The Identification of Apolipoprotein Genes in Rare Minnow (Gobiocypris
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ARTICLE IN PRESS CBC-07576; No of Pages 8 Comparative Biochemistry and Physiology, Part C xxx (2009) xxx–xxx Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc 1 The identification of apolipoprotein genes in rare minnow (Gobiocypris rarus) and 2 their expression following perfluorooctanoic acid exposure 3 Xuemei Fang a,b, Yanhong Wei a, Yang Liu a, Jianshe Wang a, Jiayin Dai a,⁎ 4 a Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, PR China 5 b Department of Chemistry and Life Science, Suzhou College, Suzhou 234000, PR China 6 article info abstract 7 8 Article history: Apolipoproteins play important roles in lipid transport and uptake in vertebrates, and they are associated 22 9 Received 2 July 2009 with pathogenesis of many cardiovascular diseases. However, the diverse apolipoproteins in individual fish 23 10 Received in revised form 23 September 2009 species have not been extensively characterized. Partial cDNA sequences encoding ApoA-IV, ApoE, ApoM, 24 11 Accepted 24 September 2009 25 12 ApoL, and ApoO, and full-length cDNA sequences encoding ApoA-I were cloned from rare minnow Available online xxxx 26 151314 (Gobiocypris rarus). Sequence analysis showed that these genes, as well as fragments of other known apolipoprotein genes (ApoC-I, ApoC-II, ApoB) of rare minnow had a high similarity (91–96%) to their 27 16 Keywords: 28 17 Apolipoprotein orthologues in the spotted barbel Hemibarbus mylodon (Teleostei:Cypriniformes). The expression of these α PROOFγ α 29 18 HNFα nine genes and their possible upstream genes, PPAR , PPAR , and HNF4 , were investigated in rare minnow 19 PFOA after subacute exposure to perfluorooctanoic acid (PFOA) for 14 days. Results showed that the expression of 30 20 PPAR mRNA for ApoA-I, ApoC-II, and ApoM was significantly downregulated in all PFOA-treated animals. Only fish 31 21 Rare minnow receiving the highest dose of PFOA showed downregulation of the expression of ApoA-IV and ApoC-I, while 32 fish treated with 10 mg PFOA/L showed upregulation of expression of ApoE. Expression of ApoB, ApoO, and 33 ApoL was unchanged between control and treated groups. In addition, the expression of PPARα was 34 increased in all dosed fish, while theED mRNAs for PPARγ and HNF4α were significantly altered with 30 and 35 3 mg PFOA/L doses, respectively. Therefore, subacute exposure to PFOA resulted in alteration of expression of 36 apolipoproteins and related genes. These changes in gene expression may further influence lipid metabolism 37 or other physiological functions in fish. 38 © 2009 Published by Elsevier Inc. 39 43 4041 42 44 1. Introduction fish apolipoproteins are also required for normal embryonic and 61 ontogenic development, tissue regeneration (Lange et al., 2005; Zhou 62 45 Apolipoproteins, the protein component of variousRECT plasma et al., 2005; Harel et al., 1990), and innate immunomodulation 63 46 lipoproteins, are synthesized in the liver, intestine, adipose tissues, (Concha et al., 2004). Although gene sequences for different members 64 Q2 47 and other tissues. The specific functions of apolipoproteins are to of the apolipoprotein family have been isolated from several fish 65 48 transport and redistribute lipids among various tissues, to act as species (Kondo et al., 2005), the diverse apolipoproteins in individual 66 49 cofactors for enzymes involved in lipid metabolism, and to maintain fish species have not been extensively characterized. In addition, little 67 50 lipoprotein structure (Mahley et al., 1984). Aberrant apolipoprotein information about the involvement of apolipoproteins during envi- 68 51 levels have been shown to lead to some diseases, such as ronmental stress, such as exposure to various pollutants, has been 69 52 atherosclerosis, stroke, and coronary heart disease (Kawakami and obtained in this lower vertebrate. 70 53 Yoshida, 2009). Thus, apolipoproteins and their mimetic peptides Exposure to some persistent organic pollutants, such as poly- 71 54 have been used in therapy for atherosclerotic lesions (Van Craeyveld chlorinated biphenyl (PCB), organochlorine, and perfluoroalkyl acids 72 55 et al., 2009), Alzheimer's disease (Handattu et al., 2009), and diabetes (PFAAs) affects the level of apolipoproteins and disrupts the balance 73 56 (Patel et al., 2009). of lipid metabolism in many organisms (Boon et al., 1984; Sakr et al., 74 57 Fish use lipids rather than carbohydrates as their main energy 2007; Lee et al., 2008). Perfluorooctanoic acid (PFOA), which is used to 75 58 source, and therefore lipid metabolism and lipoprotein physiology synthesize fluoropolymers during the manufacture of a variety of 76 59 UNCOR 77 may be more important for their homeostasis (Kondo et al., 2005). In products, is a prominent PFAA detected in abiotic and biotic matrices 60 addition to the well-known roles in lipid transport and uptake, several worldwide (Lau et al., 2007). In humans, a positive relationship 78 between serum PFOA and total cholesterol, low-density lipoprotein 79 (LDL), and very low-density lipoprotein (VLDL) was seen in 80 81 ⁎ Corresponding author. Fax: +86 10 64807099. occupationally exposed workers (Sakr et al., 2007). In mice, exposure E-mail address: [email protected] (J. Dai). to PFOA reduces the total level of A-I apolipoprotein in whole serum 82 1532-0456/$ – see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.cbpc.2009.09.008 Please cite this article as: Fang, X., et al., The identification of apolipoprotein genes in rare minnow (Gobiocypris rarus) and their expression following perfluorooctanoic acid exposure, Comp. Biochem. Physiol. Part C (2009), doi:10.1016/j.cbpc.2009.09.008 ARTICLE IN PRESS 2 X. Fang et al. / Comparative Biochemistry and Physiology, Part C xxx (2009) xxx–xxx 83 and in the high-density lipoprotein (HDL) and LDL subfractions (Xie 2.2. Total RNA isolation and reverse transcriptase reactions 134 84 et al., 2003). PFOA dissociates apolipoprotein B48 from lipoprotein 85 particles and decreases secretion of VLDL from cultured rat hepato- Total RNAs were extracted from the individual liver samples 135 86 cytes (Okochi et al., 1999). These physiological functions of PFOA may using an RNeasy Mini kit (Qiagen, Hilden, Germany) and treated with 136 87 be related to the activation of peroxisome proliferator-activated RNase-free DNase I (Qiagen) to remove any remaining genomic DNA. 137 88 receptor (PPAR) because PFOA is a PPARs agonist, and PPARs play Isolated RNA was quantified based on the A260 value. The purity of the 138 89 important roles in regulating a number of genes that encode enzymes RNA was determined from the 28S:18S rRNA ratio on a MOPS/ 139 90 involved in lipid metabolism. In addition, in mammals, PPARα, PPARγ, formaldehyde gel. Approximately 1 µg of total RNA from each sample 140 91 and hepatocyte nuclear factor 4α (HNF4α) are known to regulate was reverse transcribed using an oligo-(dT) 15 primer (Promega, 141 92 genes involved in lipid transport, especially those encoding apolipo- Madison, WI, USA) and M-MuLV reverse transcriptase, as described by 142 93 proteins (Mandard et al., 2004; Gonzalez 2008; Xie et al., 2009; Yue the manufacturer (New England Biolabs, Ipswich, MA, USA). 143 94 and Mazzone 2009). 95 Administration of PFOA to fish has been shown to alter the ex- 2.3. Amplification of rare minnow apolipoprotein fragments 144 96 pression of the function of proteins involved in intracellular fatty acid 97 transport (Wei et al., 2008). However, the effect of subacute PFOA Because there was no sequence information about rare minnow 145 98 exposure on the expression of fish apolipoproteins and their upstream ApoA-IV, ApoE, ApoM, ApoL, ApoO,orHNF4α, oligonucleotides for 146 99 genes is still unknown. Rare minnow (Gobiocypris rarus), which is PCR were mainly designed using the previously known sequences of 147 100 small in size, has a high fertilization rate, a short embryonic develop- the apolipoprotein and HNF4α genes of zebrafish (Danio rerio) and 148 101 ment period, and a sensitivity to aquatic pollutants, has been used as a spotted barbel (Hemibarbus mylodon; Teleostei:Cypriniformes). Con- 149 102 good native Chinese test species in toxicological tests by the served regions were identified, and primers for ApoA-IV, ApoE, ApoM, 150 103 Environmental Protection Agency of China since 2000 (Zhou et al., ApoL, ApoO, and HNF4α were subsequentlyOF designed (Table 1). The 151 104 1995). This study focused on the cloning and sequencing of the gene amplification products from the PCR reactions were cloned into a 152 105 products coding for rare minnow apolipoproteins, and investigated pGEM®-T Easy Vector (Promega) and sequenced. 153 106 the effect of PFOA exposure on the expression of apolipoproteins. In 107 addition, the expression of PPARα, PPARγ, and HNF4α, genes upstream 2.4. Cloning of the full-length cDNA of ApoA-I 154 108 of apolipoproteins in mammals, was also examined after PFOA 109 exposure to determine whether these genes play the same role in The 5′-and3′-ends of the ApoA-I cDNA were amplified using 155 110 the regulation of apolipoprotein expression in fish. 5′-RACE and 3′-RACE reactions with a BD SMART™ RACE cDNA 156 Amplification kit (BDPRO Biosciences Clontech, San Jose, CA, USA). 157 111 2. Materials and methods The gene-specific primers (GSPs) were designed and synthesized 158 according to the partial sequences of ApoA-I ESTs (GenBank accession 159 112 2.1. Fish, exposure, and sampling procedure numbers EE392715.1) from our rare minnow adult liver cDNA library. 160 D ′ 161 The GSPs for ApoA-I are as follows: GSP1, 5 -CATGGCTCCCTTCTC 113 Nine-month-old male rare minnows with an average body mass of CATCTGTTCCTT-3′ and GSP2, 5′- GCCCATTAGAGAGAATGTGAGTCCTG- 162 114 1.3±0.3 g were obtained from a laboratory hatchery.