Comparative embryotoxicity and proteotoxicity of three carrier solvents to zebrafish (Danio rerio) embryos.

The present study examines the effects of ethanol (ETOH), dimethyl sulfoxide (DMSO), and acetone on zebrafish embryos and the implications of the observed results on the use of these solvents to zebrafish early life stage tests. The embryos were exposed to different concentrations (0.0, 0.0001, 0.001, 0.01, 0.1, 0.05, 1, 1.5, and 2.0% v/v) of the respective solvents by diluting reagent-grade solvent with reconstituted water [DIN 38415-6-Suborganismische Testverfahren (Gruppe T) Teil 6: Giftigkeit gegenüber Fischen. Deutsches Institute für Normung e.V]. The following endpoints were investigated (mortality, hatching rate, abnormalities, heart rate, and hsp 70 induction). No effect on survival was recorded for both acetone and DMSO even up to the highest concentration. On the other hand, embryos exposed to 1.5% and 2.0% ethanol showed a significant reduction in survival rate. No developmental defects occurred with any of the solvents at the 0.1% concentration. However, starting with 1.0%, weak to very pronounced abnormalities (weak pigmentation, edema, crooked bodies, eye defect, tail defect, reduced heartbeat, and abnormal hatching) were observed depending on the solvent type and the concentration used. Ethanol has been shown to be the most embryotoxic solvent while DMSO and acetone have comparably lesser effects. Heat shock protein 70 was induced by all solvents but at different concentration ranges. DMSO has been shown to be the most potent inducer of stress proteins. Based on the study, the chemicals tested here may be used as carrier solvents in the zebrafish embryo assay at levels below 1.5, 1.5, and 1% v/v for acetone, DMSO, and ethanol, respectively. For stress protein analysis of the exposed embryos, however, the solvent levels should be below 0.1%, 0.01%, and 1.5%, respectively. Additional and separate investigations utilizing other biomarkers should be carried out to further validate the suitability of using these solvents in a typical zebrafish embryo assay.

[1]  H. Laale.  Ethanol induced notochord and spinal cord duplications in the embryo of the zebrafish, Brachydanio rerio. , 1971, The Journal of experimental zoology.

[2]  J. Rayburn,et al.  Synergism and antagonism induced by three carrier solvents with t-retinoic acid and 6-aminonicotinamide using FETAX , 1991, Bulletin of environmental contamination and toxicology.

[3]  Susan Budavari,et al.  The Merck index : an encyclopedia of chemicals, drugs, and biologicals , 1983 .

[4]  N. Love,et al.  The immunochemical detection of stress proteins in activated sludge exposed to toxic chemicals. , 2001, Water research.

[5]  H. Nawata,et al.  Enhanced synthesis of heat shock proteins and augmented thermotolerance after induction of differentiation in HL‐60 human leukemia cells , 1990, FEBS letters.

[6]  L. Hightower,et al.  Heat shock, stress proteins, chaperones, and proteotoxicity , 1991, Cell.

[7]  J. Bantle,et al.  Altered developmental toxicity caused by three carrier solvents , 1991, Journal of applied toxicology : JAT.

[8]  Itoh Hideaki,et al.  The stress (heat shock) proteins , 1991 .

[9]  L. Rensing,et al.  Heat shock response and cytotoxicity in C6 rat glioma cells: structure-activity relationship of different alcohols. , 1994, Molecular pharmacology.

[10]  Christoph Schulte,et al.  Testing Acute Toxicity in the Embryo of Zebrafish, Brachydanio rerio, as an Alternative to the Acute Fish Test: Preliminary Results , 1994 .

[11]  K. Kitchin,et al.  Further development of rodent whole embryo culture: solvent toxicity and water insoluble compound delivery system. , 1984, Toxicology.

[12]  Kayta Kobayashi,et al.  Acute effects of alcohol on larval zebrafish: a genetic system for large-scale screening , 2004, Pharmacology Biochemistry and Behavior.

[13]  P. Barr,et al.  Mammalian subtilisins: The long-sought dibasic processing endoproteases , 1991, Cell.

[14]  K. Sander,et al.  Bipartite axiation follows incomplete epiboly in zebrafish embryos treated with chemical teratogens. , 1984, The Journal of experimental zoology.

[15]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[16]  H. Köhler,et al.  Developmental and subcellular effects of chronic exposure to sub-lethal concentrations of ammonia, PAH and PCP mixtures in brown trout (Salmo trutta f. fario L.) early life stages. , 2003, Aquatic toxicology.

[17]  H. Köhler,et al.  The hepatic stress protein (hsp70) response to interacting abiotic parameters in fish exposed to various levels of pollution , 2001 .

[18]  B. Sanders,et al.  Stress proteins as biomarkers of contaminant exposure in archived environmental samples. , 1993, The Science of the total environment.

[19]  G. Dave,et al.  Toxicity of mercury, copper, nickel, lead, and cobalt to embryos and larvae of zebrafish,Brachydanio rerio , 1991, Archives of environmental contamination and toxicology.

[20]  A. Nazir,et al.  Evaluation of the No Observed Adverse Effect Level of Solvent Dimethyl Sulfoxide in Drosophila melanogaster , 2003, Toxicology mechanisms and methods.

[21]  R. Lloyd,et al.  Sublethal and chronic effects of pollutants on freshwater fish , 1994 .

[22]  S. Dyer,et al.  Synthesis and accumulation of stress proteins in tissues of arsenite‐exposed fathead minnows (Pimephales promelas) , 1993 .

[23]  A. E. Jay,et al.  Effects of organic solvents and solvent-atrazine interactions on two algae, Chlorella vulgaris and Selenastrum capricornutum , 1996 .

[24]  J. Murat,et al.  Expression of stress proteins in cultured HT29 human cell-line; a model for studying environmental aggression. , 1995, The international journal of biochemistry & cell biology.

[25]  Richard A Rabin,et al.  Ethanol effects on three strains of zebrafish: model system for genetic investigations , 2003, Pharmacology Biochemistry and Behavior.

[26]  A. Kligman.  Topical Pharmacology and Toxicology of Dimethyl Sulfoxide—Part 1 , 1965 .

[27]  R. Fujii.  The regulation of motile activity in fish chromatophores. , 2000, Pigment cell research.

[28]  K. Fent,et al.  Different expression patterns of heat shock proteins hsp 60 and hsp 70 in zebra mussels (Dreissena polymorpha) exposed to copper and tributyltin , 2000 .

[29]  U. Strähle,et al.  Ethanol impairs migration of the prechordal plate in the zebrafish embryo. , 1998, Developmental biology.

[30]  S. Saszik,et al.  Effects of embryonic exposure to ethanol on zebrafish visual function. , 2002, Neurotoxicology and teratology.

[31]  Gloria C. Li.  Induction of thermotolerance and enhanced heat shock protein synthesis in chinese hamster fibroblasts by sodium arsenite and by ethanol , 1983, Journal of cellular physiology.

[32]  G. Holcombe,et al.  Metal toxicity to embryos and larvae of seven freshwater fish species—i. cadmium , 1978, Bulletin of environmental contamination and toxicology.

[33]  R. Nagel.  DarT: The embryo test with the Zebrafish Danio rerio--a general model in ecotoxicology and toxicology. , 2002, ALTEX.

[34]  H. Satoh,et al.  Influence of Organic Solvents on the Growth of Marine Microalgae , 2001, Archives of environmental contamination and toxicology.

[35]  R. Laskowski,et al.  Heat shock proteins (HSP70) as biomarkers in ecotoxicological studies. , 1997, Ecotoxicology and environmental safety.

[36]  A. Karow,et al.  Cardiac chronotropic mechanisms of dimethyl sulphoxide: inhibition of acetylcholinesterase and antagonism of negative chronotropy by atropine. , 1976, Archives internationales de pharmacodynamie et de therapie.

[37]  V. Calabrese,et al.  Stress proteins and SH-groups in oxidant-induced cell damage after acute ethanol administration in rat. , 1996, Free radical biology & medicine.

[38]  H. Köhler,et al.  Developmental toxicity and stress protein responses in zebrafish embryos after exposure to diclofenac and its solvent, DMSO. , 2004, Chemosphere.

[39]  W. S. Fisher,et al.  Developmental Toxicity of Three Carrier Solvents Using Embryos of the Grass Shrimp, Palaemonetes pugio , 1997, Archives of environmental contamination and toxicology.

[40]  S. Henikoff,et al.  DNA Methylation Profiling Identifies CG Methylation Clusters in Arabidopsis Genes , 2005, Current Biology.

[41]  A. Abdel‐Rahman,et al.  Effects of Chronic Ethanol Feeding on Clonidine-Evoked Reductions in Blood Pressure, Heart Rate, and Their Variability: Time-Domain Analyses , 2003, Journal of Pharmacology and Experimental Therapeutics.

[42]  S. Adams,et al.  Induction of heat shock proteins, changes in liver ultrastructure, and alterations of fish behavior: are these biomarkers related and are they useful to reflect the state of pollution in the field? , 1997 .

[43]  Wolfgang Gebauer,et al.  Comparison of testing acute toxicity on embryo of zebrafish, Brachydanio rerio and RTG-2 cytotoxicity as possible alternatives to the acute fish test , 1995 .

[44]  K. Rokutan,et al.  A non-toxic heat shock protein 70 inducer, geranylgeranylacetone, suppresses apoptosis of cultured rat hepatocytes caused by hydrogen peroxide and ethanol. , 2001, Journal of hepatology.

[45]  G. Vernet,et al.  Ultrastructural response of the liver of rainbow trout, Oncorhynchus mykiss, sac-fry exposed to acetone , 1998 .

[46]  K. Brust,et al.  Toxicity of aliphatic amines on the embryos of zebrafish Danio rerio - experimental studies and QSAR , 2001 .

[47]  D. Davis,et al.  “Hormesis”—An Inappropriate Extrapolation from the Specific to the Universal , 2004, International journal of occupational and environmental health.

[48]  Todd H. Dresser,et al.  Teratogenic assessment of four solvents using the frog embryo teratogenesis assay—xenopus (FETAX) , 1992, Journal of applied toxicology : JAT.