneys and liver of fish exposed to 5 ppm CdCl2·H2O treated with nanonaringenin (Cd + NNn) and bulk naringenin (Cd + BNn). * indicates significant differences between control and Cd group. Different letters indicate significant differences between Cd and the treated groups (p ≤ 0.05).
Figure 5

Mean (±SD) CAT activity (U/g of wet tissue) in kidneys and liver of fish exposed to 5 ppm CdCl2·H2O treated with nanonaringenin (Cd + NNn) and bulk naringenin (Cd + BNn). * indicates significant differences between control and Cd group. Different letters indicate significant differences between Cd and the treated groups (p ≤ 0.05).

3.2.3 SOD activity

An exposure period of 21 days to Cd caused a significant (p ≤ 0.05) decrease in the SOD activity in the kidney of fish (83.86 ± 0.25 U/mL) in comparison to the control group (85.93 ± 0.51 U/mL). Treatment with nano naringenin (Cd + NNn) significantly (p ≤ 0.05) enhanced the SOD activity in comparison to the group exposed to Cd only. However, treatment with bulk naringenin (Cd + BNn) did not show any significant effect on the SOD activity. Thus, the treatment with nano naringenin (Cd + NNn) was significantly (p ≤ 0.05) more effective than the bulk naringenin (Cd + BNn). The group exposed to nano naringenin only (NNn) was comparable to the control group (Figure 6). The SOD activity in liver was significantly (p ≤ 0.05) enhanced in the group exposed to Cd only (84.06 ± 0.55 U/mL) in comparison to the control (79.34 ± 1.08 U/mL). In the groups exposed to Cd and treated with nanonaringenin (Cd + NNn) and bulk naringenin (Cd + BNn), a significant (p ≤ 0.05) decrease was observed in the SOD activity in comparison to the group exposed to Cd only. Further, the group exposed to nanonaringenin only (NNn) was comparable to the control group (Figure 6).

Figure 6 
                     Mean (±SD) SOD activity (U/mL) in kidneys and liver of fish exposed to 5 ppm CdCl2‧H2O treated with nanonaringenin (Cd + NNn) and bulk naringenin (Cd + BNn). * indicates significant differences between control and Cd group. Different letters indicate significant differences between Cd and the treated groups (p ≤ 0.05).
Figure 6

Mean (±SD) SOD activity (U/mL) in kidneys and liver of fish exposed to 5 ppm CdCl2‧H2O treated with nanonaringenin (Cd + NNn) and bulk naringenin (Cd + BNn). * indicates significant differences between control and Cd group. Different letters indicate significant differences between Cd and the treated groups (p ≤ 0.05).

3.2.4 Metallothionein concentration in liver (ng/g)

The metallothionein concentration in the liver of fish from the control group (126.33 ± 6.98 ng/g) was comparable to the group exposed to nanonaringenin only (NNn). A significant (p ≤ 0.05) increase in the metallothionein levels (170.65 ± 11.05 ng/g) was observed on Cd exposure in comparison to the control. Further, treatment with bulk naringenin (Cd + BNn) and nanonaringenin (Cd + NNn) significantly (p ≤ 0.05) reduced the metallothionein in comparison to the group exposed to Cd only. However, there was no significant difference observed within the treated groups (Cd + NNn and Cd + BNn) (Figure 7).

Figure 7 
                     Mean (±SD) metallothionein concentration (ng/g) in liver of fish exposed to 5 ppm CdCl2‧H2O treated with nanonaringenin (Cd + NNn) and bulk naringenin (Cd + BNn). Different letters indicate significant differences between Cd and the treated groups (p ≤ 0.05).
Figure 7

Mean (±SD) metallothionein concentration (ng/g) in liver of fish exposed to 5 ppm CdCl2‧H2O treated with nanonaringenin (Cd + NNn) and bulk naringenin (Cd + BNn). Different letters indicate significant differences between Cd and the treated groups (p ≤ 0.05).

3.2.5 Heat shock protein (HSP-70) in gills (pg/mL)

Exposure to Cd (5 ppm) for 21 days resulted in a significant (p ≤ 0.05) increase in the HSP70 concentration (251.66 ± 19.36 pg/g) in the gills of the fish exposed to Cd only when compared to the control (152.88 ± 11.815 pg/g). A significant (p ≤ 0.05) decline was observed in the HSP70 concentration in the group treated with nanonaringenin (NNn). However, treatment with bulk naringenin (BNn) did not show a significant effect on the HSP70 levels. The HSP70 concentration in the group exposed to nanonaringenin (NNn) only was not significantly different from the control (Figure 8).

Figure 8 
                     Mean (±SD) heat shock protein (HSP-70) concentration (pg/mL) in gills of fish exposed to 5 ppm CdCl2‧H2O treated with nanonaringenin (Cd + NNn) and bulk naringenin (Cd + BNn). Different letters indicate significant differences between Cd and the treated groups (p ≤ 0.05).
Figure 8

Mean (±SD) heat shock protein (HSP-70) concentration (pg/mL) in gills of fish exposed to 5 ppm CdCl2‧H2O treated with nanonaringenin (Cd + NNn) and bulk naringenin (Cd + BNn). Different letters indicate significant differences between Cd and the treated groups (p ≤ 0.05).

4 Discussion

Naringenin is one of the potent dietary phytochemicals endowed with several biological and pharmacological properties. Despite its promising biological activity, naringenin’s low bioavailability has remained a major hurdle in its use. Therefore, to enhance the bioavailability, a novel green synthesis of nanoparticles of naringenin was successfully attempted in the present study. Our results on the characterization of the nanoparticles of naringenin clearly showed the presence of nanoparticles of naringenin with an average aggregate size of 165.1 nm and a poly dispersity index (PDI) of 0.396 with two contiguous peaks. Approximately similar nanoparticle size of 166 nm (PDI = 0.291) has been reported previously for titanium oxide nanoparticles [27] and probiotic nanoparticles [28]. The electron micrographs in the present study clearly showed variable shapes of the synthesized nanoparticles of naringenin, cluster widespread, spherical accumulated, and tubular bacillary. The nanoparticles produced were stable and efficacious in combating the Cd-induced toxicity. Similar results have been reported for a nanoformulation of curcumin, as nanocurcumin and its potent antibacterial activity [29]. The results on the bioaccumulation of Cd in tissues showed that the exposure of fish to Cd (5 ppm) for three weeks significantly increased the concentration of Cd in the liver, kidney, and gills in Cd-exposed groups in comparison to the control group. The Cd concentration in kidney and liver was invariably higher followed by the gills and the flesh. Previous experimental studies on Cd exposure to fish by Kumar et al. [8,30] and Al-Anazi et al. [11] at a concentration of 5 ppm also reported a similar accumulation pattern of Cd in tissues of C. batrachus and O. niloticus, respectively. Further, in line with this, De Smet and Blust [31] reported that cadmium accumulated in tissues of common carp, Cyprinus carpio, in the following order: kidney > liver > gills > muscles. Firat et al. [32] also reported that an exposure of Cd to O. niloticus for 28 days showed a significant increase in the Cd levels in the liver and gills being the highest in the liver. The treatment with nanonaringenin (NNn) was efficacious in reducing the Cd concentration both in the liver and kidneys, while the bulk naringenin was effective only in the kidneys. It has been postulated that flavonoids and naringenin in particular are active metal chelators [33]. Thus, naringenin reduces the Cd burden with displacement of metal cofactors and/or Cd binding with enzymes [17]. Previous literature has reported that Cd toxicity is primarily attributed to the oxidative damage to cellular organelles via generation of ROS [13]. In the present study, toxicity of Cd was reflected via the oxidative stress induced in the fish which was evident by enhanced lipid peroxidation in the liver and modulated activity of the major antioxidant enzymes, CAT and SOD in the liver and kidneys. Exposure to Cd enhanced the lipid peroxidation in the liver which was mirrored as a significant increase in the hepatic MDA levels. These results are in consensus with the previous findings from experimental studies on Cd-induced oxidative stress, in rats [17,18,27], poultry [34], and various fish species, e.g., O. niloticus [11], C. batrachus [8], Japanese flounder (Paralichthys olivaceus) [35], South American catfish (Rhamda quelen) [36], and S. aurata [10]. In addition, the CAT activity was also altered, being enhanced in the kidney but decreased in the liver. In contrast, the SOD activity was decreased in the kidney and increased in the liver. A similar pattern was reported in several experimental studies on Cd exposure on fish, O. niloticus [11,37], C. batrachus [8], P. olivaceus [35], R. quelen [36], and S. aurata [10]. These findings are consistent with the hypothesis that Cd enhances oxygen-free radical production which stimulates CAT activity, to decompose H2O2 to molecular oxygen and water and maintains cell homeostasis [38]. CAT activity is often correlated to SOD activity [35]. Indeed, both enzymes function together and constitute the first line of defense against oxidative stress [8,39]. SOD catalyzes the destruction of superoxide radical by dismutation and H2O2 formation which explains a parallel increase in the lipid peroxidation with an increase in the SOD activity in the liver and kidney of Cd-exposed fishes. On the other hand, a decrease in the activity of CAT and SOD on Cd has also been previously reported, as acute exposure to Cd leads to an increase in lipid peroxides, with a decrease in levels of CAT and SOD enzymes in liver of mice [40].

The treatment with both bulk naringenin (BNn) and nanonaringenin (NNn) showed a profound protective effect against the Cd-induced oxidative stress. The treatments reversed the Cd-induced lipid peroxidation and oxidative modification of the enzyme activities. A line of studies clearly indicates that naringenin offers protection against oxidant stress through its strong antioxidant potential [17,18,41]. As previously mentioned, naringenin can act as a metal chelator and prevent the Fenton reaction which subsequently decreases the formation of hydroxyl radicals [42]. It is well-documented that naringenin effectively scavenges the free radicals owing to the electron donating properties of the 4-hydroxyl group in its β-ring, which thereby protects the membrane and inhibits the lipid peroxidation [43]. In addition, the lipophilic nature of naringenin helps it to adhere to the cell membrane thus masking the effect of the free radicals on the membrane integrity [17]. Previous studies [17,18] on the antioxidative potential of naringenin have also reported a similar restoration of the Cd-induced impairment in the antioxidant enzyme activities as observed in the present study. Induction of metallothioneins (MTs) in the liver, as the main form of storage and detoxication of metals in fish, is well-established [6]. In the present study, a significant induction in the MT-2 levels was observed in the Cd-exposed fish, which related to the higher metal accumulation in this organ. This revealed a major role of MT in metal homeostasis and detoxification. Similar findings were reported by Bervoets et al. [44], Suresh et al. [45], and Le Croizier et al. [46] who found significant correlation between Zn, Cu, and Cd accumulation and MT induction in liver of fish. In the present study, treatment with both bulk naringenin and nano naringenin significantly reduced the hepatic MT-2 levels. The effect of the two treatments was comparable. The hepatoprotective nature of naringenin against cadmium toxicity has been reported earlier [18].

The results of the present study on the effect of naringenin on Cd bioaccumulation in liver clearly correlate to the results on the reduction of MT levels in the liver. In the present study, the stress proteins (HSP70) in the fish gills were significantly induced in response to exposure to Cd. Studies of stress protein response in organs with high metabolic activity, such as, liver and gills, are of high interest. Gills are important as they are the organs involved in the uptake of the waterborne metals. HSPs are commonly used by environmental toxicologists as biochemical markers of exposure to various physical, chemical, and biological stressors, such as temperature change, tissue trauma, metal toxicity, radiation, and infection [47,48]. Elevated levels of various heat shock proteins have been measured in tissues of fish exposed to environmental contaminants, such as heavy metal [49,50]. HSP70 used as a biomarker in the present study is one of the first HSPs to be expressed under the minutest of environmental stress [47]. Treatment with naringenin, bulk (BNn), and NNn showed a significant decrease in the HSP70 levels in the gills being more profound with NNn.

5 Conclusion

While metal nanoparticles are being increasingly used in many sectors of the economy, there is a growing interest in the biological and environmental benign modes of biosynthesis. The formulation used in the present study had no stabilizers or surfactants, and the finished product entirely consisted of naringenin. Further, an evaluation of the biological parameters assessed clearly showed that the nanoformulation retained the antioxidant and metal-chelating properties of naringenin and did not chemically modify the compound. Thus, it can be stated that naringenin is a potent antioxidant with free radical-scavenging and metal-chelating properties which could mitigate the potential hazards of heavy metal toxicity in fish and humans. Nanosization of the compound could further enhance its bioavailability and efficacy to be used as a nutraceutical in fish feeds and as well as for human consumption.


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  1. Funding information: The authors extend their appreciation to King Abdulaziz City for Science and Technology (KACST), Riyadh, Saudi Arabia, for funding this project (registration no: 122-36).

  2. Author contributions: Promy Virk: conceptualization, methodology, project administration, manuscript writing; Nouf Abdallah Mreat Al-Ghamdi: data curation, writing – original draft preparation; Manal Awad: supervision, visualization, investigation; Awatif Heindi: supervision; Mai Elobeid: reviewing and editing.

  3. Conflict of interest: Authors state no conflict of interest.

  4. Data availability statement: The authors declare that all data were generated in-house and that no paper mill was used.

  5. Ethical approval: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed by the authors.

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Received: 2021-03-05
Revised: 2021-05-30
Accepted: 2021-06-01
Published Online: 2021-07-15

© 2021 Nouf Abdallah Mreat Al-Ghamdi et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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