Measurement of DNA integrity and structure in Xenopus embryos in the presence of hydroxyurea, actinomycin-D, and triethylenemelamine using the fluorescent probe Hoechst 33258.
Cell health assay of water quality (CHAWQ) is an assay using intracellular biomarkers measured by optical techniques. CHAWQ uses embryos of the South African clawed frog, Xenopus laevis, and optical transducers of intracellular biomarkers to obtain rapid assessment of toxicity to frog embryos. Since the biomarkers are common to all cells, CHAWQ can indicate toxicity of different classes of chemicals. Among the biomarkers used are 1) the change in synthesis rate, 2) the structure, or 3) the environment of DNA. Measurement of DNA to detect genotoxicants has previously used extracted DNA or flow cytometry to detect alterations in DNA content or configuration. We report the use of viable frog embryos and the fluorescent probe Hoechst 33258 to detect the effect of three DNA-active chemicals--actinomycin-D, hydroxyurea, and triethylenemelamine (TEM)--on DNA in intact embryos. We found that we can detect changes in the DNA in the presence of toxicants at concentrations comparable to longer-term assays but following a much shorter time of drug exposure. Actinomycin-D caused a fluorescence decrease, TEM caused a fluorescence increase, whereas hydroxyurea gave a biphasic response. Hydroxyurea caused a decrease at low concentrations and an increase at higher concentrations. Concentration-response data for TEM, hydroxyurea, and actinomycin-D generated EC50 values of 0.1 mg/ml, 1.4 mg/ml, and 6.34 micrograms/ml, respectively.
Department of Zoology, Oklahoma State University, Stillwater, USA.
B K
BK
Stringer
J T
JT
Blankemeyer
D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity*; D006690:Bisbenzimidazole; D002417:Cattle; D004247:DNA / Q000187:drug effects*; D003609:Dactinomycin / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D004625:Embryo, Nonmammalian / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D005456:Fluorescent Dyes; D006657:Histones / Q000187:drug effects; D006918:Hydroxyurea / Q000633:toxicity*; D008297:Male; D013723:Teratogens / Q000633:toxicity*; D013950:Thymus Gland; D014265:Triethylenemelamine / Q000633:toxicity*; D014982:Xenopus laevis / Q000196:embryology
D000970:Antineoplastic Agents; D005456:Fluorescent Dyes; D006657:Histones; D013723:Teratogens; D003609:Dactinomycin; D004247:DNA; D014265:Triethylenemelamine; D006690:Bisbenzimidazole; D006918:Hydroxyurea