Effects of chronic dietary cadmium on midgut superoxide dismutase (SOD) and catalase (CAT) in larvae from two Lymantria dispar populations

Abstract

INTRODUCTION: Cadmium (Cd) levels in the environment have increased during decades of intensive industrial development and urbanization. Lymantria dispar has proved to be a suitable organism indicator to monitor Cd pollution in the forest ecosystems. Since insects accumulate heavy metals predominantly in the gut, it is not surprising that several enzymes in the midgut of L. dispar larvae, including antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), have been marked as promising biomarkers of Cd presence. Namely, Cd indirectly induces oxidative stress in the cell. However, long-term exposure of the population to pollution often results in increased tolerance and changed sensitivity of biomarkers. OBJECTIVES: We aimed to determine specific enzyme activities and isoform patterns of SOD and CAT in the midgut of Lymantria dispar larvae after chronic treatment with Cd. To assess these parameters as biomarkers of Cd exposure, we compared the responses of two populations with different histories of an exposure to pollution. METHOD / DESIGN: Egg masses of L. dispar were collected from two localities in Serbia - the uncontaminated forest in Kosmaj Mountain, which is a protected natural resource, and a polluted site near the busy Ibar highway. Larvae were fed wheat germ diet containing 0, 50 or 100 μg Cd/g dry food starting from hatching until they were killed on the 3rd day of the 4th instar. Specific activities of SOD and CAT in the midgut homogenates were determined by spectrophotometric assays. Enzyme isoforms were separated by native polyacrylamide gel electrophoresis. Statistical analyses were performed in GraphPad Prism 7 (GraphPad Software, Inc., USA), where enzyme activities were analyzed by one-way ANOVA followed by Tukey’s post-hoc test. The level of statistical significance was p<0.05. RESULTS: Specific activity of SOD was higher in control larvae from the polluted locality compared to the control group from the uncontaminated forest. An exposure to both Cd concentrations decreased SOD activity in larvae from the polluted site. Three SOD isoforms were detected in control groups from both populations. While isoform SOD-2 was absent in the population from Kosmaj after the treatment with higher Cd concentration, both SOD-2 and SOD-3 disappeared in all Cd-treated larvae from the site near the highway. In the population from the unpolluted locality specific activity of CAT was reduced at 100 μg Cd/g dry food, whereas in another population a decrease in enzyme activity was noticed at both Cd concentrations. The same pattern of Cd influence was observed for CAT isoform activity. Only one CAT isoform was present in both control and experimental larvae from both populations. CONCLUSIONS: Higher SOD activity in control larvae originating from the site near the highway compared to those from the uncontaminated forest probably indicated the presence of traffic-related pollution that caused oxidative stress. However, neither SOD nor CAT showed activation in response to Cd treatment. A decrease in SOD and CAT activity in both Cd-treated groups in the population from the polluted site could have been a result of the trade-off in favour of the other more efficient defense mechanism(s). Such trade-off might have led to the diminished expression of isoforms SOD-2 and SOD-3. Thus, a decrease in SOD and CAT activities after Cd exposure could be seen as an adaptive strategy of L. dispar population living in chronically polluted habitat. These parameters, with SOD isoform pattern, could be used as biomarkers of Cd exposure in contaminated environments.