Avaliação toxicológica da exposição à Cannabis e cocaína na gravidez em cordão umbilical humano: validação de método analítico e prospecção de biomarcadores proteicos de toxicidade / Toxicological assessment of Cannabis and cocaine exposure during pregnancy in human umbilical cord tissue: analytical method validation and prospection of protein biomarkers related to fetotoxicity

O abuso de drogas atinge aproximadamente 35 milhões de pessoas em todo planeta, sendo um problema alarmante em decorrência de graves danos à saúde, como a dependência química e intoxicações fatais. No Brasil, o número de usuários tem crescido principalmente para o consumo de produtos da Cannabis e cocaína, drogas amplamente consumidas, inclusive entre mulheres em período gestacional, trazendo à tona um novo grupo de risco. A exposição gestacional a drogas de abuso está diretamente relacionada a malformações fetais e complicações de saúde para mãe e bebê nos períodos pré- e pós-natal. Tradicionalmente, a avaliação toxicológica da exposição é realizada pela detecção da droga parental e de seus produtos de biotransformação em matrizes materno-fetais por meio de métodos bioanalíticos. Entretanto, estes ensaios não fornecem informações acerca dos impactos fisiológicos ocasionados pela exposição, deixando uma lacuna no que tange às informações sobre os mecanismos e moléculas subjacentes envolvidos em processos de toxicidade. Desse modo, o desenvolvimento de análises toxicológicas mais robustas utilizando tecnologia de ponta, que possam comprovar o uso drogas e também elucidar aspectos de toxicidade é de suma importância, pois auxiliam na compreensão do impacto biológico relativo à exposição humana a xenobióticos. Neste trabalho foram desenvolvidos ensaios bioanalíticos, utilizando o tecido do cordão umbilical para a avaliação da exposição in utero à canabinoides. Foi desenvolvido e validado método QuECheRS adaptado como preparo de amostra, no qual etapas simultâneas de extração e hidrólise alcalina de canabinoides são alcançadas, utilizando cromatografia em fase gasosa acoplada a espectrômetro de massas para detecção de delta-9-tetraidrocanabinol (THC), canabinol (CBN), 11-hidroxi-delta-9-tetraidrocanabinol (11-OHTHC) e 11-nor-9-carboxi-tetrahidrocanabinol (THC-COOH). Também foram desenvolvidas metodologias utilizando LC-MS/MS e Trapped Ion Mobility Mass Spectrometry para análise de proteoma de cordão umbilical humano em diferentes regiões, no intuito de identificar biomarcadores proteicos relativos à fetotoxicidade do uso de drogas na gravidez. Até o presente momento, QuECheRS é utilizado pela primeira vez como abordagem bioanalítica para avaliação de drogas ilícitas em matrizes teciduais materno-fetais e mostrou-se satisfatório para detecção de produtos da Cannabis. Nos ensaios proteômicos, foram identificados potenciais biomarcadores de fetotoxicidade, como as moléculas ACTA 2, Collagen alpha-1 (XVIII), SMC1A, KNL1, KMT2A, em tecidos expostos à Cannabis e/ou cocaína. Tais macromoléculas estão correlacionadas a malformações embriogênicas e complicações de saúde na vida intra-uterina. As metodologias desenvolvidas neste trabalho podem ser úteis para uma melhor avaliação da toxicidade do uso de drogas na gravidez, fornecendo novas pistas sobre a exposição e/ou efeitos tóxicos significativos considerados na avaliação de risco

Drug abuse affects approximately 35 million people worldwide and can be considered a significant burden on society due to severe health problems, e.g. drug addiction and fatal poisonings. In Brazil, the number of users has been growing related to Cannabis and cocaine products, drugs widely used, including among women in gestational period, bringing up a new risk group. Gestational exposure to drugs of abuse is directly related to fetal malformations and health complications for mother and babies in the pre- and postnatal periods. Traditionally, toxicological assessment of exposure is performed by detecting the parent drug and its biotransformation products in maternal-fetal matrices using bioanalytical methods. However, these assays do not provide information about the physiological impacts caused by exposure, leaving a lack of information about the pathways and molecules involved in toxicity processes. Thus, the development of robust toxicological analyzes using cutting-edge technologies in order to prove drug use and also elucidate aspects of toxicity is very important, as they help in understanding the biological impact of human exposure to xenobiotics. Herein, bioanalytical methods using umbilical cord tissue to assess in utero exposure to cannabinoids were developed. A QuECheRS method was developed fully validated as a sample preparation technique for simultaneous extraction and alkaline hydrolysis of cannabinoids, using gas chromatography coupled to mass spectrometry to detect the analytes delta-9-tetrahydrocannabinol (THC), cannabinol (CBN), 11-hydroxydelta-9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH). LC-MS/MS based proteomics and Trapped Ion Mobility Mass Spectrometry were also developed in order to identify protein biomarkers related to fetotoxicity of drug use in pregnancy. Our works represents the first use of QuECheRS for evaluation of illicit drugs in maternal-fetal tissue and was suitable for detection of Cannabis products. In the proteomic assays, potential biomarkers of fetotoxicity were identified in the exposed tissues, such as ACTA 2, Collagen alpha-1 (XVIII), SMC1A, KNL1, KMT2A. These proteins are related to embryogenic malformations and health complications in intrauterine life. The methodologies developed in this project may be useful for a better assessment of the toxicity of drug use in pregnancy, providing new clues about exposure and/or significant toxic effects that should be considered in the risk assessment

Simultaneous accelerated solvent extraction and hydrolysis of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid glucuronide in meconium samples for gas chromatography-mass spectrometry analysis.

Cannabis misuse during pregnancy is associated with severe impacts on the mother and baby health, such as newborn low birth weight, growth restriction, pre-term birth, neurobehavioral and developmental deficits. In most of the cases, drug abuse is omitted or denied by the mothers. Thus, toxicological analyzes using maternal-fetal matrices takes place as a suitable tool to assess drug use. Herein, meconium was the chosen matrix to evaluate cannabis exposure through identification and quantification of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic (THCCOOH). Accelerated solvent extraction (ASE) was applied for sample preparation technique to simultaneously extract and hydrolyze conjugated THCCOOH from meconium, followed by a solid-phase extraction (SPE) procedure. The method was developed and validated for gas chromatography-mass spectrometry (GC-MS), reaching hydrolysis efficiency of 98%. Limits of detection (LOD) and quantification (LOQ) were, respectively, 5 and 10 ng/g. The range of linearity was LOQ to 500 ng/g. Inter and intra-batch coefficients of variation were <8.4% for all concentration levels. Accuracy was in 101.7-108.9% range. Recovery was on average 60.3%. Carryover effect was not observed. The procedure was applied in six meconium samples from babies whose mothers were drug users and showed satisfactory performance to confirm fetal cannabis exposure.

Ortleppascaris sp. and your host Rhinella marina: A proteomic view into a nematode-amphibian relationship.

The success of the helminth-host relationship depends on a biochemical molecular arsenal. Perhaps the proteome is the largest and most important set of this weaponry, in which the proteins have a crucial role in vital processes to the parasite/host relationship, from basic metabolism and energy production to complex immune responses. Nowadays, the bioproducts expressed by the parasites are under the "spotlight" of immunoassays and biochemical analysis in helminthology, especially in proteomic analysis, which has provided valuable information about the physiology of the infecting agent. Looking into this point of view, why not turn to the infected agent as well? This study characterised the proteomic profile of fluid-filled fibrous cysts of encapsulated Ortleppascaris sp. larvae in the hepatic parenchyma of their intermediate host, the amphibian Rhinella marina. The proteins were separated by two-dimensional electrophoresis and identified by MS with the aid of Peptide Mass Fingerprint. A total of 54 molecules were analysed in this system, revealing a complex protein profile with molecules related to basic metabolic processes of the parasite, energy production, oxi-reduction and oxidative stress processes as well as molecules related to the host response. This study contributes to proteomic studies of protein markers of the development, infectivity, virulence and co-existence of helminths and their hosts.

Taxonomic status and redescription of the genus Brasicystis Thatcher, 1979 (Digenea: Didymozoidae).

Brasicystis bennetti Thatcher, 1979 was first described from specimens obtained from the subcutaneous tissues of the mouth and operculum of Plagioscyion squamosissimus from the Amazon River in Brazil, however since 2008, Brasicystis has been considered a genus inquirendum. This study reviews some of the diagnostic characters from the original description of B. bennetti from the Amazon Delta, and redescribes the genus and species with a discussion of their taxonomic status. Ultrastructural and molecular approaches complement the data presented on this monotypic genus. The diagnosis of the tribe Didymozoini Monticelli, 1888 is amended to incorporate the genus Brasicystis, which is redescribed and revalidated here, with the proposal of an amended key.