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Developmental neurotoxicity testing: Locomotor activity in zebrafish an alternative?
Abstracts / Toxicology Letters 196S (2010) S37–S351
P207-008 Melatonin increases gene expression of superoxide dismutase and catalase in brain of APP transgenic mice after chronic exposure to aluminum T. García, M. Gómez, J.L. Esparza, M. Cabré, J.L. Domingo Universitat Rovira i Virgili, Spain Aluminum (Al) has been associated with pro-oxidant effects, as well as with various serious neurodegenerative disorders such as Alzheimer’s disease (AD). On the other hand, melatonin (Mel) is a well known antioxidant, which can directly act as free radical scavenger, or indirectly by inducing the expression of some genes linked to the antioxidant defence. In this study, 5-months old APP female transgenic (Tg2576) (Tg) and wild-type mice were fed with Al lactate supplemented in the diet (1 mg Al/g diet). Concurrently, animals received also oral Mel (10 mg/kg) until the end of the study at 11 months of age. Four treatment groups were included for both Tg and wild-type mice: control, Al only, Mel only, and Al + Mel. At the end of the treatment period, cortex, cerebellum and hippocampus were removed and processed to analyze the gene expression of CuZnSOD, and CAT by real-time RT-PCR. The results of the current study suggest that melatonin exerted an antioxidant action by inducing synthesis of new protein, with an upregulation in the gene expression of the antioxidant enzymes CuZnSOD and CAT evaluated in presence of Al and Mel, independently on the animal model. doi:10.1016/j.toxlet.2010.03.736
P207-009 The proximal axonopathy caused by 3,3-imnodipropionitrile (IDPN) in rats is not associated with a disto-proximal advancement of neurofilament depletion in motor terminals 1 , N. Garcia 2 , J. C. Soler-Martín 1 , Ú. Vilardosa 1 , S. Saldana-Ruíz ˜ 1 Llorens 1
Universitat de Barcelona, Spain, 2 Universitat Rovira i Virgili, Spain
IDPN causes an axonopathy in which neurofilaments accumulate in swollen proximal parts of axons of large myelinated neurones. This proximal axonopathy has been associated to an impairment of the slow component of the axonal transport, but the molecular mechanisms involved have not been identified. The slow anterograde transport of neurofilaments is now known to be the end result of both anterograde and retrograde movements that alternate with long pauses. Proximal accumulation of neurofilaments could result from a shift in the dominant direction of the movement, from anterograde to retrograde. In a previous study, we demonstrated loss of neurofilaments in the vestibular afferent endings of rats exposed to IDPN, suggesting that IDPN could really change the direction of the net transport. If so, a progressive loss of neurofilaments should occur in a disto-proximal order in nerve terminals of all affected neurons at the early stages of IDPN exposure. To test this prediction, adult male Long-Evans rats were exposed to 0 or 15 mM in the drinking water for 1, 3 or 5 weeks. The Levator auris longus (LAL) muscle was used to assess by immunohistochemistry the neurofilament distribution in motor nerve terminals. Muscles were immunolabeled with anti-neurofilament, and anti-PGP9.5, anti-S100 or anti-syntaxin antibodies. Acetylcholine receptors were stained with fluorescent conjugates of alpha-bungarotoxin. Muscles were examined by confocal microscopy. The predicted disto-proximal progressive loss of neurofilaments was not observed. Although some motor end plates
S219
(<20%) from treated animals lacked the neurofilament network that characterizes normal end plates, neurofilaments persisted in the entire final segment of the axon, in contrast to what occurred in vestibular afferents. The results do not support the hypothesis that IDPN acts by changing the predominant direction of neurofilament transport from anterograde to retrograde. Work supported by the Ministry of Science and Innovation, Spain (BFU2006-0343). doi:10.1016/j.toxlet.2010.03.737
P207-010 Developmental neurotoxicity testing: Locomotor activity in zebrafish an alternative? I. Waalkens-Berendsen 1 , C. De Esch 1 , H. Van Der Linde 2 , D. De Groot 1 , R. Willemsen 2 , A. Wolterbeek 1 1
TNO Quality of Life, Netherlands, 2 Erasmus MC, Netherlands
An increased demand on developmental neurotoxicity testing for screening and regulatory purposes is noticed. The zebrafish has a number of characteristics to make it a powerful alternative for this testing. In Zebrafish certain stereotypic behaviour has been characterised and several behavioural assessments have been developed. In this experiment the general (motor) activity pattern of individual zebrafish larvae was studied in periods of light and in darkness using a 96-well microtiter plate and a video-tracking system. First, locomotion of zebrafish larvae of different ages (Zodiac strain, 5, 6 and 7 days post-fertilization (dpf)) was being studied, followed by a comparison of the activity of different wild type strains (Zodiac, AB and TL, 6 dpf). Finally, locomotion was measured in zebrafish larvae (AB strain, 6 dpf) after exposure to different concentrations of ethanol. After 10–15 min acclimatisation in the dark, activity was recorded in visible light for 10 min followed by 10 min period of darkness and finally by another 10 min of light. Locomotor activity was initially low in the first period of light but increased to a more or less steady state after 10 min. This was followed by a sharp increase in activity when the light was turned off, followed by a sharp decrease in activity when returned to light again. Small differences in activity where found between larvae of different ages; six day old zebrafish showed the highest activity throughout the whole session. In addition, the activity of Zodiac larvae was significantly higher in both light periods, while the AB strain larvae showed a markedly lower activity throughout the whole session. Finally, acute ethanol increased the locomotor activity at 2% and severely decreased activity at 4%. These results showed that locomotor activity is a reliable measure of (impaired) neurodevelopment in zebrafish. doi:10.1016/j.toxlet.2010.03.738
P207-008 Melatonin increases gene expression of superoxide dismutase and catalase in brain of APP transgenic mice after chronic exposure to aluminum T. García, M. Gómez, J.L. Esparza, M. Cabré, J.L. Domingo Universitat Rovira i Virgili, Spain Aluminum (Al) has been associated with pro-oxidant effects, as well as with various serious neurodegenerative disorders such as Alzheimer’s disease (AD). On the other hand, melatonin (Mel) is a well known antioxidant, which can directly act as free radical scavenger, or indirectly by inducing the expression of some genes linked to the antioxidant defence. In this study, 5-months old APP female transgenic (Tg2576) (Tg) and wild-type mice were fed with Al lactate supplemented in the diet (1 mg Al/g diet). Concurrently, animals received also oral Mel (10 mg/kg) until the end of the study at 11 months of age. Four treatment groups were included for both Tg and wild-type mice: control, Al only, Mel only, and Al + Mel. At the end of the treatment period, cortex, cerebellum and hippocampus were removed and processed to analyze the gene expression of CuZnSOD, and CAT by real-time RT-PCR. The results of the current study suggest that melatonin exerted an antioxidant action by inducing synthesis of new protein, with an upregulation in the gene expression of the antioxidant enzymes CuZnSOD and CAT evaluated in presence of Al and Mel, independently on the animal model. doi:10.1016/j.toxlet.2010.03.736
P207-009 The proximal axonopathy caused by 3,3-imnodipropionitrile (IDPN) in rats is not associated with a disto-proximal advancement of neurofilament depletion in motor terminals 1 , N. Garcia 2 , J. C. Soler-Martín 1 , Ú. Vilardosa 1 , S. Saldana-Ruíz ˜ 1 Llorens 1
Universitat de Barcelona, Spain, 2 Universitat Rovira i Virgili, Spain
IDPN causes an axonopathy in which neurofilaments accumulate in swollen proximal parts of axons of large myelinated neurones. This proximal axonopathy has been associated to an impairment of the slow component of the axonal transport, but the molecular mechanisms involved have not been identified. The slow anterograde transport of neurofilaments is now known to be the end result of both anterograde and retrograde movements that alternate with long pauses. Proximal accumulation of neurofilaments could result from a shift in the dominant direction of the movement, from anterograde to retrograde. In a previous study, we demonstrated loss of neurofilaments in the vestibular afferent endings of rats exposed to IDPN, suggesting that IDPN could really change the direction of the net transport. If so, a progressive loss of neurofilaments should occur in a disto-proximal order in nerve terminals of all affected neurons at the early stages of IDPN exposure. To test this prediction, adult male Long-Evans rats were exposed to 0 or 15 mM in the drinking water for 1, 3 or 5 weeks. The Levator auris longus (LAL) muscle was used to assess by immunohistochemistry the neurofilament distribution in motor nerve terminals. Muscles were immunolabeled with anti-neurofilament, and anti-PGP9.5, anti-S100 or anti-syntaxin antibodies. Acetylcholine receptors were stained with fluorescent conjugates of alpha-bungarotoxin. Muscles were examined by confocal microscopy. The predicted disto-proximal progressive loss of neurofilaments was not observed. Although some motor end plates
S219
(<20%) from treated animals lacked the neurofilament network that characterizes normal end plates, neurofilaments persisted in the entire final segment of the axon, in contrast to what occurred in vestibular afferents. The results do not support the hypothesis that IDPN acts by changing the predominant direction of neurofilament transport from anterograde to retrograde. Work supported by the Ministry of Science and Innovation, Spain (BFU2006-0343). doi:10.1016/j.toxlet.2010.03.737
P207-010 Developmental neurotoxicity testing: Locomotor activity in zebrafish an alternative? I. Waalkens-Berendsen 1 , C. De Esch 1 , H. Van Der Linde 2 , D. De Groot 1 , R. Willemsen 2 , A. Wolterbeek 1 1
TNO Quality of Life, Netherlands, 2 Erasmus MC, Netherlands
An increased demand on developmental neurotoxicity testing for screening and regulatory purposes is noticed. The zebrafish has a number of characteristics to make it a powerful alternative for this testing. In Zebrafish certain stereotypic behaviour has been characterised and several behavioural assessments have been developed. In this experiment the general (motor) activity pattern of individual zebrafish larvae was studied in periods of light and in darkness using a 96-well microtiter plate and a video-tracking system. First, locomotion of zebrafish larvae of different ages (Zodiac strain, 5, 6 and 7 days post-fertilization (dpf)) was being studied, followed by a comparison of the activity of different wild type strains (Zodiac, AB and TL, 6 dpf). Finally, locomotion was measured in zebrafish larvae (AB strain, 6 dpf) after exposure to different concentrations of ethanol. After 10–15 min acclimatisation in the dark, activity was recorded in visible light for 10 min followed by 10 min period of darkness and finally by another 10 min of light. Locomotor activity was initially low in the first period of light but increased to a more or less steady state after 10 min. This was followed by a sharp increase in activity when the light was turned off, followed by a sharp decrease in activity when returned to light again. Small differences in activity where found between larvae of different ages; six day old zebrafish showed the highest activity throughout the whole session. In addition, the activity of Zodiac larvae was significantly higher in both light periods, while the AB strain larvae showed a markedly lower activity throughout the whole session. Finally, acute ethanol increased the locomotor activity at 2% and severely decreased activity at 4%. These results showed that locomotor activity is a reliable measure of (impaired) neurodevelopment in zebrafish. doi:10.1016/j.toxlet.2010.03.738