Effect of diazinon on behavior of Japanese medaka (Oryzias latipes) and gene expression of tyrosine hydroxylase as a biomarker

This paper reports on the development of a biomarker used to monitor abnormal behaviors caused by diazinon in Japanese medaka (Oryzias latipes) as a model organism. Tyrosine hydroxylase (TH) activity in tissues was measured and the TH enzyme production in specific organs using a in situ cytochemical technique was monitored. These data were comparatively analyzed with those from semi-quantitative RT-PCR utilizing medaka TH gene that could be a potential biomarker for neuronal modulations and behaviors. For monitoring experiments at behavioral and molecular biological levels, the fish were treated under different sublethal conditions of diazinon (O, O-diethyl O-[6-methyl-2-(1-methylethyl)-4-pyrimidinyl] phosphorothioate) and their behavioral responses were observed. There were no significant differences in activity of TH head and body portions when the fish were exposed to lower concentrations (0.5-10 ppb) of diazinon including control treatment (0 ppb) for 24 hr. In temporal change of TH activity at 100 ppb diazinon treatment, however, the activity of body portion appeared to be inhibited during the first 30 min exposure but later seemed to recover slightly after 1 hr. TH appeared to be expressed mainly in the olfactory bulb, midbrain and brain stem regions as assessed by in situ immunohistochemistry. The treatment (1000 ppb) significantly suppressed TH protein production in the olfactory bulb, midbrain and brain stem regions. In kidney from the body portion the higher concentration treatment (1000 ppb) caused little suppression compared with the control. The RT-PCR showed that a production of TH mRNA transcript was significantly inhibited at 5 ppm diazinon treatment in the body portion. It was concluded that a suppression of TH activity would be one of the causes for the abnormal behaviors of the medaka that could be quantitatively monitored using an image processing system. This study provides molecular and neurobehavioral bases of a biomonitoring system for toxic chemicals using a model organism such as fish.