Immunomodulatory protein from ganoderma microsporum protects against oxidative damages and cognitive impairments after traumatic brain injury.

A traumatic brain injury (TBI) causes abnormal proliferation of neuroglial cells, and over-release of glutamate induces oxidative stress and inflammation and leads to neuronal death, memory deficits, and even death if the condition is severe. There is currently no effective treatment for TBI. Recent interests have focused on the benefits of supplements or natural products like Ganoderma. Studies have indicated that immunomodulatory protein from Ganoderma microsporum (GMI) inhibits oxidative stress in lung cancer cells A549 and induces cancer cell death by causing intracellular autophagy. However, no evidence has shown the application of GMI on TBI. Thus, this study addressed whether GMI could be used to prevent or treat TBI through its anti-inflammation and antioxidative effects. We used glutamate-induced excitotoxicity as in vitro model and penetrating brain injury as in vivo model of TBI. We found that GMI inhibits the generation of intracellular reactive oxygen species and reduces neuronal death in cortical neurons against glutamate excitotoxicity. In neurite injury assay, GMI promotes neurite regeneration, the length of the regenerated neurite was even longer than that of the control group. The animal data show that GMI alleviates TBI-induced spatial memory deficits, expedites the restoration of the injured areas, induces the secretion of brain-derived neurotrophic factors, increases the superoxide dismutase 1 (SOD-1) and lowers the astroglial proliferation. It is the first paper to apply GMI to brain-injured diseases and confirms that GMI reduces oxidative stress caused by TBI and improves neurocognitive function. Moreover, the effects show that prevention is better than treatment. Thus, this study provides a potential treatment in naturopathy against TBI.

Swimming exercise prevents hippocampal dendritic spine changes and memory loss caused by aging: An application of a new semi-automated spine analysis software.

Dendritic spines are small, ratchet-like protrusions on neuronal dendrites that form synapses for receiving neuronal messages. Dendritic spine morphology is associated with synapse function. If neurons degrade or are damaged, the spine morphology of neurons changes. Given that most commercially available spine analysis software is expensive and complex, this study investigated a semi-automated spine analysis software, CTSpine, and used previously published data to verify the accuracy of the analysis results of this software. We also applied CTSpine to understand whether aging causes alterations in the hippocampal spine morphology and whether physical exercise can impede dendritic spine changes in 20 male Sprague Dawley rats. The spines of pyramidal cells in the hippocampal Cornu Ammonis 1 (CA1) region in the aging group were more enriched in filopodium type pattern than those in the control group, whereas the spines of the exercised aging group showed a similar pattern to that of the control. No significant changes were observed in neuronal dendritic spines in other hippocampal regions. However, long-term hippocampal memory was considerably decreased in the aging group, which was reversed to some extent in the exercised aging group. CTSpine, a self-developed semi-automatic spine analysis software, showed results similar to those noted in published data and can be effectively applied to the study of dendritic patterns, including neurodevelopment and disease.

Cypin binds to tubulin heterodimers and microtubule protofilaments and regulates microtubule spacing in developing hippocampal neurons.

Cytosolic PSD-95 interactor (cypin) is a multifunctional, guanine deaminase that plays a major role in shaping the morphology of the dendritic arbor of hippocampal and cortical neurons. Cypin catalyzes the Zn2+-dependent deamination of guanine to xanthine, which is then metabolized to uric acid by xanthine oxidase. Cypin binds to tubulin heterodimers via its carboxyl terminal region (amino acids (aa) 350-454), which contains a collapsin response mediator protein (CRMP) homology domain (aa 350-403). Moreover, this region alone is not sufficient to facilitate microtubule polymerization; therefore, additional cypin regions must be involved in this process. Here, we asked whether cypin binds to fully formed microtubules and how overexpression of cypin regulates the microtubule cytoskeleton in dendrites of cultured hippocampal neurons. Protein-protein docking strategies confirm that the cypin homodimer binds to tubulin heterodimers via amino acids within aa 350-454. Biochemical pull-down data suggest that aa 1-220 are necessary for cypin binding to soluble tubulin heterodimers and to taxol-stabilized microtubules. Molecular docking of the cypin homodimer to soluble tubulin heterodimers reveals a consistently observed docking pose using aa 47-71, 113-118, 174-178, and 411-418, which is consistent with our biochemical data. Additionally, overexpression of cypin in hippocampal neurons results in decreased spacing between microtubules. Our results suggest that several protein domains facilitate cypin-mediated polymerization of tubulin heterodimers into microtubules, possibly through a mechanism whereby cypin dimers bind to multiple tubulin heterodimers.

Effects of Bioceramic Material and Colored Light Irradiation on Learning and Memory in Aging Rats.

Aging is characterized by molecular damage from free radicals, leading to neural dysfunction and memory impairment. This study investigated using bioceramic material and colored light to mitigate neurodegenerative symptoms in aging rats. We assessed the effects of different color light spectrums on D-galactose-induced aging rats using the Morris water maze, novel object recognition, and open field tests. Findings revealed that bioceramic material with various light wavelengths improved activity, recognition, and memory in aging rats. Significant enhancements were observed in the open field and novel object recognition tests, with a trend toward improvement in the Morris water maze. These effects are attributed to the antioxidant properties and microcirculation enhancement associated with bioceramic materials. Color stimulation may impact enzymes, human physiology, psychological activity, and the autonomic nervous system. This study highlights the significance of exploring novel interventions for neurodegenerative symptoms and memory deficits in aging rats. Results indicate that bioceramic material with different colored light spectrums positively influences cognitive function. These findings contribute to our understanding of the therapeutic potential of bioceramic materials and emphasize the need for further research in this area.

Tear Proteomics Approach to Monitoring Sjögren Syndrome or Dry Eye Disease.

Sjögren syndrome (SS) or dry eye disease (DED) is one of the most complicated ocular surface diseases. The goal of this study is to elucidate the relationship of the changes in clinical indices of tear film (TF) homeostasis with respect to tear components to allow for SS-DED monitoring and avoid stably controlled SS-DED patients from re-entering a vicious cycle. This prospective case-control study compared stable SS-DED patients with non-SS-DED control from several aspects, including clinical indices for TF homeostasis, 2 DED diagnostic biomarkers (MMP-9 and lactoferrin), and the proteome of flush tears. Compared with non-SS-DED controls, stably controlled SS-DED subjects had less tear secretion and higher ocular surface inflammation, a higher concentration ratio of tear MMP-9/lactoferrin, a more diverse tear proteome, and lower spectral intensities of lipocalin-1, lacritin, and prolactin-inducible protein among the abundant tear proteins. For stable SS-DED patients, the concentration ratio of tear MMP-9/lactoferrin and the corrected lipocalin-1 signal was positively correlated with ocular inflammation and TF stability, respectively. MMP-9 released from stressed ocular surface epithelium and lipocalin-1 secreted from the energetic lacrimal gland are two tear biomarkers responding well to TF homeostasis. The tear proteomics approach through flush tears is a promising method for monitoring SS-DED patients with a standardized sampling procedure and lactoferrin-corrected analysis.

Acute exposure to DEHP metabolite, MEHP cause genotoxicity, mutagenesis and carcinogenicity in mammalian Chinese hamster ovary cells.

Di-(2-ethylhexyl) phthalate (DEHP), the common plasticizer used in the production of polyvinyl chloride, can be converted to the more potent metabolite mono-ethylhexyl phthalate (MEHP). Epidemiological studies have shown an association with elevated induction of rat hepatic cancer and reproductive toxicity in response to MEHP exposure. However, the mechanism of genotoxicity and carcinogenicity induced by MEHP treatment remains unclear. As a means to elucidate the mechanisms of action, lethality and mutagenicity in the adenine phosphoribosyltransferase (aprt+/-) gene induced in several CHO cell types by MEHP were assessed. Dose-response relationships were determined in the parental AA8 cell line, its nucleotide repair-deficient UV5 and base repair-deficient EM9 subclones, and also in AS52 cells harboring the bacterial guanine-hypoxanthine phosphoribosyltransferase (gpt) gene and its derived AS52-XPD-knockdown and AS52-PARP-1-knockdown cells. Treatment of AS52 with MEHP led to intracellular production of reactive oxygen species (ROS) and DNA strand breaks in a dose-dependent manner. Separately, mutations in the gpt gene of AS52 cells were characterized and found to be dominated by G:C to A:T and A:T to G:C transitions. Independent AS52-mutant cell (ASMC) clones were collected for the sequential in vivo xenograft tumorigenic studies, 4 of total 20 clones had aggressive tumor growth. Moreover, microarray analysis indicated miR-let-7a and miR-125b downregulated in ASMC, which might raise oncogenic MYC and RAS level and activate ErbB pathway. Comparative evaluation of the results indicates that the principal mechanism of this mutagenic action is probably to be through generation of ROS, causing base excision damage resulting in carcinogenicity.

Exposure to PM2.5 causes genetic changes in fetal rat cerebral cortex and hippocampus.

PM2.5 travels along the respiratory tract and enters systemic blood circulation. Studies have shown that PM2.5 increases the incidence of various diseases not only in adults but also in newborn infants. It causes chronic inflammation in pregnant women and retards fetal development. In this study, pregnant rats were exposed to PM2.5 for extended periods of time and it was found that PM2.5 exposure increased immune cells in mother rats. In addition, cytokines and free radicals rapidly accumulated in the amniotic fluid and indirectly affected the fetuses. The authors collected cerebral cortex and hippocampus samples at E18 and analyzed changes of miRNA levels. Expression levels of cortical miR-6315, miR-3588, and miR-466b-5p were upregulated, and positively correlated with the genes Pkn2 (astrocyte migration), Gorab (neuritogenesis), and Mobp (allergic encephalomyelitis). In contrast, PM2.5 decreased expression of miR-338-5p and let-7e-5p, both related to mental development. Further, PM2.5 exposure increased miR-3560 and let-7b-5p in the hippocampus, two proteins that regulate genes Oxct1 and Lin28b that control ketogenesis and glycosylation, and neural cell differentiation, respectively. miR-99b-5p, miR-92b-5p, and miR-99a-5p were decreased, leading to reduced expression of Kbtbd8 and Adam11 which reduced cell mitosis, migration, and differentiation, and inhibited learning abilities and motor coordination of the fetus. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1412-1425, 2017.

Protective effects of ascorbic acid against the genetic and epigenetic alterations induced by 3,5-dimethylaminophenol in AA8 cells.

Exposure to monocyclic aromatic alkylanilines (MAAs), namely 2,6-dimethylaniline (2,6-DMA), 3,5-dimethylaniline (3,5-DMA) and 3-ethylaniline (3-EA), was significantly and independently associated with bladder cancer incidence. 3,5-DMAP (3,5-dimethylaminophenol), a metabolite of 3,5-DMA, was shown to induce an imbalance in cytotoxicity cellular antioxidant/oxidant status, and DNA damage in mammalian cell lines. This study was designed to evaluate the protective effect of ascorbic acid (Asc) against the cytotoxicity, reactive oxygen species (ROS) production, genotoxicity and epigenetic changes induced by 3,5-DMAP in AA8 Chinese Hamster Ovary (CHO) cells. In different cellular fractions, 3,5-DMAP caused alterations in the enzyme activities orchestrating a cellular antioxidant balance, decreases in reduced glutathione levels and a cellular redox ratio as well as increases in lipid peroxidation and protein oxidation. We also suggest that the cellular stress caused by this particular alkylaniline leads to both genetic (Aprt mutagenesis) and epigenetic changes in histones 3 and 4 (H3 and H4). This may further cause molecular events triggering different pathological conditions and eventually cancer. In both cytoplasm and nucleus, Asc provided increases in 3,5-DMAP-reduced glutathione levels and cellular redox ratio and decreases in the lipid peroxidation and protein oxidation. Asc was also found to be protective against the genotoxic and epigenetic effects initiated by 3,5-DMAP. In addition, Asc supplied protection against the cell cycle (G1 phase) arrest induced by this particular alkylaniline metabolite.

Ganoderma tsuage promotes pain sensitivity in aging mice.

Advances in modern medicine have extended human life expectancy, leading to a world with a gradually aging society. Aging refers to a natural decline in the physiological functions of a species over time, such as reduced pain sensitivity and reaction speed. Healthy-level physiological pain serves as a warning signal to the body, helping to avoid noxious stimuli. Physiological pain sensitivity gradually decreases in the elderly, increasing the risk of injury. Therefore, geriatric health care receives growing attention, potentially improving the health status and life quality of the elderly, further reducing medical burden. Health food is a geriatric healthcare choice for the elderly with Ganoderma tsuage (GT), a Reishi type, as the main product in the market. GT contains polysaccharides, triterpenoids, adenosine, immunoregulatory proteins, and other components, including anticancer, blood sugar regulating, antioxidation, antibacterial, antivirus, and liver and stomach damage protective agents. However, its pain perception-related effects remain elusive. This study thus aimed at addressing whether GT could prevent pain sensitivity reduction in the elderly. We used a galactose-induced animal model for aging to evaluate whether GT could maintain pain sensitivity in aging mice undergoing formalin pain test, hot water test, and tail flexes. Our results demonstrated that GT significantly improved the sensitivity and reaction speed to pain in the hot water, hot plate, and formalin tests compared with the control. Therefore, our animal study positions GT as a promising compound for pain sensitivity maintenance during aging.

The role of PSD-95 and cypin in morphological changes in dendrites following sublethal NMDA exposure.

Focal swelling or varicosity formation in dendrites and loss of dendritic spines are the earliest indications of glutamate-induced excitotoxicity. Although it is known that microtubule dynamics play a role in varicosity formation, very little is known about the proteins that directly impact microtubules during focal swelling and dendritic spine loss. Our laboratory has recently reported that the postsynaptic protein PSD-95 and its cytosolic interactor (cypin) regulate the patterning of dendrites in hippocampal neurons. Cypin promotes microtubule assembly, and PSD-95 disrupts microtubule organization. Thus, we hypothesized that cypin and PSD-95 may play a role in altering dendrite morphology and spine number in response to sublethal NMDA-induced excitotoxicity. Using an in vitro model of glutamate-induced toxicity in rat hippocampal cultures, we found that cypin overexpression or PSD-95 knockdown increases the percentage of neurons with varicosities and the number of varicosities along dendrites, decreases the size of varicosities after sublethal NMDA exposure, and protects neurons from NMDA-induced death. In contrast, cypin knockdown or PSD-95 overexpression results in opposite effects. We further show that cypin regulates the density of spines/filopodia: cypin overexpression decreases the number of protrusions per micrometer of dendrite while cypin knockdown results in an opposite effect. Cypin overexpression and PSD-95 knockdown attenuate NMDA-promoted decreases in protrusion density. Thus, we have identified a novel pathway by which the microtubule cytoskeleton is regulated during sublethal changes to dendrites.

PSD-95 alters microtubule dynamics via an association with EB3.

Little is known about how the neuronal cytoskeleton is regulated when a dendrite decides whether to branch or not. Previously, we reported that postsynaptic density protein 95 (PSD-95) acts as a stop signal for dendrite branching. It is yet to be elucidated how PSD-95 affects the cytoskeleton and how this regulation relates to the dendritic arbor. Here, we show that the SH3 (src homology 3) domain of PSD-95 interacts with a proline-rich region within the microtubule end-binding protein EB3. Overexpression of PSD-95 or mutant EB3 results in a decreased lifetime of EB3 comets in dendrites. In line with these data, transfected rat neurons show that overexpression of PSD-95 results in less organized microtubules at dendritic branch points and decreased dendritogensis. The interaction between PSD-95 and EB3 elucidates a function for a novel region of EB3 and provides a new and important mechanism for the regulation of microtubules in determining dendritic morphology.

Ganoderma tsugae prevents cognitive impairment and attenuates oxidative damage in d-galactose-induced aging in the rat brain.

Lingzhi has long been regarded as having life-prolonging effects. Research in recent years has also reported that Lingzhi possesses anti-tumor, anti-inflammatory, immunomodulatory, hepatoprotective, and anti-lipogenic effects. The D-galactose (D-gal, 100 mg/kg/day)-induced aging Long-Evans rats were simultaneously orally administered a DMSO extract of Ganoderma tsugae (GTDE, 200 µg/kg/day) for 25 weeks to investigate the effects of GTDE on oxidative stress and memory deficits in the D-galactose-induced aging rats. We found that GTDE significantly improved the locomotion and spatial memory and learning in the aging rats. GTDE alleviated the aging-induced reduction of dendritic branching in neurons of the hippocampus and cerebral cortex. Immunoblotting revealed a significant increase in the protein expression levels of the superoxide dismutase-1 (SOD-1) and catalase, and the brain-derived neurotrophic factor (BDNF) in rats that received GTDE. D-gal-induced increase in the lipid peroxidation product 4-hydroxynonenal (4-HNE) was significantly attenuated after the administration of GTDE, and pyrin domain-containing 3 protein (NLRP3) revealed a significant decrease in NLRP3 expression after GTDE administration. Lastly, GTDE significantly reduced the advanced glycosylation end products (AGEs). In conclusion, GTDE increases antioxidant capacity and BDNF expression of the brain, protects the dendritic structure of neurons, and reduces aging-induced neuronal damage, thereby attenuating cognitive impairment caused by aging.

Diagnosing Fungal Keratitis and Simultaneously Identifying Fusarium and Aspergillus Keratitis with a Dot Hybridization Array.

Fungal keratitis (FK) is one of the most common microbial keratitis, which often leads to poor prognosis as a result of delayed diagnosis. Several studies implied that early differentiation of the two major FK, Fusarium and Aspergillus keratitis, could be helpful in selecting effective anti-fungal regimens. Therefore, a novel dot hybridization array (DHA) was developed to diagnose FK and differentiate Fusarium and Aspergillus keratitis in this study. One hundred forty-six corneal scrapes obtained from one hundred forty-six subjects impressed with clinically suspected FK were used to evaluate the performance of the DHA. Among these patients, 107 (73.3%) patients had actual FK confirmed by culture and DNA sequencing. We found that the DHA had 93.5% sensitivity and 97.4% specificity in diagnosing FK. In addition, this array had 93.2% sensitivity and 93.8% specificity in diagnosing Fusarium keratitis, as well as 83.3% sensitivity and 100% specificity in diagnosing Aspergillus keratitis. Furthermore, it had 83.9% sensitivity and 100% specificity in identifying Fusarium solani keratitis. Thus, this newly developed DHA will be beneficial to earlier diagnosis, more precise treatment, and improve prognosis of FK, by minimizing medical refractory events and surgical needs.

Administration of Lactobacillus paracasei HB89 mitigates PM2.5-induced enhancement of inflammation and allergic airway response in murine asthma model.

PM2.5 causes abnormal immune response and asthma in animals. In this study, a Balb/c mouse animal model was exposed to PM2.5 to induce asthma. Lactobacillus paracasei HB89 was fed at the same time, in order to observe whether L. paracasei HB89 mitigates respiratory tract allergies stimulated by PM2.5. The results showed that PM2.5 stimulated a significant increase in white blood cells and immunoglobulin (IgE) in OVA-induced allergic Balb/c mice, and IgE in the blood further triggered the release of histamine in the lung immune cells. This not only increased overall immune cell counts, but the lymphocyte counts also increased significantly, resulting in significant inhibitions of cytokines INF-r and TGF-ß, and induction of IL-4, IL-5, IL-13 and IL-17a. After feeding with HB89, apart from the absence of observable changes in body weight, the total white blood cell count in the animal blood and IgE response were also be reduced; the proliferation of immune cells in the lungs caused by PM2.5 was slowed down; and histamine and cytokines INF-r and TGF-ß were secreted in large quantities, but IL- 4, IL-5, IL-13, IL-17a were inhibited, which effectively reduced the possibility of asthma induction.

In Vitro and In Vivo Analysis of the Effects of 3,5-DMA and Its Metabolites in Neural Oxidative Stress and Neurodevelopmental Toxicity.

3,5-Dimethylaniline (3,5-DMA), a monocyclic aromatic amine, is widely present in a spectrum of sources including tobacco, dyes, combustion products, and suspended particulates. 3,5-DMA and its metabolites form superoxides, resulting in apoptosis or oncogenesis. Data of a direct effect of 3,5-DMA on the nervous system, especially the developing brain, are lacking. Therefore, we investigated the effects of 3,5-DMA and its metabolites on fetal neurite growth and brain development using in vitro cell cultures of primary cortical neurons to observe whether these compounds caused neuronal cytotoxicity and affected neurite structural development. With increasing concentrations of 3,5-DMA (10, 50, 100, 500, 1000 µM) and its major metabolite 5-dimethylaminophenol (3,5-DMAP) (10, 50, 100, 500, 1000 µM), reactive oxygen species (ROS), cytotoxicity, and DNA damage increased significantly in the cells and dendritic arborization decreased. The addition of 5 mM N-acetylcysteine, an ROS scavenger, reduced ROS in the cells and alleviated the neuronal damage. In vivo studies in Sprague Dawley pregnant rats suggested that exposure to 3,5-DMA (10, 30, 60, 100 mg/kg/day) subcutaneously from GD15 to GD17 led to fetal cerebral cortex thinning. BrdU labeling showed that 3,5-DMA reduced the number and generation of cortical cells. To detect the laminar position of newly generated neurons, cortex layer markers such as Satb2, Ctip2, and Tbr1 were used. 3,5-DMA perturbed the cortical layer distribution in developing fetal rats. In summary, this is the first study to provide evidence for 3,5-DMA and its metabolites causing anomalies of the fetal central nervous system development through ROS production.

Antioxidants and selenocompounds inhibit 3,5-dimethylaminophenol toxicity to human urothelial cells.

Exposure to alkyl anilines may lead to bladder cancer, which is the second most frequent cancer of the urogenital tract. 3,5-dimethylaniline is highly used in industry. Studies on its primary metabolite 3,5-dimethylaminophenol (3,5-DMAP) showed that this compound causes oxidative stress, changes antioxidant enzyme activities, and leads to death of different mammalian cells. However, there is no in vitro study to show the direct effects of 3,5-DMAP on human bladder and urothelial cells. Selenocompounds are suggested to decrease oxidative stress caused by some chemicals, and selenium supplementation was shown to reduce the risk of bladder cancer. The main aim of this study was to investigate whether selenocompounds organic selenomethionine (SM, 10 µmol/L) or inorganic sodium selenite (SS, 30 nmol/L) could reduce oxidative stress, DNA damage, and apoptosis in UROtsa cells exposed to 3,5-DMAP. 3,5-DMAP caused a dose-dependent increase in intracellular generation of reactive oxygen species, and its dose of 50 µmol/L caused lipid peroxidation, protein oxidation, and changes in antioxidant enzyme activities in different cellular fractions. The comet assay also showed single-strand DNA breaks induced by the 3,5-DMAP dose of 50 µmol/L, but no changes in double-strand DNA breaks. Apoptosis was also triggered. Both selenocompounds provided partial protection against the cellular toxicity of 3,5-DMAP. Low selenium status along with exposure to alkyl anilines can be a major factor in the development of bladder cancer. More mechanistic studies are needed to specify the role of selenium in bladder cancer.

The Effect of Ganoderma Microsporum immunomodulatory proteins on alleviating PM2.5-induced inflammatory responses in pregnant rats and fine particulate matter-induced neurological damage in the offsprings.

Fine particulate matter 2.5 (PM2.5) induces free radicals and oxidative stress in animals, leading to a range of illnesses. In this study, Ganoderma Microsporum immunomodulatory (GMI) proteins were administered to alleviate PM2.5-induced inflammatory responses in mother rats, and PM2.5-induced inflammatory responses and neurological damage in their offspring. The results suggested that GMI administration decreased the risk of neurological disorders in mother rats and their offspring by reducing the white blood cell count, lessening inflammatory responses and PM2.5-induced memory impairment, and preventing dendritic branches in the hippocampi from declining and microRNAs from PM2.5-induced modulation.

Anti-cancer effects of 3,5-dimethylaminophenol in A549 lung cancer cells.

Exposure to 3,5-dimethylaminophenol (3,5-DMAP), the metabolite of the 3-5-dimethylaniline, was shown to cause high levels of oxidative stress in different cells. The aim of the present work was to observe whether this metabolite can lead to cytotoxicity, oxidative stress, DNA damage and cell cycle changes in non-small cell lung cancer A549 cells. 3,5-DMAP caused a dose-dependent increase in cytotoxicity, generation of superoxide (O2-.), inductions in the enzyme activities orchestrating cellular antioxidant balance, increases in lipid peroxidation as well as DNA damage. However, 3,5-DMAP showed significantly lower cytotoxicity towards human lung fibroblast (HLF) cells. 3,5-DMAP also led to molecular events, like inducing apoptotic markers (ie. p53, Bad, Bax and cytochrome c); decreasing anti-apoptotic proteins (Bcl-2) and alterations in cell cycle. Our findings indicate that the cytotoxicity caused by this particular alkylaniline metabolite led to initiation of caspase 3-mediated apoptosis. Furthermore, 3,5-DMAP attenuated carcinogenic properties like migration capacity of A549 cells and eventually inhibited growth of A549 cells in an in vivo mouse model. Tumor sections showed that 3,5-DMAP down-regulated c-Myc expression but up-regulated p53 and cytochrome c, all of which might result in tumor growth arrest. Co-treatment with N-acetylcysteine provided reductions in cytotoxicity and positively modulated genetic events induced by 3,5-DMAP in A549 cells. In conclusion, our findings demonstrate 3,5-DMAP may be a potential anti-cancer drug in cancer, due to its self redox cycling properties.

Causation by Diesel Exhaust Particles of Endothelial Dysfunctions in Cytotoxicity, Pro-inflammation, Permeability, and Apoptosis Induced by ROS Generation.

Epidemiological studies suggest that an increase of diesel exhaust particles (DEP) in ambient air corresponds to an increase in hospital-recorded myocardial infarctions within 48 h after exposure. Among the many theories to explain this data are endothelial dysfunction and translocation of DEP into vasculature. The mechanisms for such DEP-induced vascular permeability remain unknown. One of the major mechanisms underlying the effects of DEP is suggested to be oxidative stress. Experiments have shown that DEP induce the generation of reactive oxygen species (ROS), such as superoxide anion and H2O2 in the HUVEC tube cells. Transcription factor Nrf2 is translocated to the cell nucleus, where it activates transcription of the antioxidative enzyme HO-1 and sequentially induces the release of vascular permeability factor VEGF-A. Furthermore, a recent study shows that DEP-induced intracellular ROS may cause the release of pro-inflammatory TNF-α and IL-6, which may induce endothelial permeability as well by promoting VEGF-A secretion independently of HO-1 activation. These results demonstrated that the adherens junction molecule, VE-cadherin, becomes redistributed from the membrane at cell-cell borders to the cytoplasm in response to DEP, separating the plasma membranes of adjacent cells. DEP were occasionally found in endothelial cell cytoplasm and in tube lumen. In addition, the induced ROS is cytotoxic to the endothelial tube-like HUVEC. Acute DEP exposure stimulates ATP depletion, followed by depolarization of their actin cytoskeleton, which sequentially inhibits PI3K/Akt activity and induces endothelial apoptosis. Nevertheless, high-dose DEP augments tube cell apoptosis up to 70 % but disrupts the p53 negative regulator Mdm2. In summary, exposure to DEP affects parameters influencing vasculature permeability and viability, i.e., oxidative stress and its upregulated antioxidative and pro-inflammatory responses, which sequentially induce vascular permeability factor, VEGF-A release and disrupt cell-cell junction integrity. While exposure to a low dose of DEP actin triggers cytoskeleton depolarization, reduces PI3K/Akt activity, and induces a p53/Mdm2 feedback loop, a high dose causes apoptosis by depleting Mdm2. Addition of ROS scavenger N-acetyl cysteine suppresses DEP-induced oxidative stress efficiently and reduces subsequent damages by increasing endogenous glutathione.

Diesel Exhaust Particles Contribute to Endothelia Apoptosis via Autophagy Pathway.

Epidemiological studies suggest that an increase of PM2.5 diesel exhaust particles (DEP) in ambient air corresponds to increased myocardial infarctions and atherosclerosis. When exposed to DEP, endothelial cells exhibit increases in oxidative stress and apoptosis, but the role of autophagy in this DEP-induced cell death remains unclear. Here, we suggest that acute DEP exposure produces intracellular reactive oxygen species (ROS) leading to induction of DEP internalization, endothelial dysfunction, and pro-inflammation in an in vitro human umbilical vein endothelial cells (HUVEC) model. This study found that increases in intracellular oxidative stress and cellular internalization of DEP occurred within 2 h of exposure to DEP. After 2 h of DEP exposure, Mdm2 expression was increased, which triggered cellular autophagy after 4 h of DEP exposure and suppressed cellular senescence. Unfortunately, phagocytized DEP could not be eliminated by cellular autophagy, which led to a continuous buildup of ROS, an increased release of cytokines, and an increased expression of anchoring molecules. After 12 h of DEP exposure, HUVEC reduced Mdm2 expression leading to increased p53 expression, which triggered apoptosis and ultimately resulted in endothelial dysfunction. On the other hand, when cells lacked the ability to induce autophagy, DEP was unable to induce cell senescence and most of the cells survived with only a small percentage of the cells undergoing necrosis. The results presented in this study clearly demonstrate the role cellular autophagy plays in DEP-induced atherosclerosis.