Robust methods for predicting the transition states of chemical reactions: new approaches that focus on key coordinates

A new method for optimizing transition state and minima structures using redundant internal coordinates is presented. The new method is innovative because it allows the user to select a few key reduced coordinates, whose Hessian components will be accurately computed by finite differencing; the remaining elements of the Hessian are approximated with a quasi-Newton method. Usually the reduced coordinates are the coordinates that are involved in bond breaking/forming. In order to develop this method, several other innovations were made, including ways to (a) select the key reduced coordinates automatically, (b) guess the transition state quickly and efficiently, (c) choose dihedrals so that the “linear angle problem” is avoided, (d) robustly convert redundant internal coordinates to Cartesian coordinates. These, and other technical developments (e.g., new quasi-Newton Hessians, new trust-radius updates), were validated using a database of 7000 initial transition-state guesses for a diverse set of 140 chemical reactions

Research into the respiratory immunodisturbing mechanisms of chemical allergens by means of in vitro test systems and transcriptomics

Chemical-induced respiratory allergy poses significant challenges for the toxicologist and regulator, not least because there are, as yet, no validated or widely accepted methods available for the identification of chemicals that have the potential to cause respiratory sensitization. This may be attributable to the fact that there remains uncertainty about the complete mechanisms through which respiratory sensitization to chemicals can be acquired. Toxicological assessment of most xenobiotics mainly relies on in vivo animal tests, having their economical and ethical drawbacks. Therefore an in vitro screening model for the identification of respiratory allergens is needed. Besides the choice of an appropriate cell model, it is also important to study relevant mechanisms and derive endpoints in the context of respiratory sensitization. For this purpose, transcriptomics, proteomics, and metabolomics are promising tools to better understand the underlying molecular and biological mechanisms through which allergic sensitization to chemicals is induced and regulated. The major goal of this thesis was to investigate the alterations in gene expression of human bronchial (BEAS-2B) and alveolar (A549) epithelial cells, and THP-1 macrophages after exposure to respiratory sensitizers and respiratory non-sensitizing chemicals, and to identify genes that are able to discriminate between both groups of compounds. The cells were exposed during 6, 10, and 24 hours to the respiratory sensitizers ammonium hexachloroplatinate IV, hexamethylene diisocyanate, and trimellitic anhydride, the irritants acrolein and methyl salicylate, and the skin sensitizer 1-chloro-2,4-dinitrobenzene at subcytotoxic levels. Overall changes in gene expression were evaluated using Agilent Whole Human Genome 4x44K oligonucleotide arrays. In each cell model, a Fisher Linear Discriminant Analysis was used to obtain a ranking of genes that reflected the potential to discriminate between respiratory sensitizers and respiratory non-sensitizing chemicals. In a first attempt to develop an in vitro respiratory sensitization assay, the 20 most discriminative genes that were able to distinguish between both groups of chemicals were discussed for each cell model. However, the exact role of these marker genes in the respiratory sensitization process and how they are influenced in vivo after exposure to respiratory allergens is yet unknown, and it is likely that different pathways are involved in sensitization of the respiratory tract. Secondly, the 1000 most discriminating genes for each cell model were used to identify canonical pathways which may contribute to a better understanding of the underlying mechanisms of respiratory sensitization. Using pathway analysis, platelet-derived growth factor signaling was found as being a possible important pathway involved in the respiratory sensitization process in THP-1 macrophages. Within the BEAS-2B cell model, the phosphatase and tensin homolog (PTEN) signaling pathway was identified and might be specific for respiratory sensitization. None of the canonical signaling pathways activated in A549 cells were specific for respiratory sensitization. In conclusion, this study demonstrated the feasibility and utility of transcriptomics approaches to identify selective markers that can discriminate respiratory sensitizers from respiratory nonsensitizers. Canonical pathways which might be specific for respiratory sensitization were identified. For the development of an in vitro assay for respiratory sensitization, these findings will need to be expanded using a larger set of chemicals, with priority on gene expression studies and analysis of mechanisms of respiratory sensitization in the BEAS-2B cell model

Gene expression profiles reveal distinct immunological responses of cobalt and cerium dioxide nanoparticles in two in vitro lung epithelial cell models

Fragmentary knowledge exists on cellular signaling responses underlying possible adverse health effects of CoO- and CeO2-nanoparticles (NP)s after inhalation.We aimed to perform a time kinetic study of gene expression profiles induced by these NPs in alveolar A549 and bronchial BEAS-2B epithelial cells, and investigated possible immune system modulation. The kinetics of the cell responses induced by the NPs were different between the lung epithelial models. Both CoO- and CeO2-NP exposure induced mainly downregulation of gene transcription. BEAS-2B cells were found to be more sensitive, as they showed a higher number of differentially expressed transcripts (DET) at a 10-fold lower NP-concentration than A549 cells. Hierarchical clustering of all DET indicated that the transcriptional responses were heterogeneous among the two cell types and two NPs. Between 1% and 14% DET encoding markers involved in immune processes were observed.The transcriptional impact of the metal oxide NPs appeared to be cell-dependent, both at the general and immune response level, whereas each lung epithelial cell model responded differently to the two NP types. Thus, the study provides gene expression markers and immune processes involved in CoO- and CeO2-NP-induced toxicity, and demonstrates the usefulness of comprehensive-omics studies to differentiate between NP responses.Peer Reviewe

Exploring the substrate selectivity of human sEH and M. tuberculosis EHB using QM/MM

The mechanisms of human soluble epoxide hydrolase (sEH) and the corresponding epoxide hydrolase enzyme from Mycobacterium tuberculosis (EHB) are studied computationally, using the quantum mechanics/molecular mechanics (QM/MM) method. To do this, we modeled the alkylation and the hydrolysis steps of three substrates: trans-1,3-diphenylpropene oxide, trans-stilbene oxide and cis-stilbene oxide. Studying the regioselectivity for trans-1,3-diphenylpropene oxide, we determined that both enzymes prefer ring opening via attack on the benzylic carbon. In agreement with experimental studies, our computations show that the rate-limiting step is hydrolysis of the ester intermediate, with reaction barriers of approximately 13 to 18 kcal/mol. Using the barrier energies of this rate-limiting step, the three epoxides were ranked in order of reactivity. Though the reactivity order was correctly predicted for sEH, the predicted order for EHB did not correspond to experimental observations. Next, the electrostatic contributions of individual residues on the barrier height of the rate-limiting step were also studied. This revealed several residues important for catalysis. The secondary tritium kinetic isotope effect for the alkylation step was determined using a cluster model for the active site of sEH. The calculated value was 1.27, suggesting a late transition state for the rate-limiting step. Finally, we analyzed the reactivity trends using reactivity indicators from conceptual density functional theory, allowing us to identify ease of electron transfer as the primary driving force for the reaction