A Black Cohosh Extract Causes Hematologic and Biochemical Changes Consistent with a Functional Cobalamin Deficiency in Female B6C3F1/N Mice.

Black cohosh rhizome, available as a dietary supplement, is most commonly marketed as a remedy for dysmenorrhea and menopausal symptoms. A previous subchronic toxicity study of black cohosh dried ethanolic extract (BCE) in female mice revealed a dose-dependent ineffective erythropoiesis with a macrocytosis consistent with the condition known as megaloblastic anemia. The purpose of this study was to investigate potential mechanisms by which BCE induces these particular hematological changes. B6C3F1/N female mice (32/group) were exposed by gavage to vehicle or 1,000 mg/kg BCE for 92 days. Blood samples were analyzed for hematology, renal and hepatic clinical chemistry, serum folate and cobalamin, red blood cell (RBC) folate, and plasma homocysteine and methylmalonic acid (MMA). Folate levels were measured in liver and kidney. Hematological changes included decreased RBC count; increased mean corpuscular volume; and decreased reticulocyte, white blood cell, neutrophil, and lymphocyte counts. Blood smear evaluation revealed increased Howell-Jolly bodies and occasional basophilic stippling in treated animals. Plasma homocysteine and MMA concentrations were increased in treated animals. Under the conditions of our study, BCE administration caused hematological and clinical chemistry changes consistent with a functional cobalamin, and possibly folate, deficiency. Further studies are needed to elucidate the mechanism by which BCE causes increases in homocysteine and MMA.

Comparative inhalation toxicity of ethyltoluene isomers in rats and mice.

The C9 alkylbenzenes, composed mostly of ethyltoluenes and trimethylbenzenes, comprise 75-90% of the naphtha fraction of crude oil. Occupational and environmental exposure to C9 alkylbenzenes occur via inhalation. We conducted short-term inhalation studies on the ethyltoluene isomers (2-, 3- or 4-) to select one isomer for more comprehensive studies. Male Hsd:Sprague Dawley rats and female B6C3F1/N mice (n = 10) were exposed by nose-only inhalation to 2-, 3- or 4-ethyltoluene (0, 1000 or 2000 ppm) or cumene (a reference compound: 0, 500 or 1000 ppm) 3 h/day, 5 days/week, for 2 weeks. Clinical observations included abnormal gait and delayed righting reflex. Rats and mice exposed to 2000 ppm 2-ethyltoluene and mice exposed to 2000 ppm 4-ethyltoluene were euthanized early in moribund condition; no exposure-related deaths were observed with 3-ethyltoluene or cumene. Histopathology of selected tissues revealed that the nose and liver (rats and mice) and lung (mice only) to be toxicity targets. In the mouse lung, all compounds except 4-ethyltoluene produced bronchial and bronchiolar hyperplasia. In rats and mice, 2-ethyltoluene was the only compound to produce lesions in the nose and liver: in mice, squamous metaplasia and neutrophilic inflammation of the respiratory epithelium and atrophy and degeneration of the olfactory epithelium were observed in the nose and centrilobular hypertrophy and necrosis were observed in the liver. In rats, 2-ethyltoluene exposure produced atrophy of the olfactory epithelium in the nose and centrilobular necrosis in the liver. Based on mortality, body weight effects and histopathology, the 2-ethyltoluene isomer was the most potent isomer.

Evaluation of an in vitro screening model to assess phosgene inhalation injury.

Therapeutic development against exposure to toxic gases is hindered by the lack of appropriate models to evaluate candidate compounds prior to animal efficacy studies. In this study, an in vitro, air-liquid interface exposure model has been tested to examine its potential application for screening treatments for phosgene (carbonyl chloride)-induced pulmonary injury. Epithelial cultures on Transwell® inserts, combined with a Vitrocell® exposure apparatus, provided a physiologically relevant exposure environment. Differentiated human bronchial epithelial (16HBE) cultures were exposed for 8 min to phosgene ranging from 0 to 64 ppm and assessed for changes in transepithelial electrical resistance (TEER, epithelial barrier integrity), cellular viability (XTT) and post-exposure (PE) cellular metabolic energy status. Exposure to phosgene concentrations ≥8 ppm caused dose-dependent and significant decreases in TEER and XTT which did not recover within 24-h PE. In addition, at 64 ppm the rate of oxidative glutamine metabolism was significantly inhibited at 6 and 24 h after exposure. Glycolytic activities (glucose utilization and lactate production) were also inhibited, but to a lesser extent. Decreased glycolytic function can translate to insufficient energy sources to counteract barrier function failure. Consistent and sensitive markers of phosgene exposure were TEER, cell viability and decreased metabolism. As such, we have assessed an appropriate in vitro model of phosgene inhalation that produced quantifiable alterations in markers of lung cell metabolism and injury in human airway epithelial cells. Data indicate the suitability of this model for testing classes of anti-edemagenic compounds such as corticosteroids or phosphodiesterase inhibitors for evaluating phosgene therapeutics.

ICP-MS characterization of seven North American snake venoms.

Snake venoms are inherently complex. They are mixtures of multiple enzymes, peptides, lipids, carbohydrates, nucleosides, and metal ions. Metal ions make up a small portion of a snake's venom but play outsized roles in enzyme function and stability. Unlike enzyme primary structure, which is easily predicted from genomic sequences, a venom's metal ion content must be measured directly. We leveraged the high throughput and sensitivity of inductively coupled plasma mass spectrometry to analyze the metal ion content of seven North American snake venoms. All venoms were collected from snakes reared at one location, so we could discount variation from environmental or geographical factors. We profiled 71 metal isotopes. Selenium isotopes were consistently high across all venoms tested. When each venom's toxicity was graphed as a function of each different metal isotope, the only strong relationships between metal content and toxicity were for magnesium isotopes.

Cellular mechanisms of mainstream cigarette smoke-induced lung epithelial tight junction permeability changes in vitro.

Mainstream cigarette smoke increases the permeability of human airways; however, the mechanism for this increased permeability is poorly defined. Tight junctions between adjacent epithelial cells constitute the physiological barrier to fluid and macromolecules in epithelium. These structures are highly regulated by phosphorylation and their association with the cytoskeleton. The goal of these studies was to identify the signal transduction pathways that regulate smoke-induced permeability. Using a physiologically relevant air-liquid interface exposure system, electrically tight monolayers of the human bronchial epithelial cell-line Calu-3 were exposed to fresh, whole mainstream cigarette smoke. This exposure results in a regulated, dose-dependent loss of epithelial barrier function in the lung epithelial monolayers. With cigarette smoke exposure, transepithelial electrical resistance (TER) is decreased and albumin flux is increased, indicating a loss in barrier function to ions and macromolecules, respectively; however, both largely recover in 30 min. Smoke-induced losses of macromolecular barrier function are the result of multicellular junctional reorganization, resulting in increased leak volume rather than leak frequency. Inhibiting Rho kinase (ROCK) significantly reduces the smoke-induced permeability to both ions and macromolecules, while inhibiting protein tyrosine kinases (PTK) only reduces smoke-induced macromolecular permeability. Interestingly, inhibiting myosin light chain kinase (MLCK) exacerbates smoke-induced permeability, indicating that MLCK and ROCK have opposing regulatory roles. Our results demonstrate that the smoke-induced loss of epithelial barrier function in human bronchial epithelium is a regulated process rather than a cytotoxic response. Additionally, our results indicate that activation of PTK and ROCK and inactivation of MLCK contribute to the increased airway permeability caused by mainstream cigarette smoke.

Transient receptor potential (TRP) channels as a therapeutic target for intervention of respiratory effects and lethality from phosgene.

Phosgene (CG), a toxic inhalation and industrial hazard, causes bronchoconstriction, vasoconstriction and associated pathological effects that could be life threatening. Ion channels of the transient receptor potential (TRP) family have been identified to act as specific chemosensory molecules in the respiratory tract in the detection, control of adaptive responses and initiation of detrimental signaling cascades upon exposure to various toxic inhalation hazards (TIH); their activation due to TIH exposure may result in broncho- and vasoconstriction. We studied changes in the regulation of intracellular free Ca(2+) concentration ([Ca(2+)]i) in cultures of human bronchial smooth muscle cells (BSMC) and human pulmonary microvascular endothelial cells (HPMEC) exposed to CG (16ppm, 8min), using an air/liquid interface exposure system. CG increased [Ca(2+)]i (p<0.05) in both cell types, The CG-induced [Ca(2+)]i was blocked (p<0.05) by two types of TRP channel blockers, SKF-96365, a general TRP channel blocker, and RR, a general TRPV (vanilloid type) blocker, in both BSMC and HPMEC. These effects correlate with the in vivo efficacies of these compounds to protect against lung injury and 24h lethality from whole body CG inhalation exposure in mice (8-10ppm×20min). Thus the TRP channel mechanism appears to be a potential target for intervention in CG toxicity.

Ethanol withdrawal upregulates kainate receptors in cultured rat hippocampal neurons.

We have previously demonstrated that kainate receptors (KA-Rs) are acutely inhibited by ethanol (EtOH). Here we show that KA-Rs are also affected by long-term EtOH exposure. Whole-cell recordings of pharmacologically isolated KA-R-mediated currents in cultured hippocampal neurons revealed that exposure to 80 mM EtOH for 3 days followed by a 24 h withdrawal period increased KA-R current densities. Quantitative confocal microscopy showed that expression of GluR6/7 subunits increases after ethanol withdrawal in these neurons. Since KA-Rs control hippocampal excitability and seizure generation, we postulate that upregulation of these receptors may have a role in the pathophysiology of alcohol withdrawal syndrome.

Cytoskeletal modulation and tyrosine phosphorylation of tight junction proteins are associated with mainstream cigarette smoke-induced permeability of airway epithelium.

Cigarette smoke increases the permeability of the lung epithelium. Consequences of increased permeability include increased access of toxins and pathogens from the air spaces to the interstitium and even the blood stream, and leakage of fluids into the air spaces. The mechanisms for permeability alterations have not been elucidated for airway epithelia. By analogy with other types of epithelia, we hypothesized that changes in the phosphorylation status and function of tight junction (TJ) or cytoskeletal proteins might mediate the smoke-induced permeability changes. We investigated the effects of exposure to mainstream cigarette smoke (MS) on cultures of Calu-3 cells, an airway epithelial cell line. Specifically, MS exposure caused increases in phosphorylation of the myosin-binding subunit (MBS) of myosin phosphatase and myosin light chain (MLC), proteins involved in the regulation of actin polymerization. These results implicate activation of Rho kinase (ROCK), consistent with previously reported data indicating that inhibition of ROCK activation suppressed MS-induced increases in permeability. MS exposure also increased polymerized (filamentous) actin (f-actin) content and caused redistribution of the TJ proteins from the normal apical circumferential band to a more basal location. The translocation of the TJ proteins was spatially associated with local increases in both f-actin and macromolecular permeability. Finally, MS exposure increased tyrosine phosphorylation of occludin but not ZO-1 and decreased association between the two TJ proteins. These results indicate that MS exposure causes alterations in cytoskeletal and TJ structure and function, resulting in increased macromolecular permeability that may contribute to the adverse health effects of MS.