Neurological impacts from inhalation of pollutants and the nose-brain connection.

The effects of inhaled particles have focused heavily on the respiratory and cardiovascular systems. Most studies have focused on inhaled metals, whereas less information is available for other particle types regarding the effects on the brain and other extra-pulmonary organs. We review here the key available literature on nanoparticle uptake and transport through the olfactory pathway, the experimental data from animal and in vitro studies, and human epidemiological observations. Nanoparticles (<0.1 µm in one dimension) may easily reach the brain from the respiratory tract via sensory neurons and transport from the distal alveoli into the blood or lymph as free particles or inside phagocytic cells. These mechanisms and subsequent biologic responses may be influenced by the chemical composition of inhaled particles. Animal studies with ambient particulate matter and certain other particles show alterations in neuro-inflammatory markers of oxidative stress and central neurodegeneration. Human observations indicate motor, cognitive, and behavioral changes especially after particulate metal exposure in children. Exposure to co-pollutants and/or underlying disease states could also impact both the biokinetics and effects of airborne particles in the brain. Data are needed from the areas of inhalation, neurology, and metal toxicology in experimental and human studies after inhalation exposure. An increased understanding of the neurotoxicity associated with air pollution exposure is critical to protect susceptible individuals in the workplace and the general population.

Prevalence of obesity in 6- and 9-year-old children living in Central-North Italy. Analysis of determinants and indicators of risk of overweight.

The objective of our article is to survey the prevalence of overweight and obesity among 6- and 9-year-old children in Emilia-Romagna, a region of Central-North Italy, and to study the eating habits and behaviours of these children and their families. During 2003 and 2005, we analysed a stratified sample of the general population of children attending pre-school (2681 children aged 6 years) and primary school (2955 children aged 9 years). Their height and weight were measured by healthcare workers. In the 6-year-old children, information concerning their eating habits was collected by means of a questionnaire completed by their parents. The prevalence of overweight was 16.5% in 6-year-old children and 20.6% in 9-year-old children. The increase of overweight from 6- to 9-year-old children was observed in males (13.5% in 6-year-old/21.3% in 9-year-old boys), but not in females. The prevalence of obesity was 8.9% in children aged 6 years and 9.0% in those aged 9 years, and it was higher in comparison with Italian surveys carried out in 1993 and in 2001: 7.5% in 6-year-old and 7.8% in 9-year-old children in 1993, and 6.6% in 6-year-old and 7.2% in 9-year-old children in 2001. In pre-school children, overweight and obesity were closely influenced by the education level, occupation and nutritional status of the parents.

Nanometer size diesel exhaust particles are selectively toxic to dopaminergic neurons: the role of microglia, phagocytosis, and NADPH oxidase.

The contributing role of environmental factors to the development of Parkinson's disease has become increasingly evident. We report that mesencephalic neuron-glia cultures treated with diesel exhaust particles (DEP; 0.22 microM) (5-50 microg/ml) resulted in a dose-dependent decrease in dopaminergic (DA) neurons, as determined by DA-uptake assay and tyrosine-hydroxylase immunocytochemistry (ICC). The selective toxicity of DEP for DA neurons was demonstrated by the lack of DEP effect on both GABA uptake and Neu-N immunoreactive cell number. The critical role of microglia was demonstrated by the failure of neuron-enriched cultures to exhibit DEP-induced DA neurotoxicity, where DEP-induced DA neuron death was reinstated with the addition of microglia to neuron-enriched cultures. OX-42 ICC staining of DEP treated neuron-glia cultures revealed changes in microglia morphology indicative of activation. Intracellular reactive oxygen species and superoxide were produced from enriched-microglia cultures in response to DEP. Neuron-glia cultures from NADPH oxidase deficient (PHOX-/-) mice were insensitive to DEP neurotoxicity when compared with control mice (PHOX+/+). Cytochalasin D inhibited DEP-induced superoxide production in enriched-microglia cultures, implying that DEP must be phagocytized by microglia to produce superoxide. Together, these in vitro data indicate that DEP selectively damages DA neurons through the phagocytic activation of microglial NADPH oxidase and consequent oxidative insult.

Vanilloid (capsaicin) receptors influence inflammatory sensitivity in response to particulate matter.

The signs of airway inflammation and hyperresponsiveness that occur in animals exposed to air pollutants are often strain- and species-specific. To investigate the underlying causes of this phenomenon, BALB/c and C57bl/6 mice were exposed intratracheally to residual oil fly ash (ROFA, 3 mg/kg) and examined after 24 h for signs of airway inflammation. BALB/c showed significantly higher numbers of neutrophils and increased airway hyperresponsiveness in response to methacholine challenge, whereas B6 mice showed no significant change in either inflammatory endpoint. To determine the underlying cause of this strain specificity, cultures of dorsal root ganglion (DRG) sensory neurons, which innervate the upper airways in situ, were explanted from both BALB/c and B6 fetal mice. After 5-7 days in culture, they were exposed to ROFA, other urban and industrial particulate matter (PM; e.g., oil fly ash, woodstove, Mt. St. Helen, St. Louis, Ottawa, coal fly ash) or to prototype irritants (e.g., capsaicin 3-10 microM, pH 5.0 and 6.5). In all instances (except for woodstove), DRG neurons from BALB/c mice released significantly higher levels of the pro-inflammatory cytokine IL-6 into their nutrient media relative to neurons from B6 mice. This cytokine release could be significantly reduced for all PM treated cultures (except woodstove) by pretreatment of cultures with capsazepine (CPZ), a competitive antagonist of vanilloid receptors. DRG neurons, cultured from BALB/c and B6 neonates, were loaded with Fluo-3 AM and exposed to the prototype irritants, acid pH (5.0, 6.5), or capsaicin (3, 10 microM). Analysis of their increases in intracellular calcium showed that significantly higher numbers of BALB/c neurons responded to these prototype irritants, relative to B6 neurons. Morphometric analysis of BALB/c neurons, histochemically stained with cobalt to label neurons bearing capsaicin-sensitive receptors, showed a significantly higher level of stained neurons relative to B6 neurons. Finally, semiquantitative RT-PCR showed a higher expression of VR1 receptor mRNA in DRG and spinal cord taken from neonatal BALB/c mice relative to B6 mice. Taken together, these data suggest that capsaicin and acid-sensitive irritant receptors, located on somatosensory cell bodies and their nerve fiber terminals, subserve PM-induced airway inflammation and are quantitatively different in responsive and nonresponsive mouse strains.

Residual oil fly ash and charged polymers activate epithelial cells and nociceptive sensory neurons.

Residual oil fly ash (ROFA) is an industrial pollutant that contains metals, acids, and unknown materials complexed to a particulate core. The heterogeneous composition of ROFA hampers finding the mechanism(s) by which it and other particulate pollutants cause airway toxicity. To distinguish culpable factors contributing to the effects of ROFA, synthetic polymer microsphere (SPM) analogs were synthesized that resembled ROFA in particle size (2 and 6 microm in diameter) and zeta potential (-29 mV). BEAS-2B human bronchial epithelial cells and dorsal root ganglion neurons responded to both ROFA and charged SPMs with an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the release of the proinflammatory cytokine interleukin-6, whereas neutral SPMs bound with polyethylene glycol (0-mV zeta potential) were relatively ineffective. In dorsal root ganglion neurons, the SPM-induced increases in [Ca(2+)](i) were correlated with the presence of acid- and/or capsaicin-sensitive pathways. We hypothesized that the acidic microenvironment associated with negatively charged colloids like ROFA and SPMs activate irritant receptors in airway target cells. This causes subsequent cytokine release, which mediates the pathophysiology of neurogenic airway inflammation.

Particulate matter initiates inflammatory cytokine release by activation of capsaicin and acid receptors in a human bronchial epithelial cell line.

Recent experiments have shown that human bronchial epithelial cells (i.e., BEAS-2B) release pro-inflammatory cytokines (i.e., IL-6 and TNFalpha) in a receptor-mediated fashion in response to the neuropeptides, substance P (SP), calcitonin gene-related protein (CGRP), and the prototype botanical irritant capsaicin. In the present experiments, we examined the relevance of these receptors to particulate matter (PM)-associated cellular inflammation. BEAS-2B cells, exposed to residual oil fly ash particles (ROFA), responded with an immediate (<30 s) increase in intracellular calcium levels ([Ca2+]i), increases of key inflammatory cytokine transcripts (i.e., IL-6, IL-8, TNFalpha) within 2 h exposure, and subsequent release of IL-6 and IL-8 cytokine protein after 4 h exposure. Pretreatment of BEAS-2B cells with pharmacological antagonists selective for the SP or CGRP receptors reduced the ROFA-stimulated IL-6 cytokine production by approximately 25 and 50%, respectively. However, pretreatment of these cells with capsazepine (CPZ), an antagonist for capsaicin (i.e., vanilloid) receptors, inhibited the immediate increases in [Ca2+]i, diminished transcript (i.e., IL-6, IL-8, TNFalpha) levels and reduced IL-6 cytokine release to control levels. BEAS-2B cells exposed to ROFA in calcium-free media failed to demonstrate increases of [Ca2+]i and showed reduced levels of cytokine transcript (i.e., IL-6, IL-8, TNFalpha) and IL-6 release, suggesting that ROFA-stimulated cytokine formation was partially dependent on extracellular calcium sources. A final set of experiments compared the inflammatory properties of the soluble and acidic insoluble components of ROFA. BEAS-2B cells, exposed to ROFA or ROFA that had been filtered through a 0.2-micrometer pore filter, produced equivocal IL-6. BEAS-2B cells exposed to pH 5.0 media for 15 min released moderate amounts of IL-6, 4 h later. This cytokine release could be blocked by amiloride, a pH receptor antagonist, but not by CPZ. BEAS-2B cells, pretreated with amiloride before ROFA exposure, showed a partial (approximately 25%) reduction of IL-6. Together, these data indicate that the acidic, soluble components of ROFA initiate cytokine release in BEAS-2B cells through activation of both capsaicin- and pH-sensitive irritant receptors.

Neuropeptides and capsaicin stimulate the release of inflammatory cytokines in a human bronchial epithelial cell line.

The role of neuropeptides in initiating and modulating airway inflammation was examined in a human bronchial epithelial cell line (i.e. BEAS-2B). At a range of concentrations, exposure of BEAS-2B cells to Substance P (SP) or calcitonin gene related protein resulted in immediate increases in intracellular calcium ([Ca(2+)](i)), the synthesis of the transcripts for the inflammatory cytokines, IL-6, IL-8 and TNFalpha after 2 h exposure, and the release of their proteins after 6 h exposure. Addition of thiorphan (100 nM), an inhibitor of neutral endopeptidase, enhanced the levels of SP-stimulated cytokine release. Stimulation of IL-6 by SP occurred in a conventional receptor-mediated manner as demonstrated by its differential release by fragments SP 4-11 and SP 1-4 and by the blockage of IL-6 release with the non-peptide, NK-1 receptor antagonist, CP-99 994. In addition to the direct stimulation of inflammatory cytokines, SP (0.5 microM), in combination with TNFalpha (25 units/ml), synergistically stimulated IL-6 release. BEAS-2B cells also responded to the botanical irritant, capsaicin (10 microM) with increases in [Ca(2+)](i) and IL-8 cytokine release after 4 h exposure. The IL-8 release was dependent on the presence of extracellular calcium. Capsaicin-stimulated increases of [Ca(2+)](i) and cytokine release could be reduced to control levels by pre-exposure to capsazepine, an antagonist of capsaicin (i.e. vanilloid) receptor(s) or by deletion of extracellular calcium from the exposure media. The present data indicate that the BEAS-2B human epithelial cell line expresses neuropeptide and capsaicin-sensitive pathways, whose activation results in immediate increases of [Ca(2+)](i) stimulation of inflammatory cytokine transcripts and the release of their cytokine proteins.

Neuropeptide denervation alters both the elicitation and induction phases of contact hypersensitivity in mice.

The effects of permanent disruption of neuropeptide transmission on the induction (i.e., sensitization) and elicitation (i.e., challenge) phases of contact hypersensitivity (CHS) are described. BALB/c mice were chemically denervated of neuropeptide (i.e., tachykinin) containing sensory C fibers by an acute injection of capsaicin (50 mg/kg) on postnatal day (PND) 2 to 3. As young adults (PND 45-60), these mice and their control littermates were sensitized by topical application of 0.1% 2,4-dinitrofluorobenzene (DNFB) or vehicle. Treatment groups generated from this exposure regimen consisted of untreated, controls (O/O), denervated, controls (CAP/O), untreated, sensitized (O/DNFB), and denervated, sensitized (CAP/DNFB). The elicitation phase of CHS was evaluated in these animals by measuring ear thickness in response to a DNFB challenge. In DNFB-sensitized groups, ear thickness was significantly increased over controls but was additionally increased 2.4-fold in CAP/DNFB compared to O/DNFB mice. The induction phase of CHS was next assessed in young adult mice by measuring lymph node cell (LNC) proliferation. For this, mice were sensitized for 3 consecutive days before their draining, auricular nodes were removed. The LNC were dissociated and cultured for 24 h with tritiated thymidine to assess LNC proliferation. As expected, significantly higher numbers of LNC occurred in both DNFB-sensitized groups (CAP/DNFB, O/DNFB) compared to the unsensitized, controls (CAP/O, O/O). However, LNC proliferation in CAP/DNFB was significantly higher than O/DNFB animals. Flow cytometry on similarly exposed mice failed to demonstrate any significant difference in the population of CD4CD8 or CD3CD45R LNC cells from neuropeptide-denervated (CAP/O, CAP/DNFB) mice or their respective treatment mates (O/O, O/DNFB), suggesting that alterations in T or B cell populations did not underlie these changes. Finally, cytokine release from the LNC from these treatment groups was examined. For this, the auricular lymph nodes were removed from animals, 2 to 4 h after the animals were administered a single application of a sensitizing concentration (0.1%) of DNFB or acetone vehicle. LNC, dissociated from these nodes, were cultured for 24 h. The nutrient media was removed from these cultured cells and examined for the release of proinflammatory cytokines, interleukin (IL)-1beta, IL-2 and tumor necrosis factor (TNF)alpha, by ELISA. There were no significant increases in IL-2. However, IL-1beta release was significantly increased in CAP/DNFB mice over O/DNFB by 18-fold and by over 30-fold compare to O/O controls. Levels of TNFalpha were significantly increased in both O/DNFB and CAP/DNFB mice over the nonsensitized controls (O/O, CAP/O). CAP/DNFB values were approximately double that of O/DNFB. There was no significant difference in IL-1beta or TNFalpha release between the nonsensitized controls (O/O, CAP/O). Collectively, these data indicate that neuropeptide denervation by neonatal administration of capsaicin alters both the induction and elicitation phases CHS and may modify sensitivity to chemically induced CHS.

Acetylcholinesterase and neuropathy target esterase inhibitions in neuroblastoma cells to distinguish organophosphorus compounds causing acute and delayed neurotoxicity.

The differential inhibition of the target esterases acetylcholinesterase (AChE) and neuropathy target esterase (NTE, neurotoxic esterase) by organophosphorus compounds (OPs) is followed by distinct neurological consequences in exposed subjects. The present study demonstrates that neuroblastoma cell lines (human SH-SY5Y and murine NB41A3) can be used to differentiate between neuropathic OPs (i.e., those inhibiting NTE and causing organophosphorus-induced delayed neuropathy) and acutely neurotoxic OPs (i.e., those highly capable of inhibiting AChE). In these experiments, concentration-response data indicated that the capability to inhibit AChE was over 100x greater than the capability to inhibit NTE for acutely toxic, nonneuropathic OPs (e.g., paraoxon and malaoxon) in both cell lines. Inhibition of AChE was greater than inhibition of NTE, without overlap of the concentration-response curves, for OPs which are more likely to cause acute, rather than delayed, neurotoxic effects in vivo (e.g., chlorpyrifos-oxon, dichlorvos, and trichlorfon). In contrast, concentrations inhibiting AChE and NTE overlapped for neuropathy-causing OPs. For example, apparent IC50 values for NTE inhibition were less than 9.6-fold the apparent IC50 values for AChE inhibition when cells were exposed to the neuropathy-inducing OPs diisopropyl phosphorofluoridate, cyclic tolyl saligenin phosphate, phenyl saligenin phosphate, mipafox, dibutyl dichlorovinyl phosphate, and di-octyl-dichlorovinyl phosphate. In all cases, esterase inhibition occurred at lower concentrations than those needed for cytoxicity. These results suggest that either mouse or human neuroblastoma cell lines can be considered useful in vitro models to distinguish esterase-inhibiting OP neurotoxicants.

Cell culture models of interspecies selectivity to organophosphorous insecticides.

In toxicology, the need to reduce uncertainties in human risk assessment is met by understanding why species and individuals within that species respond differently to chemical exposure. This kind of information is needed when extrapolating data from experimental (i.e., whole animal) systems to the human condition in terms of risk assessment. In 1993 the Neurotoxicology Division of the Environmental Protection Agency funded several investigators to examine this phenomenon (i.e., interspecies selectivity) using cell culture models. Organophosphorous (OP) insecticides were examined since they are characterized by an extremely divergent interspecies response. In 1995, a symposium entitled Novel Insights into Chemical Neurotoxicity, sponsored by the Society for In Vitro Biology featured this research. In it, a historical overview of the phenomenon of interspecies selectivity to OP insecticides was given, current explanations for it were discussed and contemporary in vitro models being used to explain it, were described. Data from these studies have helped to redefine the underlying mechanisms that characterize and influence the cross-species response to insecticides. These experiments have refocused the explanation of this phenomenon to include cellular metabolism, target enzyme baseline activities, and receptor-mediated electrophysiological and second-messenger events. Several investigators on this panel also reported on the use of subcellular markers (e.g., target esterases, second messengers, ionic fluxes) to differentiate neuropathy-causing OP compounds from acetylcholinesterase inhibitors. After these presentations, technical considerations used in the designed of in vitro neurotoxicity studies were discussed.

Characterization of the MDCK cell line for screening neurotoxicants.

The adoption of cell culture models to screen putative neurotoxicants is recognized in view of the consequences of nerve damage by environmental chemicals. Developing cell culture models that mimic certain properties of the blood-brain barrier (BBB) would be especially useful in view of the barrier's strategic role in the neurotoxic process. The present study evaluates a kidney epithelial cell line for its functional and enzymatic resemblance to cerebral endothelial cells. Madin-Darby canine kidney (i.e., MDCK) cells display morphological (i.e., ultrastructurally defined tight junctions), enzymatic (acetylcholinesterase, butyrylcholinesterase, gamma-glutamyl transpeptidase, superoxide dismutase, alkaline phosphatase, lactate dehydrogenase), and antigenic cell markers (i.e., Factor VIII), also found in cerebral endothelial cells. In addition, MDCK cells develop electrical resistance which is increased in response to conditioned media from astroglial cell lines (i.e., C6). These results suggest that the MDCK cell line might be useful for identifying neurotoxic chemicals that affect BBB integrity through similar endpoints.

Neuropeptide modulation of chemically induced skin irritation.

This study addresses the hypothesis that the early symptoms of chemically induced skin irritation are neurally mediated. Several approaches were used to affect nerve transmission in adult Balb/c female mice. These included general anesthesia (i.e., sodium pentobarbital), systemic capsaicin treatment, and pretreatment with specific pharmacological antagonists of the neuropeptides substance P (SP) and neurokinin A (NKA). After these treatments, a strongly irritating dose of dinitrofluorobenzene (DNFB) was applied to the ear and its swelling was measured over several postexposure times as an index of tissue irritation. Ear swelling in Nembutal (30 mg/kg)-anesthetized mice was depressed 62 and 76% at 4 and 24 hr postexposure compared to DNFB-treated unanesthetized animals measured at the same time points. Multiple injections of capsaicin (cumulative dose 30 mg/kg) depressed DNFB-ear swelling relative to non-capsaicin, DNFB-treated controls by 15, 40 (ip), and 44 and 43% (sc) at 4 and 24 hr postexposure, respectively. In mice exposed to acute or multiple injections of the SP antagonist CP-96,345 before DNFB application, ear swelling was depressed (relative to DNFB-treated animals) by 64 and 36% (acute, sc, 10 mg/kg) and 91 and 88% (multiple, ip, cumulative 35 mg/kg) at 0.5 and 1 hr postexposure, respectively. Mice exposed to the NKA antagonist, SR 48968, alone and in combination with the SP antagonist CP-96,345 were also examined after DNFB application. Ear swelling was diminished in mice pretreated with the NKA antagonist (1.0 mg/kg) by 17, 24, 34, and 40% at 0.5, 1, 4, and 24 hr postexposure. When used in combination with the SP antagonist, DNFB-induced ear swelling was reduced by 95% compared to unantagonized, DNFB-exposed mice at the 0.5- and 1-hr time points and remained significantly depressed by 33 and 46% at 4 and 24 hr postexposure. Taken in concert, these data suggest that neuropeptides, especially the tachykinins SP and NKA, modulate the early stages of chemically induced skin irritation.

Esterase comparison in neuroblastoma cells of human and rodent origin.

1. Activities of acetylcholinesterase (AChE), neuropathy target esterase (NTE), and carboxylesterase (CbxE) were compared in neuroblastoma cells of human origin (SH-SY5Y) and murine origin (NB41A3). 2. Mouse neuroblastoma cells had lower specific activities of NTE and CbxE than did human neuroblastoma cells; specific activities in the murine cells correlated with specific activities in mouse brain. 3. AChE activities in mouse and human neuroblastoma cells were considerably lower than AChE activities in mouse or hen brain. 4. Inhibition of esterases did not demonstrate interspecies differences for 12 of the 17 anti-esterase compounds tested with human and mouse neuroblastoma cells.

Neuropathy target esterase inhibition by organophosphorus esters in human neuroblastoma cells.

Certain organophosphorus compounds (OPs) produce a delayed neuropathy (OPIDN) in man and some animal species. Capability to cause OPIDN is generally predicted in animal models by early and irreversible inhibition of neuropathy target esterase (NTE, neurotoxic esterase). In this study, NTE inhibition in response to OP exposure was examined in cell culture, using the human SH-SY5Y neuroblastoma cell line. Cells were exposed for 1 hr to equimolar (1 x 10(-5) M) concentrations of 6 OPs associated with OPIDN in vivo (including 2 protoxicants and 4 active (-P = O) toxicants), and 8 OPs that do not produce delayed neuropathy in animal models (including 5 protoxicants and 3 -P = O compounds). The -P = O compounds that cause OPIDN in animal models inhibited NTE > 60% at the test concentration; -P = O compounds that do not cause OPIDN in animal models inhibited NTE < 30%. Protoxicants did not inhibit NTE at the test concentration, reflecting their limited metabolism in the human cell line. These results indicate that human neuroblastoma cells have potential use in the initial screening of bioactive OPs with capability for causing OPIDN.

Differential cytotoxic sensitivity in mouse and human cell lines exposed to organophosphate insecticides.

Neuroblastoma cell lines were used to examine the differential interspecies response (i.e., species selectivity) to organophosphates (OPs). Baseline activities of the major target esterases, i.e., cholinesterase, carboxylesterase, and neurotoxic esterase, were assayed in mouse and several human neural candidate cell lines. These activities were found to be variable within individual cell lines and among the various tested cell lines. Cytotoxicity data using the neutral red fluorometric assay were collected on both human (SH-SY5Y) and mouse (NB41A3) neuroblastoma clones exposed to a variety of OP insecticides. IC50 data indicated that the tested mouse cell line was consistently more sensitive than the human cell line to equimolar doses of various OP compounds (e.g., mipafox, parathion, paraoxon, DFP, leptophos oxon, fenthion, and fenitrothion). This difference in cytotoxic sensitivity was most pronounced in response to compounds requiring metabolic bioactivation (i.e., protoxicants). Cytotoxicity data also demonstrated that the NB41A3 mouse neuroblastoma cell line was more metabolically competent than the SH-SY5Y human cell line in converting the protoxicant parathion to its neurotoxic metabolite, paraoxon. B-lymphoblastoids, genetically engineered with human P450 cDNAs, demonstrated higher cytotoxic sensitivity to parathion than unengineered cells, indicating that cytochrome P450-associated monooxidase activity could also influence cytotoxic sensitivity to parathion in culture. These data suggest that interspecies-selectivity in response to OP-related cytotoxicity is influenced by intercellular differences in metabolism and baseline esterase activities.

In vitro screening batteries for neurotoxicants.

The need to develop, validate and utilize in vitro models to test chemicals for neurotoxic potential is widely appreciated. This lecture discusses the major advantages of using cell and tissue culture, the various in vitro models amenable for neurotoxicity studies, and the distinction between mechanistic and screening models. Considerations for designing screening batteries to evaluate neurotoxicants are discussed. Topics such as choice of appropriate cell models and endpoints (i.e., cytotoxic and neurotoxic), and technical considerations in the design of the battery are also presented.

Triphenyl phosphite-induced ultrastructural changes in bovine adrenomedullary chromaffin cells.

Primary cultures of bovine adrenomedullary chromaffin cells were treated with the phosphorus acid ester triphenyl phosphite (TPP), a chemical capable of producing Type II organophosphorus compound-induced delayed neurotoxicity (OPIDN), and the morphological changes were assessed by transmission electron and scanning microscopy. Following a 24-hr incubation with 100 microM TPP nearly all mitochondria were either disrupted or swollen and glycogen buildup within the cytoplasm was evident. The viability of cells treated with TPP and cultured on coverslips for scanning electron microscopy was very low. By scanning electron microscopy, the filopodia of these cells appeared contracted. The surface texture was very irregular and giant globular bodies were evident. Parallel studies were carried out with the cholinergic compound O,O-diethyl 4-nitrophenyl phosphate (paraoxon) and the Type I delayed neurotoxicant O,O-diisopropylphosphorofluoridate (DFP). Transmission and scanning electron microscopy revealed that treatment with these organophosphorus compounds did not produce the ultrastructural effects that were seen with TPP. The morphological data were confirmed biochemically by assessing the viability of the mitochondria via measurement of [3H]adenosine incorporation into ATP. Treatment with 100 microM TPP for 4 or 24 hr caused a marked inhibition (90% relative to controls) of adenosine incorporation. Neither 100 microM paraoxon nor 100 microM DFP had an inhibitory effect on incorporation. The effect of TPP was time-dependent with significant biochemical effects as early as 60 min. In contrast, ultrastructural changes were not seen until 24 hr. Morphologically, the 60-min incubations showed no perturbation in mitochondrial integrity. Our results support a specific effect of the triphenylphosphite, TPP, a Type II OPIDN compound, not a general toxic effect of organophosphorus compounds since the cholinergic agent paraoxon and the Type I delayed neurotoxic compound DFP did nto alter the cells ultrastructurally or compromise the mitochondria biochemically. The apparent target for TPP toxicity is the mitochondria.

Myelin basic protein-mRNA used to monitor trimethyltin neurotoxicity in rats.

Trimethyltin (TMT) is an alkyltin that targets neurons of the limbic system. A gene probe (i.e., mRNA) for myelin basic protein (MBP), a major component of central nervous system myelin, was used to monitor this toxic neuropathy in Sprague-Dawley rats. Animals were administered a single intraperitoneal injection of TMT-hydroxide at a neuropathic (8.0 mg/kg/body wt) or nonneuropathic (0.8 mg/kg/body wt) dose and sampled at 1, 3, or 7 days postexposure to correlate the progression of hippocampal neuropathology with probe (i.e., MBP-mRNA) levels. Microscopic examination of the brain showed only moderate but progressive damage over the 7-day postexposure period in animals treated with the neuropathic dose. Neuronal loss was first observed in the dendate gyrus and CA4 at 1 day postexposure, and progressed to the CA3c sector at 3 and 7 days postexposure. Elsewhere in the brain, minimal involvement of the entorhinal cortex neurons occurred 3 days postexposure and intensified by 7 days. No histological damage was seen at the nonneuropathic (0.8 mg/kg) dose. For gene probe analysis, the brain was divided into anterior and posterior halves. In rats treated with the neuropathic dose of TMT, the anterior brain showed progressive depressions of MBP-mRNA levels over the 1-, 3-, and 7-day postexposure period that correlated with increasing hippocampal neuropathology. The posterior brain showed no significant changes in MBP-mRNA levels with respect to that of controls over the same time period. At the nonneuropathic dose (0.8 mg/kg) a significant depression of MBP-mRNA levels occurred in the anterior brain at 7 days postexposure in the absence of overt histological damage.

Murine susceptibility to organophosphorus-induced delayed neuropathy (OPIDN).

This study reports that CD-1 strain mice are neuropathologically and biochemically responsive to acute doses of tri-ortho-cresyl phosphate (TOCP). Young (25-30 g) male and female animals were exposed (po) to a single dose of TOCP (580-3480 mg/kg) and sampled for neurotoxic esterase (NTE) activity at 24 and 44 hr postexposure and for neuropathic damage 14 days later. Biochemically, high intragroup variability existed at the lower doses, and at higher levels of TOCP exposure (i.e., greater than or equal to 1160 mg/kg), mean brain NTE inhibition never exceeded 68%. Hen and mouse brain NTE activity, assayed in vitro for sensitivity to inhibition by tolyl saligenin phosphate (TSP), the active neurotoxic metabolite of TOCP, showed similar IC50 values. Histologically, highly variable spinal cord damage was recorded throughout treatment groups and mean damage scores followed a dose-response pattern with no apparent correlation to threshold (i.e., greater than or equal to 65%) inhibition of brain NTE activity. Topographically, axonal degeneration in the mouse spinal cord predominated in the lateral and ventral columns of the upper cervical cord. Unlike the rat, which displays degeneration in the upper cervical cord's dorsal columns (i.e., gracilis fasciculus) in response to TOCP intoxication, treated mice showed minimal damage to this tract. To examine this discrepancy further, ultrastructural morphometric analysis of axon diameters in the cervical cord was performed in control mice and rats. These results indicated that in both species, the largest diameter (greater than or equal to 4 microns) axons are housed in the ventral columns of the cervical spinal cord, suggesting that axon length and diameter may not be the only criteria underlying fiber tract vulnerability in OPIDN.