xvEPA United States Environmental Protection Agency Health Effects Research Laboratory Research Triangle Park NC 27711 Research and Development EPA-600/S1-81-013 Mar 1981 Project Summary Chronic Toxicity of Lead and Cadmium: II. Changes in the Central Nervous System of the Fi Generation of Rats After Chronic Intoxication With Lead and Cadmium Z. S. Herman, K. Kmieciak-Kolada, R. Szkilnik, R. Brus, J. J. Jonek, K. Ludyga, R. Winter, J. Bodztony, B. Hebrowska, K. Kaminski, D. Piskorska, and J. Wyrebowska Lead (Pb) and cadmium (Cd) are hazardous trace elements found in the present environment of man. Taking into account possible individual and synergistic effects of chronic exposure to Pb and/or Cd, we have previously studied the effect on the central ner- vous system in rats chronically expos- ed to drinking water containing Pb and/or Cd in trace amounts. PreviousT ly, we have shown that a brief Pb and/or Cd exposure affects the subtle functions of the nervous system. In this paper we present the changes in behavior and biogenic amine concen- trations in discrete brain areas of rats from the Fi generation. Forty-day old male and female rats received one of the following treat- ments in their drinking water: Group I (control), 0.005 M acetate buffered drinking water; Group II, 5 ppm Pb; Group III, 50 ppm Pb; Group IV, 0.1 ppm Cd; Group V, 5 ppm Pb and 0.1 ppm Cd; Group VI, 50 ppm Pb and 5 ppm Cd. Pb and Cd acetate salts were dissolved in drinking water buffered to pH 7 with an 0.005 M acetate buffer. Animals were exposed for 40 days and pregnant females continued on these regimens throughout gestation and lactation. Rats born from these animals (Fi generation) were similarly exposed for 40 days beginning 20 days after birth. Each control and experimental group of animals of the F, generation consisted of 1O rats. All evaluations described were made at 30, 60 and 90 days of age. This Project Summary was develop- ed by Health Effects Research Labora- tory, Research Triangle Park, NC, to announce key findings of the research project which is fully documented in a separate report of the same title (see Project Report ordering information at back). Behavior Locomotor activity from all treatment groups during Pb/Cd exposure is report- ed as a percentage of control values (Table 1). Increased diurnal and noctur- nal locomotor activity was evident in 30- day old rats treated with 5 ppm Pb, while at 60 and 90 days a decrease in these activities was observed. Fifty ppm Pb treatment increased diurnal locomotor ------- Table 1. Locomotor Activity Changes (Percent of Control)* Following Exposure to Lead and/or Cadmium Control Measurement Time Day Night Age /Days} 30 60 90 30 60 90 {Counts/ 12 hr± SD.J 929 ±11 8 1096 ±105 950 ± 89 1690±182 1708±175 1670 ±152 5 ppm Pb 181" 87 80 280" 44* 65C 50 ppm Pb 142 161 120 90 138 62 b b b c 0 1 ppm Cd 163 68 73 186 116 95 b c c b 5 ppm Cd 84 107 124C 183* 85 112 5 ppm Pb + 01 ppm Cd 132C 82 63C 161* 88 54* 50 ppm Pb + 5 ppm Cd 87 61* 99 132C 48* 91 a - n = 10 for all groups * -p<0.01 c -p<0.05 activity in 30-, 60-, and 90-day old rats. In 90-day old rats there was an evident decrease in nocturnal activity. Cd (0.1 ppm) also increased locomotor activity of 30-day old rats, while the diurnal activity in 60- and 90-day old animals decreased. Changes in locomotor activity after Cd exposure (5 ppm) were seen as an increase of nocturnal activity in 30- day old rats. After simultaneous treat- ment with lower doses of Pb and Cd, an increase in activity of 30-day old animals was observed and a decrease in diurnal and nocturnal activity of 90-day old rats was observed. Higher doses of Pb and Cd in 60-day old rats produced a depres- sion in locomotor activity. Biogenic Amines In Discrete Areas Of Brain Regional brain NA and DAconcentra- tions are presented in Tables 2 and 3. Overall, the intoxication by Pb caused a decrease in NA content in the examined brain areas while Cd caused an increase. Pb and Cd applied concomitantly in lower doses caused an increase in NA in 30- and 90-day old animals. Higher doses usually caused a decrease of NA in 30- and 60-day old rats and an increase in 90-day old animals. After 5 ppm Pb, DA level in the striatum increased in 30- and 60-day animals. Exposure to 50 ppm Pb de- creased striatal DA in 30- and 60-day old rats and increased DA in 90-day old rats. Cd (0.1 ppm) increased striatal DA concentrations in 30-day old rats and decreased it in 60- and 90-day old ani- mals. Cd (5 ppm) increased DA in 60-day old rats. Combined Pb and Cd exposure to lower concentrations significantly in- creased DA in 30-day old rats, while exposure to 50 ppm Pb and 5 ppm Cd increased striatal DA in 30- and 60-day, but not in 90-day old rats. In general, treatment with 5 ppm Pb decreased 5-HT and increased 5-HIAA in specific areas of the brain (Tables 4 and 5). Thirty-day old rats exposed to 50 ppm Pb showed an increase in 5-HT and 5-HIAA in the brain stem while a de- crease in 5-HT was observed in the limbic system. In 60- and 90-day old rats, exposed to 50 ppm Pb, 5-HT and 5- HIAA levels were increased in specific areas of the brain. Increased striatal 5-HIAA was noted in animals receiving 0.1 ppm Cd. In 60- day old rats, a decrease in 5-HT in the hypothalamus, limbic system, and brain stem were seen. Decreased 5-HIAA in the hypothalamus and striatum was also observed in these 60-day old animals. In 90-day old rats, an increase of striatal 5-HT and a decrease of 5- HIAA in the hypothalamus, limbic sys- tem, and striatum were seen. An increase in brain stem 5-HTand 5- HIAA was observed in 30-day old rats treated with 5 ppm Cd. In 60-day old rats, the level of 5-HT decreased in the Table 2. Noreadrenalin (NA) Changes (Percent of Control^ Following Chronic Exposure to Lead and/or Cadmium Brain Area Hypothalamus Rons plus Medulla Limbic System Striatum Age (Days) 30 60 90 30 60 90 30 60 90 30 60 90 Control N A Concentra- tion (fjg/g ± SE1 2.28 ±0.08 2. 40 ±0.07 2. 30 ±0.06 0.69 ±0.02 0.69 ±0.02 0.67 ±0.01 0.61 ±0.03 0.59 ±0.01 0.55 ±0.01 0.32 ±0.04 0.32 ±0.04 0.31 ±0.01 5 ppm Pb 96 141C 95 101 132* 120* 60* 135* 136* 135* 137* 135* 50 ppm Pb 54* 56* 114 96 99 46° 50° 54* 110 105 113 80C 0. 1 ppm Cd 105 129* 103 89 109 109 80° 126C 103 85C 71* 107 5 ppm Cd 77C 115C 103 90 112 88 79° 113 96 84C 93 104 5 ppm Pb + 0.1 ppm Cd 120° 92 98 89 95 720° 71* 94 122C 162* 98 131* 50 ppm Pb + 5 ppm Cd 90 89 98 74* 95 103* 103 64° 125C 84C 100 140C * - n = 8 for all groups * - p <0.01 c -p<0.05 Table 3. Dopamine (DA) Changes (Percent of Control}* Following Chronic Exposure to Lead and/or Cadmium Brain Area Striatum Age (Days) 30 60 90 Control NA Concentra- tion ffig/g ± S.E.I 5.58 ±0.33 5.66 ±0.20 5. 14 ±0.20 5 ppm Pb 298* 120C 118 50 ppm Pb 70 62 223 b b 0. 1 ppm Cd 402 70 72 b b b 5 ppm Cd 93 122* 85C 5 ppm Pb + 0.1 ppm Cd 265* 90 87 SO ppm Pb + 5 ppm Cd 133* 121C 89 * - n — 8 for all groups * - p <0.01 c - p <0.05 ------- Table 4. 5-Hydroxytryptamine (5-HT) Changes (Percent of Control)* Following Chronic Exposure to Lead and/or Cadmium Control 5-HT Age Concentration 5 ppm 50 ppm 0 1 ppm 5 ppm 5 ppm Pb + 50 ppm Pb IDaysl /fjg/g±SE) Pb Cd Cd 0 t ppm Cd +5 ppm Cd Hypothalamus Rons plus Medulla Limbic System Striatum 30 60 90 30 60 90 30 60 90 30 60 90 2.1 2.0 2.0 0.98 0.96 0.91 0.95 0.93 0.95 0.92 0,98 0.93 ± ± ± ± ± ± ± ± ± ± ± ± 0.1 0.1 0.1 0.04 0.1 0.03 0.04 0.06 0.05 0.02 0.08 0.10 93 97 92 125C 59C 86 86 70* 142* 98 51* 107 90 103 138" 155C 4T 40C 70C 75C 131* 101 73C 37* 95 82C 95 108 85 109 104 84C 102 110 87 133* 104 102 101 155" 81C 97 96 88C 114 97 106 100 113 92 89 112 185* 120C 767* 128* 134* 88C 115C 140* 114 104 107 119C 76C 76C 100 81C 69* 130* 119C 67* 8 - n = 8 for each group * -p<0.01 c -p<0.05 Table 5. 5-Hydroxyindoleacetic Acid (5-HIAA) Changes (Percent of Control)"' Following Chronic Exposure to Lead and/or Cadmium Brain Area Hypothalamus Rons plus Medulla Limbic System Striatum Age (Days/ 30 60 90 30 60 90 30 60 90 30 60 90 Control 5-HIAA Concentration (ug/g±SE! 2.5 2.5 2.3 0.63 0.61 0.65 0.62 0.63 0.59 0.95 0.09 1.0 ± ± ± ± ± ± ± ± ± ± ± ± 0.2 0.1 0.1 0.03 0.05 0.02 0.04 0.04 0.03 0.03 0.05 0.1 Sppm Pb 97 72* 70* 141* 165* 106 91 136* 101 149* 125* 104 SO ppm Pb 96 97 86 170* 214* 110 97 127* 88 174* 98 152* 0 1 ppm Cd 92 76° 43* 115C 105 129* 80C 107 57* 237* 69* 65* 5 ppm Cd 54* 119C 89 159* 145* 93 151* 130* 117C 98 97 126* 5 ppm Pb + 0 7 ppm Cd 102 88 77C 73* 143* 173* 50* 115C 119C 95 103 101 50 ppm Pb + 5 ppm Cd 76 c 96 74 138 183 97 161 127 93 105 b b b b b 83° 77 c 8 - n = 8 for each group * -p<0.01 c -p<0.05 Table 6. Specific Biochemical Changes (Percent of Control)" Following Exposure to Lead and/or Cadmium ALA- dehydratase blood Acetyl- cholinesterase brain Monoamine oxidase brain Free Erythrocyte Porphyrins Age IDaysl 30 60 90 30 60 90 30 60 90 30 60 90 Control Mean ±S E 121 48 49 5.8 6.7 6.0 64 69 60 183 103 120 ± ± ± ± ± ± ± ± ± ± ± ± 25 18 17 1.3 0.5 1.1 15 19 15 58 23 15 5 ppm Pb 32* 52* 42* 146* 100 62* 330* 50* 50* 327* 448* 339* 50 ppm Pb 15 73 71 70 100 100 25 64 72 391 412 224 b b b b b b b b b b 0 1 ppm Cd — — 752" 151 180* 160* 158* — — 5 ppm ca . — — — . 148* 180* 100 158* 316* — — 5 ppm Pb + 0 / ppm Cd 21* 25* 50* 81° 42* 100 61* 58* 63* 247* 473* 360* 50 ppm Pb + 5 ppm Cd 90 46* 63* 55* 80C 72* 100* 170* 176* 290* 110 318* - n = 8 for each group * -p<0.01 c -p<0.05 brain stem and limbic system while the 5-HIAA level increased in the brain stem and limbic system. Increased 5- HIAA levels were found in the limbic system and Striatum of 90-day old ani- mals. Effects of the combined Pb-Cd expo- sures were primarily synergistic or protective and were not related to age or dose. Biochemical Changes ALA-dehydratase activity in the blood was significantly decreased and free erythrocyte porphyrins were signifi- cantly increased in Pb-treated rats (Table 6). AChE and MAO activity in whole brain were variously affected in rats intoxicated by Cd and/or PB (Table 6). LDH activity and AlPh activity were not significantly changed in any exam- ined group of animals. Conclusion Our behavioral studies have shown that both Pb and Cd alter locomotor activity. In control animals, age had no effect on either diurnal or nocturnal activity. In animals treated with Pb or Cd singly there was no pattern to the alterations in locomotor activity with dose or age at testing. When treated with the Pb-Cd combination, 30-day activity was generally elevated; whereas, 60- and 90-day activity was depressed. These CNS mediated activity changes seen in laboratory animals are similarto the clinical syndrome of Pb intoxication in children. > US GOVERNMENT PRINTING OFFICE 1961-757-012/7034 ------- Z 5. Herman. K. Kmieciak-Kolada, R. Szkilnik, R. Brus, J.J. Jonek, K. Ludyga, R. Winter, J. Bodziony, B. Hebrowska. K. Kaminski, D. Piskorska, and J. Wyrebowska are with the Departments of Pharmacology, Biochemistry, Cytlogy and Histology, Central Animal Farm of Silesian Medical Academy, Katawice, Poland. John Laskey is the EPA Project Officer (see below). The complete report, entitled "Chronic Toxicity of Lead and Cadmium: II. Changes in the Central Nervous System of the Fi Generation of Rats After Chronic Intoxication With Lead and Cadmium," (Order No. PB 81-150 989; Cost: $5.00, subject to change) will be available only from: National Technical Information Service 5285 Port Royal Road Springfield, v'A 221'61 Telephone: 703-487-4650 The EPA Project Officer can be contacted at: Health Effects Research Laboratory U.S. Environmental Protection Agency Research Triangle Park, NC 27711 United States Environmental Protection Agency Center for Environmental Research Information Cincinnati OH 45268 Pottage and Environmental Protection Agency EPA 335 Official Business Penalty for Private Use $300 00003?* ------- |