Effects of mercury intoxication on the response of horizontal cells of the retina of thraira fish (Hoplias malabaricus)

Methyl mercury (MeHg) is highly neurotoxic, affecting visual function in addition to other central nervous system functions. The effect of mercury intoxication on the amplitude of horizontal cell responses to light was studied in the retina of the fish Hoplias malabaricus. Intracellular responses were recorded from horizontal cells of fish previously intoxicated with MeHg by intraperitoneal injection (IP group) or by trophic exposure (T group). Only one retina per fish was used. The doses of MeHg chloride administered to the IP group were 0.01, 0.05, 0.1, 1.0, 2.0, and 6.0 mg/kg. The amplitudes of the horizontal cell responses were lower than control in individuals exposed to 0.01 (N = 4 retinas), 0.05 (N = 2 retinas) and 0.1 mg/kg (N = 1 retina), whereas no responses were recorded in the 1.0, 2.0, and 6.0 mg/kg groups. T group individuals were fed young specimens of Astyanax sp previously injected with MeHg corresponding to 0.75 (N = 1 retina), 0.075 (N = 8 retinas) or 0.0075 (N = 4 retinas) mg/kg fish body weight. After 14 doses, one every 5 days, the amplitude of the horizontal cell response was higher than control in individuals exposed to 0.075 and 0.0075 mg/kg, and lower in individuals exposed to 0.75 mg/kg. We conclude that intoxication with MeHg affects the electrophysiological response of the horizontal cells in the retina, either reducing or increasing its amplitude compared to control, and that these effects are related to the dose and/or to the mode of administration.

Hematological parameters in Nile Tilápia, Oreochromis niloticus exposed to sub-letal concentrations of mercury

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytotoxicity and genotoxicity of low doses of mercury chloride and methylmercury chloride on human lymphocytes in vitro

Mercury is a xenobiotic metal that is a highly deleterious environmental pollutant. The biotransformation of mercury chloride (HgCl2) into methylmercury chloride (CH3HgCl) in aquatic environments is well-known and humans are exposed by consumption of contaminated fish, shellfish and algae. The objective of the present study was to determine the changes induced in vitro by two mercury compounds (HgCl2 and CH3HgCl) in cultured human lymphocytes. Short-term human leukocyte cultures from 10 healthy donors (5 females and 5 males) were set-up by adding drops of whole blood in complete medium. Cultures were separately and simultaneously treated with low doses (0.1 to 1000 µg/l) of HgCl2 and CH3HgCl and incubated at 37ºC for 48 h. Genotoxicity was assessed by chromosome aberrations and polyploid cells. Mitotic index was used as a measure of cytotoxicity. A significant increase (P < 0.05) in the relative frequency of chromosome aberrations was observed for all concentrations of CH3HgCl when compared to control, whether alone or in an evident sinergistic combination with HgCl2. The frequency of polyploid cells was also significantly increased (P < 0.05) when compared to control after exposure to all concentrations of CH3HgCl alone or in combination with HgCl2. CH3HgCl significantly decreased (P < 0.05) the mitotic index at 100 and 1000 µg/l alone, and at 1, 10, 100, and 1000 µg/l when combined with HgCl2, showing a synergistic cytotoxic effect. Our data showed that low concentrations of CH3HgCl might be cytotoxic/genotoxic. Such effects may indicate early cellular changes with possible biological consequences and should be considered in the preliminary evaluation of the risks of populations exposed in vivo to low doses of mercury.

Cytogenetic damage related to low levels of methyl mercury contamination in the Brazilian Amazon

The mercury rejected in the water system, from mining operations and lixiviation of soils after deforestation, is considered to be the main contributors to the contamination of the ecosystem in the Amazon Basin. The objectives of the present study were to examine cytogenetic functions in peripheral lymphocytes within a population living on the banks of the Tapajós River with respect to methylmercury (MeHg) contamination, using hair mercury as a biological indicator of exposure. Our investigation shows a clear relation between methylmercury contamination and cytogenetic damage in lymphocytes at levels well below 50 micrograms/gram, the level at which initial clinical signs and symptoms of mercury poisoning occur. The first apparent biological effect with increasing MeHg hair level was the impairment of lymphocyte proliferation measured as mitotic index (MI). The relation between mercury concentration in hair and MI suggests that this parameter, an indicator of changes in lymphocytes and their ability to respond to culture conditions, may be an early marker of cytotoxicity and genotoxicity in humans and should be taken into account in the preliminary evaluation of the risks to populations exposed in vivo. This is the first report showing clear cytotoxic effects of long-term exposure to MeHg. Although the results strongly suggest that, under the conditions examined here, MeHg is both a spindle poison and a clastogen, the biological significance of these observations are as yet unknown. A long-term follow-up of these subjects should be undertaken.

Hematological changes and cytogenotoxicity in the tilapia Oreochromis niloticus caused by sub-chronic exposures to mercury and selenium

Fish bioassays are valuable tools that can be used to elucidate the toxicological potential of numerous substances that are present in the aquatic environment. In this study, we assessed the antagonistic action of selenium (Se) against the toxicity of mercury (Hg) in fish (Oreochromis niloticus). Six experimental groups with six fish each were defined as follows: (1) control, (2) mercury (HgCl2), (3) sodium selenite (Na2Se4O3), (4) sodium selenate (Na2Se6O4), (5) mercury + sodium selenite (HgCl2 + Na2Se4O3), and (6) mercury + sodium selenate (HgCl2 + Na2Se6O4). Hematological parameters [red blood cells (RBC), white blood cells (WBC), and erythroblasts (ERB)] in combination with cytogenotoxicity biomarkers [nuclear abnormalities (NAs) and micronuclei (MN)] were examined after three, seven, ten, and fourteen days. After 7 days of exposure, cytogenotoxic effects and increased erythroblasts caused by mercury, leukocytosis triggered by mercury + sodium selenite, leukopenia associated with sodium selenate, and anemia triggered by mercury + sodium selenate were observed. Positive correlations that were independent of time were observed between WBC and RBC, ERB and MN, and NA and MN. The results suggest that short-term exposure to chemical contaminants elicited changes in blood parameters and produced cytogenotoxic effects. Moreover, NAs are the primary manifestations of MN formation and should be included in a class characterized as NA only. Lastly, the staining techniques used can be applied to both hematological characterization and the measurement of cytogenotoxicity biomarkers.

Biomarkers of exposure to metal contamination and lipid peroxidation in the benthic fish Cathorops spixii from two estuaries in South America, Brazil

Biomarkers as lipid peroxidation, metallothionein and delta-aminolevulinic acid dehydratase were determined in Cathorops spixii to compare the biological responses of this fish from estuaries with distinct anthropogenic influence. Three areas were selected in two estuaries in accordance with the levels of contamination for the polluted (Santos/So Vicente) and with the hydrodynamic characteristics for the non-polluted (Canan,ia) estuary. Water characteristics and mercury levels in C. spixii confirmed a high human influence in the polluted system. In general, the biomarkers showed differences between the estuaries, suggesting disturbances in the specific cell mechanisms due to the presence of multiple xenobiotics in the contaminated system. Therefore, these biomarkers are recommended to promote more accurate information about the exposure to pollutants. Additionally, the study of the effect of the multiple xenobiotics on resident species such as the benthic fish C. spixii can favor a better assessment of the environmental quality of these systems.

Irreversible color vision losses in patients with chronic mercury vapor intoxication

This longitudinal study addresses the reversibility of color vision losses in subjects who had been occupationally exposed to mercury vapor. Color discrimination was assessed in 20 Hg-exposed patients (mean age = 42.4 +/- 6.5 years; 6 females and 14 males) with exposure to Hg vapor during 10.5 +/- 5.3 years and away from the work place (relative to 2002) for 6.8 +/- 4.2 years. During the Hg exposure or up to one year after ceasing it, mean urinary Hg concentration was 47 +/- 35.4 mu g/g creatinine. There was no information on Hg urinary concentration at the time of the first tests, in 2002 (Ventura et al., 2005), but at the time of the follow-up tests, in 2005, this value was 1.4 +/- 1.4 mu g/g creatinine for patients compared with 0.5 +/- 0.5 mu g/g creatinine for controls (different group from the one in Ventura et al. (2005)). Color vision was monocularly assessed using the Cambridge Colour Test (CCT). Hg-exposed patients had significantly worse color discrimination (p < 0.02) than controls, as evaluated by the size of MacAdam`s color discrimination ellipses and color discrimination thresholds along protan, deutan, and tritan confusion axes. There were no significant differences between the results of the study in Ventura et al. (2005) and in the present follow-up measurements, in 2005, except for worsening of the tritan thresholds in the best eye in 2005. Both chromatic systems, blue-yellow and red-green, were affected in the first evaluation (Ventura et al., 2005) and remained impaired in the follow-up testing, in 2005. These findings indicate that following a long-term occupational exposure to Hg vapor, even several years away from the source of intoxication, color vision impairment remains irreversible.

Visual field losses in workers exposed to mercury vapor

Visual field losses associated with mercury (Hg) exposure have only been assessed in patients exposed to methylmercury. Here we evaluate the automated visual field in 35 ex-workers (30 males; 44.20+/-5.92 years) occupationaly exposed to mercury vapor and 34 controls (21 males; 43.29+/-8.33 years). Visual fields were analyzed with the Humphrey Field Analyzer II (model 750i) using two tests: the standard automated perimetry (SAP, white-on-white) and the short wavelength automated perimetry (SWAP, blue-on-yellow) at 76 locations within a 27 degrees central visual field. Results were analyzed as the mean of the sensitivities measured at the fovea, and at five successive concentric rings, of increasing eccentricity, within the central field. Compared to controls, visual field sensitivities of the experimental group measured using SAP were lower for the fovea as well as for all five eccentricity rings (p<0.05). Sensitivities were significantly lower in the SWAP test (p<0.05) for four of the five extra-foveal eccentricity rings; they were not significant for the fovea (p = 0.584) or for the 15 degrees eccentricity ring (p = 0.965). These results suggest a widespread reduction of sensitivity in both visual field tests. Previous reports in the literature describe moderate to severe concentric constriction of the visual field in subjects with methylmercury intoxication measured manually with the Goldman perimeter. The present results amplify concerns regarding potential medical risks of exposure to environmental mercury sources by demonstrating significant and widespread reductions of visual sensitivity using the more reliable automated perimetry. (C) 2007 Elsevier Inc. All rights reserved.

Electrophysiological evidence for impairment of contrast sensitivity in mercury vapor occupational intoxication

Contrast sensitivity (CS) was evaluated in 41 former workers from a lamp manufacturing plant who were on disability retirement due to exposure to mercury and 14 age-matched controls. The CS was measured monocularly using the sweep visual evoked potential (sVEP) paradigm at 6 spatial frequencies (0.2, 0.8, 2.0, 4.0, 15.0, and 30 cpd). Statistical difference (p < 0.05) was found between the controls and the patient right and left eyes for 2.0 and 4.0 cpd. According the results in those spatial frequencies the eyes were classified in best and worst. Statistical differences were found between the controls and the best eyes for 2.0 and 4.0 cpd and for 0.8, 2.0, and 4.0 cpd for their worst eyes. No correlation was found between CS results and the time of exposure (mean 8.9 yr +/- 4.1), time away from the mercury source (mean = 6.0 yr +/- 3.9), urinary mercury level at the time of work (mean = 40.6 mu g/g +/-36.3) or with the mercury level at the CS measurement time (mean = 1.6 mu g/g +/-1.1). We show the first evidence of a permanent impairment in CS measured objectively with the sVEP. Our data complement the previous psychophysical works reporting a diffuse impairment in the CS function showing a CS reduction in the low to middle spatial frequencies. In conclusion, non-reversible CS impairment was found in occupational exposure to mercury vapor. We suggest that CS measurement should be included in studies of the mercury effects of occupational exposure. (C) 2007 Elsevier Inc. All rights reserved.

Long-Term Occupational Exposure to Organic Solvents Affects Color Vision, Contrast Sensitivity and Visual Fields

The purpose of this study was to evaluate the visual outcome of chronic occupational exposure to a mixture of organic solvents by measuring color discrimination, achromatic contrast sensitivity and visual fields in a group of gas station workers. We tested 25 workers (20 males) and 25 controls with no history of chronic exposure to solvents (10 males). All participants had normal ophthalmologic exams. Subjects had worked in gas stations on an average of 9.6 +/- 6.2 years. Color vision was evaluated with the Lanthony D15d and Cambridge Colour Test (CCT). Visual field assessment consisted of white-on-white 24-2 automatic perimetry (Humphrey II-750i). Contrast sensitivity was measured for sinusoidal gratings of 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 and 20.0 cycles per degree (cpd). Results from both groups were compared using the Mann-Whitney U test. The number of errors in the D15d was higher for workers relative to controls (p<0.01). Their CCT color discrimination thresholds were elevated compared to the control group along the protan, deutan and tritan confusion axes (p<0.01), and their ellipse area and ellipticity were higher (p<0.01). Genetic analysis of subjects with very elevated color discrimination thresholds excluded congenital causes for the visual losses. Automated perimetry thresholds showed elevation in the 9 degrees, 15 degrees and 21 degrees of eccentricity (p<0.01) and in MD and PSD indexes (p<0.01). Contrast sensitivity losses were found for all spatial frequencies measured (p<0.01) except for 0.5 cpd. Significant correlation was found between previous working years and deutan axis thresholds (rho = 0.59; p<0.05), indexes of the Lanthony D15d (rho = 0.52; p<0.05), perimetry results in the fovea (rho = -0.51; p<0.05) and at 3, 9 and 15 degrees of eccentricity (rho = -0.46; p<0.05). Extensive and diffuse visual changes were found, suggesting that specific occupational limits should be created.

Oral exposure to methylmercury modifies the prostatic microenvironment in adult rats

Methylmercury (MeHg) is an environmental pollutant that is highly toxic to the central nervous system. As its effects on male reproductive system are poorly understood, this study was carried out to analyse the effects of MeHg on the rat prostate. To evaluate the MeHg toxicity on ventral prostate, three groups of adult male Wistar rats received oral doses of 0.5, 1.0 and 3.0mg/kg MeHg, respectively, on a daily basis for 14days. A fourth group was used as a control. The prostate weight was decreased in rats treated orally with 0.5mg/kg MeHg compared to controls. Also, Hg concentration increased significantly in the prostate after treatments. There were reductions in serum testosterone levels and androgen receptor immunoreactivity in animals receiving 3.0mgMeHg/kg. The stereological data showed changes in the prostatic epithelial, stromal and luminal compartments which varied according to the different doses. Histopathological alterations, such as chronic inflammation, stratified epithelial hyperplasia and epithelial inflammatory reactive atypia, were observed in the 0.5mg/kg MeHg-treated group. Epithelial atrophy was observed in the 3.0mg/kg MeHg-treated group. In conclusion, the MeHg affects prostatic homoeostasis resulting in histopathological changes that may be relevant in the pathogenesis of prostatic disease.

Morphological evidence of neurotoxicity in retina after methylmercury exposure

The visual system is particularly sensitive to methylmercury (MeHg) exposure and, therefore, provides a useful model for investigating the fundamental mechanisms that direct toxic effects. During a period of 70 days, adult of a freshwater fish species Hoplias malabaricus were fed with fish prey previously labeled with two different doses of methylmercury (0.075 and 0.75 mu g g(-1)) to determine the mercury distribution and morphological changes in the retina. Mercury deposits were found in the photoreceptor layer, in the inner plexiform layer and in the outer plexiform layer, demonstrating a dose-dependent bioaccumulation. The ultrastructure analysis of retina revealed a cellular deterioration in the photoreceptor layer, morphological changes in the inner and outer segments of rods, structural changes in the plasma membrane of rods and double cones, changes in the process of removal of membranous discs and a structural discontinuity. These results lead to the conclusion that methylmercury is able to cross the blood-retina barrier, accumulate in the cells and layers of retina and induce changes in photoreceptors of H. malabaricus even under subchronic exposure. (c) 2012 Elsevier Inc. All rights reserved.

Bixin and norbixin protect against DNA-damage and alterations of redox status induced by methylmercury exposure in vivo

Populations in the Amazon are exposed to organic mercury via consumption of contaminated foods. These ethnic groups consume a specific plant seed annatto which contains certain carotenoids. The aim of this study was to find out if these compounds (bixin, BIX and norbixin, NOR), protect against DNA-damage caused by the metal. Therefore, rats were treated orally with methylmercury (MeHg) and with the carotenoids under conditions that are relevant to humans. The animals were treated either with MeHg (30 mu g/kg/bw/day), BIX (0.110 mg/kg/bw/day), NOR (0.011.0 mg/kg/bw/day) or combinations of the metal compound and the carotenoids consecutively for 45 days. Subsequently, the glutathione levels (GSH) and the activity of catalase were determined, and DNA-damage was measured in hepatocytes and leukocytes using single cell gel electrophoresis assays. Treatment with the metal alone caused a decrease in the GSH levels (35%) and induced DNA damage, which resulted in increased DNA migration after electrophoresis in liver and blood cells, whereas no effects were seen with the carotenoids alone. When BIX or NOR were given in combination with organic mercury, the intermediate and the highest concentrations of the carotenoids (1.0 and 10.0 mg/kg/bw/day BIX and 0.1 and 1.0 mg/kg/bw/day NOR) protected against DNA-damage. Furthermore, we found with both carotenoids, a moderate increase in the GSH levels in both metal-treated and untreated animals, while the activities of catalase remained unchanged. Our results indicate that consumption of BIX and NOR may protect humans against the adverse health effects caused by exposure to organic mercury. Environ. Mol. Mutagen., 2012. (c) 2012 Wiley Periodicals, Inc.

Mercury-induced autoimmunity : Genetics and immunoregulation

The existence of immune self-tolerance allows the immune system to mount responses against infectious agents, but not against self-molecular constitutes. Although self-tolerance is a robust phenomenon, in some individuals as well as in experimental models, the self-tolerance breaks down and as a result, a self-destructive autoimmune disease emerges. The underlying mechanisms for the development of autoimmune diseases are not known, but genetic, environmental and immunological factors are suggested to be involved. In this thesis, we used murine mercury-induced autoimmunity to test this suggestion. In susceptible mice mercuric chloride induces a systemic autoimmune disease characterized by increased serum levels of IgG1 and IgE, production of anti-nucleolar autoantibodies (ANolA) and formation of renal IgG deposits. In contrast, in resistant DBA/2 (H-2d) mice, none of these characteristics develop after exposure to mercury. By crossing and backcrossing mercury-resistant DBA/2 mice to mercury susceptible strains, we found that the resistance was inherited as a dominant trait in F1 hybrids and that one gene or a cluster of genes located in the H-2 loci determined the resistance to ANolA production, whereas resistance to the other characteristics was found to be controlled by two or three non-H-2 genes. We further put forward the “cryptic peptide hypothesis” to investigate whether mercury and another xenobiotic metal use similar pathway(s) to induce the H-2 linked production of ANolA. We found that while mercury stimulated ANolA synthesis in all H-2 susceptible (H-2s, H-2q and H-2f) mouse strains, silver induced only ANolA responses in H-2s and H-2q mice, but not in H-2f mice. Further studies showed that the resistance to silver-induced ANolA production in H-2f mice was inherited as a dominant trait. We next tested the proposition that mercury induces more adverse immunological effects in mouse strains, which are genetically prone to develop autoimmune diseases, using tight-skin 1 mice, an animal model for human Scleroderma. It was found that in this strain, mercury induced a strong immune activation with autoimmune characteristics, but did not accelerate the development of dermal fibrosis, a characteristic in Tsk/1 mice. Finally we addressed the Th1/Th2 cross-regulation paradigm by examining if a Th1-type of response could interact with a Th2-type of response if simultaneous induced in susceptible mice. Our findings demonstrated that mercury-induced autoimmunity (Th2-type) and collagen-induced arthritis (CIA) (Th1-type) can interact in a synergistic, antagonistic or additive fashion, depending on at which stage of CIA mercury is administered.

Design of a systematic approach to workplace exposure medical surveillance protocols

Current toxic tort cases have increased national awareness of health concerns and present an important avenue in which public health scientists can perform a vital function: in litigation, and in public health initiatives and promotions which may result. This review presents a systematic approach, using the paradigm of interactive public health disciplines, for the design of a matrix framework for medical surveillance of workers exposed to toxic substances. The matrix framework design addresses the required scientific bases to support the legal remedy of medical monitoring for workers injured as a result of their exposure to toxic agents. A background of recent legal developments which have a direct impact on the use of scientific expertise in litigation is examined in the context of toxic exposure litigation and the attainment of public health goals. The matrix model is applied to five different workplace exposures: dental mercury, firefighting, vinyl chloride manufacture, radon in mining and silica. An exposure matrix designed by the Department of Energy for government nuclear workers is included as a reference comparison to the design matrix. ^