Chemicals
DMP (CAS No. 131-11-3, 99% purity) and other chemicals in reagent grade were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA).
Animal model
Sexually mature earthworms Lumbricus terrestris L. (Annelida: Lumbricidae), with a well-developed clitellum and the mean weight about 3-4.2 gr (fresh weight), were obtained from Soroush Rouyesh Salamat (SRS) limited Co., Shahrekord, Iran. Earthworms were acclimated to the laboratory conditions with a photoperiod of 12/12- h at 20±2 ºC for 14 days.
Exposure method
The toxic effects of DMP on earthworms were investigated by artificial soil tests according to the OECD normal method (OECD 1984). The artificial soil was prepared with 70% fine sand, 20 % kaolin clay, and 10% sphagnum peat at pH 7.0±0.2. Experiments were performed in triplicate, and operated in 1L glass jar with 500 g artificial soil and 20 earthworms. Based on the previous similar studies (Du et al., 2015; Wang et al., 2018; Song et al. 2019), the selected DMP concentrations were set as 0, 0.1, 1, 10, and 50 mg kg-1, dissolved in acetone, and sprayed on the surface of the test soil and thoroughly mixed. The DMP-treated artificial soils were placed in a well-ventilated fume hood and turned daily for 7 days in order to evaporate acetone. Then, all soil samples were rehydrated to 35 % moisture and left 1 day to equilibrate. The earthworms were cultivated for 24 h in untreated artificial soil and then they were put in the DMP treated soils.
Preparation of earthworms’ homogenates and mitochondria suspension
After 7 and 14 days, earthworms were thoroughly washed with distilled water before being placed on damp filter paper for 12h to avoid their gut contents (OECD, 1984). The earthworms were put on ice to immobile, and then were cut into pieces and mixed with chilled 50 mM potassium phosphate buffer (1:8, w/v; pH 7.0) containing 0.5 mM EDTA. Homogenization was carried out with Ultrasonic processor (JY-250, Zhejiang, China). The resulting homogenate was divided into two portions, i) for measuring malondialdehyde (MDA) in the LPO assay, carbonylated proteins (CP) content, and total protein content, and ii) centrifuged at 10,000 rpm for 10 min at 4°C to obtain the post-mitochondrial supernatant for antioxidant enzymes (CAT and GST) analyses (Velisek et al. 2011; Hu et al. 2012).
Collection of earthworm’s coelomic fluid (ECF)
In order to collect the ECF, electric shock method was conducted (Mohamed Jaabir et al. 2011; Chellathurai Vasantha et al. 2019). Earthworms suspended in the media containing Hank's balanced salt solution (HBSS) and phosphate buffered saline (PBS) (1:8, w/v, pH 7.0), and then were subjected to electric shock (3V, 30 sec, 1 min interval for recovery and repeated 5 times). Under shocks, the bio-fluid CF was collected and then filtered through Whatman number 1 filter paper. The filtrate was then centrifuged at 8000 rpm for 10 min, resulting supernatant stored at -20 °C for determination of TAC value. The sediment containing coelomocytes was gently diluted in HBSS/PBS medium and used for NRR-time assay.
Assay of oxidative stress biomarkers
Determination of malondialdehyde (MDA/LPO) level
The TBARS assay was performed to determination the MDA/LPO level in the homogenate aliquots (Buege and Aust 1978). In brief, 500 µl of aliquots was mixed with 1 ml of chilled 30% (w/v) trichlroacetic acid (TCA) and centrifuged at 5000 ×g, 10 min (Lushchak et al. 2005). Control samples contained distilled water instead of homogenate aliquot. The resulting aliquot used for measuring the amounts of MDA, by reaction with thiobarbituric acid (TBA) at 532 nm, using UV-1700 spectrophotometer (Shimadzu, Japan). The MDA level was calculated by using molar extinction coefficient of chromophore TBA (156×103 M−1cm−1) and expressed as n moles MDA per mg protein.
Determination of catalase (CAT) activity
The activity of antioxidant enzyme catalase (CAT; EC 1.11.1.6) was measured on the basis of the breakdown of hydrogen peroxide (H2O2) at 240 nm (Luck 1965). Briefly, assay mixture consisting of 3 ml H2O2 phosphate buffer and 0.50 ml of 10% supernatant of earthworm’s homogenate, and change in absorbance was recorded at 240 nm by UV-1700 Spectrophotometer (Shimadzu, Japan). The results were expressed as IU H2O2 decomposed per mg protein, following degradation of H2O2 by CAT activity present in the samples.
Determination of glutathione S-transferase (GST) activity
The determination of GST (GST; EC 2.5.1.18) activity refers to the method of Habig et al. (1974), using S-2, 4-dinitrophenyl glutathione (CDNB) as a substrate. The principle of the method is based on measurement of the conjugation of CDNB with GSH. The formation of adduct of DNB, S-2,4-dinitrophenyl glutathione (DNPG) was monitored by measuring the rate of increase in absorbance at 340 nm for 4 min. The enzyme activity was calculated from the extinction coefficient of GSH-CDNB and expressed as n moles DNPG produced per mg protein.
Determination of carbonylated proteins (CP) content
According to the mthod of Levine et al (1994), the CP content was measured spectrophotometrically in the earthworm’s homogenate supernatants. Briefly, 0.5 ml of supernatants were treated with 0.5 ml of 10 mM 2,4-dinitrophenylhydrazine (DNPH) in 2M HCl, or with 0.5 ml of 2M HCl alone for the blank. Samples were then incubated for 1 h at room temperature in the dark, and then 10% trichloroacetic acid (TCA) was added and centrifuged at 3,000 ×g for 20 min. The pellet was washed three times in ethanol/ethylacetate and solubilized in 1 ml 6M guanidine in 20 mM potassium phosphate, adjusted to pH 2.3 with trifluoroacetic acid; the resulting solution was incubated at 37 °C for 15 min. Carbonyl concentrations were determined from the difference in absorbance at 370 nm between DNPH-treated and HCl-treated samples, with ε370 = 22,000 M−1cm−1. The content of CP were expressed as n moles CP per mg protein.
Determination of total antioxidant capacity (TAC)
To measure the changes in the TAC value, the method of ferric reducing antioxidant power (FRAP) was applied in the ECF (Benzie and Strain 1996). During the assay, yellow ferric tripyridyltriazine complex (Fe (III)-TPTZ) is reduced to blue ferrous complex (Fe (II)-TPTZ) by the action of electron-donating antioxidants in ECF samples. The resulting blue color is linearly related to the total reducing capacity of antioxidants in solutions: i) Reagents: 1) Acetate buffer 300 mM pH 3.6: Weigh 3.1 g sodium acetate trihydrate and add 16 ml of glacial acetic acid and make the volume to 1L with distilled water; 2) TPTZ (2, 4, 6-tripyridyl-s- triazine), 10 mM in 40 mM HCl; 3) FeCl3. 6H2O, 20 mM. The working FRAP reagent was prepared by mixing a, b and c in the ratio of 10:1:1 just before testing. Standard was FeSO4.7H2O: 0.1-1.5 mM in methanol. ii) Procedure: FRAP solution (1.5 ml) was incubated at 37 ̊C for 5 min. Then this solution mixed with 0.1 ml of diluted ECF (1:10) and incubated at 37 ̊C for 10 min. The end product (Fe2+-TPTZ) had blue color. The absorbance of the reaction mixture was measured at 593 nm for 4 min. For construction of the calibration curve, five concentrations of FeSO4, 7H2O (0.1, 0.4, 0.8, 1, 1.12, 1.5 mM) were used and the absorbance values were measured as for sample solutions. The values obtained were expressed as mM Fe2+ produced per L.
Determination of neutral red retention time (NRR-time)
The NRR-time assay was performed to evaluate the lysosomal membrane stability in earthworm’s coelomocytes (Weeks and Svendsen 1996; Xiao et al. 2006). As healthy cell lysosomes retain the neutral red dye for some length of time, dye retention times represent a marker of membrane permeability. The filtrate of CF was centrifuged at 8000 rpm for 10 min and resulting sediment containing coelomocytes diluted in HBSS/PBS medium and gently used for this assay. The stock solution of red dye was made by dissolving 28.8 mg of dye, C15H16N4.HCl, in 1 ml of dimethyl sulfoxide (DMSO). The working solution was prepared by diluting 5 µl of stock solution in 5 ml of physiological saline. A droplet of the sediment was transferred to a glass slide before 20 μl of working dye solution was added. The slides were scanned for non-leaking and leaking coelomocytes at 2 min intervals under a light microscope with magnification of 400, ideally 1 min per slide maximum. Tests were terminated when the dye loss was evident in at least 50% of the coelomocytes after 90 min. In the majority of the control earthworms, the number of leaking cells never reached this level within the maximum observation time of 90 min.
Determination of total protein content
The total protein content was quantificated by the method of Bradford using bovine serum albumin (BSA) as standard at absorbance of 595 nm, and presented as mg protein per ml of whole body extract of earthworms (Bradford, 1976).
Statistical Analysis
The experimental results were analyzed with the SPSS software (2019 v26). The standard ANOVA techniques, followed by Tukey’s HSD post hoc test, were used to examine the differences among treatment groups and control groups. A probability level of p<0.05 was considered as significant. Data were presented as Mean±Standard Deviation (SD) (n=20).