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Toxicokinetics of cadmium in lactating ewes: Effects of a calcium dietary supplement
PosterSession 2F. Metals of change of RR' at low frequency (LF; 0.04-0.15 Hz) and high frequency (HF; 0.15-0.40 Hz). All 5 manganese steel alloy workers (grinding, welding, machining)had higher (p < 0.05 by Chi-square) InLFIInHF (l.48 ± 0.05) than 3 mild steel welders (1.32 ± 0.06), 8 age matched control males with normal cardiovascular status (1.34 ± 0.08) and published normals (1.28 ± 0.03). The contributions of exposure to copper, zinc, solvents, vibration and concurrent depression, all knownto decrease high frequency variability and to increase InLFIInHF cannot be excluded. Despite these limitations, the data from this small convenience sample suggest that power spectrum analysis of the 24 h ambulatory ECG may be a sensitive neurobiologic index that merits consideration in the health surveillance of populations exposed to manganese and other neurotoxins.
IP2F119I
OXIDATIVE STRESS: A NEUROCHEMICAL MECHANISM OF MANGANESE-INDUCED NEUROTOXICITY
S.P. Ali *, H.M. Duhart, G.W.Lipe, G.D. Newport,W. Slikker,Jr. Neurochemistry Laboratory, Division ofNeurotoxicology. National Centerfor Toxicological Research, FDA, Jefferson, AR. USA Manganese (Mn) is an essential element, the deficiency or excess of which, is known to cause neurotoxicity in experimental animals and man. The mechanism of action of Mn is still unclear. We hypothesized that Mn-induced neurochemical changes are produced via oxidative stress. Several experiments were conducted to test this hypothesis by measuring the formation of reactive oxygen species (ROS) in cerebral tissue. The fluorescent probe 2',7' -dichlorofluorescein-diacetate (DCFH-DA) was used to quantitate the formation of ROS. In vitro exposure to Mn increased the formation of ROS in brain synaptosomes of rats of different ages, however, neonatal brain was observed to be more susceptible than adult brain. In follow-up experiments two different valence states of Mn were tested. Results revealed that in vitro exposure to divalent Mn, (MnCh; 1-1000 oM) produced dose-dependent increases of ROS in striatum whereas trivalent Mn, (MnOAc; 1-1000 oM) produced similar increases at much lower concentrations. In vivo exposure to MnOAc (Mn+ 3 , 50 or 100 mg/kg, ip) produced significant increases of ROSin caudate nucleus and hippocampus,whereasonly a higher dose of MnCh (Mn+2 , 100 mg/kg, ip) produced significant effects in hippocampus. These and our previous data suggest that both divalent and trivalent manganese induce ROS, however, Mn+3 is lOO-times more potent than Mn+2 . In conclusion, these results support the hypothesis that the neurochemical changes induced by Mn may be mediated via ROS and oxidative stress.
IP2F120 I
UPTAKE ANDTRANSPORT OF CADMIUM BY CACo-2CELLS
S. Lecoeurv", A. Blais", M. Kolf-Clauw1 , J.P. Huneau2 , G. Milhaud", D. Tome2 • I Pharmacie-Toxicologie, ENVA 7 avenue du Gal de Gaulle, 94730 Maisons Alfort; 2INRA-UNHPI,INA-PG. 16 rue Claude Bernard 75231 Paris, France One of the major environmental source of cadmium, a highly toxic heavy metal, is food and drinking water. In vivo experiments have shown that only a small portion of the dietary cadmium is taken up by the intestinal mucosa and less than 1-6% of the cadmiumentering the mucosa will pass into the body. The present study was performed to further characterize cadmium accumulation and transepithelial transport. Because of limitations related to in vivo studies we chose a human intestinal cell culture model Caco-2 cells that have been used successfully to study transport of several nutrients and also essential and non essential metals. Experiments were carried out on Caco-2 cells grown on permeable support, and used after confluency, when cells express enterocytic differentiation and a high resistivity.
123
Cadmium concentration in the cells or in the media was determined by atomic absorption spectrometry. Cells were exposed to various concentrations of cadmium ranging from 0.1 to 25 /LM. Our results showed that as less as 10 /LM of cadmium in the upper compartment bathing the cells significantlydecreased resistivityof the cell monolayer after 2 hours. However in presence of 5 /LM of cadmium we did not observe any significantmodificationof the resistivityor LDH release.Time course of cadmium uptake and transepithelialtransport were measured, over the range of concentration tested both were time and concentration dependant. When cadmium was applied to cell before confluency, 48 h afterplating, as less as 0.1 /LM enhanced cells proliferation. Cells adapted to grow in presence of cadmium showed not only faster uptake rate but also higher accumulation of cadmium. Moreoverresistivitymeasurementsshowedthat these cells were able to tolerate higher concentration of the metal. This was related to induction of metallothionein synthesis. These results suggest that Caco-2 cells is a good predictive model to study intestinal absorptionconsecutiveto expositionto low doses of cadmium.
IP2F121 I
TOXICOKINETICS OF CADMIUM IN LACTATING EWES:EFFECTSOF A CALCIUM DIETARY SUPPLEMENT
Brigitte Enriquez *, Pascale Houpert, Nassim Benm'rah, Guy Milhaud. Ecole Nationale Yeterinaire d'Alfort, service de Pharmacie-Toxicologie 94704Maisons-Alfort, France Introduction: The use of sewage sludge in the pastures is the major source of intake of heavy metals by dairy cattle. So the contamination of the human consumer can happen through Cd contaminated products (meat, offal, milk). In order to protect the consumer's health, we analysed the effects of the calcium diet content on cadmium bioavailability in lactating Prealpes ewes. Material and Methods: We used 2 groups of ewes, each of them receiving the same feedstuff except for the Ca uptake (64.5 gld and 73.4 gld during the major part of lactation). Cadmium chloride was administered by intravascular (0.1 mg Cd/kg PV) and oral (gelule) route (20 mg Cd/kg PV) to each animal as boluses. The wash-out period between the two administrations was 21 days (group 1 received at first the IV injection, group 2 was administered the gelule at first). Cadmium blood concentrations levels were measured until day 216, milk levels till the end of the lactation (day 103) and urine levels till the day 36th or day 75th day of the experiment. At day 231 the ewes were sacrified and samples of liver,kidney,mammary gland, muscle and intestine were taken for Cd measurements. Results and Conclusions: It appeared that the level of calcium uptake didn't interfere with cadmium kinetics. The lactating ewes showeda low cadmiumbioavailability (0.81 ± 0.97%),a large steady state volume of distribution (50.4 ± 52.7 IIkgb.w.) and a low blood clearance (0.08 ± 0.10 IIkglh). The hepatic clearance plays a major role in the persistance of Cd in the organism. The liver keeps up to 0.15% of the dosage. The way the blood concentrationare decreasing during the "eliminationphase" gives an evidence of a entero-hepatic cycle. Converselythe fraction of the dosage eliminated through milk or urine only reached respectively 0.06 and 0.04. We also observed the changing of Cd milk clearance with time. Twoewes became ill the day after the oral bolus and were sacrified at day 80. Their symptoms, the cadmium levels in blood (8 to 10 times higher than the other ewes), the Cd urine concentration (up to -33 times higher), the retinol binding protein level (two times higher) and the Cd kidney concentration (up to 80 times higher) were in accordancewith a Cd specificacute renal toxicity.
IP2F119I
OXIDATIVE STRESS: A NEUROCHEMICAL MECHANISM OF MANGANESE-INDUCED NEUROTOXICITY
S.P. Ali *, H.M. Duhart, G.W.Lipe, G.D. Newport,W. Slikker,Jr. Neurochemistry Laboratory, Division ofNeurotoxicology. National Centerfor Toxicological Research, FDA, Jefferson, AR. USA Manganese (Mn) is an essential element, the deficiency or excess of which, is known to cause neurotoxicity in experimental animals and man. The mechanism of action of Mn is still unclear. We hypothesized that Mn-induced neurochemical changes are produced via oxidative stress. Several experiments were conducted to test this hypothesis by measuring the formation of reactive oxygen species (ROS) in cerebral tissue. The fluorescent probe 2',7' -dichlorofluorescein-diacetate (DCFH-DA) was used to quantitate the formation of ROS. In vitro exposure to Mn increased the formation of ROS in brain synaptosomes of rats of different ages, however, neonatal brain was observed to be more susceptible than adult brain. In follow-up experiments two different valence states of Mn were tested. Results revealed that in vitro exposure to divalent Mn, (MnCh; 1-1000 oM) produced dose-dependent increases of ROS in striatum whereas trivalent Mn, (MnOAc; 1-1000 oM) produced similar increases at much lower concentrations. In vivo exposure to MnOAc (Mn+ 3 , 50 or 100 mg/kg, ip) produced significant increases of ROSin caudate nucleus and hippocampus,whereasonly a higher dose of MnCh (Mn+2 , 100 mg/kg, ip) produced significant effects in hippocampus. These and our previous data suggest that both divalent and trivalent manganese induce ROS, however, Mn+3 is lOO-times more potent than Mn+2 . In conclusion, these results support the hypothesis that the neurochemical changes induced by Mn may be mediated via ROS and oxidative stress.
IP2F120 I
UPTAKE ANDTRANSPORT OF CADMIUM BY CACo-2CELLS
S. Lecoeurv", A. Blais", M. Kolf-Clauw1 , J.P. Huneau2 , G. Milhaud", D. Tome2 • I Pharmacie-Toxicologie, ENVA 7 avenue du Gal de Gaulle, 94730 Maisons Alfort; 2INRA-UNHPI,INA-PG. 16 rue Claude Bernard 75231 Paris, France One of the major environmental source of cadmium, a highly toxic heavy metal, is food and drinking water. In vivo experiments have shown that only a small portion of the dietary cadmium is taken up by the intestinal mucosa and less than 1-6% of the cadmiumentering the mucosa will pass into the body. The present study was performed to further characterize cadmium accumulation and transepithelial transport. Because of limitations related to in vivo studies we chose a human intestinal cell culture model Caco-2 cells that have been used successfully to study transport of several nutrients and also essential and non essential metals. Experiments were carried out on Caco-2 cells grown on permeable support, and used after confluency, when cells express enterocytic differentiation and a high resistivity.
123
Cadmium concentration in the cells or in the media was determined by atomic absorption spectrometry. Cells were exposed to various concentrations of cadmium ranging from 0.1 to 25 /LM. Our results showed that as less as 10 /LM of cadmium in the upper compartment bathing the cells significantlydecreased resistivityof the cell monolayer after 2 hours. However in presence of 5 /LM of cadmium we did not observe any significantmodificationof the resistivityor LDH release.Time course of cadmium uptake and transepithelialtransport were measured, over the range of concentration tested both were time and concentration dependant. When cadmium was applied to cell before confluency, 48 h afterplating, as less as 0.1 /LM enhanced cells proliferation. Cells adapted to grow in presence of cadmium showed not only faster uptake rate but also higher accumulation of cadmium. Moreoverresistivitymeasurementsshowedthat these cells were able to tolerate higher concentration of the metal. This was related to induction of metallothionein synthesis. These results suggest that Caco-2 cells is a good predictive model to study intestinal absorptionconsecutiveto expositionto low doses of cadmium.
IP2F121 I
TOXICOKINETICS OF CADMIUM IN LACTATING EWES:EFFECTSOF A CALCIUM DIETARY SUPPLEMENT
Brigitte Enriquez *, Pascale Houpert, Nassim Benm'rah, Guy Milhaud. Ecole Nationale Yeterinaire d'Alfort, service de Pharmacie-Toxicologie 94704Maisons-Alfort, France Introduction: The use of sewage sludge in the pastures is the major source of intake of heavy metals by dairy cattle. So the contamination of the human consumer can happen through Cd contaminated products (meat, offal, milk). In order to protect the consumer's health, we analysed the effects of the calcium diet content on cadmium bioavailability in lactating Prealpes ewes. Material and Methods: We used 2 groups of ewes, each of them receiving the same feedstuff except for the Ca uptake (64.5 gld and 73.4 gld during the major part of lactation). Cadmium chloride was administered by intravascular (0.1 mg Cd/kg PV) and oral (gelule) route (20 mg Cd/kg PV) to each animal as boluses. The wash-out period between the two administrations was 21 days (group 1 received at first the IV injection, group 2 was administered the gelule at first). Cadmium blood concentrations levels were measured until day 216, milk levels till the end of the lactation (day 103) and urine levels till the day 36th or day 75th day of the experiment. At day 231 the ewes were sacrified and samples of liver,kidney,mammary gland, muscle and intestine were taken for Cd measurements. Results and Conclusions: It appeared that the level of calcium uptake didn't interfere with cadmium kinetics. The lactating ewes showeda low cadmiumbioavailability (0.81 ± 0.97%),a large steady state volume of distribution (50.4 ± 52.7 IIkgb.w.) and a low blood clearance (0.08 ± 0.10 IIkglh). The hepatic clearance plays a major role in the persistance of Cd in the organism. The liver keeps up to 0.15% of the dosage. The way the blood concentrationare decreasing during the "eliminationphase" gives an evidence of a entero-hepatic cycle. Converselythe fraction of the dosage eliminated through milk or urine only reached respectively 0.06 and 0.04. We also observed the changing of Cd milk clearance with time. Twoewes became ill the day after the oral bolus and were sacrified at day 80. Their symptoms, the cadmium levels in blood (8 to 10 times higher than the other ewes), the Cd urine concentration (up to -33 times higher), the retinol binding protein level (two times higher) and the Cd kidney concentration (up to 80 times higher) were in accordancewith a Cd specificacute renal toxicity.