Brain amines and neocortical EEG in young and aged rats

1. Frontal and parieto-occipital electroencephalography (EEG) of young (4 months-old) and aged (17 and 22 months-old) Wistar rats were analyzed, both during movement and during waking immobility. 2. The levels of monoamines, serotonin and their metabolites were measured from the frontal cortex, parieto-occipital cortex, hippocampus, brainstem and midbrain. 3. In aged rats, as compared to young rats, the most apparent changes of the quantitative EEG spectrum were the decreased amplitude of alpha (5-10 Hz) and beta (10-20 Hz) frequency bands in the frontal and parieto-occipital cortices during both movement and waking immobility behavior (p less than 0.05). 4. The levels of dopamine (DA), homovanillinic acid (HVA), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) or the ratios of 5-HT/5-HIAA and DA/HVA did not differ between young and aged rats in any brain region studied, with the exceptions of brainstem DA and parieto-occipital 5-HIAA, which were elevated in aged rats (p less than 0.05). 5. In the frontal cortex, hippocampus and midbrain, noradrenaline (NA) levels of aged rats were slightly increased as compared to young rats (p less than 0.05). 6. NA levels of the parieto-occipital cortex and brainstem did not change during aging. 7. Furthermore, there were no clear correlations between the decreased amplitude of the quantitative EEG spectrum and monoamine or serotonin concentrations, or the ratios of 5-HT/5-HIAA and DA/HVA in the cerebral cortex of aging Wistar rat.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transient Hypoxia Alters Striatal Catecholamine Metabolism in Immature Brain: An In Vivo Microdialysis Study

Microdialysis probes were inserted bilaterally into the striatum of 7-day-old rat pups (n = 30) to examine extracellular fluid levels of dopamine, its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA). The dialysis samples were assayed by HPLC with electrochemical detection. Baseline levels, measured after a 2-h stabilization period, were as follows: dopamine, not detected; DOPAC, 617 +/- 33 fmol/min; HVA, 974 +/- 42 fmol/min; and 5-HIAA, 276 +/- 15 fmol/min. After a 40-min baseline sampling period, 12 animals were exposed to 8% oxygen for 120 min. Hypoxia produced marked reductions in the striatal extracellular fluid levels of both dopamine metabolites (p less than 0.001 by analysis of variance) and a more gradual and less prominent reduction in 5-HIAA levels (p less than 0.02 by analysis of variance), compared with controls (n = 12) sampled in room air. In the first hour after hypoxia, DOPAC and HVA levels rose quickly, whereas 5-HIAA levels remained suppressed. The magnitude of depolarization-evoked release of dopamine (elicited by infusion of potassium or veratrine through the microdialysis probes for 20 min) was evaluated in control and hypoxic animals. Depolarization-evoked dopamine efflux was considerably higher in hypoxic pups than in controls: hypoxic (n = 7), 257 +/- 32 fmol/min; control (n = 12), 75 +/- 14 fmol/min (p less than 0.001 by analysis of variance). These data demonstrate that a brief exposure to moderate hypoxia markedly disrupts striatal catecholamine metabolism in the immature rodent brain.     

Effects of an N-methyl-d-aspartate receptor agonist and its antagonist CPP on the levels of dopamine and serotonin metabolites in rat striatum collected in vivo by using a brain dialysis technique

3-((±)-2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) is an antagonist at the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. In the present study, levels of dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA) were measured after intracerebroventricular injection of NMDA, CPP or both in rat striatum using a brain dialysis method. The injection of NMDA produced a significant increase in DOPAC level. HVA level was also increased by NMDA injection. The level of 5-HIAA was not affected by NMDA injection. The injection of CPP had no effect on DOPAC, HVA and 5-HIAA levels. The injection of CPP restrained the increase of DOPAC and HVA levels induced by NMDA injection. The results suggest that intracerebral injection of NMDA may increase dopamine release from rat striatum, but have no effect on serotonin release. Furthermore, CPP inhibits NMDA induced release of dopamine.     

Effects of transient, global, cerebral ischemia on striatal extracellular dopamine, serotonin and their metabolites

Rat striatal extracellular fluid levels of dopamine, serotonin, 3-methoxytyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were measured before, during and after transient, global cerebral ischemia in awake rats using in vivo brain microdialysis. Before ischemia, extracellular levels of dopamine, DOPAC, HVA and 5-HIAA were detectable and consistent from sample to sample. During cerebral ischemia, there was a large increase in extracellular dopamine levels and a decrease in the extracellular levels of DOPAC, HVA, and 5-HIAA. During reperfusion, dopamine levels returned to normal as did those of DOPAC, HVA and 5-HIAA. Dialysate serotonin and 3-methoxytyramine concentrations were below detection limits except for samples collected during ischemia and early reperfusion.     

Increased Cerebrospinal Fluid Dopamine and 3,4-Dihydroxyphenylacetic Acid Levels in Huntington''s Disease: Evidence for an Overactive Dopaminergic Brain Transmission

Levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), noradrenaline (NA), 3-methoxy-4-hydroxyphenylglycol (MHPG), and 5-hydroxyindoleacetic acid (5-HIAA) in the CSF of patients with Huntington's disease (HD) were measured by HPLC. CSF DA, DOPAC, and MHPG levels were found to be increased in HD patients. Levels of HVA, 5-HIAA, and NA in the CSF of HD patients did not differ from those of controls. Changes in CSF DA and DOPAC levels were consistent with previous findings of increased DA tissue content in some brain areas of patients with HD. These results suggest that CSF DOPAC levels could be a more reliable index of overactive dopaminergic brain systems in HD than CSF HVA levels.     

Motor Activity Increases Tryptophan, 5-Hydroxyindoleacetic Acid, and Homovanillic Acid in Ventricular Cerebrospinal Fluid of the Conscious Rat

An investigation was made into the effects of running (1 h at 20 m/min) on central serotonergic and dopaminergic metabolism in trained rats. Methodology involved continuous withdrawal of cerebrospinal fluid (CSF) from the third ventricle of conscious rats and measurements of tryptophan (TRP), 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA) levels during a 2 h post-exercise period. All three compounds were increased during the hour following exercise and returned to their basal values within an hour later. CSF flow rate was stable when metabolite levels were elevated. Brain determinations indicated that CSF metabolite variations only qualitatively paralleled brain changes. Indeed, post-exercise TRP, 5-HIAA, and HVA levels were increased to a greater extent in brain when compared to CSF. It is suggested that increased serotonergic and dopaminergic metabolism, caused by motor activity, may be involved in the behavioral effects of exercise.     

Effects of kainic acid,quisqualic acid,and their antagonist,pCB-PzDA,on rat electrocorticograms and monoamine metabolite levels in rat striatum

The action of kainic acid (KA), quisqualic acid (QA), and 1-(4-chlorobenzoyl)-piperazine-2,3-dicarboxylic acid (pCB-PzDA) was investigated in the central nervous system of male Sprague Dawley rats. Intracerebroventricularly injected KA and QA (100 nmol) induced spike discharges, and pCB-PzDA (100 nmol) suppressed electrocorticograms for one hour. pCB-PzDA enhanced the KA-induced spike discharges and inhibited those induced by QA. 2,3-Di-hydroxyphenylacetic acid(DOPAC) and homovanillic acid (HVA) levels were increased transiently by 10 nmol and continuously by 100 nmol of KA. KA dose-dependently increased 5-hydroxyindoleacetic acid (5-HIAA) levels 2 hours after administration. While 10 nmol of QA slightly increased the HVA level, 100 nmol of QA significantly increased DOPAC, HVA, and 5-HIAA levels. DOPAC and HVA levels were increased by 100 nmol of pCB-PzDA, although this agent inhibited KA-induced increases in DOPAC, HVA, and 5-HIAA levels. On the other hand, while pCB-PzDA first inhibited QA-induced increases in DOPAC, HVA and 5-HIAA levels for one hour, DOPAC and HVA levels thereafter increased additively. These findings suggest that pCB-PzDA may act not only as a NMDA antagonist, but that it may also act directly on dopaminergic neurons.     

Kinetics of Drug-Induced Changes in Dopamine and Serotonin Metabolite Concentrations in the CSF of the Rat

Cerebrospinal fluid (CSF) was removed at a constant flow rate of 1 microliter/min from the third ventricle of anesthetized rats. Every 15 min, CSF dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) concentrations were determined by direct injection of CSF into a liquid chromatographic system coupled with electrochemical detection. Mean CSF concentrations of DOPAC, HVA, and 5-HIAA were 1.29 microM, 0.88 microM, and 2.00 microM, respectively. In order to determine the turnover rates of dopamine (DA) and serotonin, experiments using monoamine oxidase (MAO) inhibition were performed. Tranylcypromine (20 mg/kg i.p.) induced a sharp exponential decrease of CSF DOPAC, HVA, and 5-HIAA, with respective half-lives of 15.60 min, 16.91 min, and 77.23 min. Their respective turnover rates were 3.74, 2.22, and 1.18 nmol X ml-1 X h-1. m-Hydroxybenzylhydrazine (NSD-1015, 100 mg/kg i.p.) and monofluoromethyl-DOPA (100 mg/kg i.p.), two decarboxylase inhibitors, induced a slow exponential decrease of all three CSF metabolites. alpha-Methyl-p-tyrosine (250 mg/kg i.p.) also induced a slow exponential decrease of DOPAC and HVA. These decreases of CSF DOPAC and HVA induced by DA synthesis inhibitors may reflect the turnover of DA in vivo. Haloperidol (0.5 mg/kg i.p.) considerably enhanced CSF DOPAC and HVA without affecting 5-HIAA, confirming that dopaminergic receptors modulate DA neurotransmission in vivo. Haloperidol administered 1.5 h after NSD-1015 did not increase DOPAC and HVA, in contrast to reserpine (5 mg/kg i.p.) injected under the same conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat brain monoamine and serotonin S2 receptor changes during pregnancy

The concentrations of noradrenaline (NA), dopamine (DA), serotonin (5-HT), and their metabolites were determined in 5 brain areas of non-pregnant, 15 and 20 day pregnant and 4 day post-partum rats. Striatal 5-HT content was significantly lower in 15 and 20 day pregnant rats than in estrous controls. A significant decrease in striatal and frontal cortex 5-hydroxyindole-3-acetic acid (5-HIAA) concentration was observed in 15 day pregnant rats. Significant increases in hypothalamic and hippocampal NA levels were observed at 4 days post-partum. Frontal cortex serotonin S2 receptorKd was reduced in 4 day post-partum rats. There was no significant change in S2 receptorB _max during pregnancy. Levels of progesterone were negatively correlated with striatal DA, homovanillic acid (HVA), 5-HT, and 5-HIAA levels, hypothalamic DA, hippocampal 5-HT, and frontal cortex 5-HIAA values as well as striatal HVA to DA, and HVA to 3,4-dihydroxyphenylacetic acid (DOPAC) ratios and amygdaloid HVA to DOPAC ratios. The limbic neurotransmitter changes might possibly contribute to mood changes which occur during pregnancy and post-partum.     

Novel multifunctional neuroprotective iron chelator-monoamine oxidase inhibitor drugs for neurodegenerative diseases. In vivo selective brain monoamine oxidase inhibition and prevention of MPTP-induced striatal dopamine depletion

Several multifunctional iron chelators have been synthesized from hydroxyquinoline pharmacophore of the iron chelator, VK-28, possessing the monoamine oxidase (MAO) and neuroprotective N-propargylamine moiety. They have iron chelating potency similar to desferal. M30 is a potent irreversible rat brain mitochondrial MAO-A and -B inhibitor in vitro (IC50, MAO-A, 0.037 +/- 0.02; MAO-B, 0.057 +/- 0.01). Acute (1-5 mg/kg) and chronic [5-10 mg/kg intraperitoneally (i.p.) or orally (p.o.) once daily for 14 days]in vivo studies have shown M30 to be a potent brain selective (striatum, hippocampus and cerebellum) MAO-A and -B inhibitor. It has little effects on the enzyme activities of the liver and small intestine. Its N-desmethylated derivative, M30A is significantly less active. Acute and chronic treatment with M30 results in increased levels of dopamine (DA), serotonin(5-HT), noradrenaline (NA) and decreases in DOPAC (dihydroxyphenylacetic acid), HVA (homovanillic acid) and 5-HIAA (5-hydroxyindole acetic acid) as determined in striatum and hypothalamus. In the mouse MPTP (N-methy-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease (PD) it attenuates the DA depleting action of the neurotoxin and increases striatal levels of DA, 5-HT and NA, while decreasing their metabolites. As DA is equally well metabolized by MAO-A and -B, it is expected that M30 would have a greater DA neurotransmission potentiation in PD than selective MAO-B inhibitors, for which it is being developed, as MAO-B inhibitors do not alter brain dopamine.     

Effect of Transient Cerebral Ischaemia and Cardiac Arrest on Brain Extracellular Dopamine and Serotonin as Determined by In Vivo Dialysis in the Rat

The effects of 20-min transient, global, forebrain ischaemia and cardiac arrest on extracellular concentrations of dopamine (DA), serotonin (5-HT), and their respective metabolites, homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), were measured in vivo by dialysis of rat striatum and hippocampus. During the ischaemic period, striatal DA content increased (250-fold basal concentrations) with parallel but much less marked increases of both striatal and hippocampal 5-HT content (eight- to 10-fold). Baseline values were restored during reperfusion. Subsequent increases of DA and 5-HT levels on cardiac arrest were comparable after both sham operation and ischaemia. Significant decreases of HVA and 5-HIAA levels were observed following ischaemia or cardiac arrest. The differential effects of ischaemia on DA and 5-HT suggest selective alterations in disposition or metabolism of the two transmitters and that dopaminergic neurones may be more vulnerable to ischaemic insults.     

Simultaneous determination of catecholamines in rat brain tissue by high-performance liquid chromatography

A novel and highly sensitive method has been developed for the determination of catecholamines [noradrenaline (NA), dopamine (DA), serotonin (5-HT) and their metabolites 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA)] in brain tissue. The method uses isocratic reversed-phase HPLC with amperometric end-point detection. The calibration curve was linear over the range 10–150 pg on-column. The assay limits of detection for NA, DA, 5-HT, 5-HIAA and HVA were 3.8, 3.8, 6.8, 5 and 7.5 pg on-column, respectively. The mean inter- and intra-assay relative standard deviations (RSDs) over the range of the standard curve were less than 5%. The absolute recoveries averaged 99.1%, 99.5%, 97.7%, 99.5% and 98.8% for NA, DA, 5-HT, 5-HIAA and HVA, respectively.     

Effects of an acute dose of ethanol on dopaminergic and serotonergic systems from rat cerebral cortex and striatum

Intraperitoneal injection 10 min before sacrifice of 1.5 g ethanol/kg weight produced an increase in rat striatal levels of homovanillic acid (HVA) (p < 0.05) but did not affect the striatal concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA). A similar ethanol treatment led to decreases in 5-HT (p < 0.05) and 5-HIAA (p < 0.05) from cerebral cortex (prefrontal and anterior cingulate areas). The results point to several ethanol-linked alterations in central serotonergic and dopaminergic systems.     

Higher plasma catecholamine and metabolite levels in the early phase of nonsegmental vitiligo

The etiology of vitiligo is still being debated, although neural factors seem to play a pivotal role in its pathogenesis. In our search for a link between vitiligo and the activity of monoaminergic systems, we used high-pressure liquid chromatography and electrochemical detector (HPLC-ED) methods to measure the plasma levels of the following substances in 35 healthy subjects and in 70 patients suffering from nonsegmental vitiligo at the different stages of the disease: catecholamines [norepinephrine (NE), epinephrine (E), and dopamine (DA)], their precursor 3,4-dihydroxyphenylalanine (DOPA), their metabolites [3-methoxy-4-hydroxyphenylglycol (MHPG), normetanephrine (NMN), metanephrine (MN), and homovanillic acid (HVA)], and 5-hydroxyindolacetic acid (5-HIAA) as the major metabolite of serotonin. We found that the levels of NE, E, NMN, MN, HVA, and 5-HIAA were significantly higher in patients compared to controls. The patients at an active phase of the disease (n = 49/70) showed significantly higher levels of NE, NMN, MHPG, and HVA than ones at a stable phase. The patients with progressive vitiligo and at its more recent onset (< 1 year) showed significantly increased levels of E, NE, and MN in comparison with longer-term sufferers. No significant differences were observed when the patients were subdivided according to the type of vitiligo or their age at its onset. The higher catecholamine and metabolite levels in the early phase of the disease may reflect increased activity by monoaminergic systems, probably due to stressful events, including the onset of vitiligo itself.     

Effects of Rat Ovariectomy on CSF Monoamine Metabolite Levels and Elimination

Cerebrospinal fluid (CSF) was removed from the third ventricle of anesthetized male, female, and ovariectomized rats. CSF 3,4-dihydroxyphenylethylamine and serotonin metabolite levels [dihydroxyphenylacetic acid, homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA)] were determined on 15-min samples by liquid chromatography coupled with electrochemical detection. Monoamine oxidase inhibition was used for studying metabolite turnover in the CSF. No difference was observed between male, ovariectomized, and sham-operated female rats. However, ventricular CSF HVA and 5-HIAA levels were significantly higher in the ovariectomized than in the sham-operated rats. These differences do not reflect effects of ovariectomy on brain metabolite production but indicate slower metabolite elimination from the CSF.     

In-use stability of monoamine metabolites in human cerebrospinal fluid

Cerebrospinal fluid (CSF) concentrations of the monoamine metabolites homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA) are commonly used to provide information about central nervous system (CNS) dopaminergic and serotonergic activity. However, little attention has been given to the effects of sample handling on the concentrations of these compounds in human CSF. Using high-performance liquid chromatography (HPLC) with electrochemical detection, we observed that, in CSF stored at −80°C, concentrations of the serotonin metabolite 5-HIAA and the dopamine metabolite HVA remained unchanged through six 1-h and six 24-h freeze–thaw cycles. Exposure to bright room light (3 h, 1230 lux) resulted in a 5-HIAA concentration that was 96.3±2.0% of the initial and an HVA concentration that was 98.8±1.03% of initial. The pH of the CSF significantly increased during both freeze–thaw series and while maintained on ice (4°C). These results demonstrate the in-use stability of 5-HIAA and HVA in human CSF under commonly-encountered laboratory conditions.     

Brain Dialysis: In Vivo Metabolism of Dopamine and Serotonin by Monoamine Oxidase A but Not B in the Striatum of Unrestrained Rats

A dialysis cannula was implanted into rat striatum while the animals were anesthetized, and the area was perfused with Ringer solution while the animals were unanesthetized after at least 3 days following surgery. Concentrations of the metabolites of 3,4-dihydroxyphenylethylamine (DA) and 5-hydroxytryptamine (5-HT) in the perfusate were determined by HPLC with electrochemical detection. Levels of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the perfusate significantly decreased after pargyline administration (50 mg/kg i.p.), which may inhibit not only monoamine oxidase (MAO)-B but also MAO-A in these high doses. The level of the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) also decreased after pargyline treatment, although change in the relative level of 5-HIAA was less than that of DOPAC or HVA. To clarify the mechanisms for the metabolism of monoamines in rat striatum, highly specific MAO-A and -B inhibitors were used in the following experiments. Treatment with l-deprenyl (10 mg/kg), a specific inhibitor for MAO-B, did not cause any statistically significant change in DOPAC, HVA, and 5-HIAA levels. No significant change was found in rat striatal homogenates at 2 h after the same treatment with l-deprenyl. In contrast, low-dose treatment (1 mg/kg) with clorgyline, a specific inhibitor for MAO-A, caused a significant decrease in levels of these three metabolites in both the perfusates and tissue homogenates. In addition to the above three metabolites, the level of 3-methoxytyramine, which is an indicator of the amount of DA released, greatly increased after treatment with a low dose (1 mg/kg) of clorgyline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prenatal Exposure to Ozone Disrupts Cerebellar Monoamine Contents in Newborn Rats

Ozone (O3) is widely distributed in environments with high levels of air pollution. Since cerebellar morphologic disruptions have been reported with prenatal O3 exposure, O3 may have an effect on some neurotransmitter systems, such as monoamines. In order to test this hypothesis, we used 60 male rats taken from either, mothers exposed to 1 ppm of O3 during the entire pregnancy, or from mothers breathing filtered and clean air during pregnancy. The cerebellum was extracted at 0, 5, and 10 postnatal days. Tissues were processed in order to analyze by HPLC, dopamine (DA) levels, 3,4 dihydroxyphenilacetic acid (DOPAC) and homovanillic acid (HVA), norepinephrine (NA), serotonin, and 5-hydroxy-indole-acetic acid (5-HIAA) contents. Results showed a decrease of DA, NA, DOPAC and HVA mainly in 0 and 5 postnatal days. There were no changes in 5-HT levels, and 5-HIAA showed an increase after 10 postnatal days. DOPAC + HVA/DA ratio showed changes in 0 and 10 postnatal days, while 5-HIAA/5-HT ratio showed a slight decrease in 0 days. The data suggest that prenatal O3 exposure disrupts the cerebellar catecholamine system rather than the indole-amine system. Disruptions in cerebellar NA could lead to ataxic symptoms and also could limit recovery after cortical brain damage in adults. These finding are important given that recovery mechanisms observed in animals are also observed in humans.     

2-Guanidinoethanol increased dopamine release and 3,4-dihydroxyphenylacetic acid content,but not homovanillic acid content in the rat brain: Electroneurochemical and enzymological studies

The effects of 2-guanidinoethanol (GEt) on the release of monoamines and on the activity of their degrading enzymes were studied in order to investigate why 3,4-dihydroxyphenylacetic acid (DOPAC) increased to a much greater extent than homovanillic acid (HVA) after GEt injection into rat brain. In differential pulse voltammograms recorded using an electrochemically treated carbon fiber electrode, two distinct oxidation peaks, one at 130mV (DOPAC peak) and the other at 300 mV (5-hydroxyindoleacetic acid (5-HIAA) peak), were observed. In the hippocampus, the DOPAC peak increased markedly compared to the peak height recorded prior to the intracerebroventricular injection of GEt (6mol). Although the DOPAC peak height increased to 350% 4 hours after GEt injection, the 5-HIAA peak showed no change. In the striatum, the DOPAC peak increased to 150% 3 hours after GEt injection. Serial changes in the extracellular levels of DOPAC, HVA, and 5-HIAA were monitored in the striatum after GEt injection, using an in vivo brain micro-dialysis technique. Although the DOPAC levels strated to increase 80 minutes after GEt injection, HVA and 5-HIAA levels showed no change. On the other hand, monoamineoxidase, which metabolizes dopamine to DOPAC, was not activated and catechol-0-methyltransferase, which metabolizes DOPAC to HVA, were not inhibited by 5 mM of GEt in vitro. These data suggested that GEt increased the release of dopamine, but not of serotonin, and that GEt might restrict the DOPAC transport system.     

Fluoxetine increases extracellular levels of 3-methoxy-4-hydroxyphenylglycol in cultured COLO320 DM cells

Fluoxetine (Prozac) is a serotonin reuptake inhibitor. It increases extracellular levels of serotonin and is used in relieving the depressive symptoms of cancer patients. It has been reported that the drug may enhance the growth of certain cancer cells. This study investigates whether fluoxetine enhances the growth of a human colon cancer cell line (COLO320 DM) and if it affects the extracellular levels of serotonin or its metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA) and other monoamines and metabolites at two cell densities. The extracellular levels of serotonin, 5-HIAA and other monoamines and metabolites were measured simultaneously by high performance liquid chromatography from cell-culture media after incubation of cells both with and without fluoxetine for 3 days. The viability of COLO320 DM cells was evaluated using 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). At low cell densities (1.25x10(5) cells ml-1), fluoxetine at 1-10 microM significantly increased the extracellular levels of serotonin (p<0.005), 5-HIAA (p<0.005), and 3-methoxy-4-hydroxyphenylglycol (MHPG; p<0.001) as compared to the controls. Fluoxetine at 10-100 microM significantly inhibited the growth of COLO320 DM (p<0.005). At high cell densities (2x10(6) cells ml-1), fluoxetine at 1-10 microM significantly increased the extracellular levels of MHPG (p<0.01), and at 10 microM it significantly increased the extracellular levels of 5-HIAA (p<0.05). Fluoxetine at 100 microM significantly inhibited the growth of the cells (p<0.0001). These results suggest that fluoxetine at 1 microM of effective concentration may increase the extracellular levels MHPG, in addition to serotonin and 5-HIAA levels, yet not inhibit the growth of COLO320 DM.