ToxSci Advance Access originally published online on August 6, 2008
Toxicological Sciences 2008 106(1):180-185; doi:10.1093/toxsci/kfn158
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Behavioral, Morphological, and Biochemical Changes after In Ovo Exposure to Methylmercury in Chicks
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* Departamento de Biologia Celular, Embriologia e Genética, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, 88090-400 Santa Catarina, Brazil
Departamento de Ciências Naturais, Centro de Ciências Naturais e Exatas, Fundação Universidade Regional de Blumenau, Blumenau, 89012-900 Santa Catarina, Brazil
Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, 88090-400 Santa Catarina, Brazil
2 To whom correspondence should be addressed at Universidade Federal de Santa Catarina, Depto BEG-CCB, Florianópolis, 88090-400 Santa Catarina, Brazil. Fax: +55-(48)-3721-5148. E-mail: yararm{at}ccb.ufsc.br.
Received June 16, 2008; accepted July 23, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Methylmercury (MeHg) is an environmental pollutant known to induce neurotoxicity in several animal species, including humans. However, studies focusing the effects of MeHg poisoning in chicks were based on phenomenological approaches and did not delve into the molecular mechanisms. The purpose of this study was to evaluate the postnatal consequences of the in ovo exposure to MeHg on behavioral, morphological and biochemical parameters in chicks. At the fifth embryonic day (E5), Gallus domesticus eggs were submitted to a single injection of 0.1 µg MeHg/0.05 ml saline. After treatment, the eggs returned to the incubator until hatching (E21). From first to fifth postnatal days (PN 1–PN 5), the MeHg-treated chicks showed lower frequency of exploratory movements and a significantly higher frequency of wing and anomalous movements. Cerebellar glutathione (GSH) levels and the activities of the GSH-related enzymes GSH reductase and GSH peroxidase were significantly higher (70, 72, and 80%, respectively) in MeHg exposed chicks in comparison to controls. Mercury impregnation was densest in the granular layer, followed by the Purkinje and molecular layers of treated chicks. A significant reduction of the number of Purkinje cells, as well as a greater distance between these cells were observed in chicks of MeHg group. Our results disclose that the prehatching exposure to MeHg induced motor impairments, which were correlated to histological damage and alterations on the cerebellar GSH system's development from PN 1 to PN 5.
Key Words: methylmercury; Gallus domesticus; embryo; behavior; glutathione; cerebellar cortex.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several studies show that the developing central nervous system (CNS) at prenatal and/or early postnatal periods is more susceptible to xenobiotic-induced neurotoxicity than in adults (for review, see Costa et al., 2004
). Of particular importance, methylmercury (MeHg), a highly toxic form of mercury in the environment, is known to induce neurodevelopmental toxicity in both animals and humans (Clarkson et al., 2003; Johansson et al., 2007).
A number of synchronous mechanisms are likely associated with MeHg-induced neurotoxicity, including impairment of intracellular calcium homeostasis (Atchison, 2005), alteration of glutamate homeostasis (Aschner et al., 2000; Farina et al., 2003a; Soares et al., 2003) and oxidative stress (Franco et al., 2007; Manfroi et al., 2004). Particularly, the glutathione system, an antioxidant tool for protecting cells against oxidative damage, represents a molecular target for MeHg (Stringari et al., 2008).
MeHg can bioaccumulate in aquatic food webs, leading to elevated concentrations in birds (Scheuhammer et al., 2007). In highly contaminated areas, when MeHg is transferred from a mother bird to her eggs, it can reach concentrations associated with impaired reproduction in wild birds such as the common loon (Gavia immer), common tern (Sterna hirundo), and California clapper rail (Rallus longirostris obsoletus) (Heinz et al., in press). Because few field studies on MeHg-induced avian toxicity exist because they are costly and difficult to conduct (Heinz et al., in press), controlled laboratory breeding studies have been done in order to investigate the toxic potential of this toxicant in birds. Studies by Heinz et al. (2006) demonstrated the toxicity of MeHg to embryos of several bird species in various experimental conditions, using mainly different solvents, injection sites, and embryo ages. Heinz et al. (in press) ranked the sensitivities of the embryos of many species of birds to MeHg. Bertossi et al. (2004) showed that MeHg induces neurotoxicity in chicks. However, little is known about the molecular mechanisms related to MeHg-induced neurotoxicity in chicks. The aforementioned studies were based on phenomenological approaches. Even though motor impairment (Dietrich et al., 2005) cerebellar damage (Carvalho et al., 2007), and alterations in glutathione homeostasis (Stringari et al., 2008) have been described as important molecular mechanisms involved with MeHg-induced neurotoxicity in rodents and primates, such indications of toxicity have not been not examined in birds.
From a methodological point of view, the chick embryo (prehatch exposure) appears to be an excellent model to study developmental neurotoxicity. A single injection of a xenobiotic into the yolk sac provides long-term prehatch exposure during the entire in ovo development (Muller et al., in press). This exposure schedule mimics a long-term exposure during the prenatal period (Muller et al., in press), which is crucial for CNS development. The brain is under intense development during the prehatch period in chicks (Bertossi et al., 2004), which may render it more sensitive to toxic environmental chemicals, including MeHg.
Taking into account that (1) there are no studies on the biochemical and histological parameters involved with MeHg-induced neurotoxicity in chicks; (2) the avian model of in ovo exposure to xenobiotics represents a promising tool for evaluating developmental neurotoxicity; (3) MeHg-induced neurotoxicity is related to oxidative and histological damage in the cerebellum, (4) the relationship between cerebellar damage and motor function is a well-reported phenomenon; and (5) the glutathione antioxidant system is an important molecular target for MeHg-induced neurotoxicity in many animal species, this study evaluated the postnatal consequences of in ovo exposure to MeHg by assessing potential relationships between behavioral, morphological and biochemical parameters. This integrative approach is an attempt to broaden the current literature on neurotoxicity in birds, improving knowledge of MeHg poisoning. Many effects of developmental exposure to heavy metals can be initially silent, and more significant damage may appear only during late childhood or adulthood; thus combined behavioral, morphological and biochemical analyses may provide significant data about MeHg toxicity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
Methylmercury II chloride (MeHg) was obtained from Aldrich Chemical Co. (Milwaukee, WI). Glutathione reductase from baker's yeast and reduced glutathione were obtained from Sigma (St Louis, MO). All other chemicals were of the highest grade commercially available.
Egg treatments.
Fertile eggs of Gallus domesticus were obtained from a commercial hatchery (Macedo-Koerich S/A, Santa Catarina, Brazil). All eggs were cleaned, labeled, weighed (65.64 ± 5.36 g) and placed horizontally in an incubator at 37.5°C and 65.0% humidity. The experiments were carried out according to the guidelines of our institution's Ethics Committee—project number 254-2003/CEUA//UFSC, and were also conducted in accordance with the Guiding Principles in the Use of Animals in Toxicology, adopted by the Society of Toxicology in July 1989.
After 5 days of incubation (E5), the eggs were divided into two experimental groups: (1) MeHg group, eggs that received a single injection of 0.1 µg of MeHg/0.05 ml saline into the yolk sac; and (2) control group, eggs treated as previously described (Muller et al., in press). After the treatments, the eggs were returned to the incubator and were monitored daily until hatching (E21). The MeHg dose was based on a previous dose-response study concerning MeHg and in ovo exposures (Heinz et al., in press). Taking into account that each egg weighed 65.6 ± 5.3 g, the administered amount of MeHg (0.1 µg) allowed for a concentration of approximately 1 ppb per egg in our experimental protocol. A high MeHg dose (1 µg/egg) induced obvious signs of systemic toxicity, and significantly reduced the number of hatches (data not shown).
Behavioral repertory.
On the day of hatching, termed postnatal day 0 (PN 0), chicks of each group (N = 14) were transferred to a home cage (36 x 47 x 33 cm) warmed by dull-emitter lamps (40 W). Food and water were provided ad libitum. Chicks were housed in cages in sets of four or five individuals until PN 5. Body weight was measured daily from PN 0 to PN 5.
In order to analyze motor activity and coordination, three behavioral categories were defined: exploratory, wing, and anomalous movements. Many effects of developmental exposure to heavy metals can be initially silent, and more significant damage may appear only during late childhood or adulthood; thus combined behavioral, morphological and biochemical analyses may provide significant data about MeHg toxicity. The behaviors were observed daily, in the morning, with the chicks placed in a wooden box (40 x 60 x 50 cm), from PN 1 to PN 5. The behavior of each chick was video-recorded for 10 min. Prior to observation, the chicks were acclimatized in the wooden box for 5 min. Behavioral analyses were not carried out on PN 0 because both control and MeHg-treated young chicks moved very little on this day. The behavioral repertory was based on the studies of Burger and Gochfeld (1995).
Tissue preparation for biochemical analyses.
After the behavioral analysis (PN 5), seven chicks per group were randomly killed by decapitation. The cerebellum was dissected, weighed, and homogenized (1:5 wt/vol) in [N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid)], 25mM, buffered to pH 7.4. Tissue homogenates were centrifuged at 20,000 x g at 4°C for 30 min. The supernatants obtained were used to determine enzymatic activities of glutathione peroxidase and glutathione reductase, as well as glutathione levels. Enzymatic activities were determined based on Carlberg and Mannervik (1985) and Wendel (1981), respectively. Glutathione was evaluated according to Ellman (1959), with minor modifications (Farina et al., 2003b).
Histological analyses.
The remaining seven chicks per group (PN 5) were anesthetized by ethyl-ether inhalation (for 6 min), and killed by transcardiac perfusion with physiological saline followed by sulfite-carnoy solution. Next, the cerebellum was removed, weighed, and immersed in the same fixative solution. Tissues were dehydrated in an ethanol series and embedded in paraffin, sectioned at 6 µm, and stained with cresyl violet for morphological and morphometric analyses. Purkinje cells were counted from 10 random visual fields in four sections (x40). The cell diameter and the distance between Purkinje cells were measured with an ocular micrometer (Olympus, Japan) (x400). Mercury deposition was evaluated by the AMG (autometallography) method (Danscher, 1984), and the sections were counterstained with hematoxylin. Metal deposition was visualized by the presence of black granules in the cerebellar cortex, which represent silver surrounding the deposited mercury. The deposition was classified as mild (+), moderate (++), or intense (+++) by an investigator who was blind to the treatment assignments.
Statistical analyses.
Behavioral differences between groups were evaluated by one-way ANOVA, followed by Duncan's multiple-range tests when appropriate (Satistica packcage 6.0, USA). Differences in biochemical analyses between the control and MeHg exposed animals were evaluated by a Student's t-test for independent variables (Statistica package 6.0). Repeated measures and ANOVA were performed to detect differences in body weight and behavioral categories from PN 1 to PN 5. Differences were considered significant when p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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From hatching to PN 5, the body weight was always significantly lower in young chicks exposed to MeHg compared with those of the control group. In contrast to the control animals, there was no significant body weight gain in MeHg-exposed chicks during PN 1 and PN 5, as observed by analysis of variance with repeated measures (p < 0.001) (Fig. 1).
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In both the control and MeHg groups, exploratory movements were the most common behavior observed from PN 1 to PN 5. However, the MeHg-exposed chicks showed a lower frequency of exploratory movements (p < 0.0001) (Fig. 2) than the control animals. The number of wing movements was significantly higher in the MeHg-treated group than in the control (p < 0.0001). The number of anomalous movements was also higher in the MeHg-exposed group (p
0.0001). These behavioral analyses suggest increased motor impairment in chicks exposed to MeHg during the prehatching period.
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Because the glutathione (GSH) antioxidant system represents an important target involved with the developmental neurotoxicity induced by MeHg in mammals, we assessed the potential effects of in ovo exposure to MeHg in the cerebellar GSH system using an avian model. The levels of GSH and the activities of the GSH-related enzymes glutathione reductase (GR) and glutathione peroxidase (GPx) were significantly higher in the cerebellum of MeHg-exposed chicks in comparison to control animals (Fig. 3).
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Cresyl violet staining showed that granular, Purkinje, and molecular layers of the cerebellar cortex were easily recognized in control animals (Fig. 4). The Nissl substance was regularly distributed, and the nucleus and nucleolus were evident in Purkinje cells. As expected, the AMG autometallography of the control showed no traces of mercury. In the MeHg-exposed group, the boundaries of the cortical layer were not evident, and the Nissl substance was irregularly distributed in pellets. To determine whether the treatments contributed to variations in the cellular arrangement in the cerebellum, we used morphometric procedures (Table 1). A significant reduction of the number of Purkinje cells, as well as a greater distance between these cells was observed. In the MeHg-treated group, mercury impregnation in the cerebellar cortex was evident by the AMG method, which evaluates the presence of black granules that represent silver surrounding lead deposition. Mercury impregnation was densest in the granular layer, followed by the Purkinje and molecular layers.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our study demonstrated that in ovo exposure to MeHg decreases the initial body weight of newly hatched chicks, and also their weight gain during the first postnatal week. Significant decreases in body weight were also reported by Carratú et al. (2006)
and Wakabayashi et al. (1995) in rodents treated with MeHg, as well as in chickens exposed to lead acetate during embryogenesis (Muller et al., in press). This reinforces the idea that weight loss is a common consequence of heavy-metal poisoning. From a methodological point of view, the observed decreased body weight in MeHg-exposed chicks is important because it indicates that a single injection of this compound into the yolk sac affects a general health index in Gallus domesticus, suggesting the accuracy and effectiveness of our model. In ovo exposure to MeHg induced the appearance of anomalous movements and low locomotor (exploratory) activity in the MeHg-treated chicks, suggesting increased motor impairment. Taking into account the role of the cerebellum in motor function (Habas, 2001), as well as the higher affinity of MeHg for this structure (Franco et al., 2006), one could suppose that the observed behavioral changes elicited by in ovo MeHg exposure may be related to cerebellar damage. Our data are in agreement with this idea: a close relationship between cerebellar damage and motor impairment was observed in MeHg-exposed chicks during the first week after hatching.
Rodents are the customary model for exploring the neurotoxic effects of MeHg (Dietrich et al., 2005; Doré et al., 2001; Franco et al., 2006). Studies focusing the effects of MeHg poisoning in chicks (Bertossi et al., 2004) were based on phenomenological approaches and did not delve into the molecular mechanisms involved in the neurotoxicity elicited by MeHg poisoning. Consequently, the combined use of behavioral, biochemical, histological and morphometric procedures to evaluate this phenomenon in chicks is a new and important contribution of the present study. We used Gallus domesticus because the in ovo development occurs in the absence of maternal factors, which makes chickens an excellent developmental model animal for prehatch stress studies. Unlike mammals, where the embryo may be subjected to maternal factors throughout gestation, avian mothers can only incorporate substances into the egg before it is laid, and most of the embryonic developmental processes take place afterward. It is important to assess the MeHg developmental neurotoxicity in alternative animal models in order to better comprehend their toxicity mechanisms, because although the effects of MeHg poisoning are well known, the severity of the damage differs according to exposure time and dose, as well as animal species and age.
As previously mentioned, impairments in the motor activity induced by MeHg were observed in rodents (Dietrich et al., 2005; Franco et al., 2006) and were related to cerebellar damage. The cerebellar tissue was also explored in our study by performing morphological and biochemical analyses. A main indication of MeHg effect in the chick cerebellum was the modifications of the Purkinje-cell arrangement in the cortical layers. This evidence was confirmed by morphometric analysis, which revealed that the distance between the Purkinje cells was greater, and also that there were fewer of these cells in the MeHg group than in the control. Because the granular layer appears to be the most-affected cerebellar region in MeHg-exposed rodents (Kakita et al., 2000; Kim et al., 2000; Wakabayashi et al., 1995), our study revealed an interesting dissimilarity between chicks and rodents exposed to MeHg. In fact, conversely to observations in rodents, the Purkinje-cell layer was more sensitive to the deleterious effects induced by MeHg than was the granular layer. The mechanisms related to this phenomenon remain unsolved, and are an open question for additional studies.
The incorporation of heavy metals into cerebellar tissue during its development can result from the thinning of endothelial cells and the presence of clefts between endothelial cells, as reported by Narbaitz et al. (1995), and the delayed maturation of the vessels and imperfect acquirement of blood-brain barrier properties, as observed by Bertossi et al. (2004). With respect to MeHg, even though it is well known that MeHg L-cysteine conjugates may share a common transport step with the l-neutral amino acid carrier transport system, allowing for the crossing of cell-membrane barriers to reach its target tissues (Aschner, 1989; Clarkson et al., 2007; Simmons-Willis et al., 2002), the aforementioned structural conditions may facilitate the entrance of MeHg into the brain and, consequently, the deposition of this metal in neural tissues. The mercury impregnation in the cerebellar cortex observed in our study was both inter- and intracellular. Ultrastructural analysis of the granular, Purkinje, and molecular layers could better elucidate the subcellular profile of MeHg deposition in neural tissues.
Studies with rodents have shown that the GSH antioxidant system is an important molecular target for the neurotoxic effects of MeHg (Stringari et al., 2006, 2008). MeHg appears to hamper this antioxidant system, which can contribute to the pro-oxidative effects of the neurotoxicant (Farina et al., 2005). However, the effects of mercurials on the GSH antioxidant system appear to depend on the degree and duration of exposure. An interesting study with rodents (Woods and Ellis, 1995) showed that, during the initial phase of MeHg exposure, MeHg-induced oxidative stress stimulated the synthesis of oxidant-scavenging GSH molecules via the upregulation of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis. Woods and Ellis (1995) found that GCS mRNA levels decreased subsequent to the initial phase events, suggesting that the toxicity and the capability of stimulating GSH synthesis depend on the degree and duration of the exposure to MeHg. Literature data for Saccharomyces cerevisiae (Dormer et al., 2002) and mice (Solis et al., 2002) have also shown that pro-oxidant conditions, including mercury exposure, are able to increase the expression of GCS. However, no published data on the effects of MeHg on GPx and GR expression are available. In our study, the MeHg dose (1 µg/egg) increased the basal activity of the cerebellar antioxidant system in chicks. Both GSH levels and GSH-related enzyme (GPx and GR) activities were significantly increased in the cerebellum of MeHg-exposed chicks compared with the controls. One µg/egg is considered a large dose for Galliformes (Heinz et al., in press), indicating that the observed upregulation in the GSH antioxidant system occurs even at high MeHg exposures. In agreement with our findings, Ji et al. (2006) observed that selenium-dependent GPx activity and GSH content were significantly increased in the brains of ducks living in a mercury-contaminated area. These observations (Ji et al., 2006; present findings) indicate that mercurials are able to induce an upregulation in the avian GSH system, which could represent a pathophysiological response to counteract the deleterious pro-oxidative effects of mercury.
In summary, our results revealed that chicks are susceptible to MeHg-induced neurotoxicity after in ovo exposure. A clear correlation between behavioral impairments, and morphological and biochemical changes in the cerebellum was established in this study. Indeed, prehatching exposure to MeHg induced motor impairments, which correlated to histological damage and alterations in the cerebellar GSH system's development from PN 1 to PN 5.
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NOTES |
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1 These authors contributed equally to this work.
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