Effect of 13-Hydroperoxyoctadecadienoic Acid (13-HPODE) Treatment on the Transcriptomic Profile of Poorly-Differentiated Caco-2 Cells

Dietary lipid peroxides (LOOHs) have been linked to gut pathologies including inflammatory bowel disease and cancer. As poorly differentiated (PDiff) intestinal epithelial (Caco-2) cells represent tumor cells and could model intestinal crypt cells, we investigated the cellular response of PDiff Caco-2 cells to the most common dietary LOOH, 13-hydroperoxyoctadecadienoic acid (13-HPODE), using RNA sequencing (RNA-seq). Further, we compared the results with the transcriptomic profiles of PDiff cells exposed to linoleic acid (LA) or hydrogen peroxide (H2O2). The results showed that 13-HPODE treatment induces expression of genes related to detoxification and several metabolic pathways including glycogen and amino acid metabolism, which may create a tumorigenic environment despite the downregulation of some proliferation-related genes. 13-HPODE also enhanced peroxisome proliferator-activated receptor signaling involved in lipid metabolism, homeostasis, and inflammation. Additionally, results indicated that 13-HPODE impacts ribosome biogenesis, phagosome, and mitochondrial function through disrupted electron transport chain, which may contribute to disease development or progression. RNA-seq results were validated using qRT-PCR. This study provides an understanding of PDiff Caco-2 cell response to 13-HPODE and the mechanisms by which 13-HPODE modulates cellular processes that may contribute to disease development or progression.

Abstract 619: Alpha Keto Acids Decompose Peroxides and Prevents Oxidation of Lipoproteins

Background: Alpha keto acids are unstable and decompose rapidly. In this study, we tested the ability of alpha keto acids to reduce peroxides and inhibit oxidation of lipoproteins. Methods: Keto salicylic acid (KSA) and Keto Octanoicacid (KoA) were synthesized and their ability to reduce hydrogen peroxides as well as lipid peroxides (LOOH) was measured using 13-hydroperoxyoctadecadienoic acid (13-HPODE). Lipoproteins (LDL and HDL) were isolated from human plasma and oxidation of liporproteins was performed using copper and MPO in the presence or absence of the keto compounds. RAW 264.7 cells and HUVECS were incubated with LPS and mm-LDL respectively either in the presence or absence of the keto compounds. RNA was isolated from treated cells and real time PCR was performed to analyze IL-1α, IL-6, MCP-1 and VCAM1 gene expressions. Reactive oxygen species were evaluated using DCF fluorescence in presence and absence of the keto compounds. Results: KSA reduced both H2O2 and 13-HPODE whereas KoA is able to reduce the former but not the latter. Both compounds inhibited the lipoprotein oxidation in a dose dependent manner and were able to reduce ROS production by H2O2. KSA is able to inhibit both LPS as well as mm-LDL induced inflammation. However, KoA showed a dual effect as it induced inflammatory markers in the presence of LPS, but inhibited the mm-LDL-induced inflammatory gene expressions. Conclusion: The results of our studies suggest that these keto compounds a) inhibit both enzymatic and non enzymatic oxidation of lipoproteins; b) reduce peroxides and ROS and c) have inhibitory and inducing effect on inflammatory cytokine/gene production in presence of mm-LDL and LPS respectively. Based on these results, we predict that these keto compounds could have therapeutic potential in reducing CVD/atherosclerosis-associated inflammation.

PpoC from Aspergillus nidulans is a fusion protein with only one active haem

In Aspergillus nidulans Ppos [psi (precocious sexual inducer)-producing oxygenases] are required for the production of so-called psi factors, compounds that control the balance between the sexual and asexual life cycle of the fungus. The genome of A. nidulans harbours three different ppo genes: ppoA, ppoB and ppoC. For all three enzymes two different haem-containing domains are predicted: a fatty acid haem peroxidase/dioxygenase domain in the N-terminal region and a P450 haem-thiolate domain in the C-terminal region. Whereas PpoA was shown to use both haem domains for its bifunctional catalytic activity (linoleic acid 8-dioxygenation and 8-hydroperoxide isomerization), we found that PpoC apparently only harbours a functional haem peroxidase/dioxygenase domain. Consequently, we observed that PpoC catalyses mainly the dioxygenation of linoleic acid (18:2Δ9Z,12Z), yielding 10-HPODE (10-hydroperoxyoctadecadienoic acid). No isomerase activity was detected. Additionally, 10-HPODE was converted at lower rates into 10-KODE (10-keto-octadecadienoic acid) and 10-HODE (10-hydroxyoctadecadienoic acid). In parallel, decomposition of 10-HPODE into 10-ODA (10-octadecynoic acid) and volatile C-8 alcohols that are, among other things, responsible for the characteristic mushroom flavour. Besides these principle differences we also found that PpoA and PpoC can convert 8-HPODE and 10-HPODE into the respective epoxy alcohols: 12,13-epoxy-8-HOME (where HOME is hydroxyoctadecenoic acid) and 12,13-epoxy-10-HOME. By using site-directed mutagenesis we demonstrated that both enzymes share a similar mechanism for the oxidation of 18:2Δ9Z,12Z; they both use a conserved tyrosine residue for catalysis and the directed oxygenation at the C-8 and C-10 is most likely controlled by conserved valine/leucine residues in the dioxygenase domain.

Defects in Conidiophore Development and Conidium-Macrophage Interactions in a Dioxygenase Mutant of Aspergillus fumigatus

ABSTRACT Oxygenated fatty acids, or oxylipins, play an essential role in physiological signaling and developmental processes in animals, plants, and fungi. Previous characterization of three Aspergillus fumigatus dioxygenases (PpoA, PpoB, and PpoC), similar in sequence to mammalian cyclooxygenases, showed that PpoA is responsible for the production of the oxylipins 8R-hydroperoxyoctadecadienoic acid and 5S,8R-dihydroxy-9Z,12Z-octadecadienoic acid and that PpoC is responsible for 10R-hydroxy-8E,12Z-hydroperoxyoctadecadienoic acid. Here, Δppo mutants were characterized to elucidate the role of fungal dioxygenases in A. fumigatus development and host interactions. The ΔppoC strain displayed distinct phenotypes compared to those of other Δppo mutants and the wild type, including altered conidium size, germination, and tolerance to oxidative stress as well as increased uptake and killing by primary alveolar macrophages. These experiments implicate oxylipins in pathogen development and suggest that ΔppoC represents a useful model for studying the A. fumigatus-host interaction.