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Methylmercury-induced increase in intracellular calcium level of dissociated rat cerebellar granule cells
Poster Session 2F. Metals
132
two-dimensional uncertainty analysis models to evaluate the impacts of population heterogeneity, measurement error, sampling error, and bias. These analyses demonstrate that estimates of exposure error and bias are highly dependent on assumptions regarding absorption and distribution of MeHg, and on the methods used to quantify uncertainty. Such analyses can inform decisions regarding the sampling and interpretation of exposure biomarkers. (This work sponsored by USDOE 95EW55084 and USEPA R825358.)
IP2F154I
CORTICAL MERCURY CONCENTRATION IN NEONATAL RATS AFTERCONTINUED
TRANSPLACENTAL EXPOSURE G. De Visscher *1 , C. Joiris", I. Vihigi3 , M. Szente", M. Kirsch-Voiders 1 . 1Laboratory for Human Genetics, Free University ofBrussels; 2lAboratory for Ecotoxicology and Polar Ecology, Free University ofBrussels, Belgium; 3 Department of Comparative Physiology, Eotvos Lordnd University, Budapest; 4Department of Comparative Physiology, Attila Joszef University, Szeged, Hungary This study is a preliminary work within a INCO-COPERNICUS project on the genetic and neurophysiological effects in the neocortex of transplacental exposure to mercury compounds; it aims at the detailed estimation of mercury content in different tissues of transplacentally exposed to during a 24 day period after birth. A P + L + P protocol was applied: the dams were exposed through the drinking water to two equimolar and double doses of mercury chloride (HgClz) or methyl mercury chloride (CH3HgCl) leading to a transplacental exposure of the embryos, the pups were exposed first through the milk and later through their drinking water. Six nests of maximum eight pups were sacrificed and tissue samples of the neocortex, liver and muscle were taken and frozen for storage. The two latter tissues are used for comparison, the first being a metabolic and the second rather being a storage tissue for mercury compounds. The remainder of the rats was also frozen so that if necessary a complete mercury content of the animal could be measured. The tissues were analyses by atomic absorption to assess the real burden after a P + L + P exposure. The first results obtained only from the low dose methyl mercury chloride exposure and a small number of animals showed a high total content of mercury right after birth. Five days later this content showed a small decrease, but on the eighth day after birth there was a large decrease of the total amount of mercury in the tissue. From this day on a small decrease was observed for every time point up to day sixteen, when the experiment was terminated. This type of decline was found in all the tissues, but the mercury contents of the liver were much higher than those of the neocortex and liver, which were similar.
I P2F155I ROLEOF MITOCHONDRIA IN BIOTRANSFORMATION OF METHYLMERCURY
K. Hirayama *1 , A. Yasutake/, N. Mori 1 , M. Inouye''. Kumamoto University, 1 College ofMedical Science, 2National Institute for Minamata Disease, and 3Research Institute of Environmental Medicine, Japan Methylmercury (MeHg) biotransformation was suggested to occur via a reactive oxygen-mediated process. Since the main intracellular site of reactive oxygen generation is reported to be in mitochondria, the participation of mitochondria in MeHg degradation in vivo was studied. In the liver of rat injected with MeHg, the highest ratio of inorganic Hg (i-Hg) was found in the mitochondria fraction, and the inner membrane fraction showed a higher ratio of i-Hg than the entire mitochondria fraction. Inhibitors of the mitochondrial respiratory
chain (rotenone, antimycin, myxothizole and NaCN) affected MeHg degradation in incubation of liver slices and isolated mitochondria of rats treated with MeHg. By incubation with KOz-crown ether complex, MeHg was readily degraded in a hydrophobic solvent such as benzene but not in water. These results suggest that Oz,_ produced in mitochondria inner membrane might play an important role in MeHg degradation in vivo.
IP2F156I
METHYLMERCURY-INDUCED INCREASE IN INTRACELLULAR CALCIUM LEVELOF DISSOCIATED RAT CEREBELLAR GRANULE CELLS
M. Sakamoto *, A. Nakano, T. Adachi. National Institute for Minamata Disease, Japan The effects of methylmercury (MeHg) on the intracellular calcium level ([Caz+li) of dissociated rat cerebellar granule cells were examined using the fluorescent eaz+ indicator fura-2. Methylmercury was exposed in the form of methylmercury chloride (MMC at 2 tLM) and methylmercury-cysteine conjugate (MeHg-cys at 20 j.tM). MMC and MeHg-cys caused increases in [Caz+Ji, though with a different timecourse profile. The increase in [Caz+li by MMC appeared rapidly, while MeHg-cys caused a slower rise in [Caz+Ji. MMC (at 1-2 tLM) and MeHg-cys (at 10--50 tLM) caused increases in [Ca2+1i dose dependently. However, the time-to-onset of the increase in [Caz+Ji was shorter even at I tLM MMC than at 25 tLM MeHg-cys. On the other hand, the estimated MeHg uptake in the cells was higher when exposed as MeHg-cys than as MMC, suggesting that MeHg-cys should be better than MMC for the in vitro MeHg exposure to cells. Removal of Ca2+ from perfusion solution or several Ca z+ channel brokers did not affect the increase in [Caz+J; caused by 10 tLM MeHg-cys. These findings indicate that the MeHg-induced increase in [Caz+J; is not from extracellular Ca z+ through Ca z+ channels but from intracellular Caz+ store(s).
IP2F157I
PRE-EXPOSURE TO INORGANIC MERCURY DELAYS TOXICITY TO METHYL MERCURY CHLORIDE IN THE SPINALCHORD NEURON X NEUROBLASTOMA HYBRID CELLLINE(NSC-34)
L.A. Chapman. H.M. Chan *. Centre for Indigenous Peoples Nutrition and the Environment and the School ofDietetics and Human Nutrition, McGill University, Montreal, Quebec, H9X 3V9, Canada
The mechanisms of methyl mercury (MeHg) toxicity are not fully known, but environmental exposure to MeHg is increasing and represents a significant health concern among fish eating populations world-wide. Emphasis must be placed on elucidation of the mechanisms of MeHg and on understanding potential indicators of subclinical toxicity. NSC-34, a hybrid of NI8TG2 mouse neuroblastoma tumour cells fused to motor neuron enriched embryonic mouse spinal cord cells has been shown to be a good model for the study of MeHg toxicity. We used this model to study the effects of chronic pre-exposure to ZnCl z and HgClz on MeHg toxicity. Cell viability, expression of metallothionein (MT) and expression of glutathione (GSH) were measured. The decrease in viability during exposure to MeHgCl was modified by chronic exposure to HgClz and cell death was delayed at 4 oM and 8 uM MeHg treatments. MT in NSC-34 cells was not induced by ZnClz (15 uM), but was induced by HgClz (1-100 oM), Preincubation of cells with HgClz (1 oM for five passages) resulted in significantly higher induction of MT and altered the dose dependent response of cells exposed to MeHg for 48 hrs (0.25-16 uM) as measured by changes in cell viability. Preincubation with HgClz (l oM. 15 d) appeared to significantly decrease total GSH in NSC-34 cells, and though when the preincubation was followed by 40 hr of exposure to MeHg (0--8 uM) GSH levels were
132
two-dimensional uncertainty analysis models to evaluate the impacts of population heterogeneity, measurement error, sampling error, and bias. These analyses demonstrate that estimates of exposure error and bias are highly dependent on assumptions regarding absorption and distribution of MeHg, and on the methods used to quantify uncertainty. Such analyses can inform decisions regarding the sampling and interpretation of exposure biomarkers. (This work sponsored by USDOE 95EW55084 and USEPA R825358.)
IP2F154I
CORTICAL MERCURY CONCENTRATION IN NEONATAL RATS AFTERCONTINUED
TRANSPLACENTAL EXPOSURE G. De Visscher *1 , C. Joiris", I. Vihigi3 , M. Szente", M. Kirsch-Voiders 1 . 1Laboratory for Human Genetics, Free University ofBrussels; 2lAboratory for Ecotoxicology and Polar Ecology, Free University ofBrussels, Belgium; 3 Department of Comparative Physiology, Eotvos Lordnd University, Budapest; 4Department of Comparative Physiology, Attila Joszef University, Szeged, Hungary This study is a preliminary work within a INCO-COPERNICUS project on the genetic and neurophysiological effects in the neocortex of transplacental exposure to mercury compounds; it aims at the detailed estimation of mercury content in different tissues of transplacentally exposed to during a 24 day period after birth. A P + L + P protocol was applied: the dams were exposed through the drinking water to two equimolar and double doses of mercury chloride (HgClz) or methyl mercury chloride (CH3HgCl) leading to a transplacental exposure of the embryos, the pups were exposed first through the milk and later through their drinking water. Six nests of maximum eight pups were sacrificed and tissue samples of the neocortex, liver and muscle were taken and frozen for storage. The two latter tissues are used for comparison, the first being a metabolic and the second rather being a storage tissue for mercury compounds. The remainder of the rats was also frozen so that if necessary a complete mercury content of the animal could be measured. The tissues were analyses by atomic absorption to assess the real burden after a P + L + P exposure. The first results obtained only from the low dose methyl mercury chloride exposure and a small number of animals showed a high total content of mercury right after birth. Five days later this content showed a small decrease, but on the eighth day after birth there was a large decrease of the total amount of mercury in the tissue. From this day on a small decrease was observed for every time point up to day sixteen, when the experiment was terminated. This type of decline was found in all the tissues, but the mercury contents of the liver were much higher than those of the neocortex and liver, which were similar.
I P2F155I ROLEOF MITOCHONDRIA IN BIOTRANSFORMATION OF METHYLMERCURY
K. Hirayama *1 , A. Yasutake/, N. Mori 1 , M. Inouye''. Kumamoto University, 1 College ofMedical Science, 2National Institute for Minamata Disease, and 3Research Institute of Environmental Medicine, Japan Methylmercury (MeHg) biotransformation was suggested to occur via a reactive oxygen-mediated process. Since the main intracellular site of reactive oxygen generation is reported to be in mitochondria, the participation of mitochondria in MeHg degradation in vivo was studied. In the liver of rat injected with MeHg, the highest ratio of inorganic Hg (i-Hg) was found in the mitochondria fraction, and the inner membrane fraction showed a higher ratio of i-Hg than the entire mitochondria fraction. Inhibitors of the mitochondrial respiratory
chain (rotenone, antimycin, myxothizole and NaCN) affected MeHg degradation in incubation of liver slices and isolated mitochondria of rats treated with MeHg. By incubation with KOz-crown ether complex, MeHg was readily degraded in a hydrophobic solvent such as benzene but not in water. These results suggest that Oz,_ produced in mitochondria inner membrane might play an important role in MeHg degradation in vivo.
IP2F156I
METHYLMERCURY-INDUCED INCREASE IN INTRACELLULAR CALCIUM LEVELOF DISSOCIATED RAT CEREBELLAR GRANULE CELLS
M. Sakamoto *, A. Nakano, T. Adachi. National Institute for Minamata Disease, Japan The effects of methylmercury (MeHg) on the intracellular calcium level ([Caz+li) of dissociated rat cerebellar granule cells were examined using the fluorescent eaz+ indicator fura-2. Methylmercury was exposed in the form of methylmercury chloride (MMC at 2 tLM) and methylmercury-cysteine conjugate (MeHg-cys at 20 j.tM). MMC and MeHg-cys caused increases in [Caz+Ji, though with a different timecourse profile. The increase in [Caz+li by MMC appeared rapidly, while MeHg-cys caused a slower rise in [Caz+Ji. MMC (at 1-2 tLM) and MeHg-cys (at 10--50 tLM) caused increases in [Ca2+1i dose dependently. However, the time-to-onset of the increase in [Caz+Ji was shorter even at I tLM MMC than at 25 tLM MeHg-cys. On the other hand, the estimated MeHg uptake in the cells was higher when exposed as MeHg-cys than as MMC, suggesting that MeHg-cys should be better than MMC for the in vitro MeHg exposure to cells. Removal of Ca2+ from perfusion solution or several Ca z+ channel brokers did not affect the increase in [Caz+J; caused by 10 tLM MeHg-cys. These findings indicate that the MeHg-induced increase in [Caz+J; is not from extracellular Ca z+ through Ca z+ channels but from intracellular Caz+ store(s).
IP2F157I
PRE-EXPOSURE TO INORGANIC MERCURY DELAYS TOXICITY TO METHYL MERCURY CHLORIDE IN THE SPINALCHORD NEURON X NEUROBLASTOMA HYBRID CELLLINE(NSC-34)
L.A. Chapman. H.M. Chan *. Centre for Indigenous Peoples Nutrition and the Environment and the School ofDietetics and Human Nutrition, McGill University, Montreal, Quebec, H9X 3V9, Canada
The mechanisms of methyl mercury (MeHg) toxicity are not fully known, but environmental exposure to MeHg is increasing and represents a significant health concern among fish eating populations world-wide. Emphasis must be placed on elucidation of the mechanisms of MeHg and on understanding potential indicators of subclinical toxicity. NSC-34, a hybrid of NI8TG2 mouse neuroblastoma tumour cells fused to motor neuron enriched embryonic mouse spinal cord cells has been shown to be a good model for the study of MeHg toxicity. We used this model to study the effects of chronic pre-exposure to ZnCl z and HgClz on MeHg toxicity. Cell viability, expression of metallothionein (MT) and expression of glutathione (GSH) were measured. The decrease in viability during exposure to MeHgCl was modified by chronic exposure to HgClz and cell death was delayed at 4 oM and 8 uM MeHg treatments. MT in NSC-34 cells was not induced by ZnClz (15 uM), but was induced by HgClz (1-100 oM), Preincubation of cells with HgClz (1 oM for five passages) resulted in significantly higher induction of MT and altered the dose dependent response of cells exposed to MeHg for 48 hrs (0.25-16 uM) as measured by changes in cell viability. Preincubation with HgClz (l oM. 15 d) appeared to significantly decrease total GSH in NSC-34 cells, and though when the preincubation was followed by 40 hr of exposure to MeHg (0--8 uM) GSH levels were