The effects of iron oxide nanoparticles on antioxidant capacity and response to oxidative stress in Mozambique tilapia (Oreochromis mossambicus, Peters 1852)

For this study we used the juvenile Mozambique tilapia (Oreochromis mossambicus, Peters 1852), weighing 6.0±1.5 g and measuring 6.5±1.0 cm in average, obtained from the Safa Aquarium (Kozhikode, India). During the two-week acclimatisation period, a total of 160 fish were housed in a 180 L cement tank under a 12-hour light/dark cycle, where they were provided with a continuous supply of dechlorinated tap water. The fish were fed standard commercial pellets (Taiyo, Uthukkottai, India) on a daily basis, which contained the appropriate amount of nutrients including 41 % crude protein, 6 % crude lipids, 7 % crude fibre, 10 % carbohydrate, 15 % ash, <8.5 % phosphorus, <1.5 % water, and trace amounts of vitamins and minerals. Their health status was monitored throughout the experiment following the guidelines set forth by the Indian Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) (23).

Optimal physico-chemical characteristics of the tap water were maintained and monitored in line with the standard methods published by the American Public Health Association (APHA) (24) and the OECD guideline on acidity and alkalinity (25) as follows: temperature 28±2 °C, pH 7.0±0.5, dissolved oxygen 8.6±0.6 mg/L, water hardness 48 mg/L calcium carbonate, total organic carbon 0.04 mg/L, non-ionised ammonia 0.01 mg/L, nitrate 10 mg/L, chlorine 4 mg/L, metallic impurities <1 mg/L, and chemical oxygen demand 3 mg/L. Water analyses were done to ensure that water did not affect the experimental outcomes.

Preparation and characterisation of IONPs used in this study has been described in detail in our previous publications (26, 27). Briefly, IONPs (Fe₃O₄-NPs; Cat. No. 637106) were obtained from Sigma (Darmstadt, Germany). X-ray diffraction (XRD; Rigaku Miniflex, Tokyo, Japan) and transmission electron microscopy (TEM; Jeol/JEM, Tokyo, Japan) were performed to confirm the size and purity of IONPs. IONPs were suspended in double-distilled water and sonicated in an ultrasonic bath (GT Sonic, Guangdong, China) at a frequency of 100 kHz for 30 min to ensure uniformity. All the other chemicals used were of analytical grade and purchased from Himedia (Thane, India).

The test concentration was selected according to IONP dispersion, as mentioned in our previous publication (27). Following the acclimatisation phase, 160 fish were randomly distributed into eight 40-litre glass tanks (30 cm width, 60 cm length, and 30 cm depth), each holding 20 fish. The control group was not exposed to IONPs. Treatment groups were exposed to IONPs at 15 mg/L for 1, 3, 4, 15, 30, and 60 days (IONP1, 3, 4, 15, 30, and 60, respectively). The depuration group was first exposed to IONPs (15 mg/L) for 60 days, and then followed up in IONP-free water for another 60 days.

IONP tissue accumulation was analysed in fish groups treated for four days (IONP4), 60 days (IONP60), and in the 60-day depuration group using a method described by Arslan et al. (28) with slight modifications. Fish from each group were captured with a small dip net to avoid additional stress, euthanised, and dissected. Gill, liver, and brain were excised and rinsed with cold saline before weighing. Wet tissue samples were weighed and digested with a mixture of concentrated nitric and hydrochloric acid (3:1) at 200 °C for 1 h, and the digested material was evaporated to remove any contaminants or residues that might interfere with analytical measurements. The remaining tissue was diluted with deionised water to a known volume. Total iron content, consisting of both iron nanoparticles and any other forms of iron within the samples, was quantified with inductively coupled plasma mass spectrometry (ICP-MS; Thermo Fisher, Waltham, MA, USA). The IONP content in total iron concentration was estimated based on the known molar mass of Fe₃O₄ and atomic mass, as follows: [1] Fe3O4 mass=Fe concentration×Fe3O4 molar massFe atomic mass F{e_3}{O_4}\;mass = {{Fe\;concentration \times F{e_3}{O_4}\;molar\;mass} \over {Fe\;atomic\;mass}}

This conversion allowed us to determine the number of nanoparticles corresponding to the measured mass, which was used to calculate the bioaccumulation factor (BAF), expressed as micrograms per gram of wet tissue weight. [2] BAF=IONP concentration in tissue μg/gIONP concentration in water mg/L {\rm{BAF}} = {{{\rm{IONP}}\;{\rm{concentration}}\;{\rm{in}}\;{\rm{tissue}}\;\left( {\mu {\rm{g}}/{\rm{g}}} \right)} \over {{\rm{IONP}}\;{\rm{concentration}}\;{\rm{in}}\;{\rm{water}}\;\left( {{\rm{mg}}/{\rm{L}}} \right)}}

At the end of every treatment, fish were captured as described above, weighed, euthanised, and dissected to remove the gill, liver, and brain. The tissues were then rinsed with cold saline and weighed. The hepatosomatic index (HSI) of the fish was calculated using the following equation: [3] HSI=Liver weight mgTotal body weight mg×100 {\rm{HSI}} = {{{\rm{Liver}}\;{\rm{weight}}\;\left( {{\rm{mg}}} \right)} \over {{\rm{Total}}\;{\rm{body}}\;{\rm{weight}}\;\left( {{\rm{mg}}} \right)}} \times 100

Tissue homogenates (1 % w/v) were prepared separately in normal saline on ice using a motor-driven tissue homogeniser (Remi, Mumbai, India) and the homogenates centrifuged at 800 g for 15 min at 4 °C to collect supernatants used for biochemical analyses. Total protein was estimated using bovine serum albumin (BSA) for the standard as described elsewhere (29). Activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx) were analysed in the gill, liver, and brain tissues. Activities of marker enzymes alkaline phosphatase (ALP) in the gill and liver and acetylcholinesterase (AChE) in the brain were estimated as described below.

SOD activity was determined as described by Marklund and Marklund (30). The assay mixture contained tris-hydrochloric acid buffer (50 mmol/L, pH 7.6) consisting of 1 mmol/L EDTA, 0.2 mmol/L pyrogallol, and 100 µL of enzyme source. The increase in absorbance was measured at 420 nm against an enzyme-free blank at 10-second intervals over 3 min using an ultraviolet (UV)-visible spectrophotometer (Shimadzu, Kyoto, Japan). Enzyme activity is expressed as nmol of oxidised pyrogallol per min per mg of protein.

CAT activity was determined using the method described by Claiborne (31). A total mixture of 3 mL was composed of phosphate buffer (50 mmol/L, pH 7.0), hydrogen peroxide (19 mmol/L), and 50 µL of enzyme source. The decrease in absorbance was measured at 240 nm against an enzyme-free blank at 10-second intervals over 3 min using a UV-visible spectrophotometer (Shimadzu, Kyoto, Japan). Enzyme activity was expressed as mol of hydrogen peroxide consumed per min per mg of protein.

The activity of GR was assayed as described by Carlberg and Mannervik (32). The assay mixture comprised phosphate buffer (100 mmol/L, pH 7.6), NADPH (0.2 µmol/L), oxidised glutathione (2 mmol/L), EDTA (0.01 mol/L), and the enzyme source. The reduction of NADPH was monitored by measuring the decrease in absorbance at 340 nm against an enzyme-free blank at 10-second intervals over 3 min using a UV-visible spectrophotometer (Shimadzu). Enzyme activity is expressed as nmol of oxidised NADPH per min per mg of protein.

The activity of GPx was determined with the method described by Mohandas et al. (33) utilising hydrogen peroxide and NADPH as substrates. The assay mixture comprised phosphate buffer (100 mmol/L, pH 7.6), EDTA (0.01 mol/L), sodium azide, GR, reduced glutathione, and NADPH (0.2 µmol/L). Enzyme assay was added and NADPH reduction monitored by measuring the decrease in its absorbance at 340 nm against an enzyme-free blank at 10-second intervals over 3 min using a UV-visible spectrophotometer (Shimadzu). Enzyme activity is expressed as nmol of oxidised NADPH per min per mg of protein.

The levels of hydrogen peroxide were assessed following the method described by Pick and Keisari (34). The assay is based on the H₂O₂-mediated and horseradish peroxidase-dependent oxidation of phenol red to a product. The incubation mixture comprised phosphate buffer (50 mmol/L, pH 7.6), horseradish peroxidase (8.5 units), phenol red (0.28 nmol/L), dextrose (5.5 nmol/L), and enzyme source (100 µL). The reaction was carried out at room temperature for 30 min, after which it was terminated by adding 10 eq/L sodium hydroxide. The absorbance was measured at 610 nm against the blank. A standard curve was prepared using known concentrations of H₂O₂, and the results are expressed as nmol of generated hydrogen peroxide per mg of protein.

LPO was measured using thiobarbituric acid as outlined by Ohkawa et al. (35). A working solution was prepared by combining 15 % w/v trichloroacetic acid, 0.37 % 2-thiobarbituric acid, and 0.25 eq/L hydrochloric acid in a 1:1:1 ratio. The enzyme source was added to the working solution at a ratio of 1:2 and incubated in a boiling water bath for 10 min. Followed the measurement of absorbance at 535 nm against the blank. Malondialdehyde (MDA) solution served as the standard, and the results are expressed as nmol of MDA per mg of protein.

The activity of ALP in gill and liver tissues was determined following the method described by Bessey et al. (36). Pre-incubation involved p-nitrophenyl phosphate and glycine buffer at pH 10.5, maintained at 37 °C for 5 min. Subsequently, the sample and 0.02 eq/L NaOH were added to the mixture and incubated for 30 min. The resultant colour was measured at 420 nm using a UV-visible spectrophotometer (Shimadzu) against the blank. Upon the addition of 0.1 mL of concentrated hydrochloric acid, the mixture was thoroughly mixed and the difference in absorbance recorded as the measure of enzyme activity. The activity of ALP was determined from the calibration curve generated using the p-nitrophenol standard. Enzyme activity is expressed as µmol of liberated p-nitrophenol per min per mg of protein.

The activity of AChE in the brain tissue was determined as described by Ellman et al. (37) by monitoring the increase in yellow colour resulting from the reaction between thiocholine and dithiobisnitrobenzoate (DTNB; 0.01 mol/L). Brain tissue was homogenised and dissolved in phosphate buffer (0.1 mol/L, pH 8.0), 15 mg of sodium bicarbonate, and DTNB. Enzyme activity is expressed as nmol of hydrolysed acetylthiocholine per min per mg of protein.

Statistical analyses were run on the IBM SPSS Statistics version 21.0 for Windows (SPSS Inc., Chicago, MI, USA). Differences between the means were determined with one-way analysis of variance (ANOVA), followed by Duncan’s multiple range post-hoc test. Normality of distribution was tested with the Kolmogorov-Smirnov test and the homogeneity of variance between subsets analysed with Levene’s test. The significance was set at P<0.05 vs control. Data are presented as means ± standard deviations (SD) for 20 animals per group.

The XRD results confirmed that IONPs used in our study were free from impurities. TEM analysis revealed a crystalline structure, with irregular and roughly symmetrical morphology, ranging in size from 1 to 100 nm. The average crystallite size calculated with the Scherrer equation was 15.65 nm. These results corroborate the manufacturer’s specifications (Figure 1).

Figure 1

Fish exposed to IONPs did not exhibit significant changes in body weight. However, gill weight increased while the hepatosomatic index decreased significantly (P<0.05) after 30 and 60 days of exposure (Table 1). Brain weight remained unchanged throughout the experiment. The hepatosomatic index returned to control levels in the depuration group, while body and organ weights remained unchanged (Table 1).

The ICP-MS analysis confirms the presence of IONPs in the gill, liver, and brain tissues of the fish after 4 and 60 days of exposure (Table 2). The gill tissue exhibited the highest bioaccumulation factor, followed by the liver and brain. The 60-day depuration failed to restore IONP levels to control.

The activities of SOD (Figure 2), CAT (Figure 3), GR (Figure 4), and GPx (Figure 5) showed a significant (P<0.05) decrease in the gill, liver, and brain in a time-dependent manner compared to respective controls. Conversely, the levels of hydrogen peroxide (Figure 6) and LPO (Figure 7) increased significantly (P<0.05) in all tissues in a time-dependent manner. Upon 60-day depuration, these levels did not return to control (Figures 2–7).

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

In both gill and liver, ALP activity dropped significantly (P<0.05) in a time-dependent manner. A similar significant and time-dependent drop was observed for brain tissue AChE activity. Again, 60-day depuration did not reverse the activities of these enzymes (Figure 8).

Figure 8