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Biomedical subjects

D G Graham

Publications and source records attributed to D G Graham.

At least 55 records · Page 3Linked to original sources
PubMed1994

Fibroblasts that express aromatic amino acid decarboxylase have increased sensitivity to the synergistic cytotoxicity of L-dopa and manganese.

Manganism, a neurodegenerative disease that can follow chronic exposure to Mn, has been associated with lesions in the basal ganglia and depletion of dopamine and its metabolites in this brain region. Herein, we have tested the hypothesis that oxidation of catechols is a critical component of Mn-induced cytotoxicity. To eliminate confounding metabolic pathways, a nonneuronal cell line, Chinese hamster ovary (CHO) fibroblasts, was transfected with a cDNA for bovine aromatic amino acid decarboxylase, and a high expressing clone was isolated (CHO/AADC). Exposure of wild-type (CHO/WT) or CHO/AADC cultures to L-dopa (62 to 500 microM) resulted in intracellular accumulation of L-dopa or L-dopa and dopamine, respectively, that was concentration-dependent. Intracellular catechol levels in CHO/AADC cells were double those in CHO/WT cultures. No dopac was identified intra- or extracellularly. Addition of MnCl2 (125 to 500 microM) resulted in cytotoxicity that progressed with increasing concentrations of L-dopa or Mn. Neither L-dopa nor MnCl2 alone was toxic at these concentrations, and cytotoxicity was completely abrogated by substitution of L-tyrosine for L-dopa. Although CHO/AADC cultures were more sensitive than CHO/WT to L-dopa and Mn, this was completely accounted for by the differences in intracellular catechol levels between the two cell lines. Preformed melanin or dopac were low-potency cytotoxins only at high MnCl2 concentrations. These results indicate that Mn and intracellular L-dopa and dopamine, but not extracellular dopac or melanin, are potent synergistic cytotoxins.

Animals↗
PubMed1994

Environmental neurotoxic illness: research for prevention.

Recognition of the deleterious neurological effects of chemicals has evolved from anecdotal observation to studies of illness in persons exposed to high doses. Now, the more subtle effects of exposures to environmental neurotoxicants are being documented: reduction in intelligence, impairment in reasoning ability, shortening of attention span, and alteration of behavior. Substances to which millions of persons are exposed occupationally and in the general environment that can result in such deficits include lead, organophosphorus pesticides, certain chlorinated hydrocarbons, carbon disulfide, solvents, and mercury. The first step in the prevention of neurological impairments due to environmental exposures is to assess the toxicity of chemicals. Fewer than 10% of the 70,000 chemicals in commercial use have been evaluated for neurotoxicity. This knowledge gap needs to be narrowed by building on existing systems of toxicity testing. Concurrent with assessment of chemicals will be tiers of in vivo screening tests to measure functional and structural changes following exposures in vitro. Epidemiologic surveillance of populations at high risk will continue to inform on the ranking of suspect or known neurotoxicants. Research and researchers must become more sophisticated in the development and application of refined biologic markers so the findings can be used to detect absorption of toxicants and early neurological or neurobehavioral dysfunction before disability occurs and to protect human health and the environment.

Biomarkers↗
PubMed1994

Dimethylhexanedione impairs the movement of neurofilament protein subunits, NFM and NFL, in the optic system.

Exposure to the neurotoxic gamma-diketone 3,4-dimethyl-2,5-hexanedione (DMHD) leads to the accumulation of neurofilaments within the proximal axon and to an inhibition in the rate of anterograde transport of recently synthesized neurofilaments. These effects of DMHD are similar to those of the neurotoxic nitrile 3,3'-iminodipropionitrile (IDPN), which is also characterized by formation of neurofilamentous swellings within the proximal axon and an inhibition of transport, both of newly synthesized neurofilaments and those already in transit in more distal regions of the axon. Due to the similarities between these compounds, DMHD also might be expected to inhibit neurofilament transport in the distal axon. The objective of this study was to examine the effects of DMHD on the movement of labeled neurofilament proteins which were in transit at the time of intoxication. Proteins in the optic system, pulse labeled with 35S-methionine, underwent transport for two weeks prior to the start of intoxication. Neurofilament transport was assessed by SDS-PAGE fluorography and computerized densitometry. At two and five weeks in control animals, the peaks of NFL and NFM neurofilament subunits had broadened and flattened from their original proximal location and assumed a more uniform, proximodistal distribution (peak dispersion). In contrast, in DMHD-treated animals, the radiolabeled NFL and NFM remained near their position at the start of intoxication, retaining a peak of radiolabeled protein. A proportion of each of the subunits, however, had entered the distal axon during intoxication suggesting that a population of filaments may remain transport competent.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Decreased levels of the high molecular weight subunit of neurofilaments and accelerated neurofilament transport during the recovery phase of 2,5-hexanedione exposure.

The neurotoxicant 2,5-hexanedione (HD) causes the accumulation of neurofilaments in the distal axon and an acceleration of neurofilament transport proximal to the site of their accumulation. It has been proposed that the acceleration of transport is due to the direct reaction of HD with neurofilament proteins and, conversely, that this acceleration is a secondary response to the axon to injury. The objective of this study was to determine whether the response of axons to HD intoxication includes acceleration of neurofilament transport. Pulse labelling was used to analyze neurofilament transport in age-matched rats exposed to HD or PBS. The animals receiving HD were exposed either throughout the period of radiolabel transport, or prior to the pulse labeling of neurofilament proteins. If acceleration of the rate of neurofilament transport was due to the direct reaction of HD with proteins, then neurofilaments synthesized after the exposure period should travel at control rates, since these proteins would not have been exposed to the toxicant. After 28 days of transport, optic nerve proteins were examined using SDS-PAGE, fluorography, and computerized densitometry. In both HD-treated groups, neurofilament transport was accelerated relative to age-matched control animals. In addition, the amount of NFH was decreased relative to other neurofilament subunits. The combination of accelerated transport and a diminished proportion of NFH is similar to the observations of neurofilament axonal transport during growth and development. These observations suggest that this persistent, secondary effect is a reparative response to injury that recapitulates axonal growth and development.

Animals↗
PubMed1993

Strategies for the prevention of environmental neurotoxic illness.

Toxic chemicals in the environment can cause a wide range of neurological disease. High-dose exposures to environmental neurotoxicants have produced encephalopathy in children ingesting chips of lead-based paint, blindness in persons who ingested methanol, blindness and ataxia in persons who consumed organic mercury, spinal cord degeneration and peripheral neuropathy in persons exposed to tri-ortho-cresyl phosphate (TOCP), and Parkinsonism in persons exposed to MPTP or to manganese. Environmental neurotoxicants have also been shown to produce a wide range of subclinical neurotoxic effects, including reduction in intelligence, impairment in reasoning ability, shortening of attention span, and alternation of behavior. The first step in the prevention of environmental neurotoxicity is to test chemicals for their toxic potential. More than 70,000 chemicals are currently in commerce. However, except for pharmaceuticals, fewer than 10% of these chemicals have been tested for neurotoxicity. A logical approach to neurotoxicologic assessment of chemical substances will build on and extend currently available test systems. It will have a tiered structure. The first or screening tier will consist of tests to measure obvious structural and functional changes, often a functional observational battery. Subsequent levels of testing will be guided by the results of initial screening. Toxicologic testing must be supplemented by epidemiologic surveillance of populations exposed to known and suspect neurotoxicants. Screening programs in these populations designed to detect excessive absorption of a neurotoxic agent or subclinical neurological dysfunction can be useful in identifying affected individuals before severe disability occurs.

Biomarkers↗
PubMed1993

Covalent cross-linking of erythrocyte spectrin by carbon disulfide in vivo.

Covalent cross-linking of proteins by CS2 has been demonstrated in vitro and represents a potential mechanism for the toxicity of this compound. In the present investigation the ability of CS2 to cross-link proteins covalently in vivo is demonstrated using denaturing polyacrylamide gel electrophoresis. Intraperitoneal injection of CS2 in rats at 2 or 5 mmol/kg for 21 or 42 days produced several high-molecular-weight (approximately 410 kDa) proteins eluted from erythrocyte membranes which were not present in control animals. Limited proteolysis of the high-molecular-weight protein bands, monomeric alpha spectrin, and monomeric beta spectrin using endoproteinase glu-C, followed by peptide mapping on denaturing polyacrylamide gels, showed the high-molecular-weight proteins to be alpha,beta heterodimers. The production of multiple heterodimers exhibiting different distances of migration was consistent with the existence of several preferred sites for cross-linking. Evidence for the presence of dithiocarbamate ester and thiourea cross-linking structures in spectrin dimers was obtained using selective base hydrolysis. No spectrin dimer was detected in control animals, and dimer formation demonstrated a cumulative dose response in CS2-treated rats. The longevity of red blood cells, the cumulative dose response, and the stability of the cross-linking structures endows spectrin cross-linking with the potential to serve as a biomarker of chronic low-level exposures to CS2 and may provide a means to correlate pathological changes with existing methods of CS2 exposure monitoring. The ability of CS2 to covalently cross-link erythrocyte spectrin suggests that CS2 may also cross-link other proteins in vivo and supports covalent cross-linking of proteins as a possible molecular mechanism through which CS2 manifests toxicity. If so, then spectrin cross-linking may parallel cross-linking reactions in the axon and provide a sensitive, preneurotoxic biomarker of this molecular event.

Animals↗
PubMed1992

A seasonal eosinophilic dermatitis in cats.

Twenty cats from Nelson with distinctive crusting and erosive dermatitis of the nasal bridge and histological lesions of eosinophilic dermatitis, often with collagenolysis, were examined in 1990 and 1991. Four of the cats also had pinnal dermatitis and five had eruptive lesions on the chin. The condition was intermittent and seasonal, occurring in summer and autumn. It is probable that the lesions were caused by hypersensitivity to insect bites, as has been demonstrated in a similar clinicopathological syndrome recently recognised in Australia.

Journal Article↗
PubMed1992

The role of pyrrole formation in the alteration of neurofilament transport induced during exposure to 2,5-hexanedione.

Exposure to the gamma-diketone, 2,5-hexanedione (HD), results in the accumulation of neurofilaments within the distal axon and is associated with acceleration of neurofilament transport within the proximal axon. The epsilon-amino groups of lysyl residues react with HD forming pyrrole adducts, followed by pyrrole-mediated protein crosslinking. Both reaction steps have been proposed as mechanisms causing neurofilament accumulation and acceleration of transport. In order to assess the importance of these steps on neurofilament transport, we compared transport in the optic system of rats exposed to HD and 3-acetyl-2,5-hexanedione (AcHD), a non-toxic analog of HD which forms pyrroles but does not crosslink proteins. Control, HD-treated, and AcHD-treated rats received intraoptic injections of [35S]-methionine and were exposed to saline, HD, or AcHD by intraperitoneal injections before and during the period of neurofilament transport. Neurofilament triplet proteins in the optic nerve and tract were identified by polyacrylamide gel electrophoresis followed by fluorography. The rate of neurofilament transport was accelerated in HD-treated animals over that of controls. However, despite higher levels of protein-bound pyrroles in AcHD-treated animals, the rate of transport was indistinguishable from that of controls. These findings indicate that pyrrole formation alone is not sufficient to cause acceleration of neurofilament transport.

Animals↗
PubMed1992

Comparison of location, severity, and dose response of proximal axonal lesions induced by 3,3'-iminodipropionitrile and deuterium substituted analogs.

Administration of 3,3'-iminodipropionitrile (IDPN) to rats results in massive accumulation of tangled neurofilaments in the proximal axons of large neurons, such as in dorsal root ganglia (DRG) and ventral horns of the lumbar spinal cord (LSC). Clinically, rats develop hyperexcitability, circling, head bobbing, and retropulsion. The ultimate toxicant and the molecular mechanism are not known. In a study designed to explore potential activation and detoxification pathways, dose-related differences in location and severity of lesions were observed in rats treated with IDPN or deuterium substituted analogs, 2,2,2',2'-tetradeuterio-IDPN (2-d-IDPN) or 3,3,3',3'-tetradeuterio-IDPN (3-d-IDPN). The compounds or saline were administered intraperitoneally to three rats per group at dose levels of 3.0, 1.5, 1.0, and 0.0 mmole/kg/day for 3 days. One week after the initial dose, tissues from DRG and LSC were collected, prepared and evaluated histologically in zones extending from areas adjacent to the cell bodies, distally toward the DRG stalk or toward the lumbar spinal roots. In the low dose IDPN group, DRG and LSC lesions were most prominent in distal zones. As dosage increased, the lesions progressed in severity and in proximity to the cell bodies. At the high dose, lesions were prominent in all zones. The same general pattern occurred with both analogs, although 2-d-IDPN was less potent than IDPN and 3-d-IDPN was more potent than IDPN. The differences in potency from the secondary isotopic effect of deuterium suggest that the 3-position is important in detoxification while the 2-position is important in the bioactivation of IDPN.

Animals↗
PubMed1991

Membrane flow within the myelin sheath in IDPN neuropathy.

This report describes some aspects of beta,beta'-iminodipropionitrile (IDPN) neuropathy in rats as observed by ultrastructural methods and X-ray diffraction. Light microscopy shows gross swelling of the axons in proximal lumbar spinal roots 8 days after intraperitoneal injection of IDPN. Mean axon cross-sectional area and mean axon perimeter increased to 280% and 160% of their control values, respectively. At the same time, myelin membrane packing was not visibly disturbed. In addition, X-ray diffraction patterns, recorded under physiological conditions, demonstrate that the myelin lipid bilayer thickness and widths of the aqueous spaces between bilayers did not change. Related observations are made on posterior tibial nerve (PNS myelin) and ventral spinal cord (CNS myelin). The various observations together are interpreted in terms of a fluid myelin membrane. It is proposed that the myelin membrane flows during axon swelling even though normal membrane-membrane contacts are maintained within the sheath. Membrane flow and slippage between membranes are explained in terms of a molecular model of the myelin multilayer.

Animals↗
PubMed1989

Thirteen-week toxicity study of n-hexane in B6C3F1 mice after inhalation exposure.

B6C3F1 mice were exposed to n-hexane 6 h/day, 5 days/week for 13 weeks at concentrations of 0, 500, 1000, 4000, and 10,000 ppm and at 1000 ppm 22 h/day, 5 days/week for 13 weeks (1000C group). Toxicological endpoints assessed included clinical signs, body and organ weight changes, gross and histopathology, neuropathology, and a battery of neurobehavioral tests. All mice survived the treatment. Exposure-related effects of n-hexane included sneezing at 10,000 ppm and body weight gain depression at 1000C and 10,000 ppm. Histopathologic changes included mild inflammatory, erosive and regenerative lesions in the olfactory and respiratory epithelium of the nasal cavity at 1000C, 4000, and 10,000 ppm. The only neurobehavioral parameter affected was a decrease in locomotor activity in female mice at 1000C and 10,000 ppm. In teased fiber preparations of tibial nerve, paranodal axonal swellings were observed at 1000C or at 10,000 ppm, but not in the control groups. The severity of the peripheral nerve lesion was mild. These studies show that n-hexane has minimal toxicity to the nervous system and respiratory system of mice.

Administration, Inhalation↗
PubMed1989

Sural nerve biopsy in chronic inflammatory demyelinating polyradiculoneuropathy.

We compared histologic features of sural nerve biopsies in 14 patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) with those in other forms of neuropathy. In CIDP endoneurial pericapillary cellular infiltrates were found in 4 patients (29%), onion bulbs in 5 patients (36%), and predominant demyelination in 7 patients (50%). None of these abnormalities was specific, but cellular infiltrates and onion bulbs appear to be diagnostically useful when combined with clinical information. To detect macrophage infiltration of myelin, cell nuclei were counter-stained in 20 teased fiber preparations. Nine patients with CIDP had a significantly higher mean number of cells per centimeter of teased fiber than 11 patients with other neuropathies. Despite overlap, significant infiltration of myelin detected by this method suggests CIDP in an appropriate clinical setting.

Biopsy↗
PubMed1989

The Duke University program for integrating ethics and human values into medical education.

In its second year of development, this program blends cognitive and affective approaches to integrating ethics and human values into medical education. The core of this effort is the establishment of direct and continuing relationships between the four advisory deans and their medical student advisees through small groups that continue throughout the four years of medical school. Clinical correlation seminars, lecture/discussions, the humanities, clinical clerkships, and electives are components of this integration process. Both basic science and clinical faculty members have observed positive changes in the degree and depth of participation, discussion, and interest, as well as in the general attitudes of the students.

Administrative Personnel↗
PubMed1989

Neurofilament protein crosslinking in gamma-diketone neuropathy: in vitro and in vivo studies using the seaworm myxicola infundibulum.

Neurofilament (NF) protein crosslinking has been proposed as the ultimate pathogenetic mechanism underlying the neuropathies caused by the gamma-diketones 2,5-hexanedione (HD) and 3,4-dimethyl-2,5-hexanedione (DMHD). Mammalian models have been used to investigate this hypothesis, but alternative experimental models are needed. Myxicola infundibulum is a marine worm which is gaining popularity in neuroscience research because of its large syncytial axon. A model system using Myxicola has been developed to investigate NF crosslinking in worms exposed to neurotoxic agents whose putative mechanisms involve covalent crosslinking of NF proteins. In vitro studies using purified NF demonstrate that progressive alkylation of Myxicola NF with [2,5-14C]DMHD is accompanied by NF protein crosslinking. Rabbit anti-Myxicola NF antisera showed highly restricted activity for Myxicola axoplasm and NF and were employed for immunoblotting axoplasm from Myxicola treated in vivo with DMHD. A dramatic increase in high molecular weight material was demonstrated in the axoplasm of treated worms, as demonstrated by polyacrylamide gel electrophoresis, and the new high molecular weight bands stained with the anti-NF antisera, indicating the presence of anti-NF reactive material in the crosslinked protein. Further, there was progression of crosslinking after cessation of exposure in vivo, an observation which suggests oxidation of remaining pyrrolyl derivatives. These studies support previous observations which suggest that NF crosslinking is the molecular event which initiates NF aggregation in gamma-diketone neurotoxicity, and establish Myxicola infundibulum as a useful species in which to study certain neurotoxic compounds.

Alkylation↗
PubMed1987

Evidence that pyrrole formation is a pathogenetic step in gamma-diketone neuropathy.

Previous studies from this laboratory have demonstrated that the addition of methyl groups at the 3 and 4 positions of the 2,5-hexanedione (2,5-HD) molecule results both in more rapid pyrrole formation and in enhanced neurotoxicity. In order to define more clearly the relationship between rates of pyrrole formation and neurotoxicity, the dl and meso diastereomers of 3,4-dimethyl-2,5-hexanedione (DMHD), 3,4-diethyl-2,5-hexanedione (DEHD), and 3,4-diisopropyl-2,5-hexanedione (DiPHD) were synthesized and purified. The rates of pyrrole formation were compared with that of unsubstituted 2,5-HD, and rates of in vitro crosslinking were determined. Each of the compounds was administered to rats to determine relative neurotoxicity. Hindlimb paralysis was reached after a total administered dose of 1.6 mmol/kg of dl-DMHD, while 5.9 mmol/kg of meso-DMHD was required. Paralysis was not achieved with either diastereomer of DEHD or DiPHD, although both produced systemic toxicity. Histologic sections of spinal cords and anterior roots from rats treated with DMHD revealed large neurofilament-filled axonal swellings, while more distal sections contained axons undergoing Wallerian-type degeneration. Neither axonal swellings nor Wallerian-type degeneration were seen in sections from spinal cord or peripheral nerve of rats treated with DEHD or DiPHD. The rates of pyrrole formation were in the order dl-DMHD greater than meso-DMHD greater than 2,5-HD greater than dl-DEHD greater than meso-DEHD greater than dl-DiPHD greater than meso-DiPHD, while in vitro crosslinking rates were in the order dl-DMHD greater than meso-DMHD greater than dl-DEHD greater than meso-DEHD greater than 2,5-HD greater than dl-DiPHD greater than meso-DiPHD. Cyclic voltammetry showed that the autoxidation of pyrroles derived from DMHD, DEHD, and DiPHD occurred more readily than that derived from 2,5-HD. In addition, we report for the first time the segregation of axoplasmic organelles in animals treated with DMHD, providing further evidence that the neurofilamentous axonopathies caused by such compounds as beta,beta'-iminodipropionitrile (IDPN), 2,5-HD and CS2 share a common underlying mechanism. The strong correlations between rates of pyrrole formation, rates of in vitro crosslinking and relative neurotoxicity are seen as evidence that pyrrole formation is a step in the pathogenetic sequence of gamma-diketone neuropathy.

Animals↗