ABSTRACT
It is largely accepted that vertebrates are more susceptible to chemical insult during the early life stage. It is implied that if a chemical such as ethanol is developmentally toxic, it must interfere with, or modulate, critical signaling pathways. The probable molecular explanation for increased embryonic susceptibility is that collectively there is no other period of an animal's lifespan when the full repertoire of molecular signaling is active. Understanding the mechanism by which ethanol exposure disrupts vertebrate embryonic development is enormously challenging; it requires a thorough understanding of the normal molecular program to understand how transient ethanol exposure disrupts signaling and results in detrimental long-lasting effects. During the past several years, investigators have recognized the advantages of the zebrafish model to discover the signaling events that choreograph embryonic development. External development coupled with the numerous molecular and genetic methods make this model a valuable tool to unravel the mechanisms by which ethanol disrupts embryonic development. In this chapter we describe procedures used to evaluate and define the morphological, cellular and molecular responses to ethanol in zebrafish.
Subject(s)
Central Nervous System Depressants/toxicity , Ethanol/toxicity , Fetus/drug effects , Signal Transduction/drug effects , Zebrafish/embryology , Animals , Cell Death/drug effects , Dose-Response Relationship, Drug , Embryonic Development/drug effects , Fetus/metabolism , Fetus/pathology , Gene Expression Profiling/methods , Gene Expression Regulation, Developmental/drug effects , Gene Silencing , Models, Animal , Oligonucleotide Array Sequence Analysis , Oligonucleotides, Antisense/metabolism , Signal Transduction/genetics , Time Factors , Zebrafish/metabolismABSTRACT
Ethanol is a well-established developmental toxicant; however, the molecular and cellular mechanism(s) of toxicity remains unclear. It has been suggested that ethanol metabolism leads to oxidative stress resulting in an increase in cell death. Alcohol developmental toxicity has not been well studied in zebrafish; however, zebrafish represent an excellent vertebrate model for investigating and understanding normal and aberrant development. To evaluate ethanol metabolism dependent toxicity, chemical inhibitors of the ethanol metabolizing enzymes were utilized. Embryos co-exposed to ethanol and a combination of ethanol metabolism inhibitors led to a significant increase in the occurrence of pericardial edema. Further, in the presence of the inhibitor mixture there was an increase in developmental malformations at lower ethanol concentrations. Cell death has been implicated as a potential explanation for ethanol-dependent toxicity. Using cell death assays, ethanol significantly increased embryonic cell death. To determine if oxidative stress underlies cardiovascular dysfunction, embryos were co-exposed to ethanol and several antioxidants. The antioxidants, glutathione and lipoic acid, partially attenuated the incidence of pericardial edema. The effectiveness of the antioxidants to protect the embryos from ethanol-induced cell death was also evaluated. The antioxidants provided no protection against cell death. Thus, ethanol-mediated pericardial edema and cell death appear to be mechanistically distinct.
Subject(s)
Antioxidants/pharmacology , Central Nervous System Depressants/antagonists & inhibitors , Central Nervous System Depressants/toxicity , Ethanol/antagonists & inhibitors , Ethanol/toxicity , Zebrafish/physiology , Animals , Apoptosis/drug effects , Cell Death/drug effects , Dose-Response Relationship, Drug , Edema/chemically induced , Edema/prevention & control , Embryo, Nonmammalian , Embryonic Development/drug effects , In Situ Nick-End Labeling , Oxidative Stress/drug effects , Pericardium/drug effectsABSTRACT
Ethanol is metabolized to acetaldehyde mainly by the alcohol dehydrogenase pathway and, to a lesser extent, through microsomal oxidation (CYP2E1) and the catalase-H(2)O(2) system. Acetaldehyde, which is responsible for some of the deleterious effects of ethanol, is further oxidized to acetic acid by aldehyde dehydrogenases (ALDHs), of which mitochondrial ALDH2 is the most efficient. The aim of this study was to evaluate zebrafish (Danio rerio) as a model for ethanol metabolism by cloning, expressing, and characterizing the zebrafish ALDH2. The zebrafish ALDH2 cDNA was cloned and found to be 1892 bp in length and encoding a protein of 516 amino acids (M(r) = 56,562), approximately 75% identical to mammalian ALDH2 proteins. Recombinant zebrafish ALDH2 protein was expressed using the baculovirus expression system and purified to homogeneity by affinity chromatography. We found that zebrafish ALDH2 is catalytically active and efficiently oxidizes acetaldehyde (K(m) = 11.5 microM) and propionaldehyde (K(m) = 6.1 microM). Similar kinetic properties were observed with the recombinant human ALDH2 protein, which was expressed and purified using comparable experimental conditions. Western blot analysis revealed that ALDH2 is highly expressed in the heart, skeletal muscle, and brain with moderate expression in liver, eye, and swim bladder of the zebrafish. These results are the first reported on the cloning, expression, and characterization of a zebrafish ALDH, and indicate that zebrafish is a suitable model for studying ethanol metabolism and, therefore, toxicity.
Subject(s)
Aldehyde Dehydrogenase/genetics , Aldehyde Dehydrogenase/metabolism , Baculoviridae/metabolism , Zebrafish/metabolism , Aldehyde Dehydrogenase/biosynthesis , Amino Acid Sequence , Animals , Blotting, Western , Brain Chemistry , Central Nervous System Depressants/metabolism , Central Nervous System Depressants/toxicity , Chromosome Mapping , Cloning, Molecular , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Electrophoresis, Polyacrylamide Gel , Ethanol/metabolism , Ethanol/toxicity , Humans , Kinetics , Molecular Sequence Data , Muscle, Skeletal/metabolism , Myocardium/metabolism , Tissue DistributionABSTRACT
Ethanol is a well-established developmental toxicant; however, the mechanism(s) of this toxicity remains unclear. Zebrafish are becoming an important model system for the evaluation of chemical and drug toxicity. In this study, zebrafish embryos were utilized to compare the developmental toxicity resulting from either ethanol or acetaldehyde exposure. Embryos were exposed to waterborne ethanol concentrations for various lengths of time but encompassed the earliest stages of embryogenesis. The waterborne ethanol concentration that causes 50% mortality (LC(50)) following a 45-h ethanol exposure was approximately 340 mM (1.98% v/v). A number of reproducible endpoints resulted from ethanol exposure and included pericardial edema, yolk sac edema, axial malformations, otolith defects, delayed development, and axial blistering. When the exposure period was reduced, similar signs of toxicity were produced at nearly identical ethanol concentrations. To estimate the embryonic dose following a given waterborne ethanol concentration, a kinetic alcohol dehydrogenase (ADH) assay was adapted. The average embryonic ethanol dose was calculated to be a fraction of the waterborne concentration. Embryos exposed to waterborne acetaldehyde resulted in similar, but not identical, endpoints as those induced by ethanol. Embryos were however, almost three orders of magnitude more sensitive to acetaldehyde than to ethanol. Ethanol and acetaldehyde both negatively impact embryonic development; however, ethanol is more teratogenic based on teratogenic indices (TIs). These results demonstrate that the zebrafish model will provide an opportunity to further evaluate the mechanism of action of ethanol on vertebrate development.
Subject(s)
Abnormalities, Multiple/chemically induced , Acetaldehyde/toxicity , Embryo, Nonmammalian/abnormalities , Embryo, Nonmammalian/drug effects , Ethanol/toxicity , Zebrafish/abnormalities , Abnormalities, Multiple/pathology , Abnormalities, Multiple/physiopathology , Alcohol Dehydrogenase/analysis , Alcohol Dehydrogenase/metabolism , Animals , Biological Assay/methods , Bone and Bones/abnormalities , Bone and Bones/drug effects , Disease Models, Animal , Dose-Response Relationship, Drug , Ear, Inner/abnormalities , Ear, Inner/drug effects , Edema/chemically induced , Female , Pericardium/drug effects , Pericardium/physiopathology , Survival Rate , Teratogens/toxicity , Yolk Sac/drug effects , Yolk Sac/physiopathology , Zebrafish/embryologyABSTRACT
Ethanol is teratogenic to many vertebrates. We are utilizing zebrafish as a model system to determine whether there is an association between ethanol metabolism and ethanol-mediated developmental toxicity. Here we report the isolation and characterization of two cDNAs encoding zebrafish alcohol dehydrogenases (ADHs). Phylogenetic analysis of these zebrafish ADHs indicates that they share a common ancestor with mammalian class I, II, IV, and V ADHs. The genes encoding these zebrafish ADHs have been named Adh8a and Adh8b by the nomenclature committee. Both genes were genetically mapped to chromosome 13. The 1450-bp Adh8a is 82, 73, 72, and 72% similar at the amino acid level to the Baltic cod ADH8 (previously named ADH1), the human ADH1B2, the mouse ADH1, and the rat ADH1, respectively. Also, the 1484-bp Adh8b is 77, 68, 67, and 66% similar at the amino acid level to the Baltic cod ADH8, the human ADH1B2, the mouse ADH1, and the rat ADH1, respectively. ADH8A and ADH8B share 86% amino acid similarity. To characterize the functional properties of ADH8A and ADH8B, recombinant proteins were purified from SF-9 insect cells. Kinetic studies demonstrate that ADH8A metabolizes ethanol, with a V(max) of 13.4 nmol/min/mg protein, whereas ADH8B does not metabolize ethanol. The ADH8A K(m) for ethanol as a substrate is 0.7 mm. 4-Methyl pyrazole, a classical competitive inhibitor of class I ADH, failed to inhibit ADH8A. ADH8B has the capacity to efficiently biotransform longer chain primary alcohols (>/=5 carbons) and S-hydroxymethlyglutathione, whereas ADH8A does not efficiently metabolize these substrates. Finally, mRNA expression studies indicate that both ADH8A and ADH8B mRNA are expressed during early development and in the adult brain, fin, gill, heart, kidney, muscle, and liver. Together these results indicate that class I-like ADH is conserved in zebrafish, albeit with mixed functional properties.