The adaptive response of Saccharomyces cerevisiae to mercury exposure

The budding yeast Saccharomyces cerevisiae has been shown to possess a number of discrete but overlapping adaptive stress responses. We show here that yeast has an adaptive stress response towards mercury and that this response overlaps to some extent with the H(2)O(2) and cadmium-inducible stress responses. Expression of the yeast GSH1 gene, encoding gamma-glutamylcysteine synthetase, is known to be regulated by hydrogen peroxide; in this study we show that expression of a GSH1-lacZ reporter gene is shown to be regulated by exposure to heavy metals, such as mercury and cadmium. Other redox-active metals, including copper and iron, were found not to induce GSH1 expression. We show that mercury-mediated regulation of the GSH1 gene is not by the same mechanism used by cadmium. Moreover, our experiments suggest the possibility that the oxidative stress produced by mercury exposure is similar to that produced by treatment with H(2)O(2), consistent with our finding that the Yap1 protein is also involved in the response of yeast towards mercury.     

Subclinical mastitis in goats is associated with upregulation of nitric oxide-derived oxidative stress that causes reduction of milk antioxidative properties and impairment of its quality

The aim of this study was to verify the existence of a nitric oxide (NO) cycle in goat milk and to study how changes in it affect milk composition during subclinical mastitis. Fifteen lactating dairy goats in which one udder-half was free from bacterial infection and the contra-lateral one was naturally infected with various species of coagulase-negative staphylococci were used. In comparison to uninfected glands, subclinical mastitis was associated with a decrease in milk yield, lactose concentration, and curd yield and an increase in nitrite and nitrate concentrations and with measurements reflecting increased formation of NO-derived free-radical nitrogen dioxide. The occurrence of NO cycling in goat milk was largely confirmed. The increase in the NO-derived stress during subclinical infection was not associated with significant increase in oxidatively modified substances, 3-nitrotyrosine, and carbonyls on proteins, but with increased levels of peroxides on fat. However, the relatively modest nitrosative stress in subclinically infected glands was associated with significant reduction in total antioxidant capacity and vitamin C levels in milk. We concluded that subclinical mastitis in goats caused by coagulase-negative staphylococci imposes negative changes in milk yield, milk quality for cheese production, and negatively affects the nutritional value of milk as food. Thus, subclinical mastitis in goats should be considered as a serious economic burden both by farmers and by the dairy industry.     

Estimate of mercury and methyl mercury intake associated with fish consumption from Sagua la Grande River,Cuba

This paper provides an estimate of the weekly intake of total mercury (THg) and methyl mercury (MeHg) from consumption of fish from the Sagua la Grande River, Villa Clara, Cuba, by determining the THg levels in different fish species. The levels varied between 0.143 and 0.484 μg g^?1 on a fresh weight basis. None of the analysed fish was found to have levels above the national and international regulatory levels, although THg levels over 0.2 μg g^?1, the threshold concentration established by the World Health Organization (WHO) for the consumption by vulnerable population groups, were found in 75% of samples. The MeHg level was found to be 84% of the THg content. A Food Frequency Survey was given to 127 townspeople to estimate river fish consumption. The weekly intake of MeHg was found to be greater than the value established by the provisional tolerable weekly intake (PTWI) in 50% of children, in 80% of pregnant women, and in 75% of women in childbearing age. These weekly intakes can represent an important risk to the population's health, especially for vulnerable groups.     

氨基脲诱导的SD大鼠氧化应激与肝脏代谢损伤研究

目的 研究氨基脲(semicarbazide, SEM)诱导的SD雄性大鼠氧化应激与肝脏代谢损伤。方法 随机将44只SD雄性大鼠均分4组: 对照组(C组)与SEM低、中、高剂量组(L组、M组、H组), SEM剂量分别为0、7.5、15、30 mg/(kg·bw), 连续灌胃28 d。利用ELISA法测定大鼠血清SEM含量、血清与肝脏活性氧(reactive oxygen species, ROS)与8-异前列腺素F2α (8-isoprostane F2α, 8-iso-PGF2α)含量、肝脏代谢酶细胞色素P450(cytochromeP450, CYP450)、鸟苷二磷酸葡萄糖醛酸转移酶(uridine diphosphoglucuronosyl transferases, UGT)、谷胱甘肽硫转移酶(glutathione S-transferase, GST)与谷胱甘肽硫转移酶pi (glutathione S-transferase pi, GSTpi)指标以及大鼠尿液SEM含量。结果 对比C组, 各组血清SEM含量极显著升高(P<0.01), M组、H组血清ROS与H组肝匀浆8-iso-PGF2α含量显著上升(P<0.01) 。L组(P<0.05)、M组、H组(P<0.01)肝脏CYP450活力均显著下降。M组、H组UGT活力与GSTpi含量显著降低(P<0.05), GST活力无显著性。SEM尿液排泄量随时间波动, 第10 d达到排泄峰值。结论 SEM短期重复染毒使其在大鼠体内蓄积, 影响机体氧化应激与肝脏代谢酶活性, 氧化应激可能对不同代谢酶产生不同抑制作用。     

刺梨多酚对丙烯酰胺致肝脏氧化损伤的保护作用

目的 探究刺梨多酚(Rosa roxburghii Tratt. polyphenols, RRTP)对丙烯酰胺致肝脏氧化损伤的缓解效果及可能影响机制。方法 采用丙烯酰胺诱导小鼠肝脏氧化损伤模型, 通过苏木精-伊红(hematoxylin-eosin, HE)染色观察肝脏组织病理情况; 运用试剂盒测定血清中谷丙转氨酶(alanine transaminase, ALT)、谷草转氨酶(aspartate aminotransferase, AST)活性, 肝脏中活性氧(reactive oxygen species, ROS)水平、丙二醛(malondialdehyde, MDA)含量、超氧化物歧化酶(superoxide dismutase, SOD)和还原型谷胱甘肽(glutathione, GSH)活性; 蛋白质免疫印迹法检测肝脏组织中核因子E2相关因子2 (nuclear factor erythroid-2 related factor 2, Nrf2)和血红素加氧酶-1 (heme oxygenase-1, Ho-1)蛋白相对表达量。结果 与模型组相比, RRTP能够显著降低血清中ALT、AST活性和肝脏中ROS和MDA含量, 提高SOD和GSH活性, 并上调Nrf2和Ho-1蛋白表达量, 改善肝脏组织病理现象。结论 RRTP对丙烯酰胺诱导的肝脏毒性具有较好的改善作用, 其作用机制可能与Nrf2/Ho-1信号通路有关。     

Measuring oxidative DNA damage and DNA repair using the yeast comet assay

Chromosomal DNA damage can be a result of several processes and agents of endogenous or exogenous origin. These cause strand breaks or oxidized bases that lead to strand breaks, which relax the normally supercoiled genomic DNA and increase its electrophoretic mobility. The extent of DNA damage can be assessed by single cell gel electrophoresis, where the chromosomal DNA migration distance correlates with the extent of DNA damage. This technique has been used for a variety of applications with several organisms, but only a few studies have been reported for Saccharomyces cerevisiae. A possible reason for this absence is that low cellular DNA content could hamper visualization. Here we report an optimization of the comet assay protocol for yeast cells that is robust and sensitive enough to reproducibly detect background DNA damage and oxidative damage caused by hydrogen peroxide. DNA repair was observed and quantified as diminishing comet tail length with time after oxidative stress removal in a process well described by first‐order kinetics with a tail length half‐life of 11 min at 37 °C. This is, to our knowledge, the first quantitative measurement of DNA repair kinetics in S. cerevisiae by this method. We also show that diet antioxidants protect from DNA damage, as shown by a three‐fold decrease in comet tail length. The possibility of assessment of DNA damage and repair in individual cells applied to the model organism S. cerevisiae creates new perspectives for studying genotoxicity and DNA repair. Copyright © 2010 John Wiley & Sons, Ltd.     

Nuclear factor erythroid 2-related factor 2 antioxidant response element pathways protect bovine mammary epithelial cells against H2O2-induced oxidative damage in vitro

The experiment was conducted to determine the role of nuclear factor (erythroid-derived 2)-like factor 2 (NFE2L2, formerly Nrf2) antioxidant response element (ARE) pathway in protecting bovine mammary epithelial cells (BMEC) against H₂O₂-induced oxidative stress injury. An NFE2L2 small interfering RNA (siRNA) interference or a pCMV6-XL5-NFE2L2 plasmid fragment was transfected to independently downregulate or upregulate expression of NFE2L2. Isolated BMEC in triplicate were exposed to H₂O₂ (600 μM) for 6 h to induce oxidative stress before transient transfection with scrambled siRNA, NFE2L2-siRNA, pCMV6-XL5, and pCMV6-XL5-NFE2L2. Cell proliferation, apoptosis and necrosis rates, antioxidant enzyme activities, reactive oxygen species (ROS) and malondialdehyde (MDA) production, protein and mRNA expression of NFE2L2 and downstream target genes, and fluorescence activity of ARE were measured. The results revealed that compared with the control, BMEC transfected with NFE2L2-siRNA3 had proliferation rates that were 9 or 65% lower without or with H₂O₂, respectively. These cells also had apoptosis and necrosis rates that were 27 and 3.5 times greater with H₂O₂ compared with the control group, respectively. In contrast, transfected pCMV6-XL5-NFE2L2 had proliferation rates that were 64.3% greater or 17% lower without or with H₂O₂ compared with the control group, respectively. Apoptosis rates were 1.8 times lower with H₂O₂ compared with the control. In addition, compared with the control, production of ROS and MDA and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and glutathione-S-transferase (GST) increased markedly in cells transfected with pCMV6-XL5-NFE2L2 and without H₂O₂. However, compared with the control, production of ROS and MDA and activity of CAT and GSH-Px increased markedly, whereas activities of SOD and GST decreased in cells transfected with pCMV6-XL5-NFE2L2 and incubated with H₂O₂. Compared with the control, cells transfected with NFE2L2-siRNA3 with or without H₂O₂ had lower production of ROS and MDA and activity of SOD, CAT, GSH-Px, and GST. Cells transfected with pCMV6-XL5-NFE2L2 with or without H₂O₂ had markedly higher protein and mRNA expression of NFE2L2, heme oxygenase-1 (HMOX-1), NADH quinone oxidoreductase 1, glutamate cysteine ligase catalytic subunit, and glutamyl cystine ligase modulatory subunit compared with the control incubations. Cells transfected with NFE2L2-siRNA3 without or with H₂O₂ had markedly lower protein and mRNA expression of NFE2L2, HMOX-1, NADH quinone oxidoreductase 1, glutamyl cystine ligase modulatory subunit, and glutamate-cysteine ligase catalytic subunit compared with the control incubations. In addition, expression of HMOX-1 was 5.3-fold greater with H₂O₂ compared with the control. Overall, results indicate that NFE2L2 plays an important role in the NFE2L2-ARE pathway via the control of HMOX-1. The relevant mechanisms in vivo merit further study.     

Influence of mercury bioaccessibility on exposure assessment associated with consumption of cooked predatory fish in Spain

BACKGROUND: Predatory fish tend to accumulate high levels of mercury (Hg). Food safety assessment of these fish has been carried out on the raw product. However, the evaluation of the risk from Hg concentrations in raw fish might be modified if cooking and bioaccessibility (the contaminant fraction that solubilises from its matrix during gastrointestinal digestion and becomes available for intestinal absorption) were taken into account. Data on Hg bioaccessibility in raw predatory fish sold in Spain are scarce and no research on Hg bioaccessibility in cooked fish is available. The aim of the present study was to evaluate Hg bioaccessibility in various kinds of cooked predatory fish sold in Spain to estimate their health risk. RESULTS: Both Hg and bioaccessible Hg concentrations were analysed in raw and cooked fish (swordfish, tope shark, bonito and tuna). There were no changes in Hg concentrations during cooking. However, Hg bioaccessibility decreased significantly after cooking (42 ± 26% in raw fish and 26 ± 16% in cooked fish), thus reducing in swordfish and tope shark the Hg concentration to which the human organism would be exposed. CONCLUSION: In future, cooking and bioaccessibility should be considered in risk assessment of Hg concentrations in predatory fish. Copyright © 2011 Society of Chemical Industry