Metal Changes in Pre- and Post-metamorphic Wood Frog (Lithobates sylvaticus) Tadpoles: Implications for Ecotoxicological Studies.

The development of anuran larvae from hatchling through metamorphosis is a particularly sensitive life stage that often is studied to assess adverse effects of water pollution, such as metal contamination. As an integral part of the food chain, high metal exposure and accumulation in developing anuran larvae may not only affect their survival but also pose a threat to secondary consumers. The presented work examines metal accumulation in wood frog tadpoles (Lithobates sylvaticus) before and after reaching metamorphic climax at emergence of the forelimbs. Metal levels were determined in whole tadpoles pre- and post-metamorphic climax in tadpole tissue excluding the stomach and intestines, as well as in water, via inductively coupled plasma-mass spectrometry. Wood frog tadpoles concentrated metals in their gut coil, with a rapid decline coincident with metamorphic climax. Tadpoles raised in a diluted bitumen-contaminated environment had higher levels of vanadium, molybdenum and cadmium, but not at levels expected to negatively impact development. In conclusion, metal accumulation in wood frog tadpoles varies greatly depending on developmental stage surrounding metamorphic climax. Metabolic changes and intestinal remodelling must be considered when studying pollutants in developing anuran larvae.

Food as medicine: Selenium enriched lentils offer relief against chronic arsenic poisoning in Bangladesh.

Chronic arsenic (As) exposure is a major environmental threat to human health affecting >100 million people worldwide. Low blood selenium (Se) increases the risk of As-induced health problems. Our aim was to reduce As toxicity through a naturally Se-rich lentil diet. In a randomized, double-blind, placebo-control trial in Bangladesh, 405 participants chronically exposed to As were enrolled. The intervention arm (Se-group) consumed Se-rich lentils (55 µg Se/day); the control arm received lentils of similar nutrient profile except with low Se (1.5 µg Se/day). Anthropometric measurements, blood, urine and stool samples, were taken at baseline, 3 and 6 months; hair at baseline and 6 months after intervention. Morbidity data were collected fortnightly. Measurements included total As in all biological samples, As metabolites in urine, and total Se in blood and urine. Intervention with Se-rich lentils resulted in higher urinary As excretion (p = 0.001); increased body mass index (p ≤ 0.01), and lower incidence of asthma (p = 0.05) and allergy (p = 0.02) compared to the control group. The Se-group demonstrated increased excretion of urinary As metabolite, dimethylarsinic acid (DMA) at 6 months compared to control group (p = 0.008). Consuming Se-rich lentils can increase As excretion and improve the health indicators in the presence of continued As exposure.

Alternative activation of macrophages by IL-4 enhances the proteolytic capacity of their phagosomes through synergistic mechanisms.

Alternatively activated macrophages, generated in a T-helper 2 environment, have demonstrated roles in wound repair and tissue remodeling in addition to being charged with immune tasks. Because the hydrolytic chemistries of the phagosomal lumen are central to many of these functions, we investigated their modification after alternative activation with IL-4 and IL-13. Most significantly, we found striking up-regulation of the proteolytic levels within the phagosome of IL-4-activated macrophages. Two synergistic mechanisms were determined to underlie this up-regulation. First, IL-4-activated macrophages displayed increased expression of cathepsin S and L, providing greater proteolytic machinery to the phagosome despite unchanged rates of lysosomal contribution. Secondly, decreased phagosomal NADPH oxidase (NOX2) activity, at least partially resulting from decreased expression of the NOX2 subunit gp91(phox), resulted in a more reductive lumenal microenvironment, which in turn, enhanced activities of local cysteine cathepsins. Decreased NOX2 activity additionally increased the phagosome's ability to reduce disulfides, further enhancing the efficiency of the macrophage to degrade proteins containing disulfide bonds. Together, these changes initiated by IL-4 act synergistically to rapidly and dramatically enhance the macrophage's ability to degrade phagocytosed protein, which, we reason, better equips this cell for its roles in wound repair and tissue remodeling.

NADPH oxidase activity controls phagosomal proteolysis in macrophages through modulation of the lumenal redox environment of phagosomes.

The phagosomal lumen in macrophages is the site of numerous interacting chemistries that mediate microbial killing, macromolecular degradation, and antigen processing. Using a non-hypothesis-based screen to explore the interconnectivity of phagosomal functions, we found that NADPH oxidase (NOX2) negatively regulates levels of proteolysis within the maturing phagosome of macrophages. Unlike the NOX2 mechanism of proteolytic control reported in dendritic cells, this phenomenon in macrophages is independent of changes to lumenal pH and is also independent of hydrolase delivery to the phagosome. We found that NOX2 mediates the inhibition of phagosomal proteolysis in macrophages through reversible oxidative inactivation of local cysteine cathepsins. We also show that NOX2 activity significantly compromises the phagosome's ability to reduce disulfides. These findings indicate that NOX2 oxidatively inactivates cysteine cathepsins through sustained ablation of the reductive capacity of the phagosomal lumen. This constitutes a unique mechanism of spatiotemporal control of phagosomal chemistries through the modulation of the local redox environment. In addition, this work further implicates the microbicidal effector NOX2 as a global modulator of phagosomal physiologies, particularly of those pertinent to antigen processing.

The effect of a high-selenium lentil diet on cardiovascular risk markers in an arsenic-exposed population.

Chronic arsenic exposure is associated with a number of systemic diseases, including cardiovascular disease. Selenium has been shown to promote arsenic excretion from the body. We investigated if a high-selenium lentil diet has an effect on blood pressure and plasma lipid levels in an arsenic-exposed population by conducting a 6-month randomized controlled dietary intervention trial with 405 participants.

A high-selenium lentil dietary intervention in Bangladesh to counteract arsenic toxicity: study protocol for a randomized controlled trial.

BACKGROUND: Millions of people worldwide are exposed to dangerous levels of arsenic (above the WHO water standard of 10 ppb) in drinking water and food. Lack of nutritious foods exacerbates the adverse health effects of arsenic poisoning. The micronutrient selenium is a known antagonist to arsenic, promoting the excretion of arsenic from the body. Studies are in progress examining the potential of using selenium supplement pills to counteract arsenic toxicity. We are planning a clinical trial to test whether high-selenium lentils, as a whole food solution, can improve the health of arsenic-exposed Bangladeshi villagers. METHODS/DESIGN: A total of 400 participants (about 80 families) will be divided into two groups via computer-generated block randomization. Eligibility criteria are age (≥14) years) and arsenic concentration in the household tube well (≥100 ppb). In this double-blind study, one group will eat high-selenium lentils grown in western Canada; the other will consume low-selenium lentils grown in Idaho, USA. Each participant will consume 65 g of lentils each day for 6 months. At the onset, midterm, and end of the trial, blood, urine and stool, plus hair (day 1 and at 6 months only) samples will be collected and a health examination conducted including assessment of acute lung inflammation, body mass and height, and blood pressure. The major outcome will be arsenic excretion in urine and feces, as well as arsenic deposition in hair and morbidity outcomes as assessed by a biweekly questionnaire. Secondary outcomes include antioxidant status, lipid profile, lung inflammation status, and blood pressure. DISCUSSION: Selenium pills as a treatment for arsenic exposure are costly and inconvenient, whereas a whole food approach to lower the toxic burden of arsenic may be a practical remedy for Bangladeshi people while efforts to provide safe drinking water are continuing. If high-selenium lentils prove to be effective in counteracting arsenic toxicity, agronomic partnerships between Canada and Bangladesh will work to improve the selenium content of the Bangladeshi-grown lentil crops. Results will be presented to the community to promote informed food choices, which may include increasing selenium in their diet. TRIAL REGISTRATION: ClinicalTrials.gov NCT02429921.

High-selenium lentil diet protects against arsenic-induced atherosclerosis in a mouse model.

BACKGROUND: Cardiovascular disease (CVD) is a major cause of death worldwide, and arsenic (As) intake, mainly through drinking water, is a well-known risk factor for CVD as well as other health problems. Selenium (Se) is a known antagonist to As toxicity. OBJECTIVE: We tested the potential of high-Se lentils from the Canadian prairies as a therapeutic food to alter the outcome of As-enhanced atherosclerosis. MATERIALS AND METHODS: Male ApoE(-/-) mice exposed to a moderate level of As (200ppb) in their drinking water, and control mice on tap water received one of three lentil diets: Se-deficient (0.009mg/kg), Se-adequate (0.16mg/kg) or Se-high (0.3mg/kg). After 13weeks, lesion formation in the aortic arch and sinus were assessed. Intralesional cellular composition, serum lipid levels and hepatic oxidative stress were assessed as well. RESULTS: Arsenic-exacerbated plaque formation was reduced in the sinus and completely abolished in the aortic arch of mice on the Se-fortified lentil diet, whereas lesions were increased in As-exposed mice on both the Se-deficient and Se-adequate diets. Notably, Se deficiency contributed to proatherogenic composition of serum lipids in As-exposed mice as indicated by high-density lipoprotein:low-density lipoprotein. At least adequate Se status was crucial for counteracting As-induced oxidative stress. CONCLUSION: This study is the first to show the potential of high-Se lentils to protect against As-triggered atherosclerosis, and this invites further investigations in human populations at risk from As contamination of their drinking water.

Abnormal cannabidiol attenuates experimental colitis in mice, promotes wound healing and inhibits neutrophil recruitment.

BACKGROUND: Non-psychotropic atypical cannabinoids have therapeutic potential in a variety of inflammatory conditions including those of the gastrointestinal tract. Here we examined the effects of the atypical cannabinoid abnormal cannabidiol (Abn-CBD) on wound healing, inflammatory cell recruitment and colitis in mice. METHODS: Colitis was induced in CD1 mice by a single intrarectal administration of trinitrobenzene sulfonic acid (TNBS, 4 mg/100 µl in 30 % ethanol) and Abn-CBD and/or the antagonists O-1918 (Abd-CBD), AM251 (CB1 receptor) and AM630 (CB2 receptor), were administered intraperitoneally (all 5 mg/kg, twice daily for 3 days). The degree of colitis was assessed macro- and microscopically and tissue myeloperoxidase activity was determined. The effects of Abn-CBD on wound healing of endothelial and epithelial cells (LoVo) were assessed in a scratch injury assay. Human neutrophils were employed in Transwell assays or perfused over human umbilical vein endothelial cells (HUVEC) to study the effect of Abn-CBD on neutrophil accumulation and transmigration. RESULTS: TNBS-induced colitis was attenuated by treatment with Abn-CBD. Histological, macroscopic colitis scores and tissue myeloperoxidase activity were significantly reduced. These effects were inhibited by O-1918, but not by AM630, and only in part by AM251. Wound healing of both HUVEC and LoVo cells was enhanced by Abn-CBD. Abn-CBD inhibited neutrophil migration towards IL-8, and dose-dependently inhibited accumulation of neutrophils on HUVEC. CONCLUSIONS: Abn-CBD is protective against TNBS-induced colitis, promotes wound healing of endothelial and epithelial cells and inhibits neutrophil accumulation on HUVEC monolayers. Thus, the atypical cannabinoid Abn-CBD represents a novel potential therapeutic in the treatment of intestinal inflammatory diseases.

Arsenic Exposure Increases Monocyte Adhesion to the Vascular Endothelium, a Pro-Atherogenic Mechanism.

Epidemiological studies have shown that arsenic exposure increases atherosclerosis, but the mechanisms underlying this relationship are unknown. Monocytes, macrophages and platelets play an important role in the initiation of atherosclerosis. Circulating monocytes and macrophages bind to the activated vascular endothelium and migrate into the sub-endothelium, where they become lipid-laden foam cells. This process can be facilitated by platelets, which favour monocyte recruitment to the lesion. Thus, we assessed the effects of low-to-moderate arsenic exposure on monocyte adhesion to endothelial cells, platelet activation and platelet-monocyte interactions. We observed that arsenic induces human monocyte adhesion to endothelial cells in vitro. These findings were confirmed ex vivo using a murine organ culture system at concentrations as low as 10 ppb. We found that both cell types need to be exposed to arsenic to maximize monocyte adhesion to the endothelium. This adhesion process is specific to monocyte/endothelium interactions. Hence, no effect of arsenic on platelet activation or platelet/leukocyte interaction was observed. We found that arsenic increases adhesion of mononuclear cells via increased CD29 binding to VCAM-1, an adhesion molecule found on activated endothelial cells. Similar results were observed in vivo, where arsenic-exposed mice exhibit increased VCAM-1 expression on endothelial cells and increased CD29 on circulating monocytes. Interestingly, expression of adhesion molecules and increased binding can be inhibited by antioxidants in vitro and in vivo. Together, these data suggest that arsenic might enhance atherosclerosis by increasing monocyte adhesion to endothelial cells, a process that is inhibited by antioxidants.

In vitro and in vivo transfection of primary phagocytes via microbubble-mediated intraphagosomal sonoporation.

The professional phagocytes, such as macrophages and dendritic cells, are the subject of numerous research efforts in immunology and cell biology. The use of primary phagocytes in these investigations however, are limited by their inherent resistance to transfection with DNA constructs. As a result, the use of phagocyte-like immortalized cell lines is widespread. While these cell lines are transfection permissive, they are generally regarded as poor biological substitutes for primary phagocytes. By exploiting the phagocytic machinery of primary phagocytes, we developed a non-viral method of DNA transfection of macrophages that employs intraphagosomal sonoporation mediated by internalized lipid-based microbubbles. This approach enables the transfection of primary phagocytes in vitro, with a modest, but reliable efficiency. Furthermore, this methodology was readily adapted to transfect murine peritoneal macrophages in vivo. This technology has immediate application to current research efforts and has potential for use in gene therapy and vaccination strategies.