Search

Two emerging psychoactive substances, 2-(2,5-dimethoxy-4-methylphenyl)-N-(2-methoxybenzyl)ethanamine (25D-NBOMe) and N-(2-methoxybenzyl)-2,5-dimethoxy-4-chlorophenethylamine (25C-NBOMe), are being abused, leading to fatal and non-fatal intoxications. However, most of their adverse effects have been reported anecdotally. In the present study, cardiotoxicity was evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, rat electrocardiography (ECG), and human ether-a-go-go-related gene (hERG) assay. Expression levels of p21 (CDC42/RAC)-activated kinase 1 (PAK1), one of known biomarkers for cardiotoxicity, were also analyzed. Both 25D-NBOMe and 25C-NBOMe at 100 μM reduced cell viability in MTT assay. At 2.0 mg/kg and 0.75 mg/kg, they prolonged QT intervals in rat ECG. PAK1 was down-regulated by treatment with these two test compounds. Furthermore, potassium channels were inhibited by 25D-NBOMe treatment in hERG assay. Taken together, these results suggest that both 25D-NBOMe and 25C-NBOMe have potential cardiotoxicity, especially regarding cardiac rhythm. Further studies are needed to confirm the relationship between PAK1 down-regulation and cardiotoxicity.

The abuse of new psychoactive substances (NPS) is an emerging social problem. Methoxetamine, one of the NPS, was designed as an alternative to ketamine and it was considered an NPS candidate owing to its high addictive potential. However, cardiotoxicity of the phencyclidine analogue, methoxetamine, has not been extensively evaluated. P21 protein (Cdc42/Rac)-activated kinase 1 (PAK-1) is associated with the drug-induced cardiotoxicity and hypertrophy of cardiomyocytes. In the present study, we investigated the effects of methoxetamine on rat cardiomyocytes and PAK-1. Methoxetamine (at 10 µM) reduced cell viability and PAK-1 mRNA levels in H9c2 cells. Methoxetamine treatment (100 µM) decreased the beating rate of primary cardiomyocytes. However, 100 µM methoxetamine-induced heart rate decline was less than 100 µM PCP- or ketamine-induced heart rate decline. Meanwhile, fingolimod hydrochloride (FTY720, 1 µM), a PAK-1 activator, increased cell viability and inhibited hypertrophy induced by methoxetamine in H9c2 cells. These results suggest that methoxetamine may have harmful effects on the cardiovascular system through the regulation of the expression and function of PAK-1.

Two synthetic tryptamines, namely [3-[2-(diethylamino)ethyl]-1H-indol-4-yl] acetate (4-AcO-DET) and 3-[2-[ethyl(methyl)amino]ethyl]-1H-indol-4-ol (4-HO-MET), are abused by individuals seeking recreational hallucinogens. These new psychoactive substances (NPSs) can cause serious health problems because their adverse effects are mostly unknown. In the present study, we evaluated the cardiotoxicity of 4-AcO-DET and 4-HO-MET using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, electrocardiography (ECG), and the human ether-a-go-go-related gene (hERG) assay. In addition, we analyzed the expression level of p21 (CDC42/RAC)-activated kinase 1 (PAK1), which is known to play various roles in the cardiovascular system. In the MTT assay, 4-AcO-DET- and 4-HO-MET-treated H9c2 cells proliferated in a concentration-dependent manner. Moreover, both substances increased QT intervals (as determined using ECG) in Sprague-Dawley rats and inhibited potassium channels (as verified by the hERG assay) in Chinese hamster ovary cells. However, there was no change in PAK1 expression. Collectively, the results indicated that 4-AcO-DET and 4-HO-MET might cause adverse effects on the cardiovascular system. Further studies are required to confirm the relationship between PAK1 expression and cardiotoxicity. The findings of the present study would provide science-based evidence for scheduling the two NPSs.

Desoxypipradrol (2-DPMP), a new psychoactive substance (NPS), acts as a norepinephrine-dopamine reuptake inhibitor (NDRI). NDRIs can be addictive due to their action mechanisms similar to cocaine and methamphetamine. However, there is a lack of scientific information regarding the exact dependency of 2-DPMP. Thus, the purpose of this study was to evaluate rewarding and reinforcing effects of 2-DPMP in rodents. The effective dose range of 2-DPMP was determined by climbing behavior test. To evaluate rewarding effects of 2-DPMP, conditioned place preference (CPP) test was performed at selected doses in mice. Self-administration (SA) test was then undertaken at two doses that caused the highest effects in the CPP test. Dopamine level changes were analyzed using synaptosomes in order to investigate effects of 2-DPMP on the central nervous system (CNS). Significant responses were observed in the climbing behavior test at doses of 0.1, 0.5, and 1 mg/kg by intraperitoneal injection (i.p.). In the CPP test, mice i.p. administered 2-DPMP at 1 mg/kg showed a significant preference in drug-paired compartment. In the SA test, mice intravenously given 0.1 mg/kg/infusion showed significantly higher active lever responses. Further, dopamine was increased in a dose-dependent manner. Taken together, these results suggest that 2-DPMP may act on the CNS and induce rewarding and reinforcing effects, indicating its dependence liability.

25I NBOMe (“25-I”, “N-Bomb”), one of new psychoactive substances (NPSs), is being abused for recreational purpose. However, the liability for abuse or dependence has not been systematically studied yet. The objective of the present study was to evaluate rewarding and reinforcing effects of 25I NBOMe using conditioned place preference (CPP) and self-administration (SA) paradigms. In addition, ultrasonic vocalizations (USVs) were measured to investigate relationships between USVs and emotional state regarding dependence on psychoactive substances. To understand molecular mechanism involved in its action, dopamine (DA) level changes were analyzed using synaptosomes extracted from the striatal region of the brain. Expression level changes of SGK1 (serum/glucocorticoid regulated kinase 1) and PER2 (period circadian protein homolog 2), two putative biomarkers for drug dependence, were also analyzed. Results showed that 25I NBOMe increased both CPP (0.3 mg/kg) and SA (0.03 mg/kg/infusion) and produced higher frequencies in USVs analysis. It also increased DA levels in the striatal region and changed expression levels of SGK1 and PER2. Results of the present study suggest that 25I NBOMe might produce rewarding and reinforcing effects, indicating its dependence liability. In addition, frequencies of USV might be associated with emotional state of mice induced by psychoactive substances regarding substance dependence. This is the first systemic preclinical report on the dependence liability of 25I NBOMe and the first attempt to introduce a possible relationship between USVs and emotional state of mice regarding substance dependency. Further studies are needed to clarify the mechanism involved in 25I NBOMe dependency and determine the usefulness of USV measurement as a method for evaluating dependence liability.

Chimeric antigen receptor-modified T cells (CAR-T) have emerged as a new modality for cancer immunotherapy due to their potent efficacy against terminal cancers. CAR-Ts are reported to exert higher efficacy than monoclonal antibodies and antibody-drug conjugates, and act via mechanisms distinct from T cell receptor-engineered T cells. These cells are constructed by transducing genes encoding fusion proteins of cancer antigen-recognizing single-chain Fv linked to intracellular signaling domains of T cell receptors. CAR-Ts are classified as first-, second- and third-generation, depending on the intracellular signaling domain number of T cell receptors. This review covers the current status of CAR-T research, including basic proof-of-concept investigations at the cell and animal levels. Currently ongoing clinical trials of CAR-T worldwide are additionally discussed. Owing to the lack of existing approved products, several unresolved concerns remain with regard to safety, efficacy and manufacturing of CAR-T, as well as quality control issues. In particular, the cytokine release syndrome is the major side-effect impeding the successful development of CAR-T in clinical trials. Here, we have addressed the challenges and regulatory perspectives of CAR-T therapy.

Increased oxidative stress with elevated levels of reactive oxygen/nitrogen species (ROS/RNS) plays an important role in the pathophysiology of many disease states. Increased ROS/RNS can modulate the cellular macromolecules of DNA, lipids, and proteins, negatively affecting their normal functions. Numerous reports have described the properties and implications of oxidized DNA and lipids. However, oxidative modifications of proteins were not fully studied partially due to the requirement for specific reagents, the lack of methods to detect, purify, and identify oxidatively modified proteins, and the relatively late development of highly sensitive analytical instruments. This chapter describes the detailed procedure for systematically identifying oxidative-modified proteins in biological samples. Applications and other suggestions to this method are also described to understand the functional roles of oxidatively modified proteins in promoting endoplasmic reticulum (ER) stress and mitochondrial dysfunction, which ultimately contribute to organ damage.

Although antiviral assays have been the most widely available biological assays for interferons (IFNs), they are less sensitive and provide considerable interassay variation. In this study, we demonstrate a new reporter cell line, which is based on HeLa cells transfected with a plasmid containing a human Mx2 promoter driving a luciferase (Luc) cDNA. To characterize the specific gene expression profiles induced by interferon alpha, we analyzed the microarray results of interferon response gene expression induced by IFN-alpha2a or IFN-alpha2b treatment with HeLa cells. We found that the Mx2 gene increased the most by treatment with both IFN-alpha2a and IFN-alpha2b. Based on this result, we designed a reporter cell line, HeLa-Mx2, suitable for determination of IFN-alpha. HeLa cells were stably transfected with the luciferase gene under the control of Mx2 promoter. The expression of luciferase can be easily measured for luminescence using a 96-well luminometer and has been correlated with the concentration of added IFN and cell density. In the validation results, our reporter cell line had specificity for type I IFN, but the significant effects of a number of other cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-2, IL-5, IL-6 and GM-CSF, or type II interferon (IFN-gamma) were not observed. Moreover, the robustness of our cell line is demonstrated by the lack of an effect of the HeLa-Mx2 cell culture's age on the performance of the reporter gene assay. The reporter gene assay demonstrated reproducible dose-response curves for IFN-alpha2a in the range of 1-10,000 IU/ml. The 95% confidential limit and total coefficient of variation estimates ranged between 96 and 116 and 10.51% in the reducible range mentioned above, respectively. In conclusion, we established a stable IFN-responsible HeLa-Mx2 cell line, which has advantages with regard to simplicity, selectivity, and reliability over conventional cytopathic effect reduction assays used to quantify IFN-alpha activity.

Heavy alcohol consumption can damage various cells and organs partly through production of reactive oxygen species (ROS) and mitochondrial dysfunction. Treatment with antioxidants can significantly reduce the degree of damage. Despite well established roles of ROS in alcohol-induced cell injury, the proteins that are selectively oxidized by ROS are poorly characterized. We hypothesized that certain cysteinyl residues of target proteins are oxidized by ROS upon alcohol exposure, and these modified proteins may play roles in mitochondrial dysfunction. A targeted proteomics approach utilizing biotin-N-maleimide (biotin-NM) as a specific probe to label oxidized cysteinyl residues was employed to investigate which mitochondrial proteins are modified during and after alcohol exposure. Human hepatoma HepG2 cells with transduced CYP2E1 (E47 cells) were used as a model to generate ROS through CYP2E1-mediated ethanol metabolism. Following exposure to 100 mM ethanol for 4 and 8 h, the biotin-NM-labeled oxidized proteins were purified with agarose coupled to either streptavidin or monoclonal antibody against biotin. The purified proteins were resolved by two-dimensional gel electrophoresis and protein spots that displayed differential abundances were excised from the gel, in-gel digested with trypsin and analyzed for identity utilizing either matrix-assisted laser desorption-time of flight mass spectrometry or microcapillary reversed-phase liquid chromatography-tandem mass spectrometry. The results demonstrate that heat shock protein 60, protein disulfide isomerase, mitochondrial aldehyde dehydrogenases, prohibitin, and other proteins were oxidized after alcohol exposure. The identity of some of the proteins purified with streptavidin-agarose was also confirmed by immunoblot analyses using the specific antibody to each target protein. This method was also used to identify oxidized mitochondrial proteins in the alcohol-fed mouse liver. These results suggest that exposure to ethanol causes oxidation of various mitochondrial proteins that may negatively affect their function and contribute to alcohol-induced mitochondrial dysfunction and cellular injury.