Erick Garcia-Garcia

Impact of Ozonation on Naphthenic Acids Speciation and Toxicity of Oil Sands Process-Affected Water to Vibrio fischeri and Mammalian Immune System

Oil sands process-affected water (OSPW) is the water contained in tailings impoundment structures in oil sands operations. There are concerns about the environmental impacts of the release of OSPW because of its toxicity. In this study, ozonation followed by biodegradation was used to remediate OSPW. The impacts of the ozone process evolution on the naphthenic acids (NAs) speciation and acute toxicity were evaluated. Ion-mobility spectrometry (IMS) was used to preliminarily separate isomeric and homologous species. The results showed limited effects of the ozone reactor size on the treatment performance in terms of contaminant removal. In terms of NAs speciation, high reactivity of NAs with higher number of carbons and rings was only observed in a region of high reactivity (i.e., utilized ozone dose lower than 50 mg/L). It was also found that nearly 0.5 mg/L total NAs was oxidized per mg/L of utilized ozone dose, at utilized ozone doses lower than 50 mg/L. IMS showed that ozonation was able to degrade NAs, oxidized NAs, and sulfur/nitrogenated NAs. Complete removal of toxicity toward Vibrio fischeri was achieved after ozonation followed by 28-day biodegradation period. In vitro and in vivo assays indicated that ozonation reduced the OSPW toxicity to mice.

Ozone treatment ameliorates oil sands process water toxicity to the mammalian immune system

We evaluated whether ozonation ameliorated the effects of the organic fraction of oil sands process water (OSPW) on immune functions of mice. Ozonation of OSPW eliminated the capacity of its organic fraction to affect various mouse bone marrow-derived macrophage (BMDM) functions in vitro. These included the production of nitric oxide and the expression of inducible nitric oxide synthase, the production of reactive oxygen intermediates and the expression of NADPH oxidase subunits, phagocytosis, and the expression of pro-inflammatory cytokine genes. Ozone treatment also eliminated the ability of OSPW organic fraction to down-regulate the expression of various pro-inflammatory cytokine and chemokine genes in the liver of mice, one week after oral exposure. We conclude that ozone treatment may be a valuable process for the remediation of large volumes of OSPW.

Commercial naphthenic acids and the organic fraction of oil sands process water downregulate pro-inflammatory gene expression and macrophage antimicrobial responses.

This is the first report showing that the organic fraction of oil sands process water (OSPW-OF), and commercial naphthenic acids (C-NAs), cause immunotoxicity. The exposure of mouse bone marrow-derived macrophages (BMDM) to different amounts of C-NAs or OSPW-OF, did not affect cell viability in vitro. We examined whether exposure of BMDM to C-NAs or OSPW-OF affected various antimicrobial responses of these cells. A dose-dependent decrease in nitric oxide response was observed after treatment of BMDM with OSPW-OF, but not with C-NAs. Although OSPW-OF and C-NAs both down-regulated the respiratory burst response of BMDM, the suppression of the production of reactive oxygen intermediates was more pronounced in cells treated with OSPW-OF. Treatment with OSPW-OF or C-NAs reduced BMDM phagocytosis of zymosan and latex beads. The decrease of BMDM antimicrobial response after exposure to OSPW-OF or C-NAs, was accompanied by decreased pro-inflammatory cytokine gene expression. Oral exposure of mice to OSPW-OF caused down-regulation in the expression of genes encoding pro-inflammatory cytokines IFNγ, IL-1β and CSF-1. Our findings indicated that OSPW causes immunotoxic effects that may impair the ability of an exposed host to defend against infectious disease. Furthermore, given the differences between the effects of OSPW-OF and C-NAs, C-NAs should not be assumed to be a direct surrogate for the immunotoxic chemical species in OSPW.

Commercial naphthenic acids and the organic fraction of oil sands process water induce different effects on pro‐inflammatory gene expression and macrophage phagocytosis in mice

Naphthenic acids (NAs) are believed to be the major toxic component of oil sands process water (OSPW). Different OSPW preparations have distinct NA compositions, and additional organics, that differ from the commercial NAs (C‐NAs) often used for toxicology studies. To evaluate whether C‐NAs are an adequate model to study OSPW toxicity in complex organisms, we compared the effects of C‐NAs and the extractable organic fraction of OSPW (OSPW‐OF) on mice immune mechanisms. Mice were orally exposed to different C‐NA doses, or OSPW‐OF at the same NA dose, for up to 8 weeks, and the expression of pro‐inflammatory genes in different organs was determined using quantitative PCR. C‐NAs and OSPW‐OF altered the expression of pro‐inflammatory genes, inducing either expression down‐regulation or up‐regulation, depending on the organ examined and time after exposure. The time at which gene expression alterations occurred, and the specific sets of genes whose expression was altered, were very different between animals exposed to C‐NAs or to OSPW‐OF. We evaluated the ability of mouse peritoneal macrophages to phagocytose yeast cell wall, as a measure of the ability of mice to mount a central function of the innate immune response. Phagocytosis was significantly reduced in animals exposed to C‐NAs, but enhanced in mice exposed to OSPW‐OF. Our results indicate that studies using C‐NAs may not necessarily reflect the possible effects induced in animals by process water from tailing ponds. Copyright

The acute and sub-chronic exposures of goldfish to naphthenic acids induce different host defense responses

Naphthenic acids (NAs) are believed to be the major toxic component in oil sands process-affected water (OSPW) produced by the oil sands mining industry in Northern Alberta, Canada. We recently reported that oral exposure to NAs alters mammalian immune responses, but the effect of OSPW or NAs on the immune mechanisms of aquatic organisms has not been fully elucidated. We analyzed the effects of acute and sub-chronic NAs exposures on goldfish immune responses by measuring the expression of three pro-inflammatory cytokine genes, antimicrobial functions of macrophages, and host defense after challenge with a protozoan pathogen (Trypanosoma carassii). One week after NAs exposure, fish exhibited increased expression of pro-inflammatory cytokines (IFNγ, IL-1β1, TNF-α2) in the gills, kidney and spleen. Primary macrophages from fish exposed to NAs for one week, exhibited increased production of nitric oxide and reactive oxygen intermediates. Goldfish exposed for one week to 20 mg/L NAs were more resistant to infection by T. carassii. In contrast, sub-chronic exposure of goldfish (12 weeks) to NAs resulted in decreased expression of pro-inflammatory cytokines in the gills, kidney and spleen. The sub-chronic exposure to NAs reduced the ability of goldfish to control the T. carassii infection, exemplified by a drastic increase in fish mortality and increased blood parasite loads. This is the first report analyzing the effects of OSPW contaminants on the immune system of aquatic vertebrates. We believe that the bioassays depicted in this work will be valuable tools for analyzing the efficacy of OSPW remediation techniques and assessment of diverse environmental pollutants.

Fcγ receptors exhibit different phagocytosis potential in human neutrophils

In neutrophils, two receptors for IgG antibodies, namely FcgammaRIIA and FcgammaRIIIB are constitutively expressed, and a third one, FcgammaRI, can be upregulated by interferon-gamma. Whether FcgammaRIIIB is capable of triggering phagocytosis by itself is still controversial. The main role of FcgammaRI has not been clearly established in these cells. To address this problem, neutrophils were treated with interferon-gamma, and then phagocytosis mediated by each type of Fcgamma receptor was evaluated by flow cytometry. FcgammaRIIA was the most efficient receptor for phagocytosis. FcgammaRIIIB could mediate phagocytosis but much less efficiently than FcgammaRIIA. Both FcgammaRIIA- and FcgammaRIIIB-mediated phagocytosis were blocked by inhibitors of Src family kinases, Syk, PI 3-K, and ERK. In contrast, interferon-gamma-induced FcgammaRI was not able to mediate phagocytosis. Also, FcgammaRI did not activate ERK in the nucleus, but was however able to stimulate an efficient calcium rise. These data show that different neutrophil Fcgamma receptors possess different phagocytosis capabilities: FcgammaRIIA and FcgammaRIIIB, but not FcgammaRI, promote phagocytosis.

FcγRIIA and FcγRIIIB Mediate Nuclear Factor Activation through Separate Signaling Pathways in Human Neutrophils

Receptors for IgG Abs (Fcγ receptors) are capable of triggering diverse cell responses in leukocytes. In neutrophils, two Fcγ receptors, namely FcγRIIA and FcγRIIIB, are constitutively expressed. The signaling pathways that regulate FcγRIIA-mediated phagocytosis have been relatively well described. However, the different signaling pathways that lead to NF activation after engagement of each Fcγ receptor have only been partially described. To address this problem, neutrophils were stimulated by cross-linking selectively each type of Fcγ receptor with specific mAbs, and NF activation was then analyzed. FcγRIIIB, but not FcγRIIA, promoted a robust increase in phosphorylated ERK in the nucleus, and also efficient phosphorylation of the NF Elk-1. Complete mAb 3G8 (anti-FcγRIIIB) induced a higher response than did F(ab′)2 fragments of mAb 3G8, suggesting a possible synergistic effect of both FcγR receptors. However, mAb IV.3 (anti-FcγRIIA) alone did not cause an increase of phosphorylated ERK in the nucleus. FcγRIIIB-induced nuclear phosphorylation of ERK, and of Elk-1, was not affected by Syk, PI3K, or MEK inhibitors. In contrast, FcγRIIA- or FcγRIIIB-mediated phosphorylation of cytoplasmic ERK depended on Syk, PI3K, and MEK. Also, ERK, but not MEK, was constitutively present in the nucleus, and FcγRIIIB cross-linking did not increase the levels of nuclear ERK or MEK. These data clearly show that different neutrophil Fcγ receptors possess different signaling capabilities. FcγRIIIB, but not FcγRIIA, activates a unique signaling pathway leading to the nuclear-restricted phosphorylation of ERK and Elk-1, independently of Syk, PI3K, or MEK.

Examination of the stimulatory signaling potential of a channel catfish leukocyte immune-type receptor and associated adaptor

Expressed by various subsets of myeloid and lymphoid immune cells, channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are predicted to play a key role in the initiation and termination of teleost cellular effector responses. These type I transmembrane proteins belong to the immunoglobulin superfamily and display features of immunoregulatory receptors with inhibitory and/or stimulatory signaling potential. Expanding on our previous work, which demonstrated that putative stimulatory IpLITR-types associated with the catfish adaptor proteins IpFcRγ and FcRγ-L, this study focuses on the functional significance of this immune receptor-adaptor signaling complex. Specifically, we generated an epitope-tagged chimeric receptor construct by fusing the extracellular domain of IpLITR 2.6b with the transmembrane region and cytoplasmic tail of IpFcRγ-L. This chimera was stably expressed in a rat basophilic leukemia (RBL) cell line, RBL-2H3, and following cross-linking of the surface receptor with an anti-hemagglutinin monoclonal antibody or opsonized microspheres, the chimeric teleost receptor induced cellular degranulation and phagocytic responses, respectively. Site-directed mutagenesis of the immunoreceptor tyrosine-based activation motif encoded within the cytoplasmic tail of the chimera confirmed that these functional responses were dependent on the phosphorylated tyrosines within this motif. Using a combination of phospho-specific antibodies and pharmacological inhibitors, we also demonstrate that the IpLITR/IpFcRγ-L-induced degranulation response requires the activity of Src homology 2 domain containing protein tyrosine phosphatases, phosphatidylinositol 3-kinase, protein kinase C, and mitogen-activated protein kinases but appears independent of the c-Jun N-terminal kinase and p38 MAP kinase pathways. In addition to this first look at stimulatory IpLITR-mediated signaling and its influence on cellular effector responses, the advantage of generating RBL-2H3 cells stably expressing a functional IpLITR-adaptor chimera will be discussed.

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-κB and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Adding Complexity to Phagocytic Signaling: Phagocytosis-Associated Cell Responses and Phagocytic Efficiency

Regulation of the phagocytic process involves complex signaling pathways that lead to particle internalization and destruction. Phagocytosis, however, is not a cellular response occurring as an isolated event. Phagocytic signaling involves the regulation of many phagocytosis-associated cell responses that are important for host defense and for the resolution of the inflammatory process. In addition, due to the destructive nature of the phagocytic process, this cell response is tightly controlled, and phagocytes must respond to activation and differentiation signals that modulate their phagocytic efficiency. Presently it is unclear how all these events are coordinated. Available information, however, suggests a model in which phagocytosis-associated cell responses are regulated through signaling pathways that occur in parallel to, and partially overlap those regulating the ingestion process itself. Additionally, activation and differentiation signals appear to potentiate, or modify the utilization important signaling enzymes that regulate phagocytosis, in order to make this process more efficient.