Identification of a new M-ALPHA analog and MDMA in an illegal health product.

The widespread abuse of illicit psychoactive substances is one of the most serious public health and social problems. A suspicious airmail package was seized by Korean customs, and two psychoactive substances in the grayish-green pills in the package were detected by ultra-performance liquid chromatography. The structures of the two substances were elucidated by a combination of liquid chromatography quadrupole time-of-flight mass spectrometry, nuclear magnetic resonance spectroscopy, and comparison with reported or newly generated spectral data of the suggested structures. One of the psychoactive substances proved to be MDMA (commonly known as "Ecstasy"), and the other compound was an M-ALPHA analog bearing a hydroxyl group and an N-methylcarboxamide group. The new M-ALPHA analog was determined as 3-(benzo[d][1,3]dioxol-5-yl)-2-hydroxy-N,2-dimethyl-3-(methylamino)propanamide and named as M-ALPHA-HMCA, wherein HMCA denotes hydroxymethylcarboxamide. Although psychoactivity of this compound has not been assessed, M-ALPHA-HMCA should be considered a potential new psychoactive substance and/or a by-product of MDMA.

Simultaneous separation and determination of 20 potential adulterant antigout and antiosteoporosis pharmaceutical compounds in herbal food products using LC with electrospray ionization MS/MS and LC with quadrupole-time-of-flight MS.

An analytical method for the simultaneous and reliable determination of 20 antigout and antiosteoporosis pharmaceutical compounds in adulterated health food products was developed using liquid chromatography with electrospray ionization tandem mass spectrometry and liquid chromatography with quadrupole-time-of-flight mass spectrometry. The method was validated through the determination of specificity, linearity, limit of detection, and limit of quantification, method detection limit, method quantitation limit, precision, accuracy, recovery, and stability. The matrix effect was also determined. The validation results of the developed method are as follows: for solid and liquid blank samples, limits of detection ranged from 0.05 to 5.00 ng/mL and limits of quantification ranged from 0.15 to 15.00 ng/mL. Linearity was acceptable, and the correlation coefficients (R2 ) were ≥0.99 for all target compounds. Both intra and interday precision were less than 9.16% RSD, and accuracies ranged from 95.31 to 116.68%. Mean recoveries for different types of dietary supplements classified as powders, liquids, tablets, and capsules were found to be 80.81 to 117.62% with less than 15.00% relative standard deviation. The stability of the standard mixture solution was less than 11.72% relative standard deviation after 48 h. By the proposed method, the presence of dexamethasone was determined in seized herbal food products at concentrations that ranged from 126 to 215 µg/g.

Development of a specific fragmentation pattern-based quadrupole-Orbitrap™ mass spectrometry method to screen drugs in illicit products.

Over the past decade, illicit drugs have been founded in marketed products, which pose a risk to public health. In particular, newly designed analogues synthesized by chemical modification of parent compounds to avoid detection by authorities are frequently detected worldwide. Although many analytical methods for determination of drugs have been reported, analytical methods using high-resolution mass spectrometry, which has the advantage of rapid screening and accurate identification of new substances, are necessary to control illicit drugs in marketed products. In this study, a rapid analytical method using an Orbitrap™ mass spectrometer for identification of illicit drugs in marketed products was developed. The 32 drugs were classified as benzodiazepine-, synthetic cannabinoid-, amphetamine- and benzylpiperazine-type drugs according to their chemical structures, and from their fragmentation patterns in tandem mass spectrometry spectra of an established method. The method validation gave a limit of detection of 0.06-5.30 ng/mL and a limit of quantification of 0.18-16.50 ng/mL, high linearity (R2 > 0.994) and mean recoveries of spiked matrix-blank samples ranging from 83.7% to 117.1%. Approximately 71% of 21 samples collected over 3 years were found to individually contain one of four types of benzodiazepines or two different synthetic cannabinoids. In one case, levels as high as 827.2 mg/g were measured suggesting adulteration at high levels, which suggests that potential illicit products containing drugs should be regularly screened to protect public health.

Simultaneous determination of illegal drug substances in dietary supplements for gout and osteoporosis using ultra-performance liquid chromatography and liquid chromatography-quadrupole-time-of-flight mass spectrometry.

The aim of this study was to simultaneously determine the presence of unauthorized drug substances in health foods and herbal products used in the treatment of conditions such as gout and anti-osteoporosis. Therefore, we developed and optimised a rapid and accurate method to simultaneously measure 20 anti-gout and anti-osteoporosis drug substances using an ultra-high-performance liquid chromatography (UPLC) system equipped with a photodiode array (PDA) detector. The method was validated to fully meet internationally accepted standards. LODs and LOQs spiked in solid and liquid negative samples were ranged from 0.12 to 1.50 µg/mL, and ranged from 0.36 to 4.50 µg/mL. Linearities (R2> 0.999), stabilities (RSD ≤ 2.92%), accuracies (84.25∼106.62%, intra-day; 84.56∼105.85%, inter-day), precisions (RSD ≤ 3.71% on the intra-day; RSD ≤ 3.47% on the inter-day), recoveries spiked in various type of blank samples such as powder, liquid, tablet, and capsule were determined within 81.20-116.20 %, respectively. From a confirmation of matrix effects (88.06∼110.50% in solid blank; 89.16∼110.52% in liquid blank), it was confirmed that this method was not significantly affected by a sample matrix. The validated method was used to analyse 116 samples containing health foods, herbal products, and seized forensic samples advertised to be effective anti-gout and anti-osteoporosis agents. Of the 20 drug substances screened, dexamethasone was detected and confirmed by comparing the tandem mass spectrometry (MS/MS) fragment ion patterns of a reference standard and the sample using LC-quadrupole-time-of-flight (Q-TOF)/MS. The concentrations of adulterants in seized forensic samples ranged from 0.013 to 0.022 %.

Application of screening methods for weight-loss compounds and identification of new impurities in counterfeit drugs.

With the increasing prevalence of obesity, the use of counterfeit drugs for weight loss is widespread owing to their easy and rapid availability. Since counterfeit weight-loss drugs are not prepared under the rigorous standard of Good Manufacturing Practice (GMP), they pose a risk to public health and cause significant side effects. To counteract the risk posed by counterfeit drugs, we investigated counterfeit weight-loss drugs seized by the Incheon Customs Services using UHPLC-PDA. Five of 23 confiscated samples with distinctive pink-coloured coating contained levothyroxine, sennoside A and B, and phenolphthalein in amounts ranging from 0.03-132.40 mg/g. In addition, three unknown compounds in one of the adulterated samples containing phenolphthalein were structurally elucidated by several analytical techniques. Their accurate masses corresponded to molecular formula of C34H22O7, C34H20O6, and C20H12O3, respectively. These compounds were identified as impurities, possibly produced during the synthesis of phenolphthalein or by improper removal during purification. These impurities were detected for the first time in counterfeit drugs.

Development of a specific fragment pattern-based quadrupole-Orbitrap mass spectrometry method to screen adulterated products of phosphodiesterase-5 inhibitors and their analogues.

Recently, adulterated supplements with phosphodiesterase-5 inhibitors (PDE-5i) have frequently observed. New synthetic analogues obtained from the chemical modification of parent compounds are frequently found in illicit products despite continuous efforts to inspect for these adulterants. A rapid and accurate method based on quadrupole-Orbitrap mass spectrometry was developed for simultaneously confirming and quantifying 85 PDE-5i and derived analogues present in illicit products for erectile dysfunction (ED). Common ions of PDE-5i according to their similar structures were proposed based on MS/MS fragmentations. These common ions could be an important diagnosis of their presence targets or new emerging analogues in supplements. Several validation parameters were employed, resulting in a limit of detection and quantification of 0.09-8.55 ng/mL and 0.24-17.10 ng/mL, respectively. The linear correlation coefficient (r2) was higher than 0.995, and mean recoveries of target compounds were in the range of 82-118%. A total of 187 illicit products, obtained from on/offline markets over a period of 3 years (2015-2017), were screened by the established method. Approximately 53% of them were adulterated with PDE-5i or derived analogues at concentrations of 0.1-726.0 mg/g in the illicit products. In the interests of public health, this study describes a rapid and accurate method to determine PDE-5i and new emerging analogues in adulterated products.

Screening of illegal sexual enhancement supplements and counterfeit drugs sold in the online and offline markets between 2014 and 2017.

The worldwide spread of illegal sexual enhancement products is posing a threat to public health. The aim of this study was to investigate illegal products claiming to be effective in improving sexual performance through the online or offline markets between 2014 and 2017; these products include foods, dietary supplements, counterfeit drugs, and herbal medicines. These samples were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the presence of 80 PDE-5 inhibitors (PDE-5i) and analogues. The developed method was validated as follows: LODs and LOQs spiked in solid- and liquid-type negative samples (0.03-3.33 ng/mL and 0.08-10.00 ng/mL), linearities (R2 > 0.997), recoveries spiked negative samples (82.2-109.3 %), accuracies (81.6-118.9 %), precisions (≥ 6.5%, RSD) of intra-day and inter-day, and stability (≥10.0%, RSD). Out of 362 measured samples, 145 were adulterated samples mostly detected in food (51%). Sildenafil group (50%) was frequently observed, followed by tadalafil group (41%). Although sildenafil and tadalafil were mainly detected in adulterated samples, their analogues were also found. In particular, new analogues have appeared steadily on illicit erectile dysfunction (ED) products even after they were first discovered. The concentration of detected samples ranged from 0.1 to 826.0 mg/g, and sildenafil of them contained a considerable amount in illicit ED products in 2014, posing a potential toxicology risk of public health. The testing method is fast and reliable making it suitable for both routine screening and up-to-date quantitative analysis of PDE-5i and their analogues in suspicious foods, dietary supplements, and counterfeit drugs.

Isolation and characterisation of a novel sildenafil analogue adulterant, desmethylpiperazinyl propoxysildenafil, in a dietary supplement.

A new sildenafil analogue was detected during routine screening of dietary supplements suspected to be adulterated with an erectile dysfunction drug(s) using HPLC-DAD. The UV spectrum of this compound was highly similar to that of sildenafil and almost identical to that of desmethylpiperazinyl sildenafil. The analogue was purified by using semi-preparative HPLC and structurally elucidated by performing mass spectrometric and NMR spectroscopic experiments. The spectral data revealed that this sildenafil analogue bears an n-propoxy group instead of an ethoxy group and possesses no methylpiperazinyl moiety. The isolated compound, structure of which was further confirmed by spectral comparison with synthetic one, was thus named as desmethylpiperazinyl propoxysildenafil.

Identification and characterization of an indazole-3-carboxamide class synthetic cannabinoid: 2-[1-(cyclohexylmethyl)-1H-indazole-3-carboxamido]-3,3-dimethylbutanoic acid (DMBA-CHMINACA).

Illicit psychoactive substances have threatened public health worldwide. An active metabolite of ADB-CHMINACA and MDMB-CHMINACA was identified for the first time in a powder-type product found in an airmail package. The structure of compound 1 was elucidated by a combination of gas chromatography-mass spectrometry (GC-MS), liquid chromatography-high resolution mass spectrometry (LC-HRMS), infrared (IR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. Compound 1 was proven to be an analogue of MDMB-CHMINACA, an indazole-based synthetic cannabinoid. The methyl ester group in MDMB-CHMINACA was replaced with a carboxylic acid group in compound 1. Compound 1 was determined as 2-[1-(cyclohexylmethyl)-1H-indazole-3-carboxamido]-3,3-dimethylbutanoic acid and named as DMBA-CHMINACA.

Determination of Grayanotoxins from Rhododendron brachycarpum in Dietary Supplements and Homemade Wine by Liquid Chromatography-Quadrupole Time-of-Flight-Mass Spectrometry and Liquid Chromatography-Tandem Mass Spectrometry.

A sensitive and specific high-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS) method combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the determination of grayanotoxins I and III in dietary supplements and homemade wine. Grayanotoxins I and III were successfully extracted using solid-phase extraction cartridges, characterized by LC-QTOF-MS, and quantitated by LC-MS/MS. The LC-MS/MS calibration curves were linear over concentrations of 10-100 ng/mL (grayanotoxin I) and 20-400 ng/mL (grayanotoxin III). Grayanotoxins I and III were found in 51 foodstuffs, with quantitative determinations revealing total toxin concentrations of 18.4-101 000 ng/mL (grayanotoxin I) and 15.3-56 000 ng/mL (grayanotoxin III). The potential of the validated method was demonstrated by successful quantitative analysis of grayanotoxins I and III in dietary supplements and homemade wine; the method appears suitable for the routine detection of grayanotoxins I and III from Rhododendron brachycarpum.

Development and validation of rapid and simultaneous method for determination of 12 hair-growth compounds in adulterated products by UHPLC-MS/MS.

Synthetic hair-growth compounds have been illegally used in diverse products to enhance the short-term efficacy of these products. In this study, a rapid and simultaneous method for the determination of hair-growth compounds in adulterated products based on ultra high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed and validated. The limit of detection (LOD) and limit of quantitation (LOQs) of the method were 0.08-43.6ng/mL and 0.27-145ng/mL for the solid-, liquid-, and cream-type samples, respectively. Good calibration linearity for all compounds was demonstrated with a correlation coefficient (r2) higher than 0.997. The intra- and inter-assay precisions were within 11%. The corresponding accuracies were 86-117% and 81-113%, respectively. The mean recoveries obtained for the solid-, liquid, and cream-type samples ranged from 87 to 114%, with a relative standard deviation (RSD) within 6%. The RSD of the stability evaluated at 4°C for 48h was less than 6%. The established method was used to screen 76 samples advertised as hair-growth treatments, from online and offline markets, over the course of two years. In 10% of the samples, four compounds, including triaminodil, minoxidil, finasteride, methyltestosterone, and testosterone-propionate were detected. The concentrations were in the range of 0.5-16.4mg/g. This technique provides a reliable platform for technical analysis for continuous monitoring of adulterated products to protect public health.

Determination of illegal adulteration of dietary supplements with synthetic hair-growth compounds by UPLC and LC-Q-TOF/MS.

In this study, we developed a UPLC-PDA and LC-Q-TOF/MS method to identify and measure the following prohibited substances that may be found in dietary supplements:triaminodil, minoxidil, bimatoprost, alimemazine, diphenylcyclopropenone, α-tradiol, finasteride, methyltestosterone, spironolatone, flutamide, cyproterone, dutasteride, and testosterone 17-propionate.The method was validated according to International Conference on Harmonization guidelines in terms of specificity, linearity, accuracy, precision, LOD, LOQ, recovery, and stability. The method was completely validated showing satisfactory data for all method validation parameters. The linearity was good (R2 > 0.999) with intra- and inter-day precision values of 0.2-3.4% and 0.3-2.9%, respectively. Moreover, the intra- and inter-day accuracies were 87-102% and 86-103%, respectively, and the precision was better than 9.4% (relative standard deviation).Hence, the proposed method is precise and has high quality,and can be utilised to comprehensively and continually monitor illegal drug adulteration in various forms of dietary supplements. Furthermore, to evaluate the applicability of the proposed method, we analysed 13 hair-growth compounds in 78 samples including food and dietary supplements. Minoxidil and triaminodil were detected in capsules at concentrations of 4.69 mg/g and 6.54 mg/g. In addition, finasteride was detected in a tablet at 13.45 mg/g. In addition, the major characteristic fragment ions were confirmed once again using LC-Q-TOF/MS for higher accuracy.

Neurotoxicity of Synthetic Cannabinoids JWH-081 and JWH-210.

Synthetic cannabinoids JWH-018 and JWH-250 in 'herbal incense' also called 'spice' were first introduced in many countries. Numerous synthetic cannabinoids with similar chemical structures emerged simultaneously and suddenly. Currently there are not sufficient data on their adverse effects including neurotoxicity. There are only anecdotal reports that suggest their toxicity. In the present study, we evaluated the neurotoxicity of two synthetic cannabinoids (JWH-081 and JWH-210) through observation of various behavioral changes and analysis of histopathological changes using experimental mice with various doses (0.1, 1, 5 mg/kg). In functional observation battery (FOB) test, animals treated with 5 mg/kg of JWH-081 or JWH-210 showed traction and tremor. Their locomotor activities and rotarod retention time were significantly (p<0.05) decreased. However, no significant change was observed in learning or memory function. In histopathological analysis, neural cells of the animals treated with the high dose (5 mg/kg) of JWH-081 or JWH-210 showed distorted nuclei and nucleus membranes in the core shell of nucleus accumbens, suggesting neurotoxicity. Our results suggest that JWH-081 and JWH-210 may be neurotoxic substances through changing neuronal cell damages, especially in the core shell part of nucleus accumbens. To confirm our findings, further studies are needed in the future.

Dependence Potential of the Synthetic Cannabinoids JWH-073, JWH-081, and JWH-210: In Vivo and In Vitro Approaches.

Synthetic cannabinoids (CBs) such as the JWH series have caused social problems concerning their abuse liability. Because the JWH series produces euphoric and hallucinogenic effects, they have been distributed illegally under street names such as "Spice" and "Smoke". Many countries including Korea have started to schedule some of the JWH series compounds as controlled substances, but there are a number of JWH series chemicals that remain uncontrolled by law. In this study, three synthetic CBs with different binding affinities to the CB1 receptor (JWH-073, 081, and 210) and Δ(9)-tetrahydrocannabinol (Δ(9)-THC) were evaluated for their potential for psychological dependence. The conditioned place preference test (unbiased method) and self-administration test (fixed ratio of 1) using rodents were conducted. Ki values of the three synthetic cannabinoids were calculated as supplementary data using a receptor binding assay and overexpressed CB1 protein membranes to compare dependence potential with CB1 receptor binding affinity. All mice administered JWH-073, 081, or 210 showed significantly increased time spent at unpreferred space in a dose-dependence manner in the conditioned place preference test. In contrast, all tested substances except Δ(9)-THC showed aversion phenomenon at high doses in the conditioned place preference test. The order of affinity to the CB1 receptor in the receptor binding assay was JWH-210 > JWH-081 >> JWH-073, which was in agreement with the results from the conditioned place preference test. However, no change in self-administration was observed. These findings suggest the possibility to predict dependence potential of synthetic CBs through a receptor binding assay at the screening level.

Quantitative determination of duloxetine and its metabolite in rat plasma by HPLC-MS/MS.

The major metabolite of duloxetine is a glucuronide conjugate of 4-hydroxy duloxetine (4-HD). However, interestingly, there have been no reports determining concentrations of 4-HD and no fully validated method has been established for measuring duloxetine and 4-HD in rat plasma. We developed a method for the simultaneous quantification of duloxetine and its metabolite in rat plasma using high-performance liquid chromatography tandem mass spectrometry. Duloxetine and 4-HD were analyzed on a reverse-phase C18 analytical column after protein precipitation of the plasma sample with methanol, using carbamazepine as an internal standard. The isocratic mobile phase of 5 mm ammonium acetate-methanol (4:6, v/v) was eluted at 0.4 mL/min. Quantification was performed on a triple-quadrupole mass spectrometer using electrospray ionization, and the ion transition monitored in selective reaction monitoring mode. The coefficient of variation for assay precision was <18.0%, and the accuracy was 84.0-118.0%. This method was successfully used to measure the concentrations of duloxetine and its metabolite in plasma following the oral administration of a single 40 mg/kg dose in rats.

Gene expression profile by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the liver of wild-type (AhR+/+) and aryl hydrocarbon receptor-deficient (AhR-/-) mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most toxic environmental pollutants that cause various biological effects on mammals. The purpose of our study was to identify the genes involved in hepatotoxicity and hepatocarcinogenesis caused by TCDD. C57BL/6 (AhR+/+, wild type) and B6.129-AhR/J (AhR-/-, knock out) mice were injected i.p. with a single treatment of TCDD at the dose of 100 microg/kg body weight. Relative liver weight was significantly increased at 72 hr after TCDD treatment without an apparent histopathological change in AhR+/+ mice (p<0.05). TCDD treatment also significantly increased activity of serum alanine aminotransferase in AhR-/- mice (p<0.05). The liver was analyzed for gene expression profiles 72 hr later. As compared with AhR-/- mice, the expression of 51 genes (>3-fold) was changed in AhR+/+ mice; 28 genes were induced, while 23 genes were repressed. Most of the genes were associated with chemotaxis, inflammation, carcinogenesis, acute-phase response, immune responses, cell metabolism, cell proliferation, signal transduction, and tumor suppression. This study suggests that the microarray analysis of genes in the liver of AhR+/+ and AhR-/- mice may help to clarify the mechanism of AhR-mediated hepatotoxicity and hepatocarcinogenesis by TCDD.

Importance of CD8(+)Vbeta8(+) T cells in IFN-gamma-mediated prevention of toxoplasmic encephalitis in genetically resistant BALB/c mice.

In our attempt to identify a major T cell population(s) that recognizes protective Toxoplasma gondii antigens and produces interferon-gamma (IFN-gamma) for prevention of toxoplasmic encephalitis (TE), we found T cell receptor Vbeta8(+) cells to be the most frequent IFN-gamma-producing population infiltrated into the brain of T. gondii-infected BALB/c mice genetically resistant to the disease. To examine the role of IFN-gamma production by this T cell population for resistance, we transferred Vbeta8(+) immune T cells purified from spleens of infected BALB/c and IFN-gamma(/) mice into infected, sulfadiazine-treated, athymic nude mice. After discontinuation of sulfadiazine treatment, control nude mice that had not received any T cells and animals that had received Vbeta8(+) T cells from IFN-gamma(/) mice all died because of reactivation of infection (TE). In contrast, animals that had received the cells from BALB/c mice survived. Thus, IFN-gamma production by Vbeta8(+) T cells plays an important role in prevention of TE in these animals. When Vbeta8(+) immune T cells were divided into CD4(+) and CD8(+) subsets, a potent protective activity was observed only in the CD8(+) subset, whereas a combination of both subsets provided greater protection than did the CD8(+)Vbeta8(+) population alone. These results indicate that the CD8(+) subset of Vbeta8(+) T cells is a major afferent limb of IFN-gamma-mediated resistance of BALB/c mice against TE, although the CD4(+) subset of the T cell population works additively or synergistically with the CD8(+)Vbeta8(+) population.

Gamma interferon production, but not perforin-mediated cytolytic activity, of T cells is required for prevention of toxoplasmic encephalitis in BALB/c mice genetically resistant to the disease.

We previously showed the requirement of both T cells and gamma interferon (IFN-gamma)-producing non-T cells for the genetic resistance of BALB/c mice to the development of toxoplasmic encephalitis (TE). In order to define the role of IFN-gamma production and the perforin-mediated cytotoxicity of T cells in this resistance, we obtained immune T cells from spleens of infected IFN-gamma knockout (IFN-gamma(-/-)), perforin knockout (PO), and wild-type BALB/c mice and transferred them into infected and sulfadiazine-treated athymic nude mice, which lack T cells but have IFN-gamma-producing non-T cells. Control nude mice that had not received any T cells developed severe TE and died after discontinuation of sulfadiazine treatment due to the reactivation of infection. Animals that had received immune T cells from either wild-type or PO mice did not develop TE and survived. In contrast, nude mice that had received immune T cells from IFN-gamma(-/-) mice developed severe TE and died as early as control nude mice. T cells obtained from the spleens of animals that had received either PO or wild-type T cells produced large amounts of IFN-gamma after stimulation with Toxoplasma gondii antigens in vitro. In addition, the amounts of IFN-gamma mRNA expressed in the brains of PO T-cell recipients did not differ from those in wild-type T-cell recipients. Furthermore, PO mice did not develop TE after infection, and their IFN-gamma production was equivalent to or higher than that of wild-type animals. These results indicate that IFN-gamma production, but not perforin-mediated cytotoxic activity, by T cells is required for the prevention of TE in genetically resistant BALB/c mice.

Differential protein expressions induced by adenovirus-mediated p16 gene transfer into Balb/c nude mouse.

To evaluate the safety of adenovirus-mediated gene transfer, we investigated differential protein expression after transducing adenoviral vector containing the p16(INK4a) tumor suppressor gene (Ad5CMV-p16) into Balb/c nude mice. We found that adenovirus-mediated p16(INK4a) gene transfer inhibited experimental lung metastasis, and that the intratumoral injection of Ad5CMV-p16 resulted in regression of A549 cell xenografted tumors in Balb/c nude mice. We investigated changes in protein expression after intratumoral injection of Ad5CMV-p16 or Ad5CMV (10(10) plaque-forming units) into A549 cell xenografted Balb/c nude mice by two-dimensional gel electrophoresis /matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Compared with the control (serum-free medium treated tumor cells) Ad5CMV-p16 gene transfer changed the expression of 29 proteins including heterogeneous nuclear ribonucleoprotein, protein phosphatase 2, 14-3-3 zeta protein, alpha-tubulin, and glutathione-S-transferase P1. Moreover, both Ad5CMV-p16 and Ad5CMV up-regulated the expression of glutathione-S-transferase P1. In addition, Ad5CMV-p16 gene transfer did not seem to increase the expression of tumorigenicity-related protein in Balb/c nude mice. Further studies will be needed to investigate the effect of Ad5CMV-p16 on normal human cells and tissues for safety evaluation. These results suggest that the p16 gene seems to have an important role in apoptosis as well as in cell cycle arrest in non-small cell lung cancer.

Oxidative DNA damage and glioma cell death induced by tetrahydropapaveroline.

A series of naturally occurring isoquinoline alkaloids, besides their distribution in the environment and presence in certain food stuffs, have been detected in human tissues including particular regions of brain. An example is salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) that not only induces neuronal cell death, but also causes DNA damage and genotoxicity. Tetrahydropapaveroline [THP; 6,7-dihydroxy-1-(3',4'-dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline], a dopamine-derived tetrahydroisoquinoline alkaloid, has been reported to inhibit mitochondrial respiration and is considered to contribute to neurodegeneration implicated in Parkinson's disease. Since THP bears two catechol moieties, the compound may readily undergo redox cycling to produce reactive oxygen species (ROS) as well as toxic quinoids. In the present study, we have examined the capability of THP to cause oxidative DNA damage and cell death. Incubation of THP with phiX174 supercoiled DNA or calf thymus DNA in the presence of cupric ion caused substantial DNA damage as determined by strand scission or formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), respectively. THP plus copper-induced DNA damage was ameliorated by some ROS scavengers/antioxidants and catalase. Treatment of C6 glioma cells with THP led to a concentration-dependent reduction in cell viability, which was prevented by the antioxidant N-acetyl-L-cysteine. When these cells were treated with 10microM THP, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were rapidly activated via phosphorylation, whereas activation of extracellular signal-regulated protein kinase (ERK) was inhibited. Furthermore, pretreatment with inhibitors of JNK and p38 MAPK rescued the glioma cells from THP-induced cytotoxicity, suggestive of the involvement of these kinases in THP-induced C6 glioma cell damage.