HgCl₂: inorganic mercury, LA: locomotor activity, MWM: Morris watermaze, PA: passive avoidance, DPOAE test: distortion product otoacoustic emission test

Regular contact with heavy metals is encountered in various human activities, which is gradually exacerbated in terms of both professional and environmental exposure (Vassallo et al., 2011[42]; Mello-Carpes et al., 2013[30]). Among these heavy metals, concerns about exposure to mercury have increased, particularly within recent decades. Mercury is found in the environment in three species:

organic mercury (methylmercury, MeHg),

Mercury is one of the most malignant and ubiquitous environmental toxins that causes a wide range of adverse health effects in humans. Due to its variety of forms, mercury is able to provoke countless effects (WHO, 1991[44]; Goyer, 1995[17]; Clarkson, 1997[11]; Peixoto et al., 2007[32]; Gao et al., 2008[15]).The effects of acute and chronic exposure to low mercury concentrations have been described for the kidneys, cardiovascular system, and neurological system (Azevedo et al., 2012[1]; Mello-Carpes et al., 2013[30]).

Inorganic mercury has been used for many years in medications, teething powders, skin creams and germicidal solutions. Mercury impacts the developing central nervous system (CNS), and induces subtle behavioral abnormalities, (learning deficits as well as memory deficits mostly, at dose levels below those related to obvious symptoms of neurotoxicity) (Newland et al., 2004[31]; Rasmussen and Newland, 2001[33]; Weiss et al., 2005[43]; Liu et al., 2009[28]). It has been reported that nervous system symptoms such as paraesthesia, fatigue, progressive weakness and neuropsychiatric disorders may be related to inorganic mercury exposure (Teixera et al., 2014[40]). In addition to that, organic mercury, the most common form of intoxication in humans, is progressively metabolized by intestinal microflora to inorganic mercury at a low rate per day (Bernhoft, 2012[3]; Teixera et al., 2014[40]). A human neonatal study performed by Gao et al. (2008[15]) revealed that increased prenatal methylmercury exposure was associated with decreased behaviour ability. This knowledge provides some support for the hypothesis that neurodevelopmental risks ensue from prenatal MeHg exposure resulting from fish consumption.

Previous studies have shown that both acute and long-term exposure to mercury may result in alterations in both the peripheral and/or central auditory systems. Ionic and metabolic changes in the auditory system are believed to be responsible for this ototoxicity (Hoshino et al., 2012[19]). Until now, the significant majority of studies have focused on central and peripheral neurological paths of the auditory tract. However, there have been no adequate studies conducted about the sensorial part of the system. In this study, we focused on the sensorial part of the auditory system using distortion product otoacoustic emission (DPOAE) tests.

WAG/Rij rats are preferred as an animal model for absence epilepsy in humans and they have many EEG and behavioural characteristics that are common with or similar to human absence epilepsy. The WAG/Rij strain of rats has been used for many studies of genetic epilepsy, colorectal cancer, and learning and memory performance (Hutteman et al., 2011[23]; Karson et al., 2015[25]).

We designed this experiment so that female rats were continuously given HgCl₂ injections throughout their gestational and lactational periods to assess the risk to the developing brain from prolonged exposure to HgCl₂. Therefore, we assumed that the dose level was low in adult rats in this experiment. The mothers continued to receive the injections after parturition, and thereby their offspring were exposed to HgCl₂ through breast milk until weaning. We examined the effects on the CNS (or brains) of the offspring based on behavioural and DPOAE tests for possible cognitive dysfunctions, spatial learning, emotional memory performances and hearing loss when they reached 5 weeks and 5 months of age to observe age-dependency.

We describe, for the first time, the age-dependent effects of gestational and lactational chronic, low-dose inorganic mercury exposure on long-term behaviour, memory and hearing in young adult and adult WAG/Rij rats. These animals constitute a valid model of genetic absence epilepsy and depressive-like behaviour. This study was designed to verify the effects of long-term exposure to low inorganic mercury doses on behavioural in young adult and adult life; and to make estimation of the common human professional exposition was one of the objectives of our study. The model for long-term mercury exposure was previously established in rats (Wiggers et al., 2008[45]) and as is the case with occupational exposure, is attained by low-dose exposure to mercury chloride (Szász et al., 2002[39]).

We have shown that chronic exposure to low-dose mercury chloride impairs performance of different memory types, general behaviour and hearing thresholds in the early life of 5-week-old WAG/Rij rats that were exposed mercury chloride during their gestational and lactational periods. The rats in the HgCl₂-exposed group exhibited no different emotional and spatial memory performances on either the PA or MWM tests compared with their age-matched controls at 5 months. Day et al. (2005[12]) proposes that the age-dependent effects of mercury that compensatory mechanisms are compromised when dually burdened by methylmercury and aging processes. According to our results, although the age-dependent effects of mercury chloride seem to be reversible or tolerable in 5-month-old WAG/Rij rats, the DPOAE test revealed that there were seriously progressive deleterious effects of mercury chloride in exposed rats beyond all of the behavioural tests.

One of the inorganic forms of mercury is mercury chloride. While mercury is most commonly seen in inorganic form (Rosales et al., 2005[34]), the majority of research focused on intoxication effects of mercury in CNS have included others forms, for example, organic (Castoldi et al., 2001[7]; Fujimura et al., 2012[14]; Maia et al., 2009[29]) or vapour states (Yoshida et al., 2011[49]; Gul et al., 2012[18]; Yasutake et al., 2012[48]). All studies have found that this substance is extremely toxic to organisms. It has been argued in previous literature that before absorption, it is possible for inorganic mercury to methylate in the gut lumen (Rudd et al., 1980[35]) and cross the placental barrier (Yang et al., 1996[47]; Chehimi et al., 2012[8]). Accordingly, inorganic mercury can act at CNS, and mercury is especially damaging for the development of the brain (Azevedo et al., 2012[1]; Trasande et al., 2005[41]). Several researchers have underlined that mercury exposure during pregnancy (Cagiano et al., 1990[6]), the neonatal period (Yoshida et al., 2011[49]), and young life (Freire et al., 2010[13]) can lead to negative consequences and may result in brain functioning deficits in the adulthood period. According to Liang et al. (2003[27]), the developing CNS is more sensitive to damage from mercury than the adult CNS. Our results affirm that lower concentrations of HgCl₂ induce behavioural, cognitive impairment and hearing-threshold deficits in 5-week-old rats and progressive hearing thresholds deficits in 5-month-old rats.

Zanoli et al. (1994[50]) showed that a single dose (8 mg/kg) of methyl mercury administered on the 15^th gestational day in rats had negative impact on the PA task when the animals were 8 weeks old, which shows that exposure during a crucial period of development of the CNS to methyl mercury leads to learning and memory deficits. Exposure to mercury in different forms may provoke diverse impacts on the CNS. In this sense, our current findings suggest that chronic HgCl₂-treated animals exhibited worse emotional and spatial memory performances on the PA and MWM tests compared with their age-matched controls at 5 weeks. However, we found no significant alterations in the LA test. Non-significant LA results do not imply that mercury does not have detrimental and toxic effects. Brookes and Kristt (1989[5]) and Choi and Kim (1984[10]) demonstrated that HgCl₂ concentrations under 1 μg/g are not toxic to in vitro cultured CNS tissues. Yoshida et al. (2011[49]) detected noteworthy alterations in gene expression in brains of mice contacted with low levels of mercury vapour during their postnatal development, in spite of the fact that their ability to learn was not affected. Hu et al. (2012[20]) administered rice contaminated with mercury to 3-week-old rats and measured in MWM that this procedure negatively affected spatial learning and memory of the rats. When it comes to effects of mercury on human beings, the effects of mercury exposure on cognitive functions in adulthood have only been reported in a few studies, a great deal of which focused on the results of uncontrolled exposure of humans to mercury (Smith et al., 1983[38]; Sletvold et al., 2012[37]) or in rats, through the course of a short exposition (Hu et al., 2012[20]). The long-term outcomes of chronic low-dose mercury chloride exposure (regional, occupational, etc.) are less well understood, and clinical studies on this topic are very limited.

Xu et al. (2012[46]) found that inorganic mercuric chloride (HgCl₂ at 0.025-25 μM) not only caused neuronal degeneration but also perturbed neuronal excitability. Basu et al. (2007[2]) found decreases in NMDA receptor levels in the basal ganglia, cerebellum, brain stem and occipital cortex that are dependent on mercury concentration. Indicating that mercury can significantly reduce NMDA receptor levels even at dietary concentrations as low as 0.1 ppm, these findings raise both ecological and physiological concerns (Sanchez, 2015[36]).

AMPA receptor (AMPAR) membrane trafficking regulation is critical with regards to synaptic plasticity as is the case for learning and memory (Zhang, 2015[51]). The activation of NMDA receptors may contribute to better spatial and emotional memory performances. This situation may explain the deterioration in the MWM and PA test results of 5-week-old rats exposed to mercury.

Furthermore, gradually decreased levels of mercury and the ameliorating neuronal development of young rats after the end of inorganic mercury exposure might contribute to normalising cognitive and spatial functions in 5-month-old rats because the behavioural test results of 5-month-old rats were not statistically significant.

On the other hand, our results showed that there are seriously progressive effects of gestational and lactational inorganic mercury exposure. Beyond all of the behavioural tests we conducted, these seriously progressive effects may be disclosed by the DPOAE test.

The ototoxic effect of mercury has been studied previously using objective and subjective test methods; the cochleotoxic effect of mercury is a less-studied topic. We determined the cochleotoxicity using DPOAE tests. Otoacoustic emissions can be suppressed in part centrally through the superior olivary complex. Lateral and medial olivocochlear bundles' axons project from the superior olive and leave the brainstem as a ventral component to the inferior vestibular nerve. As the Oort's vestibulocochlear anastomosis, they join the cochlear nerve. Axons of the lateral olivocochlear bundle synapses with afferent neurons from the cochlea. Axons of the medial olivocochlear bundle terminate at the base of cell bodies of the outer hair cells. It is generally believed that the medial efferents counteract the amplifying effects of the outer hair cells. This effect is probably mediated by acetylcholine (Bolay et al., 2008[4]).

Many studies have reported that various insults, such as mercury toxicities and chemicals, induce neurological disorders or hearing loss in vivo and that those disorders correspond to changes in the Na⁺/K⁺-ATPase activities of the brain regions and cochlear lateral wall (Huang et al., 2007[21]; Cheng et al., 2005[9]).

Igarashi et al. (1992[24]) carried out a histochemical experiment, in which it was understood that mercury deposits usually concentrate in the cerebellum and in parts of the vestibular nerves, spiral ganglion, striavascularis, and cochlear nerves. In this experiment, there was a small mercury accumulation in the acoustic maculae and in the cochlea in the vestibule; and it was found that there was only a single instance of mercury accumulation in the organ of Corti. Our results were consistent with the results of Igarashi et al. (1992[24]).

In terms of the results of the 5-week-old rat groups, we noted that the HgCl₂ group exhibited poorer test results for all frequencies compared with the control group. The differences were statistically significant at higher frequencies. These results imply that exposure to inorganic mercury during gestational and lactation periods results in ototoxicity; this toxicity can be monitored using the DPOAE test, which is an easy and objective test method. Additionally, in the 5-month-old rats, both groups exhibited age-related changes. However, our results demonstrated that the HgCl₂-exposed group was more affected by hearing thresholds than the control group. Moreover, the largest hearing loss was recorded in the 5-month-old HgCl₂ group. This result can be interpreted as age-related hearing loss being more deteriorated by HgCl₂ exposure. Our results indicate that the DPOAE test is more sensitive than the other tests used in this study to reveal inorganic mercury toxicity, particularly for late effects in aged rats.

According to Huang et al. (2011[22]), absorption in gastrointestinal tract and accumulation of mercurial compounds in the brainstem are responsible for auditory dysfunction. The findings reached by Huang et al. (2011[22]) show that exposure to low-dose mercurial compounds (the probable concentrations for human exposure) in perinatal stages can induce irreversible ototoxicity; additional contact may end in severer toxic effects in future developmental stages. According to Liang et al. (2003[27]), mercury has targeted the sensory cochlea and vestibular apparatus of the inner ear containing the central auditory structures and hair cells. In addition to that, it has been shown by neurobiochemical studies of brainstem tissues that ototoxicity triggered by mercurial compounds could be mediated by oxidative stress damage, altered Na⁺/K⁺-ATPase activities and NOx levels of the brain regions and the cochlear lateral wall. This explanation may be the most appropriate to interpret our results.

Additional studies are necessary to better understand the exact pathophysiological mechanism of long-term mercury exposure and its neurotoxicity. For this purpose, cochlear dissection and brain slices of rats must be obtained and analyzed using immunohistochemistry techniques. Moreover, given the urbanization and industrialization of today's rapidly growing world, it is important to raise public awareness about mercury and limit exposure to mercury as a public health priority.

We have shown that chronic exposure to low-dose mercury chloride impairs the performance of different memory types and hearing in WAG/Rij rats that were exposed to mercury chloride during their gestational and lactational periods. Although age-dependent effects of mercury chloride appear to be reversible or tolerable by the behavioural tests, DPOAE test results revealed that there were seriously progressive deleterious effects of mercury chloride in rats exposed to mercury. Medical teams may find a chance to identify treatment methods and alleviate hearing changes with the early diagnosis the effects of mercury exposure. In this sense, in experimental and clinical studies that research hearing loss and heavy metal ototoxicity may benefit from DPOAE tests. However, we do not know the exact mechanisms involved in these effects; additional research is accordingly necessary. However, our results are important because information about age-dependent mercury chloride exposure with long-term outcomes on both animal models and humans is very limited in the literature.

We thank Si-Ser Hearing Centers for providing us hearing threshold assessment equipment for DPOAE tests.

The authors declare that there are no conflicts of interest.