Low-Level Human Equivalent Gestational Lead Exposure Produces Sex-Specific Motor and Coordination Abnormalities and Late-Onset Obesity in Year-Old Mice

1Removal of lead from gasoline and other environmental sources has decreased the median blood lead concentration ([BPb]) of children in the United States < 10 μg/dL: the current low level of concern. However, there is compelling cross-sectional and prospective epidemiological evidence that [BPb] in children ≤ 10 μg/dL causes cognitive decline. Recently, suggested that the BPb action level in children should be 2 μg/dL. Developmental lead exposure also has been linked to a variety of neurological and neurodegenerative disorders in children and adolescents, including attention deficit hyperactivity disorder (ADHD), auditory and language impairments, retinal deficits, neuromotor dysfunction, and schizophrenia. Few studies, however, have examined the long-term effects of low-level gestational lead exposure (GLE) despite findings that children with prenatal lead exposure have reduced cognitive functions, neuromotor and visual motor dysfunction, and altered auditory and retinal function.

2Maternal lead exposure results from inhalation, diet, and/or eating in lead-contaminated work areas. Maternal skeletal bone lead from prior exposure mobilizes during pregnancy and lactation. Lead easily crosses the placental and mammary barriers. Thus, the developing fetus and child are at risk, as evidenced by findings that fetal and maternal [BPb] are similar.

3The adverse cognitive consequences of prenatal and postnatal exposure to moderate-level ([BPb] 11–39 μg/dL) and high-level ([BPb] ≥ 40 μg/dL) lead have been studied in rodents. Several reports also link moderate-to-high-level lead exposure to altered motor activity and dopaminergic signaling. To date, there are no experimental studies on the effects and mechanisms of low-level GLE on neuromotor function, despite evidence that low-level lead exposure produces these deficits in children (vide supra).

4In this report we present a new model of human equivalent GLE and the sex-specific physiological, behavioral, and neurochemical abnormalities in year-old GLE mice. These studies were conducted because the long-term consequences of GLE are unknown and increasing evidence indicates that early developmental exposure to neurotoxicants accelerates age-related functional decline and/or produces delayed neurotoxicity. Sex differences were examined because a) early developmental lead exposure produces a heightened risk for attention, visual motor, and fine-motor deficits in males; b) male and female animals exhibit differences in exposure and susceptibility to chemicals and lead neurotoxicity; and c) this is an important and underexplored area of toxicology. Our results show that GLE produced age-related, sex-specific, and non-monotonic dose–response alterations.

5All experimental and animal care procedures were in compliance with the National Institutes of Health (NIH) Public Health Service Policy on Humane Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the University of Houston. All animals were treated humanely and with regard for alleviation of suffering. Five-week-old female and male C57BL/6 mice (Harlan Sprague Dawley, Inc., Indianapolis, IN) were housed in a room with a 12:12-hr light:dark cycle as described previously. Two animal models were used: GLE and post-natal-only lead exposure (PLE). For each, dams were mated with a single male overnight, and the presence of a vaginal plug was recorded as gestational day 0.5. Dams were weighed twice weekly until postnatal day (PN) 21 (weaning). On the day of birth (PN0), the number of pups and the sex and weight of the offspring were recorded (12–18 litters per group). On PN1, litters were culled to six pups with equal number of males and females when possible. Pups were weighed on PN7, 10, and 21. At weaning, male and female mice were independently housed four to five per cage and weighed at 2, 6, 10, and 12 months of age. Control and lead drinking bottles were weighed and replaced every other day.

6Two weeks after arrival, female mice were singly housed and randomly divided into four experimental groups: one control and three GLE groups. Control dams received tap water, and GLE dams received one of three lead acetate drinking solutions (Fisher Scientific, Pittsburgh, PA): 0.005% (27 ppm lead = low-dose GLE), 0.01% (55 ppm lead = moderate-dose GLE), or 0.02% (109 ppm lead = high-dose GLE). Lead drinking solutions were provided to dams 2 weeks prior to mating to ensure [BPb] stabilization and a lead body burden throughout gestation and until PN10. Control males were used once for breeding with lead-exposed dams. We selected the pre-natal through PN10 period for our GLE study (Figure 1) because rodent brain and retinal development during this period is equivalent to that during human gestation. Our overall goal was to compare the behavioral and neurochemical changes in low-level and high-level year-old GLE mice with those in controls. Therefore, these sets of studies were not conducted in the moderate-level GLE group.

7This model was established to compare the [BPb] profiles and body weight measures with those in GLE mice. Four weeks after arrival, female mice were singly housed and randomly divided into three experimental groups: one control and two PLE groups. Upon delivery and throughout lactation (PN0–21), PLE dams received either a 0.005% (low-dose PLE) or 0.01% (moderate-dose PLE) lead drinking solution.

8After decapitation, we measured trunk [BPb] in GLE dams after 14 days of lead pretreatment and on PN0. Trunk [BPb] was measured in GLE offspring at PN0, 10, 21, 30, 60, and 1 year of age and in PLE offspring at PN7, 14, 21, 30, 60, and 1 year of age. Values (micrograms per deciliter) represent the mean ± SE for 10–15 male and female mice per age per group. Samples were analyzed by anodic stripping voltammetry using LeadCare Kit I (sensitivity, ≤ 1 μg/dL; Environmental Sciences Associates, Inc., Chelmsford, MA).

9Exploratory activity was assessed in year-old male and female mice (six to nine per sex per group). Activity was measured in a fully enclosed Optovarimax behavioral monitor (40 × 40 × 40 cm; 16 infrared photo-receptor beams per X–Y side; Columbus Instruments, Columbus, OH) located in a quiet room with dim lighting. Data were recorded based on the number of laser-beam breaks made each 5-min period. Novel exploratory activity data were collected for 30 min after a 15-min acclimation period. Two weeks later, the same animals were given a 15-min acclimation period, weighed, and injected (ip) with 3 mg/kg d-amphetamine sulfate (Sigma A-5880; Sigma-Aldrich, St. Louis, MO), and placed in the recording chamber. Five minutes later, a 3-hr locomotor activity recording began. This dose of amphetamine avoided stereotypic behavior from competing and interfering with locomotor behavior.

10Interlimb balance and coordination were assessed in year-old male and female mice (six to eight per sex per group) with a rotarod (Columbus Instruments). Mice were trained to stay on top of a stationary rod (3-cm diameter), then at a constant speed of 5 rpm for 90 sec. Mice were tested 3 times the day after they mastered the task, with a 1-hr intertrial interval. For each trial, the rod rotated at 5 rpm for 30 sec, and the speed was increased 0.1 rpm until the mouse fell off. The three trials were averaged to obtain one rotarod latency score (seconds) for each mouse.

11Year-old male mice (six per group) were given access to Wahman-type running wheels equipped with counters that recorded the distance (meters) traveled. Running wheels were in a room designated solely for this purpose. For five consecutive dark cycles, individual mice were placed in clean cages with an attached exercise wheel and food and water.

12Four weeks after the last behavioral experiment, aged male mice (four to seven per treatment group) were sacrificed by decapitation between 1000 hours and 1200 hours to avoid possible circadian effects. Brains were rapidly removed, and striatum and forebrain samples were dissected and placed in ice-cold 0.2 N perchloric acid and frozen at −80°C. Frozen samples were homogenized and centrifuged at 4°C. The supernatant was analyzed for the concentration of dopamine ([DA])and its major metabolite 3,4-dihydroxyphenylacetic acid ([DOPAC]) by HPLC with electrochemical detection as described. DA and DOPAC are expressed as nanograms per milligram protein. [DOPAC]/[DA] ratios were used as a measure of DA utilization.

13Body weight, behavioral, and neurochemical studies used one animal per litter, and the group data were analyzed by a one-way analysis of variance (ANOVA), with or without repeated measures. Male and female [BPb] data were analyzed using a two-way ANOVA. After ANOVA analyses, post-hoc multiple comparisons used the Tukey Honestly Significant Difference test (KaleidaGraph; Synergy Software, Reading, PA; Minitab Inc., Prentice-Hall, Edgewood Cliffs, NJ). Data are presented as mean ± SE, and the difference from controls was considered significant at p < 0.05.

14Fluid consumption and body weight of GLE dams exposed to water or lead from 14 days prior to conception until birth were measured, and gestational and litter measures were recorded. All control values (Table 1) were similar to those previously reported. There were no statistical differences between control and GLE groups on any measure (data not shown).

15A two-way ANOVA revealed that [BPb] of male and female littermates were not significantly different for any GLE or PLE treatment condition. Figure 2A shows that control, and low-, moderate-, and high-dose GLE produced concentration-dependent increases in [BPb] at PN0–10, with peak [BPb] of ≤ 1, ≤ 10, 27, and 42 μg/dL, respectively. By PN30, [BPb] in GLE mice were not significantly different from controls. There were no statistically significant differences between control and PLE groups on any maternal or litter measure (; data not shown). Figure 2B shows that control, and low-, and moderate-dose PLE produced significant concentration-dependent increases in [BPb] from PN7–21 with peak [BPb] of ≤ 1, 10, and 26 μg/dL, respectively. By PN60, the [BPb] in PLE mice were not significantly different from controls.

16Figure 3 shows that GLE had no significant effect on body weight at PN0, 10, and 60, although by PN60, males in all groups weighed significantly more than females (Figure 3C). Similarly, there were no significant treatment-related differences in body weight at 6 or 10 months of age (data not shown) or at 1 year in female GLE mice (Figure 3D). In contrast, year-old low- (+26%), moderate-(+21%), and high-dose (+13%) male GLE mice weighed significantly more than controls (Figure 3D). There were no significant treatment-related effects of PLE on body weight during development, aging, or at 12 months of age (Figure 3E).

17When placed in an activity chamber for 30 min for the first time, 1-year-old control male (Figure 4A) and female mice (Figure 4B) exhibited similar levels of exploratory activity. However, male mice in the low- and high-dose GLE groups were significantly less active than control mice: total mean decrease was 52% and 35%, respectively (Figure 4A). Low-dose GLE males were significantly less active than high-dose GLE males. In contrast, there were no treatment-related differences for female mice (Figure 4B). Because year-old GLE males weighed more than age-matched control males (Figure 3D), we addressed the possibility that excess body weight made the GLE males lethargic and less active. This hypothesis was not confirmed, as there were no treatment-related differences in male running wheel activity (Figure 4C).

18Amphetamine-induced motor activity was recorded in year-old mice (Figure 5). Initial data analysis suggested that there were no differences between treatment groups, although males (Figure 5A, C) were 50% less active after amphetamine challenge than females (Figure 5B, D). Because the baseline exploratory activity in GLE males (Figure 4A) but not in females (Figure 4B) was significantly decreased, we conducted a more detailed analysis of the first 30 min of amphetamine-induced motor activity. Figure 5C and D reveals no apparent between-group differences in amphetamine-induced motor activity. However, when we subtracted the baseline activity counts from the amphetamine-induced activity counts for the first 30 min, the net motor activity revealed that low- and high-dose GLE males exhibited a significant (2-fold) overall heightened sensitivity to amphetamine stimulation (Figure 5E). GLE females exhibited no overall change in sensitivity to amphetamine-stimulated motor behavior (Figure 5F).

19The mean latency to fall from the rotarod was not different in year-old male and female control mice or female GLE mice (Figure 6). In contrast, all GLE males had significantly shorter latencies to fall from the rotarod compared with age-matched controls. In addition, the mean rotarod performance of low-dose GLE males was significantly poorer than that of high-dose GLE males.

20High-dose GLE significantly increased striatal [DA] by 23% and [DOPAC] by 58%, whereas low-dose GLE significantly elevated [DOPAC] by 17% (Figure 7A). Striatal DA utilization was unchanged in GLE mice (Figure 7C). In the forebrain, high-dose GLE significantly increased [DA] by 63% and [DOPAC] by 149%, and low-GLE significantly increased [DOPAC] by 69% (Figure 7B). However, in the forebrain low-GLE decreased [DA] by 30%, and both low- and high-dose GLE increased DA utilization (Figure 7C). Moreover, the increased forebrain DA utilization was significantly greater in low-dose (+133%) than in high-dose (+50%) GLE mice.

21We report six novel results. First, a new and toxicologically relevant murine model of human equivalent low-level GLE was established. Second, the long-term physiological, behavioral, and neurochemical effects of low-level GLE were examined in year-old male and female mice. Third, sex-specific increases in body weight were observed in year-old GLE male mice. Fourth, male-specific alterations in spontaneous and amphetamine-induced motor behaviors were found in year-old GLE mice. Fifth, alterations in striatal and forebrain DA metabolism were present in year-old male GLE male mice. Sixth, and most important, GLE produced nonmonotonic dose-dependent responses because the alterations were consistently larger in the low-dose than in the high-dose GLE group. These responses are characteristic of inverted U-shaped dose–response curves often observed in lead neuro-toxicity studies.

22One of the most compelling findings in the present study is that GLE acted as a delayed obesogen. Specifically, by 1 year of age male GLE mice were obese, and the weight gain was greater in low-and moderate-dose than in high-dose GLE mice. This nonmonotonic characteristic is supported by reports showing that lifetime exposure to 5- to 20-fold higher lead levels either did not affect body weight of 14-month-old male or female rats or decreased it in adult rats.

23Obesity and related disorders such as diabetes and cardiovascular disease have increased dramatically in children and adults during the past 25 years. Additionally, epidemiological studies show a strong association between obesity and a variety of cancers. Although the molecular mechanism responsible for the lead-induced delayed obesity is unknown, there are several candidates. First, genetic polymorphisms in the vitamin D receptor, insulin-induced gene 2 (INSIG2), and FTO genes (fat mass and obesity associated genes) have been linked to obesity and metabolic disorders. The possibility that GLE produced polymorphisms in the vitamin D receptor is suggested by findings that lead inhibits vitamin D3 receptor–regulated calcium metabolism, and vitamin D receptor polymorphisms elevate [BPb] and tibial [BPb]. Second, hypothalamic dopaminergic receptors are associated with genetic obesity in mice (el-Refai and ). Developmental studies have linked lead to hypothalamic dopaminergic dysfunction and altered hypothalamic–pituitary–adrenal axis. Third, the environmental obesogen hypothesis states that dietary and environmental chemicals disrupt endocrine signaling pathways and thereby contribute to obesity. Recent studies suggest that fetal exposure to organotins produce obesity in male rats by activating certain nuclear receptors. Thus, lead-induced delayed obesity could occur via a variety of endocrine/ metabolic mechanisms.

24The spontaneous locomotor activity and rotarod performance were decreased in GLE male mice, with greater effects seen in the low-dose than in the high-dose GLE group. These curvilinear results are consistent with “negative” dose–response curves, as high-dose GLE produced less deviation from control than did low-dose GLE. Because year-old GLE males were significantly heavier than controls, we tested whether these mice were lethargic and less active on a running wheel. Because all three groups ran comparable distances, the results indicate that the GLE-induced motor alterations were not caused by a lack of motivation or inefficient locomotion.

25We hypothesized that the novel environment differentially increased the stress level of aged GLE male mice, which subsequently altered dopaminergic signaling and decreased locomotor activity. Consistent with this proposal are recent data showing that moderate-to-high-level lead exposure during gestation and throughout lactation produced a) elevated basal corticosterone concentrations in male offspring, b) a decrease in novel exploratory behavior of males, and c) decreased fixed-interval response rates in males. However, the role of DA is still unknown, as both adult male and female rats exhibited dopaminergic dysfunction in several brain regions. In summary, our findings reveal that low-level GLE produced the most profound and enduring alterations in locomotor activity, another example of nonmonotonic responses. Furthermore, the results suggest that a coordinated dysregulation of the hypothalamic–pituitary–adrenal axis and dopaminergic systems might underlie these changes.

26Amphetamine enhances low rates of responding and depresses high rates of responding, which produces the classic inverted U-shaped dose–response effect. Because year-old GLE male but not female mice had decreased locomotor activity compared with age- and sex-matched controls, we reasoned that amphetamine would differentially increase locomotor behavior in GLE males and produce no differential locomotor effect in year-old female mice. Indeed, amphetamine increased the locomotor activity of both low- and high-dose GLE mice compared with controls; larger effects occurred in low-dose GLE mice. Although the DA transporter is a major target site of amphetamine, dopamine D3 receptors in the ventral striatum also regulate the sensitivity to the locomotor-stimulating effects of amphetamine. Although alterations in striatal DA metabolism as well as D1 and D2 dopamine receptors occur in developmentally lead-exposed rats, there are at present no studies on the dopamine D3 receptors.

27Developmental lead exposure is associated with attention-deficit/hyperactivity disorder (ADHD) in children, which is among the most common childhood and adult neurological disorders. Neuroanatomical and functional abnormalities in the prefrontal cortex are reported to underlie this deficit. Moreover, recent functional magnetic resonance imaging studies revealed a diminished activation in the frontal cortex of young adults after developmental lead exposure. It is unknown whether GLE and/or early PLE produce the greatest risk for childhood ADHD. The contribution of GLE to adult-onset ADHD, which also has a 1.6 male–female odds ratio as childhood ADHD, is unknown. Our amphetamine-stimulated locomotor results in year-old male GLE mice are consistent with the U-shaped therapeutic outcome for ADHD and suggest that low-level GLE merits consideration as a causative factor to lead-induced ADHD in children and adults.

28Both groups of aged GLE male mice were impaired on the rotarod, which is a test of interlimb balance and coordination. Low-dose GLE males were significantly more impaired than high-dose GLE males. Consistent with the negative dose–response curve, rotarod activity was unchanged in adult male rats exposed to 5- to 40-fold higher lead levels during gestation and lactation or lifetime. These rotarod results are reminiscent of the poorer fine motor control, visual motor function, and postural balance found in children, adolescents, and young adults with low-to-moderate developmental lead exposure. Interestingly, there is an increased risk of neuromotor deficits in males. Our results suggest that low-level GLE contributes to persistent neuromotor and balance deficits and is a risk factor for injuries in older males.

29Because body weight and behavioral differences were found only in year-old GLE male mice, neurochemical analyses were conducted in these mice. GLE female mice are being aged for future studies. Numerous studies have reported changes in adult rat brain DA metabolism after moderate-to-high post-natal or lifetime lead exposure. However, the present report is the first study to use a GLE model and examine neurochemical changes in aged mice. We found that low-dose GLE decreased forebrain [DA] and increased striatal and forebrain [DOPAC], whereas high-dose GLE increased both striatal and forebrain [DA] and [DOPAC]. These GLE-induced changes resulted in nonmonotonic increases in fore-brain DA utilization but no change in striatal DA utilization. The inverted U-shaped dose-response function for forebrain DA utilization correlates with the GLE-induced alterations in weight gain and neuromotor functions. For two reasons it is unlikely that these dopaminergic changes resulted from a direct effect of lead in year-old mice. The peak brain [Pb] in the low- and high-dose GLE mice on PN10 were equivalent to 0.44 and 1.2 μM, respectively, and the brain [Pb] is not different from controls after PN30 (data not shown). In addition, 3–250 μM Pb2+ is required to inhibit rat brain tyrosine hydroxylase activity, depolarization-evoked and spontaneous DA release, and DA uptake. Thus, further multidisciplinary molecular, biochemical, bioinformatic, and imaging studies are needed to support our findings and provide mechanistic insight.

30In summary, our results show that GLE mice with peak [BPb] ≤ 10 μg/dL, the current low level of concern, have permanent sex-specific motor abnormalities and late-onset obesity. The nonmonotonic dose-dependent responses reveal that low-level GLE produces the most adverse effects. These data raise complex issues for risk assessment and indicate that lifetime measures of dose–response toxicant exposure should be a component of the neurotoxic risk assessment process.