Protective effects of 2,4-dinitrophenol in okadaic acid-induced cellular model of Alzheimer's disease.

Alzheimer's disease (AD) research started several decades ago and despite the many efforts employed to develop new treatments or approaches to slow and/or revert disease progression, AD treatment remains an unsolved issue. Knowing that mitochondria loss of function is a central hub for many AD-associated pathophysiological processes, there has been renewed interest in exploring mitochondria as targets for intervention. In this perspective, the present study was aimed to investigate the possible beneficial effects of 2,4 dinitrophenol (DNP), a mitochondrial uncoupler agent, in an in vitro model of AD. Retinoic acid-induced differentiated SH-SY5Y cells were incubated with okadaic acid (OA), a neurotoxin often used as an AD experimental model, and/or with DNP. OA caused a decrease in neuronal cells viability, induced multiple mitochondrial anomalies including increased levels of reactive oxygen species, decreased bioenergetics and mitochondria content markers, and an altered mitochondria morphology. OA-treated cells also presented increased lipid peroxidation levels, and overactivation of tau related kinases (GSK3ß, ERK1/2 and AMPK) alongside with a significant augment in tau protein phosphorylation levels. Interestingly, DNP co-treatment ameliorated and rescued OA-induced detrimental effects not only on mitochondria but also but also reinstated signaling pathways homeostasis and ameliorated tau pathology. Overall, our results show for the first time that DNP has the potential to preserve mitochondria homeostasis under a toxic insult, like OA exposure, as well as to reestablish cellular signaling homeostasis. These observations foster the idea that DNP, as a mitochondrial modulator, might represent a new avenue for treatment of AD.

The 2-oxoglutarate/malate carrier extends the family of mitochondrial carriers capable of fatty acid and 2,4-dinitrophenol-activated proton transport.

AIMS: Metabolic reprogramming in cancer cells has been linked to mitochondrial dysfunction. The mitochondrial 2-oxoglutarate/malate carrier (OGC) has been suggested as a potential target for preventing cancer progression. Although OGC is involved in the malate/aspartate shuttle, its exact role in cancer metabolism remains unclear. We aimed to investigate whether OGC may contribute to the alteration of mitochondrial inner membrane potential by transporting protons. METHODS: The expression of OGC in mouse tissues and cancer cells was investigated by PCR and Western blot analysis. The proton transport function of recombinant murine OGC was evaluated by measuring the membrane conductance (Gm) of planar lipid bilayers. OGC-mediated substrate transport was measured in proteoliposomes using 14C-malate. RESULTS: OGC increases proton Gm only in the presence of natural (long-chain fatty acids, FA) or chemical (2,4-dinitrophenol) protonophores. The increase in OGC activity directly correlates with the increase in the number of unsaturated bonds of the FA. OGC substrates and inhibitors compete with FA for the same protein binding site. Arginine 90 was identified as a critical amino acid for the binding of FA, ATP, 2-oxoglutarate, and malate, which is a first step towards understanding the OGC-mediated proton transport mechanism. CONCLUSION: OGC extends the family of mitochondrial transporters with dual function: (i) metabolite transport and (ii) proton transport facilitated in the presence of protonophores. Elucidating the contribution of OGC to uncoupling may be essential for the design of targeted drugs for the treatment of cancer and other metabolic diseases.

2,4-dinitrophenol enhances cisplatin and etoposide cytotoxic activity on prostate cancer cell line.

INTRODUCTION AND OBJECTIVE: Including additional compounds that disturb the energy metabolism of cancer cells in advanced cancer therapy regimens may be an approach to overcome the problem of drug resistance and the therapeutic effectiveness of classic chemotherapeutics. One of the compounds that decouple oxidative phosphorylation, and thus alter the activity of energy-producing pathways, is 2,4-DNP (2,4- dinitrophenol). OBJECTIVE: The aim of the study was to assess the ability of the 2,4-DNP to sensitize prostate cancer cells to the action of cisplatin and etoposide, or to intensify their action. MATERIAL AND METHODS: The research was carried out on three prostate cancer cell lines (LNCaP, PC-3, DU-145. To assess the effect of cisplatin or etoposide with 2,4-DNP on prostate cancer cells, MTT assay, analysis of the cell cycle and apoptosis detection was performed. Oxidative stress was investigated by CellRox fluorescence staining and expression of genes related to antioxidant defence. In addition, analysis was conducted of the expression of genes related to cell cycle inhibition, transporters associated with multi-drug resistance and DNA repair. RESULTS: The study showed that the simultaneous incubation of 2,4-DNP with cisplatin or etoposide enhances the cytotoxic effect of the chemotherapeutic agent only in LNCaP cells (oxidative phenotype). CONCLUSIONS: The enhanced cytotoxic effect of chemotherapeutics by 2,4-DNP may be the result of disturbed redox balance, reduced ability of cells to repair DNA, and the oxidative metabolic phenotype of prostate cancer cells.

Pollution characteristics, sources, and health risks of phthalate esters in ambient air: A daily continuous monitoring study in the central Chinese city of Nanchang.

In recent years, the atmospheric pollution caused by phthalate esters (PAEs) has been increasing due to the widespread use of PAE-containing materials. Existing research on atmospheric PAEs lacks long-term continuous observation and samples from cities in central China. To investigate the pollution characteristics, sources, and health risks of PAEs in the ambient air of a typical city in central China, daily PM2.5 samples were collected in Nanchang from November 2020 to October 2021. In this study, the detection and quantification of six significant PAE contaminants, namely diethyl phthalate (DEP), di-n-butyl phthalate (DnBP), diisobutyl phthalate (DIBP), Di-2-ethylhexyl phthalate (DEHP), di-n-octyl phthalate (DnOP), and diisodecyl phthalate (DIDP), were accomplished using gas chromatography and mass spectrometry. The results revealed that the concentrations of DEP, DnBP, DEHP, and DnOP were relatively high. Higher temperatures promote the volatilization of PAEs, leading to an increase in the gaseous and particulate PAE concentrations in warm seasons and winter pollution scenarios. The results of principal component analysis show that PAEs mainly come from volatile products and polyvinylchloride plastics. Using positive matrix factorization analysis, it is shown that these two sources contribute 67.0% and 33.0% in atmosphere PAEs, respectively. Seasonally, the contribution of volatile products to both gaseous and particulate PAEs substantially increases during warm seasons. The residents in Nanchang exposed to PAEs have a negligible non-cancer risk and a potential low cancer risk. During the warm seasons, more PAEs are emitted into the air, which will increase the toxicity of PAEs and their impact on human health.

Dysregulation of CITED2 in abnormal lung development in the nitrofen rat model.

PURPOSE: CITED2 both modulates lung, heart and diaphragm development. The role of CITED2 in the pathogenesis of congenital diaphragmatic hernia (CDH) is unknown. We aimed to study CITED2 during abnormal lung development in the nitrofen model. METHODS: Timed-pregnant rats were given nitrofen on embryonic day (E) 9 to induce CDH. Fetal lungs were harvested on E15, 18 and 21. We performed RT-qPCR, RNAscope™ in situ hybridization and immunofluorescence staining for CITED2. RESULTS: We observed no difference in RT-qPCR (control: 1.09 ± 0.22 and nitrofen: 0.95 ± 0.18, p = 0.64) and in situ hybridization (1.03 ± 0.03; 1.04 ± 0.03, p = 0.97) for CITED2 expression in E15 nitrofen and control pups. At E18, CITED2 expression was reduced in in situ hybridization of nitrofen lungs (1.47 ± 0.05; 1.14 ± 0.07, p = 0.0006), but not altered in RT-qPCR (1.04 ± 0.16; 0.81 ± 0.13, p = 0.33). In E21 nitrofen lungs, CITED2 RNA expression was increased in RT-qPCR (1.04 ± 0.11; 1.52 ± 0.17, p = 0.03) and in situ hybridization (1.08 ± 0.07, 1.29 ± 0.04, p = 0.02). CITED2 protein abundance was higher in immunofluorescence staining of E21 nitrofen lungs (2.96 × 109 ± 0.13 × 109; 4.82 × 109 ± 0.25 × 109, p < 0.0001). CONCLUSION: Our data suggest that dysregulation of CITED2 contributes to abnormal lung development of CDH, as demonstrated by the distinct spatial-temporal distribution in nitrofen-induced lungs.

Phenolic compounds in water: Review of occurrence, risk, and retention by membrane technology.

Phenolic compounds are one of the main contributors to water source contamination worldwide. In this review, the data collected on Elsevier, Scopus, and Pubmed, considering papers published between 2000 and 2023, showed more than 60 different phenols have been identified in water matrix (<0.065-179,000,000 ng L-1). The highest concentration reported was in surface water canals in India. The most recurrent and studied compound was bisphenol A (n = 93) in concentrations ranging from 0.45 to 2,970,000 ng L-1. The solid phase extraction (HBL Oasis cartridge) and methanol as solvent was the method of pre-concentration most used followed by gas chromatography for the determination of phenols in water samples. The importance of drinking water guidelines incorporating more phenolic compounds was emphasized given the variety of these compounds quantified in water matrix. The human health risk assessment (HRA) was performed for the min-max concentrations of the pollutants reported in the literature. High HRA even at the lowest concentrations for 2-nitrophenol, 2,6-dichlorophenol, 3,4,5-trichlorophenol, 2,3,4,6-tetrachlorophenol, and 2,4-dinitrophenol was recognized. The cancer risk estimated was considered possible for 3-methylphenol, 2,4-dimethylphenol, 2,4,6-trichlorophenol, pentachlorophenol, and 2,4-dinitrophenol in the highest concentrations. The in-depth discussion of mechanisms, advantages, challenges, and carbon footprint of membrane technologies in water treatment and phenols retention demonstrated the great potential and trends for the production of safe drinking water, highlighting reverse osmosis, as a mature technology, and membrane distillation, as an emergent technology.

Structural basis for the binding of DNP and purine nucleotides onto UCP1.

Uncoupling protein 1 (UCP1) conducts protons through the inner mitochondrial membrane to uncouple mitochondrial respiration from ATP production, thereby converting the electrochemical gradient of protons into heat1,2. The activity of UCP1 is activated by endogenous fatty acids and synthetic small molecules, such as 2,4-dinitrophenol (DNP), and is inhibited by purine nucleotides, such as ATP3-5. However, the mechanism by which UCP1 binds to these ligands remains unknown. Here we present the structures of human UCP1 in the nucleotide-free state, the DNP-bound state and the ATP-bound state. The structures show that the central cavity of UCP1 is open to the cytosolic side. DNP binds inside the cavity, making contact with transmembrane helix 2 (TM2) and TM6. ATP binds in the same cavity and induces conformational changes in TM2, together with the inward bending of TM1, TM4, TM5 and TM6 of UCP1, resulting in a more compact structure of UCP1. The binding site of ATP overlaps with that of DNP, suggesting that ATP competitively blocks the functional engagement of DNP, resulting in the inhibition of the proton-conducting activity of UCP1.

Pollution and risk assessment of phenolic compounds in drinking water sources from South-Western Nigeria.

This study reports the occurrence and risk assessment of 2,4-dinitrophenol (2,4-DNP), phenol (PHE), and 2,4,6-trichlorophenol (2,4,6-TCP) in drinking water sources in three south-western States in Nigeria (Osun, Oyo, and Lagos). Groundwater (GW) and surface water (SW) were collected during dry and rainy seasons of a year. The detection frequency of the phenolic compounds followed the trend Phenol > 2,4-DNP > 2,4,6-TCP. The mean concentrations of 2,4-DNP, Phenol, and 2,4,6-TCP in GW/SW samples from Osun State were 639/553 µg L-1, 261/262 µg L-1, and 169/131 µg L-1 during the rainy season and 154/7 µg L-1, 78/37 µg L-1, and 123/15 µg L-1 during the dry season, respectively. In Oyo State, the mean concentrations were 165/391 µg L-1 for 2,4-DNP and 71/231 µg L-1 for Phenol in GW/SW samples, respectively, during the rainy season. Generally, in the dry season, these values decreased. In any case, these concentrations are higher than those previously reported in water from other countries. The concentration of 2,4-DNP in water posed serious ecological risks to Daphnia on the acute scale while it was algae on the chronic scale. Estimated daily intake and hazard quotient calculations suggest that 2,4-DNP and 2,4,6-TCP in water pose serious toxicity concerns to humans. Additionally, the concentration of 2,4,6-TCP in water from Osun State in both seasons of the year and in both groundwater and surface water poses significant carcinogenic risks to persons ingesting water from these sources in the State. Every exposure group studied were at risk from ingesting these phenolic compounds in water. However, this risk decreased with increasing age of the exposure group. Results from the principal component analysis indicate that 2,4-DNP in water samples is from an anthropogenic source different from that for Phenol and 2,4,6-TCP. There is a strong need to treat water from GW and SW systems in these States before ingesting while assessing their quality regularly.

Disruption of mitochondrial function augments the radiosensitivity of prostate cancer cell lines.

INTRODUCTION: Ionizing radiation is one of the most widely used therapeutic methods in the treatment of prostate cancer, but the problem is developing radioresistance of the tumour. There is evidence that metabolic reprogramming in cancer is one of the major contributors to radioresistance and mitochondria play a crucial role in this process. OBJECTIVE: The aim of the study was to assess the influence of oxidative phosphorylation uncoupling to radiosensitivity of prostate cancer cells differing in metabolic phenotype. MATERIAL AND METHODS: LNCaP, PC-3 and DU-145 cells were exposed to X-rays and simultaneously treated with 2,4-dinitrophenol (2,4-DNP). The radiosensitive of cell lines was determined by cell clonogenic assay and cell cycle analysis. The cytotoxic effect was evaluated with MTT and CVS (Crystal violet staining) assay, apoptosis detection and cell cycle analysis. The phenotype of the cells was determined by glucose uptake and lactate release, ATP level measurement as well as basal reactive oxygen species level and mRNA expression of genes related to oxidative stress defence. RESULTS: The synergistic effect of 2,4-dinitrophenol and X-ray was observed only in the case of the LNCaP cell line. CONCLUSIONS: Phenotypic analysis indicates that this may be due to the highest dependence of these cells on oxidative phosphorylation and sensitivity to disruption of their redox status.

The relationship between experimental 2,4-Dinitrophenol administration and neurological oxidative stress: in terms of dose, time and gender differences.

Although 2,4-DNP is claimed to promote fast weight reduction, it is also related with an intolerable high risk of serious side effects to various tissues. On the other hand, it is known to have neuroprotective effects. These different effects of 2,4-DNP may be due to the administration conditions. For this reason, in this study, it was aimed for the first time to clarify the oxidative changes that occur in the brain during the use of 2,4-DNP, depending on the dose, time and gender. For this purpose, 60 Wistar rats (30 male, 30 female) were divided into ten groups: control groups, short-term/long-term groups and low dose/high dose groups. Except for the control groups, 2,4-DNP was administered to the other groups by oral gavage. End of the experiment, thiobarbituric acid-reactive substances (TBARs), glutathione (GSH), nitric oxide (NOx) and ascorbic acid (AA) levels were measured in the brain tissues of sacrificed animals. 2,4-DNP administration showed attenuation impact on oxidative stress depending on both dose, time and gender. It can be said that it is more beneficial in terms of neuroprotection, especially in the short-term and male groups. In conclusion, our findings suggest that, depending on the dose, time, and gender, 2,4-DNP may be beneficial in the treatment of neurodegenerative disorders.

2,4-Dinitrophenol as an Uncoupler Augments the Anthracyclines Toxicity against Prostate Cancer Cells.

One of the strategies for the treatment of advanced cancer diseases is targeting the energy metabolism of the cancer cells. The compound 2,4-DNP (2,4-dinitrophenol) disrupts the cell energy metabolism through the ability to decouple oxidative phosphorylation. The aim of the study was to determine the ability of 2,4-DNP to sensitize prostate cancer cells with different metabolic phenotypes to the action of known anthracyclines (doxorubicin and epirubicin). The synergistic effect of the anthracyclines and 2,4-DNP was determined using an MTT assay, apoptosis detection and a cell cycle analysis. The present of oxidative stress in cancer cells was assessed by CellROX, the level of cellular thiols and DNA oxidative damage. The study revealed that the incubation of LNCaP prostate cancer cells (oxidative phenotype) with epirubicin and doxorubicin simultaneously with 2,4-DNP showed the presence of a synergistic effect for both the cytostatics. Moreover, it contributes to the increased induction of oxidative stress, which results in a reduced level of cellular thiols and an increased number of AP sites in the DNA. The synergistic activity may consist of an inhibition of ATP synthesis and the simultaneous production of toxic amounts of ROS, destroying the mitochondria. Additionally, the sensitivity of the LNCaP cell line to the anthracyclines is relatively higher compared to the other two (PC-3, DU-145).

Coupling of membrane-based bubbleless micro-aeration for 2,4-dinitrophenol degradation in a hydrolysis acidification reactor.

Micro-aeration hydrolysis acidification (HA) is an effective method to enhance the removal of toxic and refractory organic matter, but the difficulty in stable dosing control of trace oxygen limits its wide application. Membrane-based bubbleless aeration has been proved as an ideal aeration method because of its higher oxygen transfer rate, more uniform mass transfer, and lower cost than HA. However, the available information on its application in HA is limited. In this study, membrane-based bubbleless micro-aeration coupled with hydrolysis acidification (MBL-MHA) was exploited to investigate the performance of 2,4-dinitrophenol (2,4-DNP) degradation via comparing it with bubble micro-aeration HA (MHA) and anaerobic HA. The results indicated that the performances in MBL-MHA and MHA were higher than those in HA during the experiment. 2,4-DNP degradation rates under redox microenvironments caused by counter-diffusion in MBL-MHA (84.43∼97.28%) were higher than those caused by co-diffusion in MHA (82.41∼94.71%) under micro-aeration of 0.5-5.0 mL air/min. The 2,4-DNP degradation pathways in MBL-MHA were nitroreduction, deamination, aromatic ring cleavage, and fermentation, while those in MHA were hydroxylation, aromatic ring cleavage, and fermentation. Reduction/oxidation-related, interspecific electron transfer-related species, and fermentative species in MBL-MHA were more abundant than that in MHA. Ultimately, more reducing/oxidizing forces formed by more redox proteins/enzymes from these rich species could enhance 2,4-DNP degradation in MBL-MHA.

A universal dual mechanism immunotherapy for the treatment of influenza virus infections.

Seasonal influenza epidemics lead to 3-5 million severe infections and 290,000-650,000 annual global deaths. With deaths from the 1918 influenza pandemic estimated at >50,000,000 and future pandemics anticipated, the need for a potent influenza treatment is critical. In this study, we design and synthesize a bifunctional small molecule by conjugating the neuraminidase inhibitor, zanamivir, with the highly immunogenic hapten, dinitrophenyl (DNP), which specifically targets the surface of free virus and viral-infected cells. We show that this leads to simultaneous inhibition of virus release, and immune-mediated elimination of both free virus and virus-infected cells. Intranasal or intraperitoneal administration of a single dose of drug to mice infected with 100x MLD50 virus is shown to eradicate advanced infections from representative strains of both influenza A and B viruses. Since treatments of severe infections remain effective up to three days post lethal inoculation, our approach may successfully treat infections refractory to current therapies.

Bicarbonate suppresses mitochondrial membrane depolarization induced by conventional uncouplers.

Bicarbonate has been known to modulate activities of various mitochondrial enzymes such as ATPase and soluble adenylyl cyclase. Here, we found that the ability of conventional protonophoric uncouplers, such as 2,4-dinitrophenol (DNP), carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), but not that of the new popular uncoupler BAM15, to decrease mitochondrial membrane potential was significantly diminished in the presence of millimolar concentrations of bicarbonate. Thus, the depolarizing activity of DNP and FCCP in mitochondria could be sensitive to the local concentration of bicarbonate in cells and tissues. However, bicarbonate could not restore the ATP synthesis suppressed by DNP or CCCP in mitochondria. Bicarbonate neither altered the depolarizing action of DNP and FCCP on proteoliposomes with reconstituted cytochrome c oxidase, nor affected the protonophoric activity of DNP and FCCP in artificial lipid membranes as measured with pyranine-loaded liposomes, thereby showing that the bicarbonate-induced reversal of the depolarizing action of DNP and FCCP on mitochondria did not result from direct interaction of bicarbonate with the uncouplers.

A Comparative Study of the Action of Protonophore Uncouplers and Decoupling Agents as Inducers of Free Respiration in Mitochondria in States 3 and 4: Theoretical and Experimental Approaches.

Theoretical and experimental studies have revealed that that in the liver mitochondria an increase in the rate of free respiration in state 3 induced by protonophore uncouplers 2,4-dinitrophenol and сarbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone is equal to or slightly greater than the increase in respiration rate in state 4 induced by these uncouplers. In contrast to these protonophore uncouplers, the decoupler α,ω-tetradecanedioic acid, increasing the rate of respiration in state 4, does not significantly affect the rate of free respiration in state 3. We have proposed quantitative indicators that allow determining the constituent part of the rate of respiration in state 4, associated with the decoupling effect of the uncoupler. Using the example of palmitic acid, we have found out the fundamental possibility of the simultaneous functioning of uncouplers by two mechanisms: as protonophores and as decouplers. The data obtained contradict the delocalized version of Mitchell's chemiosmotic theory, but are in complete agreement with its local version. It can be assumed that the F0F1-ATP synthase and nearby respiratory chain complexes form a local zone of coupled respiration and oxidative ATP synthesis (zones of oxidative phosphorylation). The uncoupler-induced stimulation of mitochondrial free respiration of mitochondria in state 3 is mainly due to the return of protons to the matrix in local zones, where the generation of a proton motive force (Δр) by respiratory chain complexes is associated with various transport processes, but not with ATP synthesis (zones of protonophore uncoupling). In contrast, respiratory stimulation in state 4 by decouplers is realized in local zones of oxidative phosphorylation by switching the respiratory chain complexes to the idle mode of operation in the absence of ATP synthesis.

Usnic acid as calcium ionophore and mast cells stimulator.

Usnic acid (UA), a secondary lichen metabolite, has long been popular as one of natural fat-burning dietary supplements. Similar to 2,4-dinitrophenol, the weight-loss effect of UA is assumed to be associated with its protonophoric uncoupling activity. Recently, we have shown that the ability of UA to shuttle protons across both mitochondrial and artificial membranes is strongly modulated by the presence of calcium ions in the medium. Here, by using fluorescent probes, we studied the calcium-transporting capacity of usnic acid in a variety of membrane systems comprising liposomes, isolated rat liver mitochondria, erythrocytes and rat basophilic leukemia cell culture (RBL-2H3). At concentrations of tens of micromoles, UA appeared to be able to carry calcium ions across membranes in all the systems studied. Similar to the calcium ionophore A23187, UA caused degranulation of RBL-2H3 cells. Therefore, UA, being a protonophoric uncoupler of oxidative phosphorylation, at higher concentrations manifests itself as a calcium ionophore, which could be relevant to its overdose toxicity in humans and also its phytotoxicity.

Airborne phthalates in indoor environment: Partition state and influential built environmental conditions.

Exposure to phthalates has recently become a major public health concern. The information of indoor airborne phthalates and their air-particle partition in real indoor environmental condition is still limited. In this study, the gas- and PM2.5-concentrations of 7 phthalates in 40 residences were concurrently measured in summer and winter. The major phthalates (median concentration in the summer and winter, respectively) in indoor air were DMP (2442.3 and 2403.4 ng/m3), DiBP (801.0 and 640.0 ng/m3) and DnBP (5173.2 and 1379.6 ng/m3), whereas the major phthalates in PM2.5 were DiBP (1055.1 and 585.9 ng/m3) and DnBP (1658.5 and 1517.0 ng/m3) and DEHP (215.1 and 344.9 ng/m3). Air-PM2.5 partition coefficients (Kp) of DiBP, DnBP and DEHP were calculated: the summer and winter median values (m3/µg) were 0.053 and 0.011 for DiBP, 0.010 and 0.004 for DnBP, 0.021 and 0.025 for DEHP, respectively. Air-PM2.5 partition of DiBP and DnBP approached equilibrium, while that of DEHP did not reach equilibrium in either season. The impacts of built environmental conditions on phthalate concentrations were characterized. Elevated temperature resulted in accumulation of airborne phthalates. Higher air humidity led to more water absorption of aerosols in summer, facilitated mass transfer of phthalates from air to PM2.5, and resulted in greater Kp of DiBP and DnBP in the summer. Any factors such as proximity to local traffic highway and indoor smoking activities, which can increase indoor PM2.5 concentrations, resulted in significantly higher airborne phthalate concentrations. Improving ventilation was not an effective measure to reduce indoor airborne phthalate concentrations.

Simultaneous Inhibition of Glycolysis and Oxidative Phosphorylation Triggers a Multi-Fold Increase in Secretion of Exosomes: Possible Role of 2'3'-cAMP.

Exosome secretion by cells is a complex, poorly understood process. Studies of exosomes would be facilitated by a method for increasing their production and release. Here, we present a method for stimulating the secretion of exosomes. Cultured cells were treated or not with sodium iodoacetate (IAA; glycolysis inhibitor) plus 2,4-dinitrophenol (DNP; oxidative phosphorylation inhibitor). Exosomes were isolated by size-exclusion chromatography and their morphology, size, concentration, cargo components and functional activity were compared. IAA/DNP treatment (up to 10 µM each) was non-toxic and resulted in a 3 to 16-fold increase in exosome secretion. Exosomes from IAA/DNP-treated or untreated cells had similar biological properties and functional effects on endothelial cells (SVEC4-10). IAA/DNP increased exosome secretion from mouse organ cultures, and in vivo injections enhanced the levels of circulating exosomes. IAA/DNP decreased ATP levels (p < 0.05) in cells. A cell membrane-permeable form of 2',3'-cAMP and 3'-AMP mimicked the potentiating effects of IAA/DNP on exosome secretion. In cells lacking 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase; an enzyme that metabolizes 2',3'-cAMP into 2'- and 3'-AMP), effects of IAA/DNP on exosome secretion were enhanced. The IAA/DNP combination is a powerful stimulator of exosome secretion, and these stimulatory effects are, in part, mediated by intracellular 2',3'-cAMP.

pH-Dependent Grafting of Cancer Cells with Antigenic Epitopes Promotes Selective Antibody-Mediated Cytotoxicity.

A growing class of immunotherapeutics work by redirecting components of the immune system to recognize markers on the surface of cancer cells. However, such modalities will remain confined to a relatively small subgroup of patients because of the lack of universal targetable tumor biomarkers among all patients. Here, we designed a unique class of agents that exploit the inherent acidity of solid tumors to selectively graft cancer cells with immuno-engager epitopes. Our targeting approach is based on pHLIP, a unique peptide that selectively targets tumors in vivo by anchoring to cancer cell surfaces in a pH-dependent manner. We established that pHLIP-antigen conjugates trigger the recruitment of antibodies to the surface of cancer cells and induce cytotoxicity by peripheral blood mononuclear and engineered NK cells. These results indicate that these agents have the potential to be applicable to treating a wide range of solid tumors and to circumvent problems associated with narrow windows of selectivity.

Analysis of 2,4-Dinitrophenol in Postmortem Blood and Urine by Gas Chromatography-Mass Spectrometry: Method Development and Validation and Report of Three Fatalities in the United States.

2,4-dinitrophenol (2,4-DNP) is a compound used in the early 1900s as a weight-loss drug but later prohibited due to its severe adverse effects, including death. It has however been attracting interest, due to its weight-loss properties, and appears to be re-emerging in forensic casework. As 2,4-DNP is available for use in industry and as a pesticide and easily accessible online, the dissemination of this drug can be fast. The compound exerts its effects through inhibition of ATP synthesis, and corresponding thermogenic energy loss which can be fatal. A method for qualitative and quantitative analysis of 2,4-DNP in blood and urine specimens using GC-MS with hydrogen as carrier gas is described. The method was validated and displayed acceptable performance parameters with linearity (R2 higher than 0.998), inter-assay imprecision (lower than 10.6%), intra-assay imprecision (lower than 10.7%), and extraction efficiency (92.1%). Stability of 2,4-DNP in blood and urine was studied, and the drug was stable up to 30 days refrigeration or frozen. Six cases in United States suspected to be related to 2,4-DNP were analyzed. Three cases were found to be positive for 2,4-DNP. Concentrations of 2,4-DNP were in the range of 61.6-220 mg/L in urine and <3-114 mg/L in blood. Based on our findings, we suggest that medical examiners and forensic toxicologists be aware of the reappearance of 2,4-DNP, including this compound as a target in death investigations related to weight-loss drugs.