Elsevier

Environmental Pollution

Volume 251, August 2019, Pages 746-755
Environmental Pollution

A transcriptomics-based analysis of the toxicity mechanisms of gabapentin to zebrafish embryos at realistic environmental concentrations

https://doi.org/10.1016/j.envpol.2019.05.063Get rights and content

Highlights

  • The ecotoxicity of gabapentin to zebrafish embryos was measured.

  • Transcriptomic techniques were applied to illustrate the underlying mechanisms.

  • The antioxidant, immune and nervous system of zebrafish were significantly affected.

Abstract

Gabapentin (GPT) has become an emerging contaminant in aquatic environments due to its wide application in medical treatment all over the world. In this study, embryos of zebrafish were exposed to gabapentin at realistically environmental concentrations, 0.1 μg/L and 10 μg/L, so as to evaluate the ecotoxicity of this emergent contaminant. The transcriptomics profiling of deep sequencing was employed to illustrate the mechanisms. The zebrafish (Danio rerio) embryo were exposed to GPT from 12 hpf to 96 hpf resulting in 136 and 750 genes differentially expressed, respectively. The results of gene ontology (GO) analysis and the Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis illustrated that a large amount of differentially expressed genes (DEGs) were involved in the antioxidant system, the immune system and the nervous system. RT-qPCR was applied to validate the results of RNA-seq, which provided direct evidence that the selected genes involved in those systems mentioned above were all down-regulated. Acetylcholinesterase (AChE), lysozyme (LZM) and the content of C-reactive protein (CRP) were decreased at the end of exposure, which is consistent with the transcriptomics results. The overall results of this study demonstrate that GPT simultaneously affects various vital functionalities of zebrafish at early developmental stage, even at environmentally relevant concentrations.

Introduction

Gabapentin (GPT) has been used as anticonvulsant during the last three decades since its first clinical application (Chadwick, 1995). It was then gradually used for the treatment of epilepsy, neuropathic pains and uremic pruritus (Gilron and JL, 2006; Lau et al., 2016), whose wide usage leads to a huge consumption. However, GPT is difficult to be metabolized, so that a large amount of GPT is typically excreted via urine (Kasprzyk-Hordern et al., 2008). Taking Germany as an example, about 80 t GPT was discharged into German municipal sewage in 2016 alone (Henning et al., 2018a). Meanwhile, the removal efficiency of GPT at sewage treatment plants (STPs) was extremely low. In the influent and effluent of a German sewage treatment plant, Dresden Kaditz, GPT were detected at the highest concentrations of 13.2 ± 3.3 μg/L and 12 ± 2.6 μg/L respectively (Gurke et al., 2015). Its low removal efficiency in STPs is strongly related to its low biodegradability which has been proved by several studies. For instance, Herrmann et al. applied a closed bottle test, and found that theoretical oxygen demand (ThOD) achieved 7.9% ± 3.6% after 28 days test, by which GPT was classified as a not readily biodegradable compound (Herrmann et al., 2015).

Because of the low removal efficiency, GPT was frequently detected in the aquatic system (Margot et al., 2016). In Minnesota, USA, GPT was detected in 24 municipal wastewater effluent at concentrations ranging from tens of ng/L to several μg/L (Writer et al., 2013). In South Wales, the concentrations of GPT varied between 11 and 1879 ng/L in surface water (Kasprzyk-Hordern et al., 2008). GPT was also detected in Germany surface water, ground water and potable water with the highest concentrations of 3.2 μg/L, 1.3 μg/L and 0.64 μg/L respectively (Henning et al., 2018b). The ubiquitous detection of GPT in the aquatic environment makes its ecotoxicity study urgent, as existing research has proven that GPT induces neuro toxicity to rats in clinical research (Karagoz et al., 2013). However, only limited ecotoxicology data was reported. Our previous study found that GPT exposure exerted the developmental toxicity to early life stages of zebrafish, but the underlying mechanisms for these effects have yet to be clarified (Li et al., 2018).

In the present study, transcriptomics profiling of deep sequencing was used to clarify the mechanisms of GPT toxicity to zebrafish embryos for the first time. Transcriptomics techniques provide a lot of information from limited numbers of test samples, which drives traditional ecotoxicity testing into a revolutionary new stage of development (Piña and Barata, 2011). Analyzing differentially expressed genes and their enriched pathways provides a comprehensive basis for understanding the toxicological mechanisms. In addition, three biomarkers described below were applied to validate the transcriptomics results. Acetylcholinesterase (AChE) has the function to make the neurotransmitter acetylcholine hydrolyzed in cholinergic synapses of both vertebrates and invertebrate (Guilhermino et al., 2000), which is crucial in neurological functions. Lysozyme (LZM) processes many biological functions, such as antiviral, immune modulation, anti-inflammatory and antitumor (Wang et al., 2015), and has been used as a sensitive and reliable biomarker to evaluate the toxic effects of pollutants to the immune system of organisms (Zhou et al., 2017). C-reactive protein (CRP) has long been recognized as a major acute-phase protein involved in both innate and adaptive immunity (Sinha et al., 2001).

Section snippets

Chemicals

GPT (purity>98%) was supplied by the Aladdin Industrial Corporation, Shanghai, China. CaCl2·2H2O (purity>99.0%) was supplied by Sinopharm Chemical Reagent Co.,Ltd, Shanghai, China. MgSO4·7H2O (purity>99.0%), NaHCO3 (purity>99.5%) and KCl (purity>99.5%) were supplied by Shanghai Lingfeng Chemical Reagent Co.,Ltd, Shanghai, China. TRIzol was supplied by Invitrogen, Shanghai, China. DNase I was supplied by New England Biolabs, Beijing, China. PrimeScript II RTase, Random primer, dNTP mix and SYBR®

Transcriptome analysis

Deep sequencing analysis was performed to the control and treatment groups and tens of millions of reads per sample (between 49,227,972 and 65,883,878, presented in Table S2) were obtained. We regarded those reads whose quality scores surpass the Q30 value (correct base recognition rate over 99.9%) were clean reads, and the results illustrate the proportion of the clean reads was 94.47% with the GC (Guanine and Cytosine) content ranging from 45.1%∼45.52%, and the total unique mapped rate over

Conclusion

In conclusion, this study found that GPT could cause the neurotoxicity and the immune toxicity to zebrafish embryos at realistically environmental concentrations. The transcriptomics data explained the mechanisms behind the developmental toxicity and antioxidant toxicity of zebrafish embryos induced by the GPT exposure. With a comprehensive analysis of the connection between the DEGs and the enriched pathways, this study provided many evidences for understanding the underlying mechanisms of

Declarations of interest

None.

Conflicts of interest

The authors declare no conflict of interest.

Notes

This study was performed strictly referencing to the Laboratory Animal—Guideline for ethical review of animal welfare (GB/T 35,892–2018), and was authorized by Animal Care and Use Committee in School of Environmental Science and Engineering of Nanjing Tech University (Nanjing, China).

Acknowledgement

We acknowledge funding from Natural Science Foundation of Jiangsu Province (No. BK20160989), Major Research Program of Natural Science of University in Jiangsu Province (No. 16KJA610002), and Cultivation Project for International Cooperation from Nanjing Tech University. We thank the Nanjing Decode Genomics for providing technique support for RNA-seq and RT-qPCR.

References (63)

  • S.F.G. Krens et al.

    Characterization and expression patterns of the MAPK family in zebrafish

    Gene Expr. Patterns

    (2006)
  • S. Lee et al.

    Neuronal apoptosis linked to EglN3 prolyl hydroxylase and familial pheochromocytoma genes: developmental culling and cancer

    Cancer Cell

    (2005)
  • C. Liu et al.

    Aspp2 negatively regulates body growth but not developmental timing by modulating IRS signaling in zebrafish embryos

    Gen. Comp. Endocrinol.

    (2014)
  • D.R. Livingstone

    Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms

    Mar. Pollut. Bull.

    (2001)
  • M. Oliveira et al.

    Effects of short-term exposure to fluoxetine and carbamazepine to the collembolan Folsomia candida

    Chemosphere

    (2015)
  • T. Pan et al.

    Valproic acid-mediated Hsp70 induction and anti-apoptotic neuroprotection in SH-SY5Y cells

    FEBS Lett.

    (2005)
  • K. Peng et al.

    Knockdown of FoxO3a induces increased neuronal apoptosis during embryonic development in zebrafish

    Neurosci. Lett.

    (2010)
  • B. Piña et al.

    A genomic and ecotoxicological perspective of DNA array studies in aquatic environmental risk assessment

    Aquat. Toxicol.

    (2011)
  • J. Rhee et al.

    Effect of pharmaceuticals exposure on acetylcholinesterase (AchE) activity and on the expression of AchE gene in the monogonont rotifer, Brachionus koreanus

    Comp. Biochem. Physiol. C

    (2013)
  • B. Schilter et al.

    Anticonvulsant drug toxicity in rat brain cell aggregate cultures

    Toxicol. Vitro

    (1995)
  • A.M. Siebel et al.

    In vitro effects of antiepileptic drugs on acetylcholinesterase and ectonucleotidase activities in zebrafish (Danio rerio) brain

    Toxicol. Vitro

    (2010)
  • S. Sinha et al.

    Acute phase response of C-reactive protein of Labeo rohita to aquatic pollutants is accompanied by the appearance of distinct molecular forms

    Arch. Biochem. Biophys.

    (2001)
  • R.S. Sohal et al.

    Oxidative stress and aging in the Mongolian gerbil (Meriones unguiculatus)

    Mech. Ageing Dev.

    (1995)
  • S. Sudha et al.

    Chronic phenytoin induced impairment of learning and memory with associated changes in brain acetylcholine esterase activity and monoamine levels

    Pharmacol. Biochem. Behav.

    (1995)
  • E. Teixidó et al.

    Assessment of developmental delay in the zebrafish embryo teratogenicity assay

    Toxicol. Vitro

    (2013)
  • T. Wang et al.

    Effect of copper nanoparticles and copper sulphate on oxidation stress, cell apoptosis and immune responses in the intestines of juvenile Epinephelus coioides

    Fish Shellfish Immunol.

    (2015)
  • T. Weichhart et al.

    The TSC-mTOR signaling pathway regulates the innate inflammatory response

    Immunity

    (2008)
  • J.H. Writer et al.

    Widespread occurrence of neuro-active pharmaceuticals and metabolites in 24 Minnesota rivers and wastewaters

    Sci. Total Environ.

    (2013)
  • H. Zheng et al.

    Lp-PLA2 silencing protects against ox-LDL-induced oxidative stress and cell apoptosis via Akt/mTOR signaling pathway in human THP1 macrophages

    Biochem. Biophys. Res. Commun.

    (2016)
  • Y. Zhou et al.

    Oxidative damage, ultrastructural alterations and gene expressions of hemocytes in the freshwater crab Sinopotamon henanense exposed to cadmium

    Ecotoxicol. Environ. Saf.

    (2017)
  • L.B.S.J. Anne Brunet et al.

    Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase

    Science

    (2004)
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