Intern
    Prof. Dr. G. Bringmann

    Spontaneous Alkaloid Formation in Man: Involvement in the Pathogenesis of Neurodegenerative Diseases?

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    1. Key Words:

    In vivo formation and medicinal relevance of endogenous alkaloids in man; exemplarily for chloral-derived b-carbolines (e.g., 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline, ‘TaClo’): synthesis, stereochemistry, trace analysis, metabolism, and disposition; pharmacodynamic and pharmacokinetic studies; investigations on the neuropharmacological potential of a new group of highly halogenated alkaloids with dopaminergic and serotonergic neurotoxicity; occurrence in humans and presumable involvement as a causative factor in the pathogenesis of neurodegenerative disorders (e.g., Parkinson’s disease).

    2. Graphical Abstract:

    Subtopic A: Possible Formation of Chloral-Derived Mammalian Alkaloids

    Figure 1: In vivo formation of 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo), which is structurally similar to the well-established dopaminergic neurotoxin MPTP, from the biogenic amine tryptamine (Ta) and the hypnotic agent chloral (Clo), by a Pictet-Spengler type cyclization. Furthermore, in the course of intoxication or addiction with the industrial solvent trichloroethylene (TRI), an enhanced concentration of chloral might also induce the formation of TaClo.

    Subtopic B: The Neurotoxicological Potential of TaClo

     

    Figure 2: Biochemical and pharmacological characteristics of TaClo and related compounds exhibited in vitro and in vivo.

    Subtopic C: Occurrence of TaClo in Human Blood after Intake of Chloral Hydrate

    Figure 3: Employing a sensitive HPLC-MS/MS analytical device: identification of the TaClo in human blood after intake of the hypnotic chloral hydrate. – The recorded HPLC-MS/MS chromatogram is illustrated by the reconstructed ion current (RIC), and the ion traces obtained from selected reaction monitoring (SRM) experiments. The molecular ion peaks of TaClo, m/z 289 (for [35Cl3]TaClo and m/z 291 (for its [37Cl35Cl2]isotopomer), were both monitored to undergo a retro-Diels-Alder fragmentation by loss of a CH2=NH portion (-29 u) as typical of a tetrahydropyrido ring system of tetrahydro--carbolines. Detection of the resulting fragments, m/z 260 and m/z 262, with the expected statistical chlorine isotopic intensities of 100:96 confirmed the identity of the TaClo molecule. The internal standard, [D4]TaClo, added to verify the presence of endogenously occurring TaClo by retention time analysis (tR = 6.8 min) and to quantify the TaClo content detected in blood, was monitored in SRM mode by scanning the loss of a CD2=NH moiety (-31 u).

    Subtopic D: Assignment of the Absolute Configurations of the Two TaClo Enantiomers by Quantum Chemical CD Calculations

    Figure 4: LC-UV and LC-CD chromatograms of the two TaClo enantiomers after separation on a chiral reversed phase (left); LC-CD spectra of (R)-TaClo and (S)-TaClo measured in stop-flow mode exhibiting opposite curves (center); attribution of the absolute configuration, as established by comparision of the experimental CD spectra with the CD spectra quantum chemically calculated for (R)- and (S)-TaClo.

    3. Brief Description:

    The β-carbolines comprise a group of tricyclic indole derivatives. Like other compounds, among them isoquinolines, agrochemicals (e.g., rotenone, paraquat), alkanes, or heavy metals, they have been considered as potential environmental inducers of neurodegenerative processes, due to their occurrence in mammalian organisms. 1-Trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo), which is structurally closely related to the well-established synthetic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is such a genuine neurotoxic agent for dopaminergic and serotonergic neurons [1]. TaClo is readily formed in the human organism by a Pictet-Spengler type condensation of the endogenously present biogenic amine tryptamine (Ta) and the non-natural aldehyde chloral (Clo). Its spontaneous formation can occur after application of the hypnotic chloral hydrate and upon exposition to the industrial solvent trichloroethylene (TRI), which is known to be metabolized to chloral (see Figure 1).
    TaClo has seriously to be taken into account as a causative or supportive agent for the development of idiopathic Parkinson's disease (see Figure 2), because of its ability to easily penetrate the blood-brain barrier [1,2], and to severely affect the striatal dopamine [1,3] and extracellular serotonin [4] metabolism. TaClo strongly interacts with enyzmes such as tyrosine hydroxylase (TH) related to dopamine biosynthesis and metabolism [5], and is capable to inhibit dopamine [3,6] and serotonin [7] uptake. Several in vitro studies revealed TaClo to be toxic to cultured neuroblastoma cells (SK-N-NH) [8], to the dopaminergic cell line IMR-32 [7], to neuronal-like clonal pheochromocytoma PC12 cells [9], to the serotonergic cell line JAR [7], to dopaminergic SH-SY5Y, and to non-dopaminergic murine Neuro2A neuroblastoma cell lines [10], most likely by passive diffusion through the cell membrane and not by a selective uptake system [7,10]. Exposure of TaClo to dopaminergic neurons in primary cell cultures from mice led to marked biochemical defects (e.g., inhibition of dopamine uptake, reduction of cell number and size) and to distinct morphological changes (e.g., swelling of dendrites, loss of axons) [6]. While the synthetic MPTP acutely produces an irreversible syndrome in rodents and primates similar to idiopathic Parkinson's disease, TaClo can trigger a slowly developing neurodegeneration in rats with a late onset of first parkinsonian-type symptoms. This is obvious from a distinctly diminished locomotion of the animals observed nine months after the end of a seven-week subchronic exposure to small doses of TaClo [1,11]. The mechanism of TaClo-induced cell death is ascribed to the strong inhibition of complex I (NADH dehydrogenase) and complex II (succinate dehydrogenase) of the mitochondrial respiratory chain [1,3,6], to the formation of reactive oxygen species such as hydroxyl radicals [4], and to DNA damaging [12] and apoptotic [8] processes.
    In blood samples obtained from elderly patients who had been treated orally with chloral hydrate for three days up to six months, TaClo was unambiguously identified in concentrations ranging from less than 1 to 35 ng/ml, by applying a sensitive GC/MS or LC-MS/MS device (see Figure 3) [13,14]. An even enhanced TaClo level of ca. 70 ng/g of clot was detected in a young epileptic, obviously as a consequence of a long-term daily intake of 1 g of chloral hydrate over a period of nearly five years [14]. The onset of Parkinson's disease in three chronically TRI-exposed persons showing a continuous release of TRI associated with the presence of TaClo on a ng-scale [15] gave further hints for the assumption that endogenously originating chloral-derived β-carbolines (such as TaClo), even though formed only on such a small scale, may chronically trigger progressive neurodegenerative lesions in brain that become manifest with age.
    In contrast to MPTP, the tetrahydro-β-carboline TaClo is a chiral compound (see Figure 4). Separation and stereochemical attribution of the two TaClo enantiomers was achieved by applying chromatography on a chiral phase HPLC column in hyphenation with circular dichroism (CD) spectroscopy (LC-CD coupling). Assignment of the absolute configuration of TaClo was achieved by quantum chemical CD calculations (see also 'Computational Chemistry') [16]. In a human clot sample, the two TaClo enantiomers were found in equal concentrations (i.e., as a racemate) corroborating a spontaneous, non-enzymatic formation of TaClo from biogenic tryptamine and therapeutically administered chloral. In urine samples of TaClo-treated rats, by contrast, the (S)-enantiomer was found to predominate, hinting at an enantiomer-differentiating metabolism of the compound [14].

    4. Selected Publications:

    [1] P. Riederer, P. Foley, G. Bringmann, D. Feineis, R. Brückner, M. Gerlach; Biochemical and pharmacological characterization of 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline: a biologically relevant neurotoxin? Eur. J. Pharmacol. 2002, 442, 1-16.
    [2] G. Bringmann, D. Feineis, R. Brückner, R. God, C. Grote, W. Wesemann; Synthesis of radiolabelled 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo), a neurotoxic chloral-derived mammalian alkaloid, and its biodistribution in rats. Eur. J. Pharm. Sci. 2006, 28, 412-422.
    [3] G. Bringmann, D. Feineis, R. Brückner, M. Blank, K. Peters, E.-M. Peters, H. Reichmann, B. Janetzky, C. Grote, H.-W. Clement, W. Wesemann; Bromal-derived tetrahydro-β-carbolines as neurotoxic agents: chemistry, impairment of the dopamine metabolism, and inhibitory effects on mitochondrial respiration. Bioorg. Med. Chem. 2000, 8, 1467-1478.
    [4] M. Gerlach, A.-Y. Xiao, C. Heim, J. Lan, R. God, D. Feineis, G. Bringmann, P. Riederer, K.-H. Sontag; 1-Trichloromethyl-1,2,3,4-tetrahydro-β-carboline increases extracellular serotonin and stimulates hydroxyl radical production in rats. Neurosci. Lett. 1998, 257, 17-20.
    [5] F. Riederer, A. Luborzewski, R. God, G. Bringmann, J. Scholz, D. Feineis, A. Moser; Modification of tyrosine hydroxylase activity by chloral-derived β-carbolines in vitro. J. Neurochem. 2002, 81, 814-819.
    [6] B. Janetzky, G. Gille, M. Abdel-mohsen, R. God, W.-D. Rausch, G. Bringmann, H. Reichmann; Effects of highly halogenated β-carbolines on dopaminergic cells in culture and mitochondrial respiration. Drug Dev. Res. 1999, 46, 51-56.
    [7] G. Bringmann, R. Brückner, R. Mössner, D. Feineis, A. Heils, K.-P. Lesch; Effect of 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo) on human serotonergic cells. Neurochem. Res. 2000, 25, 837-843.
    [8] R.S. Akundi, A. Macho, E. Muñoz, K. Lieb, G. Bringmann, H.-W. Clement, M. Hüll, B.L. Fiebich; 1-Trichloromethyl-1,2,3,4-tetrahydro-β-carboline-induced apoptosis in the human neuroblastoma cell line SK-N-SH. J. Neurochem. 2004, 91, 263-273.
    [9] G. Bringmann, D. Feineis, M. Münchbach, R. God, K. Peters, E.-M. Peters, R. Mössner, K.-P. Lesch; Toxicity and metabolism of the chloral-derived mammalian alkaloid 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo) in PC12 cells. Z. Naturforsch. 2006, 61c, 601-610.
    [10] A. Storch, Y.-I. Hwang, G. Bringmann, D. Feineis, S. Ott, R. Brückner, J. Schwarz; Cytotoxicity of chloral-derived β-carbolines is not specific towards neuronal nor dopaminergic cells. J. Neural Transm. 2006, 113, 1895-1901.
    [11] C. Heim, K.-H. Sontag; The halogenated tetrahydro-β-carboline "TaClo": a progressively-acting neurotoxin. J. Neural Transm. 1997, 50 (Suppl.), 107-111.
    [12] G. Bringmann, M. Münchbach, D. Feineis, K. Faulhaber, H. Ihmels; Studies on single-strand scissions to cell-free plasmid DNA by the dopaminergic neurotoxin 'TaClo' (1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline). Neurosci. Lett. 2001, 304, 41-44.
    [13] G. Bringmann, R. God, S. Fähr, D. Feineis, F. Fornadi, K. Fornadi; Identification of the dopaminergic neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline in human blood after intake of the hypnotic chloral hydrate. Anal. Biochem. 1999, 270, 167-175.
    [14] G. Bringmann, M. Münchbach, D. Feineis, K. Messer, S. Diem, M. Herderich, H.-W. Clement, C. Stichel-Gunkel, W. Kuhn; 'Chiral' and 'achiral' determination of the neurotoxin TaClo (1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline) from blood and urine samples by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. J. Chromatogr. B 2002, 767, 321-332.
    [15] W. Kochen, D. Kohlmüller, P. De Biasi, R. Ramsay; The endogenous formation of highly chlorinated tetrahydro-β-carbolines as a possible causative mechanism in idiopathic Parkinson's disease. Adv. Exp. Med. Biol. 2003, 527, 253-263.
    [16] G. Bringmann, D. Feineis, R. God, K. Maksimenka, J. Mühlbacher, K. Messer, M. Münchbach, K.-P. Gulden, E.-M. Peters, K. Peters; Resolution and chiroptical properties of the neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo) and related compounds: quantum chemical CD calculations and X-ray diffraction analysis. Tetrahedron 2004, 60, 8143-8151.

    5. Cooperations and Research Grants:

    Within a special research project entitled „Neurotoxins and Neuroprotection: Importance of Radical Mechanisms and of the Inhibition of the Respiratory Chain for the Etiology of the Parkinsonian Syndrome, of Neurodegenerative, and Aging Processes“, sponsored by the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (BMBF) (completed).

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