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Biomedical subjects

D G Graham

Publications and source records attributed to D G Graham.

124 records · Page 7Linked to original sources
PubMedDate not supplied

Heinz body production and hematological changes in the hen after administration of a single oral dose of n-butyl mercaptan and n-butyl disulfide.

n-Butyl mercaptan (nBM) is a breakdown product of S,S,S,-tri-n-butyl phosphorotrithioate (DEF) and S,S,S-tri-n-butyl phosphorotrithioite (merphos) in hens and in the environment. n-Butyl disulfide (nBD) is an oxidation product of nBM. A single 500 mg/kg dose of nBM and nBD was administered in gelatin capsules to groups of five 12-month old laying hens. A third group (five hens) was given gelatin capsules. One day after administration, the hens exhibited weakness which progressed to unsteadiness and inability to stand by the third day. These signs were accompanied by a pale comb 18--24 hr after dosing, which changed to dark color at 48 hr. Treated hens improved with time. Heinz bodies and extensive erythrocyte deformation and lysis were observed in blood smears taken from hens 24 and 48 hr after treatment. Hemoglobin concentration, packed cell volume, erythrocytes, and glucose-6-phosphate dehydrogenase activity were significantly lower than controls, while methemoglobin was significantly higher. As the clinical condition of these hens improved, these hematologic changes disappeared. nBM caused an initial increase in plasma butyrylcholinesterase activity which was dose-dependent and returned to normal by the end of the 28-day experiment. Also, brain acetylcholinesterase activity was not different from that of the control at termination.

Animals↗
PubMedDate not supplied

Pyrrole oxidation and protein cross-linking as necessary steps in the development of gamma-diketone neuropathy.

It has been well documented that the gamma-diketone HD1 is the ultimate toxic metabolite of n-hexane. Furthermore, it has been shown that the pathogenetic mechanism by which HD exerts its neurotoxic effects is through binding to protein lysly residues and cyclization to pyrroles. The present study sought to determine whether the presence of pyrrole residues on NF1 proteins is sufficient to cause the NF-filled axonal swellings associated with n-hexane and other gamma-diketone neuropathies or whether pyrrole oxidation and protein cross-linking also have to occur in order for neurotoxicity to develop. We synthesized the HD analogue AcHD1 and assessed its rate of pyrrole formation in vitro, the ease of oxidation of its resulting pyrroles, and its ability to cross-link proteins in vitro. The in vivo effects of AcHD on rats were examined following daily ip1 injections. AcHD was found to have a rate of pyrrole formation comparable to that of the potent HD analogue DMHD1 at 35 degrees C. The pyrrole derived from AcHD was more resistant to oxidation than that derived from the neurotoxic compound HD. AcHD did not cross-link proteins in vitro. Pyrrole derivatives were demonstrated on hemoglobin isolated from animals treated with HD, DMHD, and AcHD. Cross-linked spectrin was detected in animals treated with HD and DMHD but not with AcHD. Rats receiving 0.1 or 0.25 mmol of AcHD/kg/day did not reach the end point of hindlimb paralysis observed in the gamma-diketone neuropathies, and the NF-filled axonal swellings seen following exposure to the neurotoxic gamma-diketones were not observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Covalent cross-linking of proteins by carbon disulfide.

Carbon disulfide is known to react with amino groups of proteins to generate dithiocarbamates (2). We observed covalent cross-linking of dithiocarbamate-derivatized proteins under physiological conditions which may occur through several mechanisms. Evidence for the structure of these covalent bridges and the reactive intermediate was obtained using 13C NMR spectroscopy in conjunction with specific isotopic labeling. On incubation at 37 degrees C oxidative coupling of dithiocarbamates generated bis(thiocarbamoyl) disulfides (3) which were reduced by cysteine. In addition, an electrophilic isothiocyanate (4) was generated from decomposition of the dithiocarbamate. Nucleophilic addition of sulfhydryl and amine moieties to the isothiocyanate produced dithiocarbamate ester (5) and thiourea linkages (6), respectively. Evidence for the presence of inter- and intramolecular cross-links was obtained using denaturing polyacrylamide gel electrophoresis under reducing conditions. The formation of isothiocyanate in neutral solution, through elimination of sulfhydryl ion, was correlated with increased pKa values of the parent amine of amino acids. Dithiocarbamates derived from terminal amino groups of proteins did not appear to generate isothiocyanate or form thiourea or dithiocarbamate ester. Both the thiourea and the dithiocarbamate ester were stable at reduced pH, whereas in alkaline media the thiourea was stable but dithiocarbamate ester was hydrolyzed. Although the disulfide and ester linkages were formed more rapidly than the thiourea, generation of the latter appeared to be irreversible, leading to its gradual accumulation over a longer period of time. Generation of isothiocyanate by CS2-derived dithiocarbamates and subsequent covalent cross-linking of proteins may provide a molecular mechanism for CS2-induced axonopathy.

Amino Acids↗
PubMedDate not supplied

The mechanism of nucleophilic substitution of alkylpyrroles in the presence of oxygen.

The mechanism of oxidation of alkylpyrroles (1a-d) by molecular oxygen in the presence of nucleophiles is explored. Contrary to previous reports, oxidation of these pyrroles resulted in dimers with both the aromatic rings intact. In the presence of additional nucleophiles these pyrroles entered into substitution reactions. With 2-mercaptoethanol the site of substitution on 1a was the 3-position rather than the side chain. The first-order rate constant for this reaction in acetonitrile with excess oxygen was found to be (7.8 +/- 1.2) x 10(-7) s-1. The rate was unaffected by the presence of either BHT or catechol. Replacing hydrogens at all the potential sites of reaction by deuterium (as in 1aD) did not reduce the rate of substitution. However, the product suffered loss of deuterium from all sites. These observations support a mechanism involving the formation of a complex 20 between the pyrrole and triplet oxygen. Electron transfer from the pyrrole to oxygen in the rate-limiting step is followed by the generation of pyrrolylmethyl intermediate 23 that can react with available nucleophiles including unoxidized pyrroles.

Butylamines↗
PubMedDate not supplied

Carbon disulfide mediated protein cross-linking by N,N-diethyldithiocarbamate.

N,N-Diethyldithiocarbamate and its disulfide are used as pesticides, in industrial processes, and as therapeutic agents, providing numerous opportunities for human exposure. Animal studies and in vitro investigations have demonstrated adverse effects following exposure to dithiocarbamates. The ability of dithiocarbamates to decompose to parent amine and CS2 suggests that these adverse effects may be mediated through release of CS2. The toxicity of CS2 is well established, and covalent cross-linking of proteins has been presented as a potential molecular mechanism of CS2 induced neuropathy. In the present investigation the ability of N,N-diethyldithiocarbamate to effect covalent cross-linking of proteins under physiological conditions is examined. Using 13C NMR, cross-linking was observed to proceed through dithiocarbamate formation on protein amino groups followed by the production of bis(thiocarbamoyl) disulfide, dithiocarbamate ester, and thiourea cross-linking structures. The presence of bis(lysyl) thiourea cross-linking structures was verified by complete protein hydrolysis in conjunction with GC/MS. Generation of inter- and intramolecular cross-linking was established using denaturing polyacrylamide gel electrophoresis under reducing conditions and revealed that cross-linking proceeded more rapidly for N,N-diethyldithiocarbamate than for equimolar CS2 under similar conditions. Covalent cross-linking of solubilized neurofilament triplet proteins, the putative neurotoxic targets, was examined. Both N,N-diethyldithiocarbamate and CS2 were able to covalently cross-link the low molecular weight component of the neurofilament triplet proteins, but neither produced intermolecular cross-linking of the medium or high molecular weight component. These results establish that N,N-diethyldithiocarbamate promoted protein cross-linking occurs under physiological conditions and proceeds through liberation of CS2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

The measurement of 2-thiothiazolidine-4-carboxylic acid as an index of the in vivo release of CS2 by dithiocarbamates.

Dithiocarbamates and their disulfides are used extensively as agricultural fungicides, as accelerators of the vulcanization process of rubber in industry, and as therapeutic agents in medicine. The widespread uses of these compounds in agriculture, industry, and medicine provide many avenues of exposure to the human population. Subchronic to chronic exposures to some dithiocarbamates have resulted in the development of neuropathy in humans and experimental animals. Decomposition to CS2 presents a potential mechanism through which the toxicity of dithiocarbamates may be mediated. The purpose of this study was to determine the potential of dithiocarbamates to release CS2 in vivo. The ability to release CS2 was assessed by measuring urinary 2-thiothiazolidine-4-carboxylic acid (TTCA), which is used in industry to measure the exposure of workers to CS2. In this study, rats were housed individually in metabolic cages and given daily equimolar ip or po doses (1.5 mmol/kg) of N,N-diethyldithio-carbamate (DEDC), disulfiram (DS), N-methyldithiocarbamate (NMDC), or CS2 for 5 days, and TTCA was measured in urine collected at 24 h intervals. For each compound administered, TTCA was produced in all of the treated animals and the amount of TTCA eliminated in urine from po administration was significantly greater than that from ip administration. The relative rates of TTCA elimination in urine were DS > DEDC approximately equal to CS2 > NMDC for both routes of administration. Following administration of N,N-diethyl[13C = S] dithiocarbamate, carbon-13 enrichment at the thiocarbonyl carbon of TTCA was demonstrated using 13C NMR. Analysis of urinary TTCA proved to be useful both for establishing the in vivo release of CS2 by dithiocarbamate containing compounds and for evaluating the bioavailability of CS2. The results appear especially relevant to disulfiram, which is given orally for sustained periods in the treatment of alcoholism and has resulted in the development of neuropathy in susceptible individuals.

Administration, Oral↗
PubMedDate not supplied

Dopamine mercapturate can augment dopaminergic neurodegeneration.

Pathological and biochemical studies have consistently associated endogenous catechol oxidation with dopaminergic neurodegeneration in Parkinson's disease (PD). Recently, it has been proposed that products of catechol oxidation, the catechol thioethers, may contribute to dopaminergic neurodegeneration. In other organ systems, thioether cytotoxicity is influenced profoundly by the mercapturic acid pathway. We have pursued the hypothesis that endogenous catechol thioethers produced in the mercapturic acid pathway contribute to dopaminergic neurodegeneration. Our results showed that the extent of in vitro metal-catalyzed oxidative damage by catechol thioethers varied with the structures of the parent catechol and thioether adduct. Catechol mercapturates uniquely produced more oxidative damage than their parent catechols. In dopaminergic cell cultures, dopamine induced apoptosis in a concentration-dependent manner from 5 to 50 microM. The apoptotic effect of dopamine was greatly enhanced by subcytotoxic concentrations of the mitochondrial inhibitor, N-methyl-4-phenylpyridinium (MPP+). Similarly, subcytotoxic levels of the mercapturate or homocysteine conjugate of dopamine significantly augmented dopamine-induced apoptosis. Finally, microsomal fractions of substantia nigra from PD patients or age-matched controls had comparable cysteine-S-conjugate N-acetyltransferase activity. These data indicate that the mercapturate conjugate of dopamine may augment dopaminergic neurodegeneration and that the mercapturate pathway exists in human substantia nigra.

Acetylcysteine↗
PubMedDate not supplied

Characterization of carbon disulfide neurotoxicity in C57BL6 mice: behavioral, morphologic, and molecular effects.

Female C57BL6 mice were exposed to 0 or 800 ppm carbon disulfide (CS2), 6 h/d, 5 d/wk for 20 weeks. The neurologic function of all mice was assessed once at the end of exposures using a functional observational battery. General health effects included a decrease in body weight gain, piloerection, hunched body posture, and ptosis. Treatment-related effects included altered gait (uncoordinated placement of hind limbs and ataxia) and impaired function on an inverted screen test. In addition, rearing and locomotor movement were decreased in treated mice. Focal to multifocal axonal swelling was seen predominantly in the muscular branch of the posterior tibial nerve, and occasionally giant axonal swelling was detected in the lumbar segment of the spinal cord. Electron microscopic examination revealed swollen axons with massive accumulation of neurofilament proteins within the axoplasm. Covalent cross-linking of erythrocyte spectrin (surrogate protein to neurofilament protein) was demonstrated in mice exposed to CS2 but not in mice receiving filtered air. These data provide supportive evidence that covalent cross-linking of neurofilament proteins is a significant feature of the axonal swellings in mice produced by inhalation exposure to CS2.

Administration, Inhalation↗
PubMedDate not supplied

Cross-species extrapolation in hydrocarbon neuropathy.

Not all species demonstrate the same vulnerability to hydrocarbon neuropathy. These differences are related to axonal length and diameter as well as to variations in toxicokinetics. While cross-species extrapolation is made difficult by these factors, understanding the basis for the differences provides insight into pathogenesis.

Animals↗
PubMedDate not supplied

In vitro and in vivo studies of the molecular pathogenesis of n-Hexane neuropathy.

In vivo intoxication of rats with 2,5-hexanedione and 3,4-dimethyl-2,5-hexanedione, which results in axonal swellings filled with neurofilaments, has been combined with in vitro exposure of proteins and model amines to the gamma-diketones in attempts to explain the molecular pathogenesis of n-hexane neuropathy and related neurofilament neuropathies. The heretofore untested gamma-diketone, 3,4-dimethyl-2,5-hexanedione is proposed as a "missing link" between beta, beta'-iminodipropionitrile, which produces proximal aggregates of neurofilaments, and 2,5-hexanedione, acrylamide, and carbon disulfide, which result in neurofilament-filled swellings in the distal axon.

Animals↗
PubMedDate not supplied

Delayed neurotoxicity induced by organophosphorus compounds in the wild mallard duckling: effect of leptophos.

Feeding of 260 ppm of leptophos to mallard ducklings caused delayed neurotoxicity similar to that reported for hens. Thus leptophos caused ataxia, with subsequent paralysis, loss of appetite, and slow-down in the growth rate of the treated birds. Spinal lesions were identical in morphology and distribution to those seen in hens following leptophos administration. The severity of histologic changes correlated both with the clinical condition and the duration of intoxication.

Animals↗