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

pH, Temperature, Hydration of the Stratum Corneum of the Dorsal Hand and the Cord Area, and Acid Mantle Formation during Early Days of life in High-risk Newborns

PURPOSE: The study was done to explore variations in physiologic parameters of the stratum corneum (SC) of the dorsal hand and cord area during early days of life in high-risk newborns. METHODS: In this longitudinal study, a total of 77 high-risk newborns were assessed for pH, temperature and hydration of the SC of the dorsal hand and the cord area on the 1st, 2nd, 3rd, 5th and 7th days of life. RESULTS: Results showed that an acid mantle (AM) was formed in 57.1% for dorsal hand and 35.1% for cord area, implying significant delay regardless of decrease in pH (F=103.60, p<.001), and hydration (F=4.00, p=.003) across days in both areas. Peripheral hypothermia with low hydration level was also observed in both areas. There was a positive relation between hydration and temperature (.14

Effects of Aspartame on Maternal-Fetal and Placental Weights, Length of Umbilical Cord and Fetal Liver: A Kariometric Experimental Study / Efectos del Aspartame sobre el Peso Materno-Fetal y Placentario, Largo del Cordón Umbilical e Hígado Fetal: Un Estudio Cartométrico Experimental

Aspartame is a synthetic sweetener consumed by more than half the adult population in 75 countries. Their metabolites can be toxic, principally to the liver and retina, and there are few studies on the use of aspartame in gestation. Twenty pregnant rats were weighed and allocated randomly (n=5 per group) to receive 14 mg/kg aspartame or water by oral-gastric drip. Treated Tl: aspartame diluted in water at room temperature; Treated T2: aspartame diluted in water heated to 40° C; control Cl: water at room temperature; and control C2: water heated to 40° C. Placentas were weighed, umbilical cords measured and 1000 nuclei of fetal hepatocytes (250 from each group) were analyzed morphometrically utilizing the technique of kariometry, with application of the Mann-Whitney U-Test. There were reductions in mean placental and maternal-fetal weights, in umbilical-cord length, and the majority of kariometric parameters of the hepatocytes in the group treated with aspartame diluted in distilled water at room temperature. Reduction of placental and maternal-fetal weights occurred, shortening of the umbilical cord, and decrease in kariometric parameters in fetal hepatocyte nuclei after administration of aspartame diluted in distilled water at 40°C temperature. The use of aspartame during gestation can be prejudicial to the fetus.

El aspartame es un endulzante sintético consumido por más de la mitad de la población adulta, en 75 países. Sus metabolitos pueden ser tóxicos, principalmente en el hígado y retina y hay algunos estudios sobre el aspartame en el embarazo. Veinte ratas preñadas fueron pesadas y distribuidas aleatoriamente (n=5 por grupo) y recibieron 14 mg/Kg de aspartame o agua por vía oral- gástrica. Tratamiento 1: aspartame diluido en agua a temperatura ambiente; Tratamiento T2: aspartame diluido en agua tibia a 40 °C; control Cl: agua a temperatura ambiente, y control C2: agua tibia a 40° C. Las placentas fueron pesadas, el cordón umbilical medido y 1000 núcleos de hepatocitos fetales (250 de cada grupo) se analizaron morfométricamente utilizando la técnica de canometría con aplicación del Test U de Mann-Whitney U-Test. En el grupo tratado con aspartame diluido en agua a temperatura ambiente, hubo reducción en los pesos promedios de la placenta y materno-fetal, largo del cordón umbilical y en la mayoría de los parámetros cartométricos de los hepatocitos. Lo mismo ocurrió en el grupo tratado con aspartame diluido en agua a 40 °C. El uso del aspartame durante las gestación puede ser perjudicial para el feto.

Natural compounds, fraxin and chemicals structurally related to fraxin protect cells from oxidative stress

Coumarins comprise a group of natural phenolic compounds found in a variety of plant sources. In view of the established low toxicity, relative cheapness, presence in the diet and occurrence in various herbal remedies of coumarins, it appears prudent to evaluate their properties and applications further. The purpose of this study is to investigate cellular protective activity of coumarin compound, fraxin extracted from Weigela florida var. glabbra, under oxidative stress, to identify genes expressed differentially by fraxin and to compare antioxidative effect of fraxin with its structurally related chemicals. Of the coumarins, protective effects of fraxin against cytotoxicity induced by H2O2 were examined in human umbilical vein endothelial cells (HUVECs). Fraxin showed free radical scavenging effect at high concentration (0.5 mM) and cell protective effect against H2O2-mediated oxidative stress. Fraxin recovered viability of HUVECs damaged by H2O2- treatment and reduced the lipid peroxidation and the internal reactive oxygen species level elevated by H2O2 treatment. Differential display reverse transcription-PCR revealed that fraxin upregulated antiapoptotic genes (clusterin and apoptosis inhibitor 5) and tumor suppressor gene (ST13). Based on structural similarity comparing with fraxin, seven chemicals, fraxidin methyl ether (29.4% enhancement of viability), prenyletin (26.4%), methoxsalen (20.8 %), diffratic acid (19.9%), rutoside (19.1%), xanthyletin (18.4%), and kuhlmannin (18.2%), enhanced more potent cell viability in the order in comparison with fraxin, which showed only 9.3% enhancement of cell viability. These results suggest that fraxin and fraxin-related chemicals protect HUVECs from oxidative stress.