The profound effects of microcystin on cardiac antioxidant enzymes, mitochondrial function and cardiac toxicity in rat
Introduction
Public health concerns about toxic cyanobacteria have recently increased in many countries, owing to the frequent occurrence of cyanotoxins in both drinking and recreational waters. Among cyanotoxins, microcystins (MCs) are the most common all over the world, with molecular weights ranging between 900 and 1000 Da. To date, more than 80 structural variants have been identified (Sivonen and Jones, 1999), differing primarily in the two variable l-amino acids. So far, human illnesses attributed to cyanobacterial toxins can be categorized into gastroenteritis and related diseases, allergic and irritation reactions, and liver diseases (Bell and Codd, 1994, Chorus et al., 2000, Hitzfeld et al., 2000). In Brazil, tragic deaths of 60 hemodialysis patients are confirmed as a result of contamination of cyanobacterial toxins in the water supply used in hemodialysis unit (Jochimsen et al., 1998, Carmichael et al., 2001, Azevedo et al., 2002).
It is widely considered that hypovolemic shock leads to deaths from microcystin intoxication (Theiss et al., 1988). However, recent studies suggest that there may be also an important cardiogenic component involved. Leclaire et al. (1995) report simultaneous sharp decreases in both heart rate and blood pressure in rats administrated with MC-LR at a lethal dose, suggesting a fundamental dysfunction of the normal compensatory responses of the heart and vasculature to hypotension. Meanwhile, the sustained decreases in both cardiac output and stroke volume in the treated rats indicate that microcystins impair the blood-pumping function of heart. In accordance with in vivo findings, the electrophysiological studies on isolated rat heart indicate that cyanotoxins administration decreases both heart rate and myocardial force contraction (Mason and Wheeler, 1942). Moreover, systolic arrest in the isolated frog heart is recorded after cyanotoxins exposure (Ostensvik et al., 1981). Recently, histopathological alterations in both acute and chronic toxic experiments also verify the cardiotoxicity from MC-LR exposure (Zhang et al., 2002, Milutinović et al., 2006). Therefore, the cardiotoxicity could be another potential contributing factor to the deaths associated microcystin intoxication.
Cardiovascular diseases are well known to be directly or indirectly related to oxidative damage. Myocardium possesses several features such as abundant mitochondria and rich polyunsaturated fatty acids in the mitochondrial and plasma membranes, all of which make the myocardium vulnerability to free radical attack (Kowaltowski and Vercesi, 1999). Oxidative stress is associated with the pathophysiology of many cardiomyopathies, such as anthracycline-mediated cardiomyopathy (Singal et al., 1997, Xu et al., 2001) and alcoholic cardiomyopathy (Edes et al., 1986). Nowadays, accumulating evidences imply that MC-dependent damage is accompanied by oxidative stress in liver, kidney and intestinal mucosa in mammals (Botha et al., 2004, Ding et al., 1998, Ding et al., 2001, Moreno et al., 2003, Moreno et al., 2005). Moreover, studies strongly indicate that mitochondria are the vulnerable target of MC. To satisfy the huge energy demands of heart, cardiac mitochondria occupy about 40% of total intracellular volume of cardiomyocytes, and mitochondrial defects indeed lead to cardiomyopathy and heart failure (Goffart et al., 2004). It seems that oxidative damage and mitochondrial dysfunction are probably associated with the cardiotoxic effects from MC exposure. In the present experiments, we studied the acute responses of antioxidant system in heart and the physiological changes in mitochondria electron transport chain (ETC), with discussion on the underlying mechanism of MC-induced cardiotoxicity.
We conducted intravenous injection of extracted MC in rats at two doses, and our main purposes were: (1) to monitor the clinic characteristics of heart from MC intoxication, (2) to evaluate the roles of oxidative stress and mitochondrial dysfunction in cardiotoxic effects by MC. In this study, we determined MC contents in liver and heart, monitored heart rate, blood pressure, and clinic biomarkers, and examined pathological alterations after exposure of MC. Also, activities and mRNA transcription levels of main antioxidant enzymes (GSH-Px, GST, SOD and CAT), and levels of GSH and MDA (as a measurement of LPO) were measured in cardiac muscle; while activities of complex I, II, III in ETC and MDA levels were measured in mitochondria.
Section snippets
Toxin
The cyanobacterial material used in this experiment was collected from surface blooms (phytoplankton cells) of Lake Dianchi, Yunnan in China during May and June 2006. According to microscopic examinations, the predominant species was Microcystis aeruginosa. Methods on MC extract and the analyses of the cyanobacterial material were according to Li et al. (2005). Crude extract concentrations were determined by comparing the peak areas of the test samples with those of the standards available
Animal deaths
No mortality occurred in both the 0.16LD50 dose and the control groups. In the 1LD50 group, five rats died, while the other five survived at 24 h post-exposure. The MC-treated rats could be distinguished from the controls by reduced motor activity and reduced resistance at handling.
MC contents in liver and heart of rats
In the control group, no MC were detectable in liver and heart. Table 1 shows MC contents in liver and heart of the dead rats and the survived ones in the 1LD50 dose group. In the dead rats, MC content in liver was up
Discussion
Ostensvik et al. (1981) attribute the drop in mean blood pressure from MC intoxication to sequestration of blood in the liver. However, diminution of the physiological cardiac reserve may compromise the normal response to circulatory insufficiency as well. Leclaire et al. (1995) suggest that there may be an important cardiogenic component involved in the death caused by MC intoxication.
In the present study, accumulation of MC in livers and hearts was identified by LC–MS. In accordance with
Conflict of Interest
We declare that we have no conflict of interest.
Acknowledgments
We would like to thank Xuezheng Zhang, Dapeng Li, Rong Tang and Yanyan Zhao from the Fisheries College of Huazhong Agricultural University, for their assistance in the experiment. This work was supported by funds from the National Basic Research Program of China (973 Program) (Grant No. 2008CB418101).
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