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Lead induced thiamine deficiency in the brain decreased the threshold of electroshock seizure in rat
Toxicology 133 (1999) 105 – 113
Lead induced thiamine deficiency in the brain decreased the threshold of electroshock seizure in rat Jae Hoon Cheong a, Dong Ook Seo b, Jae Ryun Ryu b, Chan Young Shin b, Yun Tae Kim b, Hyoung-Chun Kim c, Won-Ki Kim d, Kwang Ho Ko b,* b
a College of Pharmacy, Samyook Uni6ersity, Seoul 139 -742, South Korea Centre for Biofunctional Molecules, POSTECH, Laboratory of Pharmacology, College of Pharmacy, Seoul National Uni6ersity, San 56 -1, Shillim-Dong, Kwanak-Gu, Seoul 151 -742, South Korea c College of Pharmacy, Kang Won National Uni6ersity, Chunchon 200 -701, South Korea d Department of Pharmacology, College of Medicine, Di6ision of Neuroscience, Medical Research Center, Ewha Women’s Uni6ersity, Seoul 158 -710, South Korea
Received 9 October 1998; accepted 15 January 1999
Abstract Many neurological disorders that occur frequently in lead intoxicated animals, have also been observed in thiamine deficient animals. To test whether lead intoxication could decrease the thiamine status and thresholds of electroshock seizure in rats, 3-week-old Wistar rats were treated with lead or lead plus thiamine. For comparison, a thiamine deficient group was included. Thiamine contents and transketolase activity, one of the thiamine dependent enzymes in the brain regions were significantly lowered by lead intoxication and thiamine deficiency. In both cases, thresholds of the electroshock seizure were significantly decreased. Thiamine supplementation reversed these signs and decreased the brain lead concentration in the lead treated group. The results from the present study suggest that the increased seizure susceptibility induced by lead intoxication in rats may be mediated at least in part through the changes of thiamine status. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lead; Thiamine; Transketolase; Rat brain; Seizure threshold
1. Introduction
* Corresponding author. Tel.: +82-2-8807848; fax: +82-28858211. E-mail address: [email protected] (K. Ho Ko)
Although acute lead poisoning has declined dramatically in recent years, poisoning by chronic and lower level exposures to lead is still a big health problem. Moreover, chronic intoxication is
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J. Hoon Cheong et al. / Toxicology 133 (1999) 105–113
more dangerous than acute poisoning because in many cases, chronic intoxication may cause more severe and even irreversible damage. Among the various tissues affected by lead poisoning, the central nervous system is the most sensitive target (Alperstein et al., 1991; Goyer, 1992). Encephalopathy characterized by demyelination, abnormal conduction velocity, decrement of IQ score and seizures is among the frequently observed complications of lead poisoning. Seizures are the most severe signs of lead poisoning. Alterations of seizure threshold and seizure responsiveness in lead exposed rats have been reported (Silbergeld et al., 1979). Significant reductions of the doses required to produce seizures were found in lead-exposed rats for the convulsant drugs such as picrotoxin and strychnine. The intensity of maximal electroshock seizures was increased in lead-exposed rats (Silbergeld and Hruska, 1980). However, the mechanism of lead neurotoxicity such as seizure and demyelination was not clear. It has been suggested that lead toxicity may be mediated through interactions between lead and endogenous substances with high affinity to lead. Several researchers focused on the molecules containing –SH group (Needleman and Bellinger, 1991). Thiamine, which is known as vitamin B1, is an endogenous –SH containing molecule. In addition, it has been reported that thiamine can reduce the absorption of lead in gastrointestinal tract and enhance the elimination of lead from soft tissues including brain, kidney and liver (Ghazaly, 1991). Neurological defects such as peripheral neuritis and encephalopathy that occur frequently in lead intoxicated animals, have also been observed in thiamine deficient animals. Therefore, it may be hypothesized that lead induced neurological defects are mediated at least in part through the alterations of thiamine status. In the present study, we investigated possible relationship between alterations of seizure sensitivity by lead intoxication and thiamine status. Although, most of the current problems regarding lead intoxication to date revolve around chronic and lower level exposures, for the mechanistic study of lead intoxication, we employed relatively acute and high level exposure to lead in the
present study with some obvious reasons. Less time and fewer animals are required for the experimentation, and the alterations of factors related to lead intoxication such as thiamine status may be more clearly investigated. The data obtained from the acute poisoning study may be extrapolated to the cases of chronic and lower level of lead exposures and also give impulses for the chronic studies in human as well as in animals. In this study, we examined whether lead administration influences the thiamine content and thiamine dependent biochemical reaction such as transketolase activity in the brain of 3-week-old rats, and whether the changes of the seizure threshold induced by lead intoxication is related to the changes of thiamine status. It was also tested whether administration of excessive thiamine could reverse the toxic manifestation of lead in the lead intoxicated rats.
2. Methods
2.1. Materials Lead acetate was purchased from Yakuri Chemical (Osaka, Japan). Thiamine tetrahydrofurfuryl disulfide (99.8%) was kindly donated by Il-Dong Pharmaceutical Co. (Seoul, South Korea). Thiamine deficient diet was purchased from Sankyo Farm Co., (Sendai, Japan). The contents of thiamine in the thiamine deficient diet was below the detection limit (0.25 mg/kg of rat chow) while those in normal diet was 7.29 mg of thiamine per 1 kg of rat chow. No constituents other than thiamine were altered in the thiamine deficient diet. All other reagents including thiamine hydrochloride and thiamine pyrophosphate were purchased from Sigma (St. Louis, MO).
2.2. Animal treatment Wistar rats were supplied from the Laboratory Animal Center of Seoul National University (Seoul, South Korea). Four groups of 3-week-old rats were used; (1) control group; (2) lead treated group; (3) lead plus thiamine treated group; and (4) thiamine deficient group. Until experimental
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animals became 3-week-old, lead and/or thiamine were administered to them through milk by giving lead and/or thiamine to their dams. Dams of the control group received a normal diet and tap water, and those of lead treated group received a normal diet and deionized and distilled water containing 0.2% lead acetate. Lead was not detected in the normal rat chow and the concentration of lead in the drinking water was reported to be below 0.04 ppm (Seoul Metropolitan Government, South Korea). Dams of the lead plus thiamine treated group were fed a thiamine sufficient diet containing 152.39 mg thiamine tetrahydrofurfuryl disulfide per 1 kg of rat chow ad libitum, and given deionized and distilled water containing 0.2% lead acetate. Dams of the thiamine deficient group were fed a thiamine deficient diet and tap water. All the treatments to dams were initiated upon parturition. Five dams were used for each treatment group. Only four animals were randomly selected from each dam and the brains were pooled. For each experimental group, litters from five different dams, namely five replications were employed. For the determination of lead concentration and thiamine status in rat brain, animals were sacrificed by decapitation. Brains were rapidly removed and dissected into three regions; telencephalon, brain stem (diencephalon/midbrain and pons/medulla), and cerebellum by the method of Glowinski and Iverson (1966) just before the determinations. 3. Measurement of lead concentration Separated brain tissues were lyophilized and digested overnight in an acid mixture of nitric acid and sulfuric acid (1:1, v/v). To the resulting yellowish clear solution with ten volumes of perchloric acid and nitric acid were added (4:1, v/v), and heated on a hot plate to remove organic materials and sulfuric acid. Lead concentration was measured by ICP (inductively coupled plasma) Mass spectrometry (VG Plasmaquad) (Taylor and Garbino, 1988). Nominal recoveries for lead were 9791.2% and recovery efficiency for lead was determined at 1 ppm. The lower limit of sensitivity was 0.1 mg/ml and
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lead concentration was expressed as ng/g wet tissue.
4. Quantitation of thiamine content Thiamine content in brain tissue was measured by the thiochrome method (Rindi and deGiuseppe, 1961). Briefly, isolated brain tissues were homogenized in ice-cold 5% trichloroacetic acid, centrifuged, and the resulting supernatant fraction was digested by a-amylase at 48–50°C for 30 min. After the supernatant was washed with water-saturated ethylether, cyanogen bromide and ice-cold 15% sodium hydroxide was added to produce thiochrome and further extracted by isobutanol. Fluorescence intensity of thiochrome was measured using a spectrofluorometer (FP-777, Jasco International Co. Ltd.) at 425 nm with exciting at 358 nm (Edwin, 1979) and was used to calculated thiamine contents. Thiamine contents were expressed as mg/g wet tissue.
5. Determination of transketolase activity Transketolase activity was estimated by measuring the rate of sedoheptulose-7-phosphate formation during the incubation of tissue homogenate in the presence of excess ribose-5phosphate substrate (Dreyfus and Moniz, 1962). Brain tissues were homogenized in ten volumes of ice-cold 0.04 M glycylglycine hydrochloride buffer, pH 7.6. The enzyme reaction was started by addition of 24 mM ribose-5-phosphate substrate to the homogenate and continued at 37.5°C for 30 min. After the reaction was terminated by the addition of 20% TCA, the mixture was centrifuged, and a clear and colorless supernatant fraction was obtained. An aliquot of the supernatant was desiccated under vacuum and the resulting residue was dissolved in redistilled water/concentrated sulfuric acid (2.5/6, v/v). Subsequently, the resulting solution was heated at 100°C for 4 min, with thorough agitation, and 3% aqueous cystein solution was added with shaking. Differences in absorbance between
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510 and 540 nm was measured spectrophotometrically and converted to total heptose concentration using known concentrations of sedoheptulose as standards. The activities of transketolase were expressed as nmol heptulose formed/min per mg protein. Protein content of tissue homogenate was measured by the method of Lowry et al. (1951) using bovine serum albumin as standard.
6. Measurement of electroshock seizure threshold Seizure was evoked by constant current stimulator and the resulting seizure was determined by overt hindlimb extension. To determine the electroshock seizure threshold, convulsive current 50 (CC50) which elicits convulsion in 50% of animals was calculated by a ‘staircase’ procedure (Browning et al., 1990). Individual animal was treated with electroshocks of 0.2 s stimulus duration to determine the current – convulsion relationship. If an animal showed convulsion, the next animal was given with 2 mA decrements in current intensity. If an animal did not show convulsion, the next animal was given with 2 mA increments in current intensity. In this way, the current – convulsion relationship was generated and CC50 value was determined by Litchfield – Wilcoxons II method (Litchfield and Wilcoxon, 1949). For each treatment group, 20 pups from five different dams were prepared and the animals were sacrificed right after the determination of the electroshock threshold.
7. Statistical analysis Data were expressed as the mean9 S.E.M. For statistical evaluation of data, One-way ANOVA was used. When statistically significant differences were found, Newman–Keuls’ test was used as a post-hoc test to determine the statistical differences between groups. For the statistical evaluation of the differences in CC50 values, Litchfield–Wilcoxon II method was employed. Differences were considered statistically significant when PB 0.05 was found.
8. Results
8.1. Measurement of lead concentration Lead concentrations in each brain region were summarized in Table 1. Lead concentrations in all brain regions of the lead treated group were higher than those of the control group, and those of the lead plus thiamine treated group were significantly decreased from those of the lead treated group.
9. Quantitation of thiamine content Thiamine contents in each brain region were shown in Fig. 1. Thiamine contents in all brain regions of the lead treated group and the thiamine deficient group were lower than those of the con-
Table 1 Lead concentration in brain regions of 3-week-old ratsa Lead concentration (mg/g wet tissue)
Telencephalon Brain stem Cerebellum
Control group
Lead treated group
Lead plus thiamine treated group
Thiamine deficient group
0.36 9 0.03 0.33 9 0.03 0.37 9 0.05
1.099 0.09b 1.3390.08b 0.929 0.06b
0.79 9 0.06y 0.69 90.04y 0.70 9 0.03x
0.31 9 0.05 0.30 9 0.08 0.28 9 0.09
Each value represents the mean 9S.E.M. (n = 5). One-way ANOVA revealed a significant difference (PB0.01) among the groups in each brain regions examined (df= 19, F value = 41.8 for telencephalon, 98.1 for brain stem and 40.3 for cerebellum, respectively). b Indicates a significant difference from the control group which determined by Newman–Keuls’ test as described in Section 2 (a, PB0.01). x and y indicate a significant difference from the lead treated group (x PB0.05 and y PB0.01). a
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11. Electroshock seizure threshold Electroshock seizure threshold was shown in Fig. 3. Electroshock seizure thresholds of the lead treated group and thiamine deficient group in 3-week-old rats were significantly lower than those of the control group, and thiamine treatment reversed this decrease back to the level of the control group.
12. Discussion In the present study, the relationship between lead intoxication and thiamine deficiency was in-
Fig. 1. Total thiamine contents in brain regions of 3-week-old rats. Each bar represents the mean 9 S.E.M (n= 5). Panels A, B and C show the results obtained from telencephalon, brain stem and cerebellum, respectively. One-way ANOVA revealed statistically significant differences (P B 0.01) among the groups in each brain regions examined (df =19, F= 31.46, 85.03 and 18.61 for A, B and C, respectively). *indicates a significant difference from the control group which determined by Newman–Keuls’ test as described in Section 2 (*PB0.05 and **PB 0.01). + indicates a significant difference from the lead treated group ( + PB 0.05 and + +P B 0.01). , control group, , lead treated group, b, lead plus thiamine treated group; d, thiamine deficient group.
trol group. In the lead plus thiamine treated group, thiamine contents were recovered to those of the control group.
10. Determination of transketolase activity Activities of transketolase in each brain region were shown in Fig. 2. The activities of transketolase of the lead treated group as well as the thiamine deficient group were significantly lower than those of the control group. In the lead plus thiamine treated group, transketolase activities were higher than those of the lead treated group.
Fig. 2. Transketolase activities in brain regions of 3-week-old rats. Each bar represents the mean9S.E.M. (n = 5). Panels A, B and C show the results obtained from telencephalon, brain stem and cerebellum, respectively. One-way ANOVA revealed statistically significant differences (PB0.01) among the groups in each brain regions examined (df = 19, F= 14.7, 53.5 and 88.5 for A, B and C, respectively). *indicates a significant difference from the control group which determined by Newman – Keuls’ test as described in Section 2 (*PB0.05 and **PB0.01). +indicates a significant difference from the lead treated group ( +PB 0.05 and + +PB 0.01). , control group, , lead treated group, b, lead plus thiamine treated group, d, thiamine deficient group.
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Fig. 3. Effects of thiamine on lead induced decrease of electroshock seizure threshold in 3-week-old rats. Each bar represents CC50 +upper C.L. (95% confidence limits). *indicates a significant difference from control group (P B0.05). +indicates a significant difference from lead-treated group (PB 0.05). CON; control group, LEAD; lead treated group, LEAD+THIAMINE; lead plus thiamine treated group, THIAMINE DEF; thiamine deficient group.
vestigated. Three-week-old, neonatal rats were used in this study, because lead toxicity to nervous system is generally more severe in young animals. In addition, it has been reported that manifestations of childhood lead poisoning are sustained to the adult period (Needleman et al., 1990) In the present study, contents of thiamine in the brain of rats were decreased by lead intoxication (Fig. 1). The reduced thiamine content in the brain may induce pathological consequences since thiamine is an essential factor in brain energy metabolism and biosynthetic pathways (Gaitonde et al., 1983; Gibson et al., 1984; Parker et al., 1984). In addition, thiamine is involved in the maintenance of normal membrane function and nerve conduction. Thiamine depletion affects neurons and their functions in selected areas of the central nervous system (Cooper and Pincus, 1979). Neurological defects, such as peripheral neuritis and encephalopathy, have been recognized in association with thiamine deficiency (Takashashi, 1981). Transketolase activities in the brain were decreased by lead intoxication (Fig. 2). Transketolase, one of the thiamine dependent enzymes, is
distributed in brain as well as liver, erythrocytes and leukocytes (Gaitonde et al., 1983). Among the thiamine dependent enzymes, transketolase activity is the most sensitive to thiamine deficiency (Friedrich, 1988). Transketolase activity has been widely used as a biochemical parameter to assess thiamine status in tissues (Friedrich, 1988; Hass, 1988). Transketolase is one of the enzymes involved in the hexose monophosphate (HMP) shunt generating NADPH required for lipid synthesis. Transketolase and possibly the entire HMP shunt are important in the development and maintenance of the myelin sheath (Clarke and Sokoloff, 1993) and the oligodendroglial metabolism in the CNS (Dreyfus and Moniz, 1962). The reduced transketolase activity induced by lead intoxication observed in the present study may alter these functions in the brain. Other thiamine dependent biochemical factors such as pyruvate dehydrogenase and a-ketoglutarate dehydrogenase, although less sensitive to thiamine deficiency than transketolase, may also contribute to energy impairment and morphological changes in thiamine deficiency. We suppose that the depletion of thiamine by lead intoxication resulted in decrement
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of transketolase activity as well as other neurochemical changes. The neurochemical changes caused by thiamine deficiency may mediate some of the neural symptoms of lead intoxication including the increased sensitivity to seizure. Electroshock seizure threshold was decreased by lead intoxication (Fig. 3). The lowering of electroshock seizure threshold by lead intoxication accompanied the concomitant decrement of thiamine content in the brain. The magnitude of decrement of electroshock seizure threshold induced by lead intoxication was nearly identical with that accompanied by thiamine deficiency. These results suggest that the changes of electroshock seizure threshold induced by lead intoxication may be related, at least in part, to the reduction of thiamine status in brain. It has been reported that reduced body weight may decrease the threshold for electroshock seizure. The body weight of the experimental groups were as follow; 5897.14 for control group, 34.7795.96 for lead treated group, 35.479 9.66 for lead plus thiamine treated group and 31.8497.80 for thiamine deficient group. The body weight and the threshold for electroshock seizure of lead treated group and thiamine deficient group was significantly lowered compared with control group. However, in this study, the threshold for electroshock seizure was not statistically different between control group and lead plus thiamine treated group in spite of the significant differences in the body weight between the two groups. The result may suggest that in this particular experiment the reduction of body weight would not be a main factor for the decreased seizure threshold in lead treated group and thiamine deficient group. Factors which influence the development of electroshock seizure pattern include synaptogenesis, myelination, maturation of monoaminergic systems, and electrolyte and amino acid changes in rat brain (Fox, et al., 1979). Demyelination and delays in myelin formation have been reported following lead intoxication and thiamine deficiency (Krigman et al., 1974; Wiggins, 1982). It has been demonstrated that the intensity of maximal electroshock seizures was increased in lead exposed rats (Silbergeld and Hruska, 1980).
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Taken together, the changes of electroshock seizure threshold and intensity induced by lead intoxication may be related to the changes of myelin formation due to thiamine deficiency. The decreased level of thiamine content and transketolase activity in the brain of the lead treated group returned toward control level in the lead plus thiamine treated group (Figs. 1 and 2). It has been reported that administration of excessive thiamine eliminates lead from soft tissues in lead intoxicated rodents (Ghazaly, 1991; Kim et al., 1991). It has been suggested that thiamine may facilitate the removal of lead from body fluids and other tissues by the formation of readily excretable complexes (Flora and Tandon, 1986). Alternatively, dietary thiamine may interfere with the absorption of lead into tissues by currently unknown mechanism, possibly via the formation of a lead-thiamine or lead-thiamine metabolite complex although it is unlikely that insoluble precipitates of lead-thiamine complex may be formed (Sasser et al., 1984). The complex reaction between lead and thiamine is reported to be mediated by –SH ligand of thiamine (Sasser et al., 1984). Thiamine has only one –SH ligand so it is unlikely to form large insoluble aggregates with lead. There have been no reports regarding the precipitation of dietary thiamine by lead. However, thiamine has also been shown to form a complex in vitro with other heavy metals such as, copper and cadmium (Cramer et al., 1981). Although it is not clear yet, a similar complex of lead and thiamine may be formed in the body, which could result in decreased tissue deposition. In the present study, recovery of transketolase activities in lead intoxicated animals toward control level with thiamine treatment may be interpreted as a result of replenishment of thiamine for transketolase activities. In addition, thiamine supplementation in lead treated group caused increase in seizure threshold back to the control level. Chelating therapy such as CaEDTA treatment has been widely used for the treatment of lead poisoning. However, the efficacy of such treatment was unsatisfactory in removing lead from soft tissues such as brain and liver (Cory-Slechta and Weiss, 1989). It was reported that simulta-
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neous administration of thiamine improved the efficiency of CaEDTA treatment in removing lead from the body (Goyer and Cherian 1978; Tandon et al., 1987). Taken these reports together with the results from the present study, it may be possible to use thiamine as a preventive or therapeutic agent, or as a complement to other preventive or therapeutic agents against lead intoxication. Thiamine is a soluble vitamin and has been assumed to be generally non-toxic mainly due to the ready excretion of excess thiamine from the body. In this study, each animal ingested 14.5 mg thiamine per 1 kg of body weight everyday. In many multi-vitamin supplements for human use which are commercially available at present, more than 1 mg of thiamine per 1 kg of body weight were added for everyday intake. Therefore, there would be no safety problems for the use of excess thiamine as an antidote for lead intoxication. In summary, it was shown that lead reduced thiamine level in the brain that might be responsible for the increased sensitivity to seizure in lead intoxicated animals. Thiamine supplementation reversed the increased sensitivity to seizure in lead intoxicated animals by decreasing lead concentration and recovering thiamine status in brain.
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Lead induced thiamine deficiency in the brain decreased the threshold of electroshock seizure in rat Jae Hoon Cheong a, Dong Ook Seo b, Jae Ryun Ryu b, Chan Young Shin b, Yun Tae Kim b, Hyoung-Chun Kim c, Won-Ki Kim d, Kwang Ho Ko b,* b
a College of Pharmacy, Samyook Uni6ersity, Seoul 139 -742, South Korea Centre for Biofunctional Molecules, POSTECH, Laboratory of Pharmacology, College of Pharmacy, Seoul National Uni6ersity, San 56 -1, Shillim-Dong, Kwanak-Gu, Seoul 151 -742, South Korea c College of Pharmacy, Kang Won National Uni6ersity, Chunchon 200 -701, South Korea d Department of Pharmacology, College of Medicine, Di6ision of Neuroscience, Medical Research Center, Ewha Women’s Uni6ersity, Seoul 158 -710, South Korea
Received 9 October 1998; accepted 15 January 1999
Abstract Many neurological disorders that occur frequently in lead intoxicated animals, have also been observed in thiamine deficient animals. To test whether lead intoxication could decrease the thiamine status and thresholds of electroshock seizure in rats, 3-week-old Wistar rats were treated with lead or lead plus thiamine. For comparison, a thiamine deficient group was included. Thiamine contents and transketolase activity, one of the thiamine dependent enzymes in the brain regions were significantly lowered by lead intoxication and thiamine deficiency. In both cases, thresholds of the electroshock seizure were significantly decreased. Thiamine supplementation reversed these signs and decreased the brain lead concentration in the lead treated group. The results from the present study suggest that the increased seizure susceptibility induced by lead intoxication in rats may be mediated at least in part through the changes of thiamine status. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lead; Thiamine; Transketolase; Rat brain; Seizure threshold
1. Introduction
* Corresponding author. Tel.: +82-2-8807848; fax: +82-28858211. E-mail address: [email protected] (K. Ho Ko)
Although acute lead poisoning has declined dramatically in recent years, poisoning by chronic and lower level exposures to lead is still a big health problem. Moreover, chronic intoxication is
0300-483X/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 9 9 ) 0 0 0 1 6 - 5
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more dangerous than acute poisoning because in many cases, chronic intoxication may cause more severe and even irreversible damage. Among the various tissues affected by lead poisoning, the central nervous system is the most sensitive target (Alperstein et al., 1991; Goyer, 1992). Encephalopathy characterized by demyelination, abnormal conduction velocity, decrement of IQ score and seizures is among the frequently observed complications of lead poisoning. Seizures are the most severe signs of lead poisoning. Alterations of seizure threshold and seizure responsiveness in lead exposed rats have been reported (Silbergeld et al., 1979). Significant reductions of the doses required to produce seizures were found in lead-exposed rats for the convulsant drugs such as picrotoxin and strychnine. The intensity of maximal electroshock seizures was increased in lead-exposed rats (Silbergeld and Hruska, 1980). However, the mechanism of lead neurotoxicity such as seizure and demyelination was not clear. It has been suggested that lead toxicity may be mediated through interactions between lead and endogenous substances with high affinity to lead. Several researchers focused on the molecules containing –SH group (Needleman and Bellinger, 1991). Thiamine, which is known as vitamin B1, is an endogenous –SH containing molecule. In addition, it has been reported that thiamine can reduce the absorption of lead in gastrointestinal tract and enhance the elimination of lead from soft tissues including brain, kidney and liver (Ghazaly, 1991). Neurological defects such as peripheral neuritis and encephalopathy that occur frequently in lead intoxicated animals, have also been observed in thiamine deficient animals. Therefore, it may be hypothesized that lead induced neurological defects are mediated at least in part through the alterations of thiamine status. In the present study, we investigated possible relationship between alterations of seizure sensitivity by lead intoxication and thiamine status. Although, most of the current problems regarding lead intoxication to date revolve around chronic and lower level exposures, for the mechanistic study of lead intoxication, we employed relatively acute and high level exposure to lead in the
present study with some obvious reasons. Less time and fewer animals are required for the experimentation, and the alterations of factors related to lead intoxication such as thiamine status may be more clearly investigated. The data obtained from the acute poisoning study may be extrapolated to the cases of chronic and lower level of lead exposures and also give impulses for the chronic studies in human as well as in animals. In this study, we examined whether lead administration influences the thiamine content and thiamine dependent biochemical reaction such as transketolase activity in the brain of 3-week-old rats, and whether the changes of the seizure threshold induced by lead intoxication is related to the changes of thiamine status. It was also tested whether administration of excessive thiamine could reverse the toxic manifestation of lead in the lead intoxicated rats.
2. Methods
2.1. Materials Lead acetate was purchased from Yakuri Chemical (Osaka, Japan). Thiamine tetrahydrofurfuryl disulfide (99.8%) was kindly donated by Il-Dong Pharmaceutical Co. (Seoul, South Korea). Thiamine deficient diet was purchased from Sankyo Farm Co., (Sendai, Japan). The contents of thiamine in the thiamine deficient diet was below the detection limit (0.25 mg/kg of rat chow) while those in normal diet was 7.29 mg of thiamine per 1 kg of rat chow. No constituents other than thiamine were altered in the thiamine deficient diet. All other reagents including thiamine hydrochloride and thiamine pyrophosphate were purchased from Sigma (St. Louis, MO).
2.2. Animal treatment Wistar rats were supplied from the Laboratory Animal Center of Seoul National University (Seoul, South Korea). Four groups of 3-week-old rats were used; (1) control group; (2) lead treated group; (3) lead plus thiamine treated group; and (4) thiamine deficient group. Until experimental
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animals became 3-week-old, lead and/or thiamine were administered to them through milk by giving lead and/or thiamine to their dams. Dams of the control group received a normal diet and tap water, and those of lead treated group received a normal diet and deionized and distilled water containing 0.2% lead acetate. Lead was not detected in the normal rat chow and the concentration of lead in the drinking water was reported to be below 0.04 ppm (Seoul Metropolitan Government, South Korea). Dams of the lead plus thiamine treated group were fed a thiamine sufficient diet containing 152.39 mg thiamine tetrahydrofurfuryl disulfide per 1 kg of rat chow ad libitum, and given deionized and distilled water containing 0.2% lead acetate. Dams of the thiamine deficient group were fed a thiamine deficient diet and tap water. All the treatments to dams were initiated upon parturition. Five dams were used for each treatment group. Only four animals were randomly selected from each dam and the brains were pooled. For each experimental group, litters from five different dams, namely five replications were employed. For the determination of lead concentration and thiamine status in rat brain, animals were sacrificed by decapitation. Brains were rapidly removed and dissected into three regions; telencephalon, brain stem (diencephalon/midbrain and pons/medulla), and cerebellum by the method of Glowinski and Iverson (1966) just before the determinations. 3. Measurement of lead concentration Separated brain tissues were lyophilized and digested overnight in an acid mixture of nitric acid and sulfuric acid (1:1, v/v). To the resulting yellowish clear solution with ten volumes of perchloric acid and nitric acid were added (4:1, v/v), and heated on a hot plate to remove organic materials and sulfuric acid. Lead concentration was measured by ICP (inductively coupled plasma) Mass spectrometry (VG Plasmaquad) (Taylor and Garbino, 1988). Nominal recoveries for lead were 9791.2% and recovery efficiency for lead was determined at 1 ppm. The lower limit of sensitivity was 0.1 mg/ml and
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lead concentration was expressed as ng/g wet tissue.
4. Quantitation of thiamine content Thiamine content in brain tissue was measured by the thiochrome method (Rindi and deGiuseppe, 1961). Briefly, isolated brain tissues were homogenized in ice-cold 5% trichloroacetic acid, centrifuged, and the resulting supernatant fraction was digested by a-amylase at 48–50°C for 30 min. After the supernatant was washed with water-saturated ethylether, cyanogen bromide and ice-cold 15% sodium hydroxide was added to produce thiochrome and further extracted by isobutanol. Fluorescence intensity of thiochrome was measured using a spectrofluorometer (FP-777, Jasco International Co. Ltd.) at 425 nm with exciting at 358 nm (Edwin, 1979) and was used to calculated thiamine contents. Thiamine contents were expressed as mg/g wet tissue.
5. Determination of transketolase activity Transketolase activity was estimated by measuring the rate of sedoheptulose-7-phosphate formation during the incubation of tissue homogenate in the presence of excess ribose-5phosphate substrate (Dreyfus and Moniz, 1962). Brain tissues were homogenized in ten volumes of ice-cold 0.04 M glycylglycine hydrochloride buffer, pH 7.6. The enzyme reaction was started by addition of 24 mM ribose-5-phosphate substrate to the homogenate and continued at 37.5°C for 30 min. After the reaction was terminated by the addition of 20% TCA, the mixture was centrifuged, and a clear and colorless supernatant fraction was obtained. An aliquot of the supernatant was desiccated under vacuum and the resulting residue was dissolved in redistilled water/concentrated sulfuric acid (2.5/6, v/v). Subsequently, the resulting solution was heated at 100°C for 4 min, with thorough agitation, and 3% aqueous cystein solution was added with shaking. Differences in absorbance between
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510 and 540 nm was measured spectrophotometrically and converted to total heptose concentration using known concentrations of sedoheptulose as standards. The activities of transketolase were expressed as nmol heptulose formed/min per mg protein. Protein content of tissue homogenate was measured by the method of Lowry et al. (1951) using bovine serum albumin as standard.
6. Measurement of electroshock seizure threshold Seizure was evoked by constant current stimulator and the resulting seizure was determined by overt hindlimb extension. To determine the electroshock seizure threshold, convulsive current 50 (CC50) which elicits convulsion in 50% of animals was calculated by a ‘staircase’ procedure (Browning et al., 1990). Individual animal was treated with electroshocks of 0.2 s stimulus duration to determine the current – convulsion relationship. If an animal showed convulsion, the next animal was given with 2 mA decrements in current intensity. If an animal did not show convulsion, the next animal was given with 2 mA increments in current intensity. In this way, the current – convulsion relationship was generated and CC50 value was determined by Litchfield – Wilcoxons II method (Litchfield and Wilcoxon, 1949). For each treatment group, 20 pups from five different dams were prepared and the animals were sacrificed right after the determination of the electroshock threshold.
7. Statistical analysis Data were expressed as the mean9 S.E.M. For statistical evaluation of data, One-way ANOVA was used. When statistically significant differences were found, Newman–Keuls’ test was used as a post-hoc test to determine the statistical differences between groups. For the statistical evaluation of the differences in CC50 values, Litchfield–Wilcoxon II method was employed. Differences were considered statistically significant when PB 0.05 was found.
8. Results
8.1. Measurement of lead concentration Lead concentrations in each brain region were summarized in Table 1. Lead concentrations in all brain regions of the lead treated group were higher than those of the control group, and those of the lead plus thiamine treated group were significantly decreased from those of the lead treated group.
9. Quantitation of thiamine content Thiamine contents in each brain region were shown in Fig. 1. Thiamine contents in all brain regions of the lead treated group and the thiamine deficient group were lower than those of the con-
Table 1 Lead concentration in brain regions of 3-week-old ratsa Lead concentration (mg/g wet tissue)
Telencephalon Brain stem Cerebellum
Control group
Lead treated group
Lead plus thiamine treated group
Thiamine deficient group
0.36 9 0.03 0.33 9 0.03 0.37 9 0.05
1.099 0.09b 1.3390.08b 0.929 0.06b
0.79 9 0.06y 0.69 90.04y 0.70 9 0.03x
0.31 9 0.05 0.30 9 0.08 0.28 9 0.09
Each value represents the mean 9S.E.M. (n = 5). One-way ANOVA revealed a significant difference (PB0.01) among the groups in each brain regions examined (df= 19, F value = 41.8 for telencephalon, 98.1 for brain stem and 40.3 for cerebellum, respectively). b Indicates a significant difference from the control group which determined by Newman–Keuls’ test as described in Section 2 (a, PB0.01). x and y indicate a significant difference from the lead treated group (x PB0.05 and y PB0.01). a
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11. Electroshock seizure threshold Electroshock seizure threshold was shown in Fig. 3. Electroshock seizure thresholds of the lead treated group and thiamine deficient group in 3-week-old rats were significantly lower than those of the control group, and thiamine treatment reversed this decrease back to the level of the control group.
12. Discussion In the present study, the relationship between lead intoxication and thiamine deficiency was in-
Fig. 1. Total thiamine contents in brain regions of 3-week-old rats. Each bar represents the mean 9 S.E.M (n= 5). Panels A, B and C show the results obtained from telencephalon, brain stem and cerebellum, respectively. One-way ANOVA revealed statistically significant differences (P B 0.01) among the groups in each brain regions examined (df =19, F= 31.46, 85.03 and 18.61 for A, B and C, respectively). *indicates a significant difference from the control group which determined by Newman–Keuls’ test as described in Section 2 (*PB0.05 and **PB 0.01). + indicates a significant difference from the lead treated group ( + PB 0.05 and + +P B 0.01). , control group, , lead treated group, b, lead plus thiamine treated group; d, thiamine deficient group.
trol group. In the lead plus thiamine treated group, thiamine contents were recovered to those of the control group.
10. Determination of transketolase activity Activities of transketolase in each brain region were shown in Fig. 2. The activities of transketolase of the lead treated group as well as the thiamine deficient group were significantly lower than those of the control group. In the lead plus thiamine treated group, transketolase activities were higher than those of the lead treated group.
Fig. 2. Transketolase activities in brain regions of 3-week-old rats. Each bar represents the mean9S.E.M. (n = 5). Panels A, B and C show the results obtained from telencephalon, brain stem and cerebellum, respectively. One-way ANOVA revealed statistically significant differences (PB0.01) among the groups in each brain regions examined (df = 19, F= 14.7, 53.5 and 88.5 for A, B and C, respectively). *indicates a significant difference from the control group which determined by Newman – Keuls’ test as described in Section 2 (*PB0.05 and **PB0.01). +indicates a significant difference from the lead treated group ( +PB 0.05 and + +PB 0.01). , control group, , lead treated group, b, lead plus thiamine treated group, d, thiamine deficient group.
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Fig. 3. Effects of thiamine on lead induced decrease of electroshock seizure threshold in 3-week-old rats. Each bar represents CC50 +upper C.L. (95% confidence limits). *indicates a significant difference from control group (P B0.05). +indicates a significant difference from lead-treated group (PB 0.05). CON; control group, LEAD; lead treated group, LEAD+THIAMINE; lead plus thiamine treated group, THIAMINE DEF; thiamine deficient group.
vestigated. Three-week-old, neonatal rats were used in this study, because lead toxicity to nervous system is generally more severe in young animals. In addition, it has been reported that manifestations of childhood lead poisoning are sustained to the adult period (Needleman et al., 1990) In the present study, contents of thiamine in the brain of rats were decreased by lead intoxication (Fig. 1). The reduced thiamine content in the brain may induce pathological consequences since thiamine is an essential factor in brain energy metabolism and biosynthetic pathways (Gaitonde et al., 1983; Gibson et al., 1984; Parker et al., 1984). In addition, thiamine is involved in the maintenance of normal membrane function and nerve conduction. Thiamine depletion affects neurons and their functions in selected areas of the central nervous system (Cooper and Pincus, 1979). Neurological defects, such as peripheral neuritis and encephalopathy, have been recognized in association with thiamine deficiency (Takashashi, 1981). Transketolase activities in the brain were decreased by lead intoxication (Fig. 2). Transketolase, one of the thiamine dependent enzymes, is
distributed in brain as well as liver, erythrocytes and leukocytes (Gaitonde et al., 1983). Among the thiamine dependent enzymes, transketolase activity is the most sensitive to thiamine deficiency (Friedrich, 1988). Transketolase activity has been widely used as a biochemical parameter to assess thiamine status in tissues (Friedrich, 1988; Hass, 1988). Transketolase is one of the enzymes involved in the hexose monophosphate (HMP) shunt generating NADPH required for lipid synthesis. Transketolase and possibly the entire HMP shunt are important in the development and maintenance of the myelin sheath (Clarke and Sokoloff, 1993) and the oligodendroglial metabolism in the CNS (Dreyfus and Moniz, 1962). The reduced transketolase activity induced by lead intoxication observed in the present study may alter these functions in the brain. Other thiamine dependent biochemical factors such as pyruvate dehydrogenase and a-ketoglutarate dehydrogenase, although less sensitive to thiamine deficiency than transketolase, may also contribute to energy impairment and morphological changes in thiamine deficiency. We suppose that the depletion of thiamine by lead intoxication resulted in decrement
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of transketolase activity as well as other neurochemical changes. The neurochemical changes caused by thiamine deficiency may mediate some of the neural symptoms of lead intoxication including the increased sensitivity to seizure. Electroshock seizure threshold was decreased by lead intoxication (Fig. 3). The lowering of electroshock seizure threshold by lead intoxication accompanied the concomitant decrement of thiamine content in the brain. The magnitude of decrement of electroshock seizure threshold induced by lead intoxication was nearly identical with that accompanied by thiamine deficiency. These results suggest that the changes of electroshock seizure threshold induced by lead intoxication may be related, at least in part, to the reduction of thiamine status in brain. It has been reported that reduced body weight may decrease the threshold for electroshock seizure. The body weight of the experimental groups were as follow; 5897.14 for control group, 34.7795.96 for lead treated group, 35.479 9.66 for lead plus thiamine treated group and 31.8497.80 for thiamine deficient group. The body weight and the threshold for electroshock seizure of lead treated group and thiamine deficient group was significantly lowered compared with control group. However, in this study, the threshold for electroshock seizure was not statistically different between control group and lead plus thiamine treated group in spite of the significant differences in the body weight between the two groups. The result may suggest that in this particular experiment the reduction of body weight would not be a main factor for the decreased seizure threshold in lead treated group and thiamine deficient group. Factors which influence the development of electroshock seizure pattern include synaptogenesis, myelination, maturation of monoaminergic systems, and electrolyte and amino acid changes in rat brain (Fox, et al., 1979). Demyelination and delays in myelin formation have been reported following lead intoxication and thiamine deficiency (Krigman et al., 1974; Wiggins, 1982). It has been demonstrated that the intensity of maximal electroshock seizures was increased in lead exposed rats (Silbergeld and Hruska, 1980).
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Taken together, the changes of electroshock seizure threshold and intensity induced by lead intoxication may be related to the changes of myelin formation due to thiamine deficiency. The decreased level of thiamine content and transketolase activity in the brain of the lead treated group returned toward control level in the lead plus thiamine treated group (Figs. 1 and 2). It has been reported that administration of excessive thiamine eliminates lead from soft tissues in lead intoxicated rodents (Ghazaly, 1991; Kim et al., 1991). It has been suggested that thiamine may facilitate the removal of lead from body fluids and other tissues by the formation of readily excretable complexes (Flora and Tandon, 1986). Alternatively, dietary thiamine may interfere with the absorption of lead into tissues by currently unknown mechanism, possibly via the formation of a lead-thiamine or lead-thiamine metabolite complex although it is unlikely that insoluble precipitates of lead-thiamine complex may be formed (Sasser et al., 1984). The complex reaction between lead and thiamine is reported to be mediated by –SH ligand of thiamine (Sasser et al., 1984). Thiamine has only one –SH ligand so it is unlikely to form large insoluble aggregates with lead. There have been no reports regarding the precipitation of dietary thiamine by lead. However, thiamine has also been shown to form a complex in vitro with other heavy metals such as, copper and cadmium (Cramer et al., 1981). Although it is not clear yet, a similar complex of lead and thiamine may be formed in the body, which could result in decreased tissue deposition. In the present study, recovery of transketolase activities in lead intoxicated animals toward control level with thiamine treatment may be interpreted as a result of replenishment of thiamine for transketolase activities. In addition, thiamine supplementation in lead treated group caused increase in seizure threshold back to the control level. Chelating therapy such as CaEDTA treatment has been widely used for the treatment of lead poisoning. However, the efficacy of such treatment was unsatisfactory in removing lead from soft tissues such as brain and liver (Cory-Slechta and Weiss, 1989). It was reported that simulta-
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neous administration of thiamine improved the efficiency of CaEDTA treatment in removing lead from the body (Goyer and Cherian 1978; Tandon et al., 1987). Taken these reports together with the results from the present study, it may be possible to use thiamine as a preventive or therapeutic agent, or as a complement to other preventive or therapeutic agents against lead intoxication. Thiamine is a soluble vitamin and has been assumed to be generally non-toxic mainly due to the ready excretion of excess thiamine from the body. In this study, each animal ingested 14.5 mg thiamine per 1 kg of body weight everyday. In many multi-vitamin supplements for human use which are commercially available at present, more than 1 mg of thiamine per 1 kg of body weight were added for everyday intake. Therefore, there would be no safety problems for the use of excess thiamine as an antidote for lead intoxication. In summary, it was shown that lead reduced thiamine level in the brain that might be responsible for the increased sensitivity to seizure in lead intoxicated animals. Thiamine supplementation reversed the increased sensitivity to seizure in lead intoxicated animals by decreasing lead concentration and recovering thiamine status in brain.
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