Evaluating Hemolytic and Photo Hemolytic Potential of Organophosphorus by In Vitro Method as an Alternative Tool Using Human Erythrocytes.

AIMS: The present study aims to determine the phototoxic and haemolytic activity of organophosphorus. The use of alternative in vitro assays with human erythrocytes is suggested to predict the polluting effect of these products on health. METHODOLOGY: Human erythrocytes from Toluca Blood Bank were used. Sodium dodecyl sulfate was employed as a positive control. Additionally, the haemolysis percentage of three organophosphate (Acetate, Chlorpyrifos, Malathion, Methamidophos, Methyl Parathion) induced photo haemolysis formulated with surfactants on a concentration of 2 x 109 erythrocytes were evaluated. Finally, the products were classified as irritant or phototoxic. RESULTS: Results showed that the HC50 red blood cells were similar for each organophosphate (Malathion and Methamidophos) indicating very irritant action with ratio classification (L/D) of 0.041 and 0.053, respectively. On the other hand, Chlorpyrifos was classified as an irritant with L/D= 0.14. On the other hand, the HC50 obtained photo hemolysis assays irradiated red blood cells was similar for each organophosphate (Acetate, Chlorpyrifos, Malathion, Methamidophos, Methyl Parathion) indicating no phototoxic action. CONCLUSION: As a conclusion, it can be said that the parameters of haemolysis and denaturation of proteins are good indicators to classify organophosphorus formulated with surfactants as irritating or phototoxic.

Perturbation Theory Machine Learning Modeling of Immunotoxicity for Drugs Targeting Inflammatory Cytokines and Study of the Antimicrobial G1 Using Cytometric Bead Arrays.

ChEMBL biological activities prediction for 1-5-bromofur-2-il-2-bromo-2-nitroethene (G1) is a difficult task for cytokine immunotoxicity. The current study presents experimental results for G1 interaction with mouse Th1/Th2 and pro-inflammatory cytokines using a cytometry bead array (CBA). In the in vitro test of CBA, the results show no significant differences between the mean values of the Th1/Th2 cytokines for the samples treated with G1 with respect to the negative control, but there are moderate differences for cytokine values between different periods (24/48 h). The experiments show no significant differences between the mean values of the pro-inflammatory cytokines for the samples treated with G1, regarding the negative control, except for the values of tumor necrosis factor (TNF) and Interleukin (IL6) between the group treated with G1 and the negative control at 48 h. Differences occur for these cytokines in the periods (24/48 h). The study confirmed that the antimicrobial G1 did not alter the Th1/Th2 cytokines concentration in vitro in different periods, but it can alter TNF and IL6. G1 promotes free radicals production and activates damage processes in macrophages culture. In order to predict all ChEMBL activities for drugs in other experimental conditions, a ChEMBL data set was constructed using 25 biological activities, 1366 assays, 2 assay types, 4 assay organisms, 2 organisms, and 12 cytokine targets. Molecular descriptors calculated with Rcpi and 15 machine learning methods were used to find the best model able to predict if a drug could be active or not against a specific cytokine, in specific experimental conditions. The best model is based on 120 selected molecular descriptors and a deep neural network with area under the curve of the receiver operating characteristic of 0.904 and accuracy of 0.832. This model predicted 1384 G1 biological activities against cytokines in all ChEMBL data set experimental conditions.

TcVac1 vaccine delivery by intradermal electroporation enhances vaccine induced immune protection against Trypanosoma cruzi infection in mice.

The efforts for the development and testing of vaccines against Trypanosoma cruzi infection have increased during the past years. We have designed a TcVac series of vaccines composed of T. cruzi derived, GPI-anchored membrane antigens. The TcVac vaccines have been shown to elicit humoral and cellular mediated immune responses and provide significant (but not complete) control of experimental infection in mice and dogs. Herein, we aimed to test two immunization protocols for the delivery of DNA-prime/DNA-boost vaccine (TcVac1) composed of TcG2 and TcG4 antigens in a BALB/c mouse model. Mice were immunized with TcVac1 through intradermal/electroporation (IDE) or intramuscular (IM) routes, challenged with T. cruzi, and evaluated during acute phase of infection. The humoral immune response was evaluated through the assessment of anti-TcG2 and anti-TcG4 IgG subtypes by using an ELISA. Cellular immune response was assessed through a lymphocyte proliferation assay. Finally, clinical and morphopathological aspects were evaluated for all experimental animals. Our results demonstrated that when comparing TcVac1 IDE delivery vs IM delivery, the former induced significantly higher level of antigen-specific antibody response (IgG2a + IgG2b > IgG1) and lymphocyte proliferation, which expanded in response to challenge infection. Histological evaluation after challenge infection showed infiltration of inflammatory cells (macrophages and lymphocytes) in the heart and skeletal tissue of all infected mice. However, the largest increase in inflammatory infiltrate was observed in TcVac1_IDE/Tc mice when compared with TcVac1_IM/Tc or non-vaccinated/infected mice. The extent of tissue inflammatory infiltrate was directly associated with the control of tissue amastigote nests in vaccinated/infected (vs. non-vaccinated/infected) mice. Our results suggest that IDE delivery improves the protective efficacy of TcVac1 vaccine against T. cruzi infection in mice when compared with IM delivery of the vaccine.

Antibiotics susceptibility of quinolones against Salmonella spp. strains isolated and molecularly sequenced for gyrA gene.

Drug-resistant Salmonella is frequently detected in most parts of the world, and its rate of resistance has increased significantly in recent years. However, this study aimed to evaluate the minimum inhibitory concentration (MIC, determined with the Kirby-Bauer method) of quinolones in 86 Salmonella spp. strains isolated from pigs. Both the inside and outside of the QRDR region of strains were sequenced. The DNA sequence of the QRDR region of Salmonella spp. revealed the mutations S83F, D87N and S83Y. The region outside the QRDR showed a mutation in L582G. Forty-five isolates of Salmonella ssp. were categorized as quinolone-resistant; out of these, 16 corresponded to Salmonella enterica and isolates showed intermediate resistance (6.25%) to nalidixic acid. Three isolats (18.6%) were resistant to ampicillin; two (12.5%) were resistant to carbenicillin. Moreover, three (18.7%) isolates were resistant to gentamicin, nitrofurantoin and pefloxacin, and 8 (50%) were resistant to trimethoprim/sulfamethoxazole. Six percent of Salmonella spp. strains showed less resistance to antimicrobial agents compared to S. Thyphimurium (18%). The resistance to individual quinolones varied by serotypes. For S. anatum and S. Reading, it was 12.25%, and for S. choreaeaesuis, S. typhimurium monofasica, 6.25%. In contrast, S. agona, S. bredeney and S. london were sensitive to these antibiotics. In conclusion, quinolones have become the drugs of choice for the treatment of severe Salmonella infections. The study of mutations outside the QRDR region opens up new insights about the resistance of Salmonella to fluoroquinolones.

PTML Model for Proteome Mining of B-Cell Epitopes and Theoretical-Experimental Study of Bm86 Protein Sequences from Colima, Mexico.

In this work, we developed a general perturbation theory and machine learning method for data mining of proteomes to discover new B-cell epitopes useful for vaccine design. The method predicts the epitope activity εq(cqj) of one query peptide (q-peptide) under a set of experimental query conditions (cqj). The method uses as input the sequence of the q-peptide. The method also uses as input information about the sequence and epitope activity εr(crj) of a peptide of reference (r-peptide) assayed under similar experimental conditions (crj). The model proposed here is able to classify 1 048 190 pairs of query and reference peptide sequences from the proteome of many organisms reported on IEDB database. These pairs have variations (perturbations) under sequence or assay conditions. The model has accuracy, sensitivity, and specificity between 71 and 80% for training and external validation series. The retrieved information contains structural changes in 83 683 peptides sequences (Seq) determined in experimental assays with boundary conditions involving 1448 epitope organisms (Org), 323 host organisms (Host), 15 types of in vivo process (Proc), 28 experimental techniques (Tech), and 505 adjuvant additives (Adj). Afterward, we reported the experimental sampling, isolation, and sequencing of 15 complete sequences of Bm86 gene from state of Colima, Mexico. Last, we used the model to predict the epitope immunogenic scores under different experimental conditions for the 26 112 peptides obtained from these sequences. The model may become a useful tool for epitope selection toward vaccine design. The theoretical-experimental results on Bm86 protein may help the future design of a new vaccine based on this protein.

Effects of astaxanthin in mice acutely infected with Trypanosoma cruzi.

During Trypanosoma cruzi infection, oxidative stress is considered a contributing factor for dilated cardiomyopathy development. In this study, the effects of astaxanthin (ASTX) were evaluated as an alternative drug treatment for Chagas disease in a mouse model during the acute infection phase, given its anti-inflammatory, immunomodulating, and anti-oxidative properties. ASTX was tested in vitro in parasites grown axenically and in co-culture with Vero cells. In vivo tests were performed in BALB/c mice (4-6 weeks old) infected with Trypanosoma cruzi and supplemented with ASTX (10 mg/kg/day) and/or nifurtimox (NFMX; 100 mg/kg/day). Results show that ASTX has some detrimental effects on axenically cultured parasites, but not when cultured with mammalian cell monolayers. In vivo, ASTX did not have any therapeutic value against acute Trypanosoma cruzi infection, used either alone or in combination with NFMX. Infected animals treated with NFMX or ASTX/NFMX survived the experimental period (60 days), while infected animals treated only with ASTX died before day 30 post-infection. ASTX did not show any effect on the control of parasitemia; however, it was associated with an increment in focal heart lymphoplasmacytic infiltration, a reduced number of amastigote nests in cardiac tissue, and less hyperplasic spleen follicles when compared to control groups. Unexpectedly, ASTX showed a negative effect in infected animals co-treated with NFMX. An increment in parasitemia duration was observed, possibly due to ASTX blocking of free radicals, an anti-parasitic mechanism of NFMX. In conclusion, astaxanthin is not recommended during the acute phase of Chagas disease, either alone or in combination with nifurtimox.

Experimental-Theoretic Approach to Drug-Lymphocyte Interactome Networks with Flow Cytometry and Spectral Moments Perturbation Theory.

We can combine experimental techniques like Flow Cytometry Analysis (FCA) with Chemoinformatics methods to predict the complex networks of interactions between organic compounds and targets in the immune system. In this work, we determined experimentally the values of EC50 = 17.82 µg/mL and Cytotoxicity = 20.6 % for the anti-microbial / anti-parasite drug Dermofural over Balb/C CD9 lymphocytes using flow cytometry. After that, we developed a new Perturbation-theory model for Drug-Cell Target Interactome in Lymphocytes based on dispersion-polarization moments of drug structure. The models correctly classifies 34591 out of 42715 (Accuracy = 80.9%) cases of perturbations in assay endpoints of 11492 drugs (including both train and validation series). Each endpoint correspond to one out of 2616 assays, 38 molecular and cellular targets, 77 standard type measures, in four possible (human and rodents).

Experimental and computational studies of fatty acid distribution networks.

Unbalanced uptake of Omega 6/Omega 3 (ω-6/ω-3) ratios could increase chronic disease occurrences, such as inflammation, atherosclerosis, or tumor proliferation, and methylation methods for measuring the ruminal microbiome fatty acid (FA) composition/distribution play a vital role in discovering the contribution of food components to ruminant products (e.g., meat and milk) when pursuing a healthy diet. Hansch's models based on Linear Free Energy Relationships (LFERs) using physicochemical parameters, such as partition coefficients, molar refractivity, and polarizability, as input variables (Vk) are advocated. In this work, a new combined experimental and theoretical strategy was proposed to study the effect of ω-6/ω-3 ratios, FA chemical structure, and other factors over FA distribution networks in the ruminal microbiome. In step 1, experiments were carried out to measure long chain fatty acid (LCFA) profiles in the rumen microbiome (bacterial and protozoan), and volatile fatty acids (VFAs) in fermentation media. In step 2, the proportions and physicochemical parameter values of LCFAs and VFAs were calculated under different boundary conditions (cj) like c1 = acid and/or base methylation treatments, c2 = with/without fermentation, c3 = FA distribution phase (media, bacterial, or protozoan microbiome), etc. In step 3, Perturbation Theory (PT) and LFER ideas were combined to develop a PT-LFER model of a FA distribution network using physicochemical parameters (V(k)), the corresponding Box-Jenkins (ΔV(kj)) and PT operators (ΔΔV(kj)) in statistical analysis. The best PT-LFER model found predicted the effects of perturbations over the FA distribution network with sensitivity, specificity, and accuracy > 80% for 407 655 cases in training + external validation series. In step 4, alternative PT-LFER and PT-NLFER models were tested for training Linear and Non-Linear Artificial Neural Networks (ANNs). PT-NLFER models based on ANNs presented better performance but are more complicated than the PT-LFER model. Last, in step 5, the PT-LFER model based on LDA was used to reconstruct the complex networks of perturbations in the FA distribution and compared the giant components of the observed and predicted networks with random Erdos-Rényi network models. In short, our new PT-LFER model is a useful tool for predicting a distribution network in terms of specific fatty acid distribution.

QSPR and flow cytometry analysis (QSPR-FCA): review and new findings on parallel study of multiple interactions of chemical compounds with immune cellular and molecular targets.

The immune system helps to halt the infections caused by pathogenic microbial and parasitic agents. The ChEMBL database lists very large datasets of cytotoxicity of organic compounds but notably, a large number of compounds have unknown effects over molecular and cellular targets in the immune system. Flow Cytometry Analysis (FCA) is a very important technique to determine the effect of organic compounds over these molecular and cellular targets in the immune system. In addition, multi-target Quantitative Structure- Property Relationship (mt-QSPR) models can predict drug-target interactions, networks. The objectives of this paper are the following. Firstly, we carried out a review of general aspects and some examples of applications of FCA to study the effect of drugs over different cellular targets. However, we focused more on methods, materials, and experimental results obtained in previous works reported by our group in the study of the drug Dermofural. We also reviewed different mt-QSPR models useful to predict the immunotoxicity and/or the effects of drugs over immune system targets including immune cell lineages or proteins. Secondly, we included new results not published before. Initially, we used ChEMBL data to train and validate a new model but with emphasis in the effect of drugs over lymphocytes. Lastly, we report unpublished results of the computational and FCA study of a new nitro-vinyl-furan compound over thymic lymphocytes T helpers (CD4+) and T cytotoxic (CD8+) population.

Model for high-throughput screening of drug immunotoxicity--study of the anti-microbial G1 over peritoneal macrophages using flow cytometry.

Quantitative Structure-Activity (mt-QSAR) techniques may become an important tool for prediction of cytotoxicity and High-throughput Screening (HTS) of drugs to rationalize drug discovery process. In this work, we train and validate by the first time mt-QSAR model using TOPS-MODE approach to calculate drug molecular descriptors and Linear Discriminant Analysis (LDA) function. This model correctly classifies 8258 out of 9000 (Accuracy = 91.76%) multiplexing assay endpoints of 7903 drugs (including both train and validation series). Each endpoint correspond to one out of 1418 assays, 36 molecular and cellular targets, 46 standard type measures, in two possible organisms (human and mouse). After that, we determined experimentally, by the first time, the values of EC50 = 21.58 µg/mL and Cytotoxicity = 23.6% for the anti-microbial/anti-parasite drug G1 over Balb/C mouse peritoneal macrophages using flow cytometry. In addition, the model predicts for G1 only 7 positive endpoints out 1251 cytotoxicity assays (0.56% of probability of cytotoxicity in multiple assays). The results obtained complement the toxicological studies of this important drug. This work adds a new tool to the existing pool of few methods useful for multi-target HTS of ChEMBL and other libraries of compounds towards drug discovery.

Entropy model for multiplex drug-target interaction endpoints of drug immunotoxicity.

Entropy measures are universal parameters useful to codify biologically-relevant information in many systems. In our previous work, (Gonzalez-Diaz, H., et al. Chem. Res. Toxicol. 2003, 16, 1318-1327), we introduced the molecular structure information indices called 3D-Markovian electronic delocalization entropies (3D-MEDNEs) to study the quantitative structure-toxicity relationships (QSTR) of drugs. In a second part, (Cruz-Monteagudo, M. et al. Chem. Res. Toxicol., 2008, 21 (3), 619-632), we extended 3D-MEDNEs to numerically encode toxicologically-relevant information present in Mass Spectra of the serum proteome. These works demonstrated that the idea behind classic drug QSTR models can be extended to solve more general problems in toxicological chemical research. For instance, there are not many reports of multi-target QSTR (mt-QSTR) models useful to predict multiplexed endpoints of drugs in a high number of cytotoxicity assays. In this work, we train and validate for the first time a QSTR model that correctly classifies 8,806 out of 9,001 (Accuracy = 91.1%) multiplexing assay endpoints of 7903 drugs (including both training and validation series). Each endpoint corresponds to one out of 1443 assays, 32 molecular and cellular targets, 46 standard type measures, in two possible organisms (human and mouse). We have also determined experimentally, for the first time, the values of EC50 = 8.21 µg/mL and Cytotoxicity = 26.25 % for the antimicrobial / antiparasitic drug G1 on Balb/C mouse thymic macrophages using flow cytometry. In addition, we have used the new model to predict G1 endpoints in 1,283 assays finding a low average probability of p(1) = 0.50% in 152 cytotoxicity assays. Last, we have used the model to predict average probability of the interaction of G1 with different proteins in macrophages. Interestingly, the Macrophage colony-stimulating factor receptor, the Macrophage colony-stimulating factor 1 receptor, the Macrophage migration inhibitory factor, Macrophage scavenger receptor types I and II, and the Macrophage-stimulating protein receptor, have also very low average predicted probabilities of p(1) = 0.092, 0.038, 0.077, 0.026, 0.2, 0.106, respectively. Both experimental and theoretical results show a moderate thymic macrophage cytotoxicity of G1. The obtained results are significant because they complement the immunotoxicology studies of this important drug.

Immunotoxicity, flow cytometry, and chemoinformatics: review, bibliometric analysis, and new QSAR model of drug effects over macrophages.

Bibliometric methods for analyzing and describing research output have been supported internationally by the establishment and operation of organizations such as the Institute for Scientific Information (ISI) or Scimago Ranking Institutions (SRI). This study provides an overview of the research performance of major World countries in the field cytokines, Citometric bead assays and QSAR, the most important journals in which they published their research articles, and the most important academic institutions publishing them. The analysis was based on Thomson Scientific's Web of Science (WoS), and Scimago group calculated bibliometric indicators of publication activity and actual citation impact. Studying the time period 2005-2010, and shows the visibility of Medicinal Chemistry Bioorganic in this thematic noting that the visibility of a journal must take into account not only the impact factor, but the prestige, popularity and representativeness of the theme that addresses the same making a comprehensive assessment of bibliometric indicators.

ANN multiplexing model of drugs effect on macrophages; theoretical and flow cytometry study on the cytotoxicity of the anti-microbial drug G1 in spleen.

Multiplexed biological assays provide multiple measurements of cellular parameters in the same test. In this work, we have trained and tested an Artificial Neural Network (ANN) model for the first time, in order to perform a multiplexing prediction of drugs effect on macrophage populations. In so doing, we have used the TOPS-MODE approach to calculate drug molecular descriptors and the software STATISTICA to seek different ANN models such as: Linear Neural Network (LNN), Radial Basis Function (RBF), Probabilistic Neural Networks (PNN) and Multi-Layer Perceptrons (MLP). The best model found was the LNN, which correctly classified 8258 out of 9000 (Accuracy = 93.0%) multiplexing assay endpoints of 7903 drugs (including both training and test series). Each endpoint corresponds to one out of 1418 assays, 36 molecular or cellular targets, 46 standard type measures, in two possible organisms (human and mouse). Secondly, we have determined experimentally, for the first time, the values of EC(50) = 11.41 µg/mL and Cytotoxicity = 27.1% for the drug G1 over Balb/C mouse spleen macrophages using flow cytometry. In addition, we have used the LNN model to predict the G1 activity in 1265 multiplexing assays not measured experimentally (including 152 cytotoxicity assay endpoints). Both experimental and theoretical results point out a low macrophage cytotoxicity of G1. This work breaks new ground for the 'in silico' multiplexing screening of large libraries of compounds. The results obtained are very significant because they complement the immunotoxicology studies of this important anti-microbial/anti-parasite drug.

Evaluación de la toxicidad del desodorante RLV en ojos / Evaluation of RLV deodorant toxicity in eyes

Se realizó la evaluación del posible efecto irritante de un formulado (RLV) que se empleará como desodorante y que contiene como principio activo la hexamina, la cual es empleada como antiséptico urinario. Este formulado se aplicó por vía oftálmica en 6 conejos de la raza Nueva Zelandia, durante 7 días. Las valoraciones se basan en las observaciones macroscópicas de los posibles efectos adversos que se presentan en las estructuras oculares. Para esta evaluación nos basamos en el método propuesto por Draize, así como las guías de la OECD, de acuerdo con los resultados obtenidos la forma farmacéutica elaborada a una concentración del 3 (por ciento) para el desodorante, no ocasiona irritación en las estructuras oculares

Evaluación de la toxicidad del desodorante RLV en ojos / Evaluation of RLV deodorant toxicity in eyes

Se realizó la evaluación del posible efecto irritante de un formulado (RLV) que se empleará como desodorante y que contiene como principio activo la hexamina, la cual es empleada como antiséptico urinario. Este formulado se aplicó por vía oftálmica en 6 conejos de la raza Nueva Zelandia, durante 7 días. Las valoraciones se basan en las observaciones macroscópicas de los posibles efectos adversos que se presentan en las estructuras oculares. Para esta evaluación nos basamos en el método propuesto por Draize, así como las guías de la OECD, de acuerdo con los resultados obtenidos la forma farmacéutica elaborada a una concentración del 3 (por ciento) para el desodorante, no ocasiona irritación en las estructuras oculares(AU)

Evaluación dermotoxicológica de la hexamina / Dermatoxicologic evaluation of methenamine

Se realizó la evaluación del posible efecto irritante de un formulado que se empleará como desodorante y que contiene como principio activo la hexamina, la cual es empleada como antiséptico urinario. Este formulado se aplicó por vía oftálmica en 6 conejos de la raza Nueva Zelandia durante 7 d. Las valoraciones se basan en las observaciones macroscópicas de los posibles efectos adversos que se presentan en las estructuras de la piel