Gas chromatography-tandem mass spectrometry analysis of red blood cells from Göttingen minipig following whole-body vapor exposure to VX.

A method to detect fluoride ion generated O-ethyl methylphosphonofluoridate (VX-G) in Göttingen minipig red blood cells (RBC) following whole-body exposure to VX vapor utilizing a gas chromatograph-tandem mass spectrometer (GC-MS-MS) has been developed. Dose-response curves for VX exposure were generated after applying the fluoride ion reactivation assay to the RBC fraction of serially collected whole blood samples that were taken after whole-body exposures that varied in both duration and concentration. GC-MS-MS analysis of minipig RBC samples following 180-min exposures at two different concentrations was a more precise indicator for severity of exposure than the analysis of acetylcholinesterase (AChE) inhibition for the same samples. AChE enzyme activity recovered faster than indicated by the apparent elimination rate of VX-G. GC-MS-MS analyses of RBC samples following VX exposure demonstrate this technique has both adequate sensitivity and specificity to indicate the severity of exposure.

Quantification of sarin and cyclosarin metabolites isopropyl methylphosphonic acid and cyclohexyl methylphosphonic acid in minipig plasma using isotope-dilution and liquid chromatography- time-of-flight mass spectrometry.

An analysis method for determining isopropyl methylphosphonic acid (IMPA) and cyclohexyl methylphosphonic acid (CMPA), the metabolic hydrolysis products of toxic organophosphorus nerve agents isopropyl methylphosphonofluoridate (sarin, GB) and cyclohexyl methylphosphonofluoridate (cyclosarin, GF), respectively, has been developed and validated using high-performance liquid chromatography-mass spectrometry with negative ion electrospray ionization with time-of-flight detection (LC-ESI-MS-TOF). The linear range of quantitation was 5 to 125 ng/mL in plasma with a method detection limit of 2 ng/mL for each compound. This method was developed to determine the amount of metabolic hydrolysis that was formed during and after nerve agent exposure in minipigs to account for a major pathway of GB and GF elimination that had not been previously characterized in the bloodstream, particularly during low-level whole-body inhalation experiments. Metabolic hydrolysis accounted for 70% to 90% of the recoverable agent in the bloodstream during exposure, when compared to both unbound and cholinesterase bound agent recovered by fluoride ion reactivation analysis for the same samples. The estimated half-life of IMPA and CMPA in plasma was determined to be 44 and 61 min, respectively. The method utilizes the mass selectivity of LC-ESI-MS-TOF using a bench-top instrument to achieve a detection limit that is consistent with reported LC-MS-MS methods analyzing blood samples.

The effects of repeated low-dose sarin exposure.

This project assessed the effects of repeated low-dose exposure of guinea pigs to the organophosphorus nerve agent sarin. Animals were injected once a day, 5 days per week (Monday-Friday), for 2 weeks with fractions (0.3x, 0.4x, 0.5x, or 0.6x) of the established LD(50) dose of sarin (42 microg/kg, s.c.). The animals were assessed for changes in body weight, red blood cell (RBC) acetylcholinesterase (AChE) levels, neurobehavioral reactions to a functional observational battery (FOB), cortical electroencephalographic (EEG) power spectrum, and intrinsic acetylcholine (ACh) neurotransmitter (NT) regulation over the 2 weeks of sarin exposure and for up to 12 days postinjection. No guinea pig receiving 0.3, 0.4 or 0.5 x LD(50) of sarin showed signs of cortical EEG seizures despite decreases in RBC AChE levels to as low as 10% of baseline, while seizures were evident in animals receiving 0.6 x LD(50) of sarin as early as the second day; subsequent injections led to incapacitation and death. Animals receiving 0.5 x LD(50) sarin showed obvious signs of cholinergic toxicity; overall, 2 of 13 animals receiving 0.5 x LD(50) sarin died before all 10 injections were given, and there was a significant increase in the angle of gait in the animals that lived. By the 10th day of injection, the animals receiving saline were significantly easier to remove from their cages and handle and significantly less responsive to an approaching pencil and touch on the rump in comparison with the first day of testing. In contrast, the animals receiving 0.4 x LD(50) sarin failed to show any significant reductions in their responses to an approaching pencil and a touch on the rump as compared with the first day. The 0.5 x LD(50) sarin animals also failed to show any significant changes to the approach and touch responses and did not adjust to handling or removal from the cage from the first day of injections to the last day of handling. Thus, the guinea pigs receiving the 0.4 and 0.5 x LD(50) doses of sarin failed to habituate to some aspects of neurobehavioral testing. Spectral analysis of EEG data suggested that repeated sarin exposure may disrupt normal sleeping patterns (i.e., lower frequency bandwidths). While these EEG changes returned to relative normalcy 6 days after the last injection in animals receiving 0.4 x LD(50) sarin, these changes were still observed in the animals that received 0.5 x LD(50) sarin. Ten to twelve days after the last sarin injection (in 0.4 x LD(50) group only), neurochemical data showed that striatal choline levels were reduced in comparison to the saline group. At this time, atropine sulfate (5 mg/kg, i.p.) challenge resulted in a transient elevation in striatal ACh levels in animals exposed to repeated 0.4 x LD(50) sarin as well as in control animals. No evidence of brain or heart pathology was found in any guinea pig that survived all 10 sarin injections.

Quantitation of fluoride ion released sarin in red blood cell samples by gas chromatography-chemical ionization mass spectrometry using isotope dilution and large-volume injection.

A new method for measuring fluoride ion released isopropyl methylphosphonofluoridate (sarin, GB) in the red blood cell fraction was developed that utilizes an autoinjector, a large-volume injector port (LVI), positive ion ammonia chemical ionization detection in the SIM mode, and a deuterated stable isotope internal standard. This method was applied to red blood cell (RBC) and plasma ethyl acetate extracts from spiked human and animal whole blood samples and from whole blood of minipigs, guinea pigs, and rats exposed by whole-body sarin inhalation. Evidence of nerve agent exposure was detected in plasma and red blood cells at low levels of exposure. The linear method range of quantitation was 10-1000 pg on-column with a detection limit of approximately 2-pg on-column. In the course of method development, several conditions were optimized for the LVI, including type of injector insert, injection volume, initial temperature, pressure, and flow rate. RBC fractions had advantages over the plasma with respect to assessing nerve agent exposure using the fluoride ion method especially in samples with low serum butyrylcholinesterase activity.

Ferritin binding in the developing mouse brain follows a pattern similar to myelination and is unaffected by the jimpy mutation.

We have previously provided evidence that ferritin binds selectively to white matter tracts in adult mouse and human brains. In cell culture experiments, ferritin binding is specifically localized to oligodendrocytes. The goal of the present study is to test the hypothesis that the developmental pattern for ferritin binding will coincide with the onset and progression of myelination. The first evidence of ferritin binding in the mouse brain is at 12 days of age and occurs within the brainstem. Ferritin binding persisted in the brainstem and expanded to the corpus callosum by 15-16 days of age. By 23-24 days of age ferritin binding had further extended to the striatal white matter. By adulthood, ferritin binding was strongly and selectively expressed throughout all white matter tracts. To begin to identify which factors may be involved in the induction of ferritin-binding proteins on oligodendrocytes, brains from the myelin mutant jimpy mice and unaffected littermates were examined at postnatal days 16-18. Jimpy mice were chosen because their oligodendrocytes fail to produce myelin or accumulate iron. Thus, using jimpy mice would elucidate whether these factors are necessary for ferritin-binding protein expression. Both the jimpy mutants and their controls exhibited saturable ferritin binding with similar binding densities and dissociation constants. Dissociation constants for ferritin binding in the unaffected littermates and jimpy mutant mice were 0.38 +/- 0.04 and 0.32 +/- 0.06 nM, respectively and binding densities were similar (1.1 +/- 0.09 and 0.96 +/- 0.12 fmol/mg, respectively). Our results demonstrate that expression of ferritin binding is dependent on the age of the oligodendrocytes and not dependent upon iron accumulation by oligodendrocytes or myelin production. We propose that iron delivery to oligodendrocytes is predominantly via ferritin and this method of iron uptake is unique to oligodendrocytes in the brain.

The dose-response effects of repeated subacute sarin exposure on guinea pigs.

The present study assessed the effects of repeated subacute exposure to the organophosphorous nerve agent, sarin. Guinea pigs were injected five times per week (Monday-Friday) for 2 weeks with fractions of the established LD(50) dose of sarin (42 microg/kg sc). The animals were assessed for the development of cortical EEG seizures. Changes in body weight, red blood cell (RBC) acetylcholinesterase (AChE) levels and neurobehavioral reactions to a functional observational battery were monitored over the 2 weeks of sarin exposure and for an extended postinjection period. There were dose-related changes in body weight and RBC AChE levels. No guinea pigs receiving 0.3, 0.4 or 0.5 x LD(50) of sarin showed signs of cortical EEG seizures despite decreases in RBC AChE levels to as low as 10% of baseline. Seizures were evident in animals receiving 0.6 x LD(50) of sarin as early as the second day, and subsequent injections led to incapacitation and death. Animals receiving 0.5 x LD(50) sarin showed obvious signs of cholinergic toxicity, which included a significant increase in their angle of gait. Overall, 2/13 animals receiving 0.5 x LD(50) sarin died before all 10 injections were given. By the 10th day of injections, the animals receiving saline were significantly easier to remove from their cages and handle as compared to the first day of injections. They were also significantly less responsive to an approaching pencil and touch on the rump in comparison to the first day of testing. In contrast, the animals receiving 0.4 x LD(50) sarin failed to show any significant reductions in their responses to an approaching pencil and a touch on the rump as compared to the first day. The 0.5 x LD(50) sarin animals failed to show any significant changes to the approach response and touch response and did not adjust to handling or cage removal from the first day of injections to the last day of handling. In summary, the guinea pigs receiving the 0.4 x LD(50) and 0.5 x LD(50) doses of sarin failed to habituate to some aspects of the functional observational battery testing.

Oligodendrocyte progenitor cells internalize ferritin via clathrin-dependent receptor mediated endocytosis.

We previously demonstrated ferritin binding is specific to white matter in mouse and human brain tissue and is not found within Multiple Sclerotic plaques. These results suggest that ferritin receptors are selectively expressed on oligodendrocytes. The present studies were designed to test the hypothesis that oligodendrocyte progenitor cells selectively bind ferritin and internalize it by methods consistent with receptor-mediated endocytosis. Using a cell culture system enriched for oligodendrocyte progenitor cells, we determined, that oligodendrocyte progenitor cells bind ferritin in a saturable and competitive manner with a K(d) of 5 nM and a receptor density of 0.06 fmol bound/20,000 cells. FITC tagged ferritin is internalized by A2B5, O4 or CNPase expressing cells in the culture, but not by GFAP+ cells. The uptake of ferritin into the oligodendrocyte progenitors was inhibited by treating the cells with inhibitors of receptor mediated endocytosis (hypertonic medium, potassium deficient medium, ATP depletion, sulfhydryl reagents). In addition exogenous ferritin decreased iron responsive element/iron regulatory protein binding indicating that the iron within the internalized ferritin is released and contributes to the intracellular iron pool. Given the relatively high amount of iron that can be delivered via ferritin, and the selective distribution of ferritin receptors in the white matter tracts in vivo, we propose that ferritin is a major source of iron for oligodendrocytes.

Distribution of transferrin and ferritin binding in normal and multiple sclerotic human brains.

Delivery of iron to the brain traditionally has been considered the responsibility of transferrin. However, transferrin receptors in brain are located primarily within gray matter areas rather than in the iron rich white matter tracts. In this report we present the first demonstration of ferritin binding sites in human brain and provide evidence that these binding sites are primarily in white matter tracts. This distribution of ferritin binding is opposite of that seen for the distribution of the transferrin receptor in normal adult human brain. Ferritin binds to human brain tissue in a competitive and saturable manner with a dissociation constant of 0.35 nM and a binding site density of 116.7 fmol/mg protein. In brain tissue from multiple sclerotic (MS) patients the normal pattern of transferrin and ferritin binding distributions is disrupted. Ferritin binding is absent in the lesion itself and in the immediate periplaque region within the white matter but returns to normal as the distance from the lesion becomes greater. In direct contrast to ferritin binding, transferrin binding in the MS tissue is present in the white matter tracts, but only in the periplaque region. The periplaque region also contains transferrin receptor positive cells (as determined by immunocytochemistry) morphologically consistent with oligodendrocytes. Gray matter binding of transferrin in MS patients appears normal. These data provide the initial evidence of ferritin binding in human brain, address the enigma of the apparent absence of an iron delivery system to the iron-rich white matter, and suggest loss of ferritin binding is involved in or is a consequence of demyelination associated with MS.

Receptor for interleukin 13 is a marker and therapeutic target for human high-grade gliomas.

Glioblastoma multiforme (GBM) is an incurable brain tumor. Due to the striking heterogeneity that characterizes GBM, there is no known tumor-specific antigen or receptor that is expressed by a majority of GBM patients. We found that virtually all studied human GBM specimens (23 samples) abundantly expressed a receptor for interleukin (IL)-13 in situ, whereas normal human brain had few, if any, IL-13-binding sites. The GBM-associated IL-13 receptor was both quantitatively and qualitatively different from and, thus, more restrictive than the shared signaling receptor of normal tissue: it was IL-4 independent. The receptor for IL-13 was overexpressed by a majority of cancer cells in situ. Furthermore, cytotoxins targeted to this more restrictive IL-13R produced cures in animals bearing xenografts of human high-grade gliomas. Thus, unexpectedly, the receptor for an immune regulatory cytokine may be a long sought marker and, concomitantly, a unique imaging site and therapeutic target for GBM, the most malignant and the most heterogeneous of brain tumors.

Characterization and distribution of ferritin binding sites in the adult mouse brain.

Studies on iron uptake into the brain have traditionally focused on transport by transferrin. However, transferrin receptors are not found in all brain regions and are especially low in white matter tracts where high iron concentrations have been reported. Several lines of research suggest that a receptor for ferritin, the intracellular storage protein for iron, may exist. We present, herein, evidence for ferritin binding sites in the brains of adult mice. Autoradiographic studies using 125I-recombinant human ferritin demonstrate that ferritin binding sites in brain are predominantly in white matter. Saturation binding analyses revealed a single class of binding sites with a dissociation constant (K(D)) of 4.65 x 10(-9) M and a binding site density (Bmax of 17.9 fmol bound/microg of protein. Binding of radiolabeled ferritin can be competitively displaced by an excess of ferritin but not transferrin. Ferritin has previously been shown to affect cellular proliferation, protect cells from oxidative damage, and deliver iron. The significance of a cellular ferritin receptor is that ferritin is capable of delivering 2,000 times more iron per mole of protein than transferrin. The distribution of ferritin binding sites in brain vis-à-vis transferrin receptor distribution suggests distinct methods for iron delivery between gray and white matter.