Mechanism Of Cell Damage Research Articles

Amyloid-Like Aggregates of the Yeast Prion Protein Ure2 Enter Vertebrate Cells by Specific Endocytotic Pathways and Induce Apoptosis

BackgroundA number of amyloid diseases involve deposition of extracellular protein aggregates, which are implicated in mechanisms of cell damage and death. However, the mechanisms involved remain poorly understood.Methodology/Principal FindingsHere we use the yeast prion protein Ure2 as a generic model to investigate how amyloid-like protein aggregates can enter mammalian cells and convey cytotoxicity. The effect of three different states of Ure2 protein (native dimer, protofibrils and mature fibrils) was tested on four mammalian cell lines (SH-SY5Y, MES23.5, HEK-293 and HeLa) when added extracellularly to the medium. Immunofluorescence using a polyclonal antibody against Ure2 showed that all three protein states could enter the four cell lines. In each case, protofibrils significantly inhibited the growth of the cells in a dose-dependent manner, fibrils showed less toxicity than protofibrils, while the native state had no effect on cell growth. This suggests that the structural differences between the three protein states lead to their different effects upon cells. Protofibrils of Ure2 increased membrane conductivity, altered calcium homeostasis, and ultimately induced apoptosis. The use of standard inhibitors suggested uptake into mammalian cells might occur via receptor-mediated endocytosis. In order to investigate this further, we used the chicken DT40 B cell line DKOR, which allows conditional expression of clathrin. Uptake into the DKOR cell-line was reduced when clathrin expression was repressed suggesting similarities between the mechanism of PrP uptake and the mechanism observed here for Ure2.Conclusions/SignificanceThe results provide insight into the mechanisms by which amyloid aggregates may cause pathological effects in prion and amyloid diseases.

Assessment of autophage, apoptosis and dna damage/repair process in cryopreserved ovarian cortex

OBJECTIVE: The genomic integrity of oocytes subjected to ovarian tissue cryopreservation has not been thoroughly inverstigated. To understand potential causes and mechanisms of somatic and germ cell damage due to ovarian tissue cryopreservation, biomarker expression for autophagy, apoptosis and DNA damage/repair was evaluated in fresh, slow frozen-thawed, and vitrified-warmed bovine cortical sections. DESIGN: Experimental laboratory study. MATERIALS AND METHODS: Fresh ovaries were processed into thin cortical sections (5x5x1mm). These prepared cortical sections were divided into three groups (fresh, slow freezing, vitrification). For slow freezing, 1.5M DMSO based cryoprotectant was used. For vitrification, 20% ethylene glycol plus 20% DMSO based vitrification solution with a two step equilibration method was used. After thawing tissue was process for immunohistochemical localization of DNA damage (H2AX) and repair (RAD 51) markers. In parallel, samples were lysed and analyzed by Western blot for the expression of apoptosis (PARP) and autophage (LC3B) markers. RESULTS: In the fresh control group, oocytes within primordial and primary follicles exhibited nuclear staining for H2AX and cytoplasmic staining for RAD 51. However, H2AX staining in the nucleus disappeared and nuclear localization for RAD 51 increased in both vitrified and slow frozen groups. By Western blot analysis, slow frozen tissue showed activation of autophage as indicated by cleavage of LC3B. In contrast, vitrified tissue exhibited acute activation of apoptosis as evidenced by PARP cleavage. CONCLUSION: These findings indicate that oocytes within primordial and primary follicles generate an acute DNA repair process in response to DNA damage induced by tissue cryopreservation. Slow freezing/thawing appears to elicit autophage (cell survival pathway) initially followed by apoptosis, whereas the pathway to autophage is bypassed upon warming of vitrified tissue possibly due to the effects of high concentration of cryoprotectants.

N-methyl-aspartate receptor and neuronal nitric oxide synthase activation mediate bilirubin-induced neurotoxicity.

Hyperbilirubinemia may lead to neurotoxicity and neuronal death. Although the mechanisms of nerve cell damage by unconjugated bilirubin (UCB) appear to involve a disruption of the redox status and excitotoxicity, the contribution of nitric oxide (NO·) and of N-methyl-D-aspartate (NMDA) glutamate receptors is unclear. We investigated the role of NO· and NMDA glutamate receptors in the pathways of nerve cell demise by UCB. Neurons were incubated with 100 micromol/L UCB, in the presence of 100 micromol/L human serum albumin for 4 h at 37ºC, alone or in combination with N-ω-nitro-L-arginine methyl ester (L-NAME) (an inhibitor of neuronal nitric oxide synthase [nNOS]), hemoglobin (an NO· scavenger) or (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) (an NMDA-receptor antagonist). Exposure to UCB led to increased expression of nNOS and production of both NO· and cyclic guanosine 3',5'-monophosphate (cGMP), along with protein oxidation and depletion of glutathione. These events concurred for cell dysfunction and death and were counteracted by L-NAME. Moreover, the UCB-induced loss of neuronal viability was abolished by hemoglobin, whereas the activation of nNOS and production of both NO· and cGMP were counteracted by MK-801, resulting in significant protection from cell dysfunction and death. These results reinforce the involvement of oxidative stress by showing that nerve cell damage by UCB is mediated by NO· and therefore is counteracted by NO· inhibitors or scavengers. Our findings strongly suggest that the activation of nNOS and neurotoxicity occur through the engagement of NMDA receptors. These data reveal a role for overstimulation of glutamate receptors in mediating oxidative damage by UCB.

Further Phenomena

We thank our colleagues for their clear position statement. In conclusion: “Cord blood is not the fountain of eternal youth in our time!”—and the ethical implications of “combined donations” should not be underestimated. The statement: “Autologous stem cells from cord blood have poor prospects for use in regenerative medicine, because they have to be cryopreserved until use [and it isn’t possible to produce clinically relevant amounts of non-hematopoietic stem cells from cryopreserved material],” however, cannot be left unchallenged. In addition to the two main mechanisms of cell damage during cryopreservation (namely osmotic dehydration and intracellular ice formation), further phenomena occur during the cooling, storage, and rewarming of biological cells (for example, toxicity of the cryoprotectives, “cold shock,” pH shifts, phase transitions and separations, lipid peroxidation, vesiculation, protein denaturation, “free” radicals, thermoelastic stress, embrittlement, crack formation, devitrification, recrystallization), all of which are the current subjects of cryobiological research. Such known damaging factors are likely to be responsible for the authors’ lack of success in culturing relevant amounts of non-hematopoietic stem cells (1). In a scenario of growth rates of 13% or 19% however, it is possible to grow non-hematopoietic stem cells from cord blood. The authors seem to have been using a similar cryopreservation procedure as that published 15 years ago (2) and have not introduced variations with regard to known factors of influence (for example, the type and concentration of the cryoprotective, cooling and rewarming rates, cell concentration, and methods to remove the cryoprotective. We should therefore not overemphasize the detail “cryopreservation does not turn out well”—this was assumed before 1949 (3). Instead we should better concentrate on ethical aspects when advising our patients not to store cord blood for autologous use.

Mechanisms of immune-mediated liver injury.

Hepatic inflammation is a common finding during a variety of liver diseases including drug-induced liver toxicity. The inflammatory phenotype can be attributed to the innate immune response generated by Kupffer cells, monocytes, neutrophils, and lymphocytes. The adaptive immune system is also influenced by the innate immune response leading to liver damage. This review summarizes recent advances in specific mechanisms of immune-mediated hepatotoxicity and its application to drug-induced liver injury. Basic mechanisms of activation of lymphocytes, macrophages, and neutrophils and their unique mechanisms of recruitment into the liver vasculature are discussed. In particular, the role of adhesion molecules and various inflammatory mediators in this process are explored. In addition, the authors describe mechanisms of liver cell damage by these inflammatory cells and critically evaluate the functional significance of each cell type for predictive and idiosyncratic drug-induced liver injury. It is expected that continued advances in our understanding of immune mechanisms of liver injury will lead to an earlier detection of the hepatotoxic potential of drugs under development and to an earlier identification of susceptible individuals at risk for predictive and idiosyncratic drug toxicities.

Microcalorimetric and microscopic studies on the inhibitory activities of methylene blue/TiO 2 nanocomposites on Staphylococcus aureus and the mechanism of cell damage

The effect of methylene blue/TiO 2 nanocomposites on Staphylococcus aureus was evaluated by a LKB-2277 Bioactivity Monitor at 37.0 °C. By analyzing the thermogenic power–time curves, kinetic parameters such as the growth rate constant ( k), the maximum heat power of growth phase ( P m), generation time ( t G) and the inhibitory ratio ( I) were determined. The k and P m decrease while t G and I increases with increasing concentrations of the methylene blue/TiO 2 nanocomposites added into the microbe suspension. Both k and I have linear correlations with the concentration of the methylene blue/TiO 2 nanocomposites. The effect of the methylene blue sensitized-TiO 2 nanocomposites on the cell damage of S. aureus was investigated by microscopic method.

Therapeutical approach to dry eye syndrome

Dry eye disease or dysfunctional tear syndrome is among the most frequently established diagnoses in ophthalmology. It can be defined as a disorder of the tear film resulting in changes in the ocular surface. There are many factors causing dry eye and they, can be related to deficiency in any of the components of the tear film. It has been suggested that dry eye is an inflammatory disorder that affects the ocular surface and lacrimal gland. Inflammation is the most important mechanism of corneal and conjunctival cell damage, which is responsible for the symptoms and signs of ocular surface pathology. Hormonal imbalance (particularly androgens), neural dysfunction, increased levels of pro-inflammatory cytokines and loss of immune homeostasis of the lacrimal gland and ocular surface could be possible mechanisms in the pathogenesis of dry eye disease. The aim of this paper was to review the advances in the pathogenesis and management of the dry eye disease. The appropriate dry eye treatment presupposes knowledge of all existing pharmacologic and non-pharmacologic therapeutic modalities. The mainstay of therapy is still artificial tears, with anti-inflammatory therapy and punctual occlusion therapy as second and third line therapies.

Characterization of Micro-/Nano-Scale Ice Crystal Formation in Cryo-Biomedical Engineering: A Review

The micro- and nano-scale freezing has been a focus in many fields of scientific research and engineering practices, especially in those involved with cryobiology physics. Although a series of theoretical models and experimental techniques have been available to tackle the tough issues that associate with the freezing phase change process, the topic to quantify the ice crystal formation and its subsequent effects on the biological materials were not sufficiently addressed at all, especially when facing the newly emerging area of nano-cryobiology technology. Not only the interactions between the ice front and the cell, but also the heat and solute balance during freezing produce impact on the output of a cryosurgery or cryopreservation. There is currently a strong lack of the generalized methods which are indispensable to grasp the physical pictures from micro or nanoscale aspect and thus help achieve an intensive understanding of the mechanisms of cell damage during process of ice crystallization. This review article is dedicated to present an overview on the theoretical and experimental strategies potentially appropriate for quantifying the ice crystal formation in cryobiology area. Attentions will be focused on several major theoretical directions such as the thermodynamic theory, the kinetic theory, and the density functional theory etc., concerning the micro or nano phase change and the ice nucleation activities. Interpretation of the effects of external conditions such as freezing rate, interfacial effect, size effects, electric field and restricted space etc. on the ice growth will be given. Numerical simulation method and experimental techniques will also be outlined. The review does not intend to give an exhaustive knowledge of all the themes, but rather to provide a most basic background for further probing the emerging nano-cryobiology field and thus promote innovation in future research and practices of cryobiology.

Bistability of Mitochondrial Respiration Underlies Paradoxical Reactive Oxygen Species Generation Induced by Anoxia

Increased production of reactive oxygen species (ROS) in mitochondria underlies major systemic diseases, and this clinical problem stimulates a great scientific interest in the mechanism of ROS generation. However, the mechanism of hypoxia-induced change in ROS production is not fully understood. To mathematically analyze this mechanism in details, taking into consideration all the possible redox states formed in the process of electron transport, even for respiratory complex III, a system of hundreds of differential equations must be constructed. Aimed to facilitate such tasks, we developed a new methodology of modeling, which resides in the automated construction of large sets of differential equations. The detailed modeling of electron transport in mitochondria allowed for the identification of two steady state modes of operation (bistability) of respiratory complex III at the same microenvironmental conditions. Various perturbations could induce the transition of respiratory chain from one steady state to another. While normally complex III is in a low ROS producing mode, temporal anoxia could switch it to a high ROS producing state, which persists after the return to normal oxygen supply. This prediction, which we qualitatively validated experimentally, explains the mechanism of anoxia-induced cell damage. Recognition of bistability of complex III operation may enable novel therapeutic strategies for oxidative stress and our method of modeling could be widely used in systems biology studies.

Gadolinium blocks membrane permeabilization induced by nanosecond electric pulses and reduces cell death

It has been widely accepted that nanosecond electric pulses (nsEP) are distinguished from micro- and millisecond duration pulses by their ability to cause intracellular effects and cell death with reduced effects on the cell plasma membrane. However, we found that nsEP-induced cell death is most likely mediated by the plasma membrane disruption. We showed that nsEP can cause long-lasting (minutes) increase in plasma membrane electrical conductance and disrupt electrolyte balance, followed by water uptake, cell swelling and blebbing. These effects of plasma membrane permeabilization could be blocked by Gd(3+) in a dose-dependent manner, with a threshold at sub-micromolar concentrations. Consequently, Gd(3+) protected cells from nsEP-induced cell death, thereby pointing to plasma membrane permeabilization as a likely primary mechanism of lethal cell damage.

Mechanisms of Noise-Induced Hair Cell Death

Mechanisms of Noise-Induced Hair Cell Death

Modeling of Bubble Expansion-Induced Cell Mechanical Profile in Laser-Assisted Cell Direct Writing

Cell damage due to the mechanical impact during laser-assisted cell direct writing has been observed and is a possible hurdle for broad applications of fragile cell direct writing. The objective of this study is to numerically investigate the bubble expansion-induced cell mechanical loading profile in laser-assisted cell direct writing. Some conclusions have been drawn as follows. The cell velocity increases initially and then smoothes out gradually with a constant ejection velocity. Both the cell acceleration and pressure can be very high at the beginning period of bubble expansion and then quickly approach zero in an oscillation manner. A high viscosity can lead to an observable velocity increment at the initial stage, but the ejection velocity decreases. The pressure magnitude decreases when the cell-bubble distance is large, and a larger initial pressure induces a larger cell pressure as expected. This study serves as a foundation to further investigate the cell damage mechanism in laser-assisted cell direct writing to improve the effectiveness and efficiency of cell direct writing techniques.

Cholesterol overloading leads to hepatic L02 cell damage through activation of the unfolded protein response

Reported data indicate that cholesterol loading in the liver can cause hepatic injury. To explore the possible mechanisms of cell damage resulting from cholesterol overloading in hepatocytes, cell apoptosis, the unfolded protein response (UPR) and the correlation between them were assessed in the cholesterol-overloaded normal human hepatic cell line L02. L02 cells were incubated with 200 microg/ ml of low density lipoprotein (LDL) for 24 h with or without 20 microg/ml 58035, an inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT). In the LDL+58035 group, the intracellular cholesterol level was dramatically increased, which was measured by an enzymatic combined high performance liquid chromatography assay. Expression of immunoglobulin-binding protein, X-box binding protein 1, activating transcription factor 6, activating transcription factor 4, CCAAT/enhancer-binding protein homologous protein-10, markers of endoplasmic reticulum stress (ERS)/ UPR, were up-regulated as determined using reverse transcription-polymerase chain reaction (RT-PCR) or Western blot analysis. The rate of cell apoptic death increased 21.3+/-2.4%. Meanwhile, the active caspase-3 protein expression was increased 8.4-fold compared to the active caspase-3 protein expression in the controls. Furthermore, 4-phenylbutyric acid, an inhibitor of UPR, partly reduced cell apoptosis and activation of caspase-3. This study suggests that cholesterol overloading in hepatic L02 cells induces ERS and activates the UPR which, in part, leads to the apoptotic damage of cells.

Effects of maternal phenylketonuria on the development of the offspring's eye

AbstractPhenylketonuria (PKU) is an autosomal recessive disease caused by phenylalanine‐4‐hydroxylase (P‐4‐H) deficiency, which is a liver‐specific enzyme that catalyzes the hydroxylation of l‐phenylalanine (Phe) to l‐tyrosine (Tyr). The deficiency of this enzyme leads to the accumulation of Phe in the tissues and plasma of patients. Among the clinical symptoms of this disease are mental retardation and other neurological features. The mechanisms of retinal cell damage are still poorly understood. In order to evaluate the damage in offspring of PKU mothers, fifty pregnant rats were used and arranged into two main groups, i.e., a control group and a group consisting of rats with experimentally induced PKU. Induction of PKU was carried out by daily intragastrical administration of 30 mg DL–alpha methylphenylalanine/150 g b.w. plus 60 mg/kg b.w. Phe at 12 h intervals throughout pregnancy till parturition as well as throughout lactation period till 14 days post‐partum. Morphometric analysis of retina in 1, 7 and 14‐day‐old pups of PKU mothers revealed a marked reduction of retinal thickness. Histological observations revealed numerical reduction of pigment cells. Apoptic cell death of retinal pigment cells, and of nuclear layer and ganglionic cells were observed. The nerve layer showed apparent vacuolar degeneration. At transmission electron microscope (TEM) level, pigment epithelium showed distortion of apical microvilli, vacuolation of cytoplasm and reduction of cytoplasm organelles. The Brush's membrane showed apparent thickening. In 7‐day‐old pups of PKU mothers, photoreceptor inner segment showed apparent degeneration. In 14‐day‐old pups of PKU mothers, both the inner and outer photoreceptor segment were degenerated and the stacked membrane of the outer segment appeared vacuolated and degenerated.

Preclinical photodynamic therapy research in Spain 3: Localization of photosensitizers and mechanisms of cell deathin vitro

Photodynamic therapy (PDT) is a subject of increasing biomedical research and represents a very promising therapeutic modality for palliative or even curative treatment of some superficial or endoscopically accessible tumors. In addition to the first photosensitizers (PSs) applied (hematoporphyrin-based drugs), second generation PSs with improved photophysical and photobiological properties are now studied using cell cultures, experimental tumors and clinical trials. On the other hand, there is a growing interest in the analysis of cell death mechanisms by apoptosis, which is especially relevant in oncology, because many anticancer drugs work, at least in part, by triggering apoptosis in neoplastic cells both in vitro and in vivo. The evaluation of cell death mechanisms is an important parameter to determine the efficacy and the potential toxicity of a treatment, allowing better adjustment of protocol. Using cell cultures, our research team has studied the mechanisms of cell damage and death implicated in the photodynamic processes, as well as the relationship between the cellular localization of the PS and the organelle damage during photosensitization. The results obtained in our laboratory provide a deeper understanding on the action mechanisms that lead to cell inactivation by PDT, and also allow selection of PSs with higher potential for clinical application than those currently in use.

Research Highlights

Research Highlights

Induction of autophagic cell death and radiosensitization by the pharmacological inhibition of nuclear factor–kappa B activation in human glioma cell lines

The intrinsic radioresistance of certain cancer cells may be closely associated with the constitutive activation of nuclear factor-kappa B (NF-kappaB) activity, which may lead to protection from apoptosis. Recently, nonapoptotic cell death, or autophagy, has been revealed as a novel response of cancer cells to ionizing radiation. In the present study, the authors analyzed the effect of pitavastatin as a potential inhibitor of NF-kappaB activation on the radiosensitivity of A172, U87, and U251 human glioma cell lines. The pharmacological inhibition of NF-kappaB activation was achieved using pitavastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Growth and radiosensitivity assays were performed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Hoechst 33258 staining, supravital acridine orange staining, and electron microscopy were performed utilizing 3 glioma cell lines with or without pitavastatin pretreatment to identify apoptosis or autophagy after irradiation. The growth of these 3 glioma cell lines was not significantly inhibited by pitavastatin at a concentration of up to 1 microM. Treatment with 0.1 microM of pitavastatin enhanced radiation-induced cell death in all glioma cell lines, with different sensitivity. Apoptosis did not occur in any pretreated or untreated (no pitavastatin) cell line following irradiation. Instead, autophagic cell changes were observed regardless of the radiosensitivity of the cell line. An inhibitor of autophagy, 3-methyladenine suppressed the cytotoxic effect of irradiation with pitavastatin, indicating that autophagy is a result of an antitumor mechanism. Using the most radiosensitive A172 cell line, the intracellular localization of p50, a representative subunit of NF-kappaB, was evaluated through immunoblotting and immunofluorescence studies. The NF-kappaB of A172 cells was immediately activated and translocated from the cytosol to the nucleus in response to irradiation. Pitavastatin inhibited this activation and translocation of NF-kappaB. Autophagic cell death rather than apoptosis is a possible mechanism of radiation-induced and pitavastatin-enhanced cell damage, and radiosensitization by the pharmacological inhibition of NF-kappaB activation may be a novel therapeutic strategy for malignant gliomas.

Interaction of Fullerene with Model Cell Membranes: a Computer Simulation Study

Nano-sized fullerene aggregates can enter cells and alter their functions, but the mechanisms of cell damage is unclear. In our previous work [1] we used coarse-grained molecular dynamics simulations to characterize the thermodynamics and kinetics of permeation of fullerene clusters through a model membrane. We also showed that high fullerene concentrations induce changes in the structural and elastic properties of the lipid bilayer, but these are not sufficient to cause a direct mechanical damage to the membrane. Now we explore the effect of fullerene on model membranes including an ion channel protein, Kv1.2, using computer simulations with both a coarse-grained and an atomistic representation. We also investigate the effects of a naturally abundant organic compound, gallic acid, on fullerene-membrane interactions. Recent work [2] has shown that gallic acid-coated fullerenes cause cell contraction. We use computer simulations to describe possible mechanisms of cell damage.[1] Wong-ekkabut et al., Nature Nanotech (2008), 3, 363.[2] Salonen et al., Small, in press.

In vitro effect of malathion and diazinon on oocytes fertilization and embryo development in porcine

Diazinon and malathion are commonly used organophosphate insecticides in agriculture, industry, and in veterinary medicine as an ectoparasiticide. The importance to carry out in vitro reproductive toxicology assays lies on the need of knowing the alterations these insecticides may cause at cellular level, since they are endocrine disruptors that interfere with reproductive functions. The aim of this study was to evaluate in vitro oocyte viability, fertilization, and embryo development with different concentrations of diazinon and malathion. For in vitro fertilization (IVF), porcine oocytes and sperm were co-incubated for 7 h with increasing concentrations (50, 100, and 500 microM) of diazinon and malathion. For embryo development, fertilized oocytes were cultured in medium containing the same insecticide concentrations during 96 h for embryo development and 144 h for morulae formation. Diazinon did not affect oocyte viability and embryo divisions but decreased IVF (fertilization inhibition(50) = 502 microM) and morulae formation (morulae inhibition(50) = 344 microM). Malathion affected all the studied parameters: lethal concentration(50) = 1 mM, fertilization inhibition(50) = 443 microM, development inhibition(50) = 375 microM, and morulae inhibition(50) = 216 microM. The results of this study indicate that diazinon and malathion used in commercial formulation could be toxic, producing impairment in in vitro fertilization and embryo development. This is an approach for further investigations to find out cell damage mechanisms produced by these widely used insecticides.

Update of Cell Damage Mechanisms in Thiamine Deficiency: Focus on Oxidative Stress, Excitotoxicity and Inflammation

Thiamine deficiency (TD) is a well-established model of Wernicke's encephalopathy. Although the neurologic dysfunction and brain damage resulting from the biochemical consequences of TD is well characterized, the mechanism(s) that lead to the selective histological lesions characteristic of this disorder remain a mystery. Over the course of many years, various structural and functional changes have been identified that could lead to cell death in this disorder. However, despite a concerted effort to explain the consequences of TD in terms of these changes, our understanding of the pathophysiology of this disorder remains unclear. This review will focus on three of these processes, i.e. oxidative stress, glutamate-mediated excitotoxicity and inflammation and their role in selective vulnerability in TD. Since TD inhibits oxidative metabolism, a feature of many neurodegenerative disease states, it represents a model system with which to explore pathological mechanisms inherent in such maladies, with the potential to yield new insights into their possible treatment and prevention.