Around their means (Fig. 2A, B, C). Treatment with high Cd concentrations resulted in the doubling of SOD activities at 12 h (Fig. 2A). After 12 h, SOD activities in gills decreased and remained higher than the control levels at 48 h. Enzyme activity increased initially significantly at 8 h for GPx, and reached the highest levels at 12 h (Fig. 2C). The activity of GPx increased maximally to 295 of the control in group A, followed by a decrease at 24 h in all Cdtreated groups. CAT activities also increased dramatically at 8 h of Cd exposure (Fig. 2B). However, this stimulation was only transient and the CAT activities dropped back to levels similar to those found in the control after 24 h in groups A and C.Ultrastructural analysis with transmission electron microscopyAfter the exposure period, crabs were rinsed and three to five tissue pieces from the middle of the gill lamellae (approximately 1 mm) were cut and then fixed in glutaraldehyde. After fixation, tissues were rinsed twice in buffer immediately and post-fixed in 1 osmium tetroxide, and then dehydrated in a graded ethanol series and embedded in thin viscosity resin. Ultrathin sections were cut with an ultramicrotome (Leica UC-6), stained with uranyl acetate and lead citrate, and examined using a transmission electron microscope (JEM-1011) at an accelerating voltage of 80 kV.Oxidative stress indicesFor the investigation of the hypothetical role of ROS formation due to Cd toxicity, levels of H2O2 were determined in crab gills at control conditions and in the presence of Cd (Fig. 3A). At control conditions, a small change of H2O2 was observed, most likely reflecting the basal rate of H2O2 formation that occurred as a byproduct of aerobic metabolism. In the presence of Cd the production of H2O2 was significantly increased at 24 h in the treatment groups. The time-course analysis of H2O2 Docosahexaenoyl ethanolamide chemical information content showed that the lowest dose generated approximately 3.3 times more H2O2 than the control, while the highest dose generated approximately 3.5 times more H2O2 than the control at 96 h of exposure. It is evident that Cd exposure results in the robust generation of H2O2. Lipid CP21 supplier peroxidation levels in the gills of crabs, measured as the content of MDA, are given in Fig. 3B. At the absence of Cd, there were no significant changes in lipid peroxidation levels. However, with Cd there was a remarkable increase in lipid peroxidation levels which correlated positively with exposure time and concentration of Cd. The lipid peroxidation level increased to 147 , 163 and 185 of the control at 96 h for group A, group B and group C, respectively.Statistical analysisData were expressed as means 6 SD and computed statistically using one-way analysis of variance (ANOVA). The post hoc least significant difference (LSD) test was performed for an inter-group comparison. Probability values of p,0.05 were considered as statistically significant.Results Cd concentration assay in waterThe Cd concentration in water from each treatment group was analyzed during the experiment. The exposure experiment lasted for 96 h and the Cd concentration of the exposed groups was determined every day. As shown in Table 2, at 0 h, no significant changes in the Cd concentration from each treatment groups were observed compared to the nominal exposure concentration. However, the Cd concentrations in water in all groups treated with Cd declined significantly (p,0.05) after 24 h of acute Cd exposure compared with the nominal exposure co.Around their means (Fig. 2A, B, C). Treatment with high Cd concentrations resulted in the doubling of SOD activities at 12 h (Fig. 2A). After 12 h, SOD activities in gills decreased and remained higher than the control levels at 48 h. Enzyme activity increased initially significantly at 8 h for GPx, and reached the highest levels at 12 h (Fig. 2C). The activity of GPx increased maximally to 295 of the control in group A, followed by a decrease at 24 h in all Cdtreated groups. CAT activities also increased dramatically at 8 h of Cd exposure (Fig. 2B). However, this stimulation was only transient and the CAT activities dropped back to levels similar to those found in the control after 24 h in groups A and C.Ultrastructural analysis with transmission electron microscopyAfter the exposure period, crabs were rinsed and three to five tissue pieces from the middle of the gill lamellae (approximately 1 mm) were cut and then fixed in glutaraldehyde. After fixation, tissues were rinsed twice in buffer immediately and post-fixed in 1 osmium tetroxide, and then dehydrated in a graded ethanol series and embedded in thin viscosity resin. Ultrathin sections were cut with an ultramicrotome (Leica UC-6), stained with uranyl acetate and lead citrate, and examined using a transmission electron microscope (JEM-1011) at an accelerating voltage of 80 kV.Oxidative stress indicesFor the investigation of the hypothetical role of ROS formation due to Cd toxicity, levels of H2O2 were determined in crab gills at control conditions and in the presence of Cd (Fig. 3A). At control conditions, a small change of H2O2 was observed, most likely reflecting the basal rate of H2O2 formation that occurred as a byproduct of aerobic metabolism. In the presence of Cd the production of H2O2 was significantly increased at 24 h in the treatment groups. The time-course analysis of H2O2 content showed that the lowest dose generated approximately 3.3 times more H2O2 than the control, while the highest dose generated approximately 3.5 times more H2O2 than the control at 96 h of exposure. It is evident that Cd exposure results in the robust generation of H2O2. Lipid peroxidation levels in the gills of crabs, measured as the content of MDA, are given in Fig. 3B. At the absence of Cd, there were no significant changes in lipid peroxidation levels. However, with Cd there was a remarkable increase in lipid peroxidation levels which correlated positively with exposure time and concentration of Cd. The lipid peroxidation level increased to 147 , 163 and 185 of the control at 96 h for group A, group B and group C, respectively.Statistical analysisData were expressed as means 6 SD and computed statistically using one-way analysis of variance (ANOVA). The post hoc least significant difference (LSD) test was performed for an inter-group comparison. Probability values of p,0.05 were considered as statistically significant.Results Cd concentration assay in waterThe Cd concentration in water from each treatment group was analyzed during the experiment. The exposure experiment lasted for 96 h and the Cd concentration of the exposed groups was determined every day. As shown in Table 2, at 0 h, no significant changes in the Cd concentration from each treatment groups were observed compared to the nominal exposure concentration. However, the Cd concentrations in water in all groups treated with Cd declined significantly (p,0.05) after 24 h of acute Cd exposure compared with the nominal exposure co.
