A transcriptomics-based analysis of the toxicity mechanisms of gabapentin to zebrafish embryos at realistic environmental concentrations⋆
Graphical abstract
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.
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This paper has been recommended for acceptance by Dr. Sarah Harmon.