Luigi Manzo

Neurotoxicity and molecular effects of methylmercury.

The neurotoxicity of high levels of methylmercury (MeHg) and the high susceptibility of the developing brain are well established both in humans and experimental animals. Prenatally poisoned children display a range of effects varying from severe cerebral palsy to subtle developmental delays. Still unknown is the lowest dose that impairs neurodevelopment. The primary source of human exposure is the fish. The data obtained so far from epidemiological studies on fish-eating populations are not consistent. A reference dose of 0.1 microg MeHg/kg per day has been established by the U.S. Environmental Protection Agency based on a study on Iraqi children exposed to MeHg in utero. However, these exposures occurred at high level for a limited period of time, and consequently were not typical of lower chronic exposure levels associated with fish consumption. Major obstacles for estimation of a threshold dose for MeHg include the delayed appearance of the neurodevelopmental effects following prenatal exposure and limited knowledge of cellular and molecular processes underlying these neurological changes. In this respect, a strategy which aims at identifying sensitive molecular targets of MeHg at environmentally relevant levels may prove particularly useful to risk assessment. Here some examples of MeHg molecular effects occurring at low doses/concentrations are presented.

Neuronal cell death: a demise with different shapes

It is not surprising that initially simple death programmes, developed early during phylogeny, undergo complex modifications in mammalian cells. A further consequence of the increased complexity could be that an increasing number of feedback loops gives rise to many possibilities of initiation, control and execution (Fig. 1a, Fig. 2).Multiple pathways probably cooperate to ensure the removal of injured cells, and positive feedback loops amplify death signals to prevent survival of ‘undead’ cells. In complex cellular systems such as neuronal networks, one can even speculate that execution of the death programme is predominantly apoptotic in certain subcellular regions, whereas other subroutines prevail in other cellular domains. The main implication of this standpoint is the exclusion of a single, predominant and molecularly defined commitment step. It seems likely that accumulation of damage incompatible with cell survival would require disruption of several vital functions. Once such a threshold is trespassed, other positive feedback loops would ensure the progression of the death programme and the safe disposal of the injured cell. Also, it is apparent that the morphological appearance of cell death (apoptosis or necrosis) is not linked to a putative commitment point, but it is rather the result of a more-or-less complete execution of subroutines of the death programme. Overall, the arguments presented here support the view that in neuronal death, individual, intricately interconnected pathways self-amplify or delete each other in an inflationary process, which results in the many shapes of cell death.

Early acute necrosis, delayed apoptosis and cytoskeletal breakdown in cultured cerebellar granule neurons exposed to methylmercury

Cerebellar granule cells (CGCs) are a sensitive target for methylmercury (MeHg) neurotoxicity. In vitro exposure of primary cultures of rat CGCs to MeHg resulted in a time‐ and concentration‐dependent cell death. Within 1 hr exposure, MeHg at 5–10 μM caused impairment of mitochondrial activity, de‐energization of mitochondria and plasma membrane lysis, resulting in necrotic cell death. Lower MeHg concentrations (0.5–1 μM) did not compromise cell viability, mitochondrial membrane potential and function at early time points. Later, however, the cells progressively underwent apoptosis and 100% cell death was reached by 18 hr treatment. Neuronal network fragmentation and microtubule depolymerization were detected as early as within 1.5 hr of MeHg (1 μM) exposure, long before the occurrence of nuclear condensation (6–9 hr). Neurite damage worsened with longer exposure time and proceeded to the complete dissolution of microtubules and neuronal processes (18 hr). Microtubule stabilization by taxol did not prevent MeHg‐induced delayed apoptosis. Similarly ineffective were the caspase inhibitors z‐VAD‐fluoromethylketone and z‐DEVD‐chloromethylketone, the L‐type calcium channel inhibitor nifedipine, the calcium chelator EGTA and BAPTA, and the NMDA receptor antagonist MK‐801. On the other hand, insulin‐like growth factor‐I partially rescued CGCs from MeHg‐triggered apoptosis. Altogether these results provide evidence that the intensity of MeHg insult is decisive in the time of onset and the mode of neuronal death that follows, i.e., necrosis vs. apoptosis, and suggest that cytoskeletal breakdown and deprivation of neurotrophic support play a role in MeHg delayed toxicity. J. Neurosci. Res. 59:775–787, 2000

Neurobehavioural and Molecular Changes Induced by Methylmercury Exposure During Development

There is an increasing body of evidence on the possible environmental influence on neurodevelopmental and neurodegenerative disorders. Both experimental and epidemiological studies have demonstrated the distinctive susceptibility of the developing brain to environmental factors such as lead, mercury and polychlorinated biphenyls at levels of exposure that have no detectable effects in adults. Methylmercury (MeHg) has long been known to affect neurodevelopment in both humans and experimental animals. Neurobehavioural effects reported include altered motoric function and memory and learning disabilities. In addition, there is evidence from recent experimental neurodevelopmental studies that MeHg can induce depression-like behaviour. Several mechanisms have been suggested fromin vivo- andin vitro-studies, such as effects on neurotransmitter systems, induction of oxidative stress and disruption of microtubules and intracellular calcium homeostasis. Recentin vitro data show that very low levels of MeHg can inhibit neuronal differentiation of neural stem cells. This review summarises what is currently known about the neurodevelopmental effects of MeHg and consider the strength of different experimental approaches to study the effects of environmentally relevant exposurein vivo andin vitro.

Neurotoxic and Molecular Effects of Methylmercury in Humans

Mercurials are global environmental pollutants deriving from natural processes and anthropogenic activities. Most human exposure to mercury occurs through the intake of fish, shellfish, and sea mammals contaminated with methylmercury. Methylmercury is bioaccumulated and biomagnified in the aquatic food chain and reaches its highest levels in top predatory fish. The neurotoxic hazard posed by methylmercury to humans and the unique susceptibility of the developing brain have been well documented following the mass poisonings occurring in Japan and Iraq. Adult cases of methylmercury poisoning are characterized by the delayed onset of symptoms and by the focal degeneration of neurons in selected brain regions (for example, cerebral cortex and cerebellum). Why the fetus displays different neuropathological effects and a higher sensitivity to methylmercury relative to the adult is still unknown. Depending on the degree of in utero exposure, methylmercury may result in effects ranging from fetal death to subtle neurodevelopmental delays. On the basis of epidemiological studies performed in populations having moderate chronic methylmercury exposure, no definitive consensus has been reached to date on the safety level of maternal exposure during pregnancy. Among the multiple mechanisms believed to contribute to methylmercury neurotoxicity, methylmercury-induced microtubule alterations, oxidative damage, impairment of calcium homeostasis, and the potentiation of glutamatergic neurotransmission are presented in this review.

Human developmental neurotoxicity of methylmercury : Impact of variables and risk modifiers

Methylmercury (MeHg) is a widespread environmental and food toxicant which has long been known to affect neurodevelopment in both humans and experimental animals. Risk assessment for MeHg is mainly based on human data coming from the massive episodes of poisoning in Japan and Iraq, as well as from large scale epidemiological studies concerning childhood development and neurotoxicity in relation to in utero exposure in various fish eating communities around the world. Despite the extensive literature and research, the threshold dose for MeHg neurotoxic effects is still unclear, in particular when it comes to subtle effects on neurobehaviour. In this article clinical and epidemiological findings concerning the neurodevelopmental toxicity of MeHg are reviewed. Much attention is focussed on the potential impact of factors, such as diet and nutrition, gender, pattern of exposure and co-exposure to other neurotoxic pollutants, which may modulate MeHg toxic effects. These factors, together with the notion that some symptoms may ensue or exacerbate with aging, contribute to the difficulties in the definition of safe levels for developmental exposure.

Effects of water-soluble functionalized multi-walled carbon nanotubes examined by different cytotoxicity methods in human astrocyte D384 and lung A549 cells

The widespread projected use of functionalized carbon nanotubes (CNTs) makes it important to understand their potential harmful effects. Two cell culture systems, human A549 pneumocytes and D384 astrocytoma cells, were used to assess cytotoxicity of multi-walled CNTs (MWCNTs) with varying degrees of functionalization. Laboratory-made highly functionalized hf-MW-NH(2) and less functionalized CNTs (MW-COOH and MW-NH(2)) were tested in comparison with pristine MWCNTs, carbon black (CB) and silica (SiO(2)) by MTT assay and calcein/propidium iodide (PI) staining. Purity and physicochemical properties of the test nanomaterials were also determined. In both MTT and calcein/PI assays, highly functionalized CNTs (hf-MW-NH(2)) caused moderate loss of cell viability at doses >or=100 microg/ml being apparently less cytotoxic than SiO(2). In preparations treated with CB or the other nanotube types (pristine MWCNTs, MW-COOH and the less functionalized amino-substituted MW-NH(2)) the calcein/PI test indicated no loss of cell viability, whereas MTT assay apparently showed apparent cytotoxic response, occurring not dose-dependently at exceedingly low CNT concentrations (1 microg/ml). The latter nanomaterials were difficult to disperse showing higher aggregate ranges and tendency to agglomerate in bundle-like form in cell cultures. In contrast, hf-MW-NH(2) were water soluble and easily dispersible in medium; they presented lower aggregate size range as well as considerably lower length to diameter ratios and low tendency to form aggregates compared to the other CNTs tested. The MTT data may reflect a false positive cytotoxicity signal possibly due to non-specific CNT interaction with cell culture components. Thus, these properties obtained by chemical functionalization, such as water solubility, high dispersibility and low agglomeration tendency were relevant factors in modulating cytotoxicity. This study indicates that properties obtained by chemical functionalization, such as water solubility, high dispersibility and low agglomeration tendency are relevant factors in modulating cytotoxicity of CNTs.

Antioxidants J811 and 17?-estradiol protect cerebellar granule cells from methylmercury-induced apoptotic cell death

Cerebellar granule cells (CGC) have provided a reliable model for studying the toxicity of methylmercury (MeHg), a well‐known neurotoxicant contaminating the environment. In the present study we report that doses of MeHg ranging from 0.1 μM to 1.5 μM activated apoptosis, as shown by cell shrinkage, nuclear condensation, and formation of high‐molecular‐weight DNA fragments. Nevertheless, caspase‐3‐like activity was not significantly induced, and the broad caspase inhibitor Z‐VAD‐FMK was not capable of protecting the cells. This argues for a minor role of caspases in the intracellular pathways leading to MeHg‐induced cell death in CGC. Instead, proteolytic fragments obtained by specific calpain cleavage of procaspase‐3 and α‐fodrin were increased consistently in samples exposed to MeHg, pointing to a substantial activation of calpain. Notably, two antioxidants, 17β‐estradiol (10 μM) and the Δ8,9‐dehydro derivative of 17α‐estradiol J811 (10 μM), protected from MeHg damage, preventing morphological alterations, chromatin fragmentation, and activation of calpain. These findings underscore the key role of oxidative stress in MeHg toxicity, placing it upstream of calpain activation. The shielding effect of the 17β‐estradiol and the radical scavenger J811 is potentially relevant for the development of therapeutic strategies for MeHg intoxication. J. Neurosci. Res. 62:557–565, 2000.

Carbon Monoxide Cardiotoxicity

Cardiac dysfunction including arrhythmias and myocardial ischemia have often been reported in carbon monoxide poisoning; scattered punctiform hemorrhages throughout the heart have been documented in autopsy samples. An appropriate diagnostic approach is crucial to assess carbon monoxide cardiac damage. This evaluation may be confounded by several factors, including the absence of overt symptoms and of specific ischemic changes in the electrocardiogram. In experimental studies, laboratory animals can develop cardiac changes similar to those seen in humans and therefore proved to be useful models to study the effects and the mechanisms of cardiac damage due to carbon monoxide. These investigations, as well as others performed in vitro, provide support for a direct action of carbon monoxide on the heart, in addition to systemic hypoxia produced by carboxyhemoglobin formation. This review focuses on the diagnostic aspects of carbon monoxide cardiotoxicity. Experimental results obtained in animals and in vitro models are also discussed.

Neurodevelopmental toxicity of methylmercury: Laboratory animal data and their contribution to human risk assessment

Methylmercury (MeHg) is one of the most significant public health hazards. The clinical findings in the victims of the Japanese and Iraqi outbreaks have disclosed the pronounced susceptibility of the developing brain to MeHg poisoning. This notion has triggered worldwide scientific attention toward the long-term consequences of prenatal exposure on child development in communities with chronic low level dietary exposure. MeHg neurodevelopmental effects have been extensively investigated in laboratory animals under well-controlled exposure conditions. This article provides an updated overview of the main neuromorphological and neurobehavioral changes reported in non-human primates and rodents following developmental exposure to MeHg. Different aspects of MeHgs effects on the immature organism are reported, with particular reference to the delayed onset of symptoms and the persistency of central nervous system (CNS) injury/dysfunction. Particular attention is paid to the comparative toxicity assessment across species, and to the degree of concordance/discordance between human and animal data. The contribution of animal studies to define the role of potential effect modifiers and variables on MeHg dose-response relationships is also addressed. The ultimate goal is to discuss the relevance of laboratory animal results, as a complementary tool to human data, with regard to the human risk assessment process.