Neurotoxicity of methylmercury : analysis of molecular mechanisms and behavioral alterations

MethyImercury (MeHg) is a global pollutant that is toxic for the nervous system, especially during development. Contaminated fish is the main route of human exposure. Recent studies have indicated that neurological alterations in humans may occur even after exposure to fairly low levels of MeHg. Thus, the in vitro and in vivo studies reported in this thesis have focused on the neurotoxic effects of low doses of MeHg. A major aim was to characterize the intracellular pathways leading to neuronal and glial cell death. Previous reports have indicated that cerebellar granule cells (CGC) are a preferential target of MeHg and that astrocytes are also affected. Primary culture of CGC and the human astrocytoma D384 cell line were used as experimental models to investigate the cytotoxic effects of MeHg by analyzing cellular morphology and activation of pathways characteristic of apoptotic cell death. Doses of MeHg ranging from 0. 1 to 1.5 µM induced apoptosis in CGC, as shown by cell shrinkage, dose-dependent nuclear condensation and high-molecular weight (HMW)-DNA fragmentation. However, MeHg did not induce activation of caspases, the major effector proteases activated during apoptosis. We detected a substantial activation of the calpains, Ca2+-activated proteases.

Antioxidant compounds protected CGC from MeHg damage, pointing to a key role of oxidative stress in MeHg-induced cell death. CGC undergo apoptosis when exposed to hydrogen peroxide (H202) or colchicine. While WO, did not activate caspases, colchicine induced cytochrome c-mediated activation of caspase 3. Together these data suggest that the activation of caspases is not always required to induce morphological changes and pattern of DNA fragmentation consistent with apoptosis. We therefore have examined possible caspase-independent pathways leading to chromatin rearrangement, focusing on the mitochondrial Apoptosis-Inducing Factor (AIF). This flavoprotein, by translocating into the nucleus, induces both chromatin condensation and HMW-DNA fragmentation in a caspase-independent manner. Translocation of AIF into the nucleus was observed in CGC exposed to MeHg for 16 h, suggesting that AIF may be responsible for the caspase-independent chromatin rearrangement induced by MeHg.

The mechanisms of MeHg-induced cell death were also investigated in glial cells. Since MeH, increases free radical generation and lysosomes are sensitive to oxidative stress, we looked at lysosome stability in D384 cells exposed to MeHg and WO, Lysosomal rupture was detected shortly after MeHg exposure in cells maintaining plasma membrane integrity. Disruption of lysosomes was also evident after exposure to H2O2 The lysosomal alterations induced by MeHg and WO, were persistent and preceded a decrease of the mitochondrial membrane potential and the appearance of apoptosis. Our data support the hypothesis that lysosomal membranes represent a possible target of agents causing oxidative stress, and that lysosomal hydrolytic enzymes may act as executor/regulator factors in apoptosis induced by MeHg and H2O2 In conclusion, our in vitro studies show that MeHg induces cell death by caspase-independent apoptotic pathways both in CGC and in glial D-384 cells and that oxidative stress plays a key role in MeHg cytotoxicity.

Another aim of this work was to study the effects of prenatal exposure to low level of MeHg on behavior during early ontogeny in rats. For this purpose, locomotor activity, learning and memory were evaluated. Prenatal exposure to MeHg induced a slight increase in spontaneous locomotor activity in male and female rats at 14 days, but not at 21 days. When rats where stimulated with the dopamine D, receptor agonist U91356A, a lower dopamine-mediate locomotor activity was observed in the 21 day-old male rats exposed to MeHg, with no significant difference in the females of the same age. These results indicate that gender plays an important role in the susceptibility to MeHg, in agreement with previous observations in adult rats. Furthermore, our data show that MeHg alters dopamine receptor-mediated locomotor activity, and that dopamine D, receptors are involved. The gender-dependent behavioral alterations were evident at the age of 21 days, a time period crucial for the ontogeny of dopamine receptors in the rat brain. Therefore, we performed additional studies on 21 day-old male rats using apomorphine, a non-selective agonist of dopamine receptors. Apomorphine-stimulated locomotor activity was significantly higher in exposed animals. This result further confirmed that MeHg induces changes in dopaminergic transmission. MeHg-exposed rats were also analyzed with the Morris swim maze test at 2 months of age and displayed a deficit in retention as compared to control rats. Our in vivo studies show that prenatal exposure to MeHg impairs dopaniine-mediated locomotor activity and memory functions.

List of scientific papers

I. Dare E, Gotz ME, Zhivotovsky B, Manzo L, Ceccatelli S (2000). Antioxidants J811 and 17beta-estradiol protect cerebellar granule cells from methylmercury-induced apoptotic cell death. J Neurosci Res. 62(4): 557-65.
https://pubmed.ncbi.nlm.nih.gov/11070499

II. Dare E, Gorman AM, Ahlbom E, Gotz M, Momoi T, Ceccatelli S (2001). Apoptotic morphology does not always require caspase activity in rat cerebellar granule neurons. Neurotoxicity Research. 3: 501-14.

III. Fonfria E, Dare E, Benelli M, Sunol C, Ceccatelli S (2002). Methylmercury induces release of Apoptosis-Inducing Factor (AIF) from mitochondria in cerebellar granule cells. [Submitted]

IV. Dare E, Li W, Zhivotovsky B, Yuan X, Ceccatelli S (2001). Methylmercury and H(2)O(2) provoke lysosomal damage in human astrocytoma D384 cells followed by apoptosis. Free Radic Biol Med. 30(12): 1347-56.
https://pubmed.ncbi.nlm.nih.gov/11390179

V. Gimenez-Llort L, Ahlbom E, Dare E, Vahter M, Ogren S, Ceccatelli S (2001). Prenatal exposure to methylmercury changes dopamine-modulated motor activity during early ontogeny: age and gender-dependent effects. Environ Toxicol Pharmacol. 9(3): 61-70.
https://pubmed.ncbi.nlm.nih.gov/11167150

VI. Dare E, Fetissov S, Hokfelt T, Ogren SO, Ceccatelli S (2002). Effects of prenetal exposure to methylmercury on dopamine-mediated locomotor actvivty and dopamine D1 and D2 receptor expression. [Manuscript]