Alteration of hepatocellular antioxidant gene expression pattern and biomarkers of oxidative damage in diazinon-induced acute toxicity in Wistar rat: A time-course mechanistic study

Abstract

In the present survey, the plasma level of diazinon after acute exposure was measured by HPLC method at a time-course manner. In addition, the impact of diazinon on the expression of the key genes responsible for hepatocellular antioxidative defense, including PON1, GPx and CAT were investigated. The increase in oxidative damages in treated rats was determined by measuring LPO, protein carbonyl content and total antioxidant power in plasma. After administration of 85 mg/kg diazinon in ten groups of male Wistar rats at different time points between 0-24 hours, the activity of AChE enzyme was inhibited to about 77.94 %. Significant increases in carbonyl groups and LPO after 0.75 and 1 hours were also observed while the plasma antioxidant power was significantly decreased. Despite the dramatic reduction of GP_X and PON1 gene expression, CAT gene was significantly upregulated in mRNA level by 1.1 fold after 4 hours and 1.5-fold after 24 hours due to diazinon exposure, compared to control group. Furthermore, no significant changes in diazinon plasma levels were found after 4 hours in the treated rats. The limits of detection and quantification were 137.42 and 416.52 ng/mL, respectively. The average percentage recoveries from plasma were between 90.62 % and 95.72 %. In conclusion, acute exposure to diazinon increased oxidative stress markers in a time-dependent manner and the changes were consistent with effects on hepatic antioxidant gene expression pattern. The effect of diazinon even as a non-lethal dose was induced on the gene expression of antioxidant enzymes. The change in antioxidant defense system occurs prior to diazinon plasma peak time. These results provide biochemical and molecular evidence supporting potential acute toxicity of diazinon and is beneficial in the evaluation of acute toxicity of other organophosphorus pesticides as well.

Keywords: HPLC; biomarker; diazinon; gene expression; molecular toxicity; oxidative stress; pesticide.

Table 1. Sequences of different primers used for Real-Time PCR reactions

Figure 1. Effect of acute exposure to diazinon (DZN) on plasma acetyl cholinesterase (AChE) activity in terms of inhibition percentage of control in rat. The values are expressed as the percentage of control and are the mean ± SEM in six rats in each group. *** (P< 0.001) significantly different from control.

Figure 2. Changes in plasma levels of oxidative stress biomarkers after 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12 and 24 hours of 85 mg/kg diazinon administration. Values are shown as mean ± SEM of six animals in each group, *** (P< 0.001) significantly different compared with control; (A) lipid peroxidation (TBARS), (B) total thiol molecules (TTM), (C) ferric-reducing antioxidant power (FRAP), (D) protein carbonyl.

Figure 3. Correlation between plasma diazinon levels and oxidative stress markers

Figure 4. Effects of diazinon on mRNA expression pattern of PON1, CAT, GPx genes in liver tissues. Animals were exposed to 85 mg/kg diazinon orally, after treatment (4 and 24 hours); mRNA expression was measured with real time PCR. Data are expressed as means±SEM. ** (P< 0.01) and *** (P< 0.001) significantly different compared with control.

Figure 5. Histopathologic sections of liver, H&E stain, Stars: central veins, Arrows: hepatocyte cords, LL (classical liver lobule). A & B: histopathological sections of normal liver (Control), C & D: micrographs of liver 4 hours post-treatment. E & F: micrographs of liver 24 hours post-treatment.

Figure 6. Histopathologic sections of the liver in 4 (A) and 24 (B) hours post-treatment, H & E stain. Mononuclear inflammatory cells (Star).

Figure 7. Standard calibration curves of diazinon

Figure 8. Chromatograms of diazinon in standard solutions of 400 ng/mL (A), 6400 ng/mL (B) and in rat plasma at 4 hours following gavage administration (C).

Figure 9. Variations of diazinon levels in rat plasma samples after 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12 and 24 hours of DZN (85 mg/kg, p.o.) administration. Values are shown as mean ± SEM of six animals in each group.