Inhibition of protein synthesis by products of lipid peroxidation

Effects of lipid peroxidation products on in vivo and in vitro protein synthesis have been studied. Malondialdehyde (MDA), a product, and a routinely used index of lipid peroxidation, inhibits in vivo protein synthesis in the two mosses, Tortula ruralis and Cratoneuron filicinum, and in pea (Pisum sativum) leaf discs. When wheat germ supernatant or poly(A)-rich mRNA of T. ruralis was incubated with MDA its subsequent activity in a cell-free protein-synthesizing system was reduced. When MDA was added directly to the in vitro protein-synthesizing mixture containing moss polyribosomes, the inhibition of amino acid incorporation was small. However, when simultaneous lipid peroxidation was allowed to occur along with in vitro protein synthesis there was a strong inhibition of amino acid incorporation and MDA accumulated in the reaction mixture indicating that products of lipid peroxidation other than, and apparently more toxic than, MDA were involved. It was concluded that lipid peroxidation inhibits protein synthesis probably by releasing toxic products which may react with and inactivate some components of the protein-synthesizing complex.     

Pseudomonas aeruginosa: A risk factor for fertility in male mice

The present study was designed to evaluate the effect of P. aeruginosa on reproductive potential of male mice via a series of in vitro and in vivo experiments. In vitro studies involved sperm parameters, Mg²⁺ATPase activity and acrosome status. In vivo study employed male mice which in the right vas deferens received 20?μl of either PBS (Group I) or 10⁴ cfu of P. aeruginosa (Group II). The animals were sacrificed on day 3, 7 and 14 and various parameters viz. body weight, TSI (%), bacterial load, spermiogram {i.e. sperm count, motility (%), viability (%) and morphology}, lipid peroxidation and tissue histopathology were evaluated. The results revealed that cell free supernatant of P. aeruginosa resulted in reduced motility, viability, Mg²⁺dependent ATPase activity and premature acrosomal loss of mouse spermatozoa in vitro. In vivo study showed that in comparison to group I, group II revealed significant alterations in all the parameters on all the days of sacrifice. Further, when reproductive organs of right and left side of mice in group II were compared, the right side demonstrated more devastating effects in terms of altered TSI (%) of testis and cauda epididymis, higher bacterial counts, azoospermia, increased malondialdehyde levels and severe inflammation in tissue histopathology in comparison to left side where bacteria disseminated in reduced numbers, thereby, resulting in insignificant changes in TSI (%), spermiogram, malondialdehyde levels and tissue histology. This study demonstrates that the colonization of P. aeruginosa in male genital tract could be a risk factor for fertility.     

Inhibition of Photosystem II Precedes Thylakoid Membrane Lipid Peroxidation in Bisulfite-Treated Leaves of Phaseolus vulgaris

Exposure of leaves to SO₂ or bisulfite is known to induce peroxidation of thylakoid lipids and to inhibit photosynthetic electron transport. In the present study, we have examined the temporal relationship between bisulfite-induced thylakoid lipid peroxidation and inhibition of electron transport in an attempt to clarify the primary mechanism of SO₂ phytotoxicity. Primary leaves of bean (Phaseolus vulgaris L. cv Kinghorn) were floated on a solution of NaHSO₃, and the effects of this treatment on photosynthetic electron transport were determined in vivo by measurements of chlorophyll a fluorescence induction and in vitro by biochemical measurements of the light reactions using isolated thylakoids. Lipid peroxidation in treated leaves was followed by monitoring ethane emission from leaf segments and by measuring changes in fatty acid composition and lipid fluidity in isolated thylakoids. A 1 hour treatment with bisulfite inhibited photosystem II (PSII) activity by 70% without modifying Photosystem I, and this inhibitory effect was not light-dependent. By contrast, lipid peroxidation was not detectable until after the inhibition of PSII and was strongly light dependent. This temporal separation of events together with the differential effect of light suggests that bisulfite-induced inhibition of PSII is not a secondary effect of lipid peroxidation and that bisulfite acts directly on one or more components of PSII.     

Inhibitory effects of α- and β-carotene on croton oil-induced or enzymatic lipid peroxidation and hydroperoxide production in mouse skin epidermis

• 1.1. The effects of carotenes (α- and β-) on edema, MDA contents and peroxidizability ofcroton oil-treated mouse skin epidermis, hydroperoxide production and enzymatic lipid peroxidation of epidermal homogenates were studied. Edema was determined as ear punch weight and the intensity of lipid peroxidation was measured using malondialdehyde formation.
• 2.2. Carotenes (α- and β-) significantly suppressed edema formation, hydroperoxide production, lipid peroxidation caused by croton oil, Fe + 3-ADP/NADPH or paraquat/NADPH in vivo as well as in vitro.
• 3.3. These results indicate that both α- and β-carotene have chemopreventive effects on croton oil-induced tumor promotion in skin tumorigenesis by scavenging oxygen free radicals, indirectly determined as carotene inhibition of lipid peroxidation and hydroperoxide formation.

     

Effect of copper on pro- and antioxidative reactions in radish (Raphanus sativus L.) in vitro and in vivo

The generation of superoxide radicals, lipid peroxidation (as measured by malone dialdehyde formation) and the activity of selected antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase) were assessed in radish (Raphanus sativus L.), in response to elevated concentrations of copper ions in the culture medium in vitro and in vivo. Experiments were performed on 7-day-old seedlings and 5-week-old calluses grown on media supplemented with CuSO₄ in concentrations of 10, 100 and 1000 μМ. The exposure to elevated Cu concentrations in the medium significantly reduced both callogenesis and the proliferation of radish calluses in vitro. Cu treatment resulted in the increased generation of the superoxide radical (O₂⁻) in radish seedlings and calluses indicating the occurrence of oxidative stress in radish cells, whereas the level of lipid peroxidation (LPO) remained unchanged. Both in calluses and in radish seedlings in vivo, the relative level of oxidative stress was maximal at micromolar Cu concentrations and became attenuated with increasing Cu concentrations. Stronger oxidative stress occurred in the radish seedlings in vivo, compared with radish calluses in vitro. The observed lower sensitivity of calluses to Cu-induced oxidative stress and their ability to proliferate upon exposure to Cu concentrations of up to 1000 μМ demonstrate the potential of in vitro cell-selection to obtain metal-tolerant radish plant lines.     

An inter-laboratory validation of methods of lipid peroxidation measurement in UVA-treated human plasma samples

Abstract

Lipid peroxidation products like malondialdehyde, 4-hydroxynonenal and F₂-isoprostanes are widely used as markers of oxidative stress in vitro and in vivo. This study reports the results of a multi-laboratory validation study by COST Action B35 to assess inter-laboratory and intra-laboratory variation in the measurement of lipid peroxidation. Human plasma samples were exposed to UVA irradiation at different doses (0, 15 J, 20 J), encoded and shipped to 15 laboratories, where analyses of malondialdehyde, 4-hydroxynonenal and isoprostanes were conducted. The results demonstrate a low within-day-variation and a good correlation of results observed on two different days. However, high coefficients of variation were observed between the laboratories. Malondialdehyde determined by HPLC was found to be the most sensitive and reproducible lipid peroxidation product in plasma upon UVA treatment. It is concluded that measurement of malondialdehyde by HPLC has good analytical validity for inter-laboratory studies on lipid peroxidation in human EDTA-plasma samples, although it is acknowledged that this may not translate to biological validity.     

Lipid storage and lipophagy regulates ferroptosis

The synthesis, storage, and degradation of lipids are highly regulated processes. Impaired lipid metabolism is implicated in inflammation and cell death. Although ferroptosis is a recently described form of regulated cell death driven by lipid peroxidation, the impact of lipid droplets on ferroptosis remains unidentified. Here, we demonstrate that lipophagy, the autophagic degradation of intracellular lipid droplets, promotes RSL3-induced ferroptotic cell death in hepatocytes. Lipid droplet accumulation is increased at the early stage but decreased at the late stage of ferroptosis in mouse or human hepatocytes. Importantly, either genetically enhancing TPD52-dependent lipid storage or blocking ATG5-and RAB7A-dependent lipid degradation prevents RSL3-induced lipid peroxidation and subsequent ferroptosis in vitro and in vivo. These studies support an antioxidant role for lipid droplets in cell death and suggest novel strategies for the inhibition of ferroptosis by targeting the lipophagy pathway.     

Evidence for the accumulation of peroxidized lipids in membranes of senescing cotyledons

Fluorescent products of lipid peroxidation accumulate with age in microsomal membranes from senescing cotyledons of Phaseolus vulgaris. The temporal pattern of accumulation is closely correlated with a rise in the lipid phase transition temperature reflecting the formation of gel phase lipid. Increased levels of fluorescent peroxidation products are also detectable in total lipid extracts of senescent cotyledons. Lipoxygenase activity increases with advancing age by about 3-fold on a fresh weight basis and 4-fold on a dry weight basis indicating that the tissue acquires elevated levels of lipid hydroperoxides. As well, levels of glutathione and superoxide dismutase activity decline on a dry weight basis as the cotyledons age, rendering the tissue more susceptible to oxidative damage. Catalase activity rises initially and then declines during senescence, but peroxidase activity rises steeply. Thus, apart from this increase in peroxidase, which would scavenge H₂O₂ only if appropriate cosubstrates were available, the defense mechanisms for coping with activated oxygen species (O₂⁻, H₂O₂, OH) are less effective in the older tissue. The observations support the contention that formation of gel phase lipid in senescing membranes is attributable to lipid peroxidation and suggest that the reactions of lipid peroxidation are utilized by the cotyledons to mediate deteriorative changes accompanying the mobilization and transport of metabolites from the storage tissue to the developing embryo.     

Schistosoma mansoni: Phenol oxidase's role in eggshell formation

Phenol oxidase may be involved in the formation of the eggshell in Schistosoma mansoni. ³H-Labeled female S. mansoni proteins were polymerized in vitro following incubation with S. mansoni phenol oxidase and excess l-tyrosine. Peroxidase inhibitors, autoxidation inhibitors, inhibitors of lipid peroxidation, and inactive analogs of phenol oxidase inhibitors did not inhibit eggshell formation. Fluorescent substances found in eggshell hydrolysates were similar to those formed from the reaction of phenol oxidase-generated quinones with protein-bound lysine. These observations support the classical concept of phenol oxidase-catalyzed protein hardening. However, fluorescent globules of egg material were still formed after the administration of 200 mg/kg of the phenol oxidase inhibitor diethyldithiocarbamate. These globules could not be destroyed by inhibitors of autoxidation and lipid peroxidation.     

Host phospholipid peroxidation fuels ExoU-dependent cell necrosis and supports Pseudomonas aeruginosa-driven pathology

Regulated cell necrosis supports immune and anti-infectious strategies of the body; however, dysregulation of these processes drives pathological organ damage. Pseudomonas aeruginosa expresses a phospholipase, ExoU that triggers pathological host cell necrosis through a poorly characterized pathway. Here, we investigated the molecular and cellular mechanisms of ExoU-mediated necrosis. We show that cellular peroxidised phospholipids enhance ExoU phospholipase activity, which drives necrosis of immune and non-immune cells. Conversely, both the endogenous lipid peroxidation regulator GPX4 and the pharmacological inhibition of lipid peroxidation delay ExoU-dependent cell necrosis and improve bacterial elimination in vitro and in vivo. Our findings also pertain to the ExoU-related phospholipase from the bacterial pathogen Burkholderia thailandensis, suggesting that exploitation of peroxidised phospholipids might be a conserved virulence mechanism among various microbial phospholipases. Overall, our results identify an original lipid peroxidation-based virulence mechanism as a strong contributor of microbial phospholipase-driven pathology.     

10E12Z CLA alters adipocyte differentiation and adipocyte cytokine expression and induces macrophage proliferation

The trans-10, cis-12 (10e12z) conjugated linoleic acid (CLA) isomer of CLA is responsible for loss of lipid storage or adipose tissue in vitro or in vivo. This isomer also induces inflammatory signaling in both mouse and human adipocytes in vitro. However, when these events occur and whether they are significant enough to affect other cell types are unclear. In these experiments, the 3T3-L1 cell line has been used to examine the interaction between inflammatory signaling and decreased differentiation or lipid storage induced by 10e12z CLA. In assays measuring both lipid accumulation and gene expression, differentiating 3T3-L1 cells exhibit concurrent induction of inflammatory signaling, as measured by cyclooxygenase-2 expression, and a decrease in adipocyte marker gene expression. Furthermore, in fully differentiated adipocytes, as identified in microarray assays and confirmed with real-time polymerase chain reaction, 10e12z CLA also significantly affected expression of both matrix metalloprotein-3 (MMP-3), collagen VI α 3 ColVI alpha 3 (VIα3) and the cytokine epiregulin, demonstrating that the effects of 10e12z broadly impact adipocyte function. In agreement with other experimental systems, 10e12z CLA inhibited RAW 264.7 cell proliferation; however, in response to adipocyte-conditioned media, 10e12z-CLA-treated adipocytes induced proliferation of this cell line, suggesting that the effect of 10e12z CLA is context dependent. These results are largely consistent with the known activation of the inflammatory mediator nuclear factor-κB in adipocytes in vitro and in vivo by 10e12z CLA treatment and demonstrate that adipose is an important target tissue of this isomer that impacts other cell types.     

Replication-Competent Influenza A and B Viruses Expressing a Fluorescent Dynamic Timer Protein for In Vitro and In Vivo Studies

Influenza A and B viruses (IAV and IBV, respectively) cause annual seasonal human respiratory disease epidemics. In addition, IAVs have been implicated in occasional pandemics with inordinate health and economic consequences. Studying influenza viruses in vitro or in vivo requires the use of laborious secondary methodologies to identify infected cells. To circumvent this requirement, replication-competent infectious influenza viruses expressing an easily traceable fluorescent reporter protein can be used. Timer is a fluorescent protein that undergoes a time-dependent color emission conversion from green to red. The rate of spectral change is independent of Timer protein concentration and can be used to chronologically measure the duration of its expression. Here, we describe the generation of replication-competent IAV and IBV where the viral non-structural protein 1 (NS1) was fused to the fluorescent dynamic Timer protein. Timer-expressing IAV and IBV displayed similar plaque phenotypes and growth kinetics to wild-type viruses in tissue culture. Within infected cells, Timer’s spectral shift can be used to measure the rate and cell-to-cell spread of infection using fluorescent microscopy, plate readers, or flow cytometry. The progression of Timer-expressing IAV infection was also evaluated in a mouse model, demonstrating the feasibility to characterize IAV cell-to-cell infections in vivo. By providing the ability to chronologically track viral spread, Timer-expressing influenza viruses are an excellent option to evaluate the in vitro and in vivo dynamics of viral infection.     

Intervertebral Disc Tissue Engineering with Natural Extracellular Matrix-Derived Biphasic Composite Scaffolds

Tissue engineering has provided an alternative therapeutic possibility for degenerative disc diseases. However, we lack an ideal scaffold for IVD tissue engineering. The goal of this study is to fabricate a novel biomimetic biphasic scaffold for IVD tissue engineering and evaluate the feasibility of developing tissue-engineered IVD in vitro and in vivo. In present study we developed a novel integrated biphasic IVD scaffold using a simple freeze-drying and cross-linking technique of pig bone matrix gelatin (BMG) for the outer annulus fibrosus (AF) phase and pig acellular cartilage ECM (ACECM) for the inner nucleus pulposus (NP) phase. Histology and SEM results indicated no residual cells remaining in the scaffold that featured an interconnected porous microstructure (pore size of AF and NP phase 401.4±13.1 μm and 231.6±57.2 μm, respectively). PKH26-labeled AF and NP cells were seeded into the scaffold and cultured in vitro. SEM confirmed that seeded cells could anchor onto the scaffold. Live/dead staining showed that live cells (green fluorescence) were distributed in the scaffold, with no dead cells (red fluorescence) being found. The cell—scaffold constructs were implanted subcutaneously into nude mice and cultured for 6 weeks in vivo. IVD-like tissue formed in nude mice as confirmed by histology. Cells in hybrid constructs originated from PKH26-labeled cells, as confirmed by in vivo fluorescence imaging system. In conclusion, the study demonstrates the feasibility of developing a tissue-engineered IVD in vivo with a BMG- and ACECM-derived integrated AF-NP biphasic scaffold. As well, PKH26 fluorescent labeling with in vivo fluorescent imaging can be used to track cells and analyse cell—scaffold constructs in vivo.     

Radiolabeled cholesteryl ethers: A need to analyze for biological stability before use

Radiolabeled cholesteryl ethers are widely used as non-metabolizable tracers for lipoproteins and lipid emulsions in a variety of in vitro and in vivo experiments. Since cholesteryl ethers do not leave cells after uptake and are not hydrolyzed by mammalian cellular enzymes, these compounds can act as markers for cumulative cell uptakes of labeled particles. We have employed [³H]cholesteryl oleoyl ether to study the uptake and distribution of triglyceride-rich emulsion particles on animal models. However, questionable unexpected results compelled us to analyze the stability of these ethers. We tested the stability of two commercially available radiolabeled cholesteryl ethers - [³H]cholesteryl oleoyl ether and [³H]cholesteryl hexadecyl ether from different suppliers, employing in vitro, in vivo and chemical model systems. Our results show that, among the two cholesteryl ethers tested, one ether was hydrolyzed to free cholesterol in vitro, in vivo and chemically under alkaline hydrolyzing agent. Free cholesterol, unlike cholesteryl ether, can then re-enter the circulation leading to confounding results. The other ether was not hydrolyzed to free cholesterol and remained as a stable ether. Hence, radiolabeled cholesteryl ethers should be analyzed for biological stability before utilizing them for in vitro or in vivo experiments.     

Arsenic‐induced NFκβ transactivation through Erks‐ and JNKs‐dependent pathways in mouse epidermal JB6 cells

Carnosine, a alanylLhistidine dipeptide with antioxidant properties is present at high concentrations in skeletal muscle tissue. In this study, we report on the antioxidant activity of carnosine on muscle lipid and protein stability from both in vitro and in vivo experiments. Carnosine inhibited lipid peroxidation and oxidative modification of protein in muscle tissue prepared from rat hind limb homogenates exposed to in vitro Fenton reactant (Fe²⁺, H₂O₂)generated free radicals. The minimum effective concentrations of carnosine for lipid and protein oxidation were 2.5 and 1 mM, respectively. Histidine and alanine, active components of carnosine, showed no individual effect towards inhibiting either lipid or protein oxidation. Skeletal muscle of rats fed a histidine supplemented diet for 13 days exhibited a marked increase in carnosine content with a concomitant reduction in muscle lipid peroxidation and protein carbonyl content in skeletal muscle caused by subjecting rats to a Fenitrilotriacetate administration treatment. This significant in vitro result confirms the in vivo antioxidant activity of carnosine for both lipid and protein constituents of muscle under physiological conditions.     

Iron Overload and the Predisposition of Cells to Antioxidant Consumption and Peroxidative Damage

We have investigated the effects of iron overload in vivo on the tocopherol levels and the extent of lipid peroxidation in rat liver microsomes and their response to subsequent oxidative stress in vitro. The results demonstrate a direct correlation between consumption of antioxidant defences and the induction and extent of malondialdehyde production in microsomes prepared from iron-loaded rats. The data are consistent with the requirement for iron (II)/iron (III) ratios in lipid peroxidation in control microsomes.     

High-density lipoprotein endocytosis in endothelial cells

AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescencemicroscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type Ⅰ mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrincoated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.     

Farrerol alleviates collagenase‐induced tendinopathy by inhibiting ferroptosis in rats

Tendinopathy is mainly characterized by local pain, functional limitation and decreased athletic ability, which seriously affects the quality of life of patients and the career of athletes. Farrerol (FA), one of the main active compounds extracted from Rhododendron and plants in the Rhododendron family, has a wide range of pharmacological activities, such as immunomodulatory, anti‐inflammatory and antiviral effects. However, the effect of FA on tendinopathy is unclear. Here, we investigated the pharmacological effect and mechanism of FA in tendon injury through collagenase‐induced tendinopathy in vivo and RSL3‐induced tenocytes injury in vitro. The results showed that FA alleviated the infiltration of inflammatory cells, promoted tenogenesis and improved mechanical properties of the Achilles tendon in rats. In addition, ferroptosis inducer RSL3 inhibits the tenogenesis in vitro and in vivo, which accelerates the progression of tendinopathy. Moreover, FA effectively inhibited iron accumulation and alleviated ferroptosis in the Achilles tendon. Using in vitro experiments, we found that FA antagonized ferroptosis by reducing lipid peroxidation and iron accumulation in tenocytes. Finally, we found that glutathione peroxidase 4 silencing could block the protective effect of FA on ferroptosis of tenocytes. Therefore, the results of this study suggest that FA can relieve collagenase‐induced tendinopathy by inhibiting ferroptosis, and reveal that FA may be a potentially effective drug for the treatment of tendinopathy in the future.     

In vitro growth environment produces lipidomic and electron transport chain abnormalities in mitochondria from non-tumorigenic astrocytes and brain tumours

The mitochondrial lipidome influences ETC (electron transport chain) and cellular bioenergetic efficiency. Brain tumours are largely dependent on glycolysis for energy due to defects in mitochondria and oxidative phosphorylation. In the present study, we used shotgun lipidomics to compare the lipidome in highly purified mitochondria isolated from normal brain, from brain tumour tissue, from cultured tumour cells and from non-tumorigenic astrocytes. The tumours included the CT-2A astrocytoma and an EPEN (ependymoblastoma), both syngeneic with the C57BL/6J (B6) mouse strain. The mitochondrial lipidome in cultured CT-2A and EPEN tumour cells were compared with those in cultured astrocytes and in solid tumours grown in vivo. Major differences were found between normal tissue and tumour tissue and between in vivo and in vitro growth environments for the content or composition of ethanolamine glycerophospholipids, phosphatidylglycerol and cardiolipin. The mitochondrial lipid abnormalities in solid tumours and in cultured cells were associated with reductions in multiple ETC activities, especially Complex I. The in vitro growth environment produced lipid and ETC abnormalities in cultured non-tumorigenic astrocytes that were similar to those associated with tumorigenicity. It appears that the culture environment obscures the boundaries of the Crabtree and the Warburg effects. These results indicate that in vitro growth environments can produce abnormalities in mitochondrial lipids and ETC activities, thus contributing to a dependency on glycolysis for ATP production.