EM - IUEM - Técnicas Laboratoriais Forenses
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- Analysis of tobacco and cannabis smoke and its toxicological impact in neuroblastoma tumour cellsPublication . Grafinger, Katharina; Quintas, Alexandre; Costa, Joana Couceiro daThe dried products of Cannabis sativa have been consumed for their psychoactive effects for thousands of years. Almost five hundred different natural cannabinoids could be identified in Cannabis sativa with Δ9-THC being the most psychoactive one. The most effective way of consumption is by smoking, because it avoids digestion and the first pass in the liver. Usually so called "joints" are rolled in a 1:1 ratio of dried cannabis and tobacco filler. The reason for this is that cannabis has a lower burning efficiency and that tobacco is cheaper. Processed Nicotina tabaccum contains about 0.6- 9 % nicotine; an oily, volatile liquid alkaloid. Pyrolysis of tobacco and cannabis leads to the formation of new products whose cytotoxicity is not yet established. This study investigates the effect of smoke of tobacco, cannabis and their mixture in similar conditions as humans uptake it. For production of smoke an in-house developed smoking device was used which trapped the water-soluble and organic-soluble molecules separately. Qualitative analysis of the smoke was performed using GC-MS and measurement of cytotoxicity on neuroblastoma tumour cells SH-SY5Y was performed using the MTT assay. SH-SY5Y cell line selectively expresses Cannabinoid 1 (CB1) receptor and Nicotinic acetylcholine receptor (nAChR). The results allow us to identify several pyrolysated products which are hydroxylated forms of nicotine and Δ9-THC. Moreover, MTT analysis strongly suggests different toxicological impact between smoke of tobacco, cannabis and their mixture. In fact our results points smoke from the mixture of tobacco and cannabis having the higher impact on cell viability followed by the smoke of tobacco which in turn is more toxic than smoke of cannabis, the least toxic compound out of these three. This study shows that at 100 μM cannabis has a cell viability of 80 % while tobacco and the mixture have around 40 %.
- 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.
- 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.
- “Charlie” (buphedrone/ethcathinone) behavioural patterns after stimulated binge administrationPublication . Sola Sancho, Sofía; Lopes, Álvaro TeixeiraSince the entry of XXI century, the consume, availability and variety of new psychoactive substances also called “legal highs” “legal drugs” or “design drugs,” have been rising enormously. This tendency has increased the concern of both, health care providers and legal authorities about this family of drugs. This unique study is a first toxicological approach to a drug named “Charlie”. Effects for a repeated administration of a singular dose of 45mg/kg has been assessed. “Charlie” is a combination of two synthetic cathinones (Buphedrone and Ethcathinone) with no toxicological or behavioural studies done, until the date. For developing the study a binge administration was performed in a ten days study with the combination of the drug with ethanol in a dose of 2g/kg. For the toxicological assessment, adolescent male CD1 mice were used and four behavioural tests were chosen, Functional Observational Battery (FOB), Open Field (OF) test, Marble burying test and T-maze test. Results showed the significant differences between controls and mice under the influence of “Charlie” or under the mixture of “Charlie” and ethanol at all performed tests. The combination cathinone-ethanol showed mayor effects in the variables for FOB and OF than when the drug was administered alone ad different behavioural patterns in Marble and T-Maze tests. The study of the weights and food and water intake before and during the experiments was also assessed and results pointed the variations in weight and food intake during the days of the experiment suffering a marked decrease in those days. With this study it is demonstrated the influence of “Charlie” in the behaviour and physiological conditions of the individuals under its effects by using behavioural tasks for the assess and opening a new “file” in the cathinones behavioural assessment.
- 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.
- 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
- A contribution for medico-legal estimation of age in portuguese population using the London atlas of tooth developmentPublication . Pavlovic, Strahinja; Pereira, Cristiana Palmela; Santos, Rui Filipe Vargas de SousaChronological age estimation from the dental parameters is becoming one of the fundaments of Forensic Science. Numerous body traits can be used for this purpose, but dental parameters are considered the most reliable ones. The London atlas of tooth development is the most recent developed method for dental age estimation and represents a modification of the previous older methods. The aim of this study was to evaluate the accuracy of the London atlas in a sample of a Portuguese population. The sample included 736 radiographic images (498 females and 238 males) of Portuguese origin, patients of Dental Clinic of Superior Institute of Health Sciences Egas Moniz and Dental Medicine Faculty, Univeristy of Lisbon. The age range of the individuals was between 3 and 24 years. Several tests were applied in order to evaluate the London atlas. All the statistical analysis were based on the paired t-test. The results showed that there was no statistically significant difference between left and right side of the jaw (p > 0.05). Both sides showed an average overestimation of age by one month approximately. Moreover, the significant difference between chronological and estimated age was not observed in the females. However, the significant difference was observed in a sample coming from males (right: p = 0.008; left: p = 0.003). Additionally, there was a significant difference in a population coming from Institute Egas Moniz (p <0.05). Our results indicated that the London atlas can be potentially used as a tool for age estimation, especially in cases when all teeth finished their development. However, the difference between sexes clearly suggests that separate charts should be made for each sex. Further studies, which will have as a final goal development of a new method for age estimation using dental parameters, are needed.
- Contribution to portuguese urban fire investigations: forensic fire debris analysisPublication . Oliverio, Stefania; Noronha, João Paulo; Carvalho, AntónioFire scene investigation and fire debris analysis (FDA) are two tightly linked and mutually exclusive key elements of a forensic arson investigation. While the fire scene investigation is performed by a trained fire investigator, FDA is a task undertaken by a fire debris analyst, which is normally a forensic chemist with expert knowledge in analytical chemistry. To solve a case efficiently and successfully, cooperation and information- and knowledge-exchange are necessary, especially since this branch of forensic science is defined as one of the most complex and arduous ones due to the highly destructive nature of fires that severely compromises the integrity of the evidence collected at the crime scene. The task of analysing the fire debris for the detection of ignitable liquid residues (ILRs) is further complicated by the large influence and existence of interfering products. Most ignitable liquids (ILs) are made of hydrocarbons, which are also compounds that have been found in the substrate of common household or construction materials as well as being created during the processes of combustion and pyrolysis. The purpose of this study was to identify and characterize background, combustion and pyrolysis products of commonly encountered substrate materials in fire scenes in Portugal and observe the effects on ILRs identification by using headspace solid-phase micro-extraction linked to gas chromatography-mass spectrometry (HS-SPME-GC-MS). The results show that both the background of the substrates and the combustion and pyrolysis products formed during a fire include many compounds that constitute common ILs, such as alkanes and aromatics, that especially in the presence of low amount of ILRs constitute a problem in the conclusive identification of ILs. Even though detailed analysis of the chromatographic and mass-spectral patterns and the combinations of compounds detected in the materials can avoid misinterpretation, in some cases, awareness and knowledge of interfering products is essential.
- Evaluation of current and novel methods for the storage of DNA-extractsPublication . Sjoukema, Pieterjan; Barroso, Maria Helena; Carvalho, RaquelSecure preservation of DNA-extracts has been an issue at forensic laboratories for a long time. DNA-extracts are commonly stored at either -20°C or -80°C, but storage at these temperatures is not without risk of failure, environment unfriendly and costly. The Laboratório de Polícia Científica (LPC; Forensic Science Laboratory) of the Polícia Judiciária (PJ; Judicial Police) in Lisbon, Portugal is currently looking into new ways of storing their DNA-extracts at room temperature. Both DNAstable® and GenTegra-DNA®, two commercially available products for the storage of DNA-extracts at ambient temperatures, have been evaluated. DNA samples with extremely low concentrations of DNA (0.1 ng/μL, 0.05 ng/μL and 0.025 ng/μL) and DNA-extracts extracted from buccal swabs with the SwabSolution™ kit (Promega, Madison, USA) have been stored in standard polypropylene tubes, DNAstable® tubes and GenTegra-DNA® tubes for 8 days at four different conditions, i.e., -20°C, 4°C, at room temperature and at 60°C. Besides this, randomly selected DNA-extracts that have been stored at -20°C since 2006, 2009 or 2013 have been reanalyzed with the currently at the LPC used techniques in order to assess the level of degradation after long-term storage at -20°C. Results showed that DNA degrades over time at -20°C, with recovery levels found as low as 20% after three years of storage at -20°C. DNAstable® and GenTegra-DNA® both showed to be able to preserve DNA dried at room temperature and 60°C. Given the short period of storage full DNA-profiles have also been obtained from samples stored in standard polypropylene tubes at room temperature. Significant differences were found between DNA-extracts stored at 60°C with either DNAstable® or GenTegra-DNA® and those stored in unprotected tubes, showing that both DNAstable® and GenTegra-DNA® are capable of storing DNA at room temperature for long periods of time with a similar or even higher level of protection compared to those achieved with conventional storage at -20°C. Nevertheless, more research is needed before a switch can be made from frozen storage at -20°C to the storage of DNA-extracts at room temperature with products such as DNAstable® and GenTegra-DNA®.
- 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.