砷暴露诱导人肝细胞氧化应激过程中NADPH氧化酶作用机制

目的：探讨砷暴露诱导细胞氧化应激的分子机制。方法：采用人正常肝细胞进行亚砷酸钠和砷酸钠的暴露处理,并设相应对照组,采用SOD模拟物MnTMPyP和还原型谷胱甘肽（reducedglutathione,GSH）预处理,检测细胞超氧阴离子（02。）和细胞整体ROS的水平。WestemBlot方法检测细胞氧化／抗氧化重要酶微粒体谷胱甘肽硫转移酶（microsomalglutathioneS-transferase-l,Mgst．1）、半胱氨酸双加氧酶l（cysteinedioxygenasel,Cd01）和NADPH氧化酶的催化亚基NOX4的表达。针对NADPH氧化酶,采用特异性抑制剂（diphenyleneiodoniumchloride,DPI）进行预处理,观察对砷暴露引起的细胞ROS水平及细胞凋亡的影响。结果：砷暴露能够显著诱导细胞超氧阴离子的产生,提高细胞整体ROS水平,其中三价砷（亚砷酸钠,A矿）诱导氧化应激作用显著强于五价砷（砷酸钠,As5＋）。亚砷酸钠能够显著提高NOX4的表达。针对NADPH氧化酶的抑制剂DPI能够显著抑制砷暴露引起的细胞ROS水平升高以及细胞凋亡的增加。结论：NADPH氧化酶是砷暴露诱导人肝细胞的作用靶点,砷能够通过NADPH氧化酶产生大量超氧阴离子,提高ROS水平,造成氧化应激,诱导人正常肝细胞凋亡。     

Genotoxicity Induced by Foetal and Infant Exposure to Magnetic Fields and Modulation of Ionising Radiation Effects

Background

Few studies have investigated the toxicity and genotoxicity of extremely low frequency magnetic fields (ELF-MF) during prenatal and neonatal development. These phases of life are characterized by cell proliferation and differentiation, which might make them sensitive to environmental stressors. Although in vitro evidences suggest that ELF-MF may modify the effects of ionizing radiation, no research has been conducted so far in vivo on the genotoxic effects of ELF-MF combined with X-rays.

Aim and methods

Aim of this study was to investigate in somatic and germ cells the effects of chronic ELF-MF exposure from mid gestation until weaning, and any possible modulation produced by ELF-MF exposure on ionizing radiation-induced damage. Mice were exposed to 50 Hz, 65 μT magnetic field, 24 hours/day, for a total of 30 days, starting from 12 days post-conception. Another group was irradiated with 1 Gy X-rays immediately before ELF-MF exposure, other groups were only X-irradiated or sham-exposed. Micronucleus test on blood erythrocytes was performed at multiple times from 1 to 140 days after birth. Additionally, 42 days after birth, genotoxic and cytotoxic effects on male germ cells were assessed by comet assay and flow cytometric analysis.

Results

ELF-MF exposure had no teratogenic effect and did not affect survival, growth and development. The micronucleus test indicated that ELF-MF induced a slight genotoxic damage only after the maximum exposure time and that this effect faded away in the months following the end of exposure. ELF-MF had no effects on ionizing radiation (IR)-induced genotoxicity in erythrocytes. Differently, ELF–MF appeared to modulate the response of male germ cells to X-rays with an impact on proliferation/differentiation processes. These results point to the importance of tissue specificity and development on the impact of ELF-MF on the early stages of life and indicate the need of further research on the molecular mechanisms underlying ELF-MF biological effects.     

Kinetic Characterization of the Oxidation of Esculetin by Polyphenol Oxidase and Peroxidase

Esculetin has been described as an inhibitor of tyrosinase and polyphenol oxidase and, therefore, of melanogenesis. In this work, we demonstrate that esculetin is not an inhibitor but a substrate of mushroom polyphenol oxidase (PPO) and horseradish peroxidase (POD), enzymes which oxidize esculetin, generating its o-quinone. Since o-quinones are very unstable, the usual way of determining the enzymatic activity (slope of recordings) is difficult. For this reason, we developed a chronometric method to characterize the kinetics of this substrate, based on measurements of the lag period in the presence of micromolar concentrations of ascorbic acid. The catalytic constant determined was of the same order for both enzymes. However, polyphenol oxidase showed greater affinity (a lower Michaelis constant) than peroxidase for esculetin. The affinity of PPO and POD towards oxygen and hydrogen peroxide was very high, suggesting the possible catalysis of both enzymes in the presence of low physiological concentrations of these oxidizing substrates. Taking into consideration optimum pHs of 4.5 and 7 for POD and PPO respectively, and the acidic pHs of melanosomes, the studies were carried out at pH 4.5 and 7. The in vivo pH might be responsible for the stronger effect of these enzymes on L-tyrosine and L-3,4-dihydroxyphenylanaline (L-DOPA) (towards melanogenesis) and on cumarins such as esculetin towards an alternative oxidative pathway.     

Cloning of the Pumpkin Ascorbate Oxidase Gene and Analysis of a Cis-Acting Region Involved in Induction by Auxin

A genomic clone encoding ascorbate oxidase was isolated frompumpkin (Cucurbita sp.)- This gene is consisted of four exonsand three introns. Analyses of the promoter fusion to ß-glucuronidasereporter gene by transient expression assay in pumpkin fruittissues suggested the existence of a cis-acting region responsiblefor auxin regulation. (Received November 28, 1996; Accepted March 8, 1997)     

阳离子脂质体介导基因转染肿瘤细胞

使用基因转运载体运载肿瘤细胞进行转染是基因治疗的关键环节之一。Lipo-fectamine2000和DOTAP作为商品转染试剂,具有较高的转染效率。为了进一步发掘其作为基因转运载体的应用潜力,该文研究了Lipofectamine2000和DOTAP的粒径、Zeta电位及形态,并分别与绿色荧光蛋白基因（pGFP—N2）、荧光素酶基因（pGL3）结合,形成脂质体／DNA复合物,通过载入人喉癌细胞（Hep-2）和人肺癌细胞（NCI—H460）,考察了其转染效率和细胞毒性。结果表明,脂质体Lipofectamine2000与DOTAP都能有效压缩DNA,形成复合物。Lipofectamine2000与DOTAP井目比,转染效率高,与DNA最佳转染比例范围为2：1～4：1。毒性实验显示,在N／P大于3／l时,Lipofectamine2000与DOTAP对癌细胞具有一定的细胞毒性。细胞种类对脂质体的转染效率有很大影响,Lipo—fectamine2000对Hep－2细胞的转染效率比NcI—H460高。     

光和底物对乙醇酸氧化酶的基因表达的诱导

从菠菜中提纯了乙醇酸氧化酶并制备其抗体,经免疫双扩散、Ｗｅｓｔｅｒｎｂｌｏｔ和Ｎｏｒｔｈｅｒｎｂｌｏｔ证实水稻和豌豆黄化苗中不存在乙醇酸氧化酶。在黑暗中,底物可促进该酶基因的表达,而在黄化苗光照初期,推测光可能是不经过底物促进该酶基因的表达。     

Enzymatic Cross-Linking of Casein Facilitates Gel Structure Weakening Induced by Overacidification

Casein (α_S1, α_S2, β, κ) is the major protein fraction in milk and, together with heat denatured whey proteins, responsible for gel network formation induced by acidification. Rheological measurements during gelation typically reveal a maximum storage modulus (G') at a pH close to the isoelectric point (pI) of casein (~4.6). With further decreasing pH gel stiffness decreases because of increased electrostatic repulsion, which is referred to as overacidification. In this study we investigated the effect of casein cross-linking with microbial transglutaminase on gel structure weakening induced by acidification to pH below the pI. Although enzymatic cross-linking increased the maximum stiffness (G' _MAX ) of casein gels the reduction of G' during overacidification, expressed as ratio of the plateau value (G' _FINAL ) to G' _MAX , was more pronounced. Almost no soluble protein was detected in the serum of gels from cross-linked casein, whereas considerable amounts of α_S- and κ-casein were released from reference gels below the pI. This suggests that covalent cross-linking of casein retains charged molecules within the gel network and therefore causes a higher reduction of protein-protein interactions because of higher electrostatic repulsion. Furthermore, higher amounts of uncross-linked β-casein, which was the only casein type not found in the serum, resulted in higher G' _FINAL to G' _MAX ratios, underlining the important contribution of β-casein to acid gel formation and prevention of gel structure weakening.     

Volume and Shape Changes of Human Erythrocytes Induced by Electrical Fields

The effects of electrical fields on the shape and volume of normal and abnormal red blood cells (RBC) are discussed. If an electrical field of 2-3 kV/cm is applied to the suspension of RBC, normal biconcave erythrocytes transform into spherocytes and their mean volume will increase from the usual volume of 90 µm³ to 160-170/µm³ in several minutes. There are two possible explanations for this phenomenon. The one is the membrane perforation by the potential induced by electrical field and subsequent influx of water. The other Is the mechanical surface traction caused by applied fields. The second mechanism will be discussed mainly in this paper.     

Enzymatic Improvement of Food Flavor IV. Oxidation of Aldehydes in Soybean Extracts by Aldehyde Oxidase

Aldehyde oxidase (aldehyde: oxygen oxidoreductase, EC 1.2.3.1) was partially purified from bovine liver. The enzyme irreversibly oxidized various aldehydes to the corresponding acids by using dissolved oxygen as an electron acceptor. Although the Km value for n-hexanal was low (6 µm), that for acetaldehyde was high (20 mm).

Medium-chain aldehydes such as hexanal and pentanal appear to be mainly responsible for green beany odor of soybean products. A great reduction in the beany odor was observed after the soybean extract was incubated with aldehyde oxidase under aerobic conditions. Dissolved oxygen was utilized as the electron acceptor throughout the enzyme-catalyzed oxidation of aldehydes and none of other cofactors were found to be required.

It has been shown that bovine liver mitochondrial aldehyde dehydrogenase oxidizes the soybean protein-bound aldehyde with a rate comparable to that for free n-hexanal (Agric. Biol. Chem., 43, in press). Comparative studies of aldehyde oxidase and aldehyde dehydrogenase with respect to oxidation-rates of free aldehydes and the soybean protein-bound aldehydes indicated that aldehyde oxidase acted on the bound aldehyde with a much slower rate.     

亚慢性砷中毒对大鼠肝脏自噬的抑制作用

目的:在建立亚慢性砷暴露模型的基础上,探讨砷暴露对SD大鼠肝脏自噬水平的影响.方法:出生21天断乳雄性SD大鼠分为不同水平砷暴露组,通过饮水给与NaAsO2的方式染毒;全自动生化分析仪检测血清肝功能项目的变化;HE染色检测肝脏组织病理学改变;透射电镜法超微结构的改变.Western blot方法检测自噬相关蛋白Beclin1、LC3表达水平变化.结果:砷暴露组肝脏出现显著的异常,同时伴随肝组织形态学改变.于是此同时,LC3Ⅱ/LC3Ⅰ比值以及beclin1的表达水平均随着砷作用浓度的升高而降低.结论:亚慢性砷暴露在诱导肝脏损伤的同时可伴随自噬水平的抑制,这种水平的改变可能参与了砷的肝脏毒性.     

Oxidation of N-Methyl-1,2,3,4-Tetrahydroisoquinoline into the N-Methyl-Isoquinolinium Ion by Monoamine Oxidase

N-Methyl-1,2,3,4-tetrahydroisoquinoline (NMTIQ) was found to be oxidized by monoamine oxidase (MAO) into N-methylisoquinolinium ion, which was proved to inhibit enzymes related to the metabolism of catecholamines, such as tyrosine hydroxylase, aromatic-L-amino acid decarboxylase, and MAO. NMTIQ was oxidized by both types A and B MAO in human brain synaptosomal mitochondria. Oxidation was dependent on the amount of MAO sample and the reaction time. Enzyme activity with respect to NMTIQ reached optimum at a pH of approximately 7.25, as was the case with other substrates. Type A MAO had higher activity for this substrate than type B. The Km and Vmax values of the oxidation by types A and B MAO were 571 +/- 25 microM and 0.29 +/- 0.06 pmol/min/mg protein, and 463 +/- 43 microM and 0.16 +/- 0.03 pmol/min/mg protein, respectively. The Vmax values of types A and B MAO for NMTIQ were much smaller than those for other substrates such as kynuramine. NMTIQ was the first tetrahydroisoquinoline shown to be oxidized into the isoquinolinium ion by MAO in the brain.     

Effects of Melatonin on Memory and Learning Deficits Induced by Exposure to Thinner

The neurotoxic effects of thinner, a mixture including aromatic compounds (in particular, toluene) and widely used as an industrial solvent, were examined. Exposure of rats to high inhalation concentrations (3000 p.p.m.) of thinner for 45 days (1 h per day) significantly influenced the cognitive functions and levels of neural cell adhesion molecules (NCAM) in the hippocampus, cortex, and cerebellum of experimental animals. These exposures also caused dramatic increases in levels of LPO (malondialdehyde and 4-hydroxyalkenals) in these cerebral structures, while melatonin administration significantly reduced the LPO amounts in these brain regions. The level of NCAM (180 kDa) decreased significantly in the hippocampus and cortex of thinner-exposed rats. Furthermore, thinner-exposed rats showed cognitive deficits in the passive avoidance and Morris water maze tasks; these negative effects were considerably compensated in rats additionally chronically treated with melatonin. It is concluded that treatment with melatonin prevents the development of learning and memory deficits caused by thinner exposure, possibly by reducing oxidative stress and normalizing the neural plasticity.     

NADPH氧化酶参与水杨酸诱导的蚕豆气孔关闭过程

水杨酸(SA)可以浓度依赖的方式诱导蚕豆叶片的气孔关闭,1～1000μmol·L~(-1)SA所诱导的气孔关闭可以再开放,而10~(-2)mol·L~(-1)的SA导致的气孔关闭则否。质膜NADPH氧化酶抑制剂二亚苯基碘(DPI)可削弱SA作用的45%～60%。表明SA诱导的气孔关闭可能与H_2O_2的产生有关。以H_2O_2荧光探针H_2DCFDA结合显微注射技术直接检测保卫细胞内产生H_2O_2的结果显示,100μmol·L~(-1)SA可引起保卫细胞内荧光素(DCF)荧光快速增强。在DPI存在的情况下,经SA处理的保卫细胞,仅在其叶绿体部位产生H_2O_2,而质膜附近的DCF荧光增强则受到抑制。表明叶绿体可能是保卫细胞内产生H_2O_2的主要部位,质膜NADPH氧化酶也可能参与SA诱导H_2O_2的产生。     

Low Level Laser Therapy Reduces the Development of Lung Inflammation Induced by Formaldehyde Exposure

Lung diseases constitute an important public health problem and its growing level of concern has led to efforts for the development of new therapies, particularly for the control of lung inflammation. Low Level Laser Therapy (LLLT) has been highlighted as a non-invasive therapy with few side effects, but its mechanisms need to be better understood and explored. Considering that pollution causes several harmful effects on human health, including lung inflammation, in this study, we have used formaldehyde (FA), an environmental and occupational pollutant, for the induction of neutrophilic lung inflammation. Our objective was to investigate the local and systemic effects of LLLT after FA exposure. Male Wistar rats were exposed to FA (1%) or vehicle (distillated water) during 3 consecutive days and treated or not with LLLT (1 and 5 hours after each FA exposure). Non-manipulated rats were used as control. 24 h after the last FA exposure, we analyzed the local and systemic effects of LLLT. The treatment with LLLT reduced the development of neutrophilic lung inflammation induced by FA, as observed by the reduced number of leukocytes, mast cells degranulated, and a decreased myeloperoxidase activity in the lung. Moreover, LLLT also reduced the microvascular lung permeability in the parenchyma and the intrapulmonary bronchi. Alterations on the profile of inflammatory cytokines were evidenced by the reduced levels of IL-6 and TNF-α and the elevated levels of IL-10 in the lung. Together, our results showed that LLLT abolishes FA-induced neutrophilic lung inflammation by a reduction of the inflammatory cytokines and mast cell degranulation. This study may provide important information about the mechanisms of LLLT in lung inflammation induced by a pollutant.     

茉莉酸甲酯和机械损伤对茶树叶片多酚氧化酶时序表达的影响

以温室中培养的龙井43茶树2年生扦插苗为实验材料,采用分光光度计法测定了正常茶树叶片中多酚氧化酶(PPO)活性变化的日节律,以及茉莉酸甲酯(MeJA)处理和机械损伤诱导不同叶位叶片PPO活性的时序变化.结果表明:正常茶树叶片中PPO活性高峰出现在4:00;MeJA处理茶树30 h后,其叶片PPO活性与对照间呈现显著差异,于处理后174 h达到最高峰;半定量RT-PCR检测结果发现,MeJA诱导茶树叶片PPO基因表达量最高峰出现在处理后126 h,比生化测定结果提前了48 h.机械损伤可诱导处理叶及其下叶位叶片PPO活性迅速升高,表现出一定系统性;机械损伤叶片与系统诱导叶片间PPO活性随时间变化的趋势基本平行,但机械损伤叶片中PPO活性始终高于系统诱导叶片.本研究证明,MeJA和机械损伤均可诱导茶树叶片中PPO活性的显著增强,进一步证实茉莉酸(JA)信号转导途径在茶树的直接防御反应中具有重要作用.     

Diffusion Tensor Imaging of Brain Abnormalities Induced by Prenatal Exposure to Radiation in Rodents

We assessed brain abnormalities in rats exposed prenatally to radiation (X-rays) using magnetic resonance imaging (MRI) and histological experiments. Pregnant rats were divided into 4 groups: the control group (n = 3) and 3 groups that were exposed to different radiation doses (0.5, 1.0, or 1.5 Gy; n = 3 each). Brain abnormalities were assessed in 32 neonatal male rats (8 per group). Ex vivo T₂-weighted imaging and diffusion tensor imaging (DTI) were performed using 11.7-T MRI. The expression of markers of myelin production (Kluver–Barrera staining, KB), nonpyramidal cells (calbindin-D28k staining, CaBP), and pyramidal cells (staining of the nonphosphorylated heavy-chain neurofilament SMI-32) were histologically evaluated. Decreased brain volume, increased ventricle volume, and thinner cortices were observed by MRI in irradiated rats. However, no abnormalities in the cortical 6-layered structure were observed via KB staining in radiation-exposed rats. The DTI color-coded map revealed a dose-dependent reduction in the anisotropic signal (vertical direction), which did not represent reduced numbers of pyramidal cells; rather, it indicated a signal reduction relative to the vertical direction because of low nerve cell density in the entire cortex. We conclude that DTI and histological experiments are useful tools for assessing cortical and hippocampal abnormalities after prenatal exposure to radiation in rats.     

Rapid Decrease of GAD 67 Content Before the Convulsion Induced by Hyperbaric Oxygen Exposure

Exposure to hyperbaric oxygen (HBO) can lead to seizures, the etiology of which is not completely understood. Glutamic acid decarboxylase (GAD) plays a very important role in maintaining excitatory-inhibitory balance of the central nervous system (CNS). In the present study we investigated the effects of HBO on the activity and content of GAD in vivo and in primarily cultured neurons to probe in detail its effect on the formation of convulsion induced by HBO exposure. The results obtained from in vivo and in vitro experiments were identical. In the latent period before the onset of seizure, the GAD activity followed a rise-and-fall pattern with the prolongation of HBO exposure. At the time of the onset of seizure, GAD activity descended to the normal level. Besides, in the latent period, GAD content also reduced. Such reduction came from a GAD subtype, GAD67, while the content of another GAD subtype, GAD65, remained almost unchanged. Our investigations indicated that GAD is indeed an enzyme highly sensitive to the effect of HBO exposure. The rapid reduction in GAD67 content may be very closely related to seizures induced by HBO exposure. Quan Li and Meili Guo are contributed equally to the study.     

Changes Induced by Abscisic Acid and Light in the Redox State of Ascorbate in the Apoplast of Epicotyls of Vigna angularis

Levels of ascorbic acid (AA) and dehydroascorbic acid (DHA)were examined in epicotyl segments and intact epicotyls undervarious conditions. It appears that not only the redox stateof an AA-DHA system but also the level of AA plus DHA in theapoplast might be affected by growth conditions. (Received March 26, 1994; Accepted June 4, 1994)     

Dopamine Affects the Change of Pain-Related Electrical Activity Induced by Morphine Dependence

Morphine is among the most effective analgesics. However, many evidences suggest that, besides the well-know analgesic activity, repeated opioids treatment can induce some side effects such as dependence, hyperalgesia and tolerance. The mechanism of noxious information transmission in the central nervous system after dependence is not clear. An important neurotransmitter, dopamine (DA) participates not only in the process of opioid dependence but also in pain modulation in the central nervous system. In the present study we observed changes of electrical activities of pain-excitation neurons (PENs) and pain-inhibition neurons (PINs) in the caudate nucleus (Cd) following the development of morphine dependence. We also observed the role of DA on these changes. Our results revealed that both the latency of PEN discharges and the inhibitory duration of PIN discharges decreased, and the net increased values of PEN and PIN discharges increased in the Cd of morphine dependent rats. Those demonstrated that electrical activities of both PENs and PINs increased in morphine dependent rats. DA inhibited the electrical activities of PENs and enhanced those of PINs in morphine dependent rats.     

Impairment of Central Chemoreception in Neonatal Rats Induced by Maternal Cigarette Smoke Exposure during Pregnancy

It has been postulated that prenatal cigarette smoke exposure (CSE) increases the risk for sudden infant death syndrome. The victims of infant death syndrome suffer from respiratory abnormalities, such as central apnea, diminished chemoreflex and alteration in respiratory pattern during sleep. However, no experimental evidence on CSE model exists to confirm whether prenatal CSE gives rise to reduction of neonatal central chemoreception in in vitro preparations in absence of peripheral sensory feedback. The aim of the present study was to test the hypothesis that maternal CSE during pregnancy depresses central chemoreception of the neonatal rats. The pregnant rats were divided into two groups, control (n = 8) and CSE (n = 8). Experiments were performed on neonatal (0–3days) rat pups. Fictive respiratory activity was monitored by recording the rhythmic discharge from the hypoglossal rootlets of the medullary slices obtained from the neonatal rats. The burst frequency (BF) and integrated amplitude (IA) of the discharge were analyzed. Their responses to acidified artificial cerebrospinal fluid (aCSF) were tested to indicate the change of the central chemosensitivity. Under condition of perfusing with standard aCSF (pH 7.4), no significant difference was detected between the two groups in either BF or IA (P>0.05). Under condition of perfusing with acidified aCSF (pH 7.0), BF was increased and IA was decreased in both groups (P<0.01). However, their change rates in the CSE group were obviously smaller than that in the control group, 66.98 ± 10.11% vs. 143.75 ± 15.41% for BF and −22.38 ± 2.51% vs. −44.90 ± 3.92% for IA (P<0.01). In conclusion, these observations, in a prenatal CSE model, provide important evidence that maternal smoking during pregnancy exerts adverse effects on central chemoreception of neonates.