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Paraquat, one of the most widely used herbicides, is highly toxic to humans and animals. There is much information regarding its toxic effects on the lungs, but less is known about its toxicity in other organs. Paraquat is thought to play pivotal roles in the pathophysiology of acute renal failure and the progression of chronic kidney disease. We investigated the effects of paraquat on gene expression in the kidneys of rats treated with paraquat using a DNA array system, and the gene up-regulation observed was confirmed by quantitative real-time RT-PCR. Rats were sacrificed at 3, 24 h after the first injection (20 mg/kg), and at 3 h after the second injection. Expression of six genes had increased significantly by 3 h after the first injection: metallothionein-1 (MT-1), phosphoenolpyruvate carboxykinase, Na/K-transporting ATPase beta1 subunit, glutamate oxaloacetic transaminase, glutathione-S-transferase, and heme oxygenase-1 (HO-1). The transcription levels of MT-1 and HO-1 showed the biggest increases, but the increases did not continue until 24 h after injection, and the second injection had less effect than the first. Up-regulation of MT-1 and HO-1 mRNA levels was confirmed at the protein level. We observed a paraquat-induced increase of these proteins at 3 h post-injection, whereas this level did not continue until 24 h, as observed in RNA levels. The MT-1 protein in kidneys had been consumed. In addition, the protein level due to the second injection did not increase to the same level as that due to the first injection. These results suggest that protection against paraquat injury is mediated by induction of expression of some genes, and suppression on the induction of MT-1 and HO-1 may explain the injury observed due to paraquat intake. This is the first report of inducible pathways of defense against paraquat-induced oxidative stress in the kidney.

Paraquat (PQ) is a well-known pneumotoxicant and provides an established model of oxidative stress. This study focused on the transcriptional response to PQ-driven oxidative stress in rat lungs during an early phase post-injection. Rats were sacrificed at 3 h and 24 h after PQ injection (i.p., 20 mg/ kg b.w.), and at 3 h after a second injection (i.p., 20 mg/kg b.w.). The left lungs were rapidly excised and used immediately for RNA preparation. The lung tissues did not show any pathological damage microscopically. Differential expression of RNAs in the lung at 3 h was investigated using a DNA array system. Fifteen genes showed a >1.7-fold change in expression level, which was confirmed by real-time PCR. Five genes related to oxidative stress, TRX, HO-1, GST-Yc, NQO-1, and RL/IF-1, and one gene, CLK3, whose function is unknown, showed a significant increase in their expression due to PQ injection. Two genes, HO-1 and NQO-1, that showed 3-and 2-fold increases at 3-h post-injection, were localized by immunohistochemistry. HO-1 was expressed in the bronchial epithelial cells, some type II cells and macrophages of control lungs, and the cells, especially the bronchial epithelial cells, were strongly stained 3 h following PQ treatment. Immunohistochemical analysis of NQO-1 also showed an increase in positive staining in the bronchial epithelial cells of PQ-treated lung sections. The expression of CYP2C6, 2C7, and 2C12, which are specific to or dominant in female liver, decreased markedly, while the male-specific CYP2C13 and 2C11 showed an increase or no effect. Further investigation is needed to clarify the role of these CYP2C family genes on the early phase of PQ toxicity. These results indicate that the acclimation to oxidative stress is already a highly complex process at the onset of PQ-induced damage and that the genes described herein may prove to be major contributors to the subsequent pulmonary fibrosis.

The use of capillary electrophoresis for simultaneous separation and detection of the two bipyridylium herbicides, paraquat and diquat, was investigated. Both herbicides were extracted from fortified sera with disposable ODS-silica cartridges. Separation was carried out using a capillary tube (50 microns i.d., 750 mm) of fused silica containing 10 mM glycine-HCl buffer (pH 3.0), 40 mM NaCl and 20% methanol as the carrier. Paraquat and diquat were completely separated in 10 min at an applied potential of 20 kV. On-column UV monitoring allowed detection of both herbicides simultaneously. The assay sensitivity was 0.05 micrograms/mL (signal-to-noise ratio, 2:1), which probably increases with increase in the sample volume of serum. Analytical recovery of both herbicides added to serum was about 97% at concentrations of 0.5-2.0 micrograms/mL.