Mutation Research, 4 7 ( 1 9 7 7 ) 1 - - 5 2 © Elsevier/North-Holland

Biomedical Press

CAFFEINE

J. T I M S O N

Department of Medical Genetics, The Medical School, The University, Manchester, M13 9PL (Great Britain) (Received 26 July 1977) ( A c c e p t e d 14 O c t o b e r 1 9 7 7 )

Contents Summary .................................................... Introduction .................................................. Metabolism .................................................. Effects of caffeine on DNA ........................................ Synthesis .................................................. Effects on DNA structure ....................................... Effects on DNA repair ......................................... D N A r e p a i r in b a c t e r i a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D N A r e p a i r in o t h e r m i c r o - o r g a n i s m s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA repair in Drosophila ....................................... D N A r e p a i r in m a m m a l i a n cells in c u l t u r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . D N A r e p a i r in h i g h e r p l a n t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caffeine and repair mechanisms ................................... Other biochemical and physiological effects ............................. RNA synthesis and protein synthesis ................................ Effects on enzymes ........................................... Effects on cyclic AMP ......................................... Effects on viruses ............................................ M i s c e l l a n e o u s e f f e c t s o n cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antitumour effects ........................................... M i t o s i s , cell v i a b i l i t y , a n d p l a n t cell-wall f o r m a t i o n . . . . . . . . . . . . . . . . . . . . . . . . Mitosis ................................................... Cell v i a b i l i t y : c a f f e i n e a l o n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell v i a b i l i t y : c a f f e i n e in c o m b i n a t i o n w i t h o t h e r a g e n t s . . . . . . . . . . . . . . . . . . . P l a n t cell-wall f o r m a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutagenic effects .............................................. Caffeine alone: micro-organisms ................................... In combination with other agents: micro-organisms ...................... Mitotic crossing-over in plants .................................... Caffeine alone: Drosophila ...................................... In combination with other agents: Drosophila .......................... C h i n e s e h a m s t e r cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mammals .................................................. Production of chromosomal aberrations ................................ Caffeine alone: plant material ....................................

2 2 5 7 7 8 9 9 10 10 10 12 12 12 12 13 13 13 14 15 15 15 17 17 19 19 19 20 22 22 22 23 23 24 24

In combination with other agents: plant material . . . . . . . . . . . . . . . . . . . . . . . . Caffeine alone: animal material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In combination with other agents: animal material . . . . . . . . . . . . . . . . . . . . . . . Caffeine alone: human material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In combination with other agents: human material . . . . . . . . . . . . . . . . . . . . . . Comparison of the effects on plant and animal chromosomes. . . . . . . . . . . . . . . . Teratogenic and carcinogenic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Comparison with related compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Theophylline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Theobromine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Theophylline and theobromine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Paraxanthine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Substituted caffeines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Monomethylxanthines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methyluric acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xanthine, hypoxanthine, and uric acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure--activity relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

24 26 28 29 30 30 31 34 34 35 35 36 36 36 37 37 37 38 39 39

Summary Most o f the p o p u l a t i o n o f t h e w o r l d is e x p o s e d t o caffeine to a greater or lesser e x t e n t since it o c c u r s in a n u m b e r o f plants used in t h e p r e p a r a t i o n o f widely c o n s u m e d drinks, and has in a d d i t i o n a limited t h e r a p e u t i c use. Chrom o s o m a l a b n o r m a l i t i e s are i n d u c e d b y caffeine in b o t h p l a n t cells and in m a m malian cells in c u l t u r e and it also has s o m e a n t i - m i t o t i c activity. D N A - r e p a i r processes sensitive to caffeine have been d e m o n s t r a t e d in a n u m b e r o f cell systems and it has also been s h o w n t o a f f e c t a wide range o f o t h e r cellular processes. Caffeine has p o t e n t m u t a g e n i c effects in Escherichia coli and o t h e r m i c r o - o r g a n i s m s b o t h w h e n acting alone and in c o m b i n a t i o n with o t h e r m u t a g e n s . H o w e v e r its m u t a g e n i c activity in D r o s o p h i l a has b e e n d i s p u t e d a n d the available evidence suggests t h a t it is n e i t h e r m u t a g e n i c in m a m m a l s n o r synergistic with o t h e r m u t a g e n s a l t h o u g h at very high doses it appears t o have s o m e t e r a t o g e n i c activity in m a m m a l s .

Introduction Caffeine, 1 , 3 , 7 - t r i m e t h y l x a n t h i n e , has the empirical f o r m u l a : CsH~IN402. It o c c u r s n a t u r a l l y in a n u m b e r o f plants {see Gibbs [ 1 2 1 ] f o r a list o f species in w h i c h caffeine has been identified). Most i m p o r t a n t l y f o r m a n caffeine o c c u r s in several plants f r o m w h i c h s t i m u l a t i n g beverages are prepared. A l m o s t all t h e a n c i e n t agricultural civilisations have h a d their o w n c a f f e i n e - c o n t a i n i n g beverage p l a n t and it is believed t h a t paleolithic m a n discovered t h e c h i e f sources o f caffeine in all parts o f t h e w o r l d [ 2 1 7 ] . In t h e A m a z o n region a d r i n k called guaranfi is p r e p a r e d f r o m t h e seeds o f either Paullinia cupana or P. sorbilis in the Central A m a z o n area w h e r e these plants are c u l t i v a t e d [ 3 1 5 ] and a n o t h e r drink, y o c o , is p r e p a r e d f r o m the bark o f P. y o c o in the N o r t h West A m a z o n

area. The bark of P. yoco contains 3--6% caffeine. Another beverage plant of great antiquity is Ilex paraguensis from which mat~ is prepared. This plant is native to the mountains of Paraguay and is extensively cultivated in Argentina and Southern Brazil. The caffeine content of mat~ can be as high as 0.7% [315]. Cola nitida is a tree native to West Africa and is now widely cultivated in Brazil, India, and Jamacia. A kind of tea is prepared from the powdered seeds [315]. The cola-flavoured drinks are in part manufactured from the nuts of Cola acuminata. The natives of the Sudan chew these as guru nuts. A 12 oz (340 ml) bottle of a cola drink contains 35--55 mg of caffeine [130]. Chocolate and the cocoa drinks are prepared from the seeds of Theobroma cacao which contain about 1% theobromine and a lesser a m o u n t of caffeine [315]. Cocoa contains up to 50 mg of caffeine per cup [130]. Probably the most widely consumed non-alcoholic drink is tea which is regularly drunk by at least half the population of the world. Tea is prepared from the leaves of Camellia sinensis, a bush native to Assam and Southern China and now very extensively cultivated in 'India, Sri Lanka, and m a n y other coutries in Asia and East Africa [315]. At first tea seems to have been employed as a medicine but its use as a beverage began at least as early as 600 C.E. [315]. The caffeine content of tea leaves is about 2% and the average cup of tea contains about 100--150 mg of caffeine [130]. Coffee is prepared from the fruits of species of Coffea especially C. arabica which provides about 90% of the world's supply of coffee. The beans of C. arabica contain 0.7--1.5% caffeine [315]. The plant is a native of Ethiopia and is also widely cultivated in South America especially in Brazil, Columbia and Costa Rica. Other Coffea species used include C. liberica, C. robusta, and C. canephora. The last is a native of the Congo region now cultivated in Indonesia [315]. An average cup of coffee contains about 100-150 mg of caffeine [130,253]. The caffeine content of 5 samples of instant coffee ranged from 3--7.7% [352]. Man has, therefore, been exposed to the effects of caffeine for m a n y generations both in primitive and advanced cultures. In addition to its widespread occurrence as a constituent of non-alcoholic drinks caffeine has a limited use in medicine [33,130,253,263]. It is rarely used alone being more usually prescribed in combination with other drugs on which it is believed to have an enhancing effect [33] and also because it may counteract undesirable effects of the main drug such as. drowsiness. Tables 1, 2, and 3 give a selection of some c o m m o n l y used preparations which include caffeine. Recently it has been successsfully used in combination with isoxsupine and acetominophen in the t r e a t m e n t of premenstrual tension and primary dysmenorrhoea (111). Caffeine is also used on a limited scale in veterinary medicine mainly as a cardiac and respiratory stimulant [39]. It seems reasonable, therefore, to assume t h a t most of the world's population is exposed to a greater or lesser extent to caffeine. Indeed it is possible to regard it as an addictive drug and it is interesting to note t h a t when rats were forced to consume caffeine in amounts approximately equal to those consumed by heavy coffee drinkers they prefered water containing caffeine in a subsequent flee-choice situation [350]. Since there is no reason to suppose that patients being treated with caffeine-containing preparations will reduce their normal intake of caffeine, if any mutagenic, teratogenic, carcinogenic or toxic

4

TABLE 1 USE OF CAFFEINE

IN A N A L G E S I C

PREPARATIONS

Caffeine content

Other ingredients

1

64.8 mg

Acetylsalicylic acid

259.2 mg

2

15 mg

Acetylsalicylic acid Phenacetin Quinine sulphate

200 mg 200 mg 15 mg

3

15 or 30 mg

Acetylsalicylic acid Phenacetin

230 mg 150 mg

4

30 mg

Acetylsalicylic acid Codeine phosphate Phenacetin

225 mg 8 mg 150 mg

5

50 mg

Aeetylsalicylie acid Codeine phosphate Phenacetin Phenobarbitone

200 10 200 25

S o u r c e : R e f . 3 3 , 1 - - 3 p. 2 3 0 ; 4 , 5

mg mg mg mg

p. 1 1 1 0 .

TABLE 2 PREPARATIONS

CONTAINING

Caffeine content

CAFFEINE

U S E D IN T H E T R E A T M E N T

OF MIGRAINE

Other ingredients

1

100 mg

Ergotamine tartrate

1 mg

2

100 mg

Ergotamine tartrate C y c l i z i n e . HCI

2 mg 50 m g

3

100 mg

Ergotamine tartrate Phenacetin H y o s c y a m i n e • SO 4 Atropine • SO 4

1 mg 130 mg 87.5 mg 12.5 mg

S o u r c e : R e f . 3 3 , p. 7 0 3 .

TABLE 3 PREPARATIONS CONTAINING CAFFEINE CHITIS, HAY FEVER, AND EMPHYSEMA

USED

IN

THE

Caffeine content

Other ingredients

1

Sodium benzoate Sodium iodide Sodium salyeilate E p h e d r i n e • HCI

2.24% 3.588% 2.24% 0.452%

Potassium iodide Sodium benzoate

300 mg 60 mg

Sodium iodide Thcophylline

333 mg 125 mg

4%

2

45 mg

3

133 mg

S o u r c e : R e f . 3 3 , p. 3 5 0 .

TREATMENT

OF

ASTHMA,

BRON-

effect of caffeine occurs in man such patients would constitute a high risk group. The pharmacological actions of caffeine are diverse. They include stimulation of m a n y centres in the central nervous system [33,253,263], diuretic action on the kidney [33,253], and a stimulating effect on striated muscle [253]. Caffeine has been shown to cause a dose--dependent increase in the rate and force of contraction in the isolated rabbit heart [288]. Laux [193] has found that caffeine induces a dose-dependent suppression of antibody synthesis in both in vivo and in vitro systems. The immune response of the mouse to sheep erythrocytes was suppressed by caffeine. Cell-associated protein synthesis was inhibited but not DNA or RNA synthesis. It was suggested that the inhibition of antibody production was due to the inhibition of protein synthesis [194]. Side-effects of caffeine medication include nausea, insomnia, headache, and tachycardia [33,253]. Large doses may cause excitement, muscle tremor, and tinnitus [33]. Caffeine is known to cause an increase in gastric secretion especially of pepsin and free hydrochloric acid and may cause gastric ulceration [33,253]. The fatal dose in man is estimated to be about 10 g, perhaps less in patients with cardiac disease [33,130]. Such doses are rarely, if ever, achieved. Although caffeine has been used in the treatment of acute alcoholic intoxication [130] and it is traditionally believed that the effects of caffeine are antagonistic to those of ethanol this belief is but folk-lore and is n o t based on scientific data. Alstott [ 13 ] in experiments with rabbits and rats found that the impairment of performance was significantly greater after caffeine and ethanol than after ethanol alone. It was also found that the rates of metabolism of the two compounds were independent and in toxicity tests that the effects of caffeine and ethanol were additive in causing death. However caffeine pretreatment shortened the barbital-induced sleeping time in rats [1]. In man it has been shown that habitual consumers of caffeine-containing beverages develop an acquired tolerance to caffeine which is not found in individuals who are not habitual caffeine consumers [69]. Withdrawal of caffeine from habitual users may result in severe headaches [85]. This finding provides some rationale for the inclusion of caffeine in a number of analgesics (Table 1), although this practice may not be entirely w i t h o u t risk [21]. Metabolism In man caffeine is rapidly absorbed from the gastrointestinal tract [20] and under experimental conditions has been detected in the plasma within 5 min of ingestion [132]. Peak plasma levels were found 50--75 min after taking the dose of caffeine. Caffeine is almost entirely transformed in man with only about 1% being excreted unchanged in the urine [20,71]. The metabolic halflife of caffeine in the plasma of 12 male subjects injected intravenously with 7 mg caffeine per kg b o d y weight averaged 3.5 h (range 2.5--4.5 h) [20]. After ingestion of 100 mg of caffeine by 3 subjects the half-life varied from 3.5-6.5 h in 2 subjects and from 6.0--10.5 h in the third [132]. The rapidity of caffeine metabolism ensures that there is little or no day-to-day accumulation of the drug. In 5 h u m a n subjects who consumed 8 cups of coffee each containing about 80 mg of caffeine over a period of 7 h it was estimated that about

180 mg of the drug was present in the body 1 h after the last cup of coffee. The drug had virtually disappeared from the body by the following morning [20]. Habitual consumers of caffeine-containing beverages, however, require about 7 days of total abstention from all sources of caffeine before it is completely absent from blood extracts [358]. The available data suggest that caffeine is distributed in the body tissues in proportion to their water content [20]. Caffeine will equilibrate freely between the plasma and the tissue water in both ovary and testes in man and also between maternal plasma and the human 7--8 week old fetus [127]. Caffeine and its metabolites have been identified in human amniotic fluid [311]. Thus in habitual caffeine consumers the gonads and the fetus in utero will be continuously bathed in fluctuating concentrations of caffeine. The first step in the metabolism of caffeine in man appears to be the removal of the 3-methyl group producing paraxanthine (1,7-dimethylxanthine) [71, 358]. Paraxanthine appears in both the plasma and the red blood cells of " d e c a f f e i n a t e d " subjects within 3 h of the oral administration of caffeine [358] and some is excreted in the urine [71]. Most of the paraxanthine is further metabolised into 1-methylxanthine and 7-methylxanthine both of which appear in the urine [71]. Much of the 1-methylxanthine becomes 1-methyluric acid and is excreted as such but 7-methyluric acid is not found in the urine after caffeine ingestion although it is found after the ingestion Ofotheobromine [71]. A minor pathway of caffeine metabolism may be the removal of the 7-methyl group to form theophylline (1,3-dimethylxanthine) since although this is not excreted after caffeine ingestion its breakdown product 1,3-dimethyluric acid is excreted [71], and theophylline has been detected as a caffeine metabolite in amniotic fluid [311]. Theobromine (3,7-dimethylxanthine) was not detected as a metabolite of caffeine in the urine [71] but has been detected as a metabolite of caffeine in the amniotic fluid [311]. The demethylation of caffeine in man does not appear to go b e y o n d the m o n o m e t h y l stage since there was no accumulation of xanthine in the urine or increase in the uric acid excretion [71]. In the dog [20] in so far as it has been investigated the metabolism of caffeine appears to be essentially the same as in man. When 14C-labelled caffeine was orally administered to 6-day pregnant New Zealand White rabbits at a dose rate of 3.5 mg per kg body weight caffeine and its metabolites (paraxanthine, theophylline, 1-methylxanthine, and 1,3-dimethyluric acid) were found after 6 h in both the maternal plasma and the pre-implantation blastocyst [97]. After caffeine was fed to rabbits, 1,3-dimethyluric acid and 1-methyluric acid were detected in the urine [362]. In the rat it has been shown that caffeine rapidly penetrates into the intracellular compartments of the tissues and accumulates in the cytoplasm [112]. 1-Methyl [14C]-labelled caffeine and its metabolites have been found to penetrate into the fetal tissues in pregnant rats and to accumulate in the fetal brain [113]. Following administration of [3H,14C]-caffeine to male CD-1 mice at a dose rate of 25 mg per kg body weight excretion of identifiable metabolites occurred rapidly and was 80% complete within 8 h [45]. Between 3 and 6% of the caffeine was recovered unchanged from the urine. The major metabolites were paraxanthine, 3-methylxanthine, 7-methylxanthine, 1,3-dimethyluric acid, and 1-methyluric acid [45].

After a single oral dose of 5 or 25 mg per kg body weight of [3H,'4C]caffeine the drug appeared in all tissues examined in CD-1 mice within 5 rain although a distribution in proportion to tissue water was only achieved about 1 h after dosing [46]. The m a x i m u m organ half-life was 3 h and the main tissue metabolite identified was paraxanthine [46]. From the available data, therefore, it seems reasonable to conclude that the metabolism of caffeine in all mammals investigated is essentially the same. Aspergillus niger can metabolise caffeine producing theophylline and 3-methylxanthine [153]. Theophylline was also the first product in the metabolism of caffeine by a strain of Penicillium roqueforti isolated from a caffeine-containing medium [292]. This strain was able to grow on media containing 0.04 M caffeine as sole nitrogen source. Growth was accompanied by complete removal of caffeine from the medium [292]. P. roqueforti and a species of Stemphylium were able to utilise caffeine, theobromine or xanthine as sole source of either carbon or nitrogen [189]. It has been reported that no enzymatic pathway exists in E. coli for the demethylation of caffeine [181], and neither caffeine nor theophylline was metabolised by Staphylococcus aureus although both compounds were taken up by the cells [53]. However Bacillus coagulans can utilise caffeine as sole source of nitrogen or carbon [189]. Pseudomonas putida strain 40, isolated by enrichment on caffeine as the sole source of carbon and nitrogen, can grow on 0.5% (2.6 × 10 .2 M) caffeine [374]. It will grow on any N-methyl derivative of xanthine containing one or more methyl groups at the 1, 3 or 7 positions. The caffeine is first metabolised by the action of an enzyme which is capable of removing hydrolytically all 3 m e t h y l groups with the production of methyl alcohol and xanthine. The m e t h y l alcohol is further metabolised to carbon dioxide and the xanthine follows the coventional pathways of purine degradation [374]. It would seem that the metabolic pathways followed by caffeine in microorganisms are diverse and different from those in mammals. This may be part of the reason for the difference in mutagenic response to caffeine between mammals and micro-organisms discussed later in this review. Effects o f caffeine on DNA

Synthesis Caffeine, at least in E. coli, does not behave as a purine analogue in the purine biosynthetic pathway [75] nor is it incorporated to any appreciable extent into the nucleic acids of the bacterium [180]. However there is some evidence that in certain circumstances caffeine may affect the rate of DNA synthesis. 4 doses of caffeine at the rate of 50 mg per kg b o d y weight depressed DNA synthesis in the mouse liver particularly in young (4-week old) animals, and in older (54 weeks) animals in which proliferation of the liver cells had been induced by partial h e p a t e c t o m y [232]. The semi-conservative synthesis of DNA in larvae of Drosophila melanogaster was strongly inhibited by caffeine although repair replication was unaffected [34]. Caffeine can also reduce DNA synthesis in Paramecium aurelia [308]. A significant decline in the DNA synthesis of E. coli B cells in vivo was caused by 8 mM caffeine but this may have been solely due to caffeine-death which at this concentration was f o u n d to be

18% per cell generation [136]. A temporary reduction in DNA synthesis was produced by caffeine in ultraviolet-irradiated E. coli B/r try- [204]. The depression of DNA synthesis in mouse S-180 ascites turnout cells caused by X-irradiation (500 R) is greatly reduced by the injection of 1.0 ml of 10 .3 M caffeine 10 min before irradiation [35]. It is suggested that the caffeine may act here by raising the intracellular levels of cyclic AMP. After low ultraviolet irradiation (200 erg/mm 2 or less) caffeine caused a specific block in the S phase of mouse L cells [80,81]. In mouse l y m p h o m a cells (L5178Y) caffeine at concentrations of 1--6 mM did not affect overall DNA synthesis but the DNA strands synthesised in the presence of caffeine were smaller than those of the control [199]. In L5178Y cells, LS929 mouse fibroblasts, and V79 Chinese hamster cells high doses of caffeine or theophylline (greater than 0.3 mg/ml) inhibited DNA synthesis when measured as uptake of [3H]thymidine into the DNA [200]. Caffeine greatly reduced the uptake of uridine and t h y m i n e in CHO-K1 cells and life-cycle analysis revealed that such cells traversed normally through one complete cycle but were blocked in the second cycle near the S-G2 boundary [356]. At a concentration of 10 .2 M caffeine was found to inhibit the synthesis of 7-ray-induced unscheduled DNA in HeLa Zh-63 cells [294] but not the unscheduled DNA synthesis induced in HeLa cells after ultraviolet irradiation [65]. Normal DNA synthesis in UV-irradiated HeLa cells was reduced to 48% of control by 10 .3 M caffeine [65]. In normal human lymphocytes caffeine at a concentration of 2 × 10 -3 M had no effect on the incorporation of tritiated thymidine [117]. At high concentrations, i.e. more than 1% (5.2 × 10 -2 M) caffeine did significantly decrease the incorporation of [3H]thymidine in Vicia faba although at 0.1% for 12 h it had no effect on DNA synthesis [389]. In Saccharomyces cerevisiae caffeine inhibited DNA synthesis and did not increase the intracellular cAMP level [339]. In extracts of E. coli caffeine inhibited some but not all of the purine nucleoside phosphorylases of both the ribose and deoxyribose series [181]. In cell-free extracts of cultured hmnan embryonic lung cells caffeine at concentrations greater than 1 mM inhibited the incorporation of 3H-TTP into DNA and inhibited DNA polymerase activity in the cells [375]. Caffeine, therefore, may be said to affect DNA synthesis at least in some organisms and cells under certain conditions. It would seem probable, however, that the effect is often at least in part a secondary one. Important in this respect is the inhibition by caffeine of the uptake of DNA precursors into the cell. It has been shown that the inhibition of [3H]thymidine uptake in L5178YuK cells by high doses of caffeine or theophylline was partly caused by decreased uptake of the labelled precursor into the cells and partly by a decreased rate of DNA synthesis [201]. Similar results have been obtained with 8-ethoxycaffeine in Vicia faba [ 250 ].

Effects on D N A structure The effect of 0.1% caffeine for 24 h on the nuclei of the phycomycete, Thamnidium elegans, was to enlarge and disperse the chromatic material so that the chromosomes became clearly visible [272]. Chick fibroblasts treated in vitro for 2--2.5 h with caffeine at 25 or 50 mM showed beaded and filamentous structures within the nuclei suggesting t h a t differential condensation of interphase chromatin had taken place [120]. Electron microscopy showed

different degrees of coiling within the microfibrils. On native DNA extracted from mouse L1210 cells nuclease S, extracted from Aspergillus oryzae was only effective in the presence of caffeine [57]. It was suggested t h a t this effect was due to the local unwinding of the DNA by caffeine. It was also suggested that caffeine may preferentially interact with the adenine--thymine base pairs in the DNA. Caffeine also greatly enhanced the phleomycin-stimulated degradation of T2 DNA by Neurospora endonuclease and had some activity w i t h o u t phleomycin [307]. It was suggested that caffeine binds preferentially to singlestrand DNA and increases local denaturation by binding to the DNA at sites provided by phleomycin--thymine complexes. The ability of caffeine to bind to DNA has been demonstrated and it was found t h a t this affinity was greater when the DNA was in a coil rather than helical form [337]. In UV-irradiated DNA treated with low concentrations of caffeine it has been shown that the caffeine molecules bind to the DNA near the region of the UV-induced conformational changes [192]. There is some evidence which suggests that the binding of caffeine to nucleic acids is caused by costacking of the c o m p o u n d with the nucleic acid bases particularly adenosine [265]. It has been suggested that the reduction of meiotic recombination in Schizosaccharomyces p o m b e induced by caffeine could be due to a lack of exonuclease degradation resulting from the interaction of caffeine with the DNA [211]. However the demonstration that caffeine in mammalian cells is rapidly demethylated must cast some d o u b t on the importance of in vivo binding of caffeine to DNA [131]. While caffeine increased the rate of spontaneous DNA breakdown which occurred during normal growth of E. coli B s - l l it had no such effect on the EXR strain Bs-2 nor in the parental B strain [138].

Effects on D N A repair While caffeine may only indirectly affect DNA synthesis and its effects on DNA structure have aroused only limited interest it is clear that it has a major effect in many cells and organisms on DNA-repair mechanisms. D N A repair in bacteria Caffeine will inhibit the repair of UV lesions of DNA in host-cell-reactivating (hcr ÷) strains of E. coli [142]. Considerable strain differences in effect were observed. Ultraviolet resistant strains of E. coli were not affected by caffeine at 8 mM which suggested that caffeine selectively blocked excision repair without substantially affecting recombination repair [140]. It has been f o u n d that caffeine and 8-chlorocaffeine appear to act by inhibiting the excision enzyme of the dark-repair system in the radiation-reisistant strain of E. coli B/r [212]. The enzyme responsible for both dark reactivation and host-cell reactivation is believed to be the same and is inhibited by the presence of caffeine [226]. It is normally present in the bacterial cell and is induced neither by UV-irradiation nor phage infection. The interference by caffeine with the dark-repair enzyme has been suggested as a mechanism for the enhancement of uV-induced mutation in E. coli B/r try- [204]. The rate of photoenzymatic repair obtained with continuous illumination of UV-irradiated E. coli Bs. 1 cells is much decreased in the presence of 16 mg/ml (8.3 × 10 -2 M) caffeine [143]. This effect is fully reversible when the caffeine is diluted out. Caffeine prevented the excision of

10 pyrimidine dimers from the DNA of UV-irradiated E. coli N17-9 and N14-4 [229], and the excision of UV-induced thymine-containing dimers from E. coli T- by binding to the excision enzyme [ 301]. Caffeine-resistant mutants isolated from E. coli K12 UV-repair deficient strains appeared to have increased resistance to UV and some at least could partially repair UV damage in their DNA [75]. Such caffeine-resistant mutants and others from E. coli strains B, B/r, and 15 may carry alterations in the excision-repair pathway [135]. The repair functions controlled by the phages T4D {in E. coli B) and VIr (in Proteus mirabilis, P.G. 273 thr-, and P.G. 686 thr-) are insensitive to 0.1% caffeine [371]. However caffeine t r e a t m e n t of VIr or the bacterium inhibited host-cell reactivation of the phage after UV-irradiation or t r e a t m e n t with nitrogen mustard [372]. When caffeine is used to inhibit the repair system in E. coli B there is an increased yield of the phage T4D [310]. E. coli B derivatives differing in their ability to repair DNA damage induced by UV-irradiation showed corresponding responses to the photo effects sustained in lysergic acid diethylamide-sensitised organisms and recovery from this t r e a t m e n t was inhibited by caffeine at 1.5 mg/ml [259]. However caffeine affects the repair mechanisms of E. coli it does not seem to be associated with its known affect on cAMP [51]. Caffeine has been shown to inhibit the action of the UV-specific endonuclease which is probably an adenosine 5'-triphosphate-dependent enzyme in E. coli [293]. Caffeine has been shown to inhibit the excision-repair capacity of Salmonella typhimurium trp C3 [369].

DNA repair in other micro-organisms A similar inhibitory effect of caffeine on repair mechnisms has been observed in a number of micro-organisms including strain UVS-1 of the bluegreen alga, Anacystis nidulans [18];Eudorina elegans strain 1193 [163]; the plasmodia of Physarum polycephalum subline M3cV [214]; Schizosaccharomyces pombe [96]; and Paramecium aurelia [308]. In Anabaena doliolum the inhibiting action of caffeine on the repair of UV lesions has enabled it to be used as a screening agent in the isolation of strains of the alga of different UV sensitivities [312]. In two strains of Micrococcus radiodurans which is very resistant to radiation damage caffeine failed to sensitise the cells to irradiation which suggests t h a t in this organism the very accurate repair mechanism is not caffeine-sensitive [318]. DNA repair in Drosophila Repair replication in the larvae of Drosophila melanogaster has been reported to be insensitive to the presence of caffeine [34]. The repair systems of D. melanogaster oocytes following treatment with the alkylating agents EMS and DEB were also unaffected by caffeine [359]. However a caffeinesensitive repair mechanism seems to occur in both the male and female genomes of D. melanogaster following X-irradiation [223--225,359]. The action of caffeine on this repair mechanism may be concentration-related [3881. DNA repair in mammalian cells in culture The sedimentation profile of DNA synthesised in UV-irradiated Chinese hamster cells was affected by caffeine which had no such affect on unirradiated

11 cells [336]. It was suggested that caffeine inhibited an S-phase specific postreplication dark-repair mechanism. Ultraviolet-irradiated Chinese hamster cells Cl-I also showed inhibition of the post-replication repair of DNA by caffeine and chlorocaffeine [242]. When Chinese hamster cells, M3-1 and V79-79, were subjected to UV in fractionated doses there was a greater survival of the cells if an S phase was allowed between doses than after a single dose and caffeine at 1 mM between doses did not appear to inhibit the between-doses repair [333]. Damage to the DNA in Chinese hamster cells, V79-379A, caused by cisplatinum(II)-diamine-dichloride t r e a t m e n t can be repaired by a caffeine-sensitive post-replication repair process [341]. Caffeine enhances the lethal, mutagenic and cytogenetic effects of N-methyl-N-nitrosourea (MNU) on Chinese hamster cells possibly by inhibiting the repair of MNU-induced damage [274]. Caffeine has been shown to inhibit the post-replication repair of N-acetoxy-2acetylaminofluorene-damaged DNA in Chinese hamster cells [335]. In mouse L cells, caffeine at 2 mM completely inhibits DNA repair after MNU treatment [107] or UV-irradiation [108]. The caffeine effect is n o t seen if ionising radiation is used instead of ultraviolet light [270]. The evidence suggests that mouse L cells contain repair mechanisms similar to those found in bacteria which are capable of acting on UV damage [268,270]. HeLa cells appear to react differently to caffeine when compared with other mammalian cells in culture. While caffeine potentiated the lethal effects of sulphur mustard and MNU in Chinese hamster cells it did n o t do so in HeLa cells [276]. At 10-2M and, 10 -3 M caffeine did not inhibit repair replication in HeLa cells although it did reduce normal DNA synthesis in one study [65]. Exactly the reverse results were obtained in another investigation using HeLa Zh-63 cells [387]. This apparent discrepancy may be due to cell line differences. When five cultured human cell lnes (T-1 kidney; Chang liver; H. Ep. No. 2; HeLa-S3; HeLa-0) were treated with caffeine immediately after UV-irradiation the surviving fraction was reduced which may indicate interference with a repair process [291]. In normal human lymphocytes caffeine at 2 × 10 .3 M did not decrease the repair replication of DNA following UV-irradiation [117]. It is suggested, however, that caffeine inhibits the joining of newly formed short strands of DNA in UV-irradiated cells. Treatment with 10 -3 M caffeine enhanced the chromosome damage induced by mitomycin C, methyl methanesulphonate, and X-rays in human lymphocytes and these results are consistent with the hypothesis that caffeine inhibits the filling of gaps in newly synthesised DNA [40,41]. Similar results have been obtained in human lymphocytes irradiated with gamma rays at doses of 10--30 krad and post-irradiation treated with 6 × 10 -4 M caffeine [9,10]. Caffeine has no effect on human fibroblasts from excision-deficient Xeroderma pigmentosum (XP) patients after UV-irradiation but it enhances the lethal action of UV in fibroblasts from an excision-proficient variant (XP7TA) [17]. The time taken in XP variant cells to convert low moleccular weight DNA synthesised after UV-irradidation to high molecular weight DNA is much longer than in normal cells and this slow conversion was drastically inhibited by caffeine which has no such affect in normal cells [202]. Cells from patients with XP excision-repair deficiency were intermediate both with regard to the time necessary for conversion of low molecular weight DNA to high molecular weight DNA and the caffeine inhibition of this pro-

12 cess. Caffeine also blocks repair of UV-irradiated adenovirus 2 when the irradiated virus infects fibroblasts of an excision-proficient XP cell strain [74]. Interference by caffeine with the repair mechanisms associated with semiconservative replication during S-phase in heteroploid human embryonic lung cells, strain L-132 exposed to MNNG has been observed [38]. There is, however, some evidence to suggest that normal human skin cells (NHSF) may perform an alternative replicative repair of UV lesions which is not caffeine-sensitive [ 109].

DNA repair in higher plants It has been shown that caffeine inhibits the repair of gamma-radiationinduced chromatid breaks in barley [377]. Later investigations using a caffeine concentration of 1 mg/ml which did n o t reduce seedling growth suggested that repair processes at the DNA level were being inhibited following gamma irradiation [7,8]. At non-toxic doses caffeine did not inhibit the repair of singlestrand breaks in the DNA o f barley seeds induced by MNU, MMS, or EMS, b u t at higher doses caffeine did inhibit the repair of such breaks induced by MNU [343]. An increase in the a m o u n t of chromosome damage after irradiation in buckwheat root tip cells by caffeine has been reported which may be due to interference with a caffeine-sensitive repair process [ 183]. Caffeine and repair mechanisms Caffeine-sensitive DNA repair mechanisms are present in E. coli and a number of other micro-organisms. Similar mechanisms have been found in some but not all cultured mammalian cells, Drosophila, and some higher plants. Not all DNA-repair mechanisms are caffeine-sensitive and the differences in cafffeine-response found between different E. coli strains and different cell cultures may be a reflection of the relative importance and efficiency of the different repair mechanisms in the strains and cell lines. The caffeine-sensitive repair mechanisms found in bacteria are of the excision-repair type but caffeine has n o t been shown to inhibit excision repair of any type of damage in any mammalian cell [203,271,276]. In mammalian cells in culture in which a caffeine-sensitive repair mechanism has been demonstrated it is of the postreplication repair type. This seems to be usual in cultured rodent cells [201, 203] b u t many human cell lines appear to lack a caffeine-sensitive repair process and it is perhaps significant that where caffeine-sensitive repair mechanisms have been found in human cells it is in cells cultured from patients with XP, a disease characterised by the development of multiple skin cancers induced by sunlight [273]. In rodent cells caffeine specifically inhibits postreplication repair [201] and it has been shown that this effect is n o t mediated via cyclic AMP [88]. Since postreplication repair involves newly synthesised DNA strands it is an S-phase-specific process. Therefore when a particular biological effect {mutation, chromosomal aberration etc.,) is shown to be produced by caffeine being present during the S-phase it may be regarded as good, b u t not conclusive, evidence of the involvement of postreplication repair in the production of the effect [203]. Other biochemical and physiological effects

RNA synthesis and protein synthesis The effect of caffeine on RNA and protein synthesis has attracted less atten-

13 tion than its effects on DNA synthesis and repair. When caffeine was added to the growth medium of UV-irradiated E. coli B/r try- it caused only a slight reduction in the rates of RNA and protein synthesis [204]. The synthesis of both protein and RNA in yeast and E. coli was inhibited to the extent of 50-60% by caffeine at concentrations of 0.1--0.3% [29]. The inhibition started within a few minutes of the caffeine being added to the growth media [267]. Other workers, however, report but little effect of caffeine on the synthesis of RNA in yeast [339]. There is, therefore, some confusion in the rather limited literature available a b o u t the effect of caffeine on RNA and protein synthesis in both E. coli and yeast. This may be due to strain differences. Caffeine was f o u n d to be w i t h o u t effect on RNA synthesis in the mouse liver [232]. In Vicia faba a 2-h exposure to 0.1% caffeine resulted in the inhibition of protein synthesis to about 60% of the control and of RNA synthesis to about 20--30% of control [389]. In Dictyostelium discoideum wild-type line NC4 caffeine alone at 5 X 10 -3 M reduced RNA synthesis below the control level [43]. After irradiation caffeine reduced RNA synthesis to near the non-irradiated control level. As in the case of DNA synthesis part of the effect of caffeine may be on the uptake of the labelled precursors.

Effects on e n z y m e s Caffeine at 10 -2 M almost completely inhibited the activity of rabbit-muscle phosphorylase [168]. This inhibitory effect could be completely reversed by 5'-AMP at low concentrations. Plant phosphorylases and a number of other enzymes including nucleoside phosphorylase, deoxyribonuclease, xanthine oxidase, G6PD hexokinase, and phosphoglucomutase were n o t affected by caffeine. The action of cyclic 3'5'-nucleotide phosphodiesterase was found to be competitively inhibited by caffeine [48]. Theophylline was about six times as potent. Caffeine at 10 mM inhibited cyclic 3'5'-nucleotide phosphodiesterease activity 40% in normal and 80% in neoplastic rat m a m m a r y cells grown in monolayer culture [66]. When purine-requiring mutants of Bacillus subtilis were cultured for 16 h in a medium containing 250 pg/ml of caffeine the specific activity of adenylosuccinate lyase was increased to twice t h a t of the control [243]. Two other enzymes, PRPP amidotransferase and IMP dehydrogenase, were unafffected by caffeine in this system. Effects on cyclic A M P Caffeine inhibits cyclic AMP phosphodiesterase [48] and this action can lead to a rise in the level of intracellular cAMP which can cause a delay or inhibition of cell division. This has been observed in T e t r a h y m e n a [373] and in phytohaemagglutinin-stimulated mouse spleen cells [236]. In h u m a n lymphocytes cAMP phosphodiesterease is inhibited by caffeine between 2 X 10 -4 M and 10 -2 M with 50% inhibition occuring at about 2 X 10 -3 M [191]. In Saccharomyces cerevisiae, however, caffeine neither increased the intracellular cAMP level [339] nor p r o m o t e d its action [338]. Where it occurs the increase in intracellular cAMP may explain the action of caffeine on cells particularly in relation to mitotic delay. Effects on viruses Caffeine stimulated the production of tobacco mosaic virus at low concentra-

14 tions by up to 80% in Nicotiana tabaccum tissue cultures [188], but at higher concentrations the production of both the virus and the host tissue was inhibited. Although caffeine did n o t affect the replication of Vaccinia virus grown in chick embryo fibroblasts it did block multiple reactivation and reduced the number of recombinants [306]. It was suggested t h a t this effect was due to the action of caffeine on the repair mechanisms of the host cells. Caffeine has been shown to block the repair of UV-irradiated adenovirus 2 when the irradiated virus infects XP fibroblasts from a XP variant [74]. The recovery of the herpes simplex virus synthesis in African green m o n k e y kidney cells CV-1 after UV-irradiation was inhibited by caffeine [70]. The inhibition by caffeine of the induction of endogenous C-type virus by IUdR has been observed [383], although the incorporation of 12SIUdR was n o t affected. Caffeine has been shown to inhibit host-cell reactivation in the T1 phage [286] and in the VIr phage of Proteus mirabilis [372]. The rate of reproduction of the T4Dv- phage in E. coli B was doubled when caffeine was used to inhibit the bacterial repair system [310]. The partial degradation of the DNA of the lambda phage which occurs when the phage infects a bacterium following UV irradiation was blocked by caffeine [281]. The major effect of caffeine on viruses, therefore, appears to be associated with the inhibition by caffeine of the repair mechanisms of the host cells.

Miscellaneous effects on cells Caffeine inhibits the aggregation of human blood platelets [227] and rabbit peritoneal exudate polymorphonuclear leucocytes [190]. These effects may be due to its action on cAMP phosphodiesterase. Caffeine at a concentration of 6 mM increased both the percentage motility and the grade of forward progression of ejaculated human sperm and also increased its longevity by up to 5 h [289]. Caffeine was particularly effective in cases where the control motility was low. However a later report found no significant effect of caffeine on h u m a n sperm motility at concentrations between 10-2M and 10 -6 M [84]. Epithelial cells from embryonic or newborn rat lenses showed a degree of differentiation at 24 h after t r e a t m e n t with 10 -6 M to 10 -3 M caffeine similar to that f o u n d in the control at 72 h [72]. This effect was not found with cAMP or theophylline. Caffeine inhibited cilary regeneration in T e t r a h y m e n a [373] and flagellar regeneration in Chlamydomonas reinhardii [100]. Caffeine depressed the normal recovery of Eudorina elegans from 4NQO-induced inhibition of colony-forming ability [162]. The malignant transformation of mouse A31714 cells by 4-nitroquinoline-l-oxide (4NQO) was inhibited by caffeine between 0.25 and 1.5 mM [156]. The inhibition was proportional to the concentration and occurred only when the caffeine was added to the culture soon after exposure to 4NQO. Pretreatment with caffeine was w i t h o u t effect. In cultures of Syrian hamster cells caffeine enhanced the transformation induced by the carcinogens, benzo(a)pyrene, N-acetoxy-2-fluorenylacetamide, and N-methyl-N'-nitro-N-nitrosoguanidine [83]. Caffeine also enhanced the transformation of hamster e m b r y o cells infected with oncogenic h u m a n adenovirus type 12 [196], and the frequency of SV40 transformation of C3H2K mouse cells both alone and synergistically with UV light [152]. In Saccharomyces cerevisiae the glucose suppression of sporulating ability was reversed by the

15 addition of caffeine to the medium [338,339]. Caffeine can also stabilise an unstable strain of S. cerevisiae R602 which spontaneously produces respirationdeficient mutants at a high frequency [238]. This effect is only a temporary one and disappears when the caffeine is removed. A similar effect was f o u n d in UV-induced unstable strains of Schizosaccharomyces pombe [87]. Again caffeine in the medium greatly reduced the yield of mutations but not the inheritance of the instability itself. In strains of Aspergillus nidulans which are unstable at mitosis since they carry a chromosome segment in duplicate (one in the normal position and one translocated onto another chromosome) caffeine at concentrations not inducing gene mutations increased the production of variants arising during vegetative growth from deletion of either one of the duplicated segments [47,279]. Caffeine did not change the proportions of the different deletion types nor at the concentrations used did it induce breaks in the same segments of a balanced diploid strain [279]. A concentration of 200 mg/1 caffeine has been shown to inhibit the development of a sea urchin [86]. When sea-urchin ova were fertilised with X-irradiated sperm the streak phase in first cleavage was prolonged [179]. This prolongation was reduced when caffeine at 2 mM was added to the medium. In Arbacia punctulata early cleavage stages of the zygote were retarded by caffeine [54]. This was considered to be caused by the inhibition of oxygen uptake by the caffeine. The fertilisation of the egg of A. punctulata can be prevented by caffeine [ 55]. This effect may be connnected with the changes induced by caffeine on the cell surface [56]. After a few days in a caffeine solution of 8.75 × 10 -s M the dark blue-green ciliate Stentor coeruleus lost all visible pigmentation [324]. This effect was reversed when the organism was placed in a caffeine-free medium.

Antitumour effects Post-treatment with caffeine diminishes 4NQO-induced lung t u m o u r production in the mouse [245]. Caffeine at a dose rate of I g/kg reduces the induction of mouse-lung adenomas initiated by morpholine plus sodium nitrite from 17.1 to 6.0 adenomas per mouse [228]. In combination with various psychotropic drugs caffeine significantly enhanced the a n t i t u m o u r effect of 1,3-bis(2-chloroethyl)-l-nitrosourea against murine leukaemia L1210 [67]. The action of cytoxan, N-mustard and X-ray against implanted plasmacytomas in Golden Syrian hamsters was much increased when 1% caffeine was added to the drinking water [115]. A similar effect was observed with malignant melanomas treated with X-rays, cytoxan and phenylalanine mustard [116]. The incidence of skin cancers induced in mice by mercury vapour lamp treatment drops to 50% from 90% when the skin is pretreated with 0.2% caffeine [384]. If caffeine is administered during the second and third day of larval life in Drosophila strain cl tu 48a it inhibits the development of spontaneous melanotic tumours [119]. Mitosis, cell viability, and plant cell-wall formation

Mitosis Caffeine has been shown to inhibit or delay mitosis in a wide range of cell types acting either alone or in combination with other agents. In a few cases

16 an increase in mitotic rate has been r e p o r t e d after caffeine t reat m ent . In mouse L cells 2 mM caffeine causes a small delay in the progression o f G1 cells t o S and G2 cells to M so t h a t the doubling time of such cells is increased from 16 h to up to 20 h [80]. In the same system after low ultraviolet irradiation (200 erg/mm 2) caffeine caused a specific delay in the progress of the cells through S [81]. This may be due to DNA repair inhibition since there was no additional delay in G1 to S or G2 to M. X-Irradiation o f mouse S-180 ascites t u m o u r cells in vivo (adult male Swiss albino) reduced the mitotic index to a m i ni m um after 1 h and control levels were n o t achieved after 7 h [35--37]. Caffeine at a dose rate o f 1 ml of 1 0 - 3 M injected intraperitoneally 10 min before irradiation shortened the period of mitotic inactivity and enhanced recovery so t h a t after 7 h the mitotic index of the caffeine-treated cells was m uch higher than bot h saline controls and unirradiated cells. This caffeine effect was n o t seen if the caffeine t r e a t m e n t was given 20 min before irradiation or immediately after irradiation. It was suggested t hat the effect of caffeine in this case was to increase the intracellular levels o f cAMP which was believed to have a regulat o r y effect controlling DNA synthesis and mitosis. Mouse spleen cells stimulated in vitro with phytohaemagglutinin (PHA) when t reat ed with caffeine were blocked in the G1 stage in spite of showing signs o f PHA stimulation [237]. It was suggested t h a t the initiation of mitosis was prevented in this system by the inhibition by caffeine of cAMP hydrolysing phosphodiesterase which prevented the d r o p in cAMP level in the cell necessary for the initiation of mitosis [236]. Caffeine at 1 0 - 4 M inhibited the mitosis o f PHA-stimulated hum an lymphocytes in vitro from male subjects but stimulated the mitosis o f l y m p h o c y t e s from females [329]. In the same system caffeine at 10 -3 M inhibited mitosis in l y m p h o c y t e s from b o t h sexes although the effect was greater in the malederived cultures. When caffeine at 250 pg/ml was added at the 48th h of a 72-h h u m an l y m p h o c y t e culture the mitotic index fell to about 50% of t he control [366]. Caffeine at 1 mM had little inhibitory effect on mitosis in T e t r a h y m e n a b u t it co mp letely blocked division at 8 mM [373]. The inhibited cells did not recover spontaneously during continued exposure to caffeine but the inhibition was reversible by removal to fresh caffeine-free medium. This action led t o a s y n ch r o n o u s wave of division peaking at one generation time after return to a caffeine-free medi um which suggested t ha t the cells were being blocked at a definite stage in division. Caffeine-inhibited mitosis in Oedogonium acmandriurn where the percentage o f cells in division fell as the c o n c e n t r a t i o n of caffeine was increased [285]. Gradual recovery f r o m the effects of caffeine occurred on removal of the t re a ted cells to a caffeine-free medium. Delay in the cell cycle (prolonged S and G2) occurred after caffeine (10 -4 M) t r e a t m e n t of r o o t tip cells o f irradiated b u c k w h e a t [183]. Caffeine depressed the mitotic index in Callisia fragrans nodal roots [282] and in the root-meristem cells of Vicia faba [11]. In Allium 0.4% (2.08 × 10 .2 M) caffeine for 24 h totally suppressed mitosis while concentrations o f 0.2, 0.1 and 0.04% depressed the mitotic rate [165]. With concentrations of caffeine of 0.02% or less the rate of mitosis was normal or even higher than normal. After 24 h t r e a t m e n t with 0.4% caffeine t he mitosis-free period was a b o u t 12 h. Caffeine at 0.1% in Knops solution casued a fall in the mitotic index in Allium sativum t o a b o u t 25% o f the cont rol after 10 h expo-

17 sure [ 52]. This appeared to be mainly due to an increase in the length of the time taken to complete mitosis. In Allium cepa 0.1% caffeine caused a fall in the mitotic index from 13.2 to 1.1 after 10 h exposure at 25°C [124]. Again this effect appears to be due to caffeine increasing the cell cycle time [125]. In Triticum vulgare caffeine was f o u n d to specifically inhibit cytokinesis but at low concentration {0.01%) it did not inhibit mitosis [264]. Caffeine caused a delay in the start of mitosis in Crepis capillaris root cells [92]. There has been little experimental investigation of the effect of caffeine on meiosis. Concentrations of 200 pg/ml or greater suppressed the entry of mouse ova in vitro into meiosis [154], and there was a significant decrease in the number of metaphase I cells in the testes of rats fed up to 0.3% caffeine [262]. Caffeine can either decrease or increase the mitotic index in various systems and the effect appears to depend on the concentration used. An affect on DNA synthesis seems unlikely but it is possible t h a t caffeine may cause changes in the cAMP cell concentration and thus indirectly affect the mitotic rate. When caffeine acts with other agents its effect may often be due to its action on the DNA-repair mechanisms discussed above. The observed effect of caffeine on mitosis in a particular system may therefore be the resultant of its actions on the cAMP levels and DNA-repair mechanisms.

Cell viability: caffeine alone A caffeine concentration of 8 mM caused the death o f between 30 and 50% of cells in the E. coli strains B and 15 [136]. The strains Bs-1, Bs-8, and Bs-12, all t h y m i n e excision-deficient strains, were resistant to this lethal effect. Caffeine-resistant mutants of E. coli B and 15 were isolated. Such mutants were f o u n d to have altered sensitivities to the lethal effects of UV-irradiation [135]. Caffeine-resistant mutants (Caf a) have also been obtained from E. coli K12 [75], and some of these appeared to also have increased resistance to UVirradiation. Caffeine at a concentration of 6000 pg/ml for 16 h killed 79% of cells in cultures of Chinese hamster cells [158], and toxicity in HeLa and human-skin fibroblast cultures has been observed [206]. At a concentration of 10-2M caffeine kills all cells in human l y m p h o c y t e cultures [329]. The toxicity of caffeine in Paramecium aurelia is age-related with caffeine being more toxic to older cells [308]. Caffeine has been found to have no lethal effect on growing amoebae of Dictyostelium discoideum although some inhibition of growth was observed [207]. Anabaena doliolum is fairly tolerant of caffeine since at 200 pg/ml virtually no spores are killed [313]. The sublethal and lethal concentrations of caffeine for another alga, Fischerella muscicola, were 5 mM and 10 mM respectively [304]. Oedogonium acmandrium was killed by exposure to caffeine only at the relatively high concentration of 1% for 7 days [285], and the same concentration was lethal for the root cells of Allium [165]. Caffeine was found to be a p o t e n t inhibitor of growth in Aspergillus niger and A. terreus [303]. Cell viability: caffeine in combination with other agents Caffeine has been shown to cause amplification of phleomycin-induced death in E. coli B [137,139]. The effect was equivalent to increasing the dosage of phleomycin 10 times. A decrease in survival o f UV-irradiated E. coli

18 B/r was observed after addition of caffeine to the plating medium [59]. Caffeine significantly decreased the mean lethal dose of mustard gas necessary to reduce the survival of E. coli strains B/r and Bs-1 to 37% of the control [195]. In Salmonella typhimurium LT-2 caffeine causes decreased survival after UVirradiation and abolishes the casein hydrolysate enhancement of survival [368]. The lethal effect of a number of chemical mutagens and UV in L cells was potentiated by caffeine [357]. L cells plated after treatment with mitomycin C on to caffeine-containing media had half the survival of cells w i t h o u t caffeine but if the cells were first allowed to pass through late G1 or early S the caffeine effect was lost [269]. In mouse P-388 cells grown in suspension culture caffeine at 5 X 10 -4 M strongly enhanced the cytotoxic effect of mitomycin C [251]. Caffeine also potentiated the lethal effects of sulphur mustard and MNU in Chinese hamster cells but not in HeLa cells [274,276]. A caffeine-sensitive step in the repair of DNA damage was suggested as a possible mechanism. A similar enhancement of toxicity due to cis platinum(II) diamine dichloride by caffeine in Chinese hamster cells may also be related to the inhibition of DNA repair [341]. A 2--5 fold reduction in cell survival in X-irradiated cultures of both Chinese hamster and HeLa cells by the addition of caffeine after irradiation has been reported [216]. However in two lines of HeLa cells caffeine in the post-irradiation medium failed to reduce the surviving fraction after UVirradiation [365]. Caffeine increased the mortality after UV-irradiation in all age groups of Paramecium aurelia [308]. The treatment of UV-irradiated conidia of the uvs ÷ strain of Aspergillus nidulans with caffeine enhanced the lethal UV effect [157]. The vegetative cells of the slime mould Dictyostelium discoideum strain NC-4 were relatively resistant to killing by gamma rays, UVL, and alkylating agents when compared with the m u t a n t strain ys-13. Caffeine treatment increased the killing of NC-4 by gamma and UV radiation at 1 mg/ml but decreased the gamma-ray killing rate in ys-13 [261]. A similar effect was found for the killing of these two strains with EMS. The post-irradiation survival of cells of Saccharomyces ellipsoides was reduced by caffeine [155]. After UVirradiation the caffeine supplementation of the plating medium led to a decreased survival in Schizosaccharomyces p o m b e [60]. No effect of caffeine on nitrous acid or nitrosomethylurethane induced lethality was found. In Saccharomyces cerevisiae it has been shown that the effect of caffeine on the liquid-holding recovery after treatment with diepoxybutane varies with the strain of yeast [390]. Candida albicans exhibited greater susceptibility to inactivation by UV radiation at 37°C than at 25°C [284]. It was observed that caffeine potentiation of the post-irradiation inactivation was considerable at 37°C but it had little effect at 25 ° C. Pretreatment of the spores of Anabaena doliolure with caffeine was antagonistic to UV-induced lethality but post-irradiation was synergistic [313]. Caffeine has been shown to sensitise the X-radiation induced degeneration of Drosophila testes due to the killing of the stem cells [219]. Caffeine, therefore, exhibits two distinct actions with regard to cell viability. In some systems it is itself lethal usually at relatively high concentrations while in others it is synergistic and less c o m m o n l y antagonistic with other killing agents. The effect in combination with other agents may in some cases be caused by caffeine inhibition of repair processes. There is at present no satisfactory explanation at the molecular level of caffeine lethality.

19

Plant cell-wall formation An important and characteristic property of caffeine which is confined to plant cells is its ability to cause a failure of cell-wall formation following mitosis. This results in the formation first of binucleate and later if the treatment is continued of polynucleate cells. This may be followed by nuclear fusions leading to polyploid cells. This effect has been observed in Allium root cells where the threshold concentration lies between 0.02 and 0.04% [165]. Exposure of Allium cepa roots to 0.1% caffeine for as little as 1 h is sufficient to create a binuclear cell population [128]. Longer exposure results in more binucleate cells as does an increase in the temperature from 15 to 25°C [124]. In this system it has been shown that the division cycle time increases by about 15% in the tetranucleate cells, the increase being largely due to the extra time taken to pass through mitosis which is increased by about 50%[123]. The nuclei of the polynucleate cells are more or less synchronised [129]. Similar results have been obtained with Allium sativum [ 52,79,182], Phalaris canariensis [208], Triticum vulgate [264], and Oedogonium acmandrium [285]. In Callisia fragrans caffeine treatment at 0.1% caused the formation of only a few binucleate cells [282]. This phenomenon, therefore, appears to be fairly general in plant cells although there are some species-differences in response at a given concentration. Mutagenic effects The mutagenic activity of caffeine in bacteria and fungi is well established. Its ability to cause point mutations in higher organisms is less well d o c u m e n t e d and as will be seen there are a number of conflicting reports of its effect in Drosophila while in mammals mutagenic activity although suspected and indeed expected does not seem to have been unequivocally demonstrated. Caffeine provides an almost classic case of the dangers of extrapolation of results from micro-organisms to man and of regarding chromosome damage as an indication of point mutagenicity.

Caffeine alone: micro-organisms In E. coli the mutagenic activity of caffeine in a streptomycin-dependent strain has b e e n known for a long time [76--78]. Mutation of the bacterium back to streptomycin independence was demonstrated at a caffeine concentration of 0.6% (3.12 × 10 -2 M) for 48 h when the number of mutants per 108 cells was 31.1 compared with a control rate of 9.8. In the chemostat E. coli B m u t a t i o n to T5 phage resistance was greatly increased by 150 mg/1 of caffeine [247]. This effect could be almost completely suppressed by the addition of guanosine at 500 mg/1 to the system [248]. In a similar experiment the mutation rate of E. coli B to T5 resistance was increased from the control rate o f 1.55 per l 0 s bacteria to 19.00 by caffeine at 150 mg/1 [180]. Mutation of E. coli B to T1 phage resistance by 2% caffeine for 9 h has been demonstrated [118]. At a concentration of 250 g/ml caffeine was found to be only slightly mutagenic in E. coli 15h÷m - when tested for reversion to methionine independence [133]. Under conditions of continuous culture there appears to be a delay of about 3.4 generation times after caffeine is added before mutants of

20

E. coli B/1 resistant to T5 appear [184]. This delay is n o t altered by different concentrations o f either the caffeine or the bacteria. Later it was found that the n m t a t i o n rate was directly pr opor t i onal to the growth rate o f the bacteria in most cases [185]. In glucose-limited c he most at cultures caffeine-induced T5 resistance was f o und to be p r o p o r t i o n a l to the growth rate o f the cont i nuous culture and it was suggested t hat this result was consistent with the mutational event occurring as a mistake during DNA replication [361]. In glucose-limited cultures o f E. coli K12-W6 the caffeine-induced m u t a t i o n rates were proportional to the generation rates but in methionine-limited cultures t h e y were i n d e p e n d e n t o f the generation rate [186]. It was suggested t hat caffeine may be able to induce two different kinds o f mutational response in E. coli depending on the en v ir o nm ent of the cells. Caffeine has been shown to act as a frameshift mutagen in E. coli K12 strain ND160 [63]. The mutagenic effects in the ND160 system of commercial instant coffee products have been shown to be correlated with their caffeine c o n t e n t [64]. F u r t h e r tests have shown that ND160 is n o t unique among lac Z frameshift mutants in reverting when treated with caffeine. Caffeine m a y also act as an antimutagen in E. coli. In stationary phase cultures o f E. coli 15 the his- to his* m u t a t i o n rate is reduced by more than 90% in the presence of 0.008 M caffeine [134]. Caffeine did n o t act as a mutagen in host-mediated assays when 3 histidine mutants [110] or the G-46 m u t a n t [198] of Salmonella typhimurium were used in Swiss albino mice. Over a range o f concentrations caffeine was shown to be mutagenic in Klebsiella pneumoniae [ 3 5 4 , 3 5 5 ] . In an unstable strain (R602) o f Saccharomyces cerevisiae which spontaneously produces high frequencies of a respiration-deficient (RD) m u t a n t caffeine considerably reduced the p r o d u c t i o n of RD m ut ant s although the effect disappeared when the caffeine was removed [238]. However in normal yeast caffeine was f ound to be highly effective in the p r o d u c t i o n o f respiratory deficiency [205]. Both biochemical and morphological mutants o f Ophiostoma multiannulatum were obtained with caffeine at a c o n c e n t r a t i o n o f 0.2% [ 105]. A thorough investigation d e m o n s t r a t e d t hat caffeine was acting as a true mutagen on this fungus and not simply as a selective killing agent [ 1 0 6 ] . It was also f o u n d t ha t the range of biochemical mutants obtained with caffeine was essentially the same as that p r o d u c e d by UV-radiation. However caffeine was n o t f ound to be mutagenic in the O phi ost om a back m u t a t i o n test perhaps because of the short t r e a t m e n t period used [385]. Caffeine was shown to be an effective mutagen in the blue-green alga, Plectonema boryanum [305]. In combination with other agents: micro-organisms While caffeine alone has been f o u n d to be mutagenic in E. coli its effect when acting in com bi na t i on with UV light is even more striking. Addition of caffeine to the growth m edi um of UV-irradiated E. coli B/r try- immediately after irradiation caused the p r o d u c t i o n o f a b o u t ten times as m a n y try + mutants compared with UV alone [204]. Since incubation o f the bacteria in caffeine before irradiation has no effect and the ability of the irradiated culture to respond to caffeine was com pl e t e l y lost 20 min after irradiation it was suggested that the effect was due to caffeine interference with a dark-repair e n z y m e system. When caffeine at 500 pg/ml was added t o UVL treated E. coli B/r cells the n u m b e r of mutants obtained was greatly in excess of the com bi ned

21 totals of caffeine or UVL alone [296]. The effect of caffeine can be largely reversed by treating the cells with photoreactivating white light [297,301], or by the substitution of t h y m i n e analogues in the DNA of the organism [298]. It was suggested t h a t the caffeine-induced increase in m u t a n t frequency was due to the prevention of excision of radiation-induced dimers by the caffeine. The m u t a t i o n of E. coli B/r to high-level streptomycin resistance also showed synergism between UVL and caffeine but not between X-radiation and caffeine [82]. The synergistic effect of caffeine with UVL in E. coli can show variation with the locus being studied. The reversion frequency after UVL of the try locus was doubled by caffeine while that of the his locus increased by 7.3 times and that of p r o by 12.4 times [260]. In three di-auxotrophs of E. coli B/r: 36-10, 36-18, and 36-32, all try-leu-, addition of caffeine to the plating m e d i u m after UVL caused increases in the frequencies of all three possible revertants [59]. In the hcr- strain of E. coli B/r caffeine following UVL caused no enhancement of the try* revertant yield, but in the h c r ÷ strain there was a dose enhancement by caffeine of the try ÷ revertant frequent in media containing no or only t r y p t o p h a n while in media containing both a low level of t r y p t o p h a n and an additional pool of other amino acids caffeine caused a preferential enhancement of try ÷ revertant frequency above purely dose enhancement level [61]. The yield of try ÷ mutants in an hcr ÷ strain of E. coli B/r treated with nitrous acid was enhanced by caffeine but when an hcr- strain was used caffeine had an antimutagenic effect [62]. Similar results with other hcr ÷ and hcrstrains of E. coli following UVL have been obtained [23,301,342]. The antagonistic effects of caffeine and UV on mutation induction in hcr- strains of E. coli has been explained on the basis of caffeine inhibition of an error-prone (post-replication) repair process [370]. The optimum concentration of caffeine for its synergistic effect with UVL in E. coli B/r has been estimated at 1.804 mM [300]. Caffeine at 0.1% and higher concentrations decreased the frequency of UVL-induced $3 mutants in E. coli B/phr-/MC2 [151]. Caffeine post-treatment reduced the tryptophan-revertant frequency in E. coli B/r WP-2 resulting from DEB t r e a t m e n t but it increased the frequency of DEBinduced m u t a t i o n to high-level streptomycin resistance [42]. In the same system caffeine increased the frequency of try ÷ revertants after EMS t r e a t m e n t in the h c r ÷ strain. In chloramphenicol-inhibited E. coli B/r where protein synthesis is inhibited caffeine acts as a mutagen [126]. As in the control cultures the mutagenicity of caffeine in this system can be suppressed by adenosine. In S a l m o n e l l a t y p h i m u r i u m LT-2 (trp C3) after UV-irradiation caffeine appears to act as an antimutagen in the trp- to trp ÷ m u t a t i o n [368]. However in S. t y p h i m u r i u m strains S L l 1 5 6 and S L 1 1 5 6 / R l 1 5 6 / R - U t r e c t caffeine acts synergistically with UVL in producing trp ÷ revertants [215]. Caffeine also acts synergistically in the production of mutations in A n a b a e n a d o l i o l u m [312], P l e c t o m e n a b o r y a n u m [305], D i c t y o s t e l i u m d i s c o i d e u m [207], and P h y s a r u m p o l y c e p h a l u m [146,147]. Caffeine had an antimutagenic effect on the production of the petite m u t a t i o n in yeast by ethidium bromide [149] and on the yield of reversions at the adenine 2-1 locus after irradiation [220]. In S c h i z o s a c c h a r o m y c e s p o m b e caffeine decreased the frequency of forward mutations at the ad-6 and ad-7 loci induced by UVL and NG and the UV-induced reverse mutations of his- mutants [209]. In M i c r o c o c c u s radiodurans wild-type and

22 Ts5 (temperature-sensitive) strains UVL failed to act as a mutagen and caffeine was unable to sensitise the cells to UVL [318].

Mitotic crossing-over in plants Light-green plants of Glycine max with the genotype Y1 ~Y~ produce on the leaves double spots which resemble the genotypes Y,IYII and Y,~YI~. This is believed to be due to somatic or mitotic crossing-over [349]. The frequency of mitotic crossing-over in this plant was increased many times by the application of 0.0125% caffeine to the seeds for a few h [345,346]. Caffeine also increased the rate of point mutation in y ~ y ~ plants [347]. Sodium azide increased the point m u t a t i o n frequency in G. max but n o t that of mitotic crossing-over and synergism between sodium azide and caffeine in the production of either point mutations or mitotic crossing-over could not be demonstrated [348]. Caffeine at a concentration of 0.5 mM for 1 h almost doubled the rate of mitotic crossing-over in in vitro cell cultures of Nicotiana tabaccum cv. John Williams Broadleaf [49], and induced a greater increase in mitotic crossing-over in two strains of Ustilago rnaydis at 0.1% [150]. Caffeine alone: Drosophila The question of the mutagenicity of caffeine in Drosophila melanogaster must be regarded as unresolved. It has been described as mildly mutagenic in Canton S females and Muller 5 males as demonstrated by an increase in the percentage of sex-linked lethals following caffeine treatment [14]. Yet in similar experiment with Canton S and Oregon R males no mutagenic effect could be observed [378]. Caffeine treatment of the larvae has been reported to cause the preferential death of males and to induce sex-linked recessive lethals in females [256]. Other workers could find no evidence for mutagenesis in caffeine-treated male larvae [ 12,58]. Caffeine did not significantly alter the frequency of X-radiation-induced recessive lethal mutations in D. melanogaster but it was found to be a weak mutagen alone [231]. Addition of 0.115% caffeine to the medium induced recessive sex-linked lethal m u t a t i o n in both radiosensitive Canton S and radio-stable D-32 stocks [295]. However evidence which suggests that caffeine does not induce sex-linked lethal mutations in D. melanogaster has been presented [101]. There is almost no data on the mutagenic effect of caffeine in other insects. It failed to induce d o m i n a n t lethal mutations in Bornbyx mori [234]. In combination with other agents: Drosophila It has been claimed that in Oregon-R stocks although caffeine alone is not mutagenic it is synergistic with gamma-radiation in the production of sexlinked lethals [381]. An increase in the number of X-radiation induced dominant lethals has been observed following caffeine treatment in some but not all strains of D. melanogaster [222,223]. It has been suggested that this may reflect the action of caffeine on the DNA-repair systems. No such enhancing effect of caffeine on the yield of d o m i n a n t lethals occurred when D. melanogaster was treated with EMS or DEB but enhancement after X-irradiation was again found [359]. It can be seen, therefore, t h a t while some of the data support the hypothesis

23 that caffeine acts as a mutagen in D. melanogaster and some workers have concluded t h a t it is indeed mutagenic [99,187] other data suggest t h a t it is not mutagenic in this organism. Much would seem to depend on the stock used, the means of administration, the sex treated, and perhaps the medium on which the flies are grown. It has been demonstrated t h a t different strains of D. melanogaster have different sensitivitiies to caffeine in terms of toxicity and that these are not correlated with MMS sensitivity [241]. It seems safe to say at this point only that caffeine appears not to be strongly mutagenic in D. melanogaster but t h a t it may act as a weak mutagen in certain circumstances. It may also at least under certain conditions act synergistically with other mutagenic agents perhaps most convincingly with X-rays.

Chinese hamster cells Although caffeine was found to be efficient in producing chromosomal aberrations in Chinese hamster cells it was found to be of low efficiency in the induction of m u t a t i o n to a u x o t r o p h y in this system [158]. Caffeine at 6000 ng/ml for 16 h killed 79% of the cells but produced no mutants. It has been reported that caffeine acts as an antimutagen in Chinese hamster cells and reduces the frequency of both spontaneous and UV-induced mutations [334]. The effect with UV only occurs if the caffeine is present in the culture medium during the first post-irradiation division [336]. The reality of this effect has been challenged [233] and other workers have found that if caffeine is present only during the period allowed for expression of mutants then the frequency of mutants is increased but that if it is present during the whole of the postirradiation incubation period then the frequency of mutants is decreased [16]. Evidence has been presented which suggested that in V79 cells caffeine delayed the expression of UV-induced mutants and that the delay was dependent on the dose of caffeine used [102]. Mutation frequency was increased in MNUtreated Chinese hamster cells by incubation after alkylation in media containing 0.75 mM of caffeine [274,275,277]. Caffeine at concentrations of 50-200 mg/ml had little effect on the m u t a n t frequency induced by 0.1--2.5 mg/ml AcAAF in Chinese hamster cells strain V79-4 [235]. The frequency of mutatin to ouabain-resistance after EMS treatment was enhanced in Chinese hamster cells [340]. The effects of caffeine on the mutation of Chinese hamster cells appears to be very dependent on the exact conditions and timing of the exposure to caffeine. Recently it has been suggested t h a t caffeine does n o t increase the m u t a t i o n frequency but instead converts sublethal to lethal damage in alkylated and UV-irradiated Chinese hamster cells strain V79 [103]. Other workers have obtained results which support this suggestion [302]. They f o u n d that after EMS treatment or UV-irradiation of V79 cells caffeine appeared to partly block a repair process which led to enhancement of cell killing but t h a t the error frequency in the repaired lesions was not affected by caffeine.

Mammals Experiments which mice were given caffeine at 0.1% dissolved in their drinking water from the time of mating of their parents showed no significant difference in the m u t a t i o n rate from the spontaneous m u t a t i o n rate [213], and such mice on this regime t h r o u g h o u t life continued to breed satisfactorily.

24 Essentially similar results were obtained with 0.3% caffeine in drinking water [50]. The d o m i n a n t lethal test in male Swiss (CD-1) mice showed that caffeine at 168 mg/kg body weight was not mutagenic [93]. Acute and chronic administration of caffeine to mice also failed to produce mutations or to act synergistically with known mutagens [94,95]. The C3H inbred strain of mice tolerated caffeine at a dose rate of 0.25 g/kg b o d y weight w i t h o u t evidence of mutagenicity by the d o m i n a n t lethal test [4]. No mutagenic effect of caffeine was found in mice given caffeine in their drinking water either for short periods before mating [327] or over four generations [328]. Rats treated before and during mating with 0.05 and 0.7 g/kg caffeine for four generations showed no mutagenic effects [89]. Although it has been suggested that caffeine may be mutagenic in mammals [141] and man [257] the direct evidence for this is almost nil and the indirect evidence seems largely based on extrapolation from lower organisms where there is no d o u b t about the mutagenic action of caffeine and on the clastogenic effect of caffeine in mammalian cells which will be considered later. It has been suggested that while caffeine may well be potentially mutagenic in mammals including man this mutagenicity may never be expressed in the p h e n o t y p e or offspring because the mutagenic threshold may be the same as the antimitotic threshold and therefore the production of a mutation by caffeine would be an extremely rare event [331]. Equally possibly the clastogenic effects at the mutagenic threshold may prevent multiplication of a caffeine-mutated mammalian cell. Such effects together with the relatively short half-life of caffeine in the body may represent an evolved mechanism for the avoidance of the mutagenic effect of caffeine in man which allows him to indulge freely in his addiction to the drug. Production of chromosomal aberrations

Caffeine alone: plant material Caffeine at 0.02--0.4% can cause chromosome disturbances in the root tips of Allium cepa [165]. The first effect noticed was chromosome stickiness and the formation of anaphase bridges. Structural chromosome changes including fragmentation, sister-chromatid reunion, and reciprocal translocations were found in over 30% of mitoses after 24 h in 0.1% caffeine followed by a period in water. A caffeine concentration equivalent to drinking one cup of coffee produced 6.9 aberrations per 100 cells compared with 1.6 per 100 cells in the control in onion root tips [287]. In Allium sativum root tips caffeine at 0.1 and 0.5% induced the formation of chromosome bridges, fragments, chromatid exchanges, ring chromosomes, and micronuclei [182]. When the root tips of Allium proliferum were treated with caffeine at 10 -2 M and 2 X 10 -2 M most of the chromosomal aberrations produced were obtained in cells exposed in late G2 and prophase [170]. These aberrations were mainly " s u b c h r o m a t i d " and chromatid exchanges. It was f o u n d t h a t the effect was dependent on the ATP concentration in the cells with very few aberrations being produced when ATP production was suppressed by anoxia or sodium azide. Chromosome breakage induced by caffeine has been demonstrated in Vicia faba [161], where the frequency of aberration per cell was directly proportional to the caffeine concentration and the duration of the treatment. It has been reported t h a t caffeine is

25 a more effective clastogen in V. faba at 14°C than at 35°C [252]. The induction of chromatid aberrations in V. faba by caffeine has been confirmed but no significant increase in the number of sister-chromatid exchanges was caused by caffeine in this material [175]. Chromosome damage by caffeine has also been reported in Pisum sativum [167], Callisia fragrans [282], Hordeum vulgare at 600--1000 pg/ml [377], and Coreopsis tinctoria [25].

In combination with other agents: plant material In the root meristems of V. [aba caffeine significantly increased the number of chromosomal aberrations per cell produced by the combined action of EDTA and ethyl alcohol if the caffeine was applied with or immediately after the combined t r e a t m e n t [290]. In V. faba vat. minor a chromosome aberration yield four times that of DEB alone was produced with caffeine post-treatment [319]. Simultaneous t r e a t m e n t with caffeine was less effective. Later work showed that the caffeine treatment was most effective if it was present when the cells went through the first S phase after alkylation [320]. Caffeine did n o t significantly affect the yield of aberrations in V. faba produced by X-rays or gamma-radiation [114,321,322]. However the yield of chromosomal aberrations induced by X-rays in Secale cereale seeds was signifcantly increased by caffeine post-treatment [321,323]. Chromosomal damage was greatly increased by caffeine in V. faba var. minor treated with mitomycin C, TEPA, thioTEPA, maleic hydrazide, and 4NQO [171,172]. The potentiating effect was only observed when the cells were exposed to caffeine during the S phase. In V. faba post-treatment with caffeine as potentiating agent reduced by a factor of about 4--5 the thioTEPA concentration needed to produce a given chromosomal aberration frequency [316]. Post-treatment for 5 h with 2.5 X 10 -3 M caffeine of root tips of V. faba increased the frequency of chromosome aberrations produced by maleic hydrazide and certain monofunctional alkylating agents independently of the temperature during caffeine exposure [178,317]. Posttreatment was equally effective 1 and 6 h after alkylation. With di- and tri-functional alkylating agents, however, caffeine potentiation of chromosome damage was much more effective at 25°C than at 10°C and when the post-treatment with caffeine was given 5--6 h after exposure rather than immediately after alkylation. The synergistic effect of caffeine on chromosomal aberrations produced in V. faba by maleic hydrazide, mitomycin C, and thioTEPA has been confirmed at 5 X 10 -4 M caffeine [144]. The potentiation factor was higher for thioTEPA than for m i t o m y c i n C. The threshold level for potentiation after mitomycin C was found to be 2--3 X 10-4M caffeine and the most sensitive period was confirmed as the middle to late S phase. In contrast with earlier reports [321,322] 2-h treatments with 5 X 10 -3 M caffeine strongly enhanced the frequencies of chromosomal aberrations obtained in V. faba root tips 5--6 h after X-irradiation [175]. The caffeine treatment was effective only when the time between irradiation and caffeine exposure was less than 60 min. The aberration frequency was higher at 27°C than at 15°C but was n o t greatly influenced by the addition of sodium azide. The frequency of sister-chromatid exchanges caused in V. faba chromosomes by thioTEPA and other agents was not much increased by caffeine t r e a t m e n t which significantly increased the frequency of chromosome aberrations [176,177]. The stability of chromatid

26 bridges induced by gamma-irradiation of V. faba and Crepis capillaris was increased by caffeine post-irradiation treatment [91] and in C. capillaris the appearance of gaps in the chromosomes was inhibited by caffeine[90]. When C. capillaris root chromosomes were irradiated with 400 R gamma-radiation and treated with caffeine at the same time or 4 o r 6 h after irradiation the number of cells with visible aberrations was increased [92]. However caffeine treatment 2 h after irradiation was without effect and it was suggested that the action of caffeine may have been to cause a delay in the initiation of cell mitosis. In Hordeum vulgate caffeine treatment of the cells before or 10 min after gamma-irradiation greatly increased the chromosome aberration yield [377], but when caffeine treatment was 20 or 30 min after irradiation the aberrations found were simply the sum of those expected from radiation and caffeine. It was suggested t h a t caffeine inhibited repair or rejoining of chromatid breaks after gamma-radiation. When ungerminated barley seeds in the G~ stage were treated with 10 -2 M caffeine for 2 h at 20°C and then dried back to 13% water c o n t e n t before gamma-irradiation caffeine was found to act synergistically with the radiation in the production of chromosomal aberrations in the anaphase cells of the embryonic root tips [283]. Caffeine enhanced the chromosome damage induced by both gamma-radiation and alkylating agents in germinating barley seeds probably by inhibition of the repair mechanisms [7,8]. After gamma-irradidation of barley seeds caffeine in the oxygenated water prevented part of the oxygen-dependent post-irradiation chromosom~ damage but it also counteracted the protective effect of oxygenfree hydration [164]. In this system, therefore, the action of caffeine was very different in oxic and anoxic conditions. Post-irradiation treatment with 10 -4 M caffeine of the root-tip cells of both diploid and tetraploid strains of buckwheat (Fagopyrum esculentum) increased the yield of chromosome aberrations significantly [183]. Caffeine was more effective as a synergistic agent in the tetraploid strain. It was suggested t h a t caffeine acted by inhibiting the repair of chromosome breaks and that the cells of the tetraploid buckwheat may have a more effective repair mechanism. In Allium proliferum caffeine enhanced the frequency of chromosomal aberrations induced by thioTEPA, mitomycin C, and maleic hydrazide [144,172], and by thioTEPA and mitomycin C in Nigella damascena [ 144].

Caffeine alone: animal material The effect of caffeine on the chromosomes of Drosophila melanogaster has mainly been studied in terms of chromosome loss and nondisjunction. As is the case with point m u t a t i o n in this organism there are conflicting reports in the literature regarding the action of caffeine. X-Chromosome loss has been reported following caffeine t r e a t m e n t of D. melanogaster larvae although the nondisjunction rate was somewhat reduced [256]. Feeding of 2--6 h old adult male D. melanogaster with a ring X-chromosome on media containing 0.123% caffeine led to a significant increase in the number of XO males but not in the number of translocations [230]. A large increase was also found in the number of nondisjunction females (XXY) after this treatment [229]. It was suggested t h a t these effects could be due to chromosome stickness of the type reported in Allium cepa chromosomes [165]. There is evidence t h a t the effect of caf-

27 feine on D. melanogaster is d e p e n d e n t on the stage in the life cycle at which it is administered. Inclusion of caffeine at 0.01 M in the f o o d on which the larvae are reared led to increased frequencies of c h r o m o s o m e loss in bot h males and females [58], b u t there was some evidence t h a t the f r e q u e n c y of nondisjunction was lowered by caffeine t r e a t m e n t at least in the males. In the same study it was f o u n d t h a t t r e a t m e n t o f adult flies with caffeine either by feeding or by injection caused on significant effect on c h r o m o s o m e loss or nondisjunction. Reviewing the data then available Bateman [24] in 1969 concluded that while caffeine could induce the loss of sex c h r o m o s o m e s in both sexes the m a x i m u m effect was to double the spontaneous incidence which is far from impressive. The data on nondisjunction was variable and no firm conclusion was considered possible. In general larval feeding with caffeine was more effective than adult feeding. Later data t e nd to confirm these conclusions. The addition of 0.115% caffeine to t he m edi um induced X -chrom osom e nondisjunction in females o f b o t h radio-sensitive Canton S, and radio-stable D-32 stocks of D. melanogaster to the same e x t e n t [295]. Three types of male D. rnelanogaster, normal X, ring X, and short X, treated with 0.5% caffeine during the third instar showed increases in c h r o m o s o m e loss but the effect on nondisjunction was inconclusive [286]. Undernourished cultures of D. melanogaster showed twice the f r e q u e n c y of XO males when com pa red with well fed controls [98]. The addition o f caffeine to the f o o d o f the undernourished group reduced the XOmale f r e q u e n c y in p r o p o r t i o n to the caffeine c o n c e n t r a t i o n and at high concentrations of caffeine the f r e q u e n c y of XO males was less than that of the controls. There is some evidence t ha t caffeine inhibits the normal crossing-over in meiosis in D. melanogaster [ 3 7 9 , 3 8 0 ] . Caffeine has been shown to be efficient in producing c h r o m o s o m e aberrations in cultured Chinese hamster cells [ 158]. When treated with caffeine at 37°C Chinese hamster cells were affected during S-phase [169, 170]. A pron o u n c e d ch r o ma t i d fragmentation was the main effect which was independ e n t o f ATP cell c onc e nt r at i on. In t he same system when the cells were treated at 17°C mo s t o f the aberrations were p r o d u c e d in cells treated during prophase or G2 and a b o u t half of these were " s u b c h r o m a t i d " and chromatid exchanges. At ][7°C the effect was ATP-dependent. In endoreduplicated Chinese hamster cells caffeine p r o d u c e d a significant increase in chrom osom al aberrations [258]. Some o f the caffeine-induced damage f o u n d in this system would be difficult to d e t e c t in normal diploid cells which led to the suggestion t h a t t he effect o f caffeine on c h r o m o s o m e s may be greater than previously reported. While caffeine has been shown to induce chrom osom al aberrations in mammalian cells in culture similar results have n o t been observed when whole animals have been used. Caffeine did n o t induce bone-marrow depression or micronucleus f o r m a t i o n in mice although this system has some value as a means o f evaluation of drugs as inducers of c h r o m o s o m a l aberrations [ 221]. No unusual c h r o m o s o m e changes were f o u n d in the testes of C3H mice treated with the m a x i m u m tolerated dose of caffeine [2]. Chronic caffeine t r e a t m e n t (245 days or longer) o f C3H and 101 × C3H F~ hybr i d mice also gave no evidence of caffeine induction of c h r o m o s o m a l aberrations in spermatogenesis [ 5]. The in vivo t r e a t m e n t of the $2 sarcoma of white mice with 0.25 g/kg o f caffeine p r o d u c e d no evidence o f an increase in c h r o m o s o m a l aberrations over t hat of t he cont rol

28 [ 3]. Caffeine also failed to induce a significant number of chromosomal aberrations in a cell line established from a male rat breast (MCT 1) at concentrations equivalent to 8--32 times the transitory peak level found in man [32]. There are, therefore, some differences in response to caffeine as a clastogenic agent in animal cells.

In combination with other agents: animal material Caffeine has been shown to act synergistically with UVL in Drosophila melanogaster in the production of nondisjunction and crossing-over [344]. However caffeine had no significant effect on the yield of induced crossovers on the right arm of chromosome II in D. melanogaster during spermatogenesis when added to the medium after irradiation [220]. When young D. melanogaster females were treated with caffeine prior to mating with males irradiated with 2000 R X-irradiation there was an increase in the frequency of sex chromosome loss [223]. Caffeine t r e a t m e n t of the radio-resistant D. melanogaster stock R512 increased the X-ray response to that of the normal stock. It was suggested that caffeine reduced the efficiency of the system in D. melanogaster which repairs X-irradiation-induced chromosomal damage. In a similar experiment it was f o u n d that when the females were allowed to feed on 0.2% caffeine the frequency of chromosome loss was higher than the control but it was lower than the control with 1% initially [388]. The latter group later showed a chromosome loss frequency above the control level. Caffeine failed to alter the yield of translocations recovered from male D. melanogaster irradiated as 36-h pupae and mated to caffeine treated females [309]. Mating of caffeine treated females to irradiated male D. melanogaster, however, led to an enhancement of chromosome loss and a decrease in the frequency of chromosomal rearrangement [224,225]. The results are interpreted as showing that caffeine inhibits the repair processes active in the oocytes. There is some evidence of a sex difference in response t o the interaction of caffeine and X-irradiation. In D. melanogaster yw/scSy caffeine alone increase the frequency of chromosome loss in males but had no effect on females [15]. However treatm e n t of irradiated flies with caffeine led to an increase in the radiation-induced chromosome loss in females but had no effect in the males. Caffeine post-treatment of Chinese hamster cells irradiated with UVL decreased the frequency of sister-chromatid exchanges induced by the UV but increased the frequency of chromatid aberrations of the deletion type [160]. In UV-irradiated Chinese hamster cells, strain CI-I, caffeine potentiated the production of UV-induced chromosomal aberrations possibly possibly by inhibition of the repair mechanisms [242]. Caffeine caused a doubling of the number of aberrant anaphases in Chinese hamster cells after X-irradiation [216]. The effect was the same whether the caffeine was added immediately after irradiation or up to 60 min later. In Chinese hamster cells, strain CHO-KI, caffeine has been shown to inhibit the restoration of X-ray-induced chromosomal damage [356]. MNU caused chromatid aberrations in 15% of treated Chinese hamster cells by 48 h alkylation [274]. Post-alkylation incubation in caffeine increased the number of cells showing chromosomal damage to a maxim u m of 86% by 40 h. It was again suggested that a caffeine-sensitive step o c c u r s in the repair of DNA damage which was responsible for the observed

29 effects [277]. Caffeine has also been shown to increase greatly the frequency of chromosomal aberrations produced in Chinese hamster cells by m i t o m y c i n C, TEPA, thioTEPA, maleic hydrazide, and 4NQO [144,171,172,316]. The clastogenic effects of mitomycin C and thioTEPA in Chinese hamster cells were enhanced by caffeine at concentrations which caused few or no aberrations [ 19]. In Chinese hamster cells, strain V79-379A, cis Pt(II) diamine dichloride caused chromosomal aberrations the yield and severity of which was greatly increased by non-toxic concentrations of caffeine [341]. The time of appearance of the chromosomal abnormalities suggested that t h e y arose as a consequence of DNA synthesis on a damaged template and the action of caffeine may be to inhibit a DNA-repair mechanism. Caffeine has also been shown to potentiate chromatid aberrations in the bone-marrow cells of Chinese hamsters induced by cyclophosphamide [159,278]. On the other hand caffeine has been observed to have a protective effect in the bone-marrow cells of rats against the chromosome-damaging effect of the alkylating agent dipine [239]. The caffeine reduces the number of chromosomal aberrations produced by dipine by about 30%. Caffeine also exerts a protective effect against the action of dipine in rat hepatocytes [240]. The administration of caffeine at 1% in their drinking water to mice treated with MMS enhances the chromosomal damage found in the bone-marrow cells [104]. In ascites t u m o u r cells of the mouse caffeine did not increase the number of chromosomal aberrations per damaged cell caused by hydrogen peroxide or DMS but the peak time for aberrations after treatment was found to be about 10 h earlier [290]. Caffeine at 5 × 10 -4 M enhanced the frequency of chromosomal aberrations induced by thioTEPA and m i t o m y c i n C in Potorous tridactylis cells in culture [144].

Caffeine alone: human material No increase in the incidence of chromatid breaks was found in HeLa cells continuously exposed to up to 20 mg/ml caffeine for up to 9 weeks, i.e. 48 cell divisions [326]. This is a long-term exposure to caffeine at levels higher than the transitory peaks normally obtained in man by coffee drinking. However at higher doses of up to 160 mg/ml of caffeine for 4 weeks HeLa cells showed a significant increase in the frequency of chromosomal breaks and rearrangements [30,31]. Thus caffeine can cause chromosome damage in HeLa cells but only at concentrations 8--32 times the transitory peak level normally experienced in man. Caffeine-induction of chromosome and chromatid breaks has been shown at relatively high concentrations in both HeLa cells and cultured h u m a n lymphocytes [255]. A high frequency of chromatid aberrations, mainly gaps and breaks, was induced by caffeine in cultured human lymphocytes and human embryonic lung cells in culture, again at relatively high caffeine concentrations [197]. The induction of the damage appeared to be confined to the S-phase especially the latter part of S with no effect in G~ or G2. Cultured human lymphocytes taken from volunteers on a regime of 800 mg of caffeine daily for one m o n t h showed no significant increase in chromosomal damage [363]. The highest recorded level of caffeine in the plasma was 30 mg/ml and in vitro exposure of lymphocytes from untreated donors to this level of caffeine was also without effect on the chromosomes. It was possible to produce chromosome damage, however, by a single exposure of the lym-

30 phocytes to 250--750 mg/ml caffeine at the 48th h of culture. L y m p h o c y t e s from 17 healthy donors exposed to 750 mg/ml at the 48th h of a 72-h culture had an average of 2.08 chromatid breaks, 0.40 chromatid gaps, and 0.23 chromosome breaks per metaphase [365]. The effect of caffeine was highly significant. Of control metaphases 1.65% (S.D. 2.2) had at least one chromatid gap while 51.9% (S.D. 14.9) of the caffeine-treated cells showed this type of aberration. For both sexes the damage was non-randomly distributed among the chromosomes with in both cases chromosomes 3 and 16 showing twice as much damage as expected. In an a t t e m p t to examine the mechanism of caffeine clastogenicity in human lymphocytes no evidence was obtained which would indicate that caffeine was acting as a purine analogue, or as an inhibitor of phosphodiesterase, or as a stimulator of adenylsuccinate (S-AMP) lyase, or as a labiliser of lyosomes, nor as a clastogen which could be inhibited by an antimutagen [364].

In combination with other agents: human material Single-strand DNA breaks were induced in cultured human lymphocytes by gamma-irradiation in doses of 10--30 krad, but almost complete rejoining of the breaks occurred after 60 min [9]. When caffeine at 6 X 10-4M and 6 × 10-3M was present in the medium rejoining was prevented. The effect was almost entirely confined to cells irradiated in the S--G2 phases and negligible in the G0--G~ stage [10]. The effect was assumed to be due to DNA-repair inhibition by the caffeine, and may represent the first stage in the synergistic action of caffeine in the production of chromosomal aberrations. At 10 -3 M caffeine enhanced the number of chromosomal breaks and exchanges induced in cultured human lymphocytes by X-irradiation at 50, 100 and 200 R but has no effect on the number of gaps and constrictions [40]. In the same system caffeine enhances all types of chromosome damage induced by mitomycin C (at 0.1 and 0.5 mg/ml) and MMS (at 2 X 10 -'~ M and 2 × 10 -4 M). Autoradiographic studies with 3H- and 14C-labelled caffeine did not support the hypothesis that caffeine was binding to the DNA [41], and the results are best explained on the basis of caffeine inhibition of a post-replication repair process concerned in particular with the filling in of gaps in newly synthesised DNA. Caffeine did not potentiate the chromosome damage induced in human (LU 106) cells by thioTEPA and mitomycin C [174]. However caffeine post-treatment did potentiate the chromosomal aberration frequency induced by mitomycin C in a normal h u m a n fibroblast strain and in two xeroderma pigmentosum strains (XP4LO and XP7TA) but at different threshold values [145]. Comparison o f the effects on plant and animal chromosomes In general it would seem to be the case that caffeine alone and acting together with other clastogenic agents has rather similar effects on animal and plant chromosomes. In the few cases where a direct comparison has been made, however, there are differences which are of interest. When a comparison of the chromosome-damaging effects of caffeine in the root tips of Allium proliferum and cultured Chinese hamster cells was made it was discovered that some of the differences in response were due to the treatment temperature and that the plant and animal cells responded in a more similar manner at the same tempera-

31 ture [4]. However some differences remained. In particular the frequencies of chromatid breaks and the degree of incompleteness of the exchanges were considerably higher in the hamster cells that in the Allium cells. A comparison of the potentiating effect of caffeine on chemically-induced chromatid aberrations between Vicia faba root tip cells and mouse ascites t u m o u r cells showed that in V. faba cells simultaneous and post-treatment with caffeine significantly increased the aberration peaks and the number of aberrations per damaged cell but that this effect could not be demonstrated in the ascites cells [9]. Two distinct types of chromosome-breaking effect have been attributed to caffeine and caffeine derivatives, and other methylated oxypurines, the Ostertag and Kihlman effects [173,246]. The Ostertag effect consists of a fragmentation of the chromosomes amounting at times to a pulverisation and occurs in cultured mammalian cells treated with caffeine during the S-phase [170,254, 255,257]. This effect has been demonstrated in a number of different mammalian cells in culture including human cells [30,158,197]. The Ostertag effect has not been found in plant cells even when the treatment temperature was raised to 35°C [67]. The Kihlman effect is seen in plant cells and in cultured mammalian cells at temperatures below 30°C and can be produced at any stage of the cell cycle although G2 and prophase are the most sensitive stages [173]. Apart from the temperature difference the Kihlman effect seems to be dependent on the cellular ATP level while the Ostertag effect is dependent on DNA synthesis. The type of aberration produced is also different. The Kihlman effect yields "sub-chromatid" and chromatid exchanges with few breaks while the Ostertag effect gives few exchanges but many chromatid breaks [173]. Thus the evidence is considerable t h a t caffeine has at least two distinct modes of clastogenic action. The Kihlman effect seems to be of more general occurrence while the Ostertag effect is found only in many but not all [31,32] cultured mammalian cells and may be a response to the culture conditions. In plant cells the m a x i m u m Kihlman effect was obtained between 10°C and 15°C while in Chinese hamster cells t r e a t m e n t between 20°C and 25°C induced the highest frequency of aberrations [246]. Caffeine clearly acts as a clastogenic agent both in plants and in animal cells in culture although the latter responds only at concentrations well above that normally found in man, There is no direct evidence of clastogenic effects at normal concentrations in whole animals and even in Drosophila the evidence must be regarded as inconclusive. Caffeine can clearly have a potentiating effect with other clastogenic agents in many but n o t all systems and the weight of the evidence suggests that this is mainly, perhaps entirely, due to caffeine inhibition of the DNA-repair mechanisms. The effect of caffeine on chromosomes has recently been reviewed in much more detail than is possible here by Kihlman [178]. Teratogenic and carcinogenic effects Early work showed t h a t the normal development of the sea urchin could be inhibited by caffeine at 200 mg/1 [86]. At caffeine concentrations of M/40 and higher the physiological processes underlying the differentiation mechanisms of the zygote of Arbacia puncutulata were completely inhibited [54]. Using the

32 cl tu 4sa strain of Drosophila which spontaneously develops melanotic tumours it was found that caffeine both increased the mortality of the y o u n g larvae and if administered during the second or third day of larval life it caused a decrease in the number of tumerous individuals [119]. Outside this period caffeine was n o t effective. When Chinese hamsters were treated with 0.02 g/100 ml in their drinking water for 60 days the resulting litters showed a significant change in the sex ratio from the control value of 49.2% females to 61.4% females [360]. Intraperitoneal injections of 1% caffeine to give a dose level of 0.25 mg/g body weight (which is close to the m a x i m u m tolerated dose) o f pregnant mice during the 7th--15th day period of pregnancy caused embryonic death and malformed foetuses [244]. Foetal malformation rates of 18--43% were obtained when the injection was given between the 10th and 14th day. Before that time there was little or no effect. Most of the malformations observed were of the skeletal system especially digital defects and cleft palate. Caffeine at non-teratogenic doses administered to pregnant SAF/ICR mice caused a significant increase in the numbers of offspring with cleft palate produced by X-rays at 200 rad [382]. It was suggested t h a t in this case caffeine might be acting as a co-teratogen. A specific malformation, ectrodactyly, has been produced by caffeine treatment of pregnant mice [26]. Previous exposure to caffeine appears to reduce the teratogenic effect on mice [325]. When pregnant mice (A/J strain) were injected subcutaneously once on the 13th day of gestation with 150 mg/ kg of caffeine the frequency at term of foetal death, external malformations, and subcutaneous haematomas was significantly lower in the group which had drunk a 0.05% caffeine solution for some weeks after weaning than in the group which had drunk tap water. When the experiment was repeated with 250 mg/kg caffeine the group with caffeine pretreatment had a lower frequency of foetal death but n o t of malformations or haematomas. It was suggested that the previous caffeine consumption led to an increased rate of caffeine degradation. While there are, therefore, a number of reports of the teratogenic action of caffeine in mice failure to obtain such effects has also been reported [328,353]. A population of CD-1 mice was bred for four generations with continuous ingestion of caffeine. The highest rate of intake was 25--39 mg/kg/day which is equivalent to 19--30 cups of coffee per day in m a n - - a level which is rarely likely to be achieved. No consistant dose-related effect was found on fertility, age of sexual maturity, mean litter size, weight of offsprin at weaning, sex ratio, or foetal abnormalities [328]. The main difference between this and the other reports was the route of administration of the caffeine which was oral over a long period of time while in the other cases it was by injection at one time. This inevitably means that the transitory peak level of caffeine in the animal's body was much higher in the studies reporting positive findings. However the last study [328] reflects a more direct parallel with caffeine consumption in man. Caffeine has been reported as inhibiting the growth of rats [86] but later work showed no effect of caffeine on growth at a daily dose rate of 50--60 mg/kg b o d y weight [22]. There was also no significant effect on the reproductive capacity of the animals measured in terms of average litter size. However daily intraperitoneal injection of 4--16 mg of caffeine t h r o u g h o u t the pregnancy of Hulzer rats led to a significant increase in

33 resorbtion and a decrease in the birth weight of the offspring produced when compared with saline-treated controls [122]. Developmental malformations were n o t observed in the offspring at these doses. A specific malformation (ectrodactyly) was f o u n d in the offspring of pregnant rats treated with caffeine at 75--150 mg/kg, the effect being proportional to the dose [26]. Later work showed that this specific malformation induction by caffeine in rats was in part under genetic control since it occurred in some strains but not in others [27]. The caffeine effect was considered to be teratogenic and not mutagenic since no case of the malformation was found in 60 offspring of the caffeinedamaged rats. However failure to cause teratogenic affects by the application of caffeine to pregnant rats has also been reported [353]. Caffeine has been shown to decrease the number of functional tubules in the testis of the rat and to affect spermatogenesis [262]. However in C57BL × C3H FI mice caffeine did not induce abnormalities of the sperm [376]. Using pure bred Russian rabbits the lethal dose of caffeine was f o u n d to be 0.2 g/kg body weight [314]. After habituation to caffeine at lower doses, however, this dose could be given on several days in succession w i t h o u t being lethal and up to 0.15 g/kg body weight was tolerated for months without apparant ill-effect. If larger doses than this were given daily for some time then retrogressive changes appeared in the gonads which if the caffeine was not withdrawn could lead to complete sterility. The ovary was found to be more sensitive to caffeine than the testis. Animals on doses which did n o t lead to atrophy of the gonads showed effects during reproduction. When males were so treated 75% o f the y o u n g produced died during the first week after birth while when it was the female which was treated 72% of the embryos died in utero. Ectrodactyly was found in 6 of 64 rabbits treated in utero with caffeine at 100 mg/kg maternal b o d y weight [27]. Although caffeine consumption by Chinese hamsters has been observed to alter the sex ratio [360] a survey of 17352 children from 6837 families produced no evidence of a relationship between the sex ratio of the children and their parents' coffee-drinking habits [351]. An association between coffee drinking and cancer of the lower urinary tract has been demonstrated [68] and it was suggested t h a t caffeine might be implicated. In this study, however, it was also f o u n d t h a t the incidence of cancer was twice as high in women than in men among coffee drinkers and an alternative explanation is that the effect of caffeine was to increase the rate of mitosis of the cancer cells in women and to decrease it in men [330] in the same manner as in h u m a n l y m p h o c y t e s [329]. In a survey of a number of studies which a t t e m p t e d to correlate the consumption of coffee and tea with the occurrence of hypertension, gastrointestinal ulcers, cancers of the colon, rectum, and bladder, cardiac infarction, acute and chronic hepatitis, and cirrhosis of the liver it was found that all these conditions showed a positive correlation with cigarette smoking and t h a t there was negligible evidence for the involvement of caffeine [ 148]. More recently, however, a study in which the effect of cigarette smoking was excluded showed an increased risk of myocardial infarction in women drinking six or more cups of coffee daily [218]. Coffee, of course, contains many other compounds which could be involved in the aetiology of myocardial infarction and some clinical studies and experiments with rats suggest that chronic caffeine intake may

34 diminish the lethality of infarcts [280]. There seems to be a widespread assumption that coffee drinking is a measure of caffeine intake. This may quite often be false. Many of the non-coffee drinking groups may drink tea and so have a considerable caffeine intake. Indeed it would seem to be difficult outside of certain religious groups, usually small in numbers, to find a non-caffeine consuming control group. If a difference in the incidence of a disease under investigation was demonstrated between coffee and tea drinkers it would probably be useful to identify the chemical differences between the two beverages. Caffeine has been shown to enhance the transformation of cultured Syrian hamster cells induced by a number of chemical carcinogens [83]. However caffeine at 1 g/kg body weight reduced by 65% the number of adenomas per mouse induced by morpholine plus sodium nitrite [228], and post-treatment with caffeine diminished 4NQO-induced lung tumourigenesis in mice [245]. The incidence of skin cancers on the ear of the mouse induced by UVL was reduced from 90% to 50% by pretreatment of the skin with a 0.2% caffeine solution [384]. In whole animals therefore caffeine would seem to have a protective effect against at least some carcinogens. It is clear that the relationship between caffeine and carcinogenesis is at present confused and might be a field of interesting and profitable research. Comparison with related compounds It is possible to compare the relative activities of caffeine and its chemically related compounds in an a t t e m p t to learn something of the relationship between the chemical structure and the biological activities of caffeine.

Theophy lline In a number of investigations the activity of caffeine has been directly compared with t h a t of theophylline (1,3-dimethylxanthine). Caffeine greatly reduced the suppression of DNA synthesis following X-irradiation (500 R) of mouse S-180 ascites t u m o u r cells but theophylline was ineffective [35]. The incorporation of [3H]thymidine into the DNA of mouse l y m p h o m a L5178YuK cells, LS929 mouse fibroblasts and V-79-4 Chinese hamster cells was inhibited more by theophylline than by caffeine in both UV-irradiated and non-irradiated cells [201]. Theophylline was a more effective inhibitor than caffeine of cyclic 3',5'-AMP phosphodiesterase in h u m a n blood lymphocytes [191] and in both normal and carcinomatous h u m a n lung tissue [73]. At 10 -a M theophylline inhibited cyclic guanosine phosphate diesterease more than caffeine but at 10 -s M caffeine potentiated the action of this enzyme more than theophylline, again in both normal and carcinomatous tissue [73]. Theophylline was a more effective inhibitor of the aggregation of human blood platelets in the presence of prostaglandin E1 than caffeine [227]. However in the absence of the prostaglandin caffeine still exhibited a small inhibitory effect but theophylline was inactive. Caffeine was a more effective mutagen than theophylline in E. coli B/r t- to t ÷ [22]. It was also more effective in producing abnormal anaphases in Allium cepa [166], and abnormal metaphases in Vicia faba [175], and in enhancing the frequency of chromosomal aberrations in V. faba after X-irradiation. In Saccharomyces cerevisiae caffeine was more effective in

35 TABLE 4 COMPARISON OF CAFFEINE, THEOBROMINE

AND THEOPHYLLINE

Activity

System

Order of potency

Ref.

M u t a t i o n to T 5 r e s i s t a n c e Mutagenic (synergism with UVL) Antimitotic Antimitotic

E. coli E. coli Vicia faba Human lymphocytes

C > TP > T B C > TP > T B C=TP> TB TP > C > TB

247, 248 82, 300 11 331

Cell-w aU f o r m a t i o n Polymorphonuclear leucocyte aggregation inhibition 3',5-Nucleotide phosphodiesterase inhibition E n h a n c e d activity of sAMP lyase

Allium

C = T P = TB

165

Rabbit

C > TP > TB

190

Purified enzyme

Bacillus subtilis

TP > TB > C C > TP = TB

48 243

Effect on cortical granules 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 Cardiac stimulation Diuresis

Arbacia Man Man Man

C > TB > T P C > TP > TB TP > TB > C TP > TB > C

55 130, 253 130 130

C, c a f f e i n e ; 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 .

reversing the supression of sporulation induced by glucose than theophylline [338]. Caffeine but not theophylline accelerated the differentiation of rat-lens epithelial cells [ 72]. Theophylline was less active than caffeine in the inhibition of rabbit-muscle phosphorylase [168].

Theobromine Theobromine (3,7-dimethylxanthine) was shown to cause chromosome breakage and trinucleate cell formation in Oedogonium acmandrium while caffeine caused chromosome stickiness and clumping and the production of binucleate cells [285]. Theophylline and theobromine In a number of investigations the activities of caffeine, theophylline and theobromine have been directly compared. These are summarised in Table 4.

TABLE 5 COMPARISON OF CAFFEINE, THEOBROMINE,

THEOPHYLLINE

AND PARAXANTHINE

Activity

System

Order of potency

References

Mutagenic Clastogenic Induction of exchange-type aberrations in p r o p h a s e a n d G 2

E. c o l i Human lymphocytes Vicia faba C h i n e s e h a m s t e r cells

C C C C

247,248 366 316 316

Vicia f a b a Vicia faba

C > TB > T P > P C > TP = T B > P C > TP= TB > P

174, 316 174,316 316

Human lymphocytes

TP > P > C > TB

366

Potentiation of thiotepa-induced chromatid aberrations Potentiation of maleic hydrazideinduced chromosomal aberrations Antimitotic

L C h i n e s e h a m s t e r ceils

C, c a f f e i n e ; T P , t h e o p h y U i n e ; T B , t h e o b r o m i n e ; P, p a r a x a n t h i n e .

> > > >

TP > P > TB TB > TP > P TP= TB=P TP = TB = P

36

The results indicate no single, universal order of biological activity of the three compounds although in general theobromine is the least active. The mutagenic and clastogenic activities of theobromine and theophylline have been recently reviewed [332]. Paraxanthine In Table 5 the studies in which caffeine has been directly compared with paraxanthine (1,7-dimethylxanthine), theophylline and theobromine are summarised. Again no general order of biological activity can be found. S u b s t i t u t e d caffeines

Table 6 lists some investigations in which the effect of caffeine has been directly compared with that of some 8-substituted caffeines. There is again variation in the order of p o t e n c y but in general 8-methoxycaffeine is the least active except in the production of exchange-type aberrations in G2-prophase in plant root tips and Chinese hamster cells below 30°C where it has about the same activity as 8-ethoxycaffeine and both are more active than caffeine itself [174,316]. M o norne th y lx an th ines

While caffeine has a significant clastogenic action in human l y m p h o c y t e cultures none of the m o n o m e t h y l x a n t h i n e s showed any clastogenic activity [366]. The order of potency in the suppression of mitosis in h u m a n lymphocyte cultures at 750 mg/ml was 1-methylxanthine > 3-methylxanthine =

TABLE 6 COMPARISON OF CAFFEINE AND SUBSTITUTED CAFFEINES Activity

System

Order of potency

References

M u t a t i o n to T 5 r e s i s t a n c e Mutation (synergism with UVL) Back mutation

E. coli

C > M O C > CC

248

E. coli Ophiostoma multiannulatum

CC > C > E O C

300

EOC ) C

385

Allium proliferum Chinese hamster cells a t 3 7 ° C Chinese hamster cells a t 1 7 ° C Vicia faba

EOC ) C

169, 170

EOC = C

169, 170

EOC = MOC ) C EOC = MOC ) C

169, 170, 174, 316 174, 175, 316

Embryonic mouse skin Mouse S-180 sarcoma cells Vicia faba C h i n e s e h a m s t e r cells

C ) EOC

28

C ) EOC C ) E O C ) CC ) M O C CC > C > E O C > M O C

28 174,316 174,316

Vicia faba

C > E O C > CC = M O C

316

Induction of chromosomal aberrations

Induction of chromosomal abnormalities Potentiation of thiotepainduced chromosomal aberrations Potentiation of maleic hydrazide-induced chromosomal aberrations

C, C a f f e i n e ; E O C , 8 - e t h o x y c a f f e i n e ; M O C , 8 - m e t h o x y c a f f e i n e ; CC, 8 - ¢ h l o r o c a f f e i n e .

37 TABLE 7 COMPARISON OF CAFFEINE AND METHYLURIC ACIDS Activity

System

Order of potency

References

M u t a t i o n to T 5 r e s i s t a n c e Mutation (synergism with UVL) Production of c h r o m o s o m a l aberrations

E. coil E. coli Vicia faba

C > TMU C > 13DMU > 3 MU TMU > C

248 300 174, 175, 316

P o t e n t i a t i o n of t h i o t e p a - i n d u c e d chromosomal aberrations Potentiation of maleic hydrazideinduced chromosomal aberrations P o t e n t i a t i o n of X - r a y - i n d u c e d chromosomal aberrations

Vicia faba Chinese h a m s t e r cells

C > TMU C > TMU

174,316 174, 316

Vicia faba

C > TMU

316

Vicia faba

C > TMU

175

C, c a f f e i n e ; T M U , 1 , 3 , 7 , 9 - t e t r a m e t h y l u r i c acid; 1 3 D M U , 1 , 3 - d i m e t h y l u r i c acid; 3 M U , 3 - m e t h y l u r i c acid.

7 - m e t h y l x a n t h i n e > caffeine [366]. These results suggest that there is no close correlation between clastogenic and antimitotic activity.

Methy luric acids Table 7 lists some studies in which a comparison o f the effects of caffeine and some methyluric acids has been made. Xanthine, hypoxanthine and uric acid The action of caffeine on the mitotic rate of human lymphocytes in 72-h cultures may be directly compared with that of xanthine (2,6-dioxypurine), hypoxanthine (6-oxypurine), and uric acid (2,6,8-trioxypurine) [ 2 6 6 , 3 3 1 ] . The results are summarised in Table 8.

Structure--activity relationships The biological activities of caffeine and it related c o m p o u n d s are multiple and complex and it is not possible to relate them to the chemical structures in any simple manner. In E. coli mutagenesis the methyl group at position I seems to be the most important both when caffeine is acting alone [247, 248] and synergistically with UVL [82,300] although methylation at the other positions is not without effect. In the demethylation process in man the methyl group at position I is the most stable [71]. Position 3 methylaTABLE 8 E F F E C T OF C A F F E I N E , X A N T H I N E , H Y P O X A N T H I N E MITOSIS OF C U L T U R E D H U M A N L Y M P H O C Y T E S C o n c e n t r a t i o n (M)

10 -2 10 -3 10 4

A N D U R I C A C I D ON T H E R A T E

M i t o t i c r a t e (% c o n t r o l ) Caffeine

Xanthine

Hypoxanthine

Uric acid

0 41 111

84 117 217

59 96 98

7 89 119

A d a p t e d f r o m : T i m s o n [ 3 3 1 ] , a n d Price a n d T i m s o n [ 2 6 6 ] .

OF

38 tion may be the most effective in clastogenic activity in human lymphocytes in culture [366], and in the potentiation of thioTEPA and maleic hydrazide induced chromosomal aberrations in Vicia faba root tips and cultured Chinese hamster cells [174,316]. However in the induction of exchange-type aberrations in V. faba and Chinese hamster cells methylation at all three positions appears to be necessary for the m a x i m u m effect [316]. In antimitotic activity in human lymphocytes methylation at position I is most important [331,366], and the same may be the case in V. faba [11]. 8-Ethoxycaffeine is more active than caffeine as a mutagen in Ophiostoma multiannulatum [385], and in the induction of chromosomal aberrations in Allium proliferum, V. faba, and Chinese hamster cells [169,170,175] but not in embryonic mouse skin or mouse S-180 cells [28]. Caffeine is more active than EOC when used together with UVL on E. coli [300] or with thioTEPA or maleic hydrazide to produce chromosomal aberrations in V. faba and Chinese hamster cells [174,316]. It is possible that these results are due to caffeine being a more active inhibitor of repair processes than EOC. Discussion

The biological activities of caffeine are diverse and in a particular system may be multiple and complex. Perhaps as a result of mans daily contact with the compound, which for m a n y is in fact a drug of mild addiction, much interest has been taken in its affects on biological processes at all levels of organisation from the molecular to the whole animal. It can be said with certainty t h a t caffeine is mutagenic in micro-organisms, with less certainty that it may be mildly mutagenic in Drosophila and with near certainty that it is n o t mutagenic in mammals at least at the transitory peak levels achieved in normal h u m a n consumption. Although the question of caffeine's mutagenicity in mammals and man has been discussed in the past [6,173] and the available evidence suggests that caffeine is not mutagenic in mammals yet there persists an almost subconscious feeling that it might, perhaps ought, to be mutagenic in mammals. This may in part be due to the well established mutagenicity in lower organisms and the clear demonstration of clastogenic activity in mammalian cells in culture. It is tempting to suggest that it may also in part be due to mans longstanding addiction to caffeine coupled with a feeling that such addiction should carry a penalty. However there are strong arguments against caffeine being mutagenic in man. In the first place caffeine is rapidly metabolised in mammals [71] and mammalian cells [131] and this is not the case in E. coli which cannot demethylate the methylxanthines [180]. Secondly caffeine has both clastogenic and antimitotic activity against mammalian cells and the threshold of these effects may be no higher than the mutagenic threshold which would make the probability of survival and reproduction of a caffeinem u t a t e d cell rather low. Thirdly the long exposure of the human race to caffeine and other methylxanthines may have led to the evolution of some caffeine-resistance in man. The case for caffeine mutagenicity in man is therefor non-proven. It is to be hoped that caffeine will continue to be investigated both extensively and intensively wherever possible and that future studies will include comparative work with the other methylxanthines in particular theo-

39 bromine, theophylline, and paraxanthine. In this way a better understanding of the structure--activity relationships of the methylxanthines may be built up which should eventually lead to a resolution of the presently unanswered questions about the bioloigical activities of caffeine.

Acknowledgements I wish in particular to thank Professor Bengt Kihlman of the Royal Agricultural College of Sweden, Uppsala, Sweden, who has been kind enough to read this review in draft and who made many helpful suggestions. I would also like to thank John S. Wassom, Director of the Environmental Mutagen Information Centre~ Oak Ridge, Tenn., U.S.A. for help with the literature search, and Dr. R. Ross, Keeper of Botany at the British Museum (Natural History), London, U.K. for help with the nomenclature of the caffeine-containing plants.

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