Craig E. Wheelock

Challenges in regulating pesticide mixtures

This paper introduces the field of mixture toxicity and the challenges in regulating pesticide mixtures. Even though pesticides are unique chemical stressors designed to have biological activity that can affect a number of nontarget species, they are intentionally placed into the environment in large quantities. Currently, methods and terminology for evaluating mixture toxicity are poorly established. The most common approach used is the assumption of additive concentration, with the concentrations adjusted for potency to a reference toxicant. Using this approach, the joint action of pesticides that have similar chemical structures and modes of toxic action can be predicted. However, this approach and other modeling techniques often provide little insight into the observed toxicity produced by mixtures of pesticides from different classes. Particularly difficult to model are mixtures that involve a secondary toxicant that changes the toxicokinetics of a primary toxicant. This may result in increased activation or a change in the persistence of the primary toxicant within the organism and may be responsible for a several-fold increase or decrease in toxicity. At present, the ecological effects caused by mixtures of pesticides are given little consideration in the regulatory process. However, mixtures are being considered in relation to human health in the pesticide registration process, setting a precedent that could be followed for ecological protection. Additionally, pesticide mixtures may be regulated through toxicity testing of surface water under the Clean Water Act. The limits of our basic knowledge of how mixtures interact are compromising both these avenues for regulating mixtures. We face many challenges to adequately protecting the environment from mixture toxicity; these challenges include understanding the interactions of toxicants within an organism, identifying the mixtures that most commonly occur and cause adverse effects, and developing a regulatory structure capable of minimizing environmental impacts.

Structural refinement of inhibitors of urea-based soluble epoxide hydrolases

The soluble epoxide hydrolase (sEH) is involved in the metabolism of arachidonic, linoleic, and other fatty acid epoxides, endogenous chemical mediators that play an important role in blood pressure regulation and inflammation. 1,3-Disubstituted ureas, carbamates, and amides are new potent and stable inhibitors of sEH. However, the poor solubility of the lead compounds limits their use. Inhibitor structure-activity relationships were investigated to better define the structural requirements for inhibition and to identify points in the molecular topography that could accept polar groups without diminishing inhibition potency. Results indicate that lipophilicity is an important factor controlling inhibitor potency. Polar groups could be incorporated into one of the alkyl groups without loss of activity if they were placed at a sufficient distance from the urea function. The resulting compounds had a 2-fold higher water solubility. These findings will facilitate the rational design and optimization of sEH inhibitors with better physical properties.

Application of ’omics technologies to biomarker discovery in inflammatory lung diseases

Inflammatory lung diseases are highly complex in respect of pathogenesis and relationships between inflammation, clinical disease and response to treatment. Sophisticated large-scale analytical methods to quantify gene expression (transcriptomics), proteins (proteomics), lipids (lipidomics) and metabolites (metabolomics) in the lungs, blood and urine are now available to identify biomarkers that define disease in terms of combined clinical, physiological and patho-biological abnormalities. The aspiration is that these approaches will improve diagnosis, i.e. define pathological phenotypes, and facilitate the monitoring of disease and therapy, and also, unravel underlying molecular pathways. Biomarker studies can either select predefined biomarker(s) measured by specific methods or apply an “unbiased” approach involving detection platforms that are indiscriminate in focus. This article reviews the technologies presently available to study biomarkers of lung disease within the ’omics field. The contributions of the individual ’omics analytical platforms to the field of respiratory diseases are summarised, with the goal of providing background on their respective abilities to contribute to systems medicine-based studies of lung disease. Summary of the application of ’omics-based analytical platforms for biomarker discovery in inflammatory lung diseases http://ow.ly/mjGGc

Exosomes from human macrophages and dendritic cells contain enzymes for leukotriene biosynthesis and promote granulocyte migration

BACKGROUND Leukotrienes (LTs) are potent proinflammatory lipid mediators with key roles in the pathogenesis of asthma and inflammation. Recently, nanovesicles (exosomes), released from macrophages and dendritic cells (DCs), have become increasingly appreciated as messengers in immunity. OBJECTIVE We investigated whether exosomes from human macrophages, DCs, and plasma contain enzymes for LT biosynthesis and studied potential roles for exosomes in transcellular LT metabolism and granulocyte chemotaxis. METHODS The presence of LT pathway enzymes and LT biosynthesis in exosomes and cells was analyzed by Western blot, immunoelectron microscopy, and enzyme activity assays. Surface marker expression was evaluated by flow cytometry, and granulocyte migration was assessed in a multiwell chemotaxis system. RESULTS Exosomes from macrophages and DCs contain functional enzymes for LT biosynthesis. After incubation of intact cells with the LT biosynthesis intermediate LTA(4), LTB(4) was the major product of macrophages, whereas DCs primarily formed LTC(4). However, in exosomes from both cell types, LTC(4) was the predominant LTA(4) metabolite. Exosomal LTC(4) formation (per milligram protein) exceeded that of cells. In macrophages and DCs, TGF-β1 upregulated LTA(4) hydrolase along with increased LTB(4) formation also in the exosomes. Moreover, TGF-β1 modified the expression of surface marker proteins on cells and exosomes and reduced the exosome yield from macrophages. On Ca(2+)-ionophore and arachidonic acid stimulation, exosomes produced chemotactic eicosanoids and induced granulocyte migration. Interestingly, active LTA(4) hydrolase and LTC(4) synthase were present also in exosomes from human plasma. CONCLUSION Our findings indicate that exosomes can contribute to inflammation by participation in LT biosynthesis and granulocyte recruitment.

Advances in eicosanoid research, novel therapeutic implications.

Eicosanoids are a family of oxygenated metabolites of arachidonic acid, including the prostaglandins, thromboxanes, leukotrienes and lipoxins. These lipid mediators play essential roles in normal cellular homeostasis as well as in a number of disease states. This review will focus on recent advances in the field of eicosanoids and highlight specific discoveries and achievements. Emphasis will be placed on structure and receptor biology, which are of significant pharmacological and clinical relevance.

PfEMP1-DBL1α amino acid motifs in severe disease states of Plasmodium falciparum malaria

An infection with Plasmodium falciparum may lead to severe malaria as a result of excessive binding of infected erythrocytes in the microvasculature. Vascular adhesion is mediated by P. falciparum erythrocyte membrane protein-1 (PfEMP1), which is encoded for by highly polymorphic members of the var-gene family. Here, we profile var gene transcription in fresh P. falciparum trophozoites from Ugandan children with malaria through var-specific DBL1α-PCR amplification and sequencing. A method for subsectioning region alignments into homology areas (MOTIFF) was developed to examine collected sequences. Specific PfEMP1-DBL1α amino acid motifs correlated with rosetting and severe malaria, with motif location corresponding to distinct regions of receptor interaction. The method is potentially applicable to other families of variant proteins and may be useful in identifying sequence-phenotype relationships. The results suggest that certain PfEMP1 sequences are predisposed to inducing severe malaria.

12- and 15-lipoxygenases in human carotid atherosclerotic lesions: Associations with cerebrovascular symptoms

Lipoxygenase (ALOX) enzymes are implicated in both pro- and anti-atherogenic processes. The aim of this study was to investigate mRNA expression of 12- and 15-lipoxygenases (ALOX12, ALOX12B, ALOX15, ALOX15B) and the atypical ALOXE3 in human carotid atherosclerotic lesions, in relation to cerebrovascular symptoms and risk factors. The Biobank of Karolinska Endarterectomies (BiKE) collection of human carotid plaque tissue and associated clinical data was utilized (n=132). Lesion mRNA levels were analyzed by TaqMan qPCR (n=132) and microarray hybridization (n=77). Of the investigated mRNAs, only ALOX15B (15-LOX-2; epidermis-type 15-LOX) was readily detected in all plaque samples by qPCR, and thus suitable for quantitative statistical evaluation. ALOX12, ALOX12B, ALOX15 and ALOXE3 were detected with lower frequency and at lower levels, or virtually undetected. Microarray analysis confirmed ALOX15B as the most abundant 12- or 15-lipoxygenase mRNA in carotid lesions. Comparing plaques with or without attributable cerebrovascular symptoms (amaurosis fugax, transient ischemic attack, or stroke), ALOX15B mRNA levels were higher in symptomatic than asymptomatic plaques (1.31 [1.11-1.56], n=102; and 0.79 [0.55-1.15], n=30, respectively; p=0.008; mean [95% CI], arbitrary units). Multiple regression analysis confirmed symptomatic/asymptomatic status as a significant determinant of ALOX15B mRNA levels, independently of potentially confounding factors. Immunohistochemical analyses showed abundant ALOX15B expression in macrophage-rich areas of carotid lesions, and lipidomic analyses demonstrated the presence of typical ALOX15B products in plaque tissue. In summary, we observed associations between high ALOX15B expression in carotid lesions and a history of cerebrovascular symptoms. These findings suggest a link between ALOX15B and atherothrombotic events that merits further investigation.

Evaluation of α-cyanoesters as fluorescent substrates for examining interindividual variation in general and pyrethroid-selective esterases in human liver microsomes

Carboxylesterases hydrolyze many pharmaceuticals and agrochemicals and have broad substrate selectivity, requiring a suite of substrates to measure hydrolytic profiles. To develop new esterase substrates, a series of α-cyanoesters that yield fluorescent products upon hydrolysis was evaluated for use in carboxylesterase assays. The use of these substrates as surrogates for Type II pyrethroid hydrolysis was tested. The results suggest that these novel analogs are appropriate for the development of high-throughput assays for pyrethroid hydrolase activity. A set of human liver microsomes was then used to determine the ability of these substrates to report esterase activity across a small population. Results were compared against standard esterase substrates. A number of the esterase substrates showed correlations, demonstrating the broad substrate selectivity of these enzymes. However, for several of the substrates, no correlations in hydrolysis rates were observed, suggesting that multiple carboxylesterase isozymes are responsible for the array of substrate hydrolytic activity. These new substrates were then compared against α-naphthyl acetate and 4-methylumbelliferyl acetate for their ability to detect hydrolytic activity in both one- and two-dimensional native electrophoresis gels. Cyano-2-naphthylmethyl butanoate was found to visualize more activity than either commercial substrate. These applications demonstrate the utility of these new substrates as both general and pyrethroid-selective reporters of esterase activity.

Dietary nitrate reduces resting metabolic rate: a randomized, crossover study in humans

BACKGROUND Nitrate, which is an inorganic anion abundant in vegetables, increases the efficiency of isolated human mitochondria. Such an effect might be reflected in changes in the resting metabolic rate (RMR) and formation of reactive oxygen species. The bioactivation of nitrate involves its active accumulation in saliva followed by a sequential reduction to nitrite, nitric oxide, and other reactive nitrogen species. OBJECTIVE We studied effects of inorganic nitrate, in amounts that represented a diet rich in vegetables, on the RMR in healthy volunteers. DESIGN In a randomized, double-blind, crossover study, we measured the RMR by using indirect calorimetry in 13 healthy volunteers after a 3-d dietary intervention with sodium nitrate (NaNO₃) or a placebo (NaCl). The nitrate dose (0.1 mmol · kg⁻¹ · d⁻¹) corresponded to the amount in 200-300 g spinach, beetroot, lettuce, or other vegetable that was rich in nitrate. Effects of direct nitrite exposure on cell respiration were studied in cultured human primary myotubes. RESULTS The RMR was 4.2% lower after nitrate compared with placebo administration, and the change correlated strongly to the degree of nitrate accumulation in saliva (r² = 0.71). The thyroid hormone status, insulin sensitivity, glucose uptake, plasma concentration of isoprostanes, and total antioxidant capacity were unaffected by nitrate. The administration of nitrite to human primary myotubes acutely inhibited respiration. CONCLUSIONS Dietary inorganic nitrate reduces the RMR. This effect may have implications for the regulation of metabolic function in health and disease.

Diverse roles of 2-arachidonoylglycerol in invasion of prostate carcinoma cells: Location, hydrolysis and 12-lipoxygenase metabolism

Endogenous 2‐arachidonoylglycerol (2‐AG) is antiinvasive in androgen‐independent prostate carcinoma (PC‐3) cells. Invasion of PC‐3 cells is also inhibited by exogenously added noladin ether, a non‐hydrolyzable analog of 2‐AG. In contrast, exogenous 2‐AG has the opposite effect. Cell invasion significantly increased with high concentrations of exogenous 2‐AG. In PC‐3 cells, arachidonic acid (AA) and 12‐hydroxyeicosatetraenoic acid (12‐HETE) concentrations increased along with exogenously added 2‐AG, and 12‐HETE concentrations increased with exogenously added AA. Invasion of PC‐3 cells also increased with exogenously added AA and 12(S)‐HETE but not 12(R)‐HETE. The exogenous 2‐AG‐induced invasion of PC‐3 cells was inhibited by 3‐octylthio‐1,1,1‐trifluoropropan‐2‐one (OTFP, an inhibitor of 2‐AG hydrolysis) and baicalein (a 12‐LO inhibitor). Western blot and RT‐PCR analyses indicated expression of 12‐HETE producing lipoxygenases (LOs), platelet‐type 12‐LO (P‐12‐LO) and leukocyte‐type 12‐LO (L‐12‐LO), in PC‐3 cells. These results suggest that exogenous 2‐AG induced, rather inhibited, cell invasion because of its rapid hydrolysis to free AA, and further metabolism by 12‐LO of AA to 12(S)‐HETE, a promoter of PC cell invasion. The results also suggest that PC‐3 cells and human prostate stromal (WPMY‐1) cells released free AA, 2‐AG, and 12‐HETE. In the microenvironment of the PC cells, this may contribute to the cell invasion. The 2‐AG hydrolysis and concentration of 2‐AG in microenvironment are critical for PC cells fate. Therefore, inhibitors of 2‐AG hydrolysis could potentially serve as therapeutic agents for the treatment of prostate cancer.