2008 Progress Report: Metals Neurotoxicity Research Project

EPA Grant Number: R831725C004
Subproject: this is subproject number 004 , established and managed by the Center Director under grant R831725
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Harvard Center for Children’s Environmental Health and Disease Prevention Research
Center Director: Hu, Howard
Title: Metals Neurotoxicity Research Project
Investigators: Maher, Tim , Weisskopf, Marc
Institution: Harvard University
EPA Project Officer: Callan, Richard
Project Period: June 1, 2004 through May 31, 2009 (Extended to May 31, 2011)
Project Period Covered by this Report: June 1, 2008 through May 31,2009
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2003) RFA Text |  Recipients Lists
Research Category: Human Health , Children's Health

Objective:

In project 4 (R831725C004), we focused on the long term effect of Perinatal exposure to different concentrations of manganese (as MnCl2) and lead (as Pb acetate) individually on neurochemistry and behavior in male and female Sprague- Dawley rats. During the past year, we completed the three different concentrations of MnCl2 (1.25, 5 and 10 mg/ml), and for Pb acetate we have completed a wide range of doses in order to especially obtain blood Pb levels lower than 10 µg/dL.

Progress Summary:

Three different concentrations of MnCl2 (1.25, 5 and 10 mg/ml) were chosen, with the highest dose selected based on the maximal tolerated concentration that the rats would consume and the lowest concentration based on the exposure that would increase metal level in the blood or brain tissues. For Pb acetate, we have completed a wide range of Pb acetate concentrations in order to obtain blood Pb levels that were lower than 10 µg/dL (level that was previously suggested not to produce neurotoxicity). These were grouped as low, medium and high:
 
The low concentration, Pb acetate 2.5-25 µg/ml (blood Pb level < 10 µg/dL).
The medium concentration, Pb acetate 100-250 µg/ml (blood Pb level 10-40 µg/dL).
The highest concentration, Pb acetate 2500-4000 µg/ml (blood Pb level > 40 µg/dL).
 
All exposures to the metal occurred via the drinking water to the dams during gestation and lactation.
 
i. Effect of metal exposures to the dam and offspring during gestation and lactation periods
The mean body weights of dams exposed to MnCl2 and Pb acetate were similar to control, except for the highest concentration of MnCl2 (10 mg/ml) and Pb acetate (2500 and 4000 µg/ml), which tended to have lower body weights than controls. Gestational length and number of offspring born were not affected by treatments. Furthermore, the effect of the MnCl2 and Pb acetate exposures during lactation on the dam’s body weight and body weights of the offspring during the growth development (PND1, 7, 14, 21, 28, 35, 42, 49 and 56) were evaluated. While the average the body weight of the dams during lactation was higher than during gestation, dams exposed to MnCl2 (10 mg/ml) had lower body weights during lactation than gestation. A similar effect on gestational body weight was also seen for the highest concentrations of MnCl2 and Pb acetate exposures. Growth development of offspring exposed to MnCl2 10 mg/ml had statistically significant lower body weights than the control at every PND determined (PND1 until PND 56) for male rats and female rats (except PND 49 which did not reach the statistical significance). MnCl2 5 mg/ml also affected body weight of female rat more than the male rat offspring (body weight on PND 14, 21, 35, 42 of female rats and PND 42 of male rats were statistically significant lower than the control). Exposure to MnCl2 (1.25 mg/ml) did not impair weight gain and growth of the offspring, and actually tended to increase the body weight at some PNDs (PND 35 for male rats and PND 14, 21, 35 and 49 for female rats). Similarly, exposure to Pb acetate (4000 µg/ml) also led to significantly lower body weights than the controls at every PND determined (PND1 until PND 56) for male rats and female rats (except for PND 49 and 56 which did not reach the statistical significance). Exposure to the next lower level of Pb acetate (2500 µg/ml) also led to decreased body weights, however only at some PNDs where statistical significance was reached (PND 28, 35, 42 for male rats, and PNDs 21, 28, 35 and 42 for female rats).
 
ii. Effect of metal exposures to the blood and brain metal levels in the offspring at PND21
 
Blood Mn levels in the offspring at PND21were dose-dependently increased following exposures: 3.91±0.47 (MnCl2 1.25 mg/ml), 7.85±0.84 (MnCl2 5 mg/ml) and 8.84±1.37 (MnCl2 10 mg/ml). In addition, Mn exposures also increased Mn level in every brain area determined in this study, including cortex, hippocampus, brain stem and rest of brain. There were statistically significant correlations between Mn level in the blood and in different brain areas (p< 0.001, except p < 0.002 for cortex Mn level), with r2 values of 0.629 (cortex), 0.652 (hippocampus), 0.680 (brain stem) and 0.642 (rest of brain). Manganese was found more highly distributed in the brain stem than cortex (~ 1.28 fold) and rest of the brain (~1.32 fold).
 
Blood Pb level in the offspring at PND 21 was dose-dependently increased following exposures. The low concentrations produced blood values in the range of 1.02±0.11 to 8.76±1.35 µg/dL (Pb acetate 2.5-25 µg/ml), the medium concentrations in the range of 19.16±1.18 to 35.83±5.83 µg/dL (Pb acetate 100-250 µg/ml), and the high concentrations in the range of 45.14±8.88 to 188.80±38.82 µg/dL (Pb acetate 2500-4000 µg/ml). Moreover, Pb exposures increased Pb level in every brain tissue determined in this study, including cortex, hippocampus, brain stem and rest of brain. There were statistically significant correlations between Pb level in the blood and the different brain areas (p< 0.001), with r2 values of 0.676 (cortex), 0.4877 (hippocampus), 0.515 (brain stem) and 0.628 (rest of brain). Although Pb was found increased in different areas of the brain, it was not evenly augmented. Pb was accumulated more in hippocampus and brain stem than cortex and rest of brain. There was an approximately 2-fold increase in the hippocampus compared to cortex and rest of brain (r2= 0.969 between Pb level in the cortex and hippocampus, and r2 = 0.968 between Pb level in rest brain and hippocampus, p< 0.001). There was approximately a 1-1.4 fold increase in the brain stem compared to Pb level in the cortex and rest of the brain.
 
iii. Effect of metal exposures to learning and memory
Learning and memory was determined using the Morris water maze (MWM) when rats were at PND35. Control rats demonstrated appropriate learning during the acquisition trial for 4 days. During the probe trial rats demonstrated recall of the platform position which was previously located in north quadrant (NQ) of the swimming pool. The control had the highest frequency of swimming into the NQ (8 ± 0.4 entries), time swimming in NQ (25 ± 1 sec or 42 ± 2 %). The product of frequency of entries into the NQ and duration in NQ was 207 entries-sec. As there was not statistically significant difference for the male versus female rats in the acquisition trial and probe trial, the data from those experiments were combined.
 
While Pb acetate exposure at the lowest concentrations (Pb acetate 2.5-25 µg/ml) did not result in altered learning and memory, an effect from exposure to higher concentrations (Pb acetate 100-250 µg/ml and 2500-4000 µg/ml) was observed. During the acquisition trial, Pb acetate 2500-4000 µg/ml significantly increased time to platform on days 2 and 3 (p< 0.05) (Figure 1). In addition, the probe trial data as evidenced by duration in NQ, % time in NQ and frequency X duration in NQ supported the impairment of memory from this exposure. Exposure to Pb acetate 100-250 µg/ml also led to memory impairment, as evidenced by duration in NQ, % time in NQ and frequency X duration in NQ (p< 0.05) (Figure 4.2).
 
During the acquisition trial, exposure to MnCl2 10 mg/ml significantly increased time to platform on days 2, 3 and 4 (p< 0.05), while there was no significant effect following exposure to MnCl2 1.25 and 5 mg/ml (Figure 4.3). Moreover, the probe trial data of MnCl2 10 mg/ml demonstrated impairment of memory (frequency in NQ, duration in NQ, frequency X duration in NQ, and % time in NQ), (p< 0.05) (Figure 4). In addition, the probe trial data for the lower MnCl2 1.25 and 5 mg/ml demonstrated memory impairment as evidenced by duration in NQ and % time in NQ (p< 0.05) (Figure 4.4). For all groups, the swimming speed and cue test performance were not significantly different implying that the observed effects were not the result of motor or visual system dysfunctions.
 
 
Figure 4.1:  Acquisition trial for 4 days of Pb acetate exposures (control n=29, Pb acetate 2.5-25 µg/ml n=27, Pb acetate 100-250 µg/ml n=33, Pb acetate 2500-4000 µg/ml n=28; p<0.05)
 
 
Figure 4.2: Probe trial of Pb acetate exposures (control n=29, Pb acetate 2.5-25 µg/ml n= 27, Pb acetate 100-250 µg/ml n= 33, Pb acetate 2500-4000 µg/ml n= 28; *p<0.05).
 
 
Figure 4.3: Acquisition trial for 4 days of MnCl2 exposures (control n=29, MnCl2 1.25 mg/ml n=16, MnCl2 5 mg/ml n= 15, MnCl2 10 mg/ml n=16; *p<0.05).
 
 
Figure 4.4: Probe trial of MnCl2 exposures (control n=29, MnCl2 1.25 mg/ml n=16, MnCl2 5 mg/ml n= 15, MnCl2 10 mg/ml n=16; *p<0.05).
 
 
iii. Effect of metal exposures on impulsivity and hyperactivity
The elevated-plus maze (EPM) was performed in rats on PND 56 as an indicator of impulsivity and hyperactivity. Time to first entry, frequency of entries and duration in the open arm were used as measures of impulsivity. Velocity was the major determinant of hyperactivity. There were no differences between control males and females in any parameter evaluated, except for the latency of the first occurrence to open arm. Female rats had a shorter latency than male rats (p< 0.05) to enter the open arm with an average of 144 ± 51 sec for female and 323 ± 69 sec for male rats. Increased impulsivity and hyperactivity were observed from exposure to Pb acetate 2.5-25 µg/ml in both male and female rats. In male rats, Pb acetate 2.5-25 µg/ml had a shorter latency of the first occurrence to open arms than the control, and had a higher percentage of open arm entries, while in female rats the impulsivity was evidenced from the higher % frequency of open arm entries and % duration spent in open arm (there was a tendency for decreased latency to open arm). Hyperactivity resulting from of Pb acetate 2.5-25 µg/ml exposure was seen in males (5.0 ± 0.1 cm/sec) and females (5.8 ±0.2 cm/sec) compared to control males (4.2 ± 0.3 cm/sec) and control females (4.7 ± 0.2 cm/sec). Medium level exposures to Pb acetate (100-250 µg/ml), demonstrated increased impulsivity and hyperactivity only in female rats (% duration spent in open arms; 9 ± 2 versus control of 4 ± 1, p< 0.05) and velocity (5.4 ± 0.2 cm/sec versus control female 4.6 ±  0.2 cm/sec, p< 0.05; Table 1).
 
In contrast contrast to the observed effects of exposure to Pb acetate, MnCl2 exposures only had minor effects on impulsivity and hyperactivity (Table 4.2). In some cases on females were significantly affected. Levels of 1.25 and 5 mg/ml appeared to be most effective on velocity, however at the higher dose (10 mg/ml) no significant effect was observed. Exposures to MnCl2 appeared to produce an inverted-U shaped response, especially as related to velocity. This might suggest that while with low to medium exposures these behaviors could be influenced, the higher exposures produce other toxic manifestations that mask the lower exposure effects (Table 2).
 
 
Table 4.1: Effect of Pb acetate exposures on the EPM test
 
 
Table 4.2: Effect of MnCl2 exposures on the EPM test
 
 
iv. Effect of metal exposures on neurochemistry
Microdialysis was used to determine the effects of metal exposures on the dopaminergic and glutamatergic neurotransmission in the prefrontal cortex and hippocampus, respectively. Using conscious rats at PND 85-90 microdialysates were collected before, during and after depolarization with retrodialysis of high K+ artificial CSF (KCl = 60 mM, for 1 hour). The neurotransmitter levels after stimulation with high K+ aCSF were compared with the individual rat’s basal neurotransmitter levels (the mean of the three basal values before treatment) and calculated as percent change from the baseline. The total effect of the high K+ aCSF stimulation for 60 minutes was performed using the area under the curve during stimulation (time versus % neurotransmitter change) using the trapezoidal rule (Δ basal percentage x time).
 
There were no significant differences between male and female control rats for basal dopamine and glutamate levels. However, female control rats had a higher dopaminergic response, but lower glutamatergic response compared with control male rats to the high K+ aCSF stimulation. Exposure to MnCl2 (10 mg/ml) in female rats resulted in increased basal levels of DA, but had no effect in male rats. Exposure to MnCl2 (1.25 and 10 mg/ml) in female rats demonstrated less of an increase in DA to high K+ (no data is available yet for MnCl2 5 mg/ml for female rats) (Figure 4.5), but had no effect in male rats. For the glutamatergic system, the basal glutamate levels were not affected by MnCl2 exposure in male or female rats, however MnCl2 10 mg/ml exposed male and female rats had a smaller increase in glutamate following high K+ (Figures 4.6, 4.7).
 
For Pb acetate, exposure to 100-250 µg/ml and 2500-4000 µg/ml had no effect on either basal or K+evoked DA levels and both male and female rats. We have no data yet in the 2.5-25 µg/ml exposed animals. However, there was the gender effect of Pb acetate on the basal glutamate levels and K+evoked changes. Pb acetate 100-250 µg/ml and 2500-4000 µg/ml exposed female rats had significantly higher basal glutamate levels than the controls, but there was no effect in male rats. In addition, the K+evoked glutamate levels were significant lower than the control from all Pb acetate exposure groups (2.5- 25 µg/ml, 100-250 µg/ml and 2500-4000 µg/ml) in male rats (Figure 4.8). This was not observed in the female rats.
 
 
Figure 4.5: Microdialysis profile of dopamine from Prefrontal cortex of female rats (control n=8, MnCl2 1.25 mg/ml n=4, MnCl2 10 mg/ml n=6).
 
 
Figure 4.6: Microdialysis profile of glutamate from Hippocampus of male rats (control n=6, MnCl2 1.25 mg/ml n=4, MnCl2 5 mg/ml n=4, MnCl2 10 mg/ml n=4).
 
 
Figure 4.7: Microdialysis profile of glutamate from Hippocampus of female rats (control n=7, MnCl2 1.25 mg/ml n=4, MnCl2 5 mg/ml n=4, MnCl2 10 mg/ml n=6).
 
 
Figure 4.8: Microdialysis profile of glutamate from Hippocampus of male rats (control n=6, Pb acetate 2.5-25 µg/ml n=9, Pb acetate 100-250 µg/ml n=7, Pb acetate 2500-4000 µg/ml n=10).
 
 
 
Significance
These results provide the evidence that exposure to MnCl2 and Pb acetate during gestation and lactation causes subtle neurobehavioral and neurochemical changes resulting from a wide range of the exposure levels. The data also emphasized the significance of the low doses of MnCl2 and Pb acetate exposures, as impulsivity and hyperactivity were observed from both male and female rats with Pb acetate 2.5-25 µg/ml (blood Pb level lower than 10 µg/dL). In contrast, these neurobehavioral deficits were not shown from high concentration of Pb acetate (2500-4000 µg/ml) that produced very high Pb level in blood and brain tissues. Additionally, low doses of MnCl2 of 1.25 and 5 mg/ml, but not 10 mg/ml produced hyperactivity. However, we cannot conclude that these neurobehavioral deficits did not occur with the high levels of MnCl2 and Pb acetate, as other toxic effects resulting from high level exposures can obscure the impulsivity and hyperactivity. Learning and memory were also affected from MnCl2 and Pb acetate exposures. Learning and memory impairment was shown from the medium to high level exposures of Pb acetate (Pb acetate of 100-250 µg/ml and 2500-4000 µg/ml), and MnCl2 of each level (MnCl2 1.25, 5 and 10 mg/ml).
 
In addition to the neurobehaviors, the results from the microdialysis experiments demonstrate the neurochemical changes from the exposure to these metals. Both dopaminergic and glutamatergic neurotransmissions were affected from MnCl2 exposure, although a gender effect was apparent. Exposure to MnCl210 mg/ml in male and female rats had a smaller increase of K+evoked glutamate levels than controls. This deficit might relate to the learning and memory impairment observed in male and female rats exposed to higher MnCl2 levels. In contrast, there was a gender effect of MnCl2 exposure on the basal and K+evoked DA levels. The deficit was found only in female rats as evidenced by higher basal DA and decreased K+ evoked DA levels. That the deficit in dopaminergic neurotransmission was found only in female rats might be related to the hyperactivity found from MnCl2 1.25 and 5 mg/ml in female rats.
 
Exposure to Pb acetate did not appear to affect our measure of dopaminergic neurotransmission at either the basal or K+ evoked stages in either male or female rats. This result did not correlate with the behavioral data for impulsivity or hyperactivity from Pb acetate 100-250 µg/ml of female rats. However, as we lacked the microdialysis results from DA levels of Pb acetate 2.5-25 µg/ml of male and female rats, we are unable to explain the neurobehavioral deficits from this group of rat in terms of dopaminergic neurotransmitter changes at this time. The gender effect on glutamatergic neurotransmission was observed from higher basal glutamate level of Pb acetate 100-250 µg/ml and 2500-4000 µg/ml of female rats, but not male rats. Additionally, a decrease of K+ evoked glutamate level was observed from Pb acetate exposure in male rats, but was not shown in female rats. However, from the MWM experiment, both male and female rats demonstrated impairment during the acquisition and probe trials.

Journal Articles:

No journal articles submitted with this report: View all 12 publications for this subproject

Supplemental Keywords:

children, Native American, tribal, mixtures, lead, PBPK, community, Superfund, intervention, environmental management, environmental management, international cooperation, Scientific Discipline, Waste, Health, RFA, Risk Assessment, Health Risk Assessment, Children's Health, Hazardous Waste, Biochemistry, Environmental Chemistry, Hazardous, neurodevelopmental toxicity, developmental toxicity, fate and transport , children's environmental health, mining wastes, human health risk, mining waste, community-based intervention, metal contamination, metal wastes, metals,, RFA, Health, Scientific Discipline, INTERNATIONAL COOPERATION, ENVIRONMENTAL MANAGEMENT, Waste, Environmental Chemistry, Health Risk Assessment, Hazardous Waste, Biochemistry, Children's Health, Hazardous, Risk Assessment, community-based intervention, fate and transport , developmental toxicity, Human Health Risk Assessment, neurodevelopmental toxicity, children's environmental health, mining waste, metal wastes, metals, human health risk, metal contamination

Progress and Final Reports:

Original Abstract
  • 2004 Progress Report
  • 2005 Progress Report
  • 2006
  • 2007 Progress Report
  • 2009
  • Final

  • Main Center Abstract and Reports:

    R831725    Harvard Center for Children’s Environmental Health and Disease Prevention Research

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R831725C001 Metals, Nutrition, and Stress in Child Development
    R831725C002 Exposure Assessment of Children and Metals in Mining Waste: Composition, Environmental Transport, and Exposure Patterns
    R831725C003 Manganese, Iron, Cadmium, and Lead Transport from the Environment to Critical Organs During Gestation and Early Development in a Rat Model
    R831725C004 Metals Neurotoxicity Research Project