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A Proteomic Study on Liver Impairment in Rat Pups Induced by Maternal Microcystin-LR Exposure*

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A Proteomic Study on Liver Impairment in Rat Pups Induced by Maternal Microcystin-LR Exposure* Environmental Pollution 212 (2016) 197e207 Contents lists available at ScienceDirect Environmental Pollution journal homepage: www.elsevier.com/locate/envpol A proteomic study on liver impairment in rat pups induced by maternal microcystin-LR exposure* * Sujuan Zhao, Ping Xie , Jun Chen, Luyi Liu, Huihui Fan Donghu Experimental Station of Lake Ecosystems, State Key Laboratory of Freshwater Ecology and Biotechnology of China, Institute of Hydrobiology, Chinese Academy of Sciences, Donghu South Road 7, Wuhan 430072, PR China article info abstract Article history: There is mounting evidence indicating that microcystins (MCs) are heptapeptide toxins. Recent studies Received 1 September 2015 have also shown that MCLR can transfer from mother to offspring, but it is unclear whether maternal Received in revised form MCLR can influence the liver of offspring or not. In this study, pregnant SD rats were injected intra- 14 December 2015 peritoneally with a saline solution (control) or 10 mg/kg MCLR per day from gestational day 8 (GD8) to Accepted 23 December 2015 postnatal day 15 (PD15) for a total of 4 weeks. 2-DE and MALDI-TOF-TOF mass spectrometry were used to Available online xxx screen for MCLR target proteins in the livers of rat pups. Our results demonstrated that MCLR could accumulate in the livers of neonatal rats. Proteomics studies also showed that MCLR significantly Keywords: fl MCLR in uenced many proteins, including those involved in the cytoskeleton, metabolism and particularly Rat pups oxidative stress. In addition, MCLR induced cellular structural damage and resulted in the production of Proteomics intracellular reactive oxygen species (ROS) and lipid peroxidation. Moreover, protein phosphatase (PP) Liver activity was inhibited and some serum biochemistry parameters were altered. These results suggest an Oxidative damage early molecular mechanism behind the hepatotoxicity induced by maternal MC exposure and highlight the importance of monitoring MC concentrations in new-born mammals. © 2016 Elsevier Ltd. All rights reserved. 1. Introduction fishermen living around Lake Chaohu, China (Chen et al., 2009), which demonstrates that chronic exposure is a health risk. Microcystins (MCs) are a family of cyclic heptapeptide toxins The mechanisms of MC toxicity have not been fully elucidated naturally produced by freshwater cyanobacteria, and their pro- but most likely involve protein phosphatase inhibition. Micro- duction has become a worldwide ecological issue that results in cystins act by inhibiting Ser/Thr protein phosphatase-1 and -2A water eutrophication and health threats to livestock and humans (PP1, PP2A) (Mackintosh et al., 1990), which leads to cytoskeletal (Paerl and Huisman, 2009). More than 80 structural analogues of disruption (Eriksson et al., 1989) and subsequent cell death (Boe MCs have been identified; of these, MCLR is the most toxic and et al., 1991). It has also been widely reported that MCs can induce most commonly encountered cyanobacterial toxin (Figueiredo de the production of reactive oxygen species (ROS), which may cause et al., 2004). There is evidence that prolonged exposure to MC-LR DNA damage (Nong et al., 2007). may induce neoplastic nodular formations that can be precursors Currently, mounting evidence has indicated that MCs have of primary liver cancer (PLC) (Yu, 1995; Ueno et al., 1996; Li et al., embryonic toxicity in both fish and mammals. The main effects of 2009). MCLR is classified as possibly carcinogenic to humans exposure to MCs during the early life stages of fish are interference (Group 2B) by the International Agency for Research on Cancer with developmental processes and organ functions (Palikova et al., (IARC) (2010). Recently, MCs were detected in the serum of 2003). In mammals, it has been demonstrated that MCLR can damage the placental barrier directly, allowing MCLR to enter the embryo (Wei et al., 2002). There is some evidence that MCs can inhibit growth, affect actin and microtubule organization and alter * This paper has been recommended for acceptance by David Carpenter. the morphology of mammalian embryos (Rao et al., 1998; * Corresponding author. Donghu Experimental Station of Lake Ecosystems, State Key Laboratory for Freshwater Ecology and Biotechnology of China, Institute of Sepulveda et al., 1992; Frangez et al., 2003). Moreover, Bu et al. Hydrobiology, the Chinese Academy of Sciences, Donghu South Road 7, Wuhan (2006) found that foetal livers exhibit petechial haemorrhaging 430072, PR China. Fax: þ86 186 27 68780622. and hydropic degeneration when exposed to 6 and 12 mg/kg E-mail address: [email protected] (P. Xie). http://dx.doi.org/10.1016/j.envpol.2015.12.055 0269-7491/© 2016 Elsevier Ltd. All rights reserved. 198 S. Zhao et al. / Environmental Pollution 212 (2016) 197e207 microcystin from GD 6 to GD15. Recently, Li et al. (2011b) suggested delivered dose of MCLR decreased as the pregnancy progressed that chronic exposure to microcystin may be associated with because the weight of the mother at GD8 was used to calculate the increased serum enzyme levels and liver damage among the MCLR dose, and the body weight increased from this point studied children from the Three Gorges Reservoir Region. Although throughout pregnancy. liver is the target organ and is therefore the most extensively In rats, blastocyst implantation in the uterus occurs until the 5th studied organ in research on MCs, the molecular mechanisms of gestational day, and organogenesis occurs starting from the 6th day MC-induced susceptibility to liver damage in offspring are largely (Manson and Kang, 1989). Thus, GD8 (during the stable organo- unknown. genesis stage) was chosen for the start of exposure. Given that both Pregnancy is an important and critical phase, during which gestation and lactation are more sensitive periods of life than the organogenesis can occur (Manson and Kang, 1989). Because subtle normal physiological phase, the MCLR exposure period included 14 anomalies that can appear during animal development may be days of pregnancy and 14 days of lactation. 10 mg MCLR/kg BW/day undetectable using conventional cytopathology and biochemical was chosen for this study based on the protocol of Li et al. (2012). analysis, a proteomics-based approach has been employed to study During the 4 weeks (28 days) of maternal MCLR exposure, the dams the effects of MC exposure (Li et al., 2012; Li et al., 2011a; Zhao et al., delivered naturally, and litters were killed on postnatal day 15. 2012a; Fu et al., 2005). However, there are few reports on the effects of MCLR during gestation and lactation (Chernoff et al., 2000). Thus, 2.3. MCLR concentration and effect of MCLR on pup livers in this study, we investigate hepatotoxicity of MCLR in rat pups, aiming to understand the mechanisms of MCLR-induced liver After blood collection (approximately 0.35 mL per rat), the pups damage in offspring. were sacrificed, and the livers were removed and weighed. The liver/body weight ratio was calculated. The liver index was calcu- 2. Materials and methods lated as follows: liver weight (g)/body weight (g) Â 100%. The livers of 10 pups from different dams in both groups were randomly 2.1. Chemicals chosen and analysed. The extraction and quantitative analysis of the MCLR content in the rat liver (0.2 g lyophilized sample) was The cyanobacterial toxin MCLR was isolated and purified from performed as published by Wang et al. (2008). freeze-dried surface blooms collected from Lake Dianchi using the methods previously published by Dai et al. (2008). MCLR was 2.4. Light microscopy, electron microscopy and serum biochemical separated using semi-preparative high-performance liquid chro- analyses matography (Waters 600E, USA), and pure MCLR was obtained. The MC content was determined using liquid chromatography- In preparation for light microscopy, liver samples were fixed in eelectrospray ionizationemass spectrometry (LCeESIeMS, 10% buffered formalin for 24 h at 4e8 C and then immediately Thermo Electron Corporation, Waltham, MA). The MCLR (purity dehydrated in a graded series of ethanol, immersed in xylol and >95%; the remaining components were primarily pigments) con- embedded in paraffin wax using an automatic processor. 4-mm centration was determined using UV spectra and retention times. A sections were mounted. Following deparaffinization, the sections commercial microcystin-LR standard (Wako Pure Chemical In- were rehydrated, stained with hematoxylin and eosin, and subse- dustries, Japan, purity > 95%) was used to compare the peak areas quently subjected to pathological assessment. of test samples (Fig. S1AeD in the Supporting Information). All In preparation for transmission electron microscopy, samples other chemicals used in this study were of analytical grade, and the were fixed in 2.5% glutaraldehyde fixative (in pH 7.4 phosphate chemicals used for electrophoresis were obtained from Bio-Rad buffer for 10 h at 4 C) and post-fixed in 1% osmium tetroxide Laboratories (Hercules, California, USA). fixative (in pH 7.4 phosphate buffer for 0.5 h at 4 C). After rinsing with phosphate buffer, the specimens were dehydrated in a graded 2.2. Animals ethanol series of 50e100% and then embedded in Epon 812. Ultra- thin sections were prepared with glass knives on an LKB-V ultra- Ten female SpragueeDawley [Crl:CD(SD)] rats and 10 male SD microtome (Nova, Sweden), stained with uranyl acetate and lead rats (both 12 weeks of age) were supplied by the Wuhan University citrate and examined by transmission electron microscopy using a Laboratory Animal Research Center (Hubei, China). The rats were Tecnai G2 20 TWIN (FEI, USA). Three samples from each dam were housed under controlled conditions of a 12 h light/dark cycle, randomly chosen for histological and transmission electron mi- 50 ± 5% humidity and 20 ± 1 C. The animals were allowed free croscopy analysis. access to food and water and were treated humanely to minimize Three pups from each dam were randomly chosen for plasma suffering. All animal procedures were approved by the Institutional biochemical analyses, and blood samples were collected and Animal Care and Use Committee (IACUC) and were in accordance centrifuged at 850 Â g for 10 min.
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