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(KAS-1117-680545) Activity and Age-Dependent Sensitivity of in situ Hepatic and Gill CYP1A Activity in Living Medaka (Oryzias latipes), the See-Thorough Variety.
Kashiwada, S1, 4, Tatarazako, N1, Arizono, K2, Shiraishi, H1, Ozato, K3, Wakamatsu, Y3, Hinton, D4, 1 National Institute for Environmental Studies, Japan, Tsukuba, Ibaraki, Japan4 Duke University, Durham, NC, USA2 Kumamoto Prefecture University, Kumamoto, Kumamoto, Japan3 Nagoya University, Nagoya, Aichi, Japan
ABSTRACT- To develop in situ EROD measurement method, the see-through (ST II) medaka (Oryzias latipes), which has transparent body skin, was utilized. Measurement of EROD detects fluorescence from resorufin which is a major metabolite of ethoxyresorufin by CYP1A, so that ST II medaka is expected suitable for detection of fluorescence. Transparence body skin allows observation internal organs easy. Fluorescence detection of EROD activity was achieved without any disturbance in liver and gills of ST II larvae exposed to 3-methylcholanthrene (MC) and ethoxyresorufin for 24 h simultaneously. EROD activity in liver and gills was in proportion to dose of 3-methylcholanthrene (MC), and reached peak at 40 ug/L and then decreased probably due to toxicity, meanwhile, in vitro hepatic EROD activity in adult medaka was reached peak at 100 ug/L. Larvae medaka exhibited comparatively high sensitivity to chemical exposure than adult. We also tried in situ EROD measurement in adult ST II medaka but adult ST II was not suitable to detect fluorescence for thick muscle and skin. Post-hatch ST II embryo was observed intrinsic oxidation activity turn resorufin from ethoxyresorufin without MC exposure, however, it was dramatically reduced and not detected in eight weeks larvae. Further research for the reason of intrinsic background and reduction of EROD activity were not pursued. It would be assumed that post-hatch embryo stage may need more essential metabolic activity for life. No inducible hepatic in situ EROD activity was observed before hatching. It seems that liver may begin work for EROD activity after hatching. This research developed in situ EROD measurement method, which is more cruelty-free, time and cost effective, using living the see-through medaka larvae.
Key words: medaka, EROD, see-through medaka, in situ
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