Drug interactions with caffeine are known. Co-administration of caffeine was found to increase acetaminophen-induced hepatotoxicity by enhancing the production of a reactive metabolite ( Sato & Izumi, 1989 ).
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Pharmacokinetic differences have been observed in mice after oral administration of caffeine, which may account for interstrain variation in toxicity studies ( Arnaud et al. , 1989 ). In rabbits, two subpopulations could be described, with slow or rapid caffeine metabolizing capacity. Animals with slow metabolism exhibited saturation kinetics with high doses of caffeine and inhibition of caffeine metabolism by paraxanthine. Rabbits appear to be the best model to study the inter- and intrasubject variability in caffeine disposition observed in man ( Dorrbecker et al. , 1987 ).
A decreased half-time was reported when 10 mg/kg bw caffeine were administered to pregnant rats in drinking-water on day 18 of gestation ( Nakazawa et al. , 1985 ); however a 25% decrease in mean total demethylation was demonstrated in rats between 19 and 21 days of pregnancy, with a breath test using [14C-1,3,7-methyl]caffeine at a dose of 4 mg/kg, with an immediate return to normal values one day after birth ( Arnaud & Getaz, 1986 ). Rabbits receiving 8–22 mg/kg bw per day caffeine through 29 days of gestation exibited increased plasma concentrations in the last half of gestation, demonstrating that there is an increased half-time ( Dorrbecker et al. , 1988 ).
Non-linear kinetics, shown in rats by disproportionate increases in the dose-concentration relationship, indicate a limited capacity to absorb and metabolize caffeine at doses of 10–25 mg/kg bw ( Aldridge et al. , 1977 ; Latini et al. , 1978 ).
The effects of caffeine on the rodent testis are reviewed in detail below and are not covered here. Caffeine also induced thymic atrophy at a dietary level of 0.5% (approximately 150 mg/kg bw) when fed for eight weeks to rats ( Gans, 1984 ).
The sensitivity of rats to the lethal effects of caffeine increased with age; caffeine was more toxic in male than in female rats ( Tarka, 1982 ).
The acute oral LD 50 of caffeine is 200 mg/kg bw in rats, 127 mg/kg bw in mice, 230 mg/kg bw in hamsters and in guinea-pigs and 246 mg/kg bw in rabbits; the intraperitoneal LD 50 s of caffeine are 200 mg/kg bw in rats and 235 mg/kg bw in guinea-pigs; and the intravenous LD 50 s of caffeine are 105 mg/kg bw in rats, 100 mg/kg bw in mice and 175 mg/kg bw in dogs. The toxicity of caffeine was determined after daily administration via intragastric cannula to female albino rats over 100 days (equivalent to 1/10 of the animals’ life span). Rats given daily doses slightly above the maximal LD 50 (110 mg/kg bw) exhibited a stressor reaction in the form of hypertrophy of the adrenal cortex and atrophy of the adrenal cortex and thymus gland. Some animals manifested a psychotic-like mutilation, gastric ulcers, hypertrophy of the salivary glands, liver, heart, kidneys and lungs, inhibition of oogenesis, minor changes in organ water levels, and an occasional death apparently from bronchopneumonia. Although no major change in growth rates or eating and drinking habits was apparent, some polydypsia and diuresis, thyroiditis, occasional dermatitis, some degree of nephritis, and loss of red pulp in the spleen were seen ( Tarka, 1982 ).
Many other developmental neurotoxicology studies, mostly in rats, have evaluated the effect of prenatal administration of caffeine on behavioural and neurochemical measures in neonates. These studies were reviewed by Sobotka et al. (1979) . The effects are not consistent across studies: thus, caffeine may cause subtle changes in discrete neuronal subsystems but is not a neurotoxicant in the sense of disrupting primary neuronal systems.
Sprague-Dawley rats were administered 5–75 mg/kg bw caffeine daily by gavage on days 3–19 of gestation and their offspring were observed for behavioural and developmental effects for nine weeks after birth. Dose-related developmental effects included delayed incisor eruption, delayed vaginal opening and decreased body weight. Active avoidance behaviour was also significantly decreased with the highest doses of caffeine ( West et al. , 1986 ).
When CD-COBS rats were administered 80 mg/kg bw caffeine orally as a single dose or as four doses every three hours on day 12 of gestation, the peak blood levels of caffeine and the area under the blood concentration-time curve were doubled with the single-dose as compared to multiple-dose regime ( Jiritano et al. , 1985 ). [The Working Group noted that this finding is consistent with that of the preceding study.]
Wistar rats received total daily administrations of 10 or 100 mg/kg bw caffeine by gavage, either as a single dose or as four doses every three hours, on days 6–20 of gestation. While a dose-related decrease in fetal weight and an increase in the delay in ossification were observed with both modes of administration, the major malformation, ectrodactyly, was observed only in the group given 100 mg/kg bw as a single dose ( Smith et al. , 1987 ).
In another study by Collins et al. (1987) , the previously reported delay in sternebral ossification was confirmed in day-20 fetuses of rats drinking caffeine-containing water from gestation day 0 to day 20. Among offspring that were raised to postnatal day 6, the delay in ossification was nearly reversed. The authors concluded that the reversal would have been complete if a longer postnatal period had been studied.
In order to establish a no-effect level, caffeine was administered to Osborne-Mendel rats by gavage; offspring had dose-related increases in the frequency of ectrodactyly and delayed ossification. A no-effect level for terata was 40 mg/kg bw caffeine per day, although a significant increase in the frequency of delayed sternebral ossification was observed with 6 mg/kg bw per day ( Collins et al. , 1981 ). When administered in the drinking-water at a wider dose range (10–204 mg/kg bw per day), caffeine did not induce dose-related gross anomalies. Sternebral ossification was seen less frequently in all treated groups than in controls, except with the lowest dose ( Collins et al. , 1983 ). [The Working Group concluded that caffeine was less toxic to the developing embryo and fetus when given in drinking-water than by gavage; this pattern of exposure to caffeine — small doses throughout the day — is closely similar to human exposure to caffeine.]
Developmental toxicity : Teratogenicity was reported in SMA mice given single intraperitoneal injections of 250 mg/kg bw caffeine on one of days 7–14 of gestation. Significant increases in the incidence of fetal resorptions, cleft palate and digital defects were observed, depending on the day of treatment ( Nishimura & Nakai, 1960 ).
Friedman et al. (1979) found that feeding caffeine in the diet to immature Osborne-Mendel rats at levels of 1% for three weeks and 0.5% [approximately 300 mg/kg bw per day] for 14–75 weeks produced severe testicular atrophy and aspermatogenesis. Analogous results were observed in Holtzman rats. [The Working Group noted the excessive doses used in the study.]
Ax et al. (1976) reported that when roosters were fed 0.1% caffeine [about 100 mg/kg bw per day] in a standard ration, hens inseminated with sperm from the roosters had significantly reduced numbers of fertile eggs. Semen and sperm counts were markedly reduced 17–21 days after treatment, and no semen could be collected after 30 days. These effects were reversible on removal of dietary caffeine.
Female monkeys ( Macaca fascicularis ), 12–14 per group, received 0, 10–15 or 25–35 mg/kg bw caffeine in the drinking-water daily on seven days a week for a minimum of eight weeks prior to mating with untreated males. Miscarriages and some stillbirths were reported during two cycles of pregnancy in the caffeine-treated groups, and birthweights of male infants was also significantly lower in these groups in comparison to controls. The effects were dose-related but occurred with both levels of caffeine. No malformation was observed in any of the offspring ( Gilbert et al. , 1988 ). [The Working Group noted that the exclusion criteria for stillbirths were not unequivocal.]
Male and female Sprague-Dawley rats were given cocoa powder (containing 2.50–2.58% theobromine and 0.19% caffeine) in the diet at concentrations of 0, 1.5, 3.5 and 5.0% for three generations ( Hostetler et al. , 1990 , see p. 430 of the monograph on theobromine). No consistent dose-related effect was observed in any reproductive index; nonreproductive toxicity was observed at the two highest dose levels.
As reported in an abstract, female rats [strain unspecified] in a two-generation reproduction study received daily oral administrations of 4, 20 or 126 mg/kg bw caffeine for seven days before mating and through to 20 days of lactation. The F 1 offspring received the same treatment. When mature, F 1 offspring were mated with untreated animals. Pregnancy rate and reproduction were normal in F 0 females and F 1 males; among F 1 females, however, the pregnancy rate was normal, but there were decreases in the numbers of corpora lutea, implants and fetuses at the high dose. F 2 fetuses of these high-dose females were small and oedematous ( Bradford et al. , 1983a ).
Groups of male and female Wistar rats were administered 10 mg/kg bw caffeine in the drinking-water daily through five successive sets of litters. Progressively reduced growth and increased neonatal mortality (significant) were observed in the offspring over sequential pregnancies ( Dunlop & Court, 1981 ).
In a similar study, no effect on pregnancy rate was observed in F 0 and F 1 mice, but among F 0 groups there was a significant decrease in the number of live pups per litter at the two highest dose levels. There was no significant change in reproductive organ weight, sperm motility or density or in the frequency of sperm abnormalities ( Reel et al. , 1984 ).
Reproductive effects : CD-1 mice were administered 0.012, 0.025 or 0.05% caffeine in the drinking-water (daily caffeine intake, 21.9, 43.8 or 87.5 mg/kg bw) for seven days prior to mating and during a subsequent 100-day cohabitation period. Offspring were removed when one-day old. The last set of litters from the high-dose group and the F 1 generation of untreated controls were maintained on caffeine to 90 days of age and mated within their respective groups. Following treatment of the F 0 mice, no effect on pregnancy rate was observed but there was a decrease in the number of live pups per litter at the high dose. There was no effect on any parameter in a cross-mated trial between control and high dose animals. Among the F 1 males at termination of the study, there was no effect of caffeine on the weight of the testis or epididymus relative to body weight; there was a significant decrease in sperm motility, an increase in sperm density and no change in the proportion of abnormal sperm ( Gulati et al. , 1984 ).
(iv) Genetic and related effects
The genetic and related effects of caffeine have been reviewed (Bateman, 1969; Adler, 1970; Fishbein et al., 1970; Anon., 1973; Mulvihill, 1973; Kihlman, 1974; Thayer & Palm, 1975; von Kreybig & Czok, 1976; Kihlman, 1977; Timson, 1977; Legator & Zimmering, 1979; Lachance, 1982; Tarka, 1982; Haynes & Collins, 1984; Dalvi, 1986; Grice, 1987; Rosenkranz & Ennever, 1987), as have its antimutagenic effects (Clarke & Shankel, 1975).
The results described in this section are listed in on p. 336, with the evaluation of the Working Group, as positive, negative or inconclusive, as defined in the footnotes. The results are tabulated separately for the presence and absence of an exogenous metabolic system. The lowest effective dose (LED), in the case of positive results, or the highest ineffective dose (HID), in the case of negative results, are shown, together with the appropriate reference. The studies are summarized briefly below.
Table 13.
Genetic and related effects of caffeine.
Effects on DNA structure and DNA synthesis: Caffeine interacts in different ways with DNA structure and metabolism. There is some evidence of intercalation of caffeine in double-stranded DNA (Richardson et al., 1981; Tornaletti et al., 1989). Caffeine impairs the helical structure of DNA (Ts’o et al., 1962), causes a slight increase in the rate of its elongation (Bowden et al., 1979) and lowers its melting-point. There may be local unwinding of DNA, as suggested by susceptibility to single-strand-specific nuclease digestion (Chetsanga et al., 1976).
It has been known since 1964 that caffeine interacts with DNA primarily at single-stranded regions; however, in the initial studies very high concentrations of methylxanthine were used (Byfield et al., 1981). In ultraviolet-irradiated DNA treated with low concentrations of caffeine, the caffeine molecules bind to the DNA near the region of the radiation-induced conformational changes. Caffeine binds to single-stranded (denatured) DNA regions, and it seems to bind preferentially to A-T-rich regions. This might be due to costacking, particularly with adenine (Kihlman, 1977).
Co-incubation of caffeine with single-strand-specific endonuclease induced some breakage, whereas no breakage occurred when DNA was incubated with either caffeine or endonuclease alone (Sleigh & Grigg, 1974; Chetsanga et al., 1976). Denatured (single-stranded) DNA has a higher affinity for caffeine than does native (double-stranded) DNA (Ts’o & Lu, 1964). In human lymphocytes, 3H-labelled caffeine did not bind in situ to chromosome preparations after heat or alkali denaturation (Brøgger, 1974).
There are many studies on the effects of caffeine on enzymes involved in DNA metabolism and on nucleotide pools. The RNA-dependent DNA polymerase activity of murine and avian oncogenic viruses was inhibited by caffeine (Srinivasan et al., 1979). There were conflicting reports of inhibition of Escherichia coli polymerase I polymerizing activity (Solberg et al., 1978; Balachandran & Srinivasan, 1982); however, caffeine inhibited nuclease activities of E. coli DNA polymerase (Solberg et al., 1978). DNA polymerase activity in human embryonic lung cells was inhibited by caffeine (Wragg et al., 1967). Caffeine inhibited three different exonucleases of E. coli (Roulland-Dussoix, 1967), thymidine kinase (at high concentrations; Sandlie et al., 1980) and some, but not all, of the purine nucleoside phosphorylases of both the ribose and deoxyribose series (Koch & Lamont, 1956); thymidine phosphorylase was not affected (Sandlie et al., 1980). Effects on nucleotide pools are discussed below (p. 335).
In E. coli, caffeine did not behave like a purine analogue in the purine biosynthesis pathway (Delvaux & Devoret, 1969). The effects of caffeine on DNA synthesis differed according to the assay system used. Caffeine did not inhibit DNA synthesis in vitro (Grigg, 1968), but DNA synthesis was inhibited in cell-free extracts of cultured human embryonic lung cells (Wragg et al., 1967), in Paramoecium aurelia (Smith-Sonneborn, 1974) and in Saccharomyces cerevisiae (Tsuboi & Yanagishima, 1975), but not in Tetrahymena pyriformis (Lakhanisky et al., 1981).
In Drosophila melanogaster larvae, caffeine strongly inhibited semi-conservative DNA synthesis but had no effect on repair replication (Boyd & Presley, 1974). Post-replication repair-deficient mutants were affected only minimally by caffeine (Boyd & Shaw, 1982).
In a study with partially hepatectomized mice in vivo, caffeine (given intraperitonealy at 50 mg/kg per day for four days) depressed the synthesis of DNA (as measured by 3H-thymidine incorporation) but not of RNA in the liver (Mitznegg et al., 1971). 3H-Thymidine incorporation into DNA was also depressed in mouse bone-marrow cells (Singh et al., 1984).
Caffeine increased the number of replication sites in the DNA of Chinese hamster V79 cells and in HeLa cells (Painter, 1980) and slightly increased the rate of DNA elongation in V79 cells, which qualitatively and reproducibly correlated with an increased cloning efficiency (Bowden et al., 1979). The pattern of condensation in DNA in chicken fibroblasts was changed by caffeine (Ghosh & Ghosh, 1972), which also partially inhibited cell-cycle progression from G1 through to M phase in mouse S–180 ascites cells (Boynton et al., 1974). Caffeine inhibited DNA synthesis in Chinese hamster CHO-K1 cells (Waldren & Patterson, 1979), V79 cells, mouse lymphoma L5178Y cells and mouse LS929 cells (Lehmann, 1973). An important element in this inhibition of DNA synthesis is reduced precursor uptake by cells: there were large reductions in the uptake of uridine and thymidine in Chinese hamster ovary (CHO)-K1 cells (Waldren, 1973) and that of thymidine in L5178Y-UK cells (Lehmann & Kirk-Bell, 1974). In CHO-K1 cells treated with caffeine, one complete cell cycle was possible, but in the second cycle there was a block near the G2/S interface (Waldren, 1973).
In a test for differential cytotoxicity using wild-type and DNA repair-deficient strains of CHO cells, it was concluded that caffeine was probably not a DNA damaging agent, because no differential retardation of growth was observed (Hoy et al., 1984).
In human HeLa cells, caffeine inhibited RNA but not DNA synthesis (Kuhlmann et al., 1968). 14C-Caffeine was not incorporated into the DNA of human lymphocytes (Brøgger, 1974), but it reduced the size of DNA segments synthesized by excision repair-defective xeroderma cells (Buhl & Regan, 1974).
Prokaryotes: Evidence of caffeine-induced DNA damage was observed in the Bacillus subtilis rec assay (weak responses) and in the E. coli repair test.
The mutagenic activity of caffeine was first observed in a streptomycin-dependent strain of E. coli in the 1940s (see ); however, other studies in E. coli gave positive and negative results. Its mutagenic activity was confirmed using phage-resistance and a reverse mutation assay. Caffeine was shown to induce frameshift mutations (Clarke & Wade, 1975). In most cases, the mutation rate was directly proportional to the growth rate (Kubitschek & Bendigkeit, 1964), and this is consistent with the hypothesis that a mutational event occurs as a mistake during DNA replication (Webb, 1970). Caffeine may also act as an antimutagen in E. coli (Grigg & Stuckey, 1966), perhaps by reducing growth rate (Barfknecht & Shankel, 1975).
Caffeine was consistently nonmutagenic in many studies in all the Salmonella typhimurium his reversion tester strains and in S. typhimurium forward mutation assays. It was, however, mutagenic to Xanthomonas phaseoli, Klebsiella pneumoniae and Bacillus subtilis.
Lower eukaryotes (including fungi): Caffeine was generally mutagenic in algae (Plectonema boryanum) and fungi (Physarum polycephalum, Dictyostelium discoideum, Ophiostoma multiannulatum). Some negative findings were observed in fungi (Ophiostoma reverse mutation) and yeast (Schizosaccharomyces pombe).
Caffeine induced aneuploidy (monosomics) in Saccharomyces cerevisiae.
Studies in the yeast S. pombe revealed a significant decrease in the frequency of meiotic recombination and an increase in that of mitotic gene conversion between closely linked heteroallelic markers. It was suggested that the reduction of meiotic crossing-over may be caused by an interaction of caffeine with DNA, which inhibits DNA degradation (Ahmad & Leupold, 1973; Loprieno et al., 1974). As a result of this interaction, more stable pairing might occur at the level of mismatched bases, thereby generating an increase in mitotic gene conversion.
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Featured content:What are the uses of NBR?Key Questions to Ask When Ordering pmk oil cas numberPlants: Caffeine increased the rate of point mutations in plants (Glycine max). It also induced chromosomal aberrations in many studies in plants (e.g., Allium, Hordeum and Vicia species), with only a few exceptions. The incidence of aberrations was modified by ATP (Kihlman et al., 1971a) and low temperature (Osiecka, 1976). Sister chromatid exchange was induced in Vicia faba, and mitotic recombination was induced in a number of studies in plants (e.g., Glycine max and Nicotiana tabacum).
Insects: Results of tests for sex-linked recessive lethal mutation in Drosophila melanogaster were equivocal, but chromosomal aberrations were induced when the exposed cells were in G2 or early mitosis, and there was evidence of recombinogenic effects. Predominantly positive responses were induced in tests for aneuploidy in D. melanogaster, although the frequencies were low. Dominant lethal responses were not observed in Bombyx mori.
Mammalian cells in vitro: DNA strand breakage was not induced by caffeine.
In V79 cells, mutation was not induced at the hprt locus, and there was no increase in the frequency of ouabain-resistant mutants. Also, caffeine failed to induce forward mutations either to auxotrophy at a variety of loci in CHO-K1 cells or at the tk locus in mouse lymphoma L5178Y cells. Caffeine has been reported to be antimutagenic to V79 cells, in which it reduces the fractions of both induced and spontaneous mutations.
Sister chromatid exchange was induced in some studies but not in others. Its induction may well be related to an inhibition of the poly(ADP-ribose) polymerase; this inhibition could delay the rejoining of DNA strand breaks induced by bromodeoxyuridine (Natarajan et al., 1981). Inhibition of this enzyme is associated with the induction of sister chromatid exchange (Levi et al., 1978; Morgan & Cleaver, 1982).
Micronuclei have been induced by caffeine in a cell line and in cultured mouse preimplantation embryos. The sensitivity of different cell lines to the induction of chromosomal aberrations by caffeine clearly varies widely. When treated with caffeine, CHO cells responded with large increases in the frequency of chromosomal aberrations that were dependent upon treatment during S-phase (Kihlman et al., 1971a,b; Kihlman, 1977). In mice deficient in folate, caffeine strongly increased the frequency of micronucleated cells (MacGregor, 1990).
Caffeine enhanced the frequency of cell transformation in several virus-induced systems but not in an assay for colony morphology in primary Syrian hamster embryo cells.
Human cells in vitro: The growth of HeLa cells was inhibited by concentrations of caffeine above 300 µg/ml (Ostertag et al., 1965); exposure of these cells for 2 h to 1% caffeine had virtually no effect on cell cycle time (Kuhlmann et al., 1968).
Caffeine did not induce unscheduled DNA synthesis or hprt locus mutations in human cells.
Caffeine weakly induced sister chromatid exchange in most of eight studies with human leukocytes and in all three published studies with leukocytes or lymphoblastoid cells from patients with xeroderma pigmentosum. Dose-dependent increases were obtained in only two of the studies (Ishii & Bender, 1978; Guglielmi et al., 1982).
In contrast, numerous reports have described the induction of chromosomal aberrations in human leukocytes and in HeLa cell lines. In cultured human lymphocytes from people with the heritable fragility condition, caffeine enhanced the expression of fragile sites (Ledbetter et al., 1986; Smeets et al., 1989).
Mammals in vivo: In a large number of studies in mammals in vivo, caffeine usually failed to induce significant responses. While single-strand breaks were induced in mouse liver and kidney, there was no significant effect in host-mediated assays (incubation of bacteria in the intraperitoneal cavity or in-vitro testing against bacteria from the urine of dosed rats), in an assay for specific locus in a mouse germ-line cell or in a mouse spot test.
Variable responses were obtained, however, with respect to sister chromatid exchange: of seven reports, two gave negative results, one gave a weak positive result and four a significant positive response.
In a large number of studies on the possible clastogenic effects of caffeine, almost uniformly negative responses were obtained in tests for micronuclei, bone-marrow metaphases and dominant lethal mutation. Negative results were also seen in a translocation test, and chromosomal aberrations were not induced in metaphase-I cells of mouse spermatogenesis. In addition, no sperm abnormality was induced in mice. Among this wealth of negative data, three significant positive responses were seen in the micronucleus test; in each case, the doses were in the toxic range.
Effects of methylxanthines on relevant targets other than DNA: In this section, we consider the effects of the methylxanthines, caffeine, theophylline and theobromine, on non-DNA targets but which potentially lead indirectly to DNA damage, mutation and modification of the activities of xenobiotics (including carcinogens) co-administered with methylxanthines. These aspects have been reviewed (Kihlman, 1977; Roberts, 1978; Byfield et al., 1981; Haynes & Collins, 1984; Roberts, 1984; Althaus & Richter, 1987; Boothman et al., 1988).
Non-DNA targets that are important to the genotoxic and related effects of methylxanthines are (i) cytochrome P450s (see p. 322 et seq.), (ii) cAMP metabolism, (iii) DNA metabolism, chromatin structure and function and (iv) nucleotide pools.
(1) cAMP metabolism
It is well established that methylxanthines can inhibit the phosphodiesterase involved in the degradation of cyclic nucleotides (Leonard et al., 1987), i.e., the intracellular messengers that control a wide variety of phenomena not related to survival per se. The majority of the studies were performed in vitro with caffeine concentrations higher than levels encountered by humans in vivo.
In mouse B-16 melanoma cells, Kolb and Mansfield (1980) found that theophylline inhibited DNA synthesis, reduced cell growth rate, elevated intracellular cAMP levels and changed cell morphology. Since these effects are also caused by cAMP and its potentiators in other cell lines, the inhibition of DNA synthesis is assumed to be a secondary effect of the increased level of cAMP resulting from inhibition of cAMP phosphodiesterase by theophylline. cAMP is known to inhibit cell growth and the transport of metabolites; it also mediates contact inhibition, the formation of cytoskeletal structures and increases cell adhesiveness (Rajaraman & Faulkner, 1984). Therefore, the reduced uptake of 3H-thymidine by L5178Y cells observed by Lehmann and Kirk-Bell (1974) may also, in part, be mediated by the increased cAMP concentration (Kolb & Mansfield, 1980).
cAMP does not, however, mimic the effects of caffeine on chromosomal structure nor on the gap filling process in radiation-damaged DNA. Furthermore, in some plant cells in which methylxanthines induce chromosome damage, the presence of cAMP is equivocal (Kihlman, 1977). Therefore, the effects of caffeine on cAMP levels appear not to be involved in the induction of chromosomal aberrations.
(2) DNA metabolism, chromatin structure and function
The effects of methylxanthines in cells treated with mutagenic agents can be summarized as follows (Roberts, 1984):
(i) reversal of agent-induced depression of DNA synthesis;
(ii) reversal of agent-induced inhibition of replicon initiation;
(iii) decrease in size of replicons (also in the absence of DNA damage);
(iv) inhibition of elongation of nascent DNA (to high-molecular-weight, template-sized DNA);
(v) time-dependent incision of template DNA;
(vi) time-dependent formation of DNA double-strand breaks;
(vii) inhibition of excision of base damage;
(viii) induction of protein synthesis; and
(ix) prevention of S phase delay and G2 arrest (induction of premature mitosis).
These aspects are considered together because they appear to result from two interrelated actions of methylxanthines affecting chromatin: interaction with single-stranded DNA and inhibition of poly(ADP-ribosyl)ation reactions.
Methylxanthines, in particular caffeine, interact with DNA primarily at single-stranded regions. In living cells, there is only indirect evidence for such interaction (Althaus & Richter, 1987). The finding that the production of chromosomal aberrations by methylxanthines in bean root tips is strongly dependent on temperature, with a sharp maximum around 12°C, led to the suggestion that chromosomal aberrations may be the result of an influence of the methylated oxypurines on macromolecular hydration structures (Kihlman, 1977).
Inhibition of poly(ADP-ribose)polymerase was determined in nucleotide-permeable human lymphocytes following three days of stimulation with 2 μg/ml L-phytohaemagglutinin: theophylline (2 mM) gave 89% inhibition, theobromine (1 mM), 81%, and caffeine (2 mM), 35% (Althaus & Richter, 1987). This is an important finding because poly(ADP-ribosyl)ation reactions are distributed ubiquitously among higher eukaryotes and have been demonstrated in a number of plants and lower eukaryotes. Various lines of evidence indicate an involvement of poly(ADP-ribosyl)ation in the normal cell cycle of mammalian cells and, in particular, in the molecular events occurring during S phase. Distinct changes in the levels of biosynthetic activity of poly(ADP-ribose) were observed in cellular differentiation processes. The presence of poly(ADP-ribosyl)ation in yeast is controversial. No activity has so far been found in prokaryotic organisms. An inhibition of poly(ADP-ribosyl)ation reactions by methylxanthines may result in genetic effects and in the modulation of genetic effects induced by other agents (ionizing radiation, ultraviolet light, and mutagenic and carcinogenic chemicals), because these reactions are involved in all major chromatin functions, i.e., DNA repair, DNA replication and transcriptional activity. They influence the local organization of chromatin and, in particular, the architecture of active chromatin domains as a consequence of altered protein interactions.
Important acceptor proteins for poly(ADP-ribose) are histone H2B and histone H1, which are involved in the nucleosomal organization of chromatin and of polynucleosome structures, respectively. The production of DNA strand breaks, either directly (ionizing radiation) or enzymatically in the process of DNA-excision repair, is required for the stimulation of poly(ADP-ribose) biosynthesis. In excision repair, several steps have been shown to be affected by poly(ADP-ribose)polymerase inhibitors: incision and ligation are inhibited, excision is reduced and repair synthesis is usually stimulated. Inhibition by methylxanthines of poly(ADP-ribose) synthesis usually results in reduced repair. Many unrepaired lesions are lethal, and reduced survival of damaged cells is observed.
There is a positive correlation between the sister chromatid exchange-inducing potential and the inhibitory effects of chemicals that reduce the activity of poly(ADP-ribose)polymerase. There is no concomitant increase in the frequency of chromosomal aberrations or point mutations (e.g., hprt mutants). In contrast, in cells with damaged DNA, ADP-ribosylation inhibitors significantly increased the frequency of chromosomal aberrations induced by alkylating agents and other types of chemical mutagens, and also by ultraviolet or ionizing radiation, and raised the incidence of hprt mutants (which can result from deletions) but not of ouabain-resistant mutants (resulting mostly from amino acid substitutions). Overall, an altered poly(ADP-ribose) metabolism can have specific effects on genetic phenomena such as DNA excision repair (Roberts, 1978), clastogenicity and mutagenicity (Roberts, 1984; Althaus & Richter, 1987), but also on neoplastic transformation (Roberts, 1984; Boothman et al., 1988). In the last case, controversial results have been reported.
(3) Nucleotide pools
It has been established that genetic effects can be produced not only by radiation and chemical attack upon DNA, but also by disturbances in deoxyribonucleotide precursor pools. Some studies indicate that the purine analogue, caffeine, may affect DNA precursor metabolism (Haynes & Collins, 1984). Caffeine is known to inhibit enzymes of purine metabolism and may thereby alter the normal base ratio in the DNA precursor pool, thus causing errors in pairing. Caffeine doses enhance the killing action of ultraviolet light. It inhibits both de-novo synthesis and the utilization of exogenous purines in cultured CHO cells. Furthermore, caffeine inhibited incorporation of thymidine into DNA both in prokaryotic and eukaryotic cells. In E. coli, it has been suggested that this inhibition could be caused by a caffeine-induced inhibition of thymidine kinase or, more likely, an effect of caffeine on the DNA synthesis process itself. It was shown that, although thymidine kinase is inhibited by caffeine in E. coli cells, intracellular concentrations of thymidine triphosphate which one would consequently expect to decrease, actually increased significantly. Thus, the major effect of caffeine on the nucleotide pool appears to be the result of inhibition of processes that involve thymidine triphosphate. In these experiments, intracellular concentrations of the other nucleoside triphosphate pools were only slightly increased by caffeine. The finding that chronic exposure to caffeine led to sister chromatid exchange in human peripheral blood lymphocytes (Guglielmi et al., 1982) was interpreted to be a result of inhibition of DNA synthesis brought about by inhibition of de-novo synthesis of endogenous purines and also the transport and use of exogenous purines.
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