Neurotoxicologyand Teratology, Vol. 12, pp. 99-104. ~ PergamonPress plc, 1990. Printed in the U.S.A.
0892-0362/90 $3.00 + .00
Behavioral Antagonism Between Lead and Cadmium J A C K R. N A T I O N , * C A T H Y A. G R O V E R , * G E R A L D R. B R A T T O N ~ " A N D J U A N A. S A L I N A S *
*Department of Psychology, Texas A & M University, College Station, TX 77843 ~Department of Veterinary Anatomy, Texas A & M University, College Station, TX 77843 R e c e i v e d 15 J u n e 1989
NATION, J. R., C. A. GROVER, G. R. BRATTON AND J. A. SALINAS. Behavioral antagonism between lead and cadmium. NEUROTOXICOL TERATOL 12(2) 99-104, 1990.--Adult male rats were exposed to one of four dietary conditions for a period of 60 days. Group Control-Diet received a diet containing no added lead or cadmium, group Lead-Diet received a diet that contained 500 ppm added lead, group Cadmium-Diet received a diet that contained 100 ppm added cadmium, and group Lead-Cadmium-Diet received a diet that contained both 500 ppm added lead and 100 ppm added cadmium. Subsequent to exposure, animals were tested in a Digiscan activity monitor. Animals were then sacrificed and metal concentrations were determined in blood and brain. The results from this experiment showed that lead alone increased movement and vertical activity. Cadmium alone decreased movement and increased rest time. Cotreatment with lead and cadmium failed to produce behavioral differences relative to controls; thus, it seems that the changes in activity caused by one metal are antagonized by the other. Results from the analyses of residues in tissues revealed that blood lead concentrations were lower in the cotreatment condition than the lead along condition. However, brain residue accumulations were not different for these two exposure conditions. There was no evidence that the presence of lead attenuated increases in cadmium residues in blood or brain. Overall, the residue data agree with a central, as contrasted with a peripheral, account of lead/cadmium interaction effects, at least as relates to behavior. Because lead and cadmium were additive with regard to producing decreased body weights, it seems that the toxic effect of these metals is antagonized by cotreatment in some instances, and augmented in others. Antagonism
Cadmium
Cotreatment
Lead
IN a recent study of fixed-interval (FI) performance, Nation et al. (17) observed that the joint effects of exposure to both cadmium and lead were less than the effects of either metal presented in isolation. Specifically, facilitated operant responding associated with recurrent dietary exposure to 500 ppm lead or 100 ppm cadmium was not evident in animals exposed to the same dose levels in combination. Moreover, similar antagonistic effects were observed with respect to toxicant-induced changes in neurochemistry. Increases in brain levels of dopamine (DA), serotonin [5-hydroxytryptamine (5-HT)], and their respective metabolites that were produced by exposure to lead or cadmium alone were attenuated by the cotreatment of the metals. And identical antagonism was evinced when lead and cadmium presented alone produced decreases in transmitter activity in selected brain regions; e.g., even though both lead and cadmium produced significantly lower turnover rates for 5-HT in the frontal cortex when they were presented alone, turnover rates associated with the combined treatment of the metals were not different from controls. As counterintuitive as these findings may seem, they are in agreement with an earlier in vitro report (8) which found that cadmium inhibited lead-related increases in the spontaneous release of peripheral acetylcholine. Speculative accounts of such phenomena point to the possible competition between lead and cadmium at calcium uptake sites that are integrally involved in both evoked transmitter release, and the ultimate concentrations of intracellular ionized calcium that modulate spontaneous release
[refer to (2) for a recent review of these putative chemical interactions]. And it may be that such changes in central neurotransmission underlie the behavioral and neurochemical anomalies observed by Nation et al. in their in vivo investigation of lead/cadmium interactions. But one must also consider that the metal antagonism may derive from peripheral rather than central influences. That is, it is possible that the locus of competition between lead and cadmium is at the level of the gastrointestinal tract rather than nervous system. Perhaps one metal blocks or antagonizes the intestinal absorption of the other and therein limits the distribution of the toxicant, thus decreasing residue accumulations in tissues. Because tissue concentrations of lead and cadmium were unavailable in the Nation et al. report, a peripheral rationale for the apparent antagonism cannot be ruled out. For peripheral blockade to be considered as a principal mechanism underlying lead/cadmium antagonism, it would need to be demonstrated that blood levels of lead/cadmium residues would be sharply reduced by joint exposure to the metals. Moreover, such diminished blood concentrations in the cotreatment condition should be translated into decreases in concentrations of lead and cadmium in the brain, otherwise arguments focusing on noncentral issues would be greatly compromised. The present investigation provides added information on the consequences of simultaneous exposure to lead and cadmium. In an effort to extend the range of behaviors affected by cotreatment manipulations, a general activity index was used to determine
99
100
NATION, GROVER, BRATTON AND SALINAS
whether or not effects similar to those observed for schedulecontrolled responding would exist for such behavioral dimensions as vertical and horizontal activity, distance traveled, movement time, turns, and stereotypy. In addition, the issue of central versus peripheral antagonism was addressed in this study where blood and brain residues of lead and cadmium were assessed for animals exposed to control diets, diets containing either 500 ppm lead or 100 ppm cadmium, or a diet containing both 500 ppm lead and 100 ppm cadmium. METHOD
Animals The animals used in this study were 32 adult male SpragueDawley rats (Holtzman Company, Madison, WI) approximately 50 days old at the beginning of the experiment. Initial animal weights ranged between 180 and 200 g. Eight of the animals (group Control-Diet) were maintained ad lib on a diet of laboratory chow that contained no added chemicals. Eight other animals (group Lead-Diet) were placed on an identical feeding regimen that offered lab chow containing 500 ppm lead (as lead acetate). And eight animals (group Cadmium-Diet) were maintained on an ad lib diet that contained 100 ppm cadmium (as cadmium chloride). The remaining eight animals (group Lead-CadmiumDiet) were fed the same diet of lab chow as the other animals, but their food contained both 500 ppm lead and 100 ppm cadmium.
Preparation of Food For contaminant-treated food, pellets of semipurified Teklad Laboratory chow (Harlan Sprague-Dawley, Inc., Madison, WI) were ground in a small food mill then transferred to a large stainless steel food mixer in 10-kg batches. Two liters of distilled, deionized water containing the appropriate amount(s) of the contaminant(s) (lead acetate, cadmium chloride) were added to the mixer, and the mixing process was continued until the mixture appeared homogenous. Mixing was continued 20-30 rain to ensure complete distribution of lead and/or cadmium in the food. The food then was repelleted with a laboratory pelleter (Model CL Laboratory Pellet Mill, California Pellet Mill Co., San Francisco) and stored at 0 . 0 5 , or a significant Groups × Days interaction effect, F(27,252) = 1.21, p > 0 . 0 5 . The main effect for Days was found to be significant, F(9,252) -- 77.97, p < 0 . 0 1 . Post hoc analyses (Tukey's) of weekly intake means indicated that all groups uniformly increased their consumption of food over the course of the experiment (ps 0 . 0 5 . Subsequent comparisons of group means revealed that group LeadCadmium-Diet animal body weights were lower ( p < 0 . 0 5 ) than the remaining three groups, which did not differ (ps>0.05).
Activity Measures The results from the analyses of the various activity measures showed that exposure to lead produced a behavioral pattern defined by a general increase in activity. Conversely, animals exposed to cadmium showed a decrease in behavioral activation relative to control animals. Of particular interest to the present study was the finding that animals exposed to both lead and cadmium exhibited behavioral profiles that were not significantly different from controls. Table 1 provides a summary of the group differences on the various activity measures. Statistical confirmation of the differences in group means was provided by a 4 Groups (Control-Diet, Lead-Diet, Cadmium-Diet, Lead-Cadmium-Diet) x 6 Intervals (1--6, successive five-minute test intervals) repeated measures ANOVA. The results indicated that group Lead-Diet exhibited greater numbers of movements, F(3,28) = 2.86, p < 0 . 0 5 , than the remaining three groups, which did not differ on this measure. On the measure of movement time, group Lead-Diet moved longer than the remaining three groups, and group Cadmium-Diet moved for a shorter time than controls, F(3,28) = 2.97, p < 0 . 0 5 . Consistent with this pattern of results, group Lead-Diet exhibited less rest time than the remaining three groups, and group Cadmium-Diet engaged in rest more than Controls, F ( 3 , 2 8 ) = 2 . 8 7 , p < 0 , 0 5 . It is important to note that group Lead-Cadmium-Diet was not different from group ControlDiet on any of the above measures. Group separation was also evident in terms of vertical responding. On the measure of vertical activity, group Lead-Diet had a higher mean value than the remaining three groups, F ( 3 , 2 8 ) = 3.91, p < 0 . 0 1 . Groups Control-Diet, Cadmium-Diet, and LeadCadmium-Diet were not different on this measure (ps>0.05). An identical pattern of results was obtained for the number of vertical movements measure, F ( 3 , 2 8 ) = 5.35, p < 0 . 0 0 5 , where increased activity was once again evident only for group Lead-Diet.
Lead and Cadmium Concentrations in Tissues The mean concentrations of lead and cadmium residues in
Group Activity
Control
Pb
Cd
Pb + Cd
p
Horizontal activity Total distance No. of movements Movement time (sec) Rest time (see) Vertical activity No. of vertical movements Vertical time (sec) Stereotypy count No. of stereotypy Stereotypy time (sec) Clockwise revolutions Anticlockwise revolutions Margin time (sec) Center time (sec) Time spent in comers: Left-front Right-front Left-rear Right-rear
7542 4451 196" 349* 1456" 696* 86*
9336 4899 371t 440t 1367t 911t 120t
7236 3291 289* 286~ 1519~ 604* 75*
7464 3996 299* 331"~t 1474"~ 635* 83*
--0.05 0.05 0.05 0.01 0.004
326 3036 208 259 13 12
397 3963 236 301 12 12
294 3179 218 307 8 10
316 3300 206 267 14 13
-------
1394 405
1356 444
1469 330
1361 438
---
24 98 263 90
32 36 66 155
31 21 179 133
25 45 165 170
-----
Note: Row means that do not have a common symbol are significantly different.
blood and brain are presented for each of the four groups in Table 2. With respect to the analyses performed on blood, a 4 Groups (Control-Diet, Lead-Diet, Cadmium-Diet, Lead-Cadmium-Diet) one-way A N O V A test of differences in lead residue concentrations reached an acceptable level for statistical significance, F ( 3 , 2 8 ) = 355.90, p < 0 . 0 0 0 1 . Subsequent individual comparisons of group means revealed that lead residues were greater for group Lead-Diet than for group Lead-Cadmium-Diet (p
0892-0362/90 $3.00 + .00
Behavioral Antagonism Between Lead and Cadmium J A C K R. N A T I O N , * C A T H Y A. G R O V E R , * G E R A L D R. B R A T T O N ~ " A N D J U A N A. S A L I N A S *
*Department of Psychology, Texas A & M University, College Station, TX 77843 ~Department of Veterinary Anatomy, Texas A & M University, College Station, TX 77843 R e c e i v e d 15 J u n e 1989
NATION, J. R., C. A. GROVER, G. R. BRATTON AND J. A. SALINAS. Behavioral antagonism between lead and cadmium. NEUROTOXICOL TERATOL 12(2) 99-104, 1990.--Adult male rats were exposed to one of four dietary conditions for a period of 60 days. Group Control-Diet received a diet containing no added lead or cadmium, group Lead-Diet received a diet that contained 500 ppm added lead, group Cadmium-Diet received a diet that contained 100 ppm added cadmium, and group Lead-Cadmium-Diet received a diet that contained both 500 ppm added lead and 100 ppm added cadmium. Subsequent to exposure, animals were tested in a Digiscan activity monitor. Animals were then sacrificed and metal concentrations were determined in blood and brain. The results from this experiment showed that lead alone increased movement and vertical activity. Cadmium alone decreased movement and increased rest time. Cotreatment with lead and cadmium failed to produce behavioral differences relative to controls; thus, it seems that the changes in activity caused by one metal are antagonized by the other. Results from the analyses of residues in tissues revealed that blood lead concentrations were lower in the cotreatment condition than the lead along condition. However, brain residue accumulations were not different for these two exposure conditions. There was no evidence that the presence of lead attenuated increases in cadmium residues in blood or brain. Overall, the residue data agree with a central, as contrasted with a peripheral, account of lead/cadmium interaction effects, at least as relates to behavior. Because lead and cadmium were additive with regard to producing decreased body weights, it seems that the toxic effect of these metals is antagonized by cotreatment in some instances, and augmented in others. Antagonism
Cadmium
Cotreatment
Lead
IN a recent study of fixed-interval (FI) performance, Nation et al. (17) observed that the joint effects of exposure to both cadmium and lead were less than the effects of either metal presented in isolation. Specifically, facilitated operant responding associated with recurrent dietary exposure to 500 ppm lead or 100 ppm cadmium was not evident in animals exposed to the same dose levels in combination. Moreover, similar antagonistic effects were observed with respect to toxicant-induced changes in neurochemistry. Increases in brain levels of dopamine (DA), serotonin [5-hydroxytryptamine (5-HT)], and their respective metabolites that were produced by exposure to lead or cadmium alone were attenuated by the cotreatment of the metals. And identical antagonism was evinced when lead and cadmium presented alone produced decreases in transmitter activity in selected brain regions; e.g., even though both lead and cadmium produced significantly lower turnover rates for 5-HT in the frontal cortex when they were presented alone, turnover rates associated with the combined treatment of the metals were not different from controls. As counterintuitive as these findings may seem, they are in agreement with an earlier in vitro report (8) which found that cadmium inhibited lead-related increases in the spontaneous release of peripheral acetylcholine. Speculative accounts of such phenomena point to the possible competition between lead and cadmium at calcium uptake sites that are integrally involved in both evoked transmitter release, and the ultimate concentrations of intracellular ionized calcium that modulate spontaneous release
[refer to (2) for a recent review of these putative chemical interactions]. And it may be that such changes in central neurotransmission underlie the behavioral and neurochemical anomalies observed by Nation et al. in their in vivo investigation of lead/cadmium interactions. But one must also consider that the metal antagonism may derive from peripheral rather than central influences. That is, it is possible that the locus of competition between lead and cadmium is at the level of the gastrointestinal tract rather than nervous system. Perhaps one metal blocks or antagonizes the intestinal absorption of the other and therein limits the distribution of the toxicant, thus decreasing residue accumulations in tissues. Because tissue concentrations of lead and cadmium were unavailable in the Nation et al. report, a peripheral rationale for the apparent antagonism cannot be ruled out. For peripheral blockade to be considered as a principal mechanism underlying lead/cadmium antagonism, it would need to be demonstrated that blood levels of lead/cadmium residues would be sharply reduced by joint exposure to the metals. Moreover, such diminished blood concentrations in the cotreatment condition should be translated into decreases in concentrations of lead and cadmium in the brain, otherwise arguments focusing on noncentral issues would be greatly compromised. The present investigation provides added information on the consequences of simultaneous exposure to lead and cadmium. In an effort to extend the range of behaviors affected by cotreatment manipulations, a general activity index was used to determine
99
100
NATION, GROVER, BRATTON AND SALINAS
whether or not effects similar to those observed for schedulecontrolled responding would exist for such behavioral dimensions as vertical and horizontal activity, distance traveled, movement time, turns, and stereotypy. In addition, the issue of central versus peripheral antagonism was addressed in this study where blood and brain residues of lead and cadmium were assessed for animals exposed to control diets, diets containing either 500 ppm lead or 100 ppm cadmium, or a diet containing both 500 ppm lead and 100 ppm cadmium. METHOD
Animals The animals used in this study were 32 adult male SpragueDawley rats (Holtzman Company, Madison, WI) approximately 50 days old at the beginning of the experiment. Initial animal weights ranged between 180 and 200 g. Eight of the animals (group Control-Diet) were maintained ad lib on a diet of laboratory chow that contained no added chemicals. Eight other animals (group Lead-Diet) were placed on an identical feeding regimen that offered lab chow containing 500 ppm lead (as lead acetate). And eight animals (group Cadmium-Diet) were maintained on an ad lib diet that contained 100 ppm cadmium (as cadmium chloride). The remaining eight animals (group Lead-CadmiumDiet) were fed the same diet of lab chow as the other animals, but their food contained both 500 ppm lead and 100 ppm cadmium.
Preparation of Food For contaminant-treated food, pellets of semipurified Teklad Laboratory chow (Harlan Sprague-Dawley, Inc., Madison, WI) were ground in a small food mill then transferred to a large stainless steel food mixer in 10-kg batches. Two liters of distilled, deionized water containing the appropriate amount(s) of the contaminant(s) (lead acetate, cadmium chloride) were added to the mixer, and the mixing process was continued until the mixture appeared homogenous. Mixing was continued 20-30 rain to ensure complete distribution of lead and/or cadmium in the food. The food then was repelleted with a laboratory pelleter (Model CL Laboratory Pellet Mill, California Pellet Mill Co., San Francisco) and stored at 0 . 0 5 , or a significant Groups × Days interaction effect, F(27,252) = 1.21, p > 0 . 0 5 . The main effect for Days was found to be significant, F(9,252) -- 77.97, p < 0 . 0 1 . Post hoc analyses (Tukey's) of weekly intake means indicated that all groups uniformly increased their consumption of food over the course of the experiment (ps 0 . 0 5 . Subsequent comparisons of group means revealed that group LeadCadmium-Diet animal body weights were lower ( p < 0 . 0 5 ) than the remaining three groups, which did not differ (ps>0.05).
Activity Measures The results from the analyses of the various activity measures showed that exposure to lead produced a behavioral pattern defined by a general increase in activity. Conversely, animals exposed to cadmium showed a decrease in behavioral activation relative to control animals. Of particular interest to the present study was the finding that animals exposed to both lead and cadmium exhibited behavioral profiles that were not significantly different from controls. Table 1 provides a summary of the group differences on the various activity measures. Statistical confirmation of the differences in group means was provided by a 4 Groups (Control-Diet, Lead-Diet, Cadmium-Diet, Lead-Cadmium-Diet) x 6 Intervals (1--6, successive five-minute test intervals) repeated measures ANOVA. The results indicated that group Lead-Diet exhibited greater numbers of movements, F(3,28) = 2.86, p < 0 . 0 5 , than the remaining three groups, which did not differ on this measure. On the measure of movement time, group Lead-Diet moved longer than the remaining three groups, and group Cadmium-Diet moved for a shorter time than controls, F(3,28) = 2.97, p < 0 . 0 5 . Consistent with this pattern of results, group Lead-Diet exhibited less rest time than the remaining three groups, and group Cadmium-Diet engaged in rest more than Controls, F ( 3 , 2 8 ) = 2 . 8 7 , p < 0 , 0 5 . It is important to note that group Lead-Cadmium-Diet was not different from group ControlDiet on any of the above measures. Group separation was also evident in terms of vertical responding. On the measure of vertical activity, group Lead-Diet had a higher mean value than the remaining three groups, F ( 3 , 2 8 ) = 3.91, p < 0 . 0 1 . Groups Control-Diet, Cadmium-Diet, and LeadCadmium-Diet were not different on this measure (ps>0.05). An identical pattern of results was obtained for the number of vertical movements measure, F ( 3 , 2 8 ) = 5.35, p < 0 . 0 0 5 , where increased activity was once again evident only for group Lead-Diet.
Lead and Cadmium Concentrations in Tissues The mean concentrations of lead and cadmium residues in
Group Activity
Control
Pb
Cd
Pb + Cd
p
Horizontal activity Total distance No. of movements Movement time (sec) Rest time (see) Vertical activity No. of vertical movements Vertical time (sec) Stereotypy count No. of stereotypy Stereotypy time (sec) Clockwise revolutions Anticlockwise revolutions Margin time (sec) Center time (sec) Time spent in comers: Left-front Right-front Left-rear Right-rear
7542 4451 196" 349* 1456" 696* 86*
9336 4899 371t 440t 1367t 911t 120t
7236 3291 289* 286~ 1519~ 604* 75*
7464 3996 299* 331"~t 1474"~ 635* 83*
--0.05 0.05 0.05 0.01 0.004
326 3036 208 259 13 12
397 3963 236 301 12 12
294 3179 218 307 8 10
316 3300 206 267 14 13
-------
1394 405
1356 444
1469 330
1361 438
---
24 98 263 90
32 36 66 155
31 21 179 133
25 45 165 170
-----
Note: Row means that do not have a common symbol are significantly different.
blood and brain are presented for each of the four groups in Table 2. With respect to the analyses performed on blood, a 4 Groups (Control-Diet, Lead-Diet, Cadmium-Diet, Lead-Cadmium-Diet) one-way A N O V A test of differences in lead residue concentrations reached an acceptable level for statistical significance, F ( 3 , 2 8 ) = 355.90, p < 0 . 0 0 0 1 . Subsequent individual comparisons of group means revealed that lead residues were greater for group Lead-Diet than for group Lead-Cadmium-Diet (p
