Protective effect of oleanolic acid on liver injury induced by acute exposure to mercury chloride and its possible mechanism / 环境与职业医学

Background Acute exposure to mercury chloride (HgCl2) can cause liver damage. Whether oleanolic acid (OA) as a hepatoprotective drug can protect against liver injury induced by acute exposure to HgCl2 and related mechanism of action remain unclear. Objective To investigate the protective effect and possible mechanism of OA on liver injury in mice caused by acute exposure to HgCl2. Methods Forty SPF C57BL/6 male mice were randomly divided into four groups with 10 mice in each group according to body weight. The four groups were named control group, OA group (300 mg·kg−1), HgCl2 group (5 mg·kg−1), and OA + HgCl2 group (300 mg·kg−1 OA + 5mg·kg−1 Hgcl2). Soybean oil and OA solution were administered intragastric once a day for two consecutive days. HgCl2 solution was injected intraperitoneally 2 h after the second intragastric administration. Mice were sacrificed after 48 h, and their serum and liver were collected. Liver coefficient was calculated. The changes of liver structure and iron deposition were observed by hematoxylin-eosin (HE) staining and Prussian blue staining. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total superoxide dismutase (T-SOD), reduced glutathione (GSH), malondialdehyde (MDA), and tissue iron content were measured with commercial kits. Western blotting was used to detect nuclear factor erythroid-2 related factor 2 (Nrf2), heme oxygenase 1 (HO-1), glutathione peroxidase 4 (Gpx4), transferrin receptor 1 (TFR1,) and solute carrier family 7 member 11 (SLC7A11). Results The AST and ALT levels of the HgCl2 group were (76.447±9.695) U·g−1 and (98.563±24.673)U·g−1, respectively, which were higher than those of the control group (P<0.05). After the OA pretreatment, the liver coefficient and the above indexes were decreased to (4.769±0.237)%, (57.086±10.087) U·g−1, and (87.294±27.181)U·g−1, respectively. The liver coefficient and AST level of the OA + HgCl2 group were significantly different from those of the HgCl2 group (P<0.05). After acute exposure to HgCl2, the hepatocytes of mice were disordered, accompanied by inflammatory infiltration, positive blue particles appeared in Prussian blue staining of liver tissue, and the above changes in liver tissue were alleviated after the OA pretreatment. The iron content in the HgCl2 group was (3.646±0.238) μmol·g−1, which was higher than that in the control group, (2.948±0.308) μmol·g−1. After the OA pretreatment, the iron content decreased to (3.429±0.415) μmol·g−1. Compared with the control group, acute exposure to HgCl2 resulted in decreased levels of GSH and T-SOD, decreased protein expression levels of Nrf2, HO-1, SLC7A11, and Gpx4, increased level of MDA, and increased protein expression level of TFR1 (P<0.05). After the OA pretreatment, all indicators were improved including increased GSH level, decreased MDA level, increased Nrf2, HO-1, and SLC7A11 protein expression levels, and decreased TFR1 protein expression level; compared with the HgCl2 group, the differences were statistically significant (P<0.05). Conclusion Acute HgCl2 exposure could induce liver injury in mice, and its mechanism may involve iron overload and ferroptosis. OA may alleviate the liver injury caused by acute HgCl2 exposure by affecting iron overload and the ferroptosis-related protein expression.

Ação protetora de Duguetia furfuracea (A. St.-Hil.) Saff. contra a toxicidade do cloreto de mercúrio em Escherichia coli / Protective action of Duguetia furfuracea (A. St.-Hil.) Saff. against toxicity due to mercury chloride in Escherichia coli / Acción protectora de Duguetia furfuracea (A. St.-Hil.) Saff. contra la toxicidad por cloruro de mercurio en Escherichia coli

Introdução: o mercúrio constitui um dos metais mais tóxicos e sua contaminação tem sido alvo de muitos estudos que buscam desvendar os mecanismos envolvidos em sua toxicidade e seus efeitos deletérios para os seres vivos. A espécie Duguetia furfuracea (A. St.-Hil.) Saff., conhecida popularmente como araticum-bravo, ata-brava e ata de lobo, tem sido utilizada na medicina popular como anti-reumáticas, para o tratamento de disfunções renais, dores na coluna e no estômago, e contra pediculose. Objetivo: avaliar o efeito citoprotetor do extrato etanólico e frações de D. furfuracea frente ao metal pesado cloreto de mercúrio (HgCl 2). Métodos: a caracterização dos metabólitos secundários foi realizada através de prospecção fitoquímica, na qual se observam a alteração de cor ou formação de precipitados após adição de reagentes específicos. A Concentração Inibitória Mínima (CIM) foi determinada pelo método de microdiluição em caldo e a Concentração Bactericida Mínima (CBM) verificada utilizando placas de petri com HIA (Heart Infusion Agar) para transferência das soluções incubadas em placas de microdiluição. Resultados: a prospecção fitoquímica revelou a presença de taninos, flavonoides e alcaloides. Todas as amostras apresentaram um CIM ≥ 1024 µg/mL. De acordo com os resultados obtidos na CBM, o extrato etanólico e as frações de D. furfuracea, exceto a fração hexânica, apresentaram atividade citoprotetora à bactéria E. coli frente ao metal pesado cloreto de mercúrio, sendo essa ação atribuída às propriedades quelantes dos flavonoides. Conclusões: os dados obtidos indicam a espécie D. furfuracea como uma fonte promissora no combate a metais pesados, apresentando-se como protetora de seres procariontes. Este é o primeiro relato do uso de plantas medicinais como agente citoprotetor em modelo bacteriano.

Introduction: mercury is one of the most toxic metals. Contamination by mercury has been the object of many studies aimed at unraveling the mechanisms involved in its toxicity and its harmful effects on living beings. The species Duguetia furfuracea (A. St.-Hil.) Saff., popularly known asaraticum-bravo’, ‘ata-brava’ and ‘ata de lobo’, has been used in folk medicine as antirheumatic and for the treatment of renal dysfunction, spinal pain, stomach ache and pediculosis. Objective: evaluate the cytoprotective effect of the ethanolic extract and fractions of D. furfuracea against the heavy metal mercury chloride (HgCl 2). Methods: characterization of secondary metabolites was performed by phytochemical screening, in which color change and precipitate formation are examined after the addition of specific reagents. Minimum inhibitory concentration (MIC) was determined by broth microdilution, and minimum bactericidal concentration (MBC) was verified using petri plates with HIA (heart infusion agar) to transfer the solutions incubated on microdilution plates. Results: phytochemical screening revealed the presence of tannins, flavonoids an alkaloids. All samples exhibited a MIC ≥ 1024 µg/mL. MBC results showed that the ethanolic extract and all fractions of D. furfuracea except for the hexane fraction have cytoprotective activity by the bacterium E. coli against mercury chloride, an action attributed to the chelating properties of flavonoids. Conclusions: data point to the promising value of the species D. furfuracea against heavy metals, presenting as protectors of procaryote beings. This is the first report on the use of medicinal plants as cytoprotective agents on the bacterial model.

Introducción: el mercurio es uno de los metales más tóxicos y su contaminación ha sido objeto de muchos estudios que tratan de desentrañar los mecanismos implicados en su toxicidad y sus efectos nocivos en los seres vivos. La especie Duguetia furfuracea (A. St.-Hil.) Saff., popularmente conocido como ‘araticum-bravo’, ‘ata-brava’ y ‘ata de lobo’, se ha utilizado en la medicina popular como un anti-reumático para el tratamiento de la disfunción renal, dolor vertebral y de estómago, y contra la pediculosis. Objetivo: evaluar el efecto citoprotector del extracto de etanol y fracciones de D. furfuracea al metal pesado cloruro de mercurio (HgCl2). Métodos: la caracterización de metabolitos secundarios se realizó mediante la prospección fitoquímica, en la que se observa el cambio de color o la formación de precipitados después de la adición de los reactivos específicos. La Concentración Inhibitoria Mínima (CIM) se determinó mediante microdilución en caldo y la Concentración Bactericida Mínima (CBM) verificada usando placas de petri con HIA ( Heart Infusion Agar) para transferencia de las soluciones incubadas en placas de microdilución. Resultados: la prospección fitoquímica reveló la presencia de taninos, flavonoides y alcaloides. Todas las muestras presentaron una CIM ≥ 1024 µg/mL. De acuerdo con los resultados obtenidos en la CBM, el extracto de etanol y fracciones de D. furfuracea, excepto la fracción de hexano, mostraron actividad citoprotectora de la bacteria E. coli delante del cloruro de mercurio, siendo esa acción atribuida a las propiedades quelantes de los flavonoides. Conclusiones: los datos obtenidos indican que la especie D. furfuracea como una fuente prometedora para combatir los metales pesados, que se presentan como protectores de seres procariotas. Este es el primer informe del uso de plantas medicinales como agente citoprotector en el modelo bacteriano.

Accidental Inorganic Mercury Chloride Poisoning in a 2-Year Old Child.

Inorganic mercury poisoning is uncommon, but when it occurs it can result in severe, life threatening features and acute renal failure. A 2-year old well thriving child presented with alleged history of accidental ingestion of inorganic mercury chloride. He presented with evidence of corrosive trauma to the gastrointestinal tract mucosa, but with normal renal function at admission, which was managed with BAL and other supportive treatment. But he developed non-oliguric renal failure after admission, which also improved gradually. On follow-up, two months later, the patient’s renal function was normal; indicating that renal failure caused by acute inorganic mercury poisoning produced no permanent renal damage. We have hereby presented a case of mercury intoxication in a 2-year old child, with an excellent clinical improvement and normalization of laboratory results.

Comparative effects of organic and inorganic mercury on in vivo dopamine release in freely moving rats

The present study was carried out in order to compare the effects of administration of organic (methylmercury, MeHg) and inorganic (mercury chloride, HgCl 2 ) forms of mercury on in vivo dopamine (DA) release from rat striatum. Experiments were performed in conscious and freely moving female adult Sprague-Dawley (230-280 g) rats using brain microdialysis coupled to HPLC with electrochemical detection. Perfusion of different concentrations of MeHg or HgCl 2 (2 muL/min for 1 h, N = 5-7/group) into the striatum produced significant increases in the levels of DA. Infusion of 40 muM, 400 muM, or 4 mM MeHg increased DA levels to 907 ± 31, 2324 ± 156, and 9032 ± 70 percent of basal levels, respectively. The same concentrations of HgCl 2 increased DA levels to 1240 ± 66, 2500 ± 424, and 2658 ± 337 percent of basal levels, respectively. These increases were associated with significant decreases in levels of dihydroxyphenylacetic acid and homovallinic acid. Intrastriatal administration of MeHg induced a sharp concentration-dependent increase in DA levels with a peak 30 min after injection, whereas HgCl 2 induced a gradual, lower (for 4 mM) and delayed increase in DA levels (75 min after the beginning of perfusion). Comparing the neurochemical profile of the two mercury derivatives to induce increases in DA levels, we observed that the time-course of these increases induced by both mercurials was different and the effect produced by HgCl 2 was not concentration-dependent (the effect was the same for the concentrations of 400 muM and 4 mM HgCl 2 ). These results indicate that HgCl 2 produces increases in extracellular DA levels by a mechanism differing from that of MeHg.

Effect of Glutathione on Apoptosis Induced by Methyl Mercury Chloride / 대한산업의학회지

OBJECTIVES: This study was performed to evaluate the critical role of glutathione(GSH) in methyl mercury chloride(MeHgCl)induced cell apoptosis. METHODS: The effect of GSH in MeHgCl induced cell apoptosis was observed in mouse macrophage-derived RAW 264.7 cells in vitro. The cells were cultured in Dulbecco's modified Eagle's medium(DMEM). RESULTS: MeHgCl exerted a dose dependent cytotoxicity,as demonstrated by the MTT assay, which is an assay dependent partially on the mitochondrial function. Moreover, in the presence of NAC, a GSH precursor, the MeHgCl induced cytotoxicity was significantly decreased whereas BSO, a specific GSH synthesis inhibitor,increased the MeHgCl induced cytotoxicity.The MeHgCl induced DNA fragmentation and chromatin condensation was consistent with the morphological alterations. The MeHgCl treated cells exhibited increasing annexin V-FITC binding to the phos-phatidylserine(PS)translocated from the inner to the outer leaflet of the plasma membrane and those cells with NAC pretreatment significantly exhibited decreasing annexin V-FITC binding compared to the cells treated with MeHgCl only. However BSO pretreatment markedly exhibited the increasing annexin V-FITC binding. The MeHgCl treated cells generated ROS, which was evidenced by the oxidation of dihydroethidine and the generation of the fluorescent product, ethidium. In addition, BSO pretreatment further enhanced the extent of ROS generation caused by MeHgCl whereas NAC pretreatment decreased the amount of ROS generation. MeHgCl led to a dose dependent decrease in the GSH content. Although MeHgCl exposure significantly reduced the GSH level, those cells that had a NAC pretreatment contained a higher level of GSH compared to the cells treated with MeHgCl only. In contrast, BSO pretreatment futher enhanced the extent of GSH depletion caused by MeHgCl. CONCLUSIONS: These results indicate that MeHgCl reduced the GSH content and impaired the defense against oxidative damage caused by ROS formation in RAW 264.7 cells. It is possible that these factors leads to the activation of the apoptosis signaling pathway. Ultimately these results suggest that GSH plays a crucial role in protecting the activity against MeHgCl induced apoptosis.

A Study on Factors Related to NO Synthesis by Mercurial Compounds in the EMT-6 cell / 대한산업의학회지

The effects of several factors on the nitrite synthesis were observed in the culture of EMT-6 cells which are originated from mammary adenocarcinoma of Balb/c mouse. The cells were cultured in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum. Amounts of nitrite in the culture media after 24 and 36 hours of culture were about 2 fold, and 3-fold of those measured after 12 hours respectively. There were very close associations between the amounts of nitrite measured in the culture media. A significant nitrite synthesis by EMT-6 cells occurred when IL-1 was added to the culture medium with other cytokines as IFN gamma or TNF alpha . One of each cytokines were less effective as an inducer of nitrite than the combinations of cytokines. When mercury chloride or cinnamate was added in the culture medium, the nitrite synthesis was dose-dependently decreased by the concentration of these materials. The viability of EMT-6 cells were kept on 95% or above in 36 hours after beginning of culture without any specific additives except cytokines. While after 48 hours it went down to 85% or less. These viability were decreased by the prolongation of culture time (48 hours or more), the addition of TNF alpha to cytokine mixture, and the higher concentrations of mercury chloride or cinnamate to culture medium. Simultaneous addition of the equimolar dose of selenium completely prevented mercurial compounds-induced inhibitions of nitrite syntheses. But the single addition of selenium neither influenced the viability of cells nor the productions of nitrite. These results suggests that the disorder of cell mediated immunity by mercurial compounds could be related to the inhibition of nitric oxide synthesis and selenium decreased the cytotoxicity of mercurial compounds.

Effect of Mercury Chloride on Peritoneal Macrophage or EMT-6 cell from Balb/c mice / 대한산업의학회지

The effect of treatment of mercury chloride on the nitrite and nitrate synthesis was observed in peritoneal macrophages from Balb/c mice and EMT-6 cells in vitro. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with cytokines. Amounts of nitrite and nitrate in the culture media after 24 and 36 hours of culture were about 2-fold, and 3-fold of those measured after 12 hours respectively. There were very close associations between the amounts of nitrite and nitrate measured in the culture media, according to culture time. The survival rate of peritoneal macrophages was significantly decreased by mercury chloride added into the media in dose-dependent manner, however the survivals of EMT-6 cells were not influenced by mercury chloride concentration in media. Nitrite and nitrate syntheses were dose-dependently decreased by mercury chloride added in culture media. These results reported here suggest that the disorder of cell mediated immunity by mercurials could be related to the inhibition of nitric oxide synthesis which seems to be caused by the inhibition of metabolism of cells.

Effect of Mercury Chloride on Peritoneal Macrophage or EMT-6 cell from Balb/c mice / 대한산업의학회지

The effect of treatment of mercury chloride on the nitrite and nitrate synthesis was observed in peritoneal macrophages from Balb/c mice and EMT-6 cells in vitro. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with cytokines. Amounts of nitrite and nitrate in the culture media after 24 and 36 hours of culture were about 2-fold, and 3-fold of those measured after 12 hours respectively. There were very close associations between the amounts of nitrite and nitrate measured in the culture media, according to culture time. The survival rate of peritoneal macrophages was significantly decreased by mercury chloride added into the media in dose-dependent manner, however the survivals of EMT-6 cells were not influenced by mercury chloride concentration in media. Nitrite and nitrate syntheses were dose-dependently decreased by mercury chloride added in culture media. These results reported here suggest that the disorder of cell mediated immunity by mercurials could be related to the inhibition of nitric oxide synthesis which seems to be caused by the inhibition of metabolism of cells.

NO2- and ATP synthesis in the EMT-6 cell stimulated by mercury chloride / 예방의학회지

Effect of Mercury chloride on the synthesis of NO2- and ATP were observed in EMT-6 cells which were culture with cytokines(IL-1alpha and IFN-gamma) and various concentrations of mercury chloride from 0.05 to 0.08 M. Viability of EMT-6 cells were observed above 90% in almost groups. There were not significant differences in the viability between mercury supplemented groups and control group. It suggests viability of EMT-6 cells were not influenced by these concentrations of mercury chloride. Results of the synthesis of nitrite showed significant time and group effect. There is a significant interaction effect between concentration of mercury chloride and culture time. The effect of various concentration of mercury chloride is not the same for all levels of culture time. There were significant differences in the synthesis of nitrite between mercury chloride supplemented groups and control group, and the synthesis of nitrite in EMT-6 cell by the supplement of mercury chloride was significantly decreased in a dose-dependent manner. Results of the synthesis of ATP showed a significant group effect, and the time main effect and the Group x Time interaction were also significant. There were significant differences in the synthesis of ATP between mercury chloride supplemented groups and control group, and the synthesis of ATP in EMT-6 cell by the supplement of mercury chloride was significantly decreased in a dose-dependent manner. These results suggest that the disorder of cell mediated immunity by mercury chloride could be related to the inhibition of nitric oxide synthesis which will be caused by the decreased synthesis of ATP.

A Study of Cytotoxical Mechanism of Mercurial Compounds in RAW264.7 Cell Line / 대한산업의학회지

The effects of glucose on the productions of ATP and nitrite which are inhibited by mercury compounds, were examined in a cell culture system of RAW 264.7 cells. The cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) with cytokines, IL-1 and TNF for 24 hours. The viablility of RAW 264.7 cells at the end of culture was significantly decreased by mercury chloride or methylmercury chloride added into the media in dose-dependent manner, however the viability of RAW 264.7 cells were influenced in the concentrations lese than 0.8micrometer of mercury chloride or 0.4micrometer of methylmercury chloride. The addition of 4.5 g/l glucose to normal DMEM lowered the pH of media to the range of 6.7-6.8 after 48 hours of culture, but not for the cell survivals. This supplement of glucose to the media also prevented the inhibitions of ATP and nitrite syntheses which were caused by mercurial compounds. These results suggest that the disorder of cell mediated immunity by mercurials could be related to the inhibition of nitric oxide synthesis which seams to be caused by the inhibition of ATP synthesis, especially related to the citric acid cycle.

Effect of Mercury Chloride on Humoral and Cell-mediated Immune Responses in Mice / 예방의학회지

The in vivo and in vitro humoral and cell-mediated immune responses of lymphocytes of BALB/c mouse exposed to mercury chloride(HgC12) were investigated. In vitro exposure of the splenocytes to mercury chloride produced overt cytotoxicity in 3 hours period. The IC50(the concentration required to inhibit a splenocyte viability by 50%) for mercury chloride was >0.1mM for cytotoxicity. In vivo mercury chloride exposed mice were significantly depressed delayed type hypersensitivity(DTH) response to sheep red blood cells(SRBC) in a dose-dependent manner compared with control group. Mercury chloride inhibited the proliferative responses of splenocytes to lipopolysaccharide. pokeweed mitogen, concanavalin A and phytohemagglutinin in a dose-dependent manner. Hemagglutinin response to SRBC in mercury chloride exposed mice was significantly depressed in a dose-dependent manner compared with control group. After 7 weeks of mercury chloride exposure in vivo. mercury chloride induced an increase of nonspecific serum IgG1 and IgE levels in BALB/c mice.

HgCl2 Dysregulates the Immune Response of Balb/c Mice / 예방의학회지

The studies reported here were undertaken to investigate the effects of mercury chloride on immune system of Balb/c mouse employing a flexible tier of in vitro and in vivo assays. Mercury chloride inhibited the proliferative responses of spleen cells to lipopolysaccharide, pokeweed mitogen, and phytohemagglutinin as a dose-dependent manner. This inhibitory effect was observed not only when HgCl2 was added 2nd or 3rd day of 3 days culture period but also when spleen cells was pretreated with HgC12 for 2 hours. Mercury chloride, however, potentiated the production of IgM and IgG from spleen cells. During the HgCl2 administration by drinking for 3 weeks, the weight gain of mice was significantly blunted than that of control group mice, while no overt signs related to mercury toxicity were noted in any mice of experimental group. There was no change in thymus and spleen weights, and in histological findings of kidney, bone marrow of femur, thymus, spleen, and politeful lymph node after 3 weeks of mercury exposure. However, HgC12 induced a significant increase of total serum IgM, IgG including IgG1, IgG2a and IgG2b, and IgE in Balb/c mice. Treatment in vivo with anti-IL-4 monoclonal antibody significantly abrogated the HgCl2-induced increase in total serum IgG1 and IgE. Whereas HgCl2 potentiated total serum IgM and IgG, there was, there was no difference in total serum hemagglutinin to SRBC(Sheep Red Blood cell) between experimental and control group mice when these mice were immunized with SRBC. All these findings observed in Balb/c mice suggest that mercury perturbates well-orchestrated regulation of immune responses before developing histopathological changes in lymphoid tissues.

Effects and related mechanism of low-dosed methyl-mercury chloride in promoting apoptosis of intestine epithelial cells of fetal mice / 第三军医大学学报

Objective To study the effects and related mechanism of low-dosed methyl-mercury chloride (MMC) on the epithelial apoptosis of fetal mice intestine. Methods Pregnant mice on E12.5 d and E13.5 d were injected with different doses of MMC (0, 1, 2, 4 mg/kg) intraperitoneally. After 48 h, their duodenum and colon were dissected out and sliced into the paraffin sections. Some sections were stained with HE to count the numbers of apoptotic bodies (NAB) with stereological method. The others were stained with the immunohistochemical method to observe the expression of apoptosis-associated proteins Bcl-2, Bax and immediate early gene c-fos. Results ①In all experimental groups, the NAB in the epithelium were higher than that of control groups (P