Maria Elena Crespo-López

Mercury and human genotoxicity: Critical considerations and possible molecular mechanisms

Mercury compounds versatility explains their numerous applications in diverse areas of industry. The growing use of this metal has resulted in a significant increase of environment contamination and episodes of human intoxication, arousing the concern of international organisms. Meanwhile, consequences of these intoxication outbreaks are still not fully understood, especially if we consider long-term effects of chronic exposure to relatively low levels of mercury compounds. In the present manuscript, studies about the genotoxicity of mercury compounds, performed in vitro, in vivo, and/or including epidemiologic studies of human populations were reviewed. Some mercury compounds are known as teratogenic agents, especially affecting the normal development of the central nervous system; however, the connection between mercury exposure and carcinogenesis remains controversial. Since 1990s, epidemiological studies have begun to include an increasing number of human subjects, making the results more reliable: thus, increased genotoxicity was demonstrated in human populations exposed to mercury through diet, occupation or by carrying dental fillings. In fact, concentrations of methylmercury causing significant genotoxic alterations in vitro below both safety limit and concentration were associated with delayed psychomotor development with minimal signs of methylmercury poisoning. Based on mercurys known ability to bind sulfhydryl groups, several hypotheses were raised about potential molecular mechanisms for the metal genotoxicity. Mercury may be involved in four main processes that lead to genotoxicity: generation of free radicals and oxidative stress, action on microtubules, influence on DNA repair mechanisms and direct interaction with DNA molecules. All data reviewed here contributed to a better knowledge of the widespread concern about the safety limits of mercury exposure.

Mercury in the Tapajós River basin, Brazilian Amazon: A review

This paper presents a review about mercury contamination and human exposure in the Tapajós River basin (Brazil), one of the major tributaries of the Amazon impacted by traditional gold mining from the mid 1980s. The most recent review in this region was published more than ten years ago and since then many articles about environment and especially human populations have revealed new aspects of mercury toxicology. Additionally, new biomarkers of mercury exposure and toxicity have been studied in these populations. However, there are still many open, about both mercurys biogeochemical cycle and mercury health risks. Further environmental and human risk research directions are proposed.

Role of serotonin in zebrafish (Danio rerio) anxiety: relationship with serotonin levels and effect of buspirone, WAY 100635, SB 224289, fluoxetine and para-chlorophenylalanine (pCPA) in two behavioral models.

Serotonin (5-HT) is a neurotransmitter that is involved in many behavioral functions, including the organization of defense, and its putative pathological correlate, anxiety and stress disorders. Recently, behavioral tests for anxiety have been proposed in zebrafish. Exposure to the novel tank test or to the light/dark test increased extracellular fluid 5-HT content in the brain; anxiety-like behavior correlated positively with 5-HT content in the novel tank test, while the correlation was negative in the light/dark test. Acute treatment with a low dose of fluoxetine was anxiolytic in the geotaxis test and anxiogenic in the scototaxis test, while treatment with a higher dose produced a hyperlocomotor effect in both tasks. Buspirone and WAY 100635 were anxiolytic in both tests, while SB 224289 was anxiolytic in the geotaxis and slightly anxiogenic in the scototaxis test. Serotonin depletion with pCPA was anxiogenic in the geotaxis and anxiolytic in scototaxis. These results underline the differential sensitivity of these tasks to assess serotonergic agents; alternatively, serotonin might regulate zebrafish behavior differently in the novel tank test and in the light/dark test.

Comparative study of human exposure to mercury in riverside communities in the Amazon region

Four populations in the Amazon area were selected for a comparative study of mercury-exposed and non-exposed populations: São Luiz do Tapajós, Barreiras, Panacauera, and Pindobal Grande. The highest mercury levels in human hair samples were found in São Luiz do Tapajós and Barreiras, greatly exceeding the limits established by the World Health Organization. Panacauera showed an intermediate level below 9 microg/g. This was the first comparative and simultaneous evaluation of mercury exposure in the Amazon area. Also, thanks to this type of monitoring, we were able to eliminate the uncertainties about the reference dose. On the basis of these data, we can conclude that the mercury levels detected in exposed populations of the Tapajós River basin may be dangerous not only because they are above the World Health Organization limits, but also because the simultaneous mercury detection in non-exposed populations with similar characteristics provided a valid control and revealed lower mercury levels. Our results support the importance of continuous monitoring in both exposed and non-exposed populations.

Possible role of serotoninergic system in the neurobehavioral impairment induced by acute methylmercury exposure in zebrafish (Danio rerio)

Adult zebrafish were treated acutely with methylmercury (1.0 or 5.0 μg g(-1), i.p.) and, 24h after treatment, were tested in two behavioral models of anxiety, the novel tank and the light/dark preference tests. At the smaller dose, methylmercury produced a marked anxiogenic profile in both tests, while the greater dose produced hyperlocomotion in the novel tank test. These effects were accompanied by a decrease in extracellular levels of serotonin, and an increase in extracellular levels of tryptamine-4,5-dione, a partially oxidized metabolite of serotonin. A marked increase in the formation of malondialdehyde, a marker of oxidative stress, accompanied these parameters. It is suggested that methylmercury-induced oxidative stress produced mitochondrial dysfunction and originated tryptamine-4,5-dione, which could have further inhibited tryptophan hydroxylase. These results underscore the importance of assessing acute, low-level neurobehavioral effects of methylmercury.

Methylmercury intoxication activates nitric oxide synthase in chick retinal cell culture

The visual system is a potential target for methylmercury (MeHg) intoxication. Nevertheless, there are few studies about the cellular mechanisms of toxicity induced by MeHg in retinal cells. Various reports have indicated a critical role for nitric oxide synthase (NOS) activation in modulating MeHg neurotoxicity in cerebellar and cortical regions. The aim of the present study is to describe the effects of MeHg on cell viability and NOS activation in chick retinal cell cultures. For this purpose, primary cultures were prepared from 7-day-old chick embryos: retinas were aseptically dissected and dissociated and cells were grown at 37 degrees C for 7-8 days. Cultures were exposed to MeHg (10 microM, 100 microM, and 1 mM) for 2, 4, and 6 h. Cell viability was measured by MTT method and NOS activity by monitoring the conversion of L-[H3]-arginine to L-[H3]-citrulline. The incubation of cultured retina cells with 10 and 100 microM MeHg promoted an increase of NOS activity compared to control (P < 0.05). Maximum values (P < 0.05) were reached after 4 h of MeHg incubation: increases of 81.6 +/- 5.3 and 91.3 +/- 3.7%, respectively (data are reported as mean +/- SEM for 4 replicates). MeHg also promoted a concentration- and time-dependent decrease in cell viability, with the highest toxicity (a reduction of about 80% in cell viability) being observed at the concentration of 1 mM and after 4-6 h of incubation. The present study demonstrates for the first time the modulation of MeHg neurotoxicity in retinal cells by the nitrergic system.

Genotoxicity of mercury: Contributing for the analysis of Amazonian populations

Mercury is an important source of environmental contamination affecting human beings throughout the world and especially in the Amazon. Riverside populations have been chronically exposed to relatively high levels of methylmercury for many years. Long-term effects of mercury exposure are not well known, but human genotoxicity was already showed in both in vitro and epidemiological studies. However, to date, only two studies were carried out in Amazonian populations with conflicting results and without comparing to a non-exposed population. Aiming to highlight this question and avoid interference factors, this work analyzed in vitro genotoxicity of mercury in blood lymphocytes of Amazonian individuals by two methods (micronucleus and chromosomal aberrations). Deleterious effects of low levels (1-500 μg/l or 0,004-2 μM) of methylmercury were only detected with the method to detect chromosomal aberrations. Mitotic index (proportion of cells in metaphase) was the parameter most sensible. Thus, this technique was applied for the analysis of an Amazonian non-exposed population (Panacauera) with similar social-economical characteristics of the exposed populations studied elsewhere. The mean of the mitotic index for Panacauera population was 0.0814 ± 0.0097. Inter-individual variability of this index had no relation with sex or age. This value was above those registered for some groups of exposed populations. This fact points to mercury as the main responsible for inhibiting the cell cycle and/or the loss of proliferative capacity of the cells. These results already support mitotic index as an essential parameter for the early diagnose of mercury genotoxicity in humans, and especially in Amazonian populations.

Chronic Ethanol Exposure during Adolescence in Rats Induces Motor Impairments and Cerebral Cortex Damage Associated with Oxidative Stress

Binge drinking is common among adolescents, and this type of ethanol exposure may lead to long-term nervous system damage. In the current study, we evaluated motor performance and tissue alterations in the cerebral cortex of rats subjected to intermittent intoxication with ethanol from adolescence to adulthood. Adolescent male Wistar rats (35 days old) were treated with distilled water or ethanol (6.5 g/kg/day, 22.5% w/v) during 55 days by gavage to complete 90 days of age. The open field, inclined plane and the rotarod tests were used to assess the spontaneous locomotor activity and motor coordination performance in adult animals. Following completion of behavioral tests, half of animals were submitted to immunohistochemical evaluation of NeuN (marker of neuronal bodies), GFAP (a marker of astrocytes) and Iba1 (microglia marker) in the cerebral cortex while the other half of the animals were subjected to analysis of oxidative stress markers by biochemical assays. Chronic ethanol intoxication in rats from adolescence to adulthood induced significant motor deficits including impaired spontaneous locomotion, coordination and muscle strength. These behavioral impairments were accompanied by marked changes in all cellular populations evaluated as well as increased levels of nitrite and lipid peroxidation in the cerebral cortex. These findings indicate that continuous ethanol intoxication from adolescence to adulthood is able to provide neurobehavioral and neurodegenerative damage to cerebral cortex.

Chronic ethanol exposure during adolescence through early adulthood in female rats induces emotional and memory deficits associated with morphological and molecular alterations in hippocampus

There is increasing evidence that heavy ethanol exposure in early life may produce long-lasting neurobehavioral consequences, since brain structural maturation continues until adolescence. It is well established that females are more susceptible to alcohol-induced neurotoxicity and that ethanol consumption is increasing among women, especially during adolescence. In the present study, we investigated whether chronic ethanol exposure during adolescence through early adulthood in female rats may induce hippocampal histological damage and neurobehavioral impairments. Female rats were treated with distilled water or ethanol (6.5 g/kg/day, 22.5% w/v) by gavage from the 35th–90th day of life. Ethanol-exposed animals displayed reduced exploration of the central area and increased number of fecal boluses in the open field test indicative of anxiogenic responses. Moreover, chronic high ethanol exposure during adolescence induced marked impairments on short-term memory of female rats addressed on social recognition and step-down inhibitory avoidance tasks. These neurobehavioral deficits induced by ethanol exposure during adolescence through early adulthood were accompanied by the reduction of hippocampal formation volume as well as the loss of neurons, astrocytes and microglia cells in the hippocampus. These results indicate that chronic high ethanol exposure during adolescence through early adulthood in female rats induces long-lasting emotional and memory deficits associated with morphological and molecular alterations in the hippocampus.

Glutamate Induces Glutathione Efflux Mediated by Glutamate/Aspartate Transporter in Retinal Cell Cultures

This study was undertaken in order to characterize the role of the glutamate/aspartate transporter (GLAST) in the glutathione (GSH) efflux induced by glutamate. Our results demonstrated that retinal cell cultures exhibit two mechanisms of GSH release, one Na+-independent and other Na+-dependent. Glutamate and aspartate induced GSH efflux only in presence of Na+. Treatment with PCD (L-trans-Pyrrolidine-2,4-dicarboxylate), a transportable glutamate uptake blocker, increased GSH release indicating that GSH can be carried by glutamate transporters in retinal cell cultures. Added to this, treatment with zinc ion cultures, a recognized inhibitor of GLAST blocked GSH efflux evoked by glutamate. Treatment with NMDA antagonist (MK-801) did not have any effect on the GSH release induced by glutamate. These results suggest that glutamate induces GLAST-mediated release of GSH from retinal cell cultures and this could represent an important mechanism of cellular protection against glutamate toxicity in the CNS.