Early Life Exposure to Methylmercury Influences Later Life of Development and Maternal Toxicity in Rats

Research Article

Austin J Pharmacol Ther. 2014; 2 (4). 1022

Early Life Exposure to Methylmercury Influences Later Life of Development and Maternal Toxicity in Rats

Gandhi DN1* and Dhull Dinesh K2

11Department of Neurobehavioral Toxicology, National Institute of Occupational Health, Meghaninagar, Ahmedabad-380016, India

22Department of Neurophysiology ( Glial Cells), National Institute of Mental Health and Neurosciences, (NIMHANS), Banglore- 560029, India

*Corresponding author: : Gandhi DN, Department of Neurobehavioral Toxicology, National Institute of Occupational Health, Meghaninagar, Ahmedabad-380 016, Gujarat, India

Received: February 15, 2014; Accepted: March 02, 2014; Published: May 03, 2014


Methylmercury (MeHg) is recognized as one of the most hazardous environmental pollutants. The aim of the present study was to find out whether and how early life of exposure to this neurotoxicant influences later life of developmental and maternal toxicity. The study was carried out on Wistar rats, the progeny of rat mothers exposed to MeHg (0.5, 1.0 and 1.5mg ⁄kg ⁄day) from gestational day (GD) 5 to till parturition (PND0). The following reproductive and developmental parameters were assessed: body weight, body weight gain(%), deaths, abortions or early deliveries, implantations, postimplantation, resorption, gestational length percentage viability, gait abnormality and hyper activity. The results obtained in the study showed no deaths, abortions, or early deliveries, enhanced maternal toxicity, which included deaths and decreased body weight gain (30.2%) and food consumption. The number of nonviable implants decreased significantly following exposure to 1.5mg⁄kg⁄day MeHg-treatment group, with the percentage of postimplantation loss (44.8%). In contrast, MeHg caused up to 33.3% of resorbed litters without showing sign of maternal toxicity such as gait alterations and hyperactivity in rats treated with 1.5mg⁄ kg⁄day. Average gestation length (days) was significantly affected with 1.0 and 1.5mg⁄ kg⁄day MeHg-treatment groups. The data suggests that gestational exposure would enhance the dose-dependent MeHg–induced embryo⁄fetal and maternal toxicity as a form of teratogenic action. Further studies of exposure to MeHg at present dose levels during critical windows of development induce a number of adverse health outcomes for offspring. Such effects may contribute to increased disease risks observed in human population.

Key words: Methylmercury (MeHg); Gestational exposure; Postnatal development; Maternal toxicity; Rat


In the 1950s and 60s neurological disease was noted in many people living around Minamata Bay in Japan. People of all ages were affected, but effects were most severe in infants and children. The disease was traced to methylmercury (MeHg) pollution in the bay that accumulated to high levels in fish (10–40 ppm). The principal sources of exposure to Hg in the general population are ingestion and inhalation of Hg compounds. Due to its ubiquitous presence in the environment, health concerns are increasing. Methyl mercury (MeHg), an organic methylated form of mercury, exists in aquatics receiving industrial wastes containing mercury. The health impact of water contamination of MeHg continues to draw concern, since accidental poisoning that occurred in Minamata, Japan, Niigata and Iraq [1]. Faroe Islands cohort study reported MeHg related deficits in neurological and cognitive functions in school–age children [2]. The epidemiological and animal studies demonstrate that the fetuses are more vulnerable than mothers, as the sensitivity of the nervous system to MeHg toxicity is the highest during developmental stages [3,4].

Methylmercury is an embryotoxic and can induce teratogenic effects in golden hamsters [5,6], cats [7], rats [6,8], and mice [9–12]. Exposure events during critical windows of fetal and postnatal (PN) development pose a serious risk for adverse health outcomes later life [13]. Exposure to toxic elements such as mercury or arsenic during gestation and lactation may potentially cause adverse effects on the development of foetuses and neonates [14–17]. Developmental delays in acquiring motor skills associated with low to moderate prenatal MeHg exposure are known [18]. The fetus is especially susceptible to MeHg–induced embryo⁄fetal toxicity, and neurobehavioral effects including learning deficits in healthy animals exposed during gestation [3]. Behavioural alterations were observed, even at MeHg levels below those causing morphological abnormalities [19]. Several studies on the developmental effects of MeHg on rats and mice have provided specific hypotheses regarding the mechanisms of action of MeHg. It is likely that maternal fish intake–related MeHg exposure during pregnancy, at levels safe for mothers, may affect the developing nervous system of the foetus. This possibility is supported by the data from studies of the victims of mass MeHg poisonings in Japan [1].

In addition, the dam is primarily determined the development of major regulatory system underlying behaviour and physiology in the neonatal rat [20]. However, our earlier study [21] indicates maternal and embryo⁄foetal toxicity when high dose of MeHg (2.0mg⁄kg⁄ day) was given by gavages during GD5 till parturition to pregnant rats caused hundred percentage of resorption of the F1 generationoffspring. So it further worsened MeHg toxicity even before birth, adding up the impact throughout life. However, the impact depends on exposure, duration, route as well as form of exposure. Our goal was to develop a rat model of early life MeHg exposure through which we could identify critical windows of exposure that might result in adverse impacts on the development of the nervous system later in life. Thus, the study presented here was designed to further explore the adverse developmental outcomes following early life low dose MeHg exposure has detrimental impact on later life of development and maternal toxicity leads to change in neurobehavioral outcomes.

Materials and methods

Study design

Mated female Wistar rats were dosed daily with MeHg from GD5 to till parturition. The effects of exposure observed during the pre weaning period (PND1 to PND21) of life in offspring.

Ethical issues

In all the experiments was performed in accordance the guidelines of the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA), India. The Institutional Animal Ethics Committee approved the study design.


All experiments were performed on rats, white WISTARS. Sixty–four 64; mature male and female Wistar albino rats weighing 180–200g were obtained from the institutes breeding colony. After one–week acclimation in the laboratory, female rats were mated with males [2:1] overnight and examined the following morning for vaginal smears. Vaginal smears were taken daily between 9 a.m. to 10 a.m. from mated female rats. On the day when spermatozoa in the vaginal smear were found, the female was weighted and this day was regarded as the first day of gestation (GD0). During the experimental period, animal room was maintained at temperature 22 ± 2°c; relative humidity 65 ± 5%, (12h light⁄dark cycle), with free access to food and water.

Randomization, Mating and Treatment

Pregnant rats

Proestrus virgin female rats were mated proven–fertile male rats (2:1) overnight. The day of mating confirmed by the presence of sperm–positive vaginal smears was designated as gestation day (GD) 0. At GD0 female’s pregnant animals (dams) were randomly assigned to four groups of rats, and housed individually. Out of thirty GD 0 females, twenty–eight pregnant females delivered the pups; rest of the two females in higher dose did not deliver. Date of birth was designated as postnatal day (PND) 0. (Table: 1).

Preparation of dose formulation

The dose formulation for each group was prepared separately in order to maintain a constant dose volume of not more than 5–ml⁄kgbody weight. Methyl mercury chloride (CH3ClHg) 99.9% pure, CAS no.115–09–3; batch size 8151x, Sigma–Aldrich GmbH) was obtained from Sigma Aldrich, U.S.A. The doses were based on data showing that at this exposure level, the Hg concentration in newborn rats was comparable to that found in human infants from populations with high dietary fish consumption [22,23]. Controls were treated with saline solution.

Prenatal methyl mercury exposure

Twenty eight–28; GD0 pregnant females (F0 Generation) were assigned, based on body weight, to each of the following groups using prior to MeHg treatment initiation: Vehicle Control (n=7); 0.5 mg MeHg⁄kg body weight⁄day (n= 8); 1.0 mg MeHg⁄kg body weight⁄ day (n= 7); 1.5 mg MeHg⁄kg body weight⁄day (n= 6). Throughout gestation, all pregnant rats were weighed and examined for signs of toxicity daily. The females were dosed, orally by gavage, form GD5 to till parturition.

Evaluations of F1 Generation

General condition

Beginning on GD21, dams were inspected frequently between 0800 and 2000h for birth until delivery, each presumably pregnant female was checked twice daily for completion of or difficulties in parturition. The day of parturition was defined as postnatal day (PND0), meaning the maximum resolution for gestational length was half a day. The pups were counted, examined for gross malformation and weighed individually. The body weight of pups and maternal behaviors were recorded daily during nursing. The offspring was considered the experimental unit. After parturition, the neonates were observed for mortality and signs of toxicity.

Assessment of the reproduction success

The offspring were evaluated for survival, growth, development and behaviors. When parturition was complete, the numbers of stillborn, implantation, postimplantation, resorption and live pups in each litter were recorded. Following variables were observed: Birth measures: the offspring were examined on PND1 for morphological anomalies (e.g., missing digits, facial malformations), sex by relative anogenital distance and culled pseudo–randomly to twenty animals each and balanced for sex (20 females and 20 males) to the extent possible.

Assessment of the offspring’s morphological development

Gestation length was calculated at birth and the following offspring data were collected on PND1: Pups size, sex ratio (as percent males), body weight for each pup and the number of malformed offspring. On PND1, the pups were identified within each groups of treatment and were assessed for Males Body length (mm); Females Body Length (mm); Males tail Length (mm); Females tail Length (mm); Pups viability at birth; Pups affected per dam; Pups mortality at birth (PND1–4) and the pups from each litter were weighed on PND1, 3,5,7,9,14,15,21,28.

Maternal toxicity & Behaviours

A maternal toxicity and behaviors were observed daily in the home cage of each dam and her litter between gestational day and post–delivery (PND) 1 till 14.

Terminal Evaluations

Females that did not mate were euthanized approximately 3 weeks after the completion of the mating period and were subjected to necropsy. The pregnancy status of animals that did not mate was confirmed and recorded in datasheet. Dams whose whole litters were born dead or died prior to weaning were also recorded. The dams with normal pups were euthanized approximately 3 weeks after weaning of the pups. The method of euthanasia was carbon dioxide asphyxiation followed by exsanguinations from the abdominal aorta. The number of implantation site scars was recorded for the pregnant animals.

Statistical analysis

Data were analysed by one–way analysis of variance (ANOVA) followed by Duncan test. The level of statistical significance was set at p<0.05. All data are expressed as means ± S.E.M.


Pregnant females (dams) were divided into four groups of 30 animals: control (with free access to fresh tap water), 0.5mg⁄kg⁄ day MeHg; 1.0mg⁄kg⁄day MeHg and 1.5mg⁄kg⁄day MeHg by oral gavages. Dam’s body weight was noted every day during gestation. After birth the number of pups for each group was as follows: control (N = 66), MeHg 0.5mg⁄kg⁄day (N = 80); MeHg 1.0mg⁄kg⁄day (N =73) and MeHg 1.5mg⁄kg⁄day (N = 43) per each groups of exposure. We have randomly selected either sex of twenty offspring per each groups of exposure to achieves the developmental,morphological milestonesand reproductive test.

A summary of the distribution and fate of all mated rats of the study is given in Table 1. During pregnancy, the treatment groups did not differ in water and food intake, and in the rate of the body mass increase. The pregnant rats treated with 0.5, 1.0 and 1.5mg⁄kg⁄ day MeHg from GD5 to till parturition produced neither maternal toxicity nor any noticeable signs or symptoms. The behaviour of the treated rats was similar to that of the control rats. On day 4 of gestation, the maternal body weight (g) of control (227.7±16.2) with respect to treatment groups was 0.5mg⁄kg⁄day MeHg (225.2±10.7); 1.0mg⁄kg⁄day MeHg (215.4±10.0) and 1.5mg⁄kg⁄day MeHg (182.8±3.5) remained almost within the range. On day 20 of gestation, the maternal body weight gain (g) of control and three dose levels (0.5, 1.0 and 1.5mg⁄kg⁄day) MeHg exposed dams were 111.2±4.1; 115.4±3.4; 111.0±2.0 and 79.4±2.1. Maternal weight gain of dams during gestation and weight gain during treatment was significantly reduced in high dose (1.5mg⁄kg⁄day) MeHg treatment group [F (3,20) = 3.43, p<0.05] without any sign of anxiety, hind limb ataxia or gait alterations. The 0.5 and 1.0mg⁄kg⁄day MeHg–treatment groups did not differ from the control group in the level of food and water consumption and body weight gain, whereas maternal body weight during gestational period (GD0–20) significantly reduced in 1.5 mg⁄ kg⁄day MeHg–treatment group [F (3,144) = 6.629, p< 0.01] (Figure 1 and 2).