A Comparison of ToxCast Test Results with In Vivo and Other In Vitro Endpoints for Neuro, Endocrine, and Developmental Toxicities: A Case Study Using Endosulfan and Methidathion.

INTRODUCTION: The U.S. Environmental Protection Agency's (EPA's) Toxicity Forecaster (ToxCast) is a potential tool for chemical prioritization, hazard identification, and risk assessment. We conducted a case study to compare ToxCast data with endpoints from other in vitro and in vivo studies for two data-rich pesticides: endosulfan and methidathion. METHODS: ToxCast assays for endocrine disruption, development (zebrafish), and neurotoxicity were qualitatively compared to traditional neurotoxicity, developmental and reproductive toxicity findings. We also used in vitro-in vivo extrapolation to convert half-maximal activity concentrations in active ToxCast assays to rat oral equivalent doses, and quantitatively compared these to the lowest observable effect level (LOEL) from in vivo studies. RESULTS: Endosulfan was inactive for GABAA R, unlike in vivo; but active with dopamine transporter assays and was neurotoxic in zebrafish as expected. Methidathion was not active for these endpoints in vivo or in vitro. Acetylcholinesterase inhibition was ToxCast-inactive, although both pesticides are inhibitors in vivo. ToxCast results were generally inactive for endosulfan estrogen receptor agonism and androgen receptor antagonism unlike in vivo. Calculated oral equivalent doses for estrogen receptor and androgen receptor pathways and for zebrafish assays for both compounds were generally consistent with in vivo LOELs. Endosulfan showed neurotoxicity and both pesticides showed developmental effects in the zebrafish assays, although methidathion is not developmentally toxic in vivo. CONCLUSIONS: ToxCast's predictions showed concordance on some endpoints and nonconcordance, consisting mainly of false inactives, in several critical endpoints, likely due to a lack of metabolic activation and limitations in assay design. Zebrafish assays were good predictors of developmental toxicity and neurotoxicity for endosulfan.

Insecticide Transfer Efficiency and Lethal Load in Argentine Ants.

Trophallaxis between individual worker ants and the toxicant load in dead and live Argentine ants (Linepithema humile) in colonies exposed to fipronil and hydramethylnon experimental baits were examined using accelerator mass spectrometry (AMS). About 50% of the content of the crop containing trace levels of 14C-sucrose, 14C-hydramethylnon, and 14C-fipronil was shared between single donor and recipient ants. Dead workers and queens contained significantly more hydramethylnon (122.7 and 22.4 amol/µg ant, respectively) than did live workers and queens (96.3 and 10.4 amol/µg ant, respectively). Dead workers had significantly more fipronil (420.3 amol/µg ant) than did live workers (208.5 amol/µg ant), but dead and live queens had equal fipronil levels (59.5 and 54.3 amol/µg ant, respectively). The distribution of fipronil differed within the bodies of dead and live queens; the highest amounts of fipronil were recovered in the thorax of dead queens whereas live queens had the highest levels in the head. Resurgence of polygynous ant colonies treated with hydramethylnon baits may be explained by queen survival resulting from sublethal doses due to a slowing of trophallaxis throughout the colony. Bait strategies and dose levels for controlling insect pests need to be based on the specific toxicant properties and trophic strategies for targeting the entire colony.

Structural studies of the final enzyme in the alpha-aminoadipate pathway-saccharopine dehydrogenase from Saccharomyces cerevisiae.

The 1.64 A structure of the apoenzyme form of saccharopine dehydrogenase (SDH) from Saccharomyces cerevisiae shows the enzyme to be composed of two domains with similar dinucleotide binding folds with a deep cleft at the interface. The structure reveals homology to alanine dehydrogenase, despite low primary sequence similarity. A model of the ternary complex of SDH, NAD, and saccharopine identifies residues Lys77 and Glu122 as potentially important for substrate binding and/or catalysis, consistent with a proton shuttle mechanism. Furthermore, the model suggests that a conformational change is required for catalysis and that residues Lys99 and Asp281 may be instrumental in mediating this change. Analysis of the crystal structure in the context of other homologous enzymes from pathogenic fungi and human sources sheds light into the suitability of SDH as a target for antimicrobial drug development.

An engineered bifunctional high affinity iron uptake protein in the yeast plasma membrane.

High affinity iron uptake in fungi is supported by a plasma membrane protein complex that includes a multicopper ferroxidase enzyme and a ferric iron permease. In Saccharomyces cerevisiae, this complex is composed of the ferroxidase Fet3p and the permease Ftr1p. Fe(II) serves as substrate for Fe-uptake by being substrate for Fet3p; the resulting Fet3p-produced Fe(III) is then transported across the membrane via Ftr1p. A model of metabolite channeling of this Fe(III) is tested here by first constructing and kinetically characterizing in Fe-uptake two Fet3p-Ftr1p chimeras in which the multicopper oxidase/ferroxidase domain of Fet3p has been fused to the Ftr1p iron permease. Although the bifunctional chimeras are as kinetically efficient in Fe-uptake as is the wild type two-component system, they lack the adaptability and fidelity in Fe-uptake of the wild type. Specifically, Fe-uptake through the Fet3p, Ftr1p complex is insensitive to a potential Fe(III) trapping agent - citrate - whereas Fe-uptake via the chimeric proteins is competitively inhibited by this Fe(III) chelator. This inhibition does not appear to be due to scavenging Fet3p-produced Fe(III) that is in equilibrium with bulk solvent but could be due to leakiness to citrate found in the bifunctional but not the two-component system. The data are consistent with a channeling model of Fe-trafficking in the Fet3p, Ftr1p complex and suggest that in this system, Fet3p serves as a redox sieve that presents Fe(III) specifically for permeation through Ftr1p.

A reduction of nonpyramidal cells in sector CA2 of schizophrenics and manic depressives.

BACKGROUND: Recent studies have suggested that there may be a preferential decrease of "nonpyramidal" neurons (NPs) in several corticolimbic regions of schizophrenic (SZ) brain. The current study was undertaken to determine whether a change in the density of pyramidal neurons (PNs) and NPs might be present in the hippocampal formation (HIPP) of SZ brain. METHODS: A spatial counting approach in which the location of each and every PN and NP in the stratum pyramidale of sectors CA1-4 was applied to 11 normal control (CONs) and 10 SZs matched for age and postmortem interval, as well as 4 manic depressive (MD) subjects matched for age. RESULTS: The data indicate that the CONs had approximately 10-20 times as many PNs than NPs in the various HIPP subfields. When the CON data were compared to those for the SZs, both the total number and density of PNs were found to be similar in all four sectors, while NPs were found to be selectively reduced by approximately 40% in CA2 of the SZ group. When the data were broken down according to patients with and without neuroleptic exposure, drug-free SZs showed a significant reduction in the density of NPs in CA2. The 4 MD cases both with and without neuroleptic exposure also showed a similar reduction of NPs in sector CA2. CONCLUSIONS: Taken together, the results of this study suggest that there may be a highly selective decrease in the number of NPs in sector CA2 that could play a contributory role in the pathophysiology of the major psychoses.

Seroprevalence of cytotoxin-associated gene A positive Helicobacter pylori strains in Changle, an area with very high prevalence of gastric cancer in south China.

BACKGROUND: Helicobacter pylori, especially the CagA-positive strains, are closely associated with peptic ulcers and gastric cancers. We performed a large scale gastric cancer screening project and examined the prevalence of H. pylori and CagA-positive strains in Changle, China, an area with one of the World's highest gastric cancer mortality. We also compared the prevalence with that in Hong Kong which has one-tenth of the gastric cancer mortality of that in Changle. METHODS: A total of 2424 subjects in Changle and 523 subjects in Hong Kong had endoscopic examination and venesection. Sera were tested for anti-H. pylori antibody and anti-CagA antibody and correlated with endoscopic findings. RESULTS: In Changle, 80. 9% of the subjects were H. pylori carriers. Out of 551 carriers, 408 (74%) were positive for anti-CagA antibody. A total of 76% and 87% of the asymptomatic and gastric cancer patients were positive for anti-CagA antibody, respectively (P > 0.05). Compared to Hong Kong, there was a significantly (P < 0.0001) higher prevalence of CagA-positive strains in asymptomatic subjects in Changle (76%) than in Hong Kong (28%), but not in peptic ulcers or gastric cancers. CONCLUSIONS: Subjects in Changle had a high prevalence of H. pylori infection and a high prevalence of the CagA-positive strains. The contrast in the prevalence of CagA-positive strains, in asymptomatic subjects in two areas with differing gastric cancer mortality, supports the pathogenic role of CagA-positive strains in gastric carcinogenesis.

Orexins: a role in medullary sympathetic outflow.

Orexin A and B, also known as hypocretin 1 and 2, are two recently isolated hypothalamic peptides. As orexin-containing neurons are strategically located in the lateral hypothalamus, which has long been suspected to play an important role in feeding behaviors, initial studies were focused on the involvement of orexins in positive food intake and energy metabolism. Recent studies implicate a more diverse biological role of orexins, which can be manifested at different level of the neuraxis. For example, canine narcolepsy, a disorder with close phenotypic similarity to human narcolepsy, is caused by a mutation of hypocretin receptor 2 gene. Results from our immunohistochemical and functional studies, which will be summarized here, suggest that the peptide acting on neurons in the rostral ventrolateral medulla augment sympathoexcitatory outflow to the spinal cord. This finding is discussed in the context of increased sympathetic activity frequently associated with obesity.

Endomorphins decrease heart rate and blood pressure possibly by activating vagal afferents in anesthetized rats.

Endomorphin 1 (10, 30, 100 nmol/kg) administered intravenously (i.v. ) to urethane-anesthetized rats consistently and dose-dependently lowered heart rate (HR) and mean arterial pressure (MAP); the decrease in blood pressure recovered faster as compared to the HR. The effects of endomorphin 2 were qualitatively similar. Naloxone (2 mg/kg, i.v.) completely antagonized the bradycardia and hypotension caused by endomorphin 1. Pretreatment of the rats with atropine methylnitrate, atropine sulfate (2 mg/kg, i.v.) or bilateral vagotomy nearly abolished the bradycardia and attenuated the hypotensive effect of endomorphin 1. Our studies suggest that the bradycardia effect following systemic administration of the new opioid peptide may be explained by activation of vagal afferents and the hypotensive effect may be secondary to a reduction of cardiac output and/or a direct vasodilation.

Cocaine- and amphetamine-regulated transcript-immunoreactivity in the rat sympatho-adrenal axis.

Distribution of cocaine- and amphetamine-regulated transcript-like immunoreactivity (CART-LI) was studied in the rat spinal cord, sympathetic ganglia and adrenal glands by immunohistochemical methods, utilizing a polyclonal antiserum raised against the CART peptide fragment 55-102. CART-LI was detected in nerve fibers and in basket-like terminals surrounding many postganglionic neurons of the superior cervical ganglion (SCG), stellate, paravertebral and prevertebral ganglia. Postganglionic neurons exhibited low or non-detectable levels of CART-LI. Surgical sectioning of the cervical sympathetic trunk for 6-7 days resulted in a nearly complete loss of CART-LI fibers and terminals in the SCG. In the adrenal gland, CART-LI nerve fibers formed a plexus underneath the capsule, some of which bifurcated and made a sharp turn toward the adrenal medulla, where clusters of chromaffin cells were intensely labeled. The detection of CART-LI in sympathetic ganglia and adrenal glands extends the previous observation of the presence of CART-LI in sympathetic preganglionic neurons and further supports the notion that CART peptide(s) may function as a signaling molecule in the sympatho-adrenal axis.

Release of endomorphin-2 like substances from the rat spinal cord.

Release of endomorphin (ENDO)-2 like substances from the dorsal horn of the isolated rat spinal cord was measured by the immobilized-antibody microprobe technique. Spinal cords were removed from anesthetized 4-6 week old rats and superfused with oxygenated Krebs solution at room temperature. Glass microprobes coated with ENDO-2 antibodies were inserted into the dorsal horn of the lumbar spinal cord 1.5 mm lateral to the midline to a depth 2.5 mm below the dorsal surface of the cord. Each probe remained in situ for 10 min periods before, during and after electrical stimulation applied to the dorsal root entry zone of the same spinal segment. There was no detectable basal release of immunoreactive endomorphin-2 like substance (irENDO) from the dorsal horns during the pre-stimulation, nor following the stimulation period. A significant release of irENDO was measured during the electrical stimulation. These results provide the first evidence of a irEndo release that is correlated spatially with the dorsal horn laminae I and II where ENDO-2-immunoreactive fibers are concentrated in the dorsal horn in response to electrical activation of primary afferent fibers.

Orexin A-like immunoreactivity in the rat brain.

Distribution of orexin-A-like immunoreactivity (ORX-LI) in rat brains was investigated with the use of a rabbit polyclonal antibody against the full length peptide orexin A. Virtually all the ORX-LI cell bodies were observed in the lateral hypothalamus at the level of median eminence. The large majority of ORX-LI neurons appeared spherical or fusiform, 20-30 microm in diameter and issued two to five cell processes with few secondary branchings. Numerous ORX-LI fibers were observed in subregions of the hypothalamus. ORX-LI cell processes were sparsely distributed in the cortex, hippocampus and thalamus. Many varicose ORX-LI cell processes were situated close to the 3rd and lateral ventricles, some of which appeared to be protruding into the lumen. As a corollary, orexin A may be released into the ventricles and interact with neurons in distant targets, in addition to influencing the activity of neurons with which ORX-LI axons make synaptic contacts.

Out-of-plane motion compensation in multislice spin-echo MRI.

In magnetic resonance imaging (MRI), it is well-known that patient motion plays a significant role in the degradation of image quality. Although the case of translational in-plane motion (x-y-motion) has been studied by several researchers, the effect of rigid, translational out-of-plane motion (z-motion) has not yet been completely analyzed due to its more complex nature. Out-of-plane motion introduces blurring along the slice-selection direction in addition to motion artifacts. Here, the authors present a model to represent the effect of out-of-plane motion on multislice MR data. The inversion of this model not only results in the correction of the artifacts due to out-of-plane motion, but also reduces blurring in the slice-selection direction, yielding higher resolution images. Because of the shift-varying nature of the authors' model, they propose to use a nonlinear postprocessing method, projection onto convex sets (POCS), for its inversion, provided that the motion kernel and the slice-selection profile are known. The proposed method has been tested on simulated data and then applied to actual MR data to demonstrate the feasibility of the technique in real imaging situations.

Helicobacter pylori sero-prevalence in asthma.

Asthma is a very common respiratory disease which is characterized by the presence of cytokine-mediated airway inflammation leading to smooth muscle contraction, oedema and progressive airway damage in some cases. In light of our recent finding of an increased sero-prevalence of Helicobacter pylori in bronchiectasis, we have determined serum levels of H. pylori-specific IgG in asthmatic and control subjects. Altogether 90 consecutive asthmatic [mean age +/- SD 42.6+/-16 years and 52 female (F)] and 97 healthy control subjects (mean age +/- SD 43.2+/-13.3 years and 51 female (F); P = 0.78 and 0.39 respectively) were recruited prospectively. H. pylori sero-prevalence was not significantly different between asthmatic and control subjects (P>0.05). Serum H. pylori IgG levels did not correlate with FEV1 % predicted, FVC % predicted or duration of asthma (P>0.05). Similar to the results of previously published sero-epidemiological studies, there was a weak correlation between serum H. pylori IgG with increasing age (r = 0.43, P = 0.004). Despite the sero-epidemiological association of H. pylori infection with many inflammatory conditions, our data showed no such association for middle age asthmatic patients with mild intermittent asthma in our locality.

Enhanced sensitivity to molecular diffusion with intermolecular double-quantum coherences: implications and potential applications.

Apparent molecular self-diffusion rates for (1)H intermolecular double-quantum coherences (iDQCs) were measured in solvents covering a wide range of intrinsic diffusion coefficients at 1.5, 9.4 and 14T, and water iDQC diffusion-weighted images were obtained at 1.5T in human brains and at 9.4T in rat brains. Conventional single quantum coherence (SQC) measurements were also made in the same samples. Experimental results indicate that iDQCs are approximately twice as sensitive to diffusion as SQC. A general theoretical expression was derived, and a model was proposed to explain the phenomenon. Potential applications in DWI and brain fMRI were also discussed.

Sensitivity of Bloom syndrome fibroblasts to mitomycin C.

Lymphocytes and fibroblasts from people with Bloom syndrome, an autosomal recessive disorder associated with a predisposition to a wide variety of cancers, are known to be hypersensitive to ethylating agents as measured by sister chromatid exchange induction. Recently, hypersensitivity to cell killing by mitomycin C has also been reported in Bloom syndrome fibroblasts from three donors. We report here results which confirm the hypersensitivity of Bloom syndrome fibroblasts as measured by cell killing but show that they have a normal sensitivity to mitomycin C as measured by sister chromatid exchange induction. These results are discussed in terms of their relevance to the diversity of response of Bloom syndrome cells to mutagens, and the nature of the primary defect in Bloom syndrome.

Functional MR imaging of vision in the deaf.

RATIONALE AND OBJECTIVES: Early loss of a sensory modality has been associated with cortical reorganization in both animal models and humans. The purpose of this study was to map visual activation with functional magnetic resonance (MR) imaging and to document possible developmental reorganization in the temporal lobe caused by early deafness. MATERIALS AND METHODS: Six prelingual, profoundly deaf subjects were compared with a similar group of six hearing subjects. Three visual tasks were performed by both groups: attention to movement in the field-of-view periphery, shape matching, and mental rotation. Echo-planar coronal MR imaging was performed at 1.5 T. RESULTS: Regions of interest encompassing the middle and posterior aspects of the superior and middle temporal gyri demonstrated a significantly (P < .05) increased activation in deaf subjects compared with hearing subjects, particularly on the right side (P < .05) and during the tasks involving motion. The most specific effect was noted during the mental-rotation task. CONCLUSION: These results support the hypothesis that portions of the temporal lobe usually involved in auditory processing are more active during certain visual tasks in deaf compared with hearing subjects. Cortical reorganization may be an important factor in the deaf population when considering the physiology of temporal lobe lesions and predicting surgical outcomes. Functional MR imaging may be helpful during preoperative assessment in individuals with deafness.

Characteristics and pitfalls of contrast-enhanced, T1-weighted magnetization transfer images of the brain.

RATIONALE AND OBJECTIVES: This study was undertaken to clarify the difference in signal pattern on contrast material-enhanced T1-weighted magnetic resonance (MR) magnetization transfer (MT) images between enhancing and nonenhancing lesions in various intracranial diseases and to determine the necessity of nonenhanced MT images for evaluating lesional contrast enhancement. MATERIALS AND METHODS: MR images of 116 patients who underwent nonenhanced T1-weighted imaging, nonenhanced MT imaging, and contrast-enhanced MT imaging were reviewed. The increase in signal intensity of lesions relative to normal brain was compared between nonenhanced T1-weighted images and contrast-enhanced MT images. Signal intensity of lesions was compared with that of the striate nucleus and white matter on contrast-enhanced MT images. True enhancement was determined by comparison with nonenhanced MT images. RESULTS: In all, 143 lesions, including 86 enhancing and 57 nonenhancing lesions, were identified among 63 patients. Almost all (99%) of the enhancing lesions were hyperintense to striate nucleus on contrast-enhanced MT images, and most (>87%) showed moderate to marked signal intensity increase from nonenhanced T1-weighted images to contrast-enhanced MT images. Most (>95%) of the nonenhancing lesions showed mild or no increase in relative signal intensity, and most (75%) were iso- or hypointense to striate nucleus on contrast-enhanced MT images. A few nonenhancing lesions (4%-6%), however, showed increase in signal intensity that was indistinguishable from true enhancement without comparison to non-enhanced MT images. CONCLUSION: Nonenhanced MT images should be obtained to assess pathologic enhancement accurately.

MR imaging of rabbit hip cartilage with a clinical imager and specifically designed surface coils.

RATIONALE AND OBJECTIVES: The authors' purpose was to develop a magnetic resonance (MR) imaging technique for examining the hip joint of a rabbit with a clinical MR imager. MATERIALS AND METHODS: Fourteen hips of 10 male New Zealand white rabbits were examined on a 1.5-T MR imager with a specially designed surface coil. Field of view was 3-8 cm, matrix was 256-512 x 192-256, section thickness was 0.9-2.0 mm, and spacing was 0.3-0.5 mm. The hips were sectioned and evaluated by means of light microscopy. Measurements of cartilage thickness from MR images were correlated with those from histologic specimens. RESULTS: The resolution obtainable with a 1.5-T imager was adequate for imaging articular cartilage when a débrided rabbit hip specimen was placed in the center of a 3.5-cm single-loop coil. Rabbit hip cartilage had a trilaminar appearance on MR images. The coefficient of correlation between cartilage-thickness measurements was .81. CONCLUSION: MR imaging of rabbit hip cartilage can be performed on 1.5-T clinical MR imaging unit. Limitations due to the field strength can be overcome with efficiently designed surface coils.

Corticosteroids and azathioprine do not prevent radiation-induced lung injury.

The case of a man who presented with dyspnea and a dry cough six weeks after mediastinal radiotherapy for malignant thymoma is described. The patient was on prednisone (30 mg/day) and azathioprine (100 mg/day) throughout the course of radiation. The respiratory difficulties developed as the dose of prednisone was gradually decreased to 20 mg/day postradiation. Chest x-ray showed bilateral pulmonary infiltrates. Computed tomography scan of the thorax confirmed bilateral ground glass opacities, with well-defined lateral margin on the right side corresponding to the field of radiation. However, the airspace opacities extended beyond the radiation field into the periphery of the lungs together with mild airway dilation on the left side compatible with bronchiolitis obliterans organizing pneumonia (BOOP) or cryptogenic organizing pneumonia. Bronchoalveolar lavage performed on the nonirradiated area showed an intense lymphocytosis. No cause of BOOP other than radiation was found. Treatment with high dose corticosteroids (80 mg/day) resulted in rapid clinical and radiological improvement, and resolution of chest x-ray abnormalities. Focal mediastinal radiation therapy may induce diffuse lung injury including BOOP. In addition, the concurrent use of moderate dose prednisone and azathioprine during the periradiotherapy period does not prevent the development of either BOOP or classic radiation pneumonitis.

3D visual presentation of shoulder joint motion.

The 3D visual presentation of biodynamic events of human joints is a challenging task. Although the 3D reconstruction of high contrast structures from CT data has been widely explored, then there is much less experience in reconstructing the small low contrast soft tissue structures from inhomogeneous and sometimes noisy MR data. Further, there are no algorithms for tracking the motion of moving anatomic structures through MR data. We represent a comprehensive approach to 3D musculoskeletal imagery that addresses these challenges. Specific imaging protocols, segmentation algorithms and rendering techniques are developed and applied to render complex 3D musculoskeletal systems for their 4D visual presentation. Applications of our approach include analysis of rotational motion of the shoulder, the knee flexion, and other complex musculoskeletal motions, and the development of interactive virtual human joints.