Transcriptomic point of departure determination: a comparison of distribution-based and gene set-based approaches.

A key step in assessing the potential human and environmental health risks of industrial and agricultural chemicals is to determine the toxicity point of departure (POD), which is the highest dose level that causes no adverse effect. Transcriptomic POD (tPOD) values have been suggested to accurately estimate toxicity POD values. One step in the most common approach for tPOD determination involves mapping genes to annotated gene sets, a process that might lead to substantial information loss particularly in species with poor gene annotation. Alternatively, methods that calculate tPOD values directly from the distribution of individual gene POD values omit this mapping step. Using rat transcriptome data for 79 molecules obtained from Open TG-GATEs (Toxicogenomics Project Genomics Assisted Toxicity Evaluation System), the hypothesis was tested that methods based on the distribution of all individual gene POD values will give a similar tPOD value to that obtained via the gene set-based method. Gene set-based tPOD values using four different gene set structures were compared to tPOD values from five different individual gene distribution methods. Results revealed a high tPOD concordance for all methods tested, especially for molecules with at least 300 dose-responsive probesets: for 90% of those molecules, the tPOD values from all methods were within 4-fold of each other. In addition, random gene sets based upon the structure of biological knowledge-derived gene sets produced tPOD values with a median absolute fold change of 1.3-1.4 when compared to the original biological knowledge-derived gene set counterparts, suggesting that little biological information is used in the gene set-based tPOD generation approach. These findings indicate using individual gene distributions to calculate a tPOD is a viable and parsimonious alternative to using gene sets. Importantly, individual gene distribution-based tPOD methods do not require knowledge of biological organization and can be applied to any species including those with poorly annotated gene sets.

A System Biology Approach Reveals New Targets for Human Thyroid Gland Toxicity in Embryos and Adult Individuals.

Compounds of natural or synthetic origin present in personal care products, food additives, and packaging may interfere with hormonal regulation and are called endocrine-disrupting chemicals (EDCs). The thyroid gland is an important target of these compounds. The objective of this study was to analyze public data on the human thyroid transcriptome and investigate potential new targets of EDCs in the embryonic and adult thyroid glands. We compared the public transcriptome data of adult and embryonic human thyroid glands and selected 100 up- or downregulated genes that were subsequently subjected to functional enrichment analysis. In the embryonic thyroid, the most highly expressed gene was PRMT6, which methylates arginine-4 of histone H2A (86.21%), and the downregulated clusters included plasma lipoprotein particles (39.24%) and endopeptidase inhibitory activity (24.05%). For the adult thyroid gland, the most highly expressed genes were related to the following categories: metallothionein-binding metals (56.67%), steroid hormone biosynthetic process (16.67%), and cellular response to vascular endothelial growth factor stimulus (6.67%). Several compounds ranging from antihypertensive drugs to enzyme inhibitors were identified as potentially harmful to thyroid gland development and adult function.

Toxicogenomic Analysis.

The biological functions of a cell may change in response to exposure to toxic agents. Toxicogenomics employs the recent developments in genomics, transcriptomics, and proteomics to study how a chemical impacts gene/protein expression and cell functions. We describe a method for transcriptomic analysis by RNA sequencing based on Illumina HiSeq, NextSeq, or NovaSeq Systems followed by real-time qPCR validation. We also depict a method for proteomic analysis by "one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis" (1D SDS-PAGE) and a sample preparation procedure for "liquid chromatography in tandem with mass spectrometry" (LC-MS/MS), and we present some generic points to consider during LC-MS/MS.

Evaluation of seeds ethanolic extracts of Triplaris gardneriana Wedd. using in vitro and in vivo toxicological methods.

Triplaris gardneriana Wedd. is a tree used in folk medicine to treat venereal diseases and inflammation as well as a source of biological compounds with antioxidant capacity. In order to assess the safety of these bioactive compounds, the present study aimed to determine the toxicity of an ethanolic extract of T. gardneriana, (EETg). Toxicological tests included hemolytic activity, toxicity toward the brine shrimp Artemia, cytotoxicity against breast cancer cells (MCF7) and acute oral toxicity in rodents. In addition, toxicogenomics techniques were used to determine genome expression in MCF7 cells exposed to EETg. The results showed that the extract exhibits approximately 60% of hemolytic activity at the highest tested concentration (64 µg/ml) and toxicity against nauplii of Artemia sp. (LC50 of 67.85 µg/ml). Further, EETg appears to be cytotoxic to MCF7 (cell viability reduced to 40% at 250 µg/ml after 24 hr). Genomic data demonstrated differential expression of 14 genes. Data analysis indicated possible altered pathways (e.g., xenobiotic metabolism), possible adverse health risks (e.g., hepatotoxicity), and drugs with similar gene expression profile (e.g., antimicrobials). The investigation provides important information on potentially adverse aspects of EETg, which need to be considered prior to the therapeutic utilization of this plant.Abbreviations: EETg: ethanolic extract of T. gardneriana seeds; MCF7: michigan cancer foundation-7 which refers to a human breast cell line (adenocarcinoma); NGS: next-generation sequencing; edgeR: empirical analysis of digital gene expression data in R; Consensus: consensus path database; FDR: false discovery rate; NCBI: national center for biotechnology information; KEGG: kyoto encyclopedia of genes and genomes; Ingenuity: ingenuity pathway analysis software; CMAP: connectivity map; OECD: organization for economic co-operation and development; HL-60: human promyelocytic leukemia cells; PC3: prostate cancer cells.

In vitro toxicological characterisation of the antifungal compound soybean toxin (SBTX).

Soybean toxin (SBTX) is a protein isolated from soybean seeds and composed of two polypeptide subunits (17 and 27 kDa). SBTX has in vitro activity against phytopathogenic fungi such as Cercospora sojina, Aspergillus niger, and Penicillium herguei, and yeasts like Candida albicans, C. parapsilosis, Kluyveromyces marxiannus, and Pichia membranifaciens. The present study aimed to analyze in vitro whether SBTX causes any side effects on non-target bacterial and mammalian cells that could impede its potential use as a novel antifungal agent. SBTX at 100 µg/mL and 200 µg/mL did not hinder the growth of the bacteria Salmonella enterica (subspecies enterica serovar choleraesuis), Bacillus subtilis (subspecies spizizenii) and Staphylococcus aureus. Moreover, SBTX at concentrations up to 500 µg/mL did not significantly affect the viability of erythrocytes, neutrophils, and human intestinal Caco-2 cells. To study whether SBTX could induce relevant alterations in gene expression, in vitro DNA microarray experiments were conducted in which differentiated Caco-2 cells were exposed for 24 h to 100 µg/mL or 200 µg/mL SBTX. SBTX up-regulated genes involved in cell cycle and immune response pathways, but down-regulated genes that play a role in cholesterol biosynthesis and platelet degranulation pathways. Thus, although SBTX did not affect bacteria, nor induced cytotoxity in mammalian cells, it affected some biological pathways in the human Caco-2 cell line that warrants further investigation.

Correlations between Risk Factors for Breast Cancer and Genetic Instability in Cancer Patients-A Clinical Perspective Study.

Molecular epidemiological studies have identified several risk factors linking to the genes and external factors in the pathogenesis of breast cancer. In this sense, genetic instability caused by DNA damage and DNA repair inefficiencies are important molecular events for the diagnosis and prognosis of therapies. Therefore, the objective of this study was to analyze correlation between sociocultural, occupational, and lifestyle risk factors with levels of genetic instability in non-neoplastic cells of breast cancer patients. Total 150 individuals were included in the study that included 50 breast cancer patients submitted to chemotherapy (QT), 50 breast cancer patients submitted to radiotherapy (RT), and 50 healthy women without any cancer. Cytogenetic biomarkers for apoptosis and DNA damage were evaluated in samples of buccal epithelial and peripheral blood cells through micronuclei and comet assay tests. Elder age patients (61-80 years) had higher levels of apoptosis (catriolysis by karyolysis) and DNA damage at the diagnosis (baseline damage) with increased cell damage during QT and especially during RT. We also reported the increased frequencies of cytogenetic biomarkers in patients who were exposed to ionizing radiation as well as for alcoholism and smoking. QT and RT induced high levels of fragmentation (karyorrhexis) and nuclear dissolution (karyolysis) and DNA damage. Correlations were observed between age and karyorrhexis at diagnosis; smoking and karyolysis during RT; and radiation and karyolysis during QT. These correlations indicate that risk factors may also influence the genetic instability in non-neoplastic cells caused to the patients during cancer therapies.

Prediction of Non-Genotoxic Carcinogenicity Based on Genetic Profiles of Short Term Exposure Assays.

Non-genotoxic carcinogens are substances that induce tumorigenesis by non-mutagenic mechanisms and long term rodent bioassays are required to identify them. Recent studies have shown that transcription profiling can be applied to develop early identifiers for long term phenotypes. In this study, we used rat liver expression profiles from the NTP (National Toxicology Program, Research Triangle Park, USA) DrugMatrix Database to construct a gene classifier that can distinguish between non-genotoxic carcinogens and other chemicals. The model was based on short term exposure assays (3 days) and the training was limited to oxidative stressors, peroxisome proliferators and hormone modulators. Validation of the predictor was performed on independent toxicogenomic data (TG-GATEs, Toxicogenomics Project-Genomics Assisted Toxicity Evaluation System, Osaka, Japan). To build our model we performed Random Forests together with a recursive elimination algorithm (VarSelRF). Gene set enrichment analysis was employed for functional interpretation. A total of 770 microarrays comprising 96 different compounds were analyzed and a predictor of 54 genes was built. Prediction accuracy was 0.85 in the training set, 0.87 in the test set and increased with increasing concentration in the validation set: 0.6 at low dose, 0.7 at medium doses and 0.81 at high doses. Pathway analysis revealed gene prominence of cellular respiration, energy production and lipoprotein metabolism. The biggest target of toxicogenomics is accurately predict the toxicity of unknown drugs. In this analysis, we presented a classifier that can predict non-genotoxic carcinogenicity by using short term exposure assays. In this approach, dose level is critical when evaluating chemicals at early time points.

Risk assessment of Soulatrolide and Mammea (A/BA+A/BB) coumarins from Calophyllum brasiliense by a toxicogenomic and toxicological approach.

Calophyllum brasiliense (Calophyllaceae) is a tropical rain forest tree distributed in Central and South America. It is an important source of tetracyclic dipyrano coumarins (Soulatrolide) and Mammea type coumarins. Soulatrolide is a potent inhibitor of HIV-1 reverse transcriptase and displays activity against Mycobacterium tuberculosis. Meanwhile, Mammea A/BA and A/BB, pure or as a mixture, are highly active against several human leukemia cell lines, Trypanosoma cruzi and Leishmania amazonensis. Nevertheless, there are few studies evaluating their safety profile. In the present work we performed toxicogenomic and toxicological analysis for both type of compounds. Soulatrolide, and the Mammea A/BA + A/BB mixture (2.1) were slightly toxic accordingly to Lorke assay classification (DL50 > 3000 mg/kg). After a short-term administration (100 mg/kg/daily, orally, 1 week) liver toxicogenomic analysis revealed 46 up and 72 downregulated genes for Mammea coumarins, and 665 up and 1077 downregulated genes for Soulatrolide. Gene enrichment analysis identified transcripts involved in drug metabolism for both compounds. In addition, network analysis through protein-protein interactions, tissue evaluation by TUNEL assay, and histological examination revealed no tissue damage on liver, kidney and spleen after treatments. Our results indicate that both type of coumarins displayed a safety profile, supporting their use in further preclinical studies to determine its therapeutic potential.

Toxicogenomics to Evaluate Endocrine Disrupting Effects of Environmental Chemicals Using the Zebrafish Model.

The extent of our knowledge on the number of chemical compounds related to anthropogenic activities that can cause damage to the environment and to organisms is increasing. Endocrine disrupting chemicals (EDCs) are one group of potentially hazardous substances that include natural and synthetic chemicals and have the ability to mimic endogenous hormones, interfering with their biosynthesis, metabolism, and normal functions. Adverse effects associated with EDC exposure have been documented in aquatic biota and there is widespread interest in the characterization and understanding of their modes of action. Fish are considered one of the primary risk organisms for EDCs. Zebrafish (Danio rerio) are increasingly used as an animal model to study the effects of endocrine disruptors, due to their advantages compared to other model organisms. One approach to assess the toxicity of a compound is to identify those patterns of gene expression found in a tissue or organ exposed to particular classes of chemicals, through new technologies in genomics (toxicogenomics), such as microarrays or whole-genome sequencing. Application of these technologies permit the quantitative analysis of thousands of gene expression changes simultaneously in a single experiment and offer the opportunity to use transcript profiling as a tool to predict toxic outcomes of exposure to particular compounds. The application of toxicogenomic tools for identification of chemicals with endocrine disrupting capacity using the zebrafish model system is reviewed.

Food safety knowledge on the Bt mutant protein Cry8Ka5 employed in the development of coleopteran-resistant transgenic cotton plants.

Insecticidal Cry proteins from Bacillus thuringiensis (Bt) have been exploited in the development of genetically modified (GM) crops for pest control. However, several pests are still difficult to control such as the coleopteran boll weevil Anthonomus grandis. By applying in vitro molecular evolution to the cry8Ka1 gene sequence, variants were generated with improved activity against A. grandis. Among them, Cry8Ka5 mutant protein showed coleoptericidal activity 3-fold higher (LC50 2.83 µg/mL) than that of the original protein (Cry8Ka1). Cry8Ka5 has been used in breeding programs in order to obtain coleopteran-resistant cotton plants. Nevertheless, there is some concern in relation to the food safety of transgenic crops, especially to the heterologously expressed proteins. In this context, our research group has performed risk assessment studies on Cry8Ka5, using the tests recommended by Codex as well as tests that we proposed as alternative and/or complementary approaches. Our results on the risk analysis of Cry8Ka5 taken together with those of other Cry proteins, point out that there is a high degree of certainty on their food safety. It is reasonable to emphasize that most safety studies on Cry proteins have essentially used the Codex approach. However, other methodologies would potentially provide additional information such as studies on the effects of Cry proteins and derived peptides on the indigenous gastrointestinal microbiota and on intestinal epithelial cells of humans. Additionally, emerging technologies such as toxicogenomics potentially will offer sensitive alternatives for some current approaches or methods.

La medicina genómica: un cambio de paradigma de la medicina moderna retos para la bioética y el derecho / Genomic medicine; a paradigm shift in modern medicine challenges for bioethics an the law / A medicina genômica: uma mudança de paradigma da medicina moderna desafios para a bioética e para o direito

La medicina genómica y sus diferentes formas de aplicación medicinas personalizada o individualizada, de la que hacen parte la farmacogenómica, toxicogenómica y nutrigenómica; y la medicina predictiva, regenerativa o de reemplazo, molecular y reproductiva), sin lugar a dudas han transformado a la medicina moderna y se constituyen en un nuevo paradigma. Este artículo pretende hacer una revisión de las distintas formas de la medicina genómica, desde sus beneficios para la salud humana y cambios sustanciales en el abordaje del proceso salud-enfermedad, así como de las problemáticas y paradojas asociadas que deben ser abordadas desde la Bioética y el Derecho...

Genomic medicine and its different application forms, such as personalized (to which pharmacogenomics, toxicogenomics, nutrigenomics and predictive medicine belong), regenerative, molecular and reproductive medicines, have undoubtedly transformed modern medicine, becoming a new paradigm. The present study aims at reviewing the different forms of genomic medicine, from its benefits to human health, up to the problems and paradoxes that must be approached from bioethics and law...

A medicina genômica e suas diferentes formas de aplicação (medicinas personalizada ou individualizada, da qual fazem parte a farmacogenômica, a toxicogenômica e a nutrigenômica; e a medicina preditiva, regenerativa ou de substituição, molecular e reprodutiva) sem dúvidas têm transformado a medicina moderna e constituem um novo paradigma. Este artigo pretende fazer uma revisão das distintas formas da medicina genômica, desde seus benefícios para a saúde humana e mudanças substanciais na abordagem do processo saúde-doença, bem como das problemáticas e paradoxos associados que devem ser abordados a partir da Bioética e do Direito...

Personalized therapy: an interdisciplinary challenge.

Genomics must be combined with proteomics and metabolomics to rationalize a therapeutic strategy that considers gene expression, protein expression and metabolic profiles in the target organ to gain insight into other pathways implicated in the same or contributory tissues. Multidisciplinary strategies such as this provide an interactive process by which findings are translated into novel therapies.