Western blot analysis for 4-hydroxy-2-nonenal (HNE)-modified proteins in paraquat-treated mice

Abstract

Paraquat (PQ) is widely used in agriculture as a non-selective contact herbicide. Ingestion of PQ results in multiple organ failure within one week, although the primary damage induced by PQ occurs in the lungs. It is known that reactive oxygen species (ROS) play an important role in pathological changes in PQ poisoning, although the exact mechanism of PQ toxicity has not been completely elucidated. In this study, we investigated changes in 4-hydroxy-2-nonenal (HNE)-modified proteins as markers of lipid peroxidation in PQ-treated mice. C57BL/6J mice were given PQ (10 or 50 mg/kg) intraperitoneally. After 24 h, blood and tissues were collected under isofluorene anesthesia. For histochemical studies, frozen tissue sections were immunostained with an anti-HNE monoclonal antibody. Immunoreactivity using the anti-HNE antibody was strongly increased in the kidney after PQ treatment. The expression levels of HNE-modified proteins in tissue homogenates were analyzed by Western blotting. Tissue homogenates were separated by 12.5% SDS–PAGE and transferred onto a polyvinylidene difluoride membrane. The membrane was immunostained with an anti-HNE antibody. Reactive bands were visualized with diaminobenzidine (DAB). Then the membrane was immunostained again with an anti-actin antibody and visualized with New Fuchsin. An anti-actin antibody-positive band was used to correct the protein content in tissue homogenates. The intensity of the 41-kDa protein band in the kidney and that of the 51-kDa protein band in the lung were significantly increased. These findings suggest an important role of ROS in the development of paraquat toxicity in the kidney and lung. On the other hand, an increase in peroxidation was not observed in the liver protein. It has been reported that oxidative stress after paraquat administration involved nitration of proteins by the activation of nitric oxide synthase (NOS). Therefore, paraquat may exert its toxicological action on different organs by different mechanisms.