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Biomedical subjects

D G Graham

Publications and source records attributed to D G Graham.

At least 37 records · Page 2Linked to original sources
PubMed1998

Covalent modification of hemoglobin by carbon disulfide: III. A potential biomarker of effect.

Although the neurotoxicity of CS2 has been recognized for over a century, presently there is no accepted biomarker of effect for CS2 exposure. Previous investigations have supported covalent cross-linking of erythrocyte spectrin as a potential preneurotoxic marker reflective of the biochemical changes occurring within the axon. In the present investigation, the potential of using CS2 promoted modification of hemoglobin as a dosimeter for quantifying exposure to CS2 was evaluated. Liquid chromatography was used to isolate and measure alpha and beta chains of globin in blood obtained from rats exposed to CS2 by inhalation as a function of exposure level and duration. The degree of globin modification was compared to light microscopic and ultrastructural changes in the central and peripheral nervous systems to determine the temporal relationship of globin modification to the structural changes in the axon. Samples obtained from rats exposed to CS2 contained a globin chain not present in control samples. Analysis of the peak corresponding to the new chain using electrospray mass spectrometry was consistent with the generation of a single dithiocarbamate ester or thiourea intramolecular cross-link in the alpha 1 major chain. This altered globin chain was detectable both at the subneurotoxic level of exposure and prior to axonal structural changes at the neurotoxic levels of exposure used. The extent of modification was positively correlated to the exposure level and duration for all conditions examined. These findings support hemoglobin as a potential preneurotoxic biomarker of effect for CS2 possessing several practical advantages relative to the use of CS2-mediated spectrin cross-linking.

Administration, Inhalation↗
PubMed1998

Carbon disulfide neurotoxicity in rats: V. Morphology of axonal swelling in the muscular branch of the posterior tibial nerve and spinal cord.

The study objectives were to examine the morphological progression and dose response of carbon disulfide (CS2) distal axonopathy in the muscular branch of the posterior tibial nerve (MBPTN) and spinal cord. Male and female F344 rats were exposed to 0, 50, 500 or 800 ppm CS2 by inhalation, 6 hours/day, 5 days per week, for 2, 4, 8 or 13 weeks. At 8 weeks, in the MBPTN, single fascicles contained individual swollen axons. By 13 weeks, multiple fascicles had giant swollen axons with thin myelin sheaths and occasional degenerated and regenerated axons. At 8 weeks, in the spinal cord, white matter changes in cervical segments 1 and 2 consisted of prominent multifocal axonal swelling in the fasciculus gracilis nerve tracts. In lumbar segments 1 and 2, multifocal axonal swelling was first present at 8 weeks in the lateral and ventro-medial funiculus. By 13 weeks, axonal swelling was diffuse in the fasciculus gracilis nerve tracts of the cervical spinal cord and the lateral and ventral funiculus nerve tracts in the lumbar spinal cord. Compared to the spinal cord, where axonal swelling was present in rats exposed to 800 and 500 ppm, in the muscular branch of the posterior tibial nerve, axonal swelling was only present at 800 ppm at both 8 and 13 weeks. Electron microscopic examination demonstrated marked accumulations of neurofilaments in swollen axons in the spinal cord and MBPTN. Axonal swelling was not present in the spinal cord at 50 ppm, or in the MBPT at 50 and 500 ppm. Axonal swelling was not present at earlier time points of 2 and 4 weeks in either the spinal cord or MBPTN.

Administration, Inhalation↗
PubMed1997

Neurotoxicity of endogenous cysteinylcatechols.

Progression of Parkinson's disease has been associated with several biochemical changes in the substantia nigra including increased oxidative challenge, catechol oxidation, and inhibition of mitochondrial complex I activity. Cysteinylcatechols, formed by nucleophilic addition of cysteine to oxidized catechols, have been identified as markers of catechol oxidation in brain tissue. We have examined the neurotoxicity of a series of cysteinylcatechols. Of the compounds examined, only 5-S-cysteinyl-3,4-dihydroxyphenylacetate (cysdopac) was specifically cytotoxic to differentiated P19 neuroglial cultures. Cysdopac also was neurotoxic to pyramidal neurons in organotypic cultures of hippocampus, and this effect was ablated by selective N-methyl-D-aspartate (NMDA) receptor antagonists. In vitro, cysdopac was a potent inhibitor of mitochondrial complex I activity. However, electrophysiologic experiments failed to demonstrate NMDA receptor agonist activity for cysdopac, nor did cysdopac inhibit glutamate uptake. These results showed that cysdopac was the most potent neurotoxin of this series of cysteinylcatechols and suggest that cysdopac may function as an indirect excitotoxin, potentially via inhibition of mitochondrial respiration.

Animals↗
PubMed1997

CS2-mediated cross-linking of erythrocyte spectrin and neurofilament protein: dose response and temporal relationship to the formation of axonal swellings.

Using model proteins, a mechanism for CS2-mediated covalent cross-linking of proteins has been demonstrated previously. The biologic importance of CS2-promoted protein cross-linking is apparent as a possible dosimeter of CS2 exposure and as a potential mechanism to account for the identical neuropathies produced by 2,5-hexanedione and CS2. The present investigation examines the utility of erythrocyte spectrin cross-linking as a biomarker of effect for inhalation exposure to CS2 and examines the ability of CS2 to cross-link neurofilament proteins, a potential neurotoxic target. Rats were exposed to CS2 via inhalation at control, 50-, 500-, and 800-ppm levels for 2, 4, 8, and 13 weeks and spectrin dimer formation was quantified using denaturing gel electrophoresis and densitometry. Neurofilament preparations were also obtained from spinal cords and examined for cross-linking using Western blotting methods. The results obtained for protein cross-linking were compared to morphologic changes in the cervical and lumbar spinal cord using light and electron microscopy. The spectrin dimer exhibited a cumulative dose response and was detectable at both the 50-ppm level employed that did not produce axonal swellings and prior to the development of axonal swellings for the 500- and 800-ppm levels used. Neurofilament protein cross-linking involved all three subunits and the temporal relationship of cross-linking was consistent with a contributing role in the development of axonal swellings. These results establish the sensitivity of spectrin cross-linking for evaluating inhalation exposures and extend the similarities observed for 2,5-hexanedione and CS2 in both clinical settings and in vitro models to their effects exerted on neurofilaments in the axon.

Administration, Inhalation↗
PubMed1997

Immunohistochemical detection of 4-hydroxy-2-nonenal adducts in Alzheimer's disease is associated with inheritance of APOE4.

Cumulative oxidative damage, including lipid peroxidation, is a central component of cellular aging and is thought to play a role in the pathogenesis of late-onset Alzheimer's disease (AD). Lipid peroxidation produces several cytotoxic aldehydes, one of the most potent being 4-hydroxy-2-nonenal (HNE). We have shown previously that HNE is a potent neurotoxin that covalently modifies and cross-links neuronal cytoskeletal protein in neuroglial cultures, suggesting that HNE may contribute to the pathogenesis of AD. In addition to aging, inheritance of the epsilon 4 allele of APOE is the other major risk factor for development of late-onset AD; however, the mechanisms through which aging and apolipoprotein E isoforms may collaborate in the onset or progression of AD are not known. We tested the hypothesis that HNE may yield a particular type of protein modification, pyrrole adduction, and that this may contribute to the pathogenesis of AD. Our data demonstrated that HNE formed pyrrole adducts with protein. Polyclonal antiserum was raised that specifically recognized HNE pyrrole adducts, and immunohistochemical analysis was performed on hippocampus and temporal cortex of 10 patients with histologically verified AD. Pyramidal neuron cytoplasm was immunoreactive in 4 of 4 APOE4 homozygotes, 2 of 3 APOE3/4 heterozygotes, and none of 3 APOE3 homozygotes (P < 0.05). The pattern of staining was highly suggestive of neurofibrillary tangles as the primary immunoreactive structure. These data suggest that differences in neuronal protein modification by HNE may account in part for the APOE-associated stratification of risk for late-onset AD.

Aged↗
PubMed1996

Crosslinking of apolipoprotein E by products of lipid peroxidation.

Apolipoprotein E (APOE) genotype and advancing aging are interacting ri sk factors in the expression of late onset and sporadic Alzheimer's Disease (AD). We tested the hypothesis that 2 products of lipid peroxidation, malondialdehyde (MDA) and 4 hydroxy-2-nonenal (HNE), covalently modify APOE and alter its metabolism. In vitro, both HNE and MDA crosslinked purified APOE3 and APOE4. HNE was a more potent crosslinker than MDA, and purified APO3 was more susceptible to crosslinking by HNE than was purified APOE4. In P19 neuroglial cultures, oxidative stress with lipid peroxidation led to increased intracellular accumulation of anti-HNE and anti-APOE immunoreactive proteins of approximately 50 kDa. Intercellular accumulation of the 50 kDa APOE-immunoreactive protein (APOE-50) was not prevented by cyclohexamide, suggesting formation by post-translational mechanisms. In CSF, a 50 kDa APOE-immunoreactive protein co-migrated with proteins most immunoreactive for HNE and MDA adducts, containing NaB3H4-reducible bonds. These proteins were in CSF from adult subjects (with or without dementia), and in AD patients homozygous for APOE3 or APOE4 alleles. These data suggest that HNE covalently crosslinks APOE in P19 neuroglial cultures to form a 50 kDa protein, and that similar modifications of APOE appear to occur in vivo.

Adult↗
PubMed1996

E-4-hydroxy-2-nonenal is cytotoxic and cross-links cytoskeletal proteins in P19 neuroglial cultures.

Lipid peroxidation increases with age in brain and is elevated further in Alzheimer's disease. E-4-hydroxy-2-nonenal and malondialdehyde are products of lipid peroxidation that can adduct and cross-link protein. Neurofibrillary tangles, a feature of Alzheimer's disease composed mostly of tau protein, contain cross-linked and ubiquitin-conjugated protein. In P19 neuroglial cultures, E-4-hydroxy-2-nonenal was a potent cytotoxin that cross-linked cytoskeletal proteins, including tau into high molecular weight species that were conjugated with ubiquitin. Malondialdehyde formed monoadducts with cell protein but did not cross-link and was not cytotoxic. A non-crosslinking analogue of E-4-hydroxy-2-nonenal was not cytotoxic. E-4-Hydroxy-2-nonenal may contribute to neurodegeneration and neurofibrillary tangle formation in Alzheimer's disease.

3,4-Methylenedioxyamphetamine↗
PubMed1995

In vitro acrylamide exposure alters growth cone morphology.

Acrylamide intoxication leads to degeneration of the longest axons of the central and peripheral nervous systems in humans and laboratory animals. Axonal derangements resulting from in vivo acrylamide exposure are first noted within synapses of the longest axons before involving more proximally located axonal segments or shorter axons, thus illustrating the specificity of acrylamide for the terminal axonal regions. As a possible model system for investigating the mechanism of toxicity of acrylamide on the distal axon, we exposed neurite-extending chick dorsal root ganglion (DRG) cells to acrylamide in vitro and then examined growth cones for alterations in morphology and function. Exposing DRG explants to media containing from 0.125 to 1.0 mM acrylamide for 16 hr leads to specific and dose-responsive alterations of growth cone morphology including: a nearly total loss of filopodial elements, the preservation of highly active but two-dimensional lamellar structures, an inappropriate extension of the axonal cytoskeleton into the forward region of most growth cones, and a frequent breakdown of the central and peripheral growth cone domains. The sulfhydryl alkylating agents ethacrynic acid, iodoacetamide, and iodoacetic acid were tested and none produced acrylamide-like morphological alterations at any dose. DRG cultures were also exposed to the neurotoxic acrylamide analogs glycidamide, N-hydroxy-methacrylamide (HM-ACR), and methacrylamide (M-ACR). At concentrations of 0.25 to 1.0 mM, glycidamide exposure resulted in acrylamide-like growth cone alterations. HM-ACR exposure also resulted in growth cones that were acrylamide-like but only at concentrations > 1.5 mM. M-ACR did not produce acrylamide-like growth cones at doses of up to 16.6 mM. Thus, in vitro exposure of DRG explants to acrylamide and two neurotoxic acrylamide analogs leads to reproducible and specific morphological alterations that are dose-dependent and separable from the effects of sulfhydryl alkylation.

Acrylamide↗
PubMed1995

The effect of acrylamide and other sulfhydryl alkylators on the ability of dynein and kinesin to translocate microtubules in vitro.

Chronic exposure to acrylamide leads to a dying-back axonopathy afflicting the longest axons of all tested mammalian and avian species. Prior to the onset of acrylamide-induced axonal degeneration, alterations in axonal fast transport have been consistently reported to be more severe for the retrograde than the anterograde direction. The putative retrograde motor protein, dynein, is compromised by exposure to the sulfhydryl-alkylating agent N-ethylmaleimide (NEM) at concentrations far below those required to inactivate kinesin, the putative anterograde motor protein. Since acrylamide is capable of alkylating protein sulfhydryl moieties, we tested whether a direct exposure of purified kinesin or dynein to acrylamide would result in an impairment of either enzyme's ability to translocate microtubules. Motor activity was assayed by sequentially adsorbing either kinesin or dynein to acid-washed coverslips, treating with an alkylating agent or control solution, adding microtubules and ATP, and finally imaging and quantifying the binding and gliding of microtubules using video-enhanced differential interference contrast (VE-DIC) microscopy. In comparison to controls, incubation of dynein with NEM, ethacrynic acid, or iodoacetic acid resulted in dose-dependent decreases in the amount and rate of microtubule gliding, but increases in irreversible high-affinity microtubule binding. In contrast, exposure of dynein to 1-100 mM solutions of acrylamide did not significantly alter either the binding or gliding of microtubules (a molar/hour exposure to acrylamide equivalent to 50 times that which causes retrograde transport deficits in vivo). Likewise, kinesin motility parameters were not significantly affected by acrylamide concentrations up to 100 mM while NEM solutions > 100 microM led to significant losses in the ability of kinesin to bind MT. These data indicate that acrylamide does not significantly interact with bound (adsorbed) kinesin or dynein, implying that the mechanism by which acrylamide interferes with fast axonal transport in vivo is by interaction with other factor(s) that govern the movement of vesicles.

Acrylamide↗
PubMed1995

Dialkyldithiocarbamates inhibit tyrosine hydroxylase activity in PC12 cells and in fibroblasts that express tyrosine hydroxylase.

Dithiocarbamates and CS2 have been associated with neurobehavioural changes suggestive of central dopaminergic dysfunction. Diethyldithiocarbamate (DEDC), dimethyldithiocarbamate (DMDC), and methyldithiocarbamate (MDC) were examined for their ability to inhibit tyrosine hydroxylase (TH) activity in PC12 cells and transfected CHO fibroblasts that expressed TH (CHO/TH) activity when tetrahydrobiopterin (BH4) was added to medium. DEDC or DMDC did not significantly alter viability of PC12 cells or CHO/TH cells at < or = 100 microM for 18 h; the EC50 for each compound was approximately 5 mM in both cell lines. In contrast, the EC50 for MDC was 41 or 74 microM in PC12 or CHO/TH cultures, respectively. There was no change in immunodetectable levels of TH in PC12 or CHO/TH cells following exposure to subcytotoxic concentrations of dithiocarbamates. DEDC and DMDC (5 to 100 microM) produced concentration-dependent reductions in PC12 cell dopamine and dopac levels as well as in dopa levels in CHO/TH cultures. Reduction of PC12 catechols was not due to altered vesicular storage. In vitro PC12 TH activity was 80.2 +/- 3.4% or 82.4 +/- 2.9% of control following exposure to 100 microM DEDC or DMDC, respectively, and was not fully restored by incubation with Fe2+. These results show that DEDC and DMDC, but not MDC, are low potency cytotoxins that decrease TH activity in cultured cells through mechanisms other than inhibition of BH4 biosynthesis or iron chelation.

Analysis of Variance↗
PubMed1995

Intermediates in the Paal-Knorr synthesis of pyrroles. 4-Oxoaldehydes.

The mechanism of pyrrole formation between a 4-ketoaldehyde, such as 4-oxohexanal (4), and a primary amine is examined. In organic solvents 4 readily formed the imine 6 that decomposed to pyrrole 9. In phosphate buffer (pH 7.4) the presence of deuteriums in the dideuterio (4-d2) and hexadeuterio (4-d6) analogs retarded the reaction rate by factors of 1.9 and 2.6, which are much less than the isotope effects exhibited by reactions involving cleavage of the carbon-hydrogen bond. Moreover, the deuterium labels from the uncyclized ketoaldehyde remained intact. These results suggest that the hemiaminal 5 rather than the enamine 8 is the intermediate undergoing cyclization. Due to the absence of a methyl substituent at one of the carbonyls the rate of pyrrole formation of 4-oxohexanal was 2 orders of magnitude larger than that of 2,5-hexanedione. The higher rate of pyrrole formation may account for the increased rate of pyrrole-mediated cross-linking of proteins caused by gamma-ketoaldehydes relative to gamma-diketones.

Aldehydes↗
PubMed1995

Characterization of protein adducts produced by N-methyldithiocarbamate and N-methyldithiocarbamate esters.

The toxicity of N-methyldithiocarbamate may be mediated through decomposition to more biologically active compounds. Two principal products, CS2 and methyl isothiocyanate, have the potential to interact covalently with macromolecules in biological systems. In this investigation the ability of N-methyldithiocarbamate to generate methyl isothiocyanate and CS2 under physiological conditions resulting in acylation and covalent cross-linking of proteins was examined using 13C NMR and GC/MS. Two N-methyldithiocarbamate esters, S-methyl N-methyldithiocarbamate and (N-acetyl-S-methylthiocarbamoyl)cysteine, were also investigated to evaluate the acylating ability of sulfhydryl conjugates of N-methyldithiocarbamate. The predominant and most stable adduct produced by the free dithiocarbamate and its S-substituted esters was methylthiourea on epsilon-lysyl and N-terminal alpha-amino groups. Derivatization on N-terminal amino groups progressed more rapidly for the dithiocarbamate than for its mercapturate. Methylurea protein adducts were also produced by the dithiocarbamate and its esters, suggesting production of methyl isocyanate in the decomposition of N-methyldithiocarbamate. Covalent cross-linking of beta-lactoglobulin by N-methyldithiocarbamate resulting from its decomposition to CS2 was observed using denaturing polyacrylamide gel electrophoresis. These results demonstrate the ability of a monoalkyldithiocarbamate to acylate protein amino groups and effect covalent cross-linking. These processes represent molecular mechanisms that may contribute to the toxicity of this class of compounds.

Cross-Linking Reagents↗
PubMed1995

Covalent crosslinking of neurofilament proteins by oxidized catechols as a potential mechanism of Lewy body formation.

Brainstem Lewy bodies (LB) are neuronal inclusions that are closely related to Parkinson's disease (PD). The filamentous component of LB from patients with PD contains biochemically altered neurofilaments (NF). Herein we have tested the hypothesis that the oxidized products of catechols may covalently crosslink NF. Neurofilaments were incubated in the presence of oxidized L-dopa, dopamine, or dopac and then analyzed by SDS-PAGE and protein staining or immunoblotting with monoclonal antibodies specific for neurofilament subunit proteins. Oxidized catechols yielded the same pattern of NF protein crosslinking as known covalent crosslinking agents. Coincubation of NF and catechols with N alpha-acetyl-L-lysine (NAL) produced strong reactivity on immunoblots probed with a polyclonal antiserum specific for NAL crosslinked to protein (antiserum 1400/3). Crosslinking of NAL to model proteins by oxidized dopac was followed by antibody capture assays using antiserum 1400/3. Increasing immunoreactivity was observed for 0.01 to 1.0 mM dopac and was augmented by Cu2+, Fe2+, Fe3+, Mn2+, or Mn3+ up to 0.1 mM. These results show that the products of catechol oxidation can covalently crosslink neurofilaments, that the crosslinking mechanism can involve lysine, and that copper, iron, and manganese ions can accelerate catechol-mediated protein crosslinking.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1995

Pathogenetic studies of hexane and carbon disulfide neurotoxicity.

Two commonly employed solvents, n-hexane and carbon disulfide (CS2), although chemically dissimilar, result in identical neurofilament-filled swellings of the distal axon in both the central and peripheral nervous systems. Whereas CS2 is itself a neurotoxicant, hexane requires metabolism to the gamma-diketone, 2,5-hexanedione (HD). Both HD and CS2 react with protein amino functions to yield initial adducts (pyrrolyl or dithiocarbamate derivatives, respectively), which then undergo oxidation or decomposition to an electrophile (oxidized pyrrole ring or isothiocyanate), that then reacts with protein nucleophiles to result in protein cross-linking. It is postulated that progressive cross-linking of the stable neurofilament during its anterograde transport in the longest axons ultimately results in the accumulation of neurofilaments within axonal swellings. Reaction with additional targets appears to be responsible for the degeneration of the axon distal to the swellings.

Animals↗
PubMed1994

Metabolism of 3,3'-iminodipropionitrile and deuterium-substituted analogs: potential mechanisms of detoxification and activation.

3,3'-Iminodipropionitrile (IDPN), a neurotoxicant that causes an excitatory CNS syndrome and a proximal axonopathy, is metabolized to beta-aminopropionitrile (BAPN), cyanoacetic acid (CAA), and beta-alanine (beta-ala) in rats. None of these metabolites are neurotoxic, suggesting that they are products of detoxification. To investigate potential pathways of activation and detoxification, we treated groups of rats with saline, IDPN, or deuterium-substituted analogs, 2,2,2',2'-tetradeuterio-IDPN (2-d-IDPN) or 3,3,3',3'-tetradeuterio-IDPN (3-d-IDPN) at 3 mmol/kg/day for 3 days. beta-Alanine was isolated from urine by ion-exchange chromatography and analyzed by GC-MS and HPLC. The position of the deuterium labels on beta-ala indicates that beta-ala is derived by hydrolysis of IDPN and that the carboxylate of beta-ala is derived from the nitrile of IDPN. Excretion of CAA, measured by GC-MS, is markedly reduced in 3-d-IDPN-treated rats. Since 3-d-IDPN is more potent than equimolar doses of IDPN, the diminished excretion of CAA may be due to an isotopic effect in retarding the hydrolysis of IDPN to BAPN and CAA. The finding that 2-d-IDPN is less potent than IDPN suggests that activation of IDPN may occur near the 2-position. Although nitrile hydrolysis is established, the mechanism is not yet clear.

Acetates↗