Tissue-specific transcriptome recovery on withdrawal from chronic alcohol exposure in zebrafish
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.
References (72)
- et al.
Whole-body cortisol increases after direct and visual contact with a predator in zebrafish, Danio rerio
Aquaculture
(2007) - et al.
Modeling withdrawal syndrome in zebrafish
Behavioural Brain Research
(2010) - et al.
Ethanol effects on the developing zebrafish: Neurobehavior and skeletal morphogenesis
Neurotoxicology and Teratology
(2004) - et al.
A dose for the wiser is enough: The alcohol benefits for associative learning in zebrafish
Progress in Neuro-Psychopharmacology and Biological Psychiatry
(2014) - et al.
Role and mechanisms of autophagy in alcohol-induced liver injury
Advances in Pharmacology
(2019) - et al.
Behavioral and biochemical effects of ethanol withdrawal in zebrafish
Pharmacology, Biochemistry, and Behavior
(2018) - et al.
Reversible loss of reproductive fitness in zebrafish on chronic alcohol exposure
Alcohol
(2016) - et al.
Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish
Behavioural Brain Research
(2009) Apoptosis and gene expression: Perspectives on alcohol-induced brain damage
Alcohol
(1994)- et al.
Alcoholic liver disease: Pathogenesis and new therapeutic targets
Gastroenterology
(2011)
Effects of acute and chronic ethanol exposure on the behavior of adult zebrafish (Danio rerio)
Pharmacology, Biochemistry, and Behavior
Prefrontal cortex expression of chromatin modifier genes in male WSP and WSR mice changes across ethanol dependence, withdrawal, and abstinence
Alcohol
Activation of neural stem cells from quiescence drives reactive hippocampal neurogenesis after alcohol dependence
Neuropharmacology
Oxidative stress response and gene expression with atrazine exposure in adult female zebrafish (Danio rerio)
Chemosphere
Semaphorin 5A is a bifunctional axon guidance cue regulated by heparan and chondroitin sulfate proteoglycans
Neuron
Alcohol myopathy: Impairment of protein synthesis and translation initiation
The International Journal of Biochemistry & Cell Biology
Sex hormone activity in alcohol addiction: Integrating organizational and activational effects
Progress in Neurobiology
The effects of temperature reduction on gene expression and oxidative stress in skeletal muscle from adult zebrafish. Comparative Biochemistry and Physiology
Toxicology & Pharmacology
Differences of acute versus chronic ethanol exposure on anxiety-like behavioral responses in zebrafish
Behavioural Brain Research
Molecular basis of alcoholism
Handbook of Clinical Neurology
The adult central nervous cholinergic system of a neurogenetic model animal, the zebrafish Danio rerio
Brain Research
Chronic alcohol exposure induced gene expression changes in the zebrafish brain
Behavioural Brain Research
Low dose of chronic ethanol exposure in adult zebrafish induces hepatic steatosis and injury
Biomedicine & Pharmacotherapy
Neuroendocrine regulation of the stress response in adult zebrafish, Danio rerio
Progress in Neuro-Psychopharmacology & Biological Psychiatry
iPhone® applications as versatile video tracking tools to analyze behavior in zebrafish (Danio rerio)
Pharmacology, Biochemistry, and Behavior
Alterations and recovery of rat brain gangliosides and glycosidases following long-term exposure to alcohol and rehabilitation during development
Brain Research
Ethanol alters acetylcholinesterase activity and gene expression in zebrafish brain
Toxicology Letters
Behavioral effects of taurine pretreatment in zebrafish acutely exposed to ethanol
Neuropharmacology
Impaired binocular depth inversion in patients with alcohol withdrawal
Journal of Psychiatric Research
Liver cirrhosis
Lancet
An integrative analysis of ethanol tolerance and withdrawal in zebrafish (Danio rerio)
Behavioural Brain Research
The effect of chronic alcohol abuse on sexual function
Clinics in Endocrinology and Metabolism
Semaphorin signaling guides cranial neural crest cell migration in zebrafish
Developmental Biology
Semaphorins and their signaling mechanisms
Chronic ethanol exposure alters DNA methylation in neural stem cells: Role of mouse strain and sex
Molecular Neurobiology
FASTQC A quality control tool for high throughput sequence data
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2020, Data in BriefCitation Excerpt :The control groups were also subjected to transfers but in ethanol-free tanks. Post completion of the alcohol program, the fish were maintained in an ethanol free holding tank for 9 weeks to induce withdrawal [1,2]. The zebrafish were anesthetized with Tricaine (Sigma, USA) and brain and liver tissues were isolated.
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Current address: Department of Biology, College of Science, University of Jeddah, Jeddah, Saudi Arabia