Electron paramagnetic resonance investigation of in vivo free radical formation and oxidative stress induced by 2,4-dichlorophenol in the freshwater fish Carassius auratus

State Key Laboratory of Pollution Control and Resources Reuse, School of Environment, Nanjing University, Nanjing 210093, China.
Environmental Toxicology and Chemistry (Impact Factor: 3.23). 10/2005; 24(9):2145-53. DOI: 10.1897/04-640R.1
Source: PubMed


In the present study, electron paramagnetic resonance coupled with spin-trapping technique was used, with alpha-phenyl-N-tert-butylnitrone (PBN) as a spin-trapping agent, to investigate free radical generation in freshwater fish with acute 2,4-dichlorophenol (2,4-DCP) poisoning. The PBN-radical adducts were detected in fish liver samples following treatments of 2,4-DCP (0.025, 0.05, 0.5, 5, or 25 mg/kg) 24 h after intraperitoneal (i.p.) injection and 2,4-DCP (0.5 mg/kg) at 2, 4, 8, 24, or 72 h after i.p. injection in Carassius auratus. The hyperfine splitting constants for the PBN-radical adducts are aN = 13.7 G, aH = 1.8 G, and g = 2.0058, which is consistent with those of PBN/hydroxyl radical (*OH). The results indicate that the hydroxyl radical is probably produced during acute intoxication of 2,4-DCP. The relative similarity in the kinetics (from 2 to 72 h) of superoxide dismutase activity induction and *OH generation implies that the generation of *OH possibly depends on the superoxide anion (O2*-). Superoxide anion (O2*-) might be the precursor radical undergoing the Haber-Weiss reaction to form *OH. Possible pathways for radical chain reactions in the formation of the hydroxyl radical in vivo after 2,4-DCP administration are proposed. Other parameters with respect to antioxidant defense (e.g., superoxide dismutase and catalase) and oxidative damage (lipid peroxidation level) indicate that the fish were subjected to oxidative stress induced by 2,4-DCP and that the mechanisms of oxidative stress possibly involve the in vivo stimulation of hydroxyl radical formation.

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    • "ROS levels were determined by electron paramagnetic resonance (EPR), according to the method described by Luo et al. [16] and Jiang et al. [17]. About 0.1 g of liver tissues were quickly homogenized with a cold glass homogenizer in 1.0 ml of 50 mM PBN dissolved in DMSO. "
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    ABSTRACT: Microcystins (MCs) are a group of cyclic hepatotoxic peptides produced by cyanobacteria. Microcystin-LR (MC-LR) contains Leucine (L) and Arginine (R) in the variable positions, and is one of the most common and potently toxic peptides. MC-LR can inhibit protein phosphatase type 1 and type 2A (PP1 and PP2A) activities and induce excessive production of reactive oxygen species (ROS). The underlying mechanism of the inhibition of PP1 and PP2A has been extensively studied. The over-production of ROS is considered to be another main mechanism behind MC-LR toxicity; however, the detailed toxicological mechanism involved in over-production of ROS in carp (Cyprinus carpio L.) remains largely unclear. In our present study, the hydroxyl radical (•OH) was significantly induced in the liver of carp after a relatively short-term exposure to MC-LR. The elevated reactive oxygen species (ROS) production may play an important role in the disruption of microtubule structure. Pre-injection of the antioxidant N-acetyl-cysteine (NAC) provided significant protection to the cytoskeleton, however buthionine sulfoximine (BSO) exacerbated cytoskeletal destruction. In addition, the elevated ROS formation induced the expression of apoptosis-related genes, including p38, JNKa, and bcl-2. A significant increase in apoptotic cells was observed at 12 - 48 hours. Our study further supports evidence that ROS are involved in MC-LR induced damage to liver cells in carp, and indicates the need for further study of the molecular mechanisms behind MC-LR toxicity.
    PLoS ONE 12/2013; 8(12):e84768. DOI:10.1371/journal.pone.0084768 · 3.23 Impact Factor
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    • "PBN adduct extraction was performed in an incubation system with a continuous purging of N 2 (Shi et al. 2005a; 2005b) . Spectra were recorded with an EPR spectrometer (EMX 10/12 X-band, Bruker, Rheinfelden, Germany) at room temperature with the following operation conditions: center field, 3470 G; scan range, 200 G; modulation frequency, 100 kHz; modulation amplitude, 0.5 G; receiver gain, 5 Â 10 4 scans, five times; and microwave power, 20 mW (Luo et al. 2005; Sun et al. 2008). "
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    ABSTRACT: Oxidative stress parameters and some antioxidant defense systems in the liver of Carassius auratus exposed to glyphosate and its formulation (Roundup®) have been studied. Fish were exposed to glyphosate and its formulation at concentrations of 0.032, 0.16, 0.8, and 4.0 mg L−1, all calculated on glyphosate basis, for 11 days. Hydroxyl radical generation as determined by electron spin resonance spectroscopy of its spin-adduct with α-phenyl-t-butyl nitrone increased with the concentration of Roundup®. Superoxide dismutase activities were decreased relative to control by 21%–46% when exposed to glyphosate and 45%–52% when exposed to Roundup®, suggesting that the formulation is more toxic than glyphosate alone. Catalase showed no difference between both groups.
    Toxicological and Environmental Chemistry 08/2013; 95(7). DOI:10.1080/02772248.2013.863889 · 0.83 Impact Factor
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    • "A six line spectrum of three groups with two hyperfine coupling splitting peaks was observed. According to previous literature [4] [5] [13] [16], the trapped ROS was likely to be the hydroxyl radical (·OH) and the levels could be expressed with the second couplet intensity of the triplets in the EPR spectra. Figure 2 showed the kinetics of ·OH generation over "

    Computational Water, Energy, and Environmental Engineering 01/2013; 02(02):20-25. DOI:10.4236/cweee.2013.22B004
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