In vitro studies of drug transformations: application to forensic toxicology

The forensic toxicologist faces challenges in the detection of drugs and poisons in biological samples due to transformations which occur both during life and after death. For example, changes can result from drug metabolism during life or from the use of formalin solution for post mortem embalming purposes. The former requires the identification of drug metabolites and the latter the identification of chemical reaction products in order to know which substances had been administered. The work described in this thesis was aimed at providing ways of tackling these challenges and was divided into two parts. Part 1 investigated the use of in vitro drug metabolism by human liver microsomes (HLM) to obtain information on drug metabolites and Part 2 investigated the chemical reactions of drugs and a carbamate pesticide with formalin solution and formalin-blood. The initial aim of part I was to develop an in vitro metabolism method using HLM, based on a literature review of previous studies of this type. MDMA was chosen as a model compound to develop the HLM method because its metabolism was known and standards of its metabolites were commercially available. In addition, a sensitive and selective method was developed for the identification and quantitation of hydrophilic phase I drug metabolites using LC/MS/MS with a conventional reverse-phase (C18) column. In order to obtain suitable retention factors for polar drug metabolites on this column, acetyl derivatives were evaluated for converting the metabolites to more lipophilic compounds and an optimal separation system was developed. Acetate derivatives were found to be stable in the HPLC mobile phase and to provide good chromatographic separation of the target analytes. In vitro metabolism of MDMA and, subsequently, of other drugs involved incubation of 4 µg drug substance in pH 7.4 buffer with an NADPH generating system (NGS) at 37oC for 90 min with addition of more NGS after 30 min. The reaction was stopped at 90 min by the addition of acetonitrile before extraction of the metabolites. Acetate derivatives of MDMA metabolites were identified by LC/MS/MS using multiple reaction monitoring (MRM). Three phase I metabolites (both major and minor metabolites) of MDMA were detected in HLM samples. 3,4-dihydroxy-methamphetamine and 4-hydroxy-3-methoxymethamphetamine were found to be major metabolites of MDMA whereas 3,4-methylenedioxyamphetamine was found to be a minor metabolite. Subsequently, ten MDMA positive urines were analysed to compare the metabolite patterns with those produced by HLM. An LC/MS method for MDMA and its metabolites in urine samples was developed and validated. The method demonstrated good linearity, accuracy and precision and insignificant matrix effects, with limits of quantitation of 0.025 µg/ml. Moreover, derivatives of MDMA and its metabolites were quantified in all 10 positive human urine samples. The urine metabolite pattern was found to be similar to that from HLM. The second aim of Part 1 was to use the HLM system to study the metabolism of some new psychoactive substances, whose misuse worldwide has necessitated the development of analytical methods for these drugs in biological specimens. Methylone and butylone were selected as representative cathinones and para-methoxyamphetamine (PMA) was chosen as a representative ring-substituted amphetamine, because of the involvement of these drugs in recent drug-related deaths, because of a relative lack of information on their metabolism, and because reference standards of their metabolites were not commercially available. An LC/MS/MS method for the analysis of methylone, butylone, PMA and their metabolites was developed. Three phase I metabolites of methylone and butylone were detected in HLM samples. Ketone reduction to β-OH metabolites and demethylenation to dihydroxy-metabolites were found to be major phase I metabolic pathways of butylone and methylone whereas N-demethylation to nor-methylone and nor-butylone were found to be minor pathways. Also, demethylation to para-hydroxyamphetamine was found to be a major phase I metabolic pathway of PMA whereas β-hydroxylation to β-OH-PMA was found to be a minor pathway. Formaldehyde is used for embalming, to reduce decomposition and preserve cadavers, especially in tropical countries such as Thailand. Drugs present in the body can be exposed to formaldehyde resulting in decreasing concentrations of the original compounds and production of new substances. The aim of part II of the study was to evaluate the in vitro reactions of formaldehyde with selected drug groups including amphetamines (amphetamine, methamphetamine and MDMA), benzodiazepines (alprazolam and diazepam), opiates (morphine, hydromorphone, codeine and hydrocodone) and with a carbamate insecticide (carbosulfan). The study would identify degradation products to serve as markers for the parent compounds when these were no longer detectable. Drugs standards were spiked in 10% formalin solution and 10% formalin blood. Water and whole blood without formalin were used for controls. Samples were analysed by LC/MS/MS at different times from the start, over periods of up to 30 days. Amphetamine, methamphetamine and MDMA were found to rapidly convert to methamphetamine, DMA and MDDMA respectively, in both formalin solution and formalin blood, confirming the Eschweiler-Clarke reaction between amine-containing compounds and formaldehyde. Alprazolam was found to be unstable whereas diazepam was found to be stable in both formalin solution and water. Both were found to hydrolyse in formalin solution and to give open-ring alprazolam and open-ring diazepam. Other alprazolam conversion products attached to paraformaldehyde were detected in both formalin solution and formalin blood. Morphine and codeine were found to be more stable than hydromorphone and hydrocodone in formalin solution. Conversion products of hydromorphone and hydrocodone attached to paraformaldehyde were tentatively identified in formalin solution. Moreover, hydrocodone and hydromorphone rapidly decreased within 24 h in formalin blood and could not be detected after 7 days. Carbosulfan was found to be unstable in formalin solution and was rapidly hydrolysed within 24 h, whereas in water it was stable up to 48 h. Carbofuran was the major degradation product, plus smaller amounts of other products, 3-ketocarbofuran and 3-hydrocarbofuran. By contrast, carbosulfan slowly hydrolysed in formalin-blood and was still detected after 15 days. It was concluded that HLM provide a useful tool for human drug metabolism studies when ethical considerations preclude their controlled administration to humans. The use of chemical derivatisation for hydrophilic compounds such as polar drug metabolites for analysis by LC/MS/MS with a conventional C18 column is effective and inexpensive, and suitable for routine use in the identification and quantitation of drugs and their metabolites. The detection of parent drugs and their metabolites or conversion and decomposition products is potentially very useful for the interpretation of cases in forensic toxicology, especially when the original compounds cannot be observed.

Sociabilidades e hedonismos: etnografia entre jovens usuários de substâncias psicoativas sintéticas Fortaleza Ceará

This dissertation shows a comparison between practices related to the body, to identities and to the health of young drug takers of synthetic and manipulated psychoactive substances living in Fortaleza. Through ethnographic accompaniment of parties and two social nets of young people I was able to verify the differences and similarities that make these addicted singular and plural in their notion of ethos and world vision. Notions of being hippie/alternative and being punk are present between these nets. Because their use and circulation are developed in environments usually distinct, it is possible to trace singular aspects between the nets and its actions. In parallel, on the attempt to build a triangulated data, I had information related to youth and drugs in local media and in governmental institutions bound to health mental and city security policies. With this junction of data I present a perspective to questions evolving formal and informal important controls to the conduction of their lives and identities. I present this as a sequel to well developed studies in Brasil about youth in interface to drug use, although still not very explored in the state of Ceará.

Toxicological impact of JWH-018 and its phase I metabolite N-(3-hydroxypentyl) on human cell lines

"The emergence and abuse of synthetic cannabinoids has been increasing as an alternative to cannabis, mainly among youth. As their appearance on the drug market has been recent, the pharmacological and toxicological profiles of these psychoactive substances are poorly understood. Current studies suggest that they have stronger effects compared to their natural alternatives and their metabolites retain affinity towards CB1 receptors in CNS. Since studies on its toxicological properties are scarce, the effects of the drug in human derived cell lines were investigated. The present study was designed to explore the toxicological impact of parent drug versus phase I metabolites of synthetic cannabinoids on human cells with and without CB1 receptor. The human cell line of neuroblastoma SH-SY5Y and human kidney cell line HEK-293T were exposed to JWH-018 and to its N-(3-hydroxypentyl) metabolite. Cell toxicity was evaluated using the MTT and LDH assay. Additionally, a dual staining methodology with fluorescent Annexin V-FITC and propidium iodide was performed to address the question of whether JWH-018 N-(3-hydroxypentyl) metabolite is inducing cell death through apoptosis or necrosis, in HEK293T and SH-SY5Y cell lines. The obtained results show that JWH-018 does not cause a statistically significant decrease in cell viability, in contrast to its N-(3-hydroxypentyl) metabolite, which at ≥25μM causes a significant decrease in cell viability. Both cell lines are affected by JWH-018 metabolite. Our results point to higher toxicity of JWH-018 metabolite when compared to its parent drug, suggesting a non-CB1 receptor mediated toxicological mechanism. Comparing the results from Annexin V/PI with MTT and LDH assays of SH-SY5Y and HEK293T in the presence of the synthetic cannabinoid metabolite, emerges the picture that cellular viability decreases and associated death is occurring through necrosis."

Characterization of polymorphisms of genes ADH2, ADH3, ALDH2 and CYP2E1 and relationship to the alcoholism in a Colombian population

Objective: Identify and characterize polymorphisms of genes ADH2, ADH3, ALDH2 and CYP2E1 in a Colombian population residing in the city of Bogotá and determine its possible relationship to the alcoholism. Methods: ADH2, ADH3, ALDH2, and CYP2E1 genotypes a population of 148 individuals with non-problematic alcohol and 65 individuals with alcoholism were determined with TaqMan probes and PCR-RFLP. DNA was obtained from peripheral blood white cells. Results: Significant difference was found in family history of alcoholism and use of other psychoactive substances to compare alcoholics with controls. When allelic frequencies for each category (gender) were considered, frequency of A2 allele carriers in ADH2 was found higher in male patients than controls. In women, the relative frequency for c1 allele in CYP2E1 was lower in controls than alcoholics. The ALDH2 locus is monomorphic. No significant differences in allele distributions of the loci examined to compare two populations were observed, however when stratifying the same trend was found that these differences tended to be significant. Conclusions: This study allows us to conclude the positive association between family history of alcoholism and alcoholism suggesting that there is a favorable hereditary predisposition. Since substance dependence requires interaction of multiple genes, the combination of genotypes ADH2*2, CYP2E1*1 combined with genotype homozygous ALDH2*1 found in this study could be leading to the population to a potential risk to alcoholism.

Characterization of polymorphisms of genes ADH2, ADH3, ALDH2 and CYP2E1 and relationship to the alcoholism in a Colombian population

Objective: Identify and characterize polymorphisms of genes ADH2, ADH3, ALDH2 and CYP2E1 in a Colombian population residing in the city of Bogotá and determine its possible relationship to the alcoholism. Methods: ADH2, ADH3, ALDH2, and CYP2E1 genotypes a population of 148 individuals with non-problematic alcohol and 65 individuals with alcoholism were determined with TaqMan probes and PCR-RFLP. DNA was obtained from peripheral blood white cells. Results: Significant difference was found in family history of alcoholism and use of other psychoactive substances to compare alcoholics with controls. When allelic frequencies for each category (gender) were considered, frequency of A2 allele carriers in ADH2 was found higher in male patients than controls. In women, the relative frequency for c1 allele in CYP2E1 was lower in controls than alcoholics. The ALDH2 locus is monomorphic. No significant differences in allele distributions of the loci examined to compare two populations were observed, however when stratifying the same trend was found that these differences tended to be significant. Conclusions: This study allows us to conclude the positive association between family history of alcoholism and alcoholism suggesting that there is a favourable hereditary predisposition. Since substance dependence requires interaction of multiple genes, the combination of genotypes ADH2*2, CYP2E1*1 combined with genotype homozygous ALDH2*1 found in this study could be leading to the population to a potential risk to alcoholism.

'What if you live on top of a bakery and you like cakes?'- Drug use and harm trajectories before, during and after the emergence of Silk Road

BACKGROUND: Cryptomarkets are digital platforms that use anonymising software (e.g. Tor) and cryptocurrencies (e.g. Bitcoin) to facilitate peer-to-peer (P2P) trade of goods and services. Their emergence has facilitated access to a wide range of high-quality psychoactive substances, according to surveys of users. In this paper, we ask the question 'How does changing access to drugs through cryptomarkets affect the drug use and harm trajectories of their users?'

METHODS: We conducted a digital ethnography spanning 2012-2014, a period that included the seizure of the original Silk Road marketplace and forum by law enforcement. Using encrypted online chat, we interviewed 17 people who reported using Silk Road to purchase illicit drugs. The interviews were in-depth and unstructured, and also involved the use of life history timelines to trace trajectories. Transcripts were analysed thematically using NVivo.

RESULTS: For some, Silk Road facilitated initiation into drug use or a return to drug use after cessation. Typically, participants reported experiencing a glut of drug consumption in their first months using Silk Road, described by one participant as akin to 'kids in a candy store'. There was evidence that very high availability reduced the need for drug hoarding which helped some respondents to moderate use and feel more in control of purchases made online. Cryptomarket access also appeared to affect solitary and social drug users differently. Most participants described using other cryptomarkets after the closure of Silk Road, albeit with less confidence.

CONCLUSION: In the context of high levels of drug access, supply and diversity occurring within a community regulated environment online, the impacts of cryptomarkets upon drug use trajectories are complex, often posing new challenges for self-control, yet not always leading to harmful outcomes. A major policy challenge is how to provide support for harm reduction in these highly volatile settings.

Comportamentos desviantes na adolescência : consumo de álcool, tabaco e drogas ilícitas

Relatório de Estágio, apresentado à Escola Superior de Educação do Instituto Politécnico de Castelo Branco para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Intervenção Social Escolar – Especialização em Crianças e Jovens em Risco.

Representaciones sociales y prácticas de actividad física en docentes de educación física, localidad de Suba UPZ 71

Debido a la problemática del sedentarismo e inactividad física en edad escolar y sus implicaciones en la aparición de enfermedades crónicas no transmisibles, surge un interés por realizar análisis cualitativos que tengan en cuenta los contextos sociales en los que se da esta problemática, con el objetivo de conocer, desde los actores inmersos en las comunidades, cómo se está llevando a cabo y cómo se entiende la actividad física; por tanto, se planteó en esta investigación un diseño narrativo en el cual se indagó, por medio de entrevistas a profundidad, acerca de las representaciones sociales y prácticas de actividad física que desarrollan 9 docentes de educación física pertenecientes a 3 colegios distritales de la Unidad de Planeamiento Zonal Tibabuyes, en la localidad de Suba en Bogotá, Colombia. Resultados: Los docentes entienden la actividad física como todo tipo de movimiento humano, así mismo, se evidencia que las prácticas deportivas son las que predominan en el ámbito escolar. En relación con las condiciones sociales, se encontró que el aspecto familiar es un limitante para el desarrollo de las prácticas físicas del estudiante. Las políticas escolares y distritales le restan importancia a las clases de educación física en el currículo escolar y la inseguridad, debido al consumo de sustancias psicoactivas en el sector, deteriora los espacios deportivos y recreativos como parques y zonas verdes.

Etapas de cambio comportamental frente al consumo de drogas ilegales, tabaco y alcohol en escolares de 9 a 17 años de Bogotá – Colombia

Objetivo: El propósito del estudio fue describir estadísticamente las etapas de cambio comportamental frente al consumo de sustancias psicoactivas –SPA– (alcohol, tabaco y drogas ilegales) en escolares entre 9 y 17 años de Bogotá- Colombia, pertenecientes al estudio FUPRECOL. Método: Se trata de un estudio descriptivo y transversal en 6.965 niños y adolescentes entre 9 y 17 años, pertenecientes a 24 instituciones educativas oficiales de Bogotá - Colombia. La medición de los procesos de cambio propuestos por el Modelo Transteórico (MTT), aplicados al consumo de drogas, tabaco y alcohol se aplicaron de manera auto-diligenciada mediante un cuestionario estructurado. Resultados: De la muestra evaluada, el 58,4% fueron mujeres con un promedio de edad 12,74 ± 2.38 años. En la población en general, frente al consumo de drogas, el 6% de los escolares se encontraban en etapa de pre-contemplación, 44 % en contemplación; 30% en preparación/acción, 20% en mantenimiento. Con relación al consumo de alcohol, el 5% de los niños y adolescentes se encontraban en etapa de pre-contemplación, 36 % en contemplación; 12% en preparación/acción, 46% en mantenimiento. Frente al tabaco, el 4% de los niños y adolescentes se encontraban en etapa de pre-contemplación, 33 % en contemplación; 12% en preparación/acción, 51% en mantenimiento. Conclusiones: En los escolares evaluados, un importante porcentaje se ubica en la etapa de mantenimiento frente a la intención de consumo de tabaco y alcohol. Frente al consumo de drogas ilegales los niños y adolescentes están en la etapa de contemplación. Se requieren esfuerzos mayores para fomentar programas preventivos que enseñen sobre el riesgo del abuso/dependencia de este tipo de sustancias psicoactiva sobre la salud; dándole prioridad en las agendas y políticas públicas dentro del ámbito escolar.

Temporal evolution of emerging contaminants, priority substances and heavy metals most frequently detected in reclaimed water and grounwater in a volcanic aquifer

[EN] The presence of emerging contaminants has been previously described in reclaimed water and groundwater of Gran Canaria (Spain). Despite of the environmental risk associated to irrigation with reclaimed water (R), this practice is necessary considering sustainability of the hydrological cycle in semiarid zones, especially regarding agricultural activity. The aim of this study was: i) to analyse the evolution during two years of contaminants of emerging concern, priority substances (2008/105/EC) and heavy metals in reclaimed water (R) and in a volcanic aquifer in the NE of Gran Canaria where a golf course has been irrigated with R since 1976 and ii) to relate this presence with physicochemical water properties and hydrogeological media. Reclaimed water and groundwater (GW) were monitoring quarterly from July 2009 to September 2011. Sorption and degradation processes in soil account for more compounds being detected in R. Diazinon and chlorfenvinphos were detected always in R and terbuthylazine, terbutryn and diuron at 90% of frequency. Considering all the samples, the most frequent compounds were chlorpyrifos ethyl, fluorene, phenanthrene and pyrene. Although their concentrations were frequently below 50 ngL-1, some contaminants, were occasionally detected at higher concentrations. Chlorpyrifos ethyl and diuron are priority substances detected frequently and at high concentrations so they must be included in monitoring studies. Geology and location seem to be related to the emerging compounds presence due to occasional contamination events (not related to R irrigation) and therefore not to an existence of a dangerous diffuse contamination level. Thus, it is preferable to select wells with less stable chemical water quality, in order to monitor the risk of emerging compounds presence. Considering the relationship between contaminant presence, chemical water quality, seasonal variation, hydrogeological characteristics and wells location we can conclude that chlorpyrifos ethyl and diuron were the most dangerous priority substances in terms of GW quality so they must be included in all of the monitoring studies, at least in Canary Islands.

Assessing exposures to emerging environmental contaminants in children

The period of developmental vulnerability to toxicants begins at conception and extends through gestation, parturition, infanthood and childhood to adolescence. The concern is that children: (1) may experience quantitatively and qualitatively different exposures, and (2) may have different sensitivity to chemical pollutants. Traditional toxicological studies are inappropriate for assessing the results of chronic exposure at very low levels during critical periods of development. This paper will discuss (1) the health effects associated with exposure to selected emerging organic pollutants, including brominated flame retardants, perfluorinated compounds, organophosphate pesticides and bisphenol A; (2) difficulties in monitoring these substances in children, and (3) suggest techniques and strategies for overcoming these difficulties. Such biomonitoring data can be used to identify where policies should be directed in order to reduce exposure, and to document policies that have successfully reduced exposure.

A Preference for Mephedrone: Drug Markets, Drugs of Choice, and the Emerging “Legal High” Scene

This study focuses on individuals' preferences for mephedrone, a new psychoactive substance that has emerged in several countries. We examine the reasons for mephedrone preferences, and describe the positive and negative effects of the drug experience, route of administration and consumers' views about the legality of mephedrone. Data were collected through semi-structured interviews with 45 adults who had used mephedrone since January 2010. Respondents resided in one of two jurisdictions that were characterized by different legislative controls over mephedrone. The findings suggest the importance of macro-level drug market factors that shaped people's preferences for mephedrone. Additionally, respondents' preferences were guided by pharmacological properties that helped them conceal the effects of mephedrone in public and semi-public spaces. Respondents were not deterred by the (impending) change from legal to illicit drug. The findings have implications for the study of localized drug markets, and in particular, legislative controls over emerging legal highs.

“Law on Transboundary Movement of Hazardous Substances through Sea

This study pertain to legal control of pollution from transboundary movement of hazardous substances through sea. It is an emerging area in international maritime law. There is growing environmental awareness that oceans are no longer an inexhaustible resource. This has resulted in a complex system of integrated ocean policies and international legal frame work in this regard. Considering the peculiar nature of hazardous substances, the development of law in this regard has been haphazard. The legal framework in this area is not comprehensive and lacks coherence. India is geo-strategically located in the central part of Indian Ocean through which many international sea routes lie. India is also fast emerging as a maritime hub. Indian legal framework in this respect also suffers from serious pitfalls. In this backdrop, this study makes a sincere attempt to identify and analyse the legal intricacies in this area, in order to evolve a better regime for control of pollution pursuant to transboundary movement of hazardous substances through sea

Effects of priority and emerging pollutants on river biofilms

L'activitat humana representa una de les majors causes d'entrada d'una gran varietat de substàncies en els ecosistemes fluvials. L'objectiu principal d'aquest treball es investigar els efectes que els tòxics orgànics poden exercir en els biofilms fluvials. El riu Llobregat ha estat sotmès a fortes pressions, fet que l'ha portat a uns nivells molt elevats de contaminació. En aquest estudi s'ha observat una influència dels plaguicides presents al riu en la distribució de la comunitat de diatomees, així com efectes en el biofilm a nivell funcional i estructural. Experiments amb canals experimentals han mostrat que l'herbicida diuron i el bactericida triclosan poden ocasionar una cadena d'efectes en els biofilms, incloent efectes directes i també efectes indirectes en les relacions entre els components del biofilm. Experiments amb cultius algals han mostrat que aquests tòxics, aplicats en barreja, poden tenir una major toxicitat de la prevista pels models, resultant en efectes sinèrgics.

An integrated approach to evaluate emerging contaminants in drinking water

The chemical stock of emerging contaminants in Brazilian drinking water is of great interest due to the poor water quality at surface water intakes. In addition, little is known about the effect of some contaminants, such as endocrine disrupting chemicals (EDCs), which may be present in both raw and treated water. The aim of this work was to evaluate selected emerging contaminants in Brazilian waters using both chemical and biological analyses. Sampling sites were established in different municipalities based upon raw water quality data. Estrone, 17 beta-estradiol, estriol, 17 alpha-ethinylestradiol, bisphenol A, 4-n-octylphenol and 4-n-nonylphenol were determined in the samples by liquid chromatography-tandem mass spectrometry. A yeast assay using a Saccharomyces cerevisiae bioluminescent bioreporter was used to evaluate the estrogenic activity of the water samples. The first integrated results revealed similarities between the two individual approaches, since higher values for the bioassay were accompanied by significant concentrations of some selected compounds in surface water samples. No estrogenicity was observed for drinking water samples. Our results also indicate that the usual paradigm of evaluating water quality by measuring selected EDCs in a given water sample via chemical analysis, needs to be reviewed since the observed estrogenicity of a water sample is now a better guiding parameter to the selection of samples and substances to be chemically investigated in further analysis. So far, the data bank produced in this work, i.e., the comparison between chemical burden and observed estrogenicity, is not yet sufficiently robust to fully guide this decision. (C) 2011 Elsevier B.V. All rights reserved.