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

Short Review of Calcium Disodium Ethylene Diamine Tetra Acetic Acid as a Food Additive.

Calcium disodium ethylenediaminetetraacetate (Calcium Disodium EDTA, C10H12CaN2Na2O8.2H2O) is a derivative of EthylenediamineTetraacetic Acid and is an approved food additive (E385). It is used as preservative, sequestrant, flavouring agent, and colour retention agent in foods. As a drug it is used for the reduction of blood and mobile depot lead in the treatment of acute and chronic lead poisoning. Calcium Disodium EDTA is very poorly absorbed from the gastrointestinal tract following ingestion. The compound is metabolically inert and no accumulation in the body has been found. Acute, short-term, sub chronic and chronic toxicity studies carried out with Calcium Disodium EDTA in laboratory animals found that the compound is nephrotoxic at high doses. In similar high doses, application of Calcium Disodium EDTAcan result in complexation of zinc ions, thus interfering with the zinc homeostasis and causing developmental toxicity. No evidence exists suggesting the compound exerts genotoxic or carcinogenic effects. Overall, Calcium Disodium EDTAseems to be safe for use as a food additive, as the noted toxic doses are higher than can be achieved via the addition of Calcium Disodium EDTA to food. However, human data is limited and the gross of available (human and animal) data, as well as the ADI, stems from several decades ago. Caution should also be taken when Calcium Disodium EDTA is administered as treatment for lead poisoning, as the exposure increases greatly. Until 2020, EFSA will carry out new risk assessments, and subsequently the Commission will revise the list of food additives and the conditions of use specified therein. The deadline for food additives other than colours and sweeteners is 31 December 2018, which seems appropriate regarding the non-acute need for reevaluation of Calcium Disodium EDTA as food additive.

Acute oral toxicity study of the crude ethanolic leaf extract of ficus pseudopalma blanco (moraceae) in sprague dawley rats.

Ficus pseudopalma Blanco is an ornamental plant endemic to the Philippines, especially in the island of Luzon. It is commonly used to treat kidney stones and diabetes and used for edible fruits. The leaves are cooked and eaten as vegetable despite the absence of studies on its possible toxic effect. This study was conducted to assess any toxicity of its leaf extract. Acute oral toxicity of the crude ethanolic leaf extract of F. pseudoplama was performed according to the guidelines set by OECD 425 on six 8-12 week old female Sprague Dawley rats weighing from 160-210g. One rat was treated with normal saline solution that served as the control. Toxicological and pharmacological observations were completed for 14 days. On day 14, all test animals were sacrificed via cervical dislocation and subjected to gross necropsy; liver samples were subjected to histopathological examination. Gross examination of the rodent’s organs was all normal and regarded as unremarkable. Toxicological screening showed that the experimentally treated rats behaved almost normally as the control. Histopathological examination showed no area of hepatic zonal necrosis and tumor formation was identified, no cytological aypia and sinusoid congestion, intact and uninterrupted hepatic lobular architecture, and portal tracts and vessels were unremarkable. These findings strongly suggest that the leaf extract is non toxic and safe for consumption up to 2000 mg/kg BW and may therefore be used for future nutraceuticals and drug development.