Functional roles of superoxide and hydrogen peroxide generated by mitochondrial DNA mutation in regulating tumorigenicity of HepG2 cells

Mitochondria are a major source of reactive oxygen species (ROS). Recent studies have estimated that mitochondrial DNA mutations inducing the overproduction of ROS are associated with human cancer. However, a substantial challenge in elucidating their diverse roles in regulating tumorigenesis is the lack of methods for probing ROS in living systems with molecular specificity. In this study, we reported the application of two fluorescent probes, 2‐chloro‐1,3‐dibenzothiazolinecyclohexene and naphthofluorescein disulfonate, which showed high selectivity for superoxide (O₂^•−) and hydrogen peroxide (H₂O₂). They were capable of detecting and visualizing O₂^•− and H₂O₂ overproduction caused by a mutation in the gene encoding nicotinamide adenine dinucleotide dehydrogenase subunit 6 (ND6) in HepG2 cells. The levels of O₂^•− and H₂O₂ in mitochondria isolated from HepG2 cells were found to be 0·63 ± 0·07 and 1·13 ± 0·05 μM, respectively. Using assays of tumorigenesis in mouse models, we found that treatment of the mice with different ROS scavengers suppressed tumour growth. These findings suggested that ROS generated by ND6 gene mutation do play an important role in regulating tumorigenesis and H₂O₂ may be a key modulator. Copyright © 2011 John Wiley & Sons, Ltd.     

Role of reactive oxygen species in cardiac preconditioning: Study with photoactivated rose bengal in isolated rat hearts

Oxygen radical scavengers have been shown to prevent the development of ischemic preconditioning, suggesting that reactive oxygen species (ROS) might be involved in this phenomenon. In the present study, we have investigated whether direct exposure to ROS produced by photoactivated Rose Bengal (RB) could mimic the protective effects of ischemic preconditioning.

Methods In vitro generation of ROS from photoactivated RB in a physiological buffer was first characterised by ESR spectroscopy in the presence of 2,2,6,6-tetramethyl-1-piperidone (oxoTEMP) or 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In a second part of the study, isolated rat hearts were exposed for 2.5 min to photoactivated RB. After 5 min washout, hearts underwent 30 min no-flow normothermic ischemia followed by 30 min of reperfusion.

Results and Conclusions The production of singlet oxygen (¹O₂) by photoactivated RB in the perfusion medium was evidenced by the ESR detection of the nitroxyl radical oxoTEMPO. Histidine completely inhibited oxoTEMPO formation. In addition, the use of DMPO has indicated that (i) superoxide anions (O^·-₂) are produced directly and (ii) hydroxyl radicals (HO^·) are formed indirectly from the successive O^·-₂ dismutation and the Fenton reaction. In the perfusion experiments, myocardial post-ischemic recovery was dramatically impaired in hearts previously exposed to the ROS produced by RB photoactivation (¹O₂, O^·-₂, H₂O₂ and HO^·) as well as when ¹O₂ was removed by histidine (50 mM) addition. However, functional recovery was significantly improved when hearts were exposed to photoactivated RB in presence of superoxide dismutase (10⁵ IU/L) and catalase (10⁶ IU/L).

Further studies are now required to determine whether the cardioprotective effects of Rose Bengal in presence of O^·-₂ and H₂O₂ scavengers are due to singlet oxygen or to other species produced by Rose Bengal degradation.     

The possible advantage of myrmecochory in diplochorous species: A test on two Mediterranean Euphorbia species

Myrmecochory commonly complements the advantages of ballistic dispersal in diplochorous species. We studied the role of the elaiosome in two populations of the two diplochorous Mediterranean spurges Euphorbia boetica and E. nicaeensis, which share an efficient ballistic dispersal mechanism followed by secondary removal by ants. They differ in elaiosome persistence, as most E. boetica seeds lose the elaiosome during explosive dispersal. Self-assessed dietary preferences with seeds with and without elaiosomes of each species showed differences in behaviour among and within ant species. In general, the absence of elaiosome entailed a decrease in the number of disperser ant species interacting with the seeds, whereas the number of predatory ants remains invariable. However, in one population of E. nicaeensis, experimental elimination of the elaiosome did not affect seed removal by mutualistic ants. On the other hand, analysis of refuse piles of the granivorous Messor marocanus and M. bouvieri suggests that they act as seed predators in E. boetica, whereas unintentional dispersal can be important in E. nicaeensis. We suggest, therefore, that the presence of the elaiosome in the seeds of the studied spurges increases the interaction with disperser ant species, but the possible dispersal advantage is not apparent and is spatially variable.     

Copper increases the damage to DNA and proteins caused by reactive oxygen species

Copper [Cu(II)] is an ubiquitous transition and trace element in living organisms. It increases reactive oxygen species (ROS) and free-radical generation that might damage biomolecules like DNA, proteins, and lipids. Furthermore, ability of Cu(II) greatly increases in the presence of oxidants. ROS, like hydroxyl (·OH) and superoxide (·O₂) radicals, alter both the structure of the DNA double helix and the nitrogen bases, resulting in mutations like the AT→GC and GC→AT transitions. Proteins, on the other hand, suffer irreversible oxidations and loss in their biological role. Thus, the aim of this investigation is to characterize, in vitro, the structural effects caused by ROS and Cu(II) on bacteriophage λ DNA or proteins using either hydrogen peroxide (H₂O₂) or ascorbic acid with or without Cu(II). Exposure of DNA to ROS-generating mixtures results in electrophoretic (DNA breaks), spectrophotometric (band broadening, hypochromic, hyperchromic, and bathochromic effects), and calorimetric (denaturation temperature [T _d], denaturation enthalpy [ΔH], and heat capacity [C _p] values) changes. As for proteins, ROS increased their thermal stability. However, the extent of the observed changes in DNA and proteins were distinct, depending on the efficiency of the systems assayed to generate ROS. The resulting effects were most evident when Cu(II) was present. In summary, these results show that the ROS, ·O₂ and ·OH radicals, generated by the Cu(II) systems assayed deeply altered the chemical structure of both DNA and proteins. The physiological relevance of these structural effects should be further investigated.     

Scavenging of reactive oxygen species by N‐substituted indole‐2 and 3‐carboxamides

Indole‐2 and 3‐carboxamides (IDs) are proposed to be selective cyclooxygenase inhibitors. Since cyclooxygenase‐1 may be involved in reactive oxygen species (ROS) production, we hypothesize that these indole derivatives have antioxidative properties. We have employed chemiluminescence (CL) and electron spin resonance (ESR) spin trapping to examine this hypothesis. We report here the results of a study of reactivity of 10 selected indole derivatives towards ROS. The following generators of ROS were applied: potassium superoxide (KO₂) as a source of superoxide radicals (O₂^·?), the Fenton reaction (Co‐EDTA/H₂O₂) for hydroxyl radicals (HO^·), and a mixture of alkaline aqueous H₂O₂ and acetonitrile for singlet oxygen (¹O₂). Hydroxyl radicals were detected as 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) spin adduct, whereas 2,2,6,6‐tetramethyl‐piperidine (TEMP) was used as a detector of ¹O₂. Using the Fenton reaction, 0.5 mmol/L IDs were found to inhibit DMPO‐?H radical formation in the range 7–37%. Furthermore the tested compounds containing the thiazolyl group also inhibited the ¹O₂‐dependent TEMPO radical, generated in the acetonitrile + H₂O₂ system. About 20% inhibition was obtained in the presence of 0.5 mmol/L IDs. 1 mmol/L IDs caused an approximately 13–70% decrease in the CL sum from the O₂^·? generating system (1 mmol/L). The aim of this paper is to evaluate these indole derivatives as antioxidants and their abilities to scavenge ROS. Copyright © 2004 John Wiley & Sons, Ltd.     

Cadmium modulates NADPH oxidase activity and expression in sunflower leaves

The production of reactive oxygen species (ROS) and the ways by which ROS are generated are very important facts related to heavy metal toxicity in plants. In this work, superoxide anion (O₂ ^·−) generation diminished in cadmium treated sunflower (Helianthus annuus L.) leaf discs, and this reduction was time and Cd-concentration dependent. In line with these findings, we observed that NADPH-dependent oxidase activity was significantly inhibited by 0.1 and 0.5 mM Cd²⁺ treatments and the expression of the NADPH oxidase putative gene related to O₂ ^·− synthesis in sunflower leaves was 83 % inhibited by 0.1 mM CdCl₂ and almost completely depleted by 0.5 mM CdCl₂.     

Cadmium-induced subcellular accumulation of O2·− and H2O2 in pea leaves

Cadmium is a toxic metal that produces disturbances in plant antioxidant defences giving rise to oxidative stress. The effect of this metal on H₂O₂ and O₂^·? production was studied in leaves from pea plants growth for 2 weeks with 50 µm Cd, by histochemistry with diaminobenzidine (DAB) and nitroblue tetrazolium (NBT), respectively. The subcellular localization of these reactive oxygen species (ROS) was studied by cytochemistry with CeCl₃ and Mn/DAB staining for H₂O₂ and O₂^·?, respectively, followed by electron microscopy observation. In leaves from pea plants grown with 50 µm CdCl₂ a rise of six times in the H₂O₂ content took place in comparison with control plants, and the accumulation of H₂O₂ was observed mainly in the plasma membrane of transfer, mesophyll and epidermal cells, as well as in the tonoplast of bundle sheath cells. In mesophyll cells a small accumulation of H₂O₂ was observed in mitochondria and peroxisomes. Experiments with inhibitors suggested that the main source of H₂O₂ could be a NADPH oxidase. The subcellular localization of O₂^·? production was demonstrated in the tonoplast of bundle sheath cells, and plasma membrane from mesophyll cells. The Cd‐induced production of the ROS, H₂O₂ and O₂^·?, could be attributed to the phytotoxic effect of Cd, but lower levels of ROS could function as signal molecules in the induction of defence genes against Cd toxicity. Treatment of leaves from Cd‐grown plants with different effectors and inhibitors showed that ROS production was regulated by different processes involving protein phosphatases, Ca²⁺ channels, and cGMP.     

Involvement of reactive oxygen species during early stages of ectomycorrhiza establishment between <Emphasis Type="Italic">Castanea sativa</Emphasis> and <Emphasis Type="Italic">Pisolithus tinctorius</Emphasis>

Evidence for the participation of reactive oxygen species (ROS) and antioxidant systems in ectomycorrhizal (ECM) establishment is lacking. In this paper, we evaluated ROS production and the activities of superoxide dismutase (SOD) and catalase (CAT) during the early contact of the ECM fungus Pisolithus tinctorius with the roots of Castanea sativa (chestnut tree). Roots were placed in contact with P. tinctorius mycelia, and ROS production was evaluated by determining the levels of H₂O₂ and O₂ ^·− during the early stages of fungal contact. Three peaks of H₂O₂ production were detected, the first two coinciding with O₂ ^·− bursts. The first H₂O₂ production peak coincided with an increase in SOD activity, whereas CAT activity seemed to be implicated in H₂O₂ scavenging. P. tinctorius growth was evaluated in the presence of P. tinctorius-elicited C. sativa crude extracts prepared during the early stages of fungal contact. Differential hyphal growth that matched the H₂O₂ production profile with a delay was detected. The result suggests that during the early stages of ECM establishment, H₂O₂ results from an inhibition of ROS-scavenging enzymes and plays a role in signalling during symbiotic establishment.     

Oxidative reactions in axenically seedling roots of olive (Olea europaea, L.)

The production of reactive oxygen species (ROS) plays important roles in the life cycle and in the stress response and defence mechanisms of plants. Various enzyme systems are involved in the formation of ROS in the apoplast, including plasmalemma NADPH oxidase and apoplastic peroxidases. The production of O ₂ ^·? and apoplastic peroxidase and exogenous NADH oxidation activities are all strongly dependent on the age of roots??the younger the root, the greater the activity. Apoplastic production of ROS is shown in the root by using specific histochemical probes, this ROS production is growing zone dependent. In the present study, using olive seedlings, differences were also observed between cultivars, especially in O ₂ ^·? production by the Verdial cultivar which was well above that of other cultivars studied. In all the cultivars, treatment of roots with methyl jasmonate (MeJA) or methyl salicylate (MeSA) increased O ₂ ^·? production. Similar results were observed for peroxidase activity, but not for the oxidation of exogenous NADH which was either unaffected (MeJA) or even partially inhibited (MeSA). A conclusion was that MeJA or MeSA induced apoplastic production of ROS does not use exogenous NADH. Treatment with diphenylene iodonium (DPI) reduced the formation of O ₂ ^·? , but affected neither peroxidase nor NADH oxidation activities. Cyanide inhibited O ₂ ^·? production and peroxidase and NADH oxidation activities. Treatment with MnCl₂ had a strong stimulatory effect on peroxidase and NADH oxidation activities, but much less on O ₂ ^·? production. Finally, azide greatly reduced all activities, but especially O ₂ ^·? production. Together, these results indicate a relationship between oxidative activities and the processes of root growth, and that those activities are also dependent on the cultivar, as well as an involvement of peroxidases and plasmalemma NADPH oxidase in apoplast ROS production which is sensitive to DPI, azide, and cyanide but relatively insensitive to MnCl₂, while exogenous NADH oxidation is linked to peroxidase activity.     

Anatomy of the Latex System in the Dormant Embryo of Euphorbia marginata Purch : Correlative Analysis of the Histological Organization and Topographic Distribution of Laticifers

The anatomy, morphology and topography of laticifers in themature embryo of Euphorbia marginata have been studied by conventionaland fluorescence microscopy. The mature embryo consists of awell-developed embryonic axis and two cotyledons and it containsdifferentiating vascular tissue. In the hypocotyl, six procambialstrands are present which branch and extend into the cotyledons,whereas in the root they are fused in a triarch structure. Inthe embryonic axis, the latex system consists of stelar andcortical components that are first anastomized in a cotyledonaryplexus and then are joined above in a cotyledonary system. Thestelar and cortical systems are linked by horizontal branchesto a nodal latex plexus placed under the epicotylary meristem.All the vascular laticifers, 24 in number, end unbranched inthe apex of the radicle. The cortical laticifers are profuselybranched toward the epidermis in tiny veins as far as the sub-epidermallevel. The cotyledonary laticifers follow the vascular tissueand form a network surrounding the mesophyllary areoles. Thetopography of the latex system leads one to hypothesize thatthe criteria which determine the constitution of the latex systemlareonly the essential peculiarities of idioblasts, but also correlativephenomena between growing laticifers and surrounding tissues.The stelar system appears to be under the control of vascularpolar growth, while the cortical and cotyledonary latex systemsseem to be subjected both to endogenous polar stimuli and toextra-embryonal influences. Euphorbia marginata, anatomy, embryo, laticifers     

镉胁迫下紫花苜蓿幼苗内源一氧化氮和活性氧的生成

以"甘农三号"紫花苜蓿幼苗为材料,在水培条件下,研究了不同浓度镉(Cd)胁迫下紫花苜蓿根、茎和叶内源一氧化氮(NO)和活性氧(ROS)的生成机制以及根系活力的变化.结果表明:在0~2.0 mmol·L-1范围内,随着Cd浓度的增加,幼苗内NO含量呈现先升高后降低的趋势,最后可维持在略高或持平于对照的水平.幼苗内一氧化氮合成酶(NOS)活性、硝酸还原酶(NR)活性、亚硝酸根离子(NO2-)含量和类胡萝卜素(Car)含量的变化与NO含量变化规律相似却又不全相同.NOS和NR是影响幼苗茎中NO含量的主要因素,NOS、NO2-和NR则是影响叶中NO含量的主要因素,而根中NO含量主要与NOS活性和NO2-含量有较大相关性.随着Cd浓度的增加,幼苗内过氧化氢(H2 O2)含量、丙二醛(MDA)含量、超氧阴离子(O-2·)含量和相对电导率(REC)呈现显著升高趋势,说明高浓度的Cd处理会使ROS大量积累,细胞膜遭破坏,细胞质外流,进而引发膜脂过氧化.随着Cd浓度的增加,紫花苜蓿根系活力的变化为先升高后降低,指示了低浓度Cd处理会促进植物代谢,增强其生命力;而高浓度Cd会致使植株代谢受抑制,细胞受损害.NO和ROS的相关性不大,说明二者虽同为自由基,但它们产生和变化方式大有差别.     

Increase of reactive oxygen species (ROS) in endothelial cells by shear flow and involvement of ROS in shear-induced c-fos expression

Intracellular reactive oxygen species (ROS) may participate in cellular responses to various stimuli including hemodynamic forces and act as signal transduction messengers. Human umbilical vein endothelial cells (ECs) were subjected to laminar shear flow with shear stress of 15, 25, or 40 dynes/cm² in a parallel plate flow chamber to demonstrate the potential role of ROS in shear-induced cellular response. The use of 2′,7′-dichlorofluorescin diacetate (DCFH-DA) to measure ROS levels in ECs indicated that shear flow for 15 minutes resulted in a 0.5- to 1.5-fold increase in intracellular ROS. The levels remained elevated under shear flow conditions for 2 hours when compared to unsheared controls. The shear-induced elevation of ROS was blocked by either antioxidant N-acetyl-cysteine (NAC) or catalase. An iron chelator, deferoxamine mesylate, also significantly reduced the ROS elevation. A similar inhibitory effect was seen with a hydroxyl radical (^·OH) scavenger, 1,3-dimethyl-2-thiourea (DMTU), suggesting that hydrogen peroxide (H₂O₂), ^·OH, and possibly other ROS molecules in ECs were modulated by shear flow. Concomitantly, a 1.3-fold increase of decomposition of exogenously added H₂O₂ was observed in extracts from ECs sheared for 60 minutes. This antioxidant activity, abolished by a catalase inhibitor (3-amino-1,2,4-triazole), was primarily due to the catalase. The effect of ROS on intracellular events was examined in c-fos gene expression which was previously shown to be shear inducible. Decreasing ROS levels by antioxidant (NAC or catalase) significantly reduced the induction of c-fos expression in sheared ECs. We demonstrate for the first time that shear force can modulate intracellular ROS levels and antioxidant activity in ECs. Furthermore, the ROS generation is involved in mediating shear-induced c-fos expression. Our study illustrates the importance of ROS in the response and adaptation of ECs to shear flow. J. Cell. Physiol. 175:156–162, 1998. © 1998 Wiley-Liss, Inc.     

Structural and Functional Grounds for Ephedra sinica Expansion in Mongolian Steppe Ecosystems

Morphological and structural characteristics of photosynthetic organs, diurnal changes in photosynthetic and transpiration rates, and the efficiency of water use were studied in three plant species from mountain-steppe ecosystems in Mongolia, Ephedra sinica Stapf, Stipa glareosa P. Smirn., and Allium polyrhizum Furcz. ex Regel. The species studied differed in the structural and functional mechanisms for the adaptation of photosynthetic apparatus to arid conditions. E. sinica has thick, vertical assimilating shoots, which are characterized by a high density (620 mg/cm³) and a small proportion of photosynthetic tissues (13%). The proportion of meso-phyll in the leaves of A. polyrhizum and S. glareosa was two and three times higher, respectively. The low content of phototrophic tissues in E. sinica shoot was compensated for by a high photosynthetic activity of single chloroplasts (25 mg CO₂/(10⁹ chloroplast h)), which was six times higher, than in two other species. Daily course of photosynthesis and transpiration inE. sinica differed from those of A. polyrhizum and S. glareosa by the absence of the midday depression. E. sinica had the highest efficiency of water use (45 mg CO₂/g H₂O) due to a low transpiration rate (0.25 g/g fr wt h). It is concluded that, in E. sinica, the main strategy for adaptation to arid stress is to develop in the shoot a few photosynthesizing cells of high assimilation activity. Such structural organization of photosynthetic organs in ephedra contributes to its higher efficiency of water use and stability of physiological characteristics under changing environmental conditions. These specific features of the structure of assimilating organs and their functional features contribute to a greater expansion of E. sinica with increasing climate aridization in Mongolia.     

Signaling Effects of Nitric Oxide, Salicylic Acid, and Reactive Oxygen Species on Isoeuphpekinensin Accumulation in Euphorbia pekinensis Suspension Cells Induced by an Endophytic Fungal Elicitor

Nitric oxide (NO), salicylic acid (SA), and reactive oxygen species (ROS) are important signal molecules that mediate plant resistance reactions and play important roles in secondary metabolism. To research the signal transduction pathway of the endophytic fungal elicitor from Fusarium sp. E5 promoting secondary metabolism in Euphorbia pekinensis suspension cells, the changes in NO, SA, ROS, and isoeuphpekinensin contents in the cells were investigated after elicitor addition to the cell suspension culture. The elicitor did not change H₂O₂ or O₂ ^? contents notably, whereas NO and SA contents were enhanced. Both the NO donator sodium nitroprusside (SNP) and SA enhanced isoeuphpekinensin content in the absence of the fungal elicitor, whereas the NO scavenger cPTIO and SA biosynthesis inhibitor cinnamic acid (CA) inhibited isoeuphpekinensin accumulation in the presence of the elicitor. In addition, cPTIO inhibited SA production induced by the fungal elicitor. CA did not inhibit NO production, but it significantly inhibited isoeuphpekinensin accumulation. The results demonstrated that in Euphorbia pekinensis suspension cells the endophytic fungal elicitor induced increased NO content and SA production, which promoted isoeuphpekinensin accumulation. ROS are clearly not involved in the endophytic fungus–host interaction signaling pathway.     

毛白杨细胞质抗坏血酸过氧化物酶基因的克隆及表达分析

利用同源克隆技术得到1个毛白杨细胞质抗坏血酸过氧化物酶基因,命名为PcAPX。该基因编码249个氨基酸残基,预测分子量为33.01kD。采用原核表达技术在大肠杆菌中表达并纯化该蛋白并进行酶活性分析,结果表明:重组PcAPX蛋白对抗坏血酸(AsA)和过氧化氢(H2O2)有很高的活性,其对抗坏血酸的米氏常数(Km)和最大反应速度(Vmax)分别为(0.71±0.03)mmol·L-1和(0.41±0.02)mmol·L-1·min-1·mg-1;对过氧化氢的Km和Vmax分别为(0.60±0.21)mmol·L-1和(0.35±0.12)mmol·L-1·min-1·mg-1,表明PcAPX对AsA和H2O2拥有较高的催化底物的能力和催化效率。利用实时定量RT-PCR分析毛白杨PcAPX基因的表达模式,结果表明其在老叶中表达量高于新叶、韧皮部、形成层和根部。该研究结果将进一步促进毛白杨APX基因家族成员参与植物生长调控的研究。     

Accumulation of non-utilizable starch in laticifers of Euphorbia heterophylla and E. myrsinites

Starch biosynthesis and degradation was studied in seedlings and mature plants of Euphorbia heterophylla L. and E. myrsinites L. Mature embryos, which lack starch grains in the non-articulated laticifers, develop into seedlings that accumulate starch rapidly when grown either in the light or the dark. Starch accumulation in laticifers of dark-grown seedlings was ca. 47 and 43% of total starch in light-grown controls in E. heterophylla and E. myrsinites, respectively. In light-grown seedlings, starch was present in laticifers as well as parenchyma of stems and leaves, whereas in dark-grown seedlings starch synthesis was almost exclusively limited to laticifers. In 7-month-old plants placed into total darkness, the starch in chyma was depleted within 6 d, whereas starch in laticifers was not mobilized. The starch content of latex in plants during development of floral primordia, flowering, and subsequent fruit formation remained rather constant. The results indicate that laticifers in seedlings divert embryonal storage reserves to synthesize starch even under stress conditions (darkness) in contrast to other cells, and that starch accumulated in laticifers does not serve as a metabolic reserve. The laticifer in Euphorbia functions in the accumulation and storage of secondary metabolites yet retains the capacity to produce, but not utilize starch, a primary metabolite.     

亚硝酸盐胁迫对罗氏沼虾血细胞及其抗氧化酶活力的影响

【背景】亚硝酸盐是虾类集约化养殖过程中最常见的毒性污染物之一,研究亚硝酸盐胁迫对罗氏沼虾血细胞的毒性以及抗氧化酶在抗胁迫防御中的作用,能够为罗氏沼虾养殖过程中的亚硝酸盐中毒防治提供理论参考。【方法】以不同浓度(0、1、5和10 mg·L~(-1))的亚硝态氮(NO_2~--N)对罗氏沼虾进行胁迫,于胁迫后的0、6、12、24和48 h取样,应用流式细胞术检测血细胞活性氧(ROS)含量和细胞凋亡率,同时测定血细胞总数(THC)和胞内抗氧化酶活力。【结果】1 mg·L~(-1)NO_2~--N在48 h内对血细胞ROS含量、凋亡率和THC均无显著影响。5 mg·L~(-1)NO_2~--N胁迫24 h,血细胞ROS含量显著上升,THC显著下降,胁迫48 h凋亡率显著提高。10 mg·L~(-1)NO_2~--N胁迫6 h,血细胞ROS含量和凋亡率均显著上升,胁迫12 h THC显著下降。血细胞的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GPx)的活力均不同程度地被NO_2~--N胁迫所诱导,CAT活力主要在胁迫前期提高,而GPx活力在胁迫后期提高。【结论与意义】亚硝酸盐存在浓度和时间毒性效应,一定浓度的亚硝酸盐会诱导虾血细胞产生ROS,这些ROS的过量产生诱导了血细胞发生凋亡,继而导致THC下降,这一氧化胁迫过程可能是亚硝酸盐对罗氏沼虾产生细胞毒性的重要机制之一。抗氧化酶活力的诱导表明抗氧化酶在亚硝酸盐胁迫过程中发挥防御作用。     

Regulation of cation transport pathways and glycolytic enzyme activity by alterations in red cell volume

In the presence of NH₄Cl and hypotonic solutions, Rana balcanica red cells respond by increasing their volume. The stimulation of cellular volume by hypotonicity is more rapid than that of NH₄Cl, while the maximum value is less than that observed in the presence of NH₄Cl. Depending on the cause of swelling, (net uptake of NH₄Cl or decrease in external osmolality) cells show specific responses. The NH₄Cl treatment causes a significant increase in intracellular Na⁺, from 5·14±0·78 to 29·84±0·47 mmoles l⁻¹ cell, while hypotonicity leads to a significant decrease of this cation, to 3·85±0·25 mmoles l⁻¹ cell in relation to the control, after 30 min of incubation of Rana balcanica erythrocytes. In addition, amiloride significantly reverses the NH₄Cl effect with respect to intracellular Na⁺. Both treatments cause a significant K⁺ loss in comparison with controls. Two glycolytic enzymes glyceraldehyde phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) of Rana balcanica haemolysate were found to respond to the NH₄Cl effect by significantly decreasing their activity. Copyright © 1999 John Wiley & Sons, Ltd.