Effects of lead on paraoxonase ensymes in HEPG2 cells an THP-1 differentiated macrophage cells

Abstract

Lead, an ubiquitous contaminant in the environment, is recognized as one of the important health problems. Its toxicities manifests in various pathological symptoms and diseases including cardiovascular diseases. The aim of this study was to explore effects of lead on paraoxonase (PON) enzymes. Effects of lead on PON1, PON2 and PON3 were assessed in HepG2 cells while effect of lead on PON2 was also assessed in THP-1 differentiated macrophage cells. Cell viability was determined by exposing cells to various concentrations (0, 0.05, 0.1, 0.5, 1, 10, 100 and 1000 µg/ml) of lead acetate for 24, 48 and 72 hours. Reactive oxygen species (ROS) generation and lipid peroxidation were assessed by exposing cells to various concentrations (0, 0.05, 0.1, 0.5, 1, 10 and 100 µg/ml) of lead acetate for 1 hour. Effects of lead acetate on PON1, PON2, and PON3 activities were determined using specific substrates after HepG2 cells or THP-1 differentiated macrophage cells were exposed to various concentrations (0, 0.05, 0.1, 0.5, 1, 10 and 100 µg/ml) of lead acetate for 24, 48 and 72 hours. PON protein and mRNA expressions were assessed by Western blot analysis and real time RT-PCR, respectively. The results showed that lead did not significantly decrease cell viability of both cells types at concentrations of lead acetate up to 100 µg/ml. Significant increase of ROS was shown in both cell types. Significant decrease of PON2 activity after lead exposure was observed in a concentration- and time-dependent manner in HepG2 cells but not in THP-1 differentiated macrophage cells. No effect of lead was observed on PON1 activity while decreases of PON3 activity were observed only at 24 hours of lead exposure in HepG2 cells. Modulation of PON2 activity by lead exposure was not associated to the modulation of both PON2 protein and PON2 mRNA expression. Calcium could restore the inhibitory effect of lead on PON2 activity suggesting that lead decreased PON2 activity via replacement of Ca2+ in the structure of PON2 enzyme. In contrast to PON2, modulation of PON3 activity by lead exposure was partly associated to the decrease of PON3 protein and mRNA expression.