Science of the Total Environment 743 (2020) 140796

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Science of the Total Environment

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Transcriptome analysis reveals the mechanism of common carp braininjury after exposure to lead

Yue Zhang a,b, Peijun Zhang c, Peng Yud, Xinchi Shang a,b, Yuting Lu a,b, Yuehong Li a,b,⁎a College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, Chinab Ministry of Education Laboratory of Animal Production and Quality Security, Jilin Agricultural University, Changchun 130118, Chinac Health Monitoring and Inspection Center of Jilin Province, Changchun 130062, Chinad College of Electronic and Information Engineering, Changchun University of Science and Technology, Changchun 130022, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Fluorine accumulates in brain tissuesand lead to memory loss of commoncarp.

• 1186 genes were differentially expressedin the fluorine-exposed carp.

• Long-term depression and Ion channelswere involved in fluorine-induced injury.

⁎ Corresponding author at: College of Animal Science anUniversity, Changchun 130118, China.

E-mail address: liyhong12@163.com (Y. Li).

https://doi.org/10.1016/j.scitotenv.2020.1407960048-9697/© 2020 Published by Elsevier B.V.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 15 February 2020Received in revised form 29 June 2020Accepted 5 July 2020Available online 07 July 2020

Editor: Henner Hollert

Keywords:LeadCarpTranscriptome analysisBrain damage

Lead, a widespread industrial pollutant, has been known as a powerful neurotoxin that could affect the centralnervous system. Accumulating evidences demonstrated that lead exposure could result in the damage of braintissues both in fish and human. However, the mechanism of lead induced brain injury has not been fully eluci-dated. The purpose of this study was to clarify the possible mechanism of common carp brain injury after expo-sure to lead through transcriptome analysis. Transcriptome analysis showed that 2141 differentially expressedgenes were identified. Among these, 502 genes were up-regulated and 1639 geneswere down-regulated. Mean-while, GO enrichment analysis showed Transport, biological_process, DNA-templated (regulation of transcrip-tion) and signal transduction contained the most differential genes in the biological process. Furthermore,KEGG pathway enrichment analysis showed Ion channels, GnRH signaling pathway, cell adhesion molecules,Wnt signaling pathway, and calcium signaling pathwaywere significantly enriched. In addition, 10 differentiallyexpressed genes were selected for qRT-PCR detection, and the results demonstrated that the selected genes ex-hibited the same trends with the RNA-Seq results, which indicates the transcriptome sequencing data is reliable.In conclusion, the above results provide a theoretical basis for clarifying the relationship between lead exposureand brain injury in common carp and for further studying of the genes related to lead poisoning.

© 2020 Published by Elsevier B.V.

d Technology, Jilin Agricultural

1. Introduction

With the development of industry and transportation, the pollutionof heavy metals to environment is more and more serious (Yu et al.,

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2008). Meanwhile, the pollution of heavy metals, such as lead, in wateris very prominent in China (Yu et al., 2016). The life activities of aquaticorganisms such as fish are inseparable from the water environment.Therefore, fish are more vulnerable to lead pollution in the water.Lead, known as one of the major heavy metals, could cause toxic effectson fish and mammals (Chahid et al., 2014). Previous studies showedthat lead exposure could cause the injury of liver, kidney, and gills(Lee et al., 2019). Furthermore, fish can directly affect humans throughfood intake and result in the accumulation of heavy metals in humanbody. Therefore, it is an important task for us to investigate the patho-genesis of lead poisoning and clarify the possible mechanism.

Previous studies demonstrated that lead exposure could affect thecardiovascular system, reproductive system, and immune system (HsuandGuo, 2002; Lee et al., 2019). Also, lead exposure could affect the cen-tral nervous system and it got more and more attention (Lidsky andSchneider, 2003). Lead is a powerful neurotoxin, which can causenerve damage and behavior disorder in human and animals (Assiet al., 2016). The toxicity of lead to the central nervous system mainlyaffects the development of blood-brain barrier, neurons, and glial cells(Patrick, 2006). Lead has a strong nerve affinity and can be accumulatedin nerve tissue. Previous studies showed that lead exposure could resultin the injury of learning and memory ability (Nava-Ruiz et al., 2012;Neuwirth et al., 2017). Although lead is known as a potentneurotoxicant, information regarding its threat to fish brain and under-lying mechanisms remains unclear. Transcriptome analysis has beenwidely used in the animals, such as fish, chicken, and other animals(Wang et al., 2020; Zheng et al., 2020). In this study, we aimed to inves-tigate the effects of lead on brain injury of common carp and clarify themechanism by transcriptome analysis.

2. Materials and methods

2.1. Animals and treatment

The common carp (weighting 56 ± 8 g) were obtained from theXinli Reservoir (Changchun, China) and cultured in laboratory tanks(90× 55×45 cm)with continuous aeration. The carpsweremaintainedat 24± 2 °C in laboratory tanks (90 × 55 × 45 cm)with continuous aer-ation for two weeks to adaptive the environment. Eighty common carpwere randomly divided into the control group and the lead-exposedgroup. The carps of lead-treated group were received 1 mg/L lead ace-tate (Sigma, USA) for 60 days. The dose of lead used in this study wasbased on our previous study (Dai et al., 2018) and other study (Giriet al., 2018). All animal experiments were performed according to theUS NIH guidelines for the care and use of laboratory animals and ap-proved by the Institutional Animal Care and Use Committee of Jilin Ag-ricultural University. In this study, twenty fish in each group were usedformemory experiment; ten fish in each groupwere used for the detec-tion of lead accumulation in blood and brain; three fish in each groupwere used for transcriptome analysis; three fish in each group wereused for qRT-PCR experiment.

2.2. Determination of lead accumulation in blood and brain

60 days after lead exposure, the brain and blood were collected.Then, the brain tissues were digested at 95 °C with HNO3 until the tis-sues were completely dissolved. And the concentration of lead inbrain tissues and blood were measured by using Atomic AbsorptionspectrometerAA-6300 (Shimadzu, Japan).

2.3. Protocol for the memory experiment

The effect of lead exposure on memory loss was measured as de-scribedpreviously (Lefevre et al., 2017). In brief, 60days after lead expo-sure, the carps were deprived of food for 3 days. Then, the carps weretrained in maze for five days. Subsequently, after a 2 days break, the

carps were subjected for individual trials. The carps were trainedevery 2 days for five times. After the training, each carp of the twogroups were tested once in the maze, and the time to find food andnumber of errors were recorded.

2.4. Transcriptome sequencing

Total RNA of brain tissue was extracted with Trizol reagent accord-ing to the instructions of the kit (Takara, Dalian, China). The RNAamount and purity of each sample was quantified using NanoDropND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity wasassessed byAgilent 2100with RINnumber N 7.0. Poly(A) RNA is purifiedfrom total RNA (5 μg) using poly-T oligo-attachedmagnetic beads usingtwo rounds of purification. Then the poly(A) RNA was fragmented intosmall pieces using divalent cations under high temperature. Then thecleaved RNA fragments were reverse-transcribed to create the cDNA li-brary in accordingwith the protocol for the TruSeq® StrandedmRNA Li-brary Prep kit (Illumina, San Diego, CA, USA) (Meng et al., 2019). Briefly,we used to synthesizeU-labeled second-strandedDNAswith E. coliDNApolymerase I, RNase H and dUTP. An A-base is then added to the bluntends of each strand, preparing them for ligation to the indexed adapters.Each adapter contains a T-base overhang for ligating the adapter to theAtailed fragmented DNA. Single-or dual-index adapters are ligated tothe fragments, and size selection was performed with AMPureXPbeads. After the heatlabile UDG enzyme treatment of the U-labeledsecond-stranded DNAs, the ligated products are amplified with PCR bythe following conditions: initial denaturation at 95 °C for 3min; 8 cyclesof denaturation at 98 °C for 15 s, annealing at 60 °C for 15 s, and exten-sion at 72 °C for 30 s; and then final extension at 72 °C for 5min. The av-erage insert size for the final cDNA library was 300 bp (±50 bp). Thesequencing strategy was pair-end 150 bp for Hiseq4000. NCBI Cyprinuscarpio (common carp) RefSeq GCF_000951615.1 was used as the refer-ence (ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/951/615/GCF_000951615.1_common_carp_genome/GCF_000951615.1_common_carp_genome_genomic.fna.gz). The RNA-seq data sets have been de-posited in the Gene Expression Omnibus (GEO) database under acces-sion number GSE151339.

2.5. Data analysis

Cutadapt software was used to remove the reads that containedadaptor contamination. And after removed the low quality bases andundetermined bases, we used HISAT2 software to map reads to the ge-nome (NCBI Cyprinus carpio RefSeq GCF_000951615.1, ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/951/615/GCF_000951615.1_common_carp_genome/GCF_000951615.1_common_carp_genome_genomic.fna.gz). The mapped reads of each sample were assembledusing StringTie. Then, all transcriptomes from all samples were mergedto reconstruct a comprehensive transcriptome using gffcompare soft-ware. After the final transcriptome was generated, StringTie andballgown were used to estimate the expression levels of all transcriptsand perform expression level for mRNAs by calculating FPKM. The dif-ferentially expressed mRNAs were selected with fold change N2 orfold change b0.5 and FDR b 0.05 by R package DESeq2.

2.6. Gene ontology and enrichment analysis

GOseq software was used to annotate the GO function to reveal thebiological function of the differential gene; KEGG was used to analyzethe signal pathwaymainly involved in the differential gene. GO enrich-ment analysis of the DGEs was performed with the GOseq R package.KOBAS software was used to test the statistical enrichment of DEGs inthe KEGG pathway analysis. P b 0.05 was considered significant for GOand KEGG enrichment.

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2.7. Verification by quantitative real-time PCR (qRT-PCR)

We selected 10 differentially expressed genes to verify the RNA-Seqresults. These genes (related to Transporters, Ion channels, GnRH signal-ing pathway, Cell adhesion molecules and Calcium signaling pathway)were measured by qRT-PCR and the same RNA samples were used fortranscriptome analysis. PCR was conducted using SYBR Green Real-time PCR kit according to the manufacturer's instructions. PCR reactionwas carried out in ABI 7500 real-time PCR system. The relative gene ex-pression was calculated using the 2−△△CT method.

2.8. Statistical analysis

All data of this study were expressed as mean ± SD and analyzedusing SPSS 18.0. Statistical analyses were tested by using one-wayANOVA followed by Dunn's test. P b 0.05 was considered as statisticalsignificance.

3. Results

3.1. Lead accumulation in blood and brain tissues

In this study, we firstly detected the accumulation of lead in bloodand brain tissues. As shown in Fig. 1, after 60 days lead exposure, thelevels of lead in blood and brain tissues increased significantly thanthe control group, which indicated lead was accumulated in blood andbrain tissues.

Fig. 1. Lead accumulation in blood and brain tissues. 60 days after lead exposure, the brainand blood were collected. Then, the brain tissues were digested at 95 °C with HNO3 untilthe tissues were completely dissolved. And the concentration of lead in brain tissues andblood were measured by using Atomic Absorption spectrometerAA-6300 (Shimadzu,Japan). The data of this study are presented asmean± SD of three parallelmeasurements.P b 0.05 indicates a significant difference between the two groups.

3.2. Effects of lead exposure on memory loss

The effects of lead exposure on memory loss of common carps weremeasured. The results showed that the carps of lead group used longertime to find the food. And the numbers of errors were higher than thecontrol carps (Fig. 2). These results indicated lead exposure could resultin memory loss of common carps.

3.3. Differential expression analysis

The PCA score plot showed that the experimental and control groupswere clearly separated and that the three RNA-seq samples as biologicalreplicates were clustered, with the main principal component (PC)scores as follows: PC1 = 68.02%, PC2 = 12.93% (Fig. 3C). The resultsshowed that a total of 2141 genes in brainwere differentially expressed,including 502 up-regulated genes and 1639 down-regulated genes(Fig. 3A, B). From the results of heatmap, a large number of geneswere up-regulated, such as LOC109057022, LOC095435 andLOC109064506 and a large number of genes were down-regulated,such as LOC109085545, orc4 and c34h14orf159 (Fig. 4).

3.4. GO analysis and KEGG pathway enrichment based on DEGs

To obtain insight into the biological processes that could be differen-tially regulated with lead treatment, GO enrichment analysis was

Fig. 2. Effects of lead exposure onmemory loss. 60days after lead exposure, the carpsweredeprived of food for 3 days. Then, the carps were trained in maze for five days.Subsequently, after a 2 days break, the carps were subjected for individual trials. Thecarps were trained every 2 days for five times. After the training, each carp of the twogroups were tested once in the maze, and the time to find food and number of errorswere recorded. The data of this study are presented as mean ± SD of three parallelmeasurements. P b 0.05 indicates a significant difference between the two groups.

Fig. 3. (A, B)Volcano plot of distribution trends for differentially expressed genes in lead exposure and control groups. Each dot represents one gene. Red dots represent up-regulated genesand blue dots represent down-regulated genes. Gray dots represent genes with no differential expression. (C) Principal component analysis among samples. Pink circles indicate samplestreatedwith lead,while blue circles indicate sampleswithout lead treatment. (For interpretation of the references to colour in thisfigure legend, the reader is referred to theweb version ofthis article.)

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performed in this study. GO enrichment analysis were included threemajor functional categories: biological processes, cellular componentsand molecular functions. Transport, biological_process, DNA-templated (regulation of transcription) and signal transduction con-tains themost differential genes in the biological processes. Membrane,integral component of membranes, nucleus and cytoplasm contains themost differential genes in the cellular components. Metal ion binding,

nucleotide binding, ATP binding and transferase activity contains themost differential genes in molecular functions (Fig. 5).

KEGG databasewas used to compare and comment the differentiallyexpressed unigenes sequence. The most significant 20 enrichmentKEGG pathwayswere analyzed, which indicates that the enriched path-ways played important roles in brain damage related signaling path-ways and the five largest enriched pathways were Transporters, Ion

Fig. 4.Heatmapof expression levels for all DEGs among six samples, representing three biological replicates. The transition fromblue to red strips represents an increase in gene expressionlevels. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5. GO categorization of the unigenes of transcriptome in brain. Each annotated sequence is assigned at least one GO term of the following: biological process, cellular component, ormolecular function.

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channels, GnRH signaling pathway, Cell adhesion molecules and Cal-cium signaling pathway (Fig. 6).

3.5. qRT-PCR validation

To confirm the results of RNA-Seq in this study, 10 genes involved inTransporters, Ion channels, GnRH signaling pathway, Cell adhesionmol-ecules and Calcium signaling pathway were selected and qRT-PCR wasapplied to test the expression of these genes. The results showed thatthe expression of cga, camk4, and fzr were increased. The expressionof mpz, pkd2, gabrb3, ncam1, and negr1 were decreased. And the ex-pression of these genes was consistent with the RNA-Seq results,which indicated the transcriptome sequencing datawas reliable (Fig. 7).

4. Discussion

Lead has a long history in industrial application and is a widespreadindustrial pollutant (Cheng andHu, 2010). Studies have shown that leadcan cause a series of physiological and biochemical changes (Tong et al.,2000). Lead is a powerful neurotoxin and studies showed exposure oflead could affect the central nervous system (Papanikolaou et al.,2005). In this study, through transcriptome analysis technology, weaimed to reveal themechanism of common carp brain injury after expo-sure to lead. The results showed that 2141 differential genes werefound. The KEGG function annotation revealed that the differentialgenes were significantly enriched in the pathways of Ion channels,GnRH signaling pathway, cell adhesion molecules, Wnt signaling path-way, and calcium signaling pathway.

Lead is an environmental toxicant that could affect both fish andhuman. Human are exposed to lead mainly through poisonings andconsumption of fish and other seafood (Castro-Gonzalez and Mendez-

Armenta, 2008). Lead exposure can cause toxic effects on various tis-sues, such as brain, kidney, and liver (Gurer et al., 2001). In fish, thetoxic effects of lead mainly caused by bioaccumulation (Malik et al.,2010). Previous studies demonstrated that lead could accumulate inthe brain tissues and result in the injury of brain tissues of fish(Shahsavani et al., 2011). However, the molecular mechanism underly-ing this remains poorly understood. Many previous studies suggestedthat lead could induce the ROS production, which subsequently causestissues injury (Ahamed and Siddiqui, 2007). In this study, transcriptomeanalysis showed that 2141 differentially expressed genes were identi-fied. And KEGG pathway enrichment analysis showed Ion channels,GnRH signaling pathway, cell adhesion molecules, Wnt signaling path-way, and calcium signaling pathway were significantly enriched.These pathways were related to lead-induced brain injury in commoncarp.

Wnt signaling pathway is a complex network of protein action,whose function ismost common in embryonic development and cancer,but also involved in the normal physiological process of adult animals(Logan and Nusse, 2004; Wang et al., 2014). A large body of studiesdemonstrated thatWnt signaling pathwaywas involved in the develop-ment of central nervous system (CNS) (Lambert et al., 2016). Further-more, it could regulate the function of CNS (Ille and Sommer, 2005).Previous studies demonstrated that activated Wnt signaling pathwaycould enhance cognitive function and reverse cognitive deficits inmouse Alzheimer's disease model (Vargas et al., 2014). It well knownthat exposure of lead could result in learning and memory deficits.This indicates a good agreement between lead-induced learning andmemory deficits and inhibition of Wnt signaling pathway.

Ion channel is a passive transport channel of inorganic ions acrossmembrane (Daiguji, 2010). Ion channels activity, the ability of cells toregulate the speed of corresponding substances entering and leaving

Fig. 6. KEGG assignment of unigenes in the brain transcriptome of common carp.

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cells through the opening and closing of ion channels, is of great signif-icance for the realization of various functions of cells (Xie et al., 2016).Studies showed that its activity is critical to the excitability and functionof cells and tissues (Kramer et al., 2005). Recently, accumulating evi-dences showed that many human and animal diseases were related tothe ion channels (Wemmie et al., 2013). A previous study demonstratedthat acid sensing ion channels were closely related to brain injury(Xiong et al., 2007). Acid sensing ion channels can be used as a pharma-cological target for neurodegenerative diseases (Wemmie et al., 2006).

Fig. 7. Comparison of differentially expressed genes and qRT-PCR. The X-axis displays 10 selectemean ± SD of three parallel measurements.

Calcium ion (Ca2+) is the second messenger in many biological pro-cesses. It is a key chemical involved in neurotransmitters and the releaseof intercellular signals in the nervous system (Braet et al., 2004). Exces-sive or insufficient calcium can cause various diseases, such as depres-sion. Some studies have confirmed that calcium signaling pathway isessential in the regulation of brain function (Braet et al., 2004). Itcould affect the learning and memory of animals (Bezprozvanny,2010; Gomez et al., 2001). Similarly, in the present study, functional en-richment showed ion channels and calcium signaling pathways were

d genes and Y-axis represents relative fold change. The data of this study are presented as

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enriched, which were closely related to the brain injury. The relation-ship between calcium signaling pathway and lead exposure remainsto be elucidated.

In summary, our results showed that exposure of lead could resultsinmemory loss of common carp. And the data of transcriptome analysisreveals a large number of differentially expressed geneswere identified.Among these genes, we found the genes related to Ion channels, GnRHsignaling pathway, cell adhesion molecules, Wnt signaling pathway,and calcium signaling pathway were involved in the brain injury expo-sure to lead. The present study provides a valuable resource in the toxiceffect of lead in common carp.

CRediT authorship contribution statement

Yue Zhang: Conceptualization, Investigation, Writing - originaldraft. Peijun Zhang: Investigation. Peng Yu: Investigation. XinchiShang: Investigation. Yuting Lu: Investigation. Yuehong Li: Conceptu-alization, Writing - original draft.

Declaration of competing interest

All authors declare that they have no conflict of interest.

Acknowledgement

Theworkwas supported by theNational Natural Science Foundationof China (no. 30972191), Jilin Province Industrial Technology Researchand Development Special Project (no. 2019C059-5) and Scientific Tech-nological Development Plan Project in Jilin Province of China (no.20190201179JC).

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