Hydroxyl Radical-Mediated Oxidation of Serotonin: Potential Insights into the Neurotoxicity of Methamphetamine

Abstract: When incubated with a hydroxyl radical (HO^?)-generating system (ascorbic acid/Fe²⁺-EDTA/O₂/H₂O₂), 5-hydroxytryptamine (5-HT; serotonin) is rapidly oxidized initially to a mixture of 2,5-, 4,5-, and 5,6-dihydroxytryptamine (DHT). The major reaction product is 2,5-DHT, which at physiological pH exists as its keto tautomer, 5-hydroxy-3-ethylamino-2-oxindole (5-HEO). Rapid autoxidation of 4,5-DHT gives tryptamine-4,5-dione (T-4,5-D), which reacts with the C(3)-centered carbanion of 5-HEO to give 3,3′-bis(2-aminoethyl)-5-hydroxy-[3,7′-bi-1H-indole]-2,4′,5′-3H-trione (7). The latter slowly cyclizes to 3′-(2-aminoethyl)-1′,6′,7′,8′-tetrahydro-5-hydroxyspiro[3H-indole-3,9′-[9H]pyrrolo[2,3-f]quinoline]-2,4′,5′(1H)- trione (9). A minor amount of T-4,5-D dimerizes to give 7,7′-bi-(5-hydroxytryptamine-4-one) (7,7′-D). In the presence of GSH, the reaction of T-4,5-D with 5-HEO is diverted and, in the presence of sufficient concentrations of this tripeptide, completely blocked. This is because GSH preferentially reacts with T-4,5-D to give 7-S-glutathionyltryptamine-4,5-dione (11). The results of this investigation suggest that 5,6-DHT, 5-HEO, 7, and 9 are products unique to the HO^?-mediated oxidation of 5-HT. Thus, the observation of other investigators that 5,6-DHT is formed in the brains of rats following a large dose of methamphetamine (MA) suggests that this drug might evoke HO^? formation. However, the present in vitro study indicates that 5,6-DHT is a rather minor, unstable product of the HO^?-mediated oxidation of 5-HT and suggests that detection of 5-HEO, 7/9, and 11 in rat brain following MA administration could provide additional support for HO^? formation. Furthermore, one or more of the intermediates and major products of oxidation of 5-HT by HO^? might, in addition to 5,6-DHT, contribute to the MA-induced degeneration of serotonergic neurons.     

Effects of serotonin depletion byp-chlorophenylalanine,p-chloroamphetamine or 5,7-dihydroxytryptamine on central dopaminergic neurons: Focus on tuberoinfundibular dopaminergic neurons and serum prolactin

Three serotonin (5-HT) neurotoxins,p-chlorophenylalanine (PCPA, 125 and 250 mg/kg, i.p.),p-chloroamphetamine (PCA, 10 mg/kg, i.p.) and 5,7-dihydroxytryptamine (5,7-DHT, 200 µg/rat, i.c.v.) were used to examine whether depletion of central 5-HT has an effect on central dopaminergic (DA) neuronal activities or on prolactin (PRL) secretion. Adult ovariectomized Sprague-Dawley rats primed with estrogen (polyestradiol phosphate, 0.1 mg/rat, s.c.) were treated with one of three neurotoxins and then decapitated in the morning after 3–7 days. Blood sample and brain tissues were collected. The acute effect of PCA (from 30 to 180 min) was also determined. The concentrations of 5-HT, DA and their metabolites, 5-hydroxyindoleacetic acid and 3,4-dihydroxyphenylacetic acid, in the median eminence, striatum and nucleus accumbens were determined by HPLC-electrochemical detection. All three toxins significantly depleted central 5-HT stores by 11–20%. Except for PCPA, neither PCA nor 5,7-DHT had any significant effect on basal DA neuronal activities or PRL secretion. PCA also exhibited an acute effect on the release and reuptake of 5-HT and DA. In summary, depletion of central 5-HT stores to a significant extent for 3–7 days did not seem to affect basal DA neuronal activity and PRL secretion.     

Autoreceptor-mediated inhibition of 3H-5-hydroxytryptamine release from rat brain cortex slices by analogues of 5-hydroxytryptamine

Rat brain cortex slices preincubated with ³H-5-hydroxytryptamine (³H-5-HT) were superfused with physiological salt solution containing paroxetine, an inhibitor of 5-hydroxytryptamine (5-HT) uptake. The effects of various indolethylamines on the electrically evoked tritium overflow (containing 66.3% unmetabolized ³H-5-HT) were investigated (the percentage of unmetabolized ³H-5-HT was not altered by the indolethylamines or metitepin). 6,7-Dihydroxytryptamine (6,7-DHT) did not affect the stimulation-evoked tritium overflow, whereas the latter was inhibited by the other tryptamine derivatives investigated; when the compounds were compared to each other on the basis of their inhibitory potencies the following rank order was obtained: unlabelled 5-HT > 5-methoxytryptamine > 4-HT > 6-HT > 5,6-DHT > tryptamine > 7-HT > 5,7-DHT. The inhibitory effects of these compounds were antagonized by metitepin. It is concluded that the indolethylamines inhibit the stimulation-evoked ³H-5-HT release by activating the presynaptic 5-HT autoreceptors on the 5-HT neurones of the rat brain cortex. Similarities may exist between these receptors and the postsynaptic 5-HT_l binding sites of this brain area.     

Inhibition of the uptake of 5-hydroxytryptamine, noradrenaline and dopamine into rat brain homogenates by various hydroxylated tryptamines

Recent work has shown that intracerebral injections of 5,6-dihydroxytryptamine (5,6-DHT) lead to a fairly selective and long lasting depletion of 5-HT in the rat CNS (BAUMGARTEN, BJORKLUND, LACHENMAYER, NOBIN and STENEVI, 1971; DALY, FUXE and JONSSON, 1973). This effect appears to result from a degeneration of the serotonin-containing neurons (BAUMGARTEN and LACHENMAYER, 1972a). 5,6-DHT does, however, to a lesser extent affect both NA and dopamine (DA) containing nerve terminals (BAUMGARTEN et al., 1971). In an attempt, therefore, to find compounds having a more specific toxic action we have investigated several other hydroxylated tryptamines. In order to obtain information about the differential affinities of these analogues for neuronal uptake sites we have examined their effects on the uptake of [³H]5-HT and (±)-[³H]NA into synaptosomes in homogenates of rat hypothalamus and of [³H]DA uptake into a similar preparation from the rat corpus striatum. It is known that the uptake of these putative transmitters in rat brain homogenates is predominantly into the synaptosome fraction (KANNENGIESSER, HUNT and RAYNAUD, 1973; COYLE and SNYDER, 1969).     

Intrastriatal Injection of 5,7-Dihydroxytryptamine Decreased 5-HT Levels in the Striatum and Suppressed Locomotor Activity in C57BL/6 Mice

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 5,7-dihydroxytryptamine (5,7-DHT) on striatal levels of dopamine (DA), 5-hydroxytryptamine (5-HT), and their metabolites, as well as on locomotor activity were investigated in C57BL/6 mice. The results showed that MPTP significantly increased locomotor activity and decreased striatal DA levels. However, injection of the serotonergic neurotoxin 5,7-DHT in the striatum, either alone or following high doses of MPTP, significantly decreased locomotor activity, and concomitantly decreased striatal levels of 5-HT and 5-HIAA. This study suggests that the increased locomotor activity may be due to increased striatal serotonergic activity which overcompensates for the DA deficiency. The locomotor hypoactivity, induced by 5,7-DHT, might be due to the decreased striatal levels of 5-HT and 5-HIAA.     

The metabolism of dopamine in rat caudate nucleus can be increased by in vivo “dialysis” perfusion cannulae

The development of dialysis cannula techniques coupled with high performance liquid chromatography and electrochemical detection (HPLC-EC) has provided a means to continuously sample extracellular fluid from deep brain structures $\text{in vivo}$. The present studies show that with HPLC-EC analysis of the acid metabolites of dopamine (DA) and 5-hydroxytryptamine (5-HT) in samples from dialysis cannulae implanted in the caudate nucleus of anaesthetized rats, it is possible to determine the time course of the response of dopamine- and 5-HT containing neurones to administration of monoamine oxidase inhibitors and haloperidol. The tissue concentrations of the DA and 5-HT metabolites were also determined at the conclusion of each individual experiment in both the caudate nucleus containing a cannula and in that without a cannula. In perfusion experiments where no drug was administered the content of the DA metabolites, but not that of the 5-HT metabolite, were significantly elevated in the caudate nucleus containing the cannula as compared with the contralateral tissue. These increases occurred whether the cannula was perfused or not, suggesting that the presence of the cannula may have been causing a lesion which altered the activity of the DA neurones. These results emphasize the importance of tissue analysis in conjunction with the dialysis experiments, especially where perfusion sample contents of the monoamine metabolites are being measured as a reflection of the effects of behavioural manipulation or drug treatment on endogenous neuronal activity.     

HPLC-EC analysis of catechols and indoles in rat brain dialysates

This short article reviews HPLC-EC methodology that we are currently applying to measure DA, 5-HT and their acid metabolites in rat brain dialysates collected in vivo. HPLC-EC systems based on standard-bore HPLC columns are described which are sufficiently sensitive to allow detection of the monoamine transmitters and their metabolites in regional brain dialysates collected at 10-20 min intervals. A large reduction in sample requirement was achieved by "down-scaling" the conventional HPLC-EC assay to incorporate small-bore HPLC columns. The small-bore systems allowed monoamines to be detected in samples collected over 1 to a few minutes.     

Turning in MFB-lesioned rats and antagonism of neuroleptic-induced catalepsy after lisuride and LSD.

The effects of lisuride, d-lysergic acid diethyl amide (LSD) and apomorphine were studied in rats with unilateral destruction of nigro-striatal nerve terminals either with 6-hydroxydopamine (6-OHDA) or 5, 6-dihydroxytryptamine (5,6-DHT). Lisuride at the dose of 50 μg kg^?1 i.p. induced contralateral turning for more than 4 hours while the circling induced by LSD (200 μg kg^?1) and apomorphine (1 mg kg^?1) persisted for only one hour. Lisuride, a compound stimulating both dopamine (DA) and 5-hydroxytryptamine (5-HT) receptors induced a more intense turning in 6-OHDA than in 5,6-DHT lesioned rats. This might indicate a modulation of 5-HT on rotational behavior. Haloperidol (1 mg kg^?1 i.p.) antagonized both lisuride- and LSD-induced turning. LSD, and much more persistently lisuride, counteracted the prochlorperazine-induced catalepsy. These findings correlate with the biochemical data indicating that lisuride is a very potent agonist at central dopaminergic receptors.     

Regulation of pyridoxal-5′-phosphate level by biogenic amines in mouse brain

We investigated the relationship between the concentration of pyridoxal-5′-phosphate (PLP) and biogenic amine in mouse brain. The production of PLP from pyridoxal (PL) by pyridoxal kinase (PLK) was inhibited by the addition of dopamine (DA), norepinephrine (NE) and 5-hydroxytryptamine (5-HT), but not by that of epinephrine and N-acetyl-serotonin. DA and NE were combined with PLP by a non-enzymatic reaction, whereas 5-HT was bound only slightly with PLP. The conjugated product of PLP with DA was also detected by HPLC analysis when PLK activity was assayed using PL as a substrate in the presence of DA. In an in vivo investigation, the depletion of DA and 5-HT in mouse brain after an intraperitoneal injection of 5 mg/kg reserpine, led to slight elevation of the PLP level to 120% of the control level. By contrast, the increase in DA in the brain caused by intraperitoneal administration of 150 mg/kg L-DOPA caused the PLP concentration to decrease to 70% of the control level. However, no change in PLK activity in the brain was observed when the mice were treated with either reserpine or L-DOPA. These results suggested that the level of PLP in mouse brain was partly regulated by the concentration of biogenic amines, such as DA, NE and 5-HT, without apparent induction of PLK.     

A Comparison of the Distribution of Neurotransmitters in Brain Regions of the Rat and Guinea-Pig Using a Chemiluminescent Method and HPLC with Electrochemical Detection

Six brain areas of rats and guinea-pigs, killed by microwave irradiation, were used for the concomitant measurement of the levels and regional distribution of cholinergic, biogenic amine, and amino acid neurotransmitters and metabolites. Acetylcholine (ACh) and choline (Ch) were quantified by chemiluminescence; noradrenaline (NA), dopamine (DA), 5-hydroxytryptamine (5-HT), and their metabolites by HPLC with electrochemical detection (HPLC-EC); and six putative amino acid neurotransmitters by HPLC-EC following derivatisation. The levels and regional distribution of these transmitters and their metabolites in the rat were similar to those reported in previous studies, except that biogenic amine transmitter levels were higher and metabolite concentrations were lower. The guinea-pig showed a similar regional distribution, but the absolute levels of ACh were lower in striatum and higher in hippocampus, midbrain-hypothalamus, and medulla-pons. In all areas, the levels of Ch were higher and those of NA, 5-HT, and taurine were lower than in the rat. The most marked differences between the rat and guinea-pig were in the relative proportion of DA metabolites and 5-HT turnover, as estimated by metabolite/transmitter ratios. This study can be used as a basis for a comprehensive understanding of the central effects of drugs on the major neurotransmitter systems.     

Rotational responses to serotonergic and dopaminergic agonists after unilateral dihydroxytryptamine lesions of the medial forebrain bundle: co-operative interactions of serotonin and dopamine in neostriatum.

Rats with unilateral 5,7-DHT lesions, but not 5,6-DHT lesions, showed rotational responses to 5-HTergic drugs (5MeODMT and fenfluramine) that were qualitatively similar to those induced by DAergic drugs (apomorphine and amphetamine) after 6-OHDA lesions. However, 5,7-DHT-lesioned rats also themselves showed rotational responses to DAergic drugs. The merits and limitations of a unilateral 5,7-DHT-lesioned rotating rat model for studying 5-HTergic function are discussed. It is suggested that 5-HT and DA may function in a co-operative manner in the striatum. These findings may be important for the rational pharmacotherapy of Parkinson's disease in which 5-HT as well as DA has been shown to be substantially depleted.     

Nicotine-Induced Changes in Neurotransmitter Levels in Brain Areas Associated with Cognitive Function

Nicotine, one of the most widespread drugs of abuse, has long been shown to impact areas of the brain involved in addiction and reward. Recent research, however, has begun to explore the positive effects that nicotine may have on learning and memory. The mechanisms by which nicotine interacts with areas of cognitive function are relatively unknown. Therefore, this paper is part of an ongoing study to evaluate regional effects of nicotine enhancement of cognitive function. Nicotine-induced changes in the levels of three neurotransmitters, dopamine (DA), serotonin (5-HT), norepinepherine (NE), their metabolites, homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA), and their precursor, L-DOPA, were evaluated in the ventral and dorsal hippocampus (VH and DH), prefrontal and medial temporal cortex (PFC and MTC), and the ventral tegmental area (VTA) using in vivo microdialysis in awake, freely moving, male Sprague-Dawley rats. The animals were treated with acute nicotine (0.5 mg/kg, s.c.) halfway through the 300-min experimental period. The reuptake blockers, desipramine (100 microM) and fluoxetine (30 microM), were given to increase the levels of NE and 5-HT so that they could be detected. Overall, a nicotine-induced DA increase was found in some areas, and this increase was potentiated by desipramine and fluoxetine. The two DA metabolites, HVA and DOPAC, increased in all the areas throughout the experiments, both with and without the inhibitors, indicating a rapid metabolism of the released DA. The increase in these metabolites was greater than the increase in DA. 5-HT was increased in the DH, MTC, and VTA in the presence of fluoxetine; its metabolite, 5-HIAA, was increased in the presence and absence of fluoxetine. Except in the VTA, NE levels increased to a similar extent with desipramine and fluoxetine. Overall, nicotine appeared to increase the release and turnover of these three neurotransmitters, which was indicated by significant increases in their metabolites. Furthermore, DA, and especially HVA and DOPAC, increased for the 150 min following nicotine administration; 5-HT and NE changes were shorter in duration. As gas chromatography experiments showed that nicotine levels in the brain decreased by 75% after 150 min, this may indicate that DA is more susceptible to lower levels of nicotine than 5-HT or NE. In conclusion, acute nicotine administration caused alterations in the levels of DA, 5-HT, and NE, and in the metabolism of DA and 5-HT, in brain areas that are involved in cognitive processes.     

In vivo release of endogenous dopamine, 5-hydroxytryptamine and some of their metabolites from rat caudate nucleus by phenylethylamine

The in vivo release of endogenous 3,4-dihydroxyphenylethylamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 3-methoxytyramine (3-MT), and of 5-hydroxytryptamine (5-HT) and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), has been measured in the caudate nucleus of the anesthetized rat. A push-pull cannula was implanted into the brain, and the tissue perfused with artificial CSF or artificial CSF containing 5×10^–4 M phenylethylamine. The perfusate was collected and analyzed for DA, 5-HT and their metabolites by high performance liquid chromatography with electrochemical detection (HPLC-ECD). DA was released by phenylethylamine at rates significantly greater than its basal rate. 3-MT and 5-HT were undetectable in perfusates collected under basal conditions, but could be detected readlly during phenylethylamine stimulation. DOPAC, HVA and 5-HIAA concentrations were not significantly affected by phenylethylamine. The results suggest (1) that phenylethylamine may exert its behavioural effects through increased release of both DA and 5-HT, and (2) that in vivo measurements of the acid metabolites alone may not be indicative of the release of the amines.Special Issue Dedicated to Dr. Abel Lajtha.     

Developmental Changes in Biogenic Amine Levels in the Central Nervous System and the Haemolymph of the Eastern Death's Head Hawk Moth,Acherontia styx (Lepidoptera: Sphingidae)

In an effort to understand the role of biogenic amines in insect development, changes in the levels of octopamine (OA), dopamine (DA), epinephrine (E), norepinephrine (NE), and serotonin (5-HT) in the brain, the optic lobes and the haemolymph of different developmental stages of Acherontia styx were analyzed using HPLC with electrochemical detector. In the brain, OA was the most abundant monoamine. DA, OA, and E levels in larvae peaked around the wandering stage (W). A dramatic increase in DA, 5-HT, and E levels was observed in the brain of the adult as compared to the pupal stage. NE, however, was not detected in the brain of most stages of the insect, except in the brain of 20-day-old pupae and adults. A 3-fold increase in OA levels was observed in the optic lobes of the adult as compared to late pupal stage. No changes were observed for E, DA, and 5-HT. NE was not detected in the optic lobes. In the haemolymph of 5^th instar larvae, OA was also the most abundant amine. Both DA and OA peaked prior to the onset of the W stage. In contrast, E and NE concentrations decreased with development of the 5^th instar larvae. 5-HT was not detected in the haemolymph. Finally, different profiles for amine levels were observed for the two forms of the 5^th instar larvae (green vs brown). These results are interpreted in the light of the role of biogenic amines and their relation to development in the nervous system of lepidopteran insects.     

The effect of 5,6-dihydroxytryptamine on post-decapitation convulsions

Previous data (1) have shown that L-DOPA increases the duration of the clonic phase of post-decapitation convulsions (PDC) in mice. It was suggested that this effect is produced by depleting 5-hydroxytryptamine (5-HT) in the inhibitory bulbospinal pathways and thus enhancing reflex activity in the spinal cord. If this were true then L-DOPA administration should not influence clonic PDC in animals whose 5-HT pathways were destroyed. We therefore tested the effects of L-DOPA on mice 3 weeks after pretreatment with the 5-HT neurotoxin, 5,6-dihydroxytryptamine (5, 6-DHT) (50 μg/kg, intracerebroventricularly). All mice were given the peripheral decarboxylase inhibitor, Ro 4-4602. 5,6-DHT halved the brain 5-HT levels and significantly increased the duration of clonic PDC. The administration of L-DOPA (320 mg/kg i.p.) to 5,6 DHT treated mice did not produce any further significant increases in duration. The administration of 5-hydroxytryptophan (5-HTP) (100 mg/kg, i.v.) to 5,6-DHT treated mice, however, increased 5-HT to above control levels and reduced convulsions to control levels. Administration of both 5-HTP and L-DOPA to 5,6-DHT treated mice resulted in 5-HT levels and convulsion times which were also not significantly different from the controls. These data give additional indication that intact 5-HT nerve terminals are necessary for L-DOPA to prolong the duration of clonic PDC.     

Regional Heterogeneity of Nicotine Effects on Neurotransmitters in Rat Brains <Emphasis Type="Italic">in vivo</Emphasis> at Low Doses

In our recent studies on nicotine-induced changes in neurotransmitters in brain areas associated with cognitive function using a nicotine dose of 0.5 mg/kg administered subcutaneously to conscious freely moving rats, we found changes in dopamine, norepinephrine, and serotonin, and their metabolites, in the areas examined. For the present report we examined changes in these neurotransmitters following administration of lower nicotine doses, to test regional differences in nicotine response and possible threshold levels for some effects of nicotine. The doses used were 0.15 mg/kg and 0.03 mg/kg nicotine administered subcutaneously. Nicotine levels in the brain reached peak values in less than 10 min and decreased with a half-life of about 60 min (0.15 mg/kg) or 30 min (0.03 mg/kg) to values below detection limits (1 ng/g), by the later time points of the 0.03 mg/kg experiments. Nicotine-induced dopamine (DA) increase (and increase in DA metabolites) and decrease in 5-HT levels at 0.15 mg/kg were significant in the cortex, less so in the hippocampus. Norepinephrine (NE) increase at 0.15 mg/kg was much less significant than found previously at 0.5 mg/kg. At a low nicotine dose (0.03 mg/kg), the significant changes observed were a decrease in 5-HT in the hippocampus and small increases of DA and NE in the prefrontal cortex and of NE in the medial temporal cortex. In the nucleus accumbens DA, NE, and 5-HT and their metabolites in the ventral tegmental area, mostly DA and metabolites were increased. We conclude that in areas of cognitive function nicotine-induced DA changes are more concentration dependent than changes in NE or 5-HT, and that there are regional differences in neurotransmitter changes induced by nicotine, with NE changes detectable only in the cortex and 5-HT changes only in the hippocampus at a low nicotine dose, indicating significant regional variation in sensitivity to nicotine-induced neurotransmitter changes in brain areas associated with cognitive function. The decrease in 5-HT shows that nicotine also has indirect effects caused by neurotransmitters released by nicotine. The effects of low nicotine dose are more significant in areas of reward function, indicating differences in sensitivity between cognitive and reward functions.     

IN VITRO STUDIES ON THE INTERACTION OF BRAIN MONOAMINE OXIDASE WITH 5,6- AND 5,7-DIHYDROXYTRYPTAMINE12

[¹⁴C]5,6-Dihydroxytryptamine ([¹⁴C] 5,6-DHT) and [¹⁴C]5,7-dihydroxytryptamine ([¹⁴C]5,7-DHT) were deaminated to toluene-isoamylalcohol extractable products when incubated with homogenates of rat hypothalamus or pons-medulla oblongata. [¹⁴C]5,6-Dihydroxyindole acetic acid ([¹⁴C]5.6-DHIAA) and [¹⁴C]5,7-dihydroxyindole acetic acid ([¹⁴C]5,7-DHIAA) were detected as MAO metabolites by TLC besides non-identified components. The conversion of [¹⁴C]5,6-DHT and [¹⁴C]5,7-DHT obeyed, at least initially, Michaelis-Menten kinetics (K_m 5,7-DHT: 0.5 × 10^?3M; K_m 5,6-DHT: 1.25 × 10^?3M). Inhibition of the reaction by the MAO A inhibitor, clorgyline, resulted in a typical double sigmoidal inhibition curve indicating that both amines are metabolized by both types of MAO (A and B). In deprenyl inhibition studies, however, 5,7- and 5,6-DHT seemed to be preferred substrates of MAO A. Incubation of rat brain homogenates with [¹⁴C]5,6-DHT and [¹⁴C]5,7-DHT or with the MAO metabolites [¹⁴C]5,6-DHIAA and [¹⁴C]5,7-DHIAA caused a time-dependent break-down of the dihydroxylated indole compounds with subsequent binding of radioactivity to perchloric acid insoluble tissue components. 5,6-DHT inactivated MAO in rat brain homogenates parallel to its decomposition and extensive protein binding. The inactivation of MAO by 5,6-DHT and the extensive binding of radioactivity to protein were antagonized by dithiothreitol (DTT), glutathione (GSH) and L-ascorbic acid. Reduction of [O₂] in the incubation medium slightly attenuated the inactivation of MAO by 5,6-DHT. Catalase or superoxide dismutase failed to prevent MAO from being inactivated by 5,6-DHT. The results suggest that oxidation products of 5,6-DHT, e.g. its corresponding o-quinone, are involved in the inactivation of MAO in vitro and mainly responsible for the binding of radioactivity to brain proteins in vitro. Similar mechanisms may also be operative in the in vivo neurotoxicity of 5,6-DHT. The lack of inactivation of MAO by 5,7-DHT in vitro correlated with a low degree of radioactivity binding (from [¹⁴C]5,7-DHT) to homogenate protein pellets; the binding to proteins was barely influenced by GSH, cysteine, DTT and l -ascorbic acid. These latter findings do not provide a plausible explanation for the mechanism(s) involved in the well known in vivo neurotoxicity of 5,7-DHT.     

Influence of chronic inorganic lead exposure on regional dopamine and 5-hydroxytryptamine turnover in rat brain

The results of previous behavioral studies utilizing chronic exposure to low amounts of inorganic lead (Pb) have suggested alterations in the function of biogenic amine neuronal systems. The following study was performed to provide evidence for the possible bases of these changes in pharmacological responsiveness in exposed animals. Dams were administered 0.2% Pb acetate in drinking water to expose their offspring to Pb via the maternal milk. Males were weaned to the same drinking solution. At 120–140 days a tracer dose of 1.0 mCil-[³H]2,6-tyrosine (³H-TYR) and 0.5 mCil-[³H(G)]tryptophan (³H-TRP) was injected through an indwelling jugular catheter, and norepinephrine (NE), dopamine (DA), 5-hydroxytryptamine (5-HT) and their respective precursors and metabolites were quantified by liquid chromatography with electrochemical detection with column eluate collected for liquid scintillation counting. At this level of exposure (blood lead (PbB) at day 90 in exposed animals=43.1±1.7 g/dl) no changes were observed in concentration Nf NE or DA mr DA metabolites in any brain region. However, DA turnover was decreased in Pb-exposed animals in nucleus accumbens and frontal cortex. No changes in 5-HT content and turnover were observed in any brain region, but 5-hydroxyindoleacetic acid (5-HIAA) levels were decreased in 6 of the 9 brain regions examined. These findings are consistent with observations of an attenuated behavioral responsiveness to d-amphetamine (AMPH) in exposed animals, and suggest that the changes in DA and 5-HT neurons noted by other workers at higher levels of exposure persist when PbBs are in the range of 40 g/dl.     

Diurnal alterations of catecholamines, indoleamines and their metabolites in specific brain regions of the mouse

1. The diurnal variations of regional brain concentrations of dopamine (DA), norepinephrine (NE), serotonin (5-HT) and metabolites were determined in unperturbed male CD-1 mice. Determinations were made every 4 hr for 24 hr. 2. The most striking and significant variations in biogenic amines were seen in the hypothalamus, where concentrations of NE, DA and 5-HT varied in a rhythmic pattern and as much as two-fold during this period. 3. In some cases, daily alterations in parent biogenic amines were reflected by concurrent changes in their metabolites. 4. Since concentrations of neurotransmitters in the brain are often used as an indicator of stress and/or toxicity, these data should provide an accurate data base allowing for more accurate interpretation of results.