Affimer Tagged Cubosomes: Targeting of Carcinoembryonic Antigen Expressing Colorectal Cancer Cells Using In Vitro and In Vivo Models.

Nanomedicines, while having been approved for cancer therapy, present many challenges such as low stability, rapid clearance, and nonspecificity leading to off-target toxicity. Cubosomes are porous lyotropic liquid crystalline nanoparticles that have shown great premise as drug delivery vehicles; however, their behavior in vivo is largely underexplored, hindering clinical translation. Here, we have engineered cubosomes based on the space group Im3m that are loaded with copper acetylacetonate as a model drug, and their surfaces are functionalized for the first time with Affimer proteins via copper-free click chemistry to actively target overexpressed carcinoembryonic antigens on LS174T colorectal cancer cells. Unlike nontargeted cubosomes, Affimer tagged cubosomes showed preferential accumulation in cancer cells compared to normal cells not only in vitro (2D monolayer cell culture and 3D spheroid models) but also in vivo in colorectal cancer mouse xenografts, while exhibiting low nonspecific absorption and toxicity in other vital organs. Cancerous spheroids had maximum cell death compared to noncancerous cells upon targeted delivery. Xenografts subjected to targeted drug-loaded cubosomes showed a 5-7-fold higher drug accumulation in the tumor tissue compared to the liver, kidneys, and other vital organs, a significant decrease in tumor growth, and an increased survival rate compared to the nontargeted group. This work encompasses the first thorough preclinical investigation of Affimer targeted cubosomes as a cancer therapeutic.

Review of evidence relating to occupational exposure limits for alpha-diketones and acetoin, and considerations for deriving an occupational exposure limit for 2,3-pentanedione.

Alpha-diketones, notably diacetyl, have been used as flavoring agents. When airborne in occupational settings, exposures to diacetyl have been associated with serious respiratory disease. Other α-diketones, such as 2,3-pentanedione, and analogues such as acetoin (a reduced form of diacetyl), require evaluation, particularly, in light of recently available toxicological studies. The current work reviewed mechanistic, metabolic, and toxicology data available for α-diketones. Data were most available for diacetyl and 2,3-pentanedione, and a comparative assessment of their pulmonary effects was performed, and an occupational exposure limit (OEL) was proposed for 2,3-pentanedione. Previous OELs were reviewed and an updated literature search was performed. Respiratory system histopathology data from 3-month toxicology studies were evaluated with benchmark dose (BMD) modelling of sensitive endpoints. This demonstrated comparable responses at concentrations up to 100 ppm, with no consistent overall pattern of greater sensitivity to either diacetyl or 2,3-pentanedione. In contrast, based on draft raw data, no adverse respiratory effects were observed in comparable 3-month toxicology studies that evaluated exposure to acetoin at up to 800 ppm (highest tested concentration), indicating that acetoin does not present the same inhalation hazard as diacetyl or 2,3-pentanedione. To derive an OEL for 2,3-pentanedione, BMD modelling was conducted for the most sensitive endpoint from 90-day inhalation toxicity studies, namely, hyperplasia of nasal respiratory epithelium. On the basis of this modelling, an 8-hour time-weighted average OEL of 0.07 ppm is proposed to be protective against respiratory effects that may be associated with chronic workplace exposure to 2,3-pentanedione.

Counterions determine uptake and effects of aluminum in human intestinal and liver cells.

Aluminum (Al) is highly abundant in the biosphere and can occur in different physico-chemical states. It is present in human food and undergoes transitions between dissolved and particulate species during the passage of the gastrointestinal tract. Moreover, in a complex matrix such as food different inorganic and organic counterions can affect the chemical behavior of Al following oral uptake. In this work, the effects of different counterions, namely chloride, citrate, sulfate, lactate and acetylacetonate, on Al uptake and toxicity in the human intestine are studied. The respective Al salts showed different dissolution behavior in biological media and formed nanoscaled particles correlating in reverse with the amount of their dissolved fraction. The passage through the intestinal barrier was studied using a Caco-2 Transwell® system, showing counterion-dependent variance in cellular uptake and transport. In addition, Al toxicity was investigated using Al species (Al3+, metallic Al0 and oxidic γAl2O3 nanoparticles) and counterions individually or in mixtures on Caco-2 and HepG2 cells. The strongest toxicity was observed using a combination of Al species, depending on solubility, and the lipophilic counterion acetylacetonate. Notably, only the combination of both led to toxicity, while both substances individually did not show toxic effects. A toxification of previously non-toxic Al-species by the presence of acetylacetonate is shown here for the first time. The dependency on the concentration of free Al ions was demonstrated using sodium hydrogen phosphate, which was able to counteract the toxic effects by complexing free Al ions. These findings, using Al salts as an example for a common food contaminant, underline the importance of a consideration of the chemical properties of human nutrition, especially dissolution and hydrophobicity, which can significantly influence the cellular uptake and effects of xenobiotic substances.

Evaluation of Cytotoxic and Mutagenic Effects of the Synthetic Cathinones Mexedrone, α-PVP and α-PHP.

Mexedrone, α-PVP and α-PHP are synthetic cathinones. They can be considered amphetamine-like substances with a stimulating effect. Actually, studies showing their impact on DNA are totally absent. Therefore, in order to fill this gap, aim of the present work was to evaluate their mutagenicity on TK6 cells. On the basis of cytotoxicity and cytostasis results, we selected the concentrations (35-100 µM) to be used in the further analysis. We used the micronucleus (MN) as indicator of genetic damage and analyzed the MNi frequency fold increase by flow cytometry. Mexedrone demonstrated its mutagenic potential contrary to the other two compounds; we then proceeded by repeating the analyzes in the presence of extrinsic metabolic activation in order to check if it was possible to totally exclude the mutagenic capacity for α-PVP and α-PHP. The results demonstrated instead the mutagenicity of their metabolites. We then evaluated reactive oxygen species (ROS) induction as a possible mechanism at the basis of the highlighted effects but the results did not show a statistically significant increase in ROS levels for any of the tested substances. Anyway, our outcomes emphasize the importance of mutagenicity evaluation for a complete assessment of the risk associated with synthetic cathinones exposure.

S-(+)-Pentedrone and R-(+)-methylone as the most oxidative and cytotoxic enantiomers to dopaminergic SH-SY5Y cells: Role of MRP1 and P-gp in cathinones enantioselectivity.

Cathinone derivatives are the most representative group within new drugs market, which have been described as neurotoxic. Since cathinones, as pentedrone and methylone, are available as racemates, it is our aim to study the neuronal cytotoxicity induced by each enantiomer. Therefore, a dopaminergic SH-SY5Y cell line was used to evaluate the hypothesis of enantioselectivity of pentedrone and methylone enantiomers on cytotoxicity, oxidative stress, and membrane efflux transport (confirmed by in silico studies). Our study demonstrated enantioselectivity of these cathinones, being the S-(+)-pentedrone and R-(+)-methylone the most oxidative enantiomers and also the most cytotoxic, suggesting the oxidative stress as main cytotoxic mechanism, as previously described in in vitro studies. Additionally, the efflux transporter multidrug resistance associated protein 1 (MRP1) seems to play, together with GSH, a selective protective role against the cytotoxicity induced by R-(-)-pentedrone enantiomer. It was also observed an enantioselectivity in the binding to P-glycoprotein (P-gp), another efflux protein, being the R-(-)-pentedrone and S-(-)-methylone the most transported enantiomeric compounds. These results were confirmed, in silico, by docking studies, revealing that R-(-)-pentedrone is the enantiomer with highest affinity to MRP1 and S-(-)-methylone and R-(-)-pentedrone are the enantiomers with highest affinity to P-gp. In conclusion, our data demonstrated that pentedrone and methylone present enantioselectivity in their cytotoxicity, which seems to involve different oxidative reactivity as well as different affinity to the P-gp and MRP1 that together with GSH play a protective role.

Pentafluoro-3-hydroxy-pent-2-en-1-ones Potently Inhibit FNT-Type Lactate Transporters from all Five Human-Pathogenic Plasmodium Species.

The protozoan parasite Plasmodium falciparum causes the most severe and prevailing form of malaria in sub-Saharan Africa. Previously, we identified the plasmodial lactate transporter, PfFNT, a member of the microbial formate-nitrite transporter family, as a novel antimalarial drug target. With the pentafluoro-3-hydroxy-pent-2-en-1-ones, we discovered PfFNT inhibitors that potently kill P. falciparum parasites in vitro. Four additional human-pathogenic Plasmodium species require attention, that is, P. vivax, most prevalent outside of Africa, and the regional P. malariae, P. ovale and P. knowlesi. Herein, we show that the plasmodial FNT variants are highly similar in terms of protein sequence and functionality. The FNTs from all human-pathogenic plasmodia and the rodent malaria parasite were efficiently inhibited by pentafluoro-3-hydroxy-pent-2-en-1-ones. We further established a phenotypic yeast-based FNT inhibitor screen, and found very low compound cytotoxicity and monocarboxylate transporter 1 off-target activity on human cells, particularly of the most potent FNT inhibitor BH267.meta, allowing these compounds to proceed towards animal model malaria studies.

The interplay between autophagy and apoptosis mediates toxicity triggered by synthetic cathinones in human kidney cells.

Synthetic cathinones abuse remains a serious public health problem. Kidney injury has been reported in intoxications associated with synthetic cathinones, but the molecular mechanisms involved have not been explored yet. In this study, the potential in vitro nephrotoxic effects of four commonly abused cathinone derivatives, namely pentedrone, 3,4-dimethylmethcatinone (3,4-DMMC), methylone and 3,4-methylenedioxypyrovalerone (MDPV), were assessed in the human kidney HK-2 cell line. All four derivatives elicited cell death in a concentration- and time-dependent manner, in the following order of potency: 3,4-DMMC >> MDPV > methylone ≈ pentedrone. 3,4-DMMC and methylone were selected to further elucidate the mechanisms behind synthetic cathinones-induced cell death. Both drugs elicited apoptotic cell death and prompted the formation of acidic vesicular organelles and autophagosomes in HK-2 cells. Moreover, the autophagy inhibitor 3-methyladenine significantly potentiated cell death, indicating that autophagy may serve as a cell survival mechanism that protects renal cells against synthetic cathinones toxicity. Both drugs triggered a rise in reactive oxygen and nitrogen species formation, which was completely prevented by antioxidant treatment with N­acetyl­L­cysteine or ascorbic acid. Importantly, these antioxidant agents significantly aggravated renal cell death induced by cathinone derivatives, most likely due to their autophagy-blocking properties. Taken together, our results support an intricate control of cell survival/death modulated by oxidative stress, apoptosis and autophagy in synthetic cathinones-induced renal injury.

Flavorings-Related Lung Disease: A Brief Review and New Mechanistic Data.

Flavorings-related lung disease is a potentially disabling and sometimes fatal lung disease of workers making or using flavorings. First identified almost 20 years ago in microwave popcorn workers exposed to butter-flavoring vapors, flavorings-related lung disease remains a concern today. In some cases, workers develop bronchiolitis obliterans, a severe form of fixed airways disease. Affected workers have been reported in microwave popcorn, flavorings, and coffee production workplaces. Volatile α-dicarbonyl compounds, particularly diacetyl (2,3-butanedione) and 2,3-pentanedione, are implicated in the etiology. Published studies on diacetyl and 2,3-pentanedione document their ability to cause airway epithelial necrosis, damage biological molecules, and perturb protein homeostasis. With chronic exposure in rats, they produce airway fibrosis resembling bronchiolitis obliterans. To add to this knowledge, we recently evaluated airway toxicity of the 3-carbon α-dicarbonyl compound, methylglyoxal. Methylglyoxal inhalation causes epithelial necrosis at even lower concentrations than diacetyl. In addition, we investigated airway toxicity of mixtures of diacetyl, acetoin, and acetic acid, common volatiles in butter flavoring. At ratios comparable to workplace scenarios, the mixtures or diacetyl alone, but not acetic acid or acetoin, cause airway epithelial necrosis. These new findings add to existing data to implicate α-dicarbonyl compounds in airway injury and flavorings-related lung disease.

Molecular Toxicological Mechanisms of Synthetic Cathinones on C2C12 Myoblasts.

Synthetic cathinones are popular psychoactive substances that may cause skeletal muscle damage. In addition to indirect sympathomimetic myotoxicity, these substances could be directly myotoxic. Since studies in myocytes are currently lacking, the aim of the present study was to investigate potential toxicological effects by synthetic cathinones on C2C12 myoblasts (mouse skeletal muscle cell line). We exposed C2C12 myoblasts to 3-methylmethcathinone, 4-methylmethcathinone (mephedrone), 3,4-methylenedioxymethcathinone (methylone), 3,4-methylenedioxypyrovalerone (MDPV), alpha-pyrrolidinovalerophenone (α-PVP), and naphthylpyrovalerone (naphyrone) for 1 or 24 h before cell membrane integrity, ATP content, mitochondrial oxygen consumption, and mitochondrial superoxide production was measured. 3,4-Methylenedioxymethamphetamine (MDMA) was included as a reference compound. All investigated synthetic cathinones, as well as MDMA, impaired cell membrane integrity, depleted ATP levels, and increased mitochondrial superoxide concentrations in a concentration-dependent manner in the range of 50⁻2000 µM. The two pyrovalerone derivatives α-PVP and naphyrone, and MDMA, additionally impaired basal and maximal cellular respiration, suggesting mitochondrial dysfunction. Alpha-PVP inhibited complex I, naphyrone complex II, and MDMA complex I and III, whereas complex IV was not affected. We conclude that, in addition to sympathetic nervous system effects and strenuous muscle exercise, direct effects of some cathinones on skeletal muscle mitochondria may contribute to myotoxicity in susceptible synthetic cathinone drugs users.

Small molecule diketone flavorants diacetyl and 2,3-pentanedione promote neurotoxicity but inhibit amyloid ß aggregation.

We investigated the effects of the small molecule flavorants diacetyl, 2,3-pentanedione and acetoin on neuronal cell viability and ß amyloid aggregation and morphology. Two neuroblastoma cell lines, SH-SY5Y and Neuro 2a (N2a) were exposed to diacetyl, 2,3-pentanedione and acetoin, while Thioflavin T fluorescence kinetics and transmission electron microscopy were used to assess effects on Aß1-42 fibril and aggregate formation and morphology respectively. Diacetyl was intrinsically toxic to both SH-SY5Y and N2a cells, with time and concentration-dependent reductions in cell viability occurring over 24 h and 48 h incubation periods. 2.3-Pentanedione evoked a similar concentration-dependent loss of cell viability in N2a cells at 48 h, but exhibited lessened toxicity in SH-SY5Y cells over 24 h, and minimal loss of cell viability by 48 h. Diacetyl inhibited Aß1-42 aggregation kinetics, reduced aggregate and fibril density and rendered Aß1-42 into amorphous small aggregates. 2,3-Pentanedione also reduced overall aggregate formation, but to a lesser extent than diacetyl and retaining the presence of a meshwork of Aß1-42 aggregates and fibrils. Acetoin was innocuous to neuronal cells and did not alter Aß1-42 fibril density or morphology. These findings highlight the intrinsic neurotoxicity of small molecule diketone flavorants. While providing further insight into their molecular interactions with amyloidogenic proteins, the neurotoxicity of such flavorants is a significant finding and warrants further investigation.

Mechanisms of hepatocellular toxicity associated with new psychoactive synthetic cathinones.

Synthetic cathinones are a new class of psychostimulant substances. Rarely, they can cause liver injury but associated mechanisms are not completely elucidated. In order to increase our knowledge about mechanisms of hepatotoxicity, we investigated the effect of five frequently used cathinones on two human cell lines. Bupropion was included as structurally related drug used therapeutically. In HepG2 cells, bupropion, MDPV, mephedrone and naphyrone depleted the cellular ATP content at lower concentrations (0.2-1mM) than cytotoxicity occurred (0.5-2mM), suggesting mitochondrial toxicity. In comparison, methedrone and methylone depleted the cellular ATP pool and induced cytotoxicity at similar concentrations (≥2mM). In HepaRG cells, cytotoxicity and ATP depletion could also be demonstrated, but cytochrome P450 induction did not increase the toxicity of the compounds investigated. The mitochondrial membrane potential was decreased in HepG2 cells by bupropion, MDPV and naphyrone, confirming mitochondrial toxicity. Bupropion, but not the other compounds, uncoupled oxidative phosphorylation. Bupropion, MDPV, mephedrone and naphyrone inhibited complex I and II of the electron transport chain, naphyrone also complex III. All four mitochondrial toxicants were associated with increased mitochondrial ROS and increased lactate production, which was accompanied by a decrease in the cellular total GSH pool for naphyrone and MDPV. In conclusion, bupropion, MDPV, mephedrone and naphyrone are mitochondrial toxicants impairing the function of the electron transport chain and depleting cellular ATP stores. Since liver injury is rare in users of these drugs, affected persons must have susceptibility factors rendering them more sensitive for these drugs.

Conditioned taste avoidance, conditioned place preference and hyperthermia induced by the second generation 'bath salt' α-pyrrolidinopentiophenone (α-PVP).

BACKGROUND: α-Pyrrolidinopentiophenone (α-PVP) has been reported to be rewarding in a variety of pre-clinical models. Given that a number of drugs of abuse have both rewarding and aversive effects, the balance of which influences addiction potential, the present study examined the aversive properties of α-PVP by assessing its ability to induce taste avoidance. This assessment was made in a combined taste avoidance/place conditioning design that also allowed an evaluation of the relationship between α-PVP's aversive and rewarding effects. METHODS: Male Sprague-Dawley rats were exposed to a novel saccharin solution, injected with one of four doses of α-PVP (0, 0.3, 1.0 and 3.0mg/kg) (IP) and placed on one side of a place conditioning apparatus. The next day, they were injected with vehicle, given access to water and placed on the other side. Following four conditioning cycles, saccharin avoidance and place preferences were then assessed. The effects of α-PVP on body temperature were also examined. RESULTS: α-PVP induced dose-dependent taste avoidance as well as significant increases in time spent on the drug-paired side (although this effect was not dependent on dose). α-PVP also induced dose- and time-dependent hyperthermia. CONCLUSIONS: α-PVP induced significant taste avoidance whose strength relative to the psychostimulants methylenedioxypyrovalerone (MDPV) and cocaine paralleled their relative binding to the dopamine transporter. Similar to other drugs of abuse, α-PVP has both aversive and rewarding effects. It will be important to assess how various experiential and subject variables impact these effects and their balance to predict abuse liability.

Pathology of diacetyl and 2,3-pentanedione airway lesions in a rat model of obliterative bronchiolitis.

Inhalation of diacetyl vapors by workers has been associated with obliterative bronchiolitis (OB), a poorly understood fibroproliferative disease of the small airways. Significant insights into the pathogenesis of OB have been obtained through the use of a rat model. Inhalation exposure of rats to diacetyl or 2,3-pentanedione, a related flavoring agent, can cause severe injury to the airway epithelium and underlying basement membrane. Repeated exposure to diacetyl or 2,3-pentanedione leads to aberrant repair, fibroproliferation and partial to complete occlusion of the airway lumen. Fibroproliferative lesions in rat airways were found to include both intraluminal polyps and circumferential intramural lesions. Intraluminal polyps have been observed to form secondary attachments spanning the airway lumen causing increasing obstruction. These airway lesions in rats are accompanied by inflammation in the form of peribronchial and perivascular infiltrates of lymphocytes, eosinophils and neutrophils. Diacetyl-induced OB lesions in the rat are similar to OB lesions in humans and provide a good model for studying the pathogenesis of this disease.

Chemical Reactivity and Respiratory Toxicity of the α-Diketone Flavoring Agents: 2,3-Butanedione, 2,3-Pentanedione, and 2,3-Hexanedione.

Occupational exposure to 2,3-butanedione (BD) vapors has been associated with severe respiratory disease leading to the use of potentially toxic substitutes. We compared the reactivity and respiratory toxicity of BD with that of two structurally related substitutes, 2,3-pentanedione (PD) and 2,3-hexanedione (HD). Chemical reactivity of the diketones with an arginine substrate decreased with increasing chain length (BD > PD > HD). Animals were evaluated the morning after a 2-week exposure to 0, 100, 150, or 200 ppm BD, PD, or HD (postexposure) or 2 weeks later (recovery). Bronchial fibrosis was observed in 5/5 BD and 5/5 PD rats at 200 ppm and in 4/6 BD and 6/6 PD rats at 150 ppm in the postexposure groups. Following recovery, bronchial fibrosis was observed in all surviving rats exposed to 200 ppm BD (5/5) or PD (3/3) and in 2/10 BD and 7/9 PD rats exposed to 150 ppm. Bronchial fibrosis was observed only in 2/12 HD-exposed rats in the 200 ppm postexposure group. Patchy interstitial fibrosis affected lungs of recovery groups exposed to 200 ppm PD (3/3) or BD (1/5) and to 150 ppm PD (4/9) or BD (7/10) and correlated with pulmonary function deficits. BD and PD were more reactive and produced more bronchial fibrosis than HD.

Update on flavoring-induced lung disease.

PURPOSE OF REVIEW: Since the initial report of bronchiolitis obliterans in microwave popcorn workers, exposures to flavoring substances have been identified in a variety of food and flavor manufacturing facilities and in the consumer market. Attempts to decrease the risk of lung disease have included the use of flavoring substitutes; however, these chemicals may cause similar injury. This article reviews recent flavoring exposures and data on the pathogenesis, clinical characteristics, and surveillance of flavoring-induced lung disease. RECENT FINDINGS: Diacetyl and 2,3-pentanedione exposures have occurred in food production facilities that make cookies, cereal, chocolate, and coffee. Airborne levels often exceed proposed occupational exposure limits. Cases of biopsy-proven bronchiolitis obliterans in heavy popcorn consumers have also been reported. New data demonstrate the presence of diacetyl and 2,3-pentanedione in flavored nicotine liquids used in electronic nicotine delivery systems. SUMMARY: Diacetyl substitutes cause similar peri-bronchiolar fibrotic lesions in animal studies. Their use may continue to place workers at risk for flavoring-induced lung disease, which may present in forms beyond that of fixed airflow obstruction, contributing to delays in identifying and treating patients with flavoring-induced lung disease. Engineering controls, medical surveillance and personal protective equipment can limit flavorings exposure and risk for lung disease.

Respiratory morbidity in a coffee processing workplace with sentinel obliterative bronchiolitis cases.

RATIONALE: Obliterative bronchiolitis in former coffee workers prompted a cross-sectional study of current workers. Diacetyl and 2,3-pentanedione levels were highest in areas for flavoring and grinding/packaging unflavored coffee. METHODS: We interviewed 75 (88%) workers, measured lung function, and created exposure groups based on work history. We calculated standardized morbidity ratios (SMRs) for symptoms and spirometric abnormalities. We examined health outcomes by exposure groups. RESULTS: SMRs were elevated 1.6-fold for dyspnea and 2.7-fold for obstruction. The exposure group working in both coffee flavoring and grinding/packaging of unflavored coffee areas had significantly lower mean ratio of forced expiratory volume in 1 s to forced vital capacity and percent predicted mid-expiratory flow than workers without such exposure. CONCLUSION: Current workers have occupational lung morbidity associated with high diacetyl and 2,3-pentanedione exposures, which were not limited to flavoring areas.