Theobromine and theophylline

Theobromine and theophylline

Mutation Research, 32 (1975) 1 6 9 - 1 7 8 © Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands 169 THEOBROMINE A N D T...

551KB Sizes 5 Downloads 22 Views

Mutation Research, 32 (1975) 1 6 9 - 1 7 8 © Elsevier Scientific Publishing Company,

Amsterdam--Printed

in The Netherlands

169

THEOBROMINE A N D T H E O P H Y L L I N E

J. TIMSON

Department of Medical Genetics, The Medical School, The University, Manct~ester, Mac3 9PL, (Great Britain) ( R e c e i v e d J u n e i 7 t h , 1975) ( R e v i s e d r e c e i v e d S e p t e m b e r 3 r d , 1975) ( A c c e p t e d S e p t e m b e r i 2 t h , 1975)

CONTENTS Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physiological and biochemical activity . . . . . . . . . . . . . . . . . . . . . . . Chromosome abnormalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitotic inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutagenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Comparison with 1elated compounds . . . . . . . . . . . . . . . . . . . . . . . . Caffeine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Paraxanthine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Monomethylxanthines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methyluric acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xanthine, hypoxanthine and uric acid . . . . . . . . . . . . . . . . . . . . . . Structure-activity relationships . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

169 17o 17 ° 171 171 172 172 173 173 173 174 174 174 175 175 175 176

SUMMARY

Theobromine and theophylline have a limited therapeutic use and in addition they occur in plants used in the preparation of a number of widely consumed drinks. Thus most of the population must be exposed to both compounds. Chromosome abnormalities are caused by both theobromine and theophylline in plant cells and in mammalian cells in culture, and both have anti-mitotic activity. While they are fairly potent mutagens in Escherichia coli and other lower organisms the rather scanty available evidence suggests that they are not mutagenic in mammals. The difference in mutagenic activity m a y be due to the reported inability of E. coli to demethylate these compounds, a process which occurs readily in mammals including man. The structure-activity relationships of these compounds are complex but the available evidence suggests that methylation at position I is the most important for both mutagenic activity and the anti-mitotic effect while methylation at position 3 is of most importance in the action on chromosomes.

17o

j. TIMSON

INTRODUCTION

T h e o b r o m i n e (3,7-dimethylxanthine) a n d t h e o p h y l l i n e ( I , 3 - d i m e t h y l x a n t h i n e ) b o t h have the empirical formula, CTHsN402. Like the chemically r e l a t e d caffeine (I,3,7-trimethylxanthine) t h e y occur n a t u r a l l y in a n u m b e r of p l a n t s 11. F o r m a n it is i m p o r t a n t t h a t t h e y are found in p l a n t s used in t h e m a n u f a c t u r e of some w i d e l y cons u m e d drinks. T h e o b r o m i n e is p r e s e n t in t e a 1~, m a t d 11, a n d all t h e c o c o a - b a s e d beveragesll, 41,4s, while t h e o p h y l l i n e is f o u n d in t e a 11,48. T h e o b r o m i n e is also present in chocolate w i t h plain chocolate c o n t a i n i n g o. 4 o.7% t h e o b r o m i n e a n d m i l k chocolate o.2-o.5°/o 41, I t is reasonable, therefore, to assume t h a t a l m o s t e v e r y o n e is exposed, at least to some e x t e n t , to one or b o t h compounds. B o t h t h e o b r o m i n e a n d t h e o p h y l l i n e h a v e found a l i m i t e d a p p l i c a t i o n in medicine. T h e o b r o m i n e has been used as a diuretic, as a cardiac s t i m u l a n t , a n d for the d i l a t i o n of c o r o n a r y a n d p e r i p h e r a l arteries 1. The r e c o m m e n d e d dose is 3 o o - 6 o o rag. Side effects, m a i n l y n a u s e a a n d anorexia, h a v e been o b s e r v e d only with large doses 1. F o r p h a r m a c e u t i c a l use t h e o b r o m i n e is p r e p a r e d from the dried ripe seed of Theobroma cacao or synthetically~% T h e o p h y l l i n e has a d i u r e t i c effect g r e a t e r t h a n theob r o m i n e or caffeine b u t of short d u r a t i o n ~. I t has also been used as a r e l a x a n t of inv o l u n t a r y muscle a n d has been quite successfully used in t h e t r e a t m e n t of a s t h m a a n d b r o n c h i t i s 1. T h e o p h y l l i n e is a v a l u a b l e d r u g in t h e e m e r g e n c y t r e a t m e n t of congestive h e a r t failure 1~. The r e c o m m e n d e d dose is 6o 2oo mg w i t h a m a x i m u m of I g m in 24 hs. I t can cause gastric irritation, n a u s e a a n d v o m i t i n g 1 a n d being more toxic t h a n either t h e o b r o m i n e or caffeine it has occasionally p r o v e d f a t a l 12. I t has been r e p o r t e d t h a t severe t h e o p h y l l i n e t o x i c i t y can occur with n o r m a l doses when the p a t i e n t has an i m p a i r e d t h e o p h y l l i n e d e g r a d a t i o n ability 17. F o r t h e r a p e u t i c use it is n o r m a l l y p r e p a r e d s y n t h e t i c a l l y using urea as t h e s t a r t i n g c o m p o u n d 39. U n d e r labo r a t o r y conditions it has been shown t h a t t h e o p h y l l i n e can suppress t u m o u r growth in r a t s 18 which could lead to an extension of its m e d i c a l use. Continued use of either t h e o b r o m i n e or t h e o p h y l l i n e m a y lead to t h e d e v e l o p m e n t of tolerance especially to t h e diuretic action ~2. Cross-tolerance between these c o m p o u n d s a n d caffeine also O c c u r s 12.

METABOLISM

In m a n b o t h t h e o b r o m i n e a n d t h e o p h y l l i n e are r e a d i l y a b s o r b e d b y the oral a n d all o t h e r routes 1~. A small p o r t i o n of either c o m p o u n d is e x c r e t e d u n c h a n g e d in the urine b u t m o s t is m e t a b o l i s e d to m e t h y l x a n t h i n e s a n d m e t h y l u r i c acids 7. T a b l e I gives a s u m m a r y of the results o b t a i n e d b y CORNISH AND CHRISTMAN7 b y analysis of t h e urine of i n d i v i d u a l s on a diet free from coffee, t e a a n d cocoa to w h o m a I g dose of t h e o b r o m i n e or t h e o p h y l l i n e was a d m i n i s t e r e d . Caffeine in the same i n v e s t i g a t i o n also y i e l d e d a m i x t u r e of m e t h y l x a n t h i n e s a n d m e t h y l u r i c acids in t h e urine. Similar results were o b t a i n e d when t h e o p h y l l i n e alone was i n v e s t i g a t e d 2. I t is suggested t h a t in m a n d e m e t h y l a t i o n can occur at t h e I, 3, a n d 7 positions a n d t h a t the most stable p o s i t i o n is I followed b y 7 a n d 3 respectively. H o w e v e r it a p p e a r s t h a t the d e m e t h y l a tion of t h e o b r o m i n e a n d t h e o p h y l l i n e does n o t go b e y o n d the m o n o m e t h y l x a n t h i n e stage since no a c c u m u l a t i o n of x a n t h i n e in the urine or increase in uric acid excretion was found 7. The m e t a b o l i c p a t h w a y s of t h e o b r o m i n e a n d theophy!line are clearly

THEOBROMINE TABLE

171

AND THEOPHYLLINE

I

METHYLXANTHINES AND METHYLURIC ACIDS EXCRETED IN 4 8 H PERIOD AFTER INGESTION OF I GRAM OF THEOBROMINE OR THEOPHYLLINE All v a l u e s a s p e r c e n t a g e of o r i g i n a l i n g e s t e d c o m p o u n d

required to give the excretory product.

Compound excreted

Compound ingested Theobromine Theophylline

Theobr omine 3-methylxanthine 7-methylxanthine 7-methyluric acid Theophylline i-methyluric acid 1,3-dimethyluric acid

12.o 5 19.85 28. o 5 3.9o

Total recovered

63.85

Adapted

13-3 °

9.9o 18.6o 35.oo 76.8o

f r o m : CORNISH AND CHRISTMAN 7.

different. This may be because the methyl groups in theophylline are both on the pyrimidine ring leaving the imidazole ring unsubstituted. It is suggested that this may be the reason why oxidation without demethylation is a major metabolic pathway for theophylline but not for theobromine z. The metabolism of theophylline in rats and rabbits has been shown to be very similar to that in man 49. PHYSIOLOGICAL AND BIOCHEMICAL ACTIVITY

Both theobromine and theophylline delayed or inhibited fertilisation between the gametes of Arbacia punctulata 4 and theophylline inhibits maturation of mouse oocytes in vitro 5. The effect of local subcutaneous injection of theophylline on wound healing in rats was to suppress the rate of cell proliferation and migration 52. Both theobromine and theophylline inhibited the aggregation of rabbit polymorphonuclear leucocytes in vitro 27. Theophylline will also inhibit platelet aggregation a4. It is well established that theophylline can cause an accumulation of cyclic AMP by the inhibition of phosphodiesterase in a number of systems 3,5,I6,~s,28,31,33,34,45,~2. Theobromine may also inhibit this enzyme but is much less active ~. In E. coli it has been found that both theobromine and theophylline will inhibit some of the purine nucleoside phosphorylases of both the ribose and deoxyribose types 2". Theophylline has also been shown to inhibit phosphorylase from rabbit muscle 22, and to modify the activity of L-serine dehydratase extracted from the gut of Lumbr~cus terres~ris 3~. CHROMOSOME ABNORMALITIES

Theobromine and theophylline both induce breaks in the chromosomes of HeLa cells 4°, and in human lymphocytes in cultureS°, 51. Theophylline may have been involved in the induction of chromatid breaks in the cultured lymphocytes of the mother of a stillborn triploid girl (69, xxx) 15 but it would be unsafe to extrapolate from a single case. In plants theophylline has been shown to induce fragment formation and translocations in the chromosomes of A l l i u m cepa root tips19, 2° although it is less active than caffeine while theobromine has been found to cause chromosome breakage in Oedogonium acmandrium 43. When Drosophila males were fed theobromine or theo-

172

j. TIMSON

phylline for 24 h before m a t i n g there was a significant increase in the n u m b e r of x o males in t h e p r o g e n y of t h e t h e o p h y l l i n e - t r e a t e d flies b u t n o t in the p r o g e n y of those t r e a t e d w i t h t h e o b r o m i n e 35. B o t h t h e o b r o m i n e a n d t h e o p h y l l i n e have been shown to p o t e n t i a t e t h e p r o d u c t i o n of c h r o m a t i d a b e r r a t i o n s b y t h i o t e p a in Viciafaba root t i p , a n d in Chinese h a m s t e r cell cultures 24. T h e o p h y l l i n e was more active t h a n t h e o b r o mine while caffeine was more active t h a n either. The p o t e n t i a t i o n for all conlpomld~ was c o n s i d e r a b l y g r e a t e r in Vicia faba t h a n in the Chinese h a m s t e r cells. MITOTIC INHIBITION

T h e o b r o m i n e a n d t h e o p h y l l i n e b o t h cause an inhibition of mitosis in h u m a n l y m p h o c y t e s s t i m u l a t e d w i t h p h y t o h a e m a g g l u t i n i n in culture*7,*a,s°, 5~. T h e o p h y l l i n e was more a n t i - m i t o t i c t h a n t h e o b r o m i n e b o t h when a d d e d at the s t a r t of a 72-h culture47, 48 or after 4 8 h 5°,51. T h e o p h y l l i n e is c y t o t o x i c to h u m a n l y m p h o c y t e s in culture at c o n c e n t r a t i o n s of IO aM a n d h i g h e r ~ , .8. I t has also been found to inhibit e p i d e r m a l mitosis in t h e G~ phase in the mouse ear at a c o n c e n t r a t i o n of 5" I o - 3 M (ref. 33). Theob r o m i n e acts as a m i t o t i c i n h i b i t o r in Oedogonium acmandrium4"L In Allium cepa root tips b o t h t h e o b r o m i n e a n d t h e o p h y l l i n e s u p p r e s s e d cell wall f o r m a t i o n a l t h o u g h t h e y d i d not i n h i b i t spindle function2L Their action, therefore, is not d i r e c t l y c o m p a r a b l e w i t h t h a t of colchicine. A similar a c t i v i t y has been o b s e r v e d in t h e roots of Arena sativa a n d Pisum sativum where t e t r a p l o i d a n d tri- a n d q u a d r i n u c l e a t e cells were p r o d u c e d 13. Bi- a n d t r i n u c l e a t e cells were f o r m e d in Oedogonium acmandrium t r e a t e d with t h e o b r o m i n e 43. The effect of these c o m p o u n d s on mitosis is clearly complex. I t is c o m p l i c a t e d b y t h e t o x i c i t y of t h e o p h y l l i n e , t h e a c t i o n of b o t h b u t especially theop h y l l i n e on the m e t a b o l i s m of cyclic AMP, a n d t h e i r clastogenic action. W h i l e at relat i v e l y high c o n c e n t r a t i o n s t h e y inhibit mitosis, at lower c o n c e n t r a t i o n s in p l a n t s only cell p l a t e f o r m a t i o n is affected. T h e o p h y l l i n e at high c o n c e n t r a t i o n s has been shown to i n h i b i t D N A synthesis in m a m m a l i a n cell lines 29 a l t h o u g h unlike o t h e r i n h i b i t o r s of D N A synthesis it a p p e a r s to have little or no effect on p r o p h a g e f o r m a t i o n in E. coli:~. MUTAGENICITY

N e i t h e r t h e o b r o m i n e nor t h e o p h y l l i n e was m u t a g e n i c in mice at a dose of 380 m g / k g in t h e d o m i n a n t lethal test 9. B o t h c o m p o u n d s are however n m t a g e n i e in Eztglena gracilis 6,44. A u x o t r o p h i c m u t a n t s , adenine -, t r y p t o p h a n - or riboflavin-requiring, were p r o d u c e d b y the action of theobrolnine at a c o n c e n t r a t i o n of 15 mg/25 mi, or t h e o p h y l l i n e at a c o n c e n t r a t i o n of i 2 rag/25 ml (ref. 6). A u x o t r o p h i c m u t a n t s were also p r o d u c e d in Ophiostoma multiannulatum b y t h e o p h y l l i n e 1°. In this work after t r e a t m e n t with 0.6% t h e o p h y l l i n e o.6~0 of the eonidia isolated were m u t a n t s comp a r e d with 0.06% or less in t h e control. T h e o b r o m i n e a n d t h e o p h y l l i n e b o t h act as m u t a g e n s in E. coll. A t a c o n c e n t r a t i o n of 15o ml/1 t h e o p h y l l i n e raised the m u t a t i o n r a t e of E. coli to T 5 resistance from the control r a t e of 1. 5 • IO-8/h to i i . IO-S/h, while t h e o b r o m i n e at the same c o n c e n t r a t i o n raised the m u t a t i o n rate to 7.5" IO-~/h (ref. 37). Guanosine was shown to act as an a n t i m u t a g e n in this s y s t e m a n d at a c o n c e n t r a t i o n of 500 mg/1 was able to suppress almost c o m p l e t e l y the m u t a g e n i c a c t i v i t y of either t h e o b r o m i n e or t h e o p h y l l i n e '~s. T h e o p h y l l i n e has also been shown to p r o d u c e m u t a tion from m e t h i o n i n e d e p e n d e n c e to m e t h i o n i n e i n d e p e n d e n c e in the I5h+m strain

173

THEOBROMINE AND THEOPHYLLINE

of E. coli ~4. Neither theobromine nor theophylline is incorporated to any great extent into the DNA of E. coli and it is suggested that this organism is unable to remove the methyl groups 25. The addition of theophylline to the growth medium of ultra violet light irradiated E. coli B/r try- resulted in a tenfold or greater increase in the frequency of try + mutants a°. Mutational synergism between low doses of ultra violet light and both theobromine and theophylline has been demonstrated in the mutation of E. coli B/r to high level streptomycin resistance TM. The magnitude of the synergism was inversely related to the ultra violet light dose, and only occurred when the chemical treatment followed the radiation. No mutagenic synergism was found between these compounds and X-irradiation 8. Theophylline was a more potent mutagen enhancer than theobromine and both were less active than caffeine. It is clear that while both theobromine and theophylline are mutagenic in lower organisms there seems to be no evidence that they have any such activity in mammals. Caffeine appears to have a similar restricted range of mutagenic activity ~3. It is possible that the reason for this difference is that while mammals easily and rapidly demethylate these compounds~,4L E. coil at least appears to be unable to d.o so 25. However this can be no more than an hypothesis on the present data and research into the demethylation ability of other organisms, such as Euglena, which are known to respond to the mutagenic action of these compounds would be interesting. COMPARISON WITH RELATED COMPOUNDS

It is possible to compare the relative activities of theobromine and theophylline with each other and with a number of chemically related compounds in an attempt to determine the relationships between their chemical structure and their biological activities. Caffeine In a number of investigations the activity of caffeine has been directly compared with that of theobromine and theophylline. These are summarised in Table II. From these results it is clear that there is no simple, universal order of biological activity in these compounds, although in general theobromine appears to be the least active. Paraxanthine A summary is given in Table I I I of the studies in which theobromine, theoTABLE i[ C O M P A R I S O N OF T H E O B R O M I N E ,

THEOPHYLLINE

AND CAFFEINE

Activity

System

Order of Potency

M u t a g e n i c ( s y n e r g i s m w i t h u l t r a violet light) Antimitotic

E. coli human lymphocytes

C>TP>TB

8, 46

TP>C>TB

47, 48

rabbit purified enzyme man

C>TP>TB TP > TB > C

27 3

C>TP >TB

39

P o l y m o r p h o n u c l e a r leucocyte aggregation inhibition 3'5-nucleotide phosphodiesterase inhibition A c t i o n on c e n t r a l n e r v o u s s y s t e m T B , t h e o b r o m i n e ; T P , t h e o p h y l l i n e ; C, caffeine.

[email protected]~re nces

174

J. TIMSON

TABLE III COMPARISON

OF THEOBROMINE,

THEOPHYLLINE,

PARAXANTHINE

AND CAFFEINE

Activity

System

Order of potency

References

Mutagenic

E. coli

C>TP>P>TB

37

Chinese h a m s t e r cells

C>TP>TB>P

Vicia faba

C>TP>TB>P

Clastogenic

Human lyre p h o c y t e s

C>TB>TP>P

51

Antimitotic

lHyummpahno c y t e s

TP>P>C>TB

51

P o t e n t i a t i o n of t h i o t e p a induced chromatid aberrations

24

root t i p s

TB, t h e o b r o m i n e ; TP, t h e o p h y l l i n e ; P, l l a r a x a n t h i n e ; C, caffeine. TABLE IV EFFECT

OF THEOBROMINE,

THEOPtIYLLINE,

AND URIC ACID ON THE RATE

Concentration (M) IO-~ IO ~ lO -4

OF MITOSIS

CAFFEINE, OF HUMAN

3litctic rate (o~ control) TheobroTheophyl- Caffeine mine line 3 zo6 98

o o ~6

o 41 111

A d a p t e d from: TIMSON 4s, a n d PRICE AND

XANTHINE,

HYPOXANTHINE,

LYMPHOCYTES

IN CULTURE

Xanthine

Hypoxanthine

83. 9 117. 4 216.8

58.6 9,5.6 98.2

Uric acid 6. 5 88.6 119.o

T I M S O N 42.

phylline, caffeine, and paraxanthine (I,7-dimethylxanthine) have been directly compared. Again no general order of biological activity can be found.

Monomethylxanthines All three monomethylxanthines (i-methylxanthine, 3-methylxanthine, 7-methylxanthine) depressed the mitotic rate in human lymphocytes in culture with Imethylxanthine being the most active al. None of the monomethylxanthines showed clastogenic activity in this system '5I. Methyluric acids Both 1,3-dimethyluric acid and 3-methyluric acid showed a synergistic mutagenic effect with ultra violet light in E. coli although both were less active than eithei theobromine or theophylline 46.

Xanthine, hypoxanthine and uric acid The effects on the rate of mitosis of human lymphocytes in 72-h culture of theobromine, theophylline and caffeine 4~ may be directly compared with the effects ot xanthine (2,6-dioxypurine), hypoxanthine (6-oxypurine) and uric acid (2,6,8-trioxypurine) ~. The results are summarised in Table IV.

THEOBROMINE AND THEOPHYLLINE

175

Structure-activity relationships The biological activities of theobromine and theophylline are complex and it is not possible to relate them to the chemical structure of the compounds in any simple manner. As mutagens in E. coli the methyl group at position I seems most importantS,37, 46 although methylation at the other positions is not without effect. It is worth noting that in the demethylation process in man the methyl group at position I is the most stable r. Methylation at position 3 seems to be most active both in the potentiation of chromatid aberrations induced by thiotepa in Chinese hamster cells and in Vicia faba root tips2L and in clastogenic activity in human lymphocytes in culture 51 although again the methyl groups at the other positions are not inactive. The most active methyl group in the reduction of the mitotic rate in human lymphocytes in culture is clearly at position I (refs. 48, 51). Unsubstituted xanthine is without significant effect even at Io-2M while it actually stimulates mitosis at Io-4M although theophylline is still strongly anti-mitotic at this concentration4~, 4s. At io-~M none of the oxypurines nor theobromine is anti-mitotic while caffeine reduces the mitotic rate to about 4o°/~ of the control value and theophylline is cytotoxic16,42, 'tS. It is therefore possible that methylation at position 7 in some manner prevents the full expression of the effect of the methyl group at position I. This is supported by the observation that I-methylxanthine is a more active mitosis suppressor than any of the dimethylxanthines or caffeine "~1. DISCUSSION

The biological activities of theobromine and theophylline are diverse. Both appear to have been neglected b y investigators compared with caffeine perhaps because it has been thought that they would inevitably be less active than caffeine. While this is often the case it is by no means always correct3,4s, 51. Like caffeine these compounds appear to be mutagenic only in lower organisms and the mutagenic risk to man from either compound on the limited available evidence would seem to be negligible. Their clastogenic and anti-mitotic activity, however, has been demonstrated in human cells in culture although the major effects are seen at concentrations rarely achieved in man except perhaps in therapeutic use. An investigation of the chromosomes of a number of patients who have been on long term treatment with either compound would be interesting. It is to be hoped that future investigations of the activity of caffeine wiil wherever possible include comparative work with theobromine, theophylline and paraxanthine. In this way it would be possible to build up a body of data which could lead to a better understanding of the structure-activity relationships of the methylxanthines which m a y confirm or refute the suggestions made above. ACKNOWLEDGEMENT

I should like to thank JOHN S. WASSOM,Director of the Environmental Mutagen Information Cnter, Oak Ridge, Tennessee, for help with the literature search.

176

j. TIMSON

REFERENCES 1 BLACOW, N. ~V. (Ed.), Martindale. The Extra Pharmacopoeia, 26th Edition. The Pharmaceutical Press, London, 1972 , pp. 360 362. 2 BRODIE, ]~. B., J. AXELROD AND J. REICttENTHAL, Metabolism of theophyllipe (t,3-dimethylxanthine) in man, J. Biol. Chem., 194 119521 215-222. 3 BUTCHER, R. ~vV. AND E. V~r. SUTHERLANI), Adenosine 3 ' , 5 ' - p h o s p h a t e in biological materials. I. Purification and properties of cyclic 3",5'-nucleotide phosphodiesterase and use of the enzyme to characterise adenosine 3 ' , 5 ' - p h o s p h a t e in h u m a n urine. J. Biol. Chem., 237 119621 1244--1250. 4 CHEYEY, R. H., C o m p a r a t i v e effect of m e t h y l a t e d x a n t h i n e s on the fertilisation capacity and life s p a n of Arbacia ganletes, .]. Cell Comp. Physiol., 48 11956 ) 253-269. 5 CHO, \¥. K., S. STERN ANt~ J. D. BIGGERS, i n h i b i t o r y effect of d i b u t y r y l cAMP on mouse oocytc matllration in vitro, J. Exptl. Zcol., 187 11974) 383 386. 6 COME, T. V. AND D. M. TRAVIS, I n d u c t i o n of a u x o t r o p h i c m u t a t i o n s in Euglena gracilis, J. Hered., 60 (I969) 39-41. 7 CORNISH, ~[. H. AND A. A. CHRISTMAN, A s t u d y of the metabolism of theobromine, theophylline and caffeine in man, J. Biol. Chem., 228 11957) 315-323 • b~ DONESON, i. N. AND D. M. SHANI¢-EL, Mutational synergism between radiations and nlethylated purines in Escherichia coil, .[. Baeteriol., b~7 119641 61-67. 9 EPSTEIN, S. S. AND H. SHAFNER, Chemical m u t a g e n s in the h u m a n e n v i r o n m e n t , Nature, 219 (1968) 385-387 . IO FRIES, N. AND B. KIHLMAN, Fungal m u t a t i o n s obtained with n / e t h y l x a n t h i n e s , Nature, 11~2 (1948 ) 573-576. 11 GIBBS, R. D., Chemctaxonomy cf flowering plants, Vol. l, McGill-Queens's University Press, Montreal and London, 1974, p. 2~5. 12 GOODMAN, L. S. AXD A. GILMAN, The Pharmacological Basis of Therapeutics, 3rd ed., Macmillan, New York, 1965. 13 GOSSELIN, A. Action, sur la nlitose des vdgdtaux, de deux alcaloides puriques, C.R. Acad. 5"ci. Paris, 21o (194 ° ) 544-546 . 14 GREER, S. B., G r o w t h inhibitors and their a n t a g o n i s t s as m u t a g e n s and a n t i m u t a g e n s in Escherichia cell, J. Ge.n. Microbiol., 18 11958 ) 543 564. I 5 HALBRECHT, [., L. ]~OMLOS, i ~'. SHABTAV, M. SOLOMON AND J. A. B e c K , Triploidy 6% X X X in a stillborn girl. Clin. Genet., 4 (19731 21o--212. 16 HIRSHORN, ]~., J. GROSSMAN AND G. WEISSMAN, Effect of cyclic 3',5'-adenosine m o n o p h o s p h a t e and theophylline on l y m p h o c y t e transfornlation, Prec. See. E,J)tl. Bit.1. Meal., 133 (197 o) 1361-1365 . 17 JAcons, M. H. a x n R. M. SEXIER, Theophylline toxicity due to impaired theophylline degradation, A'mer. Rev. Resp. Dis., IiO (19741 342-345 . 18 1KVLLEr~, R., Suppression of n o r m a l and enhanced t u m o u r g r o w t h in rats by agents interfering with intraceUular cyclic nucleotides, L/re Sci., i i (19721 485-491. 19 I(IHLMAX, B. AN[) A. LEVAN, The cytological effect of caffeine, Hereditas, 35 11949) lO9 - I I [. 2o I(IHLtV~A×, B., The effect of purine derivatives oi1 chronlosomes, [Iereditas 35, 119491 393 396. 21 ]~ItfLMAN, g . , A s u r v e y of purine derivatives as inducers of chronlosome changes, Hereditas, 38 (I952) 115 127. 22 I(IHLMAN, B. AND t(. OVERGAARD-HANSEN, I n h i b i t i o n of muscle p h o s p h o r y l a s e by nlethylated purines, Exptl. Cell Res., 8 (I955) 252 255. 23 KIHLMAN, B., Effects of caffeine on the genetic material, Mutation Res., 26 119741 53 7 I. 24 t(IHLMAN, B. A., S. STURELID, B. HARTLEY-AsP AND K. NILSSON, The e n h a n c e m e n t by caffeine of the frequencies of chronlosomal aberrations induced in plant and animal cells by chemical and physical agents, M2*tation Res., 26 11974) lO5 122. 25 I
THEOBROMINE AND THEOPHYLLINE

177

3I I, IxS, R. B. AND J. B. PETER, Cyclic A M P i n h i b i t i o n of c y t o t o x i n ( " l y m p h o t o x i n " ) e l a b o r a t i o n b y s t i l n u l a t e d l y m p h o c y t e s , Cell Immunol., 8 (1973) 332-335. 32 MACDONNELL, P. C., AND e . 142. TILLINGHAST, Metabolic sources of a m m o n i a in t h e e a r t h w o r m , Lumbricus terrestris L., J. Exptl. Zool., 185 (1973) 145 152. 33 MARKS, F. AND W. REBIEN, Cyclic 3 ' , 5 ' - A M P a n d t h e o p h y l l i n e i n h i b i t e p i d e r m a l m i t o s i s in G~ phase, Naturwissenschc~ften, 59 (1972) 41-42. 34 MILLS, D. C. B. AND J. B. SMITH, T h e influence on platelet a g g r e g a t i o n of d r u g s t h a t affect t h e a c c u m u l a t i o n of a d e n o s i n e 3'-5"-cyclic n l o n o p h o s p h a t e in platelets, Biochem. J. 121 (1971) 185-196. 35 MITTLER, S. AND J. E. MITTLER, T h e o b r o m i n e a n d t h e o p h y l l i n e a n d c h r o m o s o m e a b e r r a t i o n s in Drosophila melanogaster, Genetics, 60 (1968) 2c 5. 36 •OACK, D. AND S. I{LAUS, Einfluss y o n P u r i n - u n d P y r i m i d i n d e r i v a t e n a u f die P r o p h a g e n i n d u k t i o n in Escherichia col* C6oo T44, Z. Allg. Mikrobiol., 12 (1972) 583-591. 37 NOVICK, A. AND L. SZILARD, E x p e r i m e n t s on s p o n t a n e o u s a n d chemically i n d u c e d m u t a t i o n s of b a c t e r i a growing in t h e c h e m o s t a t , Cold ,Spring Harb)ur Syrup, Quant. Biol., 16 (1951) 33734338 NovIcK, A. AND L. SZlLARD, A n t i - m u t a g e n s , Nature, 17o (1952) 926-927. 39 OSOL, A., R. PRATT AND M. D. ALTSCHULE, The United States Dispensatory and Physicians' Pharmacology, 26th ed., J. B. L i p p i n c o t t Co., P h i l a d e l p h i a a n d Toronto, 1967, pp. 1172-1174. 4 ° OSTERTAG, W., Koffein- u n d T h e o p h y l l i n m u t a g e n e s e bei Zell- u n d L e u k o z y t e n k u l t u r e n des M e n s c h e n , Mutation Res., 3 (1966) 249 267. 41 PEARSON', D., The Chemical Analysis of Foods, 6 t h ed., J. & A. Churcill, L o n d o n , 197 o, pp. 298296. 42 PRICE, D. J. AND J. TIMSON, T h e effect of o x y p u r i n e s on t h e m i t o t i c rate of p h y t o h a e m a g g l u t i n i n - s t i m u l a t e d h u m a n l y m p h o c y t e s in vitro, Cytobios 4 (1971) 87-9143 SARMA, Y. S. t{. I~. AND S. N. TR1PATHI, Effects of caffeine a n d t h e o b r o m i n e on a green alga, Oedogonium acmandrium Elfving, Nucleus, 16 (1973) 167-172. 44 SCHIFF, J. A., H. LYMAN AND G, t{. I{USSELL, Isolation of m u t a n t s f r o m Euglena gracilis, Methods Enzymol., 23 ( P a r t A) (1971) 143 162. 45 SERCK-HANSSEN, G., Effects of t h e o p h y l l i n e a n d propanolol on a c e t y l c h o l i n e - i n d u c e d release of m e d u l l a r y c a t e c h o l a m i n e s , Biochem. Pharmacol., 23 (1974) 2225 2234. 46 SIDEROPOULOS, A. S., J. t~. LUMB AND D. M. SHANKEL, T h e influence of a n a l o g u e s of x a n t h i n e on t h e m u t a g e n i c effect of u l t r a violet light, Molec. Gen. Genetics, lO2 (1968) lO2 lO 7. 47 TIMSON, J., Effect of t h e o b r o m i n e , t h e o p h y l l i n e a n d caffeine on t h e m i t o s i s of h u m a n l y m p h o cytes, E M S Newsl., 6 (1972) 21. 48 TIMSON, J., Effect of t h e o b r o m i n e , t h e o p h y l l i n e a n d caffeine on t h e m i t o s i s of h u m a n l y m p h o cytes, Mutation Res., 15 (1972) 197-2Ol. 49 WEINFELD, H. AND A. A. CHRISTMAN, T h e m e t a b o l i s m of caffeine a n d t h e o p h y l l i n e , J. Biol. Chem., 200 (1953) 345-355. 50 WEINSTE1N, D., I. MAIJER, M. L. KATZ AND S. KAZMER, T h e effect of caffeine on c h r o m o s o m e s of h u m a n l y m p h o c y t e s . A search for t h e m e c h a n i s m of action, Mutation Res., 2o (1973) 155125 . 51 WEINSTEIN, D., I. MAUER, M. L. KATZ AND S. I42AZMER, T h e effect of m e t h y l x a n t h i n e s on c h r o n l o s o m e s of h u n l a n l y n l p h o c y t e s in culture, Mutation Res., 31 (1975) 57-61. 52 WILLIAMS, J. P. G., I n t e r r e l a t i o n of epithelial glycogen, cell proliferation a n d cellular migration w i t h cyclic a d e n o s i n e m o n o p h o s p h a t e in epithelial w o u n d healing, Cell Differentiation, i (1972 ) 317-323 •