Iranian Scientific Fisheries Journal

fa مقاله علمی – پژوهشی:‌ فعالیت کمپلمان‌‌ها در کشت اولیه سلول‌‌های طحال و رأس کلیه ماهی شانک عربی (Acanthopagrus arabicus) در مواجهه با دگزامتازون Activity of complements in the primary culture of spleen and head kidney cells of Arabian yellow fin sea bream (Acanthopagrus arabicus) exposed to dexamethasone آلودگي محيطهاي آبي آلودگي محيطهاي آبي پژوهشي Research داروها آلاینده‌‌های نوظهوری هستند که به دلیل تخلیه‌‌ آنها به اکوسیستم‌‌های آبی از طریق فاضلاب در سراسر جهان، نگرانی‌‌های زیادی را به‌خود جلب کرده‌‌اند. با این‌حال، مطالعات محدودی در مورد اثرات این آلاینده‌‌ها بر حیوانات آبزی وجود دارد. دگزامتازون یک گلوکوکورتیکوئید مصنوعی قوی با اثرات ضد التهابی و سرکوب‌کننده سیستم ایمنی است. مطالعه حاضر به بررسی تأثیر دگزامتازون بر فعالیت اجزاء کمپلمان سلول‌های رأس کلیه و طحال ماهی شانک عربی (<a name="_Hlk175087857">Acanthopagrus arabicus</a>) در شرایط آزمایشگاهی پرداخت. برای این منظور، سلول‌‌های طحال و رأس کلیه کشت شده در مواجهه با غلظت‌‌های مختلف دگزامتازون (غلظت‌های آزمایشی 3، 1-10×3، ۲-10×3 و 3-10×3 میکروگرم بر میلی‌‌لیتر برای طحال و غلظت‌‌های 10 × 3، 3، 1-10×3 و ۲-10×3 میکروگرم بر میلی‌‌لیتر برای رأس کلیه) قرار گرفتند. پس از ۱۲، ۲۴ و 48 ساعت، اثرات سمی دگزامتازون بر اجزاء سیستم کمپلمان بررسی شد. نتایج نشان داد که حساسیت سلول‌‌های طحال و رأس کلیه کشت شده به دگزامتازون به طور وابسته به دوز افزایش می‌‌یابد. دگزامتازون به طور معنی‌‌داری محتوای C3 سلول‌‌های کشت شده را کاهش داد درحالی‌که تغییر قابل‌توجهی در محتوای C4 و ACH50 سلول‌‌های کشت شده ایجاد نکرد. همچنین تأثیر دگزامتازون بر سلول‌‌های رأس کلیه بیش از طحال بود. بر اساس نتایج، جنبه سرکوب‌کننده سیستم ایمنی دگزامتازون عمدتاً به دلیل اثرات سرکوب‌کننده آن بر سطح جزء C3 کمپلمان‌‌هاست. توصیه می‌‌شود در آینده، تأثیرات آلاینده دارویی دگزامتازون بر آبزیان بیشتری از جمله ماهیان مدل نیز انجام شود. Introduction Emerging anthropogenic pollutants in wastewater treatment plant (WWTP) effluents have been shown to pose a particular risk to aquatics and potentially affect the aquatic environment from the molecular to the ecosystem level (Santos et al., 2010). Micropollutants are emerging pollutants that include pharmaceuticals and personal care products (PPCPs). Due to the widespread consumption of a wide range of micropollutants and their discharge into wastewater worldwide, their occurrence and fate in aquatic environments has become a research topic in recent decades (Ryu et al., 2014). The common wastewater treatment process is not able to completely remove these compounds from the wastewater, and as a result, a significant amount of these compounds enters the aquatic water environment (Rehman et al., 2015). Dexamethasone is a potent synthetic glucocorticoid with anti-inflammatory and immunosuppressive effects that is used to treat many diseases including allergies, asthma, covid-19, rheumatic problems, and skin diseases (Sanders et al., 2020). Today, the increasing use of dexamethasone from sources such as hospitals is a global concern. High levels of this drug, which is a cortisone derivative, have been detected in wastewater (Herrero et al., 2013). As one of the important components of the innate immune system in ectothermic vertebrates, including fishes, the complement system has been less studied. Evaluation of serum complement activity is considered as a valuable tool to diagnose the health status of fish. Complement in the non-specific immune response to a compound can have a direct effect such as killing the pathogen by lysing it (Ellis, 1999). <a name="_Hlk181357087">Method</a>ology Fifteen mature Arabian sea bream, A. arabicus were collected from the Bahrakan Port in Abadan. Fish were transported to the 300 L tanks. After seven days of adaptation, the fish were anesthetized using 2-phenoxy ethanol (0.35 ml/L). Then, the fish were dissected under aseptic conditions and their spleens and kidney heads were separated (<a href="#_ENREF_18" title="Wen, 2008 #76">Wen et al., 2008</a>). The separated tissues were immediately washed three times with 100 ml antibiotic medium (Leibovitz's L-15 (L-15) with 400 IU/ml penicillin, 400 µg/ml streptomycin and 200 µg/ml amphotericin B) each time for 30 minutes. The spleen cell culture was performed according to Huang et al. (2009). The head kidney cell culture followed the protocols described by Ribera et al. (2020). Dexamethasone cytotoxicity was assessed by MTT method according to Momeni et al. (2010). Cultivated spleen and head kidney cells were plated in 24-well microplates (105 cells / ml L-15 medium / well) and incubated at 28°C for 24h. The cell viability was assessed using trypan blue exclusion test. Then, 200 μL of fresh L-15 culture medium with different experimental concentrations of dexamethasone was added to each well and microplates were then incubated at 28°C for 48 h. Each treatment runs in five replicates. For this purpose, the cell suspensions were collected at 0, 12, 24 and 48 hours of experiment and transferred to microtubes for further analysis. The level of C3 and C4 was measured using immunoturbidimetry method by a double–antibody sandwich ELISA kits. ACH50 was measured by hemolytic method according to Sunyer and Tort (1995). Results Measurement of dexamethasone in hospital wastewater: To measure dexamethasone in hospital wastewater, samples were taken from the wastewater of Taleghani Hospital in Abadan, and after scanning the dexamethasone samples and determining the maximum absorption wavelength at 213 nm for it, the concentration of dexamethasone was measured using an HPLC device. The obtained concentration was 86 ng/l. Primary culture of head kidney and spleen cells: Using the trypan blue test, the survival rate of spleen and head kidney cells was determined to be 95%. Then, cells derived from the spleen and head kidney were incubated in L-15 culture medium for two weeks at 28°C. After 48 hours, cells started to grow and colony formation started 5 days after incubation. The cells grew well and completely occupied the culture dish within 12 days. All the time, the cells were floating in the culture medium as round cells and did not stick to the bottom of the culture container. The trypan blue test showed that more than 90% of the cells were alive and healthy during the test. Twelve days after incubation, the cells were passaged. The second passage was performed fifteen days after incubation and then the cells were used for cytotoxicity assay. The C3 contents notably decreased in spleen cells exposed to all concentrations of dexamethasone except for the lowest concentration (3 μM; P < 0.05). In the head kidney, the C3 contents only notably decreased in cells treated with the higher concentrations of dexamethasone (3×10 and 3×102 μM; P < 0.05). The complement C4 has no obvious change following exposure of spleen and head kidney cells to all concentrations of dexamethasone except for the highest concentration. The C4 content of spleen and head kidney cells significantly decreased after 24 h exposure to highest concentration of dexamethasone (P < 0.05).  The concentrations of dexamethasone (except the highest concentration of dexamethasone) caused a significant change in the ACH50 content of spleen cells compared to the controls (P > 0.05). The exposure of spleen cells to 3 mM dexamethasone led to an 11% decrease in alternative complement activity after 48 hours of exposure (P < 0.05). ACH50 activity in head kidney cells treated with 3×102 nM and 3 μM of dexamethasone showed no difference compared to control (P > 0.05). A significant decrease in ACH50 was observed after exposure of head kidney cells to 3×10 and 3×102 μM of dexamethasone (P < 0.05). The amount of ACH50 activity in head kidney cells treated with 3 mM dexamethasone decreased by 27% after 48 hours of exposure (P < 0.05) Discussion and Conclusion This study is the first study that reports the suppressive effects of dexamethasone on the activity of complement components of head kidney cells and spleen of Arabian yellow fin sea bream (Acanthopagrus arabicus) in cell culture medium. Dexamethasone is commonly known as an immunosuppressant drug that is toxic to immune cells and inhibits the function of the immune system (Ribas et al., 2016). The complement system plays an important role in innate and acquired immunity by facilitating the function of phagocytizing cells and cell lysis. The components of this system, which are activated in a cascade manner, lead to the destruction of pathogens by targeting the membrane components of invading agents (Boshra et al., 2006). C3 is an important component of the complement system, whose stimulation activates other components. This component belongs to the acute phase cellular proteins and is the first component of the complement system to be activated (Bayne et al., 2001). In the present study, dexamethasone led to a significant decrease in C3 levels in spleen and head kidney cells. On the other hand, the level of C4 in spleen and head kidney cells was suppressed only by the highest concentration of dexamethasone. In the study of Zach et al. (1993), corticosteroids can suppress the expression level of genes related to the synthesis and activity of C3 through the glucocorticoid (GR) and mineralocorticoid (MR) receptors. Most Osteichthyes have three corticosteroid receptors, including two glucocorticoid receptors (GR1 and GR2) and one mineralocorticoid receptor (MR), which are encoded by three different genes (Zach et al., 1993). This can explain the decrease in complement levels under the influence of dexamethasone (corticosteroid) in the present study. In the present study, dexamethasone did not affect the activity of alternative complement component (ACH50) of spleen and head kidney cells, and only its highest concentration decreased ACH50 levels after 48 hours. Milla et al. (2018) did not observe any changes in gene expression, protein quantity or activity of proteins related to innate immunity (such as ACH50 level) up to two weeks after exposure of Perca fluviatilis to 11-deoxycorticosterone (corticosteroid). While after two weeks, a significant increase in the ACH50 level of the spleen was observed. They stated that acute exposure to corticosteroids (as immunosuppressants) does not seem to have a significant effect on some innate immune proteins (Milla et al., 2018). Acknowledgment This work was supported by the Khorramshahr University of Marine Science and Technology. دگزامتازون, سیستم کمپلمان, آلاینده‌های دارویی, Acanthopagrus arabicus Dexamethasone, Innate immune factors, pharmaceutical pollutants, Arabian seabream 1 16 http://isfj.ir/browse.php?a_code=A-10-2924-1&slc_lang=fa&sid=1 Fatemeh Nozhat فاطمه نزهت yfnozhat@yahoo.com 100319475328460039506 100319475328460039506 No گروه زیست شناسی دریا، دانشکده علوم دریایی و اقیانوسی، دانشگاه علوم و فنون دریایی خرمشهر، خرمشهر، ایران Mohammad Ali Salari-Aliabadi محمد علی سالاری علی آبادی salari@kmsu.ac.ir 100319475328460039507 100319475328460039507 Yes گروه زیست شناسی دریا، دانشکده علوم دریایی و اقیانوسی، دانشگاه علوم و فنون دریایی خرمشهر، خرمشهر، ایران Negin Salamat نگین سلامات neginsalamat@kmsu.ac.ir 100319475328460039508 100319475328460039508 No گروه زیست شناسی دریا، دانشکده علوم دریایی و اقیانوسی، دانشگاه علوم و فنون دریایی خرمشهر، خرمشهر، ایران Hoda Mojiri_Forushani هدی مجیری فروشانی hmojiriforushani@abadanums.ir 100319475328460039509 100319475328460039509 No گروه فارماکولوژی، دانشکده پزشکی، دانشگاه علوم پزشکی آبادان، آبادان، ایران Hasan Morovvati حسن مروتی hmorovvati@ut.ac.ir 100319475328460039510 100319475328460039510 No گروه علوم پایه، دانشکده دامپزشکی، دانشگاه تهران، تهران، ایران