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Cadmium exposure in infancy: Effects on activity and social behaviors of juvenile rats
Neurotoxicology and Teratology, Vol. 10, pp. 135-142. ©PergamonPress plc, 1988. Printedin the U.S.A.
0892-0362/88$3.00 + .00
Cadmium Exposure in Infancy: Effects on Activity and Social Behaviors of Juvenile Rats W I L L I A M R. H O L L O W A Y , JR. A N D D O N A L D H . T H O R
Research Department, E. R. Johnstone Training and Research Center, Bordentown, N J 08505 R e c e i v e d 4 N o v e m b e r 1986 HOLLOWAY, W. R., JR. AND D. H. THOR. Cadmium exposure in infancy: Effects on activity and social behaviors of juvenile rats. NEUROTOXICOL TERATOL 10(2) 135-142, 1988.--In two experiments male and female infant rats were given single injections of cadmium chloride (0, 1, 2, 3 or 4 mg CdJkg) on Day 5 or 6. Animals receiving the 3 and 4 mg/kg doses had high mortality rates at weaning; survivors were extremely underweight and were not used in postweaning tests. Male subjects receiving 2 mg/kg in infancy were significantly more active after weaning than littermates who had received 0 or 1 mg/kg doses, and on Day 29 they also engaged in significantly more rough and tumble play with a nontreated partner than did rats in the other groups. This effect of early cadmium exposure was also evident when males were tested with similarly treated subjects on Day 44: rats in the 2 mg/kg group had higher pinning frequencies than rats in the 0 or 1 mg/kg groups. In contrast, females in the 1 and 2 mg/kg groups did not have increased activity or rough and tumble play fighting. These data are consistent with the few correlational studies in human children which suggest changes in social behaviors associated with elevated tissue cadmium levels. Cadmium Infancy Heavy metal Pin
Juvenile Crossover
Rat Rough and tumble play Investigation
Activity
Social behaviors
discrimination task is enhanced by cadmium exposure in infancy [53]. One group of investigators [14] reported decreased locomotor activity and a deficit in homing behavior of 7-day-old rats who received 1 or 2 mg/kg cadmium on Day 6. When subsequently tested at 60--70 days of age, cadmium-treated rats took longer to enter the dark compartment in a passive avoidance task and had a deficit in performance three days later in a retention test. In another study using rats [31] lever press responding during the transition from FR 25 to FR 75 was increased in adults that had been given a single injection of CdC12 (3 mg CdCl~/kg) one day after birth. Others have found no effects of cadmium during gestation on performance in a spatial discrimination task, although locomotor activity was depressed [9]. Studies of nervous system pathology are also limited. A single dose of cadmium (2 mg/kg) given on Day 4 after birth produced transient cerebellar damage without affecting bodyweight [68]. A 4 mg/kg dose, which decreased bodyweight and subsequently induced behavioral hyperactivity, caused necrosis and hemorrhage in cerebral cortex, cerebellar hemispheres and the caudate-putamen area of the brain 4 days after treatment. On Day 19 the cortical lesions were still visible but the caudate-putamen and cerebellum appeared normal. Others [34] report a necrosis of the neostriatum and corpus callosum following a single 3 mg/kg dose of cadmium on Day 5, an effect blocked by coadministration of diethyldithiocarbamate. No nervous system damage was observed at a 2.75 mg/kg dose. There is no information to our knowledge, other than correlational studies with children noted above, about the influ-
CADMIUM is a toxic metal found in some pesticides, fertilizers and sewage sludge, and is also a by-product of coal combustion [6,40]. It occurs naturally with other heavy metals such as lead and zinc; blood and hair levels of lead and cadmium are often positively correlated. Although high doses of cadmium lead to a number of disorders [17], including testicular necrosis, nervous system damage, anosmia, kidney failure, hypertension and pulmonary dysfunction, relatively little is known regarding effects of low level exposure to this material during infancy. Except at high doses, cadmium does not effectively cross the placenta, although it can affect levels of other trace metals in the fetus (e.g., [9,54]) and neonates [60]. In correlational studies with children, researchers have reported that elevated hair cadmium is related to several behavioral and electrophysiological variables (e.g., [19-22, 29, 39, 40, 58, 59]), including increases in classroom acting out behavior; frequency of borderline intelligence, mental retardation, and learning disability; number of evoked potential peak measures from occipital and parietal derivations; and decreases in WlSC-R IQs and Wide Range Achievement Test scores in reading, spelling and arithmetic. Despite these findings with humans there are very few investigations of the effects of low level cadmium exposure on behavioral or neural development in experimental animals. With some exceptions [51,52], studies with rats typically have found later hyperactivity following exposure during infancy [41, 44, 53, 68]. In contrast, mature rats have depressed locomotion when given cadmium in adulthood [18,55]. There is evidence that reversal learning of a visual
135
136
H O L L O W A Y A N D THOR
ence of early cadmium exposure on subsequent social behaviors of developing subjects, despite the fact that deficits in social competence are often a major criterion in diagnosis of childhood behavior disorders. The following experiments report the effects of a single cadmium exposure during the first week after birth on growth and survival, locomotor activity, and rough and tumble play fighting behavior of juvenile rats. METHOD
Two experiments using essentially the same procedures were conducted. To avoid redundancy the methods for each are presented together, with differences in methodologies noted where they occur.
Subjects Male and female Long Evans hooded rats born in our breeding colony were used as subjects. All litters (Experiment 1 n=9; Experiment 2 n= 14) were culled to nine pups (at least 4 males and 4 females/litter) on Day 1 (day of birth= D a y 0). Litters remained undisturbed until Day 5 (Experiment 1) or Day 6 (Experiment 2). Food and water were available ad lib via external sources and the lights were on a 12:12 LD cycle with lights on at 2:00 p.m.
Experimental Treatment On Day 5 (Experiment 1) or Day 6 (Experiment 2) after birth all pups and mothers were weighed. The four heaviest males and four heaviest females were assigned to one of four cadmium dose groups and individually identified by toe clip. Each male and female pup was injected subcutaneously (SC) on the back with either 0, I, 2 or 4 mg Cd/kg body weight (Experiment 1) or 0, 1, 2 or 3 mg Cd/kg (Experiment 2). Cadmium chloride (Alfa Products, Danvers, MA; 99% pure) was dissolved in the vehicle (0.9% NaCI), and injected in the volume 2/xl/g body weight. All doses are expressed in terms of elemental cadmium. All pups were then returned to their mothers. In Experiment 1 pups in each litter were weighed on days 5, 6, 7, 8, 10, 15, and 20. An assessment of stomach milk content was made for each rat on Days 6, 7 and 8 using a modified version of the procedure described elsewhere [43]. Each pup was given a score of 0 if no milk was visible, 1 if milk was present but did not extend completely across the abdomen, and 2 if the milk band totally crossed the abdominal area. In Experiment 2 pups were weighed on Days 6, 7, 10, 15 and 20. An assessment of stomach milk content was made on Days 6 and 7. Litters were placed in clean cages on Day 10. Mothers and the uninjected male pup were removed from each litter on Day 22 and the remaining subjects transferred into clean cages.
Behavioral Testing Locomotor activity. On Day 23 (Experiment 1) or Day 25 (Experiment 2), 1-3 hr after lights on, subjects from a litter were placed in unfamiliar plastic cages (41 x 51 x 22 cm) containing clean shavings. Number of cage quadrants entered by each subject during minutes 0-5, 10-15, and 20-25 after placement into the cages were recorded. Testing was blind only with regard to cadmium treatment. At the conclusion of testing subjects were weighed and placed into clean plastic cages with like sex littermates. Social interaction test. On Day 28 subjects were isolated in clean plastic cages (41x51x22 cm) with food and water
available. Approximately 20 hr later, the number of cage quadrants entered by each subject in 2 min was recorded. Immediately thereafter a novel juvenile of the same sex as the subject was introduced into the cage. The duration and frequency of social investigation of the intruder by the subject, frequency of crossover by the subject and frequency and duration of pinning by the subject were recorded for 10 rain on an event recorder. These measures have been described in detail elsewhere [ 11-13]. Social investigation was recorded whenever the subject's head was in close proximity (<2 cm) and directed toward the intruder's body (excluding the tail) and the subject was sniffing, following or otherwise investigating the intruder. A crossover occurred whenever the resident's body passed over or under the intruder's body. Pinning occurred when one rat was on its back with the other standing over him. The group-housed stimulus animals were of the same age and sex as the subject. When housed in groups, juvenile rats seldom initiate rough and tumble play but will respond appropriately when solicited by another rat [35]. Enough stimulus animals were available so that each was only used once each day. Testing was done in dim red light during the last half of the LD cycle. On Day 44 a second social interaction test was performed with subjects from Experiment 2, in which unfamiliar rats that received the same cadmium treatment in infancy were observed together. Subjects were isolated in plastic cages and members of one litter were arbitrarily designated residents, members of the other litter as intruders. Approximately 20 hr later, during the dark phase of the LD cycle, subjects designated as intruders were placed in the cage of subjects assigned to the resident group. Members of each pair had received the same cadmium dosage on Day 6. The number and duration of pins by each rat were recorded for the next 20 min after which each rat was weighed. RESULTS
The data from these experiments were analyzed in two different ways. Because the highest cadmium dose was higher in Experiment 1 (4 mg/kg) than in Experiment 2 (3 mg/kg), growth and survival data from these experiments were analyzed separately. Since subjects from the 3 and 4 mg/kg doses were not used in the postweaning behavioral testing due to high mortality and/or stunted growth, data for 25-min activity on Day 23 and rough and tumble play on Day 29 (from both experiments) were analyzed in single A N O V A s with Replication included as a factor in the analyses. Sex and Cadmium Dose were treated as within litter variables. Furthermore, only litters for which all data were available were included in the analyses. Thus, one litter from Experiment 1 and two litters from Experiment 2 were not included in the analyses due to deaths before weaning. Although by doing this our overall N and the d f f o r the error terms were reduced, we were able to statistically utilize the within litter nature of the design and account for the between litter variability.
Growth and Survival--Experiment 1 Body weights and survival frequencies of the rats on Day 20 are presented in Table 1. With the exception of one female death in the 2 mg/kg group, all subjects in the 0, 1, and 2 mg groups survived until weaning. In the 4 mg/kg group, however, mortality rates were extremely high; only 56% and 33% of the males and females were alive on day 10, 22% and 33%, respectively on Day 20. Body weight on Day 20 reflects the
CADMIUM AND SOCIAL BEHAVIOR
137
TABLE 1 BODY WEIGHT(MEAN -+ SE) AND SURVIVAL(IN PARENTHESES)ON DAY 20 OF NEONATAL RATS FROM EXPERIMENTS 1 AND 2 THATRECEIVED CdCI~IN INFANCY Dose (mg Cd/kg)
Experiment 1" Male Female Experiment 2T Male Female
0
1
2
3
4
46.1 ± 1.7 (100) 44.5 ± 1.3 (100)
45.0 ± 1.4 (100) 45.3 ± 1.8 (100)
45.3 ± 1.3 (100) 44.9 ± 1.5 (88.9)
--
26.1 ± 2.3 (22.2) 28.1 ± 8.9 (33.3)
41.6 _+_1.3 (100) 40.7 ± 1.2 (100)
41.7 ± 1.2 (100) 39.7 ± 1.2 (100)
38.1 ± 1.3 (100) 38.9 ± 1.1 (100)
--
30.8 ± 2.45 (78.6) 30.9 ± 2.25 (85.7)
*n=9 on Day 5 when CdC12 was administered. tn= 14 on Day 6 when CdClz was administered. 5Differs significantly (p<0.05) from the 0 mg group.
mortality pattern. There were no body weight differences among 0, 1, and 2 mg/kg groups. In contrast, rats receiving 4 mg/kg Cd weighed much less than all other groups. Because only two male and three females in the 4 mg/kg group survived, statistical comparisons were not made. Pups that received 0, l, or 2 mg Cd/kg had normal growth and did not differ in weight on any day [12.8, 14.7, 16.9 and 18.9, mean weight (g) of male and female rats collapsed across sex and cadmium treatment on days 5, 6, 7 and 8 days of age, respectively]. In contrast, rats in the 4 mg Cd/kg group had no increase in body weight, regardless of sex [12.8, 12.7, 12.8 and 12.9, mean weight (g) of male and female rats in the 4 mg Cd/kg group on days 5, 6, 7 and 8]. This was reflected in a significant Age x Treatment Interaction, F(9,72)=44.5, p<0.001. Similar results were found for the stomach index scores, where pups in the 4 mg/kg group had scores significantly lower than the other three groups on all test days after treatment [1.9_+0.1, 2.0_+0, 1.9_+0.1 and 0.7_+0.3, mean stomach scores of pups (mean_+ SE) collapsed across sex and test day, who received 0, 1, 2 or 4 mg Cd/kg on Day 5], F(3,24)=85.1, p<0.001. Nearly all pups in the 0, 1 and 2 mg/kg groups had the maximum milk index score, indicating a full stomach; most pups in the 4 mg/kg group had either an empty or partially full stomach.
Growth and Survival--Experiment 2 Three male and two females receiving 3 mg/kg died before Day 20. One male and one female died between Days 6 and 10; the others three deaths occurred between Days 10 and 15. Body weights on Day 20 are in Table 1. Due to deaths in some cells, data from four litters were omitted from the analyses of body weight. The effect of cadmium treatment was significant, F(3,27)=36.1, p<0.001: rats receiving 3 mg/kg weighed less than the other three groups (p<0.05), which didn't differ from each other. There were no differences in body weights of the pups on Day 6 (14.3, 14.3, 14.0, 14.2 for pups, collapsed across sex, in the 0, 1, 2 and 3 mg/kg groups, respectively). On Day 7, 24 hr after treatment, the depression in body weight of rats in the 3 mg/kg group was evident (16.6, 16.5, 16.0 and 15.3 g for rats in the 0, 1, 2 and 3
MALES
FEMALES
-o~'w~_ °c°40. 30.
--~-
01
2
01
2
Dose (rag Cd/kg) FIG. 1. Cagequadrantsentered (mean±SE) for 23-25-day-oldmale and female rats (n=20/group) who received cadmium in infancy.
mg/kg groups). This was reflected in the significant Age × Dose Interaction, F(3,39)=18.1, p<0.001. Whereas all subjects had stomach scores of 2 on Day 6, indicating a full stomach; on Day 7, stomach scores were significantly depressed (p<0.01) in males receiving 3 mg Cd/kg (2.0_+0, 2.0_+0, 1.9_+0.1 and 1.4_+2 for male rats in the 0, 1, 2 and 3 mg/kg groups, respectively), but only slightly depressed in females receiving this dose (2.0_+0, 2.0_+0, 2.0_+0 and 1.6_+0.2 for female rats in the 0, 1, 2 and 3 mg/kg groups).
Activity and Body Weight--Day 23 Data from 8 litters in Experiment I and 12 litters in Experiment 2 were used. Body weight. Males weighed more than females, F(I,18)= 7.47, p<0.025 (see Table 2). Also significant were effects of Dose, and the Sex x Dose, Replication x Dose, and Replication x Dose x Sex Interactions, Fs(2,36)=3.96, 3.93, 5.37 and 4.61, respectively, all ps<0.05. The primary
138
HOLLOWAY AND THOR TABLE 2 BODY WEIGHTS (MEAN -+ SE) OF MALE AND FEMALE RATS FROM EXPERIMENTS 1 AND 2 THAT RECEIVED CdC12IN INFANCY Dose (mg Cd/kg) Age
Sex
Experiment 1
Day 23*
Experiment 2
Experiment 1
Day 29t
Experiment 2
0
1
Male Female Male Female
60.4 55.7 60.8 58.2
_+ 1.6 ± 1.7 ± 2.6 ± 2.1
57.9 57.9 59.1 54.7
Male Female Male Female
114.8 101.0 84.5 79.0
_+ 3.4 ± 3.7 ± 3.6 _+ 2.0
110.7 104.3 80.3 74.5
± ± ± ±
2 1.5 2.3 2.6 2.1
59.3 56.8 54.9 56.3
_+ 2.8 ± 3.8 _+ 2.5 ± 3.2
112.1 101.9 75.6 72.4
± 1.6 ± 1.4 ± 2.7 _+ 2.2 ± ± ± ±
2.8 2.8 3.05 3.15
*n=20, 8 from Replication 1 and 12 from Experiment 2. tn=18, 8 from Replication 1 and 10 from Experiment 2. 5Differs significantly (,o<0.05) from the 0 mg group. MALES
FEMALES
MALES
FEMALES
80
6O v
~
20.
40. c3
== "E
20
E
0 0 1 2
0
1 2
0
1
2
0
1 2
Cadmium Dose (mg C d / k g )
FIG. 2. Pinning duration (mean_+SE) and pinning frequency (mean±SE) on Day 29 for male and female rats (n= 18/group) who received cadmium in infancy.
reason for the triple interaction was that in Replication 1 there was no effect of cadmium dose in males or females. H o w e v e r in Replication 2, the 2 mg/kg dose depressed body weights in males but not their female littermates. Activity. Total number of cage quadrants entered by each subject during minutes 0-5, 11-15, and 20-25 of the observation period was used in the analyses. Only the Dose Effect and Dose x Sex Interaction were significant, Fs(2,36)=4.07 and 4.39, respectively, ps<0.05. Using tests for simple effects [64], activity of males in the 2 mg/kg group was increased by early exposure to cadmium (p<0.01). The activity of females was not affected by cadmium exposure (see Fig. 1).
Social Behavior and Body Weight--Day 29 Due to inadvertent loss of females from 2 litters in Experiment 2 between Day 23 and Day 29 only 10 litters from Experiment 2 are represented. Body weight. The main effects of Sex, F(1,16)=31.86, p<0.001, and Cadmium Dose, F(2,32)=5.76, p<0.01, were
significant (see Table 2). H o w e v e r the effect of Replication, F(1,16)=67.86, p<0.001, and the Dose x Replication Interaction, F(2,32)= 3.66, p<0.05, were also significant. Subjects from Replication 1 weighed significantly more than those from Replication 2. Furthermore, the depressant effect of cadmium on body weight was only observed in Replication 2. Unlike Day 23, the effect of cadmium on body weight was evident in both males and females (Table 2). Investigation frequency and duration. No main effects or interactions were significant for Investigation Frequency. For Investigation Duration, only the main effect of Dose was significant, F(2,32)=4.65, p<0.025, with rats in both 1 and 2 mg cadmium groups having depressed investigation relative to controls [143.5___7.4, 122.6+_5.8, and 127.5-+7.1 seconds investigation ( m ean + SE) for subjects in the 0, 1 and 2 mg/kg groups, respectively]. Crossover frequency. Males engaged in more crossovers than females [25.5_+1.6 vs. 19.6_-_1.4, respectively (mean+-SE)], F(1,16)=8.35, p<0.025. No other effects were significant.
CADMIUM AND SOCIAL BEHAVIOR
Pinning frequency and duration. Effects were similar for each behavior. Although in both cases the effects of cadmium dose were significant (ps<0.005), the significant Dose × Sex Interactions [Fs(2,32)=8.33, p<0.005 and 4.53, p<0.025 for pinning frequency and duration, respectively] best describe the results. F o r both behaviors, cadmium exposure in infancy increased pinning by male subjects (ps<0.01), but had no effect on pinning by their female littermates (Fig. 2). F o r both pinning frequency and duration the effect of Replication was significant, F(1,16)=12.5 and 9.5, ps<0.01, respectively. Subjects in Replication 1 had higher pinning frequencies and longer pinning durations than those in Replication 2. No interactions involving Replication were significant. Males had marginally higher pinning frequencies than females (19.7_+ 1.7 vs. 15.2_+ 1.3, respectively), F(1,16) = 4.36, p < 0.10, and significantly longer pinning durations than their female littermates [51.8_+ 5.4 vs. 35.2_+3.7 sec (mean_+SE)], F(1,16) = 5. 08, p<0.05. Social Interaction Test--Day 45 Male subjects from Experiment 2 were tested at 45 days of age with a rat from another litter that received the same treatment in infancy. Unfamiliar juvenile rats 44 days of age will form a dominance structure when placed together for 20 min (Holloway and Thor, unpublished), i.e., one rat pins much more than its partner, especially after the initial 5 minutes of interaction. Consequently, data were analyzed with Cadmium Dose and Dominance status (subject with greatest pin duration designated as dominant) both treated as within group factors. Twelve litters had subjects in each cadmium dose group, allowing us to test six pairs at each cadmium dose. Body weight. Only the effect of Dominance was significant, F(1,5)=9.4, p<0.05, with dominant weighing more than the submissive rats [183.6_+6.7 vs. 163.2_+4.8 g, respectively (mean_+SE)]. Although we have previously not found body weight to be a determining factor in dominance of juveniles, others have suggested that body weight is a critical variable [37]. Pinning duration. Because pinning duration was used to designate dominance status, the effect of Dominance was, by definition, significant, F(1,5)=9.4, p<0.05: dominant subjects spent about four times as much time pinning the subordinant rat as vice versa [200.0_+22.1 vs. 58.8_+9.6 sec (mean_+SE)]. Although pinning duration increased with cadmium dose [119.2_+25.5, 127.5_+33:7 and 141.6_+29.9 sec (mean_+SE) for rats in the 0, 1 and 2 mg/kg groups, respectively], the effect was not significant. Pinning frequency. As with pinning duration, dominant rats had higher pinning frequencies than their subordinant partner, 48.8_+3.6 vs. 20.2_+3.5 (mean_+SE), F(1,5)=20.9, p<0.01. In addition, pinning frequency increased significantly with cadmium dose [26.3_+4.8, 36.6_+6.8 and 40.5-+5.9 (mean_+SE) for rats in the 0, 1 and 2 mg/kg groups, respectively], F(2,10)=4.59, p<0.05. Using Dunnett's t, only rats in the 2 mg/kg group differed significantly (p<0.05) from the controls. The Dominance × Cadmium Interaction was not significant. DISCUSSION
The results from these two studies allow several conclusions. The dose-response curve for cadmium as regards growth and survival was steep in both males and females. The toxicity of this substance in the neonate was very appar-
139 ent. Mortality and growth deficits were high in rats receiving 3 or 4 mg/kg, but cadmium doses of I and 2 mg/kg produced virtually no such effects. Few animals receiving 4 mg/kg cadmium were alive on Day 20. Behavioral effects during the juvenile period were also significant. Male subjects receiving 2 mg/kg cadmium had increased locomotor activity in a 25-min test in a novel chamber and engaged in more rough and tumble play fighting behavior (i.e., pinning) than controls. Social investigation and crossover (a measure of play solicitation) were not affected. Unexpectedly, females receiving the same dosages did not have altered activity or play fighting behavior in either Experiment 1 or Experiment 2, even though female growth and mortality were equally affected at the higher doses of the chemical. The effects of a single cadmium dose on growth and survival are consistent with several recent reports (e.g., [44,68]). The growth effects were observed during the first 24 hr after treatment, as evidenced by weight loss and stomachs that were either empty or only partially filled with milk. There were no deaths during the first 24 hr after exposure; most deaths occurred 5-15 days after treatment. The steep dose-response curve is typical of cadmium and may reflect the finite metallothionein binding capacity of the subjects. These small proteins avidly bind several trace minerals, including cadmium and zinc, and are present in relatively high concentrations in the neonatal rat [66]. Although metallothionein binding capacity does not appear to explain the developmental changes in cadmium-induced gonadal toxicity observed in rats [67], effects on growth and survival may be due to inability of the limited metallothionein pool to accommodate the high doses of cadmium. Increased locomotor activity, similar to what we observed in our male subjects that received early cadmium exposure, has been reported in several independent investigations. In some cases this increase occurred simultaneously with depressed body weight [44,68]. The few survivors of the 3 and 4 mg/kg dose groups were extremely underweight and hyperactive, entering nearly 100 cage quadrants during the test period. The mechanisms responsible for this behavioral change in subjects receiving the lower doses of cadmium have received little attention. The results of the few available studies have tentatively suggested the answer may lie in the altered turnover rates of the transmitters norepinephrine, dopamine and serotonin following early exposure to cadmium [41], There is also some suggestion that apomorphineinduced activity may be affected by early cadmium exposure [51]. It is also possible that like lead [23], cadmium affects the nervous tissue indirectly by destroying or altering in some way the endothelial cells of the developing brain vasculature, leading to nervous system damage [32]. If this is the case, however, the effects are not due to enduring neuropathology but to more subtle changes deriving from transient nervous system lesions. F o r example, others [68] have reported limited necrosis and hemorrhage in restricted areas of the cerebellum of some rats receiving a single injection of 2 mg Cd/kg on Day 4 after birth; no tissue damage was evident 19 days after the cadmium exposure. In other work [44] no evidence of nervous system pathology was observed in rat pups given 2 rag Cd/kg on Day 5 or in adult rats who received 3 mg CdCIJkg (1.84 mg Cd/kg) on Day 1 after birth [31]. Another possible mechanism for these effects may only indirectly involve cadmium; depending instead on the interaction of cadmium with other essential metals such as zinc, copper and iron. In adults cadmium competes with
140
H O L L O W A Y A N D THOR
these essential metals in tissues (e.g., [38]). In infants, a single cadmium injection (10 ~mole CdCIJkg) on Day 9 resulted in depressed levels of zinc in liver, kidney, cerebrum, cerebellum, heart and blood at 20 days of age [60] and increased copper levels in cerebellum. While major deficiencies in these essential metals during development result in growth retardation, it isn't known if the tissue redistribution of these metals following cadmium exposure in infancy is sufficient to affect behavioral development in the rat. The elevation in pinning, one index of rough and tumble play fighting behavior in juvenile rats, following early cadmium treatment may be a feature common in rats after heavy metal exposure in infancy. This is a relatively unique response, since there are few reports of experimental manipulations that increase play fighting behavior (e.g., [2, 3, 11,25, 35, 36, 61]). In other work we have found increased play fighting in juvenile rats after exposure to low levels of lead via the mother's milk [13]. This increased pinning following lead exposure occurred in the absence of depressed body weights. Several years ago other investigators made the casual observation that lead-treated juvenile rats engaged in more "aggression" than controls [28,48]. Since subsequent studies have revealed that juvenile rats rarely engage in adult-like aggression (e.g., [56]), those investigators were in all probability observing increased rough and tumble play fighting in their lead-exposed subjects. Although many pharmacological and lesion studies of rough and tumble play fighting have been conducted (see [37] and [62] for reviews), the neurochemical regulation of play is still not clear. Evidence to date suggests that purinergic, opioid, cholinergic and catecholaminergic mechanisms are involved (e.g., [2, 10, 12, 24, 36, 37, 62]), and that the medial septal area [3] and parafasicular nuclei of the thalamus [50] may play important roles in the expression of play fighting in males and females. The amygdala also appears to play a role in play fighting, but only in males [24]. The sex difference we observed in cadmium's effect on activity and pinning was unexpected, but was found in both experiments. Although others have reported activity increases in juvenile rats after cadmium exposure during infancy, no sex differences were reported [44,68]. In early work (reviewed in [5]) with adult rats, males were more susceptible to cadmium-induced mortality [15,30], and to the increase in hexobarbital sleeping time observed after cadmium exposure [8]. In contrast, females were reported to be more sensitive to cadmium-induced hypertension than males [49]. Play fighting in juvenile rats exhibits a sexual dimorphism (males > female) under some testing situations (e.g.,
[25,63]). This sex difference is not dependent on circulating male hormone, but rather on an organizational effect of androgen around the time of birth [27]. Cadmium treatment was on Day 5 or 6, near the end of the perinatal sensitive period for androgen organizational effects on play fighting, and may have altered in some way the neural systems involved with establishment of the male pattern of play fighting. Beatty et al. [4] found that castration of male rats as late as Day 6 after birth significantly reduced levels of juvenile play fighting. The neural effects of cadmium at the 2 mg/kg dose are transient at this age and dose [68] but may have permanently altered underlying structures subsequently involved with play fighting behavior. It is worth noting that sex differences are also present for a number of behavioral and cognitive disorders in children, including attention deficit disorder where boys are typically affected much more than girls [57]. Much of the research on heavy metal pollutants has ultimately focused on defining effects of low level lead exposure of children on school performance and the incidence of the Hyperactivity with Attention Deficit Disorder (e.g., [45]). A limited number of more recent studies have indicated that other minerals present in the environment, such as cadmium, may also adversely affect academic performance and possibly socialization behaviors of school age children (e.g., [22,42]). Because these elements occur with lead, and in the case of cadmium have body levels often correlated with lead, the interactive effects of these substances is also of interest. In several studies investigators have been able to classify learning disabled students with extremely high reliability (95%) on the basis of the configuration of the body burdens of several metal pollutants [39]. While rough and tumble play of juvenile rats may not be completely homologous with similar social behaviors of human children, there are enough similarities across species regarding the morphology and control mechanisms of rough and tumble play (including humans) [26] to indicate that effects of low levels of metal pollutants other than lead on social interaction patterns of children should be assessed. This paradigm, using rats, also provides the opportunity to study the interactive effects of various pollutants (e.g., lead and cadmium), in conjunction with other experimental manipulations, on subsequent social and intellectual competence of the developing organism. ACKNOWLEDGEMENTS The authors wish to thank Dr. Herman Spitz for his comments on this paper and Mrs. Patricia Conlow for typing the manuscript. Mr. Larry Carr maintained the animals and the colony room.
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CADMIUM AND SOCIAL BEHAVIOR
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141 32. Nolan, C. V. and Z. A. Shaikh. The vascular endothelium as a target tissue in acute cadmium toxicity. Life Sci 39: 1403-1409, 1986. 33. Normansell, L. and J. Panksepp. Effects of clonidine and yohimbine on the social play of juvenile rats. Pharmacol Bioehem Behav 22: 881-883, 1985. 34. O'Callaghan, J. P. and D. B. Miller. Diethyldithiocarbamate increases distribution of cadmium to brain but prevents cadmium-induced neurotoxicity. Brain Res 370: 354--358, 1986. 35. Panksepp, J. and W. W. Beatty. Social deprivation and play in rats. Behav Neural Biol 30: 197-206, 1980. 36. Panksepp, J., J. Jalowiec, F. G. De Eskinazi and P. Bishop. Opiates and play dominance in juvenile rats. Behav Neurosci 99: 441-453, 1985. 37. Panksepp, J., S. Siviy and L. Normansell. The psychobiology of play: Theoretical and methodological perspectives. Neurosci Biobehav Rev 8: 465--492, 1984. 38. Petering, H. G., H. Choudhury and K. L. Stemmer. Some effects of oral ingestion of cadmium on zinc, copper, and iron metabolism. Environ Health Perspect 28: 97-106, 1979. 39. Pihl, R. O. and M. Parkes. Hair element content in learning disabled children. Science 198: 204-206, 1977. 40. Piscator, M. Dietary exposure to cadmium and health effects: Impact of environmental changes. Environ Health Perspect 63: 127-132, 1985. 41. Rastogi, R. B., Z. Merali and R. L. Singhal. Cadmium alters behavior and the biosynthetic capacity for catecholamines and serotonin neonatal rat brain. J Neurochem 28: 789-794, 1977. 42. Rimland, B. and G. E. Larson. Hair mineral analysis and behavior: An analysis of 51 studies. J Learn Disabil 16: 279-285, 1983. 43. Ruppert, P. H., K. F. Dean and L. W. Reiter. Neurobehavioral toxicity of triethyltin in rats as a function of age at postnatal exposure. Neurotoxicology 5: %22, 1984. 44. Ruppert, P. H., K. F. Dean and L. W. Reiter. Development of locomotor activity of rat pups exposed to heavy metals. Toxicol Appl Pharrnacol 78: 6%77, 1985. 45. Rutter, M. and R. R. Jones (Eds.). Lead Versus Health. Sources and Effects o f Low Level Lead Exposure. New York: John Wiley and Sons, 1984. 46. Sasser, L. B. and G. E. Jarboe. Intestinal absorption and retention of cadmium in neonatal rat. Toxicol Appl Pharmacol 41: 423-431, 1977. 47. Sasser, L. B., B. J. Kelman, A. A. Levin and R. K. Miller. The influence of maternal cadmium exposure or fetal cadmium injection on hepatic metallothionein concentrations in the fetal rat. Toxicol Appl Pharmacol 80: 29%307, 1985. 48. Sauerhoff, M. W. and I. A. Michaelson. Hyperactivity and brain catecholamines in lead-exposed developing rats. Science 182: 1022-1024, 1973. 49. Schroeder, H. A. and W. H. Vinton, Jr. Hypertension induced in rats by small doses of cadmium. Am J Physiol 202: 515-518, 1962. 50. Siviy, S. M. and J. Panksepp. Dorsomedial diencephalic involvement in the juvenile play of rats. Behav Neurosci 99: 1103-1113, 1985. 51. Smith, M. J., B. Garber and R. O. Pihl. Altered behavioral response to apomorphine in cadmium exposed rats. Neurobehav Toxicol Teratol 5: 161-165, 1983. 52. Smith, M. J., R. O. Phil and B. Farrell. Long term effects of early cadmium exposure on locomotor activity in the rat. Neurobehav Toxicol Teratol 7: 1%22, 1985. 53. Smith, M. J., R. O. Pihl and B. Garber. Postnatal cadmium exposure and long term behavioral changes in the rat. Neurobehav Toxicol Teratol 4: 283-287, 1982. 54. Sowa, B. and E. Steibert. Effect of oral cadmium administration to female rats during pregnancy on zinc, copper, and iron content in placenta, foetal liver, kidney, intestine, and brain. Arch Toxicol 56: 256-262, 1985. 55. Squibb, R. E., Jr. and R. L. Squibb. Effects of food toxicants on voluntary wheel running in rats. J Nutr 109: 767-772, 1979.
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56. Takahashi, L. K. and R. K. Lore. Play fighting and the development of agonistic behavior in male and female rats. Aggress Behav 9: 217-227, 1983. 57. Taylor, E. A. Childhood hyperactivity. Br J Psychiatry 149: 562-573, 1986. 58. Thatcher, R. W., M. L. Lester, R. McAlaster and R. Horst. Effects of low levels of cadmium and lead on cognitive functioning in children. Arch Environ Health 37: 159-166, 1982. 59. Thatcher, R. W., R. McAlaster and M. L. Lester. Evoked potentials related to hair cadmium and lead in children. Ann N Y Acad Sci 425: 384-390, 1984. 60. Thomas, D. J. and P. Mushak. Effects of cadmium exposure on zinc and copper distribution in neonatal rats. Arch Toxicol 58: 130-135, 1986. 61. Thor, D. H. and W. R. Holloway, Jr. Social play in juvenile rats during scopolamine withdrawal. Physiol Behav 32: 217-220, 1984. 62. Thor, D. H. and W. R. Holloway, Jr. Social play in juvenile rats: A decade of methodological and experimental research. Neurosci Biobehav Rev 8: 455-464, 1984.
HOLLOWAY AND THOR
63. Thor, D. H. and W. R. Holloway, Jr. Sex and social play in juvenile rats (R. norvegicus). J Comp Psychol 98: 276-284, 1984. 64. Winer, B. J. Statistical Principles in Experimental Design. New York: McGraw-Hill, 1971. 65. Wong, K-L., R. Cachia and C. D. Klaassen. Comparison of the toxicity and tissue distribution of cadmium in newborn and adult rats after repeated administration. Taxicol Appl Pharmacol 56: 317-325, 1980. 66. Wong, K-L. and C. D. Klaassen. Isolation and characterization of metallothionein which is highly concentrated in newborn rat liver. J Biol Chem 254: 12399-12403, 1979. 67. Wong, K-L. and C. D. Klaassen. Age difference in the susceptibility to cadmium-induced testicular damage in rats. Toxicol Appl Pharmacol 55: 456-466, 1980. 68. Wong, K-L. and C. D. Klaassen. Neurotoxic effects of cadmium in young rats. Toxicol Appl Pharmacol 63: 330-337, 1982.
0892-0362/88$3.00 + .00
Cadmium Exposure in Infancy: Effects on Activity and Social Behaviors of Juvenile Rats W I L L I A M R. H O L L O W A Y , JR. A N D D O N A L D H . T H O R
Research Department, E. R. Johnstone Training and Research Center, Bordentown, N J 08505 R e c e i v e d 4 N o v e m b e r 1986 HOLLOWAY, W. R., JR. AND D. H. THOR. Cadmium exposure in infancy: Effects on activity and social behaviors of juvenile rats. NEUROTOXICOL TERATOL 10(2) 135-142, 1988.--In two experiments male and female infant rats were given single injections of cadmium chloride (0, 1, 2, 3 or 4 mg CdJkg) on Day 5 or 6. Animals receiving the 3 and 4 mg/kg doses had high mortality rates at weaning; survivors were extremely underweight and were not used in postweaning tests. Male subjects receiving 2 mg/kg in infancy were significantly more active after weaning than littermates who had received 0 or 1 mg/kg doses, and on Day 29 they also engaged in significantly more rough and tumble play with a nontreated partner than did rats in the other groups. This effect of early cadmium exposure was also evident when males were tested with similarly treated subjects on Day 44: rats in the 2 mg/kg group had higher pinning frequencies than rats in the 0 or 1 mg/kg groups. In contrast, females in the 1 and 2 mg/kg groups did not have increased activity or rough and tumble play fighting. These data are consistent with the few correlational studies in human children which suggest changes in social behaviors associated with elevated tissue cadmium levels. Cadmium Infancy Heavy metal Pin
Juvenile Crossover
Rat Rough and tumble play Investigation
Activity
Social behaviors
discrimination task is enhanced by cadmium exposure in infancy [53]. One group of investigators [14] reported decreased locomotor activity and a deficit in homing behavior of 7-day-old rats who received 1 or 2 mg/kg cadmium on Day 6. When subsequently tested at 60--70 days of age, cadmium-treated rats took longer to enter the dark compartment in a passive avoidance task and had a deficit in performance three days later in a retention test. In another study using rats [31] lever press responding during the transition from FR 25 to FR 75 was increased in adults that had been given a single injection of CdC12 (3 mg CdCl~/kg) one day after birth. Others have found no effects of cadmium during gestation on performance in a spatial discrimination task, although locomotor activity was depressed [9]. Studies of nervous system pathology are also limited. A single dose of cadmium (2 mg/kg) given on Day 4 after birth produced transient cerebellar damage without affecting bodyweight [68]. A 4 mg/kg dose, which decreased bodyweight and subsequently induced behavioral hyperactivity, caused necrosis and hemorrhage in cerebral cortex, cerebellar hemispheres and the caudate-putamen area of the brain 4 days after treatment. On Day 19 the cortical lesions were still visible but the caudate-putamen and cerebellum appeared normal. Others [34] report a necrosis of the neostriatum and corpus callosum following a single 3 mg/kg dose of cadmium on Day 5, an effect blocked by coadministration of diethyldithiocarbamate. No nervous system damage was observed at a 2.75 mg/kg dose. There is no information to our knowledge, other than correlational studies with children noted above, about the influ-
CADMIUM is a toxic metal found in some pesticides, fertilizers and sewage sludge, and is also a by-product of coal combustion [6,40]. It occurs naturally with other heavy metals such as lead and zinc; blood and hair levels of lead and cadmium are often positively correlated. Although high doses of cadmium lead to a number of disorders [17], including testicular necrosis, nervous system damage, anosmia, kidney failure, hypertension and pulmonary dysfunction, relatively little is known regarding effects of low level exposure to this material during infancy. Except at high doses, cadmium does not effectively cross the placenta, although it can affect levels of other trace metals in the fetus (e.g., [9,54]) and neonates [60]. In correlational studies with children, researchers have reported that elevated hair cadmium is related to several behavioral and electrophysiological variables (e.g., [19-22, 29, 39, 40, 58, 59]), including increases in classroom acting out behavior; frequency of borderline intelligence, mental retardation, and learning disability; number of evoked potential peak measures from occipital and parietal derivations; and decreases in WlSC-R IQs and Wide Range Achievement Test scores in reading, spelling and arithmetic. Despite these findings with humans there are very few investigations of the effects of low level cadmium exposure on behavioral or neural development in experimental animals. With some exceptions [51,52], studies with rats typically have found later hyperactivity following exposure during infancy [41, 44, 53, 68]. In contrast, mature rats have depressed locomotion when given cadmium in adulthood [18,55]. There is evidence that reversal learning of a visual
135
136
H O L L O W A Y A N D THOR
ence of early cadmium exposure on subsequent social behaviors of developing subjects, despite the fact that deficits in social competence are often a major criterion in diagnosis of childhood behavior disorders. The following experiments report the effects of a single cadmium exposure during the first week after birth on growth and survival, locomotor activity, and rough and tumble play fighting behavior of juvenile rats. METHOD
Two experiments using essentially the same procedures were conducted. To avoid redundancy the methods for each are presented together, with differences in methodologies noted where they occur.
Subjects Male and female Long Evans hooded rats born in our breeding colony were used as subjects. All litters (Experiment 1 n=9; Experiment 2 n= 14) were culled to nine pups (at least 4 males and 4 females/litter) on Day 1 (day of birth= D a y 0). Litters remained undisturbed until Day 5 (Experiment 1) or Day 6 (Experiment 2). Food and water were available ad lib via external sources and the lights were on a 12:12 LD cycle with lights on at 2:00 p.m.
Experimental Treatment On Day 5 (Experiment 1) or Day 6 (Experiment 2) after birth all pups and mothers were weighed. The four heaviest males and four heaviest females were assigned to one of four cadmium dose groups and individually identified by toe clip. Each male and female pup was injected subcutaneously (SC) on the back with either 0, I, 2 or 4 mg Cd/kg body weight (Experiment 1) or 0, 1, 2 or 3 mg Cd/kg (Experiment 2). Cadmium chloride (Alfa Products, Danvers, MA; 99% pure) was dissolved in the vehicle (0.9% NaCI), and injected in the volume 2/xl/g body weight. All doses are expressed in terms of elemental cadmium. All pups were then returned to their mothers. In Experiment 1 pups in each litter were weighed on days 5, 6, 7, 8, 10, 15, and 20. An assessment of stomach milk content was made for each rat on Days 6, 7 and 8 using a modified version of the procedure described elsewhere [43]. Each pup was given a score of 0 if no milk was visible, 1 if milk was present but did not extend completely across the abdomen, and 2 if the milk band totally crossed the abdominal area. In Experiment 2 pups were weighed on Days 6, 7, 10, 15 and 20. An assessment of stomach milk content was made on Days 6 and 7. Litters were placed in clean cages on Day 10. Mothers and the uninjected male pup were removed from each litter on Day 22 and the remaining subjects transferred into clean cages.
Behavioral Testing Locomotor activity. On Day 23 (Experiment 1) or Day 25 (Experiment 2), 1-3 hr after lights on, subjects from a litter were placed in unfamiliar plastic cages (41 x 51 x 22 cm) containing clean shavings. Number of cage quadrants entered by each subject during minutes 0-5, 10-15, and 20-25 after placement into the cages were recorded. Testing was blind only with regard to cadmium treatment. At the conclusion of testing subjects were weighed and placed into clean plastic cages with like sex littermates. Social interaction test. On Day 28 subjects were isolated in clean plastic cages (41x51x22 cm) with food and water
available. Approximately 20 hr later, the number of cage quadrants entered by each subject in 2 min was recorded. Immediately thereafter a novel juvenile of the same sex as the subject was introduced into the cage. The duration and frequency of social investigation of the intruder by the subject, frequency of crossover by the subject and frequency and duration of pinning by the subject were recorded for 10 rain on an event recorder. These measures have been described in detail elsewhere [ 11-13]. Social investigation was recorded whenever the subject's head was in close proximity (<2 cm) and directed toward the intruder's body (excluding the tail) and the subject was sniffing, following or otherwise investigating the intruder. A crossover occurred whenever the resident's body passed over or under the intruder's body. Pinning occurred when one rat was on its back with the other standing over him. The group-housed stimulus animals were of the same age and sex as the subject. When housed in groups, juvenile rats seldom initiate rough and tumble play but will respond appropriately when solicited by another rat [35]. Enough stimulus animals were available so that each was only used once each day. Testing was done in dim red light during the last half of the LD cycle. On Day 44 a second social interaction test was performed with subjects from Experiment 2, in which unfamiliar rats that received the same cadmium treatment in infancy were observed together. Subjects were isolated in plastic cages and members of one litter were arbitrarily designated residents, members of the other litter as intruders. Approximately 20 hr later, during the dark phase of the LD cycle, subjects designated as intruders were placed in the cage of subjects assigned to the resident group. Members of each pair had received the same cadmium dosage on Day 6. The number and duration of pins by each rat were recorded for the next 20 min after which each rat was weighed. RESULTS
The data from these experiments were analyzed in two different ways. Because the highest cadmium dose was higher in Experiment 1 (4 mg/kg) than in Experiment 2 (3 mg/kg), growth and survival data from these experiments were analyzed separately. Since subjects from the 3 and 4 mg/kg doses were not used in the postweaning behavioral testing due to high mortality and/or stunted growth, data for 25-min activity on Day 23 and rough and tumble play on Day 29 (from both experiments) were analyzed in single A N O V A s with Replication included as a factor in the analyses. Sex and Cadmium Dose were treated as within litter variables. Furthermore, only litters for which all data were available were included in the analyses. Thus, one litter from Experiment 1 and two litters from Experiment 2 were not included in the analyses due to deaths before weaning. Although by doing this our overall N and the d f f o r the error terms were reduced, we were able to statistically utilize the within litter nature of the design and account for the between litter variability.
Growth and Survival--Experiment 1 Body weights and survival frequencies of the rats on Day 20 are presented in Table 1. With the exception of one female death in the 2 mg/kg group, all subjects in the 0, 1, and 2 mg groups survived until weaning. In the 4 mg/kg group, however, mortality rates were extremely high; only 56% and 33% of the males and females were alive on day 10, 22% and 33%, respectively on Day 20. Body weight on Day 20 reflects the
CADMIUM AND SOCIAL BEHAVIOR
137
TABLE 1 BODY WEIGHT(MEAN -+ SE) AND SURVIVAL(IN PARENTHESES)ON DAY 20 OF NEONATAL RATS FROM EXPERIMENTS 1 AND 2 THATRECEIVED CdCI~IN INFANCY Dose (mg Cd/kg)
Experiment 1" Male Female Experiment 2T Male Female
0
1
2
3
4
46.1 ± 1.7 (100) 44.5 ± 1.3 (100)
45.0 ± 1.4 (100) 45.3 ± 1.8 (100)
45.3 ± 1.3 (100) 44.9 ± 1.5 (88.9)
--
26.1 ± 2.3 (22.2) 28.1 ± 8.9 (33.3)
41.6 _+_1.3 (100) 40.7 ± 1.2 (100)
41.7 ± 1.2 (100) 39.7 ± 1.2 (100)
38.1 ± 1.3 (100) 38.9 ± 1.1 (100)
--
30.8 ± 2.45 (78.6) 30.9 ± 2.25 (85.7)
*n=9 on Day 5 when CdC12 was administered. tn= 14 on Day 6 when CdClz was administered. 5Differs significantly (p<0.05) from the 0 mg group.
mortality pattern. There were no body weight differences among 0, 1, and 2 mg/kg groups. In contrast, rats receiving 4 mg/kg Cd weighed much less than all other groups. Because only two male and three females in the 4 mg/kg group survived, statistical comparisons were not made. Pups that received 0, l, or 2 mg Cd/kg had normal growth and did not differ in weight on any day [12.8, 14.7, 16.9 and 18.9, mean weight (g) of male and female rats collapsed across sex and cadmium treatment on days 5, 6, 7 and 8 days of age, respectively]. In contrast, rats in the 4 mg Cd/kg group had no increase in body weight, regardless of sex [12.8, 12.7, 12.8 and 12.9, mean weight (g) of male and female rats in the 4 mg Cd/kg group on days 5, 6, 7 and 8]. This was reflected in a significant Age x Treatment Interaction, F(9,72)=44.5, p<0.001. Similar results were found for the stomach index scores, where pups in the 4 mg/kg group had scores significantly lower than the other three groups on all test days after treatment [1.9_+0.1, 2.0_+0, 1.9_+0.1 and 0.7_+0.3, mean stomach scores of pups (mean_+ SE) collapsed across sex and test day, who received 0, 1, 2 or 4 mg Cd/kg on Day 5], F(3,24)=85.1, p<0.001. Nearly all pups in the 0, 1 and 2 mg/kg groups had the maximum milk index score, indicating a full stomach; most pups in the 4 mg/kg group had either an empty or partially full stomach.
Growth and Survival--Experiment 2 Three male and two females receiving 3 mg/kg died before Day 20. One male and one female died between Days 6 and 10; the others three deaths occurred between Days 10 and 15. Body weights on Day 20 are in Table 1. Due to deaths in some cells, data from four litters were omitted from the analyses of body weight. The effect of cadmium treatment was significant, F(3,27)=36.1, p<0.001: rats receiving 3 mg/kg weighed less than the other three groups (p<0.05), which didn't differ from each other. There were no differences in body weights of the pups on Day 6 (14.3, 14.3, 14.0, 14.2 for pups, collapsed across sex, in the 0, 1, 2 and 3 mg/kg groups, respectively). On Day 7, 24 hr after treatment, the depression in body weight of rats in the 3 mg/kg group was evident (16.6, 16.5, 16.0 and 15.3 g for rats in the 0, 1, 2 and 3
MALES
FEMALES
-o~'w~_ °c°40. 30.
--~-
01
2
01
2
Dose (rag Cd/kg) FIG. 1. Cagequadrantsentered (mean±SE) for 23-25-day-oldmale and female rats (n=20/group) who received cadmium in infancy.
mg/kg groups). This was reflected in the significant Age × Dose Interaction, F(3,39)=18.1, p<0.001. Whereas all subjects had stomach scores of 2 on Day 6, indicating a full stomach; on Day 7, stomach scores were significantly depressed (p<0.01) in males receiving 3 mg Cd/kg (2.0_+0, 2.0_+0, 1.9_+0.1 and 1.4_+2 for male rats in the 0, 1, 2 and 3 mg/kg groups, respectively), but only slightly depressed in females receiving this dose (2.0_+0, 2.0_+0, 2.0_+0 and 1.6_+0.2 for female rats in the 0, 1, 2 and 3 mg/kg groups).
Activity and Body Weight--Day 23 Data from 8 litters in Experiment I and 12 litters in Experiment 2 were used. Body weight. Males weighed more than females, F(I,18)= 7.47, p<0.025 (see Table 2). Also significant were effects of Dose, and the Sex x Dose, Replication x Dose, and Replication x Dose x Sex Interactions, Fs(2,36)=3.96, 3.93, 5.37 and 4.61, respectively, all ps<0.05. The primary
138
HOLLOWAY AND THOR TABLE 2 BODY WEIGHTS (MEAN -+ SE) OF MALE AND FEMALE RATS FROM EXPERIMENTS 1 AND 2 THAT RECEIVED CdC12IN INFANCY Dose (mg Cd/kg) Age
Sex
Experiment 1
Day 23*
Experiment 2
Experiment 1
Day 29t
Experiment 2
0
1
Male Female Male Female
60.4 55.7 60.8 58.2
_+ 1.6 ± 1.7 ± 2.6 ± 2.1
57.9 57.9 59.1 54.7
Male Female Male Female
114.8 101.0 84.5 79.0
_+ 3.4 ± 3.7 ± 3.6 _+ 2.0
110.7 104.3 80.3 74.5
± ± ± ±
2 1.5 2.3 2.6 2.1
59.3 56.8 54.9 56.3
_+ 2.8 ± 3.8 _+ 2.5 ± 3.2
112.1 101.9 75.6 72.4
± 1.6 ± 1.4 ± 2.7 _+ 2.2 ± ± ± ±
2.8 2.8 3.05 3.15
*n=20, 8 from Replication 1 and 12 from Experiment 2. tn=18, 8 from Replication 1 and 10 from Experiment 2. 5Differs significantly (,o<0.05) from the 0 mg group. MALES
FEMALES
MALES
FEMALES
80
6O v
~
20.
40. c3
== "E
20
E
0 0 1 2
0
1 2
0
1
2
0
1 2
Cadmium Dose (mg C d / k g )
FIG. 2. Pinning duration (mean_+SE) and pinning frequency (mean±SE) on Day 29 for male and female rats (n= 18/group) who received cadmium in infancy.
reason for the triple interaction was that in Replication 1 there was no effect of cadmium dose in males or females. H o w e v e r in Replication 2, the 2 mg/kg dose depressed body weights in males but not their female littermates. Activity. Total number of cage quadrants entered by each subject during minutes 0-5, 11-15, and 20-25 of the observation period was used in the analyses. Only the Dose Effect and Dose x Sex Interaction were significant, Fs(2,36)=4.07 and 4.39, respectively, ps<0.05. Using tests for simple effects [64], activity of males in the 2 mg/kg group was increased by early exposure to cadmium (p<0.01). The activity of females was not affected by cadmium exposure (see Fig. 1).
Social Behavior and Body Weight--Day 29 Due to inadvertent loss of females from 2 litters in Experiment 2 between Day 23 and Day 29 only 10 litters from Experiment 2 are represented. Body weight. The main effects of Sex, F(1,16)=31.86, p<0.001, and Cadmium Dose, F(2,32)=5.76, p<0.01, were
significant (see Table 2). H o w e v e r the effect of Replication, F(1,16)=67.86, p<0.001, and the Dose x Replication Interaction, F(2,32)= 3.66, p<0.05, were also significant. Subjects from Replication 1 weighed significantly more than those from Replication 2. Furthermore, the depressant effect of cadmium on body weight was only observed in Replication 2. Unlike Day 23, the effect of cadmium on body weight was evident in both males and females (Table 2). Investigation frequency and duration. No main effects or interactions were significant for Investigation Frequency. For Investigation Duration, only the main effect of Dose was significant, F(2,32)=4.65, p<0.025, with rats in both 1 and 2 mg cadmium groups having depressed investigation relative to controls [143.5___7.4, 122.6+_5.8, and 127.5-+7.1 seconds investigation ( m ean + SE) for subjects in the 0, 1 and 2 mg/kg groups, respectively]. Crossover frequency. Males engaged in more crossovers than females [25.5_+1.6 vs. 19.6_-_1.4, respectively (mean+-SE)], F(1,16)=8.35, p<0.025. No other effects were significant.
CADMIUM AND SOCIAL BEHAVIOR
Pinning frequency and duration. Effects were similar for each behavior. Although in both cases the effects of cadmium dose were significant (ps<0.005), the significant Dose × Sex Interactions [Fs(2,32)=8.33, p<0.005 and 4.53, p<0.025 for pinning frequency and duration, respectively] best describe the results. F o r both behaviors, cadmium exposure in infancy increased pinning by male subjects (ps<0.01), but had no effect on pinning by their female littermates (Fig. 2). F o r both pinning frequency and duration the effect of Replication was significant, F(1,16)=12.5 and 9.5, ps<0.01, respectively. Subjects in Replication 1 had higher pinning frequencies and longer pinning durations than those in Replication 2. No interactions involving Replication were significant. Males had marginally higher pinning frequencies than females (19.7_+ 1.7 vs. 15.2_+ 1.3, respectively), F(1,16) = 4.36, p < 0.10, and significantly longer pinning durations than their female littermates [51.8_+ 5.4 vs. 35.2_+3.7 sec (mean_+SE)], F(1,16) = 5. 08, p<0.05. Social Interaction Test--Day 45 Male subjects from Experiment 2 were tested at 45 days of age with a rat from another litter that received the same treatment in infancy. Unfamiliar juvenile rats 44 days of age will form a dominance structure when placed together for 20 min (Holloway and Thor, unpublished), i.e., one rat pins much more than its partner, especially after the initial 5 minutes of interaction. Consequently, data were analyzed with Cadmium Dose and Dominance status (subject with greatest pin duration designated as dominant) both treated as within group factors. Twelve litters had subjects in each cadmium dose group, allowing us to test six pairs at each cadmium dose. Body weight. Only the effect of Dominance was significant, F(1,5)=9.4, p<0.05, with dominant weighing more than the submissive rats [183.6_+6.7 vs. 163.2_+4.8 g, respectively (mean_+SE)]. Although we have previously not found body weight to be a determining factor in dominance of juveniles, others have suggested that body weight is a critical variable [37]. Pinning duration. Because pinning duration was used to designate dominance status, the effect of Dominance was, by definition, significant, F(1,5)=9.4, p<0.05: dominant subjects spent about four times as much time pinning the subordinant rat as vice versa [200.0_+22.1 vs. 58.8_+9.6 sec (mean_+SE)]. Although pinning duration increased with cadmium dose [119.2_+25.5, 127.5_+33:7 and 141.6_+29.9 sec (mean_+SE) for rats in the 0, 1 and 2 mg/kg groups, respectively], the effect was not significant. Pinning frequency. As with pinning duration, dominant rats had higher pinning frequencies than their subordinant partner, 48.8_+3.6 vs. 20.2_+3.5 (mean_+SE), F(1,5)=20.9, p<0.01. In addition, pinning frequency increased significantly with cadmium dose [26.3_+4.8, 36.6_+6.8 and 40.5-+5.9 (mean_+SE) for rats in the 0, 1 and 2 mg/kg groups, respectively], F(2,10)=4.59, p<0.05. Using Dunnett's t, only rats in the 2 mg/kg group differed significantly (p<0.05) from the controls. The Dominance × Cadmium Interaction was not significant. DISCUSSION
The results from these two studies allow several conclusions. The dose-response curve for cadmium as regards growth and survival was steep in both males and females. The toxicity of this substance in the neonate was very appar-
139 ent. Mortality and growth deficits were high in rats receiving 3 or 4 mg/kg, but cadmium doses of I and 2 mg/kg produced virtually no such effects. Few animals receiving 4 mg/kg cadmium were alive on Day 20. Behavioral effects during the juvenile period were also significant. Male subjects receiving 2 mg/kg cadmium had increased locomotor activity in a 25-min test in a novel chamber and engaged in more rough and tumble play fighting behavior (i.e., pinning) than controls. Social investigation and crossover (a measure of play solicitation) were not affected. Unexpectedly, females receiving the same dosages did not have altered activity or play fighting behavior in either Experiment 1 or Experiment 2, even though female growth and mortality were equally affected at the higher doses of the chemical. The effects of a single cadmium dose on growth and survival are consistent with several recent reports (e.g., [44,68]). The growth effects were observed during the first 24 hr after treatment, as evidenced by weight loss and stomachs that were either empty or only partially filled with milk. There were no deaths during the first 24 hr after exposure; most deaths occurred 5-15 days after treatment. The steep dose-response curve is typical of cadmium and may reflect the finite metallothionein binding capacity of the subjects. These small proteins avidly bind several trace minerals, including cadmium and zinc, and are present in relatively high concentrations in the neonatal rat [66]. Although metallothionein binding capacity does not appear to explain the developmental changes in cadmium-induced gonadal toxicity observed in rats [67], effects on growth and survival may be due to inability of the limited metallothionein pool to accommodate the high doses of cadmium. Increased locomotor activity, similar to what we observed in our male subjects that received early cadmium exposure, has been reported in several independent investigations. In some cases this increase occurred simultaneously with depressed body weight [44,68]. The few survivors of the 3 and 4 mg/kg dose groups were extremely underweight and hyperactive, entering nearly 100 cage quadrants during the test period. The mechanisms responsible for this behavioral change in subjects receiving the lower doses of cadmium have received little attention. The results of the few available studies have tentatively suggested the answer may lie in the altered turnover rates of the transmitters norepinephrine, dopamine and serotonin following early exposure to cadmium [41], There is also some suggestion that apomorphineinduced activity may be affected by early cadmium exposure [51]. It is also possible that like lead [23], cadmium affects the nervous tissue indirectly by destroying or altering in some way the endothelial cells of the developing brain vasculature, leading to nervous system damage [32]. If this is the case, however, the effects are not due to enduring neuropathology but to more subtle changes deriving from transient nervous system lesions. F o r example, others [68] have reported limited necrosis and hemorrhage in restricted areas of the cerebellum of some rats receiving a single injection of 2 mg Cd/kg on Day 4 after birth; no tissue damage was evident 19 days after the cadmium exposure. In other work [44] no evidence of nervous system pathology was observed in rat pups given 2 rag Cd/kg on Day 5 or in adult rats who received 3 mg CdCIJkg (1.84 mg Cd/kg) on Day 1 after birth [31]. Another possible mechanism for these effects may only indirectly involve cadmium; depending instead on the interaction of cadmium with other essential metals such as zinc, copper and iron. In adults cadmium competes with
140
H O L L O W A Y A N D THOR
these essential metals in tissues (e.g., [38]). In infants, a single cadmium injection (10 ~mole CdCIJkg) on Day 9 resulted in depressed levels of zinc in liver, kidney, cerebrum, cerebellum, heart and blood at 20 days of age [60] and increased copper levels in cerebellum. While major deficiencies in these essential metals during development result in growth retardation, it isn't known if the tissue redistribution of these metals following cadmium exposure in infancy is sufficient to affect behavioral development in the rat. The elevation in pinning, one index of rough and tumble play fighting behavior in juvenile rats, following early cadmium treatment may be a feature common in rats after heavy metal exposure in infancy. This is a relatively unique response, since there are few reports of experimental manipulations that increase play fighting behavior (e.g., [2, 3, 11,25, 35, 36, 61]). In other work we have found increased play fighting in juvenile rats after exposure to low levels of lead via the mother's milk [13]. This increased pinning following lead exposure occurred in the absence of depressed body weights. Several years ago other investigators made the casual observation that lead-treated juvenile rats engaged in more "aggression" than controls [28,48]. Since subsequent studies have revealed that juvenile rats rarely engage in adult-like aggression (e.g., [56]), those investigators were in all probability observing increased rough and tumble play fighting in their lead-exposed subjects. Although many pharmacological and lesion studies of rough and tumble play fighting have been conducted (see [37] and [62] for reviews), the neurochemical regulation of play is still not clear. Evidence to date suggests that purinergic, opioid, cholinergic and catecholaminergic mechanisms are involved (e.g., [2, 10, 12, 24, 36, 37, 62]), and that the medial septal area [3] and parafasicular nuclei of the thalamus [50] may play important roles in the expression of play fighting in males and females. The amygdala also appears to play a role in play fighting, but only in males [24]. The sex difference we observed in cadmium's effect on activity and pinning was unexpected, but was found in both experiments. Although others have reported activity increases in juvenile rats after cadmium exposure during infancy, no sex differences were reported [44,68]. In early work (reviewed in [5]) with adult rats, males were more susceptible to cadmium-induced mortality [15,30], and to the increase in hexobarbital sleeping time observed after cadmium exposure [8]. In contrast, females were reported to be more sensitive to cadmium-induced hypertension than males [49]. Play fighting in juvenile rats exhibits a sexual dimorphism (males > female) under some testing situations (e.g.,
[25,63]). This sex difference is not dependent on circulating male hormone, but rather on an organizational effect of androgen around the time of birth [27]. Cadmium treatment was on Day 5 or 6, near the end of the perinatal sensitive period for androgen organizational effects on play fighting, and may have altered in some way the neural systems involved with establishment of the male pattern of play fighting. Beatty et al. [4] found that castration of male rats as late as Day 6 after birth significantly reduced levels of juvenile play fighting. The neural effects of cadmium at the 2 mg/kg dose are transient at this age and dose [68] but may have permanently altered underlying structures subsequently involved with play fighting behavior. It is worth noting that sex differences are also present for a number of behavioral and cognitive disorders in children, including attention deficit disorder where boys are typically affected much more than girls [57]. Much of the research on heavy metal pollutants has ultimately focused on defining effects of low level lead exposure of children on school performance and the incidence of the Hyperactivity with Attention Deficit Disorder (e.g., [45]). A limited number of more recent studies have indicated that other minerals present in the environment, such as cadmium, may also adversely affect academic performance and possibly socialization behaviors of school age children (e.g., [22,42]). Because these elements occur with lead, and in the case of cadmium have body levels often correlated with lead, the interactive effects of these substances is also of interest. In several studies investigators have been able to classify learning disabled students with extremely high reliability (95%) on the basis of the configuration of the body burdens of several metal pollutants [39]. While rough and tumble play of juvenile rats may not be completely homologous with similar social behaviors of human children, there are enough similarities across species regarding the morphology and control mechanisms of rough and tumble play (including humans) [26] to indicate that effects of low levels of metal pollutants other than lead on social interaction patterns of children should be assessed. This paradigm, using rats, also provides the opportunity to study the interactive effects of various pollutants (e.g., lead and cadmium), in conjunction with other experimental manipulations, on subsequent social and intellectual competence of the developing organism. ACKNOWLEDGEMENTS The authors wish to thank Dr. Herman Spitz for his comments on this paper and Mrs. Patricia Conlow for typing the manuscript. Mr. Larry Carr maintained the animals and the colony room.
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