Aging unmasks adverse effects of gestational exposure to methylmercury in rats

The consequences of developmental exposure to methylmercury on behavior in aged animals were investigated. Methylmercury exposure was arranged by placing 0, 0.5 or 6.4 ppm Hg in the drinking water of female rats at least 4 weeks before mating and continuing until post-natal (PN) day 16. Brain Hg concentrations in cohorts of low- and high-dose offspring were 0.5 and 9.1 ppm at birth and 0.04 and 0.52 ppm at weaning (described in another report). Lever pressing of female offspring was maintained under a Multiple Differential Reinforcement of High Rate 9:4 Extinction schedule of food reinforcement (Mult DRH 9:4 EXT). Under the DRH 9:4 schedule, a food reinforcer was delivered when nine responses occurred within 4 s. Under the Extinction schedule, responding had no programmed consequences. No exposure-related differences in reinforcement rate under the DRH schedule or discrimination between the DRH and extinction components were apparent initially. At 950 days of age, the overall response rates of controls had shown a gradual decline over the previous 500 days to about 80% of their beginning levels, but, otherwise, most controls were healthy. A gradual decline in the reinforcement rate began to appear in low- and high-dose rats at about 500 and 800 days of age, respectively. Microanalyses of the nine-response burst maintained by the DRH schedule revealed that the lever-press duration increased, the inter-response time (IRT) was unaffected, and the time between response bursts increased. Overall, the nine-response burst remained intact as a coherent response unit. The increased time between response bursts caused the decline in reinforcement rate. All rats displayed these effects as they aged, but the mercury-exposed rats did so sooner.     

甲基汞对大鼠的行为致畸效应研究

目的探讨妊娠期甲基汞暴露对Wistar大鼠的母体毒性及仔代的行为致畸效应.方法 Wistar孕鼠80只于妊娠第6～9天采用甲基汞0.00、0.01、0.05和2.00mg·kg-1@d-1连续灌胃染毒.分别进行母体毒性、胚胎毒性、仔鼠早期生理发育和神经行为发育指标、仔鼠迷宫和程序控制行为测试、亲仔两代大鼠脑组织形态学观察和单胺类神经递质(去甲肾上腺素、多巴胺、5-羟色胺)的测定.整个实验采用双盲法.结果未观察到明显的母体毒性;3个剂量组胎仔的体重、尾长均低于对照组(P＜0.01);各剂量组仔鼠的体重增长、早期生理及神经行为发育滞后于对照组(P＜0.05);各剂量组仔鼠迷宫错误次数均比对照组多(P＜0.05),具有剂量-效应关系(rs=0.257,P＜0.05);程序控制行为学习成绩比对照组降低(P＜0.05),有剂量-效应关系(rs=-0.727 3,P＜0.01);各剂量组母鼠和仔鼠脑组织均未见形态学改变,但脑组织单胺类神经递质含量均比对照组明显增高(P＜0.05),有剂量-效应关系(s=0.712 4～0.925 7,P＜0.01).结论甲基汞在不引起可观察到母体毒性剂量下,就可产生胚胎毒性,影响仔鼠神经系统的发育,导致神经行为功能的改变.     

Prenatal methylmercury exposure results in dendritic spine dysgenesis in rats

To test the prenatal neurotoxic effect of low doses of methylmercury chloride (MM), the toxic agent was given by gavage to pregnant Wistar rats on days 6 to 9 after conception in doses of 0.025, 0.05, 0.5 and 5.0 mg/kg/day. The offspring of these animals were subjected to a routine developmental and behavioral testing battery. In essence, functional changes including impaired swimming behavior, increased auditory startle amplitude, increased passiveness and increased locomotor stereotypy compared to controls were the result of prenatal MM exposure, tested from the second to the seventh month postnatally. Further, in each group, ten prenatally treated rats were investigated histopathologically at the age of twelve months. The most striking effect was a distinct difference in the morphology of the dendritic spines of the pyramidal neurons in the somatosensory cortex demonstrated by Golgi impregnation. The spine abnormalities in the experimental animals consisted of a reduction of stubby and mushroom-shaped spines and a predominance of long and tortuous spines. Dendritic spine dysgenesis implies defective development and might be the pathological feature of the impaired behavior and learning of the MM-exposed animals.     

Developmental exposure to methylmercury alters behavioral sensitivity to D-amphetamine and pentobarbital in adult rats

Female rats were exposed to 0, 0.5, or 6.4 ppm methylmercury in their drinking water before mating, and throughout gestation and lactation. When the female offspring were 4-6 months old, they were trained to respond under a multiple differential reinforcement of high rate (DRH) 9:4-- Extinction schedule of reinforcement. No differences among exposure groups were apparent in steady-state behavior. Drug challenges were conducted with multiple doses of D-amphetamine, scopolamine, pentobarbital, haloperidol, and dizocilpine, drugs selected for their different pharmacological effects. The ED(50) values for amphetamine's reinforcement rate-reducing effects for the control, 0.5-, and 6.4-ppm groups were 3.1, 1.9, and 0.9 mg amphetamine/kg body weight, respectively, demonstrating an increased sensitivity to D-amphetamine in methylmercury-exposed rats. Rats in the 6.4-ppm group also demonstrated a relative insensitivity to pentobarbital. Further, these exposed rats exhibited an inverted U-shaped dose-effect curve under the pentobarbital dose-effect determination, while controls showed only a declining curve. Exposed rats did not respond differentially to haloperidol, scopolamine, or dizocilpine, suggesting specificity. The present data suggest an involvement of catecholaminergic and GABAergic activity in methylmercury's neurotoxicity.     

Lactational exposure to methylmercury in the hamster

 Syrian Golden hamster dams were administered ²⁰³Hg-labelled methyl mercury (MeHg; 1.6 μmol/kg) 1 day after parturition and milk was collected twice during the 1st week. The excretion of ²⁰³Hg in milk and the uptake, retention and tissue distribution of ²⁰³Hg in the pups was studied using gamma counting. The fraction of inorganic Hg in milk and in the kidneys of the pups was determined following separation of inorganic Hg and MeHg by ion exchange chromatography. The concentration of ²⁰³Hg in milk on the 1st day after MeHg administration was 0.12 nmol/g. ²⁰³Hg was mainly (80–90%) excreted as MeHg during the first 6 days of lactation. The whole body and tissue concentration of ²⁰³Hg in the pups increased for 10–15 days and decreased thereafter. The content of ²⁰³Hg in the pelt and the fraction of inorganic Hg in the kidney increased throughout the study period (4 weeks). The excretion of MeHg in milk corresponded to at least 5% of the dose administered to the dam. Our study demonstrates that breast milk may be a significant source of MeHg exposure during the critical neonatal period. Received: 28 June 1994 / Accepted: 24 October 1994     

Operant behavior performance changes in rats after prenatal methylmercury exposure

Methylmercury chloride (MMC) in doses of 0.005 – 0.05 mg/kg was force fed to pregnant rats at the sixth, seventh, eighth, and ninth day after conception. When 4 months of age the offspring were tested in lever boxes with the operant conditioning program “differential reinforcement of high rates” (DRH). Food reinforcement was made contingent to the fulfillment of performance requirements which were incrementally increased in three steps: In the first test session the animals were required to press the lever twice in 1 sec for one food reward (DRH $\text{2}\text{1}$); in the following test sessions four lever presses in 2 sec (DRH $\text{4}\text{2}$) and eight lever presses in 4 sec (DRH $\text{8}\text{4}$) were required for one reward, respectively. Animal performance in this behavioral paradigm was expressed as the group mean (n = 10) of the ratio of correct, i.e., rewarded, responses to total lever presses per session. MMC-induced performance deficits were found in the 4 × 0.05 and 4 × 0.01 mg/kg dose groups. DRH-learning differences were ascertained by an analysis of variance. The lowest effective dose of 4 × 0.01 mg/kg points to operant conditioning techniques as a suitable tool in environmental toxicology.     

Gestational exposure to methylmercury and n-3 fatty acids: effects on high- and low-rate operant behavior in adulthood

Fish in the diet is the major source of methylmercury (MeHg) exposure, but eating fish also provides important nutrients. Many fish species contain essential long chain polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), an omega-3 (or n-3) fatty acid, that is important for neural development and function. To examine interactions between MeHg and n-3 fatty acids, female Long-Evans rats were exposed, in utero, to 0, 0.5, or 5 ppm MeHg via drinking water, approximating exposures of 0, 40, and 400 mug/kg/day. They also received pre- and postnatal exposure to a diet containing either fish oil or coconut oil, creating a 2 (Diet)x3 (MeHg) full factorial design, with 6-8 rats per cell. The diets were high or marginal, respectively, in n-3 fatty acids but approximately equal in n-6 fatty acids. No exposure-related effects on developmental milestones or growth were noted. Behavior was evaluated using a series of rapidly increasing fixed ratio (FR) schedules of sucrose reinforcement; 1, 5, 25 and 75 lever presses were required for sucrose delivery, with three sessions provided at each requirement. This phase was followed by four sessions of a differential-reinforcement-of-low-rate-behavior (DRL) schedule, in which presses preceded by 10 s (or more) without a press were reinforced. Subsequently, several progressive ratio (PR) schedules that increased response requirements throughout a single session by a rate of 5%, 10%, or 20% were imposed. Rats exposed during gestation to MeHg had significantly higher response rates than controls under the large FR schedules, during the first session of DRL, and the PR 5% schedule, but neither fish oil nor coconut oil modified MeHg's effects. This finding is consistent with hypotheses that developmental MeHg exposure produced perseverative responding or altered the sensitivity of behavior to its reinforcing consequences and that certain reinforcement contingencies can unmask MeHg's effects.     

Blood and brain mercury levels after chronic gestational exposure to methylmercury in rats.

Female rats were exposed to 0, 0.5, or 6 ppm Hg (as methylmercuric chloride, 10 rats/group) in drinking water. For half the rats, exposure began 4 weeks before mating and for the others, exposure began 7 weeks before mating. All mating was done with an unexposed male. Maternal exposure continued to post-natal day (PN) 16. Blood and whole-brain mercury concentrations were determined in pups on PN 0 (birth) and PN 21 (weaning). Maternal water consumption was monitored daily during gestation and lactation. Maternal water consumption increased 2- to 3-fold through gestation for all groups. Mercury levels in blood and brain were unrelated to the duration of exposure before mating, although reproductive success appeared to be so related. Mercury levels in both media were closely related to consumption during gestation, but apparently maternal exposure during lactation did not result in exposure to the nursing pups. Brain mercury in offspring decreased between birth and weaning from 0.49 to 0.045 ppm in the low-dose rats and from 9.8 to 0.53 ppm in the high-dose rats. The brain increased in weight only about 5.5-fold during this time, indicating that there was minimal mercury exposure and some net loss from brain during this period. Brain:blood ratios averaged about 0.14 at birth and 0.24 at weaning, suggesting differential loss from neural and non-neural tissue. These ratios are higher than those reported in studies using less chronic exposure conditions or with adult rats. Brain concentrations of mercury in females in the low-dose group were about 10-15% higher than those seen in their male siblings. At the higher dose, the males had slightly higher levels of mercury in the brain than did their female siblings at birth. The relationship between brain concentration (in ppm) and cumulative mercury consumption, also expressed on a ppm basis (cumulative mercury consumed divided by maternal body weight at parturition), was not linear but was well described by a power-function relationship: Hg = A*(cum exposure)b where the exponent, b, was 1.12 and 1.17 for blood and brain, respectively, at birth. This exponent was indistinguishable from 1.0 for both media at weaning, indicating that the relationship between exposure and blood and brain levels became linear.     

Acute exposure to methylmercury opens the mitochondrial permeability transition pore in rat cerebellar granule cells.

Cerebellar granule cells are preferentially targeted during methylmercury (MeHg) poisoning. Following acute MeHg exposure, granule cells in culture undergo an increase in intracellular Ca2+ concentration ([Ca2+]i) that apparently contributes to cell death. This effect consists of several temporally and kinetically distinct phases. The initial elevation arises from release of Ca2+(i) stores; the second phase results from entry of Ca2+(e). In these experiments, we tested the hypothesis that release of mitochondrial Ca2+ through the mitochondrial permeability transition pore (MTP) contributes to the initial release of Ca2+(i). Neonatal rat cerebellar granule cells in culture and single cell microfluorimetry were used to examine MeHg-induced changes in [Ca2+]i and mitochondrial membrane potential (Psi(m)). Pretreatment with the MTP inhibitor cyclosporin A (CsA, 5 microM) delayed the initial phase of increased [Ca2+]i induced by 0.2 and 0.5 microM MeHg, but not 1.0 microM MeHg. CsA (5 microM) also delayed the irreversible loss of Psi(m) induced by 0.5 microM MeHg. Ca2+(e) was not required for either effect, because the effect of CsA on the first phase increase in [Ca2+]i and loss of Psi(m) was not altered in nominally Ca2+-free buffer. Increasing concentrations of MeHg (0.2-2.0 microM) caused increasing incidence of cell death at 24 h postexposure. Treatment with CsA provided protection against cytotoxicity at lower MeHg concentrations (0.2-0.5 microM), but not at higher MeHg concentrations (1.0-2.0 microM). Thus, the MTP appears to play a significant role in the cellular effects following acute exposure of cerebellar granule neurons to MeHg.     

Gestational and lactational exposure to heptachlor does not alter reproductive system development in rats

Like other organochlorine insecticides, heptachlor is lipophilic and accumulates in fatty tissues. Earlier studies suggested that in utero exposure to heptachlor decreased fertility in the offspring; neonatal exposure to the closely related insecticide chlordane reportedly delayed puberty and disrupted estrous cycling in females. We hypothesized that in utero and lactational exposure to heptachlor would disrupt the development of the reproductive system in males and females, resulting in altered timing of puberty and abnormal gonadal histology and reproductive hormone levels. Timed pregnant Sprague-Dawley rats were treated by oral gavage with heptachlor in corn oil at doses of 0.5 and 5.0 mg/kg or an equivalent volume of corn oil alone daily from gestational day 8 through post-natal day (PND) 21, the day of weaning (n = 7-8/group). Litters were standardized to 4 males and 4 females on the day of birth. Two of the dams in the 5.0 mg/kg/d group died. Pups in the highest dose group weighed significantly less than those in the other 2 groups on PND 0. All but 1 litter of the 5.0 mg/kg/d group died within the first 4 post-natal days. Age at eye opening was delayed with increasing heptachlor dose. There was no effect of treatment on pup weight gain in the surviving litters, on anogenital distance, age at puberty, nipple retention past the infantile period in males, estrous cycling, serum sex steroid concentrations, reproductive organ weights, or testicular or ovarian histology. These results suggest that heptachlor exposure during gestation and lactation does not disrupt development of the reproductive system in rats.     

Gestational exposure to nicotine and monoamine oxidase inhibitors influences cocaine-induced locomotion in adolescent rats

Rationale Many pregnant women continue to smoke, despite a strong association between maternal smoking and neurobehavioral deficits in the offspring. Although gestational nicotine (GN) treatment in rodents is used as the primary animal model of maternal smoking, tobacco smoke contains more than 4,000 constituents, including monoamine oxidase inhibitors (MAOIs). Objectives The aim of this study was to determine whether there are interactions between the effects of gestational exposure to nicotine and MAOIs on cocaine-induced locomotor sensitization in adolescent rats. Materials and methods Pregnant rats were implanted on day 4 of gestation with osmotic minipumps delivering saline, nicotine (3 mg/kg per day), the MAOIs clorgyline and deprenyl (1 and 0.25 mg/kg per day, respectively), or nicotine/clorgyline/deprenyl (GMN). Adolescent female offspring were tested for cocaine-induced locomotor sensitization. Animals were treated with saline or cocaine (5 or 15 mg/kg, intraperitoneally) daily from postnatal (P) days 32–36 and challenged with cocaine (15 mg/kg) on P51 (day 20). Results Group differences were observed in chronic but not acute effects of cocaine. Whereas gestational MAOI treatment, with or without nicotine, increased ambulatory response to cocaine on day 5, the opposite was found for vertical activity. Different adaptive responses were observed on cocaine challenge day. GNM animals exhibited enhanced locomotor activity in the cocaine-associated environment before cocaine challenge on day 20. In contrast, only GN animals exhibited significant locomotor sensitization to the cocaine challenge. Conclusions Gestational nicotine and MAOIs both influence brain development. Such interactions may sensitize adolescents to drug abuse and should be considered in animal models of maternal smoking.     

Spatial alternation deficits following developmental exposure to Aroclor 1254 and/or methylmercury in rats.

Polychlorinated biphenyls (PCBs) and methylmercury (MeHg) are ubiquitous environmental contaminants that alter cognitive function in both humans and animals. Because PCBs and MeHg often occur together in the environment, it is important to understand whether these two contaminants have the potential to interact, causing additive or greater than additive effects. The current study examined the combined effects of gestational and lactational exposure to Aroclor 1254 (A1254), a commercial PCB mixture, and MeHg on a series of spatial alternation tasks including cued spatial alternation (CA), non-cued spatial alternation (NCA), and delayed spatial alternation (DSA) in rats using standard two-lever operant testing chambers. Pregnant Long-Evans rats received either 6 mg/kg A1254 pipetted onto a Keebler Vanilla Wafer cookie (PCB-only group), 0.5 ppm. MeHg dissolved in the drinking water (MeHg-only group), 6 mg/kg A1254 + 0.5 ppm. MeHg (PCB + MeHg group), or corn oil vehicle and normal tap water (control group) beginning 28 days prior to mating and continuing through postnatal day 16. One male and one female from each litter began testing on spatial alternation at approximately 110 days of age. Animals were reinforced for pressing the lever opposite that pressed on the previous trial. In general, animals exposed to A1254 and/or MeHg were impaired relative to control rats on the NCA and DSA tasks. Significant reductions in NCA performance were observed in the MeHg-only and PCB + MeHg groups, while significant reductions in DSA performance were observed in the PCB-only and MeHg-only groups. The PCB + MeHg group showed a similar magnitude reduction in performance on DSA, but this difference was not statistically significant due to increased variability in that group. The reductions in DSA performance were observed across most of the delays, indicating that memory impairments were not likely the cause of the deficit. Instead, the DSA deficits following exposure to A1254 and/or MeHg are indicative of either an associative or attentional impairment. The results from the current study indicate that combined exposure to PCBs and MeHg does not exacerbate the PCB- or MeHg-induced impairments on spatial alternation tasks.     

Gestational exposure to cocaine alters cocaine reward

Exposure of the developing foetus to drugs of abuse during pregnancy may lead to persistent abnormalities of brain systems involved in drug addiction. Mice prenatally exposed to cocaine (25 mg/kg), physiological saline or non-treated during the last 7 days of pregnancy were evaluated in adulthood for the rewarding properties of cocaine (3, 25 and 50 mg/kg), using the conditioned place preference procedure. Dams treated with physiological saline gained significantly less weight over the course of gestation than controls; no other differences were observed in the maternal and offspring data. All the animals developed preference to 3 and 25 mg/kg of cocaine, but those treated prenatally with cocaine did not develop preference after receiving the highest cocaine dose. In these mice, the motor activity in response to 50 mg/kg showed a small decrease. Although a reduced response to the highest cocaine dose can be argued, we suggest that the lack of preference obtained is more likely attributable to an increased sensitivity to the environmental cues associated during training to an aversive effect of this cocaine dose. The aversive properties of cocaine seem to be more apparent and to prevail over the rewarding action of the highest dose in exposed animals. These findings indicate that recurrent gestational cocaine exposure results in permanent (mal)adaptations of the structure and function of brain reward systems.     

Gestational toluene exposure effects on spontaneous and amphetamine-induced locomotor behavior in rats

Gestational Toluene Exposure Effects on Spontaneous and Amphetamine-Induced Locomotor Behavior in Rats. Bowen, S.E., Mohammadi, M.H., Batis, J.C., and Hannigan, J.H. Neurotoxicology and Teratology, XX, 2006. The abuse of volatile organic solvents (inhalants) continues to be a major health concern throughout the world. Toluene, which is found in many products such as glues and household cleaners, is among the most commonly abused organic solvents. The neurobehavioral teratogenic sequelae of solvent abuse (i.e., repeated, brief inhalation exposures to very high concentrations of solvents) have not been examined thoroughly. In a preclinical model of inhalant abuse, timed-pregnant Sprague-Dawley rats were exposed to 0, 8000, or 12,000 parts per million (ppm) for 15 min twice daily from gestation day 8 (GD8) through GD20. In the first experiment, separate groups of offspring were observed individually in an open-field on postnatal day 22 (PN22), PN42 or PN63. In the second experiment, other offspring given identical prenatal toluene exposures were observed in an "open-field" following an acute i.p. injection of amphetamine (0, 0.56, 1.78 mg/kg) on PN28. Automated measurements of distance traveled and ambulatory time were recorded. Prenatal toluene exposure resulted in small alterations in spontaneous activity compared to non-exposed rats. Prenatal exposure to 12,000 ppm toluene resulted in significant hyposensitivity to the locomotor stimulatory effects of the amphetamine challenge in male but not female rats on PN28. The results demonstrate that prenatal exposure to abuse patterns of high concentrations of toluene through inhalation can alter spontaneous and amphetamine-induced locomotor behavior in rats. The expression of these effects also appears to depend upon the postnatal age of testing. These results imply that abuse of organic solvents during pregnancy in humans may also produce long-lasting effects on biobehavioral development.     

Postnatal behavioral effects in mice after prenatal exposure to methylmercury

CFW mice were injected with methylmercury hydroxide (1, 2, 3, 5 or 10 mg/kg as mercury) on Day 8 of gestation. Mice treated with 3, 5 or 10 mg/kg averaged $\text{1}\text{3}$ fewer pups than controls. Pups from these treated animals weighed less than controls and the weight differences persisted through weaning but were no longer significant at 56 days of age. Mice exposed to methylmercury in utero showed significant differences from controls in their behavior in a 2-way active avoidance shuttle box and in a punishment situation but not when tested in an open field, a water escape runway or a conditioned suppression paradigm. Neither the mothers nor progency of the mice exposed prenatally to methylmercury showed behavioral deficits.     

Effects of prenatal methylmercury exposure on urinary proximal tubular enzyme excretion in neonatal rats

The basal developmental pattern of excretion of 3 proximal tubular enzymes was determined in 8-h urinary specimens from neonatal rats. Gammaglutamyltransferase (GGT), alkaline phosphatase (ALP), and N-acetyl-beta-glucosaminidase (NAG) activities were measured at 3, 6, 9 and 12 days after birth. Subsequently, methylmercury chloride (CH3HgCl), known to induce foetotoxic changes in the proximal tubule was administered on days 8, 10 and 12 of gestation at 3 or 6 mg/kg and its effects on the enzyme activities were examined. Dose-related increases in the 3 enzyme activities occurred at dose levels that produced no maternal or postnatal toxicity, nor overt morphological malformation of the kidney. The peak of enzyme activities averaged about 200% and 130% of the control values for GGT, ALP, and NAG respectively, and occurred on days 3 and 6 in the treated groups. Urinary enzyme activities returned to the control levels from days 6 to 12. Our data point to the possibility of detecting CH3HgCl-induced prenatal effect on the kidney by measuring the 8-h urinary excretion of enzymes by rats in the early postnatal period.     

Combined exposure to lead,inorganic mercury and methylmercury shows deviation from additivity for cardiovascular toxicity in rats

Environmental exposure to metal mixtures in the human population is common. Mixture risk assessments are often challenging because of a lack of suitable data on the relevant mixture. A growing number of studies show an association between lead or mercury exposure and cardiovascular effects. We investigated the cardiovascular effects of single metal exposure or co‐exposure to methylmercury [MeHg(I)], inorganic mercury [Hg(II)] and lead [Pb(II)]. Male Wistar rats received four different metal mixtures for 28 days through the drinking water. The ratios of the metals were based on reference and environmental exposure values. Blood and pulse pressure, cardiac output and electrical activity of the heart were selected as end‐points. While exposure to only MeHg(I) increased the systolic blood pressure and decreased cardiac output, the effects were reversed with combined exposures (antagonism). In contrast to these effects, combined exposures negatively affected the electrical activity of the heart (synergism). Thus, it appears that estimates of blood total Hg levels need to be paired with estimates of what species of mercury dominate exposure as well as whether lead co‐exposure is present to link total blood Hg levels to cardiovascular effects. Based on current human exposure data and our results, there may be an increased risk of cardiac events as a result of combined exposures to Hg(II), MeHg(I) and Pb(II). This increased risk needs to be clarified by analyzing lead and Hg exposure data in relation to cardiac electrical activity in epidemiological studies. Copyright © 2014 John Wiley & Sons, Ltd.