How potential endocrine disruptor deltamethrin effects antioxidant enzyme levels and total antioxidant status on model organisms

Experimental organism

The narrow clawed freshwater crayfish (A. leptodactylus Eschscholtz, 1823), invertebrate model organism, was used as representative organism of fresh water ecosystems. Live organisms (N = 128) were obtained from a fisherman during the fishing season. The mean weight and the length of crayfish were 30.57 ± 5.26 g and 10.64 ± 0.48 cm, respectively.

Test chemical

Deltamethrin (China, Changzhou Kangmei Chem. Ind.Co. Ltd.), technical grade (98%) was used in the experiment and obtained from the Hacettepe University Insecticide Test Lab. The stock solution was dissolved in DMSO and stored at +4 °C till the dosing time.

Acclimatisation period and experimental design

Crayfish were acclimatised to laboratory medium for fifteen days before the trials and were fed with fresh trout during that period. Cleaning of the tanks were done twice in a day and the tanks were aerated constantly. All experiments were carried out according to the rules of “Guide for the Care and Use of Laboratory Animals” published by the US National Institutes of Health (NIH publication No. 85–23, revised 1996). After adaptation period, crayfish were stocked to experimental aquariums containing with 100 L of dechlorinated tap water. Animals were selected randomly for control and experiment groups, each stocked as 16 crayfish/8 aquaria's. The water composition was: DO₂ 6.96 ± 0.10 mg/L; pH = 7.74 ± 0.03; conductivity 0.298 ± 0.01 mS/cm. Sublethal deltamethrin concentration was selected as the 1/10 of 96 h LC50 values, 0.05 μg/L. The stability of deltamethrin concentrations in the aquarium water was provided by renewing every 48 h. Exposure lasted for one week. Haemolymph and tissue samples were taken after 48 h and 7 days.

Haemolymph sampling and analysis

Following the exposure to deltamethrin, haemolymph was taken on the base of the 2nd walking leg under ice anaesthesia using disposable syringe. Hemolymph samples were used for counting the total haemocytes and TAS determinations. THCs were performed with Thoma counting chamber according to Benli [18].

After haemolymph samples were centrifuged at 3,500 rpm, 10 min at 4 °C, TAS (mmol/L) were determined by a Randox commercial kits (Randox Laboratories Ltd., Crumlin, UK). The principle of the assay is incubating 2,2′-azinobis (3-ethylbenzothiazoline 6-sulfonate) (ABTS) with ferrylmyoglobin radical, formed by the activation of a peroxidase (metmyoglobin) with hydrogen peroxide (H₂O₂), to produce the radical cation ABTS+ [19].

Tissue sampling and analysis

Crayfish were sacrificed under ice anaesthesia and tissues were dissected; gill, muscle and hepatopancreas tissue samples were taken and stored for antioxidant enzyme analysis at −80 °C.

Superoxide Dismutase Activity (SOD)

SOD activity was done by the method of Sun et al. [20] with the Superoxide Dismutase Assay Kit (Cayman Chemical Company). Tissue samples were homogenized in Tris-HCl containing EDTA (1/10; w/v). SOD activity was inhibited by nitro blue tetrazolium reduction, with xanthine-xanthine oxidase used as a superoxide generator, and 1 international unit (IU) is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. Tissue SOD activity was stated as U/mg protein.

Catalase Activity (CAT)

Catalase activity was determined by Catalase Assay Kit (Cayman Chemical Company). The basis of the method is the reaction of the enzyme with methanol, in the presence of an optimal concentration of H₂O₂ [21]. Crayfish tissues were homogenized with 0.05 M phosphate buffer at pH 7.0. CAT activity was expressed as units representing the calculated rate constant (K)/mg protein.

Glutathione Peroxidase Activity (GPx)

GPx activity was measured according to the protocol of the Glutathione Peroxidase Assay Kit (Cayman Chemical Company). Tissue samples were homogenized in Tris-HCl containing EDTA (1/10; w/v). Tissue Glutathione Peroxidase Activity results were stated as nanomoles of NADPH oxidized per minute per mg protein, using an extinction coefficient for NADPH at 340 nm.

Two replicates were performed for all tests. Besides protein concentrations for all tissues were performed by the method of Lowry et al. [22].

Statistical analyses

The results are means ± SD. Analysis of data concerning the differences between groups was made using non-parametric Mann Whitney-U Test and Kruskal Wallis with GRAPHPAD InStat 3 Statistic Programme.

Haemolymph analysis

Following exposure to 0.05 μg/L of deltamethrin for 48 h and 7 days, the THCs values were decreased significantly when compared to control groups (p < 0.05). Means (THC/mL) of the control values were 80,989.58 ± 13,390 and 79,427.08 ± 9,909 as control and control (solvent), respectively. The decrease was around 27% after 48 h (50,885.42 ± 3,971.8) and 50% after 7 days (34,895.83 ± 3,125.8) when compared to control groups. There is no significant difference between duration times of exposure groups. The results are shown in Figure 1.

Figure 1:

Total haemocyte counts of crayfish after exposure to 0.005 μg/L of deltamethrin for 48 h and 7 days. Means in the figure with different small letters are significantly different (p < 0.05).

Haemolymph TAS levels of deltamethrin exposed crayfish were slightly increased when compared to control groups but not statistically significantly different. The haemolymph TAS results are shown in Figure 2. Means of the haemolymph TAS values for control and control (solvent), 48 h exposed group and 7 days exposed group were 1.50 ± 0.02, 1.52 ± 0.05, 1.60 ± 0.06 and 1.57 ± 0.04 mmol/L, respectively. There was no significant correlation between THCs and TAS (r² = 0.045).

Figure 2:

Haemolymph total antioxidant status of crayfish after exposed to 0.005 μg/L of deltamethrin for 48 h and 7 days.

Antioxidant enzyme activities of tissue samples

SOD enzyme activities of gill tissues were significantly increased when compared to control groups (p < 0.05). Means of gill tissue SOD enzyme activities for control and control (solvent), 48 h exposed group and 7 days exposed group were 4.33 ± 0.42, 4.54 ± 0.89, 5.26 ± 1.04 and 5.13 ± 0.45 U/mg protein, respectively. Gill tissue SOD activities were increased about 17%. SOD activities of hepatopancreas tissues were not altered statistically. Means of the hepatopancreas tissue SOD enzyme activities for control and control (solvent), 48 h exposed group and 7 days exposed group were 1.50 ± 0.59, 1.48 ± 0.46, 1.74 ± 0.34 and 1.55 ± 0.23 U/mg protein, respectively. SOD activities of muscle tissues were significantly rised (p < 0.05) after exposed to deltamethrin. Means of muscle tissue SOD enzyme activities for control and control (solvent), 48 h exposed group and 7 days exposed group were 3.69 ± 0.20, 3.91 ± 0.14, 5.40 ± 0.33 and 4.64 ± 0.27 U/mg protein, respectively. The SOD activity results of gill, hepatopancreas and muscle tissues are depicted in Figure 3.

Figure 3:

SOD levels of gill, hepatopancreas and muscle tissues of crayfish after exposed to 0.005 μg/L of deltamethrin for 48 h and 7 days. Means in the figure for each tissue with different small letters are significantly different (p < 0.05).

CAT enzyme activities of gill tissues were significantly increased when compared to control groups (p < 0.05). Means of gill tissue CAT enzyme activities for control and control (solvent), 48 h exposed group and 7 days exposed group were 0.059 ± 0.024, 0.048 ± 0.001, 0.09 ± 0.003 and 0.08 ± 0.001 mmol/min/mg protein, respectively. CAT activities were increased about 31% after 48 h and 20% after 7 days. Hepatopancreas CAT levels were increased significantly after 48 h (p < 0.05), but returned back to control levels after 7 days. Means of hepatopancreas tissue CAT enzyme activities for control and control (solvent), 48 h exposed group and 7 days exposed group were 0.077 ± 0.010, 0.081 ± 0.003, 0.176 ± 0.089 and 0.070 ± 0.001 mmol/min/mg protein, respectively. The CAT activities of muscle tissues were not affected by deltamethrin exposure. Means of muscle tissue CAT enzyme activities for control and control (solvent), 48 h exposed group and 7 days exposed group were 0.021 ± 0.05, 0.022 ± 0.01, 0.020 ± 0.004 and 0.019 ± 0.040 mmol/min/mg protein, respectively. The CAT activity results of gill, hepatopancreas and muscle tissues are shown in Figure 4.

Figure 4:

CAT levels of gill, hepatopancreas and muscle tissues of crayfish after exposed to 0.005 μg/L of deltamethrin for 48 h and 7 days. Means in the figure for each tissue with different small letters are significantly different (p < 0.05).

GPx enzyme activities of gill tissues were significantly increased when compared to control groups (p < 0.05). Means of gill tissue GPx enzyme activities for control and control (solvent), 48 h exposed group and 7 days exposed group were 1.136 ± 0.12, 1.120 ± 0.02, 1.48 ± 0.158 and 1.831 ± 0.03 mmol/min/mg protein, respectively. GPx activities were increased about 25% after 48 h and 38% after 7 days. Means of hepatopancreas tissue GPx enzyme activities for control and control (solvent), 48 h exposed group and 7 days exposed group were 0.382 ± 0.05, 0.401 ± 0.01, 0.530 ± 0.193 and 0.501 ± 0.039 mmol/min/mg protein, respectively. Hepatopancreas GPx activities were increased significantly after the exposure to deltamethrin during the both exposure period (p < 0.05). Means of muscle tissue GPx enzyme activities for control and control (solvent), 48 h exposed group and 7 days exposed group were 0.416 ± 0.05, 0.402 ± 0.20, 0.424 ± 0.052 and 0.432 ± 0.055 mmol/min/mg protein, respectively. Muscle GPx activities were not altered after deltamethrin exposure. The GPx activity results of gill, hepatopancreas and muscle tissues are shown in Figure 5.

Gill tissues SOD, CAT and GPx activities, hepatopancreas CAT and GPx activities and muscle SOD activities increased significantly compared to control groups (p < 0.05), while hepatopancreas SOD activities and muscle CAT and GPx activities were not altered significantly.

Figure 5:

GPx levels of gill, hepatopancreas and muscle tissues of crayfish after exposed to 0.005 μg/L of deltamethrin for 48 h and 7 days. Means in the figure for each tissue with different small letters are significantly different (p < 0.05).