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482 Neurotoxicity of 3-monochloropropane-1,2-diol in rats
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Poster Session P25. Neurotoxicity NEUROTOXICITY OF 3-MONOCHLOROPROPANE-1,2-DIOL IN RATS
K. Kim, C. Song, Y. Park, S. Koh, J. Kim, S. Kim, Y. Kim, H. Jung, D. Cho, K. Kil. Department of General Toxicology, National Institute of Toxicological Research, Korea Food and Drug Administration, Seoul 122–704, Korea 3-Monochloro-1,2-propanediol (3-MCPD) is a contaminant of acidhydrolyzed vegetable protein. Several reports have suggested that chronic exposure to 3-MCPD could produce neurotoxicity in vitro or neurobehavioral aspects of experimental animals. The present study further explored the in vitro neurotoxic effects of 0.1–100 µM 3-MCPD on PC12 and N18D3 cell lines. In addition, to investigate the effects of repeated ingestions of 3-MCPD on neurobehavioral impairments parameters in rats, motor activity, landing foot splay, and grip strength tests were preformed, following the treatment of 3-MCPD at doses of 10, 20, and 30 mg/kg/day for 11 weeks. We demonstrated that no significant neurotoxic effects in vitro and in neurobehavior were observed in the 3-MCPD-treated rats compared to saline-treated control rats, whereas, acrylamide, used as a positive control, induced significant increases of all neurobehavioral deficit parameters in both male and female rats. On the other hand, body weight gain was significantly decreased in high dose 3-MCPDtreated male rats as well as in acrylamide-treated rats. Taken together, these results suggest that 3-MCPD, at the dose levels of this study, does not produce in vitro neurotoxicity or neuromotor deficits.
hydroxytryptamine, 5-HT) and its metabolites, 5-hydroxyindoleacetic acid (5-HIAA) and 5-hydroxytryptophol (5-HTPL) in the locus coeruleus were studied in rats using the microdialysis method. Repeated administration of tryptophan (50 mg/kg, i.p.) for 3 days caused an increase in the levels of 5-HIAA, but not 5-HT, in the locus coeruleus, while administration of ethanol (1.25 g/kg) had no effect on the levels of 5-HT and its metabolites. Simultaneous administration of twice a day with tryptophan and ethanol for 3 consecutive days produced an increase of 5-HIAA level in the locus coeruleus, but not 5-HTPL level. This may imply that the induced microsomal enzymes (CYP2E1) by repeated administration of ethanol accelerate the metabolism of tryptophan. However, a significant increase in 5HTPL level induced by concurrent administration was not observed. This may be explained by the fact that an increased 5-HIAA affects only aldehyde dehydrogenase and/or CYP2E1, which is capable of metabolizing 5-HIAL to 5-HIAA because the metabolism of 5-HIAL to 5-HTPL is not promoted by brain alcohlol dehydrogenase. In addition, a time lag in the increased 5-HIAA levels between tryptophan alone and tryptophan plus ethanol was not observed. Moreover, teeth-chattering was significantly detected in the tryptophan plus ethanol-treated rats when compared with the tryptophan-treated rats, but not in the saline-treated controls. These results may suggest that the increased levels of 5-HIAA and 5-HTPL in the locus coeruleus induced by tryptophan are potentiated by ethanol, and that these levels are partly responsible for behavioral activation. 485
483
NEUROTOXICITY AND REACTIVITY OF 1,2-DIACETYLBENZENE (1,2-DAB) WITH MOTOR AND CYTOSKELETAL PROTEINS IN RAT SPINAL CORD AND SCIATIC NERVES
M.I. Sabri, S.B. Hashemi, P.S. Spencer. Center for Research on Occupational and Environmental Toxicology and Department of Neurology, Oregon Health & Science University, Portland, OR, USA Rats treated with 1,2-DAB, a gamma diketone-like aromatic hydrocarbon, develop in spinal motor neurons giant proximal axonal swellings filled with 10-nm neurofilaments (NF), a prominent early feature of neurodegeneration in amyotrophic lateral sclerosis. The underlying mechanism of NF accumulation in axonal swellings is not understood. This study investigated in vitro and in vivo the effect of 1,2-DAB or 1,3-DAB (a non-neurotoxic isomer) on motor and cytoskeletal proteins in the central and peripheral nervous system of rats. Animals were systemically treated with 20 mg/kg/day 1,2DAB, 1,3-DAB or vehicle for ten days. 1,2-DAB but not 1,3-DAB treated rats showed signs of neurotoxicity featured by blue discoloration and hind limb paralysis. Spinal cord (SC) and sciatic nerves (SN) homogenates were immunoblotted using monoclonal antibodies to kinesin, dynein, NFM and tau. Native protein bands showed 50≥75% reduction of both kinesin and NFM in SN of 1,2-DAB but not 1,3-DAB treated rats. Dynein and tau were reduced ≤50%. In SC, reduction of kinesin, NFM, dynein and tau was also detected. In vitro treatment of SC tissue with 1,2-DAB (1–10 mM) for 30 min at 370 C revealed a concentration-dependent loss of dynein > kinesin > tau. High molecular weight polymers were seen in SC treated with 5mM and 10 mM 1,2-DAB. In summary, 1,2-DAB reacts and depletes motor proteins kinesin and dynein and NFM in SN. A deficit of kinesin may cause blockade of anterograde axonal transport and thereby explains the accumulation of 10-nm NF in the proximal axons of 1,2-DAB treated animals. Supported by NIEHS grants ES10338 and ES11384, and the State of Oregon’s Worker’s Benefit Fund. 484
EFFECT OF REPEATED ADMINISTRATION OF TRYPTOPHAN AND ETHANOL ON 5-HIAA METABOLITE IN THE LOCUS COERULEUS IN RATS
K. Hoshi 1 , M. Hayashi 2 , T. Bandoh 1 . 1 Department of Clinical Pharmacology, Hokkaido College of Pharmacy, Otaru, Japan, 2 Chitose City Hospital, Pharmacy, Chitose, Japan The effects of consecutive administration of tryptophan alone or in combination of tryptophan and ethanol on serotonin (5-
NEUROLOGICAL AND NEUROPHYSIOLOGICAL FOLLOW-UP ON WORKERS WITH SEVERE CHRONIC EXPOSURE TO TOLUENE
P. Urban 1 , E. Lukáš 1 , D. Pelclová 2 , Z. Fenclová 2 , Z. Dlasková 2 . 1 National, Institute of Public Health, Prague, 2 Department of Occupational Medicine, 1st Medical Faculty, Charles University, Prague, Czech Republic Background: Since the 1980s, we have surveyed a group of 58 rotogravure printers with a very high level of exposure to toluene. The mean airborne concentration of toluene in the plant was about 2,000 mg/m3 . The blood concentration of toluene at the end of a working shift ranged from 2–26 mg/l. The group mean of the concentration of hippuric acid in urine was about 33 mmol/l. Most of the workers experienced acoustic pseudohallucinations during the repeated episodes of acute subintoxication from toluene. The plant was closed in 1992. The objective of this study was to describe the current neurological status of the former printers and its development. Subjects: In 2003, we managed to re-examine ten workers from the original group. They underwent a comprehensive clinical and laboratory check-up. This included neurological and neurophysiological examinations, the results of which are presented here. All subjects were men, aged 56±7 yrs, duration of exposure 17±6 yrs, and the mean time elapsed since exposure cessation was 12±3.5 yrs. Results: (1) Psychoorganic syndrome and/or other signs of CNS damage were found in 8 workers; (2) EEG was abnormal in 6 workers; (3) VEP abnormality was found in 3 workers; (4) The mean Bowman Color Confusion Index was 1.36±0.33. (5) Signs of a toxic polyneuropathy were observed in 3 workers. This diagnosis was confirmed by nerve conduction studies in 2 of them. (6) There was no significant change in the health status of the workers when compared with the situation in the 1980s. Conclusions: (1) Symptoms and signs compatible with the diagnosis of a chronic toxic encephalopathy could still be found in 80 % of former rotogravure printers, about twelve years after their removal from severe long-term exposure to toluene. (2) The abnormal findings did not show any significant development over time. This suggests that CNS damage due to toluene may not be fully reversible, but does not seem to be progressive, upon cessation of exposure. (3) The few observed cases of incipient peripheral polyneuropathy were attributable to alcohol abuse rather than toluene exposure. (4) On the basis of the present re-examination, compensation for a persistent occupational disease was recommended for 4 workers. Acknowledgement: The study was supported by grants MSM J13/98 111100002, 111100005, and CEZ:L31/98:23795.001.
Poster Session P25. Neurotoxicity NEUROTOXICITY OF 3-MONOCHLOROPROPANE-1,2-DIOL IN RATS
K. Kim, C. Song, Y. Park, S. Koh, J. Kim, S. Kim, Y. Kim, H. Jung, D. Cho, K. Kil. Department of General Toxicology, National Institute of Toxicological Research, Korea Food and Drug Administration, Seoul 122–704, Korea 3-Monochloro-1,2-propanediol (3-MCPD) is a contaminant of acidhydrolyzed vegetable protein. Several reports have suggested that chronic exposure to 3-MCPD could produce neurotoxicity in vitro or neurobehavioral aspects of experimental animals. The present study further explored the in vitro neurotoxic effects of 0.1–100 µM 3-MCPD on PC12 and N18D3 cell lines. In addition, to investigate the effects of repeated ingestions of 3-MCPD on neurobehavioral impairments parameters in rats, motor activity, landing foot splay, and grip strength tests were preformed, following the treatment of 3-MCPD at doses of 10, 20, and 30 mg/kg/day for 11 weeks. We demonstrated that no significant neurotoxic effects in vitro and in neurobehavior were observed in the 3-MCPD-treated rats compared to saline-treated control rats, whereas, acrylamide, used as a positive control, induced significant increases of all neurobehavioral deficit parameters in both male and female rats. On the other hand, body weight gain was significantly decreased in high dose 3-MCPDtreated male rats as well as in acrylamide-treated rats. Taken together, these results suggest that 3-MCPD, at the dose levels of this study, does not produce in vitro neurotoxicity or neuromotor deficits.
hydroxytryptamine, 5-HT) and its metabolites, 5-hydroxyindoleacetic acid (5-HIAA) and 5-hydroxytryptophol (5-HTPL) in the locus coeruleus were studied in rats using the microdialysis method. Repeated administration of tryptophan (50 mg/kg, i.p.) for 3 days caused an increase in the levels of 5-HIAA, but not 5-HT, in the locus coeruleus, while administration of ethanol (1.25 g/kg) had no effect on the levels of 5-HT and its metabolites. Simultaneous administration of twice a day with tryptophan and ethanol for 3 consecutive days produced an increase of 5-HIAA level in the locus coeruleus, but not 5-HTPL level. This may imply that the induced microsomal enzymes (CYP2E1) by repeated administration of ethanol accelerate the metabolism of tryptophan. However, a significant increase in 5HTPL level induced by concurrent administration was not observed. This may be explained by the fact that an increased 5-HIAA affects only aldehyde dehydrogenase and/or CYP2E1, which is capable of metabolizing 5-HIAL to 5-HIAA because the metabolism of 5-HIAL to 5-HTPL is not promoted by brain alcohlol dehydrogenase. In addition, a time lag in the increased 5-HIAA levels between tryptophan alone and tryptophan plus ethanol was not observed. Moreover, teeth-chattering was significantly detected in the tryptophan plus ethanol-treated rats when compared with the tryptophan-treated rats, but not in the saline-treated controls. These results may suggest that the increased levels of 5-HIAA and 5-HTPL in the locus coeruleus induced by tryptophan are potentiated by ethanol, and that these levels are partly responsible for behavioral activation. 485
483
NEUROTOXICITY AND REACTIVITY OF 1,2-DIACETYLBENZENE (1,2-DAB) WITH MOTOR AND CYTOSKELETAL PROTEINS IN RAT SPINAL CORD AND SCIATIC NERVES
M.I. Sabri, S.B. Hashemi, P.S. Spencer. Center for Research on Occupational and Environmental Toxicology and Department of Neurology, Oregon Health & Science University, Portland, OR, USA Rats treated with 1,2-DAB, a gamma diketone-like aromatic hydrocarbon, develop in spinal motor neurons giant proximal axonal swellings filled with 10-nm neurofilaments (NF), a prominent early feature of neurodegeneration in amyotrophic lateral sclerosis. The underlying mechanism of NF accumulation in axonal swellings is not understood. This study investigated in vitro and in vivo the effect of 1,2-DAB or 1,3-DAB (a non-neurotoxic isomer) on motor and cytoskeletal proteins in the central and peripheral nervous system of rats. Animals were systemically treated with 20 mg/kg/day 1,2DAB, 1,3-DAB or vehicle for ten days. 1,2-DAB but not 1,3-DAB treated rats showed signs of neurotoxicity featured by blue discoloration and hind limb paralysis. Spinal cord (SC) and sciatic nerves (SN) homogenates were immunoblotted using monoclonal antibodies to kinesin, dynein, NFM and tau. Native protein bands showed 50≥75% reduction of both kinesin and NFM in SN of 1,2-DAB but not 1,3-DAB treated rats. Dynein and tau were reduced ≤50%. In SC, reduction of kinesin, NFM, dynein and tau was also detected. In vitro treatment of SC tissue with 1,2-DAB (1–10 mM) for 30 min at 370 C revealed a concentration-dependent loss of dynein > kinesin > tau. High molecular weight polymers were seen in SC treated with 5mM and 10 mM 1,2-DAB. In summary, 1,2-DAB reacts and depletes motor proteins kinesin and dynein and NFM in SN. A deficit of kinesin may cause blockade of anterograde axonal transport and thereby explains the accumulation of 10-nm NF in the proximal axons of 1,2-DAB treated animals. Supported by NIEHS grants ES10338 and ES11384, and the State of Oregon’s Worker’s Benefit Fund. 484
EFFECT OF REPEATED ADMINISTRATION OF TRYPTOPHAN AND ETHANOL ON 5-HIAA METABOLITE IN THE LOCUS COERULEUS IN RATS
K. Hoshi 1 , M. Hayashi 2 , T. Bandoh 1 . 1 Department of Clinical Pharmacology, Hokkaido College of Pharmacy, Otaru, Japan, 2 Chitose City Hospital, Pharmacy, Chitose, Japan The effects of consecutive administration of tryptophan alone or in combination of tryptophan and ethanol on serotonin (5-
NEUROLOGICAL AND NEUROPHYSIOLOGICAL FOLLOW-UP ON WORKERS WITH SEVERE CHRONIC EXPOSURE TO TOLUENE
P. Urban 1 , E. Lukáš 1 , D. Pelclová 2 , Z. Fenclová 2 , Z. Dlasková 2 . 1 National, Institute of Public Health, Prague, 2 Department of Occupational Medicine, 1st Medical Faculty, Charles University, Prague, Czech Republic Background: Since the 1980s, we have surveyed a group of 58 rotogravure printers with a very high level of exposure to toluene. The mean airborne concentration of toluene in the plant was about 2,000 mg/m3 . The blood concentration of toluene at the end of a working shift ranged from 2–26 mg/l. The group mean of the concentration of hippuric acid in urine was about 33 mmol/l. Most of the workers experienced acoustic pseudohallucinations during the repeated episodes of acute subintoxication from toluene. The plant was closed in 1992. The objective of this study was to describe the current neurological status of the former printers and its development. Subjects: In 2003, we managed to re-examine ten workers from the original group. They underwent a comprehensive clinical and laboratory check-up. This included neurological and neurophysiological examinations, the results of which are presented here. All subjects were men, aged 56±7 yrs, duration of exposure 17±6 yrs, and the mean time elapsed since exposure cessation was 12±3.5 yrs. Results: (1) Psychoorganic syndrome and/or other signs of CNS damage were found in 8 workers; (2) EEG was abnormal in 6 workers; (3) VEP abnormality was found in 3 workers; (4) The mean Bowman Color Confusion Index was 1.36±0.33. (5) Signs of a toxic polyneuropathy were observed in 3 workers. This diagnosis was confirmed by nerve conduction studies in 2 of them. (6) There was no significant change in the health status of the workers when compared with the situation in the 1980s. Conclusions: (1) Symptoms and signs compatible with the diagnosis of a chronic toxic encephalopathy could still be found in 80 % of former rotogravure printers, about twelve years after their removal from severe long-term exposure to toluene. (2) The abnormal findings did not show any significant development over time. This suggests that CNS damage due to toluene may not be fully reversible, but does not seem to be progressive, upon cessation of exposure. (3) The few observed cases of incipient peripheral polyneuropathy were attributable to alcohol abuse rather than toluene exposure. (4) On the basis of the present re-examination, compensation for a persistent occupational disease was recommended for 4 workers. Acknowledgement: The study was supported by grants MSM J13/98 111100002, 111100005, and CEZ:L31/98:23795.001.