Particulate matter neurotoxicity in culture is size-dependent.

Exposure to particulate matter (PM) air pollution produces inflammatory damage to the cardiopulmonary system. This toxicity appears to be inversely related to the size of the PM particles, with the ultrafine particle being more inflammatory than larger sizes. Exposure to PM has more recently been associated with neurotoxicity. This study examines if the size-dependent toxicity reported in cardiopulmonary systems also occurs in neural targets. For this study, PM ambient air was collected over a 2 week period from Sterling Forest State Park (Tuxedo, New York) and its particulates sized as Accumulation Mode, Fine (AMF) (>0.18-1µm) or Ultrafine (UF) (<0.18µm) samples. Rat dopaminergic neurons (N27) were exposed to suspensions of each PM fraction (0, 12.5, 25, 50µm/ml) and cell loss (as measured by Hoechst nuclear stain) measured after 24h exposure. Neuronal loss occurred in response to all tested concentrations of UF (>12.5µg/ml) but was only significant at the highest concentration of AMF (50µg/ml). To examine if PM size-dependent neurotoxicity was retained in the presence of other cell types, dissociated brain cultures of embryonic rat striatum were exposed to AMF (80µg/ml) or UF (8.0µg/ml). After 24h exposure, a significant increase of reactive nitrogen species (nitrite) and morphology suggestive of apoptosis occurred in both treatment groups. However, morphometric analysis of neuron specific enolase staining indicated that only the UF exposure produced significant neuronal loss, relative to controls. Together, these data suggest that the inverse relationship between size and toxicity reported in cardiopulmonary systems occurs in cultures of isolated dopaminergic neurons and in primary cultures of the rat striatum.

Comparison of methods for the detection of coliphages in recreational water at two California, United States beaches.

Methods for detection of two fecal indicator viruses, F+ and somatic coliphages, were evaluated for application to recreational marine water. Marine water samples were collected during the summer of 2007 in Southern California, United States from transects along Avalon Beach (n=186 samples) and Doheny Beach (n=101 samples). Coliphage detection methods included EPA method 1601 - two-step enrichment (ENR), EPA method 1602 - single agar layer (SAL), and variations of ENR. Variations included comparison of two incubation times (overnight and 5-h incubation) and two final detection steps (lysis zone assay and a rapid latex agglutination assay). A greater number of samples were positive for somatic and F+ coliphages by ENR than by SAL (p<0.01). The standard ENR with overnight incubation and detection by lysis zone assay was the most sensitive method for the detection of F+ and somatic coliphages from marine water, although the method takes up to three days to obtain results. A rapid 5-h enrichment version of ENR also performed well, with more positive samples than SAL, and could be performed in roughly 24h. Latex agglutination-based detection methods require the least amount of time to perform, although the sensitivity was less than lysis zone-based detection methods. Rapid culture-based enrichment of coliphages in marine water may be possible by further optimizing culture-based methods for saline water conditions to generate higher viral titers than currently available, as well as increasing the sensitivity of latex agglutination detection methods.

Synthesis and biological evaluation of bivalent ligands for the cannabinoid 1 receptor.

Dimerization or oligomerization of many G-protein-coupled receptors (GPCRs), including the cannabinoid 1 (CB1) receptor, is now widely accepted and may have significant implications for medications development targeting these receptor complexes. A library of bivalent ligands composed of two identical CB1 antagonist pharmacophores derived from SR141716 linked by spacers of various lengths were developed. The affinities of these bivalent ligands at CB1 and CB2 receptors were determined using radiolabeled binding assays. Their functional activities were measured using GTP-γ-S accumulation and intracellular calcium mobilization assays. The results suggest that the nature of the linker and its length are crucial factors for optimum interactions of these ligands at CB1 receptor binding sites. Finally, selected bivalent ligands (5d and 7b) were able to attenuate the antinociceptive effects of the cannabinoid agonist CP55,940 (21) in a rodent tail-flick assay. These novel compounds may serve as probes that will enable further characterization of CB1 receptor dimerization and oligomerization and its functional significance and may prove useful in the development of new therapeutic approaches to G-protein-coupled receptor mediated disorders.

Nanosize titanium dioxide stimulates reactive oxygen species in brain microglia and damages neurons in vitro.

BACKGROUND: Titanium dioxide is a widely used nanomaterial whose photo-reactivity suggests that it could damage biological targets (e.g., brain) through oxidative stress (OS). OBJECTIVES: Brain cultures of immortalized mouse microglia (BV2), rat dopaminergic (DA) neurons (N27), and primary cultures of embryonic rat striatum, were exposed to Degussa P25, a commercially available TiO(2) nanomaterial. Physical properties of P25 were measured under conditions that paralleled biological measures. FINDINGS: P25 rapidly aggregated in physiological buffer (800-1,900 nm; 25 degrees C) and exposure media (approximately 330 nm; 37 degrees C), and maintained a negative zeta potential in both buffer (-12.2 +/- 1.6 mV) and media (-9.1 +/- 1.2 mV). BV2 microglia exposed to P25 (2.5-120 ppm) responded with an immediate and prolonged release of reactive oxygen species (ROS). Hoechst nuclear stain was reduced after 24-hr (>or=100 ppm) and 48-hr (>or=2.5 ppm) exposure. Microarray analysis on P25-exposed BV2 microglia indicated up-regulation of inflammatory, apoptotic, and cell cycling pathways and down-regulation of energy metabolism. P25 (2.5-120 ppm) stimulated increases of intracellular ATP and caspase 3/7 activity in isolated N27 neurons (24-48 hr) but did not produce cytotoxicity after 72-hr exposure. Primary cultures of rat striatum exposed to P25 (5 ppm) showed a reduction of immunohistochemically stained neurons and microscopic evidence of neuronal apoptosis after 6-hr exposure. These findings indicate that P25 stimulates ROS in BV2 microglia and is nontoxic to isolated N27 neurons. However, P25 rapidly damages neurons at low concentrations in complex brain cultures, plausibly though microglial generated ROS.

The cellular and genomic response of an immortalized microglia cell line (BV2) to concentrated ambient particulate matter.

Ambient particulate matter (PM) damages pulmonary tissue through oxidative stress (OS) pathways. Several reports indicate that the brain is another affected target of PM exposure. Since microglia (brain macrophages) are critical to OS-mediated neurodegeneration, the cellular and genomic response of immortalized mouse microglia (BV2) was examined in response to fine (or= 250 microg/ml) and depolarized mitochondrial membranes (>or= 6 microg/ml) within 15 min of exposure. HP and LP CAPs (>or= 25 microg/ml) differentially affected the endogenous scavengers, glutathione and nonprotein sulfhydryl in BV2 microglia after 1.5 h of exposure. Both HP and LP CAPs stimulated the release of proinflammatory cytokines tumor necrosis factor (TNF) alpha and interleukin (IL)-6 after 6 h of exposures. Microarray analysis of BV2 microglia exposed to either HP or LP CAPs (75 microg/ml, 4 h) identified 3200 (HP CAPs) and 160 (LP CAPs) differentially expressed (up- and downregulated) genes relative to media controls. Of the 3200 genes significantly affected by HP CAPs, the most prominent upregulated gene probes related to inflammatory pathways associated with Toll-like receptor signaling, MAPK signaling, T- and B-cell receptor signaling, apoptosis, and various proinflammatory cytokines and their receptors. LP CAPs significantly affected 160 genes that related to pathways associated with cellular maintenance and division, cell cycling and nuclear events. These data suggest that HP CAPs, which contained higher levels of nickel and vanadium than LP CAPs, appear to be more inflammatory and selectively upregulated the expression of inflammatory and innate immunity pathways in BV2 microglia.