A Rodent Model of Human-Dose-Equivalent 5-Fluorouracil: Toxicity in the Liver, Kidneys, and Lungs.

5-Fluorouracil (5-FU) is a chemotherapy drug widely used to treat a range of cancer types, despite the recurrence of adverse reactions. Therefore, information on its side effects when administered at a clinically recommended dose is relevant. On this basis, we examined the effects of the 5-FU clinical treatment on the integrity of the liver, kidneys, and lungs of rats. For this purpose, 14 male Wistar rats were divided into treated and control groups and 5-FU was administered at 15 mg/kg (4 consecutive days), 6 mg/kg (4 alternate days), and 15 mg/kg on the 14th day. On the 15th day, blood, liver, kidney, and lung samples were collected for histological, oxidative stress, and inflammatory evaluations. We observed a reduction in the antioxidant markers and an increase in lipid hydroperoxides (LOOH) in the liver of treated animals. We also detected elevated levels of inflammatory markers, histological lesions, apoptotic cells, and aspartate aminotransferase. Clinical treatment with 5-FU did not promote inflammatory or oxidative alterations in the kidney samples; however, histological and biochemical changes were observed, including increased serum urea and uric acid. 5-FU reduces endogenous antioxidant defenses and increases LOOH levels in the lungs, suggesting oxidative stress. Inflammation and histopathological alterations were also detected. The clinical protocol of 5-FU promotes toxicity in the liver, kidneys, and lungs of healthy rats, resulting in different levels of histological and biochemical alterations. These results will be useful in the search for new adjuvants to attenuate the adverse effects of 5-FU in such organs.

Untargeted Metabolomics Studies of H9c2 Cardiac Cells Submitted to Oxidative Stress, ß-Adrenergic Stimulation and Doxorubicin Treatment: Investigation of Cardiac Biomarkers.

One of the biggest challenges in the search for more effective treatments for diseases is understanding their etiology. Cardiovascular diseases (CVD) are an important example of this, given the high number of deaths annually. Oxidative stress (the imbalance between oxidant and antioxidant species in biological system) is one of the factors responsible for CVD occurrence, demanding extensive investigation. Excess of reactive oxygen species (ROS) are primarily responsible for this condition, and clinical and scientific literature have reported a significant increase in ROS when therapeutic drugs, such as doxorubicin and isoproterenol, are administered. In this context, the aim of this study is the investigation of potential biomarkers that might be associated with oxidative stress in cardiomyocytes. For this purpose, H9c2 cardiomyocytes were submitted to oxidative stress conditions by treatment with doxorubicin (DOX), isoproterenol (ISO) and hydrogen peroxide (PER). Metabolomics analyses of the cell extract and the supernatant obtained from the culture medium were then evaluated by CE-ESI(+)-TOF-MS. Following signal processing, statistical analyses, and molecular features annotations, the results indicate changes in the aspartate, serine, pantothenic acid, glycerophosphocholine and glutathione metabolism in the cell extract.

Comprehending Cardiac Dysfunction by Oxidative Stress: Untargeted Metabolomics of In Vitro Samples.

Cardiovascular diseases (CVDs) are noncommunicable diseases known for their complex etiology and high mortality rate. Oxidative stress (OS), a condition in which the release of free radical exceeds endogenous antioxidant capacity, is pivotal in CVC, such as myocardial infarction, ischemia/reperfusion, and heart failure. Due to the lack of information about the implications of OS on cardiovascular conditions, several methodologies have been applied to investigate the causes and consequences, and to find new ways of diagnosis and treatment as well. In the present study, cardiac dysfunction was evaluated by analyzing cells' alterations with untargeted metabolomics, after simulation of an oxidative stress condition using hydrogen peroxide (H2O2) in H9c2 myocytes. Optimizations of H2O2 concentration, cell exposure, and cell recovery times were performed through MTT assays. Intracellular metabolites were analyzed right after the oxidative stress (oxidative stress group) and after 48 h of cell recovery (recovery group) by ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) in positive and negative ESI ionization mode. Significant alterations were found in pathways such as "alanine, aspartate and glutamate metabolism", "glycolysis", and "glutathione metabolism", mostly with increased metabolites (upregulated). Furthermore, our results indicated that the LC-MS method is effective for studying metabolism in cardiomyocytes and generated excellent fit (R2Y > 0.987) and predictability (Q2 > 0.84) values.

Analysis of secondary structure in proteins by chemical cross-linking coupled to MS.

Chemical cross-linking is an attractive technique for the study of the structure of protein complexes due to its low sample consumption and short analysis time. Furthermore, distance constraints obtained from the identification of cross-linked peptides by MS can be used to construct and validate protein models. If a sufficient number of distance constraints are obtained, then determining the secondary structure of a protein can allow inference of the protein's fold. In this work, we show how the distance constraints obtained from cross-linking experiments can identify secondary structures within the protein sequence. Molecular modeling of alpha helices and beta sheets reveals that each secondary structure presents different cross-linking possibilities due to the topological distances between reactive residues. Cross-linking experiments performed with amine reactive cross-linkers with model alpha helix containing proteins corroborated the molecular modeling predictions. The cross-linking patterns established here can be extended to other cross-linkers with known lengths for the determination of secondary structures in proteins.

Histochemical and immunohistochemical study of the glomerular development in human fetuses.

Few studies exist that establish the normal morphological patterns of glomerular development, though this is one of the organs that continues to evolve morphologically during the postnatal period up to 4 weeks after birth. In our study one kidney from each autopsy of a total of 86 autopsies was analyzed [15 weeks to 40 weeks of gestational age (GA)]. We examined the variation in the nephrogenic zone thickness, the area and diameter of the glomerular tuft, the area and diameter of the glomerular capsule, and the immunohistochemical markers, anti-CD31 and anti-CD34 antibodies, which accompany the development of the glomerular microvasculature. Periodic acid-methenamine silver (PAMS) stain was used for the morphological and morphometrical analyses, and it was particularly useful in fetuses in which autolysis had occurred. The length of the nephrogenic zone (NZ) decreased with the increase of the GA (P < 0.001) according to the formula: GA = 36.5 - (0.05 x length of NZ). The areas of the Bowman capsule (P < 0.0001), the capillary tuft (P < 0.0001), and the capillary tuft diameter (P = 0.00393) of the intermediary glomeruli increased with the advance of GA, with a positive significant correlation. The same parameters of the juxtamedullary and superficial glomeruli had no correlation with the advance of GA. The cells of the primary structures in the "S" shape of the primitive nephrons were negative for CD31 and CD34. Staining for both antibodies was found, for all GAs, in the endothelial cells of the mature glomeruli tuft and in the renal interstitial vessels. The data obtained in this work contribute to the evaluation of renal maturity in autopsied fetuses and are particularly important in fetuses when autolysis has occurred, to which the parameters used in this study can also be applied. The establishment of normal morphometric and immunohistochemical parameters for the evaluation of renal maturity increases the diagnostic precision of renal pathological alterations in aborted material and perinatal autopsy.

Assessment of renal maturity by assisted morphometry in autopsied fetuses.

BACKGROUND: The kidney is one organ which presents distinct morphology depending on GA; the classic references for kidney measurements were established several years ago and it is not certain if they are still accurate. AIM: To evaluate the renal maturity using a computer-assisted morphometry in autopsied fetuses within 20 to 40 weeks of GA. STUDY DESIGN: Microscopic hematoxylin and eosin stained renal sections from 67 stillborns were utilized for the morphometric analyses. RESULTS: It was possible to promote an adjustment in the formula used to calculate the number of mature glomeruli layers to the GA (GLN = 0.212GA + 0.0169) in autopsy material and it was different from the literature (Sr = 0.709; p < 0.001). Regarding the nephrogenic zone, it decreases with the GA as confirmed by the regression equation NZ = 490.7-(11.9 x GA) (p < 0.001; Sr = -0.685). CONCLUSION: In conclusion, our data contribute to the evaluation of renal maturity and GA in autopsied fetuses, showing particular importance in autolyzed fetuses, in which the parameters used in the present study can still be applied. The correction of the formula for counting mature glomeruli layers for each GA and the addition of new morphometric parameters for the evaluation of renal maturity increase the precision for the analysis of spontaneous abortion and autopsy material, improving the correlation with pathological processes in clinical findings and in the other organs.