Stage-dependent ethoxyresorufin-O-deethylase (EROD) in vivo activity in medaka (Oryzias latipes) embryos.
Using medaka (Oryzias latipes) embryos, this study aimed to quantitatively characterize the stage-dependent in vivo ethoxyresorufin-O-deethylase (EROD) as indicator of cytochrome P4501A (CYP1A) activity. Embryos were challenged for 24-h to an agonist (β-naphthoflavone [BNF], 2.5, 5, 10, and 20 μg L(-1)) or to its combination (2.5 μg L(-1)) with an antagonist (α-naphthoflavone [ANF], 25, 50, 100, and 200 μg L(-1)), initiated at four different developmental time points (1, 3, 6, and 9 d post-fertilization [dpf]). Respective induction and competitive inhibition were evaluated over fluorescent images of whole embryo (nonorgan-specific [NOS] EROD activity) and gallbladder (organ-specific [OS] EROD activity). Both flavonoids showed signs of stability in solution. Generally speaking, the mean fluorescence intensity (MFI) values for NOS EROD increased with BNF concentration and exposure challenge. BNF co-exposure with ⩾50 μg ANF L(-1) during the 1-2 and 3-4 dpf challenges lowered NOS EROD to undetectably induced levels. Significant increments in MFIs for OS-EROD were seen from exposures to ⩾2.5 μg BNF L(-1), peaking during the 6-7 dpf challenge regardless of BNF concentration. The simultaneous BNF/ANF incubation showed competitive inhibition for OS EROD activity, although levels were generally detectably induced during all challenges and at all ANF concentrations. The morphometric in vivo gallbladder analysis indicated significant dilation in the 10 dpf-old embryos co-exposed to BNF and 200 μg ANF L(-1). This quantitative approach can be used successfully at 4 dpf at the NOS-EROD or OS-EROD levels, although the NOS-EROD response was sensitive enough to induction or inhibition, even at 2 dpf.
Laboratory for Ecotoxicology, Department of the Environment, National Institute for Agricultural and Food Research and Technology, A-6, Km. 7.5, E-28040 Madrid, Spain. Electronic address: miguelgd@inia.es.
Miguel
M
González-Doncel
Laboratory for Ecotoxicology, Department of the Environment, National Institute for Agricultural and Food Research and Technology, A-6, Km. 7.5, E-28040 Madrid, Spain.
Gregoria
G
Carbonell
Laboratory for Ecotoxicology, Department of the Environment, National Institute for Agricultural and Food Research and Technology, A-6, Km. 7.5, E-28040 Madrid, Spain.
Laura
L
San Segundo
Laboratory of Forest Soils, Department of Forest Ecology, National Institute for Agricultural and Food Research and Technology, A-6, Km. 7.5, E-28040 Madrid, Spain.
Salvador
S
Sastre
Laboratory for Ecotoxicology, Department of the Environment, National Institute for Agricultural and Food Research and Technology, A-6, Km. 7.5, E-28040 Madrid, Spain.
Eulalia M
EM
Beltrán
Laboratory for Ecotoxicology, Department of the Environment, National Institute for Agricultural and Food Research and Technology, A-6, Km. 7.5, E-28040 Madrid, Spain.
Carlos
C
Fernández-Torija
D000818:Animals; D001571:Benzoflavones; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism*; D009990:Oryzias / Q000378:metabolism*; D010078:Oxazines; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D010078:Oxazines; C007649:ethoxyresorufin; C011512:alpha-naphthoflavone; D019324:beta-Naphthoflavone; D019363:Cytochrome P-450 CYP1A1
Cytochrome P450; Fish embryo toxicity; Gallbladder; Morphometry; Quantitative fluorescence; Risk assessment