Document Type : Anatomy- Histology
1 Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.
2 Department of Aquatic Health and Diseases, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.
Article Title [Persian]
زمینه مطالعه: به واسطه تولید و کاربرد فراوان نانوذرات، مواجهه انسان و اکوسیستم ها با آن افزایش یافته است. افزایش استفاده از نانوذرات منجر به بالارفتن میزان ورود این مواد به محیط های آبی شده است.
هدف: مطالعه حاضر به منظور بررسی اثر محافظتی اسپیرولینا پلاتنسیس (Spirulina platensis) بر آسیب کبدی و استرس اکسیداتیو ناشی از نانوذرات اکسید آهن در کفال خاکستری (Mugil cephalus) انجام شد.
روش کار: ماهیان جوان کفال خاکستری به طور تصادفی در 12 گروه مساوی تقسیم شدند: گروه 0: کنترل 96 ساعت، گروه 1: کنترل 60 روز، گروه 2: 15 میلیگرم به کیلوگرم وزن نانوذرات اکسیدآهن به مدت 96 ساعت، گروه 3: 15 میلیگرم به کیلوگرم وزن نانوذرات اکسیدآهن به مدت 60روز، گروه 4: 300 میلیگرم به کیلوگرم وزن جلبک اسپیرولینا به مدت 96 ساعت،گروه 5: 300 میلیگرم به کیلوگرم وزن جلبک اسپیرولینا به مدت 60 روز، گروه 6: 15 میلیگرم به کیلوگرم وزن نانوذرات اکسیدآهن به همراه 300میلیگرم به کیلوگرم وزن جلبک اسپیرولینا بصورت همزمان به مدت 96 ساعت، گروه 7: 15 میلیگرم به کیلوگرم وزن نانوذرات اکسیدآهن به همراه 300میلیگرم به کیلوگرم وزن جلبک اسپیرولینا بصورت همزمان به مدت 60 روز، گروه 8: 15 میلیگرم به کیلوگرم وزن نانوذرات اکسیدآهن به مدت 2 هفته و سپس آغاز دریافت روزانه 300 میلیگرم به کیلوگرم وزن جلبک اسپیرولینا به مدت 96 ساعت، گروه 9: 15 میلیگرم به کیلوگرم وزن نانوذرات اکسیدآهن به مدت 2 هفته و سپس آغاز دریافت روزانه 300 میلیگرم به کیلوگرم وزن جلبک اسپیرولینا به مدت 60روز، گروه 10: 300 میلیگرم به کیلوگرم وزن جلبک اسپیرولینا به مدت 2 هفته و سپس آغاز دریافت 15میلیگرم به کیلوگرم وزن نانوذرات اکسیدآهن به مدت 96 ساعت،گروه 11: 300 میلیگرم به کیلوگرم وزن جلبک اسپیرولینا به مدت 2 هفته و سپس آغاز دریافت 15میلیگرم به کیلوگرم وزن نانوذرات اکسیدآهن به مدت 60روز، در پایان آزمایش، نمونههای بافت کبد جدا و برای تعیین گلوتاتیون احیا شده (GSH)، سوپراکسید دیسموتاز (SOD)، کاتالاز (CAT) و مالون دیآلدئید (MDA) تجزیه و تحلیل شدند.
نتایج: در مطالعه حاضر، سطوح MDA در کبد به طور معنی داری افزایش یافت در حالی که فعالیت آنتی اکسیدانی SOD و GSH در کبد در گروه مواجهه شده باا IONPs در مقایسه با گروه کنترل به طور معنی داری کاهش یافت. درمان با اسپیرولینا پلاتنسیس به طور معنی دار باعث کاهش فعالیتهای MDA و CAT و افزایش SOD و GSH کبدی در مقایسه با ماهیهای تحت درمان با IONPs شد.
نتیجهگیری نهایی: این نتایج به وضوح نشان داد که SOD و GSH در مهار تشکیل رادیکال های آزاد اکسیژن در بافت کبد نقش دارند. آسیب کبدی ناشی از IONPs در ماهیان محافظت شده با اسپیرولینا باعث بهبود معنادار در تمام پارامترهای قبلی شد. درمان با اسپیرولینا پلاتنسیس از طریق مهار رادیکال های آزاد و فعالیت های ضد التهابی و همچنین بازسازی مکانیسم های سیستم دفاعی آنتی اکسیدان های درون زا، اثر محافظتی در برابر صدمات کبدی و استرس اکسیداتیو در کبد ماهیهای مسموم با IONPs نشان میدهد.
ecause of their capacity to target a specific spot inside the body, iron oxide nanoparticles (IONPs) have been suggested for various applications (Mody et al., 2014). Providing medicine or gene, enhancing contrast in magnetic resonance imaging (MRI), separating cells, immobilizing proteins, and so on (Vidya and Chitra, 2019; Yarjanli et al., 2017). Six distinct types of iron oxide are known to exist; they include wustite (FeO), hematite (α-Fe2O3), β-Fe2O3, ε-Fe2O3, maghemite (γ-Fe2O3), and magnetite (Fe3O4) (Cornell and Schwertmann, 2003). Natural sources of nano-sized iron oxides include volcanoes and forest fires, where they are produced as magnetite and maghemite crystalline formations. Many IONPs are intentionally created and employed in many applications (Vidya and Chitra, 2019). In biomedical applications, magnetite (Fe3O4) and maghemite (Fe2O3) are widely utilized. Both compounds’ physicochemical characteristics are similar (Estelrich et al., 2015).
Metal removal from aqueous solutions and environmental cleanup employ iron oxide nanoparticles made from magnetite and maghemite (Grover et al., 2012). Due to their widespread manufacturing and usage, nanoparticle exposure to ecosystems and people has increased. There are four ways that nanoparticles may come into contact with living things: via the air, water or food, dermal contact, and sediment deposition (Vidya and Chitra, 2019). Applications of nanomaterials have increased their release into aquatic environments. The primary means by which nanoparticles enter aquatic environments include effluents from wastewater treatment facilities, during use and transit, and runoff from roofs and other roof-related activities (Garner and Keller, 2014). The hydrodynamic radius and surface charge of nanoparticles influence the accessibility of tissues and their absorption by cells when it reaches aquatic species. IONPs are digested after being absorbed by cells, releasing free iron ions into the cells (Malvindi et al., 2014). Iron deposition in the targeted tissue due to the widespread use of IONPs may be hazardous, affect cellular functions and metabolism, and cause significant alterations in the physiology, morphology, and behavior of animals exposed to it. Because of the great biocompatibility of iron oxide nanoparticles, they may readily interact with biomolecules such as proteins, lipids, and nucleic acids (Gokduman et al., 2018).
Iron oxide exposure has been linked to changes in hematological, biochemical, ionoregulatory, and enzymological parameters as well as mortality, delayed hatching, and malformation in Indian major carp, zebrafish, and Labeo rohita (Zhu et al., 2012; Keerthika et al., 2017). Metals may be linked to oxidative tissue damage because they serve as catalysts in oxidative processes (Badi et al., 2022). Exogenous chemicals have the potential to generate highly reactive oxygen species (ROS), which may oxidize biomolecules and cause cell death and tissue damage. Most biomolecules, including proteins, lipids, and lipoproteins, may react with ROS. Malondialdehyde (MDA) indicates oxidative stress in cells and tissues because lipid peroxidation is a well-established mechanism of cellular harm. MDA testing is often used as a lipid peroxidation biomarker. In both people and model systems, higher lipid peroxidation levels have been linked to several disorders.
Detoxification mechanisms, including antioxidant responses like reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT), are triggered by oxidative stress (Kim and Kang, 2015; Kim et al., 2017). Therefore, the reactions of fish exposed to metals to antioxidants may be a sensitive measure of oxidative stress. The most popular and generally accessible spirulina species is S. platensis (Arthrospira platensis), which is edible and has great nutritional and medicinal properties (Hussein et al., 2019). Spirulina or S. platensis contains essential fatty acids, polysaccharides, glycolipids, sulfolipids, vitamins, proteins (up to 70% of dry weight), lipids (5%-8% of dry weight), carbohydrates (up to 14%), some vital minerals (like calcium, iron, zinc, magnesium, manganese, and selenium), carbohydrates (up to 14%), and essential minerals (Hussein et al., 2019). Due to the presence of enzymatic and non-enzymatic antioxidants that mitigate the effects of reactive oxygen species, spirulina prevents cell damage (Farag et al., 2016). Carotenoids, tocopherols (TOH), ascorbic acid, glutathione (GSH), and chlorophyll are examples of non-enzymatic antioxidant compounds.
In contrast, SOD, CAT, glutathione peroxidase (GPX), peroxiredoxin (PrxR), and ascorbate peroxidase (AXP) are examples of enzymatic antioxidants (Abd El-Baky et al., 2009). The current research aimed to evaluate the histological and biochemical changes caused by IONPs in important tissues such as fish liver and grey mullet since the number of nanoparticles exposed in the aquatic environment has been steadily increasing. The research assessed the antioxidant and healing properties of spirulina therapy both before and after IONP exposure.
2. Materials and Methods
Iron oxide nanoparticle
IONPs were provided by ARMINANO Co. in Iran. The particles were pre-characterized before the experiment (SEM: TESCAN, MIRA3-LMU). They were pure, and the Mean±SD size was confirmed as 17.45±2.88nm. The test dose of nanoparticles (15mg/kg) was chosen based on Vidya and Chitra (2019).
Dried S. platensis powder
The substances that an aquarium system produces as dried algae powder (spirulina), are described below (Karaj, Iran). Spirulina was produced, dried at 32°C, and mixed to create a homogenized powder. Based on Karadeniz et al. (2009) study, a dose of S. platensis (300 mg/kg) was chosen.
Juvenile fish of the grey mullet (Mugil cephalus) (Mean±SD weight: 22.79±1.14g) and (Mean±SD height: 123.22±1.94mm) were caught from the fish farm of Chabahar Science Research Institute, southeast of Iran, in September 2020. The obtained fish was immediately transported to the Fish Biology Laboratory at Chabahar Maritime University.
They were kept in 40-L plastic tanks of seawater with 10 fish each, with excellent aeration and light, for two weeks before the experimental investigation to acclimate them to the laboratory environment. Before the study, the experimental fish were given commercial marine fish pellets at a rate of 5% of wet weight twice daily at 10:00 and 15:00 to assist the fish in adjusting to their new habitat. Feces and leftover food were aspirated, and there was a twice-weekly water exchange. Standard water temperature (26°C±1.4°C), total dissolved solids (TDS) (41.92±0.64) ppm, salinity (39.85±0.38) ppt, the dissolved oxygen concentration of water (70%) and pH (7.8 to 8.24) were maintained.
Fish were grouped into 12 as follows:
Group 0: control group, toxicant-free (96h, short term)
Group 1: control group, toxicant-free (60d, long term)
Group 2: IONPs at 15mg/kg for 96h (short term)
Group 3: IONPs at 15mg/kg for 60d (long term)
Group 4: S. platensis at 300mg/kg for 96h (short term)
Group 5: S. platensis at 300mg/kg for 60d (long term)
Group 6: IONPs at 15mg/kg+S. platensis at 300mg/k for 96h (short term)
Group 7: IONPs at 15mg/kg+S. platensis at 300mg/k for 60d (long term)
Group 8: IONPs at 15mg/kg (for 2 weeks) followed by S. platensis at 300mg/kg for 96h (short term)
Group 9: IONPs at 15mg/kg (for 2 weeks) followed by S. platensis at 300mg/kg for 60d (long term)
Group 10: S. platensis at 300mg/kg (for 2 weeks) followed by IONPs at 15mg/kg for 96h (short term)
Group 11: S. platensis at 300 mg/kg (for 2 weeks) followed by IONPs at 15mg/kg for 60d (long term)
Fish were captured after each treatment session using small dip nets to minimize stress on the animal, and the liver tissues were removed for histological analysis.
After collecting tissues, they were fixed in buffered formalin at a 10% concentration. Tissues were dehydrated in progressively higher concentrations of alcohol and then cleaned in xylene until they were transparent. To thoroughly eliminate xylene, tissues were placed in molten paraffin wax for one hour before being impregnated with wax. To create sections with a thickness ranging from 4 to 6 microns, blocks were cut in a rotary microtome. Hematoxylin and eosin were used to stain the sections, which were then examined under the microscope (Rahmati et al., 2022). Under a light microscope (CX21; Olympus, Tokyo, Japan), the structural changes to the liver were examined and contrasted with the control tissue. Dino-Lite digital lenses were used for the photomicrographs, and DinoCapture software was used for the survey.
During the experiment (days 3, 60), fish were anesthetized with carnation (Dianthus) powder (5g/L), and their liver tissues were separated and collected in aluminum foil and stored at -80°C until use.
Determination of stress oxidative and antioxidant in liver
The isolated sections of the liver were immediately washed in ice-cold saline. It is then homogenized in potassium phosphate 50mM (pH=7.4) and reaches a concentration of 10%. They were then centrifuged at 4000 rpm for 15 minutes at 4°C. The obtained supernatant is used to measure spectrophotometrically and ELISA applying commercial kits based on the manufacturer’s instructions.
All data were presented as Mean±SEM and analyzed in SPSS software, version 26. Variance comparisons between the means of the control and experiment treatment data were made by 1-way ANOVA. Tukey’s post hoc test was used to compare the median of each group. The difference in mean values among the groups and histological analysis was studied utilizing a nonparametric test. P≤0.05 was regarded as significant.
Effects on MDA Levels
MDA level in the liver was considerably higher in the IONPs groups (48%) compared to the control (P≤0.05). The administration of spirulina with IONPs for 60 days considerably decreased the levels of MDA in the liver compared with the IONPs alone group, which means its protective and healing impacts (Figure 1).
Effects on GSH levels
IONPs caused a considerable depletion in GSH levels in the liver (60%) compared with the control group. S. platensis treatment with IONPs did produce a considerable increase in the level of GSH in the liver (Figure 2).
Effects on CAT levels
IONPs administration considerably increased the levels of CAT by 48% in the liver compared with the control group (P≤0.05). Treatment of fish with S. platensis reduced the levels of CAT in liver compared with the IONPs group, but have no significant differences with control group (Figure 3).
Effects on SOD levels
IONPs administration reduced the levels of SOD by 41% in the liver compared to the control group. Treatments of fish with S. platensis increased the levels of SOD by 173% in the liver compared to the control group (P≤0.05). The administration of spirulina with IONPs for 60 days caused a significant increase in the levels of SOD in the liver compared with the IONPs group (Figure 4).
The histopathology of the livers in the control group was normal (Figure 5a).
Liver sections of animals treated with IONPs for 3 days, revealed a rise of the melanomacrophage centers and hyperemia (Figure 5b), which was more severe in the 60-day group (Figure 6a) (Figures 6b, 7a).
A substantial degree of vacuole degradation was detected in several samples (Figure 7a, 7b).
It is interesting to note that S. platensis therapy stopped the histopathological changes caused by IONPs. Lesions seen in the livers of S. platensis plus IONPs intoxicated animals were much less than those seen in the IONPs alone group (Figure 8a, 8b).
The severity of the liver lesions seen in the gray mullet’s liver after various treatments are shown in Table 1.
Numerous studies have revealed both in vitro and in vivo toxicity of IONPs. However, some studies report that IONPs are not poisonous and may even be beneficial (Yarjanli et al., 2017). Enzymatic activity that removes iron ions from their surface may produce ROS (Jarockyte et al., 2016). This study aimed to examine the hepatic toxic effects of IONPs in grey mullet and how S. platensis could mitigate these effects. Ions produced by IONPs may participate in Fenton’s reaction and produce ROS from superoxide and H2O2 (Dashtipour et al., 2015). IONPs may harm the balance between the body’s antioxidant and oxidant defense mechanisms (Elmallah et al., 2017). Numerous physiological processes, including DNA synthesis, cell cycle control, transcription factor activation, mitochondrial permeability transition, and apoptosis, are impacted by oxidative stress (Yarjanli et al., 2017).
The levels of oxidative stress indices and MDA in the liver were dramatically elevated in the IONPs-treated group, whereas the activity of antioxidant SOD and GSH in the liver was significantly lowered. The mean levels of MDA and CAT activities in the liver rose considerably in the IONPs group compared to the control group. Compared to fish treated with IONPs alone, spirulina administration considerably lowered liver MDA and CAT activity. These findings demonstrated that SOD and GSH prevent producing oxygen free radicals in liver tissue (Karadeniz et al., 2009). These reductions may indirectly result in oxidative DNA or mitochondrial damage in cells (Yarjanli et al., 2017). However, the MDA levels of fish given IONPs containing S. platensis reverted to normal. Therefore, it is hypothesized that S. platensis inhibits lipid peroxidation due to its free radical scavenging function (Amin et al., 2006). According to Amin et al. (2006), S. platensis protects against hepatocyte-induced lipid peroxidation and has an inhibitory effect on cytochrome P450-associated monooxygenase activities.
The mean values of liver GSH concentration were considerably lower in the IONPs group compared to the control group. Compared to the control and IONPs groups, treatment with spirulina dramatically improved liver GSH levels. GSH is the most prevalent antioxidant in all cellular compartments (Darenskaya et al., 2018). GSH serves essential functions in an organism, such as eliminating xenobiotics (Karadeniz et al., 2009). According to Nunez et al. (2012), there is a correlation between iron buildup, low GSH levels, and oxidative stress. The buildup of iron decreases GSH levels and induces oxidative stress. The low amount of GSH increases the level of transferrin receptor (TfR), which transports iron into the cell and facilitates iron buildup (Karadeniz et al., 2009). Based on the information presented above, we argue that when iron buildup occurs, low GSH contributes to further iron accumulation and oxidative stress production. The liver GSH level in the IONPs-treated group was considerably lower than in the control group. Pongrac et al. (2016) revealed that the GSH level of cells exposed to IONPs decreased significantly. However, the same parameter for IONPs was elevated in the S. platensis-treated groups compared to the other groups. It demonstrates that this scenario is the consequence of the antioxidant properties of S. platensis. The S. platensis administration contributes to the maintenance of GSH levels. Karadeniz and Cemek (2006) reported the preventive activity of S. platensis against lead-induced plasma lipid peroxidation and oxidative damage in rats.
The findings were consistent with those of Hussein et al. (2019), who indicated that oxidative stress and apoptosis might play a role in neurotoxicity, which disrupted GSH production. In this research, a decrease in GSH levels may be attributable to its conjugation with certain metabolites (Wu & Dhanasekaran, 2020). Hassan et al. (2012), who investigated spirulina’s efficacy in combating oxidative stress in rats, found a comparable rise in GSH levels. Supplementation with spirulina significantly decreased aflatoxin-induced oxidative stress, as shown by an increase in GSH content. Abd El-Baky et al. (2009) noted that under oxidative stress, S. platensis maintained the activity of cellular antioxidant enzymes and raised the quantity of reduced glutathione in these cells.
In addition, these biochemical findings are complemented by the histological study of fish livers. IONPs brought about vacuolar degeneration, increased melanomacrophage centers (MMCs), and hyperemia in liver tissue. S. platensis demonstrated liver cell protection and regeneration. The synergistic impact of hepatoprotective action and antioxidant activity reduces the beginning and progression of hepatocellular disorders. Because of IONPs, membrane function abnormalities that result in a huge inflow of water and Na+ may cause the swelling of sinusoids (Abdelhalim and Jarrar, 2012). Degeneration is caused by ion and fluid homeostasis that increase intracellular water volume (Schrand et al., 2010). The vacuolated cytoplasm of the hepatocytes of fish treated with IONPs may indicate acute and subacute liver damage caused by these NPs. Some investigations show that hepatic dysplasia and carcinomatous lesions exhibit nuclear polymorphism (Abdelhalim and Jarrar, 2012). These findings imply that IONPs may interact with proteins and enzymes of the hepatic interstitial tissue, interfering with the antioxidant defense system and leading to ROS production, which may mimic an inflammatory response (Abdelhalim and Jarrar, 2012).
Some swollen hepatocytes that were exposed to IONPs had a fatty alteration. The fatty change in hepatocytes may be related to lipid peroxidation, which causes endoplasmic damage and lipoprotein separation and indicates improper fat metabolism (Abdelhalim and Jarrar, 2012). Changes caused by IONPs in our study may suggest liver toxicity. Some hepatocytes of fish treated with IONPs displayed relative necrosis. Apoptotic modification may be accompanied by organelle swelling, particularly mitochondria, endoplasmic reticulum, and rupture of lysosomes, which may result in amorphous eosinophilic cytoplasm as the initial sign in the sequence of hepatocytes necrosis preceding the shrinking and dissolution of nuclei (Abdelhalim and Jarrar, 2012). Changes seen in hepatocytes may indicate the oxidative stress caused by glutathione deficiency.
According to the data, IONPs are rather hazardous, but spirulina improved all parameters and protected liver damage and oxidative stress after exposure to IONPs in grey mullet. The protective effect of spirulina may be mediated by increasing antioxidants. Therefore, spirulina can be a therapeutic agent in liver damage induced by IONPs and other diseases related to oxidative stress.
Compliance with ethical guidelines
All ethical principles are considered in this article. The participants were informed of the purpose of the research and its implementation stages. They were also assured about the confidentiality of their information and were free to leave the study whenever they wished, and if desired, the research results would be available to them. A written consent has been obtained from the subjects. principles of the Helsinki Convention was also observed.
The paper was extracted from the PhD dissertation of the first author, Department of Basic Sciences, Faculty of Veterinary Medicine University of Tehran.
All authors equally contributed to preparing this article.
Conflict of interest
The authors declared no conflict of interest.
This research was supported by University of Tehran. We thank Chabahar Maritime University and the Department of Marine Biology for their help and support.