the effect of copper on (3h)-tryptophan metabolism in organ cultures of rat pineal glands

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Metabolic Brain Disease [mebr] PP274-346419 October 27, 2001 16:19 Style file version Nov. 19th, 1999

Metabolic Brain Disease, Vol. 16, Nos. 3/4, December 2001 (c© 2001)

The Effect of Copper on (3H)-Tryptophan Metabolismin Organ Cultures of Rat Pineal Glands

Paresh Parmar1 and Santy Daya1,2

Received April 20, 2001; accepted June 21, 2001

Copper toxicity has been implicated in various neurodegenerative disorders such as Wilson’sdisease and Alzheimer’s disease. Free copper in the brain is toxic and leads to neuronaland cellular damage, through free radical generation. Melatonin has been investigated as apossible copper ion chelator. Melatonin could prevent copper-induced neuronal and cellulardamage through binding with copper and preventing copper-induced free radical genera-tion. The effect of copper on pineal indolamine synthesis has not been studied extensively.In the present study, copper (2 mg/kg) and melatonin (12 mg/kg) were administered dailyto Wistar rats for a 2-week and 6-week period. Pineal organ culture was utilized to monitorpineal indolamine synthesis. The pineals from the 2-week copper/melatonin-treated groupshowed a statistically significant decrease in 5-methoxytryptophol synthesis (p < 0.01),compared to the pineals from the copper-treated group. Conversly, in the 6-week experiment,5-methoxytryptophol synthesis was increased in both the copper- and copper/melatonin-treated groups. There was a statistically significant decrease in theN-acetyl serotoninlevel in the pineals from the 6-week copper-treated animals, as compared to the control-and copper/melatonin-treated group (p < 0.01). These results imply that copper reducesN-acetyltransferase activity, which results in a decrease inN-acetyl serotonin synthesis.Melatonin when coadministered with copper appears to prevent the N-acetyltransferase in-hibition by copper. Copper exerts contradictory effects on 5-methoxytryptophol synthesis.Further investigations need to be carried out to examine the effects of copper on the pinealenzymes.

Key words: melatonin; copper toxicity; pineal glands; indolamines.

INTRODUCTION

Copper has been found to be involved in the neuropathology of various neurodegen-erative disorders e.g. Wilson’s disease, Menkes disease, Parkinson’s disease, Alzheimer’sdisease, amyotrophic lateral sclerosis, and Prion disease (Sayreet al., 1999; Waggoneret al., 1999). Brain copper concentrations in patients dying of neurological Wilson’s dis-ease is higher than that of similar patients with hepatic Wilson’s disease (Walshe and Gibbs,1987). Copper is an essential trace and transition metal. It is the third most abundant traceelement found in the body, after iron and zinc. The cupric state (Cu2+) is found most often inbiological systems and copper plays a vital role as a cofactor and as an allosteric component

1Department of Pharmacology, School of Pharmacy, Rhodes University, Grahamstown, South Africa.2To whom correspondence should be addressed at Faculty of Pharmacy, Rhodes University, P.O. Box 94,Grahamstown 6140, South Africa. E-mail: [email protected]

1990885-7490/01/1200-0199/0C© 2001Plenum Publishing Corporation

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200 Parmar and Daya

of several cuproenzymes (Frieden, 1986; Uauyet al., 1998). In vivo, any copper not boundto a specific copper-protein is toxic.

In mammals, copper can be absorbed from the stomach to the distal small intestine.At least one-half of the amount of copper reaching the small intestine reappears in thebile as strongly bound compounds, and is lost in the stools. The distribution of copperthroughout the body is mediated by ceruloplasmin, albumin, transcuprein, and other quan-titatively less important copper-binding proteins (Wapnir, 1998). Copper ions are active inoxidation–reduction reactions and result in many pathologic conditions that are consistentwith oxidative damage to membranes or molecules.

β-Adrenoreceptor (β-receptor) function is impaired by oxidative stress, in various tis-sues (Van Der Vliet and Bast, 1992). The pineal gland has a rich supply ofβ-adrenergicreceptors. Theseβ-receptors are stimulated by norepinephrine, which then results in therapid synthesis of N-acetyltransferase (NAT). NAT is the rate-limiting step in the productionof melatonin (Feuer, 1990). In isolated tissue experiments with the administration of hydro-gen peroxide,β-adrenergic responsiveness is reduced. Airway tissue treated with hydrogenperoxide induced a reduction in smooth muscle relaxation by isoprenaline, which wasexpressed as a higher EC50 value and a decreased maximal response (Krameret al., 1987).

Peroxidation of lipid membranes leads to altered membrane viscosity, which affectsthe coupling of the receptor to the effector system. Oxidative stress, acting on membranes,increases the accessibility ofβ-receptors to hydrophobic, but not hydrophilic ligands. How-ever, more extensive oxidative stress decreases maximal binding toβ-receptors for bothhydrophobic and hydrophilic ligands (Kanekoet al., 1991). Thus it can be postulated thatcopper toxicity could lead to a decrease in NAT activity, which would result in a decrease inmelatonin production. Melatonin, a chain-breaking antioxidant, functions as a free radicalscavenger and repairs damaged macromolecules (restores membrane lipids, but not mem-brane proteins) (Poeggeleret al., 1994). Melatonin is a potent hydroxyl radical scavenger.Hydroxyl radicals are considered the most toxic and damaging radical of all the free radicalsproduced (Reiteret al., 1994).

Tryptophan is taken up from the blood stream into the pinealocytes, where it is utilized inthe synthesis of pineal indoles. Most of the tryptophan is converted to 5-hydroxytryptophanvia tryptophan hydroxylase in the mitochondria (Horiet al., 1976). In the synthesis of sero-tonin from tryptophan, this appears to be the rate-limiting step. 5-Hydroxytryptophan isconverted viaL-amino acid decarboxylase to serotonin (5HT) (Snyderet al., 1965). Sero-tonin can undergo various metabolic transformations, but specifically it can be acetylatedto N-acetylserotonin by the enzyme serotoninN-acetyltransferase (NAT), with acetyl coen-zyme A, a cofactor, being the acetyl donor (Weissbachet al., 1960).N-acetylserotonin isthe precursor of melatonin (Kleinet al., 1971).N-acetylserotonin is then converted to mela-tonin by O-methylation in the 5-position by the enzyme hydroxyindole-O-methyltransferase(HIOMT). Serotonin may also be methoxylated by HIOMT to form 5-methoxytryptamine.S-Adenosylmethionine donates the methyl to the serotonin. The pineal gland is responsiblefor the synthesis of melatonin (Reiter, 1989). The pineal gland contains high concentrationsof copper, zinc, and manganese, as compared to other anatomical brain structures (Wong andFritze, 1969). PINA, a novel splice variant of the ATP7B gene disrupted in Wilson’s disease,is expressed in pinealocytes. It is suggested that PINA may function as a copper transporterin rat pinealocytes, and studies by Borjiginet al.(1999a,b, 1999) suggest a potential role ofrhythmic copper metabolism in pineal and/or retinal circadian function. PINA expression

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The Effect of Copper on (3H)-Tryptophan Metabolism 201

patterns and regulation in vitro and in vivo parallel that of NAT expression, and both areregulated byβ-adrenergic stimulation via cAMP. LEC rat pineals (where PINA is deleted)display a defect in NAT protein and activity, and is independent of the PINA mutation(Borjigin et al., 1999). The high copper concentration in the pineal gland may be linked tothe regulation of NAT and PINA genes.

This study was conducted to determine the effects of copper and copper/melatoninadministration on pineal indolamine synthesis, over a 2-week and 6-week period inrats.

MATERIALS AND METHODS

Chemicals and Reagents

(3H) tryptophan (specific activity 55 mCi/mL) was obtained from Amersham Interna-tional, England. The culture medium, BGJb culture medium, was purchased from Gibco,Europe, and fortified with the antibiotics streptomycin and benzyl penicillin (Hoechst,South Africa). The aluminium TLC plates coated with silica gel 60, Type F254 (0.25mm), were purchased from Merck, Darmstadt, Germany. Beckman Ready-Sol multipur-pose liquid scintillation fluid was purchased from Beckman RIIC, Scotland. The indolestandards, melatonin (aMT),N-acetylserotonin (aHT), 5-methoxyindole acetic acid (MA),5-methoxytryptophol (ML), 5-hydroxyindoleacetic acid (HA), 5-hydroxytryptophol (HL)and serotonin (HT) were purchased from Sigma, St Louis, MO, U.S.A. All other reagentsand chemicals were obtained locally and were of the highest purity available.

Animals

Male Wistar rats weighing 250–300 g were used in the experiments. The rats wererandomly assigned to one of five groups of 5 rats per cage. The animals were housed inopaque plastic cages with metal grid floors and covers, under a diurnal lighting cycle 12 hlight:12 h dark, with food and water ad libitum. The intensity of the light during the 12-hlight phase was approximately 300 Watts/cm2. The temperature of the animal room wasmaintained between 20 and 25◦C, and the cages were cleaned daily. The food was providedad libitum and contained 5–8 mg of copper/kg. A control group (n = 5) received the drugvehicle ethanol:0.9% saline (40:60), for 2 weeks. A second control group (n = 5) receivedthe drug vehicle for 6 weeks. A copper-treated group (n = 5) received 2 mg/kg Cu2+

(copper chloride-CuCl2) for 2 weeks and another copper-treated group (n = 5) received2 mg/kg Cu2+ for 6 weeks. A copper/melatonin-treated group (n = 5) received 2 mg/kgCu2+ and 12 mg/kg melatonin for 2 weeks. Similarly, another copper/melatonin-treatedgroup (n = 5) received 2 mg/kg copper and 12 mg/kg melatonin for 6 weeks. The ratswere injected daily with the vehicle Cu2+ and Cu2+/melatonin. Copper/melatonin groupswere injected on opposite intraperitoneal sites to prevent any interaction of the copperwith the melatonin. The rats were sacrificed on Day 15 (for the 2-week group) and Day43 (for the 6-week group), respectively. The pineal glands were removed with a pair offorceps.

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Organ Culture of Rat Pineal Gland

The pineal glands were placed individually into sterile 75× 10 mm Kimble tubescontaining 52µL BGJb culture medium. Eight microlitres of (3H) tryptophan (specificactivity 55 mCi/mL) was added to each tube. The tubes were then saturated with carbogenand sealed. The tubes were then placed at 37◦C in the dark for 24 h in a Forma scientificmodel 3028 incubator. After the 24-h incubation period, the reaction was terminated by theremoval of the pineal glands from the solution.

Pineal Indole Separation

A modification of the technique employed by Klein and Notides (1969) was used toseparate the radiolabelled indoles. Five microlitres of the culture medium was spotted on aplate and, with the aid of a gentle stream of nitrogen, the spot was dried. Ten microlitresof the standard solution was spotted onto the culture medium, and dried with a gentlestream of nitrogen. The standard solution was prepared as follows: 0.1 mg of each standardindolamine was dissolved in a test tube containing 95% ethanol and 1% ascorbic acid. Theascorbic acid serves as an antioxidant. The plates were spotted under subdued light to preventphotooxidation of the indolamines. The plates were then placed in a TLC tank containingchloroform:methanol:glacial acetic acid (97:7:1). The plates were developed twice in thisdirection, and allowed to develop until the solvent front had migrated approximately 9 cm.The plates were allowed to dry, and then placed at 90◦ (right angles) to the first direction ofdevelopment, to develop once in ethyl acetate. Following this, the plates were allowed to dry,and then sprayed with Van Urks reagent, to allow for the visualization of the developed spots.Van Urks reagent is prepared by adding 1 g 4-dimethylamino-benzaldehyde to 50 mL 25%HCl, followed by the addition of 50 mL 95% ethanol. The sprayed plates were subsequentlydried in an oven at 60◦C for 10 min to allow for the color development of the spots. The spotswere then cut and individually placed into scintillation vials. To the vials, 1 mL absoluteethanol and 3 mL of Beckmans Ready-Sol scintillation fluid was added. The vials werevortexed on a Vortex rotor mixer for 30 s. The radioactivity of each metabolite was thenmeasured in a Beckman LS 2800 scintillation counter. The results (an average of threeestimations) obtained were expressed as DPM/10µL/pineal gland (mean± SEM) for eachindole. The background counts were negligible.

Statistical Analysis

Results from control, copper-treated and copper/melatonin-treated animals werecompared using ANOVA. Comparisons between groups were determined using theStudent-Newman–Keuls test. Comparisons between mean values were significantly dif-ferent ifp values were<0.05.

RESULTS

As shown in Table 1, copper/melatonin administration, significantly decreases (p <0.01) pineal (3H)-5-methoxytryptophol synthesis in pineals from the 2-week copper-treated

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Table 1.The Effect of 2-Week Copper Treatment on Rat Pineal Indole Metabolism

Mean± SEM (n = 3)

Pineal metabolites Controla Coppera Copper/Melatonina

N-acetyl serotonin (aHT) 25190± 2661 34929± 8737 22804± 81545-Hydroxyindole acetic acid (HA) 1275± 171 1612± 254 2067± 2795-Hydroxytryptophol (HL) 4311± 155 3432± 410 3151± 421Melatonin (aMT) 5843± 593 6885± 31 5157± 7685-Methoxyindole acetic acid (MA) 10847± 954 12336± 1213 8856± 9995-Methoxytryptophol (ML) 36930± 7083 31390± 7018 13392± 2145∗

aDPM/10µL/pineal.∗Significantly different from control and copper-treated 5-methoxytryptophol values,p < 0.01.

animals. It appears that melatonin administration decreases ML levels following 2 weeksof copper treatment, compared to pineal glands obtained from control and copper-treatedanimals.

The mean values of the pineal glands obtained from the 6-week copper-treated animals(Table 2) were significantly different between the aHT control and copper pineals (p <0.01). The mean copper aHT and copper/melatonin aHT values are significantly different(p < 0.01). The ML levels were increased in both the copper-treated and copper/melatonin-treated rat pineals, and were significantly different to that of the control (p < 0.01).

DISCUSSION

The rat pineal gland in organ culture is able to metabolise (3H)-tryptophan to vari-ous pineal indoles. As shown in Table 1, the 2-week copper administration did not affectML synthesis, but a significant difference between copper ML and copper/melatonin MLsynthesis (p < 0.01) was obtained. It appears that melatonin administration decreased MLsynthesis, possibly by interacting with HIOMT.

In the 6-week experiments (Table 2), the aHT level was decreased by copper andthis was significantly different atp < 0.01 between the copper and control pineals, which

Table 2.The Effects of 6-Week Copper Treatment on Rat Pineal Indole Metabolism

Mean± SEM (n = 3)

Pineal metabolites Controla Coppera Copper/Melatonina

N-acetyl serotonin (aHT) 15779± 3391 9453± 947∗ 18609± 1861∗∗

5-Hydroxyindole acetic acid (HA) 2347± 275 3534± 495 5191± 5925-Hydroxytryptophol (HL) 2776± 163 2318± 188 4687± 1015Melatonin (aMT) 1613± 96 1364± 159 2957± 13295-Methoxyindole acetic acid (MA) 4640± 781 7974± 2257 5014± 20615-Methoxytryptophol (ML) 8714± 1453 18023± 3022∗ 17644± 1885∗

aDPM/10µL/pineal.∗Significantly different from control value,p < 0.01.∗∗Significantly different from copper-treated rat value,p < 0.01.

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implies that copper administration decreases the aHT synthesis. Melatonin administrationtogether with copper in the copper/melatonin-treated rats reversed the effect of copper,as the values obtained were similar to the control. Copper administration increased theML concentration relative to control values (p < 0.01). Coadministration of melatonindid not reverse the effect of copper on ML levels, since the pineal ML concentrationof copper/melatonin-treated rats was not significantly different from that of the copper-treated rat values (Table 2). It appears that copper affects the NAT enzyme, resulting ina decrease of aHT production from serotonin. This may imply that melatonin is interact-ing with copper and preventing the inhibition of the NAT activity. Further investigationsare needed to determine NAT activity in the copper and copper/melatonin administeredpineals.

ML synthesis in 6-week pineals was different to that of 2-week pineals. After 6 weeks,copper and copper/melatonin administration increased ML synthesis significantly (p <0.01). Further investigations are needed to determine HIOMT activity in the 6-week exper-iments with copper and copper/melatonin administration.

The pineal gland has a high concentration of copper (Wong and Fritze, 1969). SincePINA is a copper transporter in the pineal gland, and it’s expression parallels that of NAT,it appears that at night when PINA activity is at its maximum (NAT levels peak at night),copper is rapidly transported into the pinealocytes. aHT levels were decreased by copper inthe 6-week copper-treated pineals. The aHT level approximates that of the control, whencopper/melatonin was administered. Copper may interact with NAT, through nonspecificbinding, and thus decreases NAT activity. Copper might inhibit the NAT enzyme, renderingit unavailable to convert serotonin toN-acetylserotonin.

It has been shown that NAT activity appears to be insensitive to elevated levels of copper(Borjigin, personal communication). These authors also showed that melatonin synthesisand release is normal in LEC rats, a Wilson’s disease model. Thus, it appears that elevatedcopper does not affect melatonin synthesis (Borjigin, personal communication). The presentfindings are in agreement with the findings of Borjiginet al., since no change in melatoninlevels were observed.

ACKNOWLEDGMENTS

This study was made possible by grants from the South African Medical ResearchCouncil and the National Research Foundation to Professor S. Daya.

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the effect of copper on (3h)-tryptophan metabolism in organ cultures of rat pineal glands

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