Elsevier

Alcohol

Volume 91, March 2021, Pages 29-38
Alcohol

Tissue-specific transcriptome recovery on withdrawal from chronic alcohol exposure in zebrafish

https://doi.org/10.1016/j.alcohol.2020.10.001Get rights and content

Highlights

  • The zebrafish transcriptome is altered in response to chronic alcohol exposure, and shows some recovery upon withdrawal.

  • Brain transcripts recover their expression profile to a greater extent compared to liver transcripts.

  • We find evidence for distinct gender-dependent tolerance to alcohol exposure in both brain and liver tissues.

  • We identify gender- and tissue-specific genes and pathways involved in chronic alcoholism and recovery on withdrawal.

Abstract

Alcohol consumption can lead to a wide range of systemic disorders brought about by transcriptional changes. Recent studies have documented altered behavior and physiology in zebrafish exposed to alcohol. In this work, we have identified the changes in the zebrafish transcriptome in response to chronic alcohol exposure. We have further followed the extent of transcriptional recovery upon withdrawal from alcohol and found evidence of tissue-specific responses. Our results indicate a greater extent of recovery of the brain transcriptome compared to the liver. We identify two distinct classes of genes in response to withdrawal from alcohol exposure – those that recover their pre-alcohol expression profile versus those that retain altered expression even after the fish are removed from the alcohol environment. Finally, we have examined gender-specific responses to alcohol exposure in zebrafish and find evidence for distinct alcohol tolerance levels. Upon chronic alcohol exposure, a higher percentage of genes show perturbation in expression profile in males compared to females. Female fish also recover better with more genes regaining the control expression level upon withdrawal from alcohol. Overall, our work identifies genes and pathways perturbed by exposure to alcohol, and demonstrates the extent of gender- and tissue-specific transcriptional changes associated with chronic alcoholism and withdrawal.

Introduction

Alcohol is a common psychoactive substance consumed globally. Alcoholism is a disease that is characterized by uncontrolled compulsive drinking, despite its noxious effect on the health and economic status of the individual. Excessive drinking can lead to a host of complications such as liver cirrhosis (Schuppan & Afdhal, 2008), neurodegeneration, hypertension (Saunders, Beevers, & Paton, 1982), infertility (Van Thiel & Lester, 1979), and depression (Weissman & Myers, 1980), among others. Pregnant women who consume alcohol risk exposing their children to the negative effects of fetal alcohol syndrome (Ikonomidou et al., 2000). Chronic exposure to alcohol can alter various signal transduction pathways and lead to neurotoxicity (Mayfield et al., 2002). Long-term alcohol exposure can produce changes in brain function that can lead to alcohol dependence, tolerance, and other behavioral effects. These changes are likely to occur due to altered gene expression arising from cellular responses to alcohol exposure (Nestler, 2000). The state of withdrawal emerges when prolonged exposure to alcohol is discontinued and is associated with symptoms such as anxiety, insomnia, tremors, autonomic nervous hyperactivity, increased blood pressure and heart rate, motor dysfunction, and convulsions, depending on the severity of withdrawal (Cachat et al., 2010; Finn & Crabbe, 1997; Heilig, Egli, Crabbe, & Becker, 2010). Withdrawal from alcohol is also understood to be a dynamic process likely involving specific transcriptional responses that reflect gene expression changes (Hashimoto & Wiren, 2008).

Alcoholism is associated with brain defects, cognitive dysfunction, and behavioral impairments, and can cause neuroadaptations in the nervous system that involve remodeling of synapses (Most, Ferguson, & Harris, 2014; Oscar-Berman & Marinković, 2007; Wilke, Sganga, Barhite, & Miles, 1994) and that affect central nervous system (CNS) activity. These adaptations are a result of several mechanisms that increase the excitability of neurons in the brain. When alcohol intake is stopped, these changes in the CNS remain for a few days and result in hyperexcitability of the CNS, which manifests as alcohol withdrawal symptoms such as tremors, seizures, and hallucinations, depending on the severity of withdrawal (Davies, 2003; Finn & Crabbe, 1997).

Consumption of alcohol also affects the gut epithelium and allows lipopolysaccharide (LPS), usually restricted to the gut, to enter the bloodstream and activate Toll-like receptors (TLRs) expressed on various tissues, including the liver Kupffer cells. A signaling cascade is initiated resulting in the release of proinflammatory cytokines in the bloodstream, which cross the blood-brain barrier and reach the brain (Most et al., 2014). Recovery from alcohol exposure is associated with a partial reversal of structural and functional brain damage, which indicates that the brain is capable of repair (Crews et al., 2005; Hayes et al., 2018; Prasad, 1993), but it is not yet known whether a part of the transcriptome undergoes reversible changes or whether specific sets of genes are activated to initiate repair.

Most alcohol metabolism occurs in the liver, making it prone to alcohol-induced damage, which includes oxidative stress (Cederbaum, Lu, & Wu, 2009), steatosis (Passeri, Cinaroglu, Gao, & Sadler, 2009), alcoholic liver disease (ALD) (Gao & Bataller, 2011; O'shea, Dasarathy, & McCullough, 2010), liver cirrhosis, and a spectrum of other diseases. Cessation of alcohol consumption, i.e. withdrawal, can lead to a multitude of symptoms that are involved in the onset of alcohol withdrawal syndrome. As alcohol is a direct hepatotoxin, it is important to examine its effect on gene expression in the liver and the extent of recovery post-withdrawal. Transcriptome profiling has provided insights into the dynamics of the body's stress response (Jin et al., 2010; Malek, Sajadi, Abraham, Grundy, & Gerhard, 2004; Pavlidis, Theodoridi, & Tsalafouta, 2015) and can be pivotal in understanding the molecular mechanisms governing physiological responses to chronic alcohol exposure and subsequent withdrawal.

Zebrafish have the “classical” vertebrate neurotransmitter systems that guide behavioral activity in response to stress and anxiety (Barcellos et al., 2007; Egan et al., 2009; Mueller, Vernier, & Wullimann, 2004), as well as drug and alcohol toxicity (Carvan, Loucks, Weber, & Williams, 2004; Kily et al., 2008; Rico, Rosemberg, Dias, Bogo, & Bonan, 2007). In earlier work (Dewari, Ajani, Kushawah, Kumar, & Mishra, 2016), we assayed the effect of chronic alcohol exposure and subsequent withdrawal on behavior and embryonic development in zebrafish. We demonstrated the negative impact of chronic alcohol exposure on reproductive fitness and behavior, measured by a decrease in fecundity and heightened anxiety in novel-tank diving assays. A 9-week withdrawal program restored the reproductive capacity of the zebrafish and diminished the anxiety and stress induced by alcohol exposure.

Here we have performed gene expression profiling of brain and liver tissues from male and female zebrafish exposed to alcohol and analyzed transcriptional changes associated with long-term alcohol exposure as well as withdrawal. We document distinct classes of genes based on the recovery of their transcriptional pattern and find evidence for tissue-specific responses to the effect of alcohol. Our results suggest that while some metabolic processes may recover, many of the toxic effects of chronic alcohol exposure are not corrected upon withdrawal from chronic alcoholism, especially in the liver. The genes and pathways identified in this work shed light on the molecular perturbations caused by alcohol and provide a resource to study distinct gender- and tissue-specific responses to chronic alcoholism.

Section snippets

Experimental setup

As described earlier (Dewari et al., 2016), 120 naïve wild type (shortfin, ‘AB’ strain) zebrafish (Danio rerio) consisting of 60 males and 60 females were divided into four groups, i.e., male control, male alcohol-exposed, female control, and female alcohol-exposed. Each group had 30 fish and was maintained in a 20-L water tank. For chronic exposure to alcohol, the fish were transferred every afternoon into a new tank containing 0.5% ethanol and were maintained in the same tank for the next

Chronic alcohol exposure causes alterations in transcript levels in zebrafish tissues

Male and female zebrafish were subjected to 9 weeks of alcohol exposure followed by an equivalent time of withdrawal (Methods), followed by RNA-seq analysis from brain and liver tissues. Sequencing reads were mapped against the zebrafish genome (danRer10) with an alignment efficiency of ~85%. Abundance estimates for transcripts of both brain and liver tissue were calculated using RSEM, and the transcripts per million (TPM) values were obtained. The transcripts for each tissue were filtered for

Discussion

We have investigated the impact of chronic alcoholism using adult zebrafish as a model. Phenotypically, alcohol-exposed fish show behavioral changes and impaired fecundity, which are restored following a withdrawal program. Earlier studies have identified differential gene expression changes in response to a chronic 21-day alcohol exposure in the zebrafish brain (Pan, Kaiguo, Razak, Westwood, & Gerlai, 2011), biochemical and behavioral changes in response to withdrawal (da Silva Chaves et al.,

Author contributions

Sofia Banu: Formal analysis, Investigation, Visualization, Writing – Original draft; Surabhi Srivastava: Methodology, Project administration, Formal analysis, Writing – Original draft, Writing – Review & Editing; Arif Mohammed: Methodology, Investigation; Gopal Kushawah: Investigation; Divya Tej Sowpati: Conceptualization, Methodology, Supervision, Writing – Review & Editing; Rakesh K. Mishra: Conceptualization, Supervision, Funding acquisition, Writing – Review & Editing.

Research data

The data that support the findings of this study are openly available as a GEO dataset with Accession GSE143416.

Declaration of competing interest

None.

Acknowledgments

This work was supported by Council of Scientific and Industrial Research (CSIR), India to RKM.

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    1

    Current address: Department of Biology, College of Science, University of Jeddah, Jeddah, Saudi Arabia

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