Carlos P. Roca

A systems toxicology approach reveals the Wnt-MAPK crosstalk pathway mediated reproductive failure in Caenorhabditis elegans exposed to graphene oxide (GO) but not to reduced graphene oxide (rGO)

Abstract The potential hazards of graphene nanomaterials were investigated by exposing the nematode Caenorhabditis elegans to graphene oxide (GO) and reduced graphene oxide (rGO). The underlying mechanisms of the nano–bio interaction were addressed with an integrated systems toxicology approach using global transcriptomics, network-based pathway analysis, and experimental validation of the in-silico-derived hypotheses. Graphene oxide was found to reduce the worms’ reproductive health to a greater degree than rGO, but it did not affect survival (24 h endpoint). Comparative analysis of GO vs. rGO effects found that the wingless-type MMTV integration site family (Wnt) pathway and the mitogen-activated protein kinase (MAPK) pathway were evoked in GO- but not in rGO-exposed worms. We therefore hypothesized that crosstalk between the Wnt and MAPK pathways is responsible for C. elegans’ reproductive sensitivity to GO exposure. By targeting the individual components of the Wnt-MAPK crosstalk pathway (with qPCR gene expression and mutant reproduction analysis), we found a signaling cascade of MOM-2 → MOM-5 → MOM-4 → LIT-1 → POP-1 → EGL-5. Specifically, the activation of POP-1 (the TCF protein homolog) and subsequent repression of the Wnt/β-catenin target gene (EGL-5), analyzed with target-gene-specific RNAi in POP-1 mutant [pop-1(q645)] worms, were the central mechanisms of reduced reproductive potential in the worms exposed to GO. Our results highlight the distinct biological and molecular mechanisms of GO and rGO exposure and the role of Wnt-MAPK pathway crosstalk in regulating GO-induced reproductive failure in in vivo systems, and they will contribute to the development of efficient and innocuous graphene applications as well to improvements in mechanism-based risk assessment.

Effect assessment of engineered nanoparticles in solid media - Current insight and the way forward.

Engineered Nanoparticles (ENPs) present novel/added challenges to the established effect assessment modus operandi, requiring an update of used methods. ENPs are dimensionally and physically different from conventional chemicals, which imply that the metrics with which we relate effect and the type of effect responses are different from that of the conventional approach. Effects on organisms are often preceded by changes on the sub-organismal level (cell, genes), dedicated tools have vast potential to detect earlier (and link to) effects on higher levels of organization. High-throughput screening (HTS) is rapid, cost-effective and specific. One way forward is to link HTS to population outcomes, targeting a systems toxicology approach. Although the benefits of integrating various levels of information may seem obvious, this is an even more decisive aspect when rapid answers are needed for ENPs. Here we rank the available tools/methods, highlight main study gaps and list priority needs and the way forward.

High-throughput transcriptomics reveals uniquely affected pathways: AgNPs, PVP-coated AgNPs and Ag NM300K case studies

Understanding the mode of action of nanomaterials (NMs) aids in improving predictions and environmental risk assessment. In the present study, a high-throughput (HTP) microarray was used to study Enchytraeus crypticus gene expression. Four Ag materials (Ag NM300K, PVP-coated AgNPs, AgNPs, and AgNO3) were tested at reproduction effect concentrations, EC20 and EC50, to anchor gene expression responses to higher effect level. The results showed that while PVP-AgNPs and AgNPs had similar responses, Ag NM300K caused effects via a differentiated transcriptomic profile, with uniquely affected processes (e.g. transcytosis). For the AgNPs, the EC50 negatively affected apoptosis, which can lead to accumulation of abnormal cells and cause apical damage (reproduction). Mechanisms which are known to be related to Ag toxicity and which were observed here for the various Ag forms included apoptosis regulation, cell redox homeostasis, impairment of energy production and response to DNA damage. This HTP genomic tool enabled discrimination between Ag materials, which is not possible via standard tests (i.e. survival and reproduction endpoints). Moreover, gene expression analysis provided information regarding the mechanisms of toxicity of NMs and the pathways uniquely affected by NMs. An adverse outcome pathway (AOP) was drafted for the first time for Ag NMs; this AOP can and should be used as a basis for further research.

Variation-preserving normalization unveils blind spots in gene expression profiling

RNA-Seq and gene expression microarrays provide comprehensive profiles of gene activity, but lack of reproducibility has hindered their application. A key challenge in the data analysis is the normalization of gene expression levels, which is currently performed following the implicit assumption that most genes are not differentially expressed. Here, we present a mathematical approach to normalization that makes no assumption of this sort. We have found that variation in gene expression is much larger than currently believed, and that it can be measured with available assays. Our results also explain, at least partially, the reproducibility problems encountered in transcriptomics studies. We expect that this improvement in detection will help efforts to realize the full potential of gene expression profiling, especially in analyses of cellular processes involving complex modulations of gene expression.

Chapter 6:Nanoinformatics for Safe-by-Design Engineered Nanomaterials

Nanotechnology, often referred to as the next industrial revolution, has a considerable socioeconomic impact. Its benefits are expected to have significant effects on almost all industrial sectors and areas of society. In a recent communication, the European Commission defined the term nanomaterial ...

High-throughput tool to discriminate effects of NMs (Cu-NPs, Cu-nanowires, CuNO3, and Cu salt aged): transcriptomics in Enchytraeus crypticus

Abstract The current testing of nanomaterials (NMs) via standard toxicity tests does not cover many of the NMs specificities. One of the recommendations lays on understanding the mechanisms of action, as these can help predicting long-term effects and safe-by-design production. In the present study, we used the high-throughput gene expression tool, developed for Enchytraeus crypticus (4 × 44k Agilent microarray), to study the effects of exposure to several copper (Cu) forms. The Cu treatments included two NMs (spherical and wires) and two copper-salt treatments (CuNO3 spiked and Cu salt field historical contamination). To relate gene expression with higher effect level, testing was done with reproduction effect concentrations (EC20, EC50), using 3 and 7 days as exposure periods. Results showed that time plays a major role in the transcriptomic response, most of it occurring after 3 days. Analysis of gene expression profiles showed that Cu-salt-aged and Cu-nanowires (Nwires) differed from CuNO3 and Cu-nanoparticles (NPs). Functional analysis revealed specific mechanisms: Cu-NPs uniquely affected senescence and cuticle pattern formation, which can result from the contact of the NPs with the worms’ tegument. Cu-Nwires affected reproduction via male gamete generation and hermaphrodite genitalia development. CuNO3 affected neurotransmission and locomotory behavior, both of which can be related with avoidance response. Cu salt-aged uniquely affected phagocytosis and reproductive system development (via different mechanisms than Cu-Nwires). For the first time for Cu (nano)materials, the adverse outcome pathways (AOPs) drafted here provide an overview for common and unique effects per material and linkage with apical effects.

Corrigendum: Variation-preserving normalization unveils blind spots in gene expression profiling

This corrects the article DOI: 10.1038/srep42460.

Identifying conserved UV exposure genes and mechanisms

Studies have been showing how changes in ultraviolet (UV) affect the terrestrial system, mostly focusing on higher plants and indirect effects, e.g. UV changed food quality/decomposition. Much less attention has been given to direct effect on terrestrial species, although the negative effects have been recognized for some earthworms. Further, the actual mechanisms of UV toxicity to soil invertebrates are even less understood. We here studied the effect of UV on the soil oligochaete Enchytraeus crypticus, and attempted to identify the possible mechanisms of toxicity using high-throughput gene expression. Applying a UV dose equivalent to UV during the winter months in northern Europe we observed an 80% decrease in reproduction. For these organisms, approximately 5% of the genes were differentially expressed. Among the observations was an activation of the DNA repair mechanisms, nucleotide excision repair, which correlated with survival of the organisms. An observed repressing of apoptosis seems to have deleterious effects (e.g. because it may lead to the accumulation of aberrant cells) leading to a decline in reproduction. The mechanisms activated by UV were similar to those mechanisms activated in humans, showing conservation across species.

Mechanisms of (photo)toxicity of TiO2 nanomaterials (NM103, NM104, NM105): using high-throughput gene expression in Enchytraeus crypticus

Titanium dioxide (TiO2) based nanomaterials (NMs) are among the most produced NMs worldwide. When irradiated with light, particularly UV, TiO2 is photoactive, a property that is explored for several purposes. There are an increasing number of reports on the negative effects of photoactivated TiO2 on non-target organisms. We have here studied the effect of a suite of reference type TiO2 NMs (i.e. NM103, NM104, and NM105 and compared these to the bulk) with and without UV radiation to the oligochaete Enchytraeus crypticus. High-throughput gene expression was used to assess the molecular mechanisms, while also anchoring it to the known effects at the organism level (i.e., reproduction). Results showed that the photoactivity of TiO2 (UV exposed) played a major role in enhancing TiO2 toxicity, activating the transcription of oxidative stress, lysosome damage and apoptosis mechanisms. For non-UV activated TiO2, where toxicity at the organism level (reproduction) was lower, results showed potential for long-term effects (i.e., mutagenic and epigenetic). NM specific mechanisms were identified: NM103 affected transcription and translation, NM104_UV negatively affected the reproductive system/organs, and NM105_UV activated superoxide anion response. Results provided mechanistic information on UV-related phototoxicity of TiO2 materials and evidence for the potential long-term effects.

A novel normalization approach unveils blind spots in gene expression profiling

RNA-Seq and gene expression microarrays provide comprehensive profiles of gene activity, but lack of reproducibility has hindered their application. A key challenge in the data analysis is the normalization of gene expression levels, which is currently performed following an implicit assumption that most genes are not differentially expressed. Here, we present a mathematical approach to normalization that makes no assumption of this sort. We have found that variation in gene expression is much greater than currently believed, and that it can be measured with available technologies. Our results also explain, at least partially, the problems encountered in transcriptomics studies. We expect this improvement in detection to help efforts to realize the full potential of gene expression profiling, especially in analyses of cellular processes involving complex modulations of gene expression.RNA-Seq and gene expression microarrays provide comprehensive profiles of gene activity, by measuring the concentration of tens of thousands of mRNA molecules in single assays. However, lack of accuracy and reproducibility have hindered the application of these high-throughput technologies. A key challenge in the data analysis is the normalization of gene expression levels, which is required to make them comparable between samples. This normalization is currently performed following approaches resting on an implicit assumption that most genes are not differentially expressed. Here we show that this assumption is unrealistic and likely results in failure to detect numerous gene expression changes. We have devised a mathematical approach to normalization that makes no assumption of this sort. We have found that variation in gene expression is much greater than currently believed, and that it can be measured with available technologies. Our results also explain, at least partially, the problems encountered in transcriptomics studies. We expect this improvement in detection to help efforts to realize the full potential of gene expression profiling, especially in analyses of cellular processes involving complex modulations of gene expression, such as cell differentiation, toxic responses and cancer.