Intracellular potassium depletion enhances apoptosis induced by staurosporine in cultured trigeminal satellite glial cells.

PURPOSE: Satellite glial cells (SGC) surrounding neurons in sensory ganglia can buffer extracellular potassium, regulating the excitability of injured neurons and possibly influencing a shift from acute to neuropathic pain. SGC apoptosis may be a key component in this process. This work evaluated induction or enhancement of apoptosis in cultured trigeminal SGC following changes in intracellular potassium [K]ic. MATERIALS AND METHODS: We developed SGC primary cultures from rat trigeminal ganglia (TG). Purity of our cultures was confirmed using immunofluorescence and western blot analysis for the presence of the specific marker of SGC, glutamine synthetase (GS). SGC [K]ic was depleted using hypo-osmotic shock and 4 mM bumetanide plus 10 mM ouabain. [K]ic was measured using the K+ fluorescent indicator potassium benzofuran isophthalate (PBFI-AM). RESULTS: SGC tested positive for GS and hypo-osmotic shock induced a significant decrease in [K]ic at every evaluated time. Cells were then incubated for 5 h with either 2 mM staurosporine (STS) or 20 ng/ml of TNF-α and evaluated for early apoptosis and late apoptosis/necrosis by flow cytometry using annexin V and propidium iodide. A significant increase in early apoptosis, from 16 to 38%, was detected in SGC with depleted [K]ic after incubation with STS. In contrast, TNF-α did not increase early apoptosis in normal or [K]ic depleted SGC. CONCLUSION: Hypo-osmotic shock induced a decrease in intracellular potassium in cultured trigeminal SGC and this enhanced apoptosis induced by STS that is associated with the mitochondrial pathway. These results suggest that K+ dysregulation may underlie apoptosis in trigeminal SGC.

High-throughput toxicity testing of chemicals and mixtures in organotypic multi-cellular cultures of primary human hepatic cells.

High-throughput screening (HTS) of liver toxicants can bridge the gap in understanding adverse effects of chemicals on humans. Toxicity testing of mixtures is time consuming and expensive, since the number of possible combinations increases exponentially with the number of chemicals. The combination of organotypic culture models (OCMs) and HTS assays can lead to the rapidly evaluation of chemical toxicity in a cost and time-effective manner while prioritizing chemicals that warrant additional investigation. We describe the design, assembly and toxicant response of multi-cellular hepatic organotypic culture models comprised of primary human or rat cells assembled in 96-well plates (denoted as µOCMs). These models were assembled using automated procedures that did not affect hepatocyte function or viability, rendering them ideal for large-scale toxicity evaluations. Rat µOCMs were assembled to obtain insights into deviations from human toxicity. Four test chemicals (acetaminophen, ethanol, isoniazid, and perfluorooctanoic acid) were added to the µOCMs individually or in mixtures. HTS assays were utilized to measure cell death, apoptosis, glutathione depletion, mitochondrial membrane damage, and cytochrome P450 2E1 activity. The µOCMs exhibited increased toxicant sensitivity compared to hepatocyte sandwich cultures. Synergistic and non-synergistic interactions were observed when the toxicants were added as mixtures. Specifically, chemical interactions in the µOCMs were manifested by changes in apoptosis and decreased glutathione. The µOCMs accurately predicted hepatotoxicity for individual and mixtures of toxicants, demonstrating their potential for large-scale toxicity evaluations in the future.

Synthesis and Evaluation of Doxorubicin-Loaded Gold Nanoparticles for Tumor-Targeted Drug Delivery.

Doxorubicin is an effective and widely used cancer chemotherapeutic agent, but its application is greatly compromised by its cumulative dose-dependent side effect of cardiotoxicity. A gold nanoparticle-based drug delivery system has been designed to overcome this limitation. Five novel thiolated doxorubicin analogs were synthesized and their biological activities evaluated. Two of these analogs and PEG stabilizing ligands were then conjugated to gold nanoparticles, and the resulting Au-Dox constructs were evaluated. The results show that release of native drug can be achieved by the action of reducing agents such as glutathione or under acidic conditions, but reductive drug release gave the cleanest drug release. Gold nanoparticles (Au-Dox) were prepared with different loadings of PEG and doxorubicin, and one formulation was evaluated for mammalian stability and toxicity. Plasma levels of doxorubicin in mice treated with Au-Dox were significantly lower than in mice treated with the same amount of doxorubicin, indicating that the construct is stable under physiological conditions. Treatment of mice with Au-Dox gave no histopathologically observable differences from mice treated with saline, while mice treated with an equivalent dose of doxorubicin showed significant histopathologically observable lesions.

Investigating acetaminophen hepatotoxicity in multi-cellular organotypic liver models.

In vivo studies clearly demonstrate the participation and subsequent death of non-parenchymal liver cells (NPCs) with corresponding hepatocyte effects. This results in a critical need to investigate how major liver cell types function cohesively during hepatotoxicity. However, virtually no studies replicate these phenomena in vitro. We report the design of multi-cellular three-dimensional (3D) organotypic liver models of primary rat hepatocytes, liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs). LSECs and KCs were separated from hepatocytes by a detachable membrane that emulates the physical and chemical properties of the Space of Disse. Acetaminophen (APAP)-induced changes to cellular function and phenotype were investigated. LSECs exhibited approximately 40% cell death at 20mM APAP. KCs exhibited decreased interleukin-10 and increased tumor necrosis factor-alpha and interferon-gamma secretion. The secretion of these proteins altered hepatocyte function and signaling. Both LSECs and KCs maintained phenotypic markers. At 20mM APAP, the 3D models exhibited aspartate aminotransferase to alanine aminotransferase ratios from 2.1-2.5 and 45% glutathione depletion, corresponding to what is seen in vivo. At 10 and 20mM APAP, the 3D models exhibited cell death, primarily through necrosis. Therefore, the 3D cultures described in this report demonstrate significant potential as realistic models for hepatotoxicity studies.

Cefazolin concentration in surgically created wounds treated with negative pressure wound therapy compared to surgically created wounds treated with nonadherent wound dressings.

OBJECTIVE: To compare cefazolin concentrations in biopsied tissue samples collected from surgically created wounds treated with negative pressure wound therapy to those collected from surgically created wounds treated with nonadherent dressings. STUDY DESIGN: Prospective, controlled, experimental study. ANIMALS: Adult female spayed Beagles (n = 12). METHODS: Full thickness cutaneous wounds were created on each antebrachium (n = 24). Immediately after surgery, cefazolin (22 mg/kg intravenously [IV]) was administered to each dog and continued every 8 hours during the study. The right wound was randomly assigned to group I or group II whereas the wound on the contralateral antebrachium was assigned to the other group. Group I wounds were treated with negative pressure wound therapy (NPWT) and group II wounds were treated with nonadherent dressings for 3 days. Dressings were changed and tissue biopsies obtained from wound beds at 24 hours intervals for both groups. Cefazolin wound tissue and plasma concentrations were measured by liquid chromatography mass spectrometry (LC-MS/MS). Blood samples for measuring plasma cefazolin concentrations were collected before biopsy sampling. At the time of surgery and at each subsequent bandage change, wound beds were swabbed and submitted for aerobic and anaerobic culture. RESULTS: After initiating cefazolin treatment, wound tissue antibiotic concentrations between treatment groups were not significantly different at any sampling time. Similarly, after initiating cefazolin treatment, plasma cefazolin concentrations were not significantly different at any sampling time for individual dogs. CONCLUSIONS: Using a canine experimental model, NPWT treatment of surgically created wounds does not statistically impact cefazolin tissue concentrations when compared with conventional nonadherent bandage therapy.

Temporal clinical chemistry and microscopic renal effects following acute uranyl acetate exposure.

Military use of depleted uranium (DU) has renewed interest in the toxicology of this metal. In this study, the nephrotoxicity of single exposure DU was assessed with and without pre-exposure stress. Adult male Sprague-Dawley rats (n=288) were administered a single IM dose of 0, 0.1, 0.3 or 1.0 mg/kg DU. Corticosterone concentrations (ng/ml, mean+/-SD) were 763.65+/-130.94 and 189.80+/-90.81 for swim stressed and unstressed rats. Serum and kidney uranium concentration, hematocrit, chemistry, and renal histology were assessed on sacrifice days 1, 3, 7 and 30 post-DU-dosing. Dose related increases in serum and kidney uranium were noted. DU concentration peaked day 1 in the kidney and days 3-7, in the serum. Dose-related elevations of Cr and BUN concentrations were seen on days 3 and 7. A decline in serum albumin coincided with Cr and BUN suggesting protein losing nephropathy. Dose related acute tubular necrosis and proliferative glomulonephritis were seen. Tubular regeneration in low dose rats was almost complete by day 30. High dose rats had extensive tubular necrosis and delayed regeneration with focal residual chronic interstitial nephritis and cortical scarring. Glomular changes were reversed in all treatment groups by day 30. Stress exposure had no impact on any measured renal parameter.

The effect of stress on the temporal and regional distribution of uranium in rat brain after acute uranyl acetate exposure.

Long-term exposure to depleted uranium (DU) has been shown to increase brain uranium and alter hippocampal function; however, little is known about the short-term kinetics of DU in the brain. To address this issue, temporal and regional distribution of brain uranium was investigated in male Sprague-Dawley rats treated with a single intraperitoneal injection of 1 mg uranium/kg as uranyl acetate. Due to the inherent stress of combat and the potential for stress to alter blood-brain barrier permeability, the impact of forced swim stress on brain uranium distribution was also examined in this model. Uranium in serum, hippocampus, striatum, cerebellum, and frontal cortex was quantified by inductively coupled plasma-mass spectrometry (ICP-MS) at 8 h, 24 h, 7 d, and 30 d after exposure. Uranium entered the brain rapidly and was initially concentrated in hippocampus and striatum. While multiple phases of uranium clearance were observed, overall clearance was relatively slow and the uranium content of hippocampus, cerebellum, and cortex remained elevated for more than 7 d after a single exposure. Prior exposure to stress significantly reduced hippocampal and cerebellar uranium 24 h post-exposure and tended to reduce uranium in all brain regions 7 d after exposure. The application of stress appeared to increase brain uranium clearance, as initial tissue levels were similar in stressed and unstressed rats.

Altered expression of transcripts for alpha-tubulin and an unidentified gene in the spinal cord of phenyl saligenin phosphate treated hens (Gallus gallus).

Phenyl saligenin phosphate (PSP) induces a central-peripheral distal axonopathy in domestic fowl that develops 7-21 days after a single exposure. Neurotoxic esterase (NTE) is the initial molecular target for this neurotoxicity. PSP has to covalently bind to NTE and chemically "age" for induction of axonopathy. It was hypothesized that exposure to PSP results in early changes in spinal cord gene expression that do not occur with phenylmethylsulfonyl fluoride, a non-neuropathic compound that also inhibits NTE, or DMSO controls. Targeted display was used to screen approximately 15,000 gel bands. Three candidate genes were identified, but only the transcript designated P1 showed decreased expression following PSP exposure (2 mg/kg i.m.) in subsequent Northern blot and in situ hybridization experiments in samples taken <48 h after exposure. Additional experiments revealed that a approximately 2.5 kb alpha-tubulin transcript had decreased expression at 12-48 h after PSP exposure, with maximum change at 48 h (33%, p = 0.0479). A approximately 4.5 kb alpha-tubulin transcript had increased expression at 12 h (38%, p = 0.0125) and decreased expression at 48 h (28%, p = 0.0576). In situ hybridization on spinal cord revealed neuronal expression of P1 and alpha-tubulin transcripts. Decreased expression of transcripts for P1 and alpha-tubulin was present at 12 and 48 h, respectively. This decrease occurred in all neurons, not just those whose axons degenerate. Results suggest that (1) in PSP-induced OPIDN (organophosphorus-induced delayed neurotoxicity) some gene transcript expression changes are associated with initiation of axonopathy, and (2) PSP modulates spinal cord gene expression in neuronal types that do not undergo axonal degeneration.