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- Toxicological profile of JWH-018 and JWH-018 N-(3-Hydroxypentyl) metabolite in the HEK 293 T cell linePublication . Sultan, Haider; Quintas, Alexandre; Dias, Mário JoãoThe rise in use of synthetic cannabinoids as herbal smoking mixtures has been attributed to a number of reasons; primary among them being their “legality” and their promotion as being safer than the natural alternatives. As their emergence has been quite recent, their pharmacological and toxicological profiles are not known completely. One of the earliest synthetic cannabinoid to be detected in these herbal mixtures was JWH-018. In the present study, the cell toxicity of JWH-018 and its N-(3-hydroxypentyl) metabolite was investigated using human cell line HEK-293 T. The cell viability was assessed using the MTT assay after treatment with standard JWH-018 and its N-hydroxymetabolite in a concentration range between 1ng/ml to 5000ng/ml of both respectively. The results show that the highest concentration of 5000ng/ml shows statistically significant decrease in cell viability for both the parent compound and the metabolite when compared to cell controls. In addition, the metabolite also showed a statistically significant decrease in cell viability at a concentration of 2500ng/ml. There were also significant differences observed between the smaller concentrations (1ng/ml and 10ng/ml) when compared to the highest concentration (5000ng/ml) for both the parent compound and metabolite. The cytotoxic effect was seen at levels which are above the expected tissue levels after use of JWH-018 either by ingestion or smoking. The cytotoxicity of both the parent compound and the metabolite highlights the adverse effects these drugs may have with consequent need for further research and regulation.
- Synthetic cannabinoid method development with Saccharomyces cerevisiae and Escherichia coli proteome patternsPublication . Olipoh, George; Quintas, Alexandre; Costa, Isabel MargaridaThe emergence and abuse of new synthetic cannabinoids has been on the rise because legal regulations against particular derivatives of cannabinoids tend to cause the introduction of new variants as the law is made for specific molecules and not for a group of molecules. They come with no clinical indications of use on their labels, and vaguely described intoxicating effects hence they were not banned. Consequently, they are also referred to as ‘Smart Drugs’ or more formally ‘New Psychoactive Substances’ (NPSS). Many classified synthetic cannabinoid agonists remain available to users in many countries mainly through ‘smart’ and online shops. The challenges posed to the regulation of these products are due to their ever-changing composition, adulterants and variable vehicles of distribution. Synthetic cannabinoids are famous for their recreational effects similar to that of marijuana, which has neuropsychiatric effects due to the action of its primary active ingredient, Δ9-tetrahydrocannabinol (Δ9-THC), binding to endogenous cannabinoid receptors. This study was conducted first to find the best microorganism model for the development of a new screening method for the analysis of synthetic cannabinoids by using the proteome expression patterns obtained from proteins of Saccharomyces cerevisiae and E. coli. The research also sought to find an efficient protein extraction and concentration method for expressed microorganism proteins for two-dimension gel electrophoresis. Saccharomyces cerevisiae and Escherichia coli were studied in this work. S. cerevisiae rather than E. coli proved to be a better microorganism model for to be exploited for findings in this research. Diclofenac and JWH-018 standard were used as test drugs. The Amicon® membrane filter concentration method was found to be the most efficient with protein yield greater than 20mg/mL for 500mL cell culture volume. Even though twodimension gels did not show a large number of protein spots, the application of more concentrated protein solution from S. Cerevisiae is paramount to get a better proteomic pattern for use as a sensor.
- Identification of HIV-1 transmission clusters: application in epidemiological studies and forensic casesPublication . Zaković, Suzana; Taveira, NunoIn this study, HIV-1 transmission clusters were analysed, to identify transmission networks in Angola and investigate their viral and host correlates, and to identify relatedness of strains in a potential HIV-1 criminal transmission in Portugal. Two methods of cluster analysis, visual inspection and automatic search of reliable nodes on the phylogenetic trees, were applied on the partial pol gene from 365 HIV-1 infected Angolans. Afterwards, HIV-1 diversity and transmission dynamics were analysed in identified transmission networks. It was found that 44.65% of analyzed individuals were part of the transmission networks. The majority (73%) from 63 identified clusters were pairwise, and the largest clusters (n=3) were comprised of 7 individuals, suggesting that current drivers of local epidemics are large number of small clusters. Besides, slow transmission dynamics was identified (median=27 months), which is, together with prevalence of small clusters, concordant with main mode of HIV transmission in Angola, heterosexual transmission. Extreme diversity of subtypes and gradual changes of subtype distribution were identified. This was observed in increased frequency of subtypes A (19.75%), and recombinant CRF02_AG (11.11%), which could indicate on their preferential transmission, and in frequency of CRF18_cpx and CRF37_cpx, that had established successful transmission in the country in recent years. Moreover, potential criminal HIV-1 transmission, involving a father and a son, was analyzed with phylogenetic analysis. Based on analysis of three HIV-1 gene fragments, this relationship could not be refuted. However, although epidemiological link was confirmed, deliberate transmission could not be proved, nor the presence of the third party could be excluded. Understanding the epidemiology of HIV infection is a prerequisite to address public health issues and implement HIV preventive measurements. This study has showed that transmission clusters identification, is a useful tool to approach these issues through analysis of transmission dynamics, viral phenotypic and genotypic properties, trends of current HIV diversity and HIV forensics.
- Chemical characterization of "legal highs" containing 4-fluoroamphetamine and synthetic cathinonesPublication . Rodriguez Zancajo, Victor Manuel; Lopes, Álvaro TeixeiraThe number of “Smart Shops” and online distributors of “Legal highs” have proliferated in the last years, they are becoming a thread to public health and an international motive of concern. Unfortunately, there is very little chemical or toxicological literature on these substances. This study presents the results of the analysis of fourteen “legal highs” that were purchased in “Smart Shops” in Portugal in February 2013. These substances were analysed by a wide variety of sophisticated analytical methods including Fourier transform infrared (FTIR), Gas chromatography couple with mass spectrometry (GCMS), Proton nuclear magnetic resonance (1H NMR) and Wave-length dispersive X-ray fluorescence (WD-XRF). The next synthetic cathinones were detected: flephedrone, buphedrone, ethcathinone, 4-methylethcathinone, dimethylmethcathinone and methedrone. 4-fluoroamphetamine was also identified and all were confirmed by at least one different technique. Analytical data of these substances are presented. Methedrone, which was present in three products, is a cathinone derivative responsible for some fatalities. A particularly high toxicity and narrow therapeutic window linked with the methoxy group have been attributed to methedrone. Data obtained indicate products of high purity with small amounts of caffeine. Active ingredients were found on its own or in mixtures in the different products, and great inconsistencies and erroneous information were observed after evaluation of the packages of products. Legal status of these substances is changing continuously, and most of the chemicals that have been detected in this study were banned in Portugal some weeks after the acquisition of the products. Thus, they belong now to the illegal market of stimulants of abuse. Elemental composition of the products is also presented, obtained by XRF analysis. X-ray irradiation resulted in an unanticipated change in colour at different degree on the products. It was demonstrated that this change in colour did not chemically altered the compounds present in the samples, making XRF a suitable technique for producing inorganic profiles of samples containing cathinones.
- Application of SERS with raman reporter-labelled metallic nanoparticles for latent fingermark enhancementPublication . Echeonwu, Bobmanuel Chimaroke; Croxton, Ruth S.The importance of human fingermark identification and individualization for forensic purposes cannot be overemphasized. Consequently, the need for robust and effective techniques explains the continued and intensified research in this field of forensics despite the fact that there are already numerous operational techniques available to forensic identification experts. Novel approaches have recently been adopted to understand the chemistry of latent fingermarks and produce reagents targeting some of these constituents of latent fingermarks. Nanotechnology and surface-enhanced Raman spectroscopy (SERS) are gaining more attention in this area of endeavor, where nanoparticle-based reagents are increasingly being developed and optimized for application in latent fingermark enhancement and chemical imaging of such latent fingermarks. In this study, surface-enhanced Raman spectroscopy has been used to investigate the applicability of Raman reporter-labeled metallic nanoparticles to the enhancement of latent fingermarks. Rhodamine 6G and 4-mercaptobenzoic acid have been investigated as Raman reporters adsorbed on either gold or silver nanoparticles. The functionalized nanoparticles were deposited onto latent fingermark samples and characteristic Raman vibrational bands of the reporter molecules when adsorbed on nanoparticles (1076 cm-1 and 1583 cm-1 for 4-mercptobenzoic acid; 610 cm-1, 1360 cm-1 1505 cm-1 and 1648 cm-1 for rhodamine 6G) were monitored and showed very low variability of peak position between samples (coefficient of variation generally < 0.5%). Adsorption of reporter molecules on nanoparticles was characterized using UV-vis absorption spectroscopy as well as the behaviour of characteristic Raman shifts of the molecules which gave an indication of successful adsorption. Rhodamine 6G in this study however had a weak adsorption onto silver nanoparticles as a result of suboptimal amounts of the dye molecule used. Preferential deposition of the Raman reporter-labeled nanoparticles on fingermark ridges was also observed with fingermarks deposited on non-porous surface (glass slide). This study demonstrates the potential for SERS chemical imaging of latent fingermarks based on characteristic SERS vibrational bands from reporter molecules adsorbed on metallic nanoparticles and deposited onto latent fingermarks.
- A comprehensive study of herbal blends from portuguese smart-shops: isolation, analysis and toxicological impactPublication . Lopes, Inês Costa; Quintas, Alexandre; Soeiro, RitaIn the last few years, several samples were found to contain different combinations of multiple synthetic cannabinoids, fact which may end up in a serious health threat since these substances are not fully studied and the toxic as well as the pharmacological effects are not known. The isolation and quantification of psychoactive substances from the herbal blends, known as ‘Spice’, was performed by HPLC-UV and the identification by LC-MS/MS. The HPLC-UV method was validated for the quantification and has proven to be fit for purpose. The sample 1 has shown the coexistence of JWH-122 (29,07mg/g) and JWH-210 (175,9mg/g) while the sample 2 was found to contain one single synthetic cannabinoid, the JWH-018 (149,7mg/g). The sample 3 presented the same multiple psychoactive substances as sample 1, JWH-122 (7,3mg/g) and JWH-210 (129,9mg/g). Since these drugs are mainly smoked, a smoking machine was created* in order to characterize the chemical composition of the pyrolysis. The GC-MS analysis performed to the pyrolysis solutions has shown no degradation of the JWH-018, drug used for the assay. The toxicity impact of the characterized substances was assessed on human neuroblastoma SH-SY5Y cells, applying a combination of multiple synthetic cannabinoids (JWH-122 and JWH-210) and single one (JWH-018). The preliminary results indicate that these psychoactive substances are not toxic to the cells in the concentrations ranging from 0μM to 50μM. *Designed by Dr. Alexandre Quintas and is under a patenting process
- Mapping portuguese soils using spectroscopic techniques with a machine learning approachPublication . Stafford, Hannah; Família, Carlos; Faria, MafaldaSoil analysis is an important part of forensic science as it can provide vital links between a suspect and a crime scene based on its characteristics. The use of soil in a forensic context can be characterised into two categories: intelligence purposes or court purposes. The core basis of the comparison of sites to determine the provenance is that soil composition, type etc. vary from one place to another. The aim of this project is to ‘map’ soils and predict the location of a sample of unknown origin based on the chemometric profiles of Fourier transform infrared (FTIR) spectra, micro x-ray fluorescence profiles and visible spectra. Thirty one samples were collected in triplicate from Monsanto Park in Lisbon for each predetermined collection point on a defined grid. Full FTIR spectra (400-4000cm-1), Visible (1100-401cm-1) spectra, UV (400-200cm-1) spectra and μXRF profiles were collected for all samples. A subset of 43 discriminant features was selected from a total of 1430 using the Boruta feature selection algorithm from the FTIR, μXRF and visible spectra. These discriminant features acted as input data that was used to create a neural network which allowed the prediction of Cartesian co-ordinates (or location) of the samples with a high degree of accuracy (86%) and has shown to be a very useful approach to predict soil location.
- Assessment of neuronal cytotoxicity of JWH-073 and JWH-250Publication . Pereira, Carlos Victor Montefusco; Quintas, Alexandre; Costa, Joana Couceiro daSynthetic cannabinoids from marijuana herbal blends like ‘Spice’ and ‘K2’ are drawing the attention of drug of abuse organizations, including the UNODC1, the EMCCDA2 and emergency hospital all over the world. This concern rises from clinical episodes of psychotropic effects that go beyond the regular range of marijuana and THC – namely, panic attacks, psychosis, catatonia, addiction and withdrawal symptoms. Our study addressed two emergent synthetic cannabinoids (napthtoylindoles) denominated JWH-073 and JWH-250 that are currently detected on ‘Spice’-like products, in order to observe their cell toxicity profile on neuronal cells in vitro model (SH-SY5Y). Using 0.2% DMSO as negative control, MTT and LDH results revealed that within concentrations of 1, 5, 10, 25, 37.5 and 50 μM, JWH-250 is identified as ‘toxic’ in a statistically significant manner at higher concentrations. This work did not detect any statistically significant toxicity from JWH-073. This data suggests to extend these studies on new synthetic cannabinoids to neuronal cells with increased concentrations, as well as the application of assays assessing apoptosis (conditions and signalling), neuronal function and activity (as cell membrane potential assay) within differentiated cells as neurons and glia. At the same time, the evaluation of herbal mixtures of more than one cannabinoids and plant types is advisable in order to understand synergic effects.
- The use of wavelength-dispersive x-ray fluorescence (WDXRF) spectroscopy and multivariate techniques for the assessment of illegal dyes in spicesPublication . Adusei, Emmanuel; Gonçalves, Luísa LimaSudan dyes are synthetic azo and diazo compounds that are banned for use in food worldwide including the European Community due to their potential toxicity to humans. The ability of WDXRF spectroscopic technique to predict the levels of adulteration of paprika and sweet pepper suspected to be adulterated with Sudan I-IV, Para Red and Sunset Yellow FCF dyes was investigated in this study. Logistic regression and discriminant analysis classification models were developed to predict the type of adulteration using WDXRF spectral features such as the Compton and Rayleigh scatter intensities and the Compton and Rayleigh ratios. Prediction of the levels of adulteration was assessed by using multiple regression analysis. 83% of the 210 adulterated samples were correctly classified by the logistic regression with 90% sensitivity, 75%specificity with a prediction power of 92% into respective adulteration groups. 86% and 90% correct prediction were obtained for discriminant analysis models with 94% sensitivity and 74% specificity. Three multiple regression models were performed for each data set. Models based on the Compton and Rayleigh ratios, Compton and Rayleigh scatter intensities as well as a full model based on both ratios and scatter intensities were developed and compared with each other. The full model revealed to be the best model to predict the levels of adulteration with an adjusted R2 between 95.1 to 99.0% with the errors of estimate between 1.6 to 3.7%.
- Use of aspartic acid racemization in fingerprints as a molecular clockPublication . Adelantado Sánchez, Carlos; Quintas, Alexandre; Soeiro, RitaAspartic acid is the chemical, amongst amino acids, whose racemization process is considered to be the fastest and the most reliable way to conduct an ageing study in some living-being tissues. Racemization is a natural-occurring event in which L-stereoisomer of aspartic acid is converted into D-stereoisomer in an equilibrium dependent on factors namely pH, environmental conditions and, most critically, on temperature.