Crowding Effects of Polystyrene Nanoparticles on Lactate Dehydrogenase Activity in Hydra Attenuata

Abstract

Plastics pervade our environment and potentially release important quantities of plastic nanoparticles (NPs) from degradation in the environment. The purpose of this study was to examine the crowding effects of polystyrene NPs on lactate dehydrogenase (LDH) in vitro and following exposure to Hydra attenuata. First, LDH activity was measured in vitro in the presence of filamentous (F-)actin and NPs (50 and 100 nm diameter) to determine changes in viscosity and the fractal kinetics of LDH. The fractal dimension (fD) was also determined using the rescaled range analysis procedure. Secondly, these changes were examined in hydra exposed to NPs for 96h to concentrations of NPs. The data revealed that the addition of F-actin increased the rate of LDH at low substrate (pyruvate) concentrations compared to LDH alone with a gradual decrease in the rate with the addition of pyruvate, which is characteristic of the fractal behavior of enzymes in crowded environments. The addition of 50 and 100 nm NPs also produced these changes, which suggest that NPs could change the space properties of the LDH reaction. The fD was reduced to 0.85 and 0.91 with 50 and 100 nm NPs compared to 1.093 with LDH alone. Decrease in the fD was related with increased amplitudes and frequency in viscosity waves in the reaction media. Exposure of hydra to NPs confirmed the increase in LDH activity and the fD was significantly correlated with LDH activity (r = -0.5). Correction of LDH activity (residuals) still revealed an increase in LDH activity in hydra suggesting increased anaerobic metabolism by NPs. In conclusion, the presence of NPs in the intracellular space decreased the fD, which could influence LDH activity in organisms exposed to NPs.

Keywords: Hydra attenuata; fractal dimension; lactate dehydrogenase; nanoplastics; polystyrene.

Figure 1

The fractal kinetic behavior of LDH in the presence of F-actin and 50 nm nanoparticles (NPs). LDH activity was determined alone and in the presence of F-actin (A) and 50/100 nm diameter NP (B). The data represent the mean with standard error for N = 3 replicates per treatment. The treatments were all significant from controls. The star symbol indicates difference between the crowding agents (F-actin, 50 and 100 nm NPs) and LDH alone. * p < 0.05.

Figure 2

Change in viscosity by F-actin and NPs during the LDH reaction. The viscosity probe 9-(dicyanovinyl) julolidine (DCVJ) was added during the LDH reaction in the presence of 0.4 mM pyruvate. Fluorescence was measured at 450 nm excitation and 520 nm emission.

Figure 3

Rate change in LDH activity in hydra exposed to polystyrene NPs. LDH activity was determined with increasing amounts of pyruvate (A) and changes in viscosity (B). The data represent the mean with the standard error for N = 9 individuals per treatment. The star symbol represents significant difference compared to controls for the same pyruvate concentration. * p < 0.05.

Figure 4

LDH activity and fractal dimension of LDH activity from Hydra exposed to NPs. Hydra were exposed to 50 nm NPs for 96 and analyzed for LDH activity (A) and the correlation with the fractal dimension (fD) (B). The data represents the mean with the standard error for N = 9 individuals per treatment. The star symbol * indicates significance from controls. * p < 0.05.