Ana Luiza Ziulkoski

Simultaneous determination of omeprazole, hydroxyomeprazole and omeprazole sulphone in human plasma by isocratic HPLC-DAD: application to the phenotyping of CYP2C19 and CYP3A4 in brazilian volunteers

A simple HPLC-DAD method using a reverse phase column and isocratic elution for the simultaneous determination of omeprazole (OME), 5-hydroxyomeprazole (HOME) and omeprazole sulphone (OMES) was developed. The proposed method was used to study CYP2C19 and CYP3A4 genetic polymorphisms using OME as the probe drug in a group of Brazilian volunteers. OME, HOME and OMES were extracted from plasma samples with Tris buffer pH 9.5 (0.2 mol L-1) and ethyl acetate. HPLC separation was achieved using a Shim-Pack RP-18e (150 ´ 4.6 mm i.d., 5 µm) column, with acetonitrile phosphate buffer pH 7.6 (24:76) as mobile phase and total run time of 15 min. Retention times were 2.7 min for internal standard (sulpiride), 4.1 min for HOME, 11.6 min for OME and 12.6 min for OMES. Detection (UV at 302 nm) of analytes was linear in the range from 25 to 1000 ng mL-1. Extraction recoveries were in the range of 64.3 to 73.2% for all analytes. A group of 38 Brazilian healthy volunteers was phenotyped with this method, after a single oral dose of 20 mg omeprazole. The method presented adequate accuracy and precision, with limit of quantification of 25 ng mL-1 for omeprazole and metabolites, which allowed the identification of ultra-rapid metabolizers for both CYP2C19 and CYP3A4 and took advantage of the selective identification offered by diode-array detectors.

Evaluation of genotoxicity and cytotoxicity of water samples from the Sinos River Basin, southern Brazil.

Some water bodies in the Sinos River Basin (SRB) have been suffering the effects of pollution by residential, industrial and agroindustrial wastewater. The presence of cytotoxic and genotoxic compounds could compromise the water quality and the balance of these ecosystems. In this context, the research aimed to evaluate the genotoxicity and cytotoxicity of the water at four sites along the SRB (in the cities of Santo Antônio da Patrulha, Parobé, Campo Bom and Esteio), using bioassays in fish and cell culture. Samples of surface water were collected and evaluated in vitro using the Astyanax jacuhiensis fish species (micronucleus test and comet assay) and the Vero lineage of cells (comet assay and cytotoxicity tests, neutral red - NR and tetrazolium MTT). The micronucleus test in fish showed no significant differences between the sampling sites, and neither did the comet assay and the MTT and NR tests in Vero cells. The comet assay showed an increase in genetic damage in the fish exposed to water samples collected in the middle and lower sections of the basin (Parobé, Campo Bom and Esteio) when compared to the upper section of the basin (Santo Antônio da Patrulha). The results indicate contamination by genotoxic substances starting in the middle section of the SRB.

Cytotoxicity assays as tools to assess water quality in the Sinos River basin.

Cytotoxicity assays using cell cultures may be an alternative to assess biological toxicity of surface waters and may help to improve the control of water quality. This study compared two methods to prepare culture media for the exposure of Hep-2 cells to water samples collected from the Rolante River, an important affluent of the Sinos River. The toxicity was evaluated using the MTT and neutral red assays. Two methods were used to prepare culture media. In method 1, the sample was diluted at 1:1, 1:10, 1:100, 1:1000, 1:10.000 (v/v, sample/medium) in a standard culture medium; in method 2, water samples were used as the solvent for the culture medium, which was prepared at concentrations of 100, 80, 60, 40 and 20%. Semi-confluent cultures were then exposed to the media test for 24 hours, and cytotoxicity was determined immediately using the MTT and NR assays. Mitochondrial activity (MTT) was significantly lower at all concentrations in both methods, except at 1:1000 in method 1. However, the lysosome viability (NR) results revealed cytotoxicity only in the 1:1 sample of method 1. Both culture preparation methods were efficient and sensitive to the MTT assay, but method 2 seemed to be more adequate for the NR assay. The Rolante River has cytotoxic contaminants to Hep-2 cells, which may be one of the explanations for the poor water quality of the Sinos River basin.

Relation between CYP2C19 phenotype and genotype in a group of Brazilian volunteers

The CYP2C19 gene presents polymorphism affecting the pharmacokinetics of several drugs of clinical importance. The purpose of this study was to investigate the correlation between CYP2C19 genotype and metabolic phenotype in a group of 38 Brazilian volunteers, evaluating the phenotype prediction capacity of the genotyping procedure. For CYP2C19 phenotyping, omeprazole was used as the probe drug, using the hydroxylation metabolic ratio as the phenotypic indicator. Venous blood samples were drawn before and three hours after an oral administration of 20 mg omeprazole. The plasma concentrations of omeprazole and hydroxy-omeprazole were determined by high performance liquid chromatography. The genotyping assay was carried out using a Real-Time-PCR-based assay, identifying the alleles *1 (completely functional), *2, *3 and *4 (null). The phenotyping procedure estimated the presence of 4 poor, 34 extensive and 1 ultra-extensive metabolizer. The genotyping identified 4 poor, 23 extensive and 11 intensive metabolizers. The groups of volunteers classified according to the number of active alleles of CYP2C19 had significant differences in the metabolic ratios of omeprazole hydroxylation. However, volunteers exhibiting the same number of active alleles presented different phenotypes. Therefore, the phenotyping of CYP2C19 is a more promising alternative to dose individualization of CYP2C19 substrate drugs.

Determination of amitriptyline and its main metabolites in human plasma samples using HPLC-DAD: application to the determination of metabolic ratios after single oral dose of amitriptyline

A simple and sensitive HPLC-DAD method for the simultaneous determination of amitriptyline, nortriptyline, E-10-hydroxyamitriptyline, Z-10-hydroxyamitriptyline, E-10-hydroxynortriptyline, Z-10-hydroxynortriptyline and desmethylnortriptyline in human plasma samples was developed and validated. The method employs a two step liquid-liquid extraction and a reversed phase separation with isocratic elution. Precision assays showed R.S.D % lower than 12.1% and accuracy was in the range of 93.1 to 102.5%. Lowest Limit of detection was 5 ng mL-1 for all analytes. Metabolic ratios of amitriptyline demethylation were evaluated in individuals genotyped for CYP2C19, with clear differences between volunteers with zero or two active alleles. The method is suitable for therapeutic drug monitoring of patients under amitriptyline treatment, also allowing the indication of CYP2C19 activity.

Monitoring the Genotoxic and Cytotoxic Potential and the Presence of Pesticides and Hydrocarbons in Water of the Sinos River Basin, Southern Brazil

The Sinos River is one of the most polluted rivers in Brazil. The purpose of this work was to monitor the presence of some pesticides and hydrocarbons as well as the genotoxic and cytotoxic potential on HEp-2 cells from water samples collected at seven sites in the Sinos River Basin (SRB), southern Brazil. Nine samples were taken from the three main rivers in the SRB and used as a solution to dilute the HEp-2 cell culture medium after microfiltration. Twenty-four pesticides and 19 hydrocarbons were measured. Cytotoxicity was assessed by methyl thiazolyl tetrazolium (MTT) and neutral red (NR) assays, in which cells were exposed to different concentrations of the water samples for 24xa0h. Genotoxicity of the microfiltrated raw water samples was assessed by comet assay after 6 and 24xa0h of exposure. Among the chemicals analyzed, only the 2,4-D, dichloromethane, tetrachloroethene, chloroform, bromodichloromethane, styrene, and toluene were detected, but they were all lower than the limit established by Brazilian regulations. Twenty samples from a total of 60 had a cytotoxic effect in the MTT assay and 30 in the NR assay. The comet assay indicated the presence of genotoxic substances in the water at the seven locations monitored. Temporal and spatial variation was observed in the cytotoxicity and genotoxicity assays. Results indicated that the water in all stretches of the SRB is contaminated and it can cause harmful effects to humans and to the aquatic biota. This HEp-2 cell-line approach can be an additional tool for environmental monitoring.

Thirdhand tobacco smoke: procedures to evaluate cytotoxicity in cell cultures.

Abstract The risks associated to tobacco smoking are not ceased with smoke extinction. Many toxic compounds remain in the environment after the cigarette is extinguished and accumulated in the air or on surfaces. However, little is known about the risks of this exposure. The aim of this study was to evaluate procedures to collect thirdhand smoke (THS) and prepare the samples to perform three in vitro toxicity tests. Cellulose papers and cotton wipes were used to impregnate with nicotine solution and smoke cigarette in a chamber or in smoker’s home. Samples were immersed in methanol or Dulbecco’s modified Eagle’s medium (DMEM) to expose Hep-2 cells. MTT, neutral red uptake (NRU) and trypan blue assays were performed. The concentration of nicotine in DMEM extract of THS in paper and cotton was similar to those in methanol extract (pu2009>u20090.05). Alterations in the mitochondrial and lysosomal functions were found in both paper and cotton samples; however, the cytotoxic effect was not always observed. There was a decrease of 21–31% in MTT assay and 38–56% in NRU assay (pu2009<u20090.003). There was a dose-response relationship between the amount of cigarettes and lysosomal viability; the correlation was higher for cotton samples (ru2009=u2009−0.843, pu2009<u20090.001). As a dose-response relationship was found only in NRU assay, this test may be a more suitable choice rather than the MTT assay. Paper and wipe sampling can be reliable markers of tobacco smoke contamination. Moreover, these materials, if properly prepared, can be used as substrate providers to perform cellular assays.

Acompanhamento farmacoterapêutico em pacientes dislipidêmicos de um lar de idosos da cidade de Novo Hamburgo-RS

INTRODUCTION: Since the last century, life expectancy and the incidence of disease in the elderly have increased, especially chronic diseases. OBJECTIVE: This study aims to evaluate the pharmacotherapeutic follow-up(PF) in dyslipidemic patients of a nursing home in Novo Hamburgo city, state of Rio Grande do Sul, Brazil. METHODOLOGY: This is a quantitative, observational study with a longitudinal retrospective design, evaluating 50 elderly patients who live in a nursing home (80.2 ± 7.64 years old, 32 women). We assessed the lipid profile of these patients (total cholesterol, triglycerides, HDL and LDL) before and after one-year PF. The analysis was conducted through descriptive statistics and Student t test or U of Mann Whitney test for paired samples. RESULTS: 56% of the patients presented changes in lipid profile in the beginning of the study and 30% one year later, with significant improvement of the lipid profile after the monitoring. Moreover, total cholesterol levels showed a favorable decrease after a year of monitoring (206 ± 53 vs. 180 ± 43 mg/dL; P = 0.009). Most patients diagnosed with dyslipidemia were using drugs, for at least three months, to treat this pathology (statins and fibrates). The majority of these patients used them correctly. CONCLUSIONS: The patients had significant improvement in their lipid profile after one year of monitoring.

Cellular effects of thirdhand tobacco smoke from smokers’ homes

Abstract After the cigarette is extinguished, many toxic compounds remain in the environment and accumulate in the air or on surfaces. This exposure is termed thirdhand smoke (THS) and its risks are poorly known. The aim of the study was to evaluate the cellular effects of THS from smokers’ homes. Papers were placed in nine smoker’s home and three nonsmoker’s homes. An area equivalent to the paper size was cleaned with a cotton wipe. A549, Hep-2 and 3T3 cells were exposed to THS for 24u2009h and cellular functions were assessed by MTT, neutral red (NR) reuptake and trypan blue exclusion assays. High levels of nicotine were found in samples from smokers’ homes. Cellular proliferation was similar in almost all samples after THS exposure. Few changes in the cellular functions were observed, mainly higher mitochondrial activity, in paper samples. We are able to detect markers of THS collected from smokers’ homes, but a clear evidence of cellular toxicity cannot be demonstrated by the present assays. This is the first study to evaluate the cellular effects of THS samples collected from smokers’ homes.

Dispositivos poliméricos cardiovasculares: comportamento termomecânico e viabilidade celular

In recent decades new synthetic materials have been developed with adequate biofunctionality and biocompatibility to become a biomaterial. Biostable polymers have widespread use in the biomedical field, and many advances in polymeric biomaterials have been made in the search for improvements to cardiovascular implants. Currently, the most commonly used synthetic materials for the production of vascular grafts are PTFE and PET, due to their chemical stability after implant. In this work, a study of the thermal and mechanical properties of the commercial devices based on PET and PTFE is reported, as well as their cytotoxicity in mouse fibroblast cells, 3T3- NIH, through tests for the evaluation of cell viability (MTT test and VN). These materials showed high thermal stability (over 300 ° C), even after 270 days in vitro degradation and elastic behavior (maximal strain value of 186±22% by PET and 65±19% by PTFE). Cell viability by VN and MTT of PTFE device was more than 80%, thus, classified as non-cytotoxic. For PET device, VN test showed no cytotoxic effect, however the results obtained by MTT indicated that it causes alteration of mitochondrial function, independent of dose and time measured.