Lipid peroxidation and antioxidant responses in zebrafish brain induced by Aphanizomenon flos-aquae DC-1 aphantoxins
Introduction
Toxicogenic cyanobacteria-secreted paralytic shellfish poisons (PSPs) are frequently encountered in freshwater systems worldwide as a result of human activities (Ballot et al., 2010, Ledreux et al., 2010). Blooms dominated by Aphanizomenon flos-aquae DC-1 have occurred in Dianchi Lake, in Yunnan province, China, in most years in recent decades, as a result of the favorable nutrient supply and water temperature (Xing et al., 2007, Wu et al., 2010). At present, it is worrying that the prevalent algae in these blooms have been shown to secrete neurotoxic PSPs (Liu et al., 2006), which can damage the brain ultrastructure and influence locomotor function in zebrafish (Danio rerio) (Zhang et al., 2013a, Zhang et al., 2013b). This lake is an important source of freshwater for drinking and agricultural irrigation, and an important location for recreational activities and as a tourist destination for a population of over five million in the vicinity of Kunming city, Yunnan province (Liu et al., 2006). The blooms thus increase the potential danger to human health and environmental safety, and are having adverse effects on the local economy because of the risk of paralytic shellfish poisoning.
PSPs are potent alkaloid neurotoxins, discovered principally in the marine ecosystem where they are synthesized by dinoflagellates, but also in freshwater cyanobacteria and in bacteria (Ballot et al., 2010, Ledreux et al., 2010). PSPs can accumulate to a very high concentration in aquatic biota with no apparent harm to animals that filter-feed on these dinoflagellates and cyanobacteria (Zaccaroni and Scaravelli, 2008). However, subsequent human consumption of PSP-contaminated foodstuffs commonly leads to the development of paralytic shellfish poisoning, with significant morbidity and mortality (Ferrão-Filho and Kozlowsky-Suzuk, 2011). There is currently no effective antidote for PSP intoxication, and artificial respiration and fluid therapy remain the only available treatments (Wiese et al., 2010).
Concerning the effects of PSP exposure on fish, previous studies showed that PSPs could be transferred from toxic algae to a high-trophic-level fish (Kwong et al., 2006, Jiang et al., 2007). When PSPs entered fish in these ways, they delayed hatching and led to malformations and mortality in zebrafish (Oberemm et al., 1999, Lefebvre et al., 2004), and accumulated in the muscles of Geophagus brasiliensis (Clemente et al., 2010). Along with the physiological effects, PSPs altered the sensorimotor function and spontaneous swimming behavior of larval zebrafish and herring (Lefebvre et al., 2004, Lefebvre et al., 2005). All these results demonstrate that PSPs exert potent toxicities in fish, not only by inducing the physiological effects, but also by causing behavioral changes in both larval and adult fish (Ferrão-Filho and Kozlowsky-Suzuk, 2011).
Lipid peroxidation is considered to represent an imbalance, with increased reactive oxygen species (ROS) and decreased activity of the antioxidant protective system (Boveris et al., 2008). The spectrum of ROS includes superoxide radicals, hydrogen peroxide, and hydroxyl radicals. The main protective system against ROS and their toxic by-products includes enzymes such as SOD, CAT, GPx and GST, as well as non-enzymatic systems such as GSH (Halliwell and Gutteridge, 2007, Melegari et al., 2012).
Several recent studies have demonstrated that extracted saxitoxin (STX) or PSPs from Cylindrospermopsis raciborskii may induce oxidative stress, lipid peroxidation in cultured neuro-2A cells and in brains of the fish Hoplias malabaricus, and antioxidative responses in the hepatopancreas of clams (da Silva et al., 2011, Melegari et al., 2012). These results also show that PSPs could induce oxidative stress, lipid peroxidation and antioxidant defense responses in both nervous cells and the nervous system, as well as in other non-nervous organs.
To the best of our knowledge, the effects of sublethal doses of A. flos-aquae DC-1 aphantoxins or PSPs on oxidative stress, lipid peroxidation and the antioxidant defense responses in the zebrafish brain have not yet been established. Therefore, we investigated the changes in oxidative ROS and MDA, and antioxidative GSH, SOD, CAT and GPX in zebrafish brain at 1–24 h after exposure to sublethal doses of A. flos-aquae DC-1 aphantoxins or PSPs. The results of this study will improve our understanding of biochemical changes and physiological function in the zebrafish brain, and the effects of neurotoxic stress induced by A. flos-aquae DC-1 aphantoxins or PSPs. These results will also enable us to further understand the mechanisms responsible for brain damage and antioxidation induced by neurotoxins or PSPs.
Section snippets
Chemicals
Reference standards for PSP toxins including STX group toxins (dcSTX, STX, neoSTX) and gonyautoxin (GTX) group toxins (GTX1–5, dcGTX2, 3) were purchased from the National Research Council in Canada, (Halifax, Nova Scotia, Canada). Chemical kits for SOD, CAT, GPx, GSH, ROS and MDA were provided by the Nanjing Bioengineering Institute, China. All other chemicals were of the highest grade available from commercial sources, unless otherwise indicated.
Algal culture, toxin extraction, purification and analysis
The algae collected from Lake Dianchi water
Analysis of Aphanizomenon flos-aquae DC-1 toxins
HPLC-fluorescence derivatization (FLD) analysis revealed that the toxins extracted from cultured A. flos-aquaeDC-1 algae contained the three toxic components neoSTX, GTX1 and GTX5, which were identical to the respective control standards. The total content of PSP toxin from A. flos-aquae DC-1 was 9.52 ng/mg dry cell weight. The overall toxicity of the samples was 6.51 ng STXeq/mg dry cell, according to the relative toxicity ratios of GTX1, GTX5 and neoSTX versus STX, respectively. Moreover,
Discussion
We confirmed that A. flos-aquae DC-1 aphantoxins or PSPs increased ROS and MDA levels, increased activity of antioxidant enzymes SOD, CAT and GPx, and decreased levels of antioxidant GSH in zebrafish brain, demonstrating oxidative stress, lipid peroxidation and antioxidant responses, respectively. These changes were time and dose dependent. The results suggest that the neurotoxicity of aphantoxins or PSPs increases ROS and MDA levels and decreases GSH in zebrafish brain, and these changes might
Conclusions
In conclusion, this study demonstrated that sublethal doses of A. flos-aquae DC-1 toxin or PSPs caused oxidative stress, lipid peroxidation, and antioxidant defense response in zebrafish brains. Both high-and low-dose toxins increased the ROS and MDA levels, indicative of oxidative stress and lipid peroxidation. Further analysis revealed that GSH was depleted and SOD, CAT and GPx activities were increased, suggesting elimination of ROS and MDA through the antioxidant defense response in
Acknowledgments
The study was financially supported by the National High-Tech Research and Development Program of China (2013AA065804) and the National Basic Research Programs of China (2008CB418001).
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