Protective effect of hydroferrate fluid, MRN-100, against lethality and hematopoietic tissue damage in γ-radiated Nile tilapia, Oreochromis niloticus

摘要

MRN-100 was prepared in distilled water (DW) with the concentration of Fe2+ and Fe3+ ions at about 2 × 10?12 mol/l. MRN-100 is obtained from phytosin, a plant extract that contains iron and neutral lipid compounds and can be found in rice, wheat, or radish seeds. To start, 1 unit of phytosin is dispersed in 100 ml DW, and ferric chloride as FeCl₃?6H₂O is added. The lipid compounds are removed through a liquid–liquid extraction using a separation funnel. The remaining liquid is filtered with No. 5 filter paper and the filtrate is evaporated and condensed in a water bath. The iron compound obtained is subjected to fractional determination with respect to bivalent ferrate and trivalent ferrate in order to generate MRN-100 [13, 21]. Briefly, the sample's Fe (II) quantity is determined using the o-phenanthrolin method. Hydroxylamine-HCl 10% solution is added to the sample liquid to reduce Fe (III) to Fe (II) beforehand. Subsequently, all of the ferrate quantities are determined, followed by the determination of the quantity of Fe (III). As a result, the iron compounds thus obtained turned out to be bivalent and tervalent ferrates. MRN-100 was provided by ACM Co., Ltd, Japan. Radiation was carried out with a γ-ray source of 4-MCi 137 Cs (Radiotherapy Department, Mansoura University Hospital, Egypt). The radiation employed for the whole-body radiation was a single dose of 15 Gy with a target object distance of 27 cm and an exposure rate of 200 R/min. During radiation, each group was kept in small oblong-shaped glass vessels containing 2 l of aged tap water. Immediately after radiation, fish were transferred into large, 200-l tanks, and 10% of the water was changed every day until the end of the experiment. Survival of fish was monitored twice-daily at morning and at night for 27 days post-radiation. The Oreochromis niloticus (6–8 weeks old, ～50 ± 15 g body weight, length ～16.5 ± 10 cm) were purchased from a fish farm specializing in Tilapia (Iman Farm, Kafr El-Sheikh, Egypt) and acclimatized for 1 week prior to experimentation. The fish were placed in tanks (54 fish per tank); each tank contained 200 l of aged dechlorinated tap water. The number of fish per liter used in the current study was within the proximity of another published study [22]. The fish were kept outdoors throughout the experimental period where the water in all aquaria was aerated continuously and temperatures were kept at ～ 22 ± 2oC. The fish were fed standard laboratory floating pellets twice a day (at 8 AM and 1 PM). A total of 216 Oreochromis niloticus were randomly divided into four groups (G1–G4) of 54 fish per group. Of these, 47 fish from each group were used for daily recording of survival post-exposure to radiation. (After 1 week, 5–7 fish of each group were used for hematology and histology studies. At 4 weeks, on the last day of the survival study, 5–7 fish of each group were used for follow-up hematology studies). G1 served as the control with no administration of MRN-100 or radiation; G2 was exposed to whole-body γ-radiation only; G3 and G4 were treated with doses of 1 ml and 3 ml of MRN-100 per liter of water, respectively, for 1 week, then exposed to radiation, while continuing to receive MRN-100 for 27 days. The effect of radiation and MRN-100 on the survival rate of fish was examined by recording the dead fish among the four groups daily for 27 days, while biochemical and histopathological analysis was performed on the surviving fish at 1 and 4 weeks post-exposure to γ-radiation. For biochemical analysis, 5–7 fish from each group were collected at 1 and 4 weeks post-irradiation and were treated with a mixture of clove oil and alcohol for 2–3 min. Each fish was put on a clean towel and 1–2 ml of blood was withdrawn from the caudal vein using a syringe containing EDTA. The tubes were thoroughly shaken to avoid clotting of blood and used for blood analysis, which include: white blood cell count (WBC), red blood cell count (RBC), hemoglobin content (HGB), hematocrit percentage (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and platelet count (PLT). Blood analysis was evaluated according to the protocol provided by the kit's manufacturer. In addition, 1 ml of blood from each fish was left to coagulate, then centrifuged and the sera were collected to evaluate the level of serum glutamic pyruvic transaminase (SGPT) in accordance with the protocol provided by the kit's manufacturer. A range of organs, including the hepatopancreas, spleen and gills of each group, were examined for histopathological changes at 1 week post-exposure to γ-irradiation. Organs were fixed in 10% formalin solution, sectioned, and fixed overnight in cassettes. The paraffin-embedded tissues were sectioned on a microtome to a thickness of 4 μm, stained with hematoxylin and eosin (H&E) and observed under light microscopy.