Journal of Neurochemistry Raven Press, New York 0 1984 International Society for Neurochemistry

Lesch-Nyhan Syndrome, Caffeine Model: Increase of Purine and Pyrimidine Enzymes in Rat Brain

M. D. Minana, M. Portoles, A. Jorda, and S. Grisolia

Instituto de Investiguciones Citolbgicas de la Ccrja de Ahorros de Valencia, Valencia, Spuin

Abstract: Rats ingesting high doses of caffeine reproduce the self-destructive behaviour observed in the Lesch- Nyhan syndrome. This syndrome includes a deficit in hypoxanthine-guanine phosphoribosyltransferase. We have observed, however, that the activity of hypoxan- thine-guanine phosphoribosyltransferase increases in di- rect proportion to the concentration of caffeine found in rat brain. It appears, therefore, that the caffeine model is not a true model for the Lesch-Nyhan syndrome, or al- ternatively, that the deficit in hypoxanthine-guanine phosphoribosyltransferase is coincidental and not a main key to the multifarious aspects of the syndrome. partic-

ularly the self-mutilation. The possibility that levels of dopamine are increased in the caffeine model are dis- cussed as a basis for the destructive behaviour. We have found also that ingestion of large amounts of caffeine in- creases the activities in rat brain of adenosine deaminase, purine nucleoside phosphorylase, aspartate carbamoyl- transferase, dihydroorotase, and dihydroorotate oxidase. Key Words: Purines-Pyrimidines-Lesch-Nyhan syn- drome-Caffeine. Miiiana M. D. et al. Lesch-Nyhan syndrome, caffeine model: Increase of purine and pyrim- idine enzymes in rat brain. J . Neurochem. 43, 1556-1560 ( 1984).

The Lesch-Nyhan syndrome is an X-linked ge- netic disorder associated with a virtually complete deficiency of the enzyme of purine metabolism, hy- poxanthine-guanine phosphoribosyltransferase (HGPRTase; 1MP:pyrophosphate phosphoribosyl- transferase; EC 2.4.2.8) (Seegmiller et al., 1967). It is characterized clinically by hyperuricemia, exces- sive production of uric acid, and certain character- istic neurological features including self-mutilation, choreathetosis, spasticity, and mental retardation (Nyhan et al., 1965; Kelley and Wyngaarden, 1983). Increases in the levels of enzymes that participate in de novo synthesis of purines have been detected (Seegmiller et al., 1967; Kogut et al., 1970; Raivio and Seegmiller, 1973; Zoref and Sperling, 1980). Further, there is evidence that de novo biosynthesis of pyrimidines is increased (Kennedy, 1978; Mar- tinez-Ramon et al., 1979).

The greater synthesis of purines and pyrimidines as well as the increased activity of adenine phos- p hori bo s y 1 t r an sfera s e AMP : p y ro - phosphate phosphoribosyltransferase; EC 2.4.2.7)

( APRTa s e ;

(Lloyd et a l . , 1981; Kelley and Wyngaarden, 1983) are attributed to a greater content of 5-phos- phoribosyl- I-pyrophosphate (PRPP), a substrate common to both pathways (Torrelio and Paz, 1979). In addition, an increase in the activity of ribose- phosphate pyrophosphokinase (EC 2.7.6.1) has been reported (Reem, 1975; Brosh et al., 1976).

It is known that rats administered large doses of caffeine reproduce the self-destructive behaviour observed in the Lesch-Nyhan syndrome (Boyd et al., 1965). Waldren and Patterson (1979) found that caffeine had an inhibitory effect on the biosynthesis of purines and concluded that this inhibition oc- curred in an early step of the de novo biosynthesis. Rumsby et al. (1982) have shown that caffeine also inhibits pyrimidine biosynthesis and found that the blockage occurred in the fifth of sixth step of the pathway. These authors (Waldren and Patterson, 1979; Rumsby e t al . , 1982) suggested that PRPP must be implicated in the effect of caffeine, since it is the only substrate used in both the purine and the pyrimidine pathways.

Received December 20, 1983; revised April 17, 1984; accepted April 27, 1984.

Address correspondence and reprint requests to M. D. Mifiana, Instituto de Investigaciones Citologicas de la Caja de Ahorros de Valencia, Arnadeo de Saboya, 4 Valencia-10. Spain.

Abbreviations used: ADA, Adenosine deaminase; APRTase,

Adenine phosphoribosyltransferase; ATCase, Aspartate carba- moyltransferase; DHOase, Dihydroorotase; Dopamine, 3,4-Di- hydroxyphenylethylamine; HGPRTase, Hypoxanthine-guanine phosphoribosyltransferase; PNP, Purine nucleoside phosphory- lase; PRPP, 5-Phosphoribosyl-l-pyrophosphate; TTP, Thymidine triphosphate.

1556

CAFFEINE AND LESCH-NYHAN SYNDROME 1557

Because there is a neurological disorder in the Lesch-Nyhan syndrome, we studied the activities of the enzymes HGPRTase, adenosine deaminase (ADA; adenosine aminohydrolase; EC 3.5.4.4) , purine nucleoside phosphorylase (PNP; purine-nu- c1eoside:orthophosphate ribosyltransferase; EC 2.4.2.1 ) , aspartate carbamoyltransferase ( ATCase ; carbamoy1phosphate:L-aspartate carbamoyltrans- ferase; EC 2.1.3.2), dihydroorotase (DHOase; L- 5,ti-dihydroorotate aminohydrolase; EC 3.5.2.3; and dihydroorotate oxidase; EC 1.3.3.1) in brains of rats treated with caffeine and with signs of self- mutilation. We found an increase in the HGPRTase activity, which was, in addition, correlated with the caffeine levels of plasma and brain.

MATERIALS AND METHODS Materials

PRPP, thymidine triphosphate (TTP), carbamoyl phos- phate disodium salt, and xanthine oxidase were pur- chased from Sigma Chemical. All other chemicals were of the highest purity available.

Treatment of animals Male and female Wistar rats weighing 150-200 g were

used. The animals were fed a standard diet and caffeine in their drinking water. The caffeine was administered gradually so that the animals became accustomed to it: 2 g/L the first 3 days, 4 g/L the next 3 days, and finally 8 g/L from 4 to 6 days, depending on the time needed for the rats to show symptoms of self-mutilation. Sucrose 100 g/L was added to the caffeine solutions. Pair-fed control rats were given the same diet and sucrose solution without caffeine.

During the experimental period, the rats were housed in plastic boxes, one animal per box at 22"C, 60% hu- midity, on a 12-h light/dark cycle. Rats were sacrificed by decapitation, and the brains were quickly removed and used immediately.

Enzyme assays Dihydroorotate oxidase was assayed as described by

Kennedy (1973). The tissue was homogenized in nine vol- umes of 0.25 M sucrose and the homogenate was used directly to test enzyme activities. The incubation mixture contained 200 pmol of Tris-HC1 (pH 8); 2 pmol of sodium dihydroorotate; and enzyme in a total volume of 2 ml. After incubation for 30 min at 37"C, 1 ml of 2 M HCIO, was added and centrifuged. The absorption at 280 nm, compared with a suitable blank, was measured.

ATCase was assayed as described by Kennedy (1978). The brain was homogenized in four volumes of 0.25 M sucrose and the homogenate was centrifuged for 10 min at 20,000 g. The supernatant portion was assayed. The assay system contained 200 kmol of Tris-HC1 (pH 8.5); 20 Fmol of sodium aspartate (pH 7); 2.8 pmol of disodium carbamoylphosphate; and enzyme in a final volume of 2 ml. The incubation was carried out at 37°C for 20 min and was stopped by addition of 0.2 ml of 4 M HC10,. After centrifugation, a portion of the supernatant was an- alysed (Prescott and Jones, 1969).

The DHOase activity of brain tissue was assayed as described by Mori et ai. (1975).

HGPRTase was estimated using hypoxanthine as sub- strate and oxidizing the hypoxanthine remaining after ter- mination of the HGPRTase reaction to uric acid with xan- thine oxidase (EC 1.2.3.2) (Kennedy, 1978). The brains were homogenized in three volumes of 0.05 M Tris-HC1 (pH 7.4), containing 1 mM mercaptoethanol and 0.1 mM EDTA and the crude homogenate was centrifuged for 15 min at 16,000 g (Gutensohn and Guroff, 1972). The su- pernatant solution was assayed essentially as described by Kennedy (1978), except that 3.3 mM TTP was in- cluded.

For measurement of PNP and ADA activities, the brain was homogenized in four volumes of 0.25 M sucrose and the homogenate was centrifuged for 10 min at 20,000 g. The supernatant portion was assayed. PNP activity was estimated using inosine as substrate and measuring by HPLC the hypoxanthine produced (Halfpenny and Brown, 1980). When the ADA activity was estimated, adenosine was the substrate and the inosine formed was measured by HPLC (Hartwich et al., 1978).

Caffeine in plasma and brain was measured according to the method of Blanchard et al. (1980). Protein was determined by the method of Lowry et al. (1951), with bovine serum albumin as standard.

Statistical significance between experimental and con- trol conditions was measured by Student's t test.

RESULTS Table 1 shows the amount of food and drink in-

gested by the animals during the treatment. There was a gradual decrease in their consumption of food and fluid, but this decrease was less pronounced than when the animals were given 8 g caffeine/L from the beginning (not shown).

The concentration of caffeine found in the plasma of male rats was 19.6 2 3.9 Fg/ml plasma whereas that of the female rats, interestingly, was 27.8 t 5.2 pg/ml plasma.

It is also of much interest that the concentration of caffeine in brain was 1.5 t 0.1 times that found in plasma, assuming, as is generally believed, that 80% of the tissue is water. The concentration in the female brains was also higher (Table 2).

Figure 1 shows an increase in the activities of the enzymes of the salvage pathway of purines, HGPRTase, ADA, and PNP. This increase is greater in females than in males, possibly due to the greater

TABLE 1. Effects of caffeine on food andfluid intakes

Food intake (giday) Fluid intake (mllday) Caffeine

(dL) Males Females Males Females

2 18.9 5 1.4 16.0 _f 4.0 23.0 5 2.9 18.9 k 3.8 4 11.6 ? 1.8 9.8 2 1 . 1 17.5 2 2.9 14.7 2 1.5 8 9 . 3 r 1 . 9 8 . 1 5 2 . 1 13.421.5 1 0 . 3 k 2 . 0

Caffeine was administered to female (n = 25) and male rats (n = 20) in their drinking fluid. The experimental conditions were as described in the text. Values are means t SD.

J . Neurochem., Vol. 43, No. 6 , 1984

1558 M . D. MINANA ET A L .

TABLE 2. Levels of caffeine in plasma and brains of male and female rats

~

Caffeine concentration Males Females

kg/g brain 24.5 2 4.9 31.9 i 7.8 pgiml plasma 19.6 '-e 3.9 27.8 i 5.2

Levels of caffeine in plasma and brains of male (n = 20) and female rats (n = 25). The rats were treated as described in the text. Values are means '-e SD.

concentration of caffeine found in the brains of the female rats.

In Fig. 2 a correlation can be seen between the increased activity of HGPRTase and the concentra- tion of caffeine in brain.

In Fig. 3 an increase can also be seen in the ac- tivity of three enzymes of the pathway of de n o w biosynthesis of pyrimidines: ATCase, DHOase, and dihydroorotate oxidase. Once again, a greater in- crease is observed in the females than in the males.

From these experiments, it is apparent that fe- males are more sensitive to caffeine than males.

DISCUSSION

The experiments presented here show that ani- mals ingesting caffeine for a period of time varying

' q 3 - T . .~ a PI,? 1. .a:.

FIG. 1. Levels of ADA, PNP, and HGPRTase of brains of con- trol (0) and caffeine-treated (El) rats. The rats were treated as described in the text. Enzyme activities were assayed as detailed in Materials and Methods. ADA values are expressed in nmol/min/mg protein, PNP and HGPRTase in pmollhimg protein. Values are means ? SD obtained from seven to nine animals. 'p < 0.05; **p < 0.01 ; '**p < 0.001,

A

A

A

0.0 ' I I I I I

'1 10 20 30 (10 io

CAFFEINE ,' G9 BRAIN

FIG. 2. Relationship between HGPRTase activity and caf- feine concentration. Specific activities are expressed as pmollhirng protein. Each point refers to one brain from a treated animal; 0 pg caffeineig brain denotes control rats (n = 10). Values are the averages of duplicate determinations.

from 10 to 12 days (just until they show signs of self-mutilation) have increased levels of ATCase, DHOase, and dihydroorotate oxidase in the brain. This finding is consistent with the increased enzyme levels observed in fibroblasts and erythrocytes of patients with Lesch-Nyhan syndrome (Kennedy, 1978; Torrelio and Paz, 1979).

0,2 40

30

20 0 . 1

5 10 z - t 0 a

w b- 0 x

' 2 0 0 . 0 :

_I 40 _1

5

II \

50 , 1

\ 0 . 2

0

a. 30

0.1 20

10

0 0 . 0 D~HYDRO-OROTATE DHOASE ATCASE

OXIDASE

FIG. 3. Activities of ATCase, DHOase, and dihydroorotate ox- idase of brains of control (0) and caffeine-treated (El) rats. The rats were treated as described in the text. Enzyme activ- ities were assayed as detailed in Materials and Methods. ATCase values are expressed in pmolihimg protein, DHOase and dihydroorotate oxidase in nmollhimg protein. Values are means ~t SD obtained from seven to nine animals. *p < 0.05; "D < 0.01.

J . Neurochem., Vol. 43, No . 6, 1984

CAFFEINE A N D LESCH-NYHAN SYNDROME 1559

We have also observed increased activity of HGPRTase, ADA, and PNP, enzymes involved in the salvage pathway of the biosynthesis of purines. Earlier studies (Ferrer et al., 1982) showed in- creases in the levels of carbamoyl phosphate syn- thetase (ammonia) (EC 6.3.4.16) and of ornithine carbamoyltransferase (EC 2.1.3.3) in livers of rats ingesting high doses of caffeine.

Waldren and Patterson (1979) found that caffeine had an inhibitory effect on the first steps of the purine biosynthetic pathway. However, it is un- likely that it acts on the phosphoribosylpyrophos- phate amidotransferase (EC 2.4.2.14), as, in the presence of 20 mM caffeine in cell-free extracts (Ehmann et al., 1975), its activity was 85% of normal. In addition, it has been shown that caffeine does not have an inhibitory effect on phosphoribo- syltransferase activity (Lau and Henderson, 1972). Rumbsy et al. (1982) have found that 40 mM caf- feine inhibited the phosphoribosylpyrophosphate synthetase by only 10-20%.

In the Lesch-Nyhan syndrome with self-destruc- tive behaviour, there is a deficient activity of HGPRTase in all tissues. As shown here, adminis- tering high doses of caffeine in the rat can repro- duce the same behaviour, but the enzyme activity in brain is increased. That is to say, self-mutilation is produced in both cases in spite of the opposite change in activity of this enzyme. This suggests that the self-destructive behaviour reflects factors other than the activity of this particular enzyme.

Lloyd et al. (1981) have shown a 3,4-dihydroxy- phenylethylamine (dopamine) deficiency in the basal ganglia of Lesch-Nyhan brains. Mueller et al. (1982) and Mueller and Nyhan (1982) suggested that the self-mutilation in the Lesch-Nyhan syndrome could be due to a dopamine receptor supersensi- tivity. In rats treated with caffeine, there is an in- crease in adrenal dopamine (Snider and Waldeck, 1974). Ungerstedt et al. (1981) showed that rota- tional behaviour is inhibited by dopamine antago- nistic drugs.

It is now generally agreed that many of the effects of methylxanthines, including caffeine, are me- diated through action on adenosine receptors (Fred- holm, 1980; Rall, 1980). Fredholm (1982) showed that after 1 week of treatment with caffeine the number of adenosine receptors in rat cerebral cor- tical membranes was increased. Interestingly, Green et al. (1982) have suggested that adenosine may be involved in the modulation of dopaminergic function in the striatum.

Thus, it is possible that the self-destructive be- haviour observed in the Lesch-Nyhan syndrome and in rats given high doses of caffeine may have a similar mechanism, being mediated by a dopami- nergic component that could, in turn, be modulated by adenosine receptors.

Adenosine depresses cell firing in the mammalian

CNS (Phillips and Wu, 1981) and inhibits release of neurotransmitters in various central and peripheral neuronal tissues (Fredholm and Hedqvist, 1980). The central stimulant effects of methylxanthines such as caffeine are linked to a blockage of adeno- sine receptors (Daly et al., 1981).

Increased neurological activity induced by caf- feine presumably underlies an increased need for nucleotide synthesis, which implies a great activity of the enzymes catalysing the steps of the purine salvage pathways. At the same time, the increased activity of the enzymes catalysing the first steps of the pyrimidine biosynthetic pathway does not nec- essarily indicate increased biosynthesis. Further studies on the control of purine and pyrimidine bio- synthesis are necessary in approaching these ques- tions.

Acknowledgments: This work was supported in part by the International Molecular Program of the IIC- KUMC. We thank Dr. W. L. Nyhan for his interest in this work and F. Thompson for critically reading the manuscript. We also wish to thank the referee for his helpful comments. M. D. Miriana is a fellow of “Seccion de Metabolopatias” of the Instituto de Investigaciones Citologicas.

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