Polarographic Measurements of Spontaneous and Mitochondria‐Promoted Oxidation of 5,6‐and 5,7‐Dihydroxytryptamine

Abstract

Spontaneous oxygen consumption by 5,6-and 5,7-DHT (dihydroxytryptamine), reiated indoleethylamines, and 6-hydroxydopamine and oxygen consumption by these compounds in the presence of rat liver mitochondria were measured by the polarographic oxygen electrode technique. 5,6-and 5,7-DHT react with oxygen at very different rates (2.7 nmol O,/min and 33.4 nrnol O,/min, respectively) when incubated in buffer, pH 7.2, at a concentration of 1 mM and with different kinetic characteristics. While the oxidation of 5,7-DHT obeys a reaction of second-order type, the oxidation of 5,6-DHT is more complex and characterized by autocatalytic promotion. Colowed quinoidal oxidation products appeared during the degradation of both indoleamines. Glutathione, ascorbate, dithiothreitol, cysteine, albumin, and superoxide dismutase partially prevented 5,6-and 5,7-DHT from oxidative destruction. Catalase saved oxygen only in the case of 5,6-DHT by recycling of 0, released from near-stoichiometrically formed H20, during oxidation of 5,6-DHT.' 5,7-DHT did not generate H,O, in measurable amounts. Oxygen consumption rates of 5,6-and 5,7-DHT were enhanced after addition of rat liver mitochondria to the incubation medium; this resulted in an accelerated formation of quinoidal products. This stimulatory effect on the oxidation rates of both 5,6-and 5,7-DHT was blocked by cyanide, but not rotenone, and was abolished by boiling of the mitochondria fraction. The observed increase in oxygen consumption in the presence of mitochondria was found not to be influenced by monoamine oxidase-dependent deamination of 5,6-and 5,7-DHT. It is postulated that 5,6-and 5,7-DHT are capable of participating in the electron transfer of the mitochondria1 respiration chain beyond complex 111. Results obtained in determinations of ADP:O ratios in respiratory control experiments exclude a possible interference of 5,6-DHT, 5,7-DHT, and 6-OH-DA with phosphorylating sites. During the activated state of respiration, no signs of electron transfer inhibition by 5,6-and 5,7-DHT were detectable. A comparison and evaluation of the autoxidation rates of various hydroxylated indoleethylamines, of their affinity to the 5-HT transport sites, and their neurotoxic potency in vivo reveals that interaction of these compounds with oxygen at restricted reaction velocity is a prerequisite for efficient toxicity in monoaminergic neurons following active accumulation in these neurons via the high-affinity uptake systems.

Influence of peroxidase on the oligomerization of 5,6- and 5,7-dihydroxytryptamine investigated by matrix-assisted laser desorption/ionization mass spectrometry

“…Neither the MH ion of 5,6-DHT nor the ions related to its oligomerization products are detectable. These results show the high reactivity of 5,6-DHT in the presence of peroxidase H 2 O 2 and may be explained as due to the reaction pattern proposed by Klemm et al 25 5,6-DHT easily autooxidizes (see Scheme 2), generating an electrophilic o-quinone and other oxidation products which undergo extensive covalent binding with protein nucleophiles both in vitro 26 and in vivo. 27 The data obtained here may be explained by the formation, catalytically induced by H 2 O 2 , of such oxidation products and their fast reaction with peroxidase.…”

“…Formation of simple oligomers up to octamers was observed and, even after 360 minutes, the reaction environment remained reddish-brown in color, with the production of only small amounts of precipitate. This comparison shows that only in the reaction of peroxidase H 2 O 2 are reactive species, like those indicated by Klemm et al, 25 produced. Instead, the reaction of 5,7-DHT peroxidase H 2 O 2 leads to the formation of oligomers.…”

Serotonin neurotoxins — past and present

“…Two intracellular catalytic mechanisms have been proposed to explain the evolvement of the strong neurotoxicity potential of 5,7-DHT in vivo following its accumulation in monoaminergic neurons by transporters: 1) mitochondria-(i.e., cytochrome c-) mediated speeding of the autoxidation process of 5,7-DHT (transfer of electrons from 5,7-DHT to the cytochrome-c segment of the respiratory chain (Klemm et al, 1980), and 2) interaction of the hydroperoxide intermediate(s) of autoxidized 5,7-DHT (and its MAO-metabolites) with GSH-peroxidase and formation of trihydroxyindoles which autoxidize (via semiquinone radical intermediates) to give the 4,7-p-quinone of 5-OH-tryptamine (Sinhababu and Borchardt 1985; Tabatabaei and Dryhurst 1998).…”

“…Indole dimers then undergo autoxidation and intermolecular coupling to eventually yield insoluble melanoid polymeric products (Tabatabaie and Dryhurst, 1998). We have shown that rat brain mitochondria accelerate the autoxidation process of 5,6-DHT (Klemm et al, 1980). Under these conditions, oxidation of 5,6-DHT to the corresponding o-quinone is likely to occur.…”

“…In SH-deficient milieu, intramolecular cyclization involving the side chain yields aminochrome I which rearranges to form the more stable 5,6-dihydroxyindole. 5,6-Dihydroxyindole (a natural intermediate in enzyme catalyzed melanogenesis) undergoes autoxidation to form the corresponding p-quinoneimine or to yield 5,6-o-indolequinone via semiquinone radical intermediates, a process during which H 2 O 2 is generated (Klemm et al, 1980;Blank et al, 1998). The semi-quinone radical intermediates serve as driving forces in the autoxidation process via generation of O 2 -.…”